Red Light-Induced Phosphorylation of Plasma Membrane H+-ATPase in Stomatal Guard Cells

Abstract

Stomatal opening is stimulated by red and blue light. Blue light activates plasma membrane (PM) H⁺-ATPase by phosphorylating its penultimate residue, threonine, via a blue light photoreceptor phototropin-mediated signaling pathway in guard cells. Blue light-activated PM H⁺-ATPase promotes the accumulation of osmolytes and, thus, the osmotic influx of water into guard cells, driving stomatal opening. Red light-induced stomatal opening is thought to be dependent on photosynthesis in both guard cell chloroplasts and mesophyll cells; however, how red light induces stomatal opening and whether PM H⁺-ATPase is involved in this process have remained unclear. In this study, we established an immunohistochemical technique to detect the phosphorylation level of PM H⁺-ATPase in guard cells using whole leaves of Arabidopsis (Arabidopsis thaliana) and unexpectedly found that red light induces PM H⁺-ATPase phosphorylation in whole leaves. Red light-induced PM H⁺-ATPase phosphorylation in whole leaves was correlated with stomatal opening under red light and was inhibited by the plant hormone abscisic acid. In aha1-9, a knockout mutant of one of the major isoforms of PM H⁺-ATPase in guard cells, red light-dependent stomatal opening was delayed in whole leaves. Furthermore, the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibited red light-induced PM H⁺-ATPase phosphorylation as well as red light-induced stomatal opening in whole leaves. Our results indicate that red light-induced PM H⁺-ATPase phosphorylation in guard cells promotes stomatal opening in whole leaves, providing insight into the photosynthetic regulation of stomatal opening.

Figure 1.

Establishment of an immunohistochemical technique using whole leaves to detect PM H⁺-ATPase in guard cells. Phosphorylation (A) and amount of PM H⁺-ATPase (B) in guard cells in whole leaves from gl1 and phot1 phot2 are shown. Phosphorylation or amount of the protein was detected using anti-pThr or anti-H⁺-ATPase antiserum, respectively. Mature leaves harvested from dark-adapted plants were illuminated with red light (600 µmol m^–2 s^–1) for 30 min (R30) followed by blue light (5 µmol m^–2 s^–1) superimposed on red light for 2.5 min (R + B2.5) or kept in the dark (D30). Typical fluorescence and corresponding bright-field images (top) and quantification of the fluorescence intensities (bottom) are shown. Arrowheads indicate guard cells. Data represent means of relative values from three independent measurements with sd. A, Daggers denote that the mean is statistically significantly higher than D30 of gl1 set to 1. N.S., Not significant (one-tailed one-sample Student’s t test: ^†, P < 0.05; ^††, P < 0.01; ^†††, P < 0.005; and N.S., P = 0.516). The asterisk indicates that the mean of R + B2.5 is statistically significantly higher than that of R30 in gl1 (one-tailed Student’s test: *, P < 0.005). B, The means were not statistically significantly different from D30 of gl1 set to 1 (two-tailed one-sample Student’s t test: N.S., P > 0.45). Bars = 50 µm.

Figure 2.

Light-induced stomatal opening in whole leaves. A, Mature leaves harvested from dark-adapted Col-0 plants (0 min) were illuminated with red light (RL), both red and blue light (RB), or kept in the dark (Dk) for 60, 120, or 180 min. Light intensities were 600 µmol m^–2 s^–1 for red light and 5 µmol m^–2 s^–1 for blue light. Data represent means of representative values from three independent measurements with sd. Asterisks denote statistically significant increases in the mean compared with that of 0 min (one-tailed Dunnett’s test: *, P < 0.05; **, P < 0.01; and ***, P < 0.001). Hash signs indicate that the mean of RB is statistically significantly higher than that of RL at each time point (one-tailed Student’s t test: ^#, P < 0.05). B, Light-induced stomatal opening in whole leaves from gl1 and phot1 phot2. Mature leaves harvested from dark-adapted plants were illuminated with red light (R60), both red and blue light (RB60), or kept in the dark (D60) for 60 min. Light intensities were the same as in A. Data represent means of representative values from four independent measurements with sd. Different letters indicate statistically significant differences among means (Tukey’s test: P < 0.05).

Figure 3.

Initial responses to red light in guard cells in whole leaves. Mature leaves were harvested from dark-adapted plants (0 min) and kept in the dark (Dk) for 60 min or illuminated with red light (RL; 600 µmol m^–2 s^–1) for the indicated times. A, Immunohistochemical detection of the phosphorylation of PM H⁺-ATPase in guard cells using anti-pThr antiserum. Leaves were illuminated for 5, 10, 30, or 60 min. Typical fluorescence and bright-field images (top) and quantification of the fluorescence intensities (bottom) are shown. Arrowheads indicate guard cells. Data represent means of relative values from three independent measurements with sd. Daggers denote that the mean is statistically significantly higher than at 0 min set to 1. N.S., Not significant (one-tailed one-sample Student’s t test: ^†, P < 0.05; ^††, P < 0.005; and N.S., P > 0.07). Bar = 50 µm. B, Measurement of red light-induced stomatal opening. Leaves were illuminated for 15, 30, 45, or 60 min. Data represent means of representative values from three independent measurements with sd. Asterisks denote statistically significant increases of the mean compared with that of 0 min (one-tailed Dunnett’s test: *, P < 0.05; **, P < 0.01; ***, P < 0.005; and N.S., P = 0.717).

Figure 4.

