小麦热激转录因子基因TaHsfA2f生物学特性及耐热性调控作用
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摘要
植物热激转录因子(heat shock transcription factor, Hsf)在植物体响应热胁迫和其他逆境胁迫信号转导通路中发挥重要的调节作用,数量因作物不同差异较大。小麦(Triticum aestivum) Hsf家族成员数量多,包括多个亚家族,生物学特性和功能复杂多样。本研究从小麦中同源克隆了A2亚族成员TaHsfA2f(GenBank No. MK045331)的完整开放阅读框序列,序列长1 062 bp,编码353个氨基酸,蛋白质序列含DNA结合结构域(DNA-binding domain, DBD)、核定位信号序列(nuclear localization signal, NLS)(MRKELEDAMSNKRRRR)、核输出信号序列(nuclear export signal, NES)(LKRDKGLLM)和激活域(aromatic, large hydrophobic and acidic amino residues, AHA)(DDFWEDLLHE)。通过转化烟草(Nicotiana tabacum)表皮细胞发现,正常条件下Ta HsfA2f蛋白质定位在细胞核。同源分析表明,TaHsfA2f与多种作物的HsfA2e蛋白相似性较高。组织特异性表达分析表明,TaHsfA2f在小麦中组成型表达。显著性分析表明,成熟根中高表达,幼茎和幼叶中较低表达(P<0.05)。叶片中TaHsfA2f的表达分别受热胁迫、水杨酸和过氧化氢处理上调(P<0.05),于处理60 min和120 min时达峰值。TaHsfA2f可不同程度提高转基因拟南芥(Arabidopsis thaliana)植株的基础耐热性和获得耐热性,在热胁迫条件下上调一系列热相关蛋白基因的表达。本研究为进一步解析小麦A2亚族基因特性与调控功能提供理论依据。
Plant heat shock transcription factor(Hsf) is important regulating factor of signal transduction pathway activated transcriptively by genes under heat stress and other stresses. Hsf can specifically bind to heat shock elements(HSE) in the upstream promoter region of heat shock protein genes to realize the regulation to these genes expression. Plant Hsfs belong to multi-genes family, the members are different among varieties. Hsfs are divided into 3 classes of A, B and C, each class including several subclasses. Many previous works were mainly focused on class A, especially subclass A1 and A2. There are more than 56 members in wheat(Triticum aestivum) Hsf family reported in previous research, containing multiple subclass members, showing diverse characteristics and functions. Recently, the Hsf family members were found to reach to 82 based on wheat genome sequence. In this paper, The TaHsfA2 f was isolated from wheat young leaves treated by heat shock at 37 ℃ for 1.5 h using homologous cloning methods. Sequence analysis showed that the coding sequence(CDS) of TaHsfA2 f(GenBank No. MK045331) was 1 062 bp encoding a protein of353 amino acids. The amino acid sequence analysis demonstrated that TaHsfA2 f contained a DNA-binding domain(DBD), a nuclear localization signal(NLS) of MRKELEDAMSNKRRRR peptide, a nuclear export signal(NES) of LKRDKGLLM peptide and an aromatic, large hydrophobic and acidic amino residues(AHA)of DDFWEDLLHE peptide. Through transient reporter assays with tobacco(Nicotiana tabacum) epidermal cells, it was found that the TaHsfA2 f protein was subcellular localized in the nuclei under normal growth conditions. Homologous analysis showed that Ta HsfA2 f protein shared higher identities with the HsfA2 e proteins from some other crops such as barley(Hordeum vulgare), Sargassum mongolicus, Arabidopsis thaliana and so on. qRT-PCR analysis showed that TaHsfA2 f was expressed in the majority of tissues and organs of wheat with higher expression level in mature roots and lower expression level in young shoots(P<0.05).TaHsfA2 f expression in leaf were up-regulated by heat shock at 37 ℃, salicylic acid(SA) and H2 O2(P<0.05),respectively, and the peak values appeared at 60 min and 120 min after treatments. Through genetic transformation in wide type A. thaliana and assays of thermotolerances of transgenic line 10_19, line 13_9 and line 20_14 of overexpressing TaHsfA2 f, the results revealed that overexpression of TaHsfA2 f could improve both the basal and acquired thermotolerances of transgenic A. thaliana, and relative Hsp genes expression were upregulated to different degrees at 8 h after 2 different heat regimes treatments. Compared to WT, under heat stress, the seedlings growth potential of 3 transgenic lines were better and chlorophyll contents of the rosette leaves were higher and relative electric conductivity(REC) were lower, providing physiological evidences for phenotypes. These results could provide theoretical evidence for further understanding of biological characteristics and functions of subclass HsfA2 members of wheat Hsf family.
