Advances in Botanical Research, Volume 42

Chemical Manipulation of Antioxidant Defences in Plants ROBERT EDWARDS, MELISSA BRAZIER-HICKS, DAVID P. DIXON AND IAN ...

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Chemical Manipulation of Antioxidant Defences in Plants

ROBERT EDWARDS, MELISSA BRAZIER-HICKS, DAVID P. DIXON AND IAN CUMMINS

School of Biological and Biomedical Sciences, University of Durham, Durham DH1 3LE, United Kingdom

I. Oxidative Stress In Plants and Exposure to Xenobiotics . . . . . . . . . . . . . . . A. Mechanisms of Chemically Imposed Oxidative Damage. . . . . . . . . . . . B. Counteracting Oxidative Damage Caused by Xenobiotics . . . . . . . . . . II. A Central Protective Role for GSH and GSTs . . . . . . . . . . . . . . . . . . . . . . . . A. Plant GSTs as Multifunctional GSH-Dependent Proteins . . . . . . . . . . B. GSTs and Xenobiotic Detoxification in Plants . . . . . . . . . . . . . . . . . . . . C. Roles for GSTs in Counteracting Oxidative Stress . . . . . . . . . . . . . . . . . D. Inducibility of GSTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Safeners. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Mode of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Safener Enhancement of GSTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Safener Enhancement of Other Xenome Components. . . . . . . . . . . . . . IV. Herbicide Safeners, the Xenome, and Cell Signalling . . . . . . . . . . . . . . . . . . A. Functions for an Inducible Plant Xenome? . . . . . . . . . . . . . . . . . . . . . . . . B. What is the Relationship between Safener-Responsive and Other Plant Signalling Pathways?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Do All Safeners Work the Same Way?. . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Safeners and Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Selective Enhancement of GSTs with Antioxidant Functions . . . . . . . B. Safener-Inducible GSTs are Active GPOXs . . . . . . . . . . . . . . . . . . . . . . . C. Safener induction of DHARs and GSTLs. . . . . . . . . . . . . . . . . . . . . . . . . D. Coordinated Upregulation of Antioxidant Metabolism . . . . . . . . . . . . VI. Safeners and Signal Transduction in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Gene Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Primary Signalling Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Advances in Botanical Research, Vol. 42 Incorporating Advances in Plant Pathology Copyright 2005, Elsevier Ltd. All rights reserved.

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0065-2296/05 $35.00 DOI: 10.1016/S0065-2296(04)42001-1

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VII. Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT Synthetic xenobiotics such as herbicides elicit the generation of reactive oxygen intermediates (ROIs) in plants, which induce antioxidant defences, notably the production of glutathione (GSH) and glutathione transferases (GSTs). GSTs also play an important role in catalysing the conjugation of xenobiotics with GSH, which leads to their detoxification. We have been interested in the multiple roles of GSTs in counteracting xenobiotic-induced stress in plants and the induction of these enzymes by herbicide safeners, which act without the need for large-scale ROI accumulation. Safeners are compounds that enhance herbicide tolerance in cereals and are known to induce the expression of diVerent classes of xenobiotic-detoxifying enzymes, which we have collectively termed the xenome. It has been proposed that safeners act by enhancing the detoxification of xenobiotics. However, we propose that safeners primarily protect plants by inducing a specific subset of antioxidant responses that relate to the endogenous functions of the xenome. Using GSTs as an example of multifunctional protective proteins, we examine this paradigm for safener action and what it tells us about antioxidant signalling pathways in plants.

I. OXIDATIVE STRESS IN PLANTS AND EXPOSURE TO XENOBIOTICS Reactive oxygen intermediates (ROIs) are a consequence of aerobic metabolism and in plants are constantly generated as a consequence of photosynthesis (Alscher et al., 1997). Under basal conditions, ROI generation is kept under control, but imposed biotic or abiotic stress leads to a massive increase in ROIs and the potential for oxidative damage to cellular constituents (Mittler, 2002). The classical cascade of oxidative reactions first involves the production of singlet oxygen (1O2), hydroxyl ions (OH), superoxide (O 2 ), and hydrogen peroxide (H2O2). These oxidising species then lead to the formation of organic hydroperoxides and the generation of cytotoxic alkenals (Fig. 1). In addition to their roles as damaging oxidants, ROIs are increasingly implicated in cellular signalling pathways that activate stress responses (Laloi et al., 2004; Scandalios, 2002). The plant cell, therefore, needs to closely regulate ROI metabolism to ensure signalling functions are not lost in the process of quenching their formation (Mittler, 2002). ‘‘Natural’’ conditions known to impose oxidative stress in plants include high levels of illumination, extremes of temperature, limited water availability, wounding, and infection (Laloi et al., 2004; Mittler, 2002). In addition, due to anthropogenic activities, plants are also exposed to oxidative stress imposed by organic and inorganic

CHEMICAL MANIPULATION OF ANTIOXIDANT DEFENCES

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Fig. 1. Imposition of oxidative stress by xenobiotics and the protective roles of glutathione transferases (GSTs). (top) The mechanisms of reactive oxygen intermediates (ROIs) generation by xenobiotics are shown, as well as (bottom) the toxic consequences to the cell. Reactions in enclosures show the intervention of GSTs in detoxifying oxidation products. ADH, alcohol dehydrogenase; APX, ascorbate peroxidase; DHAR, dehydroascorbate peroxidase; GPOX, glutathione peroxidase; GRX, glutaredoxin; GST, glutathione transferase; GSTL, lambda class of GSTs; ROI, reactive oxygen intermediates; TRX, thioredoxin.

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compounds (xenobiotics) released into the environment as pollutants or cropprotection agents (Sandermann, 2003). Irrespective of the cause of ROI generation, the defences that plants use to regulate ROIs and downstream oxidative damage involve a coordinated change in primary metabolism and the production of antioxidants. There is an increasing realization that these responses are tailored to the individual stress (Laloi et al., 2004). However, one of the most commonly conserved and best studied responses to oxidative insult in plants is the enhanced synthesis of the thiol tripeptide glutathione ( -glu-cys-gly; GSH) along with the accumulation of GSH-dependent proteins (May et al., 1998; Noctor and Foyer, 1998). In this chapter, we examine how synthetic xenobiotics can cause oxidative damage and the antioxidant responses they elicit, focussing on the central roles of GSH and the glutathione transferases (GSTs). In particular, we are interested in the observation that cytoprotective mechanisms can be induced by a specific group of chemicals called herbicide safeners, which elicit GSH and GST production without overt ROI generation or cellular oxidation occurring. Safeners are well known to enhance the expression of xenobiotic metabolising enzymes in plants, and we have determined that a consequence of this is that they also induce a subset of endogenous antioxidant secondary metabolism. Based on these findings, we now propose a paradigm in which the mechanisms by which plants regulate the detoxification of synthetic compounds are integrally linked to an essential component of the oxidative stress responses. To explore this new model, we need to first define the ways in which xenobiotics cause oxidative stress in plants and the protective mechanisms used in defence. A. MECHANISMS OF CHEMICALLY IMPOSED OXIDATIVE DAMAGE

Although herbicides have diverse chemistries and diVerent sites of action depending on their class, the ways in which they invoke phytotoxicity by causing uncontrolled oxidation in the cell are surprisingly similar. This is most clearly seen with the photobleaching herbicides, which act either by directly interfering with electron transport in photosystems I and II or by disrupting synthesis of the light-harvesting chlorophyll and carotenoid pigments (Edwards and Dixon, 2000). The result of such inhibition is the light-driven formation of singlet oxygen and superoxide, resulting in lipid peroxidation of the chloroplast and other organelles and causing the rapid degradation of cellular membranes and the release of toxic aldehydes and cell death (Fig. 1). Herbicides that inhibit essential but non-photosynthetic reactions do not cause rapid photobleaching. However, over a longer exposure time, the loss of cell turgor and the general inhibition of organised


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metabolism are all classically caused by the oxidation of cellular membranes and a build-up of toxic products. A clear demonstration of this was seen when hydroperoxide generation was determined in diVering populations of the weed black-grass (Alopecurus myosuroides) treated with a range of herbicides with diVering mechanisms of action (Cummins et al., 1999). Plants showing sensitivity to herbicides accumulated hydroperoxides irrespective of the mode of action of the applied compound. In contrast, populations that showed tolerance to diVerent classes of herbicides (multiple herbicide resistance [MHR]) showed much reduced levels of hydroperoxides accumulating when similarly treated. This has led us to propose that although chemicals may interact with diVering target proteins to initiate their primary disruptive eVects, that ultimately cytotoxicity results from uncontrolled oxidation, principally of membranes, due to the derailing and deregulation of primary metabolism (Fig. 1). B. COUNTERACTING OXIDATIVE DAMAGE CAUSED BY XENOBIOTICS

With the caveat that plants have had to develop unique protective responses to photooxidation, the chemistry of ROI generation and counteracting antioxidant action is conserved in animals and plants. Thus, we can learn a good deal about generic responses to xenobiotics from the well-developed literature in animals (Hayes and Mclellan, 1999). Protection against xenobiotic-induced damage is primarily exhibited at two levels: (a) Direct biotransformation of the xenobiotic to more polar and less toxic compounds that are suitable for export from the cell and ultimately excretion and (b) mopping up toxic intermediates formed from ROI generation. In animals, both levels of protection are enhanced after an initial exposure to cytotoxic drugs as a result of the induction of genes encoding key components of drug and antioxidant metabolism. Although there are obvious diVerences in the routes by which xenobiotic metabolites are ultimately processed by internalisation rather than excretion in plants as compared with animals, this dual level of protection is surprisingly conserved across the phyla (Sandermann, 1994). In particular, in both plants and animals, exposure to xenobiotics elicits a concerted increase in the cellular concentrations of GSH and changes in the expression of GSHdependent enzymes (Coleman et al., 1997; Hayes and Mclellan, 1999). As we will see, GSH can act as a cytoprotectant at two levels: acting to conjugating electrophilic xenobiotics to form polar nontoxic peptide derivatives and serving as a multifunctional antioxidant. In both cases, these activities are catalysed through the action of GSH-dependent proteins, notably the GSTs.

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II. A CENTRAL PROTECTIVE ROLE FOR GSH AND GSTs The utility of GSH in counteracting oxidative stress lies in the multifunctional activity of the sulphur atom of the cysteinyl residue (Mannervik et al., 1989). Thus, the sulphydryl group of GSH can act as a nucleophile, reductant, and scavenging agent for free radicals (Fig. 1). As a nucleophile, the negatively charged thiolate anion can be used in conjugation reactions with electrophilic compounds. When used as a reductant, GSH undergoes disulphide formation with itself to form GSSG. When acting as a radical scavenging agent, the thiyl radical of GSH then undergoes conversion to GSSG with the concomitant release of superoxide. The importance of GSSG formation is that this oxidised form can undergo reduction and recycling back to GSH through the action of glutathione reductase (Noctor and Foyer, 1998). In turn, GSH is used to maintain the ascorbic acid pool in its reduced state through the reduction of dehydroscorbate (Fig. 1). To achieve its central protective role in conjugating toxic electrophilic xenobiotics and counteracting the oxidative damage they impose, plant cells contain a range of GSH-dependent enzymes. The functions of these proteins can largely be divided into the directed detoxification of reactive xenobiotics or stress metabolites arising from endogenous metabolism, and functions in redox homeostasis/disulphide exchange. One particularly important group of stress-responsive GSH-dependent proteins showing both these activities are the GSTs. As shown in Fig. 1, these proteins exert a range of protective activity, which is particularly important in counteracting xenobiotic-imposed oxidative stress. A. PLANT GSTs AS MULTIFUNCTIONAL GSH-DEPENDENT PROTEINS

The plant GSTs (EC 2.5.1.18) are soluble proteins encoded by a superfamily of genes, which in plants can be divided into six classes (Dixon et al., 2002a,b), namely the phi (GSTF), tau (GSTU), theta (GSTT), zeta (GSTZ), and lambda (GSTL) GSTs, and the dehydroascorbate reductases (DHARs). Using a unified classification system, GSTs can be uniquely identified with respect to originating species, enzyme class, and polypeptide composition (Edwards et al., 2000). For example, the first phi class GST identified in maize (Zea mays) is termed ‘‘ZmGSTF1.’’ This classification system is proving to be useful in view of the large size of the plant GST superfamily. While animals contain zeta and theta GSTs, GSTFs, GSTUs, GSTLs, and the DHARs are found only in plants. GST polypeptides have a typical molecular mass of around 25 kDa and a modular active site composed of a


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conserved GSH-binding domain (G-site) and a more variable hydrophobic ligand-binding site (H-site). This conservation in GST structure is seen throughout nature, and its flexibility in design has allowed these proteins to fulfil multiple functions (Sheehan et al., 2001), as indicated for the plant enzymes in Fig. 1. In plants, the GSTFs and GSTUs use GSH in nucleophilic substitution/addition reactions in the detoxification of herbicides (Edwards and Dixon, 2000). The thiolate species of GSH is used to drive these reactions, with a conserved serine in the G-site helping to stabilise the formation of this reactive species. A similar active site chemistry is used in the GSTZs and GSTTs, albeit the S-glutathionylated conjugates in these cases are produced as reaction intermediates and do not accumulate as end products (Dixon et al., 2002b). Thus, the GSTZs catalyse the GSH-dependent isomerisation of maleylacetoacetate to fumarylacetoacetate in the course of tyrosine catabolism (Thom et al., 2001), whereas GSTTs form GSOH as an intermediate in the reduction of fatty acid hydroperoxides to the respective monohydroxy alcohols (Dixon et al., 1999). When acting in the reduction of hydroperoxides, the GSTs are termed glutathione peroxidases (GPOXs). As we will see, the GPOX activity of GSTs is of fundamental importance in counteracting oxidative stress (Fig. 1). The GST-GPOXs are distinct from the classic selenium-dependent GPOXs, which have been shown to be active in preventing membrane oxidation in plants (Yoshimura et al., 2004). All of the GSTFs, GSTUs, GSTTs, and GSTZs studied are soluble dimeric proteins composed of identical or closely related subunits (Dixon et al., 2002b; Frova, 2003). In contrast, the GSTLs and DHARs are monomeric proteins that diVer from all the other family members in containing a cysteine residue in the G-site in place of the serine (Dixon et al., 2002a). The consequence of this substitution is that DHARs and GSTLs cannot use GSH in conventional S-glutathionylation reactions but use the tripeptide in redox reactions. Finally, GSTs in both animals and plants have the ability to bind toxic xenobiotics and natural products (Sheehan et al., 2001). In the case of the plant proteins, GSTUs and GSTFs have been shown to bind bioactive flavonoids (Cummins et al., 2003; Mueller et al., 2000; Smith et al., 2003) and porphyrins (Lederer and Bo¨ger, 2003). It is, therefore, quite possible that the detoxifying role of GSTs in the presence of GSH can extend to reducing the bioavailability of xenobiotics and cytotoxins released due to cellular oxidation through ligand binding in addition to their known catalytic functions (Fig. 1). B. GSTs AND XENOBIOTIC DETOXIFICATION IN PLANTS

GSTs are part of a coordinated detoxification system for pollutants and pesticides in plants, which we have termed the xenome, defining it as the

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‘‘biosystem responsible for the detection, transport, and biotransformation of xenobiotics in the cell.’’ The xenome can be considered as consisting of two parts, which are responsible for detection and signalling and transport and biotransformation (Fig. 2). The signalling component is considered later in this chapter, but with respect to xenobiotic biotransformation and transport, the xenome consists of a large group of proteins that carry out four tiers of metabolism. In phase 1 metabolism, the parent xenobiotic undergoes hydrolytic or oxidative modification to introduce or reveal functional groups (OH, NH2, COOH), which can undergo further biotransformation. The best characterised phase 1 enzymes are the cytochrome P450 (CYP) mixed function oxidases, which are membrane-bound haem-containing proteins that catalyse a diverse range of oxidoreductive reactions on both endogenous and xenobiotic substrates (Werck-Reichhart and Feyereisen, 2000). In phase 2 metabolism, GSTs or type 1 glucosyltransferases (GTs) are responsible

Fig. 2. The plant xenome. Organisation of the four phases (circled numbers) of xenobiotic metabolism in plants and their transcriptional regulation. GST, glutathione transferase; GT, glucosyltransferase; MT, malonyltransferase.


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for conjugating the herbicide or its phase 1–activated metabolites with GSH or glucose, respectively (Cole and Edwards, 2000). Glucose conjugates may then be further modified by malonylation, before deposition of the polar conjugates into the vacuole in phase 3 of metabolism. There appears to be more than one mechanism for vacuolar import, although it is recognised that the adenosine triphosphate (ATP) binding cassette (ABC) transporter proteins are responsible for the transport of S-glutathionylated conjugates (Foyer et al., 2001). Finally in phase 4 metabolism, the polar derivatives from the vacuole undergo reexport into the cytoplasm and incorporation into endogenous metabolism, with the resulting residues frequently associated with cell wall components or other macromolecules (Skidmore, 2000). Overall, some idea of the complexity of the plant xenome can be obtained from Arabidopsis thaliana, the genome of which encodes 350 CYPs, 107 type 1 GTs, 55 GSTs, and 129 ABC-like proteins. As shown in Fig. 2, GSTs occupy a central role in the xenome and in many crops are primarily responsible for the rapid metabolism of major classes of herbicides including the chloro-s-triazines, chloroacetanilides, diphenyl ethers, and members of the sulphonylureas and aryloxyphenoxypropionates (Edwards and Dixon, 2000). As GSTs are up to 20 times more abundant in crops than in nondomesticated plants (Hatton et al., 1999) the metabolism of these herbicides is much slower in competing weeds. GSTs are, therefore, a major determinant for the selectivity of postemergence herbicides. An interesting feature of GST-mediated conjugation of herbicides in crops are the classes of enzyme predominantly responsible for the detoxification of each type of herbicide. Thus, in maize and sorghum, the detoxification of chloroacetanilide herbicides is carried out by the phi class of GSTs, whereas in soybean, wheat, and rice, this activity is largely attributable to the tau class of enzymes (Edwards and Dixon, 2000). Interestingly, this partitioning of activity reflects the relative abundance of the respective GSTF and GSTU expressed sequence tags (ESTs) in the diVerent species (Frova, 2003) and demonstrates that GSTs have catalytic functions that span the diVerent classes. C. ROLES FOR GSTs IN COUNTERACTING OXIDATIVE STRESS

Acting at a secondary level of defence, members of the GST superfamily can oVer diVering mechanisms of cellular protection from xenobiotic-imposed oxidative damage. A number of studies have pointed to the importance of the activity of specific GSTs in quenching the accumulation of cytoxic alkenals released following lipid peroxidation (Fig. 1). Based on what is known in animal cells, this can be achieved through their GST-GPOX

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activity directed toward fatty acid hydroperoxides, as well as the direct conjugation of alkenals (Hayes and Mclellan, 1999). The importance of the GPOX activity of plant GSTs has been demonstrated in three in vivo studies. Interestingly, in each case, although the constitutively expressed GSTTs have the greatest GPOX activity when assayed in vitro, it has been the GSTUs and GSTFs with this activity that have been shown to provide plants with demonstrable protection from oxidative stress in vivo. Considering the evidence in turn: (a) When a GSTF from tobacco with GPOX activity was overexpressed, the transgenic tobacco seedlings showed enhanced tolerance to biotic stress and changes in GSH metabolism, consistent with the enzyme acting as a GPOX (Roxas et al., 1997). (b) In black-grass plants, a GSTF with very high GPOX activity but a restricted ability to detoxify herbicides was found to be singularly expressed in populations showing MHR traits (Cummins et al., 1999). The expression of this GSTF was associated with reduced levels of hydroperoxides formed on exposure of the weed to several classes of herbicides, further reinforcing the role of this enzyme in quenching oxidative damage caused by herbicides rather than directly detoxifying them. (c) An intriguing link between GST-GPOXs and apoptosis has been revealed by expressing a GSTU from tomato in yeast (Kampranis et al., 2000). Transgenic expression of the tomato GSTU suppressed bax-induced apoptosis in the yeast apparently by preventing early signalling events associated with hydroperoxide formation. Subsequent studies in yeast revealed that six distinct GSTUs from tomato could protect the cells from hydroperoxideinduced damage, with each protein showing a distinct spectrum of activity toward diVerent oxidants (Kilili et al., 2004). Several of these GSTUs were also able to conjugate and detoxify the cytotoxic alkenals formed from hydroperoxides, suggesting a dual protective function by both reducing the hydroperoxides and mopping up the associated alkenal degradation products. Interestingly, similar dual activities have been demonstrated with both specific GSTFs and GSTUs from wheat (Cummins et al., 1999). Acting in a completely distinct way, the GSTLs and DHARs also function to counteract oxidative stress. A protein subsequently identified as an outlying member of the GST superfamily (Dixon et al., 2002a) was identified as the major extractable DHAR in rice (Urano et al., 2000). The DHARs have a defined role in maintaining the pool of reduced ascorbate through recycling dehydroascorbate formed from the dismutation of monodehydroascorbate (Foyer and Mullineaux, 1998). As such, a specific member of the GST superfamily, the DHARs, has a direct role in maintaining cellular redox status during oxidative stress (Fig. 1). The similar cysteinyl-based active site chemistry of the GSTLs is also suggestive of functions for these proteins in redox homeostasis. To date, the activity of the GSTLs has remained elusive,


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although GSTLs have been shown to be active glutathione-dependent thioltransferases (Dixon et al., 2002a). Other GSH-dependent proteins with this activity such as the glutaredoxins have functions in making and breaking protein disulphide bonds (Fig. 1). Therefore, the GSTLs probably have similar functions to the glutaredoxins, in making and breaking mixed disulphides between GSH and protein, which form during oxidative stress in the cell (Fig. 1). D. INDUCIBILITY OF GSTs

One of the characteristic features of the GSTs is their inducibility by conditions known to elicit oxidative stress. Taking Arabidopsis as an example, specific GSTs have been shown to be induced by dehydration and wounding (Kiyosue et al., 1993; Yang et al., 1998); exposure to the plant hormones (Smith et al., 2003), heavy metals (Smith et al., 2004), oxidants (Chen and Singh, 1999), or salicylic acid (Sappl et al., 2004); and after infection (Wagner et al., 2002). The GSTs most commonly identified as being induced in Arabidopsis using either diVerential messenger RNA (mRNA) analysis or proteomics are GSTUs or GSTFs. Similarly, these two classes of GSTs have been shown to be elicited by multiple biotic and abiotic stresses in many other plant species (reviewed by Marrs [1996] and Edwards et al. [2000]). In the many reports of inducible GSTs has been the consistent observation that a group of chemicals called herbicide safeners are particularly active in inducing GSTs, with this enhancement demonstrated in maize (Irzyk et al., 1995; Jepson et al., 1994), wheat (Cummins et al., 1997), and related Triticum species (Riechers et al., 2003), barley (Scalla and Roulet, 2002), rice (Deng and Hatzios, 2002a), sorghum (Gronwald and Plaisance, 1998), and Arabidopsis (DeRidder et al., 2002; Loutre et al., 2003; Smith et al., 2004).

III. SAFENERS Safeners, also known as herbicide antidotes, are a diverse group of agrochemicals with world-wide usage and enhance herbicide tolerance in cereal crops but not in competing weeds (Davies and Caseley, 1999). As such, they enhance herbicide selectivity, and some important safeners, which are referred to in the text, as well as their companion crops are shown in Fig. 3. Unlike the ‘‘safening’’ seen with sublethal prior applications of herbicides or other phytotoxins that enhance crop tolerance to subsequent herbicide applications, herbicide safeners provide protection without any overt damage.

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Fig. 3.

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Herbicide safeners.

A. MODE OF ACTION

The mechanisms by which safeners protect cereals from herbicide damage has been the subject of debate for more than 30 years, with two major hypotheses emerging (Hatzios, 2003): (a) Safeners act as herbicide


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antagonists by competing for binding at target site proteins, and (b) safeners reduce the bioavailability of herbicides at their site of action either by reducing their translocation or by enhancing their degradation. There has been some rekindling of interest in the antagonist theory following the observation that the safener dichlormid competes for protein binding in maize with the chloroacetanilide herbicides it ‘‘safens’’ against (Walton and Casida, 1995). However, the subsequent purification and identification of the safener-binding protein as a putative methyltransferase of unknown function was not consistent with it being a herbicide target site, which weakened the argument that the safener was acting as an antagonist (Scott-Craig et al., 1998). Instead, the observation that safeners accelerate the metabolism of diVerent classes of herbicides in large grained cereals such as rice, maize, wheat, sorghum, and barley has consistently supported the theory that safeners reduce the bioavailability of herbicides by enhancing their detoxification (reviewed by Davies and Caseley [1999]). B. SAFENER ENHANCEMENT OF GSTs

The enhancement of herbicide metabolism by safeners is now known to be achieved through the induction of key xenome components. Early studies concentrated on the safener-enhanced metabolism of chloroacetanilide herbicides in maize. In this crop, chloroacetanilides are predominantly detoxified by S-glutathionylation, and safeners such as dichlormid and benoxacor were found to increase the rates of herbicide conjugation (Davies and Caseley, 1999). This enhanced detoxification was due to the safenermediated induction of a specific phi class GST (ZmGSTF2), which was highly active in conjugating chloroacetanilide substrates (Holt et al., 1995; Irzyk et al., 1995; Jepson et al., 1994). Similarly, GSTFs active toward chloroacetanilides and related herbicides were shown to be selectively induced by safeners in sorghum (Gronwald and Plaisance, 1998) and barley (Scalla and Roulet, 2002). The dominance of GSTFs as safener-inducible herbicide-detoxifying enzymes originally suggested that this class of GSTs was predominantly responsible for herbicide metabolism in maize and other cereals (Droog, 1997). However, the detailed analysis of safener-treated maize seedlings revealed the presence of inducible ZmGSTUs in maize, which were active in metabolising chloroacetanilide and diphenyl ether herbicides (Dixon et al., 1998). Safener-inducible GSTUs with activity toward herbicides have now been described in rice (Deng and Hatzios, 2002b), wheat (Cummins et al., 1997; Pascal and Scalla, 1999; Riechers et al., 1997) and the wheat progenitor species Triticum tauschii (Riechers et al., 1997). The GSTs in wheat have been the subject of study (Cummins et al., 2003) and show some interesting

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diVerences for the corresponding enzymes in maize. Thus, GSTFs are the most abundant class of GSTs in maize at the level of the proteome (Irzyk and Fuerst, 1993) and are highly expressed as the respective transcripts (McGonigle et al., 2000). In wheat, GSTUs predominate (Cummins et al., 1997), although careful fractionation of the GSH-dependent proteins present also revealed the presence of GSTFs. Intriguingly, despite the diVerences in their relative abundance, the overall organization of GSTUs and GSTFs appears to be similar in maize and wheat with homologues of the major detoxifying GSTs identified in both species (Cummins et al., 2003). In cereals, GSTs are well known for their enhancement by safeners, and this is associated with increased herbicide tolerance. Because safeners do not promote significant protection against herbicides in any plant other than large-grained cereals, it has always been assumed that these compounds do not induce xenome components in wild grasses or dicotyledonous plants. This assumption is almost certainly incorrect. In the case of the nondomesticated weed black-grass, it has been demonstrated that GSTFs can be induced by the safener fenchlorazole ethyl (Fig. 3) in wild type populations (Cummins et al., 1999). In contrast, in MHR populations, these GSTs were already fully induced and unresponsive to safener treatment. Similarly, in dicotyledonous plants, GSTs have been shown to be induced by safeners. Thus, in Arabidopsis, a specific tau class enzyme AtGSTU19 was shown to be selectively induced by treatment with benoxacor (Fig. 3), with the GST shown to be active in detoxifying chloroacetanilide herbicides (DeRidder et al., 2002; Smith et al., 2004). C. SAFENER ENHANCEMENT OF OTHER XENOME COMPONENTS

With a considerable body of evidence pointing to the enhancement of GSTs by safeners, it is also clear that other xenome components are induced by these chemicals. For example, CYP-catalysed activities directed toward a range of herbicides have been shown to be enhanced in extracts from safener-treated wheat (reviewed by Davies and Caseley [1999]). GTs (Brazier et al., 2002) and ABC proteins (Gaillard et al., 1994; Theodoulou et al., 2003) in wheat and barley have also been shown to be induced by safeners. Thus, all of the three phases involved in the detoxification of xenobiotics are safener responsive (Fig. 2). As seen with the GSTs, it was presumed that the induction of CYPs, GTs, and ABC proteins was specific to cereal crops and not seen in dicotyledonous plants. As determined with the GSTs, this view is now challenged by the observation that a herbicidedetoxifying CYP was induced by safeners in tobacco cell cultures (Yamada et al., 2000).


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IV. HERBICIDE SAFENERS, THE XENOME, AND CELL SIGNALLING Although safeners clearly can enhance the expression of plant xenome components, which results in an increased rate of xenobiotic detoxification, the biological significance and mechanisms employed by this signalling system are far from obvious and pose three major questions, which are now considered in turn. A. FUNCTIONS FOR AN INDUCIBLE PLANT XENOME?

Because plants have only had to detoxify synthetic compounds for a relatively short period in their evolution, the plant xenome, and in particular its inducible components, must have arisen to carry out other important endogenous roles in metabolism. Drawing parallels with what is known about drug metabolism in animals, the obvious explanation for an inducible xenome in plants would be to counteract phytotoxins generated by invading microbial pathogens or allelopathic plants. This fascinating area of plant-pathogen and plant-plant interactions has received little attention. In the case of the GTs, it has been demonstrated that the mycotoxin deoxynivalenol from pathogenic Fusarium species is detoxified in plants by glucosylation and that the enzyme responsible is induced on exposure to the toxin (Poppenberger et al., 2003). However, in other cases, such a function for an inducible xenome is less clearcut. Thus, GSTs in Arabidopsis are known to be selectively induced in response to microbial infection (Wagner et al., 2002). Similarly, although several allelochemicals can be detoxified by GSTs in vitro (Cummins et al., 1997), there are no reports of the respective conjugates being identified in vivo. We conjecture that the emphasis on considering plant CYPs, GSTs, GTs, and ABC proteins in the context of detoxifying foreign compounds may be misleading when considering the functions of the xenome in plant metabolism. Instead, we should consider that although animals can use these enzymes only to detoxify bioactive compounds, plants can also use these enzymes in the synthesis of such products through their involvement in secondary metabolism. Thus, the endogenous functions of the xenome relate to the synthesis, conjugation, and intracellular transport of secondary metabolites, such as alkaloids, phenolics, and terpenoids. As such, changes in xenome composition on safener treatment which are known to enhance the detoxification of xenobiotics might also be expected to alter the accumulation of natural products. To examine this possibility, wheat seedlings were treated with and without the safener cloquintocet mexyl (Fig. 4). The plants were then left for 24 h

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Fig. 4. High-performance liquid chromatography (HPLC) mass spectrometry (MS) of phenolic metabolites from control and safener (cloquintocet mexyl) treated wheat. Wheat seedlings (10 days old) grown in the presence or absence of cloquintocet mexyl (10 mg/L1) were extracted in methanol and analysed by HPLCMS for UV-Vis–absorbing metabolites. The identities of phenolic metabolites was confirmed by photodiode array spectral analysis and time-of-flight MS after electrospray ionization.

before extraction and analysis of phenolic metabolites present by highperformance liquid chromatography (HPLC) with the compounds identified by diode array detection and mass spectrometry. The results showed a major shift in phenolic metabolism on safener treatment. Two unidentified ultraviolet (UV) absorbing metabolites eluting at 18.5 min and 21.5 min and tricinglucuronide disappeared, whereas peaks corresponding to ferulic acid and its ester, as well as the flavonoid C-glucoside isoorientin were seen to accumulate. This experiment demonstrates that safeners can cause specific reprogramming of plant secondary metabolism, in this instance leading to a selective accumulation of a phenylpropanoid and a flavonoid C-glucoside.


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B. WHAT IS THE RELATIONSHIP BETWEEN SAFENER-RESPONSIVE AND OTHER PLANT SIGNALLING PATHWAYS?

As discussed earlier, most xenobiotics trigger plant defence responses such as GST induction by acting to generate ROIs through perturbing oxidative metabolism. Induction by safeners is distinct from this, as these compounds cause no overt injury (Davies and Caseley, 1999), and our studies have shown that their action is not associated with large-scale ROI generation (unpublished observation, 1999). This suggests that safeners must be interfering with signalling events that are downstream of the primary ROI generation to elicit a subset of specific responses. The selectivity of induction by safeners as compared with chemicals that exert a general stress response was graphically demonstrated in a detailed proteomic study in Arabidopsis using GSTs as biomarkers (Smith et al., 2004). The induction of GSTs by the safener benoxacor was much more restricted than determined with copper salts (Smith et al., 2004). Similarly, the GST polypeptides induced by salicylic acid (Sappl et al., 2004) were distinct from those enhanced by benoxacor treatment. To examine the diVerential regulation of xenome components in Arabidopsis in further detail, we examined the expression of messenger RNAs (mRNAs) representing all six classes of AtGSTs following exposure to a cellular reductant (GSH), an oxidant (t-butyl hydroperoxide), a chemical toxin (1-chloro-2,4dinitrobenzene [CDNB]), an auxin analogue (2,4-dichlorophenoxyacetic acid [2,4-D]), and the maize safener dichlormid. Relative transcript abundance was determined by semiquantitative polymerase chain reaction (PCR) after normalizing each reaction using the actin 1 gene as an internal control (Dixon et al., 2002a) (results are shown in Fig. 5). DiVerential regulation of the six GSTs in response to the diVering treatments was clearly determined. Thus, although the theta AtGSTT1 was unaVected by any of the chemicals, AtGSTL1 was induced by all treatments. The safener dichlormid (Fig. 3) gave a modest induction of the phi (AtGSTF3), tau (AtGSTU2) and AtDHAR2 transcripts but had no eVect on the zeta AtGSTZ1. As such, this induction was not identical to that caused by the chemical toxin CDNB, which enhanced the expression of AtGSTU2 and AtDHAR2 but had no eVect on AtGSTF3. Similarly, the induction by GSH and 2,4-D diVered from that seen with dichlormid for these three GSTs, with only t-butyl hydroperoxide giving a similar spectrum of activation. This suggests that dichlormid was acting in a similar way to organic hydroperoxides, but this cannot be entirely correct because AtGSTZ1 was induced by t-butyl hydroperoxide but not by the safener. From this and other proteomic studies (Smith et al., 2004), we can conclude that the safening of GSTs is far from a generic ‘‘chemical stress’’ response, but a selective activation of discrete xenome proteins regulated through a unique pathway.

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Fig. 5. Transcript abundance as determined by quantitative polymerase chain reaction (PCR) of glutathione transferase (GST) family members in Arabidopsis cell cultures treated with chemicals. The percentage induction was determined by reference to cells treated with water. Treatments are as follows: GSH, glutathione; butyl


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C. DO ALL SAFENERS WORK THE SAME WAY?

By definition, safeners enhance herbicide tolerance in various cereal crops, and in every case reported, this is associated with the induction of GSTs and enhanced rates of detoxification (Davies and Caseley, 1999; Hatzios, 2003). However, there is considerable diversity in the chemistries that invoke safening, with each chemical being associated with a diVerent species (Fig. 3). The reasonable question that follows is do all these compounds act at the same point in the chain of signalling events that leads to the induction of GSTs? If there was specific binding of the safener to a receptor protein as proposed for the dichloroacetamide safeners in maize (Scott-Craig et al., 1998), the chemical diversity of these compounds would suggest ligand interactions with many receptors. This in turn would lead to every safener having the potential to diVerentially regulate GSTs. If, on the other hand, all safeners induce a similar metabolic perturbation in the cell, which then becomes the source of the signal, then all compounds would be expected to give an identical induction of GSTs. To test these two extreme scenarios of safener-mediated signalling, wheat and Triticum precursors were exposed to the safeners fenchlorazole ethyl, cloquintocet mexyl, and dichlormid, which have diverse chemistries and were developed for use in wheat and maize, respectively (Fig. 3). Modern bread wheat (Triticum aestivum) has a hexaploid AABBDD genome, whereas the progenitor species from which the genome was derived are the diploid Triticum tauschii (DD), and tetraploid Triticum turgidum (AABB), Triticum urartu contributing the AA genome. The reasons for including the progenitor species was that it has been demonstrated that diVerent safener-inducible GSTs have been donated from each genome, most notably from the DD genotype (Xu et al., 2002). In this experiment, we were, therefore, able to examine the induction of both GSTUs and GSTFs by the safeners in closely related Triticum species that have donated diVerent xenomes to modern wheat. The plants were exposed to safeners for 48 h and the complement of phi and tau GSTs present determined essentially as described previously (Cummins et al., 2003). Proteins in crude plant extracts were resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and then immunoblotted with antisera raised to a tau wheat TaGSTU1-1 and a phi class maize ZmGSTF1-2. These antisera had been shown to recognise wheat GSTUs in a class-specific manner (Cummins et al., 1997, 2003). A single 25-kDa polypeptide corresponding to TaGSTU1 and closely related GSTs H2O2, tert-butyl hydroperoxide; CDNB, 1-chloro-2,4-dinitrobenzene, dichlormid; NAA, naphthalene acetic acid; 2,4-D, 2,4-dichlorophenoxyacetic acid. Details of the experimental protocol have been published previously (Dixon et al., 2002a).

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(Cummins et al., 1997) was recognised by the anti-TaGSTU serum in each species. Treatment with cloquintocet mexyl gave negligible enhancement of TaGSTUs, whereas dichlormid and fenchlorazole ethyl gave good induction of the respective polypeptides in T. dicoccum, T. urartu, and T. aestivum but had no eVect on the DD progenitor T. tauschii. With the anti-GSTF serum, two polypeptides of 24 and 28 kDa could be identified in safener-treated plants, with the smaller GST subunit also being detected in the untreated plants. The observed induction of the GSTFs was both species and chemical specific. As determined with the GSTUs, cloquintocet mexyl gave only a weak enhancement of GSTFs. In contrast, fenchlorazole ethyl induced GSTFs in the progenitor species irrespective of genome, but not in hexaploid T. aestivum, whereas dichlormid was active in all but T. tauschii. These results clearly demonstrate that although safeners elevate total GST content in Triticum species, this is achieved through the diVerential induction of diVering isoenzymes. This observation points to safeners with diVering chemistries interacting with diVerent cellular signalling components rather than having a common single point of interaction.

V. SAFENERS AND OXIDATIVE STRESS Based on the evidence assembled throughout this chapter, we now propose a new paradigm for safener action, namely that the eVect of safeners on the xenome in general, and GSTs in particular, represents the activation of a subset of the oxidative stress response that is present in all plants to counteract cytotoxicity. Because ROI-induced signalling induced by ‘‘natural’’ biotic and abiotic stress is already known to proceed via multiple pathways (Laloi et al., 2004; Mittler, 2002), there is plenty of scope for safeners to interfere with the existing complex network of transduction components to produce responses that are both chemical and species specific. For reasons that we do not understand, safener responsiveness is greatly amplified in cereals as compared with other plants and can be exploited in crop protection to provide enhanced tolerance to herbicides. We presume that safener responsiveness is associated with an undefined useful agronomic trait associated with oxidative stress tolerance, which has been selected during domestication and breeding. We base this proposal on the evidence provided in the following sections. A. SELECTIVE ENHANCEMENT OF GSTs WITH ANTIOXIDANT FUNCTIONS

The fact that GSTs are known to conjugate synthetic xenobiotics and that their increased expression following safener treatment protects plants due to increased herbicide detoxification is entirely logical, but there have been few


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attempts to quantify this relationship. In fact, a review of the literature suggests that a correlation between safener-enhanced detoxification and increased herbicide tolerance has not been unequivocally established for any herbicide/safener/crop combination. To illustrate this point, safeners such as fenchlorazole ethyl (Fig. 3) are required to be coapplied with the aryloxyphenoxypropionate herbicide fenoxaprop ethyl to prevent unacceptable damage to wheat or barley (Tal et al., 1993). However, the safener promoted only a 40% increase in the overall rates of fenoxaprop ethyl detoxification, which proceeds via S-glutathionylation of the herbicide (Tal et al., 1993). One possibility to explain the disproportionate increase in herbicide tolerance is that a relatively modest enhancement in detoxification can provide major protection from chemical injury if the enhancement of the metabolising enzymes occurs either in or around susceptible tissues only. Intriguingly, a report has shown that safener-inducible GSTs that detoxify the herbicide dimethenamid accumulate in the dermal layers surrounding the coleoptiles in T. tauschii (Riechers et al., 2003). Because dimethenamid is selectively toxic to the coleoptiles, the expression of these detoxifying GSTUs around the site of herbicide action would aVord major protection while having only a modest eVect on the overall rates of metabolism in the whole plant. However, in other cereal crops such as maize, both safenerinducible GSTUs (Dixon et al., 1998) and GSTFs (Jepson et al., 1994) are expressed in multiple tissue types, suggesting that the expression of GSTs around herbicide target sites is not a universal phenomenon. Instead, the inability to explain the safener-mediated enhancement of tolerance on increased detoxification alone has led us to propose that induced GSTs must have secondary protective activities. Specifically, as shown in Fig. 1, we suggest that GSTs have two distinct activities in counteracting fatty acid oxidation and mixed disulphide formation, respectively, and that both these tiers of cytoprotection are activated by safeners. In doing so, they counteract the downstream toxicity caused by herbicide action and prevent phytotoxicity. B. SAFENER-INDUCIBLE GSTs ARE ACTIVE GPOXs

All of the safener-inducible GSTFs reported in cereals are highly active GPOXs. Thus, the major inducible GSTFs in maize (Dixon et al., 1997), wheat (Cummins et al., 2003), sorghum (Gronwald and Plaisance, 1998), and barley (Scalla and Roulet, 2002) actively reduced fatty acid hydroperoxides to the corresponding monohydroxy alcohols (Fig. 1). In cereals, this GST-GPOX activity is particularly associated with GSTFs, rather than the corresponding inducible GSTUs, which have much lower activities toward organic hydroperoxides (Cummins et al., 2003). Interestingly, the safener-inducible GSTFs in

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wheat (Cummins et al. 2003) and sorghum (Gronwald and Plaisance, 1998) were also active in conjugating toxic alkenals released from fatty acid peroxidation (Fig. 1). Therefore, the induction of these GSTFs by safeners would promote both the primary quenching of the hydroperoxides and the detoxification of the alkenal degradation products (Fig. 1). Interestingly, the possibility that S-glutathionylated alkenal metabolites accumulate in oxidatively stressed plant cells in a similar manner to that determined in animals cells (Hayes and Mclellan, 1999) would help explain the requirement for the co-induction of ABC transporter proteins along with GSTFs as they would be required for increased vacuolar deposition. C. SAFENER INDUCTION OF DHARs AND GSTLs

In addition to the GSTF-GPOX response, safeners induce GSTLs and DHARs (Fig. 4). These GSTs can have no possible role in herbicide metabolism, but the GSTLs in particular are highly responsive to safeners. Thus, GSTLs were first identified in maize because of their massive induction by benzenesulphonamide safeners and were termed the In2.1 proteins (Hershey and Stoner, 1991). Later studies also showed that a wheat GSTL termed cla40 was strongly induced by the safener cloquintocet mexyl but lacked any detoxifying activity toward xenobiotics (Theodoulou et al., 1999). Our PCR studies in Arabidopsis have shown that AtGSTL1, which is predicted to be expressed in the cytoplasm, is highly induced by the safener dichlormid (Fig. 3). AtDHAR2 is also modestly induced by this safener (Fig. 3). Because neither AtGSTLs nor AtDHARs can detoxify herbicides, any protective eVect the safener-inducible GSTs can exert must be mediated through their activities as redox proteins. In the case of the DHARs, we propose that safener induction would help promote the eYcient recycling of dehydroascorbate, thus maintaining the pool of reduced ascorbic acid required to prevent ROI generation (Fig. 1). In the case of the GSTLs, based on the current evidence, we propose that these proteins are induced because of their thioltransferase activity, which is required under oxidising conditions to maintain protein thiols in their biologically active form (Fig. 1). D. COORDINATED UPREGULATION OF ANTIOXIDANT METABOLISM

In addition to inducing the expression of GSTs, safeners are known to elevate GSH content in maize and other cereals (Farago et al., 1994). Early studies also demonstrated that this eVect was not confined to monocotyledonous plants, with dichlormid enhancing GSH content in


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tobacco cell suspension cultures (Rennenberg et al., 1982). GSH accumulation is stimulated by safeners enhancing the expression of enzymes of primary sulphur assimilation (adenosine 50 -phosphosulphate sulphotransferase and ATP-sulphurylase) and -glutamylcysteine synthetase, which is the penultimate enzyme in the GSH biosynthetic pathway (Farago et al., 1994). Our studies on the eVects of safeners on xenome metabolism now suggest that a further level of antioxidant protection is induced through the reprogramming of secondary metabolism to produce phenylpropanoid and flavonoid antioxidants (Fig. 4). These compounds have broad-ranging antioxidant activities and are widely used as cytoprotectants in plants during biotic and abiotic stress (Grassman et al., 2002). We now speculate that other classes of secondary metabolites with antioxidant functions will be similarly enhanced by safeners.

