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

Transcription

1 New Phytol. (1996), 134, 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 of Biological Sciences, Institute of Environmental and Biological Sciences^ Lancaster University,, Lancaster LAI 4YQ, UK {Received 19 September 1995; accepted 27 June 1996) SUMMARY A method which determines the null point for stomatal aperture has been used to estimate the apoplastic concentrations of potassium and calcium adjacent to the stomatal guard cells of Commelina communis. These two ions are contributors to important aspects of stomatal physiology : the determination of guard cell turgor (K^) and intracellular signalling (Ca^^) when the guard cells respond to the various stimuli that effect changes in stomatal aperture. We obtained estimates of apopiastic K^ concentrations in the range of mol m"^, which are in general agreement with those of Bowling (1987). Ca^^ concentrations appear to be in the region of 0-05 mol m~^ adjacent to the guard cells even though much higher concentrations (up to 4 mol m~^) may be delivered to the leaf in the xylem sap. Thus it is suggested that the gradient in apoplastic Ca"^ may be very large over short distances, and may be strictly controlled by cells within the epidermis. Key words: Commelina communis, apoplast, calcium, potassium, stomata. 60 % of the exposed surface. Small drops of glassdistilled water, or of 1 mol m"^^ CaClg solution, were Movement of potassium ions into and out of guard placed in the depressions and potassium activity was cells plays a major part in achieving the osmotic measured using K-sensitive microelectrodes. The changes that are required for stomatal movements potassium activities determined in this way were (Tallman, 1992), and changes in the concentration of surprisingly low, being in the region of 50 mmol m~^. free calcium in the cytosol of the guard cells are Bowling (1987) also used K-sensitive microbelieved to be involved in the signal transduction electrodes pressed into the cell walls just beneath the sequence that leads to an efflux of K"^ and stomatal cuticle of the abaxial epidermis of Commelina closure (McAinsh, Brownlee & Hetherington, 1990; communis. He found K"*^ activities ranging from 3 to 100 mol m~^ depending both on the precise position Assman, 1993 ; Ward & Schroeder, 1994). Studies of guard cells have assumed considerable importance in of the electrode above the stomatal complex, and on recent years because the accessibility of stomata whether the stomata were open or closed. C. within the leaf epidermis, and also the ease with communis was one of the three species used in Blatt's which changes in aperture can be measured, allow determinations and there was thus a 1000-fold investigations of processes that would be very elusive discrepancy between the two methods. There are no plasmodesmatal connections between in most other plant tissues. Although much is now known about intracellular concentrations of K"^ and mature guard cells and the adjacent subsidiary cells; Ca^"^ within the stomatal complex, there remains consequently ions must enter and leave these cells considerable uncertainty about the apoplastic con- via the apoplast. When stomata of C. communis are centrations. Blatt (1985) used glass capillaries to cut wide open, the K concentrations within the guard through the epidermis and create a depression cells have been estimated to be as high as fim. in diameter bordered by epidermal and 600 mol m"^, falling to below 100 mol m"^^ when the mesophyll cells, the epidermis contributing up to stomata close (MacRobbie, 1987). There has been much research on the stomata of C. communis using isolated epidermis, and 'normal' stomatal behaviour * To whom correspondence should be addressed. (e.g. expected responses to light, COg and abscisic INTRODUCTION t.mansfield(s) lancaster.ac.uk

