Scarecrow-like 3 promotes gibberellin signaling by antagonizing master growth repressor DELLA in Arabidopsis.

The diterpenoid phytohormone gibberellin (GA) controls diverse developmental processes throughout the plant life cycle. DELLA proteins are master growth repressors that function immediately downstream of the GA receptor to inhibit GA signaling. By doing so, DELLAs also play pivotal roles as integrators of internal developmental signals from multiple hormone pathways and external cues. DELLAs are likely nuclear transcriptional regulators, which interact with other transcription factors to modulate expression of GA-responsive genes. DELLAs are also involved in maintaining GA homeostasis through feedback up-regulating expression of GA biosynthesis and receptor genes. However, the molecular mechanisms by which DELLAs restrict growth and development are largely unknown. This study reveals an important step of the mechanism. Previous microarray studies identified scarecrow-like 3 (SCL3) as a direct target gene of DELLA in Arabidopsis seedlings. SCL3 expression is induced by DELLA and repressed by GA. Unexpectedly, a scl3 null mutant displays reduced GA responses and elevated expression of GA biosynthesis genes during seed germination and seedling growth, indicating that SCL3 functions as a positive regulator of GA signaling. SCL3 seems to act as an attenuator of DELLA proteins. Transient expression, ChIP, and co-IP studies show that SCL3 autoregulates its own transcription by directly interacting with DELLA. Our data further show that SCL3 and DELLA antagonize each other in controlling both downstream GA responses and upstream GA biosynthetic genes. This work is beginning to shed light on how this complex regulatory network achieves GA homeostasis and controls GA-mediated growth and development in the plant.

The AtGenExpress hormone and chemical treatment data set: experimental design, data evaluation, model data analysis and data access.

We analyzed global gene expression in Arabidopsis in response to various hormones and in related experiments as part of the AtGenExpress project. The experimental agents included seven basic phytohormones (auxin, cytokinin, gibberellin, brassinosteroid, abscisic acid, jasmonate and ethylene) and their inhibitors. In addition, gene expression was investigated in hormone-related mutants and during seed germination and sulfate starvation. Hormone-inducible genes were identified from the hormone response data. The effects of each hormone and the relevance of the gene lists were verified by comparing expression profiles for the hormone treatments and related experiments using Pearson's correlation coefficient. This approach was also used to analyze the relationships among expression profiles for hormone responses and those included in the AtGenExpress stress-response data set. The expected correlations were observed, indicating that this approach is useful to monitor the hormonal status in the stress-related samples. Global interactions among hormones-inducible genes were analyzed in a pairwise fashion, and several known and novel hormone interactions were detected. Genome-wide transcriptional gene-to-gene correlations, analyzed by hierarchical cluster analysis (HCA), indicated that our data set is useful for identification of clusters of co-expressed genes, and to predict the functions of unknown genes, even if a gene's function is not directly related to the experiments included in AtGenExpress. Our data are available online from AtGenExpressJapan; the results of genome-wide HCA are available from PRIMe. The data set presented here will be a versatile resource for future hormone studies, and constitutes a reference for genome-wide gene expression in Arabidopsis.

Preventing unwanted breakups: using polygalacturonases to regulate cell separation.

Cell separation is an important biological process in plants that is precisely regulated both spatially and temporally. Key separation events include abscission of organs such as leaves and fruit and dehiscence events such as pod shatter in canola and other Brassicas. Polygalacturonases (PGs) are enzymes essential for the degradation of pectin, an important component of the adhesive material between cells. Although there are around 70 PG genes with overlapping expression domains, recent analysis has revealed the function of several PGs in specific aspects of Arabidopsis reproductive development. Upstream regulators that control the expression domain of some of these PGs during reproductive development have also been identified. This information provides new strategies to control unwanted cell separation events in various crops.

ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE1 (ADPG1), ADPG2, and QUARTET2 are Polygalacturonases required for cell separation during reproductive development in Arabidopsis.

Cell separation is thought to involve degradation of pectin by several hydrolytic enzymes, particularly polygalacturonase (PG). Here, we characterize an activation tagging line with reduced growth and male sterility caused by increased expression of a PG encoded by QUARTET2 (QRT2). QRT2 is essential for pollen grain separation and is part of a small family of three closely related endo-PGs in the Arabidopsis thaliana proteome, including ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE1 (ADPG1) and ADPG2. Functional assays and complementation experiments confirm that ADPG1, ADPG2, and QRT2 are PGs. Genetic analysis demonstrates that ADPG1 and ADPG2 are essential for silique dehiscence. In addition, ADPG2 and QRT2 contribute to floral organ abscission, while all three genes contribute to anther dehiscence. Expression analysis is consistent with the observed mutant phenotypes. INDEHISCENT (IND) encodes a putative basic helix-loop-helix required for silique dehiscence, and we demonstrate that the closely related HECATE3 (HEC3) gene is required for normal seed abscission and show that IND and HEC3 are required for normal expression of ADPG1 in the silique dehiscence zone and seed abscission zone, respectively. We also show that jasmonic acid and ethylene act together with abscisic acid to regulate floral organ abscission, in part by promoting QRT2 expression. These results demonstrate that multiple cell separation events, including both abscission and dehiscence, require closely related PG genes.

Potential sites of bioactive gibberellin production during reproductive growth in Arabidopsis.

