Spatial and Temporal Calcium Signaling and Its Physiological Effects in Moso Bamboo under Drought Stress

Abstract

Elevations in cytosolic free calcium concentration constitute a fundamental signal transduction mechanism in plants; however, the particular characteristics of calcium ion (Ca²⁺) signal occurrence in plants is still under debate. Little is known about how stimulus-specific Ca²⁺ signal fluctuations are generated. Therefore, we investigated the identity of the Ca²⁺ signal generation pathways, influencing factors, and the effects of the signaling network under drought stress on Phyllostachys edulis (Carrière) J. Houz. Non-invasive micro testing and laser confocal microscopy technology were used as platforms to detect and record Ca²⁺ signaling in live root tip and leaf cells of P. edulis under drought stress. We found that Ca²⁺ signal intensity (absorption capacity) positively correlated with degree of drought stress in the P. edulis shoots, and that Ca²⁺ signals in different parts of the root tip of P. edulis were different when emitted in response to drought stress. This difference was reflected in the Ca²⁺ flux and in regional distribution of Ca²⁺. Extracellular Ca²⁺ transport requires the involvement of the plasma membrane Ca²⁺ channels, while abscisic acid (ABA) can activate the plasma membrane Ca²⁺ channels. Additionally, Ca²⁺ acted as the upstream signal of H₂O₂ in the signaling network of P. edulis under drought stress. Ca²⁺ was also involved in the signal transduction process of ABA, and ABA can promote the production of Ca²⁺ signals in P. edulis leaves. Our findings revealed the physiological role of Ca²⁺ in drought resistance of P. edulis. This study establishes a theoretical foundation for research on the response to Ca²⁺ signaling in P. edulis.

1. Introduction

Calcium ions (Ca²⁺) are a primary signaling element for diverse cell processes in response to environmental cues. Ca²⁺ is a vital regulatory molecule for response to stress in plant growth and development [1,2]. When plants are affected by various physical stimuli, such as temperature, drought, salt, light, gravity, or chemical substances, such as plant hormones and pathogenic inducers, extracellular and intracellular sources of calcium release Ca²⁺ into the cytoplasm through Ca²⁺ channels. The spatio-temporal activity of membrane-localized Ca²⁺ channels or transporters causes an increase in cytosolic free calcium ion concentration (Ca²⁺)cyt, resulting in specific signals [3,4]. Almost all of the extracellular stimuli can lead to changes in intracellular (Ca²⁺)cyt. However, different stress stimuli can lead to variations in the pattern of Ca²⁺ spatial-temporal changes. There are significant differences in time, frequency, amplitude, and regional distribution. Studies have illustrated that plants may rely on different forms of Ca²⁺ to reflect the specificity of different stimulation signals to achieve signal transduction [5].

Although Ca²⁺ signaling has been extensively studied in other gramineous plants, such as rice, little is known about the Ca²⁺ signal identities and functions of the clonal plant Phyllostachys edulis (Carrière) J. Houz. Therefore, a better understanding of the calcium signal characteristics of bamboo under drought stress is an important prerequisite for the study of clonal habits of bamboo using signal transduction methods.

At present, research has shown that root tip cells of P. edulis seedlings transport Ca²⁺ from the extracellular region, cell walls, nucleus, and other calcium stores under drought stress. With increased duration of drought stress, the distribution of and changes in Ca²⁺ will produce regular fluctuations [6]. The more pronounced the drought stress, the greater the Ca²⁺ distribution in the root tip. Exogenous application of calcium fertilizer can relieve the physiological effects of drought stress and improve drought resistance in P. edulis [7]. Although roots constitute the most direct organ of water absorption, both Ca²⁺ uptake velocity and density of the different organ changed under drought stress [8]. Moreover, stomatal opening and closing behaviors were also regulated by Ca²⁺ signals at the cellular scale [9].

