Evaluation of the open time of calcium channels at variation potential generation in wheat leaf cells

Abstract

The role of ions in the generation and mechanism of propagation of variation potential (VP) has been widely investigated. It is likely that Ca²⁺ influx via calcium channels is an initial stage of VP; however, development of long-term membrane depolarization requires prolonged open times of calcium channels. We investigated depolarization time in the present study. It was shown that local burning induced VP in wheat seedling and the electrical response was suppressed under EGTA presence. Depolarization formation, which may indicate open time of calcium channels at VP generation, was observed up to 30 s after reaction induction when calcium ions were added to initially calcium-free medium. Long-term calcium channel open time may be the reason for long membrane depolarization at VP and may also be connected with the type of channels participating in wound reaction propagation.

Abbreviations

AP

action potential

APW

artificial pond water

EGTA

ethylene glycol tetraacetic acid

E_m

transmembrane electrical potential

VP

variation potential

Introduction

Coordination of plant responses to external wounding is based on activation of a number of signaling systems. The electrophysiological system plays a leading role in the coordination of wound reactions; however, little is known about its functioning. In particular, the process of variation potential generation (VP), defined as slow transitional depolarization of cell membranes extending out to the zone of local damaging irritation^1-3, is poorly understood. According to the literature, variation potential is not a self-propagating signal, rather it is a local electrical reaction, induced by signal transmission from the wounded area¹. The signal can be either a hydraulic wave or a chemical agent^4-7. The ionic nature of changes in membrane potential at VP remains almost unknown. The main hypothesis about the mechanism of the wound-induced electric reaction generation is that it is caused by a temporary inactivation of the plasma membrane's proton pump.^6,8-10 Passive fluxes of Ca²⁺, Cl⁻ and K⁺ may also play a role in variation potential formation^11-13. It is probable that calcium ion influx is a key component in the process of VP generation in cells. Increase in the intracellular concentration of free calcium leads to inactivation of the proton pump and activation of anionic channels.^8,11-13 The role of calcium ions as inductors of electric reaction development was also shown for another type of the plant cell electric signal – the action potential (AP).¹⁴ In comparison to AP, cell depolarization at VP is much longer, most likely caused by a more lengthy duration of plasma membrane proton pump inactivation. The reason for the long-term inactivation of H⁺-ATPase may be based on long-term increase in the content of calcium in cytosol that probably is connected with the open times of calcium channels.

Accordingly, the objective of this study was to elucidate the participation of calcium ions’ fluxes in VP initiation and to estimate the open time of calcium channels in the electrical reaction system.

Results and Discussion

Electrical activity was registered in parenchymal cells of wheat leaves after wounding. Leaf tip burning led to generation of variation potential with amplitude of 75 ± 4 mV (Fig. 1). Parameters of VP measured in wheat leaf cells were comparable with VPs registered in other higher plants.^{2,8,10-11,15-16}

Figure 1.

Average dynamics of the variation potential in wheat leaf cells (n = 10). Gray shade indicates standard error of the values. The distance between wounding zone and E_m measurement was approximately 5 cm. Initiation of the burn wound is indicated by the black arrow.

In calcium-free medium, VP amplitude decreased considerably, equaling 13 ± 8% of the control value (Fig. 2a). VP generation was also suppressed in the presence of La³⁺ (Fig. 2b) or Gd³⁺ (Fig. 2c). Depolarization amplitudes were 4 ± 1% and 14 ± 1% (at La³⁺ and Gd³⁺, respectively). These results show that VP generation required Ca²⁺ influx from external medium and that VP is probably connected with activation of calcium channels.

Figure 2.

Typical records of changes of membrane potential of wheat leaf cells at the leaf tip burn in presence of EGTA (1 mM) (A) and calcium channel blockers: lanthanum chloride (5 mM) (B) and gadolinium chloride (5 mM) (C) (n = 5–7). The distance between wounding zone and E_m registration was approximately 5 cm. Initiation of the burn wound is indicated by the black arrow.

Our results are in agreement with data from the literature that showed that blocking of Ca²⁺-channels by gadolinium or by lanthanum led to the decrease in amplitude of the electric reaction following wounding in different plants.^2,8 Participation of calcium influx in VP generation was also shown in experiments with EGTA, where incubation of plant seedlings in the presence of calcium chelator led to a decrease in the amplitude of VP induced by a burn.^8,11-12 Replacement of calcium ions with magnesium ions in the solution surrounding tomato stems caused reduction in the quantity of fast electrical reaction impulses following a burn wound.¹⁷

We conclude that calcium influx from extracellular spaces is necessary for VP development. Calcium ions serve as the inductor of VP formation, activating anionic channels and inactivating proton pumps.^8,13 A similar role for calcium has been shown for the formation of action potential, another type of electrical reaction. The long-term duration of membrane depolarization at VP in comparison with AP is connected to the long inactivation of H⁺ -ATPase, which can be caused by elongated prolonged entrance of calcium ions into cells, i.e., long open time of calcium channels.

