Calcium signatures are decoded by plants to give specific gene responses


  • Helen J. Whalley,

    1. Cell Signalling Group, Paterson Institute for Cancer Research, The University of Manchester, Manchester, UK
    Search for more papers by this author
  • Marc R. Knight

    Corresponding author
    1. Durham Centre for Crop Improvement Technology, School of Biological and Biomedical Sciences, Durham University, Durham, UK
    • Cell Signalling Group, Paterson Institute for Cancer Research, The University of Manchester, Manchester, UK
    Search for more papers by this author

(Author for correspondence: tel +44 191 334 1224; email


Calcium is a ubiquitous cellular second messenger communicating information about the outside world to plant cells. In plants, many stimuli lead to a transient rise in intracellular calcium concentration, which is thought to activate the appropriate response (McAinsh & Pittman, 2009). It is certainly vital for survival that a plant is able to respond appropriately to any given stimulus. This leads to a conundrum, however: how is the cell able to distinguish between calcium elevations elicited by different stimuli? One attractive hypothesis is that the specific characteristics of different calcium elevations (‘calcium signatures’) might encode specific information in plants (Allen et al., 2001; Love et al., 2004; Miwa et al., 2006; McAinsh & Pittman, 2009; Dodd et al., 2010; Short et al., 2012). By correlating cytosolic free calcium concentration ([Ca2+]c) signature profiles to the expression of genes, researchers have postulated that that such higher-order information is encoded in [Ca2+]c signatures produced in response to, for example, ozone (Short et al., 2012), elicitors of defence (Lecourieux et al., 2005) or nod factors (Miwa et al., 2006). [Ca2+]c signature profiles are also hypothesized to encode information that controls stomatal aperture (Allen et al., 2001). Circadian and diurnal oscillations of [Ca2+]c have also been proposed to specify information on timing of cellular processes (Love et al., 2004). We have previously demonstrated that calcium is an intermediate between stimulus perception and gene expression in a number of situations, for example oxidative stress, cold and drought (Knight et al., 1996, 1997; Clayton et al., 1999; Rentel & Knight, 2004; Whalley et al., 2011). Comparison of the specific characteristics of the calcium signatures produced by each of these different stresses shows them to be substantially different (e.g. number of phases, magnitude and duration), consistent with the idea of calcium signature-encoded stimulus-specific information. A more direct approach to correlating patterns of calcium signatures to specific responses is to test the effect of signatures that are artificially imposed on plant cells. Reports in the literature have been few, but in one study, artificially imposed [Ca2+]c oscillations were shown to be able to be decoded by guard cells, with specific frequencies and amplitudes being required to mediate closure (Allen et al., 2001). We wished to combine this powerful approach of imposing calcium signatures upon plants with global measurement of gene expression to address the broader question of whether novel calcium signatures might lead to different transcriptomic responses. As the frequency of calcium oscillations has been shown to control the specificity of activation of certain transcription factors in mammalian cells (Dolmetsch et al., 1998), and oscillations in intracellular calcium have been reported in response to several stimuli in plants (Campbell et al., 1996; Moyen et al., 1998; Allen et al., 2001; Miwa et al., 2006), we hypothesized that different characteristics of calcium oscillations might define different transcriptomic responses in plants. The parameters of oscillations (amplitude, frequency and number of pulses) can be easily defined and compared, making this type of calcium signature ideal for addressing the question of whether calcium signatures can encode specificity to downstream gene expression.