When there are no environmental pressures present, the majority of the Ca²⁺ in plant cells is distributed in the extracellular compartments, cell wall, vacuoles, and endoplasmic reticulum, with less concentrated distribution in the cytoplasm to prevent the precipitation of calcium and phosphoric acid [10]. To regulate this lower Ca²⁺ concentration in the cytoplasm, plant cells will actively export Ca²⁺. When a stimulus signal reaches a cell, plasma Ca²⁺ channels transiently increase Ca²⁺ permeability. When the cytoplasmic Ca²⁺ concentration increases to a certain threshold, it binds to calmodulin (CaM) to form Ca-CaM compounds, and thus activate CaM. Activated CaM further activates various key enzymes in the plant, which further phosphorylates and dephosphorylates phospholipase, nicotinamide adenine dinucleotide (NAD) kinase, and Ca²⁺-ATPase. In addition, it amplifies the initial stimulation signal and subsequently causes the cells to produce a physiological response corresponding to the signal, such as cell division, material synthesis, etc. [2,11].

Under drought stress, a complex signaling network is formed by a communication mechanism between regulatory signals. Ca²⁺ signaling can be combined with calcium receptors, such as CaM, to amplify the signal and transmit the oscillation to initiate stomatal closure and production of reactive oxygen species [12,13]. Previously, reactive oxygen species were considered toxic byproducts of plant metabolism. However, recent studies have shown that reactive oxygen species also have an important part to play in cellular signal transduction and regulation networks [14,15,16]. Abscisic acid (ABA) is a root chemical signal that plays an important role in regulating stomatal movement of plants under drought stress. H₂O₂ can be used as a downstream signal of ABA to activate Ca²⁺ channels in the plasma membrane. Involvement of ABA can induce stomatal closure by increasing Ca²⁺ concentration in guard cells [17]. Sha et al. used 25% PEG-6000 to simulate water stress in maize plants, inducing CaM gene expression in the leaves [18]. The study found that exogenous ABA treatment can also induce significant CaM gene expression, and that H₂O₂ is involved in ABA-induced CaM gene expression in the late regulation period.

The emergence of new technologies has made it possible to study changes in Ca²⁺ signaling in response to environmental stress in plants. Non-invasive microelectrode technology (NMT) and laser confocal microscopy are effective techniques for detecting Ca²⁺ signals. Antoine et al. demonstrated the influx of Ca²⁺ over the course of fertilization in maize with non-invasive microelectrode technology [19]. In addition, a live-cell Ca²⁺ imaging platform has been used to detect Ca²⁺ signals in the cytoplasm and nucleus of Arabidopsis thaliana (L.) Heynh. This technique was also used to observe the spatial-temporal distribution of Ca²⁺ in living cells of A. thaliana under stimulated adverse environmental conditions [20]. These studies use a method to generate Ca²⁺ signaling pathways by treating experimental materials with Ca²⁺ inhibitors. Ethylene glycol-bis (2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA), lanthanum chloride (LaCl₃), and chlorpromazine (CPZ) are the most commonly used reagents. At present, laser confocal scanning microscopy has been used to study Ca²⁺ fluorescence localization in P. edulis cells under drought stress; however, most of these studies have been limited to the root tip [7] and few studies have been conducted on Ca²⁺ localization in cells located in the leaves of P. edulis using this technique.

To bridge this scientific gap, in this study we investigated the spatial-temporal location and flux velocity of Ca²⁺ ions by inducing drought stress in P. edulis with 20% PEG-6000. Using non-invasive micro-test technology (NMT) and laser confocal microscopy, we demonstrated the regularity of cellular Ca²⁺ dynamics in response to drought stress and provided measures of Ca²⁺ signaling in P. edulis leaf cells. We further studied the Ca²⁺ signaling pathway and analyzed the communication of the signal network pathway between Ca²⁺, H₂O₂, and ABA in leaf cells using Ca²⁺ inhibitors (Ca²⁺ channel blockers) and ABA. The aim of this study was to reveal the physiological role of Ca²⁺ in drought resistance and establish a theoretical foundation for the cellular response to Ca²⁺ signaling in P. edulis.

