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Comparison of the responses to NaCl stress of two pea cultivars using split-root system
Scientia Horticulturae 123 (2009) 164–169
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Comparison of the responses to NaCl stress of two pea cultivars using split-root system Houneida Attia a,1,*, Sarra Nouaili a,b,1, Abdelaziz Soltani b, Mokhtar Lachaaˆl a a b
Unite´ de Physiologie et Biochimie de la Tole´rance au Sel des Plantes, Faculte´ des Sciences de Tunis, Campus Universitaire, 2092 Tunis El Manar, Tunisia Laboratoire d’Adaptation des Plantes aux Stress Abiotiques, Centre de Biotechnologie a` la, Technopole de Borj-Ce´dria (CBBC), BP 901, 2050 Hammam-Lif, Tunisia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 March 2009 Received in revised form 31 August 2009 Accepted 1 September 2009
Two pea (Pisum sativum L.) cultivars were compared: cv Lincoln and cv Douce de Provence. Seedlings grown for 14 d on standard medium were challenged for 21 d with salt using a split-root system. This protocol allowed salt-treated plants to absorb nutrients through a part of their root system maintained in control medium (C), the other part of the root system being placed in medium added with 75 mM NaCl (S). Full salt treatment (S/S) resulted in severe but non-lethal growth inhibition, high concentration of Na+ and Cl in leaves, and decrease in leaf K+ and chlorophyll contents. The two latter effects were more pronounced in Lincoln than in D. Provence. Growth inhibition was partially (Lincoln) or totally (D. Provence) alleviated in S/C configuration, and K+ content was less diminished than in full salt treatment. S/C treatment mitigated Na+ and Cl accumulation in Lincoln, but not in D. Provence. Thus, in the latter cultivar, growth inhibition by salt in S/S condition likely did not result from excessive Na+ and Cl accumulation in leaves. Increased electrolyte leakage from leaf tissues evidenced damages to leaf cell plasma membrane of both cultivars in S/S condition. However, damages to chloroplasts, as inferred from chlorophyll loss, were much pronounced in Lincoln than in D. Provence. Antioxidant enzymic activities in leaves were measured as proxies for oxidative stress. Catalase activity was stimulated by S/S treatment in both cultivars, but superoxide dismutase (Fe and Cu/Zn isoforms) and gaiacol peroxidase activities were augmented only in Lincoln. The absence of superoxide dismutase activity stimulation by salt in D. Provence could signify either that constitutive activity was sufficient to ensure protection against oxidative stress, or that intrinsic salt tolerance of this cultivar mitigated cellular oxidative stress. Thus, intraspecific variability for salt response exists between pea cultivars presenting similar growth sensitivity to salt. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Antioxidant enzyme activity Pisum sativum L. Salinity Split-root
1. Introduction Salinity is one of the major abiotic stresses that affect plant productivity (Gueta-Dahan et al., 1997). Through its osmotic effect, salt in the soil causes stomatal closure, which reduces the CO2/O2 ratio in leaves and inhibits CO2 fixation. Other detrimental effects are due to salt accumulated in plant tissues. However, direct Na+ and Cl toxicity does not seem to be the sole cause of salt-induced growth reduction (Hu et al., 2005). Indeed, salt tolerance depends on the provision to plants of several macro-elements, among which N, K, and Ca are the most important ones (Grattan and Grieve, 1999). Salt has been shown to depress the chloroplast content in K+, NO3 and SO42 (Schro¨ppel-Meier and Kaiser, 1988). In barley,
Abbreviations: D. Provence, Douce de Provence; GPX, gaiacol peroxidase; SOD, superoxide dismutase; ROS, reactive oxygen species. * Corresponding author. Tel.: +216 71 872 600; fax: +216 71 871 666. E-mail address: [email protected] (H. Attia). 1 These authors have participated equally to this work. 0304-4238/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2009.09.002
the efficiency of salt treatment to stimulate K+ efflux and deplete cellular K+ pools correlated with cultivar sensitivity to salinity (Chen et al., 2005). In Arabidopsis and tomato mutants, hypersensitivity to salt is frequently associated with loss of capacity to absorb K+ (Borsani et al., 2001; Zhu et al., 1998). Split-root experiments have shown that inhibition of K+ uptake at high salinity level limited growth of Crithmum maritimum (Ben Hamed et al., 2008). Similar experiments indicated that K+ and Ca2+ were limiting for Arabidopsis growth when they were presented to roots together with NaCl, but not when they could be absorbed by roots placed in control medium, the other part of the root system being submitted to NaCl (Attia et al., 2008b). Salt stress increases the rate of reactive oxygen species (ROS: superoxide, hydrogen peroxide, hydroxyl radical and singlet oxygen) formation, inducing oxidative stress in plants (Parvaiz et al., 2008). Oxidative stress is thought to arise in chloroplasts primarily from unbalance between absorbed light energy and metabolic energy use, because these conditions favour leakage of electrons to oxygen (Hasegawa et al., 2000), and from other
H. Attia et al. / Scientia Horticulturae 123 (2009) 164–169
