利用BiFC技术研究拟南芥GPX3和RRTF1与DOS1体内相互作用
摘要
逆境胁迫可以分为非生物胁迫与生物胁迫两大类,严重影响着植物的正常生长发育,其中许多非生物胁迫都可以诱导植物体内产生大量的活性氧(Reactive Oxygen Species, ROS),因此ROS可能是各种胁迫信号的交叉点。近年来,科学家们通过DNA微阵列技术分析了植物体内大量受ROS调控的基因,对这些基因的功能进行了研究,并得到了许多在ROS信号转导中起重要作用的基因元件。
实验室前期的研究发现,拟南芥GPX家族中的GPX3(Glutathione peroxidase 3)编码一个双功能蛋白,不仅能够清除植株体内过量的H_2O_2,而且可以直接或间接调节上游氧化信号,传递和激活下游信号分子;AtDOS1(Drought overly sensitive 1)则是通过酵母双杂交实验证实的能与GPX3发生强烈体外互作的蛋白因子,DOS1定位于细胞核中,具有核定位信号(NLS)和保守序列WWE domain(Trp-Trp-Glu),在植物应答干旱和氧化胁迫中发挥重要作用。进一步的研究表明,DOS1可能为植物氧化胁迫信号转导途径中一个重要的转录调节因子,并且在H_2O_2胁迫下,DOS1会从细胞核向细胞质跨膜扩散,从而参与胁迫信号的传递。AtRRTF1(ROS related transcription factor 1)则是另一个通过芯片分析得到的可能与AtDOS1存在相互作用的基因,该基因编码带有锌指结构的转录因子,其表达受H_2O_2诱导,在生物及非生物胁迫信号转导中起中心作用。酵母双杂交实验也显示RRTF1和DOS1之间存在体外相互作用。但目前我们仍然缺少GPX3与DOS1以及RRTF1与DOS1之间存在体内互作的证据,并且对于DOS1在氧化胁迫下的跨膜运动及机理也不甚明了。
本论文通过双分子荧光互补实验(BiFC)研究了GPX3与DOS1之间以及RRTF1与DOS1之间的体内互作关系,发现在野生型拟南芥叶肉细胞原生质体内,均能检测到这两对蛋白之间发生相互作用的黄色荧光信号,说明GPX3与DOS1以及RRTF1与DOS1都存在体内互作。进一步的DAPI染色实验证明DOS1与RRTF1发生互作的部位在细胞核内。在gpx3突变体原生质体中,没有检测到较为明显的RRTF1与DOS1之间互作的荧光,表明GPX3可能影响DOS1与RRTF1之间的相互作用。为确定GPX3与DOS1以及RRTF1与DOS1之间的体内互作是否影响氧化胁迫下DOS1的跨膜运动,从而影响DOS1传递胁迫信号,我们将实验室已有的DOS1::GFP转化WT植株(WT-DOS1::GFP)分别和gpx3,rrtf1 T-DNA插入纯合突变体进行杂交,筛选出带有DOS1-GFP标签的gpx3和rrtf1突变体植株(gpx3-DOS1::GFP和rrtf1-DOS1::GFP),用激光共聚焦扫描电子显微镜分别观察氧化胁迫下两种突变体中DOS1的跨膜运动与WT中的差异,经过统计分析发现GPX3对DOS1的抗氧化胁迫运动有一定影响,在缺失GPX3的情况下,更有利于拟南芥下表皮条保卫细胞中的DOS1在受到H_2O_2和ABA作用时发生从细胞核向细胞质扩散的现象;而RRTF1表达下调时,DOS1的跨膜运动与正常情况相比没有明显的差别,说明RRTF1可能与DOS1的扩散关系不大。
综上所述,GPX3与DOS1以及RRTF1与DOS1之间均存在体内互作,并且GPX3在一定程度上影响DOS1与RRTF1的体内相互作用;同时,GPX3与DOS1之间的体内互作可能影响DOS1在氧化胁迫下的运动。
Adverse stresses can be classified as abiotic ones and biotic ones, which seriously affect plant growth and development. The abiotic stresses are diverse. They can trigger the production of reactive oxygen species (ROS), which may be the cross point of various stress signals. In recent years, scientists have analyzed the expression patterns of large numbers of genes by DNA microarray, investigated the functions of the genes and found many important cis-elements acting in ROS signal transduction.
Previous results from our laboratory revealed that GPX3 (Glutathione peroxidase 3), a member of GPX family, encodes a functional protein. It can not only scavenge H_2O_2, but also regulate upstream ROS signals and activate and transfer downstream signals. AtDOS1 (Drought overly sensitive 1) has been identified to interact with GPX3 in vitro very strongly through the yeast two hybrid experiment. It localizes in nucleus, containing NLS and WWE domain, and plays a key part in plants response to drought and oxidative stress. DOS1 appears to be one of the important transcriptional regulatory factors in oxidative signal transduction in plants. Moreover, DOS1 can diffuse from the nucleus to the cytoplasm under H_2O_2 stress and transfer the redox signals. AtRRTF1 (ROS related transcription factor 1) is another gene which may interact with AtDOS1 in gene chips. It encodes a transcriptional factor containing zinc structure. The expression of AtRRTF1 is induced by H_2O_2 treatments. RRTF1 plays central roles in signal transductions of biotic and abiotic stresses. Yeast two hybrid experiment also showed that RRTF1 interacting with DOS1 in vitro. Yet, until now we still have no evidence about the in vivo interaction between GPX3 and DOS1 as well as RRTF1 and DOS1. We still have little knowledge about the diffusion mechanisms of DOS1 in Arabidopsis under oxidative stress.
