TY - JOUR
AU - Yue, Ming
AB - Abstract Heterotrimeric G proteins have been shown to transmit ultraviolet B (UV-B) signals in mammalian cells, but whether they also transmit UV-B signals in plant cells is not clear. In this paper, we report that 0.5 W m−2 UV-B induces stomatal closure in Arabidopsis (Arabidopsis thaliana) by eliciting a cascade of intracellular signaling events including Gα protein, hydrogen peroxide (H2O2), and nitric oxide (NO). UV-B triggered a significant increase in H2O2 or NO levels associated with stomatal closure in the wild type, but these effects were abolished in the single and double mutants of AtrbohD and AtrbohF or in the Nia1 mutants, respectively. Furthermore, we found that UV-B-mediated H2O2 and NO generation are regulated by GPA1, the Gα-subunit of heterotrimeric G proteins. UV-B-dependent H2O2 and NO accumulation were nullified in gpa1 knockout mutants but enhanced by overexpression of a constitutively active form of GPA1 (cGα). In addition, exogenously applied H2O2 or NO rescued the defect in UV-B-mediated stomatal closure in gpa1 mutants, whereas cGα AtrbohD/AtrbohF and cGα nia1 constructs exhibited a similar response to AtrbohD/AtrbohF and Nia1, respectively. Finally, we demonstrated that Gα activation of NO production depends on H2O2. The mutants of AtrbohD and AtrbohF had impaired NO generation in response to UV-B, but UV-B-induced H2O2 accumulation was not impaired in Nia1. Moreover, exogenously applied NO rescued the defect in UV-B-mediated stomatal closure in the mutants of AtrbohD and AtrbohF. These findings establish a signaling pathway leading to UV-B-induced stomatal closure that involves GPA1-dependent activation of H2O2 production and subsequent Nia1-dependent NO accumulation. Heterotrimeric G proteins, composed of α-, β-, and γ-subunits, are a key intracellular signaling molecule in both mammalian and plant systems. Classically, upon signal reception by a receptor coupled to the heterotrimer, the Gα-subunit separates from the Gβγ dimer, and either Gα or the Gβγ dimer can act as a functional unit and induce downstream signaling (Oldham and Hamm, 2008). In contrast to mammalian cells, where multiple α, β, and γ genes exist, there is only one prototypical Gα (GPA1), one Gβ (AGB1), and two known Gγ (AGG1 and AGG2) genes in Arabidopsis (Arabidopsis thaliana; Temple and Jones, 2007). Despite the comparative simplicity of players, G proteins have been shown to participate in multiple signaling pathways in Arabidopsis, including developmental processes, phytohormone responses, and responses to biotic and abiotic environmental signals such as pathogens, ozone, drought, and light (Assmann, 2005; Temple and Jones, 2007; Warpeha et al., 2007; Okamoto et al., 2009; Nilson and Assmann, 2010). Depletion of the stratospheric ozone layer results in increased levels of the sun’s UV-B radiation (280–315 nm) at the Earth’s surface. Although this influx of shortwave photons with high energy implies serious effects for all living organisms (Frohnmeyer and Staiger, 2003), UV-B is also a key environmental signal that initiates diverse responses in a range of organisms (Jansen and Bornman, 2012). Thus, understanding the mechanism of UV-B signal transduction in cells is very important. In recent years, significant progress has been made in identifying the molecular players and understanding the early mechanisms and functions of the UV-B perception and signaling pathway in plants. The perception of UV-B by UV RESISTANCE LOCUS8 (UVR8) followed by the interaction among UVR8, CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), and ELONGATED HYPOCOTYL5 (HY5) has emerged as a primary mechanism of the UV-B response that is crucial for UV-B acclimation and tolerance (Rizzini et al., 2011; Christie et al., 2012; Heijde and Ulm, 2012; Jansen and Bornman, 2012). However, few of the molecular players involved in UV-B signal transduction are currently known. In mammalian cells, heterotrimeric G proteins have been shown to mediate various UV-B-induced cellular responses, such as secretion of heparin-binding epidermal growth factor (HB-EGF), activation of mitogen-activated protein kinases, cyclooxygenase2 expression, and apoptosis in human keratinocytes (Seo et al., 2004, 2007; Seo and Juhnn, 2010), suggesting that G proteins are important molecular players in UV-B signal transduction. However, at present, whether G proteins participate in the responses of