Plant Signalling: Calcium First andSecond

Colin Brownlee

The cloning of a receptor protein for extracellularcalcium that signals via cytosolic calcium oscillationsin stomatal guard cells of Arabidopsis has provideddefinitive evidence that calcium ions, delivered viathe cell walls or apoplast, can play a ‘first messenger’role in plants.

Plant cells are surrounded by cell walls that presentan aqueous continuum, the apoplast, through whichhormones and other potential regulatory factors canpass [1]. In plants, a function for apoplastic Ca2+ asan extracellular messenger has been indicated fromstudies with stomatal guard cells, though compo-nents of the pathway that may link changes in extra-cellular Ca2+ with specific changes in intracellularCa2+ have until now been elusive. A recent report byHan et al. [2] describes a novel cell surface receptorfor apoplastic Ca2+ and firmly places Ca2+ on the listof extracellular ‘first messengers’ in plants.

Pairs of guard cells in the leaf epidermis form poresthrough which water and gas exchange occurs. Thepore size can be regulated by factors in the apoplastthat alter the turgor pressure inside the guard cells,including the plant hormone abscisic acid (ABA), CO2,auxin and elevated extracellular Ca2+ [3,4]. CytosolicCa2+ is an important component of the signalingnetwork that regulates stomatal aperture [4]. Increasesin apoplastic Ca2+ have been shown to bring aboutincreases in cytosolic Ca2+ in guard cells in isolatedepidermal strips from Commelina communis leaves[5]. Increasing extracellular Ca2+ from 0.01 mM tohigher than 0.1 mM was found to lead to repetitiveelevations in cytosolic Ca2+ which had a period ofseveral minutes.

The pattern of these oscillatory increases in cytoso-lic Ca2+ varies with the external Ca2+ concentration.Their significance has been the source of muchspeculation. It is possible that they encode stimulus-specific information in much the same way as cytosolicCa2+ oscillations in animal cells [6]. Alternatively,increased external Ca2+ may simply promote Ca2+ influxand consequent elevation of cytosolic Ca2+. Delays inthe activation of efflux mechanisms could give rise tooscillatory elevations of cytosolic Ca2+ that reflect dis-turbance of the cytosolic Ca2+ homeostatic machinery.

Several lines of evidence suggest, however, thatthese repetitive Ca2+ increases do indeed encode spe-cific information. For example, similar repetitive Ca2+

increases occur in response to ABA [7,8] and differentpatterns of Ca2+ oscillations can give rise to differentstomatal closure responses [9]. Arabidopsis det-3

mutants are defective in the ability to generate oscilla-tory Ca2+ increases in response to elevated externalCa2+ and also in the stomatal closure response to ele-vated external Ca2+, though interestingly they showednormal responses to ABA [8]. Another Arabidopsismutant that showed altered patterns of Ca2+ oscilla-tions in response to elevated external Ca2+ and to ABA,gca2, was also found to be defective in the stomatalclosure response to elevated external Ca2+. This workstrongly suggested that specific information determin-ing stomatal aperture may be encoded in the frequencyof cytosolic Ca2+ elevations in guard cells.

Han et al. [2] have recently provided further strongevidence that external Ca2+ may indeed act as aphysiological signal in plants, and that repetitiveincreases in cytosolic Ca2+ arise specifically inresponse to activation of a cell surface receptor. Theycloned a calcium sensing receptor (CAS) by expressinga cDNA library from Arabidopsis leaves in animalHEK293 cells and screening for external Ca2+-inducedelevations of cytosolic Ca2+. The CAS protein shows nosequence similarity to the well characterized Ca2+

receptors of the G-protein-coupled receptor family inanimals [10]. CAS appears to have a single transmem-brane domain and its putative intracellular carboxyl ter-minus has a rhodanese-like sequence which maycontribute to protein–protein interactions. The putativeextracellular amino terminus has several acidic residuesthat may function in low-affinity Ca2+ binding.

