龙须菜中rbc L和hsp70对高温和植物激素的响应
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摘要
龙须菜已在我国沿海从南到北广泛栽培,但其栽培周期受夏季高温的限制。本研究采用qRT-PCR和Western blot技术研究了龙须菜核酮糖-1, 5-二磷酸羧化酶/加氧酶大亚基(rbcL)和热休克蛋白70(HSP70)对高温胁迫及3种抗逆植物激素的响应。结果显示:①高温(33°C)显著抑制了龙须菜rbc L转录和蛋白的表达水平,而100μmol/L水杨酸(SA100)和50μmol/L茉莉酸甲酯(MJ50)的添加可减轻高温的不利影响。在SA100和MJ50组中,rbc L转录表达量分别为高温组的1.31倍和1.32倍(3 h),rbcL蛋白表达量分别为高温组的1.36倍和2.10倍(24 h);并且这2种激素也能一定程度上缓解高温对藻体Rubisco活性的抑制作用,恢复Rubisco活化状态。但是50μmol/L脱落酸(ABA50)的添加则基本上抑制了rbcL表达及Rubisco活性。②3种激素进一步促进了高温诱导的龙须菜HSP70的表达,在激素添加后,其转录水平升高0.53～1.00倍(3 h),蛋白水平升高0.93～2.45倍(24 h)。可见,植物激素SA、MJ和ABA在调控由高温引起的光合作用酶的抑制和热休克蛋白的诱导表达方面发挥了一定的作用。
Seaweed Gracilariopsis lemaneiformis is mainly used as the bait for abalone and raw material for agar production in China. This macroalga is widely cultivated along the coasts of China from south to north, but its cultivation period is limited by high temperature in summer. In the current study, the effects of heat and phytohormone treatments on Rubisco large subunit(rbcL) and heat shock protein 70(HSP70) were investigated by using quantitative real-time PCR and Western blot technologies in G. lemaneiformis. The results showed that transcript and protein levels of rbc L in G. lemaneiformis were inhibited by heat stress(33 °C), however, 100 μmol/L salicylic acid(SA100) and 50 μmol/L methyl jasmonate(MJ50) treatments reduced the inhibition of high temperature on the expression of rbcL. In SA100 and MJ50 groups, the transcriptional expression of rbc L increased to1.31-and 1.32-fold at 3 h, and the protein levels of rbcL were enhanced to 1.36-and 2.10-fold at 24 h compared to the heat-stress group, respectively. Moreover, SA100 and MJ50 effectively alleviated the inhibition effect of heat stress on Rubisco activity, and recovered the Rubisco activation states. However, the addition of 50 μmol/L abscisic acid(ABA50) mainly inhibited rbc L expression and Rubisco activity. Heat stress stimulated the transcript and protein levels of HSP70; hsp70 transcript expression further rose by 0.53–1.00 times at 3 h, and its protein expression enhanced by 0.93–2.45 times at 24 h in the heat-stress group after applying SA100, MJ50 or ABA50. In conclusion, SA, MJ and ABA play a certain role in regulating the inhibition of photosynthesis enzyme and the induction of heat shock proteins caused by heat stress.
Seaweed Gracilariopsis lemaneiformis is mainly used as the bait for abalone and raw material for agar production in China. This macroalga is widely cultivated along the coasts of China from south to north, but its cultivation period is limited by high temperature in summer. In the current study, the effects of heat and phytohormone treatments on Rubisco large subunit(rbcL) and heat shock protein 70(HSP70) were investigated by using quantitative real-time PCR and Western blot technologies in G. lemaneiformis. The results showed that transcript and protein levels of rbc L in G. lemaneiformis were inhibited by heat stress(33 °C), however, 100 μmol/L salicylic acid(SA100) and 50 μmol/L methyl jasmonate(MJ50) treatments reduced the inhibition of high temperature on the expression of rbcL. In SA100 and MJ50 groups, the transcriptional expression of rbc L increased to1.31-and 1.32-fold at 3 h, and the protein levels of rbcL were enhanced to 1.36-and 2.10-fold at 24 h compared to the heat-stress group, respectively. Moreover, SA100 and MJ50 effectively alleviated the inhibition effect of heat stress on Rubisco activity, and recovered the Rubisco activation states. However, the addition of 50 μmol/L abscisic acid(ABA50) mainly inhibited rbc L expression and Rubisco activity. Heat stress stimulated the transcript and protein levels of HSP70; hsp70 transcript expression further rose by 0.53–1.00 times at 3 h, and its protein expression enhanced by 0.93–2.45 times at 24 h in the heat-stress group after applying SA100, MJ50 or ABA50. In conclusion, SA, MJ and ABA play a certain role in regulating the inhibition of photosynthesis enzyme and the induction of heat shock proteins caused by heat stress.
