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Panicle architecture directly affects crop productivity and is a key target of high-yield rice breeding. Panicle length strongly affects panicle architecture, but the underlying regulatory mechanisms are largely unknown. Here, we show that two quantitative trait loci (QTLs), PANICLE RACHIS LENGTH5 (Prl5) and PRIMARY BRANCH LENGTH6 (Pbl6), independently regulate panicle length in rice. Prl5 encodes a gibberellin biosynthesis enzyme, OsGA20ox4. The expression of Prl5 was higher in young panicles resulting in panicle rachis elongation. Pbl6 is identical to ABERRANT PANICLE ORGANIZATION 1 (APO1), encoding an F-box-containing protein. We found a novel function that higher expression of Pbl6 is responsible for primary branch elongation. RNA-seq analysis revealed that these two genes independently regulate panicle length at the level of gene expression. QTL pyramiding of both genes increased panicle length and productivity. By combining these two genes in various combinations, we designed numerous panicle architecture without trade-off relationship.

Growth and development are tightly co-ordinated events in the lifetime of living organisms. In temperate bamboo plants, spring is the season when environmental conditions are suitable for the emergence of new shoots. Previous studies demonstrated that bamboo plants undergo an energy-consuming 'fast stem growth' phase. However, the events during the initiation of stem elongation in bamboo are poorly understood. To understand the onset of bamboo stem growth, we performed hormone and transcriptome profiling of tissue regions in newly elongating shoots of the Moso bamboo Phyllostachys edulis. The growth hormones auxins, cytokinins and gibberellins accumulated in the shoot apex, while the stress hormones ABA, salicylic acid (SA) and jasmonic acid (JA) are predominantly found in the lower part of the stem. The mature basal part of the stem showed enrichment of transcripts associated with cell wall metabolism and biosynthesis of phenylpropanoid metabolites, such as lignin. In the young upper stem region, expression of cell formation- and DNA synthesis-related genes was enriched. Moreover, the apical region showed enhanced expression of genes involved in meristem maintenance, leaf differentiation and development, abaxial/adaxial polarity and flowering. Our findings integrate the spatial regulation of hormones and transcriptome programs during the initiation of bamboo stem growth., (© The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Domestication of crops based on artificial selection has contributed numerous beneficial traits for agriculture. Wild characteristics such as red pericarp and seed shattering were lost in both Asian (Oryza sativa) and African (Oryza glaberrima) cultivated rice species as a result of human selection on common genes. Awnedness, in contrast, is a trait that has been lost in both cultivated species due to selection on different sets of genes. In a previous report, we revealed that at least three loci regulate awn development in rice; however, the molecular mechanism underlying awnlessness remains unknown. Here we isolate and characterize a previously unidentified EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family member named REGULATOR OF AWN ELONGATION 2 (RAE2) and identify one of its requisite processing enzymes, SUBTILISIN-LIKE PROTEASE 1 (SLP1). The RAE2 precursor is specifically cleaved by SLP1 in the rice spikelet, where the mature RAE2 peptide subsequently induces awn elongation. Analysis of RAE2 sequence diversity identified a highly variable GC-rich region harboring multiple independent mutations underlying protein-length variation that disrupt the function of the RAE2 protein and condition the awnless phenotype in Asian rice. Cultivated African rice, on the other hand, retained the functional RAE2 allele despite its awnless phenotype. Our findings illuminate the molecular function of RAE2 in awn development and shed light on the independent domestication histories of Asian and African cultivated rice.

