Type 2C protein phosphatase clade D family members dephosphorylate guard cell plasma membrane H+-ATPase

Abstract

Plasma membrane (PM) H+-ATPase in guard cells is activated by phosphorylation of the penultimate residue, threonine (Thr), in response to blue and red light, promoting stomatal opening. Previous in vitro biochemical investigation suggested that Mg2+- and Mn2+-dependent membrane-localized type 2C protein phosphatase (PP2C)-like activity mediates the dephosphorylation of PM H+-ATPase in guard cells. PP2C clade D (PP2C.D) was later demonstrated to be involved in PM H+-ATPase dephosphorylation during auxin-induced cell expansion in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether PP2C.D phosphatases are involved in PM H+-ATPase dephosphorylation in guard cells. Transient expression experiments using Arabidopsis mesophyll cell protoplasts revealed that all PP2C.D isoforms dephosphorylate the endogenous PM H+-ATPase. We further analyzed PP2C.D6/8/9, which display higher expression levels than other isoforms in guard cells, observing that pp2c.d6, pp2c.d8, and pp2c.d9 single mutants showed similar light-induced stomatal opening and phosphorylation status of PM H+-ATPase in guard cells as Col-0. In contrast, the pp2c.d6/9 double mutant displayed wider stomatal apertures and greater PM H+-ATPase phosphorylation in response to blue light, but delayed dephosphorylation of PM H+-ATPase in guard cells; the pp2c.d6/8/9 triple mutant showed similar phenotypes to those of the pp2c.d6/9 double mutant. Taken together, these results indicate that PP2C.D6 and PP2C.D9 redundantly mediate PM H+-ATPase dephosphorylation in guard cells. Curiously, unlike auxin-induced cell expansion in seedlings, auxin had no effect on the phosphorylation status of PM H+-ATPase in guard cells.

Figure 1.

Effect of transiently expressed PP2C.Ds on phosphorylation status of PM H⁺-ATPase in MCPs. Arabidopsis MCPs transiently expressing GFP-tagged PP2C.Ds were incubated in incubation buffer at room temperature for 14 h. The phosphorylated PM H⁺-ATPase and amount of PM H⁺-ATPase were detected by immunoblot using anti-phospho-Thr947 antibody (Anti-pThr₉₄₇) and a specific antibody for PM H⁺-ATPase (anti-H⁺-ATPase), respectively. GFP-tagged PP2C.Ds were detected by immunoblot using the anti-GFP antibody. Numbers on the right side represent the positions of molecular weight markers.

Figure 2.

Expression and localization of PP2C.D6, PP2C.D8 and PP2C.D9. A, Relative expression levels of PP2C.Ds in transcriptional inhibitors-untreated GCPs. Expression level of each PP2C.D was obtained from eFP browser (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi;  Yang et al., 2008). B, Expression of PP2C.D6, PP2C.D8 and PP2C.D9 in the epidermis. The epidermal tissues were obtained from rosette leaves of 4-week-old transgenic plants expressing the GUS reporter gene under control of PP2C.D6 and PP2C.D9 promoters. GUS staining was performed at 37°C for 16 h. Bars = 10 µm. C, Subcellular localization of PP2C.D6, PP2C.D8, and PP2C.D9. Gene constructs encoding GFP-fused PP2C.D6 or PP2C.D8 or PP2C.D9 under 35SCaMV promoter were transformed into guard cells of broad bean by particle bombardment. Typical fluorescent images from GFP obtained by a confocal microscope and images of guard cells taken with phase-contrast microscopy are shown. Bars = 10 µm.

Figure 3.

