Journal of Experimental Botany, Vol. 51, No. 351, pp. 1721-1732,
October 2000
© 2000 Oxford University Press
Original Papers |
Solute balance of a maize (Zea mays L.) source leaf as affected by salt treatment with special emphasis on phloem retranslocation and ion leaching
1 Albrecht von Haller Institut für Pflanzenwissenschaften, Abteilung Biochemie der Pflanze, Untere Karspüle 2, D-37073 Göttingen, Germany
2 Christian-Albrechts-Universität, Institut für Pflanzenernährung und Bodenkunde, Olshausenstrasse 40-60, D-24118 Kiel, Germany
3 Lehrstuhl für Biotechnologie, Biozentrum der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany
Received 23 July 1999; Accepted 16 May 2000
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Abstract |
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Strategies for avoiding ion accumulation in leaves of plants grown at high concentration of NaCl (100 mol m-3) in the rooting media, i.e. retranslocation via the phloem and leaching from the leaf surface, were quantified for fully developed leaves of maize plants cultivated hydroponically with or without salt, and with or without sprinkling (to induce leaching). Phloem sap, apoplastic fluid, xylem sap, solutes from leaf and root tissues, and the leachate were analysed for carbohydrates, amino acids, malate, and inorganic ions. In spite of a reduced growth rate Na+ and Cl- concentrations in the leaf apoplast remained relatively low (about 4–5 mol m-3) under salt treatment. Concentrations of Na+ and Cl- in the phloem sap of salt-treated maize did not exceed 12 and 32 mol m-3, respectively, and thus remained lower than described for other species. However, phloem transport rates of these ions were higher than reported for other species. The relatively high translocation rate of ions found in maize may be due to the higher carbon translocation rate observed for C4 plants as opposed to C3 plants. Approximately 13–36% of the Na+ and Cl- imported into the leaves through the xylem were exported by the phloem. It is concluded that phloem transport plays an important role in controlling the NaCl content of the leaf in maize. Surprisingly, leaching by artificial rain did not affect plant growth. Ion concentrations in the leachate were lower than reported for other plants but increased with NaCl treatment.
Key words: Apoplast, maize, leaching, phloem transport, salt tolerance.
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Introduction |
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Soil salinity poses serious limitations to agriculture in many areas around the world. Salinity can reduce plant growth by osmotic stress, ion toxicity, and nutritional disturbances (Greenway and Munns, 1980
; Läuchli, 1986; Cheeseman, 1988). There are large differences in responses to salinity between species, for example, a 50% yield reduction occurs for beans at 60 mol m-3 and for sugar beet at 260 mol m-3 monovalent salts (Maas and Hoffman, 1977). Salt tolerance of plants is a complex phenomenon that involves morphological and developmental as well as physiological and biochemical processes (Greenway and Munns, 1980; Hanson and Hitz, 1982; Cheeseman, 1988; Munns, 1993). Salt exclusion in the root, sequestration in the leaf cell vacuoles, ion retranslocation from the leaves (Lessani and Marschner, 1978) and possibly ion leaching from the leaf apoplast by precipitation (Arens, 1934) are mechanisms to reduce ionic burden of plants under saline conditions. In glycophytes sensitivity to salinity is associated with an inability to prevent or tolerate elevated NaCl concentrations in the shoots, where it accumulates in the vacuoles. High NaCl concentrations in the vacuole requires the accumulation of compatible osmolytes in the cytosol. When the vacuolar storage capacity is surpassed, rising cytosolic concentrations of NaCl may result in enzyme inhibition and turgor loss (Rausch et al., 1996).
It has been suggested that reduced plant growth under saline conditions is at least partly due to high salt concentrations building up in the apoplast of growing tissues (Oertli, 1968). Speer and Kaiser have shown that under salt treatment apoplastic ion concentrations of pea (164 mol m-3, salt-sensitive) were much higher than in spinach (56 mol m-3, salt-tolerant) (Speer and Kaiser, 1991). When rice plants are subjected to mildly saline conditions (50 mol m-3 NaCl), the leaves show strong symptoms of water deficiency and NaCl concentrations in the apoplastic water space of about 600 mol m-3 (Flowers et al., 1991). Various mechanisms may contribute to avoid toxic ion concentrations in the leaf symplast and apoplast, the relative significance varying with plant species as well as with the solute under consideration: (1) removal from the equilibrium by precipitation either in the apoplast (Fink, 1992) or in the vacuoles of idioblasts; (2) guttation; (3) abscission of entire leaves; (4) incorporation into epidermis (Sangster and Hodson, 1986) and trichomes (De Silva et al., 1996), (5) phloem export; and (6) leaching from the leaf apoplast (Arens, 1934). Leaching is defined as the removal of inorganic or organic metabolites from plant tissue into moisture such as rain, fog or dew (Tukey, 1970). Most investigations discuss leaching in relation to forest decline (Mengel et al., 1987; Pfirrmann et al., 1990; Turner and Tingey, 1990) or nutrient cycling in nutrient-limited ecosystems (Tukey Jr, 1969; Tukey Jr and Mecklenburg, 1964). However, in older literature the so-called ‘cuticulary excretion’ (Arens, 1934) was considered to be an important mechanism for avoiding high salt concentrations in the leaf (Tukey, 1970). The relevance of leaching from the leaf for apoplastic solute balance is still debated (Pennewiss et al., 1997).