引文
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[9]Hayat Q,Hayat S,Irfan M,et al.Effect of exogenous salicylic acid under changing environment:a review[J].Environmental and Experimental Botany,2010,68(1):14-25.
[10]Wang L J,Chen S J,Kong W F,et al.Salicylic acid pretreatment alleviates chilling injury and affects the antioxidant system and heat shock proteins of peaches during cold storage[J].Postharvest Biology and Technology,2006,41(3):244-251.
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[12]Sánchez-Vallet A,López G,Ramos B,et al.Disruption of abscisic acid signaling constitutively activates Arabidopsis resistance to the necrotrophic fungus Plectosphaerella cucumerina[J].Plant Physiology,2012,160(4):2109-2124.
[13]王重彬,邹同雷,孙雪,等.水杨酸和茉莉酸甲酯对高温龙须菜(Gracilariopsis lemaneiformis)理化及基因表达的影响[J].海洋与湖沼,2015,46(5):1132-1138.Wang C B,Zou T L,Sun X,et al.Effects of salicylic acid and methyl jasmonate on physicochemical property and gene expression of Gracilariopsis lemaneiformis under heat stress[J].Oceanologia et Limnologia Sinica,2015,46(5):1132-1138(in Chinese).
[14]Wang F J,Wang C B,Zou T L,et al.Comparative transcriptional profiling of Gracilariopsis lemaneiformis in response to salicylic acid-and methyl jasmonatemediated heat resistance[J].PLoS ONE,2017,12(5):e0176531.
[15]朱招波,孙雪,徐年军,等.水杨酸对龙须菜抗高温生理的影响[J].水产学报,2012,36(8):1304-1312.Zhu Z B,Sun X,Xu N J,et al.Effects of salicylic acid on the resistance of Gracilaria/Gracilariopsis lemaneiformis to high temperature[J].Journal of Fisheries of China,2012,36(8):1304-1312(in Chinese).
[16]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the2?￠￠CTmethod[J].Methods,2001,25(4):402-408.
[17]Wang Y,Feng Y Q,Wang H F,et al.Physiological and proteomic analyses of two Gracilaria lemaneiformis strains in response to high-temperature stress[J].Journal of Applied Phycology,2016,28(3):1847-1858.
[18]Lu K X,Cao B H,Feng X P,et al.Photosynthetic response of salt-tolerant and sensitive soybean varieties[J].Photosynthetica,2009,47(3):381-387.
[19]潘璐,刘杰才,李晓静,等.高温和加富CO2温室中黄瓜Rubisco活化酶与光合作用的关系[J].园艺学报,2014,41(8):1591-1600.Pan L,Liu J C,Li X J,et al.Correlation between rubisco activase and photosynthesis of cucumber in greenhouse under high temperature and elevated CO2[J].Acta Horticulturae Sinica,2014,41(8):1591-1600(in Chinese).
[20]Parry M A J,Andralojc P J,Khan S,et al.Rubisco activity:effects of drought stress[J].Annals of Botany,2002,89(7):833-839.
[21]Crafts-Brandner S J,Law R D.Effect of heat stress on the inhibition and recovery of the ribulose-1,5-bisphosphate carboxylase/oxygenase activation state[J].Planta,2000,212(1):67-74.
[22]Zhou Y H,Yu J Q,Mao W H,et al.Genotypic variation of Rubisco expression,photosynthetic electron flow and antioxidant metabolism in the chloroplasts of chillexposed cucumber plants[J].Plant and Cell Physiology,2006,47(2):192-199.
[23]Janda T,Gondor O K,Yordanova R,et al.Salicylic acid and photosynthesis:signalling and effects[J].Acta Physiologiae Plantarum,2014,36(10):2537-2546.
[24]Chen Y E,Cui J M,Li G X,et al.Effect of salicylic acid on the antioxidant system and photosystem II in wheat seedlings[J].Biologia Plantarum,2016,60(1):139-147.