Plant heat shock transcription factor(Hsf) is important regulating factor of signal transduction pathway activated transcriptively by genes under heat stress and other stresses. Hsf can specifically bind to heat shock elements(HSE) in the upstream promoter region of heat shock protein genes to realize the regulation to these genes expression. Plant Hsfs belong to multi-genes family, the members are different among varieties. Hsfs are divided into 3 classes of A, B and C, each class including several subclasses. Many previous works were mainly focused on class A, especially subclass A1 and A2. There are more than 56 members in wheat(Triticum aestivum) Hsf family reported in previous research, containing multiple subclass members, showing diverse characteristics and functions. Recently, the Hsf family members were found to reach to 82 based on wheat genome sequence. In this paper, The TaHsfA2 f was isolated from wheat young leaves treated by heat shock at 37 ℃ for 1.5 h using homologous cloning methods. Sequence analysis showed that the coding sequence(CDS) of TaHsfA2 f(GenBank No. MK045331) was 1 062 bp encoding a protein of353 amino acids. The amino acid sequence analysis demonstrated that TaHsfA2 f contained a DNA-binding domain(DBD), a nuclear localization signal(NLS) of MRKELEDAMSNKRRRR peptide, a nuclear export signal(NES) of LKRDKGLLM peptide and an aromatic, large hydrophobic and acidic amino residues(AHA)of DDFWEDLLHE peptide. Through transient reporter assays with tobacco(Nicotiana tabacum) epidermal cells, it was found that the TaHsfA2 f protein was subcellular localized in the nuclei under normal growth conditions. Homologous analysis showed that Ta HsfA2 f protein shared higher identities with the HsfA2 e proteins from some other crops such as barley(Hordeum vulgare), Sargassum mongolicus, Arabidopsis thaliana and so on. qRT-PCR analysis showed that TaHsfA2 f was expressed in the majority of tissues and organs of wheat with higher expression level in mature roots and lower expression level in young shoots(P<0.05).TaHsfA2 f expression in leaf were up-regulated by heat shock at 37 ℃, salicylic acid(SA) and H2 O2(P<0.05),respectively, and the peak values appeared at 60 min and 120 min after treatments. Through genetic transformation in wide type A. thaliana and assays of thermotolerances of transgenic line 10_19, line 13_9 and line 20_14 of overexpressing TaHsfA2 f, the results revealed that overexpression of TaHsfA2 f could improve both the basal and acquired thermotolerances of transgenic A. thaliana, and relative Hsp genes expression were upregulated to different degrees at 8 h after 2 different heat regimes treatments. Compared to WT, under heat stress, the seedlings growth potential of 3 transgenic lines were better and chlorophyll contents of the rosette leaves were higher and relative electric conductivity(REC) were lower, providing physiological evidences for phenotypes. These results could provide theoretical evidence for further understanding of biological characteristics and functions of subclass HsfA2 members of wheat Hsf family.
引文
李慧聪,李国良,郭秀林.2014.玉米热激转录因子基因ZmHsf-Like对逆境胁迫响应的信号途径[J].作物学报,40(4):622-628.(Li H C,Li G L,Guo X L.2014.Signal transduction pathway of ZmHsf-Like gene responding to different abiotic stresses[J].Acta Agronomica Sinica,40(4):622-628.)
李慧聪,李国良,郭秀林.2015.玉米热激转录因子基因(ZmHsf06)的克隆、表达和定位分析[J].农业生物技术学报,23(1):41-51.(Li H C,Li G L,Guo X L.2015.Cloning,expression characteristics and subcellular-location of heat shock transcription factor ZmHsf06 in Zea mays[J].Journal of Agricultural Biotechnology,23(1):41-51.)
