GIBBERELLIN INSENSITIVE DWARF1 Research Articles

Gibberellin homeostasis and plant height control by EUI and a role for gibberellin in root gravity responses in rice

The rice Eui (ELONGATED UPPERMOST INTERNODE) gene encodes a cytochrome P450 monooxygenase that deactivates bioactive gibberellins (GAs). In this study, we investigated controlled expression of the Eui gene and its role in plant development. We found that Eui was differentially induced by exogenous GAs and that the Eui promoter had the highest activity in the vascular bundles. The eui mutant was defective in starch granule development in root caps and Eui overexpression enhanced starch granule generation and gravity responses, revealing a role for GA in root starch granule development and gravity responses. Experiments using embryoless half-seeds revealed that RAmy1A and GAmyb were highly upregulated in eui aleurone cells in the absence of exogenous GA. In addition, the GA biosynthesis genes GA3ox1 and GA20ox2 were downregulated and GA2ox1 was upregulated in eui seedlings. These results indicate that EUI is involved in GA homeostasis, not only in the internodes at the heading stage, but also in the seedling stage, roots and seeds. Disturbing GA homeostasis affected the expression of the GA signaling genes GID1 (GIBBERELLIN INSENSITIVE DWARF 1), GID2 and SLR1. Transgenic RNA interference of the Eui gene effectively increased plant height and improved heading performance. By contrast, the ectopic expression of Eui under the promoters of the rice GA biosynthesis genes GA3ox2 and GA20ox2 significantly reduced plant height. These results demonstrate that a slight increase in Eui expression could dramatically change rice morphology, indicating the practical application of the Eui gene in rice molecular breeding for a high yield potential.

Gibberellin Metabolism, Perception and Signaling Pathways in Arabidopsis

Bioactive gibberellins (GAs) are diterpene phytohormones that modulate growth and development throughout the whole life cycle of the plant. Arabidopsis genes encoding most GA biosynthesis and catabolism enzymes, as well as GA receptors (GIBBERELLIN INSENSITIVE DWARF1, GID1) and early GA signaling components have been identified. Expression studies on the GA biosynthesis genes are beginning to reveal the potential sites of GA biosynthesis during plant development. Biochemical and genetic analyses demonstrate that GA de-represses its signaling pathway by binding to GID1s, which induce degradation of GA signaling repressors (DELLAs) via an ubiquitin-proteasome pathway. To modulate plant growth and development, the GA pathway is also regulated by endogenous signals (other hormones) and environmental cues (such as light, temperature and salt stress). In many cases, these internal and external cues directly affect GA metabolism and bioactive GA levels, and indirectly alter DELLA accumulation and GA responses. Importantly, direct negative interaction between DELLA and PIF3 and PIF4 (2 phytochrome interacting transcription factors) appears to integrate the effects of light and GA on hypocotyl elongation.

The GID1-Mediated Gibberellin Perception Mechanism Is Conserved in the Lycophyte Selaginella moellendorffii but Not in the Bryophyte Physcomitrella patens

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.

GA action: turning on de-DELLA repressing signaling

Phytohormone gibberellins (GA) are a large family of tetracyclic diterpenoids and play the important roles in modulation of plant growth and development throughout the plant life cycle. GA depresses its signaling by the GA-promoted destabilization of the DELLA protein growth repressors via 26S proteasome pathway. Recent evidences indicate that the DELLA proteins interact with multiple environmental and other hormonal response pathways and confer plant growth restraint. Furthermore, the discovery of rice GIBBERELLIN INSENSITIVE DWARF1 (GID1) and three Arabidopsis AtGID1 homologs as soluble GA receptors opens new prospects for understanding of de-DELLA repressing system in GA signaling.

Gibberellin Receptor and Its Role in Gibberellin Signaling in Plants

Gibberellins (GAs) are a large family of tetracyclic, diterpenoid plant hormones that induce a wide range of plant growth responses. It has been postulated that plants have two types of GA receptors, including soluble and membrane-bound forms. Recently, it was determined that the rice GIBBERELLIN INSENSITIVE DWARF1 (GID1) gene encodes an unknown protein with similarity to the hormone-sensitive lipases that has high affinity only for biologically active GAs. Moreover, GID1 binds to SLR1, a repressor of GA signaling, in a GA-dependent manner in yeast cells. Based on these observations, it has been concluded that GID1 is a soluble receptor mediating GA signaling in rice. More recently, Arabidopsis thaliana was found to have three GID1 homologs, AtGID1a, b, and c, all of which bind GA and interact with the five Arabidopsis DELLA proteins.

