植物络合素合酶及其基因研究进展
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摘要
植物络合素(phytochelatin, PC)能与重金属产生络合反应,在植物解除重金属毒害过程中起关键作用,其在生物体内的合成受植物络合素合酶(phytochelatin synthase, PCS)控制。深入研究植物络合素合酶对理解植物对抗重金属胁迫的机制,以及运用基因工程手段创造工程植物来修复重金属污染环境具有重要意义。本文从PCS的双功能酶特性、基因表达和酶学特征、物种分布和亲缘关系、催化活性中心及激活模式5个方面作简要评述。PCS的主要功能为催化合成植物络合素,同时具有肽酶的副功能;PCS基因的表达是组成型的,在少数物种中,其表达也受Cd~(2+)等重金属离子诱导,而酶的活性受部分金属离子调控,其中以Cd~(2+)效果最为明显。PCS分布广泛,不是植物所独有的,在酵母、线虫等物种中同时存在,然而该酶在植物中同源性较高,在其他物种中较低,暗示其在植物对环境适应过程中具有某些重要作用。PCS的催化中心位于N-基端,其中与藻青菌中Cys70、His183和Asp201相对应的3个氨基酸在所有物种中为严格保守;PCS的重金属活化被认为与其含半胱氨酸有关,包含重金属直接接触活化和形成间接中间物活化2种假说。最后对PCS的多功能性、基因表达特性、酶激活机制及此基因的利用进行了展望,旨在为与PCS相关的研究提供参考。
Phytochelatin(PC), which is produced under catalyzation of phytochelatin synthase(PCS), plays a key role in phytoremediation as its ability of conjugating heavy metal ion in plants. Intensive research on phytochelatin synthase is of great significance to understand the mechanism of heavy metal resistance in plants, as well as inspires on strategies for creating engineering plants to ameliorate soil contaminated by heavy metal. This review was based on five aspects including bifunctional enzyme property, gene expression and enzymology feature, species distribution and phylogeny, catalytic active center, and activating mode. PCS mainly catalyzes the process of phytochelatin synthesis, as well as acts as peptidase. The gene expression of PCS is constitutive, whereas it can be regulated by heavy metal ion like Cd~(2+)in a few species, and PCS is activated by some metal ions in which Cd~(2+)is the most effective. It is widely distributed and can be found in organism kingdom such as yeast and nematode besides plants where PCS family members have closer genetic relationship than in other species, which infer its roles in plant's adaption to environment.The catalytic center of PCS is located at N-terminal, and the three amino acid residues corresponding to Cys70, His183 and Asp201 in cyanobacterium absolutely conserved in all species. The heavy metal activation of PCS is believed to be related to cysteine, including two hypotheses of direct contact activation of heavy metals and formation of indirect intermediates. At the last part of this review, further discussions and prospects are given on multiple functions, gene expression, activating mechanism of PCS, as well as application regarding to the genes, aiming to provide some insights and references for further study.
Phytochelatin(PC), which is produced under catalyzation of phytochelatin synthase(PCS), plays a key role in phytoremediation as its ability of conjugating heavy metal ion in plants. Intensive research on phytochelatin synthase is of great significance to understand the mechanism of heavy metal resistance in plants, as well as inspires on strategies for creating engineering plants to ameliorate soil contaminated by heavy metal. This review was based on five aspects including bifunctional enzyme property, gene expression and enzymology feature, species distribution and phylogeny, catalytic active center, and activating mode. PCS mainly catalyzes the process of phytochelatin synthesis, as well as acts as peptidase. The gene expression of PCS is constitutive, whereas it can be regulated by heavy metal ion like Cd~(2+)in a few species, and PCS is activated by some metal ions in which Cd~(2+)is the most effective. It is widely distributed and can be found in organism kingdom such as yeast and nematode besides plants where PCS family members have closer genetic relationship than in other species, which infer its roles in plant's adaption to environment.The catalytic center of PCS is located at N-terminal, and the three amino acid residues corresponding to Cys70, His183 and Asp201 in cyanobacterium absolutely conserved in all species. The heavy metal activation of PCS is believed to be related to cysteine, including two hypotheses of direct contact activation of heavy metals and formation of indirect intermediates. At the last part of this review, further discussions and prospects are given on multiple functions, gene expression, activating mechanism of PCS, as well as application regarding to the genes, aiming to provide some insights and references for further study.
