.leurnal *{ :. Phytopathology J Phytopathol 159.707112 (2011) @ 20ll Blackwell Verlag GmbH School of Biological Sciences, Universiti Saiw Malaysia, Puhu, Pinang, Malaysia Peroxidase Activity after Viral Infection and Whitefly Infestation in Juvenileand Mature lraves af Solanum lycopersicum Herraov Drnuct, Touovrrsu Serno2, Arnreo Aru Hesslxl, Ar. Tnrrnxr Azzl, RoNero Er,rnreuEMonem.s3, SuHen-l Ar Herrnol, Furao Mrert' and Sazlr,v AntsAxAna Authors' addressesl rSchool of Biological Scienas, Universiti Sains Malaysia, 11800hrlau, Pioang, Malaysia; zFaculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan; 3Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; aDepartment of Medical Microbiolory, University of Malaya, Kuala Lumpur, Malaysia (correspondence to H. Diang. E-mait [email protected]) Reeived Deenber 8,2010; acepted July 12,20ll Keyword* peroxidase activity, leafage, Tonuto yellow leaf curl virus, Bemisia tobaci Abstuact Whiteffy infestation and the begomoviruses that they transmit have besn shown to afect the activities of plant defence proteins, but with no relation to hetero- phylly, a proccss of great importancc undcdying the overall biology of plants. Hereo we have assess€d the efrects of Totruto yellow leaf anl virus IYLCV) infection on Solawn lycapersicun peroxidase (POD) activity and have examined whether leaves of different ages exhibit diferential POD activity in response to infection and infestation with Bemkiq tabaci B biotype. Wc uscd leaf discs of two ages (luvcnile and mature) with two diferent infection statuscs (infected and healthy) to exarninc the activity of the tomato plant peroxidase using guaiacol as a zubstrate and taking exposure time into aocouat. S. lycopersicum showed increased POD activity in the presence of TYLCV. The activity of the enzyme was higher in matura than in juvenile leaves, In Seneral, beth infecrcd. and healthy leaves exhibited greater POD activity during whitefly infestation. In the infested juvenilc leaves, POD activity was much lower in thc healthy loavos and increasod gradually with period of exposure to B. tabaci B infestation. [n contrast, the activity of the enzyrne remained low in infested mature leaves in both the pr€sence and absence of tho virus even with increarcd €rpoffirc timc. Dctormi- nation of th€ distribution of an ins€ct pest is critical for sampling and management. I-eaf age is presumed to be associated with the within-host distribution of the geminivirus vector B. tabaci. Juvenile leaves will uzually attract more insects due to increased nutri- tional value and weaker defences. Our results high- licht tha importance qf leaf age/positiqn on the whitefly - host plant - geminivirus interactions and have important implications for sampling and control strategies. doi: 10. I I I lfi .I 439-M34.2011.01830.x Introducdon Plants have the special characteristic that they producc leaves of different sizes during growth (Cevahir et al. 2004); gsnerally, young leaveswhich are small become maturc lcavos that are large. This variation in sizc occurring along the axis of an individual plant, known as heterophylly, is associatedwith meny physiological changes. For example, the expression of scveral genes (Chandler and Robertson 1994; Vega et al. 2002'1, pigment cont€nt (Heloir et al. 1998), photosynthetic activity (Hutchison et al. 1990) and nitrogen conc€n- tration (tlan ct al. 2008) has becn shown ts vary sig- nificantly with leaf age. Plants are also known to defcnd themselves against herbivores (Duffey and Stout 1996; Romeo et al. 1996) and pathogens (McKenzie et at. 2002). The degreesto whieh they expr€ss defensive traits are dependent on the age of their leaves (Coley 1980). In general, young leaves suffer significantly greatsr Srazing demage from bsrbivorous insects than mature leaves (Reichle et al. 1973),which is presumably becausc they are nutrition- ally richer and are less tough than mature leaves (Coley et al. 2006). Susceptibility