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Energy fluxes and driving forces for photosynthesis in Lemna minor exposed to herbicides
Aquatic Botany 90 (2009) 172–178
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Energy fluxes and driving forces for photosynthesis in Lemna minor exposed to herbicides Philippe Eullaffroy a,b,*, Ce´cile Frankart a, Aziz Aziz a, Michel Couderchet a, Christian Blaise b a b
Laboratoire Plantes Pesticides et De´veloppement Durable (PPDD), UPRES 2069 (URVVC), Universite´ de Reims Champagne-Ardenne, BP 1039, 51687 Reims Cedex 2, France Environnement Canada, Centre Saint-Laurent, 105 rue McGill, Montre´al, Qc, H2Y 2E7 Canada
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 April 2008 Received in revised form 17 September 2008 Accepted 19 September 2008 Available online 27 September 2008
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’P0 Þ 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. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Chlorophyll fluorescence Toxicity Photosynthesis Duckweed Water
1. Introduction Along with other environmental problems, agricultural and viticultural activities also contribute to degradation of aquatic ecosystems. Over the past decade pesticide application has become the predominant method to control pests and diseases in crop production. Phytosanitary products are continuously discharged into natural aquatic systems mainly through water runoff processes and may subsequently be hazardous to aquatic nontarget organism. Aquatic plant toxicity tests are frequently conducted to assess environmental risk and are often performed to obtain information on pollutant-induced toxic effects (Blaise et al., 2000; Eullaffroy and Vernet, 2003; Ku¨ster and Altenburger, 2007). Since the photosynthetic process is the key phase of plant
* Corresponding author at: Laboratoire Plantes Pesticides et De´veloppement 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: [email protected] (P. Eullaffroy). 0304-3770/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2008.09.002
metabolism and is known to be very sensitive to environmental changes, it has often been studied to evaluate the health status of plants. Among the tools used to study effects of environmental changes on the photosynthetic apparatus, chlorophyll a fluorescence is often proposed as a simple, rapid and sensitive method (Strasser et al., 2004; Ku¨ster and Altenburger, 2007). It gives precise data regarding photochemical efficiency and adverse effects in plants exposed to stressful conditions such as frost or xenobiotic exposure (Geoffroy et al., 2004; Strauss et al., 2006) and has been successfully used to monitor changing physiological states 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 fluorescence kinetics known as the Kautsky effect (Kautsky and Hirsch, 1931). These kinetics exhibit a sequence of phases named O, J, I and P reflecting the successive but overlapping filling-up of photosystem II (PSII) electron acceptor pools as QA, QB and PQ whose oxydoreduction states are closely controlled by PSII functions (Bukhov et al., 2004; Laza´r, 2006; Dewez et al., 2007). It is generally accepted that the rapid initial fluorescence rise from the initial O
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(F0), minimal Chl a fluorescence yield, to J, the first intermediate step at 2 ms, indicates reduction of the primary electron acceptor of PSII, QA and corresponds to the peak concentrations of QAQB and QAQB (QA and QB are the two first electron acceptors of PSII) (Zhu et al., 2005). Moreover, there is evidence that J transient is also influenced by the S-state of the watersplitting system (Hsu, 1993). The origin of the following J–I and I–P phases is still unclear. These transients seem to be related to the PSII heterogeneity (Lavergne and Briantais, 1996; Strasser and Stirbet, 1998; Stirbet et al., 1998) and recent work showed that I may correspond to an increase in concentrations of QAQB2 and P to the maximum concentrations of QAQB2 and PQH2 (plastoquinol pool) (Zhu et al., 2005; To´th et al., 2007). The I–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 the thermal phase of Chl fluorescence induction whereas O–J corresponds to the photochemical phase (Samson et al., 1999; Schreiber, 2002). This polyphasic fluorescence kinetics is affected by environmental conditions and can reflect the response of plants to various stresses and hence relate to their health status (Bussotti et al., 2007; Ku¨ster et al., 2007; Eullaffroy et al., 2007). Based on OJIP transients Strasser and his team have developed a test called the ‘‘JIP test’’ (Strasser and Strasser, 1995; Strasser et al., 2000) that quantifies the in vivo energy fluxes passing through the photosystems and evaluates plant photosynthetic performance. Besides assessing the function of PSII, this test also reflects the rate of the electron chain within the thylakoid membrane and the subsequent functioning of the Ferredoxin-NADP+ oxidoreductase and Calvin Cycle (Schansker et al., 2003). The process of all of the photosynthetic reactions should be reflected in the shape of the OJIP fluorescence kinetics. A number of parameters are derived from the fluorescence transients to quantify the flow of energy through the reaction centre (RC) of PSII (Strasser and Strasser, 1995; Force et al., 2003; Strasser et al., 2004). Many ecotoxicological studies have used the maximum quantum yield ratio (FV/FM) to evaluate the health status of a plant. However recent work showed that this ratio is not always the most suitable parameter to display contaminant toxicity or plant sensitivity (Force et al., 2003; Christen et al., 