Cell- and stimulus-type-specific intracellular-free Ca 2+ signals in Arabidopsis thaliana

One sentence summary: Enhancer-trap targeting of aequorin to specific cell types identifies stimulus- and cell-specific [Ca 2+ ] i signalling in Arabidopsis. ABSTRACT Appropriate stimulus-response coupling requires 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 signalling 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, H 2 O 2 , NaCl and cold in plants and also in circadian timing. We use GAL4 transactivation of aequorin in enhancer trap lines of Arabidopsis thaliana to test the hypothesis that stimulus- and cell-specific information can be encoded in the pattern of dynamical alterations in the concentration of intracellular-free ([Ca 2+ ] i ) . We demonstrate that mechanically-induced increases in [Ca 2+ ] i are largely restricted to the epidermal pavement cells of leaves, that NaCl induces oscillatory [Ca 2+ ] i signals in spongy mesophyll and vascular bundle cells, but not other cell types and detect circadian rhythms of [Ca 2+ ] i only in the spongy mesophyll. We demonstrate stimulus-specific [Ca 2+ ] i dynamics in response to touch, cold and H 2 O 2 , 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 Ca 2+ reporter dyes in chlorophyll-containing tissues to identify cell- and stimulus-specific complexity of [Ca 2+ ] i dynamics in leaves of Arabidopsis and determine from which tissues stress and circadian-regulated [Ca 2+ ] i signals arise.


INTRODUCTION
Ca 2+ is an important second messenger involved in a wide range of responses in plants (Dodd et al., 2010). Fluorescent reporters of Ca 2+ have revolutionized the study of Ca 2+ signaling, permitting measurement of the intracellular concentration of Ca 2+ ([Ca 2+ ] i ) with high sub-cellular spatial and temporal resolution. In plants, the use of fluorescent reporters has identified stimulus-induced increases in [Ca 2+ ] i that encode information in the temporal and sub-cellular distribution of the [Ca 2+ ] i signal (Dodd et al., 2010).
However, the utility of fluorescent Ca 2+ 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 more, in situ measurements of [Ca 2+ ] 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 and co-workers introduced recombinant aequorin as a reporter of [Ca 2+ ] in plant systems (Knight et al., 1991). Aequorin is a bioluminescent reporter and therefore has an intrinsically high signal to noise ratio, and requires no damaging excitation light and is therefore 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 [Ca 2+ ] cyt signalling in mechanical stimulation, cold, salinity and pathogen stress and in the daily timing of plants (Campbell et al., 1996;Knight et al., 1996;Knight et al., 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 [Ca 2+ ] i signals arise.
We describe targeting of the in vivo Ca 2+ 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 A. thaliana, the yeast GAL4 transcriptional activator is inserted 6 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 GREEN FLUORESCENT PROTEIN (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 cellspecific expression of any transgene downstream from another GAL4 UAS. These GAL4 UAS-driven transgenes are introduced by super transformation or crossing.
We test the hypothesis that there is cell-and tissue-specificity of the [Ca 2+ ] i signalling networks in A. thaliana. 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 H 2 O 2 because all cause a rapid increase in [Ca 2+ ] i followed by dynamical alterations of [Ca 2+ ] i when measured in seedlings in which aequorin has been targeted to the whole seedling (Lynch et al., 1989;Knight et al., 1996;Knight et al., 1997;Price et al., 2004;Monshausen et al., 2009). Additionally we investigate from which cells diel oscillations of [Ca 2+ ] i arise. Diel and circadian oscillations of [Ca 2+ ] 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 [Ca 2+ ] cyt peak at around 300 nM about 8 h after dawn (Love et al., 2004). The circadian clock and PHYTOCHROME Aand CRYPTOCHROME1 and 2-dependent signalling pathways drive daily rhythms of [Ca 2+ ] cyt (Dalchau et al., 2010) with cyclic adenosine diphosphate ribose (cADPR) acting as an intermediary (Dodd et al., 2007) (Johnson et al., 1995;Wood et al., 2000;Wood et al., 2001;Love et al., 2004;Dodd et al., 2006;Dodd et al., 2010;Zhu et al., 2013). In this study we identify cell-and stimulus-specific [Ca 2+ ] i signals in the leaf and other tissues of A. thaliana, suggesting the presence of cell-specific signaling cassettes in plants.

