茶树越冬芽在休眠与萌发时期的物质交流变化及其分子调控
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摘要
为揭示不同萌发物候型茶树的休眠机制,以特早生茶树品种龙井43和中生茶树品种碧云为材料,利用钙黄素处理茶树茎段,检测越冬芽在休眠与萌发时期与其他器官的物质交流情况。利用同源比对鉴定胼胝质水解相关基因,并分析其序列特征及在冬季不同时期的表达模式。结果表明,越冬芽在茶树生长阶段和休眠阶段都存在着与着生茎段和母叶间的物质交流;从茶树越冬芽休眠形成到解除的不同时期,其物质交流存在"强-弱-强"的变化规律,但龙井43的与碧云相比存在较短的物质交流减弱时期;两种茶树的物质交流变化模式与鉴定到的茶树胼胝质水解正向调控相关基因CsGLU1的表达模式密切相关;启动子序列分析进一步证实CsGLU1启动子区有多个与激素信号以及低温和休眠响应相关转录因子结合的保守序列。茶树越冬芽在休眠和非休眠状态下都存在与茎和母叶之间的物质交流,且物质交流强弱与茶树越冬芽休眠状态改变密切相关。CsGLU1可能是参与胼胝质水解调控,改变茶树越冬芽物质交流水平,进而影响茶树休眠状态的关键基因。这对明确茶树越冬芽休眠状态变化和深入揭示不同萌发物候型茶树休眠机理有重要意义。
Early-sprouting cultivar Longjing 43 and late-sprouting cultivar Biyun were employed in this study to disclose the dormancy mechanism in tea plant with different sprouting phenophases. The levels of substance exchange were monitored by detecting the fluorescence signal in calcein treated overwintering buds. The glucanase related genes were identified by sequence homology analysis. Their characteristics and expression patterns during different time of winter were further analyzed. The substance exchanges were detected either in stem-bud unit or mother leaf-stem unit. From the initial formation to release in dormancy, the substance exchange in overwintering buds showed strong-weak-strong variation patterns in both cultivars, however, the duration of weak exchange stage was much shorter in Longjing 43 than in Biyun. Moreover, there was a close correlation between substance exchange variation pattern and the expression pattern of CsGLU1, a gene identified in tea plant with positive callose hydrolyzation activity. On the basis of promoter sequence analysis, plenty of transcription factor binding sequences related to hormone signaling, cold stimulation and dormancy regulation were found in CsGLU1 promoter region, which validates its putative functions in dormancy regulation. In conclusion, overwintering buds of tea plant have substance exchange with stem and mother leaf both in dormancy and non-dormancy status, furthermore, the variation of substance exchange level was consistent to the changes of dormancy status. CsGLU1 is a callose hydrolyzation related gene, which is supposed to be a key gene regulating tea plant dormancy transition through affecting the substance exchange in overwintering buds. The study provides meaningful results for understanding the changes of dormancy statuses in overwintering buds and deeply exploring the regulation mechanism in tea plant with different sprouting phenophase.
Early-sprouting cultivar Longjing 43 and late-sprouting cultivar Biyun were employed in this study to disclose the dormancy mechanism in tea plant with different sprouting phenophases. The levels of substance exchange were monitored by detecting the fluorescence signal in calcein treated overwintering buds. The glucanase related genes were identified by sequence homology analysis. Their characteristics and expression patterns during different time of winter were further analyzed. The substance exchanges were detected either in stem-bud unit or mother leaf-stem unit. From the initial formation to release in dormancy, the substance exchange in overwintering buds showed strong-weak-strong variation patterns in both cultivars, however, the duration of weak exchange stage was much shorter in Longjing 43 than in Biyun. Moreover, there was a close correlation between substance exchange variation pattern and the expression pattern of CsGLU1, a gene identified in tea plant with positive callose hydrolyzation activity. On the basis of promoter sequence analysis, plenty of transcription factor binding sequences related to hormone signaling, cold stimulation and dormancy regulation were found in CsGLU1 promoter region, which validates its putative functions in dormancy regulation. In conclusion, overwintering buds of tea plant have substance exchange with stem and mother leaf both in dormancy and non-dormancy status, furthermore, the variation of substance exchange level was consistent to the changes of dormancy status. CsGLU1 is a callose hydrolyzation related gene, which is supposed to be a key gene regulating tea plant dormancy transition through affecting the substance exchange in overwintering buds. The study provides meaningful results for understanding the changes of dormancy statuses in overwintering buds and deeply exploring the regulation mechanism in tea plant with different sprouting phenophase.
