Avoidance of sodium accumulation by the stomatal guard cells of the halophyte Aster tripolium

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Journal of Experimental Botany, Vol. 48, No. 38, pp. 77-711, March 1997 Journal of Experimental Botany Avoidance of sodium accumulation by the stomatal guard cells of the halophyte Aster tripolium L.

1 Journal of Experimental Botany, Vol. 48, No. 38, pp , March 1997 Journal of Experimental Botany Avoidance of sodium accumulation by the stomatal guard cells of the halophyte Aster tripolium L..R.R. Perera 1, D.L.R. De Silva 2 and T.A. Mansfield Institute of Environmental and Biological Sciences, Division of Biological Sciences, Lancaster University, Lancaster LA14YQ, U Received 19 April 1996; Accepted 2 September 1996 Abstract X-ray microanatysis has revealed that the sodium content of the stomatal guard cells of Aster tripolium remains much lower than that of other leaf cells when the plants are grown at high salinity. Large amounts of sodium did, in contrast, accumulate in epidermal and subsidiary cells, and particularly in the mesophyll tissue, suggesting that a mechanism exists to limit the extent of its entry into guard cells. Even in plants grown at high salinity, the content of potassium was much higher than that of sodium in the guard cells, consistent with the view that this is a major ion involved in determining stomatal movements in this halophyte. Determinations were also made for the nonhalophyte Commelina communis, and it was found that the guard cells accumulated large amounts of sodium when it was presented to them as an alternative to potassium. It is suggested that the acquisition by the guard cells of some ability to restrict the intake of sodium ions may be an important component of sodium-driven regulation of transpiration, and hence of salinity tolerance, in A. tripolium. ey words: Salinity tolerance, sodium, potassium, stomata, Aster tripolium. Introduction The guard cells of the halophyte Aster tripolium L. have unusual ionic responses. The stomata on isolated epidermis open when they are supplied with potassium ions, and in this respect they resemble those of glycophytes. They do, however, close when presented with sodium ions at concentrations (around 5 mol m~ 3 ) likely to be found in the apoplast of leaves when plants are grown under saline conditions (Perera et ai, 1994). In the glycophyte Commelina communis L., the stomata of which have been extensively studied, irreversible opening and eventual damage to the guard cells occurred when the stomata were presented with + instead of + (Jarvis and Mansfield, 198; Clint, 1984). It has previously been suggested by this laboratory that the response to + in A. tripolium is important in controlling transpiration, and hence the rate of delivery of salt to the leaves. There could thus be a 'top-down' regulatory mechanism which, it is hypothesized, would operate in this way: when the capacity of the shoot tissues to accumulate salt in cell vacuoles is exceeded, there is an increase in the + concentration in the apoplast, including that around the guard cells; this causes partial stomatal closure and reduces transpiration, resulting in increased water use efficiency and restricting the flow of salt into the leaves (Perera et ai, 1994). A knowledge of the distribution of ions in the epidermis and other tissues, and the relative concentrations of ions in different cells, was necessary to give a clearer picture of the relevance of the unusual stomatal behaviour of A. tripolium. The results are reported here of X-ray microanalysis of tissues and cells of plants grown with or without 3 mol m~ 3 Cl in the rhizosphere, suggesting that the guard cells have acquired some ability to discriminate between + and +, although + is not totally excluded. Materials and methods Seeds of A. tripolium L. were germinated by placing them on closed plastic Petri dishes lined with filter papers moistened 1 Present address: Division of Botany, Open University of Sri Lanka, wala, Nugegoda, Sri Lanka. 2 To whom correspondence should be addressed. Fax: Oxford University Press 1997 Downloaded from on 28 November 217

78 Perera et al. with distilled water, and placed in a growth room (day/night temperature 24/2 C, 16 h day length at a quantum flux density of 5/i.mol m~ 2 s" 1 ) for 7 d.

