Aquatic Botany 90 (2009) 172–178

Energy fluxes and driving forces for photosynthesis in Lemna minor exposedto herbicides

Philippe Eullaffroy a,b,*, Cecile Frankart a, Aziz Aziz a, Michel Couderchet a, Christian Blaise b

a Laboratoire Plantes Pesticides et Developpement Durable (PPDD), UPRES 2069 (URVVC), Universite de Reims Champagne-Ardenne, BP 1039, 51687 Reims Cedex 2, Franceb Environnement Canada, Centre Saint-Laurent, 105 rue McGill, Montreal, Qc, H2Y 2E7 Canada

A R T I C L E I N F O

Article history:

Received 28 April 2008

Received in revised form 17 September 2008

Accepted 19 September 2008

Available online 27 September 2008

Keywords:

Chlorophyll fluorescence

Toxicity

Photosynthesis

Duckweed

Water

A B S T R A C T

Analysis of fast chlorophyll fluorescence rise OJIP was carried out to assess the impact of diuron, paraquat

and flazasulfuron on energy fluxes and driving forces for photosynthesis in Lemna minor. Results showed

that diuron and paraquat treatment produced major changes in electron transport in active reaction

centres (RCs). However, diuron had a more pronounced effect on the yield of electron transport per

trapped exciton (c0) than on the yield of primary electron transport ð’P0Þ showing that dark reactions are

more sensitive to diuron than light-dependent reactions. In contrast, paraquat treatment effects were not

due to a target-specific action on those dark and light reactions. Paraquat also induced a marked surge in

the total absorption of photosystem II (PSII) antenna chlorophyll per active RC displaying a large increase

of the dissipation of excess energy through non-photochemical pathways (thermal dissipation

processes). Flazasulfuron induced a slight decrease of both the total driving force for photosynthesis

and the quantum yield of electron transport beyond QA� combined to a small but significant increase of

the non-photochemical energy dissipation per RC (DI0/RC). We conclude that energy fluxes and driving

force for photosynthesis generate useful information about the behaviour of aquatic plant photosystems

helping to localize different target sites and to distinguish heterogeneities inside the PSII complexes.

Regardless of the active molecule tested, the DFABS, ’E0, DI0/RC and/or ET0/RC parameters indicated a

significant variation compared to control while ’P0(FV/FM) showed no significant inhibition suggesting

that those parameters are more sensitive for identifying a plant’s energy-use efficiency than the

maximum quantum yield of primary PS II photochemistry alone.

� 2008 Elsevier B.V. All rights reserved.

Contents lists available at ScienceDirect

Aquatic Botany

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1. Introduction

Along with other environmental problems, agricultural andviticultural activities also contribute to degradation of aquaticecosystems. Over the past decade pesticide application has becomethe predominant method to control pests and diseases in cropproduction. Phytosanitary products are continuously dischargedinto natural aquatic systems mainly through water runoffprocesses and may subsequently be hazardous to aquatic non-target organism. Aquatic plant toxicity tests are frequentlyconducted to assess environmental risk and are often performedto obtain information on pollutant-induced toxic effects (Blaiseet al., 2000; Eullaffroy and Vernet, 2003; Kuster and Altenburger,2007). Since the photosynthetic process is the key phase of plant

* Corresponding author at: Laboratoire Plantes Pesticides et Developpement

Durable, UPRES 2069 (URVVC), Universite de Reims Champagne-Ardenne, BP 1039,

51687 Reims Cedex 2, France. Tel.: +33 3 26 91 85 25; fax: +33 3 26 91 32 84.

E-mail address: philippe.eullaffroy@univ-reims.fr (P. Eullaffroy).