The classical way to analyze characteristics of ionic channels is with a patch-clamp method.¹⁸ However, use of whole plants as the objects of the research has a number of methodological difficulties, including the presence of cell walls, small size cells and their integration in the symplast. In this regard, the indirect way of an assessment of the open time of calcium channels was offered. Electric reaction was induced by a leaf burn in the calcium-free medium and then Ca²⁺ was added to the solution at various time intervals after the irritation.

In the absence of the burn wound stimulus, increasing at calcium concentration increasing in the washing solution to standard values (10⁻³ M) membrane depolarization was not observed; on the contrary, there was a slight hyperpolarization (8 ± 4 mV) (Fig. 3).

Figure 3.

Typical record of changes of membrane potential of wheat leaf cells upon addition of calcium ions to the initially calcium-free medium. Final concentration of the calcium ions in the washing solution was 10⁻³ M (n = 4). The moment of calcium ions addition is indicated by the gray arrow.

Calcium ions were added to the medium after 4 different time intervals after VP induction and led to the development of membrane depolarization (Fig. 4). This effect was observed with addition of calcium ions at 10, 20, and 30 s after wounding. At 40 s, calcium addition to the medium did not lead to noticeable changes in electric activity of cells. It should also be noted that VP amplitude depended on the time between reaction induction and calcium addition, i.e., increased time interval led to decreased VP amplitude.

Figure 4.

Changes of membrane potential of wheat leaf cells induced by leaf tip burn in the calcium-free medium followed by addition of calcium ions in the washing solution on different time intervals after wounding. (A) Typical records. (B) Average magnitudes (n = 4–6). The distance between the wounding zone and E_m measurement was approximately 5 cm. Initiation of the wound is indicated by the black arrow. The moment of addition of calcium ions is indicated by the gray arrow.

These results indicate long-term (at least 30 s) influx of calcium ions to the cytoplasm at VP. The long-term influx can be caused by large open time of calcium channels participating in VP generation. However, we cannot exclude an alternative explanation for our results, in which long-term Ca²⁺ influx reflects prolonged action of VP-induced signals and repeated activation of calcium channels. This signal could be a hydraulic wave or caused by chemical agent propagation.^4-7 If the signal is hydraulic, long-term Ca²⁺ influx could be caused by prolonged mechanical deformation, which activates mechanosensitive channels. If it has a chemical basis, the long-term influx reflects prolonged increase of the chemical agent concentration, which can activate ligand-dependent channels.

Conclusion

We conclude from our results that there is evidence showing that the prolonged depolarization of a membrane at VP, in contrast to a membrane at AP, is caused by the long open time of calcium channels and, as a result, concomitant increase in cytosol calcium concentration. The duration (about 30 s) of calcium channels being open at VP can be connected with participation of ligand-activated or mechanosensitive ionic channels in the reaction development. This is in contrast to fast potential-dependent calcium channels, in which activation takes place with AP formation.

Materials and Methods

Seedlings of wheat (Triticum aestivum L.) were cultivated hydroponically in a Binder KBW 240 (Binder GmbH, Tuttlingen, Germany) plant growth chamber at 24°C under a 16/8 h (light/dark) photoperiod. Seedlings used in experiments were 14–21 d-old with a second leaf length of 15–20 cm.

The microelectrode technique was used for intracellular membrane potential (E_m) measurements (microscope Olympus BX51); automatic manipulators Luigs and Neumann; amplifier Multiclamp 700B (Axon Inst., Molecular Devices, Sunnyvale, CA, USA); PC).¹² Data acquisition speed was 3 points/s and low pass filter frequency was 10 Hz. The plant was placed beside the microscope and the leaf was fixed horizontally. Part of the leaf was placed in a cuvette filled with artificial pond water (APW), containing 0.1 mМ NaCl, 1 mМ KCl, and 0.5 mМ CaCl₂ in distilled water (pH = 6–7). In separate experiments, APW in the cuvette was changed to solutions with calcium chelator or calcium channel blockers as described below.

Micropipettes were fabricated from 1.2 mm (O.D.) borosilicate glass tubing (Sutter Instruments, Novato, CA, USA). Capillaries were pulled in a horizontal pipette puller and filled with 100 mM KCl. The tip diameter was approximately 0.5 μm. The electrode was moved by motorized manipulators, with its location controlled visually on the microscope. Electrical measurements were carried out in parenchymal cells in the 2^nd–3^rd layer below the epidermis. The reference electrode was immersed into cuvette with APW. The VP was induced by open-flame burning of the leaf tip (apical 2–3 mm) for 2 s.⁷ Distance between the registration zone and wounded area was 5 cm.¹²

Measurements of VP parameter changes after exposure to calcium chelator (EGTA) (1 mM) and calcium channel blockers lanthanum chloride (5 mM) and gadolinium chloride (5 mM) were used for estimation of calcium ion participation in VP generation.^8,11,12 Plants were incubated in the presence of the inhibitor for at least 1 h prior to wounding.

For estimation of calcium channel open time at VP generation, the seedling was incubated in the presence of calcium chelator (1 mM) for 1 h. VP was induced in the calcium-free media and then at different time intervals—10, 20, 30 and 40 s—after wounding, calcium ions (CaCl₂, 10⁻³ M) were added to the cuvette.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by the Russian Scientific Fund (Project No. 14-26-00098).