VI. SAFENERS AND SIGNAL TRANSDUCTION IN PLANTS The mechanisms by which safeners induce GSTs and other xenome components has received surprisingly little attention as compared with signalling systems regulating hormone action, wounding, and infection. This situation may well now change based on the observation that safeners function to induce gst genes in Arabidopsis (DeRidder et al., 2002; Smith et al., 2004), which will permit safening pathways to be studied using molecular genetics. Interestingly, the promoters from safener-inducible gst genes in cereals are also chemically inducible in dicotyledonous plants. Thus, the promoter of the lambda class GST gene termed In2-1, which was induced by benzenesulphonamide safeners was shown to be similarly responsive when controlling the expression of a reporter gene in Arabidopsis (De Veylder et al., 1997). When the safener-inducible promoter of the maize zmgstf2 gene was used to control the expression of -glucuronidase in potato, the gene was found to be fully responsive to application of the safener R329148 in the foliage (Robertson et al., 2000). Interestingly, the promoter was also constitutively activated in stem, root, and tuber tissue, which had some resonance with the selective developmental expression of zmgstf2 in maize roots (Jepson et al., 1994). These experiments reveal that the safener signal transduction pathway must be conserved between monocotyledonous and dicotyledonous plants and that the pathway is also subject to developmental control.

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R. EDWARDS ET AL. A. GENE REGULATION

At the level of gene regulation, although progress in identifying how the promoters of gst genes are regulated by plant hormones and development, the corresponding elements leading to induction by xenobiotics are yet to be reported. Thus, gstu gene promoters have been shown to contain auxin regulatory elements (Droog et al., 1995; Takahashi et al., 1995), although gstz genes are regulated through ethylene-responsive elements. Similarly, other gst promoter elements have been described, which are regulated either by infection or by fungi or are co-regulated with genes involved in anthocyanin biosynthesis (Marrs, 1996). Chemically inducible GSTs in animals contain regulatory elements in the promoters of the respective genes termed xenobiotic responsive elements (XREs) and antioxidant response elements (AREs) (Hayes and Pulford, 1995). These elements bind to regulatory proteins that promote gene transcription when the cell is exposed to xenobiotics (XRE) or oxidative stress (ARE). In the case of genes containing AREs in their promoters, activation follows the binding of the transcription factor Nrf2, which is selectively released during oxidative stress (Hayes and McMahon, 2001). Based on the selective induction of plant gst genes by safeners, it might be anticipated that they also contain ARE or XRE regulatory elements, but they have not been identified in any plant gst promoters. One promoter element identified in stress-inducible plant gsts is the 20-bp octopine synthase (ocs) element, also termed the activating sequence-1 (as-1) element (Xiang et al., 1996). In tobacco, the promoters of tau class gsts containing the as-1 cis-acting element confer inducibility to plant hormones, with as-1 behaving in a similar way to ARE elements (Droog et al., 1995; Ulmasov et al., 1995; Xiang et al., 1996). It, therefore, appears that the spectrum of chemicals (including xenobiotics) that activate gst promoters is dependent on the number and organization of as-1 elements (Ulmasov et al., 1995; Xiang et al., 1996). Whether organized arrays of as-1– like promoters regulate the safener inducibility of plant gsts is yet to be determined but would seem highly likely based on the regulation of these genes by other chemicals.

B. PRIMARY SIGNALLING EVENTS

From the evidence presented here, it seems highly unlikely that all safeners initiate signalling in the same way. If this were the case. then the induction of GSTs would not be safener specific (Fig. 6). We, therefore, can only speculate that signalling is mediated by one of the following mechanisms.


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Fig. 6. DiVerential induction of glutathione transferases (GSTs) by herbicide safeners in wheat and Triticum progenitor species. Seedlings of Triticum tauschii (Tt; genome DD), Triticum dicoccum (Td; genome AABB), Triticum urartu (Tu; genome AA) and Triticum aestivum (Ta; genome AABBDD) were treated for 48 h with either water (control) or 10 mg/L1 of the safeners fenchlorazole ethyl, cloquintocet mexyl, or dichlormid (see Fig. 3) essentially as described by Cummins et al. (2003). Total protein extracts were then normalised and separated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis before probing with antisera raised to the tau class wheat TaGSTU1-1 or the phi class maize ZmGSTF1-2. The molecular mass of the immunodetected GST subunits is shown in each case.

1. That safeners, or their bioactivated metabolites, bind to proteins that either directly or indirectly regulate the promoters of gst genes. The action of binding could be to either initiate activation or remove an endogenous

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Fig. 7. Potential signalling mechanism used by safeners in plant cells. Two safeners (X1-Cl and X2-Cl) show diVerential induction of glutathione transferase (GST) genes by either (a) each alkylating sulphydryl groups on specific receptor proteins (R1-SH, R2-SH), with the respective adducts then initiating distinct transduction events, or (b) initiating reactive oxygen intermediate (ROI) production and downstream oxidative events such as alkenal generation with these chemicals then initiating innate antioxidant signalling pathways. In the latter case, we conclude that the metabolic disruption caused by each safener must occur in a unique manner, which in the diagram is shown as occurring in distinct compartments.

repressor. In this model, we propose that each safener would interact with a diVerent regulatory protein. 2. That safeners, or their metabolites, act as either specific inhibitors or activators of enzymes that produce signalling molecules. This perturbation in the signalling agent then initiates the transduction events leading to gst gene activation. Candidate reactions for safener-mediated disruption would be pathways involved in ROI generation or downstream events leading to the production of stress metabolites like alkenals (Fig. 1), which are known to induce gsts in animals (Tjalkens et al., 1998). An important caveat for this latter model is that the metabolic disruption caused by the safener is both limited in scope and shows potential to cause multiple signalling outcomes either through compartmentation or through speed or scale of the response. Perhaps one important clue about the action of safeners is that they all contain electrophilic carbon atoms due to the presence of adjacent chlorine groups. As such, these compounds will have a tendency to alkylate-sulphydryl


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groups, which if regulatory either in the form of protein residues or metabolites could initiate the signalling cascade in a chemical-specific manner. Scenarios by which safeners might diVerentially elicit gst genes are shown in Fig. 7.

VII. FUTURE PERSPECTIVES Herbicide safeners comprise a fascinating group of chemicals that in addition to their applications in crop protection have utility in studying and manipulating oxidative stress tolerance in plants. They also represent a set of novel tools to unravel plant cell signalling networks relating to oxidative stress responses. Future challenges include identifying the primary interactions of these compounds with signalling components and the identification of the transduction chain, which ultimately results in gene induction. In this respect, the discovery that Arabidopsis is responsive to safeners now presents us with the opportunity of applying molecular genetic techniques to identify the genes involved. In addition, it will be of interest to apply proteomic techniques to identify proteins that interact with safeners. These studies in turn will provide additional new insights into the networking of oxidative signalling in plants which is already proving to be multilayered (Laloi et al., 2004). In addition to using safeners as chemical probes to study oxidative stress responses, these enzymes may also have utility in manipulating plant tolerance to adverse environmental conditions, including organic and inorganic pollutants. As such, safeners may extend their roles in counteracting xenobiotics from crop protection into phytoremediation.

ACKNOWLEDGMENTS The new paradigm on safening and the study of the xenome described in this chapter are subject of a personal development fellowship awarded to R. E. by the Biotechnology and Biological Sciences Research Council (grant no. 12/RDF/20613).

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The Impact of Molecular Data in Fungal Systematics

P. D. BRIDGE*, B. M. SPOONER{ AND P. J. ROBERTS{

*British Antarctic Survey, High Cross, Madingley Road, Cambridge, CB3 0ET, United Kingdom { Mycology Section, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, United Kingdom

I. Introduction to Fungal Systematics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Kingdom Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Traditional Systematic Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Development of Fungal Molecular Systematics . . . . . . . . . . . . . . . . . . . . . . . . A. Historical Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. DNA Regions Used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Phylogeny Reconstruction and Dating Radiations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Higher Level Systematics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Species Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Hydnellum Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Ascochyta Species. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Below Species Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Cryptic Systematics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Constraints to Molecular Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Rates of Sequence Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Reference Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Biogeography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Maintenance of Infrastructure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Advances in Botanical Research, Vol. 42 Incorporating Advances in Plant Pathology Copyright 2005, Elsevier Ltd. All rights reserved.

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0065-2296/05 $35.00 DOI: 10.1016/S0065-2296(04)42002-3

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ABSTRACT Molecular data has been used in fungal systematics since the 1970s, and its rate of incorporation has increased significantly in recent years. In phylogeny molecular data has already been used to clarify major evolutionary lines, and has aided in the delineation of higher taxonomic groups including the kingdom Fungi, and the main phyla within it. Molecular data has been used at all taxonomic levels and has allowed for a greater phylogenetic signal to be represented within systematic groups. At the higher levels this has led to the re‐evaluation of some orders and families, and at lower taxonomic levels it has helped in the identification of species, particular populations and possibly individuals. There are however some limitations to the widespread use of molecular data. Some of these relate to the comparability and utility of methods between diVerent fungal groups, some relate to the wide diversity of life cycles adopted by fungi, and others are due to the paucity of comparable definitive evolutionary markers. A significant limitation to the wider application of molecular data is the restricted range of data currently available, and the relation of this to the as yet unquantified numbers of undescribed species. Despite these limitations molecular data has had a very significant effect on our understanding of fungal systematics, and many further systematic aspects are likely to be elucidated in the future.

I. INTRODUCTION TO FUNGAL SYSTEMATICS A. THE KINGDOM FUNGI

The kingdom Fungi is a large, diverse group of organisms that range in size from simple yeast cells of less than 5 m in diameter, up to complex fruiting bodies that may reach 1 m in size, and to diVuse colonial organisations that are reported to exist over areas of more than 800 Ha (Anon, 1992; Barnard, 2000). The concept of a ‘‘fungus’’ has developed over many years, and the historic definition of fungi as nonphotosynthetic plants has been shown to be both too simplistic and phylogenetically inaccurate. The definition of the fungal kingdom has been refined as more functional, structural, chemical, and molecular information has become available and the kingdom is now loosely defined as nonphotosynthetic eukaryotes with cell walls containing chitin and -glucans, and a wholly absorptive nutrition. Fungi do not have amoeboid pseudopodial stages and may occur as both single-celled and multicelled organisms. Fungal cells contain mitochondria with flattened cristae, and Golgi bodies or individual cisternae are present (Carlile et al., 2001; Hawksworth et al., 1995). As this definition of the fungi has been developed, various organisms have become included or excluded from the kingdom. One example of this is the zoosporic Oomycetes, where the cell wall is largely composed of cellulose and glucans. This group includes wellknown plant pathogens such as species of Pythium and Phytophthora and the aquatic pathogen Saprolegnia, and this group is now accommodated in the

IMPACT OF MOLECULAR DATA IN FUNGAL SYSTEMATICS

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Straminipila (also spelled Stramenopila; see Leipe et al. (1994) and Dick (2001)) or Chromista (Kirk et al., 2001). Conversely, obligate mammalian parasites in the genus Pneumocystis, previously considered to be protozoan, have been placed as a basal group in the fungi (Pneumocystidiomycetes), largely on the basis of cell wall composition and analysis of DNA sequences (Pixley et al., 1991; Sjamsuridzal et al., 1997). Phyla within the fungi are defined primarily on the basis of life cycles, mode of reproduction, and cell wall and septum structure. Currently the kingdom consists of the Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, and Glomeromycota. Historically the Chytridiomycota has been placed in groups now included in the Straminipila, but it has been retained in the fungi on the basis of members having chitin in the cell walls and lacking mastigonemes, and DNA sequence analysis (Hawksworth et al., 1995). One important feature of the fungi is that many have life cycles that consist of two or more stages. These are primarily diVerentiated by the mode of reproduction and may be asexual, where growth and reproduction is by mitosis, or sexual, where reproduction involves meiosis. A fungus either producing asexual diaspores or no spores at all is termed anamorphic or imperfect, and the sexual state when present is termed the teleomorph or the perfect state. The mode of sexual reproduction is a major characteristic for assigning individual fungi to phyla, and a fungus that is known only from its vegetative state cannot be easily placed in a phylum using traditional morphological analysis. Historically such fungi were placed in an additional phylum, the ‘‘Deuteromycota.’’ This placement, however, suggests a phylogenetic relationship between vegetative fungi that has been demonstrated to be incorrect. Vegetative fungi are now considered as mitosporic forms of the major phyla, and the term ‘‘Deuteromycota’’ should not be used (Reynolds and Taylor, 1993). When anamorph and teleomorph forms both exist and occur separately, there are generally dual names, with one name being used for the anamorph and a diVerent name being used for the teleomorph. In some cases, the morphology of the anamorph can be very basic, and so apparently similar anamorphic forms may produce diVerent and sometimes unrelated teleomorphs. This occurs commonly in the Ascomycetes, and examples of common anamorphic genera with more than one genus of teleomorph include Phoma, Aspergillus, and Paecilomyces. The term holomorph is used for any specimen where the teleomorph and one or more anamorphic state is present, and in nearly all cases, the holomorph name is the same as that of the teleomorph. Although biochemical and molecular criteria are now used to define the fungi as a kingdom, the systematics of fungi has developed historically from

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observational characteristics, particularly those involved in growth and reproduction. Among the most important characteristics are those associated with the type of spore, the way in which it is produced, and the way in which it is released. As a result, it can be extremely diYcult to identify a fungus by classic methods if spores or spore-bearing structures are not produced, a major limitation to systematics, particularly with environmental or culture-based studies. Fungal nomenclature is governed by the International Code of Botanical Nomenclature (Greuter et al., 2000), and reference collections of type, and authenticated, material are maintained as dried specimens in herbaria. Although this is generally satisfactory for the larger fungi, smaller specimens and microfungi do not always retain key features or do not preserve as scientifically useful specimens when dried, and most microfungi are routinely maintained in culture. However, this is only practical when the fungi can be grown in pure culture, and for the many species that presently cannot be cultured, the reference material may be limited to dried material, often associated with material from a host organism. Dried material can be used to obtain both chemical and molecular data (Bruns et al., 1990; Paterson and Hawksworth, 1985), although the recovery of these will be dependent on the age of the material and its storage history. B. TRADITIONAL SYSTEMATIC TOOLS

Below the level of phylum, fungal systematics is largely based on life cycle and developmental characteristics, the gross morphology of the fruiting body, features of spore production and discharge, ultrastructure of walls, septa, and other structures, together with features of the host, habitat, and niche. An important feature of fungal systematics is that characteristics used to group or separate one group of fungi may be used diVerently in another group. An example of this is host specificity, where this characteristic is of major importance in defining species in many rust (Uredinales) and smut (Ustilaginales) fungi. In Fusarium, however, many diVerent host-specific populations may occur within a single species, and the host-specific groups are often further subdivided into cultivar-specific races (Armstrong and Armstrong, 1958). Two major limitations to the use of the traditional micromorphological and life cycle–based characteristics are the lack of specificity shown by some fungi, and a high level of phenotypic plasticity. Although highly host-specific pathogenic forms exist in Fusarium oxysporum, the species is a common soil organism and can occur on a very wide range of materials as a spoilage organism. Similarly, other genera such as Penicillium and Trichoderma can


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occur on a wide range of substrates, and individual species, such as Trichoderma viride, have been reported from both the polar regions and the tropics (McRae and Seppelt, 1999; Wijesekera et al., 1996). Many fungi can show considerable variation in morphological characteristics, both in culture and in the environment, and morphological features that may be good characteristics for species of some genera may be highly variable in species of other genera. In the genus Ganoderma, high phenotypic plasticity at the macroscopic level and the uniformity of microscopic characters have resulted in considerable taxonomic uncertainty, and Moncalvo (2000) cites these, together with subjective interpretation of features, as ‘‘responsible for the creation of numerous unnecessary names.’’ Similar statements have been made for other fungal genera, and although physiological, biochemical, or mating characteristics may aid in the delineation of individual species, they do not generally give phylogenetically reliable information. One possible exception to this is the use of secondary metabolites. As individual metabolites can be assigned to particular biosynthetic pathways or families, it has been suggested that these may in turn reflect phylogenetic origin (Frisvad, 1994). This approach has been used with lichen-forming fungi but has not been generally applied to other fungi with the exception of a few genera including Aspergillus, Fusarium, and Penicillium. However, studies on the correlation between metabolite production and molecular characters have suggested that metabolite production may be subject to both selective constraints and adaptive evolution from an ancestral organism, and therefore, metabolite production may not reflect recent evolutionary divergence (Varga et al., 2003; Ward et al., 2002). A small number of morphological and structural characteristics do, however, appear to be of fundamental phylogenetic importance, and this is most evident in the separation of the fungi into their constituent phyla. These divisions are predominantly based on the type and structure of the cell wall and hyphal septa, as well as the type and formation of the meiotic spores. These fungal phyla are largely supported by molecular analysis (Berbee and Taylor, 1993), and in some instances, it has been possible to identify particular DNA sequences that are specific to them (Gardes and Bruns, 1993). Lichens, often presumed to be a very distinct and separate group of organisms by nonmycologists, have long been regarded as a mixed bag of fungi, with most species assigned to various orders within the Ascomycota. Molecular evidence confirms this disposition, suggesting that the association of fungi with algae and/or cyanobacteria to form lichens may have occurred on at least five occasions in evolutionary history (Gargas et al., 1995; Grube and Winka, 2002; Lutzoni et al., 2001).

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II. DEVELOPMENT OF FUNGAL MOLECULAR SYSTEMATICS A. HISTORICAL PERSPECTIVE

The first attempts to use data derived from DNA in fungal systematics were in the late 1970s when techniques for giving overall measures of DNA sequence similarity were used for fungi (de Bertoldi et al., 1973). These methods had largely been developed in bacterial systematics, and they were generally introduced for the study of yeasts and microfungi. The simplest technique was to obtain an estimate of the proportion of the total genome that was made up of guanine/cytosine base pairs. The measure, termed mol% G þ C, gave a definite value that was used as an exclusionary character, that is, organisms with diVerent mol% G þ C values must be diVerent, but organisms with the same value were not necessarily similar. The method was used for a small number of fungal studies (de Bertoldi et al., 1973; Kuninaga and Yokosawa, 1980), but in general it was only widely applied in yeast systematics (Meyer and PhaV, 1970). Another method of determining overall DNA similarity that was also applied to fungal groups was the determination of overall DNA/DNA hybridisation values. This was achieved by extracting total DNA and hybridising single strands from two diVerent organisms together. The amount of annealing obtained was then compared to the annealing of homologous DNA samples, and a percentage hybridisation value was obtained. In bacteria, where there is a relatively simple genome, cutoV levels of more than 70–75% hybridisation could be used to define species, and lower levels could be used to define genera (Priest and Austin, 1993). However, such distinctions were not so apparent with the larger fungal genome, where considerable background similarity resulted in higher overall hybridisation levels, and the method was not widely used outside studies on speciation in yeasts (van der Walt, 1980). The first widespread use of molecular methods for fungal systematics came about with the development of methods for restriction fragment length polymorphism (RFLP) analysis with labelled probes. DiVerent target regions were used at diVerent taxonomic levels, but the most common approaches were to use probes for RFLP analysis of multicopy regions. In the fungi, the most commonly used were the nuclear ribosomal RNA (rRNA) gene cluster and total mitochondrial DNA (Pipe et al., 1995; Spitzer et al., 1989; Vilgalys and Cubeta, 1994). Again these methods were largely exclusionary, as similar RFLPs could only indicate common restriction sites and could not elucidate sequence diVerences between sites. DNA fragments obtained in such studies could be isolated and sequenced, and this


39

methodology was used together with systematic and phylogenetic techniques to generate trees showing relationships and possible phylogenies of taxa (Blanz and Unseld, 1987). The early sequence-based analyses were made from relatively short (100–400 bp) fragments of DNA, often from the 5S rRNA subunit (Blanz and Unseld, 1987), and it was the introduction of the polymerase chain reaction (PCR) and automated DNA sequencing that allowed more extensive sequence comparisons to be made. The overall development of the techniques and instrumentation has not been the only factor in the advance of molecular systematics for fungi, and further advances have been made from the use of the sequence information generated. Fungi commonly occur in close association with other organisms, and many species cannot be grown in pure culture in the laboratory (as discussed elsewhere). These factors can be major constraints in molecular studies, because it is often not possible to get DNA preparations that comprise solely DNA from the fungus of interest. Comparison of sequences from common DNA regions has allowed the identification of short sequences that occur only in fungi or within subgroups of the fungi, such as the Basidiomycetes or arbuscular mycorrhizal fungi (Di Bonito et al., 1995; Gardes and Bruns, 1993). These sequences can then be used to specifically amplify the fungal component of mixed DNA preparations, and so it has become possible to obtain PCR products from the fungal partner both in close associations and in environmental samples. This has allowed the generation of sequence data from many lichen-forming fungi, fungal mycorrhizae and endophytes, obligate pathogens, and fungi in soil and water samples (Bridge and Hawksworth, 1998; Lanfranco et al., 1998; Viaud et al., 2000; Ward et al., 1998). B. DNA REGIONS USED

Fungal molecular systematics has historically been based on analysis of the rRNA gene cluster and to a lesser extent mitochondrial DNA. Other gene regions have been used in some studies, but at the present time sequences from the rRNA gene cluster are the most extensive data set available, particular for the analysis of relationships between diVerent taxonomic groups (Edel, 1998). The fungal rRNA gene cluster, in common with other eukaryotic organisms, is a multiply repeated cluster that comprises the genes for the small ribosomal subunit, the large ribosomal subunit, and the gene for the 5.8S subunit. The 5.8S subunit gene is located between the small and large subunit genes, and the three genes are separated by two internally transcribed spacers. The individual gene clusters are separated by intergenic

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spacers that can contain extensive repeated elements and that in some, but not all, fungi also contain the 5S subunit gene (Hillis and Dixon, 1991). It is evident that diVerent regions of DNA in fungi can show considerably diVerent rates of evolution, and there are also suggestions that the rate of change of a single region may vary between fungal taxa (Bruns et al., 1991). It is, therefore, diYcult to select a single gene region that will provide a common level of phylogenetic or systematic resolution across the fungi, and such comparisons may require the adoption of multigene approaches as used with other organisms. In very general terms, sequence diVerences in the large and small subunit genes can often be used to diVerentiate families and genera, and diVerences in the internally transcribed spacers can often diVerentiate between species. In other instances, the sequences of the intergenic spacers can be used at species level or in some cases at a subspecific level (Bainbridge, 1994; Bridge et al., 2003; Bruns et al., 1991). These distinctions are generalisations, and in some cases there may be very little diVerence between internally-transcribed spacer (ITS) sequences between species, and in other cases there may be little homology within a species. A similar situation can occur with mitochondrial sequences where total mitochondrial RFLPs may be characteristic of both taxa and individual organisms (see later discussion). Within the mitochondrial genome, the most widely used sequences have again been those that include the genes for ribosomal subunits. These are not arranged in a cluster in the mitochondria and so occur as distinct individual genes. There are a number of complications to the use of mitochondrial sequences in fungi. The first is that, in general, they are subject to a faster rate of change than nuclear sequences (Bruns et al., 1991), and the second concerns the mode of mitochondrial inheritance. In common with animals, mitochondrial inheritance in fungi is generally unilinear, although bilinear inheritance, leading to a mosaic of mitochondrial types in a single organism, has been reported, as has mitochondrial recombination (Borst and Grivell, 1978; Kohn, 1992; May and Taylor, 1988). Where unilinear inheritance does occur, it may also involve some level of selection for a single mitochondrial type (de la Bastide and Horgen, 2003). The extensive use of single genes or DNA regions in systematics has been criticised, and it is now becoming increasingly common to use more than one gene. The simplest examples of this have been the use of both the rRNA spacer regions and one or more of the subunit genes (Begerow et al., 2002; Scorzetti et al., 2002; Sivakumaran et al., 2003) or the use of parts of both the small and the large subunit genes (Sugiyama et al., 2002; Voigt et al., 1999). Although this approach uses only one gene cluster, it does have the advantage of diVerent rates of change between the two regions. Precise figures for substitution rates in these genes in fungi are not available, but


41

gamma substitution rates of 1.65 for the LSU and 1.33 for the SSU were estimated by Wang et al. (1999) for eukaryotes overall. Other combinations of DNA regions that have been used have included both nuclear and mitochondrial subunit genes (Hofstetter et al., 2002) or have targeted entirely diVerent genes, two of the most widely used being the -tubulin genes and elongation factor 1a (Li and Edlind, 1994; Myburg et al., 2002; Shearer, 1995; Voigt and Wo¨ stemeyer, 2001). There have not been many publications that have used non-rRNA genes in large systematic studies, but two examples of this are the phylogeny of 54 genera of Zygomycetes made on the basis of actin and elongation factor genes (Voigt and Wo¨ stemeyer, 2001) and a higher level phylogeny of the major fungal lineages based on - and tubulin gene sequences (Keeling, 2003). Some of the other genes that have been used in systematic studies include chitin synthase, histone, peroxidase, cytochrome oxidase, and the mating loci (Barve et al., 2003; Maijala et al., 2003; Martin and Tooley, 2002; Mehmann et al., 1994; Myburg et al., 2002). C. DATA ANALYSIS

The great majority of fungal molecular data have been analysed by standard methods that result in some form of tree representation. For band data both distance and association coeYcients have been used, with trees drawn from clustering methods such as unweighted pair-group arithmetic average (UPGMA) (Bridge and Saddler, 1998). The most common methods of analysis of sequence data have been maximum parsimony analyses and distance-based approaches such as nearest neighbour. Some studies have used other methodologies, and the most common of these has been maximum likelihood (Bridge and Saddler, 1998; Pankhurst, 1991; SwoVord and Olson, 1990). There are inherent limitations and assumptions made when any of these or other methods of analysis are used. In general, these have not been recognised as well in mycology as in some other biological groups, and very few mycological data sets have been analysed by the alternative methods being developed in other areas of biology. Bayesian analysis involving Monte Carlo methods has been used in some instances (Maier et al., 2003), but these methods require considerable computational time and capacity and can result in very long run times for only restricted data sets. Two areas that are of particular concern in mycology are the lack of an adequate fossil record for phylogeny reconstruction and the determination of specific and subspecific taxa. Although standard methods can be used to reconstruct phylogeny from fungal molecular data, the validation of such phylogenies remains problematic (see later discussion). There is very limited

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information regarding fossil fungi, so there is little information on what taxa may have become extinct. Methods such as maximum parsimony assume that all current taxa have descended through the shortest possible lineages from an ancestral organism. This methodology assumes that extinctions have occurred relatively evenly throughout these lineages, and that the range of taxa included in the study is representative of the full range of extant organisms (Bridge and Saddler, 1998; SwoVord and Olson, 1990). In the case of the fungi, we know that many species cannot be easily detected in the environment, so it cannot always be assumed that the known taxa are entirely representative of the complete lineage. The lack of knowledge regarding extinctions can also cause problems because major parts of some lineages may have become extinct, and this could then aVect the most parsimonious route. Hennig (1966) pointed out that there were some shortcomings with using cladistic methods at the level of species and below, and a number of techniques such as the phylogenetic species concept (PSC) (Nixon and Wheeler, 1990) and the application of population aggregation analysis (PAA) (Davis and Nixon, 1992) have been investigated for other organisms. Recent consideration of PSC suggests that it may not cope well with plasticity (Goldstein and de Salle, 2000), and intuitively PAA could lead to the subdivision of some currently accepted fungal species concepts. However, PSC has recently been applied to fungal molecular data on a number of occasions when clear lineages based on sequence data could be determined (Zervakis et al., 2004). Analysis at the species level and below is further complicated in fungi where there can be major diVerences in biological or morphological species concepts between diVerent groups. One example of this is the genus Armillaria, where diVerent groups defined by sequence analysis within single species can be interfertile (Pe´ rez-Sierra et al., 2004; Sierra et al., 1999). The most common alternative to cladistic/phylogenetic approaches is to group individuals on the basis of overall similarity, such as common RFLP fragments, overall sequence similarity, and so on. This can, however, bring further concerns with the relatively small diVerences that can occur at the specific and subspecific levels, whereby a small amount of correlated variation becomes obscured by a much larger amount of random variation. Under these circumstances, methods that use some form of data reduction can provide better results, and it has been suggested that placing an emphasis on correlated variation may help to filter out random diVerences (Bridge, 1998). One such methodology that has been used increasingly with molecular data at these taxonomic levels is principle component analysis (PCA). In


43

PCA, correlated variations are combined into individual vectors that are then used to plot the positions of organisms in multidimensional (usually two- or three-dimensional) models. PCA has the advantage of using correlated variation, but the disadvantages are that it is a nonhierarchical method, and that it may be diYcult to accurately interpret the final scatter plots (Bridge, 1998; Dudzinski, 1975). A further concern in data analysis is the combination of two or more data sets from diVerent genes. Although in many instances, such approaches have led to congruent trees, investigation of the exact relationships between the data by partition homogeneity or convergence tests can raise concerns over the validity of combining the data, as indicated for sequences from Phytophthora by Martin and Tooley (2002). DiVerences in substitution rates for the genes studied may also lead to complications in combining data sets in multiple gene studies (Pupko et al., 2002), although the eVect of this in fungal studies has not yet been widely considered.

III. PHYLOGENY RECONSTRUCTION AND DATING RADIATIONS The introduction of molecular methods has resulted in some major changes to the phylogeny of the fungi. The classic definition of fungi as non-photosynthetic plants has now been discounted, and fungi have been shown to have evolved as a monophyletic group from the lineage that also gave rise to animals. This finding has been supported by analysis of a number of regions including the genes for rRNA SSU, elongation factor, actin, and - and -tubulin (Baldauf and Palmer, 1993; Bruns et al., 1991; Wainwright et al., 1993). Current phylogenies place fungi as a sister group to the microsporidiales, branching below the metazoan clade (Baldauf et al., 2000). The relationship of fungi and microsporidiales is, however, controversial, and phylogenies based on tubulin genes suggest that the microsporidiales may have arisen from a common ancestor to the Zygomycetes (Keeling, 2003). It is generally accepted that a systematic framework should be representative of the underlying phylogeny of the organisms in question. In the fungi, there are a number of limitations on arriving at a truly phylogenetic classification. One of the major constraints is the shortage of material available for dating radiations and apparent phylogenetic lines. Although some 950 species of fossil fungi have been described (Kalgutkar and Jansonius, 2000), this total represents a sparse sampling of the world’s ancient mycota and an adequate fossil record is not available.

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The reconstruction of fungal evolution has been restricted because of this scarce and fragmentary fossil record. Although the earliest recognisable fossil fungi have been obtained from the Devonian period (Remy et al., 1994), this date (400 MYA) is after the major radiations of the eukaryotic kingdoms and so, on its own, provides little information about the time scale for the origin of fungi. Molecular phylogenies, largely made from rRNA 18S gene, have shown the fungi to form a monophyletic group that diverged from the animal lineage at around the same time as the plants (Knoll, 1992; Wang et al., 1999). The timing of this divergence is, however, uncertain, and it has been suggested that it occurred at some time prior to the Cambrian era. Berbee and Taylor (2001) used a date of 965 (140) MYA based on the rRNA trees produced by Doolittle et al. (1996), whereas Wang et al. (1999) suggested 1576 (88) MYA in a study based on 50 genes. Subsequent divergences within the fungi are diYcult to date because of these discrepancies in starting dates; however, the early Devonian fossils are identifiable as mycorrhizal fungi, placing their divergence prior to that date, and clear Basidiomycete features have been recorded from material from the beginning of the Permian period (Dennis, 1970), suggesting an Ascomycete/Basidiomycete split possibly in the Carboniferous. Berbee and Taylor (2001) used these fossil records and others as calibration points to arrive at a substitution rate of 1.26% per 100 million years for the 18S rRNA, a figure comparable with the 1.33 gamma value determined by Wang et al. (1999).

IV. HIGHER LEVEL SYSTEMATICS As mentioned previously, the molecular re-evaluation of the fungi has resulted in a number of significant changes, and two of the most significant have been the removal of the Oomycetes to the Straminipila, as well as the inclusion of Pneumocystis and related organisms. The current concept of the fungi, therefore, consists of two sister groups comprising the Ascomycetes and Basidiomycetes, together with the Zygomycetes, Glomeromycetes, and Chytridiomycetes. Although Ascomycetes and Basidiomycetes clearly form two distinct groups, the relationships among the other phyla are less certain and these groups may yet prove to be polyphyletic. There is some evidence for both single and mixed lineages, and the Mucorales, Glomerales, and Mortierellales have all been suggested as distinct lines that are basal to the Ascomycete/Basidiomycete ancestry (Bruns et al., 1993; Voigt and Wo¨ stemeyer, 2001; Voigt et al., 1999). This suggestion has been supported


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by Schu ler et al. (2001) who considered that the glomeralean lineage was suYciently distinct to be considered a single phylum. Molecular methods have also had a significant impact below the major division level. Based on their superficial morphology, agarics (the gilled mushrooms and toadstools) would seem to be a well-defined group within the kingdom Fungi. However, even based on classic morphology, doubts have long been expressed on their homogeneity (Singer, 1951). These doubts have now been confirmed by molecular analysis, suggesting that ‘‘mushrooms and toadstools’’ include a wide range of fungi that have evolved multiple times from diverse ancestors (Hibbett et al., 1997). More extensive research has taken this further, resulting in a radical reorganisation of the agarics, together with the gasteroid fungi (e.g., puVballs, false truZes, and the like). Some of this is clearly reflected in the last two editions of Ainsworth and Bisby’s Dictionary of the Fungi, a long-standing and well-respected reference work for mycologists. The dictionary, which provides inter alia a taxonomic overview of all fungi and fungus-like organisms down to the level of genus, has undergone an extraordinary change between its eighth edition (Hawksworth et al., 1995) and its ninth edition (Kirk et al., 2001). The 32 orders of the class Basidiomycetes listed in the eighth edition have been replaced by just 16 orders in the ninth edition, almost entirely as a result of molecular research over a period of just 5–6 years. Some of these results have been summarised in a substantial 14-author paper (Moncalvo et al., 2002). Amongst the many surprises for the traditional taxonomist has been the incorporation of puVballs (Lycoperdon species) and stilt puVballs (Battarraea and Tulostoma species) within the family Agaricaceae, alongside everyday cultivated and field mushrooms (Agaricus species) and parasol mushrooms (Macrolepiota species). Previously, the puVballs, though often considered to have evolved from distant agaricoid ancestors, were thought of as radically distinct, in terms of their macromorphology, their basidiospores (very diVerent from those of the agarics), and their raindrop-mediated spore dispersal mechanism. As such, in the eighth edition of the Dictionary of Fungi (Hawksworth et al., 1995), they were placed in their own family (Lycoperdaceae) in their own order (Lycoperdales) within the Basidiomycetes. The equally distinct stilt puVballs were placed in a separate family (Tulostomataceae) in a separate order (Tulostomatales). By the ninth edition (Kirk et al., 2001), both orders had been subsumed within the order Agaricales, and in the summary by Moncalvo et al. (2002), both orders had been subsumed within the family Agaricaceae, making puVballs even closer to the true mushrooms than toadstools.

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V. SPECIES LEVEL The successful use of molecular techniques for the systematics of fungi at the level of species can be highly dependent on the technique used and the fungal group under consideration. In some instances, such as in various Colletotrichum species, there may be little or no variation in ITS sequences within a species and very little diVerence between species (Sreenivasaprasad et al., 1992). Although this then provides a definitive character for the species, problems may arise in interpreting small diVerences in sequences. In these instances, greater discrimination may be obtained by targeting other DNA regions or techniques. At the other extreme, there can be considerable variation within ITS sequences within a species. In the Basidiomycete fungus Rhizoctonia solani, diVerent individuals within the species that are vegetatively compatible can show up to 34% ITS sequence variation (Kuninaga et al., 1997), and at these levels it may be extremely diYcult to eVectively align the sequences under comparison. Alternatively, other DNA regions may be more appropriate for identifying the currently accepted species, and one example of this is provided by the agaric genus Armillaria, where IGS sequences have been used to define individual species (Coetzee et al., 2000). Some of the potential outcomes to using molecular methods at the species level with fungi can be demonstrated by looking at specific examples, as demonstrated in Hydnellum, a genus of stipitate-hydnoid (toothed) Basidiomycetes, and the coelomycetous (anamorphic) Ascomycete genus Ascochyta. A. HYDNELLUM SPECIES

The stipitate-hydnoid genus Hydnellum comprises a group of species producing large fruiting bodies whose spore-producing surfaces are borne on vertical spines (teeth). The group is ectomycorrhizal and associated with both conifers and deciduous trees (Pegler et al., 1997). Although a number of individual ‘‘species’’ have been described, based mainly on macromorphological distinctions, some are poorly characterised and it has been suggested that certain pairs of similar-looking species are not just closely related but may be synonymous. Seven Hydnellum species are accepted as occurring in the United Kingdom, and most are considered rare or endangered (Ing, 1992; Lizon, 1995). Some are associated only with conifers (Hydnellum peckii, Hydnellum aurantiacum, Hydnellum ferrugineum), some with deciduous trees (Hydnellum spongiosipes) and some with both types of tree (Hydnellum caeruleum, Hydnellum concrescens, Hydnellum scrobiculatum) (Pegler et al., 1997). Within the seven species, two pairs are similar morphologically: Hydnellum concrescens/H. scrobiculatum (found with both conifers and deciduous trees)


47

and H. ferrugineum/H. spongiosipes (the former with conifers, the latter with deciduous trees). There are six further synonyms in Hydnellum for the seven U.K. species, including Hydnellum diabolus, considered by some to be synonymous with H. peckii. This latter synonymy has not been widely accepted, however, and H. diabolus is generally considered a distinct species that does not occur in the United Kingdom. As some of the Hydnellum species are listed in the provisional U.K. Red Data List of Fungi (Ing, 1992) as rare or endangered, it is important that the individual species can be accurately described and delimited in order to ascertain their true conservation status. Molecular methods have been used to determine species concepts and investigate relationships within species pairs (Bridge and Panchal, 2004). In common with many other ectomycorrhizal fungi, species of Hydnellum have not been grown in artificial culture, and this limits the range of molecular techniques available. Although the fungi produce distinct fruit bodies, these grow indeterminately and thus will often incorporate external debris including moss, leaves, and twigs (Pegler et al., 1997). In addition, the fruit body often has small arthropods associated with it and in the latter stages of its life may also contain nematodes (Bridge, 2002, unpublished observation). Under these circumstances, it is very important to ensure that any DNA that is extracted and characterised has been derived from the fungus, and so the choice of method is limited to those that show some specificity for fungal DNA. A number of PCR primers have been developed for the amplification of rRNA gene clusters, and a number of these have enhanced specificity for fungi in general, and Basidiomycetes in particular (Gardes and Bruns, 1993). As part of an English Nature–funded project (Bridge and Panchal, 2004), total DNA was extracted from fresh collections and herbarium material of species of Hydnellum, and PCR was undertaken with the primers ITS1F (enhanced fungal specificity) and ITS4B (enhanced Basidiomycete specificity) (Gardes and Bruns, 1993). The amplification products from these reactions contained a single 700-bp product that included both of the ITS regions and the 5.8S gene. The sequences of the PCR products from 14 sequences from U.K. collections were analysed, together with reference sequences from H. diabolus and Hydnellum geogenium (AF351863 and AF351868), the latter being a distinct non-U.K. species that was included as a marker. The aligned sequences were recovered in an average distance tree in five distinct lines, each corresponding to a single species or species pair (Fig. 1). Line 1 consisted of sequences from specimens of the species pair H. concrescens/H. scrobiculatum, line 2 contained sequences from H. caeruleum, line 3 included H. peckii and H. geogenium, line 4 consisted of sequences

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Fig. 1. species.

P. D. BRIDGE ET AL.

Average distance tree of complete ITS/5.8S sequences from Hydnellum

from the H. ferrugineum/H. spongiosipes species pair, and line 5 contained the sequences from H. aurantiacum. The sequence from H. diabolus appeared as a single line when all sequences were aligned together, but when sequences were aligned in smaller subgroups, it showed its closest match to the H. peckii line. Overall, each of the seven U.K. species could be diVerentiated from each other and thus could be considered distinct species. On the basis of the sequences analysed in this study, there was up to 10% ITS sequence variation within a species, around 15–20% variation between closely related species and species pairs, and approximately 30% variation between the diVerent species lines. The 30% figure is high for a study of this type because it is generally recognised that sequences should be 60–70% similar to ensure accurate alignment. B. ASCOCHYTA SPECIES

The genus Ascochyta consists of some 350 species of anamorphic fungi that are largely associated with plants and cause various diseases of important crops (Boerema et al., 1993; Sutton, 1980). Individual species are generally pathogenic to individual plant species, although this is often an example of host preference rather than host specificity, as many can also cause diseases on alternative hosts (Sutton and Waterstone, 1966). Primary distinction of Ascochyta species


49

is on the basis of the host plant, the disease caused, and the morphology of the conidia and teleomorph. Despite their economic importance and their ease of culture, there are few rDNA sequences currently available for species of Ascochyta. A study of the species involved in diseases of legumes has provided ITS sequences for seven named species and varieties and three morphologically similar species of Phoma (Fatehi, 2000; Fatehi et al., 2003). Analysis of the ITS sequence variation of the diVerent Ascochyta species showed a maximum of 5% variation between any pair of sequences, with most species pairings being approximately 2% diVerent. However, there was also little variation (3–5%) between these species and the morphologically similar, but distinct, species of Phoma. Within each species, variation ranged from 0% to 1%, and this indicates that the ITS regions are very conserved between these fungi (Fatehi, 2000). An alternative gene sequence that has been examined for some members of this genus is the mating type locus (MAT) gene, where the sequences for the high mobility group (HMG) protein of the MATI-2 gene were found to be suYciently variable to delineate a number of species and to suggest the separation of the chickpea pathogen A. rabei, from the other legume associated species (Barve et al., 2003). An alternative DNA region that has been used with filamentous fungi is the mitochondrial genome, and one particular technique that has provided taxonomic information at various levels is obtaining RFLPs from A þ T–rich DNA that is presumed to comprise the total mitochondrial genome (Sagawa et al., 1998; Smith and Anderson, 1989; Typas et al., 1992). When applied to the species of Ascochyta, mitochondrial RFLPs show considerably more variation than the rRNA ITS sequences. Overall, between one and seven diVerent mitochondrial RFLPs were obtained for isolates of the Ascochyta and Phoma species when generated by two diVerent restriction enzymes. Although each species showed a number of RFLPs, no single pattern was shown by isolates of more than one species, so the individual RFLPs were characteristic of subgroups of the species (Fatehi, 2000). In most of the species examined in that study, there were no clear correlations between the RFLP subgroups and other characteristics (geography, pathogenicity, etc.) with one exception. The exception was the group of three species Ascochyta pinodella, Ascochyta pinodes, and Phoma medicaginis var. pinodella. These species had identical ITS sequences but diVered in their conidial morphology. All isolates that had been assigned to each name showed the same mitochondrial RFLPs, and the RFLPs were diVerent for each species name (Fatehi et al., 2003). Observations to date suggest that mitochondrial sequences can change at a faster rate than nuclear sequences (Brown et al., 1982). One interpretation of the above results is, therefore, that these three species’ names indicate recently evolved taxa that have developed from a single

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common ancestor. The species are all pathogenic on peas and beans, and these are crops that have themselves evolved rapidly through selective breeding over the last 2000 years, and there could possibly be a link between the evolution of the crop and the evolution of the pathogen. What becomes less clear under these circumstances, however, is at what point the three taxa are considered to be separate species, rather than recent adaptations of a single species. In this particular instance, there was some morphological support for the separation of the taxa, and although there were some uncertainties regarding their teleomorphs and the original type material, in this case the three species epithets were retained (Fatehi et al., 2003).

