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Efficient leaf ion partitioning, an overriding condition for abscisic acid‐controlled stomatal and leaf growth responses to NaCl salinization in two legumes

Efficient leaf ion partitioning, an overriding condition for abscisic acid‐controlled stomatal... Abstract Two tree medics contrasting in salt tolerance, Medicago arborea and Medicago citrina, were compared to evaluate the relative importance of abscisic acid on leaf growth and stomatal responses to salt stress. Plants were grown for 30 d in solution culture with 1, 50, 100 or 200 mM NaCl. Salinized plants of M. citrina had lower Na+ and Cl– uptake and maintained better leaf growth than M. arborea. In M. citrina, stomatal conductance was only slightly affected by salt and, in consequence, the salt treatment had no significant influence, neither on the CO2 fixation rate nor the transpiration rate in these plants. Moreover, leaf photosynthetic pigments and soluble protein in M. citrina were increased by the presence of NaCl, while a decrease of both parameters with salt was found in M. arborea. However, leaf and xylem ABA increased only in salt‐treated M. citrina, while no differences were found among treatments in M. arborea. The role of ion compartmentation, gas exchange parameters and ABA concentrations in relation to salt tolerance in M. arborea and M. citrina is discussed. Key words: Abscisic acid, chloride compartmentation, leaf growth, Medicago arborea, Medicago citrina, salinity, sodium compartmentation, stomatal closure. Received 31 March 2003; Accepted 3 June 2003 Introduction Inhibition of leaf growth is the first response to excess salt in sensitive plants. The understanding of the mechanisms involved in the conservation of this parameter in salt‐tolerant forage legumes is of primary importance because of the strong dependence of yield on leaf production and expansion. In Gramineae species subjected to short‐term salinity, leaf growth inhibition is mainly the result of an ABA‐mediated response to the osmotic component of salt stress (Munns and Termaat, 1986). In the Na+ excluder Phaseolus vulgaris, ABA mediates in both the short‐ and long‐term responses to Na+ toxicity and the signalling of the salt‐induced water deficit (Montero et al., 1998; Sibole et al., 1998, 2000). In this species, a high correlation between both leaf expansion rate and leaf and xylem ABA was found, suggesting a role of ABA in Na+ signalling. Carbon assimilation is central to leaf growth and productivity. Under saline conditions, photosynthetic carbon assimilation is severely restricted by reduced leaf expansion. Moreover, in salt‐sensitive species, both the stomatal and non‐stomatal components of CO2 are affected by NaCl. The role of ABA in the control of stomatal closure is well established, and rapid progress with biochemical, electrophysiological and molecular approaches, is ongoing (Schroeder et al., 2001). However, the dominance of ABA on stomatal regulation in whole plants suffering root stress has not yet been elucidated. Previous work with the Na+ excluder Phaseolus vulgaris showed a high correlation between stomatal conductance and either leaf or xylem ABA in plants submitted to either short‐ or long‐term salt stress (Sibole et al., 1998, 2000; Montero et al., 1998). The aim of the present study was to examine if the bean model of an ABA regulation of leaf expansion and stomatal responses can also be extended to other, more salt‐tolerant, non‐excluder species of the same family. Two tree medic species were chosen that contrast in their tolerance to NaCl. Medicago arborea is originally from the Greek islands and is currently grown as a commercial drought‐tolerant forage crop (Lambert et al., 1989). Medicago citrina is a closely related species, endemic of some small western Mediterranean islets. Under saline conditions, most of the early physiological changes experienced by plants are primarily in response to the osmotic component of salt. However, even with osmoregulation underway, salt‐treated plants face a build‐up of toxic ion concentrations in their tissues, which is considered a key limiting factor for plant growth in saline soils (Munns et al., 1995). This study was focused on this second phase, in which the response of the plant is predominantly to the effects of ion accumulation. Materials and methods Plant growth conditions Homogenous sized seeds were scarified in H2SO4 (96% w/w): 30 min M. arborea and 60 min M. citrina. At the emergence of the second trifoliate leaf, uniformly sized seedlings were selected and transplanted to 4.0 l pails (4 plants per container) filled with aerated modified 50% Hoagland’s solution (Epstein, 1972). The saline treatments were 50, 100, 150, and 200 mM NaCl, and were administered in a step‐wise fashion, adding daily increments of 50 mM until the desired concentration was reached. The experiments were conducted in a greenhouse under controlled conditions, with the following regimes: temperature: min/max 17/35 °C; relative humidity: min/max 30/70%. Growth parameters Plants were transferred to the laboratory the night before harvest. Leaf area was measured with a leaf area meter (D‐T Area Meter, Delta‐T Devices, Burwell, Cambridge, England). Tissue dry weight was determined after drying the samples in an air‐forced oven at 60 °C. for 48 h. The roots were washed three times in distilled water before drying. Leaf water content Water content of individual leaves of the main axis occupying positions 2, 4, 8, and 12 from the shoot apex was determined according to the following equation: LS=FW–DW/DW; where FW refers to the fresh weight and DW is dry weight. Gas exchange measurements Gas exchange measurements were made at noon using a closed system infrared gas analyser (LI 6200, Li‐Cor, Lincoln, NE, USA) on leaf 8 from the apex. This leaf was chosen to ensure the measurement of a fully expanded productive leaf that was not prone to senescence in the salt treatments. Measurements were taken under the following conditions: photon flux density, 946±17 µmol m–2 s–1; ambient CO2 concentration, 350±2 µmol mol–1 air; relative humidity, 40±0.5%; air temperature, 32±0.2 °C. Net photosynthesis (A), stomatal conductance (gs), internal CO2 concentration (Ci) and transpiration rate were calculated according to Farquhar and Sharkey (1982). Photosynthetic pigments and soluble protein concentration Samples of roots, and the leaves used for gas exchange measurements (leaf 8), were immediately frozen with liquid nitrogen and stored at –80 °C. Photosynthetic pigments and soluble protein were extracted using bicine buffer with pH 8 (Lawlor et al., 1989), and photosynthetic pigment concentrations were quantified according to Lichtenthaler and Wellburn (1983). Protein concentration was determined following the method described by Bradford (1976). Collection of xylem sap Sap collection was conducted at noon in plants individually grown in 1.0 l containers to avoid root tangling. Xylem sap was collected at midday using a pressure chamber (Soilmoisture, Santa Barbara, CA). Roots were washed, blotted dry and carefully inserted into a plastic bag. Afterwards, shoots were excised 5–6 cm above the shoot base using a sharp blade. The detopped plants were pressurized gradually and held at c. 0.7 MPa for 5 min to collect sufficient sap for analysis. After discarding the first drops (20–30 µl), sap was collected with a micropipette. Xylem sap pH was recorded with an Ingold micro‐pH electrode (Ingold, SA, Urdorf, Switzerland) and immediately frozen in liquid N2. In this work, data from plants grown at salt concentrations greater than 100 mM are not presented, as it was impossible to obtain sufficient xylem sap from M. arborea in a feasible, accurate manner as described above. Ion concentrations 0.1 g of finely ground oven‐dried tissue was digested overnight with 25 ml of 0.1 M HNO3 at room temperature. Xylem sap for ion analysis was used without further purification. Contents of Na+, K+, and Ca2+ of the cold acid extract and xylem sap were determined by inductively coupled plasma atomic emission spectrometry (Perkin‐Elmer Plasma‐2000). Chloride content of the cold acid extract was determined using a selective ion electrode potentiometer (Ingold, SA, Urdorf, Switzerland). Xylem sap Cl– was determined by HPLC (Waters, Mildorf, MA) equipped with a 4.6×150 mm IC‐PAK Anion HC column. Ion concentrations were calculated on a tissue‐water basis from the fresh and dry weights of the same leaf. Abscisic acid concentration Plant tissue was immediately frozen in liquid nitrogen, freeze‐dried, and finely ground with a mortar and pestle. 