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Mitogen-activated protein kinase (MAPK) cascades transmit environmental signals and induce stress and defence responses in plants. These signalling cascades are negatively controlled by specific Ser/Thr protein phosphatases of the type 2C (PP2C) and dual-specificity phosphatase (DSP) families that inactivate stress-induced MAPKs; however, the interplay between phosphatases of these different types has remained unknown. This work reveals that different Arabidopsis MAPK phosphatases, the PP2C-type AP2C1 and the DSP-type MKP1, exhibit both specific and overlapping functions in plant stress responses. Each single mutant, ap2c1 and mkp1, and the ap2c1 mkp1 double mutant displayed enhanced stress-induced activation of the MAPKs MPK3, MPK4, and MPK6, as well as induction of a set of transcription factors. Moreover, ap2c1 mkp1 double mutants showed an autoimmune-like response, associated with increased levels of the stress hormones salicylic acid and ethylene, and of the phytoalexin camalexin. This phenotype was reduced in the ap2c1 mkp1 mpk3 and ap2c1 mkp1 mpk6 triple mutants, suggesting that the autoimmune-like response is due to MAPK misregulation. We conclude that the evolutionarily distant MAPK phosphatases AP2C1 and MKP1 contribute crucially to the tight control of MAPK activities, ensuring appropriately balanced stress signalling and suppression of autoimmune-like responses during plant growth and development.

Analogues of vertebrate natriuretic peptides (NPs) present in plants, termed plant natriuretic peptides (PNPs), comprise a novel class of hormones that systemically affect salt and water balance and responses to plant pathogens. Several lines of evidence indicate that Arabidopsis thaliana PNP (AtPNP-A) affects cellular redox homeostasis, which is also typical for the signaling of its vertebrate analogues, but the molecular mechanism(s) of this effect remains elusive. Here we report identification of catalase 2 (CAT2), an antioxidant enzyme, as an interactor of AtPNP-A. The full-length AtPNP-A recombinant protein and the biologically active fragment of AtPNP-A bind specifically to CAT2 in surface plasmon resonance (SPR) analyses, while a biologically inactive scrambled peptide does not. In vivo bimolecular fluorescence complementation (BiFC) showed that CAT2 interacts with AtPNP-A in chloroplasts. Furthermore, CAT2 activity is lower in homozygous atpnp-a knockdown compared with wild type plants, and atpnp-a knockdown plants phenocopy CAT2-deficient plants in their sensitivity to elevated H2O2, which is consistent with a direct modulatory effect of the PNP on the activity of CAT2 and hence H2O2 homeostasis. Our work underlines the critical role of AtPNP-A in modulating the activity of CAT2 and highlights a mechanism of fine-tuning plant responses to adverse conditions by PNPs.

Rapid response to tissue damage Damaged plants are susceptible to microbial attack. In response to physical damage, plants proactively generate signal peptides to activate their immune systems. Hander et al. examined wound responses in the model plant Arabidopsis thaliana . They identified a metacaspase that releases an immunomodulatory signal peptide from its precursor form within 30 seconds of the damage. The metacaspase itself was activated by a burst of calcium released by tissue damage. Science , this issue p. eaar7486

Plant elicitor peptide 1 (Pep1) is a versatile immune modulator involved in plant defense against herbivores and pathogens. A recent study uncovers that arabidopsis (Arabidopsis thaliana) releases Pep1 from the carboxyl-terminus of the tonoplast-resident precursor protein, PROPEP1, cleaved by Ca(2+)-activated metacaspases upon cell membrane rupture in the damaged tissues.

The first line of inducible plant defence, pattern-triggered immunity (PTI), is activated by the recognition of exogenous as well as endogenous elicitors. Exogenous elicitors, also called microbe-associated molecular patterns, signal the presence of microbes. In contrast, endogenous elicitors seem to be generated and recognized under more diverse circumstances, making the evaluation of their biological relevance much more complex. Plant elicitor peptides (Peps) are one class of such endogenous elicitors, which contribute to immunity against attack by bacteria, fungi, as well as herbivores. Recent studies indicate that the Pep-triggered signalling pathways also operate during the response to a more diverse set of stresses including starvation stress. In addition, in silico data point to an involvement in the regulation of plant development, and a study on Pep-mediated inhibition of root growth supports this indication. Importantly, Peps are neither limited to the model plant Arabidopsis nor to a specific plant family like the previously intensively studied systemin peptides. On the contrary, they are present and active in angiosperms all across the phylogenetic tree, including many important crop plants. Here we summarize the progress made in research on Peps from their discovery in 2006 until now. We discuss the two main models which describe their likely function in plant immunity, highlight the studies supporting additional roles of Pep-triggered signalling and identify urgent research tasks to further uncover their biological relevance.

