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Journal of Experimental Botany, Vol. 48, No. 308, pp. 693-706, March 1997 Journal of Experimental Botany Ultrastructural and physiological changes in root cells of Sorghum plants [Sorghum bicolor x S. sudanensis cv. Sweet Sioux) induced by NaCI Hans-Werner Koyro1 Botanisches Institut der Tiera'rztlichen Hochschule Hannover, BOnteweg 176, D-30559 Hannover, Germany Received 20 December 1995; Accepted 12 September 1996 Abstract The xerophytic, but salt-sensitive Sorghum cultivar 'Sweet Sioux' is known as an ion excluder with a high K/Na selectivity at the plasmalemma and tonoplast of epidermal root cells. The aim of this study is the correlation of salt-effected changes in physiological parameters with structural and ultrastructural changes in root cells. The investigation was carried out with root cells because these cells are most directly exposed to the growth medium. Sorghum bicolor xS. sudanensis cv. Sweet Sioux plants were grown under steady-state conditions on nutrient solutions with or without 40 mol m " 3 NaCI. Sorghum sustained this treatment but showed several salt-induced structural and physiological changes which were studied in various cell types of the root tip. (1) NaCI salinity led to a shorter growth region and to salt-induced alterations in the chemical and physical properties of the cell walls in the root tips. (2) Salt treatment also increased the membrane surface in root cells: root cells showed an increase in the quantity of vesicles in the epidermis and in the middle cortex cells. Additionally, some of the epidermis cells of salt-treated plants revealed a characteristic build-up of transfer cells, suggesting an increase in membrane surfaces to increase the uptake and storage of substances. (3) The number of mitochondria increased in the epidermal and in the cortex cells after salt stress thus indicating an additional supply of energy for osmotic adaptation and for selective uptake and transport processes. (4) In the epidermal cytoplasm NaCI stress led to a significant decrease of the P, K, Ca, and S concentra1 tions accompanied by an increase of Na concentration. Electron micrographs show an increase in electron optical contrast within the cytosol and in the matrix of the mitochondria. These results are discussed with regard to the possibility of influence on the part of metabolic functions. (5) The NaCI concentrations were seen to increase and the K concentrations to decrease during salt stress in the vacuoles of the epidermis and cortex cells. The salt-induced increase in vacuolar NaCI concentrations of epidermis and cortex cells are in the region 2 cm behind the root tip, which is sufficient for an osmotic balance towards the growth medium. Additional solutes are necessary 0.5 mm behind the root tip to facilitate osmotic adaptation. The results show ultrastructural changes caused by an Na-avoiding mechanism characterized by a high level of energy consumption. The exclusion of Na from the symplast seems to lead additionally to a decrease in cytoplasmic concentrations of such essential elements as Mg, P, S, and Ca and is thus responsible directly (via energy supply in mitochondria, homeostasis, selectivity of K over Na) or indirectly (via enzyme conformation, cytoplasmic hydration) for the ultrastructural degradation indicated. The salinity-induced multiplicity of structural and functional changes within cell compartments constitutes a group of indicators for the limited NaCI tolerance of Sorghum. Key words: Sorghum bicolor x S. sudanensis, structure, salt tolerance, NaCI, Ca-deficiency. ultra- Introduction Sorghum plants are cultivated mainly in sub-humid and semi-arid climate regions (Corlett et al., 1994; Gorz et al., Present address: Zentrale Biotechnische Betriebseinheit im Strahlenzentrum der Justus-Liebig-Universitat Giessen, D-35392 Giessen, Germany. Fax: +49 641 993 0099. E-mail: Hans-Werner.Koyro©strz.uni_giessen.de © Oxford University Press 1997 694 Koyro 1987; Hodson and Evans, 1995). Osmotic adaptation to drought results from the accumulation of organic compounds (for example, sucrose, glucose and fructose) in the vacuoles and with proline or glycinebetaine as an osmoprotectant, in the cytoplasm (Basnayake et al, 1995; Bhaskaran et al, 1985; Jones, 1978; Weimberg et al., 1984). The metabolic costs of drought-induced osmotic stress seem to be compensated for by a proportionally high reduction in biomass