2. Materials and Methods

2.1. Plant Materials and Treatment

The sprouting seed materials used in this study were taken from the parent P. edulis from Guilin, Guangxi in September 2017. The thousand seed weight of these seed materials is equal to 22.75 ± 0.35 g. The P. edulis seeds were treated in November 2017. The seeds were soaked in warm water for 24 h at 50 °C, and then removed and disinfected with 5‰ potassium permanganate solution for 5 min. The sterilized seeds were then repeatedly washed with distilled water and germinated in an incubator in a dark environment set to a constant temperature of 25 °C. After the seeds germinated, the seedlings were selected for regular, even growth and placed in a Petri dish with pad disinfectant (lower layer) and filter paper (upper layer). The dishes were cultured at a constant temperature of 28 °C in an illumination incubator (PRX-1000B, Safe, Ningbo, China). The Petri dishes were set to point the roots vertically downwards. Proper humidity was maintained in the Petri dishes and any seeds found to be growing mold were removed. When the vertical root length reached approximately 3 cm, the seedlings were transferred to a Seed Germination Pouch (Phytotc CYG-98LB, size: 30 cm × 25 cm, Beijing Bioconsumable Tech., Ltd., Beijing, China) and cultured with a 12-h light period with a light intensity of 120–150 mmol m⁻² s⁻¹ and a temperature of 22 ± 2 °C. Figure 1 gives more details about the performance of the experimental materials.

For each experimental treatment, ten strains of P. edulis seedlings were selected in duplicate. To substitute drought stress, 20% polyethylene glycol-6000 (PEG, Coolaber, Beijing, China) was used for 5 min, 10 min, 15 min, and 30 min. Distilled water was used for the control (CK). In addition, Ca²⁺ inhibitors and ABA (Sigma-Aldrich, Shanghai, China) were used to treat P. edulis seedlings. The Ca²⁺ production pathway and its distribution were studied, and the effects of Ca²⁺ signaling on the H₂O₂/ABA signaling network were analyzed under drought stress. The Ca²⁺ inhibitors used in the experiment were extracellular Ca²⁺ chelating agent EGTA, Ca²⁺ channel blocker LaCl₃, and CaM antagonist CPZ (Sigma-Aldrich, Shanghai, China). The dosage of additives was added according to the method of Lu [21].

2.2. Laser Confocal Microscopy Luminescence Imaging

Here, we present detailed instructions for laser confocal microscopy luminescence imaging of cytosolic Ca²⁺ and H₂O₂ concentration and distribution in root tip and leaf cells of P. edulis seedlings.

2.2.1. Esterified Fluorescent Probe Stock Solution Configuration

The calcium ion fluorescent probe Fluo-8 (AAT Bioquest, Sunnyvale, CA, USA) was fully dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, Shanghai, China) to a concentration of 1 mmol/L, −20 °C dark storage reserve.

2′,7′-dichloro fluorescin diacetate (H2DCFDA, Sigma-Aldrich, Shanghai, China) was made up of 50 mmol/L DMSO mother liquor, which was stored in a separate container and frozen.

2.2.2. Fluorescent Labeling

As to the root tip cells and leaf cells Ca²⁺ fluorescent labeling, we put the lower epidermis of the leaves of P. edulis seedlings flat on transparent tape and used a surgical blade to gently scrape the upper epidermis off the leaves. Then we placed the samples of lower epidermis in Hank’s balanced salt solution (without calcium ions) containing 20 μmol/L Fluo-8. We incubated them for 40 min in the dark at room temperature and rinsed the lower epidermis of P. edulis leaves with a buffer several times. We then incubated them again at room temperature for 20 min, ensuring that the esterification probe was fully dissolved. Finally we placed them on a glass slide and added 0.5 mL of Hank’s buffer to complete slice preparation.

As to the H₂O₂ fluorescent labeling of P. edulis leaf cells, the lower epidermis of P. edulis seedling leaves was first laid flat on transparent tape, and then the upper epidermis of the leaves was removed using a surgical blade and placed in MES, Free acid, monohydrate (Coolaber, Beijing, China) buffer to a final concentration of 50 μmol/L and incubated at room temperature in the dark for 15 min. We rinsed the lower epidermis samples several times with a buffer and placed them on glass slides. We then added 0.5 mL of buffer to complete slice preparation.

2.2.3. Laser Confocal Microscope Observation

Localization of Ca²⁺ fluorescence in P. edulis root tips and leaves: The prepared in vivo test slices were placed on a laser confocal microscope (LSM510, LeicaDM4, Berlin, Germany) for observation and scanning. Fluorescence intensity was controlled and all parameters were kept constant during the test (parameter settings: excitation wavelength 488 nm, BP 505-530, Pinhole 280, DG 581, AO 0.1, AG 1.34). At least three fields from different repeats were selected for each test. After the results were stable, one field of view was used for the analysis.

