Relationship between NaCl- and H2O2-Induced Cytosolic Ca2+ Increases in Response to Stress in Arabidopsis

Salinity is among the environmental factors that affect plant growth and development and constrain agricultural productivity. Salinity stress triggers increases in cytosolic free Ca2+ concentration ([Ca2+]i) via Ca2+ influx across the plasma membrane. Salinity stress, as well as other stresses, induces the production of reactive oxygen species (ROS). It is well established that ROS also triggers increases in [Ca2+]i. However, the relationship and interaction between salinity stress-induced [Ca2+]i increases and ROS-induced [Ca2+]i increases remain poorly understood. Using an aequorin-based Ca2+ imaging assay we have analyzed [Ca2+]i changes in response to NaCl and H2O2 treatments in Arabidopsis thaliana. We found that NaCl and H2O2 together induced larger increases in [Ca2+]i in Arabidopsis seedlings than either NaCl or H2O2 alone, suggesting an additive effect on [Ca2+]i increases. Following a pre-treatment with either NaCl or H2O2, the subsequent elevation of [Ca2+]i in response to a second treatment with either NaCl or H2O2 was significantly reduced. Furthermore, the NaCl pre-treatment suppressed the elevation of [Ca2+]i seen with a second NaCl treatment more than that seen with a second treatment of H2O2. A similar response was seen when the initial treatment was with H2O2; subsequent addition of H2O2 led to less of an increase in [Ca2+]i than did addition of NaCl. These results imply that NaCl-gated Ca2+ channels and H2O2-gated Ca2+ channels may differ, and also suggest that NaCl- and H2O2-evoked [Ca2+]i may reduce the potency of both NaCl and H2O2 in triggering [Ca2+]i increases, highlighting a feedback mechanism. Alternatively, NaCl and H2O2 may activate the same Ca2+ permeable channel, which is expressed in different types of cells and/or activated via different signaling pathways.


Introduction
The presence of high salinity affects almost every aspect of plant growth and development, and causes enormous losses in agricultural production worldwide.It is estimated that about 10 million hectares of agricultural land is abandoned every year because of high salinity, and salt stress affects as much as a quarter to a third of global agricultural land, particularly land which has been irrigated [1][2][3].Given the continued increase in human population occurring in the world, it is estimated that crop production must be increased 50% by 2025 to stave off large-scale food shortages [4].Thus, it is crucial to understand how plants respond to salt stress.
Many studies have been carried out to dissect the molecular and genetic mechanisms of the plant response to salt (NaCl) stress, often using the model organism Arabidopsis thaliana [5][6][7].Excess NaCl is toxic to plants, causing cellular ion imbalances and hyperosmotic stress [1][2][3]7].NaCl stress also triggers a calcium signaling cascade in plants, leading to transcriptional regulation and subsequent physiological and developmental responses [1].Although the molecular nature of initial perception of salt stress is unknown, it has been well established that salt stress triggers a transient increase in cytosolic Ca 2+ concentration ([Ca 2+ ] i ) that lasts about 2 min [8,9].This increase has been proposed to represent a salt sensory process in plants [3,10].
In plants, Ca 2+ as a secondary messenger is a key element to understanding a sophisticated network of signaling pathways responding to a large array of abiotic and biotic stimuli, including salt stress [11][12][13].These specific Ca 2+ signatures are formed by the tightly regulated activities of Ca 2+ channels and transporters in different tissues, organelles and membranes [13][14][15][16], and the changes in [Ca 2+ ] i are detected by cytosolic Ca 2+ sensors.More than 250 Ca 2+ -binding EF-hand proteins have been identified in Arabidopsis [17], including the calmodulin (CaM), the calmodulin-like (CML), the Ca 2+dependent protein kinase (CDPK), and the calcineurin B-like (CBL) protein families.These cytosolic Ca 2+ sensors decode and relay the information encoded within [Ca 2+ ] i signatures, allowing the plant to tightly bring about the appropriate adaptation to its ever-changing environment.
The salinity stress-induced increase in [Ca 2+ ] i leads to the activation of SOS3/CBL4, which functions as the primary Ca 2+ sensor of [Ca 2+ ] i changes under salt stress [3].Upon activation, SOS3/CBL4 interacts with the C-terminal region of a CBLinteracting protein kinase (CIPK) called SOS2/CIPK24, which in turn activates a plasma membrane Na + /H + antiporter SOS1 that transports sodium ions out of the cell [3].This salt signaling pathway reinforces the concept that the salt-induced [Ca 2+ ] i increase is an essential component for bringing about the plant response to salt tress.
Interestingly, after salt stress treatment there is an overproduction of reactive oxygen species (ROS), such as hydrogen peroxide (H 2 O 2 ) [18][19][20][21][22]. The time constants for saltinduced increases in [Ca 2+ ] i and ROS are about 3 sec and 400 sec, respectively, as estimated from previous studies [8,21].It appears that the increase in [Ca 2+ ] i occurs earlier than the ROS elevation after salt stress treatment.Considering ROS have also been shown to trigger increases in [Ca 2+ ] i [21,[23][24][25][26], it is possible that ROS-induced [Ca 2+ ] i increases might serve as a feed forward mechanism in the salt stress signal transduction pathway.However, less is known about the relationship and interaction between the salt stress-induced [Ca 2+ ] i increases and the [Ca 2+ ] i increases evoked by ROS, which are produced in response to either salt stress specifically or other stresses in general [1,27].
In this study, we have systematically analyzed the relationship and interaction between salt stress-induced [Ca 2+ ] i increases and the ROS-induced [Ca 2+ ] i increases in Arabidopsis.We found the increases in [Ca 2+ ] i induced by both stimuli were higher than these induced by either single stress, suggesting that NaCl and H 2 O 2 have an additive effect on [Ca 2+ ] i .We have also found that NaCl-induced [Ca 2+ ] i increases might inhibit both NaCl-and H 2 O 2 -gated channels by a feedback mechanism, but more NaCl-gated channels; a similar response was seen when the H 2 O 2 -induced [Ca 2+ ] i increases were analyzed.These data suggest responses seen involve both feedback inhibitory mechanisms, as well as an interaction between two stimuli-mediated Ca 2+ signaling pathways.

