全 文 :书!"#$%&
,2016,36(5):0979-0986
犃犮狋犪犅狅狋.犅狅狉犲犪犾.犗犮犮犻犱犲狀狋.犛犻狀.
!"#$:10004025(2016)05097908 犱狅犻:10.7606/j.issn.10004025.2016.05.0979
%&(
:20151012;)*&%+(:20160328
,-./
:
()*+%,-
(31270428)
0123
:
./0
(1980-),1,2345678,9:,;<=>?@#$%67。Email:wanglingli_521@163.com
ABCD
:
EFG
,
HI
,
458J:
,
;<=>?@#$%KLM8N%67
。Email:lwenzhe@nwu.edu.cn
45犝犞犅6789:;<=>?
9:@ABCDE
!"#
1,2,
$%&
2,3,
()
2
(1OP%Q 8R+%S,T!OP044000;2!"U% 8R+%%Q,!V710069;3WX:Y%Q 8R+%%Q,Z[WX
471022)
F G:\](犆犪犿狆狋狅狋犺犲犮犪犪犮狌犿犻狀犪狋犪Decne.)^ _`ab]+(Nyssaceae)\]_(犆犪犿狆狋狅狋犺犲犮犪),cdef$]gh;
kl\]ghmnopqrstuv\]gwxhy
|67O} UVB~28
\]
8h~,18\]
2h、4h、6h8hh~,12d
\]h
、MDA、 (Fpro)SOD,o 、 ¡¢£\]g,¤
UVB~\]8¥¦§8¨©$hª«,o¬\]gc\]®¯ UVB~°±h²³´$。?bµ
:(1)28\]¶UVB
8h~12d,·¸µ¹º»,¼ MDA、Fpro\]g
½¾¿
,
ÀÁ
8hUVB~28\]´8·ÃhÄÅÆÇ。(2)18\]¶UVB~12d,
ÈÉ
UVB~ÊËh¾¿,Ìͺ»,Fproµ¹¾¿;
2~6hh MDAK¸
ε¹ÏÐ
,
ÑÒÓÈÊ˾¿ÔÕÖ×Ø
;
8hUVB~h MDA·¸µ¹¾¿;SODÈ
ÊËhÙÚÔÛܺ
、
ÕÖ
、
ÝܺhÞß×Ø
,
ÀÁ
8hh UVB~à8\]á´8ÂÄ
ÅÆÇ
。(3)18\] 、 ¡¢£\]gÈÉ
UVB~ÊËhÙÚ½Ôâ¾×Ø,¼ã
8h
~h\]g½äl
,
å£ ¡£\]gµ¹l`¢£
。
æç?bèÁ
,
¾Ã UVB~
\]éêÂàhÆÇ
,
¼\]AëìÞ8o§8¨©íî
,
oàï´8\]g𫯾à UVBh
ÄÅ
。
HIJ
:
\]
;UVB~;\]g;8¥¦
KLMN$
:Q945.79; !O>PQ:A
犈犳犳犲犮狋狊狅犳犈狀犺犪狀犮犲犱犝犾狋狉犪狏犻狅犾犲狋犅犚犪犱犻犪狋犻狅狀狅狀犘犺狔狊犻狅犾狅犵犻犮犪犾犐狀犱犻犮犲狊犪狀犱
犆犪犿狆狋狅狋犺犲犮犻狀犆狅狀狋犲狀狋犻狀犆犪犿狆狋狅狋犺犲犮犪犪犮狌犿犻狀犪狋犪犇犲犮狀犲
WANGLingli1,2,ZHOUXiaojun2,3,LIU Wenzhe2
(1DepartmentofLifeScience,YunchengUniversity,Yuncheng,Shanxi044000,China;2ColegeofLifeScience,Northwest
University,Xian,710069,China;3SchoolofLifeSciences,LuoyangNormalUniversity,Luoyang,Henan471022,China)
犃犫狊狋狉犪犮狋:犆犪犿狆狋狅狋犺犲犮犪犪犮狌犿犻狀犪狋犪 Decne.(Nyssaceae)isa majorsourceofanticancercamptothecin
(CPT).ItisimperativetoinduceCPTaccumulationinordertodevelopCPTproductionstrategiestosatis
fyclinicalusesofCPT.Inthisstudy,twoyearold犆.犪犮狌犿犻狀犪狋犪weredealt8heachdaywithUVBradi
ationfor12days,andoneyearold犆.犪犮狌犿犻狀犪狋犪wererespectivelyarrangedtoradiate2h,4h,6hand8h
withUVBradiationeachdayfor12days.Thecontentsofchlorophyl,MDAandfreeproline(Fpro),the
activityofSODinleafandCPTcontentinyoungleaves,youngshootsandrootwereseparatelymeasured
afterUVBtreatment.InordertorevealthatcamptothecinisthedefenseproductofUVBstress,the
effectsofUVBradiationonthephysiologicalindicesandsecondarymetaboliteswereanalyzed.Theresults
showedthat:(1)intwoyearold犆.犪犮狌犿犻狀犪狋犪,chlorophylcontentwassignificantlydecreased,MDA,
FproandCPTcontentsweresignificantlyincreasedafter8hUVBtreatmentdaily.Itindicatedthat8h
UVBradiationcausedgreatstressinfluencesontwoyearold犆.犪犮狌犿犻狀犪狋犪.(2)Inoneyearold犆.犪犮狌
犿犻狀犪狋犪,withthetimeofUVBradiationincreasing,chlorophylcontentwasgradualydecreased;Fpro
contentwassignificantlyincreased;MDAcontenthadnosignificantlydifferencebetween2hand6hUV
Bradiation,butsignificantlyincreasedafter8hradiationcomparedwiththecontrol;SODactivityde
creasedfirstly,thenincreased,lastlydecreasedwiththetimeofUVBradiationprolongingeveryday.It
showedthat8hUVBradiationalsocausedstressinfluencesononeyearold犆.犪犮狌犿犻狀犪狋犪.(3)CPTcon
tentsinvegetativeorgansofoneyearold犆.犪犮狌犿犻狀犪狋犪weregradualyincreasedwiththetimeofUVBra
diationprolonging,andthecontentswerethehighestafter8hUVBradiationeachday.Moreover,CPT
contentincreasedmoreobviouslyinyoungleavesandyoungshootsthanthoseinroots.Itconfirmedthat
enhancedUVBradiationcausedcertaindamageto犆.犪犮狌犿犻狀犪狋犪,and犆.犪犮狌犿犻狀犪狋犪respondedtothis
stressby not only changing physiologicalindices,but also changing secondary metabolism to
accumulateCPT.