The endogenous Arabidopsis (Arabidopsis thaliana) peptides, AtPeps, elicit an innate immune response reminiscent of pattern-triggered immunity. Detection of various danger signals, including microbe-associated molecular patterns (MAMPs), leads to elevated transcription of PROPEPs, the AtPep precursors, and PEPRs, the AtPep receptors. It has been hypothesized that AtPeps are involved in enhancing pattern-triggered immunity. Following this idea, we analyzed the relationship between MAMP- and AtPep-elicited signaling. We found that the perception of MAMPs enhanced a subsequent AtPep-triggered production of reactive oxygen species (ROS). Intriguingly, other components of AtPep-triggered immunity like Ca2+ influx, mitogen-activated protein kinase phosphorylation, ethylene production, and expression of early defense genes, as well as ROS-activated genes, remained unchanged. By contrast, treatment with methyl jasmonate promoted an increase of all analyzed AtPep-triggered responses. We positively correlated the intensities of generic AtPep-triggered responses with the abundance of the two AtPep receptors by generating constitutively expressing PEPR1 and PEPR2 transgenic lines and by analyzing pepr1 and pepr2 mutants. Further, we show that enhanced, as well as basal, ROS production triggered by AtPeps is absent in the double mutant of the respiratory burst oxidase homologs D and F (rbohD rbohF). We present evidence that the enhancement of AtPep-triggered ROS is not based on changes in the ROS detoxification machinery and is independent of mitogen-activated protein kinase and Ca2+ signaling pathways. Taken together, these results indicate an additional level of regulation besides receptor abundance for the RbohD/RbohF-dependent production of AtPep-elicited ROS, which is specifically operated by MAMP-triggered pathways.

International audience; Sensing of potential pathogenic bacteria is of critical importance for immunity. In plants, this involves plasmamembrane-resident pattern recognition receptors, one of which is the FLAGELLIN SENSING 2 (FLS2) receptor kinase. Ligand-activated FLS2 receptors are internalized into endosomes. However, the extent to which these spatiotemporal dynamics are generally present among pattern recognition receptors (PRRs) and their regulation remain elusive. Using live-cell imaging, we show that at least three other receptor kinases associated with plant immunity, PEP RECEPTOR 1/2 (PEPR1/2) and EF-TU RECEPTOR (EFR), internalize in a ligand-specific manner. In all cases, endocytosis requires the coreceptor BRI1-ASSOCIATED KINASE 1 (BAK1), and thus depends on receptor activation status. We also show the internalization of liganded FLS2, suggesting the transport of signaling competent receptors. Trafficking of activated PRRs requires clathrin and converges onto the same endosomal vesicles that are also shared with the hormone receptor BRASSINOSTERIOD INSENSITIVE 1 (BRI1). Importantly, clathrin-dependent endocytosis participates in plant defense against bacterial infection involving FLS2-mediated stomatal closure and callose deposition, but is uncoupled from activation of the flagellin-induced oxidative burst and MAP kinase signaling. In conclusion, immunity mediated by pattern recognition receptors depends on clathrin, a critical component for the endocytosis of signaling competent receptors into a common endosomal pathway.