CAS was found to be predominantly expressed inleaves, stems and flowers and in guard cells in the epi-dermis. Expression of a constitutive 35s::CAS–GFPfusion gene produced a protein that located to the cellsurface in onion epidermal cells and Arabidopsis guardcells. Reducing CAS expression with a constitutivelyexpressed antisense construct led to significant dis-ruption of external Ca2+-induced cytosolic Ca2+

increase. Interestingly, ABA-induced stomatal closurewas unaffected in these antisense plants, suggestingthat ABA-induced cytosolic Ca2+ oscillations arise byseparate initial pathways (see Figure 1). However, theoverall similarities in the patterns of ABA- and externalCa2+-induced cytosolic Ca2+ oscillations [7–9] sug-gests that they involve similar mechanisms down-stream of the receptor.

It will be of great interest to determine whether ABA-induced cytosolic Ca2+ increases are compromised inCAS antisense plants. Han et al. [2] provided prelimi-nary evidence based on inhibitors that CAS-inducedcytosolic Ca2+ elevation requires release of Ca2+ fromintracellular stores via the production of IP3. However,this contrasts with earlier work by McAinsh et al. [5]who found that external Ca2+-induced elevations ofcytosolic Ca2+ did not require phospholipase C, but didinvolve Ca2+ influx.

Han et al. [2] argue that the transpiration rate maybe a major factor affecting the rate of delivery of Ca2+

to the stomatal guard cell apoplast. High transpiration

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Current Biology, Vol. 13, R923–R924, December 2, 2003, ©2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/j.cub.2003.11.016

The Marine Biological Association of the UK, The Laboratory,Citadel Hill, Plymouth PL1 2PB, UK.

and evaporation from the stomatal pore would lead toincreased apoplastic Ca2+, leading to stomatal closureand consequent reduction in apoplastic Ca2+, provid-ing a feedback mechanism for regulating apoplasticCa2+. This argument implies that stomata are majorregulators of their own apoplastic Ca2+. However,stronger control of apoplastic Ca2+ is likely to beexerted by other cell types in the leaf (Figure 1). TheCa2+ in the xylem sap that is delivered to the leafapoplast has been shown to be in the millimolar range(see [11] for example).

As guard cells studied so far do not remain openwhen external Ca2+ rises above 0.01 mM, mechanismsmust exist to reduce the apoplastic Ca2+ concentrationaround the guard cell. Ruiz and Mansfield [11] reportedevidence that the subsidiary cells surrounding theguard cells perform this task in Commelina by takingup Ca2+ and precipitating it as calcium oxalate in theirvacuoles. In calcicole species, which grow on soilswith high concentrations of available Ca2+, the xylemsap Ca2+ concentration can be even higher. There isgood evidence that, in these plants, the palisade meso-phyll cells together with the specialized epidermal cellsknown as trichomes play a major role in removingapoplastic Ca2+ [12,13].

It will be of great interest to elucidate the signal trans-duction pathway downstream of CAS and its interac-tion with other proteins and transduction pathwaysinvolving Ca2+, such as ABA signalling. Imaging studiesof apoplastic Ca2+ will be required to further determine

the role of apoplastic Ca2+ as a universal external firstmessenger in plants. As CAS was at least partially func-tional when expressed in animal cells [2], it may interactwith components of the Ca2+ signal transductionmachinery that are conserved between animals andplants. External Ca2+ in animal cells carries out a rangeof signalling roles involved in Ca2+ homeostasis, regu-lation of hormone secretion, programmed cell deathand cell proliferation [10]. CAS antisense plants werealso shown to lack the gross morphological responses(bolting) to Ca2+ deficiency at their roots [2]. CAS andapoplastic Ca2+ may therefore carry out a range offunctions relating to the physiology, development andmorphogenesis of plants.

References1. Roelfsema, M.R.G., and Hedrich, R. (2002). Studying guard cells in

the intact plant: modulation of stomatal movements by apoplasticfactors. New Phytol. 153, 425-431.

