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First published online October 2, 2003; 10.1104/pp.103.026518 Plant Physiology 133:829-837 (2003) © 2003 American Society of Plant Biologists Interaction of Cadmium with Glutathione and Photosynthesis in Developing Leaves and Chloroplasts of Phragmites australis (Cav.) Trin. ex Steudel1Institute of Agroenvironment and Forest Biology, Via Salaria Km 29,300 00016 Monterotondo scalo (Rome), Italy (F.P., M.A.I., A.M.); and Department of Plant Biology and Environmental Biotechnology, University of Perugia, Borgo XX Giugno, 06121 Perugia, Italy (S.P.)
We investigated how the presence of cadmium (Cd) at the emergence of Phragmites australis Trin. (Cav.) ex Steudel plants from rhizomes interacted with leaf and chloroplast physiological and biochemical processes. About 8.5 nmol Cd mg–1 chlorophyll was found in leaves, and 0.83 nmol Cd mg–1 chlorophyll was found in chloroplasts of plants treated with 50 µM Cd. As a result, a 30% loss of chlorophyll was measured concomitantly with a comparable percentage reduction in light-saturated photosynthesis. Rubisco content and activity were lowered by 10% and 60%, respectively. Antioxidant activity was stimulated by Cd treatment and was associated with an increase in the glutathione and pyridine pools, and with a larger pool of reduced glutathione. It is suggested that the glutathione pool and its predominance in the reduced state protected the activity of many key photosynthetic enzymes against the thiophilic binding of Cd. Chloroplast ultrastructure was not significantly altered with 50 µM treatment and the efficiency of photosystem II, measured as the fluorescence ratio Fv/Fm, remained high because F0 and Fm were proportionally decreased. In plants treated with 100 µM Cd, all effects were exacerbated, but Fv/Fm remained close to that of control leaves and the glutathione and pyridine nucleotides pools were lowered. The results suggest that glutathione exerted a direct important protective role on photosynthesis in the presence of Cd.
Most plants respond to cadmium (Cd) present in the root environment: the metal ion is absorbed on cortical cell walls or it is channeled into roots, where it is then subsumed into the closest vacuoles or loaded into the xylem for transport into leaves (Sanità di Toppi and Gabbrielli, 1999  ). The amount of this metal retained in the roots or transported to leaves differs significantly between species. Some Brassicaceae (e.g. Thlaspi caerulescens) are known to accumulate up to 1% (w/w) Cd on a dry weight basis and tolerate Cd in the aboveground tissues (Lombi et al., 2000). Unfortunately, this tolerance capacity is limited to a restricted number of species and genera, and thus, even a few micromoles of Cd in the root environment are toxic to most plants. Cd often damages root tips (visible browning), reduces nutrient and water uptake, impairs photosynthesis, and inhibits growth (Das et al., 1998). The basis of these dys-functions include irreversible changes to protein conformation by forming metal thiolate bonds (Dafré et al., 1996) and alteration of cell wall and membrane permeability by binding to nucleophilic groups (Ramos et al., 2002). Furthermore, because Cd induces antioxidant responses in all plant organs (Iannelli et al., 2002), it is also assumed that it directly or indirectly causes the formation of active oxygen species, thus interfering with the redox status and inducing significant chlorophyll loss (i.e. visible chlorosis; Baryla et al., 2001; Schutzendubel et al., 2001). Currently, it is not known clearly how this metal, which is not capable of carrying on a Fenton-type reaction, is involved in the formation of active oxygen species and thus in the most deleterious membrane damage (Navari-Izzo and Quartacci, 2001). It could be hypothesized (Atal et al., 1991) that photochemistry is somehow altered in the presence of Cd, with the consequent formation under high light of chlorophyll triplets in the photosystem antennae and then in a cascade of singlet oxygen, superoxide ions, and other reactive oxygen species. However, this does not imply that photochemistry is the cause of the high sensitivity of photosynthesis to Cd. Results of transient fluorescence analysis are, in fact, contradictory as are the analyses of various metabolic dys-functions thought to limit photosynthetic activities (Malik et al., 1992; Mendelssohn et al., 2001).