The methylesterification status of cell wall homogalacturonans, mediated through the action of pectin methylesterases (PMEs), influences the biophysical properties of plant cell walls such as elasticity and porosity, important parameters for cell elongation and water uptake. The completion of seed germination requires cell wall extensibility changes in both the radicle itself and in the micropylar tissues surrounding the radicle. In wild-type seeds of Arabidopsis (Arabidopsis thaliana), PME activities peaked around the time of testa rupture but declined just before the completion of germination (endosperm weakening and rupture). We overexpressed an Arabidopsis PME inhibitor to investigate PME involvement in seed germination. Seeds of the resultant lines showed a denser methylesterification status of their cell wall homogalacturonans, but there were no changes in the neutral sugar and uronic acid composition of the cell walls. As compared with wild-type seeds, the PME activities of the overexpressing lines were greatly reduced throughout germination, and the low steady-state levels neither increased nor decreased. The most striking phenotype was a significantly faster rate of germination, which was not connected to altered testa rupture morphology but to alterations of the micropylar endosperm cells, evident by environmental scanning electron microscopy. The transgenic seeds also exhibited an apparent reduced sensitivity to abscisic acid with respect to its inhibitory effects on germination. We speculate that PME activity contributes to the temporal regulation of radicle emergence in endospermic seeds by altering the mechanical properties of the cell walls and thereby the balance between the two opposing forces of radicle elongation and mechanical resistance of the endosperm.

Plants perceive UV-B radiation as an informational signal by a pathway involving UVR8 as UV-B photoreceptor, activating photomorphogenic and acclimation responses. In contrast, the response to UV-B as an environmental stress involves mitogen-activated protein kinase (MAPK) signalling cascades. Whereas the perception pathway is plant specific, the UV-B stress pathway is more broadly conserved. Knowledge of the UV-B stress-activated MAPK signalling pathway in plants is limited, and its potential interplay with the UVR8-mediated pathway has not been defined. Here, we show that loss of MAP kinase phosphatase 1 in the mutant mkp1 results in hypersensitivity to acute UV-B stress, but without impairing UV-B acclimation. The MKP1-interacting proteins MPK3 and MPK6 are activated by UV-B stress and are hyperactivated in mkp1. Moreover, mutants mpk3 and mpk6 exhibit elevated UV-B tolerance and partially suppress the UV-B hypersensitivity of mkp1. We show further that the MKP1-regulated stress-response MAPK pathway is independent of the UVR8 photoreceptor, but that MKP1 also contributes to survival under simulated sunlight. We conclude that, whereas UVR8-mediated acclimation in plants promotes UV-B-induced defence measures, MKP1-regulated stress signalling results when UV-B protection and repair are insufficient and damage occurs. The combined activity of these two mechanisms is crucial to UV-B tolerance in plants.

A primary component of plant defense is the detection of pathogen-associated molecular patterns (PAMPs) by plasma membrane-localized pathogen recognition receptors. PAMP perception results in rapid and transient activation of phosphorylation-dependent signaling pathways that lead to a wide array of defense-related responses, including extensive changes in gene expression. In Arabidopsis, several kinases, including the mitogen-activated protein kinases (MAPKs) MPK6 and MPK3, are rapidly activated after PAMP treatment, and are thought to positively regulate a wide array of defense-related responses. In contrast, negative regulation of PAMP responses by downstream phosphatases remains poorly understood. Here we report the identification of Arabidopsis MAP Kinase Phosphatase 1 (MKP1) as a negative regulator of diverse PAMP responses, including activation of MPK6 and MPK3, transient production of extracellular reactive oxygen species, accumulation of a subset of PAMP-regulated transcripts, and inhibition of seedling growth. In agreement with the enhanced PAMP response phenotypes observed in the mkp1 mutant, we found that mkp1 seedlings and adult plants are more resistant to the virulent bacterial pathogen Pseudomonas syringae pv. tomato (Pto) DC3000. Further genetic analysis revealed that MPK6, but not MPK3, is required for the mkp1-dependent increase in resistance to Pto and enhanced PAMP-induced growth inhibition observed in mkp1 seedlings. Together, our data support a role for MKP1 as a negative regulator of MPK6-mediated PAMP responses.

Reversible phosphorylation is a crucial regulatory mechanism that controls the activity of proteins. In mitogen-activated protein kinase (MAPK) signaling cascades, the cellular response depends on the intensity and duration of the MAPK activation, which is determined by balanced phosphorylation-dephosphorylation. MAPK phosphatases (MKPs), a subgroup of the dual-specificity phosphatases, are major negative regulators of MAPKs. The plant MKP family members are highly diverse in their structure and biological functions, and can be classified into five groups by sequence analysis. We review the recent progress made by genetic studies in identifying the physiological role of plant MKPs in a multitude of cellular processes, including cytoskeleton rearrangement, stress responses and phytohormone signaling, and examine the importance of negative regulators in plant MAPK signaling networks.