synthesis (Auge et al., 1995; McCree, 1986; Osmond, 1980). In contrast to these results, vanadate-sensitive ATPase activity in Sorghum roots increases 5-fold under salt stress (40 mol m~3 NaCl) while the rate of ATPase activity in nitrate- and azide-sensitive tissue is six times higher (Koyro et al., 1993). It is not clear whether the energy requirement induced by salt treatment can be compensated for by growth reduction. Much effort has been invested in the conjunctive use of saline irrigation waters in semi-arid regions to grow Sorghum plants (Sharma et al., 1994). However, Sorghum is resistant to low water potential in the soil caused by drought, but highly sensitive to NaCl salinity (Amzallag, 1994; Weimberg et al., 1984; Yamane, 1993). This suggests a direct effect of Na + or Cl~ on the metabolism. The root tip acts as a finely-tuned sensor for different kinds of stress (Colmer et al., 1994; Roth and Bergmann, 1988). The outer cortex and especially the epidermis cells have a key function in the uptake of minerals into the symplast of the plant. Direct exposure of the root cortex led us to expect structural changes in these cells, which may explain the low salt tolerance of the Sorghum species. At the tissue level, roots show a broad spectrum of structural adaptations to environmental changes. NaCl salinity or Fe deficiency can, for example, lead to reduced growth and to a shorter elongation zone (Bernstein et al., 1993; Wilson and Robards, 1978; Clarkson et al., 1987). A diminished O2 supply can induce development of an aerenchyma (Stelzer and LSuchli, 1977). A considerable amount of data is available on saltinduced changes at the cellular level. Comparative investigations have been conducted with drought-sensitive Sorghum and salt-tolerant Spartina plants grown under steady-state conditions with or without 40 mol m~3 NaCl (Koyro et al., 1993). Salt treatment caused an increase of epidermal IMP (intramembraneous particle) frequencies in the plasmalemma and tonoplast. This is interpreted by the authors to mean an increase in membrane- bound enzymes (such as ATPases). This supposition was confirmed by a salt-induced increase in ATPase activity (F-, T- and P-ATPase) per ng protein. In Suaeda maritima and A triplex hastata the root endodermis develops faster and the Casparian strip was significantly bigger after the addition of NaCl to the nutrient solution (Kramer et al, 1978; Hajibagheri et al, 1985). Moreover, Nishizawa and Mori (1984) have dem- onstrated the presence of vesicular (pinocytotic) ion transport from the plasmalemma to the tonoplast. There can also be differences in the behaviour of single cells within the same tissue with regard to environmental stress. Several authors have detected transfer cells in solitary epidermal or hypodermal cells (Kramer et al, 1977, 1978; Gunning, 1977) and Fricke et al. (1994) has demonstrated a correlation between position and ion composition of single leaf epidermal cells in barley. Huang and Van Steveninck (1990) have shown that salinity causes many plastids in cortical root cells to adopt varying amoeboid shapes, but have also determined variations in the density of the cytoplasm. Functional differentiation is usually accompanied by differences in (ultra) structure. The salt-induced increase in the activity of all three ATPase types in Sorghum root tips and, simultaneously, changes in the IMP-frequencies in the tonoplast and plasmalemma of epidermis cells constitute only one example of such an interrelationship (Koyro et al, 1993). Many analytical studies have been carried out on the correlation between the mineral content of Sorghum plants and attendant specific soil conditions (Richardson and McCree, 1985; Press and Stewart, 1987). Studies of element distribution at the cellular level are, by comparison, rare. The aim of the present paper is to describe salt-induced changes in cell structures and cytoplasmic and vacuolar element concentrations in epidermis and cortex cells of Sorghum roots in order to identify the physiological reason for sensitivity to NaCl salinity and to pinpoint those metabolic processes in the plant where genes for salt tolerance might be expressed (Munns, 1993). Materials and methods Plant growth Seeds of Sorghum bicolor x S. sudanensis (L.) Moench cv. Sweet Sioux were washed for several minutes with warm tap water and imbibed for 1 d in the dark (25 °C) in an aerated 0.2 mol m~ 3 CaSO 4 solution. After this, the