Cell- and Stimulus Type-Specific Intracellular Free Ca²⁺ Signals in Arabidopsis^,,

Enhancer trap targeting of aequorin to specific cell types identifies stimulus- and cell-specific signaling via cytosolic-free calcium concentration in Arabidopsis.

Abstract

Appropriate stimulus-response coupling requires that each signal induces a characteristic response, distinct from that induced by other signals, and that there is the potential for individual signals to initiate different downstream responses dependent on cell type. How such specificity is encoded in plant signaling is not known. One possibility is that information is encoded in signal transduction pathways to ensure stimulus- and cell type-specific responses. The calcium ion acts as a second messenger in response to mechanical stimulation, hydrogen peroxide, NaCl, and cold in plants and also in circadian timing. We use GAL4 transactivation of aequorin in enhancer trap lines of Arabidopsis (Arabidopsis thaliana) to test the hypothesis that stimulus- and cell-specific information can be encoded in the pattern of dynamic alterations in the concentration of intracellular free Ca²⁺ ([Ca²⁺]_i)_. We demonstrate that mechanically induced increases in [Ca²⁺]_i are largely restricted to the epidermal pavement cells of leaves, that NaCl induces oscillatory [Ca²⁺]_i signals in spongy mesophyll and vascular bundle cells, but not other cell types, and detect circadian rhythms of [Ca²⁺]_i only in the spongy mesophyll. We demonstrate stimulus-specific [Ca²⁺]_i dynamics in response to touch, cold, and hydrogen peroxide, which in the case of the latter two signals are common to all cell types tested. GAL4 transactivation of aequorin in specific leaf cell types has allowed us to bypass the technical limitations associated with fluorescent Ca²⁺ reporter dyes in chlorophyll-containing tissues to identify the cell- and stimulus-specific complexity of [Ca²⁺]_i dynamics in leaves of Arabidopsis and to determine from which tissues stress- and circadian-regulated [Ca²⁺]_i signals arise.

Ca²⁺ is an important second messenger involved in a wide range of responses in plants (Dodd et al., 2010). Fluorescent reporters of Ca²⁺ have revolutionized the study of Ca²⁺ signaling, permitting measurement of the intracellular concentration of Ca²⁺ ([Ca²⁺]_i) with high subcellular spatial and temporal resolution. In plants, the use of fluorescent reporters has identified stimulus-induced increases in [Ca²⁺]_i that encode information in the temporal and subcellular distribution of the [Ca²⁺]_i signal (Dodd et al., 2010). However, the utility of fluorescent Ca²⁺ indicators in plants has been usually restricted to a few specific cell types, including the guard cells, root hairs, and pollen tubes, because these tissues are low in chlorophyll. In other tissues, chlorophyll fluorescence reduces the signal-to-noise ratio to unacceptable levels. Furthermore, in situ measurements of cytosolic-free Ca²⁺ concentration ([Ca²⁺]_cyt) signals in single cells in tissues below the plant surface using fluorescence or confocal microscopy has proved technically challenging, even in root tissues (Zhu et al., 2013), due to light scatter and fluorescence by the cell walls. To overcome some of these difficulties, Knight et al. (1991) introduced recombinant aequorin as a reporter of Ca²⁺ concentration in plant systems. Aequorin is a bioluminescent reporter and, therefore, has an intrinsically high signal-to-noise ratio, and it requires no damaging excitation light and, therefore, is suited to measurements in chlorophyll-containing tissue. The major limitation of the use of aequorin is low light emission, meaning that imaging even at the subtissue level has proved elusive (Zhu et al., 2013), and subcellular imaging in plants is not possible. Aequorin has found high utility and has identified roles for [Ca²⁺]_cyt signaling in mechanical stimulation, cold, salinity, and pathogen stress and in the daily timing of plants (Campbell et al., 1996; Knight et al., 1996, 1997; Gong et al., 1998; Kawano et al., 1998; Baum et al., 1999; Love et al., 2004; Kosuta et al., 2008; Monshausen et al., 2009; Zhu et al., 2013). However, in leaves and shoots, it is not known from which tissues the stress- and circadian-regulated [Ca²⁺]_i signals arise.

We describe targeting of the in vivo Ca²⁺ reporter aequorin to spongy mesophyll, trichome, vascular, and epidermal pavement cells using GAL4-mediated transactivation in enhancer trap lines (Gardner et al., 2009). In the GAL4 enhancer trap system as implemented in Arabidopsis (Arabidopsis thaliana), the yeast GAL4 transcriptional activator is inserted randomly into the genome under the control of a minimal promoter (Kiegle et al., 2000). When the insertion is located near an endogenous enhancer, GAL4 is expressed. GAL4 expression is visualized by combining on the same insertion cassette endoplasmic reticulum-targeted GFP downstream of a GAL4 upstream activation sequence (UAS). In some cases, cell-specific GFP expression can be detected in individual lines. These cell-specific driver lines can be used to deliver cell-specific expression of any transgene downstream from another GAL4 UAS. These GAL4 UAS-driven transgenes are introduced by supertransformation or crossing. Previously, GAL4 transactivation of aequorin has been described for root tissues (Kiegle et al., 2000) and guard cells (Dodd et al., 2006).

