207 1. Introduction Ion fluxes across cellular membranes are known to play the key role in triggering and mediating defense mecha- nisms in plants, however little data are currently available on ion signatures generated during plant-virus interac- tions. Changes in K+ fluxes after virus inoculation may be mediated by depolarization-activated outward-rectifying K+ channels (Shabala et al., 2010). In animals, the immunosuppressant drug mycophenolic acid (MPA) [(4E)-6-(4-Hydroxy-6-methoxy-7-methyl-3- oxo-1,3-dihydro-2-benzofuran-5-yl)-4-methylhex-4-enoic acid] depletes cellular guanine nucleoside (GN) by inhibi- tion of inosine monophosphate dehydrogenase, modulat- ing the activity of K ATP channels (Li et al., 2000). MPA is also an effective antiviral drug in plants such as grapevine (Panattoni et al., 2007; Skiada et al., 2009; Luvisi et al., 2012 a; Skiada et al., 2013; Panattoni et al., 2014; Guaz- zelli et al., 2015), but no reports about its effect on K- conducting ion channels in plants are available. In terms of metabolic dependence of MPA action in plant cells, antiviral drug translocation across membranes was linked to the free energy available in a proton elec- trochemical potential difference (Luvisi et al., 2012 b). Nowadays, compounds that influence K ATP channel ac- tivity are currently available for clinical use, and include diazoxide (DO) (7-Chloro-3-methyl-4H-1,2,4-benzothia- diazine 1,1-dioxide) (Babenko et al., 1998; 2000) whose effectiveness was also confirmed in plants to investigate the inhibition of mitochondrial K ATP channels (Chiandussi et al., 2002). With regard to the potential effect due to GN depletion by MPA, cyclic derivatives of GN can be involved in plant K-conducting ion channels. In fact, inward-rectifier potas- sium ion channels have been cloned from Hordeum vul- gare, Nicotiana tabacum and Arabidopsis thaliana (Leng et al., 1999). These channels open in the presence of cy- clic nucleotides such as cyclic guanosine monophosphate (cGMP), and are therefore referred to as cyclic nucleotide gated channels (Leng et al., 2002). The cGMP is thought to be present in the cytosol of plant cells, and to be in- volved in signal transduction pathways which regulate many aspects of cellular metabolism (Assmann, 1995). The activity of antiviral drugs in grapevine cells was recently investigated using electrophysiological methods (Panattoni et al., 2013 a). In the present paper we investi- gate the effects of MPA on depolarization induced by ex- tracellular K+ in foliar samples of Vitis vinifera cv. Sangio- vese. This grapevine cultivar is affected by various plant viruses (Rizzo et al., 2012; Rizzo et al., 2015) and the ef- Effects of extracellular K+ on grapevine membrane potential as influenced by the antiviral mycophenolic acid. An electrophysiological study A. Luvisi1 (*), E. Rinaldelli 2, A. Panattoni 1 1 Dipartimento di Scienze Agrarie, Alimentari e Agro-ambientali, Università di Pisa, Via del Borghetto, 80, 56124 Pisa, Italy. 2 Dipartimento di Scienze delle Produzioni Agroalimentari e dell’Ambiente, Università degli Studi di Firenze, Viale delle Idee, 30, 50019 Sesto Fiorentino (FI), Italy. Key words: chemotherapy, virus host interaction, Vitis vinifera. Abstract: Mycophenolic acid (MPA) is an effective antiviral drug in plants, and its action in modulating the activity of KATP channels is already known in animals. In the present work an electrophysiological study was carried out to investi- gate MPA effects on plant K+ channels, through the measurement of trans-plasma membrane potential in samples of Vitis vinifera cv. Sangiovese treated with extracellular K+. Tests confirmed that the administration of MPA (in preincubated samples or in those maintained under chemical treatment) can reduce the membrane depolarization induced by K+. However, MPA-induced alteration in membrane potential was sensitive to the KATP channel opener diazoxide, as well to treatments with guanosine. This result confirms the effectiveness of MPA in influencing KATP channel activity as well as inhibiting activity of the inward-rectifier potassium ion channel which could be mediated by guanosine depletion induced by MPA. Adv. Hort. Sci., 2015 29(4): 207-212 (*) Corresponding author: aluvisi@agr.unipi.it Received for publication 28 September 2015 Accepted for publication 5 November 2015 208 Adv. Hort. Sci., 2015 29(4): 207-212 fects of antiviral drugs where investigated (Panattoni et al., 2011; Luvisi et al., 2011; Panattoni et al., 2013 b). In addi- tion, the effects of GN- or DO- treatments were evaluated in order to investigate the effect of GN depletion caused by MPA or its effects on K ATP channels. 