Shoot Cadmium Accumulation and Photosynthetic Performance of ...

JOURNAL OF PLANT NUTRITION Vol. 27, No. 5, pp. 775–795, 2004

Shoot Cadmium Accumulation and Photosynthetic Performance of Barley Plants Exposed to High Cadmium Treatments Andon Vassilev,1,* Fernando C. Lidon,2 Jose´ C. Ramalho,3 Maria do Ceú Matos,4 and Maria G. Bareiro4 1

Department of Plant Physiology and Biochemistry, Agricultural University of Plovdiv, Plovdiv, Bulgaria 2 GDEH, UBIA, Faculdade de Cieˆncias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 3 Instituto de Investigaçaõ Cientı´ fica Tropical-CEPTA, Tapada da Ajuda, Lisboa, Portugal 4 Plant Physiology Department, Estaçaõ Agrono´mica NacionalINIA, Quinta do Marqueˆs, Av. da Republic, Oeiras, Portugal

ABSTRACT Barley (Hordeum vulgare L., cv. Ribeka) plants grown in sand culture were exposed for 10 days to high cadmium (Cd) treatments in order to

*Correspondence: Andon Vassilev, Assistant Professor, Agricultural University of Plovdiv, Department of Plant Physiology and Biochemistry, 12 Mendeleev St., Plovdiv 4000, Bulgaria; Fax: 359 32 633 157; E-mail: [email protected] or [email protected]. 775 DOI: 10.1081/PLN-120030613 Copyright & 2004 by Marcel Dekker, Inc.

0190-4167 (Print); 1532-4087 (Online) www.dekker.com

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Vassilev et al. study both shoot Cd accumulation and photosynthetic performance. Cadmium inhibited root dry mass and induced changes in biomass allocation pattern without any effect on biomass accumulation at the whole plant level. The maximal shoot Cd concentration— 41 8 mg Cd kg1 DW—without any visual toxicity symptoms on the shoots was found at 28 mg Cd kg1 sand. Reduced leaf gas exchange, photosynthetic pigments content and electron transport activity but not altered lipid peroxidation status of thylakoids was, however, detected at the highest treatment—42 mg Cd kg1 sand. The results indicated good tolerance of barley growth and photosynthetic machinery to Cd, but the shoot Cd accumulation achieved even in the artificial conditions was considered insufficient for short-term phytoextraction. Key Words: Barley; Cadmium; Chlorophyll a fluorescence; Leaf gas exchange; Lipid peroxidation; Photosynthetic electron transport; Photosynthetic pigments.

INTRODUCTION Cadmium is a nonessential element and a heavy metal pollutant of the environment, resulting from various mining, industrial, and agricultural activities. As it has a high mobility in the soil-plant system, extensive research has been conducted during the last decades, focused mainly on Cd accumulation in crops and its consequences to human health[1] and Cd phytotoxicity.[2] Recently, a new emerging technology called phytoremediation has been proposed as a low-cost and environmentally sustainable technique for removal of heavy metals from contaminated soils.[3–5] There is some evidence that Cd phytoextraction could be easily implemented due to both lower Cd contamination and higher Cd mobility in the soil-plant system as compared, for example, to lead (Pb) or zinc (Zn).[6] Besides metal hyperaccumulators plants, nonaccumulating Cd but high biomass crops are also considered for phytoextraction purposes, but it has been suggested that the success of this approach might be limited by Cd-induced phytotoxicity problems.[5] Ebbs and Kochian[7] have recently reported that barley could tolerate high Cd concentrations present in the solution and accumulate elevated Cd concentrations in shoots. This suggestion is somewhat different from the results of our previous work,[8,9] where we found that barley is able to grow well on Cd-contaminated soil, but not to accumulate Cd in the shoots at levels sufficient for short-term phytoextraction. As higher Cd

