DOI: 10.3724/SP.J.1006.2016.01541

Acta Agronomica Sinica (作物学报) 2016/42:10 PP.1541-1550

Differential Proteomic Analysis and Photosynthetic Characteristics of Winter Rapeseed under Low Temperature Stress

Isolation and identification of the differentially expressed proteins in winter rapeseed at low temperature, laid a foundation for revealing the mechanism of cold resistance of winter rapa. The strong cold resistant winter rape Longyou 7 was used as experimental material. Two dimensional electrophoresis, mass spectrometry and retrieval techniques were used to compare the proteomic differences at low temperature (4℃, -4℃) and normal atmospheric temperature (25℃/20℃), and functions were analyzed by KO and KEGG. It was observed that Longyou 7 had subsided growth point, creeping stem, and closed or semi-closed stomata under low temperature. The 2-DE and PDQuest8.0.1 software analysis showed that the number of protein spots were 726 and 738, respectively. Compared with the normal temperature treatment, at the 4℃ treatment showed differential expression at 10 protein spots while didn't at five protein. Eleven proteins were identified by MS MALDI-TOF-TOF mass spectrometry analysis. They are involved in carbohydrate metabolism, rugan metabolism, amino acid metabolism, organic acid metabolism, nucleic acid metabolism, signal transduction in cell and communication and other cellular processes. we found the high activity of antifreeze protein in Longyou 7 leaf protein extraction solution after low temperature treatment through ice crystal morphology microscopic observation. Five out of the 11 proteins identified were associated with photosynthesis. Under low temperature, ribulose 1,5-bisphosphate carboxylase (RuBPCase) activity and photosynthetic rate decreases in leaves of Longyou 7. Under the low tem-perature, the proteome of winter rape was significantly changed, and the specific protein was expressed. The decrease of Pn in leaves was related to the expression inhibition and the activity decrease of RuBPCase. The decrease of Pn in leaves was mainly caused by non stomatal limitation. High activity of antifreeze protein plays an important role in cold resistance of winter rape.

Key words:Winter rape,Low temperature stress,Protein proteomics,Photosynthetic characteristics

ReleaseDate:2017-01-12 13:30:55

[1] 刘自刚, 张长生, 孙万仓, 杨宁宁, 王月, 何丽, 赵彩霞, 武军艳, 方彦, 曾秀存. 不同生态区冬前低温下白菜型冬油菜不同抗寒品种(系)比较. 作物学报, 2014, 40: 346-354

Liu Z G, Zhang C S, Sun W C, Yang N N, Wang Y, He L, Zhao C X, Wu J Y, Fang Y, Zeng X C. Comparison of winter rapeseed varieties (lines) with different cold resistance planted in the northern-extending regions in China under low temperature be-fore winter. Acta Agron Sin, 2014, 40: 346-354 (in Chinese with English abstract)

[2] 王学芳, 孙万仓, 李孝泽, 武军艳, 马维国, 康艳丽, 曾潮武, 蒲媛媛, 叶剑, 刘红霞, 曾军, 张亚红. 河西走廊种植冬油菜的环境效应. 作物学报, 2008, 34: 2210-2217

Wang X F, Sun W C, Li X Z, Wu J Y, Ma W G, Kang Y L, Zeng C W, Pu Y Y, Ye J, Liu H M, Zeng J, Zhang Y H. The environment effect of planting winter rape in Hexi Corridor. Acta Agron Sin, 2008, 34: 2210-2217 (in Chinese with English abstract)

[3] 王学芳, 孙万仓, 李孝泽, 武军艳, 刘红霞, 曾潮武, 蒲媛媛, 张朋飞, 张俊杰. 我国北方风蚀区冬油菜抗风蚀效果. 生态学报, 2009, 29: 6572-6577

Wang X F, Sun W C, Li X Z, Wu J Y, Liu H X, Zeng C W, Pu Y Y, Zhang P F, Zhang J J. Wind erosion-resistance of fields plante with winter rapeseed in the wind erosion region of northern China. Acta Ecol Sin, 2009, 29: 6572-6577 (in Chinese with English abstract)

Sun W C, Ma W G, Lei J M, Liu Q, Yang R Y, Wu J Y, Wang X F, Ye J, Zeng J, Zhang Y H, Kang Y L, Guo X J, Wei W H, Yang J, Pu Y Y, Zeng C W, Liu H X. Study on adaptation and introduc-tion possibility of winter rapeseed to dry and cold areas in north-west china. Sci Agric Sin, 2007, 40: 2716-2726 (in Chinese with English abstract)

[4] 孙万仓, 马卫国, 雷建民, 刘秦, 杨仁义, 武军艳, 王学芳, 叶剑, 曾军, 张亚宏, 康艳丽, 郭秀娟, 魏文惠, 杨杰, 蒲媛媛, 曾潮武, 刘红霞. 冬油菜在西北旱寒区的适应性和北移的可行性研究. 中国农业科学, 2007, 40: 2716-2726

