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Previous studies have suggested a role for jasmonates in the promotion of stomatal closure (Raghavendra and Reddy, 1987; Gehring et al., 1997). This report describes whole-cell patch-clamp experiments that demonstrate that methyl jasmonate (Me-JA) has concentration-dependent effects on guard cell K⁺ channels and promotes stomatal closure by biasing potassium transport at the plasma membrane for K⁺ efflux.

Stomatal closure is brought about by a reduction in guard cell solutes. Potassium salts are abundant in guard cells (Outlaw, 1983) and many regulators of stomatal movements, such as ABA, exert their activity, at least in part, through the modulation of potassium fluxes (MacRobbie, 1981; Blatt, 1990). Concentrations of jasmonates increase in response to stresses that alter water status and necessitate a change in stomatal aperture (Creelman and Mullet, 1995; Lehmann et al., 1995). In the majority of species investigated, jasmonates have been implicated in the control of stomatal movements by their effects on transpiration (Leshem et al., 1994; Wang, 1999), gas exchange (Lee et al., 1996; Herde et al., 1997), and stomatal aperture (Raghavendra and Reddy, 1987; Gehring et al., 1997). However, little is known about the mechanisms by which jasmonates affect stomatal behavior.

To investigate the effect of Me-JA on guard cell plasma membrane K⁺ channels in patch-clamp experiments, Vicia faba guard cell protoplasts were prepared by enzymatic digestion (Lemtiri-Chlieh, 1996). Protoplasts were perfused with 10 mM KCl, 1 mM CaCl₂, 2 mM MgCl₂, 5 mM MES, and sometimes 20 mM potassium Glu (pH 5.5, 480 or 520 mOsmol kg¹). Patch pipettes prepared from Kimax-51 glass (Kimble/Kontes, Vineland, NJ) contained 200 mM potassium Glu, 2 mM K₂ATP, 2 mM MgCl₂, and 10 mM HEPES buffer (pH 7.6-8, 520 mOsmol kg¹). Once the whole-cell configuration was obtained, the membrane was clamped close to K⁺ equilibrium potential (E_K) and no current measurements made for 2 min. Recordings were only made when the seal resistance exceeded 1 G. Currents were monitored using a Dagan 3900A or Axopatch 200A Integrating Patch Clamp Amplifier (Dagan Corporation, Minneapolis; Axon Instruments Inc., Union City, CA). Voltage commands were given and signal recordings assessed by microcomputer using pClamp 6.0 software (Axon Instruments Inc.). The microcomputer and amplifier were connected via an input/output device and current traces low pass filtered at 5 kHz before analog to digital conversion. Liquid junction potentials were corrected for as described by Neher (1992). Me-JA (Duchefa Biochemie, Haarlem, The Netherlands) was a racemic mixture and solubilized in ethanol. Student's t tests and analysis of variance were performed to compare current characteristics and channel conductance before and after Me-JA application.

Me-JA was added to the bath solution at three different concentrations (0.1, 1, and 50 µM). At 0.1 µM, Me-JA led to an increase in the magnitude of the outward potassium current (IK_out) and a decrease in the magnitude of the inward potassium current (IK_in; Fig. 1). These changes in current magnitude reflected significant changes in the chord conductance of K_out and K_in (K_out, 64 mV: P = 0.02; K_in, 196 mV: P = 0.001) as illustrated in Table I, which contains mean relative conductance values for both channels (the ratio of the conductance post- to prior Me-JA application). No voltage sensitivity of either channel to 0.1 µM Me-JA could be detected within the voltage ranges described in Table I (K_out: P = 0.91; K_in: P = 0.735).

View larger version (31K): [in this window] [in a new window]   Figure 1.   Effect of 0.1 µM Me-JA on IKout and IKin. Data from a typical guard cell protoplast. a, Electrical recordings made in response to a series of 600-ms voltage pulses (above) ranging in 20 mV steps from 64 to 196 mV (holding potential 76 mV). Recordings were made before (left) and at the times indicated after Me-JA had been added to the bath. Scale: vertical, 250 pA; horizontal, 200 ms. b, IKout and IKin (the time-dependent component of the current traces in a) plotted as a function of membrane potential. Cross-referenced to part a by symbol.

