冬青卫矛叶片质外体适应冬季胁迫的蛋白质组学分析

植物的质外体在感知外界信号和胁迫应答中起重要作用。该研究采用生理生化和蛋白质组学方法,对秋季和冬季冬青卫矛叶片的理化指标、微观结构以及叶片质外体液体中的蛋白变化进行比较分析,探索冬青卫矛叶片质外体响应冻胁迫的分子机制,以期为植物抗冻分子机制研究提供依据。结果表明:(1)冬季冬青卫矛叶片中MDA、可溶性糖含量以及SOD、POD活性均显著升高,气孔张开度减小,叶片厚度变小。(2)冬季冬青卫矛质外体液体中的蛋白质种类和含量显著高于秋季。(3)冬青卫矛叶片质外体液体中共鉴定到838个肽段和194个蛋白质;与秋季相比,冬季冬青卫矛叶片质外体液体中共筛选到43种差异积累蛋白(DAPs),其中26个蛋白质显著上调,17个蛋白质显著下调;蛋白表达模式显示,胚胎发育晚期丰富蛋白质、铁超氧化物歧化酶、过氧化物酶、丝氨酸羧肽酶等在冬季表达量较高,推测它们可能是冬季胁迫响应敏感的蛋白质。(4)KEGG富集分析显示,差异蛋白主要与应激防御、细胞壁修饰、抗病、自由基清除、甘油脂类代谢、淀粉和蔗糖代谢、次生代谢物的生物合成等生物学过程相关。(5)验证实验结果表明,冬季冬青卫矛8个差异积累蛋白与其对应的基因的表达趋势一致。研究认为,冬季冬青卫矛质外体液体中积累的蛋白可通过清除活性氧、促进单糖、寡糖和游离氨基酸等渗透调节物的生成而增强对环境的适应;推测冬青卫矛质外体中积累的单糖和寡糖可能通过增加质外体液体的浓度从而降低冰点,进而提高冬青卫矛对冬季胁迫的耐受性。

Proteomic Analysis of Apoplast Fluid in the Leaves of Euonymus japonicus Revealed the Molecular Mechanism of Adaptation to Winter Stress

Plant apoplast plays an important role in sensing external signals and stress responses. In this study, we used physiological and biochemical, and proteomic methods to analyze the physical and chemical indexes, microstructure, and protein changes in the apoplast fluid of Euonymus japonicus leaves in autumn and winter, and to explore the molecular mechanism of the response in the apoplast fluid of E. japonicus leaves to freezing stress, to provide a basis for the study of plant antifreeze molecular mechanism. The results showed that: (1) the contents of MDA and soluble sugar in the leaves of E. japonicus in winter increased significantly, the activities of SOD and POD increased significantly, the stomatal conductance decreased, and the leaf thickness decreased. (2) The types and contents of proteins in the apoplast fluid of E. japonicus leaves in winter were significantly higher than those in autumn. (3) A total of 838 peptides and 194 proteins were identified in the apoplast fluid of E. japonicus leaves. Compared with autumn, a total of 43 differentially accumulated proteins (DAPs) were screened in the apoplast fluid of E. japonicus leaves in winter, among which 26 DAPs were significantly up-regulated and 17 DAPs were significantly down-regulated. The analysis of protein expression patterns showed that the late embryogenesis abundant (LEA) protein, iron superoxide dismutase (Fe-SOD), peroxidase, and serine carboxypeptidase were highly expressed in winter, suggesting that they may be sensitive to a winter stress response. (4) KEGG enrichment analysis showed that DAPs were mainly related to biological processes such as stress defense, cell wall modification, disease resistance, free radical scavenging, glycerol lipid metabolism, starch and sucrose metabolism, and biosynthesis of secondary metabolites. (5) The results of verification experiments showed that the expression trends of 8 DAPs were consistent with those of their corresponding genes in winter. It was speculated that the proteins accumulated in the apoplast fluid of E. japonicus leaves in winter could enhance the adaptation to the environment by scavenging reactive oxygen species and promoting the production of osmotic regulators such as monosaccharides, oligosaccharides, and free amino acids, and the accumulation of monosaccharides and oligosaccharides in the apoplast may reduce the freezing point by increasing the concentration of apoplast fluid, thereby improving the tolerance of E. japonicus to winter stress.