Towards Understanding the Involvement of H+-ATPase in Programmed Cell Death of Psammosilene tunicoides after Oxalic Acid Application

Psammosilene tunicoides is a unique perennial medicinal plant species native to the Southwestern regions of China. Its wild population is rare and endangered due to over-excessive collection and extended growth (4–5 years). This research shows that H+-ATPase activity was a key factor for oxalate-inducing programmed cell death (PCD) of P. tunicoides suspension cells. Oxalic acid (OA) is an effective abiotic elicitor that enhances a plant cell’s resistance to environmental stress. However, the role of OA in this process remains to be mechanistically unveiled. The present study evaluated the role of OA-induced cell death using an inverted fluorescence microscope after staining with Evans blue, FDA, PI, and Rd123. OA-stimulated changes in K+ and Ca2+ trans-membrane flows using a patch-clamp method, together with OA modulation of H+-ATPase activity, were further examined. OA treatment increased cell death rate in a dosage-and duration-dependent manner. OA significantly decreased the mitochondria activity and damaged its electron transport chain. The OA treatment also decreased intracellular pH, while the FC increased the pH value. Simultaneously, NH4Cl caused intracellular acidification. The OA treatment independently resulted in 90% and the FC led to 25% cell death rates. Consistently, the combined treatments caused a 31% cell death rate. Furthermore, treatment with EGTA caused a similar change in intracellular pH value to the La3+ and OA application. Combined results suggest that OA-caused cell death could be attributed to intracellular acidification and the involvement of OA in the influx of extracellular Ca2+, thereby leading to membrane depolarization. Here we explore the resistance mechanism of P. tunicoides cells against various stresses endowed by OA treatment.


Introduction
Oxalic acid (OA) is a simple dicarboxylic acid that widely exists in biological systems and plays functional role in plants. OA's chemical nature as a potent metal chelator has received more attention for its physiological functions in metabolism and signalling pathways in plant cells [1]. Moreover, OA has been recorded as an effective elicitor, improving plants' resistance against adverse effects of phytopathogens. For instance, OA induced systemic resistance of tomatoes against Botrytis cinerea and Sclerotinia sclerotiorum (S. sclerotiorum) in sunflower [2,3]. In addition, after plants are challenged to biotic stress, study also suggested that OA may influence inward K+ channels and Ca 2+ channels by mediating the activation of H + -ATPase.

Effects of OA on Suspension Cell Viability of P. tunicoides
Previous studies have reported that differential expression of genes contributing to PCD triggered by exogenous OA in tomatoes [30]. Current study analyzed the effects of OA on suspension-cultured cells of P. tunicoides using live/dead staining methods with Evans blue and FDA (Figure 1). The percentage of cell death was measured every hour between 0-8 hours (h). It was found that 1 mM OA treatment had significant influence on plant cells death. The death rate of 90%±10 was observed after the 8 h of OA treatment. Compared with 100 µM OA treatment, the rate of cell death was about 54% ± 4 by within 8 h, as shown in (Figure 1). Identical results were recorded even after 24 h of treatment. However, with decline of OA concentration to 10 µM, during the entire course of 8 h of treatment, no visible alteration in cell death rate could be observed. The evidence related to PCD, such as vacuole shrinkage, plasma membrane invagination and the formation of cysts, was clear and in agreement with published results [31,32]. These dose-dependent data supported published hypothesis that OA may influence the viability of cells to a measurable extent, and in some cases, even lead to PCD in Panax ginseng cells [33].

