小麦叶绿素酶及脱镁叶绿素水解酶活性测定条件的优化
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摘要
叶绿素降解是植物叶片衰老最明显的标志。为探明小麦叶绿素降解途径中叶绿素酶和脱镁叶绿素水解酶2个关键酶反应的最佳环境条件,对其反应的最佳环境温度及时间进行了探讨。结果表明,小麦叶片叶绿素酶的最适反应温度和最佳反应时间分别是40℃和80 min,脱镁叶绿素水解酶的最佳反应温度和最佳反应时间分别是35℃和90 min。该研究结果可为2种酶活性的测定提供重要参考。
Chlorophyll degradation is the most obvious sign in leaf senescence. To find out the catalysis character of two key enzymes in chlorophyll degradation pathway, chlorophyllase(CLH)and pheophytinase(PPH), the optimum environmental condition for determination of CLH and PPH activity in wheat leaves was studied in aspect of temperrature and reaction time. The results showed that the optimum reaction temperrature and time for CLH were 40 ℃ and 80 min, and its for PPH were 35 ℃ and 90 min. The results can provide important reference for the determination for two enzymes.
Chlorophyll degradation is the most obvious sign in leaf senescence. To find out the catalysis character of two key enzymes in chlorophyll degradation pathway, chlorophyllase(CLH)and pheophytinase(PPH), the optimum environmental condition for determination of CLH and PPH activity in wheat leaves was studied in aspect of temperrature and reaction time. The results showed that the optimum reaction temperrature and time for CLH were 40 ℃ and 80 min, and its for PPH were 35 ℃ and 90 min. The results can provide important reference for the determination for two enzymes.
引文
[1]张雪茹,冯美臣,李广信,等.低温胁迫下冬小麦叶绿素含量高光谱估测[J].山西农业科学,2017,45(4):526-529.
[2]KUSABA M,ITO H,MORITA R,et al. Rice NON-YELLOW COLORING1 is involved in light-harvesting complex II and grana degradation during leaf senescence[J]. Plant Cell,2007,19:1362-1375.
[3] HARPAZ-SAAD S,AZOULAY T,ARAZI T,et al. Chlorophyllase is a rate-limiting enzyme in chlorophyll catabolism and is posttranslationally regulated[J]. Plant Cell,2007,19:1007-1022.
[4] SHIMODA Y,ITO H,TANAKA A. Arabidopsis STAY-GREEN,Mendel's green cotyledon gene,encodes magnesium-dechelatase[J].Plant Cell,2016,28:2147-2160.
[5]CHEN J,ZHU X,REN J,et al. Suppressor of overexpression of CO1negatively regulates dark-induced leaf degreening and senescence by directly repressing pheophytinase and other senescence-associated genes in Arabidopsis[J]. Plant Physiol,2017,173:1881-1891.
[6] LIRA B S,ROSADO D,ALMEIDA J. Pheophytinase knockdown impacts carbon metabolism and nutraceutical content under normal growth conditions in tomato[J]. Plant Cell Physiol,2016,57:642-653.
[7]ZHANG J,YU G H. Functional characterization and hormonal regulation of the Pheophytinase gene LpPPH controlling leaf senescence in perennial ryegrass[J]. J Exp Bot,2016,67:935-945.
[8]王伟,徐跃进,万正杰.黄瓜衰老过程中叶绿素降解相关酶基因c DNA片段的克隆与表达初步研究[J].园艺学报,2011,38(6):1104-1110.
[9] PRU譕INSK譧A,ANDERS I,AUBRY S,et al. In vivo participation of red chlorophyll catabolite reductase in chlorophyll breakdown[J].The Plant Cell,2007,19:369.
[10]AZOULAY SHEMER T,HARPAZ-SAAD S,BELAUSOV E,et al.Citrus chlorophyllase dynamics at ethylene-induced fruit color-break:A study of chlorophyllase expression,posttranslational processing kinetics,and in situ intracellular localization[J]. Plant Physiol,2008,148:108.
[11]GUPTA S,GUPTA S,KUMAR N. Role of chlorophyllase in chlorophyll homeostasis and post-harvest breakdown in Piper betle L.leaf[J]. Indian J Biochem Bio,2011,48:353-360.
[12] KAEWSUKSAENG S,URANO Y,AIAMLA-OR S,et al. Effect of UV-B irradiation on chlorophyll-degrading enzyme activities and postharvest quality in stored lime(Citrus latifolia Tan.)fruit[J].Postharvest Biol Technol,2011,61:124-130.
