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Direct measurement of sodium and potassium in the transpiration stream of salt‐excluding and non‐excluding varieties of wheat

Direct measurement of sodium and potassium in the transpiration stream of salt‐excluding and... Abstract The xylem‐feeding insect Philaenus spumarius was used to analyse sodium and potassium fluxes in the xylem of intact, transpiring wheat plants. Two cultivars were compared: the salt‐excluding (Chinese Spring) and the non‐excluding (Langdon). Chinese Spring accumulated much less sodium in its leaves than the salt‐sensitive Langdon. After 7 d in 150 mol m−3 NaCl, the sodium concentration in the leaf sap of Langdon reached over 600 mol m−3. This was some three‐fold greater than that in Chinese Spring. Similar findings have previously been reported from these cultivars. The reduced ion accumulation was specific to sodium; accumulation of K+ was unaffected by NaCl in Chinese Spring, such that it developed a much lower leaf Na+/K+ ratio than Langdon. The spittlebug, P. spumarius was used to sample xylem sap from both cultivars. This approach showed that the leaf xylem sap of Chinese Spring had much lower levels of sodium than that of Langdon. In the 150 mol m−3 NaCl treatment, sodium levels in the leaf xylem reached only 2–3 mol m−3 in Chinese Spring, compared with 8–10 mol m−3 in Langdon. Transpiration rates were found to be similar in the two varieties. The lower leaf xylem content alone was thus sufficient to account for the reduced accumulation of sodium in leaves of Chinese Spring. The mechanisms by which xylem sodium might be lowered are discussed and it is concluded that sodium is probably excluded from the xylem in the root of Chinese Spring. Triticum aestivum, Triticum turgidum, Philaenus spumarius, xylem transport, sodium exclusion. Introduction Soil salinity is a major problem for agriculture throughout the world, and millions of hectares of agricultural land show decreased yields because of high salinity (Richards, 1995; Flowers and Yeo, 1995). In regions such as Pakistan, over a quarter of the cultivatable land is compromised by high salinity (Ahmad, 1990) and the problem is accelerating because of the declining quality of the irrigation water (Binzel and Reuveni, 1994). Most of the major food crops of the world are highly sensitive to soil salinity; they show reduced growth rate and yield, and may have stunted fruits, leaves and stems (Bernstein, 1975). Salinity inhibits plant growth by a range of mechanisms, including osmotic effects, direct ion toxicity and interference with the uptake of nutrients, particularly K+ (Greenway and Munns, 1980; Leigh and Wyn Jones, 1984; Zhu et al., 1998). Sodium chloride may cause disruption of cytoplasmic components such as microtubules, microfibrils, spherosomes, and ribosomes (Mansour et al., 1993). Some crops are more tolerant of salt, and can maintain their yield well under saline conditions. Such crops will become increasingly important for dealing with salinity problems worldwide (Epstein et al., 1980). In addition, molecular techniques may offer new approaches to improve plant productivity in saline and other sub‐optimal environments (Hasegawa et al., 2000). These are likely to be successful only if they have a sound basis in stress physiology (Blum et al., 1996) and are backed up by studies of salt uptake and distribution within the plant. For higher plants to be salt‐tolerant, the toxic sodium ion must be isolated from sensitive enzymes in the cytoplasm (Gorham et al., 1985). Protection of leaf mesophyll cells is particularly important because these are the primary source of photoassimilates and, ultimately, yield. Certain cultivars of wheat have an enhanced ability to exclude sodium from their leaves. For example, Triticum aestivum cv. Chinese Spring excludes sodium almost entirely from its leaves. In contrast, Triticum turgidum cv. Langdon accumulates sodium in its leaves and is less salt‐tolerant (Gorham et al., 1987, 1990). The xylem is the major route for the transport of ions, including sodium, into the shoot. Regulation of xylem solute content will therefore be an important component of the regulation of shoot solute composition and salt tolerance. Some plants increase their salt tolerance by excluding Na+ from the xylem in the root. Others actively sequester Na+ from the xylem into cells bordering the transpiration stream in the shoot (Blom‐Zandstra et al., 1998) while others effectively compartmentalize sodium within vacuoles in the stem and leaf (Leigh and Storey, 1993). Recirculation of Na+ from the shoot may also take place via the phloem. In the more salt‐tolerant variety of wheat, Chinese Spring, sodium exclusion has been proposed to occur at sites of xylem loading within the endodermal membranes of the root (Gorham et al., 1990). Despite the importance of xylem transport for plant nutrition in general and for salt tolerance in particular, mineral nutrient dynamics have hardly ever been measured in the xylem of intact, transpiring plants. Direct extraction of xylem sap from such plants is confounded by the large negative pressures that exist there (Tyree, 1997; Steudle, 2000). Various indirect methods for xylem sap have been used, including the collection of root exudates (Jeschke et al., 1992) and facilitated exudation with the pressure bomb (Wolf et al., 1991; Gollan et al., 1992). These methods can yield useful comparative information. However, the solute composition they measure may not reflect that of the transpiration stream in the intact plant since the flux is lower and the driving forces different from those under transpiring conditions. Novel approaches for the extraction of xylem sap from intact transpiring plants, using xylem‐feeding insects, have recently been developed (Andersen et al., 1989; Malone et al., 1999). Philaenus spumarius (Cercopidae), more commonly known as the spittlebug, is an abundant insect in the summer in the UK. It is highly polyphagous and able to feed on over 500 different host plants. In addition, it feeds from mature xylem at the full hydraulic tension of the transpiration stream (Malone et al., 1999). Experiments in which insects were fed on stem sections perfused with artificial sap indicate that the sodium and potassium content of Philaenus excreta is similar to that of the xylem sap. For example, when the K+ concentration of the perfusion solution was 3.98±0.26 mol m−3 the excreta of the insect was 4.00±1.44 mol m−3. Similarly, with a Na+ concentration in the perfusion solution of 1.25±0.07 mol m−3, excreta had an almost identical Na+ concentration of 1.20±0.22 mol m−3 (Watson, 