Rao Weiyi, Fan Jizhou, Sun Zongping, Wang Chun, Guan Junjie, Duan Yingying, Yu Maoyun, Peng Daiyin, Xing Shihai
The First Clinical School, Anhui University of Chinese Medicine, Hefei, China.
Anhui Province Key Laboratory of Environmental Hormone and Reproduction, Anhui Province Key Laboratory of Embryo Development and Reproductive Regulation, Fuyang Normal University, Fuyang, China.
Physiol Plant. 2025 May-Jun;177(3):e70343. doi: 10.1111/ppl.70343.
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.
赖氨酸二羟基异丁酰化（Khib）是植物对低温胁迫响应中最近发现的一种翻译后修饰，其作用尚不清楚。在此，我们对霍山石斛进行了全面的叶绿体蛋白质组范围的二羟基异丁酰化分析，以探索Khib修饰在响应低温胁迫中的潜在功能。我们在3281个叶绿体蛋白中总共鉴定出13040个Khib位点，这些位点在低温胁迫下受到显著差异调节。发现Khib修饰的蛋白与质子跨膜转运体活性、ATP酶偶联离子跨膜转运体活性和活性单原子离子跨膜转运体活性高度相关。鉴定出的响应低温胁迫而差异调节的Khib位点主要集中在AKhib、KKhib和EKhib基序内。值得注意的是，霍山石斛中的果糖-1,6-二磷酸醛缩酶2（DhFBA2）是参与卡尔文-本森循环的关键叶绿体代谢酶，在低温处理后显示出显著的Khib修饰，在八个Khib位点发生修饰。对DhFBA2编码基因进行定点诱变并在酵母中表达，结果表明K338位点的Khib修饰对于维持DhFBA2酶活性和增强低温耐受性至关重要。分子动力学模拟和表面静电势分析进一步表明，K338位点的Khib修饰通过将DhFBA2的表面亲和力降低到最佳状态来改善其结构稳定性，从而促进其活性并提高低温抗性。
