Jasmonate inhibits plant growth and reduces gibberellin levels via microRNA5998 and transcription factor MYC2.

Jasmonate (JA) and gibberellins (GAs) exert antagonistic effects on plant growth and development in response to environmental and endogenous stimuli. Although the crosstalk between JA and GA has been elucidated, the role of JA in GA biosynthesis remains unclear. Therefore, in this study, we investigated the mechanism underlying JA-mediated regulation of endogenous GA levels in Arabidopsis (Arabidopsis thaliana). Transient and electrophoretic mobility shift assays showed that transcription factor MYC2 regulates GA inactivation genes. Using transgenic plants, we further evaluated the contribution of MYC2 in regulating GA inactivation genes. JA treatment increased DELLA accumulation but did not inhibit DELLA protein degradation. Additionally, JA treatment decreased bioactive GA content, including GA4, significantly decreased the expression of GA biosynthesis genes, including ent-kaurene synthase (AtKS), GA 3ß-hydroxylase (AtGA3ox1), and AtGA3ox2, and increased the expression of GA inactivation genes, including GA 2 oxidase (AtGA2ox4), AtGA2ox7, and AtGA2ox9. Conversely, JA treatment did not significantly affect gene expression in the myc2 myc3 myc4 triple mutant, demonstrating the MYC2-4-dependent effects of JA in GA biosynthesis. Additionally, JA post-transcriptionally regulated AtGA3ox1 expression. We identified microRNA miR5998 as an AtGA3ox1-associated miRNA; its overexpression inhibited plant growth by suppressing AtGA3ox1 expression. Overall, our findings indicate that JA treatment inhibits endogenous GA levels and plant growth by decreasing the expression of GA biosynthesis genes and increasing the expression of GA inactivation genes via miR5998 and MYC2 activities.

DELLA degradation by gibberellin promotes flowering via GAF1-TPR-dependent repression of floral repressors in Arabidopsis.

Flowering is the developmental transition from the vegetative to the reproductive phase. FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), and LEAFY (LFY) are floral integrators. These genes are repressed by several floral repressors including EARLY FLOWERING3 (ELF3), SHORT VEGETATIVE PHASE (SVP), TEMPRANILLO1 (TEM1), and TEM2. Although gibberellin (GA) promotes flowering by activating the floral integrator genes, the exact molecular mechanism remains unclear. DELLAs are negative regulators in GA signaling and act as coactivators of the transcription factor GAI ASSOCIATED FACTOR 1 (GAF1). GAs convert the GAF1 complex from a transcriptional activator to a repressor. Here, we show that GAF1 functions in the GA-dependent flowering pathway by regulating FT and SOC1 expression in Arabidopsis thaliana. We identified four flowering repressors, ELF3, SVP, TEM1, and TEM2, as GAF1-target genes. In response to GAs, GAF1 forms a transcriptional repressor complex and promotes the expression of FT and SOC1 through the repression of four flowering repressor genes, ELF3, SVP, TEM1, and TEM2.

DELLA-GAF1 complex is involved in tissue-specific expression and gibberellin feedback regulation of GA20ox1 in Arabidopsis.

KEY MESSAGE: The GAF1 transcription factor is shown to bind to the promoter of the Arabidopsis GA-biosynthetic enzyme GA20ox1 and, in association with DELLA protein, promotes GA20ox1 expression, thereby contributing to its feedback regulation and tissue specificity. Gibberellins (GAs) are phytohormones that promote plant growth and development, including germination, elongation, flowering, and floral development. Homeostasis of endogenous GA levels is controlled by GA feedback regulation. DELLAs are negative regulators of GA signaling that are rapidly degraded in the presence of GAs. DELLAs regulate several target genes, including AtGA20ox2 and AtGA3ox1, encoding the GA-biosynthetic enzymes GA 20-oxidase and GA 3-oxidase, respectively. Previous studies have identified GAI-ASSOCIATED FACTOR 1 (GAF1) as a DELLA interactor, with which DELLAs act as transcriptional coactivators; furthermore, AtGA20ox2, AtGA3ox1, and AtGID1b were identified as target genes of the DELLA-GAF1 complex. Among the five Arabidopsis GA20ox genes, AtGA20ox1 is the most highly expressed gene during vegetative growth; its expression is controlled by GA feedback regulation. Here, we investigated whether AtGA20ox1 is regulated by the DELLA-GAF1 complex. The electrophoretic mobility shift and transactivation assays showed that three GAF1-binding sites exist in the AtGA20ox1 promoter. Using transgenic plants, we further evaluated the contribution of the DELLA-GAF1 complex to GA feedback regulation and tissue-specific expression. Mutations in two GAF1-binding sites obliterated the negative feedback regulation and tissue-specific expression of AtGA20ox1 in transgenic plants. Thus, our results showed that GAF1-binding sites are involved in GA feedback regulation and tissue-specific expression of AtGA20ox1 in Arabidopsis, suggesting that the DELLA-GAF1 complex is involved in both processes.