引文
[1]冯保民,麻密.植物络合素及其合酶在重金属抗性中的功能研究进展.应用与环境生物学报,2003(6):657-661.FENG B M,MA M.Research advance of phytochelatins and phytochelatin synthase on heavy metal tolerance.Chinese Journal of Applied and Environmental Biology,2003(6):657-661.(in Chinese with English abstract)
[2]BRUNETTI P,ZANELLA L,PROIA A,et al.Cadmium tolerance and phytochelatin content of Arabidopsis seedlings over-expressing the phytochelatin synthase gene AtPCS1.Journal of Experimental Botany,2011,62(15):5509-5519.
[3]VATAMANIUK O K,MARI S,LU Y P,et al.AtPCS1,a phytochelatin synthase from Arabidopsis:Isolation and in vitro reconstitution.Proceedings of the National Academy of Sciences of the USA,1999,96(12):7110-7115.
[4]GRILL E,L?FFLER S,WINNACKER E L,et al.Phytochelatins,the heavy-metal-binding peptides of plants,are synthesized from glutathione by a specificγ-glutamylcysteine dipeptidyl transpeptidase(phytochelatin synthase).Proceedings of the National Academy of Sciences of the USA,1989,86(18):6838-6842.
[5]高华,张玉秀,柴团耀.植物络合素和植物络合素合酶的研究.西北植物学报,2001,21(4):779-790.GAO H,ZHANG Y X,CHAI T Y.Research advances in phytochelatins and phytochelatin synthase.Acta Botanica Boreali-Occidentalia Sinica,2001,21(4):779-790.(in Chinese with English abstract)
[6]CLEMENS S,PER?OH D.Multi-tasking phytochelatin synthases.Plant Science,2009,177(4):266-271.
[7]KüHLENZ T,SCHMIDT H,URAGUCHI S,et al.Arabidopsis thaliana phytochelatin synthase 2 is constitutively active in vivo and can rescue the growth defect of the PCS1-deficient cad1-3mutant on Cd-contaminated soil.Journal of Experimental Botany,2014,65(15):4241-4253.
[8]LI A M,YU B Y,CHEN F H,et al.Characterization of the Sesbania rostrata phytochelatin synthase gene:Alternative splicing and function of four isoforms.International Journal of Molecular Sciences,2009,10(8):3269-3282.
[9]LIU Z L,GU C S,CHEN F D,et al.Heterologous expression of a Nelumbo nucifera phytochelatin synthase gene enhances cadmium tolerance in Arabidopsis thaliana.Applied Biochemistry and Biotechnology,2012,166(3):722-734.
[10]LEE B D,HWANG S.Tobacco phytochelatin synthase(NtPCS1)plays important roles in cadmium and arsenic tolerance and in early plant development in tobacco.Plant Biotechnology Reports,2015,9(3):107-114.
[11]WANG F J,WANG Z B,ZHU C.Heteroexpression of the wheat phytochelatin synthase gene(TaPCS1)in rice enhances cadmium sensitivity.Acta Biochimica et Biophysica Sinica,2012,44(10):886-893.
[12]BLUM R,BECK A,KORTE A,et al.Function of phytochelatin synthase in catabolism of glutathioneconjugates.The Plant Journal,2007,49(4):740-749.
[13]CHEN J J,ZHOU J M,GOLDSBROUGH P B.Characterization of phytochelatin synthase from tomato.Physiologia Plantarum,1997,101(1):165-172.
[14]LEE S,KORBAN S.Transcriptional regulation of Arabidopsis thaliana phytochelatin synthase(AtPCS1)by cadmium during early stages of plant development.Planta,2002,215(4):689-693.
[15]TSUJI N,NISHIKORI S,IWABE O,et al.Characterization of phytochelatin synthase-like protein encoded by alr0975from a prokaryote,Nostoc sp.PCC 7120.Biochemical and Biophysical Research Communications,2004,315(3):751-755.