Effects of OA on Respiratory Electron Transporter Chains
It is common knowledge that mitochondria is the power-generating organelles of a cell. The respiratory electron transport chains provide the driving forces for metabolism and generate redox signals, regulating every aspect of plant biology by controlling enzyme gene expression [34,35]. Kinetic data indicate that mitochondrion undergoes significant changes in membrane integrity before classical signs of apoptosis manifest. These changes concern both inner and outer mitochondrial membranes, disrupting inner transmembrane potential and releasing inter-membrane proteins through the outer membrane [36]. This study examined the mitochondrial membrane potential (∆Ψ m ) after treatment with 1 mM OA using the mitochondrial marker, rhodamine (Rd) 123. The decrease in green fluorescence intensity was observed in OA-treated cells after 15 min, becoming even more evident until 75 min, due to the collapse of ∆Ψ m and loss of mitochondrial membrane integrity, as seen in (Figure 2). The diffuse, high level of cytoplasmic fluorescence was likely due to the loss of mitochondrial membrane integrity, and an inability to specifically accumulate Rd123 occurred in mitochondria under these conditions. The ratio of fluorescence intensities began to increase significantly in the cytoplasm. However, it could not reach the nadir as in its beginning. The images revealed that the mitochondrial self-repairing mechanism might work in the process, but the mitochondrial membrane integrity had already been lost and could not resume completely.
It is common knowledge that mitochondria is the power-generating organelles of a cell. The respiratory electron transport chains provide the driving forces for metabolism and generate redox signals, regulating every aspect of plant biology by controlling enzyme gene expression [34,35]. Kinetic data indicate that mitochondrion undergoes significant changes in membrane integrity before classical signs of apoptosis manifest. These changes concern both inner and outer mitochondrial membranes, disrupting inner transmembrane potential and releasing inter-membrane proteins through the outer membrane [36]. This study examined the mitochondrial membrane potential (∆Ψm) after treatment with 1 mM OA using the mitochondrial marker, rhodamine (Rh) 123. The decrease in green fluorescence intensity was observed in OA-treated cells after 15 min, becoming even more evident until 75 min, due to the collapse of ∆Ψm and loss of mitochondrial membrane integrity, as seen in (Figure 2). The diffuse, high level of cytoplasmic fluorescence was likely due to the loss of mitochondrial membrane integrity, and an inability to specifically accumulate Rh123 occurred in mitochondria under these conditions. The ratio of fluorescence intensities began to increase significantly in the cytoplasm. However, it could not reach the nadir as in its beginning. The images revealed that the mitochondrial self-repairing mechanism might work in the process, but the mitochondrial membrane integrity had already been lost and could not resume completely. Figure 2. A series of images of cells were undergoing disruption of respiratory electron transport chains after treating 1 mmol/L of OA. Rd123 stained the cells. The green fluorescence of Rd123 in the mitochondria was evident (A). After the treatment of 1 mM OA, the fluorescence intensity began to decrease after 15 min (B). The boundary became obscure, and the fluorescence intensity continued to drop after 30 min (C). After 60 min the fluorescence intensity in the mitochondria decreased markedly (D). The fluorescence intensity disappeared almost entirely after 75 min (E). The bright-field photo of (E) picture (F).

Effects of OA on Nuclear Membrane Integrity
In the early stage, cell membrane permeability is an essential index for distinguishing apoptosis-like PCD from necrosis. However, it will gradually further become leaky with the time-lapse of cells undergoing programmed death. Propidium iodide (PI), which only stains the nucleus of a late stage of PCD but is capable of degrading the nucleus of necrotic Figure 2. A series of images of cells were undergoing disruption of respiratory electron transport chains after treating 1 mmol/L of OA. Rd123 stained the cells. The green fluorescence of Rd123 in the mitochondria was evident (A). After the treatment of 1 mM OA, the fluorescence intensity began to decrease after 15 min (B). The boundary became obscure, and the fluorescence intensity continued to drop after 30 min (C). After 60 min the fluorescence intensity in the mitochondria decreased markedly (D). The fluorescence intensity disappeared almost entirely after 75 min (E). The bright-field photo of (E) picture (F).

Effects of OA on Nuclear Membrane Integrity
In the early stage, cell membrane permeability is an essential index for distinguishing apoptosis-like PCD from necrosis. However, it will gradually further become leaky with the time-lapse of cells undergoing programmed death. Propidium iodide (PI), which only stains the nucleus of a late stage of PCD but is capable of degrading the nucleus of necrotic cells quickly, was employed and added to the media containing suspension cells treated by OA. As indicated in (Figure 3), the fluorescence intensity of the PI-staining nucleus began to appear only in the cells after 1 hour of OA stress and then increased and dispersed around the nucleus gradually ( Figure 3B-E). Here, the staining of the nucleus represented the PI diffusion across the plasma membrane. However, its physical rupture did not occur after the loss of membrane integrity ( Figure 3F). The result of PI staining demonstrated again the hallmark of PCD caused by OA, just as indicated by Rd123 staining of mitochondria. by OA. As indicated in (Figure 3), the fluorescence intensity of the PI-staining nucleus began to appear only in the cells after 1 hour of OA stress and then increased and dispersed around the nucleus gradually ( Figure 3B-E). Here, the staining of the nucleus represented the PI diffusion across the plasma membrane. However, its physical rupture did not occur after the loss of membrane integrity ( Figure 3F). The result of PI staining demonstrated again the hallmark of PCD caused by OA, just as indicated by Rd123 staining of mitochondria. Figure 3. Serial fluorescence images loss of nuclear membrane integrity after the treatment of 1 mmol/L OA stained by PI. The nuclear membrane maintained integration, and weak red fluorescence could be observed (A). After the treatment of 1 mM OA, the fluorescence began to appear (after 1 h) (B). The fluorescence intensity increased because the nuclear membrane integrity was lost (after 3 h) (C). The fluorescence intensity in cytoplasm, especially the location of nucleus, continued to increase (after 5 h) (D). A large amount of PI stained the nucleus, and the fluorescence intensity was evident (after 7 h) (E). Microscopic was photographed under the white light field from E cells, and the cells were relatively intact (F).