We test the hypothesis that there is cell and tissue specificity of the [Ca²⁺]_i signaling networks in Arabidopsis. We use trichome, spongy mesophyll, epidermal pavement, vascular bundle, and guard cell-specific enhancer trap lines that we described previously (Dodd et al., 2006; Gardner et al., 2009) to investigate the responses of cell types to NaCl, cold, mechanical stimulation, and hydrogen peroxide (H₂O₂) because all cause a rapid increase in [Ca²⁺]_i followed by dynamic alterations of [Ca²⁺]_i when measured in seedlings in which aequorin has been targeted to the whole seedling (Lynch et al., 1989; Price et al., 1994; Knight et al., 1996, 1997; Monshausen et al., 2009). Additionally, we investigate from which cells diel oscillations of [Ca²⁺]_i arise. Diel and circadian oscillations of [Ca²⁺]_cyt were first detected in seedlings in which aequorin was constitutively expressed (Johnson et al., 1995) and later demonstrated to be occurring in the leaves (Love et al., 2004). The daily oscillations of [Ca²⁺]_cyt peak at around 300 nm about 8 h after dawn (Love et al., 2004). The circadian clock and PHYTOCHROME A-, CRYPTOCHROME1-, and CRYPTOCHROME2-dependent signaling pathways drive daily rhythms of [Ca²⁺]_cyt (Dalchau et al., 2010) with cyclic ADP ribose (cADPR) acting as an intermediary (Dodd et al., 2007). The core circadian clock gene CIRCADIAN CLOCK ASSOCIATED1 (CCA1) is required for circadian oscillations of [Ca²⁺]_cyt and cADPR, because cca1-1 loss of function abolishes the rhythms of both [Ca²⁺]_cyt and cADPR, resulting in constitutively high [Ca²⁺]_cyt, whereas overexpression of CCA1 results in constitutively low cADPR concentration and [Ca²⁺]_cyt (Dodd et al., 2007; Xu et al., 2007).

Despite the central importance of [Ca²⁺]_i signaling in the regulation of leaf biology, study of the dynamics of stimulus-induced and circadian-regulated [Ca²⁺]_i signals in leaves has usually been limited to the stomatal guard cells, or data have only been gathered on the responses at the whole seedling level or from images of whole leaves, summing the responses of different cell types (Johnson et al., 1995; Wood et al., 2000, 2001; Love et al., 2004; Dodd et al., 2006, 2010; Zhu et al., 2013). In this study, we identify cell- and stimulus-specific [Ca²⁺]_i signals in the leaf and other tissues of Arabidopsis, suggesting the presence of cell-specific signaling cassettes in plants.

RESULTS

GAL4 Transactivation of Aequorin in Cell-Specific GAL4-GFP Enhancer Trap Lines

Multiple independent lines transformed with pBINYFPAEQ were obtained for each of the enhancer trap lines used in this study. More than five independent transformants per GAL4 enhancer trap line were analyzed, and only those lines with the highest total aequorin activity were selected for further analysis. Consistent with previous reports of aequorin transformation under the control of the Cauliflower Mosaic Virus 35S (CaMV35S) promoter or using the GAL4 transactivation system (Dodd et al., 2006; Gardner et al., 2009), we observed no gross alterations in the visible phenotype of the plants when they were transformed with yellow fluorescent protein fused to apoaequorin (YFPAPOAEQUORIN). The tissue-specific localization of GFP in the selected enhancer trap lines and, therefore, the restriction of the GAL4 transactivation in all the cell-specific driver lines analyzed here was described previously (Gardner et al., 2009; see Fig. 6A). In this study, YFPAPOAEQUORIN was used to determine in which cells aequorin was expressed in each of the enhancer trap lines. The YFPAPOAEQUORIN fluorescence was detected only in the specific cells marked by GFP in the GAL4 driver lines. GFP/yellow fluorescent protein (YFP) fluorescence was not detected in other cells (Supplemental Figs. S1–S4). GFP and YFPAPOAEQUORIN were cotargeted to the living vascular cells, including the bundle sheath cells in KC274 (Supplemental Fig. S1; see also Fig. 6A in Gardner et al., 2009), the epidermal pavement cells of leaves in KC464 (Supplemental Fig. S2; see also Fig. 6A in Gardner et al., 2009), the leaf spongy mesophyll cells in JR11-2 (Supplemental Fig. S3; see also Fig. 6A in Gardner et al., 2009), and the trichomes in KC380 (Supplemental Fig. S4; see also Fig. 6A in Gardner et al., 2009). We also confirmed the localization of YFPAPOAEQUORIN specifically to mature stomatal guard cells in E1728, as described previously (Dodd et al., 2006). In all lines, the expression was restricted to the specific cells in the aerial tissues, with the exception of KC274, in which expression in the vascular tissues was detected in roots, shoots, and leaves.

