Sodium Influx and Potassium Efflux Currents in Sunflower Root Cells Under High Salinity

Helianthus annuus L. is an important oilseed crop, which exhibits moderate salt tolerance and can be cultivated in areas affected by salinity. Using patch-clamp electrophysiology, we have characterized Na+ influx and K+ efflux conductances in protoplasts of salt-tolerant H. annuus L. hybrid KBSH-53 under high salinity. This work demonstrates that the plasma membrane of sunflower root cells has a classic set of ionic conductances dominated by K+ outwardly rectifying channels (KORs) and non-selective cation channels (NSCCs). KORs in sunflower show extreme Na+ sensitivity at high extracellular [Ca2+] that can potentially have a positive adaptive effect under salt stress (decreasing K+ loss). Na+ influx currents in sunflower roots demonstrate voltage-independent activation, lack time-dependent component, and are sensitive to Gd3+. Sunflower Na+-permeable NSCCs mediate a much weaker Na+ influx currents on the background of physiological levels of Ca2+ as compared to other species. This suggests that sunflower NSCCs have greater Ca2+ sensitivity. The responses of Na+ influx to Ca2+ correlates well with protection of sunflower growth by external Ca2+ in seedlings treated with NaCl. It can be, thus, hypothesized that NaCl tolerance in sunflower seedling roots is programmed at the ion channel level via their sensitivity to Ca2+ and Na+.


INTRODUCTION
Sunflower (Helianthus annuus L.) is an important crop that is widely used in the oil industry and animal feeding. Global sunflower production increased more than twice since 2000 (Pilorgé, 2020). It is the third highest oilseed produced in the world, the fourth vegetable oil and the third protein feed source among oilseed crops. Although sunflower plants exhibit medium salt Na + influx through NSCCs can be inhibited by increased external [Ca 2+ ] . This phenomenon is widely used in agriculture to ameliorate NaCl toxicity (Bressan et al., 1998). We have previously found that high external Ca 2+ levels inhibit both Na + entry and K + efflux channels, thereby blocking both Na + toxic influx and loss of K + (Shabala et al., 2006). In the recent past, blockade of Na + influx by external Ca 2+ has only been investigated in Arabidopsis thaliana Shabala et al., 2006). Therefore, it is still unclear whether other plants share this mechanism.
In the present investigations, using patch-clamp electrophysiology, we have characterized the NSCC-like Na + conductance and determined its Ca 2+ sensitivity in root protoplasts of H. annuus L. seedlings (hybrid KBSH-53), which is widely cultivated in arid regions of India. To our knowledge, this is the first electrophysiological study of any ion currents in sunflower as well as properties of Na + influx and K + efflux conductances in this species.
The hydroponic cultivation system was used for sunflower root growth measurements. Germinated seeds (germination: 2 days on wetted filter paper) were cultivated during 7 days in vertical polycarbonate sheets. Each root was directed to a separate channel of polycarbonate sheets in order to prevent root entanglement (Green House Polycarbonate Sheets; Greenhouse Megastore, United States). Polycarbonate sheets were mounted vertically in large square glass vessel and dipped into the medium (volume: 2 L), which was stirred with a stream of air (air compressor Barbus Aquael OXYPRO; China). The medium contained 5% original Murashige and Skoog nutrient composition (MS; Duchefa #M0221; Murashige and Skoog, 1962), рН 6.0 (adjusted by KOH). Treatments (CaCl 2 , NaCl, etc.) were added to this medium as required. All growth solutions were replaced every day (for freshness). Root length (main root) was measured after 7 days of treatment.

