CPK12: A Ca2+-dependent protein kinase balancer in abscisic acid signaling

Ca²⁺ is believed to be a critical second messenger in ABA signal transduction. Ca²⁺-dependent protein kinases (CDPKs) are the best characterized Ca²⁺ sensors in plants. Recently, we identified an Arabidopsis CDPK member CPK12 as a negative regulator of ABA signaling in seed germination and post-germination growth, which reveals that different members of the CDPK family may constitute a regulation loop by functioning positively and negatively in ABA signal transduction. We observed that both RNA interference and overexpression of CPK12 gene resulted in ABA-hypersensitive phenotypes in seed germination and post-germination growth, suggesting a high complexity of the CPK12-mediated ABA signaling pathway. CPK12 stimulates a negative ABA-signaling regulator (ABI2) and phosphorylates two positive ABA-signaling regulators (ABF1 and ABF4), which may partly explain the ABA hypersensitivity induced by both downregulation and upregulation of CPK12 expression. Our data indicate that CPK12 appears to function as a balancer in ABA signal transduction in Arabidopsis.     

Hierarchy and roles of pathogen-associated molecular pattern-induced responses in Nicotiana benthamiana

Our current understanding of pathogen-associated molecular pattern (PAMP)-triggered immunity signaling pathways in plants is limited due to the redundancy of several components or the lethality of mutants in Arabidopsis (Arabidopsis thaliana). To overcome this, we used a virus-induced gene silencing-based approach in combination with pharmacological studies to decipher links between early PAMP-triggered immunity events and their roles in immunity following PAMP perception in Nicotiana benthamiana. Two different calcium influx inhibitors suppressed the reactive oxygen species (ROS) burst: activation of the mitogen-activated protein kinases (MAPKs) and PAMP-induced gene expression. The calcium burst was unaffected in plants specifically silenced for components involved in ROS generation or for MAPKs activated by PAMP treatment. Importantly, the ROS burst still occurred in plants silenced for the two major defense-associated MAPK genes NbSIPK (for salicylic acid-induced protein kinase) and NbWIPK (for wound-induced protein kinase) or for both genes simultaneously, demonstrating that these MAPKs are dispensable for ROS production. We further show that NbSIPK silencing is sufficient to prevent PAMP-induced gene expression but that both MAPKs are required for bacterial immunity against two virulent strains of Pseudomonas syringae and their respective nonpathogenic mutants. These results suggest that the PAMP-triggered calcium burst is upstream of separate signaling branches, one leading to MAPK activation and then gene expression and the other to ROS production. In addition, this study highlights the essential roles of NbSIPK and NbWIPK in antibacterial immunity. Unexpectedly, negative regulatory mechanisms controlling the intensity of the PAMP-triggered calcium and ROS bursts were also revealed by this work.     

Calcium-dependent protein kinase 29 modulates PIN-FORMED polarity and Arabidopsis development via its own phosphorylation code

PIN-FORMED (PIN)-mediated polar auxin transport (PAT) is involved in key developmental processes in plants. Various internal and external cues influence plant development via the modulation of intracellular PIN polarity and, thus, the direction of PAT, but the mechanisms underlying these processes remain largely unknown. PIN proteins harbor a hydrophilic loop (HL) that has important regulatory functions; here, we used the HL as bait in protein pulldown screening for modulators of intracellular PIN trafficking in Arabidopsis thaliana. Calcium-dependent protein kinase 29 (CPK29), a Ca²⁺-dependent protein kinase, was identified and shown to phosphorylate specific target residues on the PIN-HL that were not phosphorylated by other kinases. Furthermore, loss of CPK29 or mutations of the phospho-target residues in PIN-HLs significantly compromised intracellular PIN trafficking and polarity, causing defects in PIN-mediated auxin redistribution and biological processes such as lateral root formation, root twisting, hypocotyl gravitropism, phyllotaxis, and reproductive development. These findings indicate that CPK29 directly interprets Ca²⁺ signals from internal and external triggers, resulting in the modulation of PIN trafficking and auxin responses.

Ca²⁺-dependent protein kinase 29 directly phosphorylates the hydrophilic loop of PIN-FORMED proteins to modulate their intracellular trafficking and Arabidopsis development.     