[13] SYTYKIEWICZ H,SPRAWKA I,CZERNIEWICZ P,et al. Biochemical characterisation of chlorophyllase from leaves of selected Prunus species-a comparative study[J]. Acta Biochim Pol,2013,60:457-465.
[14]唐蕾.银杏叶生长过程中的叶绿素酶活性变化[J].植物生理学报,2002,38(6):561-563.
[15]刘彩云,刘洪祥,杜传印.白肋烟和马里兰烟叶绿素酶活性测定及其基因表达的实时定量PCR分析[J].中国农学通报,2011,27(3):139-142.
[16]刘振,唐蕾,王海鸥,等.低温贮藏和短波紫外线辐照对芥蓝采后叶绿素降解及脱镁叶绿素酶活性的影响[J].食品工业科技,2013,34(15):332-334.
[17]AIAMLA-OR S,NAKAJIMA T,SHIGYO M. Pheophytinase activity and gene expression of chlorophyll-degrading enzymes relating to UV-B treatment in postharvest broccoli(Brassica oleracea L. Italica Group)florets[J]. Postharvest Biol Technol,2012,63:60-66.
[18] MCFEETERS R F,CHICHESTER C O,WHITAKER J R. Purification and properties of chlorophyllase from Ailanthus altissima(Tree-of-Heaven)[J]. Plant Physiol,1971,47:609.
[19] M魱NGUEZ-MOSQUERA M I,GANDUL-ROJAS B. Measurement of chlorophyllase activity in olive fruit(Olea europaea)[J]. Journal of Biochemistry,1994,116:263-268.
[20]李小玲,华智锐.外源茉莉酸甲酯对盐胁迫下黄芩种子萌发及幼苗生理特性的影响[J].山西农业科学,2016,44(11):1603-1607.
[21] FANG Z Y J,SOLOMOS T,FANG Z Y. Chlorophyllase activities and chlorophyll degradation during leaf senescence in non-yellowing mutant and wild type of Phaseolus vulgaris L.[J]. J Exp Bot,1998,49:503-510.
[22] JACOB-WILK D,HOLLAND D,GOLDSCHMIDT E E. Chlorophyll breakdown by chlorophyllase:isolation and functional expression of the Chlase1 gene from ethylene-treated Citrus fruit and its regulation during development[J]. Plant J,2010,20:653-661.
[23] FERNANDEZ-LOPEZ J A,ALMELA L,ALMANSA M S,et al.Partial purification and properties of chlorophyllase from chlorotic Citrus limon leaves[J]. Phytochemistry,1992,31:447-449.
[24]TREBITSH T,GOLDSCHMIDT E E,RIOV J. Ethylene induces de novo synthesis of chlorophyllase,a chlorophyll degrading enzyme,in Citrus fruit peel[J]. Proceedings of the National Academy of Sciences,1993,90:9441.
[25] SCHELBERT S,AUBRY S,BURLA B. Pheophytin pheophorbide Hydrolase(Pheophytinase)is involved in chlorophyll breakdown during leaf senescence in Arabidopsis[J]. Plant Cell,2009,21:767-785.
[2]KUSABA M,ITO H,MORITA R,et al. Rice NON-YELLOW COLORING1 is involved in light-harvesting complex II and grana degradation during leaf senescence[J]. Plant Cell,2007,19:1362-1375.
[3] HARPAZ-SAAD S,AZOULAY T,ARAZI T,et al. Chlorophyllase is a rate-limiting enzyme in chlorophyll catabolism and is posttranslationally regulated[J]. Plant Cell,2007,19:1007-1022.
[4] SHIMODA Y,ITO H,TANAKA A. Arabidopsis STAY-GREEN,Mendel's green cotyledon gene,encodes magnesium-dechelatase[J].Plant Cell,2016,28:2147-2160.
[5]CHEN J,ZHU X,REN J,et al. Suppressor of overexpression of CO1negatively regulates dark-induced leaf degreening and senescence by directly repressing pheophytinase and other senescence-associated genes in Arabidopsis[J]. Plant Physiol,2017,173:1881-1891.
[6] LIRA B S,ROSADO D,ALMEIDA J. Pheophytinase knockdown impacts carbon metabolism and nutraceutical content under normal growth conditions in tomato[J]. Plant Cell Physiol,2016,57:642-653.
[7]ZHANG J,YU G H. Functional characterization and hormonal regulation of the Pheophytinase gene LpPPH controlling leaf senescence in perennial ryegrass[J]. J Exp Bot,2016,67:935-945.
