Stephen G. Thomas

Genetic Characterization and Functional Analysis of the GID1 Gibberellin Receptors in Arabidopsis

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 SCFSLY1 E3 ubiquitin ligase that targets the DELLA protein for degradation.

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 SCFSLY1-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.

Global Analysis of DELLA Direct Targets in Early Gibberellin Signaling in Arabidopsis

Bioactive gibberellins (GAs) are phytohormones that regulate growth and development throughout the life cycle of plants. DELLA proteins are conserved growth repressors that modulate all aspects of GA responses. These GA-signaling repressors are nuclear localized and likely function as transcriptional regulators. Recent studies demonstrated that GA, upon binding to its receptor, derepresses its signaling pathway by binding directly to DELLA proteins and targeting them for rapid degradation via the ubiquitin-proteasome pathway. Therefore, elucidating the signaling events immediately downstream of DELLA is key to our understanding of how GA controls plant development. Two sets of microarray studies followed by quantitative RT-PCR analysis allowed us to identify 14 early GA-responsive genes that are also early DELLA-responsive in Arabidopsis thaliana seedlings. Chromatin immunoprecipitation provided evidence for in vivo association of DELLA with promoters of eight of these putative DELLA target genes. Expression of all 14 genes was downregulated by GA and upregulated by DELLA. Our study reveals that DELLA proteins play two important roles in GA signaling: (1) they help establish GA homeostasis by direct feedback regulation on the expression of GA biosynthetic and GA receptor genes, and (2) they promote the expression of downstream negative components that are putative transcription factors/regulators or ubiquitin E2/E3 enzymes. In addition, one of the putative DELLA targets, XERICO, promotes accumulation of abscisic acid (ABA) that antagonizes GA effects. Therefore, DELLA may restrict GA-promoted processes by modulating both GA and ABA pathways.

DELLA Proteins and Gibberellin-Regulated Seed Germination and Floral Development in Arabidopsis

RGA (repressor of ga1-3) and GAI (gibberellin insensitive) are negative regulators of plant hormone gibberellin (GA) signaling in Arabidopsis. The GA-deficient mutant ga1-3 is a nongerminating, extreme dwarf that flowers late and produces male-sterile flowers. The rga and gai null alleles interact synergistically to rescue vegetative growth and floral initiation in ga1-3, indicating that RGA and GAI are major repressors for these processes. However, rga and gai in combination cannot rescue seed germination or floral development in ga1-3. RGA and GAI belong to the DELLA subfamily within the GRAS family of plant regulatory proteins. Three additional DELLA proteins RGL1, RGL2, and RGL3 are present in Arabidopsis. Previous studies provided evidence that RGL2 and possibly RGL1 control seed germination. To investigate further the function of the RGL genes, we examined the expression profiles of all 5 DELLA protein genes by real-time PCR. RGA and, to a lesser extent, GAI mRNAs were expressed ubiquitously in all tissues, whereas RGL1, 2, and 3 transcripts were present at high levels only in germinating seeds and/or flowers and siliques. Using the newly isolated rgl1, rgl2, and rgl3 T-DNA insertion mutants, we demonstrated that RGL2 is the major repressor in seed germination. We further provided evidence that RGA, RGL1, and RGL2 are all involved in modulating floral development. Interestingly, RGL2 expression is regulated not only at the transcript level. We showed that RGL2 protein in imbibed seeds is rapidly degraded by GA treatment and that the F-box protein SLY1 is required for RGL2 degradation to occur.

Gibberellin biosynthesis and its regulation

The GAs (gibberellins) comprise a large group of diterpenoid carboxylic acids that are ubiquitous in higher plants, in which certain members function as endogenous growth regulators, promoting organ expansion and developmental changes. These compounds are also produced by some species of lower plants, fungi and bacteria, although, in contrast to higher plants, the function of GAs in these organisms has only recently been investigated and is still unclear. In higher plants, GAs are synthesized by the action of terpene cyclases, cytochrome P450 mono-oxygenases and 2-oxoglutarate-dependent dioxygenases localized, respectively, in plastids, the endomembrane system and the cytosol. The concentration of biologically active GAs at their sites of action is tightly regulated and is moderated by numerous developmental and environmental cues. Recent research has focused on regulatory mechanisms, acting primarily on expression of the genes that encode the dioxygenases involved in biosynthesis and deactivation. The present review discusses the current state of knowledge on GA metabolism with particular emphasis on regulation, including the complex mechanisms for the maintenance of GA homoeostasis.