Localization of H₂O₂ fluorescence in root tips and leaves of P. edulis: The prepared in vivo test slices were placed on a laser confocal microscope (LSM510) for observation and scanning (parameter settings: excitation wavelength 488 nm, BP 505-530, Pinhole 386, DG 768, AO 0, AG 1). At least three fields from different repeats were selected for each test. After the results were stable, one field of view was used for analysis.

2.3. Measurement of Ca²⁺ Flux

Net Ca²⁺ flux was measured using non-invasive micro-test technology (Physiolyzer, Younger USA LLC, Amherst, MA 01002, USA; Xuyue (Beijing) Sci. & Tech. Co., Ltd., Beijing, China). NMT non-invasively measures Ca²⁺ fluxes with a high temporal and spatial resolution. It measures the concentration gradient of Ca²⁺ by means of selective microsensor oscillation between two points in the root tip of P. edulis seedlings (Figure 2).

After different test treatments, the roots were rinsed with redistilled water and immediately incubated in measuring solution to equilibrate for 10 min. Then, the roots were transferred to a measuring chamber containing 10−15 mL of a fresh measuring solution. Ions were monitored in the following solutions: 0.1 mM KCl, 0.1 mM CaCl₂, 0.1 mM MgCl₂, 0.5 mM NaCl, 0.3 mM MES, and 0.2 mM Na₂SO₄, following adjustment of the pH to 6.0. The measuring chamber was mounted on the micromanipulator, and the flux microsensor was positioned close to the root tip at four points: at the pileorhiza, meristematic zone, elongation zone, and mature zone (0 μm, 200 μm, 500 μm, and 800 μm from the root apex) of P. edulis seedlings.

The system setup parameters in the experiment are as follows. The Ca²⁺ flux microsensor (Φ4.5 ± 0.5 μm, XY-CGQ-01, Xuyue (Beijing) Sci. &Tech. Co., Ltd., Beijing, China) was filled with a backfilling solution (100 mM CaCl₂) to a length of approximately 1.0 cm from the tip. The micropipettes were front filled with 40–50 μm columns of selective liquid ion-exchange cocktails (Ca²⁺ LIX, XY-SJ-Ca, YoungerUSA LLC, Amherst, MA, USA). An Ag/AgCl wire microsensor holder YG003-Y11 (Younger USA) was inserted in the back of the microsensor to make electrical contact with the electrolyte solution. YG003-Y11 (Younger USA) was used as the reference microsensor. Prior to the flux measurement, the flux microsensor was calibrated with a measuring solution having different concentrations of Ca²⁺ 0.1 mM and 0.01 mM. The electrodes with a Nernstian slope > 22 mV per decade were used in this study. Only the same flux microsensor was calibrated again according to the same procedure and standards after each test. Data was discarded if the post-test calibrations failed. The following figure shows the schematic and actual measurement of Ca²⁺ flux.

The data for Ca²⁺ fluxes were calculated by Fick’s law of diffusion as follows:

J = −D·(dc/dx)

(1)

where dx (30 μm) is the distance the flux microsensor moved repeatedly from one point to another perpendicular to the surfaces of the samples at a frequency of ca. 0.3 Hz.

2.4. Statistical Analysis

Data were analyzed by single factorial analysis of variance and statistical correlation analysis. The significance of differences among means was evaluated using the least significant difference test, with a family wise error rate of 0.05, using the Statistical Package for Social Sciences, v18.0 (SPSS Inc., Chicago, IL, USA). Significant differences are marked with alphabet.

3. Results and Discussion

3.1. Variation in the Flux and Distribution of Ca²⁺ among Different Parts of the Root Tips of P. edulis Seedlings under Drought Stress

The transient net Ca²⁺ flux was measured from different regions along the root axis using NMT in four different areas, the root apex and root hair zone, including pileorhiza, the meristematic zone, elongation zone, and mature zone (Figure 3a). Responses in the root apex Ca²⁺ flux are shown in Figure 3b. The results showed that there was a significant difference in the ability of P. edulis seedlings to absorb Ca²⁺ from different parts of the root tip after drought stress. The Figure 3b showed the most uptake of Ca²⁺ in pileorhiza. The pileorhiza was the area with the strongest Ca²⁺ signal response ability, and the Ca²⁺ concentration in the root pileorhiza was the highest. The elongation zone was the region with the strongest Ca²⁺ efflux. The intensity of Ca²⁺ uptake capacity at different parts of the root tip from strong to weak was: pileorhiza, mature area, meristematic zone, and elongation zone. The Figure 3c shows the fluorescence localization of P. edulis root tip cells by laser confocal microscopy. To better observe the distribution of Ca²⁺ in the pileorhiza, the corresponding heat map is shown in Figure 3d. Combined with Figure 3b, it can be concluded that there were differences in the responsiveness of different parts of the root tip of P. edulis to drought stress, which is reflected in both the Ca²⁺ flux and regional distribution.