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 CaMV35S promoter or using the GAL4 transactivation system (Dodd et al., 2006;Gardner et al., 2009), we observed no gross alterations in 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 previously described in Gardner et al. (2009) (see Figure 6A Gardner et al., 2009). 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/YFP fluorescence was not detected in other cells (Supplemental Figs. S1-S4). GFP and YFPAPOAEQUORIN were co-targeted to the living vascular cells, including the bundle sheath cells in KC274 (Supplemental Fig. S1; see also  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 previously reported 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 ER 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 co-localisation of YFPAPOAEQUORIN and GFP to the same cell types, consistent with the enhancer trap expression pattern.

Localization of Nycthemeral and Circadian Oscillations of [Ca 2+ ] 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  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) sucrose (Johnson et al., 1995) (no rhythm detected from 6 out of 6 plant clusters) ( were unable to calibrate the AEQUORIN signal during the long time course circadian analysis, but in previous studies we estimated that the peak [Ca 2+ ] i is around 300 nM (Love et al., 2004).
To determine whether the changes observed in AEQUORIN light emission over time were due to changes in [Ca 2+ ] i or APOAEQUORIN expression, total AEQUORIN was quantified for all the lines at 7-9 h and 21-23 h after dawn in LD cycles. 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 non-significant 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 [Ca 2+ ] i rather than AEQUORIN pool size (Supplemental Table S1). In Arabidopsis seedlings expressing CaMV35S::APOAEQUORIN, NaCl results in a [Ca 2+ ] cyt signature which consists in a rapid rise of [Ca 2+ ] 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 [ Ca

Using GAL4 Transactivation of Aequorin to measure [Ca 2+ ] i in Specific Cell Types
We describe a suite of cell-specific aequorin reporter lines that permit measurement of have the potential to be both an output, and an input in to the circadian oscillator (Johnson et al., 1995;Dodd et al., 2007). To understand the role of [Ca 2+ ] cyt in the circadian network it is necessary to determine specifically in which cells [Ca 2+ ] cyt functions in the circadian clock. All plant cells are believed to have circadian rhythms which are cell autonomous Yakir et al., 2011). There is evidence of tissue-specific circadian behaviour, for example the vascular bundle circadian clock appears to have a different genetic architecture compared to other tissues (Para et al., 2007). We previously reported that in the timing of cab expression 1-1 mutant the circadian rhythms of [Ca 2+ ] 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 [Ca 2+ ] 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 [Ca 2+ ] cyt rhythms arise from leaf tissues (Love et al., 2004 (Love et al., 2004) and therefore it is possible that we did not detect relatively low amplitude changes in [Ca 2+ ] 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 [Ca 2+ ] i that occur in light and dark cycles have a higher amplitude of oscillations than the free running circadian rhythm of [Ca 2+ ] i that occur in LL (Fig. 1A) and potentially nycthermeral [Ca 2+ ] i oscillations were detected from aequorin targeted specifically to the trichomes, guard cells, epidermal pavement cells and vascular bundle (Fig. 1B-E). It appears therefore that daily oscillations of [Ca 2+ ] i are common to all leaf cell types and that we were unable to detect low-amplitude circadian oscillations of [Ca 2+ ] i from celltypes 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 [Ca 2+ ] i can occur in all cell types is that only the spongy mesophyll cells reported a pattern of [Ca 2+ ] 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 this is not the case for circadian rhythms of [Ca 2+ ] i in Arabidopsis.

