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Relative importance of osmotic-stress and ion-specific effects on ABA-mediated inhibition of leaf expansion growth in Phaseolus vulgaris

1998; Wiley; Volume: 21; Issue: 1 Linguagem: Inglês

10.1046/j.1365-3040.1998.00249.x

1365-3040

Elena Montero, Catalina Cabot, Charlotte Poschenrieder, Juan Barceló,

Seed Germination and Physiology

The osmotic and ion-specific components of salt-induced inhibition of leaf expansion growth were investigated in beans grown from 12 h to several days in either NaCl-containing solution cultures, an isosmotic concentrated macronutrient solution, or a vermiculite–compost mixture with low Na+ but high Cl– availability. Inhibition of leaf expansion and leaf ABA increase was more intense in the NaCl than in the isosmotic macronutrient treatment. Root Na+ was highly correlated to inhibition of leaf expansion and leaf or xylem sap ABA. When Na+ was sequestered in soil, salinized plants showed no reduction in leaf expansion or ABA increase, regardless of the presence of high leaf Cl– concentrations. Stomatal conductance exhibited an exponential relationship with the reciprocal value of xylem sap ABA. Our results indicate that an ion-specific effect caused by Na+ in roots may account for an ABA-mediated reponse of both stomatal closure and leaf expansion inhibition. Salinity, particularly when NaCl is the predominant salt, is one of the most important causes of yield decrease in crop plants, especially in arid and semi-arid regions (Epstein 1983). The effects of increased salinization on plant growth are complex and not well understood. The bicomponent nature of salt stress, causing both a decrease of the substrate osmotic potential and ion-specific toxicity, makes investigations of the mechanisms of salt-induced growth inhibition extremely difficult. Disparate observations on plant response to salinity have been explained by the hypothesis of a two-phase response model (Munns & Termaat 1986). According to this model, growth in salt-stressed plants is initially reduced by a salt-induced water-stress effect, which, by inducing a root-derived signal, causes an inhibition of the leaf expansion rate. The inhibition of leaf expansion growth during this first phase is not ion-specific but caused by an effect of the salt outside the plant, i.e. by a decrease of the osmotic potential in the substrate. Specific ion toxicity inside plants would only be responsible for further inhibition of leaf expansion in a second, later phase, when a considerable salt concentration has accumulated in the leaves (Munns & Termaat 1986; Munns 1993). There is now considerable experimental evidence that an increase of xylem sap ABA may signal soil water status or resistance to water flow, causing stomatal closure in water-stressed plants (Tardieu & Davies 1993). Experiments with maize and sunflower (Zhang & Davies 1990) suggest that the increase in xylem ABA content may quantitatively account not only for stomatal closure but also for the reduction in leaf expansion under water stress. In contrast, bioassay experiments by Munns (1992) do not support a role of xylem sap ABA in the inhibition of leaf expansion growth of water- or salt-stressed plants. Recent investigations with maize exposed for a few hours or days to PEG 6000 or NaCl suggest that water stress induced by the osmotic treatment may be responsible for the observed increase of leaf ABA which, in turn, would cause the inhibition of leaf expansion growth in the osmotic-stressed plants (Chazen et al. 1995). Unfortunately, only leaf ABA, not xylem sap ABA, was analysed in this study. The relative importance of osmotic and ion-specific toxicity effects in the response of a salt-sensitive plant such as bean, with poor capacity to exclude Cl– from the transpiration stream, is not clearly established. Even less clear is the role of xylem ABA as a possible root-derived signal in stomatal closure and inhibition of leaf expansion growth in bean. In contrast to experiments with other plant species, no correlation between xylem sap ABA and stomatal conductance could be established in two water-stressed bean cultivars (Trejo & Davies 1991). In the present study, an attempt was made to establish the relative importance of osmotic and ion-specific toxicity effects on leaf growth inhibition in salt-stressed beans and its possible relation to stomatal conductance in xylem ABA, using plants cultivated either in isosmotic solutions with or without NaCl or in substrate with