海水胁迫对菠菜(Spinacia olerancea L.)叶绿体活性氧和叶绿素代谢的影响

»29 »8 ù2009 M83 ÿÐACTAECOLOGICASINICAVol.29,No.8Aug., 2009htp:/www.ecologica.cnÁ “:SEÚnp VÄ SùÁ'ù “(20050307031); Ê 81 SÐÁ'ù “(BK2006140)là ° ù:2008-05-09; ©è ° ù:2008-12-10YßTCorrespondingauthor.E-mail:srguonjau.edu.cnZ £ çk(SpinaciaoleranceaL.)= s8= s Í¥Y孙 锦1, 2,贾永霞1,郭世荣1,李 娟1(1. 2Ø j<vÐóÐý, 2Ø 210095;2.Å Õ 8 j< SÐý vùî î, §Ü 730070)K1:Z £ ùË ÖÕõ=k 0Z £ ÖÕ §3|'¹ k,¨ £ ¡ZE,ùî Z £ çk(SpinaciaoleranceaL.)= s8(ROS)= s Í(Chl)¥Y。²TV ü,Z £ ç/, 2ñk ÖÕ= s8 =Ñ| Ö0(O-·2 )Á 3 Îq、VÄ _(H2O2)d= (MDA)c A÷ 6Ú,i Oõ=k¥4Úv¿ §3|'õ=k= s8 =¥ÑÄþ Ä ½(SOD)、 FÊÓ ÖVÄþ ½(APX)、!ÅtÎð ½(GR) FÊÓ Ö(AsA)、Îð!Åt(GSH)c (®¿ §3|',7VÄþ ½(POD)Ú¿ §3|'Ä4J,Ú(MV)ÉB P2ñk ÖÕ= s8 =O-·2Á 3Î qF y、H2O2c 4Ú、 ÉVÄF×, b“4AsA üA® k= s8 =ROS £ Ü,ϳ ®MV/î¥ø×¥ ÉVÄ 。Z £ ç/, 2ñk ÖÕ= Ë= s Íb(Chlb)、= s Ía(Chla)# î -8þÉð= s Í Ö(Pchl)、 Ãðú(Mg-protoIX)、ðú(Proto) Uúð(Uro)c üA®,7 ä Íð(PBG)-¼û Ö(ALA)Æ,Chlî sE¸,i Oõ=k¥ sEñv¿ §3|'MVÉBF Õ sEñ,7AsAsϳ ®Z £ çMV/î¥E¸T¨。Z £ ç üA4Ú õ=k= Ë= s Í ½(Chlase)7 §3|' ÀµY,MV) Øõ=kChlase¥Yñv¿ §3|',AsA2ñ ÖÕ= ËChlase Àµ üAY。 ²T ª ü,Z £ç/,k= s8 =ROSÐChl á MM1,ÇYV= s8 ¥Ä PChl³,7 O PChlî¥PBG_UroÄç sE。 0Z £ ÖÕ §3|'= s8 b“ROSö1YVSODAsA-GSHÖÌ“d, b“ ? ï <,h ROS= s8 ¥Ä å Chlî¥ sEñ,i OZ £ ç Chlase¥Y l;7Z £ ùË ÖÕõ=k= s8 b“ROSö1G ¿SODPOD,ROS¥ b“ ? ïµK,V7Á ROSv Æ,= s8 ¥Ä å Chlî¥ sEñø×,i OZ £ çA÷4Ú Chlase,F Chl³。1oM:Z £k(SpinaciaoleranceaL.);= s8;= s ÍÓcI|:1000-0933(2009)08-4361-11 Ïms Ë|:Q143, Q945, Q948 ÓDS M :AEffectsofseawaterstressonmetabolismofreactiveoxygenspeciesandchlorophylinchloroplastsofspinach(SpinaciaoleranceaL.)SUNJin1, 2, JIAYong-Xia1, GUOShi-Rong1, LIJuan11ColegeofHorticulture, NanjingAgriculturalUniversity, Nanjing210095, China2VegetableInstitute, GansuAcademyofAgriculturalSciences, Lanzhou730070, ChinaActaEcologicaSinica, 2009, 29(8):4361 