2. Materials and Methods Plant material Virus-free (with regard to viruses included in the Eu- ropean Commission directive 2005/43/EC), eight-year- old V. vinifera cv. Sangiovese were used for electrophysi- ological tests. The sanitary condition of each plant was confirmed by RT-PCR (Nakaune and Nakano, 2006; Faggioli et al., 2013). Symptomless plants were used. In June 2013, fully expanded leaves were excised and fresh freehand samples (3-5 mm diameter) were cut with an ethanol-cleaned razor blade for testing (Rinaldelli et al., 2012; 2014). Measurement of membrane potential (Em) For chemical assays (Table 1), preincubation for 1 h in basal solution (BS) or chemical-BS solutions (MPA-BS, GN-BS, DO-BS at 0.5, 1.0, 2.0 mM) adjusted to pH 5.6 with TRIS (2-Amino-2-hydroxymethyl-propane-1,3-di- ol) was followed by preparation of leaf segments accord- ing to Luvisi et al. (2012 b). Step 1 for Em measurements was conducted perfusing aerated BS or chemical-BS through the chamber that fixes the sample at a flow rate of 10.0 x 10-3 L min-1. The measuring electrodes used were micropipettes (tip diameter < 1µm) obtained from single-barreled borosilicate capillaries (World Precision Instruments, Sarasota, USA) as described in Rinaldelli et al. (2012). Insertion of the microelectrodes took place in the central zone of the mesophyll by way of a microma- nipulator (World Precision Instruments, Sarasota, USA). Successful microelectrode impalement was determined by rapid attainment of a steady value without subsequent decay (Ober and Sharp, 2003), followed by a stabilized Em for 5 min. Em after stabilization was recorded to evaluate the effect of chemicals on membrane potential. Membrane signal steadiness was calculated considering the number of successful cell impalements out of those attempted (%). Step 2 began after Em stabilization, using solutions enriched with KCl. Two procedures were carried out in order to investigate the effects induced by extracellular K+ following MPA treatments. The first procedure was carried out on sam- ples in which treatment involved only the preincubation step (Li et al., 2000). Thus, samples were preincubated in MPA-BS, followed by Em measurement under BS (step 1) and K-BS (step 2). In this test, two concentrations of KCl (5, 10 mM) were evaluated. The effect of chemical treat- ment was calculated considering Em after BS stabilization and maximum Em achieved after K-BS administration, expressed as Δ Em (Table 1). The second procedure was carried out following a conventional electrophysiological approach. In order to evaluate the interference of chemi- cals (MPA, GN or DO) on effects induced by K+, tests were carried out using chemical-BS solution both in preincuba- tion and in step 1, while in step 2 the solution was enriched with KCl (chemical-K-BS) at 10 mM (concentration cho- sen considering results of the first procedure). The inter- ference of chemicals on membrane depolarization induced by K+ was calculated considering Em after chemical-BS Table 1 - Solutions used in membrane potential tests Chemical assay Solution Abbreviation Membrane potential (Em) CaCl 2 5.0x10-4 M, K 2 SO 4 2.5x10-3 M, MES 5.0x10-3 M BS Em BS BS with MPA MPA-BS Em MPA BS with Guanosine GN-BS Em GN BS with Diazoxide DO-BS Em DO Extracellular K+ assay Solution Abbreviation Membrane potential (Em) Variation of membrane potential (%) BS with KCl K-BS Em K Em K - Em BS = Δ Em K MPA-BS with KCl MPA-K-BS Em MPA-K Em MPA-K - Em MPA = Δ Em MPA-K GN-BS with KCl GN-K-BS Em GN-K Em GN-K - Em GN = Δ Em GN-K DO-BS with KCl DO-K-BS Em DO-K Em DO-K - Em DO = Δ Em DO-K MPA-BS with Guanosine MPA-GN-BS Em MPA-GN MPA-BS with Diazoxide MPA-DO-BS Em MPA-DO MPA-GN-BS with KCl MPA-GN-K-BS Em MPA-GN-K Em MPA-GN-K - Em MPA-GN = Δ Em MPA-GN-K MPA-DO-BS with KCl MPA-DO-K-BS Em MPA-DO-K Em MPA-DO-K - Em MPA-DO = Δ Em MPA-DO-K 209 Luvisi et al., Effects of extracellular K+ on grapevine membrane potential stabilization and maximum Em achieved after chemical- K-BS administration, expressed