The New Rga Locus Encodes a Negative Regulator of Gibberellin Response in Arabidopsis Thaliana

We have identified a new locus involved in gibberellin (GA) signal transduction by screening for suppressors of the Arabidopsis thaliana GA biosynthetic mutant ga1-3. The locus is named RGA for repressor of ga1-3. Based on the recessive phenotype of the digenic rga/ga1-3 mutant, the wild-type gene product of RGA is probably a negative regulator of GA responses. Our screen for suppressors of ga1-3 identified 17 mutant alleles of RGA as well as 10 new mutant alleles at the previously identified SPY locus. The digenic (double homozygous) rga/ga1-3 mutants are able to partially repress several defects of ga1-3 including stem growth, leaf abaxial trichome initiation, flowering time, and apical dominance. The phenotype of the trigenic mutant (triple homozygous) rga/spy/ga1-3 shows that rga and spy have additive effects regulating flowering time, abaxial leaf trichome initiation and apical dominance. This trigenic mutant is similar to wild type with respect to each of these developmental events. Because rga/spy/ga1-3 is almost insensitive to GA for hypocotyl growth and its bolting stem is taller than the wild-type plant, the combined effects of the rga and spy mutations appear to allow GA-independent stem growth. Our studies indicate that RGA lies on a separate branch of the GA signal transduction pathway from SPY, which leads us to propose a modified model of the GA response pathway.     

Cytosolic activity of SPINDLY implies the existence of a DELLA-independent gibberellin-response pathway

Specific plant developmental processes are modulated by cross-talk between gibberellin (GA)- and cytokinin-response pathways. Coordination of the two pathways involves the O-linked N -acetylglucosamine transferase SPINDLY (SPY) that suppresses GA signaling and promotes cytokinin responses in Arabidopsis. Although SPY is a nucleocytoplasmic protein, its site of action and targets are unknown. Several studies have suggested that SPY acts in the nucleus, where it modifies nuclear components such as the DELLA proteins to regulate signaling networks. Using chimeric GFP–SPY fused to a nuclear-export signal or to a glucocorticoid receptor, we show that cytosolic SPY promotes cytokinin responses and suppresses GA signaling. In contrast, nuclear-localized GFP–SPY failed to complement the spy mutation. To examine whether modulation of cytokinin activity by GA and spy is mediated by the nuclear DELLA proteins, cytokinin responses were studied in double and quadruple della mutants lacking the activities of REPRESSOR OF GA1-3 (RGA) and GA-INSENSITIVE (GAI) or RGA, GAI, RGA Like1 (RGL1) and RGL2. Unlike spy , the della mutants were cytokinin-sensitive. Moreover, when GA was applied to a cytokinin-treated quadruple della mutant it was able to suppress various cytokinin responses. These results suggest that cytosolic SPY and GA regulate cytokinin responses via a DELLA-independent pathway(s).     

Cross talk between gibberellin and cytokinin: the Arabidopsis GA response inhibitor SPINDLY plays a positive role in cytokinin signaling

SPINDLY (SPY) is a negative regulator of gibberellin (GA) responses; however, spy mutants exhibit various phenotypic alterations not found in GA-treated plants. Assaying for additional roles for SPY revealed that spy mutants are resistant to exogenously applied cytokinin. GA also repressed the effects of cytokinin, suggesting that there is cross talk between the two hormone-response pathways, which may involve SPY function. Two spy alleles showing severe (spy-4) and mild (spy-3) GA-associated phenotypes exhibited similar resistance to cytokinin, suggesting that SPY enhances cytokinin responses and inhibits GA signaling through distinct mechanisms. GA and spy repressed numerous cytokinin responses, from seedling development to senescence, indicating that cross talk occurs early in the cytokinin-signaling pathway. Because GA3 and spy-4 inhibited induction of the cytokinin primary-response gene, type-A Arabidopsis response regulator 5, SPY may interact with and modify elements from the phosphorelay cascade of the cytokinin signal transduction pathway. Cytokinin, on the other hand, had no effect on GA biosynthesis or responses. Our results demonstrate that SPY acts as both a repressor of GA responses and a positive regulator of cytokinin signaling. Hence, SPY may play a central role in the regulation of GA/cytokinin cross talk during plant development.     