It is well established that many plants, as soon as Cd enters roots or arrives in leaves, stimulate sulfate absorption (Nocito et al., 2002 We have investigated how the presence of Cd at the emergence of Phragmites australis plants from rhizomes interacted with glutathione and photosynthesis. Analysis of the redox state (including GSH and GSSG and pyridine nucleotides), chloroplast ultrastructure, antioxidant activities of chloroplasts and leaves, as well as analysis of gas exchange and fluorescence of leaves, suggest that developing leaves, and thus photosynthesis, could cope with mild Cd toxicity. Increased GSH concentration, a very mobile antioxidant and a targeted thiol, seems to be the optimal defense strategy, together with phytochelatins, in the preservation of key photosynthetic thiolic enzymes from Cd inactivation.
Table I shows that about 8.5 nmol Cd mg–1 total chlorophyll were translocated from roots to leaves of P. australis plants that had emerged in the presence of 50 µM Cd, and from there, 0.83 nmol Cd was passed to chloroplasts. When Cd in the root environment was 100 µM, then 21 nmol of this metal arrived in leaves and 3 nmol reached the chloroplasts (Table I). A similar distribution of Cd between leaves and chloroplasts has been found in other species (Siedleka and Krupa, 1999 ; Ramos et al., 2002). The leaf content of Fe, Ca, and Zn significantly increased with respect to controls in the presence of 50 µM Cd, whereas at 100 µM, Cd, Fe, and Zn did not change with respect to controls and Ca was slightly and significantly reduced (Table I).
In Table II, we have summarized the most relevant results from photosynthesis and fluorescence measurements. Photosynthesis was measured by varying the concentration of CO2 in the leaf cuvette and maintaining the photosynthetic photon flux density (PPFD) incident on the leaf surface at 800 µmol m–2 s–1 or by varying PPFD and maintaining CO2 at 350 µbar bar–1. On a leaf area basis, Cd decreased photosynthesis at 350 µbar bar–1 CO2 and 800 µmol m–2 s–1 PPFD by around 28% of the control values (22.5 µmol m–2 s–1) in the presence of 50 µM Cd, and by about 60% in presence of 100 µM Cd. Maximum photosynthesis measured under saturating light and CO2 was reduced with respect to controls by 40% at 50 µM Cd and by 50% at 100 µM Cd. The initial slope of the photosynthesis curve at low internal CO2 (40–150 µbar bar–1) was also strongly reduced: by 60% and 83% in 50 and 100 µM Cd leaves, respectively. The slope of the photosynthetic response at low PPFD did not change between control and 50 µM Cd leaves, and was slight lower than the slope of the control in 100 µM Cd leaves. However, photosynthesis on a chlorophyll basis showed no statistically significant difference between control and 50 µM Cd, and a slight increase from control with 100 µM Cd. This indicates that photosynthesis was mostly limited by chlorophyll content and thus most likely by the photochemical capacity with 50 µM Cd. There were no changes in the CO2 compensation point or apparent photorespiration with any treatment. Dark respiration only increased strongly in leaves exposed to 100 µM Cd. Transpiration was the same in control and 50 µM Cd leaves, but decreased (but not significantly) in 100 µM Cd leaves, suggesting that stomata closed in response to high Cd concentration. The increase in the internal CO2 concentration with the increase of Cd in leaves was perhaps an indication that metabolism was limited by something other than the supply of CO2, even at the highest Cd treatment. Photochemical and nonphotochemical quenching of fluorescence decreased and increased with respect to the increase in Cd concentration, in accordance with the lower photosynthesis and higher dissipation as heat of the absorbed energy.
Estimation of photosystem (PS) II quantum yield and PSII efficiency by fluorescence showed that decreases in
Table III shows a large data set, including enzymatic and nonenzymatic antioxidants, glutathione and glutathione-related enzymes, and pyridine nucleotides, all determined in chloroplasts and in leaves. Generally, Cd-treated leaves showed higher values than control leaves, with a maximum in 50 µM Cd-treated leaves, and a slight decrease in 100 µM with respect to 50 µM Cd-treated leaves. Furthermore, measurements carried out on chloroplasts showed higher values than measurements in leaves, particularly for SOD, APX, and GST activities, which were much higher than activities measured in leaves with increasing Cd concentrations. In contrast, CAT activity was not affected by the presence of Cd, GR increased only with 100 µM Cd, and GPX increased only in chloroplasts of the two Cd treatments, whereas ascorbate content was not affected by Cd. However, Cd exposure strongly increased total glutathione, particularly GSH, in chloroplasts at 50 µM Cd and to a lesser extent in leaves. Cd exposure also increased the pyridine nucleotide content, whereas total NADP (NADPH and NADP) and NAD (NADH and NAD) almost doubled in leaves and chloroplasts.