Retranslocation of ions via the phloem has to be considered as a potentially important mechanism to prevent salt accumulation in fully expanded leaves (Lessani and Marschner, 1978; Munns et al., 1986, 1988; Wolf et al., 1990; Durand and Lacan, 1994; Gouia et al., 1994; Jeschke et al., 1995). It is still controversial whether high rates of ion retranslocation and low salt levels in the leaves indicate salt-tolerant or salt-sensitive plants (Lessani and Marschner, 1978; Läuchli and Wieneke, 1979; Munns, 1988; Cramer et al., 1994b). On the one hand high ion retranslocation activity prevents salt accumulation in fully expanded leaves, but on the other hand it represents a threat to younger leaves which are phloem sinks. One way of studying phloem transport is the direct measurement of ion concentrations in sieve tube sap obtained via aphid stylet technique (Downing, 1980; Munns et al., 1986; Wolf et al., 1990). With this method pure phloem sap can be obtained from intact plants and from such plants where the collection of phloem sap with the stem incision technique is not successful, like the important culture plant maize.
It is the objective of this analysis to evaluate the relative importance of phloem retranslocation and leaching from the leaf for solute balance of maize source leaves under saline and control conditions. For this purpose, concentrations of organic acids, inorganic ions, carbohydrates, and amino acids in roots, xylem sap, leaves, leaf apoplast, phloem sap, and in the leachate were compared between control and salt-stressed maize plants. The phloem translocation rates of metabolites and ions were also investigated.
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Materials and methods |
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Plant material, growth conditions, and collection of leachate
Maize (Zea mays L. cv. Helix) plants were grown hydroponically in constantly aerated nutrient solution (0.7 mol m-3 K2SO4, 0.1 mol m-3 KCl, 2.0 mol m-3 Ca(NO3)2, 0.5 mol m-3 MgSO4, 0.1 mol m-3 KH2PO4, 10 mmol m-3 H3BO3, 0.5 mmol m-3 MnSO4, 0.5 mmol m-3 ZnSO4, 0.2 mmol m-3 CuSO4, 0.01 mmol m-3 (NH4)6Mo7O24, 120 mmol m-3 FeEDTA, pH 6.5) in a growth cabinet (15 h day length, 23/18 °C day/night temperature, 60–70% relative humidity, 300–330 µmol photons m-2 s-1 light intensity at canopy level). The solution (5.0 l per pot) was changed every other day from day 8 after germination to day 31. NaCl was added to the nutrient solution in daily increments of 25 mol m-3, starting on day 18 after germination. Plants were exposed for 8 d to 100 mol m-3 NaCl. Half the plants in the growth chamber without or with salt treatment were sprinkled twice a day (morning and evening) with de-ionized water (pH 4.0 adjusted with H2SO4), in an amount equivalent to 10 mm rain per day. For this purpose a pump with a pressure of 5 bar (ASV Stübbe GmbH, Vlotho, Germany) and special jets (1 mm diameter, Hellmuth Bahrs GmbH, Brüggen, Germany) were used. Leachates were collected in a bowl beneath the upper plant part of four plants. To avoid contamination like dust or guttation drops, leaves were cleaned before sprinkling. Leaves 4 and 5 were used for the collections of sieve tube sap and leaf samples. These leaves showed no signs of senescence or salt injury.
Collection of sieve tube sap
Sieve tube sap was obtained from severed stylets of the oat–bird cherry aphid, Rhopalosiphum padi (L.). About 10 aphids were caged for 2–3 h on the mid-portion of the leaf. Then, their stylets were cut by a laser beam (Barlow and McCully, 1972; Lohaus et al., 1995). The exudating phloem sap was collected in microcapillaries (total volume 0.5 µl). Evaporation from the exuding phloem sap was prevented since the front edge of the microcapillary was in close contact with the leaf surface and the end was surrounded by a cap. The humidity around the capillary was adjusted to about 80%. Under these conditions evaporation from reference capillaries was undetectable. Exudation rates per stylet were 100–250 nl h-1, as determined by drawing the sieve tube sap into a calibrated 0.5 µl disposable microcapillary. The samples were diluted with HPLC water to a volume of 100 µl, and stored at -80 °C. Samples were taken in the second half of the light period.
Determination of apoplastic air and water volume
For determination of apoplastic air volume (Vair) leaf segments of 11.4 cm2 were weighed, infiltrated with high-viscosity silicone fluid (polymethylsiloxane; viscosity 5 cs; 
0.904 g cm-3; Dow Corning, Poole, UK), for which the plasma membrane is impermeable, under vacuum in a 50 ml PE syringe, and reweighed. The leaf surface was blotted dry with thin tissues, and the weight increase (corrected for the density of silicone oil) was used to determine Vair.
Apoplastic water volume was determined as described previously (Husted and Schjoerring, 1995). Leaf segments cut from washed and blotted dry leaves were weighed and infiltrated with 0.05 mol m-3 indigo carmine (indigo-5,5'-disulphonic acid; Sigma, St Louis, MO, USA). The vacuum infiltration was carried out using a 50 ml syringe. The leaf segments were then centrifuged at 715 g for 4 min at 4 °C, and the extinction of the dye before infiltration (Einf) and in the apoplastic washing fluid (EAWF) was measured spectrophotometrically in 150 µl samples at 610 nm. The relative apoplastic water volume (Vapo, cm3 H2O cm-3 tissue) was calculated as:
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Collection of apoplastic fluid
Apoplastic fluid was collected with the infiltration technique (according to Mühling and Sattelmacher, 1995; Lohaus et al., 1995). After infiltration, the leaves were centrifuged for 3 min at 715 g at 4 °C. Calculation of apoplastic solute concentration was carried out by multiplication of the solute concentration in the apoplastic wash fluid by (Vair+Vapo)Vapo-1. Samples were taken in the second half of the light period.
Cytoplasmic contamination of the apoplastic fluids was determined by measuring the activity of malate dehydrogenase relative to that in the bulk of leaf extracts (Tetlow and Farrar, 1993; Lohaus et al., 1995).