VI. BELOW SPECIES LEVEL Molecular methods have had a considerable impact in the delineation and identification of taxa at below the species level in fungi (Fungaro et al., 1996; Harlton et al., 1995; Maurer et al., 1997; Vilgalys and Cubeta, 1994). In many situations, such as the description of a plant pathogen, the assessment of environmental activities, or the use of a fungus in a specific application, the identification of a fungus to a particular functional or sibling group may be of greater practical benefit than the identification to species level. A wide variety of methods have been used to identify subspecific taxa in the fungi, the three most widely used being ITS and IGS sequence variation, mitochondrial DNA relatedness, and DNA fingerprinting methods. ITS and IGS spacer analysis and mitochondrial DNA RFLPs have been discussed earlier and can be used for species delineation. A number of DNA fingerprinting techniques have been used, and historically these included hybridisation and probe methods (see earlier discussion), and in current studies they all involve the amplification of some part or parts of the fungal genome. The three most widely used have been random amplification of polymorphic DNA (RAPD) (Welsh and McClelland, 1990; Williams et al., 1990), amplification of repetitive sequences (rep-PCR) (Arora et al., 1996; Edel et al., 1995; Latge´ et al., 1998), and amplification fragment length polymorphism (AFLP) (Mu¨ eller et al., 1996; Vos et al., 1995). For each of these general methods, there have been a number of variations to the general principles to give further methodologies including variable number tandem repeat PCR (VNTR-PCR) (Bridge et al., 1997a), microsatellite amplification (Buscot et al., 1996; Meyer et al., 1992), and sequence characterised amplified regions (SCARs) (Naqvi and Chattoo, 1996). When selecting methods for use at subspecific level, it is important to consider the basic biology and life cycle of the fungus being studied. The variability of life cycle seen in the fungi can result in diVerent interpretations


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being made for results from the same methodology for diVerent organisms. Probably the most important factor is whether meiosis occurs in the group under consideration. If meiosis occurs, then the resulting progeny and subsequent populations may be heterogeneous for some of the common nuclear genome-based fingerprinting methods such as RAPDs, AFLPs, and rep-PCR, whereas if meiosis does not occur, as in an apparently anamorphic fungus, these characteristics will remain largely homogeneous. Species of the insect pathogenic fungal genera Beauveria and Metarhizium occur almost exclusively in their anamorphic (mitotic) forms. Both genera contain species that are widespread in distribution and occur on a wide range of hosts. In Beauveria bassiana, subspecific groups have been identified on the basis of probe-derived RFLPs of the nuclear rRNA gene region, and these subgroups show some correlation with broad host groupings. When RAPD patterns were obtained from the same isolates, they were shown to delineate the same groupings, demonstrating that both rRNA and RAPDs could be used to reliably identify the same subspecific populations (Maurer et al., 1997). In the genus Metarhizium, the two species Metarhizium anisopliae and Metarhizium flavoviride occur on a wide range of insects world-wide. There have been a number of suggestions of geographically isolated and host-restricted populations within these species, and a number of criteria have been investigated (Bridge et al., 1997b; Fungaro et al., 1996). Driver and Milner (1998) identified two major lineages corresponding to these species based on rRNA ITS sequence analysis, with each lineage consisting of a number of subgroups that they identified as varieties. The diVerent varieties varied in their host range and distribution with some, such as M. anisopliae var. anisopliae, occurring world-wide on various hosts, some such as M. anisopliae var. acridum occurring on a restricted host range, and others such as M. flavoviride var. frigidum occurring only at relatively low temperatures. In each case, RAPD patterns were found to correspond to the major ITS sequence-based groupings, and in most cases the presumed genetic diversity of the population was supported by the level of RAPD variation in each group (Driver and Milner, 1998). The Basidiomycete bracket fungus Ganoderma boninense is a pathogen of oil palm, where it occurs within the palm tissue as a dikaryotic mycelium. This mycelium gives rise to a sporophore on the outside of the palm, and this produces monokaryotic basidiospores by meiosis. The individual basidiospores can be isolated and germinated to produce monokaryotic mycelium in culture, and colonies derived from individual basidiospores from within a single sporophore can each show distinct RAPD and AFLP patterns (Bridge et al., 2003; Pilotti et al., 2000). Although these amplification fingerprints can be diVerent for each single spore, they can also show some degree of

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homology. This situation is evident when progeny from a single sporophore are considered in isolation; however, it is often diYcult or impossible to identify the sibling groups by these methods when they occur in the environment together with spores from other unrelated sporophores (Pilotti et al., 2000). This diversity is, however, further complicated in the environment by the mating system present in Ganoderma. Ganoderma boninense has a tetrapolar mating system within a single sibling group (single sporophore). As a result there is a 1 in 4 possibility of mating between homothallic mycelia from two diVerent spores. However, the mating system is multiallelic, and there appear to be no barriers to mating between mycelia derived from diVerent sibling groups (Sanderson et al., 2000). This system favours outcrosses by 4 to 1, so the RAPD or AFLP obtained from dikaryotic mycelium is most likely to be derived from two sibling groups. As a result, it is necessary to identify the individual alleles within any characterisation method to follow recent descent and origin. The level of complexity seen for molecular markers in fungi can, however, be utilised to identify either individuals or small groups of closely related individuals. RAPD analysis has been used to identify subpopulations in isolates of Beauveria brongniartii occurring as a pathogen of the European cockchafer (Melolontha melolontha). A study of the subpopulations of B. brongniartii in Valle d’Aosta in Italy found that diVerent subpopulations could be identified at diVerent sites in the valley and in surrounding areas. One subpopulation outside the valley was, however, atypical, in that it appeared to have the same RAPD banding pattern as a subpopulation found closer to the head of the valley. Isolates obtained from the head of the valley had been used in field trials at the atypical site some years previously, and one interpretation of these results could be that the introduced population had become established (Cravanzola et al., 1997). In a large study on G. boninense in oil palm plantations in Malaysia and Papua New Guinea, Miller et al. (1999) and Pilotti et al. (2000) have shown that individual isolates may be traced within the plantation system by analysis of mitochondrial DNA, and that this feature was correlated with somatic compatibility between individual isolates. This has allowed the analysis of individual fungi across areas of oil palm estates, and the combination of mitochondrial DNA information with true monokaryotic matings has allowed the detection of individual alleles at diVerent times and locations within a single plantation/estate (Pilotti et al., 2000). The ability to obtain information at this level is of fundamental importance in determining the spread of a fungus, and in this case, where the fungus is an economically important plant pathogen, the results have suggested that basidiospores may have a key role in the spread of the organism (Pilotti and Bridge,


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2002). This information can then be used to suggest possible disease control strategies. Similar approaches have also been used in various fungal ecology studies, where the spread and maintenance of populations may be linked with ecosystem function (Swedjemark and Stenlid, 1995), and in conservation studies (Bridge, 2003, unpublished), where such information can be fundamental in developing a species conservation plan. In many instances, it is possible to detect variation in molecular characteristics within fungal species. The interpretation of this variation is, however, more diYcult. In some instances, a certain amount of variation may be due to methodology, such as with RAPD analysis (Lamboy, 1994a,b). In other instances, the amount of variation due to methodology is unclear, and it is, therefore, important to consider whether the variation observed is correlated with other characters. In some instances, the diversity seen with one marker may be mirrored by use of a diVerent marker, as was the case in some of the examples discussed earlier. In the case of B. bassiana, interspecific groups identified by RAPD correlated with those obtained from rRNA RFLP analysis (Maurer et al., 1997); in Metarhizium, individual varieties showed variable but distinct RAPD patterns (Driver and Milner, 1998); and a similar situation occurred with mitochondrial RFLPs in Ascochyta (Fatehi, 2000; Fatehi et al., 2003). In instances where other molecular markers are not available, there may be strong correlations with other factors such as host or distribution. One example of this is rRNA ITS variation in the lichenised fungus Parmelia sulcata. This organism was common in urban areas but decreased as air quality declined. Analysis of the populations of P. sulcata from country areas where air quality has not declined shows the presence of three populations defined by ITS length polymorphisms. Parallel collecting of P. sulcata in the United Kingdom and Spain showed that where it was recolonising urban areas as air quality improved, the new colonist apparently always belonged to the same single ITS length group (Crespo et al., 1999). This would suggest that the ITS length polymorphism correlated with either an improved ability to spread and colonise or to an improved ability to tolerate atmospheric pollutants. However, no other markers were available to test these hypotheses.

VII. CRYPTIC SYSTEMATICS One area of systematics where molecular methodologies are making an important impact is in the characterisation of cryptic taxa. As discussed later, only a relatively small proportion of the predicted numbers of fungi are currently described, and of these many do not grow in culture or produce

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characteristic features. As a result, there are many fungal taxa that cannot be adequately examined by traditional methods. Molecular techniques potentially provide a methodology for characterising some of these cryptic groups and for incorporating them into a systematic framework. One area where this approach has been demonstrated is the use of molecular methods to assess fungal diversity in soils and other environmental samples. Historically, soil fungi have been characterised by direct observation or by culture techniques sometimes linked to baiting methods. Studies have shown that when total fungal DNA is extracted from soil samples and characterised, the taxa that are identified do not always reflect those obtained from microscopy and culture (Gardes and Bruns, 1996; Lawley et al., 2004; Viaud et al., 2000). Such findings can have significant implications for biodiversity and ecology studies but in general have little impact on systematics. However, one other result of molecular diversity studies has been the identification of novel fungal DNA sequences. These new sequences can often be aligned with sequences from known fungi and have sometimes been placed as separate distinct groups in phylogenies, indicating that they may represent undescribed taxa that are closely related to known groups. However, in some instances sequences found in this manner have not been unequivocally aligned with any known groups, and it has been suggested that they may be basal organisms or represent lineages yet to be determined (Amaral Zettler et al., 2002). It should, however, be remembered that systematically useful DNA sequences are available for only some 20% or less of known fungi, and it is likely that some of these unidentified sequences will be from known taxa that have not yet been investigated (see later discussion).

VIII. CONSTRAINTS TO MOLECULAR METHODS A. RATES OF SEQUENCE EVOLUTION

There has been considerable debate over whether a phylogeny or systematic arrangement derived from molecular data is truly representative of the ancestry of the organisms in question or whether it represents the ancestry of that gene, that is, a gene tree (Bruns et al., 1991; Cotton and Page, 2003). As this has been extensively discussed, it will not be considered directly here, apart from some limited fungal examples. It should be remembered that some of the sequences commonly used in fungal systematics occur as multiple copies within the nucleus, and there is some evidence to suggest that not all copies are identical. This is further compounded where fungi are binucleate or multinucleate, and additional copies may also be present. Several


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studies have highlighted individual cases of multiple rRNA sequences occurring in an individual isolate or specimen (Fatehi and Bridge, 1998; Sanders et al., 1995; Zervakis et al., 2004). These may be isolated examples, but there have not been any wide ranging studies in this area. A similar situation that has not been widely examined for fungal data is where individual genes within a gene family have arisen through a historic duplication eVect. Bowen et al. (1992) examined chitin synthase genes in a wide range of ascomycetes and found that individuals contained mixed genes from two or three gene families. By separating the individual gene families, they were able to demonstrate that the same groupings and lines of descent were apparent in each family, suggesting one or more early duplication events. One related situation is the apparent diVerence in rates of change of some DNA regions in diVerent fungi. One example of this is in the use of mitochondrial information. Simple total mitochondrial DNA RFLPs have been obtained for fungi in many instances. In some cases, the RFLPs obtained have been highly variable within a species, with individual RFLP patterns being correlated with hereditary lines within subspecific populations (Miller et al., 1999; Whittaker et al., 1994). In other cases, the mitochondrial RFLPs have shown much greater levels of conservation, with a small number of individual RFLP patterns being associated with large subpopulations in a single species (Varga et al., 1993). One reason for this is that there is not just a single mechanism for mitochondrial inheritance. Although the usual mechanism in fungi is unilinear inheritance from the major cytoplasmic donor (Kohn, 1992), unilinear inheritance from the minor cytoplasmic donor (paternal), bilinear inheritance, and recombination have all been reported (Earl et al., 1981; Jin and Horgern, 1994; Milgroom and Lipari, 1993; Taylor, 1986). There are various other examples of situations where molecular information appears relevant at diVerent taxonomic levels in diVerent groups, one being the study of introns within the rRNA subunit genes. Introns in these genes have been used as markers of phylogeny in some lichen-associated fungi (Gargas et al., 1995) and as markers of subspecific populations in some mitosporic Ascomycete fungi (Neuve´ glise et al., 1997). B. REFERENCE MATERIAL

A major limitation to the implementation of molecular systematics in fungi is the range and availability of reliable reference material for comparison and interpretation. It has been estimated that only 17% of the 80,000þ accepted fungal species can be grown in pure culture (Hawksworth, 1991). These organisms provide a source of easily handled material for generating DNA sequences, but they form only a limited part of the fungal kingdom.

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Dried specimens of many free-living fungi are available, and although these can provide DNA sequences (Bruns et al., 1990; Taylor and Swann, 1994), the age, preparation, handling, and storage of the material can all aVect the recovery of DNA (Cubero et al., 1999). As a result, it is not always possible to obtain DNA of suYcient quality for sequencing studies from dried collections. Many fungi occur in close association with other organisms. In simple situations with a single fungus and a single host or associated organism, the fungal DNA may be obtained by the use of primers specific to the fungal DNA. This approach has been used successfully in obtaining fungal DNA from lichen specimens and in obtaining the DNA of a single fungal pathogen from plant or animal tissue (Crespo et al., 1999; Gardes and Bruns, 1993). However, some fungi occur in close association with others, so attempts to obtain DNA from a particular fungal component may result in the purification of DNA from other fungi. There are many examples of this, but three particular areas for concern include fungal endophytes in plant material, lichenicolous fungi occurring on lichens, or fast-growing microfungi on larger fungi. The second and third of these instances are also concerns when isolating fungi into pure culture, particularly when all the fungal partners grow as largely undiVerentiated mycelium. Assuming that good DNA sequences can be obtained in a systematic study, their use in any wider study is dependent on the availability of suitable reference sequences. The primary sources of reference sequences for most fungal systematists are the public-access sequence databases held at NCBI and EMBL. These are, however, a very limited resource, as of the accepted fungal species, less than 14,000 (16%) are represented by any sequence data. Concerns have been raised about accuracy of some of these sequences, and a study has suggested that up to 20% of the fungal rRNA gene associated sequences may be compromised or incorrect, further reducing the number of reference sequences available (Bridge et al., 2003). These problems have been recognised by many mycologists and information on known contaminant sequences is becoming increasingly available (e.g., see http://www. tu-darmstadt.de/fb/bio/bot/schuessler/amphylo/amphylo_contam.html). The shortage of reference material is a major constraint to the use of direct molecular methods with environmental samples, as the isolation of a novel sequence will not necessarily indicate the presence of a novel organism, but merely one that is not currently in the database. C. BIOGEOGRAPHY

The extent of geographical distribution for fungi remains largely unknown. For the larger fungi, there are some clear distinctions between tropical and temperate species, and there are also some major diVerences in species


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distribution associated with continents and other geographic features (Zervakis et al., 2004). The exact nature of these distributions is, however, uncertain, and there has been an increased reporting of species normally considered rare or nonnative in the United Kingdom on woodchip mulches (Shaw et al., 2004). What is not clear, however, is whether these reports relate to an influx of larger fungi from other regions or whether the fungi were always present in a dormant or nonfruiting state, and this is a situation that may be resolved through molecular detection and characterisation studies. Many of the filamentous fungi and yeasts are considered cosmopolitan in occurrence, with some species occurring in polar, temperate, and tropical regions, so their radiations cannot be easily associated with continental breakup and drift. This is further compounded by some studies on airborne particles that suggest that fungal spores may be transported over significant distances (Chalmers et al., 1996). It is not known to what extent this leads to isolation and speciation in the microfungi, and molecular studies at both the population and the phylogenetic level could be important tools for determining this (Goodwin et al., 1994; Nuytinck et al., 2004; Pe´ rez-Sierra et al., 2004; Zervakis et al., 2004). The widespread distribution of fungi may also have implications for the estimation of overall species numbers. Host specificity can be used to associate fungi with known plant or animal radiations, but this assumes that the host specificity is an ancestral trait, and that it is always maintained. This assumption has not been proven, and in the case of the entomoparasitic genus Cordyceps, one molecular phylogeny has suggested a possible drastic host shift (Nikoh and Fukatsu, 2000). Another entomoparasitic species, Verticillium lecanii, is found almost exclusively on arthropods, but it has also been found in mineral crusts in Antarctic rock in the absence of any potential arthropod hosts (Hughes and Lawley, 2003). The ITS sequence of the Antarctic collection, however, shows no diVerences from that of tropical insect pathogenic isolates (Hughes, 2003, personal communication), a finding that may suggest that this collection was not a separate evolutionary line. Some fungi, such as rusts or vascular pathogens, can develop cultivar specificity over a short period, so it may not be appropriate to assume that host specificity is always an indication of phylogeny. D. MAINTENANCE OF INFRASTRUCTURE

There has been considerable discussion regarding the apparent decline of systematics in biological research. In the United Kingdom, this concern has resulted in reports in the House of Lords and various associated submissions (House of Lords, 2002). In mycology, where there is a relatively small number of practicing systematists, the loss of individual posts or reference

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collections can have a major eVect. Historically, many systematic mycologists have specialised in the study of specific groups of fungi, and the loss of these positions can result in a significant loss to the larger mycological community. In the United Kingdom, there is already very limited research into many fungal groups outside of the agricultural and food areas, and the capacity to obtain accurate identifications is becoming very limited. This problem is not confined to the U.K. but also applies to many other countries and has significant implications for the future support of molecular methodologies.

IX. CONCLUSION The introduction of molecular methods in systematic mycology has had a major impact in defining taxa and relationships at all systematic levels. This eVect has increased rapidly over the last 15 years or so as molecular methods have become more available to systematists. Molecular methodologies have played a key role in defining the fungal kingdom and are still providing new information for determining major systematic divisions. At lower taxonomic levels, new orders, genera, and species are being described and existing taxa are frequently redefined. The availability of molecular data is important to place these taxa within a possible evolutionary framework and to determine potential relationships between them. The available information for the fungi is, however, very limited in relation to the estimated size of the fungal kingdom, and the use of molecular detection methods is becoming increasingly important for detecting natural diversity and identifying new taxa.

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Cytoskeletal Regulation of the Plane of Cell Division: An Essential Component of Plant Development and Reproduction

HILARY J. ROGERS

School of Biosciences, CardiV University, P.O. Box 915 CardiV CF10 3TL, United Kingdom

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 A. Cytoskeletal Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 II. How is the Plane of Cell Division Defined? .. ... ... ... ... ... ... ... ... ... ... ... . 75 A. Mechanical Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 B. Plant Growth Regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 C. Cell-Cell Signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 D. Light Perception. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 E. Cell Cycle Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 III. Implementation of the Decision on the Orientation of Division . . . . . . . . . 85 A. The Pre-Prophase Band. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 B. The Phragmoplast and Cytokinesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 IV. Consequences of the Orientation of a Cell Division . . . . . . . . . . . . . . . . . . . . 90 A. Coordinating the Plane of Cell Division . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 V. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

ABSTRACT In this chapter, the role of the cytoskeleton in regulating the plane of cell division is examined as three interlinked processes. In the first, the cell perceives internal or external signals, which direct the plane and orientation of its future division. Such signals can be acting at short range such as mechanical stress or cell-cell signalling or Advances in Botanical Research, Vol. 42 Incorporating Advances in Plant Pathology Copyright 2005, Elsevier Ltd. All rights reserved.

0065-2296/05 $35.00 DOI: 10.1016/S0065-2296(04)42003-5

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may come from a longer range coordination of plant development, such as plant growth regulators. The central role of the cortical microtubule (MT) array is discussed, and a range of mutants and model systems are considered, which help to understand the role of the cytoskeleton in determining cell shape, cell polarisation, and division. Once signals have been perceived by the cell, the correct plane is marked, initially through a specialised MT array, the pre-prophase band (PPB). Because this structure disappears before the process of cytokinesis begins, much attention has been paid to how the cytokinetic apparatus positions the new plate to coincide with the positioning of PPB. The role of actin patches is discussed, again in light of useful mutants. In the final section, the consequences of the orientation for plant morphology are considered, and how the cytoskeleton may be involved in coordinating the plane of cell division across whole organs of the plant.

I. INTRODUCTION Normal morphogenesis requires cell division to proceed in an organised fashion to produce specialised organs and cell types. The correct plane of cell division is crucial to the development of new structures and in the diVerentiation of specialised cell types (Smith, 2001). However, we still have a lot to learn about the signals dictating the plane of cell divisions, how these signals are transduced within the cell, and when the plane of cell division is fixed. Substantial progress has been made in understanding the role of the cytoskeleton in this process. Several well-studied cytoskeletal structures are thought to play an important role in transducing external signals into the positioning of the cell division plane. The cytoskeleton is intimately involved in the implementation of the external or internal cues determining the orientation of division, largely through the positioning of the pre-prophase band (PPB) and in the guidance of the newly developing cell wall. Ultimately the positioning of the cell wall can be vital for the function of the daughter cells because the geometry of the daughter cells is often retained after cytokinesis and aVects the orientation of cell elongation. This in turn can determine the orientation of future cell divisions, tissue morphology, and the shape of the whole plant. In this chapter, I consider how the plane and orientation of a cell division is determined, how that decision is implemented, and the consequences of that decision for the development of tissues, organs, and the whole plant. A particularly important aspect of the plane of cell division is the production of nonidentical daughter cells. For example, the very first division in a zygote, which gives rise ultimately to the suspensor and embryonic tissues, is asymmetrical, as are some of the divisions that occur during formation of the stomatal complex. An asymmetrical division is also needed to form the vegetative and generative cells during microgametogenesis. I will, therefore,

CYTOSKELETAL REGULATION OF THE PLANE OF CELL DIVISION

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use asymmetrical divisions as an important example of how the plane of division can aVect both whole organ and the cell function. A. CYTOSKELETAL PROTEINS

Microtubules (MTs), composed of tubulin, and microfilaments (MFs), composed of actin, are the best-characterised cytoskeletal structures within the plant cell. MTs form part of four distinct structures during the cell cycle: the interphase array, the PPB (a transient structure that predicts the future plane of cell division), the mitotic spindle, and the phragmoplast. MFs form a cortical array and are associated with the PPB and the phragmoplast. Both tubulin and actin are encoded by gene families in all higher plants, and members of the gene families show diVerential gene expression (Baird et al., 2000; Bernatsky and Tanksley, 1986; Fosket et al., 1993; McDowell et al., 1996; McElroy et al., 1990; Rogers et al., 1993; Shah et al., 1983; Thangavelu et al., 1993). Tubulin gene products are also subject to posttranslational modifications increasing the number of isoforms (Luduena, 1998; Smertenko et al., 1997, 1998). However, in most cases it is still not clear whether diVerent tubulin and actin isoforms perform distinct roles or whether the multiple isoforms are required for fine-tuned regulation of cytoskeletal protein production. The cytoskeleton is a dynamic structure undergoing constant cycles of assembly and disassembly during the cell cycle (Cleary et al., 1992; Hush et al., 1994; Zhang et al., 1990, 1993). Cytoskeletal binding proteins play an important role in modulating the dynamics of the cytoskeletal structures. One class of these includes motor proteins: dyneins associated with MTs, myosins associated with MFs, and kinesins, which can associate with both MTs and MFs. Kinesins are able to move vesicles or other MTs relative to the MT to which they are bound or move the MT relative to other larger cellular structures (Asada and Collings, 1997; Asada and Shibaoka, 1994; Asada et al., 1997; Song et al., 1997). Another class of cytoskeletal binding proteins are structural and are involved in cross-linking MTs to each other or to other cellular structures. They can also play an important role in stimulating MT polymerisation (Bokros et al., 1995, Chan et al., 1996, 1999; Cyr and Palevitz, 1989; Durso and Cyr, 1994; Durso et al., 1996; Hugdahl et al., 1995; Jiang and Sonobe, 1993; Marc et al., 1996; Schellenbaum et al., 1993; Smertenko et al., 2000; Stoppin et al., 1996) or stabilising existing MTs (Cyr and Palevitz, 1989; Mizuno, 1995; Rutten et al., 1997). Actin-binding proteins (ABPs) such as profilins, actin-depolymerising factor (Lopez et al., 1996), villin (Vidali et al., 1999), and annexins

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(Calvert et al., 1996) play a similar role in modulating MF stability (Staiger et al., 1997; Vidali and Hepler, 1997). MTs are organised into their various arrays by MT-organising centres (MTOCs). However, as plant cells do not have centrosomes, it has been diYcult to identify the molecules specifically associated with MTOCs.

-Tubulin, which plays an important role in animal cells, is not exclusively localised to the MTOC in plants (Binarova et al., 1998, 2000; Panteris et al., 2000), and antibodies to other nucleation proteins from animal cells such as 6C6 (Chevrier et al., 1992) and MPM-2 (Vaughn and Harper, 1998) have also failed to reveal conclusively MTOC-specific proteins. An MTOC candidate protein has been identified in Arabidopsis (Whittington et al., 2001). MOR1 encodes a protein with significant homology with a known class of MT-associated proteins (MAPs). Mutants in MOR1 show severe disruption of cortical MT arrays, but their mitotic and cytokinetic MT arrays are not aVected, indicating that other MT-associated proteins remain to be identified. Progress has been made in understanding actin nucleation in plants (Deeks and Hussey, 2003). The Arp2/3 complex initiates G-actin polymerisation into F-actin, and in yeast it is required for the formation of actin patches associated with polarised growth (Winter et al., 1997). In Arabidopsis, mutants in the ARP2 and ARP3 orthologues (WURM and DISTORTED1, respectively) show defects in cell shape, attributable to misdirected expansion of various cell types including trichomes and epidermal cells (Mathur et al., 2003). There is also a lack of diVerentiation of hypocotyl stomatal complexes in 74% of mutant seedlings, indicating defects in cell diVerentiation as well as expansion. Thus, an Arp2/3 complex may be performing a similar role in plants in F-actin polymerisation to that found in animals. In animal cells, the protein HSPC300 has been implicated in activation of the Arp2/3 complex, and analysis of maize brick mutants (Frank and Smith, 2002) suggests that the maize protein BRICK1, which shows homology to HSPC300, may perform a similar function. Interestingly, in bric1k (brk1) mutants, 20–40% of stomatal subsidiary cells fail to form normally (Gallagher and Smith, 2000). In wild type plants, stomatal subsidiary cells develop as a result of asymmetrical divisions of subsidiary mother cells (SMCs) (Fig. 1B). The defect in the brk1 mutants is due to a failure of the SMCs to polarise correctly before dividing. It is thought that the polarisation of SMCs is controlled by signalling from the guard mother cells (GMCs). In mosaic plants with wild type and mutant sectors, two further Brk mutants: Brk2 and Brk3, which are part of a common pathway, act non–cell autonomously between the SMCs and the GMCs (Frank et al., 2003). Thus, wild type GMCs can rescue mutant SMCs. How this is achieved


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Fig. 1. Diagram of stomatal development. (A) Meristemoid mother cells (also called stomatal initials) (MMCs) divide asymmetrically to produce a meristemoid (M, shaded grey) and a neighbouring epidermal cell (N). The meristemoid cell can convert into a guard mother cell (GMC), which in turn divides to form the two guard cells of the stoma (S). The meristemoid can also divide again to form another neighbouring cell before conversion to a GMC. New meristemoids can also arise from neighbouring cells, but in the wild type, the newly formed meristemoid is always oriented away from the existing stoma. SDD1 and TMM regulate the orientation of this division to ensure that stomata do not form from immediately adjacent cells. FLP appears to act downstream of TMM and prevents stomatal clustering, but its role is less clear. (B) Epidermal cells divide asymmetrically to form a GMC and a neighbouring cell (N), subsidiary mother cells divide asymmetrically to form stomatal subsidiary cells (SC) and an epidermal cell (E). The GMC divides symmetrically to form the stoma (S).

is not clear. It could either be by diVusion or active transport of the BRK protein. Alternatively, the action of BRK proteins may in some way enable correct communication between the SMC and the GMC to aVect the correct polarisation and division. Thus, genes that are presumed to be involved

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in actin organisation are important in regulating the correct plane of cell division. MT orientation is closely linked to the orientation of cellulose deposition (Gunning and Hardham, 1982), which in turn is a key component of the orientation of cell growth and division. To regulate cellulose microfibril deposition, cortical MTs must be associated to the plasma membrane and reorient in response to external or internal cues. Gardiner et al. (2001) isolated a candidate protein for this interaction. Using a monoclonal antibody raised against a 90-kDa tubulin-binding protein, they isolated a cDNA clone with strong homology to phospholipase D (PLD), including a calciumdependent lipid-binding domain identical to the Arabidopsis PLD delta. The protein colocalises with cortical MTs and treatment with Taxol, a drug that hyperstabilises MT polymers, results in a corresponding reorganisation of its distribution. Depolymerisation of MTs did not aVect its detection in the plasma membrane, whereas detergent extraction did, indicating that this protein is an excellent candidate for forming a bridge between the plasma membrane and cortical MTs with putative functions in the transduction of external signals to the cytoskeletal arrays. It is thought that cortical MTs are constantly turning over, so a reorientation could involve either disassembly of existing arrays and reassembly in a diVerent orientation or merely a detachment from the plasma membrane, reorientation, and reattachment in a diVerent position. Evidence for the reorientation of intact polymers comes from experiments with TBY-2 tobacco cells in which protoplasts were treated with Taxol (Wymer et al., 1996). Taxol did not inhibit the restoration of organised arrays as cell walls reformed on the protoplasts, indicating that disassembly of the MT polymers was not necessary for their reorientation. The process was, however, energy requiring and the authors postulate that motor proteins may be involved. A candidate for regulating the formation of cortical MTs following cell division is the AtKTN1 gene (Burk et al., 2001; Burk and Ye, 2002). This gene encodes a protein with significant homology to katanin an MT-severing protein first isolated from sea-urchin oocytes (McNally and Vale, 1993), which also has in vitro MT-severing activity. Based on the phenotype of Atktn1 (also known as fra2) mutants and on immunolocalisation, the major role of AtKTN1 is in the depolymerisation of the perinuclear MT array following cytokinesis so the tubulin can be reused to assemble the cortical array. These mutants show defects in cell elongation linked to aberrant cortical MT arrays and cellulose microfibrils.


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II. HOW IS THE PLANE OF CELL DIVISION DEFINED? The plane of cell division is determined by a combination of external and internal cues. These then need to be transduced to the cell division machinery that will implement the decision. One interesting question is whether a cell becomes polarised before division or whether the fates of the two daughter cells are determined after division has taken place. Some of these external cues act over long distances such as the plant growth regulators (PGRs), whereas others such as mechanical stress and cell-cell signalling are more local. Light is also an important regulator, and in single cell systems such as fucoid zygotes, much has been learned about the signal transduction mechanisms resulting in the generation of polarity and the plane of cell division. Mutants in Arabidopsis and maize have also been particularly useful in indicating important long-range and short-range signalling pathways. A. MECHANICAL STRESS

It has been known for many years that the application of mechanical stress to whole plant tissues can aVect the plane of cell divisions (Brown and Sax, 1962). Lynch and Lintilhac (1997) showed that tobacco protoplasts respond to mechanical stress by orienting their cell divisions along the paths of principal stresses. Isolated spherical protoplasts divide in random orientation, but if mechanical force is applied, the dominant division plane of cells immediately close to the force is parallel to the force, whereas protoplasts further away from the force position their plane of cell division at right angles to it. This is important because it had been known for more than a century that most cells tend to divide across the shortest path (i.e., in a barrel-shaped cell the most common plane of division is perpendicular to the long axis) (Smith, 2001). These results with isolated protoplasts support the hypothesis that mechanical forces may play an important part in determining this ‘‘path of least resistance.’’ The fact that protoplasts respond in this way also suggests that a functional cell wall is not necessary for this signal transduction pathway. Although the pathway has not been determined, parallels with animal systems suggest that the mechanical stress signal may be perceived by integrins, a class of transmembrane proteins also found in plants (Schindler et al., 1989). In animals, these proteins are associated with the cytoskeleton (Wang et al., 1993), and thus, might provide a direct link to the nucleus and alterations in gene expression. However, cytoskeletal association of integrins has not been demonstrated in plants. Although colocalisation of proteins binding to integrin antibodies and cytoskeletal proteins

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was shown in living onion epidermal cells (Reuzeau, 1997), the importance of cytoskeleton-integrin-ECM signalling complexes in plants has been questioned (Brownlee, 2002) because genes homologous to animal integrins have not been found in the Arabidopsis genome (The Arabidopsis Genome Initiative, 2000). However, there is evidence that mechanical pressure in whole tissues results in the reorientation of cortical MTs. Thus, when leaf epidermal cells of Lolium rigidum were subjected to pressure, the MT arrays visualised by immunofluorescence reoriented from mainly transversal to mainly longitudinal (Cleary and Hardham, 1993). Likewise, epidermal cells of maize coleoptiles responded to mechanical forces by rapid reorientation (Zandomeni and Schopfer, 1994), and this response was mediated by MTs associated with the plasma membrane. B. PLANT GROWTH REGULATORS

Auxin has been implicated in almost all aspects of plant growth and development and has been shown to aVect both elongation and division at the cellular level. Auxins can change cell-wall properties and alter MT orientation (Shibaoka, 1994). However, it is not clear that there is always a direct link between MT reorientation and changes in cell elongation. In several plant tissue systems (e.g., maize coleoptiles) (Nick et al., 1990), IAA induces a reorientation of cortical MTs to a transversal axis. However, disruption of the MTs using colchicine does not always aVect the changes in cell growth. Therefore, two mechanisms may be involved in auxin-regulated cell expansion, one colchicine sensitive and the other insensitive (Shibaoka, 1994). At a tissue level, the eVects of auxin on the orientation of cell division are clearly seen in the formation of lateral roots. Lateral root initiation requires dynamic auxin gradients (Benkova et al., 2003) and for a lateral root to form the orientation of cell division has to change from anticlinal to periclinal, so auxin must be directly or indirectly aVecting this change in the plane of cell division. Two genes ALF1 and ALF4 are implicated in this process. In alf1-1 mutants, there is hyperproliferation of lateral roots (Celenza et al., 1995) and a failure to develop an apical hook in dark grown seedlings, both features that can be mimicked by growth on auxin-containing media. This led to the conclusion that this gene regulates levels of free auxin in the root, thus modulating lateral root formation. In alf4-1 mutants, lateral roots fail to develop and this mutant is not rescued by addition of exogenous auxin or by the alf1-1 mutation, suggesting that ALF4 may be required by pericycle cells either to sense auxin or to respond to it. In the latter case, ALF4 may form part of the signal transduction pathway from the growth regulator to changes in the orientations of cell division, perhaps via reorientation of


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MTs. Cloning of the ALF4 gene may, therefore, provide another clue to how auxin aVects the plane of cell division, at least in the pericycle cells. Several mutants have helped in our understanding of how auxins interface with the orientation of cell division (Fig. 3). An interesting mutant class, fass mutants (Torres-Ruiz and Jurgens, 1994), provides evidence that auxin is involved in pattern formation during embryogenesis. Although these mutants reach maturity, they are characterised by stunted growth and have proximo-distally compressed organs. At a cellular level, this is reflected in a disorganised MT cortical array and the failure to develop a PPB, possibly due to a defect in MTOCs (McClinton and Sung, 1997). fass mutants have defects in auxin homeostasis (Fisher et al., 1996), and FASS may act as a negative regulator, keeping auxin at wild type levels. The FASS protein itself may be an auxin-conjugating enzyme. The link to the cytoskeletal eVects and plane of cell division, however, remains obscure. Other mutants have also helped to build a model for the role of auxin in the regulation of patterning during embryogenesis. Mutations in MONOPTEROS (MP) interfere with the formation of the body axis in embryos (Hardtke and Berleth, 1998) and MP encodes a transcription factor, which binds to auxin-inducible promoters. bodenlos (bdl) mutants are aVected in primary root formation and apical-basal patterning in the Arabidopsis embryo (Hamann et al., 1999). The BODENLOS protein interacts directly with MONOPTEROS in a yeast 2-hybrid assay and both are expressed early in embryogenesis (Hamann et al., 2002). The implication is that BODENLOS inhibits MONOPTEROS action during root meristem initiation. axr6 mutants are disrupted in their embryonic development by aberrant patterns of cell division, resulting in defects in the cells of the suspensor, root, and hypocotyl precursors, as well as provasculature (Hobbie et al., 2000). The AXR6 gene has been cloned (Hellmann et al., 2003) and encodes a component of the auxin-stimulated, ubiquitin-mediated degradation complex, which removes Aux/IAA auxin response proteins. However, insights into the precise links between auxin action, changes in MT orientation, and changes to the orientation of cell division remain elusive. Further mutants may need to be discovered to reveal the genes regulating this link. GA is known to aVect cytoskeletal organisation in vacuolated diVerentiated cells by promoting transversally oriented MTs (Baluska et al., 1993), which in turn are thought to aVect the organisation of cellulose microfibrils and hence the viscoelastic properties of the cell wall (Taylor and Cosgrove, 1989). The eVect of GA may also require auxin action (Shibaoka, 1994). It is generally assumed that in meristems, the eVect of GA on the plane of cell division is related to a stimulation of cell division, resulting in a higher frequency of lateral divisions. Evidence from a Nicotiana plumbaginifolia

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mutant hyp2 though suggests that GA may have a more direct eVect on the plane of cell division in meristems probably via an eVect on cell shape (Traas et al., 1995a). The hyp2 mutants are characterised by abnormal postembryonic cell elongation, with both the hypocotyl and the root having shorter larger cells and an increased number of cell files in the root. Both these characteristics were partially ameliorated by the addition of GA. Thus, GA appears to stimulate longitudinal cell expansion, whereas HYP2 inhibits lateral cell expansion. Whether HYP2 is itself involved in GA signal transduction remains to be determined. Ethylene is known to inhibit cell elongation, promote radial expansion, and inhibit cell division, and the results on morphology can be clearly seen in the classic ‘‘triple response.’’ Nicolas et al. (2001) showed that the inhibition of cell division in lupin hypocotyls is selective, so axial division is inhibited while radial divisions are not. This implies that ethylene is having a specific eVect on the orientation of cell divisions. It has been proposed that ethylene influences the orientation of cell expansion through a direct interaction with the MTs (Shibaoka, 1994), and it was even suggested that MTs may have an ethylene-binding site (Steen and Chadwick, 1981). Given our understanding of the ethylene-signalling pathway, however, it would seem more likely that the ethylene signal is transduced via the receptor and associated signalling cascade. However, the directional eVect on cell division suggests that as well as interacting with the cortical MTs, ethylene signalling may also aVect spindle MTs, thus regulating the orientation of cell division (Nicolas et al., 2001). Although we have seen changes in cell shape when we have treated TBY-2 cells with ethylene, we have not seen any changes in the orientation of cell division. This suggests that in isolated cells or short chains, the changes in cell shape are not suYcient to reorient the plane of cell division. Cytokinins are clear stimulators of cell division when added exogenously to cell cultures and interact directly with the cell cycle machinery through transcriptional induction of D-type cyclins (Francis and Sorrell, 2001; Riou-Khamlichi et al., 1999). In whole tissues, exogenously applied kinetin can inhibit auxin-induced stem elongation, and this eVect is partially alleviated by MT-disrupting agents and by cellulose synthesis inhibitors. Early work also showed that kinetin application results in a reorientation of MTs longitudinally to the cell axis (Shibaoka, 1994). This could then aVect cellulose deposition and the orientation of cell expansion. However, the role of cytokinins in influencing the orientation of cell division is less clearcut. A change in cell size and shape can aVect the plane of cell division as suggested by the auxin pathway mutants described earlier. Thus, a shortening of cells tending toward the isodiametric could stimulate a change in the plane of cell division from periclinal to anticlinal. However, plants in


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which cytokinin levels are elevated through mutation (Chin-Atkins et al., 1996) or expression of the ipt gene (Vanloven et al., 1993) do not show gross developmental or cellular abnormalities. The amp1 mutant described by Chin-Atkins et al. (1996) has a sixfold increase in cytokinin levels compared to wild type and has enlarged apical meristems, increased leaf number, and delayed senescence; however, plants grow to maturity. Tobacco plants expressing the ipt gene under a heat-shock promoter showed an increase in stem thickening, debudding of axillary shoots, and inhibition of root development. Although some perturbation of development was noted, it is not clear that this is linked directly to a perturbation in planes of cell division. A possible link between eVects of cytokinin and the cytoskeleton is indicated in the petunia mutant trapu (Dubois et al., 1996). In this mutant, the cortical tubules do not form parallel arrays in elongating cells, the PPB is absent from dividing cells, and the whole plant is stunted. Application of cytokininstimulated growth of the mutant, although its morphology was still abnormal. Thus, the gene identified by this mutant, TRA1, may mediate cytokinin signals regulating cell elongation and cell shape, although cloning of the gene will be required to fully understand its function. C. CELL-CELL SIGNALLING

The plane of cell division is influenced at least in part by the position of the cell in a tissue, and this eVect could result from communication with adjacent cells. Work on the positioning of stomata has provided evidence that at least in these cells, cell-cell signalling is an important regulator of the plane of cell division. Two mutants were discovered in a screen for abnormal stomatal morphology (Yang and Sack, 1995). These are too many mouths (tmm) and four lips ( flp). In both these mutants, the spacing of stomata is aberrant, with extra stomata produced where epidermal cells would be expected. Stomata, in Arabidopsis, arise from three rounds of asymmetrical cell divisions (Fig. 1A). In the first, a stomatal initial or meristemoid mother cell gives rise to a meristemoid and a neighbouring epidermal cell. In the second and third divisions, meristemoids divide to produce both epidermal cells and GMCs. Unlike the wild type in which meristemoids and stomata are evenly spaced in between epidermal cells, in tmm mutants stomata are arranged in clusters of varying stages of development from meristemoids to fully formed stomata. In flp mutants, clusters of stomata are also formed, but the clusters are smaller, and the overall increase in stomatal number is less. In both mutants, the arrangement of the clusters resembles the distribution of single stomata in the wild type. A third mutant stomatal density and distribution1-1 (sdd1-1) also has additional stomata; however, only a small

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proportion of them are arranged as clusters (Berger and Altmann, 2000). The SDD1 gene has been cloned and encodes a subtilisin-like Ser protease, which is strongly expressed in meristemoid and GMCs (von Groll et al., 2002); its role may be to cleave a proteinaceous signalling molecule. The SDD1 protein is exported into the apoplast and is probably associated with the plasma membrane. Interestingly overexpression of SDD1 in wild type or in the flp mutant background results in the opposite phenotype to the mutants (i.e., lower numbers of stomata), whereas overexpression of SDD1 in tmm mutants has no eVect on the phenotype. The authors, therefore, concluded that the action of SDD1 requires the presence of TMM. Further insights into TMM and FLP action were obtained from studies of the tmm and flp mutant phenotypes in a range of plant organs (Geisler et al., 1998). Thus, the TMM protein appears to regulate both meristemoid formation and stomatal patterning. However, loss of TMM function has varying eVects in diVerent tissues ranging from an increase to a virtual elimination of stomata. By contrast, FLP gene product does not appear to regulate meristemoid initiation and may act downstream of TMM. The TMM gene has been cloned by positional cloning and encodes a leucine-rich repeat (LRR) containing receptor-like protein (Nadeau and Sack, 2002). This structure is similar to another well-studied signalling protein CLAVATA2 (CLV2) (Jeong et al., 1999). Both TMM and CLV2 lack a cytoplasmic kinase domain needed for a functional transmembrane receptor and are, therefore, called LRR-containing receptor-like proteins. CLV2 associates with an LRR receptor–like kinase CLV1 to form a receptor complex, so if TMM performs a similar role, presumably other factors are involved. The expression pattern of TMM is consistent with the mutant phenotype: TMM is expressed in many cells, neighbouring stomata, meristemoids, or GMCs (Nadeau and Sack, 2002) and is expressed more highly in recently produced cells, its expression thereafter declining. However, TMM does not appear in itself to mark cells for an asymmetrical division, rather the data suggest that TMM is part of a receptor complex that senses positional cues during epidermal development. Thus, cells expressing TMM define a population of cells from which meristemoids will arise, and TMM in some way regulates the orientation of the asymmetrical divisions, which define stomatal spacing. Further studies on tmm mutants (Geisler et al., 2000) and on wild type cells used sequential dental resin impressions of stomatal development to determine how the tmm mutation disrupts patterning. Their results strongly suggest that spacing of stomata is largely determined by the orientation of the cell division next to a preexisting stoma. Thus, in wild type, divisions are oriented so that the newly formed meristemoid is positioned away from the preexisting stoma. This positioning was found to be independent of cell