犓犲狔狑狅狉犱狊:犆犪犿狆狋狅狋犺犲犮犪犪犮狌犿犻狀犪狋犪;ultravioletB(UVB)radiation;camptothecin(CPT)content;physi
ologicalindex
犆犪犿狆狋狅狋犺犲犮犪犪犮狌犿犻狀犪狋犪Decne.belongstothe
Nyssaceaefamilyandisaperennialdeciduousplant
thatisuniquetoChina,andismainlydistributed
alongtheYangtzeRiverandSouthwestprovinces
ofChina[1].Theplantisknowntobotanyand
medicinebecauseitsvariousorganscontaintheal
kaloidcamptothecin (CPT)anditsderivatives
whichhaveimportantbiologicalactivities.
CPT,apentacyclicquinolinealkaloid,isan
effectivemedicineincancertreatment,whichwas
firstisolatedfromthestemof犆.犪犮狌犿犻狀犪狋犪,and
thestructuresofthisalkaloidweredeterminedby
Walandhiscolaborators[2].CPTisknownforits
remarkableanticanceractivitytoinhibittheeu
karyoticDNAtopoisomeraseI[3].Italsoinhibits
retrovirusessuchasthehumanimmunodeficiency
virus(HIV)andtheequineinfectiousanemiavi
rus[4].CPTisavaluablecompoundasachemicalpre
cursoroftopotecanandirinotecanwhichwereapproved
bytheUSFoodandDrugAdministrationin1996for
thetreatmentofovarianandcolorectalcancers[5].
Althoughmuchisknownaboutmanyfactors
whichaffecttheaccumulationofCPTin犆.犪犮狌犿犻
狀犪狋犪,stilonlylittleisknownabouttheeffectof
UVBradiationonCPTaccumulation.Becauseof
thepromisingclinicalusesofCPT,itisimportant
toinvestigatethefactorsaffectingCPTyieldin
plantmaterial.Inthepaststudy,droughtcanin
creasedCPTcontentinleavesof犆.犪犮狌犿犻狀犪狋犪[6].
Methyljasmonicacidandthetreatmentsofyeast
extractonleafdiscspunchedfrom犆.犪犮狌犿犻狀犪狋犪
seedlingspromotedthemRNAexpressionoftryp
tophandecarboxylase (TDC),akeyenzymein
volvedinCPTbiosynthesis[7].Similarly,methyl
jasmonicacidandyeastextracttreatmentson犆.
犪犮狌犿犻狀犪狋犪celsuspensionculturesincreasedCPT
accumulation[8].ItcanenhanceCPTproductionby
ethanoladditioninthesuspensioncultureofthe
endophyte,犉狌狊犪狉犻狌犿狊狅犾犪狀犻[9].Wang犲狋犪犾[10]re
portedCPTcontentdecreasedafter10daysUVB
radiation,andincreasedafter40daysUVBradia
tion.ButtheydidnotstudythechangeofCPT
contentbetween10days UVBradiation.This
studyinvestigatedtheeffectsofUVBradiation
between12daysontheaccumulationofCPTin犆.
犪犮狌犿犻狀犪狋犪,whichwouldprovideabasisformaxi
mizingCPTyieldanddesigninganaffectiveCPT
productionsystem.
089 ! " # $ % & 36ñ
1 Materialsandmethods
1.1 犘犾犪狀狋犿犪狋犲狉犻犪犾狊
Oneyearoldandtwoyearold犆.犪犮狌犿犻狀犪狋犪
plantsweregrownfromseeds.Seedsof犆.犪犮狌犿犻
狀犪狋犪wereselectedtogatherfromBotanicalGarden
ofXi’aninNovemberof2012and2013,thenre
spectivelyembeddedinwetsandat25℃inMarch
thenextyear.Aftertheseedsgeminated,theuni
formseedlingswereselectedandtransplantedinto
flowerpotsinthebotanicalgardenofNorthwestU
niversity(Xi’an,China).
1.2 犝犞犅狉犪犱犻犪狋犻狅狀
SupplementalUVBradiationwasprovidedby
filteredGucunbrand(GucunInstrumentFactory,
Shanghai,China)30 W sunlamps.Lampswere
suspendedaboveandperpendiculartotheplanted
rowsandfilteredwitheither0.13mmthickcelu
losediacetate(transmissiondownto290nm)for
UVBirradianceor0.13mmpolyesterplasticfilms
(absorbsalradiationbelow320nm)asacontrol.
Thedesiredirradiationwasobtainedbychanging
thedistancebetweenthelampsandtheplants.
