Synergistic derepression of gibberellin signaling by removing RGA and GAI function in Arabidopsis thaliana

RGA and GAI are negative regulators of the gibberellin (GA) signal transduction pathway in Arabidopsis thaliana. These genes may have partially redundant functions because they are highly homologous, and plants containing single null mutations at these loci are phenotypically similar to wild type. Previously, rga loss-of-function mutations were shown to partially suppress defects of the GA-deficient ga1-3 mutant. Phenotypes rescued include abaxial trichome initiation, rosette radius, flowering time, stem elongation, and apical dominance. Here we present work showing that the rga-24 and gai-t6 null mutations have a synergistic effect on plant growth. Although gai-t6 alone has little effect, when combined with rga-24, they completely rescued the above defects of ga1-3 to wild-type or GA-overdose phenotype. However, seed germination and flower development defects were not restored. Additionally, rga-24 and rga-24/gai-t6 but not gai-t6 alone caused increased feedback inhibition of expression of a GA biosynthetic gene in both the ga1-3 and wild-type backgrounds. These results demonstrate that RGA and GAI have partially redundant functions in maintaining the repressive state of the GA-signaling pathway, but RGA plays a more dominant role than GAI. Removing both RGA and GAI function allows for complete derepression of many aspects of GA signaling.     

Functional analysis of SPINDLY in gibberellin signaling in Arabidopsis

The Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) protein negatively regulates the gibberellin (GA) signaling pathway. SPY is an O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) with a protein-protein interaction domain consisting of 10 tetratricopeptide repeats (TPR). OGTs add a GlcNAc monosaccharide to serine/threonine residues of nuclear and cytosolic proteins. Determination of the molecular defects in 14 new spy alleles reveals that these mutations cluster in three TPRs and the C-terminal catalytic region. Phenotypic characterization of 12 spy alleles indicates that TPRs 6, 8, and 9 and the catalytic domain are crucial for GA-regulated stem elongation, floral induction, and fertility. TPRs 8 and 9 and the catalytic region are also important for modulating trichome morphology and inflorescence phyllotaxy. Consistent with a role for SPY in embryo development, several alleles affect seedling cotyledon number. These results suggest that three of the TPRs and the OGT activity in SPY are required for its function in GA signal transduction. We also examined the effect of spy mutations on another negative regulator of GA signaling, REPRESSOR OF ga1-3 (RGA). The DELLA motif in RGA is essential for GA-induced proteolysis of RGA, and deletion of this motif (as in rga-delta17) causes a GA-insensitive dwarf phenotype. Here, we demonstrate that spy partially suppresses the rga-delta17 phenotype but does not reduce rga-delta17 or RGA protein levels or alter RGA nuclear localization. We propose that SPY may function as a negative regulator of GA response by increasing the activity of RGA, and presumably other DELLA proteins, by GlcNAc modification.     

Mutations at the SPINDLY locus of Arabidopsis alter gibberellin signal transduction.

Three independent recessive mutations at the SPINDLY (SPY) locus of Arabidopsis confer resistance to the gibberellin (GA) biosynthesis inhibitor paclobutrazol. Relative to wild type, spy mutants exhibit longer hypocotyls, leaves that are a lighter green color, increased stem elongation, early flowering, parthenocarpy, and partial male sterility. All of these phenotypes are also observed when wild-type Arabidopsis plants are repeatedly treated with gibberellin A3 (GA3). The spy-1 allele is partially epistatic to the ga1-2 mutation, which causes GA deficiency. In addition, the spy-1 mutation can simultaneously suppress the effects of the ga1-2 mutation and paclobutrazol treatment, which inhibit different steps in the GA biosynthesis pathway. This observation suggests that spy-1 activates a basal level of GA signal transduction that is independent of GA. Furthermore, results from GA3 dose-response experiments suggest that GA3 and spy-1 interact in an additive manner. These results are consistent with models in which the SPY gene product regulates a portion of the GA signal transduction 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).     