Autophosphorylation Affects Substrate-Binding Affinity of Tobacco Ca2+-Dependent Protein Kinase1.

Protein kinases regulate diverse physiological processes. Because many kinases preserve inherent autophosphorylation capability, autophosphorylation appears to be one of the most important mechanisms for cellular signaling. However, physiological functions of autophosphorylation are still largely unknown, other than the self-activation by phosphorylation of activation loop in the catalytic domain. REPRESSION OF SHOOT GROWTH (RSG) is the transcription factor involved in gibberellin (GA) feedback regulation. The tobacco (Nicotiana tabacum) Ca2+-dependent protein kinase, NtCDPK1, phosphorylates RSG, resulting in the negative regulation of RSG. NtCDPK1 was previously shown to be autophosphorylated in a Ca2+-dependent manner. Here, we investigated the functional importance of autophosphorylation in NtCDPK1. Ser-6 and Thr-21 were identified as autophosphorylation sites of NtCDPK1. Autophosphorylation not only reduced the binding affinity of NtCDPK1 for RSG, but also inhibited the homodimerization of NtCDPK1. Furthermore, autophosphorylation decreased the phosphorylation efficiency of RSG yet increased that of myelin basic protein. Ser-6 and Thr-21 of NtCDPK1 were phosphorylated in response to GAs in plants. The substitution of these autophosphorylation sites with Ala enhanced the NtCDPK1 overexpression-induced sensitization of seeds to a GA biosynthetic inhibitor during germination. These results suggest new functions of autophosphorylation in CDPKs, namely, autophosphorylation can prevent the excessive phosphorylation of substrates and alter the substrate preference of CDPKs.

Gibberellin Induces an Increase in Cytosolic Ca2+ via a DELLA-Independent Signaling Pathway.

DELLA proteins play a central role in gibberellin (GA) signaling. GA triggers DELLA degradation via the ubiquitin-proteasome pathway, thereby promoting plant growth. An increase in cytosolic Ca2+ ([Ca2+]cyt) was observed previously after several hours of GA application. Recent studies also suggest the existence of a DELLA-independent GA response. However, the effect of DELLA on the GA-induced increase in [Ca2+]cyt remains unknown. This study reexamined the effects of GAs on [Ca2+]cyt using the Ca2+ sensor protein aequorin in Arabidopsis (Arabidopsis thaliana). [Ca2+]cyt increased within a few minutes of GA treatment, even in transgenic plants expressing a mutated degradation-resistant version of REPRESSOR OF ga1-3 and in della pentuple mutant plants. In addition, it was also revealed that Ca2+ is not involved in DELLA degradation. These results suggest that the GA-induced increase in [Ca2+]cyt occurs via a DELLA-independent pathway, providing important information on the GA signaling network.

DELLA-GAF1 Complex Is a Main Component in Gibberellin Feedback Regulation of GA20 Oxidase 2.