引文
[1]Ruttink T,Arend M,Morreel K,Storme V,Rombaut s S,Fromm J,Bhalerao R,Boerjan W,Rohde A.A molecular timetable for apical bud formation and dormancy induction in poplar.Plant Cell,2007,19:2370–2390
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[9]晏嫦妤,李家贤,黄华林,何雨媚.茶树休眠的研究进展.安徽农业科学,2012,40:10387–10389Yan C Y,Li J X,Huang H L,He Y M.Research progress of tea plant dormancy.J Anhui Agric Sci,2012,40:10387–10389(in Chinese with English abstract)
[10]钱利生,沈生荣,潘根生.茶树新梢内源激素的HPLC分析及日变化.茶叶科学,1996,(2):135–139Qian L S,Shen S R,Pan G S.HPLC analysis and diurnal variation of the endogenous hormones in shoots of tea plants.J Tea Sci,1996,(2):135–139(in Chinese with English abstract)
[11]黄亚辉,粟本文,郑红发,曾贞,刘霞林.茶树春梢萌动期间内源激素含量的变化.植物生理学通讯,2001,37:306–307Huang Y H,Su B W,Zheng H F,Zeng Z,Liu X L.Changes of endohormone in prouting shoot of tea plant.Plant Physiol Commun,2001,37:306–307(in Chinese with English abstract)
[12]禹利君,史云峰,肖海云,刘富知,刘仲华.不同物候型茶树内源GA3和ABA的变化及其对腋芽萌发调控的影响.作物学报,2008,34:277–283Yu L J,Shi Y F,Xiao H Y,Liu F Z,Liu Z H.Dynamic changes of endogenous GA3 and ABA contents in tea culticars with different phenological characters and their impact on the regulation axillary buds sprouting.Acta Agron Sin,2008,34:277–283(in Chinese with English abstract)
[13]Wang X,Hao X,Ma C,Cao H,Yue C,Wang L,Zeng J,Yang Y.Identification of differential gene expression profiles between winter dormant and sprouting axillary buds in tea plant(Camellia sinensis)by suppression subtractive hybridization.Tree Genet Genom,2014,10:1149–1159
[14]王新超,杨亚军,马春雷,金基强,曹红利.茶树细胞周期蛋白基因的克隆与表达.西北植物学报,2011,31:2365–2372Wang X C,Yang Y J,Ma C L,Jin J Q,Cao H L.Cloning and expression analysis of cyclin gene(Cs CYC1)of tea plant.Acta Bot Boreali-Occident Sin,2011,31:2365–2372(in Chinese with English abstract)
[15]王新超,马春雷,杨亚军,金基强,马建强,曹红利.茶树细胞周期蛋白依赖激酶(Cs CDK)基因c DNA全长克隆与分析.园艺学报,2012,39:333–342Wang X C,Ma C L,Yang Y J,Jin J Q,Ma J Q,Cao H L.c DNA cloning and expression analysis of cyclin-dependent kinase(Cs CDK)gene in tea plant.Acta Hort Sin,2012,39:333–342(in Chinese with English abstract)
[16]Doxey A C,Yaish M W,Moffatt B A,Griffith M,Mc Conkey B J.Functional divergence in the Arabidopsis beta-1,3-glucanase gene family inferred by phylogenetic reconstruction of expression states.Mol Biol Evol,2007,24:1045–1055
[17]Rinne P L,Paul L K,Vahala J,Kangasjarvi J,van der Schoot C.Axillary buds are dwarfed shoots that tightly regulate GA pathway and GA-inducible 1,3-beta-glucanase genes during branching in hybrid aspen.J Exp Bot,2016,67:5975–5991
[18]Chang S,Puryear J,Cairney J.A simple and efficient method for isolating RNA from pine trees.Plant Mol Biol Rep,1993,11:113–116