Effects of OA on Cytoplasmic pH of P. tunicoides
To enhance normal cell function, pHcyt oscillations are usually maintained within a narrow range. Several cellular processes, such as cytoskeletal organization, vesicle fusion, and enzyme activities, are sensitive to pH and might be regulated by differences in pHcyt [37]. Cytosol acidification and the corresponding medium alkalinization are early events occurring in cells [38,39]. The pH values in rape oilseed decreased rapidly and were markedly lower than 5.63 measured before OA treatment [13]. Therefore, to characterize OAinduced events within plant cells, the question of whether the pHcyt oscillations could be stimulated by OA or not was further sophisticated. A fluorescent indicator of pHcyt, BCECF-AM, which can release BCECF within the cells after hydrolysis by intracellular esterase, was used to detect the changes in pHcyt. The fluorescent intensity and emission from BCECF accumulated in the cytoplasm may change in a pH-dependent manner, and hence, pHcyt can be mapped by analyzing fluorescence ratio imaging. OA-induced alterations in the fluorescence intensity and pHcyt are shown in Figure 4. Once OA was added to the cell suspension (final concentration 1 mM and 100 µM), the fluorescence intensity Figure 3. Serial fluorescence images loss of nuclear membrane integrity after the treatment of 1 mmol/L OA stained by PI. The nuclear membrane maintained integration, and weak red fluorescence could be observed (A). After the treatment of 1 mM OA, the fluorescence began to appear (after 1 h) (B). The fluorescence intensity increased because the nuclear membrane integrity was lost (after 3 h) (C). The fluorescence intensity in cytoplasm, especially the location of nucleus, continued to increase (after 5 h) (D). A large amount of PI stained the nucleus, and the fluorescence intensity was evident (after 7 h) (E). Microscopic was photographed under the white light field from E cells, and the cells were relatively intact (F).

Effects of OA on Cytoplasmic pH of P. tunicoides
To enhance normal cell function, pH cyt oscillations are usually maintained within a narrow range. Several cellular processes, such as cytoskeletal organization, vesicle fusion, and enzyme activities, are sensitive to pH and might be regulated by differences in pH cyt [37]. Cytosol acidification and the corresponding medium alkalinization are early events occurring in cells [38,39]. The pH values in rape oilseed decreased rapidly and were markedly lower than 5.63 measured before OA treatment [13]. Therefore, to characterize OA-induced events within plant cells, the question of whether the pH cyt oscillations could be stimulated by OA or not was further sophisticated. A fluorescent indicator of pH cyt , BCECF-AM, which can release BCECF within the cells after hydrolysis by intracellular esterase, was used to detect the changes in pH cyt . The fluorescent intensity and emission from BCECF accumulated in the cytoplasm may change in a pH-dependent manner, and hence, pH cyt can be mapped by analyzing fluorescence ratio imaging. OAinduced alterations in the fluorescence intensity and pH cyt are shown in Figure 4. Once OA was added to the cell suspension (final concentration 1 mM and 100 µM), the fluorescence intensity began to decrease rapidly (from 0.132 ± 0.0086 to 0.07 ± 0.004 for 1 mM, and 0.131 ± 0.0062 to 0.097 ± 0.0058 for 100 µM, respectively) within 30 min, which meant pH cyt began to drop drastically over a short time.
The results indicated that the treatment of OA led to acidification within the cytoplasm, which was dependent on its concentration. Furthermore, it was clear that 1 mM OA influenced cytoplasmic acidification was more significantly than 100 µM OA did. At the same time, pHcyt had no significant difference in the presence of 10 µM OA.