Genetic Characterization and Functional Analysis of the GID1 Gibberellin Receptors inArabidopsis

We investigated the physiological function of three Arabidopsis thaliana homologs of the gibberellin (GA) receptor GIBBERELLIN-INSENSITIVE DWARF1 (GID1) by determining the developmental consequences of GID1 inactivation in insertion mutants. Although single mutants developed normally, gid1a gid1c and gid1a gid1b displayed reduced stem height and lower male fertility, respectively, indicating some functional specificity. The triple mutant displayed a dwarf phenotype more severe than that of the extreme GA-deficient mutant ga1-3. Flower formation occurred in long days but was delayed, with severe defects in floral organ development. The triple mutant did not respond to applied GA. All three GID1 homologs were expressed in most tissues throughout development but differed in expression level. GA treatment reduced transcript abundance for all three GID1 genes, suggesting feedback regulation. The DELLA protein REPRESSOR OF ga1-3 (RGA) accumulated in the triple mutant, whose phenotype could be partially rescued by loss of RGA function. Yeast two-hybrid and in vitro pull-down assays confirmed that GA enhances the interaction between GID1 and DELLA proteins. In addition, the N-terminal sequence containing the DELLA domain is necessary for GID1 binding. Furthermore, yeast three-hybrid assays showed that the GA-GID1 complex promotes the interaction between RGA and the F-box protein SLY1, a component of the SCF(SLY1) E3 ubiquitin ligase that targets the DELLA protein for degradation.

Rice GIBBERELLIN INSENSITIVE DWARF1 Is a Gibberellin Receptor That Illuminates and Raises Questions about GA Signaling

Recently, gibberellins (GAs) joined the list of plant hormones with a known receptor protein with the report by [Ueguchi-Tanaka et al. (2005)][1] that the GIBBERELLIN INSENSITIVE DWARF1 (GID1) protein of rice has the expected properties of the long-sought GA receptor. This exciting discovery not

Transfer of a Dwarf Gene from Brassica rapa to Oilseed B. napus, Effects on Agronomic Traits, and Development of a ‘Perfect’ Marker for Selection

Lodging can be a serious problem in oilseed Brassica napus. Dwarf genes have been used to reduce lodging in other crops, and their use in wheat and rice was a major factor in the success of the ‘Green Revolution’. We previously reported on a single, semi-dominant, gibberellin insensitive dwarf mutant of B. rapa (Brrga1-d), and provided evidence for homology of this gene to the wheat ‘Green Revolution’ dwarf gene. In this paper, we report using interspecific hybridization of B. rapa and B. oleracea and embryo rescue to resynthesize B. napus containing the Brrga1-d dwarf gene. The dwarf gene was backcrossed into two parents of a commercial hybrid combination and evaluated as inbred and hybrid lines in field experiments. The Brrga1-d gene reduced plant height and lodging in inbred and hybrid lines of B. napus, even when present as a single dose in heterozygous genotypes. Seed yields of inbred lines homozygous for Brrga1-d were reduced compared to near isogenic wild-type inbreds, presumably due to damage by insects caused by a slight delay in flowering time. However, yields of hybrids that were homozygous or heterozygous for Brrga1-d were similar to those of near isogenic wild-type hybrids. In addition, we report on a ‘perfect’ PCR marker for selection of the Brrga1-d gene that is based on detecting the nucleotide mutation causing the dwarf phenotype.

Gid1, a gibberellin‐insensitive dwarf mutant, shows altered regulation of probenazole‐inducible protein (PBZ1) in response to cold stress and pathogen attack

A recessive gibberellin (GA)-insensitive dwarf mutant of rice, gibberellin-insensitive dwarf1 (gid1), has been identified, which shows a severe dwarf phenotype and contains high concentrations of endogenous GA. To elucidate the function of gid1, proteins regulated downstream of gid1 were analysed using a proteomic approach. Proteins extracted from suspension-cultured cells of gid1 and its wild type were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Of a total of 962 proteins identified from the suspension-cultured cells, 16 were increased and 14 were decreased in gid1 compared with its wild type. Among the proteins hyper-accumulated in gid1 were osmotin, triosephosphate isomerase, probenazole inducible protein (PBZ1) and pathogenesis-related protein 10. Of these four genes, only the expression of PBZ1 was increased by exogenous GA3 application. Expression of this gene was also enhanced in shoots of the wild type by cold stress or by rice blast fungus infection. Under normal growth conditions, there was more PBZ1 protein in gid1 than in the wild type. In addition, gid1 showed increased tolerance to cold stress and resistance to blast fungus infection. The entcopalyl diphosphate synthase (OsCPS) genes, which encode enzymes at the branch point between GA and phytoalexin biosynthesis, were expressed differentially in gid1 relative to the wild type. Specifically, OsCPS1, which encodes an enzyme in the GA biosynthesis pathway, was down-regulated and OsCPS2 and OsCPS4, which encode enzymes in phytoalexin biosynthesis, were up-regulated in gid1. These results suggest that the expression of PBZ1 is regulated by GA signalling and stress stimuli, and that gid1 is involved in tolerance to cold stress and resistance to blast fungus.