Thespectralirradiancefromthelampswasdeter
minedwithanOptronicsModel742(OptronicsLa
boratories,Orlando,FL,USA)spectroradiome
ter.Thespectralirradiancewasweightedwitha
generalizedplantresponsespectrumandnormal
izedat300nmtoobtainthedesiredlevelofbiolog
icalyeffectiveUVBradiation.Thelampheighta
bovetheplantswasadjustedtomaintainadistance
of0.15mbetweenthelampsandthetopofthe
plantsandprovidedsupplementalirradiancesof
2.1effectiveuw·cm-2.Twoyearoldplantswere
irradiatedfor12daysand8hdaily.Inorderto
furtherilustratetheeffectsofsupplementalUVB
irradiance,oneyearoldplantswereirradiatedfor
12daysandrespectivelyarranged2h,4h,6hand
8hradiationdaily.FilteredUVBradiationwas
regardedasthecontrol(CK).
1.3 犇犲狋犲狉犿犻狀犪狋犻狅狀狅犳狆犺狔狊犻狅犾狅犵犻犮犪犾狆犪狉犪犿犲狋犲狉狊
Physiologicalparametersweredeterminatedin
leavesof犆.犪犮狌犿犻狀犪狋犪.Chlorophylwasextracted
with96% alcohol,andchlorophyl contentwas
measuredaccordingtothemethodofZhang[11].
Malonaldehyde(MDA)contentwasdetermined
accordingtothemethodofHeathandPacker[12].
Fprocontentwasextractedfromleavesin3%
aqueoussulphosalicylicacidandestimatedusing
ninhydrinreagent[13].Superoxidedismutase (SOD)
activitywasdeterminedaccordingtothemethodof
GiannopolitisandRies[14].
1.4 犆犘犜犮狅狀狋犲狀狋犪狀犪犾狔狊犻狊
1.4.1 犎犘犔犆犪狀犪犾狔狊犻狊 TheHPLCsystemconsis
tedofaHPLCpump (LC10ATvp),areversed
phasecolumn(VPODS,150mm×4.6mm,5μm)
andaUVVISdetector(SPD10Avp)forthede
tectionofCPTat254nm[15].Sampleinjectionvol
umewas10μLaccordingtothepresumablealka
loidcontent.Theflowratewas1.0mL· min-1.
Themobilephaseusedwasmethanol/water(62/
38,V/V).Columntemperaturewas25℃.Under
thiscondition,the HPLCchromatogramsofthe
standardandsamplesolutionswereshowedinFig.1.
CPTstandardwaskindlysuppliedbyDr.H.Bischof
ofBoehringerIngelheimPharmaKG.inGermany.
Fig.1 TheHPLCchromatogramsofthestandard
andsample(peak1:camptothecin)
1.4.2 犛狋犪狀犱犪狉犱犮狌狉狏犲 CPTstandard2.5mgwas
putin50mLvolumetricflask,suitableamountof
chromatographicmethanolwasadded,andmetered
volumeaftertheultrasonichelpingdissolve.The
solutionwasshakedandfilteredwith0.45μmmi
croporousmembrane,then0.05mg·mL-1stand
ardsolutionwasgot.2,3,5,8and10mLofCPT
standardsolutionwerepreciselymeasuredandput
in10mLvolumetricflask,dilutedwithchromato
graphicmethanol,thenrespectivelymeteredvol
1895ò ./0,ó:¾ÃUVB~\]8¥¦\]ghª«(ôF)
ume.ThestandardcurveforCPTwasconstructed
byseparateinjectionof10μLoftheabovemen
tionedstandardsolutionaccordingtotheabove
chromatographyconditions.Theregressionequa
tionbetweenpeakarea犢andtheconcentrationof
camptothecin犡 (μg·mL
-1)was:犢=16686犡
6577.5,犚2=0.9993.
1.4.3 犇犲狋犲狉犿犻狀犪狋犻狅狀狅犳犆犘犜犮狅狀狋犲狀狋 Thesam
plesweredriedintheshadeandgroundedina
mortar.100mgofthesampleswastransferredtoa
centrifugetubeand4mLmethanolwasadded.Af
terextractedwithsonicationfor10minatroom
temperature,30 mL waterand40 mL dichlo
romethanewereaddedandthiswasmixedvigor
ouslyfor5minonamagneticalstirrer[16],centrif
ugationfor10minat2000r·min-1yieldedtwo
phases.Thedichloromethanephase which was
provedtocontainCPT,wasrecoveredandevapo
ratedtodrynessinvacuumusingarotavapor.The
remainingresiduewasredissolvedinHPLCgrade
methanol(1mL),filteredwith0.45μm micro
porousmembranethengotsamplesolutionwhich
wasusedforthedeterminationofCPTcontent.
Samplepeaks withthesameretentiontimeto
standardswereverifiedbyspectralscananalysis.
2 Results
2.1 犈犳犳犲犮狋狊狅犳犝犞犅狉犪犱犻犪狋犻狅狀狅狀狋狑狅狔犲犪狉狅犾犱
犆.犪犮狌犿犻狀犪狋犪
Thecontentsofchlorophyl,MDAandFpro
aswelasSODactivityweremeasuredaftertwo
yearoldplantswereirradiatedwithUVBradia
tion.TheresultsshowedinTable1,chlorophyla
andbcontentswerereduced,chlorophyla/bratio
hadalesserextentincreaseunderenhancedUVB.
MDAandFprocontentsincreased.Theactivityof
SODwasreduced.CPTcontentsinyoungleaves,
youngshootsandrootof犆.犪犮狌犿犻狀犪狋犪hadobvi
ouslyincreasedafter UVBtreatmentcompared
withthecontrol,CPTcontentinroothasnosig
nificantdifferencecomparedwiththecontrol.