plant cells to UV-B is not known. Stomata embedded in the epidermis of terrestrial plants are important for CO2 absorption and water transpiration and are possible points of entry for pathogens. Thus, the regulation of stomatal apertures is extremely important for the survival of plants. Phenotypic analyses of Arabidopsis mutants lacking the Gα- or Gβ-subunit show that these G proteins are involved in stomatal movement regulated by abscisic acid (ABA; Wang et al., 2001; Pandey and Assmann, 2004; Liu et al., 2007; Fan et al., 2008; Zhang et al., 2011), sphingosine-1-P (Coursol et al., 2003, 2005), phosphatidic acid (PA; Mishra et al., 2006), extracellular calmodulin (ExtCaM; Chen et al., 2004; Li et al., 2009), extracellular ATP (Hao et al., 2012), and the pathogen-associated molecular pattern flg22 (Zhang et al., 2008), suggesting that plant G proteins respond to various stimuli as key regulators of stomatal movement. On exposure to UV-B radiation, many plant species exhibit decreases in stomatal conductance and/or aperture under growth chamber, greenhouse, and field conditions (Musil and Wand, 1993; Nogués et al., 1999; Jansen and Noort, 2000). However, in some species, UV-B has been reported to induce either stomatal opening or stomatal closure, perhaps depending on the metabolic state of guard cells (Jansen and Noort, 2000). Furthermore, UV-B-inhibited photosynthesis is partially caused by stomatal limitation (He et al., 2004). Thus, understanding the mechanism of stomatal movement regulated by UV-B is extremely important for improving the resistance of plants to enhanced UV-B radiation, but, to date, it is poorly understood. Recently, compelling evidence emerged that hydrogen peroxide (H2O2) and nitric oxide (NO) function as signaling molecules in plants, mediating a range of responses to environmental stress including UV-B radiation (Neill et al., 2002; Qiao and Fan, 2008; Wilson et al., 2008). Increasing evidence also points to the role for H2O2 and NO as essential components in guard cell signaling. For example, both H2O2 and NO have been implicated in ABA-, salicylic acid (SA)-, ethylene-, ExtCaM-, and darkness-induced stomatal closure. Furthermore, several main cellular players in stomatal movement, such as mitogen-activated protein kinases, protein phosphatases, cytoskeleton, and ion channels, have already been identified as likely targets downstream of H2O2 or NO (Neill et al., 2008; Wang and Song, 2008; Huang et al., 2009; Li et al., 2009; Wilkins et al., 2011; Yemets et al., 2011). G protein signaling to the membrane-bound H2O2 synthetic enzyme, NADPH oxidase, has been implicated in the development of disease resistance and the apoptotic hypersensitive response in rice (Oryza sativa; Suharsono et al., 2002). Production of reactive oxygen species (ROS) in response to the air pollutant ozone is also impaired in a mutant lacking the Gα subunit (Joo et al., 2005). The heterotrimeric G proteins also participate in ROS metabolism in plant cells (Wei et al., 2008; Zhao et al., 2010). During stomatal movement, G proteins mediate H2O2 production induced by ABA (Zhang et al., 2011), ExtCaM (Chen et al., 2004; Li et al., 2009), and extracellular ATP (Hao et al., 2012) as well as NO production induced by ExtCaM in guard cells (Li et al., 2009). In addition, phospholipase Dα and its product PA, which interact with GPA1 during ABA inhibition of stomatal opening (Mishra et al., 2006), also promote ABA-induced ROS production (Zhang et al., 2009). These observations suggest that G proteins may be key regulators of H2O2 and NO production in plant cells, including guard cells. With regard to the stomatal movement regulated by UV-B radiation, our previous studies showed that H2O2 and NO generation are required for UV-B-induced stomatal closure (He et al., 2005, 2011a, 2011b). However, whether the UV-B-induced production of H2O2 and NO in guard cells is also regulated by G proteins remains unknown. In this study, we use Arabidopsis mutants (e.g. GPA1 null mutants gpa1-1 and gpa1-2; Nia1-2, Nia2-1, and Nia1-2/Nia2-5, which are defective in NO production; and AtrbohD, AtrbohF, and AtrbohD/AtrbohF, which are defective in producing H2O2) and pharmacological reagents to show that the G protein is involved in the regulation of UV-B-induced stomatal closure in Arabidopsis via sequential elucidation of H2O2 and NO, two key regulators of UV-B regulation of stomatal movements. Our results