Materials and Methods

Plant growth and treatment

Seeds of Arabidopsis thaliana (L.) Heynh. Columbia (Col-0) accession constitutively expressing apoaequorin under the 35S promoter (35S::Aeq) were obtained from Lehle Seeds (Round Rock, TX, USA) and grown as previously described (Whalley et al., 2011). Seven-day-old 35S::Aeq seedlings were reconstituted by floating on water containing 10 μM coelenterazine (stock 1% (v/v) methanol; LUX Biotechnology Ltd, Edinburgh, UK) in the dark at 21°C for 12–24 h. Seedlings were then transferred to an electroporation cuvette containing SM media (0.1 mM each of KCl, CaCl2, MgCl2), rested for at least 2 h and treated with electrical stimulation inside a dark box, and the resulting luminescence was recorded as described previously (Whalley et al., 2011). The following voltage regimes were used to produce each calcium signature (the length of the voltage pulse is shown in brackets) – signature 1: 9 V (0.5 s), 9 V (0.7 s), 10 V (0.4 s), 10 V (0.7 s), 10 V (0.7 s), 10 V (1.2 s), 10 V (1.5 s), 10 V (1.7 s), 10 V (2 s), 10 V (2.2 s); signature 2: 5 V (0.5 s), 6 V (0.5 s), 6 V (0.7 s), 6 V (0.9 s), 7 V (0.5 s), 7 V (0.5 s), 7 V (0.6 s), 7 V (0.7 s), 7 V (0.7 s), 7 V (0.8 s); signature 3: 9 V (0.5 s), 10 V (0.5 s), 10 V (0.7 s), 10 V (0.9 s), 10 V (1.2 s), 10 V (1.4 s), 10 V (1.6 s), 10 V (1.8 s), 10 V (2 s), 10 V (2.2 s). Variation in voltage magnitude and duration of pulse was needed to counteract the increasing attenuation of sensitivity of the seedlings to electrical stimulation with consecutive transients. Total aequorin for calibration was measured by freezing seedlings (Whalley et al., 2011), and this was calibrated into [Ca2+]c as described previously (Knight & Knight, 2000). Tissue was harvested by briefly drying on tissue paper and flash-freezing in liquid nitrogen 45 min after the end of each treatment. Luminescence was confirmed to be calcium-dependent by testing the effect of calcium inhibitors (e.g. see Supporting Information, Fig. S1), and the lack of artefactual luminescence of the media was tested by imaging (e.g. Fig. S2).

cDNA synthesis and labelling

RNA was extracted using the RNeasy Plant Total RNA kit (Qiagen) and the quality of RNA samples was assessed using an Agilent 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA, USA). Between 1 and 2 μg total RNA was labelled using reverse transcriptase Superscript III (Invitrogen) and the Genisphere 3DNA 900 indirect labelling kit (Genisphere Inc., Hatfield, PA, USA). For each treatment, cDNA was labelled and hybridized twice with channel reversal on the second replicate.

Microarray preparation and hybridization

Microarray slides printed with the Operon Arabidopsis v3 AROS oligo set (obtained from Dr David Galbraith, University of Arizona, Tucson, AZ, USA) were baked, cross-linked, pre-hybridized, washed and dried, before hybridization of cDNA as previously described (Whalley et al., 2011). Hybridized slides were scanned using a Perkin Elmer ScanArray Express HT (Perkin Elmer, Wellesley, MA, USA) and the resulting image files were transferred into the analysis program BlueFuse Version 3.0 or 3.2 (BlueGnome, Cambridge, UK) and analysed as described previously (Whalley et al., 2011). Following automatic and manual exclusion of bad data spots in BlueFuse, the data were global median normalized in Excel. Data for the microarray experiments described herein are publicly available from with accession number E-MEXP-2663. Analysis of 500 bp of upstream sequences (downloaded from the TAIR website: was performed using the oligo analysis, pattern assembly and DNA pattern matching tools available online at the Regulatory Sequence Analyses Tools site (RSAT: according to the developers' instructions (van Helden, 2003).

