Aequorin-based luminescence imaging reveals differential calcium signalling responses to salt and reactive oxygen species in rice roots.

It is well established that both salt and reactive oxygen species (ROS) stresses are able to increase the concentration of cytosolic free Ca(2+) ([Ca(2+)]i), which is caused by the flux of calcium (Ca(2+)). However, the differences between these two processes are largely unknown. Here, we introduced recombinant aequorin into rice (Oryza sativa) and examined the change in [Ca(2+)]i in response to salt and ROS stresses. The transgenic rice harbouring aequorin showed strong luminescence in roots when treated with exogenous Ca(2+). Considering the histological differences in roots between rice and Arabidopsis, we reappraised the discharging solution, and suggested that the percentage of ethanol should be 25%. Different concentrations of NaCl induced immediate [Ca(2+)]i spikes with the same durations and phases. In contrast, H₂O₂ induced delayed [Ca(2+)]i spikes with different peaks according to the concentrations of H₂O₂. According to the Ca(2+) inhibitor research, we also showed that the sources of Ca(2+) induced by NaCl and H₂O₂ are different. Furthermore, we evaluated the contribution of [Ca(2+)]i responses in the NaCl- and H₂O₂-induced gene expressions respectively, and present a Ca(2+)- and H₂O₂-mediated molecular signalling model for the initial response to NaCl in rice.

Introduction

Rice (Oryza sativa L.) is the staple food for more than half of the world’s population. Salinity is one of the most common abiotic stresses encountered by rice, which is classified as a salt-sensitive crop in early stages of development, and limits its productivity (Lutts ; Todaka ). To improve the rice yield under saline conditions, it is important to understand the molecular mechanisms involved in how rice responds to salt stress (Kumar ).

Many studies have been carried out to dissect the genetic and molecular mechanisms of how plants respond to salt stress. A well-defined pathway is the Salt Overly Sensitive (SOS) signalling pathway, which comprises SOS3, SOS2 and SOS1 (Zhu, 2000), and is required to mediate the highly complex regulatory networks involved in plant response to salinity (Ji ). Importantly, the SOS signal transduction cascade is activated by a calcium (Ca2+) spike, which is caused by the flux of Ca2+. This salt stress triggered increase of cytosolic free Ca2+ ([Ca2+]i) is considered to be the first recorded response to salt stress (Knight ; Tracy ). Ca2+ is an essential second messenger in the sophisticated network of plant signalling pathways responding to a large array of external stimuli, including salt stress (Hetherington and Brownlee, 2004; Pandey ; Dodd ). The Ca2+ channels and transporters activated by these stimuli form specific Ca2+ signatures, and these changes in Ca2+ signatures are transmitted by protein sensors that preferably bind Ca2+. The binding of Ca2+ results in conformational changes which modulate their activity or their ability to interact with other proteins, and activate the expression of downstream salt response genes through a Ca2+ signalling cascade (Rentel and Knight, 2004; Dodd ; Kudla ; Batistic and Kudla, 2012).

Furthermore, salt stress also increases the level of reactive oxygen species (ROS) predominantly represented by H2O2 (Bienert ; Hong ; Miller ). Although toxic by nature, ROS are now considered as important signalling molecules in many biological processes, including biotic and abiotic stress tolerance (Mittler ; Schippers ). In Arabidopsis, Respiratory Burst Oxidase Homolog F (RBOHF, an NADPH oxidase catalysing ROS production) is required for shoot sodium homeostasis during salt stress (Jiang ). Furthermore, the RBOHF-dependent salinity-induced ROS accumulation is regulated by protein phosphorylation in a Ca2+-dependent manner (Drerup ). Interestingly, ROS have also been shown to trigger an increase of [Ca2+]i (Mori and Schroeder, 2004). Previous limited evidence implied that NaCl-gated Ca2+ channels and H2O2-gated Ca2+ channels may differ (Jiang ). However, the different mechanisms between NaCl- and H2O2-induced [Ca2+]i changes are yet to be explored. Also, SALT-RESPONSIVE ERF1 (SERF1) is reported to function as a central hub to regulate ROS-dependent signalling during the initial response to salt stress in rice (Schmidt ). Therefore, it is attractive to investigate the relationship among Ca2+, H2O2 and SERF1 in the early salt-stress signalling cascade.

