OsGGC2, Gγ Subunit of Heterotrimeric G Protein, Regulates Plant Height by Functionally Overlapping with DEP1 in Rice.

Plant heterotrimeric G proteins have been shown to regulate the size of various organs. There are three types of Gγ subunits in plants: type A, consisting of a canonical Gγ domain; type B, possessing a plant-specific domain at the N-terminus of the Gγ domain; and type C, possessing a plant-specific domain at the C-terminal of the Gγ domain. There is one type A, one type B, and three type C of the five γ-subunits in the rice genome. In type C Gγ subunits, GS3, which controls grain size; DEP1, which controls plant height and panicle branching; and their homolog OsGGC2, which affects grain size, have been reported; however, the function of each gene, their interactions, and molecular mechanisms for the control of plant height have not yet been clarified. In this study, we generated loss-of-function mutants of DEP1 and OsGGC2, which have high homology and similar expression, and investigated their phenotypes. Since both dep1 and osggc2 mutants were dwarfed and the double mutants showed a synergistic phenotype, we concluded that both DEP1 and OsGGC2 are positive regulators of plant height and that their functions are redundant.

gw2 mutation increases grain width and culm thickness in rice (Oryza sativa L.).

Grain size is one of the most important agricultural traits in rice. To increase grain yield, we screened a large grain mutant from mutants with the 'Koshihikari' background. As a result, we obtained a mutant, KEMS39, that has a large grain size and increased yield. Cultivation tests revealed that this mutant had improved lodging resistance with thicker internodes. Next-generation sequencing analysis revealed the presence of a 67 bp deletion in the GW2 mRNA, owing to a mutation in the 3' splice site of the sixth intron of the GW2 gene. To determine whether this mutation was responsible for the larger grain and thicker internodes, we performed gene editing and obtained a mutant with a 7 bp deletion, including this 3' splice site. As this gw2 mutant had large grains and thicker internodes, the causal gene of KEMS39 was determined as GW2. Thicker internodes are attributed to the pleiotropic effect of gw2 mutation. On the basis of these results, we conclude that gw2 mutation has the potential to be an important genetic resource with the ability to achieve a well-balanced and high-yielding effect that simultaneously improves grain productivity and lodging resistance.

Proteomics Analysis of Plasma Membrane Fractions of the Root, Leaf, and Flower of Rice.

The plasma membrane regulates biological processes such as ion transport, signal transduction, endocytosis, and cell differentiation/proliferation. To understand the functional characteristics and organ specificity of plasma membranes, plasma membrane protein fractions from rice root, etiolated leaf, green leaf, developing leaf sheath, and flower were analyzed by proteomics. Among the proteins identified, 511 were commonly accumulated in the five organs, whereas 270, 132, 359, 146, and 149 proteins were specifically accumulated in the root, etiolated leaf, green leaf, developing leaf sheath, and developing flower, respectively. The principle component analysis revealed that the functions of the plasma membrane in the root was different from those of green and etiolated leaves and that the plasma membrane protein composition of the leaf sheath was similar to that of the flower, but not that of the green leaf. Functional classification revealed that the root plasma membrane has more transport-related proteins than the leaf plasma membrane. Furthermore, the leaf sheath and flower plasma membranes were found to be richer in proteins involved in signaling and cell function than the green leaf plasma membrane. To validate the proteomics data, immunoblot analysis was carried out, focusing on four heterotrimeric G protein subunits, Gα, Gß, Gγ1, and Gγ2. All subunits could be detected by both methods and, in particular, Gγ1 and Gγ2 required concentration by immunoprecipitation for mass spectrometry detection.

Identification of Heterotrimeric G Protein γ3 Subunit in Rice Plasma Membrane.

Heterotrimeric G proteins are important molecules for regulating plant architecture and transmitting external signals to intracellular target proteins in higher plants and mammals. The rice genome contains one canonical α subunit gene (RGA1), four extra-large GTP-binding protein genes (XLGs), one canonical ß subunit gene (RGB1), and five γ subunit genes (tentatively named RGG1, RGG2, RGG3/GS3/Mi/OsGGC1, RGG4/DEP1/DN1/OsGGC3, and RGG5/OsGGC2). RGG1 encodes the canonical γ subunit; RGG2 encodes the plant-specific type of γ subunit with additional amino acid residues at the N-terminus; and the remaining three γ subunit genes encode the atypical γ subunits with cysteine abundance at the C-terminus. We aimed to identify the RGG3/GS3/Mi/OsGGC1 gene product, Gγ3, in rice tissues using the anti-Gγ3 domain antibody. We also analyzed the truncated protein, Gγ3∆Cys, in the RGG3/GS3/Mi/OsGGC1 mutant, Mi, using the anti-Gγ3 domain antibody. Based on nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, the immunoprecipitated Gγ3 candidates were confirmed to be Gγ3. Similar to α (Gα) and ß subunits (Gß), Gγ3 was enriched in the plasma membrane fraction, and accumulated in the flower tissues. As RGG3/GS3/Mi/OsGGC1 mutants show the characteristic phenotype in flowers and consequently in seeds, the tissues that accumulated Gγ3 corresponded to the abnormal tissues observed in RGG3/GS3/Mi/OsGGC1 mutants.

