Yukimoto Iwasaki

A Novel Cytochrome P450 Is Implicated in Brassinosteroid Biosynthesis via the Characterization of a Rice Dwarf Mutant, dwarf11, with Reduced Seed Length

We have characterized a rice (Oryza sativa) dwarf mutant, dwarf11 (d11), that bears seeds of reduced length. To understand the mechanism by which seed length is regulated, the D11 gene was isolated by a map-based cloning method. The gene was found to encode a novel cytochrome P450 (CYP724B1), which showed homology to enzymes involved in brassinosteroid (BR) biosynthesis. The dwarf phenotype of d11 mutants was restored by the application of the brassinolide (BL). Compared with wild-type plants, the aberrant D11 mRNA accumulated at higher levels in d11 mutants and was dramatically reduced by treatment with BL, implying that the gene is feedback-regulated by BL. Precise determination of the defective step(s) in BR synthesis in d11 mutants proved intractable because of tissue specificity and the complex control of BR accumulation in plants. However, 6-deoxotyphasterol (6-DeoxoTY) and typhasterol (TY), but not any upstream intermediates before these compounds, effectively restored BR response in d11 mutants in a lamina joint bending assay. Multiple lines of evidence together suggest that the D11/CYP724B1 gene plays a role in BR synthesis and may be involved in the supply of 6-DeoxoTY and TY in the BR biosynthesis network in rice.

The heterotrimeric G protein α subunit acts upstream of the small GTPase Rac in disease resistance of rice

We used rice dwarf1 (d1) mutants lacking a single-copy Gα gene and addressed Gαs role in disease resistance. d1 mutants exhibited a highly reduced hypersensitive response to infection by an avirulent race of rice blast. Activation of PR gene expression in the leaves of the mutants infected with rice blast was delayed for 24 h relative to the wild type. H2O2 production and PR gene expression induced by sphingolipid elicitors (SE) were strongly suppressed in d1 cell cultures. Expression of the constitutively active OsRac1, a small GTPase Rac of rice, in d1 mutants restored SE-dependent defense signaling and resistance to rice blast. Gα mRNA was induced by an avirulent race of rice blast and SE application on the leaf. These results indicated the role of Gα in R gene-mediated disease resistance of rice. We have proposed a model for the defense signaling of rice in which the heterotrimeric G protein functions upstream of the small GTPase OsRac1 in the early steps of signaling.

A large-scale collection of phenotypic data describing an insertional mutant population to facilitate functional analysis of rice genes

In order to facilitate the functional analysis of rice genes, we produced about 50,000 insertion lines with the endogenous retrotransposon Tos17. Phenotypes of these lines in the M2 generation were observed in the field and characterized based on 53 phenotype descriptors. Nearly half of the lines showed more than one mutant phenotype. The most frequently observed phenotype was low fertility, followed by dwarfism. Phenotype data with photographs of each line are stored in the Tos17 mutant panel web-based database with a dataset of sequences flanking Tos17 insertion points in the rice genome (http://tos.nias.affrc.go.jp/). This combination of phenotypic and flanking sequence data will stimulate the functional analysis of rice genes.

Function of the α subunit of rice heterotrimeric G protein in brassinosteroid signaling.

The alpha subunit of plant heterotrimeric G proteins (Galpha) plays pivotal roles in multiple aspects of development and responses to plant hormones. Recently, several lines of evidence have shown that Galpha participates in brassinosteroid (BR) responses in Arabidopsis and rice plants. In this study, we conducted a comprehensive analysis of the roles of the rice Galpha in the responses to BR using a defective mutant of the Galpha gene, T65d1. Decreased sensitivity to 24-epi-brassinolide (24-epiBL) in the T65d1 mutant was observed in many processes examined, e.g. in the inhibition of root growth and the promotion of coleoptile elongation. The T65d1 mutant also showed similar phenotypes to those of BR-deficient mutants, such as the specifically shortened second internode and the constitutive photomorphogenic growth phenotype under dark conditions. However, a negative feedback effect by 24-epiBL on the expression of BR biosynthetic genes was observed in the T65d1 mutant, and the levels of BR intermediates did not fluctuate in this mutant. To determine the epistatic relationship between the T65d1 mutant and d61-7, a weak allele of a rice BR receptor mutant, the two mutants were crossed. The T65d1/d61-7 double mutant showed no epistasis in the elongation inhibition of the internodes, the internode elongation pattern, the leaf angle and the morphological abnormality of leaf, except for the vertical length of seed and the seed weight. Our results suggest that the rice Galpha affects the BR signaling cascade but the Galpha may not be a signaling molecule in BRI1-meditated perception/transduction.

