Ca²⁺-Dependent and -Independent Abscisic Acid Activation of Plasma Membrane Anion Channels in Guard Cells of <em>Nicotiana tabacum</em>^{<a href="#fn1" rid="fn1" class=" fn">1</a>}

Abstract

The aperture of stomata is determined by two guard cells that surround the stomatal pore. Guard cells are devoid of plasmodesmata (Wille and Lucas, 1984) and thus function virtually autonomously. During drought, stomata close to reduce the loss of water via transpiration, a response that depends on the phytohormone abscisic acid (ABA; Assmann and Shimazaki, 1999; Schroeder et al., 2001; Roelfsema and Hedrich, 2005). ABA triggers the efflux of K ^{salts from the guard cell, thereby reducing the osmotic potential of the cell and causing stomatal closure. The efflux of K salts is driven through depolarization of the plasma membrane, which allows K extrusion through outward rectifying K channels. Two mechanisms could mediate the ABA-induced depolarization of guard cells: activation of anion channels and/or inhibition of H-ATPases in the plasma membrane.}

In guard cells of Vicia faba studied in intact plants, ABA triggers a transient activation of R- and S-type anion channels (Roelfsema et al., 2001). A peak in anion channel activity is found after approximately 5 min; during prolonged stimulation, the activity of anion channels levels off, but a prestimulus value is only observed after removal of ABA. This led to the suggestion that the first phase of this ABA response induces fast stomatal closure, while the second phase inhibits reopening of the stomata (Roelfsema and Hedrich, 2005). The extent to which ABA may inhibit plasma membrane H^{-ATPases is not known in detail, but ABA was shown to inhibit blue light-induced activation of H-ATPases in guard cells (}Zhang et al., 2004).

Various lines of evidence predicted an important role of the cytoplasmic Ca ^{concentration in ABA activation of anion channels in guard cells (}Hetherington and Brownlee, 2004). Both R- and S-type anion channels in V. faba guard cells are stimulated by cytoplasmic Ca ⁽Schroeder and Hagiwara, 1989; Hedrich et al., 1990), and ABA can induce cytoplasmic Ca ^{transients in guard cells in isolated epidermal strips or epidermal fragments of} Commelina communis (McAinsh et al., 1990; Gilroy et al., 1991) as well as in Arabidopsis (Arabidopsis thaliana; Allen et al., 1999, 2001). In contrast to these data, Levchenko et al. (2005) found that ABA activates anion channels without provoking cytoplasmic Ca ^{changes in guard cells of} V. faba. The differences in results may be due to different experimental conditions, because Levchenko et al. (2005) measured guard cells in intact plants, while previous reports were mainly conducted with isolated epidermal peels. Alternatively, the differences may relate to differences between plant species. In some species, ABA-induced stomatal closure could involve alterations in the cytoplasmic Ca ^{concentration of guard cells, whereas in others, stomata close in a Ca-independent fashion.}

In search of species-specific variation in the anion channel response to ABA, guard cell responses of Nicotiana tabacum were compared with those previously obtained for V. faba (Levchenko et al., 2005). In contrast to V. faba, this plant species can be transformed easily and thus has the advantage that it can be used for molecular biological approaches. The size of N. tabacum stomata is similar to that of V. faba and C. communis, which is a prerequisite for long-term intracellular recordings. For these reasons, N. tabacum and its close relative, Nicotiana benthamiana, already were the species of choice in several studies (Armstrong et al., 1995; Leyman et al., 1999; Hunt et al., 2003; von Caemmerer et al., 2004). We found that ABA activates anion channels in guard cells of intact plants, in a similar manner as previously observed for V. faba. However, in contrast to previous results with V. faba, the majority of N. tabacum guard cells showed an ABA-induced rise in the cytoplasmic Ca ^{concentration. Patch clamp experiments with} N. tabacum guard cell protoplasts revealed that cytoplasmic Ca ^{as well as ABA enhance anion channel activity. These data suggest species-specific differences concerning the role of cytoplasmic Ca changes in guard cell responses to ABA.}

Notes

^{This work was supported by the Deutsche Forschungsgemeinschaft (grants to R.H., M.R.G.R., and P.D.).}

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Rainer Hedrich (ed.grubzreuw-inu.kinatob@hcirdeh).

www.plantphysiol.org/cgi/doi/10.1104/pp.106.092643