Whole-Plant Measure of Temperature-Induced Changes in the Cytosolic pH of Potato Plants Using Genetically Encoded Fluorescent Sensor Pt-GFP

Cytosolic pH (pHcyt) regulates a wide range of cellular processes in plants. Changes in pHcyt occurring under the effect of different stressors can participate in signal transmission. The dynamics of pHcyt under the action of external factors, including significant factors for open ground crops such as temperature, remains poorly understood, which is largely due to the difficulty of intracellular pH registration using standard methods. In this work, model plants of potato (one of the essential crops) expressing a fluorescent ratiometric pH sensor Pt-GFP were created. The calibration obtained in vivo allowed for the determination of the pHcyt values of the cells of the leaves, which is 7.03 ± 0.03 pH. Cooling of the whole leaf caused depolarization and rapid acidification of the cytosol, the amplitude of which depended on the cooling strength, amounting to about 0.2 pH units when cooled by 15 °C. When the temperature rises to 35–40 °C, the cytosol was alkalized by 0.2 pH units. Heating above the threshold temperature caused the acidification of cytosol and generation of variation potential. The observed rapid changes in pHcyt can be associated with changes in the activity of H+-ATPases, which was confirmed by inhibitory analysis.


Introduction
Potato is one of the most important crops grown in open ground conditions. Cultivation in open ground conditions exposes potato plants to a greater influence of uncontrollable external factors than growing in greenhouses under a controlled level of humidity, degree of irrigation and other conditions [1][2][3]. In addition, traditional potato breeding was aimed at increasing productivity, quality and improving resistance to diseases; so modern varieties have limited resistance to abiotic stresses, for example, salinity, drought and cold stress [4]. Temperature has a significant influence on the development of potato plants and affects their productivity [5,6].
Temperature changes affect the activity of a number of processes, including photosynthesis and starch metabolism [7,8], which ultimately can lead to plant death or crop yield reduction. For example, a temperature rise of 1 • C above 35 • C could result in 1. .29% loss of maize yield [9], and maize productivity also decreases by 0.3% on cold days occurring during the growing season [10]. Each 1 • C of global warming (increase in temperature from 11 • C to 19 • C) decreases wheat yield by 4.1-6.4% [11]; and under cold stress (2 • C/8 • C) there is a 35-78% decrease in wheat grain yield [12]. Potatoes are sensitive to temperature changes too. Potatoes have a wide range of temperature resistance, so the temperature minimum for tuberization is 4-18 • C (for different cultivars this threshold is different), and the maximum is 30 • C [5,13,14]. However, when the temperature rises by 1 • C in the range of 19.1-27.7 • C, potato yield reduces by 11% [15]. A 19-29% decline in potato yield in 2038-2067 is predicted with an increase in maximum (of 0.7, 1.2, and 2.1 • C) and minimum (of 0.6, 1.3, and 2.0 • C) temperatures and a decrease in irrigation [16]. A decrease in yield is mediated via changes in metabolism, the direct regulation of the activity of key enzymes and the initiation of a plant defense reaction, which occurs with the participation of signaling systems. One of the ways of regulating enzymes and transporters is by a change in the cytosolic pH, the value of which is 7.2-7.5; however, more acidic values have also been noted, for example, 6.62 [17][18][19][20]. The cytosolic pH can shift due to the action of various factors. For example, alkalization is observed in cases of penetration of pathogen fungus Alternaria alternata hyphae into the plant cell (by 1.5-2 units) [21], gravistimulation (by 0.4 units) [22], the external addition of indole-3-acetic acid (IAA) (by 0.2 units) [23] and anoxia, which acidifies cytosol by 0.8-1.2 units [24]. Cold stress is indicated to provoke acidification of the cytosol [25]. It should be noted that the majority of research on the effect of temperature changes on cytosolic pH has been carried out on cell cultures [26] or in the cells of individual organs [25], and the pH has not been studied in the whole organs or plant. The effect of heating on cytosolic pH has not yet been reliably determined. Temperatures in the range of 30 • C to 55 • C cause a metabolic response for many plant species [27]. Therefore, a change in the intracellular pH caused by temperature can be a signal/messenger of various processes in the plant, including those transmitted over long distances [17].
In plant cells, there is a complex of pH regulation systems, including buffer systems of phosphate and carbonate anions; the biochemical pH-stat that functions due to the pH-sensitive processes of carboxylation and decarboxylation of organic acids; and the biophysical pH-stat including H + -ATPases of tonoplast and plasmalemma, proton pyrophosphatases and H + -cotransporters [28][29][30]. The H + -ATPases are the most important participants in the regulation of intracellular pH [31]. Their activity can be regulated by a plethora of factors such as blue light [32], the fusicoccin toxin [33], the oral secretion of Spodoptera littoralis [34], phytohormones [31,35], drought [36] and temperature changes [37]. Cooling leads to a decrease in the H + -ATPases activity in the initial hours of treatment [38]. Heating induces a different response from the proton pumps, depending on the duration of the treatment and the strength of the temperature stimulus [39,40]. The main way of regulating the activity of H + -ATPases is through changes in membrane lipids [37,41] and phosphorylation, or dephosphorylation of seven amino acid residues of the C-terminus [35]. Moreover, the binding of H + -ATPase to the regulatory 14-3-3 protein is required for the effective functioning of the proton pump; phosphorylation of the penultimate threonine residue of the C-terminus of H + -ATPase provides this bond [42]. The H + -ATPases also have an electrogenic function and contribute the most to the membrane's potential generation and potential changes [34,43,44]. Changes in cytosolic pH and electrical potential may be related: the generation of damage-induced variation potential is mediated by a decrease in the activity of the H + -ATPases [44,45], which also leads to acidification of the cytosol [39].
The importance of cytosolic pH for the regulation of many cellular processes has been the reason for creating of various methods for its measurement, which has different degrees of invasiveness and toxicity for the plant and the duration of observation. Glass, liquid-membrane ion-sensitive and vibrating electrodes have previously been successfully used for continuous pH measurement during long-term (several hours) observations of processes [46][47][48][49]. Intracellular pH can also be measured using optical (fluorescent) methods, which are less invasive. The optical methods include the use of chemical fluorescent dyes and genetically encoded fluorescent probes. Fluorescent molecular probes need to be loaded into the cells, which is associated with certain difficulties. Furthermore, the analysis of long-term (from several tens of minutes to several hours) processes is limited by photobleaching and photodamage of the molecular probes [48,50], which generally limits their successful application. The pH-sensitive genetically encoded sensors based on GFPs, which include Pt-GFP, pHusion and pHluorin, overcome these limitations: these sensors are non-toxic and are constantly expressed in cells, so they are more suitable for whole-plant imaging [23,51]. GFP contains the protein β-barrel, which surrounds the fluorescence-emitting chromophore. Chromophore is formed as a result of the cyclization of the internal tripeptide S65-Y66-G67. Spectral properties of GFP and its mutants depend on pH due to the presence of tyrosine residue (Y66) in the chromophore [52]. Chromophore has different forms, dependent on the pH. The neutral form (A) of the chromophore can be transformed into the anionic form (B) with an increase in pH by changing the protonation of amino acid residues and then by the modulation of conformation [53]. The neutral form of GFP has an absorption maximum at 390 nm; the anionic form-at 475 nm. Dependence on the pH of the GFP's spectral properties allows the use of GFPs as ratiometric pH sensors. The ratiometric fluorescent sensor Pt-GFP from Ptilosarcus gurneyi has sensitivity to a larger pH range among other popular pH sensors [51,54]. S65 residue of Pt-GFP chromophore was replaced by Q65 residue, which is accompanied by a more pronounced peak of excitation at 475 nm [55]. Both absorption peaks of Pt-GFP (at 390 and 475 nm) are expressed at low pH; alkalization leads to a smoothing of the peak at 390 nm and an increase in the second absorption peak [51]. The sensor is characterized by a low sensitivity to cooling [23] and sodium chloride [51].
The majority of research on cytosolic pH was carried out using Arabidopsis model plants [23,51,56]. Therefore, the creation and usage of model plants based on common crops are important for identifying patterns in their responses to biotic and abiotic factors in order to further improve their cultivation conditions. This work describes the creation of potato plants with the genetically encoded Pt-GFP sensor and their application for the registration of pH shifts under the local and systemic action of a temperature stimulus.

