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.     

Overlapping functions of YDA and MAPKKK3/MAPKKK5 upstream of MPK3/MPK6 in plant immunity and growth/development

Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE3(MAPK3 or MPK3) and MPK6 play important signaling roles in plant immunity and growth/development. MAPK KINASE4(MKK4)and MKK5 function redundantly upstream of MPK3 and MPK6 in these processes. YODA(YDA), also known as MAPK KINASE KINASE4(MAPKKK4), is upstream of MKK4/MKK5 and forms a complete MAPK cascade(YDA–MKK4/MKK5–MPK3/MPK6) in regulating plant growth and development. In plant immunity, MAPKKK3 and MAPKKK5 function redundantly upstream of the same...     

Antagonistic interactions between two MAP kinase cascades in plant development and immune signaling

Mitogen‐activated protein kinase (MAPK) signaling plays important roles in diverse biological processes. In Arabidopsis, MPK3/MPK6, MKK4/MKK5, and the MAPKKK YODA (YDA) form a MAPK pathway that negatively regulates stomatal development. Brassinosteroid (BR) stimulates this pathway to inhibit stomata production. In addition, MPK3/MPK6 and MKK4/MKK5 also serve as critical signaling components in plant immunity. Here, we report that MAPKKK3/MAPKKK5 form a kinase cascade with MKK4/MKK5 and MPK3/MPK6 to transduce defense signals downstream of multiple plant receptor kinases. Loss of MAPKKK3/MAPKKK5 leads to reduced activation of MPK3/MPK6 in response to different pathogen‐associated molecular patterns (PAMPs) and increased susceptibility to pathogens. Surprisingly, developmental defects caused by silencing of YDA are suppressed in the mapkkk3 mapkkk5 double mutant. On the other hand, loss of YDA or blocking BR signaling leads to increased PAMP‐induced activation of MPK3/MPK6. These results reveal antagonistic interactions between a developmental MAPK pathway and an immune signaling MAPK pathway.     

Activation of the Arabidopsis thaliana mitogen-activated protein kinase MPK11 by the flagellin-derived elicitor peptide, flg22

Mitogen-activated protein kinases (MAPK) mediate cellular signal transduction during stress responses, as well as diverse growth and developmental processes in eukaryotes. Pathogen infection or treatments with conserved pathogen-associated molecular patterns (PAMPs) such as the bacterial flagellin-derived flg22 peptide are known to activate three Arabidopsis thaliana MAPK: MPK3, MPK4, and MPK6. Several stresses, including flg22 treatment, are known to increase MPK11 expression but activation of MPK11 has not been shown. Here, we show that MPK11 activity can, indeed, be increased through flg22 elicitation. A small-scale microarray for profiling defense-related genes revealed that cinnamyl alcohol dehyrogenase 5 requires MPK11 for full flg22-induced expression. An mpk11 mutant showed increased flg22-mediated growth inhibition but no altered susceptibility to Pseudomonas syringae, Botrytis cinerea, or Alternaria brassicicola. In mpk3, mpk6, or mpk4 backgrounds, MPK11 is required for embryo or seed development or general viability. Although this developmental deficiency in double mutants and the lack of or only subtle mpk11 phenotypes suggest functional MAPK redundancies, comparison with the paralogous MPK4 reveals distinct functions. Taken together, future investigations of MAPK roles in stress signaling should include MPK11 as a fourth PAMP-activated MAPK.     

Identification of additional MAP kinases activated upon PAMP treatment

Mitogen-activated protein (MAP) kinase cascades play important roles in plant immunity. Upon pathogen associated molecular pattern (PAMP) treatment, MPK3, MPK6 and MPK4 are quickly activated by upstream MKKs through phosphorylation. Western blot analysis using α-phospho-p44/42-ERK antibody suggests that additional MPKs with similar size as MPK4 are also activated upon PAMP perception. To identify these MAP kinases, 7 candidate MPKs with similar sizes as MPK4 were selected for further analysis. Transgenic plants expressing these MPKs with a ZZ-3xFLAG double tag of 17 kD were generated and analyzed by western blot. MPK1, MPK11 and MPK13 were found to be phosphorylated upon treatment with flg22. Our study revealed additional MAPKs being activated during PAMP-triggered immunity.     