Effects of NH4Cl and Fusicoccinon on Cytoplasmic pH and Cell Viability of P. tunicoides
Previous studies have demonstrated that intracellular alkalization is probably associated with PCD or abscission of plant cells [40,41]. Two experimental models were established to explore further the physiological roles of cytoplasmic acidification in the progress of cell death following OA treatment. The first could decrease pHcyt, causing the temporary intracellular acidification without altering extracellular pH. The second could increase the pHcyt to relieve intracellular acidification. ( Figure 5) illustrates pHcyt changes in 5 min application followed by removal of NH4Cl, compared with continuous application of NH4Cl. The present dose-dependent mode detected in the pHcyt was similar to that of the OA-induced cell death rate. After removal of NH4Cl, cytoplasmic acidification was indicated by the decrease of fluorescence intensity. Compared with the standard condition, the cell death rate was much higher under the cytoplasmic acidification treatment (data not shown). Fusicoccin (FC), a strong activator of the H + -ATPase, enhanced H + export from the intracellular. FC may caused cytoplasmic alkalinization, and The results indicated that the treatment of OA led to acidification within the cytoplasm, which was dependent on its concentration. Furthermore, it was clear that 1 mM OA that influenced cytoplasmic acidification was more significant than 100 µM OA did. At the same time, pHcyt had no significant difference in the presence of 10 µM OA.

Effects of NH 4 Cl and Fusicoccinon on Cytoplasmic pH and Cell Viability of P. tunicoides
Previous studies have demonstrated that intracellular alkalization is probably associated with PCD or abscission of plant cells [40,41]. Two experimental models were established to explore further the physiological roles of cytoplasmic acidification in the progress of cell death following OA treatment. The first could decrease pHcyt, causing the temporary intracellular acidification without altering extracellular pH. The second could increase the pHcyt to relieve intracellular acidification. ( Figure 5) illustrates pHcyt changes in 5 min application followed by removal of NH 4 Cl, compared with continuous application of NH 4 Cl. The present dose-dependent mode detected in the pHcyt was similar to that of the OA-induced cell death rate. After removal of NH 4 Cl, cytoplasmic acidification was indicated by the decrease of fluorescence intensity. Compared with the standard condition, the cell death rate was much higher under the cytoplasmic acidification treatment (data not shown). Fusicoccin (FC), a strong activator of the H + -ATPase, enhanced H + export from the intracellular. FC may cause cytoplasmic alkalinization, and could weaken the pH cyt alteration by OA as seen in ( Figure 5A). Moreover, the addition of FC reduced the percentage of dead cells from 90.5 ± 5.7% to 31.61 ± 9.35% after being treated by OA. The results showed that cytoplasmic acidification was an inevitable precondition to the PCD induced by OA.
could weaken the pHcyt alteration by OA as seen in ( Figure 5A). Moreover, the addition of FC reduced the percentage of dead cells from 90.5 ± 5.7% to 31.61 ± 9.35% after being treated by OA. The results showed that cytoplasmic acidification was an inevitable precondition to the PCD induced by OA.

Effects of EGTA and La 3+ on Cytoplasmic pH of OA-Treated Cells
Elicitor-induced Ca 2+ spiking is one of the earliest events that act as a master messenger for almost all downstream response reactions. Medium alkalinization is thought to result from elicitor-induced depolarization of the plasma membrane and is associated with Ca 2+ influx/Clefflux [39]. In Chara coralline, a lowered pHcyt increased cytosolic free Ca 2+ ([Ca 2+ ]cyt) affinity, activating Cl − efflux. OA could chelate Ca 2+ to form Ca oxalate crystals. The formation is regarded as a highly controlled cellular process rather than a simple precipitation phenomenon. Specialized mechanisms must be present in crystal idioblasts to deal with the enormous fluxes of Ca 2+ [42]. In order to investigate the role of extracellular Ca 2+ in the process of acidification induced by OA, the cells were treated with ethylene glycol tetraacetic acid (EGTA) (a Ca 2+ chelator) and La 3+ (a Ca 2+ channel blocker) before the addition of OA, respectively. From the results of fluorescent intensity, both EGTA and La 3+ have a minor effect on the decrease of pHcyt (Figure 6), which suggested extracellular Ca 2+ may neither participate in the modification of the pHcyt nor position downstream of acidification induced by OA.