Genetically encoded fluorescent Ca2+ probes are useful tools to non-invasively describe Ca2+ signatures in plants (Monshausen, 2012). The fluorescence resonance energy transfer (FRET)-based probes yellow cameleon YC2.1 (Miyawaki ) and the improved YC3.6 (Nagai ) are currently used to monitor the [Ca2+]i in the cytoplasm. With high quantum yield, FRET-based probes are suitable to measure the Ca2+ signatures in the cellular or subcellular resolutions (Monshausen, 2012). Aequorin, a photoprotein derived from the luminescent jellyfish Aequoria victoria, reacts specifically with Ca2+ and emits blue light at ~460nm (Shimomura ). Although the aequorin-based probe gives a low quantum yield, it is more suitable for cell population or whole plant measurement of [Ca2+]i (Monshausen, 2012). Since the transformation of recombinant aequorin in plant systems (Knight ), it has proved to be a useful tool for non-invasive investigation of Ca2+-mediated signalling in response to various stresses in whole seedlings (Zhu ). Specific stimuli can trigger unique Ca2+ signatures, which are decoded subsequently by intracellular Ca2+ sensors, leading to the activation of downstream events (Luan, 2009). Kurusu established a transgenic rice cell line expressing apoaequorin, and characterized the regulation mechanism of microbe-associated molecular pattern-induced [Ca2+]i transients. In spite of the progress achieved by rice cell lines, stable transgenic rice expressing apoaequorin is needed to investigate the changes of [Ca2+]i that appear in response to various environmental stimuli.

In this paper, we introduced recombinant aequorin, as a reporter of [Ca2+]i, into rice. Transgenic rice harbouring aequorin showed strong luminescence in roots when treated with exogenous Ca2+. We also showed that NaCl and H2O2 treatments induce different [Ca2+]i spikes, and may employ different Ca2+ channels. Furthermore, we present a Ca2+- and H2O2-mediated molecular signalling model for the initial response to NaCl in rice.

Materials and methods

Vector construction and transformation of rice

In order to improve the aequorin expression vector for transgenic research in rice, the coding region of apoaequorin in pMAQ2 (Knight ) was transferred to 35S-pCAMBIA1301 (Zhou ) through XbaI and PstI. Plasmids were introduced into Agrobacterium tumefaciens EHA105 by electroporation. Rice transformation was performed by the Agrobacterium-mediated method, as previously described (Chen ).

Plant materials and growth conditions

Rice (Oryza sativa L. cv. Nipponbare) seeds were sterilized with 75% ethanol and planted in a square plate containing half-strength Murashige and Skoog salts (MS; Gibco), and 1.5% (w/v) agar (Becton Dickinson). Seedlings were grown vertically in the growth chamber conditioned with 16h of light at 28°C and 8h of dark at 22°C for five days. The seedlings were then sprayed with coelenterazine for reconstitution of aequorin before subsequent experiments began.

Root cell death detection

A root cell death assay was performed as previously described (Qin ). Roots of five-day-old seedlings were submerged in different concentrations of NaCl solution for 30 s and then stained with 1% Evans blue solution for 10min, washed by distilled water for 2h, and then photographed.

Southern-blot analysis of transgenic rice

Genomic DNA of transgenic rice was isolated following the instructions of a Plant Genomic DNA Kit (TIANGEN) and the purified DNA was digested with restriction enzyme EcoRI. 2 µg of digested DNA was separated on 0.8% agarose gel. After electrophoresis, the digested DNA was transferred to Hybond-N+ nylon membrane (Amersham Pharmacia) and hybridized with a 32P-dCTP-labelled hygromycin-resistant gene probe. The blots were washed at 65°C under stringent conditions and analysed using Typhoon-8600. The primers used to amplify the probe are listed in Supplementary Table S1.

RT-PCR analysis

For the examination of apoaequorin expression in different tissues, the shoot, shoot base and root of five-day-old transgenic rice seedlings were selected, and the PCR was conducted with 28 cycles for both apoaequorin and OsACTIN. For the examination of NaCl and H2O2 induced gene expression, the roots of five-day-old rice seedlings were selected. Ca2+ channel blocker pre-treatment was performed by 1mM LaCl3 treatment for 30min. NaCl treatment was performed by 0.15M NaCl treatment for 1h. H2O2 treatment was performed by 1mM H2O2 treatment for 1h. The relative expression levels were calculated according to the 2-∆∆Ct method (Livak and Schmittgen, 2001). Each experiment was carried out with three independent biological replications. For RT experiments, 5 μg of total RNA was denatured at 65°C for 5min followed by quick chill on ice in a 14 μl reaction containing 1 μl oligo (dT)12–18 (500 μg ml-1) primer, and 1μl of 10mM dNTP mixture (10mM each dATP, dGTP, dCTP, and dTTP at neutral pH). After addition of 4 μl 5× reaction buffer (Promega), the reaction was incubated at 37°C for 2min, and 1 μl (200 units) of M-MLV RTa (Promega) was added to the reaction and incubated at 42°C for another 50min. For inactivation, the reaction was heated at 70°C for 15min. The primers are listed in Supplementary Table S1.