Collection of xylem sap
For xylem sap collection a hand-made Passioura-type root pressure bomb (cf. Passioura, 1980) was used, with the intact root system of a maize plant tightly pressure sealed into the bomb lid. For each new maize plant used, the bomb was filled with fresh nutrient solution identical to the culture medium (with or without NaCl). Air pressure was applied to the water phase up to pressure values of 0.09 MPa for control plants and 0.45 MPa for salt-treated plants in order to increase the leaf xylem pressure to slightly above atmospheric values. Xylem sap was collected from leaves 3 to 6, either from the cut leaf blade or directly from a xylem vessel of a single vascular bundle by using the minimal-invasive xylem pressure probe technique (Balling and Zimmermann, 1990). The latter yielded 2–5 µl of xylem sap for each sample.
Photosynthesis and transpiration
Net photosynthesis in the light and transpiration during the day (Sharkey et al., 1986) of source leaves were measured with a portable infrared gas analyser (LCA3; ADC, Hoddesdon, UK).
Measurements of ions and metabolites
To determine tissue contents of water-soluble metabolites and ions leaf and root material were sampled periodically. After shock freezing in liquid nitrogen, approximately 0.25 g of tissue were ground in a mortar under liquid nitrogen. After addition of 5 ml chloroform : methanol (3 : 7, v/v) the sample was homogenized until it was completely thawed and then kept on ice for 30 min. Then the homogenate was extracted twice with 3 ml water. The aqueous phases were combined and evaporated in a rotatory evaporator. The dried residue was dissolved in 2 ml ultrapure H2O (Millipore, Germany) and stored at -80 °C until analysis.
Concentrations of anions and malate in sieve tube sap, apoplastic fluid, root pressure exudate, leaf leachate, and leaf or root samples were analysed by ion chromatography (DX500, Dionex, Idstein, Germany) using an IonPac anion exchange column (AS4 and AS4a together, 4x200 mm, Dionex, Idstein, Germany) connected with a conductivity detector module (CD 20, Dionex, Idstein, Germany). The ions were eluted with 1.8 mol m-3 Na2CO3 and 1.7 mol m-3 NaHCO3 for 20 min. Cations were analysed by ion chromatography (DX500, Dionex, Idstein, Germany) using an IonPac anion exchange column (CS12A, 4x200 mm, Dionex, Idstein, Germany) connected with a conductivity detector module (CD 20, Dionex, Idstein, Germany). The ions were eluted with 30 normal m-3 H2SO4 for 16 min. Amino acids were analysed by HPLC (Pharmacia/LKB, Freiburg, Germany) using the fluorescent o-phthaldialdehyde precolumn derivatization (according to Riens et al., 1991). Sugars were assayed amperometrically (according to Lohaus et al., 1995).
Recovery of metabolites and ions
The recovery of the main metabolites and ions during the extraction processes described above was measured previously (Weiner, 1990; Winzer et al., 1996). With maize extracts the recovery was between 90% and 107% for malate, asparagine, glutamine, aspartate, glutamate, alanine, and with sugar beet extracts the recovery was between 85% and 112% for sucrose, malate, glutamate, glutamine, aspartate, asparagine, serine, potassium, and chloride.
Statistics
Statistical analyses were done with a ANOVA by SigmaStat (Jandel Scientific).
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Results |
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Plant growth
Root growth was less adversely affected by NaCl than that of shoot, leading to a shift in the root-to-shoot ratio in favour to the root. Sprinkling did not significantly affect plant fresh matter production in salt-grown and control plants (Table 1
). Net assimilation rate, chlorophyll content of leaves and specific leaf weight did not vary significantly between treatments. Transpiration rate was decreased by the salt treatment.
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Solute concentrations in leaves and roots
Concentrations of organic and inorganic solutes in roots and leaves were affected by salinization, while sprinkling evoked only minor changes (Table 2). In leaves, Na+ and Cl– concentrations increased almost by the same increment (40 µmol g-1 FW) with salt treatment. The concentration of K+ was slightly reduced under the same conditions. Malate and nitrate concentrations decreased significantly in the presence of salt while the effect on calcium, sucrose, and starch concentrations was insignificant. In roots, exposure to salt increased Na+ concentration (approximately 70 µmol g-1 FW) more strongly than that of Cl– (approximately 35 µmol g-1 FW), while the concentrations of K+, Ca2+ and malate were strongly reduced. Salt stress enhanced the total amino acid concentration about 1.6-fold in leaves and roots. In leaves this effect was strongly reduced by sprinkling.
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Solute concentration in the apoplastic fluid
The gas- and the water-filled volume of the leaf apoplast (Table 3), which are important parameters for the calculation of apoplastic ion concentrations from apoplastic washing fluids, were neither affected by salinization nor by sprinkling. Under all conditions the ratio Vair to Vapo was about two.
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The apoplastic fluid collected by vacuum infiltration was checked for cytoplasmic contaminations by determining malat dehydrogenase (MDH) activity. The activity of this enzyme in maize leaves varied between 95 µmol g-1 FW min-1 (control dry) and 75 µmol g-1 FW min-1 (salt dry). No difference could be detected when leaves were extracted with water instead of buffer of pH 7.4. The MDH activity was stable over at least 4 h. The corresponding activities in the apoplastic fluid were 0.05 µmol g-1 FW min-1 (control, dry or sprinkled) and 0.2 µmol g-1 FW min-1 (salt, dry or sprinkled), respectively. Thus, only 0.06% and 0.3% of the corresponding cytoplasmic metabolites would appear in the apoplastic fluid due to cell rupture.
Na+ and Cl- concentrations in the apoplastic fluid increased by about 2.4–2.8 mol m-3 in response to the salt treatment (Table 4). In either case, K+ was the dominant cation despite the fact that its concentration was slightly reduced (1.0–1.3 mol m-3) by salt treatment. The K+ concentration never exceeded 8 mol m-3. This fact also confirms that the apoplastic fluid was not or slightly contaminated with cytoplasm since damage of membranes would lead to higher apoplastic K+ concentration due to the high K+ concentration (approximately 100 mol m-3) in the cytoplasm. The concentration of total amino acids was increased about 3-fold by salt treatment, but the concentration of the main organic acid, malate, was decreased. The effects of sprinkling were insignificant; only total amino acid and sucrose concentrations were reduced upon sprinkling under salt conditions.
