SULTR2;1 Adjusts the Bolting Timing by Transporting Sulfate from Rosette Leaves to the Primary Stem.

Sulfur (S) is an essential macronutrient for plant growth and metabolism. SULTR2;1 is a low-affinity sulfate transporter facilitating the long-distance transport of sulfate in Arabidopsis. The physiological function of SULTR2;1 in the plant life cycle still needs to be determined. Therefore, we analyzed the sulfate transport, S-containing metabolite accumulation and plant growth using Arabidopsis SULTR2;1 disruption lines, sultr2;1-1 and sultr2;1-2, from seedling to mature growth stages to clarify the metabolic and physiological roles of SULTR2;1. We observed that sulfate distribution to the stems was affected in sultr2;1 mutants, resulting in decreased levels of sulfate, cysteine, glutathione (GSH) and total S in the stems, flowers and siliques; however, the GSH levels increased in the rosette leaves. This suggested the essential role of SULTR2;1 in sulfate transport from rosette leaves to the primary stem. In addition, sultr2;1 mutants unexpectedly bolted earlier than the wild-type without affecting the plant biomass. Correlation between GSH levels in rosette leaves and the bolting timing suggested that the rosette leaf GSH levels or limited sulfate transport to the early stem can trigger bolting. Overall, this study demonstrated the critical roles of SULTR2;1 in maintaining the S metabolite levels in the aerial part and transitioning from the vegetative to the reproductive growth phase.

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.

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.

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.

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.

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.

Post-translational methylation of high mobility group box 1 (HMGB1) causes its cytoplasmic localization in neutrophils.

High mobility group box 1 (HMGB1) protein plays multiple roles in transcription, replication, and cellular differentiation. HMGB1 is also secreted by activated monocytes and macrophages and passively released by necrotic or damaged cells, stimulating inflammation. HMGB1 is a novel antigen of anti-neutrophil cytoplasmic antibodies (ANCA) observed in the sera of patients with ulcerative colitis and autoimmune hepatitis, suggesting that HMGB1 is secreted from neutrophils to the extracellular milieu. However, the actual distribution of HMGB1 in the cytoplasm of neutrophils and the mechanisms responsible for it are obscure. Here we show that HMGB1 in neutrophils is post-translationally mono-methylated at Lys42. The methylation alters the conformation of HMGB1 and weakens its DNA binding activity, causing it to become largely distributed in the cytoplasm by passive diffusion out of the nucleus. Thus, post-translational methylation of HMGB1 causes its cytoplasmic localization in neutrophils. This novel pathway explains the distribution of nuclear HMGB1 to the cytoplasm and is important for understanding how neutrophils release HMGB1 to the extracellular milieu.

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.