Yohsuke Takahashi

Repression of shoot growth, a bZIP transcriptional activator, regulates cell elongation by controlling the level of gibberellins.

Cell expansion, a developmental process regulated by both endogenous programs and environmental stimuli, is critically important for plant growth. Here, we report the isolation and characterization of RSG (for repression of shoot growth), a transcriptional activator with a basic leucine zipper (bZIP) domain. To examine the role of RSG in plant development, we generated transgenic tobacco plants expressing a dominant-negative form of RSG, which repressed the activity of full-length RSG. In transgenic plants, this expression severely inhibited stem internode growth, specifically cell elongation. These plants also had less endogenous amounts of the major active gibberellin (GA) in tobacco, GA1. Applying GAs restored the dwarf phenotypes of transgenic tobacco plants that expressed the dominant-negative form of RSG. To investigate the function of RSG in the regulation of the endogenous amounts of GAs, we identified a target for RSG. RSG bound and activated the promoter of Arabidopsis GA3, one of the genes encoding enzymes involved in GA biosynthesis. Moreover, the dominant-negative form of RSG decreased expression of the GA3 homolog in transgenic tobacco plants. Our results show that RSG, a bZIP transcriptional activator, regulates the morphology of plants by controlling the endogenous amounts of GAs.

Involvement of 14-3-3 Signaling Protein Binding in the Functional Regulation of the Transcriptional Activator REPRESSION OF SHOOT GROWTH by Gibberellins

REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) transcriptional activator with a basic Leu zipper domain that regulates endogenous amounts of gibberellins (GAs) by the control of a GA biosynthetic enzyme. The 14-3-3 signaling proteins have been suggested to suppress RSG by sequestering it in the cytoplasm. Here, we show that RSG phosphorylation on Ser-114 is important for 14-3-3 binding. We found that GA levels regulate the intracellular localization of RSG. RSG translocated into the nucleus in response to a reduction in GA levels. GA treatment could reverse this nuclear accumulation. The GA-induced disappearance of RSG–green fluorescent protein from the nucleus did not depend on protein degradation. By contrast, the mutant RSG (S114A) that could not bind to 14-3-3 continued to be localized predominantly in the nucleus after GA application. Analysis of the mRNA levels of GA biosynthetic genes showed that the feedback regulation of the GA 20-oxidase gene was inhibited in transgenic plants expressing a dominant negative form of RSG. Our results suggest that RSG is negatively modulated by GAs by 14-3-3 binding and might be involved in GA homeostasis.

A Tobacco Calcium-Dependent Protein Kinase, CDPK1, Regulates the Transcription Factor REPRESSION OF SHOOT GROWTH in Response to Gibberellins

The homeostasis of gibberellins (GAs) is maintained by negative feedback in plants. REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) transcriptional activator that has been suggested to play a role in GA feedback by the regulation of GA biosynthetic enzymes. The 14-3-3 signaling proteins negatively regulate RSG by sequestering it in the cytoplasm in response to GAs. The phosphorylation on Ser-114 of RSG is essential for 14-3-3 binding of RSG. Here, we identified tobacco Ca2+-dependent protein kinase (CDPK1) as an RSG kinase that promotes 14-3-3 binding to RSG by phosphorylation of Ser-114 of RSG. CDPK1 interacts with RSG in a Ca2+-dependent manner in vivo and in vitro and specifically phosphorylates Ser-114 of RSG. Inhibition of CDPK repressed the GA-induced phosphorylation of Ser-114 of RSG and the GA-induced nuclear export of RSG. Overexpression of CDPK1 inhibited the feedback regulation of a GA 20-oxidase gene and resulted in sensitization to the GA biosynthetic inhibitor. Our results suggest that CDPK1 decodes the Ca2+ signal produced by GAs and regulates the intracellular localization of RSG.

AGF1, an AT-Hook Protein, Is Necessary for the Negative Feedback of AtGA3ox1 Encoding GA 3-Oxidase

Negative feedback is a fundamental mechanism of organisms to maintain the internal environment within tolerable limits. Gibberellins (GAs) are essential regulators of many aspects of plant development, including seed germination, stem elongation, and flowering. GA biosynthesis is regulated by the feedback mechanism in plants. GA 3-oxidase (GA3ox) catalyzes the final step of the biosynthetic pathway to produce the physiologically active GAs. Here, we found that only the AtGA3ox1 among the AtGA3ox family of Arabidopsis (Arabidopsis thaliana) is under the regulation of GA-negative feedback. We have identified a cis-acting sequence responsible for the GA-negative feedback of AtGA3ox1 using transgenic plants. Furthermore, we have identified an AT-hook protein, AGF1 (for the AT-hook protein of GA feedback regulation), as a DNA-binding protein for the cis-acting sequence of GA-negative feedback. The mutation in the cis-acting sequence abolished both GA-negative feedback and AGF1 binding. In addition, constitutive expression of AGF1 affected GA-negative feedback in Arabidopsis. Our results suggest that AGF1 plays a role in the homeostasis of GAs through binding to the cis-acting sequence of the GA-negative feedback of AtGA3ox1.

