Running title: Short-term salt stress effects on antioxidant enzymes

Transcription

1 1 Running title: Short-term salt stress effects on antioxidant enzymes Correspondence to: Dr. José A. Hernández Centro de Edafología y Biología Aplicada del Segura CSIC Departamento de Nutrición y Fisiología Vegetal Apdo E Murcia SPAIN FAX: jahernan@cebas.csic.es

2 2 Short-term effects of salt stress on antioxidant systems and leaf water relations of pea plants Hernández JA and 1 Almansa MS Departamento de Nutrición y Fisiología Vegetal, Centro de Edafología y Biología Aplicada del Segura, CSIC, Apartado 4195, E Murcia, Spain, 1 Department of Applied Biology, Plant Biology Div., Universidad Miguel Hernández, Campus de Orihuela E Alicante (Spain). ABSTRACT In pea (Pisum sativum L.) plants the effect of short-term salt stress and recovery on growth, water relations and the activity of some antioxidant enzymes was studied. Leaf growth was interrupted by salt addition. However, during recovery, growth was restored, although there was a delay in returning to control levels. Salt stress brought about a decrease in osmotic potential and in stomatal conductance, but at 48 h and 24 h post-stress, respectively, both parameters recovered control values. In pea leaves, a linear increase in the Na + concentration was observed in salt treated plants. In the recovered plants, a slight reduction in the Na + concentration was observed, probably due to a dilution effect since the plant growth was restored and the total Na + content was maintined in leaves after the stress period. A significant increase of SOD activity occurred after 48 h of stress and after 8h of the recovery period (53% and 42%, respectively), and it reached control values at 24 h post-stress. APX activity did not change during the stress period, and after only

3 3 8h post-stress it was increased by 48% with respect to control leaves. GR showed a 71% increase after 24h of salt stress and also a significant increase was observed in the recovered plants. A strong increase of TBARS was observed after 8h of stress (180% increase), but then a rapid decrease was observed during the stress period. Surprisingly, TBARS again increased at 8h post-stress (78% increase), suggesting that plants could perceive the elimination of NaCl from the hydroponic cultures as another stress during the first hours of recovery. These results suggest that short-term NaCl stress produces reversible effects on growth, leaf water relations and on SOD and APX activities. This work also suggests that both during the first hours of imposition of stress and during the first hours of recovery an oxidative stress was produced. Abbreviations: ASC, ascorbate; AOS, activated oxygen species; ASC-GSH cycle, ascorbate-glutathione cycle; APX, ascorbate peroxidase; gi, stomatal conductance; GSH, glutathione, reduced form; GR, glutathione reductase; O.- 2, superoxide radical; SOD superoxide dismutase; TBARS, thiobarbituric acid-reactive substances INTRODUCTION Salinity is one the major limiting environmental factors in crop production. Under salt stress, plants have to cope with water stress, imposed by the low external water potential, and with ion toxicity, due to accumulation inside the plant (Greenway and Munns 1980). Although many studies have indicated that salt stress induces the expression of specific genes and metabolic modifications (Bohnert and Jensen 1996, Delumeau et al. 2000, Hernández et al. 2000) the mechanisms of salt tolerance in plants are not fully understood. Salt stress, in addition to the known components of

4 4 osmotic stress and ion toxicity, is also manifested as an oxidative stress, and all of these factors contribute to its deleterious effects (Gueta-Dahan et al. 1997). However, ion content and salt tolerance are not often correlated, and several studies indicate that acquisition of salt tolerance may also be a consequence of improving resistance to oxidative stress (Hernández et al. 1993, 1995, 1999, 2000, 2001, Streb and Feieranbed 1996, Gosset et al. 1996, Gueta-Dahan et al. 1997, Gómez et al. 1999). In previous work we suggested that the induction of antioxidant defences is at least one component of the tolerance mechanism of peas (Pisum sativum L.) to long-term salt stress (Hernández et al., 2000). Recently, we reported that salt stress produced an O and H 2 O 2 -mediated oxidative stress in the apoplast of pea leaves, which brought about necrotic leaf lesions, localised initially on minor veins (Hernández et al. 2001). Salt stress causes stomatal closure, which reduces the CO 2 /O 2 ratio in leaves and inhibits CO 2 fixation. These conditions increase the rate of activated oxygen species (AOS such as superoxide, hydrogen peroxide, hydroxyl radical and singlet oxygen) formation, via enhanced leakage of electrons to oxygen. AOS attack proteins, lipids and nucleic acids, and the degree of damage depends on the balance between the formation of AOS and its removal by the antioxidative scavenging systems. The effects of various environmental stresses in plants are known to be mediated, at least in part, by an enhanced generation of AOS and/or the inhibition of the systems that defend against them (Hernández et al. 1993, 1995, Mitler and Zilinskas 1994, Alscher et al. 1997, Noctor and Foyer, 1998, Shalata and Tal, 1998). To mitigate and repair damage initiated by AOS, plants have developed a complex antioxidant system. The primary components of this system include carotenoids, ascorbate, glutathione, tocopherols and enzymes such as superoxide dismutase (SOD, EC ), catalase (EC ), glutathione peroxidase (GPX, EC ), peroxidases and the enzymes involved in