The role of gibberellin signalling in plant responses to abiotic stress

Plant hormones are small molecules that regulate plant growth and development, as well as responses to changing environmental conditions. By modifying the production, distribution or signal transduction of these hormones, plants are able to regulate and coordinate both growth and/or stress tolerance to promote survival or escape from environmental stress. A central role for the gibberellin (GA) class of growth hormones in the response to abiotic stress is becoming increasingly evident. Reduction of GA levels and signalling has been shown to contribute to plant growth restriction on exposure to several stresses, including cold, salt and osmotic stress. Conversely, increased GA biosynthesis and signalling promote growth in plant escape responses to shading and submergence. In several cases, GA signalling has also been linked to stress tolerance. The transcriptional regulation of GA metabolism appears to be a major point of regulation of the GA pathway, while emerging evidence for interaction of the GA-signalling molecule DELLA with components of the signalling pathway for the stress hormone jasmonic acid suggests additional mechanisms by which GA signalling may integrate multiple hormone signalling pathways in the response to stress. Here, we review the evidence for the role of GA in these processes, and the regulation of the GA signalling pathway on exposure to abiotic stress. The potential mechanisms by which GA signalling modulates stress tolerance are also discussed.

Molecular mechanism for the interaction between gibberellin and brassinosteroid signaling pathways in Arabidopsis

Plant development is modulated by the convergence of multiple environmental and endogenous signals, and the mechanisms that allow the integration of different signaling pathways is currently being unveiled. A paradigmatic case is the concurrence of brassinosteroid (BR) and gibberellin (GA) signaling in the control of cell expansion during photomorphogenesis, which is supported by physiological observations in several plants but for which no molecular mechanism has been proposed. In this work, we show that the integration of these two signaling pathways occurs through the physical interaction between the DELLA protein GAI, which is a major negative regulator of the GA pathway, and BRASSINAZOLE RESISTANT1 (BZR1), a transcription factor that broadly regulates gene expression in response to BRs. We provide biochemical evidence, both in vitro and in vivo, indicating that GAI inactivates the transcriptional regulatory activity of BZR1 upon their interaction by inhibiting the ability of BZR1 to bind to target promoters. The physiological relevance of this interaction was confirmed by the observation that the dominant gai-1 allele interferes with BR-regulated gene expression, whereas the bzr1-1D allele displays enhanced resistance to DELLA accumulation during hypocotyl elongation. Because DELLA proteins mediate the response to multiple environmental signals, our results provide an initial molecular framework for the integration with BRs of additional pathways that control plant development.

Genetic Analysis Reveals That C19-GA 2-Oxidation Is a Major Gibberellin Inactivation Pathway in Arabidopsis

Bioactive hormone concentrations are regulated both at the level of hormone synthesis and through controlled inactivation. Based on the ubiquitous presence of 2β-hydroxylated gibberellins (GAs), a major inactivating pathway for the plant hormone GA seems to be via GA 2-oxidation. In this study, we used various approaches to determine the role of C19-GA 2-oxidation in regulating GA concentration and GA-responsive plant growth and development. We show that Arabidopsis thaliana has five C19-GA 2-oxidases, transcripts for one or more of which are present in all organs and at all stages of development examined. Expression of four of the five genes is subject to feed-forward regulation. By knocking out all five Arabidopsis C19-GA 2-oxidases, we show that C19-GA 2-oxidation limits bioactive GA content and regulates plant development at various stages during the plant life cycle: C19-GA 2-oxidases prevent seed germination in the absence of light and cold stimuli, delay the vegetative and floral phase transitions, limit the number of flowers produced per inflorescence, and suppress elongation of the pistil prior to fertilization. Under GA-limited conditions, further roles are revealed, such as limiting elongation of the main stem and side shoots. We conclude that C19-GA 2-oxidation is a major GA inactivation pathway regulating development in Arabidopsis.

Update on Gibberellin Signaling. A Tale of the Tall and the Short

Bioactive gibberellins (GAs) are plant hormones that control growth and development throughout the life cycle of the plant. Isolation and characterization of mutants that are impaired in GA biosynthesis have been crucial in both understanding the role of GAs in regulating plant development and elucidating the GA biosynthetic pathway (Davies, 1995; Hedden and Phillips, 2000). The broad spectrum of developmental processes controlled by GAs is clearly illustrated by the phenotypes of GA biosynthesis mutants. In general, these GA-deficient mutants exhibit a common phenotype: a dark-green dwarf plant that is defective in leaf expansion and stem elongation (Fig. 1). In many species, GA-deficient mutants also display defects during seed germination, altered floral initiation time, and are impaired in development of flower, fruit, and seed. In recent years our understanding of GA metabolism has advanced rapidly with the cloning of