0304-3770/$ – see front matter � 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.aquabot.2008.09.002

metabolism and is known to be very sensitive to environmentalchanges, it has often been studied to evaluate the health status ofplants. Among the tools used to study effects of environmentalchanges on the photosynthetic apparatus, chlorophyll a fluores-cence is often proposed as a simple, rapid and sensitive method(Strasser et al., 2004; Kuster and Altenburger, 2007). It givesprecise data regarding photochemical efficiency and adverseeffects in plants exposed to stressful conditions such as frost orxenobiotic exposure (Geoffroy et al., 2004; Strauss et al., 2006) andhas been successfully used to monitor changing physiologicalstates of the photosynthetic system (for a review see Schreiber,2004; Papageorgiou et al., 2007). At high excitation irradiance,dark-adapted leaves show a characteristic polyphasic fluorescencekinetics known as the Kautsky effect (Kautsky and Hirsch, 1931).These kinetics exhibit a sequence of phases named O, J, I and Preflecting the successive but overlapping filling-up of photosystemII (PSII) electron acceptor pools as QA, QB and PQ whose oxydo-reduction states are closely controlled by PSII functions (Bukhovet al., 2004; Lazar, 2006; Dewez et al., 2007). It is generallyaccepted that the rapid initial fluorescence rise from the initial O

P. Eullaffroy et al. / Aquatic Botany 90 (2009) 172–178 173

(F0), minimal Chl a fluorescence yield, to J, the first intermediatestep at 2 ms, indicates reduction of the primary electronacceptor of PSII, QA and corresponds to the peak concentrationsof QA

�QB and QA�QB

� (QA and QB are the two first electronacceptors of PSII) (Zhu et al., 2005). Moreover, there is evidencethat J transient is also influenced by the S-state of the water-splitting system (Hsu, 1993). The origin of the following J–I andI–P phases is still unclear. These transients seem to be related tothe PSII heterogeneity (Lavergne and Briantais, 1996; Strasserand Stirbet, 1998; Stirbet et al., 1998) and recent work showedthat I may correspond to an increase in concentrations ofQA�QB

2� and P to the maximum concentrations of QA�QB

2� andPQH2 (plastoquinol pool) (Zhu et al., 2005; Toth et al., 2007). TheI–P phase is also known to be influenced by photosystem I (PSI)activity (Schansker et al., 2005). J–I and I–P phases are,respectively referenced as the first and second part of thethermal phase of Chl fluorescence induction whereas O–Jcorresponds to the photochemical phase (Samson et al., 1999;Schreiber, 2002). This polyphasic fluorescence kinetics isaffected by environmental conditions and can reflect theresponse of plants to various stresses and hence relate to theirhealth status (Bussotti et al., 2007; Kuster et al., 2007; Eullaffroyet al., 2007). Based on OJIP transients Strasser and his team havedeveloped a test called the ‘‘JIP test’’ (Strasser and Strasser,1995; Strasser et al., 2000) that quantifies the in vivo energyfluxes passing through the photosystems and evaluates plantphotosynthetic performance. Besides assessing the function ofPSII, this test also reflects the rate of the electron chain withinthe thylakoid membrane and the subsequent functioning of theFerredoxin-NADP+ oxidoreductase and Calvin Cycle (Schanskeret al., 2003). The process of all of the photosynthetic reactionsshould be reflected in the shape of the OJIP fluorescence kinetics.A number of parameters are derived from the fluorescencetransients to quantify the flow of energy through the reactioncentre (RC) of PSII (Strasser and Strasser, 1995; Force et al.,2003; Strasser et al., 2004). Many ecotoxicological studies haveused the maximum quantum yield ratio (FV/FM) to evaluate thehealth status of a plant. However recent work showed that thisratio is not always the most suitable parameter to displaycontaminant toxicity or plant sensitivity (Force et al., 2003;Christen et al., 2007). In this study we used the OJIP test toappraise pesticide-induced effects via the change in thebiophysical parameters quantifying the energy flow throughPSII. Parameters related to energy absorption (trapping andconversion) are used as a measure of pesticide sensitivity and tolocalize the ‘‘effect sites’’ of the selected herbicide. Since mostpesticides used in agriculture are herbicides (44% of the worldpesticides market, Kiely et al., 2004), we studied the efficiency ofthe OJIP test to rapidly yield information on the specific effect ofdifferent herbicides toward the photosynthetic apparatus.Diuron, paraquat and flazasulfuron were chosen because oftheir differing modes of action, i.e. PSII, photosystem I (PSI) andprotein biosynthesis inhibitors, respectively (Boger and Sand-mann, 1998; Tomlin, 2000). Herein, we show the advantages ofusing the OJIP test to evaluate contaminant toxicity and thehealth status of a plant through the use of a number ofparameters obtained from the same measurement rather thanthat from a single parameter such as the well known FV/FM ratio.