Gibberellins (GAs) are phytohormones that regulate many aspects of plant growth and development, including germination, elongation, flowering, and floral development. Negative feedback regulation contributes to homeostasis of the GA level. DELLAs are negative regulators of GA signaling and are rapidly degraded in the presence of GAs. DELLAs regulate many target genes, including AtGA20ox2 in Arabidopsis (Arabidopsis thaliana), encoding the GA-biosynthetic enzyme GA 20-oxidase. As DELLAs do not have an apparent DNA-binding motif, transcription factors that act in association with DELLA are necessary for regulating the target genes. Previous studies have identified GAI-ASSOCIATED FACTOR1 (GAF1) as such a DELLA interactor, with which DELLAs act as coactivators, and AtGA20ox2 was identified as a target gene of the DELLA-GAF1 complex. In this study, electrophoretic mobility shift and chromatin immunoprecipitation assays showed that four GAF1-binding sites exist in the AtGA20ox2 promoter. Using transgenic plants, we further evaluated the contribution of the DELLA-GAF1 complex to GA feedback regulation. Mutations in four GAF1-binding sites abolished the negative feedback of AtGA20ox2 in transgenic plants. Our results showed that GAF1-binding sites are necessary for GA feedback regulation of AtGA20ox2, suggesting that the DELLA-GAF1 complex is a main component of the GA feedback regulation of AtGA20ox2.

DELLAs function as coactivators of GAI-ASSOCIATED FACTOR1 in regulation of gibberellin homeostasis and signaling in Arabidopsis.

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.

Scaffold Function of Ca2+-Dependent Protein Kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation.

A molecular mechanism to ensure signaling specificity is a scaffold. REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) transcription factor that is involved in gibberellin feedback regulation. The 14-3-3 proteins negatively regulate RSG by sequestering it in the cytoplasm in response to gibberellins. The N. tabacum Ca2+-dependent protein kinase NtCDPK1 was identified as an RSG kinase that promotes 14-3-3 binding of RSG by phosphorylation of RSG. CDPKs are unique sensor responders of Ca2+ that are only found in plants and some protozoans. Here, we report a scaffolding function of CDPK. 14-3-3 proteins bound to NtCDPK1 by a new mode. Autophosphorylation of NtCDPK1 was necessary for the formation of the binding between NtCDPK1 and 14-3-3 but not for its maintenance. NtCDPK1 formed a heterotrimer with RSG and 14-3-3. Furthermore, we found that NtCDPK1 transfers 14-3-3 to RSG after phosphorylation of RSG and that RSG dissociates from NtCDPK1 as a complex with 14-3-3. These results suggest that NtCDPK1 is an interesting scaffolding kinase that increases the specificity and efficiency of signaling by coupling catalysis with scaffolding on the same protein.

Alteration of substrate specificity: the variable N-terminal domain of tobacco Ca(2+)-dependent protein kinase is important for substrate recognition.

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. Ca(2+)-dependent protein kinases (CDPKs) play central roles in Ca(2+) 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.

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.

Neucrin is a novel neural-specific secreted antagonist to canonical Wnt signaling.

A gene encoding a novel secreted protein in mice and humans was identified, and named Neucrin. Mouse Neucrin consists of 343 amino acids with a cysteine-rich domain in its carboxyl terminal region. The positions of 10 cysteine residues in the cysteine-rich domain are similar to those of Dickkopfs (Dkks), secreted Wnt antagonists. However, whereas Dkks have two cysteine-rich domains, Neucrin has only one. Neucrin as well as Dkks bound to LDL receptor-related protein 6 and inhibited the stabilization of cytosolic beta-catenin, indicating that Neucrin is an antagonist of canonical Wnt signaling. Mouse Neucrin expression was not detected in any major tissues in the adult, but was detected in developing neural tissues, including the brain and spinal cord. The expression pattern of Neucrin is distinct from that of any Dkk. Neucrin is a unique secreted Wnt antagonist that is predominantly expressed in developing neural tissues.

Autophosphorylation of Ser-6 via an intermolecular mechanism is important for the rapid reduction of NtCDPK1 kinase activity for substrate RSG.