disturbances of electron transport chain (Diaz-Vivancos et al., 2008). However, it may also originate from other dysfunctions such as excess salt accumulation in leaves, and mineral deficiency (Tewari et al., 2004). Indeed, salt-induced K+ deprivacy induces antioxidant response (Attia et al., 2008a), and it has been shown to activates NAD(P)H oxidases responsible for ROS production (Cakmak, 2005). Various genes and proteins, expression of which is activated in response to salt stress, have been identified, some of them ensuring protection against ROS (Hasegawa et al., 2000). To mitigate and repair damages initiated by ROS, plants have developed a complex antioxidant system which includes enzymes such as superoxide dismutase, catalase, various peroxidases, and many other enzymes involved in the ascorbate-glutathione cycle (Foyer and Halliwell, 1976). In pea, the components of this antioxidant defence system are found in different subcellular compartments (Hernandez et al., 1993; Jime´nez et al., 1997). Induction of several of these components (Hernandez et al., 1999) has been shown to correlate with long-term salt tolerance in this species (Hernandez et al., 2000). In the present study, two pea cultivars were compared for their salt response. A split-root system was devised for salt application, aimed at distinguishing the effects of the salt in the root medium from those of the salt accumulated in the tissues. Effects of salt were investigated on plant growth and ion accumulation in leaves, and several proxies for salt-induced stress in leaves were used, including chlorophyll content decrease, damages to membrane permeability, and antioxidant enzymes. 2. Materials and methods 2.1. Plant material Seeds of two pea (Pisum sativum L.) commercial cultivars largely used in Tunisia were obtained from Laboratoire des Le´gumineuses a` graines of Tunisian National Institute for Agronomic Research. Douce de Provence (D. Provence thereafter) has a shorter reproductive cycle (45 d) than Lincoln (60 d). 2.2. General culture conditions Seeds were surface sterilized with 96% (v/v) ethanol for 3 min, and with 2% (w/v) sodium hypochlorite for further 5 min. After rinsing they were sown on vermiculite. Vigorous seedlings were selected and grown in aerated distilled water for 7 d. Then, plants were transplanted to aerated nutrient solution of Long Ashton (Hewitt, 1966) for another 7 d. Thereafter, this solution is named control (C) medium. The growth chamber conditions were set at 25/18 8C day/night temperature, 80% relative humidity and 150 mmol m2 s1 of light intensity with a 14-h photoperiod. 2.3. Salt treatments Fourteen-day-old plants grown on C medium were used. Their root systems were separated in two even parts, each placed in a pot filled with an aerated liquid medium. Three lots of plants were prepared, distinguished according to the nature of the root medium: C/C plants with control medium in both pots, S/S plants with C medium supplemented with 75 mM NaCl (S medium) in both pots, and C/S plants with C medium in one pot and S medium in the other pot. Eight plants of each treatment were harvested 3 weeks later. 2.4. Biomass measurements and inorganic ions assays At the harvest, individual plants were divided into leaf, stem and root fractions. The fresh weights (FW) were immediately
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determined. The samples were then dried in a forced-draft oven at 70 8C for 48 h and dry weights (DW) were determined. Ions were extracted with 0.5% HNO3. Cations were assayed by flame photometry (Eppendorf) with butane-air flame for K+ and Na+, and acetylene-air flame for Ca2+, and Cl by coulometry (Butcher Cotlove chloridometer), according to manufacturers’ instructions. 2.5. Total chlorophyll content analysis Total chlorophylls were extracted from fresh leaves in 80% acetone and assayed photometrically according to Arnon (1949). 2.6. Electrolyte leakage Electrolyte leakage was determined as described by DionisioSese and Tobita (1998). Leaf discs were cut into 2–3 mm pieces and placed in test tubes containing 10 mL distilled water. The tubes were incubated in a water bath at 32 8C for 2 h and the initial electrical conductivity of the medium (EC1) was measured. The samples were autoclaved at 121 8C for 20 min to release all electrolytes, then cooled to 25 8C, and the final electrical conductivity (EC2) was measured. The electrolyte leakage (EL, %) was calculated as EL = 100 EC 1/EC2 2.7. Enzyme extraction and assays All operations were performed at 0–4 8C. Leaves (0.2 g) were homogenized with a mortar and pestle in 0.6 mL of ice cold pH 8 50 mM phosphate buffer containing 50 mM Tris–HCl pH 7, 1% (w/v) polyvinyl pyrrolidone, 0.05% TritonX-100 (w/v), 0.5 mM phenylmethylsulphonyl fluoride, 1 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol, 20 mM MgCl2 and 50 mM KCl. The homogenate was centrifuged at 13,000 g for 30 min at 4 8C and the supernatant was used for enzymatic activity measurements. Protein concentration was determined according to Bradford (1976) using bovine serum albumin as a standard. Enzyme activities were analyzed after native gel electrophoresis of the supernatant. For superoxide dismutase (SOD, EC 1.15.1.1) and gaiacol peroxidase (GPX, EC 1.11.1.7), stacking gel was 5% acrylamide in pH 6.8, 0.5 M Tris–HCl buffer, and resolving gel was 5% acrylamide in pH 8.8, 1.5 M Tris–HCl buffer. Catalase (EC 1.11.1.6) was identified on 6% acrylamide gels. A same pH 8.3, 23 mM Tris–glycine running buffer was used for the three enzymatic systems. The migration was performed at 4 8C under 120 V, with a pH 4.0, 8 mM glycine buffer as the running buffer, using Bio-Rad Miniprotean system. SOD activity was revealed by incubating the gels in the dark, using 2.5 mM nitroblue tetrazolium in pH 7.8, 50 mM phosphate buffer, and then developed for 20 min under moderate light, using a mixture of 0.6 mM riboflavin and 0.4% (w/v) N,N,N,N0 ,N0 tetramethylethylenediamine in pH 7.8, 50 mM phosphate buffer (Beauchamp and Fridovich, 1971). The identity of SOD isoforms was determined using SOD inhibitors: KCN for Cu/Zn-SOD and H2O2 for both Cu/Zn-SOD and Fe-SOD (Pan and Yau, 1992). To show peroxidase activity, the gels were incubated for 30 min in the dark in 50 mL of pH 4, 100 mM acetate buffer containing 1% (w/v) gaiacol. They were then transferred to 50 mL acetate buffer (100 mM, pH 5) containing 25 mg of 3-amino-9-ethylcarbazole, 25 mL of N,N-dimethylformamide (99%, v/v), 1 mL 0.1 M CaCl2 and 0.5 mL H2O2 (30%, v/v). Gel staining for catalase activity was performed according to Chandlee and Scandalios (1983) after pretreatment in 0.01% (v/v) H2O2 for 10 min. The staining mixture contained 1% (w/v) FeCl3 and 1% (w/v) K3Fe(CN)6 in distilled water.