as Δ Em (Table 1). Preincubation and electrophysiological tests were carried out at 22±0.5°C under light (30 watt m-2). All tests were conducted on 15 healthy or infected samples. Plots are representative of 15 equivalent experiments. Measurements were performed under Faraday cage to protect tests from external radio frequency interference. Statistical analysis The effects of treatments on Δ Em were elaborated us- ing Sigma-Plot software (version 11; Systat Software, San Jose, CA). The software was used to perform one- or two- way analysis of variance (ANOVA) in a random design and pairwise multiple comparisons on significant effects and interactions using the Holm-Sidak method. Data ex- pressed in percent were converted to arcsin values. P ≤ 0.05 was considered to be significant. 3. Results and Discussion Em measurement in chemical preincubated samples Foliar samples were preincubated in chemical-BS so- lutions and membrane potential was measured with mi- croelectrodes. Preincubation with MPA-BS solution did not change the resting membrane potential with regard to the chosen concentration (Table 2). However, MPA seems to interfere with the signal steadiness at 1.0 and 2.0 mM, causing a reduction of successful impalement by 37.9 and 64.7 %, respectively. Assays carried out with BS enriched by GN or DO showed no effect on mem- brane potential and did not interfere with the steadiness of the signal. With regard to the effects induced by extracellular K+, Em measurement was carried out perfusing aerated BS solution throughout the chamber with the fixed sample (step 1), followed by perfusion of K-BS solution (step 2), and recording the depolarization due to K+ treatment. In MPA-preincubated samples, the antiviral drug can cause a differential response to potassium effects, as shown in Fig- ure 1. The cell response confirms the Nernstian changes in diffusion potential due to increased K+-concentration or activation of K+ channels. As reported in Table 3, KCl caused different depolar- ization according to each concentration, but the preincuba- Table 2 - Effect of sample preincubation with chemical administered at different concentrations (0.5, 1.0, 2.0 mM) on membrane potential (Em, mV) or membrane potential signal steadiness on samples of Vitis vinifera cv. Sangiovese, expressed as % of successful microelectrode impalement Treatment Em (mV) Signal steadiness (%) BS -109.5±6.0 a (z) 53.6 MPA-BS (mM) 0.5 -110.6±7.2 a 51.7 1.0 -110.0±6.9 a 33.3 2.0 -107.6±5.7 a 18.8 GN-BS (mM) 0.5 -111.6±8.5 a 50.0 1.0 -112.0±9.5 a 48.4 2.0 -109.0±7.0 a 53.6 DO-BS (mM) 0.5 -110.9±7.8 a 45.5 1.0 -113.5±8.5 a 48.4 2.0 -108.3±7.1 a 51.7 (z) Values in the same column followed by the same letter do not differ significantly according to Duncan’s multiple range test (P ≤0.05). BS= Basal solution; MPA= Mycophenolic acid; GN= Guanosine; DO= Diazoxide. Table 3 - Depolarization induced in samples of Vitis vinifera cv. San- giovese by extracellular K+ at 5 or 10 mM following MPA-BS preincubation compared to untreated sample Δ Em (%) K-BS (mM) 5 10 BS 18.2±3.5a (z)A (y) 30.7±3.7 aB MPA-BS (mM) 0.5 17.9±4.1 aA 29.8±3.2 aB 1.0 12.0±3.2 bA 23.3±2.0 bB 2.0 11.1±2.3 bA 16.4±4.1 cB (z) values in the same column followed by the same letter do not differ significantly according to Duncan’s multiple range test (P ≤0.05). (y) values in the same line followed by the same letter do not differ sig- nificantly according to Duncan’s multiple range test (P ≤0.05). The effect was calculated considering Em after BS stabilization and maximum Em achieved after K-BS administration, expressed as Δ Em (%). BS = basal solution; MPA = mycophenolic acid. Fig. 1 - Effects induced on samples of Vitis vinifera cv. Sangiovese by extracellular K+ (KCl 10 mM) subsequent to MPA 1.0 mM treatment compared to untreated sample. BS = basal solution; MPA = mycophenolic acid. Plot is representative of 15 equiva- lent experiments. 