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Osmotic adjustment, water relations and gas exchange in pepper plants grown under NaCl or KCl
Environmental and Experimental Botany 52 (2004) 161–174
Osmotic adjustment, water relations and gas exchange in pepper plants grown under NaCl or KCl M.C. Mart´ınez-Ballesta, V. Mart´ınez, M. Carvajal∗ Departamento de Nutrición y Fisiolog´ıa Vegetal, Centro de Edafolog´ıa y Biolog´ıa Aplicada del Segura, CSIC. P.O. Box 4195, 30080 Murcia, Spain Accepted 28 January 2004
Abstract Pepper plants (Capsicum annuum L. cv. Orlando) were used to compare the effects of NaCl and KCl on osmotic adjustment, water relations, and gas exchange. Thus, two different saline treatments, 60 mM NaCl and 60 mM KCl, were applied and different measurement times (1, 2, 3 and 10 days) were assayed in order to determine the effect of the treatment duration on the parameters studied. Reductions in root hydraulic conductance, stomatal conductance and net assimilation of CO2 were observed after NaCl and KCl addition. Mineral composition of leaf sap was also determined and it was observed that Cl− and NO3 − were the main anions used by pepper plants to achieve the osmotic adjustment. Also, salinity induced a decrease in the concentrations of Ca2+ and Mg2+ in leaves. Osmotic regulation by organic solutes was also determined, by analysis of the contents of sugars and amino acids. It appeared that sucrose was the main carbohydrate accumulated by the plants in order to maintain turgor. However, the degree of osmotic adjustment observed indicated that changes in leaf turgor occurred after either saline treatment, for all application times, suggesting that pepper plants could not adjust their water relations sufficiently. Thus, Na+ and K+ exerted a toxic effect on pepper plants mainly by affecting the plant water relations, although the effect of Na+ on water relations parameters was more significant than that of K+ . © 2004 Elsevier B.V. All rights reserved. Keywords: Capsicum annuum; Net assimilation of CO2 ; Osmotic adjustment; Root hydraulic conductance; Stomatal conductance; Water relations
1. Introduction
Abbreviations: DTT, dl-dithiothreitol; L0 , water hydraulic conductance; Gs , stomatal conductance; ACO2 , net assimilation of CO2 ; Jv , sap flow; ψw , water potential; ψ , osmotic potential; ψp , turgor potential ∗ Corresponding author. Tel.: +34-968-396310; fax: +34-968-396213. E-mail address: [email protected] (M. Carvajal).
High levels of saline ions in the apoplast alter the aqueous and ionic thermodynamic equilibria, which results in hyperosmotic stress, ionic imbalance and toxicity (Cramer et al., 1986). Thus, the exposed plants have to minimise water loss and thereby maintain a favourable water status for development (Sohan et al., 1999). Cells must then develop a sufficiently low osmotic potential to reverse the flow of water, either through the uptake of ions from the medium or by
0098-8472/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2004.01.012
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synthesis and transport of organic compounds. Thus, osmotic adjustment helps plant cells to withstand salt stress and water deficits by maintaining sufficient turgor for growth (Carvajal et al., 1999). It involves the regulation of the intracellular levels of organic compounds, many of which are compartmented mainly in the cytoplasm, whereas inorganic ions (principally Na+ , K+ and Cl− ) are sequestered in the vacuole or cytoplasm (Jeschke et al., 1986; Voetberg and Sharp, 1991). Wyn Jones (1981) reported that the osmotic adjustment by salt accumulation needs less energy and carbon than the adjustment by organic solutes. But, Leigh and Storey (1993) proposed that this capacity to include salts is a beneficial trait only when the absorption of salts is accompanied by an ability of the plant to regulate internal Na+ and Cl− concentrations. In addition, ions or charged metabolites are encountered (Hasegawa et al., 2000). The accumulation of these osmolytes is believed to facilitate “osmotic adjustment” to a greater extent. The effects of salinity on root hydraulic conductance (L0 ) have been reported widely (Shalhevet et al., 1976; Munns and Passioura, 1984; Evlagon et al., 1990; Navarro et al., 2000; Martinez-Ballesta et al., 2000), and it has been suggested that they are due to the hyperosmotic stress and ionic imbalance caused by the high apoplastic concentrations of Na+ and Cl− . Also, net photosynthesis is affected strongly by NaCl-saline conditions and this is related directly to a reduction in stomatal conductance as well as to low intercellular CO2 levels (Downton et al., 1985; Ouerghi et al., 2000; Bayuelo-Jimenez et al., 2003). Thus, in saline conditions, decreases in CO2 assimilation, stomatal conductance and water potential in leaves of Citrus trees have been shown (Lloyd et al., 1989, 1990; Walker, 1982; Garc´ıa-Sanchez et al., 2002). The application of iso-osmotic concentrations of NaCl and KCl to pepper plants could help in discriminating the effects of specific ion toxicities during salt stress. In root cells, both salts seem to have similar effects on aquaporin functionality and H+ -ATPase activity (Mart´ınez-Ballesta et al., 2002). Therefore, the aim of the present work was to study the effects of two different salts, NaCl and KCl, on the physiology of the whole plant. For this, water relations, gas exchange and osmotic adjustment of the plants were assessed at different times after the addition of the salts.