In this paper, the interactions between GPX3 and DOS1 as well as RRTF1 and DOS1 in vivo were investigated using the method of splite YFP (BiFC). There was yellow fluorescence (interaction indicator) in protoplasts of WT plants transformed with GPX3-contained vector and DOS1-contained vector, as well as RRTF1-contained vector and DOS1-contained vector, indicating that DOS1 may interact with GPX3 and RRTF1 in vivo. DAPI staining analysis showed that the place in which DOS1 interacts with RRTF1 is the cell nucleus. No obvious interaction between DOS1 and RRTF1 in vivo was found in protoplasts from gpx3 plants suggesting that GPX3 may influence such interaction in vivo. To further determine if the interaction between DOS1 with GPX3 and RRTF1 affect the diffusion of DOS1 across nucleus membrane and the transferring of stress signals by DOS1 under oxidative stress, we obtained Arabidopsis gpx3 and rrtf1 T-DNA insertion mutants with DOS1-GFP tag (gpx3-DOS1::GFP, rrtf1-DOS1::GFP) by using hybridization technology between WT-DOS1::GFP and gpx3, rrtf1 mutants. Then, differences in DOS1 diffusion across nucleus membrane in the two mutants and WT were analyzed in confocal microscopy and statistical analysis. The results showed that GPX3 may influence the DOS1’s diffusing movement to a certain extent. Without the GPX3, the DOS1 in guard cells would be more inclined to diffuse from the nucleus to the cytoplasm after treatments with H_2O_2 or ABA. But when the expression of RRTF1 was down-regulated, there was no obvious difference for the DOS1 diffusing movement in rrtf1 mutant with DOS1-GFP tag compared to the WT with DOS1-GFP tag. These datas indicated that RRTF1 may not affect the diffusion of DOS1 under oxidative stress.
To sum up, there exists in vivo interaction between GPX3 and DOS1 as well as RRTF1 and DOS1. GPX3 may affect the interaction between RRTF1 and DOS1. The in vivo interaction between GPX3 and DOS1 may modulate the diffusing movement of DOS1 across nucleus membrane under oxidative stress in Arabidopsis.
实验室前期的研究发现,拟南芥GPX家族中的GPX3(Glutathione peroxidase 3)编码一个双功能蛋白,不仅能够清除植株体内过量的H_2O_2,而且可以直接或间接调节上游氧化信号,传递和激活下游信号分子;AtDOS1(Drought overly sensitive 1)则是通过酵母双杂交实验证实的能与GPX3发生强烈体外互作的蛋白因子,DOS1定位于细胞核中,具有核定位信号(NLS)和保守序列WWE domain(Trp-Trp-Glu),在植物应答干旱和氧化胁迫中发挥重要作用。进一步的研究表明,DOS1可能为植物氧化胁迫信号转导途径中一个重要的转录调节因子,并且在H_2O_2胁迫下,DOS1会从细胞核向细胞质跨膜扩散,从而参与胁迫信号的传递。AtRRTF1(ROS related transcription factor 1)则是另一个通过芯片分析得到的可能与AtDOS1存在相互作用的基因,该基因编码带有锌指结构的转录因子,其表达受H_2O_2诱导,在生物及非生物胁迫信号转导中起中心作用。酵母双杂交实验也显示RRTF1和DOS1之间存在体外相互作用。但目前我们仍然缺少GPX3与DOS1以及RRTF1与DOS1之间存在体内互作的证据,并且对于DOS1在氧化胁迫下的跨膜运动及机理也不甚明了。
本论文通过双分子荧光互补实验(BiFC)研究了GPX3与DOS1之间以及RRTF1与DOS1之间的体内互作关系,发现在野生型拟南芥叶肉细胞原生质体内,均能检测到这两对蛋白之间发生相互作用的黄色荧光信号,说明GPX3与DOS1以及RRTF1与DOS1都存在体内互作。进一步的DAPI染色实验证明DOS1与RRTF1发生互作的部位在细胞核内。在gpx3突变体原生质体中,没有检测到较为明显的RRTF1与DOS1之间互作的荧光,表明GPX3可能影响DOS1与RRTF1之间的相互作用。为确定GPX3与DOS1以及RRTF1与DOS1之间的体内互作是否影响氧化胁迫下DOS1的跨膜运动,从而影响DOS1传递胁迫信号,我们将实验室已有的DOS1::GFP转化WT植株(WT-DOS1::GFP)分别和gpx3,rrtf1 T-DNA插入纯合突变体进行杂交,筛选出带有DOS1-GFP标签的gpx3和rrtf1突变体植株(gpx3-DOS1::GFP和rrtf1-DOS1::GFP),用激光共聚焦扫描电子显微镜分别观察氧化胁迫下两种突变体中DOS1的跨膜运动与WT中的差异,经过统计分析发现GPX3对DOS1的抗氧化胁迫运动有一定影响,在缺失GPX3的情况下,更有利于拟南芥下表皮条保卫细胞中的DOS1在受到H_2O_2和ABA作用时发生从细胞核向细胞质扩散的现象;而RRTF1表达下调时,DOS1的跨膜运动与正常情况相比没有明显的差别,说明RRTF1可能与DOS1的扩散关系不大。
综上所述,GPX3与DOS1以及RRTF1与DOS1之间均存在体内互作,并且GPX3在一定程度上影响DOS1与RRTF1的体内相互作用;同时,GPX3与DOS1之间的体内互作可能影响DOS1在氧化胁迫下的运动。
Adverse stresses can be classified as abiotic ones and biotic ones, which seriously affect plant growth and development. The abiotic stresses are diverse. They can trigger the production of reactive oxygen species (ROS), which may be the cross point of various stress signals. In recent years, scientists have analyzed the expression patterns of large numbers of genes by DNA microarray, investigated the functions of the genes and found many important cis-elements acting in ROS signal transduction.