Dose-dependence and kinetics of NaCl-and H 2 O 2induced [Ca 2+ ] i increases
To analyze whether and how increases in [Ca 2+ ] i induced by NaCl and H 2 O 2 interact each other in Arabidopsis, we attempted initially to identify optimum concentrations of NaCl and H 2 O 2 , which ideally could be applied to generate about half of the maximum amplitude of [Ca 2+ ] i for potential up-and downregulation.In addition, we attempted to establish the kinetics of NaCl-and H 2 O 2 -induced increases in [Ca 2+ ] i for administrating these stresses in different sequential combinations.First, to analyze NaCl-induced increases in [Ca 2+ ] i , we treated Arabidopsis seedlings expressing aequorin with solutions containing 0 to 600 mM NaCl.Aequorin bioluminescence images were taken every 10 sec for 500 sec, and the peak [Ca 2+ ] i was calculated and analyzed, as NaCl induces a transient increase in [Ca 2+ ] i [8,9].Plants grown on the halfstrength MS medium had an average basal [Ca 2+ ] i of 80 ± 21 nM (Figure 1A and C).As expected, the [Ca 2+ ] i increased in response to NaCl treatment (Figure 1A).The magnitudes of [Ca 2+ ] i increases were dependent on the concentration of NaCl, higher concentration of NaCl evoked a larger increase in [Ca 2+ ] i .The NaCl concentration needed for a half-maximal response was ~200 mM, which was chosen as an optimum concentration to subsequently analyze the interaction with Then, we determined the temporal dynamics of NaClinduced [Ca 2+ ] i increases under the imposed experimental conditions as a control for further comparison (Figure 1B).We found that [Ca 2+ ] i increased immediately after the application of 200 mM NaCl, reached a peak of ~1 µM at about 20 sec, and then declined gradually (Figure 1B).Note that, the peaking time might be shorter than 20 sec based on previous studies [8,9].Nevertheless, imaging aequorin bioluminescence for less than 10 sec resulted in images with low a signal-noise ratio in our system.Thus, the temporal resolution was about 10 sec, which is sufficient for the current study.At about 200 sec, the [Ca 2+ ] i was reduced to a new resting level of under 200 nM.
Similarly, we analyzed increases in [Ca 2+ ] i in response to H 2 O 2 .Seedlings were treated with different concentrations of H 2 O 2 from 0 to 15 mM and [Ca 2+ ] i was analyzed.As expected, H 2 O 2 induced increases in [Ca 2+ ] i in a dose-dependent manner (Figure 1C).The H 2 O 2 concentration for a half-maximal response was around 4 mM with the magnitude of [Ca 2+ ] i similar to that induced by 200 mM NaCl.We then determined the temporal dynamics of the [Ca 2+ ] i increase induced by 4 mM H 2 O 2 .Following treatment with 4 mM H 2 O 2 , [Ca 2+ ] i increased and reached a peak of ~1 µM at 50 sec (Figure 1D).It took another 100 sec for the [Ca 2+ ] i to reach a new basal level of just under 200 nM.Taken together, it seems that increases in [Ca 2+ ] i occur faster in response to NaCl than H 2 O 2 , but are reset to a resting level 150 sec after treatment.

