Introduction Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol151.Issue1.2000:

Plant Science 151 2000 9 – 18 Repression of cystathionine g-synthase in Arabidopsis thaliana produces partial methionine auxotrophy and developmental abnormalities Jungsup Kim, Thomas Leustek Biotechnology Center for Agriculture and the En6ironment, Rutgers Uni6ersity, Cook College, 59 Dudley Road, New Brunswick, NJ 08901 - 8520 , USA Received 2 July 1999; received in revised form 1 September 1999; accepted 1 September 1999 Abstract Cystathionine g-synthase CGS, a key enzyme in methionine biosynthesis, was repressed in transgenic Arabidopsis thaliana by antisense expression of CGS RNA. CGS activity was reduced by 5 – 9-fold in the antisense plants resulting in severe growth stunting, morphological abnormalities and an inability to flower. Feeding the plants methionine Met or Met metabolites reversed the morphological effects of CGS repression. There was little change in the content of free Met and S-methylmethionine despite the need for exogenously applied Met for growth. The overall amino acid content was significantly increased. The CGS antisense transgene is inherited as a single recessive locus. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords : Antisense repression; Arabidopsis thaliana; Cystathionine g-synthase; Methionine biosynthesis www.elsevier.comlocateplantsci

1. Introduction

The sulfur containing amino acid methionine Met is a fundamental metabolite in plant cells. It is both a protein constituent and the precursor of S-adenosyl- L -methionine SAM the primary bio- logical methyl-group donor. Met is of equal im- portance to animals that lack the ability to synthesize this amino acid and must obtain it from their diet or from enteric bacteria. The nutritional value of some crops, legumes in particular, is limited by low Met content [1]. Despite its bio- chemical and agronomic importance the regula- tion of Met synthesis in higher plants is not well understood. Met is a 4-carbon amino acid synthesized from independently derived components Fig. 1. The sulfur atom is derived from Cys. The carbon skele- ton is derived from Asp as are the amino acids Lys and Thr. The immediate precursor of both Met and Thr is O-phosphohomoserine OPH. The first Met-specific reaction is catalyzed by cys- tathionine g-synthase CGS which condenses Cys and OPH to form cystathionine. Next, cystathion- ine b-lyase carries out b-cleavage to form homo- cysteine Hcy. Met is produced by transmethyl- ation of homocysteine. Aside from its incorpora- tion into proteins Met is the precursor of S- methylmethionine SMM, a compound that is an intermediate in the synthesis of dimethylsulfonio- propionate DMSP in some angiosperms [2,3], and SAM, which is synthesized by Met adenosyl- transferase SAM synthetase. The Met pathway Abbre6iations : ACC, 1-aminocyclopropane-1-carboxylic acid; CGS, cystathionine g-synthase; DMS, dimethylsulfide; DMSP, dimethylsul- foniopropionate; Hcy, homocysteine; Kan ® , kanamycin resistant; MTA, 5-methylthioadenosine; MTHB, 4-methylthio-2-hydroxy bu- tyric acid; MTOB, 4-methylthio-2-oxobutanoic acid; OPH, O-phos- phohomoserine; PITC, phenylisothiocyanate; SAH, S-adenosyl- L -homocysteine; SAM, S-adenosyl- L -methionine; SAT, serine acetyl- transferase; SMM, S-methylmethionine; TS, threonine synthase. Corresponding author. Tel.: + 1-732-9320312, ext. 326; fax: + 1- 732-9328165, ext. 326. E-mail address : leustekaesop.rutgers.edu T. Leustek 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 1 8 8 - 0 enzymes are distributed between plastids and the cytosol [4]. Plastids contain all the Asp-family enzymes, the complete Cys pathway, CGS and cystathionine b-lyase. Met synthase and SAM syn- thetase are exclusively cytosolic as are the enzymes for SMM synthesis [5]. Met synthesis is regulated at multiple levels. A general mechanism for control of the Asp-family amino acids centers on feedback inhibition of Asp kinase AK, the first enzyme of the Asp pathway, by Lys, Thr, and SAM [6]. Combined treatment with Lys and Thr is herbicidal because they re- press the Asp pathway blocking the synthesis of the carbon skeleton and causing Met starvation [7]. A Met-specific control mechanism centers on the competition between CGS and Thr synthase TS for their common substrate OPH. TS activity is stimulated by SAM and it has a much higher affinity for OPH than does CGS. Thus, it has been proposed that CGS may compete poorly for OPH when Met hence SAM is abundant [8,9]. By contrast, when Met is limiting and TS less active CGS has a greater ability to compete for OPH. There is also evidence that when Met is limiting CGS expression is induced. For example, com- bined treatment with Thr and Lys causes CGS activity to increase, whereas Met treatment causes it to decrease [10,11]. With the recent cloning of the CGS cDNA from Arabidopsis thaliana [12] it became possible to study its function in transgenic plants. Repression of CGS activity was found to limit the ability of A. thaliana to grow autonomously without exoge- nous application of Met. The CGS-repressed plants show an abnormal morphology that is in- herited as a recessive trait.

2. Materials and methods