YFPAPOAEQUORIN fluorescence was present in the cytosol and nucleus for all lines (Supplemental Figs. S1–S5), as reported previously by Dodd et al. (2006) for the E1728 GAL4-GFP enhancer trap line expressing YFPAPOAEQUORIN in guard cells. The GFP signal was excluded from the nucleoplasm due to an endoplasmic reticulum-targeting sequence in the mGFP5 variant used in the enhancer trap (Haseloff et al., 1997). The different subcellular localization allowed confirmation that the spectral separation of the GFP and YFP emission spectra was sufficient to conclude that there was colocalization of YFPAPOAEQUORIN and GFP to the same cell types, consistent with the enhancer trap expression pattern.

Localization of Nycthemeral and Circadian Oscillations of [Ca²⁺]_i

In 12-h-light and 12-h-dark cycles (LD), rhythms of aequorin bioluminescence were detected from the spongy mesophyll of JR11-2 (Fig. 1A), the trichomes of KC380 (Fig. 1B), the vascular cells of KC274 (Fig. 1C), the epidermal cells of KC464 (Fig. 1D), and the guard cells of E1728 (Fig. 1E). However, only the nycthemeral [Ca²⁺]_i oscillations reported from the spongy mesophyll cells had the dynamics of a peak in the middle to end of the photoperiod and anticipation of dawn in the night typical for nycthemeral rhythms of [Ca²⁺]_cyt in LD reported by CaMV35S:AEQUORIN (Love et al., 2004). In constant light (LL), free-running circadian rhythms of [Ca²⁺]_i with a period close to 24 h were detected in the JR11-2 enhancer trap line expressing YFPAPOAEQUORIN in spongy mesophyll cells in the presence of distilled water and mannitol (period in water, 22.1 ± 0.6 h, eight out of 12 plant clusters relative amplitude of error < 0.5, period in mannitol 22.6 ± 0.5 h, five out of six plant clusters relative amplitude of error < 0.5; Fig. 1A; Supplemental Fig. S6). The circadian oscillations of [Ca²⁺]_i had the characteristics of circadian oscillations of [Ca²⁺]_cyt detected previously in plants constitutively expressing CaMV35S::APOAEQUORIN, including peaking in the middle to end of the subjective day (Love et al., 2004), a declining amplitude (Dalchau et al., 2010), and being abolished by 3% (w/v) Suc (Johnson et al., 1995; no rhythm detected from six out of six plant clusters; Fig. 1A; Supplemental Fig. S6). No circadian oscillations of [Ca²⁺]_i were detected in LL under any of the experimental conditions tested when GAL4-transactivated YFPAPOAEQUORIN was targeted specifically to the trichome, vascular, epidermal pavement, and guard cells (no rhythms detected from four out of four plant clusters tested for each line; Fig. 1, B–E). Due to the low signal-to-noise ratio of the enhancer trap lines, we were unable to calibrate the AEQUORIN signal during the long-time-course circadian analysis, but in previous studies, we estimated that the peak [Ca²⁺]_i is around 300 nm (Love et al., 2004).

Figure 1.

Daily [Ca²⁺]_i oscillations in the spongy mesophyll cells of the JR11-2 enhancer trap line (A), in the trichome cells of the KC380 enhancer trap line (B), in the vascular cells of the KC274 enhancer trap line (C), in the epidermal cells of the KC464 enhancer trap line (D), and in the guard cells of the E1728 enhancer trap line (E). Agar media supplements are as follows: white symbols, deionized water (dH₂O); gray symbols, 90 mm mannitol (Man) control; black symbols, 90 mm Suc. Light conditions are as follows: black bars, darkness; white bars, light; gray bars, subjective dark under LL. n ≥ 4. Data are presented as means ± se.

To determine whether the changes observed in AEQUORIN light emission over time were due to changes in [Ca²⁺]_i or APOAEQUORIN expression, total AEQUORIN was quantified for all the lines at 7 to 9 h and 21 to 23 h after dawn in LD. This is the condition in which the amplitude of light emission was greatest and in which all the lines had cyclic light emission. There was no significant difference in total AEQUORIN pool size between the time points, representing the timing of the minima and maxima of the diel oscillations of light signal. The nonsignificant trend was for the AEQUORIN pool to be largest when the diel light emission was smallest. These data indicate that the oscillations in light emission we detected are a consequence of changes in [Ca²⁺]_i rather than AEQUORIN pool size (Supplemental Table S1).

Analysis of Stimulus-Induced [Ca²⁺]_i Increases in Spongy Mesophyll, Pavement Epidermis, Vasculature, Trichomes, and Guard Cells

Mechanical stimulation resulted in an immediate and rapid rise of [Ca²⁺]_i levels in all plants tested of KC464 in which YFPAPOAEQUORIN was targeted to the epidermal pavement cells (n = 12; Supplemental Fig. S7), with a mean peak value of 646.6 ± 76.2 nm (Fig. 2A). The magnitude of the mechanical stimulation-induced increase in [Ca²⁺]_i was smaller in all other tissues (approximately 250–370 nm; Fig. 2). For all tissues other than the epidermal pavement cells, not all the plants responded to the mechanical stimulation: seven out of 12, three out of 10, four out of 10, and seven out of 13 plants responded to mechanical stimulation in guard, mesophyll, trichome, and vascular cell enhancer trap lines, respectively (Supplemental Figs. S8–S11). In all tissues, when a response to mechanical stimulation was detected, it comprised an immediate transient elevation of [Ca²⁺]_i that returned to resting within 20 s of stimulation (Fig. 2). Occasionally, following the transient, subsequent [Ca²⁺]_i changes of low amplitude were detected in epidermal pavement cells (Supplemental Fig. S7).