to soveral pa&ogens has besn repor&edto decrease with increasing leaf age (Koch and Mew 1991; Roumen et al. 1992). Insect fmding and pathogcns are known to induce the exprcssion of pathogcncsis-related (PR) proteins (McKenzie et al. 2002). [n fact, the most important components of plant protective $ystems are enzymatic defences (Fink and Scandalios2004 Rubio et al. 2002). These include peroxidases, which are knowa to have a number of functions, including roles in defenceagainst infections (Hammerschmi& et al. 1982; Trevisan et al. 2ffi3) and insect infcstation (Quecini et al, 2007), Thu$, increas€d antirfeeding efects are prcdicted with high POD activity. There has been a great deal of research regarding peroxidases due to their biochemical roles
School of Biological Sciences, Universiti Saiw Malaysia, Puhu, Pinang, Malaysia
Peroxidase Activity after Viral Infection and Whitefly Infestation in Juvenile andMature lraves af Solanum lycopersicum
Herraov Drnuct, Touovrrsu Serno2, Arnreo Aru Hesslxl, Ar. Tnrrnxr Azzl, RoNero Er,rnreuE Monem.s3, SuHen-lAr Herrnol, Furao Mrert' and Sazlr,v AntsAxAna
Authors' addressesl rSchool of Biological Scienas, Universiti Sains Malaysia, 11800 hrlau, Pioang, Malaysia; zFaculty ofPharmaceutical Sciences, Fukuoka University, Fukuoka, Japan; 3Faculty of Tropical Medicine, Mahidol University,Bangkok, Thailand; aDepartment of Medical Microbiolory, University of Malaya, Kuala Lumpur, Malaysia(correspondence to H. Diang. E-mait [email protected])
AbstuactWhiteffy infestation and the begomoviruses that theytransmit have besn shown to afect the activities ofplant defence proteins, but with no relation to hetero-phylly, a proccss of great importancc undcdying theoverall biology of plants. Hereo we have assess€d theefrects of Totruto yellow leaf anl virus IYLCV)infection on Solawn lycapersicun peroxidase (POD)activity and have examined whether leaves of differentages exhibit diferential POD activity in response toinfection and infestation with Bemkiq tabaci Bbiotype. Wc uscd leaf discs of two ages (luvcnile andmature) with two diferent infection statuscs (infectedand healthy) to exarninc the activity of the tomatoplant peroxidase using guaiacol as a zubstrate andtaking exposure time into aocouat. S. lycopersicumshowed increased POD activity in the presence ofTYLCV. The activity of the enzyme was higher inmatura than in juvenile leaves, In Seneral, bethinfecrcd. and healthy leaves exhibited greater PODactivity during whitefly infestation. In the infestedjuvenilc leaves, POD activity was much lower in thchealthy loavos and increasod gradually with period ofexposure to B. tabaci B infestation. [n contrast, theactivity of the enzyrne remained low in infestedmature leaves in both the pr€sence and absence oftho virus even with increarcd €rpoffirc timc. Dctormi-nation of th€ distribution of an ins€ct pest is criticalfor sampling and management. I-eaf age is presumedto be associated with the within-host distribution ofthe geminivirus vector B. tabaci. Juvenile leaves willuzually attract more insects due to increased nutri-tional value and weaker defences. Our results high-licht tha importance qf leaf age/positiqn on thewhitefly - host plant - geminivirus interactions andhave important implications for sampling and controlstrategies.
doi: 10. I I I lfi . I 439-M34.2011.01830.x
IntroducdonPlants have the special characteristic that they produccleaves of different sizes during growth (Cevahir et al.2004); gsnerally, young leaves which are small becomematurc lcavos that are large. This variation in sizcoccurring along the axis of an individual plant, knownas heterophylly, is associated with meny physiologicalchanges. For example, the expression of scveral genes(Chandler and Robertson 1994; Vega et al. 2002'1,pigment cont€nt (Heloir et al. 1998), photosyntheticactivity (Hutchison et al. 1990) and nitrogen conc€n-tration (tlan ct al. 2008) has becn shown ts vary sig-nificantly with leaf age.