张玉杰,张园园,张华宁,等.2018.小麦热激转录因子基因TaHsfA2e特性及耐热性功能初探[J].作物学报,44(12):1818-1828.(Zhang Y J,Zhang Y Y,Zhang H N,et al.2018.Characterization and regulatory roles in thermotolerance of wheat heat shock transcription factor gene TaHsfA2e[J].Acta Agronomica Sinica,44(12):1818-1828.)
赵立娜,张华宁,段硕楠,等.2017.玉米Zm Hsf04基因的克隆和特性及其对耐热性的调控[J].农业生物技术学报,25(9):1411-1422.(Zhao L N,Zhang H N,Duan S N,et al.2017.Cloning and characterization of Maize(Zea mays)Zm Hsf04 gene and its regulating role in thermotolerance[J].Journal of Agricultural Biotechnology,25(9):1411-1422.)
赵立娜,刘子会,段硕楠,等.2018.小麦热激转录因子基因Ta Hsf B2d的克隆和特性及其对耐热性调控[J].作物学报,44(1):53-62.(Zhao L N,Liu Z H,Duan S N,et al.2018.Cloning and characterization of heat shock transcription factor gene Ta Hsf B2d and its regulating role in thermotolerance[J].Acta Agronomica Sinica,44(1):53-62.)
Aranda M A,Escaler M,Thomas C L,et al.1999.A heat shock transcription factor in pea is differentially controlled by heat and virus replication[J].The Plant Journal,20(2):153-161.
Charng Y Y,Liu H C,Liu N Y,et al.2007.A heat-induced transcription factor,HsfA2,is required for extension of acquired thermotolerance in Arabidopsis[J].Plant Physiology,143(1):251-262.
Chauhan H,Khurana N,Agarwal P,et al.2013.A seed preferential heat shock transcription factor from wheat provides abiotic stress tolerance and yield enhancement in transgenic Arabidopsis under heat stress environment[J].PLoS One,8(11):e79577.
Duan S N,Liu B H,Zhang Y Y,et al.2019.Genome-wide identification and abiotic stress-responsive pattern of heat shock transcription factor family in Triticum aestivum L[J].BMC Genomics,20:1-20
Guo M,Liu H J.Ma X.2016.The plant heat stress transcription factors(HSFs):Structure,regulation and function in response to aboitic stresses[J].Frontiers in Plant Science,7(273):1-14.
Jiang Y L,Zheng Q Q,Chen L,et al.2018.Ectopic overexpression of maize heat shock transcription factor gene ZmHsf04 confers increased thermo and saltstress tolerance in transgenic Arabidopsis[J].Acta Physiologiae Plantarum,40(9):1-12.
Heerklotz D,D?ring P,Bonzelius F.2001.The balance of nuclear import and export determines the intrancellular distribution and function of tomato heat stress transcription factor Hsf A2[J].Molecular and Cell Biology,21(5):1759-1768.
Hu X J,Chen D D,Mclntyre C L,et al.2018.Heat shock factor C2a serves as a proactive mechanism for heat protection in developing grains in wheat via an ABA-mediated regulatory pathway[J].Plant Cell&Environment,41(1):79-98.
Ikeda M,Ohme-Takagi M.2009.A novel group of transcriptional repressors in Arabidopsis[J].Plant and Cell Physiology,50(5):970-975
Kang S,Eltahir E A B.2018.North China Plain threatened by deadly heatwaves due to climate change and irrigation[J].Nature Communications,9:2894-2903
Lin Y X,Jiang H Y,Chu Z X,et al.2011.Genome-wide identification,classification and analysis of heat shock transcription factor family in maize[J].BMC Genomics,12(1):76-89.
Li H C,Li G L,Liu Z H,et al.2014.Cloning,localization and expression of ZmHsf-Like in Zea mays[J].Journal of Integrative Agriculture,13(6):1230-1238.
Li H C,Zhang H N,Li G L,et al.2015.Expression of maize heat shock transcription factor gene ZmHsf06 enhances the thermotolerance and drought-stress tolerance of transgenic Arabidopsis[J].Functional and Plant Biology,42(11):1080-1090.
Liu H,Charng Y.2013.Common and distinct functions of Arabidopsis Class A1 and A2 heat shock factors in diverse abiotic stress responses and development[J].Plant Physiology,163(1):276-90.
Liu H C,Liao H T,Charng Y Y.2011.The role of class A1heat shock factors(HSFA1s)in response to heat and other stresses in Arabidopsis[J].Plant and Cell Environment,34(5):738-751.
