Differential effects of salinity on leaf elongation kinetics of three grass species

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1 Plant and Soil 253: , Kluwer Academic Publishers. Printed in the Netherlands. 233 Differential effects of salinity on leaf elongation kinetics of three grass species Grant R. Cramer Department of Biochemistry, Mail Stop 200, University of Nevada, Reno, NV 89557, USA Received 4 April 2002; accepted in revised form 22 November 2002 Key words: Hordeum vulgare, Hordeum jubatum, leaf elongation, salinity, Zea mays Abstract This study focuses on the inhibitory effect of salinity on the leaf extension of three different grass species: Hordeum jubatum L., Hordeum vulgare L. and Zea mays L. Leaf elongation rates (LER) were measured on the third leaf of the plants. NaCl was added to the hydroponic solution (0, 40, 80 and 120 mm) and changes in LER were measured over time with a displacement transducer. Salinity inhibited LER immediately in all three species, and a new, but lower steady-state LER was reached within 5 h. The decrease in LER was proportional to the salinity level. Differences in salt tolerance (% of control LER) were evident between genotypes within 5 h after salinization, but the relative salt tolerance of the plant at this stage was not necessarily indicative of the long-term salt tolerance of the species. In general, H. jubatum was more tolerant than maize, which was more tolerant than barley to these short-term salinity stresses. In contrast, barley is more salt tolerant than maize over the long term. The mechanisms of inhibition of LER by salinity, as tested by the applied-tension technique, varied with the species examined, affecting either the apparent yield threshold, the hydraulic conductance of the whole plant or both. The cell wall extensibility was not significantly affected by salinity in the three species tested in this study. Introduction Leaf growth in a grass is confined to its basal region, which is enclosed by sheaths of subtending leaves. Growth is largely unidirectional, with a basal meristem producing parallel files of cells (MacAdam et al., 1989). Cells within a file are displaced away from their origin (the meristematic region) as a result of continued production and elongation of new cells within the same file. After the growth of younger cells displaces a local cell past the distal margin of the meristem, cell elongation continues in the absence of cell division (Tardieu et al., 2000). The result of the coordinated sequence of division, expansion and displacement is that the monocot leaf consists of cell files characterized by a gradient in age, and the distance between a cell and its origin is a function of both age and developmental stage. Because of this unidirectional developmental gradient, the leaf growth zone of grasses is well suited FAX No: cramer@unr.edu for the study of biophysical and biochemical processes associated with growth (Gandar and Hall, 1988; Schnyder and Nelson, 1987). Cell expansion is a function of water uptake and cell wall extension. It involves both biochemical and physical processes. The current view is that a biochemical loosening of the cell wall under turgor pressure initiates cell expansion followed by water and solute uptake (Boyer, 2001; Cosgrove, 1997). Mathematical models of growth with a mechanistic approach, enable one to separate growth into its regulatory components and pinpoint where experimentation should be directed. Such a mechanistic growth model is well developed at the cellular level and can be simply described by the following equation (Green et al., 1971): 1 v v t = E p, (1) where, v, t, E, and p are cell volume, time, cell wall yielding properties, and turgor pressure, respectively.