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Phloem sap
Phloem sap composition reflected the salt treatment (Table 5), i.e. the Na+ concentration increased from 0.7 mol m-3 in control plants to 11.9 mol m-3 in salt-treated plants, while the Cl- concentration increased from 8.7 mol m-3 to 31.6 mol m-3. The concentrations of most other solutes increased slightly, i.e. K+ by 12 mol m-3. Nitrogen was transported in the phloem sap exclusively in the form of amino-N and the nitrogen concentration increased 1.6-fold with salt treatment. The concentrations of NO-3, Ca2+ and hexoses in the phloem sap were under the detection limit of approximately 0.01 mol m-3 which corresponds to a concentration in the phloem sap of about 0.5 mol m-3. Sprinkling of salt-treated plants had no effect on the concentrations of inorganic ions, but led to a decrease in amino acid concentration.
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Phloem translocation rates
In order to convert phloem solute concentration (Table 5
) into the corresponding translocation rates (Table 6), it was assumed that differences between leaf carbon content calculated from assimilated carbon in the leaf tissue (Table 6) and accumulated carbon in the leaf tissue (Fig. 1) were due to carbon translocated via the phloem. The parts of maize leaves studied in these experiments were fully expanded. The consumption of assimilates by these parts of the leaves should therefore be restricted to maintenance metabolism only. The rate of net CO2 assimilation was virtually constant over the entire illumination period, and neither the salt nor the sprinkling treatment evoked significant effects (Table 6). Figure 1 shows the accumulation of carbon compounds during the illumination period and their subsequent reduction in the dark period. In the control and the salt-treated plants 3645 µatom C g-1 FW were fixed during the 15 h light period. The difference in the carbon content of the major metabolites between the beginning and the end of the photoperiod amounted to 550–600 µatom C g-1 FW (Fig. 1; Table 6). Thus, only 16–17% of the carbon-assimilation products remained in the leaves during the day, mainly as starch, sucrose, and malate. The translocation rate of carbon was about 203 µatom C g-1 FW h-1 in control and salt-treated plants, and was about 5% lower in sprinkled plants. Carbon was transported predominantly (>90%) as sucrose.
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control sprinkled, 
salt dry, 
salt sprinkled) harvested at different times in the course of a diurnal cycle. The data are expressed as the carbon content of the various metabolites (starch, sucrose, malate, amino acids) and are mean values from five individual series of measurements with three measurements each. Coefficients of variation: control dry, 30–36%; control sprinkled, 22–41%; salt dry, 22–42%; salt sprinkled, 36–42%.As a consequence of the pressure flow-driven solute flux in the sieve tubes, phloem transport of photosynthates is intimately associated with the export of ions from the source leaves. Translocation rates of ions (Table 6
) can be calculated from the molar ratios of carbon to ions in the phloem sap (Table 5). Exposure to NaCl resulted in an increased translocation rate of Na+ (17-fold), Cl– (3-fold), amino acids (1.5-fold), and K+ (1.2-fold) in the light period.
Xylem sap analysis
Analysis of the xylem sap samples showed that salt treatment increased the concentration of Na+ by about 2.0 mol m-3 and that of Cl- by about 5.4 mol m-3 (Table 7). The concentration of the other ions and amino-N were also increased under salinization. Interestingly, with the exception of potassium in control plants no significant differences in ion concentrations were observed between samples collected from the cut leaf blade and those obtained directly from single xylem vessels by means of the xylem pressure probe despite the difference in sucrose concentration. In the case of salt-treated plants it was not possible to obtain a sufficient number of samples from single xylem vessels.
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Ion concentration in the leachate
Dominant ions in the leachate of control plants were Ca2+ and NO-3 (Table 8). Salt treatment led to a considerable increase of cation concentration in the leachate, and Cl- became the dominant anion. In comparison to total leaf ion content, the amounts of ions leaching out were low. However, if free ions in the apoplastic solution are used as a reference, it became apparent that approximately 0.1% of K+, 0.8% of Na+ and up to 5% of Ca2+ions present in the apoplast are leached daily (Table 8). In order not to overestimate Ca2+ leaching it has to be considered that Ca2+ concentrations in the apoplastic solution are rather low due to sorption processes.
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Discussion |
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Overall response of maize to salinity
It is well established that carbon partitioning between shoots and roots is flexible and highly responsive to the environment (Dalton et al., 1997
; Fageria et al., 1997). Salt treatment of maize led to an increase in the relative root size, since growth reduction was stronger in shoots than in roots (Table 1). This is in general agreement with previous findings with wheat and maize (Lewis et al., 1989), with rice (Misra et al., 1997) as well as with tomato (Dalton et al., 1997). The precise mechanism by which salt affects carbon partitioning remains unclear. However, the higher retranslocation of nitrogen compounds via the phloem (Table 6; Gilbert et al., 1998) may be taken as an indication of a relative increase of the root sink strength, although a reduced shoot sink strength due to decreasing growth rates can not be excluded as an alternative explanation.
Since net CO2 assimilation on a fresh weight basis was unaffected by salt treatment or sprinkling (Table 1) it may be concluded that shoot growth rate is not limited by net photosynthesis. The lower carbon assimilation per plant observed in later growing stages is a reflection of a reduced leaf area and is in agreement with earlier results (Cramer et al., 1994a: Lewis et al., 1989), which showed that salinity affects maize leaf growth rate more strongly and earlier than net photosynthesis. The reduction of shoot growth upon exposure to salinity can not be explained by shortage of nutrient supply since the concentrations of K+ and amino-N in the xylem sap were increased by salt treatment (Table 7). This is in general agreement with findings of Munns who showed that K+ concentration in the xylem sap of barley did not decline until the external NaCl concentration exceeded 100 mol m-3 (Munns, 1985).