The stomatal apparatus consists of a pair of guard cells and regulates gas exchange between the leaf and atmosphere. In guard cells, blue light (BL) activates H(+)-ATPase in the plasma membrane through the phosphorylation of its penultimate threonine, mediating stomatal opening. Although this regulation is thought to be widely adopted among kidney-shaped guard cells in dicots, the molecular basis underlying that of dumbbell-shaped guard cells in monocots remains unclear. Here, we show that H(+)-ATPases are involved in the regulation of dumbbell-shaped guard cells. Stomatal opening of rice was promoted by the H(+)-ATPase activator fusicoccin and by BL, and the latter was suppressed by the H(+)-ATPase inhibitor vanadate. Using H(+)-ATPase antibodies, we showed the presence of phosphoregulation of the penultimate threonine in Oryza sativa H(+)-ATPases (OSAs) and localization of OSAs in the plasma membrane of guard cells. Interestingly, we identified one H(+)-ATPase isoform, OSA7, that is preferentially expressed among the OSA genes in guard cells, and found that loss of function of OSA7 resulted in partial insensitivity to BL. We conclude that H(+)-ATPase is involved in BL-induced stomatal opening of dumbbell-shaped guard cells in monocotyledon species., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Molecular mechanisms of hybrid breakdown associated with sterility (F2 sterility) are poorly understood as compared with those of F1 hybrid sterility. Previously, we characterized three unlinked epistatic loci, hybrid sterility-a1 (hsa1), hsa2, and hsa3, responsible for the F2 sterility in a cross between Oryza sativa ssp. indica and japonica. In this study, we identified that the hsa1 locus contains two interacting genes, HSA1a and HSA1b, within a 30-kb region. HSA1a-j (japonica allele) encodes a highly conserved plant-specific domain of unknown function protein (DUF1618), whereas the indica allele (HSA1a-i(s)) has two deletion mutations that cause disruption of domain structure. The second gene, HSA1b-i(s), encodes an uncharacterized protein with some similarity to a nucleotide-binding protein. Homozygous introgression of indica HSA1a-i(s)-HSA1b-i(s) alleles into japonica showed female gamete abortion at an early mitotic stage. The fact that the recombinant haplotype HSA1a-j-HSA1b-i(s) caused semi-sterility in the heterozygous state with the HSA1a-i(s)-HSA1b-i(s) haplotype suggests that variation in the hsa1 locus is a possible cause of the wide-spectrum sterility barriers seen in F1 hybrids and successive generations in rice. We propose a simple genetic model to explain how a single causal mechanism can drive both F1 and F2 hybrid sterility., (Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.)

Grain weight is an important crop yield component; however, its underlying regulatory mechanisms are largely unknown. Here, we identify a grain-weight quantitative trait locus (QTL) encoding a new-type GNAT-like protein that harbors intrinsic histone acetyltransferase activity (OsglHAT1). Our genetic and molecular evidences pinpointed the QTL-OsglHAT1's allelic variations to a 1.2-kb region upstream of the gene body, which is consistent with its function as a positive regulator of the traits. Elevated OsglHAT1 expression enhances grain weight and yield by enlarging spikelet hulls via increasing cell number and accelerating grain filling, and increases global acetylation levels of histone H4. OsglHAT1 localizes to the nucleus, where it likely functions through the regulation of transcription. Despite its positive agronomical effects on grain weight, yield, and plant biomass, the rare allele elevating OsglHAT1 expression has so far escaped human selection. Our findings reveal the first example, to our knowledge, of a QTL for a yield component trait being due to a chromatin modifier that has the potential to improve crop high-yield breeding.

Rice internodes are vital for supporting high-yield panicles, which are controlled by various factors such as cell division, cell elongation and cell wall biosynthesis. Therefore, formation and regulation of the internode cell-producing intercalary meristem (IM) are important for determining the shape of internodes. To understand the regulation of internode development, we analysed a rice dwarf mutant, dwarf 50 (d50). Previously, we reported that parenchyma cells in the elongated internodes of d50 ectopically deposit cell wall phenolics. In this study, we revealed that D50 encodes putative inositol polyphosphate 5-phosphatase (5PTase), which may be involved in phosphoinositide signalling required for many essential cellular functions, such as cytoskeleton organization, endocytosis and vesicular trafficking in eukaryotes. Analysis of the rice genome revealed 20 putative 5PTases including D50. The d50 mutation induced abnormally oriented cell division, irregular deposition of cell wall pectins and thick actin bundles in the parenchyma cells of the IM, resulting in abnormally organized cell files of the internode parenchyma and dwarf phenotype. Our results suggest that the putative 5PTase, encoded by D50, is essential for IM formation, including the direction of cell division, deposition of cell wall pectins and control of actin organization., (© 2012 Blackwell Publishing Ltd.)

