Exogenous treatment with salicylic acid attenuates cadmium toxicity in pea seedlings

Plant Physiology and Biochemistry 47 (2009) 224–231

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Research article

Exogenous treatment with salicylic acid attenuates cadmium toxicity in pea seedlings Losanka P. Popova a, *, Liliana T. Maslenkova a, Rusina Y. Yordanova a, Albena P. Ivanova a, Aleksander P. Krantev a, Gabriella Szalai b, Tibor Janda b a b

Acad. M. Popov Institute of Plant Physiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Street, Bl. 21, Sofia 1113, Bulgaria Agricultural Research Institute at the Hungarian Academy of Sciences, H-2462 Martonvasar, POP 19, Hungary

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 October 2008 Accepted 9 November 2008 Available online 18 November 2008

The present study investigated the possible mediatory role of salicylic acid (SA) in protecting plants from cadmium (Cd) toxicity. The exposure of pea plants to increasing Cd concentrations (0.5, 1.0, 2.0 and 5.0 mM) during early stages of their establishment, caused a gradual decrease in shoot and root fresh weight accumulation, the rate of CO2 fixation and the activity of ribulose-1,5-bisphosphate carboxylase (RuBPC, E.C. 4.1.1.39), the effect being most expressed at higher Cd concentrations. In vivo the excess of Cd-induced alterations in the redox cycling of oxygen-evolving centers and the assimilatory capacity of the pea leaves as revealed by changes in thermoluminescence emission after flash illumination. The levels of some important parameters associated with oxidative stress, namely lipid peroxidation, electrolyte leakage and proline production were increased. Seed pretreatment with SA alleviated the negative effect of Cd on growth, photosynthesis, carboxylation reactions, thermoluminescence characteristics and chlorophyll content, and led to decrease in oxidative injuries caused by Cd. The data suggest that the beneficial effect of SA during an earlier growth period could be related to avoidance of cumulative damage upon exposure to cadmium thus reducing the negative consequences of oxidative stress caused by heavy metal toxicity. In addition, the observed high endogenous levels of SA after treatment with Cd suggests that SA may act directly as an antioxidant to scavenge the reactive oxygen species and/ or indirectly modulate redox balance through activation of antioxidant responses. Taken together these evidences could explain at some extend the protective role of SA on photochemical activity of chloroplast membranes and photosynthetic carboxylation reactions in Cd-stressed pea plants. Ó 2008 Elsevier Masson SAS. All rights reserved.

Key words: Antioxidants Cadmium Fatty acids Photosynthesis Pisum sativum L. Salicylic acid Thermoluminescence

1. Introduction Cadmium (Cd) is a highly toxic trace element which enters the environment mainly from industrial processes and phosphate fertilizers. Cadmium is easily taken up by plant roots and can be loaded into the xylem for its transport into leaves. A large number of studies have demonstrated the toxic effect of Cd on plant metabolism, such as decrease uptake of nutrient elements [45], changes in nitrogen metabolism [8], inhibition of photosynthesis through effects on the chlorophyll metabolism and chloroplasts structure [19], the activity of both photosystem II and the enzymes of photosynthetic carbon

Abbreviations: A, net CO2 assimilation rate; DTT, dithiothreitol; FA, fatty acids; FAME, fatty acids methyl esters; Gs, stomatal conductance; MDA, malondialdehyde; o-ANI, o-anisic acid; o-HCA, o-hydroxy-cinnamic acid (o-coumaric acid); p-HBA, para-hydroxy-benzoic acid; PSII, photosystem II; RuBPC, ribulose-1,5-bisphosphate carboxylase; SA, salicylic acid; TCA, trichloroacetic acid; TL, thermoluminescence; WUE, water use efficiency. * Corresponding author. Tel.: þ359 (2) 979 26 06; fax: þ359 (2) 873 99 52. E-mail address: [email protected] (L.P. Popova). 0981-9428/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2008.11.007

metabolism [3,27,48]. The toxicity of Cd has been related with the increase of lipid peroxidation and alterations in the antioxidant system [43,45]. Cadmium also produces alterations in the functionality of membranes by inducing changes in their lipid composition [23] and this can affect some enzymatic activities associated with membranes such as Hþ-ATPase [17]. Different plant species and varieties show a wide range of plasticity in Cd tolerance, reaching from the high degree of sensitivity to the hyper accumulating phenotype of some tolerant higher plants. Legume plants are less tolerant to Cd toxicity than cereals and grasses [31]. A first barrier against Cd stress is retention of Cd in the cell wall [36]. Once Cd enters the cytosol other detoxification mechanisms are induced, primarily the formation of complexes between Cd and phytochelatins (PCs) and their subsequent compartmentalization [12,52]. Other mechanisms that plants have developed to cope with damages caused by cadmium are related with some stress signaling molecules, such as salicylic acid, jasmonic acid and ethylene. All these compounds were induced by Cd treatment, which suggest that they are involved in cell response to Cd toxicity [42].