[19]Hao X,Horvath D P,Chao W S,Yang Y,Wang X,Xiao B.Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant(Camellia sinensis(L.)O.Kuntze).Int J Mol Sci,2014,15:22155–22172
[20]Jian L C,Li P H,Sun L H,Chen T H H.Alterations in ultrastructure and subcellular localization of Ca2+in poplar apical bud cells during the induction of dormancy.J Exp Bot,1997,48:1195–1207
[21]刘芳,王家艳,王晓丽,周蕴薇.细叶百合鳞茎在低温解除休眠过程中茎尖细胞超微结构的变化.园艺学报,2013,40:1110–1118Liu F,Wang J Y,Wang X L,Zhou Y W.The apical bud cell ultra-structure changes of Lilium pumilum bulbs during breaking dormancy under refrigerated condition.Acta Hort Sin,2013,40:1110–1118(in Chinese with English abstract)
[22]Ruonala R,Rinne P L,Kangasj?rvi J,van der Schoot C.CENL1expression in the rib meristem affects stem elongation and the transition to dormancy in Populus.Plant Cell,2008,20:59–74
[23]Knox J P,Benitez-Alfonso Y.Roles and regulation of plant cell walls surrounding plasmodesmata.Curr Opin Plant Biol,2014,22:93–100
[24]Paul L K,Rinne P L,van der Schoot C.Refurbishing the plasmodesmal chamber:a role for lipid bodies?Front Plant Sci,2014.5:40
[25]Nagar P K,Kumar A.Changes in endogenous gibberellin activity during winter dormancy in tea(Camellia sinensis(L.)O.Kuntze).Acta Physiol Plant,2000,22:439–443
[26]Cao H,Wang L,Yue C,Hao X,Wang X,Yang Y.Isolation and expression analysis of 18 Csb ZIP genes implicated in abiotic stress responses in the tea plant(Camellia sinensis).Plant Physiol Biochem,2015,97:432–442
[27]Wang X C,Zhao Q Y,Ma C L,Zhang Z H,Cao H L,Kong Y M,Yue C,Hao X Y,Chen L,Ma J Q,Jin J Q,Li X,Yang Y J.Global transcriptome profiles of Camellia sinensis during cold acclimation.BMC Genom,2013,14:415
[28]郝心愿,曹红利,杨亚军,王新超,马春雷,肖斌.茶树生长素响应因子基因Cs ARF1的克隆与表达分析.作物学报,2013,39:389–397Hao H X,Cao C H,Yang Y J,Wang X C,Ma C L,Xiao B.Cloning and expression analysis of auxin response factor gene(Cs ARF1)in tea plant(Camellia sinensis[L.]O.Kuntze).Acta Agron Sin,2013,39:389–397(in Chinese with English abstract)
[29]Jimenez S,Lawton-Rauh A L,Reighard G L,Abbott A G,Bielenberg D G.Phylogenetic analysis and molecular evolution of the dormancy associated MADS-box genes from peach.BMC Plant Biol,2009,9:81
[2]Cooke J E,Eriksson M E,Junttila O.The dynamic nature of bud dormancy in trees:environmental control and molecular mechanisms.Plant Cell Environ,2012,35:1707–1728
[3]Rinne P L,van der Schoot C.Symplasmic fields in the tunica of the shoot apical meristem coordinate morphogenetic events.Development,1998.125:1477–1485
[4]Nathalie D,Annika J,Baba K,Schrader J,Sj?din A,Bhalerao R R,Resman L,Trygg J,Moritz T,Bhalerao R P.Environmental and hormonal regulation of the activity–dormancy cycle in the cambial meristem involves stage-specific modulation of transcriptional and metabolic networks.Plant J,2007,50:557–573