2 234 A more rigorous description of growth includes both the mechanical and hydraulic aspects of growth (Boyer et al., 1985): 1 v v t = ml m + L ( o s Y) (2) This is a variation of an equation first derived by Lockhart (1965), where m, L, o, s,andyrepresent the cell wall extensibility, the hydraulic conductance, the xylem water potential, the cell osmotic potential, and the yield threshold, respectively. The term, ml/(m+l), is often referred to as the growth coefficient and is comparable to E in Equation (1). The term, ( o s Y), is the driving force for cell expansion. The yield threshold is the minimum p at which cells expand. Although cell expansion is three-dimensional, it can be adequately described in one dimension (change in length) if the forces applied are small (Nonami and Boyer, 1990b). On the basis of Equation (2), the rates of cell expansion (or leaf extension) can be plotted as a function of ( o s ). Such a plot is theoretically linear, with the x intercept equal to Y (when Y equals o s, growth is zero) and the slope equal to the growth coefficient (Green et al., 1971). This mathematical description of cell expansion provides a model from which one can analyze growth. A variety of techniques (and variations) have been used for the analysis of cell elongation and plant growth (Boyer et al., 1985; Cosgrove, 1987; Cramer and Bowman, 1991; Frensch and Hsiao, 1995; Green et al., 1971; Nonami and Boyer, 1990a, b; Okamoto et al., 1989). All of these techniques potentially suffer from artifacts, the most obvious being that the measurements require changes in growth rate. In addition, growth rates must be at steady-state to be theoretically valid. It takes time to reach steady-state growth and one can argue that techniques used to change the growth rate might cause additional changes in the growth parameters before reaching steady-state growth. Nevertheless, they serve as useful tools, when their limitations are recognized. Applied-tension techniques have been developed to modify elongation rates with intact plants (Cramer and Bowman, 1991; Nonami and Boyer, 1990b). The elongation rates are modified with weights to generate a turgor-like, unidirectional force in the leaf. Leaf elongation rate as a function of applied force is then measured with a LVDT (linear variable differential transformer), which is a displacement transducer. The method of Nonami and Boyer (1990b) differs slightly from that of Cramer and Bowman (1991). Plastic extension is plotted against the increased force applied to the leaves, which enables the determination of one growth coefficient: m. Cramer and Bowman s method (1991) takes a similar approach, but has the added advantage of determining the apparent yield threshold and the growth coefficient, which includes both L and m. Soil salinity decreases rates of leaf expansion to various degrees in all crop plants, but it is not known exactly how this occurs. The primary cause of reduced growth could be located in the photosynthetic or the growing tissue of the leaf. In either case, an inhibiting factor could affect cell expansion in the growth zone of the leaf. Greenway studied the effects of salinity on the growth and ion transport of barley (Greenway, 1962a, 1963, 1965; Greenway and Pitman, 1965; Greenway et al., 1965). He not only investigated the uptake of several different ions, but also in several varieties of barley differing in salt tolerance. He established that lower Na + and Cl contents were associated with salttolerant varieties. He then contributed significantly to our understanding of the nature of their transport into and within the plant. At the time, these pioneering studies were considered to be short-term experiments, covering the response of plants over a week or two. Today, many investigations have focused on much shorter response times, from minutes to a day. When salinity is applied to the root medium, leaf elongation is immediately inhibited for maize (Cramer, 1992b; Cramer and Bowman, 1991; Munns et al., 2000b), rice (Yeo et al., 1991) and barley (Munns et al., 2000a; Thiel et al., 1988). There is a rapid hydraulic signal (lowered water potential) that reduces the effective turgor of the growth zone (Cramer and Bowman, 1991; Thiel et al., 1988). With time, osmotic adjustment occurs in the growth zone and turgor recovers. Leaf elongation also recovers to a new steady-state rate, but this rate is below the control rate. Turgor has completely recovered, yet steady-state growth is inhibited below that of the control. However, the apparent yield threshold has increased, causing a reduced effective turgor force for cell elongation. By 24 h of salinization, other secondary events are occurring (Cramer, 1992a). The yield threshold is still increased relative to controls, but cell wall extensibility is reduced as well. In addition, high Na/Ca can reduce hydraulic conductance. The differences in salt tolerance between two maize cultivars are attributed to differences in effective turgor, resulting from differences in the increase of the apparent yield threshold.