Figure 2 shows transmission electron microscopy micrographs of developing leaves of control and Cd-treated plants. The chloroplasts from Cd-untreated plants were typical mesophyll chloroplasts (Fig. 2A) with a well-organized internal membrane structure and normally developed grana and stroma thylakoids. Little or no starch was present. In 50 µM Cd-treated plants (Fig. 2B), most of the chloroplasts were undamaged or only slightly damaged. A small proportion of chloroplasts were lenticular in shape. In 100 µM Cd-treated plants, the chloroplast thylakoid system was affected (Fig. 2C), with an apparent swelling of the thylakoid membrane, fewer grana, and abundant starch grains.
In this experiment, we grew P. australis plants from rhizomes in the presence of 50 µM Cd and, after 4 weeks, we found 8.5 nmol Cd mg–1 chlorophyll in leaves and 0.83 nmol Cd mg–1 chlorophyll in chloroplasts. In general, a typical sign of Cd toxicity is visible loss of chlorophylls, as also observed in our Cd-treated leaves. This has been interpreted as the effect of strong oxidation on the photochemical apparatus (Somashekaraiah et al., 1992 ), the effect of interference of Cd with Fe root uptake (Siedleka and Krupa, 1999), or reduction of chloroplast density and size (Baryla et al., 2001). Our measurements, reported in Table I, excluded the possibility of loss of chlorophylls being caused by low levels of Fe in leaves, but did not exclude that Cd could have replaced Mg in chlorophylls (Küpper et al., 1998). The reduced amount of active chlorophylls in our Cd-treated leaves with respect to control leaves resulted in a decrease in F0 values. This parameter is used to obtain the Fv/Fm ratio, an estimation of PSII efficiency in dark-adapted leaves (Fracheboud et al., 1999). Surprisingly, this ratio, in our experiment, remained high, even after exposure of chloroplasts to high Cd concentrations. In fact, Fm decreased proportional to F0 (Table II), thus explaining the maintenance of a high ratio and possibly indicating balanced damage to, or close regulation of, energy harvesting and energy conversion capacities. However, in a short-term Cd treatment, we took small leaf discs from fully mature control plants and exposed them to 8.5 nmol Cd mg–1 chlorophyll, the amount found in the 50 µM Cd-treated leaves. After a few days, Fv/Fm decreased to 40% of the initial value and of the control value. Thus, in contrast to the previous result, Cd was very damaging for PSII, and chlorosis occurred on leaf surfaces in association with the decrease in Fv/Fm. It must be stressed that the two experiments were performed with newly emerged leaves in contact with Cd from their emergence, or with fully mature leaves. This confirmed that P. australis had an intrinsic capacity to defend chloroplasts against toxicity caused by nanomolar amounts of Cd (Fediuc and Erdei, 2002), but also indicated that this capacity was higher in developing than in mature leaves. A comparable growth-stage response of toxicity to Cd has been found in different sections of a single leaf of rye (Secale cereale; Krupa and Moniak, 1998).
Although Cd did not cause a reduction in Fv/Fm, it did induce a reduction in photosynthetic electron transport. Differences in the fluorescence parameters, specifically of
On the other hand, analysis of parameters elaborated from gas exchange and biochemical measurements, reported in Table II, indicated that the major interference of Cd with photosynthesis was a reduction in maximal extractable Rubisco activity (60% less than controls), which could only slightly be attributed to a reduction in Rubisco content (10% less than controls). Because Cd is typically associated with widespread oxidative activity, as indirectly confirmed in this experiment by antioxidant stimulation (Table III), the effects on Rubisco content could be considered a result of oxidative activity (Romero-Puertas et al., 2002
Another major effect observed in this investigation was that nanomoles of Cd in leaves and in chloroplasts increased the GSH level by about 37 and six times, respectively, above levels in the controls. This was a predictable result because it is known that Cd acts as a strong sink for thiols, which increases the demand for sulfate absorption (Nocito et al., 2002
Doubling (100 µM Cd) the concentration of Cd in the growth medium almost doubled the concentration of Cd in leaves, and in chloroplasts, Cd concentrations were as high as 3 nmol mg–1 chlorophyll. This high metal concentration reduced photosynthesis and chlorophylls (50%–60%), as well as Rubisco content (35%) and activity (70%). Notably, the decrease in photosynthesis seems to follow (as with 50 µM Cd) that of chlorophyll and of maximum extractable Rubisco activity. Despite this, the efficiency of PSII was still close to control values. In contrast, chloroplasts had visible alterations in thylakoids and a large starch accumulation. It is likely, as previously argued, that Cd bound to the cytosolic thiol-dependent enzyme F-1,6-P2ase and reduced sucrose synthesis, thus limiting phosphate recycling between cytosol and chloroplasts (Sharkey, 1990 In conclusion, this experiment showed that leaves of P. australis can avoid Cd-induced irreversible damage to photochemistry when they emerge in the presence of 50 µM of the metal and can develop defense mechanisms to cope with the affinity of Cd for thiols. The high concentration of GSH, a very mobile molecule, seems to be an optimal response defense strategy: it provides widespread antioxidant protection to leaves and throughout the plant and, most importantly, it increases the thiol concentration in the cytoplasm and chloroplasts. Enhancing the number of targetable thiols probably helps to preserve the activity of key photosynthetic enzymes and to sustain photosynthesis until a higher Cd concentration (100 µM) overloads this defense capacity.
