Masao Ishimoto

Bruchid resistance of transgenic azuki bean expressing seed α‐amylase inhibitor of common bean

Various species of bruchid beetles including Callosobruchus chinensis, C. maculatus and C. analis cause post‐harvest damage of azuki bean seeds, an important East Asian grain legume. The α‐amylase in the midguts of these insects is inhibited by the α‐amylase inhibitor (αAI) present in common bean seeds. Transformation of azuki bean with the αAI gene driven by the promoter of phytohemagglutinin results in high levels of αAI in the seeds and the complete block of bruchid development on the seeds. Zabrotes subfasciatus, a South and Central American bruchid that is a storage pest of common bean, develops normally on the transgenic azuki bean.

Protective mechanism of the Mexican bean weevil against high levels of alpha-amylase inhibitor in the common bean.

[alpha]-Amylase inhibitor ([alpha]AI) protects seeds of the common bean (Phaseolus vulgaris) against predation by certain species of bruchids such as the cowpea weevil (Callosobruchus maculatus) and the azuki bean weevil (Callosobruchus chinensis), but not against predation by the bean weevil (Acanthoscelides obtectus) or the Mexican bean weevil (Zabrotes subfasciatus), insects that are common in the Americas. We characterized the interaction of [alpha]AI-1 present in seeds of the common bean, of a different isoform, [alpha]AI-2, present in seeds of wild common bean accessions, and of two homologs, [alpha]AI-Pa present in seeds of the tepary bean (Phaseolus acutifolius) and ([alpha]AI-Pc in seeds of the scarlet runner bean (Phaseolus coccineus), with the midgut extracts of several bruchids. The extract of the Z. subfasciatus larvae rapidly digests and inactivates [alpha]AI-1 and [alpha]AI-Pc, but not [alpha]AI-2 or [alphha]AI-Pa. The digestion is caused by a serine protease. A single proteolytic cleavage in the [beta] subunit of [alpha]AI-1 occurs at the active site of the protein. When degradation is prevented, [alpha]AI-1 and [alpha]AI-Pc do not inhibit the [alpha]-amylase of Z. subfasciatus, although they are effective against the [alpha]-amylase of C. chinensis. [alpha]AI-2 and [alpha]AI-Pa, on the other hand, do inhibit the [alpha]-amylase of Z. subfasciatus, suggesting that they are good candidates for genetic engineering to achieve resistance to Z. subfasciatus.

Accumulation of high levels of free amino acids in soybean seeds through integration of mutations conferring seed protein deficiency

Soybean (Glycine max [L.] Merr.) seeds are rich in protein, most of which is contributed by the major storage proteins glycinin (11S globulin) and β-conglycinin (7S globulin). Null mutations for each of the subunits of these storage proteins were integrated by crossbreeding to yield a soybean line that lacks both glycinin and β-conglycinin components. In spite of the absence of these two major storage proteins, the mutant line grew and reproduced normally, and the nitrogen content of its dry seed was similar to that for wild-type cultivars. However, protein bodies appeared underdeveloped in the cotyledons of the integrated mutant line. Furthermore, whereas free amino acids contribute only 0.3–0.8% of the seed nitrogen content of wild-type varieties, they constituted 4.5–8.2% of the seed nitrogen content in the integrated mutant line, with arginine (Arg) being especially enriched in the mutant seeds. Seeds of the integrated mutant line thus appeared to compensate for the reduced nitrogen content in the form of glycinin and β-conglycinin by accumulating free amino acids as well as by increasing the expression of certain other seed proteins. These results indicate that soybean seeds are able to store nitrogen mostly in the form of either proteins or free amino acids.

Oryzacystatins exhibit growth-inhibitory and lethal effects on different species of bean insect pests, Callosobruchus chinensis (Coleoptera) and Riptortus clavatus (Hemiptera).

Oryzacystatins I and II, cysteine proteinase inhibitors in rice seeds, caused growth retardation of different species of bean insect pests, Callosobruchus chinensis (Coleoptera) and Riptortus clavatus (Hemiptera), when added to their diets at concentrations of 0.3-0.5% (w/w). At concentrations of up to 1%, almost all insects died. Our results suggest the usefulness of cystatin for insect pest control and also the critical role of cysteine proteinase in the digestive events of insects.

Molecular characterization of a bean α-amylase inhibitor that inhibits the α-amylase of the Mexican bean weevil Zabrotes subfasciatus

Abstract. Cultivated varieties of the common bean (Phaseolus vulgaris L.) contain an α-amylase inhibitor (αAI-1) that inhibits porcine pancreatic α-amylase (PPA; EC 3.2.1.1) and the amylases of certain seed weevils, but not that of the Mexican bean weevil, Zabrotes subfasciatus. A variant of αAI-1, called αAI-2, is found in certain arcelin-containing wild accessions of the common bean. The variant αAI-2 inhibits Z. subfasciatus α-amylase (ZSA), but not PPA. We purified αAI-2 and studied its interaction with ZSA. The formation of the αAI-2-ZSA complex is time-dependent and occurs maximally at pH 5.0 or below. When a previously isolated cDNA assumed to encode αAI-2 was expressed in transgenic tobacco seeds, the seeds contained inhibitory activity toward ZSA but not toward PPA, confirming that the cDNA encodes αAI-2. The inhibitors αAI-1 and αAI-2 share 78% sequence identity at the amino acid level and they differ in an important region that is part of the site where the enzyme binds the inhibitor. The swap of a tripeptide in this region was not sufficient to change the specificity of the two inhibitors towards their respective enzymes. The three-dimensional structure of the αAI-1/PPA complex has just been solved and we recently obtained the derived amino acid sequence of ZSA. This additional information allows us to discuss the results described here in the framework of the amino acid residues of both proteins involved in the formation of the enzyme-inhibitor complex and to pinpoint the amino acids responsible for the specificity of the interaction.