Liu Z G, Sun W C, Fang Y, Li X C, Yang N N, Wu J Y, Zeng X C, Wang Y. Effects of low temperature on the photosynthetic appa-ratus of winter oilseed rape. Sci Agric Sin, 2015, 48: 672-682 (in Chinese with English abstract)

[5] 刘自刚, 孙万仓, 方彦, 李学才, 杨宁宁, 武军艳, 曾秀存, 王月. 夜间低温对白菜型冬油菜光合机构的影响. 中国农业科学, 2015, 48: 672-682

[6] Kumar A, Bimolata W, Kannan M, Kirti P B, Qureshi I A, Ghazi I A. Comparative proteomics reveals differential induction of both biotic and abiotic stress response associated proteins in rice during Xanthomonas oryzae pv. oryzae infection. Funct Integr Genom, 2015, 15: 425-437

Liu Z G, Sun W C, Yang N N, Wang Y, He L, Zhang C X, Shi P F, Yang G, Li X C, Wu J Y, Fang Y, Zeng X C. Morphology and physiological characteristics of cultivars with different levels of cold-resistance in winter rapeseed (Brassica campestris L.) during cold acclimation. Sci Agric Sin, 2013, 46: 4679-4687 (in Chinese with English abstract)

Zhang T G, Wang Y Y, Wang J, Wang N, Zhang Y, Sun W C, Chen Q Q, Xia H J. Cloning and real-time expression analysis of a novel MAPK kinase gene BnMKK2 in Brassica napus. Bull Bot Res, 2012, 32: 578-583 (in Chinese with English abstract)

[7] 刘自刚, 孙万仓, 杨宁宁, 王月, 何丽, 赵彩霞, 史鹏飞, 杨刚, 李学才, 武军艳, 方彦, 曾秀存. 冬前低温胁迫下白菜型冬油菜抗寒性的形态及生理特征. 中国农业科学, 2013, 46: 4679-4687

Zhang T G, Wang N, Wang J, Wang Y Y, Zhang Y, Sun W C, Chang Y, Xia H J. Cloning and expression analysis of a BnMKK4 gene from Brassica napus L. Plant Physiol J, 2012, 48: 491-498 (in Chinese with English abstract)

[8] 张腾国, 王圆圆, 王娟, 王宁, 张艳, 孙万仓, 陈琼琼, 夏惠娟. 油菜BnMKK2基因的克隆及表达分析. 植物研究, 2012, 32: 578-583

[9] 张腾国, 王宁, 王娟, 王圆圆, 张艳, 孙万仓, 常燕, 夏惠娟. 油菜BnMKK4全长基因的克隆及表达分析. 植物生理学报, 2012, 48: 491-498

Jia L Y, Zhang T G, Wang J, Wang Y Y, Xia H J. Isolation and sequence analysis of BnMPK6 gene promoter from Brassica napus (rape) Longyou 6. J Northwest Nor Univ (Nat Sci), 2012, 48: 86-90 (in Chinese with English abstract)

[10] 贾凌云, 张腾国, 王娟, 王圆圆, 夏惠娟. 陇油6号油菜BnMPK6基因启动子分离及序列分析. 西北师范大学学报(自然科学版), 2012, 48: 86-90

Zhang T G, Wang J, Wang Y Y, Wang N, Chang Y, Zhang Y, Chen Q Q, Sun W C. Cloning and expression analysis of BnHMGB2 gene in Brassica napus. Bull Bot Res, 2012, 32: 724-730 (in Chinese with English abstract)

Huang-pu H Y, Guan C Y. A preliminary analysis of differential proteins between Brassica napus resistance to sclerotinia scle-rotiorum near-isogenic lines and susceptible parent. Sci Agric Sin, 2010, 43: 2000-2007 (in Chinese with English abstract)

[11] 张腾国, 王娟, 王圆圆, 王宁, 常燕, 张艳, 陈琼琼, 孙万仓. 油菜BnHMGB2基因的克隆及表达分析. 植物研究, 2012, 32: 724-730

[12] 黄甫海燕, 官春云. 甘蓝型油菜抗菌核病近等基因系和感病亲本蛋白差异初步研究. 中国农业科学, 2010, 43: 2000-2007

Gong F S, Zhang J B. Plant Physiology Experiment, 2nd edn. Beijing: Higher Education Press, 1995. pp 90-92

[13] Bradford M M. A rapid method for the quantification of micro-gram quantities of protein utilizing the principle of protein. dye binding. Anal Biochem, 1976, 72: 248-254

Li Y, Zhang G, Zhang Y X, Hao Z. Effect of salicylic acid on Euonymus japonicus stem cold resistance and electrical impedance spectroscopy parameters. Chin J Ecol, 2010, 29: 460-466 (in Chinese with English abstract)

[14] Katayama H, Nagasu T, Oda Y. Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectr, 15: 1416-1421

Fan C F, Bi Y, Wang Y F, Ren Y L, Yang Z M, Wang Y. Effect of salicylic acid on Muskmelon postharvest diseases and phenyl-propanoid metabolism. Sci Agric Sin, 2012, 45: 584-589 (in Chinese with English abstract)