4371.Abstract:Theefectsofseawaterstresonmetabolismofreactiveoxygenspecies(ROS)andchlorophyl(Chl)inchloroplastoftwospinachcultivars, Yuanyespinach(seawatersensitivecultivar)andHelanNo.3 (seawatertolerantcultivar)wereinvestigatedbyhydroponics.Theresultsshowedthatproductionrateofsuperoxideradical(O-·2), contentofhydrogenperoxide(H2O2)andmalonaldehyde(MDA)inchloroplastoftwospinachcultivarswereremarkablyincreasedbyseawaterwithhigherrateincv.Yuanye;Underthestresofseawater, theactivityofanti-oxidationenzymesuchassuperoxidedismutase(SOD), ascorbicacidperoxidase(APX)andglutathionereductase(GR), andthecontentofhtp:/www.ecologica.cnantioxidantsuchasascorbicacid(AsA)andglutathionereduced(GSH)inchloroplastofcv.Yuanyewerelowerthanthoseofcv.HelanNo.3, whileperoxidase(POD)activityinchloroplastofcv.Yuanyewashigherthanthatofcv.HelanNo.3;Thesupplementofmethylviologen(MV), thephoto-oxidant, enhancedtheproductionrateofO-·2, thecontentofH2O2 andMDAinchloroplastoftwospinachcultivars, whiletheadditionofAsA, theeliminatorforROS, decreasedROSlevelandaleviatedoxidizationofplasmolemma.Thecontentofchlorophylb(Chlb), chlorophyla(Chla)andprecursorofChlsuchasprotochlorophyl(Pchl), Mg-protoporphyrin (Mg-ProtoIX), protoporphyrin (Proto )anduroorphyrinogen (Uro)wasremarkablydecreased, butthecontentofporphobilinogen(PBG)and-aminolevulinicacid(ALA)wasincreasedbythestressofseawater, whichleadtoinhibitionforChlsynthesis, andtheinhibitionwasaggravatedbyMVandeliminatedbyAsA.Chlorophylase(Chlase)activityintheleavesofcv.YuanyewasimprovedwhileitwasnotinfluencedintheleavesofHelanNo.3underseawaterstress.TheChlaseactivityintheleavesofcv.YuanyewasmoregreatlyafectedbyMVtreatmentthanthatofHelanNo.3, however, ChlaseactivityoftwocultivarswasnotinfluencedbyAsA.TheseresultssuggestthatROSiscloselyrelatedtoChlmetabolism, whichnotonlyinjureplasmolemmabutalsoinhibittheprocesoftransformationofPBGtoUro