Protein phosphatase 1 positively regulates stomatal opening in response to blue light in Vicia faba

Abstract

Phototropins, plant blue light receptors, mediate stomatal opening through the activation of the plasma membrane H(+)-ATPase by unknown mechanisms. Here we report that type 1 protein phosphatase (PP1) positively regulates the blue light signaling between phototropins and the H(+)-ATPase in guard cells of Vicia faba. We cloned the four catalytic subunits of PP1 (PP1c) from guard cells and determined the expression of the isoforms in various tissues. Transformation of Vicia guard cells with PP1c isoforms that had lost enzymatic activity by one amino acid mutation, or with human inhibitor-2, a specific inhibitor protein of PP1c, suppressed blue light-induced stomatal opening. Addition of fusicoccin, an activator of the plasma membrane H(+)-ATPase, to these transformed guard cells induced normal stomatal opening, suggesting that the transformations did not affect the basic mechanisms for stomatal opening. Tautomycin, an inhibitor of PP1, inhibited blue light-induced H(+) pumping, phosphorylation of the plasma membrane H(+)-ATPase in guard cell protoplasts, and stomatal opening. However, tautomycin did not inhibit the blue light-dependent phosphorylation of phototropins. We conclude that PP1 functions downstream of phototropins and upstream of the H(+)-ATPase in the blue light signaling pathway of guard cells.

Fig. 1.

Amino acid sequences and expression patterns of Vicia PP1c isogenes. (A) Alignment of the deduced amino acid sequences of VfPP1c isoforms. Identical amino acids appear in negative print. Arrowhead indicates the conserved His residue in the catalytic site. The sequences were aligned by using CLUSTAL X. (B) Expression of Vicia PP1c isogenes in guard cell protoplasts (GCPs), mesophyll cell protoplasts (MCPs), leaves, stems, and roots determined by RT-PCR. Actin mRNA was used as an internal standard.

Fig. 2.

Inhibition of blue light-dependent stomatal opening by expression of mutant forms of PP1c in guard cells. (A) Protein phosphatase activity of VfPP1c-1 and VfPP1c-1-H137N prepared from E. coli cells. Phosphatase activity was measured by using [³²P]-labeled myelin basic protein in the absence (control) or presence of 10 nM tautomycin. Each value was expressed as percentage of VfPP1c-1 (55,498 ± 2,914 nmol·min⁻¹·mg⁻¹ protein). Data are means ± SE (n = 3). (B) Images of guard cells in the bright field (a, c, e, and g) and fluorescence microscope (b, d, f, and h). Typical cases of guard cells transformed with VfPP1c-1:sGFP (a–d) or VfPP1c-1-H137N:sGFP (e–h) are shown. Gene constructs were introduced into Vicia guard cells by particle bombardment. Epidermal strips were detached and preincubated for 2.5 h in darkness, and then illuminated by red light (RL, 150 μmol·m⁻²·s⁻¹) with or without blue light (BL, 10 μmol·m⁻²·s⁻¹) for 2.5 h. (Scale bar, 10 μm.) (C and D) Half stomatal apertures measured next to transformed and nontransformed guard cells. Results are means ± SE (n = 45, pooled from triplicate experiments) from VfPP1c-1:sGFP or VfPP1c-1-H137N:sGFP (C) and VfPP1c-3:sGFP or VfPP1c-3-H121N:sGFP (D) transformants. Asterisks indicate significant differences between nontransformed and transformed guard cells (P < 0.01).

Fig. 3.