2.2 犝犞犅狉犪犱犻犪狋犻狅狀犲犳犳犲犮狋狊狅狀犮犺犾狅狉狅狆犺狔犾犮狅狀狋犲狀狋狊
犻狀狅狀犲狔犲犪狉狅犾犱犆.犪犮狌犿犻狀犪狋犪
TheeffectsofenhancedUVBonchlorophyl
犜犪犫犾犲1 犈犳犳犲犮狋狊狅犳犝犞犅狉犪犱犻犪狋犻狅狀狅狀狆犺狔狊犻狅犾狅犵犻犮犪犾犻狀犱犻犮犲狊
犪狀犱犆犘犜犮狅狀狋犲狀狋犻狀狋狑狅狔犲犪狉狅犾犱犆.犪犮狌犿犻狀犪狋犪
Index CK UVB(8h)
Chla/(mg·g-1) 1.507±0.125 1.193±0.266
Chlb/(mg·g-1) 0.646±0.102 0.505±0.142
Chla+Chlb/(mg·g-1) 2.152±0.165 1.698±0.075
Chla/Chlb 2.333±0.212 2.364±0.179
MDA/(nmol·g-1) 1.104±0.083 1.170±0.133
Fpro/(μg·g-1) 1.987±0.137 3.046±0.153
SOD/(U·g-1) 231.9±10.11 222.4±12.18
CPT/%
Youngleaves 0.219±0.017 0.313±0.019
Youngshoots 0.163±0.007 0.274±0.004
Root 0.045±0.008 0.067±0.013
Note:Eachassaywasrepeatedthreetimesfromthreeinde
pendentexperiments.Thedataarethemeans±SEMofthreerepli
cates.Asterisk()indicatesstatisticalysignificantdifference(犘<
0.05;ANOVA,Tukeytest).
contentsinoneyearold犆.犪犮狌犿犻狀犪狋犪wereshown
inFig.2.Thesametotwoyearoldplant,chloro
phylcontentsdecreasedafterUVBradiation,and
withthetreatmenttimeprolonging,thechloro
phylaandchlorophyl bcontentsgradualyde
creased.Thetotalchlorophylcontentof2h,4h,
6hand8hUVBtreatmentdailywasrespectively
reducedby9.4%,10.8%,15.7%,26.8%than
thecontrol.While8h UVBtreatmentdaily,
chlorophylaandbcontentsdecreased26.4%and
27.8%,respectively.Chlorophyla/bratiohada
littlechangecomparedwiththecontrol,itissug
gestedthatthedifferenceofdestroyedextentabout
chlorophylaandbisnotobvious.
2.3 犝犞犅狉犪犱犻犪狋犻狅狀犲犳犳犲犮狋狊狅狀犕犇犃,犉狆狉狅犮狅狀
狋犲狀狋狊犪狀犱犛犗犇犪犮狋犻狏犻狋狔犻狀狅狀犲狔犲犪狉狅犾犱犆.犪犮狌犿犻狀犪狋犪
UVBradiationhadobviouseffectsonMDA,
FprocontentandSODactivityin犆.犪犮狌犿犻狀犪狋犪.
WiththetimeofUVBradiationincreasing,Fpro
content gradualyincreased;SOD activity de
creasedfirstly,thenincreased,lastlydecreased;
MDAcontentkeptincreasingwiththetreatment
timeprolonging,however,therewerenosignifi
cantdifferencebetween6hUVBradiation,MDA
contentwassignificantlyincreasedafter8hUVB
treatment(Fig.3).UVBradiationofshorttime
inducedtheproductionofreactiveoxygenspecies
(ROS),suchasO2-· ,H2O2,andreducedSODac
tivity.Meanwhile,Fprocontentwassignificantly
increasedinordertodefenseUVBstress.When
289 ! " # $ % & 36ñ
Eachassaywasrepeatedthreetimesfromthreeindependent
experiments.Thedataarethemeans±SEMof
threereplicates.Differentlettersindicatestatisticaly
significantdifferences(犘<0.05;ANOVA,Tukeytest).
Thesameasbelow
Fig.2 EffectofUVBradiationonchlorophylcontent
inoneyearold犆.犪犮狌犿犻狀犪狋犪
Fig.3 EffectsofUVBradiationonMDA,Fprocontents
andSODactivityinoneyearold犆.犪犮狌犿犻狀犪狋犪
Fig.4 EffectofUVBradiationonCPTcontent
inoneyearold犆.犪犮狌犿犻狀犪狋犪
thetimeofUVBtreatmentreachedto6hdaily,
SODactivityincreasedinordertoeliminateover
muchreactiveoxygenspeciesandFprocontent
keptinarelativebalanceatthesametime.When
thetimeofUVBtreatmentreachedto8hdaily,
SODactivitydecreasedandFprocontentsignifi
cantlyincreased.
2.4 犝犞犅狉犪犱犻犪狋犻狅狀犲犳犳犲犮狋狊狅狀犆犘犜犮狅狀狋犲狀狋狊犻狀狅狀犲
狔犲犪狉狅犾犱犆.犪犮狌犿犻狀犪狋犪
UVBradiationalsohadobviouseffectson
CPTcontentsinyoungleaves,youngshootsand
rootsofoneyearold犆.犪犮狌犿犻狀犪狋犪(Fig.4).When
thetimeofenhancedUVBtreatmentreachedto8
hdaily,theCPTcontentsweresignificantlyin
creasedcomparedwiththecontrol.Withthetime
ofUVBradiationprolonging,CPTcontentsin
youngleavesandshootsincreasedfasterthanthat
intheroot,andCPTcontentofyoungleavesin
creasedto0.230%from0.106% ofthecontrol
groupafter8hradiationdaily,youngstemin
creasedfrom0.064%to0.158%,andtheincrease
ofCPTcontentinroothadalittlechange,in
creasedfrom0.036%to0.065%.
3 Discussion
UVBradiationhasmanyeffectsonplants.
