Cystathionine ␥-synthase, the first committed enzyme of methionine biosynthesis in higher plants, is encoded by the CGS1 gene in Arabidopsis thaliana. We have shown previously that the stability of the CGS1 mRNA is negatively regulated in response to methionine application [Chiba, Y., Ishikawa, M., Kijima, F., Tyson, R. H., Kim, J., Yamamoto, A., Nambara, E., Leustek, T., Wallsgrove, R. M. & Naito, S. (1999) Science 286, 1371-1374]. To determine whether methionine itself is the effector of the CGS1 exon 1-mediated posttranscriptional regulation, we carried out transfection experiments. The results suggested that, rather than methionine, Sadenosyl-L-methionine (AdoMet), or one of its metabolites, acts as the effector of this regulation. To further identify the actual effector, we exploited the wheat germ in vitro translation system. The effects of various metabolites and analogs of AdoMet were tested by using RNA carrying a CGS1 exon 1-reporter fusion. These tests identified AdoMet as the effector of this regulation. Sadenosyl-L-ethionine, an analog of AdoMet, also had effector activity. A. thaliana mto1 mutants, which are deficient in this regulation, showed a much reduced response to AdoMet in vitro, with a leaky allele showing a less reduced response. RNA translated in vitro in the presence of AdoMet contained a 5-truncated RNA species, similar to the one that we previously suggested was an in vivo degradation intermediate of CGS1 mRNA. Together, the results show that the basic reactions of CGS1 exon 1-mediated posttranscriptional regulation occur in the wheat germ in vitro translation system, and that AdoMet acts as the effector. C ystathionine ␥-synthase (CGS; EC 4.2.99.9) catalyzes the first committed step of methionine biosynthesis in higher plants (1) (Fig. 1), which is considered to be the key regulatory step in methionine biosynthesis (2-5). Unlike many of the key-step enzymes in metabolic pathways, CGS is not an allosteric enzyme (2). CGS is encoded in Arabidopsis thaliana by the CGS1 gene (gene ID At3g01120, GenBank accession no. AB010888) (6, 7). We have previously shown (8) that CGS1 expression involves feedback regulation at the level of mRNA stability in response to methionine application in vivo. A. thaliana mto1 mutants are deficient in this feedback regulation, and overaccumulate CGS1 mRNA, CGS protein, and soluble methionine. Seven independently isolated mto1 mutants were found to carry single-base changes within the first exon of CGS1, giving rise to amino acid sequence changes (8, 9).Transient and transgenic expression experiments using CGS1 exon 1-reporter fusions (8, 10) showed that the exon 1 coding sequence of CGS1 is necessary and sufficient for its posttranscriptional regulation, in response to exogenous application of methionine. In vitro mutagenesis of CGS1 exon 1 revealed that it is its amino acid sequence that has a role in this regulation. We have identified a stretch of 11-13 amino acid residues, termed the MTO1 region, located Ϸ80 residues from the N terminus of CGS, and coverin...

Cystathionine ␥-synthase (CGS) catalyzes the first committed step of Met biosynthesis in plants. We have previously shown that expression of the gene for CGS is feedback-regulated at the level of mRNA stability, and that the amino acid sequence encoded by the first exon of the CGS gene itself is responsible for the regulation (Chiba, Y., Ishikawa, M., Kijima, F., Tyson, R. H., Kim, J., Yamamoto, A., Nambara, E., Leustek, T., Wallsgrove, R. M., and Naito, S. (1999) Science 286, 1371-1374). To identify the functional region within CGS exon 1, deletion analysis was performed. The results showed that the 41-amino acid region of exon 1 highly conserved among plants is necessary and sufficient for the regulation. Analyses of in vivo and in vitro generated mutations that abolish the regulation identified the functionally important amino acids as 11-13 residues within this conserved region. The importance of these residues was confirmed by deletion analysis within the conserved region. These studies identified the functional region of CGS exon 1 required for the posttranscriptional autoregulation of the CGS gene as (A)RRNCSNIGVAQ(I), with uncertainty of the first and last residues. This sequence is almost perfectly conserved among CGS sequences of higher plants but cannot be found elsewhere in the public databases.