β-氨基丁酸处理对采后桃果实还原势的影响及抗病性的诱导作用
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摘要
采用10 mmol/Lβ-氨基丁酸(β-aminobutyric acid,BABA)和病原菌Rhizopus stolonifer处理采后‘白凤’水蜜桃,以此研究BABA处理诱导水蜜桃防卫反应的模式,并从还原势变化角度分析相关诱导抗性机理。结果显示:经10 mmol/L BABA处理的桃果实在20℃下贮藏2 d后,伴随着果实的发病,其还原性内源信号分子NO含量以及PpNPR1-like、PpCHI和PpGNS基因表达丰度均明显高于对照组;而BABA+病原菌处理组PpNPR1-like、PpCHI和PpGNS表达丰度在贮藏前3 d整体上显著高于BABA处理和病原菌接种果实(P<0.05),在整个贮藏期间3种基因的表达丰度均处于较高水平。此外,与对照组相比,BABA处理和BABA+病原菌接种均可显著诱导NO的积累,并同时提升桃果实中磷酸戊糖途径关键酶葡萄糖-6-磷酸脱氢酶和6-磷酸葡萄糖酸脱氢酶活力,促使果实中还原型辅酶Ⅱ(nicotinamide adenine dinucleotide phosphate,NADPH)和还原型谷胱甘肽大量生成,并降低NADP~+和氧化型谷胱甘肽含量,因而有效提高果实组织还原势。通过这些结果可推测,10 mmol/L BABA可通过诱导Priming反应的方式赋予桃果实在受病原菌侵染时更强的抗病性,从而抑制采后软腐病的发生;同时,BABA处理可通过诱导还原性信号分子积累并提高磷酸戊糖途径关键酶活力,进而提升桃果实还原势、活化相关转录因子,以此诱导PRs基因的表达。
The present study was conducted to investigate the pattern of disease resistance induced by 10 mmol/L β-aminobutyric acid(BABA) treatment in postharvest peach(Prunus persica Batsch cv ‘Baifeng') fruits and to analyze the underlying mechanism with respect to the change in redox status. The results showed that the peach fruits treated with 10 mmol/L BABA alone demonstrated remarkably higher concentrations of the endogenous reductive signaling molecule nitric oxide(NO) and higher expression levels of PpNPR1-like, PpCHI and PpGNS genes after 2 days of storage at 20 ℃ when compared with the control, accompanied by an increase in disease incidence. Meanwhile, the fruits simultaneously treated with BABA and inoculated with the pathogen Rhizopus stolonifer exhibited significantly higher expression levels of PpNPR1-like, PpCHI and PpGNS genes during the first three days of storage than did those receiving either treatment alone(P < 0.05), and maintained high levels throughout the storage period. Moreover, either BABA treatment alone or the combined treatment stimulated the accumulation of NO and simultaneously elevated the activities of glucose 6-phosphatedehydrogenase and 6-phosphogluconate dehydrogenase, which are recognized as key enzymes in the pentose phosphate pathway(PPP), thereby contributing to enhanced production of nicotinamide adenine dinucleotide phosphate(NADPH) and glutathione(GSH) and lower contents of NADP~+ and oxidized glutathione and consequently enhanced redox status. Therefore, 10 mmol/L BABA could confer peach fruits with enhanced resistance to R. stolonifera infection by activating the priming defense, thereby reducing the incidence of soft rot disease. At the same time, BABA treatment could induce the accumulation of the reductive signaling molecule and increase the activities of the key enzymes in PPP and consequently enhance the reduction potential and activate relevant transcription factors, thereby inducing the expression of PR gene.