Althoughtherearesomesignificanteffectsabout
UVBradiationonplantgrowthanddevelopment
incertainspeciesandecosystems,itturnedout
thattheoveraldamagingeffectsofaboveambient
UVBaremodestordifficulttodetectundernatu
ralconditions[17].Anexaminationofmorethan
200plantspeciesrevealsthatroughly20%aresen
sitive,50% aremildlysensitiveortolerantand
30% arecompletelyinsensitiveto UVBradia
tion[18].UVBradiationactsasakindofenviron
mentalstress,andtheplantwouldhavetoadaptto
UVBradiationtominimumthedamagewhenthe
stresslevelreachtoalimitation.Withashortpe
riodofUVBradiationin犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪
seedlings,rootswereelongatedandchlorophyl
andsolubleproteincontentwereincreasedinleaf,
butprolongedUVBradiationinhibitedtheelonga
tedrootlength,causedleafchlorophylcontent,
solubleproteincontentdecrease[19].Theeffectsof
enhanced UVB radiation on plantphysiology,
morphology,growthandbiomasshavebeeninves
tigatedextensively.EnhancedUVBradiationmay
exertanadverseinfluenceonthephysiologicaland
biochemicalprocessesofplants.Inthisstudy,we
reportedtheresponsesof犆.犪犮狌犿犻狀犪狋犪plantto
3895ò ./0,ó:¾ÃUVB~\]8¥¦\]ghª«(ôF)
enhancedUVBradiation.Thisissupportedbythe
earlierfindingsofintraspecificresponsesinfla
vonoid metabolismin犆狌犮狌犿犻狊狊犪狋犻狏狌狊[20],soy
bean[21]and犃.狋犺犪犾犻犪狀犪[22]andinflavonoidcon
tentandchlorophylcontentdecreasedinrice[23].
UVBradiationhadobviouseffectsonchloro
phylcontentsof犆.犪犮狌犿犻狀犪狋犪.TheUVBradia
tionmechanismofdecreasingchlorophylcontents
wasstilnotclear.Changesinchlorophylcontents
haveoftenbeenusedasanindextoassessthede
greesofUVBradiationsensitivity.Inthisstudy,
chlorophylcontentsin犆.犪犮狌犿犻狀犪狋犪leaveswere
alsosensitivetoenhancedUVBradiation.UVB
radiationsignificantlydecreasedchlorophyl con
tents,primarilybecauseUVBradiationdestroyed
thestructureofthechloroplast,inhibitedthesyn
thesisofnewchlorophylanddestroyedthestruc
tureofchloroplastenvelope membraneandin
creasedthedegradationofchlorophyl.Yang犲狋
犪犾.[24]reportedthatUVBradiationreducedthe
photosyntheticpigmentinleaves (includingthe
contentsofchlorophylandcarotenoid,especialy
thecontentofchlorophyla).Zhangindicatedthat
UVBradiationofdifferentintensitycanmakethe
growth,chlorophylcontentsdecreasein犞犻犮犻犪犳犪
犫犪seeding[25].Theseresultsshowedthatenhanced
UVBradiationmadetheplantchlorophyl dam
age,changedtheproportionofchlorophylaand
chlorophylb,affectedtheformationofphotosyn
theticproteincomplexes,andinhibitedtheforma
tionoforganeles.
LittlewasknownoftheresponsesofSODac
tivityandMDAcontentstoenhancedUVBradia
tioninthepast.Theprimaryexplanationofcel
membrane’sdamageinducedbyUVBwasthefree
radicaltheory.UVBradiationmayinducethepro
ductionofreactiveoxygenspecies(ROS),suchas
O2-· ,H2O2,andchangetheSODactivity,result
inmembranelipidperoxidation,whichfurtherlead
tothechangesofmembranestructureandfinaly
altermembranepermeability.Asaresult,thecels
ofplantareinjuredandMDAcontentincreased.
SODisoneoftheprotectiveenzymestodefense
theinjuryofROS.Innormalcircumstances,the
generationandeliminationofROSinplantsarein
astateofdynamicbalance.Inourstudy,SODac
tivityfirstlydecreased,thenincreased,lastlyde
creased,whichisconsistentwiththechangeof
SODactivityunderlowtemperaturestresscondi
tion[26].ThestressofUVBinducedtheproduc
tionofacertainamountofROSunderthetreat
mentof4hdaily,inordertoeliminateROStime
ly,theactivityofSODthatisintrinsicalin犆.犪犮狌
犿犻狀犪狋犪decreased.Meanwhile,Fprocontentsig
nificantlyincreasedbetween4hradiationdaily
comparedwiththecontrol,whichindicatedthein
creasedFproimprovedtheresistancetotheUVB
stress.AcertainamountofUVBradiationmay
increasethecontentofROSsuchasO2-·inplant,
andSODcancatalyzeO2-· disproportionationto
generateH2O2andO2,enhancetheactivityofan
tioxidantenzymesystem[27].Feng犲狋犪犾.[28]alsore
portedthatenhancedUVBradiationinlowdose
orshorttermtreatmentcanbrieflystimulatethe
activityofSODandPODtoincrease.In犆.犪犮狌犿犻
狀犪狋犪,whenthetimeofUVBtreatmentwas4-6
h,alargeamountofROSwasgenerated,SODac
tivitysignificantlyincreasedtoeliminatesupera
bundantROS,meanwhileFproandMDAcontents
hadnosignificantlydifferences.Thissuggested
thathigherSODactivitycaninhibitmembranelip
idoxidation,andkeepMDAcontentinabalance.