Ma H,Wang C T,Yang B.et al.2016.CarHSFB2,a class Bheat shock transcription factor,is involved in different developmental processes and various stress responses in chickpea(Cicer Arietinum L.)[J].Plant Molecular Biology Reporter,34:1-14.
Mishra S K,Tripp J,Winkelhaus S,et al.2002.In the complex family of heat stress transcription factors,HsfA1has a unique role as master regulator of thermotolerance in tomato[J].Genes and Develepment,16:1555-1567.
Nishizawa A,Yabuta Y,Yoshida E,et al.2006.Arabidopsis heat shock transcription factor A2 as a key regulator in response to several types of environmental stress[J].The Plant Journal,48(4):535-547.
Nover L,Scharf K D,Gagliardi D,et al.1996.The HSF world:Classification and properties of plant heat stress transcription factors[J].Cell Stress Chaperones,1(4):215-223.
Nover L,Bharti K,D?ring P,et al.2001.Arabidopsis and the heat stress transcription factor world:How many heat stress transcription actors do we need?[J].Cell Stress Chaperones,6(3):177-189.
Ogawa D,Yamaguchi K,Nishiuchi T.2007.High-level overexpression of the Arabidopsis HsfA2 gene confers not only increased themotolerance but also salt/osmotic stress tolerance and enhanced callus growth[J].Journal of Experimental Botany,58(12):3373-3383.
Qin D D,Wu H Y,Peng H R,et al.2008.Heat stress-responsive transcriptome analysis in heat susceptible and tolerant wheat(Triticum aestivum L.)by using wheat genome array[J].BMC Genomics,9:432-450.
Scharf K D,Heider H,H?hfeld I,et al.1998.The tomato Hsf system:HsfA2 needs interaction with HsfA1 for efficient nuclear import and may be localized in cytoplasmic heat stress granules[J].Molecular and Cell Biology,18(4):2240-2251.
Schramm F,Ganguli A,Kiehlmann E,et al.2006.The heat stress transcription factor HSFA2 serves as a regulatory amplifier of a subset of genes in the heat stress response in Arabidopsis[J].Plant and Molecular Biology,60(5):759-772.
Shim D,Hwang J U,Lee J,et al.2009.Orthologs of the class A4 heat shock transcription factor HsfA4a confer cadmium tolerance in wheat and rice[J].Plant Cell,21(12):4031-4043.
Skylas DJ,Cordwell SJ,Hains PG,et al,2002.Heat shock of wheat during grain filing:Proteins associated with heattolerance[J].Journal of Cereal Science,35(2):175-188.
Tang R M,Zhu W J,Song X Y,et al.2016.Genome-wide identification and function analyses of heat shock transcription factor in potato[J].Frontiers in Plant Science,7:1-18.
Zhang S X,Xu Z S,Li P S,et al.2013.Overexpression of TaHSF3 in transgenic Arabidopsis enhances tolerance to extreme temperatures[J].Plant and Molecular Biology Report,(31):688-697.
Zhu X,Thalor S K,Takahashi Y,et al.2012.An inhibitory effect of the sequence-conserved upstream open-reading frame on the translation of the main open-reading frame of HsfB1 transcripts in Arabidopsis[J].Plant Cell and Environment,35(11):2014-2030.
Xue G P,Sadat S,Drenth J,2014.The heat shock factor family from Triticum aestivum in response to heat and other major abiotic stresses and their role in regulation of heat shock protein genes[J].Journal of Experimental Botany,65(2):539-557.
Xue G P,Drenth J,McIntyre C L.2015.TaHsfA6f is a transcriptional activator that regulates a suite of heat stress protection genes in wheat(Triticum aestivum L.)including previously unknown Hsf targets[J].Journal of Experimental Botany,66(3):1025-1039.
Wardlaw I F,Wrigley C W.1994.Heat tolerance in temperate cereals:An overview[J].Australian Journal of Plant Physiology,21(6):695-703.
李慧聪,李国良,郭秀林.2015.玉米热激转录因子基因(ZmHsf06)的克隆、表达和定位分析[J].农业生物技术学报,23(1):41-51.(Li H C,Li G L,Guo X L.2015.Cloning,expression characteristics and subcellular-location of heat shock transcription factor ZmHsf06 in Zea mays[J].Journal of Agricultural Biotechnology,23(1):41-51.)