5 5 the ascorbate-glutathione cycle (ASC-GSH cycle; Foyer and Halliwell 1976): ascorbate peroxidase (APX, EC ), dehydroascorbate reductase (DHAR, EC ), monodehydroascorbate reductase (MDHAR, EC ) and glutathione reductase (GR, EC ) (Noctor and Foyer 1998). Many components of this antioxidant defence system can be found in different subcellular compartments (Jiménez et al. 1997, Gómez et al. 1999, Hernández et al. 2000). In previous work, carried out in this laboratory, the response of antioxidant systems to long-term salt stress has been studied (Hernández et al. 1993,1995, 1999, 2000, 2001, Gómez et al. 1999). However, data concerning the effects of short-term salt stress and recovery on the activity of antioxidant enzymes are scarce. In this work, using pea plants, the effects of short-term salt stress (70 mm NaCl, up to 48 h) and recovery (up to 48 h) on the activity of some antioxidant enzymes, growth and water relations were studied. This work suggests that, both during the first hours of imposition of stress and during the first hours of recovery, an oxidative stress was produced. Material and Methods Growth of plants in salt stress conditions Pea seeds were surface sterilised (ethanol (96 %, v/v) 3 min and sodium hypochlorite (10 %, v/v) 5 min), germinated and grown in vermiculite. Vigorous seedlings were selected for hydroponic culture in a growth chamber (ASL). First, plants were cultivated in aerated distilled water for 7 days (Hernández et al. 1993). Then, plants were transplanted to aerated optimum nutrient solution for another 7 days. The growth chamber was set at 24/18ºC, 80% relative humidity and 200 µmol m -2 s -1 of light intensity with a 16 h photoperiod. After this period, 70 mm NaCl was added to the nutrient solution and plant leaves were sampled at 0 h, 8 h, 24 h and 48 h after the

6 6 NaCl was added. Then, plants were returned to aerated optimum nutrient solution without NaCl, and leaves were sampled at 8 h, 24 h and 48 h post-stress, corresponding to 56 h, 72 h and 96 h of growth. Plant growth and water relations Plant growth was followed by measuring the leaf area of one leaf of each plant at different time periods after salinisation and after recovery. Leaf profiles were copied on transparent paper sheets, and areas were later quantitated by integration using a graphic table and Autocad program (Ortiz et al. 1994). For each time period 4 leaves were analysed. Leaf water content was determined by weighing tissue before (fresh weight) and after complete lyophilisation (dry weight) using an analytical balance. Stomatal conductance (gi) was determined on fully expanded intact leaves with a portable porometer (model Licor 1600), using 6 plants in each experiment. For the determination of osmotic potentials, leaves were immediately frozen in liquid nitrogen and kept at 80ºC. Leaves were thawed, and the sap was obtained by extrusion, using a polypropylene syringe provided with a nylon filter at the tip. Osmotic potentials were determined using a Wescor Inc. C-52 sample chamber. Mineral Composition Lyophilised leaves was powdered and subjected to digestion with HNO 3 :HClO 4 (2:1, Chapman and Pratt 1961). The resulting solutions were diluted appropiately and analysed for Na +, K + and Ca 2+ by atomic absorption spectrometry.