Aequorin reconstitution and luminescence imaging

Seedlings were grown on half-strength MS medium for five days. Reconstitution of aequorin was performed in vivo by spraying seedlings with 10 µM coelenterazine and followed by incubation at 21°C in the dark for 12–16h. For surfactant treatment, 0.01% or 0.1% of silwet L-77 (Sigma) was added to the coelenterazine solution. For Ca2+ inhibitor treatments, rice roots were treated with different concentrations of GdCl3, LaCl3, neomycin and thapsigargin, respectively for 30min before 0.25M NaCl and 1mM H2O2 treatment. Treatments and aequorin luminescence imaging were performed at room temperature using a ChemiPro HT system as described previously (Jiang ). The recording was started about 5 s prior to treatment and luminescence images were acquired for 3min. For the analysis of time courses of increase in [Ca2+]i, each exposure time was 30 s and the images were taken continuously for several minutes. To avoid the interference of chloroplast auto-fluorescence signal in the aequorin luminescence imaging, all the treatments were performed in the complete darkness. To record the chloroplast auto-fluorescence signal, seedlings were first exposed to strong light for 1min. After that, the light was turned off and the chloroplast auto-fluorescence was recorded. WinView/32 and Meta Morph 7.7 were used to analyse recorded luminescence images.

Results

Production and characterization of transgenic rice expressing apoaequorin

In order to monitor [Ca2+]i responses in rice, we developed transgenic rice over-expressing apoaequorin under the control of cauliflower mosaic virus (CMV) 35S promoter (Fig. 1A). Three independent transgenic lines (AQ-2, AQ-3 and AQ-5) harbouring one copy of apoaequorin were selected by Southern blot analysis, and the homozygous T3 generations of these lines were used for subsequent experiments (Fig. 1B). In addition, the heterologous expression of apoaequorin had no effects on the growth and life cycle of transgenic rice (data not shown). After the reconstitution of aequorin by spraying seedlings with coelenterazine, the aequorin luminescence of these seedlings were recorded using a photo-counting camera by treating plants with exogenous Ca2+ (see Materials and methods for detail). Ca2+-treated seedlings showed strong and diverse luminescence in roots, and AQ-3 with the strongest luminescence was selected for further analysis (Fig. 1D). To our surprise, the aequorin-based luminescence signal was only observed in roots and we failed to detect any signal in shoots when treated with Ca2+ (Fig. 1D compared to bright-field in Fig. 1C and chloroplast auto-fluorescence in Fig. 1E). To confirm the expression of apoaequorin in the whole plant, we extracted RNA from different tissues of transgenic seedlings, and the expression of apoaequorin was examined using reverse transcription-polymerase chain reaction (RT-PCR). The results showed that apoaequorin is expressed in all the selected tissues (Supplementary Fig. S1A). It is likely that the leaf wax prevents the permeating of coelenterazine (Supplementary Fig. S1B). To test this hypothesis, we added surfactant (Silwet L-77, Sigma) while spraying coelenterazine. Both luminescence signals in roots and dotted signals in shoots were observed (Supplementary Fig. S1C–H). These results showed that transgenic rice expressing apoaequorin was able to reflect the [Ca2+]i level in rice roots.

Fig. 1.

Transgenic rice harbouring aequorin showed strong and diverse luminescence in roots. (A) The construction of apoaequorin expression vector for transgenic research in rice. (B) Southern-blot analysis of five independent lines (AQ-1 to AQ-5) of transgenic rice. (C-E) Ca2+-treated seedlings showed strong and diverse luminescence exclusively in roots. (C) Bright-field image. (D) Pseudocolour image of aequorin luminescence in roots. (E) Pseudocolour image of chloroplast auto-fluorescence. Red rectangles indicate the places of shoots, and there is no aequorin luminescence signal in shoots shown in (D). The relationship between luminescence intensity and the pseudocolour images are scaled by pseudocolour bars.

