增強UV-B輻射對喜樹生理指標及喜樹堿含量的影響

王玲麗，周曉君，劉文哲

(1 運城學院 生命科學系，山西運城 044000；2 西北大學 生命科學學院，西安 710069;3 洛陽師范學院 生命科學學院，河南洛陽 471022)

增強UV-B輻射對喜樹生理指標及喜樹堿含量的影響

王玲麗1，2，周曉君2，3，劉文哲2*

(1 運城學院 生命科學系，山西運城 044000；2 西北大學 生命科學學院，西安 710069;3 洛陽師范學院 生命科學學院，河南洛陽 471022)

摘要:喜樹(Camptotheca acuminata Decne.)隸屬于藍果樹科(Nyssaceae)喜樹屬(Camptotheca),為抗癌藥物喜樹堿的主要資源，提高喜樹堿的積累以滿足臨床需求是喜樹堿開發的重要途徑。該研究運用UV-B輻射對2年生喜樹進行每天8 h輻射處理，對1年生喜樹分別設置每天2 h、4 h、6 h和8 h的輻射處理，連續處理12 d后分別測定各處理喜樹葉的葉綠素、MDA、游離脯氨酸 (Fpro)含量和SOD活性，以及幼葉、幼枝和根中喜樹堿含量，分析UV-B輻射對喜樹生理指標和次生代謝物的影響，以揭示喜樹堿為喜樹適應UV-B輻射逆境的防御產物。結果顯示：(1)2年生喜樹經UV-B每天8 h輻射處理12 d后，葉綠素含量較對照顯著降低，而MDA、Fpro和喜樹堿含量均增加，說明每天8 h UV-B輻射對2年生喜樹產生了較強的脅迫傷害。(2)1年生喜樹經UV-B輻射處理12 d后，隨著每天UV-B輻射時間的增加，葉綠素含量不斷降低，Fpro含量顯著增加；每天2～6 h處理的MDA含量與對照無顯著差異，但總體隨處理時間增加呈上升趨勢；每天8 h UV-B輻射的MDA含量較對照顯著增加；SOD活性隨每天處理時間的延長呈先下降、后上升、再下降的變化趨勢，說明每天8 h的UV-B輻射對一年生喜樹也產生了脅迫傷害。(3)1年生喜樹幼葉、幼枝和根中喜樹堿含量隨著每天UV-B輻射時間的延長均呈遞增趨勢，而且每天8 h輻射處理的喜樹堿含量均最高，其中幼葉和幼枝中喜樹堿含量顯著高于根中含量。實驗結果表明，增強UV-B輻射對喜樹造成了一定的傷害，而喜樹通過改變生理以及次生代謝機制，以進一步產生喜樹堿來響應增強UV-B的脅迫。

關鍵詞:喜樹；UV-B輻射；喜樹堿含量；生理指標

Camptotheca acuminataDecne.belongstotheNyssaceaefamilyandisaperennialdeciduousplantthatisuniquetoChina,andismainlydistributedalongtheYangtzeRiverandSouthwestprovincesofChina[1].Theplantisknowntobotanyandmedicinebecauseitsvariousorganscontainthealkaloidcamptothecin(CPT)anditsderivativeswhichhaveimportantbiologicalactivities.

CPT,apentacyclicquinolinealkaloid,isaneffectivemedicineincancertreatment,whichwasfirstisolatedfromthestemofC. acuminata,andthestructuresofthisalkaloidweredeterminedbyWallandhiscollaborators[2].CPTisknownforitsremarkableanti-canceractivitytoinhibittheeukaryoticDNAtopoisomeraseI[3].Italsoinhibitsretrovirusessuchasthehumanimmunodeficiencyvirus(HIV)andtheequineinfectiousanemiavirus[4].CPTisavaluablecompoundasachemicalprecursoroftopotecanandirinotecanwhichwereapprovedbytheUSFoodandDrugAdministrationin1996forthetreatmentofovarianandcolorectalcancers[5].