Previous results from our laboratory revealed that GPX3 (Glutathione peroxidase 3), a member of GPX family, encodes a functional protein. It can not only scavenge H_2O_2, but also regulate upstream ROS signals and activate and transfer downstream signals. AtDOS1 (Drought overly sensitive 1) has been identified to interact with GPX3 in vitro very strongly through the yeast two hybrid experiment. It localizes in nucleus, containing NLS and WWE domain, and plays a key part in plants response to drought and oxidative stress. DOS1 appears to be one of the important transcriptional regulatory factors in oxidative signal transduction in plants. Moreover, DOS1 can diffuse from the nucleus to the cytoplasm under H_2O_2 stress and transfer the redox signals. AtRRTF1 (ROS related transcription factor 1) is another gene which may interact with AtDOS1 in gene chips. It encodes a transcriptional factor containing zinc structure. The expression of AtRRTF1 is induced by H_2O_2 treatments. RRTF1 plays central roles in signal transductions of biotic and abiotic stresses. Yeast two hybrid experiment also showed that RRTF1 interacting with DOS1 in vitro. Yet, until now we still have no evidence about the in vivo interaction between GPX3 and DOS1 as well as RRTF1 and DOS1. We still have little knowledge about the diffusion mechanisms of DOS1 in Arabidopsis under oxidative stress.
In this paper, the interactions between GPX3 and DOS1 as well as RRTF1 and DOS1 in vivo were investigated using the method of splite YFP (BiFC). There was yellow fluorescence (interaction indicator) in protoplasts of WT plants transformed with GPX3-contained vector and DOS1-contained vector, as well as RRTF1-contained vector and DOS1-contained vector, indicating that DOS1 may interact with GPX3 and RRTF1 in vivo. DAPI staining analysis showed that the place in which DOS1 interacts with RRTF1 is the cell nucleus. No obvious interaction between DOS1 and RRTF1 in vivo was found in protoplasts from gpx3 plants suggesting that GPX3 may influence such interaction in vivo. To further determine if the interaction between DOS1 with GPX3 and RRTF1 affect the diffusion of DOS1 across nucleus membrane and the transferring of stress signals by DOS1 under oxidative stress, we obtained Arabidopsis gpx3 and rrtf1 T-DNA insertion mutants with DOS1-GFP tag (gpx3-DOS1::GFP, rrtf1-DOS1::GFP) by using hybridization technology between WT-DOS1::GFP and gpx3, rrtf1 mutants. Then, differences in DOS1 diffusion across nucleus membrane in the two mutants and WT were analyzed in confocal microscopy and statistical analysis. The results showed that GPX3 may influence the DOS1’s diffusing movement to a certain extent. Without the GPX3, the DOS1 in guard cells would be more inclined to diffuse from the nucleus to the cytoplasm after treatments with H_2O_2 or ABA. But when the expression of RRTF1 was down-regulated, there was no obvious difference for the DOS1 diffusing movement in rrtf1 mutant with DOS1-GFP tag compared to the WT with DOS1-GFP tag. These datas indicated that RRTF1 may not affect the diffusion of DOS1 under oxidative stress.
To sum up, there exists in vivo interaction between GPX3 and DOS1 as well as RRTF1 and DOS1. GPX3 may affect the interaction between RRTF1 and DOS1. The in vivo interaction between GPX3 and DOS1 may modulate the diffusing movement of DOS1 across nucleus membrane under oxidative stress in Arabidopsis.
引文
[1]彭珂珊,徐宣斌,胡晋辉等.干旱是西部地区生态系统受损的关键因素[J].石家庄经济学院学报, 2002, 25(3): 257-262.
[2] Fujita M, Fujita Y, Noutoshi Y, et al.. Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks[J]. Current Opinion Plant Biology, 2006, 9(4): 436-442.
[3]陈珂,蒋祺,类延宝等.植物对干旱胁迫的生理响应[J].安徽农业科学, 2009, 37(5): 1907-1908.
[4]冯建成,杨彬元.早稻新品种区域试验初报[J].福建农业科技, 2007, (2): 4-6.
[5]周乐明,魏新林,李中秀等.早杂新组合不同生态点品比试验[J].江西农业学报, 2006, 18(4): 45-47.
