ZmCPK1, a calcium‐independent kinase member of the Zea mays CDPK gene family,functions as a negative regulator in cold stress signalling

Calcium‐dependent protein kinases (CDPKs) have been shown to play important roles in plant environmental stress signal transduction. We report on the identification of ZmCPK1 as a member of the maize (Zea mays) CDPK gene family involved in the regulation of the maize cold stress response. Based upon in silico analysis of the Z. mays cv. B73 genome, we identified that the maize CDPK gene family consists of 39 members. Two CDPK members were selected whose gene expression was either increased (Zmcpk1) or decreased (Zmcpk25) in response to cold exposure. Biochemical analysis demonstrated that ZmCPK1 displays calcium‐independent protein kinase activity. The C‐terminal calcium‐binding domain of ZmCPK1 was sufficient to mediate calcium independency of a previously calcium‐dependent enzyme in chimeric ZmCPK25‐CPK1 proteins. Furthermore, co‐transfection of maize mesophyll protoplasts with active full‐length ZmCPK1 suppressed the expression of a cold‐induced marker gene, Zmerf3 (ZmCOI6.21). In accordance, heterologous overexpression of ZmCPK1 in Arabidopsis thaliana yielded plants with altered acclimation‐induced frost tolerance. Our results identify ZmCPK1 as a negative regulator of cold stress signalling in maize.     

MAPK phosphatase MKP2 mediates disease responses in Arabidopsis and functionally interacts with MPK3 and MPK6

Mitogen‐activated protein kinase (MAPK) cascades have important functions in plant stress responses and development and are key players in reactive oxygen species (ROS) signalling and in innate immunity. In Arabidopsis, the transmission of ROS and pathogen signalling by MAPKs involves the coordinated activation of MPK6 and MPK3; however, the specificity of their negative regulation by phosphatases is not fully known. Here, we present genetic analyses showing that MAPK phosphatase 2 (MKP2) regulates oxidative stress and pathogen defence responses and functionally interacts with MPK3 and MPK6. We show that plants lacking a functional MKP2 gene exhibit delayed wilting symptoms in response to Ralstonia solanacearum and, by contrast, acceleration of disease progression during Botrytis cinerea infection, suggesting that this phosphatase plays differential functions in biotrophic versus necrotrophic pathogen‐induced responses. MKP2 function appears to be linked to MPK3 and MPK6 regulation, as indicated by BiFC experiments showing that MKP2 associates with MPK3 and MPK6 in vivo and that in response to fungal elicitors MKP2 exerts differential affinity versus both kinases. We also found that MKP2 interacts with MPK6 in HR‐like responses triggered by fungal elicitors, suggesting that MPK3 and MPK6 are subject to differential regulation by MKP2 in this process. We propose that MKP2 is a key regulator of MPK3 and MPK6 networks controlling both abiotic and specific pathogen responses in plants.     

Mitogen‐activated protein kinase 3 and 6 regulate Botrytis cinerea‐induced ethylene production in Arabidopsis

Plants challenged by pathogens, especially necrotrophic fungi such as Botrytis cinerea, produce high levels of ethylene. At present, the signaling pathways underlying the induction of ethylene after pathogen infection are largely unknown. MPK6, an Arabidopsis stress‐responsive mitogen‐activated protein kinase (MAPK) was previously shown to regulate the stability of ACS2 and ACS6, two type I ACS isozymes (1‐amino‐cyclopropane‐1‐carboxylic acid synthase). Phosphorylation of ACS2 and ACS6 by MPK6 prevents rapid degradation of ACS2/ACS6 by the 26S proteasome pathway, resulting in an increase in cellular ACS activity and ethylene biosynthesis. Here, we show that MPK3, which shares high homology and common upstream MAPK kinases with MPK6, is also capable of phosphorylating ACS2 and ACS6. In the mpk3 mutant background, ethylene production in gain‐of‐function GVG‐NtMEK2^DD transgenic plants was compromised, suggesting that MPK6 and MPK3 function together to stabilize ACS2 and ACS6. Using a liquid‐cultured seedling system, we found that B. cinerea‐induced ethylene biosynthesis was greatly compromised in mpk3/mpk6 double mutant seedlings. In contrast, ethylene production decreased only slightly in the mpk6 single mutant and not at all in the mpk3 single mutant, demonstrating overlapping roles for these two highly homologous MAPKs in pathogen‐induced ethylene induction. Consistent with the role of MPK3/MPK6 in the process, mutation of ACS2 and ACS6, two genes encoding downstream substrates of MPK3/MPK6, also reduced B. cinerea‐induced ethylene production. The residual levels of ethylene induction in the acs2/acs6 double mutant suggest the involvement of additional ACS isoforms, possibly regulated by MAPK‐independent pathway(s).     

