Effect of Methanol on Photosynthesis and Chlorophyll Fluorescence of Flag Leaves of Winter Wheat

Avariable onine at www.sclenceairect.comAgricultural Sciences in ChinaScienceDirect2008. 7(4); 432-437April 2008Effect of Methanol on Photosynthesis and Chlorophyll Fluorescence of FlagLeaves of Winter WheatZHENG Yue-jin*, YANG Yue qin*, LIANG Shan-shan and YI Xian-fengCollege of Agriculture, Henan University of Science and Technology, Luoyang 471003, P.R.ChinaAbstractPhotosynthesis and chlorophyll a fluorescence parameters, photochemical efficiency of PS II (Fv/Fm), photochemicalquenching of PS II (qP), nonphotochemical quenching of PS I (NPQ), maximum activity of PS I (Fv/Fo) as well as electrontransport rate (ETR), and quantum yield of PS II (OPS II) were measured on flag leaves of the winter wheat treated bymethanol at different concentrations. The results revealed that photosynthesis was greatly improved by methanol, asindicated by higher photosynthetic rates and stomatal conductance. The enhancement effect of methanol onphotosynthesis was maintained for 3-4 days. Different methanol concentration treatments also increased intercellular CO2concentration and transpiration rates. No significant decline was found in FvFm, Fv/Fo, and OPS II, which revealed nophotoinhibition during methanol application in different methanol concentrations. Methanol showing no apparentinhibitory effects indicated higher potential photosynthetic capacity of flag leaves of winter wheat. However, the increasein photosynthesis was not followed by an increase in the photosynthetic activity (Fv/Fm), and fluorescence parametersdid not indicate an improvement in intercellular CO2 concentration and PS II photochemical eficiency compared with thecontrol, thereby encouraging us to propose that lower leaf temperatures caused by applied methanol would reduce bothdark respiration and photorespiration (most importantly), thus, increasing net CO2 uptake and photosynthetic rates.Key words: methanol, photosynthetic activity, chlorophyll fluorescence, flag leaf, winter wheatstress conditions (Fv/Fm) (Waldhoff et al. 2002),INTRODUCTIONphotochemical quenching of PS II (qP), andnonphotochemical quenching of PS II (NPQ), as wellIn recent years, the technique of chlorophyl1as electron transport rate (ETR) and quantum yield offluorescence has become ubiquitous in plantPS II (PPS II).ecophysiology studies (Xu et al. 2005; Liu et al.2006).Organic solvents have been found to be effective onNo investigation into the photosynthetic performancealgal growth in the late 1980s (Stratton 1987; Strattonof plants under field conditions seems complete withoutand Smith 1988) and the early 1990s (Nonomura andsome fluorescence data. This trend has been fuelled,Benson 1992; Ei Jay 1996). Methanol has been provedto a large degree, by the introduction of a number ofto show great positive effects on photosynthesishighly user-riendly (portable) chlorophyll fluorometers.(Nonomura and Benson 1992; Li and Yi 2004). However,Chlorophyll a fluorescence has been used to evaluateunderstanding the effects of methanol on plants is stillphotosynthetic performance under natural and some中国煤化TIsed conclusions wereTYHCNMHGReceived 24 October, 2006 Accepted 22 August, 2007Correspondence Y1 Xian feng. E mail: yxeng1975@ 126.com. Tel: +86 379 64282340. Pax: +86 379-64282340. * These authors contributed eqully to this work.02008.CAS.Arghts rered PubshelebetereneEfet of Methanol on Photosyahesis and Chlorophyl Fluorescence of Flag Leaves of Winter Wheat433made concerning the effects on the photosyntheticProtocolsactivity and the increase of biomass. A lot ofinvestigations have shown that the production andFlag leaves of winter wheat were evenly sprayed byphotosynthetic activity of algae were increased at lowmethanol in 0 (distilled water), 2, 5, and 8%concentration of methanol (Theodoridou et al.2002).concentrations from 10:00 to 11:00 am in naturalThe same effects were also found in higher plant speciesconditions at the milk stage in the field. Photosyntheticwhen sprayed with 10- 50% methanol (Nonomura andrate, stomatal conductance (GS), transpiration rate andBenson 1992; Li et al.1995; Li and Yi 2004) and whenintercellular CO, concentration were measured usingcultured in 0.2-5 mmol LI methanol (Gout et al.2000).portable photosynthesis systems (CITRA-1, PP