Global lysine crotonylation profiling reveals metabolic and stress-responsive mechanisms in Reynoutria japonica

Abstract

Reynoutria japonica (Huzhang), also known as Japanese knotweed, is a traditionally valued medicinal herb in Asian medicine. Historically introduced to Europe and England for ornamental purposes, it has since become widely regarded as an invasive species due to its aggressive growth and adaptability. Understanding the mechanisms underlying its robust growth and environmental adaptability is therefore of both horticultural and ecological interest. Lysine crotonylation (Kcr) is a newly discovered post-translational modification implicated in diverse biological processes, but its roles in non-histone proteins, especially within medicinal plant R. japonica, remain poorly understood. Here, we present the first comprehensive proteome-wide profiling of Kcr in R. japonica. Using high-resolution liquid chromatography–tandem mass spectrometry (LC-MS/MS) coupled with immunoaffinity enrichment, we identified 18,914 Kcr sites across 5842 proteins and characterized six conserved sequence motifs, constituting the largest plant crotonylome described to date. Functional enrichment revealed that Kcr-modified proteins are primarily associated with critical metabolic pathways, including carbon fixation, photosynthesis, fatty acid degradation, the tricarboxylic acid (TCA) cycle, and protein translation. Notably, abundant Kcr modifications were found on enzymes responsible for the biosynthesis of secondary metabolites such as resveratrol and anthraquinones. Additionally, stress-responsive changes in global Kcr modification were observed, with H2B carrying the highest number of Kcr sites and showing a marked reduction under stress. These findings provide novel insights into the functional significance of Kcr in plant metabolic regulation and stress adaptation.