1999). The aim of the study was, firstly, to use the non‐invasive Philaenus method to measure and compare Na+ and K+ fluxes in two varieties of wheat. Secondly, to test the hypothesis that under salt stress, the Na+ concentration in the xylem of leaves of Chinese Spring was significantly lower than that in Langdon. Materials and methods Plant growth conditions Seeds of Triticum turgidum cv. Langdon and Triticum aestivum cv. Chinese Spring (henceforth abbreviated to ‘Spring’) were germinated for 3 d in the dark at 23 °C. The seedlings were then transferred to a plastic mesh above a thoroughly aerated solution of 0.5 mol m−3 CaCl2. After a further 2 d in darkness at 23 °C, the seedlings were illuminated at 200 μmol m−2 (PPFD at plant height) for 2 d. Thirty seedlings of each cultivar were placed in 15 l tanks of aerated half‐strength Long Ashton solution in a growth cabinet at 23 °C and 18/6 h light/dark. Salt stress was imposed by adding NaCl in increments of 50 mol m−3 d−1, to a final concentration of 0, 50, 100 or 150 mol m−3 NaCl. Addition was programmed so that all treatments achieved their final salt concentration on the same day, designated day 0. Data from the 100 mol m−3 NaCl salt treatment was usually intermediate between those for 50 and 150; this data set was omitted from the figures to improve clarity. Bulk tissue Na+ and K+ Plants of each cultivar were removed from treatments at appropriate time intervals and divided into root and first leaf. The root material was blotted dry briefly using soft tissue paper. Tissue segments were frozen at −70 °C in a microfuge tube, then thawed. A hole was made in the bottom of the tube with a fine needle and the tube was nested inside a similar tube. The assemblage was centrifuged for 5 min to collect the sap in the lower tube. This sap was diluted as necessary for determination of [Na+] and [K+] by atomic emission using a flame photometer (Corning 400, Corning Inc., USA). Sampling of xylem sap Adult P. spumarius were caged on the first leaves of the wheat plants using specially constructed plastic chambers. Every hour during the sampling period (typically lasting 6 h d−1) chamber lids were removed and the insects’ excreta was collected from the floor of the cage using a Hamilton syringe. The amount of excreta varied between insects and with conditions, but usually about 50 μl of excreta was collected at each sampling interval. The fluid was stored in a microfuge tube at –70 °C prior to determination of [Na+] and [K+] as above. Transpiration rate Gravimetric measurements of transpiration rate were made on plants of both cultivars growing in 0 or 100 mol m−3 NaCl. Individual plants were placed into 150 ml plastic bottles containing the appropriate solution aerated via fine polyethylene tubes. ‘Blank’ bottles, containing the treatment solution but no plants, were also set up to control for evaporation from the bottles. Plants were left for 24 h to equilibrate then, after briefly removing the air‐line, they were weighed on a balance placed inside the growth cabinet, each 45 min for 15 h. The leaves were then excised and their total surface area measured using a planimeter (Li‐Cor area meter, model 3100, Li‐Cor, USA). Statistical analysis Statistical analysis was carried out using the Mintab® v.12.1 for Windows package (Minitab Inc.). Analyses of variance were carried out on the data using a general linear model. Results Ion accumulation in roots After exposure to 50 or 150 mol m−3 NaCl, the concentration of Na+ in bulk root sap increased progressively (Fig. 1). By 2–4 d after salt addition, the sodium concentration in root sap equalled that of the medium; by 7 d it was approximately double that of the medium, with roots incubated in 150 mol m−3 NaCl having about 300 mol m−3 sodium in their sap. The kinetics and magnitude of this sodium accumulation were similar in the two varieties. The concentration of potassium in root sap increased rapidly during the first days in the nutrient solution, but then remained relatively constant. Salt stress had no effect on root sap potassium levels in general, except that there was a significant depression of [K+] in Spring at the highest level of NaCl (P<0.001 using GLM model) (Fig. 1). Fig. 1. View largeDownload slide Concentration of sodium and potassium in bulk sap extracted from root tissue of Chinese Spring and Langdon varieties of wheat exposed to different levels of sodium chloride (•=0, ▪=50, or ▴=150 mol m−3 NaCl). NaCl was added in 50 mol m−3 steps, all treatments achieved final salt concentration on day 0. Each point represents the mean of five roots ±standard error. Fig. 1. View largeDownload slide Concentration of sodium and potassium in bulk sap extracted from root tissue of Chinese Spring and Langdon varieties of wheat exposed to different levels of sodium chloride (•=0, ▪=50, or ▴=150 mol m−3 NaCl). NaCl was added in 50 mol m−3 steps, all treatments achieved final salt concentration on day 0. Each point represents the mean of five roots ±standard error. Ion accumulation in the first leaf After addition of salt, sodium concentration in the leaf sap increased with time (Fig. 2). However, as reported previously (Gorham et al., 1987, 1990) and in marked contrast to the pattern in roots, Na+ accumulation was much lower in leaves of Spring than in those of Langdon (P<0.001 using the GLM model). This was evident at both 50 and 150 mol m−3 levels of applied salt (Fig. 2). By 7 d in 150 mol m−3 NaCl, sodium in the leaf sap of Langdon had reached levels of over 600 mol m−3. This is over 3‐fold greater than in the corresponding tissue of Spring. K+ in leaf sap reached about 200 mol m−3 by the third day in the nutrient solution, then remained constant (Fig. 2). At low levels of NaCl stress (0 and 50 mol m−3) there was little difference between the two varieties. However, at the highest level of salt stress leaf K+ was reduced by up to 50% in Langdon (P<0.001 using the GLM model) whereas it was unchanged in Spring. Spring's ability to exclude sodium from the leaves whilst maintaining potassium content is reflected most strongly in the ratio of Na+/K+ in the leaf sap (Fig. 3 ). Na+/K+ ratios were calculated for both roots and shoots using the data in Figs 1 and 2. In roots, the ratio increased progressively in both varieties following exposure to salt. In leaves of Langdon the ratio also increased progressively during salt stress. However, in leaves of Spring the Na+/K+ ratio remained very low throughout the experiment. Fig. 2. View largeDownload slide Concentration of sodium and potassium in bulk sap extracted from leaf tissue of Chinese Spring and Langdon varieties of wheat