[16]RIGOUIN C,VERMEIRE J J,NYLIN E,et al.Characterization of the phytochelatin synthase from the human parasitic nematode Ancylostoma ceylanicum.Molecular and Biochemical Parasitology,2013,191(1):1-6.
[17]GARCIA-GARCIA J D,GIRARD L,HERNáNDEZ G,et al.Zn-bis-glutathionate is the best co-substrate of the monomeric phytochelatin synthase from the photosynthetic heavy metalhyperaccumulator Euglena gracilis.Metallomics,2014,6(3):604-616.
[18]LEE S,KANG B S.Expression of Arabidopsis phytochelatin synthase 2 is too low to complement an AtPCS1-defective cad1-3 mutant.Molecules and Cells,2005,19(1):81-87.
[19]CLEMENS S,KIM E J,NEUMANN D,et al.Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast.The EMBO Journal,1999,18(12):3325-3333.
[20]COUSELO J L,NAVARRO-AVI?óJ,BALLESTER A.Expression of the phytochelatin synthase TaPCS1 in transgenic aspen,insight into the problems and qualities in phytoremediation of Pb.International Journal of Phytoremediation,2010,12(4):358-370.
[21]OVEN M,PAGE J E,ZENK M H,et al.Molecular characterization of the homo-phytochelatin synthase of soybean Glycine max:Relation to phytochelatin synthase.Journal of Biological Chemistry,2002,277(7):4747-4754.
[22]STROI?SKI A,ZIELEZI?SKA M.Cadmium and oxidative stress influence on phytochelatin synthase activity in potato tuber.Acta Physiologiae Plantarum,2001,23(2):157-160.
[23]KIM Y J,CHANG K S,LEE M R,et al.Expression of tobacco cDNA encoding phytochelatin synthase promotes tolerance to and accumulation of Cd and as in Saccharomyces cerevisiae.Journal of Plant Biology,2005,48(4):440-447.
[24]LI J C,GUO J B,XU W Z,et al.RNA interferencemediated silencing of phytochelatin synthase gene reduce cadmium accumulation in rice seeds.Journal of Integrative Plant Biology,2007,49(7):1032-1037.
[25]KNYAZEV A V,KULUEV B R,CHEMERIS A V,et al.Effect of cadmium on promoter activity of rice phytochelatin synthase gene in transgenic tobacco plants.Russian Journal of Plant Physiology,2013,60(5):701-705.
[26]DAS N,BHATTACHARYA S,BHATTACHARYYA S,et al.Identification of alternatively spliced transcripts of rice phytochelatin synthase 2 gene OsPCS2 involved in mitigation of cadmium and arsenic stresses.Plant Molecular Biology,2017,94(1/2):167-183.
[27]VESTERGAARD M,MATSUMOTO S,NISHIKORI S,et al.Chelation of cadmium ions by phytochelatin synthase:Role of the cystein-rich C-terminal.Analytical Sciences,2008,24(2):277-281.
[28]HA S B,SMITH A P,HOWDEN R,et al.Phytochelatin synthase genes from Arabidopsis and the yeast Schizosaccharomyces pombe.The Plant Cell,1999,11(6):1153-1164.
[29]HEISS S,WACHTER A,BOGS J,et al.Phytochelatin synthase(PCS)protein is induced in Brassica juncea leaves after prolonged Cd exposure.Journal of Experimental Botany,2003,54(389):1833-1839.
[30]GASIC K,KORBAN S S.Expression of Arabidopsis phytochelatin synthase in Indian mustard(Brassica juncea)plants enhances tolerance for Cd and Zn.Planta,2007,225(5):1277-1285.
[31]GOTO F,ARASHIMA Y,SHIMADA H,et al.The expression analysis of phytochelatin synthase gene in Athyrium yokoscense//FUKADA H.Plant and Cell Physiology.Oxford,England:Oxford University Press,2004:S168.
[32]SHUKLA D,KESARI R,MISHRA S,et al.Expression of phytochelatin synthase from aquatic macrophyte Ceratophyllum demersum L.enhances cadmium and arsenic accumulation in tobacco.Plant Cell Reports,2012,31(9):1687-1699.
