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Variation in oxidative stress and photochemical activity in Arabidopsis thaliana leaves subjected to cadmium and excess copper in the presence or absence of jasmonate and ascorbate
Chemosphere 66 (2007) 421–427 www.elsevier.com/locate/chemosphere
Variation in oxidative stress and photochemical activity in Arabidopsis thaliana leaves subjected to cadmium and excess copper in the presence or absence of jasmonate and ascorbate Waldemar Maksymiec *, Małgorzata Wo´jcik, Zbigniew Krupa Department of Plant Physiology, Maria Curie-Skłodowska University, 20-033 Lublin, Poland Received 26 January 2006; received in revised form 30 May 2006; accepted 12 June 2006 Available online 24 July 2006
Abstract We have presented changes in the photosynthetic apparatus activity of Arabidopsis thaliana plants occurring within 15–144 h of 100 lM Cu or Cd action with regard to jasmonate (JA) as well as expression of the oxidative stress and non-enzymic defense mechanisms. The inhibitory effect of both heavy metals related to developing dissipative processes and lipid peroxide formation was expressed in darkadapted state after the longest time as a decrease in potential quantum yield of PSII. In dark- and light-adapted state the heavy metals affected the enzymic phase of photosynthesis already from the 15th hour, which was related to the lipid peroxide formation. Photochemical quenching decrease was induced after 48th hour and did not show a close correlation with the JA pathway. Blockade of endogenously formed JA by propyl gallate decreased the effect of Cu and Cd on both the whole photosynthetic apparatus starting from the 48th hour and on the primary photochemistry of PSII after 144 h. In the case of Cu the effect was related to a lipid peroxidation decrease and to an increase in glutathione and phytochelatin (PC) levels, but in the case of Cd to lipid peroxidation, O 2 and especially to PCs increase. The obtained results indicated that JA after the longest time might enhance the sensitivity of A. thaliana to Cu and Cd stress. Asc enhanced toxic action of Cu and Cd after 15 h, but after a longer time it diminished the influence of Cd (but not Cu) on photosynthetic activity. 2006 Elsevier Ltd. All rights reserved. Keywords: Cd; Cu; Glutathione; Heavy metals; Jasmonic acid; Stress
1. Introduction Heavy metal contamination has disastrous effects on plant productivity and threatens human and animal health. One of the major effects of heavy metals on plants is enhanced production of reactive oxygen species (ROS), Abbreviations: Apx, ascorbate peroxidase; Asc, ascorbate; CAT, catalase; Chl, chlorophyll; Fv/Fm, potential quantum yield of PSII; GSH, glutathione; JA, jasmonic acid; NPQ, non-photochemical quenching of fluorescence; MDA, malondialdehyde; MJ, methyl jasmonate; PCs, phytochelatins; PG, propyl gallate; qP, photochemical quenching coefficient; Rfd, Chl fluorescence decrease ratio; ROS, reactive oxygen species; SOD, superoxide dismutase; %X, the additional energy emitted per energy absorbed in the antenna PSII due to the closure of a fraction of reaction centers. * Corresponding author. Fax: +48 81 537 59 01. E-mail address: [email protected] (W. Maksymiec). 0045-6535/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2006.06.025
which usually damage membranes, nucleic acids and chloroplast pigments (Somashekaraiah et al., 1992; Chen and Goldsbrough, 1994; Dra˛z_ kiewicz et al., 2004). In consequence, decreased photosynthetic activity and/or growth of tissues are followed by reduction of plant productivity (Ouzounidou, 1996; Maksymiec, 1997; Siedlecka et al., 2001). The insufficiently utilized assimilatory force by Calvin cycle slowed down due to heavy metal stress may, in consequence, enhance proton gradient formed in chloroplasts and increase nonphotochemical dissipation of light energy and/or decrease photochemical efficiency (Maksymiec and Baszyn´ski, 1996; Maksymiec, 1997). Redox changes in the photosystems related with proton gradient forming and glutathione (GSH) level can modify the ability of cells to destroy hydrogen peroxide. The Asc–GSH cycle is indispensable for efficient removal of ROS (Noctor et al., 1998). Recently, it was demonstrated that Cu affects this
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pathway both in roots (Gupta et al., 1999) and leaves (Cuypers et al., 2000; Dra˛z_ kiewicz et al., 2003). There is, however, no information about how the pools of Asc and GSH function in plants when one of them is inadequate. Their levels are various in plants exposed to excess of heavy metals (Cuypers et al., 2000; Dra˛z_ kiewicz et al., 2003; Wo´jcik and Tukiendorf, 2004). It was suggested that Asc pool may function effectively as a redox couple in the absence of a sufficient GSH pool (May et al., 1998). On the other hand Asc can enhance toxic effects of some heavy metals (data not shown). Xiang and Oliver (1998) showed that GSH might be involved in jasmonate (JA) signal transduction during Cu of Cd stress. JAs are formed from lipid peroxides as a consequence of enhanced lipoxygenase activity, but it is quite possible that the formation may be a prolonged consequence of heavy metal-induction of oxidative stress. However, only few and not clear data are known about molecular events effecting the course of JAs. Some data indicate an increase in lipid peroxides content in leaves following almost immediately Cu application (Cuypers et al., 2000; Tripathi et al., 2006). Because Cu was significantly accumulated only 2 d after starting the supply thereof, it can be concluded that lipid peroxidation points to the appearance of a signaling response. At prolonged exposure to Cd, lipid peroxides were also formed (Metwally et al., 2003). In such a case, the authors indicated that Cd toxicity may be alleviated by salicylic acid, a known signaling factor that blocks the JA pathways. Recent investigations have indicated that JA signaling may be involved in the expression of heavy metal induced stress. They concern the inductive action of Cu or Cd ions on JA accumulation and some JA-responsive events such as vegetative storage proteins 2 and mitogen-activated protein kinase (Mira et al., 2002; Agrawal et al., 2003; Maksymiec et al., 2005) and also the interactive action between heavy metals and methyl jasmonate (MJ) (Maksymiec and Krupa, 2002b). Some data show the JA influence on increased H2O2 accumulation (Maksymiec and Krupa, 2002a; Maksymiec and Krupa, 2006). The above mentioned data suggest that heavy metals may act either directly on oxidative processes or through induction of the signaling pathways. In the present work, the involvement of JA and Asc as well as GSH in the toxicity mechanism of Cu and Cd was investigated. The photosynthetic apparatus was studied because there is a close correlation between the photosynthetic activity, JA and Asc–GSH levels related to ROS accumulation efficiency, but so far this relationship has not been investigated in detail. 2. Material and methods 2.1. Plant material and growth conditions Seeds of Arabidopsis thaliana L., cv. Heynh wild type Columbia were sown in doubly autoclaved soil and left for about 10 d for germination. Then the seedlings were