2007). In this study we used the OJIP test to appraise pesticide-induced effects via the change in the biophysical parameters quantifying the energy flow through PSII. Parameters related to energy absorption (trapping and conversion) are used as a measure of pesticide sensitivity and to localize the ‘‘effect sites’’ of the selected herbicide. Since most pesticides used in agriculture are herbicides (44% of the world pesticides market, Kiely et al., 2004), we studied the efficiency of the OJIP test to rapidly yield information on the specific effect of different herbicides toward the photosynthetic apparatus. Diuron, paraquat and flazasulfuron were chosen because of their differing modes of action, i.e. PSII, photosystem I (PSI) and protein biosynthesis inhibitors, respectively (Bo¨ger and Sandmann, 1998; Tomlin, 2000). Herein, we show the advantages of using the OJIP test to evaluate contaminant toxicity and the health status of a plant through the use of a number of parameters obtained from the same measurement rather than that 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 the Ardennes area of France. Before initiating experiments, the fronds
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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 containing sterile 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 continuous irradiance (100 mmol photons m2 s1) provided by cool white fluorescent lamps (Sylvania Gro Lux F36W, Germany). The plants were sub-cultured twice a week. 2.2. Pesticide application Pesticide treatments were performed on duckweeds with diuron, paraquat (Sigma, France) and flazasulfuron (used as Katana1Zeneca Sopra, France). All pesticide concentrations shown here refer to the active ingredient. All stock solutions were prepared just prior to initiating the experiments. Each experiment was replicated three times. Plants were exposed to phytosanitary products for 48 h. The experiments were conducted under static conditions. 2.3. Toxicity assessment Chl a fluorescence transients were recorded at room temperature from the upper face of duckweed fronds with a PEA fluorometer (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 lightemitting diodes with an excitation light intensity of 600 W m2 at 650 nm. The fast fluorescence kinetics was measured in a time span from 10 ms to 1 s. Each transient was analyzed by utilizing the following 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. The following equations were used to assess the photosynthetic performance of duckweeds (Strasser et al., 2000; Appenroth et al., 2001; Strauss et al., 2006): The rate of QA reduction in the first ms, M 0 ¼ 4ðF 300 F 0 Þ=ðF M F 0 Þ, The variable fluorescence at the J step, VJ = (F2 ms F0)/(FM F0), The fraction of inhibited centres, FIC = (VJcontaminated VJcontrol)/ (1 VJcontrol), The specific energy fluxes (per reaction centre, RC): for absorption, ABS=RCð¼ M 0 ð1=V J Þð1=’P0 ÞÞ, trapping, TR0/RC (= M0(1/VJ)), electron transport, ET0/RC (= M0(1/VJ)c0), and dissipation, DI0/RC (=(ABS/RC) (TR0/RC)), The maximum efficiency of PSII photochemistry in the darkadapted state, ’P0 ð¼ F V =F M ¼ ðF M F 0 Þ=F M ¼ TR0 =ABSÞ, The efficiency with which a trapped exciton can move an electron into the transport chain further than QA, c0 (=1 VJ = ET0/TR0), The quantum yield of electron transport, ’E0 ð¼ ’P0 c0 Þ, The performance index, PIABS ð¼ ðRC=ABSÞ½ð’P0 =ð1 ’P0 ÞÞ ½ðc0 =ð1 c0 ÞÞ, expressing the three functional steps of photosynthesis, i.e. absorption of light energy (ABS), trapping of excitation energy (TR) and conversion of excitation energy to electron transport (ET), The amount of active PSII RCs per absorption, RC=ABSð¼ ð’P0 V J Þ=M 0 Þ, The driving force for photosynthesis, DFABS = log PIABS = DFRC + DFw + DFc with: DFRC = log (RC/ABS), DF ’ ¼ logð’P0 =ð1 ’P0 ÞÞ and DF c ¼ logðc0 =ð1 c0 ÞÞ.
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The recorded fluorescence transients were plotted in a logarithmic time scale. At least 12 fronds were measured for each type of treatment. 2.4. Statistical analysis Data presented in this study are the means standard deviation. The statistical differences between controls and contaminated samples were analyzed by the Mann and Whitney test (Mann and Whitney, 1947), p values <0.05 were considered significant (asterisks [*] indicate significant values). Analyses were performed with Sigma Stat 2.03 (SPSS Inc., USA) software. 3. Results 3.1. Toxicity Fig. 1 illustrates a typical fluorescence rise from control and herbicide-treated duckweeds. To better reveal the changes in the shape of the transients, the curves were plotted as the relative variable fluorescence, Vt = (Ft F0)/(FM F0) on a logarithmic time scale (insert of Fig. 1). The kinetics of an untreated Lemna are composed of four clear transients. First, O (F0), was the origin of the Chl fluorescence rise. Then, kinetics develop to reach a maximum (P, i.e. FP = FM) attained after about 1-s irradiation. From the position of these two characteristic points on the time scale, we identified the two other transients known as the J-step (peak) and the I-step after 2 and 30 ms, respectively. As an example of how the polyphasic rise may be modified in plants treated with chemicals, we show in Fig. 1 that the OJIP rise is significantly affected in fronds exposed 48 h to 20 mg L1 of diuron. Diuron increased the magnitude of the J-step while the following I-step tended to disappear. The sigmoid shape of the O–J and J–I phases was lost based on the mode of action of diuron (i.e. electron transfer blockage from QA to QB which limits electron flow in PSII) and interpreted as an accumulation of QA (Strasser et al., 