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lineage because the dental resin impressions showed that most stomata were in contact with at least one clonally unrelated cell. The cell lineage hypothesis requires a complex of clonally derived cells. The distribution also appeared not to be directly dictated by position. Thus, cells adjacent to stomata were just as likely as other cells to divide asymmetrically and form meristemoid mother cells. However, the plane of division of the meristemoid mother cell was related to its position so that the new meristemoid was positioned away from the existing stoma (Fig. 1A). How and when the asymmetry is initiated was revealed by cytological studies (Zhao and Sack, 1999), showing that before division of the meristemoid cell the nucleus and PPB locate to one side of the cell while the vacuole is on the other side. Thus, the cell is already set up for the asymmetrical division. This implies that the fate of the daughter cells is defined before division rather than after. How this asymmetry is specified, however, remains to be determined. Cytological studies have also addressed the issue of when the fate of a cell neighbouring a stoma, meristemoid, or GMC (termed a neighbouring cell ) is determined (Geisler et al., 2003). Again, nuclear positioning appears to be actively regulated: Neighbouring cells with distally located nuclei were more likely to divide asymmetrically, but this was not a defining characteristic, as some cells with distal nuclei did not divide but diVerentiated into pavement cells. Interestingly, TMM does not appear to be linked to nuclear positioning because no diVerences were detected in nuclear distribution in tmm mutants. Because in tmm mutants the asymmetry of division in neighbouring cells appears to be randomised, the importance of nuclear positioning in relation to the asymmetrical division is called into question. Thus, the role of intercellular signalling may be to position the new cell wall itself rather than the nucleus. D. LIGHT PERCEPTION

Fucoid zygotes have been used extensively to study the eVects of light on the polarity of cell division. In fucoid algae, as in higher plants, the first mitotic division is polar. In fucoid algae, a rhizoidal region extends from the zygote by tip growth and an asymmetrical cell division perpendicular to the rhizoid results in two daughter cells: the thallus and the rhizoid, with diVerent morphologies and cell fates (Quatrano and Shaw, 1997). When zygotes are exposed to directional light, the rhizoid forms from the shaded side (photopolarisation), so the plane of cell division is directly regulated by light (Fig. 2). The polarisation occurs with a clear chronology (Hable and Kropf, 1998). The first event is adhesion of the zygote to the substratum via the uniform secretion of mucilage. This immobilises the zygote, presumably allowing it to respond to directional stimuli. Almost simultaneously

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Fig. 2. Photo-polarisation in fucoid zygotes. The first event after exposure to polarised light is the uniform deposition of mucilage (M1) followed by a second secretion from the shaded side (M2) and the formation of a calcium gradient (Ca2þ) either from external calcium or release of calcium from internal stores. It is during this period that inhibitors of protein tyrosine kinases are most eVective at inhibiting axis formation. Independent studies agree that actin filaments (A) condense near the site of rhizoid outgrowth and form a subapical ring near the rhizoid tip. However, the role of actin earlier in photo-polarisation is less well established.

photo-polarisation occurs, followed by a second secretory phase, this time localised to the future rhizoid pole. During the polarisation phase, the polarisation can be changed by changing the direction of the stimulus until around 8–10 h after fertilisation. Calcium gradients have been identified within the photo-polarised zygotes, with elevated calcium levels on the shaded side of the cell (Brownlee and Wood, 1986). Gradients are detected within 1 h of light exposure in Silvetia compressa zygotes, reaching a peak after 2 h and then declining by the time of rhizoid growth initiation (Pu and Robinson, 1998). The source of the calcium is unclear. Pu and Robinson (2003) used a self-referencing ion-specific probe to measure local calcium fluxes in polarising S. compressa cells and found that the increase in calcium derives at least in part from extracellular sources. However, Love et al. (1997) showed that neither removal of external calcium from the medium, nor high concentrations of EGTA or lanthanum chloride had any eVect on photo-polarisation, whereas blockers of intracellular calcium release, such as nifedipine, reduced photo-polarisation by up to 75%, suggesting that


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internal calcium release was the major contributor to the calcium gradient. These conflicting data may arise from the diVerent methodologies used and clearly need to be resolved. How the channels are activated by light is also not clear, although the signal transduction may involve cyclic guanosine monophosphate (cGMP) because stimulation with blue light increased cGMP levels by twofold in Silvetia compressa zygotes, and blocking this increase also blocked photo-polarisation (Robinson and Miller, 1997). The role of the cytoskeleton in photo-polarisation has been the focus of several studies, again with some conflicting results. MTs appear not to be required as depolymerisation with oryzalin had no eVect on photopolarisation (Hable and Kropf, 1998). Actin does seem to play an important role, but results are conflicting. Love et al. (1997) found that inhibition of actin polymerisation with cytochalasin B, cytochalasin D, or latrunculin B had no eVect on photo-polarisation but inhibited the fixation of the polar axis. However, both Pu et al. (2000) and Hable and Kropf (1998) also used latrunculin B on P. compressa zygotes and found that even low levels abolished photo-polarisation and the formation of the calcium gradient. Furthermore, the distribution of actin during the formation of the gradient appeared uniform using microinjected fluorescent phalloidin, suggesting that an asymmetry in cortical actin is not involved in the early stages of the generation of the calcium gradient. There is, however, some controversy over the distribution of the actin filaments. Alessa and Kropf (1999) detected localised patches of actin filaments on the shaded pole, and these repositioned to a new pole if the light direction was altered during the polarisation phase. However, Pu et al. (2000) did not observe any actin patch formation and attribute the diVerences in results to the methodology used. Again, further work is required to resolve the role of actin. This is an important issue as one model for the formation of the polarity was the anchoring of calcium channels by actin filaments onto the shaded side of the zygote. However, this would require an asymmetry of the actin filaments to precede the calcium gradient, which was not evident from the results of Pu et al. (2000). One possibility is that although the actin filaments are distributed uniformly in the early stages, the motor molecule myosin is able to regulate the positioning of calcium channels. The role of actin later in the polarisation process seems less controversial. Both Pu et al. (2000) and Alessa and Kropf (1999) found a condensation of actin filaments near the site of rhizoid outgrowth at the time of its initiation and the formation of a subapical actin ring near the rhizoid tip. Some light has been shed on how the calcium gradient eVects the polarisation. KN-93, an inhibitor of calcium/calmodulindependent kinase II (CaM kinase II), abolished rhizoid germination on

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S. compressa zygotes almost completely (Pu and Robinson, 2003) when administered continuously but had no eVect if administered as a pulse only during the light stimulation. This suggests that CaM kinase II may play an important role in the transduction of the calcium signal to downstream eVectors but is not required for setting up the calcium gradient, as might be expected. Interestingly, the inhibitor also had little eVect if administered later, just before germination, suggesting that the CaM kinase is not required for secretion of new cell-wall materials or that it has already done its job before germination. Protein phosphorylation also plays an important role in polarisation (Corellou et al., 2000). Inhibitors of protein tyrosine kinases (PTKs), such as genistein, inhibited axis formation and associated events such as polar secretion. The timing of the genistein administration was important. Later treatments were less eYcient at inhibiting germination than earlier treatments, suggesting that PTKs may be particularly important during photopolarisation. However, genistein was also able to prevent axis formation in the dark, suggesting that it acts downstream of the signalling pathways from diVerent environmental stimuli. However, genistein did not appear to aVect actin localisation, so these experiments imply that actin localisation per se is not suYcient for photo-polarisation. Corellou et al. (2000) suggest a new model for the transduction of the environmental signal into polarisation in fucoid zygotes involving PTKs in the formation of a protein complex that then interacts with actin filaments to initiate signalling pathways leading to the polarisation process. Clearly, fucoid algae provide a powerful tool to analyse the induction of polarisation and consequent planes of asymmetrical cell division; however, there appear to be some concerning controversies in the data, which will need to be resolved before a clear picture emerges. It is also not clear whether the mechanisms resulting in photo-polarisation of fucoid zygotes will be applicable to polarisation and fixing of planes of cell division in higher plants. E. CELL CYCLE GENES

Entry into mitosis requires the activity of the cyclin-dependent kinase (CDK) p34cdc2. MAPs are potential substrates for p34cdc2. This provides a direct link between the control of cell division and the cytoskeleton (Wick and Rogers, 2001). The maize homologue of CDK p34cdc2 has been localised to the nucleus during interphase and to the PPB during late G2/M (Colasanti et al., 1993), suggesting a close association with the cytoskeleton. Another interesting observation is the eVect of expressing a fission yeast cell cycle regulatory gene, Spcdc25, in tobacco (McKibbin et al., 1998).


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Spcdc25 is a tyrosine phosphatase, and in fission yeast it acts on the CDK to activate it in late G2 by dephosphorylation of Tyr-15 (Kumagai and Dunphy, 1991). Induction of Spcdc25 expression in tobacco roots resulted in an increase in the frequency of lateral root primordia (McKibbin et al., 1998), and we have shown the same result when this gene is expressed in Arabidopsis (S. Li, C. Rosser, D. Francis, and H. J. Rogers, 2002, unpublished data, 2002). In addition, the cells within the tobacco root primordia were significantly smaller. This suggests that Spcdc25 may be interacting with the plant cell cycle machinery in a similar fashion to its role in fission yeast, because overexpression of Spcdc25 in fission yeast also results in a small cell phenotype (Russell and Nurse, 1986). Because the initiation of lateral roots requires a change in the plane of cell division, there may be a link between the regulation of key cell cycle checkpoints, cell size, and the orientation of cell divisions, a conclusion supported by our experiments with tobacco cell cultures. We expressed Spcdc25 in the tobacco TBY-2 cell line, and in addition to obtaining a reduction in mitotic cell size, we also observed eVects on the plane of cell division (C. B. Orchard, R. J. Herbert, D. Francis, and H. J. Rogers, unpublished data, 2003).

III. IMPLEMENTATION OF THE DECISION ON THE ORIENTATION OF DIVISION Once the signals determining the plane and position of division have been perceived by the cell, they need to be transduced to the cell cycle machinery. In many cells, perhaps the most important structure in this early part of the process is nuclear positioning and PPB. The PPB disappears before the onset of mitosis. However, a memory of its position is retained, and during the process of cytokinesis, the cytoskeleton plays a major role in the positioning of the new cell plate in the position marked by the PPB. Again, mutants have been very helpful in defining the genes regulating cytokinesis, and it has been suggested that this last process in the division of the cell is subject to a series of checkpoints, analogous to those that regulate entry into mitosis. A. THE PRE-PROPHASE BAND

Perhaps the most important cytoskeletal structure in the determination of the cell division plane is the PPB (Mineyuki, 1999; Wick, 1991a,b). This structure becomes transiently visible before the onset of mitosis as a ring encircling the nucleus. It then narrows and disappears as the mitotic spindle forms. In cells that are going to divide symmetrically, the PPB forms across

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the centre of the cell, taking the shortest path. Before its appearance, in highly vacuolated cells, the nucleus is suspended by transvacuolar cytoplasmic strands, which then pull the nucleus into the centre of the cell. These strands are formed by continuous MT strands (Flanders et al., 1990), although they also contain MFs. Analogous structures are seen in small densely cytoplasmic cells as MTs radiate from the nucleus in all directions (Wick, 1991a). Thus, in these cells, it would seem that these MT structures are the first predictors of the plane of cell division. The PPB is chiefly composed of a ring of MTs aligned parallel to the band, although MFs are associated with the MTs, also oriented in the same manner. The MF ring is essential both for narrowing the PPB and for positioning it to the correct site (Eleftheriou and Palevitz, 1992; Mineyuki and Palevitz, 1990). The nucleus and the PPB form a close association (Wick and Duniec, 1983), perhaps via the MTs or MFs known as the phragmosome, and it may be as a result of communication from the nucleus (Dixit and Cyr, 2002a) that timing of PPB disassembly is controlled. This communication may be mediated by an increase in phosphorylation of PPB-associated proteins because blocking phosphoprotein phosphatase activity with okadaic acid resulted in a disruption of the association between mitotic spindle assembly and PPB breakdown (Zhang et al., 1992). However, using inhibitors of protein kinases also aVected PPB breakdown in tobacco BY-2 cells (Katsuta and Shibaoka, 1992). This suggests that both phosphorylation and dephosphorylation are required for the correct disassembly of the PPB. Although the substrates of these enzymes are not known, MAPs are likely candidates. The key feature of the PPB is that its position predicts the site of the new cell plate despite that it disappears well before the onset of cytokinesis. Much eVort has, therefore, been put into understanding how a memory of the positioning of the PPB is retained by the cell. In addition to predicting the orientation of cytokinesis, the PPB also predicts the orientation and position of the mitotic spindle. Thus, the positioning of the new cell plate can be seen as a three-stage process: First, the PPB forms a close association with the premitotic nucleus (Mineyuki, 1999), anchoring it or positioning it. This then determines spindle orientation and position, orienting it perpendicularly to the PPB. This in turn dictates the orientation of the new cell plate as this arises from the remnants of the spindle (see later discussion) and attaches to sites marked by the PPB on the cell cortex. Evidence for a key role for the PPB in this process comes chiefly from studies in which the PPB was disrupted or forms abnormally. Granger and Cyr (2001) used a GFP-labeled MT reporter protein to follow mitoses in TBY-2 cells with abnormal PPBs. In a population of dividing cells, they found 2% in which the PPB was abnormal. Comparing these with cells in which the PPB formed normally,


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they were able to show that the PPB does associate with the premitotic nucleus, because in cells where the PPB was positioned between the equator and the pole of the cell, the nucleus followed the position of the PPB. Disruption of the PPB in fern protonema using light treatments or centrifugation also showed a close relationship between orientation of the PPB and the phragmoplast (Mineyuki et al., 1991; Murata and Wada, 1991). A number of molecules have been implicated in marking the cell cortex. A large body of evidence points to a zone depleted of actin (Cleary, 1995; Cleary et al., 1992; Liu and Palevitz, 1992). There may also be a deposition of vesicles at the site of the PPB (Galatis et al., 1984). Fusion of vesicles and localised cell-wall thickening at the PPB have been detected (Galatis et al., 1982). However, inhibition of secretion using brefeldin A during PPB formation in TBY-2 cells did not inhibit correct phragmoplast positioning, indicating that Golgi secretion is not required for this process (Dixit and Cyr, 2002b). Further studies have also revealed the association of p34cdc2 kinase with the PPB (Colasanti et al., 1993; Mews et al., 1997), suggesting that it too may have a role in marking the cell wall perhaps by phosphorylation of unknown wall substrates. A guanosine triphosphatase (GTPase) from Saccharomyces pombe, Spg1p, was shown to form an important link between cytokinesis and mitosis (Schmidt et al., 1997; Sohrmann et al., 1998). If homologues of Spg1p exist in plants, these may provide another clue to the missing link between the PPB and the positioning of cytokinesis. In fission yeast, Spg1p interacts with Cdc7p, a protein kinase that associates with the spindle body early in mitosis. In wild type cells, Cdc7p always segregates to only one spindle pole late in the anaphase, and deregulation of septum formation was found to correlate with a symmetrical distribution of Cdc7p. Spg1p is required for the correct distribution of Cdc7p, so Cdc7p may be at the top of a GTPase-regulated protein kinase signalling cascade resulting in the signal to form a septum. Although the role of the PPB in marking the site of the new cell plate seems widely accepted in most plant cell divisions, analyses of the PPB of the first microspore division suggest that cell-wall marking may not be its only role. It has been argued that in this cell division, it is the asymmetry and thus the placement of the new cell plate that determines embryogenic fate (Zaki and Dickinson, 1990). However, a study by Simmonds and Keller (1999) suggests that it is the degree of integrity of the PPB that may be the key factor. This additional role of the PPB in ensuring correct wall consolidation had already been proposed (Mineyuki and Gunning, 1990) based on data from time-lapse photography showing that cell-wall maturation does not occur if the attachment site had not been previously marked by the PPB. In the male gametophyte, the lack of a PPB may be an important factor in the

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movement of the male germ unit down the pollen tube, because the wall between the vegetative and generative cells remains incomplete. B. THE PHRAGMOPLAST AND CYTOKINESIS

At the end of mitosis, the phragmoplast positions the new cell plate, which is formed during cytokinesis. The phragmoplast is composed of a network of MTs derived from the mitotic spindle (Gunning and Wick, 1985; Wick et al., 1981; Zhang et al., 1993) onto which vesicles carrying wall material fuse. The MTs expand centrifugally from the centre of the plate until they make contact with the existing cell wall. To complete cytokinesis, the new cell plate needs to connect to the existing cell wall in the correct position identified by the PPB. However, although MTs form an integral part of the phragmoplast and are essential for the correct fusion of vesicles onto the growing plate, MFs are thought to position the growing phragmoplast (Smith, 1999). How this precise positioning is achieved, however, is not completely clear. Actin-containing cytoplasmic strands have been detected between the phragmoplast and the cell cortex (Valster and Hepler, 1997), which may guide the phragmoplast to the correct cortical sites. However, evidence from studies on the stomatal complex indicate that the cortical sites may be defined much earlier. Actin-dependent migration of the nucleus during G1 to a defined cortical site has been observed in the formation of the stomatal complex (Kennard and Cleary, 1997) and an actin patch then appears to form at this cortical site (Cleary, 1995). However, nuclear migration may not be essential for the formation of the actin patch. In the monocot stomatal complex, subsidiary cells, which are localized laterally to the guard cells, are formed from asymmetrical divisions of SMCs. In a small subset of SMCs, the actin patch formed without nuclear migration (Cleary and Mathesius, 1996). Thus, the role of nuclear migration in the marking of the cortical sites for phragmoplast fusion remains uncertain. The role of the actin patch, though, seems to be better defined. When the cell enters mitosis, the spindle and phragmoplast appear to stay close to the actin patch, and this seems to be essential for correct positioning of the new cell wall (Pickett-Heaps et al., 1999). Two maize mutants have provided further insights into the role of the actin patch in the regulation of plane of cell division. These two mutants, discordia 1 and 2 (Gallagher and Smith, 1999), aVect two asymmetrical cell divisions of the leaf epidermis required in the formation of the stomatal complex and in silica-cork cell pairs. These mutations appear not to aVect symmetrical divisions, as other cells of the epidermis are normal. In both asymmetrical divisions, early events of mitosis appear normal: The PPB forms correctly, actin patches are present, the


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spindle forms and contacts the actin patches, the nuclei contact the actin patch, and actin filaments connect the phragmoplast to the actin patch. Nuclear migration also appears normal. It is, however, the later stages of phragmoplast formation that are disrupted in the dcd mutants. Thus, the phragmoplast forms correctly, but its guidance to the correct cortical sites is impaired. The authors postulate that the DCD proteins may be involved either in mediating the connection of the actin filaments to the phragmoplast or to the actin patch or may be motor proteins required for pulling the phragmoplast into position. Alternatively, they may mediate attachment of the daughter nuclei to the phragmoplast assisting the positioning of the new cell walls. These mutants thus illustrate that this process requires the coordination of many proteins to achieve the distinctive shapes of cells produced by asymmetrical divisions. It also shows that diVerent proteins may be required for making the connection between the phragmoplast and the existing cell wall in diVerent cell types. Analysis of an Arabidopsis mutant gnom (allelic to emb30) suggests that vesicle transport and delivery may be a key regulator of division plane. In these mutants, cell shape is irregular and they appear to be impaired, in both division plane and cell expansion throughout their development (Mayer et al., 1993; Shevell et al., 1994), including defects in a number of typically asymmetrical divisions. The GNOM gene has been cloned (Shevell et al., 1994) and encodes a protein containing a conserved domain known as Sec7. This domain is found in yeast and mammalian proteins, some of which are involved in vesicle transport. Thus, the primary defect in gnom mutants may be in some aspect of vesicle transport, which aVects correct cytokinesis, perhaps again involved in interactions with the cell wall for correct positioning of the new cell plate. Another mutant provides more clues to the components involved in the fusion of the phragmoplast with the cell wall. tan1 maize mutants show abnormalities in cytokinesis, namely a severe reduction in longitudinal divisions. This spatial defect is linked to a failure of phragmoplasts to contact cortical sites previously marked by the PPB. However, it seems that the defect may not aVect longitudinal divisions specifically but all dividing cells of the leaf primordium. It is suggested that transverse divisions may be less dependent on a fully functional cytokinetic mechanism (Cleary and Smith, 1998). The TAN gene has been cloned (Smith et al., 2001) and found to encode a highly basic protein, distantly related to the basic MT-binding domain of vertebrate APC proteins. The TAN1 transcript is highly expressed in dividing tissues, the protein binds to MTs in vitro. An anti-TAN antibody also labels cytoskeletal structures in leaf primordia. Given the phenotype of tan1 mutants and the properties of the TAN1 protein, it seems likely that

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TAN1 interacts directly or indirectly with the cell cortex to regulate the positioning of MTs. Interestingly, although at the cellular level, tan1 mutants are aberrant, leaf shape remains normal. Careful measurements of cells in the meristematic prediVerentiation zone at the base of elongating leaf blades (Mitkovski and Sylvester, 2003) reveal that tan1 mutants are aberrant in cell expansion and division. The combined defects result in the same final dimensions of the cells as in the wild type. This explains why whole organs in the mutant appear similar to wild type and suggests an additional role for the TAN1 protein during cell expansion. Some cells undergoing morphogenetically critical longitudinal or asymmetrical divisions appear to use additional actin to help ensure correct placement of daughter cells. For instance, in certain cells of the developing stomatal complex of various monocots, dense arrays of MFs extend between the mitotic nucleus and the adjacent cortical region (Cho and Wick, 1990, 1991; Cleary, 1995; Cleary and Mathesius, 1996). Disruption of these MFs leads to misoriented spindles or mitotic nuclei that drift away from the division site and ultimately to incorrectly placed cell plates (Cho and Wick, 1990, 1991).

IV. CONSEQUENCES OF THE ORIENTATION OF A CELL DIVISION Green (1994) identified three geometrical issues in shoot development: form (or shape), pattern (the arrangement of elements in relation to each other), and organogenesis, which changes the pattern to make a new form or shape with its own patterns within it. Orientation in cell division and direction of expansion form the basis for the shape of the plant organ. In many cells, expansion of the cell is controlled by hoops of cellulose microfibrils, in turn governed by a transverse alignment of MTs. This arrangement limits increases in girth, thus promoting elongation in response to nondirectional turgor pressure (Green, 1980). Green (1994) postulated that the direction of expansion is a result of the balance of two forces. The first is a tendency for MTs to avoid bending and to pack closely, favouring alignment with the long axis of a cell. The second is a process of ‘‘self-cinching ’’ in which the MTs form hoops round the cell. This latter process would be energy requiring, would be assisted by MT-binding proteins, and favour alignment of the MTs with the short axis of the cell. In addition, intercellular communication between cells results in coalignment of MTs in adjacent cells, resulting in aligned cellulose microfibrils. Thus, there is a coordination of orientation within a tissue. However, it is important to recognise that MTs in interphase cells are not static: They are highly dynamic and are constantly changing


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their orientation (Takesue and Shibaoka, 1998). These changes, as has been discussed earlier, are able to influence MT orientation and hence direction of expansion. A. COORDINATING THE PLANE OF CELL DIVISION

An important feature of the control over plane of cell division is the apparent coordination of this process within tissues. For example, during development, the positioning of the cell division plane at critical stages can aVect the whole developmental programme of an organ, or if early enough in development, that of the whole plant. This occurs both in the sporophyte and in gametophytes, although the genes involved may be subtly diVerent. For example, the first mitotic division of the microspore is characterised by a highly asymmetrical division, producing the generative and vegetative cells. In cultured microspores, various treatments can disrupt this asymmetrical division, converting it to a symmetrical one and giving rise to aberrant development or somatic embryos instead of pollen (Eady et al., 1995; Touraev et al., 1997; Zhao et al., 1996). Thus, the whole course of pollen development may be diverted by the disruption of the cell division plane of one division event. Although cell division is central to plant development, it would seem that cell division per se is not responsible for the control of cell fate. This is exemplified by transgenic plants in which the expression of cell cycle genes has been altered (Doerner et al., 1996; Hemerly et al., 1995). Although cell size or cell number is aberrant in these plants, the overall tissue organisation is not. Correct plane of cell division may not even be essential for the normal development of a tissue. Examples of this eVect are the maize tangled mutant, in which leaves develop normally despite an alteration in the plane of cell division (Smith et al., 1996), and the Arabidopsis fass mutant, in which the axes of the embryo are not disrupted despite aberrations in cytokinesis (Torres-Ruiz and Jurgens, 1994). Likewise, the ton1 and ton2 mutants (Traas et al., 1995b) lack PPBs and ordered cortical MT arrays in most cell types, but although the plants are short, thick, and misshapen, the organs are all formed in their correct relative positions. The TON2 gene was cloned and found to encode a protein with homology to the B-type subunit of type 2A protein phosphatases (PP2A) (Camilleri et al., 2002). The B-type subunit of PP2As in other organisms regulates the substrate specificity, subcellular localisation, and activity of the PP2A complex. How TON2 interacts with the cytoskeleton to regulate MTs is not known, but its identity as a phosphatase regulator supports the role for kinase/phosphatase regulation of the cytoskeleton, discussed earlier. Interestingly, in the tangled mutant, the

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cortical MT array in adjoining cells appears to be oriented in the same manner even if the division plane is diVerent (Cleary and Smith, 1998), suggesting that this cytoskeletal structure may be important in maintaining correct overall shape. However, there are other mutants which suggest that correct plane of division is sometimes crucial to organ formation. Thus, in the scarecrow (scr) mutant of Arabidopsis, which is defective in the periclinal division of endodermis/cortex, the endodermis and cortex cell types fail to diVerentiate properly (DiLaurenzio et al., 1996). Another relationship between cell fate and plane of cell division is where cell fate dictates the rate and the plane of cell division. For example, in the root epidermis it has been proposed (Berger et al., 1998) that the TTG gene controls the epidermal cell fate and the production of anticlinal longitudinal divisions. Two Arabidopsis mutants may contribute to our understanding of this process in the male gametophyte. In Arabidopsis stud mutants, no walls are formed after telophase II of cytokinesis, resulting in a microspore containing four nuclei (Hulskamp et al., 1997). These four nuclei then appear to undergo relatively normal divisions to form four vegetative and four generative nuclei within the one cell. This poses the interesting problem of how polarity is maintained in this multinucleate cell. The authors suggest two possibilities. One is that there is a linear gradient maintaining polarity across the whole cell. However, this would result in an alignment of the four units within the cell, and this has not yet been checked. The other possibility is that there is a gradient radiating from each nucleus. Further investigation of this mutant may be useful in establishing whether either of these two models is correct. Another interesting feature is that in this mutant, only one pollen tube is produced for each giant pollen grain, indicating that the control of this process is at a cellular level. This mutant appears not to aVect either somatic cell divisions or meiotic divisions in the female gametophyte, although in the latter, aberrant depositions of callose are often observed. This suggests that STUD may be involved in cell-plate callose deposition and that this requirement is particularly stringent in male meiosis. This also raises the possibility that STUD may be related to KNOLLE, a cytokinesis-specific syntaxin (Lauber et al., 1997). In knolle mutants, cross walls fail to complete during embryo development with the production of enlarged cells containing polyploid nuclei (Lukowitz et al., 1996). Cloning of the STUD gene will be required to fully understand its role. Another mutant sidecar pollen (scp) produces two cells in the microspore, but only one continues development while the other remains as an additional vegetative cell (Chen and McCormick, 1996). This suggests perhaps another layer of control in this process. In the scp mutant, the pollen tube can form from either the functional or the nonfunctional vegetative cell but, not both, indicating


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that there is tight control on the number of pollen tubes produced, but that the presence of a functional germ unit is not required. A fuller understanding of the role of these two genes awaits their isolation. Another aspect of the coordination of cell division planes within an organ is the precise positioning of the cell division plane in relation to the positions of adjacent cell junctions. Thus, in many tissues, there is an avoidance of plane positions that would result in nonstaggered cells. This eVect does not occur when new cell divisions are stimulated by wounding and planes of cell division often line up from cell to cell. This diVerence has been ascribed to the lack of synchrony of cell divisions within tissues compared to the synchronous divisions stimulated by wounding (Lloyd, 1991). How the cell planes align after wounding is also interesting. Lining up of actin strands has been observed in such cells (Goodbody and Lloyd, 1990), suggesting that there might be preferred points of attachment for the actin strands to the cell wall. The shape of the cell may also be important in determining the plane of division. Thus, it has been suggested (Cleary and Smith, 1998; Lloyd, 1991) that there is a default division plane, resulting in the minimal path being adopted. This is supported by evidence from laser microsurgery that cytoplasmic strands anchoring the nucleus in premitotic cells are under tension (Goodbody et al., 1991). In elongated cells, this results in transverse cell plates, which produce periclinal divisions. Evidence from the tan1 mutant in maize (discussed earlier) further suggests that the default division plane may be less dependent on a fully functional cytokinetic machinery. In isodiametric cells, however, the minimal path is less obvious, resulting in the choice of planes being potentially wider. It has been suggested, therefore (Lloyd, 1991), that a change in plane of division is more easily achieved in isodiametric cells that have either failed to elongate or in which the rate of cell division is higher and thus the cells have had less time to elongate.

V. CONCLUSION In this chapter, I have tried to bring together data from a wide range of sources to address two questions: (1) How does the cytoskeleton respond to external and internal signals regulating the plane of cell division and (2) how does it actually position division in the correct place? Our understanding of the cytoskeletal apparatus participating in cell division has made substantial progress thanks at least in part to a plethora of mutants isolated from Arabidopsis and other species. It has been educational, however, to note that mutants with striking eVects on the cytoskeleton and cell shape do not always reveal genes with a direct link to the cytoskeleton (Table 1, Fig. 3).

TABLE I Identified Genes AVecting Plane of Cell Division

Gene/gene group

Cellular mutant phenotype

Whole organ/plant mutant phenotype

ALF4

Reduced cell expansion in shoots

Lateral roots fail to develop, small bushy plants, male sterile

AMP1

Increased cell proliferation

AtKTN1 (also known as FRA2)

Defects in cell elongation: reduction in cell length and an increase in cell width in all organs

Enlarged apical meristems, increased leaf number and delayed senescence Global alteration in plant morphology; defective in the mechanical strength of interfascicular fibers in the inflorescence stem

EVect on cytoskeleton/putative cytoskeletal role of protein May form part of the signal transduction pathway from the growth regulator to changes in the orientations of cell division No direct link/eVect

Aberrant cortical MT arrays, and cellulose microfibrils; gene has putative function in depolymerisation of the perinuclear MT array following cytokinesis

Gene function/ homology to known genes

Reference

Cloned but no homology to genes of known function

Celenza et al., 1995, 2003

Homology to glutamate carboxypeptidases linked to small signal molecules Protein with significant homology to katanin, an MT-severing protein

Chin-Atkins et al., 1996

Burk and Ye, 2002; Burk et al., 2001

AUXIN RESISTANT 6 (AXR6)

Aberrant patterns of cell division in the embryo from the two cell stage

Arrest growth soon after germination, lack a root and hypocotyl, severe defects in cotyledon vascular pattern

Probably no direct link to cytoskeleton

BRICK1 (BRK1)

Stomatal subsidiary cells fail to form normally, lack of epidermal cell lobes in leaves

No serious abnormalities reported

Presumed to be involved in actin organisation

BODENLOS (BDL)

Abnormal early divisions of embryo from the two cell stage


DISCORDIA 1 and 2 (DCD 1,2)

Disruption of asymmetric cell divisions in leaf epidermis

Primary root meristem not formed but postembryonic roots develop and seedlings grow to fertile adult plants Abnormal epidermal cell pattern

FASS (FS)

Aberrations in cytokinesis

Stunted plant proximo-distally compressed organs

Normal PPB and spindles, failure to position phragmoplast correctly (probably via MFs) to PPB marked sites Disorganised MT cortical array and failure to develop a PPB

Encodes a component of the auxinstimulated, ubiquitinmediated degradation complex Shows homology to HSPC300, which activates the Arp2/3 complex in animals Encodes an auxin response protein

Hobbie et al., 2000

Not cloned

Gallagher and Smith, 1999

Not cloned

McClinton and Sung, 1997; Torres Ruiz and Jurgens, 1994; Traas et al., 1995

Frank and Smith, 2002; Gallagher and Smith, 2000

Hamann et al., 1999, 2002

(continues)

TABLE I (continued )

Gene/gene group


Whole organ/plant mutant phenotype

EVect on cytoskeleton/putative cytoskeletal role of protein

GNOM allelic to EMB30

Aberrant asymmetric division of the zygote, producing two nearly equal-sized cells

Extreme phenotype: meristems of the shoot and the root fail to form

Defect in vesicle transport aVecting correct cytokinesis

HYP2

Pattern formation in the root meristem is perturbed; extra anticlinal cell divisions in all cell types Enlarged cells containing polyploid nuclei

Shorter thicker cells throughout plant

Unknown

Abnormal seedlings with disturbed radial organization of tissue layers Seedlings lack basal structures such as hypocotyl, radicle, and root meristem

Cross walls fail to complete during embryo development Probably no direct link to cytoskeleton

Seedlings are extremely squat, do not develop flowers and eventually die

Severe disruption of cortical MT arrays; mitotic and cytokinetic MT arrays are not aVected

KNOLLE

MONOPTEROS (MP)

MOR1

Failure to establish division patterns that would normally generate basal body structures Severe disruption of cortical MT arrays


Reference

Protein contains an Sec7 domain found in yeast and mammalian vesicle transport proteins Not cloned

Mayer et al., 1993; Shevell et al., 1994

Cytokinesisspecific syntaxin

Lauber et al., 1997; Lukowitz et al., 1996

Transcription factor, which binds to auxininducible promoters Homology to and ancient family of MT-associated proteins

Hardtke and Berleth, 1998

Traas et al., 1995

Whittington et al., 2001

SCARECROW (SCR)

Defective in periclinal divisions of endodermis/ cortex

SIDECAR POLLEN (SCP)

Aberrant cell division pattern during pollen development Defect in male cytokinsis after meiosis telophase II

STUD (STD)

TANGLED (TAN )

Alteration in the plane of cell division throughout leaf development

TONNEAU (TON1)

Aberrations in division plane/cytokinesis

TOO MANY MOUTHS (TMM) and FOUR LIPS (FLP) and STOMATAL DENSITY AND DISTRIBUTION1-1 (SDD1-1)

Aberrant spacing of stomata, forming clusters of stomata in flp and tmm mutants

Loss of ground tissue layer and radial pattern defect in root and shoot Partial to complete male infertility


Belongs to the VHIID family of transcriptional regulators

DiLaurenzio et al., 1996; Silverstone et al., 1998

Unknown

Not cloned

Chen and McCormick, 1996

Tetra nucleate microspores, partial male fertility Normal leaf shape

Defective cytokinesis in male meiosis

Not cloned

Hulskamp et al., 1997

Defect in phragmoplast guidance (probably via MFs) to PPB marked sites Disorganised MT cortical array and failure to develop a PPB TMM has no eVect on nuclear positioning

Distantly related to the basic MT-binding domain of vertebrate APC proteins B-type subunit of type 2A protein phosphatases SDD1 encodes a subtilisin-like Ser protease, may cleave a proteinaceous signalling molecule; TMM encodes a leucine-rich repeat (LRR) containing receptor-like protein

Cleary and Smith, 1998; Smith et al., 2001

Stunted plant proximo-distally compressed organs No serious abnormalities reported

Camilleri et al., 2002; Nacry et al., 1998; Traas et al., 1995 Berger and Altmann, 2000; Nadeau and Sack, 2002; von Groll et al., 2002; Yang and Sack, 1995

(continues)

TABLE I (continued )

Gene/gene group


TRAPU (TRA 1)

Disorganised meristems, rounded cells

TTG (TRANSPARENT TESTA GLABRA)

Production of anticlinal longitudinal divisions

WURM and DISTORTED1

Defects in cell shape; misdirected expansion of various cell types; also defects in cell diVerentiation

Whole organ/plant mutant phenotype Shortened, thickened organs, no flowering Lack trichomes, anthocyanins, and seed coat mucilage, root epidermal cells in all positions diVerentiate into root-hair cells Trichome expansion is randomized, pavement cells fail to produce lobes, hypocotyl cells curl out of the normal epidermal plane, and root hairs are sinuous

EVect on cytoskeleton/putative cytoskeletal role of protein


Reference

Disorganised MT cortical array and failure to develop a PPB Probably no direct link to cytoskeleton

Not cloned

Dubois et al., 1996

Putative role in signal transduction, protein-protein interacting region

Berger et al., 1998; Walker et al., 1999

Complex initiates G-actin polymerisation into F-actin

Homology to the yeast Arp2/3 complex

Mathur et al., 2003


99

Fig. 3. Diagram illustrating the process of plant morphogenesis and the roles of the cytoskeletal structures. (Based in part on Dubois et al., 1996.) Plant growth regulators (PGRs) and microtubules (MTs) are included. An attempt has been made to place the mutants described in the text at the appropriate point in the pathway, although in many cases this remains very speculative, and some genes may be acting simultaneously at diVerent points as shown.

Nevertheless, some of the processes in determining the plane of cell division are emerging. Despite the complexity of this process, revealed by each new mutant described, our understanding of the role of the PPB, how it marks the future site of division, and how the phragmoplast positions the new cell plate seems to be growing rapidly. However, there still seem to be substantial gaps in our understanding of how external or internal cues regulate changes to the plane of cell division and how these signals are perceived by the cytoskeleton. In most cells, mechanical forces may be an essential regulator of the plane of cell division, as shown by the elegant experiments of Lynch and Lintilhac (1997), perhaps providing a default pathway. However, to form complex organs, the planes of cell division need to be carefully manipulated through

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long-range and local cell-cell signalling. External signals need to cross the plasma membrane, and data from stomatal and shoot meristem development indicate that one mechanism at least operates through receptor-like kinases. Reorientation of MTs seems to be a common feature in the response to a wide range of diVerent signals including the major PGRs and mechanical stress. Thus, in many cell types, this may be the first cytoskeletal component to respond to a division plane signal. However, how receptor-like proteins are linked to the cortical MTs remains to be fully elucidated. Perhaps the missing interface between the external signal and changes to the MT cortical array may be provided by the isolation of a phospholipase D (Gardiner et al., 2001), which interacts both with the plasma membrane and with cortical MTs. Further work on this protein is urgently needed to establish its importance in relation to diVerent external signals and in diVerent cell systems. Fucoid algae provide an interesting system to study the intracellular fixation of signals dictating a cellular asymmetry, and the resolution of the conflicting data on the role of actin filaments will be important in getting a full picture of this system. However, it would seem that the signalling in this system may be substantially diVerent to that discovered in stomatal and shoot development. Is this type of signalling through calcium gradients important in higher plants? Clearly, there are parallels to pollen tube and root tip growth, but is calcium signalling also involved in cells that go on to divide? If so, are there multiple independent signalling systems that converge on the cytoskeleton? If this is the case, it seems likely that a whole plethora of cytoskeletal-interacting proteins are waiting to be discovered to complete the link. Even more complex perhaps are the long-range signals provided by PGRs and environmental signals in multicellular systems. Is each type of asymmetrical division regulated diVerently according to its function? The formation of a stomatal cell must clearly respond to a completely diVerent set of environmental and developmental cues to a microspore, but do the pathways converge on the cytoskeleton or above it? The analysis of mutants has brought us a long way toward understanding many of these processes. As the corresponding genes are cloned, we will gain new insights, and their function can be tested through in vivo and in vitro studies of protein functions and interactions.

ACKNOWLEDGMENTS I would like to thank Dr. Dennis Francis and Dr. Martin Day for their helpful comments on the manuscript. Unpublished work mentioned in this review was funded by the NuYeld Foundation, CardiV University, and University College Worcester.


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Nitrogen and Carbon Metabolism in Plastids: Evolution, Integration, and Coordination with Reactions in the Cytosol

ALYSON K. TOBIN* AND CAROLINE G. BOWSHER{

*School of Biology, Sir Harold Mitchell Building, University of St Andrews, St Andrews, Fife, KY 169TH Scotland, United Kingdom { School of Biological Sciences, University of Manchester, 3.614 Stopford Building, Oxford Road, Manchester M13 9PT, United Kingdom

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Endosymbiotic Origin of Plastids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Gene Migration and the Evolution of Nuclear-Encoded Plastidic Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Aims and Scope of this Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Nitrogen Assimilation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Nitrite Reductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Glutamine Synthetase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Glutamate Synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Evolution of Nitrogen Assimilation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Integration of Plastid Nitrogen Metabolism . . . . . . . . . . . . . . . . . . . . . . . III. Carbon Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Calvin Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Glycolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Oxidative Pentose-Phosphate Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Terpenoid Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Advances in Botanical Research, Vol. 42 Incorporating Advances in Plant Pathology Copyright 2005, Elsevier Ltd. All rights reserved.

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0065-2296/05 $35.00 DOI: 10.1016/S0065-2296(04)42004-7

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A. K. TOBIN AND C. G. BOWSHER

ABSTRACT Plastids are diverse organelles that diVer in form and function depending on their location within a plant. Their evolutionary origin, as free-living cyanobacteria, has left remnants of autonomy, and whereas the majority of the genetic control now lies within the nucleus, in terms of metabolism the plastid is fundamental to the life of the cell. This chapter describes the involvement of the plastid in carbon and nitrogen metabolism, in particular nitrate and ammonium assimilation, the Calvin cycle, oxidative pentose-phosphate pathway, glycolysis, and terpenoid biosynthesis. We have selected these pathways because they provide an opportunity to describe the metabolic interchange between plastids and cytosol and show duplication of some or all of the reactions in these two subcellular compartments. We discuss current knowledge of the likely ancestry of the genes encoding these pathways and consider how this has contributed to the compartmentation of nitrogen and carbon metabolism within the cell.

I. INTRODUCTION Plastids are double-membrane–bound organelles unique to plants and present in all living plant cells, except for pollen. They exist in a variety of forms, from the photosynthetic chloroplast, to the pigment-storing chromoplast of flowers and fruit, and the starch-accumulating amyloplasts found in storage organs. The capacity for interconversion from one form to another, together with malleability of both form and function in response to environmental signals such as light, makes this a highly plastic type of organelle. With this diversity, what defines a plastid is that it originates from a proplastid. Also common to all plastids are plastid DNA, a protein-rich soluble phase, termed the stroma, and a surrounding double membrane, the ‘‘plastid envelope.’’ Variations on this basic form include a complex arrangement of internal membranes (thylakoids) in chloroplasts, large starch grains in amyloplasts, and the crystalline prolamellar body of etioplasts found in the leaves of dark-grown tissue. Despite this variety, all plastids in a particular species of plant share the same DNA, inherited in most cases through the maternal parent, indicating that the nuclear genome exerts the greatest influence over plastid morphology and function. This nuclear control has been a continual process, whereby DNA from the plastid genome has become assimilated into the nucleus during the course of evolution.

