烤烟成熟期烟叶GS同工酶活性与氮素运转的关系
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摘要
为研究不同氮效率烤烟品种成熟期叶片谷氨酰胺合成酶同工酶活性和相关生理指标的动态变化,深入理解烤烟叶片氮素代谢的生理生化机制。以氮效率不同的3个烤烟品种中烟100、K326和NC89为材料进行盆栽试验。在不同叶龄时期取第12片叶(自下向上数),采用Western blot方法,测定叶片的叶肉和主脉谷氨酰胺合成酶同工酶蛋白亚基含量,同时对叶片NH_4~+浓度、总氮、质外体NH_4~+浓度以及氨气挥发量进行测定。结果表明,烤烟叶片叶肉中的GS主要以GS2同工酶为主,其蛋白亚基含量随着叶龄的增长逐渐下降,而叶脉中GS1同工酶占主导地位,其蛋白亚基含量呈先升高后下降的趋势。在叶龄45～65 d,叶肉和主脉的GS同工酶活性均表现为NC89>K326>中烟100,且品种间差异达显著水平。叶肉和主脉中GS1同工酶活性与总氮和叶片铵浓度呈负相关,与质外体铵浓度和氨气挥发量呈正相关。而叶肉中GS2同工酶活性与总氮和叶片铵浓度呈正相关,与质外体铵浓度和氨气挥发量呈负相关。叶脉GS2活性仅与总氮和氨气挥发量有显著相关性。氮低效烤烟品种成熟期叶片中两种谷氨酰胺合成酶同工酶活性均较低,氮素转移和再利用能力差,导致植株吸收的氮素以氨气形式挥发损失量大,叶片衰老速度较快。而氮高效品种氮素同化和再利用能力较强,氨气挥发量小,易发生贪青晚熟。
A pot experiment was conducted to study the dynamic changes of glutamine synthetase isoenzyme activity and the related physiological indexes in leaves of flue-cured tobacco with different nitrogen efficiencies at maturity stage, and understand the physiological and biochemical mechanism of nitrogen metabolism in flue-cured tobacco leaves. Three different flue-cured tobacco varieties Zhongyan 100, K326, and NC89 with different nitrogen efficiencies were used as materials, and the 12 th leaves were taken at different leaf ages. Western blot method was used to determine the content of glutamine synthetase isoenzyme isoforms in leaf mesophyll and main veins. At the same time, the concentrations of NH_4~+, total nitrogen, apoplastic NH_4~+ and ammonia volatilization were determined. The results show, GS2 was the main form of GS isoenzyme in mesophyll of flue-cured tobacco leaves, the protein subunit content gradually decreased with the age of the leaf. The GS1 isoenzyme dominated in the veins, and its protein subunits showed a trend of increasing first and then decreasing. From 45 to 65 days of leaf age, the GS isoenzyme activities of leaf mesophyll and main vein were all expressed as NC89 > K326 > Zhongyan 100, and the difference among varieties was significant. The activities of GS1 in mesophyll and main veins were negatively correlated with total nitrogen and leaf ammonium concentrations, and positively correlated with apoplastic ammonium concentration and ammonia volatilization. The GS2 activity in mesophyll was positively correlated with total nitrogen and leaf ammonium, and negatively correlated with apoplastic ammonium concentration and ammonia volatilization. The GS2 activity of leaf veins was only significantly correlated with the total nitrogen and ammonia volatilization. The activity of two glutamine synthetase isoenzymes in leaves of nitrogen-inefficient flue-cured tobacco varieties at the mature period was lower, and the ability of nitrogen transfer and reuse was poor, resulting in most of the nitrogen absorbed by plants evaporated in the form of ammonia, and the leaves had a faster rate of aging. However, nitrogen efficient varieties had strong nitrogen reutilization capacity, so the ammonia volatilization was small and the aging was postponed.