2 78 Perera et al. with distilled water, and placed in a growth room (day/night temperature 24/2 C, 16 h day length at a quantum flux density of 5/i.mol m~ 2 s" 1 ) for 7 d. They were subsequently grown in vermiculite in full strength Hoagland's solution (Hoagland and Arnon, 1938) under the same growth room conditions and 35% minimum relative humidity for 5 weeks. Salinity was then imposed on half the plants at a rate of 5 mol m~ 3 d" 1 by adding Cl to the nutrient solution to give a final concentration of 3 mol m~ 3. Controls consisted of plants maintained at the basic nutrient solution without salt additions. Treatments were continued until the plants were about 8-weeks-old. Preparation of the specimens for X-ray microanalysis 5x5 mm 2 pieces of fresh material from the youngest fully expanded leaf of the salt-treated and control plants of A. tripolium were mounted in the grooves of aluminium specimen holders using Tissue Tek II OCT compound (Miles, perville, Illinois, USA). The mounted specimens were then quench frozen in nitrogen slush (temperature < 21 C), and stored in liquid nitrogen until they were planed in an ultramicrotome (Reichert Jung, FC 4E) at a temperature of -13 C to give a flat surface. After planing they were stored in liquid nitrogen in a jar kept in a dewar filled with liquid nitrogen. The planed frozen specimens were transferred to the scanning electron microscope (SEM) (Jeol JSM 84A) without contact with the atmosphere, and were examined at a temperature lower than 145 C. The residual water present in the form of ice was removed by freeze etching the specimens for 2 min at 87 C. This procedure, which was routinely carried out for all the specimens only to dry the surface contamination, does not carry a great risk of recrystallization of ice within the samples (Robards and Sleytr, 1985). After sublimation the samples were cooled to 145 C. Furthermore, the heating and cooling of the specimens were done very rapidly to make sure that this procedure is 'recrystallization safe' (Robards and Sleytr, 1985). The specimens were then given a uniform coating (5 nm) of aluminium to provide stability under the electron beam. X-ray microanalysis was performed in the SEM equipped with a Link LZ5 detector and a Link system 86 series II analyser. The accelerating voltage was 15 kv. Analysis was carried out by repeated scans across an area of 1 ^m 2 in the vacuolar region for a 2 s preset time. Different cell types in the cross-section were scanned for sodium, potassium and chlorine. Each cell was scanned in three different places (in the middle and at either end). The net counts were expressed as percentage relative to net aluminium count, which was considered as 1% (Huang et al., 1994). Abaxial epidermal tissues were also examined under the SEM for the distribution of the above elements. In this case the epidermal peels were washed in deionized water and mounted cuticular surface upwards on to the polished surfaces of carbon stubs using thin layers of Tissue Tek II OCT compound; they were then immediately frozen in N 2 -slush, and used for X-ray microanalysis after coating with aluminium. For comparison with A. tripolium, isolated epidermal peels of C. communis were used to determine the distribution of sodium, potassium and chlorine when they were presented with Cl or G. Here the abaxial epidermal pieces (5x5 mm) were incubated for 3 h under CO 2 -free air in 1 mol m~ 3 MES buffer (ph adjusted to 6.15 with either OH or OH appropriately) containing 5 mol m~ 3 G or Cl. After the incubation the epidermal pieces were washed in distilled water and prepared for X-ray microanalysis in the same manner as the epidermal peels of A. tripolium. It is known that the X-ray counting efficiency of sodium is lower than that of potassium or chlorine (Van Steveninck et al., 1982) and, therefore, the relative counting efficiencies were determined by analysing frozen agar standards containing known proportions of sodium, potassium and chlorine. The counting efficiencies for potassium and chlorine were close to unity, while that for sodium was very low. Thus, a correction factor of x 13 was applied for all the counts for sodium. Results Tables 1 and 2 show the mean net X-ray counts together with the percentages (ratioed against aluminium, i.e. as 1%) for sodium, potassium and chlorine. Three measurements were made on each cell and the mean net counts given in Tables 1 and 2 represent at least 12 separate determinations. As can be seen in Table 2, percentage X-ray counts for in guard cells of plants grown in 3 mol m~ 3 Cl were consistently much lower than those for the subsidiary cells or epidermal cells (this species does not have any anatomically distinct subsidiary cells, but the term subsidiary is used here for convenience to denote the cells immediately adjacent to the guard cells, while epidermal cells are those which are not attached to guard cells) this difference being even more pronounced in leaf sections than epidermal strips. so the total X-ray counts for, and particularly Cl in the subsidiary and epidermal cells of the leaf sections were higher than those of the isolated epidermis. This may be because the use of leaf sections allows the analyses to be carried out more satisfactorily in the interior of the cells. On the other hand, lower X-ray counts obtained for epidermal peels may be due to the fact that a certain amount of these elements was lost during the preparation of the specimens, as they were washed thoroughly in deionized water. Furthermore, it is prudent not to make any direct comparison on the X-ray counts obtained from leaf sections with those from epidermal strips. In plants grown in 3 mol m~ 3 Cl, the proportional increase in percentage sodium counts in epidermal and subsidiary cells with respect to guard cells was about 2-3-fold in epidermal strips, while in leaf sections the increase was about 4-5-fold in upper epidermis and 18-fold in lower epidermis. The most striking feature of the data is that in leaf epidermis + ions are mostly confined to the epidermal and subsidiary cells, and to a large degree are excluded from the guard cells. Chloride seems to be the major balancing anion in the leaves at high salinity: X-ray counts for Cl in guard cells were, however, generally lower than those for. This suggests that an organic anion such as malate may balance the + content of the guard cells, as in some other species (Assman and Zeiger, 1987). For control plants that had not been exposed to salinity the analyses revealed that in all cell types there was very little sodium or chloride and seemed to be the major cation in all the cells examined in the leaves (Table 1). Downloaded from on 28 November 217