Effects of EGTA and La 3+ on Cytoplasmic pH of OA-Treated Cells
Elicitor-induced Ca 2+ spiking is one of the earliest events that act as a master messenger for almost all downstream response reactions. Medium alkalinization is thought to result from elicitor-induced depolarization of the plasma membrane and is associated with Ca 2+ influx/Cl − efflux [39]. In Chara coralline, a lowered pH cyt increased cytosolic free Ca 2+ ([Ca 2+ ]cyt) affinity, activating Cl − efflux. OA could chelate Ca 2+ to form Ca oxalate crystals. The formation is regarded as a highly controlled cellular process rather than a simple precipitation phenomenon. Specialized mechanisms must be present in crystal idioblasts to deal with the enormous fluxes of Ca 2+ [42]. In order to investigate the role of extracellular Ca 2+ in the process of acidification induced by OA, the cells were treated with ethylene glycol tetraacetic acid (EGTA) (a Ca 2+ chelator) and La 3+ (a Ca 2+ channel blocker) before the addition of OA, respectively. From the results of fluorescent intensity, both EGTA and La 3+ have a minor effect on the decrease of pH cyt (Figure 6), which suggested extracellular Ca 2+ may neither participate in the modification of the pH cyt nor position downstream of acidification induced by OA. could weaken the pHcyt alteration by OA as seen in ( Figure 5A). Moreover, the addition of FC reduced the percentage of dead cells from 90.5 ± 5.7% to 31.61 ± 9.35% after being treated by OA. The results showed that cytoplasmic acidification was an inevitable precondition to the PCD induced by OA.

Effects of EGTA and La 3+ on Cytoplasmic pH of OA-Treated Cells
Elicitor-induced Ca 2+ spiking is one of the earliest events that act as a master messenger for almost all downstream response reactions. Medium alkalinization is thought to result from elicitor-induced depolarization of the plasma membrane and is associated with Ca 2+ influx/Clefflux [39]. In Chara coralline, a lowered pHcyt increased cytosolic free Ca 2+ ([Ca 2+ ]cyt) affinity, activating Cl − efflux. OA could chelate Ca 2+ to form Ca oxalate crystals. The formation is regarded as a highly controlled cellular process rather than a simple precipitation phenomenon. Specialized mechanisms must be present in crystal idioblasts to deal with the enormous fluxes of Ca 2+ [42]. In order to investigate the role of extracellular Ca 2+ in the process of acidification induced by OA, the cells were treated with ethylene glycol tetraacetic acid (EGTA) (a Ca 2+ chelator) and La 3+ (a Ca 2+ channel blocker) before the addition of OA, respectively. From the results of fluorescent intensity, both EGTA and La 3+ have a minor effect on the decrease of pHcyt (Figure 6), which suggested extracellular Ca 2+ may neither participate in the modification of the pHcyt nor position downstream of acidification induced by OA. Figure 6. Effects of EGTA and La 3+ on 1 mM OA-treated cell pHcyt. Figure 6. Effects of EGTA and La 3+ on 1 mM OA-treated cell pH cyt .

The Changes Induced by OA in K + and Ca 2+ Inward
Exogenous molecules can modulate transporters located at the plasma membrane, and these elicitor-induced ion fluxes are immediate responses of plant cells. The cytoplasmic acidification and related medium alkalinization are thought to be due to elicitor-induced depolarization of the plasma membrane and subsequent K + /H + exchanger, with Ca 2+ influx, which is generally observed in the earliest responses of plant cells to avirulent pathogens [39]. In addition, the induction of this altered biochemical balance depends on an H + -pumping ATPase activity, and the stimulation of its activity and growth by FC and the inhibition by vanadate also support the idea that plasma membrane H + -ATPases play a role in the maintenance of pH cyt [43,44]. The experiment used the electrophysiological approach to determine the position of OA in the alterations of K + , Ca 2+ channel and H + -ATPases. The K + channel and Ca 2+ channel holding potential on P. unicoides cells are −50 mV and +20 mV, respectively, similar to Arabidopsis results in patch-clamp experiments. High dose OA, which caused evident PCD, induced a decrease of inward K + current from 350 pA to 150 pA and increased inward Ca 2+ wind from 50 pA to 300 pA after 10 min. To investigate the relationship between OA and H + -ATPase, the effects of H + -ATPases activator FC on the channel currents were also tested. The addition of FC, which effectively weakens the altered current process, caused inward K + current to rise to 280 pA and inward Ca 2+ current to fall to 150 pA, respectively. However, both channels' current was altered significantly compared with that of the normal condition. These results suggested that the OA may regulate the inward K + channel and inward Ca 2+ channel by mediating H + -ATPases activity.

