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.     

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.     

New insights into gibberellin signaling in regulating flowering in Arabidopsis

In angiosperms,floral transition is a key developmental transition from the vegetative to reproductive growth,and requires precise regulation to maximize the reproductive success.A complex regulatory network governs this transition through integrating flowering pathways in response to multiple exogenous and endogenous cues.Phytohormones are essential for proper plant developmental regulation and have been extensively studied for their involvement in the floral transition.Among various phytohormones,gibberellin(GA)plays a major role in affecting flowering in the model plant Arabidopsis thaliana.The GA pathway interact with other flowering genetic pathways and phytohormone signaling pathways through either DELLA proteins or mediating GA homeostasis.In this review,we summarize the recent advances in understanding the mechanisms of DELLA-mediated GA pathway in flowering time control in Arabidopsis,and discuss its possible link with other phytohormone pathways during the floral transition.     

Transcriptional regulation of gibberellin metabolism genes by auxin signaling in Arabidopsis

Auxin and gibberellins (GAs) overlap in the regulation of multiple aspects of plant development, such as root growth and organ expansion. This coincidence raises questions about whether these two hormones interact to regulate common targets and what type of interaction occurs in each case. Auxins induce GA biosynthesis in a range of plant species. We have undertaken a detailed analysis of the auxin regulation of expression of Arabidopsis (Arabidopsis thaliana) genes encoding GA 20-oxidases and GA 3-oxidases involved in GA biosynthesis, and GA 2-oxidases involved in GA inactivation. Our results show that auxin differentially up-regulates the expression of various genes involved in GA metabolism, in particular several AtGA20ox and AtGA2ox genes. Up-regulation occurred very quickly after auxin application; the response was mimicked by incubations with the protein synthesis inhibitor cycloheximide and was blocked by treatments with the proteasome inhibitor MG132. The effects of auxin treatment reflect endogenous regulation because equivalent changes in gene expression were observed in the auxin overproducer mutant yucca. The results suggest direct regulation of the expression of GA metabolism genes by Aux/IAA and ARF proteins. The physiological relevance of this regulation is supported by the observation that the phenotype of certain gain-of-function Aux/IAA alleles could be alleviated by GA application, which suggests that changes in GA metabolism mediate part of auxin action during development.     

Fusion genetic analysis of gibberellin signaling mutants

A fusion genetic strategy was used to identify gibberellin (GA) signaling mutants in transgenic Arabidopsis expressing the beta-glucuronidase (GUS) and firefly luciferase (LUC) reporter genes under control of the GA-responsive GASA1 promoter. Initial analyses determined the spatial and temporal patterns of reporter expression, and showed that reporter induction by GA was antagonized by ABA. gamma-Irradiated M2 progeny with altered reporter activities were identified by LUC bioimaging followed by GUS assays and northern hybridization of the endogenous GASA1 mRNA. Genetic analysis showed that three mutants, which overexpressed both reporters and endogenous GASA1, were caused by recessive (goe1 and goe2, for GASA over-expressed) and semi-dominant (goe3) mutations at different loci. These mutants are altered in their sensitivity to GA and the GA biosynthetic inhibitor paclobutrazol, and in the expression of several GA signaling related genes.     

Quantitative phosphoproteomics of early elicitor signaling in Arabidopsis

Perception of general elicitors by plant cells initiates signal transduction cascades that are regulated by protein phosphorylation. The earliest signaling events occur within minutes and include ion fluxes across the plasma membrane, activation of MAPKs, and the formation of reactive oxygen species. The phosphorylation events that regulate these signaling cascades are largely unknown. Here we present a mass spectrometry-based quantitative phosphoproteomics approach that identified differentially phosphorylated sites in signaling and response proteins from Arabidopsis cells treated with either flg22 or xylanase. Our approach was sensitive enough to quantitate phosphorylation on low abundance signaling proteins such as calcium-dependent protein kinases and receptor-like kinase family members. With this approach we identified one or more differentially phosphorylated sites in 76 membrane-associated proteins including a number of defense-related proteins. Our data on phosphorylation indicate a high degree of complexity at the level of post-translational modification as exemplified by the complex modification patterns of respiratory burst oxidase protein D. Furthermore the data also suggest that protein translocation and vesicle traffic are important aspects of early signaling and defense in response to general elicitors. Our study presents the largest quantitative Arabidopsis phosphoproteomics data set to date and provides a new resource that can be used to gain novel insight into plant defense signal transduction and early defense response.     

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.     

油菜素内酯、赤霉素与光共同调控拟南芥的细胞伸长和光形态建成

在植物的生长发育过程中,植物激素发挥着重要的作用. 最新研究对油菜素内酯、赤霉素两类植物激素与光的信号通路共同调控植物的细胞伸长和光形态建成的分子机制给予了精确的阐述,这也为提高农作物产量提拱了理论基础.     