Patch-Clamp Measurements
Conventional patch-clamp and protoplast isolation techniques were used Demidchik et al., 2010). The standard bathing solution contained (in mM): 0.3 KCl, 2 Tris, adjusted to pH 6.0 with 1 MES, and 600 mOsm kg −1 , with D-sorbitol. Other salines are indicated in figure legends. A freshly prepared mixture of this solution was applied in whole-cell outside-out patches. The pipette solution (PS) contained the following composition (mM): 70 KGluconate, 10 KCl, 1 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N0,N0-tetraacetic acid (BAPTA) and 0.475 mM CaCl 2 (10 nM free Ca 2+ ), pH 7.2 with 2 Tris, and 1 MES. To examine the sensitivity of wholecell outward current to cation channel blockers (TEA + and Gd 3+ ), 10 mM TEACl or 100 μM GdCl 3 were added to the bathing solution. The size of protoplasts was measured using Nikon NIS-Elements software and used to calculate the mA/ m 2 current densities. Typical transmembrane currents are from the same cell (including Gd 3+ blockade test). Liquid junction potentials were calculated by JPCalc, which is included in Axon Clampex 10.6 software (Molecular Devices, United States) and corrected. The voltage was held at −90 mV, then square 7.6 s-long or 1.5 s-long depolarizing or hyperpolarizing voltage pulses were applied. Currents were measured using PC-ONE Patch/ Whole Cell Clamp (CORNERSTONE Series) amplifier (Dagan Corporation, United States) controlled by Digidata 1,320/Clampex 10.6 (Molecular Devices, United States). Current-voltage (I-V) and other curves were plotted and analyzed using SigmaPlot 10.0 (Systat Software Inc., United States).

Effect of Ca 2+ on Sunflower Seedling Growth in High Salinity Conditions
Root growth tests were carried out using seedlings of H. annuus L. KBSH-53 in vertical hydroponic chambers in controlled environment (Figure 1). The effect of 80 and 120 mM NaCl on the length of main root was examined (preliminary tests showed that 40 mM NaCl did not modify plant growth). Measurements were carried out against two levels of Ca 2+ (0.2 and 2 mM) in the cultivation solution containing 5% MS (original composition). Growth in NaClfree solutions (control conditions) containing 2 mM CaCl 2 was approximately 25% slower than growth on the background of 0.2 mM CaCl 2 (p = 0.007; seven independent trials; each trial included 9-10 plants). Addition of 80 mM NaCl along with 0.2 mM CaCl 2 resulted in approximately 5-fold decrease of root length (p < 0.001; 12 independent trials; each trial included 9-10 plants). At the same time, 120 mM NaCl induced 6.5-fold delay in growth. Increase of external Ca 2+ level from 0.2 to 2 mM significantly improved plant growth in the presence of NaCl. In this case, application of 80 NaCl did not induce statistically significant decrease of root length (p = 0.235; 11 independent trials; each trial included 8-10 plants) while the effect of 120 mM NaCl was twice smaller (p = 0.008; eight independent trials; each trial contained eight plants; comparison with 0.2 mM CaCl 2 ). Overall, these data show that Ca 2+ (the physiological range) has a strong ameliorative effect on the growth of H. annuus L. KBSH-53 roots in salinized conditions.

Protoplast Isolation and Obtaining Gigaohmic Resistance Patches
No reports have been published about transmembrane currents of H. annuus L. or protocols for protoplast isolation for patchclamp tests in this species. To our knowledge, several attempts have been made to isolate sunflower protoplasts suitable for patch-clamp studies but none of them were successful for implementation in routine electrophysiological practice. In most cases, protoplast isolation from sunflower required overnight treatment by enzymes and did not yield viable protoplasts from any tissues apart from hypocotyl (Lenee and Chupeau, 1986;Kativat et al., 2012). We have developed protocols for H. annuus L. root patch-clamp analyses that were based on previous protocols elaborated for A. thaliana and Triticum aestivum Demidchik et al., 2010;Straltsova et al., 2015;Sosan et al., 2016;Makavitskaya et al., 2018). Ten osmolality levels were examined (300-750 mOsm kg −1 ; 50 mOsm kg −1 step) in 10 replicates. Round shaped viable protoplasts were observed only at 600 and 650 mOsm kg −1 but the density of viable protoplasts was approximately six times higher at 600 mOsm kg −1 comparing to 650 mOsm kg −1 (up to 55 ± 4 viable protoplasts per 1 ml of the enzyme solution; mean ± SE; n = 10). Experimental work on protoplasts reported here was carried out using the osmolality level of 600 mOsm kg −1 .