3 Table 1. X-ray microanalvsis data of A. tnpolium grown with no added Cl Counts shown for sodium are the values after correction for counting efficiency. Salinity tolerance in Aster tripolium 79 a counts ± SE counts+ SE 19±13 8±5 15±9 1±1 6± ±4 23±9 13±3 16O± ± ± ± ± ±68 136O± ± ±63 346± ±8 11±5 86±44 14±3 16±6 91 ±42 1±4 54±21 19±6 4±9 16±7 18± Table 2. X-ray microanalvsis data of A. tripolium grown with 3 mol m l Counts shown for sodium are the values after correction for counting efficiency. Cl Cl 73O±19O 337 ±33 382±31 812±53 351± ±32 22 ±3 415O±9O 458 ±55 41± 1 64±14 85O±19O ± O±18 25 ±15 338± ±13 887O±116O 619±6O 79 ±2 142 ±56 314±56 124±19 119± ± ± O± ± ± ±78 471± ± ± ±1 145 ±85 176± In plants grown with 3 mol m 3 Cl, all cells displayed increases in the : ratio, but the rise was less marked in the guard cells than elsewhere (Table 3). It appears that the guard cells may be more successful than most other cell types both in retaining and in partially excluding during exposure to high salinity. The percentage X-ray counts for sodium in mesophyll cells were also high in salt-treated plants (Table 2). This implies that the mesophyll cells of Cl-treated plants absorb and retain, presumably for osmoregulatory purposes, some of the salt transported to the leaf tissue via the xylem. For comparative purposes, some determinations for Commelina communis were made, but because this is a glycophyte the plants could not be grown in saline conditions. Therefore epidermal peels were treated directly with 5 mol m~ 3 C1 or Cl and the contents of, and Cl after 3 h incubation were determined (Table 4). When + was made available to guard cells of C. communis they accumulated it to a point where the + concentration was more than 3 times that of +, even though there appeared to be an abundant supply of + available in most of the other cells of the epidermis. Discussion A large part of our knowledge of the ionic relations of stomatal guard cells is derived from studies of two species, Downloaded from on 28 November 217

4 71 Perera etal. Table 3. / ratios of different cells of leaf tissue of Aster tripolium grown with no added sodium or 3 mol m~ 3 Cl Omol nt 3 Cl molm- 3 CI 85 I Commelina communis and Vicia faba. These are both glycophytes, and when their stomata open, the guard cells accumulate + ions, the balancing of which is shared by Cl" and malate, the latter making a substantial contribution if Cl~ is in short supply (Tallman, 1992). In C. communis, studies using isolated epidermis have shown that substitution of an external supply of + with +, in whole or in part, can disrupt the ability of stomata to close in response both to environmental signals (darkness and CO 2 ) and to abscisic acid (Jarvis and Mansfield, 198). If the supply of + to the guard cells is sufficiently prolonged they are eventually irreversibly damaged (Clint, 1984), and the data here (Table 4) suggest that an inability to exclude may be responsible. There may even be a preference for + over +, because the data in Table 4 indicate that large concentrations of + were available in the surrounding cells. These supplies of + in subsidiary and epidermal cells are those normally drawn upon by the guard cells of this species, for they drop dramatically when stomata open (Penny and Bowling, 1974). It is clear that the stomata of halophytes must have evolved mechanisms to overcome the disabling effect of + that occurs in C. communis. In Cakile maritima L. and Suaeda maritima L. (Eshel et al., 1974; Flowers et al., 1989) the stomata appear to be able to use + alongside +, or as an alternative to +, for driving the turgor changes in the guard cells. In the case of C. maritima, Eshel et al. (1974) found using X-ray microanalysis that when plants had been grown on Hoagland's nutrient solution, during stomatal opening potassium accumulated in the guard cells which were almost sodium-free. In contrast, plants grown in the presence of Cl contained high amounts of sodium in the guard cells and the content of potassium had dropped to a very low level. This suggests there is a facultative mechanism which can adapt according to the ionic status of the leaves. The situation found in A. tripolium is clearly different. The guard cells accumulate some sodium when the plants are grown at high salinity, but does not completely replace. The proportional increase in sodium within the guard cells was smaller than that in the neighbouring cells of high salinity plants. It is known from previous studies (Perera et al., 1994) that supplies of Cl to the guard cells strongly inhibit stomatal opening in A. tripolium, and the present results suggest that this effect occurs despite the entry of some + into the guard cells. The data enable us to suggest that the guard cells function essentially like those of glycophytes, making use of potassium and associated anions to regulate their turgor. Sodium ions do not replace potassium, but the guard cells have acquired the important ability to detect the concentration of sodium adjacent to or inside them, and respond in a manner which regulates water relations and the further uptake of salt into the shoot. Acknowledgements LRR Perera thanks the Open University of Sri Lanka for leave of absence, and the Association of Commonwealth Table 4. X-ray microanalysis data of different cells of lower epidermis of Commelina communis incubated for 3 h in the presence of either 5 mol m' 3 Cl or Cl 5 mol m" 3 Cl Guard cell Inner lateral subsidiary cell Outer lateral subsidiary cell Terminal subsidiary cell Epidermal cell 5 mol m" 3 Cl Guard cell Inner lateral subsidiary cell Outer lateral subsidiary cell Terminal subsidiary cell Epidermal cell 42 ±15 5l± ±8 27±14 676± ± ±48 23 ±5 238 ± ± ±18 478± ± ± ±29 17± ±49 65 ± ± a ± ±42 4± ± ± ±26 34OO±35O 421± Downloaded from on 28 November 217