Tobacco (Nicotiana tabacum) Ca2+-dependent protein kinase 1 (NtCDPK1) is involved in feedback regulation of the plant hormone gibberellin through the phosphorylation of the transcription factor, REPRESSION OF SHOOT GROWTH (RSG). Previously, Ser-6 and Thr-21 were identified as autophosphorylation sites in NtCDPK1. Autophosphorylation of Ser-6 and Thr-21 not only decreases the binding affinity of NtCDPK1 for RSG, but also inhibits the homodimerization of NtCDPK1. Furthermore, autophosphorylation decreases the phosphorylation efficiency of RSG. We demonstrated that Ser-6 and Thr-21 of NtCDPK1 are phosphorylated in response to GAs in plants. The substitution of these autophosphorylation sites with Ala enhances the NtCDPK1 overexpression-induced sensitization of seeds to a GA biosynthetic inhibitor during germination. These findings suggested that autophosphorylation of Ser-6 and Thr-21 prevents excessive phosphorylation of RSG. In this study, we attempted to determine which autophosphorylation site is responsible for the functional regulation of NtCDPK1. Ser-6 was autophosphorylated within 1 min, whereas Thr-21 required over 5 min to be completely autophosphorylated. Furthermore, we found that Ser-6 and Thr-21 were autophosphorylated by inter- and intramolecular mechanisms, respectively, which may be reflected in the faster autophosphorylation of Ser-6. Although both autophosphorylation sites were involved in the reduction of the binding affinity of NtCDPK1 for RSG and the inhibition of NtCDPK1 homodimerization, autophosphorylation of Ser-6 alone was sufficient to decrease the kinase activity of NtCDPK1 for RSG. These results suggest that autophosphorylation of Ser-6 is important for the rapid reduction of NtCDPK1 kinase activity for RSG, whereas that of Thr-21 may play an auxiliary role.

DELLA-dependent and -independent gibberellin signaling.

DELLA proteins act as negative regulators in gibberellin (GA) signal transduction. GA-induced DELLA degradation is a central regulatory system in GA signaling pathway. Intensive studies have revealed the degradation mechanism of DELLA and the functions of DELLA as a transcriptional regulator. Meanwhile, recent studies suggest the existence of a DELLA-independent GA signaling pathway. In this review, we summarized the DELLA-independent GA signaling pathway together with the well-analyzed DELLA-dependent pathway.

Direct determination of estrogenic and antiestrogenic activities using an enhanced plant two-hybrid system.

This paper reports a simple, low-cost, and extremely sensitive reporter-gene assay system for comprehensive analysis of estrogenic activity using transgenic Arabidopsis thaliana: the EPTH system. It had the capability to detect 17beta-estradiol at a concentration of 10 pM. The system was rendered 5 times more sensitive than a previous system [Tojo, T.; Tsuda, K.; Wada, T.; Yamazaki, K. Ecotoxicol. Environ. Saf. 2006, 64, 106-114) (1)] by increasing the copy number of the transactivation domain fused to a nuclear receptor co-activator. The system can efficiently detect other estrogenic and antiestrogenic substances. Estrogenic activities were determined in treated sewage samples from four distinct sewage farms using the system. Results showed that the system can detect estrogenic activity directly and more efficiently than a yeast two-hybrid system without any manipulation for extraction and condensation of hydrophobic compounds and aseptic treatment. Furthermore, the system also is useful as a powerful tool for discovery of a new category of natural estrogenic substances that are undetectable by previous plant and yeast systems.

Binding of GID1 to DELLAs promotes dissociation of GAF1 from DELLA in GA dependent manner.

Gibberellins (GAs) are important phytohormones for plant growth and development. DELLAs are members of the plant-specific GRAS protein family and act as repressors of GA signaling. DELLAs are rapidly degraded in the presence of GAs. GA-GID1-DELLA complexes are recognized and ubiquitinated by the SCF(SLY) complex. The sleepy1 (sly1) F-box mutant exhibits dwarfism and low-germination phenotypes due to high accumulation of DELLAs. Overexpression of GID1 in the sly1 mutant partially rescues these phenotypes without degradation of DELLAs suggesting that proteolysis independent regulation of DELLAs exists in GA signaling. But the molecular mechanisms of non-proteolytic regulation of DELLA are largely unknown. Recently we identified a DELLA binding transcription factor, GAI-ASSOCIATED FACTOR1 (GAF1). GAF1 also interacts with co-repressor TOPLESS RELATED (TPR) in nuclei. DELLAs and TPR act as coactivator and corepressor of GAF1, respectively. GAs converts the GAF1 complex from transcriptional activator to repressor via degradation of DELLAs. The overexpression of ΔPAM, lacking of DELLAs binding region of GAF1, partially rescue dwarf phenotypes of GA deficient or GA insensitive mutant. In this study, we investigate the relationship between non-proteolytic regulation of DELLAs and GA signaling via DELLA-GAF1 complex using modified yeast two-hybrid system.