Discussion
Previous results demonstrated the accumulation of OA, which is essential for the pathogenicity of fungi. OA can acidify the infected plant tissues to activate many fungal enzymes and protein kinase of host plant cells at low pH and degrade the plant cell wall via acidity or chelation of the cell wall Ca 2+ [37][38][39]. The research revealed that OA maintained its toxicity even the pH decline by OA treatment, and suggested that acidification was not the only mode of OA action bringing about deleterious effects during PCD in A. thaliana [16]. Likewise, we demonstrated that OA, even when its pH was adjusted equally to the suspension cells media, could induce PCD in P. tunicoides. These results suggested that OA itself functioned as an inducer of PCD; its acidity may accelerate the PCD process.
Recently, plant mitochondria as cellular stress sensors and central organelles in PCD have attracted increasing interest [45]. The outer organelle membrane disrupted and released proteins, such as cytochrome C (cyt c ), into the cytosol, triggering caspase activation or performing other functions relevant to PCD activation of catabolic proteases and nucleases [46,47]. These changed cytosol circumstances and activated proteases and nucleases may influence the energy metabolism, disrupt the nuclear membrane, then break down the inside nuclear DNA. Mitochondria also generate ROS through electron-transfer intermediates intimately involved in cell death signalling pathways [4,48]. In the process of OA-induced PCD, the disruption of respiratory electron transport chains and loss of nuclear membrane integrity were detected by specific fluorescent indicators Rd123 and PI. Mitochondria undergo significant changes before classic characteristics of the nuclear membrane appear. The main cellular organelle structure was damaged, and related possible signalling pathways induced by high-dose OA caused the entire PDC. We confirmed the OA toxicity to the cells.
It was observed that cytosolic pH regulation of anion channels plays a specific role in the cytosolic pH regulation in plant cells by providing an anion shunt conductance [48]. Anion channels/transporters seem to act as key players in signalling pathways leading to the adaptation of plant cells to abiotic and biotic stresses in control of metabolism and the maintenance of electrochemical gradients. Previous research suggested that increase of anion current was a required upstream event in the signalling pathway leading to oxalate-induced cell death [16]. (2',7'-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluoresceinacetoxymethyl (BCECF-AM) was employed to detect the possible cytoplasmic pH (pH cyt ) oscillations in the exposure of the suspension-cultured cell to OA. The decrease in time and dose-dependent florescent density decrease indicated the intracellular acidification in the process. It mimicked pH cy drops using the ammonium chloride (NH 4 Cl) method leading to more cell death than the usual condition, suggesting that the cytosolic acidification induced by OA may be critical for initiation PCD. The pretreatment of FC (as an activator of H + -ATPase, cytosolic alkalinization) could efficiently inhibit the cell death induced by OA with a drop of the cell death percentage by 70%; the evidence could further confirm the pH cyt drop role played in the OA-induced cell death. In mammals, many experiments have demonstrated that cytoplasmic acidification is a feature of apoptosis. Several agents leading to cytoplasmic alkalinization through activation of ion channels and pumps could prevent apoptosis stimulated by intra-or extracellular elements [49].
Interestingly, some similar results were obtained in the field of plant science. The hypothesis predicts that biotic and abiotic stresses-induced cytoplasmic acidification triggers the synthesis of phytoalexins and other secondary metabolites. Cytoplasmic acidification, which caused DNA breakdown, active caspase-like enzymes, and ROS, might act as messages involved in triggering defense responses and related PCD [50].
Intracellular acidification, combined with K + and Ca 2+ flux, was regarded as an early marker of an elicitation process leading to PCD. Several studies suggested that changes in pH cyt resulting from ion fluxes and H + -ATPase play a role [51]. Recently, members of 2ligand-gated ion channel families, glutamate receptor-like channels (GLRs) and cyclic nucleotide-gated channels (CNGCs) were implicated in immune responses. Nevertheless, more precise data are necessary to understand their direct involvement in creating Ca 2+ signals during immune responses [52]. These results supported the view that the ion fluxes are related to the early signalling for PCD. Inward and outward rectifying K + channels carrying K + ions across the membrane played a critical role in regulating biochemical balance. The triggering of the HR in tobacco cells by specific bacterial pathogens required the activation of a plasma membrane K + /H + exchanger, which needs H + -ATPase function [53]. Combined with these results, it might suggest that the addition of OA functioned on the K + /H + exchanger, which decreased the K + influx (Figure 7) and showed OA played a prominent inhibition role on the inward K + current paralleled by the drop of efflux of H + . The accumulated H + in the cytoplasm may contribute to the pH cyt reduction. In the process, OA may also influence the phosphorylation of the H + -ATPase, which not only altered the activity of the K + /H + exchanger but mediated the H + extrusion from the vacuolar proton pool. The results of FC as the activator of the H + -ATPase, which is bound to the 14-3-3 family regulatory protein associated with the phosphorylation-dependent C-terminal end, played a role in OA-induced cytoplasmic acidification, and related PCD could further support our hypothesis [54]. Elicitor-induced Ca 2+ spiking was one of the earliest events that acted as a master messenger for most downstream response reactions. Some studies reported that [Ca 2+ ] cyt elevation down-regulates inward-rectifying K + channels and proton pumps in the plasma membrane of guard cells [55,56]. Results also showed that OA could activate the inward Ca 2+ channel effectively by mediating the activity of H + -ATPase. The induced [Ca 2+ ] cyt elevation may originate from the extracellular Ca 2+ influx or efflux of some organelles such as endoplasmic reticulum, mitochondria, which could regulate [Ca 2+ ] cyt through Ca 2+ -ATPase and Ca 2+ /H + exchanger [57]. EGTA (a Ca 2+ chelator with at least 10 4 fold greater affinity than OA) and La 3+ (a Ca 2+ channel blocker) were used to pre-incubate the OA-treated cells, which could remove the possible role of extracellular Ca 2+ . The slight effect on the decrease of pH cyt suggested that extracellular Ca 2+ might not be the primary mechanism participating in the regulation of pH cyt, and the chelation of OA might not be the primary function in the process of PCD. The Ca 2+ store deletion is possible as the primary source of the [Ca 2+ ] cyt elevation. More related genic and proteinic studies are needed to illustrate these points and the oxalic acid-induced cell signal significance. studies are needed to illustrate these points and the oxalic acid-induced cell signal significance.