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.

Transformation of azuki bean by Agrobacterium tumefaciens

Stable transformation and regeneration was developed for a grain legume, azuki bean (Vigna angularis Willd. Ohwi & Ohashi). Two constructs containing the neomycin phosphotransferase II gene (nptII) and either the β-glucuronidase (GUS) gene or the modified green fluorescent protein [sGFP(S65T)] gene were introduced independently via Agrobacterium tumefaciens-mediated transformation. After 2 days of co-cultivation on MS medium supplemented with 100 μM acetosyringone and 10 mg l−1 6-benzyladenine, seedling epicotyl explants were placed on regeneration medium containing 100 mg l−1 kanamycin. Adventitious shoots developing from explant calli were excised onto rooting medium containing 100 mg l−1 kanamycin. Rooted shoots were excised and repeatedly selected on the same medium containing kanamycin. Surviving plants were transferred to soil and grown in a green house to produce viable seeds. This process took 5 to 7 months after co-cultivation. Molecular analysis confirmed the stable integration and expression of foreign genes.

A red fluorescent protein, DsRed2, as a visual reporter for transient expression and stable transformation in soybean

Fluorescent proteins such as green fluorescent protein (GFP) from Aequorea victoria are often used as markers for transient expression and stable transformation in plants, given that their detection does not require a substrate and they can be monitored in a nondestructive manner. We have now evaluated the red fluorescent protein DsRed2 (a mutant form of DsRed from Discosoma sp.) for its suitability as a visual marker in combination with antibiotic selection for genetic transformation of soybean [Glycine max (L.) Merrill]. Transient and stable expression of DsRed2 in somatic embryos was readily detected by fluorescence microscopy, allowing easy confirmation of gene introduction. We obtained several fertile transgenic lines, including homozygous lines, that grew and produced seeds in an apparently normal manner. The red fluorescence of DsRed2 was detected by fluorescence microscopy without background fluorescence in both leaves and seeds of the transgenic plants. Furthermore, in contrast to seeds expressing GFP, those expressing DsRed2 were readily identifiable even under white light by the color conferred by the transgene product. The protein composition of seeds was not affected by the introduction of DsRed2, with the exception of the accumulation of DsRed2 itself, which was detectable as an additional band on electrophoresis. These results indicate that DsRed2 is a suitable reporter (even more suitable than GFP) for genetic transformation of soybean.

Molecular cloning and characterization of two soybean protein disulfide isomerases as molecular chaperones for seed storage proteins.

Protein disulfide isomerase family proteins play important roles in the folding of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum. In this study, we cloned two similar protein disulfide isomerase family genes from soybean leaf (Glycine max L. Merrill. cv Jack). The cDNAs encode proteins of 525 and 551 amino acids, named GmPDIL‐1 and GmPDIL‐2, respectively. Recombinant versions of GmPDIL‐1 and GmPDIL‐2 expressed in Escherichia coli exhibited oxidative refolding activity for denatured RNaseA. Genomic sequences of both GmPDIL‐1 and GmPDIL‐2 were cloned and sequenced. The comparison of soybean genomic sequences with those of Arabidopsis, rice and wheat showed impressive conservation of exon–intron structure across plant species. The promoter sequences of GmPDIL‐1 apparently contain a cis‐acting regulatory element functionally linked to unfolded protein response. GmPDIL‐1, but not GmPDIL‐2, expression was induced under endoplasmic reticulum‐stress conditions. GmPDIL‐1 and GmPDIL‐2 promoters contain some predicted regulatory motifs for seed‐specific expression. Both proteins were ubiquitously expressed in soybean tissues, including cotyledon, and localized to the endoplasmic reticulum. Data from coimmunoprecipitation experiments suggested that GmPDIL‐1 and GmPDIL‐2 associate with proglycinin, a precursor of the seed storage protein glycinin, and the α′‐subunit of β‐conglycinin, a seed storage protein found in cotyledon cells under conditions that disrupt the folding of glycinin or β‐conglycinin, suggesting that GmPDIL‐1 and GmPDIL‐2 are involved in the proper folding or quality control of such storage proteins as molecular chaperones.

Characterization of glutamate decarboxylase mediating γ-amino butyric acid increase in the early germination stage of soybean (Glycine max [L.] Merr)

Glutamate decarboxylase (GAD) is an enzyme that catalyzes the production of gamma-amino butyric acid (GABA) from glutamate through a decarboxylation reaction. A full-length cDNA encoding glutamate decarboxylase (GmGAD1) was isolated from germinating soybean seeds (Glycine max [L.] Merr.). The GmGAD1 gene had a 1512-bp open reading frame, which encodes 503 amino acids. According to its sequence similarity with other GAD genes, GmGAD1 was classified into GAD1 in the plant GAD family. Recombinant GmGAD1 protein expressed in E. coli catalyzed alpha-decarboxylation of glutamic acid. The levels of GABA were rapidly increased in soybean seeds during the early imbibition period (6 h) of germination or during the soaking treatment, whereas mRNA of GmGAD1 gene was not detected in these materials. The GmGAD1 protein was observed in seeds of various states such as developing, matured and soaking. These data suggest that the increased levels of GABA during the early stage of germination or soaking treatment were mediated by GmGAD1 protein synthesized in developing soybean seeds.