IodoTMT-labeled redox proteomics reveals the involvement of oxidative post-translational modification in response to para-hydroxybenzoic acid and hydrogen peroxide stresses in poplar

Poplar is widely planted as an economic and ecological tree species. However, accumulation of the phenolic acid allelochemical para-hydroxybenzoic acid (pHBA) in soil is a severe threat to the growth and productivity of poplar. pHBA stress leads to excessive production of reactive oxygen species (ROS). However, it is unclear which redox-sensitive proteins are involved in the pHBA-induced cellular homeostasis regulatory mechanism. We here identified reversible redox-modified proteins and modified cysteine (Cys) sites in exogenous pHBA-and hydrogen peroxide (H2O2)-treated poplar seedling leaves by using the iodoacetyl tandem mass tag-labeled redox proteomics method. In total, 4786 redox modification sites were identified in 3176 proteins, with 104 and 91 proteins being differentially modified at 118 and 101 Cys sites in response to pHBA and H2O2 stresses, respec-tively. The differentially modified proteins (DMPs) were predicted to be mainly localized in the chloroplast and cytoplasm, with most proteins being enzymes with catalytic activities. The KEGG enrichment analysis of these DMPs revealed that proteins related to the MAPK signaling pathway, soluble sugar metabolism, amino acid metabolism, photosynthesis, and phagosome pathways were extensively regulated by redox modifications. Moreover, combined with our previous quantitative proteomics data, 8 proteins were upregulated and oxidized under both pHBA and H2O2 stresses. Reversible oxidation of Cys sites in these proteins might be actively responsible for the regulation of tolerance to pHBA-induced oxidative stress. Based on the aforementioned re-sults, a redox regulatory model activated by pHBA-and H2O2-induced oxidative stress was proposed. This study conducts the first redox proteomics analysis of poplar in response to pHBA stress and provides a new insight into the mechanistic framework of reversible oxidative post-translational modifications to gain a better under-standing of pHBA-induced chemosensory effects on poplar.