3 235 During these early events, photosynthesis does not appear to be limiting growth (Cramer et al., 1994; Yeo et al., 1991). In a review of how drought, salinity and temperature limit cell expansion (Cramer and Bowman, 1993), it was found that there were no universal mechanisms of control. The mechanisms of control vary with genotype and the stress imposed. For example, the response of the cell wall extensibility to osmotic stress is highly variable and depends on the genotype and the duration of the stress. Hydraulic conductivity is almost always reduced for all three stresses, yet often this effect is secondary to earlier events. In addition, turgor is almost never affected by these stresses in expanding cells. Thus, when one examines how cell expansion is controlled in a particular genotype, one must examine all of the growth parameters of the Lockhart equation (Lockhart, 1965). In this report, we use this approach to describe how salinity inhibits cell expansion of the leaves of three grass species known to differ in salt tolerance (Maas and Hoffman, 1977; Suhayda et al., 1992). Materials and methods Plant material and growth conditions Seeds of Hordeum jubatum L. and barley (Hordeum vulgare L. cv. Harrington ) were collected as described (Suhayda et al., 1992) and were a gift from Dr. R. E. Redmann at the University of Saskatechewan. The maize hybrids (Zea mays L. Pioneer hybrid 3578 and 3772) were a gift from Pioneer Hi-Bred International, Inc, and the other barley cultivar, CM72, was a gift from the Agronomy Department at the University of California, Davis. All plants were grown under similar conditions as described before (Cramer and Bowman, 1991). Seedlings in a vermiculite medium were irrigated daily with 0.25 Hoagland solution and grown under constant conditions (25 C, 255 µmol m 2 s 1 PAR). One day before experiments were started, seedlings with their third leaf beginning to emerge from the sheath, were transferred to and the roots immersed in an aerated 0.25 Hoagland solution in an 800-ml plastic container. NaCl was added to the nutrient solutions from a concentrated stock solution (4 M) to final concentrations of 0, 40, 80 and 120 mm. Maize, barley and H. jubatum reached the third leaf stage at 8,12, and14 days after planting, respectively. Leaf elongation measurements Elongation of the third leaf was measured with a displacement transducer (LVDT) system (Cramer and Bowman, 1991). In experiments where weights were added to the LVDT, the plant was fixed at the crown to a plant support grid with rapid-drying glue (Duro Super Glue, Loctite Corp, Cleveland, OH, USA) to prevent slippage of the plant. The glue did not affect the elongation of the third leaf. Water relations measurements After 5 h of salt treatment, when growth rates were at steady-state, the elongation zone of the third leaf was excised. Growth zones from four different plants were frozen in liquid N 2 in a plastic 1-cm 3 syringe. After thawing, sap was expressed out of the syringe and its osmolality measured with a Wescor 5500 vapor pressure osmometer. The s (MPa)oftheleafwas calculated by dividing the osmolality (mmol kg 1 )by negative 400. The water potential of the xylem sap ( o ) of the third leaf was measured with a pressure chamber according to Boyer (1967). To take into account the influences of ions in the xylem on o (due to salinity), xylem sap was collected and its osmolality measured. The negative balancing pressure was summed with s of the xylem sap to calculate the water potential of the leaf. Apparent turgor of the leaf was calculated by subtracting s of the leaf from o. Determination of growth parameters Plots of relative elongation rate (RER) versus turgor pressure are used to estimate the growth parameters of the Lockhart equation and were determined using an applied-tension technique (Cramer and Bowman, 1991). Estimations of the growth parameters by the applied-tension technique are comparable to values derived from the pressure-block technique (Cramer and Schmidt, 1995) and the guillotine psychrometer technique (Nonami and Boyer, 1990a, b). Figure 1 gives an example of how the growth parameters were estimated from the leaf response to applied tension (from the added weight). The elastic extension, E,was calculated as the difference in length between the time of weight removal and the length shortly thereafter. The rate of linear extension (steady-state growth rate) was calculated as the slope of the linear regression between 20 and 47 min after the weight was added (represented by the dotted line in Figure 1). Different