The architecture of rice is greatly influenced by the growth of tillers, i.e. vegetative shoot branches. OsSPL14, a member of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, and strigolactones (SLs) are known to suppress outgrowth of tiller buds. OsSPL14 also regulates panicle development. We show that OsSPL14 mRNA accumulates in leaf primordia during the vegetative phase and in the primordia of bracts, or modified leaves, in the panicles, but not in the meristems. OsSPL14 is a target of miR156, and accumulation of OsSPL14 transcripts is negatively regulated by miR156. The enhancement of the expression level of OsSPL14 by the introduction of the mOsSPL14 gene, in which the miR156 cleavage site is mutated, resulted in an increase in the plastochron, an acceleration of flowering and a decrease in tiller number in the wild type and in dwarf10-2, an SL-deficient mutant. Our analysis suggests that OsSPL14 and SLs function in parallel pathways to suppress tiller growth. SLs exuded from roots trigger germination of root parasitic plants that can cause severe damage to crop productivity. SL-deficient mutants, however, exhibit an excess branching phenotype which is usually undesirable for productivity. Our results indicate that OsSPL14 can be used to manipulate the branching patterns of SL-deficient mutants. We also confirmed that this strategy is applicable to Arabidopsis. A greater understanding of the OsSPL14 and SL pathways and their interactions may help in the production of root parasite-resistant crops.

Reproductive isolation plays an important role in speciation as it restricts gene flow and accelerates genetic divergence between formerly interbreeding population. In rice, hybrid breakdown is a common reproductive isolation observed in both intra and inter-specific crosses. It is a type of post-zygotic reproductive isolation in which sterility and weakness are manifested in the F(2) and later generations. In this study, the physiological and molecular basis of hybrid breakdown caused by two recessive genes, hbd2 and hbd3, in a cross between japonica variety, Koshihikari, and indica variety, Habataki, were investigated. Fine mapping of hbd2 resulted in the identification of the causal gene as casein kinase I (CKI1). Further analysis revealed that hbd2-CKI1 allele gains its deleterious function that causes the weakness phenotype by a change of one amino acid. As for the other gene, hbd3 was mapped to the NBS-LRR gene cluster region. It is the most common class of R-gene that triggers the immune signal in response to pathogen attack. Expression analysis of pathogen response marker genes suggested that weakness phenotype in this hybrid breakdown can be attributed to an autoimmune response. So far, this is the first evidence linking autoimmune response to post-zygotic isolation in rice. This finding provides a new insight in understanding the molecular and evolutionary mechanisms establishing post-zygotic isolation in plants.

Plant height is one of the most important traits in crop improvement. Therefore revealing the mechanism of plant elongation and controlling plant height in accordance with breeding object is important. In this study we analyzed a novel dwarf mutant, ssd1, of which phenotype is different from typical GA- or BR-related dwarf phenotype. ssd1 exhibits pleiotropic defects in elongation of various organs such as stems, roots, leaves, and flowers. ssd1 also shows abnormal cell files and shapes, which suggests defects of normal cell division in the mutant. Map-based cloning and complementation test demonstrated that the dwarf phenotype in ssd1 mutant was caused by insertion of retrotransposon in a gene, which encodes plant-specific protein with unknown biochemical function. A BLAST search revealed that SSD1-like genes exist in diverse plant species, including monocots and dicots, but not fern and moss. Our results demonstrate that SSD1 controls plant elongation by controlling cell division in higher plants.

Indole-3-acetic acid (IAA) plays a critical role in many aspects of plant growth and development; however, complete pathways of biosynthesis, localization and many aspects of functions of IAA in rice remain unclear. Here, we report the analysis of a rice tryptophan- (Trp-) and IAA-deficient mutant, tryptophan deficient dwarf1 (tdd1), which is embryonic lethal because of a failure to develop most organs during embryogenesis. Regenerated tdd1 plants showed pleiotropic phenotypes: dwarfing, narrow leaves, short roots and abnormal flowers. TDD1 encodes a protein homologous to anthranilate synthase beta-subunit, which catalyses the first step of the Trp biosynthesis pathway and functions upstream of Trp-dependent IAA biosynthesis. TDD1-uidA and DR5-uidA expression overlapped at many sites in WT plants but was lacking in tdd1, indicating that TDD1 is involved in auxin biosynthesis. Both Trp and IAA levels in flowers and embryos were much lower in tdd1 than in wild type (WT). Trp feeding completely rescued the mutant phenotypes and moderate expression of OsYUCCA1, which encodes a key enzyme in Trp-dependent IAA biosynthesis, also rescued plant height and root length, indicating that the abnormal phenotypes of tdd1 are caused predominantly by Trp and IAA deficiency. In tdd1 embryos, the expression patterns of OSH1 and OsSCR, which mark the presumptive apical region and the L2 layer, respectively, are identical to those in WT, suggesting a possibility either that different IAA levels are required for basic pattern formation than for organ formation or that an orthologous gene compensates for TDD1 deficiency during pattern formation.