Stomatal phenotypes of pp2c.d6, pp2c.d8, and pp2c.d9 single mutants. A, Schematic representation of the structures of PP2C.D6 (At3g51370), PP2C.D8 (At4g33920), and PP2C.D9 (At5g06750) and the location of the T-DNA insertions in pp2c.d6, pp2c.d8, and pp2c.d9. Boxes and lines represent exons and introns, respectively. B, Expression of PP2C.D6, PP2C.D8, and PP2C.D9 analyzed by RT-PCR in Col-0, pp2c.d6, pp2c.d8, and pp2c.d9. Total RNA was extracted from rosette leaves of 4-week-old plants. TUB2 was used as an internal control. C, Stomatal opening in response to light in Col-0, pp2c.d6, and pp2c.d9. Epidermal tissues from dark-adapted plants in the basal buffer were illuminated with light (blue light at 10 µmol m⁻² s⁻¹ superimposed on red light at 50 µmol m⁻² s⁻¹) or kept in the dark for 3 h. Values represent mean ± se (n = 5); measurement of 30 stomata in each experiment. Asterisk indicates statistically significant difference from corresponding Col-0 (Student’s t test; *P < 0.05). D, Immunohistochemical detection of the phosphorylation of PM H⁺-ATPase in response to blue light in Col-0, pp2c.d6, pp2c.d8, and pp2c.d9 guard cells. Epidermal tissues from dark-adapted plants were illuminated with red light (50 µmol m⁻² s⁻¹) for 20 min (Red), after which blue light (10 µmol m⁻² s⁻¹) was superimposed on the red light for 2.5 min (Red + Blue). The phosphorylated PM H⁺-ATPase was detected by the immunohistochemical method using anti-pThr₉₄₇ antibody. Typical fluorescent images (left) and relative intensities of the fluorescent signals (right) are shown. Arrowheads indicate the position of the stomata. Bars = 10 µm. Data indicate mean ± sd (n = 5) with measurement of 30 stomata in each sample.

Figure 4.

Stomatal phenotypes in pp2c.d6/9, pp2c.d6/8, and pp2c.d8/9 double mutants. A, Expression of PP2C.D6, PP2C.D8 and PP2C.D9 analyzed by RT-PCR in Col-0 and pp2c.d6/9, pp2c.d6/8 and pp2c.d8/9. Other details are the same as in Figure  3B. B, Stomatal opening in response to light in Col-0 and pp2c.d6/9, pp2c.d6/8, pp2c.d8/9, and pp2c.6/8/9. Other details are the same as in Figure  3C. Asterisk indicates statistically significant difference from corresponding Col-0 (Student’s t test; *P < 0.05, **P < 0.01). C, Immunohistochemical detection of the phosphorylation of PM H⁺-ATPase in response to blue light in Col-0 and pp2c.d6/9, pp2c.d6/8, pp2c.d8/9, and pp2c.6/8/9 guard cells. Other details are the same as in Figure  3D. Asterisk indicates statistically significant difference from corresponding Col-0 (Student’s t test; *P < 0.05, **P < 0.01).

Figure 5.

Time course of PM H⁺-ATPase dephosphorylation after blue light illumination in Col-0 and pp2c.d6/9 guard cells. Epidermal tissues from dark-adapted plants were pre-illuminated with red light (50 µmol m⁻² s⁻¹) for 20 min, then a blue light pulse (10 µmol m⁻² s⁻¹, 30 s) was superimposed on the red light. The epidermal tissues were fixed at the indicated time points and phosphorylated PM H⁺-ATPase was detected by immunohistochemical method. Typical fluorescent images (upper) and relative intensities of the fluorescent signals (lower) are shown. Arrowheads indicate the position of the stomata. Bar = 10 µm. Data indicate mean ± se (n = 5) with measurement of 30 stomata in each sample. Asterisk indicates statistically significant difference from corresponding Col-0 (Student’s t test; *P < 0.05, **P < 0.01).

Figure 6.

Stomatal opening in response to light in Col-0 and pGC1::GFP-PP2C.D9/Col-0. Values represent mean ± sd; measurement of 30 stomata in each experiment. Double asterisk indicates statistically significant difference from corresponding Col-0 (Student’s t test; **P < 0.01). Experiments were repeated on three occasions with similar results. Other details are the same as in Figure  3C.

Figure 7.

In vitro phosphatase assay using recombinant PP2C.D9 protein for the dephosphorylation of yeast-expressed PM H⁺-ATPase (AHA2). Microsomal membrane fractions isolated from yeast expressing AHA2 were used for in vitro phosphatase assays. The microsomes were incubated with or without recombinant His-PP2C.D9 for 30 min at 24°C. His-PP2C.D9 was detected by immunoblot using the anti-His antibody.

Figure 8.

Effect of exogenous auxin on phosphorylation status of PM H⁺-ATPase in guard cells. Epidermal tissues from dark-adapted plants were incubated with 0.25% (v/v) DMSO (Dark), 10-µM FC, or 10-µM IAA for 20 min in the dark. FC and IAA were dissolved in DMSO. Typical fluorescent images (upper) and relative intensities of the fluorescent signals (lower) are shown. Arrowheads indicate the positions of the stomata. Bars = 10 µm. Data indicate mean ± se (n = 3) with measurement of 30 stomata in each sample. Double asterisk indicates statistically significant difference from dark sample (Student’s t test; *P < 0.01).