A. ENDOSYMBIOTIC ORIGIN OF PLASTIDS

Plastids originated from free-living cyanobacteria that became engulfed in a eukaryotic host cell (Margulis, 1970). Two major plastid lineages arose: One led to the green algae and their descendants, the land plants, the other to the

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red algae (rhodophytes), phytoplankton, and macrophytes that share an evolutionary lineage through their plastids, but not through their host cells. Land plants acquired their plastids by a single endosymbiotic event. Secondary symbiosis enabled other groups, including the Euglenophytes, to obtain green plastids from the common green algal ancestor associating with diVerent host cells. Secondary symbiosis also allowed the red algal primary symbiont to become incorporated into a range of eukaryotic cells to produce the cryptophytes, haptophytes, heterokonts, and some dinoflagellates (Falkowski et al., 2004). B. GENE MIGRATION AND THE EVOLUTION OF NUCLEAR-ENCODED PLASTIDIC PROTEINS

Given the evolutionary origin of plastids and their retention of DNA, it is interesting to find that only a small proportion of plastid enzymes remain encoded within their own genome. Higher plant plastids have retained only 50–100 protein-encoding genes, whereas the cyanobacteria Synechocystis and Nostoc punctiforme encode, respectively, more than 3000 and 5000 proteins (Timmis et al., 2004). Thus, plastids contain only 1–5% as many protein-encoding genes as a comparatively small cyanobacterial genome (Martin and Herrmann, 1998). Given that plastids are estimated to contain at least 1000, and possibly as many as 5000 diVerent proteins, the vast majority of these proteins are encoded within the nucleus and the corresponding DNA sequences have migrated there from the plastid genome during the course of evolution. So, although the genes have been ‘‘lost’’ to the nucleus, many of the enzymes involved in the original cyanobacterial functions, such as adenosine triphosphate (ATP) synthesis and amino acid biosynthesis, are still present in the plastid (Martin and Herrmann, 1998). Martin and Herrmann (1998) argue persuasively that the duplication and/ or migration of plastid genes to the nucleus is a widespread and frequent evolutionary event. According to these authors, this is the first step in the evolution of a nuclear-encoded plastidic protein. The next step is then believed to be the acquisition of a targeting sequence that allows the protein to be moved into the plastid. Some proteins of cyanobacterial origin have become ‘‘stranded’’ in the higher plant cytosol because their genes have migrated to the nucleus and they have yet to gain the plastid targeting sequence. If localisation in the cytosol confers some metabolic advantage to the plant, it is likely that this protein will be retained, even if an isoform also exists in the plastid. In time, the plastidic isoform might become redundant and its gene will become a pseudogene. If both isoforms are of use, then they will both be retained, and either parallel, or interacting

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metabolic pathways can result, with, for example, full glycolytic pathways occurring in both compartments, or partial reactions taking place in the plastid and intermediates being exported to the cytosol for further metabolism. In this way, the specific location of an enzyme can enhance the metabolic flexibility of a higher plant, where compartmentation allows reactions to be retained within an organelle, and control of metabolite transport can finetune the interactions between metabolic pathways of the plastid and cytosol. C. AIMS AND SCOPE OF THIS CHAPTER

Plastids have been described as biosynthesis and assimilatory powerhouses, being the site of a number of major metabolic pathways, including photosynthetic CO2 fixation, starch synthesis, fatty acid synthesis, pigment synthesis, nitrogen assimilation, and amino acid biosynthesis. Compartmentation of metabolism, either solely or in part, allows enzymes and associated metabolites of a particular pathway to be concentrated (Bowsher and Tobin, 2001). As such, this can prevent incompatible metabolic processes from occurring simultaneously or can enhance the likelihood of favourable interactions (Emes and Dennis, 1997). In this chapter, we consider the role of the plastid in nitrogen and carbon assimilation in higher plants, having chosen these pathways because of their central involvement in autotrophy. We have decided to emphasise the role of compartmentation, with respect to plastidcytosolic interactions and for this reason have selected those pathways where there is significant interchange between these two subcellular locations. In so doing, we aim to provide an insight into why certain reactions are exclusive to the plastid, whereas others are shared with the cytosol. Our examples are of primary nitrate and ammonium assimilation, glycolysis, the Calvin cycle, oxidative pentose-phosphate pathway (OPPP), and terpenoid biosynthesis (Fig. 1).

II. NITROGEN ASSIMILATION The pathway of nitrite and ammonium assimilation is shown in Fig. 2. All reactions of primary assimilation and amino acid biosynthesis shown are catalysed by enzymes either that are exclusively located within the plastid as a single isoform or that have additional isoforms located in the cytosol or other organelles. In some cases, there are diVerences exhibited between tissues from diVerent sources (e.g., photosynthetic and nonphotosynthetic). Overall, the properties of the enzymes of nitrate assimilation and ammonium assimilation have been well characterised and extensively reviewed


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Fig. 1. Overview of pathways of nitrogen and carbon metabolism in higher plant plastids, showing interconnections between pathways and partitioning with the cytosol. Reactions shown in italics are unique to chloroplasts; all other reactions are found in chloroplasts and in non‐photosynthetic plastids. Abbreviations: DHAP, dihydroxyacetone phosphate; PEP, phosphoenolpyruvate; 3‐PGA, 3‐phosphoglycerate.

Fig. 2. The pathway of nitrogen assimilation in plastids. GS, glutamine synthetase; GOGAT, glutamate synthase; NR, nitrate reductase; NiR, nitrite reductase.

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(Crawford, 1995; Ireland and Lea, 1999; Lea and Ireland, 1999). The focus of the information presented here is the localisation of these enzymes, particularly their compartmentation in the plastids and the insight that this gives in terms of specific functionality. A. NITRITE REDUCTASE

Within the plastid, nitrite reductase (NiR) catalyses the reduction of nitrite to ammonium. Although the leaf and root form in Pisum sativum are immunologically indistinguishable and have similar inhibition properties, light absorption spectra, and electron-paramagnetic signals (Bowsher et al., 1988), other studies have clearly identified multiple isoforms in leaves and roots (Kutscherra et al., 1987). NiR is encoded by a single gene in Spinacia oleracea and Hordeum vulgare (Back et al., 1988; Duncanson et al., 1993). This is confirmed by the isolation of an H. vulgare mutant nir1 lacking NiR activity and protein in leaves and roots. Unless plants are grown on low nitrogen, the nir1 mutation is lethal (Duncanson et al., 1993; Wray, 1993). In contrast, there are at least two genes in Zea mays (Lahners et al., 1988) and four in Nicotiana tabacum (Kronenberger et al., 1993). The six-electron reduction of nitrite catalysed by NiR requires reduced ferredoxin as the electron donor. In leaves, reduced ferredoxin is generated photosynthetically (Fig. 3). In nonphotosynthetic cells, NADPH generated via the OPPP serves as the source of reducing power through the action of ferredoxin NADPþ oxidoreductase (FNR) to generate reduced ferredoxin (Fig. 3) (Bowsher et al., 1989, 1993). Nitrate is an important environmental signal for the induction of transcription of NiR (Rastogi et al., 1993; Seith et al., 1991, 1994; Wang et al., 2000; Wray, 1993). Nitrate also rapidly induces the necessary genes to generate reductant in nonphotosynthetic cells, including glucose-6-phosphate dehydrogenase (G6PDH) (Knight et al., 2001) and 6-phosphogluconate dehydrogenase (6PGDH) (Redinbaugh and Campbell, 1998) of the OPPP (see Section III.C.2.a), ferredoxin, and FNR (Bowsher et al., 1993; Wang et al., 2000). B. GLUTAMINE SYNTHETASE

Ammonium is incorporated into glutamine by the enzyme glutamine synthetase (GS). Immunologically and kinetically distinct GS isoforms have been identified in monocots and dicots. GS belongs to a small gene family, with diVerent subunits being encoded by diVerent genes (Lam et al., 1996; Oliveira and Coruzzi, 1999). In most species, GS is encoded by a minimum of four functional genes encoding one plastidic and at least three cytosolic


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Fig. 3. Nitrogen assimilation in plastids: interactions with photosynthesis, photorespiration, and the oxidative pentose phosphate pathway. Reactions in italics are unique to chloroplasts, reactions in bold are unique to plastids in non‐photosynthetic tissue: all others are common to both. Abbreviations: Fd‐GOGAT, ferredoxin‐ dependent glutamate synthase; Fdred, reduced ferredoxin; Fdox, oxidized ferredoxin; FNR, ferredoxin NADPþ oxidoreductase; GS1, cytosolic glutamine synthetase; GS2, plastidic glutamine synthetase; NADH GOGAT, NADH‐dependent glutamate synthase; NiR, nitrite reductase; 2‐OG, 2‐oxoglutarate; PS, photosynthesis.

polypeptides (Forde and Cullimore, 1988; Forde and Woodall, 1995). There is extensive evidence that genes for the chloroplast and cytosolic isoforms are expressed in diVerent cell types (Carvalho et al., 1992; Edwards et al., 1990; Kamachi et al., 1992; Peat and Tobin, 1996; Tobin and Yamaya, 2001). The balance of GS isoforms changes in response to the environment and/ or development (Cock et al., 1991; Li et al., 1993; Sakakibara et al., 1992). There is good evidence that the plastidic and cytosolic forms of GS also have distinct functional roles (Ireland and Lea, 1999). Tobin and Yamaya (2001) reviewed the cellular compartmentation of the two forms of GS. There are low levels of the plastidic form of GS (i.e., GS2) in nongreen tissue, and this increases with photosynthetic development (Tobin et al., 1985). The increase in GS2 levels is in parallel with an increase in photorespiratory capacity and photorespiration (Tobin et al., 1985). GS2 represents the major form of GS present in leaves and has been shown, on the basis of comprehensive

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biochemical, genetic, and molecular data, to reassimilate ammonium released during photorespiration (Fig. 3) (Edwards and Coruzzi, 1989; Keys et al., 1978; Kozaki and Takeba, 1996; Lea and Forde, 1994; Migge et al., 1996; Wallsgrove et al., 1987). In fully-expanded rice leaves, GS2 is primarily localised in the mesophyll and parenchyma cells (Tobin and Yamaya, 2001). Work with isolated Arabidopsis thaliana mitochondria and chloroplasts has suggested that GS2 may be dual targeted (Taira et al., 2004). Furthermore, transgenic A. thaliana plants generated to express a GS-negative green fluorescent protein (GFP) reporter were constructed and the mitochondria identified as a site of GFP targeting (Taira et al., 2004). This is in contrast to previous biochemical and localisation studies, and thus, this observation, if confirmed, would have important implications for the recycling of photorespiratory ammonium. Such dual targeting of GS2 must be investigated further in a wider range of plant species. As photorespiratory ammonium assimilation represents the major role for GS2 in leaves, it has been diYcult to characterise whether GS2 in chloroplasts is also involved in general nitrogen metabolism. GS2 in Z. mays and H. vulgare leaf tissue has been shown to be induced in response to nitrate, suggesting that it also has a role in primary ammonium assimilation (Sakakibara et al., 1992; Tobin and Yamaya, 2001). In contrast, GS2 barley mutants are able to assimilate nitrate, suggesting that the cytosolic GS1 is able to support primary assimilation (Blackwell et al., 1987). An alternative reason for this nitrate induction of GS2 in leaves could be that this would result in increased Rubisco synthesis (Stitt and Krapp, 1999), enhance photorespiratory flux, and therefore lead to an increased need for capacity to support photorespiratory ammonium assimilation (Tobin and Yamaya, 2001). In root tissue, there are diVerences between plant species relating to the presence or absence of plastidic GS. There is a known distinction in plants between those that are primarily root assimilators of nitrate and those that assimilate nitrate in the shoots (Andrews, 1986). Temperate legumes are usually root assimilators of nitrate, growing in soils with low external nitrate concentrations, whereas tropical and subtropical species are predominantly shoot assimilators. Plants in nitrate-rich temperate soils primarily exhibit root plastidic nitrate and ammonium assimilation (Woodall and Forde, 1996). These diVerences in the site of assimilation have been suggested to be an adaptation, perhaps relating to low temperature tolerance. Oryza sativa has little or no plastidic GS (Tobin and Yamaya, 2001). In contrast, H. vulgare roots have small but clearly detectable levels of plastid GS (Peat and Tobin, 1996; Tobin and Yamaya, 2001). Furthermore, immunolocalisation studies indicate that GS is found to varying degrees in diVerent H. vulgare root plastid types (Peat and Tobin, 1996). The significance of


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this has yet to be determined but may be indicative of specialisation of function. Emes and Fowler (1983) first reported the nitrate induction of plastidic GS in P. sativum roots, with a twofold to fivefold increase in activity. Subsequent studies confirmed the induction to be at the messenger RNA (mRNA) and protein level in Z. mays roots (Redinbaugh and Campbell, 1993; Sakakibara et al., 1992). Such induction of root plastid GS and its coordination with NR and NiR genes supports the suggestion that in roots the plastidic GS2 is directly responsible for the assimilation of ammonium generated as a result of the action of NiR in root plastids (Fig. 3). C. GLUTAMATE SYNTHASE

Glutamate is the major constituent of total amino acids extracted from mature leaves, and it is the nitrogen donor for most transamination reactions in amino acid metabolism, being synthesised by glutamate synthase (GOGAT) in conjunction with GS in a cyclic manner (Miflin and Lea, 1980). There are two distinct forms of GOGAT, diVering in their specificity for an electron donor, ferredoxin-GOGAT (Fd-GOGAT) and NADH-GOGAT (Suzuki and Gadal, 1984). These exist as multiple isozymes, but all forms are localised within the plastid (Chen and Cullimore, 1989; Suzuki and Gadal, 1984). Both NADH-GOGAT and Fd-GOGAT contain the same prosthetic groups in the N-terminal domain, but in the C-terminal domain NADH-GOGAT also contains an additional iron sulphur cluster and flavin, which are thought to be involved in electron acceptance from NADH (Curti et al., 1995). Fd-GOGAT is most abundant in mature photosynthetic tissue (Matoh and Takahashi, 1982), and NADH-GOGAT is most abundant in immature nonphotosynthetic tissue (Matoh and Takahashi, 1982). Two distinct Fd-GOGAT genes (Coschigano et al., 1998) and a single NADHGOGAT gene (Lam et al., 1997) have been identified in A. thaliana. Studies have shown that in wild type A. thaliana, Fd-GOGAT represents about 95% of the total GOGAT activity in the leaves and 68% in the roots (Somerville and Ogren, 1980; Suzuki and Rothstein, 1997), with the remaining activity being NADH-GOGAT (Somerville and Ogren, 1980). The isoform-specific patterns of expression and localisation suggest distinct physiological roles (Fig. 3). This has been further supported by studies incorporating a range of biochemical, genetic, and molecular approaches. 1. Fd-GOGAT In the 1980s, a number of conditional lethal photorespiratory mutants defective in Fd-GOGAT were isolated and characterised (Kendall et al., 1986; Somerville and Ogren, 1982). These gls photorespiratory mutants

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were originally isolated based on them exhibiting conditional chlorosis in air, but recovering in 1% CO2. From analysis of these plants, it was proposed that in leaves Fd-GOGAT had a major, if not exclusive, role in photorespiratory ammonium metabolism (Fig. 3). This role is further supported by immunological and localisation studies (Tobin and Yamaya, 2001). Analysis of A. thaliana has led to the identification of two genes encoding Fd-GOGAT, GLU1, and GLU2 (Coschigano et al., 1998). GLU1 levels are abundant in leaves, low in roots, and stimulated by light and sucrose. In contrast, GLU2, which is not susceptible to sucrose or light induction, is constitutively expressed at low levels in leaves and high levels in roots (Coschigano et al., 1998; Oliveira et al., 1997). Upon further study, it is clear that the gls mutation specifically aVects GLU1 and not GLU2. Furthermore, the gls mutants failed to respond to exogenously supplied inorganic nitrogen, suggesting that in addition to reassimilating photorespiratory ammonium, GLU1 is involved in nitrogen assimilation and its role cannot be compensated for by GLU2 (Coschigano et al., 1998). This confirms that in A. thaliana, GLU1 and GLU2 have distinct metabolic roles. GLU2 has been suggested to be a housekeeping Fd-GOGAT gene that may synthesise basal levels of glutamate needed for protein biosynthesis (Coschigano et al., 1998). To date, evidence for multiple Fd-GOGAT isoforms in other higher plants has yet to be reported. Future localisation studies could provide great insight into distinguishing specific functionality. In roots, Fd-GOGAT activity changes in levels and distribution during root development (Ishiyama et al., 1998; Matoh and Takahashi, 1982; Peat, 1996). It is evenly distributed at high levels in the root tip and decreasing in the older cells, being mainly found in the central cylinder and secondary root primordia (Ishiyama et al., 1998). Such distribution, together with a lack of response to ammonium induction (Tobin and Yamaya, 2001), suggests that Fd-GOGAT may be involved in remobilising glutamine transported from N-depleted shoots (Ishiyama et al., 1998). Analysis of the barley mutant LaPr 85/73 deficient in Fd-GOGAT has confirmed the importance of this enzyme in nitrite assimilation in roots (Joy et al., 1992). Although there was no detectable change in root Fd-GOGAT activity in this mutant, there was an increased concentration of glutamine in the root and an increase in export of glutamine to the shoot. If the mutation only aVects the leaf Fd-GOGAT, this would suggest that a disruption of glutamine metabolism in the leaf aVects the export of glutamine from the root (Tobin and Yamaya, 2001). Alternatively, any small but undetectable changes in levels of Fd-GOGAT in the root must have a marked impact on the balance of glutamine metabolism, resulting in more glutamine being made available for export to the shoot (Tobin and Yamaya, 2001). It is


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clear from work with H. vulgare roots that Fd-GOGAT levels increase in response to nitrate. This increase would correspond to the increase in NiR and the supply of reduced ferredoxin via FNR (see Section II.A). Previous studies have supported a link of reductant supply to Fd-GOGAT in pea root plastids (Fig. 3) (Bowsher et al., 1992). 2. NADH-GOGAT Until recently, much less was known about the function of NADH-GOGAT in higher plants. Genetic studies of the Fd-GOGAT–deficient gls mutant (see Section II.C.1) indicated that NADH-GOGAT activity was unable to replace Fd-GOGAT, suggesting it has a distinct metabolic role. But the lack of an equivalent NADH-GOGAT mutant meant that roles were proposed based on the available protein localisation and expression data (Fig. 3). Although present in leaves, NADH-GOGAT is more prevalent in nonphotosynthetic tissues, seeds, roots (Ireland and Lea, 1999; Matoh and Takahashi, 1982), and cotyledons (Hecht et al., 1988). Based on the expression of NADH-GOGAT in vascular cells of root and developing leaves, as well as in O. sativa root epidermal cells, it has been proposed to function in glutamine export to the phloem in senescing tissues and roots (Goto et al., 1998; Hayakawa et al., 1994; Ishyama et al., 1998). The observations that NADH-GOGAT levels are stimulated in O. sativa roots by low levels of ammonium (Hirose et al., 1997), by nitrate in A. thaliana (Wang et al., 2000), and by nitrate-starved N. tabacum (Lancien et al., 1999) are indicative that NADH-GOGAT has a role in primary nitrogen assimilation in roots (Fig. 3). The identification and analysis of an A. thaliana T-DNA–tagged mutant (glt1-T) disrupted in the GLT1 gene encoding NADH-GOGAT has provided valuable additional information to confirm the role of this enzyme in higher plant plastid metabolism (Lancien et al., 2002). Under nonphotorespiratory growth conditions, no NADH-GOGAT activity was detected and the fresh weight and glutamate levels decreased 20% and 70%, respectively, whereas glutamine accumulated. Such a response might be anticipated if NADH-GOGAT is involved in primary ammonium assimilation. The failure of Fd-GOGAT to compensate for the loss of NADH-GOGAT activity suggests that as they are localised diVerently they have non-overlapping roles. These observations also provide further evidence for the proposed role of NADH-GOGAT in nitrogen remobilisation. Interestingly, threonine levels were also found to accumulate in the glt1-T mutant, and this, together with an observed decrease in glutamate and aspartate, has also led to the suggestion that NADH-GOGAT may coordinate metabolism to respond to such amino acid changes (Lancien et al., 2002). Clearly, there is now a need for further studies to verify this.

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NADH-GOGAT also appears to be important in nitrogen fixation, with levels increasing with the onset of nitrogen fixation in nodules (Suzuki and Gadal, 1984). Ammonium assimilation derived from symbiosis was specifically impaired in a Medicago truncatula antisense transgenic line, with less than 50% wild type NADH-GOGAT levels (Schoenbeck et al., 2000). D. EVOLUTION OF NITROGEN ASSIMILATION

The available evidence suggests that components involved in nitrogen assimilation in higher plants are derived from a cyanobacterium-like organism. Such results support the hypothesis that plastids from higher plants are derived from a cyanobacterium-like endosymbiont and that most of the endosymbiont genome was transferred to the nucleus of the host during evolution, as discussed in Section I (Douglas and Turner, l991; Margulis, 1970; Martin and Herrmann, 1998; Weeden, 1981). Comparison of the nuclear-encoded NiR from higher plants with cyanobacterial NiR indicates that they are highly homologous (Luque et al., 1993). This supports the suggestion that higher plant NiR is derived from a cyanobacterium-like organism. GS evolution has been widely studied as it has been identified as one of the oldest functioning genes (Kumada et al., 1993) and as a useful molecular clock (Pesole et al., 1991). When producing a phylogenetic tree for GS from alignment of sequences from archaebacteria, eubacteria, and eukaryotes, there are three forms of GS: GSI, GSII, and GSIII. These include sequences for archaebacteria/eubacteria, eubacteria/eukaryotes, and eubacteria, respectively (Inokuchi et al., 2002). As such, land plants and algae were originally identified as GSII type only. That is, in A. thaliana, there are four GSII genes encoding the cytosolic GS1 isoenzymes and one GSII gene encoding the plastidic GS2 isoenzyme. Similarly in Z. mays there is at least one GSII gene encoding a plastidic GS2 isoenzyme and one encoding a cytosolic GS1 isoenzyme (Inokuchi et al., 2002). This suggests that the isoenzymes located in the plastid and cytosol have arisen as a result of a gene duplication event. Interestingly, a search of available expressed sequence tag (EST) banks for A. thaliana and Z. mays identified putative GSI sequences that were used to screen an M. truncatula cDNA library and led to the identification of a GSI-like gene (Mathis et al., 2000). Subsequently, further screening of EST banks identified GSI-related sequences also present in P. sativum and B. napus. This has been presented as evidence for paralogous evolution of the GSI and GSII genes (Mathis et al., 2000). The phylogenetic tree of GOGAT indicates two forms, GOGATI and GOGATII, composed of eubacteria/eukaryotes and archaebacteria/


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eubacteria, respectively (Inokuchi et al., 2002). The higher plant Fd-GOGAT and NADH-GOGAT, though encoded by diVerent nuclear genes, appear to be related in evolutionary terms (Sakakibara et al., 1991). They are both found in the GOGATI alignment and it would appear that they have an endosymbiotic origin (Inokuchi et al., 2002). When investigating the evolutionary relationship of the components of the nitrogen assimilatory pathway, some interesting observations have been made. Nitrate reductase (NR) has three forms, based on phylogenetic analysis, with one form containing the cytosolic higher plant NR together with algal NR but not the cyanobacterial form (Stolz and Basu, 2002). The available evolutionary evidence indicates higher plant NR originated from the host (eubacterial) cell. It would appear that the cyanobacterial NR gene was lost by the chloroplast, whereas NiR was retained (Stolz and Basu, 2002). One might expect this to indicate some functional advantage in separating the location of NR and NiR during nitrate assimilation. For instance, by locating NR in the cytosol, it may be able to quickly respond to nitrate as it enters the cell and to coordinate the responses of the other nitrate assimilatory enzymes/genes and link these to other metabolic processes within the cell, regardless of location. A study of GS and GOGAT from 37 species, including 8 species of archaebacteria, 25 species of eubacteria, and 4 species of eukaryotes (including A. thaliana), indicates that the genes have evolved paralogously, having frequently been transferred horizontally among the three domains during early evolutionary life (Inokuchi et al., 2002). This may well reflect a competitive advantage contributing to the diVerent physiological roles seen by diVerent isoforms in diVerent intercellular and intracellular locations. E. INTEGRATION OF PLASTID NITROGEN METABOLISM

Nitrogen assimilation is localised, at least in part, in the plastid. However, the plastid cannot operate in isolation, with precursors (e.g., nitrate), intermediates (e.g., ammonium and carbon skeletons) and end products all needing to be exchanged with the cytosol. As such, plastid nitrogen assimilation is only possible because of the presence of a whole battery of transport proteins in the inner plastid envelope membrane. A number of recent reviews have comprehensively characterised the information available (Flu¨ gge, 1998, 1999; Weber and Flu¨ gge, 2002; Weber et al., 2004). The metabolic capacity of a plant will constantly be changing with, for example, plant development, environmental conditions, and availability of nutrients. All these factors can have an impact on the capacity for nitrogen metabolism. To operate eYciently, nitrogen assimilation must be coordinated

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with other metabolic processes, including nitrogen storage and remobilisation (Lea and Miflin, 2003), amino acid synthesis and use (Noctor et al., 2002), and carbon metabolism (Foyer et al., 2001). By necessity, integration with metabolism occurs at a number of levels. This is beyond the scope of this chapter, but to explore this topic further, readers are referred to Stitt et al. (2002).

III. CARBON METABOLISM Plastids, in the form of chloroplasts, are well recognised as being responsible for the reductive biosynthesis of intermediary carbohydrates from CO2. The Calvin cycle is the primary carboxylating process in plants and operates solely in the chloroplast. It has been well characterised and its regulation understood for many years (Macdonald and Buchanan, 1997; Robinson and Walker, 1981). A number of the enzymes and metabolites in the Calvin cycle are also involved in other metabolic pathways operating either solely in the plastid or also in other locations. As such, plastid compartmentation can be seen as a means of coordinating these processes, and this is essential to avoid futile cycling. To illustrate the importance of plastid compartmentation in metabolism, we examine the operation of the Calvin cycle, glycolysis, and the OPPP. A. CALVIN CYCLE

1. Evolutionary Aspects The 13 enzyme reactions of the Calvin cycle (or the reductive pentosephosphate pathway) in the chloroplast form the major route through which carbon enters metabolism. The complexity of metabolism can be seen in S. oleracea plastids. Of the enzymes involved in the Calvin cycle, five have isoforms in glycolytic and gluconeogenic pathways of the cytosol. As the primary carboxylating pathway in higher plants, the enzymes of the Calvin cycle are all located in the stroma of the chloroplast (Macdonald and Buchanan, 1997). The Calvin cycle has been well studied in terms of origin of genes, and apart from the large subunit of Rubisco, the genes are all nuclear-encoded (Fig. 4). It is clear from studies that there is a wide variation in the original source of Calvin cycle genes, with gene duplications, losses, and transfers all taking place (Martin and Herrmann, 1998; Martin and Schnarrenberger, 1997) (Fig. 4). Rubisco, phosphoglycerate kinase (PGK) (Brinkmann and Martin, 1996), transketolase (TKL) (Flechner et al., 1996), ribulose-5-phosphate 3-epimerase (RPE) (Kopriva et al., 2000), and phosphoribulokinase (PRK) all have subunits encoded by a gene of


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Fig. 4. Evolutionary origins of the genes of the Calvin cycle and the Oxidative Pentose Phosphate Pathway (OPPP) in plastids. For reasons of clarity, the OPPP is simplified in this diagram. The products of the TA reactions, erythrose 4‐P and fructose 6‐P can re‐enter the pathway at the TKL and FBP stages, respectively. (Shaded subunits are of cyanobacterial origin; solid black subunits have arisen from duplication of nuclear gene; white subunits are of unknown origin). Abbreviations: FBA, fructose bisphosphate aldolase; FBPase, fructose 1,6‐bisphosphatase; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; G6PDH, glucose 6‐phosphate dehydrogenase; NGAPDH, NADP dependent glyceraldehydes non‐phosphorylating 3‐phosphate dehydrogenase; 6PGDH, 6‐phosphogluconate dehydrogenase; PGK, phosphoglycerate kinase; PRK, phosphoribulokinase; RPE, ribulose-5‐phosphate 3‐epimerase; RPI, Ribose phosphate isomerase; SBP, sedoheptulose bisphosphatase; TA, Transaldolase; TKL, transketolase; TPI, triose phosphate isomerase.

cyanobacterial origin. Triose phosphate isomerase (TPI) subunits are encoded by a gene of mitochondrial origin (Martin and Schnarrenberger, 1997). The plastidic fructose bisphosphatase enzyme (FBPase) shows no resemblance to the known cyanobacterial enzyme (Rogers and Keeling, 2004), indicating that the gene for the ancestral cytosolic enzyme has been duplicated and the protein targeted to the plastid, resulting in deletion of the original cyanobacterial enzyme. Ribose-5-phosphate isomerase (RPI), sedoheptulose1,7-bisphosphatase (SBP), and fructose-1,6-bisphosphate aldolase (FBA) are encoded by genes whose origins remain uncertain (Martin and Herrmann, 1997; Martin and Schnarrenberger, 1997; Rogers and Keeling, 2004). The

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available evidence suggests that SBP, TKL, RPE, and RPI are found exclusively in the plastid with no cytosolic isoforms. 2. Calvin Cycle: Interactions/Regulation During the operation of the Calvin cycle, carbon is withdrawn at diVerent places for sucrose and starch synthesis (Stitt, 1997). That is, triose phosphates are exported to the cytosol for conversion into sucrose, whereas fructose-6-phosphate is used for starch synthesis. This has been confirmed by studies of transgenic plants with altered levels of Calvin cycle enzymes. In transgenic plants with decreased levels of FBA (Haake et al., 1998) or FBPase (Kossmann et al., 1995), there is a marked decrease in starch levels while sugar levels remain high. Starch synthesis is inhibited because these enzymes are located downstream of the site where carbon is withdrawn for sucrose synthesis, but upstream of where carbon is removed for starch. In contrast, decreased levels of TKL as a result of generating antisense plants leads to a decrease in leaf sugar levels and little eVect on starch levels (Henkes et al., 2001). The operation of TKL as the first step in the Calvin cycle downstream of fructose-6-phosphate means that high rates of starch synthesis will be supported. B. GLYCOLYSIS

Glycolysis is a ubiquitous metabolic pathway observed in virtually all living cells. During the operation of glycolysis, orthophosphate and ADP are utilised, NADþ is reduced to NADH, and ATP is generated. In plants, glycolysis leads to the formation of numerous and varied chemical constituents. Glycolysis has been defined as the sequence of reactions leading to the overall conversion of glucose, or other monosaccharides, to pyruvic acid. However, the pathway is not necessarily a single enzyme sequence from start to end. Glycolysis in plants is extremely flexible with a number of distinctive features compared with the process in other organisms. This includes multiple potential entry and exit points (Dennis et al., 1997; Plaxton, 1996). In addition, there are alternative enzyme steps allowing particular steps to be either bypassed or linked into alternative branches of the pathway (ap Rees 1988; Plaxton, 1996). Plant glycolysis has been discussed in full in several excellent reviews (ap Rees, 1985, 1988; Dennis et al., 1997; Givan, 1999; Miernyk, 1990; Plaxton, 1996). 1. Localisation In higher plant cells, the available evidence supports the existence of two complete, or almost complete, and spatially separated glycolytic pathways. The dual location of glycolysis in the plastid and cytosol raises interesting


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questions regarding the importance of plastid compartmentation. There are significant diVerences in terms of the pathways roles, structure, regulation, and compartmentation in diVerent phyla and within diVerent cells of the same type (Plaxton, 1996). Although some degree of flexibility of plant glycolysis has been demonstrated, it is clear that this is primarily in the cytosolic pathway and it appears to be important under conditions of nutritional phosphate deficiency (Plaxton, 1996). As such, an alternative pyrophosphate-dependent phosphofructokinase (PFK) and an NADPdependent nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (NGA3PDH) that bypasses the formation of ATP by PGK are unique to the cytosol (Fig. 5). In contrast, it would appear that glycolysis operating in the plastid is a far more conservative pathway (Plaxton, 1996).

Fig. 5. Evolutionary origins of the genes of the glycolytic pathway in plastids and cytosol. (Shaded subunits are of cyanobacterial origin; solid black subunits have arisen from duplication of nuclear gene; striped subunits are of mitochondrial origin; white subunits are of unknown origin). Abbreviations: ENO, enolase; FBA, fructose bisphosphate aldolase; FBPase, fructose 1,6 bisphosphatase; G6PI, glucose 6-phosphate isomerase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NGAPDH, NADP dependent glyceraldehydes non-phosphorylating 3-phosphate dehydrogenase; PFK, phosphofructokinase; PFP, pyrophosphate-dependent fructose 6-phosphate 1-phosphotransferase; ; PGK, phosphoglycerate kinase; PGM, phosphoglyceratemutase; PK, pyruvate kinase; TPI, triose phosphate isomerase.

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Initial studies on plastids isolated from developing Ricinus communis endosperm established the presence of nearly all of the glycolytic enzymes (Miernyk and Dennis, 1982, 1983; Simcox et al., 1977). Subsequent studies on other nonphotosynthetic plastids confirmed the presence of most of the glycolytic pathway enzymes, although the levels of phosphoglyceromutase (PGM) were low or undetectable in some studies (Emes and Tobin, 1993). For instance, in the nonphotosynthetic flower buds of Brassica oleracea, plastid PGM had a much lower specific activity than that of other glycolytic enzymes (Journet and Douce, 1985). Root plastids from P. sativum (Trimming and Emes, 1993) and Acer pseudoplatanus amyloplasts (Frehner et al., 1990) had no detectable PGM. Studies on the P. sativum shoot chloroplast confirmed a similar situation (Stitt and ap Rees, 1979). Further studies on A. pseudoplatanus plastids also determined that the activities of ATP-dependent PFK and NAD GAPDH were too low to account for the physiological rate necessary to convert starch to triose phosphates (Frehner et al., 1990). The apparent contribution of the plastid pathway is clearly aVected by the metabolic demands of the tissue and therefore varies with the tissue type and age. For example, enolase from R. communis seed plastids increases during development, reaching approximately 30% of the total cell activity when oil synthesis is at its highest rate. In contrast, only 10% of glycolytic activity is plastidic in germinating seed plastids, 7% in chloroplasts of immature leaves, and 0% in chloroplasts from mature leaves. Fatty acid synthesis occurs in developing R. communis seed plastids, and the operation of the entire pathway of glycolysis would be necessary for the conversion of hexoses to long-chain fatty acids present in the plastid. In contrast, in amyloplasts it would appear that hexose phosphates derived from starch are exported from the amyloplast and metabolised primarily via the cytosolic-glycolytic pathway (Frehner et al., 1990). The dual location of glycolysis in the cytosol and plastids has led to comparisons of the kinetic and regulatory properties of the isoenzymes involved. Such studies were initially hampered by a need to develop suitable methods for the eVective separation of these diVerent isoforms and being able to assign the isoforms to particular locations. However, it is clear from such studies that the plastid enzymes have distinct properties and appear to be more highly regulated when compared with their cytosolic counterpart. In studies of spinach leaf PFK, the chloroplast form is inhibited by orthophosphate, but this inhibition is relieved by PEP or 3-PGA, whereas the cytosolic form is stimulated by orthophosphate (Dennis and Greyson, 1987; Kelly and Latzko, 1977a,b). In contrast, in R. communis endosperm, plastid PFK is activated by orthophosphate at suboptimal pH levels and inhibited by ATP,


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whereas the cytosolic isoform is unaVected by orthophosphate but is very sensitive to 3-PGA (Garland and Dennis, 1980). Such kinetic diVerences in sensitivity to allosteric activators between the two isoforms are presumably of physiological significance, perhaps adjusting the balance of triose phosphates between the plastid and cytosol. When pyruvate kinase (PK) was first isolated from S. oleracea leaves, two isoforms were isolated, which, though not distinguished by location, were clearly regulated diVerently, with one form activated by aspartate and one form inhibited by glutamate (Baysdorfer and Bassham, 1984). In R. communis endosperm, the aYnity of the plastid PK isoform for ADP is an order of magnitude greater than that of the cytosolic enzyme. Also, the plastid isoform requires sulphydryl protection for stability, suggesting that its environment is very diVerent from that of the cytosolic isoform (Ireland et al., 1980). There appear to be tissue-specific plastid PK isoforms in higher plants, with PK from developing seed leucoplasts being immunologically distinct from the leaf chloroplast form (McHugh et al., 1995). Such diVerences may reflect diVerences in the primary role of plastidic PK. Depending on tissue type and metabolic demands, glycolysis may operate in the cytosol and in the plastid. It is clear that this pathway is not operating in isolation within these distinct locations and that there is interchange of glycolytic intermediates from one compartment to another. A number of translocators have been identified on the plastid envelope to allow exchange of specific glycolytic intermediates to and from the plastid (Flugge, 1999). 2. Evolutionary Aspects All of the glycolytic enzymes are nuclear encoded, and in terms of the evolutionary origins of the genes, again there is marked variation throughout the pathway (Martin and Herrmann, 1997) (Fig. 5). The evolution of enzymes overlapping with the Calvin cycle has already been discussed (see Section III. A.1). The plastidic subunits of glucose-6-phosphate isomerase (G6PI) (Hattori et al., 1995; Nowitzki et al., 1998), GAPDH, PGK, and PGM are each encoded by a gene of cyanobacterial origin. Enolase subunits are encoded by genes of uncertain origin. Plastidic PK is a heterotetramer, with a subunit encoded by a gene of cyanobacterial origin and by duplication of a nuclear gene (Blakeley et al., 1991; Hattori et al., 1995; Plaxton, 1989). A notable diVerence observed between the plastid and the cytosolic glycolytic pathway is that cytosolic G6PI appears to have a mitochondrial origin (Nowitzki et al., 1998). In most organisms, the glycolytic pathway has evolved by gene duplication, giving rise to diVerent isoenzymes (Fothergill-Gillmore, 1986). As such, selection may have produced enzymes optimal for the metabolic demands and

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chemical diVerences of specific plant tissues. Exceptions to this rule also support the argument that multiple genes are not necessarily a requirement to meet metabolic demands. Several reports have mentioned the absence of particular glycolytic enzymes in chloroplasts. For instance, Van Der Straten et al. (1991) have shown that at the gene level, A. thaliana has an incomplete set of glycolytic enzymes in the plastids. In chloroplasts, glycolytic enzymes are primarily involved in photosynthetic carbon fixation and not glycolysis. If enolase is one of the few steps not involved in the Calvin cycle, then it would follow that without a glycolytic role, it is not needed in the chloroplast. It has been suggested, however, that in isolated P. sativum chloroplasts enolase might have a role in carbohydrate breakdown (Stitt and ap Rees, 1979). C. OXIDATIVE PENTOSE-PHOSPHATE PATHWAY

Although glycolysis is the major catabolic route for hexoses, all plant cells possess an alternative route, the OPPP. These two pathways are linked through common intermediates, glucose-6-phosphate (Glu6P), fructose-6phosphate, and glyceraldehyde-3-phosphate. Generally, the OPPP is divided into two stages. The first is the irreversible oxidative decarboxylation of Glu6P to ribulose-5-phosphate, resulting in the generation of two molecules of NADPH per molecule of Glu6P. The second stage is reversible, resulting in the formation of fructose-6-phosphate and glyceraldehyde-3-phosphate, which in principle can then be further metabolised by glycolysis. The OPPP has three main functions in plants. First, it generates NADPH, which is used as a reductant in biosynthetic circumstances when NADPH is not being generated by photosynthesis. It is, therefore, particularly important in nonphotosynthetic tissues and in processes such as assimilation of inorganic nitrogen and fatty acid biosynthesis, as well as in maintaining redox potential to protect against oxidative stress (Debnam et al., 2004; Emes and Dennis, 1997). Studies using isolated P. sativum root plastids, for example, have established that nitrite reduction (see Section II.A) (Bowsher et al., 1989) and glutamate synthesis (see Section II.C.1) (Bowsher et al., 1992) are dependent on NADPH generated by the flux of carbon through the OPPP. Second, it generates ribose-5-phosphate, which is necessary for the synthesis of nucleotides and nucleic acids. Third, it generates erythrose-4-phosphate, which is required for the synthesis of shikimic acid, the precursor of aromatic rings. As such, the reversible nonoxidative section of the pathway is the source of carbon skeletons for the synthesis of nucleotides, aromatic amino acids, phenylpropanoids, and derivatives (Herrmann and Weaver, 1999). The second stage of the OPPP is important because it enables any excess pentose phosphate produced during NADPH generation to be returned to


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the glycolytic sequence (ap Rees, 1985). It also allows ribose-5-phosphate and erythrose-4-phosphate to be formed from glyceraldehyde-3-phosphate and fructose-6-phosphate without the need to oxidise carbohydrate. Varying amounts of ribose-5-phosphate and erythrose-4-phosphate will be withdrawn from the OPPP depending on whether they are being used for nucleotide synthesis and phenylpropanoid production, respectively. The nonoxidative section of the OPPP may be regarded as a pool of intermediates close to dynamic equilibrium and capable of being adjusted as intermediates are withdrawn from the pool (Dennis and Blakeley, 2000). 1. Localisation Knowledge of the OPPP enzymes at the molecular level is not as advanced as that of other components of carbon metabolism. However, it is clear that all or part of the OPPP is duplicated in the plastids and the cytosol (Dennis and Miernyk, 1982). Interestingly, all the enzymes of the pathway have been identified in a range of plastid types, and any absences of enzymes seem to be within the cytosol. All studies have confirmed the presence of G6PDH (Graeve et al., 1994; Knight et al., 2001; von Schaewen et al., 1995; Wendt et al., 2000) and 6PGDH (Krepinsky et al., 2001; Redinbaugh and Campbell, 1998) in both the plastid and the cytosol. However, depending on species, tissue types, developmental stage, and environmental conditions, the distribution of the enzymes involved in the later nonoxidative steps is less clear (Kruger and von Schaewen, 2003). The nonoxidative enzymes have been shown to be present in diVerent plastid types, for example, chloroplasts (Schnarrenberger et al., 1973), etioplasts (Schnarrenberger et al., 1975), chromoplasts (Thom et al., 1998), root plastids (Emes and Fowler, 1979), and R. communis endosperm (Nishimura and Beevers, 1979). In 1995, Schnarrenberger made a comprehensive study of the nonoxidative enzymes in S. oleracea leaves, and the results indicated that they were restricted to plastids. Subsequent studies confirmed the absence of cytosolic counterparts in S. oleracea and P. sativum leaves, as well as in Z. mays and P. sativum roots (Debnam and Emes, 1999). Although more than 50% of nonoxidative enzymes were reported in the cytosol of N. tabacum leaves and roots (Debnam and Emes, 1999), this might be an artefact of the way the organelles were separated. That is, diVerential centrifugation of N. tabacum leaf extracts was used for organelle isolation and this gives low plastid yield and might reflect a selective loss of enzymes. In chloroplasts isolated from N. tabacum, mesophyll protoplasts fractionated on sucrose density gradients, 95% of plastids recovered were intact with negligible cytosolic contamination, and approximately 90% of the transketolase activity was found to be associated with the chloroplasts (Henkes

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et al., 2001). Experiments with R. communis endosperm (Nishimura and Beevers, 1979), Glycine max root nodules (Hong and Copeland, 1990), and B. oleracea flower buds (Journet and Douce, 1985) all suggest that there is cytosolic activity of the nonoxidative enzymes. 2. Nature and Roles of Multiple Isoforms of OPPP Enzymes The presence of multiple isoforms is possibly indicative of specific functions. Selected examples of particular OPPP enzymes are presented to illustrate diVerences between isoforms and the possible implications this has for metabolic regulation. a. Oxidative Steps. As has already been stated, G6PDH exists in multiple isoforms. The cytosolic enzyme is very stable and has been fully purified (Fickenscher and Scheibe, 1986; Srivastava and Anderson, 1983). In contrast, the plastidic enzyme has proved to be very unstable and therefore diYcult to purify. It is really only with the use of molecular techniques that a more comprehensive understanding of the plastidic G6PDH has been possible. Two classes of plastidic G6PDH, P1 and P2, have now been identified (Knight and Emes, 1996; Knight et al., 2001; Wendt et al., 2000). The P1 form of G6PDH is expressed at high levels in green tissues (von Schaewen et al., 1995; Wendt et al., 2000), whereas the P2 form of G6PDH is expressed throughout the plant but with highest steady-state transcript levels being found in the stems and roots (Knight et al., 2001; Wendt et al., 2000). Plastidic G6PDH has long been known to be reductively inactivated in the light by the ferredoxin/thioredoxin system (Buchanan, 1991). This enzymemediated inactivation is due to the reversible dithiol disulphide interchange of two highly conserved regulatory cysteine residues (Wenderoth et al., 1997; Wendt et al., 1999). Although both the P1 and the P2 forms are inactivated by DTT and inhibited by increased NADPH/NADPþ, the P2 form appears to be less sensitive to both types of modulation. That is, the P2 form of G6PDH has been shown to have a more than eightfold higher Ki value for NADPH. Such kinetic diVerences may reflect diVering metabolic requirements of the cells in which they are expressed. In illuminated chloroplasts, ferredoxin is reduced directly via photosynthetic electron flow, and the OPPP needs to be inactivated in the light to avoid potentially futile interactions with the Calvin cycle. In contrast, nonphotosynthetic tissue needs to maintain a flux through the plastidic OPPP to drive ferredoxin-dependent reactions such as nitrite reduction (see Section II.A) and glutamate synthesis (see Section II.C.1), even when there are high stromal NADPH/NADPþ levels. The involvement of the P2 form of G6PDH in the provision of NADPH for these ferredoxin-dependent reactions has been implicated,