Additive effect of NaCl and H 2 O 2 on triggering increases in [Ca 2+ ] i
To investigate thoroughly the relationship and/or interaction between [Ca 2+ ] i increases triggered by NaCl and H 2 O 2 , Arabidopsis seedlings were treated with 200 mM NaCl or 4 mM H 2 O 2 separately, or 200 mM NaCl together with 4 mM H 2 O 2 .Note that, although salt-induced ROS production could be detected within ~2 min after salt treatment [21] the estimated half-time to the peak of ROS production is more than 5 min.Note also that, we did not detect a second peak of [Ca 2+ ] i within 5 min after salt stress treatment (Figure S1A), suggesting that salt-induced ROS could not trigger a detectable increase in [Ca 2+ ] i under the current experimental conditions.Because we measured [Ca 2+ ] i changes within 5 min after salt treatment, the effect of salt-induced ROS on [Ca 2+ ] i should not interfere apparently.The [Ca 2+ ] i increases recorded after single treatments were consistent with the results described above (Figure 2A-C).NaCl and H 2 O 2 induced similar increases in [Ca 2+ ] i (Figure 2E).When plants were treated with both stimuli simultaneously, the peaks of [Ca 2+ ] i were much larger than that induced by each individual stimulus (Figure 2D and E), showing an additive effect.To further analyze how salt-induced ROS affects [Ca 2+ ] i increases in response to salt stress within 500 sec, we carried out an experiment by using the NADPH oxidase inhibitor DPI [28] and ROS scavengers ascorbic acid and glutathione [27], and found that neither of these reagents significantly affected [Ca 2+ ] i increases induced by NaCl (Figure S1).These results suggest that the NaCl-and H 2 O 2 -induced [Ca 2+ ] i increases may be largely independent events.In other words, NaCl and H 2 O 2 might activate different Ca 2+ permeable channels.

