光温处理对小豆苗期生理性状及叶绿素合成前体的影响
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摘要
探讨不同低温和光照条件下小豆苗期的冷害发生机制及引起初生叶黄化和叶绿素合成的受阻位点,旨在为小豆耐寒新品种选育及栽培提供理论依据。选择2个对温度和光照反应不同的日本小豆品种,利用人工气候室再现小豆苗期的低温障碍,研究低温遮光处理(昼夜10～13°C, 2%遮光) 18 d和28 d对小豆苗期H_2O_2、SOD、CAT、APX、叶绿素的影响;利用植物生长箱再现小豆苗期的黄化障碍,研究不同的低温处理长度(1 d、3 d、5 d、7 d;10°C, 50μmol m~(–2) s~(–1))和暗处理长度(25°C,黑暗1 d、3 d、5 d、7 d)对绿化后(24 h、25°C、62.5μmol m~(–2) s~(–1))叶绿素合成能力及受阻位点的影响。苗期小豆耐低温和不耐低温品种的最大差异是长期低温遮光处理的H_2O_2含量和SOD活性。长期低温遮光处理后不耐低温品种的H_2O_2含量是耐低温品种的约66倍,但随着绿化处理时间的延长,抗氧化酶活性和叶绿素含量急剧下降直至8 h后消失。与低温处理相比,暗处理才是造成叶绿素合成能力差异的主要原因。对叶绿素合成中间产物的研究表明,从ALA向Proto Ⅸ的转化可能受阻,最终导致叶绿素合成受阻,叶绿素含量下降。说明H_2O_2含量和SOD活性可能与小豆苗期耐冷性关系更密切。引起小豆苗期叶绿素合成受阻位点是Proto Ⅸ的转化。
The aim of this study was to discuss the mechanism of chilling injury and the blocked site of chlorophyll synthesis in primary leaf, providing theoretical basis for breeding and cultivation of cold-resistant cultivars. Two Japanese adzuki bean varieties with different temperature and light reactions were used to study the effects of short-term(18 d) and long-term(28 d) low temperature shading treatments(10–13°C, 2% shading) on H_2O_2, SOD, CAT, APX, and Chl of seedlings in the artificial climate chamber, and the effects of the treatment durations of low temperature(1 d, 3 d, 5 d, 7 d, 10°C, 50 μmol m~(–2) s~(–1)) and dark(25°C,1 d, 3 d, 5 d, 7 d) under chlorophyll synthesis blocked site(25°C, and under illumiation at 62.5 μmol m~(–2) s~(–1) for 24 h). The content of H_2O_2 and activity of SOD had significant difference between cold resistant and susceptible varieties during seedling stage. The content of H_2O_2 in susceptible variety was 66 folds of the resistant one, and with the greening treatment antioxidant activity and content of chlorophyll were rapidly dropped until totally vanished after 8 h. The main cause of difference in chlorophyll synthesis between the two varieties was the dark treatment but not the low temperature treatment. It was suggested that the transformation from δ-ALA to Proto Ⅸ might be blocked in chloroplast stroma, which eventually inhibited chlorophyll synthesis and decreased chlorophyll content. It is suggested that H_2O_2 content and SOD activity may be more closely related to the cold tolerance of adzuki bean at seedling stage. The transformation of Proto Ⅸ is the blocking site in chlorophyll synthesis which causes etiolated seedlings of adzuki bean.