AlthoughmuchisknownaboutmanyfactorswhichaffecttheaccumulationofCPTinC. acuminata,stillonlylittleisknownabouttheeffectofUV-BradiationonCPTaccumulation.BecauseofthepromisingclinicalusesofCPT,itisimportanttoinvestigatethefactorsaffectingCPTyieldinplantmaterial.Inthepaststudy,droughtcanincreasedCPTcontentinleavesofC. acuminata[6].MethyljasmonicacidandthetreatmentsofyeastextractonleafdiscspunchedfromC. acuminataseedlingspromotedthemRNAexpressionoftryptophandecarboxylase(TDC),akeyenzymeinvolvedinCPTbiosynthesis[7].Similarly,methyljasmonicacidandyeastextracttreatmentsonC. acuminatacellsuspensionculturesincreasedCPTaccumulation[8].ItcanenhanceCPTproductionbyethanoladditioninthesuspensioncultureoftheendophyte, Fusarium solani[9].Wanget al[10]reportedCPTcontentdecreasedafter10daysUV-Bradiation,andincreasedafter40daysUV-Bradiation.ButtheydidnotstudythechangeofCPTcontentbetween10daysUV-Bradiation.ThisstudyinvestigatedtheeffectsofUV-Bradiationbetween12daysontheaccumulationofCPTinC. acuminata,whichwouldprovideabasisformaximizingCPTyieldanddesigninganaffectiveCPTproductionsystem.

1Materials and methods

1.1Plant materials

One-year-oldandtwo-year-oldC. acuminataplantsweregrownfromseeds.SeedsofC. acuminatawereselectedtogatherfromBotanicalGardenofXi’aninNovemberof2012and2013,thenrespectivelyembeddedinwetsandat25 ℃inMarchthenextyear.Aftertheseedsgeminated,theuniformseedlingswereselectedandtransplantedintoflowerpotsinthebotanicalgardenofNorthwestUniversity(Xi’an,China).

1.2UV-B radiation

SupplementalUV-BradiationwasprovidedbyfilteredGucunbrand(GucunInstrumentFactory,Shanghai,China) 30Wsunlamps.Lampsweresuspendedaboveandperpendiculartotheplantedrowsandfilteredwitheither0.13mmthickcellulosediacetate(transmissiondownto290nm)forUV-Birradianceor0.13mmpolyesterplasticfilms(absorbsallradiationbelow320nm)asacontrol.Thedesiredirradiationwasobtainedbychangingthedistancebetweenthelampsandtheplants.ThespectralirradiancefromthelampswasdeterminedwithanOptronicsModel742 (OptronicsLaboratories,Orlando,FL,USA)spectroradiometer.Thespectralirradiancewasweightedwithageneralizedplantresponsespectrumandnormalizedat300nmtoobtainthedesiredlevelofbiologicallyeffectiveUV-Bradiation.Thelampheightabovetheplantswasadjustedtomaintainadistanceof0.15mbetweenthelampsandthetopoftheplantsandprovidedsupplementalirradiancesof2.1effectiveuw·cm-2.Two-year-oldplantswereirradiatedfor12daysand8hdaily.InordertofurtherillustratetheeffectsofsupplementalUV-Birradiance,one-year-oldplantswereirradiatedfor12daysandrespectivelyarranged2h, 4h, 6hand8hradiationdaily.FilteredUV-Bradiationwasregardedasthecontrol(CK).

1.3Determination of physiological parameters

PhysiologicalparametersweredeterminatedinleavesofC. acuminata.Chlorophyllwasextractedwith96%alcohol,andchlorophyllcontentwasmeasuredaccordingtothemethodofZhang[11].Malonaldehyde(MDA)contentwasdeterminedaccordingtothemethodofHeathandPacker[12].Fprocontentwasextractedfromleavesin3%aqueoussulphosalicylicacidandestimatedusingninhydrinreagent[13].Superoxidedismutase(SOD)activitywasdeterminedaccordingtothemethodofGiannopolitisandRies[14].