The present study was conducted to investigate the pattern of disease resistance induced by 10 mmol/L β-aminobutyric acid(BABA) treatment in postharvest peach(Prunus persica Batsch cv ‘Baifeng') fruits and to analyze the underlying mechanism with respect to the change in redox status. The results showed that the peach fruits treated with 10 mmol/L BABA alone demonstrated remarkably higher concentrations of the endogenous reductive signaling molecule nitric oxide(NO) and higher expression levels of PpNPR1-like, PpCHI and PpGNS genes after 2 days of storage at 20 ℃ when compared with the control, accompanied by an increase in disease incidence. Meanwhile, the fruits simultaneously treated with BABA and inoculated with the pathogen Rhizopus stolonifer exhibited significantly higher expression levels of PpNPR1-like, PpCHI and PpGNS genes during the first three days of storage than did those receiving either treatment alone(P < 0.05), and maintained high levels throughout the storage period. Moreover, either BABA treatment alone or the combined treatment stimulated the accumulation of NO and simultaneously elevated the activities of glucose 6-phosphatedehydrogenase and 6-phosphogluconate dehydrogenase, which are recognized as key enzymes in the pentose phosphate pathway(PPP), thereby contributing to enhanced production of nicotinamide adenine dinucleotide phosphate(NADPH) and glutathione(GSH) and lower contents of NADP~+ and oxidized glutathione and consequently enhanced redox status. Therefore, 10 mmol/L BABA could confer peach fruits with enhanced resistance to R. stolonifera infection by activating the priming defense, thereby reducing the incidence of soft rot disease. At the same time, BABA treatment could induce the accumulation of the reductive signaling molecule and increase the activities of the key enzymes in PPP and consequently enhance the reduction potential and activate relevant transcription factors, thereby inducing the expression of PR gene.
引文
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[5]汪开拓,郑永华,唐文才,等.茉莉酸甲酯处理对葡萄果实NO和H2O2水平及植保素合成的影响[J].园艺学报,2012,39(8):1559-1566.DOI:10.16420/j.issn.0513-353x.2012.08.015.
[6]WANG K T,LIAO Y X,KAN J Q,et al.Response of direct or priming defense against Botrytis cinerea to methyl jasmonate treatment at different concentrations in grape berries[J].International Journal of Food Microbiology,2015,194:32-39.DOI:10.1016/j.ijfoodmicro.2014.11.006.
[7]COHEN Y,RUBIN A E,KILFIN G.Mechanisms of induced resistance in lettuce against Bremia lactucae by DL-β-amino-butyric acid(BABA)[J].European Journal of Plant Pathology,2010,126(4):553-573.DOI:10.1007/s10658-009-9564-6.
[8]HAMIDUZZAMAN M,JAKAB G,BARNAVON L,et al.β-Aminobutyric acid-induced resistance against downy mildew in grapevine acts through the potentiation of callose formation and jasmonic acid signaling[J].Molecular Plant-Microbe Interactions,2005,18(8):819-829.DOI:10.1094/MPMI-18-0819.
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[10]WANG L,ZHANG H,JIN P,et al.Enhancement of storage quality and antioxidant capacity of harvested sweet cherry fruit by immersion withβ-aminobutyric acid[J].Postharvest Biology and Technology,2016,118:71-78.DOI:10.1016/j.postharvbio.2016.03.023.
[11]廖云霞,费良航,夏明星,等.不同浓度β-氨基丁酸处理对葡萄果实抗病性的诱导模式研究[J].食品科学,2018,39(17):221-228.DOI:10.7506/spkx1002-6630-201817036.
[12]WANG K T,LIAO Y X,XIONG Q,et al.Induction of direct or priming resistance against Botrytis cinerea in strawberries byβ-aminobutyric acid and their effects on sucrose metabolism[J].Journal of Agricultural and Food Chemistry,2016,64(29):5855-5865.DOI:10.1021/acs.jafc.6b00947.
[13]COHEN Y,VAKNIN M,MAUCH-MANI B.BABA-induced resistance:milestones along a 55-year journey[J].Phytoparasitica,2016,44(4):513-538.DOI:10.1007/s12600-016-0546-x.
[14]龚波林.以溴化钾-溴酸钾-2’,7’-二氯荧光素为试剂对痕量水杨酸的间接荧光测定法[J].分析化学,2001,29(9):1055-1057.DOI:10.3321/j.issn:0253-3820.2001.09.019.
[15]MURPHY M E,NOACK E.Nitric oxide assay using hemoglobin method[J].Methods in Enzymology,1994,233:240-250.DOI:10.1016/S0076-6879(94)33027-1.