Altered Expression of SPINDLY Affects Gibberellin Response and Plant Development

Gibberellins (GAs) are plant hormones with diverse roles in plant growth and development. SPINDLY (SPY) is one of several genes identified in Arabidopsis that are involved in GA response and it is thought to encode an O-GlcNAc transferase. Genetic analysis suggests that SPY negatively regulates GA response. To test the hypothesis that SPY acts specifically as a negatively acting component of GA signal transduction, spy mutants and plants containing a 35S:SPY construct have been examined. A detailed investigation of the spy mutant phenotype suggests that SPY may play a role in plant development beyond its role in GA signaling. Consistent with this suggestion, the analysis of spy er plants suggests that the ERECTA (ER) gene, which has not been implicated as having a role in GA signaling, appears to enhance the non-GA spy mutant phenotypes. Arabidopsis plants containing a 35S:SPY construct possess reduced GA response at seed germination, but also possess phenotypes consistent with increased GA response, although not identical to spy mutants, during later vegetative and reproductive development. Based on these results, the hypothesis that SPY is specific for GA signaling is rejected. Instead, it is proposed that SPY is a negative regulator of GA response that has additional roles in plant development.     

SPINDLY 与赤霉素的信号转导

SPINDLY(SPY)作为一负调节子参与GA的信号转导,34肽重复结构(TPR)与C-端区域对其正常功能都十分重要。SPY基因在植物中呈组成型表达,其蛋白主要出现在细胞核部位。SPY蛋白与动物中的氧连N-乙酰葡萄糖胺转移酶(OGT)具有广泛的同源性,两者可能有着类似的作用机制。本文主要介绍GA突变体、SPY基因、SPY蛋白及其在大麦中的同源物HvSPY的结构与功能相关方面的一些研究进展。     

Two O-linked N-acetylglucosamine transferase genes of Arabidopsis thaliana L. Heynh. have overlapping functions necessary for gamete and seed development

The Arabidopsis SECRET AGENT (SEC) and SPINDLY (SPY) proteins are similar to animal O-linked N-acetylglucosamine transferases (OGTs). OGTs catalyze the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to Ser/Thr residues of proteins. In animals, O-GlcNAcylation has been shown to affect protein activity, stability, and/or localization. SEC protein expressed in Escherichia coli had autocatalytic OGT activity. To determine the function of SEC in plants, two tDNA insertional mutants were identified and analyzed. Although sec mutant plants did not exhibit obvious phenotypes, sec and spy mutations had a synthetic lethal interaction. This lethality was incompletely penetrant in gametes and completely penetrant postfertilization. The rate of both female and male sec spy gamete transmission was higher in plants heterozygous for both mutations than in plants heterozygous for sec and homozygous for spy. Double-mutant embryos aborted at various stages of development and no double-mutant seedlings were obtained. These results indicate that OGT activity is required during gametogenesis and embryogenesis with lethality occurring when parentally derived SEC, SPY, and/or O-GlcNAcylated proteins become limiting.     

The Arabidopsis F-box protein SLEEPY1 targets gibberellin signaling repressors for gibberellin-induced degradation

The nuclear DELLA proteins are highly conserved repressors of hormone gibberellin (GA) signaling in plants. In Arabidopsis thaliana, GA derepresses its signaling pathway by inducing proteolysis of the DELLA protein REPRESSOR OF ga1-3 (RGA). SLEEPY1 (SLY1) encodes an F-box-containing protein, and the loss-of-function sly1 mutant has a GA-insensitive dwarf phenotype and accumulates a high level of RGA. These findings suggested that SLY1 recruits RGA to the SCFSLY1 E3 ligase complex for ubiquitination and subsequent degradation by the 26S proteasome. In this report, we provide new insight into the molecular mechanism of how SLY1 interacts with the DELLA proteins for controlling GA response. By yeast two-hybrid and in vitro pull-down assays, we demonstrated that SLY1 interacts directly with RGA and GA INSENSITIVE (GAI, a closely related DELLA protein) via their C-terminal GRAS domain. The rga and gai null mutations additively suppressed the recessive sly1 mutant phenotype, further supporting the model that SCFSLY1 targets both RGA and GAI for degradation. The N-terminal DELLA domain of RGA previously was shown to be essential for GA-induced degradation. However, we found that this DELLA domain is not required for protein-protein interaction with SLY1 in yeast (Saccharomyces cerevisiae), suggesting that its role is in a GA-triggered conformational change of the DELLA proteins. We also identified a novel gain-of-function sly1-d mutation that increased GA signaling by reducing the levels of the DELLA protein in plants. This effect of sly1-d appears to be caused by an enhanced interaction between sly1-d and the DELLA proteins.     