seeds were then plotted on plastic grids and germinated for 1 d in large plastic containers (10 1) in a 0.2 mol m~ 3 CaSO 4 solution. Subsequently, the seedlings were cultivated under photoperiodic conditions (16 h light/8 h dark) in a growth cabinet (Koyro et al, 1993). After 1 week, the plants were placed in a nutrient solution with 1 mol m " 3 K + (Epstein, 1972). Three weeks later, one batch of plants received 40 mol m~ 3 NaCl (added within 2 d to the growth medium). The remainder of the culture served as a control. The nutrient solutions were renewed weekly. Light microscopy (Lm) The light-microscopical investigation was performed with a Zeiss photomicroscope III equipped with a Normarsky lens. The photomicroscope's Hg-HP lamp (HBO 50 W) suffices to induce fluorescence. Fresh material was sectioned by hand with a razor blade 1-2 cm behind the root apex (n = 20). Sections were stained by mounting them in aniline blue (Hughes and Ultrastrvctural and physiological changes in Sorghum Gunning, 1980). A Zeiss UG exciter filter and Zeiss 41 barrier filter (passed A >410nm) were used to detect the fluorescence induced by aniline blue (specifically for callose). Background autofluorescence was substracted photographically from the aniline blue-induced fluorescence. A sequence (>10 sections) of 1-10 fim thin cross-sections was cut in fixed, dehydrated and resin embedded root tips in order to investigate the influence of NaCl on the cell development of root tissues, thus enabling morphometric analysis of equivalent sections from each treatment (n = 32 cells). Root tips were therefore fixed for 4 h with 1% glutaraldehyde in 50mol m~ 3 cacodylate buffer (pH 7.2). To imitate the osmotic differences of the nutrient solutions, 40 mol m~ 3 NaCl was added to the fixative of salt-treated plants. After washing the root tips three times in buffer (with and without NaCl) the tips were post-fixed for 4 h with a 1% OsO 4 solution in buffer. After a few washings in distilled water, the root tips were dehydrated in acetone (Sitte, 1962). They were then transferred stepwise from acetone to propylenoxid (25%, 50%, 75%, 100% propylenoxid), and replaced at the end of this procedure by resin (Technovit, Kulzer). Embedded material was sectioned with a glass knife on an LKB ultramicrotome (Ultrotome I). The sections were stained with methylene blue (in 2% ethanol) on a heating plate (60 °C). Transmission electron microscopy (TEM) Root tips were prefixed with 1% glutaraldehyde in 50 mol m" 3 cacodylate buffer (pH 7.2). 40 mol m~ 3 NaCl was added to the fixation solution of salt-treated plants. The washing of the roots, post-fixation with Os0 4 , and dehydration were performed in the same way as for light microscopical examination. After this, roots were embedded in ERL^1206 resin (Spurr, 1969), and polymerized at 70 °C for 8 h. Ultrathin cross-sections (30-70 nm) were cut 0.5-1 mm behind the root apex with the Ultrotome I (LKB) or Ultracut E (Reichert-Jung) ultramicrotome. The sections were stained for 8 min with 1% uranyl acetate and for 4 min with 5% lead citrate before they were observed in a transmission electron microscope (Zeiss EM 10A or Zeiss EM 902). The number of mitochondria, vesicles and plastids per cell was calculated in more than 30 cells per treatment (10 sections per cell) from three cultures. Electron microscopy on freeze-fractured root tips Root tips (n= 16 per treatment) were placed in Balzers specimen carriers and frozen within 515 ms under high pressure (> 210 MPa) in a Balzers HPM 010. Freezing the specimen by rapid immersion, with a 2 m s~l entry velocity of the specimen into liquid propane (Reichert K.F 80) was employed as an alternative procedure (Kaeser et ai, 1989). The frozen root tips were fractured longitudinally, etched for 1 min (2.4 x 10" 4 Pa; 163 K), shadowed with platinum-carbon (1 nm Pt/C layer) at 45°, and, stabilized with carbon at 90° (25 nm C layer, Balzers BA 360 M). The specimens were subsequently thawed, rinsed with water, cleaned for 72 h with chromosulphuric acid and for 24 h with sodium hypochlorite. Afterwards, the replicas were washed several times with distilled water, mounted on pioloform coated copper grids, and viewed in a transmission Zeiss EM 10a electron microscope. Micrographs were taken 0.5-1 mm behind the root tip. X-ray microanalyses (EDXA) Root tips (15 tips per treatment) were mounted on a Cu-specimen stub together with droplets of standard solutions (40, 80, 120 mol m " 3 K + , Na + , Cl", 20, 40, 80 mol m " 3 Ca 2 + , Mg2+, PO 3 ~, SOJ". The roots were cut transversely with a 695 blade cooled with liquid nitrogen on a cryo-stage under high vacuum conditions, then transferred to the column of an ETEC Autoscan SEM equipped with a KEVEX Si/Li detector for X-ray count harvesting. The electron beam was focused 0.5 mm behind the root tip on cytoplasm and vacuoles of epidermal cells and on vacuoles of middle cortex cells, and 2 cm behind the root tip on vacuoles of both cell types (15 sections per treatment and stage). Subsequent procedure of the quantification have been described elsewhere (Koyro, 1989; Stelzer et al, 1988). Results Figure 1 shows a typical cross-section of controls and NaCl-treated roots (« = 32). Intercellulars are in the process of development between all cortex cells of root controls 400 ^m behind the root tip. Actively dividing regions are visible in the endodermal and epidermal cell layers. In all cortex cells, with the exception of the endodermis and epidermis cells, a multitude of small vacuoles is present. In this region the typical elements of the stele are visible, showing only some vesicles in the metaxylem element vessels and in the sieve-tube. Advanced development of nearly all tissues is one consequence of the salinization: the middle cortex cells show a large central vacuole; the endodermal and epidermal cells have large vesicles, the metaxylem vessel element no longer contains living contents. The reduced degree of enlargement in cells adapted to 40 mol m~3 NaCl was not the result of absorption of lignin and suberin into the cell walls. Observations made with solutions of Sudan III or Phloroglucinol/HCl did not show any of these substances at 1 or 2 cm from the root tip (results not shown). However, in contrast to the control roots, an additional deposition of callose into the cell walls of the outer cortex cells and of the endodermis was generally observed after salt treatment (Fig. 2a, b). Changes in wall structure can influence the elongation of the cell. The observations made by transmission electron microscopy with freeze-fracture replicas of epidermal cells show differences in the wall in response to salinity treatment (Fig. 3). At 1 mm behind the root tip of the controls, thin microfibrils were arranged mainly (in 87% of the cells investigated) in a parallel array. In corresponding cells of the salt-treated roots, the microfibrils almost exclusively (91% of the investigated cells) formed a closely meshed network. Comparison of the ultrastructures in conventional fixated and embedded samples was undertaken between 0.75 and 1 mm behind the root tip. The observations showed conspicuous differences between the control and salinity treatment. In the epidermis and cortex cells of control roots the stroma of the plastids, the matrix of the mitochondria, the nucleoplasm and the cytoplasm all appear to have a comparable high density (Fig. 4a, b). No uniform picture was presented by salt-treated epidermis 696 Koyro Fig. 1. Light micrographs (Normarsky optics) of cross-sections 400 pm from the junction of the meristem and root cap. There are conspicuous differences in the state of development of root tissues of the (a) control and (b) NaCl-treated plants (bar, 100 ^m); r: epidermis; c: cortex; ic: intercellulars; ed: endodermis; ph: phloem; mx: metaxylem vessel element. Ultrastructural and physiological changes in Sorghum 697 Fig. 2. Cross-sections of Sorghum roots 1-2 cm behind the root tip, stained with aniline blue, (a) Aniline blue-induced fluorescence can be seen in the stele, but not in the cortex of controls, (b) The aniline blue positive deposits show up clearly in the outer cortex cells of NaCl-treated plants; r: epidermis; c: cortex; ed: endodermis. 