Calcium-dependent protein kinase CPK6 positively functions in induction by yeast elicitor of stomatal closure and inhibition by yeast elicitor of light-induced stomatal opening in Arabidopsis

Abstract

Yeast elicitor (YEL) induces stomatal closure that is mediated by a Ca(2+)-dependent signaling pathway. A Ca(2+)-dependent protein kinase, CPK6, positively regulates activation of ion channels in abscisic acid and methyl jasmonate signaling, leading to stomatal closure in Arabidopsis (Arabidopsis thaliana). YEL also inhibits light-induced stomatal opening. However, it remains unknown whether CPK6 is involved in induction by YEL of stomatal closure or in inhibition by YEL of light-induced stomatal opening. In this study, we investigated the roles of CPK6 in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening in Arabidopsis. Disruption of CPK6 gene impaired induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening. Activation by YEL of nonselective Ca(2+)-permeable cation channels was impaired in cpk6-2 guard cells, and transient elevations elicited by YEL in cytosolic-free Ca(2+) concentration were suppressed in cpk6-2 and cpk6-1 guard cells. YEL activated slow anion channels in wild-type guard cells but not in cpk6-2 or cpk6-1 and inhibited inward-rectifying K(+) channels in wild-type guard cells but not in cpk6-2 or cpk6-1. The cpk6-2 and cpk6-1 mutations inhibited YEL-induced hydrogen peroxide accumulation in guard cells and apoplast of rosette leaves but did not affect YEL-induced hydrogen peroxide production in the apoplast of rosette leaves. These results suggest that CPK6 positively functions in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening in Arabidopsis and is a convergent point of signaling pathways for stomatal closure in response to abiotic and biotic stress.

Figure 1.

Induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening in the wild type and cpk6-1 and cpk6-2 mutants. A, YEL-induced stomatal closure in the wild type (WT) and cpk6-1 and cpk6-2 mutants. B, Inhibition by YEL of light-induced stomatal opening in the wild type and cpk6-1 and cpk6-2 mutants. Averages from three independent experiments (90 total stomata per bar) are shown. Error bars represent ses (n = 3).

Figure 2.

YEL activation of I_Ca currents in wild-type and cpk6-2 GCPs. A, I_Ca currents in wild-type (WT) GCPs treated without YEL (top trace) or with 50 µg mL^–1 YEL (bottom trace). B, I_Ca currents in cpk6-2 GCPs treated without YEL (top trace) or with 50 µg mL^–1 YEL (bottom trace). C, Current-voltage relationship for YEL activation of I_Ca currents in wild-type GCPs (n = 5) as recorded in A (white circles, control; black circles, 50 µg mL^–1 YEL). D, Current-voltage relationship for YEL activation of I_Ca currents in cpk6-2 GCPs (n = 5) as recorded in B (white circles, control; black circles, 50 µg mL^–1 YEL). A ramp voltage protocol from +20 to –180 mV (holding potential, 0 mV; ramp speed, 200 mV s^–1) was used. After making whole-cell configuration, GCPs were recorded 16 times to get averages for control. After adding YEL extracellularly, the GCPs were recorded for 16 times to get averages for the YEL treatment. The interpulse period was 1 min.

Figure 3.

YEL-induced transient [Ca²⁺]_cyt elevations in wild-type and cpk6-2 guard cells expressing YC3.6. A, A representative trace of fluorescence emission ratios (535/480 nm) showing 50 µg mL^–1 YEL-induced transient [Ca²⁺]_cyt elevations in wild-type (WT) guard cells. B, A representative trace of fluorescence emission ratios (535/480 nm) showing 50 µg mL^–1 YEL-induced transient [Ca²⁺]_cyt elevations in cpk6-2 guard. C, Percentage of number of guard cells showing different number of transient [Ca²⁺]_cyt elevations in wild-type and cpk6-2 guard cells. [Ca²⁺]_cyt elevations were counted when changes in fluorescence emission ratios were more than or equal to 0.1 from the baseline.

Figure 4.

YEL activation of S-type currents in wild-type (WT) and cpk6-2 GCPs. A, S-type currents in wild-type GCPs treated without (top trace) or with 50 µg mL^–1 YEL (bottom trace). B, S-type currents in cpk6-2 GCPs treated without (top trace) or with 50 µg mL^–1 YEL (bottom trace). C, Steady-state current-voltage relationship for YEL activation of S-type currents in wild-type GCPs as recorded in A (white circles, control; black circles, YEL). D, Steady-state current-voltage relationship for YEL activation of S-type currents in cpk6-2 GCPs as recorded in B (white circles, control; black circles, YEL). The voltage protocol was stepped up from +35 mV to –145 mV in 30-mV decrements (holding potential, +30 mV). GCPs were treated with YEL for 2 h before recordings. Each datum point was obtained from five GCPs. Error bars represent ses.

Figure 5.

YEL inhibition of K_in currents in wild-type (WT) and cpk6-2 GCPs. A, K_in currents in wild-type GCPs treated without (top trace) or with 50 µg mL^–1 YEL (bottom trace). B, K_in currents in cpk6-2 GCPs treated without (top trace) or with 50 µg mL^–1 YEL (bottom trace). C, Steady-state current-voltage relationship for YEL inhibition of K_in currents in wild-type GCPs as recorded in A (white circles, control; black circles, YEL). D, Steady-state current-voltage relationship for YEL inhibition of K_in currents in cpk6-2 GCPs as recorded in B (white circles, control; black circles, YEL). The voltage protocol was stepped up from 0 to –200 mV in 20-mV decrements (holding potential, –40 mV). GCPs were treated with YEL for 2 h before recordings. Each datum point was obtained from at least seven GCPs. Error bars represent ses.

Figure 6.

YEL-induced H₂O₂ production in apoplast of leaf tissues and H₂O₂ accumulation in apoplast of leaf tissues and guard cells. A, H₂O₂ production induced by 50 µg mL^–1 YEL in apoplast of leaf tissues of the wild type (WT) and cpk6-2 mutant. B, Total and maximal H₂O₂ production induced by YEL as recorded in A. The luminescence was measured between 1 and 20 min after adding YEL, where the luminescence at each sampling point was integrated for 2 s. “Total RLU” is the sum of luminescence between 1 and 20 min, and “max RLU” is the highest luminescence. C, H₂O₂ accumulation induced by 50 µg mL^–1 YEL in the apoplast of leaf tissues of the wild type and cpk6-2 mutant. D, H₂O₂ accumulation induced by 50 µg mL^–1 YEL in guard cells of the wild type and cpk6-2 mutant. H₂O₂ accumulation was expressed as the percentage of 2',7'-dichlorofluorescein (DCF) fluorescence levels. Averages from three independent experiments (more than 150 total guard cells per bar in total) are shown.