establish a linear signaling cascade in which the Gα protein transmits UV-B signals to elicit H2O2, which then elicits NO in guard cells to regulate UV-B-dependent stomatal closure. RESULTS Optimization of UV-B Radiation on Stomatal Movement in Arabidopsis Leaves Previous studies have shown that UV-B can induce stomatal closure or opening (Jansen and Noort, 2000). To determine the effect of UV-B radiation on stomatal movement in Arabidopsis leaves, freshly prepared leaves were first floated on MES-KCl buffer under light for 3 h to induce stomatal opening, and then the light was supplemented with 0.3 to 1.0 W m−2 UV-B radiation for another 4 h. Under these experimental conditions, exposure to all tested doses of UV-B radiation induced decreases in stomatal aperture that were evident at 0.3 W m−2, substantial at 0.5 W m−2, and maximal at 0.7 W m−2 (Fig. 1A). To determine the optimal time of UV-B radiation to induce stomatal closure, the time courses of stomatal closure regulated by 0.5, 0.7, and 0.9 W m−2 UV-B radiation were determined: the maximum effects of the three doses of UV-B were all achieved at 3 h (Fig. 1B). To determine whether UV-B-induced stomatal closure was caused by widespread, nonspecific damage to guard cells, we further assayed the viability of guard cells irradiated by 0.5, 0.7, and 0.9 W m−2 UV-B for 4 h with the fluorescent indicator dyes fluorescein diacetate (FAD; which passes through cell membranes and is hydrolyzed by intracellular esterase to produce a polar compound that accumulates inside the cell and exhibits green fluorescence when excited by blue light) and propidium iodide (PI; which passes through damaged cell membranes and intercalates with DNA and RNA to form a bright red fluorescent complex seen in the nuclei of dead cells; Supplemental Fig. S1). The results showed that guard cells treated by 0.5 and 0.7 W m−2 UV-B for 4 h were marked by FAD (Supplemental Fig. S1, B and C) but not by PI (data not shown). However, the guard cells irradiated by 0.9 W m−2 UV-B for 4 h could be marked by PI (Supplemental Fig. S1H) and not by FAD (Supplemental Fig. S1D), indicating that the stomatal closure induced by 0.3 to 0.7 W m−2 UV-B was not caused by the loss of guard cell viability. Based on these results, we chose 0.5 W m−2 UV-B irradiation for 3 h for subsequent experiments. This dose of UV-B was the biologically effective radiation, normalized to 300 nm, according to Caldwell (1971) and corresponds to 3.45 µmol m−2 s−1, which is close to the fluence rate of UV-B in sunlight (Brown and Jenkins, 2008). Figure 1. Open in new tabDownload slide UV-B induced stomatal closure in Arabidopsis leaves of wild-type ecotype Ws. A, Leaves with open stomata were kept in MES buffer under light alone (0) or with the indicated doses of UV-B for 4 h. B, Leaves with open stomata were kept in MES buffer under light alone (Light) or with 0.5, 0.7, and 0.9 W m−2 UV-B (UV-B) for the indicated times. After treatment, stomatal apertures were observed in epidermal fragments from abaxial surfaces of the treated leaves. Each assay was repeated at least three times. The data are presented as means ± se (n = 150). Means with different letters are significantly different at P < 0.01. Figure 1. Open in new tabDownload slide UV-B induced stomatal closure in Arabidopsis leaves of wild-type ecotype Ws. A, Leaves with open stomata were kept in MES buffer under light alone (0) or with the indicated doses of UV-B for 4 h. B, Leaves with open stomata were kept in MES buffer under light alone (Light) or with 0.5, 0.7, and 0.9 W m−2 UV-B (UV-B) for the indicated times. After treatment, stomatal apertures were observed in epidermal fragments from abaxial surfaces of the treated leaves. Each assay was repeated at least three times. The data are presented as means ± se (n = 150). Means with different letters are significantly different at P < 0.01. Heterotrimeric G Protein α-Subunit Is Involved in UV-B Promotion of Stomatal Closure To assess whether G proteins might mediate UV-B signaling in guard cells, we first investigated stomatal responses to pertussis toxin (PTX), an inhibitor of the G protein α-subunit, and cholera toxin (CTX), an activator of the G protein α-subunit (Chen et al., 2004). As shown in Figure 2A, 0.5 W m−2 UV-B-induced stomatal closure was completely inhibited when leaves were incubated with PTX. Meanwhile, similar to UV-B, CTX significantly induced stomatal closure