Innate immunity represents the first line of inducible defense against microbial infection in plants and animals1–3. In both kingdoms, recognition of pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs), such as flagellin, initiates convergent signalling pathways involving MAP kinase (MAPK) cascades and global transcriptional changes to boost immunity1–4. Although Ca2+ has long been recognized as an essential and conserved primary mediator in plant defense responses, how Ca2+ signals are sensed and relayed into early MAMP signalling is unknown5,6. Here, we use a functional genomic screen and genome-wide gene expression profiling to show that four calcium-dependent protein kinases (CDPKs) are Ca2+ sensor PKs critical to transcriptional reprogramming in plant innate immune signalling. Unexpectedly, CDPKs and MAPK cascades act differentially in four MAMP-mediated regulatory programs to control early genes involved in synthesis of defense peptides and metabolites, cell wall modifications and redox signalling. Transcriptome profile comparison suggests that CDPKs are the convergence point of signalling triggered by most MAMPs. Double, triple and quadruple cpk mutant plants display progressively diminished oxidative burst and gene activation induced by flg22, as well as compromised pathogen defense. In contrast to negative roles of calmodulin (CAM) and a CAM-activated transcription factor in plant defense7,8, the present study reveals Ca2+ signalling complexity and demonstrates key positive roles of specific CDPKs in initial MAMP signalling.

Nutrient signalling integrates and coordinates gene expression, metabolism and growth. However, its primary molecular mechanisms remain incompletely understood in plants and animals. Here we report novel Ca2+ signalling triggered by nitrate with live imaging of an ultrasensitive biosensor in Arabidopsis leaves and roots. A nitrate-sensitized and targeted functional genomic screen identifies subgroup III Ca2+-sensor protein kinases (CPKs) as master regulators orchestrating primary nitrate responses. A chemical switch with the engineered CPK10(M141G) kinase enables conditional analyses of cpk10,30,32 to define comprehensive nitrate-associated regulatory and developmental programs, circumventing embryo lethality. Nitrate-CPK signalling phosphorylates conserved NIN-LIKE PROTEIN (NLP) transcription factors (TFs) to specify reprogramming of gene sets for downstream TFs, transporters, N-assimilation, C/N-metabolism, redox, signalling, hormones, and proliferation. Conditional cpk10,30,32 and nlp7 similarly impair nitrate-stimulated system-wide shoot growth and root establishment. The nutrient-coupled Ca2+ signalling network integrates transcriptome and cellular metabolism with shoot-root coordination and developmental plasticity in shaping organ biomass and architecture.

Ca2+ has long been recognized as a conserved second messenger and principal mediator in plant immune and stress responses. How Ca2+ signals are sensed and relayed into diverse primary and global signaling events is still largely unknown. Comprehensive analyses of the plant-specific multigene family of Ca2+-dependent protein kinases (CDPKs) are unraveling the molecular, cellular and genetic mechanisms of Ca2+ signaling. CDPKs, which exhibit overlapping and distinct expression patterns, sub-cellular localizations, substrate specificities and Ca2+ sensitivities, play versatile roles in the activation and repression of enzymes, channels and transcription factors. Here, we review the recent advances on the multifaceted functions of CDPKs in the complex immune and stress signaling networks, including oxidative burst, stomatal movements, hormonal signaling and gene regulation.