Plants are also known to defcnd themselves againstherbivores (Duffey and Stout 1996; Romeo et al. 1996)and pathogens (McKenzie et at. 2002). The degrees towhieh they expr€ss defensive traits are dependent onthe age of their leaves (Coley 1980). In general, youngleaves suffer significantly greatsr Srazing demage frombsrbivorous insects than mature leaves (Reichle et al.1973), which is presumably becausc they are nutrition-ally richer and are less tough than mature leaves(Coley et al. 2006). Susceptibility to soveral pa&ogenshas besn repor&ed to decrease with increasing leaf age(Koch and Mew 1991; Roumen et al. 1992).
Insect fmding and pathogcns are known to inducethe exprcssion of pathogcncsis-related (PR) proteins(McKenzie et al. 2002). [n fact, the most importantcomponents of plant protective $ystems are enzymaticdefences (Fink and Scandalios 2004 Rubio et al. 2002).These include peroxidases, which are knowa to have anumber of functions, including roles in defence againstinfections (Hammerschmi& et al. 1982; Trevisan et al.2ffi3) and insect infcstation (Quecini et al, 2007), Thu$,increas€d antirfeeding efects are prcdicted with highPOD activity. There has been a great deal of researchregarding peroxidases due to their biochemical roles
Drnxc et al.
(Jackson and Ricardo 194) and status as a potentialsourcc of plant molccular natkcrs (Huang ct al. 2000'2003). In addition, manipulation of peroxidase geneshas been suggested as a control strategy against insectpests @owd and Lagrimini 1997; Privalle et al. 1999).
The variation of plant defence with leaf age canpotentially affect the population dynamics of herbivo-rous insects, particularly those in which both imma1s.and adult stages fecd on foliar compounds. This istypical of whiteflies in which the adult stage prefersthe underside of leaves to feed, hide and heed. The Bbiotype of Bemisia tabaci complex (designated 'B) isthe most ssrious worldwide agricultural whiteffy pet(Perring et al. 1993; Brown 1994). 3'cauFs ageaoo.system damage due to its high dispsrsal pot€ntid(Cock 1993; Brown et al. 2000) allowing for the rapiddevelopment of resistance. This feature, combined wlththe innate ability of th adult $tagp to transit rturrr'ous ge,rniniviruscs @rown and Nelson 1989; Brownand Poulos 1990; Kring et al. 1991), renders the pri-mary control tool, insecticides, increasingly ineffcctual.
Whiteflies alone have been shown to cause accumula-tion of specific PR proteins in tomato plants (Mayeret al. 1996). McKenzie et al. (2002) examined theresponso of PR proteins in tomato plaats to Tornatomottle virus (IoMoV) and reported increased enzymaticactivity of many PR proteins in viruliferous whitefly'infcsted plants. However, these studies did not take leafage into consideration. The production and levels of PRprotcins are functions of development (Miller 1983) andleaf age (Cevahir et al. 2004). To address the validity ofplaat physiology al$essmenq including plant defence,Marron et al. (2008) argued for the need to take intoaeount the position on the plant (stage of developmentand decre€ of maturity), as theffe may be influ€ntielphysiological trait$. In the present study, we examinedthe responses of Soloum lycopersictmt peroxidase toinfection with the B-v€ctor€d geminivirus Tonwto yellowleaf canl virus (TYI-CU and to B infestatioa, eaking intoasount both leaf age and expozure time.
Materlah and MethodsExeerlmeqtrt ptmtSolanum lycopersicmt cv. Hezuo 903 was gtown undergreenhouse conditions (27 * zoc, LD 14: l0 h photo-period and 75 * 5o/o humidity).