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2.8. Statistics Statistical analyses were performed with StatisticaTM Software, using ANOVA and Newman–Keuls test for post hoc mean comparison. 3. Results 3.1. Growth and salt accumulation After 21 d of culture in S/S configuration, the whole plant biomass was 68% (Lincoln) and 76% (D. Provence) of control (C/C) plants (Fig. 1). C/S configuration alleviated the salt-induced growth inhibition, partially in cv Lincoln, and totally in cv D. Provence. Large amounts of Na+ and Cl were accumulated in S/S plants, exceeding 1.4 mmol g1 DW in leaves of both cultivars (Fig. 2). C/S treatment strongly limited Na+ and Cl accumulation in Lincoln leaves, as compared to full salt treatment. On the contrary, in D. Provence leaves, it led to Na+ and Cl accumulation almost as high as in S/S treatment. 3.2. Potassium and calcium In leaves of both cultivars, the accumulation of K+ (Fig. 2) was depressed by S/S treatment as compared to C/C treatment. That of Ca2+ was not significantly modified. Culture in C/S configuration partially alleviated the restrictions imposed by salt to K+ accumulation. 3.3. Total chlorophylls Total chlorophylls were much more affected by salt in Lincoln than in D. Provence (Fig. 3). The chlorophyll content of D. Provence leaves was reduced to by S/S treatment to 70% of its control value (chlorophyll content of C/C plant leaves), and only to 88% in C/S configuration. In Lincoln leaves the corresponding values were 40% (S/S) and 70% (C/S) of C/C values. 3.4. Electrolyte leakage The extent of membrane damage was estimated by electrolyte leakage from leaf tissues. This index was strongly increased by salt (S/S) in leaves of both cultivars (Fig. 4), and no significant difference between them could be evidenced. 3.5. Antioxidant enzymes Three bands of SOD activity were obtained on non-denaturing PAGE gels (Fig. 5A). The band with the lowest mobility could be attributed to Fe-SOD activity, and the fastest migrating band to Cu/ Zn-SOD activity. Salt treatment (S/S) increased significantly SOD and GPX activities in Lincoln leaves, but no appreciable change in band intensity could be observed in D. Provence (Fig. 5A and B). Finally, catalase activity was stimulated by S/S treatment in both cultivars (Fig. 5C). 4. Discussion The two cultivars presented similar growth sensitivity to salt, and similar accumulation of Na+ and Cl in their leaves when grown in full salt medium. However, they differed for three other traits: salt-induced chlorophyll loss was more important in Lincoln than in D. Provence; SOD and GPX activities were salt-inducible in Lincoln, and mostly constitutive in D. Provence; Na+ and Cl accumulation was limited in C/S configuration in Lincoln, but not in D. Provence. Comparing Lincoln responses to C/C, C/S and S/S
Fig. 1. Effect of salt treatments on whole plant biomass. Plants were 14-d old at the start of the experiment. The root system of each plant was separated in two parts, each one placed in a pot filled with an aerated liquid medium. C/C: plants with C medium (no NaCl) in both pots. S/S: plants with S medium (75 mM NaCl) in both pots. C/S: plants with half of their roots in C medium and the other half in S medium. The plants were harvested 21 d latter. Means of 8 replicates and confidence intervals for p = 0.05. Means sharing a same letter are not significantly different at p = 0.01 (ANOVA, and post hoc mean comparison with Newman–Keuls test). Lincoln and D. Provence are two cultivars.