2 464 D. L. R. De Silva, S. J. Honour and T. A. Mansfield acid) is only found when K^ concentrations in the bathing medium are in the region of 50 mol m"^ (Travis & Mansfield, 1979). If this requirement is at all indicative of the way in which availability of K"^ influences stomatal behaviour in vivo, then Bowling's determinations appear more appropriate. Blatt estimated that his procedure damaged cells adjacent to the depression in which his measurements were made, and it can be suggested that this wounding of the tissue might have led to a major change in the ionic relations of the cells. In this paper we describe attempts to estimate apoplastic K^ and Ca^"^ concentrations using a simple null point method. When the abaxial epidermis is stripped carefully from leaves of C. communis on which the stomata are open, those stomata remain open to approximately the same extent after the epidermis has been placed in an appropriate incubation medium. We have used media with a range of K"^ and Ca^^ concentrations and have determined whether the stomata open or close, and for each cation have estimated a 'null point' at which no change in aperture would have occurred. We report here some preliminary results obtained using the technique, and discuss the strengths and weaknesses of the experimental approach. ing ambient air through soda lime, was fed into the chamber. The incubation tank was filled with distilled water, maintained at 25 C, ensuring that the relative humidity within the chamber was > 85 % (during incubation of epidermal strips, relative humidity around the strips approaches 100%). Incubations and determinations of stomatal apertures After 2 h in the pre-incubation chamber, the abaxial epidermes were quickly removed according to the method described by Weyers & Travis (1981). Epidermal strips were cut into 5x10 mm pieces and floated on 10 mol m"^ MES (2-[N-morpholino] ethanesulphonic acid) (Sigma, Poole, Dorset, UK) adjusted to ph 6-15 using KOH. Immediately after detachment, one strip from each leaf was mounted in MES buffer on a microscope slide, and 15 randomly selected apertures were measured using a Leitz Periplan microscope fitted with a projection facility. The other epidermal strips were then incubated in 5-cm closed Petri dishes containing a range of KCl and/or CaCU concentrations in 10 cm^ of 10 mol m~^ MES, adjusted to ph 6-15 using KOH. The temperature of the water bath was maintained at 25 C and photon flux density beneath the Petri dish lid was 290/imol m"^^ s"^. COg-free air was supplied to each dish at a rate of 100 cm^ min~^ via hypodermic MATERIALS AND METHODS needles dipping into the medium. After 1 and 3 h, three strips of epidermis (each from a difterent leaf) Plant material were removed from each dish, and 15 stomatal Commelina communis L. was grown from seed in a apertures (randomly selected) were measured on heated glasshouse (minimum temperature 20 C, each strip. All experiments were conducted on three day length 16 h), in Fisons' Levington compost for separate occasions and thus there were wk, until the third true leaf was beginning to measurements of aperture for each data point. expand. During this period the seedlings were irrigated with tap water containing no added RESULTS nutrients. The plants were then transferred to a controlled environment room (25 C, day length Mean abaxial stomatal aperture on the excised leaves 16 h, r.h. 6 5 %, quantum flux density after 2 h in high humidity, high irradiance and COg250 /^mol m~^ s~^) and kept well watered throughout. free air at C lay between 12 and 14 /im on all Twice a week they were watered with Long Ashton occasions. Mean stomatal apertures and also the nutrient solution, as described by Hewitt (1966). percentage changes from the initial value after 1 and 3 h incubation were plotted against the concentration of KCI. Pre-incubation After both 1 and 3 h in high KCl concentrations When the plants were 4 wk old, the youngest fully (100 mol m~^ or more) stomata opened more widely expanded leaf from each of six plants was excised, than the initial value. At KCl concentrations below and the petiole submerged in distilled water in a 60 mol m~^, stomatal aperture narrowed. Second specimen tube. The excised leaves were then kept for order regressions were fitted to both sets of data and 2 h in pre-incubation conditions designed to en- the KCl concentration effecting no change in courage stomatal opening and to be as close as stomatal aperture was about 60 mol m"^ after 1 h of possible to the conditions subsequently experienced incubation (Fig. 1 a). Continued incubation up to 3 h in the epidermal strip bioassay. The leaves were moved the null point to around 75 mol m~^ KCl placed on top of an incubation tank identical and (Fig. 1 b). Although apoplastic calcium levels have adjacent to the bioassay tanks, beneath a transparent also been implicated in stomatal movements in Perspex propagation cover which transmitted C.communis, there was no added calcium in the 86% of the incident quantum flux density incubation medium. Therefore a factorial experi(320 ^mol m"^ s"^). COg-free air, obtained by pass- ment was carried out to determine the effects oi