Experfinent 1Six individual plants (,|*-S-leaf stagc, c. l0 cm inheigh$ were infected with TYLCV (isolate Yl0, aninfectious clone of TYLCV originally identified inShanghai) using the inoculation technique described byCui et al. (2004). Briefly, the inoculate was grown at28oC for 2 days, after which a fine needle was used toinject a volume of 0.2 ml of culture into either stemsor petioles. Another set of the sam€ number of physio-logicany similar plants that were not inf€cted served asa control. All plants wcre then watsred wcry 14 daysand allowcd to grow until the S6leaf stage and usedfor experiments. After l0 days postinjection, plants
were inspected for infection symptoms. Juvenile leaves(JIx) wcre esllcctcd from the tips of hcalthy andinfected plants and stored at -80"C to avoid enzlmedegradation stop its activity.
Experimmt 2Eight hcalthy plants (4-5-leafstage, c. l0 cm in height)werp divided into two groups of four plants each andplaccd into clean glass cages measuring60 x 60 x 60 crn. One group was infested with a popu-lation of B whiteflies. Thirty females and ten males perleaf were released into the resp€ctive treatment clipcagpe (five cagef p€r plant). The other group with thesame number and type of clip cages but without white-fties scrvd as a control. These plants were treatedaccoding to the same procedures as the infectedplsnts" Seven and 2l days after the release of white-ffics, l€af discs were collected from the tip (JLs) andbottom (mature lcaves, MLs) of (a) healthy plantsinfested with B, p) infected plants infested with B, (c)control hcalthy plants and (d) control infected plantsand stored as previously until analyses.
hotein cxtrection rnd qumtfrcetiooFor each leaf disc type, proteins were extracted with50 ml of prccooled extraction buffer (200 mlrr potassiumphosphate, pH 6.0, containing 12.5% polyvinylpynoli-done). Homog€nate$ wene entrifuged at 12 2M.54 g at4"C for 15 min. Clear supornatants werc immediatelystored at -80'C. Proteins were quantified in triplicateusing a DTX 880 Multimode spectrophotometer (Beck-man Coulter [nc., Brea, CA, USA) at 595 nm (Bradfordle76).
Mcranrffi dFOD *"livityPeroxidase activity was established sp€ctrophotometri-cally and in triplicate at 470 nm using 20 m*r guaiacoland 15 mu HzOz as substrates, thus sligbtly adaptingthe techdquc of Trevisan et al. (199?). For each leafdisc type, 185 pl of the reaction mixture (100% guaia-cnl,30Yo hy&ogen peroxide, and 0.2 u PBS, pH 6.9)was added in triplicate to the wells of 96well plates.A volume of 15 pl of a given crude extract was addedto each of the three wells containing tle reaction mix-ture. After brief shaking, the 9Gwell plates wereplaced into a Bio-Tek Plate Reader conncctod to acomputer and analysed using the KC4 V3.4 softwarepackage @io-Tek, Winooski, VT, USA). Increases inabsorption were monitord for 5 min at intervals of60 s.
Deta colhctionInfection was confirmed by polymerase chain reaction(PCR). In both studies, leaf discs were sampled at thesame period from all plants following the procedurereported previously (Unyayar and Qekig 2005). Wecalsulated mean POD activity using the formula: PODactivity : As
- A3rRun time x Protcin concentration,
where A5 and Ao are th€ mean absorbances of threereplicates of measurement at t : 5 min and t : 60 s,
On Bemislo labaailnvatiort ia China
respectiv€ly, the latter of which is considersd as thefirst rcading. POD activity uras cxpterscd in mg/tilprotein per min. The differences in FOD activityr€spornes between juvenile and mature leaves follow-ing infection and white,fly infestations were comparedstatistically by analysis of varianoe (euove) from thesvsrerol3 statistical software package (Systat 2004).In all analyses, P < 0.05 was taken to indicate statisti-cal significance.
ResultsStatus of expcrhcotd phrtsFrom l0 days poetidoction, all tomato plants injectedwith the infectious clones of TYLCV displayed symp-toms, such as leaf blade curling and general reductionsin haf size and plant height. Infection was confirmedby FCR In both tnJected and healthy plants (Ftg. t).