2. Material and methods 2.1. Plant culture Seeds of pepper (Capsicum annuum L. cv. Orlando) were germinated on wet filter paper, in the dark at 29 ◦ C, for 15–20 days. After this time, the seedlings were placed in 0.5 litre trays containing continuously-aerated Hoagland nutrient solution: KNO3 (3 mM), Ca(NO3 )2 (2 mM), NH4 H2 PO4 (0.5 mM), MgSO4 (0.5 mM), KCl (50 M), H3 BO3 (25 M), MnSO4 (2 M), ZnSO4 (2.0 M), CuSO4 (0.5 M), H2 MoO4 (0.5 M), Fe-ethylendiaminodi(o-hydroxyphenylacetic) acid (Fe-EDDHA) (20 M). Five days later, plants were divided in two groups for the experiments. One group of plants was transferred and grown in nine containers (of 15 litre capacity, with 8 plants per container) for L0 and gas exchange measurements. Another group of plants was transferred into three containers (of 15 litre capacity, with 10 plants per container) to measure leaf water relations and ion and organic solute concentrations. Both groups of plants were grown with Hoagland solution (pH 5.5), in a controlled environment with a 16 h photoperiod, at 25 ◦ C, 75% relative humidity (RH) and a photosynthetically active radiation (PAR) of 500 mol m−2 s−1 , and 8 h in the dark, at 18 ◦ C and 60% RH. Nutrient solution was changed every 4 days and pH was checked and adjusted daily. The plants were subjected to the following treatments; control (Hoagland), NaCl (60 mM) and KCl (60 mM). The measurements were carried out 1, 2, 3 and 10 days after treatment application. 2.2. Root hydraulic conductance Hydraulic conductance (L0 ) of roots was measured by pressurising the roots using the Schölander chamber (Jackson et al., 1996). For this, the aerial parts of the plant were removed, leaving the base of the stem which was sealed, with silicone grease, into a tapered glass tube. The plant was placed into a pressure chamber, with the same nutrient solution that it was grown in, and a gradual increase of pressure (from 0.1 to 0.5 MPa) was applied to detached roots. The range of the generated sap flows included a flow equivalent to the whole-plant transpiration flow. In the pressure chamber, it is assumed that there is a balance between
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the negative pressure in the xylem of the cut leaf and the pressure that forces water from the cells into the vessels. Following a different time at each pressure, the xylem sap for solute analysis was collected using Pasteur pipettes and transferred to Eppendorf tubes. The Eppendorf tubes and the roots of the plant were weighed. Sap flow (Jv ) was calculated as mg (g root FW)−1 h−1 and plotted against pressure, the slope being the L0 value. Mercuric chloride (50 M) was added to the nutrient solution of plants from which shoots had been excised for the measurement of the initial values of L0 . After 5 min, the plants were transferred to a nutrient solution without Hg and the L0 was measured 1 h later. Mercury was scavenged from roots with DTT (5 mM). After 1 h, L0 was measured as described above.
2.5. Ion analysis of xylem sap
2.3. Gas exchange
Organic solutes were measured in the leaf sap. For sugar analysis, the sap was filtered through a sep-pak C18 cartridges to remove the green colour. Glucose, fructose and sucrose concentrations were determined directly by HPLC, using a LiCrospher 100 NH2 5 m column coupled with a differential refractrometer detector. The mobile phase was acetonitrile:water (85:15), with a flow rate of 1.5 ml min−1 . All data were registered and processed using the Borwing 1.21 computer programme. Quantification was carried out by comparing peak areas with those of known standards. Total amino acids were determined by the ninhydrin method (Rosen, 1957).
Net CO2 assimilation (ACO2 ) and adaxial stomatal conductance, Gs , of leaves (more stomata were detected on the adaxial surface of the leaves, data not shown) were measured using a portable photosynthesis system (Li-COR LI-6200) attached to an infra-red CO2 analyser (IRGA LI-6250). Measurements were made on the most recent fully-expanded leaves, during the middle of the photoperiod. 2.4. Leaf water relations The leaf water potential (ψw ) of the most recent fully-expanded leaves was measured using a pressure chamber technique (Turner, 1988). The same leaves were then put in Eppendorf tubes with holes at the bottom and rapidly frozen with liquid nitrogen. These tubes were then centrifuged twice, at 4000 × g for 4 min (4 ◦ C), using a Hettich-Universal32R centrifuge, in such a way that all sap was extracted from samples. The osmotic potential (ψ0 ) of the leaf sap was calculated, after measuring sap osmolarity with an automatic freezing-point depression osmometer (Digital Osmometer, Roebling, Berlin), by the van’t Hoff equation (Nobel, 1991): ψ0 = −nRT where n is mosmol, R = 0.083 and T = t a (K). Turgor potential (ψp ) was calculated as the difference between leaf water potential and osmotic potential.
For the ion analysis, the xylem sap collected was diluted and injected into a Dionex-D-100 ion chromatograph. For the anions, an Ionpac AS12A (4 mm × 250 mm) (10–32) column and guard column were used. The flow rate was 1.5 ml min−1 with an eluent of 2.7 mM Na2 CO3 /0.3 mM NaHCO3 . For the cation analysis, an lonpac CS12A (4 mm × 250 mm) (10–32) column and guard column were used. The flow rate was 1 ml min−1 , with an eluent of 11 mM H2 SO4 . The ions were detected with a conductivity detector and quantified by comparing peak areas with those of known standards. 2.6. Organic solute analysis of the xylem sap
3. Results Root hydraulic conductance values for different saline treatments and different times of salt application were determined (Fig. 1). A great reduction in L0 for plants treated with KCl or NaCl, compared to control plants, can be appreciated (Fig. 1a). The decrease was similar for both salinity treatments. In the control treatment, L0 values fell sharply with HgCl2 addition (Fig. 1b) at all times of measurement. Using DTT as a scavenger for Hg2+ , L0 of control plants was restored to values similar to the pre-treatment value (Fig. 1c). There were no significant differences in L0 in NaClor KCl-treated plants after Hg2+ addition, with regard to their controls, on any day of measurement. Root hydraulic conductivity decreased with increas-
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Fig. 1. Hydraulic conductance (L0 ) of roots of control pepper plants and plants treated with 60 mM NaCl or 60 mM KCl (a); after HgCl2 (50 M) (b) and DTT (2 mM) (c) treatments. Measurements were carried out 1, 2, 3 and 10 days after saline treatment addition. Number of roots, 5; error bars, ±S.E.
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Fig. 2. Net assimilation of CO2 (ACO2 ) and stomatal conductance (Gs ) of leaves of control pepper plants and plants treated with 60 mM NaCl or 60 mM KCl. Measurements were carried out 1, 2, 3 and 10 days after saline treatment addition. Number of roots, 6; error bars, ±S.E.
ing time of measurement, being greater in control plants. Net assimilation of CO2 (ACO2 ) and stomatal conductance (Gs ) were measured for leaves in order to study gas exchange (Fig. 2). Values of ACO2 and Gs decreased in both saline treatments compared to control plants, but this reduction was higher in NaCl-plants. Net assimilation of CO2 showed a tendency to increase with time, but Gs showed a tendency to decrease with time.
Leaf water potential, Ψ w , decreased after NaCl or KCl addition, with regard to control plants (Fig. 3), the decrease being greater for NaCl-treated plants. A tendency of Ψ w to increase with the time of measurement was observed in control plants. The presence of NaCl or KCl in the nutrient solution reduced slightly the values for osmotic potential, Ψ , compared to control plants. There was a general increase of Ψ with time in all treatments. Turgor potential, ψp , was reduced with respect to control plants after NaCl or KCl
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Fig. 3. Water potential (ψw ), osmotic potential (ψ ) and turgor (ψp ) of leaves of control plants and plants treated with 60 mM NaCl or 60 mM KCl. Measurements were carried out 1, 2, 3 and 10 after saline treatment addition. Number of roots, 5; error bars, ±S.E.