The crosstalk between NaCl-and H 2 O 2 -induced [Ca 2+ ] i increases
To further characterize the potential interaction between the two stimuli-triggered [Ca 2+ ] i signals, plants were successively treated either with the same stimulus or the other.When the Arabidopsis seedlings were treated with 200 mM NaCl, the level of [Ca 2+ ] i increased quickly to reach a peak and decreased to the new resting level after 150 sec (Figure 3A), as described in Figure 1B.A subtle increase in [Ca 2+ ] i could be detected in seedlings after washing with deionized water at 200  sec (Figure 3A and B; green).Then, NaCl was added again, which caused a small increase in [Ca 2+ ] i .It decayed from 300 sec to a level similar to the previous resting level (Figure 3A  and B).Compared to the first NaCl treatment, which led to a large [Ca 2+ ] i increase to ~1 µM, the 2 nd NaCl treatment resulted in a [Ca 2+ ] i increase that was only a fraction of the size of the first [Ca 2+ ] i increase.This observation suggests that the NaClactivated Ca 2+ permeable channel (NaC) might be desensitized or adapted by unknown signaling elements upstream of NaC activation.To test whether the NaC is desensitized or adapted, we waited for 3 hr and were able to detect a normal ( ~1 µM) [Ca 2+ ] i increase in response to NaCl, suggesting that the NaC is most likely desensitized (data not shown).
Subsequently we analyzed whether the hydrogen peroxideactivated Ca 2+ permeable channel (HpC) was affected by the initial NaCl treatment.The second NaCl treatment was replaced by 4 mM H 2 O 2 at 300 sec (Figure 3C).Interestingly, the peak of [Ca 2+ ] i induced by H 2 O 2 was clearly greater than that induced by 200 mM NaCl (P < 0.001).After 450 sec, the [Ca 2+ ] i decreased to a new basal level under 200 µM (Figure 3C and D).The lower inhibition of HpC than NaC by the initial NaCl treatment suggests that the initial high level of [Ca 2+ ] i , which resulted from NaC activation (called NaC [Ca 2+ ] i microdomain/puff) subsequently inhibited NaC more than HpC (Figure 3).
By analogy, we used H 2 O 2 as the first stimuli to treat the seedlings and then analyzed the second treatment using H 2 O 2 or NaCl (Figure 4).When H 2 O 2 was added to the Petri dish, at 300 sec after the first H 2 O 2 treatment, the [Ca 2+ ] i level stabilized at 178 ± 32 nM, similar to previous resting levels (Figure 4A  and B).But when we used 200 mM NaCl to replace H 2 O 2 at 300 sec, the peak values of [Ca 2+ ] i were 381 ± 23 nM, small but significantly higher than that seen with the second H 2 O 2 treatment (Figure 4B and D).Similarly, our results suggest that the high [Ca 2+ ] i , which resulted from the initial HpC activation (called HpC [Ca 2+ ] i microdomain), inhibited HpC more than NaC (Figures 4 and 5).

