Comparative phosphoproteomic analysis reveals that phosphorylation of sucrose synthase GhSUS2 by Ca2+-dependent protein kinases GhCPK84/93 affects cotton fiber development.

Cotton fiber elongation is a critical growth phase that affects final fiber length. Morphological analysis indicated an asynchronous fiber elongation pattern between two cotton varieties, J7-1 and J14-1. Through phosphoproteomic analysis, a total of 89 differentially-phosphorylated proteins (DPPs) were identified in elongating fibers between J7-1 and J14-1. Gene ontology (GO) analysis showed that these DPPs were mainly enriched in sucrose synthase activity, transferase activity, and UDP-glycosyltransferase activity. In J14-1, the phosphorylation level of GhSUS2, a key sucrose synthase in the sucrose metabolism pathway, was significantly higher than that in J7-1. We further revealed that GhSUS2 positively regulates fiber elongation, and GhSUS2-silenced transgenic cotton displayed the phenotype of 'short fibers' compared with the controls. During fiber development, the residue Ser11 in the GhSUS2 protein is phosphorylated by the Ca2+-dependent protein kinases GhCPK84 and GhCPK93. Phosphorylated GhSUS2 is localized in the cytoplasm, whereas unphosphorylated GhSUS2 is localized in the plasma membrane. Moreover, abscisic acid (ABA) could promote the transcription and translation of GhCPK84 and GhCPK93, thereby enhancing the phosphorylation of GhSUS2 to impede fiber elongation. Thus, our data demonstrates that GhSUS2 plays a positive role in fiber development, but its phosphorylation by GhCPK84 and GhCPK93 hinders fiber elongation of cotton.

Benzotriazole Ultraviolet Stabilizers (BUVSs) as Potential Protein Kinase Antagonists in Rice.

The ubiquitous occurrence of benzotriazole ultraviolet stabilizers (BUVSs) in the environment and organisms has warned of their potential ecological and health risks. Studies showed that some BUVSs exerted immune and chronic toxicities to animals by disturbing signaling transduction, yet limited research has investigated the toxic effects on crop plants and the underlying mechanisms of signaling regulation. Herein, a laboratory-controlled hydroponic experiment was conducted on rice to explore the phytotoxicity of BUVSs by integrating conventional biochemical experiments, transcriptomic analysis, competitive sorption assays, and computational studies. The results showed that BUVSs inhibited the growth of rice by 6.30-20.4% by excessively opening the leaf stomas, resulting in increased transpiration. BUVSs interrupted the transduction of abscisic acid (ABA) signal through competitively binding to Ca2+-dependent protein kinase (CDPK), weakening the CDPK phosphorylation and further inhibiting the downstream signaling. As structural analogues of ATP, BUVSs acted as potential ABA signaling antagonists, leading to physiological dysfunction in mediating stomatal closure under stresses. This is the first comprehensive study elucidating the effects of BUVSs on the function of key proteins and the associated signaling transduction in plants and providing insightful information for the risk evaluation and control of BUVSs.

Autophosphorylation-based Calcium (Ca2+) Sensitivity Priming and Ca2+/Calmodulin Inhibition of Arabidopsis thaliana Ca2+-dependent Protein Kinase 28 (CPK28).

Plant calcium (Ca2+)-dependent protein kinases (CPKs) represent the primary Ca2+-dependent protein kinase activities in plant systems. CPKs are composed of a dual specificity (Ser/Thr and Tyr) kinase domain tethered to a calmodulin-like domain (CLD) via an autoinhibitory junction (J). Although regulation of CPKs by Ca2+ has been extensively studied, the contribution of autophosphorylation in controlling CPK activity is less well understood. Furthermore, whether calmodulin (CaM) contributes to CPK regulation, as is the case for Ca2+/CaM-dependent protein kinases outside the plant lineage, remains an open question. We therefore screened a subset of plant CPKs for CaM binding and found that CPK28 is a high affinity Ca2+/CaM-binding protein. Using synthetic peptides and native gel electrophoresis, we coarsely mapped the CaM-binding domain to a site within the CPK28 J domain that overlaps with the known site of intramolecular interaction between the J domain and the CLD. Peptide kinase activity of fully dephosphorylated CPK28 was Ca2+-responsive and was inhibited by Ca2+/CaM. Using in situ autophosphorylated protein, we expand on the known set of CPK28 autophosphorylation sites, and we demonstrate that, unexpectedly, autophosphorylated CPK28 had enhanced kinase activity at physiological concentrations of Ca2+ compared with the dephosphorylated protein, suggesting that autophosphorylation functions to prime CPK28 for Ca2+ activation and might also allow CPK28 to remain active when Ca2+ levels are low. Furthermore, CPK28 autophosphorylation substantially reduced sensitivity of the kinase to Ca2+/CaM inhibition. Overall, our analyses uncover new complexities in the control of CPK28 and provide mechanistic support for Ca2+ signaling specificity through Ca2+ sensor priming.

Ca2+ signaling in plant manganese uptake: CPK21/23 kinases phosphorylate and activate manganese transporter NRAMP1.

This brief article highlights the results of Fu et al. (Proc Natl Acad Sci USA 119:e2204574119, 2022), who recently found that manganese (Mn) deficiency triggers long-lasting multicellular Ca2+ oscillations in the elongation zone (EZ) of Arabidopsis roots and revealed a Ca2+-CPK21/23-NRAMP1 axis as an important mechanism for plant tolerance and adaptation to low Mn.

The Ca(2+) -dependent protein kinase CPK3 is required for MAPK-independent salt-stress acclimation in Arabidopsis.

Plants use different signalling pathways to respond to external stimuli. Intracellular signalling via calcium-dependent protein kinases (CDPKs) or mitogen-activated protein kinases (MAPKs) present two major pathways that are widely used to react to a changing environment. Both CDPK and MAPK pathways are known to be involved in the signalling of abiotic and biotic stresses in animal, yeast and plant cells. Here, we show the essential function of the CDPK CPK3 (At4g23650) for salt stress acclimation in Arabidopsis thaliana, and test crosstalk between CPK3 and the major salt-stress activated MAPKs MPK4 and MPK6 in the salt stress response. CPK3 kinase activity was induced by salt and other stresses after transient overexpression in Arabidopsis protoplasts, but endogenous CPK3 appeared to be constitutively active in roots and leaves in a strictly Ca(2+) -dependent manner. cpk3 mutants show a salt-sensitive phenotype comparable with mutants in MAPK pathways. In contrast to animal cells, where crosstalk between Ca(2+) and MAPK signalling is well established, CPK3 seems to act independently of those pathways. Salt-induced transcriptional induction of known salt stress-regulated and MAPK-dependent marker genes was not altered, whereas post-translational protein phosphorylation patterns from roots of wild type and cpk3 plants revealed clear differences. A significant portion of CPK3 was found to be associated with the plasma membrane and the vacuole, both depending on its N-terminal myristoylation. An initial proteomic study led to the identification of 28 potential CPK3 targets, predominantly membrane-associated proteins.

Xanthine oxidase inhibitor allopurinol improves atrial electrical remodeling in diabetic rats by inhibiting CaMKII/NCX signaling.

AIMS: Atrial fibrillation (AF) is a common arrhythmia which is associated with higher risk of stroke, heart failure and all-cause mortality. Abnormal Ca2+ handling in diabetes mellitus (DM) can cause delayed depolarization involved with increased NCX activity. Complicated mechanisms are involved in atrial remodeling, of which CaMKII may be a key node signal. Therefore, we intend to explore whether CaMKII activation induces atrial electrical remodeling by regulating NCX expression in this study. MAIN METHODS: Adult male SD rats were used to establish a diabetic rat model, divided into three groups: the control group, DM group and allopurinol group. Hemodynamic and ECG indicators were recorded, after which electrophysiological studies were conducted. The protein expression of CaMKII, p-CaMKII, XO, MnSOD and NCX was measured by Western blot and immunohistochemistry. H&E and Masson staining were applied for observing myocardial fibrosis. HL-1 cells were cultured for the measurement of ROS generation. KEY FINDINGS: The arrangement of atrial myocytes was disordered and the collagen volume fraction of the atrium tissue was elevated in the DM group compared with the control group, and improved by allopurinol. Higher incidence of inducible AF, reduced conduction velocity and higher conduction inhomogeneity were observed in diabetic rats. These electrophysiological abnormalities were accompanied by higher oxidative stress and protein expression of p-CaMKII and NCX. Allopurinol prevented the development of these abnormal changes. SIGNIFICANCE: Allopurinol can improve atrial electrical remodeling by inhibiting CaMKII activity and protein expression of NCX. These data indicate xanthine oxidase inhibition can reduce oxidative stress and ameliorate atrial electrical remodeling.

Overexpression of Zinc Finger Transcription Factor ZAT6 Enhances Salt Tolerance.