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phytoaccumulation might be achieved by means of different soil amendments,[10] plant performance at induced high Cd loading in the tissues, should be studied in more detail. Consequently, we found it would be useful to clarify the ability of barley to tolerate as well as accumulate Cd in the shoots. In general, barley can withstand Cd accumulation until the metal reaches the toxic threshold level in the tissue, suffering growth reduction and toxicity at higher levels. The impact of Cd on photosynthesis is considered to be one of the most important factors limiting plant growth.[11] On the other hand, the photosynthetic performance may serve as a useful criterion for evaluation of plant tolerance to Cd, as many Cd-induced physiological disturbances may finally be focused on photosynthesis.[12] Cadmium effects on photosynthesis have been intensively studied during the last three decades,[13,14] but some unclear aspects regarding primary and secondary effects still remain. In addition, it has been recently reported that Cd is able to induce over-production of oxy radicals in plant cells,[15–17] although it is a nonredox active metal unlike iron (Fe) or copper (Cu). The mode of this Cd action is still not understood, but its consequences, including membrane lipid peroxidation and enzyme inactivation, are known to have a great impact on cell physiology and photosynthesis. Impairments in leaf gas exchange due to limited access of CO2 was the earliest suggestion for Cd-exposed plants, but the expression of the stomata limitation seems to be species- and conditions-dependent.[18–20] Also, leaf chlorosis is widely reported, and might be due to the inhibition of chlorophyll biosynthesis,[21] Fe and magnesium (Mg) deficiency,[22] Mg substitution in the chlorophyll molecule[23] as well as chlorophyll degradation resulting from oxidative damage.[24] Interestingly, Baryla et al.[19] have recently reported that leaf chlorosis in Cd-exposed oilseed rape was due to neither of the mentioned reasons, but was attributable to a marked decrease in chloroplast density caused by a reduction in the number of chloroplasts per cell. Furthermore, Cd-induced disorders in chloroplast and thylakoid ultrustructure[25,26] and the negative effects on PSII and PSI activities, analyzed in vitro[27,28] have also been well documented. Conversely, in vivo studies by chlorophyll a fluorescence techniques have led to the conclusion that the photochemical reactions are not sensitive or less sensitive to Cd than Calvin’s cycle reactions.[29–31] In the present article, the influence of increasing sand Cd levels on dry mass accumulation, shoot Cd accumulation, and photosynthesis of barley plants are presented. The Cd concentrations used in this study were higher than in most other investigations, since the objective was to evaluate barley

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responses to severe Cd contamination, found at old smelter sites[32] or achieved by means of chemically assisted Cd phytoextraction.

MATERIALS AND METHODS Plant Material and Growth Conditions Barley (Hordeum vulgare L. cv. Ribeka) plants were grown in sand culture in a glasshouse under natural conditions of light, temperature, and humidity following the protocol described elsewhere.[20] Twenty days after emergence, the plants were given 125 mL of nutrient solution containing: 0, 2.5, 5, and 7.5 mM Cd (corresponding to 0, 14, 28, and 42 mg Cd kg1 sand, respectively). Metal ions were applied as 3CdSO48H2O and plants were exposed to Cd treatment for 10 days. Sand moisture was maintained during the experiment at 75–80% of maximum water holding capacity gravimetrically using deionized water. Plants were harvested 10 days after treatment.

Dry Mass Determination After harvest, the plants were separated into shoots and roots. The roots were thoroughly washed with deionized water. Dry mass of shoots and roots was determined after drying samples at 70 C to constant weight.

Mineral Analysis The concentrations of Cd in roots and Cd, Fe, and Mg in shoots were determined after dry mineralization of samples by atomic absorption spectrophotometry (Perkin-Elmer 5000 Atomic Absorption Spectrophotometer). Three replicate plant tissue samples, each 1 g dry mass, were put in an oven at 500 C for 24 h. Each sample was dissolved in 5 mL 20% HCl and the solution was used for analyses.

Leaf Gas Exchange Measurements Leaf gas-exchange measurements included net photosynthesis (A), stomatal conductance (gs), transpiration rate (E ), and intercellular CO2 concentration (ci). They were performed on the second youngest fully

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expanded leaf, using a Li-6200 infrared gas analyzer (Li-Cor, Inc., Lincoln, NE). The measurements were carried out in a growth chamber, under controlled conditions of temperature (25 1 C), air humidity (75 1%), photosynthetic photon flux density (PPFD, 800 mmol m2 s1) and external CO2 concentration (ca. 380 mmol mol1). Photosynthetic capacity (Amax) was measured, using a Clark-type leafdisc oxygen electrode (LD2/2, Hansatech, Kings Lynn, UK), in leaf pieces (2 cm2) placed under saturating conditions of light (800 mmol m2 s1, provided by a Bjorkman lamp, Hansatech) and CO2 (ca. 7%, supplied by 400 mL KHCO3, 2 M), at a stabilized temperature of 25 C.