2. Material and methods

2.1. Plant material

Duckweed (Lemna minor L.) was collected from ponds in theArdennes area of France. Before initiating experiments, the fronds

were disinfected by immersion in NaOCl 1% (v/v) and Tween 0.01%(v/v) for 3 min and then rinsed with distilled water for 5 min.Duckweeds were maintained in large PVC aquaria containingsterile growth medium (pH 6.5) adapted from Chollet (1993).Nutrient medium consisted of Ca2+, 2.65 mM; Mg2+, 0.16 mM; Na+,0.15 mM; K+, 0.35 mM; HCO3

�, 4.46 mM; SO42�, 0.61 mM; Cl�,

0.11 mM; NO3�, 0.35 mM. Stock and contaminated cultures were

maintained in a growth chamber at 22 � 2 8C under continuousirradiance (100 mmol photons m�2 s�1) provided by cool whitefluorescent lamps (Sylvania Gro Lux F36W, Germany). The plantswere sub-cultured twice a week.

2.2. Pesticide application

Pesticide treatments were performed on duckweeds with diuron,paraquat (Sigma, France) and flazasulfuron (used as Katana1-Zeneca Sopra, France). All pesticide concentrations shown here referto the active ingredient. All stock solutions were prepared just priorto initiating the experiments. Each experiment was replicated threetimes. Plants were exposed to phytosanitary products for 48 h. Theexperiments were conducted under static conditions.

2.3. Toxicity assessment

Chl a fluorescence transients were recorded at room tempera-ture from the upper face of duckweed fronds with a PEAfluorometer (Plant Efficiency Analyzer, Hansatech1, England).All measurements were performed with 30 min dark-adapted L.

minor.The PEA saturating flash was provided by an array of six light-

emitting diodes with an excitation light intensity of 600 W m�2 at650 nm. The fast fluorescence kinetics was measured in a timespan from 10 ms to 1 s. Each transient was analyzed by utilizing thefollowing data: F0, fluorescence intensity at 50 ms (open RCs), FM (=FP), maximum fluorescence intensity (closed RCs), F300 and FJ,fluorescence intensity at 300 ms and 2 ms, respectively. Thefollowing equations were used to assess the photosyntheticperformance of duckweeds (Strasser et al., 2000; Appenrothet al., 2001; Strauss et al., 2006):

� T

he rate of QA reduction in the first ms,M0 ¼ 4ðF300 � F0Þ=ðFM � F0Þ, � T he variable fluorescence at the J step, VJ = (F2 ms � F0)/(FM � F0), � T he fraction of inhibited centres, FIC = (VJcontaminated � VJcontrol)/

(1 � VJcontrol),

� T he specific energy fluxes (per reaction centre, RC): for

absorption, ABS=RCð¼ M0ð1=V JÞð1=’P0ÞÞ, trapping, TR0/RC (=

M0(1/VJ)), electron transport, ET0/RC (= M0(1/VJ)c0), anddissipation, DI0/RC (=(ABS/RC) � (TR0/RC)),

� T he maximum efficiency of PSII photochemistry in the dark-

adapted state, ’P0ð¼ FV=FM ¼ ðFM � F0Þ=FM ¼ TR0=ABSÞ,

� T

he efficiency with which a trapped exciton can move anelectron into the transport chain further than QA

�, c0

(=1 � VJ = ET0/TR0),

� T he quantum yield of electron transport, ’E0

ð¼ ’P0�c0Þ,

� T

he performance index, PIABSð¼ ðRC=ABSÞ½ð’P0=ð1� ’P0

ÞÞ�½ðc0=ð1�c0ÞÞ�, expressing the three functional steps of photo-synthesis, i.e. absorption of light energy (ABS), trapping ofexcitation energy (TR) and conversion of excitation energy toelectron transport (ET),

� T he amount of active PSII RCs per absorption,

RC=ABSð¼ ð’P0V JÞ=M0Þ,

� T

he driving force for photosynthesis, DFABS = log -PIABS = DFRC + DFw + DFc with: DFRC = log (RC/ABS), DF’ ¼logð’P0

=ð1� ’P0ÞÞ and DFc ¼ logðc0=ð1�c0ÞÞ.