7 7 Enzyme extraction and assays All operations were performed at 0-4ºC. For total extracts, leaves (1 g) were homogenised with a mortar and pestle in 2 ml of ice-cold 50mM K-phosphate buffer ph 7.8, 0.1 mm EDTA containing 5 mm cysteine, 1% (w/v) PVP, 0.1 mm PMSF and 0.2% Triton X-100 (v/v). For APX activity 20 mm ascorbate was added. The homogenate was centrifuged at g for 20 min and the supernatant fraction was filtered through Sephadex G-50 M PD-10 columns (Pharmacia Biotech AB, Uppsala) equilibrated with the same buffer used for the homogenisation, with or without 5 mm ASC. APX, GR and SOD, were assayed according to Hernández et al. (1999). Protein was estimated according to Bradford (1976). The extent of lipid peroxidation was estimated by determining the concentration of thiobarbituric acid-reactive substances (TBARS). The leaf samples were immediately frozen in liquid nitrogen. Leaf material (200 mg) was homogenized in 2 ml 0.1% TCA solution. The homogenate was centrifuged at g for 10 min and 0.5 ml of the supernatant obtained was added to 1.5 ml 0.5% TBA in 20% TCA. The mixture was incubated at 90º C in a shaking water bath for 20 min, and the reaction was stopped by placing the reaction tubes in an ice-water bath. Then, the samples were centrifuged at g for 5 min, and the absorbance of the supernatant was read at 532 nm. The value for non-specific absorption at 600 nm was subtracted (Cakmak and Horst 1991). The amount of TBARS (red pigment) was calculated from the extinction coefficient 155 mm -1 cm -1 (Cakmak and Horst 1991). Results In control plants, leaf area had increased by 18.5% after 96 h of the growth period in relation to time zero of growth. However, in salt-treated plants leaf growth was interrupted by salt addition (Fig 1A). After 8 h of recovery, leaf growth was also

8 8 restored, but it was delayed in relation to control plants (approx. 8.2 % of delay) (Fig 1 A). After 48 h of salt stress, there was a decrease of the water content. This decrease even was higher after 8 h of recovery, but finally it reach control values at the end of the recovery period (Fig 1B). During the salinisation period, the maximum decrease of the osmotic potential ocurred after 48 h of salt stress (nearly 31% decrease). However, the osmotic potential rapidly recovered control values after 24 h of the post-stress period (Fig 2A). A reduction in stomatal conductance values (gl) occurred in salt-stressed plants. However, when salt was eliminated from the nutrient medium, gl started to increase in the recovered plants, and returned to control values 24 h after the poststress period (Fig 2B). In control plants, the levels of Na + were kept around mmol kg DW -1. However, in salt-treated plants a linear increase in the Na + concentration was observed (up to 7.4- fold of increase after 48 h of stress) (Fig 2C). A slight reduction in the Na + concentration was observed in the recovered plants, although this value was still high (5.4-fold higher than in control plants). However, no significant changes were found for Ca 2+ and K + contents (data not shown). A significant increase of SOD activity occurred after 48 h of stress and after 8 h of the recovery period (53 % and 42 % increase, respectively) (Fig 3A). This activity reached control values after 24 h and 48 h post-stress. APX activity did not change during the stress period, although in this situation it could be necessary for H 2 O 2 elimination. After only 8 h of the post-stress period APX had increased by 46 % in relation to control plants (Fig 3B). Regarding GR activity, this enzyme significantly increased in salt-treated plants, showing a 71% rise after 24 h of stress, and a significant increase was also observed in the recovered plants (Fig 3C).