We confirmed this difference in the results of Ying [7], which used laser confocal microscopy on fluorescence localization in the root tip. This study showed that Ca²⁺ absorption intensity in the elongation zone was weakest under drought stress, while Ying’s study showed that under drought stress, the Ca²⁺ in the root tip of P. edulis was mainly distributed in the pileorhiza and elongation zone; the distribution in the meristem area was relatively lower. This was due to the presence of a large number of small vacuoles in the cells of the elongation zone, which were not present in meristem zone. The vacuoles, as a calcium bank in plant cells, may provide a large amount of Ca²⁺ for P. edulis cytoplasm under drought stress. This leads to a greater concentration of Ca²⁺ in the elongate zone.

3.2. Effects of Drought Stress Duration on Ca²⁺ Absorption Regularity in the Pileorhiza of P. edulis

Under different durations of PEG-induced drought stress, we observed different conductivity, which represents leaf cell permeability (Figure 4a). An increase in drought stress duration caused electrolyte leakage in the cells, which in turn led to an increase in leaf conductivity. The cell membrane permeability of P. edulis seedlings increased, and there was a positive correlation between time and membrane permeability. The difference in conductivity increase between the 10- and 15-min treatments was higher than that in the 5–10 min period (difference in conductivity = 15.2%). The relative electrical conductivity of P. edulis seedlings under drought stress was 40.4% higher in the 30-min treatment than in the 15 min treatment. We observed significant correlation between Ca²⁺ flux and drought stress time (p < 0.01, Pearson correlation coefficient (r) = 0.967) (Figure 4b). All the flux data in Figure 4b represents stable and optimal real-time flux that can respond to Ca²⁺ concentration in response to drought stress. Relative to the control group, Ca²⁺ flux in pileorhiza of P. edulis seedlings gradually shifted from efflux to influx. Ten minutes of PEG treatment was found to be a “threshold”, after which efflux became influx. There was also a positive correlation between leachate conductivity and time as Ca²⁺ absorption intensity in the pileorhiza (p < 0.01, r = 0.976), and leaf membrane permeability increased.

Ca²⁺ flux in the pileorhiza of P. edulis seedlings in the control group (CK) maintained an out-of-range value of approximately 152 nmol m⁻² s⁻¹. Figure 5 showed that Ca²⁺ flux in the pileorhiza was only slightly changed in the experimental group after 5 min of PEG treatment, indicating that the slight degree of drought stress did not significantly induce the stress response leading to Ca²⁺ signaling in such a short time. When treated with PEG for 30 min, the Figure 5 shows a “V-shaped” pattern of ion uptake, with net flux first increasing and then decreasing. As drought stress time increased, Ca²⁺ flux gradually returned to control group standard of efflux. It is evident that there is a positive correlation between Ca²⁺ signal and drought stress time (Figure 5). Under drought stress, there is a “stress threshold” for the Ca²⁺ signal response. When the stress level reached this threshold, the Ca²⁺ flux at the pileorhiza underwent significant changes, which may be related to the plant’s ability to withstand drought stress.

Thirty minutes of PEG simulated drought stress showed obvious Ca²⁺ absorption in the pileorhiza, and Ca²⁺ flux oscillation increased. With the increase in the duration of drought stress, Ca²⁺ absorption increased. P. edulis seedlings were able to absorb Ca²⁺ from the extracellular environment under drought stress conditions, suggesting that while the intracellular calcium stores provide Ca²⁺ to the cytoplasm, Ca²⁺ absorbed from extracellular environment was also an important way to accumulate calcium signaling ions in the pileorhiza. It is suggested that Ca²⁺, as an important signaling molecule in response to drought stress, participated in the transmission of drought signaling to the roots of P. edulis seedlings.