Living organisms must acquire new biological functions to adapt to changing and hostile environments. Deepwater rice has evolved and adapted to flooding by acquiring the ability to significantly elongate its internodes, which have hollow structures and function as snorkels to allow gas exchange with the atmosphere, and thus prevent drowning. Many physiological studies have shown that the phytohormones ethylene, gibberellin and abscisic acid are involved in this response, but the gene(s) responsible for this trait has not been identified. Here we show the molecular mechanism of deepwater response through the identification of the genes SNORKEL1 and SNORKEL2, which trigger deepwater response by encoding ethylene response factors involved in ethylene signalling. Under deepwater conditions, ethylene accumulates in the plant and induces expression of these two genes. The products of SNORKEL1 and SNORKEL2 then trigger remarkable internode elongation via gibberellin. We also demonstrate that the introduction of three quantitative trait loci from deepwater rice into non-deepwater rice enabled the latter to become deepwater rice. This discovery will contribute to rice breeding in lowland areas that are frequently flooded during the rainy season.

Epigenetic mutations confer heritable changes in gene expression that are not due to changes in the underlying sequence of the DNA. We identified a spontaneous rice mutant, Epi-d1, that shows a metastable dwarf phenotype. The phenotype is mitotically and meiotically inheritable and corresponds to the metastable epigenetic silencing of the DWARF1 (D1) gene. The silenced state is correlated with repressive histone and DNA methylation marks in the D1 promoter region but is not associated with DNA sequence alterations. Compared with other known epigenetic silenced loci in plants such as paramutable maize alleles and silent Arabidopsis genes, the Epi-d1 silencing phenomenon shows a high level of bidirectional metastable mutability. Epigenetic alleles such as Epi-d1 could thus provide for rapid adaptation under selective conditions.

In rice (Oryza sativa) and Arabidopsis thaliana, gibberellin (GA) signaling is mediated by GIBBERELLIN-INSENSITIVE DWARF1 (GID1) and DELLA proteins in collaboration with a GA-specific F-box protein. To explore when plants evolved the ability to perceive GA by the GID1/DELLA pathway, we examined these GA signaling components in the lycophyte Selaginella moellendorffii and the bryophyte Physcomitrella patens. An in silico search identified several homologs of GID1, DELLA, and GID2, a GA-specific F-box protein in rice, in both species. Sm GID1a and Sm GID1b, GID1 proteins from S. moellendorffii, showed GA binding activity in vitro and interacted with DELLA proteins from S. moellendorffii in a GA-dependent manner in yeast. Introduction of constitutively expressed Sm GID1a, Sm G1D1b, and Sm GID2a transgenes rescued the dwarf phenotype of rice gid1 and gid2 mutants. Furthermore, treatment with GA(4), a major GA in S. moellendorffii, caused downregulation of Sm GID1b, Sm GA20 oxidase, and Sm GA3 oxidase and degradation of the Sm DELLA1 protein. These results demonstrate that the homologs of GID1, DELLA, and GID2 work in a similar manner in S. moellendorffii and in flowering plants. Biochemical studies revealed that Sm GID1s have different GA binding properties from GID1s in flowering plants. No evidence was found for the functional conservation of these genes in P. patens, indicating that GID1/DELLA-mediated GA signaling, if present, differs from that in vascular plants. Our results suggest that GID1/DELLA-mediated GA signaling appeared after the divergence of vascular plants from the moss lineage.

Reproductive barriers are important for the maintenance of species identity. We discovered a reproductive barrier via hybrid breakdown among the progeny of a cross between the japonica rice cultivar Koshihikari and the indica rice cultivar Habataki. Genetic analysis indicated that the hybrid breakdown is regulated by the interaction of two recessive genes: hbd2 in Habataki and hbd3 in Koshihikari. Linkage mapping showed that hbd2 is located near the 100 cM region of chromosome 2 in Habataki, whereas hbd3 is located near the 60 cM region of chromosome 11 in Koshihikari. Construction of nearly isogenic lines for hbd2 and Hbd3 (NIL-hbd2 and NIL-Hbd3), as well as a pyramiding line (NIL-hbd2 + Hbd3), confirmed that the hybrid breakdown is induced by the interaction of these two recessive genes. Our results indicate that these genes are novel for the induction of hybrid breakdown in rice.