Micropropagation and Genetic Transformation of Potato Plants
Studies were carried out on plants of potato (Solanum tuberosum) cultivar "Nevsky". Plants were cultured in vitro on a Murashige and Skoog (MS) [57] basal medium containing 30 g L −1 (w/v) sucrose and 7 g L −1 (w/v) agar. Stem explants with one node were used for micropropagation every 3 weeks.
Transgenic S. tuberosum (cv. Nevsky) plants were produced using an Agrobacteriummediated transformation. The strain AGL0 of Agrobacterium tumefaciens was cultivated on a YEP nutrient medium supplied with 100 mg L −1 rifampicin. A. tumefaciens was transformed with pART27-ptGFP (NanoLight ® , Technologies, Malvern, PA, USA) by electroporation using a MicroPulser (Bio-Rad, Hercules, CA, USA). The T-region of the pART27-ptGFP vector contained the kanamycin resistance gene nptII and ptGFP-gene under the control of the CaMV 35S promoter and ocs terminator. The pART27-ptGFP vector also contained the aadA gene of spectinomycin resistance (Tn7 Spr/Str). Transgenic bacteria were screened for antibiotic resistance on selective growth media YEP supplied with 100 mg L −1 rifampicin and 100 mg L −1 spectinomycin.
Potato micropropagation, organogenesis induction and cultivation of regenerants were carried out under 16/8 h (light/dark) photoperiod at 25 • C with a light intensity of 60 µmol m −2 s −1 .