Effectr of Mccdon on PIOD rcdvityInfection signifcantly affected the FOD activity ofS. Iycopersia,vn (F = 5,67, df = l, P : 0.021). Healtttytomato plants infected had lower POD activity thanthose infected with TYLCV, and this difference waslikely due to the prese,nce of the virus (Fig. 2).
Frg, ! DEtcatios af Tqnqto yellow lad eurl vtrs{ (TYLCYI infwtiqnby polymerase chain r€action using leaf ertract from Solowa lyo-Trcrsiaan experimental plants. M = Mar*er; Ll : Water (negativecontrol); L2 : DNA templat€ (positive control); L3 -LE = Healthy plants; L9 - Ll4 = TYlCV-infected plants
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Fig.2 Respoase of Solanutt lycopereicwtt plant peroxidase activityto Torutto yellow leaf, curl virnr iafection. Bar chart$ with diferentlettrrs ar€ signincan$ ditrerent (P < 0,0t based on Tukey's HSDte6t
Efrocb of lerf rge on FOD rcdvtttThe POD activity of S.lyaopersieum vafied sigpifi-cantly with leaf age in both healthy (F: 9.18,df * l, P = 0.006) and infected planu (F = ll.l8,df - l, P = 0.003). The lwel of POD activity washigher in the MLs than in JLs. The difference in PODactivity between the two leaf typee rcnded to be morepronounced in the presence of TYLCV virus (Fig. 3).
Efrccbof rfift{y hfesffitr or FOD rcffyfty ln JL6The POD activity in healthy JLs of ,S. lycopersicann vavied significantly with infestation (F = 26.363, dI : 2,P < 0.001). Healthy JLs harbouring B whiteflies hadhigher FOD activity than those free of whiteflies(Table l). Pairwise comparisons using Tukey's testrevealed that the POD activity in the 2lday infestedJLs was sigaiffcantly higher than that in the 7-dayinfested leanee JLs (matrix of pairwise mean differ-snce$ = 5.575, matrix of pairwise comparison probabil-ities = 0.001). In the infected JLs harbouring Bwhitefliesn Tukey's tcst showcd that POD activityincreased significantly with increasing exposure time(matrix of pairwise mean difierences : 4.135, matrix ofpairwise comparison probabilities - 0.011) (Fie. a 9.
Efiecf of rUbily Meffidoo on P(OD .cdvlly h MIsSolannn lycopersicttn infestation sigrrificantly affectcdPOD activity in healthy MLs (F = 3.135, df : 2,
rcf @nrtrrl t*CI lftvadls lcu'rr
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Fig. 3 Responsc of Solatwn lycopu$aon baf peroxidasc activity toTonuto yellow kaf curl yrnrs infection, Bar charts of diEereot colouraad witb dr$ereat httc{B or aunbem arc *isdfreantb difereqt(P < 0.05) based on Tukey's HSD tcst
Tabh Imovl for effeots of lcef ags etrd duration of whitefly ideetation andtheir interactions on peroxidase activity of healthy and Tomatoyellow h{ cad vinrs-infected leaves of Salanran lycopersiaon
Fig. 4 Response of Salanun lyeopersicwtt leaf peroxidase activity toB whitefty infestation. Y indicate,s juvenile lcaves, and Q indicatesm&ture feaves, Grcy and laok bar charts itrdicatt healthy and Torntoyellow leaf curl vinr.r-infccted leaves, respectively. Bar charts of differ-cnt colour and with difrercnt htters or aumbcrs are signmcantly dif-ferent (P < 0.05) bssed on Tukey's HSD test
P : 0.043). Leaves infested with B whiteflics showedan inareaFe in POD aativity compared to thcir non-infested count€rparts. In healthy MLs, th€ enzymeactivity did not differ significantly between the 7 and2l-day exposure periods, but tended to be higher inthe fust period (matrix of pairwise mcen difhr-etrces = -6.657, matrix of pairwise comparison probs-bilities : 0.318). POD activity did not differsignificantly between infested and non-infested MLs{Fie.4()).