Figure 2.

Average [Ca²⁺]_i response to mechanical stimulation (1 mL of deionized water applied from 30 s to 35 s). A, Pavement epidermal cells of the KC464 enhancer trap line (n = 12). B, Guard cells of the E1728 enhancer trap line (n = 12). C, Spongy mesophyll cells of the JR11-2 enhancer trap line (n = 10). D, Trichome cells of the KC380 enhancer trap line (n = 10). E, Vascular cells of the KC274 enhancer trap line (n = 13). Data are presented as means ± se.

In Arabidopsis seedlings expressing CaMV35S::APOAEQUORIN, NaCl results in a [Ca²⁺]_cyt signature that consists of a rapid rise of [Ca²⁺]_cyt levels immediately followed by a slower relaxation (Knight et al., 1997; Kiegle et al., 2000; Tracy et al., 2008). Here, we demonstrate that it is possible to partially deconvolute the NaCl-induced [Ca²⁺]_i signatures based on the intensity and the pattern of the [Ca²⁺]_i response in the cell types providing differential contributions to the whole-plant NaCl-induced [Ca²⁺]_i signature. In epidermal cells, only one component of the NaCl-induced [Ca²⁺]_i signature was discernible, a rapid rise of [Ca²⁺]_i for approximately 20 to 30 s, peaking at a mean of 1,381.0 ± 66.6 nm (Fig. 3A). Subtracting the mean mechanical stimulation-induced increase derives a NaCl-specific [Ca²⁺]_i transient with a mean peak of 735.0 ± 66.6 nm in epidermal pavement cells. A large transient NaCl-induced increase in [Ca²⁺]_i was not detected in guard, trichome, and mesophyll cells but was detected from vascular cells (Fig. 3). The small NaCl-induced initial [Ca²⁺]_i transients detected in guard, trichome, and mesophyll cells can be ascribed to mechanical stimulation (compare Fig. 2, B–D, and Fig. 3, B–D). In all cell types except the epidermal pavement cells, sustained increases in [Ca²⁺]_i following NaCl stimulation were detected (Fig. 3). Vascular cells had a similar pattern of [Ca²⁺]_i dynamics to previous reports from plants expressing CaMV35S::APOAEQUORIN (Knight et al., 1997; Kiegle et al., 2000; Tracy et al., 2008), a transient increase in [Ca²⁺]_i that reached 778.0 ± 90.4 nm (527.8 ± 90.4 nm after correction for mechanical stimulation) followed by a gradual decrease over 150 s (Fig. 3E). The dynamics of the NaCl-induced [Ca²⁺]_i signals from mesophyll and vasculature cells were more complex, with mesophyll cells reporting a sustained [Ca²⁺]_i peaking 120 s after stimulation, whereas in the vasculature, there were prolonged relaxation kinetics following the initial transient (Fig. 3, C and E). However, for both mesophyll and vasculature, the mean average behavior obscures more complex dynamics reported by individual plants (Supplemental Figs. S12–S16). When the data from individual plants were analyzed, in both spongy mesophyll and vasculature cells, complex oscillatory [Ca²⁺]_i signals were induced by NaCl (Supplemental Figs. S14 and S16).

Figure 3.

Average [Ca²⁺]_i response to salt stress (200 mm NaCl applied from 30–35 s). A, Pavement epidermal cells of the KC464 enhancer trap line. B, Guard cells of the E1728 enhancer trap line. C, Spongy mesophyll cells of the JR11-2 enhancer trap line. D, Trichome cells of the KC380 enhancer trap line. E, Vascular cells of the KC274 enhancer trap line. Data are presented as means ± se. n = 10 in each case.

H₂O₂ induced a characteristic biphasic increase in [Ca²⁺]_i comprising a transient increase in [Ca²⁺]_i (“spike”) followed by a more prolonged elevation of [Ca²⁺]_i peaking around 40 to 50 s after injection in the range of 200 nm for all tissues tested (Fig. 4; Supplemental Figs. S17–S21). However, the peak values and dynamics of the initial [Ca²⁺]_i spike or transient (519.9 ± 58.0, 322.0 ± 77.9, 149.0 ± 31.5, 153.0 ± 4.4, and 200.8 ± 26.5 nm for epidermal, guard, mesophyll, trichome, and vasculature cells, respectively) were very similar to those obtained after mechanical stimulation. Therefore the H₂O₂-specific [Ca²⁺]_i signal comprises a slow rise in [Ca²⁺]_i followed by sustained elevation without cell-specific dynamics (Fig. 4).

Figure 4.

Average [Ca²⁺]_i response to H₂O₂ (3.3 mm H₂O₂ applied from 30 s to 35 s). A, Pavement epidermal cells of the KC464 enhancer trap line. B, Guard cells of the E1728 enhancer trap line. C, Spongy mesophyll cells of the JR11-2 enhancer trap line. D, Trichome cells of the KC380 enhancer trap line. E, Vascular cells of the KC274 enhancer trap line. Data are presented as means ± se. n = 10 in each case.