WhenthetimeofUVBtreatmentreachedto8h,
asUVBstressdeepened,SODactivitydecreased,
ROSwasaccumulatedandthelipidperoxidationof
membrane system was enhanced,accordingly
MDAandFprocontentsignificantlyincreased.We
cametoaconclusionthatUVBstressof8hdailyde
creasedtheactivityofprotectiveenzyme,causedacer
taindamageofbiomembrane.Itcanbefurthertestified
thattheleavesof犆.犪犮狌犿犻狀犪狋犪becamewitheringafter
wehadstudied10hUVBradiationdaily.
Freeprolineaccumulationhasbeenobserved
inresponsetoawiderangeofabioticandbiotic
stressesinplants.Prolineisconsideredtobeone
ofthefirstmetabolicresponsestostress,andis
perhapsasecond messenger[29].Environmental
factorsincludingwaterdeprivation,salinization,
489 ! " # $ % & 36ñ
highandlowtemperature,heavymetaltoxicity,
pathogeninfection,nutrientdeficiency,atmos
phericpolutionandUVradiationinducetheeleva
tionoftheprolinelevelinplants.In犆.犪犮狌犿犻狀犪狋犪
plant,enhancedUVBradiationmadeFprocontent
increase,itmaybeanadaptiveresponsetoresist
UVBradiationstress.Anegativecorrelationbe
tweenSODactivityandFprocontentwasfound
underUVBstressconditionin犆.犪犮狌犿犻狀犪狋犪.
MDAistheproductofmembranelipidperoxi
dation whentheplanttissuesufferedoxidative
stress,whichreflectsthedegreeofcel membrane
lipidperoxidationandtheplanttorespondto
stress.Inthispaper,MDAcontentshavenosig
nificantdifferencebetween6h UVBradiation
comparedwiththecontrol,however,MDAcon
tentwassignificantlyincreasedafter8h UVB
treatment.AlowdoseofUVBstressinducedthe
productionofacertainamountofROS,whilein
herentSODin犆.犪犮狌犿犻狀犪狋犪rapidlyeliminated
theseROS,andresultedinthedecreaseofSODac
tivity.WhenthetimeofUVBtreatmentreached
to8hdaily,alargenumberofROSweregenera
tedandcaused membranelipidperoxidation,so
MDAcontentwassignificantlyincreased.Incon
clusion,thereisacertaincorrelationamongthe
changeofSODactivity,Fprocontentand MDA
contentunderUVBstress,thedecreaseofSOD
activityisaccompaniedbytheincreaseofMDAor/
andFprocontent.
Undertheconditionofadversity,inaddition
totheplantphysiologicalindicescanbeinduced
somechanges,thesecondarymetabolismofplant
wilalsobechanged.Plantsinteractwiththeiren
vironmentbyproducingadiversearrayofseconda
rymetabolites,oneofwhichisalkaloid.Bioticand
abioticenvironmenthaveimportantrolesinthe
secondarymetabolizingofplant.Asasecondary
metabolite,CPTmayplayacrucialroleduringbi
oticandabioticstresses,whichposeagreatimpact
onalkaloidbiosynthesisandaccumulation[30].The
increaseofalkaloidaccumulationduringseedlings
developmentwasobservedandthisshowedthat
CPTmayplayadefensivefunctionfortheplants
duringthisvulnerablestageoftheirlifecycle.UV
lightresponsiveregionsinthepromoterofthe
tryptophandecarboxylase(狋犱犮)genein犆犪狋犺犪狋狉犪狀
狋犺狌狊狉狅狊犲狌狊hadbeenidentified[31].Inourstudy,
UVBradiationcanobviouslyenhanceCPTcontent
in犆.犪犮狌犿犻狀犪狋犪,wehypothesizethatUVBmight
stimulatetheexpressionof狋犱犮geneandincrease
CPTaccumulationin犆.犪犮狌犿犻狀犪狋犪.CPTaccumu
lationinducedby UVBradiationdemonstrated
thatCPT wasinvolvedinplantdefenseagainst
UVBradiation.When犆.犪犮狌犿犻狀犪狋犪sufferedUV
Bradiationinalowdose(2h,4h),CPTcontent
wasincreasedslowly;withthetimeofUVBradi
ationincreasing(6h,8h),CPTcontentwasin
creasedrapidly,whichilustratedthatthelong
timeofUVBradiationcausedcertaindamageto
犆.犪犮狌犿犻狀犪狋犪,and犆.犪犮狌犿犻狀犪狋犪defensedprima
rilybyincreasingthesecondarymetabolitesCPT.
Moreover,CPT contentsin youngleavesand
shootswereincreasedmoreobviouslythanthosein
root,itindicatedthatenhanced UVBradiation
couldpriorityimproveCPTaccumulationofaerial
organin犆.犪犮狌犿犻狀犪狋犪,becauseaerialorganmain
lysufferedUVBstress.
EnhancedUVBradiationcausedcertaindam
ageto犆.犪犮狌犿犻狀犪狋犪,notonlyaffectsthemorphol
ogyof犆.犪犮狌犿犻狀犪狋犪[10],butalsoaffectsthephysi
ologicalandbiochemicalmetabolism.Underthe
stressofUVB,犆.犪犮狌犿犻狀犪狋犪itselfdefensedthis
stressbychangingphysiologicalindices (i.e.,
SODactivity,MDAandFprocontent)andsecond
ary metabolismtoaccumulateCPT.Therefore,
thechangesofphysiologicalindicesandCPTaccu
mulationarealsotheadaptivemechanismtore
sponsetothestressofenhancedUVBradiationin
犆.犪犮狌犿犻狀犪狋犪.Furthermore,intheindustryplant
ingof犆.犪犮狌犿犻狀犪狋犪,themethodofsupplementing
8hUVBradiationdailytopromotetheincreaseof
CPTcontentisfeasible.
5895ò ./0,ó:¾ÃUVB~\]8¥¦\]ghª«(ôF)
犚犲犳犲狉犲狀犮犲狊:
[1] £+%Q£#$õö÷øù,£#$ú[M]."û:+%
üýþ
,1983.52(2):144147.