The optimization of discharging solution for luminescence imaging in rice

The discharging solution is used to estimate the amount of remaining aequorin in the calibration and is important to calculate the Ca2+ concentration based on the luminescence intensity (Knight ). Considering the histological differences in roots between rice and Arabidopsis (Rebouillat ), we re-examined the percentage of ethanol in the discharging solution for the rice experiment. We tested a series of discharging solutions with different percentages of ethanol from 0% to 50%. Low percentages of ethanol (below 15%) had no significant difference in the luminescence imaging compared with 0% ethanol. Interestingly, the average luminescence intensity of plants increased by about one fold when treated with discharging solution containing 20% ethanol compared with that treated with a low concentration of ethanol (Supplementary Fig. S2). In spite of the leap from 15% to 20%, even higher percentages of ethanol in the discharging solution had little effect in the luminescence intensity of rice, indicating the saturation of discharged aequorin. Based on these results, we suggested that in contrast with 10% ethanol which is normally used in Arabidopsis (Yuan ), the percentage of ethanol in the discharging solution for rice should be 25%.

NaCl induced an immediate [Ca2+]i spike in rice roots

In order to investigate the [Ca2+]i changes in response to salt stress in rice roots, we examined the aequorin-based luminescence under various concentrations of NaCl treatments. The intensity of [Ca2+]i-dependent luminescence signals relied on the strength of salt stimuli (Fig. 2A). Detailed analysis showed that 0.1M (or less) of NaCl failed to induce visible [Ca2+]i-dependent luminescence signals, while 0.15M NaCl successfully induced a visible concentration of luminescence signals. 0.25M NaCl had a more obvious effect than 0.2M, and was very similar to 0.5M NaCl treatments (Fig. 2A). To further investigate the salt concentration-dependent increase of [Ca2+]i in rice roots, we calculated the average luminescence intensity of rice roots in response to different concentrations of NaCl treatments. As shown in Fig. 2B, a rapid increase of luminescence intensity in response to NaCl treatment occurred within a narrow range of NaCl concentrations (0.1M to 0.25M). NaCl treatments below or above this region had minor effect on the [Ca2+]i responses in rice roots (Fig. 2B).

Fig. 2.

Aequorin-based luminescence in roots under NaCl treatments. (A) Pseudocolour images of aequorin luminescence in roots treated with different concentrations of NaCl. The relationship between luminescence intensity and the pseudocolour images are scaled by a pseudocolour bar. (B) The line chart of luminescence signal intensity of every treatment. (C) The time courses of [Ca2+]i changes induced by different concentrations of NaCl in rice roots. Data for independent experiments are shown (mean±sd; n=10; *** P<0.001; NS, not significant P>0.05; Student’s t-test).

To investigate the time courses of [Ca2+]i responses induced by different concentrations of NaCl in rice roots, the average luminescence intensity of continuous images with exposure time of 30 s were analysed and a comparison of basic parameters (amplitudes, durations and phases) of [Ca2+]i responses were made. As shown in Fig. 2C, a strong luminescence signal was detected in the first image, which collected the luminescence signal within the first 30 s. However, almost no luminescence signal was detected after the first 30 s. This indicated that salt stress immediately induced a sharp spike of [Ca2+]i within 30 s, which quickly declined to the basal level after the spike. The amplitudes of luminescence signals varied according to different concentrations of NaCl, while both the durations and phases were the same. Detailed analysis showed that 0.1M NaCl had little effect on the induction of [Ca2+]i, while 0.15M NaCl was able to induce an obvious spike of [Ca2+]i. The effect of 0.2M NaCl was not significantly different in the increase of amplitude of [Ca2+]i increase compared with 0.15M. Interestingly, increasing the concentration of NaCl to 0.25M dramatically increased the amplitude of [Ca2+]i by about two fold, but concentrations higher than 0.25M NaCl had little effect on the increasing of amplitude compared with 0.25M (Fig. 2C). These results were similar to the curves shown in Fig. 2B. It is worth noting that the spike of [Ca2+]i failed to decline to the basal level after the induction by 2M NaCl treatment (Fig. 2C). This indicated a destruction of calcium transport systems, which are responsible for maintaining low [Ca2+]i, and crucial to the living cells. As expected, Evans blue staining revealed that treatment with a high concentration of NaCl resulted in serious cell death in the rice roots (Supplementary Fig. S3).

H2O2 induced a delayed [Ca2+]i spike in rice roots

In order to investigate the [Ca2+]i changes in response to ROS stress in rice roots, we examined the aequorin-based luminescence after the application of H2O2. We collected the luminescence signals 45 times at one minute intervals after treatment with H2O2. We found clear luminescence signals in the first and second minute. Interestingly, after the weak luminescence signals in the third minute, we failed to collect any additional signals over the remaining 42min (Supplementary Fig. S4). This was different from that in Arabidopsis, which was reported to have a second peak 5–20min after the application of H2O2 (Rentel and Knight, 2004).