4 236 amounts of weights were used on different plants to estimate the steady-state growth rate at the particular applied tension force (weight). The linear regression was extrapolated to the time of the weight addition to determine the total extension component (P e +E) by taking the difference in length of this value to the length just prior to weight addition. P e, the plastic extension, was calculated as the difference between E and (P e +E). Both LER and P e were divided by the length of the growth zone to determine RER and the relative plastic extension, P. The length of the growth zone of the third leaf was measured by piercing the growth zone of plants that were treated with salinity for 5 h, with a pin every 3 mm and determining the length between holes 48 h later. Statistical analyses All data were analyzed with StatView version 5.01 (SAS Institute Inc.). All data were first analyzed by ANOVA to determine significant (p 0.05) treatment effects. Significant differences between individual means were determined using Fisher s protected least significant difference. Results Effects of salinity on growth Leaf elongation of intact plants was reduced immediately by the addition of salinity to the nutrient solution (Figure 2). Initially, LER declined rapidly in response to salinity, but then entered a recovery phase, reaching a steady-state rate after 5 h for all species and salt treatments. The steady-state response to salinity was lower than controls and was proportional to the level of salinity. In addition, this response differed between species. There was not much distinction between species at the 40 and 80 mm NaCl level (Figure 3). However, clear distinctions were found between species, but not within species at the 120 mm NaCl level. Barley was much more sensitive than maize, which was more sensitive than H. jubatum at 120 mm NaCl. Salinity effects on growth parameters In most cases the length of the growth zone of the third leaf was unaffected by salinity at all levels tested (Figure 4). However, one of the barley cultivars, CM72, Figure 1. The effect of adding weight (30 g) to the LVDT core on leaf length over time. The weight was added at 10 min and removed at 57 min. had a distinct shortening of the growth zone with salinity (reduced by 20% of control at 120 mm NaCl). The other barley cultivar, Harrington, had a less distinct reduction of the length of the growth zone (reduced by 12% of control at 120 mm NaCl). This effect is important because changes in the growth zone can affect the slope of the plots of RER versus turgor used for estimation of growth parameters, and therefore must be considered when estimating growth parameters. The lack of effect on the length of the growth zone of maize cultivars is similar to the response of another salt-stressed maize cultivar tested previously (Cramer, 1992b; Cramer and Bowman, 1991). Plotting RER (LER/length of growth zone) allows one to remove the effect of a shortened growth zone and measure the growth parameters more accurately. Measurements of the apparent turgor indicated that there was no change in turgor in all salt-treated plants measured after 5 h of treatment when leaf elongation reached a new but reduced steady-state rate. Turgor was 0.24 ± 0.03 MPa for Harrington, 0.30 ± 0.10 MPa for CM72, 0.55 ± 0.08 MPa for 3578 and 0.41 ± 0.03 MPa for It was impossible to get accurate measurements for H. jubatum, because of the difficulty in expressing enough sap from the xylem to estimate the osmotic potential of the xylem sap. Applying a tension-force (weight) to the leaf increased LER (Figure 1) and enabled estimation

5 237 Figure 2. The effect of salinity on the leaf elongation rate of three grass species. Each symbol is the average of 40 replications. Each curve was smoothed by a moving average technique before averaging. of other growth parameters (Cramer and Bowman, 1991). Increases in force increased steady-state RER (Figure 5). The growth coefficient, ml/(m+l), was measured as the slope of the lines in Figure 5. The growth coefficient was unaffected by the different salinity treatments in both maize cultivars. The growth coefficient was decreased at the 120 mm NaCl treatment level for both barley cultivars, but was unaffected

6 238 Table 1. The relative effect (% of control) of salinity on hydraulic conductance (L) Genotype Salinity (mm) Harrington CM H. jubatum indicates that the reduction in the growth coefficient by salinity for these species was due to a reduction in hydraulic conductance (Table 1). Figure 3. The effect of salinity on the average steady-state leaf elongation rate (% of control) of three grass species. at the other salinity treatments. The growth coefficient for H. jubatum was decreased by salinity with both the 80 and 120 mm NaCl treatments, but was unaffected by the 40 mm NaCl treatment. The apparent yield threshold, Y, is estimated as the x-intercept of plots of RER versus the elongation force (apparent turgor). Note that in this case the elongation force is only represented by the applied elongation force (weight), since turgor could not be estimated in H. jubatum. In addition, the applied force will be different for each genotype because each genotype has a different leaf cross-sectional area. Y was increased by each salinity level in both cultivars of maize (Figure 5). Y also increased for both barley cultivars at the 80 and 120 mm NaCl levels. However, there were no significant effects of salinity on the Y of H. jubatum. The relative plastic extension, P, of either maize cultivar was not significantly affected by salinity (Figure 6). Note, there are no units for P because the units for length of extension/length of the growth zone cancel each other out. The slope of this plot represents m (Nonami and Boyer, 1990b), the cell wall extensibility, and was not significantly affected for either maize cultivar. Since both m and ml/(m+l) were unaffected by salinity for either cultivar of maize, L, the hydraulic conductance, was also not significantly affected by salinity in maize (Table 1). Likewise, P and m were not significantly affected by salinity in barley or H. jubatum (Figure 6). This Discussion Differences between genotypes in short-term salt tolerance (LER as a % of control) could be identified over a 5-h period and at different levels of salinity. Differences were most pronounced at the highest level of salinity and did not necessarily correlate with differences at the lowest level of salinity. For example, Harrington had the least reduction in LER compared to the other genotypes at 40 mm NaCl, but had the largest reduction of LER at 120 mm NaCl. Overall, H. jubatum was more salt tolerant in the short-term than barley or maize. There was little difference between genotypes at the lower salinity levels, 40 and 80 mm NaCl. However, distinct differences were apparent at 120 mm NaCl, with the salt tolerance of H. jubatum > maize > barley. Within maize, 3578 was more salt tolerant than 3772 at all salinity levels, consistent with previous findings (Cramer et al., 1994) for both shortterm and long-term growth. Long-term growth studies also support the greater salt tolerance of H. jubatum compared to Harrington (Huang and Redmann, 1995a, b; Suhayda et al., 1992). In this short-term study, maize was shown to be more salt tolerant than barley. Both cultivars of maize or barley responded in a similar manner as the other cultivar to salinity. This ranking for maize and barley is not consistent with long-term growth studies of these species. Maize varieties are classified as more salt-sensitive (Maas and Hoffman, 1977). Others have also pointed out that short-term salt tolerance studies do not necessarily correlate with long-term studies

