Constitutive Overexpression of Cystathionine γ-Synthase in Arabidopsis Leads to Accumulation of Soluble Methionine andS-Methylmethionine

Abstract

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 ...

A single-nucleotide mutation in a gene encoding S-adenosylmethionine synthetase is associated with methionine over-accumulation phenotype in Arabidopsis thaliana.

“…Developmental regulation of Met over-accumulation in the three mto1-1, mto2-1 and mto3-2 mutants, which all affect different regulatory steps of the Met biosynthetic pathway, strongly suggests that tissue-specific accumulation of Met in older plants is regulated mainly through a translocation process rather than at the level of Met biosynthesis. This is in agreement with several reports indicating that the developmental regulation of Met levels in older plants may involve transport of excess Met from the source rosette tissues to the reproductive sink tissues , Kim et al 2002.…”

Dynamics of methionine biosynthesis

“…The fact that reduced methionine over-accumulation was observed in all three mto mutants, which all affect different regulatory steps of the pathway, suggests that tissue-specific accumulation of methionine may be regulated through a translocation process. This is in agreement with several reports showing that excess methionine may be transported from source leaf tissue to reproductive sink tissues in older plants Naito et al 1994;Kim et al 2002).…”

Methionine catabolism in Arabidopsis cells is initiated by a γ-cleavage process and leads to S -methylcysteine and isoleucine syntheses

“…Also, the AtMGL protein is highly expressed in imbibed seeds, suggesting that transcript accumulation at the dry mature stage is a prerequisite for a rapid production of MGL during the imbibition process. The discrepancy between mRNA and protein levels could not be correlated to any particular status of free Met in the different plant organs, which contain comparable amounts of free Met (20-70 nmol⅐g Ϫ1 FW) (21). Together these data suggest that MGL could participate in the regulation of Met homeostasis in all plant organs and could play an important role in the resumption of cell metabolism during germination.…”