Mitogen-Activated Protein Kinase Phosphatases Affect UV-B-Induced Stomatal Closure via Controlling NO in Guard Cells

Abstract

Ultraviolet B (UV-B) radiation induces the activation of MITOGEN-ACTIVATED PROTEIN KINASE PHOSPHATASE1 (MKP1) and its targets MPK3 and MPK6, but whether they participate in UV-B guard cell signaling is not clear. Here, evidence shows that UV-B-induced stomatal closure in Arabidopsis (Arabidopsis thaliana) is antagonistically regulated by MKP1 and MPK6 via modulating hydrogen peroxide (H₂O₂)-induced nitric oxide (NO) production in guard cells. The mkp1 mutant was hypersensitive to UV-B-induced stomatal closure and NO production in guard cells but not to UV-B-induced H₂O₂ production, suggesting that MKP1 negatively regulates UV-B-induced stomatal closure via inhibiting NO generation in guard cells. Moreover, MPK3 and MPK6 were activated by UV-B in leaves of the wild type and hyperactivated in the mkp1 mutant, but the UV-B-induced activation of MPK3 and MPK6 was largely inhibited in mutants for ATRBOHD and ATRBOHF but not in mutants for NIA1 and NIA2 mpk6 mutants showed defects of UV-B-induced NO production and stomatal closure but were normal in UV-B-induced H₂O₂ production, while stomata of mpk3 mutants responded to UV-B just like those of the wild type. The defect of UV-B-induced stomatal closure in mpk6 mutants was rescued by exogenous NO but not by exogenous H₂O₂ Furthermore, double mutant mkp1/mpk6 and the single mutant mpk6 showed the same responses to UV-B in terms of either stomatal movement or H₂O₂ and NO production. These data indicate that MPK6, but not MPK3, positively regulates UV-B-induced stomatal closure via acting downstream of H₂O₂ and upstream of NO, while MKP1 functions negatively in UV-B guard cell signaling via down-regulation of MPK6.

Figure 1.

The mkp1 mutant is hypersensitive to UV-B-induced stomatal closure. Leaves of wild-type (WT) Ws, the mkp1 mutant, and line 6 (a representative line of mkp1 mutant complemented by the expression of wild-type MKP1) with open stomata were exposed to different doses of UV-B for 3 h (A) or to 0.5 W m⁻² UV-B for different times (B), then stomatal apertures were measured in epidermal strips from abaxial surfaces of the treated leaves. The data shown are means ± se of at least three independent experiments, each with 50 stomata. Means with different letters are significantly different at P < 0.01.

Figure 2.

The mkp1 mutant is hypersensitive to UV-B-induced NO production but not to UV-B-induced H₂O₂ production in guard cells. Leaves of wild-type (WT) Ws, the mkp1 mutant, and line 6 (a representative line of mkp1 complemented by the expression of wild-type MKP1) with open stomata were exposed to 0.5 W m⁻² UV-B for the indicated times, then epidermal strips were peeled from abaxial surfaces of the treated leaves and fluorescence images and pixel intensities in guard cells preloaded with 50 µm H₂DCFDA (A and B) or 10 µM DCF-2DA (C and D) were recorded. Data of fluorescence pixel intensities (B and D) are displayed as means ± se of three replicates, each replicate with 20 stomata. Means with different letters are significantly different at P < 0.05. Bars in A and C = 10 μm.

Figure 3.

UV-B activates MPK3 and MPK6 in leaves of wild-type (WT) Col-0 and Ws and hyperactivates MPK3 and MPK6 in leaves of the mkp1 mutant. A, Leaves of wild-type Col-0 were exposed to different doses of UV-B for 2 h. B, Leaves of wild-type Col-0 were exposed to 0.5 W m⁻² UV-B for the indicated times or leaves of mutants mpk3-1 and mpk6-4 were exposed to 0.5 W m⁻² UV-B for 2 h. C, Leaves of wild-type Ws, mutant mkp1, and line 6 (a representative line of mkp1 complemented by the expression of wild-type MKP1) were exposed to 0.5 W m⁻² UV-B for the indicated times. After treatment, total proteins were extracted from the treated leaves and immunoblot analysis was performed with anti-phospho-p44/42 MPK and anti-actin (loading control) antibodies.

Figure 4.

MPK6 mediates UV-B-induced stomatal closure via inducing NO generation in guard cells, and mkp1 hypersensitivity to UV-B-induced NO production and stomatal closure depend on MPK6 activation. A, Leaves of wild-type (WT) Col-0 and Ws, single mutants mpk3-1, mpk3-2, mpk6-1, and mpk6-4, and double mutant mkp1-1/mpk6-1 (mkp1/mpk6) with open stomata were incubated in MES buffer in the absence or presence of 100 µm H₂O₂ or 100 µm SNP under light alone or with 0.5 W m⁻² UV-B for 3 h, then stomatal apertures were measured in epidermal strips from the abaxial surfaces of the treated leaves. Data presented are means ± se of at least three independent experiments, and means with different letters are significantly different at P < 0.01. B to E, Leaves of wild-type Col-0 and Ws and mutants mpk3-1, mpk3-2, mpk6-4, mpk6-1, and mkp1/mpk6 were incubated in MES buffer in the absence or presence of 100 µm H₂O₂ under light alone or with 0.5 W m⁻² UV-B for 3 h, then fluorescence images (B and D) and pixel intensities (C and E) in guard cells preloaded with 50 µm H₂DCFDA (B and C) or 10 µm DAF-2DA (D and E) were recorded. Data of fluorescence intensities are shown as means ± se of three independent experiments, and means with different letters are significantly different at P < 0.05. Bars in B and D = 10 µm.

Figure 5.

UV-B activation of MPK3 and MPK6 depends on H₂O₂ production but not NO production. A, Leaves of wild-type (WT) Col-0 and mutants AtrbohD, AtrbohF, and AtrbohD/F were exposed to light alone or with 0.5 W m⁻² UV-B for 2 h. B, Leaves of wild-type Col-0 and mutant mpk6-4 were incubated in MES buffer containing 100 µm H₂O₂ for the indicated times. C, Leaves of wild-type Col-0 and Landsberg erecta (Ler) and mutants nia1-2 (nia1), nia2-1 (nia2), and nia1-1/nia2-5 (nia1/2) were exposed to light alone or with 0.5 W m⁻² UV-B for 2 h. After treatments, total proteins were extracted from the treated leaves, and immunoblot analysis was performed with anti-phospho-p44/42 MPK and anti-actin (loading control) antibodies. The data shown are representative of up to three independent experiments.