asaresultofChldecomposition.Inseawatertolerantcultivar(cv.HelanNo.3), theROSeliminationismainlydependedonSODandAsA-GSHsystem, whichcouldaleviateoxidationinjuryofROStochloroplastmembraneandinhibitionofChlsynthesis, andChlaseactivitywaslessinfluencedbyseawaterstress;whileinseawatersensitivecultivar(cv.Yuanye), itismainlydependedonSODandPOD, whichhaslimitedabilitytoeliminateROSandleadtoaccumulatemassROStosevereoxidationinjuryofchloroplastmembraneandinhibitionofChlsynthesis, andChlaseactivitywassignificantlyimprovedbyseawaterstress, whichenhancedChldecomposition.KeyWords:seawater;spinach(SpinaciaoleranceaL.);chloroplast;reactiveoxygenspecies;chlorophyl= s Í(Chl) ¹ o K×1¥ ä Í,Ç V lv ; ?,7 OÎî“d#;“dQÏ 1 。±þ8 =Chlc ¥Ú® | %¿Chlîг¥î ÿ Ü 2 。Chl 3þî3¥ © BçCp¸,ûöYChl¥î,V7 Chlc ®Chl³ ½³Ä³ ÕQ Ë, Ï= s Í ½(Chlase)ÐChl³¥1“ á M 4 ;(ROS)9 V Chl³ 5,Yuzo© 6 ª¹ROS V ?Ð Chl4- 6Ì¥ åÊ,V7F ÎChl³。±þ®Ñ 7 、SO28 、NO2 9、 £s ç5 、 º10©Hq/,Chl³ÐROSµ1;÷ ç/,ROS ÐChl³EChlî#EÊÄû ü 。= s8 Á 3ROS¥×1% 11,÷ ç/= s8 =ROSv Æ 12, 13 ,= s8$ ? 3VÄQ,; ä ͳ,Y ±þT¨。Z £ ç/,k(SpinaciaoleranceaL.)= ËChlc ® 14, ;Î q¥® ®¿Chlc ® îÁ 15 。yN,ùîZ £ ç/k= s8ROSÐChl¥1“, üZ £ çTþ 3 Ø 3Ä¥Y µ×1 IN´。' k¨ £ ¡ZE,ùî Z £ çk= s8ROSÐChl¥Y, ù ü= s8ROSÐChl¥1“,¹Z £ ¡Tþ4 Ø G 。1 ÐZE1.1 k kʨ¥k ÖÕ¹Z £ ùË ÖÕõ=k 0Z £ ÖÕ §3|' 14;Z £ |1ÏS Êê ûZ×,9c÷ ¹26.64g·L-1,pH¹7.8,' Ö0ÐFî¹:Na+:349.98 mmol·L-1;Cl-:410.14 mmol·L-1;Mg2+:40.20 mmol·L-1;SO2-4 :21.15mmol·L-1;Ca2+:7.61 mmol·L-1;K+:6.83 mmol·L-1。1.2 ) Ø !Âk¿2007 M8À11 2Ø j<vÐ1 eÑ i =É。k±é ¡â -Ó14 , ks2sÉ:Z £ çk= s8ROS¥Y:2ñ ÖÕsY !2ñ) Ø,'1/2 Hoagland!A(vu,EC´0.14 ds·m-1,pH7.3)c40%Z £¥1/2 Hoagland!A() Ø u,EC´1.77ds·m-1,pH7.6),sYV U¹:Y:õ=kv;H: §3|'v;Y+S:õ=k) ØH+S: §3|') Ø。ç±»104362 3 ÿ Ð 29 htp:/www.ecologica.cn?,|) رé Æc40%Z £¥1/2Hoagland!AÏ,) Ø»3?»6?Ê |±é1 3ÉÄ­/¥»4»5= Ë4 |= s8,©çM1·S;Z £ ç/J,Ú(MV) FÊÓ Ö(AsA)k= ËChl¥Y。