Chlorophyll a Fluorescence and Pigment Determinations Chlorophyll fluorescence parameters were measured using a PAM 2000 system (H. Walz, Effeltrich, Germany) on leaf discs (from undamaged areas) placed inside the LD2/2 O2 electrode, under CO2 saturating conditions, at 25 C. Measurements of the minimal fluorescence from the antennae, Fo, and photochemical efficiency of PSII, Fv/Fm, were taken from overnight dark-adapted leaves. The photochemical quenching, qP,[33] the estimation of quantum yield of photosynthetic noncyclic electron transport, e,[34] and the PSII efficiency of energy conversion, Fv0 =Fm0 ,[29] were determined under photosynthetic steadystate conditions, using a PPFD of 550 mmol m2 s1 as actinic light and 4200 mmol m2 s1 as saturating flashes (with a duration of 0.8 s). Chlorophyll a and b and total carotenoids were extracted in 100% acetone, measured spectrophotometrically and calculated according to the formulae of Lichtenthaler.[35]

Photosynthetic Electron Transport Rates Determination of photosynthetic activities coupled to PSII and PSI were measured in a Clark-type oxygen electrode (LW2, Hansatech, Kings Lynn, UK), using subchloroplast fractions obtained as described by Droppa et al.,[36] with minor modifications, as described by Lidon and Henriques.[37] The electron transport rates were determined according to Droppa et al.[36] in 1 mL of reaction mixture containing 100–150 mg chl, at 25 C and with PPFD of 3000 mmol m2 s1, given by a Bjorkman lamp (Hansatech).

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Estimation of Leaf Lipid Peroxidation The leaf lipid peroxidation status was determined as 2-thiobarbituric acid (TBA) reactive compounds (mostly malondialdehyde, MDA), as described by Heath and Packer.[38]

Ethylene Production Associated with Thylakoid Membranes Ethylene production was measured in 500 mL of thylakoid extracts incubated at a light intensity of 500–600 mmol m2 s1, provided by a Bjorkman lamp in 2 mL flasks. After 2 h of incubation a 1 mL gas sample was withdrawn from the headspace gas of the incubating flask using a gas-tight syringe. Ethylene concentration in this gas sample was assayed by a Pye Unicam Series 204 gas chromatograph equipped with a Porapak Q column and a flame ionization detector (FID). Nitrogen, at a flow rate of 30 mL min1 was the carrier gas. The temperatures were set to 90 C for the oven, room temperature for the injection port, and 150 C for the detector. Ethylene was identified and quantified by comparison with the peak area from the gas samples containing a known concentration (29 mmol mol1) of ethylene standard.

Statistical Analysis Statistical analysis was performed using a one-way ANOVA (for P < 0.05). Based on the ANOVA results, a Tukey test for mean comparison was performed, for a 95% confidence level, to test for significant differences among treatments. In the tables, different letters (a, b, c) express significant differences, with a representing the highest value. A regression analysis was applied when this was more appropriate.

RESULTS Plant Growth and Cadmium Concentrations in Plant Organs The growth response of barley plants to 10-days exposure at increasing Cd levels is presented in Fig. 1A. The roots, which are in close contact with the heavy metal and are one of the primary sites of toxic action, showed a biomass decrease in all Cd treatments as might be

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Figure 1. Plant biomass (A) and Cd concentrations in roots and shoots (B) of barley plants exposed to high Cd loading. Mean values are the average of five (A) or three (B) replicates SE. Within the same parameter, values flanked by the same letters are not significantly different for P ¼ 0.05 following one-way ANOVA test. The symbol ( ) in regression equations means significance at 0.05 probability level.

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expected. For the 42 mg Cd kg1 treatment, this decrease reached 46% when compared to the control plants. At the same time, a tendency to increase shoot biomass was detected, but the total plant biomass showed no significant changes. These results are not surprising, since clear toxicity symptoms were absent, e.g., leaf chlorosis, necrosis of leaf tips, etc., in plants of 14 and 28 mg Cd kg1 treatments and slight present in the 42 mg Cd kg1 treatment. It should be mentioned that 14 and 28 mg Cd kg1 sand were not lethal concentrations for barley plants, which continued to grow for at least a month after the end of the study period (data not shown). In control plants, the Cd concentration in root and shoot tissue was similar and quite low (less than 1 mg Cd kg1 DW). However, in Cdexposed plants that changed (Fig. 1B), since Cd association (accumulation þ absorption) with roots was clearly stronger (ca. 10-fold higher) than in shoots. The association with root and the accumulation in shoot tissues followed linear patterns in relation to the external metal concentration, reaching values as high as 705 72 mg Cd kg1 in the roots and 71 10 mg Cd kg1 in the shoots, in the 42 mg Cd kg1 treatment.