transferred separately into soil filled pots (one plant per one plot). Six weeks after sowing, the plants were transferred into full strength modified Hoagland nutrient solution (0.25 mM Fe3+ was added) and cultivated at 23/ 18 C day/night temperature and photosynthetic photon flux density of 140 lmol m2 s1 under 11/13 h day/night regime. After 6 d of acclimation, the plants were treated through their cut off stems with 100 lM CuSO4 or CdSO4 and MJ (105 M) or its synthesis inhibitor – propyl gallate (PG) (100 lM) during 15 h. The PG concentration used did not significantly affect growth, pigment concentration, photosynthetic activity (Maksymiec and Krupa, 2002a) and O 2 level of control plants. Asc (2.5 mM) was added in 2.5 mM Hepes-NaOH (pH 5.9). In the experiments with prolonged time of treatment 100 lM CuSO4 or CdSO4 and 107, 106 and 105 M MJ were added to the nutrient solution on 48 or 144 h. PG and Asc were added in the abovementioned amounts. MJ was finally dissolved in 0.5 ml ethanol (also introduced to control plants). The leaves were harvested, frozen in liquid nitrogen and kept at 80 C for analyses of ROS. For thiol peptide and fluorescence analysis fresh leaves were used. 2.2. Measurement of chlorophyll fluorescence Chl fluorescence was measured at 24 C as described previously (Maksymiec and Baszyn´ski, 1999) using a PAM 101 modulated fluorometer (Walz) equipped with PDA-100 and Schott KL 1500 lamps. Minimal fluorescence yield, F 00 was obtained upon illumination with a weak beam emitting from a far-red diode. A saturating light (8800 lmol m2 s1)and actinic light (240 lmol m2 s1) were used to determine maximal fluorescence intensity ðF m ; F m0 Þ and the steady state fluorescence (Fs). The photochemical quenching coefficient (qP) was calculated according to Van Kooten and Snel (1990), and %X ¼ F v0 =F m0 ð1 qp Þ (the additional energy emitted per energy absorbed in the antenna PSII due to the closure of a fraction of reaction centers) after Demmig-Adams et al. (1996). The Chl fluorescence decrease ratio (Rfd) was determined according to Lichtenthaler and Rinderle (1988) as (FmFs)/Fs ratio, and the total non-photochemical quenching of fluorescence (NPQ) was calculated as Stern Volmer quenching, NPQ ¼ ðF m =F m0 Þ 1. 2.3. ROS assays All samples were prepared for soluble protein. ROS were analysed after rapid homogenization of the frozen leaf material in a solution containing 50 mM Na2HPO4/ KH2PO4 (pH 7.0), 0.8% (v/v) Triton X-100 and 1% (w/v) polyvinyl pyrrolidone. The homogenate was centrifuged at 16 000g for 17 min, and the enzyme activities and ROS content in the supernatant were determined immediately. All operations were performed at 4 C. Samples for O 2 radicals were prepared according to Green and Hill (1984). The reduction of nitroblue tetrazolium to farmazan
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by superoxide anion was monitored as absorbance increase at 490 nm, using 100 mM1 cm1 absorbance coefficient. Measurements were carried out with 1 ml of the reaction mixture containing 40 ll SOD (1 mg ml1) and without it. The level of lipid peroxidation products in leaves was expressed as malondialdehyde reactive metabolites (MDA). The assay was carried out at 532 nm as described by Tewari et al. (2002). The measurements were corrected for unspecific turbidity by subtracting the absorbance at 600 nm. 2.4. Quantitation of glutathione and phytochelatins Leaves were weighed and ground with a double volume of 0.1 M HCl. Homogenates were centrifuged at 4 C and 18 000g for 5 min and the supernatants were separated in a linear gradient (0–20%) of acetonitrile in 0.05% trifluoroacetic acid (TFA) for 40 min at a flow rate of 1 ml min1 in a Beckman chromatograph with Supelco column filled with Ultrasphere C-18. The column was washed with 70% acetonitrile and equilibrated with 0.05% TFA. The obtained peptide fractions were mixed with 200 lM Ellman’s reagent (5,5 0 -dithiobis-2-nitrobenzoic acid, DTNB) in 0.05 M potassium-phosphate buffer pH 7.6. Absorbance of the products of DTNB reaction with –SH groups was measured at 405 nm using Beckman detector. The retention times and peak areas were determined by a computerized program (Gould Nouveau, Beckman). 2.5. Statistical analysis The results were based on at least five replicates from two independent experiments. Sigma Stat 3.0 software was applied for calculations. Sample variability is given as the standard error of the mean values.
Fig. 1. The Chl fluorescence parameters of PSII (relative units) in darkadapted leaves: the maximal quantum yield of PSII photochemistry (Fv/ Fm), or in the dark- and light-adapted leaves: the Chl fluorescence decrease ratio (Rfd) and the non-photochemical quenching of fluorescence (NPQ) in A. thaliana plants after supply of 100 lM Cu or Cd alone, or with the addition of 105 M methyl jasmonate (MJ), 2.5 mM ascorbate (Asc) and 100 lM propyl gallate (PG). Data are means ±SE.
3. Results 3.1. Chl fluorescence parameters The Fv/Fm ratio was similar to control during the first 48 h of exposure to both Cu and Cd ions (Fig. 1). At prolonged time it decreased by about 17%. The decrease was diminished after additional PG supply and enhanced in Cu + Asc treatment (by 26%). The Rfd ratio was lower in Cu and Cd-treated plants and decreased with the time of treatment to about 50% of control. PG diminished this effect from the 48th hour, but Asc enhanced it in Cu-treated plants, and in Cd-treated plants only after 15 h. NPQ increased after Cu and Cd treatment and it was enhanced or diminished after 15 h in Cu and Cd-treated plants, respectively, by all additionally used substances (except Cd + MJ treatment). After a longer Cu-treatment, MJ increased NPQ value, but PG did not modify it. In leaves of 144 h Cd-treated plants, the additionally added PG decreased NPQ value below the control level, which was not changed by MJ, but Asc increased it to 182% of control.