treatments shows that growth inhibition and Na+/Cl content varied together, suggesting that excess ion accumulation was detrimental to growth in this cultivar. In D. Provence on the contrary, the presence of high Na+ and Cl concentration in leaves of C/S plants did not prevent growth from proceeding at rate comparable to that of plants not challenged by salt. Thus, the depressive effect of the full salt (S/S) treatment on D. Provence growth was likely not due to a toxic effect of accumulated ions. An opposite behaviour has been described for pepper (Lycoskoufis et al., 2005). In this salt tolerant species, culture in split-root (C/S) configuration hardly mitigated the adverse effects of salt, suggesting direct effect of absorbed Na+ and Cl ions. The accumulation of Na+ and Cl in leaves of glycophytes is known to impose various constraints which alter cell functional state, resulting in physiological stress (Gaspar et al., 2002). As initially hypothesized by Oertli (1968) and later confirmed (Flowers et al., 1991), in the absence of efficient internalization of the ions by leaf cells, their concentration in the leaf apoplast may reach excessive values, leading to leaf cell dehydration (Munns and Passioura, 1984) and membrane disruption (Speer and Kaiser, 1991). Furthermore, if not efficiently compartmentalized in leaf cell vacuoles, Na+ and Cl may build to toxic levels in the cytoplasm (Flowers et al., 1991; Kronzucker et al., 2006), and essential metabolic pathways such as photosynthesis may be inhibited. Ionic and osmotic disturbances may also result in stomata closure, which limits photosynthesis (Meloni et al., 2003). For both cultivars, the parallelism between growth inhibition and leaf K+ content reduction over the C/C, C/S and S/S treatments suggests that salt-induced restriction to K+ provision to leaves might be involved in the growth inhibition by salt. In contrast, Ca2+ transport to leaves was not modified by the treatments. Increase in leaf Na+ and Cl content associated with K+ content decrease upon increasing NaCl concentrations in the medium has been described in pea (Hernandez et al., 1999). Either genetic or salt-induced, deficiency in the capacity to absorb K+ and to maintain K+ cytoplasmic homeostasis is associated with sensitivity to salinity (Borsani et al., 2001; Chen et al., 2005; Munns and Tester, 2008). The salt-induced diminution of cytosolic K+ activity has been proposed to inhibit growth through a variety of mechanisms
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Fig. 2. Ion concentration in leaves of pea plants grown in split-root configuration. Treatment: See legend of Fig. 1. Means sharing a same letter are not significantly different at p = 0.01 (ANOVA, and post hoc mean comparison with Newman–Keuls test performed separately for each cultivar and each element).
including oxidative damages. Indeed, K+ deficiency enhances ROS production and oxidative damages to chloroplasts (Cakmak, 2005). More generally, oxidative damages are a possible cause of the growth reduction which accompanies starvation in N, P, K and S (Cakmak, 1994; Kumar et al., 2004). When plants were grown in salty medium, chlorophyll concentration was largely diminished in Lincoln but much less in D. Provence. The loss of chlorophylls is considered as a marker of
a cellular component of salt stress (Singh and Dubey, 1995). Thus, our results support the hypothesis that chloroplasts of Lincoln were more stressed by salt than those of D. Provence, in spite of similar extent of leaf cell plasma membrane damages in both cultivars, as judged from the magnitude of the electrolyte leakage. Salt tolerance involves resistance to oxidative stress. Indeed, antioxidative enzymic activities generally are augmented in stressed plants (Gueta-Dahan et al., 1997; Hasegawa et al.,
Fig. 3. Total chlorophyll concentration in leaves of pea plants grown in split-root configuration. See legend of Fig. 1. Means sharing a same letter are not significantly different at p = 0.01 (ANOVA, and post hoc mean comparison with Newman–Keuls test performed separately for each cultivar).
Fig. 4. Electrolyte leakage from discs of pea leaves. The plants were grown for 21 d at 0 (control: C) and 75 mM (salt: S) NaCl. Means of 8 replicates and confidence intervals for p = 0.05. Means sharing a same letter are not significantly different at p = 0.01 (ANOVA, and post hoc mean comparison with Newman–Keuls test performed separately for each cultivar). Lincoln and D. Provence are two cultivars.
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Fig. 5. Electrophoregrams of antioxidant enzymes in extracts from pea leaves. The plants were submitted to the indicated treatments for 21 d. Lincoln and D. Provence are two cultivars. (A) Superoxide dismutase, (B) gaiacol peroxidase, and (C) catalase.
2000), including pea (Hernandez et al., 1995, 1999, 2000). Futhermore, when different pea cultivars were compared, antioxidant response was induced by salt in tolerant cultivars, but not in salt-sensitive ones (Hernandez et al., 1995, 2000, 2001; Gomez et al., 1999). Thus, the rationale commonly used to interpret overexpression of antioxidant systems is that it is an adaptative response to apparition of ROS in cells. In our work, however, there was no parallelism between salt tolerance and antioxidant system inducibility. One reason for the absence of SOD and GPX induction in leaves of salt-treated D. Provence cultivar might be that the constitutive level of these activities was sufficient to cope with the imposed stress. A complementary explanation would be that the strength of cellular stress was not sufficient in D. Provence to sollicitate supplementary SOD and GPX expression. In summary, an important conclusion of our study is that an intraspecific variability exists for salt response in pea even between cultivars presenting similar growth sensitivity to salt. This variability concerns salt transport to leaves, chloroplast sensitivity to salt, and pattern of antioxidant response. Acknowledgement Authors are indebted to Professor C. Grignon for stimulating discussions on this work. References Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxydase in Beta vulgaris. Plant Physiol. 24, 1–15. Attia, H., Arnaud, N., Karray, N., Lachaal, M., 2008a. Long-term effects of mild salt stress on growth, ion accumulation and superoxide dismutase expression of Arabidopsis rosette leaves. Physiol. Plant. 132, 293–305. Attia, H., Karray, N., Rabhi, M., Lachaal, M., 2008b. Salt-imposed restrictions on the uptake of macroelements by roots of Arabidopsis thaliana. Acta Physiol. Plant. 30, 723–727. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276–287. Ben Hamed, K., Messedi, D., Ranieri, A., Abdelly, C., 2008. Diversity in the response of two potential halophytes (Batis maritima and Crithmum maritimum) to salt ¨ ztu¨rk, M., Ashraf, M., Grignon, C. (Eds.), Biosaline stress. In: Abdelly, C., O Agriculture and High Salinity Tolerance. Birkha¨user, Basel, pp. 71–80. Borsani, O., Cuartero, J., Fernandez, J.A., Valpuesta, V., Botella, M.A., 2001. Identification of two loci in tomato reveals distinct mechanisms for salt tolerance. Plant Cell 13, 873–887.