Photosynthetic Performance The photosynthetic performance of Cd-exposed barley plants was evaluated using several techniques. Leaf gas exchange and chlorophyll fluorescence measurements, at the end of the experimental period, showed some effects in Amax, A, and gs (Table 1). Cd treatment decreased A (significantly only at 28 and 42 mg Cd kg1 treatments) to ca. half of that of the control. On gs and E the effect of Cd was more gradual and not so strong, with a significant decrease (ca. 30%) for gs only in the 42 mg Cd kg1 treatment. Small, nonsignificant changes were observed in ci in response to Cd treatments. Amax was significantly decreased (ca. 35%) only in the highest Cd treatment. Chlorophyll fluorescence response showed only slight changes in Cdexposed barley plants (Table 2). Despite some tendency to change, only Fo, qP, and e showed significant reductions, and even so only in the highest Cd treatment. On the other hand, the activity of thylakoidal electron transport (Fig. 2) was more affected at lower levels of Cd than the previous parameters. Significant decreases were obtained already at 28 mg Cd kg1 for electron transport involving PSII and PSI. Nevertheless, the PSII seems to be affected at lower Cd concentrations than PSI. After 10 days’ exposure to 28 mg Cd kg1, the decrease in PSII

23.05 a 20.41 a 21.60 a 14.93 b

(100) (89) (94) (65)

Amax (mmol O2 m2 s1) 13.97 13.17 8.40 7.41

a (100) a (94) b (60) b (53)

A (mmol CO2 m2 s1) 292 259 232 203

a (100) ab (89) ab (80) b (70)

gs (mmol H2O m2 s1) 5.88 5.17 4.53 4.65

a a a a

(100) ( 88) ( 77) ( 79)

E (mmol H2O m2 s1)

290 279 312 274

a a a a

(100) (96) (108) (95)

ci (mmol mol1)

Mean values are the average of six replicates. Within the same column, values flanked by the same letters are not significantly different for P ¼ 0.05 following one-way ANOVA test. Data in parenthesis are expressed as percent of control value.

Control 14 mg Cd 28 mg Cd 42 mg Cd

Treatment

Table 1. Photosynthetic capacity (Amax), photosynthetic rate (A), stomatal conductance (gs), transpiration rate (E), and intercellular CO2 concentration (ci) of leaves of barley plants grown in Cd-contaminated sand.

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Table 2. Selected chlorophyll fluorescence parameters and quenching analysis coefficients in leaves of barley plants grown in Cd-contaminated sand. Treatment

Fo


43 a 42 a 46 b 38 c

Fv/Fm

Fv0 =Fm0

0.81 0.81 0.79 0.79

0.53 0.51 0.51 0.51

a a a a

a a a a

qP 0.536 0.466 0.480 0.433

qNP a a a b

0.695 0.795 0.776 0.760

e a a a a

0.285 0.237 0.274 0.214

a a a b

Mean values are the average of six replicates. Within the same column, values flanked by the same letters are not significantly different for P ¼ 0.05 following one-way ANOVA test.

Figure 2. Rates of photosynthetic electron transport in thylakoid membranes isolated from leaves of barley plants grown in Cd-contaminated sand. Electron transport rates were measured between: (1) H2O and 2,6-dichlorophenolindophenol (DCPIP) (PSII þ OEC, oxygen evolving complex), (2) 1,5-diphenylcarbohydrazide (DPC) and DCPIP (PSII-OEC), and (3) reduced DCPIPH2 and methyl viologen (MV) (PSI). Control values (representing 100%) were 84.9, 86.0, and 496.1 mmol O2 mg chl1 h1 for PSII þ OEC, PSII-OEC, and PSI, respectively. Mean values are the average of three replicates SE. Within the same parameter, values flanked by the same letters are not significantly different for P ¼ 0.05 following one-way ANOVA test.