L.P. Popova et al. / Plant Physiology and Biochemistry 47 (2009) 224–231

Salicylic acid (SA) as a potent signaling molecule in plants is involved in eliciting specific responses to biotic and abiotic stresses. It has been shown that SA provides protection in maize [24] and winter wheat plants [50] against low-temperature stress, induces termotolerence in mustard seedlings [10,13] or modulates plant responses to salt and osmotic stresses [7], ozone or UV light [47], drought [46] and herbicides [1]. Furthermore, SA is also known to be involved in plant protection to heavy metals. SA pretreatment alleviates Pb- and Hg-induced membrane damage in rice [35] and Cd toxicity in barley [32], maize [39] and soybean plants [15]. Although SA has a beneficial effect against different abiotic or biotic stresses, the compound itself stressed the plants. Pancheva et al. [40] showed that long-term treatment (7 days) of barley seedlings with 500 mM and 1 mM SA reduced root and seedlings growth, chlorophyll and protein contents and the rate of CO2 assimilation. At the same experimental conditions Maslenkova and Toncheva [30] observed the inhibitory effect of SA on PSII oxygen-evolving reactions. The effect of SA depended on the time of treatment duration - no changes in these parameters were observed when barley seedlings were treated with SA for 2 h, the inhibition appeared 6 h after the start of treatment. These data confirm the suggestion that SA plays different roles based on its endogenous levels in particular plant species under specific developmental and environmental conditions [40]. Although there have been many reports on the photochemical and biochemical events occurring in photosynthesis during Cd toxicity, a lot of contradictory data can be found in the literature. Probably this is due to the very heterogeneous experimental approaches, including both laboratory grown conditions and field experiments. Only limited number of studies has been carried out during the germinating stage of plants. We have attempted to study the effect of exposure of pea plants to Cd during early stages of their establishment, on the physiological and biochemical properties of pea leaves. The study reported in this paper is mainly focused on the mechanisms by which SA influences pea plants tolerance to Cd toxicity. 2. Results 2.1. Growth response to Cd toxicity Exposure to high Cd concentrations resulted in dramatic decreases in both shoot and root fresh weights (34.9 and 22.8% at 2 mM Cd and 21.4 and 13.4% at 5 mM Cd, respectively) (Table 1). Chlorophyll content showed approximately 35 and 63% reduction in 2 and 5 mM Cd-treated plants, respectively. Pretreatment with SA before exposure to Cd however showed 22.7 and 33% restoration of the chlorophyll levels. Taken together these measurements indicate that Cd concentrations above 0.5 mM impose a significant level of stress on pea plants in accordance with the increased Cd content in root tissue (Table 1). SA pretreatment reduced root accumulation of Cd in all Cd-treated variants.

225

2.2. Effect of cadmium on the free and bound o-HCA and SA contents in leaves In the present work, it was found that control leaves initially contained little o-HCA or SA in free form compared to the bound forms. Cd treatment did not affect their content. Cd (1 and 2 mM) increased the level of bound o-HCA and SA, the effect was higher expressed in 2 mM Cd-treated plants (approximately 3-fold increase, compared with the controls). The total content of the free and bound o-HCA and SA was significantly high in all Cd-treated variants (Table 2). 2.3. Gas-exchange parameters and RuBPC activity The growth inhibition of pea plants by Cd treatment was accompanied by a decrease in the rate of photosynthesis (A). The most marked reduction was observed after treatment of pea plants with 2 mM Cd, an almost 2-fold decrease as compared with the control. SA pretreatment for 6 h before exposure to Cd recovered at high extends the rate of A (Fig. 1A). The water use efficiency was also affected by cadmium treatment, undergoing a significant reduction in Cd-treated variants. Pretreatment with SA before exposure to cadmium alleviated at high extends the inhibitory effect of Cd on the values of WUE (Fig. 1B). The effects of Cd and SA on stomatal conductance followed the same tendency (Fig. 1C). Treatment of pea plants with 1 and 2 mM Cd caused a decline in the activity of RuBPC approximately by 40%. A very strong protective effect of SA was observed on RuBPC activity (Fig. 1D). 2.4. Thermoluminescence emission in Cd-stressed and SApretreated pea leaves The illumination of unfrozen dark adapted leaves from nontreated (control) pea plants with one short saturating flash induced a main TL peak at 35  C, the so-called B-band (S2Q B ) and a hardly distinguishable shoulder at temperature about 40  C (Fig. 2A). Two consecutive flashes (2Fl) generate a maximal TL  emission with B-band (S2(3)Q B ) peaking at 33 C. The amplitude of B-band decreases after three flashes (3Fl) but the shoulder at 40  C designated as afterglow (AG) band become better pronounced (Fig. 2A). The B-band results from the thermal activated recombination of the trapped electrons and positive charges on the reduced quinone acceptor (Q B ) and the S2(S3) oxidation state of the water-oxidizing complex of PSII, respectively [44]. The AG TL emission corresponds to a back electron transfer towards PSII centers initially in the S2(3)QB state [16]. A damping of period-four oscillation of B-band intensity, according to exciting flash number was observed at high concentration of heavy metal (Fig. 2C). The AG/B ratio, estimated from TL glow curve after deconvolution show a decrease from the value of 0.8 in control to 0.55 in Cdstressed plants (Fig. 2D). This tendency was much better expressed in the case of TL induction by short (30 s) FR illumination of dark adapted pea leaves (Fig. 2B). This kind of