GAL4 Transactivation of Aequorin Identifies Cell-and Stimulus-type Specific [Ca 2+ ] i Signals
The role of [Ca 2+ ] i in the response of Arabidopsis to stress signals including mechanical stimulation, H 2 O 2 , NaCl and cold was first identified based on [Ca 2+ ] cyt elevations detected in seedlings constitutively expressing aequorin under the control of the CaMV35S promoter (Knight et al. 1996;Knight et al., 1997;Knight et al., 2005) subsequently often a second slower elevation and decline of the [Ca 2+ ] cyt signal, returning to resting values after a few minutes. The temporal-dynamics of this "spikeshoulder" pattern of [Ca 2+ ] cyt signal exhibits considerable variation dependent on the signal applied (McAinsh and Pittman, 2008). In root tissues it is apparent that this whole plant response masks more complex cell-specific behaviours (Kiegle et al., 2000), notably in the endodermis and pericycle of roots in response to NaCl. It is likely hyperpolarisation activated Ca 2+ channels (Pei et al., 2000). Chloroplast-derived H 2 O 2 signals in response to high light, wounding or infection with an incompatible hypersensitive response-inducing pathogen are localised specifically to the vascular bundle (Mullineaux et al., 2006). Similarly the key high light-induced antioxidant protein ASCORBATE PEROXIDASE 2 is expressed specifically in the vascular bundle (Fryer et al., 2003). Further studies will be required to determine if the pathways that elevate [Ca 2+ ] cyt in response to chloroplast-derived H 2 O 2 signals in the vasculator are similar or different to those that generate [Ca 2+ ] cyt signals in the guard cell in response to plasma membrane localised NADPH oxidase-derived H 2 O 2 activity.
We previously suggested that the "spike-shoulder" pattern of [Ca 2+ ] cyt increase often measured using constitutively targeted aequorin might not represent the underlying [Ca 2+ ] cyt dynamics in single cells due to the integration of potential cell-specific [Ca 2+ ] cyt signals and/or out of phase oscillatory [Ca 2+ ] cyt dynamics in single cells (Dodd et al., 2006). Here, we found evidence for stimulus-and cell-type specific [Ca 2+ ] i dynamics using GAL4-transactivation of aequorin. For example, NaCl induced oscillatory [Ca 2+ ] i signals specifically in spongy mesophyll and vascular cells (Supplemental Fig. S14 and Supplemental Fig. S16). This is consistent with previous studies that found NaCl induced oscillatory [Ca 2+ ] cyt signals specifically in the endodermis and pericycle of roots (Kiegle et al., 2000) and that oscillatory [Ca 2+ ] cyt dynamics are easier to resolve when measurements are made from smaller cell populations (Tracy et al., 2008). The NaCl-induced oscillatory [Ca 2+ ] i signals in the vasculature and spongy mesophyll cells are indicative of a role in signal transduction and regulation of NaCl-regulated downstream responses. By contrast in the guard cell, NaCl appears to alter only the steady-state [Ca 2+ ] i (Fig. 3B). The instantaneous nature of the change in steady-state [Ca 2+ ] 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]. Increasing [NaCl] will cause Na + influx through non selective cation channels and could possibly result in blockage of IK in , which in turn will result in a more hyperpolarised plasma membrane potential due to reduced depolarising influx of K + (Véry et al., 1998). In other systems the blockage of guard cell IK in by cytosolic Na + has taken longer to develop than the near instantaneous effect on [Ca 2+ ] i seen here (Véry et al., 1998) and therefore other mechanisms also might be at work. For cold stimulation there was consistency in the dynamics of response across cell types, similar in dynamics described previously for whole plants, root specific-cell types and guard cell populations (Knight et al., 1996;Kiegle et al., 2000;Knight et al., 2005;Dodd et al., 2006) suggesting that there are not cell-specific responses to cold in terms of Ca 2+ signalling.

CONCLUSION
Here we report the use of GAL4-transactivation of aequorin to analyze [Ca 2+ ] i signalling in specific-cell types, including those of the leaves. We identify a high degree of specialization in Ca 2+ signaling networks within different cell types and also commonalities. Our data suggest commonality in the pathways by which cold, H 2 O 2 and possibly daily timing signals are transduced within cells, but identify cell-specific dynamics to [Ca 2+ ] 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 offer the opportunity to dissect in detail the role of Ca 2+ signals in cell-specific stimulus-response coupling.