In the majority of experiments (16 of 20), the effect of 0.1 µM Me-JA on IK_out and IK_in was detected within 15 min of exposure. This is illustrated in Figure 1 where electrical recordings made from a typical guard cell protoplast are shown. Ten minutes after the addition of 0.1 µM Me-JA, IK_out has clearly already increased. In this example, K_in is slower to respond than K_out, but a decrease in inward current at 176 and 196 mV can be detected 20 min after the addition of the growth regulator.

The activation and deactivation kinetics of both currents were examined after the addition of 0.1 µM Me-JA (Tables II and III). Although the activation time constant for IK_out was voltage dependent (P = <0.001), no effect of 0.1 µM Me-JA on the activation time constant for either current could be detected (K_out: P = 0.786; K_in: P = 0.572). Deactivation kinetics were examined after the return of the membrane potential to the holding potential from a series of activating voltage pulses. There was no significant effect of Me-JA on any of the deactivation time constants (P  0.1), regardless of the magnitude of the conditioning voltage step (data not shown, P  0.26).

The only other concentration of Me-JA tested to have a significant effect was 50 µM Me-JA (Table I). This concentration of Me-JA led to a significant reduction in the steady-state conductance of K_out (Table I; P = 0.01). Considering the effect of 0.1 µM Me-JA on this conductance, the data suggest Me-JA may have a bimodal effect on K_out. However, jasmonates have not been reported to have a corresponding dual effect on stomatal movements and unlike the other concentrations tested, 50 µM Me-JA is vastly in excess of estimates of jasmonate concentrations in planta (Creelman and Mullet, 1997). Pollen and seed germination, root growth, and cell division have all been reported to show a bimodal response to jasmonates when the concentrations tested have included those in excess of the physiological and the significance of the generally inhibitory effect of such supraoptimal concentrations is unclear.

The changes in the chord conductance of K_out and K_in evoked by 0.1 µM Me-JA would increase the capacity for K⁺ efflux across the guard cell plasma membrane and decrease the capacity for K⁺ influx. This suggests that Me-JA is capable of promoting a net loss of potassium from the guard cell cytoplasm and by this mechanism, stomatal closure. This is consistent with the findings of Raghavendra and Bhaskar Reddy (1987), who reported a significant reduction in the potassium content of isolated Commelina benghalensis guard cells after a 4-h incubation in 1 µM Me-JA. It has also been suggested that the ability of jasmonic acid to accelerate pulvinar movements in Cassia fasciculata (Bourbouloux et al., 1994) and inhibit light-induced turgor increases in Mimosa pudica pulvini (Tsurumi and Asahi, 1985) may result from comparable effects on K⁺ channels in these cells.

Single-channel studies are now required to determine if Me-JA directly affects the conductance of K_out and K_in or whether additional signal transduction components are involved. Cytosolic alkalinization precedes jasmonate-induced stomatal closure in Paphiopedilum spp. and acid loading of the guard cells eliminated this response (Gehring et al., 1997). This would suggest some overlap between jasmonate and abscisic acid signal transduction pathways (Blatt and Armstrong, 1993). The likely involvement of cytosolic calcium is entirely speculative in the absence of any measurements of jasmonate-induced increases in cytosolic calcium in guard cells. However, calcium has been implicated in other jasmonate-dependent processes (Leon et al., 1998; Kenton et al., 1999) and jasmonates are known to alter the activity of at least one calcium transport protein in mammalian systems (Starling et al., 1994).

The ability of Me-JA to alter the properties of potassium conductances in the guard cell plasma membrane has now been demonstrated. Aims for the future must be to investigate how these effects of Me-JA are mediated and under what conditions such control is important.