Integrated proteomic and phosphoproteomic analysis reveals the MAPK cascade as a key regulator of ethylene-induced latex production in Hevea brasiliensis

Natural rubber is a crucial industrial raw material globally, with wide applications in industries such as automotive and healthcare. Application of ethylene promotes latex production in the Hevea rubber tree (Hevea brasiliensis). However, its potential protein-regulation mechanism remains unclear. To elucidate the role of ethylene in latex production, we employed high-resolution proteomics and phosphoproteomics to determine the global changes in protein abundance and phosphorylation during ethylene-stimulated latex production. Using latex samples collected from ethephon-treated rubber tree, we identified more than 3,700 quantifiable proteins and 2,000 phosphorylated proteins with over 6,000 phosphorylation sites. Proteins involved in the mitogen-activated protein kinase (MAPK) cascade, metabolic pathways, and vesicular trafficking, like rubber elongation factors (REFs), small rubber particle proteins (SRPPs), and 14-3-3 proteins, exhibit pronounced robust phosphorylation dynamics upon ethephon application. Interestingly, the serine/threonine-proline (S/T-P) motif, which could be recognized by MAPKs, was highly enriched in REFs, 14-3-3, and ubiquitin-associated DENN (UDENN) domain-containing proteins, suggesting a potential role of MAPKs in ethylene-induced rubber biosynthesis. Consistently, the MAPKs show altered activation in ethephon-treated Hevea rubber trees, compared to water-treated controls. Additionally, activation of UDENN domain proteins implicated Ras-related Rab GTPases in membrane trafficking and rubber particle formation. These results provide comprehensive information on global changes in protein abundance and phosphorylation upon ethephon application in latex production, and may provide valuable clues for understanding the molecular basis of ethephon-regulated natural rubber biosynthesis in Hevea rubber trees.