Thioredoxin inhibits MPK38-induced ASK1, TGF‐β, and p53 function in a phosphorylation-dependent manner

Murine protein serine–threonine kinase 38 (MPK38) is a member of the AMP‐activated protein kinase-related serine/threonine kinase family. The factors that regulate MPK38 activity and function are not yet elucidated. Here, thioredoxin (Trx) was shown to be a negative regulator of MPK38. The redox-dependent association of MPK38 and Trx was mediated through the C‐terminal domain of MPK38. Single and double amino acid substitution mutagenesis of MPK38 (C286S, C339S, C377S, and C339S/C377S) and Trx (C32S, C35S, and C32S/C35S) demonstrated that Cys³³⁹ and Cys³⁷⁷ of MPK38 and Cys³² and Cys³⁵ of Trx are required for MPK38–Trx complex formation. MPK38 directly interacted with and phosphorylated Trx at Thr⁷⁶. Expression of wild‐type Trx, but not the Trx mutants C32S/C35S and T76A, inhibited MPK38‐induced ASK1, TGF‐β, and p53 function by destabilizing MPK38. The E3 ubiquitin–protein ligase Mdm2 played a critical role in the regulation of MPK38 stability by Trx. Treatment of cells with 1-chloro-2,4-dinitrobenzene, a specific inhibitor of Trx reductase, decreased MPK38–Trx complex formation and subsequently increased MPK38 stability and activity, indicating that Trx negatively regulates MPK38 activity in vivo. Finally, we used ASK1-, Smad3-, and p53-null mouse embryonic fibroblasts to demonstrate that ASK1, Smad3, and p53 play important roles in the activity and function of MPK38, suggesting a functional link between MPK38 and ASK1, TGF-β, and p53 signaling pathways. These results indicate that Trx functions as a physiological inhibitor of MPK38, which plays an important role in inducing ASK1-, TGF‐β-, and p53-mediated activity.     

MEKK1, MKK1/MKK2 and MPK4 function together in a mitogen-activated protein kinase cascade to regulate innate immunity in plants

Mitogen-activated protein kinase (MAPK) cascades play important roles in regulating plant innate immune responses. In a genetic screen to search for mutants with constitutive defense responses, we identified multiple alleles of mpk4 and mekk1 that exhibit cell death and constitutive defense responses. Bimolecular fluorescence complementation (BiFC) analysis showed that both MPK4 and MEKK1 interact with MKK1 and MKK2, two closely related MAPK kinases. mkk1 and mkk2 single mutant plants do not have obvious mutant phenotypes. To test whether MKK1 and MKK2 function redundantly, mkk1 mkk2 double mutants were generated. The mkk1 mkk2 double mutant plants die at seedling stage and the seedling-lethality phenotype is temperature-dependent. Similar to the mpk4 and mekk1 mutants, the mkk1 mkk2 double mutant seedlings accumulate high levels of H2O2, display spontaneous cell death, constitutively express Pathogenesis Related (PR) genes and exhibit pathogen resistance. In addition, activation of MPK4 by flg22 is impaired in the mkk1 mkk2 double mutants, suggesting that MKK1 and MKK2 function together with MPK4 and MEKK1 in a MAP kinase cascade to negatively regulate innate immune 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).     

Mitogen-activated protein kinase 4 is involved in the regulation of mitotic and cytokinetic microtubule transitions in Arabidopsis thaliana