2ñ ÖÕsY !8ñ) Ø:1/2 Hoagland!A(v u,CK);1/2 Hoagland!A+1mol·L-1 MV(CK+MV);1/2 Hoagland!A+5mmol·L-1 AsA(CK+AsA);1/2 Hoagland!A+1mol·L-1MV+5mmol·L-1 AsA(CK+MV+AsA);c40%Z £¥1/2 Hoagland!A(T);c40%Z £¥1/2Hoagland!A+1mol·L-1 MV(T+MV);c40%Z £¥1/2Hoagland!A+5mmol·L-1 AsA(T+AsA);c40%Z £¥1/2Hoagland!A+1mol·L-1 MV+5mmol·L-1 AsA(T+MV+AsA)。Z £) ت»2?,sY¨1 molMV5mmol·L-1 AsA= ë ¨ ,MV ¥Å H¨8s ¹0.05%Tween20 ·³,v¨F8s ¹0.05%Tween20¥ b £) Ø24hªMV) Ø¥Bö±é¨5mmol·L-1 AsA= ë ¨ , 6Bö±é¨© b £= ë ¨ ,72 hªÊ |±é1 3ÉÄ­/¥»4»5= ËÉChl -8þÉc = s8 =Ñ| Ö0(O-·2 )Á 3 Î q、VÄ _(H2O2)d= (MDA)c #Chlase¥©ç,i9 ØO-·2Á 3 Îq、H2O2MDAc ¥M´(M´=) Ø´/v´)。 2 k (ׯ3Q, Û 。1.3 ©ç·S#ZE1.3.1 = s8¥4 | ITakeda© 16¥ZE。¹ £= s8B,¨Percol k41500g/0 Ö23min,|4 |¥= s8MǺÉÏ, P= s8 ir1mg.ml-1,©ç= s8¥, qr90%。1.3.2 O-·2Á 3 Î q、ÑÄþ Ä ½(SOD)、VÄþ ½(POD)¨0.05mol·L-1、pH7.8¥ÖÏ A(PBS) d= s825ª,âÉ(protein)c ¨ I ®¡E;O-·2Á 3 Î q¨¦¹S© 17¥ZE©ç,ʹnmol·min-1·mg-1·Chl;SOD¨¦¹S© 18¥ZE,Å ¡ ¹:(NBT);ÄÎð50%¥ ½ ¹1ñ ïÊ(U),U·min-1·g-1·proteinV U;POD¨ß7 !pE 19,1 min Ä XâÉbÄ¥470 nm/OD´MĹBñ ½ ïÊ(U),ʹU·min-1·g-1·protein。 1.3.3 H2O2c ¨0.2mol·L-1¥HClO4 d= s83,vUchida© 20¥ZE©ç,mol·mg-1·ChlV U。1.3.4 MDAc ¨5%¥ Ø oY Ö(TCA) d= s83,vHeathPacker 21¥ZE©ç,mol·mg-1·ChlV U。1.3.5 FÊÓ ÖVÄþ ½(APX)、!ÅtÎð ½(GR)¨0.05 mol·L-1¥K2HPO4-KH2PO4Ï A(pH7.0) d= s82 5, APX¨NakanoAsada22ZE©ç, ½molAsA·min-1·g-1·proteinV U;GRvFoyerHaliwel 23¥ZE, ½molNADPH·min-1·g-1·proteinV U。1.3.6 FÊÓ Ö(AsA)c ¿8s 15¥1 è¨5%¥TCA d= s8,vÓD 24 î ZE©ç,mg·g-1·ChlV U。1.3.7 Îð!Åt(GSH)Ä!Åt(GSSG)c ¨5%Ü £ Ö d= s83,vElman 25¥ZE©ç,Êmmol·g-1·ChlV U。1.3.8 Chlî -8þÉc = s Ía(Chla)、= s Íb(Chlb)c ¨d4 |E©ç26 ,ÊV U¹mg·g-1.FW;ðú(Proto)、 Ãðú(Mg-ProtoIX)、ð= s Í Ö(Pchl)¨HodginsVanHuystee¥ZE27, Uúð(Uro) ä Íð(PBG)©ç¨Bogorad¥ZE 28,-¼û Ö(ALA)©çvÜ ùZE29 ,Ê (V U¹g·g-1·FW。1.3.9 ChlasekChl¹Q¸þ,vMargaret 30ZE©ç,ʹmolChl·min-1·g-1·FW。4363 8 ù äÆ ©:Z £ çk(SpinaciaoleranceaL.)= s8= s Í¥Y htp:/www.ecologica.cn1.4 sk ¨MicrosoftExcelSPSS10.0 Èqs,¨Duncan's¯µEÉ×1 。