210 Adv. Hort. Sci., 2015 29(4): 207-212 tion with MPA was able to cause a reduction in depolar- ization. In particular, MPA at 1.0 or 2.0 mM significantly affected KCl at 10 mM, while MPA at 5 mM seemed not to alter the effect induced by extracellular K+. KCl at 10 mM was used for the subsequent test. Em measurement in samples maintained under chemical treatment Tests carried out while maintaining chemical adminis- tration throughout steps 1 (perfusion of chemical-BS so- lution) and 2 (perfusion of chemical-BS solution enriched by KCl) of Em measurement confirmed the effective- ness of MPA in reducing the effect induced by K+ (Table 4). MPA interference was concentration-dependent and at the lower concentration MPA did not interfere with the effect on membrane potential induced by K+. Con- versely, at higher dosages (2:1 MPA:KCl), MPA causes a Δ Em MPA-K of 2.5±0.7%, with a reduction of more than 90% of potassium effects. MPA effectiveness was main- tained at equal concentration (1:1 MPA:KCl), showing Δ Em MPA-K at 9.5±1.1 %, with a reduction of almost 70 % of potassium effects. No effects on trans-plasma membrane depolarization due to K+ were registered by GN or DO. Simultaneous administration of GN with MPA (2:1) caused complete restoration of external K+ effect on mem- brane potential, as well as adding DO (1:1) (data not shown). 4. Conclusions Membrane depolarization caused by MPA (Rinaldelli et al., 2012) is a temporary effect. In fact, after 1 h of pre- incubation in MPA-BS solution, the trans-plasma mem- brane potential of the sample was not altered by the an- tiviral drug compared to the untreated control. Similarly, GN or DO did not change resting membrane potential. In- terference of signal steadiness caused by MPA at a higher concentration may be linked to cell toxicity induced by the antiviral drug (Panattoni et al., 2007; Luvisi et al., 2012 a). In fact, samples under stress conditions increase the difficulty of cell membrane measurements (Vuletic et al., 1987; Rawyler et al., 2002). With regard to effects induced by extracellular K+, pre- incubation tests showed how MPA at 1.0 mM or higher concentrations can interfere up to 10 mM of KCl. The MPA reduction of membrane depolarization caused by ex- tracellular K+ was confirmed by following tests in which chemical administration was maintained throughout all steps of measurement. The reduction of potassium effects was higher compared to administering MPA only in the preincubation step. This result is probably linked to the variation in MPA intra-/extracellular gradient due to solu- tion changes between preincubation and steps 1-2. Our results indicate that the antiviral MPA inhibited the effects of extracellular K+ in plants, through specific chan- nels. In fact, the MPA-induced alteration in membrane po- tential was sensitive to the K ATP channel opener DO, which hyperpolarized resting membrane potential in treated cells to a level similar to that achieved in control cells; results were also similar to those obtained in animal cells (Li et al., 2000). Moreover, GN was able to inhibit the MPA ac- tion against extracellular K+, suggesting that MPA could act also as an inward-rectifier potassium ion channel in- hibitor through the depletion of GN. Inhibition of the activity of K+ channels caused by MPA may be involved in programmed cell death (PCD) in Table 4 - Two-way factorial analysis of variance of Δ Em caused by chemical-K-BS (MPA-K-BS, GN-K-BS, DO-K-BS, KCl at 10 mM) on samples of Vitis vinifera cv. Sangiovese treaded chemical-BS (MPA-BS, GN-BS, DO-BS). Pairwise multiple comparison analysis with Holm-Sidak test was performed Source of Variation DF SS P Treatment (A) 2 0.836 <0.001 Concentration (B) 3 0.228 <0.001 A x B 6 0.696 <0.001 Residual 170 0.207 Total 181 1.961 Comparison for factor DM t P Comparison for A DO vs. MPA 0.149 23.461 <0.001 GN vs. MPA 0.139 21.880 <0.001 DO vs. GN 0.010 1.5888 NS Comparison for B within MPA 0.0 mM vs. 2.0 mM 0.284 22.276 <0.001 0.5 mM vs. 2.0 mM 0.250 19.586 <0.001 0.0 mM vs. 1.0 mM 0.231 18.160 <0.001 0.5 mM vs. 1.0 mM 0.197 15.470 <0.001 1.0 mM vs. 2.0 mM 0.052 4.116 <0.001 0.0 mM vs. 0.5 mM 0.034 2.690 NS Comparison for B within GN 2.0 mM vs. 0.5 mM 0.00543 0.433 NS 2.0 mM vs. 0.0 mM 0.00467 0.366 NS 1.0 mM vs. 0.5 mM 0.00276 0.220 NS 2.0 mM vs. 1.0 mM 0.00267 0.209 NS 1.0 mM vs. 0.0 mM 0.00200 0.157 NS 0.0 mM vs. 0.5 mM 0.00076 0.061 NS Comparison for B within DO 2.0 mM vs. 0.5 mM 0.0473 3.771 NS 2.0 mM vs. 0.0 mM 0.0447 3.505 NS 2.0 mM vs. 1.0 mM 0.0406 3.189 NS 1.0 mM vs. 0.5 mM 0.0067 0.531 NS 1.0 mM vs. 0.0 mM 0.0040 0.316 NS 0.0 mM vs. 0.5 mM 0.0026 0.210 NS BS= Basal solution; MPA = Mycophenolic acid; GN = Guanosine; DO = Diazoxide. 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