Table 1 Effects of Cd and SA on some physiological parameters of pea plants and data on root Cd accumulation. Dry seeds were soaked in 500 mM SA or water for 6 h, and were germinated on moist filter paper for three days. Then were transferred to hydroponic medium and were grown for 14 days without or with Cd in the medium. The data of shoot and root fresh weight and chlorophyll content are means  s.e. (n ¼ 5) and for Cd content  s.e. (n ¼ 3). Conc. (mM Cd)

0 0.5 1.0 2.0 5.0

Chlorophyll (mg/g FW)

Cd content (mg/kg DW)

SA

Shoot FW (g/plant) þSA

Root FW (g/plant) SA

þSA

SA

þSA

SA

þSA

0.51  0.04 0.45  0.04 0.47  0.02 0.18  0.03 0.11  0.04

0.54  0.04 0.55  0.01 0.48  0.05 0.22  0.05 0.17  0.07

0.22  0.05 0.21  0.03 0.16  0.04 0.05  0.01 0.03  0.00

0.21  0.07 0.21  0.04 0.19  0.04 0.08  0.01 0.03  0.01

3.05  0.3 3.07  0.3 2.49  0.2 2.25  0.2 1.13  0.4

2.76  0.1 3.04  0.3 2.80  0.2 2.76  0.2 1.49  0.4

23.59 108.38 190.85 480.82 n.d.

11.05 86.98 74.69 130.17 n.d

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Table 2 Effects of Cd and SA on the accumulation of free and bound o-HCA and SA in pea leaves. The details of treatment and growth are as in Table 1. Each value corresponds to a typical experiment among at least tree replicates. Conc. (mM Cd)

0 0.5 1.0 2.0

Free o-HCA (ng/g FW)

Bound o-HCA (ng/g FW)

Free SA (ng/g FW)

SA

þSA

SA

þSA

SA

þSA

SA

þSA

31.5  2.9 15.7  1.7 33.7  2.2 41.4  2.1

32.6  2.1 21.6  1.9 27.2  2.5 61.7  3.7

131.1  5.8 64.7  3.9 149.2  6.9 332.5  9.4

92.4  8.9 96.6  7.9 72.3  3.0 59.4  1.5

73.2  2.1 71.9  2.4 68.1  1.9 51.5  1.3

90.9  3.1 66.2  2.9 41.8  1.9 46.7  2.4

478.8  13 437.8  11 1151.0  25 1235.2  11

375.0  9 443.1  7 464.1  10 649.7  10

illumination excites mainly PSI but a part of the energy can be absorbed in PSII antenna leading to charge separation and the induction of TL B-band. TL glow curve from control plants show an intense AG emission at 45  C and B-band peaking at lower temperature as compared to the peak position after flash illumination most probably as a consequence of lumen acidification and destabilization of S2 and S3 states of oxygen-evolving complex [34]. Fig. 2B demonstrates that high Cd concentrations lead to a substantial decrease in AG emission without changes in peak temperature maximum. In the same time the B-band looks broadened and shifted to higher temperature. Short term application of 500 mM SA to the pea seeds do not exerted changes in the investigated TL parameters. However, pretreatment with SA before the imposition of high concentration of heavy metal have a stabilizing effect on photochemical reactions, judging from some restoration of AG/B ratio and B-band oscillation pattern (Fig. 2C and D).

16

[µmol CO2 m-2s-1]

A

CO2 assimilation (A)

- SA + SA

2.5. Effect of SA on hydrogen peroxides level, electrolyte leakage, lipid peroxidation and proline level No major changes were observed in hydrogen peroxides level in Cd-treated plants or pretreated with SA before exposure to Cd (Table 3). Because Cd is known to induce oxidative stress we investigated the damage to membranes by monitoring MDA content and electrolyte leakage. Relative to control, Cd-treated pea plants exhibited a higher rate of lipid peroxidation. SA alone caused a very small increase in MDA level (about 15%). Presoaking of seeds with SA before application of Cd decreased the level of MDA, the effect was more pronounced in higher applied Cd concentrations. Electrolyte leakage was also affected by Cd (the increase was approximately 45–70% compared to the control). Pretreatment with SA alleviated the effect of Cd on the values of this parameter.