Plant Hormone Receptors Share Destructive Strategy

Gibberellins (GAs) are hydrophobic carboxylic acid molecules that function to regulate plant development. Ueguchi-Tanaka et al. announce the accomplishment of what has been an elusive goal--the identification and characterization of a GA receptor. The authors began with a screen for plants insensitive to the effects of the hormone. One such mutant, gid1 (for gibberellin-insensitive dwarf1) was resistant to GA and appeared to act upstream of known GA signaling intermediates in genetic experiments. Several lines of evidence strongly supported a receptor function for GID1. GID1 bound GA in vitro with micromolar binding affinity, and mutants with reduced biological function had impaired GA binding. GID1 bound to the protein SLR1 in yeast two-hybrid assays in a GA-dependent manner. SLR is a component of GA signaling, a putative transcriptional suppressor whose activity appears to be controlled through ubiquitin-dependent degradation. Finally, transgenic plants overexpressing GID1 showed increased sensitivity to GA. Although the precise signaling mechanism for the GID1 protein, which has sequence similarity to mammalian hormone-sensitive lipases, is not yet clear, it is intriguing that the receptor seems to modulate proteolytic degradation of a negative regulator (SLR1). This mechanism is reminiscent of the mechanism by which auxin, another plant hormone, was recently shown to prompt degradation of a different transcriptional suppressor. Although the auxin receptor and GID1 are structurally distinct, both seem to be modulating targeted destruction of transcriptional regulators by the proteasome. M. Ueguchi-Tanaka, M. Ashikari, M. Nakajima, H. Itoh, E. Katoh, M. Kobayashi, T. Chow, Y. C. Hsing, H. Kitano, I. Yamaguchi, M. Matsuoka, GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberellin. Nature 437 , 693-698 (2005). [PubMed]

Rice Genomics Application to Science

As the result of Rice Genome Research, we can available many tools including molecular markers, genomic libraries, sequences data, many mutants produced by tag lines, microarray and proteome system. These progresses give us many chances to approach to science and breeding. Here we introduce rice genomics application to our research, study of Gibberellin (GA) biosynthetic, signal transduction pathway (1,2 and 3). And also we show the rice genomics application to the molecular breeding (4 and 5). We has been collected and maintained many rice mutants including dwarf and slender mutants. Dwarf mutants in plants are crucial for elucidating regulatory mechanisms for plant growth, development and response to plant hormones. We have screened GA-sensitive and insensitive rice mutants. We have cloned several genes associated with GA biosynthesis and 3 genes associated with GA signal transduction by using the rice genomics. A rice dwarf mutant Daikoku carrying the d1 gene is not only short, but a/so has broad, dark green leaves, compact panicles, and short, round grains. These phenotypes are all controlled by a recessive allele (d1) of the rice Dwarf1 (D1). The mutant was characterized as Gibberellin (GA)-insensitive, because no production of alpha-amylase in the d1 mutant was detectable by application of GA3. For clarifying the molecular mechanisms of GA signal transduction, the rice Dwarf 1 (D1) gene was isolated by a map-based cloning strategy. (1, 2). 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. Some results indicate that the product of the SLR1 gene is an intermediate of the GA signal transduction pathway and slr1-1 mutant is caused by a loss-of-function mutation of the product of the SLR1 gene, which is an ortholog of GAI, RGA, RHT, and D8 which are associate with GA signal (3). The gid1 (gibberellin insensitive dwarf1) mutant shows severe dwarf phenotype similar to that of the null allele of d18, which is a deficient mutant of GA biosynthesis. In gid1seedlings, however, endogenous Ga1 content was almost 120 times higher than that in wild plant and the application of exogenous GA3 did not restore phenotype. These results indicated that gid1 is defective in the perception to GA, and the GID1 gene may encode a positive regulator of GA signaling. Recently, we succeeded to clone the gid1 gene with Yue-ie Caroline Hsing lab. collaboration, Academia Sinica. Analyzing the function of these genes, epistatic interaction and biochemistry approach guides us the outline of GA signal transduction. And now we can control the plant height and plant type with modification of these plant hormone genes. (l) Proc Natl Acad Sci USA, 1999; 96: 10284-10289. (2) Proc Natl Acad Sci USA, 2000; 97: 11638-11643. (3) Plant Cell, 2001; 13: 999.1010. (4) Plant Cell, 2000; 12: 1591-1605. (5) Plant Cell, 2000; 12: 2473-2483.