7 Figure 4. The effect of salinity on the length of the growth zone of the leaves of three grass species. The measured distance (length in mm) between pin holes is expressed on the y axis. Any length longer than 3 mm represents growth in that segment (the base of the y axis is set to 3 mm). The position of each segment along the growth zone at the time of pin pricking is presented on the x-axis. Each symbol is the average of six replications. Error bars represent the standard error of the mean. 239

8 240 Figure 5. The effect of salinity on the relative elongation rate, RER, of the leaves of three grass species. Each symbol is the average of 10 replications. Error bars represent the standard error of the mean. (Lynch et al., 1982; Munns et al., 1995; Yeo et al., 1991). In addition to the genotypic differences in salt tolerance, the mechanisms that inhibited leaf elongation when plants were exposed to a short-term salinity stress, appear to be species specific. Salinity did not significantly affect hydraulic conductance in maize, but there appears to be an effect on hydraulic conduct-

9 241 Figure 6. The effect of salinity on the relative plastic extension, P, of the growing zone of the leaves of three grass species. Each symbol is the average of 10 replications. Error bars represent the standard error of the mean. ance that limits the LER of barley at 120 mm NaCl. Hydraulic conductance was the sole limiting factor of LER for salt-stressed H. jubatum. These results are consistent with the long-term effects of salinity on whole plant hydraulic resistance in these two species (Huang and Redmann, 1995a). The vascular tissues in the maize leaf growth zone are encased by layers of small, undifferentiated cells