Ion concentrations in the apoplast
Ion concentrations in the leaf apoplast are governed by import via the xylem, uptake into the symplast (including storage in the vacuole), as well as export through retranslocation by the phloem and leaching of solutes through rain. The influence of the salt treatment on Na+ and Cl- concentrations in the leaf apoplast of maize was less pronounced than expected based on previous data suggesting an inverse relationship between apoplastic salt concentration and salt tolerance (Speer and Kaiser, 1991). In that study, salt treatment led to an increase of the apoplastic Na+ and Cl– concentrations by 50 mol m-3 with the salt-sensitive pea but not with the salt-tolerant spinach. The NaCl concentration in the aqueous apoplastic space of rice leaves was calculated to be around 600 mol m-3 in leaves of plants whose roots were exposed to only 50 mol m-3 NaCl (Flowers et al., 1991). These rice leaves were showing symptoms of water deficiency. Maize is generally considered to be salt-sensitive (Yeo et al., 1977; Fortmeier and Schubert, 1995). A growth reduction of approximately 40% was found at 100 mol m-3 external NaCl (Table 1). However, the plants remained healthy for the duration of the experiment and the leaves showed no sign of senescence or salt injury. In agreement with earlier works it was found that the Na+ concentration in the apoplastic fluid of maize leaves increased to just 4 mol m-3 and that of Cl– to 5 mol m-3 with 100 mol m-3 external NaCl (Table 4) (Wang and Zhao, 1997). Thus the growth reduction in maize is probably not due to the accumulation of salt in the apoplastic fluid.
Ion retranslocation in the phloem and its significance for leaf ion balance
Phloem sap may be obtained either through aphid stylets or, with certain plant species such as castor bean or white lupin, by manual incision. Based on literature data, Na+ and Cl– concentration in the phloem sap of salt-treated plants can differ between 10 and 100 mol m-3 (Downing, 1980 (Aster tripolium); Munns et al., 1986 (barley); Jeschke et al., 1986 (lupin); Munns et al., 1988 (lupin); Wolf et al., 1990 (barley)). Higher concentrations of Na+ were observed in the phloem sap of salt-sensitive plants and lower concentrations in salt-tolerant plants. It was proposed that a high Na+ concentration in the phloem sap contributes substantially to the salt sensitivity of the plant (Jeschke et al., 1987). Unfortunately, in the different studies the phloem sap was obtained using different methods (manual incision or aphid stylet technique), and an influence of the collection methods on the concentration measurements can not be ruled out.
In these experiments, Na+ and Cl– concentrations in the phloem never exceeded 14 and 33 mol m-3, respectively. Na+ and Cl- concentrations in the phloem sap increased 17-fold and 4-fold, respectively, when 100 mol m-3 NaCl were added to the nutrient solution (Table 5). This increase corresponds to earlier results (Munns et al., 1986; Wolf et al., 1990), where Na+ concentrations in the phloem sap of salt-treated barley (100 mol m-3 external NaCl) of about 15–20 mol m-3 were found. An even more dramatic response was observed when NaCl concentration in the nutrient solution of maize was increased to 150 mol m-3. In this case, the Na+ concentration in the phloem sap increased to 82 or 43 mol m-3, without or with sprinkling, and the Cl- concentration rose to 158 and 154 mol m-3, respectively. However, under the latter conditions leaves did show symptoms of toxicity, i.e. leaf tips were necrotic and these plants were not used for further experiments.
Phloem retranslocation from maize leaves decreased in the order K+, Cl–, and Na+ (Table 6), all these ions being present in the leaves at high concentrations at 100 mol m-3 external NaCl (Table 2). For salt-treated plants the ratio between leaf tissue concentration and phloem concentration was 1.6 for K+, 1.7 for Cl- and 3.8 for Na+, which reflects the cellular compartmentation with most Na+ immobilized in the vacuole (Colmer et al., 1994). Similar ratios have been found for salt-treated lupin (about 1.1 for K+, 3.1 for Cl– and 3.5 for Na+) (Munns et al., 1988). The ratio between apoplastic concentration and phloem concentration increased in the same order as the ratio between leaf and phloem concentrations, K+ 0.10, Cl– 0.14, and Na+ 0.32 with 100 mol m-3 external NaCl. The lower ratio for K+ than for Na+ may be taken as an indication for a selectivity of K+ over Na+ by the systems transporting ions into the phloem. Total solute concentration in the phloem sap increased with the salt treatment and so did the osmolality. This may be a necessity to maintain sieve tube turgor at decreasing leaf water potential.
In spite of the relatively low ion concentrations in the phloem, translocation rates were somewhat higher than reported elsewhere. In barley, phloem export rates of K+, Cl–, and Na+ were shown to be in the range of 0.58, 0.24 and 0.08 µmol g-1 FW h-1 (Greenway et al., 1965). This study's data for maize for the same ions are 1.3, 0.6, and 0.22 µmol g-1 FW h-1, respectively (Table 6). The relatively high translocation rate of ions found in maize may be due to the higher carbon translocation rate observed with C4 plants (about 200 µmol C g-1 FW h-1 in maize) as opposed to C3 plants (about 60–70 µmol C g-1 FW h-1 in barley and spinach, calculated from Riens et al., 1994). It is concluded that phloem retranslocation of ions contribute significantly to the leaf solute balance, i.e. it prevents ion accumulation in apoplast and symplast. In maize this is due to the high phloem transport rates rather than to the high ion concentrations in the phloem sap.
The relatively high NaCl retranslocation in the phloem sap of mature maize leaves was not reflected by high NaCl concentrations in young leaves. In these leaves, Na+ concentrations increase only from 5.8 to 17.0 µmol g-1 FW and Cl– concentrations from 7.7 to 25.7 µmol g-1 FW with 100 mol m-3 external NaCl. Either the growth rate of this C4 plant is rapid enough to prevent ion accumulation in young leaves, or the larger part of the ions in the phloem were transported to the roots.