B

12

Water use efficiency [WUE]

12

10

10

8

8

6

6 4 4 2

2 0

C

D

Stomatal conductance (gs)

0 180

RuBPC

140

0,04

120 0,03

100 80

0,02

60 40

0,01

of the control]

[mmol CO2 m-2 s-1]

160

enzyme activity [

0,05

[µmol CO2m-2s-1/mmol H2O m-2s-1]

14

Bound SA (ng/g FW)

20 0,00

0 control

0.5 µM Cd

1.0 µM Cd

2.0 µM Cd

control

0.5 µM Cd

1.0 µM Cd

2.0 µM Cd

Fig. 1. Changes in gas-exchange parameters and RuBPC activity in pea plants treated with Cd and SA, A, net CO2 assimilation rate; B, water use efficiency, WUE; C, stomatal conductance, gs; D, ribulose-1,5,-bisphosphate carboxylase, RuBPC. In controls the activity of RuBPC was 0.66 mmol CO2/g FW min1. Variants and treatment as described in Table 1. The values are means  s.e. (n ¼ 3).

L.P. Popova et al. / Plant Physiology and Biochemistry 47 (2009) 224–231

12000

A

227

B

8000

Control 2µM Cd 5µM Cd

10000

TL intensity (arb. u.)

TL intensity (arb. u.)

7000 2 Fl 8000 B

AG

6000 3 Fl 4000 1 Fl 2000

6000 5000 4000 3000 2000 1000

0

0 10

20

30

40

50

60

70

0

10

20

Temperature (°°C) 1,2

1,0

C

Control 2µM Cd 5µM Cd 2µM Cd + SA 5µM Cd + SA

0,8

40

50

60

70

D

- SA + SA

0,8

AG/ B

TL B - band

1,0

30

Temperature (°°C)

0,6

0,6

0,4

0,4 0,2

0,2 0,0

0,0 1

2

3

4

5

6

OµM Cd

2µM Cd

5µM Cd

Flash number Fig. 2. Thermoluminescense glow curves in pea leaves induced by 1–3 flashes (A). The components representing B- and AG-band after TL curve decomposition are shown in dots; changes in TL B-band oscillation pattern (C) and in the ratio of AG to B-band integrated intensities (D) in leaves of Pisum sativum plants treated with 2 and 5 mM Cd and after pretreatment of the seeds with 500 mM SA; effect of Cd on TL curves recorded after 30 s far-red radiation (B). TL was recorded immediately after corresponding excitation, with a 0.5  C s1 heating rate.

Concentrations of the stress metabolite proline increased upon Cd exposure. The most prominent effect was at 2 mM Cd, a nearly 3-fold rise as compared with the control. SA pretreatment counteracted the Cd-induced increase in proline levels. 2.6. Effect of Cd and SA on the amount of total lypophilic extracts, FAME, and on the fatty acid/FA/composition The low concentration of Cd (0.5 mM) caused a slight increase of the total biomass, but the amount of the total lipophilic extracts and the FAME decreased. Cd (1 mM) leads to decrease of the amount of the biomass, as well of the lipid content. Surprisingly, the highest concentration we used in our experiments (2 mM Cd) increased the lipid content about two-fold in comparison with the control. SA alone increased the content of FAME (respectively the lipid content). Pretreatment with SA before exposure to Cd alleviated the loss of dry mass due to Cd supply and also increased the amount of lipids in 0.5 and 1 mM Cd-treated plants (Table 4). The data on the effect of Cd and SA on the composition of the fatty acids are presented in Table 5. Cd treatment leads to increase

of the short-chain (14:0 þ 14:1) fatty acids, as well of the sum of long-chain (20:0, 22:0 and 24:0) fatty acids. The amount of 18:3 acid decreased, but the other FA remain unchanged. More significant changes in 16:0, 16:1, 18:0 and 18:3 were observed in 0.5 mM Cd-treated plants. Treatment with SA alone had no significant effect on FA composition of pea leaves with the exception of an increase of 14:1 and 22:0 FA. As it was mentioned above, Cd leads to increase in the amounts of short-chain and long-chain FA in pea plants. Contrary, in pretreated SA plants this tendency is not expressed and the amount of these acids is closer to the control plants. At 2 mM Cd-treated plants pretreatment with SA leads to amounts of 18:2 and 18:3 acids closer to control plants. 3. Discussion This study was undertaken to identify the possible mechanisms and the influence of SA in enhancing pea plants tolerance to Cd toxicity.