9 9 A strong increase in TBARS was observed after 8 h of stress (180% increase in relation to control plants) (Fig 4). Nevertheless, a rapid decrease was observed during the stress period, suggesting an adaptation to the stress conditions. Surprisingly, the TBARS again increased after 8 h of the recovery period (78% increase) (Fig 4), in spite of the increase observed in antioxidant enzymes, suggesting that plants could interpret the elimination of NaCl as other stress period during the first hours of recovery. During the post-stress period, TBARS progressively decreased, although after 48 h of recovery this value was still 21 %higher than in control plants. Discussion It is know that salt stress affect both leaf growth and water status (Alarcón et al. 1994, Ortiz et al. 1994, Torrecillas et al.1995). The osmotic effect resulting from soil salinity may cause disturbances in the water balance of the plant, reducing turgor and inhibiting growth, as well as provoking stomatal closure and reducing photosynthesis (Poljakoff-Mayber 1982, Sánchez-Blanco et al. 1991, Alarcón et al. 1993). Plants respond by means of osmotic adjustment, normally by increasing the concentrations of Na + and Cl - in their tissues, althoug such accumulation of inorganic ions may produce important toxic effects and cell damage (Flowers and Yeo 1986). This limited osmotic adjustment was not sufficient to avoid water stress in the treated plants, and thus there was a decrease of the leaf water content after 48 h of salt stress. This decrease was even higher after 8 h of recovery, but finally it reach control values at the end of the recovery period. Wilson et al. (1989) indicated that osmotic adjustment accounted for decreases in the fresh mass/dry mass ratio, increases in the apoplastic water content, and direct solute accumulation. No significant changes in the first two parameters were observed, therefore, the leaf

10 10 osmotic potential reductions observed under saline stress were the result of the accumulation of solutes, mainly Na + (Fig 2 A and Fig 2C). A slight reduction in the Na + concentration was observed in the recovered plants, although this value was still high (5.4-fold greater than in control plants). The lower Na + level observed in the recovered plants, compared to that observed after 48 h of salt treatment, could be due to a dilution effect since plant growth was restored and the total Na + content was maintained in leaves after the stress period. The decrease in gl under salt stress, as well as the recovery after the stress was removed, has been previously described by other authors (Alarcón et al. 1994, Mittler and Zilinskas 1994, Torrecillas et al. 1995). This decrease in gl might limit photosynthesis and reduce leaf growth rate (Hernández et al. 1999). In pea plants recovered from drought (10 h after re-watering) an increase in cytosolic APX and cytosolic CuZn-SOD transcripts abundance occurred (Mittler and Zilinskas 1994). This agrees with our results, where an increase in APX activity at 8 h post-stress, and an increase in SOD activity in salt-treated plants as well as after 8 h of recovery, was observed. It is well established that APX plays a key role in the removal of H 2 O 2 in the chloroplast and cytosol of higher plants (Gillham and Dodge 1986). Recently, the presence of all the components of the ASC-GSH cycle in mitochondria and peroxisomes from pea plants has been described (Jiménez et al. 1997). APX is located in the mitochondrial and peroxisomal membrane fractions (Jiménez et al. 1997), where it seems to play a key role scavenging the H 2 O 2 that could leak from these cell organelles. H 2 O 2 can easily permeate membranes, and an important advantage of the presence of APX in the mitochondrial and peroxisomal membranes would be the degradation of leaking H 2 O 2. This membrane scavenging of H 2 O 2 could avoid an increase in the cytosolic H 2 O 2 concentration during normal metabolism and under

11 11 certain plant-stress situations, when the level of H 2 O 2 produced can be substantially enhanced (del Río et al. 1998). The increase in GR, both in salt-treated and recovered plants, could have resulted in a higher pool of GSH, which could be used in ASC regeneration. Gamble and Burke (1984) suggested that GR could play a key role in the protection against oxidative stress. In a NaCl-tolerant pea cultivar, long-term salt treatment caused a significant increase in the activities of the ASC-GSH cycle in the soluble fraction. However, in a sensitive pea cultivar, no changes in the specific activities of cytosolic APX, MDHAR or GR were observed, and cytcuzn-sod decreased by about 35% (Hernández et al., 2000), suggesting that the cytosolic compartment may be important in the antioxidant response to NaCl (Hernández et al., 2000). In cucumber leaves, catalase and GR were enhanced either by NaCl or KCl stress (Lechno et al. 1997). As K + is considered to be a compatible solute for plants, whereas Na + ions are toxic, this response suggests that the salt effect is osmotic rather than a specific ionic effect of the sodium (Lechno et al. 1997). However, in the current study, where a strong osmotic stress does not seem to be produced in the first 24 h of salt stress, the changes observed in GR activity seem to be due to a specific ionic effect rather than an osmotic effect, although we cannot rule out also the osmotic effects. In Nicotiana plumbaginifolia L. plants, short-term NaCl treatment raised the transcript levels of Gpx, Apx, SodA, SodB and SodCc, although total SOD and APX activities decreased (Savouré et al. 1999). This discrepancy between transcript levels and enzyme activities during NaCl treatment may result from a higher turnover of these enzymes and/or an increase of their inactivation by H 2 O 2 (Scandalios 1993). In previous work, we showed that long-term salt stress induced transcript levels of some antioxidant

