Plant Hormone Protocols

Methods in Molecular Biology

TM

VOLUME 141

Plant Hormone Protocols Edited by

Gregory A. Tucker Jeremy A. Roberts

HUMANA PRESS

Extraction and Purification of Enzymes

1 Extraction and Purification of an Enzyme Potentially Involved in ABA Biosynthesis Gregory A. Tucker, Pete Bass, and Ian Taylor

1. Introduction The advent of genetic engineering has provided a means to manipulate the biosynthesis of plant hormones to the advantage of agriculture. Such manipulation is very dependent on a detailed knowledge of the biosynthetic pathways involved in the production of the hormones, and more precisely about the enzymes involved in this biosynthesis. The knowledge of enzymes assists, but is not always a prerequisite for, the isolation of the genetic material, usually cDNAs, used as the genetic tools to manipulate hormone synthesis. The first such example of the manipulation of hormone biosynthesis by the application of genetic engineering was for the hormone ethene. The biosynthetic pathway for ethene was first described in apples by Adams and Yang (1). Methionine was found to be the precursor of this hormone in plant tissue and is converted via S-adenysyl-methionine (SAM) into the unique compound 1-amino-1-carboxyl cyclopropane (ACC). The conversion of SAM into ACC is carried out by a key biosynthetic enzyme, ACC synthase, and the subsequent conversion of ACC into ethene is carried out by a second enzyme, namely ACC oxidase. These two enzymes From: Methods in Molecular Biology, vol. 141: Plant Hormone Protocols Edited by: G. A. Tucker and J. A. Roberts © Humana Press Inc., Totowa, NJ

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have been extensively studied and cDNAs for both identified, although by completely different methods. ACC oxidase cDNA was initially identified by a process of differential screening of a tomatoripening cDNA library. This technique was used to identify cDNA clones from a library made from ripe tomato, which are exclusively expressed in a ripe fruit but not a green fruit. Since the biosynthesis of ethene was known to be ripening specific, it was postulated that at least some of any clones identified would encode biosynthetic enzymes. The technique identified several clones, one of which, pTOM13, appeared to have expression closely correlated with ethene synthesis. The use of this cDNA to transform tomato plants with an antisense gene resulted in genetically manipulated plants with lowered ethene production and a correspondingly reduced ability to convert ACC to ethene (2). It was thus postulated that the pTOM13 cDNA encoded the enzyme ACC oxidase, a conclusion further supported by the observation that yeast genetically engineered with pTOM13 cDNA acquired the ability to convert ACC to ethene (3). The sequence analysis of the pTOM 13 cDNA suggested a close relationship between the ACC oxidase and flavonone oxidase enzymes. This information enabled Ververdis and John (4) to successfully extract the ACC oxidase enzyme and carry out its purification and characterization. The isolation of a cDNA encoding ACC synthase was achieved by a different route. In this instance the enzyme itself was first extracted and partially purified, and then an antibody was raised against the protein. Using this antibody a cDNA expression library was screened and the corresponding cDNA isolated (5). The identity of the cDNA was confirmed by the expression of ACC synthase activity in Escherichia coli transformed with the cDNA (5). This cDNA has been used to construct an antisense gene, which has in turn been used to silence members of the endogenous ACC synthase gene family and bring about a marked reduction in ethene synthesis in transgenic plants (6). The resultant plants showed