To investigate the H2O2 concentration-dependent increase of [Ca2+]i in rice roots, we examined the aequorin-based luminescence signals and calculated the average luminescence intensity of rice roots in response to different concentrations of H2O2 treatments. Overall, the H2O2 response was quite similar to the NaCl response, when looking only at the concentration-dependent increase in luminescence (Fig. 3A). A concentration of 0.2mM H2O2 was able to induce clear luminescence signals, and the more H2O2 was applied, the stronger the luminescence signals would be (Fig. 3A). Detailed analysis showed that there was almost a linear relationship between luminescence signals and the H2O2 concentrations when the concentration of H2O2 was low (below 0.5mM). Higher concentrations of H2O2 (more than 0.5mM) were able to induce stronger luminescence signals but with a reduced rate of increase (Fig. 3B). Concentrations of H2O2 higher than 5mM had little additional effect on the luminescence changes (Supplementary Fig. S5).

Fig. 3.

Aequorin-based luminescence in roots under H2O2 treatments. (A) Pseudocolour images of aequorin luminescence in roots treated with different concentrations of H2O2. The relationship between luminescence intensity and the pseudocolour images are scaled by a pseudocolour bar. (B) The line chart of luminescence signal intensity of every treatment. (C) The time courses of [Ca2+]i changes induced by different concentrations of H2O2 in rice roots. Data for independent experiments are shown (mean±sd; n=10; NS, not significant P>0.05; Student’s t-test).

Next, we investigated the time courses of increase in [Ca2+]i induced by different concentrations of H2O2 in rice roots. H2O2 did not induce an immediate spike of [Ca2+]i as NaCl did. Our results showed that within the first 30 s, only the highest concentration of H2O2 (5mM) was able to induce any luminescence signal, this is in contrast to NaCl which within 30 s was able to induce luminescence signals for all but the lowest concentration (0.1M) (Fig. 3C compared with Fig. 2C). Furthermore, the phase of [Ca2+]i responses were also different among treatments with different concentrations of H2O2. Treatments with higher concentrations of H2O2 (more than 0.4mM) had a peak between 30 s and 1min, lower concentrations of H2O2 (below than 0.4mM) would delay the peak by about 30 s. Moreover, the spike of [Ca2+]i induced by H2O2 did not decline to the basal level as quickly as NaCl. The luminescence signals declined gradually to the basal level within 3min (Fig. 3C). These results revealed that H2O2 induces a delayed [Ca2+]i spike compared to NaCl.

The different effects of Ca2+ inhibitors in NaCl- and H2O2-induced [Ca2+]i responses and downstream gene expression

To further investigate the source of Ca2+ in NaCl- and H2O2-induced [Ca2+]i responses, we tested GdCl3-, LaCl3-, neomycin- and thapsigargin-treated plants on the [Ca2+]i increase in response to NaCl and H2O2 respectively. Gd3+ and La3+ are agonists of Ca2+, and they have been used as Ca2+ channel blockers to inhibit Ca2+ flux (Tracy ). In our experiment, GdCl3 and LaCl3 had similar inhibitory effects in NaCl- and H2O2-induced [Ca2+]i increases respectively (Fig. 4). 1mM of GdCl3 and LaCl3 almost completely inhibited the [Ca2+]i increase in response to NaCl, and inhibited about 90% of [Ca2+]i increase in response to H2O2 (Fig. 4). Furthermore, significant dosage effects were observed except for LaCl3 in H2O2-induced [Ca2+]i increase. Different concentrations of LaCl3 treatment had similar inhibitory effects in H2O2-induced [Ca2+]i increase (Fig. 4B, C). Neomycin is an inhibitor of InsP3-stimulated Ca2+ release from internal stores (Munnik ). 0.01mM of neomycin treatment had no significant inhibitory effect, while 0.1mM and 1mM of neomycin treatment inhibited about 50% of [Ca2+]i increase in response to NaCl. In the case of H2O2, 0.01mM and 0.1mM of neomycin treatment inhibited about 20% of [Ca2+]i increase, while 1mM of neomycin treatment inhibited about 50% of [Ca2+]i increase (Fig. 4B, C). Thapsigargin is an inhibitor of endoplasmic reticulum (ER) Ca2+-ATPases, and application of thapsigargin would empty the intracellular Ca2+ store in ER (Treiman ). In our experiment, thapsigargin significantly inhibited the NaCl-induced [Ca2+]i increase, and a dosage effect for inhibition was observed. By contrast, thapsigargin had no significant effect on the H2O2-induced [Ca2+]i increase (Fig. 4B, C). These results showed that the sources of Ca2+ in NaCl- and H2O2-induced [Ca2+]i responses are different.