? A mitogen-activated protein kinase kinase kinase (MAPKKK) double mutant, Arabidopsis homologue of nucleus and phragmoplast associated kinase (anp) anp2anp3, and the mitogen-activated protein kinase (MAPK) 4 mutant mpk4 of Arabidopsis thaliana show prominent cytokinetic defects. This prompted the analysis of mitotic and cytokinetic progression as a function of MAPK signalling. Mutants were compared with wild types untreated or treated with the specific MAPKK inhibitor PD98059. ? This study included phenotype analysis, expression analysis of the MPK4 promoter, immunofluorescent localization of MPK4, tubulin and MAP65-1, and time-lapse microscopic visualization of the mitotic microtubule (MT) transitions in control, mutant and inhibitor-treated cells. ? Mutant and inhibitor-treated cells showed defects in mitosis and cytokinesis, including aberrant spindle and phragmoplast formation and drastically delayed or abortive mitosis and cytokinesis. As a result, bi- and multinucleate cells were formed, ultimately disturbing the vegetative tissue patterning. MPK4 was localized to all stages of the expanding phragmoplast, in a pattern similar to that of its putative substrate MAP65-1. ? In this study, MPK4 is shown to be involved in the regulation of mitosis/cytokinesis through modulation of the cell division plane and cytokinetic progression.     

A crucial role for the phosphorylation of STRAP at Ser188 by MPK38 in STRAP-dependent cell death through ASK1, TGF-β, p53, and PI3K/PDK1 signaling pathways

Serine-threonine kinase receptor-associated protein (STRAP) is a TGF-β receptor-interacting protein that participates in the regulation of cell proliferation and cell death in response to various stresses. Here, we demonstrate that STRAP phosphorylation plays an important role in determining the pro- or anti-apoptotic function of STRAP. Murine protein serine/threonine kinase 38 (MPK38) phosphorylates STRAP at Ser¹⁸⁸ via direct interaction. Complex formation between STRAP and MPK38 is mediated by Cys¹⁵² and Cys²⁷⁰ of STRAP and Cys³³⁹ and Cys³⁷⁷ of MPK38, suggesting the redox dependency of this interaction. MPK38-mediated STRAP Ser¹⁸⁸ phosphorylation contributes to the pro-apoptotic function of STRAP by modulating key steps in STRAP-dependent ASK1, TGF-β, p53, and PI3K/PDK1 signaling pathways. Moreover, knockdown of endogenous MPK38 using an inducible MPK38 shRNA system and in vivo activation of MPK38 by treatment of HEK293 and STRAP-null MEF cells with 1-chloro-2,4-dinitrobenzene (DNCB), a specific inhibitor of Trx reductase, provide evidence that STRAP Ser¹⁸⁸ phosphorylation plays a key role in STRAP-dependent cell death. Adenoviral delivery of MPK38 in mice also demonstrates that STRAP Ser¹⁸⁸ phosphorylation in the liver is tightly associated with cell death and proliferation through ASK1, TGF-β, p53, and PI3K/PDK1 pathways, resulting in apoptotic cell death.     

Mutational evidence that the Arabidopsis MAP kinase MPK6 is involved in anther, inflorescence, and embryo development

Loss-of-function, dominant-negative, and change-of-function genetic approaches were used to investigate the role played by the Arabidopsis mitogen-activated protein (MAP) kinase MPK6 throughout development. Plants homozygous for T-DNA null alleles of MPK6 displayed reduced male fertility and abnormal anther development. In addition, a portion of the seed produced by mpk6 plants was found to contain embryos that had burst out of their seed coats. To address potential functional redundancy, a dominant-negative version of MPK6 was constructed by changing the TEY activation loop motif to the amino acid sequence AEF. Plants expressing MPK6AEF via the MPK6 native promoter were found to produce excessive stomata, consistent with the recently described role of MPK6 in stomatal patterning. A novel floral phenotype characterized by abnormal sepal development was also observed in MPK6AEF lines. The gene expression pattern of the MPK6 native promoter was determined using a YFP-MPK6 fusion construct, and expression was observed throughout most plant tissues, consistent with a role for MPK6 in multiple developmental processes. The YFP-MPK6 construct was found to rescue the fertility phenotype of mpk6 null alleles, indicating that the fusion protein retains its biological activity. It was also observed, however, that plants expressing YFP-MPK6 displayed reduced apical dominance and a shortening of inflorescence internodes. These results suggest that the YFP tag modifies the activity of MPK6 in a manner that affects inflorescence development but not anther development. Taken together, the present results indicate that MPK6 is involved in the regulation of multiple aspects of plant development.     