low Na+ but high Cl– availability. Seeds of bush beans, Phaseolus vulgaris L. cv. Contender, were sown in trays of vermiculite previously saturated with distilled water. The trays were kept in the dark at 20°C until germination and emergence of a homogeneous population had been achieved. The seedlings were then transferred to a climate-controlled growth chamber (16 h photoperiod, 25°C day, 20°C night and RH 40% day, 70% night). Irradiance measured at seedling level was 300 μmol m–2 s–1 PAR, supplied by cool white fluorescent light supplemented with incandescent lamps. Two experiments were carried out: experiment 1 was performed in order to study the possible ion-specific effects of NaCl on leaf expansion growth, while experiment 2 was conducted to distinguish Na+ from Cl– effects. In this experiment the photoperiod of the growth chamber was set to 12 h d–1. Eight-day-old seedlings were transplanted to 0·25 dm3 Erlenmeyer flasks (one plant per container) filled with aerated 50% modified Hoagland solution (Epstein 1972) with 1·0 mol m–3 NaCl. After 3 d and coincident with the beginning of a new dark period, plants were transferred to the following treatment solutions: 50% modified Hoagland with 1 mol m–3 NaCl (control), 500% modified Hoagland with 1 mol m–3 NaCl, or 50% modified Hoagland with 75 mol m–3 NaCl. The concentrated macronutrient solution and the 50% modified Hoagland solution with 75 mol m–3 NaCl were isosmotic (measured π = 0·41 MPa) (Termaat & Munns 1986). Leaf expansion growth values (increment in leaf area during the time considered divided by the time considered) were measured on six plants per treatment every 3 h. Significant differences between treatments were observed after 12 h and only these results are shown. After emergence, the seedlings were transplanted to pails (four plants per container) filled with 4 dm3 of aerated 50% modified Hoagland solution (Epstein 1972) with 1 mol m–3 NaCl. On days 7, 9 and 11 after transfer to solution culture and coincident with the beginning of a new dark period, during the expansion of the first trifoliolate leaf, 25 mol m–3 NaCl was added every 2 d to half of the pails. The solution culture was maintained at pH 6, topped up daily and changed every 3 d for the duration of the experiment. An equal number of seedlings were transplanted into 15-cm-diameter pots (one plant per pot) containing a mixture of vermiculite and compost (1:1 v/v). Pots were placed in trays containing 50% modified Hoagland nutrient solution. The salinization of these plants was performed following the same time schedule described above for the solution culture experiments. The first measurement was made 16 h after the first salt addition, while all subsequent measurements were made at 24 h intervals for 6 d after the first salt addition. Leaf area was determined daily as the product of measured length and width, according to the following equation: LA (cm2) = L×W× 0·86 for the primary leaf and LA (cm2) = L×W× 0·57 for the first trifoliolate leaf (Cabot 1985). Relative leaf growth rates were calculated as hourly and daily increments in leaf surface area in experiments 1 and 2, respectively. Before drying at 60 °C to a constant weight, root tissue was rinsed by dipping three times for a few seconds in distilled-deionized water. Root and leaf dried tissue (100 mg), finely ground, was digested in 25 cm3 of 0·1 kmol m–3 nitric acid for 12 h and filtered. The sodium content of the tissue was determined by inductively coupled plasma emission spectrophotometry (Perkin-Elmer Plasma-2000, Perkin-Elmer, Norwalk, CA, USA). The chloride content was determined in the same acid extracts using a selective ion electrode (Ingold, SA, Urdorf, Switzerland). The stomatal conductance of the abaxial surface was determined at noon using a diffusion porometer (MK3, Delta-T Devices Ltd, Cambridge, UK). Upon sampling, root and leaf tissue was immediately frozen in liquid nitrogen, freeze-dried and finely ground with a mortar and pestle. Freeze-dried tissue samples (40 mg) were extracted overnight, in the dark, at 4 °C with distilled water. ABA was analysed by radioimmunoassay (Quarrie et al. 1988). The monoclonal antibody used (AFR MAC 252), provided by Dr S. A. Quarrie, was specific for (+)- ABA. Xylem sap was collected as described by Trejo & Davies (1991), using a pressure chamber (Soil Moisture Equipment, Santa Barbara, CA, USA), and was analysed directly for ABA using the above method. The experiments were conducted three times with similar results. The data