40 mg of freeze‐dried tissue was extracted overnight with distilled water in the dark at 4 °C. ABA concentration was analysed by radio‐immunoassay (Quarrie et al., 1988). Xylem sap was analysed directly from plants treated with either 1 or 100 mM NaCl using this same method, as previous analyses demonstrated that further extraction and purification steps were not necessary (Montero et al., 1994). Statistics Data are presented as the mean ±SE for each treatment (n=6). Significant differences among treatments were analysed by ANOVA. LSD values were calculated at the P <0.05 probability level (StatView, v. 4.0, Abacus Concepts, Inc. Berkeley, CA, USA). Results Growth Leaf growth was more affected by increased salt in M. arborea than in M. citrina as shown in Fig. 1. M. citrina had no significant differences in leaf dry weight and in leaf area. In M. arborea, both parameters showed a moderate linear decline at 50 and 100 mM NaCl (of 13% and 26%, respectively, in leaf dry weight, and 26.5% and 28% in leaf area). At 200 mM NaCl, the growth reduction was much more severe (50% of controls) for M. arborea, and plants showed visual symptoms of NaCl toxicity: tip necrosis and leaf chlorosis, in the youngest shoots. Stem dry weight decreased with salt supply in both species, while root dry weight only declined at the highest NaCl treatment. At 200 mM NaCl, stem and root dry weights were 32 and 54%, and 44 and 68% of controls in M.arborea and M. citrina, respectively. Leaf water content M. arborea had higher leaf water contents than M. citrina, and this parameter increased with leaf age in both species (Fig. 2). Salinization showed a tendency although not significant, to influence leaf water content in fully expanded leaves (positions 4 and 8 from the apex). For example, leaf water content increased in M. arborea, although slightly decreased in M. citrina with increased salt supply. The decrease in water content with increasing salt supply noted in expanding leaves (position 2 from the apex) may be consequence of a salt‐induced delay of leaf emergence. Gas exchange measurements Instantaneous CO2 assimilation rate and stomatal conductance decreased along with increased NaCl in M. arborea. The response found in the CO2 assimilation rate had the same pattern observed for the leaf growth, with values 13, 22 and 51% lower at 50, 100 and 200 mM NaCl treatments, respectively, than control plants. CO2 assimilation with respect to chlorophyll a concentrations were 91.5±5.3a, 79.1±3.6b, 73.2±5.3bc, and 68.2±3.0c nmol mg–1 s–1 for 1, 50, 100, and 200 mM NaCl‐treated plants. In this species, the decline in the A values was highly correlated with gs (r2=0.72; P <0.0001) (Fig. 3), leaf soluble protein (r2=0.89; P <0.0001) and chlorophyll a (r2=0.81; P <0.0001). The relative intercellular CO2 concentration (Ci/Ca) had values of 0.84±0.02a, 0.81±0.03a, 0.72±0.03b, and 0.66±0.04b for 1, 50, 100, and 200 mM NaCl‐treated plants, respectively. In agreement with the leaf growth response observed in M.citrina, no significant differences between treatments were found in the photosynthetic parameters, as CO2 assimilation rates were 15.5±0.9a, 17.1±2.5a, 16.2±1.5a, and 16.9±3.0a µmol m–2s–1 referred to leaf area and 76.1±8.4a, 74.7±9.6a, 62.1±9.7a, and 67.7±8.2a nmol mg–1 s–1 referred to chlorophyll a concentration and stomatal conductance were 0.55±0.07a, 0.60±0.11a, 0.58±0.06a, and 0.53±0.12a mol m–2s–1 and the relative intercellular CO2 concentration (Ci/Ca) had values of 0.84±0.03a, 0.79±0.05a, 0.82±0.04a, and 0.80±0.04a for 1, 50, 100, and 200 mM NaCl‐treated plants, respectively. For each data set mentioned, values with the same letter in superscript are not significantly different (P >0.05). Ion concentrations Root and stem Na+ and Cl– concentrations increased with salt increments, although there were no differences between species. In both Medicago, the highest Na+/Cl– concentrations were in the leaves, and increased with NaCl treaments (Fig. 4A, B). Although the expanded leaf (leaf 8) had higher Na+/Cl– concentrations than those of leaf 2, as this later leaf was in the expansion phase and in most instances not completely unfolded, Na+/Cl– concentrations could be considered as substantial. For instance, at 200 mM NaCl, leaf 2 Cl– of M. arborea exceeded the concentration in the nutrient solution. In both leaves considered, M. arborea had significantly greater Na+ values than M. citrina with respect to leaf number. Leaf Cl– concentrations were greater than for Na+ (Fig. 4B). In all saline treatments, M. arborea had higher Cl– concentrations in leaf 2. However, in leaf 8, no differences between species were found for Cl– concentrations, although higher Cl– concentrations were found in M. citrina in 50 and 100 mM NaCl treatments. Only at 200 mM NaCl, was M. arborea leaf Cl– higher than M. citrina. A negative relationship between either instantaneous CO2 assimilation (Fig. 5A) or stomatal conductance (Fig. 5B) and leaf Na+ concentration was found in M. arborea. In this species, leaf Na+ concentration was correlated with chlorophyll a concentration (r2=0.51; P <0.0001) and soluble protein concentration (r2=0.74; P <0.0001). Xylem Na+/Cl– was significantly higher in 100 mM NaCl M. arborea than M. citrina (Table 1). Potassium concentration decreased with NaCl uptake in leaves, stems and roots. The decline was similar in both species for each salt treatment, when considered with respect to their controls (Fig. 4C). Significant differences in the leaf Na+:K+ ratio were only found between species at 200 mM NaCl. Leaf 2 Na+:K+ ratio had values of 2.6±0.52 in M. arborea and 1.08±0.05 in M. citrina, while leaf 8 Na+:K+ ratio had values of 4.5±0.76 in M. arborea and 3.46±0.27 in M. citrina. No differences in xylem K+ were found between 1 and 100 mM NaCl‐treated plants. However, xylem K+ was 2‐fold higher in M. arborea than in M. citrina (Table 1). Stem and root Ca2+ declined with salinization with no significant differences between species. In both species leaf Ca2+ was the cation least affected by salt (Fig. 4D). Leaf 2 Ca2+ only decreased at 200 mM NaCl, while leaf 8 Ca2+ declined along with increased salt treatments. Leaf 2 Na+:Ca2+ ratio was higher in M. arborea than M.citrina grown at 200 mM NaCl, with found values of 7.09±0.61 and 4.09±0.98 in M. arborea and M. citrina, respectively. No significant differences were found in leaf 8 Na+:Ca2+ ratio either between species or treatments, with found values of of 8.28±0.54 and 9.72±1.64 in M. arborea and M. citrina, respectively, at 200 mM NaCl. However, the pattern for xylem Ca2+ was distinct in the two species in response to the treatment considered. In M. citrina, a significant drop was noted, as xylem Ca2+ fell to less than half of the values of control in response to the 100 mM NaCl treatment. By contrast, M. arborea xylem Ca2+ was significantly higher than the control group and rose more than 3‐fold in response to 100 mM NaCl. This rise in xylem Ca2+ was also significantly higher in salt‐treated plants of M. arborea than M. citrina (Table 1). Photosynthetic pigments and protein concentrations A slight increase in chlorophyll a concentration was found in M. citrina with salt (Fig. 6A). In the 200 mM NaCl treatment, chlorophyll a significantly decreased in M. arborea with respect to the controls. In both