[16]NAGANO I,SHAPSHAK P,YOSHIOK M,et al.Increased NADPH-diaphorase reactivity and cytokine expression in dorsal root ganglia in acquired immunodeficiency syndrome[J].Journal of the Neurological Sciences,1996,136(1/2):117-128.DOI:10.1016/0022-510X(95)00317-U.
[17]RAHMAN I,KODE A,BISWAS S K.Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method[J].Nature Protocols,2007,1(6):3159-3165.DOI:10.1038/nprot.2006.378.
[18]?INDELá?L,?INDELá?OVáM,BURKETOVáL.Changes in activity of glucose-6-phosphate and 6-phosphogluconate dehydrogenase isozymes upon potato virus Y infection in tobacco leaf tissues and protoplasts[J].Plant Physiology and Biochemistry,1999,37(3):195-201.DOI:10.1016/S0981-9428(99)80034-5.
[19]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding[J].Analytical Biochemistry,1976,72(Suppl 1/2):248-254.DOI:10.1016/0003-2697(76)90527-3.
[20]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J].Methods,2001,25(4):402-408.DOI:10.1006/meth.2001.1262.
[21]CONRATH U.Molecular aspects of defencepriming[J].Trends in Plant Science,2011,16(10):524-531.DOI:10.1016/j.tplants.2011.06.004.
[22]O’BRIEN J A,DAUDI A,BUTT V S,et al.Reactive oxygen species and their role in plant defence and cell wall metabolism[J].Planta,2012,236(3):765-779.DOI:10.1007/s00425-012-1696-9.
[23]MOU Z,FAN W,DONG X.Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes[J].Cell,2003,113(7):935-944.DOI:10.1016/S0092-8674(03)00429-X.
[24]MURMU J,BUSH M J,DELONG C,et al.Arabidopsis basic leucinezipper transcription factors TGA9 and TGA10 interact with floral glutaredoxins ROXY1 and ROXY2 and are redundantly required for anther development[J].Plant Physiology,2010,154(3):1492-1504.DOI:10.1104/pp.110.159111.
[25]LINDERMAYR C,SELL S,MüLLER B,et al.Redox regulation of the NPR1-TGA1 system of Arabidopsis thaliana by nitric oxide[J].Plant Cell,2010,22(8):2894-2907.DOI:10.1105/tpc.109.066464.
[26]LEVéE V,MAJOR I,LEVASSEUR C,et al.Expression profiling and functional analysis of populus WRKY23 reveals a regulatory role in defense[J].New Phytologist,2009,184(1):48-70.DOI:10.1111/j.1469-8137.2009.02955.x.
[27]YU M,LAMATTINA L,SPOEL S H,et al.Nitric oxide function in plant biology:a redox cue in deconvolution[J].New Phytologist,2014,202(4):1142-1156.DOI:10.1111/nph.12739.
[28]KUMAR D.Salicylic acid signaling in disease resistance[J].Plant Science,2014,228:127-134.DOI:10.1016/j.plantsci.2014.04.014.
[29]AHARONI A,GALILI G.Metabolic engineering of the plant primarysecondary metabolism interface[J].Current Opinion in Biotechnology,2011,22(2):239-244.DOI:10.1016/j.copbio.2010.11.004.
[30]SIDDAPPAJI M H,SCHOLES D R,BOHN M,et al.Overcompensation in response to herbivory in Arabidopsis thaliana:the role of glucose-6-phosphate dehydrogenase and the oxidative pentose-phosphate pathway[J].Genetics,2013,195(2):589-598.DOI:10.1534/genetics.113.154351.
[31]LE HENANFF G,FARINE S,KIEFFER-MAZET F,et al.Vitis vinifera VvNPR1.1 is the functional ortholog of AtNPR1 and its overexpression in grapevine triggers constitutive activation of PRgenes and enhanced resistance to powdery mildew[J].Planta,2011,234(2):405-417.DOI:10.1007/s00425-011-1412-1.
[32]WALLY O,JAYARAJ J,PUNJA Z.Comparative resistance to foliar fungal pathogens in transgenic carrot plants expressing genes encoding for chitinase,β-1,3-glucanase and peroxidise[J].European Journal of Plant Pathology,2009,123(3):331-342.DOI:10.1007/s10658-008-9370-6.
