Interactions of Calcium with Abscisic Acid in the Control of Stomatal Aperture

Biochem. Physiol. Pflanlel1 l86, .B3- 339 (l990) Gustav Fischer Verlag Jena

Interactions of Calcium with Abscisic Acid in the Control of Stomatal Aperture*) C. J. ATKINSON 1), T. A. MANSFIELD!), M. R. McAINSW), A. M. HETHERINGTON I)

c.

BROWNLEE2),

I) Lancaster University, Division of Biological Sciences, Lancaster, U.K. 2) Plymoth Marine Laboratory, Plymoth, U.K.

Key Term Index: stomata, abscisic acid, intracellular calcium, calcium nutrition

Summary It has been recognised for many years that stomatal aperture is influenced by calcium ions. We have recently discovered that the action of abscisic acid on guard cells is dependent upon calcium. In this paper we discuss the interactions of abscisic acid with calcium ions, and in particular assess the contribution which extracellular and intracellular calcium may make to the control of stomatal aperture.

Introduction It has been known for many years that calcium ions have an inhibitory effect on stomatal opening (ILJIN 1957; FUJINO 1967). More recently, experiments with isolated epidermis and isolated guard cells have provided a more detailed picture of the ways in which calcium salts can affect the functioning of stomata (e.g. DE SILVA et al. 1985; SCHWARTZ 1985; INOUE and KA TOH 1987). Because stomatal movements depend on the turgor changes of cells in the stomatal complex, it is important to note that calcium salts also inhibit the swelling of protoplasts isolated from guard cells (FITZSIMONS and WEYERS 1986; SMITH and WILLMER 1988) (Fig. 1). This indicates that the principal effects observed are not dependent on changes in the properties of the cell walls. Much of the research so far published on this topic has been concerned specifically with the effects of calcium salts on the turgor of guard cells, and the possible role of calcium as a factor determining the pattern of stomatal behaviour in the plant as a whole has not received much attention. The long distance transport of calcium, including free calcium ions, is thought to take place by mass flow through the apoplast of the root (excepting, perhaps, the endodermis) and via the xylem (CLARKSON 1984). This means that the rate of delivery of calcium to the leaves should be related to the rate of transpiration. There is convincing evidence that solutes that enter the leaves from the xylem accumulate around the stomatal complexes. This is thought to occur because areas of the epidermis adjacent to the stomata are the termini for liquid water that passes from the xylem into the apoplast of the epidermis (TANTON and CROWDY 1972; MElDNER and SHERIFF 1976; MAIER-MAERCKER 1983). *) Presented at the FESPP-Workshop "Stomata '89", held at Berlin, September 4 to 9, 1989. BPP 186 (1990) 5/6

333

Commelina communis

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Time of day

Fig. 1. Inhibition of stomatal opening and guard cell protoplast swelling of Commelina communis by calcium ions. Isolated epidennal tissue and guard cell protoplast preparations were incubated in a range of calcium ion concentrationss (see DE SILVA, et aI., SMITH and WILLMER, 1988 for details). Fig. 2. Diurnal changes in stomatal resistance for leaves of Commelina communis grown on low and high calcium. The relative stomatal resistance was measured by viscous flow porometry for plants grown on Long Ashton nutrient solution with different amounts of calcium (low, 1 mol m- 3 ; high, 8 mol m- 3 ) . Values shown are the means of three plants per treatment ± one SE.

Research with isolated epidermis of Commelina communis has shown that the concentration range over which calcium inhibits stomatal opening is 0 to 0.5 mol m- 3 . The range of calcium concentrations (not necessarily free Ca2+) measured in xylem sap is generally higher than this. ARMSTRONG and KIRKBY (I979), for example, found concentrations in the range 5-7 mol m- 3 in xylem exudates from tomato. This suggests that the concentrations reaching the vicinity of the stomata could be of significance in determining the physiological state of the guard cells.