[25]Shi Q H,Bao Z Y,Zhu Z J,et al.Effects of different treatments of salicylic acid on heat tolerance,chlorophyll fluorescence,and antioxidant enzyme activity in seedlings of Cucumis sativa L.[J].Plant Growth Regulation,2006,48(2):127-135.
[26]Wang L J,Fan L,Loescher W,et al.Salicylic acid alleviates decreases in photosynthesis under heat stress and accelerates recovery in grapevine leaves[J].BMCPlant Biology,2010,10:34.
[27]吴文华,潘瑞炽.茉莉酸甲酯对水稻幼苗光合作用的影响[J].植物学报,1998,40(3):256-262.Wu W H,Pan R C.Effect of jasmonic acid methyl ester on the photosynthesis of rice seedlings[J].Acta Botanica Sinica,1998,40(3):256-262(in Chinese).
[28]金微微,张会慧,王炎,等.茉莉酸甲酯对干旱及复水下烤烟幼苗光合特性和抗氧化酶的影响[J].干旱区地理,2011,34(6):933-940.Jin W W,Zhang H H,Wang Y,et al.Effects of methyl jasmonate on photosynthetic characteristics and antioxidant enzyme of flue-curing tobacco seedlings under drought stress and rehydration[J].Arid Land Geography,2011,34(6):933-940(in Chinese).
[29]Pandey D M,Goswami C L,Kumar B,et al.Hormonal regulation of photosynthetic enzymes in cotton under water stress[J].Photosynthetica,2000,38(3):403-407.
[30]Schuster G,Even D,Kloppstech K,et al.Evidence for protection by heat-shock proteins against photoinhibition during heat-shock[J].The EMBO Journal,1988,7(1):1-6.
[31]Schroda M,Vallon O,Wollman F A,et al.A chloroplast-targeted heat shock protein 70(HSP70)contributes to the photoprotection and repair of photosystem IIduring and after photoinhibition[J].The Plant Cell,1999,11(6):1165-1178.
[32]Zhang H N,Li W,Li J J,et al.Characterization and expression analysis of hsp70 gene from Ulva prolifera J.Agardh(Chlorophycophyta,Chlorophyceae)[J].Marine Genomics,2012,5:53-58.
[33]Gu Y H,Zhang X,Lu N,et al.Cloning and transcription analysis of hsp70-1 and hsp70-2 of Gracilaria lemaneiformis under heat shock[J].Aquaculture,2012,358-359:284-291.
[34]Huovinen P S,Oikari A O J,Soimasuo M R,et al.Impact of UV radiation on the early development of the giant kelp(Macrocystis pyrifera)gametophytes[J].Photochemistry and Photobiology,2000,72(3):308-313.
[35]刘伟,杨锐,徐丽宁,等.坛紫菜hsp70基因克隆与表达[J].宁波大学学报(理工版),2012,25(2):17-25.Liu W,Yang R,Xu L N,et al.Cloning and expression of hsp70 for Porphyra haitanensis[J].Journal of Ningbo University(Natural Science&Engineering),2012,25(2):17-25(in Chinese).
[36]Li G Q,Zang X N,Zhang X C,et al.Cloning of ubiquitin-activating enzyme and ubiquitin-conjugating enzyme genes from Gracilaria lemaneiformis and their activity under heat shock[J].Gene,2014,538(1):155-163.
[37]Li H,Liu S S,Yi C Y,et al.Hydrogen peroxide mediates abscisic acid-induced HSP70 accumulation and heat tolerance in grafted cucumber plants[J].Plant,Cell and Environment,2014,37(12):2768-2780.
[38]Hu X L,Liu R X,Li Y H,et al.Heat shock protein 70regulates the abscisic acid-induced antioxidant response of maize to combined drought and heat stress[J].Plant Growth Regulation,2010,60(3):225-235.
[2]唐婷,郑国伟,李唯奇.植物光合系统对高温胁迫的响应机制[J].中国生物化学与分子生物学报,2012,28(2):127-132.Tang T,Zheng G W,Li W Q.Defense mechanisms of plants photosystem to heat stress[J].Chinese Journal of Biochemistry and Molecular Biology,2012,28(2):127-132(in Chinese).
[3]Luo H B,Ma L,Xi H F,et al.Photosynthetic responses to heat treatments at different temperatures and following recovery in grapevine(Vitis amurensis L.)leaves[J].PLoS ONE,2011,6(8):e23033.