698 Koyro Fig. 3. Electron micrographs of freeze-fracture replicas from epidermal cells of Sorghum root tips, (a) In control roots, the cellulose microfibrils are arranged mainly in a parallel array (bar; 100 nm). (b) In corresponding cells of the salt-treated roots, the microfibrils form a close-meshed network. Ultrastructural and physiological changes in Sorghum 699 r Fig. 4. Electron micrographs showing epidermis cells of controls (a, b) and salt-treated roots (c, d). (a, c) The survey of the epidermis cells demonstrates the identical stage of development, (b) The cytoplasm and the enclosed organells appear to be very dense. The plastids frequently store large amounts of starch, (d) Differences of density in the cytoplasm are visible between neighbouring epidermal cells. In cells where the cytoplasm appears to be less dense, the chnstae of the mitochondria are swollen (arrow), p: plastid; me: mitochondnum; the bars represent 10 fim. cells as a whole. Some cells showed no change at all in comparison with the controls (right cell, Fig. 4c, d). In many corresponding cells of salt-treated roots, the cytoplasm, the matrices of the mitochondria and the nucleo- plasm contained a lower concentration of heavy metal binding substances, which points to a decreased density in their matrices (left cell in Fig. 4c, d). The sole exception was the stroma of the plastids. 700 Koyro The walls of the epidermis cells are very thin, and differences in the ultrastructure of resin embedded samples cannot be observed after NaCl treatment. Nevertheless, in some epidermal cells of the salt-treated roots, invaginations are apparent in the plasmalemma and cell wall, giving the overall appearance of a transfer cell (Fig. 5). However, it is also possible, that the structure shown is the root cell's reaction to bacterial infection. These protuberances in the cell wall are observed only in cells with a high cytoplasmic density. The influence of salinity on the number of mitochondria, small vesicles and plastids in epidermal and middle cortex cells is listed in Table 1. NaCl treatment increases the amount of vesicles in the epidermis and in the middle cortex cells. The amount of mitochondria increased significantly only in the epidermis. The number of plastids did not change significantly in any of the recorded cells. The epidermis cells develop more slowly than the cortical cells in the root tip (Fig. 6, see also Fig. 1). An Table 1. Average number of mitochondria, vesicles and plastids in epidermal (with the exception of transfer cells) and cortical root cells of controls and NaCl-treated Sorghum plants: all values are given as mean ± SD Epidermis Mitochondria Vesicles Plastids Cortex (middle) Mitochondria Vesicles Plastids Control + 40 NaCl 12.1 ±0.7 2.1 ±0.5 2.8 ±0.4 15.8 ± 1.8 4.2±1 8 2.8±0.2 13 0 ± 1.4 0.5 + 0.2 l.8±1.2 14.3 ±0.1 3.2±0.5 1.9±0.3 accurate EDX measurement of the cytoplasm was only possible in epidermal cells 0.5 mm behind the root tip (Fig. 6). K and P are the dominant elements in the cytoplasm and vacuoles of epidermal and cortex cells (Fig. 7). P and K concentrations are always higher in the Fig. 5. Electron micrograph showing the interface between a transfer cell and a 'normal' epidermal cell from a salt-treated Sorghum root. The christae of the mitochondria are only swollen in cells where the cytoplasms appear to be less dense (arrow).The enrichment of mitochondna (me), endoplasmic reticulum and ribosomes next to the wall is characteristic of this cell type. The transfer cell shows the typical invaginations of the inner cell wall (bar. 1 ^m). Ultrastructural and physiological changes in Sorghum 701 Fig. 6. Cryo-scanning electron micrograph from transverse fracture-faces of Sorghum root tips 0.5 mm behind the tip (bar, 10 fim); c: cytoplasm, N: nuclei, v: vacuole. vacuoles of the epidermis cells, as they are in the cortex cells. Salinity leads to a small increase in Na and a significant decrease in P, K, S, and Ca in the cytoplasm (Fig. 7a). Apart from an increase in NaCl and a decrease in Ca, the vacuole shows no significant salt-induced changes in element concentrations (Fig. 7b). The composition of the vacuolar solutes is similar in the epidermis and cortex cells (Fig. 7b, d). It is obvious that salinity produces a greater increase in epidermal NaCl than in the cortical vacuoles 0.5 mm behind the root tip. K concentrations decrease after salt treatment only in cortex cells of this region. P concentrations decrease and Cl concentrations increase during cell development in epidermal and cortical vacuoles (Fig, 7c, e). Salinity causes a significant decrease in K and Ca concentrations in epidermal and cortex cells 2 cm behind the root tip. Only in this region are saltinduced increases in Na and Cl sufficient for osmotic balance towards the growth medium. Discussion Salt and drought stress was associated in root tips of Sorghum with a shortening of the growth region. The elongation zone of Sorghum leaves was also shorter in salinity despite the fact that the duration of elongation growth associated with a cell during its displacement from the