Systems,However, these investigations mainly focused onLtd, UK) 20 min after spraying. Photosynthesisinfluence of methanol on growth (Okumura et al. 2001)induction light intensity was set at 1000 umol m2 s'.and its metabolic process (Gout et al. 2000), fewThe measurement of Chl a fluorescence induction andtouched on photosynthetic performance and chlorophyllparameters was done at field temperature by using a pulsea fluorescence parameters. And at present, there is aamplitude-modulated fluorometer (FMS-2, Hansatechlack of understanding on the dependency between theLtd, Norfolk, UK) as described by Rohacek and Bartakeffect of methanol on carbon accumulation and the(1999). The minimal (dark) (Fo) and maximal (Fm)appropriate energy dissipation through the primaryfluorescence yield was obtained with weak modulatedprocess of photosynthesis (Li et al. 1995), especially,light (0.04 umol m2 s"), then witha 1 s pulse offor monocotyledons. In this study, we investigated thesaturating light (5000 umol m2s"). The ratio ofFv/Fmstimulatory as well as the inhibitory effects of methanolwas served as a measure of the maximum photochemicalon chlorophyll a fluorescence and photosynthesis whenefficiency of PS I. Photochemical quenching (qP) andthe higher plant species such as winter wheat wasnonphotochemical quenching (NPQ) were calculatedtreated by methanol at different concentrations.according to Schreiber et al. (1986). The efficiency ofenergy conversion in PS II (PS II) was calculated as(Fm'- Fs)/Fm' (Fs = stationary level of fluorescenceMATERIALS AND METHODSemission, Fm' = maximum fluorescence duringilumination) (Genty et al. 1989). The level of (1- qP)Study areawas calculated afer van Kooten and Snel(1990). Beforetaking fluorescence measurements, the leaves wereThe study was conducted in 2006 in the arid and semi-adapted to darkness for 20 min.arid transition area in Luoyang City (elevation averagedTo evaluate the electron transport condition, totalat 600 m, 34° 35N, 112° 24E), Henan Province, China.electron flow rate (.) was calculated according to KrallThe climate of experimental site was dominated by theand Edward (1992): Jp=OPS IIx PPFDxaXf, wherewarm and temperate zonal continental monsoon. ThePPFD refers to photosynthetic photon flux density, aannual average air temperature was 14.8C. The averageindicates the proportion of light that leaves absorb, andannual precipitation was at 578 mm.frepresents the proportion of light energy distrbutedto PS II.Experimental designThe noncyclic photosynthetic electron flow inphotorespiration (。) and carbon reduction (J) areEach plot in this experiment was 5 m2 and arranged inexpressed as J。=2/3 [J。-4 (P。+R,)] andJc= 1/3 Jp+the north-south direction. Winter wheat variety, Jinfeng8 P,+R)], respectively (Epron et al.1995). Rates of3, was selected and planted. A total of 12 plots wereoxidation and carboxylation are obtained from: Jp=4Vcestablished and randomly distributed, spaced 1 m apart中国煤化工19. whe P,along a transect line in a 200 m2 area. Three duplicationssymboland R。refers towere, respectively, set for the control and methanolrespira:YHC N M H Gander ight, i.e.,treatment at different concentrations.photorespiration rate.2008.CAS.Angt woPolotdbeDoreorur434ZHENG Yue-jin et al.RESULTStest whether there was a long-term effect of methanol.The results proved a 3-4 day potential of methanol toenhance flag leave photosynthesis of winter wheatPhotosynthesis(Fig.1), especially, at the concentration of 5%.As shown in Table 1, photosynthetic CO2 assimilationChlorophyll a fluorescence parameters(photosynthetic rate) of flag leaves of winter wheattreated by methanol at different concentrations (2-8%)No significant changes were observed in PS IIincreased significantly compared with the control, withphotochemical activity (chi-square= 1.985, df=3, P=the highest effect being that of 5% methanol (t= -2.573,df=13, P=0.023). No significant difference was0.576), i.e, Fv/Fm (Table 2), indicating no occurrenceobserved between three treatments (chi-square= 0.105,of photoinhibition under administration of methanol.df=2, P=0.949). Methanol application at differentSimultaneously, the maximum ratio of photochemicalconcentrations (2-8%) also apparently enhancedstomatal conductance of flag leaves (chi-square =+ Control 毋 0% .10.271, df=3, P= 0.016) (Table 1). Stomata士2%景8%conductance induced by 5% methanol was 2.42 timesas that of the control. Corresponding with stomata517tconductance, transpiration rates increased with the6ttreatments of methanol but did not show a significance(chi- square =4.827, df=3, P=0.185). The higherintercellular CO2 concentrations were also observed4twhen administered with different methanol .