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Comparison of the responses to NaCl stress of two pea cultivars using split-root system Houneida Attia a,1,*, Sarra Nouaili a,b,1, Abdelaziz Soltani b, Mokhtar Lachaaˆl a a b
Unite´ de Physiologie et Biochimie de la Tole´rance au Sel des Plantes, Faculte´ des Sciences de Tunis, Campus Universitaire, 2092 Tunis El Manar, Tunisia Laboratoire d’Adaptation des Plantes aux Stress Abiotiques, Centre de Biotechnologie a` la, Technopole de Borj-Ce´dria (CBBC), BP 901, 2050 Hammam-Lif, Tunisia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 March 2009 Received in revised form 31 August 2009 Accepted 1 September 2009
Two pea (Pisum sativum L.) cultivars were compared: cv Lincoln and cv Douce de Provence. Seedlings grown for 14 d on standard medium were challenged for 21 d with salt using a split-root system. This protocol allowed salt-treated plants to absorb nutrients through a part of their root system maintained in control medium (C), the other part of the root system being placed in medium added with 75 mM NaCl (S). Full salt treatment (S/S) resulted in severe but non-lethal growth inhibition, high concentration of Na+ and Cl in leaves, and decrease in leaf K+ and chlorophyll contents. The two latter effects were more pronounced in Lincoln than in D. Provence. Growth inhibition was partially (Lincoln) or totally (D. Provence) alleviated in S/C configuration, and K+ content was less diminished than in full salt treatment. S/C treatment mitigated Na+ and Cl accumulation in Lincoln, but not in D. Provence. Thus, in the latter cultivar, growth inhibition by salt in S/S condition likely did not result from excessive Na+ and Cl accumulation in leaves. Increased electrolyte leakage from leaf tissues evidenced damages to leaf cell plasma membrane of both cultivars in S/S condition. However, damages to chloroplasts, as inferred from chlorophyll loss, were much pronounced in Lincoln than in D. Provence. Antioxidant enzymic activities in leaves were measured as proxies for oxidative stress. Catalase activity was stimulated by S/S treatment in both cultivars, but superoxide dismutase (Fe and Cu/Zn isoforms) and gaiacol peroxidase activities were augmented only in Lincoln. The absence of superoxide dismutase activity stimulation by salt in D. Provence could signify either that constitutive activity was sufficient to ensure protection against oxidative stress, or that intrinsic salt tolerance of this cultivar mitigated cellular oxidative stress. Thus, intraspecific variability for salt response exists between pea cultivars presenting similar growth sensitivity to salt. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Antioxidant enzyme activity Pisum sativum L. Salinity Split-root
1. Introduction Salinity is one of the major abiotic stresses that affect plant productivity (Gueta-Dahan et al., 1997). Through its osmotic effect, salt in the soil causes stomatal closure, which reduces the CO2/O2 ratio in leaves and inhibits CO2 fixation. Other detrimental effects are due to salt accumulated in plant tissues. However, direct Na+ and Cl toxicity does not seem to be the sole cause of salt-induced growth reduction (Hu et al., 2005). Indeed, salt tolerance depends on the provision to plants of several macro-elements, among which N, K, and Ca are the most important ones (Grattan and Grieve, 1999). Salt has been shown to depress the chloroplast content in K+, NO3 and SO42 (Schro¨ppel-Meier and Kaiser, 1988). In barley,
Abbreviations: D. Provence, Douce de Provence; GPX, gaiacol peroxidase; SOD, superoxide dismutase; ROS, reactive oxygen species. * Corresponding author. Tel.: +216 71 872 600; fax: +216 71 871 666. E-mail address: [email protected] (H. Attia). 1 These authors have participated equally to this work. 0304-4238/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2009.09.002
the efficiency of salt treatment to stimulate K+ efflux and deplete cellular K+ pools correlated with cultivar sensitivity to salinity (Chen et al., 2005). In Arabidopsis and tomato mutants, hypersensitivity to salt is frequently associated with loss of capacity to absorb K+ (Borsani et al., 2001; Zhu et al., 1998). Split-root experiments have shown that inhibition of K+ uptake at high salinity level limited growth of Crithmum maritimum (Ben Hamed et al., 2008). Similar experiments indicated that K+ and Ca2+ were limiting for Arabidopsis growth when they were presented to roots together with NaCl, but not when they could be absorbed by roots placed in control medium, the other part of the root system being submitted to NaCl (Attia et al., 2008b). Salt stress increases the rate of reactive oxygen species (ROS: superoxide, hydrogen peroxide, hydroxyl radical and singlet oxygen) formation, inducing oxidative stress in plants (Parvaiz et al., 2008). Oxidative stress is thought to arise in chloroplasts primarily from unbalance between absorbed light energy and metabolic energy use, because these conditions favour leakage of electrons to oxygen (Hasegawa et al., 2000), and from other
