Deciphering systemic wound responses of the pumpkin extrafascicular phloem by metabolomics and stable isotope-coded protein labeling (ICPL).

In cucurbits phloem latex exudes from cut sieve tubes of the extrafascicular phloem (EFP) serving in defense against herbivores. We analyzed inducible defense mechanisms in the EFP of Cucurbita maxima after leaf damage. As an early systemic response, wounding elicited transient accumulation of jasmonates and a decrease in exudation probably due to partial sieve tube occlusion by callose. The energy status of the EFP was enhanced as indicated by increased levels of adenosine triphosphate, phosphate and intermediates of the citric acid cycle. Gas chromatography coupled to mass spectrometry also revealed that sucrose transport, gluconeogenesis/glycolysis and amino acid metabolism were up-regulated after wounding. Combining ProteoMinerTM technology for enrichment of low-abundance proteins with stable isotope-coded protein labeling (ICPL) we identified 51 wound-regulated phloem proteins. Two sucrose nonfermenting 1-related protein kinases and a 32 kDa 14-3-3 protein are candidate central regulators of stress metabolism in the EFP. Other proteins such as the silverleaf-whitefly-induced protein 1, mitogen-activated protein kinase 6 and heat shock protein 81 have known defensive functions. ICPL and western blot analyses indicated that cyclophilin 18 is a reliable marker for stress responses of the EFP. As a hint towards induction of redox-signaling we have observed delayed oxidation-triggered polymerization of the major phloem proteins 1 and 2 (PP1/PP2), which correlated with a decline in carbonylation of PP2. In sum, wounding triggered transient sieve tube occlusion, enhanced energy metabolism and accumulation of defense-related proteins in the pumpkin EFP. The systemic wound response was mediated by jasmonate and redox signaling