A pot experiment was conducted to study the dynamic changes of glutamine synthetase isoenzyme activity and the related physiological indexes in leaves of flue-cured tobacco with different nitrogen efficiencies at maturity stage, and understand the physiological and biochemical mechanism of nitrogen metabolism in flue-cured tobacco leaves. Three different flue-cured tobacco varieties Zhongyan 100, K326, and NC89 with different nitrogen efficiencies were used as materials, and the 12 th leaves were taken at different leaf ages. Western blot method was used to determine the content of glutamine synthetase isoenzyme isoforms in leaf mesophyll and main veins. At the same time, the concentrations of NH_4~+, total nitrogen, apoplastic NH_4~+ and ammonia volatilization were determined. The results show, GS2 was the main form of GS isoenzyme in mesophyll of flue-cured tobacco leaves, the protein subunit content gradually decreased with the age of the leaf. The GS1 isoenzyme dominated in the veins, and its protein subunits showed a trend of increasing first and then decreasing. From 45 to 65 days of leaf age, the GS isoenzyme activities of leaf mesophyll and main vein were all expressed as NC89 > K326 > Zhongyan 100, and the difference among varieties was significant. The activities of GS1 in mesophyll and main veins were negatively correlated with total nitrogen and leaf ammonium concentrations, and positively correlated with apoplastic ammonium concentration and ammonia volatilization. The GS2 activity in mesophyll was positively correlated with total nitrogen and leaf ammonium, and negatively correlated with apoplastic ammonium concentration and ammonia volatilization. The GS2 activity of leaf veins was only significantly correlated with the total nitrogen and ammonia volatilization. The activity of two glutamine synthetase isoenzymes in leaves of nitrogen-inefficient flue-cured tobacco varieties at the mature period was lower, and the ability of nitrogen transfer and reuse was poor, resulting in most of the nitrogen absorbed by plants evaporated in the form of ammonia, and the leaves had a faster rate of aging. However, nitrogen efficient varieties had strong nitrogen reutilization capacity, so the ammonia volatilization was small and the aging was postponed.
引文
[1]史宏志,韩锦峰.烤烟碳氮代谢几个问题的探讨.烟草科技,1998,129(2):34-36.Shi H Z,Han J F.Discussion on several problems of carbon and nitrogen metabolism in flue-cured tobacco.Tobacco Sci Technol,1998,129(2):34-36(in Chinese).
[2]严小龙,张福锁.植物营养遗传.北京:中国农业出版社,1997.pp 78-106.Yan X L,Zhang F S.Genetics of Plant Nutrition.Beijing:China Agriculture Press,1997.pp 78-106(in Chinese).
[3]Hirel B,Andrieu B,Renard S,Quillere I,Chelle M,Pommel B,Fournier C.Physiology of maize II:Identification of physiological markers representative of the nitrogen status of maize(Zea mays)leaves during grain filling.Physiol Planta,2005,124:178-188.
[4]Fei H,Chaillou S,Hirel B,Polowick P,Mahon J,Vessey J.Effects of the overexpression of a soybean cytosolic glutamine synthetase gene(GS15)linked to organ-specific promoters on growth and nitrogen accumulation of pea plants supplied with ammonium.Plant Physiol Biochem,2006,44:543-550.
[5]Li X P,Zhao X Q,He X,Zhao G Y,Li B,Liu D C,Zhang A M,Zhang X Y,Tong Y P,Li Z S.Haplotype analysis of the genes encoding glutamine synthetase plastic isoforms and their association with nitrogen-use and yield-related traits in bread wheat.New Phytol,2011,189:449-458.
[6]邓扬悟.甜瓜谷氨酰胺合成酶基因的克隆和功能研究.上海交通大学博士学位论文,上海,2010.Deng Y W.Cloning and Characterization of Glutamine Synthetase Genes in Melon.PhD Dissertation of Shanghai JiaoTong University,Shanghai,China,2010(in Chinese with English abstract).