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Fig. 4. Concentrations of anions (Cl− , NO3 − , PO4 3− and SO4 2− ) in leaf sap of control pepper plants and of plants treated with 60 mM NaCl or 60 mM KCl. Measurements were carried out 1, 2, 3 and 10 days after saline treatment addition. Number of roots, 5; error bars, ±S.E.
addition, but the decrease was greater for NaCl-treated plants. A decrease of ψp with days of measurement was observed in control plants and in plants treated with NaCl or KCl. Ion analyses for leaf sap of pepper plants treated with NaCl or KCl, at different times of measurement (24, 48 and 72 h and 10 days), were carried out in order to evaluate the relative importance of each nutrient (Fig. 4). Chloride concentration increased progressively with time of measurement in both NaCland KCl-treated plants, compared to control plants, and was higher for the NaCl treatment. Concentrations of NO3 − increased during the 3 days after NaCl or KCl addition, with regard to control plants. However, a great decrease was observed 10 days after the application of the saline treatments. There were no marked differences for PO4 3− or SO4 2− concentrations between salinised and control plants. Also, no changes in PO4 3− or SO4 2− levels were observed with the time of measurement.
Regarding cation analysis, the sap K+ concentration was significantly higher, for all treatments, as compared with Na+ and Mg2+ (Fig. 5). For the K+ concentrations, there were no differences between treatments or days of measurement. Sap concentration of Na+ increased after NaCl treatment and the Na+ content increased with the days of measurement. However, in control plants and in plants treated with KCl there were no significant differences in Na+ concentration, and it remained unchanged with the time of measurement. The concentration of Mg2+ was higher for control plants. The Mg2+ concentration declined on the 10th day after NaCl or KCl addition. However, the reduction was higher for KCl-treated plants than for NaCl-treated plants. The concentration of Ca2+ was also determined but no changes were observed (data not shown). Fructose concentration remained unchanged between treatments and with the time of measurement
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Fig. 5. Concentrations of cations (Na+ , K+ and Mg2+ ) in leaf sap of control pepper plants and of plants treated with 60 mM NaCl or 60 mM KCl. Measurements were carried out 1, 2, 3 and 10 days after saline treatment addition. Number of roots, 5; error bars, ±S.E.
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Fig. 6. Glucose, fructose and sucrose concentrations in leaf sap of control pepper plants and of plants treated with 60 mM NaCl or 60 mM KCl. Measurements were carried out 1, 2, 3 and 10 days after saline treatment addition. Number of roots, 5; error bars, ±S.E.
(Fig. 6). Glucose concentration was similar in control plants and in plants treated with NaCl, whereas it was lower for KCl-treated plants. It also remained unchanged during the days of measurement. Sucrose
concentration increased progressively over time in NaCl- and KCl-plants compared to control plants. The increase was higher in the KCl treatment than with NaCl during the first 3 days.
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Fig. 7. Total amino acids concentration in leaf sap of control pepper plants and of plants treated with 60 mM NaCl or 60 mM KCl. Measurements were carried out 1, 2, 3 and 10 days after saline treatment addition. Number of roots, 5; error bars, ±S.E.
Soluble amino acids decreased similarly for both salinity treatments with regard to control plants (Fig. 7). A general tendency of the amino acids concentration to increase within the first 3 days of measurement was observed for all treatments.
4. Discussion In both saline treatments, there was a reduction in L0 with respect to control values (Fig. 1). Strong reductions of L0 have been shown in several species by Shalhevet et al. (1976), Munns and Passioura (1984) and Evlagon et al. (1990). However, while these authors suggested that the osmotic concentration had negative effects on L0 , we hypothesised that there must have been a toxic effect of NaCl or KCl, rather than an osmotic effect, on L0 . Thus, in previous experiments we observed that large reductions in root hydraulic conductance of salinised plants were related closely to the decrease in the activity or concentration of aquaporins in the root plasma membrane (Carvajal et al., 1999, 2000), this effect being due mainly to the specific toxicity of Na+ and Cl− (Mart´ınez-Ballesta et al., 2000).
It has been shown that HgCl2 blocks the flow of water through aquaporins in plant roots and that the flow is restored by reducing agents (Maggio and Joly, 1995; Carvajal et al., 1996). However, this reagent had only a slight effect on NaCl- and KCl-treated plants (Fig 1b). In this work, the very weak response of the L0 of salt-stressed roots to the Hg treatment suggests that, in the plasma membrane, aquaporins were greatly reduced in number or, if present, were non-functional. In previous experiments (Martinez-Ballesta et al., 2000), treatments with Na+ or Cl− salt mixtures produced similar decreases in L0 , but the ameliorative effect of Ca2+ only occurred with the Na+ salts treatment, suggesting that Na+ was most likely the ion responsible for such reductions. However, in the current experiment, similar results were obtained with both salts (NaCl and KCl). Also, there were no effects of Ca2+ on the restoration of L0 in plants treated with KCl (data not shown). Therefore, Cl− could have an effect on L0 and on aquaporin functionality. A tendency of L0 to decrease with the time of measurement was observed in all treatments, which could be explained by the modifications of root structure with age that involve changes in hydraulic resistance (Mencuccini and Grace, 1996).