1995; Bo¨ger and Sandmann, 1998). The shape changes of the O–J–I–P curves were converted into quantitative changes of diverse parameters. Analysis of these changes 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 L1) 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 the selected herbicides. The effects of herbicide on the PSII reaction centre activity was quantified by the performance index, PIABS. This index is the overall expression of three functional steps (energy absorption, energy trapping and energy conversion into the electron transport) (Strauss et al., 2006). The average decrease of the PIABS after 48 h of exposure to 10 and 100 mg L1 of diuron, paraquat and flazasulfuron was 80, 30 and 0% and 99, 99, and 22%, respectively (data not shown). Fig. 2 displays the log value of the PIABS with its components. The partial (DFRC, DFw, DFc) and total driving force for photosynthesis (DFABS) can then be observed. The three herbicides tested induced a change in the three components of the total driving force. The diuron-treated fronds showed a DFABS decrease mainly due to a lower DFc related to the trapping of the excitation energy. DFRC (light energy absorption) and DFw (conversion of excitation energy to electron transport) were not affected by diuron (Fig. 2A). Paraquat induced an important decrease of DFABS comparable to the one observed in diuron-treated fronds. However, the response of DFABS differed in the major component that is affected by paraquat. In fact, DFw, was the main component of DFABS that was mostly affected by paraquat while DFRC, and DFc, were more impacted than in diuron-treated fronds (Fig. 2B). The total driving force for photosynthesis of duckweed exposed to flazasulfuron is only slightly diminished, owing mostly to a decrease in DFw when exposed to a concentration higher than 20 mg L1. The lowest flazasulfuron concentration tested induced a light increase of all the partial components of the total driving force for photosynthesis (Fig. 2C). From the fluorescence transient we can assess the yield of electron transport ð’E0 Þ which is the product of the yield of primary electron transport ð’P0 Þ, and the yield of electron transport per trapped exciton, c0 (=ET0/TR0). Diuron-, paraquatand flazasulfuron-treated fronds showed a 97, 84 and 8% inhibition of the electron transport, respectively, at the highest concentration tested (Fig. 3). However, in Lemna exposed 48 h to diuron, this inhibition was due more to a decrease in efficiency with which a trapped exciton can move an electron into the electron transport chain further than QA (c0), rather than to a decrease of the maximum quantum yield of primary photochemistry ð’P0 Þ (Fig. 3A). The decrease of ’E0 induced by paraquat was due to an inhibition in the same proportion of c0 (=ET0/TR0) and ’P0 (=TR0/ABS) (Fig. 3B). Flazasulfuron induces a slight but significant decrease in the yield of electron transport involving equally c0 (=ET0/TR0) and ’P0 ð¼ TR0 =ABSÞ (Fig. 3C). Fig. 4 is informative on effects of the studied herbicides linked to specific energy fluxes through PSII at the RC. Diuron, paraquat and 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 L1 (Fig. 4A). The same pattern was observed in fronds exposed to paraquat but with a much higher increase of ABS/RC and DI0/RC, i.e. 200 and 800%, respectively (Fig. 4B). Duckweed exposed to the highest concentration of flazasulfuron showed 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) were more pronounced in Lemna exposed to diuron than paraquat (Fig. 4A and B). There is no effect on the QA reoxidation rate in duckweed exposed to flazasulfuron (Fig. 4C). In L. minor exposed to diuron and paraquat, the decline in ET0/RC is mostly due to a
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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 c0 Þ) 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.
decrease in the transport of electrons further than QA (c0) and not from a decrease in trapping (TR0/RC). The fraction of inhibited centres (FIC) was severely increased, 94 and 57%, at 100 mg L1 of diuron and paraquat, respectively. FIC already reached about 60% in fronds exposed for 48 h at 10 mg L1 of diuron. Because ET0/RC is calculated as TR0/RC c0 the decline in ET0/RC in diuron- and paraquat-treated fronds mainly reflected a much lower probability that an electron residing on QA will enter the electron transport
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Fig. 3. Effect of different concentrations of (A) diuron, (B) paraquat, and (C) flazasulfuron in L. minor on the maximum efficiency of PSII photochemistry, ’P0 ð¼ 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, ’E0 ð¼ ’P0 c0 Þ (—).
chain since trapping showed no significant inhibition. Flazasulfuron showed a slight impact on FIC since only 5% of the centres were inhibited in fronds exposed to the highest concentration (Fig. 4C). FIC is strongly correlated to the decrease in the efficiency with which a trapped exciton by the RC will lead to a reduction of QA to QA (ET0/RC) (r2 = 0.99; data not shown).
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toxicity effects (Blaise and Fe´rard, 2005). OJIP chlorophyll a fluorescence curves were appraised to enhance our knowledge concerning toxicity and site of action of contaminants on the photosynthetic machinery through the study of energy fluxes and driving force for photosynthesis. 4.1. Effect of diuron on energy fluxes
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 M 0 ð1=V J Þð1=’P0 ÞÞ ( ); trapping, TR0 =RCð¼ M 0 ð1=V J ÞÞ ( ); electron transport, ET0 =RCð¼ M 0 ð1=V J Þc0 Þ ( );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.