[15] 龚富生, 张嘉宝. 植物生理学实验(第2版). 北京: 高等教育出版社, 1995. pp 90-92

Li C Y, Chen S S, Xu W, Li D S, Gu X, Zhu X K, Guo W S, Feng C N. Seedlings to low temperature stress of Yangmai 16 leaves anti-oxidant enzymes and osmotic adjustment substances. Acta Agron Sin, 2011, 37: 2293-2298 (in Chinese with English abstract)

[16] Deveris A L. Antifreeze glycopeptides: Interaction with ice and water. Method Enzym, 1986, 127: 293-303

[17] Liu Z, Dreybrodt W. Significance of the carbon sink produced by H2O-arbonate-CO2-aquatic phototroph interaction on land. Sci Bull, 60: 182-191

[18] Majeran W, Zybailov B, Ytterberg A J, Dunsmore J, Sun Q, van Wijk K J. Consequences of C4 differentiation for chloroplast membrane proteomes in maize mesophyll and bundle sheath cells. Mol Cell Proteom, 2008, 7: 1609-1638

[19] Guo L, Sui Z, Zhang S, Ren Y, Liu Y. Comparison of potential diatom “barcode” genes (18S and ITS rDNA, COI, rbcL) and their effectiveness in discriminating and determining species taxonomy in bacillariophyta. Int J System Evol Microbiol, 2015, 65: 1369-1380

[20] Tiong W H C, Kelly E J. Salicylic acid burn induced by wart re-mover: A report of two cases. Burns, 2009, 35: 139-140

[21] da Silveira Novelino A M, Daemon E, Soares G L. Evaluation of the acaricide effect of thymol, menthol, salicylic acid, and methyl salicylate on Boophilus microplus (Canestrini 1887) (Acari: Ixodidae) larvae. Parasitol Res, 2007, 101: 809-811

[22] 李亚, 张钢, 张玉星, 郝征. 水杨酸对大叶黄杨茎抗寒性和电阻抗图谱参数的影响. 生态学杂志, 2010, 29: 460-466

[23] 范存斐, 毕阳, 王云飞, 任亚琳, 杨志敏, 王毅. 水杨酸对厚皮甜瓜采后病害及苯丙烷代谢的影响. 中国农业科学, 2012, 45: 584-589

[24] 李春燕, 陈思思, 徐雯, 李东升, 顾骁, 朱新开, 郭文善, 封超年. 苗期低温胁迫对扬麦16叶片抗氧化酶和渗透调节物质的影响. 作物学报, 2011, 37: 2293-2298

[25] Bahari L, Yashunsky V, Braslavsky I. Microscopic investigation of antifreeze proteins activity at cryogenic temperatures. Cryobiology, 2015, 71: 570-573

[26] Braslavsky, Drori R, Celik Y, Dolev M B, Davies P L. Characteristics of the interaction of antifreeze proteins with ice crystals. Cryobiology, 2015, 71: 540

[27] Beck E H, Heim R, Hansen J. Plant resistance to cold stress: mechanisms and environmental signals triggering frost hardening and dehardening. J Biosci, 2004, 29: 102-109

[28] Szechyńska-Hebdaab M, Wąseka I, Gołębiowska-Pikaniac G, Dubasa E, Żura I, Wędzonyac M. Photosynthesis-dependent physiological and genetic crosstalk between cold acclimation and cold-induced resistance to fungal pathogens in triticale (Tritico-secale Wittm.). J Plant Physiol, 2015, 177: 30-43

[29] Hagiwara Y, Aomatsu H. Supercooling enhancement by adding antifreeze protein and ions to water in a narrow space. Int J Heat Mass Transfer, 2015, 86: 55-64

[30] Sun T, Davies P L, Walker V K. Structural basis for the inhibition of gas hydrates by α-helical antifreeze proteins. Biophys J, 2015, 109: 1698-1705

[31] Gupta R, Deswal R. Antifreeze proteins enable plants to survive in freezing conditions. J Biosci, 2014, 39: 931-944

[32] Wilkensa C, Poulsena J C N, Ramløvb H, Leggioa L L. Purifica-tion, crystal structure determination and functional characteriza-tion of type III antifreeze proteins from the European eelpout Zoarces viviparus. Cryobiology, 2014, 69: 163-168

[33] Ustun N S, Turhan S. Antifreeze proteins: characteristics, func-tion, mechanism of action, sources and application to foods. J Food Proc Preserv, 2015, 39: 3189-3197

[34] Todde G, Hovmöller S E, Laaksonen A. Influence of antifreeze proteins on the ice/water interface. J Physical Chem, 2015, 119: 2407-3413

[35] Lauersen K J, Brown A, Middleton A, Davies P L, Walker V K. Expression and characterization of an antifreeze protein from the perennial rye grass, Lolium perenne. Cryobiology, 2011, 62: 194-201