P. Eullaffroy et al. / Aquatic Botany 90 (2009) 172–178174

The recorded fluorescence transients were plotted in alogarithmic time scale. At least 12 fronds were measured for eachtype of treatment.

2.4. Statistical analysis

Data presented in this study are the means � standard deviation.The statistical differences between controls and contaminatedsamples were analyzed by the Mann and Whitney test (Mann andWhitney, 1947), p values<0.05 were considered significant (asterisks[*] indicate significant values). Analyses were performed with SigmaStat 2.03 (SPSS Inc., USA) software.

3. Results

3.1. Toxicity

Fig. 1 illustrates a typical fluorescence rise from control andherbicide-treated duckweeds. To better reveal the changes in theshape of the transients, the curves were plotted as the relativevariable fluorescence, Vt = (Ft � F0)/(FM � F0) on a logarithmic timescale (insert of Fig. 1). The kinetics of an untreated Lemna arecomposed of four clear transients. First, O (F0), was the origin of theChl fluorescence rise. Then, kinetics develop to reach a maximum(P, i.e. FP = FM) attained after about 1-s irradiation. From theposition of these two characteristic points on the time scale, weidentified the two other transients known as the J-step (peak) andthe I-step after 2 and 30 ms, respectively. As an example of how thepolyphasic rise may be modified in plants treated with chemicals,we show in Fig. 1 that the OJIP rise is significantly affected in frondsexposed 48 h to 20 mg L�1 of diuron. Diuron increased themagnitude of the J-step while the following I-step tended todisappear. The sigmoid shape of the O–J and J–I phases was lostbased on the mode of action of diuron (i.e. electron transferblockage from QA

� to QB which limits electron flow in PSII) andinterpreted as an accumulation of QA

� (Strasser et al., 1995; Bogerand Sandmann, 1998).

The shape changes of the O–J–I–P curves were converted intoquantitative changes of diverse parameters. Analysis of thesechanges by the JIP test allowed us to assess the absorption,trapping, electron transport and dissipation and then the driving

Fig. 1. Typical chlorophyll a fluorescence transients O–J–I–P in untreated and

diuron-treated (48 h, 20 mg L�1) Lemma minor, plotted on logarithmic time scale. In

the insert, the same kinetics were expressed as the relative variable fluorescence,

Vt = (Ft � F0)/(FM � F0).

force for each of those energy pathways in plants exposed to theselected herbicides.

The effects of herbicide on the PSII reaction centre activity wasquantified by the performance index, PIABS. This index is the overallexpression of three functional steps (energy absorption, energytrapping and energy conversion into the electron transport)(Strauss et al., 2006). The average decrease of the PIABS after48 h of exposure to 10 and 100 mg L�1 of diuron, paraquat andflazasulfuron was 80, 30 and 0% and 99, 99, and 22%, respectively(data not shown). Fig. 2 displays the log value of the PIABS with itscomponents. The partial (DFRC, DFw, DFc) and total driving force forphotosynthesis (DFABS) can then be observed. The three herbicidestested induced a change in the three components of the totaldriving force.

The diuron-treated fronds showed a DFABS decrease mainly dueto a lower DFc related to the trapping of the excitation energy. DFRC

(light energy absorption) and DFw (conversion of excitation energyto electron transport) were not affected by diuron (Fig. 2A).

Paraquat induced an important decrease of DFABS comparable tothe one observed in diuron-treated fronds. However, the responseof DFABS differed in the major component that is affected byparaquat. In fact, DFw, was the main component of DFABS that wasmostly affected by paraquat while DFRC, and DFc, were moreimpacted than in diuron-treated fronds (Fig. 2B).

The total driving force for photosynthesis of duckweed exposedto flazasulfuron is only slightly diminished, owing mostly to adecrease in DFw when exposed to a concentration higher than20 mg L�1. The lowest flazasulfuron concentration tested induced alight increase of all the partial components of the total driving forcefor photosynthesis (Fig. 2C).

