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.

Previously, we reported that the rice dwarf mutant, d1, is defective in the ␣ subunit of the heterotrimeric G protein (G␣). In the present study, gibberellin (GA) signaling in d1 and the role of the G␣ protein in the GA-signaling pathway were investigated. Compared with the wild type, GA induction of ␣-amylase activity in aleurone cells of d1 was greatly reduced. Relative to the wild type, the GA 3-treated aleurone layer of d1 had lower expression of Ramy1A, which encodes ␣-amylase, and OsGAMYB, which encodes a GAinducible transcriptional factor, and no increase in expression of Ca 2 ؉ -ATPase. However, in the presence of high GA concentrations, ␣-amylase induction occurred even in d1. The GA sensitivity of second leaf sheath elongation in d1 was similar to that of the wild type in terms of dose responsiveness, but the response of internode elongation to GA was much lower in d1. Furthermore, Os20ox expression was up-regulated, and the GA content was elevated in the stunted internodes of d1. All these results suggest that d1 affects a part of the GA-signaling pathway, namely the induction of ␣-amylase in the aleurone layer and internode elongation. In addition, a double mutant between d1 and another GA-signaling mutant, slr, revealed that SLR is epistatic to the D1, supporting that the G␣ protein is involved in GA signaling. However, the data also provide evidence for the presence of an alternative GA-signaling pathway that does not involve the G␣ protein. It is proposed that GA signaling via the G␣ protein may be more sensitive than that of the alternative pathway, as indicated by the low GA responsiveness of this G␣-independent pathway. H eterotrimeric G proteins are associated with the cytoplasmic face of the plasma membrane of eukaryotic cells and mediate signalings from receptors on the cell surface. The ␣-subunits of heterotrimeric G (G␣) proteins transduce signals from G protein-coupled receptors to effector proteins, accompanied by the GTPase-catalyzed hydrolysis of GTP. In mammals, a set of genes has been identified for each of the G protein subunits: more than 16 genes for the ␣ subunits, 5 genes for the ␤ subunit, and 6 genes for the ␥ subunit, most of which are expressed in a tissue-specific manner. Thus mammals contain multiforms of the G proteins that are probably involved in separate systems of signal transduction. Genomic and cDNA clones that encode polypeptides similar to the mammalian G␣ proteins have also been isolated from various plant species, including Arabidopsis (1), tomato (2), Lotus japonicas (3), rice (4), and soybean (5). However, with the exception of soybean, which has two genes, only a single gene has been identified in each plant species. Nevertheless, it has been proposed that the plant heterotrimeric G protein is involved in various signal transduction systems, including those of several plant hormones, blue and red light-mediated responses, pathogen resistance, and pathogen-related gene expression.By using a constitutive stimulator (Mas7) of GDP͞GTP exchange by G␣ proteins, Jon...

We used rice dwarf1 (d1) mutants lacking a single-copy G␣ gene and addressed G␣'s 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. G␣ mRNA was induced by an avirulent race of rice blast and SE application on the leaf. These results indicated the role of G␣ 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. Heterotrimeric G proteins, a major group of signaling molecules involved in a variety of cellular activities in mammals, are mainly responsible for various cellular responses to external signals (1). In mammals, G proteins consist of ␣, ␤, and ␥ subunits, and at least 23 ␣, 6 ␤, and 12 ␥ genes are known (2). In plants, a number of pharmacological studies suggested that heterotrimeric G proteins are involved in a variety of signaling, including light reception (3), hormone signaling (4), and regulation of ion channels (5). However, direct evidence to support these observations has been obtained only recently (6). Analysis of mutations in a gene encoding the G␣ subunit of rice termed dwarf1 (d1) showed that G␣ is involved in stem elongation and the determination of seed shape in rice (7, 8) and influences gibberellin signal transduction (9). More recently, in Arabidopsis, mutants in the single-copy G␣ subunit gene were shown to have reduced cell division in aerial tissues (10) and to lack regulation of the ion channel by the phytohormone abscisic acid in guard cells (11). Furthermore, involvement of G␣ in phytochromemediated light signal transduction of Arabidopsis was also demonstrated by the study of transgenic plants overexpressing the G␣ gene (12). Arabidopsis mutants lacking G␤ were recently shown to have effects in leaf, flower, and fruit development (13). Therefore, the importance and diverse functions of heterotrimeric G proteins in the signaling of plants are recently becoming clear; however, the molecular mechanisms of G protein signaling remain to be studied.Many studies using inhibitors and agonists of heterotrimeric G proteins in several plant species have suggested that G proteins are involved in defense signaling (14-17). Particularly, changes in cytosolic Ca 2ϩ concentrations, which are often observed in elicitor-treated plant cells, are assumed to be regulated by heterotrimeric G proteins (18). However, the roles of the heterotrimeric G protein in plant defense have not been directly tested by the use of G protein mutants.In th...

Transgenic rice containing an antisense cDNA for the ␣ subunit of rice heterotrimeric G protein produced little or no mRNA for the subunit and exhibited abnormal morphology, including dwarf traits and the setting of small seeds. In normal rice, the mRNA for the ␣ subunit was abundant in the internodes and f lorets, the tissues closely related to abnormality in the dwarf transformants. The position of the ␣-subunit gene was mapped on rice chromosome 5 by mapping with the restriction fragment length polymorphism. The position was closely linked to the locus of a rice dwarf mutant, Daikoku dwarf (d-1), which is known to exhibit abnormal phenotypes similar to those of the transformants that suppressed the endogenous mRNA for the ␣ subunit by antisense technology. Analysis of the cDNAs for the ␣ subunits of five alleles of Daikoku dwarf (d-1), ID-1, DK22, DKT-1, DKT-2, and CM1361-1, showed that these dwarf mutants had mutated in the coding region of the ␣-subunit gene. These results show that the G protein functions in the formation of normal internodes and seeds in rice.