Chloroplast crotonylome analysis of wheat seedling leaves reveals the roles of crotonylated proteins involved in salt stress response.

Lysine crotonylation (Kcr) is a novel post-translational modification. Whether it exists in wheat chloroplasts and what are its functions in salt stress response remain unclear. Here, we performed the first comprehensive chloroplast crotonylome analysis of wheat seedling leaves to reveal potential functions of Kcr proteins in salt stress response. In a total of 471 chloroplast proteins, 1290 Kcr sites were identified as significantly regulated by salt stress, mainly involved in photosynthesis, protein folding and ATP synthesis. The identified Kcr sites responded to salt stress were concentrated within KcrK and KcrF motifs, of which the conserved KcrF motif was identified in the Kcr proteins of wheat chloroplasts for the first time. Notably, ten Kcr sites were identified in fructose-1,6-bisphosphate aldolase (TaFBA6), a chloroplast key metabolic enzyme involved in the Calvin-Benson cycle. Site-directed mutagenesis of TaFBA6 showed that the Kcr at K367 is critical in maintaining its enzymatic activity and conferring salt tolerance in yeast. Further molecular dynamics simulation and surface electrostatic potential analyses indicated that the Kcr at K367 could improve the structural stability of TaFBA6 by decreasing the distribution of positive charge on protein surface to resist to alkaline environment, thereby promoting the activity of TaFBA6 and salt tolerance.