qP decreased from the 48th hour after Cu and Cd supply (Fig. 2). The influence of the additional substances was not distinct in this case, but in Cd-treated leaves MJ and Asc additionally diminished qP value after 15 and 48 h, respectively. After 144 h, the inhibitory effect of the heavy metal was diminished by Asc. The %X value increased after Cu and Cd supply. At the beginning this effect was increased by Asc (in the case of Cu) and by MJ (in Cd-treated plants), but after the longer time, this effect was inverted in the former case or not observed in the latter. PG distinctly influenced the %X value only after 48 h in Cd treated plants and was found as an inhibitory effect. 3.2. The effect of Cu, Cd and MJ on ROS accumulation The elevated O content was found after the early 2 phases of Cu treatment, where it reached 147% and 232% of control after 15 and 48 h, respectively (Fig. 3). After a longer time it was below the control level. PG usually increased O 2 accumulation except 48 h treatment. At Cd
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Fig. 4. Content of malondialdehyde (MDA) products in A. thaliana leaves treated for 48 h with 107, 106 and 105 M methyl jasmonate (MJ) or 100 lM Cu or Cd alone, or with the addition of 100 lM propyl gallate (PG). Data are means ±SE. Fig. 2. The Chl fluorescence parameters of PSII (relative units) analyzed in light-adapted leaves: the photochemical quenching (qP) of fluorescence (estimated degree of open reaction centers), and the additional energy emitted per energy absorbed in the antenna PSII due to the closure of a fraction of the reaction centers in the PSII antennae (%X) in A. thaliana leaves after supply of 100 lM Cu or Cd alone, or with the addition of 105 M methyl jasmonate (MJ), 2.5 mM ascorbate (Asc) and 100 lM propyl gallate (PG). Data are means ±SE.
excess its level decreased below control during the investigated time period and was enhanced by adding PG. The content of O 2 increased after plant treatment with MJ, especially after 144 h. The effect of Cu and Cd on lipid peroxides was different after 48 and 144 h exposition. After Cu supply their level increased 3.6-fold after 48 h, and about 9.5-fold after 144 h (Figs. 4 and 5). The formation of lipid peroxides decreased only after 144 h treatment with PG. A slightly inductive effect of Cd on MDA formation was
Fig. 5. Content of malondialdehyde (MDA) products in A. thaliana leaves treated for 144 h with 107, 106 and 105 M methyl jasmonate (MJ) or 100 lM Cu or Cd alone, or with the addition of 100 lM propyl gallate (PG). Data are means ±SE.
found, especially after 144 h. After adding PG the decrease in JA synthesis additionally enhanced this process despite a stimulatory effect of exogenous MJ on the level of lipid peroxides in control plants (Figs. 4 and 5). However, in this case accumulation of MDA was not correlated with MJ concentration. Fig. 3. Relative O 2 content (% of control) in A. thaliana leaves after treatment with 107, 106 and 105 M methyl jasmonate (MJ) or 100 lM Cu or Cd alone, or with the addition of 100 lM propyl gallate (PG). Data are means ±SE. Control values of O 2 : 3.94 ± 0.21, 1.86 ± 0.12 and 2.40 ± 0.04 nmol NBT g1 FW after 15, 48 and 144 h, respectively.
3.3. Effect on glutathione and phytochelatins accumulation PCs were not accumulated after 15 h of the treatments (Fig. 6). The concentration of GSH was increased by Cu
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It was postulated as a direct action of heavy metals on the photosynthetic electron transport. Recently an indirect toxic activity was also proposed, e.g., through Ca pool modification or down-regulation of the electron transport due to inhibited activity of the Calvin cycle (Maksymiec, 1997; Maksymiec and Baszyn´ski, 1999; Vinit-Dunand et al., 2002). According to our investigations Cu and Cd act on photosynthetic apparatus as follows:
Fig. 6. Glutathione (GSH) and phytochelatins (PCs) content after 15 h treatment with 100 lM Cu or Cd alone, or with the addition of 100 lM propyl gallate (PG), 2.5 mM ascorbate (Asc) or 105 M methyl jasmonate (MJ). Values are means of two separate time series. Standard errors were less than 15% of the value.
Fig. 7. Glutathione (GSH) and phytochelatins (PCs) content after 144 h treatment with 100 lM Cu or Cd alone, or with the addition of 100 lM propyl gallate (PG), 2.5 mM ascorbate (Asc) or 105 M methyl jasmonate (MJ). Values are means of two separate time series. Standard errors were less than 15% of the value.
and Cd supply and was additionally enhanced in Cu + PG and Cu + Asc treated plants. Asc and, to a minor degree, MJ decreased GSH accumulation in Cd treated plants. Increasing time of heavy metal treatment induced the accumulation of PCs and GSH, especially in the case of Cd (Fig. 7). PG and also Asc enhanced the effect of Cd and Cu on PCs synthesis, but MJ did only in the latter case. PCs were not detected in control plants. 4. Discussion The inhibitory effect of Cd and Cu ions excess on the photosynthetic apparatus has been known for a long time.