Plant Material and Growth Conditions
Seeds of Arabidopsis thaliana guard-cell specific GAL4 GFP enhancer trap line E1728 expressing YFPAPOAEQUORIN under the control of the GAL4 upstream activation sequence (UAS) promoter (Dodd et al., 2006), and vascular-cell specific KC274, trichome-cell specific KC380, epidermal-cell specific KC464 and spongy mesophyllcell specific JR11-2 GAL4 GFP enhancer trap lines (Gardner et al., 2009)  followed by three washes with autoclaved distilled water. Seeds were sown on 0.5 X Murashige and Skoog nutrient mixture dissolved in 0.8% agar (w/v), unless otherwise stated. Germination was synchronized by stratification at 4ºC for 2 days in the dark.
Seedlings were transferred to MLR30 growth chambers (Sanyo, Japan) and grown in 12 h light/12 h dark 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 upstream activation sequence (Kiegle et al., 2000) as described in Dodd et al. (2006). Homozygous transformants were selected by growth on media containing hygromycin 40 µg ml -1 . 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 CaCl 2 dissolved in 10% (v/v) ethanol (discharge solution), and measuring aequorin bioluminescence in a multifunctional microplate reader (FluoStar OPTIMA, BMG LabTech, Germany). Imaging of GFP and YFP to confirm colocalization of GFP and YFPAPOAEQUORIN was performed as described in Kiegle et al. (2000) and GFP and YFP were collected using an emission window of 495 -525 nm and 595 -650 nm, respectively.

Measurement of [Ca 2+ ] i and Statistical Analysis
Imaging of circadian oscillations of [Ca 2+ ] i and the growth conditions, light regimes and entrainment regime for circadian measurements were as described in (Dodd et al., (2007). Circadian rhythms of aequorin luminescence, corresponding to circadian rhythms in [Ca 2+ ] i , were analyzed using the software based on the Fast Fourier Transform-Non Linear Least Squares method (FFT-NLLS) described in Plautz et al. (1997) 2+ ] i in response to mechanical stimulation, cold, NaCl and H 2 O 2 and subsequent calibration of bioluminescence to estimate [Ca 2+ ] i were measured as follows. In order to reconstitute aequorin, 14 day old seedlings transformed with GAL4UAS:YFPAPOAEQUORIN were incubated overnight in the dark at room temperature in 500 ul of 20 µM coelenterazine free base (Nanolight, UK) dissolved in distilled water within a luminometer tube (51 mm long × 12 mm diameter, Sarstedt, Leicester, UK). Bioluminescence was measured using a photon-counting luminometer (photomultiplier tube 9899A) cooled to -20ºC with a FACT50 housing (Electron Tubes, UK). 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 4ºC distilled water onto the plants described above. Response to NaCl and H 2 O 2 was determined by injecting 1 ml of the solutions onto the plants to reach a final concentration of 200 mM 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.

Luminometry of changes in [Ca
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. [Ca 2+ ] i levels were determined according to Fricker et al. (1999).
Nycthemeral and circadian [Ca 2+ ] i oscillations were determined in four independent experiments with at least four independent replicates in each; the mean data for one of the experiments are reported. [Ca 2+ ] i levels in response to stimulation are the mean of two independent experiments with at least 10 independent replicates per treatment and line.

Supplemental Data
The following material is available in the online version of this article.

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[Ca 2+ ] i response to cold stress (1 ml of cold deionized water applied from t = 30 s to t = 35 s) in vascular cells of the KC274 enhancer trap line. Each graph describes the data obtained from an individual seedling. Table S1. Quantification of total AEQUORIN in light-dark cycles.

Supplemental
Total AEQUORIN was quantified for all the lines at 7-9 h and 21-23 h after dawn in light-dark cycles, by discharging all available AEQUORIN in the presence of 1 M CaCl 2 dissolved in 10% (v/v) ethanol. AEQUORIN bioluminescence was measured in leaf samples of 11 day old seedlings in a photon-counting luminometer (photomultiplier tube 9899A) cooled to -20ºC with a FACT50 housing (Electron Tubes, UK). Data are presented as mean ± standard error. "N" represents the number of samples analyzed per enhancer trap line and time point. There was no significant difference in total AEQUORIN pool size between the time points (P > 0.05, Mann-Whitney Rank Sum Test).