[6]许保权.杂交中籼新组合的比较试验研究[J].安徽农业科学, 2007, 35(24): 7435-7445.
[7] Lin Xiuqin, Yuan Kun, Wang Zhenhui, et al.. Advances on Comparative Proteomics of Plants under Stresses[J]. Chinese journal of tropical agriculture, 2009, 29(2): 1-4.
[8] Mahajan S, Tuteja N. Cold salinity and drought stresses: an overview[J]. Archives of Biochemistry and Biophysics, 2005, 444(2): 139-158.
[9] Yin Kui de. Research Advances on Active Oxygen Special (ROS) under Abiotic Stress[J]. Journal of Shenyang Agricultural University, 2003-04, 34(2): 147-149.
[10] Zhao Tianhong, Sun J iawe i, Fu Yu, et al.. Advances of Research on Metabolism of Plant Reactive Oxygen Species and Exogenous Regulation under Abiotic Stresses[M]. Journal of Crops, China Academic Journal Electronic Publishing House, 1994-2010.
[11]蒋明义.水分胁迫下植物体内自由基的产生与细胞的氧化损伤[J].植物学报, 1999, 41: 229- 234.
[12] Navrot N, Rouhier N, Gelhaye E, et al.. Reactive oxygen species generation and antioxidant systems in plant mitochondria[J]. Physiologia Plantarum, 2007, 68(129): 1852-2195.
[13] Chen Q, Vazquez E J, Moghaddas S, et al.. Production of reactive oxygen species by mitochondria: Central role of complexⅢ[ J]. Journal of Biological Chemistry, 2003, 40(278): 36027-36031.
[14] Ianm M. Plant mitochondrial and oxidative stress: Electron transport, NADPH turnover, and metabolism of reactive oxygen species[J]. Annual Review of Plant Physiol and Plant Physiology and Biochemistry, 2001, 1(52): 561-591.
[15]姜卫东,高光林,俞开锦等.水分胁迫对果树光合作用及同化代谢的影响研究进展[J].果树学报, 2002, 19(6): 416-420.
[16] Mittler R. Oxidativestress, antioxidants and stress tolerance[M]. Trends in Sciences, 2002, 7(9): 405-410.
[17] Halliwel L, Gutteridgejm C. Free radicals in biology and medicine[M]. Oxford: Oxford University Press, 1989, 66: 444-456.
[18] Laloi C, Apel K, Danon A, et al.. Reactive oxygen signalling:the latest news[J]. Current opinion PlantBiology, 2004, 7(3): 323-328.
[19] Wang Yanqin, Dong Haitao. Biological Function of Phosphatidic Acid as a Second Messenger in Plants[J]. Chinese Journal of Biochemistry and Molecular Biology, 2006, 22(9): 697-703.
[20] Somervolle C, Brow se J, Jaworski J G, et al.. Biochemistry and molecular biology of plants[M]. Rock ville: American Society of plant Physiologists, 2000, 122(94): 456-572.
[21] Mittler R, Vanderauwera S, Gollery M, et al.. Reactive oxygen gene network of plants[J]. Trends in Plant Science, 2004, 9(10): 490-498.
[22]陈世苹,高玉葆,梁宇等.水分胁迫下内生真菌感染对黑麦草叶内保护酶系统活力的影响[J].应用与环境生物学报, 2001, 7(4): 348-354.
[23]李明,王根轩等.干旱胁迫对甘草幼苗保护酶活性及脂质过氧化作用的影响[J].生态学报, 2002, 22(4): 503-507.
[24]陈建军,韩锦峰,汪瑞新等.水分胁迫下烟草光合作用的气孔与非气孔限制[J].植物生理学通讯, 1991, 27: 415-418.
[25] Delaunay A, Pflieger D, Barrault MB, et al.. A thiol pemxidase is an H2O2 receptor and redox-transducer in gene activation[J]. Cell, 2002, 111(4): 471-481.
[26] Zheng M, Aslund F, Storz G, et al.. Activation of the OxyR transcription factor by reversible disulfide bond formation.[J]. Science, 1998, 279(5357): 1718-1721.
[27] Yuchen Miao, Dong Lv, ChunPeng Song, et al.. An Arabidopsis Glutathione Peroxidase Functions as Both a Redox Transducer and a Scavenger in Abscisic Acid and Drought Stress Responses[J]. The Plant Cell, 2006, 12(18): 2749-2766.
[28] Graham Noctor. Ascorbate and glutathione: Keeping active oxygen under control[J]. Annual Review of Plant Physiology, 1998, 49: 249-279.
[29] WU Shun, Xiao lang tao. Metabolism and Signaling Conduction of the Reactive Oxygen Species in Plant[J]. Journal of Hunan Agricultural University, 2003, 29(5): 1161-1177.
[30] Du Xiu Min, Yin Wen Xuan, Zhao Yan Xiu, et al.. The Production and Scavenging of Reactive Oxygen Species in Plants[J]. Chinese Journal of Biotechnology, 2001, 17(2): 3333-3366.
[31] Yamasaki H, Heshiki R, Ikehara N, et al.. Leaf goldening induced by high light in Ficus micrcarpa Lf atropical[J]. Plant Research, 1995, 108: 171-180.