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based on the expression of genes encoding two P2 isoenzymes from A. thaliana following transfer from ammonium-containing medium to nitrate-containing medium (Wang et al., 2000). A similar increase has also been reported in P2 genes from roots of N. tabacum (Knight et al., 2001) and Lycopersicon esculentum (Wang et al., 2001) in response to nitrate and in response to ammonium/glutamate in H. vulgare root plastids (Esposito et al., 2001a,b). In the latter case, the P2 isoform was able to support an increased flux through the OPPP (Esposito et al., 2003; Wright et al., 1997). The P2 form of G6PDH has also been implicated in balancing the redox poise in chloroplasts (Debnam et al., 2004). Transgenic N. tabacum plants with decreased levels of P2 had an increased content of reduced glutathione, elevated ascorbate, and a much higher ratio of reduced/oxidised ascorbate. This led to greater protection from oxidative stress in antisense lines, whereas leaf discs from plants overexpressing the P2 form of G6PDH showed increased oxidative damage, as measured by lipid peroxidation. b. Nonoxidative Steps. As has already been discussed (see Section III.A.2), transgenic N. tabacum plants with decreased transketolase activity have been generated (Henkes et al., 2001). The dual involvement of transketolase in the Calvin cycle (see Section III.A.2) as well as the OPPP makes it diYcult to determine an impact resulting from functionally impaired OPPP. Although the OPPP was not strongly inhibited in these plants, a 50–70% decrease in transketolase activity resulted in a reduction in the wounding-induced stimulation of OPPP flux that occurs in wild type plants (Henkes et al., 2001). Ribulose-5-phosphate 3-epimerase (RPE) exists in chloroplastic and cytosolic isoforms in O. sativa and other species (Kopriva et al., 2000). The chloroplast enzyme is a hexamer, but the cytosolic form has a dimeric subunit. It has been suggested that this diVerence may facilitate the networking of metabolic pathways through complementary binding interactions between sequential enzymes. Such a process would be functionally useful. That is, transketolase and RPE essentially catalyse the same reaction in the OPPP and the Calvin cycle (Stitt and ap Rees, 1979). The Km and pH optima properties of the enzymes are of a similar range to those of animal and yeast cell enzymes (Tiege et al., 1998). The broad pH optima observed for transketolase and RPE could help the enzymes function in two diVerent metabolic pathways and under a range of conditions. For example, the Calvin cycle operates in the daytime, when the pH of the chloroplast stroma will be high; whereas the OPPP will operate during darkness when the pH of the stroma will be much lower. Interestingly, the enzyme substrate aYnities are not considered suYcient for eYcient catalysis in chloroplasts in vivo (Tiege et al., 1998). As such, an enzyme superstructure has been proposed in vivo with the RPE,

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transketolase, and other Calvin cycle enzymes interacting adjacent to the thylakoid membranes (Kopriva et al., 2000; Tiege et al., 1998). Such a structure could catalyse both the Calvin cycle and the OPPP by a process of metabolite channelling as described for the OPPP in P. sativum (Debnam et al., 1996). Alternatively, such an association of enzymes in vivo could alter the kinetic properties of the enzymes via an allosteric mechanism. 3. Interactions between the Plastid and Cytosol The operation of a complete OPPP in the plastids and either a complete or a partial pathway in the cytosol has implications in terms of the interaction of diVerent metabolic processes. If ribulose-5-phosphate isomerase and RPE are only in the plastid, there will be a need for ribulose-5-phosphate generated in the cytosol as a result of the oxidative steps of the OPPP to be transported into the plastid for subsequent metabolism to xylulose-5phosphate and ribose-5-phosphate. The ability of plastids to fully metabolise and utilise products produced in the cytosol is supported by a number of observations. That is, externally supplied ribose-5-phosphate is able to support nitrite reduction in P. sativum root plastids (Bowsher et al., 1989) and the plastid OPPP is capable of recycling ribose-5 phosphate to Glu6P (Hartwell et al., 1996). Similarly, ribulose-5-phosphate supplied externally to intact P. sativum root plastids can also support nitrite reduction (Debnam and Emes, 1999). A member of the phosphate translocator family of plastid inner-envelope membrane proteins with the capacity to transport pentose phosphates has been identified (Eicks et al., 2002). The existence of such a transporter is essential to increase the potential for interaction between the OPPP reactions in the plastid and cytosol (Eicks et al., 2002). It has been argued that the cytosolic and plastidic OPPP must also cooperate in the provision of reductant for biosynthesis. For instance, Z. mays mutants lacking detectable cytosolic 6PGDH had a reduced capacity to promote carbon flux through the OPPP when the demand for NADPH was increased in the plastid by treatment with nitrite (Averill et al., 1998). This suggests that in wild type roots the cytosolic OPPP is needed to meet these additional demands for NADPH. The precise mechanism of this interaction now needs to be investigated further. 4. Evolution of OPPP a. Oxidative Steps. The plastidic and cytosolic isoforms of G6PDH have a high degree of homology. It is thought that the G6PDH gene of the cyanobacterial antecedent of plastids was replaced by a nuclear-encoded copy following gene duplication of the cytosolic isoform and recruitment of a plastid-targeting sequence (Wendt et al., 1999). Wendt et al. (2000) proposed


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that the redox regulation exhibited in the plastidic form was first present in a nuclear-encoded antecedent. With specialisation into autotrophic and heterotrophic tissue, it is thought that multiple plastidic isoforms may have been preferentially selected with diVerences in their thioredoxin-mediated redox status or tolerance to NADPH. 6PGDH from chloroplasts and cytosol is also highly conserved and shows more similarity to its cyanobacterial homologues (Krepinsky et al., 2001). They appear to have arisen as a result of a duplication of the gene originally acquired from the ancestral plastid genome by endosymbiotic gene transfer. b. Nonoxidative Steps. Given the dual role of enzymes in the nonoxidative steps of the OPPP with those in the Calvin cycle, the evolution of many of these genes has already been discussed (Fig. 4; see Section III.A.1). Transaldolase has an evolutionary history of uncertain origin (Martin and Herrmann, 1998). D. TERPENOID BIOSYNTHESIS

Terpenoids are a diverse group of compounds, with more than 25,000 known types (Sacchettini and Poulter, 1997) produced from the 5-C precursor ‘‘active isoprene’’ compound, isopentenyl pyrophosphate (IPP) and its isomer, dimethylallyl pyrophosphate (DMAPP). The biological functions of terpenoids range from light harvesting in photosynthesis (e.g., carotenoids, the phytol side chain of chlorophylls), electron transport (e.g., plastoquinone), growth regulation (e.g., zeatin, gibberellins and abscisic acid [ABA]), and a vast array of volatile and toxic terpenoids involved in animal-plant interactions, both as feeding attractants and as toxic repellents. Terpenoids are classed according to the number of IPP units of which they are composed (e.g., mono- [C-10], sesqui- [C-15], di- [C20], tri- [C30] terpenoids, as well as polyterpenoids, such as rubber, and cyclised or steroidal, compounds). It is only within the last 10 years that terpenoid biosynthesis would have been featured in a review on plastid metabolism. Prior to 1993 it was universally accepted that terpenoids were produced in all organisms via a cytosolic pathway involving mevalonic acid (MVA) as an intermediate (Fig. 6). The discovery of an MVA-independent route (Rohmer et al., 1993) in some eubacteria led to a search for this ‘‘alternative pathway’’ in other organisms. This pathway, termed the DXP or DOXP pathway after its initial product 1-deoxy-D-xylulose 5-phosphate, has been discovered in eubacteria, cyanobacteria, Streptomycetes, algae, liverworts, and higher plants, where it is located in the plastids.


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Here, we discuss evidence for the plastidic DXP pathway, compare its performance in diVerent organisms, and consider the extent to which these parallel processes, in the plastid and cytosol, interact in the production of terpenoids in higher plants. 1. The DXP Pathway for Terpenoid Biosynthesis Classic studies using 14C labelling first led to the possibility of an alternative, non-MVA route for terpenoid synthesis. As the background to these early studies has been comprehensively reviewed elsewhere (Lichtenthaler, 1999), we present a brief overview simply to set the present work into context. Perhaps the first indication of a plastidic pathway came from the observation that although 14CO2 was readily incorporated into plastidic terpenoids, such as carotenoids, feeding with 14C-labelled acetate and MVA resulted in accumulation of label into cytosolic terpenoids with only very slow uptake into plastidic terpenoids (Lichtenthaler, 1999 and references cited). This series of experiments indicated distinct plastidic and cytosolic routes for terpenoid synthesis and resulted in the discovery of the plastid-located DXP pathway. The elucidation of the pathway required careful analysis of labelling patterns of radioactive and/or stable isotope-labelled substrates from which the intermediates and catalytic steps were tentatively proposed. For example, Schwender et al. (1997) fed 1-[2H]-deoxy-D-xylulose 5-phosphate and found eYcient incorporation into isoprene released from Populus, Chelidonium, and Salix species, whereas label was also incorporated into the phytol chain of chlorophylls in both a red (Cyanidium) and two species of green (Scenedesmus and Chlamydomonas) algae as well as in a higher plant (Lemna minor). This was early evidence that the DXP pathway could be used by algae and higher plants to produce terpenoids. At the time of Licthenthaler’s review (1999) the only enzymatic step in the proposed pathway that had been identified was Fig. 6. The plastidic (DXP pathway, reactions 1–7) and cytosolic (MVA pathway, reactions 9–14. Reaction 8 is common to both, with plastid and cytosolic isoenzymes) reactions for terpenoid biosynthesis in higher plants. Abbreviations: 1, 1‐deoxy‐D‐xylulose 5‐phosphate synthase (DXP synthase); 2, 1‐deoxy‐D‐xylulose 5‐phosphate reductoisomerase (DXR); 3, 4‐diphosphocytidyl‐2C‐methyl‐D‐erythritol synthase (also called 2‐C‐methyl‐D‐erythritol 4‐phosphate cytidyltransferase (MECT); 4, 4‐diphosphocytidyl‐2C‐methyl‐D‐erythritol kinase; 5, 2C‐methyl‐D‐erythritol 2,4‐ cyclodiphosphate synthase (MECS); 6, 2C‐methyl‐D‐erythritol 2,4‐cyclodiphosphate reductase (also called [E]‐4‐hydroxy‐3‐methyl‐but‐2‐enyl diphosphate synthase [HDS]); 7, Isopentenyl/dimethylallyl diphosphate synthase (IDDS); 8, isopentenyl/ dimethylallyl diphosphate isomerase (IDI); 9, acetoacetyl‐CoA thiolase; 10, 3‐ hydroxy‐3‐methyl‐glutaryl‐CoA synthase; 11, 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMGR); 12, MVA kinase; 13, phosphomevalonate kinase; 14, MVA diphosphate decarboxylase. Reaction ‘‘a’’ is a partially characterised plastidic IPP exporter (Bick and Lange, 2003).

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1–deoxy-D-xylulose 5-phosphate synthase (DXP synthase). Considerable progress has been made by the use of molecular genetic techniques in concert with isotopic labelling analyses and, more recently, the use of transgenic plants. 2. Reactions of the DXP Pathway Note that in the following sections, the superscript numbers attached to each enzyme in the subheads correspond to the numbered reactions in the pathways presented in Fig. 6. a. DXP Synthase.1 DXP synthase (1-deoxy-D-xylulose 5-phosphate synthase; DXS) catalyses the first step in the DXP pathway. The reaction is a transketolase-like decarboxylation, transferring a C-2 unit from pyruvate to D-glyceraldehyde 3-phosphate (GA3-P) to form 1-deoxy-D-xylulose 5-phosphate (DXP) (Rohmer et al., 1996). Evidence for this reaction in photosynthetic organisms came from experiments by Lichtenthaler’s group, first with the green alga Scenedesmus (Schwender et al., 1996, 1997) and later with higher plants such as Chlorella (Lichtenthaler et al., 1997; reviewed in Lichtenthaler, 1999). The reaction was verified by overexpression in Escherichia coli of DXP synthase genes from higher plant (Mentha) (Lange et al., 1998) and bacterial (E. coli) (Lois et al., 1998) sources, both of which resulted in the formation of DXP from GA3-P and pyruvate. The gene for DXP synthase, DXS, has been isolated and characterised from Mentha piperita (Lange et al., 1998), Capsicum annuum (Bouvier et al., 1998), Synechococcus leopoliensis (Miller et al., 1999), a Streptomyces species (Strain CL 190; gene originally denoted drs) (Kuzuyama et al., 2000a), A. thaliana (Araki et al., 2000; Estevez et al., 2000), L. esculentum (Lois et al., 2000), Stevia rebaudiana (Asteraceae) (Totte et al., 2003), and Morinda citrifolia (Rubiaceae) (Han et al., 2003). The higher plant gene is nuclear encoded. Plastid-targeting sequences have been identified in M. piperita (Lange et al., 1998), C. annuum (Bouvier et al., 1998), A. thaliana (Araki et al., 2000), M. truncatula (Walter et al., 2002), and S. rebaudiana (Totte et al., 2003), and translocation into chloroplasts has been shown in A. thaliana (Araki et al., 2000) and L. esculentum (Lois et al., 2000). Krushkal et al. (2003) have compared sequences for 11 DXS genes from 11 Angiosperm species and, based on sequence information, propose that not only do they contain plastid-targeting sequences, but they also show targeting to the thylakoids. This hypothesis awaits further analysis by in situ expression and/or import studies. Most early reports indicate that DXP synthase is coded for by a single gene, but there is increasing evidence of a multigene family in a number of species. Han et al. (2003) suggest there is a small gene family in M. citrifolia, whereas Walter et al. (2002) identified two


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gene classes in M. truncatula. The deduced sequences of the mature proteins, MtDXS1 and MtDXS2, share only 70% homology, and both contain plastid-targeting sequences. DXS1 is more highly and widely expressed, while DXS2 seems to be specifically associated with mycorrhizal formation in the roots and in specialised gland cells where monoterpene synthesis occurs in the leaves. Walter et al. (2002) also identified partial sequences of multiple DXS genes in database searches of several monocot and dicot species including Z. mays, L. esculentum, and N. tabacum. Given reports of cell-specific locations for terpenoid synthesis (as discussed later in this chapter), it is possible that these multiple DXS genes may be associated with cell-specific targeting and/or developmental-specific expression of the DXP pathway in higher plants. b. 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase (DXR).2 DXR catalyses the NADPH-dependent conversion of DXP to 2-C-methyl-D-erythritol 4-phosphate (MEP). It is inhibited by the antibiotic fosmidomycin, and this has proved to be useful in measurement of terpenoid production via the DXP pathway. Schwender et al. (1999) cloned a cDNA fragment of the DXR gene from A. thaliana, expressed it in E. coli, and found that the expressed protein was able to convert DXP into MEP, with its activity being inhibited by fosmidomycin. Several groups have now obtained clones of DXR from higher plants, including A. thaliana (Carretero-Paulet et al., 2002), Z. mays (Hans et al., 2004), Mentha x piperita (Lange and Croteau, 1999a; Mahmoud and Croteau, 2001), L. esculentum (Rodriguez-Concepcion et al., 2001), and Stevia rebaudiana (Totte et al., 2003). Plastid-targeting sequences have been identified in the DXR genes from Stevia rebaudiana (Totte et al., 2003) and A. thaliana (Carretero-Paulet et al., 2002). In addition, Carretero-Paulet et al. (2002) compared the A. thaliana N-terminal sequence with the equivalent region of all known plant DXRs, either derived from cDNAs or EST entries in the GenBank database. Analysis by the ChloroP program predicted a plastid targeting peptide sequence in all of the plant DXR sequences. Plastid location of DXR has been determined by immunolocalisation in Z. mays roots (Hans et al., 2004) and by transient expression of a DXR-GFP fusion protein, as well as by immunogold localisation in A. thaliana (Carretero-Paulet et al., 2002). c. 4-Diphosphocytidyl-2C-methyl-D-erythritol Synthase.3 The third reaction in the DXP pathway, the conversion of 2-C-methyl-D-erythritol 4-phosphate (MEP) to 4-diphosphocytidyl-2C-methyl-D-erythritol is catalysed by 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (also called 2-Cmethyl-D-erythritol 4-phosphate cytidylyltransferase [MECT]) (Kuzuyama

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et al., 2000b). The gene has been cloned from A. thaliana and is similar to the ispD gene coding for the enzyme in E. coli. The A. thaliana protein has been purified to homogeneity, following in vitro expression in E. coli, and shown to be enzymatically active. It has also been found to contain a plastid targeting sequence (Rohdich et al., 2000). Okada et al. (2002) produced transgenic A. thaliana plants with antisense expression of the gene (called AtMECT ) and found that pigmentation and accumulation of ent-kaurene, the plastidic precursor of gibberellin, were both reduced in comparison to wild type. Inhibition of the DXP pathway by fosmidomycin (DXR inhibitor; see Section III.D.2.b) produced the same phenotype in the wild type and it wasconcluded that 4-diphosphocytidyl-2C-methyl-D-erythritol synthase was an important step in the synthesis of terpenoid precursors for ent-kaurene synthesis. d. 4-Diphosphocytidyl-2C-methyl-D-erythritol Kinase.4 4-Diphosphocytidyl2C-methyl-D-erythritol kinase (gene designated ispE ) catalyses the ATPdependent phosphorylation of 4-diphosphocytidyl-2C-methyl-D-erythritol to 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate. The recombinant proteins from E. coli (Lu¨ ttgen et al., 2000), L. esculentum (Rohdich et al., 2000), and M. piperita (Lange and Croteau, 1999b) have been characterised, although the suggestion that the M. piperita gene product also converts isopentenyl monophosphate into IPP seems to be of little physiological relevance (Eisenreich et al., 2004). e. 2C-Methyl-D-erythritol 2,4-cyclodiphosphate Synthase.5 4-Diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate is converted to 2-C-methylD-erythritol-2,4-cyclodiphosphate (cMEPP) by 2C-methyl-D-erythritol 2,4cyclodiphosphate synthase (MECS) (gene designated ispF). Burlat et al. (2004) found that in Catharanthus roseus, mRNA cellular expression (determined by Northerns and in situ hybridisation) was identical to that of other DXP pathway genes (DXS, DXR) being highly expressed in the internal phloem parenchyma. The recombinant E. coli protein has been studied and found to catalyse the above reaction and to require Mg2þ or Mn2þ (Herz et al., 2000; Takagi et al., 2000). Experiments using 13C feeding to isolated chromoplasts of Capsicum annuum and Narcissus pseudonarcissus have demonstrated their capacity to convert MEP to cMEPP and the eYcient conversion of MEP and cMEPP to the carotene precursor phytoene (Fellermeier et al., 2001, 2003), thereby confirming the operation of a major part of the DXP pathway in these plastids. This followed earlier work from the same group (Fellermeier et al., 1999) showing that labelled DXP was incorporated into -carotene in isolated chloroplasts and chromoplasts.


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f. 2C-Methyl-D-erythritol 2,4-cyclodiphosphate Reductase.6 This stage in the DXP pathway was first elucidated by analysis of E. coli strains engineered to hyperexpress DXP pathway genes, followed by functional analysis using 13C-labelled substrates. The penultimate step was determined to be the conversion of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate to 1-hydroxy2-methyl-2-(E )-butenyl 4-diphosphate, by 2C-methyl-D-erythritol 2,4-cyclodiphosphate reductase (also called [E]-4-hydroxy-3-methylbut-2-enyl diphosphate synthase [HDS] encoded in E. coli by ispG/GcpE) (Hecht et al., 2001) and the protein is sometimes referred to as IspG and GCPE. Querol et al. (2002) identified an A. thaliana protein with homology to the IspG/GcpE gene product of E. coli. The A. thaliana protein was shown to be targeted to the plastid and able to complement a gcpE defective E. coli strain (Querol et al., 2002). Sequence analysis also indicates a plastid-targeting N-terminal sequence in the gene from L. esculentum. Expression (determined by analysis of ESTs) did not change during fruit ripening, despite a large increase in carotenoid formation, and this was interpreted as evidence that HDS is not rate limiting for carotenoid synthesis during fruit ripening (Rodriguez-Concepcion et al., 2003). However, gene expression does not provide a means of identifying either enzyme function or in vivo flux, and without further biochemical analysis, this interpretation remains speculative. g. Isopentenyl/Dimethylallyl Diphosphate Synthase (IDDS).7 The last step in the DXP pathway has been found to be catalysed by isopentenyl/ dimethylallyl diphosphate synthase (IDDS), encoded by the E. coli IspH/ LytB gene, and homologues in other organisms (Rohdich et al., 2002). This converts 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP) to isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). IPP and DMAPP can then be interconverted by isopentenyl/dimethylallyl diphosphate isomerase (IDI8). Progress in our understanding of these final steps in the DXP pathway in plants has come from virus-induced gene silencing (VIGS) experiments in Nicotiana benthamiana (Page et al., 2004). VIGS knockout of IspG and IspH (encoding 2C-methyl-D-erythritol 2,4cyclodiphosphate reductase and IDDS, respectively) resulted in albino leaves because of the loss of chlorophyll and carotenoid synthesis. Knocking out IDI caused a mottling of the leaves, indicating a partial loss of pigment synthesis. Analysis of metabolic perturbation, by feeding 14C-labelled DXP, produced results consistent with the proposed pathway (i.e., 2-C-methyl-Derythritol-2,4-cyclodiphosphate accumulated in IspG knockouts and breakdown products of HMBPP accumulated in IspH knockouts). This work confirms that the last two steps of the DXP pathway in higher plants are

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catalysed by 2C-methyl-D-erythritol 2,4-cyclodiphosphate reductase and IDDS, and that in higher plant plastids, IDI, unlike its E. coli homologue, is essential for normal terpenoid synthesis (Page et al., 2004). 3. Regulation of the DXP Pathway and Interaction with the Cytosolic MVA Pathway for Terpenoid Synthesis In the decade since the discovery of the DXP pathway, research has focused on identifying the genes and catalytic steps involved in the pathway. Studies of the regulation of pathway flux are still at a relatively early stage, although some good progress has been made using transgenic plants and inhibitors in combination with labelled substrates. DXS, the first enzyme in the DXP pathway, has been underexpressed and overexpressed in A. thaliana, resulting in changes in concentration of several terpenoids including chlorophylls, tocopherols, carotenoids, ABA, and gibberellins, indicating a major role for this enzyme in regulating plastid terpenoid production in A. thaliana (Estevez et al., 2001). Mahmoud and Croteau (2001) transformed peppermint (Mentha x piperita L.) with a homologous sense version of DXR and produced two sets of transformants. In one, where DXR expression had been lost, the leaves were chlorotic and slow growing, with lower concentrations of essential oils than in wild type. In a second set, where DXR had been overexpressed, there was a 50% increase in terpenoid production, indicating that DXR exerts significant control over DXP pathway flux. Although other study results have been interpreted as indicating a less significant role for DXR in controlling pathway flux, these must be treated with caution because it is usually gene expression that is reported, and not measurement of enzyme activity. For example, although the levels of DXS transcript and DXS protein do not increase during tomato fruit ripening (Rodriguez-Concepcion et al., 2001), without determining the activity of the enzyme, there still remains the possibility of posttranslational modification/regulation to increase flux through DXR when increased carotenoid production is occurring in this tissue. Although other transgenic approaches have been used in the study of the DXP pathway (for discussion, see Page et al., 2004; Rodriguez-Concepcion et al., 2002), these have usually generated large phenotypic diVerences, for example, by removing expression completely. Although this approach has been extremely valuable in elucidating the pathway, such large perturbations are of limited use when determining the regulation of pathway flux because of the increased likelihood of pleiotropic eVects. For further progress to be made using transgenic plants, it is important to produce plants with a range of small alterations in enzyme activity and to carry out a full analysis of flux control using established and rigorous biochemical techniques (e.g.,


145

metabolic control analysis). This is feasible now that the genetic basis has been established. Table I provides a summary of the pathways used to synthesise selected groups of terpenoid compounds in plants. This is an oversimplification that hides the fact that the cytosolic and plastidic pathways would appear to be capable of exchanging metabolites. Several studies have now been performed into this metabolic ‘‘crosstalk’’ between the MVA and DXP pathways. Laule et al. (2003) used the inhibitors fosmidomycin (which inhibits the plastid DXP pathway at DXR) and lovastatin (which inhibits the cytosolic MVA pathway at 3-hydroxy-3-methylglutaryl-CoA reductase [HMGR11]) together with pool size measurements, in A. thaliana, to determine whether blocks in one pathway could be compensated in the other pathway. Fosmidomycin inhibition caused a loss of plastid-derived terpenoids (chlorophylls, carotenoids), whereas lovastatin inhibition initially led to reduced levels of cytosolic sterols that eventually recovered back to preinhibitor levels. The conclusion was that whereas the plastidic DXP pathway could export intermediates to compensate for loss of the cytosolic pathway, this was a unidirectional process, with no evidence of import occurring into the plastid. Bick and Lange (2003) followed up this work and measured terpenoid transport in isolated chloroplasts and membrane preparations. Transport was essentially unidirectional, with IPP, GPP, FPP, and DMAPP all being

TABLE I Predominant Pathways Used for the Synthesis of Selected Groups of Terpenoids in Plantsa MVA Sterols Ubiquinone Phytol side chain of chlorophyll Carotenoids Volatile terpenoids Geraniol Menthone Thymol Toxins, deterrents Cannabinoids Taxoids Iridoid alkaloids Humulone a

DXP

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

Information summarised from Eisenreich et al. (2001) and references therein. Note that there is some crosstalk between pathways depending on species and/or tissue, as discussed in the text.

146


exported at significant rates from the chloroplast, thus supporting the observations of Laule et al. (2003). In their analysis of the A. thaliana clb4 mutant that lacks the gene for 2C-methyl-D-erythritol 2,4-cyclodiphosphate reductase6, Guttie´ rrez-Nava et al. (2004) found that the embryos, but not the parent plant, were able to produce chlorophyll. The authors suggest that chlorophyll synthesis in these mutants may be supported by import of MVA-pathway intermediates from the cytosol into the plastid, thus contrasting with the conclusions of Bick and Lange (2003). However, no measurements were made of plastid terpenoid transport in the study by Guttie´ rrez-Nava et al. (2004), and this interesting observation remains to be tested. These studies have provided new insights into the potential for traYcking terpenoid compounds between the cytosol and plastid, although the transporters involved have yet to be isolated or characterised. There is suYcient evidence from the study of Bick and Lange (2003) to indicate that novel transporters are involved and that these oVer the potential for manipulating the interchange between cytosolic and plastidic pools of terpenoids, with the aim of modifying the types of products produced by the plant. The data from Laule et al. (2003) and Bick and Lange (2003) provide a useful indicator of compartmentation but, being based on pool size measurements alone (Laule et al., 2003), cannot be used to estimate fluxes or the relative contribution of each pathway under steady-state conditions in vivo. For this, more elaborate analyses are required. These have been discussed in a review in which a detailed explanation of the methodology (13C-glucosefeeding and isotope positioning analysis by NMR) is also given (Eisenreich et al., 2004). Using this methodology, Schuhr et al. (2003) present work on Ginkgo biloba seedlings, indicating that although ginkgolides (terpenoid compounds) were derived primarily from the plastid-DXP pathway, 1% of the IPP molecules used for their synthesis had been imported from the cytosolic MVA pathway. In addition to this, it was also proposed that cytosolic farnesyl diphosphate was imported to the plastid where a chloroplastsynthesised IPP unit was added to produce the geranylgeranyl diphosphate precursor for ginkgolides. Export from the plastid DXP pathway to the cytosol has also been inferred from labelling studies. In Catharanthus roseus cell cultures, small amounts of label from 13C-DX were incorporated into sitosterol, which is synthesised predominantly via the cytosolic MVA pathway (Arigoni et al., 1997). Schuhr et al. (2003) estimated (from the data of Arigoni et al., 1997) that 2.5% of the IPP/DMAPP molecules used for sitosterol formation were produced in the plastids and exported to the cytosol. In their own detailed experiments with C. roseus cells, Schuhr et al. (2003) calculated that approximately 17% of the IPP molecules used for lutein


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(chloroplast terpenoid) synthesis had been imported from the cytosolic MVA pathway and that the DXP pathway contributed less than 5% to the synthesis of cytosolic sitosterol. Phytol synthesis was complex, with as much as 40% of IPP units appearing to be derived from cytosolic MVA. The extent to which crosstalk occurs was found to depend on the conditions and supply of exogenous substrates. The authors conclude that a simple two-compartment model may not be suYcient to account for the complex interactions involved in terpenoid biosynthesis and that more information is needed on pool sizes, metabolic regulation, and compartmentation in order to fully understand pathway regulation. 4. Evolution of the DXP Pathway Lichtenthaler (1999) has presented a putative scheme for the evolution of the DXP and MVA pathways in higher plants and algae. This is based on known endosymbiotic events and knowledge of the two pathways, and not on genome analysis. Lichtenthaler (1999) thus proposed that Euglena, which possesses only the MVA pathway, lost the DXP pathway following a second endosymbiotic event. In contrast, the Chlorophyta (examples being Chlorella, Scenedesmus, and Chlamydomonas) lost their cytosolic MVA pathway and retained the DXP pathway gained from a primary endosymbiotic event. Higher plants have retained both pathways, as have the Rhodophyta and Heterokontophyta (e.g., Ochromonas) despite the latter also undergoing a second endosymbiotic event (Lichtenthaler, 1999). Lange et al. (2000) have carried out a comprehensive survey of the evolutionary relationships between the genes of the MVA and DXP pathways from a range of organisms. Comparisons were made between genes from 35 genomes. The main conclusion for higher plant plastids was that the DXP pathway genes were acquired from the cyanobacterial ancestor, because it is only in plastid-bearing eukaryotes that the DXP pathway is found. However, it was also noted that four of the five genes for DXP pathway enzymes (DXR being the single exception) that were examined did not branch with the cyanobacterial genes following phylogenetic analysis, perhaps because of lateral transfer of DXP genes occurring between eubacteria subsequent to the endosymbiotic origin of the plastids. 5. Interactions with Other Pathways For terpenoid synthesis to occur in the plastid, there has to be a supply of the initial substrates pyruvate and glyceraldehyde-3-phosphate. The latter is an intermediate in the Calvin cycle, the OPPP, and the glycolytic pathway (Figs. 4 and 5). If these pathways cannot provide an adequate supply, then triose phosphate, in the form of 3-PGA, can be imported from the cytosol on

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the phosphate translocator and converted to glyceraldehyde-3-phosphate via the Calvin cycle. Pyruvate, for the DXP pathway, can be provided by plastidic glycolysis (Fig. 5), imported from the cytosol (Proudlove and Thurman, 1981), or generated as a side-reaction of Rubisco (Roy and Andrews, 2000).

IV. CONCLUSION Plastids are a vital part of a plant cell, and even though much of their genetic information has migrated to the nucleus, they have maintained a considerable metabolic influence that extends throughout the plant. The Calvin cycle is the basis of autotrophy, bringing a net carbon gain and providing the basic organic material required for all biosynthesis within the plant. It is, therefore, remarkable to find that some of its key enzymes, for example, FBPase, are encoded by genes that do not have any evolutionary links to the cyanobacterial ancestor of the chloroplast (see Section III.A.1). In the case of FBPase, it would seem that the gene for the ancestral cytosolic enzyme has gained a plastid targeting sequence, and the original cyanobacterial gene has been deleted (Rogers and Keeling, 2004). As yet there is no obvious explanation as to why this might have occurred. Replacement of one gene for another would have to have conferred some advantage to the cell. We can only speculate that this has perhaps allowed the Calvin cycle to function more eYciently within a plastid that has more metabolic branch points than were present in the original cyanobacteria. Another striking example of the replacement of an ancestral plastidic protein is that of the OPPP enzyme G6PDH. This is a vital enzyme in the plastid because it provides an essential link between carbon and nitrogen metabolism (see Sections II.A and II.C.1). Yet it seems that this plastid protein is encoded by a gene originating in the host cell, and that the cyanobacterial form has been lost. The crucial requirement for redox regulation of G6PDH, together with its role in regulating the redox poise within the plastid, would appear to explain why the more highly regulated cytosolic ancestor has replaced the gene of cyanobacterial origin (Wendt et al., 1999, 2000; see Section III.C.4.a). In this chapter, we have given several examples of pathways that operate in both the plastid and the cytosol. Where pathways have become duplicated, this can lead to increased metabolic flexibility, and hence an increased capacity to survive in fluctuating and unpredictable environments. A good example of this is the increase in glycolytic flux under conditions of phosphate starvation (Plaxton, 1996). Although this aVects the cytosolic,


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rather than the plastidic glycolytic pathway, it does illustrate how seemingly identical pathways have been retained during the course of evolution. Key reactions of the cytosolic pathway have their evolutionary origin in the cyanobacterial ancestor of the plastid (as discussed in Section III.B.2), and their retention in the cytosol could be simply due to the fact that they are not causing any harm to the plant. If, however, phosphate deficiency exerts a significant selection pressure on a given plant species, this single example serves to illustrate how reactions would be retained in the cytosol because they are of benefit to the plant. The cytosolic pathway does not, of course, require phosphate starvation in order to operate, and yet the balance between this and the plastid pathway is clearly going to change with external conditions. Perhaps we will gain a more complete understanding of the benefits, or otherwise, of pathway duplication by testing plants under a more diverse range of environmental conditions. Terpenoids are required for a variety of purposes and by a range of organisms (as discussed in Section III.D). Higher plants have retained both a plastidic and a cytosolic pathway for terpenoid synthesis, and unlike the previous examples of glycolysis and the OPPP, the pathways show no duplication. The series of reactions in the plastid (DXP pathway) and cytosol (MVA pathway) are entirely diVerent, and yet they both result in the synthesis of the terpenoid precursor IPP. This seems to be a remarkable duplication of eVort. It is particularly interesting to note that this duplication only occurs in higher plants and in some algal groups (Table II). Other

TABLE II Summary of the Distribution of the Mevalonic Acid (MVA) and Deoxyxylulose Phosphate (DXP) Pathways for Terpenoid Synthesis in DiVerent Organismsa

Archaebacteria Eubacteria Fungi Animals Photosynthetic eukaryotes Algae

MVA

DXP

Yes Some Yes Yes

No Yes No No

Chlorophytes Rhodophytes Heterokontophytes

No Yes Yes

Yes Yes Yes

Plastid Cytosol

No Yes

Yes No

Higher plants

a

Adapted from Eisenreich et al., 2001; Lange et al., 2000.

Comments Most use DXP only

Euglena uses MVA only

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organisms have retained either the MVA pathway or the DXP pathway, but not both. Why should photosynthetic eukaryotes be so diVerent in this respect? The plastidic pathway can be traced to the cyanobacterial ancestor, albeit with some evidence of lateral gene transfer (Lange et al., 2000). Its genes are unlikely to have been displaced by those of host cell origin, as in the examples of glycolytic and OPPP enzymes, because the reactions catalysed in the MVA pathway are so completely diVerent. This might explain why one pathway has not replaced the other. However, it seems likely that the rules of metabolic compartmentation might apply in this situation. Plastid terpenoids might be produced by the DXP pathway simply because the plastid is unable to import them from the cytosol. The phytol side chain of chlorophyll, for example, is unique to the chloroplast and that is where it is made. Transport of terpenoid compounds across the chloroplast envelope has received very little research interest (but see Bick and Lange, 2003, as discussed in Section III.D.3), and yet without this information, we cannot really understand how the cytosolic and plastidic pathways interact or why they remain distinct and separate within the cell. The present-day pathways for nitrogen and carbon assimilation within plastids clearly show remnants of their endosymbiotic ancestry. Metabolic pathways have become more complex and interrelated, partly because of the need for plants, as sedentary organisms, to survive in less than optimal conditions. This is also apparent in the plasticity of form and function of the plastid itself, ranging from a fully photosynthetic chloroplast to a nonphotosynthetic, but still biosynthetic, amyloplast. The interchangeability of these plastid forms means that particular plastid types need not be restricted to particular locations. Immature mesophyll cells, for example, contain rudimentary chloroplasts with only a limited capacity for photosynthesis (Bowsher and Tobin, 2001), and mature chloroplasts retain the capacity to divide, so a mesophyll cell is likely to contain a mixed population of plastids at any one time (Pyke, 1999). Even in photosynthetic tissue, such as the leaves, there are significant numbers of nonphotosynthetic cells (e.g., vascular and epidermal) containing plastids, so mature chloroplasts and nonphotosynthetic plastids can be present in adjacent leaf cells. All of this adds to the complexity of plastid function within a multicellular organism. We have yet to gain an understanding of the metabolic capacity of these diVerent plastid populations. Progress in proteomics will enable us to more fully characterise the heterogeneity of organelles in situ. Metabolomics approaches will also enable us to gain a better knowledge of the integration of pathways in the whole plant (Stitt and Fernie, 2003). Much will depend on


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our understanding of the compartmentation of enzymes and metabolites within and between organelles, cells, and tissues.

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Advances in

BOTANICAL RESEARCH Incorporating Advances in Plant Pathology

Editor-in-Chief J. A. CALLOW

School of Biosciences, The University of Birmingham, United Kingdom

Editorial Board A. R. HARDHAM J. S. HESLOP-HARRISON M. KREIS R. A. LEIGH E. LORD D. G. MANN P. R. SHEWRY D. SOLTIS

Australian National University, Canberra, Australia University of Leicester, United Kingdom Universite de Paris-Sud, Orsay, France University of Cambridge, Cambridge, United Kingdom University of California, Riverside, USA Royal Botanic Garden, Edinburgh, United Kingdom IACR-Long Ashton Research Station, United Kingdom University of Florida at Gainesville, USA

CONTRIBUTORS TO VOLUME 42

CAROLINE G. BOWSHER School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom MELISSA BRAZIER-HICKS School of Biological and Biomedical Sciences, University of Durham, Durham DH1 3LE, United Kingdom P. D. BRIDGE British Antarctic Survey, High Cross, Cambridge CB3 0ET, United Kingdom IAN CUMMINS School of Biological and Biomedical Sciences, University of Durham, Durham DH1 3LE, United Kingdom DAVID P. DIXON School of Biological and Biomedical Sciences, University of Durham, Durham DH1 3LE, United Kingdom ROBERT EDWARDS School of Biological and Biomedical Sciences, University of Durham, Durham DH1 3LE, United Kingdom P. J. ROBERTS Mycology Section, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, United Kingdom HILARY J. ROGERS School of Biosciences, CardiV University, CardiV CF10 3TL, United Kingdom B. M. SPOONER Mycology Section, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, United Kingdom ALYSON K. TOBIN School of Biology, Sir Harold Mitchell Building, University of St Andrews, St Andrews, Fife, KY 169TH Scotland, United Kingdom

CONTENTS OF VOLUMES 30–41

Contents of Volume 30 Nitrate and Ammonium Nutrition of Plants: Physiological and Molecular Perspectives G. FORDE and D. T. CLARKSON Secondary Metabolites in Plant–Insect Interactions: Dynamic Systems of Induced and Adaptive Responses J. A. PICKETT, D. W. M. SMILEY and C. M. WOODCOCK Biosynthesis and Metabolism of Caffeine and Related Purine Alkaloids in Plants H. ASHIHARA and A. CROZIER Arabinogalactan-Proteins in the Multiple Domains of the Plant Cell Surface M. D. SERPE and E. A. NOTHNAGEL Plant Disease Resistance: Progress in Basic Understanding and Practical Application N. T. KEEN

Contents of Volume 31 PLANT TRICHOMES Edited by D. L. Hallahan and J. C. Gray Trichome Diversity and Development E. WERKER

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Structure and Function of Secretory Cells A. FAHN Monoterpenoid Biosynthesis in Glandular Trichomes of Labiate Plants D. L. HALLAHAN Current and Potential Exploitation of Plant Glandular Trichome Productivity S. O. DUKE, C. CANEL, A. M. RIMANDO, M. R. TELLEZ, M. V. DUKE and R. N. PAUL Chemotaxonomy Based on Metabolites from Glandular Trichomes O. SPRING Anacardic Acids in Trichomes of Pelagonium: Biosynthesis, Molecular Biology and Ecological Effects D. J. SCHULTZ, J. I. MEDFORD, D. COX-FOSTER, R. A. GRAZZINI, R. CRAIG and R. O. MUMMA Specification of Epidermal Cell Morphology B. J. GLOVER and C. MARTIN Trichome Initiation in Arabidopsis A. R. WALKER and M. D. MARKS Trichome Differentiation and Morphogenesis in Arabidopsis ˆ LSKAMP and V. KIRIK M. HU Trichome Plasmodesmata: A Model System for Cell-to-Cell Movement F. WAIGMANN and P. ZAMBRYSKI


Contents of Volume 32 PLANT PROTEIN KINASES Edited by M. Kreis and J. C. Walker Plant Protein-Serine/Threonine Kinases: Classification into Subfamilies and Overview of Function D. G. HARDIE Bioinformatics: Using Phylogenetics and Databases to Investigate Plant Protein Phosphorylation E. R. INGHAM, T. P. HOLTSFORD and J. C. WALKER Protein Phosphatases: Structure, Regulation and Function S. LUAN Histidine Kinases and the Role of Two-Component Systems in Plants G. E. SCHALLER Light and Protein Kinases J. C. WATSON Calcium-Dependent Protein Kinases and Their Relatives E. M. HRABAK Receptor-Like Kinases in Plant Development K. U. TORII and S. E. CLARK A Receptor Kinase and the Self-Incompatibility Response in Brassica J. M. COCK Plant Mitogen-Activated Protein Kinase Signalling Pathways in the Limelight S. JOUANNIC, A.-S. LEPRINCE, A. HAMAL, A. PICAUD, M. KREIS and Y. HENRY

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Plant Phosphorylation and Dephosphorylation in Environmental Stress Responses in Plants K. ICHIMURA, T. MIZOGUCHI, R. YOSHIDA, T. YUASA and K. SHINOZAKI Protein Kinases in the Plant Defence Response G. SESSA and G. B. MARTIN SNF1-Related Protein Kinases (SnRKs) – Regulators at the Heart of the Control of Carbon Metabolism and Partitioning N. G. HALFORD, J.-P. BOULY and M. THOMAS Carbon and Nitrogen Metabolism and Reversible Protein Phosphorylation D. TOROSER and S. C. HUBER Protein Phosphorylation and Ion Transport: A Case Study in Guard Cells J. LI and S. M. ASSMANN

Contents of Volume 33 Foliar Endophytes and Their Interactions with Host Plants, with Specific Reference to the Gymnospermae W.-M. KRIEL, W. J. SWART and P. W. CROUS Plants in Search of Sunlight D. KOLLER The Mechanics of Root Anchorage A. R. ENNOS


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Molecular Genetics of Sulphate Assimilation M. J. HAWKESFORD and J. L. WRAY Pathogenicity, Host-Specificity, and Population Biology of Tapesia spp., Causal Agents of Eyespot Disease of Cereals J. A. LUCAS, P. S. DYER and T. D. MURRAY

Contents of Volume 34 BIOTECHNOLOGY OF CEREALS Edited by Peter Shewry Cereal Genomics K. J. EDWARDS and D. STEVENSON Exploiting Cereal Genetic Resources R. J. HENRY Transformation and Gene Expression P. BARCELO, S. RASCO-GAUNT, C. THORPE and P. A. LAZZERI Opportunities for the Manipulation of Development of Temperate Cereals J. R. LENTON Manipulating Cereal Endosperm Structure, Development and Composition to Improve End Use Properties P. R. SHEWRY and M. MORELL Resistance to Abiotic Freezing Stress in Cereals M. A. DUNN, G. O’BRIEN, A. P. C. BROWN, S. VURAL and M. A. HUGHES

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Genetics and Genomics of the Rice Blast Fungus Magnaporthe grisea: Developing an Experimental Model for Understanding Fungal Diseases of Cereals N. J. TALBOT and A. J. FOSTER Impact of Biotechnology on the Production of Improved Cereal Varieties R. G. SOLOMON and R. APPELS Overview and Prospects P. R. SHEWRY, P. A. LAZZERI and K. J. EDWARDS

Contents of Volume 35 Recent Advances in the Cell Biology of Chlorophyll Catabolism H. THOMAS, H. OUGHAM and S. HO«RTENSTEINER The Microspore: A Haploid Multipurpose Cell A. TOURAEV, M. PFOSSER and E. HEBERLE-BORS The Seed Oleosins: Structure Properties and Biological Role J. NAPIER, F. BEAUDOIN, A. TATHAM and P. SHEWRY Compartmentation of Proteins in the Protein Storage Vacuole: A Compound Organelle in Plant Cells L. JIANG and J. ROGERS Intraspecific Variation in Seaweeds: The Application of New Tools and Approaches C. MAGGS and R. WATTIER Glucosinolates and Their Degradation Products R. F. MITHEN


