Guard Cell Chloroplasts Are Essential for Blue Light-Dependent Stomatal Opening in Arabidopsis

Blue light (BL) induces stomatal opening through the activation of H[+]-ATPases with subsequent ion accumulation in guard cells. In most plant species, red light (RL) enhances BL-dependent stomatal opening. This RL effect is attributable to the chloroplasts of guard cell, the only cells in the epidermis possessing this organelle. To clarify the role of chloroplasts in stomatal regulation, we investigated the effects of RL on BL-dependent stomatal opening in isolated epidermis, guard cell protoplasts, and intact leaves of Arabidopsis thaliana. In isolated epidermal tissues and intact leaves, weak BL superimposed on RL enhanced stomatal opening while BL alone was less effective. In guard cell protoplasts, RL enhanced BL-dependent H[+]-pumping and DCMU, a photosynthetic electron transport inhibitor, eliminated this effect. RL enhanced phosphorylation levels of the H[+]-ATPase in response to BL, but this RL effect was not suppressed by DCMU. Furthermore, DCMU inhibited both RL-induced and BL-dependent stomatal opening in intact leaves. The photosynthetic rate in leaves correlated positively with BL-dependent stomatal opening in the presence of DCMU. We conclude that guard cell chloroplasts provide ATP and/or reducing equivalents that fuel BL-dependent stomatal opening, and that they indirectly monitor photosynthetic CO{2} fixation in mesophyll chloroplasts by absorbing PAR in the epidermis

BL-dependent H⁺-pumping enhanced by RL-activated guard cell photosynthesis.

<p>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 <i>phot1phot2</i> (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).</p

BL-induced phosphorylation of H⁺-ATPase in wild-type guard cell protoplasts.

<p>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 <i>t</i>-tests (**: 0.01<<i>P</i><0.05, ***: <i>P</i><0.01). n.s. indicates the statistically insignificant difference (<i>P</i>>0.43).</p

BL-dependent stomatal opening enhanced by RL-activated guard cell photosynthesis.

<p>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 <i>t</i>-tests (*: 0.05<<i>P</i><0.1, **: 0.01<<i>P</i><0.05, ***: <i>P</i><0.01).</p