Motoko Awazuhara

Insertional Inactivation of the Methionine S-Methyltransferase Gene Eliminates the S-Methylmethionine Cycle and Increases the Methylation Ratio

Methionine (Met) S-methyltransferase (MMT) catalyzes the synthesis of S-methyl-Met (SMM) from Met andS-adenosyl-Met (Ado-Met). SMM can be reconverted to Met by donating a methyl group to homocysteine (homo-Cys), and concurrent operation of this reaction and that mediated by MMT sets up the SMM cycle. SMM has been hypothesized to be essential as a methyl donor or as a transport form of sulfur, and the SMM cycle has been hypothesized to guard against depletion of the free Met pool by excess Ado-Met synthesis or to regulate Ado-Met level and hence the Ado-Met toS-adenosylhomo-Cys ratio (the methylation ratio). To test these hypotheses, we isolated insertional mmtmutants of Arabidopsis and maize (Zea mays). Both mutants lacked the capacity to produce SMM and thus had no SMM cycle. They nevertheless grew and reproduced normally, and the seeds of the Arabidopsis mutant had normal sulfur contents. These findings rule out an indispensable role for SMM as a methyl donor or in sulfur transport. The Arabidopsis mutant had significantly higher Ado-Met and lowerS-adenosylhomo-Cys levels than the wild type and consequently had a higher methylation ratio (13.8 versus 9.5). Free Met and thiol pools were unaltered in this mutant, although there were moderate decreases (of 30%–60%) in free serine, threonine, proline, and other amino acids. These data indicate that the SMM cycle contributes to regulation of Ado-Met levels rather than preventing depletion of free Met.

Metabolomics and differential gene expression in anthocyanin chemo-varietal forms of Perilla frutescens

We have investigated metabolite profiles and gene expression in two chemo-varietal forms, red and green forms, of Perilla frutescens var. crispa. Striking difference in anthocyanin content was observed between the red and green forms. Anthocyanin, mainly malonylshisonin, was highly accumulated in the leaves of the red form but not in the green form. Less obvious differences were also observed in the stems. However, there was no remarkable difference in the contents and patterns of flavones and primary metabolites such as inorganic anions, organic anions and amino acids. These results suggest that only the regulation of anthocyanin production, but not that of other metabolites, differs in red and green forms. Microscopic observation and immunohistochemical studies indicated that the epidermal cells of leaves and stems are the sites of accumulation of anthocyanins and localization of anthocyanidin synthase protein. By differential display of mRNA from the leaves of red and green forms, we could identify several genes encoding anthocyanin-biosynthetic enzymes and presumptive regulatory proteins. The possible regulatory network leading to differential anthocyanin accumulation in a form-specific manner is discussed.

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.

Composition of seed storage proteins changed by glutathione treatment of soybeans.

The application of glutathione to immature soybean cotyledons reduced the accumulation of the β subunit of β-conglycinin, and increased the accumulation of most glycinins. Both reduced and oxidized forms of glutathione had these effects. The application of an inhibitor of glutathione synthesis, buthionine sulfoximine, increased accumulation of β subunit. These results suggest that glutathione is important in affecting the composition of seed storage proteins.

Genetic and physiological approaches toward understanding the mechanisms underlying the sulfur-regulated expression of β -conglycinin genes

Accumulation of the β subunit of β -conglycinin, a major seed storage protein of soybean, is known to be upregulated by sulfate deficiency and repressed by exogenous application of methionine in the in vitro culture of immature cotyledons. Accumulation of the β subunit mRNA increased in seeds of soybean plants as the concentration of sulfate in the media was decreased. The promoter of the β subunit was capable of upregulation, suggesting that the regulation mainly occurs at the level of transcription. The level of free sulfate in seeds of soybean grown under sulfur deficiency was dramatically reduced. Immature soybean seeds cultured in vitro in a sulfate deficient medium accumulated an elevated level of the β subunit. These results suggest that the level of sulfate concentration in seeds is a main trigger for the induction of the β subunit accumulation in plants exposed to sulfur deficiency. Possible application of genetical approaches for better understanding of the regulation mechanism is discussed.

Function of the sulfate transporter Sultr2;1 in seeds of Arabidopsis thaliana

Arabidopsis thaliana has eleven putative sulfate transporter genes, many of which are expressed in a tissue specific manner. We made transgenic Arabidopsis plants (ATR2) carrying the antisense coding sequence of the low-affinity type sulfate transporter Sultr2;1 under the control of cauliflower mosaic virus (CaMV) 35S promoter. In this study, we characterized the role of Sultr2;1 in seeds using ATR2 transgenic lines.

Screening of Arabidopsis thaliana mutants sensitive to sulfur deficiency

When higher plants are exposed to sulfur deficiency, the patterns of gene expression and metabolic activities are modified to sustain normal growth.