Table 3 Effects of Cd and SA on lipid peroxidation, electrolyte leakage, peroxides and proline content in pea leaves. Variants and treatment as described in Table 1. The values are means  s.e. (n ¼ 5). Conc. (mM Cd)

0 0.5 1.0 2.0

Peroxides (nmol/g FW)

Lipid peroxidation (nmol MDA/g FW)

Electrolyte leakage (ms/g FW)

Proline (mmol/g FW)

SA

þSA

SA

þSA

SA

þSA

SA

þSA

1.45  0.12 1.46  0.09 1.48  0.14 1.50  0.21

1.49  0.24 1.47  0.18 1.50  0.17 1.37  0.20

107.60  4.9 134.05  3.8 154.40  4.2 180.20  3.0

124.00  4.1 95.70  2.7 95.64  3.0 130.50  3.2

26.95  0.90 34.70  0.70 39.20  0.85 46.20  1.20

22.50  1.52 16.90  0.49 27.80  1.71 36.10  0.87

3.17  0.21 3.31  0.19 5.28  0.25 8.96  0.45

3.43  0.17 3.44  0.21 3.33  0.24 4.30  0.19

228

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Table 4 Effects of Cd and SA on the amount of biomass, total lipophilic extracts and FAME in pea leaves. The results are means  s.e. from three parallel experiments. Conc. (mM Cd)

Dry WT (g) SA

0 0.5 1.0 2.0

0.9  0.1 1.0  0.1 0.8  0.1 0.5  0.1

Total lipophilic extract (mg/gDW)

FAME (mg/g DW)

þSA

SA

þSA

SA

þSA

1.1  0.1 1.1  0.1 1.0  0.1 0.7  0.1

289  11 135  5 144  6 220  9

204  8 373  15 225  9 207  8

7.3  0.6 5.9  0.5 4.9  0.4 14.2  1.1

11.5  0.9 6.0  0.5 6.1  0.5 9.7  0.8

We show that Cd produced a concentration-dependant reduction in the growth of pea plants, measured as fresh weight of roots and shoots (Table 1). Under our experimental conditions, pea plants did not tolerate Cd concentrations higher than 5 mM without showing any visible toxicity symptoms. This confirms the data by other authors [5] that pea plants can be considered as cadmiumsensitive species. In general, it can be said that the sensitivity of given plant species to heavy metal toxicity depends on its concentration, the treatment duration on the plant species, its age, and the plant organ examined [33]. We explain our results with the fact that pea plants were exposed to Cd at the very early stage of their development. The growth inhibition of pea plants was accompanied by a significant decrease in net photosynthesis measured as CO2 assimilation which at the highest Cd concentration (2 mM) was reduced about two times (Fig. 1A). The water use efficiency was also affected by cadmium treatment, undergoing a progressive reduction with increasing Cd concentrations (Fig. 1B). The activity of RuBPCase (Fig. 1D) and chlorophyll content (Table 1) also decreased with rising Cd concentrations. In addition to the negative effects of Cd on the photosynthetic carboxylation reactions PSII electron transport and especially oxygen-evolving complex were found to be very sensitive to the effect of Cd [11]. Different components of electron transport chain were proposed as primary targets of Cd and different mechanisms of action were discussed. Donor side [11,28] or acceptor side [3] were implicated as main sites of the inhibition. In our attempt to contribute to the understanding the effects of cadmium on PSII reactions, an analysis of the TL glow curve parameters was done in Cd-stressed pea. The traces in Fig. 2C reveal that higher Cd concentrations affect the B-band oscillation pattern and decreases the TL intensity. These results show a reduction in the number of PSII reaction centers and an increase of misses in charge separation thus suggesting that the centers can not reach their higher oxidation states, S3 and S4. Whereas the B-TL band is specific for PSII charge recombination [44], the AG band is thought to originate from a feedback of reducing equivalents from the stroma towards PSII centers initially in TL inactive S2(S3)Qb state [16]. It has been proposed AG to reflect the [NADPH þ ATP] assimilatory potential in the chloroplasts when induced by flashes. The decrease of the assimilatory potential as evidenced by a lower AG/B ratio (Fig. 2D) can be due to the reduced electron transport. In the case of high concentration of heavy metal this can be a result of harmful effect of reactive oxygen species on the thylakoid membrane composition