We have previously developed techniques and voltage-clamp protocols for the patch-clamp analysis of inwardly-and outwardlydirected conductances in higher plants, including Na + -conducting NSCCs Demidchik et al., 2010). The probability rate of observing "gigaohmic" contact required for patch-clamp measurements in sunflower protoplasts was low (2,750 protoplasts were patch-clamped; "gigaohmic" contact formed in 409 protoplasts). Approximately one-third of these protoplasts survived after addition of high NaCl concentration and maintained gigaohmic pipette resistance (139 protoplasts). A number of methods for improving patch stability were applied (different levels of external Ca 2+ , H + , use of Na + instead K + in the patch-clamp pipette, additional pipette polishing, hydrophobic coating, etc.) but none of these significantly improved the "gigaseal. "

Currents of Sunflower Root Protoplasts in Control Conditions and in Presence of NaCl
Protoplasts were patch-clamped in the sealing solution containing 20 mM CaCl 2 and 0.3 mM KCl (pH 6.0) using pipettes filled with the solution comprising of 70 mM KGluconate and 10 mM KCl (pH 7.2, 100 nM Ca 2+ ). High external Ca 2+ allowed gigaseal formation , while high intracellular (pipette) K + "mimicked" cellular K + level (Demidchik, 2014). Potassium gluconate (70 mM) in the pipette solution was used instead of KCl to avoid Cl − efflux currents, which can overlap with Na + influx conductance. Gluconate is a poorly permeable organic anion that minimizes anion efflux currents in patch-clamped root protoplasts (Makavitskaya et al., 2018). In these conditions, a moderate negative inwardly directed current was measured (Figure 2A). This current was voltage-independent and sensitive to 100 μM Gd 3+ (77.3 ± 4.5% decrease of the amplitude; ±SE; n = 5; data not shown). It showed very rapid ("instantaneous") activation kinetics. When external CaCl 2 was decreased from 20 to 0.2 mM, this current decreased by five times, demonstrating that it was mediated by Ca 2+ influx (Figures 2-4, 5; p < 0.001; n = 5). These Ca 2+ currents were similar to NSCC-mediated Ca 2+ currents previously reported in A. thaliana root protoplasts . It should be noted that in Arabidopsis, NSCCs mediating these currents were Na + -permeable . Addition of NaCl to patchclamped protoplasts in the presence of 20 mM extracellular Ca 2+ did not induce inwardly directed current (as expected for Na + influx NSCCs). The reversal potential was −91 ± 4 mV (20 mM CaCl 2 ; ±SE; n = 11) and it was not modified by NaCl addition (Figure 2).
The outward current measured in the presence of 20 mM external CaCl 2 was significantly blocked by the addition of NaCl to the bathing solution (Figure 2). The outwardly directed conductance dropped three times when 80 mM NaCl was added (Figure 5). In the conditions used in the work, the outward current could be mediated by K + efflux through KORs or by Cl − influx via anion channels (Demidcik et al., 2002(Demidcik et al., , 2014de Angeli et al., 2007;Demidchik, 2012;Hedrich, 2012). However, only K + currents can be blocked by Na + because the anion channels are insensitive to this and other alkali metals (Barbier-Brygoo et al., 2000). Moreover, the addition of K + channel blocker TEA + (30 mM TEACl) inside the patchclamp pipette instead of 80 mM K + (70 mM KGluc and 10 mM KCl) decreased the outward current by 8-9 times (p < 0.001; n = 5; data not shown) demonstrating that this current was mediated by KORs.