瓷13concentrations (chi-square = 6.967, df=3, P=0.07)(Table 1). Application of distilled water (0% methanol)had slight but no significant influence on photosyntheticDays. after aplieaioaon of methanol at dferent cntntion()process, as indicated by the net photosynthetic rate,stomata conductance, and transpiration rate. WeFig. 1 Changes in photosynthetic rates in winter wheat flag leavescontinuously measured photosynthesis for 5 days totreated with different concentration methanol.Table 1 Photosynthetic parameters of flag leaves of winter wheat treated by methanol at dffrent concentrationsPhotosynthetic parametersTeatmentsTranspiration rateStomatal conducancePhotosyothetic rateInercellular CO,Leaf temperature(mmol H2O m-3别)(umol CO, m2g1)(umol Co, m2g1)concentration (ppm)(°C)Control5.29土0.411007.63土215.6314.85+2.23282.00士8.2532.4+1.20% methanol5.03土0.32984.35 土179.2814.44土1.82286.76 土9.0331.5+1.0 .2% methanol5.76土0.321836.33土383.4817.79土3.88288.67土14.3030.6土1.85% methanol5.92土0.302439.63土289.7218.13土1.12 .302.38土2.6327.3出1.18% methanol6.16土0.292 371.63土650.6318.08 0.87300.00 5.0025.0+0.8Table 2 Chlorophyll a fluorescence parameters of flag leaves of winter wheat treated by methanol at dfferent concentrationsChlorophyll fluorescencepurametersFv/Fo6.18+0.725.92+0.685.69+0.825.73 10.555.60 +0.24Fv/Pm0.86+0.01 .0.85+0.020.85 +0.01OPS I0.20士0.020.20+0.01中国煤化工0.20+0.02 .NPQ0.35土0.100.36土0.08YHCNMHG0.31 0.051.31 +0.391.30土0.281.23 :0.15EIF84.28土2.285.34土3.7104.16士5.888.23.384.04土3.202008. CAAS. Alngt red PuinedbyEeweredeeEffeet of Methanol on Photosyathesis and Chlorophyll Fluorescence of Flag Leaves of Winter Wheat435quantum yields and concurrent nonphotochemicalsignificant change in the control and the three treatmentsprocesses, Fv/Fo, decreased slightly along the methanol(40.06, 40.81, 40.91 and 41.23% respectively) (Fig.concentrations (chi-square= 2.005, df=3, P=0.571).2). Rates of oxidation of Rubisco (V) were recordedOPS II, which reflects the actual quantum yield ofas 2.03, 1.13, 1.00, and 0.55 pmol m2 s-I in controlledelectron transport in PS II, did not show significant and treated leaves, whereas carboxylation rates ofchange (chi -square= 1.819, df=3, P=0.611) (Table 2).Rubisco (V) were 18.03, 20.37, 20.55, and 20.19 umolApplication of ditillel water (0% methanol) showedm:2 s' in the corresponding leaves. The ratio of VJvcno significant effect on Fv/Fm and中PS II comparedwas significantly declined in the methanol treatment (Fig.with the control.3). However, we did not witness the differences in V。,V。and VJVc of winter wheat leaves treated by 0%Quenching coefficientsmethanol and the control.Table 2 indicated that qP increased by 2% methanoltreatment but decreased by 8%, and no change was25 L0.12found when treated with 5% methanol. Non-200.10photochemical quenching (NPQ) is thought to protect0.08the photochemical apparatus against the destructive5t0.06 ;effects of excess light energy (Weis and Berry 1987).1000.04However, no significant decrease in NPQ was induced02by methanol with different concentrations compared0.00with the control. ETR was not improved when methanolControlP%2%5%8%was supplied at different concentrations (Table 2).Methanol concenrationsPhotosynthetic electron distributionFig. 3 Changes in the ratios of oxidation to carboxylation (Vo/VJin winter wheat flag leaves treated with different concentrationmethanol.The ratio of noncyclic photo-respiratory electron flowto total electron flow rate, J/Jp of winter wheat flagDISCUSSIONleaves accounted for 12.81, 6.84, 6.05, and 3.46%in the control and three treatments, respectively.However, the ratio of noncyclic electron flow forThe results were consistent with our previous study onphotosynthetic carbon reduction to total electron flowleaf-used lettuce (Li and Yi 2004). Treatments byrate, JJp of winter wheat flag leaves showed nomethanol at different concentrations promoted stomatalbehavior, thereby enhancing the transpiration rate andstomatal conductance (Table 1). Increase in45 |photosynthetic rate induced by methanol was similar40with those by David et al. (2003), who showed that35 t30 t十J,毋 I,the rate of oxygen evolution and photosynthetic rateof Lemna gibba increased under lower methanol20 tconcentrations. Slight increases of intercellular CO,15 tconcentration were observed when administered withdifferent concentrations of methanol, which may be%due to an increase in the availability of CO2 via thecellul=l (Nonomura andMethanol concentrstionsBenscTYH中国煤化工1 a decrease inFig. 