Satoshi Naito

S-adenosyl-l-methionine is an effector in the posttranscriptional autoregulation of the cystathionine γ-synthase gene in Arabidopsis

Cystathionine γ-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, S-adenosyl-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. S-adenosyl-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.

A 235-bp region from a nutritionally regulated soybean seed-specific gene promoter can confer its sulfur and nitrogen response to a constitutive promoter in aerial tissues of Arabidopsis thaliana

Abstract The seed-specific promoter of the β subunit gene of β-conglycinin, a major seed storage protein of soybean, is upregulated in response to both sulfur and nitrogen nutrition. This response was studied in non-seed tissues of transgenic Arabidopsis thaliana by fusing the β subunit promoter (Pβ) downstream of an enhancer from the cauliflower mosaic virus 35S RNA promoter (P35S) and using the β-glucuronidase (GUS) reporter gene. The chimeric promoter was upregulated by sulfur deficiency (ΔS), indicating that the Pβ is able to direct the ΔS response in non-seed tissues. Deletion analysis of the Pβ revealed that, in seeds, the −307–−73 bp region is sufficient for the ΔS response. Insertion of the 235 bp region into the P35S conferred the ΔS response on this constitutive promoter in both seed and non-seed tissues. The extent of upregulation by ΔS was more evident when three of the 235 bp fragments were tandemly inserted into the P35S. The findings presented here suggest that a common mechanism exists for sulfur-regulated expression of the Pβ in both seed and non-seed tissues and that this 235 bp region of the Pβ is sufficient for regulation by both sulfur and nitrogen nutrition.

Independent roles of methionine and O-acetyl-l-serine in the regulation of the β subunit gene of β-conglycinin

Abstract The gene encoding the β subunit of β-conglycinin, one of the major seed storage proteins of soybean (Glycine max [L.] Merr.) , is downregulated by methionine (Met) and upregulated by O-acetyl-l-serine (OAS). We examined the interaction between Met and OAS in the regulation of the expression of the β subunit gene in in vitro-cultured immature soybean cotyledons. Application of Met reduced the accumulation of the β subunit both in the presence and absence of OAS in the media: Application of OAS increased the accumulation of the β subunit both in the presence and absence of Met. We also studied the regulation of the β subunit gene promoter by sulfur in Arabidopsis thaliana (L.) Heynh. mutant mto1-1, that over-accumulates soluble Met, using the β-glucuronidase (GUS) gene as a reporter. When grown under sulfur-deficient conditions, the GUS activity in seeds of the transgenic plants carrying the mto1-1 mutation was lower than that of the plants which do not carry the mutation, suggesting that the β subunit gene promoter was downregulated by the mto1-1 mutation under sulfur deficiency. When the transgenic plants carrying the mto1-1 mutation were grown under control and sulfur-deficient conditions, the GUS activity was higher under sulfur-deficient conditions, suggesting that the β subunit gene promoter was upregulated by sulfur deficiency even in the mto1-1 mutant background. Furthermore, OAS contents in immature siliques were elevated both in the mto1-1 mutant and wild-type plants under sulfur deficiency. Met contents in immature siliques of the mto1-1 mutant plants increased even under sulfur deficiency. There was a strong positive correlation between the GUS activities in seeds and the endogenous [OAS]/[Met] ratio. These results suggest that OAS and Met regulate the β subunit accumulation through independent pathways.