GIBBERELLIN INSENSITIVE DWARF1 (GID1) encodes a soluble gibberellin (GA) receptor that shares sequence similarity with a hormone-sensitive lipase (HSL). Previously, a yeast two-hybrid (Y2H) assay revealed that the GID1-GA complex directly interacts with SLENDER RICE1 (SLR1), a DELLA repressor protein in GA signaling. Here, we demonstrated, by pull-down and bimolecular fluorescence complementation (BiFC) experiments, that the GA-dependent GID1-SLR1 interaction also occurs in planta. GA(4) was found to have the highest affinity to GID1 in Y2H assays and is the most effective form of GA in planta. Domain analyses of SLR1 using Y2H, gel filtration, and BiFC methods revealed that the DELLA and TVHYNP domains of SLR1 are required for the GID1-SLR1 interaction. To identify the important regions of GID1 for GA and SLR1 interactions, we used many different mutant versions of GID1, such as the spontaneous mutant GID1s, N- and C-terminal truncated GID1s, and mutagenized GID1 proteins with conserved amino acids replaced with Ala. The amino acid residues important for SLR1 interaction completely overlapped the residues required for GA binding that were scattered throughout the GID1 molecule. When we plotted these residues on the GID1 structure predicted by analogy with HSL tertiary structure, many residues were located at regions corresponding to the substrate binding pocket and lid. Furthermore, the GA-GID1 interaction was stabilized by SLR1. Based on these observations, we proposed a molecular model for interaction between GA, GID1, and SLR1.

SPINDLY (SPY) encodes an O-linked N-acetylglucosamine transferase that is considered to be a negative regulator of gibberellin (GA) signaling through an unknown mechanism. To understand the function of SPY in GA signaling in rice, we isolated a rice SPINDLY homolog (OsSPY) and produced knockdown transgenic plants in which OsSPY expression was reduced by introducing its antisense or RNAi construct. In knockdown plants, the enhanced elongation of lower internodes was correlated with decreased levels of OsSPY expression, similar to the spindly phenotype of Arabidopsis spy mutants, suggesting that OsSPY also functions as a negative factor in GA signaling in rice. The suppressive function of OsSPY in GA signaling was supported by the findings that the dwarfism was partially rescued and OsGA20ox2 (GA20 oxidase) expression was reduced in GA-deficient and GA-insensitive mutants by the knockdown of OsSPY function. The suppression of OsSPY function in a GA-insensitive mutant, gid2, also caused an increase in the phosphorylation of a rice DELLA protein, SLR1, but did not change the amount of SLR1. This indicates that the function of OsSPY in GA signaling is not via changes in the amount or stability of SLR1, but probably involves control of the suppressive function of SLR1. In addition to the GA-related phenotypes, OsSPY antisense and RNAi plants showed increased lamina joint bending, which is a brassinosteroid-related phenotype, indicating that OsSPY may play roles both in GA signaling and in the brassinosteroid pathway.

GAMYB is a component of gibberellin (GA) signaling in cereal aleurone cells, and has an important role in flower development. However, it is unclear how GAMYB function is regulated. We examined the involvement of a microRNA, miR159, in the regulation of GAMYB expression in cereal aleurone cells and flower development. In aleurone cells, no miR159 expression was observed with or without GA treatment, suggesting that miR159 is not involved in the regulation of GAMYB and GAMYB-like genes in this tissue. miR159 was expressed in tissues other than aleurone, and miR159 over-expressors showed similar but more severe phenotypes than the gamyb mutant. GAMYB and GAMYB-like genes are co-expressed with miR159 in anthers, and the mRNA levels for GAMYB and GAMYB-like genes are negatively correlated with miR159 levels during anther development. Thus, OsGAMYB and OsGAMYB-like genes are regulated by miR159 in flowers. A microarray analysis revealed that OsGAMYB and its upstream regulator SLR1 are involved in the regulation of almost all GA-mediated gene expression in rice aleurone cells. Moreover, different sets of genes are regulated by GAMYB in aleurone cells and anthers. GAMYB binds directly to promoter regions of its target genes in anthers as well as aleurone cells. Based on these observations, we suggest that the regulation of GAMYB expression and GAMYB function are different in aleurone cells and flowers in rice.