12 12 enzymes, but this induction was not correlated with the corresponding changes in the enzyme activities (Hernández et al. 2000). Salt stress is known to result in extensive lipid peroxidation (Hernández et al. 1995, 2001, Gosset et al. 1996, Gómez et al. 1999). Neither GR, APX nor SOD activities changed during the first 8 h of salt stress, suggesting a correlation between the stomatal closure and the strong peak of TBARS observed in this growth period. An increase in AOS production may result from stomatal closure causing a decrease in the CO 2 concentration inside the chloroplasts. This, in turn, might cause a decrease in the concentration of NADP + available to accept electrons from PSI/II and thus initiate O 2 reduction with the concomitant generation of AOS (Halliwell 1982). In this situation an oxidative stress is produced, as indicated by the observed strong increase in TBARS. Surprisingly, the TBARS again increased after 8 h of the recovery period, in spite of the increase observed in antioxidant enzymes, suggesting that plants could perceive the elimination of NaCl as another stress period during the first hours of recovery. Increasing evidence exists that membrane injury under salt stress is related to an increased production of highly toxic AOS (Hernández et al. 1995, 2001, Gosset et al. 1996, Gómez et al. 1999). Since lipid peroxidation is the symptom most easily ascribed to oxidative damage (Zhang and Kirkham 1994), it is often used as an indicator of increased oxidative damage (Halliwell 1982, Hernández et al. 1995, 2001, Gómez et al. 1999). Because a strong osmotic stress was not produced by short-term salt stress, at least in the first 24 h, and similarly for antioxidant enzyme changes, the increase in TBARS seems to be due more to toxic effects of salts rather than an osmotic stress, although we cannot rule out also the osmotic effects. However, at 8h of recovery, where Na+ was now present in the leaf tissue, the increase in TBARS observed could be due to both factors (toxic and osmotic effects).

13 13 In N. plumbaginifolia, short-term NaCl treatments (12 h) also produced an increase of lipid peroxidation (Savouré et al. 1999). Salt stress produced ion leakage, indicating injury to membrane integrity, which could be affected by AOS formed during leaf photosynthesis or respiration, enhancing lipid oxidation of the membranes (Lechno et al. 1997, Savouré et al. 1999). Peroxisomes are also important AOS generators, and in pea plants an important decrease in catalase activity by salt effect has been reported, although no increase in H 2 O 2 was detected neither in peroxisomes (Corpas et al. 1993) or mitochondria (Hernández et al. 1993) from pea plants. In accordance with those results, it was suggested that an increase of H 2 O 2 content could occur in the cytosol of salt-stressed plants through a simple diffusion of this molecule out of peroxisomes and mitochondria (Corpas et al. 1993, Hernández et al. 1993). Taken together, these results suggest that short-term NaCl stress produces reversible effects on leaf water relations, growth, water content and SOD and APX activities. However, during the first hours of both stress and recovery an increase in TBARS occurred, suggesting the involvement of an oxidative stress during these periods. During the first hours of recovery the induction of SOD, APX and GR cannot prevent TBARS production. Probably, the change of culture conditions (elimination of NaCl from hydroponic cultures) is perceived by plants as an hypoosmotic stress situation. Recently, Cazalé et al. (1998) reported that an oxidative burst is produced in tobacco cells in response to hypoosmotic stress. This oxidative burst could cause membrane lipid peroxidation and could explain the increase in TBARS observed at 8h of recovery.