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disturbances of electron transport chain (Diaz-Vivancos et al., 2008). However, it may also originate from other dysfunctions such as excess salt accumulation in leaves, and mineral deficiency (Tewari et al., 2004). Indeed, salt-induced K+ deprivacy induces antioxidant response (Attia et al., 2008a), and it has been shown to activates NAD(P)H oxidases responsible for ROS production (Cakmak, 2005). Various genes and proteins, expression of which is activated in response to salt stress, have been identified, some of them ensuring protection against ROS (Hasegawa et al., 2000). To mitigate and repair damages initiated by ROS, plants have developed a complex antioxidant system which includes enzymes such as superoxide dismutase, catalase, various peroxidases, and many other enzymes involved in the ascorbate-glutathione cycle (Foyer and Halliwell, 1976). In pea, the components of this antioxidant defence system are found in different subcellular compartments (Hernandez et al., 1993; Jime´nez et al., 1997). Induction of several of these components (Hernandez et al., 1999) has been shown to correlate with long-term salt tolerance in this species (Hernandez et al., 2000). In the present study, two pea cultivars were compared for their salt response. A split-root system was devised for salt application, aimed at distinguishing the effects of the salt in the root medium from those of the salt accumulated in the tissues. Effects of salt were investigated on plant growth and ion accumulation in leaves, and several proxies for salt-induced stress in leaves were used, including chlorophyll content decrease, damages to membrane permeability, and antioxidant enzymes. 2. Materials and methods 2.1. Plant material Seeds of two pea (Pisum sativum L.) commercial cultivars largely used in Tunisia were obtained from Laboratoire des Le´gumineuses a` graines of Tunisian National Institute for Agronomic Research. Douce de Provence (D. Provence thereafter) has a shorter reproductive cycle (45 d) than Lincoln (60 d). 2.2. General culture conditions Seeds were surface sterilized with 96% (v/v) ethanol for 3 min, and with 2% (w/v) sodium hypochlorite for further 5 min. After rinsing they were sown on vermiculite. Vigorous seedlings were selected and grown in aerated distilled water for 7 d. Then, plants were transplanted to aerated nutrient solution of Long Ashton (Hewitt, 1966) for another 7 d. Thereafter, this solution is named control (C) medium. The growth chamber conditions were set at 25/18 8C day/night temperature, 80% relative humidity and 150 mmol m2 s1 of light intensity with a 14-h photoperiod. 2.3. Salt treatments Fourteen-day-old plants grown on C medium were used. Their root systems were separated in two even parts, each placed in a pot filled with an aerated liquid medium. Three lots of plants were prepared, distinguished according to the nature of the root medium: C/C plants with control medium in both pots, S/S plants with C medium supplemented with 75 mM NaCl (S medium) in both pots, and C/S plants with C medium in one pot and S medium in the other pot. Eight plants of each treatment were harvested 3 weeks later. 2.4. Biomass measurements and inorganic ions assays At the harvest, individual plants were divided into leaf, stem and root fractions. The fresh weights (FW) were immediately
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determined. The samples were then dried in a forced-draft oven at 70 8C for 48 h and dry weights (DW) were determined. Ions were extracted with 0.5% HNO3. Cations were assayed by flame photometry (Eppendorf) with butane-air flame for K+ and Na+, and acetylene-air flame for Ca2+, and Cl by coulometry (Butcher Cotlove chloridometer), according to manufacturers’ instructions. 2.5. Total chlorophyll content analysis Total chlorophylls were extracted from fresh leaves in 80% acetone and assayed photometrically according to Arnon (1949). 2.6. Electrolyte leakage Electrolyte leakage was determined as described by DionisioSese and Tobita (1998). Leaf discs were cut into 2–3 mm pieces and placed in test tubes containing 10 mL distilled water. The tubes were incubated in a water bath at 32 8C for 2 h and the initial electrical conductivity of the medium (EC1) was measured. The samples were autoclaved at 121 8C for 20 min to release all electrolytes, then cooled to 25 8C, and the final electrical conductivity (EC2) was measured. The electrolyte leakage (EL, %) was calculated as EL = 100 EC 1/EC2 2.7. Enzyme extraction and assays All operations were performed at 0–4 8C. Leaves (0.2 g) were homogenized with a mortar and pestle in 0.6 mL of ice cold pH 8 50 mM phosphate buffer containing 50 mM Tris–HCl pH 7, 1% (w/v) polyvinyl pyrrolidone, 0.05% TritonX-100 (w/v), 0.5 mM phenylmethylsulphonyl fluoride, 1 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol, 20 mM MgCl2 and 50 mM KCl. The homogenate was centrifuged at 13,000 g for 30 min at 4 8C and the supernatant was used for enzymatic activity measurements. Protein concentration was determined according to Bradford (1976) using bovine serum albumin as a standard. Enzyme activities were analyzed after native gel electrophoresis of the supernatant. For superoxide dismutase (SOD, EC 1.15.1.1) and gaiacol peroxidase (GPX, EC 1.11.1.7), stacking gel was 5% acrylamide in pH 6.8, 0.5 M Tris–HCl buffer, and resolving gel was 5% acrylamide in pH 8.8, 1.5 M Tris–HCl buffer. Catalase (EC 1.11.1.6) was identified on 6% acrylamide gels. A same pH 8.3, 23 mM Tris–glycine running buffer was used for the three enzymatic systems. The migration was performed at 4 8C under 120 V, with a pH 4.0, 8 mM glycine buffer as the running buffer, using Bio-Rad Miniprotean system. SOD activity was revealed by incubating the gels in the dark, using 2.5 mM nitroblue tetrazolium in pH 7.8, 50 mM phosphate buffer, and then developed for 20 min under moderate light, using a mixture of 0.6 mM riboflavin and 0.4% (w/v) N,N,N,N0 ,N0 tetramethylethylenediamine in pH 7.8, 50 mM phosphate buffer (Beauchamp and Fridovich, 1971). The identity of SOD isoforms was determined using SOD inhibitors: KCN for Cu/Zn-SOD and H2O2 for both Cu/Zn-SOD and Fe-SOD (Pan and Yau, 1992). To show peroxidase activity, the gels were incubated for 30 min in the dark in 50 mL of pH 4, 100 mM acetate buffer containing 1% (w/v) gaiacol. They were then transferred to 50 mL acetate buffer (100 mM, pH 5) containing 25 mg of 3-amino-9-ethylcarbazole, 25 mL of N,N-dimethylformamide (99%, v/v), 1 mL 0.1 M CaCl2 and 0.5 mL H2O2 (30%, v/v). Gel staining for catalase activity was performed according to Chandlee and Scandalios (1983) after pretreatment in 0.01% (v/v) H2O2 for 10 min. The staining mixture contained 1% (w/v) FeCl3 and 1% (w/v) K3Fe(CN)6 in distilled water.