[4]Zhang X W,Li J P,Liu A L,et al.Expression profile in rice panicle:Insights into heat response mechanism at reproductive stage[J].PLoS ONE,2012,7(11):e49652.
[5]Wang W X,Vinocur B,Shoseyov O,et al.Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response[J].Trends in Plant Science,2004,9(5):244-252.
[6]Mayer M P,Bukau B.Hsp70 chaperones:cellular functions and molecular mechanism[J].Cellular and Molecular Life Sciences,2005,62(6):670-684.
[7]Sung D Y,Kaplan F,Guy C L.Plant Hsp70 molecular chaperones:protein structure,gene family,expression and function[J].Physiologia Plantarum,2001,113(4):443-451.
[8]Yoshimi T,Odagiri K,Hiroshige Y,et al.Induction profile of HSP70-cognate genes by environmental pollutants in Chironomidae[J].Environmental Toxicology and Pharmacology,2009,28(2):294-301.
[9]Hayat Q,Hayat S,Irfan M,et al.Effect of exogenous salicylic acid under changing environment:a review[J].Environmental and Experimental Botany,2010,68(1):14-25.
[10]Wang L J,Chen S J,Kong W F,et al.Salicylic acid pretreatment alleviates chilling injury and affects the antioxidant system and heat shock proteins of peaches during cold storage[J].Postharvest Biology and Technology,2006,41(3):244-251.
[11]Wasternack C,Hause B.Jasmonates:Biosynthesis,perception,signal transduction and action in plant stress response,growth and development.An update to the2007 review in Annals of Botany[J].Annals of Botany,2013,111(6):1021-1058.
[12]Sánchez-Vallet A,López G,Ramos B,et al.Disruption of abscisic acid signaling constitutively activates Arabidopsis resistance to the necrotrophic fungus Plectosphaerella cucumerina[J].Plant Physiology,2012,160(4):2109-2124.
[13]王重彬,邹同雷,孙雪,等.水杨酸和茉莉酸甲酯对高温龙须菜(Gracilariopsis lemaneiformis)理化及基因表达的影响[J].海洋与湖沼,2015,46(5):1132-1138.Wang C B,Zou T L,Sun X,et al.Effects of salicylic acid and methyl jasmonate on physicochemical property and gene expression of Gracilariopsis lemaneiformis under heat stress[J].Oceanologia et Limnologia Sinica,2015,46(5):1132-1138(in Chinese).
[14]Wang F J,Wang C B,Zou T L,et al.Comparative transcriptional profiling of Gracilariopsis lemaneiformis in response to salicylic acid-and methyl jasmonatemediated heat resistance[J].PLoS ONE,2017,12(5):e0176531.
[15]朱招波,孙雪,徐年军,等.水杨酸对龙须菜抗高温生理的影响[J].水产学报,2012,36(8):1304-1312.Zhu Z B,Sun X,Xu N J,et al.Effects of salicylic acid on the resistance of Gracilaria/Gracilariopsis lemaneiformis to high temperature[J].Journal of Fisheries of China,2012,36(8):1304-1312(in Chinese).
[16]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the2?￠￠CTmethod[J].Methods,2001,25(4):402-408.
[17]Wang Y,Feng Y Q,Wang H F,et al.Physiological and proteomic analyses of two Gracilaria lemaneiformis strains in response to high-temperature stress[J].Journal of Applied Phycology,2016,28(3):1847-1858.
[18]Lu K X,Cao B H,Feng X P,et al.Photosynthetic response of salt-tolerant and sensitive soybean varieties[J].Photosynthetica,2009,47(3):381-387.
[19]潘璐,刘杰才,李晓静,等.高温和加富CO2温室中黄瓜Rubisco活化酶与光合作用的关系[J].园艺学报,2014,41(8):1591-1600.Pan L,Liu J C,Li X J,et al.Correlation between rubisco activase and photosynthesis of cucumber in greenhouse under high temperature and elevated CO2[J].Acta Horticulturae Sinica,2014,41(8):1591-1600(in Chinese).
[20]Parry M A J,Andralojc P J,Khan S,et al.Rubisco activity:effects of drought stress[J].Annals of Botany,2002,89(7):833-839.
[21]Crafts-Brandner S J,Law R D.Effect of heat stress on the inhibition and recovery of the ribulose-1,5-bisphosphate carboxylase/oxygenase activation state[J].Planta,2000,212(1):67-74.