All the plants tested of all the enhancer trap lines (10 out 10 for each specific cell type) reported cold shock-induced [Ca²⁺]_i increases (Supplemental Figs. S22–S26), with a very large mean peak height compared with mechanical stimulation and NaCl, resulting in a peak reaching [Ca²⁺]_i values of 1,242.3 ± 40.0, 1,168.3 ± 77.3, 688.3 ± 49.1, 709.7 ± 47.4, and 895.1 ± 42.8 nm for epidermal, guard, mesophyll, trichome, and vasculature cells, respectively (595.7 ± 40.0, 810.3 ± 77.3, 357.3 ± 49.1, 339.6 ± 47.4, and 644.9 ± 42.8 nm after mechanical stimulation correction; Fig. 5). There was no second component after cold stimulus in any of the cell lines analyzed.

Figure 5.

Average [Ca²⁺]_i response to cold stress (1 mL of cold deionized water applied from 30 s to 35 s). A, Pavement epidermal cells of the KC464 enhancer trap line. B, Guard cells of the E1728 enhancer trap line. C, Spongy mesophyll cells of the JR11-2 enhancer trap line. D, Trichome cells of the KC380 enhancer trap line. E, Vascular cells of the KC274 enhancer trap line. Data are presented as means ± se. n = 10 in each case.

DISCUSSION

Using GAL4 Transactivation of Aequorin to Measure [Ca²⁺]_i in Specific Cell Types

We describe a suite of cell-specific aequorin reporter lines that permit the measurement of [Ca²⁺]_i in leaves. The strong regulation by the GAL4 UAS drove sufficiently high aequorin expression to permit reproducible measurement of [Ca²⁺]_i from relatively small populations of cells (e.g. trichomes or guard cells), cells that are rich in chlorophyll (spongy mesophyll), and tissues deep within the plant (vascular bundle). GAL4 is a yeast transcription factor with no orthologs in plants; therefore, GAL4 is unlikely to induce artifacts through the regulation of endogenous genes. This technical advance has permitted the measurement of cell- and stimulus-specific [Ca²⁺]_i signals in Arabidopsis leaves.

Using GAL4 Transactivation of Aequorin to Measure Daily [Ca²⁺]_i Rhythms in Specific Leaf Cell Types

A major motivation of this study was to determine the source of circadian oscillations of [Ca²⁺]_i within the plant. It has been suggested that circadian oscillations of [Ca²⁺]_cyt have the potential to be both an output and an input to the circadian oscillator (Johnson et al., 1995; Dodd et al., 2007). To understand the role of [Ca²⁺]_cyt in the circadian network, it is necessary to determine specifically in which cells [Ca²⁺]_cyt functions in the circadian clock. All plant cells are believed to have circadian rhythms that are cell autonomous (Dodd et al., 2004; Yakir et al., 2011). There is evidence of tissue-specific circadian behavior; for example, the vascular bundle circadian clock appears to have a different genetic architecture compared with other tissues (Para et al., 2007). We previously reported that in the timing of chlorophyll a/b binding protein expression1-1 mutant the circadian rhythms of [Ca²⁺]_cyt become uncoupled from those of CHLOROPHYLL A/B BINDING PROTEIN2 expression, demonstrating the presence of at least two circadian oscillators running at different speeds (Xu et al., 2007). We speculated that these different circadian oscillators might be located in different cell types (Xu et al., 2007). Here, using GAL4 transactivation, we demonstrate that the predominant aequorin signal in the measurement of circadian oscillations of [Ca²⁺]_i arises from the spongy mesophyll (Fig. 1A). This is consistent with our previous imaging of aequorin bioluminescence from plants constitutively expressing aequorin, which demonstrated that daily and circadian [Ca²⁺]_cyt rhythms arise from leaf tissues (Love et al., 2004).

We were unable to detect circadian oscillations of [Ca²⁺]_i in other leaf tissues. Our inability to detect circadian oscillations of [Ca²⁺]_i from epidermal pavement cells, trichomes, guard cells, and the vascular bundle does not necessarily mean that circadian oscillations of [Ca²⁺]_i are absent in those tissues. The dynamic range of circadian oscillations of [Ca²⁺]_cyt is about 200 nm (Love et al., 2004); therefore, it is possible that we did not detect relatively low-amplitude changes in [Ca²⁺]_i when aequorin was targeted to small populations of cells, but the signal-to-noise ratio was great enough when aequorin was targeted to the more populous spongy mesophyll cells. Nycthermeral oscillations of [Ca²⁺]_i that occur in light and dark cycles have a higher amplitude of oscillations than the free-running circadian rhythm of [Ca²⁺]_i that occurs in LL (Fig. 1A), and potentially nycthermeral [Ca²⁺]_i oscillations were detected from aequorin targeted specifically to the trichomes, guard cells, epidermal pavement cells, and vascular bundle (Fig. 1, B–E). It appears, therefore, that daily oscillations of [Ca²⁺]_i are common to all leaf cell types and that we were unable to detect low-amplitude circadian oscillations of [Ca²⁺]_i from cell types other than the spongy mesophyll due to the low signal-to-noise ratio from aequorin when it was targeted to smaller populations of cells. A caveat to this conclusion that daily rhythms of [Ca²⁺]_i can occur in all cell types is that only the spongy mesophyll cells reported a pattern of [Ca²⁺]_i oscillations in light and dark cycles that are consistent with those reported by constitutively targeted aequorin (Love et al., 2004). It was previously suggested that the major bioluminescent signal from tobacco (Nicotiana plumbaginifolia) transformed with aequorin arises mostly from the epidermal layers (Wood et al., 2001); our data demonstrate that this is not the case for circadian rhythms of [Ca²⁺]_i in Arabidopsis.

GAL4 Transactivation of Aequorin Identifies Cell- and Stimulus Type-Specific [Ca²⁺]_i Signals

The role of [Ca²⁺]_i in the response of Arabidopsis to stress signals, including mechanical stimulation, H₂O₂, NaCl, and cold, was first identified based on [Ca²⁺]_cyt elevations detected in seedlings constitutively expressing aequorin under the control of the CaMV35S promoter (Knight et al., 1991, 1996, 1997). The canonical [Ca²⁺]_cyt response measured in response to these stimuli in plants constitutively expressing aequorin in all tissues is a transient elevation immediately upon stimulation, to relatively high values (dependent on the strength and type of stimulus), followed by a rapid decline to intermediate values of [Ca²⁺]_cyt and subsequently often a second slower elevation and decline of the [Ca²⁺]_cyt signal, returning to resting values after a few minutes. The temporal dynamics of this “spike-shoulder” pattern of [Ca²⁺]_cyt signal exhibits considerable variation dependent on the signal applied (McAinsh and Pittman, 2009). In root tissues, it is apparent that this whole-plant response masks more complex cell-specific behaviors (Kiegle et al., 2000), notably in the endodermis and pericycle of roots in response to NaCl. It is likely that summation of the behavior of many individual cells probably obscures the underlying oscillatory nature of NaCl-induced [Ca²⁺]_cyt increases in root cells (Tracy et al., 2008).

GAL4 transactivation of aequorin to specific cell types has allowed partial deconvolution of the whole-plant [Ca²⁺]_i signal to identify cell specificity and the dynamics of [Ca²⁺]_i signals. We demonstrate that mechanical stimulation-induced [Ca²⁺]_i signals are largely restricted to the epidermal pavement cells in the leaf (Fig. 2; Supplemental Figs. S7–S11). The epidermis being the location of the major mechanical stimulation-induced [Ca²⁺]_i signals is intuitively appealing, since wind-, rain-, and animal-induced mechanical stimulation signals will first occur at the epidermis (Braam, 2005). In our study, mechanical stimulation was applied by an application of room-temperature water with minimal force; therefore, it is possible that mechanical stimulation-induced [Ca²⁺]_i signals were not detected with the same intensity from tissues below the epidermis because the physical stimulus was attenuated during penetration through the leaf. Demonstration that epidermal pavement cells have a large mechanical stimulation-induced [Ca²⁺]_i transient allowed post-data-capture correction of pavement epidermal cell [Ca²⁺]_i signals to account for mechanical stimulation-induced signals when investigating the effects of other stimuli. For example, when the contribution to the [Ca²⁺]_i dynamics made by mechanical stimulation is taken into account, all cells, including the epidermal pavement cells, had an essentially monophasic response to H₂O₂ (compare Figs. 2 and 4; Supplemental Figs. S17–S21).

The detection of H₂O₂-mediated [Ca²⁺]_i increases in guard cells in E1728 (Fig. 4B) is consistent with the extensive literature linking plasma membrane NADPH oxidase-mediated H₂O₂ increases to abscisic acid-induced stomatal closure (Webb and Robertson, 2011). In guard cells, H₂O₂ increases are detected in the cytosol within 30 s of abscisic acid treatment and are thought to activate Ca²⁺ influx through plasma membrane hyperpolarization-activated Ca²⁺ channels (Pei et al., 2000). Chloroplast-derived H₂O₂ signals in response to high light, wounding, or infection with an incompatible hypersensitive response-inducing pathogen are localized specifically to the vascular bundle (Mullineaux et al., 2006). Similarly, the key high-light-induced antioxidant protein ASCORBATE PEROXIDASE2 is expressed specifically in the vascular bundle (Fryer et al., 2003). Further studies will be required to determine if the pathways that elevate [Ca²⁺]_cyt in response to chloroplast-derived H₂O₂ signals in the vasculator are similar to or different from those that generate [Ca²⁺]_cyt signals in the guard cell in response to plasma membrane-localized NADPH oxidase-derived H₂O₂ activity.

We previously suggested that the spike-shoulder pattern of [Ca²⁺]_cyt increase often measured using constitutively targeted aequorin might not represent the underlying [Ca²⁺]_cyt dynamics in single cells, due to the integration of potential cell-specific [Ca²⁺]_cyt signals and/or out-of-phase oscillatory [Ca²⁺]_cyt dynamics in single cells (Dodd et al., 2006). Here, we found evidence for stimulus- and cell type-specific [Ca²⁺]_i dynamics using GAL4 transactivation of aequorin. For example, NaCl caused oscillatory [Ca²⁺]_i signals specifically in spongy mesophyll and vascular cells (Supplemental Figs. S14 and S16). This is consistent with previous studies that found that NaCl caused oscillatory [Ca²⁺]_cyt signals specifically in the endodermis and pericycle of roots (Kiegle et al., 2000) and that oscillatory [Ca²⁺]_cyt dynamics are easier to resolve when measurements are made from smaller cell populations (Tracy et al., 2008). The NaCl-induced oscillatory [Ca²⁺]_i signals in the vasculature and spongy mesophyll cells are indicative of a role in signal transduction and the regulation of NaCl-regulated downstream responses. By contrast, in the guard cell, NaCl appears to alter only the steady-state [Ca²⁺]_i (Fig. 3B). The instantaneous nature of the change in steady-state [Ca²⁺]_i in response to NaCl in the guard cell is suggestive of an alteration in plasma membrane potential as a consequence of the increased external NaCl concentration. Increasing NaCl concentration will cause Na⁺ influx through nonselective cation channels and could possibly result in blockage of inward rectifying K⁺ currents, which in turn will result in a more hyperpolarized plasma membrane potential due to reduced depolarizing influx of K⁺ (Véry et al., 1998). In other systems, the blockage of guard cell inward rectifying K⁺ currents by cytosolic Na⁺ has taken longer to develop than the nearly instantaneous effect on [Ca²⁺]_i seen here (Véry et al., 1998); therefore, other mechanisms also might be at work.

For cold stimulation, there was consistency in the dynamics of response across cell types, similar to the dynamics described previously for whole plants, root-specific cell types, and guard cell populations (Knight et al., 1991, 1996; Kiegle et al., 2000; Dodd et al., 2006), suggesting that there are not cell-specific responses to cold in terms of Ca²⁺ signaling.

CONCLUSION

Here, we report the use of GAL4 transactivation of aequorin to analyze [Ca²⁺]_i signaling in specific cell types, including those of the leaves. We identify a high degree of specialization in Ca²⁺ signaling networks within different cell types and also commonalities. Our data suggest commonality in the pathways by which cold, H₂O₂, and possibly daily timing signals are transduced within cells but identify cell-specific dynamics to [Ca²⁺]_i signals induced by mechanical stimulation and NaCl. The library of GAL4-mediated aequorin transactivation lines with root- and now leaf/shoot cell-specific aequorin expression offers the opportunity to dissect in detail the role of Ca²⁺ signals in cell-specific stimulus-response coupling.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Seeds of the Arabidopsis (Arabidopsis thaliana) guard cell-specific GAL4 GFP enhancer trap line E1728 expressing YFPAPOAEQUORIN under the control of the GAL4 UAS promoter (Dodd et al., 2006) and vascular cell-specific KC274, trichome cell-specific KC380, epidermal cell-specific KC464, and spongy mesophyll cell-specific JR11-2 GAL4 GFP enhancer trap lines (Gardner et al., 2009) were used in this study. Seeds were surface sterilized by washing for 1 min in 100% (v/v) ethanol and then incubating them for 10 min in 50% (v/v) sodium hypochlorite followed by three washes with autoclaved distilled water. Seeds were sown on 0.5× Murashige and Skoog nutrient mixture dissolved in 0.8% agar (w/v), unless otherwise stated. Germination was synchronized by stratification at 4°C for 2 d in the dark. Seedlings were transferred to MLR30 growth chambers (Sanyo) and grown in LD at 19°C; the light intensity was 50 µmol m⁻² s⁻¹.

Generation and Selection of Lines Expressing Aequorin in Specific Leaf Cells

KC274, KC380, KC464, and JR11-2 were transformed with pBINYFPAEQ containing the YFPAPOEQUORIN fusion downstream from a GAL4 UAS (Kiegle et al., 2000) as described by Dodd et al. (2006). Homozygous transformants were selected by growth on medium containing 40 µg mL⁻¹ hygromycin. Lines were selected for high total aequorin activity by destructive measurement of total aequorin activity in leaf samples of T1 and T2 seedlings by discharging all available aequorin in the presence of 1 m CaCl₂ dissolved in 10% (v/v) ethanol (discharge solution) and measuring aequorin bioluminescence in a multifunctional microplate reader (FluoStar OPTIMA; BMG LabTech). Imaging of GFP and YFP to confirm the colocalization of GFP and YFPAPOAEQUORIN was performed as described by Kiegle et al. (2000), and GFP and YFP were collected using emission windows of 495 to 525 nm and 595 to 650 nm, respectively.

Measurement of [Ca²⁺]_i and Statistical Analysis

Imaging of circadian oscillations of [Ca²⁺]_i and the growth conditions, light regimes, and entrainment regime for circadian measurements were as described by Dodd et al. (2007). Circadian rhythms of aequorin luminescence, corresponding to circadian rhythms in [Ca²⁺]_i, were analyzed using the software based on the fast Fourier transform-nonlinear least-squares method described by Plautz et al. (1997) using the Biological Rhythms Analysis Software System.

Luminometry of changes in [Ca²⁺]_i in response to mechanical stimulation, cold, NaCl, and H₂O₂ and subsequent calibration of bioluminescence to estimate [Ca²⁺]_i were measured as follows. In order to reconstitute aequorin, 14-d-old seedlings transformed with GAL4 UAS:YFPAPOAEQUORIN were incubated overnight in the dark at room temperature in 500 µL of 20 µm coelenterazine free base (Nanolight) dissolved in distilled water within a luminometer tube (51 mm long × 12 mm diameter; Sarstedt). Bioluminescence was measured using a photon-counting luminometer (photomultiplier tube 9899A) cooled to −20°C with a FACT50 housing (Electron Tubes). Injections were performed from a 1-mL light-tight syringe attached to a 75-mm needle inserted into a light-tight port in the luminometer sample housing at a distance of 40 mm approximately over the plants. Response to cold was determined by injecting 1 mL of 4°C distilled water onto the plants described above. Response to NaCl and H₂O₂ was determined by injecting 1 mL of the solutions onto the plants to reach final concentrations of 200 and 3.3 mm, respectively. The injection of 1 mL of room-temperature distilled water was used as a touch response control for all the treatments. All the stimuli injections were performed at 30 s over a 5-s interval followed after 300 s by the injection of 1 mL of discharge solution. Measurements were made until the detected luminescence reached 10% of the first peak after discharge injection. [Ca²⁺]_i levels were determined according to Fricker et al. (1999).

Nycthemeral and circadian [Ca²⁺]_i oscillations were determined in four independent experiments with at least four independent replicates in each; mean data for one of the experiments are reported. [Ca²⁺]_i levels in response to stimulation are means of two independent experiments with at least 10 independent replicates per treatment and line.

Supplemental Data

The following materials are available in the online version of this article.

• Supplemental Figure S1. Localization of GFP and YFPAPOAEQUORIN in the vascular cells of the KC274 enhancer trap line.

• Supplemental Figure S2. Localization of GFP and YFPAPOAEQUORIN in the pavement epidermal cells of the KC464 enhancer trap line.

• Supplemental Figure S3. Localization of GFP and YFPAPOAEQUORIN in the spongy mesophyll cells of the JR11-2 enhancer trap line.

• Supplemental Figure S4. Localization of GFP and YFPAPOAEQUORIN in the trichome cells of the KC380 enhancer trap line.

• Supplemental Figure S5. Different spatial localization of the GFP and YFPAPOAEQUORIN fluorescence signals in the cells.

• Supplemental Figure S6. Analysis of the circadian [Ca²⁺]_i oscillations in the spongy mesophyll cells of the JR11-2 enhancer trap line.

• Supplemental Figure S7. [Ca²⁺]_i response to mechanical stimulation in pavement epidermal cells.

• Supplemental Figure S8. [Ca²⁺]_i response to mechanical stimulation in guard cells.

• Supplemental Figure S9. [Ca²⁺]_i response to mechanical stimulation in spongy mesophyll cells.

• Supplemental Figure S10. [Ca²⁺]_i response to mechanical stimulation in trichome cells.

• Supplemental Figure S11. [Ca²⁺]_i response to mechanical stimulation in vascular cells.

• Supplemental Figure S12. [Ca²⁺]_i response to salt stress in pavement epidermal cells.

• Supplemental Figure S13. [Ca²⁺]_i response to salt stress in guard cells.

• Supplemental Figure S14. [Ca²⁺]_i response to salt stress in spongy mesophyll cells.

• Supplemental Figure S15. [Ca²⁺]_i response to salt stress in trichome cells.

• Supplemental Figure S16. [Ca²⁺]_i response to salt stress in vascular cells.

• Supplemental Figure S17. [Ca²⁺]_i response to H₂O₂ in pavement epidermal cells.

• Supplemental Figure S18. [Ca²⁺]_i response to H₂O₂ in guard cells.

• Supplemental Figure S19. [Ca²⁺]_i response to H₂O₂ in spongy mesophyll cells.

• Supplemental Figure S20. [Ca²⁺]_i response to H₂O₂ in trichome cells.

• Supplemental Figure S21. [Ca²⁺]_i response to H₂O₂ in vascular cells.

• Supplemental Figure S22. [Ca²⁺]_i response to cold stress in pavement epidermal cells.

• Supplemental Figure S23. [Ca²⁺]_i response to cold stress in guard cells.

• Supplemental Figure S24. [Ca²⁺]_i response to cold stress in spongy mesophyll cells.

• Supplemental Figure S25. [Ca²⁺]_i response to cold stress in trichome cells.

• Supplemental Figure S26. [Ca²⁺]_i response to cold stress in vascular cells.

• Supplemental Table S1. Quantification of total AEQUORIN in light/dark cycles.

Glossary

[Ca²⁺]_i

intracellular concentration of Ca²⁺

[Ca²⁺]_cyt

cytosolic-free Ca²⁺

UAS

upstream activation sequence

H₂O₂

hydrogen peroxide

cADPR

cyclic ADP ribose

YFPAPOAEQUORIN

yellow fluorescent protein fused to apoaequorin

YFP

yellow fluorescent protein

LD

12-h-light and 12-h-dark cycles

LL

constant light