The aim of this study was to discuss the mechanism of chilling injury and the blocked site of chlorophyll synthesis in primary leaf, providing theoretical basis for breeding and cultivation of cold-resistant cultivars. Two Japanese adzuki bean varieties with different temperature and light reactions were used to study the effects of short-term(18 d) and long-term(28 d) low temperature shading treatments(10–13°C, 2% shading) on H_2O_2, SOD, CAT, APX, and Chl of seedlings in the artificial climate chamber, and the effects of the treatment durations of low temperature(1 d, 3 d, 5 d, 7 d, 10°C, 50 μmol m~(–2) s~(–1)) and dark(25°C,1 d, 3 d, 5 d, 7 d) under chlorophyll synthesis blocked site(25°C, and under illumiation at 62.5 μmol m~(–2) s~(–1) for 24 h). The content of H_2O_2 and activity of SOD had significant difference between cold resistant and susceptible varieties during seedling stage. The content of H_2O_2 in susceptible variety was 66 folds of the resistant one, and with the greening treatment antioxidant activity and content of chlorophyll were rapidly dropped until totally vanished after 8 h. The main cause of difference in chlorophyll synthesis between the two varieties was the dark treatment but not the low temperature treatment. It was suggested that the transformation from δ-ALA to Proto Ⅸ might be blocked in chloroplast stroma, which eventually inhibited chlorophyll synthesis and decreased chlorophyll content. It is suggested that H_2O_2 content and SOD activity may be more closely related to the cold tolerance of adzuki bean at seedling stage. The transformation of Proto Ⅸ is the blocking site in chlorophyll synthesis which causes etiolated seedlings of adzuki bean.
引文
[1]徐宁,程须珍,王丽侠,王素华,刘长友,孙蕾,梅丽.用于中国小豆种质资源遗传多样性分析SSR分子标记筛选及应用.作物学报,2009,35:219-227.Xu N,Cheng X Z,Wang L X,Wang S H,Liu C Y,Sun L,Mei L.Screening and application of SSR molecular markers for genetic diversity analysis of Chinese adzuki bean germplasm resources.Acta Agron Sin,2009,35:219-227(in Chinese with English abstract).
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[6]李進才,趙習コウ,松井鋳一郎.光ストレスおよび遮光栽培におけるCattleyaとCymbidium葉の抗酸化酵素活性および色素含量の変化.園学雑,2001,70:372-379.Li J C,Zhao X K,Matsui I.Effect of light stress and shading cultivation on antioxidant enzyme activity and pigment content of cattleya and cymbidium leaves.Jpn Hortic J,2001,70:372-379(in Japanese).
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[9]Erich W,Laties G G.Quantification of hydrogen peroxide in plant extracts by the chemiluminescence reaction with luminal.Hytochemistry,1982,21:827-831.
[10]沈利星.雑草科学実験法.第5項植物の抗酸化活性測定法.東京:日本雑草学会発行,2001.pp 296-298.Shen L X.Methods of Scientific Experiment of Weeds and Determination of Antioxidant Activity of Plants.Tokyo:The Japanese Weed Society Publishers,2001.pp 296-298(in Japanese).
[11]加藤荣.光合成研究法.东京:日本共立出版社,1981.pp40-41.Katou E.Research Methods of Photosynthesis.Tokyo:Japan Co-publisher,1981.pp 40-41(in Japanese).
[12]何寧,小嶋道之,黒澤聪,加藤清明.低温遮光処理アズキ初生葉の過酸化水素含量と抗酸化酵素活性に及ぼす影響.日本作物学会記事,2006,75:360-365.He N,Kojima M Y,Kurosawa A,Kato K.Effects of chilling and shading on hydrogen peroxide content and activity of oxidation-inhibiting enzymes in the primary leaves of adzuki beans.Jpn J Crop Sci,2006,75:360-365(in Japanese).
[13]Harpaz-Saad S,Azoulay T,Arazi T,Ben-Yaakov E,Mett A,H?rtensteiner S,Gidoni D,Gal-On A,Goldschmidt E E,Eyal Y.Chlorophyllase is a rate-limiting enzyme in chlorophyll catabolism and is posttranslationally regulated.Plant Cell,2007,19:1007-1022.
[14]Von Gromoff E D,Alawady A,Meinecke L,Grimm B,Beck C F.Heme,a plastid-derived regulator of nuclear gene expression in chlamydomonas.Plant Cell,2008,20:552-567.
[15]王平荣,张帆涛,高家旭,孙小秋,邓晓建.高等植物叶绿素生物合成的研究进展.西北植物学报,2009,29:629-636.Wang P R,Zhang F T,Gao J X,Sun X Q,Deng X J.An overview of chlorophyll biosynthesis in higher plants.Acta Bot BorealiOccident Sin,2009,29:629-636(in Chinese with English abstract).