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16 Chl.a

a Pig ment con t ent ( m g g- 1 DW)

14

Chl.b

a

Total Car.

a

12 10 8 6

b a

a a

4

a a

a

b

2

b

0 0

14 28 Ex ternal Cd concentrations (mg kg-1 sand)

42

Figure 3. Content of photosynthetic pigments in barley plants grown in Cdcontaminated sand. Mean values are the average of the three replicates SE. Within the same parameter, values flanked by the same letters are not significantly different for P ¼ 0.05 following the one-way ANOVA test.

activity with OEC represented about 40% and without OEC 19%, whereas in PSI activity decreased ca. 30%. The similar inhibition of PSII activities with or without OEC at 42 mg Cd kg1 treatment shows that Cd can interact with both the donor and the acceptor side of this photosystem. In this treatment the PSI activity decreased to ca. 67% of its initial value. The photosynthetic pigments also showed a tendency to decrease, which became significant only in the 42 mg Cd kg1 treatment (Fig. 3). For this treatment chl a, chl b and total carotenoids decreased to 49, 51, and 57% of the control values, respectively. No changes were detected in chl (a/b) and (total chl/total carotenoids) ratios (data not shown).

Lipid Peroxidation Status As an indicator of leaf lipid peroxidation status the content of thiobarbituric acid reactive compounds (mostly MDA) was measured. It was evident that these compounds did not vary significantly in the leaves

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until the 28 mg Cd kg1 treatment but increased significantly at 42 mg Cd kg1 (Table 3). Since ethylene production associated thylakoid membrane degradation and mediated by oxy radicals and H2O2 is a final product of acyl lipid peroxidation,[39] it can be used as an important indicator of the thylakoid peroxidation status. The results obtained did not show any significant variation in ethylene production with Cd treatment and it was significantly lower than the control (Table 3).

DISCUSSION The inhibition of dry mass accumulation in plants suffering heavy metal stress is an effect widely observed in phytotoxicity studies. In particular, it has also been shown that Cd inhibits dry mass accumulation in barley plants.[26,40] Root growth seems to be more sensitive to Cd than shoot growth, due to its greater Cd accumulation,[41,42] but in some cases the opposite effect may occur.[16] In the present study, dry mass accumulation of barley plants (cv. Ribeka) exposed to Cd, applied 20 days after emergence, when the young plants are well-developed with functioning roots and photosynthetic apparatus, have been analyzed. The high root Cd accumulation, when compared to that of shoots (Fig. 1A), would have altered cell physiology, disturbing membrane permeability, and respiration as was observed by Llamas et al.,[43] and could have also affected the meristem functioning, as well as retarding mitotic index.[41] These lead to the observed decrease in root dry mass, even in the 14 mg Cd kg1 treatment. Due to these differential effects on root and shoot growth, plant sink-source interactions would be changed, with consequences for the pattern of dry mass allocation in plant organs. That seems to be what happens in our study, since Cd induced changes in dry mass allocation in organs of barley plants without affecting biomass accumulation at the whole plant level. Maximal shoot Cd concentration without visual toxicity symptoms reached 41 8 mg Cd kg1 DW, at 28 mg Cd kg1 treatment (Fig. 1B), while in roots more than a 10-fold increase was observed. It is noteworthy that, in the barley cv. CE9704 at similar external Cd treatment we observed higher Cd values in the shoot tissues, accompanied by the expression of toxicity symptoms.[20] In general, Cd accumulation and distribution in barley plants followed a similar pattern to that reported in previous studies,[26,40] as well as observed in other gramineae species.[44,45] All these plants showed a Cd accumulation in shoots several fold lower than that of some

1520 a 1533 a 1227 b 1200 b

Treatment


150 125 114 77

a (100) ab (83) b (76) c (51)

Fe content in shoots (mg kg1 DW) 6.71 a 8.13 a 8.03 a 11.20 b

(100) (121) (120) (167)

Lipid peroxidation status of leaves (MDA, mmol g1 FW)

58.3 44.5 50.5 48.4

a a a a

(100) (76) (87) (83)

Lipid peroxidation status of chloroplasts (mg C2H4 mg1 Chl h1)

Mean values are the average of three replicates. Within the same column, values flanked by the same letters are not significantly different for P ¼ 0.05 following one-way ANOVA test. Data in parenthesis are expressed as percent of control value.