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Leaf gas exchange of pepper plants was impaired by NaCl or KCl addition to the nutrient solution (Fig. 2). This reduction has been attributed to salt effects on stomata, with the consequent restriction of CO2 availability for carboxylation, or to the acceleration of senescence (Pessarakli, 1994). In our experiments, the rate of photosynthesis decreased in both saline treatments, compared to control plants, during the 3 days of measurements. This decrease could be due mainly to the reduction in stomatal conductance that was observed in all salinised plants, but not to injuries to the photosynthetic apparatus (Björkman and Demmig, 1987; Demmig and Björkman, 1987), supported by the fact that, after 10 days, ACO2 values of salinised plants had recovered, reaching values similar to or higher than control plant values (Fig. 2). A decrease in Gs was observed for the NaCl and KCl treatments, probably caused by closure of the stomata or a decrease of water uptake through the roots. The quick response to NaCl or KCl addition observed in pepper plants, in terms of water loss and a reduction in Gs , could indicate a certain level of adaptation of this crop to salt stress (Munns and Termaat, 1986). A general tendency of Gs to decline with time was seen for all treatments, which may reflect reported decreases of stomatal conductance with plant age (Mencuccini and Grace, 1996). It has been proposed that the reduction of leaf gas exchange in response to salinity is due to an increase in leaf Na+ concentration (Garc´ıa-Legaz et al., 1993; Walker et al., 1993). However, other authors associated reductions in photosynthetic capacity and stomatal conductance with high concentrations of Cl− (Bañuls et al., 1997; Garc´ıa-Sanchez et al., 2002). In our plants, an effect of both Na+ and Cl− could have occurred, since there were reductions with both treatments, but the reduction was greater in plants treated with NaCl. Osmotic adjustment involves the net accumulation of solutes in cells in response to a fall in the water potential of their environment. As a consequence of this net accumulation, the cell osmotic potential is lowered, and turgor pressure tends to be maintained (Blum et al., 1996). In our experiments, the NaCl and KCl treatments markedly reduced the leaf water potential and this change was not compensated for by a reduction in leaf osmotic potential (Fig. 3), the values of which showed only a small decrease compared to
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control plants. Thus, turgor was not maintained and osmotic adjustment was not sufficient to offset the reduction in leaf water potential in salt-stressed plants. The changes observed in all the water relations in the plants during the time of measurement were possibly due to changes in metabolism related to plant age. The rate of acclimation to salinity and the duration of the salt treatment are clearly key factors influencing the plant response, but plant age and size are also likely to be involved (Franco et al., 1993). Salt tolerance in plants is associated usually with the ability to restrict the uptake and/or transport of saline ions from roots to shoots (Hajibagheri et al., 1987). Cl− and NO3 − concentrations increased in leaves of NaCl- and KCl-treated plants, suggesting that these were the main anions involved in osmotic adjustment (Fig. 4). In our experiments, the saline stress decreased the Mg2+ and Ca2+ in leaves (data not shown). This effect has been reported in other species, such as Helianthus annuus (Sanchez-Raya and Delgado, 1996) and Solanum melongena (Savvas and Lenz, 1994). Lynch and Läuchli (1985) proposed that decreases in the concentrations of these cations in shoots may be related to a reduced Ca2+ release into the root xylem, possibly due to effects on the active loading of cations into the xylem vessels. Also, excess K+ may cause a deficiency of other plant nutrients, especially Mg2+ (Chapman, 1966). Günes et al. (1996) observed equal Na+ and Cl− uptake in pepper plants. In our NaCl-treated plants, Cl− contents in leaves were higher than for Na+ . This was observed also in leaves of salinised plants of other species (Downton et al., 1985; Läuchli and Wieneke, 1989; Bethke and Drew, 1992). A possible explanation could be an increase in anion flow rate or a decrease in cation flow rate, as observed in barley and wheat roots (Hiatt and Hendricks, 1967; Osmond and Popp, 1983). Osmotic adjustment can be achieved by the accumulation of inorganic ions and/or organic substances, to permit the maintenance of turgor (Morgan, 1984, 1992). After the salinity treatments were imposed, only increases in leaf sucrose concentrations, to values nearly double those observed in control plants, were observed (Fig. 6). This significant contribution highlights this carbohydrate as the main organic solute involved in the osmotic adjustment, suggesting some diversion of carbohydrates from synthetic
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growth processes. However, the production of osmotica is metabolically expensive and limits plant production by consuming significant quantities of carbon (Greenway and Munns, 1980). The alternative to producing organic osmotica is the accumulation of a high concentration of ions from the external medium, for which the energy cost is lower. Nevertheless, this causes other problems and a high concentration of osmotic ions can produce a toxic effect on the normal biochemical activities within the cell (Volkmar et al., 1998). In this work, the contribution of amino acids accumulation to osmotic adjustment was not significant, since in the NaCl and KCl treatments the amounts of amino acids were decreased with regard to control plants (Fig. 7). This could be due to a toxic effect of Na+ , K+ and/or Cl− on cytoplasmic enzymes involved in amino acids synthesis. So, we can conclude that Na+ and K+ exerted a toxic effect on pepper plants mainly by affecting the plant water relations, although the effect of Na+ on these parameters was more significant than that of K+ . Since photosynthesis is thought to be one specific turgor-dependent process, it can be suggested that the low rates of gas exchange observed in NaCland KCl-leaves were a result of changes in leaf water relations. Salinity decreased water uptake in pepper plants, so that they lost turgor and the necessary osmotic adjustment, in terms of inorganic ions and organic substances accumulation, was not achieved. At high salinity, the uptake of water by plants is reduced (Ehret and Ho, 1986) and, in conditions of high transpiration demand, water uptake must balance water consumption (Awang et al., 1993). In this experiment, the increase in sucrose content and the decreases in leaf water potential and stomatal conductance under saline conditions show that, although plants may have been adjusted osmotically, water uptake could not balance water loss and so turgor decreased.
Acknowledgements This work was supported by the Ministerio de Ciencia y Tecnolog´ıa (CICYT-AGL2000-0506-C02-02). M.C. Martinez Ballesta was funded by a grant from the Fundación Seneca, Comunidad Autónoma de la Región de Murcia (Spain). The authors thank
David Walker for correction of the English in the manuscript.
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Martinez-Ballesta, M.C., Martinez, V., Carvajal, M., 2000. Regulation of water channel activity in whole roots and in protoplasts from roots of melon plants grown under saline conditions. Aust. J. Plant Physiol. 27, 685–691. Martinez-Ballesta, M.C., Martinez, V., Carvajal, M., 2002. Aquaporin functionality in relation to H+ -ATPase activity in root cells of Capsicum annuum grown under salinity. Physiol. Plant 117, 413–420. Mencuccini, M., Grace, J., 1996. Developmental patterns of above-ground hydraulic conductance in a Scots pine (Pinus sylvestris L) age sequence. Plant Cell Environ. 19, 939–948. Morgan, J.M., 1984. Osmoregulation and water stress in higher plants. Annu. Rev. Plant Physiol. 35, 299–319. Morgan, J.M., 1992. Osmotic components and properties associated with genotypic differences in osmoregulation in wheat. Aust. J. Plant Physiol. 19, 67–76. Munns, R., Passioura, J.B., 1984. Hydraulic resistance of plants. III. Effects of NaCl in barley and lupin. Aust. J. Plant Physiol. 11, 351–359. Munns, R., Termaat, A., 1986. Whole plant responses to salinity. Aust. J. Plant Physiol. 13, 143–160. Navarro, J.M., Martinez, V., Carvajal, M., 2000. Ammonium, bicarbonate and calcium effects on tomato plants grown under saline conditions. Plant Sci. 157, 89–96. Nobel, P.S., 1991. Physicochemical and Environmental Plant Physiology. Academic Press, San Diego, CA. Osmond, C.B., Popp, M., 1983. The balance of malate synthesis and metabolism in response to ion uptake in excised wheat roots. Plant Sci. Lett. 32, 115–123. Ouerghi, Z., Gornic, G., Roudam, M., 2000. Effect of NaCl on photosynthesis of two wheat species (Triticum durum and T-aestiuum) differing in their sensitivity to salt stress. J. Plant Physiol. 156, 335–340. Pessarakli, M., 1994. Handbook of Plant and Crops Stress. Marcel Dekker, New York. Rosen, H., 1957. A modified ninhydrin colorimetric analysis for amino acids. Arch. Biochem. Biophys. 67, 10–15. Sanchez-Raya, A.J., Delgado, I.C., 1996. Mineral nutrient transport by sunflower seedlings grown under saline conditions (NaCl). J. Plant. Nutr. 19, 1463–1475. Savvas, D., Lenz, F., 1994. Influence of NaCl concentration in the nutrient solution on mineral composition of eggplants grown in sand culture. Angew. Bot. 70, 124–127. Shalhevet, J., Maas, E.V., Hoffman, G.J., Ogata, G., 1976. Salinity and the hydraulic conductance of roots. Physiol Plant. 38, 224–232. Sohan, D., Jasoni, R., Zajicek, J., 1999. Plant-water relations of NaCl and calcium-treated sunflower plant. Environ. Exp. Bot. 42, 105–111. Turner, N.C., 1988. Measurement of plant water status by the pressure chamber technique. Irrig. Sci. 9, 289–308. Voetberg, G.S., Sharp, R.E., 1991. Growth of maize primary root at low water potentials. III. Role of increased proline deposition in osmotic adjustment. Plant Physiol. 96, 1125–1130. Volkmar, K.M., Hu, Y., Steppuhn, H., 1998. Physiological responses of plants to salinity: a review. Can. J. Plant Sci. 78, 19–27.