Relationship between xylem and phloem transport
Xylem import into source leaves was estimated from the transpiration rates and ion concentrations in the xylem sap. The ratio of phloem export of nitrogen (about 40%) and potassium (about 35%) to xylem import remained unaffected by salt treatment (Table 9) since the concentrations of amino-N and K+ increased in phloem sap and also in the xylem sap (Table 7). These results correspond to the findings of Munns, who saw an increase of K+ concentrations in the xylem sap of lupine upon salt-treatment (Munns, 1988). Under control conditions, between 13% (Na+) and 36% (Cl–) of ions imported via the xylem are exported by the phloem again (Table 9). Similar results have been obtained in other studies (Munns et al., 1986; Jeschke et al., 1992; Gouia et al., 1994; Marschner, 1995; Marschner et al., 1997). Upon exposure to 100 mol m-3 NaCl in the rooting medium the xylem import of Na+ increased strongly (from 0.1 to 0.7 µmol g-1 FW of leaf h-1) and so did the Cl– import (from 0.5 to 2.0 µmol g-1 FW of leaf h-1; Table 9). Under the same conditions the phloem export of Na+ was increased more strongly than xylem import, 13% of the imported Na+ was retranslocated in control plants and 32% in salt-treated plants. For Cl– the xylem import to phloem export ratio was similar under both conditions (36% control and 28% with salt conditions). Na+ phloem export is plant species dependent. Approximately 10% of xylem-imported Na+ is exported through the phloem in barley (Munns et al., 1986), 31% in Leptochloa fusca (Jeschke et al., 1995); 37% in cotton (Gouia et al., 1994), 40–70% in white lupin (Munns et al., 1988), and 77% in bean (Gouia et al., 1994). It seems that high Na+ retranslocation from the leaves is linked to high salt sensitivity of the plants.
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Role of leaching in the ion balance of maize leaves
In older literature the so-called ‘cuticulary excretion’ (Arens, 1934) was considered to be an important mechanism for avoiding high salt concentrations in the leaf (Tukey, 1970). The influence of misting in stimulating growth as observed by these authors is in general agreement with more recent findings in Picea (Leisen and Marschner, 1990). In the experiments reported here leaching had no significant effect on plant growth. Absolute amounts of ions leached from maize leaves in the present investigation (Table 8) seem rather low compared to those reported previously (Evans et al., 1981; Foster, 1990; Mecklenburg et al., 1966; Tukey Jr, 1970). If the total ion content of maize leaves is considered as reference, leaching of ions from the leaf surface by artificial rain equivalent to 10 mm d-1 varied from 0.01% (K+) to 0.17% (Ca2+). If the ion concentration in the apoplastic fluid is taken as a reference it appears that 0.1% of the K+, 0.8% of the Na+ and even 5% of the Ca2+ is leached on a daily basis. It has to be taken into account that the very low Ca2+ concentration in the apoplastic fluid is responsible for the relatively high leaching rate for Ca2+. At least two apoplastic Ca2+ fractions are detectable: free and bound fractions (Mühling and Sattelmacher, 1995). Since only the free Ca2+ is in equilibrium with water on the leaf surface, this fraction only may be considered as a reference. This view is supported by Mecklenburg et al. who demonstrated that only the free Ca2+ fraction is the source of 45Ca efflux from bean leaves (Phaseolus vulgaris L.) into distilled water (Mecklenburg et al., 1966). As already discussed earlier, upon exposure of maize to salt, Na+ and Cl- concentrations in the apoplastic solution of leaves did not increase as much as expected. But leaching of all measured cations increased significantly. Whether this indicates damage of the cuticles which represents the rate-limiting barrier for ion efflux from foliage (Schönherr, 1976; Scherbatskoy and Tyree, 1990) needs to be examined.
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This work was supported by the Deutsche Forschungsgemeinschaft as part of the priority research program 717 ‘The apoplast of higher plants—compartment for storage, transport and reaction’. The authors are grateful to Professor HW Heldt for stimulating discussions and Professor B Osmond, Dr W Peters and Dr K Pawlowski for critical remarks on the manuscript. We also acknowledge the reviewers for helpful comments on the manuscript.
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4 To whom correspondence should be addressed. Fax: +49 551 395749. E-mail: glohaus{at}gwdg.de
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Arens K.1934. Die kutikuläre Exkretion des Laubblattes. Jahrbuch wissenschaftliche Botanik 80, 248–300.
Balling A, Zimmermann U.1990. Comparative measurements of the xylem pressure of Nicotiana plants by means of the pressure bomb and pressure probe. Planta 182, 325–338.[Web of Science]
Barlow CA, McCully ME.1972. The ruby laser as an instrument for cutting the stylets of feeding aphids. Canadian Journal of Zoology 50, 1497–1499.
Cheeseman JM.1988. Mechanisms of salinity tolerance in plants. Plant Physiology 87, 547–550.
Colmer TD, Fan TWM, Higashi RM, Läuchli A.1994. Interactions of Ca2+ and NaCl stress on the ion relations and intracellular pH of Sorghum bicolor root tips: an in vivo 31P-NMR study. Journal of Experimental Botany 45, 1037–1044.
Cramer GR, Alberico GJ, Schmidt C.1994a. Leaf expansion limits dry matter accumulation of salt-stressed maize. Australian Journal of Plant Physiology 21, 663–674.
Cramer GR, Alberico GJ, Schmidt C.1994b. Salt tolerance is not associated with sodium accumulation of two maize hybrids. Australian Journal of Plant Physiology 21, 675–692.
Dalton FN, Maggio A, Piccinni G.1997. Effect of root temperature on plant response functions for tomato: comparison of static and dynamic salinity stress indices. Plant and Soil 92, 307–319.