[32]彭金英,黄勇平.植物防御反应的两种信号转导途径及其相互作用[J].植物生理与分子生物学报, 2005, 31(4): 347-353.
[33] Anna Corina, Po-Pu Liu, Robin K. Cameron, et al.. Identification of likely orthologs of tobacco salicylic acid-binding protein 2 and their role in systemic acquired resistance in Arabidopsis thaliana[J]. The Plant Journal, 2008, 56(13): 445-456.
[34] JABS T, Tsch Pem, Coll Ing C, et al.. Elicitor-stimulated ion fluxes and O2- from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley[J]. Proceedings of the Natural Acad. Science. USA, 1997, 94: 4800-4805.
[35] Guan L M, Scandalios J G. Hydrogen peroxide-mediated catalase gene expression in response towounding[J]. Free Radical Biology and Medicine, 2000, 9(28): 1182-1190.
[36] Orozco Cárdenasml, KnarváEZVásqu EZ J, Kryan C A. Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding Ksystem in Kand methyl jasmonate[J]. The Plant Cell, 2001, 13(11): 179-191.
[37] Johannes Stratmann. Ultraviolet-B radiation coopts defense signaling pathways[J]. Trends in Plant Science, 2003, 8(11): 526-533.
[38] Karpinskis, Kreynoldsh, Kkarpinskab, et al.. System icsignalling and acclimation in response to excess excitation energy in Arabidopsis[J]. Science, 1999, 284(5414): 654-657.
[39] Mulpuri V. Rao, Hyung-il Lee, Keith R. Davis. Ozone-induced ethylene production is dependent on salicylic acid, and both salicylic acid and ethylene act in concert to regulate ozone-induced cell death[J]. The Plant Journal, 2002, 32(4): 447-456.
[40] Kschweizer P, Kmosinger E, Kmetraux J P, et al.. Heat-induced resistance to powdery m ildew(Blum eria gram in) is sphord eiGis associated with aburst of active oxygen species[J]. Physiology Molecular Plant Pathol, 1998, 52: 185-199.
[41] Wisniewski J P, Kcornill E P, Kagnel J P, et al.. The extension multigene family responds differentially to superoxide or hydrogen peroxide in tomato cell cultures[J]. FEBS Letters, 1999, 447(233): 264-268.
[42] Miguel Angel Torres, Jonathan D G Jones, Jeffery L Dang, et al.. Pathogen-induced, NADPH oxidase-derived reactive oxygen intermediates suppress spread of cell death in Arabidopsis thaliana[J]. Nature Genetics, 2005, 37: 1130-1134.
[43] Cindy M. Yamamoto, Amiya P. Sinha Hikim, Phuong N. Huynh, et al.. Redistribution of Bax Is an Early Step in an Apoptotic Pathway Leading to Germ Cell Death in Rats, Triggered by Mild Testicular Hyperthermia[J]. Biology of Reproduction, 2000, 63(46): 1683-1690.
[44] Christophe Fleury, Bernard Mignotte, Jean-Luc Vayssière, et al.. Mitochondrial reactive oxygen species in cell death signaling[J]. ScienceDirect-Biochimie, 2002, 84(23): 131-141.
[45] Levin E A, Ktenhaken R, Kdixon R, et al. H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response[J]. Cell, 1994, 79: 583-593.
[46] Desikan R, Kreynolds A, Khancock J T, et al.. Harp in and hydrogen peroxide both initiate programmed cell death but have differential effects on gene expression in Arabidopsis suspension cultures[J]. Biochemical Journal, 1998, 330: 115-120.
[47] Xu Hu, Dennis L. Bidney, Nasser Yalpani, et al.. Overexpression of a Gene Encoding Hydrogen Peroxide-Generating Oxalate Oxidase Evokes Defence Responses in Sunflower[J]. Plant Physiology, 2003, 133: 170-181.
[48] VranováE, Kin ZèD, Kbreu Segem F V. Signal transduction during oxidative stress[J]. Journal of Experimental Botany, 2002, 53(372): 1227-1236.
[49] Fath A, Kbethke P, Kbellign IV, et al.. Active oxygen and cell death in cereal aleurone cells[J]. Journal of Experimental Botany, 2002, 53(372): 1273-1282.
[50]王娟,李德全.逆境条件下植物体内渗透调节物质的积累与活性氧代谢[J].植物学通报, 2001, 18(4): 459-465.
[51] Hyun Mee Do, Jeum Kyu Hong, Ho Won Jung, et al.. Expression of Peroxidase-like Genes, H2O2 Production, and Peroxidase Activity During the Hypersensitive Response to Xanthomonas campestris pv. vesicatoria in Capsicum annuum[J]. Molecular Plant-Microbe Interactions, 2003, 16(3): 196-205.
[52] Dorey S, Baillieul F, Sa indrenan P, et al.. Tobacco class I and II catalase are differentially expressed during elicitor-induced hypersensitive cell death and localized acquired resistance[J]. Molecular Plant- Microbe Interactions, 1998, 11: 1102-1109.
[53] PG Sappl, AJ Carroll, R Clifton, et al.. The Arabidopsis glutathione transferase gene family displays complex stress regulation and co-silencing multiple genes results in altered metabolic sensitivity to oxidative stress[J]. The Plant Journal, 2009, 58(66): 53-68.