ATR and MKP1 play distinct roles in response to UV‐B stress in Arabidopsis

Ultraviolet‐B (UV‐B) stress activates MAP kinases (MAPKs) MPK3 and MPK6 in Arabidopsis. MAPK activity must be tightly controlled in order to ensure an appropriate cellular outcome. MAPK phosphatases (MKPs) effectively control MAPKs by dephosphorylation of phosphothreonine and phosphotyrosine in their activation loops. Arabidopsis MKP1 is an important regulator of MPK3 and MPK6, and mkp1 knockout mutants are hypersensitive to UV‐B stress, which is associated with reduced inactivation of MPK3 and MPK6. Here, we demonstrate that MPK3 and MPK6 are hyperactivated in response to UV‐B in plants that are deficient in photorepair, suggesting that UV‐damaged DNA is a trigger of MAPK signaling. This is not due to a block in replication, as, in contrast to atr, the mkp1 mutant is not hypersensitive to the replication‐inhibiting drug hydroxyurea, hydroxyurea does not activate MPK3 and MPK6, and atr is not impaired in MPK3 and MPK6 activation in response to UV‐B. We further show that mkp1 leaves and roots are UV‐B hypersensitive, whereas atr is mainly affected at the root level. Tolerance to UV‐B stress has been previously associated with stem cell removal and CYCB1;1 accumulation. Although UV‐B‐induced stem cell death and CYCB1;1 expression are not altered in mkp1 roots, CYCB1;1 expression is reduced in mkp1 leaves. We conclude that the MKP1 and ATR pathways operate in parallel, with primary roles for ATR in roots and MKP1 in leaves.     

A Förster resonance energy transfer sensor for live‐cell imaging of mitogen‐activated protein kinase activity in Arabidopsis

The catalytic activity of mitogen‐activated protein kinases (MAPKs) is dynamically modified in plants. Since MAPKs have been shown to play important roles in a wide range of signaling pathways, the ability to monitor MAPK activity in living plant cells would be valuable. Here, we report the development of a genetically encoded MAPK activity sensor for use in Arabidopsis thaliana. The sensor is composed of yellow and blue fluorescent proteins, a phosphopeptide binding domain, a MAPK substrate domain and a flexible linker. Using in vitro testing, we demonstrated that phosphorylation causes an increase in the Förster resonance energy transfer (FRET) efficiency of the sensor. The FRET efficiency can therefore serve as a readout of kinase activity. We also produced transgenic Arabidopsis lines expressing this sensor of MAPK activity (SOMA) and performed live‐cell imaging experiments using detached cotyledons. Treatment with NaCl, the synthetic flagellin peptide flg22 and chitin all led to rapid gains in FRET efficiency. Control lines expressing a version of SOMA in which the phosphosite was mutated to an alanine did not show any substantial changes in FRET. We also expressed the sensor in a conditional loss‐of‐function double‐mutant line for the Arabidopsis MAPK genes MPK3 and MPK6. These experiments demonstrated that MPK3/6 are necessary for the NaCl‐induced FRET gain of the sensor, while other MAPKs are probably contributing to the chitin and flg22‐induced increases in FRET. Taken together, our results suggest that SOMA is able to dynamically report MAPK activity in living plant cells.     

Two guard cell‐preferential MAPKs,MPK9 and MPK12, regulate YEL signalling in Arabidopsis guard cells

We report that two mitogen‐activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate abscisic acid (ABA)‐induced stomatal closure in Arabidopsis thaliana. Yeast elicitor (YEL) induced stomatal closure accompanied by intracellular reactive oxygen species (ROS) accumulation and cytosolic free calcium concentration ([Ca²⁺]_cyt) oscillation. In this study, we examined whether these two MAP kinases are involved in YEL‐induced stomatal closure using MAPKK inhibitors, PD98059 and U0126, and MAPK mutants, mpk9, mpk12 and mpk9 mpk12. Both PD98059 and U0126 inhibited YEL‐induced stomatal closure. YEL induced stomatal closure in the mpk9 and mpk12 mutants but not in the mpk9 mpk12 mutant, suggesting that a MAPK cascade involving MPK9 and MPK12 functions in guard cell YEL signalling. However, YEL induced extracellular ROS production, intracellular ROS accumulation and cytosolic alkalisation in the mpk9, mpk12 and mpk9 mpk12 mutants. YEL induced [Ca²⁺]_cyt oscillations in both wild type and mpk9 mpk12 mutant. These results suggest that MPK9 and MPK12 function redundantly downstream of extracellular ROS production, intracellular ROS accumulation, cytosolic alkalisation and [Ca²⁺]_cyt oscillation in YEL‐induced stomatal closure in Arabidopsis guard cells and are shared with ABA signalling.     