Plant Materials
Under aseptic conditions, selected loose, light yellow or milky P. tunicoides callus established MS liquid culture system according to callus quality and culture medium volume 1:10 (g/mL). Cultures were incubated at 25 ± 1 °C in the dark on a rotary shaker at 110 ± 5 rpm overnight, and the suspension cells of P. tunicoides were harvested.
The suspension cells of P. tunicoides were cultured in 250 mL Erlenmeyer flasks containing 100 mL of Murashige and Skoog [58] salt solution, supplemented with 0.5 mg/L 6-

Plant Materials
Under aseptic conditions, selected loose, light yellow or milky P. tunicoides callus established MS liquid culture system according to callus quality and culture medium volume 1:10 (g/mL). Cultures were incubated at 25 ± 1 • C in the dark on a rotary shaker at 110 ± 5 rpm overnight, and the suspension cells of P. tunicoides were harvested.
The suspension cells of P. tunicoides were cultured in 250 mL Erlenmeyer flasks containing 100 mL of Murashige and Skoog [58] salt solution, supplemented with 6-BA 0.5 mg/L, 2,4-D 0.5mg/L, the pH adjusted 5.8 by NaOH or HCl. Cultures were incubated at 22 ± 2 • C in the dark on a rotary shaker at 110 ± 5 rpm overnight. Cell suspensions were transferred into a new medium after 14 days using 1:5 dilutions. All experiments were performed at 22 ± 2 • C using log-phase cells (6 days after subculture).

Determination of Cell Viability and Death
For FDA staining, cells were incubated in an aqueous FDA solution (0.01% w/v) for 15 min at room temperature, and followed the Evans blue staining method procedure. Cells were observed under a fluorescence microscope (Motic AE31); only live cells appeared green. The cell viability was indicated as the ratio of live cells to total cells.
Evans blue staining was employed to determine cell death. The suspension cells were incubated in a 0.1% (w/v) aqueous solution of Evans blue for 10 min at room temperature. The cells were then washed with fresh MS media twice to remove unbound dye cells before observation by centrifugation at 600× g rpm for 5 min. Subsequently, cells were observed and counted with a bright field microscope (Motic AE31); only dead cells appeared blue. The cell death was indicated as the ratio of dead cells to total cells.
All experiments were independently replicated at least 3 times, and 500 cells were examined and analyzed statistically. Data are presented as means ± SD.