exposed to different levels of sodium chloride (•=0, ▪=50, or ▴=150 mol m−3 NaCl). NaCl was added in 50 mol m−3 steps, all treatments achieved final salt concentration on day 0. Each point represents the mean of five leaves ±standard error. Fig. 2. View largeDownload slide Concentration of sodium and potassium in bulk sap extracted from leaf tissue of Chinese Spring and Langdon varieties of wheat exposed to different levels of sodium chloride (•=0, ▪=50, or ▴=150 mol m−3 NaCl). NaCl was added in 50 mol m−3 steps, all treatments achieved final salt concentration on day 0. Each point represents the mean of five leaves ±standard error. Fig. 3. View largeDownload slide [Na+]/[K+] ratios in bulk sap from root and first leaf tissue of Chinese Spring and Langdon, exposed to different levels of sodium chloride (•=0, ▪=50, or ▴=150 mol m−3 NaCl). NaCl was added in 50 mol m−3 steps, all treatments achieved final salt concentration on day 0. Data from Figs 1 and 2. Fig. 3. View largeDownload slide [Na+]/[K+] ratios in bulk sap from root and first leaf tissue of Chinese Spring and Langdon, exposed to different levels of sodium chloride (•=0, ▪=50, or ▴=150 mol m−3 NaCl). NaCl was added in 50 mol m−3 steps, all treatments achieved final salt concentration on day 0. Data from Figs 1 and 2. Sodium and potassium in leaf xylem sap The concentration of sodium in xylem sap was estimated by analysing excreta from P. spumarius placed midway along the first leaf. The sodium content of leaf xylem sap increased with salt stress in both varieties (Fig. 4). However, this effect was much smaller in Spring than in Langdon. The level of sodium in leaf xylem was always low in Spring. Even at the highest salt treatment, sodium levels in the xylem of the leaf reached only 2–3 mol m−3 in Spring, compared with 8–10 mol m−3 in Langdon (Fig. 4). In contrast to the pattern with sodium, potassium levels in the xylem of the leaf were not affected by salt treatment (Fig. 4). Leaf xylem potassium was constant at about 3 mol m−3 in both varieties, from day 1 onwards. These results show, for the first time, that sodium levels in the leaf xylem are significantly lower in Spring than in Langdon. This effect is seen most readily from the ratio of Na+/K+ in xylem sap: in Spring this ratio remains below 1 even at the highest salt treatment, whereas in Langdon the ratio exceeds 2 at some stages in 50 mol m−3 NaCl and at all stages in 150 mol m−3. The data are consistent with bulk leaf sodium being lower in Spring because of lower delivery of sodium to the leaf rather than, for example, because of rapid sodium recycling in the phloem. Fig. 4. View largeDownload slide Concentration of sodium and potassium in P. spumarius excreta collected from insects feeding on leaves of plants of Chinese Spring and Langdon (•=0, ▪=50, or ▴=150 mol m−3 NaCl). NaCl was added in 50 mol m−3 steps, all treatments achieved final salt concentration on day 0. Each point represents the mean of at least eight samples, typically over 15 ±standard error. Fig. 4. View largeDownload slide Concentration of sodium and potassium in P. spumarius excreta collected from insects feeding on leaves of plants of Chinese Spring and Langdon (•=0, ▪=50, or ▴=150 mol m−3 NaCl). NaCl was added in 50 mol m−3 steps, all treatments achieved final salt concentration on day 0. Each point represents the mean of at least eight samples, typically over 15 ±standard error. Flux rate into the leaf Transpiration rates did not differ much between the two varieties and were not greatly affected by salt (Table 1). Thus, differences in the rate of Na+ accumulation in leaves of the two varieties must reflect the concentrations of sodium in their xylem saps, rather than the delivery rates of that sap. This conclusion ignores sodium export from the leaves via the phloem, but the present data strongly suggest that Na+ recycling does not significantly contribute to the reduction of leaf sodium in Spring. If phloem export of sodium were the major mechanism for reducing leaf sodium, then the xylem sap of Langdon and Spring would have a similar sodium concentration. However, the leaf of Langdon contained three times more Na+ than Spring and had a 3‐fold higher delivery rate of Na+. This means Na+ delivery rates alone can explain the observed differences in bulk leaf sodium between the varieties. No significant differences in the growth rate of root or shoot were noted in either variety at any of the NaCl concentrations used (data not shown), making it unlikely that any difference in ion concentration was due to differences in dilution by growth. Table 1. The mean and daily transpiration rates for 0 and 100 mol m−3 NaCl‐treated Spring and Langdon plants 144 h after the start of salt treatment Data are the mean of 16 measurements recorded every 45 min over 15 h during the light period. Sd is the standard deviation. Total leaf surface area was used in the calculation of mean transpiration rate. Treatment   Mean transpiration rate (g H2O min−1 mm−2) (×10−5)   Sd (×10−6)   Daily transpiration rate per plant (g d−1)   Sd   0 mol m−3 CS  1.53  7.64  0.59  0.13  0 mol m−3 L  1.99  5.29  0.83  0.50  100 mol m−3 CS  1.54  5.59  0.73  0.16  100 mol m−3 L  1.82  1.11  0.42  0.19  Treatment   Mean transpiration rate (g H2O min−1 mm−2) (×10−5)   Sd (×10−6)   Daily transpiration rate per plant (g d−1)   Sd   0 mol m−3 CS  1.53  7.64  0.59  0.13  0 mol m−3 L  1.99  5.29  0.83  0.50  100 mol m−3 CS  1.54  5.59  0.73  0.16  100 mol m−3 L  1.82  1.11  0.42  0.19  View Large Discussion Gorham et al. reported that under saline conditions, Spring accumulates less sodium in its leaves than does Langdon (Gorham et al., 1990). This phenomenon was also clear under the present conditions (Fig. 2). Furthermore, the effect was specific to sodium; accumulation of K+ was unaffected by NaCl in Spring, so that it developed a much lower leaf Na+/K+ ratio than Langdon (Fig. 3). In Langdon, the higher salt levels caused a distinct depression of bulk leaf K+. This depression was not correlated with any reduction in K+ delivery in the xylem and it suggests that there was increased export of K+ from leaves to roots at high external salt concentrations. The decreased K+ concentration in the leaf is compensated, in part, by increased sodium in Langdon so that the total of K+ and Na+ remained roughly constant across all salt treatments, and throughout most of the experiment. In the current work, P. spumarius was used to make direct determinations of sodium levels in the xylem sap of intact transpiring plants. This approach has the advantage that it is non‐destructive so that the dynamics of mineral delivery can be