The rice SLR1 (SLENDER RICE 1) gene encodes a DELLA protein that belongs to a subfamily of the GRAS protein superfamily and that functions as a repressor of gibberellin (GA) signaling. Based on the constitutive GA response phenotype of slr1 mutants, SLR1 has been thought to be the sole DELLA-type protein suppressing GA signals in rice. However, in rice genome databases we identified two sequences homologous to SLR1: SLR1-like1 and -2 (SLRL1 and -2). SLRL1 and SLRL2 contain regions with high similarity to the C-terminal conserved domains in SLR1, but lack the N-terminal conserved region of the DELLA proteins. The expression of SLRL1 was positively regulated by GA at the mRNA level and occurred preferentially in reproductive organs, whereas SLRL2 was moderately expressed in mature leaf organs and was not affected by GA. Transformation of SLRL1 into the slr1 mutant rescued the slender phenotype of this mutant. Moreover, overexpression of SLRL1 in normal rice plants induced a dwarf phenotype with an increased level of OsGA20ox2 gene expression and diminished the GA-induced shoot elongation, suggesting that SLRL1 acts as a repressor of GA signaling. Consistent with the fact that SLRL1 does not have a DELLA domain, which is essential for degradation of DELLA proteins, a level of SLRL1 protein was not degraded by application of gibberellic acid. However, the repressive activity of SLRL1 against GA signaling was much weaker than a truncated SLR1 lacking the DELLA domain. Based on these characteristics of SLRL1, the functional roles of SLRL1 in GA signaling in rice are discussed.

Gibberellins (GAs) are phytohormones that are essential for many developmental processes in plants. It has been postulated that plants have both membrane-bound and soluble GA receptors; however, no GA receptors have yet been identified. Here we report the isolation and characterization of a new GA-insensitive dwarf mutant of rice, gid1. The GID1 gene encodes an unknown protein with similarity to the hormone-sensitive lipases, and we observed preferential localization of a GID1-green fluorescent protein (GFP) signal in nuclei. Recombinant glutathione S-transferase (GST)-GID1 had a high affinity only for biologically active GAs, whereas mutated GST-GID1 corresponding to three gid1 alleles had no GA-binding affinity. The dissociation constant for GA4 was estimated to be around 10(-7) M, enough to account for the GA dependency of shoot elongation. Moreover, GID1 bound to SLR1, a rice DELLA protein, in a GA-dependent manner in yeast cells. GID1 overexpression resulted in a GA-hypersensitive phenotype. Together, our results indicate that GID1 is a soluble receptor mediating GA signalling in rice.

DELLA proteins are repressors of gibberellin signaling in plants. Our previous studies have indicated that gibberellin signaling is derepressed by SCF(GID2)-mediated proteolysis of the DELLA protein, SLENDER RICE1 (SLR1), in rice. In addition, the gibberellin-dependent increase of phosphorylated SLR1 in the loss-of-function gid2 mutant suggests that the SCF(GID2)-mediated degradation of SLR1 might be initiated by gibberellin-dependent phosphorylation. To confirm the role of phosphorylation of SLR1 in its gibberellin-dependent degradation, we revealed that SLR1 is phosphorylated on an N-terminal serine residue(s) within the DELLA/TVHYNP and polyS/T/V domain. However, gibberellin-induced phosphorylation in these regions was not observed in the gid2 mutant following the constitutive expression of SLR1 under the control of the rice actin1 promoter. Treatment with gibberellin induced both the phosphorylated and non-phosphorylated forms of SLR1 with similar induction kinetics in gid2 mutant cells. Both the phosphorylated and non-phosphorylated SLR1 proteins were degraded by gibberellin treatment with a similar half-life in the rice callus cells, and both proteins interacted with recombinant glutathione S-transferase (GST)-GID2. These results demonstrate that the phosphorylation of SLR1 is independent of its degradation and is dispensable for the interaction of SLR1 with the GID2/F-box protein.