Discussion
Calcium is a universal second messenger that plays an important role in signal transduction in animals and plants [25,[29][30][31].In the past 20 years, tremendous progress has been made in understanding the changes in [Ca 2+ ] i that appear in response to various abiotic and biotic stresses in plants, including salt stress, oxidative stress, drought, high and low temperatures, and pathogen elicitors [13,25,26,32].It is known that a specific stimulus can trigger unique temporal and spatial patterns of [Ca 2+ ] i , also known as [Ca 2+ ] i signatures [33].The [Ca 2+ ] i signature encodes information from the environmental stimulus which will be decoded subsequently by intracellular Ca 2+ sensors, such as calmodulins (CaMs) and calcineurin Blike proteins (CBLs), leading to the activation of downstream events [10].It is also known that the basal [Ca 2+ ] i is maintained at a concentration about 10,000-fold below the extracellular Ca 2+ concentration [29,31,34].In general, Ca 2+ channels in the plasma membrane and/or endomembranes are activated in response to environmental stimuli, leading to increases in [Ca 2+ ] i [30,32].Salt stress-induced increases in [Ca 2+ ] i have long been proposed as being involved in the process perceiving the salt signal although the properties of the saltactivated Ca 2+ permeable channel are poorly understood and its molecular nature remains to be identified [1,2,7].
In addition, various abiotic and biotic stresses lead to the production of ROS and oxidative stresses, which control many different processes in plants [27,[35][36][37].It has been well established that salt stress enhances the production of reactive oxygen species (ROS) in plants [18][19][20][21][22]. Interestingly, ROS has also been shown to activate Ca 2+ permeable channels in the plasma membrane, which in turn lead to Ca 2+ influx into the cell and thus increases in [Ca 2+ ] i [19,24,38].Note that, the salt stress-induced [Ca 2+ ] i increases precede the production of H 2 O 2 signaling molecule [39].Nevertheless, little is known about the molecular mechanisms underlying ROS perception in plant cells, and it is possible that ROS activation of Ca 2+ permeable channels may serve as a ROS perception process.
The decay of the increases in [Ca 2+ ] i induced by both NaCl and H 2 O 2 seen in this study (Figure 1B and D) as well as previous studies [8,9,15] indicates that the stimulus-activated Ca 2+ permeable channels may be inactivated via a feedback inhibitory mechanism, i.e. elevated [Ca 2+ ] i inhibits these channels, a desensitization process commonly seen for receptor ion channels in animals [31,40].It remains to be addressed whether the localized increases in [Ca 2+ ] i induced by one stimulus, called [Ca 2+ ] i microdomain [29,31], inhibit the other stimulus-activated Ca 2+ permeable channels.It is known that that NaCl induces multiple peaks of [Ca 2+ ] i under certain conditions [9], possibly because the same NaCl-sensitive channels are repetitively activated, or that NaCl might trigger H 2 O 2 production which subsequently activates another Ca 2+ channel, different from the NaCl-sensitive channel.Under our experimental conditions, we did not observe multiple peaks of [Ca 2+ ] i after salt treatment (Figure S1A).
NaCl and H 2 O 2 together induced larger increases in [Ca 2+ ] i than either NaCl or H 2 O 2 alone (Figure 2), suggesting that NaCl and H 2 O 2 may activate distinct Ca 2+ permeable channels, NaC and HpC (Figure 5).NaC and HpC are likely regulated by feedback inhibition (Figure 5), considering their desensitization seen in this study (Figure 1B and D) as well as previous reports [8,9].We demonstrated that repetitive NaCl treatments failed to trigger repetitive [Ca 2+ ] i increases (Figure 3A and B).This indicates that the NaC cannot be activated repetitively within a short period of time, i.e.NaC is possibly desensitized.We propose that a feedback inhibition may be involved in the desensitization (Figure 5).Upon NaCl treatment, the NaC opens, leading to the localized increase of [Ca 2+ ] i , NaC [Ca 2+ ] i microdomain/puff.NaC [Ca 2+ ] i in turn signals the channel to close, which prevents further [Ca 2+ ] i increases and allows the basal [Ca 2+ ] i to be reset via Ca 2+ pumps.This feedback inhibition avoids the excessive increase of [Ca 2+ ] i which could be disastrous to plant cells.The same phenomenon was also observed with the activation of HpC (Figure 5), i.e.HpC [Ca 2+ ] i microdomain inhibits HpC via a feedback mechanism.Clearly, the most significant effect is that after the initial treatment by either NaCl or H 2 O 2 , [Ca 2+ ] i increases induced by both NaCl and H 2 O 2 are reduced (Figures 3 and 4).It is most likely that localized NaC [Ca 2+ ] i and HpC [Ca 2+ ] i merge to form a relatively global [Ca 2+ ] i , which then feedback inhibits both NaC and HpC (Figure 5).We observed that Arabidopsis was unable to recover from 200 mM NaCl treatment 5 min after an initial stimulation.Similar results were observed after 4 mM H 2 O 2 treatment.In contrast, a previous study has shown that Arabidopsis is able to recover its ability to respond almost fully to cold shock 3 min after an initial cold shock [41].Note that, our work does not prove that HpC and NaC are localized in discreet and different microdomains in the plasma membrane, rather we have shown HpC and NaC may differ and interact via [Ca 2+ ] i microdomains.It is also possible that NaCl and H 2 O 2 may activate the same Ca 2+ permeable channel, which is expressed in different types of cells and/or activated via different signaling pathways, leading to the differential changes in [Ca 2+ ] i .
In general, when plants are exposed to one stress, their resistance to other stresses can be enhanced.It is most likely that stress-evoked [Ca 2+ ] i increases well as stressstimulated overproduction of ROS function as key integrators, possibly mediating stress signal perception and signal transduction.Our results demonstrate the inhibitory interaction of NaCl-and H 2 O 2 -induced [Ca 2+ ] i increases, and may predict distinct Ca 2+ permeable channels activated by NaCl and H 2 O 2 , respectively (Figure 5).In the future, it is important to analyze the pharmacological properties of these putative Ca 2+ permeable channels activated by NaCl and H 2 O 2 as described previously for MAMP-activated channels [42].Obviously, the identification of these channels or sensors will be a hallmark in the study of plant salt resistance in the future.In addition, how NaC and HpC interact to contribute to the [Ca 2+ ] i signatures and other downstream events can be further analyzed when their molecular nature is identified.

