Miyako Ueguchi-Tanaka

GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberellin.

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.

slender Rice, a Constitutive Gibberellin Response Mutant, Is Caused by a Null Mutation of the SLR1 Gene, an Ortholog of the Height-Regulating Gene GAI/RGA/RHT/D8

The rice slender mutant (slr1-1) is caused by a single recessive mutation and results in a constitutive gibberellin (GA) response phenotype. The mutant elongates as if saturated with GAs. In this mutant, (1) elongation was unaffected by an inhibitor of GA biosynthesis, (2) GA-inducible α-amylase was produced by the aleurone layers without gibberellic acid application, and (3) endogenous GA content was lower than in the wild-type plant. These results indicate that the product of the SLR1 gene is an intermediate of the GA signal transduction pathway. SLR1 maps to OsGAI in rice and has significant homology with height-regulating genes, such as RHT-1Da in wheat, D8 in maize, and GAI and RGA in Arabidopsis. The GAI gene family is likely to encode transcriptional factors belonging to the GRAS gene superfamily. DNA sequence analysis revealed that the slr1-1 mutation is a single basepair deletion of the nuclear localization signal domain, resulting in a frameshift mutation that abolishes protein production. Furthermore, introduction of a 6-kb genomic DNA fragment containing the wild-type SLR1 gene into the slr1-1 mutant restored GA sensitivity to normal. These results indicate that the slr1-1 mutant is caused by a loss-of-function mutation of the SLR1 gene, which is an ortholog of GAI, RGA, RHT, and D8. We also succeeded in producing GA-insensitive dwarf rice by transforming wild-type rice with a modified SLR1 gene construct that has a 17–amino acid deletion affecting the DELLA region. Thus, we demonstrate opposite GA response phenotypes depending on the type of mutations in SLR1.

An Overview of Gibberellin Metabolism Enzyme Genes and Their Related Mutants in Rice

To enhance our understanding of GA metabolism in rice (Oryza sativa), we intensively screened and identified 29 candidate genes encoding the following GA metabolic enzymes using all available rice DNA databases: ent-copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA 20-oxidase (GA20ox), GA 3-oxidase (GA3ox), and GA 2-oxidase (GA2ox). In contrast to the Arabidopsis genome, multiple CPS-like, KS-like, and KO-like genes were identified in the rice genome, most of which are contiguously arranged. We also identified 18 GA-deficient rice mutants at six different loci from rice mutant collections. Based on the mutant and expression analyses, we demonstrated that the enzymes catalyzing the early steps in the GA biosynthetic pathway (i.e. CPS, KS, KO, and KAO) are mainly encoded by single genes, while those for later steps (i.e. GA20ox, GA3ox, and GA2ox) are encoded by gene families. The remaining CPS-like, KS-like, and KO-like genes were likely to be involved in the biosynthesis of diterpene phytoalexins rather than GAs because the expression of two CPS-like and three KS-like genes (OsCPS2, OsCPS4, OsKS4, OsKS7, and OsKS8) were increased by UV irradiation, and four of these genes (OsCPS2, OsCPS4, OsKS4, and OsKS7) were also induced by an elicitor treatment.

Erect leaves caused by brassinosteroid deficiency increase biomass production and grain yield in rice

New cultivars with very erect leaves, which increase light capture for photosynthesis and nitrogen storage for grain filling, may have increased grain yields. Here we show that the erect leaf phenotype of a rice brassinosteroid–deficient mutant, osdwarf4-1, is associated with enhanced grain yields under conditions of dense planting, even without extra fertilizer. Molecular and biochemical studies reveal that two different cytochrome P450s, CYP90B2/OsDWARF4 and CYP724B1/D11, function redundantly in C-22 hydroxylation, the rate-limiting step of brassinosteroid biosynthesis. Therefore, despite the central role of brassinosteroids in plant growth and development, mutation of OsDWARF4 alone causes only limited defects in brassinosteroid biosynthesis and plant morphology. These results suggest that regulated genetic modulation of brassinosteroid biosynthesis can improve crops without the negative environmental effects of fertilizers.

The Gibberellin Signaling Pathway Is Regulated by the Appearance and Disappearance of SLENDER RICE1 in Nuclei

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 GA3 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.

A Rice Brassinosteroid-Deficient Mutant, ebisu dwarf (d2), Is Caused by a Loss of Function of a New Member of Cytochrome P450

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.

Crown rootless1, Which Is Essential for Crown Root Formation in Rice, Is a Target of an AUXIN RESPONSE FACTOR in Auxin Signaling

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 β-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.

Molecular Interactions of a Soluble Gibberellin Receptor, GID1, with a Rice DELLA Protein, SLR1, and Gibberellin

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. GA4 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.

Structural basis for gibberellin recognition by its receptor GID1.

Gibberellins (GAs) are phytohormones essential for many developmental processes in plants. A nuclear GA receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1), has a primary structure similar to that of the hormone-sensitive lipases (HSLs). Here we analyse the crystal structure of Oryza sativa GID1 (OsGID1) bound with GA4 and GA3 at 1.9 Å resolution. The overall structure of both complexes shows an α/β-hydrolase fold similar to that of HSLs except for an amino-terminal lid. The GA-binding pocket corresponds to the substrate-binding site of HSLs. On the basis of the OsGID1 structure, we mutagenized important residues for GA binding and examined their binding activities. Almost all of them showed very little or no activity, confirming that the residues revealed by structural analysis are important for GA binding. The replacement of Ile 133 with Leu or Val—residues corresponding to those of the lycophyte Selaginella moellendorffii GID1s—caused an increase in the binding affinity for GA34, a 2β-hydroxylated GA4. These observations indicate that GID1 originated from HSL and was further modified to have higher affinity and more strict selectivity for bioactive GAs by adapting the amino acids involved in GA binding in the course of plant evolution.

Loss-of-Function Mutations of the Rice GAMYB Gene Impair α-Amylase Expression in Aleurone and Flower Development

GAMYB was first isolated as a positive transcriptional regulator of gibberellin (GA)-dependent α-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 α-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 α-amylase in aleurone, OsGAMYB also is important for floral organ development and essential for pollen development.