3 Apoplastic K* and Ca^^ concentrations near guard cells (a) « KCi concentration (mol m~^> (jb) 15 - ^ I 10 - ya 40-5 r o 20 QJ' 0 S -20 a- S -10 y ^ BD KO concentration mo) m ) 1 r KCI concentration (mol m"^) Figure 1. (a) Response of open stomata to different KCI concentrations in the incubation medium. Mean values of stomatal aperture (K = 135) with 95 % confidence limits (main figure) and percentage changes (inset) from the initial value which is represented hy the broken horizontal line in the main figure. Epidermis of C. communis was incubated in the different KCI concentrations for 1 h. The curves were fitted using a second order polynomial by regression analysis, (b) as above, but after incubation for 3 h. added calcium on the null point for potassium. Initial experiments using mol m"^ CaCl^ indicated that the presence of CaCI^ at concentrations of 0-1 mol ni~' or more resulted in significant stomatal closure relative to the initial nnean aperture. Therefore the experiment was repeated using a smaller range (0 O'lS mol m"^) of CaCi^ concentrations, and the highest concentration of potassium was limited to 100 mol m"^. As can be seen from Figure 2, the presence of added calcium in the incubation medium did not alter the trend of the null point curve significantly, but rather Ca^* moved the intercept between 50 and 75 mol m'' KCI. Therefore, in the subsequent investigation 65 mol m"^

4 466 D.L.R.De Silva, S. jf. Honour and T. A. Mansfield E 3: ro e o KCI concentration (mol Figure 2. Response of open stomata to KCI and CaClj in various concentrations and combinations in the incubation medium. Mean values of stomatal aperture {n = 135) with 95 % confidence limits (main figure) and percentage changes (inset) from the initial value which is represented by the broken horizontal line in the main figure. (#, O, no added calcium;, D, 0-05 mol m'^ CaCl^; A, A, 0-10 mol m"^ CaCll.,; V, 0-15 mol ~ t a CO 14-4 ro E o c/ CaCL concentration (mol m"-^ Figure 3. Response of open stomata to different CaCla concentrations in the incubation medium. Mean values of stomatal aperture {n = 135) with 95 % confidence limits (main figure) and percentage changes (inset) from the initial value which is represented by the broken horizontal line in the main figure. Epidermis of C. communis was incubated in 65 mol m~^ KCI for 3 h with different concentrations of CaCla. For each point n = 135. The curves were fitted using a second order polynomial by regression analysis.

5 Apoplastic K'^ and Ca^* concentrations near guard cells KCl was added to the MES in the basic incubation medium, in the behef that this was a realistic apoplastic KCl concentration against which to assess the action of CaClj. on stomatal opening. After incubation for 1 h, no clear effect of CaClj on stomatal aperture was seen but incubation of peels for 3 h in the absence of any added CaClj (MES + 65 mol m"'"' KCl only) resulted in an increase in stomatal aperture of about 20% (Fig. 3), and the range of Ca^* concentrations led to responses that enabled a clear null point to be determined when a second order polynomial was fitted to the data. The value at which no change in aperture occurred was mol m~^ CaClj. It is important to emphasize that the zero point on the abscissa of Figure 3 refers to an absence of additional CaCl,. We were not able to exclude some contamination of water and reagents, which could have been up to 0'02 mol m~' Ca"" (A. M. Hetherington, pers. comm.). DISCUSSION The solute concentrations in the apoplast are determined by the balance of import from the xylem, absorption by the cells and export by the phloem (Grignon & Sentenac, 1991). By removing the epidermis from the leaf (resulting in cessation of communication with xyiem or phloem) it was our objective to ' freeze ' the solute content of the apoplast which occurred in the intact leaf immediately before peeling. The data obtained therefore refer to the specific conditions we imposed, which were highly favourable for stomatal opening. The technique we used for carefully stripping the epidermis does not rupture the cells and it is unlikely that release of cell contents would be a problem. Stomata responded to changes in the ionic composition of the incubation medium by opening or closing. An important question to be addressed is whether changes in stomatal aperture are acceptable indicators of changes in apoplastic solute concentrations. We make no claim that apoplastic