4. Discussion Herbicides such as diuron, paraquat and flazasulfuron are commonly used in agriculture and their residues can be easily translocated into aquatic environments through surface runoff (Kloeppel et al., 1997; Garmouna et al., 1998). The study of the physiological effects of such pollutants on non-target organisms requires criteria to distinguish their acute and chronic (sub)lethal
In diuron-treated fronds, the decrease in DFc was the most pronounced effect indicating a strong blockage which inhibits electron flow from reduced QA towards PQ, leading to a loss of capability for intact electron transport in the plant. Diminished efficiency by which a trapped exciton can move an electron into the electron transport chain further than QA was the main factor that determined the magnitude of DFABS diminution in Lemna exposed to diuron. This halogenated-phenylurea herbicide also induced a decrease of the quantum yield for electron transport beyond QA ð’E0 ¼ ’P0 c0 Þ confirming a limitation of QA reoxidation. The more pronounced effect on the yield of electron transport per trapped exciton (c0) suggested that the dark reactions after QA, represented by c0 (=ET0/TR0), are more sensitive to diuron than light-dependent reactions represented by ’P0 . This agrees with the concept that diuron occupies the binding site of QB on the reaction centre complex. As a consequence, the light 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 in an active RC (DI0/RC). Dissipation refers to the loss of absorbed energy via heat and fluorescence emission and energy transfer to other systems (Strasser et al., 2000) and is represented by equation DI0/RC = (ABS/RC) (TR0/RC). Therefore, dissipation can be thought of as the absorption of photons in excess of what can be trapped by the RC. In fronds exposed to diuron, the excitation energy in the antenna of the active RC was in excess of that required for trapping and some energy was dissipated. Since no decrease in the light reactions leading to the energy flux as trapping per active RC was observed, we concluded that energy dissipation was rising via a relative increase of light harvesting complex per RC favouring the dissipation of the energy in excess through non-photochemical pathways (thermal dissipation processes). The increase of ABS/RC especially at the highest concentration tested illustrates a change in the antenna size of PSII RC due to a change in the number of LHC complexes 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, the increase in the expression ABS/RC is mainly due to a decrease in the number of RCs per absorption (RC/ABS), as shown in the partial driving force DFRC = log (RC/ABS), respectively by the partial performance index PIRC = RC/ABS. Again, diuron also lowered photosynthetic electron transport by decreasing the electron flux per RC between the two photosystems (ET0/TR0 = c0). It is known that diuron efficiently blocks PSII-catalyzed photosynthetic electron transport at the secondary electron acceptor, QB (Van Rensen, 1982). In this case, the first electron acceptor (QA) cannot be reoxidized by the plastoquinone pool via the QB electron carrier, and consequently minimize the yield of the dark reactions. This results in inhibition of both electron transport and 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 for photosynthesis, comparable to that seen in diuron-treated fronds.
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However, all three components DFRC, DFw and DFc were significantly lowered by paraquat. As in diuron-treated plants, there was a considerable decrease of the quantum yield of electron transport beyond QA. Owing to the interaction of paraquat which deviates electron flow progressing towards NADP, results show that electron transport inhibition was not due to a target-specific action on the light reactions measured by ’P0 ð¼ TR0 =ABSÞ, nor by the dark reactions determined by c0 ð¼ ET0 =TR0 Þ. Paraquat treatment produced major changes in electron transport in active RC. Here, the diminution of the number of electron entering into the transport chain (c0) is not as important as in diuron-treated fronds, but paraquat induced a very important rise in the total absorption of PSII antenna chlorophyll by an active RC leading to a large increase of energy fluxes per RC for dissipation (DI0/RC). Our results indicate that paraquat and diuron cause an obvious decrease in the number of active PSII reaction centres and of the electron transport rate. This can lead to an inactivation of a fraction of the RC’s of PSII converted to non-QA reducing centres (Srivastava et al., 1999; Tsimilli-Michael et al., 1999) resulting in an increase of the average antenna size per remaining active reaction centre already seen in L. minor exposed to chromium (Ait Ali et al., 2006). Under these circumstances, regulation of light harvesting is necessary to balance the absorption and utilization of light energy, thereby minimizing the potential of photooxidative damage (Mu¨ller et al., 2001). Paraquat, also known as methyl-viologen, is a very effective electron acceptor that competes strongly with ferredoxin for electrons from the FeS clusters of PSI and, as a consequence, strongly suppresses cyclic electron transfer around PSI (Schansker et al., 2005). Paraquat is also known as a redox-active compound that is photoreduced by PSI and then reoxidized by transferring electrons to O2, forming reactive oxygen species (ROS) (Preston et al., 1991; Foyer et al., 1994; Bo¨ger and Sandmann, 1998). The high oxygen concentration of the chloroplast will ensure a rapid reoxidation of paraquat radicals which regenerates paraquat and produces superoxide O2 (Fuerst and Norman, 1991). Active oxygen species will diminish electron transport in the thylakoid membranes of the chloroplast resulting, therefore, in substantial changes in characteristics of Chl a fluorescence kinetics and parameters (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 driving force for photosynthesis. For the highest inhibitor concentration delta DF = 0.1 compared to 2.0 for diuron and paraquat. The main source of this slight inhibition was due to a change in the maximum quantum yield for primary photochemistry ’P0 ð¼ TR0 =ABS þ F V =F M Þ. This led, at the highest concentration, in a decrease of the number of active PSII reaction centres. At this concentration a small but significant increase of the nonphotochemical energy dissipation per RC (DI0/RC) is also seen. Little is known about the toxicity of flazasulfuron on non-target plants. It has been shown that this herbicide can reduce growth and photosynthetic activity of plants by lowering or decreasing leaf gas exchanges and photosynthetic pigment concentrations and by a marked disorganization of the leaf plastids (Frankart et al., 2003; Magne´ et al., 2006). Flazasulfuron is an effective inhibitor of acetolactate synthase, an enzyme involved in the biosynthesis of branched-chain amino acids (Bo¨ger and Sandmann, 1998). As an inhibitor of protein synthesis it may disrupt the assembly of chlorophyll–protein complexes included in light harvesting complex of PSII. The accumulation of free chlorophyll (not bound