The time-dependent component of the outward K + current was inhibited after the addition of NaCl to the bathing solution while instantaneous current remained very similar (Figure 2). It can be thus hypothesized that the residual outward current was mediated by anion channel-catalyzed Cl − influx or K + efflux via NSCCs (previously described in Shabala et al., 2006). The maximal reduction of the outward current was 4.3, as measured in the presence of 80 NaCl at 7.6 depolarizing pulses . Statistically significant (p < 0.01; ANOVA test) difference between "2 mM CaCl 2 " (circles) and "40 mM NaCl" (squares) was found at all voltage values apart from −105 mV. The difference between "2 mM CaCl 2 " (circles) and "80 mM NaCl" (triangles) was statistically significant at all voltage values (p < 0.01; ANOVA test). The standard bathing solution contained (in mM): 0.3 KCl, 2 Tris, adjusted to pH 6.0 with 1 MES, and 600 mOsM, with D-sorbitol. The pipette solution contained 70 mM K gluconate, 10 mM KCl; 100 nM Ca 2+ was adjusted with 1 mM BAPTA and 0.475 mM CaCl 2 , pH 7.2 with 2 mM Tris, 1 mM MES. 100 μM GdCl 3 was added to the bathing solution on the background of 2 mM CaCl 2 and 80 mM NaCl for 5 min before recording current-voltage curves. Frontiers in Plant Science | www.frontiersin.org (Figure 2). This reduction was 3.2 times as calculated for 1.5-s-long segments of depolarising pulses (directly comparable with pulses used in Figures 3, 4). These results demonstrate a high sensitivity of KOR to Na + and suggest a relatively low sensitivity of KOR to external Ca 2+ in salt-tolerant sunflower.

Sodium Influx Currents in Sunflower Root Protoplasts Under Low External Ca 2+
Calcium ions are blockers of plant Na + -permeable NSCCs Shabala et al., 2006). This may be the reason for no detection of Na + influx conductance in the presence of 20 mM CaCl 2 (Figure 2). However, the decrease of external Ca 2+ from 20 to 2 mM (typical soil solution level of Ca 2+ ; White and Broadley, 2003;Marschner, 2011) resulted in the increase in the inward Na + current, which correlated with a shift of reversal potential to more positive values (from −86.6 ± 3.2 mV in control to −49.8 ± 2.5 mV at 40 mM NaCl and −30.1 ± 1.8 mV at 80 mM NaCl; ±SE; n = 6-11), consistent with currents being dominated by the movement of Na + (Figure 3). This can be interpreted as weakening the Ca 2+ -induced blockade of the NSCCs. Sodium influx current showed an "instantaneous" kinetics and was voltage-independent. The shift of the reversal potential in response to NaCl to more positive values decreased the KOR-mediated outwardlydirected currents (as the activation curve moved positive). Moreover, the decrease of the external CaCl 2 destabilized patches and caused a breakdown at depolarization that did not allow depolarizing pulses longer than 1.5 s (note: 7.6 s-long pulses were applied at 20 mM CaCl 2 to record full activation of KORs). In this regard, the measurements were limited to shorter segments of the outwardly-directed K + currents (Figure 3; see also calculation of conductance change in Figure 5), and it was not possible to fully compare the data with those shown in Figure 2. The obtained data demonstrated that an addition of NaCl (both 40 and 80 mM), in the presence of 2 mM CaCl 2 , inhibited the outwardly-directed currents slightly weaker than in the presence of 20 mM CaCl 2 (Figures 3, 5). The time-dependent component of the current was almost fully inhibited. . The difference between "0.2 mM CaCl 2 " (circles) and "40 mM NaCl" (squares) as well as between "0.2 mM CaCl 2 " (circles) and "80 mM NaCl" (triangles) was statistically significant at all voltage values (p < 0.01; ANOVA test). Statistically significant (p < 0.01; ANOVA test) difference between "40 mM NaCl" (squares) and "80 mM NaCl" (triangles) was found at a voltage of more negative than −80 mV. The standard bathing solution contained (in mM): 0.3 KCl, 2 Tris, adjusted to pH 6.0 with 1 MES, and 600 mOsM, with D-sorbitol. The pipette solution contained 70 mM K gluconate, 10 mM KCl; 100 nM Ca 2+ was adjusted with 1 mM BAPTA and 0.475 mM CaCl 2 , pH 7.2 with 2 mM Tris, 1 mM MES.