Involvement of MPK4 in osmotic stress response pathways in cell suspensions and plantlets of Arabidopsis thaliana: activation by hypoosmolarity and negative role in hyperosmolarity tolerance

Three of the protein kinases activated by hypoosmotic stress in Arabidopsis thaliana cell suspensions were previously characterized [FEBS, 2002, 527, 43-50] as mitogen-activated protein (MAP) kinases and two of them corresponded to Arabidopsis mitogen-activated protein kinase 6 (MPK6) (44 kDa) and MPK3 (39 kDa). The third MAP kinase was identified here to MPK4, using a corresponding specific antibody. Like MPK6 and MPK3, MPK4 activity is clearly inhibited by apigenin and MPK4 activation by hypoosmolarity needs upstream phosphorylation events. Activation of the 3 MAP kinases, MPK3, 4 and 6, was confirmed in plantlets submitted to hypoosmotic stress. The action of a biotic signal, flagellin, was also demonstrated to induce the activations of the 3 MAP kinases. Using the mutant displaying MPK4 gene inactivation, the independence of the MPK3 and MPK6 activations towards the presence of MPK4 was demonstrated, both in hypoosmotic and flagellin signalling pathways. Although MPK4 was not activated by hyperosmolarity in cell suspensions nor in seedlings, a possible negative regulation of hyperosmolarity resistance by MPK4 is suggested, based both on phenotype and downstream gene expression studies.     

Arabidopsis map kinase 4 negatively regulates systemic acquired resistance

Transposon inactivation of Arabidopsis MAP kinase 4 produced the mpk4 mutant exhibiting constitutive systemic acquired resistance (SAR) including elevated salicylic acid (SA) levels, increased resistance to virulent pathogens, and constitutive pathogenesis-related gene expression shown by Northern and microarray hybridizations. MPK4 kinase activity is required to repress SAR, as an inactive MPK4 form failed to complement mpk4. Analysis of mpk4 expressing the SA hydroxylase NahG and of mpk4/npr1 double mutants indicated that SAR expression in mpk4 is dependent upon elevated SA levels but is independent of NPR1. PDF1.2 and THI2.1 gene induction by jasmonate was blocked in mpk4 expressing NahG, suggesting that MPK4 is required for jasmonic acid-responsive gene expression.     

Transient MPK6 activation in response to oxygen deprivation and reoxygenation is mediated by mitochondria and aids seedling survival in Arabidopsis

Mitogen-activated protein kinases (MPKs) are regulated by diverse stresses with a reactive oxygen species (ROS) component. Here, we report the rapid and transient activation of MPK3, MPK4 and MPK6 upon oxygen deprivation as well as reoxygenation in seedlings of Arabidopsis thaliana. MPK activation peaked within 2 h of oxygen deprivation and again at a higher magnitude within 5 min of reoxygenation. MPK6 was the predominant kinase regulated by oxygen availability in both aerial and root tissue, except in mpk6 mutants, which displayed compensatory activation of MPK3. A universal consequence of oxygen deprivation in eukaryotes is inhibition of the terminal step of the mitochondrial electron transport chain (mETC). We demonstrate that treatment of seedlings with the mETC inhibitors antimycin A and potassium cyanide under normoxia promotes transient MPK6 and MPK3 activation. Confocal imaging of seedlings provided evidence that both oxygen deprivation and mETC inhibitors stimulate mitochondria-associated ROS production. We found that seedling survival of prolonged oxygen deprivation was improved in transgenics that ectopically overexpress MPK3, MPK4 and MPK6, but the induction of mRNAs associated with low oxygen acclimation responses were not markedly altered in MPK6 overexpression lines or mpk6 loss-of-function mutants. However, distinctions in MPK6 activation potential were correlated with other differences in mRNAs accumulation. Our findings suggest that oxygen deprivation and reoxygenation trigger mitochondrial ROS production to activate MPK signaling, which in turn regulate reversible processes that aid survival of transient oxygen deprivation.