Characterization of Heterotrimeric G Protein γ4 Subunit in Rice.

Heterotrimeric G proteins are the molecule switch that transmits information from external signals to intracellular target proteins in mammals and yeast cells. In higher plants, heterotrimeric G proteins regulate plant architecture. Rice harbors one canonical α subunit gene (RGA1), four extra-large GTP-binding protein genes (XLGs), one canonical ß-subunit gene (RGB1), and five γ-subunit genes (tentatively designated RGG1, RGG2, RGG3/GS3/Mi/OsGGC1, RGG4/DEP1/DN1/qPE9-1/OsGGC3, and RGG5/OsGGC2) as components of the heterotrimeric G protein complex. Among the five γ-subunit genes, RGG1 encodes the canonical γ-subunit, RGG2 encodes a plant-specific type of γ-subunit with additional amino acid residues at the N-terminus, and the remaining three γ-subunit genes encode atypical γ-subunits with cysteine-rich C-termini. We characterized the RGG4/DEP1/DN1/qPE9-1/OsGGC3 gene product Gγ4 in the wild type (WT) and truncated protein Gγ4∆Cys in the RGG4/DEP1/DN1/qPE9-1/OsGGC3 mutant, Dn1-1, as littele information regarding the native Gγ4 and Gγ4∆Cys proteins is currently available. Based on liquid chromatography-tandem mass spectrometry analysis, immunoprecipitated Gγ4 candidates were confirmed as actual Gγ4. Similar to α-(Gα) and ß-subunits (Gß), Gγ4 was enriched in the plasma membrane fraction and accumulated in the developing leaf sheath. As RGG4/DEP1/DN1/qPE9-1/OsGGC3 mutants exhibited dwarfism, tissues that accumulated Gγ4 corresponded to the abnormal tissues observed in RGG4/DEP1/DN1/qPE9-1/OsGGC3 mutants.

Thermo-responsive allele of sucrose synthase 3 (Sus3) provides high-temperature tolerance during the ripening stage in rice (Oryza sativa L.).

High-temperature stress during the ripening stage leads to quality deterioration due to an increase in chalky grains in brown rice (Oryza sativa L.). In a previous study, we identified a QTL for Appearance quality of brown rice 1 (Apq1) using chromosome segment substitution lines of the indica cultivar 'Habataki' in the japonica cultivar 'Koshihikari' background and narrowed down the locus to a 48-kb region on chromosome 7. To verify the function and mechanisms of this QTL in grain appearance, in this study, we fine-mapped the gene and conducted high-temperature tolerance tests. As a result of the genetic mapping, we narrowed down the candidate region of Apq1 to a 19.4-kb region including three predicted genes. Among these, the temporal expression pattern of sucrose synthase 3 (Sus3) corresponded well with the high temperature-sensitive period during ripening, and expression of the 'Habataki' allele of Sus3 was increased under high-temperature condition. In addition, we transformed the 'Habataki' Sus3 gene into 'Nipponbare', and the transformants obtained high-temperature tolerance. Therefore, we conclude that the causal gene underlying the QTL Apq1 is the thermo-responsive Sus3 allele, and the increase in Sus3 expression under high-temperature condition during ripening leads to high-temperature tolerance in rice.

Proteomic analysis of the promotive effect of plant-derived smoke on plant growth of chickpea.