3.3. Analysis of Ca²⁺ Signal Transport Patterns in the Pileorhiza of P. edulis under Drought Stress

The study of the Ca²⁺ signaling system in plant cells was mainly carried out by pretreatment of experimental materials with Ca²⁺ signal inhibitors [21]. Regulation of Ca²⁺ channels is vital. Plasma membrane Ca²⁺-permeable channels interact with Ca²⁺ activated nicotinamide adenine dinucleotide phosphate (NADPH) to form a self-amplifying system—a ROS-Ca²⁺ hub [22]. This system could provide the transduction and amplification of the initial Ca²⁺ or reactive oxygen species (ROS) stimuli into a more sustainable response, with implications for cell growth, hormonal signaling, and stress response [23,24]. To obtain further evidence of the role of plasma membrane Ca²⁺ channels in P. edulis root tips, different Ca²⁺ inhibitors and ABA were used to further determine the Ca²⁺ signaling pathways. Ca²⁺ signal intensity varied with time in the pileorhiza of P. edulis seedlings treated with LaCl₃ (Ca²⁺ channel blocker), EGTA (extracellular Ca²⁺ chelating agent), and exogenous ABA.

To determine the factors affecting Ca²⁺ transport, Ca²⁺ flux in P. edulis root tip treated with a calcium antagonist and ABA was measured using NMT. As shown in Figure 6a, LaCl₃ was applied to the P. edulis seedlings treated with PEG-induced drought stress. The LaCl₃ treatment significantly impeded extracellular Ca²⁺ influx at the pileorhiza compared to the experimental group without LaCl₃. Extracellular Ca²⁺ channels were involved in Ca²⁺ fluid transport. The extracellular Ca²⁺ in seedlings could enter cells through Ca²⁺ channels under drought stress, which was one of the reasons for the increase of cytoplasmic Ca²⁺ concentration.

In addition, EGTA was used to treat the roots of P. edulis seedlings under PEG-simulated drought stress. Ca²⁺ flux at the pileorhiza was almost always in a state of efflux (Figure 6b), while net Ca²⁺ flux of without EGTA treatment saw a pattern of "efflux-absorption-efflux". The EGTA-chelating extracellular Ca²⁺ increased, resulting in decreases of extracellular Ca²⁺ concentration and a decrease in the ability of pileorhiza to absorb Ca²⁺; this caused an increase in Ca²⁺ excretion. Ca²⁺ uptake in the pileorhiza with the addition of EGTA was significantly lower than in the CK. It is worth noting that treating the pileorhiza with EGTA and LaCl₃ while under drought stress had the opposite effects on Ca²⁺ uptake curves. Ca²⁺ efflux rate from the pileorhiza of LaCl₃ decreased as EGTA treatment increased. LaCl₃ treatment hindered the Ca²⁺ channel in the pileorhiza, which led to a reduction of Ca²⁺ excretion from the pileorhiza.

Figure 6c shows Ca²⁺ absorption in the pileorhiza when treated with 30 μmol/L ABA for 10 min as PEG-induced drought stress treatment. Compared with the control, the ABA treatment showed a smaller oscillation in Ca²⁺ flux in the pileorhiza, and Ca²⁺ net flux reflected its strong absorption capacity. Therefore, ABA can promote the increase in Ca²⁺ absorption intensity in the pileorhiza of P. edulis seedlings. We conclude that ABA may activate the extracellular Ca²⁺ channel in the pileorhiza and promote Ca²⁺ uptake (Figure 6a,b). The efflux of ABA-induced Ca²⁺ spikes remained high among all treatments. These results further confirm that plasma membrane Ca²⁺ channels activity is involved in Ca²⁺ signaling in PEG-induced drought stress by controlling (Ca²⁺)cyt through Ca²⁺ influxes. Under drought stress, adding the Ca²⁺ channel blocker LaCl₃ and extracellular Ca²⁺ chelating agent EGTA could significantly inhibit extracellular Ca²⁺ influx. Exogenous application of ABA could increase the ability of the pileorhiza to absorb Ca²⁺.

3.4. Analysis of Ca²⁺ Signaling Pathway in Leaves under Drought Stress

In addition to chemical signal root transduction, drought stress triggered Ca²⁺ movement in leaf cells. Ca²⁺ may regulate leaf stomatal movement to control transpiration and respiration, as a self-protection and adaption measure in response to environmental stress. To simultaneously monitor cell-specific Ca²⁺ P. edulis seedling leaves, we used a confocal microscope to record Ca²⁺ fluorescence localization (Figure 7).