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to protein) could allow an additional quenching of excess absorbed light energy, by increasing energy dissipation (DI0/RC) (Gilmore and Govindjee, 1999; Mu¨ller et al., 2001). Acknowledgment This work was supported, in part, by the Europol’Agro Foundation. References Ait Ali, N., Dewez, D., Didur, O., Popovic, R., 2006. Inhibition of photosystem II photochemistry by Cr is caused by the alteration of both D1 protein and oxygen evolving complex. Photosynth. Res. 89, 81–87. Appenroth, K.-J., Sto¨ckel, J., Srivastava, A., Strasser, R.J., 2001. Multiple effects of chromate on the photosynthetic apparatus of Spirodela polyrhiza as probed by OJIP chlorophyll a fluorescence measurements. Environ. Pollut. 115, 49–64. Blaise, C., Forget, G., Trottier, S., 2000. Toxicity screening of aqueous samples using a cost-effective 72-h exposure Selenastrum capricornutum assay. Environ. Toxicol. 15, 352–359. Blaise, C., Fe´rard, J.-F., 2005. Overview of contemporary toxicity testing. 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Contents lists available at ScienceDirect
Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot
Energy fluxes and driving forces for photosynthesis in Lemna minor exposed to herbicides Philippe Eullaffroy a,b,*, Ce´cile Frankart a, Aziz Aziz a, Michel Couderchet a, Christian Blaise b a b
Laboratoire Plantes Pesticides et De´veloppement Durable (PPDD), UPRES 2069 (URVVC), Universite´ de Reims Champagne-Ardenne, BP 1039, 51687 Reims Cedex 2, France Environnement Canada, Centre Saint-Laurent, 105 rue McGill, Montre´al, Qc, H2Y 2E7 Canada
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 April 2008 Received in revised form 17 September 2008 Accepted 19 September 2008 Available online 27 September 2008
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’P0 Þ 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. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Chlorophyll fluorescence Toxicity Photosynthesis Duckweed Water
1. Introduction Along with other environmental problems, agricultural and viticultural activities also contribute to degradation of aquatic ecosystems. Over the past decade pesticide application has become the predominant method to control pests and diseases in crop production. Phytosanitary products are continuously discharged into natural aquatic systems mainly through water runoff processes and may subsequently be hazardous to aquatic nontarget organism. Aquatic plant toxicity tests are frequently conducted to assess environmental risk and are often performed to obtain information on pollutant-induced toxic effects (Blaise et al., 2000; Eullaffroy and Vernet, 2003; Ku¨ster and Altenburger, 2007). Since the photosynthetic process is the key phase of plant
* Corresponding author at: Laboratoire Plantes Pesticides et De´veloppement 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: [email protected] (P. Eullaffroy). 0304-3770/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2008.09.002
metabolism and is known to be very sensitive to environmental changes, it has often been studied to evaluate the health status of plants. Among the tools used to study effects of environmental changes on the photosynthetic apparatus, chlorophyll a fluorescence is often proposed as a simple, rapid and sensitive method (Strasser et al., 2004; Ku¨ster and Altenburger, 2007). It gives precise data regarding photochemical efficiency and adverse effects in plants exposed to stressful conditions such as frost or xenobiotic exposure (Geoffroy et al., 2004; Strauss et al., 2006) and has been successfully used to monitor changing physiological states 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 fluorescence kinetics known as the Kautsky effect (Kautsky and Hirsch, 1931). These kinetics exhibit a sequence of phases named O, J, I and P reflecting the successive but overlapping filling-up of photosystem II (PSII) electron acceptor pools as QA, QB and PQ whose oxydoreduction states are closely controlled by PSII functions (Bukhov et al., 2004; Laza´r, 2006; Dewez et al., 2007). It is generally accepted that the rapid initial fluorescence rise from the initial O
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(F0), minimal Chl a fluorescence yield, to J, the first intermediate step at 2 ms, indicates reduction of the primary electron acceptor of PSII, QA and corresponds to the peak concentrations of QAQB and QAQB (QA and QB are the two first electron acceptors of PSII) (Zhu et al., 2005). Moreover, there is evidence that J transient is also influenced by the S-state of the watersplitting system (Hsu, 1993). The origin of the following J–I and I–P phases is still unclear. These transients seem to be related to the PSII heterogeneity (Lavergne and Briantais, 1996; Strasser and Stirbet, 1998; Stirbet et al., 1998) and recent work showed that I may correspond to an increase in concentrations of QAQB2 and P to the maximum concentrations of QAQB2 and PQH2 (plastoquinol pool) (Zhu et al., 2005; To´th et al., 2007). The I–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 the thermal phase of Chl fluorescence induction whereas O–J corresponds to the photochemical phase (Samson et al., 1999; Schreiber, 2002). This polyphasic fluorescence kinetics is affected by environmental conditions and can reflect the response of plants to various stresses and hence relate