K* concentration is the sole or even the main determinant of stomatal aperture, but evidence clearly suggests that it does have a very significant influence (Travis & Mansfield, 1979). A premise upon which the widely used epidermal strip bioassay is based is that incubation medium is continuous with the apoplastic solution, and it is well established that manipulation of its composition can result directly in measurable changes in stomatal aperture (Snaith & Mansfield, 1982). It therefore appears justifiable to assume that changes in stomatal aperture can be driven by changes in apoplastic solute concentrations. Previous data have reported increased stomatal opening in C. communis in response to increasing K* concentration (Travis & Mansfield, 1979) and a reduction in opening in response to increasing Ca^+ 467 concentration (De Silva, Hetherington & Mansfield, 1985; Mansfield, Hetherington & Atkinson, 1990). Here we have found that, starting with open stomata, partial closure occurred in KCl concentrations of up to 50 mol m"^. Further opening did not begin to occur until the incubation solution contained more than 75 mol m~" KCl. The calculated 'null point', i.e. the concentration at which the K+ concentration in the incubation medium was assumed to' be sufficiently close to that of the apoplast to effect no change in stomatal aperture, was c. 65 mol m"^. This falls in the middle of the range of mol m"* estimated by Bowling (1987) and it corresponds to a K* concentration that would support about 90 % of maximal stomatal opening, interpolating the data of Travis & Mansfield (1979). Bowling concluded that there were higher apoplastic K* concentrations when stomata %vere closed than when they were open, and our estimate is higher than his value for open stomata. It would, however, be unwise to place great emphasis on this discrepancy because there may have been differences in age and nutritional status of the leaves. As can be seen from Figure 2 the null point for potassium when there was no added calcium in the medium is considerably lower than that obtained from Figure 1 b. This suggests that on another occasion, even though the plant material was supposedly the same, the apopiastic K* concentration at the outset was substantially lower than 75 mol m"^. There is ciearly a dynamic situation in the K+ concentration in the apoplast around the guard cells, and perhaps the point worth emphasizing is that our estimates are within the range determined by Bowling (1987) and are three orders of magnitude above the values obtained by Blatt (1985). Canny (1993) pointed out that although diffusion does take place within the apoplast, it occurs only very slo\vly, with solutes moving at only a fraction of their rate in water. Most exchange between the apoplast and the incubating medium wil! occur when the concentration gradient between them is highest. In the case of our determinations for Ca'* these gradients were probably much lower than those for K*. Furthermore, Ca^"^ ions are significantly less mobile than K* ions in plant cells because they may be bound b;' cell walls (Assmann, 1993). These factors may explain why after only 1 h of incubation no relationship was observed between the Ca^* concentration in the medium and stomatal aperture. Nevertheless, the presence of calcium in the apoplast appears to be essential for the promotion of closure either by darkness or by ABA (Schwartz, 1985; Atkinson et al., 1989) and concentrations above O'l mol m~^ can themselves cause stomatal closure in the light (De Silva et al, 1985), Although the relationship between apoplastic and cj'tosolic Ca^* is probably less straightforward than for K*, there is no doubt that increasing apoplastic Ca^* causes closure

6 468 D. L. R. De Silva, S. J. Honour and T. A. Mansfield of open stomata. Schwartz (1985) found that having pre-incubated detached leaves of C. communis in tap water for 2 h, subsequent incubation of abaxial epidermal strips in 1 mol m~^ CaCl, reduced aperture to < 90%. Application of 0-1 mol m~'^ CaClg was less effective, although it still closed the stomata partially, but 0-01 mol m"^^ CaCl, had no measurable effect on stomatal aperture. De Silva et al. (1985) found that 0-1 and 0-5 mol m~^ CaClg reduced abaxial stomatal aperture of C. communis to 66 % and 25 %, respectively, of the control value. We report similar responses in the current data, with CaClg concentrations of mol m~^ or more causing reductions in stomatal aperture. However, significant