CK is the Ca²⁺ fluorescence map of the lower epidermal cells treated with distilled water. Under normal water conditions, Ca²⁺ was mainly localized within the cell walls of guard cells, accessory cells, and long cells in P. edulis seedling leaves. Ca²⁺ was less commonly found to be distributed in the cytoplasm. As drought stress duration increased, Ca²⁺ concentration in the cytoplasm increased, except for in tethered cells. Among them, Ca²⁺ in long cells and guard cells increased significantly. The fluorescence intensity in the cytoplasm was also higher than that of the CK. Fluorescence intensity in the cytoplasm of accessory cells did not significantly increase. There were particularly pronounced differences in Ca²⁺ distribution in the 30 min PEG treatment. In addition to in tethered cells, the cytoplasmic fluorescence in all living cells, including guard cells, increased. Our results suggest that Ca²⁺ signals in response to PEG stress reflect distinct cellular Ca²⁺ dynamics.

With increased duration of drought stress, the fluorescence intensity in the cytoplasm of long cells increased. Only when a certain degree of drought stress was reached would Ca²⁺ in the cytoplasm of leaf guard cells escape from the cell wall and diffuse into the cytoplasm, resulting in a significant increase in the fluorescence intensity of the cytoplasm. In comparison, the Ca²⁺ in the cytoplasm of accessory cells was not obviously enhanced. To further verify that Ca²⁺ in the leaf cells of P. edulis was also transmitted through the cytoplasmic Ca²⁺ channels, three Ca²⁺ inhibitors were used to treat the seedlings under drought conditions. Ca²⁺ fluorescence localization under these conditions is shown in Figure 8.

To confirm the effect of calcium channels on Ca²⁺, we analyzed the Ca²⁺ fluorescence under three Ca²⁺ inhibitor treatments. The addition of CPZ inhibited the binding of Ca²⁺ to CAM in the seedlings; thus, the Ca²⁺ signal was not transmitted further. The results showed that after treatment with 0.1 mmol/L CPZ in the seedlings under PEG-simulated drought stress for 10 min, the brightness of the fluorescence in the cell wall and cytoplasm of the leaf cells was less than under the treatment without CPZ. Under drought stress, the Ca²⁺ in leaf cells of the P. edulis seedlings treated with 0.1 mmol/L CPZ were mainly distributed in the cell wall. The fluorescence brightness in the cell wall decreased more obviously than in other parts of the leaf.

We analyzed the distribution of Ca²⁺ in leaves treated with 3 mmol/L EGTA. Fluorescence intensity in the cell wall and cytoplasm reduced, indicating that Ca²⁺ concentration in the cell wall and cytoplasm decreased compared with the control group. This indicates that P. edulis seedling leaves can still take up Ca²⁺ from extracellular pathways under drought stress to produce calcium signals. Extracellular Ca²⁺ was chelated in the leaves under EGTA treatment, resulting in a decrease in extracellular Ca²⁺ uptake.

Treatment with LaCl₃ prevents Ca²⁺ from extracellular entry, and Ca²⁺ in the cytoplasm of leaf cells significantly reduced. Ca²⁺ was mainly concentrated in the cell walls in this treatment (Figure 8). The results of Ca²⁺ fluorescence localization in leaf cells after treatment with 0.5 mmol/L LaCl₃ for 10 min in drought-stressed P. edulis seedlings showed a decrease in Ca²⁺ fluorescence intensity in the cytoplasm of the cells. This was caused by the LaCl₃ blocking the cytoplasmic Ca²⁺ channel, blocking extracellular Ca²⁺ entry through the cell wall. This shows that Ca²⁺ in the leaves of P. edulis can be transported through Ca²⁺ channels of the plasma membrane, absorbing Ca²⁺ from outside the cell, and using the potential difference to generate calcium signals.