[54] A Wahid, M Perveen, S Gelani, et al.. Pretreatment of seed with H2O2 improves salt tolerance of wheat seedlings by alleviation of oxidative damage and expression of stress proteins[J]. Journal of plant physiology, 2007, 164(63): 283-294.
[55] Solomon M, Kbelengh IB, Kdelledonne M, et al.. The involvement of cysteine protease inhibitor genes in the regulation of programmed cell death in plants[J]. The Plant Cell, 1999, 11: 431-443.
[56] Etienn E P, Petitot A, Skhouot V, et al.. Induction of tcI 7, a gene encoding a B-subunit of proteasome, in tobacco plants treated with elicitin, salicylic acid or hydrogen peroxide[J]. FEBS Letters, 2000, 466(42): 213-218.
[57] X Wan, J Tan, S Lu, et al.. Increased tolerance to oxidative stress in transgenic tobacco expressing a wheat oxalate oxidase gene via induction of antioxidant enzymes is mediated by H2O2[J]. Physiologia plantarum, 2009, 136: 30-44.
[58] Song Lilu, Zhang Quan. Reactive oxygen gene network of plants and its regulation[J]. Chinese Bulletin of Life Sciences, 2007, 19(3): 1166-1188.
[59] Anthony R G, Henriques R, Helfer A, et al.. A protein kinase target of a PDK1 signalling pathway is involved in root hair growth in Arabidopsis[J]. EMBO Journal, 2004, 23(3): 572-581.
[60] Rentel M C, Lecourieux D, Ouaked F, et al.. OXI1 kinase isnecessary for oxidative burst-mediated signalling in Arabidopsis[J]. Nature, 2004, 427(6977): 858-861.
[61] Merlot S, Gosti F, Guerrier D, et al.. The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway[J]. The Plant Journal, 2001, 03(25): 295-303.
[62] Klans Apel, Heribert Hirt. REACTIVE OXYGEN SPECIES:Metabolism, Oxidative Stress, and Signal Transduction[M]. Annual Review of Plant Biology, 2004, 6(55): 373-399.
[63] Vandenabeele S, Vanderauwera S, Vuylsteke M, et al.. Catalase deficiency drastically affects gene expression induced by high light in Arabidopsis thaliana[J]. The Plant Journal, 2004, 01(39): 45-58.
[2] Fujita M, Fujita Y, Noutoshi Y, et al.. Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks[J]. Current Opinion Plant Biology, 2006, 9(4): 436-442.
[3]陈珂,蒋祺,类延宝等.植物对干旱胁迫的生理响应[J].安徽农业科学, 2009, 37(5): 1907-1908.
[4]冯建成,杨彬元.早稻新品种区域试验初报[J].福建农业科技, 2007, (2): 4-6.
[5]周乐明,魏新林,李中秀等.早杂新组合不同生态点品比试验[J].江西农业学报, 2006, 18(4): 45-47.
[6]许保权.杂交中籼新组合的比较试验研究[J].安徽农业科学, 2007, 35(24): 7435-7445.
[7] Lin Xiuqin, Yuan Kun, Wang Zhenhui, et al.. Advances on Comparative Proteomics of Plants under Stresses[J]. Chinese journal of tropical agriculture, 2009, 29(2): 1-4.
[8] Mahajan S, Tuteja N. Cold salinity and drought stresses: an overview[J]. Archives of Biochemistry and Biophysics, 2005, 444(2): 139-158.
[9] Yin Kui de. Research Advances on Active Oxygen Special (ROS) under Abiotic Stress[J]. Journal of Shenyang Agricultural University, 2003-04, 34(2): 147-149.
[10] Zhao Tianhong, Sun J iawe i, Fu Yu, et al.. Advances of Research on Metabolism of Plant Reactive Oxygen Species and Exogenous Regulation under Abiotic Stresses[M]. Journal of Crops, China Academic Journal Electronic Publishing House, 1994-2010.
[11]蒋明义.水分胁迫下植物体内自由基的产生与细胞的氧化损伤[J].植物学报, 1999, 41: 229- 234.
[12] Navrot N, Rouhier N, Gelhaye E, et al.. Reactive oxygen species generation and antioxidant systems in plant mitochondria[J]. Physiologia Plantarum, 2007, 68(129): 1852-2195.
[13] Chen Q, Vazquez E J, Moghaddas S, et al.. Production of reactive oxygen species by mitochondria: Central role of complexⅢ[ J]. Journal of Biological Chemistry, 2003, 40(278): 36027-36031.
[14] Ianm M. Plant mitochondrial and oxidative stress: Electron transport, NADPH turnover, and metabolism of reactive oxygen species[J]. Annual Review of Plant Physiol and Plant Physiology and Biochemistry, 2001, 1(52): 561-591.
[15]姜卫东,高光林,俞开锦等.水分胁迫对果树光合作用及同化代谢的影响研究进展[J].果树学报, 2002, 19(6): 416-420.
[16] Mittler R. Oxidativestress, antioxidants and stress tolerance[M]. Trends in Sciences, 2002, 7(9): 405-410.
[17] Halliwel L, Gutteridgejm C. Free radicals in biology and medicine[M]. Oxford: Oxford University Press, 1989, 66: 444-456.