• 0–15 h: The yield of primary photochemistry (Fv/Fm) and reduced QA pool (qP) was constant, the whole photosynthetic activity (Rfd) decreased and there was an increase in excess energy loss (%X) and non-photochemical quenching (NPQ), due to zeaxanthin cycle operation. Summarizing, it seems that during the first phase Cu and Cd affect the dark phase of photosynthesis followed by an increase in dissipative processes, allowing not disturbed electron transport through PSII. • 15–48 h: Additionally to the above presented changes, there was a slight decrease in Fv/Fm and a big decrease in qP, which indicated that Cu and Cd diminished reoxidation of Q A and started to inactivate the RC of PSII. As the effect was found especially in the light-adapted state, it may be related to developed photoinhibitory processes. • 48–144 h: At this time the protective role of non-photochemical energy dissipation gradually diminished, inhibition of dark and light phase of photosynthesis increased and the pool of oxidized quinones decreased i.e. the pool of closed reaction centers increased. Fv/Fm decreased as a result of preferential Fm decrease (data not shown). It may be attributed to inactivation of the donor side of PSII (To´th et al., 2005) and we presume that after 144 h Cu and Cd ions induced some structural changes in PSII centers. An increase in NPQ might be due to a dissociation of light-harvesting complex from PSII core (Ouzounidou and Ilias, 2005). At a higher NPQ level the %X increase, observed after 48 h, was the result of a favour decrease in the pool of the open reaction centers (qP decrease). After blockade of JA synthesis both heavy metals caused a decrease in the Fv/Fm and the whole photosynthetic processes (Rfd decrease). The balance between electron utilization and deleting by quinones in PSII was still at the same level if qP was not changed. This result indicated that JA may enhance Cu and Cd toxicity, but it probably does not disturb electron flow around QA. If Rfd value is a good indicator of the net CO2 fixation (Lichtenthaler and Babani, 2004), Cu or Cd-induced Rfd decrease, attenuated by PG, may be the consequence of MJ induced stomata closure observed in A. thaliana leaves after MJ supply (Suhita et al., 2004). However, exogenous MJ did not additionally diminish Rfd, indicating that only a definite level of JAs in the cells can determine the sensitivity of the photosynthetic apparatus to heavy metals. It is
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worth noting that either blocking of JA synthesis or its addition may stimulate O accumulation in stressed 2 plants. This is difficult to explain, but it might indicate that blockade of octadecanoid pathways at the site of PG action as well as accumulation of its final metabolite express a similar response to O 2 formation. Parallel to the Rfd decrease, induced by Cu, lipid peroxidation products were accumulated, indicating the occurrence of oxidative damage of membranes. The blockade of JA synthesis diminished this process after 144 h and, in consequence, the activity of the photosynthetic apparatus was enhanced. Because the improvement of the Calvin cycle (after adding PG) started from the 48th hour we suppose that lipid peroxidation was only partially the cause of photosynthesis inhibition. Cd ions induced accumulation of MDA products more slightly than Cu, and O 2 level decreased below the control (from the 15th hour). This result was similar to that obtained by Sko´rzyn´ska-Polit et al. (2003/4) for A. thaliana plants, however, opposite to that observed in rice plants (Shah et al., 2001), which indicates that there exists some discrepancy depending on the plant species. The improvement of the photosynthetic activity by adding PG to Cd-stressed plants might be due to induction of PCs accumulation as was also detected in other plants (Tukendorf and Rauser, 1990; Cobbett and Goldsbrough, 2002). Cd and excess Cu increased GSH accumulation in A. thaliana leaves already after 15 h. This had presumably an inductive character because it did not depend on the concentration of the metals (data not shown). Although JA is an inductor of GSH genes, when applied exogenously it did not increase GSH both in control and heavy metaltreated plants. This phenomenon indicates that besides the transcription there exist other mechanisms regulating GSH level, which confirms earlier suppositions of Xiang and Oliver (1998). PC concentration was especially high after adding MJ, Asc or PG to Cd-treated plants. In Cutreated plants MJ-induced GSH decrease may be due to its limited synthesis. Because JA is accumulated during heavy metal stress (Maksymiec et al., 2005) it is likely to enhance the inhibitory effects of metals. In our study this was found as decreased GSH accumulation, especially after 144 h of Cu action. Cu and Cd preferentially inhibit the Calvin cycle. In this case Asc is used in the Mehler cycle. Asc excess can restock the pool of Asc used and, in consequence, improve photosynthetic electron transport. However, when in excess, Asc did not increase the oxidized pool of QA in Cu-treated plants, but after a short term action it increased %X as well as NPQ. Therefore, we hypothesize that exogenous Asc after a short time specifically induces the dissipative processes through reduction of Cu2+ to Cu1+ and, in consequence, it induces the enhancement of radical processes in the chloroplast resulting in a decrease in potential quantum yield of PSII. This effect was not observed in the case of Cd (which does not change the oxidative degree), supporting the above assumption. Because
we did not observe any enhancement of O 2 accumulation by Asc added to Cu-treated plants after 15 h (data not shown), it is possible that H2O2 was preferentially accumulated. A sharp increase in NPQ value induced by Asc may also be the consequence of Asc stimulatory effect on the violaxanthin cycle if it participates in regeneration of zeaxanthin (Bratt et al., 1995), and Asc content is increased by Cu in A. thaliana leaves (Dra˛z_ kiewicz et al., 2003). Our results showed that the enzymic phase of photosynthesis was preferentially slowed down by Cu and Cd ions beginning already from the 15th hour. These changes in the photosynthetic apparatus were related to JA action and MDA, GSH and PC accumulation. After a longer time of heavy metal action JA might increase their toxic action and probably it was partially the cause of the changes induced by the metals. Asc can enhance toxic action of Cu and Cd after a short time, however, after a longer time it can diminish the influence of Cd on the photosynthetic activity. Although MJ showed a stimulatory effect on GSH gene expression, exogenously applied MJ diminished GSH accumulation after Cd or Cu supply. This may indicate that although JAs are signaling factors increasing plants resistance to any stress factors, they may also enhance the toxic effect of Cd, and especially Cu. This fact is an intriguing problem concerning gene expression during stress action and the development of the signaling activity on the level of enzymatic reaction in plant cells. Acknowledgement This study was supported by the State Committee for Scientific Research (KBN), grant No. 3PO4C05022. References Agrawal, G.K., Tamogami, S., Iwahashi, H., Agrawal, V.P., Rakwal, R., 2003. Transient regulation of jasmonic acid-inducible rice MAP kinase gene (OsBWMK1) by diverse biotic and abiotic stresses. Plant Physiol. Biochem. 41, 355–361. ˚ kerlund, H.-E., 1995. Bratt, C.E., Arvidsson, P.-O., Carlsso, M., A Regulation of violaxanthin deepoxidase activity by pH and ascorbate concentration. Photosynth. Res. 45, 169–175. Chen, G.H., Goldsbrough, P.B., 1994. Increased activity of c-glutamylcysteine synthetase in tomato cells selected for cadmium tolerance. Plant Physiol. 106, 233–239. Cobbett, C., Goldsbrough, P., 2002. Phytochelatins and metallothioneins: role in heavy metal detoxification and homeostasis. Annu. Rev. Plant Biol. 53, 159–182. Cuypers, A., Vangronsveld, J., Clijsters, H., 2000. Biphasic effect of copper on the ascorbate–glutathione pathway in primary leaves of Phaseolus vulgaris seedlings during the early stages of metal assimilation. Physiol. Plant. 110, 512–517. Demmig-Adams, B., Adams, W.W., Baker, D.H., Logan, B.A., Bowling, D.R., Verhoeven, A.S., 1996. Using chlorophyll fluorescence to asses the fraction of absorbed light allocated to the thermal dissipation of excess excitation. Physiol. Plant. 98, 253–264. Dra˛z_ kiewicz, M., Sko´rzyn´ska-Polit, E., Krupa, Z., 2003. Response of the ascorbate–glutathione cycle to excess copper in Arabidopsis thaliana (L.). Plant Sci. 164, 195–202.