[5]van der Schoot C,Rinne P L.Dormancy cycling at the shoot apical meristem:transitioning between self-organization and self-arrest.Plant Sci,2011,180:120–131
[6]Low H P,Gréco B,Tanahashi Y,Gallant J,Jones S N,BillingsGagliardi S,Recht L D,Schwartz W J.Plasmodesmata at the crossroads between development,dormancy,and defense.Can J Bot,2004,81:1182–1197
[7]Rinne P L,Welling A,Vahala J,Ripel L,Ruonala R,Kangasjarvi J,van der Schoot C.Chilling of dormant buds hyperinduces FLOWERING LOCUS T and recruits GA-inducible 1,3-beta-glucanases to reopen signal conduits and release dormancy in Populus.Plant Cell,2011,23:130–146
[8]Rinne P L,Kaikuranta P M,van der Schoot C.The shoot apical meristem restores its symplasmic organization during chillinginduced release from dormancy.Plant J,2001,26:249–264
[9]晏嫦妤,李家贤,黄华林,何雨媚.茶树休眠的研究进展.安徽农业科学,2012,40:10387–10389Yan C Y,Li J X,Huang H L,He Y M.Research progress of tea plant dormancy.J Anhui Agric Sci,2012,40:10387–10389(in Chinese with English abstract)
[10]钱利生,沈生荣,潘根生.茶树新梢内源激素的HPLC分析及日变化.茶叶科学,1996,(2):135–139Qian L S,Shen S R,Pan G S.HPLC analysis and diurnal variation of the endogenous hormones in shoots of tea plants.J Tea Sci,1996,(2):135–139(in Chinese with English abstract)
[11]黄亚辉,粟本文,郑红发,曾贞,刘霞林.茶树春梢萌动期间内源激素含量的变化.植物生理学通讯,2001,37:306–307Huang Y H,Su B W,Zheng H F,Zeng Z,Liu X L.Changes of endohormone in prouting shoot of tea plant.Plant Physiol Commun,2001,37:306–307(in Chinese with English abstract)
[12]禹利君,史云峰,肖海云,刘富知,刘仲华.不同物候型茶树内源GA3和ABA的变化及其对腋芽萌发调控的影响.作物学报,2008,34:277–283Yu L J,Shi Y F,Xiao H Y,Liu F Z,Liu Z H.Dynamic changes of endogenous GA3 and ABA contents in tea culticars with different phenological characters and their impact on the regulation axillary buds sprouting.Acta Agron Sin,2008,34:277–283(in Chinese with English abstract)
[13]Wang X,Hao X,Ma C,Cao H,Yue C,Wang L,Zeng J,Yang Y.Identification of differential gene expression profiles between winter dormant and sprouting axillary buds in tea plant(Camellia sinensis)by suppression subtractive hybridization.Tree Genet Genom,2014,10:1149–1159
[14]王新超,杨亚军,马春雷,金基强,曹红利.茶树细胞周期蛋白基因的克隆与表达.西北植物学报,2011,31:2365–2372Wang X C,Yang Y J,Ma C L,Jin J Q,Cao H L.Cloning and expression analysis of cyclin gene(Cs CYC1)of tea plant.Acta Bot Boreali-Occident Sin,2011,31:2365–2372(in Chinese with English abstract)
[15]王新超,马春雷,杨亚军,金基强,马建强,曹红利.茶树细胞周期蛋白依赖激酶(Cs CDK)基因c DNA全长克隆与分析.园艺学报,2012,39:333–342Wang X C,Ma C L,Yang Y J,Jin J Q,Ma J Q,Cao H L.c DNA cloning and expression analysis of cyclin-dependent kinase(Cs CDK)gene in tea plant.Acta Hort Sin,2012,39:333–342(in Chinese with English abstract)