in leaves under light. Furthermore, the viability assay also showed that the PTX and CTX treatments did not cause the loss of guard cell viability (Supplemental Fig. S1, E–G). These results suggest that UV-B induces Arabidopsis stomatal closure by activating Gα. Figure 2. Open in new tabDownload slide Gα mediates UV-B-induced stomatal closure. A, Leaves of wild-type Ws with open stomata were kept in MES buffer in the absence (Control) or presence of 400 ng mL−1 PTX or 400 ng mL−1 CTX under light alone (Light) or with 0.5 W m−2 UV-B (UV-B) for 3 h. B, Leaves of the wild-type (WT) Ws, gpa1-1, and gpa1-2 with open stomata were incubated in MES buffer under light alone or with 0.5 W m−2 UV-B for 3 h. C, Leaves of wild-type Ws plants or two cGα lines (constitutively overexpressing G protein α-subunit AtGPA1) were floated on MES buffer under light for 3 h to open stomata and then continually incubated under light alone or with 0.5 W m−2 UV-B for the indicated times. After treatments, stomatal apertures were measured in epidermal strips from abaxial surfaces of the treated leaves. Each assay was repeated at least three times. The data are presented as means ± se (n = 150). Means with different letters are significantly different at P < 0.01. Figure 2. Open in new tabDownload slide Gα mediates UV-B-induced stomatal closure. A, Leaves of wild-type Ws with open stomata were kept in MES buffer in the absence (Control) or presence of 400 ng mL−1 PTX or 400 ng mL−1 CTX under light alone (Light) or with 0.5 W m−2 UV-B (UV-B) for 3 h. B, Leaves of the wild-type (WT) Ws, gpa1-1, and gpa1-2 with open stomata were incubated in MES buffer under light alone or with 0.5 W m−2 UV-B for 3 h. C, Leaves of wild-type Ws plants or two cGα lines (constitutively overexpressing G protein α-subunit AtGPA1) were floated on MES buffer under light for 3 h to open stomata and then continually incubated under light alone or with 0.5 W m−2 UV-B for the indicated times. After treatments, stomatal apertures were measured in epidermal strips from abaxial surfaces of the treated leaves. Each assay was repeated at least three times. The data are presented as means ± se (n = 150). Means with different letters are significantly different at P < 0.01. To confirm these pharmacological data, we further used the Arabidopsis mutants gpa1-1 and gpa1-2 harboring the recessive transfer DNA knockout alleles of GPA1, the only prototypical Gα gene in the Arabidopsis genome (Temple and Jones, 2007), and two independent Arabidopsis transgenic lines overexpressing a constitutively active form of GPA1 (cGα): GPA1, with a point mutation of Glu-222 to Leu, which locks Gα in the active state once activated (Okamoto et al., 2001), and wild-type Arabidopsis ecotype Wassilewskija (Ws). As shown in Figure 2B and Supplemental Figure S2, 0.5 W m−2 UV-B-induced stomatal closure was completely impaired in the mutants gpa1-1 and gpa1-2, as the mutant stomata in the presence of UV-B behaved exactly like the wild-type control in the absence of UV-B. In contrast, cGα lines showed not only smaller stomatal aperture than the wild type under light but also showed faster stomatal closure induced by 0.5 W m−2 UV-B than the wild type (Fig. 2C). In the mean time, we also checked the effects of PTX and CTX on guard cell responses in gpa1 mutants. Consistent with the above results, our data showed that gpa1 mutants were insensitive to these drugs (Supplemental Fig. S3). Therefore, our results provide the genetic evidence that Gα is involved in UV-B signal transduction in guard cells. Together with the pharmacological experiment described above, these results indicate that Gα acts as a positive regulator of guard cell responses to UV-B radiation. NADPH Oxidase-Dependent H2O2 Is Required for UV-B-Induced Stomatal Closure To evaluate the potential roles of H2O2 originated from NADPH oxidases and peroxidases in 0.5 W m−2 UV-B-induced stomatal closure in Arabidopsis, we first investigated the effects of diphenylene iodonium chloride (DPI; an inhibitor of NADPH oxidase) and salicylhydroxamic acid (SHAM; an inhibitor of cell wall peroxidase) as well as catalase (CAT; the H2O2 scavenger) and ascorbic acid (ASA; an important reducing substrate for H2O2 removal) on the UV-B-induced stomatal closure and H2O2 generation (using the H2O2 fluorescent dye 2′,7′-dichlorofluorescin diacetate [H2DCFDA]) in guard cells of wild-type Arabidopsis ecotype Columbia (Col-0). Consistent with our previous results (He et al., 2005, 2011a), UV-B had a cumulative effect over time on H2O2 production in guard cells that was evident at 1 h, substantial at 2 h, and reached maximum at 3 h (Supplemental Fig. S4). Clearly, under UV-B radiation, the significant rise in H2O2 level preceded stomatal closure (Fig. 1B; Supplemental Fig. S2). Furthermore, both ASA and CAT significantly inhibited UV-B-induced stomatal closure (Fig. 3A) and scavenged the UV-B-induced H2O2 in guard cells (Fig. 3, B and C). Together, these results suggest that H2O2 is an essential signal in the guard cell response to UV-B in Arabidopsis. However, both the UV-B-induced stomatal closure (Fig. 3A) and H2O2 generation (Fig. 3, B and C) were largely inhibited by DPI but not by SHAM, in contrast to our previous results in broad bean (Vicia faba) epidermal strips (He et al., 2011a), suggesting that 0.5 W m−2 UV-B-induced stomatal closure in Arabidopsis may be mediated by H2O2 sourced from NADPH oxidases but not from peroxidases. To confirm these pharmacological data, we further used Arabidopsis single and double mutants of AtrbohD and AtrbohF, two highly expressed Atrboh genes in guard cells (Kwak et al., 2003). Stomata of either single mutants AtrbohD and AtrbohF or double mutant AtrbohD/AtrbohF failed to close under 0.5 W m−2 UV-B (Fig. 3A; Supplemental Fig. S2). Meanwhile, UV-B also failed to induce H2O2 generation in guard cells of these mutants (Fig. 3, B and C; Supplemental Fig. S4), resembling the responses of the wild type (Col-0) pretreated with DPI but not SHAM (Fig. 3). These genetic results further demonstrate that functional AtrbohD and AtrbohF proteins are required for the both H2O2 generation and stomatal closure induced by 0.5 W m−2 UV-B. Figure 3. Open in new tabDownload slide NADPH oxidase-dependent H2O2 production regulates UV-B-induced stomatal closure. Arabidopsis leaves of wild-type (WT) Col-0 with open stomata incubated in MES buffer in the absence (Control) or presence of 100 µm ASA, 100 units mL−1 CAT, 10 µm DPI, or 5 mm SHAM or leaves of mutants AtrbohD, AtrbohF, and AtrbohD/AtrbohF with open stomata incubated in MES buffer alone were exposed to light without (Light) or with 0.5 W m−2 UV-B (UV-B) for 3 h, then epidermal strips were peeled from abaxial surfaces of the treated leaves. A, Stomatal apertures were measured in epidermal strips, and the data are presented as means ± se (n = 150). Means with different letters are significantly different at P < 0.01. B and C, Fluorescence pixel intensities (B) and images (C) in guard cells preloaded with 50 µm H2DCFDA for 10 min in darkness were recorded. Data of fluorescence pixel intensities are displayed as means ± se (n = 60). Means with different letters are significantly different at P < 0.05. Bar in C = 10 µm for all images. [See online article for color version of this figure.] Figure 3. Open in new tabDownload slide NADPH oxidase-dependent H2O2 production regulates UV-B-induced stomatal closure. Arabidopsis leaves of wild-type (WT) Col-0 with open stomata incubated in MES buffer in the absence (Control) or presence of 100 µm ASA, 100 units mL−1 CAT, 10 µm DPI, or 5 mm SHAM or leaves of mutants AtrbohD, AtrbohF, and AtrbohD/AtrbohF with open stomata incubated in MES buffer alone were exposed to light without (Light) or with 0.5 W m−2 UV-B (UV-B) for 3 h, then epidermal strips were peeled from abaxial surfaces of the treated leaves. A, Stomatal apertures were measured in epidermal strips, and the data are presented as means ± se (n = 150). Means with different letters are significantly different at P < 0.01. B and C, Fluorescence pixel intensities (B) and images (C) in guard cells preloaded with 50 µm H2DCFDA for 10 min in darkness were recorded. Data of fluorescence pixel intensities are displayed as means ± se (n = 60). Means with different letters are significantly different at P < 0.05. Bar in C = 10 µm for all images. [See online article for color version of this figure.] Combining our previous results that 0.8 W m−2 UV-B-induced H2O2 generation in broad bean guard cells depends on peroxidase but not NADPH oxidase (He et al., 2011a) with our results here that 0.5 W m−2 UV-B induces H2O2 production in Arabidopsis via NADPH oxidases but not peroxidase (Fig. 3), it might be suggested that different doses of UV-B activate distinct sources of H2O2. To confirm this suggestion, we also investigated the effect of 