species, no significant differences were found in chlorophyll b, carotenes and xanthophyll concentrations among treatments (data not shown). Soluble leaf protein significantly decreased in M. arborea, while in M. citrina, it increased with salt (Fig. 6B). No differences in root protein concentration were found for either species or treatments (data not shown). Abscisic acid concentrations No differences were found in leaf ABA concentrations among salt treatments in M. arborea (Fig. 7). In M. citrina, both non‐expanded and expanded leaves had a significant increase in ABA at 100 and 200 mM NaCl. ABA did not increase in roots with NaCl, and thus, no significant differences were found in root ABA concentrations for either species or treatments. Xylem ABA significantly increased in 100 NaCl M. citrina, while no differences were found in M. arborea between treatments (Table 1). The correlation between leaf water content and leaf ABA in expanded leaves was much higher in M. citrina (r2=0.97; P=0.016) than in M. arborea (r2=0.39; P=0.030). Discussion Under the mild NaCl saline conditions used here, M. citrina had better leaf growth than M. arborea, and this response was strongly related to leaf Na+/Cl–, as both expanded (leaf 8 from the apex) and the still expanding (leaf 2 from the apex) leaves of M. arborea had overall significantly higher Na+/Cl– concentrations. The higher leaf Na+/Cl– exclusion capacity in M. citrina was not due to a better overall K+/Na+ selectivity, as leaf K+ decreased with salt in both species, and leaf Na+/K+ ratio was only higher in M. arborea than M. citrina at 200 mM NaCl. Moreover, leaf growth inhibition in M. arborea was not closely linked to leaf Ca2+, as Ca2+ was only found to decline in leaf 2 at 200 mM NaCl in both species. However, at this salt treatment, the higher leaf 2 Na+/Ca2+ ratio in M. arborea, jointly with the above‐mentioned higher Na+/K+ ratio, suggest a possible onset of an ion imbalance which could have contributed to the reduction in leaf growth found in the former species. There is evidence that ABA is involved in the regulation of leaf expansion under salt stress (Hartung et al., 1999; Cramer and Quarrie, 2002). Previous results with bean strongly suggested the possibility of a leaf growth ABA‐mediated plant response to Na+ (Montero et al., 1998; Sibole et al., 2000). However, in both Medicago species, no direct relationship was found between leaf growth and leaf ABA. No significant differences were found in root ABA among either treatments or species, and this growth regulator only increased in leaves and xylem of salt‐treated M. citrina. Although there is evidence that ABA is a root‐borne signal that participates in root–shoot communication processes (Hartung et al., 2002), it can also be synthesized in the leaves (Popova et al., 2000; Holbrook et al., 2002). In salt‐treated M. citrina, the increase in leaf ABA was correlated with decreased leaf water content, which suggests that ABA could partake in the adjustment of leaf metabolism to a decrease in water activity. Since ABA can be translocated into the phloem (Wolf et al., 1990), the higher xylem ABA concentrations found in 100 mM salt‐treated M. citrina could be the result of its retranslocation from the leaves via the phloem to xylem. Correspondingly, the lack of response in leaf ABA concentration in salt‐treated M. arborea could be related to the observed increase in leaf water content with NaCl (Hartung et al., 2002). The differences found in leaf growth between species are related to the decline in M. arborea CO2 assimilation, while no differences among treatments were found in M.citrina. In salt‐treated M. arborea, photosynthetic rate was limited by stomatal closure restricting CO2 diffusion into the mesophyll cells. However, the decline in Ci was poorly correlated with stomatal conductance (r2=0.26; P=0.088) which could be related to the presence of non‐stomatal effects. The decrease in CO2 assimilation was positively correlated with chlorophyll a and soluble protein, suggesting a regulation of photosynthetic unit size in response to NaCl stress. However, the rate of photosynthesis in salt‐treated plants also significantly decreased with respect to controls when expressed on a chlorophyll a basis. This would also indicate a decrease in the photosynthetic capacity probably due to a decline in leaf Na+/Cl– compartmentation capacity (Fig. 5). These results show a poor dependence between either CO2 assimilation rate or stomatal conductance and leaf Na+, for leaf Na+ concentrations of up to 150 mM. However, the dependence between these parameters greatly increased when leaf Na+ reached concentrations above those of the external NaCl (200 mM). This would indicate a reduced capacity for intracellular ion compartmentation in M. arborea, which would negatively affect enzyme activity (Wyn Jones and Gorham, 2002). Different studies have reported a relationship between non‐stomatal effects and the presence of high leaf Na+/Cl– concentrations (Seemann and Critchley, 1985; Bethke and Drew, 1992). Additionally, James et al. (2002) reported that non‐stomatal limitation was associated with leaf Na+ concentration above 200 mM. The higher capacity of M. citrina for intracellular ion compartmentation is even more clear when Cl– is considered, for example, at 100 mM NaCl leaf 8 Cl– is significantly higher in M. citrina than in M. arborea. There is evidence that xylem ABA regulates stomatal conductance under stress conditions (Davies and Zhang, 1991). Previous results in beans showed a high positive correlation between stomatal closure and either leaf or xylem ABA (Montero et al., 1998; Sibole et al., 2000). However, in salt‐treated M.arborea, neither leaf nor xylem ABA was related to stomata closure. Moreover, although salt‐treated plants had marked changes in xylem ion concentration, no differences in xylem pH were found among treatments and, therefore, an increase in the leaf apoplast ABA half‐life due to basification can be discarded (Wilkinson and Davies, 1997). Accumulation of apoplastic solutes has been shown to affect stomatal aperture (Ewert et al., 2000). Xylem Na+ was significantly higher in salt‐treated M. arborea and, therefore, it would be feasible that an accumulation of Na+ in the guard cell apoplastic space could disturb the K+ channels that participate in stomatal movement (Schroeder et al., 2001). For other species, it has been suggested that Na+ could contribute to stomata closure in other species. Perera et al. (1997) found a direct response of the stomata of Aster tripolium to Na+, which was not ABA‐mediated. The authors hypothesized that such a sensory system is useful to control the amount of salt delivered to the leaf by the transpirational stream. Other xylem‐transported elements implicated in regulating stomatal closure, such as Ca2+, were much higher in 100 mM salt‐treated M. arborea than in controls. However, bulk xylem calcium might not be an adequate indicator for its actual concentration in the substomatal cavity, since Ca2+ could present a marked gradient along the leaf apoplast (De Silva et al., 1998). M. citrina displayed no differences in stomatal conductance among treatments and therefore no relationship was found with the increased xylem and leaf ABA concentrations found in salt‐treated plants in this species. There is a great deal of evidence that stomatal conductance is regulated by the amount of ABA present in the apoplast of epidermal cells and is pH dependent (Wilkinson and Davies, 1997). Although xylem ABA of salt‐treated M. citrina increased, the ABA that would actually reach the guard cells may be not higher than controls. The xylem pH in salt‐treated M. citrina was about 6, which is the pH optima reported by Wilkinson and Davies (1997) for a carrier‐mediated epidermal ABA uptake. At this pH, it can be expected that the ABA that is not immediately metabolized can be compartmentalized in the epidermal and mesophyll cells (Hartung et al., 