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Variation in oxidative stress and photochemical activity in Arabidopsis thaliana leaves subjected to cadmium and excess copper in the presence or absence of jasmonate and ascorbate Waldemar Maksymiec *, Małgorzata Wo´jcik, Zbigniew Krupa Department of Plant Physiology, Maria Curie-Skłodowska University, 20-033 Lublin, Poland Received 26 January 2006; received in revised form 30 May 2006; accepted 12 June 2006 Available online 24 July 2006
Abstract We have presented changes in the photosynthetic apparatus activity of Arabidopsis thaliana plants occurring within 15–144 h of 100 lM Cu or Cd action with regard to jasmonate (JA) as well as expression of the oxidative stress and non-enzymic defense mechanisms. The inhibitory effect of both heavy metals related to developing dissipative processes and lipid peroxide formation was expressed in darkadapted state after the longest time as a decrease in potential quantum yield of PSII. In dark- and light-adapted state the heavy metals affected the enzymic phase of photosynthesis already from the 15th hour, which was related to the lipid peroxide formation. Photochemical quenching decrease was induced after 48th hour and did not show a close correlation with the JA pathway. Blockade of endogenously formed JA by propyl gallate decreased the effect of Cu and Cd on both the whole photosynthetic apparatus starting from the 48th hour and on the primary photochemistry of PSII after 144 h. In the case of Cu the effect was related to a lipid peroxidation decrease and to an increase in glutathione and phytochelatin (PC) levels, but in the case of Cd to lipid peroxidation, O 2 and especially to PCs increase. The obtained results indicated that JA after the longest time might enhance the sensitivity of A. thaliana to Cu and Cd stress. Asc enhanced toxic action of Cu and Cd after 15 h, but after a longer time it diminished the influence of Cd (but not Cu) on photosynthetic activity. 2006 Elsevier Ltd. All rights reserved. Keywords: Cd; Cu; Glutathione; Heavy metals; Jasmonic acid; Stress
1. Introduction Heavy metal contamination has disastrous effects on plant productivity and threatens human and animal health. One of the major effects of heavy metals on plants is enhanced production of reactive oxygen species (ROS), Abbreviations: Apx, ascorbate peroxidase; Asc, ascorbate; CAT, catalase; Chl, chlorophyll; Fv/Fm, potential quantum yield of PSII; GSH, glutathione; JA, jasmonic acid; NPQ, non-photochemical quenching of fluorescence; MDA, malondialdehyde; MJ, methyl jasmonate; PCs, phytochelatins; PG, propyl gallate; qP, photochemical quenching coefficient; Rfd, Chl fluorescence decrease ratio; ROS, reactive oxygen species; SOD, superoxide dismutase; %X, the additional energy emitted per energy absorbed in the antenna PSII due to the closure of a fraction of reaction centers. * Corresponding author. Fax: +48 81 537 59 01. E-mail address: [email protected] (W. Maksymiec). 0045-6535/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2006.06.025
which usually damage membranes, nucleic acids and chloroplast pigments (Somashekaraiah et al., 1992; Chen and Goldsbrough, 1994; Dra˛z_ kiewicz et al., 2004). In consequence, decreased photosynthetic activity and/or growth of tissues are followed by reduction of plant productivity (Ouzounidou, 1996; Maksymiec, 1997; Siedlecka et al., 2001). The insufficiently utilized assimilatory force by Calvin cycle slowed down due to heavy metal stress may, in consequence, enhance proton gradient formed in chloroplasts and increase nonphotochemical dissipation of light energy and/or decrease photochemical efficiency (Maksymiec and Baszyn´ski, 1996; Maksymiec, 1997). Redox changes in the photosystems related with proton gradient forming and glutathione (GSH) level can modify the ability of cells to destroy hydrogen peroxide. The Asc–GSH cycle is indispensable for efficient removal of ROS (Noctor et al., 1998). Recently, it was demonstrated that Cu affects this
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pathway both in roots (Gupta et al., 1999) and leaves (Cuypers et al., 2000; Dra˛z_ kiewicz et al., 2003). There is, however, no information about how the pools of Asc and GSH function in plants when one of them is inadequate. Their levels are various in plants exposed to excess of heavy metals (Cuypers et al., 2000; Dra˛z_ kiewicz et al., 2003; Wo´jcik and Tukiendorf, 2004). It was suggested that Asc pool may function effectively as a redox couple in the absence of a sufficient GSH pool (May et al., 1998). On the other hand Asc can enhance toxic effects of some heavy metals (data not shown). Xiang and Oliver (1998) showed that GSH might be involved in jasmonate (JA) signal transduction during Cu of Cd stress. JAs are formed from lipid peroxides as a consequence of enhanced lipoxygenase activity, but it is quite possible that the formation may be a prolonged consequence of heavy metal-induction of oxidative stress. However, only few and not clear data are known about molecular events effecting the course of JAs. Some data indicate an increase in lipid peroxides content in leaves following almost immediately Cu application (Cuypers et al., 2000; Tripathi et al., 2006). Because Cu was significantly accumulated only 2 d after starting the supply thereof, it can be concluded that lipid peroxidation points to the appearance of a signaling response. At prolonged exposure to Cd, lipid peroxides were also formed (Metwally et al., 2003). In such a case, the authors indicated that Cd toxicity may be alleviated by salicylic acid, a known signaling factor that blocks the JA pathways. Recent investigations have indicated that JA signaling may be involved in the expression of heavy metal induced stress. They concern the inductive action of Cu or Cd ions on JA accumulation and some JA-responsive events such as vegetative storage proteins 2 and mitogen-activated protein kinase (Mira et al., 2002; Agrawal et al., 2003; Maksymiec et al., 2005) and also the interactive action between heavy metals and methyl jasmonate (MJ) (Maksymiec and Krupa, 2002b). Some data show the JA influence on increased H2O2 accumulation (Maksymiec and Krupa, 2002a; Maksymiec and Krupa, 2006). The above mentioned data suggest that heavy metals may act either directly on oxidative processes or through induction of the signaling pathways. In the present work, the involvement of JA and Asc as well as GSH in the toxicity mechanism of Cu and Cd was investigated. The photosynthetic apparatus was studied because there is a close correlation between the photosynthetic activity, JA and Asc–GSH levels related to ROS accumulation efficiency, but so far this relationship has not been investigated in detail. 2. Material and methods 2.1. Plant material and growth conditions Seeds of Arabidopsis thaliana L., cv. Heynh wild type Columbia were sown in doubly autoclaved soil and left for about 10 d for germination. Then the seedlings were
