Abstract
Mitogen-activated protein kinases (MAPKs) target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3, and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses) is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phospho)proteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g., WRKY transcription factors and proteins encoded by the genes from the “PEN” pathway required for penetration resistance to filamentous pathogens). Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org).
Highlights
As sessile organisms, plants rely predominantly on physical barriers and adaptive mechanisms to adapt to biotic and abiotic stress conditions
Transgenic Arabidopsis thaliana (Col-0) plants were created with a dexamethasone (DEX)-inducible expression of this heterologous MKK5DD
Two immunoreactive bands representing phosphorylated mitogen-activated protein kinase (MAPK) are detected in plant extracts of such transgenic plants after 4–6 h of DEX treatment (Figure 1A), which is not seen in corresponding control plants (Col-0 KR) that express the kinase-inactive version of MAPK kinase 5 (MKK5) (MKK5KR)
Summary
Plants rely predominantly on physical barriers and adaptive mechanisms to adapt to biotic and abiotic stress conditions. Pathogens can trigger PTI (pattern-triggered immunity) after recognition of conserved microbe-associated molecular patterns (MAMPs) or damageassociated molecular patterns (DAMPs), which are plant endogenous components released during tissue wounding, e.g., from herbivores or lytic enzymes released by pathogens. Binding of these MAMP/DAMP ligands to surface exposed pattern recognition receptors (PRRs) leads to activation of a variety of defense reactions to counteract the negative impact of the stresses (Boller and Felix, 2009; Knogge et al, 2009; Nicaise et al, 2009; Zipfel, 2009; Ranf et al, 2011). Downstream of these receptor complexes are intracellular phosphorylation events and cascades, such as calcium-dependent protein kinases (CDPKs) (Schulz et al, 2013) and mitogen-activated protein kinase (MAPK) cascades (Pitzschke et al, 2009; Andreasson and Ellis, 2010; Tena et al, 2011; Meng and Zhang, 2013)
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