Plant-derived smoke plays a key role in seed germination and plant growth. To investigate the effect of plant-derived smoke on chickpea, a gel-free/label-free proteomic technique was used. Germination percentage, root/shoot length, and fresh biomass were increased in chickpea treated with 2000 ppm plant-derived smoke within 6 days. On treatment with 2000 ppm plant-derived smoke for 6 days, the abundance of 90 proteins including glycolysis-related proteins significantly changed in chickpea root. Proteins related to signaling and transport were increased; however, protein metabolism, cell, and cell wall were decreased. The sucrose synthase for starch degradation was increased and total soluble sugar was induced. The proteins for nitrate pathway were increased and nitrate content was improved. On the other hand, although secondary metabolism related proteins were decreased, flavonoid contents were increased. Based on proteomic and immuno-blot analyses, proteins related to redox homeostasis were decreased and increased in root and shoot, respectively. Furthermore, fructose­bisphosphate aldolase was increased; while, phosphotransferase and phosphoglycero mutase were decreased in glycolysis. In addition, phosphoglyceraldehyde­3­phosphate dehydrogenase and glutamine synthetase related genes were up-regulated. These results suggest that plant-derived smoke improves early stage of growth in chickpea with the balance of many cascades such as glycolysis, redox homeostasis, and secondary metabolism. BIOLOGICAL SIGNIFICANCE: The current study examined the effects of plant-derived smoke on root of chickpea seedlings using a gel-free/label-free proteomic technique. Based on functional categorization of results from proteomics, proteins related to glycolysis, signaling, transport, protein metabolism, cell wall, and cell were predominantly changed in chickpea. The proteins related to carbohydrate and nitrate pathways were increased, while, those of secondary metabolism were decreased. Physiological analysis indicated that flavonoid, total soluble sugar, and nitrate content were increased in root of chickpea treated with plant-derived smoke for 6 days. Moreover, accumulated protein abundance of glyceraldehyde­3­phosphate dehydrogenase and fructose-bisphosphate aldolase was in agreement with immuno-blot results, which suggests that glycolysis process might be enhanced in root of chickpea in response to plant-derived smoke.

Direct interaction between VRN1 protein and the promoter region of the wheat FT gene.

The wheat florigen gene Wheat FLOWERING LOCUS T (WFT, which is identical to VRN3) is an integrator of the vernalization, photoperiod and autonomous pathways in wheat flowering. Many studies have indicated that VERNALIZATION 1 (VRN1) directly or indirectly up-regulates WFT expression in leaves. VRN1 encodes an APETALA1/FRUITFULL-like MADS box transcription factor that is up-regulated by vernalization and aging, leading to promotion of flowering. In this study, the VRN1 protein was expressed as a His-Tag fusion protein in Escherichia coli and used in an electrophoretic mobility shift assay (EMSA). The results from the EMSA indicated that the VRN1 protein directly binds to the CArG-box in the promoter region of WFT, suggesting the direct up-regulation of WFT by VRN1 in the leaves of wheat plants.

Overexpression of SRS5 improves grain size of brassinosteroid-related dwarf mutants in rice (Oryza sativa L.).

Grain size is a trait that is important for rice (Oryza sativa L.) yield potential. Many genes regulating grain size have been identified, deepening our understanding of molecular mechanisms of grain size determination in rice. Previously, we cloned SMALL AND ROUND SEED 5 (SRS5) gene (encoding alpha-tubulin) from a small and round seed mutant and revealed that this gene regulates grain length independently of the brassinosteroid (BR) signaling pathway, although BR-related mutants set small grain. In this study, we showed that overexpression of SRS5 can promote grain length and demonstrated that the overexpression of SRS5 in BR-related mutants rescued the shortened grain length, which is an unfavorable phenotype in the yield potential of BR-related mutants, while preserving the useful semi-dwarf and erect leaf phenotypes.

Detection of Novel QTLs Regulating Grain Size in Extra-Large Grain Rice (Oryza sativa L.) Lines.

BACKGROUND: Grain size is an important trait that affects rice yield. Although many genes that contribute to grain size have been cloned from mutants or by quantitative trait locus (QTL) analysis based on bi-parental mapping, the molecular mechanisms underlying grain-size determination remain poorly understood. In this study, we identified the lines with the largest grain size and detected novel QTLs affecting the grain size. RESULTS: We screened the National Institute for Agrobiological Sciences Genebank database and identified two rice lines, BG23 with the widest grain and LG10 with the longest grain. Using these two lines, we performed QTL analysis for grain size. Eight QTLs were detected during the QTL analyses using F2 populations derived from crosses between the large-grain lines BG23 or LG10 and the middle-size grain cultivars Nipponbare and Kasalath. Both BG23 and LG10 possessed large-grain alleles of four major QTLs: GW2, GS3, qSW5/GW5, and GW8. Other three minor QTLs were derived from BG23. However, these QTLs did not explain the differences in grain size between these two lines. Additionally, four QTLs for grain length or width were detected in an F2 population derived from a cross between BG23 and LG10; this population lacked the strong effects of the four major QTLs shared by both parent plants. Of these newly detected QTLs, the effects of two QTLs, GL3b and GL6, were confirmed by progeny testing. Comparison of the length of inner epidermal cells in plants homozygous for BG23 and LG10 alleles indicated that GL3b and GL6 genes regulate cell elongation and cell division, respectively. CONCLUSIONS: In this study, we detected 12 loci including 14 QTLs regulating grain size from two lines with largest grains available in Japanese stock. Of these loci, we confirmed the effect of two gene loci and mapped their candidate region. Identification of novel genes regulating grain size will contribute to our understanding of the molecular mechanisms controlling grain size.

