不同叶位和离体失水时间对烤烟叶片保水能力的影响
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摘要
为阐明叶位和离体时间对烤烟叶片保水能力的影响。以2个田间抗旱性不同的烤烟品种豫烟6号和农大202的离体叶片为材料,采用离体叶片自然失水法,研究了不同叶位和离体时间对叶片失水率、抗氧化酶活性、激素和抗逆胁迫响应基因表达的影响。结果表明:5个不同叶位的失水率为离体0~24 h较高,24 h后失水速率逐渐变缓,叶位间差异不显著,农大202失水率显著高于豫烟6号;离体叶片内抗氧化酶(SOD、POD、CAT)活性、可溶性蛋白含量、脱落酸(ABA)含量均在离体24 h时达到最高值,丙二醛(MDA)含量也在24 h后增速变缓;4个胁迫响应基因(AREB、CDPK2、ERD10C、LEA5)相对表达量也在24 h达到最高,说明烤烟叶片离体24 h生理代谢反应最强,2个品种在离体24 h各指标差异最大。因此,烤烟叶片保水力与取样叶位关系不大,离体叶片失水24 h为测定烟草保水力的合适时间。
In order to elucidate the effect of stalk positions and natural dehydration after priming on water retention capacity of flue-cured tobacco leaves, evaluation index of drought resistance of tobacco leaves was investigated. Effects of different stalk positions and natural dehydration after priming on leaf water loss rate, antioxidant enzyme activity, hormone and stress resistance gene expression were studied through natural dehydration method with two flue-cured tobacco varieties, Yuyan 6 and Nongda 202 of different drought resistance. Results showed that the water loss rate of leaves of 5 different stalk positions was higher during 0-24 h, and gradually slowed down after 24 h, and there was no significant difference among different stalk positions, but water loss rate of Nongda 202 was significantly higher than that of Yuyan 6. Antioxidant enzyme(SOD, POD, CAT) activity, soluble protein content, abscisic acid(ABA) content in excised leaves reached the highest value 24 hours in vitro, and Malondialdehyde(MDA) content also slowed down after 24 hours. The relative expression of four stress responsive genes(AREB, CDPK2, ERD10 C, LEA5) reached the highest level after 24 hours. Physiological metabolism of excised flue-cured tobacco leaves was the strongest 24 hours in vitro, and the difference between the indexes of the two varieties was the greatest 24 hours in vitro. There was little relationship between water retention capacity and leaf position in excised flue-cured tobacco leaves, and leaf water loss 24 hours in vitro was the appropriate time to determine water retention capacity of excised tobacco leaves.
In order to elucidate the effect of stalk positions and natural dehydration after priming on water retention capacity of flue-cured tobacco leaves, evaluation index of drought resistance of tobacco leaves was investigated. Effects of different stalk positions and natural dehydration after priming on leaf water loss rate, antioxidant enzyme activity, hormone and stress resistance gene expression were studied through natural dehydration method with two flue-cured tobacco varieties, Yuyan 6 and Nongda 202 of different drought resistance. Results showed that the water loss rate of leaves of 5 different stalk positions was higher during 0-24 h, and gradually slowed down after 24 h, and there was no significant difference among different stalk positions, but water loss rate of Nongda 202 was significantly higher than that of Yuyan 6. Antioxidant enzyme(SOD, POD, CAT) activity, soluble protein content, abscisic acid(ABA) content in excised leaves reached the highest value 24 hours in vitro, and Malondialdehyde(MDA) content also slowed down after 24 hours. The relative expression of four stress responsive genes(AREB, CDPK2, ERD10 C, LEA5) reached the highest level after 24 hours. Physiological metabolism of excised flue-cured tobacco leaves was the strongest 24 hours in vitro, and the difference between the indexes of the two varieties was the greatest 24 hours in vitro. There was little relationship between water retention capacity and leaf position in excised flue-cured tobacco leaves, and leaf water loss 24 hours in vitro was the appropriate time to determine water retention capacity of excised tobacco leaves.
引文
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[9]Clarke J M,Romagosa I,Jana S,et al.Relationship of excised-leaf water loss rate and yield of durum wheat in diverse environments.[J].Canadian Journal of Plant Science,1989,69(4):1075-1081.
[10]Clarke J M,Townley smith T F.Heritability and relationship to yield of excised-leaf water retention in durum wheat[J].Crop Science,1986,26(2):289-292.
[11]Winter S R,Musik K B Porter.Evaluation of screening technique for breeding drought-resistant winter wheat[J].Crop Science,1988,28:512–516.
