Keito Nishizawa

Comprehensive Analysis of Mitochondria in Roots and Hypocotyls of Soybean under Flooding Stress using Proteomics and Metabolomics Techniques

Flooding is a serious problem for soybeans because it reduces growth and grain yield. Proteomic and metabolomic techniques were used to examine whether mitochondrial function is altered in soybeans by flooding stress. Mitochondrial fractions were purified from the roots and hypocotyls of 4-day-old soybean seedlings that had been flooded for 2 days. Mitochondrial matrix and membrane proteins were separated by two-dimensional polyacrylamide gel electrophoresis and blue-native polyacrylamide gel electrophoresis, respectively. Differentially expressed proteins and metabolites were identified using mass spectrometry. Proteins and metabolites related to the tricarboxylic acid cycle and γ-amino butyrate shunt were up-regulated by flooding stress, while inner membrane carrier proteins and proteins related to complexes III, IV, and V of the electron transport chains were down-regulated. The amounts of NADH and NAD were increased; however, ATP was significantly decreased by flooding stress. These results suggest that flooding directly impairs electron transport chains, although NADH production increases in the mitochondria through the tricarboxylic acid cycle.

Arabidopsis Vacuolar Sorting Mutants (green fluorescent seed) Can Be Identified Efficiently by Secretion of Vacuole-Targeted Green Fluorescent Protein in Their Seeds

Two Arabidopsis thaliana genes have been shown to function in vacuolar sorting of seed storage proteins: a vacuolar sorting receptor, VSR1/ATELP1, and a retromer component, MAIGO1 (MAG1)/VPS29. Here, we show an efficient and simple method for isolating vacuolar sorting mutants of Arabidopsis. The method was based on two findings in this study. First, VSR1 functioned as a sorting receptor for β-conglycinin by recognizing the vacuolar targeting signal. Second, when green fluorescent protein (GFP) fusion with the signal (GFP-CT24) was expressed in vsr1, mag1/vps29, and wild-type seeds, both vsr1and mag1/vps29 gave strongly fluorescent seeds but the wild type did not, suggesting that a defect in vacuolar sorting provided fluorescent seeds by the secretion of GFP-CT24 out of the cells. We mutagenized transformant seeds expressing GFP-CT24. From ∼3,000,000 lines of M2 seeds, we obtained >100 fluorescent seeds and designated them green fluorescent seed (gfs) mutants. We report 10 gfs mutants, all of which caused missorting of storage proteins. We mapped gfs1 to VSR1, gfs2 to KAM2/GRV2, gfs10 to the At4g35870 gene encoding a novel membrane protein, and the others to different loci. This method should provide valuable insights into the complex molecular mechanisms underlying vacuolar sorting of storage proteins.

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.

A novel plant protein disulfide isomerase family homologous to animal P5 – molecular cloning and characterization as a functional protein for folding of soybean seed-storage proteins

The protein disulfide isomerase is known to play important roles in the folding of nascent polypeptides and in the formation of disulfide bonds in the endoplasmic reticulum (ER). In this study, we cloned a gene of a novel protein disulfide isomerase family from soybean leaf (Glycine max L. Merrill. cv Jack) mRNA. The cDNA encodes a protein called GmPDIM. It is composed of 438 amino acids, and its sequence and domain structure are similar to that of animal P5. Recombinant GmPDIM expressed in Escherichia coli displayed an oxidative refolding activity on denatured RNase A. The genomic sequence of GmPDIM was also cloned and sequenced. Comparison of the soybean sequence with sequences from Arabidopsis thaliana and Oryza sativa showed significant conservation of the exon/intron structure. Consensus sequences within the promoters of the GmPDIM genes contained a cis‐acting regulatory element for the unfolded protein response, and other regulatory motifs required for seed‐specific expression. We observed that expression of GmPDIM was upregulated under ER‐stress conditions, and was expressed ubiquitously in soybean tissues such as the cotyledon. It localized to the lumen of the ER. Data from co‐immunoprecipitation experiments suggested that GmPDIM associated non‐covalently with proglycinin, a precursor of the seed‐storage protein glycinin. In addition, GmPDIM associated with the α′ subunit of β‐conglycinin, a seed‐storage protein in the presence of tunicamycin. These results suggest that GmPDIM may play a role in the folding of storage proteins and functions not only as a thiol‐oxidoredactase, but also as molecular chaperone.

Genetic improvement of the somatic embryogenesis and regeneration in soybean and transformation of the improved breeding lines

Somatic embryos of soybean [Glycine max (L.) Merrill] have been used to generate transgenic plants by particle bombardment. The induction and proliferation of somatic embryos from immature cotyledons are dependent on the genotype of the cultivar. Whereas somatic embryogenesis and plant regeneration are inefficient in most cultivars, they are efficient in the cultivar Jack. We previously established a breeding line, QF2, by the integration of null mutations of each subunit of the major seed storage proteins glycinin and β-conglycinin, but the embryogenic response of this line is insufficient to allow efficient transformation. We have now backcrossed QF2 to cultivar Jack in order to combine the null traits with competence for somatic embryogenesis. The backcrossed breeding lines selected on the basis of the absence of the major storage proteins exhibited an improved capacity for the induction and proliferation of somatic embryos compared with that of QF2. The induced somatic embryogenic tissue of these breeding lines was successfully used for the production of transgenic plants by particle bombardment. These results also indicate that somatic embryogenesis in soybean is genetically controlled and inherited in a manner independent of the null traits of the major seed storage proteins.