Results and Discussion

Using electrical stimulation (Whalley et al., 2011), we produced three different series of 10 calcium oscillations combining two different amplitudes and two different frequencies in Arabidopsis seedlings (Fig. 1). We subsequently performed whole-genome microarray analysis to identify which genes were regulated by each of the signatures. Fig. 2(a,b) shows that all three calcium signatures caused a substantial number of genes to be up- or down-regulated. The most striking observation was that the identities of the genes regulated by the three different calcium signatures were very different: 61.1, 73.7 and 79.6% of the genes up-regulated by signatures 1, 2 and 3, respectively, were unique to that signature; and 89.7, 76.0 and 93.5% of the genes down-regulated by signatures 1, 2 and 3, respectively, were unique to that signature (Fig. 2a,b; expression data for each of these six lists of genes are provided in Tables S1–S6). Given these findings, it is quite possible that different transcription factors, binding to different cis-acting DNA sequences, respond differently to the distinct calcium signatures. To investigate this possibility further, we analysed the promoters of the genes up-regulated in response to each of the three calcium signatures separately (we focused on up-regulated genes as down-regulated genes did not seem to be enriched for specific promoter sequences, probably because post-transcriptional regulation is the likely major degree of control for rapidly down-regulated genes). This analysis revealed that the most common promoter motif was different for each of the signatures (Fig. 2c). Furthermore, in most cases, each motif represented well-established transcription factor binding sites. For example, the two most common motifs for signature 1 overlap forming the W-box motif (Rushton et al., 1995). This W-box is also found in promoters of genes responding to signature 2, but less commonly than the sequence CACGT, the core sequence of a known drought-associated and ABA-associated motif, the ABRE (Hobo et al., 1999). By contrast, signature 3 revealed a completely different set of consensus motifs, the most common of which agrees with the consensus for the Site II element (Kosugi et al., 1995). The second most common motif in signature 3 genes was ‘CCGGTT’, which has been identified bioinformatically as a very strong candidate transcription factor binding site, but no function has been ascribed as yet (Yamamoto et al., 2007). Interestingly, we had already previously demonstrated that the ABRE and Site II promoter motifs are calcium-regulated in planta (Whalley et al., 2011). The W-box is known to be regulated by transcription factors, which themselves are regulated by calmodulin (Rushton et al., 2010). The preponderance of calcium-regulated motifs in the promoters of the genes induced by electrical stimulation strongly suggests that expression of the majority of the electrically induced genes is calcium-dependent. We cannot formally discount with this type of analysis, however, that some of the genes induced by electrical stimulation are not calcium-dependent. Our data suggest that these elements are differentially sensitive to different types of [Ca2+]c signature. Fig. 2 shows the potential for relatively subtle differences in calcium signatures to specify very different gene expression profiles. It has been established that, in plants, different types of environmental stresses provoke distinct calcium signatures (Dodd et al., 2010). These stimuli also provoke distinct gene expression profiles (Kilian et al., 2007). It thus seems reasonable to hypothesize that the specific characteristics of the calcium signatures in response to natural stimuli are encoding these appropriate patterns of gene expression. The major challenge in future will be to determine how these complex signatures are decoded by plant cells, what parameters of the calcium signature are being ‘read’, and the identity of the decoders. Knowing this might allow the control of stress pathways, leading to tolerance in crop plants, by breeding to produce better adapted varieties in the face of climate change.

Figure 1.

Blue lines show the resulting intracellular calcium ([Ca2+]cyt) traces when Arabidopsis seedlings were treated with the voltage regimes described in the 'Materials and Methods' section; green lines show the resulting calcium trace of untreated seedlings. (a) Signature 1, high frequency, high amplitude; (b) signature 2, high frequency, low amplitude; (c) signature 3, low frequency, low amplitude. High frequency, 40 s period; low frequency, 80 s period.

Figure 2.

Venn diagrams depicting the groups of genes whose expression increases (a) or decreases (b) by 1.5-fold or greater in response to three different calcium signatures. (c) Table showing consensus DNA sequences enriched in the promoter regions of genes up-regulated in response to three different calcium signatures, showing the frequency (mean number of motifs per gene).


We would like to thank Dr Nigel Saunders for providing protocols and equipment for microarray hybridizations and analysis, and Dr Heather Knight and Mr Alex Sargeant for critical review of this manuscript.