Hisakazu Hasegawa

The Sg-1 Glycosyltransferase Locus Regulates Structural Diversity of Triterpenoid Saponins of Soybean

Group A saponins in soybean are diversified compounds belonging to a group of triterpene saponins and are causal components for bitterness and astringent aftertastes of soy products. This work describes the identification of Sg-1, a UDP-sugar–dependent glycosyltransferase gene that is responsible for the unpleasant tastes due to allelic variation regulating the terminal sugar species in group A saponins. Triterpene saponins are a diverse group of biologically functional products in plants. Saponins usually are glycosylated, which gives rise to a wide diversity of structures and functions. In the group A saponins of soybean (Glycine max), differences in the terminal sugar species located on the C-22 sugar chain of an aglycone core, soyasapogenol A, were observed to be under genetic control. Further genetic analyses and mapping revealed that the structural diversity of glycosylation was determined by multiple alleles of a single locus, Sg-1, and led to identification of a UDP-sugar–dependent glycosyltransferase gene (Glyma07g38460). Although their sequences are highly similar and both glycosylate the nonacetylated saponin A0-αg, the Sg-1a allele encodes the xylosyltransferase UGT73F4, whereas Sg-1b encodes the glucosyltransferase UGT73F2. Homology models and site-directed mutagenesis analyses showed that Ser-138 in Sg-1a and Gly-138 in Sg-1b proteins are crucial residues for their respective sugar donor specificities. Transgenic complementation tests followed by recombinant enzyme assays in vitro demonstrated that sg-10 is a loss-of-function allele of Sg-1. Considering that the terminal sugar species in the group A saponins are responsible for the strong bitterness and astringent aftertastes of soybean seeds, our findings herein provide useful tools to improve commercial properties of soybean products.

Use of a feedback-insensitive α subunit of anthranilate synthase as a selectable marker for transformation of rice and potato

A selection system based on a mutant rice gene for a feedback-insensitive α subunit of anthranilate synthase (OASA1D) was developed for the transformation of rice and potato. Expression of OASA1D conferred resistance to the tryptophan analog 5-methyltryptophan (5MT) in transformed cells of rice and potato. The selection system based on OASA1D and 5MT was associated with a high transformation efficiency, a short time frame for the generation of transgenic plants, simple culture procedures, and it was as effective as hygromycin B selection in rice (monocotyledon) and kanamycin selection in potato (dicotyledon). Transgenic rice and potato plants established by 5MT selection had normal morphology and accumulated tryptophan when OASA1D was expressed under the control of a constitutive promoter. These results demonstrate the efficacy of OASA1D as a selectable marker and they suggest that the 5MT selection system based on this gene will prove applicable to a wide range of plant species and culture procedures.

Metabolic engineering for the production of prenylated polyphenols in transgenic legume plants using bacterial and plant prenyltransferases.

Prenylated polyphenols are secondary metabolites beneficial for human health because of their various biological activities. Metabolic engineering was performed using Streptomyces and Sophora flavescens prenyltransferase genes to produce prenylated polyphenols in transgenic legume plants. Three Streptomyces genes, NphB, SCO7190, and NovQ, whose gene products have broad substrate specificity, were overexpressed in a model legume, Lotus japonicus, in the cytosol, plastids or mitochondria with modification to induce the protein localization. Two plant genes, N8DT and G6DT, from Sophora flavescens whose gene products show narrow substrate specificity were also overexpressed in Lotus japonicus. Prenylated polyphenols were undetectable in these plants; however, supplementation of a flavonoid substrate resulted in the production of prenylated polyphenols such as 7-O-geranylgenistein, 6-dimethylallylnaringenin, 6-dimethylallylgenistein, 8-dimethylallynaringenin, and 6-dimethylallylgenistein in transgenic plants. Although transformants with the native NovQ did not produce prenylated polyphenols, modification of its codon usage led to the production of 6-dimethylallylnaringenin and 6-dimethylallylgenistein in transformants following naringenin supplementation. Prenylated polyphenols were not produced in mitochondrial-targeted transformants even under substrate feeding. SCO7190 was also expressed in soybean, and dimethylallylapigenin and dimethylallyldaidzein were produced by supplementing naringenin. This study demonstrated the potential for the production of novel prenylated polyphenols in transgenic plants. In particular, the enzymatic properties of prenyltransferases seemed to be altered in transgenic plants in a host species-dependent manner.