1998). This ABA would not participate in stomatal regulation, but could enhance the adaptation of these plants to salt (Jia et al., 1996) or have a protective role (Cramer, 2002). In M. citrina, a higher leaf Na+/Cl– compartmentation capacity would protect cell metabolism from the toxic effects of these ions. In this species, no significant differences were found in Ci/Ca and stomatal conductance between treatments, and salinized plants were able to maintain CO2 assimilation rate and leaf growth. Moreover, the negative effect of salt could have been ameliorated by the elevated chlorophyll a and soluble leaf protein concentrations found in NaCl‐treated M. citrina. Elevated chlorophyll content and increased photosynthetic capacity have been reported as a part of the cell adaptation process to salinity (Locy et al., 1996). An increase in leaf protein has been reported in salt (Wu and Seliskar, 1998) and water‐stressed plants (Pankovic et al., 1999). Moreover, an increase in leaf soluble protein has been related to an increased mesophyll conductance in drought‐tolerant varieties (Lauteri et al., 1997). By contrast, in M. arborea, soluble protein concentration decreased with salt. The strong correlation between leaf soluble protein and leaf Na+/Cl– suggests a toxic Na+/Cl– effect on leaf protein synthesis (Helal and Mengel, 1979) and/or a Na+/K+ imbalance (Serrano et al., 1999). In this study, an attempt has been made to determine whether or not changes in ABA concentrations were negatively correlated with leaf growth and stomatal responses in Na+ non‐excluder legumes, as found earlier for the Na+‐excluder bean (Montero et al., 1998; Sibole et al., 2000). As the results showed no correlation between increased ABA and either leaf growth or stomatal conductance, there is no straightforward affirmative answer. Instead, these results point to other components that may participate in leaf growth control in these two legume species when submitted to a long‐term mild salinization. The better leaf response to salt in M. citrina is related to a higher compartmentation capacity, which maintains cell metabolism and energy production required to sustain leaf growth in saline conditions. Acknowledgements Financial support from the DGICYT project BFI2001‐2475‐C02‐02 and the European Funds for Regional Development is gratefully acknowledged. View largeDownload slide Fig. 1. Leaf dry weight (A) and total leaf area (B) in M. arborea and M. citrina grown in 1, 50, 100 or 200 mM Na/Cl for 30 d. Error bars=standard error of mean (n=6). Values with the same letter are not significantly different (P >0.05). View largeDownload slide Fig. 1. Leaf dry weight (A) and total leaf area (B) in M. arborea and M. citrina grown in 1, 50, 100 or 200 mM Na/Cl for 30 d. Error bars=standard error of mean (n=6). Values with the same letter are not significantly different (P >0.05). View largeDownload slide Fig. 2. Leaf water mean content of leaves 2, 4, 8, and 12 from the apex in M. arborea (A) and M. citrina (B) grown in 1, 50, 100 or 200 mM NaCl for 30 d. Error bars=standard error of mean n=6). View largeDownload slide Fig. 2. Leaf water mean content of leaves 2, 4, 8, and 12 from the apex in M. arborea (A) and M. citrina (B) grown in 1, 50, 100 or 200 mM NaCl for 30 d. Error bars=standard error of mean n=6). View largeDownload slide Fig. 3. Relationship between instantaneous CO2 assimilation rate, stomatal conductance (A) and leaf protein concentration (B) of leaf 8 from the apex in M. arborea grown in 1, 50, 100 or 200 mM NaCl for 30 d. View largeDownload slide Fig. 3. Relationship between instantaneous CO2 assimilation rate, stomatal conductance (A) and leaf protein concentration (B) of leaf 8 from the apex in M. arborea grown in 1, 50, 100 or 200 mM NaCl for 30 d. View largeDownload slide Fig. 4. Sodium (A), Cl– (B), K+ (C), and Ca2+ (D) concentrations in tissue water of leaves 2 and 8 from the apex in M. arborea and M. citrina in 1, 50, 100 or 200 mM NaCl for 30 d. Error bars=standard error of mean (n=6). Values with the same letter are not significantly different (P >0.05). View largeDownload slide Fig. 4. Sodium (A), Cl– (B), K+ (C), and Ca2+ (D) concentrations in tissue water of leaves 2 and 8 from the apex in M. arborea and M. citrina in 1, 50, 100 or 200 mM NaCl for 30 d. Error bars=standard error of mean (n=6). Values with the same letter are not significantly different (P >0.05). View largeDownload slide Fig. 5. Relationship between instaneous CO2 assimilation rate (A) and somatal conductance (B) and leaf Na+ of leaf 8 from the apex in M. arborea grown in 50, 100 or 200 mM NaCl for 30 d. View largeDownload slide Fig. 5. Relationship between instaneous CO2 assimilation rate (A) and somatal conductance (B) and leaf Na+ of leaf 8 from the apex in M. arborea grown in 50, 100 or 200 mM NaCl for 30 d. View largeDownload slide Fig. 6. Chlorophyll a (A) and soluble protein (B) concentrations of leaf 8 from the apex in M. arborea and M. citrina in 1, 50, 100 or 200 mM NaCl for 30 d. Error bars=standard error of mean (n=6). Values with the same letter are not significantly different (P >0.05). View largeDownload slide Fig. 6. Chlorophyll a (A) and soluble protein (B) concentrations of leaf 8 from the apex in M. arborea and M. citrina in 1, 50, 100 or 200 mM NaCl for 30 d. Error bars=standard error of mean (n=6). Values with the same letter are not significantly different (P >0.05). View largeDownload slide Fig. 7. ABA concentrations of leaves 2 and 8 from the apex in M. arborea and M. citrina grown in 1, 50, 100 or 200 mM NaCl for 30 d. Error bars=standard error of means (n=6). Values with the same letter are not significantly different (P >0.05). View largeDownload slide Fig. 7. ABA concentrations of leaves 2 and 8 from the apex in M. arborea and M. citrina grown in 1, 50, 100 or 200 mM NaCl for 30 d. Error bars=standard error of means (n=6). Values with the same letter are not significantly different (P >0.05). Table 1. Xylem sap Na+, Cl–, K+, Ca2+, and ABA concentrations in M. arborea and M. citrina grown in 1 or 100 mM NaCl for 30 d For each column, values with the same letter are not significantly different (P >0.05). Species  Treatment  Na+  Cl–  Ca2+  K+  ABA  pH    NaCl (mM)  (mM)  (mM)  (mM)  (mM)  (nM)    M. arborea  1  0.052±0.04 a  0.24±0.09 a  0.10±0.10 a  15.89±0.25 a  23.1±4.60 a  6.06±0.16 a    100  5.19±0.26 b  11.1±2.19 b  0.38±0.15 b  16.41±0.25 a  36.8±12.5 a  6.13±0.11 a  M. citrina  1  0.022±0.01 a  0.32±0.09 a  0.20±0.11 b  8.46±1.03 b  18.6±9.47 a  5.99±0.26 a    100  2.41±0.45 c  6.68±1.58 c  0.08±0.02 a  9.23±0.52 b  61.7±18.5 b  5.90±0.12 a  Species  Treatment  Na+  Cl–  Ca2+  K+  ABA  pH    NaCl (mM)  (mM)  (mM)  (mM)  (mM)  (nM)    M. arborea  1  0.052±0.04 a  0.24±0.09 a  0.10±0.10 a  15.89±0.25 a  23.1±4.60 a  6.06±0.16 a    100  5.19±0.26 b  11.1±2.19 b  0.38±0.15 b  16.41±0.25 a  36.8±12.5 a  6.13±0.11 a  M. citrina  1  0.022±0.01 a  0.32±0.09 a  0.20±0.11 b  8.46±1.03 b  18.6±9.47 a  5.99±0.26 a    100  2.41±0.45 c  6.68±1.58 c  0.08±0.02 a  9.23±0.52 b  61.7±18.5 b  5.90±0.12 a  View Large References Bethke PC , Drew MC. 1992. 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A monoclonal antibody to (S)‐abscisic acid: its characterization and use in a radioimmunoassay for measuring abscisic acid in crude extracts of cereals and lupin leaves. Planta  173, 330–339. Google Scholar Schroeder JI , Allen GJ, Hugouvieux V, Kwak JM, Waner D. 2001. Guard cell signal transduction. Annual Review of Plant Physiology and Plant Molecular Biology  52, 627–658. Google Scholar Seemann JR , Critchley Ch. 1985. Effects of salt stress on the growth, ion content, stomatal behaviour and photosynthetic capacity of a salt‐sensitive species, Phaseolus vulgaris L. Planta  164, 151–162. Google Scholar Serrano R , Mulet JM, Rios G, Márquez A, de Larrinoa IF, Leube P, Mendizabal I, Pascual‐Ahuir A, Ros R, Montesinos C. 1999. A glimpse of the mechanisms of ion homeostasis during salt stress. Journal of Experimental Botany  50, 1023–1036. Google Scholar Sibole JV , Montero E, Cabot C, Poschenrieder Ch, Barceló J. 1998. 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Journal of Experimental Botany  49, 1005–1013. Google Scholar Wyn Jones G , Gorham J. 2002. Intra‐ and intercellular compartmentation of ions—a study in specificity and plasticity. In: Läuchli A, Lüttge U, eds: Salinty: environment–plants–molecules. Dordrecht: Kluwer Academic Publishers, 159–180. Google Scholar http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Experimental Botany Oxford University Press