The role of two f-box proteins, SLEEPY1 and SNEEZY, in Arabidopsis gibberellin signaling

The SLEEPY1 (SLY1) F-box gene is a positive regulator of gibberellin (GA) signaling in Arabidopsis (Arabidopsis thaliana). Loss of SLY1 results in GA-insensitive phenotypes including dwarfism, reduced fertility, delayed flowering, and increased seed dormancy. These sly1 phenotypes are partially rescued by overexpression of the SLY1 homolog SNEEZY (SNE)/SLY2, suggesting that SNE can functionally replace SLY1. GA responses are repressed by DELLA family proteins. GA relieves DELLA repression when the SCF(SLY1) (for Skp1, Cullin, F-box) E3 ubiquitin ligase ubiquitinates DELLA protein, thereby targeting it for proteolysis. Coimmunoprecipitation experiments using constitutively expressed 35S:hemagglutinin (HA)-SLY1 and 35S:HA-SNE translational fusions in the sly1-10 background suggest that SNE can function similarly to SLY1 in GA signaling. Like HA-SLY1, HA-SNE interacted with the CULLIN1 subunit of the SCF complex, and this interaction required the F-box domain. Like HA-SLY1, HA-SNE coimmunoprecipitated with the DELLA REPRESSOR OF GA1-3 (RGA), and this interaction required the SLY1 or SNE carboxyl-terminal domain. Whereas HA-SLY1 overexpression resulted in a decrease in both DELLA RGA and RGA-LIKE2 (RGL2) protein levels, HA-SNE caused a decrease in DELLA RGA but not in RGL2 levels. This suggests that one reason HA-SLY1 is able to effect a stronger rescue of sly1-10 phenotypes than HA-SNE is because SLY1 regulates a broader spectrum of DELLA proteins. The FLAG-SLY1 fusion protein was found to coimmunoprecipitate with the GA receptor HA-GA-INSENSITIVE DWARF1b (GID1b), supporting the model that SLY1 regulates DELLA through interaction with the DELLA-GA-GID1 complex.     

A gibberellin insensitive mutant of Arabidopsis thaliana

A dwarf mutant of Arabidopsis thaliana (L.) Heynh. was found to be less sensitive to applied gibberellins than the wild type, and this character was controlled by one partially-dominant gene (denoted Gai) located on chromosome 1. This mutant resembled gibberellin-deficient mutants since not only stem growth, but also apical dominanace and seed germination were reduced. However, in contrast to the latter mutants, gibberellin does not reverse these effects in the Gai mutant. The insensitivity of the mutant could be quantified in much more detail in the recombinant of this mutation with the GA deficient mutant ga-1/ga-1 . Endogenous gibberellins of the Gai mutant did not differ from the wild type either in quantity or composition. The data suggest that the gene controls a step involved in gibberellin action.     

Identification and characterization of Arabidopsis gibberellin receptors

Three gibberellin (GA) receptor genes (AtGID1a, AtGID1b and AtGID1c), each an ortholog of the rice GA receptor gene (OsGID1), were cloned from Arabidopsis, and the characteristics of their recombinant proteins were examined. The GA-binding activities of the three recombinant proteins were confirmed by an in vitro assay. Biochemical analyses revealed similar ligand selectivity among the recombinants, and all recombinants showed higher affinity to GA(4) than to other GAs. AtGID1b was unique in its binding affinity to GA(4) and in its pH dependence when compared with the other two, by only showing binding in a narrow pH range (pH 6.4-7.5) with 10-fold higher affinity (apparent K(d) for GA(4) = 3 x 10(-8) m) than AtGID1a and AtGID1c. A two-hybrid yeast system only showed in vivo interaction in the presence of GA(4) between each AtGID1 and the Arabidopsis DELLA proteins (AtDELLAs), negative regulators of GA signaling. For this interaction with AtDELLAs, AtGID1b required only one-tenth of the amount of GA(4) that was necessary for interaction between the other AtGID1s and AtDELLAs, reflecting its lower K(d) value. AtDELLA boosted the GA-binding activity of AtGID1 in vitro, which suggests the formation of a complex between AtDELLA and AtGID1-GA that binds AtGID1 to GA more tightly. The expression of each AtGID1 clone in the rice gid1-1 mutant rescued the GA-insensitive dwarf phenotype. These results demonstrate that all three AtGID1s functioned as GA receptors in Arabidopsis.     