[33]SHRI M,DAVE R,DIWEDI S,et al.Heterologous expression of Ceratophyllum demersum phytochelatin synthase,CdPCS1,in rice leads to lower arsenic accumulation in grain.Scientific Reports,2014,4:5784.
[34]SHUKLA D,KESARI R,TIWARI M,et al.Expression of Ceratophyllum demersum phytochelatin synthase,CdPCS1,in Escherichia coli and Arabidopsis enhances heavy metal-(loid)s accumulation.Protoplasma,2013,250(6):1263-1272.
[35]VATAMANIUK O K,BUCHER E A,WARD J T,et al.Anew pathway for heavy metal detoxification in animals:Phytochelatin synthase is required for cadmium tolerance in Caenorhabditis elegans.Journal of Biological Chemistry,2001,276(24):20817-20820.
[36]CLEMENS S,SCHROEDER J I,DEGENKOLB T.Caenorhabditis elegans expresses a functional phytochelatin synthase.The FEBS Journal,2001,268(13):3640-3643.
[37]ESSIG Y J,WEBB S M,STURZENBAUM S R.Deletion of phytochelatin synthase modulates the metal accumulation pattern of cadmium exposed C.elegans.International Journal of Molecular Sciences,2016,17(2):257.
[38]MARCHLER-BAUER A,LU S,ANDERSON J B,et al.CDD:A Conserved Domain Database for the functional annotation of proteins.Nucleic Acids Research,2010,39(Suppl.1):D225-D229.
[39]REA P A,VATAMANIUK O K,RIGDEN D J.Weeds,worms,and more.Papain’s long-lost cousin,phytochelatin synthase.Plant Physiology,2004,136(1):2463-2474.
[40]VIVARES D,ARNOUX P,PIGNOL D.A papain-like enzyme at work:Native and acyl-enzyme intermediate structures in phytochelatin synthesis.Proceedings of the National Academy of Sciences of the USA,2005,102(52):18848-18853.
[41]YAMAMOTO D,MATSUMOTO K,OHISHI H,et al.Refined X-ray structure of papain.E-64-c complex at 2.1-Aresolution.Journal of Biological Chemistry,1991,266(22):14771-14777.
[42]GRILL E,WINNACKER E,ZENK M H.Phytochelatins:The principal heavy-metal complexing peptides of higher plants.Science,1985,230(4726):674-676.
[2]BRUNETTI P,ZANELLA L,PROIA A,et al.Cadmium tolerance and phytochelatin content of Arabidopsis seedlings over-expressing the phytochelatin synthase gene AtPCS1.Journal of Experimental Botany,2011,62(15):5509-5519.
[3]VATAMANIUK O K,MARI S,LU Y P,et al.AtPCS1,a phytochelatin synthase from Arabidopsis:Isolation and in vitro reconstitution.Proceedings of the National Academy of Sciences of the USA,1999,96(12):7110-7115.
[4]GRILL E,L?FFLER S,WINNACKER E L,et al.Phytochelatins,the heavy-metal-binding peptides of plants,are synthesized from glutathione by a specificγ-glutamylcysteine dipeptidyl transpeptidase(phytochelatin synthase).Proceedings of the National Academy of Sciences of the USA,1989,86(18):6838-6842.
[5]高华,张玉秀,柴团耀.植物络合素和植物络合素合酶的研究.西北植物学报,2001,21(4):779-790.GAO H,ZHANG Y X,CHAI T Y.Research advances in phytochelatins and phytochelatin synthase.Acta Botanica Boreali-Occidentalia Sinica,2001,21(4):779-790.(in Chinese with English abstract)
[6]CLEMENS S,PER?OH D.Multi-tasking phytochelatin synthases.Plant Science,2009,177(4):266-271.
[7]KüHLENZ T,SCHMIDT H,URAGUCHI S,et al.Arabidopsis thaliana phytochelatin synthase 2 is constitutively active in vivo and can rescue the growth defect of the PCS1-deficient cad1-3mutant on Cd-contaminated soil.Journal of Experimental Botany,2014,65(15):4241-4253.