leaf base was longer in salinized leaves than in the control (Bernstein et ai, 1993). Additionally, transpiration, growth and photosynthesis were strongly reduced by salinity (Auge et ai, 1995; Koyro, 1989; Nagy et ai, 1995). It has been shown that the turgor, the yield threshold and the wall extensibility have an influence on the cell growth (Cosgrove, 1981). As reported for other root cells (Pritchard et al., 1991) Sorghum roots exhibit complete regulation of single-cell turgor when osmotically stressed with 40 mol m " 3 NaCl (Koyro, 1989). This result suggests an effect of NaCl in Sorghum on the yield threshold and/or on the wall extensibility. The present paper shows several salt-induced changes in the cell wall of outer cortex cells from Sorghum which could explain a change in wall rigidity. (1) Salinity induces the storage of the j3-l,3-D-glucan, callose, in the cell walls of the outer cortex cells in Sorghum. (2) Freeze etching replicas present a typical secondary cell wall in epidermis cells of controls whereas salt-treated cells present mainly primary cell walls. 702 Koyro 20 10 0 80 70 (c) 60 50 40 30 20 10 0 Na Mg Cl Ca Na Cl Ca 3 Fig. 7. Cytoplasmic (a) and vacuolar (b-e) element concentrations (mol m ) in epidermal (a-c) and cortical cells (d, e) of Sorghum roots grown under steady-state conditions on nutrient solutions without D or with • 40 mol m" 3 NaCl. Mean values and SD (vertical bars). The measurements were taken 0.5 mm (a, b and d) or 2 cm (c and e) behind the root tip. Binzel et ai, (1987) have shown that alterations in the physical properties of the cell wall take place in cells adapted to NaCl stress. Carbon is partitioned for osmotic adjustment at the expense of cell wall synthesis. There is considerable data supporting the hypothesis that the hydrolysis of glucans (i.e. xyloglucan) permits wall expansion (Cleland, 1981; Lipetz, 1970). Callose, 0-1,3-Dglucan, is an insoluble polysaccharide found in many plant tissues; it seems however, to be particularly associated with senescence, dormancy, and injury. Numerous small deposits on the wall between the pits are followed by the deposition of a continuous layer on the inner wall surface (Currier, 1957). It is possible that the deposition of callose hinders the expansion of the root cells. The arrangement of microfibrils in a parallel array (Fig. 3) in the longitudinal cell walls supports the enlargement of cells in one (i.e. longitudinal) direction (Lflttge et ai, 1989). In corresponding cells of salt-treated roots, cells show no preference for cell enlargement. The arrangement of microfibrils in the longitudinal cell walls in a closly meshed network supports the uniform cell extension. Apart from indicating changes in the quantity of organelles, it was possible to demonstrate a salt-induced increase in electron optical-contrast in the cytosol and in the matrix of the mitochondria, but not in the stroma of the plastids. Similar results have been shown for Zea mays and barley (Huang and Van Steveninck, 1990, Nir et al., 1966; Siew and Klein, 1968). It is difficult to interpret results of samples fixed with Ultrastructural and physiological changes in Sorghum glutaraldehyde and osmium, dehydrated in acetone and embedded in resin because of the possible presence of artefacts. It should, nevertheless, be possible to attribute salt-induced differences between structures directly or indirectly to physiological changes. Increase in contrast suggests an imbibed cytosol and matrix. It is believed that there is a connection between the decrease in Mg, P, S, K, and Ca concentrations in the cytoplasm, as shown for bulk frozen samples (Fig. 7), and the decrease in the electron optical contrast (Fig. 4). Dissolved ions and especially mineral nutrients such as Ca, Mg and S, alter the physical properties of the solvent water by forming hydration shells around the ion, and also alter the properties of the protein molecule through interactions (Marschner, 1993). Disulphide bonds as well as ionic or covalent bonds with Mg and Ca are essential components in the hydration, stability, and conformation of many enzymes and in hydration of the cytoplasm. A decrease in the concentration of these elements could, therefore, be responsible for an imbibition of the proteins into the cytoplasm as suggested for resin embedded Sorghum root cells. Despite many investigations of the effects of NaCl on enzyme activty (Jennings, 1976), the results indicate