[18] Laloi C, Apel K, Danon A, et al.. Reactive oxygen signalling:the latest news[J]. Current opinion PlantBiology, 2004, 7(3): 323-328.
[19] Wang Yanqin, Dong Haitao. Biological Function of Phosphatidic Acid as a Second Messenger in Plants[J]. Chinese Journal of Biochemistry and Molecular Biology, 2006, 22(9): 697-703.
[20] Somervolle C, Brow se J, Jaworski J G, et al.. Biochemistry and molecular biology of plants[M]. Rock ville: American Society of plant Physiologists, 2000, 122(94): 456-572.
[21] Mittler R, Vanderauwera S, Gollery M, et al.. Reactive oxygen gene network of plants[J]. Trends in Plant Science, 2004, 9(10): 490-498.
[22]陈世苹,高玉葆,梁宇等.水分胁迫下内生真菌感染对黑麦草叶内保护酶系统活力的影响[J].应用与环境生物学报, 2001, 7(4): 348-354.
[23]李明,王根轩等.干旱胁迫对甘草幼苗保护酶活性及脂质过氧化作用的影响[J].生态学报, 2002, 22(4): 503-507.
[24]陈建军,韩锦峰,汪瑞新等.水分胁迫下烟草光合作用的气孔与非气孔限制[J].植物生理学通讯, 1991, 27: 415-418.
[25] Delaunay A, Pflieger D, Barrault MB, et al.. A thiol pemxidase is an H2O2 receptor and redox-transducer in gene activation[J]. Cell, 2002, 111(4): 471-481.
[26] Zheng M, Aslund F, Storz G, et al.. Activation of the OxyR transcription factor by reversible disulfide bond formation.[J]. Science, 1998, 279(5357): 1718-1721.
[27] Yuchen Miao, Dong Lv, ChunPeng Song, et al.. An Arabidopsis Glutathione Peroxidase Functions as Both a Redox Transducer and a Scavenger in Abscisic Acid and Drought Stress Responses[J]. The Plant Cell, 2006, 12(18): 2749-2766.
[28] Graham Noctor. Ascorbate and glutathione: Keeping active oxygen under control[J]. Annual Review of Plant Physiology, 1998, 49: 249-279.
[29] WU Shun, Xiao lang tao. Metabolism and Signaling Conduction of the Reactive Oxygen Species in Plant[J]. Journal of Hunan Agricultural University, 2003, 29(5): 1161-1177.
[30] Du Xiu Min, Yin Wen Xuan, Zhao Yan Xiu, et al.. The Production and Scavenging of Reactive Oxygen Species in Plants[J]. Chinese Journal of Biotechnology, 2001, 17(2): 3333-3366.
[31] Yamasaki H, Heshiki R, Ikehara N, et al.. Leaf goldening induced by high light in Ficus micrcarpa Lf atropical[J]. Plant Research, 1995, 108: 171-180.
[32]彭金英,黄勇平.植物防御反应的两种信号转导途径及其相互作用[J].植物生理与分子生物学报, 2005, 31(4): 347-353.
[33] Anna Corina, Po-Pu Liu, Robin K. Cameron, et al.. Identification of likely orthologs of tobacco salicylic acid-binding protein 2 and their role in systemic acquired resistance in Arabidopsis thaliana[J]. The Plant Journal, 2008, 56(13): 445-456.
[34] JABS T, Tsch Pem, Coll Ing C, et al.. Elicitor-stimulated ion fluxes and O2- from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley[J]. Proceedings of the Natural Acad. Science. USA, 1997, 94: 4800-4805.
[35] Guan L M, Scandalios J G. Hydrogen peroxide-mediated catalase gene expression in response towounding[J]. Free Radical Biology and Medicine, 2000, 9(28): 1182-1190.
[36] Orozco Cárdenasml, KnarváEZVásqu EZ J, Kryan C A. Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding Ksystem in Kand methyl jasmonate[J]. The Plant Cell, 2001, 13(11): 179-191.
[37] Johannes Stratmann. Ultraviolet-B radiation coopts defense signaling pathways[J]. Trends in Plant Science, 2003, 8(11): 526-533.
[38] Karpinskis, Kreynoldsh, Kkarpinskab, et al.. System icsignalling and acclimation in response to excess excitation energy in Arabidopsis[J]. Science, 1999, 284(5414): 654-657.
[39] Mulpuri V. Rao, Hyung-il Lee, Keith R. Davis. Ozone-induced ethylene production is dependent on salicylic acid, and both salicylic acid and ethylene act in concert to regulate ozone-induced cell death[J]. The Plant Journal, 2002, 32(4): 447-456.
[40] Kschweizer P, Kmosinger E, Kmetraux J P, et al.. Heat-induced resistance to powdery m ildew(Blum eria gram in) is sphord eiGis associated with aburst of active oxygen species[J]. Physiology Molecular Plant Pathol, 1998, 52: 185-199.
[41] Wisniewski J P, Kcornill E P, Kagnel J P, et al.. The extension multigene family responds differentially to superoxide or hydrogen peroxide in tomato cell cultures[J]. FEBS Letters, 1999, 447(233): 264-268.