[7]卢素萍,李玉娥,刘鹏飞,赵铭钦,张渝婕,李志鹏.氮用量对南阳不同基因型烤烟烟叶品质的影响.江西农业大学学报,2016,38:67-73.Lu S P,Li Y E,Liu P F,Zhao M Q,Zhang Y J,Li Z P.Effects of nitrogen fertilizer on quality of different genotypes of flue-cured tobacco in Nanyang county.Acta Agric Univ Jiangxiensis,2016,38:67-73(in Chinese with English abstract).
[8]崔昌范,王书凯,宋立君.烤烟新品种K326和NC89特征特性及配套栽培技术规范.延边大学农学学报,1999,21(1):70-75.Cui C F,Wang S K,Song L J.Characteristics of flue-cured tobacco new varieties K326 and NC89 and their cultivation techniques.J Agric Sci Yanbian Univ,1999,21(1):70-75(in Chinese).
[9]武云杰,李飞,杨铁钊,张小全.氮素营养水平对衰老期烟叶氮代谢的影响及品种间差异.中国烟草学报,2014,20(4):41-47.Wu Y J,Li F,Yang T Z,Zhang X Q.Effects of nitrogen nutrition on nitrogen metabolism in flue-cured tobacco leaves and differences among varieties.Acta Tabac Sin,2014,20(4):41-47(in Chinese with English abstract).
[10]黄培堂.分子克隆实验指南.北京:化学工业出版社,2007.pp155-157.Huang P T.The Condensed Protocols from Molecular Cloning:a Laboratory Manual.Beijing:Chemical Industry Press,2007.pp155-157(in Chinese).
[11]Wang X,Wei Y,Shi L,Ma X,Theg S.New isoforms and assembly of glutamine synthetase in the leaf of wheat(Triticum aestivum L.).J Exp Bot,2015,66:6827-6834.
[12]邹琦.植物生理学实验指导.北京:中国农业出版社,2000.pp56-89.Zou Q.The Guidance of Plant Physiology Experiment.Beijing:China Agriculture Press,2000.pp 56-89(in Chinese).
[13]段旺军,杨铁钊,刘化冰,张小全,戴亚,李东亮.烟叶氨气补偿点的品种间差异及其与氮素代谢的关系研究.植物营养与肥料学报,2011,17:419-424.Duan W J,Yang T Z,Liu H B,Zhang X Q,Dai Y,Li D L.Differences in NH3 compensation point among tobacco(Nicotiana tabacum L.)cultivars and its relationship with nitrogen metabolism.J Plant Nutr Ferti,2011,17:419-424(in Chinese with English abstract).
[14]武云杰,杨铁钊,张小全,李飞,李丽华.不同烤烟品种烟叶衰老期氨气挥发及其与氮素代谢的相关性.中国农业科学,2013,46:4027-4034.Wu Y J,Yang T Z,Zhang X Q,Li F,Li L H.Study on correlation between ammonia volatilization and nitrogen metabolism during the tobacco leaves senescence.Sci Agric Sin,2013,46:4027-4034(in Chinese with English abstract).
[15]Nielsen K H,Schjoerring J K.Regulation of apoplastic NH4+concentration in leaves of oilseed rape.Plant Physiol,1998,118:1361-1368.
[16]范江,杨继洪,王守旗,肖志新,梁梅,李海平,邱尧.打顶对烤烟生长和各器官烟碱积累分配的影响.作物研究,2016,30(1):36-40.Fan J,Yang J H,Wang S Q,Xiao Z X,Liang M,Li H P,Qiu Y.Effect of topping on growth of flue-cured tobacco and nicotine accumulation and distribution in different organs.Crop Res,2016,30(1):36-40(in Chinese with English abstract).