that it is not ion excess of NaCl but ion deficiency (Ca, Mg, P, K) that is a possible reason for the decrease in enzyme activity in the cytoplasm. Further experiments are needed to investigate enzyme activities under more natural conditions. Hydration of the cytoplasm may also be changed by an acidification. An increase in the Na + /K + -exchange at the tonoplast has been put forward as one possible reason for a change of the pH in the cytoplasm (Garbarino and Dupont, 1989; Uribe and Luttge, 1984). Contrary to this theoretical possibility, NaCl induces a reduction of the pH gradient across the tonoplast in Sorghum bicolor root tips by vacuolar alkalization rather than cytoplasmic acidification (Colmer et al., 1994). An increase in pH degrades the roots and lowers the rate of Na and Cl exclusion in excised roots exposed to NaCl stress (Tsuchiya et al., 1995). Ca seems to be beneficial in maintaining a high pH-gradient between cytoplasm and vacuole and sustains the putative driving force for N a + transport from the cytoplasm into the vacuole via a N a + / H + channel as well as levels of cytoplasmic K + , cytoplasmic and vacuolar phosphate (Colmer et al., 1994). The decrease in Ca in cytoplasm and vacuoles of epidermis cells correlates with inhibition of membrane functions such as K + / N a + selectivity (Colmer et al., 1994). Ca can alleviate the inhibitory effect of NaCl on root growth by maintaining plasmamembrane selectivity of K over Na (Zhong and Lauchli, 1994). Salt-induced changes of the ultrastructure and element composition in epidermal root cells can be explained in terms of a Ca deficiency leading to loss of control over the cytoplasmic homeostasis. Further salinity 703 experiments are needed with varying Ca supply to investigate the sites of action of Ca. Salinity increased the amount of vesicles in the epidermal and in the middle cortex cells of Sorghum roots. It could not be established whether these vesicles were of tonoplast or cytoplasmic origin. Advance of vacuolation in the apical region of barley root apices could be effected by treatment with 50 mol m~ 3 NaCl (Huang and Van Steveninck, 1990). Water stress led to an increase of small vesicles (< 1 /xm) in root cells of Zea mays (Nishizawa and Mori, 1981) as did Ca-deficiency in potato sprouts (Hecht-Buchholz, 1979). The build-up of many small vesicles led to an increase in membrane surface. The authors interpret this increase in the number of vesicles as a limited enhancement of the transport capacity and storing activity of the afflicted cell and as a possible means of excluding excess ions from the cytoplasm. The ability of plants to control the uptake and compartmentation of Na and Cl constitutes a mechanism for salt tolerance (Flowers and LSuchli, 1983). A multiplicity of small vesicles processes a larger surface than one big vesicle, hence a higher exchange capacity (Na versus K ) . This system enables a plant cell to avoid ion toxicities, imbalances or interactions between substances in the cytoplasm. Additionally, the build-up of transfer cells was only in single epidermal cells of salt-treated plants, and never in hypodermal cells. Transfer cells have been shown to be present for several species in epidermal or hypodermal cells (Kramer et al., 1978; Kramer, 1983; Winter, 1988). It has been claimed that this is an indirect effect due to salt-induced iron deficiency (Kramer et al., 1980). There appears to be a correlation between the buildup of transfer cells and an extensive exchange of substances at the barrier between apoplast and symplast (Gunning, 1977; Hill and Hill, 1976; Thomson et al., 1988). With regard to NaCl tolerance, transfer cells have been considered to be connected with K + / N a + discrimination (Kramer, 1983) with increases in the exchange capacity between compartments. The advantage of an increase in vesicular and transfer cell (plasmalemma) surface indicates a higher and more selective uptake and storage of substances. The decrease in electron density within the matrix of the mitochondria seems to reflect a degeneration of the mitochondria and therefore a decreasing efficiency. Opik (1965, 1966), Schwab et al. (1969) describe similar changes for mitochondria. In root tips of a salt-sensitive cultivar of Agrostis stolonifera (grown on 100 mol m~ 3 NaCl) the sacculi of the mitochondria were swollen and their number per organelle was lower than in controls, whereas there was no salt effect