Two guard cell mitogen‐activated protein kinases,MPK9 and MPK12, function in methyl jasmonate‐induced stomatal closure in Arabidopsis thaliana

Methyl jasmonate (MeJA) and abscisic acid (ABA) signalling cascades share several signalling components in guard cells. We previously showed that two guard cell‐preferential mitogen‐activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signalling in Arabidopsis thaliana. In this study, we examined whether these two MAP kinases function in MeJA signalling using genetic mutants for MPK9 and MPK12 combined with a pharmacological approach. MeJA induced stomatal closure in mpk9‐1 and mpk12‐1 single mutants as well as wild‐type plants, but not in mpk9‐1 mpk12‐1 double mutants. Consistently, the MAPKK inhibitor PD98059 inhibited the MeJA‐induced stomatal closure in wild‐type plants. MeJA elicited reactive oxygen species (ROS) production and cytosolic alkalisation in guard cells of the mpk9‐1, mpk12‐1 and mpk9‐1 mpk12‐1 mutants, as well in wild‐type plants. Furthermore, MeJA triggered elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in the mpk9‐1 mpk12‐1 double mutant as well as wild‐type plants. Activation of S‐type anion channels by MeJA was impaired in mpk9‐1 mpk12‐1. Together, these results indicate that MPK9 and MPK12 function upstream of S‐type anion channel activation and downstream of ROS production, cytosolic alkalisation and [Ca²⁺]_cyt elevation in guard cell MeJA signalling, suggesting that MPK9 and MPK12 are key regulators mediating both ABA and MeJA signalling in guard cells.     

Identification and characterization of an ABA‐activated MAP kinase cascade in Arabidopsis thaliana

Abscisic acid (ABA) is a major phytohormone involved in important stress‐related and developmental plant processes. Recent phosphoproteomic analyses revealed a large set of ABA‐triggered phosphoproteins as putative mitogen‐activated protein kinase (MAPK) targets, although the evidence for MAPKs involved in ABA signalling is still scarce. Here, we identified and reconstituted in vivo a complete ABA‐activated MAPK cascade, composed of the MAP3Ks MAP3K17/18, the MAP2K MKK3 and the four C group MAPKs MPK1/2/7/14. In planta, we show that ABA activation of MPK7 is blocked in mkk3‐1 and map3k17mapk3k18 plants. Coherently, both mutants exhibit hypersensitivity to ABA and altered expression of a set of ABA‐dependent genes. A genetic analysis further reveals that this MAPK cascade is activated by the PYR/PYL/RCAR‐SnRK2‐PP2C ABA core signalling module through protein synthesis of the MAP3Ks, unveiling an atypical mechanism for MAPK activation in eukaryotes. Our work provides evidence for a role of an ABA‐induced MAPK pathway in plant stress signalling.     

Nitric oxide promotes MPK6‐mediated caspase‐3‐like activation in cadmium‐induced Arabidopsis thaliana programmed cell death

Nitric oxide (NO), a vital cell‐signalling molecule, has been reported to regulate toxic metal responses in plants. This work investigated the effects of NO and the relationship between NO and mitogen‐activated protein kinase (MAPK) in Arabidopsis (Arabidopsis thaliana) programmed cell death (PCD) induced by cadmium (Cd²⁺) exposure. With fluorescence resonance energy transfer (FRET) analysis, caspase‐3‐like protease activation was detected after Cd²⁺ treatment. This was further confirmed with a caspase‐3 substrate assay. Cd²⁺‐induced caspase‐3‐like activity was inhibited in the presence of the NO‐specific scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO), suggesting that NO mediated caspase‐3‐like protease activation under Cd²⁺ stress conditions. Pretreatment with cPTIO effectively inhibited Cd²⁺‐induced MAPK activation, indicating that NO also affected the MAPK pathway. Interestingly, Cd²⁺‐induced caspase‐3‐like activity was significantly suppressed in the mpk6 mutant, suggesting that MPK6 was required for caspase‐3‐like protease activation. To our knowledge, this is the first demonstration that NO promotes Cd²⁺‐induced Arabidopsis PCD by promoting MPK6‐mediated caspase‐3‐like activation.     