Inhibition of blue light-dependent stomatal opening by transformation of guard cells with inhibitor-2. (A) Interaction of Vicia PP1cs with inhibitor-2 determined by yeast two-hybrid assay. β-Galactosidase activity was measured by filter assay using X-gal as a substrate. (B) Inhibition of VfPP1c-1 activity by inhibitor-2:sGFP. Experimental conditions were the same as in Fig. 2A. Phosphatase activities are expressed as percentages of that measured without inhibitor-2:sGFP (65,760 ± 2,418 nmol min⁻¹·mg⁻¹ protein). Data are means ± SE (n = 3). (C) Images of guard cells in the bright field (a, c, e, and g) and fluorescence microscope (b, d, f, and h). Typical guard cells transformed with sGFP (a–d) and inhibitor-2:sGFP (e–h) are shown. Other experimental conditions are the same as in Fig. 2B. (Scale bar, 10 μm.) (D) Determination of half stomatal apertures of guard cells transformed with sGFP and inhibitor-2:sGFP as well as of nontransformed neighboring guard cells. Data represent means ± SE (n = 45, pooled from triplicate experiments). Asterisk indicates significant difference between nontransformed and transformed guard cells (P < 0.01). (E) Subcellular localization of inhibitor-2 and VfPP1c-1. Gene constructs encoding inhibitor-2:sGFP and VfPP1c-1:DsRed were cotransformed into Vicia guard cells by particle bombardment, and fluorescent images were obtained by confocal laser-scanning microscopy. (Scale bar, 10 μm.)

Fig. 4.

Response to fusicoccin of guard cells transformed with PP1c mutant and inhibitor-2. (A and C) Images of guard cells under bright field (a) and fluorescence conditions (b). Typical guard cells transformed with VfPP1c-1-H137N:sGFP (A) and inhibitor-2:sGFP (C) are shown. Epidermal strips were preincubated for 2.5 h in darkness before fusicoccin (10 μM) was added; images were taken after further incubation for 2.5 h in darkness. (Scale bars, 10 μm.) (B and D) Determination of half stomatal apertures of guard cells transformed with VfPP1c-1-H137N:sGFP (B) or inhibitor-2:sGFP (D). Data are means ± SE (n = 45, pooled from triplicate experiments).

Fig. 5.

Effects of tautomycin on H⁺ pumping, stomatal opening, and the phosphorylation of Vfphots and the plasma membrane H⁺-ATPase in response to blue light. (A) Inhibition of blue light-dependent H⁺ pumping in Vicia guard cell protoplasts by tautomycin. Guard cell protoplasts were preincubated for 30 min under the red light (RL, 600 μmol·m⁻²·s⁻¹), and then were exposed to 10 μM tautomycin for 2 h. Afterward, a pulse of blue light (BL, 100 μmol·m⁻²·s⁻¹, 30 s) was applied at the times indicated by vertical arrowheads. (B) Effect of tautomycin on ATP-dependent H⁺ pumping in microsomal vesicles from guard cell protoplasts. The basal reaction mixture (250 μl) contained membrane vesicles (10 μg of protein), 10 mM Mops-KOH (pH 7.0), 0.25 M mannitol, 5 mM MgCl₂, 1 mM EGTA, 50 mM KNO₃, 5 μg·ml⁻¹ oligomycin, and 1 μM quinacrine. Vanadate at 100 μM and tautomycin at 10 μM were added. ΔF/F, change in fluorescence divided by the initial fluorescence. (C) Inhibition of blue light-dependent stomatal opening by tautomycin. Epidermal strips were preincubated for 2.5 h in darkness with 10 μM tautomycin, and then illuminated by red light (RL, 150 μmol·m⁻²·s⁻¹) with or without blue light (BL, 10 μmol·m⁻²·s⁻¹) for 2.5 h. Data represent means ± SE (n = 65, pooled from triplicate experiments). Asterisk indicates significant difference between control and tautomycin treatments (P < 0.01). (D) Levels of phosphorylation and the amounts of Vfphot and the H⁺-ATPase. Guard cell protoplasts were treated as described for Fig. 5A with [³²P]orthophosphate. Autoradiography was carried out on Vfphots and H⁺-ATPase, which were isolated by immunoprecipitation from 200 and 100 μg of guard cell proteins, respectively. Western blotting of Vfphot and the H⁺-ATPase was performed by using polyclonal antibodies raised against to individual protein. Asterisk indicates a nonspecific protein. Experiments repeated on two occasions gave similar results.