[12]武斌,李新海,肖木辑,等.53份玉米自交系的苗期耐旱性分析[J].中国农业科学,2007,40(4):665-676.WU Bin,LI Xinhai,XIAO Muji,et al.Genetic variation in fiftythree maize inbred lines in relation to drought tolerance at seeding stage[J].Scientia Agricultura Sinica,2007,40(4):665-676.
[13]王丹丹,唐章林,荆蓉蓉,等.甘蓝型油菜遗传图谱构建及苗期耐旱相关性状的QTL定位[J].西南大学学报(自然科学版),2014,(7):8-16.WANG Dandan,TANG Zhanglin,JING Rongrong.et al.Construction of a molecular genetic map of rapeseed(Brassica napus L.)and QTL mapping of its drought tolerance-related traits[J].Journal of Southwest University(Natural Science Edition),2014,(7):8-16.
[14]H a n Y,Wa n g W,S u n J,e t a l.P o p u l u s e u p h r a t i c a X T H overexpression enhances salinity tolerance by the development of leaf succulence in transgenic tobacco plants[J].Journal of Experimental Botany,2013,64(14):4225.
[15]Lechthaler S,Robert E M R,TonnéN,et al.Rhizophoraceae mangrove saplings use hypocotyl and leaf water storage capacity to cope with soil water salinity changes[J].Frontiers in Plant Science,2016,7:1-13.
[16]Li Q,Yu B,Gao Y,et al.Cinnamic acid pretreatment mitigates chilling stress of cucumber leaves through altering antioxidant enzyme activity.[J].Journal of Plant Physiology,2011,168(9):927-934.
[17]李合生.植物生理生化实验原理和技术[M].北京:高等教育出版社,2000:280-283.LI Hesheng.Principles and techniques of plant physiological biochemical experiment[M].Beijing:Higher education press,2000:280-283.
[18]Bradford M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding[J].Analytical Biochemistry,1976,72:248–254.
[19]Zhao J,Li G,Yi G X,et al.Comparison between conventional indirect competitive enzyme-linked immunosorbent assay(ic ELISA)and simplified ic ELISA for small molecules[J].Analytica Chimica Acta,2006,571(1):79-85.
[20]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method[J].Methods,2001,25:402-408.
[21]Tang Q Y,Zhang C X.Data Processing System(DPS)software with experimental design,statistical analysis and data mining developed for use in entomological research.Insect Science.2013,20(2):254-260.
[22]Verslues P E,Agarwal M,Katiyaragarwal S,et al.Methods and concepts in quantifying resistance to drought,salt and freezing,abiotic stresses that affect plant water status.[J].Plant Journal,2006,45(4):523-539.
[23]Leprince O,Deltour R,Thorpe P C,et al.The role of free radicals and radical processing systems in loss of desiccation tolerance in germinating maize(Zea mays L.)[J].New Phytologist,2010,116(4):573-580.
[24]Leprince O,Harren F J M,Buitink J,et al.Metabolic dysfunction and unabated respiration precede the loss of membrane integrity during dehydration of germinating radicles[J].Plant Physiology,2000,122(2):597-608.
[25]Walters C,Pammenter N W,Berjak P,et al.Desiccation damage,accelerated ageing and respiration in desiccation tolerant and sensitive seeds[J].Seed Science Research,2001,11(2):135-148.
[26]Fujita Y,Yoshida T,Yamaguchishinozaki K.Pivotal role of the AREB/ABF-Sn RK2 pathway in ABRE-mediated transcription in response to osmotic stress in plants[J].Physiologia Plantarum,2013,147(1):15.
[27]Urao T,Katagiri T,Mizoguchi T,et al.Two genes that encode Ca-dependent protein kinases are induced by drought and highsalt stresses in Arabidopsis thaliana[J].Molecular Genetics and Genomics,1994,244(4):331-340.
[28]Yang L,Li W,Jiang S,et al.Group 5 LEA protein,Zm LEA5C,enhance tolerance to osmotic and low temperature stresses in transgenic tobacco and yeast[J].Plant Physiology&Biochemistry,2014,84:22.
[29]Liu X,Liu S,Wu J,et al.Overexpression of Arachis hypogaea NAC3 in tobacco enhances dehydration and drought tolerance by increasing superoxide scavenging[J].Plant Physiology&Biochemistry,2013,70(1):354-359.
[30]Chen Guoxiong,Takao Komatsud,Ma Jianfeng et al.An ATPbinding cassette subfamily G full transporter is essential for the retention of leaf water in both wild barley and rice[J].Proceedings of the National Academy of Sciences,2011,108(30):12354–12359.
