Proteome-wide profiling of lysine β-hydroxybutyrylation in Reynoutria japonica (Huzhang) and its dynamics under hydrogen sulfide stress.

Abstract

Reynoutria japonica is both an important industrial source of resveratrol and a medicinal plant notable for its strong environmental adaptability. However, its responses to hydrogen sulfide (H2S), a key environmental signaling molecule, remain largely unexplored. Lysine β-hydroxybutyrylation (Kbhb) is a newly identified short-chain fatty acylation, but its role in regulating plant stress responses is unclear. Here, we performed the first proteome-wide profiling of Kbhb in R. japonica, identifying 17,102 high-confidence sites across 6185 proteins. Motif analysis revealed six conserved motifs. Functional enrichment analysis revealed that Kbhb-modified proteins are predominantly involved in core carbon and energy metabolism, with extensive modifications on enzymes related to acetyl-CoA flux, β-hydroxybutyrate metabolism, and the biosynthesis of resveratrol and anthraquinones. Under NaHS-imposed H2S stress, short-term exposure (3 h) triggered ROS accumulation and increased global Kbhb levels, particularly on histones H3 and H2A, suggesting an early signaling or chromatin-associated response; prolonged exposure reduced chlorophyll content and decreased Kbhb abundance. Pharmacological inhibition and in planta transient assays identified RjapHDA5L and RjapHDA9L as potential candidate regulators that regulate Kbhb modification's homeostasis. Furthermore, functional validation in a yeast heterologous expression system demonstrated that increasing Kbhb levels via β-hydroxybutyrate (BHB) or HDAC inhibitor treatment significantly enhanced the enzymatic activity of isocitrate dehydrogenase (IDH), a key TCA cycle enzyme. These findings indicate that Kbhb serves as a functional activator of core metabolic enzymes. Our study provides the first global landscape of Kbhb in R. japonica and establishes a molecular foundation for understanding how Kbhb dynamics regulate plant metabolism and epigenetic adaptation under environmental stress.