Plant materials and growth
Arabidopsis thaliana ecotype Columbia-0 (Col-0) constitutively expressing intracellular aequorin (pMAQ2, a kind gift from Dr. M. Knight) under the control of the cauliflower mosaic virus 35S promoter was used [8,33].Arabidopsis plants were grown in 150 mm x 15 mm round Petri dishes in halfstrength Murashige and Skoog salts (MS; Gibco), supplemented with 1.5% (w/v) sucrose (Sigma), and 0.8% (w/v) agar (Becton Dickinson) adjusted to pH 6.0 with KOH in controlled environmental rooms at 21 ± 2°C.The fluency rate of white light was ~110 µmol m -2 sec -1 .The photoperiods were 16 h light/8 h dark cycles.Seeds were sterilized with 2.5% PPM (Plant preservative mixture; Caisson Laboratories) and stratified at 4°C for 3 days in the dark, and then transferred to growth rooms.

Aequorin reconstitution and measurement of [Ca 2+ ] i
Arabidopsis thaliana plants expressing cytosolic apoaequorin were used for [Ca 2+ ] i measurements [33,43].Seedlings were grown on half-strength Murashige and Skoog medium for 7 days.Reconstitution of aequorin was performed in vivo by spraying seedlings with 240 µL of 10 µM coelenterazine per Petri dish followed by incubation at 21°C the dark for 8 hr.Treatments aequorin luminescence imaging were performed at room temperature using a ChemiPro HT system that includes a cryogenically cooled and back-illuminated CCD camera, liquid nitrogen autofiller, camera controller, and computer-equipped WinView/32 software (Roper Scientific) as described previously [33].The CCD camera has a 1300 × 1340 pixel resolution and is cooled to −110°C by the cryogenic cooler system prior to image recording.The recording was started 50 s prior treatments and luminescence images were taken every 10 sec.The total remaining aequorin was estimated by treating plants with a discharging solution containing 0.9 M CaCl 2 in 10% (v/v) ethanol and recorded for 5 min until values were within 1% of the highest discharge value [8,15,33].WinView/32 and Meta Morph 6 were used to analyze recorded luminescence images.Experiments were carried out at room temperature (22 to 24°C).

NaCl and H 2 O 2 treatments
For stress treatments, Petri dishes were placed individually into the ChemiPro HT chamber and luminescence images were taken at 10 sec intervals starting 50 sec prior the treatment.The treatment solution (100 mL) at described concentrations of NaCl or H 2 O 2 was added into Petri dish in the dark, and luminescence was recorded continuously.For changes in bath solution, a four-channel peristaltic pump (Dynamax RP-1, Rainin) was used to perfuse Petri dish with water as indicated in the figures.Then, additional stress treatment was applied by adding 100 mL solution into Petri dish.

Calibration of calcium measurements
The cytosolic free cytosolic Ca 2+ concentrations were calculated based on the calibration equation described previously [41] with modification to the ChemiPro system.The wild-type Arabidopsis expressing aequorin were placed individually in each well in 96-well plates containing ½ MS medium, 1.5% (w/v) sucrose, and 0.8% (w/v) agar for 10 days.Kinetic luminescence measurements were performed with an automated microplate luminescence reader (Mithras LB 940, Berthold Technologies).After automatic injection of 0.2 ml of solution into the each well bioluminescence counts were integrated every 1 sec as described previously [41].The solutions containing a range of [NaCl] from 0 to 600 mM were used to treat the plants, and the peak values of [Ca 2+ ] i were calculated used the equation described previously [41].Similar measurements were carried out using the ChemiPro HT system as described above to obtain L/L max values for each treatment, where L is luminescence and L max is the total remaining counts for bioluminescence.Then, we fit these data to the previously describe equation pCa = a * (−log (L/L max )) + b, and obtained the equation pCa = 0.9057209 * (−log (L/L max )) + 4.7712743.Note that, the calculated Ca 2+ concentrations presented in the current study are similar to those reported previously [8,41].