on the ultrastructure of mitochondria in a salt-tolerant cultivar (Smith et al., 1982). Visible changes in the sacculi (Fig. 5) and ER also point to a Ca-deficiency (Fig. 7; Koyro and Stelzer, 1988) 704 Koyro and may also be an indication of increased respiratory activity. Ca is often discussed in connection with membrane integrity, cell wall composition and salt tolerance (Greenway and Munns, 1980; Lynch and Lauchli, 1988). The structural changes observed in mitochondria within epidermal cells could therefore be caused by a Ca-deficiency in the matrix. The number of mitochondria was higher in the epidermal and cortical cells of Sorghum plants treated with NaCl than in the controls. K-deficiency and NaCl salinity in salt-sensitive species can also lead to an increase in the number of microbodies as well as to an increase in the number of mitochondria (Hecht-Buchholz et al., 1971; Hecht-Buchholz, 1983; Walker and Taiz, 1988). The saltinduced increase in F-ATPase activity in Sorghum roots corresponds to the increase in the number of mitochondria in the cortex cells and, therefore, suggests an additional supply of energy for osmotic adaptation and for selective uptake and transport processes (Koyro et al., 1993). Salinity does not change the amount of plastids in all three cell types and, therefore, has no influence on the interpretation of changes in the F-ATPase activity. Koyro and coworkers discuss the high energy requirement of salt-stressed plants, explaining it as the consequence of the very selective uptake of K over Na (SKNa) and of osmotic adaptation to organic compounds. Especially 0.5 mm behind the root tip (Fig. 7a-c) organic compounds seem to be necessary for the osmotic adaptation in the cytoplasm and in the vacuoles of root cells. The salt-induced increase in the number of mitochondria and the swollen christae, when taken together with the increase of the F-ATPase activity (Koyro et al., 1993) as documented, points to increased availability of metabolic energy or to lower metabolic activity per mitochondrion. Sorghum has very efficient selective mechanisms for the avoidance of a Na-enrichment in the symplast (Koyro and Stelzer, 1988, Pei and Zhang, 1995). The export of sodium, against a gradient of the Na-concentration, from the cytoplasm into the nutrient solution, into vacuoles, or into xylem element vessels against a gradient of the Na-concentration is an energy consuming process. As shown for the halophyte Spartina townsendii, the tolerance of high Na concentrations in leaf, shoot or root minimizes the energy demand. In contrast to Spartina, the salt-excluding Sorghum species needs metabolic energy for the osmotic adaptation to organic substances, for a highly selective K-uptake and probably for the recirculation of Na from the shoot to the root. Zea mays showed a lower P level in mitochondria of root cells after salt stress (Stelzer, 1984). In the saltsensitive sorghum, bean and cotton, salinity leads to a decrease in cytoplasmic and vacuolar P concentrations of root tip cells probably as a result of leakage due to increased membrane permeability (Greenway et al., 1992; Karaki et al., 1995; Koyro, 1989; Martinez and LauchJi, 1993). It is possible that P-concentration limits the ATPase activity and the salt tolerance of sorghum. Further salinity experiments are needed with varying P supply to investigate its influence on the salt tolerance in the root tip. To summarize: the results show ultrastructural changes caused by an extreme energy-consuming Na-avoiding mechanism with extremely high energy consumption, as well as a decrease in cytoplasmic concentrations of essential elements such as Mg, P, S, and Ca, thus causing the ultrastructural degradations, indicated, either directly (via energy supply in mitochondria, homeostasis, selectivity of K over Na) or indirectly (via enzyme conformation, cytoplasmic hydration). The shown physiological and structural changes demonstrated have the potential to provide a highly valuable means of detecting early stages of NaCl stress, and may also provide opportunities for screening different varieties for their adaptation to salinity. Acknowledgements We gratefully acknowledge the permission of Professor H Moor (ETH, Zurich) to use the high-pressure apparatus in his laboratory. We thank Drs W Kaeser and R Stelzer for critical discussions and Dr G Collier for checking the manuscript. 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