The protein kinase CPK28 phosphorylates ascorbate peroxidase and enhances thermotolerance in tomato

High temperatures are a major threat to plant growth and development, leading to yield losses in crops. Calcium-dependent protein kinases (CPKs) act as critical components of Ca²⁺ sensing in plants that transduce rapid stress-induced responses to multiple environmental stimuli. However, the role of CPKs in plant thermotolerance and their mechanisms of action remain poorly understood. To address this issue, tomato (Solanum lycopersicum) cpk28 mutants were generated using a CRISPR-Cas9 gene-editing approach. The responses of mutant and wild-type plants to normal (25°C) and high temperatures (45°C) were documented. Thermotolerance was significantly decreased in the cpk28 mutants, which showed increased heat stress-induced accumulation of reactive oxygen species (ROS) and levels of protein oxidation, together with decreased activities of ascorbate peroxidase (APX) and other antioxidant enzymes. The redox status of ascorbate and glutathione were also modified. Using a yeast two-hybrid library screen and protein interaction assays, we provide evidence that CPK28 directly interacts with cytosolic APX2. Mutations in APX2 rendered plants more sensitive to high temperatures, whereas the addition of exogenous reduced ascorbate (AsA) rescued the thermotolerance phenotype of the cpk28 mutants. Moreover, protein phosphorylation analysis demonstrated that CPK28 phosphorylates the APX2 protein at Thr-59 and Thr-164. This process is suggested to be responsive to Ca²⁺ stimuli and may be required for CPK28-mediated thermotolerance. Taken together, these results demonstrate that CPK28 targets APX2, thus improving thermotolerance. This study suggests that CPK28 is an attractive target for the development of improved crop cultivars that are better adapted to heat stress in a changing climate.

The protein kinase CPK28 regulates thermotolerance in plants by targeting APX2, thus regulating cellular redox homeostasis.     

Ca2+-Dependent Protein Kinase11 and 24 Modulate the Activity of the Inward Rectifying K+ Channels in Arabidopsis Pollen Tubes

Potassium (K⁺) influx into pollen tubes via K⁺ transporters is essential for pollen tube growth; however, the mechanism by which K⁺ transporters are regulated in pollen tubes remains unknown. Here, we report that Arabidopsis thaliana Ca²⁺-dependent protein kinase11 (CPK11) and CPK24 are involved in Ca²⁺-dependent regulation of the inward K⁺ (K+_in) channels in pollen tubes. Using patch-clamp analysis, we demonstrated that K+_in currents of pollen tube protoplasts were inhibited by elevated [Ca²⁺]_cyt. However, disruption of CPK11 or CPK24 completely impaired the Ca²⁺-dependent inhibition of K+_in currents and enhanced pollen tube growth. Moreover, the cpk11 cpk24 double mutant exhibited similar phenotypes as the corresponding single mutants, suggesting that these two CDPKs function in the same signaling pathway. Bimolecular fluorescence complementation and coimmunoprecipitation experiments showed that CPK11 could interact with CPK24 in vivo. Furthermore, CPK11 phosphorylated the N terminus of CPK24 in vitro, suggesting that these two CDPKs work together as part of a kinase cascade. Electrophysiological assays demonstrated that the Shaker pollen K+_in channel is the main contributor to pollen tube K+_in currents and acts as the downstream target of the CPK11-CPK24 pathway. We conclude that CPK11 and CPK24 together mediate the Ca²⁺-dependent inhibition of K+_in channels and participate in the regulation of pollen tube growth in Arabidopsis.     

Involvement of the glutamate receptor AtGLR3.3 in plant defense signaling and resistance to Hyaloperonospora arabidopsidis

Like their animal counterparts, plant glutamate receptor‐like (GLR) homologs are intimately associated with Ca²⁺ influx through plasma membrane and participate in various physiological processes. In pathogen‐associated molecular patterns (PAMP)‐/elicitor‐mediated resistance, Ca²⁺ fluxes are necessary for activating downstream signaling events related to plant defense. In this study, oligogalacturonides (OGs), which are endogenous elicitors derived from cell wall degradation, were used to investigate the role of Arabidopsis GLRs in defense signaling. Pharmacological investigations indicated that GLRs are partly involved in free cytosolic [Ca²⁺] ([Ca²⁺]_cyt) variations, nitric oxide (NO) production, reactive oxygen species (ROS) production and expression of defense‐related genes by OGs. In addition, wild‐type Col‐0 plants treated with the glutamate‐receptor antagonist 6,7‐dinitriquinoxaline‐2,3‐dione (DNQX) had a compromised resistance to Botrytis cinerea and Hyaloperonospora arabidopsidis. Moreover, we provide genetic evidence that AtGLR3.3 is a key component of resistance against H. arabidopsidis. In addition, some OGs‐triggered immune events such as defense gene expression, NO and ROS production are also to different extents dependent on AtGLR3.3. Taken together, these data provide evidence for the involvement of GLRs in elicitor/pathogen‐mediated plant defense signaling pathways in Arabidopsis thaliana.     