presented here are means of 4–6 replicates with standard errors from a representative experiment. Plants grown in 50% Hoagland solution culture (controls) exhibited significantly higher leaf expansion rates and lower leaf ABA concentrations than the plants exposed to solutions with lower osmotic potential (Table 1). No statistically significant differences in leaf osmotic, turgor or water potentials between treatments were observed (data not shown). The 50% modified Hoagland + 75 mol m–3 NaCl treatment, in spite of being isosmotic with 500% modified Hoagland solution, caused a more severe reduction of leaf expansion rate and a significantly higher increase of ABA leaf contents. A good linear correlation (r2 = 0·88; n = 18) between leaf expansion rates and leaf ABA concentrations was found. Each point was a coupled value of ABA and expansion rate corresponding to the same leaf. No differences between leaf concentrations of Na+ for plants from the different treatments were observed (Table 1). In contrast, leaf Cl– concentrations of plants growing in 50% Hoagland + 75 mol m–3 NaCl were substantially higher than in leaves from the other treatments (Table 1). From 50 mol m–3 NaCl onwards, the progressive exposure of plants to increasing NaCl concentrations in solution culture caused an inhibition of leaf expansion rate (Fig. 1). At lower salinity no effect on leaf expansion was seen. In contrast, salinization of plants growing in vermiculite–compost mixture did not influence leaf expansion (Fig. 1). . Leaf growth rate (cm2 h–1) during the six experimental days in controls (○) and salt-stressed (○) plants grown in solution culture (a) or vermiculite compost (b). Seedlings of beans were transplanted to pails filled with 4 dm3 of aerated 50% modified Hoagland's solution and placed in a growth chamber. Salt stress was imposed by addition of 25 mol m–3 NaCl on day 7, 9 and 11 (arrows). Leaf area was measured daily as the product of length and width 0·57. Leaf growth rates were determined as daily increments in leaf surface area. The time-dependent changes in Cl– and Na+ contents in the roots and leaves of control and salt-stressed plants grown either in solution culture or in the vermiculite–compost mixture are shown in Figs 2 and 3. In both substrates, root and leaf Cl– content increased with NaCl supply, achieving values of up to 2% in dry mass. At the end of the experiment, up to 37% of the total plant Cl– content was located in the shoots (Fig. 2). Roots of plants exposed to NaCl-supplemented solution culture accumulated much more Na+ than those of plants growing in the NaCl-treated vermiculite–compost mixture (Figs 3a & b). Regardless of the substrate, no differences in leaf Na+ concentrations between control and NaCl treatments were observed (Figs 3c & d). . Chloride concentrations (g/100 g dry weight) of roots (a & b) and leaves (c & d) of salt-stressed (○) and control (○) bean plants grown in solution culture (a & c) or vermiculite–compost (b & d). Salt stress was imposed by addition of 25 mol m–3 NaCl on day 7, 9 and 11 (arrows). Tissue samples were taken for six consecutive days after the first salt addition. . Sodium concentrations (g/100 g dry weight) of roots (a & b) and leaves (c & d) of salt-stressed (○) and control (○) bean plants grown in solution culture (a & c) or vermiculite–compost (b & d). Salt stress was imposed by addition of 25 mol m–3 NaCl on day 7, 9 and 11 (arrows). Tissue samples were taken for six consecutive days after the first salt addition. In both substrates, NaCl-treated plants exhibited lower mean values of stomatal conductance than unstressed controls, but only in the solution-cultured plants were the differences statistically significant (Figs 4a & b). . Stomatal conductance (cm s–1) of leaves of salt-stressed (○) and control (○) bean plants grown in solution culture (a) or vermiculite–compost (b). Salt stress was imposed by addition of 25 mol m–3 NaCl on day 7, 9 and 11 (arrows). Stomatal conductance was measured at midday on the abaxial surface of first trifoliolate leaf using a diffusion porometer. Figure 5 displays the root and leaf ABA content for the 6 d observation period. In plants grown in the vermiculite–compost mixture, NaCl did not induce any increase of ABA, either in roots or in shoots (Figs 5b & d). In contrast, in solution culture, salt-stressed plants exhibited higher root and leaf ABA concentrations than unstressed controls. The salt-induced increase of endogenous ABA was much more intense in leaves than in roots (Figs 