Inhibition of the light-induced phosphorylation of PM H⁺-ATPase in guard cells and stomatal opening in whole leaves by the phytohormone ABA. Mature leaves harvested from dark-adapted plants were pretreated with 20 µm ABA dissolved in 0.1% (v/v) dimethyl sulfoxide (DMSO; +ABA) or 0.1% (v/v) DMSO alone (–ABA) for 30 min in the dark before light illumination. A, Immunohistochemical detection of the phosphorylation of PM H⁺-ATPase in guard cells using anti-pThr antiserum. Pretreated leaves were illuminated with red light (600 µmol m^–2 s^–1) for 30 min (R30) followed by blue light (5 µmol m^–2 s^–1) superimposed on red light for 2.5 min (R + B2.5) or kept in the dark (D30). Typical fluorescence and bright-field images (top) and quantification of the fluorescence intensities (bottom) are shown. Data represent means of relative values from four independent measurements with sd. Daggers denote that the mean is statistically significantly higher than D30 of –ABA set to 1. N.S., Not significant (one-tailed one-sample Student’s t test: ^†, P < 0.01; and N.S., P > 0.18). Asterisks indicate that the mean of R + B2.5 is statistically significantly higher than that of R30 within each treatment and that the mean of R + B2.5 of +ABA is statistically significantly lower than that of –ABA (one-tailed Student’s t test: *, P < 0.01; and **, P < 0.005). Bars = 50 µm. B, Measurement of light-induced stomatal opening under ABA treatment. Pretreated leaves were illuminated with red light (R60), both red and blue light (RB60), or kept in the dark (D60) for 60 min. Light intensities were the same as in A. Data represent means of representative values from four independent measurements with sd. Different letters indicate statistically significant differences among means (Tukey’s test: P < 0.05).

Figure 5.

Red light-induced stomatal opening in whole leaves from PM H⁺-ATPase mutants. Mature leaves from aha1-9 (A) or aha2-5 (B) and Col-0 were harvested from dark-adapted plants. The harvested leaves were kept in the dark (Dk) for 60 min or illuminated with red light (RL; 600 µmol m^–2 s^–1) for 15, 30, 45, or 60 min. Data represent means of representative values from three independent measurements with sd. A, Asterisks denote statistically significant decrements of the mean of aha1-9 compared with that of Col-0 at each time point. N.S., Not significant (one-tailed Student’s t test: *, P < 0.05; **, P < 0.001; and N.S., P > 0.07). B, The mean of aha2-5 was not statistically significantly lower than that of Col-0 at each time point (one-tailed Student’s t test: N.S., P > 0.6).

Figure 6.

Effects of the photosynthesis inhibitor DCMU on the light responses of guard cells in whole leaves. Mature leaves harvested from dark-adapted plants were pretreated with 10 µm DCMU dissolved in 0.1% (v/v) DMSO (+DCMU) or 0.1% (v/v) DMSO alone (–DCMU) for 30 min in the dark before light illumination. A, Immunohistochemical detection of the phosphorylation of PM H⁺-ATPase in guard cells using anti-pThr antiserum. Pretreated leaves were illuminated with red light (600 µmol m^–2 s^–1) for 30 min (R30) followed by blue light (5 µmol m^–2 s^–1) superimposed on red light for 2.5 min (R + B2.5) or kept in the dark (D30). Typical fluorescence and bright-field images (top) and quantification of the fluorescence intensities (bottom) are shown. Arrowheads indicate guard cells. Data represent means of relative values from five independent measurements with sd. Daggers indicate that the mean is statistically significantly higher than D30 of –DCMU set to 1. N.S., Not significant (one-tailed one-sample Student’s t test: ^†, P < 0.05; ^††, P < 0.001; and N.S., P > 0.58). Asterisks indicate that the mean of R + B2.5 is statistically significantly higher than that of R30 within each treatment and that the mean of R + B2.5 of +DCMU is statistically significantly lower than that of –DCMU (one-tailed Student’s t test: *, P < 0.05; **, P < 0.01; and ***, P < 0.005). Bars = 50 µm. B, Measurement of light-induced stomatal opening under DCMU treatment. Pretreated leaves were illuminated with red light (R60), both red and blue light (RB60), or kept in the dark (D60) for 60 min. Light intensities were the same as in A. Data represent means of representative values from four independent measurements with sd. Different letters indicate statistically significant differences among means (Tukey’s test: P < 0.05).

Figure 7.

Effects of Suc or CO₂ concentration on the phosphorylation of PM H⁺-ATPase in guard cells in isolated epidermis. Immunohistochemical detection of the phosphorylation of PM H⁺-ATPase in stomatal guard cells using anti-pThr antiserum is shown. Epidermal tissue was illuminated with red light (600 µmol m^–2 s^–1) for 30 min (R30) followed by blue light (5 µmol m^–2 s^–1) superimposed on red light for 2.5 min (R + B2.5) or kept in the dark (D30) with the indicated conditions. A, Epidermis was incubated without (–) or with 30 mm mannitol (Man) or Suc. B, Experimental basal buffer was aerated with ambient or soda lime-passed low-CO₂ air (CO₂ = 30–40 µL L⁻¹). The quantification of fluorescence intensities is shown. Data represent means of relative values from independent measurements (A, n = 4; B, n = 5) with sd. Daggers denote that the mean is statistically significantly higher than D30 of –sugar (A) or that of ambient (B) set to 1. N.S., Not significant (one-tailed one-sample Student’s t test: ^†, P < 0.05; N.S. [A], P > 0.08; and N.S. [B], P > 0.10). Asterisks indicate that the mean of R + B2.5 is statistically significantly higher than that of R30 with a corresponding control condition (one-tailed Student’s t test: *, P < 0.01).