Fig. 4.

The effects of Ca2+ inhibitors in NaCl- and H2O2-induced [Ca2+]i increases. (A) Treatment of Ca2+ inhibitors (1mM of GdCl3, LaCl3 and neomycin, 10 μM of thapsigargin) differentially reduced NaCl and H2O2 induced [Ca2+]i changes. The relationship between luminescence intensity and the pseudocolour images are scaled by a pseudocolour bar. (B, C) The effects of different concentrations of Ca2+ inhibitors in NaCl (B) and H2O2 (C) induced [Ca2+]i increases. The concentration gradient for GdCl3, LaCl3 and neomycin are 0.01mM, 0.1mM and 1mM; for thapsigargin it is 0.1 μM, 1 μM and 10 μM. Data for independent experiments are shown (mean±sd; n=10; NS, not significant P>0.05; Student’s t-test).

To evaluate the contribution of [Ca2+]i responses in the NaCl- and H2O2-induced gene expression, the expression levels of SERF1, MITOGEN-ACTIVATED PROTEIN KINASE5 (MAPK5), DEHYDRATION-RESPONSIVE ELEMENT BINDING2A (DREB2A) and STRESS-RESPONSIVE NAC1 (SNAC1), which were reported to be induced by NaCl and H2O2 in rice, were analysed by quantitative RT-PCR (qRT-PCR) (Schmidt ). In our experiment, the expression levels of these genes were greatly increased by the NaCl treatment as reported previously (Schmidt ). After the pre-treatment of LaCl3, the induction by NaCl was seriously inhibited for all the genes examined, indicating a Ca2+-dependent manner of these inductions (Fig. 5A). On the other hand, H2O2 induced three of the four genes with DREB2A as the exception. Interestingly, only the expression of MAPK5 showed Ca2+-dependent induction by H2O2. The expression of SERF1 and SNAC1 were induced by H2O2, however, pre-treatment of LaCl3 did not inhibit the induction of the expression by H2O2, indicating a Ca2+-independent manner of these inductions (Fig. 5B).

Fig. 5.

Relative expression levels of initial response genes induced by NaCl (A) and H2O2 (B). (A) H2O+H2O, untreated control. H2O+NaCl, NaCl treatment without pre-treatment. LaCl3+H2O, only LaCl3 pre-treatment, without NaCl treatment. LaCl3+NaCl, LaCl3 pre-treatment before NaCl treatment. (B) H2O+H2O, untreated control. H2O+H2O2, H2O2 treatment without pre-treatment. LaCl3+H2O, only LaCl3 pre-treatment, without H2O2 treatment. LaCl3+H2O2, LaCl3 pre-treatment before H2O2 treatment. Data for independent experiments are shown (mean±sd; n=3).

Discussion

Since the transformation of aequorin in plants (Knight ), it has proved to be a useful tool for non-invasive investigation of Ca2+-mediated signalling in response to various stresses in whole seedlings (Zhu ). Furthermore, GAL4 transactivation of aequorin in enhancer trap lines enabled the testing of the stimulus- and cell-specific [Ca2+]i signalling in specific tissues of Arabidopsis (Kiegle ; Martí ). In our experiment, the luminescence of aequorin was limited to rice roots, although the expression of apoaequorin was universal. It is unlikely that the leaf wax blocked the aequorin luminescence, because we detected chloroplast auto-fluorescence, which was emitted from the leaf cells (Fig. 1E). However, the leaf wax could have prevented the permeating of coelenterazine. To test this hypothesis, a surfactant was used to allow the coelenterazine to permeate through the leaf wax. After the addition of 0.01% surfactant, both strong luminescence signals in roots and weak luminescence signals in shoots were detected (Supplementary Fig. S1C). The luminescence signals in shoots were dotted, indicating insufficient permeation of coelenterazine. Although a higher concentration of surfactant (0.1%) increased the dotted luminescence signals in shoots, it greatly decreased the luminescence signals in roots, indicating the toxic effect of the surfactant to rice roots (Supplementary Fig. S1F). These results showed that our system is able to reflect the [Ca2+]i level only in roots, not shoots.

