花生叶片蛋白组对UV-B辐射增强的响应

A proteomic analysis of Arachis hypogaea leaf in responses to enhanced ultraviolet-B radiation

为揭示UV-B辐射增强处理降低花生光合速率和花生抵御UV-B辐射增强的分子机制,应用蛋白质双向电泳与质谱联用技术对自然光环境下补增UV-B辐射(54μW/cm2)处理24h的苗期花生叶片差异表达蛋白质变化进行了分析。结果表明:补增UV-B处理下,花生叶片中共检测到丰度变化在2.5倍以上的差异表达蛋白点39个(其中22种蛋白质表达下调,17种表达上调),经过MALDI-TOF-TOF分析及数据库检索,成功鉴定出其中的27种蛋白质。被鉴定的27种蛋白质按其功能大致可归为8类,第Ⅰ类:光合作用相关的蛋白质,包括质体蓝素、1,5-二磷酸核酮糖羧化酶小亚基、放氧复合物增强子蛋白1、PsbP结构域蛋白6和果糖二磷酸醛缩酶;第Ⅱ类:糖代谢相关蛋白质,包括苹果酸脱氢酶;第Ⅲ类:能量合成相关蛋白质,包括ATP合酶;第Ⅳ类:氨基酸代谢相关蛋白质,包括半胱氨酸合成酶;第Ⅴ类:蛋白质加工相关蛋白质,包括热激蛋白;第Ⅵ类:蛋白质翻译相关蛋白质,包括核糖体循环因子;第Ⅶ类:防御相关蛋白质,包括几丁质酶、过氧化物酶、Cu-Zn超氧化物歧化酶、二羟肉桂酸3-O-转甲基酶和类萌发素蛋白;第Ⅷ类,未知功能蛋白质。这些研究结果为进一步研究花生抵御UV-B辐射的分子机理提供了有意义的线索。

Uhraviolet-B （ UV-B, 280--320 nm） constitutes a minor part of the solar spectrum, which can be absorbed by stratospheric ozone layer. However, a global depletion of the ozone layer, largely due to the release of man-made chlorofluorocarbons, has resulted in an increase of ground-level solar UV-B radiation. UV-B can influence plant processes, either through direct damage or via various regulatory effects. Many researches have warned that excessive UV-B radiation can harm living organisms by damaging DNA, proteins, lipids, and membranes and consequently affecting plant growth, development, morphology, and productivity. Two-dimensional polyacrylamide gel electrophoresis （ 2-D PAGE） is a powerful technique for resolving hundreds of proteins in parallel. Combined with mass spectrometry （ MS ） , it allows rapid and reliable protein identification. In recent years, proteomic-based technologies have been successfully applied to systematicstudies of the stress responses many plant species, including Arabidopsis, soybean, rice, wheat, barley, potato, tomato, and many others. A wide range of abiotic stresses have been examined, such as drought, nutrition deficiency, temperature, oxidative stress, herbicides, wounding, anoxia, salt, and heavy metals. These investigations have provided a wealth of important information on the physiological processes involved in plant stress responses. To explore the molecular mechanisms of the decreased photosynthetic rate and the resistance of peanut （Arachis hypogaea ） when exposed to enhanced UV-B radiation, 2-D PAGE and MS were used to identify the differentially-expressed proteins in peanut seedling leaves in response to supplementary UV-B radiation （54 μW/cm2 ） for 24 h. A total of 39 protein spots were differentially expressed by at least 2.5 fold compared with the controls （22 proteins were down-regulated and 17 were up-regulated） after treatment with supplementary UV-B radiation. Of those protein spots, 27 were successfully identified by MALDI TOF/TOF MS after a database search. Those 27 proteins could be classified into eight categories according to their functions： class I, photosyntlesis （plastocyanin, ribulose-1,5-bisphosphate carboxylase small subunit, oxygen-evolving enhancer protein 1, PsbP domain-containing protein 6, and fructose-bisphosphate aldolase ）; class Ⅱ, carbohydrate metabolism （malate dehydrogenase） ; class III, energy synthesis （ATP synthase） ; class IV, amino acid biosynthesis （cysteine synthase） ; class V, protein biosynthesis （ribosome recycling factor）; class VI, protein processing （heat shock proteins）; class VII, defense responses （chitinase, peroxidase, Cu-Zn SOD, caffeic acid 3-O-methyhransferase, and germin-like protein） ; class VIII, unknown proteins. In conclusion, we hypothesized that the enhanced UV-B radiation caused a decrease in the photosynthesis rate of peanut leaves mainly via three mechanisms. First, enhanced UV-B may down-regulate the expression of ribosome recycling factor, which caused a decrease in the expression of subunit PsbP in photosystem Ⅱ, thus destroying the thylakoid membrane structure. Second, the reduced plastocyanin expression may have induced a decrease in photosynthetic electron transport efficiency. Third, the down-regulation of ribulose-1, 5-bisphosphate carboxylase and fructose-1,6-bisphosphate aldolase resulted in a decrease in carbon assimilation. At the same time, peanut may also enhance its resistance to UV-B stress by increasing the expressions of antioxidant enzymes and non-enzymatic antioxidants, germin-like proteins, pathogenesis-related proteins, and heat shock proteins. These results provide important information for understanding the molecular mechanisms by which A. hypogaea responds to elevated UV-B stress.