[16]Doxey A C,Yaish M W,Moffatt B A,Griffith M,Mc Conkey B J.Functional divergence in the Arabidopsis beta-1,3-glucanase gene family inferred by phylogenetic reconstruction of expression states.Mol Biol Evol,2007,24:1045–1055
[17]Rinne P L,Paul L K,Vahala J,Kangasjarvi J,van der Schoot C.Axillary buds are dwarfed shoots that tightly regulate GA pathway and GA-inducible 1,3-beta-glucanase genes during branching in hybrid aspen.J Exp Bot,2016,67:5975–5991
[18]Chang S,Puryear J,Cairney J.A simple and efficient method for isolating RNA from pine trees.Plant Mol Biol Rep,1993,11:113–116
[19]Hao X,Horvath D P,Chao W S,Yang Y,Wang X,Xiao B.Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant(Camellia sinensis(L.)O.Kuntze).Int J Mol Sci,2014,15:22155–22172
[20]Jian L C,Li P H,Sun L H,Chen T H H.Alterations in ultrastructure and subcellular localization of Ca2+in poplar apical bud cells during the induction of dormancy.J Exp Bot,1997,48:1195–1207
[21]刘芳,王家艳,王晓丽,周蕴薇.细叶百合鳞茎在低温解除休眠过程中茎尖细胞超微结构的变化.园艺学报,2013,40:1110–1118Liu F,Wang J Y,Wang X L,Zhou Y W.The apical bud cell ultra-structure changes of Lilium pumilum bulbs during breaking dormancy under refrigerated condition.Acta Hort Sin,2013,40:1110–1118(in Chinese with English abstract)
[22]Ruonala R,Rinne P L,Kangasj?rvi J,van der Schoot C.CENL1expression in the rib meristem affects stem elongation and the transition to dormancy in Populus.Plant Cell,2008,20:59–74
[23]Knox J P,Benitez-Alfonso Y.Roles and regulation of plant cell walls surrounding plasmodesmata.Curr Opin Plant Biol,2014,22:93–100
[24]Paul L K,Rinne P L,van der Schoot C.Refurbishing the plasmodesmal chamber:a role for lipid bodies?Front Plant Sci,2014.5:40
[25]Nagar P K,Kumar A.Changes in endogenous gibberellin activity during winter dormancy in tea(Camellia sinensis(L.)O.Kuntze).Acta Physiol Plant,2000,22:439–443
[26]Cao H,Wang L,Yue C,Hao X,Wang X,Yang Y.Isolation and expression analysis of 18 Csb ZIP genes implicated in abiotic stress responses in the tea plant(Camellia sinensis).Plant Physiol Biochem,2015,97:432–442
[27]Wang X C,Zhao Q Y,Ma C L,Zhang Z H,Cao H L,Kong Y M,Yue C,Hao X Y,Chen L,Ma J Q,Jin J Q,Li X,Yang Y J.Global transcriptome profiles of Camellia sinensis during cold acclimation.BMC Genom,2013,14:415
[28]郝心愿,曹红利,杨亚军,王新超,马春雷,肖斌.茶树生长素响应因子基因Cs ARF1的克隆与表达分析.作物学报,2013,39:389–397Hao H X,Cao C H,Yang Y J,Wang X C,Ma C L,Xiao B.Cloning and expression analysis of auxin response factor gene(Cs ARF1)in tea plant(Camellia sinensis[L.]O.Kuntze).Acta Agron Sin,2013,39:389–397(in Chinese with English abstract)
[29]Jimenez S,Lawton-Rauh A L,Reighard G L,Abbott A G,Bielenberg D G.Phylogenetic analysis and molecular evolution of the dormancy associated MADS-box genes from peach.BMC Plant Biol,2009,9:81