transferred separately into soil filled pots (one plant per one plot). Six weeks after sowing, the plants were transferred into full strength modified Hoagland nutrient solution (0.25 mM Fe3+ was added) and cultivated at 23/ 18 C day/night temperature and photosynthetic photon flux density of 140 lmol m2 s1 under 11/13 h day/night regime. After 6 d of acclimation, the plants were treated through their cut off stems with 100 lM CuSO4 or CdSO4 and MJ (105 M) or its synthesis inhibitor – propyl gallate (PG) (100 lM) during 15 h. The PG concentration used did not significantly affect growth, pigment concentration, photosynthetic activity (Maksymiec and Krupa, 2002a) and O 2 level of control plants. Asc (2.5 mM) was added in 2.5 mM Hepes-NaOH (pH 5.9). In the experiments with prolonged time of treatment 100 lM CuSO4 or CdSO4 and 107, 106 and 105 M MJ were added to the nutrient solution on 48 or 144 h. PG and Asc were added in the abovementioned amounts. MJ was finally dissolved in 0.5 ml ethanol (also introduced to control plants). The leaves were harvested, frozen in liquid nitrogen and kept at 80 C for analyses of ROS. For thiol peptide and fluorescence analysis fresh leaves were used. 2.2. Measurement of chlorophyll fluorescence Chl fluorescence was measured at 24 C as described previously (Maksymiec and Baszyn´ski, 1999) using a PAM 101 modulated fluorometer (Walz) equipped with PDA-100 and Schott KL 1500 lamps. Minimal fluorescence yield, F 00 was obtained upon illumination with a weak beam emitting from a far-red diode. A saturating light (8800 lmol m2 s1)and actinic light (240 lmol m2 s1) were used to determine maximal fluorescence intensity ðF m ; F m0 Þ and the steady state fluorescence (Fs). The photochemical quenching coefficient (qP) was calculated according to Van Kooten and Snel (1990), and %X ¼ F v0 =F m0 ð1 qp Þ (the additional energy emitted per energy absorbed in the antenna PSII due to the closure of a fraction of reaction centers) after Demmig-Adams et al. (1996). The Chl fluorescence decrease ratio (Rfd) was determined according to Lichtenthaler and Rinderle (1988) as (FmFs)/Fs ratio, and the total non-photochemical quenching of fluorescence (NPQ) was calculated as Stern Volmer quenching, NPQ ¼ ðF m =F m0 Þ 1. 2.3. ROS assays All samples were prepared for soluble protein. ROS were analysed after rapid homogenization of the frozen leaf material in a solution containing 50 mM Na2HPO4/ KH2PO4 (pH 7.0), 0.8% (v/v) Triton X-100 and 1% (w/v) polyvinyl pyrrolidone. The homogenate was centrifuged at 16 000g for 17 min, and the enzyme activities and ROS content in the supernatant were determined immediately. All operations were performed at 4 C. Samples for O 2 radicals were prepared according to Green and Hill (1984). The reduction of nitroblue tetrazolium to farmazan
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by superoxide anion was monitored as absorbance increase at 490 nm, using 100 mM1 cm1 absorbance coefficient. Measurements were carried out with 1 ml of the reaction mixture containing 40 ll SOD (1 mg ml1) and without it. The level of lipid peroxidation products in leaves was expressed as malondialdehyde reactive metabolites (MDA). The assay was carried out at 532 nm as described by Tewari et al. (2002). The measurements were corrected for unspecific turbidity by subtracting the absorbance at 600 nm. 2.4. Quantitation of glutathione and phytochelatins Leaves were weighed and ground with a double volume of 0.1 M HCl. Homogenates were centrifuged at 4 C and 18 000g for 5 min and the supernatants were separated in a linear gradient (0–20%) of acetonitrile in 0.05% trifluoroacetic acid (TFA) for 40 min at a flow rate of 1 ml min1 in a Beckman chromatograph with Supelco column filled with Ultrasphere C-18. The column was washed with 70% acetonitrile and equilibrated with 0.05% TFA. The obtained peptide fractions were mixed with 200 lM Ellman’s reagent (5,5 0 -dithiobis-2-nitrobenzoic acid, DTNB) in 0.05 M potassium-phosphate buffer pH 7.6. Absorbance of the products of DTNB reaction with –SH groups was measured at 405 nm using Beckman detector. The retention times and peak areas were determined by a computerized program (Gould Nouveau, Beckman). 2.5. Statistical analysis The results were based on at least five replicates from two independent experiments. Sigma Stat 3.0 software was applied for calculations. Sample variability is given as the standard error of the mean values.
Fig. 1. The Chl fluorescence parameters of PSII (relative units) in darkadapted leaves: the maximal quantum yield of PSII photochemistry (Fv/ Fm), or in the dark- and light-adapted leaves: the Chl fluorescence decrease ratio (Rfd) and the non-photochemical quenching of fluorescence (NPQ) in A. thaliana plants after supply of 100 lM Cu or Cd alone, or with the addition of 105 M methyl jasmonate (MJ), 2.5 mM ascorbate (Asc) and 100 lM propyl gallate (PG). Data are means ±SE.
3. Results 3.1. Chl fluorescence parameters The Fv/Fm ratio was similar to control during the first 48 h of exposure to both Cu and Cd ions (Fig. 1). At prolonged time it decreased by about 17%. The decrease was diminished after additional PG supply and enhanced in Cu + Asc treatment (by 26%). The Rfd ratio was lower in Cu and Cd-treated plants and decreased with the time of treatment to about 50% of control. PG diminished this effect from the 48th hour, but Asc enhanced it in Cu-treated plants, and in Cd-treated plants only after 15 h. NPQ increased after Cu and Cd treatment and it was enhanced or diminished after 15 h in Cu and Cd-treated plants, respectively, by all additionally used substances (except Cd + MJ treatment). After a longer Cu-treatment, MJ increased NPQ value, but PG did not modify it. In leaves of 144 h Cd-treated plants, the additionally added PG decreased NPQ value below the control level, which was not changed by MJ, but Asc increased it to 182% of control.