Alteration of Substrate Specificity: The Variable N-Terminal Domain of Tobacco Ca2+-Dependent Protein Kinase Is Important for Substrate Recognition

The variable N-terminal domain of CDPK1 is required for the recognition of the substrate RSG, which is a transcriptional activator involved in the gibberellin feedback regulation. This work opens the possibility of engineering the substrate specificity of CDPK by manipulation of the variable N-terminal domain, enabling a rational rewiring of cellular signaling pathways. Protein kinases are major signaling molecules that are involved in a variety of cellular processes. However, the molecular mechanisms whereby protein kinases discriminate specific substrates are still largely unknown. Ca2+-dependent protein kinases (CDPKs) play central roles in Ca2+ signaling in plants. Previously, we found that a tobacco (Nicotiana tabacum) CDPK1 negatively regulated the transcription factor REPRESSION OF SHOOT GROWTH (RSG), which is involved in gibberellin feedback regulation. Here, we found that the variable N-terminal domain of CDPK1 is necessary for the recognition of RSG. A mutation (R10A) in the variable N-terminal domain of CDPK1 reduced both RSG binding and RSG phosphorylation while leaving kinase activity intact. Furthermore, the R10A mutation suppressed the in vivo function of CDPK1. The substitution of the variable N-terminal domain of an Arabidopsis thaliana CDPK, At CPK9, with that of Nt CDPK1 conferred RSG kinase activities. This chimeric CDPK behaved according to the identity of the variable N-terminal domain in transgenic plants. Our results open the possibility of engineering the substrate specificity of CDPK by manipulation of the variable N-terminal domain, enabling a rational rewiring of cellular signaling pathways.

DELLAs Function as Coactivators of GAI-ASSOCIATED FACTOR1 in Regulation of Gibberellin Homeostasis and Signaling in Arabidopsis

This work reports the discovery of the DELLA-binding transcription factor GAF1 and shows that DELLAs and TPR act as coactivators and a corepressor with GAF1, respectively. GA converts the GAF1 complex from transcriptional activator to repressor via degradation of DELLAs. Accordingly, DELLAs turn on or off two sets of GA-regulated genes by dual functions, namely titration and coactivation. Gibberellins (GAs) are essential regulators of plant development, and DELLAs are negative regulators of GA signaling. The mechanism of GA-dependent transcription has been explained by DELLA-mediated titration of transcriptional activators and their release through the degradation of DELLAs in response to GA. However, the effect of GA on genome-wide expression is predominantly repression, suggesting the existence of unknown mechanisms of GA function. In this study, we identified an Arabidopsis thaliana DELLA binding transcription factor, GAI-ASSOCIATED FACTOR1 (GAF1). GAF1 shows high homology to INDETERMINATE DOMAIN1 (IDD1)/ENHYDROUS. GA responsiveness was decreased in the double mutant gaf1 idd1, whereas it was enhanced in a GAF1 overexpressor. GAF1 binds to genes that are subject to GA feedback regulation. Furthermore, we found that GAF1 interacts with the corepressor TOPLESS RELATED (TPR) and that DELLAs and TPR act as coactivators and a corepressor of GAF1, respectively. GA converts the GAF1 complex from transcriptional activator to repressor via the degradation of DELLAs. These results indicate that DELLAs turn on or off two sets of GA-regulated genes via dual functions, namely titration and coactivation, providing a mechanism for the integrative regulation of plant growth and GA homeostasis.

Identification of Negative cis-Acting Elements in Response to Copper in the Chloroplastic Iron Superoxide Dismutase Gene of the Moss Barbula unguiculata

Superoxide dismutases (SODs) are ubiquitous metalloenzymes that catalyze the dismutation of superoxide radicals. Chloroplasts have two isozymes, copper/zinc SOD (Cu/ZnSOD) and iron SOD (FeSOD), encoded by nuclear genes. Because bryophytes are considered as the earliest land plants, they are one of the most interesting plant models for adaptation against oxidative stress. In a previous study, we found that the FeSOD gene was expressed under Cu-deficient conditions and repressed under high-Cu-supply conditions; on the other hand, the Cu/ZnSOD gene was induced by Cu in a moss, Barbula unguiculata. The expression of Cu/ZnSOD and FeSOD is coordinately regulated at the transcriptional level depending on metal bioavailability. Here, using transgenic moss plants, we determined that the GTACT motif is a negative cis-acting element of the moss FeSOD gene in response to Cu. Furthermore, we found that a plant-specific transcription factor, PpSBP2 (for SQUAMOSA promoter-binding protein), and its related proteins bound to the GTACT motif repressed the expression of the FeSOD gene. The moss FeSOD gene was negatively regulated by Cu in transgenic Nicotiana tabacum plants, and the Arabidopsis thaliana FeSOD gene promoter containing the GTACT motif was repressed by Cu. Our results suggested that molecular mechanisms of GTACT motif-dependent transcriptional suppression by Cu are conserved in land plants.