Plant Morphology of Heterotrimeric G Protein Mutants.

The heterotrimeric G protein complex, comprising Gα, Gγ and Gγ subunits, is an evolutionarily conserved signaling molecular machine that transmits signals from transmembrane receptors to downstream target proteins. Plants conserved the core G protein elements, while developing their own regulatory systems differently from animals. Genetic evidence supports the conclusion that the heterotrimeric G proteins regulate shoot, root and epidermis development, as well as sugar sensing, hormone responsiveness and abiotic and biotic stress tolerance. This review is a compendium of the known morphological changes conferred by loss- and gain-of-function mutations of the G protein subunit genes across three higher land plant models, namely Arabidopsis, rice and maize.

Cooperative degradation of chitin by extracellular and cell surface-expressed chitinases from Paenibacillus sp. strain FPU-7.

Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene (chiW) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes (chiA to chiF and chiW) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine.

Rice transgenic plants with suppressed expression of the ß subunit of the heterotrimeric G protein.

The deficient mutant for the rice heterotrimeric G protein α subunit gene (RGA1), d1, showed dwarfism and set small seed due to a reduced cell number. Mutants for the rice heterotrimeric G protein ß subunit gene (RGB1) have not been isolated. To determine the functions of RGB1, transgenic rice plants with suppressed expression of RGB1 were studied using the RNAi method. RGB1 knock-down lines showed browning of the lamina joint regions and nodes and reduced fertility, but these abnormality were not observed in d1. Transgenic plants in which the G protein ß subunit was greatly decreased were not obtained, suggesting that the complete suppression of RGB1 mRNA may be lethal. In contrast, the d1 mutants, with complete loss of the G protein α subunit, were fertile and half the size of the WT. These studies suggest that RGB1 has different functions than RGA1.

Identification of heterotrimeric G protein α and ß subunits in rice.

Like those in mammals, heterotrimeric G protein complexes have been implicated in signal transduction pathways in plants; however, the subunits themselves have not been isolated. In this study, the rice heterotrimeric G protein subunits α (Gα) and ß (Gß) were purified by affinity chromatography using anti-Gα and -Gß antibodies and SDS-PAGE. Six and seven peptides, respectively, were identified by mass spectrometry and identified as the translation products of the Gα gene RGA1 and Gß gene RGB1. During purification, the subunits dissociated easily from the G protein complex.

Small and round seed 5 gene encodes alpha-tubulin regulating seed cell elongation in rice.

Seed size is an important trait in determinant of rice seed quality and yield. In this study, we report a novel semi-dominant mutant Small and round seed 5 (Srs5) that encodes alpha-tubulin protein. Lemma cell length was reduced in Srs5 compared with that of the wild-type. Mutants defective in the G-protein alpha subunit (d1-1) and brassinosteroid receptor, BRI1 (d61-2) also exhibited short seed phenotypes, the former due to impaired cell numbers and the latter due to impaired cell length. Seeds of the double mutant of Srs5 and d61-2 were smaller than those of Srs5 or d61-2. Furthermore, SRS5 and BRI1 genes were highly expressed in Srs5 and d61-2 mutants. These data indicate that SRS5 independently regulates cell elongation of the brassinosteroid signal transduction pathway.

Suppression of the rice heterotrimeric G protein ß-subunit gene, RGB1, causes dwarfism and browning of internodes and lamina joint regions.