Although the importance of auxin in root development is well known, the molecular mechanisms involved are still unknown. We characterized a rice (Oryza sativa) mutant defective in crown root formation, crown rootless1 (crl1). The crl1 mutant showed additional auxin-related abnormal phenotypic traits in the roots, such as decreased lateral root number, auxin insensitivity in lateral root formation, and impaired root gravitropism, whereas no abnormal phenotypic traits were observed in aboveground organs. Expression of Crl1, which encodes a member of the plant-specific ASYMMETRIC LEAVES2/LATERAL ORGAN BOUNDARIES protein family, was localized in tissues where crown and lateral roots are initiated and overlapped with beta-glucuronidase staining controlled by the DR5 promoter. Exogenous auxin treatment induced Crl1 expression without de novo protein biosynthesis, and this induction required the degradation of AUXIN/INDOLE-3-ACETIC ACID proteins. Crl1 contains two putative auxin response elements (AuxREs) in its promoter region. The proximal AuxRE specifically interacted with a rice AUXIN RESPONSE FACTOR (ARF) and acted as a cis-motif for Crl1 expression. We conclude that Crl1 encodes a positive regulator for crown and lateral root formation and that its expression is directly regulated by an ARF in the auxin signaling pathway.

A small family of plant proteins, designated PSEUDO RESPONSE REGULATORS (PRRs), is crucial for a better understanding of the molecular link between circadian rhythm and photoperiodic control of flowering time in the dicotyledonous model plant Arabidopsis thaliana. Recently, we showed that the monocotyledonous model plant Oryza sativa also has homologous members of the OsPRR family (Oryza sativa PRR). In the previous experiments with rice, we mainly characterized a japonica variety (Nipponbare). By employing an indica variety (Kasalath), in this study we further characterized OsPRRs with reference to the photoperiod sensitivity Hd (Heading date) QTL (quantitative trait loci) implicated in the control of flowering time in rice. The circadian-controlled and sequential expression profiles of the five OsPRR genes were observed not only for Nipponbare but also for Kasalath. Then each of these OsPRR genes was mapped on the rice chromosomes. Among these OsPRR genes, OsPRR37 was mapped very closely to Hd2-QTL, which was identified as the major locus that enhances the photoperiod sensitivity of flowering in Nipponbare. Furthermore, we found that Kasalath has a severe mutational lesion in the OsPRR37 coding sequence.

The regulation of floral organ number is closely associated with floral meristem size. Mutations in the gene FLORAL ORGAN NUMBER1 (FON1) cause enlargement of the floral meristem in Oryza sativa (rice), resulting in an increase in the number of all floral organs. Ectopic floral organs develop in the whorl of each organ and/or in the additional whorls that form. Inner floral organs are more severely affected than outer floral organs. Many carpel primordia develop indeterminately, and undifferentiated meristematic tissues remain in the center in almost-mature flowers. Consistent with this result, OSH1, a molecular marker of meristematic indeterminate cells in rice, continues to be expressed in this region. Although floral meristems are strongly affected by the fon1-2 mutation, vegetative and inflorescence meristems are largely normal, even in this strong allele. We isolated the FON1 gene by positional cloning and found that it encodes a leucine-rich repeat receptor-like kinase most similar to CLAVATA1 (CLV1) in Arabidopsis thaliana. This suggests that a pathway similar to the CLV signaling system that regulates meristem maintenance in Arabidopsis is conserved in the grass family. Unlike CLV1, which is predominantly expressed in the L3 layer of the shoot meristem, FON1 is expressed throughout the whole floral meristem, suggesting that small modifications to the CLV signaling pathway may be required to maintain the floral meristem in rice. In addition, FON1 transcripts are detected in all meristems responsible for development of the aerial part of rice, suggesting that genes sharing functional redundancy with FON1 act in the vegetative and inflorescence meristems to mask the effects of the fon1 mutation.

The phytohormone gibberellin (GA) controls growth and development in plants. Previously, we identified a rice F-box protein, gibberellin-insensitive dwarf2 (GID2), which is essential for GA-mediated DELLA protein degradation. In this study, we analyzed the biological and molecular biological properties of GID2. Expression of GID2 preferentially occurred in rice organs actively synthesizing GA. Domain analysis of GID2 revealed that the C-terminal regions were essential for the GID2 function, but not the N-terminal region. Yeast two-hybrid assay and immunoprecipitation experiments demonstrated that GID2 is a component of the SCF complex through an interaction with a rice ASK1 homolog, OsSkp15. Furthermore, an in vitro pull-down assay revealed that GID2 specifically interacted with the phosphorylated Slender Rice 1 (SLR1). Taken these results together, we conclude that the phosphorylated SLR1 is caught by the SCFGID2 complex through an interacting affinity between GID2 and phosphorylated SLR1, triggering the ubiquitin-mediated degradation of SLR1.