[31]Karolina M.Jarzyniak,Micha Jasin ski.Membrane transporters and drought resistance–a complex issue[J].Frontiers in Plant Science,2014,5:e687.
[32]刘国顺.烟草栽培学[M].北京:中国农业出版社,2003:221-222.LIU Guoshun.Tobacco Cultivation[M].Beijing:China Agriculture Press,2003:221-222.
[33]赵阳,王树声,张亚丽,等.增加烟草一级和二级侧根是抵御干旱的生理机制[J].植物营养与肥料学报,2017,23(2):548-555.ZHAO Yang,WANG Shusheng,ZHANG Yali,et al.Role of increasing first-and second-order lateral roots of tobacco for drought tolerance[J].Journal of Plant Nutrition and Fertilizer,2017,32(2):548-555.
[2]梁太波,王建伟,尹启生,等.水分胁迫对烤烟氨同化关键酶活性和生物量的影响[J].中国烟草学报,2012,18(3):50-54.LIANG Taibo,WANG Jianwei,YIN Qisheng,et al.Effects of water stress on ammonium assimilation and biomass of flue-cured tobacco[J].Acta Tabacaria Sinica,2012,18(3):50-54.
[3]马新蕾,房燕,王玉军,等.十个烤烟品种的抗旱性鉴定[J].中国烟草学报,2005,11(5):26-30.MA Xinlei,FANG Yan,WANG Yujun,et al.The evaluation of drought resistance of ten tobacco cultivars.Acta Tabacaria Sinica,2005,11(5):26-30.
[4]刘贞琦,伍贤进,刘振业.土壤水分对烟草光合生理特性影响的研究[J].中国烟草学报,1995,2(1):44-49.LIU Zhenqi,WU Xianjin,LIU Zhenye.The effects of soil water content on photosynthetic characteristics of tobacco[J].Acta Tabacaria Sinica,1995,2(1):44-49.
[5]裴斌,张光灿,张淑勇,等.土壤干旱胁迫对沙棘叶片光合作用和抗氧化酶活性的影响[J].生态学报,2013,(05):1386-1396.PEI Bin,ZHANG Guangcan,ZHANG Shuyong,et al.Effects of soil drought stress on photosynthetic characteristics and antioxidant enzyme activities in Hippophae Rhamnoides Linn.Seeding[J].Acta Ecologica Sinica,2013,33(5):1386-1396.
[6]任庆成,陈秀华,张生杰,等.不同烤烟品种抗旱生理特征比较研究[J].西北植物学报,2009,29(10):2019-2025.REN Qingcheng,CHEN Xiuhua,ZHANG Shengjie,et al.Comparison of drought resistance characteristics of different fluecured tobacoo varieties[J].Acta Botanica Boreali Occidentalia Sinica,2009,29(10):2019-2025.
[7]Clarke J M,Mccaig T N.Excised-leaf water retention capability as an indicator of drought resistance of Triticum genotypes.[J].Canadian Journal of Plant Science,1982,62(3):571-578.
[8]Clarke J M,Mccaig T N.Evaluation of techniques for screening for drought resistance in wheat.[J].Crop Science,1980,22(3):503-506.
[9]Clarke J M,Romagosa I,Jana S,et al.Relationship of excised-leaf water loss rate and yield of durum wheat in diverse environments.[J].Canadian Journal of Plant Science,1989,69(4):1075-1081.
[10]Clarke J M,Townley smith T F.Heritability and relationship to yield of excised-leaf water retention in durum wheat[J].Crop Science,1986,26(2):289-292.
[11]Winter S R,Musik K B Porter.Evaluation of screening technique for breeding drought-resistant winter wheat[J].Crop Science,1988,28:512–516.
[12]武斌,李新海,肖木辑,等.53份玉米自交系的苗期耐旱性分析[J].中国农业科学,2007,40(4):665-676.WU Bin,LI Xinhai,XIAO Muji,et al.Genetic variation in fiftythree maize inbred lines in relation to drought tolerance at seeding stage[J].Scientia Agricultura Sinica,2007,40(4):665-676.
[13]王丹丹,唐章林,荆蓉蓉,等.甘蓝型油菜遗传图谱构建及苗期耐旱相关性状的QTL定位[J].西南大学学报(自然科学版),2014,(7):8-16.WANG Dandan,TANG Zhanglin,JING Rongrong.et al.Construction of a molecular genetic map of rapeseed(Brassica napus L.)and QTL mapping of its drought tolerance-related traits[J].Journal of Southwest University(Natural Science Edition),2014,(7):8-16.