Effects of variations in the rhizospheric concentration of calcium We have grown Commelina communis in solution culture with two different rhizospheric concentrations of calcium, namely I and 8 mol m- 3 , and this led to plants with different calcium content (Table 1) , and the amount in the xylem sap rose about 4-fold from 0.18 to 0.82 mol m- 3 , i.e. within the range of the sensitivity of stomata on isolated epidermis to free calcium ions. However, observations of stomatal behaviour on the intact plants by diffusion and viscous flow porometry, and studies of gas exchange characteristics in an environment334

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Table 1. The Ca cOllcelllnllion in tissue of' Commelina communis L. plants grown in nutrient solution at two concentratwns of Ca. Sample description

1 mol m- 3

Bulk leaf total Ca (ftmol g -I dwt) Xylem sap (ftmol cm - 3) Abaxial epidermal tissue (f.tmol g -) dwt)

41

±

1

8 mol m- 3

0.18 ± 0.03 107 ± 25

111

±

4

82 ± 0.10 309 ± 43

controlled cuvette indicated that the stomata of the calcium-rich plants were more not less open (Fig. 2). It was also found that the plants supplied with the greater amount of calcium had produced significantly more biomass, and this may have depended on improved efficiency of carboxylation coupled with greater stomatal opening (ATKINSON et al. 1989b). These results suggest at first sight that the calcium content of the xylem sap does not exert any immediate control over the functioning of stomata, and there appears to be a contradiction with the data from studies of the effects of calcium on isolated epidermis. In an attempt to resolve this question we have examined the effects of injecting different concentrations of calcium ions directly into the xylem. The simplest procedure adopted was to supply detached leaves with a range of calcium nitrate concentrations from 0 to 16 mol m- 3 . In the case of both Commelina communis and Triticum aestivum there was a marked suppression of transpiration as the calcium concentration increased. We also succeeded in injecting 8 mol m- 3 Ca(N0 3 h directly into the xylem (in the mid-vein) of leaves that were still attached to the plant (Fig. 3). This technique had been developed and tested in previous studies in which it was necessary to deliver abscisic acid via the xylem (ATKINSON et al. 1989a). When calcium ions were supplied in this manner there were decreases in stomatal opening that were closely dependant on the duration of application. In the case of C. communis a treatment lasting 1 hour caused a rapid reduction in stomatal aperture, and this was reversed when the supply of calcium ceased. Commelina communis

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Triticum aestivum

5

11

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Time of day

Fig. 3. Typical calciulIl-li/(iuced changes in stomatal conductance for leaves ofColllll1elina communis and Triticum aestivum fed with 8 mol m- 3 Ca(N0 3 h via the xylem. Leaves were mounted within a environmentally controlled gas exchange cuvette and fed calcium through a catheter bleed system, with a hypodermic needle mounted in the mid-rib. Arrows mark the time of initiating the calcium feed. BPP 186 (1990) 5/6

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We also found that applying a solution of Ca(N03h containing a surfactant to the adaxial leaf surfaces of C. communis and T. aestivum caused stomatal closure within an hour comparable to that found in darkness (Fig. 3). A lower concentration of Ca(N03h (8 mol m~3) caused incomplete stomatal closure in both species.

o 24

6

12

18

Time of day

Fig. 4. The change in stomatal resistance for an attached leaf of Triticum aestivum supplied with 8 mol m- 3 Ca(N03h. 0.05 cm 3 of 8 mol m- 3 calcium nitrate in 0.05 % (v/v) tween was applied to the adaxial surface of an attached leaf of wheat, mounted within a viscous flow porometer. Symbols : day 1 before the calcium application (0), day 2 control (0), day 2 after exposure to calcium (_). The arrow marks the timing of the calcium application . .

These experiments showed that stomata on the intact plant are as responsive to changes in the apoplastic concentration of calcium as those on isolated epidennis. At present it is difficult to reconcile satisfactorily data that appear to be contradictory, i.e. wider stomatal opening in calcium-rich plants, but clear evidence that a high concentration of calcium delivered to the leaf via the xylem, or applied to the leaf surface, causes stomata to close. Although we applied concentrations of calcium that were higher than those we have detected in xylem sap, it is necessary to recognise that solutes in the xylem accumulate in the vicinity of the guard cells (TANTON and CROWDY 1972; MAIER-MAERCKER 1983). This means that a long-tenn exposure to a lower concentration might be simulated by the treatments we have applied. It appears that in calcium-rich plants there must either be some restriction upon the amount of calcium reaching the neighbourhood of the guard cells, or alternatively a change in sensitivity at the cellular level. Further studies of stomatal behaviour on plants grown with different supplies of rhizospheric calcium are clearly required. They are likely to lead to advances in our understanding of how cellular responses to subtle movements of calcium ions are achieved without interference from bulk movements of calcium in the plant as a whole. 336