Phosphorylation-independent binding of 14-3-3 to NtCDPK1 by a new mode.

14-3-3 pproteins play essential roles in diverse cellular processes through the direct binding to target proteins. REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) transcription factor that is involved in gibberellin (GA) feedback regulation. The 14-3-3 proteins bind to RSG depending on the RSG phosphorylation of Ser-114 and negatively regulate RSG by sequestering it in the cytoplasm in response to GAs. The Ca(2+)-dependent protein kinase NtCDPK1 was identified as an RSG kinase that promotes 14-3-3 binding of RSG by phosphorylation of RSG. 14-3-3 weakly binds to NtCDPK1 by a new mode. The autophosphorylation of NtCDPK1 was necessary for the formation of the binding between NtCDPK1 and 14-3-3 but not for its maintenance. In this study, we showed that 14-3-3 binding to NtCDPK1 does not require the autophosphorylation when RSG was bound to NtCDPK1. These data suggest that 14-3-3 binds to an unphosphorylated motif in NtCDPK1 exposed by a conformational change in NtCDPK1 but not to a phosphate group generated by autophosphorylation of NtCDPK1.

The mechanism of substrate recognition of Ca2+-dependent protein kinases.

Ca2+-dependent protein kinases (CDPKs) are encoded by a multigene family and are thought to play central roles in Ca2+ signaling in plants. Although the primary structures of CDPK isoforms are highly conserved, several studies suggested a distinct physiological function for each CDPK isoform in plants. Hence, there should be mechanisms by which individual CDPK specifically recognizes its substrate. Recently, the variable N-terminal domain of NtCDPK1 was shown to play an essential role in the specific recognition of the substrate. Because the variable N-terminal domain of other CDPKs may also be involved in the substrate recognition, the search for interacting proteins of the variable N-terminal domain would provide important clues to identify the physiological substrates of each CDPK. Additionally, manipulation of the variable N-terminal domain may enable us to engineer the substrate specificity of CDPK, leading a rational rewiring of cellular signaling pathways.

bZIP transcription factor RSG controls the feedback regulation of NtGA20ox1 via intracellular localization and epigenetic mechanism.

Gibberellins (GAs) are phytohormones that regulate growth and development throughout the life cycle of plants. Negative feedback contributes to homeostasis of GA levels. DELLA proteins are involved in this process. Since DELLA proteins do not have apparent DNA binding motifs, other DNA binding proteins might act as a mediator downstream of DELLA proteins in the GA feedback regulation. In this review, we highlight the mechanisms of GA feedback regulation, specifically the differential regulation of GA 20-oxidase (GA20ox) and GA 3-oxidase (GA3ox) by transcription factors. RSG (REPRESSION OF SHOOT GROWTH) is a tobacco (Nicotiana tabacum) transcriptional activator with a basic leucine zipper domain that controls the levels of endogenous GAs through the regulation of GA biosynthesis genes. Recently we reported that RSG not only regulates the expression of ent-kaurene oxidase gene but is also involved in the negative feedback of NtGA20ox1 by GAs. RSG plays a role in the homeostasis of GAs through direct binding to the NtGA20ox1 promoter triggered by a decrease in GA levels in the cell. Furthermore, decreases in GA levels promote modifications of active histone marks on the NtGA20ox1 promoter. We have developed a hypothetical model to explain how RSG regulates dual target genes via epigenetic regulation.

CDPK1, a calcium-dependent protein kinase, regulates transcriptional activator RSG in response to gibberellins.

The homeostasis of gibberellins (GAs) is maintained by negative-feedback regulation in plant cells. REPRESSION OF SHOOT GROWTH (RSG) is a transcriptional activator with a basic Leu zipper domain suggested to contribute GA feedback regulation by the transcriptional regulation of genes encoding 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; however, the kinase that catalyzes the reaction is unknown. Recently a Ca(2+)-dependent protein kinase (CDPK) was identified as an RSG kinase that promotes 14-3-3 binding of RSG by phosphorylation of the Ser-114 of RSG. Our results suggest that CDPK decodes the Ca(2+) signal produced by GAs and regulates the intracellular localization of RSG in plant cells.