The transcription factor RSG regulates negative feedback of NtGA20ox1 encoding GA 20-oxidase.

Gibberellins (GAs) are phytohormones that regulate growth and development throughout the life cycle of plants. RSG (REPRESSION OF SHOOT GROWTH) is a tobacco (Nicotiana tabacum) transcriptional activator with a basic leucine zipper domain that regulates the endogenous amount of GAs by control of GA biosynthetic enzymes. Negative feedback contributes to homeostasis of the GA levels. Previous studies suggested that RSG is directly or indirectly involved in the GA negative feedback of NtGA20ox1 encoding GA 20-oxidase. Using transgenic tobacco plants, we have identified a cis-acting region that is responsible for the feedback regulation of NtGA20ox1. This region contains an RSG-binding sequence. A mutation in the RSG-binding sequence abolished negative feedback of NtGA20ox1 in transgenic plants. Chromatin immunoprecipitation (ChIP) assays showed that RSG binds to the NtGA20ox1 promoter in vivo in response to a decrease in GA levels, and that this binding is abolished within 3 h after GA treatment. Furthermore, decreases in GA levels promote modifications of active histone marks in the promoter of NtGA20ox1. Our results suggest that RSG plays a role in the homeostasis of GAs through direct binding to the NtGA20ox1 promoter.

A Multiple-Stimuli-Responsive as-1-Related Element of parA Gene Confers Responsiveness to Cadmium but Not to Copper

The expression of parA, an auxin-regulated gene expressed during the culture of tobacco (Nicotiana tabacum L.) mesophyll protoplasts, is induced by cadmium. To identify the cadmium-responsive element, we examined the parA promoter using the GUS reporter gene. Cadmium responsiveness was retained in a 5[prime] deletion of the parA promoter to -78 bp, but it was nullified by further deletion to -49 bp, which implies that the region -49 to -78 bp contained a cadmium-responsive element. This region contains a sequence similar to as-1, an enhancer sequence from the cauliflower mosaic virus 35S RNA promoter that binds the nuclear factor ASF-1. We named the sequence in the parA promoter pas. Gel-shift assays revealed that pas and as-1 compete for the same DNA-binding nuclear protein(s). Since pentamers of either pas and as-1 were able to confer cadmium responsiveness on a minimal promoter but mutant as-1 was not, we propose that pas and as-1 are involved in cadmium-responsive gene expression. Neither pas nor as-1 conferred responsiveness to copper. The specificity of this response, involving the fuction of as-1- related elements including pas, is discussed.

Germin-like protein gene family of a moss, Physcomitrella patens, phylogenetically falls into two characteristic new clades

We identified 77 EST clones encoding germin-like proteins (GLPs) from a moss, Physcomitrella patens in a database search. These Physcomitrella GLPs (PpGLPs) were separated into seven groups based on DNA sequence homology. Phylogenetic analysis showed that these groups were divided into two novel clades clearly distinguishable from higher plant germins and GLPs, named bryophyte subfamilies 1 and 2. PpGLPs belonging to bryophyte subfamilies 1 lacked two cysteines at the conserved positions observed in higher plant germins or GLPs. PpGLPs belonging to bryophyte subfamily 2 contained two cysteines as observed in higher plant germins and GLPs. In bryophyte subfamily 1, 12 amino acids, in which one of two cysteines is included, were deleted between boxes A and B. Further, we determined the genomic structure of all of seven PpGLP genes. The sequences of PpGLPs of bryophyte subfamily 1 contained one or two introns, whereas those of bryophyte subfamily 2 contained no introns. Other GLPs from bryophytes, a liverwort GLP from Marchantia polymorpha, and two moss GLPs from Barbula unguiculata and Ceratodon purpureus also fell into bryophyte subfamily 1 and bryophyte subfamily 2, respectively. No higher plant germins and GLPs were grouped into the bryophyte subfamilies 1 and 2 by our analysis. Moreover, we revealed that PpGLP6 had manganese-containing extracellular superoxide dismutase activity. These results indicated that bryophyte possess characteristic GLPs, which phylogenetically are clearly distinguishable from higher plant GLPs.