From the fluorescence transient we can assess the yield ofelectron transport ð’E0

Þ which is the product of the yield ofprimary electron transport ð’P0

Þ, and the yield of electrontransport per trapped exciton, c0 (=ET0/TR0). Diuron-, paraquat-and flazasulfuron-treated fronds showed a 97, 84 and 8% inhibitionof the electron transport, respectively, at the highest concentrationtested (Fig. 3). However, in Lemna exposed 48 h to diuron, thisinhibition was due more to a decrease in efficiency with which atrapped exciton can move an electron into the electron transportchain further than QA

� (c0), rather than to a decrease of themaximum quantum yield of primary photochemistry ð’P0

Þ(Fig. 3A). The decrease of ’E0

induced by paraquat was due toan inhibition in the same proportion of c0 (=ET0/TR0) and ’P0

(=TR0/ABS) (Fig. 3B). Flazasulfuron induces a slight but significantdecrease in the yield of electron transport involving equally c0

(=ET0/TR0) and ’P0ð¼ TR0=ABSÞ (Fig. 3C).

Fig. 4 is informative on effects of the studied herbicides linkedto specific energy fluxes through PSII at the RC. Diuron, paraquatand flazasulfuron did not affect the trapping rate of the RC (TR0/RC)in treated duckweed fronds. However, diuron induced a 25%increase of the excitation energy in the antenna of an active RC(ABS/RC) resulting in a 90% increase in the effective dissipation(DI0/RC) at 100 mg L�1 (Fig. 4A). The same pattern was observed infronds exposed to paraquat but with a much higher increase ofABS/RC and DI0/RC, i.e. 200 and 800%, respectively (Fig. 4B).Duckweed exposed to the highest concentration of flazasulfuronshowed a slight loss of energy (15%) in dissipation (DI0/RC)compared to the control, but no significant increase in ABS/RC(Fig. 4C).

The decrease in ET0/RC, related to the reoxidation of reduced QA

via electron transport in an active RC, and c0 (= ET0/TR0) weremore pronounced in Lemna exposed to diuron than paraquat(Fig. 4A and B). There is no effect on the QA reoxidation rate induckweed exposed to flazasulfuron (Fig. 4C). In L. minor exposed todiuron and paraquat, the decline in ET0/RC is mostly due to a

Fig. 2. Total (DFABS = DFABS = DFRC + DFw + DFc) and partial (DFRC = log [RC/ABS];

DF’ ¼ log½’P0=ð1� ’P0

Þ�; DFc ¼ log½c0=ð1�c0Þ�) driving forces for

photosynthesis in L. minor exposed to (A) diuron, (B) paraquat, and (C)

flazasulfuron. All logarithmic values (see Section 2) are indicated as the

difference of the herbicide-treated sample minus the control.

Fig. 3. Effect of different concentrations of (A) diuron, (B) paraquat, and (C)

flazasulfuron in L. minor on the maximum efficiency of PSII photochemistry, ’P0ð¼

TR0=ABSÞ (– – –), the efficiency with which a trapped exciton can move an electron

into the transport chain further than QA�, c0 (=ET0/TR0) (- - -) and the quantum

yield of electron transport, ’E0ð¼ ’P0

�c0Þ (—).

P. Eullaffroy et al. / Aquatic Botany 90 (2009) 172–178 175

decrease in the transport of electrons further than QA� (c0) and not

from a decrease in trapping (TR0/RC). The fraction of inhibitedcentres (FIC) was severely increased, 94 and 57%, at 100 mg L�1 ofdiuron and paraquat, respectively. FIC already reached about 60%in fronds exposed for 48 h at 10 mg L�1 of diuron. Because ET0/RC iscalculated as TR0/RC �c0 the decline in ET0/RC in diuron- andparaquat-treated fronds mainly reflected a much lower probabilitythat an electron residing on QA will enter the electron transport

chain since trapping showed no significant inhibition. Flazasul-furon showed a slight impact on FIC since only 5% of the centreswere inhibited in fronds exposed to the highest concentration(Fig. 4C). FIC is strongly correlated to the decrease in the efficiencywith which a trapped exciton by the RC will lead to a reduction ofQA to QA

� (ET0/RC) (r2 = 0.99; data not shown).