Several calcium-independent protein kinases were activated by hyperosmotic and saline stresses in Arabidopsis cell suspension. Similar activation profiles were also observed in seedlings exposed to hyperosmotic stress. One of them was identified to AtMPK6 (Droillard, M. J., Boudsocq, M., Barbier-Brygoo, H., and Lauriè re, C. (2002) FEBS Lett. 527, 43-50) but the others remained to be identified. They were assumed to belong to the SNF1 (sucrose nonfermenting 1)-related protein kinase 2 (SnRK2) family, which constitutes a plant-specific kinase group. The 10 Arabidopsis SnRK2 were expressed both in cells and seedlings, making the whole SnRK2 family a suitable candidate. Using a family-specific antibody raised against the 10 SnRK2, we demonstrated that these non-MAPK protein kinases activated by hyperosmolarity in cell suspension were SnRK2 proteins. Then, the molecular identification of the involved SnRK2 was investigated by transient expression assays. Nine of the 10 SnRK2 were activated by hyperosmolarity induced by mannitol, as well as NaCl, indicating an important role of the SnRK2 family in osmotic signaling. In contrast, none of the SnRK2 were activated by cold treatment, whereas abscisic acid only activated five of the nine SnRK2. The probable involvement of the different Arabidopsis SnRK2 in several abiotic transduction pathways is discussed.Environmental stresses such as drought, cold, and salinity impose osmotic stress on plants, leading to imbalance in ionic homeostasis, oxidative damages, and growth inhibition. Understanding how plants respond to these stresses is critical to improve plant resistance. Reversible protein phosphorylation is one of the major mechanisms for mediating intracellular responses, including responses to osmotic changes. Indeed, several protein kinases have been shown to be activated by hyperosmotic stresses in different plant species. Because of the well known osmosensing pathway in yeast involving a mitogenactivated protein kinase (MAPK), 1 much interest was focused on the MAPK family in plants. In Arabidopsis, AtMPK6 and AtMPK4 were shown to be activated by hyperosmolarity, salt, cold, or drought (1, 2), whereas the tobacco SIPK was activated by hyperosmotic or salt stresses (3-5). In alfalfa, SAMK was reported to be activated by cold and drought but not NaCl (6), whereas SIMK was activated by sorbitol, KCl, and NaCl (7).In mammals, MAPK cascades are composed of MAPK, MAPKK, and MAPKKK, each component being activated by phosphorylation by the upstream kinase. The involvement of MAPKK and MAPKKK in plant osmotic response was suggested by molecular and biochemical studies. The Arabidopsis MAPKKK AtMEKK1 was transcriptionally induced by salt stress and the protein was able to complement a yeast mutant affected in osmotic signaling (8). Moreover, Mizoguchi et al. Other kinase families have been shown to play a role in osmotic signaling. Among them, SOS2 (salt overly sensitive 2), which belongs to the sucrose nonfermenting 1-related protein kinase 3 (SnRK3) family, was transcription...

In plants and animals, innate immunity is triggered through pattern recognition receptors (PRRs) in response to microbe-associated molecular patterns (MAMPs) to provide the first line of inducible defense. Plant receptor protein kinases (RPKs) represent the main plasma membrane PRRs perceiving diverse MAMPs. RPKs also recognize secondary danger-inducible plant peptides and cell-wall signals. Both types of RPKs trigger rapid and convergent downstream signaling networks controlled by calcium-activated PKs and mitogen-activated PK (MAPK) cascades. These PK signaling networks serve specific and overlapping roles in controlling the activities and synthesis of a plethora of transcription factors (TFs), enzymes, hormones, peptides and antimicrobial chemicals, contributing to resistance against bacteria, oomycetes and fungi.

SUMMARYAbscisic 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.

In Arabidopsis the stomatal defense response, a feature of the innate immunity in plants, involves oxylipin-mediated mechanisms that are independent of the phytohormone abscisic acid.

In Arabidopsis cell suspension, hyperosmotic stresses (mannitol and NaCl) were previously shown to activate nine sucrose non-fermenting 1 related protein kinases 2 (SnRK2s) whereas only five of them were also activated by abscisic acid (ABA) treatment. Here, the possible activation by phosphorylation/ dephosphorylation of each kinase was investigated by studying their phosphorylation state after osmotic stress, using the Pro-Q Diamond, a specific dye for phosphoproteins. All the activated kinases were phosphorylated after osmotic stress but the induced phosphorylation changes were clearly different depending on the kinase. In addition, the increase of the global phosphorylation level induced by ABA application was lower, suggesting that different mechanisms may be involved in SnRK2 activation by hyperosmolarity and ABA. On the other hand, SnRK2 kinases remain activated by hyperosmotic stress in ABA-deficient and ABA-insensitive mutants, indicating that SnRK2 osmotic activation is independent of ABA. Moreover, using a mutant form of SnRK2s, a specific serine in the activation loop was shown to be phosphorylated after stress treatments and essential for activity and/or activation. Finally, SnRK2 activity was sensitive to staurosporine, whereas SnRK2 activation by hyperosmolarity or ABA was not, indicating that SnRK2 activation by phosphorylation is mediated by an upstream staurosporine-insensitive kinase, in both signalling pathways. All together, these results indicate that different phosphorylation mechanisms and at least three signalling pathways are involved in the activation of SnRK2 proteins in response to osmotic stress and ABA.