2 ²TÐs2.1 Z £ çk= s8O-·2Á 3 Î q、H2O2MDAc ¥YZ £ ç/, ñk ÖÕ= s8O-·2Á 3 Î q(m1)、H2O2c (m1) Ø»3?á Î 6Ú,»6?µ/ ¯A÷Ú¿Mv,i Oõ=k¥ 6Úv¿ §3|'。 Â) Ø»6?õ=ksY4Ú40.98%32.82%, §3|'sY4Ú15.35%20.93%。Z £ ç/,2ñk ÖÕ= s8MDAc (m1)¥MÄ? pÐO-·2Á 3 Î qH2O2c M »。 Vn,Z £ ç/,k= s8O-·2Á 3 Î qF y,H2O2v Æ,Á «VÄÁþMDAc 9F,= s8 s ; 0Z £ ÖÕ §3|' O-·2Á3 Î qH2O2c ®,MDAc M ,= s8 ñ l。m1 Z £ çk= s8O-·2Á 3 Î q、H2O2MDAc ¥YFig.1 EfectsofseawaterstressonproductionrateofO-·2 , contentofH2O2 andMDAinchloroplastsofspinachY:õ=kv,controlofYuanyespinach;Y+S:õ=k40%Z £ i) Ø, 40%concentrationofseawatertreatmentofYuanyespinach;H:§3|v,controlofHelanNo.3;H+Y: §3|40%Z £ i) Ø, 40%concentrationofseawatertreatmentofHelanNo.3;mÏM3 VUµsA÷(P'D0.05),SameletersmeantnosignificantdiferenceatP<0.05levelwithinthecharts2.2 Z £ çk= s8SOD、POD¥YZ £ ç»3?,õ=k §3|'= s8 =SOD(m2)sY1Mv4Ú21.35%14.11%; ç»6?,õ=k1vA÷®49.78%,7 §3|'1vA÷4Ú30.12%。Z £ ç/,õ=k= s8 =POD(m2)ûà 6Ú, ç»3?1v4Ú54.38%, ç»6?1v4Ú118.23%;7 §3|' ç»3? vA÷4Ú89.49%, ç»6?» üA®,Ç1v4Ú21.21%。 Vn,Z £ ç/, 0Z £ ÖÕ §3|' SÔ û Ú¥SOD,O-·2 b“ ? ïv¿õ=k,7POD) Ø»6?/,H2O2 b“ ? ïl¿õ=k。2.3 Z £ çk= s8 =APX、GR¥YZ £ ç/,õ=k= s8APX(m3)A÷/,»3?»6?sY v®66.90%75.76%,7 §3|'= s8APXA÷ 6Ú,»3?»6?sY v4Ú86.03%75.52%;GR(m3)ÐAPXM »,õ=k= s8GR v®,7 §3|' v4Ú。 Vn,Z £ ç/, §3|'= s8 Ú¥APXGR,H2O2 b“ ? ï <7õ=k= s8APXGR/,H2O2 b“ ? ï Ð,V7ÁH2O2= s8 =v Æ。2.4 Z £ çk= s8AsA、GSHc GSH/GSSG1´¥YZ £ ç/,õ=k= s8AsAc (m4) ت»3?»6?sY vA÷®36.14%48.19%,7 §3|' ç»3?ÐvµsA÷,»6? vA÷®25.52%;GSH(m4)ÐAsA4364 3 ÿ Ð 29 htp:/www.ecologica.cnM »,õ=k= s8 =GSHc ) ت»3?»6?sY v®21.61%37.06%,7 §3|'»3?ÐvµsA÷,»6? v®19.97%;õ=k= s8GSH/GSSG1´(m4) ت»3?»6?sY vA÷®72.22%74.06%,7 §3|') Ø»6?ÐvµsA÷。Vn,Z £ ç/, §3|'= s8AsAGSHc ¥® üAl¿õ=k,i OGSH/GSSG1´MÄ üA,»û GSHÐGSSG­W¥ Ü。2.5 Z £ ç/MVAsAk= s8O-·2Á 3 Î q、H2O2MDAc M´¥Ym5²TA U,Z £ ç/2ñk ÖÕ= s8O-·2Á 3 Î q、H2O2MDAc M´ (v¿1;vHq/,MV) Ø¥k= s8O-·2Á 3 Î q、H2O2MDAc ¥M´9v¿1,AsA) Ø¥M´l¿1,7MV+AsA) Ø BMV) Ø®Z £ ç/,MV、AsAMV+ASA) Ø¥ f ÐvM »。 