Fig. 4. Influence of (A) diuron, (B) paraquat, and (C) flazasulfuron on the fraction of

inhibited centres (FIC = [VJcontaminated � VJcontrol]/[1 � VJcontrol]) and several JIP-test

parameters related to energy fluxes: the specific energy fluxes for absorption,

ABS=RCð¼ M0ð1=V JÞð1=’P0ÞÞ ( ); trapping, TR0=RCð¼ M0ð1=V JÞÞ ( ); electron

transport, ET0=RCð¼ M0ð1=V JÞc0Þ ( );dissipation, DI0=RCð¼ ðABS=RCÞ �ðTR0=RCÞÞ ( ) and c0 (=ET0/TR0) ( ). The data have been normalized to the

control plant. *Significant difference at P < 0.05.

P. Eullaffroy et al. / Aquatic Botany 90 (2009) 172–178176

4. Discussion

Herbicides such as diuron, paraquat and flazasulfuron arecommonly used in agriculture and their residues can be easilytranslocated into aquatic environments through surface runoff(Kloeppel et al., 1997; Garmouna et al., 1998). The study of thephysiological effects of such pollutants on non-target organismsrequires criteria to distinguish their acute and chronic (sub)lethal

toxicity effects (Blaise and Ferard, 2005). OJIP chlorophyll a

fluorescence curves were appraised to enhance our knowledgeconcerning toxicity and site of action of contaminants on thephotosynthetic machinery through the study of energy fluxes anddriving force for photosynthesis.

4.1. Effect of diuron on energy fluxes

In diuron-treated fronds, the decrease in DFc was the mostpronounced effect indicating a strong blockage which inhibitselectron flow from reduced QA towards PQ, leading to a loss ofcapability for intact electron transport in the plant. Diminishedefficiency by which a trapped exciton can move an electron intothe electron transport chain further than QA

� was the main factorthat determined the magnitude of DFABS diminution in Lemna

exposed to diuron. This halogenated-phenylurea herbicide alsoinduced a decrease of the quantum yield for electron transportbeyond QA

� ð’E0¼ ’P0

�c0Þ confirming a limitation of QA�

reoxidation. The more pronounced effect on the yield of electrontransport per trapped exciton (c0) suggested that the darkreactions after QA

�, represented by c0 (=ET0/TR0), are moresensitive to diuron than light-dependent reactions represented by’P0

. This agrees with the concept that diuron occupies the bindingsite of QB on the reaction centre complex. As a consequence, thelight independent electron flow from QA

� to QB is blocked.Regardless of the active molecule, all fronds exposed to an

herbicide showed a significant increase of the effective dissipation inan active RC (DI0/RC). Dissipation refers to the loss of absorbedenergy via heat and fluorescence emission and energy transfer toother systems (Strasser et al., 2000) and is represented by equationDI0/RC = (ABS/RC) � (TR0/RC). Therefore, dissipation can be thoughtof as the absorption of photons in excess of what can be trapped bythe RC. In fronds exposed to diuron, the excitation energy in theantenna of the active RC was in excess of that required for trappingand some energy was dissipated. Since no decrease in the lightreactions leading to the energy flux as trapping per active RC wasobserved, we concluded that energy dissipation was rising via arelative increase of light harvesting complex per RC favouring thedissipation of the energy in excess through non-photochemicalpathways (thermal dissipation processes). The increase of ABS/RCespecially at the highest concentration tested illustrates a change inthe antenna size of PSII RC due to a change in the number of LHCcomplexes per PSII RC or to the inactivation of RC’s, forming non-QA

reducing, so called ‘‘heat sink’’ or ‘‘silent reaction’’ centres. Since 90%of the electron transport is inhibited between QA

� and QB, theincrease in the expression ABS/RC is mainly due to a decrease in thenumber of RCs per absorption (RC/ABS), as shown in the partialdriving force DFRC = log (RC/ABS), respectively by the partialperformance index PIRC = RC/ABS.

Again, diuron also lowered photosynthetic electron transportby decreasing the electron flux per RC between the twophotosystems (ET0/TR0 = c0).

It is known that diuron efficiently blocks PSII-catalyzedphotosynthetic electron transport at the secondary electronacceptor, QB (Van Rensen, 1982). In this case, the first electronacceptor (QA) cannot be reoxidized by the plastoquinone pool viathe QB electron carrier, and consequently minimize the yield of thedark reactions. This results in inhibition of both electron transportand degradation of the D1 protein [i.e. RC] (Fuerst and Norman,1991; Kirilovsky et al., 1994; Bukhov et al., 2004).