1.4CPT content analysis

1.4.1HPLCanalysisTheHPLCsystemconsistedofaHPLCpump(LC-10ATvp),areversedphasecolumn(VP-ODS, 150mm×4.6mm, 5μm)andaUV-VISdetector(SPD-10Avp)forthedetectionofCPTat254nm[15].Sampleinjectionvolumewas10μLaccordingtothepresumablealkaloidcontent.Theflowratewas1.0mL·min-1.Themobilephaseusedwasmethanol/water(62/38,V/V).Columntemperaturewas25 ℃.Underthiscondition,theHPLCchromatogramsofthestandardandsamplesolutionswereshowedinFig.1.CPTstandardwaskindlysuppliedbyDr.H.BischoffofBoehringerIngelheimPharmaKG.inGermany.

Fig.1　The HPLC chromatograms of the standard and sample (peak 1: camptothecin)

1.4.2StandardcurveCPTstandard2.5mgwasputin50mLvolumetricflask,suitableamountofchromatographicmethanolwasadded,andmeteredvolumeaftertheultrasonichelpingdissolve.Thesolutionwasshakedandfilteredwith0.45μmmicroporousmembrane,then0.05mg·mL-1standardsolutionwasgot. 2, 3, 5, 8and10mLofCPTstandardsolutionwerepreciselymeasuredandputin10mLvolumetricflask,dilutedwithchromatographicmethanol,thenrespectivelymeteredvolume.ThestandardcurveforCPTwasconstructedbyseparateinjectionof10μLoftheabove-mentionedstandardsolutionaccordingtotheabovechromatographyconditions.TheregressionequationbetweenpeakareaYandtheconcentrationofcamptothecinX (μg·mL-1)was: Y=16 686X-6 577.5, R2=0.999 3.

1.4.3DeterminationofCPTcontentThesamplesweredriedintheshadeandgroundedinamortar. 100mgofthesampleswastransferredtoacentrifugetubeand4mLmethanolwasadded.Afterextractedwithsonicationfor10minatroomtemperature, 30mLwaterand40mLdichloromethanewereaddedandthiswasmixedvigorouslyfor5minonamagneticalstirrer[16],centrifugationfor10minat2 000r·min-1yieldedtwophases.ThedichloromethanephasewhichwasprovedtocontainCPT,wasrecoveredandevaporatedtodrynessinvacuumusingarotavapor.Theremainingresiduewasre-dissolvedinHPLC-grademethanol(1mL),filteredwith0.45μmmicroporousmembranethengotsamplesolutionwhichwasusedforthedeterminationofCPTcontent.Samplepeakswiththesameretentiontimetostandardswereverifiedbyspectralscananalysis．

2Results

2.1Effects of UV-B radiation on two-year-old C. acuminata

Thecontentsofchlorophyll,MDAandFproaswellasSODactivityweremeasuredaftertwo-year-oldplantswereirradiatedwithUV-Bradiation.TheresultsshowedinTable1,chlorophyllaandbcontentswerereduced,chlorophylla/bratiohadalesserextentincreaseunderenhancedUV-B.MDAandFprocontentsincreased.TheactivityofSODwasreduced.CPTcontentsinyoungleaves,youngshootsandrootofC. acuminatahadobviouslyincreasedafterUV-Btreatmentcomparedwiththecontrol,CPTcontentinroothasnosignificantdifferencecomparedwiththecontrol.

2.2UV-B radiation effects on chlorophyll contents in one-year-old C. acuminata

TheeffectsofenhancedUV-Bonchlorophyllcontentsinone-year-oldC. acuminatawereshowninFig.2.Thesametotwo-year-oldplant,chlorophyllcontentsdecreasedafterUV-Bradiation,andwiththetreatmenttimeprolonging,thechlorophyllaandchlorophyllbcontentsgraduallydecreased.Thetotalchlorophyllcontentof2h, 4h, 6hand8hUV-Btreatmentdailywasrespectivelyreducedby9.4%, 10.8%, 15.7%, 26.8%thanthecontrol.While8hUV-Btreatmentdaily,chlorophyllaandbcontentsdecreased26.4%and27.8%,respectively.Chlorophylla/bratiohadalittlechangecomparedwiththecontrol,itissuggestedthatthedifferenceofdestroyedextentaboutchlorophyllaandbisnotobvious.