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(Citrus limon L.) trees irrigated with NaCl or Na2 SO4 . Aust. J. Plant Physiol. 20, 173–185. Wyn Jones, R.J., 1981. Salt tolerance. In: Johnson, C.B. (Ed.), Physiological Processes Limiting Plant Productivity. Butterworths, London, pp. 271–292.
Osmotic adjustment, water relations and gas exchange in pepper plants grown under NaCl or KCl M.C. Mart´ınez-Ballesta, V. Mart´ınez, M. Carvajal∗ Departamento de Nutrición y Fisiolog´ıa Vegetal, Centro de Edafolog´ıa y Biolog´ıa Aplicada del Segura, CSIC. P.O. Box 4195, 30080 Murcia, Spain Accepted 28 January 2004
Abstract Pepper plants (Capsicum annuum L. cv. Orlando) were used to compare the effects of NaCl and KCl on osmotic adjustment, water relations, and gas exchange. Thus, two different saline treatments, 60 mM NaCl and 60 mM KCl, were applied and different measurement times (1, 2, 3 and 10 days) were assayed in order to determine the effect of the treatment duration on the parameters studied. Reductions in root hydraulic conductance, stomatal conductance and net assimilation of CO2 were observed after NaCl and KCl addition. Mineral composition of leaf sap was also determined and it was observed that Cl− and NO3 − were the main anions used by pepper plants to achieve the osmotic adjustment. Also, salinity induced a decrease in the concentrations of Ca2+ and Mg2+ in leaves. Osmotic regulation by organic solutes was also determined, by analysis of the contents of sugars and amino acids. It appeared that sucrose was the main carbohydrate accumulated by the plants in order to maintain turgor. However, the degree of osmotic adjustment observed indicated that changes in leaf turgor occurred after either saline treatment, for all application times, suggesting that pepper plants could not adjust their water relations sufficiently. Thus, Na+ and K+ exerted a toxic effect on pepper plants mainly by affecting the plant water relations, although the effect of Na+ on water relations parameters was more significant than that of K+ . © 2004 Elsevier B.V. All rights reserved. Keywords: Capsicum annuum; Net assimilation of CO2 ; Osmotic adjustment; Root hydraulic conductance; Stomatal conductance; Water relations
1. Introduction
Abbreviations: DTT, dl-dithiothreitol; L0 , water hydraulic conductance; Gs , stomatal conductance; ACO2 , net assimilation of CO2 ; Jv , sap flow; ψw , water potential; ψ , osmotic potential; ψp , turgor potential ∗ Corresponding author. Tel.: +34-968-396310; fax: +34-968-396213. E-mail address: [email protected] (M. Carvajal).
High levels of saline ions in the apoplast alter the aqueous and ionic thermodynamic equilibria, which results in hyperosmotic stress, ionic imbalance and toxicity (Cramer et al., 1986). Thus, the exposed plants have to minimise water loss and thereby maintain a favourable water status for development (Sohan et al., 1999). Cells must then develop a sufficiently low osmotic potential to reverse the flow of water, either through the uptake of ions from the medium or by
0098-8472/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2004.01.012
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synthesis and transport of organic compounds. Thus, osmotic adjustment helps plant cells to withstand salt stress and water deficits by maintaining sufficient turgor for growth (Carvajal et al., 1999). It involves the regulation of the intracellular levels of organic compounds, many of which are compartmented mainly in the cytoplasm, whereas inorganic ions (principally Na+ , K+ and Cl− ) are sequestered in the vacuole or cytoplasm (Jeschke et al., 1986; Voetberg and Sharp, 1991). Wyn Jones (1981) reported that the osmotic adjustment by salt accumulation needs less energy and carbon than the adjustment by organic solutes. But, Leigh and Storey (1993) proposed that this capacity to include salts is a beneficial trait only when the absorption of salts is accompanied by an ability of the plant to regulate internal Na+ and Cl− concentrations. In addition, ions or charged metabolites are encountered (Hasegawa et al., 2000). The accumulation of these osmolytes is believed to facilitate “osmotic adjustment” to a greater extent. The effects of salinity on root hydraulic conductance (L0 ) have been reported widely (Shalhevet et al., 1976; Munns and Passioura, 1984; Evlagon et al., 1990; Navarro et al., 2000; Martinez-Ballesta et al., 2000), and it has been suggested that they are due to the hyperosmotic stress and ionic imbalance caused by the high apoplastic concentrations of Na+ and Cl− . Also, net photosynthesis is affected strongly by NaCl-saline conditions and this is related directly to a reduction in stomatal conductance as well as to low intercellular CO2 levels (Downton et al., 1985; Ouerghi et al., 2000; Bayuelo-Jimenez et al., 2003). Thus, in saline conditions, decreases in CO2 assimilation, stomatal conductance and water potential in leaves of Citrus trees have been shown (Lloyd et al., 1989, 1990; Walker, 1982; Garc´ıa-Sanchez et al., 2002). The application of iso-osmotic concentrations of NaCl and KCl to pepper plants could help in discriminating the effects of specific ion toxicities during salt stress. In root cells, both salts seem to have similar effects on aquaporin functionality and H+ -ATPase activity (Mart´ınez-Ballesta et al., 2002). Therefore, the aim of the present work was to study the effects of two different salts, NaCl and KCl, on the physiology of the whole plant. For this, water relations, gas exchange and osmotic adjustment of the plants were assessed at different times after the addition of the salts.