Efficient leaf ion partitioning, an overriding condition for abscisic acid‐controlled stomatal and leaf growth responses to NaCl salinization in two legumes

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Publisher
Oxford University Press
ISSN
0022-0957
eISSN
1460-2431
DOI
10.1093/jxb/erg231
pmid
12925667
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See Article on Publisher Site

Abstract

Abstract Two tree medics contrasting in salt tolerance, Medicago arborea and Medicago citrina, were compared to evaluate the relative importance of abscisic acid on leaf growth and stomatal responses to salt stress. Plants were grown for 30 d in solution culture with 1, 50, 100 or 200 mM NaCl. Salinized plants of M. citrina had lower Na+ and Cl– uptake and maintained better leaf growth than M. arborea. In M. citrina, stomatal conductance was only slightly affected by salt and, in consequence, the salt treatment had no significant influence, neither on the CO2 fixation rate nor the transpiration rate in these plants. Moreover, leaf photosynthetic pigments and soluble protein in M. citrina were increased by the presence of NaCl, while a decrease of both parameters with salt was found in M. arborea. However, leaf and xylem ABA increased only in salt‐treated M. citrina, while no differences were found among treatments in M. arborea. The role of ion compartmentation, gas exchange parameters and ABA concentrations in relation to salt tolerance in M. arborea and M. citrina is discussed. Key words: Abscisic acid, chloride compartmentation, leaf growth, Medicago arborea, Medicago citrina, salinity, sodium compartmentation, stomatal closure. Received 31 March 2003; Accepted 3 June 2003 Introduction Inhibition of leaf growth is the first response to excess salt in sensitive plants. The understanding of the mechanisms involved in the conservation of this parameter in salt‐tolerant forage legumes is of primary importance because of the strong dependence of yield on leaf production and expansion. In Gramineae species subjected to short‐term salinity, leaf growth inhibition is mainly the result of an ABA‐mediated response to the osmotic component of salt stress (Munns and Termaat, 1986). In the Na+ excluder Phaseolus vulgaris, ABA mediates in both the short‐ and long‐term responses to Na+ toxicity and the signalling of the salt‐induced water deficit (Montero et al., 1998; Sibole et al., 1998, 2000). In this species, a high correlation between both leaf expansion rate and leaf and xylem ABA was found, suggesting a role of ABA in Na+ signalling. Carbon assimilation is central to leaf growth and productivity. Under saline conditions, photosynthetic carbon assimilation is severely restricted by reduced leaf expansion. Moreover, in salt‐sensitive species, both the stomatal and non‐stomatal components of CO2 are affected by NaCl. The role of ABA in the control of stomatal closure is well established, and rapid progress with biochemical, electrophysiological and molecular approaches, is ongoing (Schroeder et al., 2001). However, the dominance of ABA on stomatal regulation in whole plants suffering root stress has not yet been elucidated. Previous work with the Na+ excluder Phaseolus vulgaris showed a high correlation between stomatal conductance and either leaf or xylem ABA in plants submitted to either short‐ or long‐term salt stress (Sibole et al., 1998, 2000; Montero et al., 1998). The aim of the present study was to examine if the bean model of an ABA regulation of leaf expansion and stomatal responses can also be extended to other, more salt‐tolerant, non‐excluder species of the same family. Two tree medic species were chosen that contrast in their tolerance to NaCl. Medicago arborea is originally from the Greek islands and is currently grown as a commercial drought‐tolerant forage crop (Lambert et al., 1989). Medicago citrina is a closely related species, endemic of some small western Mediterranean islets. Under saline conditions, most of the early physiological changes experienced by plants are primarily in response to the osmotic component of salt. However, even with osmoregulation underway, salt‐treated plants face a build‐up of toxic ion concentrations in their tissues, which is considered a key limiting factor for plant growth in saline soils (Munns et al., 1995). This study was focused on this second phase, in which the response of the plant is predominantly to the effects of ion accumulation. Materials and methods Plant growth conditions Homogenous sized seeds were scarified in H2SO4 (96% w/w): 30 min M. arborea and 60 min M. citrina. At the emergence of the second trifoliate leaf, uniformly sized seedlings were selected and transplanted to 4.0 l pails (4 plants per container) filled with aerated modified 50% Hoagland’s solution (Epstein, 1972). The saline treatments were 50, 100, 150, and 200 mM NaCl, and were administered in a step‐wise fashion, adding daily increments of 50 mM until the desired concentration was reached. The experiments were conducted in a greenhouse under controlled conditions, with the following regimes: temperature: min/max 17/35 °C; relative humidity: min/max 30/70%. Growth parameters Plants were transferred to the laboratory the night before harvest. Leaf area was measured with a leaf area meter (D‐T Area Meter, Delta‐T Devices, Burwell, Cambridge, England). Tissue dry weight was determined after drying the samples in an air‐forced oven at 60 °C. for 48 h. The roots were washed three times in distilled water before drying. Leaf water content Water content of individual leaves of the main axis occupying positions 2, 4, 8, and 12 from the shoot apex was determined according to the following equation: LS=FW–DW/DW; where FW refers to the fresh weight and DW is dry weight. Gas exchange measurements Gas exchange measurements were made at noon using a closed system infrared gas analyser (LI 6200, Li‐Cor, Lincoln, NE, USA) on leaf 8 from the apex. This leaf was chosen to ensure the measurement of a fully expanded productive leaf that was not prone to senescence in the salt treatments. Measurements were taken under the following conditions: photon flux density, 946±17 µmol m–2 s–1; ambient CO2 concentration, 350±2 µmol mol–1 air; relative humidity, 40±0.5%; air temperature, 32±0.2 °C. Net photosynthesis (A), stomatal conductance (gs), internal CO2 concentration (Ci) and transpiration rate were calculated according to Farquhar and Sharkey (1982). Photosynthetic pigments and soluble protein concentration Samples of roots, and the leaves used for gas exchange measurements (leaf 8), were immediately frozen with liquid nitrogen and stored at –80 °C. Photosynthetic pigments and soluble protein were extracted using bicine buffer with pH 8 (Lawlor et al., 1989), and photosynthetic pigment concentrations were quantified according to Lichtenthaler and Wellburn (1983). Protein concentration was determined following the method described by Bradford (1976). Collection of xylem sap Sap collection was conducted at noon in plants individually grown in 1.0 l containers to avoid root tangling. Xylem sap was collected at midday using a pressure chamber (Soilmoisture, Santa Barbara, CA). Roots were washed, blotted dry and carefully inserted into a plastic bag. Afterwards, shoots were excised 5–6 cm above the shoot base using a sharp blade. The detopped plants were pressurized gradually and held at c. 0.7 MPa