The Role of Lysine Dihydroxyisobutyrylation in <i>Dendrobium huoshanese</i> Under Low‐Temperature by Proteomic Analysis

The involvement of lysine dihydroxyisobutyrylation (Khib), a recently identified post-translational modification in plant responses to low temperature stress, is unknown. Here, we performed a comprehensive chloroplast proteome-wide dihydroxyisobutyrylation analysis in Dendrobium huoshanense to explore the potential function of Khib modification in response to low-temperature stress. We identified a total of 13,040 Khib sites in 3281 chloroplast proteins that were significantly differentially regulated under low-temperature stress. Khib-modified proteins were found to be highly associated with proton transmembrane transporter activity, ATPase-coupled ion transmembrane transporter activity, and active monoatomic ion transmembrane transporter activity. The identified Khib sites differentially regulated in response to low-temperature stress were primarily concentrated within AKhib, KKhib, and EKhib motifs. Notably, fructose-1,6-bisphosphate aldolase 2 (DhFBA2) from D. huoshanense, a key chloroplast metabolic enzyme involved in the Calvin-Benson cycle, showed significant Khib modification following low-temperature treatment, with modification occurring at eight Khib sites. Site-directed mutagenesis of the DhFBA2-encoding gene and its expression in yeast revealed that Khib modification at the K338 site is essential for maintaining DhFBA2 enzymatic activity and enhancing low-temperature tolerance. Molecular dynamics simulation and surface electrostatic potential analysis further showed that Khib modification at K338 improved the structural stability of DhFBA2 by reducing its surface affinity to an optimal status, thus promoting its activity and improving low-temperature resistance.