Acclimatization of Potato
Well-rooted 7-day-old potato plantlets were transferred into clear polypropylene containers with a 10% solution of mineral composition of MS liquid nutrient medium. Every container had a transparent polyethylene lid (height 12 cm). After 2 days, the polyethylene lids were removed. Plantlets were cultivated on a liquid nutrient medium for 7 days. After acclimatization, potato plantlets were grown in pots with soil containing 220 mg L −1 NH 4 + NO 3 , 200 mg L −1 P 2 O 5 and 250 mg L −1 K 2 O. The pH of the soil was 5.5-6.5. Potato plantlets were cultivated in an environmentally controlled room under 16/8 h (light/dark) photoperiod at 25 • C with a cool-white and pink fluorescence tubular lamp (FLUORA OSRAM, Munich, Germany; 60 µmol m −2 s −1 ).

PCR Analysis of Transgenic Plants
The presence of the Pt-GFP gene in the plantlets was analyzed by PCR. Genomic DNA from the putative transgenic lines of the potato was isolated using the CTAB method [58].

Confocal Laser Scanning Microscopy (CLSM)
Fluorescent images and fluorescent spectra of different tissues and organs of transgenic plants were obtained by an LSM710 confocal laser scanning microscope (Carl Zeiss, Jena, Germany) with a Plan-Apochromat 20x/0.5 objective. Pt-GFP was excited at 405 nm and 488 nm, and detected between 505 nm and 525 nm or 495 nm and 600 nm (for spectra). Image data were analyzed using the software Zen 2.1 (Carl Zeiss) [59] and the open-source ImageJ 1.52p software [60].

Fluorescence Imaging of Pt-GFP in the Leaf of a Whole Plant
Fluorescence imaging of the Pt-GFP signal of the potato leaves was carried out using the fluorescence imaging system DVS-03 (ILIT RAS, Shatura, Russia). Pt-GFP fluorescence was excited by a 395/25 nm luminodiode and 490/20 nm luminodiode and was emitted by a CMOS-camera (PRIME 95B, Photometrics, USA) with a 535/43 nm filter. Fluorescent images were obtained at an exposure of 2000 ms with a frequency of 20 s. Micro-Manager 1.4 software [61] and ImageJ 1.52p software [60] were used for processing raw images.
40-day-old potato plants with leaves of 3-4 cm long were used for fluorescence imaging. Before image recording, plants were fixed on the measuring stand ( Figure S1) and adaptation lasted for 90 min. The recording was performed under actinic light illumination of 650/50 nm (40 µmol m −2 s −1 ) and an ambient temperature of 23 • C. After adaptation, but before the temperature effect on the plants, a series of fluorescence images were recorded for 5 min. There was a variety of impacts: The abaxial side of the leaves was cooled or heated using Peltier device, the polarity of which can be changed to switch from cooling to heating and back. The temperature changed periodically. In total, four temperature effects were applied with increasing strength, lasting 2 min within a period of 5 min. The tips of the leaves were heated with a ceramic cement resistor for 4 min to 52 • C; 3.
The detached leaves treated with a H + -ATPase inhibitor (2.5 mM sodium orthovanadate) or standard solution (1 mM NaCl, 0.1 mM KCl, 0.1 mM CaCl 2 ) were first cooled to 9.4 • C for 3 min, and then after 7 min heated to 42.9 • C for 3 min by the Peltier device. The treatment of the leaves with reagents was carried out by loading them through the petiole using a desiccator and vacuum pump at a pressure of 0.2 atmosphere for 5 min; 4.
H + -ATPase inhibitor or standard solution was added to 10 × 10 mm pieces cut out of the leaves and placed in Petri dishes (volume 15 mL) filled with standard solution.
Non-transgenic potato plants characterized by a low ratio F490/F395 level were used to control changes in the fluorescence of heated or cooled leaves. After incubation in buffer solutions, the leaves were fixed onto the measuring stand in such a way that the abaxial side of the leaf was immersed in 5 mL of the corresponding buffer solution. Further fluorescent images were obtained on a fluorescence imaging system, as described above. In addition, immediately after incubation of the leaves in the buffer solutions, the pH level of each solution was re-measured using a pH-meter. The equations of F490/F395 dependence on pH were calculated using GraphPad Prism 6 software. The Boltzmann fit was chosen for fitting sigmoidal curves to calibration data [62] using apparent pK 7.3 for the Pt-GFP sensor [51].