10 242 (Tang and Boyer, 2002). These small cells appear to act as a barrier to water flow to the expanding cells, resulting in a substantial water potential gradient between the expanding cells and the xylem. The water potential gradient is growth induced as a result of continued wall yielding of the expanding cells (Boyer, 2001). Nevertheless, the reduction of the LER of maize by short-term salinity was not caused by a reduction in hydraulic conductance. However, the limitation on hydraulic conductance in barley may be located in the shoot, since the root hydraulic resistance of salt-stressed barley does not increase (Munns and Passioura, 1984). The hydraulic resistances in these small, undifferentiated cells next to the xylem have been found to be a limiting factor for growth in drought-stressed soybean (Nonami et al., 1997). Perhaps in maize, these cells respond differently to salinity than the same type of cells in the Hordeum species. In maize, only an increase in the apparent yield threshold as measured by the applied-tension technique was responsible for the inhibition of LER by salinity. This is consistent with previous short-term studies of maize (Cramer, 1992b; Cramer and Bowman, 1991; Cramer and Schmidt, 1995) using two different techniques. The nature of the apparent yield threshold is unknown. Originally it was thought to be a rheological property of the cell wall (Cramer and Bowman, 1991), but no evidence to support this hypothesis could be found with in vitro assays for either maize or barley (Cramer et al., 2001). Intact, living plants are required to observe the apparent yield threshold. The apparent yield threshold may be a measure of the ability of the cells to extend and adjust. For example, with both the applied-tension technique and the pressure block technique, it takes approximately 50 to 60 min for the measurements to be made from which the apparent yield threshold is estimated. With the pressure block technique, pressure is used to block growth. The plant adjusts and starts growing again, then the pressure is increased again to compensate. At some higher pressure, the plant can no longer adjust and grow further. Others have observed two types of yield thresholds, one that is rapid and close to p and another that is adjustable over time (Boyer et al., 1985; Frensch and Hsiao, 1995). It would seem that the apparent yield threshold is similar to what Frensch and Hsiao (1995) called the minimum yield threshold. Estimates of turgor in this study could be misleading. There are at least two possible sources of error for the measurement of turgor that could affect the estimates of the apparent yield threshold. The first is that the average organ turgor measured does not accurately reflect the turgor of the specific cells limiting leaf elongation (i.e., in the epidermis). The second possible error in turgor measurement is that there is a leakage of solutes from the expanding cells into the apoplast that cannot be accounted for by the techniques used in this study. The assumption made in this study was that the cell wall solutes were negligible or similar to that in the xylem. This assumption is supported by the observation that Na +,K + and Ca 2+ concentrations have been found to be quite low (1 4, 20, and 1 mm, respectively) in the apoplast of salt-stressed maize leaves (A. Läuchli, personal communication). However, this second possible error is plausible, because ABA has been found to increase the apparent yield threshold of maize leaves similar to salinity (Cramer et al., 1998) and ABA increases the efflux of K + from maize coleoptile cells (Zocchi and De Nisi, 1996). Thus, solute concentrations in the apoplast could be significant, but not detected by previous methods, causing overestimates of turgor. More research on this subject is needed to clarify these issues. Salinity did not have any significant effects on cell wall extensibility (represented by the slope in Figure 6) for any of the species studied here. These results are consistent with previous results on short-term salt stress in maize (Cramer, 1992b; Cramer and Bowman, 1991; Cramer and Schmidt, 1995; Cramer et al., 2001) and barley (Cramer et al., 2001; Lynch et al., 1988) using a variety of different techniques (applied tension, INSTRON, and pressure block). However, it should be noted that one study (Neumann, 1993) indicated a rapid hardening of the cell wall in maize leaves after short-term salt stress. I do not have a good explanation for this particular discrepancy, except that there might be differences in the tissue types or genotypes used. Interestingly, in a subsequent study (Lu and Neumann, 1998), water deficit inhibited the LER of different species by different mechanisms, indicating that not all genotypes respond in the same way to stress. Increasing the water potential of the leaves of short-term salt-stressed plants to control levels by pressurization to the roots, restores LER to control rates for both maize (Munns et al., 2000b) and barley (Munns et al., 2000a). Drought-stressed maize plants respond to root pressurization in a similar manner (Tang and Boyer, 2002). These data support the hypothesis that salinity effects on growth in the short

11 243 term are primarily due to water deficit and not due to ion toxicity. The LER of plants with high water status is not increased by root pressurization (Hsiao et al., 1998). This pressurization technique acts to increase the water potential gradient in the growth zone (Hsiao et al., 1998; Tang and Boyer, 2002). In plants with high water status there isn t a hydraulic limitation and thus pressurization has no effect. In drought- or salt-stressed plants, the plants adjust to the lower water potential of the xylem by increased wall yielding (Tang and Boyer, 2002) and solute deposition (Fricke and Flowers, 1998). Wall yielding and solute deposition would lower the water potential of the expanding cells, maintain a water potential gradient between the expanding cells and the xylem and allow for continued water uptake. At some point wall yielding and solute deposition cannot compensate enough for the lower water potential in the xylem, the water potential gradient becomes diminished and growth slows (Tang and Boyer, 2002). Root pressurization overcomes this limitation by restoring the water potential gradient, allowing water uptake and cell expansion in the growth zone. Thus, growth is limited in drought and saltstressed plants by hydraulic factors and wall yielding factors. In summary, salinity decreased the LER of the three grass species studied including the salt tolerant H. jubatum. Differences in salt tolerance (% of control LER) were detected between genotypes within 5 h after salinization, but the relative salt tolerance at this stage was not necessarily indicative of the long-term salt tolerance of these species. The mechanisms of inhibition of LER by salinity varied with the species examined, affecting either the apparent yield threshold, the hydraulic conductance of the whole plant or both. The cell wall extensibility was not significantly affected by salinity in all species tested in this study. Thus, while salinity inhibited leaf elongation of all three grass species, the mechanism of inhibition was not universal, but different for each species. It would be prudent to measure all growth parameters when one investigates the mechanisms of growth inhibition by salinity. Acknowledgements This paper is presented in honor of Dr. Hank Greenway, whose work and writings were extremely influential upon me at the beginning of my career. I would like to thank Rana Munns, Tim Colmer and the two anonymous reviewers for their helpful comments on an earlier version of this manuscript. References Boyer J S 1967 Leaf water potentials measured with a pressure chamber. 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