Inward potassium channel in guard cells as a target for polyamine regulation of stomatal movements

Abstract

A number of studies show that environmental stress conditions such as drought, high salt, and air pollutants increase polyamine levels in plant cells. However, little is understood about the physiological function of elevated polyamine levels. We report here that polyamines regulate the voltage-dependent inward K(+) channel in the plasma membrane of guard cells and modulate stomatal aperture, a plant "sensor" to environmental changes. All natural polyamines, including spermidine, spermine, cadaverine, and putrescine, strongly inhibited opening and induced closure of stomata. Whole-cell patch-clamp analysis showed that intracellular application of polyamines inhibited the inward K(+) current across the plasma membrane of guard cells. Single-channel recording analysis indicated that polyamine regulation of the K(+) channel requires unknown cytoplasmic factors. In an effort to identify the target channel at the molecular level, we found that spermidine inhibited the inward K(+) current carried by KAT1 channel that was functionally expressed in a plant cell model. These findings suggest that polyamines target KAT1-like inward K(+) channels in guard cells and modulate stomatal movements, providing a link between stress conditions, polyamine levels, and stomatal regulation.

Figure 1

Effect of spermidine on stomatal movements. Epidermal peels were obtained from 4-week-old V. faba leaves. Stomatal pore size was measured after 2-h incubation in peel solution containing 0, 0.1, 0.5, 1.0, 3.0, and 6.0 mm spermidine. For stomatal opening assay (A), peels were collected from the plants before light cycle, and the initial stomatal pore size was typically in the range of 3.7 ± 0.6 μm. After 2-h illumination, the pore size reached 14.0 ± 2.7 μm in the control solution without spermidine. For stomatal closure assay (B), peels were collected from the plants 4 h after light cycle, and the initial pore size was 12.4 ± 0.6 μm. The data in both A and B are presented as mean ± se from seven individual experiments.

Figure 2

Regulation of stomatal aperture by natural polyamines. At various concentrations, effects on stomatal aperture of three polyamines (spermine, ○; putrescine, □; and cadaverine, ▵) were compared. The procedures of stomatal opening assay (A) and closure assay (B) are as described in Figure 1. All polyamines were used at concentrations of 0.1, 0.5, 1.0, 3.0, and 6.0 mm and presented in common log scale. Seven individual experiments were performed and the data were presented as mean ± se.

Figure 3

Effect of spermidine on I_Kin in guard cells. In V. faba guard cell protoplasts, whole-cell patch-clamp recordings revealed the typical I_Kin current (B), I_Kin current with 1 mm spermidine in pipette solution (C), and with 1 mm spermidine in bath solution (D). During the recordings, the holding potential was −50 mV, the currents were recorded at the membrane potentials from −20 to −160 mV with increment of −20 mV (A), and the current and time scales for B, C, and D are indicated by vertical and horizontal bars shown in D. The current amplitudes (mean ± se) from 14 control cells (○) and 18 cells treated with 1 mm spermidine in the pipette solution (●) are presented as current-voltage (I–V) curves in E. All data shown here were collected from whole-cell configuration with 2.5 GΩ or higher seal resistance. Spd, Spermidine.

Figure 4

Dose response of I_Kin to spermidine. The responses of I_Kin to different concentrations of spermidine in the pipette solution are shown as I–V curves (control, n = 14; 0.1 mm, n = 9; 0.5 mm, n = 12; 1 mm, n = 18; and 5 mm, n = 7). The same voltage protocol was used as in Figure 3. The data were presented as mean ± se.

Figure 5

Effects of natural polyamines on I_Kin. At −160 mV, the whole-cell current amplitudes in the presence of 1 mm tested compounds are shown as percentage of the control. CTL, control, n = 14; BUT, butylamine, n = 9; PUT, putrescine, n = 8; CAD, cadaverine, n = 9; SPD, spermidine, n = 18; SPM, spermine, n = 11. Asterisks indicate significant differences as compared with the control (P < 0.05).

Figure 6

Effect of spermidine on single-channel current of I_Kin in guard cells. The inside-out patch was obtained from V. faba guard cell protoplasts, and single-channel current of I_kin was recorded in the control bath solution for 30 s. Then the bath chamber was perfused with the solution containing 1 mm spermidine. The single-channel recording was performed again for 30 s after perfusion for 5 min. The current traces from a representative patch are shown in A before (control) and after perfusion of 1 mm spermidine. During the recording, the membrane potential was held at −100 mV. The dotted lines indicate the closing state and different opening levels. In this patch, there are at least 4 channels opening to hyperpolarized membrane potential. The analysis of P_o for this patch is shown in B. Six patches have been studied upon spermidine application. The average single-channel conductance is 13.83 ± 0.43 pS, which is not significantly different from the control (13.12 ± 0.54 pS, P > 0.05).

Figure 7

Effect of spermidine on KAT1 channel activity in a plant cell model. Whole-cell patch-clamp recording was performed on the tobacco mesophyll cells over-expressed with KAT1 gene (for details, see “Materials and Methods”). I_Kin was recorded in the cells from vector-only transformed plant (A), KAT1-expressing plant (B), and KAT1-expressing plant with 1 mm spermidine in the pipette solution during the recording (C). The current shown here was elicited at the membrane potential from −40 to −180 mV, whereas the holding potential was kept at −50 mV (D). E, I–V curve summarizing the currents from the control cells (○), KAT1-expressing cells (●), and KAT1-expressing cells with 1 mm spermidine (□). The data presented as mean ± se were collected from more than 10 individual cells in each group. The currents were normalized to pA per pF membrane capacitance.

Figure 8

HPLC analysis of polyamines in V. faba leaves. The standard HPLC procedure was used to measure polyamine levels in the extracts from 3- to 4-week-old V. faba leaves. Four analogs of natural polyamines were assayed, and an unnatural polyamine, hexanediamine, was added as an internal standard. The recording traces in A show the peaks of polyamine standards (upper trace), extracts of normal plant (middle trace), and extracts of drought-treated plant (lower trace). The calculated polyamine concentrations from three individual experiments were presented as mean ± se in micromolars per gram fresh weight and plotted in B. PUT, Putrescine; CAD, cadaverine; HDA, hexanediamine; SPD, spermidine; SPM, spermine.