stomatal opening was observed at concentrations of less than 0"05 mol m~^. Since the only variable in the experiment was the Ca""^ concentration, it is most likely to have been a depletion of apoplastic Ca^"^ which allowed the stomata to open more widely than the initial value, strongly implying that the apoplastic Ca^"^ concentration was higher than mol m~^. The fitted value at which it was estimated no change in stomatal aperture would occur was c mol m~^. This figure, though suggested as a highly conservative estimate, is also compatible with data such as those of Willmer & Mansfield (1969) and Atkinson et al. (1989), who reported that Ca^^ concentrations of at least mol m~^ were required to inhibit stomatal opening. McAinsh et al. (1995) have used C.communis to study the oscillations in cytosolic calcium (which are known to be associated with stomatal closure) that are induced by increases in Ca^"^ in the bathing medium. No oscillations occurred with 0-01 mol m"^ Ca^"^, but an increase to 0-1 mol m~^ did induce them. Our estimate of in vivo apoplastic Ca^^ is thus also compatible with these findings. The fact that significant stomatal opening of up to 20% was observed in the absence of added CaClg indicated that any stomatal closure recorded was not a result of cell damage during peeling. External Ca^"^ is believed to cause a reduction in stomatal aperture via its elevation of cytosolic free Ca^"^, which is involved in the signal transduction cascades leading to a loss of guard cell turgor (McAinsh et al., 1995). It might be argued that the null point method used here would be inappropriate if a minimum external Ca^"^ concentration were required to override the homeostatic mechanism that maintains a stable cytosolic Ca^^ concentration. We suggest that this is unlikely to have led to misinterpretation because there was no evidence of any discontinuity in the plot of stomatal aperture vs. Ca^^ concentration, i.e. there was no indication of a threshold concentration above which stomatal closure was induced. We do not suggest that the data we have obtained reflect the action of K"^ and Ca^"^ alone. As it was always an accompanying counter ion, inevitably C r concentrations varied as well as those of cations. The contribution of CA" per se to changes in stomatal aperture is not known. Other cations such as Na^ and Mg"^"^, and various anions are present in the apoplast (Canny, 1993), as well as plant growth substances such as auxins, ABA and cytokinins. If these are present in any significant concentrations in the in vivo apoplast, transfer of epidermal strips to a medium where these factors are absent may infiuence stomatal aperture. Perhaps the most important would be ABA; prior to these determinations its accumulation in the leaves was minimized by keeping the plants well watered throughout the growth period. Furthermore, any effect of ABA delivered in the xylem would have been reduced by the 2 h preincubation period in which leaves were fed with distilled water under conditions designed to induce a high transpiration rate. Thus at the time of peeling, apoplastic ABA would have been at a low level. The ph of the incubation medium is also likely to be important and we have here used only one value, viz Edwards & Bowling (1986) found that the apoplastic ph around the stomatal complex of C. communis was between 6 and 7 when the stomata were open, and hence our chosen value is considered to be realistic. It is fully acknowledged that stomata respond in a complex fashion to a wide range of environmental conditions and stimuli, and we do not wish to draw simplistic conclusions from the data presented. Nevertheless, it is indisputably the case that when concentrations of K"^ and Ca^"^ are varied independently in a medium in direct contact with the apoplast adjacent to the guard cells, there are positive and negative changes in stomatal aperture (Fig. 2). We suggest that the null points we have determined provide a useful guide to the magnitude of apoplastic concentrations, but that they should not be regarded as precise estimates. The values determined apply to leaves of a particular age held under conditions favourable for stomatal opening. A primary reason for undertaking these determinations was our continuing interest in the ways in which apopiastic Ca^"^ concentrations are controlled. In C. communis we have found concentrations of free calcium in xylem sap as high as 4 mol m~^ when there is abundant calcium in the rhizosphere (Ruiz, Atkinson & Mansfield, 1993) and we have suggested that deposition of calcium oxalate in epidermal cells is responsible for reducing the apoplastic Ca^"^ concentration as xylem sap traverses the epidermis towards the stomata (Ruiz & Mansfield, 1994). The finding here that apopiastic Ca^"^ might be as low as 0-05 mol m~^ adjacent to the guard cells demonstrates how effective this regulatory mechanism might be. It must also be emphasized that there is no fixed value for apoplastic Ca^^ in the leaf for it might change by more than an order o magnitude, from values of 1-4 mol m~^ adjacent tc