and function. Geiken et al. [20] reported that in the presence of high concentrations of Cd PSII oxygen-evolving complex was altered and disassembly of the stacked regions in chloroplasts of stressed pea plants can be seen. Our data show that MDA content and the rate of electrolyte leakage in Cd-treated plants were observed to be greater than that in nutrient solution grown control (Table 3). This confirmed that Cd toxicity in sensitive pea plants was linked to free radical processes in membrane components leading to alterations in membrane stability and increasing their permeability. The analysis of the level of lipids and FA composition in Cdtreated plants support this observation. Cadmium at concentrations 0.5 and 1.0 mM caused a decrease in the amount of the total lipophilic extract and the FAME. The highest concentration of Cd we used (2.0 mM) led to increase of the lipid content (Table 4), most probably as a reaction to harmful effect of Cd. Similar data on the effect of Cd were reported for different plant species, like: wheat [29], barley [51], tomato [6] and mustard [37]. Fatty acids composition was also affected by Cd treatment (Table 5). The levels of the short-chain (14:0 þ 14:1), as well of the sum of long-chain (20:0, 22:0 and 24:0) FA were higher in treated variants, while the amount of 18:3 acid was lower. The increase of the longchain fatty acids in pea plants after Cd-stress could be an indication that some processes of elongation of fatty acid chains are activated. On the other hand the accumulation of 16:0 and 18:0 acid, especially by low Cd treatment (0.5 mM), could be an indication that there are some alterations in biosynthesis pathway between these two acids. In 2.0 mM Cd-treated pea plants the amount of the linoleic acid (18:2) increased, but the linolenic acid (18:3) decreased. This could be an indication that the desaturase activity by the transformation 18:2 < 18: 3 was affected. More significant changes in 16:0, 16:1, 18:0, 18:1 and 18:3 were observed by low Cd concentration. Similar changes in the FA-profiles are typical to plants exposed to environmental stress. The decrease of the unsaturated FA and the increase of saturated and monoenoic acids lead to decrease the fluidity of lipid membranes and their permeability. Here we found that Cd treatment caused accumulation of the total levels of SA and o-HCA, the effect was higher expressed on their conjugated forms (Table 2). Similar effect of cadmium on SA accumulation has been reported by Pal et al. [38] and Krantev et al. [27] for maize plants. Cd content of dry seeds and root tissue was low in the absence of Cd in the growth medium and strongly increased after treatment with Cd, being approximately 20 times higher in 2 mM Cd-treated plants (Table 1). Presoaking of pea seeds for 6 h with 500 mM SA before exposure to Cd had a beneficial effect on growth, photosynthesis, carboxylation reactions, thermoluminescence characteristics and chlorophyll content, and led to decrease in oxidative injuries caused by Cd. Although SA participates in the development of stress symptoms, it is also needed for the adaptation process and the induction of stress tolerance. Most abiotic stresses increased the plant concentration of SA, which points to its involvement in stress

Table 5 Effects of Cd and SA on the fatty acid composition in pea leaves. The values obtained are means  s.e. from three parallel measurements; the standard deviations (related to peak proportions on the chromatogram) are as follows:  0.3 for 16:0 and 18:3; 0.2 for 18:2 and 0.1 for the others. Conc. (mM Cd)