[22]Zhou Y H,Yu J Q,Mao W H,et al.Genotypic variation of Rubisco expression,photosynthetic electron flow and antioxidant metabolism in the chloroplasts of chillexposed cucumber plants[J].Plant and Cell Physiology,2006,47(2):192-199.
[23]Janda T,Gondor O K,Yordanova R,et al.Salicylic acid and photosynthesis:signalling and effects[J].Acta Physiologiae Plantarum,2014,36(10):2537-2546.
[24]Chen Y E,Cui J M,Li G X,et al.Effect of salicylic acid on the antioxidant system and photosystem II in wheat seedlings[J].Biologia Plantarum,2016,60(1):139-147.
[25]Shi Q H,Bao Z Y,Zhu Z J,et al.Effects of different treatments of salicylic acid on heat tolerance,chlorophyll fluorescence,and antioxidant enzyme activity in seedlings of Cucumis sativa L.[J].Plant Growth Regulation,2006,48(2):127-135.
[26]Wang L J,Fan L,Loescher W,et al.Salicylic acid alleviates decreases in photosynthesis under heat stress and accelerates recovery in grapevine leaves[J].BMCPlant Biology,2010,10:34.
[27]吴文华,潘瑞炽.茉莉酸甲酯对水稻幼苗光合作用的影响[J].植物学报,1998,40(3):256-262.Wu W H,Pan R C.Effect of jasmonic acid methyl ester on the photosynthesis of rice seedlings[J].Acta Botanica Sinica,1998,40(3):256-262(in Chinese).
[28]金微微,张会慧,王炎,等.茉莉酸甲酯对干旱及复水下烤烟幼苗光合特性和抗氧化酶的影响[J].干旱区地理,2011,34(6):933-940.Jin W W,Zhang H H,Wang Y,et al.Effects of methyl jasmonate on photosynthetic characteristics and antioxidant enzyme of flue-curing tobacco seedlings under drought stress and rehydration[J].Arid Land Geography,2011,34(6):933-940(in Chinese).
[29]Pandey D M,Goswami C L,Kumar B,et al.Hormonal regulation of photosynthetic enzymes in cotton under water stress[J].Photosynthetica,2000,38(3):403-407.
[30]Schuster G,Even D,Kloppstech K,et al.Evidence for protection by heat-shock proteins against photoinhibition during heat-shock[J].The EMBO Journal,1988,7(1):1-6.
[31]Schroda M,Vallon O,Wollman F A,et al.A chloroplast-targeted heat shock protein 70(HSP70)contributes to the photoprotection and repair of photosystem IIduring and after photoinhibition[J].The Plant Cell,1999,11(6):1165-1178.
[32]Zhang H N,Li W,Li J J,et al.Characterization and expression analysis of hsp70 gene from Ulva prolifera J.Agardh(Chlorophycophyta,Chlorophyceae)[J].Marine Genomics,2012,5:53-58.
[33]Gu Y H,Zhang X,Lu N,et al.Cloning and transcription analysis of hsp70-1 and hsp70-2 of Gracilaria lemaneiformis under heat shock[J].Aquaculture,2012,358-359:284-291.
[34]Huovinen P S,Oikari A O J,Soimasuo M R,et al.Impact of UV radiation on the early development of the giant kelp(Macrocystis pyrifera)gametophytes[J].Photochemistry and Photobiology,2000,72(3):308-313.
[35]刘伟,杨锐,徐丽宁,等.坛紫菜hsp70基因克隆与表达[J].宁波大学学报(理工版),2012,25(2):17-25.Liu W,Yang R,Xu L N,et al.Cloning and expression of hsp70 for Porphyra haitanensis[J].Journal of Ningbo University(Natural Science&Engineering),2012,25(2):17-25(in Chinese).
[36]Li G Q,Zang X N,Zhang X C,et al.Cloning of ubiquitin-activating enzyme and ubiquitin-conjugating enzyme genes from Gracilaria lemaneiformis and their activity under heat shock[J].Gene,2014,538(1):155-163.
[37]Li H,Liu S S,Yi C Y,et al.Hydrogen peroxide mediates abscisic acid-induced HSP70 accumulation and heat tolerance in grafted cucumber plants[J].Plant,Cell and Environment,2014,37(12):2768-2780.
[38]Hu X L,Liu R X,Li Y H,et al.Heat shock protein 70regulates the abscisic acid-induced antioxidant response of maize to combined drought and heat stress[J].Plant Growth Regulation,2010,60(3):225-235.