Novel Bacterial N-Acetyltransferase Gene for Herbicide Detoxification in Land Plants and Selection Maker in Plant Transformation

Phosphinothricin (PPT) is the active ingredient in bialaphos, which specifically inhibits glutamine synthetase in land plants. We isolated a novel PPT-resistant gene from a soil bacterium, Nocardia sp., and characterized it. The encoded protein, consisting of 177 amino acids, showed significant similarity to bacterial N-acetyltransferases, and we originally designated the gene MAT (methionine sulfone N-acetyltransferase). The recombinant MAT protein exhibited functions as a methionine sulfone and PPT N-acetyltransferase in vitro. The PPT N-acetyltransferase activity reached the maximum at pH 8–8.5, indicating that the protein might optimally function in chloroplasts. We therefore constructed a MAT gene, encoding the enzyme with a chloroplast-localizing signal in its amino-terminus. Plant transformation with the construct resulted in the generation of PPT-resistant rice and Arabidopsis. Furthermore, the transformed Arabidopsis was selectable in a synthetic medium containing PPT. The MAT gene thus facilitated establishment of herbicide-resistant plants, and as a new selectable gene marker.

Stable accumulation of seed storage proteins containing vaccine peptides in transgenic soybean seeds.

There has been a significant increase in the use of transgenic plants for the large-scale production of pharmaceuticals and industrial proteins. Here, we report the stable accumulation of seed storage proteins containing disease vaccine peptides in transgenic soybean seeds. To synthesize vaccine peptides in soybean seeds, we used seed storage proteins as a carrier and a soybean breeding line lacking major seed storage proteins as a host. Vaccine peptides were inserted into the flexible disordered regions in the A1aB1b subunit three-dimensional structure. The A1aB1b subunit containing vaccine peptides in the disordered regions were sorted to the protein storage vacuoles where vaccine peptides are partially cleaved by proteases. In contrast, the endoplasmic reticulum (ER)-retention type of the A1aB1b subunit containing vaccine peptides accumulated in compartments that originated from the ER as an intact pro-form. These results indicate that the ER may be an organelle suitable for the stable accumulation of bioactive peptides using seed storage proteins as carriers.

Overexpression of RSOsPR10, a root-specific rice PR10 gene, confers tolerance against drought stress in rice and drought and salt stresses in bentgrass

RSOsPR10 was originally identified as a rice root-specific pathogenesis-related protein whose production was induced by drought and salinity stresses, but not by low temperature or abscisic acid. Our previous studies revealed that RSOsPR10 expression is up-regulated by jasmonic acid, and strongly inhibited by salicylic acid. Immunohistochemical experiments indicated RSOsPR10 is expressed in the root cortical cells. In the present study, we generated RSOsPR10-overexpressing lines of rice and bentgrass to examine the physiological roles of RSOsPR10 in plants. RSOsPR10-overexpressing rice plants were highly tolerant against drought stress, but not against salinity. In contrast, RSOsPR10-overexpressing bentgrass plants were tolerant against drought and salinity stresses. There was little difference between transgenic and wild-type plants regarding phenotype or above-ground growth rates. However, the root mass of the transgenic rice and bentgrass plants was significantly greater than that of the wild-type plants. Therefore, RSOsPR10 is likely involved in mediating environmental stress tolerance through an increase in root growth and development.

A vacuolar sorting receptor-independent sorting mechanism for storage vacuoles in soybean seeds

The seed storage proteins of soybean (Glycine max) are composed mainly of glycinin (11S globulin) and β-conglycinin (7S globulin). The subunits of glycinin (A1aB1b, A1bB2, A2B1a, A3B4, and A5A4B3) are synthesized as a single polypeptide precursor. These precursors are assembled into trimers with a random combination of subunits in the endoplasmic reticulum, and are sorted to the protein storage vacuoles. Proteins destined for transport to protein storage vacuoles possess a vacuolar sorting determinant, and in this regard, the A1aB1b subunit contains a C-terminal peptide that is sufficient for its sorting to protein storage vacuoles. The A3B4 subunit, however, lacks a corresponding C-terminal sorting determinant. In this study, we found that, unlike the A1aB1b subunit, the A3B4 subunit does not bind to previously reported vacuolar sorting receptors. Despite this difference, we observed that the A3B4 subunit is sorted to protein storage vacuoles in a transgenic soybean line expressing the A3B4 subunit of glycinin. These results indicate that a protein storage vacuolar sorting mechanism that functions independently of the known vacuolar sorting receptors in seeds might be present in soybean seeds.