Fatty dcid (WT % of total) 14:0

0 0.5 1.0 2.0

14:1

16:0

16:1

18:0

18:1

18:2

18:3

20:0

22:0

24:0

SA

þSA

SA

þSA

SA

þSA

SA

þSA

SA

þSA

SA

þSA

SA

þSA

SA

þSA

SA

þSA

SA

þSA

SA

þSA

0.3 0.3 0.5 0.4

0.4 0.3 0.2 0.4

0.3 1.1 1.4 1.0

1.6 0.8 0.5 0.9

21.9 27.9 21.9 20.2

20.5 23.8 17.6 20.0

2.0 2.3 1.8 2.1

1.6 2.1 1.8 1.8

3.2 4.6 3.6 3.8

3.5 4.3 3.1 3.7

4.3 5.0 3.9 4.1

3.9 4.8 3.1 2.9

16.6 16.6 15.1 19.1

15.7 18.0 15.6 15.7

49.0 37.3 46.3 45.7

47.9 41.6 55.3 50.3

0.4 0.8 0.6 0.5

0.5 0.7 0.4 0.6

0.5 1.2 1.8 1.2

1.8 1.0 0.5 1.5

0.2 0.6 0.7 0.4

0.5 0.5 0.1 0.4

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signaling. SA is a direct scavenger of hydroxyl radical and an ironchelating compound, thereby inhibiting the direct impact of hydroxyl radicals as well as their generation via the Fentonreaction [14]. The observed high levels of SA after treatment with Cd may act directly as an antioxidant to scavenge the reactive oxygen species and/or indirectly modulate redox balance through activation of antioxidant responses. According to Guo et al. [21] exogenous SA induced Cd tolerance in rice by enhancing the antioxidant defence activities. It has been presented evidence that not only SA but also other related compounds, such as o-HCA, could provoke protection against abiotic stress [25]. On the basis of ability of o-HCA to quench singlet molecular oxygen, it was suggested that it may play a role in the antioxidative response [18]. Several hypothetical explanations may account for the positive effect of SA for attenuating Cd stress in pea plants. i. SA prevented cumulative damage development in response to Cd. The suggestion is supported by the data of the lowered root level of Cd in SApretreated pea plants (Table 2). Similar data have been reported by Szalai et al. [49] in maize. Cd is usually accumulated in the roots, because this is the first organ exposed to heavy metals in the soil, but it is also translocated into the shoots. Obviously the lowered root level of Cd in SA-pretreated pea plants reduced the harmful effect of Cd and exerts a beneficial effect on the growth and photosynthesis. ii. SA alleviated the oxidative damages caused by Cd. The values of MDA, electrolyte leakage and proline content of SA-pretreated plants were lower compared with the Cd exposed plants (Fig. 2). iii. Pretreatment with SA exerted a protective effect on the membrane stability judging by the increased total lipids level and by changes in their FA composition. Taken together these evidences could explain at some extent the protective role of SA on photochemical activity of chloroplast membranes and photosynthetic carboxylation reactions in Cdstressed pea plants. 4. Materials and methods 4.1. Plant growth and treatment with Cd Seeds of pea (Pisum sativum L., cv. Ran) were sterilized and divided into two groups. One half of the seeds was soaked in 500 mM SA solution for 6 h, the other half of the seeds was soaked in water (control), and then the both groups were allowed to germinate on moist filter paper in the dark. Three-day-old, dark grown seedlings, were placed in polyethylene pots filled with 0.6 L modified Hoagland solution (0.3125 mM KNO3, 0.45 mM Ca(NO3)2, 0.0625 mM KH2PO4, 0.125 mM MgSO4  7H2O, 0.125 mM MgSO4  7H2O, 11.92 mM HBO3, 4.57 mM MnCl2  4H2O, 0.191 mM ZnSO4  7H2O, 0.08 mM CuSO4  5H2O, 0.024 mM (NH4)6Mo7O24  4H2O,15.02 mM FeSO4  7H2O, 23.04 mM Na2EDTA  5H2O). The nutrient solution was aerated twice a day, and changed tree times in a week. CdCl2 was added at a concentration of 0.5, 1, 2 and 5 mM. Plants were grown in a growth chamber at a day/night cycle 16 h/8 h, at 22/ 18  C, respectively, relative humidity between 50% and 60%, and 120 mmol m2 s1 PAR. After 12 days of growth the plants were harvested for analysis. The growth of the shoot and root was analyzed in terms of their fresh weight. 4.2. Gas exchange The measurements were performed by a portable photosynthesis system Li-6400 (Li-Cor, Lincoln, USA). Leaves of 5–6 plants (second well-expanded leaf pairs) were placed in a 250 cm3 camber. Quantum flux density was 870 mmol m2 s1 PAR. Flow rate through the cuvette was 18–20 cm3 s1, boundary layer resistance (ra) was 0.8 s cm1. Leaf temperature was 26  2  C.

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4.3. Enzyme extraction and RuBPC measurement Leaf tissue was ground in a mortar on ice at a ratio of 1 g fresh mass to 5 ml cold extraction medium containing 0.33 M sorbitol, 0.05 M HEPES-NaOH, 2 mM KNO3, 2 mM EDTA, 1 mM MnCl2, 1 mM MgCl2, 0.5 mM K2HPO4, 0.02 M NaCl, and 0.2 M Na-isoascorbate, pH 7.6. The homogenate was quickly filtered through four layers of cheesecloth and centrifuged at 20,000  g for 15 min, and the supernatant used directly for enzyme assay. RuBPC (E.C. 4.1.1. 39) activity was assayed from the activated crude preparation by following the incorporation of NaH14CO3 into acid stable products [41]. The assay mixture for RuBPC contained in 50 mM HEPES-NaOH (pH 8.0): 20 mmol MgCl2, 1 mmol dithiothreitol (DTT), 20 mmol NaHCO3 (containing 1.48 MBq, specific radioactivity 0.38 MBq mmol1), and the enzyme extract equivalent to 0.3–0.4 mg protein. Reaction, at 25  1  C, was initiated by addition of 2 mmol RuBP and stopped after 1 min reaction time with 6 M HCl. Reaction volume was 1 ml. The amount of fixed 14CO2 was measured in a liquid scintillation spectrometer. 4.4. Thermoluminescence measurement TL glow curves of P. sativum leaf discs were measured using the apparatus and software described earlier [34]. Luminescence was detected by a Hamamatsu H-5701-50 photomultiplier linked to an amplifier. A 4  4 cm Peltier element was used for temperature control. Dark adapted leaf samples were cooled within a few seconds and irradiated via a fiber optic either by far-red radiation by a PAM 102-FR light source for 30 s or by a single turnover flash lamp (XST 103; Walz, Effertrich, Germany) at 1  C. The leaf sample was gently pressed against the plate by a rubber ring and a Pyrex window, with the addition of 100 ml water for better thermal conduction. The rate of heating during measurements was 0.5  C s1. The TL measurements were repeated several times and representative glow curves are presented in the figures. 4.5. Determination of hydrogen peroxides level, lipid peroxidation, electrolyte leakage and proline level Fresh material (about 0.150 g) was homogenized in 0.1% cold TCA. The homogenate was centrifuged at 15,000  g for 25 min. The supernatant obtained was used for the determination of both hydrogen peroxides and lipid peroxidation levels. The endogenous hydrogen peroxides was measured spectrophotometrically (l ¼ 390 nm) by a reaction with 1 M KI. The results were calculated using the standard curve prepared with fresh hydrogen peroxide solutions [26]. The level of lipid peroxidation was measured with the method of Heath and Packer [22] with slight modifications. A 0.2–0.3 g leaf material was homogenized in 3 ml 0.1% TCA and centrifuged at 15,000  g for 30 min at 4  C. To 0.5 ml aliquot of the supernatant 0.5 ml buffer and 1 ml reagent (0.5% thiobarbituric acid, TBA in 20% TCA, w/v) were added. For a blank- 0.5 ml 0.1% TCA þ 0.5 ml buffer and 1 ml reagent. The test-tubes were heated at 95  C for 30 min and then quickly cooled in an ice bath. After cooling and centrifugation to give a clear supernatant the absorbance of the supernatant at 532 nm was read and the value for the non-specific absorption at 600 nm was subtracted. The level of malondialdehyde (MDA) was estimated by using the mM extinction coefficient of 155 mM1 cm1. The degree of membrane integrity was also assessed by the leakage of electrolytes from the whole above ground part of three plants with approximately similar sizes. The leaf segments were strictly washed, blotted dry, weighted and put in stopped vials filled with the exact volume of bidistilled and deionized water. The vials were than incubated for 20 h in the dark with continuously shaking.