to their health status (Bussotti et al., 2007; Ku¨ster et al., 2007; Eullaffroy et al., 2007). Based on OJIP transients Strasser and his team have developed a test called the ‘‘JIP test’’ (Strasser and Strasser, 1995; Strasser et al., 2000) that quantifies the in vivo energy fluxes passing through the photosystems and evaluates plant photosynthetic performance. Besides assessing the function of PSII, this test also reflects the rate of the electron chain within the thylakoid membrane and the subsequent functioning of the Ferredoxin-NADP+ oxidoreductase and Calvin Cycle (Schansker et al., 2003). The process of all of the photosynthetic reactions should be reflected in the shape of the OJIP fluorescence kinetics. A number of parameters are derived from the fluorescence transients to quantify the flow of energy through the reaction centre (RC) of PSII (Strasser and Strasser, 1995; Force et al., 2003; Strasser et al., 2004). Many ecotoxicological studies have used the maximum quantum yield ratio (FV/FM) to evaluate the health status of a plant. However recent work showed that this ratio is not always the most suitable parameter to display contaminant toxicity or plant sensitivity (Force et al., 2003; Christen et al., 2007). In this study we used the OJIP test to appraise pesticide-induced effects via the change in the biophysical parameters quantifying the energy flow through PSII. Parameters related to energy absorption (trapping and conversion) are used as a measure of pesticide sensitivity and to localize the ‘‘effect sites’’ of the selected herbicide. Since most pesticides used in agriculture are herbicides (44% of the world pesticides market, Kiely et al., 2004), we studied the efficiency of the OJIP test to rapidly yield information on the specific effect of different herbicides toward the photosynthetic apparatus. Diuron, paraquat and flazasulfuron were chosen because of their differing modes of action, i.e. PSII, photosystem I (PSI) and protein biosynthesis inhibitors, respectively (Bo¨ger and Sandmann, 1998; Tomlin, 2000). Herein, we show the advantages of using the OJIP test to evaluate contaminant toxicity and the health status of a plant through the use of a number of parameters obtained from the same measurement rather than that 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 the Ardennes area of France. Before initiating experiments, the fronds
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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 containing sterile 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 continuous irradiance (100 mmol photons m2 s1) provided by cool white fluorescent lamps (Sylvania Gro Lux F36W, Germany). The plants were sub-cultured twice a week. 2.2. Pesticide application Pesticide treatments were performed on duckweeds with diuron, paraquat (Sigma, France) and flazasulfuron (used as Katana1Zeneca Sopra, France). All pesticide concentrations shown here refer to the active ingredient. All stock solutions were prepared just prior to initiating the experiments. Each experiment was replicated three times. Plants were exposed to phytosanitary products for 48 h. The experiments were conducted under static conditions. 2.3. Toxicity assessment Chl a fluorescence transients were recorded at room temperature from the upper face of duckweed fronds with a PEA fluorometer (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 lightemitting diodes with an excitation light intensity of 600 W m2 at 650 nm. The fast fluorescence kinetics was measured in a time span from 10 ms to 1 s. Each transient was analyzed by utilizing the following 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. The following equations were used to assess the photosynthetic performance of duckweeds (Strasser et al., 2000; Appenroth et al., 2001; Strauss et al., 2006): The rate of QA reduction in the first ms, M 0 ¼ 4ðF 300 F 0 Þ=ðF M F 0 Þ, The variable fluorescence at the J step, VJ = (F2 ms F0)/(FM F0), The fraction of inhibited centres, FIC = (VJcontaminated VJcontrol)/ (1 VJcontrol), The specific energy fluxes (per reaction centre, RC): for absorption, ABS=RCð¼ M 0 ð1=V J Þð1=’P0 ÞÞ, trapping, TR0/RC (= M0(1/VJ)), electron transport, ET0/RC (= M0(1/VJ)c0), and dissipation, DI0/RC (=(ABS/RC) (TR0/RC)), The maximum efficiency of PSII photochemistry in the darkadapted state, ’P0 ð¼ F V =F M ¼ ðF M F 0 Þ=F M ¼ TR0 =ABSÞ, The efficiency with which a trapped exciton can move an electron into the transport chain further than QA, c0 (=1 VJ = ET0/TR0), The quantum yield of electron transport, ’E0 ð¼ ’P0 c0 Þ, The performance index, PIABS ð¼ ðRC=ABSÞ½ð’P0 =ð1 ’P0 ÞÞ ½ðc0 =ð1 c0 ÞÞ, expressing the three functional steps of photosynthesis, i.e. absorption of light energy (ABS), trapping of excitation energy (TR) and conversion of excitation energy to electron transport (ET), The amount of active PSII RCs per absorption, RC=ABSð¼ ð’P0 V J Þ=M 0 Þ, The driving force for photosynthesis, DFABS = log PIABS = DFRC + DFw + DFc with: DFRC = log (RC/ABS), DF ’ ¼ logð’P0 =ð1 ’P0 ÞÞ and DF c ¼ logðc0 =ð1 c0 ÞÞ.
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The recorded fluorescence transients were plotted in a logarithmic time scale. At least 12 fronds were measured for each type of treatment. 2.4. Statistical analysis Data presented in this study are the means standard deviation. The statistical differences between controls and contaminated samples were analyzed by the Mann and Whitney test (Mann and Whitney, 1947), p values <0.05 were considered significant (asterisks [*] indicate significant values). Analyses were performed with Sigma Stat 2.03 (SPSS Inc., USA) software. 