5a & c). . Abscisic acid concentrations (ng g–1 dry weight) of roots (a & b) and leaves (c & d) of salt-stressed (○) and control (○) bean plants grown in solution culture (a & c) or vermiculite–compost (b & d). Salt stress was imposed by addition of 25 mol m–3 NaCl on day 7, 9 and 11 (arrows). For ABA analysis, 40 mg freeze-dried tissue samples were extracted overnight, in the dark, at 4 °C with distilled water. ABA was analysed by radioimmunoassay. In solution-cultured plants, NaCl caused a significant increase of xylem sap ABA concentrations (data not shown). Using data from all treatments obtained by measuring xylem sap ABA and stomatal conductance every 3 h during the day after each salt addition, an exponential relationship between stomatal conductance and the reciprocal values of xylem ABA concentrations could be established (Fig. 6). In salinized plants, a highly significant correlation (r2 = 0·75; n = 26) between xylem sap ABA and leaf ABA concentrations was observed, and both leaf and xylem sap ABA concentrations were highly correlated to leaf expansion growth (Figs 7a & b). There was no significant linear correlation between either Na+ or Cl– concentrations in leaves and leaf expansion growth or leaf ABA concentrations (data not shown). However, both leaf expansion and leaf ABA were highly correlated to root Na+ concentrations (Figs 8a & b). . Correlation of the reciprocal xylem ABA (cm3 H2O ng–1 ABA) versus stomatal conductance (cm s–1). Beans were grown in solution culture or in a vermiculite–compost 1:1 mixture. Half of the plants from each growing medium were salinized with 25 mol m–3 salt additions during the expansion of the first trifoliolate leaves. Stomatal conductance and xylem sap ABA were measured every 3h during the day after each salt addition. . Correlation of leaf ABA content (a) and xylem sap ABA (b) versus leaf expanding rate. Solution cultured beans, salinized with 25 mol m–3 salt additions during the expansion of the first trifoliolate leaves showed a high linear correlation between first trifoliolate leaf expansion rates and either leaf ABA (r2 = 0·91) or xylem sap ABA (r2 = 0·76). . Correlation of root Na+ content versus leaf expansion rate (a) and leaf ABA content (b). Solution cultured beans, salinized with 25 mol m–3 salt additions during the expansion of the first trifoliolate leaves show a high linear correlation between either first trifoliate (r2 = 0·94) leaf expansion rates or leaf ABA contents (r2 = 0·95) and their respective Na+ root contents. According to the two-phase response model of Munns & Termaat (1986), the inhibition of leaf expansion growth experienced in plants after a few hours exposure to NaCl was attributed to the decreased water potential of the rooting solution rather than to the presence of a specific salt. Our observation that leaf expansion growth was more inhibited in a NaCl-containing nutrient solution than in an isosmotic concentrated macronutrient solution (Table 1), however, shows that, in beans, ion-specific effects may play an important role even during the first response phase. After a short exposure to NaCl no significant increase of Na+ or Cl– concentrations in leaves was observed, and the inhibition of leaf expansion growth cannot be explained by direct ion-toxicity effects in leaf tissues. The lack of significant differences between treatments in leaf turgor potential and the fact that stomata were closed exclude the possibility that the more intense inhibition of leaf expansion growth in NaCl-treated plants was caused by water deficits in leaves and/or inhibition of photosynthesis. There is convincing experimental evidence that a decrease of either substrate water potential or hydraulic conductivity in roots increases leaf ABA (Tardieu & Davies 1993; Chazen et al. 1995). The highly significant correlation between leaf expansion rates and ABA leaf concentrations observed in this experiment, regardless of the stress treatment, supports the hypothesis that both the osmotic and the ion-specific effects on leaf expansion growth were ABA-mediated. The observation that under mild stress conditions the salt-sensitive bean plants have a certain capacity to exclude Na+ from the shoots by recirculation of Na+ to the roots (Jacoby 1979) has led to the assumption that Cl–, rather than Na+, may be responsible for ion-specific toxicity in this species (Seemann & Critchley 1985; Marschner 1995). Results from our second experiment provide evidence against this hypothesis. Plants growing in a vermiculite–compost