Ectopic Expression of the Tetratricopeptide Repeat Domain of SPINDLY Causes Defects in Gibberellin Response

The SPINDLY (SPY) protein of Arabidopsis is a negative regulator of gibberellin (GA) response. The SPY protein has 10 copies of the tetratricopeptide repeat (TPR) at the N terminus. TPR motifs function as protein-protein interaction domains. Several spy alleles are affected only in the TPR region suggesting that protein-protein interactions mediated by this domain are important for proper GA signaling. We have used a reverse genetics approach to further investigate the role of the TPR domain. The TPR domain of SPY was overexpressed in wild-type, gai, and spy plants. Expression of the TPR domain alone is not sufficient to rescue spy mutants. Expression of the TPR domain in a wild-type background produces phenotypes similar to those caused by loss-of-function spy mutants including resistance to GA biosynthesis inhibitors, short hypocotyl length, and early flowering. The dwarfing of the floral shoot internodes caused by the gai mutation was suppressed by expression of the TRP domain. Expression of the TPR domain had no effect on the abundance of endogenous SPY mRNA. The TPR domain was found to interact with SPY both in vitro and in yeast two-hybrid assays. These data indicate that the TPR domain of SPY can participate in protein-protein interactions and that these interactions are important for the proper functioning of SPY.     

SPINDLY and GIGANTEA interact and act in Arabidopsis thaliana pathways involved in light responses, flowering, and rhythms in cotyledon movements

SPINDLY (SPY) is a negative regulator of gibberellin signaling in Arabidopsis thaliana that also functions in previously undefined pathways. The N terminus of SPY contains a protein-protein interaction domain consisting of 10 tetratricopeptide repeats (TPRs). GIGANTEA (GI) was recovered from a yeast two-hybrid screen for proteins that interact with the TPR domain. GI and SPY also interacted in Escherichia coli and in vitro pull-down assays. The phenotypes of spy and spy-4 gi-2 plants support the hypothesis that SPY functions with GI in pathways controlling flowering, circadian cotyledon movements, and hypocotyl elongation. GI acts in the long-day flowering pathway upstream of CONSTANS (CO) and FLOWERING LOCUS T (FT). Loss of GI function causes late flowering and reduces CO and FT RNA levels. Consistent with SPY functioning in the long-day flowering pathway upstream of CO, spy-4 partially suppressed the reduced abundance of CO and FT RNA and the late flowering of gi-2 plants. Like gi, spy affects the free-running period of cotyledon movements. The free-running period was lengthened in spy-4 mutants and shortened in plants that overexpress SPY under the control of the 35S promoter of Cauliflower mosaic virus. When grown under red light, gi-2 plants have a long hypocotyl. This hypocotyl phenotype was suppressed in spy-4 gi-2 double mutants. Additionally, dark-grown and far-red-light-grown spy-4 seedlings were found to have short and long hypocotyls, respectively. The different hypocotyl length phenotypes of spy-4 seedlings grown under different light conditions are consistent with SPY acting in the GA pathway to inhibit hypocotyl elongation and also acting as a light-regulated promoter of elongation.     

The rice SPINDLY gene functions as a negative regulator of gibberellin signaling by controlling the suppressive function of the DELLA protein, SLR1, and modulating brassinosteroid synthesis

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.     

SPINDLY is a nuclear-localized repressor of gibberellin signal transduction expressed throughout the plant

SPY (SPINDLY) encodes a putative O-linked N-acetyl-glucosamine transferase that is genetically defined as a negatively acting component of the gibberellin (GA) signal transduction pathway. Analysis of Arabidopsis plants containing a SPY::GUS reporter gene reveals that SPY is expressed throughout the life of the plant and in most plant organs examined. In addition to being expressed in all organs where phenotypes due to spy mutations have been reported, SPY::GUS is expressed in the root. Examination of the roots of wild-type, spy, and gai plants revealed phenotypes indicating that SPY and GAI play a role in root development. A second SPY::GUS reporter gene lacking part of the SPY promoter was inactive, suggesting that sequences in the first exon and/or intron are required for detectable expression. Using both subcellular fractionation and visualization of a SPY-green fluorescent protein fusion protein that is able to rescue the spy mutant phenotype, the majority of SPY protein was shown to be present in the nucleus. This result is consistent with the nuclear localization of other components of the GA response pathway and suggests that SPY's role as a negative regulator of GA signaling involves interaction with other nuclear proteins and/or O-N-acetyl-glucosamine modification of these proteins.     