studied over extended periods in individual intact plants. This technique should greatly facilitate studies of mineral transport dynamics in a wide range of systems. The P. spumarius approach demonstrated that xylem sap from midway along the leaf of Spring had much lower levels of sodium than that of Langdon (Fig. 4). Transpiration rates were similar in the two varieties. Thus, the lower concentration of sodium in the xylem of Spring must reflect substantially (3‐fold) lower delivery of sodium to the leaf in this cultivar. This alone can account for the reduced accumulation of sodium in leaves of Spring. This conclusion is consistent with reports that sodium export via the phloem is likely to be small (Grunberg and Taleisnik, 1991; Munns et al., 1988). These results support the hypothesis of Gorham et al. that sodium is excluded from the xylem of Spring (Gorham et al., 1990). The lower sodium content of the leaf xylem in Spring could be due to three mechanisms: reduced uptake into the root stele; recovery of Na+ from the basal xylem and sequestration into parenchyma cells bordering the transpiration pathway (Blom‐Zandstra et al., 1998); or recovery and recycling in the phloem. These recovery mechanisms make a contribution to sodium balance in some plants but seem unlikely in the experimental plants used here. In this study the P. spumarius insect feeding site was midway along the plant leaf. If recovery of Na+ along the transport pathway (either by release to parenchyma cells or transfer to the phloem) was the main mechanism for the observed reduction of leaf xylem Na+ concentration in Spring, then this recovery had to occur prior to the location of the insect. Thus massive Na+ recovery activity would be concentrated in the leaf stalk with little recovery occurring in the leaf blade. If this were the case, experiments on 22Na uptake by Spring (Gorham et al., 1990) would have been expected to show very high levels of radioactivity in the leaf stalk. This was not observed. The concentration of Na+ in leaf xylem could be reduced by re‐circulation out of the leaf in the phloem. Figure 2 shows that potassium levels in the leaf sap remained constant, or actually declined (in Langdon) despite constant K+ influx via the xylem, of about 3 mol m−3 of K+ d−1. Since the growth rate of these leaves was small, most of the daily delivery of K+ must have been recirculated from the leaf via the phloem. The phloem typically contained about 120 mol m−3 of K+ (tested on exudates from aphid stylets). It can be estimated, therefore, that the volume of sap effluxed via the phloem must be some 3/120 of that influxed via the xylem. Xylem influx can be estimated from the daily transpiration rate, as 0.59–0.83 ml d−1 (Table 1). The total volume of phloem flow in these seedlings must be about 3/120 of this, or about 0.02 ml d−1. There is much less evidence in these data for the recirculation of Na+, although phloem sap was found to contain up to 42 mol m−3 of Na+ under some circumstances (R Watson, unpublished results). If sodium is excluded effectively from the root xylem of Spring several unusual properties are implied for ion and water uptake in this plant. For example, it would mean that all incoming solutes are screened by at least one solute‐reflecting barrier before they enter the xylem. This will be possible only if there are significant apoplastic bypasses in this tissue (otherwise sodium from the medium would be swept into the xylem with the transpiration stream). Apoplastic bypasses are proposed to be present in most roots, to account for low whole‐root reflection coefficients, in accordance with the ‘composite transport’ model (Steudle and Peterson, 1998). Gorham et al. proposed that exclusion occurs at the endodermis (Gorham et al., 1990). If this is the case membrane transporters of various types are likely to be involved (Gassmann et al., 1996). Inwardly‐directed K+ transporters such as HKT1 are found throughout the root cortex, not only on the endodermal membranes (Keunecke et al., 1997). The specificity of such K+ transporters can be manipulated so as to decrease Na+ uptake and improve salt tolerance, at least in yeast cells (Rubio et al., 1995). Presumably, the major K+ transporter in Spring is much more specific than that in Langdon. In addition, outwardly directed Na+ ‘scavenging’ pumps (de Boer and Wegner, 1997; Hasegawa et al., 2000) could be present over large parts of root cortex and endodermis. Additionally, these transporters could be present along the transpirational pathway and remove the Na+ from the xylem as it moves from the root to the shoot. Such transporters have been shown to compartmentalize Na+ in vacuoles in the stem and leaf (Leigh and Storey, 1993). Unless these scavengers were extremely numerous and effective (and consumed large amounts of energy) it is unlikely that they could by themselves reduce xylem sap sodium to the low levels seen in leaves of Spring or Langdon. However, they could perhaps play a role in determining the difference between these two varieties. High specificity transporters can be inducible under certain nutrient regimes. Various transcripts were shown to be induced by salt (but not by associated stresses such as drought or osmotica) in alfalfa (Winicov and Deutch, 1994) and barley (DuPont, 1992). However, the efficient exclusion of sodium in Spring was already fully apparent within 1 d after salt addition (Fig. 4). This suggests that the highly selective K+ transporter in Spring is probably constitutive rather than salt‐inducible, although very rapid induction of some transporters by salt is possible (DuPont, 1992). A lower Na+ in the root xylem of Spring would support the hypothesis that Na+ entry to the xylem is restricted at the endodermis, whereas if there was no difference between the two varieties, removal of Na+ along the pathway would be implicated as the mechanism. In this experiment, the P. spumarius technique was only used to gain information on leaf xylem composition. The insect has been observed to feed from many locations on a variety of different plants, such as the stem, stipules and petiole (Watson, 1999), but it is unknown at present whether P. spumarius will feed from the root of a transpiring plant. In summary, the P. spumarius approach allows analysis of nutrient dynamics in the xylem of intact, transpiring plants. 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Genetic analysis of salt tolerance in Arabidopsis: evidence for a critical role of potassium nutrition. The Plant Cell  10, 1181–1191. Google Scholar © Society for Experimental Biology http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Experimental Botany Oxford University Press