Manipulation of saponin biosynthesis by RNA interference-mediated silencing of β-amyrin synthase gene expression in soybean

Soybean seeds contain substantial amount of diverse triterpenoid saponins that influence the seed quality, although little is known about the physiologic functions of saponins in plants. We now describe the modification of saponin biosynthesis by RNA interference (RNAi)-mediated gene silencing targeted to β-amyrin synthase, a key enzyme in the synthesis of a common aglycon of soybean saponins. We identified two putative β-amyrin synthase genes in soybean that manifested distinct expression patterns with regard to developmental stage and tissue specificity. Given that one of these genes, GmBAS1, was expressed at a much higher level than the other (GmBAS2) in various tissues including the developing seeds, we constructed two RNAi vectors that encode self-complementary hairpin RNAs corresponding to the distinct regions of GmBAS1 under the control of a seed-specific promoter derived from the soybean gene for the α′ subunit of the seed storage protein β-conglycinin. These vectors were introduced independently into soybean. Six independent transgenic lines exhibited a stable reduction in seed saponin content, with the extent of saponin deficiency correlating with the β-amyrin synthase mRNA depletion. Although some transgenic lines produced seeds almost devoid of saponins, no abnormality in their growth was apparent and the antioxidant activity of their seeds was similar to that of control seeds. These results suggest that saponins are not required for seed development and survival, and that soybean seeds may therefore be amenable to the modification of triterpenoid saponin content and composition through molecular biologic approaches.

The C-terminal region of α′ subunit of soybean β-conglycinin contains two types of vacuolar sorting determinants

In maturing seed cells, proteins that accumulate in the protein storage vacuoles (PSVs) are synthesized on the endoplasmic reticulum (ER) and transported by vesicles to the PSVs. Vacuolar sorting determinants (VSDs) which are usually amino acid sequences of short or moderate length direct the proteins to this pathway. VSDs identified so far are classified into two types: sequence specific VSDs (ssVSDs) and C-terminal VSDs (ctVSDs). We previously demonstrated that VSDs of α′ and β subunits of β-conglycinin, one of major storage proteins of soybean (Glycine max), reside in the C-terminal ten amino acids. Here we show that both types of VSDs coexist within this region of the α′ subunit. Although ctVSDs can function only at the very C-termini of proteins, the C-terminal ten amino acids of α′ subunit directed green fluorescent protein (GFP) to the PSVs even when they were placed at the N-terminus of GFP, indicating that an ssVSD resides in the sequence. By mutation analysis, it was found that the core sequence of the ssVSD is Ser-Ile-Leu (fifth to seventh residues counted from the C-terminus) which is conserved in the α and β subunits and some vicilin-like proteins. On the other hand, the sequence composed of the C-terminal three amino acids (AFY) directed GFP to the PSVs when it was placed at the C-terminus of GFP, though the function as a VSD was disrupted at the N-terminus of GFP, indicating that the AFY sequence is a ctVSD.

Assessment of the importance of α-amylase inhibitor-2 in bruchid resistance of wild common bean

Both α-amylase inhibitor-2 (αAI-2) and arcelin have been implicated in resistance of wild common bean (Phaseolus vulgaris L.) to the Mexican bean weevil (Zabrotes subfasciatus Boheman). Near isogenic lines (NILs) for arcelin 1–5 were generated by backcrossing wild common bean accessions with a cultivated variety. Whereas seeds of a wild accession (G12953) containing both αAI-2 and arcelin 4 were completely resistant to Z. subfasciatus, those of the corresponding NIL were susceptible to infestation, suggesting that the principal determinant of resistance was lost during backcrossing. Three independent lines of transgenic azuki bean [Vigna angularis (Willd.) Ohwi and Ohashi] expressing αAI-2 accumulated high levels of this protein in seeds. The expression of αAI-2 in these lines conferred protection against the azuki bean weevil (Callosobruchus chinensis L.), likely through inhibition of larval digestive α-amylase. However, although the seed content of αAI-2 in these transgenic lines was similar to that in a wild accession of common bean (G12953), it did not confer a level of resistance to Z. subfasciatus similar to that of the wild accession. These results suggest that αAI-2 alone does not provide a high level of resistance to Z. subfasciatus. However, αAI-2 is an effective insecticidal protein with a spectrum of activity distinct from that of αAI-1, and it may prove beneficial in genetic engineering of insect resistance in legumes.

Accumulation of β-Conglycinin in Soybean Cotyledon through the Formation of Disulfide Bonds between α′- and α-Subunits

β-Conglycinin, one of the major soybean (Glycine max) seed storage proteins, is folded and assembled into trimers in the endoplasmic reticulum and accumulated into protein storage vacuoles. Prior experiments have used soybean β-conglycinin extracted using a reducing buffer containing a sulfhydryl reductant such as 2-mercaptoethanol, which reduces both intermolecular and intramolecular disulfide bonds within the proteins. In this study, soybean proteins were extracted from the cotyledons of immature seeds or dry beans under nonreducing conditions to prevent the oxidation of thiol groups and the reduction or exchange of disulfide bonds. We found that approximately half of the α′- and α-subunits of β-conglycinin were disulfide linked, together or with P34, prior to amino-terminal propeptide processing. Sedimentation velocity experiments, size-exclusion chromatography, and two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis, with blue native PAGE followed by sodium dodecyl sulfate-PAGE, indicated that the β-conglycinin complexes containing the disulfide-linked α′/α-subunits were complexes of more than 720 kD. The α′- and α-subunits, when disulfide linked with P34, were mostly present in approximately 480-kD complexes (hexamers) at low ionic strength. Our results suggest that disulfide bonds are formed between α′/α-subunits residing in different β-conglycinin hexamers, but the binding of P34 to α′- and α-subunits reduces the linkage between β-conglycinin hexamers. Finally, a subset of glycinin was shown to exist as noncovalently associated complexes larger than hexamers when β-conglycinin was expressed under nonreducing conditions.