[8]LI A M,YU B Y,CHEN F H,et al.Characterization of the Sesbania rostrata phytochelatin synthase gene:Alternative splicing and function of four isoforms.International Journal of Molecular Sciences,2009,10(8):3269-3282.
[9]LIU Z L,GU C S,CHEN F D,et al.Heterologous expression of a Nelumbo nucifera phytochelatin synthase gene enhances cadmium tolerance in Arabidopsis thaliana.Applied Biochemistry and Biotechnology,2012,166(3):722-734.
[10]LEE B D,HWANG S.Tobacco phytochelatin synthase(NtPCS1)plays important roles in cadmium and arsenic tolerance and in early plant development in tobacco.Plant Biotechnology Reports,2015,9(3):107-114.
[11]WANG F J,WANG Z B,ZHU C.Heteroexpression of the wheat phytochelatin synthase gene(TaPCS1)in rice enhances cadmium sensitivity.Acta Biochimica et Biophysica Sinica,2012,44(10):886-893.
[12]BLUM R,BECK A,KORTE A,et al.Function of phytochelatin synthase in catabolism of glutathioneconjugates.The Plant Journal,2007,49(4):740-749.
[13]CHEN J J,ZHOU J M,GOLDSBROUGH P B.Characterization of phytochelatin synthase from tomato.Physiologia Plantarum,1997,101(1):165-172.
[14]LEE S,KORBAN S.Transcriptional regulation of Arabidopsis thaliana phytochelatin synthase(AtPCS1)by cadmium during early stages of plant development.Planta,2002,215(4):689-693.
[15]TSUJI N,NISHIKORI S,IWABE O,et al.Characterization of phytochelatin synthase-like protein encoded by alr0975from a prokaryote,Nostoc sp.PCC 7120.Biochemical and Biophysical Research Communications,2004,315(3):751-755.
[16]RIGOUIN C,VERMEIRE J J,NYLIN E,et al.Characterization of the phytochelatin synthase from the human parasitic nematode Ancylostoma ceylanicum.Molecular and Biochemical Parasitology,2013,191(1):1-6.
[17]GARCIA-GARCIA J D,GIRARD L,HERNáNDEZ G,et al.Zn-bis-glutathionate is the best co-substrate of the monomeric phytochelatin synthase from the photosynthetic heavy metalhyperaccumulator Euglena gracilis.Metallomics,2014,6(3):604-616.
[18]LEE S,KANG B S.Expression of Arabidopsis phytochelatin synthase 2 is too low to complement an AtPCS1-defective cad1-3 mutant.Molecules and Cells,2005,19(1):81-87.
[19]CLEMENS S,KIM E J,NEUMANN D,et al.Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast.The EMBO Journal,1999,18(12):3325-3333.
[20]COUSELO J L,NAVARRO-AVI?óJ,BALLESTER A.Expression of the phytochelatin synthase TaPCS1 in transgenic aspen,insight into the problems and qualities in phytoremediation of Pb.International Journal of Phytoremediation,2010,12(4):358-370.
[21]OVEN M,PAGE J E,ZENK M H,et al.Molecular characterization of the homo-phytochelatin synthase of soybean Glycine max:Relation to phytochelatin synthase.Journal of Biological Chemistry,2002,277(7):4747-4754.
[22]STROI?SKI A,ZIELEZI?SKA M.Cadmium and oxidative stress influence on phytochelatin synthase activity in potato tuber.Acta Physiologiae Plantarum,2001,23(2):157-160.
[23]KIM Y J,CHANG K S,LEE M R,et al.Expression of tobacco cDNA encoding phytochelatin synthase promotes tolerance to and accumulation of Cd and as in Saccharomyces cerevisiae.Journal of Plant Biology,2005,48(4):440-447.
[24]LI J C,GUO J B,XU W Z,et al.RNA interferencemediated silencing of phytochelatin synthase gene reduce cadmium accumulation in rice seeds.Journal of Integrative Plant Biology,2007,49(7):1032-1037.
[25]KNYAZEV A V,KULUEV B R,CHEMERIS A V,et al.Effect of cadmium on promoter activity of rice phytochelatin synthase gene in transgenic tobacco plants.Russian Journal of Plant Physiology,2013,60(5):701-705.