De Silva DLR, Hetherington AM, Mansfield TA.1996. Where does all the calcium go? Evidence of an important regulatory role for trichomes in two calcicoles. Plant, Cell and Environment 19, 880–886.
Downing N.1980. Measurements of the osmotic concentrations of stylet sap, haemolymph and honeydew from aphids under osmotic stress. Journal of Experimental Botany 33, 557–573.
Durand M, Lacan D.1994. Sodium partitioning within the shoot of soybean. Physiologia Plantarum 91, 65–71.
Evans LS, Curry TM, Lewin KF.1981. Responses of leaves of Phaseolus vulgaris L. to simulated acidic rain. New Phytologist 88, 403–420.
Fageria NK, Baligar VC, Dalton FN, Maggio A, Piccini G.1997. Upland rice genotypes evaluation for phosphorus use efficiency. Journal of Plant Nutrition 20, 499–509.
Fink S.1992. Occurence of calcium oxalate crystals in non-mycorrhizal fine roots of Picea abies (L.) Karst. Journal of Plant Physiology 140, 137–140.
Flowers TJ, Hajibagheri MA, Yeo AR.1991. Ion accumulation in the cell walls of rice plants growing under saline conditions: evidence for the Oertli hypothesis. Plant, Cell and Environment 14, 319–325.
Fortmeier R, Schubert S.1995. Salt tolerance of maize (Zea mays L.): the role of sodium exclusion. Plant, Cell and Environment 18, 1041–1047.
Foster JR.1990. Influence of pH and plant nutrient status on ion fluxes between tomato plants and simulated acid mists. New Phytologist 116, 475–485.
Gilbert GA, Gadush MV, Wilson C, Madore MA.1998. Amino acid accumulation in sink and source tissues of Coleus blumei Benth. during salinity stress. Journal of Experimental Botany 49, 107–114.
Gouia H, Ghorbal MH, Touraine B.1994. Effects of NaCl on flows of N and mineral ions and on NO3- reduction rate within whole plants of salt-sensitive bean and salt-sensitive cotton. Plant Physiology 105, 1409–1418.[Abstract]
Greenway H, Gunn A, Pitman M, Thomas DA.1965. Plant response to saline substrates. VI. Chloride, sodium, and potassium uptake and distribution within the plant during ontogenesis of Hordeum vulgare. Australian Journal of Biological Science 18, 525–540.
Greenway H, Munns R.1980. Mechanism of salt tolerance in nonhalophytes. Annual Review of Plant Physiology 31, 149–190.[Web of Science]
Hanson AD, Hitz WD.1982. Metabolic responses of mesophytes of plant water deficits. Annual Review of Plant Physiology 33, 163–203.[Web of Science]
Husted S, Schjoerring JK.1995. Apoplastic pH and ammonium concentration in leaves of Brassica napus L. Plant Physiology 109, 1453–1460.[Abstract]
Jeschke WD, Klagges S, Hilpert A, Bhatti AS, Sarwar G.1995. Partitioning and flows of ions and nutrients in salt-treated plants of Leptochloa fusca L. Kunth: Cations and chloride. New Phytologist 130, 23–35.[Web of Science]
Jeschke WD, Pate JS, Atkins CA.1986. Effects of NaCl salinity on growth development, ion transport and ion storage in white lupin (Lupinus albus L. cv. Ultra). Journal of Plant Physiology 124, 257–274.
Jeschke WD, Pate JS, Atkins CA.1987. Partitioning of K+, Na+, Mg2+, and Ca2+ through xylem and phloem component organs and nodulated white lupin under mild salinity. Journal of Plant Physiology 128, 77–93.
Jeschke WD, Wolf O, Hartung W.1992. Effect of NaCl salinity on flows and partitioning of C, N, and mineral ions in whole plants of white lupin, Lupinus albus L. Journal of Experimental Botany 43, 777–888.
Läuchli A.1986. Responses and adaptations of crops to salinity. Acta Horticulturae 190, 243–246.
Läuchli A, Wieneke J.1979. Studies on growth and distribution of Na+, K+, and Cl– in soybean varieties differing in salt tolerance. Zeitschrift für Pflanzenernährung und Bodenkunde 142, 3–13.
Leisen E, Marschner H.1990. Einfluß von Düngung und saurer Benebelung auf Nadelverlust sowie Auswaschung und Gehalte an Mineralstoffen und Kohlehydraten in Nadeln von Fichten (Picea abies (L.) Karst.). Forstwissenschaftliches Cblatt 109, 253–263.
Lessani H, Marschner H.1978. Relation between salt tolerance and long distance transport of sodium and chloride in various crop species. Australian Journal Plant Physiology 5, 27–37.
Lewis OAM, Leidi EO, Lips SH.1989. Effect of nitrogen source on growth response to salinity stress in maize and wheat. New Phytologist 111, 155–160.
Lohaus G, Winter H, Riens B, Heldt HW.1995. Further studies of the phloem loading process in leaves of barley and spinach. The comparison of metabolite concentrations in the apoplastic compartment with those in the cytosolic compartment and in the sieve tubes. Botanica Acta 108, 270–275.
Maas EV, Hoffman GJ.1977. Crop salt tolerance—current assessment. ASCE Journal Irrigation Drain Division 103, 115–134.
Marschner H.1995. Mineral nutrition of higher plants, 2nd edn. London: Academic Press Inc.
Marschner H, Kirkby EA, Engels C.1997. Importance of cycling and recycling of mineral nutrients within plants for growth and development. Botanica Acta 110, 265–273.
Mecklenburg RA, Tukey Jr HB, Morgan JV.1966. A mechanism for the leaching of calcium from foliage. Plant Physiology 41, 610–613.
Mengel K, Lutz HJ, Breininger MT.1987. Auswaschung von Nährstoffen durch sauren Nebel aus jungen intakten Fichten (Picea abies). Zeitschrift für Pflanzenernährung und Bodenkunde 150, 61–68.
