Elsevier

Plant Science

Volume 151, Issue 1, 7 February 2000, Pages 9-18
Plant Science

Repression of cystathionine γ-synthase in Arabidopsis thaliana produces partial methionine auxotrophy and developmental abnormalities

https://doi.org/10.1016/S0168-9452(99)00188-0Get rights and content

Abstract

Cystathionine γ-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.

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 biological methyl-group donor. Met is of equal importance 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 biochemical and agronomic importance the regulation 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 skeleton 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 cystathionine γ-synthase (CGS) which condenses Cys and OPH to form cystathionine. Next, cystathionine β-lyase carries out β-cleavage to form homocysteine (Hcy). Met is produced by transmethylation of homocysteine. Aside from its incorporation into proteins Met is the precursor of S-methylmethionine (SMM), a compound that is an intermediate in the synthesis of dimethylsulfoniopropionate (DMSP) in some angiosperms [2], [3], and SAM, which is synthesized by Met adenosyltransferase (SAM synthetase). The Met pathway enzymes are distributed between plastids and the cytosol [4]. Plastids contain all the Asp-family enzymes, the complete Cys pathway, CGS and cystathionine β-lyase. Met synthase and SAM synthetase 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 repress 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, combined 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 exogenous application of Met. The CGS-repressed plants show an abnormal morphology that is inherited as a recessive trait.

Section snippets

Preparation of antibodies against recombinant CGS

Recombinant A. thaliana CGS was synthesized as an S-TAG and hexa-His fusion protein expressed from vector pET30c (Novagen). A 1.4 kbp XhoI fragment from the CGS cDNA [12] (GenBank Accession Number U43709) was cloned into pET30c to produce the pET-CGS construct which was used to transform Escherichia coli strain BL21(DE3)pLysS (Novagen). Transformants were selected on LB medium with 40 μg/ml chloramphenicol and 30 μg/ml kanamycin. The culture was grown in liquid LB medium with the antibiotics at

Results

Six KanR plants were isolated from a transformation with the CGS[−] vector. All were confirmed to carry the CGS[−] transgene construct using a tissue PCR method (not shown). Four of the transformants, analyzed by genomic DNA blotting with the CGS cDNA as a probe showed a pattern of hybridization indicative of having arisen through independent single integration events. The transformants were analyzed with HindIII or BamHI, enzymes that produce, respectively, either approximately 5 or

Discussion

Gene families encode many amino acid biosynthetic enzymes in plants. CGS is unusual in that it exists as a single copy gene in A. thaliana [12]. Being a single copy gene there was a greater likelihood that its expression could be effectively repressed using an antisense RNA method. Indeed, all of the six independently isolated transgenic A. thaliana lines transformed with the CGS[−] construct showed pronounced affects on growth and a 5–9-fold reduction in the level of CGS and CGS enzyme

Acknowledgements

This work was supported by the National Science Foundation (Grant #MCB-9728661). We wish to thank Dr Satoshi Naito for providing mto1 seed and Dr Andrew Hanson for many helpful discussions.

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