PCaPs,possible regulators of PtdInsP signals on plasma membrane

In plants, Ca²⁺, phosphatidylinositol phosphates (PtdInsPs) and inositol phosphates are major components of intracellular signaling. Several kinds of proteins and enzymes, such as calmodulin (CaM), protein kinase, protein phosphatase, and the Ca²⁺ channel, mediate the signaling. Two new Ca²⁺-binding proteins were identified from Arabidopsis thaliana and named PCaP1 and PCaP2 [plasma membrane (PM)-associated Ca²⁺(cation)-binding protein 1 and 2]. PCaP1 has an intrinsically disordered region in the central and C-terminal parts. The PCaP1 gene is expressed in most tissues and the PCaP2 gene is expressed predominantly in root hairs and pollen tubes. We recently demonstrated that these proteins are N-myristoylated, stably anchored in the PM, and are bound with phosphatidylinositol phosphates, especially PtdInsP₂s. Here we propose a model for the switching mechanism of Ca²⁺-signaling mediated by PtdInsPs. Ca²⁺ forms a complex with CaM (Ca²⁺-CaM) when there is an increase in the cytosol free Ca²⁺. The binding of PCaPs with Ca²⁺-CaM causes PCaPs to release PtdInsPs. Until the release of PtdInsPs, the signaling is kept in the resting state.Key words: calcium signal, calmodulin, inositol phosphate, intrinsically disordered protein, myristoylation, phosphatidylinositol phosphate, plasma membrane     

Disturbance of chlorophyll biosynthesis at Mg branch affects the chloroplast ROS homeostasis and Ca<Superscript>2+</Superscript> signaling in <Emphasis Type="Italic">Pisum sativum</Emphasis>

Reactive oxygen species (ROS) and calcium (Ca²⁺), two crucial intracellular signaling molecules, have been reported to play important roles in chlorophyll biosynthesis. In this study, we aimed to investigate whether disturbance of chlorophyll synthesis affects chloroplast ROS and Ca²⁺ homeostases. Chlorophyll biosynthesis was inhibited at the Mg branch by virus-induced gene silencing (VIGS) of CHLI gene encoding the Mg chelatase CHLI subunit in pea (Pisum sativum). Subsequently, ROS and intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in these chlorophyll-deficient pea plants were evaluated by histochemical and fluorescent staining assays. The results showed that the superoxide anion and hydrogen peroxide were predominantly generated in chloroplasts of the yellow leaves of pea VIGS-CHLI plants. The expression of genes encoding chloroplast antioxidant enzymes (CuZn-superoxide dismutase, ascorbate peroxidase, glutathione reductase, phospholipid glutathione peroxidase, peroxiredoxin and thioredoxins) were also decreased in the leaves of VIGS-CHLI plants compared with the control plants. Additionally, the [Ca²⁺]_i were significantly reduced in the yellow leaves of VIGS-CHLI plants compared with the green leaves of VIGS-GFP control plants. The expression of genes encoding Ca²⁺ signaling related proteins (thylakoid Ca²⁺ transporter, calmodulins and calcineurin B-like protein) was down-regulated in yellow VIGS-CHLI leaves. These results indicate that inhibition of chlorophyll biosynthesis at the Mg branch by silencing CHLI affects chloroplast ROS homeostasis and Ca²⁺ signaling and down-regulates the expression of ROS scavenging genes and Ca²⁺ signaling related genes.     

Identification of five B-type response regulators as members of a multistep phosphorelay system interacting with histidine-containing phosphotransfer partners of Populus osmosensor

Background

Mechanosensing and its downstream responses are speculated to involve sensory complexes containing Ca²⁺-permeable mechanosensitive channels. On recognizing osmotic signals, plant cells initiate activation of a widespread signal transduction network that induces second messengers and triggers inducible defense responses. Characteristic early signaling events include Ca²⁺ influx, protein phosphorylation and generation of reactive oxygen species (ROS). Pharmacological analyses show Ca²⁺ influx mediated by mechanosensitive Ca²⁺ channels to influence induction of osmotic signals, including ROS generation. However, molecular bases and regulatory mechanisms for early osmotic signaling events remain poorly elucidated.

Results

We here identified and investigated OsMCA1, the sole rice homolog of putative Ca²⁺-permeable mechanosensitive channels in Arabidopsis (MCAs). OsMCA1 was specifically localized at the plasma membrane. A promoter-reporter assay suggested that OsMCA1 mRNA is widely expressed in seed embryos, proximal and apical regions of shoots, and mesophyll cells of leaves and roots in rice. Ca²⁺ uptake was enhanced in OsMCA1-overexpressing suspension-cultured cells, suggesting that OsMCA1 is involved in Ca²⁺ influx across the plasma membrane. Hypo-osmotic shock-induced ROS generation mediated by NADPH oxidases was also enhanced in OsMCA1-overexpressing cells. We also generated and characterized OsMCA1-RNAi transgenic plants and cultured cells; OsMCA1-suppressed plants showed retarded growth and shortened rachises, while OsMCA1-suppressed cells carrying Ca²⁺-sensitive photoprotein aequorin showed partially impaired changes in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) induced by hypo-osmotic shock and trinitrophenol, an activator of mechanosensitive channels.