2. Material and methods 2.1. Plant culture Seeds of pepper (Capsicum annuum L. cv. Orlando) were germinated on wet filter paper, in the dark at 29 ◦ C, for 15–20 days. After this time, the seedlings were placed in 0.5 litre trays containing continuously-aerated Hoagland nutrient solution: KNO3 (3 mM), Ca(NO3 )2 (2 mM), NH4 H2 PO4 (0.5 mM), MgSO4 (0.5 mM), KCl (50 M), H3 BO3 (25 M), MnSO4 (2 M), ZnSO4 (2.0 M), CuSO4 (0.5 M), H2 MoO4 (0.5 M), Fe-ethylendiaminodi(o-hydroxyphenylacetic) acid (Fe-EDDHA) (20 M). Five days later, plants were divided in two groups for the experiments. One group of plants was transferred and grown in nine containers (of 15 litre capacity, with 8 plants per container) for L0 and gas exchange measurements. Another group of plants was transferred into three containers (of 15 litre capacity, with 10 plants per container) to measure leaf water relations and ion and organic solute concentrations. Both groups of plants were grown with Hoagland solution (pH 5.5), in a controlled environment with a 16 h photoperiod, at 25 ◦ C, 75% relative humidity (RH) and a photosynthetically active radiation (PAR) of 500 mol m−2 s−1 , and 8 h in the dark, at 18 ◦ C and 60% RH. Nutrient solution was changed every 4 days and pH was checked and adjusted daily. The plants were subjected to the following treatments; control (Hoagland), NaCl (60 mM) and KCl (60 mM). The measurements were carried out 1, 2, 3 and 10 days after treatment application. 2.2. Root hydraulic conductance Hydraulic conductance (L0 ) of roots was measured by pressurising the roots using the Schölander chamber (Jackson et al., 1996). For this, the aerial parts of the plant were removed, leaving the base of the stem which was sealed, with silicone grease, into a tapered glass tube. The plant was placed into a pressure chamber, with the same nutrient solution that it was grown in, and a gradual increase of pressure (from 0.1 to 0.5 MPa) was applied to detached roots. The range of the generated sap flows included a flow equivalent to the whole-plant transpiration flow. In the pressure chamber, it is assumed that there is a balance between
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the negative pressure in the xylem of the cut leaf and the pressure that forces water from the cells into the vessels. Following a different time at each pressure, the xylem sap for solute analysis was collected using Pasteur pipettes and transferred to Eppendorf tubes. The Eppendorf tubes and the roots of the plant were weighed. Sap flow (Jv ) was calculated as mg (g root FW)−1 h−1 and plotted against pressure, the slope being the L0 value. Mercuric chloride (50 M) was added to the nutrient solution of plants from which shoots had been excised for the measurement of the initial values of L0 . After 5 min, the plants were transferred to a nutrient solution without Hg and the L0 was measured 1 h later. Mercury was scavenged from roots with DTT (5 mM). After 1 h, L0 was measured as described above.
2.5. Ion analysis of xylem sap
2.3. Gas exchange
Organic solutes were measured in the leaf sap. For sugar analysis, the sap was filtered through a sep-pak C18 cartridges to remove the green colour. Glucose, fructose and sucrose concentrations were determined directly by HPLC, using a LiCrospher 100 NH2 5 m column coupled with a differential refractrometer detector. The mobile phase was acetonitrile:water (85:15), with a flow rate of 1.5 ml min−1 . All data were registered and processed using the Borwing 1.21 computer programme. Quantification was carried out by comparing peak areas with those of known standards. Total amino acids were determined by the ninhydrin method (Rosen, 1957).
Net CO2 assimilation (ACO2 ) and adaxial stomatal conductance, Gs , of leaves (more stomata were detected on the adaxial surface of the leaves, data not shown) were measured using a portable photosynthesis system (Li-COR LI-6200) attached to an infra-red CO2 analyser (IRGA LI-6250). Measurements were made on the most recent fully-expanded leaves, during the middle of the photoperiod. 2.4. Leaf water relations The leaf water potential (ψw ) of the most recent fully-expanded leaves was measured using a pressure chamber technique (Turner, 1988). The same leaves were then put in Eppendorf tubes with holes at the bottom and rapidly frozen with liquid nitrogen. These tubes were then centrifuged twice, at 4000 × g for 4 min (4 ◦ C), using a Hettich-Universal32R centrifuge, in such a way that all sap was extracted from samples. The osmotic potential (ψ0 ) of the leaf sap was calculated, after measuring sap osmolarity with an automatic freezing-point depression osmometer (Digital Osmometer, Roebling, Berlin), by the van’t Hoff equation (Nobel, 1991): ψ0 = −nRT where n is mosmol, R = 0.083 and T = t a (K). Turgor potential (ψp ) was calculated as the difference between leaf water potential and osmotic potential.
For the ion analysis, the xylem sap collected was diluted and injected into a Dionex-D-100 ion chromatograph. For the anions, an Ionpac AS12A (4 mm × 250 mm) (10–32) column and guard column were used. The flow rate was 1.5 ml min−1 with an eluent of 2.7 mM Na2 CO3 /0.3 mM NaHCO3 . For the cation analysis, an lonpac CS12A (4 mm × 250 mm) (10–32) column and guard column were used. The flow rate was 1 ml min−1 , with an eluent of 11 mM H2 SO4 . The ions were detected with a conductivity detector and quantified by comparing peak areas with those of known standards. 2.6. Organic solute analysis of the xylem sap
3. Results Root hydraulic conductance values for different saline treatments and different times of salt application were determined (Fig. 1). A great reduction in L0 for plants treated with KCl or NaCl, compared to control plants, can be appreciated (Fig. 1a). The decrease was similar for both salinity treatments. In the control treatment, L0 values fell sharply with HgCl2 addition (Fig. 1b) at all times of measurement. Using DTT as a scavenger for Hg2+ , L0 of control plants was restored to values similar to the pre-treatment value (Fig. 1c). There were no significant differences in L0 in NaClor KCl-treated plants after Hg2+ addition, with regard to their controls, on any day of measurement. Root hydraulic conductivity decreased with increas-
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Fig. 1. Hydraulic conductance (L0 ) of roots of control pepper plants and plants treated with 60 mM NaCl or 60 mM KCl (a); after HgCl2 (50 M) (b) and DTT (2 mM) (c) treatments. Measurements were carried out 1, 2, 3 and 10 days after saline treatment addition. Number of roots, 5; error bars, ±S.E.