Testing the Temperature Dependence of Pt-GFP Fluorescence
The dependence of the Pt-GFP spectral properties on temperature was tested at Excitation and emission spectra of the purified Pt-GFP were obtained using Synergy Mx Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA) in black 96-well microplates with a clear glass bottom (Eppendorf, Hamburg, Germany). Heating of Pt-GFP solutions was carried out with a sequential increase in temperature from 25 • C to 30, 35, 40 and 45 • C using a Microplate Reader control unit. Excitation spectra were obtained in the range from 330 to 510 nm, and the fluorescence signal was registered at 530 nm. Fluorescence spectra were recorded in the range of 500 to 600 nm with excitation at 395 or 480 nm.

Measurements of Electric Potential in Potato Plants Using Macroelectrodes
Electrical potentials were extracellularly measured with Ag + /AgCl electrodes EVL-1MZ (Gomel Plant of Measuring Devices, Gomel, Belarus). The electrodes were connected with a high-impedance amplifier IPL-113 (Semico, Novosibirsk, Russia) and a personal computer. Measuring electrodes were set in contact with a plant by cotton threads wetted with water. The measurements of electric signals were carried out with fluorescence measurements by fluorescence imaging system DVS-03 at the same time. The scheme of electrode positions on the leaf is presented on Figure S1. The reference electrode was set in a pot with soil.

Temperature Changes of Potato Leaves
A thermal imager Testo 885 (Testo, Lenzkirch, Germany) was used for control of the temperature of the potato leaves which were cooled or heated. Received images were processed by IRSoft 4.5 software [63].

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 6 software. Data are represented as mean ± standard deviation (SD). The number of biological replicates was from 5 to 7.

Characteristics of Potato Transformants
As a result of the agrobacterial transformation of potatoes by the gene of the Pt-GFP ratiometric pH sensor, 19 regenerants were obtained. The efficiency of the genetic transformation was 13.19%. A polymerase chain reaction confirmed the presence of the Pt-GFP gene in the genome of 5 of the 19 potato regenerants (Figure 1). range from 330 to 510 nm, and the fluorescence signal was registered at 530 nm. Fluorescence spectra were recorded in the range of 500 to 600 nm with excitation at 395 or 480 nm.

Measurements of Electric Potential in Potato Plants Using Macroelectrodes
Electrical potentials were extracellularly measured with Ag + /AgCl electrodes EVL-1MZ (Gomel Plant of Measuring Devices, Gomel, Belarus). The electrodes were connected with a high-impedance amplifier IPL-113 (Semico, Novosibirsk, Russia) and a personal computer. Measuring electrodes were set in contact with a plant by cotton threads wetted with water. The measurements of electric signals were carried out with fluorescence measurements by fluorescence imaging system DVS-03 at the same time. The scheme of electrode positions on the leaf is presented on Figure S1. The reference electrode was set in a pot with soil.

Temperature Changes of Potato Leaves
A thermal imager Testo 885 (Testo, Lenzkirch, Germany) was used for control of the temperature of the potato leaves which were cooled or heated. Received images were processed by IRSoft 4.5 software [63].

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 6 software. Data are represented as mean ± standard deviation (SD). The number of biological replicates was from 5 to 7.