Conclusions

We have identified a sole MCA ortholog in the rice genome and developed both overexpression and suppression lines. Analyses of cultured cells with altered levels of this putative Ca²⁺-permeable mechanosensitive channel indicate that OsMCA1 is involved in regulation of plasma membrane Ca²⁺ influx and ROS generation induced by hypo-osmotic stress in cultured rice cells. These findings shed light on our understanding of mechanical sensing pathways.     

Cytosolic Ca2+ pulses and protein kinase activation in the signal transduction pathways leading to the plant oxidative burst

The oxidative burst, the rapid production of O₂- and H₂O₂ by plant cells in response to pathogens and Stressors, is a critical step in plant disease resistance and is controlled by several different elicitor-initiated signaling pathways. While different defense elicitors appear to activate disparate initial steps in signaling the oxidative burst, all of the elicitors tested thus far appear to stimulate pathways that converge on the same three core signaling intermediates: 1) the Ca₂₊-independent activation of a mitogen-activated protein kinase (MAPK) family member, 2) the influx of Ca₂₊ into the cytosol, deriving most critically from an internal compartment, and 3) the Ca²⁺-dependent activation of additional protein kinases including a second MAPK homologue and possibly calcium dependent protein kinases (CDPKs). Data from several recent reports are summarized to place these signaling events into a complete and updated model of signaling to the plant oxidative burst.     

Mutations in FLS2 Ser-938 Dissect Signaling Activation in FLS2-Mediated Arabidopsis Immunity

FLAGELLIN-SENSING 2 (FLS2) is a leucine-rich repeat/transmembrane domain/protein kinase (LRR-RLK) that is the plant receptor for bacterial flagellin or the flagellin-derived flg22 peptide. Previous work has shown that after flg22 binding, FLS2 releases BIK1 kinase and homologs and associates with BAK1 kinase, and that FLS2 kinase activity is critical for FLS2 function. However, the detailed mechanisms for activation of FLS2 signaling remain unclear. The present study initially identified multiple FLS2 in vitro phosphorylation sites and found that Serine-938 is important for FLS2 function in vivo. FLS2-mediated immune responses are abolished in transgenic plants expressing FLS2_S938A, while the acidic phosphomimic mutants FLS2_S938D and FLS2_S938E conferred responses similar to wild-type FLS2. FLS2-BAK1 association and FLS2-BIK1 disassociation after flg22 exposure still occur with FLS2_S938A, demonstrating that flg22-induced BIK1 release and BAK1 binding are not sufficient for FLS2 activity, and that Ser-938 controls other aspects of FLS2 activity. Purified BIK1 still phosphorylated purified FLS2_S938A and FLS2_S938D mutant kinase domains in vitro. Phosphorylation of BIK1 and homologs after flg22 exposure was disrupted in transgenic Arabidopsis thaliana plants expressing FLS2_S938A or FLS2_D997A (a kinase catalytic site mutant), but was normally induced in FLS2_S938D plants. BIK1 association with FLS2 required a kinase-active FLS2, but FLS2-BAK1 association did not. Hence FLS2-BIK1 dissociation and FLS2-BAK1 association are not sufficient for FLS2-mediated defense activation, but the proposed FLS2 phosphorylation site Ser-938 and FLS2 kinase activity are needed both for overall defense activation and for appropriate flg22-stimulated phosphorylation of BIK1 and homologs.     

A Ca2+-dependent Mechanism of Neuronal Survival Mediated by the Microtubule-associated Protein p600

In acute and chronic neurodegeneration, Ca²⁺ mishandling and disruption of the cytoskeleton compromise neuronal integrity, yet abnormalities in the signaling roles of cytoskeletal proteins remain largely unexplored. We now report that the microtubule-associated protein p600 (also known as UBR4) promotes neuronal survival. Following depletion of p600, glutamate-induced Ca²⁺ influx through NMDA receptors, but not AMPA receptors, initiates a degenerative process characterized by endoplasmic reticulum fragmentation and endoplasmic reticulum Ca²⁺ release via inositol 1,4,5-trisphosphate receptors. Downstream of NMDA receptors, p600 associates with the calmodulin·calmodulin-dependent protein kinase IIα complex. A direct and atypical p600/calmodulin interaction is required for neuronal survival. Thus, p600 counteracts specific Ca²⁺-induced death pathways through regulation of Ca²⁺ homeostasis and signaling.     

CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and Ca(2+)-permeable channels and stomatal closure

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca²⁺ in guard cell ion channel regulation. However, genetic mutants in Ca²⁺ sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca²⁺-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell–expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca²⁺ activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca²⁺-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca²⁺-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca²⁺ oscillation experiments revealed that Ca²⁺-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca²⁺-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca²⁺-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling.