Ca2+ is critical for ABA-induced inhibition of stomatal opening
We have provided direct evidence that an increase in [Ca2+]i is a component in the signalling pathways by which ABA regulates stomatal movements in A. thaliana. ABA increased [Ca2+]i in single guard cells of A. thaliana(Figure 6), and BAPTA in the extracellular medium completely abolished the ABA-induced inhibition of stomatal opening (Figure 7b). BAPTA may have limited influx of Ca2+ across the plasma membrane via an ABA- and hyperpolarization-regulated Ca2+ channel (Grabov and Blatt, 1999; Hamilton et al., 2000). However, it is likely that internal stores are also central to ABA-turgor signalling in guard cells (Grabov and Blatt, 1999). It was not possible to test further the role of elevations in [Ca2+]i in ABA signalling in A. thaliana by micro-injecting BAPTA into the cytosol, due to the relatively small changes in half stomatal aperture in this species.
The aperture responses of the stomata of the Columbia ecotype of A. thaliana to ABA, Ca2+ and other stimuli are quantitatively similar to those reported previously for the Landsberg erecta ecotype (Allen et al., 1999a; Allen et al., 1999b; Leymarie et al., 1998; Roelfsema and Prins, 1995; Webb and Hetherington, 1997). We observed ABA-induced and [Ca2+]e-induced increases of [Ca2+]i in the Columbia ecotype of similar magnitudes and forms to those reported previously in the stomata of L. erecta (Allen et al., 1999a; Allen et al., 1999b).
The higher quantum efficiency of fura compared to indo-1 and the cameleon Ca2+-indicator allowed imaging of Arabidopsis guard cells [Ca2+]i with greater spatial resolution than has previously been possible, partially because pixel binning was not required in this study (Allen et al., 1999a; Allen et al., 1999b). We were able to observe that complex spatial–temporal patterns underlie oscillations in [Ca2+]i in guard cells of Arabidopsis(Figure 5). However, the technical difficulties associated with micro-injecting the guard cells of this species precluded obtaining sufficient loaded cells to perform a detailed analysis of these patterns. For the same reasons it was not possible to analyse the effects of calcium agonists on Ca2+ signals in the guard cells of A. thaliana. Such analysis awaits the development of new variants of calcium cameleon with a sufficient quantum efficiency to allow detailed spatial analysis of Arabidopsis guard cell [Ca2+]i, or improvements in micro-injection techniques.
The temporal patterns of the [Ca2+]e-induced oscillations in [Ca2+]i in A. thaliana guard cells were not identical to those we have previously observed in C. communis (McAinsh et al., 1995). In A. thaliana, 1 mm CaCl2 induced irregular oscillations (Figure 4), whereas in C. communis 1 mm CaCl2 induced oscillations that had a characteristic biphasic, ‘spike-shoulder’ pattern. The reasons for these differences are unclear. However, a number of factors are known to affect the pattern of stimulus-induced oscillations in guard cell [Ca2+]i. These include the plasma membrane potential (Grabov and Blatt, 1998; Hetherington et al., 1998; Staxén et al., 1999); ABA (Hetherington et al., 1998; Staxén et al., 1999); [Ca2+]e (McAinsh et al., 1995); and osmotic shock (Hetherington et al., 1998).
Ca2+ is involved in ABA-induced gene expression in guard cells of A. thaliana
We have also provided evidence that elevations in [Ca2+]i form a component of the transduction chain by which ABA regulates the CDeT6-19 promoter. We have shown that ABA increases [Ca2+]i in guard cells of A. thaliana(Figure 6), and that activation of the CDeT6-19 promoter by ABA was inhibited by Ca2+ antagonists (Figure 8). Despite the semi-quantitative nature of the GUS reporter, the effects of the Ca2+ antagonists on cDeT6-19 promoter activity were marked, suggesting that Ca2+ is an important component in the ABA-nuclear signalling pathway(s). LaCl3, verapamil and BAPTA probably inhibited ABA-induced increases in [Ca2+]i by inhibiting Ca2+-influx across the plasma membrane (Grabov and Blatt, 1999; Hamilton et al., 2000). We were unable to confirm whether LaCl3, verapamil and BAPTA prevented ABA-induced increases in [Ca2+]i in A. thaliana. Such experiments would have required larger data sets than we could obtain by micro-injection of fluorescent indicators in to guard cells of A. thaliana.
The effects of the Ca2+-antagonists on ABA-induced gene expression in the guard cell are strikingly similar to the effects of these agents upon the ABA-turgor signalling pathway. For example, BAPTA inhibited both ABA-induced CDeT6-19 activity and ABA-induced inhibition of stomatal opening in A. thaliana(Figures 7 and 8). Similarly, the putative Ca2+ channel blockers, LaCl3 and verapamil both inhibited ABA-induced CDeT6-19 promoter activity (Figure 8), and have previously been shown to prevent ABA-induced inhibition of stomatal opening in C. communis (DeSilva et al., 1985). Taken together, these data suggest that an increase in [Ca2+]i is a common component of the transduction pathways by which ABA regulates CDeT6-19 promoter activity in guard cells of A. thaliana and induces reductions in guard cell turgor.
The histochemical GUS assay is semi-quantitative and does not usefully report small changes in gene expression, therefore it was not possible to determine whether there is an absolute requirement for Ca2+ during ABA-nuclear signalling. More quantitative analysis of the activity of the cDet6-19 promoter might have been achieved using a fluorescent substrate in cell homogenates. However, the techniques for purifying guard cells involve a cold shock during blending or an osmotic shock during protoplasting. The cold and osmotic signalling pathways closely overlap the ABA signalling pathways. Thus purification of the guard cells to produce homogenates would have prevented the ABA signalling pathway being studied in isolation. Our previous studies demonstrated that cold induces cDeT6-19 promoter activity (J.E.T. et al., unpublished observations). We did attempt a quantitative measure of GUS activity in situ using the ImaGene green fluorescent substrate (Molecular Probes; see Experimental procedures) and photometry. This proved unsuccessful because the substrate was unable to enter guard cells. We observed fluorescent signal only from guard cells that had previously been damaged. We concluded that the cell wall prevented entry of the substrate. Other researchers have found that this fluorescent substrate is suited to measuring only very high levels of expression in vivo, and have been unable to quantify changes in gene expression in planta (Fleming et al., 1996).
There is convincing evidence that increases in [Ca2+]i also form a component of the ABA-nuclear signalling pathways in maize leaf protoplasts (Sheen, 1996); etiolated hypocotyls of the phytochrome-deficient tomato mutant aurea (Wu et al., 1997); wheat aleurone (Napier et al., 1989); and chickpea seeds (Colorado et al., 1991). Increases in [Ca2+]i have been implicated in the regulation of a number of ABA-inducible gene promoters including rab18 (Knight et al., 1997); lti78 (Knight et al., 1997; Wu et al., 1997); HVA1 (Sheen, 1996); and kin2 (Wu et al., 1997). Therefore increases in [Ca2+]i appear to act as a component of several of the ABA-nuclear signalling pathways operating in plant cells.
ABA was more effective in activating the CDeT6-19 promoter than increased [Ca2+]e in the absence of exogenous ABA (Figure 8). This represents a difference in the ABA-nuclear signalling pathway compared to the ABA-turgor signalling pathway, because increases in [Ca2+]e and ABA both reduced guard cell turgor in Arabidopsis(Figure 7a; Roelfsema and Prins, 1995; Webb and Hetherington, 1997) and C. communis (McAinsh et al., 1995). Therefore increases in [Ca2+]e, probably by increasing [Ca2+]i(Figures 4 and 5), appear to be able to activate sufficient downstream signalling elements to strongly activate the ABA-turgor signalling pathway, but the same increases in [Ca2+]e appear to only weakly stimulate the ABA-nuclear signalling pathway. These data suggest that there are differences in the architecture of the ABA-turgor and ABA-nuclear signalling pathways operating in guard cells.
There is conflicting evidence concerning the ability of increases in [Ca2+]i to substitute for ABA in the ABA-nuclear pathways operating in other plant cell types. Increases in [Ca2+]i induced by micro-injection of Ca2+ into the cytosol (Wu et al., 1997), or by treatment with the ionophore A23187 (Sheen, 1996), resulted in increased activity of ABA-responsive reporters in heterologous expression assays in the absence of ABA. Similarly, increases in [Ca2+]e in the absence of exogenous ABA have been demonstrated to induce the appearance of ABA-responsive polypeptides (Colarado et al., 1991; Napier et al., 1989). In the present study we were able to induce only weak GUS expression driven by the ABA-responsive CDeT6-19 promoter in guard cells in the absence of ABA. Similarly, Sheen (1996) observed that the HVA1 promoter was not activated in response to 1 mm[Ca2+]e.
In animal cells, both the spatial (Hardingham et al., 1997) and temporal pattern of stimulus-induced increases in [Ca2+]i (Dolmetsch et al., 1997, Dolmetsch et al., 1998; Li et al., 1998) affect the pattern of alteration in gene expression. Particularly striking is evidence that the activation of a specific subset of pro-inflammatory transcription factors in Jurkat T cells depends on the frequency of oscillations in [Ca2+]i (Dolmetsch et al., 1998). One possibility is that elevated [Ca2+]i may be able to substitute for ABA in the ABA-nuclear signalling pathway only if the appropriate ‘Ca2+-signature’ is generated (McAinsh and Hetherington, 1998). A recent report suggests that the correct pattern of oscillations of [Ca2+]i may have a central role in driving reductions in guard-cell turgor (Allen et al., 2000).
Whilst the appropriate Ca2+-signature may be very important for the correct regulation of gene expression in a number of cell types, the failure of elevated [Ca2+]e to induce strong activity of an ABA-responsive promoter in guard cells in this and a previous study (Shen et al., 1995) could have another explanation. There may be Ca2+-independent component(s) of the ABA signal-transduction pathways that are required for full activity of the CDeT6-19 promoter. These Ca2+-independent signalling elements may be activated in response to ABA, but not activated in response to elevated [Ca2+]e or [Ca2+]i. Preliminary data suggesting that a mitogen-activated protein kinase cascade may contribute to ABA signalling in guard cells (Burnett et al., 2000) present a tantalizing clue to the identity of the Ca2+-independent signalling elements.