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2.8. Statistics Statistical analyses were performed with StatisticaTM Software, using ANOVA and Newman–Keuls test for post hoc mean comparison. 3. Results 3.1. Growth and salt accumulation After 21 d of culture in S/S configuration, the whole plant biomass was 68% (Lincoln) and 76% (D. Provence) of control (C/C) plants (Fig. 1). C/S configuration alleviated the salt-induced growth inhibition, partially in cv Lincoln, and totally in cv D. Provence. Large amounts of Na+ and Cl were accumulated in S/S plants, exceeding 1.4 mmol g1 DW in leaves of both cultivars (Fig. 2). C/S treatment strongly limited Na+ and Cl accumulation in Lincoln leaves, as compared to full salt treatment. On the contrary, in D. Provence leaves, it led to Na+ and Cl accumulation almost as high as in S/S treatment. 3.2. Potassium and calcium In leaves of both cultivars, the accumulation of K+ (Fig. 2) was depressed by S/S treatment as compared to C/C treatment. That of Ca2+ was not significantly modified. Culture in C/S configuration partially alleviated the restrictions imposed by salt to K+ accumulation. 3.3. Total chlorophylls Total chlorophylls were much more affected by salt in Lincoln than in D. Provence (Fig. 3). The chlorophyll content of D. Provence leaves was reduced to by S/S treatment to 70% of its control value (chlorophyll content of C/C plant leaves), and only to 88% in C/S configuration. In Lincoln leaves the corresponding values were 40% (S/S) and 70% (C/S) of C/C values. 3.4. Electrolyte leakage The extent of membrane damage was estimated by electrolyte leakage from leaf tissues. This index was strongly increased by salt (S/S) in leaves of both cultivars (Fig. 4), and no significant difference between them could be evidenced. 3.5. Antioxidant enzymes Three bands of SOD activity were obtained on non-denaturing PAGE gels (Fig. 5A). The band with the lowest mobility could be attributed to Fe-SOD activity, and the fastest migrating band to Cu/ Zn-SOD activity. Salt treatment (S/S) increased significantly SOD and GPX activities in Lincoln leaves, but no appreciable change in band intensity could be observed in D. Provence (Fig. 5A and B). Finally, catalase activity was stimulated by S/S treatment in both cultivars (Fig. 5C). 4. Discussion The two cultivars presented similar growth sensitivity to salt, and similar accumulation of Na+ and Cl in their leaves when grown in full salt medium. However, they differed for three other traits: salt-induced chlorophyll loss was more important in Lincoln than in D. Provence; SOD and GPX activities were salt-inducible in Lincoln, and mostly constitutive in D. Provence; Na+ and Cl accumulation was limited in C/S configuration in Lincoln, but not in D. Provence. Comparing Lincoln responses to C/C, C/S and S/S
Fig. 1. Effect of salt treatments on whole plant biomass. Plants were 14-d old at the start of the experiment. The root system of each plant was separated in two parts, each one placed in a pot filled with an aerated liquid medium. C/C: plants with C medium (no NaCl) in both pots. S/S: plants with S medium (75 mM NaCl) in both pots. C/S: plants with half of their roots in C medium and the other half in S medium. The plants were harvested 21 d latter. Means of 8 replicates and confidence intervals for p = 0.05. Means sharing a same letter are not significantly different at p = 0.01 (ANOVA, and post hoc mean comparison with Newman–Keuls test). Lincoln and D. Provence are two cultivars.