The addition of 100 μM Gd 3+ , which is a non-specific blocker of NSCCs and other plant cation channels (Demidchik and Maathuis, 2007) to the bathing solution containing 2 mM CaCl 2 and 80 mM NaCl, caused a very strong inhibition of both inward and outward currents (5-6-fold decrease of currents; Figure 3). This indicates that both currents were mediated by cation channels (not by anion channels).
Lowering the external CaCl 2 from 2 to 0.2 mM in the presence of 40 or 80 mM NaCl resulted in further increase in inwardly-directed voltage-independent Na + current (Figure 4). The reversal potential values measured after the addition of 40 and 80 mM NaCl were − 37.4 ± 2.9 mV and −26.5 ± 3.2 mV, respectively (±SE; n = 5). These values were more positive compared to those measured at 2 mM Ca 2+ , suggesting that it was due to increased permeability to Na + (in conditions of external 40 or 80 mM Na + , Na + reversal potential is positive). The outwardly-directed K + efflux conductance was equally blocked by 40 and 80 mM NaCl in the presence of 0.2 mM CaCl 2 , suggesting the saturation of the blockade at 40 mM NaCl or lower level of salt (Figures 4, 5). Interestingly, the time-dependent current component was almost fully blocked, when 40 or 80 mM NaCl were added on the background of 0.2 mM CaCl 2 .

DISCUSSION
Overall, data reported here demonstrate for the first time that H. annuus root plasma membrane has a set of ionic conductances dominated by NSCCs and KORs. Similar conductances were previously recorded in the plasma membranes of root protoplasts isolated from A. thaliana (Maathuis and Sanders, 2001;Demidchik et al., , 2010Shabala et al., 2006), Thellungiella halophila (Volkov et al., 2004;Volkov and Amtmann, 2006), Pisum sativum (Zepeda-Jazo et al., 2011), T. aestivum (Straltsova et al., 2015) and other species (Demidchik, 2014). To our knowledge, this work is the first patch-clamp and voltageclamp study on sunflower. It should be noted that previous works have touched on the topic of sunflower electrophysiology only in terms of measurements of membrane potential (Stankovic et al., 1997).  Figures 2-4). G max is the maximal value of conductance measured calculated in an individual experiment (set of IV curves). Experimental conditions and ionic species in external and pipette solutions are same as in Figures 2-4. Data are mean ± SE (n = 5-11; *** p < 0.0001; ANOVA test; comparison to control; no significant difference where unmarked).
In this investigation, the Helianthus Na + influx currents were also measured and analyzed (Figures 2-4). These currents showed voltage-independent activation, lack of time-dependent component and high sensitivity to Gd 3+ . These properties are fully in line with the characteristics of Na + -permeable NSCCs previously measured in A. thaliana (Maathuis and Sanders, 2001;Shabala et al., 2006) and T. halophile (Volkov et al., 2004;Volkov and Amtmann, 2006). However, sunflower Na + -permeable NSCCs showed a much weaker response to the decrease of extracellular Ca 2+ as compared to Arabidopsis or Thellungiella in the range of physiological Ca 2+ levels (2-0.2 mM). Thus, sunflower NSCCs has smaller Na + current density (and potentially lower number of channels per same membrane area) than Arabidopsis or Thellungiella at physiological extracellular [Ca 2+ ], potentially preventing toxic Na + influx and cell reactions induced by NaCl. This makes it possible to assume that Ca 2+ could cause greater inhibition of NSCCs in sunflower roots. Interestingly, the response of Na + influx to Ca 2+ correlated well with Ca 2+ -induced protection of root growth in sunflower seedlings treated with NaCl at different external [Ca 2+ ] (Figure 1). Growth inhibition by 80 mM NaCl was prevented by 2 mM CaCl 2 while the treatment with 0.2 CaCl 2 was not effective (Figure 1).
