Phosphoproteomic analysis of Arabidopsis membranes reveals novel elements involved in response to oligogalacturonides

Oligogalacturonides (OGs) are plant endogenous elicitors that accumulate during fungal infection and can act as danger signals to activate the plant immune response. We used 2-D DIGE proteomic analysis coupled with phospho-specific ProQ Diamond staining for a quantitative measure of both protein abundance variation and phosphorylation state changes in total microsomes of OG-treated Arabidopsis seedlings. Two proteins PCaP1 and DET3, which undergo phosphorylation changes after 10 min upon OG treatment, were further investigated. PCaP1 is a plasma membrane-associated protein that binds Ca2+ and phosphatidylinositol phosphates, major components of intracellular signaling. DE-ETIOLATED3 (DET3) encodes the subunit C of the vacuolar H+–ATPase. PCaP1 is present as a multispot protein in 2D gels. Null pcap1 and det3 mutant seedlings are compromised in OG-induced phosphorylation of the mitogen activated kinases (MAPKs) MPK3 and MPK6, and in several early responses to both OGs and a bacterial pathogen-associated molecular pattern (PAMP), flg22, indicating that PCaP1 and DET3 are required for full activation of the defense responses triggered by biotic elicitors.