Table 1　Effects of UV-B radiation on physiological indices

Note:Eachassaywasrepeatedthreetimesfromthreeindependentexperiments.Thedataarethemeans±SEMofthreereplicates.Asterisk(*)indicatesstatisticallysignificantdifference(P< 0.05;ANOVA,Tukeytest).

2.3UV-B radiation effects on MDA, Fpro contents and SOD activity in one-year-old C. acuminata

Each assay was repeated three times from three independent experiments. The data are the means ± SEM of three replicates. Different letters indicate statisticallysignificant differences (P< 0.05; ANOVA, Tukey test).The same as belowFig.2　Effect of UV-B radiation on chlorophyll content in one- year-old C. acuminata

Fig.3　Effects of UV-B radiation on MDA, Fpro contents and SOD activity in one- year-old C. acuminata

Fig.4　Effect of UV-B radiation on CPT content in one-year-old C. acuminata

2.4UV-B radiation effects on CPT contents in one-year-old C. acuminata

UV-BradiationalsohadobviouseffectsonCPTcontentsinyoungleaves,youngshootsandrootsofone-year-oldC. acuminata (Fig.4).WhenthetimeofenhancedUV-Btreatmentreachedto8hdaily,theCPTcontentsweresignificantlyincreasedcomparedwiththecontrol.WiththetimeofUV-Bradiationprolonging,CPTcontentsinyoungleavesandshootsincreasedfasterthanthatintheroot,andCPTcontentofyoungleavesincreasedto0.230%from0.106%ofthecontrolgroupafter8hradiationdaily,youngstemincreasedfrom0.064%to0.158%,andtheincreaseofCPTcontentinroothadalittlechange,increasedfrom0.036%to0.065%.

3Discussion

UV-Bradiationhasmanyeffectsonplants.AlthoughtherearesomesignificanteffectsaboutUV-Bradiationonplantgrowthanddevelopmentincertainspeciesandecosystems,itturnedoutthattheoveralldamagingeffectsofabove-ambientUV-Baremodestordifficulttodetectundernaturalconditions[17].Anexaminationofmorethan200plantspeciesrevealsthatroughly20%aresensitive, 50%aremildlysensitiveortolerantand30%arecompletelyinsensitivetoUV-Bradiation[18].UV-Bradiationactsasakindofenvironmentalstress,andtheplantwouldhavetoadapttoUV-Bradiationtominimumthedamagewhenthestresslevelreachtoalimitation.WithashortperiodofUV-BradiationinArabidopsis thalianaseedlings,rootswereelongatedandchlorophyllandsolubleproteincontentwereincreasedinleaf,butprolongedUV-Bradiationinhibitedtheelongatedrootlength,causedleafchlorophyllcontent,solubleproteincontentdecrease[19].TheeffectsofenhancedUV-Bradiationonplantphysiology,morphology,growthandbiomasshavebeeninvestigatedextensively.EnhancedUV-Bradiationmayexertanadverseinfluenceonthephysiologicalandbiochemicalprocessesofplants.Inthisstudy,wereportedtheresponsesofC. acuminataplanttoenhancedUV-Bradiation.ThisissupportedbytheearlierfindingsofintraspecificresponsesinflavonoidmetabolisminCucumis sativus[20],soybean[21]andA. thaliana[22]andinflavonoidcontentandchlorophyllcontentdecreasedinrice[23].

UV-BradiationhadobviouseffectsonchlorophyllcontentsofC. acuminata.TheUV-Bradiationmechanismofdecreasingchlorophyllcontentswasstillnotclear.ChangesinchlorophyllcontentshaveoftenbeenusedasanindextoassessthedegreesofUV-Bradiationsensitivity.Inthisstudy,chlorophyllcontentsinC. acuminataleaveswerealsosensitivetoenhancedUV-Bradiation.UV-Bradiationsignificantlydecreasedchlorophyllcontents,primarilybecauseUV-Bradiationdestroyedthestructureofthechloroplast,inhibitedthesynthesisofnewchlorophyllanddestroyedthestructureofchloroplastenvelopemembraneandincreasedthedegradationofchlorophyll.Yanget al.[24]reportedthatUV-Bradiationreducedthephotosyntheticpigmentinleaves(includingthecontentsofchlorophyllandcarotenoid,especiallythecontentofchlorophylla).ZhangindicatedthatUV-Bradiationofdifferentintensitycanmakethegrowth,chlorophyllcontentsdecreaseinVicia fabaseeding[25].TheseresultsshowedthatenhancedUV-Bradiationmadetheplantchlorophylldamage,changedtheproportionofchlorophyllaandchlorophyllb,affectedtheformationofphotosyntheticproteincomplexes,andinhibitedtheformationoforganelles.