ª üZ £ ç Pk= s8 =ROS £ Ü4Ú, ÉVÄñF×MV) ØÉB PROS9F, ÉVÄñF4365 8 ù äÆ ©:Z £ çk(SpinaciaoleranceaL.)= s8= s Í¥Y htp:/www.ecologica.cn,7AsA V b“sROS, P ÉVÄñ®。2.6 Z £ ç/MV、AsAk= ËChlb、Chla# -8þÉPchl、Mg-protoIX、Proto、Uro、PBGALAc¥YV1²TV ü,Z £ ç P2ñk ÖÕ= Ë¥Chlb、Chla、Pchl、Mg-protoIX、ProtoIX、Uroc (1Mv®,7PBGALAc 6Ú, Ï §3|'= ËUroc ¥®l¿õ=k,sY®12.50%25.34%, ª üZ £ ç/2ñk ÖÕ¥Chlî sE,i O §3|'¥ sEñl¿õ=k。vHq/,MV) ØChl -8þÉc ¥YÐZ £) ØM »,MV¥Eñv¿Z £) ØAsA) Ø Põ=kChl -8þÉc §3|'Y üA;MV+AsA P2ñk ÖÕ= ËChlb、Chla、Pchl、Mg-protoIX、ProtoIX、Uroc BMV) ص î4Ú,7 PPBGALAc ®。Z £ ç/,ò) ØChl -8þÉc ¥YÐv'M »。 Vn,Z £ ç Pk= ËChlî sE¸, §34366 3 ÿ Ð 29 htp:/www.ecologica.cn|' sEñl¿õ=k;MVÉBF sEñ,AsAsϳ ÕE¸T¨。2.7 Z £ ç/MV、AsAk= ËChlase¥YZ £ çA÷4Úõ=k= ËChlase(m6),7 §3|' Àµ üAY。vZ £ç/,MVMV+AsA) ØA÷4Úõ=k= ËChlase,7AsA Àµ üAY。 §3|'vHq/,MV、AsAMV+AsA) Ø (Chlase Àµ üAY;Z £) Ø/,MVMV+AsAA÷4ÚChlase,7AsA Àµ üAY。V1 Z £ ç/MV、AsAk= Ë= s Íî -8þÉc ¥YTable1 EffectsofMVandAsAonChlprecursorscontentofspinachleavesunderseawaterstress) ØTreatmentsChlb(mg·g-1 FW)Y HChla(mg·g-1 FW)Y HPchl(g·g-1FW)Y HMg-ProtoIX(g·g-1FW)Y HProto(g·g-1FW)Y HUro(g·g-1FW)Y HPBG(g·g-1 FW)Y HALA(g·g-1FW)Y HCK 0.202b 0.350a 0.537b 0.815a 0.379b 0.475b 0.250b 0.265b 0.646b 0.639b 13.423b14.831a10.558d10.161f0.782d 0.835fCK+MV 0.144d 0.250d 0.428e 0.695d 0.263d 0.402d 0.141d 0.197d 0.398d 0.520d 3.624e 6.607e12.806c12.145d3.454a 5.543aCK+AsA 0.253a 0.358a 0.648a 0.804a 0.418a 0.480b 0.309a 0.267b 0.721a 0.753a 15.613a15.079a11.346d10.825ef2.483b 1.546eCK+MV+AsA 0.157c 0.301c 0.467cd0.772b 0.301c 0.470b 0.192c 0.245c 0.488c 0.553cd 6.593d 9.409d11.219d11.484de3.031ab 4.948bT 0.165c 0.296c 0.482c 0.727bc0.265d 0.436c 0.141d 0.241c 0.365e 0.578c 10.022c12.977b13.633bc13.600c1.523c 1.298dT+MV 0.106e 0.256d 0.322f 0.679d 0.209e 0.303f 0.118f 0.211d 0.316f 0.467e 4.321e 7.220e15.