2 Changes in the ratis of J/J]p and JJp in winter wheat flagnonphC N M H Gon was induced,leaves treateed with didifferent concentratiowhich resulted in efficient nicotinamide adenine436ZHENG Yue-.jin et al.dinucleotide phosphate (reduced) and adenosineboth dark respiration and (most importantly)triphosphate synthesis, therefore benefitingphotorespiration, thus, increasing net CO2 uptake andphotosynthetic accumulation (Table 2). The combinationphotosynthetic rate. The effect is even maintained forof methanol as carbon source has been investigated byseveral days as revealed by a 3-4 day potential ofCossin (1964) and Gout et al. (2000). It was postulatedmethanol to enhance photosynthesis in flag leaves ofthat methanol might be incorporated into carbonwinter wheat. This hypothesis was not followed bymetabolism as a single carbon compound (Cossin 1964;David et al. (2003), who proposed that energyNonomura and Benson 1992; David et al. 2003), likedistribution and decline in nonphotochemical quenchingformaldehyde and serine as primary products (Goutwere related to the increasing net CO2 uptake of Lemnaet al.2000). But whether there is methanol oxidase orgibba under lower methanol concentrations.not in higher plants still remain controversial, despite For flag leaves of winter wheat treated by methanolits existence in methanol-used alga.at different concentrations, the ratio Fv/Fo, as theAs seen from Tables 1 and 2, the increase in themaximal quantum yield of photochemical andphotosynthetic rate induced by methanol was not concurrent non-photochemical processes of PS 1Ifollowed by an increase in the photosynthetic activity.(Rohacek and Bartak 1999), was decreased. Therefore,Actually, the PS II activity supported by the watertoxicity of methanol with higher concentrations to somespltting system and the oxygen evolution were notdegrees is shown, here, on flag leaves of winter wheat.significantly increased (Table 2). Moreover, there wasThe real effect of methanol shown as an inhibition or ano improvement in the PS II photochemical efficiencystimulation of photosynthesis has to be further(Fv/Fm) compared with the control. When comparedinvestigated at the molecular level because it appeared,with the control, no change was seen for fluorescencehere, that the effect of methanol on flag leaves of winterparameters concerming the PS II activity, such as thewheat is a complex of interactions between energyPS II quantum yield (Fv/Fm), the basal quantum yieldstorage via photosynthesis and nonphotochemicalof nonphotochemical processes (Fo/Fm), the maximumenergy-dissipative processes. The mechanism ofratio of photochemical quantum yields (Fv/Fo), and themethanol by which a decrease in leaf temperature andphotochemical quenching value (qP) under methanol-consequent depression in the photorespiration ratestreated conditions (Table 2). Although NPQ plays anneeds further investigation.important role in excessive solar energy dissipation, nochange was detected after administration of methanolAcknowledgementswith different concentrations. However, it is stillThe study was supported by the Student Researchunknown why the increase in photosynthesis was notTraining Program of Henan University of Science andlinearly correlated with the photosynthetic activity asTechnology (2007142) and Program for Science andindicated by Fv/Fm and中PS II. Therefore, theTechnology Innovation Talents in Universities of Henanimprovement in the photosynthesis treated with a higherProvince, China (2008 HASTTT003).concentration of methanol appeared to be related to othermechanisms. As reported in Table 1, most remarkableReferencesdifferences were found in leaf stomatal conductance,Cossin R.1964. The utilization of carbon-1 compounds byaccompanied by only modest changes in transpiration.plants. 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