Characteristics of Potato Transformants
As a result of the agrobacterial transformation of potatoes by the gene of the Pt-GFP ratiometric pH sensor, 19 regenerants were obtained. The efficiency of the genetic transformation was 13.19%. A polymerase chain reaction confirmed the presence of the Pt-GFP gene in the genome of 5 of the 19 potato regenerants (Figure 1).  Further experiments were carried out on the plants of transformation line 1 (NKM01). The choice of this regenerant was based on the results of a PCR analysis and the severity of the characteristic peak of Pt-GFP protein emission in the tissues of the transformed plants ( Figure S2). A detailed study of Pt-GFP localization in the potato plants was carried out using confocal laser scanning microscopy (CLSM). A bright fluorescent sensor signal was observed on CLSM images of transgenic plant cells, and it was more pronounced when excited at 488 nm ( Figure 2A). The emission spectrum of transgenic plants had a maximum at 509 nm (typical for Pt-GFP) when excited at both 405 and 488 nm. The untransformed potato plants did not possess such fluorescence. CLSM images of the cells of the untransformed plants contained a weak fluorescent signal due to the autofluorescence of tissues, which is much less than that of the transformed cells ( Figure 2).
Further experiments were carried out on the plants of transformation line 1 (NKM01). The choice of this regenerant was based on the results of a PCR analysis and the severity of the characteristic peak of Pt-GFP protein emission in the tissues of the transformed plants ( Figure S2).
A detailed study of Pt-GFP localization in the potato plants was carried out using confocal laser scanning microscopy (CLSM). A bright fluorescent sensor signal was observed on CLSM images of transgenic plant cells, and it was more pronounced when excited at 488 nm (Figure 2A). The emission spectrum of transgenic plants had a maximum at 509 nm (typical for Pt-GFP) when excited at both 405 and 488 nm. The untransformed potato plants did not possess such fluorescence. CLSM images of the cells of the untransformed plants contained a weak fluorescent signal due to the autofluorescence of tissues, which is much less than that of the transformed cells (Figure 2). The presence of the Pt-GFP sensor was confirmed in all organs of the transgenic potatoes, for example, in the leaves (abaxial epidermis, abaxial and adaxial mesophyll, vascular tissue), stem cortex, tip, absorption zone and elongation zone of the root (Figure 3). The fluorescent signal of the Pt-GFP sensor indicates the presence of this protein in the cytoplasm and cell nuclei.
To determine the pH value in potato leaf cells, a calibration curve was performed in the pH range of 4 to 9 ( Figure 4). Potato leaves were incubated in solutions with different pH levels in the presence of the protonophore, and then fluorescent images of potato tissues were obtained using the DVS-03 fluorescence imaging system or using CLSM. After image processing, the dependence of the fluorescence of the Pt-GFP protein on the pH in the cells of a whole potato leaf was obtained. The dependence of the ratio F490/F395 on pH was approximated by the Boltzmann sigmoid ( Figure 4). The results obtained using CLSM for the absorption zone of the root, stem cortex and adaxial mesophyll of the leaf are shown in Figure S3.
In the range of 6.5 to 8 pH units, a close to linear dependence of Pt-GFP fluorescence on pH was observed, which corresponds to the literature data on the sensitivity range of the Pt-GFP sensor, both in solution and in situ calibration [51,64].
The cytosolic pH of the cells of a whole potato leaf, determined by a whole-body imaging method, was 7.03 ± 0.03. pH values determined with CLSM were 7.09 ± 0.14 in The presence of the Pt-GFP sensor was confirmed in all organs of the transgenic potatoes, for example, in the leaves (abaxial epidermis, abaxial and adaxial mesophyll, vascular tissue), stem cortex, tip, absorption zone and elongation zone of the root (Figure 3). The fluorescent signal of the Pt-GFP sensor indicates the presence of this protein in the cytoplasm and cell nuclei.
To determine the pH value in potato leaf cells, a calibration curve was performed in the pH range of 4 to 9 ( Figure 4). Potato leaves were incubated in solutions with different pH levels in the presence of the protonophore, and then fluorescent images of potato tissues were obtained using the DVS-03 fluorescence imaging system or using CLSM. After image processing, the dependence of the fluorescence of the Pt-GFP protein on the pH in the cells of a whole potato leaf was obtained. The dependence of the ratio F490/F395 on pH was approximated by the Boltzmann sigmoid ( Figure 4). The results obtained using CLSM for the absorption zone of the root, stem cortex and adaxial mesophyll of the leaf are shown in Figure S3.
In the range of 6.5 to 8 pH units, a close to linear dependence of Pt-GFP fluorescence on pH was observed, which corresponds to the literature data on the sensitivity range of the Pt-GFP sensor, both in solution and in situ calibration [51,64].
The cytosolic pH of the cells of a whole potato leaf, determined by a whole-body imaging method, was 7.03 ± 0.03. pH values determined with CLSM were 7.09 ± 0.14 in the cortical cells of the absorption zone of the root, 6.79 ± 0.12 in the cortical cells of stem, and 6.75 ± 0.17 in the mesophyll cells of the leaf adaxial side.

Testing the Temperature Dependence of Pt-GFP Fluorescence
The effect of temperature on the fluorescence of purified Pt-GFP was evaluated. The emission and excitation spectra of Pt-GFP in pH 5.0 or 7.4 solutions are shown in Figure 5. An increase in the temperature of the solution caused a decrease in the intensity of the Pt-GFP fluorescence with an increase in temperature from 25 to 45 • C ( Figure 5A,B). At the same time, there was no change in the position of the excitation and emission peaks ( Figure 5C,D). It is also important to note the constancy of the fluorescence ratio of the anionic and neutral forms of the protein (F480/F395) in both alkaline and acidic solutions ( Figure 5E). Thus, the results demonstrate the possibility of using the Pt-GFP to measure of the temperature-induced pH changes in plants.

Figure 4.
In-vivo calibration of the Pt-GFP sensor in a potato leaf using a full-scale fluorescence imaging system. F490/F395 is the ratio of the fluorescence intensity of the Pt-GFP sensor when excited at wavelengths of 490 and 395 nm, with a registration of fluorescence at 535/43 nm. The leaf was incubated in a solution with an appropriate pH value in the presence of the CCCP protonophore. Data are represented as mean ± SD (n = 5).