Direct measurement of sodium and potassium in the transpiration stream of salt‐excluding and non‐excluding varieties of wheat

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0022-0957
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10.1093/jexbot/52.362.1873
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Abstract

Abstract The xylem‐feeding insect Philaenus spumarius was used to analyse sodium and potassium fluxes in the xylem of intact, transpiring wheat plants. Two cultivars were compared: the salt‐excluding (Chinese Spring) and the non‐excluding (Langdon). Chinese Spring accumulated much less sodium in its leaves than the salt‐sensitive Langdon. After 7 d in 150 mol m−3 NaCl, the sodium concentration in the leaf sap of Langdon reached over 600 mol m−3. This was some three‐fold greater than that in Chinese Spring. Similar findings have previously been reported from these cultivars. The reduced ion accumulation was specific to sodium; accumulation of K+ was unaffected by NaCl in Chinese Spring, such that it developed a much lower leaf Na+/K+ ratio than Langdon. The spittlebug, P. spumarius was used to sample xylem sap from both cultivars. This approach showed that the leaf xylem sap of Chinese Spring had much lower levels of sodium than that of Langdon. In the 150 mol m−3 NaCl treatment, sodium levels in the leaf xylem reached only 2–3 mol m−3 in Chinese Spring, compared with 8–10 mol m−3 in Langdon. Transpiration rates were found to be similar in the two varieties. The lower leaf xylem content alone was thus sufficient to account for the reduced accumulation of sodium in leaves of Chinese Spring. The mechanisms by which xylem sodium might be lowered are discussed and it is concluded that sodium is probably excluded from the xylem in the root of Chinese Spring. Triticum aestivum, Triticum turgidum, Philaenus spumarius, xylem transport, sodium exclusion. Introduction Soil salinity is a major problem for agriculture throughout the world, and millions of hectares of agricultural land show decreased yields because of high salinity (Richards, 1995; Flowers and Yeo, 1995). In regions such as Pakistan, over a quarter of the cultivatable land is compromised by high salinity (Ahmad, 1990) and the problem is accelerating because of the declining quality of the irrigation water (Binzel and Reuveni, 1994). Most of the major food crops of the world are highly sensitive to soil salinity; they show reduced growth rate and yield, and may have stunted fruits, leaves and stems (Bernstein, 1975). Salinity inhibits plant growth by a range of mechanisms, including osmotic effects, direct ion toxicity and interference with the uptake of nutrients, particularly K+ (Greenway and Munns, 1980; Leigh and Wyn Jones, 1984; Zhu et al., 1998). Sodium chloride may cause disruption of cytoplasmic components such as microtubules, microfibrils, spherosomes, and ribosomes (Mansour et al., 1993). Some crops are more tolerant of salt, and can maintain their yield well under saline conditions. Such crops will become increasingly important for dealing with salinity problems worldwide (Epstein et al., 1980). In addition, molecular techniques may offer new approaches to improve plant productivity in saline and other sub‐optimal environments (Hasegawa et al., 2000). These are likely to be successful only if they have a sound basis in stress physiology (Blum et al., 1996) and are backed up by studies of salt uptake and distribution within the plant. For higher plants to be salt‐tolerant, the toxic sodium ion must be isolated from sensitive enzymes in the cytoplasm (Gorham et al., 1985). Protection of leaf mesophyll cells is particularly important because these are the primary source of photoassimilates and, ultimately, yield. Certain cultivars of wheat have an enhanced ability to exclude sodium from their leaves. For example, Triticum aestivum cv. Chinese Spring excludes sodium almost entirely from its leaves. In contrast, Triticum turgidum cv. Langdon accumulates sodium in its leaves and is less salt‐tolerant (Gorham et al., 1987, 1990). The xylem is the major route for the transport of ions, including sodium, into the shoot. Regulation of xylem solute content will therefore be an important component of the regulation of shoot solute composition and salt tolerance. Some plants increase their salt tolerance by excluding Na+ from the xylem in the root. Others actively sequester Na+ from the xylem into cells bordering the transpiration stream in the shoot (Blom‐Zandstra et al., 1998) while others effectively compartmentalize sodium within vacuoles in the stem and leaf (Leigh and Storey, 1993). Recirculation of Na+ from the shoot may also take place via the phloem. In the more salt‐tolerant variety of wheat, Chinese Spring, sodium exclusion has been proposed to occur at sites of xylem loading within the endodermal membranes of the root (Gorham et al., 1990). Despite the importance of xylem transport for plant nutrition in general and for salt tolerance in particular, mineral nutrient dynamics have hardly ever been measured in the xylem of intact, transpiring plants. Direct extraction of xylem sap from such plants is confounded by the large negative pressures that exist there (Tyree, 1997; Steudle, 2000). Various indirect methods for xylem sap have been used, including the collection of root exudates (Jeschke et al., 1992) and facilitated exudation with the pressure bomb (Wolf et al., 1991; Gollan et al., 1992). These methods can yield useful comparative information. However, the solute composition they measure may not reflect that of the transpiration stream in the intact plant since the flux is lower and the driving forces different from those under transpiring conditions. Novel approaches for the extraction of xylem sap from intact transpiring plants, using xylem‐feeding insects, have recently been developed (Andersen et al., 1989; Malone et al., 1999). Philaenus spumarius (Cercopidae), more commonly known as the spittlebug, is an abundant insect in the summer in the UK. It is highly polyphagous and able to feed on over 500 different host plants. In addition, it feeds from mature xylem at the full hydraulic tension of the transpiration stream (Malone et al., 1999). Experiments in which insects were fed on stem sections perfused with artificial sap indicate that the sodium and potassium content of Philaenus excreta is similar to that of the xylem sap. For example, when the K+ concentration of the perfusion solution was 3.98±0.26 mol m−3 the excreta of the insect was 4.00±1.44 mol m−3. Similarly, with a Na+ concentration in the perfusion