DbcussbtrThere was an obvious relationship between leaf agcand POD activity r€sponses, with more maturs l€avesshowiug grcat€r enzyme activity. Differential defensiveresponses of young and mature leavee have beenreported previously. Cxvahir et al. (2fX)4) assessd dif-ferent physiological parameters of Gazania splmdensleaves and found increased POD activity in juvenile ascompared to adult leaves. In an investigation of theeffects of drought stress on antioxidant enzymes ofLycopersicon esculentwn (S.lycopersicwn) leaves,Unyayar and (lekig (2005) observed greater ascorbateperoxidase activity in young leaves. Although th€ dif-fcrences in POD activity between young and matureleaves are not yet clear, they could be explained on thebasis of differences in physiological traits of the two
developmental stages. Coley (1980) stressed thatmature leaves may be tough and contain substancesthat decrease digestibility, whereas young leaves arelow in defensive chemicals. Coley also suggested thatyoung leaves have greater escape ability due to theirsynchronous flushing. Thus, the higher POD activityobsen/cd in mature leavcs of S. lycopersieum may beassociated with increased amounts of defensive chemi'cals. This observation has important implications fortomato eontrol via transgenesis, Several qualitativetomato plant varieties have been developed from S. ly-copersicum. However, the productivity of these trans-genic strains is being severely affected by narrowing ofthe genetic base, which results in an increase in suscep-tibility..to various types of stress. To overcome thisissue, Unyayar and Qekig (2005) argued for the needto consider thc developmental stages of leaves, as theymay contribuG to the diferential prwention of oxida-tive damage in tomato plaats experiencing drought.Mllckeos et al. (1997) also suggested that analysingantioxidant enzymes of transgenic plants may help toidentify the critical components of the defence systsmand provide a basis for designing strategies to achieveoxidative stress tolerance.
There was clearly a relationship between infectionand POD activity, with plants conlaining the virusexhibiting gleat€r enzymatic activity than their healthycounterparts. Increas€d POD activity in response tovirus infection has be€n reported previously in manyplant species. McKenzie et al. (2002) examined thedefensive responses of B. tabaci biotype B in relationto infection. They analysed the accumulation ofdefensive proteins, including peroxidase, in healthyand ToMoV-infected plants. Their findings indicatedthat infect€d plants display $€ater PQD activity thantheir healthy counterparts. In plants, virus infectionhas often been associated with a plethora of biochemi-cal and metabolic changes (Soosaar et al. 2005). Forexampl€, in cotton platrts, cotton leaf c'url virus(CLCUV) infection results in increases in catechol, phe-nols, carotenoids, proteins, total $ugars, chlorophyll,oil content and lipase contents (Kang et al. 2003; Agh:raf et al. 269{). Such infection also decreases Ca+ +
and K' concentrations in Gossypium hirsutwn CYF846 (Nadeem et al. 2006). In this study, the enzyrneactivity in the TYlCV-infected plants was significantlygJcatsr than that in the healthy plants. Although thercasons for the observed variations in POD activitybetween infected and healthy S. lycopersiaan ar€ notyet clear, they may be due to differences in the bio-chemical and metabolic changes associated with eachphysiological status. With reference to previousreports, it is tempting to infer that the greater psroxi-dase activity in the infect€d plants represents are$pons€ to incr€ased accumulation of biochemicalelemeirts and metabolites in infectpd comlmred toh€althy plants. The simultancous increases in PODactivity and the lcvels of catechol, phcnols, carotc-noids, proteins, total $ugars, chlorophyll, oil contentand lipase enzym€ contents (Kang st al. 2CI3; Ashraf
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et al. 2004) in plants infected with begomovirus ssomto support this nrggcstion. Wc also found that PODa*ivity varied significantly with leaf age.