Distinct cell-specific expression patterns of early and late gibberellin biosynthetic genes during Arabidopsis seed germination

Gibberellins (GAs) are biosynthesized through a complex pathway that involves several classes of enzymes. To predict sites of individual GA biosynthetic steps, we studied cell type-specific expression of genes encoding early and late GA biosynthetic enzymes in germinating Arabidopsis seeds. We showed that expression of two genes, AtGA3ox1 and AtGA3ox2, encoding GA 3-oxidase, which catalyzes the terminal biosynthetic step, was mainly localized in the cortex and endodermis of embryo axes in germinating seeds. Because another GA biosynthetic gene, AtKO1, coding for ent-kaurene oxidase, exhibited a similar cell-specific expression pattern, we predicted that the synthesis of bioactive GAs from ent-kaurene oxidation occurs in the same cell types during seed germination. We also showed that the cortical cells expand during germination, suggesting a spatial correlation between GA production and response. However, promoter activity of the AtCPS1 gene, responsible for the first committed step in GA biosynthesis, was detected exclusively in the embryo provasculature in germinating seeds. When the AtCPS1 cDNA was expressed only in the cortex and endodermis of non-germinating ga1-3 seeds (deficient in AtCPS1) using the AtGA3ox2 promoter, germination was not as resistant to a GA biosynthesis inhibitor as expression in the provasculature. These results suggest that the biosynthesis of GAs during seed germination takes place in two separate locations with the early step occurring in the provasculature and the later steps in the cortex and endodermis. This implies that intercellular transport of an intermediate of the GA biosynthetic pathway is required to produce bioactive GAs.     

Evolutionary analysis of three gibberellin oxidase genes in rice, Arabidopsis, and soybean

GAs are plant hormones that play fundamental roles in plant growth and development. GA2ox, GA3ox, and GA20ox are three key enzymes in GA biosynthesis. These enzymes belong to the 2OG-Fe (II) oxygenase superfamily and are independently encoded by different gene families. To date, genome-wide comparative analyses of GA oxidases in plant species have not been thoroughly carried out. In the present work, 61 GA oxidase family genes from rice (Oryza sativa), Arabidopsis, and soybean (Glycine max) were identified and a full study of these genes including phylogenetic tree construction, gene structure, gene family expansion and analysis of functional motifs was performed. Based on phylogeny, most of the GA oxidases were divided into four subgroups that reflected functional classifications. Intron/intron average length of GA oxidase genes in rice analysis revealed that GA oxidase genes in rice experienced substantial evolutionary divergence. Segmental duplication events were mainly found in soybean genome. However, in rice and Arabidopsis, no single expansion pattern exhibited dominance, indicating that GA oxidase genes from these species might have been subjected to a more complex evolutionary mechanism. In addition, special functional motifs were discovered in GA20ox, GA3ox, and GA2ox, which suggested that different functional motifs are associated with differences in protein function. Taken together our results suggest that GA oxidase family genes have undergone divergent evolutionary routes, especially at the monocot-dicot split, with dynamic evolution occurring in Arabidopsis thaliana and soybean.     

Evolutionarily conserved DELLA-mediated gibberellin signaling in plants

Gibberellins (GAs) play important roles in many essential plant growth and development processes. A family of nuclear growth-repressing DELLA proteins is the key component in GA signaling. GA perception is mediated by GID1, and the key event of GA signaling is the degradation of DELLA proteins via the 26S proteasome pathway. DELLA proteins integrating other plant hormones signaling and environmental cue modulating plant growth and development have been revealed. GA turning on the de-DELLA-repressing system is conserved, and independently establishes step-by-step recruitment of GA-stimulated GID1-DELLA interaction and DELLA growth-repression functions during land plant evolution. These discoveries open new prospects for the understanding of GA action and DELLA-mediated signaling in plants.     

GID1-mediated gibberellin signaling in plants

Gibberellin (GA) perception is mediated by GID1 (GA-INSENSITIVE DWARF1), a receptor that shows similarity to hormone-sensitive lipases. A key event in GA signaling is the degradation of DELLA proteins, which are negative regulators of GA response that interact with GID1 in a GA-dependent manner. This GID1-DELLA GA-perception system is conserved among vascular plants but is not found in the moss Physcomitrella patens. The identification of factors in GA signaling downstream of DELLA and the development of a new concept of DELLA function beyond its role as a repressor of GA signaling are important advances. DELLA proteins appear to have at least two other distinct roles: maintaining GA homeostasis and regulating cross-talk between GA and other plant hormones.     

New targets of Arabidopsis thioredoxins revealed by proteomic analysis

Proteomics was used to search for putative thioredoxin (TRX) targets in leaves of the model plant, Arabidopsis thaliana. About forty different proteins have been found to be reduced by TRX, after TRX itself has been specifically reduced by its NADPH-dependent reductase. Twenty-one of the identified proteins were already known or recently proposed to be TRX-dependent and nineteen of the proteins were new potential targets. The identified proteins are involved in a wide variety of processes, including the Calvin cycle, metabolism, photosynthesis, folding, defense against oxidative stress and amino acid synthesis. Two proteins from the glycine cleavage complex were also identified as putative TRX targets, and a new role can be postulated in leaves for TRX in defense against herbivores and/or pathogens.