Testing the Temperature Dependence of Pt-GFP Fluorescence
The effect of temperature on the fluorescence of purified Pt-GFP was evaluated. The emission and excitation spectra of Pt-GFP in pH 5.0 or 7.4 solutions are shown in Figure 5. An increase in the temperature of the solution caused a decrease in the intensity of the Pt-GFP fluorescence with an increase in temperature from 25 to 45 °C ( Figure 5A,B). At the same time, there was no change in the position of the excitation and emission peaks ( Figure 5C,D). It is also important to note the constancy of the fluorescence ratio of the anionic and neutral forms of the protein (F480/F395) in both alkaline and acidic solutions ( Figure 5E). Thus, the results demonstrate the possibility of using the Pt-GFP to measure of the temperatureinduced pH changes in plants.

Temperature-Induced pH Shifts in the Potato Leaf
During the study, changes in the pH of the cytosol and electrical potentials were determined in the zone affected by the stress-factors, i.e., cooling or heating of a whole leaf of an intact potato plant.
An uneven decrease in the temperature of the leaf surface was observed, due to differing leaf thickness, when the whole leaf was chilled by the Peltier device ( Figure 6). The most pronounced decrease in temperature (up to 9 • C from the initial temperature of 23 • C) was observed in the thinner distal part of the leaf (area1) (Figure 6), while the thick central part of the potato leaf (area2) was cooled to 9.9 • C. The difference in the extent of cooling of a potato leaf was reflected in the change in the pH level and the electrical potential in these two zones of interest.
During the cooling of the leaf surface, a rapid decrease in the cytosolic pH occurred: it acidified after cooling started, and also quickly recovered after the end of the stimulus action and the return of the leaf temperature to the initial value. The magnitude of the pH shift directly depended on the cooling depth. At maximum cooling in area1, the pH decreased by 0.21 units, in area2-by 0.17. No changes in the F490/F395 ratio of the non-transgenic plant were observed compared to that of the transgenic plant line. The results are shown in Figure S4.
Along with the change in the intracellular pH, cooling caused a transient depolarization of the membrane of the cells of the leaf, with a maximum amplitude of 30 mV recorded in the distal part of the leaf where the most pronounced cooling occurred (area1) ( Figure 6B,D). The dynamics of the changes in pH and electrical potential were similar.  In the case of heating the whole leaf by the Peltier device, an uneven change in the surface temperature of the leaf was also noted. The thinner distal part of the leaf (area 1) was heated by 26.3 • C from the initial temperature, while the thicker central part of the leaf (area 2) was heated only by 15.4 • C (Figure 7).
Heating caused alkalization of the cytoplasm by 0.3 pH units. When a temperature was reached (35-40 • C), rapid acidification of the cytoplasm began, along with ongoing heating.
The dynamics of the electrical potential in the heated leaf, in general, corresponded to the dynamics of the cytosolic pH. First, when the leaf surface was heated, hyperpolarization occurred, and then, when the critical temperature was reached, depolarization took place with the maximum amplitude, which was 35 mV. Stimuli 3 and 4 caused the generation of variation potential in area 1, and only the last stimulus caused the same in area 2. The recovery of the electrical potential after the temperature returned to the initial values was slower compared to cooling, especially with strong heating. The amplitudes of coldand heat-induced depolarization were similar for transgenic and non-transgenic plants. Moreover, these stressors did not lead to changes in the F490/F395 ratio, which could reflect a change in pH ( Figure S4).

Registration of pH Changes at a Distance from the Zone of Local Stimulation
The next step of the research was the analysis of pH changes in the unstimulated areas of the leaf under local stimulation. In this case, only area1 was exposed to high temperatures, and changes in pH and electrical potential were recorded at different distances

Registration of pH Changes at a Distance from the Zone of Local Stimulation
The next step of the research was the analysis of pH changes in the unstimulated areas of the leaf under local stimulation. In this case, only area1 was exposed to high temperatures, and changes in pH and electrical potential were recorded at different distances from the stimulation zone. Local heating caused the propagation of an electrical signal to the unstimulated area. The electrical signal was a transient depolarization with an amplitude of 50 mV, which can be characterized as a variation potential (VP) (Figure 8). There was a decrement of VP, which was expressed in a decrease in the amplitude of the reaction with the distance from the stimulation zone. Along with the VP propagation, local heating also induced a change in the intracellular pH in the unstimulated areas of the leaf. Such changes represent a wave of the pH decreasing, lasting several tens of minutes. The amplitude of the acidification wave decreased with the distance from the local heating zone, by~0.2 pH units at a distance of 1 cm from the stimulation zone (area1) and by~0.1 pH units at a distance of 3 cm from the stimulation zone (area2). Along with the propagation of the acidification wave near the stimulation zone, there was also a short-term alkalization of small amplitude. This may be due to an increase in temperature in the area adjacent to the stimulated area. of the acidification wave near the stimulation zone, there was also a short-term alkalization of small amplitude. This may be due to an increase in temperature in the area adjacent to the stimulated area.