Misra AN, Sahu SM, Meera I, Mohapatra P, Das N, Misra M, Dalton FN, Maggio A, Piccinni G.1997. Root growth of a salt-susceptible and a salt-resistant rice (Oryza sativa L.) during seedling establishment under NaCl salinity. Journal Agronomic Crop Science 178, 9–14.
Mühling KH, Sattelmacher B.1995. Apoplastic ion concentration of intact leaves of field bean (Vicia faba) as influenced by ammonium and nitrate nutrition. Journal of Plant Physiology 147, 81–86.
Munns R.1985. Na+, K+, and Cl- in xylem sap flowing to shoots of NaCl-treated barley. Journal of Experimental Botany 36, 1032–1042.
Munns R.1993. Physiological processes limiting plant growth in saline soils: some dogmas and hypotheses. Plant, Cell and Environment 16, 15–24.
Munns R.1988. Effect of high external NaCl concentrations on ion transport within the shoot of Lupinus albus. I. Ions in xylem sap. Plant, Cell and Environment 11, 283–289.
Munns R, Tonnet ML, Shennan C, Gardner A.1988. Effect of high external NaCl concentration on ion transport within the shoot of Lupinus albus. II. Ions in phloem sap. Plant, Cell and Environment 11, 291–300.
Munns R, Fisher DB, Tonnet ML.1986. Na+ and Cl- transport in the phloem from leaves of NaCl-treated barley. Australian Journal Plant Physiology 13, 757–766.[Web of Science]
Oertli JJ.1968. Extracellular salt accumulation, a possible mechanism of salt injury in plants. Agrochimica 12, 461–469.
Passioura JB.1980. The transport of water from soil to shoot in wheat seedlings. Journal of Experimental Botany 31, 333–345.
Pennewiss K, Mühling KH, Sattelmacher B.1997. Leaching from the leaf surface and its significance for apoplastic ion balance. In: Ando T et al., eds. Plant nutrition—for sustainable food production and environment. Japan: Kluwer Academic Publishers, 87–88.
Pfirrmann T, Runkel KH, Schramel P, Eisenmann T.1990. Mineral and nutrient supply, content and leaching in Norway spruce exposed for 14 months to ozone and acid mist. Environmental Pollution 64, 229–254.[Medline]
Rausch T, Kirsch M, Löw R, Lehr A, Viereck R, Zhigang A.1996. Salt stress responses of higher plants: the role of proton pumps and Na+/H+-antiporters. Journal of Plant Physiology 148, 425–433.
Riens B, Lohaus G, Heineke D, Heldt HW.1991. Amino acid and sucrose content determined in the cytosolic, chloroplastic, and vacuolar compartments and in the phloem sap of spinach leaves. Plant Physiology 97, 227–233.
Riens B, Lohaus G, Winter H, Heldt HW.1994. Production and diurnal utilization of assimilates in leaves of spinach (Spinacia oleracea L.) and barley (Hordeum vulgare L.). Planta 192, 497–501.
Sangster AG, Hodson MJ.1986. Some factors influencing silica transport and deposition in grasses. American Journal of Botany 73, 642–643.
Scherbatskoy T, Tyree MT.1990. Kinetics of exchange of ions between artificial precipitation and maple leaf surfaces. New Phytologist 114, 703–712.
Schönherr J.1976. Water permeability of isolated cuticular membranes: the effect of pH and cations on diffusion, hydrodynamic permeability and size of polar pores in the cutin matrix. Planta 128, 113–126.
Sharkey TD, Stitt M, Heineke D, Gerhardt R, Raschke K, Heldt HW.1986. Limitation of photosynthesis by carbon metabolism II. O2-insensitive CO2 uptake results from limitation of triose phosphate utilization. Plant Physiology 81, 1123–1129.
Speer M, Kaiser WM.1991. Ion relations of symplastic and apoplastic space in leaves from Spinacia oleracea L. and Pisum sativum L. under salinity. Plant Physiology 97, 990–997.
Tetlow IJ, Farrar JF.1993. Apoplastic sugar concentration and pH in barley leaves infected with brown rust. Journal of Experimental Botany 44, 929–936.
Tukey HB.1969. Leaching of metabolites from foliage and its implication in the tropical rain forest. In: Odum HT, ed. A tropical rain forest. US Atomic Energy Commission.
Tukey HB.1970. The leaching of substances from plants. Annual Review of Plant Physiology 21, 305–324.
Tukey HB, Mecklenburg RA.1964. Leaching of metabolites from foliage and subsequent reabsorption and redistribution of the leachate in plants. American Journal of Botany 51, 737–742.[Web of Science]
Turner DP, Tingey DT.1990. Foliar leaching and root uptake of Ca, Mg, and K in relation to acid fog effects on Douglas-fir. Water Air Soil Pollution 49, 205–214.
Wang BS, Zhao KF.1997. Changes in Na and Ca concentrations in the apoplast and symplast of etiolated maize seedlings under NaCl stress. Acta Agronomica Sinica 23, 27–33.
Weiner H.1990. Untersuchungen zur C4-Photosynthese: Permeabilität von Bündelscheidezellen und Kompartimentierung von Metaboliten in Maisblättern. PhD thesis. Universität Göttingen, Germany.
Winzer T, Lohaus G, Heldt HW.1996. Influence of phloem transport, N-fertilization and ion accumulation on sucrose storage in the taproots of fodder beet and sugar beet. Journal of Experimental Botany 47, 863–870.
Wolf O, Munns R, Tonnet L, Jeschke WD.1990. Concentrations and transport of solutes in xylem and phloem along the leaf axis of NaCl-treated Hordeum vulgare. Journal of Experimental Botany 41, 1133–1141.
Yeo AR, Kramer D, Läuchli A, Gullasch J.1977. Ion distribution in salt-stressed mature Zea mays roots in relation to ultrastructure and retention of sodium. Journal of Experimental Botany 28, 17–29.
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