for 5 min to collect sufficient sap for analysis. After discarding the first drops (20–30 µl), sap was collected with a micropipette. Xylem sap pH was recorded with an Ingold micro‐pH electrode (Ingold, SA, Urdorf, Switzerland) and immediately frozen in liquid N2. In this work, data from plants grown at salt concentrations greater than 100 mM are not presented, as it was impossible to obtain sufficient xylem sap from M. arborea in a feasible, accurate manner as described above. Ion concentrations 0.1 g of finely ground oven‐dried tissue was digested overnight with 25 ml of 0.1 M HNO3 at room temperature. Xylem sap for ion analysis was used without further purification. Contents of Na+, K+, and Ca2+ of the cold acid extract and xylem sap were determined by inductively coupled plasma atomic emission spectrometry (Perkin‐Elmer Plasma‐2000). Chloride content of the cold acid extract was determined using a selective ion electrode potentiometer (Ingold, SA, Urdorf, Switzerland). Xylem sap Cl– was determined by HPLC (Waters, Mildorf, MA) equipped with a 4.6×150 mm IC‐PAK Anion HC column. Ion concentrations were calculated on a tissue‐water basis from the fresh and dry weights of the same leaf. Abscisic acid concentration Plant tissue was immediately frozen in liquid nitrogen, freeze‐dried, and finely ground with a mortar and pestle. 40 mg of freeze‐dried tissue was extracted overnight with distilled water in the dark at 4 °C. ABA concentration was analysed by radio‐immunoassay (Quarrie et al., 1988). Xylem sap was analysed directly from plants treated with either 1 or 100 mM NaCl using this same method, as previous analyses demonstrated that further extraction and purification steps were not necessary (Montero et al., 1994). Statistics Data are presented as the mean ±SE for each treatment (n=6). Significant differences among treatments were analysed by ANOVA. LSD values were calculated at the P <0.05 probability level (StatView, v. 4.0, Abacus Concepts, Inc. Berkeley, CA, USA). Results Growth Leaf growth was more affected by increased salt in M. arborea than in M. citrina as shown in Fig. 1. M. citrina had no significant differences in leaf dry weight and in leaf area. In M. arborea, both parameters showed a moderate linear decline at 50 and 100 mM NaCl (of 13% and 26%, respectively, in leaf dry weight, and 26.5% and 28% in leaf area). At 200 mM NaCl, the growth reduction was much more severe (50% of controls) for M. arborea, and plants showed visual symptoms of NaCl toxicity: tip necrosis and leaf chlorosis, in the youngest shoots. Stem dry weight decreased with salt supply in both species, while root dry weight only declined at the highest NaCl treatment. At 200 mM NaCl, stem and root dry weights were 32 and 54%, and 44 and 68% of controls in M.arborea and M. citrina, respectively. Leaf water content M. arborea had higher leaf water contents than M. citrina, and this parameter increased with leaf age in both species (Fig. 2). Salinization showed a tendency although not significant, to influence leaf water content in fully expanded leaves (positions 4 and 8 from the apex). For example, leaf water content increased in M. arborea, although slightly decreased in M. citrina with increased salt supply. The decrease in water content with increasing salt supply noted in expanding leaves (position 2 from the apex) may be consequence of a salt‐induced delay of leaf emergence. Gas exchange measurements Instantaneous CO2 assimilation rate and stomatal conductance decreased along with increased NaCl in M. arborea. The response found in the CO2 assimilation rate had the same pattern observed for the leaf growth, with values 13, 22 and 51% lower at 50, 100 and 200 mM NaCl treatments, respectively, than control plants. CO2 assimilation with respect to chlorophyll a concentrations were 91.5±5.3a, 79.1±3.6b, 73.2±5.3bc, and 68.2±3.0c nmol mg–1 s–1 for 1, 50, 100, and 200 mM NaCl‐treated plants. In this species, the decline in the A values was highly correlated with gs (r2=0.72; P <0.0001) (Fig. 3), leaf soluble protein (r2=0.89; P <0.0001) and chlorophyll a (r2=0.81; P <0.0001). The relative intercellular CO2 concentration (Ci/Ca) had values of 0.84±0.02a, 0.81±0.03a, 0.72±0.03b, and 0.66±0.04b for 1, 50, 100, and 200 mM NaCl‐treated plants, respectively. In agreement with the leaf growth response observed in M.citrina, no significant differences between treatments were found in the photosynthetic parameters, as CO2 assimilation rates were 15.5±0.9a, 17.1±2.5a, 16.2±1.5a, and 16.9±3.0a µmol m–2s–1 referred to leaf area and 76.1±8.4a, 74.7±9.6a, 62.1±9.7a, and 67.7±8.2a nmol mg–1 s–1 referred to chlorophyll a concentration and stomatal conductance were 0.55±0.07a, 0.60±0.11a, 0.58±0.06a, and 0.53±0.12a mol m–2s–1 and the relative intercellular CO2 concentration (Ci/Ca) had values of 0.84±0.03a, 0.79±0.05a, 0.82±0.04a, and 0.80±0.04a for 1, 50, 100, and 200 mM NaCl‐treated plants, respectively. For each data set mentioned, values with the same letter in superscript are not significantly different (P >0.05). Ion concentrations Root and stem Na+ and Cl– concentrations increased with salt increments, although there were no differences between species. In both Medicago, the highest Na+/Cl– concentrations were in the leaves, and increased with NaCl treaments (Fig. 4A, B). Although the expanded leaf (leaf 8) had higher Na+/Cl– concentrations than those of leaf 2, as this later leaf was in the expansion phase and in most instances not completely unfolded, Na+/Cl– concentrations could be considered as substantial. For instance, at 200 mM NaCl, leaf 2 Cl– of M. arborea exceeded the concentration in the nutrient solution. In both leaves considered, M. arborea had significantly greater Na+ values than M. citrina with respect to leaf number. Leaf Cl– concentrations were greater than for Na+ (Fig. 4B). In all saline treatments, M. arborea had higher Cl– concentrations in leaf 2. However, in leaf 8, no differences between species were found for Cl– concentrations, although higher Cl– concentrations were found in M. citrina in 50 and 100 mM NaCl treatments. Only at 200 mM NaCl, was M. arborea leaf Cl– higher than M. citrina. A negative relationship between either instantaneous CO2 assimilation (Fig. 5A) or stomatal conductance (Fig. 5B) and leaf Na+ concentration was found in M. arborea. In this species, leaf Na+ concentration was correlated with chlorophyll a concentration (r2=0.51; P <0.0001) and soluble protein concentration (r2=0.74; P <0.0001). Xylem Na+/Cl– was significantly higher in 100 mM NaCl M. arborea than M. citrina (Table 1). Potassium concentration decreased with NaCl uptake in leaves, stems and roots. The decline was similar in both species for each salt treatment, when considered with respect to their controls (Fig. 4C). Significant differences in the leaf Na+:K+ ratio were only found between species at 200 mM NaCl. Leaf 2 Na+:K+ ratio had values of 2.6±0.52 in M. arborea and 1.08±0.05 in M. citrina, while leaf 8 Na+:K+ ratio had values of 4.5±0.76 in M. arborea and 3.46±0.27 in M. citrina. No differences in xylem K+ were found between 1 and 100 mM NaCl‐treated plants. However, xylem K+ was 2‐fold higher in M. arborea than in M. citrina (Table 1). Stem and root Ca2+ declined with salinization with no significant differences between species. In both species leaf Ca2+ was the cation least affected by salt (Fig. 4D). Leaf 2 Ca2+ only decreased at 200 mM NaCl, while leaf 8 Ca2+ declined along with increased salt treatments. Leaf 2 Na+:Ca2+ ratio was higher in M. arborea than M.citrina grown at 200 mM NaCl, with found values of 7.09±0.61 and 4.09±0.98 in M. arborea and M. citrina, respectively. No significant differences were found in leaf 8 Na+:Ca2+ ratio either between species or treatments, with found values of of 8.28±0.54 and 9.72±1.64 in M. arborea and M. citrina, respectively, at 200 mM NaCl. However, the pattern for xylem Ca2+ was distinct in the two species in response to the treatment