Differential regulation of trichome formation on the adaxial and abaxial leaf surfaces by gibberellins and photoperiod in Arabidopsis thaliana (L.) Heynh.

In wild-type (WT) Columbia and Landsberg erecta ecotypes of Arabidopsis thaliana (L.) Heynh., trichomes are present on the adaxial surfaces of all rosette leaves but are absent from the abaxial surfaces of the first-formed leaves. We have determined that both long-day (LD) photoperiod and gibberellin (GA) stimulate trichome formation. WT plants grown in LD conditions produce the first abaxial trichome on earlier leaves than plants grown in short-day (SD) conditions. Photoperiod sensitivity of abaxial trichome formation on WT plants develops gradually over time, reaching the maximum sensitivity about 24 d after germination. Application of gibberellic acid to WT plants growing in SD conditions accelerates the onset of abaxial trichomes. Conversely, application of 20 to 80 mg L-1 paclobutrazol, a GA biosynthesis inhibitor, to wild-type plants suppresses trichome initiation on the abaxial epidermis. The GA-deficient mutants ga1-5 and ga4-1 and the GA-insensitive mutant gai-1 exhibit delayed onset of abaxial trichomes when grown in LD conditions. The null mutant ga1-3 produces completely glabrous leaves when grown in SD conditions. Application of gibberellic acid to glabrous ga1-3 plants consistently induces earlier formation of trichomes on the adaxial epidermis than on the abaxial epidermis, demonstrating a difference between the adaxial and abaxial surfaces in their response to GA with regard to trichome formation.     

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.     

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.     

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.     

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.     

Phenotypic Suppression of the Gibberellin-Insensitive Mutant (gai) of Arabidopsis

The semidominant gibberellin-insensitive (gai) mutant of Arabidopsis thaliana shows impairment in multiple responses to the plant hormone gibberellin A3, which include effects on seed germination, stem elongation, apical dominance, and rapid flowering in short days. Results presented here show that the gai mutation also interferes with development of fertile flowers in continuous light. Mu-tagenesis of the gai mutant resulted in recovery of 17 independent mutants in which the gibberellin-insensitive phenotype is partially or completely suppressed. Sixteen of the suppressor mutations act semidominantly to restore gibberellin responsiveness. One representative of this class, the gar1 mutation, could not be genetically separated from the gai locus and is proposed to cause inactivation of the gai gene. The exceptional gar2 mutation partially suppresses the gai phenotype, is completely dominant, and is not linked to the gai locus. The gar2 mutation may define a new gene involved in gibberellin signaling. A recessive allele of the spindly (SPY) locus, spy-5, was also found to partially suppress the gai mutant phenotype.     

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 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.     

Ethylene-mediated enhancement of apical hook formation in etiolated Arabidopsis thaliana seedlings is gibberellin dependent

Dark-grown Arabidopsis seedlings develop an apical hook by differential elongation and division of hypocotyl cells. This allows the curved hypocotyl to gently drag the apex, which is protected by the cotyledons, upwards through the soil. Several plant hormones are known to be involved in hook development, including ethylene, which causes exaggeration of the hook. We show that gibberellins (GAs) are also involved in this process. Inhibition of GA biosynthesis with paclobutrazol (PAC) prevented hook formation in wild-type (WT) seedlings and in constitutive ethylene response (ctr)1-1, a mutant that exhibits a constitutive ethylene response. In addition, a GA-deficient mutant (ga1-3) did not form an apical hook in the presence of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC). Analysis of transgenic Arabidopsis seedlings expressing a green fluorescent protein (GFP)-repressor of ga1-3 (RGA) fusion protein suggested that ACC inhibits cell elongation in the apical hook by inhibition of GA signaling. A decreased feedback of GA possibly causes an induction of GA biosynthesis based upon the expression of genes encoding copalyl diphosphate synthase (CPS; GA1) and GA 2-oxidase (AtGA2ox1). Furthermore, expression of GASA1, a GA-response gene, suggests that differential cell elongation in the apical hook might be a result of differential GA-sensitivity.