[2]BORGES A A,SANDALIO L M.Induced resistance for plant defense[J].Frontiers in Plant Science,2015,6:109.DOI:10.3389/fpls.2015.00109.
[3]TADA Y,SPOEL S H,PAJEROWSKA-MUKHTAR K,et al.Plant immunity requires conformational charges of NPR1 via S-nitrosylation and thioredoxins[J].Science,2008,321:952-956.DOI:10.1126/science.1156970.
[4]SKELLY M J,LOAKE G J.Synthesis of Redox-active molecules and their signaling functions during the expression of plant disease resistance[J].Antioxidants and Redox Signaling,2013,19(9):990-997.DOI:10.1089/ars.2013.5429.
[5]汪开拓,郑永华,唐文才,等.茉莉酸甲酯处理对葡萄果实NO和H2O2水平及植保素合成的影响[J].园艺学报,2012,39(8):1559-1566.DOI:10.16420/j.issn.0513-353x.2012.08.015.
[6]WANG K T,LIAO Y X,KAN J Q,et al.Response of direct or priming defense against Botrytis cinerea to methyl jasmonate treatment at different concentrations in grape berries[J].International Journal of Food Microbiology,2015,194:32-39.DOI:10.1016/j.ijfoodmicro.2014.11.006.
[7]COHEN Y,RUBIN A E,KILFIN G.Mechanisms of induced resistance in lettuce against Bremia lactucae by DL-β-amino-butyric acid(BABA)[J].European Journal of Plant Pathology,2010,126(4):553-573.DOI:10.1007/s10658-009-9564-6.
[8]HAMIDUZZAMAN M,JAKAB G,BARNAVON L,et al.β-Aminobutyric acid-induced resistance against downy mildew in grapevine acts through the potentiation of callose formation and jasmonic acid signaling[J].Molecular Plant-Microbe Interactions,2005,18(8):819-829.DOI:10.1094/MPMI-18-0819.
[9]ZHANG Z K,YANG D P,YANG B,et al.β-Aminobutyric acid induces resistance of mango fruit to postharvest anthracnose caused by Colletotrichum gloeosporioides and enhances activity of fruit defense mechanisms[J].Scientia Horticulturae,2013,160(27):78-84.DOI:10.1016/j.scienta.2013.05.023.
[10]WANG L,ZHANG H,JIN P,et al.Enhancement of storage quality and antioxidant capacity of harvested sweet cherry fruit by immersion withβ-aminobutyric acid[J].Postharvest Biology and Technology,2016,118:71-78.DOI:10.1016/j.postharvbio.2016.03.023.
[11]廖云霞,费良航,夏明星,等.不同浓度β-氨基丁酸处理对葡萄果实抗病性的诱导模式研究[J].食品科学,2018,39(17):221-228.DOI:10.7506/spkx1002-6630-201817036.
[12]WANG K T,LIAO Y X,XIONG Q,et al.Induction of direct or priming resistance against Botrytis cinerea in strawberries byβ-aminobutyric acid and their effects on sucrose metabolism[J].Journal of Agricultural and Food Chemistry,2016,64(29):5855-5865.DOI:10.1021/acs.jafc.6b00947.
[13]COHEN Y,VAKNIN M,MAUCH-MANI B.BABA-induced resistance:milestones along a 55-year journey[J].Phytoparasitica,2016,44(4):513-538.DOI:10.1007/s12600-016-0546-x.
[14]龚波林.以溴化钾-溴酸钾-2’,7’-二氯荧光素为试剂对痕量水杨酸的间接荧光测定法[J].分析化学,2001,29(9):1055-1057.DOI:10.3321/j.issn:0253-3820.2001.09.019.
[15]MURPHY M E,NOACK E.Nitric oxide assay using hemoglobin method[J].Methods in Enzymology,1994,233:240-250.DOI:10.1016/S0076-6879(94)33027-1.