[17]王云华,欧吉权,王志强,周忠新,欧阳敏,张楚富.黄瓜(Cucumis sativus L.)子叶发育过程中谷氨酰胺合成酶同工酶的变化.武汉大学学报(理学版),2005,51:517-520.Wang Y H,Ou J Q,Wang Z Q,Zhou Z X,Ou Y M,Zhang C F.Changes of glutamine synthetase isozymes during the development of cucumber(Cucumis sativus L.)cotyledons.J Wuhan Univ(Nat Sci Edn),2005,51:517-520(in Chinese with English abstract).
[18]Rueda-López M,Crespillo R,Cánovas F M,Avila C.Differential regulation of two glutamine synthetase genes by a single Dof transcription factor.Plant J,2008,56:73-85.
[19]Stanulovi?V S,Garcia R M,Labruyère W T,Ruijter JM,Hakvoort T B M,Lamers W H.The 3'-UTR of the glutamine-synthetase gene interacts specifically with upstream regulatory elements,contains m RNA-instbility elements and is involved in glutamine sensing.Biochimie,2006,88:1255-1264.
[20]Kumar R,Taware R,Gaur V S,Guru S K,Kumar A.Influence of nitrogen on the expression of TaDof1 transcription factor in wheat and its relationship with photo synthetic and ammonium assimilating efficiency.Mol Biol Rep,2009,36:2209-2216.
[2]严小龙,张福锁.植物营养遗传.北京:中国农业出版社,1997.pp 78-106.Yan X L,Zhang F S.Genetics of Plant Nutrition.Beijing:China Agriculture Press,1997.pp 78-106(in Chinese).
[3]Hirel B,Andrieu B,Renard S,Quillere I,Chelle M,Pommel B,Fournier C.Physiology of maize II:Identification of physiological markers representative of the nitrogen status of maize(Zea mays)leaves during grain filling.Physiol Planta,2005,124:178-188.
[4]Fei H,Chaillou S,Hirel B,Polowick P,Mahon J,Vessey J.Effects of the overexpression of a soybean cytosolic glutamine synthetase gene(GS15)linked to organ-specific promoters on growth and nitrogen accumulation of pea plants supplied with ammonium.Plant Physiol Biochem,2006,44:543-550.
[5]Li X P,Zhao X Q,He X,Zhao G Y,Li B,Liu D C,Zhang A M,Zhang X Y,Tong Y P,Li Z S.Haplotype analysis of the genes encoding glutamine synthetase plastic isoforms and their association with nitrogen-use and yield-related traits in bread wheat.New Phytol,2011,189:449-458.
[6]邓扬悟.甜瓜谷氨酰胺合成酶基因的克隆和功能研究.上海交通大学博士学位论文,上海,2010.Deng Y W.Cloning and Characterization of Glutamine Synthetase Genes in Melon.PhD Dissertation of Shanghai JiaoTong University,Shanghai,China,2010(in Chinese with English abstract).
[7]卢素萍,李玉娥,刘鹏飞,赵铭钦,张渝婕,李志鹏.氮用量对南阳不同基因型烤烟烟叶品质的影响.江西农业大学学报,2016,38:67-73.Lu S P,Li Y E,Liu P F,Zhao M Q,Zhang Y J,Li Z P.Effects of nitrogen fertilizer on quality of different genotypes of flue-cured tobacco in Nanyang county.Acta Agric Univ Jiangxiensis,2016,38:67-73(in Chinese with English abstract).
[8]崔昌范,王书凯,宋立君.烤烟新品种K326和NC89特征特性及配套栽培技术规范.延边大学农学学报,1999,21(1):70-75.Cui C F,Wang S K,Song L J.Characteristics of flue-cured tobacco new varieties K326 and NC89 and their cultivation techniques.J Agric Sci Yanbian Univ,1999,21(1):70-75(in Chinese).
[9]武云杰,李飞,杨铁钊,张小全.氮素营养水平对衰老期烟叶氮代谢的影响及品种间差异.中国烟草学报,2014,20(4):41-47.Wu Y J,Li F,Yang T Z,Zhang X Q.Effects of nitrogen nutrition on nitrogen metabolism in flue-cured tobacco leaves and differences among varieties.Acta Tabac Sin,2014,20(4):41-47(in Chinese with English abstract).