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Fig. 2. Net assimilation of CO2 (ACO2 ) and stomatal conductance (Gs ) of leaves of control pepper plants and plants treated with 60 mM NaCl or 60 mM KCl. Measurements were carried out 1, 2, 3 and 10 days after saline treatment addition. Number of roots, 6; error bars, ±S.E.
ing time of measurement, being greater in control plants. Net assimilation of CO2 (ACO2 ) and stomatal conductance (Gs ) were measured for leaves in order to study gas exchange (Fig. 2). Values of ACO2 and Gs decreased in both saline treatments compared to control plants, but this reduction was higher in NaCl-plants. Net assimilation of CO2 showed a tendency to increase with time, but Gs showed a tendency to decrease with time.
Leaf water potential, Ψ w , decreased after NaCl or KCl addition, with regard to control plants (Fig. 3), the decrease being greater for NaCl-treated plants. A tendency of Ψ w to increase with the time of measurement was observed in control plants. The presence of NaCl or KCl in the nutrient solution reduced slightly the values for osmotic potential, Ψ , compared to control plants. There was a general increase of Ψ with time in all treatments. Turgor potential, ψp , was reduced with respect to control plants after NaCl or KCl
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Fig. 3. Water potential (ψw ), osmotic potential (ψ ) and turgor (ψp ) of leaves of control plants and plants treated with 60 mM NaCl or 60 mM KCl. Measurements were carried out 1, 2, 3 and 10 after saline treatment addition. Number of roots, 5; error bars, ±S.E.
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Fig. 4. Concentrations of anions (Cl− , NO3 − , PO4 3− and SO4 2− ) in leaf sap of control pepper plants and of plants treated with 60 mM NaCl or 60 mM KCl. Measurements were carried out 1, 2, 3 and 10 days after saline treatment addition. Number of roots, 5; error bars, ±S.E.
addition, but the decrease was greater for NaCl-treated plants. A decrease of ψp with days of measurement was observed in control plants and in plants treated with NaCl or KCl. Ion analyses for leaf sap of pepper plants treated with NaCl or KCl, at different times of measurement (24, 48 and 72 h and 10 days), were carried out in order to evaluate the relative importance of each nutrient (Fig. 4). Chloride concentration increased progressively with time of measurement in both NaCland KCl-treated plants, compared to control plants, and was higher for the NaCl treatment. Concentrations of NO3 − increased during the 3 days after NaCl or KCl addition, with regard to control plants. However, a great decrease was observed 10 days after the application of the saline treatments. There were no marked differences for PO4 3− or SO4 2− concentrations between salinised and control plants. Also, no changes in PO4 3− or SO4 2− levels were observed with the time of measurement.
Regarding cation analysis, the sap K+ concentration was significantly higher, for all treatments, as compared with Na+ and Mg2+ (Fig. 5). For the K+ concentrations, there were no differences between treatments or days of measurement. Sap concentration of Na+ increased after NaCl treatment and the Na+ content increased with the days of measurement. However, in control plants and in plants treated with KCl there were no significant differences in Na+ concentration, and it remained unchanged with the time of measurement. The concentration of Mg2+ was higher for control plants. The Mg2+ concentration declined on the 10th day after NaCl or KCl addition. However, the reduction was higher for KCl-treated plants than for NaCl-treated plants. The concentration of Ca2+ was also determined but no changes were observed (data not shown). Fructose concentration remained unchanged between treatments and with the time of measurement
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Fig. 5. Concentrations of cations (Na+ , K+ and Mg2+ ) in leaf sap of control pepper plants and of plants treated with 60 mM NaCl or 60 mM KCl. Measurements were carried out 1, 2, 3 and 10 days after saline treatment addition. Number of roots, 5; error bars, ±S.E.
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Fig. 6. Glucose, fructose and sucrose concentrations in leaf sap of control pepper plants and of plants treated with 60 mM NaCl or 60 mM KCl. Measurements were carried out 1, 2, 3 and 10 days after saline treatment addition. Number of roots, 5; error bars, ±S.E.
(Fig. 6). Glucose concentration was similar in control plants and in plants treated with NaCl, whereas it was lower for KCl-treated plants. It also remained unchanged during the days of measurement. Sucrose
concentration increased progressively over time in NaCl- and KCl-plants compared to control plants. The increase was higher in the KCl treatment than with NaCl during the first 3 days.
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Fig. 7. Total amino acids concentration in leaf sap of control pepper plants and of plants treated with 60 mM NaCl or 60 mM KCl. Measurements were carried out 1, 2, 3 and 10 days after saline treatment addition. Number of roots, 5; error bars, ±S.E.
Soluble amino acids decreased similarly for both salinity treatments with regard to control plants (Fig. 7). A general tendency of the amino acids concentration to increase within the first 3 days of measurement was observed for all treatments.
4. Discussion In both saline treatments, there was a reduction in L0 with respect to control values (Fig. 1). Strong reductions of L0 have been shown in several species by Shalhevet et al. (1976), Munns and Passioura (1984) and Evlagon et al. (1990). However, while these authors suggested that the osmotic concentration had negative effects on L0 , we hypothesised that there must have been a toxic effect of NaCl or KCl, rather than an osmotic effect, on L0 . Thus, in previous experiments we observed that large reductions in root hydraulic conductance of salinised plants were related closely to the decrease in the activity or concentration of aquaporins in the root plasma membrane (Carvajal et al., 1999, 2000), this effect being due mainly to the specific toxicity of Na+ and Cl− (Mart´ınez-Ballesta et al., 2000).
It has been shown that HgCl2 blocks the flow of water through aquaporins in plant roots and that the flow is restored by reducing agents (Maggio and Joly, 1995; Carvajal et al., 1996). However, this reagent had only a slight effect on NaCl- and KCl-treated plants (Fig 1b). In this work, the very weak response of the L0 of salt-stressed roots to the Hg treatment suggests that, in the plasma membrane, aquaporins were greatly reduced in number or, if present, were non-functional. In previous experiments (Martinez-Ballesta et al., 2000), treatments with Na+ or Cl− salt mixtures produced similar decreases in L0 , but the ameliorative effect of Ca2+ only occurred with the Na+ salts treatment, suggesting that Na+ was most likely the ion responsible for such reductions. However, in the current experiment, similar results were obtained with both salts (NaCl and KCl). Also, there were no effects of Ca2+ on the restoration of L0 in plants treated with KCl (data not shown). Therefore, Cl− could have an effect on L0 and on aquaporin functionality. A tendency of L0 to decrease with the time of measurement was observed in all treatments, which could be explained by the modifications of root structure with age that involve changes in hydraulic resistance (Mencuccini and Grace, 1996).