3. Results 3.1. Toxicity Fig. 1 illustrates a typical fluorescence rise from control and herbicide-treated duckweeds. To better reveal the changes in the shape of the transients, the curves were plotted as the relative variable fluorescence, Vt = (Ft F0)/(FM F0) on a logarithmic time scale (insert of Fig. 1). The kinetics of an untreated Lemna are composed of four clear transients. First, O (F0), was the origin of the Chl fluorescence rise. Then, kinetics develop to reach a maximum (P, i.e. FP = FM) attained after about 1-s irradiation. From the position of these two characteristic points on the time scale, we identified the two other transients known as the J-step (peak) and the I-step after 2 and 30 ms, respectively. As an example of how the polyphasic rise may be modified in plants treated with chemicals, we show in Fig. 1 that the OJIP rise is significantly affected in fronds exposed 48 h to 20 mg L1 of diuron. Diuron increased the magnitude of the J-step while the following I-step tended to disappear. The sigmoid shape of the O–J and J–I phases was lost based on the mode of action of diuron (i.e. electron transfer blockage from QA to QB which limits electron flow in PSII) and interpreted as an accumulation of QA (Strasser et al., 1995; Bo¨ger and Sandmann, 1998). The shape changes of the O–J–I–P curves were converted into quantitative changes of diverse parameters. Analysis of these changes 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 L1) 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 the selected herbicides. The effects of herbicide on the PSII reaction centre activity was quantified by the performance index, PIABS. This index is the overall expression of three functional steps (energy absorption, energy trapping and energy conversion into the electron transport) (Strauss et al., 2006). The average decrease of the PIABS after 48 h of exposure to 10 and 100 mg L1 of diuron, paraquat and flazasulfuron was 80, 30 and 0% and 99, 99, and 22%, respectively (data not shown). Fig. 2 displays the log value of the PIABS with its components. The partial (DFRC, DFw, DFc) and total driving force for photosynthesis (DFABS) can then be observed. The three herbicides tested induced a change in the three components of the total driving force. The diuron-treated fronds showed a DFABS decrease mainly due to a lower DFc related to the trapping of the excitation energy. DFRC (light energy absorption) and DFw (conversion of excitation energy to electron transport) were not affected by diuron (Fig. 2A). Paraquat induced an important decrease of DFABS comparable to the one observed in diuron-treated fronds. However, the response of DFABS differed in the major component that is affected by paraquat. In fact, DFw, was the main component of DFABS that was mostly affected by paraquat while DFRC, and DFc, were more impacted than in diuron-treated fronds (Fig. 2B). The total driving force for photosynthesis of duckweed exposed to flazasulfuron is only slightly diminished, owing mostly to a decrease in DFw when exposed to a concentration higher than 20 mg L1. The lowest flazasulfuron concentration tested induced a light increase of all the partial components of the total driving force for photosynthesis (Fig. 2C). From the fluorescence transient we can assess the yield of electron transport ð’E0 Þ which is the product of the yield of primary electron transport ð’P0 Þ, and the yield of electron transport per trapped exciton, c0 (=ET0/TR0). Diuron-, paraquatand flazasulfuron-treated fronds showed a 97, 84 and 8% inhibition of the electron transport, respectively, at the highest concentration tested (Fig. 3). However, in Lemna exposed 48 h to diuron, this inhibition was due more to a decrease in efficiency with which a trapped exciton can move an electron into the electron transport chain further than QA (c0), rather than to a decrease of the maximum quantum yield of primary photochemistry ð’P0 Þ (Fig. 3A). The decrease of ’E0 induced by paraquat was due to an inhibition in the same proportion of c0 (=ET0/TR0) and ’P0 (=TR0/ABS) (Fig. 3B). Flazasulfuron induces a slight but significant decrease in the yield of electron transport involving equally c0 (=ET0/TR0) and ’P0 ð¼ TR0 =ABSÞ (Fig. 3C). Fig. 4 is informative on effects of the studied herbicides linked to specific energy fluxes through PSII at the RC. Diuron, paraquat and 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 L1 (Fig. 4A). The same pattern was observed in fronds exposed to paraquat but with a much higher increase of ABS/RC and DI0/RC, i.e. 200 and 800%, respectively (Fig. 4B). Duckweed exposed to the highest concentration of flazasulfuron showed 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) were more pronounced in Lemna exposed to diuron than paraquat (Fig. 4A and B). There is no effect on the QA reoxidation rate in duckweed exposed to flazasulfuron (Fig. 4C). In L. minor exposed to diuron and paraquat, the decline in ET0/RC is mostly due to a
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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 c0 Þ) 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.
decrease in the transport of electrons further than QA (c0) and not from a decrease in trapping (TR0/RC). The fraction of inhibited centres (FIC) was severely increased, 94 and 57%, at 100 mg L1 of diuron and paraquat, respectively. FIC already reached about 60% in fronds exposed for 48 h at 10 mg L1 of diuron. Because ET0/RC is calculated as TR0/RC c0 the decline in ET0/RC in diuron- and paraquat-treated fronds mainly reflected a much lower probability that an electron residing on QA will enter the electron transport
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Fig. 3. Effect of different concentrations of (A) diuron, (B) paraquat, and (C) flazasulfuron in L. minor on the maximum efficiency of PSII photochemistry, ’P0 ð¼ 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, ’E0 ð¼ ’P0 c0 Þ (—).
chain since trapping showed no significant inhibition. Flazasulfuron showed a slight impact on FIC since only 5% of the centres were inhibited in fronds exposed to the highest concentration (Fig. 4C). FIC is strongly correlated to the decrease in the efficiency with which a trapped exciton by the RC will lead to a reduction of QA to QA (ET0/RC) (r2 = 0.99; data not shown).
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toxicity effects (Blaise and Fe´rard, 2005). OJIP chlorophyll a fluorescence curves were appraised to enhance our knowledge concerning toxicity and site of action of contaminants on the photosynthetic machinery through the study of energy fluxes and driving force for photosynthesis. 4.1. Effect of diuron on energy fluxes
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 M 0 ð1=V J Þð1=’P0 ÞÞ ( ); trapping, TR0 =RCð¼ M 0 ð1=V J ÞÞ ( ); electron transport, ET0 =RCð¼ M 0 ð1=V J Þc0 Þ ( );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.
