Department of Ornamental Horticulture, Sichuan Agricultural University, 211 Huimin Road, Wenjiang District, Chengdu, Sichuan, 611130, People's Republic of China.
BMC Genomics. 2021 Jan 14;22(1):51. doi: 10.1186/s12864-020-07365-5.
Low-temperature severely affects the growth and development of chrysanthemum which is one kind of ornamental plant well-known and widely used in the world. Lysine crotonylation is a recently identified post-translational modification (PTM) with multiple cellular functions. However, lysine crotonylation under low-temperature stress has not been studied.
Proteome-wide and lysine crotonylation of chrysanthemum at low-temperature was analyzed using TMT (Tandem Mass Tag) labeling, sensitive immuno-precipitation, and high-resolution LC-MS/MS. The results showed that 2017 crotonylation sites were identified in 1199 proteins. Treatment at 4 °C for 24 h and - 4 °C for 4 h resulted in 393 upregulated proteins and 500 downregulated proteins (1.2-fold threshold and P < 0.05). Analysis of biological information showed that lysine crotonylation was involved in photosynthesis, ribosomes, and antioxidant systems. The crotonylated proteins and motifs in chrysanthemum were compared with other plants to obtain orthologous proteins and conserved motifs. To further understand how lysine crotonylation at K136 affected APX (ascorbate peroxidase), we performed a site-directed mutation at K136 in APX. Site-directed crotonylation showed that lysine decrotonylation at K136 reduced APX activity, and lysine complete crotonylation at K136 increased APX activity.
In summary, our study comparatively analyzed proteome-wide and crotonylation in chrysanthemum under low-temperature stress and provided insights into the mechanisms of crotonylation in positively regulated APX activity to reduce the oxidative damage caused by low-temperature stress. These data provided an important basis for studying crotonylation to regulate antioxidant enzyme activity in response to low-temperature stress and a new research ideas for chilling-tolerance and freezing-tolerance chrysanthemum molecular breeding.
低温严重影响菊花的生长和发育，菊花是一种世界知名且广泛应用的观赏植物。赖氨酸丁酰化是一种最近被鉴定的具有多种细胞功能的翻译后修饰（PTM）。然而，低温胁迫下的赖氨酸丁酰化尚未得到研究。
使用 TMT（串联质量标签）标记、敏感免疫沉淀和高分辨率 LC-MS/MS 分析了低温下菊花的全蛋白质组和赖氨酸丁酰化。结果表明，在 1199 种蛋白质中鉴定出 2017 个丁酰化位点。在 4°C 下处理 24 小时和-4°C 下处理 4 小时导致 393 个上调蛋白和 500 个下调蛋白（1.2 倍阈值和 P<0.05）。生物信息学分析表明，赖氨酸丁酰化参与了光合作用、核糖体和抗氧化系统。将菊花中的丁酰化蛋白和基序与其他植物进行比较，以获得同源蛋白和保守基序。为了进一步了解赖氨酸丁酰化在 K136 处如何影响 APX（抗坏血酸过氧化物酶），我们在 APX 中的 K136 处进行了定点突变。定点丁酰化表明 K136 上赖氨酸去丁酰化降低了 APX 活性，而 K136 上赖氨酸完全丁酰化增加了 APX 活性。
总之，我们的研究比较分析了低温胁迫下菊花的全蛋白质组和丁酰化，深入了解了丁酰化正向调节 APX 活性以减轻低温胁迫引起的氧化损伤的机制。这些数据为研究丁酰化调节抗氧化酶活性以响应低温胁迫提供了重要依据，并为菊花耐冷性和耐寒性分子育种提供了新的研究思路。