Arabidopsis MPK6 is involved in cell division plane control during early root development,and localizes to the pre‐prophase band,phragmoplast, trans‐Golgi network and plasma membrane

The proper spatial and temporal expression and localization of mitogen‐activated protein kinases (MAPKs) is essential for developmental and cellular signalling in all eukaryotes. Here, we analysed expression, subcellular localization and function of MPK6 in roots of Arabidopsis thaliana using wild‐type plants and three mpk6 knock‐out mutant lines. The MPK6 promoter showed two expression maxima in the most apical part of the root meristem and in the root transition zone. This expression pattern was highly consistent with ‘no root’ and ‘short root’ phenotypes, as well as with ectopic cell divisions and aberrant cell division planes, resulting in disordered cell files in the roots of these mpk6 knock‐out mutants. In dividing root cells, MPK6 was localized on the subcellular level to distinct fine spots in the pre‐prophase band and phragmoplast, representing the two most important cytoskeletal structures controlling the cell division plane. By combining subcellular fractionation and microscopic in situ and in vivo co‐localization methods, MPK6 was localized to the plasma membrane (PM) and the trans‐Golgi network (TGN). In summary, these data suggest that MPK6 localizing to mitotic microtubules, secretory TGN vesicles and the PM is involved in cell division plane control and root development in Arabidopsis.     

Direct Phosphorylation and Activation of a Mitogen-Activated Protein Kinase by a Calcium-Dependent Protein Kinase in Rice

The mitogen-activated protein kinase (MAPK) is a pivotal point of convergence for many signaling pathways in eukaryotes. In the classical MAPK cascade, a signal is transmitted via sequential phosphorylation and activation of MAPK kinase kinase, MAPK kinase (MKK), and MAPK. The activation of MAPK is dependent on dual phosphorylation of a TXY motif by an MKK, which is considered the sole kinase to phosphorylate and activate MAPK. Here, we report a novel regulatory mechanism of MAPK phosphorylation and activation besides the canonical MAPK cascade. A rice (Oryza sativa) calcium-dependent protein kinase (CDPK), CPK18, was identified as an upstream kinase of MAPK (MPK5) in vitro and in vivo. Curiously, CPK18 was shown to phosphorylate and activate MPK5 without affecting the phosphorylation of its TXY motif. Instead, CPK18 was found to predominantly phosphorylate two Thr residues (Thr-14 and Thr-32) that are widely conserved in MAPKs from land plants. Further analyses reveal that the newly identified CPK18-MPK5 pathway represses defense gene expression and negatively regulates rice blast resistance. Our results suggest that land plants have evolved an MKK-independent phosphorylation pathway that directly connects calcium signaling to the MAPK machinery.     

Co-regulation of indole glucosinolates and camalexin biosynthesis by CPK5/CPK6 and MPK3/MPK6 signaling pathways

Secondary plant metabolites, represented by indole glucosinolates (IGS) and camalexin, play important roles in Arabidopsis immunity. Previously, we demonstrated the importance of MPK3 and MPK6, two closely related MAPKs, in regulating Botrytis cinerea (Bc)‐induced IGS and camalexin biosynthesis. Here we report that CPK5 and CPK6, two redundant calcium‐dependent protein kinases (CPKs), are also involved in regulating the biosynthesis of these secondary metabolites. The loss‐of‐function of both CPK5 and CPK6 compromises plant resistance to Bc. Expression profiling of CPK5‐VK transgenic plants, in which a truncated constitutively active CPK5 is driven by a steroid‐inducible promoter, revealed that biosynthetic genes of both IGS and camalexin pathways are coordinately upregulated after the induction of CPK5‐VK, leading to high‐level accumulation of camalexin and 4‐methoxyindole‐3‐yl‐methylglucosinolate (4MI3G). Induction of camalexin and 4MI3G, as well as the genes in their biosynthesis pathways, is greatly compromised in cpk5 cpk6 mutant in response to Bc. In a conditional cpk5 cpk6 mpk3 mpk6 quadruple mutant, Bc resistance and induction of IGS and camalexin are further reduced in comparison to either cpk5 cpk6 or conditional mpk3 mpk6 double mutant, suggesting that both CPK5/CPK6 and MPK3/MPK6 signaling pathways contribute to promote the biosynthesis of 4MI3G and camalexin in defense against Bc.     