In Arabidopsis, the discharging solution contains 10% ethanol, which is sufficient to permeate the exogenous Ca2+ and combine the remaining aequorin in the cell (Yuan ). In our experiment, we suggested a much higher concentration of ethanol (about 25%) to discharge all the aequorin. There are several differences in root radial structure between rice and Arabidopsis. From outside in, the epidermis, the ground tissue consisting of four tissues (exodermis, sclerenchyma cell layer, midcortex or mesodermis, and endodermis), and the central cylinder are present in a rice root, compared with single cell layers of epidermis, cortex, endodermis and the central cylinder in Arabidopsis (Dolan ; Rebouillat ). These additional cell layers in rice roots make it much thicker than that of Arabidopsis, and they form barriers that inhibit the permeation of exogenous Ca2+ in discharging solutions. In this research, we used luminescence intensity instead of real [Ca2+]i in rice. Knight described the equation to determine the Ca2+ concentration based on the luminescence intensity in Arabidopsis. However, the parameters in the equation can be variable among different species. Thus, for the accurate quantification of [Ca2+]i in rice, a titration curve for analyzing the relationship between the Ca2+ concentration and luminescence intensity should be shown and real [Ca2+]i should be estimated.

We investigated the [Ca2+]i changes in response to various concentrations of salt stress, and found that it was sensitive within a very narrow range of NaCl concentrations (Fig. 2B). We observed that severe salt stress (more than 0.25M NaCl) did not increase [Ca2+]i significantly compared with salt stress by 0.25M NaCl. Rice is a salt-sensitive crop and continued exposure to about 0.15M NaCl does not allow rice to complete its life cycle (Munns and Tester, 2008). In this view, there is no need for rice to evolve an energy wasting mechanism to respond to such a high concentration of NaCl. Alternatively, it is possible that severe salt stress (more than 0.25M NaCl) does increase [Ca2+]i significantly. However, due to the saturation of our detection system, we failed to detect stronger luminescence signals when treated with high concentrations of NaCl. Moreover, we also observed that treatment with 0.1M NaCl (or less) did not induce an obvious increase of [Ca2+]i, although it is sufficient to induce the expression of many salt response genes (Schmidt ). Our evidence suggests that the induction of these salt response genes are Ca2+ dependent (Fig. 5A); it is likely that low strength salt stress induces a very tiny [Ca2+]i, response which is beyond the resolution of our detection system. It was reported that H2O2 triggered a biphasic [Ca2+]i increase in Arabidopsis seedlings, and the second peak 5 to 20min after stress application was located exclusively in the root (Rentel and Knight, 2004). However, in our experiment, we detected the luminescence signals only in the first 3min and failed to detect additional signals even with the time extended to 45min (Supplementary Fig. S4). Considering the different environments rice and Arabidopsis roots are adapted to, rice may have evolved a different mechanism to cope with ROS stress.

We investigated the time courses of [Ca2+]i changes in response to different concentrations of NaCl and H2O2 treatments in rice roots. Treatments of NaCl at different concentrations induced immediate [Ca2+]i spikes within 30 s (Fig. 2C). In contrast, H2O2 did not induce any [Ca2+]i increases within the first 30 s unless the concentration of H2O2 was high (Fig. 3C). In the view that the salt sensors are still unknown in plants, we propose that the salt sensors may be on the surface of the root and are closely coupled with Ca2+ channels. These salt sensors can respond to different concentrations of salt directly. On the other hand, it is likely that the effect of H2O2 on calcium signalling is less direct. The treatment of roots with H2O2 will in the first instance cause oxidation of the apoplast, and potentially it can affect calcium fluxes once it has been taken up by the cell through aquaporins, and thus a delay in response is observed. The H2O2- induced [Ca2+]i spikes extended for several minutes (Fig. 3C), which also indicated a complicated and indirect way of H2O2-induced [Ca2+]i response.

GdCl3 and LaCl3 are widely used to block Ca2+ channels (Tracy ). As expected, 1mM of GdCl3 and LaCl3 almost completely inhibit the [Ca2+]i increase in response to NaCl. Interestingly, although 1mM of GdCl3 and LaCl3 were able to inhibit about 90% of [Ca2+]i increase in response to H2O2, it cannot go further. There were no significant differences between 0.1mM and 1mM of blocker treatments, indicating a saturation of Ca2+ channels blocked by GdCl3 and LaCl3 (Fig. 4B, C). These results suggested that H2O2 is able to induce a [Ca2+]i increase via different channels, and a small portion of these channels are GdCl3/LaCl3 insensitive. We also examined the source of Ca2+ in NaCl- and H2O2- induced [Ca2+]i responses by treatment with neomycin, which is an inhibitor of InsP3-stimulated Ca2+ release from internal stores (Munnik ). Neomycin inhibited about 50% of [Ca2+]i increase in response to both NaCl and H2O2. These results suggested that the [Ca2+]i increases caused by NaCl and H2O2 treatments came from both internal and external stores of Ca2+. ER is an important internal Ca2+ store in the cell (Franzini-Armstrong, 1998). In our experiment, we used thapsigargin to empty the intracellular Ca2+ store in ER. As a result, NaCl induced [Ca2+]i increase was significantly inhibited, indicating a participation of ER in the NaCl-induced [Ca2+]i increase. Interestingly, thapsigargin had no significant effect on the H2O2-induced [Ca2+]i increase (Fig. 4B, C). This suggested that another internal Ca2+ store, rather than ER, may participate in the H2O2-induced [Ca2+]i increase.