Contents of Volume 36 PLANT VIRUS VECTOR INTERACTIONS Edited by R. Plumb Aphids: Non-Persistent Transmission T. P. PIRONE and K. L. PERRY Persistent Transmission of Luteoviruses by Aphids B. REAVY and M. A. MAYO Fungi M. J. ADAMS Whitefly Transmission of Plant Viruses J. K. BROWN and H. CZOSNEK Beetles R. C. GERGERICH Thrips as Vectors of Tospoviruses D. E. ULLMAN, R. MEIDEROS, L. R. CAMPBELL, A. E. WHITFIELD, J. L. SHERWOOD and T. L. GERMAN Virus Transmission by Leafhoppers, Planthoppers and Treehoppers (Auchenorrhyncha, Homoptera) E. AMMAR and L. R. NAULT Nematodes S. A. MacFARLANE, R. NEILSON and D. J. F. BROWN Other Vectors R. T. PLUMB

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Contents of Volume 37 ANTHOCYANINS IN LEAVES Edited by K. S. Gould and D. W. Lee Anthocyanins in Leaves and Other Vegetative Organs: An Introduction D. W. LEE and K. S. GOULD Le Rouge et le Noir: Are Anthocyanins Plant Melanins? G. S. TIMMINS, N. M. HOLBROOK and T. S. FEILD Anthocyanins in Leaves: History, Phylogeny and Development D. W. LEE The Final Steps in Anthocyanin Formation: A Story of Modification and Sequestration C. S. WINEFIELD Molecular Genetics and Control of Anthocyanin Expression B. WINKEL-SHIRLEY Differential Expression and Functional Significance of Anthocyanins in Relation to Phasic Development in Hedera helix L. W. P. HACKETT Do Anthocyanins Function as Osmoregulators in Leaf Tissues? L. CHALKER-SCOTT The Role of Anthocyanins for Photosynthesis of Alaskan Arctic Evergreens During Snowmelt S. F. OBERBAUER and G. STARR Anthocyanins in Autumn Leaf Senescence D. W. LEE


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A Unified Explanation for Anthocyanins in Leaves? K. S. GOULD, S. O. NEILL and T. C. VOGELMANN

Contents of Volume 38 An Epidemiological Framework for Disease Management C. A. GILLIGAN Golgi-independent Trafficking of Macromolecules to the Plant Vacuole D. C. BASSHAM Phosphoenolpyruvate Carboxykinase: Structure, Function and Regulation R. P. WALKER and Z.-H. CHEN Developmental Genetics of the Angiosperm Leaf C. A. KIDNER, M. C. P. TIMMERMANS, M. E. BYRNE and R. A. MARTIENSSEN A Model for the Evolution and Genesis of the Pseudotetraploid Arabidopsis thaliana Genome Y. HENRY, A. CHAMPION, I. GY, A. PICAUD, A. LECHARNY and M. KREIS

Contents of Volume 39 Cumulative Subject Index Volumes 1–38

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Contents of Volume 40 Starch Synthesis in Cereal Grains K. TOMLINSON and K. DENYER The Hyperaccumulation of Metals by Plants M. R. MACNAIR Plant Chromatin — Learning from Similarities and Differences J. BRZESKI, J. DYCZKOWSKI, S. KACZANOWSKI, P. ZIELENKIEWICZ and A. JERZMANOWSKI The Interface Between the Cell Cycle and Programmed Cell Death in Higher Plants: From Division unto Death D. FRANCIS The Importance of Extracellular Carbohydrate Production by Marine Epipelic Diatoms G. J. C. UNDERWOOD and D. M. PATERSON Fungal Pathogens of Insects: Cuticle Degrading Enzymes and Toxins A. K. CHARNLEY

Contents of Volume 41 Multiple Responses of Rhizobia to Flavonoids During Legume Root Infection JAMES E. COOPER Investigating and Manipulating Lignin Biosynthesis in the Postgenomic Era CLAIRE HALPIN


Application of Thermal Imaging and Infrared Sensing in Plant Physiology and Ecophysiology HAMLYN G. JONES Sequences and Phylogenies of Plant Pararetroviruses, Viruses, and Transposable Elements CELIA HANSEN and J. S. HESLOP-HARRISON Role of Plasmodesmata Regulation in Plant Development ARNAUD COMPLAINVILLE and MARTIN CRESPI

xix

AUTHOR INDEX

Numbers in bold refers to page on which full references are listed

A Adam, G., 15, 30 Adam, P., 142, 143, 154, 155, 161 Ahumada, I., 141, 144, 152, 161 Aime, M. C., 45, 63 Aitken, E. A. B., 51, 52, 64 Akagawa, T., 123, 155 Akazawa, T., 130, 154 Alabouvette, C., 50, 61 Alessa, L., 83, 101 Allen, E. R., 10, 31 Allen, R. D., 10, 31 Al-Sabarna, K., 10, 30 Alscher, R. G., 2, 27 Alt, M., 54, 65 Altmann, T., 80, 97, 101, 110 Amaral Zetter, L. A., 54, 58 Amato, G., 42, 61 Amils, R., 54, 58 Amrhein, N., 14, 29 Amslinger, S., 143, 155, 161 An, Y. Q., 71, 106 Anderson, J. B., 49, 65 Anderson, L. E., 134, 163 Ando, A., 49, 65 Andrews, M., 120, 151 Andrews, T. J., 148, 162 Anon, 34, 58 Anthony, R. G., 71, 106 Antoniw, J. F., 39, 66 Apel, C., 2, 4, 20, 27, 30 Apostolakos, P., 72, 87, 104, 107 ap Rees, T., 132, 133, 135, 151, 163 Arabidopsis Genome Initiative, 76, 109 Araki, N., 140, 151 Arie, T., 40, 48, 58 Arigoni, D., 142, 143, 145, 146, 149, 151, 153, 154, 155, 161, 162 Armstrong, G. M., 36, 58 Armstrong, J. K., 36, 58 Arora, D. K., 39, 46, 50, 51, 58, 59 Asada, T., 71, 101 Assinder, S. J., 55, 67 Atallah, M., 10, 30 Atanassova, N., 10, 30 Austin, B., 38, 64

Avelange, I., 50, 61 Averill, R. H., 136, 151 Ayliffe, M. A., 115, 164 Azimzadeh, J., 91, 97, 102 B Baboeuf, I., 140, 141, 164 Bacher, A., 142, 143, 145, 146, 149, 151, 153, 154, 155, 156, 159, 161, 162 Back, E., 118, 151, 157, 161 Backhaus, R. A., 140, 151 Badur, R., 128, 133, 135, 155 Bailey-Serres, J., 136, 151 Bainbridge, B. W., 38, 39, 49, 58, 64, 66 Baird, W. M. V., 71, 101 Baldauf, S. L., 43, 58 Baluska, F., 77, 101 Bandyopadhyay, A., 7, 11, 31, 77, 95, 105 Banerji, S., 34, 64 Barlow, P. W., 77, 101 Barnard, J., 34, 58 Barns, S. M., 44, 59 Baroni, T. J., 45, 63 Barrie, F. R., 35, 61 Barton, M. K., 77, 103 Barve, M. P., 40, 48, 58 Bassham, J. A., 131, 151 Basu, P., 125, 163 Battista, C. D., 50, 59 Baurle, I., 95, 105 Baysdorfer, C., 131, 151 Becker, T. W., 120, 160 Beevers, H., 133, 134, 160 Begerow, D., 40, 41, 58, 63 Beinsberger, S. E. I., 79, 109 Bellini, C., 78, 91, 95, 96, 97, 102, 109 Benfey, P. N., 92, 97, 103 Benkova, E., 76, 95, 101, 105 Benson, D. R., 124, 157 Berbee, M. L., 37, 43, 44, 58 Berger, D., 80, 97, 101, 110 Berger, F., 92, 98, 101 Bergfeld, R., 76, 107 Bergounioux, C., 91, 105 Berleth, T., 77, 96, 105 Bernatsky, R., 71, 101

168

AUTHOR INDEX

Berthiller, F., 15, 30 Beussman, D. J., 11, 14, 23, 28 Bick, J. A., 139, 145, 146, 150, 151 Bielawski, J. P., 37, 67 Binarova, P., 72, 101 Bird, I. F., 120, 156 Birk, C., 23, 31 Bismuth, E., 123, 158 Blackwell, R. D., 120, 122, 151, 156 Blackwod, L., 136, 152 Blakekalff, M. M. A., 5, 28 Blakeley, S. D., 131, 133, 151, 153, 159 Blanz, P. A., 38, 59 Bleeker, M., 50, 66 Blume, Y. B., 71, 101, 108 Blumenthal, J. M., 124, 162 Blundell, T. L., 98, 110 Boerema, G. H., 48, 59 Bo¨ger, P., 7, 30 Bogre, L., 72, 101 Bokros, C. L., 71, 101, 105 Boller, T., 54, 65 Bolognesi-Winfield, A. C., 98, 110 Bonfante, P., 39, 62 Borner, T., 50, 63 Boronat, A., 140, 141, 143, 144, 152, 159, 161 Borst, P., 40, 59 Botton, B., 50, 59 Bouchez, D., 78, 91, 95, 96, 97, 102, 109 Bouget, F. Y., 84, 103 Boulton, E. L., 123, 132, 151 Bouvier, F., 140, 151 Bowen, A. R., 54, 59 Bowles, D. J., 72, 102 Bowsher, C. G., 116, 118, 123, 132, 135, 136, 150, 151, 152, 155, 161 Bozzetti, M. P., 124, 160 Braun, S., 139, 140, 162 Braus, G. H., 40, 63 Brazier-Hicks, M., 11, 14, 27, 30 Bridge, P. D., 39, 40, 41, 42, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 59, 60, 61, 63, 64, 65 Bright, S. W. J., 11, 13, 21, 23, 29, 30, 120, 164 Bringer-Meyer, S., 137, 140, 161 Brinkmann, H., 126, 152, 159 Brock, I. W., 119, 152 Brown, A. E., 45, 65 Brown, C. L., 75, 101 Brown, R. C., 88, 107 Brown, W. M., 49, 59 Browning, K. S., 71, 101 Brownlee, C., 76, 82, 83, 84, 101, 103, 106 Brunner, I., 40, 63 Brunold, C., 22, 23, 29 Bruns, T. D., 36, 37, 39, 40, 43, 44, 47, 53, 54, 55, 59, 61

Brygoo, Y., 39, 53, 55, 64, 66 Buchanan, B. B., 126, 134, 152, 159 Buddie, A., 51, 59 Buker, S., 94, 102 Burdet, H.-M., 35, 61 Burk, D. H., 74, 94, 101 Burkhardt, B., 120, 163 Burkhardt, W., 118, 151 Burlat, V., 142, 152 Buscot, F., 50, 59 Bush, M., 71, 102 Butler, J. P., 75, 110 Butlin, J., 56, 65 Buttner, G., 89, 96, 106 C Caboche, M., 78, 91, 95, 96, 97, 109, 118, 157 Callaham, D. A., 71, 105 Calvert, C. M., 72, 102 Camara, B., 133, 140, 151, 163 Camilleri, C., 91, 97, 102 Cammack, R., 118, 136, 151 Campbell, W. H., 118, 121, 133, 161 Campos, N., 140, 141, 143, 144, 152, 159, 161 Cannon, P. F., 34, 44, 45, 62 Cantero, A., 140, 144, 154 Canvin, D. T., 130, 163 Cao, Y., 42, 64 Carey, M., 51, 59 Carfagna, S., 135, 153, 154 Carillo, P., 126, 163 Carlberg, I., 6, 30 Carlile, M. J., 34, 60 Carrayol, E., 120, 160 Carretero-Paulet, L., 141, 144, 152, 161 Cartwright, H. N., 72, 104 Carvalho, H., 119, 152 Caseley, J. C., 11, 13, 14, 17, 19, 28 Casida, J. E., 13, 19, 31, 32 Celenza, J. L., 76, 94, 102 Cenklova, V., 72, 101 Cerff, R., 152, 159 Chadwick, A. V., 78, 109 Chalmers, M. O., 56, 60 Chan, J., 71, 102, 108 Chandler, D., 38, 64 Chang, H. S., 145, 146, 158 Chapman, A., 77, 105 Chattoo, B. B., 50, 64 Chaudhury, A. M., 79, 94, 102 Chen, F.-L., 121, 152 Chen, W. Q., 11, 28 Chen, Y. C. S., 92, 97, 102 Chen-Wu, J. L., 54, 59 Cheresh, D. A., 75, 108 Cheung, A. Y., 71, 110

AUTHOR INDEX Chevrier, V., 72, 102 Chin-Atkins, A. N., 79, 94, 102 Cho, B. H., 11, 32 Cho, G., 43, 60 Cho, S.-O., 84, 87, 90, 102, 103 Chua, N.-H., 89, 96, 108 Chung, G. F., 52, 54, 63 Ciampaglio, C. N., 97, 108 Cigelnik, E., 40, 44, 66 Clark, I. M., 14, 22, 32 Clark, S. E., 80, 105 Clarke, E. D., 13, 29 Clatot, N., 10, 30 Cleary, A. L., 71, 76, 87, 88, 89, 90, 92, 93, 97, 102, 103, 105, 107 Clemencon, H., 40, 45, 62, 63 Clijsters, H. M. M., 79, 109 Cock, J. M., 119, 152 Coenen, A., 55, 66 Coetzee, M. P. A., 46, 60 Cohen, B. A., 57, 61 Cohen, J. D., 77, 103 Colasanti, J., 84, 87, 103 Cole, D. J., 5, 7, 9, 10, 11, 13, 14, 15, 19, 20, 21, 22, 25, 27, 28, 29, 30, 32 Coleman, J. O. D., 5, 28 Collings, D. A., 71, 74, 100, 101, 104 Colon, A., 71, 103 Comandini, O., 57, 64 Compernolle, F., 140, 141, 164 Convey, P., 53, 62 Cooke, T. J., 77, 103 Copeland, L., 134, 156 Corellou, F., 84, 103 Cornelius, M. J., 120, 156 Coruzzi, G. M., 118, 119, 120, 121, 122, 123, 152, 153, 158, 160 Coschigano, K. T., 121, 122, 152, 158 Cosgrove, D. J., 77, 109 Coshigano, K., 118, 158 Cotton, J. A., 54, 60 Courtois, M., 142, 152 Couteaudier, Y., 49, 50, 52, 63 Coutinho, T. A., 46, 60 Craig, S., 79, 94, 102 Cramer, C. L., 2, 27 Cravanzola, F., 52, 60 Crawford, N. M., 118, 123, 135, 152, 164 Crespo, A., 53, 55, 60 Croft, J. H., 55, 60, 66 Croteau, R., 140, 141, 142, 147, 149, 150, 158 Croteau, R. B., 141, 144, 159 Cubero, O. F., 53, 55, 60 Cubeta, M. A., 38, 49, 66 Cullimore, J. V., 119, 121, 124, 152, 154, 159 Cummins, I., 5, 7, 9, 10, 11, 13, 14, 15, 19, 20, 21, 22, 25, 28, 29 Cunillera, N., 141, 152

169

Curti, B., 121, 152 Curtis, I. S., 142, 160 Cyr, R. J., 71, 74, 86, 87, 100, 103, 104, 106, 110 D Dairi, T., 141, 142, 157, 163 Dales, R. B. G., 55, 60 Damianova, R., 10, 30 Danon, A., 2, 4, 20, 27, 30 David, J. C., 34, 44, 45, 62 Davies, J., 11, 13, 14, 17, 19, 28 Davies, T. G. E., 5, 28 Davis, B. G., 6, 7, 10, 11, 17, 19, 28 Davis, J. I., 42, 60 Davison, P. A., 98, 110 de Azevedo, J. L., 49, 51, 61 Debeaupuis, J., 50, 62 de Bertoldi, M., 37, 38, 60 Debnam, P. M., 118, 132, 133, 134, 135, 136, 152, 157 Deeks, M. J., 72, 103 de la Bastide, P. Y., 40, 60 de la Luz, 146, 155 del Pozo, C., 77, 105 Delrot, S., 9, 29 De Luca, V., 131, 156 Demoulin, V., 35, 61 Deng, F., 11, 13, 28 Dennis, D. T., 116, 128, 130, 131, 132, 133, 151, 153, 155, 156, 159, 160, 163 Dennis, E. S., 79, 94, 102 Dennis, R. L., 44, 60 Deppong, D. O., 81, 104 DePriest, P. T., 37, 55, 61 DeRidder, B. P., 11, 14, 17, 23, 28, 31 Deruyter, A., 2, 24, 29 DeSalle, R., 42, 61 Desjardin, E. A., 39, 60 De Veylder, L., 23, 28 d’Harlingue, A., 140, 151 Dharmasiri, N., 77, 105 Dharmasiri, S., 77, 105 Di Bonito, R., 39, 60 Dick, M. W., 34, 60 Dickinson, H. G., 87, 110 DiDonato, R., 94, 102 Diez-Juez, E., 141, 144, 161 DiLaurenzio, L., 92, 97, 103 Di Martino Rigano, V., 135, 153, 154 Dinsmore, A., 13, 29 Dixit, R., 86, 87, 103 Dixon, D. P., 4, 6, 7, 9, 10, 11, 13, 14, 15, 17, 19, 21, 23, 28, 29, 30, 32 Dixon, M. T., 39, 62 Doerner, P., 91, 103 Dolan, L., 92, 98, 101

170

AUTHOR INDEX

Dolezel, J., 72, 101 Donahue, J. L., 2, 27 Donoghue, M. J., 44, 62 Doolittle, K. W., 76, 107 Doolittle, R. F., 43, 60 Doolittle, W. F., 43, 58 Douce, R., 130, 134, 156 Douglas, S. E., 124, 153 Dowd, C. A., 7, 31 Draber, P., 71, 72, 101, 108 Dreßen, U., 126, 154 Driver, F., 51, 53, 60 Droog, F., 2, 13, 24, 28, 29 Dubois, F., 79, 98, 99, 103 Dudzinski, M. L., 42, 60 Dufaud, A., 14, 29 Duncanson, E., 118, 153 Duniec, J., 86, 110 Dunphy, W. G., 85, 105 Durand, J., 79, 98, 99, 103 Durso, N. A., 71, 103, 106 E Eady, C., 91, 103 Earl, A. J., 55, 60 Echeverria, M. A., 78, 107 Edel, V., 39, 50, 61 Edlind, T., 40, 63 Edwards, J. W., 119, 120, 153 Edwards, R., 4, 5, 6, 7, 9, 10, 11, 13, 14, 15, 17, 19, 20, 21, 22, 23, 25, 27, 28, 29, 30, 32 Eicks, M., 136, 153 Eisenreich, W., 142, 143, 145, 146, 149, 151, 153, 154, 155, 156, 159, 161, 162 Eleftheriou, E. P., 86, 103 Elliot, M. L., 39, 60 Elstner, E. F., 23, 29 Emes, M. J., 116, 118, 121, 123, 130, 132, 133, 134, 135, 136, 151, 152, 153, 155, 157, 164 Endow, S. A., 71, 109 Engler, G., 91, 105 Engler, J. D., 91, 105 Esch, J. J., 98, 110 Esposito, S., 135, 153, 154 Estelle, M., 77, 95, 105 Estevez, J. M., 140, 144, 154 F Falkowski, P. G., 115, 154 Farago, S., 22, 23, 29 Fatehi, J., 48, 49, 53, 54, 55, 60, 61 Fekete, C., 55, 66 Feldmann, K. A., 89, 92, 96, 97, 103, 108 Fell, J. W., 40, 65 Fellermeier, M., 142, 154, 156, 159

Fellous, A., 71, 108 Feng, D.-F., 43, 60 Ferario-Mery, S., 126, 154 Fernie, A. R., 132, 135, 150, 152, 163 Ferrario-Mery, S., 123, 158 Ferreira, P., 91, 105 Ferrell, K. M., 140, 157 Ferrer, A., 141, 152 Feyereisen, R., 8, 32 Fickenscher, K., 134, 154 Filgueiras, T. S., 35, 61 Fink, G. R., 76, 94, 102 Fischer, K., 136, 153, 159 Fisher, D. D., 74, 110 Fisher, R. H., 77, 103 Flachmann, R., 128, 133, 135, 155 Flanders, D. J., 86, 103 Flavell, R. B., 71, 109 Flechner, A., 126, 131, 154, 160, 162 Flores, E., 124, 159 Flu¨ gge, U.-I., 125, 131, 136, 153, 154, 155, 159, 164 Focke, M., 139, 140, 162 Fogel, R., 36, 55, 59 Foley, V. M., 7, 31 Fonseca, A., 40, 65 Ford, B. G., 119, 154 Forde, B., 120, 165 Forge, B. G., 120, 158 Fosket, D. E., 71, 103 Fothergill-Gillmore, L. A., 131, 154 Fowler, M. W., 121, 133, 153 Foyer, C. H., 4, 6, 9, 10, 29, 30, 126, 154, 160 Francis, D., 78, 84, 85, 104, 106 Frank, M. J., 72, 95, 104 Frehner, M., 130, 154 Freshour, G., 92, 97, 103 Frijters, A., 50, 66 Friml, J., 76, 101 Frisvad, J. C., 36, 61 Frova, C., 7, 9, 29 Fry, W. E., 57, 61 Fuerst, E. P., 11, 13, 14, 30, 31 Fukatsu, T., 57, 64 Fungaro, M. H. P., 49, 51, 61 Furbank, R. T., 157 Furholz, A., 145, 146, 158 G Gaber, A., 7, 32 Gadal, P., 121, 123, 124, 158, 163 Gaillard, C., 14, 29 Galatis, B., 72, 87, 104, 107 Gallagher, K., 72, 88, 95, 104 Gallego, F., 140, 141, 144, 159, 161 Gamas, P., 124, 159 Gant, J. S., 124, 162

AUTHOR INDEX Gant, S. J., 72, 102 Gantt, J. S., 119, 158 Gao, W. Y., 143, 144, 160 Gardes, M., 37, 39, 47, 53, 55, 61 Gardiner, J. C., 74, 100, 104 Gargas, A., 37, 55, 61 Garland, W. J., 131, 155 Gartner, K., 143, 161 Garvin, D. F., 135, 164 Geisler, M., 80, 81, 104 Gerttula, S. M., 89, 97, 109 Geuns, J. M. C., 140, 141, 164 Gibbon, B. C., 72, 109 Gibon, Y., 126, 163 Gilkes, A. F., 118, 153 Gillmor, C. S., 89, 96, 108, 146, 155 Gilroy, S., 74, 100, 104 Girard, P.-A., 49, 50, 52, 63 Givan, C. V., 128, 155 Glo¨ ssl, J., 15, 30 Golding, A., 132, 135, 152 Goldsbrough, P. B., 7, 11, 14, 17, 23, 28, 31 Goldstein, P. Z., 42, 61 Golovkin, M., 71, 109 Gomez, F., 54, 58 Gonzalez, D., 44, 59 Gooday, G. W., 34, 60 Goodbody, K. C., 93, 104 Goodman, H., 123, 158 Goodman, H. M., 132, 164 Goodwin, S. B., 57, 61 Goto, S., 123, 155 Graeve, K., 133, 134, 155, 164 Graf, R., 72, 107 Grandjean, O., 91, 97, 102 Granger, C. L., 86, 104 Grant, G., 94, 102 Grassman, J., 23, 29 Gray, J. C., 98, 110 Green, P. B., 90, 104 Greenland, A. J., 11, 13, 21, 23, 29, 30, 71, 108 Greyson, M. F., 130, 153 Griffen, A. M., 49, 66 Grimshaw, C., 132, 135, 152 Grini, P., 92, 97, 105 Grisafi, P. L., 76, 94, 102 Grivell, L. A., 40, 59 Groner, R., 139, 140, 162 Gronwald, J. W., 11, 13, 21, 22, 29 Groppe, K., 54, 65 Gross, W., 155 Grosse, H., 155 Grube, M., 37, 53, 55, 60, 61 Grueter, W., 35, 61 Gruissem, W., 145, 146, 158 Gryzenhout, M., 40, 64

Guegler, K., 118, 123, 135, 164 Guevara-Garcia, A., 146, 155 Guh, J. O., 11, 32 Guilfoyle, T., 24, 32 Guillaumin, J.-J., 42, 57, 64 Gunderson, J. H., 34, 63 Gunning, B. E. S., 71, 74, 87, 88, 103, 104, 106, 107, 110 Guo, W.-J., 11, 14, 17, 23, 31 Guttie´ rrez-Nava, M., 146, 155 H Ha, D. B. D., 79, 98, 99, 103 Haake, V., 128, 155 Hable, W. E., 83, 105 Hagan, I., 87, 109 Hagen, G., 24, 32 Hahn, M. G., 92, 97, 103 Hake, S., 91, 108 Halford, N. G., 84, 85, 106 Hall, J. C., 21, 32 Hall, N. P., 120, 121, 156, 164 Hallahan, D. L., 14, 22, 32 Hamann, T., 95, 105 Hamers, M. E. C., 48, 59 Han, I.-S., 71, 103 Han, S. C., 89, 97, 109 Han, Y. S., 140, 155 Hanesworth, V. R., 71, 101 Hans, J., 140, 141, 155, 164 Hardham, A. R., 74, 76, 102, 104 Hardtke, C. S., 77, 96, 105 Harlton, C. E., 49, 61 Harper, J. D. I., 72, 74, 100, 104, 109 Harper, M. A., 56, 60 Harrington, T. C., 40, 63 Hartwell, J., 136, 155 Hase, T., 119, 120, 121, 125, 162 Hasegawa, M., 42, 64, 131, 160 Hasenbein, N. G., 72, 96, 110 Hasezawa, S., 24, 31 Hass, H., 43, 64 Hatton, P. J., 9, 29 Hattori, F., 123, 131, 155 Hatzios, K. K., 11, 12, 13, 19, 28, 29 Hauschild, R., 134, 136, 148, 164 Hause, B., 72, 101, 141, 155 Hause, G., 143, 144, 160 Hauser-Hahn, I., 71, 108 Hawksworth, D. L., 34, 35, 36, 39, 44, 45, 53, 55, 59, 60, 61, 64 Hayakawa, T., 119, 122, 123, 155, 156 Hayes, J. D., 5, 10, 22, 24, 29 He, X.-L., 14, 32 Heale, J. B., 38, 49, 64, 66 Heberle-Bors, E., 91, 109 Hecht, S., 142, 143, 154, 155, 156, 159, 161

171

172

AUTHOR INDEX

Hecht, U., 123, 155 Hedges, S. B., 40, 43, 44, 66 Hehn, A., 7, 13, 14, 19, 21, 22, 25, 28 Heifetz, P. B., 145, 146, 158 Heineke, D., 155 Helariutta, Y., 92, 97, 103 Heldt, H. W., 155 Hellmann, H., 77, 105 Hemerly, A., 91, 105 Henkes, S., 128, 133, 135, 155 Hennig, W., 41, 61 Henze, K., 126, 154, 156 Hepler, P. K., 71, 72, 87, 88, 103, 105, 109, 110 Herbert, M., 133, 162 Hernandez, G., 124, 162 Herrero, A., 124, 159 Herrmann, K. M., 132, 156 Herrmann, R. G., 115, 124, 126, 127, 131, 137, 159 Hershey, H. P., 22, 29 Herz, S., 142, 156, 159 Heuser, T., 140, 160 Hibbett, D. S., 44, 59, 62 Hightower, R. C., 71, 108 Hillemann, D., 124, 157 Hillis, D. M., 39, 62 Himmerich, S., 34, 63 Hinkle, G., 43, 66 Hippeli, S., 23, 29 Hirata, A., 34, 65 Hirel, B., 120, 123, 158, 160 Hirose, N., 123, 156 Hirsch, P. R., 50, 58 Hobbie, L., 77, 95, 105 Hocart, C. H., 79, 94, 102 Hodges, M., 123, 158 Hoferichter, P., 155 Hofmann, K., 87, 108 Hofstetter, V., 40, 45, 62, 63 Hoge, H., 2, 24, 29 Hogers, R., 50, 66 Holderness, M., 52, 54, 63 Holt, D. C., 11, 13, 21, 23, 29, 30 Homes, M., 50, 66 Hong, Z. Q., 134, 156 Hopkin, J. M., 34, 64 Horbach, S., 137, 140, 161 Horgen, P., 40, 55, 60, 62 Hosted, T. J., 124, 157 House of Lords, 57, 62 Hsieh, M.-H., 121, 123, 158 Huang, C. Y., 115, 164 Huang, S. R., 71, 106 Huang, Y., 128, 153 Huchon, D., 42, 64 Huckelsby, D. P., 118, 136, 151 Hucklesby, D. P., 118, 132, 151, 152 Hugdahl, J. D., 71, 101, 105

Hughes, K. A., 57, 62 Hull, A., 94, 102 Hu¨ lskamp, M., 72, 92, 97, 98, 105, 106 Hung, C. Y., 92, 98, 101 Huntley, R., 78, 107 Huppe, H. C., 135, 165 Hurwitz, L. R., 77, 95, 105 Hush, J. M., 71, 105 Hussey, P. J., 71, 72, 103, 106, 108, 119, 158 Hwang, I., 92, 96, 105 I Iba, K., 140, 151 Igarashi, H., 71, 108 Imaishi, H., 14, 32 Imperial, S., 143, 161 Ing, B., 46, 62 Ingber, D. E., 75, 110 Inokuchi, R., 124, 125, 156 Inze´ , D., 4, 23, 28, 30, 91, 105 Ireland, R. J., 118, 119, 123, 131, 156, 158 Irzyk, G., 11, 13, 30 Irzyk, G. P., 13, 14, 30, 31 Ishiyama, K., 122, 123, 156 J Jablonkai, I., 7, 13, 14, 19, 21, 22, 25, 28 Jacobsson, S., 45, 63 Jacqmard, A., 78, 107 James, C. M., 98, 110 James, T. Y., 45, 63 Jansen, C. G., 71, 102 Jansonius, J., 43, 62 Jensen, L. C. W., 71, 102 Jeong, S., 80, 105 Jepson, I., 11, 13, 21, 23, 29, 30, 31 Jiang, C.-J., 71, 105, 106, 108 Jime´ nez, L. F., 140, 146, 154, 155 Jin, T., 55, 62 Job, D., 72, 102 John, P. C. L., 86, 87, 106, 110 Johnson, J. E., 45, 63 Jones, S. S., 13, 31 Jorgensen, J. E., 91, 103 Jost, W., 118, 157 Journet, E.-P., 130, 134, 156 Joy, K. W., 122, 156 Jullien, M., 91, 95, 96, 97, 109 Jun Wei, K., 72, 96, 110 Jurgens, G., 76, 77, 89, 91, 92, 95, 96, 101, 105, 106, 109 K Kalgutkar, R. M., 43, 62 Kamachi, K., 119, 156 Kambara, Y., 14, 32

AUTHOR INDEX Kamiya, Y., 140, 142, 154, 160 Kammerer, B., 159 Kampranis, S. C., 10, 30 Kanaboshi, H., 7, 32 Kaneda, K., 141, 142, 157, 163 Kanellopoulos, P. N., 10, 30 Katsaros, C., 87, 104 Katsuta, J., 86, 105 Katz, M. E., 115, 154 Kawabata, S., 119, 120, 121, 162 Kawaide, H., 140, 142, 154, 160 Kawasaki, H., 34, 65 Keeler, S. J., 14, 30 Keeling, P. J., 40, 43, 62, 127, 148, 156, 161 Keenan, B. G., 54, 58 Keller, W. A., 87, 108 Kellerman, J., 131, 156, 160 Kelly, G. J., 130, 156 Kendall, A. C., 120, 121, 156, 164 Kennard, J. L., 88, 105 Kernebeck, B., 72, 98, 106 Kerp, H., 43, 64 Kerry, B. R., 50, 58 Kevei, F., 55, 66 Keys, A. J., 120, 156 Khan, S., 71, 106 Kibby, G., 56, 65 Kientz, M., 95, 105 Kilili, K. G., 10, 30 Kim, C. S., 11, 32 Kim, E. Y., 11, 32 Kim, H.-H., 71, 101 Kim, K. C., 11, 32 King, S. P., 157 Kirk, D. W., 118, 153 Kirk, P. M., 34, 44, 45, 61, 62 Kis, K., 142, 143, 154, 155 Kistler, H. C., 37, 67 Kiyosue, T., 11, 30 Kloareg, B., 84, 103 Knappe, S., 136, 153 Knight, J. S., 118, 132, 133, 134, 135, 152, 157 Knoll, A. H., 43, 62, 115, 154 Knowles, V. L., 131, 159 Kobayashi, G. S., 38, 65 Kochian, L. V., 135, 164 Koeppe, M. K., 14, 30 Kohl, D. H., 136, 152 Kohn, L. M., 40, 55, 62 Kojima, S., 123, 155 Komesli, S., 72, 102 Kondi, G., 10, 30 Kopriva, S., 126, 135, 136, 157 Koprivova, A., 126, 135, 136, 157 Kossmann, J., 128, 157 Kovar, D. R., 72, 109 Kozaki, A., 120, 157 Kozakiewicz, Z., 37, 55, 66

Kramer, V., 118, 157 Krapp, A., 120, 126, 163 Krepinsky, K., 133, 137, 157 Kreuz, K., 14, 22, 23, 29 Krieger, C., 143, 161 Kroll, D. J., 26, 32 Kronenberger, J., 78, 95, 96, 97, 109, 118, 157 Kropf, D. L., 83, 101, 105 Krska, R., 15, 30 Kruger, N. J., 133, 136, 151, 157 Krushkal, J., 140, 157 Kubatova, E., 72, 101 Kubeik, A. R., 50, 67 Kubicek, C. P., 50, 63 Kuchler, K., 15, 30 Kuiper, M., 50, 66 Kuma, K.-I., 124, 125, 156 Kumada, Y., 124, 157 Kumagai, A., 85, 105 Kumar, S., 40, 43, 44, 66 Kuninaga, S., 38, 45, 62 Ku¨ ntzel, H., 55, 60 Kuryama, R., 71, 101 Kusaba, M., 24, 31 Kusumi, K., 140, 151 Kutchan, T. M., 143, 144, 160 Kutscherra, K., 118, 157 Kuzuyama, T., 140, 141, 142, 154, 157, 160, 163 L LaBrie, S. T., 118, 123, 135, 164 Lagudah, E. S., 19, 32 Laguerre, G., 50, 61 Lahners, K., 118, 157 Laloi, C., 2, 4, 20, 27, 30 Lam, E., 24, 32 Lam, H. M., 118, 158 Lam, H.-M., 121, 158 Lamb, C., 91, 103 Lambert, A.-M., 71, 72, 102, 108, 109 Lamboy, W. F., 52, 62 Lanave, C., 124, 160 Lancien, M., 123, 158 Lane, D. J., 44, 59 Lanfranco, L., 39, 62 Lange, B. M., 139, 140, 141, 142, 145, 146, 147, 149, 150, 151, 158 Lange, C., 134, 136, 148, 164 Langenkamper, G., 133, 134, 164 Langer, E., 44, 62 Langer, G., 44, 62 Lapthorn, A., 7, 28 Larson, K., 6, 30 Larsson, E., 45, 63 Lasker, B. A., 38, 65 Latge, J. P., 50, 62

173

174

AUTHOR INDEX

Latzel, C., 146, 151, 162 Latzko, E., 130, 156 Lau, S.-M. C., 14, 30 Lauber, M. H., 92, 96, 105 Laufs, P., 91, 95, 96, 97, 109 Laule, O., 145, 146, 158 Lawley, B., 53, 57, 62 Lawrence, S. L., 71, 108 Lay, V. J., 11, 13, 21, 23, 29, 30 Lazarus, C. M., 55, 60 Lea, P. J., 118, 119, 120, 121, 122, 123, 126, 151, 156, 158, 160 Leaver, C., 4, 30 Lederer, B., 7, 30 Leipe, D. D., 34, 63 Leisse, A., 132, 135, 152 Lemmon, B. E., 88, 107 Lenze, D., 155 Leo´ n, P., 140, 144, 146, 154, 155 Leonardi, M., 57, 64 Lepidi, A. A., 37, 38, 60 Lepingle, A., 118, 157 Leslie, J. D., 71, 103 Leu, W. M., 89, 96, 108 Leustek, T., 123, 158 Le´ vesque, C. A., 49, 61 Levy, J., 89, 97, 109 Li, J., 40, 63 Li, M.-G., 119, 158 Li, R., 72, 110 Libbenga, K., 2, 24, 29 Lichtenthaler, F. W., 139, 140, 162 Lichtenthaler, H. K., 139, 140, 141, 147, 158, 162 Lim, J., 121, 122, 152 Lindsey, K., 91, 103 Linthorst, H. J. M., 140, 155 Lintilhac, P. M., 75, 99, 106 Lipari, S. E., 50, 55, 63 Little, E., 43, 60 Liu, B., 74, 87, 94, 101, 106 Liu, N. Y., 77, 95, 105 Livak, K. J., 50, 67 Lizon, P., 46, 63 Lloyd, C. W., 71, 86, 93, 102, 103, 104, 106, 108 Loddenko¨ tter, B., 159 Lois, L. M., 140, 141, 143, 144, 159, 161 Lolle, S. J., 92, 97, 105 Lomer, C. J., 51, 59 Lonsdale, D. M., 71, 109 Lopez, I., 71, 106 Loukari, H., 87, 104 Loutre, C., 11, 30 Love, J., 82, 83, 106 Luckey, S. W., 26, 32 Lucyshyn, D., 15, 30 Luduena, R. F., 71, 106 Ludwig, R. A., 120, 163

Lukowitz, W., 92, 96, 105, 106 Lunn, J. E., 157 Lu¨ nsdorf, H., 55, 60 Luque, I., 124, 159 Luschnig, C., 15, 30 Lu¨ ttgen, H., 142, 156, 159 Lutz, M., 40, 58 Lutzoni, F., 37, 63 Lynch, T. M., 75, 99, 106 Lysak, M., 72, 101 M Macdonald, F. D., 126, 159 Maciver, S. K., 71, 106 Mae, T., 119, 123, 155, 156 Mahmoud, S. S., 141, 144, 159 Maier, U., 142, 154 Maier, W., 41, 63 Maijala, P., 40, 63 Makris, A. M., 10, 30 Malamy, J. E., 92, 97, 103 Mann, M., 72, 110 Mannervik, B., 6, 30 Marc, J., 71, 74, 100, 104, 106 Margulis, L., 124, 159 Marks, M. D., 98, 110 Marrs, K. A., 11, 24, 30 Marshall, W. A., 56, 60 Martin, F., 40, 42, 50, 59, 63 Martin, M., 123, 158 Martin, W., 115, 124, 126, 127, 131, 133, 137, 147, 149, 150, 152, 154, 156, 157, 158, 159, 160, 162, 164 Martinoia, E., 14, 29 Masamoto, K., 140, 151 Masclaux, C., 123, 158 Massaro, G., 135, 153, 154 Masumura, T., 10, 32 Mathesius, U., 88, 90, 102 Mathis, R., 124, 159 Mathur, J., 72, 98, 106 Mathur, N., 72, 98, 106 Matoh, T., 121, 122, 123, 159 Matt, P., 126, 163 Mauch, F., 11, 15, 32 Maurer, P., 49, 50, 52, 63 Maurino, V., 136, 153 May, G. S., 40, 63 May, M. J., 4, 30 Mayer, U., 89, 92, 95, 96, 105, 106 McCaskill, D., 140, 158 McClelland, M., 50, 67 McClinton, R. S., 77, 95, 106 McCormick, S., 92, 97, 102 McDowell, J. M., 71, 106 McElroy, D., 71, 106 McGonigle, B., 14, 30 McGovern, M., 77, 95, 105

AUTHOR INDEX McHugh, S. G., 131, 159 McKibbin, R., 84, 85, 106 McKinney, E. C., 71, 106 Mclellan, L. I., 5, 10, 22, 29 McMahon, M., 24, 29 McNally, F. J., 74, 106 McNally, J. G., 76, 107 McNeill, J., 35, 61 McRae, C. F., 36, 63 Meade, G., 7, 31 Meagher, R. B., 71, 106, 108 Medoff, G., 38, 65 Mehmann, B., 40, 63 Meiners, S., 75, 108 Melo-Oliveira, R., 118, 121, 122, 152, 158 Melzer, M., 135, 136, 164 Merrikh, H., 94, 102 Mews, M., 87, 106 Meya, G., 120, 160 Meyer, S. A., 38, 63 Meyer, W., 50, 63 Meyer, Y., 124, 159 Miao, Z.-H., 24, 32 Michniewicz, M., 76, 101 Miernyk, J. A., 128, 130, 133, 153, 160 Miflin, B. J., 120, 121, 126, 156, 158, 160 Migge, A., 120, 160 Mikawa, T., 40, 65 Miki, B. L., 131, 159 Milgroom, M. G., 50, 55, 63 Millar, A. H., 11, 17, 31 Miller, B. J., 83, 107, 140, 160 Miller, O. K., 45, 63 Miller, R. N. G., 52, 54, 63 Mills, P. R., 45, 65 Milner, R. J., 51, 53, 60 Mineyuki, Y., 85, 86, 87, 106, 107 Mitkovski, M., 90, 107 Mittler, R., 2, 20, 30 Miyao, K., 7, 32 Miyasaka, H., 7, 32 Miyata, T., 124, 125, 156 Mizuno, K., 71, 107 Moffatt, B., 118, 161 Mohlmann, T., 133, 163 Mohr, H., 118, 123, 155, 162, 163 Momany, M., 54, 59 Moncalvo, J.-M., 36, 40, 42, 45, 54, 56, 57, 62, 63, 67 Moore, R., 87, 106 Moore, R. C., 74, 110 Moose, S. P., 19, 32 Morawetz, R., 50, 63 Morby, A. P., 119, 152 Morcuende, R., 126, 163 Morejohn, L. C., 71, 101, 105 Mori, H., 71, 108 Moriyama, Y., 49, 65

175

Morrison, W. H., 74, 94, 101 Moyer, M., 118, 151 Muehlbauer, F. J., 40, 48, 58 Mu¨ eller, U. G., 50, 63 Muller, C., 126, 139, 140, 141, 162, 163 Mullineaux, P. M., 9, 10, 29 Munch, J.-C., 50, 59 Murata, N., 10, 32 Murata, T., 87, 107 Murphy, A. S., 7, 11, 31 Murray, A. J. S., 120, 151 Murray, J. A. H., 78, 107 Myburg, H., 40, 64 N Nacry, P., 78, 95, 96, 97, 109 Nadeau, J. A., 80, 81, 97, 104, 107 Nagata, T., 24, 31 Nagata, Y., 49, 65 Nagi, N., 121, 158 Nakagawa, T., 10, 32 Nakamura, T., 123, 155 Naqvi, N. I., 50, 64 Natsuaki, T., 45, 62 Nayagam, S., 123, 132, 151 Negm, F. B., 128, 153 Neuhaus, H.-E., 133, 163 Neuve´ glise, C., 55, 64 Newcomb, W., 91, 110 Nicholson, D. H., 35, 61 Nick, P., 76, 107 Nicolas, I. L., 78, 107 Nikoh, N., 57, 64 Nishida, H., 34, 65 Nishimura, M., 133, 134, 160 Niwa, Y., 140, 151 Nixon, K. C., 41, 42, 60, 64 Noctor, G., 4, 6, 30, 126, 154, 160 Nourizadeh, S. D., 7, 11, 31 Novitskaya, L., 126, 160 Nowitzki, J., 155 Nowitzki, U., 131, 160 Nurse, P., 85, 108 Nuti, M. P., 37, 38, 60 Nuytinck, J., 57, 64 O Oberwinkler, F., 40, 41, 58, 63 O’Donnell, K., 37, 40, 44, 66, 67 Oelmuller, R., 123, 155 Oeser, A., 133, 162 Ogren, W. L., 121, 163 O’Hagan, D., 7, 13, 14, 19, 21, 22, 25, 28 Ohkawa, H., 14, 32 Ohmiya, A., 24, 32 Ojima, K., 119, 123, 155, 156 Okada, K., 142, 160