Results presented here also demonstrate a high sensitivity of KOR to Na + and suggest a relatively low sensitivity of KOR to external Ca 2+ in salt-tolerant sunflower. Similar sensitivity to external Na + is known for animal KORs, such as Kv2.1 and related to Na + reaction with the high and low affinity Na + binding sites in Kv2.1 channel (Kiss et al., 1998). Potassium outwardly-directed conductances mediated by KORs in salttolerant T. halophila decreased 1.5-1.7 times after the addition of 100 mM external Na + (Volkov et al., 2004;Volkov and Amtmann, 2006). In salt-sensitive species A. thaliana, this blockade was 1.3-1.9 times both in root epidermis and leaf mesophyll cells (showing a tendency to increase with an increase in the concentration of extracellular Ca 2+ ; Shabala et al., 2006). From the present findings, we hypothesize that enhanced sensitivity of K + efflux system to Na + can play an important role for adaptation because this will decrease K + loss under salinity conditions. It fits well within the hypothesis that maintaining a high K + /Na + ratio in plant cells and prevention of K + efflux under salt stress are key mechanisms of salt tolerance in higher plants (Shabala and Cuin, 2012;Demidchik et al., 2014. Intriguingly, K + outwardly directed conductance in sunflower showed greater Na + sensitivity at higher extracellular CaCl 2 levels that can have a positive effect in conditions of salinity (as cells will lose less K + ; Figure 5). This can be explained by the influence of CaCl 2 on the Na + -induced blockade of KORs in the case of measurements which were carried out at 20 and 2 mM external Ca 2+ . In animal plasma membrane K + channels, Na + can compete with K + for binding sites within a pore region modulating channel characteristics and functions in Ca 2+ -dependent manner (Kiss et al., 1998;Sauer et al., 2013). In animals, Ca 2+ modifies the K + channel activity via action on the surface charge, reaction with the specific binding sites at extracellular loops, effect on the EF-hands and calmodulin binding sites at cytosolic side (Shah et al., 2006). We hypothesize that the elevated extracellular Ca 2+ controls the Na + block of the sunflower K + channel by increasing Na + sensitivity. Interestingly, Lemtiri-Chlieh et al. (2020) have recently reported that divalent cation Mg 2+ added to the pipette solution can change both the activity of leaf NSCCs and their sensitivity to Gd 3+ , suggesting sophisticated interactions of cations within the NSCC complex.
Involvement of root KORs (potentially encoded by Shakertype GORK) to NaCl responses and salt stress adaptation have been demonstrated in a number of species (Adem et al., 2020). It is a redox-dependent phenomenon as GORK is additionally activated by ROS (Demidchik et al., 2010). Potassium loss via GORK triggered by depolarization and ROS can lead to ionic disequilibrium, induction of autophagy, and programmed cell death (Demidchik et al., 2010. Enhanced blockade of KOR by Na + will be the simplest and "economical" mechanism for preventing K + loss that will retain the greatest amount of metabolic energy for adaptation in salinity conditions. The cell's energy balance has recently been recognized as one of the main salt stress targets (Tyerman et al., 2019). Thus targeting KORs and their Na + sensitivity regions to save energy for reparation needs offers high hopes for generation of salt-tolerant varieties by molecular breeding techniques.
In conclusion, the data presented here strongly suggest that the moderate resistance of sunflower to NaCl stress is programmed at potassium and non-selective channel level via the sensitivity of ion channels to Ca 2+ and Na + .

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
VD was responsible for research supervision, experimental design, management of experiments, data analysis, and writing the manuscript. SB and MY were involved in the preparation of plant material, research supervision, and design of experiments. PH, IN, YT, XH, and MK carried out electrophysiological experiments. VS and AV conducted hydroponics studies. AS and IS carried out routine cultivation of sunflower seedlings, maintained patch-clamp equipment, and participated in manuscript preparation. All authors contributed to the article and approved the submitted version.

FUNDING
This study was supported by joint Belarus-India Project 018/53 of the State Committee of Science and Technology of Belarus (to VD and SB), International Academic Exchange Research Project (China-Belarus; to VD, MY, and SH), Guangdong Province Pearl River Fellowship (to VD), and the Russian Science Foundation grant#15-14-30008 (VD).