Plant Material Phragmites australis (Cav.) Trin. ex Steud rhizomes were collected from a clean site in the Trasimeno Lake (Perugia, Italy), potted in three groups of four with expanded argyll, and watered with 2 liters of full-strength Hoagland nutrient solution, pH 6.0.
The Cd was added as CdSO4 to the nutrient solution to give concentrations of 0, 50, or 100 µM Cd. Air was continuously and gently bubbled through all pots, and solutions were replaced twice a week. Pots were located inside a growth chamber with controlled temperature (27°C/22°C day/night), humidity (95%/85% day/night), PPFD (800 µmol m–2 s–1 at 40 cm from white lamps), and a 16-h photoperiod. Four large leaf samples from plants that had emerged in pots containing 0, 50, and 100 µM Cd were taken after 21 d, weighed, immediately frozen in liquid nitrogen, and stored at –80°C.
Leaf discs (1 cm2) were taken from mature leaves of plants grown under the conditions of the previous experiment (growth in presence of Cd). Chlorophylls were measured on three replicate discs as described below. Six leaf discs were floated on small plates in a 50 mM phosphate buffer containing 8.5 nmol Cd mg–1 leaf disc total chlorophyll, and six replicates were placed in the same buffer solution without Cd as controls. The amount of Cd used corresponds to the amount measured in the leaves of plants exposed to 50 µM Cd (see Table I). After 3 d, the ratio of variable (Fv = Fm – F0) to maximum fluorescence (Fm), Fv/Fm, was measured on dark-adapted discs (30 min in darkness) from all treatments using the fluorometer (PAM 2000; Walz, Effeltrich, Germany).
Fresh leaf samples were ground in an ice-cold isolation medium containing 0.33 M sorbitol, 50 mM HEPES-KOH, 2 mM EDTA, 2 mM EGTA, 1 mM MgCl2, 1 mM MnCl2, and 0.2% (w/v) bovine serum albumin at pH 7.3. The homogenate was centrifuged at 2,000g for 10 min and the pellet was suspended in a second aliquot of the isolation medium and centrifuged at 6,500g for 20 min in a Percoll discontinuous gradient (40%–80%, v/v). The lower dark green band containing the intact chloroplasts was collected, after first carefully removing the upper part, and then by diluting the dark green band with an ice-cold import buffer containing 0.33 M sorbitol and 50 mM HEPES-KOH, pH 7.3, to obtain the purified fraction containing intact chloroplasts (Cline et al., 1985
Determination of Cd was carried out on leaves and chloroplasts of P. australis plants. Ca, Zn, and Fe contents were also measured in P. australis leaves. After 21 d of Cd treatment, leaf samples and chloroplasts pellets were dried at 80°C for 48 h and the dry weight was measured. Determinations of metals were made by atomic absorption spectrophotometry (Analyst 300; Perkin Elmer, Germany) on nitric-perchloric acid (3:1, v/v) digests of four replicate samples from plant tissue for Cd, Ca, Zn, and Fe and from pelleted chloroplasts for Cd.