mixture with low Na+, but high Cl– availability, accumulated high root and shoot Cl– concentrations (Figs 2b & d), while Na+ levels remained low in both organs (Figs 3b & d). These plants did not show any salt-induced inhition of growth. Only in the solution cultured-plants, with high Na+ availability leading to an accumulation of more than 1% Na+ in root dry matter (Fig. 3a), was a significant decrease of leaf expansion (Fig. 1) and a greatly significant increase of leaf ABA levels (Fig. 5c) observed. These effects were not caused by an accumulation of Na+ in the leaves. Due to the relatively short exposure time to NaCl, the plants were able to effectively exclude Na+ from the shoots, and the salt-stressed plants had the same Na+ tissue concentrations in leaves as in unstressed controls (Figs 3c & d). These results, in addition to the good correlations between root Na+ concentrations and both leaf expansion growth and leaf ABA concentrations (Figs 8a & b), suggest that the accumulation of Na+ in roots caused ion-specific toxicity effects that were responsible for increased leaf ABA and inhibition of leaf expansion growth. Several authors have observed that NaCl negatively influences root hydraulic conductivity (Azaizeh & Steudle 1991; Azaizeh et al. 1992; Chazen et al. 1995). Increased resistance to water flow induced by Na+ inside bean roots may act as a signal for inhibition of leaf expansion growth in a similar way as proposed for drought-stressed plants (Tardieu & Davies 1993). Accumulation of Na+ in the roots of the plants exposed to NaCl in solution culture in our experiment was accompanied by a substantial increase of the xylem sap ABA concentration. A significant deviation from linearity of the relation between reciprocal values of xylem sap ABA and stomatal conductance (Fig. 6) indicates that the Na+-induced increase of xylem sap ABA cannot be explained by a concentration effect due to decreased transpiration but that a significant net increase of ABA export from roots must have occurred (Tardieu & Davies 1993; Jarvis & Davies 1997). This result supports the hypothesis, already well documented for drought-stressed plants (Trejo & Davies 1991), that at least certain leaf responses of beans to salt stress are mediated by root-derived ABA, and that in salt-stressed beans ABA may act as a signal from roots to shoots. As a good correlation between xylem sap ABA and leaf ABA concentrations was observed, leaf expansion was well correlated with ABA concentrations in both xylem sap and leaves (Figs 7a & b). In salt-stressed Lupinus, Wolf et al. (1990) calculated that both xylem and phloem flows of ABA increased under saline conditions. Our experiments strongly support the hypothesis that salt-induced inhibition of leaf expansion is mediated by ABA, but they do not provide an answer to the question of whether the ABA responsible for growth inhibition was synthesized in leaves and/or translocated from the roots. This latter hypothesis is favoured by the observation that leaf ABA levels also increased in the plants of our first experiment where no salt effects on leaf water relations, stomatal conductance and leaf Na+ or Cl– concentrations (Table 1) were detectable. In conclusion, our results show that in salt-sensitive beans, even during the first phase of response to salt stress, Na+-specific ion toxicity in roots is responsible for inhibition of leaf expansion growth, and that an accumulation of Na+ in leaves is not necessary for an increase of leaf ABA and an inhibition of leaf expansion to occur. Accumulation of Cl– in leaves does not seem to play a role in the initial leaf growth inhibition. Specific Na+ toxicity inside roots seems to be responsible for the more intense inhibition of leaf expansion in NaCl-stressed plants than in plants exposed to an iso-osmotic concentrated macronutrient solution in which responses would be caused only by osmotic effects in the substrate. Although the possibility of an excessive accumulation of nutrient ion concentrations in leaves cannot be completely excluded, it seems improbabe that, during the short exposure time used in this experiment (12 h), accumulation of toxic concentrations of K+ or NO3– in leaves took place. We thank Dr W. J. Davies for providing laboratory facilities and helpful suggestions concerning the manuscript. Technical assistance with ion analysis by John V. Sibole is gratefully acknowledged. This work was supported by DGCIYT project PB-94–0738-CO2-O2 and by a postgraduate formation grant (PN89) to E.M.