There was a clear inverte relationship between infes-tation status and POD activity, with leaves harbouringB whiteflies tending to show incr€ased enzrymaticdefence compared to non-infested leaves. Similarobservations have been reported previously. Mayeret al. (2002) reported that infostation with the silverleafwhitefly Bemisia aryentifolii significantly increased theactivities of foliar chitinase and peroxidaw, in Brassicasp. and tomato plants. In an carlier study, Mayer et al.(1996) found that tomato plants harbouring B. tabaciwhitcflies showed increased levels of defensive proteins,in particular peroxidase, In a study involving B. tabacibiotype B and dwarf cherry tomato, McKenzie et al.(2002) reportd an lncrease ln POD actlvity ln theleaves of infesrcd tomato plants compared to non'infested controls. In contrast with these studies, weused juvenile and mature leaves of tomato and foundgreatcr POD aaivity in whitcfly'infe$t€d leaves' Ourfindings are in agreement with Coley's theory regard'ing plant-herbivore interactions (Coley 1980)' whichpostulated that plant species and lsaves ofvarious agesdiffer in their anti-herbivore characteristics, with oldleaves having better defence than young leaves due totheir lower palatability.
As well as provisioning insigbt into the develop-mental regulation of peroxidase-based tomato plantdefence against B whiteflies and the viruses that theycarry, the present study emphasized the sipificanceof considering leaf age when assossiag plant physio'logical traits, i.e. defence, as these vary with the levelof heterophylly. Our study was performed primarilyto detarmine the levels of peroridase activity in $. ly-copersicam after begomoviral infection and whiteflyinfestation. We observed increased POD activity afterinfectio4 during infestation and in mature leaves.Peroxidases arc defensive enzymes (Hammerschmidtet al. 1982; Trevisan et al. 2003; Quecini et al. 2007)and their increase, a biochemical symptom or events,which cause resistance (Seevers et al. l97l;' Reuveniand Ferrcira 1985). Overexpression of a peroxidasegene of inner leaf tissues in wheat epidermis resultedin enhanced resistance against a fungal pathogm thatenters plant body by direct penetration (Altpeteret al. 2005). There have been a number of recentstudies regarding the use of gene engineering andcrop transformation for plant disease and infestationreeistance (Altpeter et al. 2005), and these approachesare both anticipated to rise with decreasing effective-nesg of current control strategies. Plants have a largenumber of peroxidase isoenzymes (Welinder 1992;Quiroga et al- 2000) including tomato plants (Botellaet al. 1993). The incr€as€s in POD activity duringTYLCV infection, in the presence of whiteflies, andin mature leaf substraies in the present study mayarise by different mechanisms, thus additionalresearch works are needed to characterize the peroxi-dase (s) involved.
Ac&mwledgenenbAssistancr rcndered by some gtudents ir gratefully acknowledgrd.HD aleo thanks Jiang Mi Ling, Ibrahim Bambina Bawa and FuWel for their moral supporl
BcfcrcrcAltpetet F, Varshney A, Abderhaldea O, Douchkov D, Sauttor C,
Kumlehn J, Dudlcr R, fthweizer P. (2005) Stable expression of adcfcnse-r€lated gsne iil wheat spidermis under transcriptional coa-trol of a novel promoter codors pathoge'n lesistancc. Plant MolBiol J2:2?l-283.
Ashraf MY, Mahmood S, Sarwar G, Arhraf M, Naeem M, Zafar S.(2{XX) Physiologicol and biochemical changes in r€si$tant andsusccptible ta Cottdt leaf cwl virss (CLCuV) cotton varietics atgormimtion end early reedling rteger: chengBe in liplc, oilcort€nt, protein and soluble sugars. /nl I Eiol Biotechnol l:217-222.
Botella MA, au€sada MA, Hasegawa PM, Valpuesta V. (1993)Nucleotide sequeoc€s of two peroxidase gen€s from tomato(Lycopersicon escuhn um), Plott Physiol 1|N;665-$6.