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The amount of electrolyte leakage was measured conductometrically. Proline concentration was determined spectrophotometrically at 520 nm after Bates et al. [4]. Leaf soluble protein content was determined by the method of Bradford [9], chlorophyll was extracted by acetone and measured spectrophotometrically according to Arnon [2].

equipped with FID and capillary column SP WAX 52CB (30 m  0.25 mm). The temperature was programmed from 165 to 230  C at a rate of 4  C min1 and a 10 min hold at 230  C. The temperature of injector was 260  C, and of the detector was 280  C; the carrier gas was nitrogen (1.45  103 Pa).

4.6. Determination of Cd content

The experiments were repeated several times and the average of the values was used. The data were statistically evaluated using the standard deviation and T-test methods.

Around 1 g of dry root material was wet digested in H2SO4/ HNO3 mixture (1/5, v/v) for 24 h, and then it was treated with HNO3/HClO4 mixture (5/1, v/v). Cadmium concentration in the digest was measured by atomic absorption spectrophotometer Perkin–Elmer (Germany). 4.7. Salicylic acid extraction and analytical procedure Salicylic acid and its precursors were measured according to Meuwly and Me´traux [33]. Two grams of the 3rd leaves were ground in liquid nitrogen in a mortal and pestle, in the presence of 1 g quartz sand. The tissue powder was transferred to a centrifugation tube and mixed with 2 ml of 70% methanol containing 250 ng ortho-anisic acid (o-ANI) (used as internal standard) and 25 mg para-hydroxybenzoic acid (p-HBA) (used as extraction carrier). The extract was centrifuged at 10,000  g for 20 min. The pellet was resuspended in 2 ml 90% methanol, vortexed and centrifuged as above. The methanol content was evaporated from 2 ml of the mixed supernatants at room temperature under a vacuum. One milliliters of 5% (w/v) TCA was added to the residual aqueous phase and the mixture was centrifuged at 15,000  g for 10 min. The supernatant was gently partitioned twice against 3 ml of a 1/1 (v/v) mixture of ethyl acetate/cyclohexane. The upper organic layers contained the free phenolic portion. The aqueous phases containing the methanol-soluble bound phenolics were acid hydrolysed. 250 ng o-ANI, 25 mg p-HBA and 1.3 ml 8 N HCl were added to the aqueous phase and incubated for 60 min at 80  C before partitioning twice as above. Just prior to the HPLC analysis, the organic phases were evaporated to dryness under a vacuum and resuspended in 1 ml of the HPLC starting mobile phase. SA and o-HCA were quantified fluorimetrically (W474 scanning fluorescence detector, Waters, USA), with excitation at 317 nm and emission at 436 nm for o-HCA, followed by excitation at 305 nm and emission at 407 nm for SA. 4.8. Analysis of the total lipophilic extracts All leaf samples (6.8–13.3 g FW) were cut into small pieces and extracted twice with 65 ml chloroform/methanol (1/1, v/v). The extracts were combined and diluted with water until two layers were obtained. The lower layer was evaporated under vacuum and the obtained total lipophilic extract was kept at 30  C. Part of lipophilic extracts (50 mg from each sample) was transported in small vials with teflon screw caps. Five milliliters of 15% acetyl chloride in absolute methanol was added and the vials were heated 4 h at 55  C. After cooling the samples were diluted with water and the obtained fatty acids methyl esters (FAME) were extracted with hexane (2  5 ml). The FAME in combined hexane extracts were purified by preparative thin–layer chromatography (TLC) on 20  20 cm silica-gel G (Merck) plates (layer thickness 0.5 mm) with mobile phase hexane – acetone (95:5 v/v). The spots of the FAME were visualized under UV – light, scrapped off with the silica-gel layer and eluted with hexane. The amount of each sample was determined gravimetrically. The FAME was analyzed by gas chromatography (GC) on Hewlett Packard 5890 (Hewlett Packard, Palo Alto, California, USA)

4.9. Statistical analysis

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