Freeprolineaccumulationhasbeenobservedinresponsetoawiderangeofabioticandbioticstressesinplants.Prolineisconsideredtobeoneofthefirstmetabolicresponsestostress,andisperhapsasecondmessenger[29].Environmentalfactorsincludingwaterdeprivation,salinization,highandlowtemperature,heavymetaltoxicity,pathogeninfection,nutrientdeficiency,atmosphericpollutionandUV-radiationinducetheelevationoftheprolinelevelinplants.InC. acuminataplant,enhancedUV-BradiationmadeFprocontentincrease,itmaybeanadaptiveresponsetoresistUV-Bradiationstress.AnegativecorrelationbetweenSODactivityandFprocontentwasfoundunderUV-BstressconditioninC. acuminata.

MDAistheproductofmembranelipidperoxidationwhentheplanttissuesufferedoxidativestress,whichreflectsthedegreeofcellmembranelipidperoxidationandtheplanttorespondtostress.Inthispaper,MDAcontentshavenosignificantdifferencebetween6hUV-Bradiationcomparedwiththecontrol,however,MDAcontentwassignificantlyincreasedafter8hUV-Btreatment.AlowdoseofUV-BstressinducedtheproductionofacertainamountofROS,whileinherentSODinC. acuminatarapidlyeliminatedtheseROS,andresultedinthedecreaseofSODactivity.WhenthetimeofUV-Btreatmentreachedto8hdaily,alargenumberofROSweregeneratedandcausedmembranelipidperoxidation,soMDAcontentwassignificantlyincreased.Inconclusion,thereisacertaincorrelationamongthechangeofSODactivity,FprocontentandMDAcontentunderUV-Bstress,thedecreaseofSODactivityisaccompaniedbytheincreaseofMDAor/andFprocontent.

Undertheconditionofadversity,inadditiontotheplantphysiologicalindicescanbeinducedsomechanges,thesecondarymetabolismofplantwillalsobechanged.Plantsinteractwiththeirenvironmentbyproducingadiversearrayofsecondarymetabolites,oneofwhichisalkaloid.Bioticandabioticenvironmenthaveimportantrolesinthesecondarymetabolizingofplant.Asasecondarymetabolite,CPTmayplayacrucialroleduringbioticandabioticstresses,whichposeagreatimpactonalkaloidbiosynthesisandaccumulation[30].TheincreaseofalkaloidaccumulationduringseedlingsdevelopmentwasobservedandthisshowedthatCPTmayplayadefensivefunctionfortheplantsduringthisvulnerablestageoftheirlifecycle.UVlightresponsiveregionsinthepromoterofthetryptophandecarboxylase(tdc)geneinCathatranthus roseushadbeenidentified[31].Inourstudy,UV-BradiationcanobviouslyenhanceCPTcontentinC. acuminata,wehypothesizethatUV-BmightstimulatetheexpressionoftdcgeneandincreaseCPTaccumulationinC. acuminata.CPTaccumulationinducedbyUV-BradiationdemonstratedthatCPTwasinvolvedinplantdefenseagainstUV-Bradiation.WhenC. acuminatasufferedUV-Bradiationinalowdose(2h, 4h),CPTcontentwasincreasedslowly;withthetimeofUV-Bradiationincreasing(6h, 8h),CPTcontentwasincreasedrapidly,whichillustratedthatthelongtimeofUV-BradiationcausedcertaindamagetoC. acuminata,andC. acuminatadefensedprimarilybyincreasingthesecondarymetabolites-CPT.Moreover,CPTcontentsinyoungleavesandshootswereincreasedmoreobviouslythanthoseinroot,itindicatedthatenhancedUV-BradiationcouldpriorityimproveCPTaccumulationofaerialorganinC. acuminata,becauseaerialorganmainlysufferedUV-Bstress.