14 14 Acknowledgement Authors thank Dr. J.J. Alarcón (Dept Riego y Salinidad, CEBAS-CSIC) and Prof. A. Ros-Barceló (Dept. Plant Biology, Universidad de Murcia) for their valuable reviews of this manuscript, and Dr. D.J. Walker for correction of the English. References Alarcón JJ, Sánchez-Blanco MJ, Bolarín MC, Torrecillas A. (1993) Water relations and osmotic adjustment in Lycopersicon esculentum and L. pennellii during shortterm salt exposure and recovery. Physiol Plant 89: Alarcón JJ, Sánchez-Blanco MJ, Bolarín MC, Torrecillas A (1994) Growth and osmotic adjustment of two tomato cultivars during and after saline stress. Plant and Soil 166: Alscher RG, Donahue JL, Cramer CL (1997) Reactive oxygen species and antioxidants: relationships in green cells. Physiol Plant 100: Bohnert HJ, Jensen RG (1996) Metabolic engineering for increased salt tolerance. The next step. Aust J Plant Physiol 23: Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: Cakmak, I, Horst WJ (1991) Effect of aluminium on lipid peroxidation, superoxide dismutase, catalase and peroxidase activities in root tips of soybean (Glycine max). Physiol Plant 83: Cazalé AC, Rouet-Mayer MA, Barbier-Brygoo H, Mathieu Y, Laurière C (1998) Oxidative burst and hypoosmotic stress in tobacco cell suspensions. Plant Physiol 116:

15 15 Chapman HD, Pratt PF (1961) Methods of Analysis for Soils, Plants and Waters. Univ. Calif. Div. Agric. Sci. pp Corpas FJ, Gómez M, Hernández JA, del Río LA (1993) Metabolism of activated oxygen in peroxisomes from two Pisum sativum L. cultivars with different sensitivity to sodium chloride. J Plant Physiol 141: del Río LA, Pastori GM, Palma JM, Sandalio LM, Sevilla F, Corpas FJ, Jiménez A, López-Huertas E, Hernández JA (1998) The activated oxygen role of peroxisomes in senescence. Plant Physiol 116: Delumeau O, Morère-Le Paven MC, Montrichard F, Laval-Martin DL (2000) Effects of short-term NaCl stress on calmodulin transcript levels and calmodulin-dependent NAD kinase activity in two species of tomato. Plant Cell Environ 23: Flowers TJ, Yeo AR (1986) Ion relations of plant under drought and salinity. Aust J Plant Physiol 13: Foyer CH, Halliwell B (1976) Presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta 133: Gamble PE, Burke JJ (1984) Effect of water stress on the chloroplast antioxidant system. I. Alterations in glutathione reductase activity. Plant Physiol 76: Gillham DJ, Dodge AD. (1986) Hydrogen-peroxide-scavenging systems within pea chloroplasts. A quantitative study. Planta 167: Gómez JM, Hernández JA, Jiménez A, del Río LA, Sevilla F (1999). Differential response of antioxidative enzymes of chloroplasts and mitochondria to long-term NaCl stress of pea plants. Free Rad Res 31: S11-18 Gosset DR, Banks SW, Millhollon EP, Lucas MC (1996) Antioxidant response to NaCl stress in a control and an NaCl-tolerant cotton cell line grown in the

16 16 presence of paraquat, buthionine sulfoximine, and exogenous glutathione. Plant Physiol 112: Greenway H, Munns R (1980) Mechanisms of salt tolerance in nonhalophytes. Annu Rev Plant Physiol 31: Gueta-Dahan Y, Yaniv Z, Zilinskas, BA, Ben-Hayyim G (1997) Salt and oxidative stress: similar and specific responses and their relation to salt tolerance in Citrus. Planta 203: Halliwell B (1982) The toxic effects of oxygen on plant tissues. In Superoxide Dismutase. Vol. 1 (ed L.W. Oberley), pp , CRC Press, Inc. Boca Raton, Florida. Hernández JA, Corpas FJ, Gómez M, del Río LA, Sevilla F (1993) Salt-induced oxidative stress mediated by activated oxygen species in pea leaf mitochondria. Physiol Plant 89: Hernández JA, Olmos E, Corpas FJ, Sevilla F, del Río LA (1995) Salt-induced oxidative stress in chloroplast of pea plants. Plant Sci 105: Hernández JA, Campillo A, Jiménez A, Alarcón JJ, Sevilla F (1999) Response of antioxidant systems and leaf water relations to NaCl stress in pea plants. New Phytol 141: Hernández JA, Jiménez A, Mullineaux PM, Sevilla F (2000) Tolerance of pea (Pisum sativum L.) to long-term salt stress is associated with induction of antioxidant defences. Plant Cell Environ 23: Hernández JA, Ferrer MA, Jiménez A, Ros-Barceló A, Sevilla F (2001) Antioxidant systems and O.- 2 /H 2 O 2 production in the apoplast of Pisum sativum L. leaves: its relation with NaCl-induced necrotic lesions in minor veins. Plant Physiol 127:

17 17 Jiménez A, Hernández JA, del Río LA, Sevilla F (1997) Evidence for the presence of the ascorbate-glutathione cycle in mitochondria and peroxisomes of pea (Pisum sativum L.) leaves. Plant Physiol 114: Lechno S, Zamski E, Tel-Or E (1997) Salt stress-induced responses in Cucumber plants. J Plant Physiol 150: Mittler R, Zilinskas BA (1994) Regulation of pea cytosolic ascorbate peroxidase and other antioxidant enzymes during the progression of drought stress and following recovery from drought. Plant J 5: Noctor G, Foyer C (1998) Ascorbate and Glutathione: Keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol 49: Ortiz A, Martinez V, Cerdá A ( 1994) Effects of osmotic shock and calcium on growth and solute composition of Phaseolus vulgaris plants. Physiol Plant 91: Poljakoff-Mayber A (1982) Biochemical and physiological responses of higher plants to salinity stress. In San Prieto A, ed. Biosaline Research. A Look to the Future. Plenum Press, New York, NY Sánchez-Blanco MJ, Bolarín, MC, Alarcón JJ, Torrecillas, A. (1991). Salinity effects on water relations in Lycopersicon esculentum and its wild salt-tolerant relative species L. pennellii. Physiol Plant 83: Savouré A, Thorin D, Davey M, Hua XJ, Mauro S, Van Montagu M, Inzè D, Verbruggen, N (1999) NaCl and CuZnSO 4 treatments trigger distinct oxidative defence mechanism in Nicotiana plumbaginifolia L. Plant Cell Environ 22: Scandalios JG (1993) Oxygen stress and superoxide dismutases. Plant Physiol 101:7-12.

18 18 Shalata A, Tal M (1998) The effect of salt stress on lipid peroxidation and antioxidants in the leaf of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii. Physiol Plant 104: Streb P, Feierabend J (1996) Oxidative stress-responses accompanying photoinactivation of catalase in NaCl-treated rye leaves. Bot Acta 109: Torrecillas A, Guillaume C, Alarcón JJ, Rúiz-Sánchez MC (1995) Water relations of two tomato species under water stress and recovery. Plant Sci 105: Wilson JR, Ludlow MM, Fisher MJ, Schulze EE (1989) Adaptation to water stress of the leaf water relations of four tropical forage species. Aust J Plant Physiol 7: Zhang J, Kirkham MB (1994) Drought-stress-induced changes in activities of superoxide dismutase, catalase, and peroxidase in wheat species. Plant Cell Physiol 35:

19 19 Figure legends Fig.1.- Effect of short term NaCl stress and recovery on leaf growth (A, as % of the area at time 0) and water content (B, as % by weight) of pea leaves. Measurement were made at 0, 8 h, 24 h and 48 h of stress and after 8 h, 24 h and 48 h of post-stress (corresponding to 56 h, 72 h and 96 h of the growth period). Data are the mean ± SE of at least three different experiments. Fig 2.- Effect of short term NaCl stress and recovery on osmotic potential (A), stomatal conductance (B) and Na + concentration (C) of pea leaves. The measurements were made at the same points given in Fig 1. Fig 3.-. Effect of short term NaCl stress and recovery on antioxidant enzyme activities of pea leaves. Data are expressed as % of control values in each time period. Differences from control values were significant at: P< 0.05 (a); P< 0.01 (b); P< (c) according the Duncan s Multiple Range Test. Fig. 4.- Effect of short term NaCl stress and recovery on lipid peroxidation (given as TBARS) in pea leaves. Data are expressed as % of control values in each time period.

20 20 Leaf area (% of control) NaCl -NaCl A Control NaCl-treated Water Content (%) NaCl -NaCl B Time (h) Fig 1

21 21 -NaCl Osmotic potential (- ) Bars NaCl A Control NaCl-treated Stomatal Conductance (mmol m -2 s -1) [Na + ] mmol (kg DW) B C Time (h) Fig 2

22 22 -NaCl SOD activity (% of control) NaCl b b A APX activity (% of control) b B GR activity (% of control) b c a a C b Time (h) FIG 3

23 Fig 4 23