[16]NAGANO I,SHAPSHAK P,YOSHIOK M,et al.Increased NADPH-diaphorase reactivity and cytokine expression in dorsal root ganglia in acquired immunodeficiency syndrome[J].Journal of the Neurological Sciences,1996,136(1/2):117-128.DOI:10.1016/0022-510X(95)00317-U.
[17]RAHMAN I,KODE A,BISWAS S K.Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method[J].Nature Protocols,2007,1(6):3159-3165.DOI:10.1038/nprot.2006.378.
[18]?INDELá?L,?INDELá?OVáM,BURKETOVáL.Changes in activity of glucose-6-phosphate and 6-phosphogluconate dehydrogenase isozymes upon potato virus Y infection in tobacco leaf tissues and protoplasts[J].Plant Physiology and Biochemistry,1999,37(3):195-201.DOI:10.1016/S0981-9428(99)80034-5.
[19]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding[J].Analytical Biochemistry,1976,72(Suppl 1/2):248-254.DOI:10.1016/0003-2697(76)90527-3.
[20]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J].Methods,2001,25(4):402-408.DOI:10.1006/meth.2001.1262.
[21]CONRATH U.Molecular aspects of defencepriming[J].Trends in Plant Science,2011,16(10):524-531.DOI:10.1016/j.tplants.2011.06.004.
[22]O’BRIEN J A,DAUDI A,BUTT V S,et al.Reactive oxygen species and their role in plant defence and cell wall metabolism[J].Planta,2012,236(3):765-779.DOI:10.1007/s00425-012-1696-9.
[23]MOU Z,FAN W,DONG X.Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes[J].Cell,2003,113(7):935-944.DOI:10.1016/S0092-8674(03)00429-X.
[24]MURMU J,BUSH M J,DELONG C,et al.Arabidopsis basic leucinezipper transcription factors TGA9 and TGA10 interact with floral glutaredoxins ROXY1 and ROXY2 and are redundantly required for anther development[J].Plant Physiology,2010,154(3):1492-1504.DOI:10.1104/pp.110.159111.
[25]LINDERMAYR C,SELL S,MüLLER B,et al.Redox regulation of the NPR1-TGA1 system of Arabidopsis thaliana by nitric oxide[J].Plant Cell,2010,22(8):2894-2907.DOI:10.1105/tpc.109.066464.
[26]LEVéE V,MAJOR I,LEVASSEUR C,et al.Expression profiling and functional analysis of populus WRKY23 reveals a regulatory role in defense[J].New Phytologist,2009,184(1):48-70.DOI:10.1111/j.1469-8137.2009.02955.x.
[27]YU M,LAMATTINA L,SPOEL S H,et al.Nitric oxide function in plant biology:a redox cue in deconvolution[J].New Phytologist,2014,202(4):1142-1156.DOI:10.1111/nph.12739.
[28]KUMAR D.Salicylic acid signaling in disease resistance[J].Plant Science,2014,228:127-134.DOI:10.1016/j.plantsci.2014.04.014.
[29]AHARONI A,GALILI G.Metabolic engineering of the plant primarysecondary metabolism interface[J].Current Opinion in Biotechnology,2011,22(2):239-244.DOI:10.1016/j.copbio.2010.11.004.
[30]SIDDAPPAJI M H,SCHOLES D R,BOHN M,et al.Overcompensation in response to herbivory in Arabidopsis thaliana:the role of glucose-6-phosphate dehydrogenase and the oxidative pentose-phosphate pathway[J].Genetics,2013,195(2):589-598.DOI:10.1534/genetics.113.154351.
[31]LE HENANFF G,FARINE S,KIEFFER-MAZET F,et al.Vitis vinifera VvNPR1.1 is the functional ortholog of AtNPR1 and its overexpression in grapevine triggers constitutive activation of PRgenes and enhanced resistance to powdery mildew[J].Planta,2011,234(2):405-417.DOI:10.1007/s00425-011-1412-1.
[32]WALLY O,JAYARAJ J,PUNJA Z.Comparative resistance to foliar fungal pathogens in transgenic carrot plants expressing genes encoding for chitinase,β-1,3-glucanase and peroxidise[J].European Journal of Plant Pathology,2009,123(3):331-342.DOI:10.1007/s10658-008-9370-6.
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