Figure 1 .
Figure 1.Increases in [Ca 2+ ] i in response to NaCl and H 2 O 2 treatments.(A and C) Increases in [Ca 2+ ] i induced by several concentrations of NaCl (A) and H 2 O 2 (C) in Arabidopsis.Seedlings expressing aequorin and grown for 7 days were treated with solutions containing several concentrations of NaCl or H 2 O 2 , and aequorin images were taken every 10 sec for 500 sec.Data for four independent experiments are shown (mean ± sem; n = 64).(B and D) Time courses of increases in [Ca 2+ ] i induced by 200 mM NaCl (B) or 4 mM H 2 O 2 (D).Seedlings grown for 7 days were treated with NaCl and H 2 O 2 at time zero, and aequorin images were taken every 10 sec.Representative recordings from individual seedlings were shown.Similar results were seen in six independent experiments using 128 seedlings.doi: 10.1371/journal.pone.0076130.g001

Figure 2 .
Figure 2. Increases in [Ca 2+ ] i in response to NaCl and H 2 O 2 individually or combined.(A to D) Imaging of [Ca 2+ ] i increases in response to the treatments of water (H 2 O; A), 200 mM NaCl (B), 4 mM H 2 O 2 (A), and 200 mM NaCl and 4 mM H 2 O 2 together (D) in Arabidopsis seedlings expressing aequorin.[Ca 2+ ] i increases were analyzed by imaging bioluminescence and scaled by a pseudocolor bar.(E) Quantification of [Ca 2+ ] i increases from experiments as in (A) to (D).Data for four independent experiments are shown (mean ± sd; n = 64; *** P < 0.001; NS, not significant P > 0.05).doi: 10.1371/journal.pone.0076130.g002

Figure 3 .
Figure 3. NaCl-induced [Ca 2+ ] i increases inhibits NaCl-activated channels more than H 2 O 2 -activated channels.(A and C) Arabidopsis seedlings were subjected to a 200 mM NaCl treatment once at 0 sec, and the solution was perfused by deionized water at 200 sec.Then, a second 200 mM NaCl (A), or 4 mM H 2 O 2 (C) treatment was applied around 300 sec.Aequorin luminescence was recorded continuously through the treatments in the dark.(B and D) Quantification of [Ca 2+ ] i increases for the 2 nd NaCl (B) or 2 nd H 2 O 2 treatment (C) from experiments as in (A) to (C), respectively.Data for four independent experiments are shown (mean ± sd; n = 64; NS, not significant P > 0.05; *** P < 0.001).doi: 10.1371/journal.pone.0076130.g003

Figure 4 .
Figure 4. H 2 O 2 -induced [Ca 2+ ] i increases inhibits H 2 O 2 -activated channels more than NaCl-activated channels.(A and C) Arabidopsis seedlings were subjected to a 4 mM H 2 O 2 treatment once at 0 sec, and the solution was perfused by deionized water at 200 sec.Then, a second 4 mM H 2 O 2 (A), or 200 mM NaCl (C) treatment was applied around 300 sec.Aequorin luminescence was recorded continuously through the treatments in the dark.(B and D) Quantification of [Ca 2+ ] i increases for the 2 nd H 2 O 2 (B) or 2 nd NaCl treatment (C) from experiments as in (A) to (C), respectively.Data for four independent experiments are shown (mean ± sd; n = 64; NS, not significant P > 0.05; *** P < 0.001).doi: 10.1371/journal.pone.0076130.g004

Figure S1. H 2 O 2
Figure S1.H 2 O 2 levels do not affect [Ca 2+ ] i increases in response to NaCl treatment.(A) Arabidopsis seedlings were treated with water (Control), the NADPH oxidase inhibitor DPI (15 µM), and ROS scavenger ascorbic acid (5 mM) and glutathione (5 mM) two hours prior to the NaCl treatment.The seedlings were then subjected to a 200 mM NaCl treatment, and aequorin luminescence was recorded continuously through the treatments in the dark.(B) Quantification of peak [Ca 2+ ] i increases from experiments as in (A).Data for three independent experiments are shown (mean ± sd; n = 35 to 62; NS, not significant, P > 0.05).(PDF)