qP decreased from the 48th hour after Cu and Cd supply (Fig. 2). The influence of the additional substances was not distinct in this case, but in Cd-treated leaves MJ and Asc additionally diminished qP value after 15 and 48 h, respectively. After 144 h, the inhibitory effect of the heavy metal was diminished by Asc. The %X value increased after Cu and Cd supply. At the beginning this effect was increased by Asc (in the case of Cu) and by MJ (in Cd-treated plants), but after the longer time, this effect was inverted in the former case or not observed in the latter. PG distinctly influenced the %X value only after 48 h in Cd treated plants and was found as an inhibitory effect. 3.2. The effect of Cu, Cd and MJ on ROS accumulation The elevated O content was found after the early 2 phases of Cu treatment, where it reached 147% and 232% of control after 15 and 48 h, respectively (Fig. 3). After a longer time it was below the control level. PG usually increased O 2 accumulation except 48 h treatment. At Cd
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Fig. 4. Content of malondialdehyde (MDA) products in A. thaliana leaves treated for 48 h with 107, 106 and 105 M methyl jasmonate (MJ) or 100 lM Cu or Cd alone, or with the addition of 100 lM propyl gallate (PG). Data are means ±SE. Fig. 2. The Chl fluorescence parameters of PSII (relative units) analyzed in light-adapted leaves: the photochemical quenching (qP) of fluorescence (estimated degree of open reaction centers), and the additional energy emitted per energy absorbed in the antenna PSII due to the closure of a fraction of the reaction centers in the PSII antennae (%X) in A. thaliana leaves after supply of 100 lM Cu or Cd alone, or with the addition of 105 M methyl jasmonate (MJ), 2.5 mM ascorbate (Asc) and 100 lM propyl gallate (PG). Data are means ±SE.
excess its level decreased below control during the investigated time period and was enhanced by adding PG. The content of O 2 increased after plant treatment with MJ, especially after 144 h. The effect of Cu and Cd on lipid peroxides was different after 48 and 144 h exposition. After Cu supply their level increased 3.6-fold after 48 h, and about 9.5-fold after 144 h (Figs. 4 and 5). The formation of lipid peroxides decreased only after 144 h treatment with PG. A slightly inductive effect of Cd on MDA formation was
Fig. 5. Content of malondialdehyde (MDA) products in A. thaliana leaves treated for 144 h with 107, 106 and 105 M methyl jasmonate (MJ) or 100 lM Cu or Cd alone, or with the addition of 100 lM propyl gallate (PG). Data are means ±SE.
found, especially after 144 h. After adding PG the decrease in JA synthesis additionally enhanced this process despite a stimulatory effect of exogenous MJ on the level of lipid peroxides in control plants (Figs. 4 and 5). However, in this case accumulation of MDA was not correlated with MJ concentration. Fig. 3. Relative O 2 content (% of control) in A. thaliana leaves after treatment with 107, 106 and 105 M methyl jasmonate (MJ) or 100 lM Cu or Cd alone, or with the addition of 100 lM propyl gallate (PG). Data are means ±SE. Control values of O 2 : 3.94 ± 0.21, 1.86 ± 0.12 and 2.40 ± 0.04 nmol NBT g1 FW after 15, 48 and 144 h, respectively.
3.3. Effect on glutathione and phytochelatins accumulation PCs were not accumulated after 15 h of the treatments (Fig. 6). The concentration of GSH was increased by Cu
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It was postulated as a direct action of heavy metals on the photosynthetic electron transport. Recently an indirect toxic activity was also proposed, e.g., through Ca pool modification or down-regulation of the electron transport due to inhibited activity of the Calvin cycle (Maksymiec, 1997; Maksymiec and Baszyn´ski, 1999; Vinit-Dunand et al., 2002). According to our investigations Cu and Cd act on photosynthetic apparatus as follows:
Fig. 6. Glutathione (GSH) and phytochelatins (PCs) content after 15 h treatment with 100 lM Cu or Cd alone, or with the addition of 100 lM propyl gallate (PG), 2.5 mM ascorbate (Asc) or 105 M methyl jasmonate (MJ). Values are means of two separate time series. Standard errors were less than 15% of the value.
Fig. 7. Glutathione (GSH) and phytochelatins (PCs) content after 144 h treatment with 100 lM Cu or Cd alone, or with the addition of 100 lM propyl gallate (PG), 2.5 mM ascorbate (Asc) or 105 M methyl jasmonate (MJ). Values are means of two separate time series. Standard errors were less than 15% of the value.
and Cd supply and was additionally enhanced in Cu + PG and Cu + Asc treated plants. Asc and, to a minor degree, MJ decreased GSH accumulation in Cd treated plants. Increasing time of heavy metal treatment induced the accumulation of PCs and GSH, especially in the case of Cd (Fig. 7). PG and also Asc enhanced the effect of Cd and Cu on PCs synthesis, but MJ did only in the latter case. PCs were not detected in control plants. 4. Discussion The inhibitory effect of Cd and Cu ions excess on the photosynthetic apparatus has been known for a long time.