In the present study, we investigated the function of the heterotrimeric G protein ß-subunit (Gß) gene (RGB1) in rice. RGB1 knock-down lines were generated in the wild type and d1-5, a mutant deficient for the heterotrimeric G protein α-subunit (Gα) gene (RGA1). Both transgenic lines showed browning of the lamina joint regions and nodes that could be attributed to a reduction of RGB1 function, as the abnormality was not observed in d1-5. The RGB1 knock-down lines generated in d1-5 were shorter, suggesting RGB1 to be a positive regulator of cellular proliferation, in addition to RGA1. The number of sterile seeds also increased in both RGB1 knock-down lines. These results suggest that Gßγ and Gα cooperatively function in cellular proliferation and seed fertility. We discuss the potential predominant role of RGB1 in G protein signaling in rice.

A novel kinesin 13 protein regulating rice seed length.

The causal gene of a novel small and round seed mutant phenotype (srs3) in rice was identified by map-based cloning and named the SRS3 gene. The SRS3 gene was grouped as a member of the kinesin 13 subfamily. The SRS3 gene codes for a protein of 819 amino acids that contains a kinesin motor domain and a coiled-coil structure. Using scanning electron microscopy, we determined that the cell length of seeds in the longitudinal direction in srs3 is shorter than that in the wild type. The number of cells of seeds in the longitudinal direction in srs3 was not very different from that in the wild type. The result suggests that the small and round seed phenotype of srs3 is due to a reduction in cell length of seeds in the longitudinal direction. The SRS3 protein, which is found in the crude microsomal fraction, is highly expressed in developing organs.

The SMALL AND ROUND SEED1 (SRS1/DEP2) gene is involved in the regulation of seed size in rice.

The causal gene of a novel small and round seed mutant 1 (srs1) was identified in rice by map-based cloning and named SMALL AND ROUND SEED 1 (SRS1). The SRS1 gene is identical to the previously identified DENSE AND ERECT PANICLE 2 (DEP2). The SRS1/DEP2 gene encodes a novel protein of 1365 amino acids residues without known functional domains. In the longitudinal direction of the lemma, both cell length and cell number are reduced in srs1-1 compared to the wild type, whereas in the lateral cross section of the lemma, cell length in srs1-1 is greater than that in the wild type, but the cell number in srs1-1 is the same as that in wild type. These results suggest that the small and round seed phenotype of srs1-1 is due to the reduction in both cell length and cell number in the longitudinal direction, and the elongation of the cells in the lateral direction of the lemma. The SRS1 mRNA and proteins are abundant in wild type rice specifically in young organs, namely young leaves, internodes and panicles. Interestingly, the tissues expressing SRS1 are closely related to the tissues that exhibit abnormalities in the srs1 mutants.

Expression profile of the α subunit of the heterotrimeric G protein in rice.

Previous studies on the activity of the rice Gα promoter using a ß-Glucuronidase (GUS) reporter construct indicated that Gα expression was highest in developing organs and changed in a developmental stage-dependent manner. In this paper, GUS activity derived from the rice Gα promoter was analyzed in seeds and developing leaves. In seeds, GUS activity was detected in the aleurone layer, embryo, endosperm and scutellar epithelium. In developing leaves, the activity was detected in the mesophyll tissues, phloem and xylem of the leaf sheath and in the mesophyll tissue of the leaf blade. The activity in the aleurone layer and scutellar epithelium suggests that the Gα subunit may be involved in gibberellin signaling. The activity in the mesophyll tissues of the leaf blade suggests that the Gα subunit may be related to the intensity of disease resistance. The pattern of the activity in the developing leaf also indicates that the expression of Gα follows a developmental profile at the tissue level.

Function and expression pattern of the alpha subunit of the heterotrimeric G protein in rice.

The d1 mutant, which is deficient for the heterotrimeric G-protein alpha subunit (Galpha) gene of rice, shows dwarfism and sets small round seeds. To determine whether dwarfism in d1 is due to a reduction in cell number or to shortened cell length, the cell number of the leaf sheath, the internode, the root and the lemma was compared between Nipponbare, a wild-type rice and d1-5, a d1 allele derived from Nipponbare. Our results indicate that the cell number was reduced in all organs analyzed in d1-5. In addition, cell enlargement was found in roots and lemma of d1-5, although the organ length in d1-5 was shorter than that of wild-type rice. These results suggest that rice Galpha participates in cell proliferation in rice. Western blot analyses using anti-Galpha antibody and RT-PCR analyses indicate that Galpha is mostly expressed in the developing organs. Galpha promoter activity studies using the GUS reporter gene confirmed that the expression of Galpha was highest in developing organs. We conclude that rice Galpha participates in the regulation of cell number in a developmental stage-dependent manner.