considered. In M. citrina, a significant drop was noted, as xylem Ca2+ fell to less than half of the values of control in response to the 100 mM NaCl treatment. By contrast, M. arborea xylem Ca2+ was significantly higher than the control group and rose more than 3‐fold in response to 100 mM NaCl. This rise in xylem Ca2+ was also significantly higher in salt‐treated plants of M. arborea than M. citrina (Table 1). Photosynthetic pigments and protein concentrations A slight increase in chlorophyll a concentration was found in M. citrina with salt (Fig. 6A). In the 200 mM NaCl treatment, chlorophyll a significantly decreased in M. arborea with respect to the controls. In both species, no significant differences were found in chlorophyll b, carotenes and xanthophyll concentrations among treatments (data not shown). Soluble leaf protein significantly decreased in M. arborea, while in M. citrina, it increased with salt (Fig. 6B). No differences in root protein concentration were found for either species or treatments (data not shown). Abscisic acid concentrations No differences were found in leaf ABA concentrations among salt treatments in M. arborea (Fig. 7). In M. citrina, both non‐expanded and expanded leaves had a significant increase in ABA at 100 and 200 mM NaCl. ABA did not increase in roots with NaCl, and thus, no significant differences were found in root ABA concentrations for either species or treatments. Xylem ABA significantly increased in 100 NaCl M. citrina, while no differences were found in M. arborea between treatments (Table 1). The correlation between leaf water content and leaf ABA in expanded leaves was much higher in M. citrina (r2=0.97; P=0.016) than in M. arborea (r2=0.39; P=0.030). Discussion Under the mild NaCl saline conditions used here, M. citrina had better leaf growth than M. arborea, and this response was strongly related to leaf Na+/Cl–, as both expanded (leaf 8 from the apex) and the still expanding (leaf 2 from the apex) leaves of M. arborea had overall significantly higher Na+/Cl– concentrations. The higher leaf Na+/Cl– exclusion capacity in M. citrina was not due to a better overall K+/Na+ selectivity, as leaf K+ decreased with salt in both species, and leaf Na+/K+ ratio was only higher in M. arborea than M. citrina at 200 mM NaCl. Moreover, leaf growth inhibition in M. arborea was not closely linked to leaf Ca2+, as Ca2+ was only found to decline in leaf 2 at 200 mM NaCl in both species. However, at this salt treatment, the higher leaf 2 Na+/Ca2+ ratio in M. arborea, jointly with the above‐mentioned higher Na+/K+ ratio, suggest a possible onset of an ion imbalance which could have contributed to the reduction in leaf growth found in the former species. There is evidence that ABA is involved in the regulation of leaf expansion under salt stress (Hartung et al., 1999; Cramer and Quarrie, 2002). Previous results with bean strongly suggested the possibility of a leaf growth ABA‐mediated plant response to Na+ (Montero et al., 1998; Sibole et al., 2000). However, in both Medicago species, no direct relationship was found between leaf growth and leaf ABA. No significant differences were found in root ABA among either treatments or species, and this growth regulator only increased in leaves and xylem of salt‐treated M. citrina. Although there is evidence that ABA is a root‐borne signal that participates in root–shoot communication processes (Hartung et al., 2002), it can also be synthesized in the leaves (Popova et al., 2000; Holbrook et al., 2002). In salt‐treated M. citrina, the increase in leaf ABA was correlated with decreased leaf water content, which suggests that ABA could partake in the adjustment of leaf metabolism to a decrease in water activity. Since ABA can be translocated into the phloem (Wolf et al., 1990), the higher xylem ABA concentrations found in 100 mM salt‐treated M. citrina could be the result of its retranslocation from the leaves via the phloem to xylem. Correspondingly, the lack of response in leaf ABA concentration in salt‐treated M. arborea could be related to the observed increase in leaf water content with NaCl (Hartung et al., 2002). The differences found in leaf growth between species are related to the decline in M. arborea CO2 assimilation, while no differences among treatments were found in M.citrina. In salt‐treated M. arborea, photosynthetic rate was limited by stomatal closure restricting CO2 diffusion into the mesophyll cells. However, the decline in Ci was poorly correlated with stomatal conductance (r2=0.26; P=0.088) which could be related to the presence of non‐stomatal effects. The decrease in CO2 assimilation was positively correlated with chlorophyll a and soluble protein, suggesting a regulation of photosynthetic unit size in response to NaCl stress. However, the rate of photosynthesis in salt‐treated plants also significantly decreased with respect to controls when expressed on a chlorophyll a basis. This would also indicate a decrease in the photosynthetic capacity probably due to a decline in leaf Na+/Cl– compartmentation capacity (Fig. 5). These results show a poor dependence between either CO2 assimilation rate or stomatal conductance and leaf Na+, for leaf Na+ concentrations of up to 150 mM. However, the dependence between these parameters greatly increased when leaf Na+ reached concentrations above those of the external NaCl (200 mM). This would indicate a reduced capacity for intracellular ion compartmentation in M. arborea, which would negatively affect enzyme activity (Wyn Jones and Gorham, 2002). Different studies have reported a relationship between non‐stomatal effects and the presence of high leaf Na+/Cl– concentrations (Seemann and Critchley, 1985; Bethke and Drew, 1992). Additionally, James et al. (2002) reported that non‐stomatal limitation was associated with leaf Na+ concentration above 200 mM. The higher capacity of M. citrina for intracellular ion compartmentation is even more clear when Cl– is considered, for example, at 100 mM NaCl leaf 8 Cl– is significantly higher in M. citrina than in M. arborea. There is evidence that xylem ABA regulates stomatal conductance under stress conditions (Davies and Zhang, 1991). Previous results in beans showed a high positive correlation between stomatal closure and either leaf or xylem ABA (Montero et al., 1998; Sibole et al., 2000). However, in salt‐treated M.arborea, neither leaf nor xylem ABA was related to stomata closure. Moreover, although salt‐treated plants had marked changes in xylem ion concentration, no differences in xylem pH were found among treatments and, therefore, an increase in the leaf apoplast ABA half‐life due to basification can be discarded (Wilkinson and Davies, 1997). Accumulation of apoplastic solutes has been shown to affect stomatal aperture (Ewert et al., 2000). Xylem Na+ was significantly higher in salt‐treated M. arborea and, therefore, it would be feasible that an accumulation of Na+ in the guard cell apoplastic space could disturb the K+ channels that participate in stomatal movement (Schroeder et al., 2001). For other species, it has been suggested that Na+ could contribute to stomata closure in other species. Perera et al. (1997) found a direct response of the stomata of Aster tripolium to Na+, which was not ABA‐mediated. The authors hypothesized that such a sensory system is useful to control the amount of salt delivered to the leaf by the transpirational stream. Other xylem‐transported elements implicated in regulating stomatal closure, such as Ca2+, were much higher in 100 mM salt‐treated M. arborea than in controls. However, bulk xylem calcium might not be an adequate indicator for its actual concentration in the substomatal cavity, since Ca2+ could present a marked gradient along the leaf apoplast (De Silva et al., 1998). M. citrina displayed no differences in stomatal conductance among treatments and therefore no relationship was found with the increased xylem and leaf ABA concentrations found in salt‐treated