[14]H a n Y,Wa n g W,S u n J,e t a l.P o p u l u s e u p h r a t i c a X T H overexpression enhances salinity tolerance by the development of leaf succulence in transgenic tobacco plants[J].Journal of Experimental Botany,2013,64(14):4225.
[15]Lechthaler S,Robert E M R,TonnéN,et al.Rhizophoraceae mangrove saplings use hypocotyl and leaf water storage capacity to cope with soil water salinity changes[J].Frontiers in Plant Science,2016,7:1-13.
[16]Li Q,Yu B,Gao Y,et al.Cinnamic acid pretreatment mitigates chilling stress of cucumber leaves through altering antioxidant enzyme activity.[J].Journal of Plant Physiology,2011,168(9):927-934.
[17]李合生.植物生理生化实验原理和技术[M].北京:高等教育出版社,2000:280-283.LI Hesheng.Principles and techniques of plant physiological biochemical experiment[M].Beijing:Higher education press,2000:280-283.
[18]Bradford M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding[J].Analytical Biochemistry,1976,72:248–254.
[19]Zhao J,Li G,Yi G X,et al.Comparison between conventional indirect competitive enzyme-linked immunosorbent assay(ic ELISA)and simplified ic ELISA for small molecules[J].Analytica Chimica Acta,2006,571(1):79-85.
[20]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method[J].Methods,2001,25:402-408.
[21]Tang Q Y,Zhang C X.Data Processing System(DPS)software with experimental design,statistical analysis and data mining developed for use in entomological research.Insect Science.2013,20(2):254-260.
[22]Verslues P E,Agarwal M,Katiyaragarwal S,et al.Methods and concepts in quantifying resistance to drought,salt and freezing,abiotic stresses that affect plant water status.[J].Plant Journal,2006,45(4):523-539.
[23]Leprince O,Deltour R,Thorpe P C,et al.The role of free radicals and radical processing systems in loss of desiccation tolerance in germinating maize(Zea mays L.)[J].New Phytologist,2010,116(4):573-580.
[24]Leprince O,Harren F J M,Buitink J,et al.Metabolic dysfunction and unabated respiration precede the loss of membrane integrity during dehydration of germinating radicles[J].Plant Physiology,2000,122(2):597-608.
[25]Walters C,Pammenter N W,Berjak P,et al.Desiccation damage,accelerated ageing and respiration in desiccation tolerant and sensitive seeds[J].Seed Science Research,2001,11(2):135-148.
[26]Fujita Y,Yoshida T,Yamaguchishinozaki K.Pivotal role of the AREB/ABF-Sn RK2 pathway in ABRE-mediated transcription in response to osmotic stress in plants[J].Physiologia Plantarum,2013,147(1):15.
[27]Urao T,Katagiri T,Mizoguchi T,et al.Two genes that encode Ca-dependent protein kinases are induced by drought and highsalt stresses in Arabidopsis thaliana[J].Molecular Genetics and Genomics,1994,244(4):331-340.
[28]Yang L,Li W,Jiang S,et al.Group 5 LEA protein,Zm LEA5C,enhance tolerance to osmotic and low temperature stresses in transgenic tobacco and yeast[J].Plant Physiology&Biochemistry,2014,84:22.
[29]Liu X,Liu S,Wu J,et al.Overexpression of Arachis hypogaea NAC3 in tobacco enhances dehydration and drought tolerance by increasing superoxide scavenging[J].Plant Physiology&Biochemistry,2013,70(1):354-359.
[30]Chen Guoxiong,Takao Komatsud,Ma Jianfeng et al.An ATPbinding cassette subfamily G full transporter is essential for the retention of leaf water in both wild barley and rice[J].Proceedings of the National Academy of Sciences,2011,108(30):12354–12359.
[31]Karolina M.Jarzyniak,Micha Jasin ski.Membrane transporters and drought resistance–a complex issue[J].Frontiers in Plant Science,2014,5:e687.
[32]刘国顺.烟草栽培学[M].北京:中国农业出版社,2003:221-222.LIU Guoshun.Tobacco Cultivation[M].Beijing:China Agriculture Press,2003:221-222.
[33]赵阳,王树声,张亚丽,等.增加烟草一级和二级侧根是抵御干旱的生理机制[J].植物营养与肥料学报,2017,23(2):548-555.ZHAO Yang,WANG Shusheng,ZHANG Yali,et al.Role of increasing first-and second-order lateral roots of tobacco for drought tolerance[J].Journal of Plant Nutrition and Fertilizer,2017,32(2):548-555.