• 0–15 h: The yield of primary photochemistry (Fv/Fm) and reduced QA pool (qP) was constant, the whole photosynthetic activity (Rfd) decreased and there was an increase in excess energy loss (%X) and non-photochemical quenching (NPQ), due to zeaxanthin cycle operation. Summarizing, it seems that during the first phase Cu and Cd affect the dark phase of photosynthesis followed by an increase in dissipative processes, allowing not disturbed electron transport through PSII. • 15–48 h: Additionally to the above presented changes, there was a slight decrease in Fv/Fm and a big decrease in qP, which indicated that Cu and Cd diminished reoxidation of Q A and started to inactivate the RC of PSII. As the effect was found especially in the light-adapted state, it may be related to developed photoinhibitory processes. • 48–144 h: At this time the protective role of non-photochemical energy dissipation gradually diminished, inhibition of dark and light phase of photosynthesis increased and the pool of oxidized quinones decreased i.e. the pool of closed reaction centers increased. Fv/Fm decreased as a result of preferential Fm decrease (data not shown). It may be attributed to inactivation of the donor side of PSII (To´th et al., 2005) and we presume that after 144 h Cu and Cd ions induced some structural changes in PSII centers. An increase in NPQ might be due to a dissociation of light-harvesting complex from PSII core (Ouzounidou and Ilias, 2005). At a higher NPQ level the %X increase, observed after 48 h, was the result of a favour decrease in the pool of the open reaction centers (qP decrease). After blockade of JA synthesis both heavy metals caused a decrease in the Fv/Fm and the whole photosynthetic processes (Rfd decrease). The balance between electron utilization and deleting by quinones in PSII was still at the same level if qP was not changed. This result indicated that JA may enhance Cu and Cd toxicity, but it probably does not disturb electron flow around QA. If Rfd value is a good indicator of the net CO2 fixation (Lichtenthaler and Babani, 2004), Cu or Cd-induced Rfd decrease, attenuated by PG, may be the consequence of MJ induced stomata closure observed in A. thaliana leaves after MJ supply (Suhita et al., 2004). However, exogenous MJ did not additionally diminish Rfd, indicating that only a definite level of JAs in the cells can determine the sensitivity of the photosynthetic apparatus to heavy metals. It is
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worth noting that either blocking of JA synthesis or its addition may stimulate O accumulation in stressed 2 plants. This is difficult to explain, but it might indicate that blockade of octadecanoid pathways at the site of PG action as well as accumulation of its final metabolite express a similar response to O 2 formation. Parallel to the Rfd decrease, induced by Cu, lipid peroxidation products were accumulated, indicating the occurrence of oxidative damage of membranes. The blockade of JA synthesis diminished this process after 144 h and, in consequence, the activity of the photosynthetic apparatus was enhanced. Because the improvement of the Calvin cycle (after adding PG) started from the 48th hour we suppose that lipid peroxidation was only partially the cause of photosynthesis inhibition. Cd ions induced accumulation of MDA products more slightly than Cu, and O 2 level decreased below the control (from the 15th hour). This result was similar to that obtained by Sko´rzyn´ska-Polit et al. (2003/4) for A. thaliana plants, however, opposite to that observed in rice plants (Shah et al., 2001), which indicates that there exists some discrepancy depending on the plant species. The improvement of the photosynthetic activity by adding PG to Cd-stressed plants might be due to induction of PCs accumulation as was also detected in other plants (Tukendorf and Rauser, 1990; Cobbett and Goldsbrough, 2002). Cd and excess Cu increased GSH accumulation in A. thaliana leaves already after 15 h. This had presumably an inductive character because it did not depend on the concentration of the metals (data not shown). Although JA is an inductor of GSH genes, when applied exogenously it did not increase GSH both in control and heavy metaltreated plants. This phenomenon indicates that besides the transcription there exist other mechanisms regulating GSH level, which confirms earlier suppositions of Xiang and Oliver (1998). PC concentration was especially high after adding MJ, Asc or PG to Cd-treated plants. In Cutreated plants MJ-induced GSH decrease may be due to its limited synthesis. Because JA is accumulated during heavy metal stress (Maksymiec et al., 2005) it is likely to enhance the inhibitory effects of metals. In our study this was found as decreased GSH accumulation, especially after 144 h of Cu action. Cu and Cd preferentially inhibit the Calvin cycle. In this case Asc is used in the Mehler cycle. Asc excess can restock the pool of Asc used and, in consequence, improve photosynthetic electron transport. However, when in excess, Asc did not increase the oxidized pool of QA in Cu-treated plants, but after a short term action it increased %X as well as NPQ. Therefore, we hypothesize that exogenous Asc after a short time specifically induces the dissipative processes through reduction of Cu2+ to Cu1+ and, in consequence, it induces the enhancement of radical processes in the chloroplast resulting in a decrease in potential quantum yield of PSII. This effect was not observed in the case of Cd (which does not change the oxidative degree), supporting the above assumption. Because
we did not observe any enhancement of O 2 accumulation by Asc added to Cu-treated plants after 15 h (data not shown), it is possible that H2O2 was preferentially accumulated. A sharp increase in NPQ value induced by Asc may also be the consequence of Asc stimulatory effect on the violaxanthin cycle if it participates in regeneration of zeaxanthin (Bratt et al., 1995), and Asc content is increased by Cu in A. thaliana leaves (Dra˛z_ kiewicz et al., 2003). Our results showed that the enzymic phase of photosynthesis was preferentially slowed down by Cu and Cd ions beginning already from the 15th hour. These changes in the photosynthetic apparatus were related to JA action and MDA, GSH and PC accumulation. After a longer time of heavy metal action JA might increase their toxic action and probably it was partially the cause of the changes induced by the metals. Asc can enhance toxic action of Cu and Cd after a short time, however, after a longer time it can diminish the influence of Cd on the photosynthetic activity. Although MJ showed a stimulatory effect on GSH gene expression, exogenously applied MJ diminished GSH accumulation after Cd or Cu supply. This may indicate that although JAs are signaling factors increasing plants resistance to any stress factors, they may also enhance the toxic effect of Cd, and especially Cu. This fact is an intriguing problem concerning gene expression during stress action and the development of the signaling activity on the level of enzymatic reaction in plant cells. Acknowledgement This study was supported by the State Committee for Scientific Research (KBN), grant No. 3PO4C05022. References Agrawal, G.K., Tamogami, S., Iwahashi, H., Agrawal, V.P., Rakwal, R., 2003. Transient regulation of jasmonic acid-inducible rice MAP kinase gene (OsBWMK1) by diverse biotic and abiotic stresses. Plant Physiol. Biochem. 41, 355–361. ˚ kerlund, H.-E., 1995. Bratt, C.E., Arvidsson, P.-O., Carlsso, M., A Regulation of violaxanthin deepoxidase activity by pH and ascorbate concentration. Photosynth. Res. 45, 169–175. Chen, G.H., Goldsbrough, P.B., 1994. Increased activity of c-glutamylcysteine synthetase in tomato cells selected for cadmium tolerance. Plant Physiol. 106, 233–239. Cobbett, C., Goldsbrough, P., 2002. Phytochelatins and metallothioneins: role in heavy metal detoxification and homeostasis. Annu. Rev. Plant Biol. 53, 159–182. Cuypers, A., Vangronsveld, J., Clijsters, H., 2000. Biphasic effect of copper on the ascorbate–glutathione pathway in primary leaves of Phaseolus vulgaris seedlings during the early stages of metal assimilation. Physiol. Plant. 110, 512–517. Demmig-Adams, B., Adams, W.W., Baker, D.H., Logan, B.A., Bowling, D.R., Verhoeven, A.S., 1996. Using chlorophyll fluorescence to asses the fraction of absorbed light allocated to the thermal dissipation of excess excitation. Physiol. Plant. 98, 253–264. Dra˛z_ kiewicz, M., Sko´rzyn´ska-Polit, E., Krupa, Z., 2003. Response of the ascorbate–glutathione cycle to excess copper in Arabidopsis thaliana (L.). Plant Sci. 164, 195–202.
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