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Intracellular calcium In the preceding section we have discussed the contribution which extracellular calcium may make to the control of stomatal aperture. In the remainder of this paper we will consider how alterations to the concentration of calcium ions in the guard cell cytosol contribute to the regulation of guard cell turgor. The regulation of stomatal aperture through alterations to the cytosolic concentration of calcium ions makes an attractive hypothesis. There is already a considerable body of circumstantial evidence which implicates cytosolic calcium ions in the control of a diverse array of processes in both stomata and other plant cells (HEPLER and WAYNE 1985; MANSFIELD et al. 1990). In addition , it is now becoming apparent that plant cells , like their animal counterparts, contain the components required for an operational signal transduction apparatus based on calcium ions (POOVAIAH and REDDY 1987) . Some of these have also been shown to be present in stomatal guard cells (MANSFIELD et a\. 1990). However, of equal importance, is the fact that a substantial amount of information relating to the control of ion fluxes in stomatal guard cells is already documented (SERRANO and ZEIGER 1989). This means that the concept of a turgor control system based on cytosolic calcium ions can be integrated into a pre-existing physiological framework. As a consequence of this it is easier to test rigorously whether intracellular calcium controls stomatal aperture. Recently we have attempted to test aspects of this hypothesis. Using the fluorescent calcium indicator Fura 2 and dual excitation wavelength fluorescence microscopy we have demonstrated that the application of I !J-M ABA to detached epidennis from C. communis results in an increase in guard cell cytosolic free calcium concentration. This increase was observed in 8 out of the 10 cells tested and preceded any observable alteration in stomatal aperture. During the course of these experiments we established that the concentration of free calcium ions in the cytosol of the unstimulated cell was approximately 115 !J-M (McAINSH et a\. 1990). After perfusing the preparation with ABA the increases in free calcium concentration were variable. Importantly, in some experiments the ABA induced increases in cytosolic calcium concentration approached the levels reported to inhibit the inwardly conducting K+ channel (SCHROEDER and HAGIW A RA 1989). It is also possible that such concentrations could indirectly activate the inwardly directed K+ channel by the mechanisms discussed by Schroeder and colleagues (SCHROEDER and HAGIWARA 1989; SCHROEDER and HEDRICH 1989) . However, the recent results from Raschke's laboratory (KELLER et al. 1989) indicate that further work is required on calcium-activated anion conductances before their role in the control of guard cell turgor can be fully assessed. Nevetheless, through a coupling of some or all of the above events to an increase in cytosolic free calcium it may be possible to account for the action of ABA on stomatal guard cells. However, further work needs to be carried out before the calcium hypothesis can be said to have been fully tested. One outstanding question which requires an answer is whether the observed increase in cytosolic calcium concentration precede alterations to the concentration of K+ Cl - and H+ in the guard cell. It will be possible to answer this question using a similar experimental approach to that adopted in the calcium studies. It is our intention to investigate temporal and spatial aspects of ABA-induced alterations to the cytosolic concentration of the ions listed above. Additional questions which need to be addressed include whether calcium ions are involved in the response of stomatal guard cells to other extracellular signals such as BPP l86 (1990) 5/6

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CO2 , light, auxins and cytokinins. Also does the alteration to cytosolic free calcium participate in the control of carbon metabolism which also contributes to the turgor relations of the stomatal guard cell?

Acknowledgements We wish to acknowledge the financial assistance of the Agricultural and Food Research Council, Shell Research Ltd., The Gatsby Charitable Foundation, and Lancaster University Committee for Research.