4. Discussion Herbicides such as diuron, paraquat and flazasulfuron are commonly used in agriculture and their residues can be easily translocated into aquatic environments through surface runoff (Kloeppel et al., 1997; Garmouna et al., 1998). The study of the physiological effects of such pollutants on non-target organisms requires criteria to distinguish their acute and chronic (sub)lethal
In diuron-treated fronds, the decrease in DFc was the most pronounced effect indicating a strong blockage which inhibits electron flow from reduced QA towards PQ, leading to a loss of capability for intact electron transport in the plant. Diminished efficiency by which a trapped exciton can move an electron into the electron transport chain further than QA was the main factor that determined the magnitude of DFABS diminution in Lemna exposed to diuron. This halogenated-phenylurea herbicide also induced a decrease of the quantum yield for electron transport beyond QA ð’E0 ¼ ’P0 c0 Þ confirming a limitation of QA reoxidation. The more pronounced effect on the yield of electron transport per trapped exciton (c0) suggested that the dark reactions after QA, represented by c0 (=ET0/TR0), are more sensitive to diuron than light-dependent reactions represented by ’P0 . This agrees with the concept that diuron occupies the binding site of QB on the reaction centre complex. As a consequence, the light 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 in an active RC (DI0/RC). Dissipation refers to the loss of absorbed energy via heat and fluorescence emission and energy transfer to other systems (Strasser et al., 2000) and is represented by equation DI0/RC = (ABS/RC) (TR0/RC). Therefore, dissipation can be thought of as the absorption of photons in excess of what can be trapped by the RC. In fronds exposed to diuron, the excitation energy in the antenna of the active RC was in excess of that required for trapping and some energy was dissipated. Since no decrease in the light reactions leading to the energy flux as trapping per active RC was observed, we concluded that energy dissipation was rising via a relative increase of light harvesting complex per RC favouring the dissipation of the energy in excess through non-photochemical pathways (thermal dissipation processes). The increase of ABS/RC especially at the highest concentration tested illustrates a change in the antenna size of PSII RC due to a change in the number of LHC complexes 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, the increase in the expression ABS/RC is mainly due to a decrease in the number of RCs per absorption (RC/ABS), as shown in the partial driving force DFRC = log (RC/ABS), respectively by the partial performance index PIRC = RC/ABS. Again, diuron also lowered photosynthetic electron transport by decreasing the electron flux per RC between the two photosystems (ET0/TR0 = c0). It is known that diuron efficiently blocks PSII-catalyzed photosynthetic electron transport at the secondary electron acceptor, QB (Van Rensen, 1982). In this case, the first electron acceptor (QA) cannot be reoxidized by the plastoquinone pool via the QB electron carrier, and consequently minimize the yield of the dark reactions. This results in inhibition of both electron transport and 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 for photosynthesis, comparable to that seen in diuron-treated fronds.
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However, all three components DFRC, DFw and DFc were significantly lowered by paraquat. As in diuron-treated plants, there was a considerable decrease of the quantum yield of electron transport beyond QA. Owing to the interaction of paraquat which deviates electron flow progressing towards NADP, results show that electron transport inhibition was not due to a target-specific action on the light reactions measured by ’P0 ð¼ TR0 =ABSÞ, nor by the dark reactions determined by c0 ð¼ ET0 =TR0 Þ. Paraquat treatment produced major changes in electron transport in active RC. Here, the diminution of the number of electron entering into the transport chain (c0) is not as important as in diuron-treated fronds, but paraquat induced a very important rise in the total absorption of PSII antenna chlorophyll by an active RC leading to a large increase of energy fluxes per RC for dissipation (DI0/RC). Our results indicate that paraquat and diuron cause an obvious decrease in the number of active PSII reaction centres and of the electron transport rate. This can lead to an inactivation of a fraction of the RC’s of PSII converted to non-QA reducing centres (Srivastava et al., 1999; Tsimilli-Michael et al., 1999) resulting in an increase of the average antenna size per remaining active reaction centre already seen in L. minor exposed to chromium (Ait Ali et al., 2006). Under these circumstances, regulation of light harvesting is necessary to balance the absorption and utilization of light energy, thereby minimizing the potential of photooxidative damage (Mu¨ller et al., 2001). Paraquat, also known as methyl-viologen, is a very effective electron acceptor that competes strongly with ferredoxin for electrons from the FeS clusters of PSI and, as a consequence, strongly suppresses cyclic electron transfer around PSI (Schansker et al., 2005). Paraquat is also known as a redox-active compound that is photoreduced by PSI and then reoxidized by transferring electrons to O2, forming reactive oxygen species (ROS) (Preston et al., 1991; Foyer et al., 1994; Bo¨ger and Sandmann, 1998). The high oxygen concentration of the chloroplast will ensure a rapid reoxidation of paraquat radicals which regenerates paraquat and produces superoxide O2 (Fuerst and Norman, 1991). Active oxygen species will diminish electron transport in the thylakoid membranes of the chloroplast resulting, therefore, in substantial changes in characteristics of Chl a fluorescence kinetics and parameters (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 driving force for photosynthesis. For the highest inhibitor concentration delta DF = 0.1 compared to 2.0 for diuron and paraquat. The main source of this slight inhibition was due to a change in the maximum quantum yield for primary photochemistry ’P0 ð¼ TR0 =ABS þ F V =F M Þ. This led, at the highest concentration, in a decrease of the number of active PSII reaction centres. At this concentration a small but significant increase of the nonphotochemical energy dissipation per RC (DI0/RC) is also seen. Little is known about the toxicity of flazasulfuron on non-target plants. It has been shown that this herbicide can reduce growth and photosynthetic activity of plants by lowering or decreasing leaf gas exchanges and photosynthetic pigment concentrations and by a marked disorganization of the leaf plastids (Frankart et al., 2003; Magne´ et al., 2006). Flazasulfuron is an effective inhibitor of acetolactate synthase, an enzyme involved in the biosynthesis of branched-chain amino acids (Bo¨ger and Sandmann, 1998). As an inhibitor of protein synthesis it may disrupt the assembly of chlorophyll–protein complexes included in light harvesting complex of PSII. The accumulation of free chlorophyll (not bound
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