solution of 1.25±0.07 mol m−3, excreta had an almost identical Na+ concentration of 1.20±0.22 mol m−3 (Watson, 1999). The aim of the study was, firstly, to use the non‐invasive Philaenus method to measure and compare Na+ and K+ fluxes in two varieties of wheat. Secondly, to test the hypothesis that under salt stress, the Na+ concentration in the xylem of leaves of Chinese Spring was significantly lower than that in Langdon. Materials and methods Plant growth conditions Seeds of Triticum turgidum cv. Langdon and Triticum aestivum cv. Chinese Spring (henceforth abbreviated to ‘Spring’) were germinated for 3 d in the dark at 23 °C. The seedlings were then transferred to a plastic mesh above a thoroughly aerated solution of 0.5 mol m−3 CaCl2. After a further 2 d in darkness at 23 °C, the seedlings were illuminated at 200 μmol m−2 (PPFD at plant height) for 2 d. Thirty seedlings of each cultivar were placed in 15 l tanks of aerated half‐strength Long Ashton solution in a growth cabinet at 23 °C and 18/6 h light/dark. Salt stress was imposed by adding NaCl in increments of 50 mol m−3 d−1, to a final concentration of 0, 50, 100 or 150 mol m−3 NaCl. Addition was programmed so that all treatments achieved their final salt concentration on the same day, designated day 0. Data from the 100 mol m−3 NaCl salt treatment was usually intermediate between those for 50 and 150; this data set was omitted from the figures to improve clarity. Bulk tissue Na+ and K+ Plants of each cultivar were removed from treatments at appropriate time intervals and divided into root and first leaf. The root material was blotted dry briefly using soft tissue paper. Tissue segments were frozen at −70 °C in a microfuge tube, then thawed. A hole was made in the bottom of the tube with a fine needle and the tube was nested inside a similar tube. The assemblage was centrifuged for 5 min to collect the sap in the lower tube. This sap was diluted as necessary for determination of [Na+] and [K+] by atomic emission using a flame photometer (Corning 400, Corning Inc., USA). Sampling of xylem sap Adult P. spumarius were caged on the first leaves of the wheat plants using specially constructed plastic chambers. Every hour during the sampling period (typically lasting 6 h d−1) chamber lids were removed and the insects’ excreta was collected from the floor of the cage using a Hamilton syringe. The amount of excreta varied between insects and with conditions, but usually about 50 μl of excreta was collected at each sampling interval. The fluid was stored in a microfuge tube at –70 °C prior to determination of [Na+] and [K+] as above. Transpiration rate Gravimetric measurements of transpiration rate were made on plants of both cultivars growing in 0 or 100 mol m−3 NaCl. Individual plants were placed into 150 ml plastic bottles containing the appropriate solution aerated via fine polyethylene tubes. ‘Blank’ bottles, containing the treatment solution but no plants, were also set up to control for evaporation from the bottles. Plants were left for 24 h to equilibrate then, after briefly removing the air‐line, they were weighed on a balance placed inside the growth cabinet, each 45 min for 15 h. The leaves were then excised and their total surface area measured using a planimeter (Li‐Cor area meter, model 3100, Li‐Cor, USA). Statistical analysis Statistical analysis was carried out using the Mintab® v.12.1 for Windows package (Minitab Inc.). Analyses of variance were carried out on the data using a general linear model. Results Ion accumulation in roots After exposure to 50 or 150 mol m−3 NaCl, the concentration of Na+ in bulk root sap increased progressively (Fig. 1). By 2–4 d after salt addition, the sodium concentration in root sap equalled that of the medium; by 7 d it was approximately double that of the medium, with roots incubated in 150 mol m−3 NaCl having about 300 mol m−3 sodium in their sap. The kinetics and magnitude of this sodium accumulation were similar in the two varieties. The concentration of potassium in root sap increased rapidly during the first days in the nutrient solution, but then remained relatively constant. Salt stress had no effect on root sap potassium levels in general, except that there was a significant depression of [K+] in Spring at the highest level of NaCl (P<0.001 using GLM model) (Fig. 1). Fig. 1. View largeDownload slide Concentration of sodium and potassium in bulk sap extracted from root tissue of Chinese Spring and Langdon varieties of wheat exposed to different levels of sodium chloride (•=0, ▪=50, or ▴=150 mol m−3 NaCl). NaCl was added in 50 mol m−3 steps, all treatments achieved final salt concentration on day 0. Each point represents the mean of five roots ±standard error. Fig. 1. View largeDownload slide Concentration of sodium and potassium in bulk sap extracted from root tissue of Chinese Spring and Langdon varieties of wheat exposed to different levels of sodium chloride (•=0, ▪=50, or ▴=150 mol m−3 NaCl). NaCl was added in 50 mol m−3 steps, all treatments achieved final salt concentration on day 0. Each point represents the mean of five roots ±standard error. Ion accumulation in the first leaf After addition of salt, sodium concentration in the leaf sap increased with time (Fig. 2). However, as reported previously (Gorham et al., 1987, 1990) and in marked contrast to the pattern in roots, Na+ accumulation was much lower in leaves of Spring than in those of Langdon (P<0.001 using the GLM model). This was evident at both 50 and 150 mol m−3 levels of applied salt (Fig. 2). By 7 d in 150 mol m−3 NaCl, sodium in the leaf sap of Langdon had reached levels of over 600 mol m−3. This is over 3‐fold greater than in the corresponding tissue of Spring. K+ in leaf sap reached about 200 mol m−3 by the third day in the nutrient solution, then remained constant (Fig. 2). At low levels of NaCl stress (0 and 50 mol m−3) there was little difference between the two varieties. However, at the highest level of salt stress leaf K+ was reduced by up to 50% in Langdon (P<0.001 using the GLM model) whereas it was unchanged in Spring. Spring's ability to exclude sodium from the leaves whilst maintaining potassium content is reflected most strongly in the ratio of Na+/K+ in the leaf sap (Fig. 3 ). Na+/K+ ratios were calculated for both roots and shoots using the data in Figs 1 and 2. In roots, the ratio increased progressively in both varieties following exposure to salt. In leaves of Langdon the ratio also increased progressively during salt stress. However, in leaves of Spring the Na+/K+ ratio remained very low throughout the experiment. Fig. 2. View largeDownload slide Concentration of