References ARMSTRONG, M. J., and KIRBY, E. A.: The influence of humidity on the mineral composition of tomato plants with special reference to calcium distribution. Plant Soil 53, 427-435 (1979). ATKINSON, C. J., DAVIES, W. J., and MANSFIELD, T. A.: Changes in stomatal conductance of intact ageing wheat leaves in response to abscisic acid. J. Exp. Bot. 40,1021-1028 (1989a). ATKINSON, C. J., MANSFIELD, T. A., KEAN, A. M., and DAVIES, W. J.: Control of stomatal aperture by calcium in isolated epidermal tissue and whole leaves of Commelina communis L. New Phytol. 111,9-17 (l989b). CLARKSON, D. T.: Calcium transport between tissues and its distribution in the plant. Plant Cell Environ. 7, 449-456 (1984). DE SILVA, D. L. R., HETHERINGTON, A. M., and MANSFIELD, T. A.: Synergism between calcium ions and abscisic acid in preventing stomatal opening. New Phytol. 100, 473-482 (1985). FITZSIMONS, P. J., and WEYERS, J. D. B.: Volume changes of Commelina communis guard cell protoplasts in response to K+ and light CO2 • Physiol. Plant 66, 463-468 (1986). FUJINO, M.: Role of adenosinetriphosphate and adenosinetriphosphatase in stomatal movement. Sci. Fac. Edu. Nagasaki: Univ. 18, 1-47 (1967). HEPLER, P. K., and WAYNE, R. 0.: Calcium and plant development. Ann. Rev. Plant Physiol. 36, 397-439 (1985). INOUE, H., and KATOH, Y.: Calcium inhibits ion-stimulated opening in epidermal strips of Commelina communis L. J. Exp. Bot. 38, 142-149 (1987). ILJIN, W. S.: Drought resistance in plants and physiological processes. Ann. Rev. Plant Physiol. 8, 257-274 (1957). KELLER, B. V., HEDRICH, R., and RASCHKE, K.: Voltage-dependent anion channels in the plasma membrane of guard cells. Nature 341, 450-453 (1989). MAIER-MAERCKER, U.: The role of peristomatal transpiration in the mechanisms of stomatal movement. Plant Cell Environ. 6, 369-380 (1983). MANSFIELD, T. A., HETHERINGTON, A. M., and ATKINSON, C. J.: Some aspects of stomatal physiology. Ann. Rev. Plant Physiol. and Molec. BioI. 41, 55-75 (1990). McAINSH, M. R., BROWNLEE, C., and HETHERINGTON, A. M.: Abscisic acid-induced elevation of guard cell cytosolic Ca2+ precedes stomatal closure. Nature 343, 186-188 (1990). MEIDNER, H., and SHERIFF, D. W.: Water and Plants. Blackie, Glasgow and London 1976. POOVIAH, B. W., and REDDY, A. S. N.: Calcium messenger systems in plants. CRC Crit. Rev. Plant Sci. 6,47- 103 (1987). SCHROEDER, S. I., and HAGIWARA, S.: Cytosolic calcium regulates ion channels in the plasma membrane of Vicafaba guard cells. Nature 338,427-431 (1989). SCHROEDER, S. I., and HEDRICH, R.: Involvement of ion channels and active transport in osmoregulation and signaling of higher plant cells. Trends Biochem. Sci. 14, 187-192 (1989). SCHWARTZ, A.: Role of Ca2+ and EGTA on stomatal movements in Commelina communis L. Plant Physioi. 79, 1003-1005 (1985). SERRANO, E. E., and ZEIGER, E.: Sensory transduction and electrical signaling in guard cells. Plant Physiol. 91, 795-799 (1989).

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SMITH, G. N.. and WllLMER, C. M.: Effects of calcium and abscisic acid on volume changes of guard cells protoplasts of Commelina communis. J. Exp. Bot. 30, 1529-1539 (1988). TANTON, T. W., and CROWDY, S. H.: Water pathways in higher plants. Ill. The transpiration stream within leaves. 1. Exp. Bot. 23,619-625 (1972). Author's address: Prof. TERENCE A. MANSFIELD, Lancaster University, Division of Biological Sciences, Lancaster LA1 4YQ, U.K.

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