Mitogen-activated protein (MAP) kinase phosphatases are important negative regulators of the levels and kinetics of MAP kinase activation that modulate cellular responses. The dual-specificity phosphatase MAP KINASE PHOSPHATASE1 (MKP1) was previously shown to regulate MAP KINASE6 (MPK6) activation levels and abiotic stress responses in Arabidopsis thaliana. Here, we report that the mkp1 null mutation in the Columbia (Col) accession results in growth defects and constitutive biotic defense responses, including elevated levels of salicylic acid, camalexin, PR gene expression, and resistance to the bacterial pathogen Pseudomonas syringae. PROTEIN TYROSINE PHOSPHATASE1 (PTP1) also interacts with MPK6, but the ptp1 null mutant shows no aberrant growth phenotype. However, the pronounced constitutive defense response of the mkp1 ptp1 double mutant reveals that MKP1 and PTP1 repress defense responses in a coordinated fashion. Moreover, mutations in MPK3 and MPK6 distinctly suppress mkp1 and mkp1 ptp1 phenotypes, indicating that MKP1 and PTP1 act as repressors of inappropriate MPK3/MPK6-dependent stress signaling. Finally, we provide evidence that the natural modifier of mkp1 in Col is largely the disease resistance gene homolog SUPPRESSOR OF npr1-1, CONSTITUTIVE 1 (SNC1) that is absent in the Wassilewskija accession. Our data thus indicate a major role of MKP1 and PTP1 in repressing salicylic acid biosynthesis in the autoimmune-like response caused by SNC1.

Highlight Dysfunction of the Pep-PEPR system and its interplay with JA signalling results in increased plant susceptibility towards herbivore attack indicating that endogenous signalling also contributes to herbivore defence., A number of plant endogenous elicitors have been identified that induce pattern-triggered immunity upon perception. In Arabidopsis thaliana eight small precursor proteins, called PROPEPs, are thought to be cleaved upon danger to release eight peptides known as the plant elicitor peptides Peps. As the expression of some PROPEPs is induced upon biotic stress and perception of any of the eight Peps triggers a defence response, they are regarded as amplifiers of immunity. Besides the induction of defences directed against microbial colonization Peps have also been connected with herbivore deterrence as they share certain similarities to systemins, known mediators of defence signalling against herbivores in solanaceous plants, and they positively interact with the phytohormone jasmonic acid. A recent study using maize indicated that the application of ZmPep3, a maize AtPep-orthologue, elicits anti-herbivore responses. However, as this study only assessed the responses triggered by the exogenous application of Peps, the biological significance of these findings remained open. By using Arabidopsis GUS-reporter lines, it is now shown that the promoters of both Pep-receptors, PEPR1 and PEPR2, as well as PROPEP3 are strongly activated upon herbivore attack. Moreover, pepr1 pepr2 double mutant plants, which are insensitive to Peps, display a reduced resistance to feeding Spodoptera littoralis larvae and a reduced accumulation of jasmonic acid upon exposure to herbivore oral secretions. Taken together, these lines of evidence extend the role of the AtPep-PEPR system as a danger detection mechanism from microbial pathogens to herbivores and further underline its strong interaction with jasmonic acid signalling.

Members of the AtPep group of Arabidopsis endogenous peptides have frequently been reported to induce pattern-triggered immunity and to increase resistance to diverse pathogens by amplifying the innate immune response. Here, we made the surprising observation that dark-induced leaf senescence was accelerated by the presence of Peps. Adult leaves as well as leaf discs of Col-0 wild type plants showed a Pep-triggered early onset of chlorophyll breakdown and leaf yellowing whereas pepr1 pepr2 double mutant plants were insensitive. In addition, this response was dependent on ethylene signaling and inhibited by the addition of cytokinins. Notably, addition of the bacterial elicitors flg22 or elf18, both potent inducers of pattern-triggered immunity, did not provoke an early onset of leaf senescence.Continuous darkness leads to energy deprivation and starvation and therewith promotes leaf senescence. We found that continuous darkness also strongly induced PROPEP3 transcription. Moreover, Pep-perception led to a rapid induction of PAO, APG7 and APG8a, genes indispensable for chlorophyll degradation as well as autophagy, respectively, and all three hallmarks of starvation and senescence. Notably, addition of sucrose as a source of energy inhibited the Pep-triggered early onset of senescence. In conclusion, we report that Pep-perception accelerates dark/starvation-induced senescence via an early induction of chlorophyll degradation and autophagy. This represents a novel and unique characteristic of PEPR signaling, unrelated to pattern-triggered immunity.