Analysis of the Mechanism of Temperature Induced Changes in pH
The mechanism of pH shifts under the temperature changes is most likely to be the modulation of H + -ATPase activity, the contribution of which was determined using an inhibitor, sodium orthovanadate. When sodium orthovanadate was added to the leaf bathing solution, a decrease in pH by 0.25 was observed ( Figure 9A), and persistent inhibition of the proton pump occurred: the pH level remained low throughout the observation period. The pHcyt of leaves washed with the standard solution did not change.
An analysis of the effect of sodium orthovanadate on thermally induced changes in pH and electrical potential was performed on leaves loaded with the inhibitor or standard solution. During heating and cooling, the reactions of the leaves treated with a standard solution and the inhibitor differed ( Figure 9B). The pH changes ( Figure 9C) in the leaves

Analysis of the Mechanism of Temperature Induced Changes in pH
The mechanism of pH shifts under the temperature changes is most likely to be the modulation of H + -ATPase activity, the contribution of which was determined using an inhibitor, sodium orthovanadate. When sodium orthovanadate was added to the leaf bathing solution, a decrease in pH by 0.25 was observed ( Figure 9A), and persistent inhibition of the proton pump occurred: the pH level remained low throughout the observation period. The pH cyt of leaves washed with the standard solution did not change.
An analysis of the effect of sodium orthovanadate on thermally induced changes in pH and electrical potential was performed on leaves loaded with the inhibitor or standard solution. During heating and cooling, the reactions of the leaves treated with a standard solution and the inhibitor differed ( Figure 9B). The pH changes ( Figure 9C) in the leaves treated with the inhibitor were less pronounced than those in leaves treated with the standard solution. Sodium orthovanadate caused a decrease in pH shifts by 50% under cooling, and by 59% under heating. Along with the suppression of temperature-induced pH shifts in the presence of the inhibitor, there was a significant suppression of changes in the electrical potential ( Figure 9D).

Discussion
Potato plants genetically transformed with pH-sensitive fluorescent sensor were characterized in this work as one of the few model objects from a group of the most important agricultural crops for studying plant responses to the changes in environmental conditions that are significant for cultivation. The optimal growth temperature of the potato is considered to be between 16 and 25 °C. Therefore, high and low temperatures during the cultivation season cause physiological changes in potato plants that affect its development and can lead to a decrease in productivity: the minimum temperature for tuber initiation ranges from 4 to 18 °C, and at high temperatures (29-31 °C) tuberization decreases [5,14,65,66]. The use of a genetically encoded sensor in a plant allows for the performance of non-invasive single or multiple assessments, as well as long-term monitoring of intracellular pH, without loading of molecular probes [51]. The high uniform expression of the Pt-GFP sensor in cells of various tissues allows for the measurement of pH changes, both in individual cells using high-resolution microscopy and in the whole plant