[42] Miguel Angel Torres, Jonathan D G Jones, Jeffery L Dang, et al.. Pathogen-induced, NADPH oxidase-derived reactive oxygen intermediates suppress spread of cell death in Arabidopsis thaliana[J]. Nature Genetics, 2005, 37: 1130-1134.
[43] Cindy M. Yamamoto, Amiya P. Sinha Hikim, Phuong N. Huynh, et al.. Redistribution of Bax Is an Early Step in an Apoptotic Pathway Leading to Germ Cell Death in Rats, Triggered by Mild Testicular Hyperthermia[J]. Biology of Reproduction, 2000, 63(46): 1683-1690.
[44] Christophe Fleury, Bernard Mignotte, Jean-Luc Vayssière, et al.. Mitochondrial reactive oxygen species in cell death signaling[J]. ScienceDirect-Biochimie, 2002, 84(23): 131-141.
[45] Levin E A, Ktenhaken R, Kdixon R, et al. H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response[J]. Cell, 1994, 79: 583-593.
[46] Desikan R, Kreynolds A, Khancock J T, et al.. Harp in and hydrogen peroxide both initiate programmed cell death but have differential effects on gene expression in Arabidopsis suspension cultures[J]. Biochemical Journal, 1998, 330: 115-120.
[47] Xu Hu, Dennis L. Bidney, Nasser Yalpani, et al.. Overexpression of a Gene Encoding Hydrogen Peroxide-Generating Oxalate Oxidase Evokes Defence Responses in Sunflower[J]. Plant Physiology, 2003, 133: 170-181.
[48] VranováE, Kin ZèD, Kbreu Segem F V. Signal transduction during oxidative stress[J]. Journal of Experimental Botany, 2002, 53(372): 1227-1236.
[49] Fath A, Kbethke P, Kbellign IV, et al.. Active oxygen and cell death in cereal aleurone cells[J]. Journal of Experimental Botany, 2002, 53(372): 1273-1282.
[50]王娟,李德全.逆境条件下植物体内渗透调节物质的积累与活性氧代谢[J].植物学通报, 2001, 18(4): 459-465.
[51] Hyun Mee Do, Jeum Kyu Hong, Ho Won Jung, et al.. Expression of Peroxidase-like Genes, H2O2 Production, and Peroxidase Activity During the Hypersensitive Response to Xanthomonas campestris pv. vesicatoria in Capsicum annuum[J]. Molecular Plant-Microbe Interactions, 2003, 16(3): 196-205.
[52] Dorey S, Baillieul F, Sa indrenan P, et al.. Tobacco class I and II catalase are differentially expressed during elicitor-induced hypersensitive cell death and localized acquired resistance[J]. Molecular Plant- Microbe Interactions, 1998, 11: 1102-1109.
[53] PG Sappl, AJ Carroll, R Clifton, et al.. The Arabidopsis glutathione transferase gene family displays complex stress regulation and co-silencing multiple genes results in altered metabolic sensitivity to oxidative stress[J]. The Plant Journal, 2009, 58(66): 53-68.
[54] A Wahid, M Perveen, S Gelani, et al.. Pretreatment of seed with H2O2 improves salt tolerance of wheat seedlings by alleviation of oxidative damage and expression of stress proteins[J]. Journal of plant physiology, 2007, 164(63): 283-294.
[55] Solomon M, Kbelengh IB, Kdelledonne M, et al.. The involvement of cysteine protease inhibitor genes in the regulation of programmed cell death in plants[J]. The Plant Cell, 1999, 11: 431-443.
[56] Etienn E P, Petitot A, Skhouot V, et al.. Induction of tcI 7, a gene encoding a B-subunit of proteasome, in tobacco plants treated with elicitin, salicylic acid or hydrogen peroxide[J]. FEBS Letters, 2000, 466(42): 213-218.
[57] X Wan, J Tan, S Lu, et al.. Increased tolerance to oxidative stress in transgenic tobacco expressing a wheat oxalate oxidase gene via induction of antioxidant enzymes is mediated by H2O2[J]. Physiologia plantarum, 2009, 136: 30-44.
[58] Song Lilu, Zhang Quan. Reactive oxygen gene network of plants and its regulation[J]. Chinese Bulletin of Life Sciences, 2007, 19(3): 1166-1188.
[59] Anthony R G, Henriques R, Helfer A, et al.. A protein kinase target of a PDK1 signalling pathway is involved in root hair growth in Arabidopsis[J]. EMBO Journal, 2004, 23(3): 572-581.
[60] Rentel M C, Lecourieux D, Ouaked F, et al.. OXI1 kinase isnecessary for oxidative burst-mediated signalling in Arabidopsis[J]. Nature, 2004, 427(6977): 858-861.
[61] Merlot S, Gosti F, Guerrier D, et al.. The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway[J]. The Plant Journal, 2001, 03(25): 295-303.
[62] Klans Apel, Heribert Hirt. REACTIVE OXYGEN SPECIES:Metabolism, Oxidative Stress, and Signal Transduction[M]. Annual Review of Plant Biology, 2004, 6(55): 373-399.
[63] Vandenabeele S, Vanderauwera S, Vuylsteke M, et al.. Catalase deficiency drastically affects gene expression induced by high light in Arabidopsis thaliana[J]. The Plant Journal, 2004, 01(39): 45-58.