Lysine crotonylation coordinates osmotic stress response in Populus davidiana × P. bolleana through synergistic metabolic and transcriptional regulation

As a highly conserved post-translational modification, lysine crotonylation (abbreviated as Kcr) regulates diverse biological activities. To explore its role in plant osmotic stress resistance, we performed 4D-FastDIA-based crotonylome profiling on nuclear proteins of Populus davidiana × P. bolleana under osmotic stress. We identified 10,527 crotonylated peptides corresponding to 11,246 Kcr sites on 3606 proteins. Among these, 3020 sites on 1646 proteins were osmotic stress-responsive, with 958 upregulated and 688 downregulated. Among these modifications, H2A, H2B, H3, and H4 all exhibit crotonylation. KEGG analysis revealed 124 differentially modified proteins enriched in five pathways, including glyoxylate and dicarboxylate metabolism, suggesting their potential role in osmotic adaptation. Furthermore, 29 transcription factors (TFs) exhibited osmotic-responsive Kcr modifications. We studied 5 TFs, finding that their osmotic stress resistance significantly decreased upon removal of crotonylation, underscoring its regulatory importance. This study provides the evidence that non-histone crotonylation modulates plant osmotic resistance. In osmotic adaptation of woody plants, a vast network of non-histone Kcr modifications exists alongside traditional histone modifications. While histone Kcr modifications likely act as top-level regulators of stress responses, the extensive non-histone Kcr modifications may fulfill specific functional roles. These findings offer new insights into the mechanisms of plant stress responses. • Populus drought crotonylome: 3,020 Kcr sites on 1,646 proteins, >99.7% non-histone. • Kcr-modified proteins function in glyoxylate metabolism and TF activation, and their removal impairs drought resistance. • This work provides evidence via 4D-FastDIA that non-histone crotonylation critically regulates plant drought adaptation.