(100) (101) (81) (79)

Mg content in shoots (mg kg1 DW)

Table 3. Mg and Fe content in shoots and lipid peroxidation status of leaves and chloroplasts of barley plants grown in Cd-contaminated sand.

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Brassica sp. proposed for Cd phytoextraction.[10] For example, Brassica juncea was found to be able to accumulate more than 400 mg Cd kg1 DW in shoots.[30] On the other hand, it is well known that dicotyledons such as Brassica sp., are more sensitive to Cd than barley and other cereals, which are considered to be semi-resistant.[46] The decline in A of Cd-exposed barley plants could be due both to stomatal and nonstomatal factors. For the plants at 28 mg Cd kg1 treatment a significant decrease in A was detected, which could result from stomatal limitation, since gs decreased and Amax remained unchanged (Table 1). For exposure to higher Cd levels (42 mg Cd kg1), mesophyll impairment must have occurred, since the decrease in A was accompanied by a significant Amax drop. The observation that ci did not show significant changes, even when gs decreases and Amax is not affected (e.g., 28 mg Cd kg1), suggests that some mesophyll factor could be involved in a down-regulation process. These effects of Cd levels on leaf gas exchange differ from what was found for the other barley cv. CE9704 involved in our experimentation,[20] but confirm our earlier data collected from barley plants (cvs. Obzor and Hemus) grown from seed to seed in soil contaminated by 45 mg Cd kg1.[9] Thus, the results obtained reflect an important genetic variation between cultivars of the same species. Among the observed mesophyll effects, the impact on the photosynthetic pigments was especially evident in the 42 mg Cd kg1 treatment, when both chlorophyll and carotenoids were significantly affected (Fig. 3). That could result from mineral deficiency (e.g., Fe) induced by high Cd levels[22] or from some Cd-induced Mg substitution in the chlorophyll molecule.[23] In fact, significantly lower levels of Fe and Mg were found in the shoots of plants growing at 28 and 42 mg Cd kg1 (Table 3), despite the fact that the Fe contents were above the deficiency threshold value of 25 mg Fe kg1 DW reported for barley.[47] Those reduced contents of the photosynthetic pigments might have had some impact on the efficiency of light capture in the antennae, which agrees with the significant decrease in Fo found in the 42 mg Cd kg1 treatment (Table 2), but did not reduce the in vivo PSII photochemical efficiency (given by Fv/Fm and Fv0 =Fm0 values). Concomitantly, mesophyll effects were also detected in photosynthetic electron transport, through the changes on qP and e (Table 2) and in the in vitro thylakoid electron transport rates involving the PSI and II (Fig. 2). In the latter measurements, PSII and PSI electron transport activities were obtained without substrate limitations, so the observed potential rates indicated decreasing rate of ATP synthesis and NADP reduction in Cd-exposed barley plants at the two treatments with higher Cd concentration (Fig. 2). In a situation where no serious thylakoid

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damage could be supposed [as indicated by no increase in ethylene production (Table 3)], one possible reason enabling decrease in photochemical activities could be Cd-induced alterations in chlorophyll molecule integration into pigment-protein complexes.[48] The lower e observed in plants at 42 mg Cd kg1 treatment, clearly indicated lower efficiency of light utilization (Table 2). Obviously, it was due to a decrease in qP as Fv0 =Fm0 , a measure of PSII photochemical efficiency under steady-state light conditions,[29] was not depressed considerably. The smaller fraction of the open PSII reaction centers (qP) together with a qNP tendency to increase supports the opinion of Krupa et al.[29] that Cdinduced alterations in primary carbon metabolism may lead to downregulation of PSII activity due to reduced demand for ATP and NADPH. That also agrees with previous studies, where a lower capacity for 14C photoassimilation in Cd-treated barley plants was found,[49] and that ATP and ADP pools in leaves of Cd-treated bean plants increased.[50] Subsequently, we considered the decrease of qP as a mechanism for avoiding over-reduction of the primary electron acceptor of PSII QA. The general opinion that PSI is less sensitive to the action of Cd was only partially supported by our results. In fact, the electron transport at the PSI level was affected only by the 28 mg Cd kg1 treatment, while the electron transport involving PSII showed some impact already in the 14 mg Cd kg1 treatment. The observed results involving the in vitro PSII and PSI activities in Cd-exposed plants are in a good correspondence with those presented by Chugh and Sawhney[28] in pea seedlings and are also in line with many reports with different plant species suffering Cd stress.[51,27] To further study the mesophyll effects, lipid peroxidation was examined at both leaf and thylakoid levels in order to find if Cd induces oxygen stress as well as if this may be linked to the observed results by in vitro techniques. Lipid peroxidation at leaf level was detected through the measurement of TBA-reactive compounds content, but became significant only in the 42 mg Cd kg1 treatment (Table 3). On the other hand, we did not find any changes in lipid peroxidation status at thylakoid level, as shown by ethylene production associated with chloroplast membranes. So it seems plausible to exclude the oxidative damage as responsible for the decrease in the photosynthetic pigment contents (mostly at the 42 mg Cd kg1 treatment), contrary to what was suggested by Somashekaraiah et al.[24] under Cd-toxicity conditions. In fact, it was shown that Cd accumulation at chloroplast level is much lower than that of whole leaf tissue.[26,27] The results obtained about the lipid peroxidation status of the thylakoids are consistent with the data of Baryla et al.[19] in oilseed rape plants suffering Cd toxicity, but differ