The methylesterification status of cell wall pectins, mediated through the interplay of pectin methylesterases (PMEs) and pectin methylesterase inhibitors (PMEIs), influences the biophysical properties of plant cell walls. We found that the overexpression of a PMEI gene in Arabidopsis thaliana plants caused the stems to develop twists and loops, most strongly around points on the stem where leaves or inflorescences failed to separate from the main stem. Altered elasticity of the stem, underdevelopment of the leaf cuticle, and changes in the sugar composition of the cell walls of stems were evident in the PMEI overexpression lines. We discuss the mechanisms that potentially underlie the aberrant growth phenotypes.

[Abstract] Activation of mitogen activated protein kinases (MAPKs) is involved in many abiotic and biotic stress responses including plant defense. MAPK acitvation is based on the dual phosphorylation of threonine (T) and tyrosin (Y) residues (T-x-Y motif) in the activation loop of the MAPK protein. By determination of the phosphorylation status of a specific MAPK one can detect if the MAPK has been activated or not. This protocol describes how to analyze the phosphorylation status of Arabidopsis MAPKs MPK3 and MPK6 by using leaf disks, western blotting and a specific antibody (Figure 1). It can also be used for the analysis of MAPKs in other plant systems although some alterations regarding protein extraction might be necessary.

Plants fend off potentially damaging ultraviolet (UV)-B radiation by synthesizing and accumulating UV-B-absorbing flavonols that function as sunscreens. Regulation of this biosynthetic pathway is largely transcriptional and controlled by a network of transcription factors, among which the PRODUCTION OF FLAVONOL GLYCOSIDES (PFG) family of R2R3-MYB transcription factors was recently identified with a pivotal function. Here, we describe the response of Arabidopsis seedlings to narrow-band UV-B radiation at the level of phenylpropanoid pathway genes using whole-genome transcriptional profiling and identify the corresponding flavonol glycosides accumulating under UV-B. We further show that the bZIP transcriptional regulator ELONGATED HYPOCOTYL5 (HY5) is required for the transcriptional activation of the PFG1/MYB12 and PFG3/MYB111 genes under UV-B and visible light. A synthetic protein composed of HY5 with the VP16 activation domain is sufficient to activate PFG1/MYB12 expression in planta. However, even though myb11 myb12 myb111 triple mutants have strongly reduced CHS levels in darkness as well as in constant light, neither light- nor UV-B-inducibility seems impaired. Notwithstanding this, absence of the three PFG family transcription factors results in reduced UV-B tolerance, whereas PFG1/MYB12 overexpression leads to an increased tolerance. Thus, our data suggest that HY5-dependent regulation of PFG gene expression contributes to the establishment of UV-B tolerance.

AtPeps are a family of small peptides in Arabidopsis that are believed to act as endogenous amplifiers of the plant's innate immune response. In our recent study, (10) we showed that in Arabidopsis leaf disks, bacterial MAMPs (microbe-associated molecular patterns) such as the flagellin derived elicitor flg22, greatly enhanced the release of reactive oxygen species (ROS) triggered by a subsequent AtPep-perception. This enhanced ROS production could be a hallmark either of improved local defense or of the initiation of ROS-based systemic signaling. Here, we established a superior ROS detection system based on a new derivative of luminol (L-012). With this sensitive system we were able to show that chitin, too, acts as an enhancer of AtPep-triggered ROS, linking this specific defense response amplification also to the recognition of fungal pathogens. In addition we used the more sensitive ROS assay to transfer the experimental setup from cut leaf disks to unwounded seedlings. Thereby we revealed that wounding is not a prerequisite to enable the MAMP-induced enhancement of the AtPep-triggered ROS response.