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.

Characterization of the putative alpha subunit of a heterotrimeric G protein in rice.

A recombinant protein with a cDNA that encodes the putative α subunit of a rice heterotrimeric G protein was synthesized in Escherichia coli and purified. The recombinant protein (rGrice α) with an apparent molecular mass of 45 kDa was bound with guanosine 5′-(3-O-thio)triphosphate with an apparent association constant (kapp) of 0.36. The protein also hydrolyzed GTP and its Kcat was 0.44. rGrice α was ADP-ribosylated by activated cholera toxin.Monoclonal antibodies raised against rGrice α reacted with a 45 kDa polypeptide localized in the plasma membrane of rice seedlings. The peptide map of this polypeptide after digestion with V8 protease was identical to that of rGrice α. A 45 kDa polypeptide in the plasma membrane, as well as rGrice α, was ADP-ribosylated by activated cholera toxin. The GTPase activity of the plasma membrane was stimulated 2.5-fold by mastoparan 7 but not mastoparan 17. These properties were similar to those of the α subunits of heterotrimeric G proteins in animals, suggesting that the putative α subunit is truly the α subunit itself.

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 androundseed 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

Cooperative Degradation of Chitin by Extracellular and Cell Surface-Expressed Chitinases from Paenibacillus sp. Strain FPU-7

ABSTRACT 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.

Proteome analysis of rice root plasma membrane and detection of cold stress responsive proteins.

To investigate the function of plant plasma membrane, proteins of rice plasma membrane were analyzed and the proteins changed by cold stress were identified. Plasma membrane proteins were purified with an aqueous two-phase partitioning method from root of rice seedlings, and activity of specific H(+)-ATPase localized in plasma membranes was measured. The plasma membrane proteins were separated by SDS-PAGE or 2D-PAGE, and analyzed with nano LC-MS/MS. The number of transmembrane helices was predicted from the amino acid sequence of annotated proteins. Functional categorization revealed that the most of proteins were associated with energy production, signal transduction, protein synthesis, cell growth/division and defense. In addition, 12 cold stress responsive proteins were identified from the plasma membrane using 2D-PAGE based proteomics method. Out of them, cold shock protein-1 was significantly decreased in plasma membrane of rice under cold stress.

Identification of a protein kinase gene associated with pistillody, homeotic transformation of stamens into pistil-like structures, in alloplasmic wheat

Homeotic transformation of stamens into pistil-like structures (called pistillody) has been reported in cytoplasmic substitution (alloplasmic) lines of bread wheat (Triticum aestivum) having the cytoplasm of a wild relative species, Aegilops crassa. Our previous studies indicated that pistillody is caused by alterations of the class B MADS-box gene expression pattern associated with mitochondrial gene(s) in the Ae. crassa cytoplasm. To elucidate the nuclear gene involved in the cross-talk between pistillody-related mitochondrial gene(s) and nuclear homeotic genes, we performed cDNA subtraction analysis using cDNAs derived from young spikes of a pistillody line and a normal line. As a result, we identified a protein kinase gene, WPPK1 (wheat pistillody-related protein kinase 1), which is upregulated in the young spikes of the pistillody line. RT-PCR analysis indicated that WPPK1 is strongly expressed in pistils and pistil-like stamens in the pistillody line, suggesting that it is involved in the formation of pistil-like stamens as well as pistils. The full-length cDNA sequence for WPPK1 showed high similarity with a flowering plant PVPK-1 protein kinase, and phylogenetic analysis indicated that it is a member of AGC group protein kinases. Furthermore, a phosphorylation assay indicated that it has protein kinase activity. In situ hybridization analysis revealed that WPPK1 is expressed in developing pistils and pistil-like stamens as well as in their primordia. These indicate that in the alloplasmic line, WPPK1 plays a role in formation and development of pistil-like stamens.