张玉杰,张园园,张华宁,等.2018.小麦热激转录因子基因TaHsfA2e特性及耐热性功能初探[J].作物学报,44(12):1818-1828.(Zhang Y J,Zhang Y Y,Zhang H N,et al.2018.Characterization and regulatory roles in thermotolerance of wheat heat shock transcription factor gene TaHsfA2e[J].Acta Agronomica Sinica,44(12):1818-1828.)
赵立娜,张华宁,段硕楠,等.2017.玉米Zm Hsf04基因的克隆和特性及其对耐热性的调控[J].农业生物技术学报,25(9):1411-1422.(Zhao L N,Zhang H N,Duan S N,et al.2017.Cloning and characterization of Maize(Zea mays)Zm Hsf04 gene and its regulating role in thermotolerance[J].Journal of Agricultural Biotechnology,25(9):1411-1422.)
赵立娜,刘子会,段硕楠,等.2018.小麦热激转录因子基因Ta Hsf B2d的克隆和特性及其对耐热性调控[J].作物学报,44(1):53-62.(Zhao L N,Liu Z H,Duan S N,et al.2018.Cloning and characterization of heat shock transcription factor gene Ta Hsf B2d and its regulating role in thermotolerance[J].Acta Agronomica Sinica,44(1):53-62.)
Aranda M A,Escaler M,Thomas C L,et al.1999.A heat shock transcription factor in pea is differentially controlled by heat and virus replication[J].The Plant Journal,20(2):153-161.
Charng Y Y,Liu H C,Liu N Y,et al.2007.A heat-induced transcription factor,HsfA2,is required for extension of acquired thermotolerance in Arabidopsis[J].Plant Physiology,143(1):251-262.
Chauhan H,Khurana N,Agarwal P,et al.2013.A seed preferential heat shock transcription factor from wheat provides abiotic stress tolerance and yield enhancement in transgenic Arabidopsis under heat stress environment[J].PLoS One,8(11):e79577.
Duan S N,Liu B H,Zhang Y Y,et al.2019.Genome-wide identification and abiotic stress-responsive pattern of heat shock transcription factor family in Triticum aestivum L[J].BMC Genomics,20:1-20
Guo M,Liu H J.Ma X.2016.The plant heat stress transcription factors(HSFs):Structure,regulation and function in response to aboitic stresses[J].Frontiers in Plant Science,7(273):1-14.
Jiang Y L,Zheng Q Q,Chen L,et al.2018.Ectopic overexpression of maize heat shock transcription factor gene ZmHsf04 confers increased thermo and saltstress tolerance in transgenic Arabidopsis[J].Acta Physiologiae Plantarum,40(9):1-12.
Heerklotz D,D?ring P,Bonzelius F.2001.The balance of nuclear import and export determines the intrancellular distribution and function of tomato heat stress transcription factor Hsf A2[J].Molecular and Cell Biology,21(5):1759-1768.
Hu X J,Chen D D,Mclntyre C L,et al.2018.Heat shock factor C2a serves as a proactive mechanism for heat protection in developing grains in wheat via an ABA-mediated regulatory pathway[J].Plant Cell&Environment,41(1):79-98.
Ikeda M,Ohme-Takagi M.2009.A novel group of transcriptional repressors in Arabidopsis[J].Plant and Cell Physiology,50(5):970-975
Kang S,Eltahir E A B.2018.North China Plain threatened by deadly heatwaves due to climate change and irrigation[J].Nature Communications,9:2894-2903
Lin Y X,Jiang H Y,Chu Z X,et al.2011.Genome-wide identification,classification and analysis of heat shock transcription factor family in maize[J].BMC Genomics,12(1):76-89.
Li H C,Li G L,Liu Z H,et al.2014.Cloning,localization and expression of ZmHsf-Like in Zea mays[J].Journal of Integrative Agriculture,13(6):1230-1238.
Li H C,Zhang H N,Li G L,et al.2015.Expression of maize heat shock transcription factor gene ZmHsf06 enhances the thermotolerance and drought-stress tolerance of transgenic Arabidopsis[J].Functional and Plant Biology,42(11):1080-1090.