A chemical genetic approach demonstrates that MPK3/MPK6 activation and NADPH oxidase‐mediated oxidative burst are two independent signaling events in plant immunity

Plant recognition of pathogen‐associated molecular patterns (PAMPs) such as bacterial flagellin‐derived flg22 triggers rapid activation of mitogen‐activated protein kinases (MAPKs) and generation of reactive oxygen species (ROS). Arabidopsis has at least four PAMP/pathogen‐responsive MAPKs: MPK3, MPK6, MPK4 and MPK11. It was speculated that these MAPKs may function downstream of ROS in plant immunity because of their activation by exogenously added H₂O₂. MPK3/MPK6 or their orthologs in other plant species have also been reported to be involved in the ROS burst from the plant respiratory burst oxidase homolog (Rboh) of the human neutrophil gp91phox. However, detailed genetic analysis is lacking. Using a chemical genetic approach, we generated a conditional loss‐of‐function mpk3 mpk6 double mutant. Consistent with results obtained using a conditionally rescued mpk3 mpk6 double mutant generated previously, the results obtained using the new conditional loss‐of‐function mpk3 mpk6 double mutant demonstrate that the flg22‐triggered ROS burst is independent of MPK3/MPK6. In Arabidopsis mutants lacking a functional AtRbohD, the flg22‐induced ROS burst was completely blocked. However, activation of MPK3/MPK6 was not affected. Based on these results, we conclude that the rapid ROS burst and MPK3/MPK6 activation are two independent early signaling events in plant immunity, downstream of FLS2. We also found that MPK4 negatively affects the flg22‐induced ROS burst. In addition, salicylic acid pre‐treatment enhances the AtRbohD‐mediated ROS burst, which is again independent of MPK3/MPK6 based on analysis of the mpk3 mpk6 double mutant. The establishment of an mpk3 mpk6 double mutant system using a chemical genetic approach provides a powerful tool to investigate the function of MPK3/MPK6 in the plant defense signaling pathway.     

Histone H2B monoubiquitination regulates salt stress‐induced microtubule depolymerization in Arabidopsis

Histone H2B monoubiquitination (H2Bub1) is recognized as a regulatory mechanism that controls a range of cellular processes. We previously showed that H2Bub1 was involved in responses to biotic stress in Arabidopsis. However, the molecular regulatory mechanisms of H2Bub1 in controlling responses to abiotic stress remain limited. Here, we report that HISTONE MONOUBIQUITINATION1 (HUB1) and HUB2 played important regulatory roles in response to salt stress. Phenotypic analysis revealed that H2Bub1 mutants confer decreased tolerance to salt stress. Further analysis showed that H2Bub1 regulated the depolymerization of microtubules (MTs), the expression of PROTEIN TYROSINE PHOSPHATASE1 (PTP1) and MAP KINASE PHOSPHATASE (MKP) genes – DsPTP1, MKP1, IBR5, PHS1, and was required for the activation of mitogen‐activated protein kinase3 (MAP kinase3, MPK3) and MPK6 in response to salt stress. Moreover, both tyrosine phosphorylation and the activation of MPK3 and MPK6 affected MT stability in salt stress response. Thus, the results indicate that H2Bub1 regulates salt stress‐induced MT depolymerization, and the PTP–MPK3/6 signalling module is responsible for integrating signalling pathways that regulate MT stability, which is critical for plant salt stress tolerance.     

Novel crosstalk between ERK MAPK and p38 MAPK leads to homocysteine‐NMDA receptor‐mediated neuronal cell death

Hyperhomocysteinemia is an independent risk factor for both acute and chronic neurological disorders, but little is known about the underlying mechanisms by which elevated homocysteine can promote neuronal cell death. We recently established a role for NMDA receptor‐mediated activation of extracellular signal‐regulated kinase (ERK)‐MAPK in homocysteine‐induced neuronal cell death. In this study, we examined the involvement of the stress‐induced MAPK, p38 in homocysteine‐induced neuronal cell death, and further explored the relationship between the two MAPKs, ERK and p38, in triggering cell death. Homocysteine‐mediated NMDA receptor stimulation and subsequent Ca²⁺ influx led to a biphasic activation of p38 MAPK characterized by an initial rapid, but transient activation followed by a delayed and more prolonged response. Selective inhibition of the delayed p38 MAPK activity was sufficient to attenuate homocysteine‐induced neuronal cell death. Using pharmacological and RNAi approaches, we further demonstrated that both the initial and delayed activation of p38 MAPK is downstream of, and dependent on activation of ERK MAPK. Our findings highlight a novel interplay between ERK and p38 MAPK in homocysteine‐NMDA receptor‐induced neuronal cell death.