176 Okada, M., 124, 125, 156 Okada, N., 42, 64 O’Keefe, D. P., 14, 30 Oliveira, I., 118, 121, 158 Oliveira, I. C., 118, 122, 160 Olsen, G. J., 41, 66 On˜ ate-Sa´ nchez, L., 11, 17, 31 Opatrny, Z., 71, 108 Osborn, M., 88, 110 Oudin, A., 142, 152 Ozino, O. I., 52, 60 P Pacioni, G., 57, 64 Page, J. E., 143, 144, 160 Page, R. D. M., 54, 60 Pagel, M., 37, 63 Paine, J. A., 23, 31 Palevitz, B. A., 71, 86, 87, 103, 106 Pallett, K. E., 14, 22, 32 Palmer, J. D., 43, 58, 156 Panchal, G., 39, 46, 47, 51, 56, 59 Pankhurst, R. J., 41, 64 Panteris, E., 72, 107 Parekh, N. S., 92, 97, 105 Paris, S., 50, 62 Parker, J. S., 77, 101 Pascal, S., 13, 30 Pasquier, A., 39, 53, 66 Pastuglia, M., 91, 97, 102 Paterson, R. R. M., 36, 64 Patterson, D. J., 34, 63 Pearce, D. A., 50, 59 Peat, L. J., 119, 120, 122, 160 Peer, W. A., 7, 11, 31 Peever, T. L., 40, 48, 58 Pegler, D. N., 34, 44, 45, 46, 61, 64 Peleman, J., 50, 66 Pelzer-Reith, B., 160 Penger, A., 160 Pereira, S., 119, 152 Pe´ rez-Sierra, A., 42, 57, 64 Perotto, S., 39, 62 Pesole, G., 124, 160 Peter, C., 71, 108 Peter, U., 155 Petersen, R., 26, 32 Phaff, H. J., 38, 63 Piatti, P., 52, 60 Pickard, B. G., 76, 107 Pickett-Heaps, J. D., 88, 107 Pierro, A., 77, 95, 105 Pieters, R., 48, 59 Pietersma, M., 134, 136, 148, 164 Pilotti, C. A., 51, 52, 64, 65 Pine, E. M., 44, 62 Pipe, N. D., 38, 64 Pistilli, M., 140, 157

AUTHOR INDEX Pixley, F. J., 34, 64 Pizzirani-Kleiner, A. A., 49, 51, 61 Plaisance, K. L., 11, 13, 21, 22, 29 Plaumann, M., 133, 137, 157 Plaxton, W. C., 128, 129, 131, 148, 151, 159, 161 Podtelejnikov, A. V., 72, 110 Poduje, L., 13, 19, 31 Poppenberger, B., 15, 30 Porter, D., 34, 63 Pot, J., 50, 66 Potin, P., 84, 103 Potter, S., 11, 13, 30 Poulter, C. D., 137, 162 Pozueta-Romero, J., 130, 154 Prager, E. M., 49, 59 Preparata, G., 124, 160 Priest, F., 38, 64 Prior, C., 51, 59 Privalle, L., 118, 151, 157 Proudlove, M. O., 148, 161 Pruitt, R. E., 92, 97, 105 Pu, R. S., 82, 83, 107 Pulford, D. J., 24, 29 Punithalingam, E., 48, 49, 53, 61 Punja, Z. K., 49, 61 Pupko, T., 42, 64 Pyke, K. A., 150, 161 Pysh, L., 92, 97, 103 Q Quatrano, R. S., 81, 107 Querol, J., 143, 161 Quick, P. W., 133, 163 R Radykewicz, T., 142, 146, 154, 162 Rafalski, J. A., 50, 67 Rajapakse, C. N. K., 36, 67 Raschke, M., 142, 143, 144, 154, 160 Rashbrooke, M. C., 72, 96, 110 Rastogi, R., 118, 161 Raudaskoski, M., 40, 63 Raven, J. A., 115, 154 Rawlins, D. J., 86, 103 Reddy, A. S. N., 71, 109 Redhead, S. A., 45, 63 Redinbaugh, M. G., 118, 121, 133, 161 Reeb, V., 37, 63 Reece, K. S., 71, 106 Regnier, F. E., 11, 14, 17, 23, 31 Reichler, S., 144, 154 Reid, E. E., 130, 163 Reijans, M., 50, 66 Reindl, A., 144, 154 Reinhardt, D., 77, 105 Reins, B., 155

AUTHOR INDEX Remy, W., 43, 64 Rennenberg, H., 23, 31 Reuzeau, C., 76, 107 Reynolds, D. R., 35, 64 Riba, G., 49, 50, 52, 55, 63, 64 Richter, G., 142, 161 Rideau, M., 142, 152 Ridley, S. M., 119, 164 Riechers, D. E., 11, 13, 19, 21, 31, 32 Rigano, C., 135, 153 Rigo, K., 37, 66 Rinaldi, A. C., 57, 64 Riou-Khamlichi, C., 78, 107 Ripley, S., 53, 62 Ritchie, S., 74, 100, 104 Rivera, A., 50, 59 Robbins, P. W., 54, 59 Roberts, P. J., 39, 40, 46, 51, 56, 59, 64, 65 Robertson, N., 23, 31 Robinson, K. R., 82, 83, 107 Robinson, S. P., 126, 161 Rochefort, D. A., 124, 157 Rodrigues-Pousada, D. R., 132, 164 Rodriguez-Concepcion, M., 140, 141, 143, 144, 152, 159, 161 Roger, A. J., 43, 58 Rogers, H. J., 71, 84, 108, 109, 110 Rogers, M., 127, 148, 161 Rohdich, F., 142, 143, 145, 146, 149, 153, 154, 155, 156, 159, 161, 162 Rohmer, M., 137, 140, 161, 162 Romano, M. L., 21, 32 Romero, C., 140, 154 Rose, J., 123, 132, 151 Rothenberg, M., 71, 106 Rothstein, S. J., 118, 121, 151, 157, 161, 163 Roulet, A., 11, 21, 31 Roux, Y., 123, 158 Roxas, V. P., 10, 31 Roy, H., 148, 162 Roytrakul, S., 140, 155 Rujan, T., 147, 149, 150, 158 Russell, P., 85, 108 Rutherford, M. A., 50, 59 Rutten, T., 71, 102, 108 S Sacchettini, J. C., 137, 162 Saccone, C., 124, 160 Sack, F. D., 79, 80, 81, 97, 104, 107, 110 Saddler, G., 41, 59 Sagawa, I., 49, 65 Sagbohan, J., 51, 59 Sagner, S., 142, 146, 151, 154, 156, 159, 162 Sahm, H., 137, 140, 161 Sakakibara, H., 119, 120, 121, 125, 162 Saleh, N., 71, 108

177

Salema, R., 119, 152 Salimath, S. S., 40, 48, 58 Samac, D. M., 124, 162 Sanchez-Bravo, J., 78, 107 Sandermann, H., 4, 5, 31 Sanders, I. R., 54, 65 Sanderson, F. R., 51, 52, 64, 65 Sangwan, R. S., 79, 98, 99, 103 Sangwon, R. S., 131, 159 Sappl, P. G., 11, 17, 31 Sarfati, J., 50, 62 Sauer, M., 76, 101 Sauret-Gueto, S., 141, 144, 161 Savona, C., 159 Sax, K., 75, 101 Scalla, R., 11, 13, 21, 30, 31 Scandalios, J. G., 2, 13, 31 Schaer, B., 23, 31 Schafer, E., 76, 107 Scheibe, R., 133, 134, 154, 155, 164, 165 Scheible, W.-R., 126, 163 Schellenbaum, P., 71, 108 Schiefelbein, J., 92, 98, 101 Schindler, M., 75, 108 Schlee, D., 118, 157 Schmidt, J., 143, 144, 160 Schmidt, S., 87, 108, 109, 123, 155 Schmit, A.-C., 72, 102 Schnarrenberger, C., 126, 127, 131, 133, 137, 154, 155, 156, 157, 159, 160, 162 Schneiderbauer, 118, 161 Schneidereit, J., 125, 164 Schneitz, K., 92, 97, 105 Schoenbeck, M. A., 124, 162 Schofield, O., 115, 154 Schopfer, P., 76, 107, 110 Schuhmacher, R., 15, 30 Schuhr, C. A., 142, 146, 154, 156, 159, 161, 162 Schußler, A., 44, 65 Schwan, A. L., 21, 32 Schwarz, H., 92, 96, 105 Schwarzott, D., 44, 65 Schwender, J., 139, 140, 141, 162 Scorzetti, G., 40, 65 Scott-Craig, J. S., 13, 19, 31 Seagull, R. W., 88, 110 Seeley, E. H., 11, 14, 17, 23, 31 Seemann, M., 137, 140, 161, 162 Seifertova, D., 76, 101 Seith, B., 118, 162, 163 Sek, F. J., 87, 106 Seppelt, R. D., 36, 63 Seto, H., 140, 141, 142, 154, 157, 160, 163 Shah, D. M., 71, 108 Sharkey, D. E., 71, 106 Shaw, D. S., 55, 67 Shaw, P. J., 86, 103

178

AUTHOR INDEX

Shaw, P. J. A., 56, 65 Shaw, S. L., 81, 107 Shearer, G., 136, 152 Shearer, G., Jr., 40, 65 Sheehan, D., 7, 31 Sherman, A., 118, 153 Shevell, D. E., 89, 96, 108 Shibaoka, H., 71, 76, 77, 78, 86, 91, 101, 105, 108, 109 Shigeoka, S., 7, 32 Shimmen, T., 71, 110 Shinozaki, K., 11, 30 Shultz, C., 121, 158 Shurman, A., 118, 163 Shuster, C., 118, 162 Sieberer, T., 15, 30 Sierra, A. P., 42, 65 Silflow, C. D., 119, 158 Silva, P. C., 35, 61 Silverstone, A. L., 97, 108 Simanis, V., 87, 108, 109 Simcox, P. D., 130, 163 Simmonds, D. H., 87, 91, 108, 110 Simon, L., 44, 59 Singer, R., 44, 65 Singh, K. B., 11, 17, 28, 31 Singh, T., 39, 46, 51, 59 Sivakumaran, S., 40, 65 Sjamsuridzal, W., 34, 65 Skidmore, M. W., 9, 31 Skog, J. E., 35, 61 Slater, M., 11, 30 Smertenko, A., 71, 108 Smith, A. P., 7, 11, 14, 17, 23, 31 Smith, L. G., 70, 72, 75, 88, 89, 91, 92, 93, 95, 97, 102, 104, 108, 109 Smith, M. L., 49, 65 Smith, R. K., 10, 31 Snustad, D. P., 119, 158 Sogin, M. L., 34, 43, 44, 54, 58, 59, 63, 66 Sohrmann, M., 87, 108, 109 Somerville, C. R., 121, 163 Song, H., 71, 109 Sonnewald, U., 23, 31, 128, 133, 135, 155, 157 Sonobe, S., 71, 105, 108 Sorrell, D. A., 78, 104 Souret, F. F., 140, 157 Spek, A., 2, 24, 29 Spiteller, M., 142, 154 Spiteller, P., 142, 154 Spitzer, E. D., 38, 65 Spooner, B. M., 39, 42, 46, 51, 56, 57, 59, 64 Sreenivasaprasad, S., 45, 65 Srinivasan, N., 98, 110 Srivastava, D. K., 134, 163 Staiger, C. J., 72, 109 Stalpers, J. A., 34, 44, 45, 62 Statzell-Tallman, A., 40, 65

Steen, D. A., 78, 109 Steinberg, C., 50, 61 Steinmann, T., 92, 96, 105 Stenlid, J., 52, 66 Stephenson, G. R., 21, 32 Steppuhn, J., 91, 103 Stewart, G. R., 119, 164 Stickel, S. K., 43, 44, 59, 66 Stitt, M., 120, 126, 128, 130, 132, 133, 135, 150, 155, 163 Stolz, J. F., 125, 163 Stoner, T. D., 22, 29 Stoppin, V., 71, 109 St-Pierre, B., 142, 152 Strack, D., 140, 141, 155, 164 Sugimoto, K., 72, 96, 110 Sugiyama, J., 34, 65 Sugiyama, M., 40, 65 Sugiyama, T., 119, 120, 121, 125, 162 Suire, C., 140, 151 Sullivan, E., 37, 67 Summerbell, R. C., 40, 65 Sun, T., 97, 108 Sundaresan, V., 84, 87, 103 Sung, R., 77, 95, 106 Sunkel, C., 119, 152 Su¨ ss, K.-H., 126, 135, 136, 157, 164 Sutton, B. C., 34, 44, 45, 48, 61, 65 Suzuki, A., 121, 124, 163 Swann, E. C., 55, 66 Swarup, R., 119, 152 Swedjemark, G., 52, 66 Swofford, D. L., 41, 66 Sylvester, A. W., 90, 91, 107, 108 Szaniszlo, P. J., 54, 59 Szaro, T. M., 44, 59 T Tahiri-Alaoui, A., 39, 66 Taira, M., 120, 163 Tajiri, Y., 34, 65 Takagi, M., 140, 141, 142, 157, 163 Takahashi, E., 121, 122, 123, 159 Takahashi, S., 140, 157 Takahashi, Y., 24, 31 Takeba, G., 120, 157 Takeda, T., 7, 32 Takesue, K., 91, 109 Takeuchi, T., 45, 62 Tal, A., 21, 32 Tanaka, K., 10, 32 Tanksley, S. D., 71, 101 Tateno, Y., 124, 157 Taylor, A., 77, 109 Taylor, F. J. R., 115, 154 Taylor, J. W., 35, 36, 37, 39, 40, 43, 44, 54, 55, 58, 59, 63, 64, 66 Taylor, T. N., 43, 64

AUTHOR INDEX Tegeler, A., 133, 134, 136, 148, 165 Tehler, A., 37, 55, 61 Teichmann, T., 76, 101 Temple, S. J., 124, 162 Teren, J., 37, 66 Tetour, M., 133, 162 Thangavelu, M., 71, 109 Theodoulou, F. L., 9, 14, 29, 32 Thom, E., 133, 163 Thompson, C. J., 124, 157 Thorn, R. G., 45, 63 Thuan, T. B., 34, 65 Thurman, D. A., 148, 161 Tiege, M., 135, 136, 164 Timmis, J. N., 115, 164 Tingey, S. V., 50, 67 Tinsley, B., 94, 102 Tjaden, G., 121, 158 Tjalkens, R. B., 26, 32 Tobar, J., 94, 102 Tobin, A. K., 116, 119, 120, 122, 130, 150, 152, 153, 160, 164 Toby, G., 10, 30 Tolbert, N. E., 133, 162 Tommasini, R., 14, 29 Tonoike, H., 71, 103 Tooley, P. W., 40, 42, 63 Torres-Ruiz, R. A., 77, 91, 95, 109 Toth, B., 37, 66 Totong, R., 94, 102 Totte, N., 140, 141, 164 Touraev, A., 91, 109 Traas, J., 78, 91, 95, 96, 97, 109 Travis, S. J., 38, 65 Trehane, P., 35, 61 Trepp, G. B., 124, 162 Trewavas, A. J., 82, 83, 106 Trimming, B. A., 130, 164 Trotochaud, A. E., 80, 105 Tsang, S., 43, 60 Tsichlis, P. N., 10, 30 Tsukitani, Y., 86, 110 Turland, N. J., 35, 61 Turner, G., 55, 60 Turner, J. C., 120, 121, 156, 164 Turner, S., 124, 153 Turpin, D. H., 135, 165 Twell, D., 91, 103 Typas, M. A., 49, 66 U Ulmasov, T., 24, 32 Unseld, M., 38, 59 Urano, J., 10, 32 Ushimaru, T., 10, 32

V Valcke, R. L. M., 79, 109 Vale, R. D., 74, 106 Valois, F., 34, 63 Valster, A. H., 88, 109 Valtersson, U., 120, 163 Vance, C. P., 124, 162 Van Damme, E. J. M., 140, 141, 164 van de Lee, T., 50, 66 Van den Ende, W., 140, 141, 164 van der Heijden, R., 140, 155 Vanderkooy, A., 2, 24, 29 Van Der Straeten, 132, 164 van der Walt, J. P., 38, 66 van Heerden, A., 71, 101 Vanloven, K., 79, 109 Van Montagu, M., 4, 23, 28, 30, 91, 105, 132, 164 Vanonckelen, H. A., 79, 109 Vanoni, M. A., 121, 152 Vantard, M., 71, 72, 102, 108, 109 Vardanyan, A., 10, 30 Varga, J., 37, 55, 66 Vaucheret, H., 118, 157 Vaughn, K., 11, 21, 31 Vaughn, K. C., 72, 109 Venverloo, C. J., 93, 104 Verbeken, A., 57, 64 Verberne, M. C., 140, 155 Verduin, S. J. W., 45, 63 Vernoux, T., 4, 30 Verpoorte, R., 140, 155 Verzotti, E., 121, 152 Viaud, M., 39, 53, 66 Vidali, L., 71, 72, 110 Vieira, M. L. C., 49, 51, 61 Viklicky, V., 71, 108 Vilgalys, R., 38, 40, 42, 44, 45, 49, 54, 56, 57, 59, 62, 63, 66, 67 Villemur, R., 119, 158 Vincente, O., 91, 109 Vogler, A. P., 42, 61 Voigt, K., 40, 44, 66 Volkmann, D., 87, 106 Voll, L. M., 125, 164 Vona, V., 135, 153, 154 von Groll, U., 80, 97, 110 von Schaewen, A., 133, 134, 136, 148, 155, 157, 164, 165 Vos, P., 50, 66 Vugrek, O., 72, 96, 110 W Wada, M., 87, 107 Wadsworth, P., 71, 88, 105, 110 Wagner, U., 11, 15, 32

179

180

AUTHOR INDEX

Wainwright, P. O., 34, 43, 63, 66 Waizenegger, I., 92, 96, 105 Wakefield, A. E., 34, 64 Walbot, V., 6, 11, 29 Walker, A. R., 98, 110 Walker, C., 44, 65 Walker, D. A., 126, 161 Walker, E. L., 119, 153 Wallsgrove, R. M., 120, 121, 156, 164 Walter, M. H., 140, 141, 155, 164 Walton, J. D., 13, 19, 31, 32 Wang, A., 49, 59 Wang, D. Y.-C., 40, 43, 44, 66 Wang, N., 75, 110 Wang, R. C., 118, 123, 135, 164 Wang, X., 145, 146, 158 Wang, Y. H., 135, 164 Ward, E., 11, 13, 30, 39, 66 Ward, T. J., 37, 67 Wasteneys, G. O., 71, 72, 87, 96, 103, 110 Watanabe, H., 142, 163 Watanabe, M., 125, 162 Waterstone, J. M., 48, 65 Watkinson, S. C., 34, 60 Watson, A. T., 119, 152 Weathers, P. J., 140, 157 Weaver, L. M., 132, 156 Weber, A., 125, 164 Weber, K., 88, 110 Wederoth, I., 134, 136, 148, 164 Weeden, N. F., 124, 164 Weeds, A. G., 71, 106 Weerakoon, N. D., 74, 100, 104 Weisburg, W. G., 44, 59 Weiss, M., 41, 63 Welsh, J., 50, 67 Wenderoth, I., 133, 134, 136, 148, 164, 165 Wendt, U. A., 133, 134, 136, 148, 165 Wendt, U. K., 134, 136, 148, 164 Wenk-Siefert, I., 43, 58 Werck-Reichhart, D., 7, 8, 13, 14, 19, 21, 22, 25, 28, 32 Westhoff, P., 126, 154 Wheeler, Q. D., 41, 64 White, T. J., 39, 40, 43, 54, 59 Whitehead, D., 42, 65 Whitehead, M., 42, 65 Whittaker, S. L., 55, 67 Whittington, A. T., 72, 96, 110 Wick, S. M., 71, 84, 85, 86, 87, 88, 90, 101, 102, 103, 104, 110 Wiemken, A., 54, 65 Wijesekera, H. T. R., 36, 67 Wijesundera, R. L. C., 36, 67 Wildung, M. R., 140, 158 Williams, J. G. K., 50, 67

Willmitzer, L., 128, 157 Wilson, A. C., 49, 59 Wingfield, B. D., 40, 46, 60, 64 Wingfield, M. J., 40, 46, 60, 64 Winka, K., 37, 61 Winter, D., 72, 110 Wipf, D., 50, 59 Wohlleben, W., 124, 157 Wood, J. W., 82, 101 Woodall, J., 119, 120, 154, 165 Woollard, A., 87, 108 Wo¨ stemeyer, J., 40, 44, 66 Wozniak, M., 83, 107 Wray, J. C., 118, 163 Wray, J. G., 118, 153 Wray, J. L., 118, 165 Wright, D. P., 135, 165 Wu, R., 71, 106 Wungsintaweekul, J., 142, 154, 156, 159, 161 Wymer, C. L., 74, 110 Wysocka-Diller, J., 92, 97, 103 X Xia, G. X., 89, 96, 108 Xiang, C. B., 24, 32 Xu, F., 11, 21, 31 Xu, F. X., 19, 32 Xu, J., 7, 11, 31 Y Yamada, K., 14, 32 Yamaguchi-Shinozaki, K., 11, 30 Yamaya, T., 119, 120, 122, 123, 155, 156, 164 Yanagi, S. O., 49, 65 Yang, K. Y., 11, 32 Yang, M., 79, 80, 97, 104, 110 Ye, Z.-H., 74, 94, 101 Yokosawa, R., 38, 45, 62 Yokota, A., 34, 65 Yokota, E., 71, 110 Yoshimura, K., 7, 32 You, R., 91, 103 Young, R., 54, 59 Z Zabeau, M., 50, 66 Zaki, M. A. M., 87, 110 Zandomeni, K., 76, 110 Zanetti, G., 121, 152 Zeidler, J., 139, 140, 141, 162 Zenk, M. H., 142, 143, 144, 146, 151, 154, 156, 159, 160, 161, 162 Zervakis, G. I., 42, 54, 56, 57, 67

AUTHOR INDEX Zettler, E., 54, 58 Zhang, D., 71, 88, 110 Zhang, K., 86, 110 Zhang, M., 71, 106 Zhang, Q., 11, 21, 31 Zhao, J. P., 91, 110 Zhao, L., 81, 110

Zhong, R., 74, 94, 101 Zhu, T., 145, 146, 158 Zimmer, W., 140, 160 Zimmermann, I., 92, 97, 105 Zonia, L. E., 72, 109 Zrenner, R., 128, 155

181

SUBJECT INDEX

A ABC. See Adenosine triphosphate ABP. See Proteins Actin, 40, 43 -depolymerising factor, 71–2 MFs and, 71 nucleation, 72 polymerisation, 83 roles of, 70, 88–9 Adenosine triphosphate (ATP), 115 binding cassette (ABC), 9 AFLPs. See Amplification fragment length polymorphisms Allelochemicals, 15 Alopecurus myosuroides (black-grass), 5, 10 Ammonium assimilation of, 114, 116, 120, 124 incorporation of, 118 Amplification fragment length polymorphisms (AFLPs), 50 Amplification of repetitive sequences (rep-PCR), 50 Animals, 75 alkenals and, 26 xenobiotics and, 5 Annexins, 71–2 Antioxidant responsive elements (AREs), 24 Arabidopsis, 11, 14, 15 components of, 17, 27 genome, 23, 76 mutants in, 75, 89–91 PLD delta, 74 proteins in, 72 thaliana, 9, 120, 121–5, 140 AREs. See Antioxidant responsive elements Armillaria, 42, 45–6 Ascochyta species, 48–9 Ascomycetes, 34–5, 37, 44 Aspergillus, 35, 37 ATP. See Adenosine triphosphate Auxins, 76–7, 99 B Barley, 13, 122 Basidiomycetes, 34, 44–5 Battarraea (stilt puffballs), 45 Bdl. See mutants Beauveria, 51–3 bassiana, 50 brongniartii, 52

Benoxacor, 13, 14 Black-grass (Alopecurus myosuroides), 5, 10 Brassica pleracea, 130 Brefeldin A, 87 C Calvin cycle, 114, 116, 148 carbon metabolism and, 126–8 enzyme reactions of, 126–8 evolutionary aspects of, 126–8 interactions/regulation of, 128 Cambrian era, 43 Capsicum annuum (C. annuum), 140, 142 Carbon metabolism, 113–14, 150 Calvin cycle and, 126–8 glycolysis and, 128–32 OPPP and, 132–7 terpenoid biosynthesis and, 137–48 Catharanthus roseus, 142 CDK. See Cyclin-dependent kinase Cell(s). See also Plane of cell division -cell signaling, 79–81 cortex, 87 cycle genes, 84–5 daughter, 70, 81 elongation, 78 guard mother, 72–3, 79, 81 meristemoid mother, 73 signaling, 15–20 Silvetia compressa, 82 subsidiary mother, 72–3 cGMP. See Cyclic guanosine monophosphate Chloroacetanilides, 9, 13–14 Chloroplasts, 120, 140 Chytridiomycetes, 34, 44 cMEPP. See 2C-Methyl-D-erythritol-2, 4-cyclodiphosphate Colchicine, 76 Colletotrichum, 45 Cordyceps, 57 Crops. See also specific crops GSTs and, 5 safeners and cereal, 3, 11–14 Cyanobacteria, 114 Cyclic guanosine monophosphate (cGMP), 83 Cyclin-dependent kinase (CDK), 84–5 Cythochrome P450 (CYP), 8, 14, 15 Cytochalasin B/D, 83

184

SUBJECT INDEX

Cytokinesis, 88–90 onset of, 86 process of, 70, 74, 85 Cytokinins, 78–9 Cytoskeletal proteins, 71–4 Cytoskeleton. See also Plane of cell division PGRs and, 70, 75, 76–9, 99, 100 roles of, 69, 83–4 structures, 70, 71 Cytotoxicity, 5 D Dehydroascorbate reductases (DHARs), 6–7 At, 12, 17, 22 functions of, 10 roles of, 3, 10–11 safener-induction of, 12, 16, 22 Deuteromycota, 35 Devonian period, 43 DHARs. See Dehydroascorbate reductases Dichlormid, 12, 13, 17 Dictionary of the Fungi (Ainsworth & Bisby), 44–5 Dimethylallyl pyrophosphate (DMAPP), 137, 143 4-Diphosphocytidyl-2C-methyl-D-erythritol kinase, 142 synthase, 141–2 DMAPP. See Dimethylallyl pyrophosphate DNA fingerprinting techniques, 50 fungal systematics and, 34, 37–40 hybridisation values, 38 regions, 39–40, 54–5 sequencing, 34, 38 DXP (1-deoxy-D-xylulose 5-phosphate) pathway, 138–9, 139–40, 144–7, 149 synthase1, 140–1 DXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase), 141 Dyneins, 71 E EMBL, 56 Enzyme(s). See also Xenomes activators of, 3, 26 calvin cycle/reactions of, 126–8 GSH, 5 iso, 130 xenobiotic-detoxifying, 2 Escherichia coli, 140 ESTs. See Expressed sequence tags Ethylene, 78 European cockchafer (Melolontha melolontha), 52 Expressed sequence tags (ESTs), 9, 124

F FBA. See Fructose-1, 6-bisphosphate aldolase FBPase. See Fructose bisphosphatase Fenchlorazole ethyl, 12, 14, 21 Fructose bisphosphatase (FBPase), 127 Fructose-1, 6-bisphosphate aldolase (FBA), 127 Fungal systematics below species level in, 49–53 biogeography and, 56–7 constraints in, 54–7 cryptic taxa in, 53–4 data analysis in, 41–2 dating radiations in, 41–2 development of molecular, 37–42 DNA and, 34, 37–40 higher level, 44–5 historical perspective of, 37–9 introduction to, 34–7 phylogeney reconstruction in, 43–4 reference materials for, 55–6 sequence evolution rates in, 54–5 species level in, 45–9 tools in, 37–8 Fungi anamorphic (imperfect), 35 concept of, 34 fossil, 41–2 kingdom, 34–6 life cycles of, 34–5 sister groups of, 44–5 teleomorph (perfect), 35 Fusarium, 15 characteristics, 36–7 oxysporum, 36 G GA, 77–8 Ganoderma, 36 boninense, 51, 52 Gene(s) ALF1/4, 76–7 Arabidopsis, 23, 76 AtKTN1, 74 AXR6, 77 elongation factor, 40, 43 GLU1/2, 122 GNOM, 89 mating type locus, 48 migration, 115–16 plane of cell division and, 93, 94–8, 99 regulation, 24 SDD1, 80 STUD, 92 TAN, 89–90 Genistein, 84 Glomerales, 44

SUBJECT INDEX Glomeromycetes, 34, 44 Glucose-6-phosphate (Glu6P), 132 Glucose-6-phosphate dehydrogenase (G6PDH), 118, 134–5, 136–7 Glucose-6-phosphate isomerase (G6PI), 131 Glucosyltransferases, type 1 (GTs), 8–9 Glutamate synthase (GOGAT) Fd-, 119, 121–3 NADH, 121, 123–4 phylogenetic tree of, 124–5 Glutamine synthetase (GS) forms of, 124–5 isoforms, 119–20 role of, 118–21, 2, 120–1 Glutaredoxins, 11 Glutathione (GSH) -binding domain, 7 concentrations of, 5 enzymes, 5 production of, 2 roles of, 2–4, 6–11 xenobiotics with, 2 Glutathione peroxidases (GPOXs), 3, 7 active, 3, 21–2 role of, 9–10 Glutathione transferases (GSTs) At, 17, 18 classes of, 6, 17, 18 inducibility of, 11 oxidative stress and, 3, 9–11 PCR of, 17, 18 production of, 2 roles of, 6–11 safener enhancement of, 13–14, 20–1 theta (GSTTs), 3, 6–7 xenobiotics with, 2, 7–9, 12, 19–20 zeta (GSTZs), 3, 6–7 Glutathione transferases, lambda (GSTLs), 10 multifunctional, 3, 6–7 safener-induction of, 12, 16, 22 Glutathione transferases, phi (GSTFs), 3, 6–7 dominance of, 13–14 expression of, 21 inducible, 21–2 Zm, 13 Glutathione transferases, tau (GSTUs), 3, 6–7 expression of, 21 organization of, 13–14 Glycolysis, 114 carbon metabolism and, 128–32 evolutionary aspects of, 129, 131–2 localisation in, 128–31 GMC. See Guard mother cells GOGAT. See Glutamate synthase Golgi bodies, 34 GPOXs. See Glutathione peroxidases

185

GS. See Glutamine synthetase GSH. See Glutathione G6PDH. See Glucose-6-phosphate dehydrogenase G6PI. See Glucose-6-phosphate isomerase GSTFs. See Glutathione transferases, phi GSTLs. See Glutathione transferases, lambda GSTs. See Glutathione transferases GSTTs. See Glutathione transferases GSTUs. See Glutathione transferases, tau GSTZs. See Glutathione transferases GTPase. See Guanosine triphosphatase GTs. See Glucosyltransferases, type 1 Guanosine triphosphatase (GTPase), 87 Guard mother cell (GMC), 72–3, 79, 81 H HDS. See 4-Hydroxy-3-methylbut-2-enyl diphosphate synthase Herbicide antidotes. See Safeners Herbicides. See also Chloroacetanilides; Safeners classes of, 5 detoxification of, 11–13 photobleaching, 3, 4–5 High-performance liquid chromatography (HPLC), 16 HMBPP. See 1-Hydroxy-2-methyl-2-(E)butenyl 4-diphosphate HMG. See Proteins Holomorph, 35 Hordeum vulgare (H. vulgare), 118, 123 HPLC. See High-performance liquid chromatography H-site. See Hydrophobic ligand-binding site H2O2. See Hydrogen peroxide Hydnellum species, 46–48 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP), 143 4-Hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS), 143 Hydrogen peroxide (H2O2), 2, 3 Hydrophobic ligand-binding site (H-site), 7 Hydroxyl ions (OH ), 2 I IDDS. See Isopentenyl/dimethylallyl diphosphate synthase Integrins, 75–6 International Code of Botanical Nomeclature, 35 Inter-transcribed spacer (ITS), 40, 45, 47, 48 Isopentenyl pyrophosphate (IPP), 137, 143 Isopentenyl/dimethylallyl diphosphate synthase (IDDS), 143–4 Italy, 52

186

SUBJECT INDEX K

Kinesins, 71 L Lanthanum chloride, 82 Latrunculin B, 83 Leucine-rich repeat (LRR), 80 Lichens, 37 Light, 75 Lolium rigidum, 76 LRR. See Leucine-rich repeat Lupin hypocotyls, 78 Lycoperdon (puffballs) M Maize GSTs and, 5 mutants in, 75, 89, 90 safeners and, 5, 12, 13–14 Malaysia, 52 MAPs. See MT-associated proteins Mass spectrometry (MS), 16 Mating type locus gene (MAT), 48 MECS. See 2C-Methyl-D-erythritol 2,4-cyclodiphosphate Mentha piperita, 140, 141 MEP. See 2-C-Methyl-D-erythritol 4-phosphate Meristemoids, 73, 80–1 Messenger RNAs (mRNAs), 11, 17 Metabolites, secondary, 36–7 Metarhizium, 50–3 flavoviride, 51 2C-Methyl-D-erythritol 2, 4-cyclodiphosphate reductase, 143 synthase (MECS), 142 2-C-Methyl-D-erythritol 4-phosphate (MEP), 141 2C-Methyl-D-erythritol-2, 4-cyclodiphosphate (cMEPP), 142 Mevalonic acid (MVA), 137, 138–9, 144–7, 149 MHR. See Multiple herbicide resistance Microfilaments (MFs), 71 Microgametogenesis, 70 Microtubules (MTs), 100 cortical, 74 organization of, 72 role of, 70 transverse alignment of, 90 Mitochondria, 34 MONOPTEROS (MP), 77 Morphogenesis, 70 Mortierellales, 44 MP. See MONOPTEROS mRNAs. See Messenger RNAs

MT-associated proteins (MAPs), 72 MT-organising centres (MTOCs), 72 MTs. See Microtubules Mucorales, 44 Multiple herbicide resistance (MHR), 5, 10 Mushrooms, 44–5 Mutants amp1, 79 bodenlos, 75 bodenlos (bdl), 75 dcd, 89 discordia1/2, 88 fass, 77, 90, 99 flp (four lips), 79–80 gls, 121 maize, 75, 89, 90 in Arabidopsis, 75, 89–91 sdd1-1 (stomatal density and distribution 1-1), 79–80 sidecar pollen (scp), 92 tmm (too many mouths), 79–81 MVA. See Mevalonic acid Mycology, 41–2 Myosins, 71 N NADP-dependent nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (NGA3PDH), 129 Narcissus pseudonarcissus, 142 NCBI, 56 NGA3PDH. See NADP-dependent nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase Nicotiana benthamiana, 143 plumbaginifolia, 77–8 tabacum, 118 Nitrate reductase (NR), 125 Nitrite reductase (NiR) forms of, 124, 125 nitrogen and, 118, 119 role of, 117, 118, 119 Nitrogen, 150 assimilation/evolution of, 116–26 GS and, 118–21 metabolism, 113–50 NiR and, 118, 119 Nostoc punctiforme, 115 NR. See Nitrate reductase O O 2. See Superoxide Octopine synthase (ocs), 24 OH . See Hydroxyl ions Oomycetes, 34, 44

SUBJECT INDEX Oxidative pentose-phosphate pathway (OPPP), 116, 148–9 carbon metabolism and, 132–7 evolution of, 136–7 functions of, 132–3 localisation of, 133–4 multiple isoforms of, 134 nonoxidative steps of, 135–6 oxidative steps of, 134–5 Oxygen singlet (1O2) P PAA. See Population aggregation analysis Paecilomyces, 35 Papau New Guinea, 52 Parmelia sulcata, 53 PCA. See Principal component analysis PCR. See Polymerase chain reaction Pelvetia compressa, 82, 83 fastigiata, 83 Penicillium, 36–7 Pentose-phosphate pathway, 114 Permian period, 44 PFK. See Pyrophosphate-dependent phosphofructokinase PGK. See Phosphoglycerate kinase PGM. See Phosphoglyceromutase PGRs. See Plant growth regulators Phragmoplast, 71, 88–90 Phoma, 35, 48–9 6-Phosphogluconate dehydrogenase (6PGDH), 118 Phosphoglycerate kinase (PGK), 126–7 Phosphoglyceromutase (PGM), 130 Phospholipase D (PLD), 74 Phosphoribulokinase (PRK), 127 Photo-polarisation, 82–4 Photorespiration, 120 Phragmosome, 86 Phylogenetic species concept (PSC), 41–2 Phytophthora, 34, 42 Phytotoxicity, 4 Phytotoxins, 15 Pisum sativum (P. sativum), 118, 124 Plane of cell division. See also Cytoskeleton cell cycle genes and, 84–5 cell-cell signaling and, 79–81 consequences, 90–3 coordination of, 91–3 daughter cells and, 70, 81 definition of, 75–85 genes and, 93, 94–8, 99 introduction to, 70–1 light perception and, 81–5 mechanical stress and, 75–6 PGRs and, 70, 75, 76–9, 99, 100

187

PPB and, 85–8, 99 regulation of, 69–100 Plant(s). See also Glutathione; Glutathione transferases allelopathic, 15 land, 114–15 monocotyledonous, 15 oxidative stress to, 2–5 pathogens, 34 safener/signal transduction in, 23–7 signaling pathways, 12, 17, 18 xenobiotics and, 2–5 xenome, inducible, 15–16 Plant growth regulators (PGRs), 75, 76–79, 99, 100 Plastid(s) endosymbiotic origin of, 114–15 interactions, 136 introduction to, 114–16 proteins, nuclear-encoded, 115–16 role of, 116, 117 PLD. See Phospholipase D Pneumocystis, 34, 44 Polar secretion, 84 Polymerase chain reaction (PCR) of GSTs, 17, 18 introduction of, 38–9 primers, 47 studies, 22 variable number tandem repeat (VNTR-PCR), 50 Polypeptides, 119 Population aggregation analysis (PAA), 42 PP2A. See Type 2A protein phosphatases Pre-prophase band (PPB) plane of cell division and, 85–8, 99 position of, 70 structure of, 85–6 Principal component analysis (PCA), 42 PRK. See Phosphoribulokinase Profilins, 71 Protein(s) ABC, 14, 15 actin-binding (ABP), 71–2 BODENLOS, 77 BRK, 73 cytoskeletal, 71–4 FASS, 77, 99 green fluorescent, 120 GT, 14, 15 hign mobility group (HMG), 48 HSPC300, 72 In2.1, 22 LRR-containing receptor-like, 80 motor, 71 nuclear-encoded plastidic, 115–16 phosphorylation, 84 plant, 3, 6–7 in Arabidopsis, 72

188

SUBJECT INDEX

Protein(s) (cont. )

TMM, 80 tyrosine kinases (PTKs), 84 Protoplasts, 75–6 PSC. See Phylogenetic species concept PTKs. See Proteins Puffballs (Lycoperdon), 45 Pyrophosphate-dependent phosphofructokinase (PFK), 129 Pyruvate, 148 Pythium, 34 R Random amplification of polymorphic (RAPD), 50–2 RAPD. See Random amplification of polymorphic Reactive oxygen intermediates (ROIs), 2–4, 5 rep-PCR. See Amplification of repetitive sequences Restriction fragment length polymorphisms (RFLPs), 38, 40, 54–5 Rhizoctonia solani, 45 Rhizoids, 81–4 Ribose-5-phosphate isomerase (RPI), 127 Ribulose-5-phosphate 3-epimerase (RPE), 126–7, 135 Rice, 12 Ricinus communis (R. communis), 130–1 RNA r, 43 ribosomal, 38 ROIs. See Reactive oxygen intermediates RPE. See Ribulose-5-phosphate 3-epimerase RPI. See Ribose-5-phosphate isomerase S Saccharomyces pombe, 87 Safener(s) benzene-sulphonamide, 22 cereal crops and, 3, 11–14 cloquintocet mexyl, 15–16, 22 compounds, 12, 19–20 coordinated upregulation and, 16, 22–3 description/definition of, 11, 12, 19 enhancement of GSTs, 13–14, 20–1 herbicide, 4, 15–20, 16, 18 -induction of DHARs, 12, 16, 22 -induction of GSTLs, 12, 16, 22 maize and, 5, 12, 13–14 oxidative stress and, 3, 12, 16, 20–3 signal transduction and, 23–7 xenomes and, 8, 14 Saprolegnia, 34

SBP. See Sedoheptulose-1, 7-bisphosphatase scp. See Mutants SDS-PAGE. See Sodium dodecyl sulphate polyacrylamide gel electrophoresis Sedoheptulose-1, 7-bisphosphatase (SBP), 127 S-glutathionylation, 13 Silvetia compressa, 82 SMC. See Subsidiary mother cell Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), 19 Sorghum, 5, 13 Spain, 53 Spinacia oleracea (S. oleracea), 118, 126 Stevia rebaudiana, 140, 141 Stilt puffballs (Battarraea/Tulostoma), 45 Stomata, 80–1 Stomatal initials, 73 Straminipila, 44 Streptomycetes, 137, 140 Stroma, 114 Subsidiary mother cell (SMC), 72–3 Superoxide (O2), 2, 4 Synechococcus leopoliensis, 140 Synechocystis, 115 T Taxol, 74 Terpenoid biosynthesis, 114, 116, 149 carbon metabolism and, 137–48, 138–9 DXP pathway for, 138–9, 139–40, 144–7, 145, 149 DXP synthase1 for, 140–1 other pathways for, 127, 129, 147–8 Thallus, 81 TKL. See Transketolase Toadstools, 44–5 Tobacco, 10 cell cultures, 14 protoplasts, 75 Spcdc25, 85 TBY-2, 74, 86 Tomato, 10 TPI. See Triose phosphate isomerase Transketolase (TKL), 126–7 Trichoderma viride, 36 Triose phosphate isomerase (TPI), 127 Triticum. See also Wheat aestivum, 19–20 precursors, 19 tauschii, 13, 19 turgidum, 19–20 urartu, 19–20

SUBJECT INDEX Tubulin a, 43 b, 43 gene products, 71 MTs and, 71–4 Tulostoma (stilt puffballs), 45 Type 2A protein phosphatases (PP2A), 91 U Ultraviolet (UV), 16 United Kingdom, 53, 56 Unweighted pair-group arithmetic average (UPGMA), 41 V Verticillium lecanjii, 57 Virus-induced gene silencing (VIGS), 143 VNTR-PCR. See Polymerase chain reaction W Weeds, 11 Wheat, 12, 13–14. See also Triticum bread, 19 precursors, 19 seedlings, 15–16

X Xenobiotic responsive elements (XREs), 24 Xenobiotics animals and, 5 damage caused by, 5 GSH and, 2 GSTs and, 2, 7–9, 12, 19–20 plants and, 2–5 synthetic, 2 Xenome(s), 2 components of, 8, 13 definition of, 7–8 metabolism phases of, 8–9 plants, inducible, 15–16 safeners and, 8, 14 XREs. See Xenobiotic responsive elements Z Zea mays, 118, 124 Zygomycetes, 34, 40, 44 Zygotes fucoid, 75, 81–4, 100 Pelvetia compressa, 82, 83 Pelvetia fastigiata, 83

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CONTRIBUTORS TO VOLUME 42 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii

CONTENTS OF VOLUMES 30–41 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix

Chemical Manipulation of Antioxidant Defences in Plants ROBERT EDWARDS, MELISSA BRAZIER-HICKS, DAVID P. DIXON AND IAN CUMMINS I. II. III. IV. V. VI. VII.

Oxidative Stress In Plants and Exposure to Xenobiotics . . . . . . . . . . . . . . . . . . . . . A Central Protective Role for GSH and GSTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Safeners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Herbicide Safeners, the Xenome, and Cell Signalling . . . . . . . . . . . . . . . . . . . . . . . . Safeners and Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Safeners and Signal Transduction in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future Perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 6 11 15 20 23 27 27 27

The Impact of Molecular Data in Fungal Systematics P. D. BRIDGE, B. M. SPOONER AND P. J. ROBERTS I. II. III. IV. V. VI. VII. VIII. IX.

Introduction to Fungal Systematics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Development of Fungal Molecular Systematics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phylogeny Reconstruction and Dating Radiations . . . . . . . . . . . . . . . . . . . . . . . . . . Higher Level Systematics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Species Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Below Species Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cryptic Systematics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Constraints to Molecular Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

34 38 43 44 46 50 53 54 58 58

vi

CONTENTS

Cytoskeletal Regulation of the Plane of Cell Division: An Essential Component of Plant Development and Reproduction HILARY J. ROGERS I. II. III. IV. V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . How Is the Plane of Cell Division Defined? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Implementation of the Decision on the Orientation of Division. . . . . . . . . . . . . . . . Consequences of the Orientation of a Cell Division . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

70 75 85 90 93 100 101

Nitrogen and Carbon Metabolism in Plastids: Evolution, Integration, and Coordination with Reactions in the Cytosol ALYSON K. TOBIN AND CAROLINE G. BOWSHER I. II. III. IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nitrogen Assimilation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carbon Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

114 116 126 148 151

AUTHOR INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

167

SUBJECT INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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