Gibberellin 3-oxidase (GA3ox) catalyzes the final step in the synthesis of bioactive gibberellins (GAs). We examined the expression patterns of all four GA3ox genes in Arabidopsis thaliana by promoter-beta-glucuronidase gene fusions and by quantitative RT-PCR and defined their physiological roles by characterizing single, double, and triple mutants. In developing flowers, GA3ox genes are only expressed in stamen filaments, anthers, and flower receptacles. Mutant plants that lack both GA3ox1 and GA3ox3 functions displayed stamen and petal defects, indicating that these two genes are important for GA production in the flower. Our data suggest that de novo synthesis of active GAs is necessary for stamen development in early flowers and that bioactive GAs made in the stamens and/or flower receptacles are transported to petals to promote their growth. In developing siliques, GA3ox1 is mainly expressed in the replums, funiculi, and the silique receptacles, whereas the other GA3ox genes are only expressed in developing seeds. Active GAs appear to be transported from the seed endosperm to the surrounding maternal tissues where they promote growth. The immediate upregulation of GA3ox1 and GA3ox4 after anthesis suggests that pollination and/or fertilization is a prerequisite for de novo GA biosynthesis in fruit, which in turn promotes initial elongation of the silique.

Contribution of gibberellin deactivation by AtGA2ox2 to the suppression of germination of dark-imbibed Arabidopsis thaliana seeds.

Gibberellin levels in imbibed Arabidopsis thaliana seeds are regulated by light via phytochrome, presumably through regulation of gibberellin biosynthesis genes, AtGA3ox1 and AtGA3ox2, and a deactivation gene, AtGA2ox2. Here, we show that a loss-of-function ga2ox2 mutation causes an increase in GA(4) levels and partly suppresses the germination inability during dark imbibition after inactivation of phytochrome. Experiments using 2,2-dimethylGA(4), a GA(4) analog resistant to gibberellin 2-oxidase, in combination with ga2ox2 mutant seeds suggest that the efficiency of deactivation of exogenous GA(4) by AtGA2ox2 is dependent on light conditions, which partly explains phytochrome-mediated changes in gibberellin effectiveness (sensitivity) found in previous studies.

Activation of gibberellin biosynthesis and response pathways by low temperature during imbibition of Arabidopsis thaliana seeds.

Exposure of imbibed seeds to low temperature (typically 4 degrees C) is widely used to break seed dormancy and to improve the frequency of germination. However, the mechanism by which temperature accelerates germination is largely unknown. Using DNA microarray and gas chromatography-mass spectrometry analyses, we found that a subset of gibberellin (GA) biosynthesis genes were upregulated in response to low temperature, resulting in an increase in the level of bioactive GAs and transcript abundance of GA-inducible genes in imbibed Arabidopsis thaliana seeds. Using a loss-of-function mutant, the cold-inducible GA biosynthesis gene, AtGA3ox1, was shown to play an essential role in mediating the effect of low temperature. Besides temperature, AtGA3ox1 also is positively regulated by active phytochrome and negatively regulated by GA activity. We show that both red light and GA deficiency act in addition to low temperature to elevate the level of AtGA3ox1 transcript, indicating that multiple signals are integrated by the AtGA3ox1 gene to control seed germination. When induced by low temperature, AtGA3ox1 mRNA was detectable by in situ RNA hybridization in an additional set of cell types relative to that in red light-induced seeds. Our results illustrate that the GA biosynthesis and response pathways are activated during seed imbibition at low temperature and suggest that the cellular distribution of bioactive GAs may be altered under different light and temperature conditions.

Gibberellin biosynthesis and response during Arabidopsis seed germination.

The hormone-mediated control of plant growth and development involves both synthesis and response. Previous studies have shown that gibberellin (GA) plays an essential role in Arabidopsis seed germination. To learn how GA stimulates seed germination, we performed comprehensive analyses of GA biosynthesis and response using gas chromatography-mass spectrometry and oligonucleotide-based DNA microarray analysis. In addition, spatial correlations between GA biosynthesis and response were assessed by in situ hybridization. We identified a number of transcripts, the abundance of which is modulated upon exposure to exogenous GA. A subset of these GA-regulated genes was expressed in accordance with an increase in endogenous active GA levels, which occurs just before radicle emergence. The GA-responsive genes identified include those responsible for synthesis, transport, and signaling of other hormones, suggesting the presence of uncharacterized crosstalk between GA and other hormones. In situ hybridization analysis demonstrated that the expression of GA-responsive genes is not restricted to the predicted site of GA biosynthesis, suggesting that GA itself, or GA signals, is transmitted across different cell types during Arabidopsis seed germination.

Emission of ent-kaurene, a diterpenoid hydrocarbon precursor for gibberellins, into the headspace from plants.

ent-Kaurene is a tetracyclic hydrocarbon precursor for gibberellins (GAs) in plants and fungi. To address whether fungal GA biosynthesis enzymes function in plants, we generated transgenic Arabidopsis plants overexpressing ent-kaurene synthase (GfCPS/KS) from a GA-producing fungus Gibberella fujikuroi. GfCPS/KS catalyzes a two-step reaction corresponding to ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) activities in plants. When GfCPS/KS was overexpressed and targeted to plastids, a range of GA-deficient phenotypes of the ga1-3 and ga2-1 mutants (defective in CPS and KS, respectively) were restored to wild type. Unexpectedly, the transgenic lines overproducing GfCPS/KS emitted the GA precursor ent-kaurene into the headspace besides its accumulation in the plant body. When co-cultivated with the ent-kaurene overproducers in a closed environment, the airborne ent-kaurene was able to fully complement the dwarf phenotype of ga1-3 and ga2-1 mutants, but not that of the ga3-1 mutant (defective in ent-kaurene oxidase). These results suggest that ent-kaurene may be efficiently metabolized into bioactive GAs in Arabidopsis when supplied as a volatile. We also provide evidence that ent-kaurene is released in the headspace of wild-type Chamaecyparis obtusa and Cryptomeria japonica plants, suggesting the occurrence of this hydrocarbon GA precursor as a volatile in nature.