sodium and potassium in bulk sap extracted from leaf tissue of Chinese Spring and Langdon varieties of wheat exposed to different levels of sodium chloride (•=0, ▪=50, or ▴=150 mol m−3 NaCl). NaCl was added in 50 mol m−3 steps, all treatments achieved final salt concentration on day 0. Each point represents the mean of five leaves ±standard error. Fig. 2. View largeDownload slide Concentration of sodium and potassium in bulk sap extracted from leaf tissue of Chinese Spring and Langdon varieties of wheat exposed to different levels of sodium chloride (•=0, ▪=50, or ▴=150 mol m−3 NaCl). NaCl was added in 50 mol m−3 steps, all treatments achieved final salt concentration on day 0. Each point represents the mean of five leaves ±standard error. Fig. 3. View largeDownload slide [Na+]/[K+] ratios in bulk sap from root and first leaf tissue of Chinese Spring and Langdon, exposed to different levels of sodium chloride (•=0, ▪=50, or ▴=150 mol m−3 NaCl). NaCl was added in 50 mol m−3 steps, all treatments achieved final salt concentration on day 0. Data from Figs 1 and 2. Fig. 3. View largeDownload slide [Na+]/[K+] ratios in bulk sap from root and first leaf tissue of Chinese Spring and Langdon, exposed to different levels of sodium chloride (•=0, ▪=50, or ▴=150 mol m−3 NaCl). NaCl was added in 50 mol m−3 steps, all treatments achieved final salt concentration on day 0. Data from Figs 1 and 2. Sodium and potassium in leaf xylem sap The concentration of sodium in xylem sap was estimated by analysing excreta from P. spumarius placed midway along the first leaf. The sodium content of leaf xylem sap increased with salt stress in both varieties (Fig. 4). However, this effect was much smaller in Spring than in Langdon. The level of sodium in leaf xylem was always low in Spring. Even at the highest salt treatment, sodium levels in the xylem of the leaf reached only 2–3 mol m−3 in Spring, compared with 8–10 mol m−3 in Langdon (Fig. 4). In contrast to the pattern with sodium, potassium levels in the xylem of the leaf were not affected by salt treatment (Fig. 4). Leaf xylem potassium was constant at about 3 mol m−3 in both varieties, from day 1 onwards. These results show, for the first time, that sodium levels in the leaf xylem are significantly lower in Spring than in Langdon. This effect is seen most readily from the ratio of Na+/K+ in xylem sap: in Spring this ratio remains below 1 even at the highest salt treatment, whereas in Langdon the ratio exceeds 2 at some stages in 50 mol m−3 NaCl and at all stages in 150 mol m−3. The data are consistent with bulk leaf sodium being lower in Spring because of lower delivery of sodium to the leaf rather than, for example, because of rapid sodium recycling in the phloem. Fig. 4. View largeDownload slide Concentration of sodium and potassium in P. spumarius excreta collected from insects feeding on leaves of plants of Chinese Spring and Langdon (•=0, ▪=50, or ▴=150 mol m−3 NaCl). NaCl was added in 50 mol m−3 steps, all treatments achieved final salt concentration on day 0. Each point represents the mean of at least eight samples, typically over 15 ±standard error. Fig. 4. View largeDownload slide Concentration of sodium and potassium in P. spumarius excreta collected from insects feeding on leaves of plants of Chinese Spring and Langdon (•=0, ▪=50, or ▴=150 mol m−3 NaCl). NaCl was added in 50 mol m−3 steps, all treatments achieved final salt concentration on day 0. Each point represents the mean of at least eight samples, typically over 15 ±standard error. Flux rate into the leaf Transpiration rates did not differ much between the two varieties and were not greatly affected by salt (Table 1). Thus, differences in the rate of Na+ accumulation in leaves of the two varieties must reflect the concentrations of sodium in their xylem saps, rather than the delivery rates of that sap. This conclusion ignores sodium export from the leaves via the phloem, but the present data strongly suggest that Na+ recycling does not significantly contribute to the reduction of leaf sodium in Spring. If phloem export of sodium were the major mechanism for reducing leaf sodium, then the xylem sap of Langdon and Spring would have a similar sodium concentration. However, the leaf of Langdon contained three times more Na+ than Spring and had a 3‐fold higher delivery rate of Na+. This means Na+ delivery rates alone can explain the observed differences in bulk leaf sodium between the varieties. No significant differences in the growth rate of root or shoot were noted in either variety at any of the NaCl concentrations used (data not shown), making it unlikely that any difference in ion concentration was due to differences in dilution by growth. Table 1. The mean and daily transpiration rates for 0 and 100 mol m−3 NaCl‐treated Spring and Langdon plants 144 h after the start of salt treatment Data are the mean of 16 measurements recorded every 45 min over 15 h during the light period. Sd is the standard deviation. Total leaf surface area was used in the calculation of mean transpiration rate. Treatment   Mean transpiration rate (g H2O min−1 mm−2) (×10−5)   Sd (×10−6)   Daily transpiration rate per plant (g d−1)   Sd   0 mol m−3 CS  1.53  7.64  0.59  0.13  0 mol m−3 L  1.99  5.29  0.83  0.50  100 mol m−3 CS  1.54  5.59  0.73  0.16  100 mol m−3 L  1.82  1.11  0.42  0.19  Treatment   Mean transpiration rate (g H2O min−1 mm−2) (×10−5)   Sd (×10−6)   Daily transpiration rate per plant (g d−1)   Sd   0 mol m−3 CS  1.53  7.64  0.59  0.13  0 mol m−3 L  1.99  5.29  0.83  0.50  100 mol m−3 CS  1.54  5.59  0.73  0.16  100 mol m−3 L  1.82  1.11  0.42  0.19  View Large Discussion Gorham et al. reported that under saline conditions, Spring accumulates less sodium in its leaves than does Langdon (Gorham et al., 1990). This phenomenon was also clear under the present conditions (Fig. 2). Furthermore, the effect was specific to sodium; accumulation of K+ was unaffected by NaCl in Spring, so that it developed a much lower leaf Na+/K+ ratio than Langdon (Fig. 3). In Langdon, the higher salt levels caused a distinct depression of bulk leaf K+. This depression was not correlated with any reduction in K+ delivery in the xylem and it suggests that there was increased export of K+ from leaves to roots at high external salt concentrations. The decreased K+ concentration in the leaf is compensated, in part, by increased sodium in Langdon so that the total of K+ and Na+ remained roughly constant across all salt treatments, and throughout most of the experiment. In the current work, P. spumarius was used to make direct determinations of sodium levels in the xylem sap of intact transpiring plants. This