Bradford MM. (1976) A rapid aad sensitive mcthod for quantitationof microgram quartitiee of proGin utilizing the principle ofproteindye-bindirg. Atul Bioclem 72:.24&-254.
Bro*n JK. (1904) Tb€ sta$s of Sanrirro t4fuci (Cffir..) ar a peet andvoctor in world agroecosystflis. FIO Pl@rt Prot Bull 4Lil-32.
Brown JK, Nelson MR. (1989) Two whitefly-trarsnritted egmaivirusesisolatod from pepper affectod with tigre drsor*. Phytopatlmlogy7*90E.
Brown JK, Poulos BT. (1990) Serreoo golden moseb virus a aewlyidertified whiteffy-transmited geminivirus of pe,pper sod tomrtoia the United Statcs and Mexicf. Plort Dis 74:72O.
Brown JK, Frohlich DR, Cooper AD, Bcdford ID, Markham PCi.(2m0) Gonetic analyrie of ,arirto (Homiptera: Ahyrodidae)populations by isoelec'tric focuring electrophoresir. Blochem Gea3&13-25.
Cevahir G, Yentllr S, Yazgaa M, Onal M, Yilmaar N. (2004) Pcr-oxidase activity in relatiol to anthocyadn and chlorophyll contentin juvenib and adult leavea of 'tnini-stst" Gazn,k sple&ns. PakJ Bott6:ffi3-ffi.
Chandler PM, Robertson M. 094) Gone cxpre*sion rcgulated byabrcisic acid and its rclation to strcss toleraoce. Am Rev PlantPhysiol Plut Mol Eiol6:,113-141.
Cock MJW. (193) Benitio tafuci, an Update 198G1992 on theCouon Whitefly wirh o, Antttated Bibltogaphy. Ascot, UK,CAB IIBC, 78 pp.
Coley P. (1980) Efccts of leaf age and plant life history patterns oohorhivory. Natwc N:545-546.
Coley PD, Batcmrn ML, Kursar TA. (2000 Tlrc efrects of plantquality on catcrpilbr growth ard defensc agaiast natural enemics.Oikos 113:,219-228.
Cui )L Tao )t, Xo Y, Fauquet CM, Zrou X. (2004) A DNAb€fiaassociated tith Tomato yellow leaf ctrl China virus is rcquircd fiorsymptom induction. J Virol 7t:13966-1t974.
Dovd PB Lagrinioi LM. (1994 Examiaation of diferent tobacco(Nicottou spp.) types under- and overproduciug tobacco anionicpcroxidase for thcir leaf reoistance to Helicowrpa tea. J Ckm kol23:2357-2370.
Duffcy SS, Stout MJ. (196) Antinutritive and toric components ofplant defense against insecto, Arch Insect Biochent Physiol 32:1-11.
Fink RC, Saruidaliog JG. (2002) Molceular arolutioa &dd strueturc-function relationships of the superoxide dismutase gene families iaAngiosperms and their relationship to other eukaryotic aadprokaryotic rupcroxide dismutases. Arch Biochem Biophys !ll}'l9-36.
Hammcrschmidt R, Nucklcs EM, Kuc J. (1982) Associction ofcnhanced pcroxidase activity with iaduced systemic resistance ofcrrcumber to Colletotrichtm, lagenarium. Phytiol Plont Patholfr:73-82.
Hal Q, Kawasaki T, Nakano T, Chib6 Y. (2m8) l:af-age cffeetr onseasooal variability ia photorynthaio parameters aad its relationshipa with leaf mass psr area and leaf nitogen concent ationwithin a Pkus densifora worm. Ttee Piysiol2t;551*558.
DIENG et al.712
Heloir MC. Fourniour C, Barbier M, J€andet P' Bessis R' (1998)
Endogenous polyauines concentrations in juvenile, adult aad
micropropagateO grapevine (Vttis vinikrc L' cv' Pinot noir)' "i'irtl:61-42.