[2] WALLME,WANIMC,COOKECE,犲狋犪犾.Plantantitu
moragents,theisolationandstructureofcamptothecin,ano
velalkaliodalleukaemiaandtumorinhibitorfrom犆犪犿狆狋狅狋犺犲犮犪
犪犮狌犿犻狀犪狋犪 [J].犑狅狌狉狀犪犾狅犳 犃犿犲狉犻犮犪狀 犆犺犲犿犻犮犪犾犛狅犮犻犲狋狔,
1966,88(16):38883890.
[3] KJELDSENE,SVEJSTRUPJQ,GROMOVAII,犲狋犪犾.
Camptothecininhibitsboththecleavageandrelegationreac
tionsofeukaryoticDNAtopoisomeraseI[J].犑狅狌狉狀犪犾狅犳犕狅
犾犲犮狌犾犪狉犅犻狅犾狅犵狔,1992,228(4):10251030.
[4] PRIELE,SHOWWALTERSD,ROBERTSM,犲狋犪犾.The
topoisomeraseIinhibitor,camptothecin,inhibitsequineinfec
tionsanemiavirusreplicationinchronicalyinfectedCF2Th
cels[J].犑狅狌狉狀犪犾狅犳犞犻狉狅犾狅犵狔,1991,65(8):41374141.
[5] GREEMERSJP,DESPASR,FAVALLIG,犲狋犪犾.Topote
can,anactivedruginthesecondlinetreatmentofepithelialo
variancancer:resultsofalargeEuropeanphaseIIstudy[J].
犑狅狌狉狀犪犾狅犳犆犾犻狀犻犮犪犾犗狀犮狅犾狅犵狔,1996,14(12):30563061.
[6] LIUZJ.Droughtinducedin狏犻狏狅synthesisofcamptothecinin
犆犪犿狆狋狅狋犺犲犮犪犪犮狌犿犻狀犪狋犪seedlings[J].犘犺狔狊犻狅犾狅犵犻犪犘犾犪狀狋犪
狉狌犿,2000,110(4):483488.
[7] L?PEZMEYERM,NESSLERCL.Tryptophandecarboxylaseis
encodedbytwoantonomouslyregulatedgenesin犆犪犿狆狋狅狋犺犲犮犪犪犮狌
犿犻狀犪狋犪whicharediferentialyexpressedduringdevelopmentand
stress[J].犜犺犲犘犾犪狀狋犑狅狌狉狀犪犾,1997,11(6):11671175.
[8] SONGSH,BYUNSY.Characterizationofcelgrowthand
camptothecinproductionincelculturesof犆犪犿狆狋狅狋犺犲犮犪犪犮狌
犿犻狀犪狋犪[J].犑狅狌狉狀犪犾狅犳 犕犻犮狉狅犫犻狅犾狅犵狔犪狀犱 犅犻狅狋犲犮犺狀狅犾狅犵狔,
1998,8(6):631638.
[9] VENUGOPALANA,SRIVASTAVAS.Enhancedcamptoth
ecinproductionbyethanoladditioninthesuspensioncultureof
theendophyte,犉狌狊犪狉犻狌犿狊狅犾犪狀犻 [J].犅犻狅狉犲狊狅狌狉犮犲犜犲犮犺狀狅犾狅
犵狔,2015,188:251257.
[10] .ÿ!,EFG.¾ÃUVB~\] "8$#$8
$ghª«
[J].#$+%%&,2011,29(6):712717.
WANGHX,LIU WZ,EffectsofenhancedUVBradiation
onbiomassandcontentsofcamptothecinand10hydroxy
camptothecinin犆犪犿狆狋狅狋犺犲犮犪犪犮狌犿犻狀犪狋犪 [J].犘犾犪狀狋犛犮犻犲狀犮犲
犑狅狌狉狀犪犾,2011,29(6):712717.
[11] %&.#$8%æç()[M].Õÿ:Õÿ+%*+üý
þ
,1985:134135.
[12] HEATH RI,PACKERL.Photoperoxidationinisolated
chloroplasts:I.Kineticsandstoichiometryoffattyacidper
oxidation[J].犃狉犮犺犻狏犲狊狅犳犅犻狅犮犺犲犿犻狊狋狉狔犪狀犱犅犻狅狆犺狔狊犻犮狊,
1968,125(1):189198.
[13] BATESLS,WALDRONRP,TEAREID.Rapiddetermi
nationoffreeprolineforwaterstressstudies[J].犘犾犪狀狋犪狀犱
犛狅犻犾,1973,39(1):205208.
[14] GIANNOPOLITISCN,RIESSK.Superoxidedismutases.I.
Purificationandquantitativerelationshipwithwatersolublepro
teininseedlings[J].犘犾犪狀狋犘犺狔狊犻狅犾狅犵狔,1977,59(2):315318.
[15] ./0.\],-./h?@\]g0NÞßh67
[D].!V:!"U%,2006.
[16] VANHENGELAJ,HARKESMP,WICHERSHJ,犲狋犪犾.
Characterizationofcalusformationandcamptothecinproduc
tionbycellinesof犆犪犿狆狋狅狋犺犲犮犪犪犮狌犿犻狀犪狋犪[J].犘犾犪狀狋犆犲犾犾,
犜犻狊狊狌狉犲犪狀犱.犗狉犵犪狀犆狌犾狋狌狉犲,1992,28(1):1118.
[17] SEARLESPS,FLINTSD,CALDWELLM M.Ametaa
nalysisofplantfieldstudiessimulatingstratosphericozone
depletion[J].犗犲犮狅犾狅犵犻犪,2001,127(1):110.