2. Han, S., Tang, R., Anderson, L.K., Woerner, T.E., and Pei, Z-M.(2003). A cell surface receptor mediates extracellular Ca2+ sensingin guard cells. Nature 425, 196-200.

3. Hetherington, A.M., and Woodward, F.I. (2003). The role of stomatain sensing and driving environmental change. Nature 424, 901-908.

4. Hetherington, A.M. (2001). Guard cell signaling. Cell 107, 711-714.5. McAinsh, M.R., Webb, A.A.R., Taylor, J.E., and Hetherington, A.M.

(1995). Stimulus-induced oscillations in guard cell cytosolic freecalcium. Plant Cell 7, 1207-1219.

6. Berridge, M.J., Bootman, M.D., and Roderick, H.L. (2003). Calciumsignalling:dynamics, homeostasis and remodelling. Nat. Rev. Mol.Cell Biol. 4, 517-529

7. Staxen, I., Pical, C., Montgomery, L.T., Gray, J.E., Hetherington,A.M., and McAinsh, M.R. (1999). Abscisic acid induced oscillationsin guard cell cytosolic free calcium that involve phosphoinositide-specific phospholipase C. Proc. Natl. Acad. Sci. USA 96, 1779-1784.

8. Allen, G.J,, Chu, S.P., Schumaker, K., Shimazaki, C.T., Vafeados, D.,Kemper, A., Hawke, S.D., Tallman, G., Tsien, R.Y., Harper, J.F., et al.(2000). Alteration of stimulus-specific guard cell calcium oscillationsand stomatal closing in Arabidopsis det3 mutant. Science 289,2338-2342.

9. Allen, G.J., Chu, S.P., Harrington, C.L., Schumaker, K., Hoffmann,T., Tang, Y.Y., Grill, E., Schroeder, J.I. (2001). A defined range ofguard cell calcium oscillation parameters encodes stomatal move-ments. Nature 411, 1053-1057.

10. Brown, E.M., and MacLeod, R.J. (2001). Extracellular calciumsensing and extracellular calcium signaling. Physiol. Rev. 81, 239-295.

11. Ruiz, L.P., and Mansfield, T.A. (1994). A postulated role for calciumoxalate in the regulation of calcium ions in the vicinity of stomatalguard cells. New Phytol. 127, 473-481.

12. DeSilva, D.L.R., Hetherington, A.M., and Mansfield, T.A. (1998). Theregulation of apoplastic calcium in relation to intracellular signallingin stomatal guard cells. Z. Pflanzenern. Bodenk. 161, 533-539

13. DeSilva, D.L.R., Mansfield, T.A., and McAinsh, M.R. (2001). Changesin stomatal behaviour in the calcicole Leontodon hispidus due tothe disruption by ozone of the regulation of apoplastic Ca2+ by tri-chomes. Planta 214, 158-162.

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Figure 1. Ca2+ signalling in plants.

Ca2+ delivered from the xylem sap (red arrows) undergoesprogressive dilution from the xylem sap to the guard cells (Gc).Ca2+ is removed from the apoplast by cells of the palisademesophyll (Pm), the trichomes (T) and the subsidiary cells (Sc)surrounding each pair of guard cells. Ca2+ in the apoplastaround the guard cells is likely to be lower than 0.1 mM, com-pared with levels of several mM in the xylem sap. Inset:apoplastic Ca2+ is sensed by the novel CAS receptor located atthe cell surface. Activation of CAS gives rise to cytosolic Ca2+

oscillations that are similar in magnitude and frequency tothose observed in response to ABA application. Whether theseoscillations are the result of activation of independent Ca2+

signal transduction pathways or whether they converge down-stream of Ca2+ or ABA sensing is not known.

ABA

Ca2+

or

Ca2+

T Sc Gc

Pm

Cas

>1.0 mM<0.1 mM

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