3.5. Analysis of the Effects of Ca²⁺ Signals on H₂O₂ and ABA Signaling Pathways under Drought Stress

The H₂O₂ fluorescence localization map of P. edulis leaves is shown in Figure 9. H₂O₂ concentration in the leaf cytoplasm increased with drought stress time (Figure 9a). P. edulis seedling leaves treated with different Ca²⁺ inhibitors under PEG-simulated drought stress were subjected to laser confocal technology for H₂O₂ fluorescence localization to study the relationship between Ca²⁺ signaling and H₂O₂ in the stress signaling pathway (Figure 9b). Compared with CK, plants treated with 0.1 mmol/L CPZ showed lower leaf fluorescence, indicating that CPZ could also prevent H₂O₂ signal transduction. The calcium signals were unable to be transmitted normally, resulting in the decrease of H₂O₂ in the leaf cells. This indicates that the regulation of H₂O₂ activity requires the participation of Ca²⁺. Both 0.5 mmol/L LaCl₃ and 3 mmol/L EGTA inhibited the production of H₂O₂ in guard cells, accessory cells, and long cells in leaves of P. edulis seedlings under drought stress. It is inferred that the Ca²⁺ signal is generated upstream of the active oxygen signal in the drought stress signaling network of P. edulis, and that H₂O₂ activity in leaf cells requires Ca²⁺ participation.

Laser confocal microscopy was used to detect the Ca²⁺ and H₂O₂ fluorescence signal in living leaf cells. The results indicated that the exogenous application of ABA under drought stress led to an increase in the concentration of Ca²⁺ and H₂O₂ in the mesophyll cells of P. edulis seedlings. Interestingly, there was a positive correlation between Ca²⁺ and H₂O₂ dynamics. ABA might activate Ca²⁺ channels of the plasma membrane and promote the production of Ca²⁺ signals in the pileorhiza of P. edulis seedlings.

H₂O₂ fluorescence intensity in guard cells was significantly higher in the ABA-treated group than in the group not treated with ABA (Figure 10). In plants, ABA is involved in many stress responses [25,26,27]. Regulatory systems of reactive oxygen species (ROS) are known to be integrated with other pathways involving Ca²⁺ signaling, protein kinases, and hormones pathways to regulate the defense mechanism in plants [28]. The existence of ABA-induced genes, which are expressed in stomatal guard cells, allows us to closely examine the role of Ca²⁺ [29]. In rice, the Ca²⁺/CaM-dependent protein kinase OsDMI3 has been shown to be required for ABA-induced antioxidant defense [30]. These results demonstrated that ABA also plays a key role in the signal transduction of Ca²⁺ in the signaling network of the leaves of P. edulis treated with drought stress. Ca²⁺ not only acted as the upstream signal of H₂O₂, but was also involved in the signal transduction process of ABA. ABA could promote the production of Ca²⁺ signaling in leaves and stimulate the burst of H₂O₂, a reactive oxygen species, in the guard cells. P. edulis may enhance drought tolerance via ABA-induced stomatal closure by ROS production.

4. Conclusions

This study indicated that the conductivity of P. edulis leaves increased with the degree of drought stress induced. Ca²⁺ is an important signaling molecule in response to drought stress in the root tip of P. edulis, especially in the transmission of stress signals and resistance to drought stress. Under drought stress, root tip cells of P. edulis can be used to transport Ca²⁺ from the extracellular environment to the cytoplasm, Ca²⁺ channels participate in extracellular Ca²⁺ transportation, and ABA may activate Ca²⁺ channels in the plasma membrane and promote Ca²⁺ signal production in the pileorhiza of P. edulis. In P. edulis leaves, Ca²⁺ can also be transported through the Ca²⁺ channels of the plasma membrane under drought stress, absorbing Ca²⁺ from outside the cell and generating Ca²⁺ signals through potential difference. The responsiveness of Ca²⁺ signals to drought stress in leaves of P. edulis from strong to weak was shown as follows: (1) long cells; (2) guard cells; (3) accessory cells; and (4) plug cells. Ca²⁺ acts as the upstream signal of H₂O₂ in the signal network of the mesophyll cells of the P. edulis under drought stress. It is also involved in ABA signal transduction process. ABA could promote Ca²⁺ signal production and stimulate H₂O₂ bursts in P. edulis leaves.

This study provides a method for the spatial and temporal localization of Ca²⁺ signaling and flux in P. edulis. Further research on Ca²⁺ signaling is essential, as it may help shed light on the physiology of P. edulis under stress. There are also areas for improvement in this study. The first is that processing the mesophyll samples may damage the cells or put them under stress conditions, thus affecting the Ca²⁺ fluorescence of the leaves under the laser confocal microscope. Autofluorescence of lignin can also have an effect on Ca²⁺ fluorescence. It is particularly important to improve this methodology in the future.