Functional analysis of HvSPY,a negative regulator of GA response,in barley aleurone cells and <Emphasis Type="Italic">Arabidopsis</Emphasis>

SPINDLY (SPY) is an important regulator of plant development, and consists of an N-half tetratricopeptide repeat (TPR) domain containing 10 TPR motifs and a C-half catalytic domain, similar to O-GlcNAc transferase (OGT) of animals. The best characterised role of SPY is a negative regulator of GA signalling, and all known spy alleles have been isolated based on increased GA response. Of the eight alleles that directly affect the TPR domain, all alter TPRs 6, 8 and/or 9. To test the hypothesis that a subset of TPRs, including 6, 8 and 9, are both essential and sufficient for the regulation of GA response, we overexpressed the full-length barley (Hordeum vulgare L.) SPY protein (HvSPY) and several deletion mutants in barley aleurone cells and in Arabidopsis wild type (WT) and spy-4 plants. Transient assays in barley aleurone cells, that also express endogenous HvSPY, demonstrated that introduced HvSPY and HvTPR inhibited GA₃-induced α-amylase expression. With the exception of HvSPYΔ1–5, the other deletion proteins were partially active in the barley assay, including HvSPYΔ6–9 which lacks TPRs 6, 8 and 9. In Arabidopsis, analysis of seed germination under a range of conditions revealed that 35S:HvSPY increased seed dormancy. Hvspy-2, which lacks parts of the eighth and ninth TPRs, was able to partially complement all aspects of the spy-4 phenotype. In the presence of AtSPY, 35S:HvTPR caused some phenotypes consistent with a decrease in GA signalling, including increased seed sensitivity to paclobutrazol and delayed flowering. These plants also possessed distorted leaf morphology and altered epidermal cell shape. Thus, despite genetic analysis demonstrating that TPRs 6, 8 and 9 are required for regulation of GA signalling, our results suggest that these TPRs are neither absolutely essential nor sufficient for SPY activity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evidence that the Arabidopsis nuclear gibberellin signalling protein GAI is not destabilised by gibberellin

Plant growth is regulated by bioactive gibberellin (GA), although there is an unexplained diversity in the magnitude of the GA responses exhibited by different plant species. GA acts via a group of orthologous proteins known as the DELLA proteins. The Arabidopsis genome contains genes encoding five different DELLA proteins, the best known of which are GAI and RGA. The DELLA proteins are thought to act as repressors of GA-regulated processes, whilst GA is thought to act as a negative regulator of DELLA protein function. Recent experiments have shown that GA induces rapid disappearance of nuclear RGA, SLR1 and SLN1 (DELLA proteins from rice and barley), suggesting that GA signalling and degradation of DELLA proteins are coupled. However, RGL1, another Arabidopsis DELLA protein, does not disappear from the nucleus in response to GA treatment. Here, we present evidence suggesting that GAI, like RGL1, is stable in response to GA treatment, and show that transgenic Arabidopsis plants containing constructs that enable high-level expression of GAI exhibit a dwarf, GA non-responsive phenotype. Thus, GAI appears to be less affected by GA than RGA, SLR1 or SLN1. We also show that neither of the two putative nuclear localisation signals contained in DELLA proteins are individually necessary for nuclear localisation of GAI. The various DELLA proteins have different properties, and we suggest that this functional diversity may explain, at least in part, why plant species differ widely in their GA response magnitudes.     

The Arabidopsis SLEEPY1 gene encodes a putative F-box subunit of an SCF E3 ubiquitin ligase

The Arabidopsis SLY1 (SLEEPY1) gene positively regulates gibberellin (GA) signaling. Positional cloning of SLY1 revealed that it encodes a putative F-box protein. This result suggests that SLY1 is the F-box subunit of an SCF E3 ubiquitin ligase that regulates GA responses. The DELLA domain protein RGA (repressor of ga1-3) is a repressor of GA response that appears to undergo GA-stimulated protein degradation. RGA is a potential substrate of SLY1, because sly1 mutations cause a significant increase in RGA protein accumulation even after GA treatment. This result suggests SCF(SLY1)-targeted degradation of RGA through the 26S proteasome pathway. Further support for this model is provided by the observation that an rga null allele partially suppresses the sly1-10 mutant phenotype. The predicted SLY1 amino acid sequence is highly conserved among plants, indicating a key role in GA response.     

DELLA Proteins and GA Signalling in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Gibberellin (GA) is a classical plant hormone involved in many aspects of plant growth and development. A family of five homologs called the DELLA proteins, comprised of GAI, RGA, RGL1, RGL2 and RGL3, were recently found to act as critical GA signal mediators in Arabidopsis. Reports have shown that GAI and RGA are coupled together to repress stem elongation growth whereas RGL2 is a major negative regulator of seed germination. GA down-regulates DELLA proteins through protein degradation likely via the proteasome pathway. The conserved and functionally important DELLA domain is responsible for protein stability in response to GA.