Universities for a scholarship, and we are grateful to Dr Andrew Malloch for advice. References Assman SM, Zeiger E. 1987. Guard cell bioenergetics. In: Zeiger E, Farquhar GD, Cowan 1R, eds.

5 Universities for a scholarship, and we are grateful to Dr Andrew Malloch for advice. References Assman SM, Zeiger E Guard cell bioenergetics. In: Zeiger E, Farquhar GD, Cowan 1R, eds. Stomatal function. Stanford University Press, Clint G Ionic relations of stomatal guard cells. PhD thesis, University of Cambridge, U. Eshel A, Waisel Y, Ramati A The role of sodium in stomatal movements of a halophyte: a study by X-ray microanalysis. In: Wehrmann J, ed. Proceedings of the 7th international colloquium on plant analysis and fertilizer problems. German Society of Plant Nutrition, Hannover. Flowers TJ, Hajibagheri MA, Leach RP, Rogers WJ, Yeo AR Salt tolerance in the halophyte Suaeda maritima. In: Plant water relations and growth under stress. Proceedings of the Yamada Conference XXII, Osaka, Japan, Hoagland DR, Aroon DI The water culture method for growing plants without soil. Circular of California Agriculture Experimental Station 347, and Report of Smithsonian Institute 193, 461. Miscellaneous publication no. 3514: Salinity tolerance in Aster tnpolium 711 Huang CX, Canny MJ, Oates, McCully ME Planing frozen hydrated plant specimens for SEM observation and EDX microanalysis. Microscopy Research and Technique 28, Janis RG, Mansfield TA Reduced stomatal responses to light, carbon dioxide and abscisic acid in the presence of sodium ions. Plant, Cell and Environment 3, Penny MG, Bowling DJF A study of potassium gradients in epidermis of intact leaves of Commelina communis L. in relation to stomatal opening. Planta 119, Perera LRR, Mansfield TA, Malloch AJC Stomatal responses to sodium ions in Aster tripolium: a new hypothesis to explain salinity regulation in above-ground tissues. Plant, Cell and Environment 17, Robards AW, Sleytr UB Low temperature methods in biological electron microscopy. Amsterdam, Elsevier. Tallman G The chemiosmotic model of stomatal opening revisited. Critical Reviews in Plant Sciences 11, Van Steveninck RFM, Van Steveninck ME, Stelzer R, Lauchli A Studies on the distribution of and Cl in two species of lupins {Lupinus luteus and Lupinus angustifohus) differing in salt tolerance. Physiologia Plantarum 56, Downloaded from on 28 November 217

Estimations of apoplastic concentrations of K"^ and Ca^+ in the vicinity of stomatal guard cells

New Phytol. (1996), 134, 463-469 Estimations of apoplastic concentrations of K"^ and Ca^+ in the vicinity of stomatal guard cells BY D. L. R. D E S I L V A, SARAH J. HONOUR AND T. A. MANSFIELD* Division