plants in this species. There is a great deal of evidence that stomatal conductance is regulated by the amount of ABA present in the apoplast of epidermal cells and is pH dependent (Wilkinson and Davies, 1997). Although xylem ABA of salt‐treated M. citrina increased, the ABA that would actually reach the guard cells may be not higher than controls. The xylem pH in salt‐treated M. citrina was about 6, which is the pH optima reported by Wilkinson and Davies (1997) for a carrier‐mediated epidermal ABA uptake. At this pH, it can be expected that the ABA that is not immediately metabolized can be compartmentalized in the epidermal and mesophyll cells (Hartung et al., 1998). This ABA would not participate in stomatal regulation, but could enhance the adaptation of these plants to salt (Jia et al., 1996) or have a protective role (Cramer, 2002). In M. citrina, a higher leaf Na+/Cl– compartmentation capacity would protect cell metabolism from the toxic effects of these ions. In this species, no significant differences were found in Ci/Ca and stomatal conductance between treatments, and salinized plants were able to maintain CO2 assimilation rate and leaf growth. Moreover, the negative effect of salt could have been ameliorated by the elevated chlorophyll a and soluble leaf protein concentrations found in NaCl‐treated M. citrina. Elevated chlorophyll content and increased photosynthetic capacity have been reported as a part of the cell adaptation process to salinity (Locy et al., 1996). An increase in leaf protein has been reported in salt (Wu and Seliskar, 1998) and water‐stressed plants (Pankovic et al., 1999). Moreover, an increase in leaf soluble protein has been related to an increased mesophyll conductance in drought‐tolerant varieties (Lauteri et al., 1997). By contrast, in M. arborea, soluble protein concentration decreased with salt. The strong correlation between leaf soluble protein and leaf Na+/Cl– suggests a toxic Na+/Cl– effect on leaf protein synthesis (Helal and Mengel, 1979) and/or a Na+/K+ imbalance (Serrano et al., 1999). In this study, an attempt has been made to determine whether or not changes in ABA concentrations were negatively correlated with leaf growth and stomatal responses in Na+ non‐excluder legumes, as found earlier for the Na+‐excluder bean (Montero et al., 1998; Sibole et al., 2000). As the results showed no correlation between increased ABA and either leaf growth or stomatal conductance, there is no straightforward affirmative answer. Instead, these results point to other components that may participate in leaf growth control in these two legume species when submitted to a long‐term mild salinization. The better leaf response to salt in M. citrina is related to a higher compartmentation capacity, which maintains cell metabolism and energy production required to sustain leaf growth in saline conditions. Acknowledgements Financial support from the DGICYT project BFI2001‐2475‐C02‐02 and the European Funds for Regional Development is gratefully acknowledged. View largeDownload slide Fig. 1. Leaf dry weight (A) and total leaf area (B) in M. arborea and M. citrina grown in 1, 50, 100 or 200 mM Na/Cl for 30 d. Error bars=standard error of mean (n=6). Values with the same letter are not significantly different (P >0.05). View largeDownload slide Fig. 1. Leaf dry weight (A) and total leaf area (B) in M. arborea and M. citrina grown in 1, 50, 100 or 200 mM Na/Cl for 30 d. Error bars=standard error of mean (n=6). Values with the same letter are not significantly different (P >0.05). View largeDownload slide Fig. 2. Leaf water mean content of leaves 2, 4, 8, and 12 from the apex in M. arborea (A) and M. citrina (B) grown in 1, 50, 100 or 200 mM NaCl for 30 d. Error bars=standard error of mean n=6). View largeDownload slide Fig. 2. Leaf water mean content of leaves 2, 4, 8, and 12 from the apex in M. arborea (A) and M. citrina (B) grown in 1, 50, 100 or 200 mM NaCl for 30 d. Error bars=standard error of mean n=6). View largeDownload slide Fig. 3. Relationship between instantaneous CO2 assimilation rate, stomatal conductance (A) and leaf protein concentration (B) of leaf 8 from the apex in M. arborea grown in 1, 50, 100 or 200 mM NaCl for 30 d. View largeDownload slide Fig. 3. Relationship between instantaneous CO2 assimilation rate, stomatal conductance (A) and leaf protein concentration (B) of leaf 8 from the apex in M. arborea grown in 1, 50, 100 or 200 mM NaCl for 30 d. View largeDownload slide Fig. 4. Sodium (A), Cl– (B), K+ (C), and Ca2+ (D) concentrations in tissue water of leaves 2 and 8 from the apex in M. arborea and M. citrina in 1, 50, 100 or 200 mM NaCl for 30 d. Error bars=standard error of mean (n=6). Values with the same letter are not significantly different (P >0.05). View largeDownload slide Fig. 4. Sodium (A), Cl– (B), K+ (C), and Ca2+ (D) concentrations in tissue water of leaves 2 and 8 from the apex in M. arborea and M. citrina in 1, 50, 100 or 200 mM NaCl for 30 d. Error bars=standard error of mean (n=6). Values with the same letter are not significantly different (P >0.05). View largeDownload slide Fig. 5. Relationship between instaneous CO2 assimilation rate (A) and somatal conductance (B) and leaf Na+ of leaf 8 from the apex in M. arborea grown in 50, 100 or 200 mM NaCl for 30 d. View largeDownload slide Fig. 5. Relationship between instaneous CO2 assimilation rate (A) and somatal conductance (B) and leaf Na+ of leaf 8 from the apex in M. arborea grown in 50, 100 or 200 mM NaCl for 30 d. View largeDownload slide Fig. 6. Chlorophyll a (A) and soluble protein (B) concentrations of leaf 8 from the apex in M. arborea and M. citrina in 1, 50, 100 or 200 mM NaCl for 30 d. Error bars=standard error of mean (n=6). Values with the same letter are not significantly different (P >0.05). View largeDownload slide Fig. 6. Chlorophyll a (A) and soluble protein (B) concentrations of leaf 8 from the apex in M. arborea and M. citrina in 1, 50, 100 or 200 mM NaCl for 30 d. Error bars=standard error of mean (n=6). Values with the same letter are not significantly different (P >0.05). View largeDownload slide Fig. 7. ABA concentrations of leaves 2 and 8 from the apex in M. arborea and M. citrina grown in 1, 50, 100 or 200 mM NaCl for 30 d. Error bars=standard error of means (n=6). Values with the same letter are not significantly different (P >0.05). View largeDownload slide Fig. 7. ABA concentrations of leaves 2 and 8 from the apex in M. arborea and M. citrina grown in 1, 50, 100 or 200 mM NaCl for 30 d. Error bars=standard error of means (n=6). Values with the same letter are not significantly different (P >0.05). Table 1. Xylem sap Na+, Cl–, K+, Ca2+, and ABA concentrations in M. arborea and M. citrina grown in 1 or 100 mM NaCl for 30 d For each column, values with the same letter are not significantly different (P >0.05). Species  Treatment  Na+  Cl–  Ca2+  K+  ABA  pH    NaCl (mM)  (mM)  (mM)  (mM)  (mM)  (nM)    M. arborea  1  0.052±0.04 a  0.24±0.09 a  0.10±0.10 a  15.89±0.25 a  23.1±4.60 a  6.06±0.16 a    100  5.19±0.26 b  11.1±2.19 b  0.38±0.15 b  16.41±0.25 a  36.8±12.5 a  6.13±0.11 a  M. citrina  1  0.022±0.01 a  0.32±0.09 a  0.20±0.11 b  8.46±1.03 b  18.6±9.47 a  5.99±0.26 a    100  2.41±0.45 c  6.68±1.58 c  0.08±0.02 a  9.23±0.52 b  61.7±18.5 b  5.90±0.12 a  Species  Treatment  Na+  Cl–  Ca2+  K+  ABA  pH    NaCl (mM)  (mM)  (mM)  (mM)  (mM)  (nM)    M. arborea  1  0.052±0.04 a  0.24±0.09 a  0.10±0.10 a  15.89±0.25 a  23.1±4.60 a  6.06±0.16 a    100  5.19±0.26 b  11.1±2.19 b  0.38±0.15 b  16.41±0.25 a  36.8±12.5 a  6.13±0.11 a  M. citrina  1  0.022±0.01 a  0.32±0.09 a  0.20±0.11 b  8.46±1.03 b  18.6±9.47 a  5.99±0.26 a    100  2.41±0.45 c  6.68±1.58 c  0.08±0.02 a  9.23±0.52 b  61.7±18.5 b  5.90±0.12 a  View Large References Bethke PC , Drew MC. 1992. 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Journal of Experimental BotanyOxford University Press

Published: Sep 1, 2003

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