[8]王伟,徐跃进,万正杰.黄瓜衰老过程中叶绿素降解相关酶基因c DNA片段的克隆与表达初步研究[J].园艺学报,2011,38(6):1104-1110.
[9] PRU譕INSK譧A,ANDERS I,AUBRY S,et al. In vivo participation of red chlorophyll catabolite reductase in chlorophyll breakdown[J].The Plant Cell,2007,19:369.
[10]AZOULAY SHEMER T,HARPAZ-SAAD S,BELAUSOV E,et al.Citrus chlorophyllase dynamics at ethylene-induced fruit color-break:A study of chlorophyllase expression,posttranslational processing kinetics,and in situ intracellular localization[J]. Plant Physiol,2008,148:108.
[11]GUPTA S,GUPTA S,KUMAR N. Role of chlorophyllase in chlorophyll homeostasis and post-harvest breakdown in Piper betle L.leaf[J]. Indian J Biochem Bio,2011,48:353-360.
[12] KAEWSUKSAENG S,URANO Y,AIAMLA-OR S,et al. Effect of UV-B irradiation on chlorophyll-degrading enzyme activities and postharvest quality in stored lime(Citrus latifolia Tan.)fruit[J].Postharvest Biol Technol,2011,61:124-130.
[13] SYTYKIEWICZ H,SPRAWKA I,CZERNIEWICZ P,et al. Biochemical characterisation of chlorophyllase from leaves of selected Prunus species-a comparative study[J]. Acta Biochim Pol,2013,60:457-465.
[14]唐蕾.银杏叶生长过程中的叶绿素酶活性变化[J].植物生理学报,2002,38(6):561-563.
[15]刘彩云,刘洪祥,杜传印.白肋烟和马里兰烟叶绿素酶活性测定及其基因表达的实时定量PCR分析[J].中国农学通报,2011,27(3):139-142.
[16]刘振,唐蕾,王海鸥,等.低温贮藏和短波紫外线辐照对芥蓝采后叶绿素降解及脱镁叶绿素酶活性的影响[J].食品工业科技,2013,34(15):332-334.
[17]AIAMLA-OR S,NAKAJIMA T,SHIGYO M. Pheophytinase activity and gene expression of chlorophyll-degrading enzymes relating to UV-B treatment in postharvest broccoli(Brassica oleracea L. Italica Group)florets[J]. Postharvest Biol Technol,2012,63:60-66.
[18] MCFEETERS R F,CHICHESTER C O,WHITAKER J R. Purification and properties of chlorophyllase from Ailanthus altissima(Tree-of-Heaven)[J]. Plant Physiol,1971,47:609.
[19] M魱NGUEZ-MOSQUERA M I,GANDUL-ROJAS B. Measurement of chlorophyllase activity in olive fruit(Olea europaea)[J]. Journal of Biochemistry,1994,116:263-268.
[20]李小玲,华智锐.外源茉莉酸甲酯对盐胁迫下黄芩种子萌发及幼苗生理特性的影响[J].山西农业科学,2016,44(11):1603-1607.
[21] FANG Z Y J,SOLOMOS T,FANG Z Y. Chlorophyllase activities and chlorophyll degradation during leaf senescence in non-yellowing mutant and wild type of Phaseolus vulgaris L.[J]. J Exp Bot,1998,49:503-510.
[22] JACOB-WILK D,HOLLAND D,GOLDSCHMIDT E E. Chlorophyll breakdown by chlorophyllase:isolation and functional expression of the Chlase1 gene from ethylene-treated Citrus fruit and its regulation during development[J]. Plant J,2010,20:653-661.
[23] FERNANDEZ-LOPEZ J A,ALMELA L,ALMANSA M S,et al.Partial purification and properties of chlorophyllase from chlorotic Citrus limon leaves[J]. Phytochemistry,1992,31:447-449.
[24]TREBITSH T,GOLDSCHMIDT E E,RIOV J. Ethylene induces de novo synthesis of chlorophyllase,a chlorophyll degrading enzyme,in Citrus fruit peel[J]. Proceedings of the National Academy of Sciences,1993,90:9441.
[25] SCHELBERT S,AUBRY S,BURLA B. Pheophytin pheophorbide Hydrolase(Pheophytinase)is involved in chlorophyll breakdown during leaf senescence in Arabidopsis[J]. Plant Cell,2009,21:767-785.
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