Rice gibberellin-insensitive dwarf mutant gene Dwarf 1 encodes the alpha-subunit of GTP-binding protein.

A rice Dwarf 1 gene was identified by using a map-based cloning strategy. Its recessive mutant allele confers a dwarf phenotype. Linkage analysis revealed that a cDNA encoding the alpha-subunit of GTP-binding protein cosegregated with d1 in 3,185 d1 segregants. Southern hybridization analysis with this cDNA as a probe showed different band patterns in several d1 mutant lines. In at least four independent d1 mutants, no gene transcript was observed by Northern hybridization analysis. Sequencing analysis revealed that an 833-bp deletion had occurred in one of the mutant alleles, which resulted in an inability to express GTP-binding protein. A transgenic d1 mutant with GTP-binding protein gene restored the normal phenotype. We conclude that the rice Dwarf 1 gene encodes GTP-binding protein and that the protein plays an important role in plant growth and development. Because the d1 mutant is classified as gibberellin-insensitive, we suggest that the GTP-binding protein might be associated with gibberellin signal transduction.

Brassinosteroid/Sterol synthesis and plant growth as affected by lka and lkb mutations of Pea

The dwarf pea (Pisum sativum) mutants lka and lkb are brassinosteroid (BR) insensitive and deficient, respectively. The dwarf phenotype of the lkb mutant was rescued to wild type by exogenous application of brassinolide and its biosynthetic precursors. Gas chromatography-mass spectrometry analysis of the endogenous sterols in this mutant revealed that it accumulates 24-methylenecholesterol and isofucosterol but is deficient in their hydrogenated products, campesterol and sitosterol. Feeding experiments using 2H-labeled 24-methylenecholesterol indicated that the lkb mutant is unable to isomerize and/or reduce the Delta24(28) double bond. Dwarfism of the lkb mutant is, therefore, due to BR deficiency caused by blocked synthesis of campesterol from 24-methylenecholesterol. The lkb mutation also disrupted sterol composition of the membranes, which, in contrast to those of the wild type, contained isofucosterol as the major sterol and lacked stigmasterol. The lka mutant was not BR deficient, because it accumulated castasterone. Like some gibberellin-insensitive dwarf mutants, overproduction of castasterone in the lka mutant may be ascribed to the lack of a feedback control mechanism due to impaired perception/signal transduction of BRs. The possibility that castasterone is a biologically active BR is discussed.

Modification by gibberellin of the growth-temperature relationship in mutant and normal genotypes of several cereals

High-resolution growth measurements were conducted using a linear variable displacement transformer in conjunction with a temperature-programmed meristem-cooling collar. Chilling and rewarming profiles were determined for a range of Gramineae, in the presence and absence of varying concentrations of gibberellic acid (GA3). In wheat (Triticum aestivum L.) seedlings, the growth-constraining temperature (Pe) was progressively lowered by increasing GA3 concentration, with a difference of-4.8°C between controls and material treated with 10(-4) M GA3. Dwarf-5 maize (Zea mays L.) seedlings had a higher Pe than tall segregates and the difference was markedly reduced by exposure to a saturating concentration of GA3. A similar effect was observed with Tanginbozu dwarf rice (Oryza sativa L.). The growth ratetemperature responses of Rht3 gibberellin-insensitive dwarf wheat seedlings were unaffected by GA3 and the Pe values for these segregates were around 5° C higher than for normals. Slender (s1) barley (Hordeum vulgare L.) genotypes had Pe values of-7° C, compared with +4° C for wild-type material, and did not show positive hysteresis for growth rate during the rewarming phase. These studies indicate that GA3 modifies the thermal sensitivity of meristem function in Gramineae in a manner which enhances low-temperature growth.

Characterization of a Gibberellin-Insensitive Dwarf Wheat, D6899

The effects of gibberellin in two wheat varieties, Nainari 60 and D6899, have been studied. D6899 is a dwarf wheat having a single locus mutation, the Rht 3 gene ("Tom Thumb" gene), which is located on chromosome 4A. When compared with the standard height wheat variety, Nainari 60, D6899 does not have a slowdown in cellular metabolism such as respiration rate, protein content, rate of protein synthesis, and uptake of amino acids. The content of ATP is even higher in D6899. However, all of the gibberellin-mediated physiological processes that we have studied, including leaf elongation, synthesis and release of hydrolytic enzymes, and secretion of phosphate ions and reducing sugars in aleurone layers, are retarded in D6899. D6899 and Nainari 60 have essentially the same uptake and metabolism of gibberellin and their levels of endogenous inhibitors such as abscisic acid do not differ drastically. The dosage-response curve of the gibberellin-mediated alpha-amylase production indicates that a rate-limiting step, which is common to many of the diverse gibberellin responses, is partially blocked in D6899 wheat.