[26]DAS N,BHATTACHARYA S,BHATTACHARYYA S,et al.Identification of alternatively spliced transcripts of rice phytochelatin synthase 2 gene OsPCS2 involved in mitigation of cadmium and arsenic stresses.Plant Molecular Biology,2017,94(1/2):167-183.
[27]VESTERGAARD M,MATSUMOTO S,NISHIKORI S,et al.Chelation of cadmium ions by phytochelatin synthase:Role of the cystein-rich C-terminal.Analytical Sciences,2008,24(2):277-281.
[28]HA S B,SMITH A P,HOWDEN R,et al.Phytochelatin synthase genes from Arabidopsis and the yeast Schizosaccharomyces pombe.The Plant Cell,1999,11(6):1153-1164.
[29]HEISS S,WACHTER A,BOGS J,et al.Phytochelatin synthase(PCS)protein is induced in Brassica juncea leaves after prolonged Cd exposure.Journal of Experimental Botany,2003,54(389):1833-1839.
[30]GASIC K,KORBAN S S.Expression of Arabidopsis phytochelatin synthase in Indian mustard(Brassica juncea)plants enhances tolerance for Cd and Zn.Planta,2007,225(5):1277-1285.
[31]GOTO F,ARASHIMA Y,SHIMADA H,et al.The expression analysis of phytochelatin synthase gene in Athyrium yokoscense//FUKADA H.Plant and Cell Physiology.Oxford,England:Oxford University Press,2004:S168.
[32]SHUKLA D,KESARI R,MISHRA S,et al.Expression of phytochelatin synthase from aquatic macrophyte Ceratophyllum demersum L.enhances cadmium and arsenic accumulation in tobacco.Plant Cell Reports,2012,31(9):1687-1699.
[33]SHRI M,DAVE R,DIWEDI S,et al.Heterologous expression of Ceratophyllum demersum phytochelatin synthase,CdPCS1,in rice leads to lower arsenic accumulation in grain.Scientific Reports,2014,4:5784.
[34]SHUKLA D,KESARI R,TIWARI M,et al.Expression of Ceratophyllum demersum phytochelatin synthase,CdPCS1,in Escherichia coli and Arabidopsis enhances heavy metal-(loid)s accumulation.Protoplasma,2013,250(6):1263-1272.
[35]VATAMANIUK O K,BUCHER E A,WARD J T,et al.Anew pathway for heavy metal detoxification in animals:Phytochelatin synthase is required for cadmium tolerance in Caenorhabditis elegans.Journal of Biological Chemistry,2001,276(24):20817-20820.
[36]CLEMENS S,SCHROEDER J I,DEGENKOLB T.Caenorhabditis elegans expresses a functional phytochelatin synthase.The FEBS Journal,2001,268(13):3640-3643.
[37]ESSIG Y J,WEBB S M,STURZENBAUM S R.Deletion of phytochelatin synthase modulates the metal accumulation pattern of cadmium exposed C.elegans.International Journal of Molecular Sciences,2016,17(2):257.
[38]MARCHLER-BAUER A,LU S,ANDERSON J B,et al.CDD:A Conserved Domain Database for the functional annotation of proteins.Nucleic Acids Research,2010,39(Suppl.1):D225-D229.
[39]REA P A,VATAMANIUK O K,RIGDEN D J.Weeds,worms,and more.Papain’s long-lost cousin,phytochelatin synthase.Plant Physiology,2004,136(1):2463-2474.
[40]VIVARES D,ARNOUX P,PIGNOL D.A papain-like enzyme at work:Native and acyl-enzyme intermediate structures in phytochelatin synthesis.Proceedings of the National Academy of Sciences of the USA,2005,102(52):18848-18853.
[41]YAMAMOTO D,MATSUMOTO K,OHISHI H,et al.Refined X-ray structure of papain.E-64-c complex at 2.1-Aresolution.Journal of Biological Chemistry,1991,266(22):14771-14777.
[42]GRILL E,WINNACKER E,ZENK M H.Phytochelatins:The principal heavy-metal complexing peptides of higher plants.Science,1985,230(4726):674-676.