Discussion
Potato plants genetically transformed with pH-sensitive fluorescent sensor were characterized in this work as one of the few model objects from a group of the most important agricultural crops for studying plant responses to the changes in environmental conditions that are significant for cultivation. The optimal growth temperature of the potato is considered to be between 16 and 25 • C. Therefore, high and low temperatures during the cultivation season cause physiological changes in potato plants that affect its development and can lead to a decrease in productivity: the minimum temperature for tuber initiation ranges from 4 to 18 • C, and at high temperatures (29-31 • C) tuberization decreases [5,14,65,66]. The use of a genetically encoded sensor in a plant allows for the performance of non-invasive single or multiple assessments, as well as long-term monitoring of intracellular pH, without loading of molecular probes [51]. The high uniform expression of the Pt-GFP sensor in cells of various tissues allows for the measurement of pH changes, both in individual cells using high-resolution microscopy and in the whole plant using whole-body imaging. As demonstrated in Figures 6 and 7, whole-body imaging allows for the study of the spatial heterogeneity of responses to temperature factors.
Some literature data indicate the need for careful use of GFP-based sensors in the study of temperature-dependent processes, because GFP fluorescence can also be temperature dependent [67,68]. At the same time, the properties of Pt-GFP were previously analyzed at temperatures from 4 to 22 • C [25]: Pt-GFP did not change its properties in this temperature range. Furthermore, Pt-GFP remains stable when heated to 80.5 • C [55]. In our work, we have shown that the fluorescence of Pt-GFP remains almost unchanged at temperatures from 25 to 45 • C. When the protein is heated, the ratio between the fluorescence of the anionic and neutral forms of Pt-GFP remains unchanged and the pH sensitivity of the sensor is maintained ( Figure 5F).
The usage of potato as a model plant allows for the determination of the features of shifts in intracellular pH caused by such an important abiotic factor as a temperature change. The acidification of the cytosol of potato cells caused by cooling corresponds to the direction of cold-induced pH shifts for Vigna radiata and Arabidopsis thaliana, according to early research of cell suspensions [25,26,[69][70][71]. The registered magnitude of pH shift in potato plants (by 0.2 units) ( Figure 6) is generally slightly smaller than that of V. radiata (by 0.6 pH units) [26], V. radiata and T. aestivum (by 0.1-0.6 pH units) [69], A. cherimola (by 0.72 pH units) [72], and A. thaliana (by 0.1 pH units) [25]. The differences can be due to both the greater strength and duration of cooling performed in the works [72], and the fact that, in the studies [26,69], the pH was measured in a cell suspension, and not in the whole plant.
The study of the dynamics of temperature-induced changes in the intracellular pH revealed their high rate (Figures 6 and 7). This indicates that the pH changes are primarily associated with the "biophysical pH-stat"-a change in the activity of H + -transporting systems, including H + -ATPases of the tonoplast and plasmalemma [28]. The participation of the H + -ATPases of plasmalemma is also indicated by the changes in the electrical potential, consistent with the pH shifts ( Figures 6 and 7), to the formation of which the proton pump contributes the most in plant cells [44]. The key role of H + -ATPases in temperature-induced changes in pH and electrical potentials is evidenced by the experiments with proton-pump inhibition by sodium orthovanadate (Figure 9). Inhibition of H + -ATPase activity reduced the amplitude of pH and electrical potential changes compared to the standard solution treatment ( Figure 9C,D). However, the interpretation of in vivo experiments with sodium orthovanadate should be performed with caution, because it can also affect the activity of other enzyme activity, for example, phosphatases [35]. The set of the data obtained in our work indicates the contribution of the proton pump to temperature-induced pH shifts.
A decrease in H + -ATPase activity may be associated with the influx of calcium ions into the cell, which is the central event of the cold-induced cell response [73], also inducing the development of depolarization [38,42]. Another way that the cold influences the activity of the ATPase can be an increase in membrane rigidity [37,[74][75][76]. Along with the H + -ATPase of plasmalemma, the vacuolar H + -ATPase is also apparently involved in the response to cold stress, since an increase in the vacuolar pH was registered along with a decrease in the cytosolic pH [69,71].
An increase in temperature to sub-threshold values leads to cytosol alkalinization (Figure 7). This can be caused directly by the effect of temperature on the membrane lipid matrix [76], or by the regulation of activity by reversible phosphorylation of the proton pump [77].
Heating to the extent of exceeding the threshold temperature leads to the formation of a deep depolarization wave, propagating beyond the stimulated zone-a variation potential [74,78]. The formation of VP is associated with the inactivation of the proton pump occurring in case of the threshold temperature being exceeded [40], or after prolonged treatment with sub-threshold temperatures [41,79]. The generation and propagation of VP are accompanied by transient acidification of cytosol (Figures 7 and 8). In the case of heat shock, the inactivation of the proton pump and the subsequent acidification of the cytosol are the result of earlier rapid events: changes in the properties of membranes, and activation of the signaling cascade involving lipids messenger, calcium release and ROS generation [76,80]. The pH changes associated with the propagation of long-distance stress signals were described earlier, and represent a transient acidification of the cytoplasm, along with apoplastic alkalinization [17,39,[81][82][83][84][85][86]. On maize [39] and pea plants [85], cytosol acidification was observed directly during the generation of an electrical impulse. At the same time, the results of this research demonstrate the prolonged acidification wave after electrical impulse generation. The formation of the wave presumably can be caused by the activation of regulatory/signaling systems during the generation of VP.
In general, the results of the research indicate both local and systemic changes in pH under temperature changes. pH changes can act as regulators of the activity of physiological processes and gene activation, e.g., they are shown to regulate the activity of photosynthesis by long-distance stress signals [44,87,88]. They can be a result of other propagating signals or be an important basic condition accompanying the dominant signaling cascade [17]. Changes in pH can be coordinated with other stress signals, such as a calcium signal [89], or they can induce the next signal [90]. The pH level determines the functioning of enzymes and many other processes, thus determining the pH as a signal. The study of the generation and propagation of such a signal is important for an understanding of the relationship of the processes in a plant cell and fine-tuning their regulation.

Conclusions
The created model potato plants expressing a pH-sensitive fluorescent sensor allow for the recording of the dynamics of intracellular pH on whole plants. There are rapid local and systemic changes in cytosolic pH in response to temperature changes. A change in the activity of the H + -ATPases contributes to them. Further research should focus on the mechanisms of modulation of H + -ATPases activity, as well as on the role of pH changes in the formation of resistance to stressors.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/agriculture11111131/s1, Figure S1: The scheme of experiment in the fluorescence imaging system DVS-03, Figure S2: Emission spectra of transgenic potato plants and non-transgenic potato plants, Figure S3: In vivo calibration of Pt-GFP fluorescence ratio (F488/F405), Figure S4: Cold-and heat-induced changes in F490/F395 ratio and electrical potential of non-transgenic potato leaf cells.

Conflicts of Interest:
The authors declare no conflict of interest.