Hoyeun Kim

Role of O-acetyl-l-serine in the coordinated regulation of the expression of a soybean seed storage-protein gene by sulfur and nitrogen nutrition

Abstract. The composition of seed storage proteins is regulated by sulfur and nitrogen supplies. Under conditions of a low sulfur-to-nitrogen ratio, accumulation of the β-subunit of β-conglycinin, a sulfur-poor seed storage protein of soybean (Glycine max [L.] Merr.), is elevated, whereas that of glycinin, a sulfur-rich storage protein, is reduced. Using transgenic Arabidopsis thaliana [L.] Heynh., it was found that the promoter from the gene encoding the β-subunit of β-conglycinin up-regulates gene expression under sulfur deficiency and down-regulates gene expression under nitrogen deficiency. To obtain an insight into the metabolic control of this regulation, the concentrations of metabolites related to the sulfur assimilation pathway were determined. Among the metabolites, O-acetyl-l-serine (OAS), one of the precursors of cysteine biosynthesis, accumulated to higher levels under low-sulfur and high-nitrogen conditions in siliques of transgenic A. thaliana. The pattern of OAS accumulation in response to various levels of sulfur and nitrogen was similar to that of gene expression driven by the β-subunit promoter. Elevated levels of OAS accumulation were also observed in soybean cotyledons cultured under sulfur deficiency. Moreover, OAS applied to in-vitro cultures of immature soybean cotyledons under normal sulfate conditions resulted in a high accumulation of the β-subunit mRNA and protein, whereas the accumulation of glycinin was reduced. These changes were very similar to the responses observed under conditions of sulfur deficiency. Our results suggest that the level of free OAS mediates sulfur- and nitrogen-regulation of soybean seed storage-protein composition.

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.

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.

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.

Effects of exogenous ABA application on sulfate and OAS concentrations, and on composition of seed storage proteins in in vitro cultured soybean immature cotyledons

Composition of seed storage proteins in in vitro cultured cotyledons is known to be affected by exogenous application of abscisic acid (ABA) and sulfur deficiency. In this paper, we analyzed effects of exogenous ABA on regulation of soybean seed storage protein accumulation in relation to that of sulfur deficiency. When exogenous ABA was applied at 10 µM and 100 µM, accumulation of the β subunit of β-conglycinin, a major seed storage protein, increased, whereas that of glycinin, another major seed storage protein, decreased. Free sulfate concentrations in cotyledons were not significantly affected by application of 10 µM ABA, but doubled by application of 100 µM ABA. As we have previously shown that concentrations of O-acetyl-L-serine (OAS), a precursor of cysteine biosynthesis, in in vitro cultured soybean cotyledons increased under sulfur deficiency, effects of exogenous ABA application on OAS concentration in soybean cotyledons were determined. The concentrations of OAS increased in accordance with the increase of exogenous ABA concentrations. These results suggested that exogenous application of ABA induces changes in soybean storage protein composition through the increase of OAS concentration.

Formation of Tissue Masses on Floral Inflorescence in A. thaliana Plants That Accumulate Reduced Levels of MT2a mRNA

Abstract Eight independent transgenic lines of Arabidopsis thaliana were generated carrying homozygous insertions of the cauliflower mosaic virus 35S RNA promoter driving antisense MT2a ORF or the heat shock-protein 81-1 promoter driving sense MT2a ORF. Six transgenic lines showed lower levels of MT2a mRNA accumulation than the wild type plants and among them, three lines produced abnormal tissue masses at the base of floral stalks at low frequencies. Such structures had never been observed in transgenic plants with wild type levels of MT2a transcript accumulation or in the wild type plants. Moreover, we isolated a line from ethyl methane sulfonate (EMS)-mutagenized 5,000 M2 plants that exhibited abnormal tissue masses at the base of floral stalks at low frequencies. The phenotype was inherited at least for four successive generations. This line was found to accumulate MT2a mRNA at a reduced level in the aerial parts. A MT2a promoter::β-glucuronidase (GUS) gene was introduced into the mutant line and it was found that GUS activity was reduced in the mutant plants, suggesting that the mutation(s) in the line downregulated the MT2a promoter in trans. These results suggest that the reduction in MT2a mRNA accumulation is the cause of the abnormal tissue masses observed. MT2a is likely to be involved in the regulation of tissue formation in A. thaliana and possible mechanisms underlying the effects were discussed.