[10]黄培堂.分子克隆实验指南.北京:化学工业出版社,2007.pp155-157.Huang P T.The Condensed Protocols from Molecular Cloning:a Laboratory Manual.Beijing:Chemical Industry Press,2007.pp155-157(in Chinese).
[11]Wang X,Wei Y,Shi L,Ma X,Theg S.New isoforms and assembly of glutamine synthetase in the leaf of wheat(Triticum aestivum L.).J Exp Bot,2015,66:6827-6834.
[12]邹琦.植物生理学实验指导.北京:中国农业出版社,2000.pp56-89.Zou Q.The Guidance of Plant Physiology Experiment.Beijing:China Agriculture Press,2000.pp 56-89(in Chinese).
[13]段旺军,杨铁钊,刘化冰,张小全,戴亚,李东亮.烟叶氨气补偿点的品种间差异及其与氮素代谢的关系研究.植物营养与肥料学报,2011,17:419-424.Duan W J,Yang T Z,Liu H B,Zhang X Q,Dai Y,Li D L.Differences in NH3 compensation point among tobacco(Nicotiana tabacum L.)cultivars and its relationship with nitrogen metabolism.J Plant Nutr Ferti,2011,17:419-424(in Chinese with English abstract).
[14]武云杰,杨铁钊,张小全,李飞,李丽华.不同烤烟品种烟叶衰老期氨气挥发及其与氮素代谢的相关性.中国农业科学,2013,46:4027-4034.Wu Y J,Yang T Z,Zhang X Q,Li F,Li L H.Study on correlation between ammonia volatilization and nitrogen metabolism during the tobacco leaves senescence.Sci Agric Sin,2013,46:4027-4034(in Chinese with English abstract).
[15]Nielsen K H,Schjoerring J K.Regulation of apoplastic NH4+concentration in leaves of oilseed rape.Plant Physiol,1998,118:1361-1368.
[16]范江,杨继洪,王守旗,肖志新,梁梅,李海平,邱尧.打顶对烤烟生长和各器官烟碱积累分配的影响.作物研究,2016,30(1):36-40.Fan J,Yang J H,Wang S Q,Xiao Z X,Liang M,Li H P,Qiu Y.Effect of topping on growth of flue-cured tobacco and nicotine accumulation and distribution in different organs.Crop Res,2016,30(1):36-40(in Chinese with English abstract).
[17]王云华,欧吉权,王志强,周忠新,欧阳敏,张楚富.黄瓜(Cucumis sativus L.)子叶发育过程中谷氨酰胺合成酶同工酶的变化.武汉大学学报(理学版),2005,51:517-520.Wang Y H,Ou J Q,Wang Z Q,Zhou Z X,Ou Y M,Zhang C F.Changes of glutamine synthetase isozymes during the development of cucumber(Cucumis sativus L.)cotyledons.J Wuhan Univ(Nat Sci Edn),2005,51:517-520(in Chinese with English abstract).
[18]Rueda-López M,Crespillo R,Cánovas F M,Avila C.Differential regulation of two glutamine synthetase genes by a single Dof transcription factor.Plant J,2008,56:73-85.
[19]Stanulovi?V S,Garcia R M,Labruyère W T,Ruijter JM,Hakvoort T B M,Lamers W H.The 3'-UTR of the glutamine-synthetase gene interacts specifically with upstream regulatory elements,contains m RNA-instbility elements and is involved in glutamine sensing.Biochimie,2006,88:1255-1264.
[20]Kumar R,Taware R,Gaur V S,Guru S K,Kumar A.Influence of nitrogen on the expression of TaDof1 transcription factor in wheat and its relationship with photo synthetic and ammonium assimilating efficiency.Mol Biol Rep,2009,36:2209-2216.