EnhancedUV-BradiationcausedcertaindamagetoC. acuminata,notonlyaffectsthemorphologyofC. acuminata[10],butalsoaffectsthephysiologicalandbiochemicalmetabolism.UnderthestressofUV-B, C. acuminataitselfdefensedthisstressbychangingphysiologicalindices(i.e.,SODactivity,MDAandFprocontent)andsecondarymetabolismtoaccumulateCPT.Therefore,thechangesofphysiologicalindicesandCPTaccumulationarealsotheadaptivemechanismtoresponsetothestressofenhancedUV-BradiationinC. acuminata.Furthermore,intheindustryplantingofC. acuminata，themethodofsupplementing8hUV-BradiationdailytopromotetheincreaseofCPTcontentisfeasible.

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(編輯:潘新社)

EffectsofEnhancedUltraviolet-BRadiationonPhysiologicalIndicesandCamptothecinContentinCamptotheca acuminataDecne

WANGLingli1，2,ZHOUXiaojun2，3,LIUWenzhe2*

(1DepartmentofLifeScience,YunchengUniversity,Yuncheng,Shanxi044000,China; 2CollegeofLifeScience,NorthwestUniversity,Xi’an, 710069,China;3SchoolofLifeSciences,LuoyangNormalUniversity,Luoyang,He’nan471022,China)

Abstract:Camptotheca acuminata Decne. (Nyssaceae) is a major source of anticancer camptothecin (CPT). It is imperative to induce CPT accumulation in order to develop CPT production strategies to satisfy clinical uses of CPT. In this study, two-year-old C. acuminata were dealt 8 h each day with UV-B radiation for 12 days, and one-year-old C. acuminata were respectively arranged to radiate 2 h，4 h，6 h and 8 h with UV-B radiation each day for 12 days. The contents of chlorophyll, MDA and free proline (Fpro), the activity of SOD in leaf and CPT content in young leaves, young shoots and root were separately measured after UV-B treatment. In order to reveal that camptothecin is the defense product of UV-B stress, the effects of UV-B radiation on the physiological indices and secondary metabolites were analyzed. The results showed that: (1) in two-year-old C. acuminata, chlorophyll content was significantly decreased, MDA, Fpro and CPT contents were significantly increased after 8 h UV-B treatment daily. It indicated that 8 h UV-B radiation caused great stress influences on two-year-old C. acuminata. (2) In one-year-old C. acuminata, with the time of UV-B radiation increasing, chlorophyll content was gradually decreased; Fpro content was significantly increased; MDA content had no significantly difference between 2 h and 6 h UV-B radiation, but significantly increased after 8 h radiation compared with the control; SOD activity decreased firstly, then increased, lastly decreased with the time of UV-B radiation prolonging every day. It showed that 8 h UV-B radiation also caused stress influences on one-year-old C. acuminata. (3) CPT contents in vegetative organs of one-year-old C. acuminata were gradually increased with the time of UV-B radiation prolonging, and the contents were the highest after 8 h UV-B radiation each day. Moreover, CPT content increased more obviously in young leaves and young shoots than those in roots. It confirmed that enhanced UV-B radiation caused certain damage to C. acuminata, and C. acuminata responded to this stress by not only changing physiological indices, but also changing secondary metabolism to accumulate CPT.

Key words:Camptotheca acuminata; ultraviolet-B (UV-B) radiation; camptothecin (CPT) content; physiological index

文章編號:1000-4025(2016)05-0979-08

doi:10.7606/j.issn.1000-4025.2016.05.0979

收稿日期:2015-10-12;修改稿收到日期:2016-03-28

基金項目:國家自然科學基金(31270428)

作者簡介:王玲麗(1980-)，女，在讀博士研究生，講師，主要從事結構植物學研究。 E-mail：wanglingli_521@163.com *通信作者:劉文哲，教授，博士生導師，主要從事結構植物學與繁殖生態學研究。E-mail: lwenzhe@nwu. edu.cn

中圖分類號:Q945.79;

文獻標志碼:A