[16]Okuda T Y,Matuda A,Yamanaka S,Sagisaka S.Abrupt increase in the level of hydrogen peroxide in leaves of winter wheat is caused by cold treatment.Plant Physiol,1991,97:1265-1267.
[17]Fridovich I.Biological effects of the superoxide radicals.Arch Biochem Biophys,1986,247:1-11.
[18]重岡成.活性酸素代謝の応答機構.光合成生物の活性酸素代謝の応答機構.生物工学会誌,2001,79:303-322.Shigeoka N R.The response mechanism of photosynthetic biological reactive oxygen metabolism.Jpn J Bioengineer,2001,79:303-322(in Japanese).
[19]Azevedo R A,Alas R M,Smith R J,Lea P J.Response of antioxidant enzymes to transfer from elevated carbon dioxide to air and ozone fumigation,in the leaves and roots of wild-type and a catalase-deficient mutant of barley.Physiol Plant,1998,104:280-292.
[20]Foyer C H,Mullineaux P.Causes of photooxidative stress and amelioration of defense systems in plants.Acta Ophthalmol,1994,44:276-315.
[21]宮尾光恵,水澤直樹.強光環境から身を守る植物の防御機構.化学と生物,1999,37:396-400.Miyao H E,Mizusawa N K.The plant defense mechanism obtained from the strong light environment.Jpn Chem Biol,1999,37:396-400(in Japanese).
[22]王宝增.叶绿素降解代谢的研究进展.生物学教学,2010,35(2):7-9.Wang B Z.Research progress of chlorophyll degradation metabolism.Biol Teach,2010,35(2):7-9(in Chinese with English abstract).
[23]Gopal K,Pattanayak G K,Biswal A K,Reddy V S,Tripathy B C.Light-dependent regulation of chlorophyll b biosynthesis in chlorophyllide a oxygenase overexpressing tobacco plants.Biochem Biophys Res Commun,2005,326:466-471.
[24]田中歩,平島真澄,田中亮一.クロロフェル代謝と植物の生育.化学と生物,2004,42:2-26.Tanaka A,Hirasima M S,Tanaka L Y.The development of chlorophyll metabolism and plant.Chem Biol,2004,42:2-26(in Japanese with English abstract).
[2]村田吉平.北海道における作物育種.札幌:北海道協同組合通信社,1998.pp 139-155.Murata K P.Hokkaido Plant Breeding.Sapporo:Hokkaido Agricultural Cooperatives Publisher,1998.pp 139-155(in Japanese).
[3]新免輝夫.現代植物生理学.东京:環境応答,1991.pp142-158.Shinme T.Modern Plant Physiology.Tokyo:Institute of Society Press,1991.pp 142-158(in Japanese).
[4]Northen R T.Home Orchid Growing,3rd edn.New York:Van Nostrand Reinhold,1970.pp 19-28.
[5]横田明穂.植物ストレスにおける応答.東京:学会出版センター,2002.pp 209-224.Yokota M H.Introduction to Plant Molecular Physiology.Tokyo:Institute of Society Press,2002.pp 209-224(in Japanese).
[6]李進才,趙習コウ,松井鋳一郎.光ストレスおよび遮光栽培におけるCattleyaとCymbidium葉の抗酸化酵素活性および色素含量の変化.園学雑,2001,70:372-379.Li J C,Zhao X K,Matsui I.Effect of light stress and shading cultivation on antioxidant enzyme activity and pigment content of cattleya and cymbidium leaves.Jpn Hortic J,2001,70:372-379(in Japanese).
[7]Salin W L.Toxic oxygen species and protective systems of the chloroplast.Physiol Plant,1988,72:681-689.
[8]Shen W K,Nada S,Tachibana S.Oxygen radical generation in chilled leaves of cucumber(Cucumis sativus L.)cultivars with different tolerances to chilling temperatures.Engei Gakkai Zasshi,2008,68:780-787.
[9]Erich W,Laties G G.Quantification of hydrogen peroxide in plant extracts by the chemiluminescence reaction with luminal.Hytochemistry,1982,21:827-831.
