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.

SummaryTwo genes in the rice genome were identi®ed as those encoding the g subunits, g1 and g2, of heterotrimeric G proteins. Using antibodies against the recombinant proteins for the a, b, g1, and g2 subunits of the G protein complexes, all of the subunits were proven to be localized in the plasma membrane in rice. Gel ®ltration of solubilized plasma membrane proteins showed that all of the a subunits were present in large protein complexes (about 400 kDa) containing the other subunits, b, g1, and g2, and probably also some other proteins, whereas large amounts of the b and g (g1 and g2) subunits were freed from the large complexes and took a 60-kDa form. A yeast two-hybrid assay and coimmunoprecipitation experiments showed that the b subunit interacted tightly with the g1 and g2 subunits, and so the b and g subunits appeared to form dimers in rice cells. Some dimers were associated with the a subunit, because few b, g1, and g2 subunits were present in the 400-kDa complexes in a rice mutant, d1, which was lacking in the a subunit. When a constitutively active form of the a subunit was prepared by the exchange of one amino acid residue and introduced into d1, the mutagenized subunit was localized in the plasma membrane of the transformants and took a free, and not the 400-kDa, form.

SUMMARYIn the present study, we investigated the function of the heterotrimeric G protein b-subunit (Gb) 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 a-subunit (Ga) 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 Gbc and Ga cooperatively function in cellular proliferation and seed fertility. We discuss the potential predominant role of RGB1 in G protein signaling in rice.

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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.