Rd 123 and PI Staining Procedures
Rd123 staining is plasma membrane-permeable and aggregates within mitochondria. The excited fluorescence intensity Rd123 is proportionate to the potential of electron transport chains, reflecting the functional integrity of mitochondria. We added 100 mL of Rd123 stock solution (10 mg/L) to 1 mL of cells suspension to a final concentration of 1 g/mL, and incubated at 25 • C for 15 min in a dark environment. The suspension cells were rinsed 3 times with new MS media to remove excess Rd123.
For PI staining to examine the integrity of the plasma membrane, 20 L of PI stock solution (20 g/L), which is a membrane-impermeable DNA/RNA stain, was added to 1 mL of cells suspension, and the cells were gently centrifuged and incubated in controlled darkness at 27 • C for 15 min. Subsequently, the suspension cells were rinsed 3 times with new MS media to remove excess PI. PI-negative staining cells are live cells, and PI-positive staining cells are primary cells in the late stages of PCD. All experiments were repeated 3 times.

Intracellular pH (pH i ) Measurement
We dissolved 50 µg of BCECF-AM in 8.4 µL DMSO to a final concentration of 10 mmol/L and stored it at −20 • C in a controlled dark environment as stock. Further, the working solution was prepared by adding 2 µL of stock solution to 1 mL of suspension cells to a final concentration of 20 µmol/L; after incubating in the working solution for 15 min at 25 ± 1 • C, the suspension cells were rinsed 3 times in a new MS medium to remove excess BCECE-AM. These cells fluorescence emission images were acquired using a cooled charged-coupled device (CCD) camera on an inverted microscope (Motic AE31). The fluorescence intensity was calculated using software IPP 6.0 through the analysis of fluorescence images. The CCD camera was also used for bright-field images collection.
All experiments were repeated 3 times with different samples, and representative images were presented.

Patch Clamp and Data Acquisition
Protoplasts of P. tunicoides were isolated as described [59]. In the whole-cell voltageclamp, the K + and Ca 2+ currents of P. tunicoides cells were recorded with an EPC-9 amplifier (Heka Instrument) described [60]. Pipettes were pulled with a vertical puller (Narishige) modified for two-stage pulls. Data were analyzed using PULSEFIT 8.7, IGOR 3.0, and ORIGIN 7.0 software. The standard solution for potassium current measurements contained 10 mM K-glutamate, 1 mM CaCl 2 , 2 mM MgCl 2 , 10 mM Mes, pH 5.5, in the bath and 80 mM K-glutamate, 1.1 mM EGTA, 5 mM Mg-ATP, 20 mM KCl, 10 mM Hepes, pH 7.2, in the pipette. The standard solution for calcium current measurements contained 100 mM CaCl 2 , 0.1 mM DTT, and 10 mM MES-Tris, pH 5.6, in the bath and 10 mM BaCl 2 , 0.1 mM DTT, 4 mM EGTA, and 10 mM HEPES-Tris, pH 7.1, in the pipette. D-sorbitol was used to adjust the osmolality of pipette and bath solutions to 400 and 500 mmol/kg, respectively.

Conclusions
The regulation and execution processes of PCD, particularly the processes induced by abiotic factors, remain unknown. PCD is a crucial process in plant development, senescence or immunity and plays an important role in the plant stress response. The processes and biochemical and molecular pathways of plant PCD induced by abiotic stress are very important for understanding the tolerance/resistance of plants to abiotic stress, enabling plant tolerance to be increased in the future by manipulating the inhibition of PCD. In a global environment with climate changes, susceptible and tolerant genotypes/species are highly desirable. Although some of the biochemical, molecular, and morphological mechanisms are known, in this paper, we focused on the PCD process, mechanisms, and induced by OA in P. tunicoides, which is a unique perennial medicinal plant species, including the cytoplasmic pH oscillations that are essential in the process independent of the extracellular Ca 2+ . It is conceivable that OA is correlated to ion channels located at the plasma membrane in triggering responses of plant cells to various environmental perturbations. Our study suggests that OA may influence inward K + channels and Ca 2+ channels by mediating the activation of H + -ATPase.
Author Contributions: X.J., methodology, software, formal analysis, investigation, resources; M.A.M., data curation, writing-original draft preparation; Y.Q., validation, writing-review and editing, visualization; Z.Z., supervision, project administration, funding acquisition. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement:
The data presented in this study are available on request from the corresponding authors.