Recently, the molecular processes controlling early stress perception and signalling have been explored and several genes have been identified as initial response genes in rice (Dai Yin ; Schmidt ). Among these genes, SERF1 has a critical role and functions as a central hub in the ROS-dependent signalling during the initial response to salt stress in rice (Schmidt ). In our research, we examined NaCl- and H2O2- induced expression levels of SERF1 and other initial response genes with or without LaCl3. As expected, SERF1 and all the other initial response genes examined were induced by NaCl treatment. Furthermore, the induction of all these genes was seriously inhibited by the pre-treatment of LaCl3 (Fig. 5A). In the H2O2 treatment experiments, expression of the initial response genes SERF1 and SNAC1 were induced. However, the induction of these genes was not inhibited by the pre-treatment of LaCl3 (Fig. 5B). Considering the fact that a [Ca2+]i spike is the first response to salt stress (Knight ), and salt-induced ROS accumulation is regulated by Ca2+ (Drerup ), we propose a Ca2+ and H2O2 mediated molecular signalling model for the initial response to NaCl in rice (Fig. 6). Salt stress immediately induces a [Ca2+]i spike, and the increase of [Ca2+]i triggers a ROS burst probably through the activation of NADPH oxidases. The increased H2O2 induces the expression of SERF1 and downstream initial response genes. In addition, H2O2 also induces a [Ca2+]i spike, and the increase of [Ca2+]i is needed for the expression of some initial response gene(s), such as MAPK5. In our experiment, H2O2 did not induce the expression of DREB2A significantly as previously described (Schmidt ). We reasoned that 1h of H2O2 treatment may not be long enough to induce the expression of DREB2A. In the previous report, 3h of H2O2 treatment was able to induce the expression of DREB2A, while 30min of H2O2 treatment had no obvious effect (Schmidt ). Considering the fact that 1h of NaCl treatment is sufficient to induce the expression of DREB2A (Fig. 5A), a complex network rather than a linear structure may exist to regulate the expression of DREB2A (Kim ).

Fig. 6.

Proposed role of Ca2+ and H2O2 during the initial response to NaCl. NaCl as a primary signal induces a [Ca2+]i spike, and the increase of [Ca2+]i triggers a ROS burst. The increased concentration of H2O2 induces the expression of SERF1 and downstream initial response genes (solid arrows). In addition, H2O2 also induces a [Ca2+]i spike, and the increase of [Ca2+]i is needed for the expression of some initial response gene(s) (dotted arrows).

In summary, based on the aequorin luminescence, we established a calcium reporter line in rice. Using this, we tested the different [Ca2+]i responses to NaCl and H2O2 in rice roots. Ca2+ inhibitor treatments revealed that both external and internal Ca2+ stores are involved in the [Ca2+]i increases induced by NaCl and H2O2. Further research suggested that the internal Ca2+ store in ER participates in the NaCl-induced [Ca2+]i increase, while another internal Ca2+ store, rather than ER may participate in the H2O2- induced [Ca2+]i increase. In addition, we dissected the early salt-stress signalling cascade and presented a Ca2+- and H2O2-mediated molecular signalling model for the initial response to NaCl in rice.

Supplementary data

Supplementary data can be found at JXB online.

Supplementary Fig. S1. Leaf wax prevents the permeating of coelenterazine.

Supplementary Fig. S2. The optimization of discharging solution for luminescence imaging in rice.

Supplementary Fig. S3. Treatment with high concentration of NaCl resulted in serious cell death in the rice roots.

Supplementary Figure S4 H2O2 does not induce a second peak of [Ca2+]i response.Supplementary Fig. S4. H2O2 does not induce a second peak of [Ca2+]i response.

Supplementary Fig. S5. High concentrations of H2O2 have little additional effect on the luminescence changes.

Supplementary Table S1. The sequences of primers used in this research.