treatments shows that growth inhibition and Na+/Cl content varied together, suggesting that excess ion accumulation was detrimental to growth in this cultivar. In D. Provence on the contrary, the presence of high Na+ and Cl concentration in leaves of C/S plants did not prevent growth from proceeding at rate comparable to that of plants not challenged by salt. Thus, the depressive effect of the full salt (S/S) treatment on D. Provence growth was likely not due to a toxic effect of accumulated ions. An opposite behaviour has been described for pepper (Lycoskoufis et al., 2005). In this salt tolerant species, culture in split-root (C/S) configuration hardly mitigated the adverse effects of salt, suggesting direct effect of absorbed Na+ and Cl ions. The accumulation of Na+ and Cl in leaves of glycophytes is known to impose various constraints which alter cell functional state, resulting in physiological stress (Gaspar et al., 2002). As initially hypothesized by Oertli (1968) and later confirmed (Flowers et al., 1991), in the absence of efficient internalization of the ions by leaf cells, their concentration in the leaf apoplast may reach excessive values, leading to leaf cell dehydration (Munns and Passioura, 1984) and membrane disruption (Speer and Kaiser, 1991). Furthermore, if not efficiently compartmentalized in leaf cell vacuoles, Na+ and Cl may build to toxic levels in the cytoplasm (Flowers et al., 1991; Kronzucker et al., 2006), and essential metabolic pathways such as photosynthesis may be inhibited. Ionic and osmotic disturbances may also result in stomata closure, which limits photosynthesis (Meloni et al., 2003). For both cultivars, the parallelism between growth inhibition and leaf K+ content reduction over the C/C, C/S and S/S treatments suggests that salt-induced restriction to K+ provision to leaves might be involved in the growth inhibition by salt. In contrast, Ca2+ transport to leaves was not modified by the treatments. Increase in leaf Na+ and Cl content associated with K+ content decrease upon increasing NaCl concentrations in the medium has been described in pea (Hernandez et al., 1999). Either genetic or salt-induced, deficiency in the capacity to absorb K+ and to maintain K+ cytoplasmic homeostasis is associated with sensitivity to salinity (Borsani et al., 2001; Chen et al., 2005; Munns and Tester, 2008). The salt-induced diminution of cytosolic K+ activity has been proposed to inhibit growth through a variety of mechanisms
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Fig. 2. Ion concentration in leaves of pea plants grown in split-root configuration. Treatment: See legend of Fig. 1. Means sharing a same letter are not significantly different at p = 0.01 (ANOVA, and post hoc mean comparison with Newman–Keuls test performed separately for each cultivar and each element).
including oxidative damages. Indeed, K+ deficiency enhances ROS production and oxidative damages to chloroplasts (Cakmak, 2005). More generally, oxidative damages are a possible cause of the growth reduction which accompanies starvation in N, P, K and S (Cakmak, 1994; Kumar et al., 2004). When plants were grown in salty medium, chlorophyll concentration was largely diminished in Lincoln but much less in D. Provence. The loss of chlorophylls is considered as a marker of
a cellular component of salt stress (Singh and Dubey, 1995). Thus, our results support the hypothesis that chloroplasts of Lincoln were more stressed by salt than those of D. Provence, in spite of similar extent of leaf cell plasma membrane damages in both cultivars, as judged from the magnitude of the electrolyte leakage. Salt tolerance involves resistance to oxidative stress. Indeed, antioxidative enzymic activities generally are augmented in stressed plants (Gueta-Dahan et al., 1997; Hasegawa et al.,
Fig. 3. Total chlorophyll concentration in leaves of pea plants grown in split-root configuration. See legend of Fig. 1. Means sharing a same letter are not significantly different at p = 0.01 (ANOVA, and post hoc mean comparison with Newman–Keuls test performed separately for each cultivar).
Fig. 4. Electrolyte leakage from discs of pea leaves. The plants were grown for 21 d at 0 (control: C) and 75 mM (salt: S) NaCl. Means of 8 replicates and confidence intervals for p = 0.05. Means sharing a same letter are not significantly different at p = 0.01 (ANOVA, and post hoc mean comparison with Newman–Keuls test performed separately for each cultivar). Lincoln and D. Provence are two cultivars.
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Fig. 5. Electrophoregrams of antioxidant enzymes in extracts from pea leaves. The plants were submitted to the indicated treatments for 21 d. Lincoln and D. Provence are two cultivars. (A) Superoxide dismutase, (B) gaiacol peroxidase, and (C) catalase.
2000), including pea (Hernandez et al., 1995, 1999, 2000). Futhermore, when different pea cultivars were compared, antioxidant response was induced by salt in tolerant cultivars, but not in salt-sensitive ones (Hernandez et al., 1995, 2000, 2001; Gomez et al., 1999). Thus, the rationale commonly used to interpret overexpression of antioxidant systems is that it is an adaptative response to apparition of ROS in cells. In our work, however, there was no parallelism between salt tolerance and antioxidant system inducibility. One reason for the absence of SOD and GPX induction in leaves of salt-treated D. Provence cultivar might be that the constitutive level of these activities was sufficient to cope with the imposed stress. A complementary explanation would be that the strength of cellular stress was not sufficient in D. Provence to sollicitate supplementary SOD and GPX expression. In summary, an important conclusion of our study is that an intraspecific variability exists for salt response in pea even between cultivars presenting similar growth sensitivity to salt. This variability concerns salt transport to leaves, chloroplast sensitivity to salt, and pattern of antioxidant response. Acknowledgement Authors are indebted to Professor C. Grignon for stimulating discussions on this work. References Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxydase in Beta vulgaris. Plant Physiol. 24, 1–15. Attia, H., Arnaud, N., Karray, N., Lachaal, M., 2008a. Long-term effects of mild salt stress on growth, ion accumulation and superoxide dismutase expression of Arabidopsis rosette leaves. Physiol. Plant. 132, 293–305. Attia, H., Karray, N., Rabhi, M., Lachaal, M., 2008b. Salt-imposed restrictions on the uptake of macroelements by roots of Arabidopsis thaliana. Acta Physiol. Plant. 30, 723–727. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276–287. Ben Hamed, K., Messedi, D., Ranieri, A., Abdelly, C., 2008. Diversity in the response of two potential halophytes (Batis maritima and Crithmum maritimum) to salt ¨ ztu¨rk, M., Ashraf, M., Grignon, C. (Eds.), Biosaline stress. In: Abdelly, C., O Agriculture and High Salinity Tolerance. Birkha¨user, Basel, pp. 71–80. Borsani, O., Cuartero, J., Fernandez, J.A., Valpuesta, V., Botella, M.A., 2001. Identification of two loci in tomato reveals distinct mechanisms for salt tolerance. Plant Cell 13, 873–887.
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