Figure 1.
BL-dependent stomatal opening enhanced by RL-activated guard cell photosynthesis.
A–D, BL-induced stomatal opening in isolated epidermal tissues of Arabidopsis. A, Stomatal aperture after 2 h incubation in darkness (Dark) or under BL at various fluence rates (B2, B5, B10, and B20 represent 2, 5, 10, and 20 µmol m−2 s−1 of BL, respectively). B, Stomatal aperture after 2 h incubation in darkness or under BL (B5) superimposed on RL at various fluence rates (R15, R55, R100, and R300 represent 15, 55, 100, and 300 µmol m−2 s−1 of RL, respectively). C, Stomatal aperture after 2 h incubation in darkness or under 5 µmol m−2 s−1 BL (B5), 60 µmol m−2 s−1 RL (R60), or 5 µmol m−2 s−1 BL plus 55 µmol m−2 s−1 RL (R55+B5). D, Stomatal aperture after 2 h incubation in darkness or under simultaneous irradiation with BL and RL (R55+B5), with or without DCMU. Data shown are means ± SE of four (A) or three (B–D) independent experiments. For each light condition in an experiment, the apertures of 45 stomata were determined. Asterisks indicate the statistically significant differences compared to Dark control (without DCMU in D), assessed by Student's t-tests (*: 0.05<P<0.1, **: 0.01<P<0.05, ***: P<0.01).
Figure 2.
BL-dependent H+-pumping enhanced by RL-activated guard cell photosynthesis.
A–C. H+-pumping induced by the irradiation with BL with (B and C, [R55+B5]) or without RL irradiation (A, [B5]) in wild-type guard cell protoplasts. Guard cell protoplasts (50 µg protein) were irradiated with 5 µmol m−2 s−1 BL with or without 2 to 3 h pre-irradiation and background irradiation with 55 µmol m−2 s−1 RL. DCMU (dissolved in DMSO) was administered to the protoplasts before pre-irradiation with RL at a final concentration of 10 µM (0.01% DMSO) (C). D, Maximum rates of H+-pumping by guard cell protoplasts under different light conditions as in A to C. Data represent means ± SD of three independent experiments. E–I. pH changes induced by irradiation with BL or RL in wild-type (E, G, and I) and phot1phot2 (F and H) guard cell protoplasts. Guard cell protoplasts (50 µg protein) were dark-adapted for 1 h before 5 µmol m−2 s−1 BL (E, F, and I [B5]) or 60 µmol m−2 s−1 RL (G and H [R60]) was applied where indicated by arrows. DCMU was administered before dark-adaptation at 10 µM (I).
Figure 3.
BL-induced phosphorylation of H+-ATPase in wild-type guard cell protoplasts.
Guard cell protoplasts (60 µg protein) were irradiated with 5 µmol m−2 s−1 BL with or without 2 h pre-irradiation and background irradiation with 55 µmol m−2 s−1 RL (R55+B5 and B5, respectively). DCMU (dissolved in DMSO) was administered before pre-irradiation with RL at 10 µM (0.01% DMSO). In samples collected at 0, 3, 5, and 10 min after BL exposure, the phosphorylation status of the H+-ATPase was determined by protein blotting with a 14-3-3 protein. Each lane contained 5 or 7 µg guard cell proteins for protein blotting (upper panels) and immunoblotting (lower panels). A, Representative example of similar results obtained in five independent experiments. B, Quantification of the binding of 14-3-3 proteins to the H+-ATPase. The 14-3-3 binding before or 3 min after BL exposure was determined. Values presented are means of five independent experiments with SDs. Asterisks indicate the statistically significant differences, assessed by Student's t-tests (**: 0.01<P<0.05, ***: P<0.01). n.s. indicates the statistically insignificant difference (P>0.43).
Figure 4.
Light-induced increases of photosynthetic rate and stomatal conductance in intact leaves of Arabidopsis.
A, Fluence rate-dependency of photosynthetic rate and stomatal conductance in response to RL. B and C, Fluence rate-dependency of BL-induced changes in stomatal conductance in response to RL. D and E, Repeated BL-induced changes in stomatal conductance in a control leaf (D) and a DCMU-treated leaf (E). Photosynthetic rate and stomatal conductance are indicated by red and blue traces, respectively. The timing of fluence rate increases (red and blue arrows) and light-off (black arrows) is indicated next to the corresponding track. The RL irradiation scheme is shown on top of each graph. The irradiation with BL at 5 µmol m−2 s−1 lasted for 10 min in all cases (light-off markers were omitted for clarity).