[10]沈利星.雑草科学実験法.第5項植物の抗酸化活性測定法.東京:日本雑草学会発行,2001.pp 296-298.Shen L X.Methods of Scientific Experiment of Weeds and Determination of Antioxidant Activity of Plants.Tokyo:The Japanese Weed Society Publishers,2001.pp 296-298(in Japanese).
[11]加藤荣.光合成研究法.东京:日本共立出版社,1981.pp40-41.Katou E.Research Methods of Photosynthesis.Tokyo:Japan Co-publisher,1981.pp 40-41(in Japanese).
[12]何寧,小嶋道之,黒澤聪,加藤清明.低温遮光処理アズキ初生葉の過酸化水素含量と抗酸化酵素活性に及ぼす影響.日本作物学会記事,2006,75:360-365.He N,Kojima M Y,Kurosawa A,Kato K.Effects of chilling and shading on hydrogen peroxide content and activity of oxidation-inhibiting enzymes in the primary leaves of adzuki beans.Jpn J Crop Sci,2006,75:360-365(in Japanese).
[13]Harpaz-Saad S,Azoulay T,Arazi T,Ben-Yaakov E,Mett A,H?rtensteiner S,Gidoni D,Gal-On A,Goldschmidt E E,Eyal Y.Chlorophyllase is a rate-limiting enzyme in chlorophyll catabolism and is posttranslationally regulated.Plant Cell,2007,19:1007-1022.
[14]Von Gromoff E D,Alawady A,Meinecke L,Grimm B,Beck C F.Heme,a plastid-derived regulator of nuclear gene expression in chlamydomonas.Plant Cell,2008,20:552-567.
[15]王平荣,张帆涛,高家旭,孙小秋,邓晓建.高等植物叶绿素生物合成的研究进展.西北植物学报,2009,29:629-636.Wang P R,Zhang F T,Gao J X,Sun X Q,Deng X J.An overview of chlorophyll biosynthesis in higher plants.Acta Bot BorealiOccident Sin,2009,29:629-636(in Chinese with English abstract).
[16]Okuda T Y,Matuda A,Yamanaka S,Sagisaka S.Abrupt increase in the level of hydrogen peroxide in leaves of winter wheat is caused by cold treatment.Plant Physiol,1991,97:1265-1267.
[17]Fridovich I.Biological effects of the superoxide radicals.Arch Biochem Biophys,1986,247:1-11.
[18]重岡成.活性酸素代謝の応答機構.光合成生物の活性酸素代謝の応答機構.生物工学会誌,2001,79:303-322.Shigeoka N R.The response mechanism of photosynthetic biological reactive oxygen metabolism.Jpn J Bioengineer,2001,79:303-322(in Japanese).
[19]Azevedo R A,Alas R M,Smith R J,Lea P J.Response of antioxidant enzymes to transfer from elevated carbon dioxide to air and ozone fumigation,in the leaves and roots of wild-type and a catalase-deficient mutant of barley.Physiol Plant,1998,104:280-292.
[20]Foyer C H,Mullineaux P.Causes of photooxidative stress and amelioration of defense systems in plants.Acta Ophthalmol,1994,44:276-315.
[21]宮尾光恵,水澤直樹.強光環境から身を守る植物の防御機構.化学と生物,1999,37:396-400.Miyao H E,Mizusawa N K.The plant defense mechanism obtained from the strong light environment.Jpn Chem Biol,1999,37:396-400(in Japanese).
[22]王宝增.叶绿素降解代谢的研究进展.生物学教学,2010,35(2):7-9.Wang B Z.Research progress of chlorophyll degradation metabolism.Biol Teach,2010,35(2):7-9(in Chinese with English abstract).
[23]Gopal K,Pattanayak G K,Biswal A K,Reddy V S,Tripathy B C.Light-dependent regulation of chlorophyll b biosynthesis in chlorophyllide a oxygenase overexpressing tobacco plants.Biochem Biophys Res Commun,2005,326:466-471.
[24]田中歩,平島真澄,田中亮一.クロロフェル代謝と植物の生育.化学と生物,2004,42:2-26.Tanaka A,Hirasima M S,Tanaka L Y.The development of chlorophyll metabolism and plant.Chem Biol,2004,42:2-26(in Japanese with English abstract).