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from the response of another barley cultivar (cv. CE9704) involved in our studies. We have established diminished total fatty acids content and increased ethylene production in the thylakoids of plants from cv. CE9704 and characterized it as more sensitive to Cd than cv. Ribeka (Vassilev et al., unpublished data). In conclusion, the results obtained in this study show a good tolerance of the photosynthesis machinery of barley plants (cv. Ribeka), which is able to withstand shoot Cd concentrations up to ca. 41 mg Cd kg1 DW, but not higher concentrations. At the whole plant level, dry mass accumulation of barley plants was not significantly affected even at higher shoot Cd concentrations (ca. 70 mg Cd kg1 DW) but a toxicity symptoms like leaf chlorosis and necrosis of leaf tips became visible, accompanying some physiological disturbances. However, in spite of the good ability of this barley cultivar to tolerate Cd, the level of Cd accumulation in its shoots would not be sufficient for short-term phytoextraction. With a shoot Cd concentration of about 40 mg kg1 and maximum straw yield of about 5 t ha1,[8] barley could remove a maximum of 200 g Cd ha1 yr1, which is 5 to 10 times lower than was proposed for willow[6] or pennycress.[52] As usual, Cd content in seeds is many times less than in shoots,[9] so their contribution to Cd removal would be negligible. ABBREVIATIONS A Amax Chl ci DCPIP DCPIPH2 DPC E Fm Fo Fv/Fm and Fv0 =Fm0 gs MDA

Net photosynthetic rate at ambient CO2 Photosynthetic capacity at CO2 and light saturating conditions Chlorophyll Internal calculated CO2 concentration 2,6-Dichlorophenolindo-phenol Reduced 2,6-dichlorophenolindo-phenol 1,5-Diphenyl-carbohydrazide Transpiration rate Maximal fluorescence in dark adapted leaves Minimal chlorophyll fluorescence of antennae in dark adapted leaves Photochemical efficiency of PSII in dark adapted leaves and under photosynthetic steady-state conditions, respectively Stomatal conductance to water vapor Malondialdehyde

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OEC PPFD PSII and PSI qNP qP TBA e

791

Oxygen evolving complex Photosynthetic photon flux density Photosystem II and photosystem I Nonphotochemical quenching Photochemical quenching 2-Thiobarbituric acid Estimation of the quantum yield of photosynthetic noncyclic electron transport

ACKNOWLEDGMENTS The authors thank Tech. Eng. Carlos S. Carvalho and Ana P. Ramos for technical assistance, Engs. Jose´ M. Semedo and Nuno C. Marques (all from EAN, Oeiras, Portugal) for help with gas-exchange measurements as well as the Dept. Plant Physiology in EAN for the working facilities provided. The authors also thank Prof. J. Vangronsveld (Limburgs University Centrum, Diepenbeek, Belgium) and Dr. P. Kettlewell (Harper Adams Agricultural College, England) for the critical reading of the manuscript and the valuable corrections. The European Scientific Foundation (GPoll Programme) is gratefully acknowledged for providing a grant to A. Vassilev.

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