GAMYB was first isolated as a positive transcriptional regulator of gibberellin (GA)-dependent alpha-amylase expression in barley aleurone cells, and its molecular and biochemical properties have been well characterized. However, the role of GAMYB elsewhere in the plant is not well understood. To investigate the molecular function of GAMYB outside of the aleurone cells, we isolated loss-of-function mutants from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17. Through PCR screening using primers for rice GAMYB (OsGAMYB) and Tos17, we isolated three independent mutant alleles that contained Tos17 inserted in the exon region. No alpha-amylase expression in the endosperm was induced in these mutants in response to GA treatment, indicating that the Tos17 insertion had knocked out OsGAMYB function. We found no significant defects in the growth and development of the mutants at the vegetative stage. After the phase transition to the reproductive stage, however, shortened internodes and defects in floral organ development, especially a defect in pollen development, were observed. On the other hand, no difference was detected in flowering time. High-level OsGAMYB expression was detected in the aleurone cells, inflorescence shoot apical region, stamen primordia, and tapetum cells of the anther, but only low-level expression occurred in organs at the vegetative stage or in the elongating stem. These results demonstrate that, in addition to its role in the induction of alpha-amylase in aleurone, OsGAMYB also is important for floral organ development and essential for pollen development.

We characterized a rice dwarf mutant, ebisu dwarf (d2). It showed the pleiotropic abnormal phenotype similar to that of the rice brassinosteroid (BR)-insensitive mutant, d61. The dwarf phenotype of d2 was rescued by exogenous brassinolide treatment. The accumulation profile of BR intermediates in the d2 mutants confirmed that these plants are deficient in late BR biosynthesis. We cloned the D2 gene by map-based cloning. The D2 gene encoded a novel cytochrome P450 classified in CYP90D that is highly similar to the reported BR synthesis enzymes. Introduction of the wild D2 gene into d2-1 rescued the abnormal phenotype of the mutants. In feeding experiments, 3-dehydro-6-deoxoteasterone, 3-dehydroteasterone, and brassinolide effectively caused the lamina joints of the d2 plants to bend, whereas more upstream compounds did not cause bending. Based on these results, we conclude that D2/CYP90D2 catalyzes the steps from 6-deoxoteasterone to 3-dehydro-6-deoxoteasterone and from teasterone to 3-dehydroteasterone in the late BR biosynthesis pathway.

Gibberellin (GA) regulates growth and development in plants. We isolated and characterized a rice GA-insensitive dwarf mutant, gid2. The GID2 gene encodes a putative F-box protein, which interacted with the rice Skp1 homolog in a yeast two-hybrid assay. In gid2, a repressor for GA signaling, SLR1, was highly accumulated in a phosphorylated form and GA increased its concentration, whereas SLR1 was rapidly degraded by GA through ubiquitination in the wild type. We conclude that GID2 is a positive regulator of GA signaling and that regulated degradation of SLR1 is initiated through GA-dependent phosphorylation and finalized by an SCF(GID2)-proteasome pathway.

The slender rice1 mutant (slr1) shows a constitutive gibberellin (GA) response phenotype. To investigate the mode of action of SLR1, we generated transgenic rice expressing a fusion protein consisting of SLR1 and green fluorescent protein (SLR1-GFP) and analyzed the phenotype of the transformants and the subcellular localization of GFP in vivo. SLR1-GFP worked in nuclei to repress the GA signaling pathway; its overproduction caused a dwarf phenotype. Application of GA(3) to SLR1-GFP overproducers induced GA actions such as shoot elongation, downregulation of GA 20-oxidase expression, and upregulation of SLR1 expression linked with the disappearance of the nuclear SLR1-GFP protein. We also performed domain analyses of SLR1 using transgenic plants overproducing different kinds of truncated SLR1 proteins. The analyses revealed that the SLR1 protein can be divided into four parts: a GA signal perception domain located at the N terminus, a regulatory domain for its repression activity, a dimer formation domain essential for signal perception and repression activity, and a repression domain at the C terminus. We conclude that GA signal transduction is regulated by the appearance or disappearance of the nuclear SLR1 protein, which is controlled by the upstream GA signal.