The committing step in Met and S-adenosyl-l-Met (SAM) synthesis is catalyzed by cystathionine ␥-synthase (CGS). Transgenic Arabidopsis plants overexpressing CGS under control of the cauliflower mosaic virus 35S promoter show increased soluble Met and its metabolite S-methyl-Met, but only at specific stages of development. The highest level of Met and S-methyl-Met was observed in seedling tissues and in flowers, siliques, and roots of mature plants where they accumulate 8-to 20-fold above wild type, whereas the level in mature leaves and other tissues is no greater than wild type. CGS-overexpressing seedlings are resistant to ethionine, a toxic Met analog. With these properties the transgenic lines resemble mto1, an Arabidopsis, CGS-mutant inactivated in the autogenous control mechanism for Met-dependent downregulation of CGS expression. However, wild-type CGS was overexpressed in the transgenic plants, indicating that autogenous control can be overcome by increasing the level of CGS mRNA through transcriptional control. Several of the transgenic lines show silencing of CGS resulting in deformed plants with a reduced capacity for reproductive growth. Exogenous feeding of Met to the most severely affected plants partially restores their growth. Similar morphological deformities are observed in plants cosuppressed for SAM synthetase, even though such plants accumulate 250-fold more soluble Met than wild type and they overexpress CGS. The results suggest that the abnormalities associated with CGS and SAM synthetase silencing are due in part to a reduced ability to produce SAM and that SAM may be a regulator of CGS expression.Met is derived from Asp as are the amino acids Lys, Thr, and Ile. The committing step in Met synthesis occurs when the side chain of O-phosphohomoserine (OPH) condenses with the thiol group of Cys to form cystathionine (Fig. 1), an irreversible reaction catalyzed by CGS (EC 4.2.99.9). Cystathionine is cleaved to form homocysteine, which is then methylated with 5-methyltetrahydrofolate to form Met. The major metabolic fates of Met include its incorporation into protein, adenosylation to form SAM, and methylation to form S-methyl Met (SMM) (Fig. 1).CGS competes with TS for OPH, their common substrate. Thus, TS may exert some control over the rate with which OPH is channeled toward Met (Bartlem et al., 2000; Fig. 1). TS is allostrically regulated by SAM (Curien et al., 1998) suggesting that Met synthesis could influence TS activity. Even so, several lines of evidence indicate that CGS controls the rate of Met synthesis. CGS activity decreases when Met is fed to the aquatic angiosperm Lemna paucicostata and increases when Met synthesis is blocked by inhibition of aspartokinase, the first enzyme in the biosynthesis of the Asp family of amino acids (Thompson et al., 1982). In the Arabidopsis mutant mto1, CGS is overexpressed, resulting in overaccumulation of soluble Met (Inaba et al., 1994;Chiba et al., 1999). Finally, antisense-RNA repression of CGS expression results in growth deformities stemming ...

In the S-methylmethionine cycle of plants, homocysteine methyltransferase (HMT) catalyzes the formation of two molecules of methionine from homocysteine and S-methylmethionine, and methionine methyltransferase (MMT) catalyzes the formation of methionine from S-methylmethionine using S-adenosylmethionine as a methyl group donor. Somewhat surprisingly, two independently isolated knockdown mutations of HMT2 (At3g63250), one of three Arabidopsis thaliana genes encoding homocysteine methyltransferase, increased free methionine abundance in seeds. Crosses and flower stalk grafting experiments demonstrate that the maternal genotype at the top of the flower stalk determines the seed S-methylmethionine and methionine phenotype of hmt2 mutants. Uptake, transport and inter-conversion of [(13)C]S-methylmethionine and [(13)C]methionine in hmt2, mmt and wild-type plants show that S-methylmethionine is a non-essential intermediate in the movement of methionine from vegetative tissue to the seeds. Together, these results support a model whereby elevated S-methylmethionine in hmt2 vegetative tissue is transported to seeds and either directly or indirectly results in the biosynthesis of additional methionine. Manipulation of the S-methylmethionine cycle may provide a new approach for improving the nutritional value of major grain crops such as rice, as methionine is a limiting essential amino acid for mammalian diets.

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SummaryHomoserine kinase (HSK) produces O-phospho-L-homoserine (HserP) used by cystathionine c-synthase (CGS) for Met synthesis and threonine synthase (TS) for Thr synthesis. The effects of overexpressing Arabidopsis thaliana HSK, CGS, and Escherichia coli TS (eTS), each controlled by the 35S promoter, were compared. The results indicate that in Arabidopsis Hser supply is the major factor limiting the synthesis of HserP, Met and Thr. HSK is not limiting and CGS or TS control the partitioning of HserP. HSK overexpression had no effect on the level of soluble HserP, Met or Thr, however, when treated with Hser these plants produced far more HserP than wild type. Met and Thr also accumulated markedly after Hser treatment but the increase was similar in HSK overexpressing and wild-type plants. CGS overexpression was previously shown to increase Met content, but had no effect on Thr. After Hser treatment Met accumulation increased in CGS-overexpressing plants compared with wild type, whereas HserP declined and Thr was unaffected. Arabidopsis responded differentially to eTS expression depending on the level of the enzyme. At the highest eTS level the Thr content was not increased, but the phenotype was negatively affected and the T1 plants died before reproducing. Comparatively low eTS did not affect phenotype or Thr/Met level, however after Hser treatment HserP and Met accumulation were reduced compared with wild type and Thr was increased slightly. At intermediate eTS activity seedling growth was retarded unless Met was supplied and CGS expression was induced, indicating that eTS limited HserP availability for Met synthesis.