approach has the advantage that it is non‐destructive so that the dynamics of mineral delivery can be studied over extended periods in individual intact plants. This technique should greatly facilitate studies of mineral transport dynamics in a wide range of systems. The P. spumarius approach demonstrated that xylem sap from midway along the leaf of Spring had much lower levels of sodium than that of Langdon (Fig. 4). Transpiration rates were similar in the two varieties. Thus, the lower concentration of sodium in the xylem of Spring must reflect substantially (3‐fold) lower delivery of sodium to the leaf in this cultivar. This alone can account for the reduced accumulation of sodium in leaves of Spring. This conclusion is consistent with reports that sodium export via the phloem is likely to be small (Grunberg and Taleisnik, 1991; Munns et al., 1988). These results support the hypothesis of Gorham et al. that sodium is excluded from the xylem of Spring (Gorham et al., 1990). The lower sodium content of the leaf xylem in Spring could be due to three mechanisms: reduced uptake into the root stele; recovery of Na+ from the basal xylem and sequestration into parenchyma cells bordering the transpiration pathway (Blom‐Zandstra et al., 1998); or recovery and recycling in the phloem. These recovery mechanisms make a contribution to sodium balance in some plants but seem unlikely in the experimental plants used here. In this study the P. spumarius insect feeding site was midway along the plant leaf. If recovery of Na+ along the transport pathway (either by release to parenchyma cells or transfer to the phloem) was the main mechanism for the observed reduction of leaf xylem Na+ concentration in Spring, then this recovery had to occur prior to the location of the insect. Thus massive Na+ recovery activity would be concentrated in the leaf stalk with little recovery occurring in the leaf blade. If this were the case, experiments on 22Na uptake by Spring (Gorham et al., 1990) would have been expected to show very high levels of radioactivity in the leaf stalk. This was not observed. The concentration of Na+ in leaf xylem could be reduced by re‐circulation out of the leaf in the phloem. Figure 2 shows that potassium levels in the leaf sap remained constant, or actually declined (in Langdon) despite constant K+ influx via the xylem, of about 3 mol m−3 of K+ d−1. Since the growth rate of these leaves was small, most of the daily delivery of K+ must have been recirculated from the leaf via the phloem. The phloem typically contained about 120 mol m−3 of K+ (tested on exudates from aphid stylets). It can be estimated, therefore, that the volume of sap effluxed via the phloem must be some 3/120 of that influxed via the xylem. Xylem influx can be estimated from the daily transpiration rate, as 0.59–0.83 ml d−1 (Table 1). The total volume of phloem flow in these seedlings must be about 3/120 of this, or about 0.02 ml d−1. There is much less evidence in these data for the recirculation of Na+, although phloem sap was found to contain up to 42 mol m−3 of Na+ under some circumstances (R Watson, unpublished results). If sodium is excluded effectively from the root xylem of Spring several unusual properties are implied for ion and water uptake in this plant. For example, it would mean that all incoming solutes are screened by at least one solute‐reflecting barrier before they enter the xylem. This will be possible only if there are significant apoplastic bypasses in this tissue (otherwise sodium from the medium would be swept into the xylem with the transpiration stream). Apoplastic bypasses are proposed to be present in most roots, to account for low whole‐root reflection coefficients, in accordance with the ‘composite transport’ model (Steudle and Peterson, 1998). Gorham et al. proposed that exclusion occurs at the endodermis (Gorham et al., 1990). If this is the case membrane transporters of various types are likely to be involved (Gassmann et al., 1996). Inwardly‐directed K+ transporters such as HKT1 are found throughout the root cortex, not only on the endodermal membranes (Keunecke et al., 1997). The specificity of such K+ transporters can be manipulated so as to decrease Na+ uptake and improve salt tolerance, at least in yeast cells (Rubio et al., 1995). Presumably, the major K+ transporter in Spring is much more specific than that in Langdon. In addition, outwardly directed Na+ ‘scavenging’ pumps (de Boer and Wegner, 1997; Hasegawa et al., 2000) could be present over large parts of root cortex and endodermis. Additionally, these transporters could be present along the transpirational pathway and remove the Na+ from the xylem as it moves from the root to the shoot. Such transporters have been shown to compartmentalize Na+ in vacuoles in the stem and leaf (Leigh and Storey, 1993). Unless these scavengers were extremely numerous and effective (and consumed large amounts of energy) it is unlikely that they could by themselves reduce xylem sap sodium to the low levels seen in leaves of Spring or Langdon. However, they could perhaps play a role in determining the difference between these two varieties. High specificity transporters can be inducible under certain nutrient regimes. Various transcripts were shown to be induced by salt (but not by associated stresses such as drought or osmotica) in alfalfa (Winicov and Deutch, 1994) and barley (DuPont, 1992). However, the efficient exclusion of sodium in Spring was already fully apparent within 1 d after salt addition (Fig. 4). This suggests that the highly selective K+ transporter in Spring is probably constitutive rather than salt‐inducible, although very rapid induction of some transporters by salt is possible (DuPont, 1992). A lower Na+ in the root xylem of Spring would support the hypothesis that Na+ entry to the xylem is restricted at the endodermis, whereas if there was no difference between the two varieties, removal of Na+ along the pathway would be implicated as the mechanism. In this experiment, the P. spumarius technique was only used to gain information on leaf xylem composition. The insect has been observed to feed from many locations on a variety of different plants, such as the stem, stipules and petiole (Watson, 1999), but it is unknown at present whether P. spumarius will feed from the root of a transpiring plant. In summary, the P. spumarius approach allows analysis of nutrient dynamics in the xylem of intact, transpiring plants. 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Journal of Experimental Botany – Oxford University Press

Published: Sep 1, 2001

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