Between 0.2 and 0.5 g of fresh leaf was ground to a fine powder with a mortar and pestle under liquid nitrogen. Proteins were then extracted at 4°C by grinding with 50 mM of ice-cold phosphate buffer, pH 7.0, containing 0.1% (w/v) AA, 0.1% (v/v) Triton X-100, and 1% (w/v) polyvinyl pyrrolidine. The homogenate was centrifuged at 4°C for 20 min at 12,000g. The clear supernatant fraction was used for the enzyme assays. Protein concentration was quantified as described by Bradford (1976
Rubisco was extracted from frozen samples, separated, and identified by SDS-PAGE using Rubisco from spinach (Spinacia oleracea; Sigma, St. Louis) as standard, as described by Laemmli (1970
Chloroplasts and frozen leaf tissue were ground with a mortar and pestle under liquid N2. Eighty percent (v/v) acetone was added to extract pigments and, after centrifugation of the supernatant for 10 min at 10,000g, O.D. was measured at 470, 646.8, and 663.2 nm using a spectrophotometer (Perkin Elmer). The extinction coefficients and the equations reported by Lichtenthaler (1987
The AA content was determined by HPLC according to Olmos and Hellin (1996
The concentrations of GSH and GSSG were spectrophotometrically determined with an enzyme-recycling assay at 412 nm (Griffith, 1980
Frozen leaf samples were ground with a mortar and pestle under liquid nitrogen and were extracted with ethanol-water (1:1, v/v) containing 0.1 M NaOH or 0.1 M HCl (Carrier and Neve, 1979
Total APX (EC 1.11.1.11) activity was determined by measuring the oxidation rate of ascorbate at 290 nm according to Asada (1992
GST (EC 2.5.1.18) was measured spectrophotometrically at 340 nm by monitoring the increase in absorbance due to the formation of the conjugate, S-2,4-dinitro (phenylglutathione;
GR (EC 1.6.4.2) activity was assayed spectrophotometrically after the formation of thiobenzoic acid at 412 nm, according to Smith et al. (1988
GPX (EC 1.11.1.9) activity was measured spectrophotometrically after the decrease in A340 of NADPH (
The central part of a P. australis leaf was enclosed in the cuvette of a gas exchange system (HCM 1000; Walz), configured for simultaneous measurement of chlorophyll fluorescence (PAM 101 modulated fluorometer; Walz). The relative humidity of air entering the cuvette was set at 50% and air and cuvette temperatures were 25°C or 7°C. CO2 partial pressure was varied between 0 and 700 µbar bar–1 at 50 µbar bar–1. A white light source (KL 1500; Schott, Mainz, Germany) was used to vary the incident PPFD on the leaf surface between 0 and 2,000 µmol m–2 s–1 PPFD. The leaf absorbance (Aleaf) was measured with a spectroradiometer (Li-1800; Li-Cor, Lincoln, NE). Photosynthesis and stomatal conductance were calculated according to von Caemmerer and Farquhar (1981
Fresh tissues (1–2 mm2) were taken from last fully expanded leaves and were immediately treated for microscopy observations (TEM 400 T; Philips, Eindhoven, The Netherlands), as reported in Loreto et al. (2001
Data reported in the tables and figures were all analyzed using the statistical program SPSS 6.0 (SPSS, Chicago). Significance of difference was tested at P = 0.05 using ANOVA and post hoc LSD. The data are means ± SE from four determinations.
The authors wish to thank Dr. Lucia Fiore and Angelo De Martino for helping with the biochemical analyses. Received May 7, 2003; returned for revision May 30, 2003; accepted June 20, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.026518.
1 This work was supported by the Consiglio Nazionale delle Ricerche of Italy. * Corresponding author; e-mail angelo.massacci{at}ibaf.cnr.it; fax 39–06–9064492.
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ABSTRACT
RESULTS
CO2 int), dark respiration (Rdark), internal CO2 concentration (CO2 int), Rubisco activity (RuBPCase), initial slope of CO2 assimilation rate curve response to photosynthetic photon flux density (
A/
F/Fm), photochemical (qP) and nonphotochemical (qN and NPQ) fluorescence quenching of absorbed light energy, basal (F0), maximal (Fm), and fluorescence ratio (Fv/Fm with Fv = Fm-F0) of dark-adapted leaves
= 39.4 mM–1 cm–1) and was expressed as micromoles H2O2 per milligram of protein per minute according to Chance and Maehly (1955
etlík I, Küpper F, Spiller M, Prá
il O (2002) Heavy metal-induced inhibition of photosynthesis: targets of in vivo heavy metal chlorophyll formation. J Phycol 38: 429–441