[18] TERAMURAAH.EffectsofultravioletBradiationonthe
growthandyieldofcropplants[J].犘犺狔狊犻狅犾狅犵犻犪犘犾犪狀狋犪狉狌犿,
1983,58(3):415427.
[19] HAN W,HANR.EffectofdifferenttimesofUVBradia
tiononseedlinggrowthof犃狉犪犫犻犱狅狆狊犻狊狋犺犪犾犻犪狀犪[J].犆犺犻狀犲狊犲
犅狌犾犾犲狋犻狀狅犳犅狅狋犪狀狔,2015,50(1):4046.
[20] MURALINS,TERAMURAAH.Intraspecificdifferences
in犆狌犮狌犿犻狊狊犪狋犻狏狌狊sensitivitytoultravioletBradiation[J].
犘犺狔狊犻狅犾狅犵犻犪犘犾犪狀狋犪狉狌犿,1986,68(4):673677.
[21] D’SURNEYSJ,TSCHAPLINSKITJ,EDWARDSNT,犲狋
犪犾.Biologicalresponsesoftwosoybeancultivarsexposedto
enhancedUVBradiation[J].犈狀狏犻狉狅狀犿犲狀狋犪犾犪狀犱犈狓狆犲狉犻
犿犲狀狋犪犾犅狅狋犪狀狔,1993,33(3):347356.
[22] FISCUSEL,PHILBECKR,犲狋犪犾.Growthof犃狉犪犫犻犱狅狆狊犻狊fla
vonoidmutantsundersolarradiationandUVfilters[J].犈狀狏犻狉狅狀
犿犲狀狋犪犾犪狀犱犈狓狆犲狉犻犿犲狀狋犅狅狋犪狀狔,1999,41(3):231245.
[23] TERAMURAAH,ZISKALH,SZTEINAE.Changesin
growthandphotosyntheticcapacityofricewithincreasedUVB
radiation[J].犘犺狔狊犻狅犾狅犵犻犪犘犾犪狀狋犪狉狌犿,1991,83(3):373380.
[24] 123,4 5,.67.¾ÃUVB~89:;jABA
hª«
[J].8N%&,2000,20(1):3942.
YANGJH,CHENT,WANG XL.EfectofenhancedUVB
radiationonendogenousABAandfreeprolinecontentsinwheat
leaves[J].犃犮狋犪犈犮狅犾狅犵犻犮犪犛犻狀犻犮犪,2000,20(1):3942.
[25] %è,?0@,ó.ÌAÃBUVB~ÄÅCD
"8ÚEFGhª«
[J].![:YU%%&
(
)*+%ý
),2010,35(1):105110.
ZHANGHX,WUNB,HULT,犲狋犪犾.Theeffectsofdif
ferenttreatmentswith UVBradiationstressonseedling
growthandcharacterofchlorophylfluorescenceof犞犻犮犻犪犳犪
犫犪L..[J].犑狅狌狉狀犪犾狅犳犛狅狌狋犺狑犲狊狋犆犺犻狀犪犖狅狉犿犪犾犝狀犻狏犲狉狊犻狋狔
(NaturalScienceEdition),2010,35(1):105110.
[26] HIJ,%KL,1
M.»NÄÅ\] "SOD、MDA
hª«
[J].OP+%67,2002,15(2):197202.
FENGJC,ZHANGYJ,YANGTZ.Effectoflowtempera
turestressonthemembranelipidperoxidationandthecon
centrationoffreeprolinein犆犪犿狆狋狅狋犺犲犮犪犪犮狌犿犻狀犪狋犪seedling
[J].犉狅狉犲狊狋犚犲狊犲犪狉犮犺,2002,15(2):197202.
[27] 4 5,Q
[J].U±+%%&,1999,19(4):453455.
CHENT,REN H X,WANGXL.Influenceofenhanced
UVBradiationonantioxidantsystemsinwheatleaves[J].
犃犮狋犪犛犮犻犲狀狋犻犪犲犆犻狉犮狌犿狊狋犪狀狋犻犪犲,1999,19(4):453455.
[28] HV,VWG,HXY,ó.¾ÃUVB~ZD[\]¨
©hª«
[J].#$%&,1999,41(8):833836.
FENGGN,ANLZ,FENGHY,犲狋犪犾.Effectsofenhanced
UVBradiationonproteinmetabolismofbeanleaves[J].犃犮
狋犪犅狅狋犪狀犻犮犪犛犻狀犻犮犪,1999,41(8):833836.
[29] HAREPD,CRESSW A,Metabolicimplicationsofstress
inducedprolineaccumulationinplants[J].犘犾犪狀狋犌狉狅狑狋犺
犚犲犵狌犾犪狋犻狅狀,1997,21(2):79102.
[30] ^Y_,` £a,` 2a,ó.\] "£\]g10b,]gcdh«¯Gefg
[J].#$%&,2003,45(7):
809814.
ZUYG,TANGZH,YUJH,犲狋犪犾.Differentresponsesof
camptothecinand10hydroxycamptothecintoheatshockin
犆犪犿狆狋狅狋犺犲犮犪犪犮狌犿犻狀犪狋犪seedlings[J].犃犮狋犪犅狅狋犪狀犻犮犪犛犻狀犻犮犪,
2003,45(7):809814.
[31] OUWERKERKPBF,HALLARDD,VERPOORTER,犲狋犪犾.
IdentificationofUVBlightresponsiveregionsinthepromoterof
thetryptophandecarboxylasegenefrom犆犪狋犺犪狉犪狀狋犺狌狊狉狅狊犲狌狊[J].
犘犾犪狀狋犕狅犾犲犮狌犾犪狉犅犻狅犾狅犵狔,1999,41(4):491503.
(
!"
:
#$%
)
689 ! " # $ % & 36ñ