7 Apoplastic K^ and concentrations near guard cells the xylem to < 0-1 mol m ^ near the guard cells, i.e. a linear distance of /*m in some cases. The control of apoplastic Ca^* in the epidermis might be essential to permit its use in intracellular signalling in guard cells in all plant species, but might assume special importance in calcicoles (De Silva & Mansfield, 1994). 469 Grignon C, Sentenac H, ph and ionic conditions in the apoplast. Annual Review of Plant Physiology and Plant Molecular Biology 42: Hew^itt EJ Sand and water culture methods used in the study of plant mineral nutrition. Technical communication No. 22: Commonwealth Agricultural Bureau, Farnham, Bucks. MacRobbie EAC Ionic relations of guard cells. In: Zeiger E, Farquhar GD, Cowan IR, eds. Stomatal Function. Stanford, California: Stanford University Press, Mansfield TA, Hetherington AM, Atkinson CJ Some current aspects of stomatal physiology. Annual Review of Plant ACKNOWLEDGEMENT Physiology and Plant Molecular Biology 41: McAinsh MR, Brownlee C, Hetherington AM Abscisic We are grateful to the Natural Environment Research acid induced elevation of guard cell cytosolic Ca^"*^ precedes stomatal closure. Nature 343: Council for their support of this research, and to Professor McAinsh MR, Webb AAR, Taylor JE, Hetherington AM. A. M. Hetherington for his advice Stimulus-induced oscillations in guard cell cytosolic free calcium. Plant Cell 7: Ruiz LP, Mansfield TA A postulated role for calcium REFERENCES oxalate in the regulation of calcium ions in the vicinity of Assmann SM Signal transduction in guard cells. Annual stomatal guard cells. New Phytologist 127: Review of Cell Biology 9: Ruiz LP, Atkinson CJ, Mansfield TA Calcium in the Atkinson CJ, Mansfield TA, Kean AM, Davies WJ xylem and its influence on the behaviour of stomata. Control of stomatal aperture by calcium in isolated epidermal Philosophical Transactions of the Royal Society of London Series tissue and whole leaves of Commelina communis L. New B 341: Phytologist 111: Snaith PJ, Mansfield TA Stomatal sensitivity to abscisic Blatt MR Extracellular potassium activity' in attached acid: can it be defined? Plant, Cell and Environment 5: leaves and its relation to stomatal function. Journal of Schwartz A Role of Ca^* and EGTA on stomatal Experimental Botany 36: movements in Commelina communis L. Plant Physiology 79: Bowling DJF Measurement of the apoplastic activity of K'^ and c r in the leaf epidermis of Commelina communis in relation Tallman G The chemiosmotic model of stomatal opening to stomatal activity. Journal of Experimental Botanv 38: revisited. Critical Reviews in Plant Sciences 11: Travis AJ, Mansfield TA Stomatal responses to light and Canny MJ The transpiration stream in the leaf apoplast: COg are dependent on KCl concentration. Plant, Cell and vv'ater and solutes. Philosophical Transactions of the Royal Environment 46: Society of London Series B 341: Ward JM, Schroeder JI Calcium-activated K^ channels De Silva DLR, Hetherington AM, Mansfield TA and calcium-induced calcium release by slow vacuolar ion Synergism between calcium ions and abscisic acid in preventing channels in guard cell vacuoles implicated in the control of stomatal opening. New Phytologist 100: stomatal closure. Plant Cell 6: De Silva DLR, Mansfield TA The stomatal physiology of Weyers JDB, Travis AJ Selection and preparation of leaf calcicoles in relation to calcium delivered in the xylem sap. epidermis for experiments on stomatal physiology. Journal of Proceedings of the Royal Society of London Series B 257: Experimental Botany 32: Edwards MC, Bowling DJF The growth of rust germ Willmer CM, Mansfield TA A critical examination of the use of detached epidermis in studies of stomatal physiology. tubes towards stomata in relation to ph gradients. Physiological New Phytologist 68: and Molecular Plant Pathology 29:

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