4.2. Effect of paraquat on energy fluxes

Paraquat induced an important decrease of the driving force forphotosynthesis, comparable to that seen in diuron-treated fronds.

P. Eullaffroy et al. / Aquatic Botany 90 (2009) 172–178 177

However, all three components DFRC, DFw and DFc weresignificantly lowered by paraquat. As in diuron-treated plants,there was a considerable decrease of the quantum yield of electrontransport beyond QA

�. Owing to the interaction of paraquat whichdeviates electron flow progressing towards NADP, results showthat electron transport inhibition was not due to a target-specificaction on the light reactions measured by ’P0

ð¼ TR0=ABSÞ, nor bythe dark reactions determined by c0ð¼ ET0=TR0Þ.

Paraquat treatment produced major changes in electrontransport in active RC. Here, the diminution of the number ofelectron entering into the transport chain (c0) is not as importantas in diuron-treated fronds, but paraquat induced a very importantrise in the total absorption of PSII antenna chlorophyll by an activeRC leading to a large increase of energy fluxes per RC for dissipation(DI0/RC).

Our results indicate that paraquat and diuron cause an obviousdecrease in the number of active PSII reaction centres and of theelectron transport rate. This can lead to an inactivation of a fractionof the RC’s of PSII converted to non-QA reducing centres (Srivastavaet al., 1999; Tsimilli-Michael et al., 1999) resulting in an increase ofthe average antenna size per remaining active reaction centrealready seen in L. minor exposed to chromium (Ait Ali et al., 2006).Under these circumstances, regulation of light harvesting isnecessary to balance the absorption and utilization of light energy,thereby minimizing the potential of photooxidative damage(Muller et al., 2001).

Paraquat, also known as methyl-viologen, is a very effectiveelectron acceptor that competes strongly with ferredoxin forelectrons from the FeS clusters of PSI and, as a consequence,strongly suppresses cyclic electron transfer around PSI (Schanskeret al., 2005). Paraquat is also known as a redox-active compoundthat is photoreduced by PSI and then reoxidized by transferringelectrons to O2, forming reactive oxygen species (ROS) (Prestonet al., 1991; Foyer et al., 1994; Boger and Sandmann, 1998). Thehigh oxygen concentration of the chloroplast will ensure a rapidreoxidation of paraquat radicals which regenerates paraquat andproduces superoxide O2

� (Fuerst and Norman, 1991). Activeoxygen species will diminish electron transport in the thylakoidmembranes of the chloroplast resulting, therefore, in substantialchanges in characteristics of Chl a fluorescence kinetics andparameters (e.g. c0) (Ekmekci and Terzioglu, 2005; Eullaffroy et al.,2007).

4.3. Effect of flazasulfuron on energy fluxes

This herbicide induced a very slight decrease of the total drivingforce for photosynthesis. For the highest inhibitor concentrationdelta DF = �0.1 compared to �2.0 for diuron and paraquat. Themain source of this slight inhibition was due to a change inthe maximum quantum yield for primary photochemistry’P0ð¼ TR0=ABSþ FV=FMÞ. This led, at the highest concentration,

in a decrease of the number of active PSII reaction centres. At thisconcentration a small but significant increase of the non-photochemical energy dissipation per RC (DI0/RC) is also seen.Little is known about the toxicity of flazasulfuron on non-targetplants. It has been shown that this herbicide can reduce growthand photosynthetic activity of plants by lowering or decreasing leafgas exchanges and photosynthetic pigment concentrations and bya marked disorganization of the leaf plastids (Frankart et al., 2003;Magne et al., 2006). Flazasulfuron is an effective inhibitor ofacetolactate synthase, an enzyme involved in the biosynthesis ofbranched-chain amino acids (Boger and Sandmann, 1998). As aninhibitor of protein synthesis it may disrupt the assembly ofchlorophyll–protein complexes included in light harvestingcomplex of PSII. The accumulation of free chlorophyll (not bound

to protein) could allow an additional quenching of excess absorbedlight energy, by increasing energy dissipation (DI0/RC) (Gilmoreand Govindjee, 1999; Muller et al., 2001).

Acknowledgment

This work was supported, in part, by the Europol’AgroFoundation.

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