Liu H,Charng Y.2013.Common and distinct functions of Arabidopsis Class A1 and A2 heat shock factors in diverse abiotic stress responses and development[J].Plant Physiology,163(1):276-90.
Liu H C,Liao H T,Charng Y Y.2011.The role of class A1heat shock factors(HSFA1s)in response to heat and other stresses in Arabidopsis[J].Plant and Cell Environment,34(5):738-751.
Ma H,Wang C T,Yang B.et al.2016.CarHSFB2,a class Bheat shock transcription factor,is involved in different developmental processes and various stress responses in chickpea(Cicer Arietinum L.)[J].Plant Molecular Biology Reporter,34:1-14.
Mishra S K,Tripp J,Winkelhaus S,et al.2002.In the complex family of heat stress transcription factors,HsfA1has a unique role as master regulator of thermotolerance in tomato[J].Genes and Develepment,16:1555-1567.
Nishizawa A,Yabuta Y,Yoshida E,et al.2006.Arabidopsis heat shock transcription factor A2 as a key regulator in response to several types of environmental stress[J].The Plant Journal,48(4):535-547.
Nover L,Scharf K D,Gagliardi D,et al.1996.The HSF world:Classification and properties of plant heat stress transcription factors[J].Cell Stress Chaperones,1(4):215-223.
Nover L,Bharti K,D?ring P,et al.2001.Arabidopsis and the heat stress transcription factor world:How many heat stress transcription actors do we need?[J].Cell Stress Chaperones,6(3):177-189.
Ogawa D,Yamaguchi K,Nishiuchi T.2007.High-level overexpression of the Arabidopsis HsfA2 gene confers not only increased themotolerance but also salt/osmotic stress tolerance and enhanced callus growth[J].Journal of Experimental Botany,58(12):3373-3383.
Qin D D,Wu H Y,Peng H R,et al.2008.Heat stress-responsive transcriptome analysis in heat susceptible and tolerant wheat(Triticum aestivum L.)by using wheat genome array[J].BMC Genomics,9:432-450.
Scharf K D,Heider H,H?hfeld I,et al.1998.The tomato Hsf system:HsfA2 needs interaction with HsfA1 for efficient nuclear import and may be localized in cytoplasmic heat stress granules[J].Molecular and Cell Biology,18(4):2240-2251.
Schramm F,Ganguli A,Kiehlmann E,et al.2006.The heat stress transcription factor HSFA2 serves as a regulatory amplifier of a subset of genes in the heat stress response in Arabidopsis[J].Plant and Molecular Biology,60(5):759-772.
Shim D,Hwang J U,Lee J,et al.2009.Orthologs of the class A4 heat shock transcription factor HsfA4a confer cadmium tolerance in wheat and rice[J].Plant Cell,21(12):4031-4043.
Skylas DJ,Cordwell SJ,Hains PG,et al,2002.Heat shock of wheat during grain filing:Proteins associated with heattolerance[J].Journal of Cereal Science,35(2):175-188.
Tang R M,Zhu W J,Song X Y,et al.2016.Genome-wide identification and function analyses of heat shock transcription factor in potato[J].Frontiers in Plant Science,7:1-18.
Zhang S X,Xu Z S,Li P S,et al.2013.Overexpression of TaHSF3 in transgenic Arabidopsis enhances tolerance to extreme temperatures[J].Plant and Molecular Biology Report,(31):688-697.
Zhu X,Thalor S K,Takahashi Y,et al.2012.An inhibitory effect of the sequence-conserved upstream open-reading frame on the translation of the main open-reading frame of HsfB1 transcripts in Arabidopsis[J].Plant Cell and Environment,35(11):2014-2030.
Xue G P,Sadat S,Drenth J,2014.The heat shock factor family from Triticum aestivum in response to heat and other major abiotic stresses and their role in regulation of heat shock protein genes[J].Journal of Experimental Botany,65(2):539-557.
Xue G P,Drenth J,McIntyre C L.2015.TaHsfA6f is a transcriptional activator that regulates a suite of heat stress protection genes in wheat(Triticum aestivum L.)including previously unknown Hsf targets[J].Journal of Experimental Botany,66(3):1025-1039.
Wardlaw I F,Wrigley C W.1994.Heat tolerance in temperate cereals:An overview[J].Australian Journal of Plant Physiology,21(6):695-703.