The purpose of the present investigation is to examine the function of the C2H2-type zinc finger transcription factor of Arabidopsis thaliana 6 (ZAT6) in salt stress tolerance in cells of rice (Oryza sativa L.), cotton (Gossypium hirsutum L.) and slash pine (Pinus elliottii Engelm.). Cells of O. sativa, G. hirsutum, and P. elliottii overexpressing ZAT6 were generated using Agrobacterium-mediated genetic transformation. Molecular and functional analysis of transgenic cell lines demonstrate that overexpression of ZAT6 increased tolerance to salt stress by decreasing lipid peroxidation and increasing the content of abscisic acid (ABA) and GA8, as well as enhancing the activities of antioxidant enzymes such as ascorbate peroxidise (APOX), catalase (CAT), glutathione reductase (GR), and superoxide dismutase (SOD). In rice cells, ZAT6 also increased expression of Ca2+-dependent protein kinase genes OsCPK9 and OsCPK25 by 5-7 fold under NaCl stress. Altogether, our results suggest that overexpression of ZAT6 enhanced salt stress tolerance by increasing antioxidant enzyme activity, hormone content and expression of Ca2+-dependent protein kinase in transgenic cell lines of different plant species.

Microbe Associated Molecular Pattern Signaling in Guard Cells.

Stomata, formed by pairs of guard cells in the epidermis of terrestrial plants, regulate gas exchange, thus playing a critical role in plant growth and stress responses. As natural openings, stomata are exploited by microbes as an entry route. Recent studies reveal that plants close stomata upon guard cell perception of molecular signatures from microbes, microbe associated molecular patterns (MAMPs), to prevent microbe invasion. The perception of MAMPs induces signal transduction including recruitment of second messengers, such as Ca(2+) and H2O2, phosphorylation events, and change of transporter activity, leading to stomatal movement. In the present review, we summarize recent findings in signaling underlying MAMP-induced stomatal movement by comparing with other signalings.

The Arabidopsis Ca²âº-dependent protein kinase CPK27 is required for plant response to salt-stress.

Ca(2+)-dependent protein kinases (CDPKs) play vital roles in plant adaptations to environmental challenges. The precise regulatory mechanism of CDPKs in mediating salt stress still remains unclear, although several CDPK members have been identified to be involved in salt stress accumulation in various plants, such as Arabidopsis thaliana and Oryza sativa. Here, we investigated the function of an Arabidopsis CDPK, CPK27, in salt stress-signaling. CPK27 is a membrane-localized protein kinase; its expression was induced by NaCl. cpk27-1, a T-DNA insertion mutant of CPK27, was much more sensitive to salt stress than wild-type plants in terms of seed germination and post-germination seedling growth. In ion-flux assay, cpk27-1 mutants exhibited a lower capacity than wild-type plants to extrude Na(+) and import H(+) after a long-term salt treatment (110mM NaCl for 10days). Moreover, the content of Na(+) was higher and K(+) was lower in cpk27-1 mutants than in wild-type plants under salt stress. In addition, the level of salt-elicited H2O2 production was higher in cpk27-1 mutants than in wild-type plants Col after a short-term NaCl shock and long-term salt treatment. Collectively, our results suggest that CPK27 is required for plant adaptation to salt stress.

CLONING OF A cDNA ENCODING A 66-kDa Ca2 +-DEPENDENT PROTEIN KINASE (CDPK) FROM DUNALIELLA TERTIOLECTA (CHLOROPHYTA).

A cDNA clone encoding a Ca2 +-dependent protein kinase (DtCPK1) with a calculated molecular mass of 65,746 Da was isolated by sequential immuno- and hybridization-screening from a cDNA library of the halotolerant green alga, Dunaliella tertiolecta Butcher (Chlorophyceae). Primary structure analysis of DtCPK1 revealed a long variable domain preceding a catalytic domain, an autoinhibitory junction domain, and a C-terminal calmodulin-like domain containing 4 EF-hand motifs. Database searches showed that DtCPK1 has a high similarity to CCK1, a CDPK from the green alga, Chlamydomonas eugamentos Moewus. The N-terminal long variable domain of DtCPK1 contains neither the N-myristoylation motif, which is found in many CDPKs, nor the PEST motif, which is associated with rapid protein turnover and found in one CDPK subfamily. However, a putative Ca2 +-dependent lipid binding domain that might be responsible for the association of cytosolic DtCPK1 with the cell membrane was identified in the variable domain. Three CDPKs, with molecular masses of 62, 54, and 47 kDa respectively, were observed in an in-gel protein kinase assay of D. tertiolecta cells extract. No change in the activities of these CDPKs were observed for up to 30 min after D. tertiolecta cells had been subjected to a hypoosmotic shock. An antibody raised against a CDPK purified from D. tertiolecta and used to isolate the DtCPK1 cDNA clone cross-reacted strongly with the 62-kDa CDPK but weakly with the 54-kDa CDPK in a Western blot, indicating that the 62-kDa CDPK is identical to DtCPK1. There was no change in the intensity of these bands after hypoosmotic shock, implying that the cellular level of the enzyme protein is not associated with hypoosmotic shock. These results indicate that CDPK is activated only by the increase in cytosolic-free Ca2 + concentration in vivo.

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.