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Leaf gas exchange of pepper plants was impaired by NaCl or KCl addition to the nutrient solution (Fig. 2). This reduction has been attributed to salt effects on stomata, with the consequent restriction of CO2 availability for carboxylation, or to the acceleration of senescence (Pessarakli, 1994). In our experiments, the rate of photosynthesis decreased in both saline treatments, compared to control plants, during the 3 days of measurements. This decrease could be due mainly to the reduction in stomatal conductance that was observed in all salinised plants, but not to injuries to the photosynthetic apparatus (Björkman and Demmig, 1987; Demmig and Björkman, 1987), supported by the fact that, after 10 days, ACO2 values of salinised plants had recovered, reaching values similar to or higher than control plant values (Fig. 2). A decrease in Gs was observed for the NaCl and KCl treatments, probably caused by closure of the stomata or a decrease of water uptake through the roots. The quick response to NaCl or KCl addition observed in pepper plants, in terms of water loss and a reduction in Gs , could indicate a certain level of adaptation of this crop to salt stress (Munns and Termaat, 1986). A general tendency of Gs to decline with time was seen for all treatments, which may reflect reported decreases of stomatal conductance with plant age (Mencuccini and Grace, 1996). It has been proposed that the reduction of leaf gas exchange in response to salinity is due to an increase in leaf Na+ concentration (Garc´ıa-Legaz et al., 1993; Walker et al., 1993). However, other authors associated reductions in photosynthetic capacity and stomatal conductance with high concentrations of Cl− (Bañuls et al., 1997; Garc´ıa-Sanchez et al., 2002). In our plants, an effect of both Na+ and Cl− could have occurred, since there were reductions with both treatments, but the reduction was greater in plants treated with NaCl. Osmotic adjustment involves the net accumulation of solutes in cells in response to a fall in the water potential of their environment. As a consequence of this net accumulation, the cell osmotic potential is lowered, and turgor pressure tends to be maintained (Blum et al., 1996). In our experiments, the NaCl and KCl treatments markedly reduced the leaf water potential and this change was not compensated for by a reduction in leaf osmotic potential (Fig. 3), the values of which showed only a small decrease compared to
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control plants. Thus, turgor was not maintained and osmotic adjustment was not sufficient to offset the reduction in leaf water potential in salt-stressed plants. The changes observed in all the water relations in the plants during the time of measurement were possibly due to changes in metabolism related to plant age. The rate of acclimation to salinity and the duration of the salt treatment are clearly key factors influencing the plant response, but plant age and size are also likely to be involved (Franco et al., 1993). Salt tolerance in plants is associated usually with the ability to restrict the uptake and/or transport of saline ions from roots to shoots (Hajibagheri et al., 1987). Cl− and NO3 − concentrations increased in leaves of NaCl- and KCl-treated plants, suggesting that these were the main anions involved in osmotic adjustment (Fig. 4). In our experiments, the saline stress decreased the Mg2+ and Ca2+ in leaves (data not shown). This effect has been reported in other species, such as Helianthus annuus (Sanchez-Raya and Delgado, 1996) and Solanum melongena (Savvas and Lenz, 1994). Lynch and Läuchli (1985) proposed that decreases in the concentrations of these cations in shoots may be related to a reduced Ca2+ release into the root xylem, possibly due to effects on the active loading of cations into the xylem vessels. Also, excess K+ may cause a deficiency of other plant nutrients, especially Mg2+ (Chapman, 1966). Günes et al. (1996) observed equal Na+ and Cl− uptake in pepper plants. In our NaCl-treated plants, Cl− contents in leaves were higher than for Na+ . This was observed also in leaves of salinised plants of other species (Downton et al., 1985; Läuchli and Wieneke, 1989; Bethke and Drew, 1992). A possible explanation could be an increase in anion flow rate or a decrease in cation flow rate, as observed in barley and wheat roots (Hiatt and Hendricks, 1967; Osmond and Popp, 1983). Osmotic adjustment can be achieved by the accumulation of inorganic ions and/or organic substances, to permit the maintenance of turgor (Morgan, 1984, 1992). After the salinity treatments were imposed, only increases in leaf sucrose concentrations, to values nearly double those observed in control plants, were observed (Fig. 6). This significant contribution highlights this carbohydrate as the main organic solute involved in the osmotic adjustment, suggesting some diversion of carbohydrates from synthetic
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growth processes. However, the production of osmotica is metabolically expensive and limits plant production by consuming significant quantities of carbon (Greenway and Munns, 1980). The alternative to producing organic osmotica is the accumulation of a high concentration of ions from the external medium, for which the energy cost is lower. Nevertheless, this causes other problems and a high concentration of osmotic ions can produce a toxic effect on the normal biochemical activities within the cell (Volkmar et al., 1998). In this work, the contribution of amino acids accumulation to osmotic adjustment was not significant, since in the NaCl and KCl treatments the amounts of amino acids were decreased with regard to control plants (Fig. 7). This could be due to a toxic effect of Na+ , K+ and/or Cl− on cytoplasmic enzymes involved in amino acids synthesis. So, we can conclude that Na+ and K+ exerted a toxic effect on pepper plants mainly by affecting the plant water relations, although the effect of Na+ on these parameters was more significant than that of K+ . Since photosynthesis is thought to be one specific turgor-dependent process, it can be suggested that the low rates of gas exchange observed in NaCland KCl-leaves were a result of changes in leaf water relations. Salinity decreased water uptake in pepper plants, so that they lost turgor and the necessary osmotic adjustment, in terms of inorganic ions and organic substances accumulation, was not achieved. At high salinity, the uptake of water by plants is reduced (Ehret and Ho, 1986) and, in conditions of high transpiration demand, water uptake must balance water consumption (Awang et al., 1993). In this experiment, the increase in sucrose content and the decreases in leaf water potential and stomatal conductance under saline conditions show that, although plants may have been adjusted osmotically, water uptake could not balance water loss and so turgor decreased.
Acknowledgements This work was supported by the Ministerio de Ciencia y Tecnolog´ıa (CICYT-AGL2000-0506-C02-02). M.C. Martinez Ballesta was funded by a grant from the Fundación Seneca, Comunidad Autónoma de la Región de Murcia (Spain). The authors thank
David Walker for correction of the English in the manuscript.
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