0.7 W m−2 UV-B on H2O2 generation in Arabidopsis. Indeed, when the dose of UV-B was up to 0.7 W m−2, it induced H2O2 generation in guard cells and stomatal closure not only in the wild type but also in the single and double mutants of AtrbohD and AtrbohF, and these effects of UV-B could be inhibited by SHAM but not by DPI (Supplemental Fig. S5), suggesting that higher doses of UV-B induce H2O2 generation in Arabidopsis guard cells also via peroxidase but not NADPH oxidases. Nia1-Dependent NO Is Involved in UV-B-Induced Stomatal Closure To evaluate the potential role of the nitrate reductase (NR) source of NO in 0.5 W m−2 UV-B-induced stomatal closure in Arabidopsis, we used both pharmacological and genetic approaches. Stomata of single mutant Nia1-2 and double mutant Nia1-2/Nia2-5 failed to close upon 0.5 W m−2 UV-B treatment, resembling the response of the wild type (Col-0) pretreated with the NR inhibitor tungstate or the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (c-PTIO), whereas the single mutant Nia2-1 responded normally to the UV-B radiation like the wild type (Fig. 4A; Supplemental Fig. S2). Furthermore, we measured NO changes using the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2DA). As shown in Figure 4, B and C, and Supplemental Figure S6, UV-B induced a dramatic increase in NO level in wild-type and Nia2-1 guard cells but not in mutant Nia1-2 and Nia1-2/Nia2-5 guard cells or wild-type guard cells pretreated with c-PTIO or tungstate. These results not only confirm the essential role of NR source of NO in the mediation of UV-B-induced stomatal closure but also indicate that it is Nia1, but not the Nia2 isoform of NR, that is responsible for NO synthesis during the UV-B-induced stomatal closure in Arabidopsis. Figure 4. Open in new tabDownload slide Nia1-dependent NO generation is required for UV-B-induced stomatal closure. Arabidopsis leaves of wild-type (WT) Col-0 with open stomata incubated in MES buffer in the absence (Control) or presence of 200 µm c-PTIO or 1 mm tungstate and leaves of single mutants Nia1-2 and Nia2-1 or double mutant Nia2-5/Nia1-2 with open stomata incubated in MES buffer alone were exposed to light without (Light) or with 0.5 W m−2 UV-B (UV-B) for 3 h. A, Stomatal apertures were measured in epidermal strips, and the data are displayed as means ± se (n = 150). Means with different letters are significantly different at P < 0.01. B and C, Fluorescence pixel intensities (B) and images (C) in guard cells preloaded with 10 µm DAF-2DA for 30 min in darkness were recorded. Data of fluorescence pixel intensities are displayed as means ± se (n = 60). Means with different letters are significantly different at P < 0.05. Bar in C = 10 µm for all images. [See online article for color version of this figure.] Figure 4. Open in new tabDownload slide Nia1-dependent NO generation is required for UV-B-induced stomatal closure. Arabidopsis leaves of wild-type (WT) Col-0 with open stomata incubated in MES buffer in the absence (Control) or presence of 200 µm c-PTIO or 1 mm tungstate and leaves of single mutants Nia1-2 and Nia2-1 or double mutant Nia2-5/Nia1-2 with open stomata incubated in MES buffer alone were exposed to light without (Light) or with 0.5 W m−2 UV-B (UV-B) for 3 h. A, Stomatal apertures were measured in epidermal strips, and the data are displayed as means ± se (n = 150). Means with different letters are significantly different at P < 0.01. B and C, Fluorescence pixel intensities (B) and images (C) in guard cells preloaded with 10 µm DAF-2DA for 30 min in darkness were recorded. Data of fluorescence pixel intensities are displayed as means ± se (n = 60). Means with different letters are significantly different at P < 0.05. Bar in C = 10 µm for all images. [See online article for color version of this figure.] Gα Modulates Both H2O2 and NO Production in UV-B-Induced Stomatal Closure To investigate the relationship between Gα and H2O2 or NO in UV-B-induced stomatal closure, we used the following experiments. First, we employed pharmacological drugs to assess whether Gα mediates UV-B-induced stomatal closure via the regulation of H2O2 or NO production in guard cells. As shown in Figures 5 and 6, the Gα inhibitor PTX obviously prevented not only the H2O2 generation (Fig. 5, A and B) but also the NO production (Fig. 6, A and B) induced by UV-B in guard cells of the wild type (Col-0), consistent with the inhibitory effect of PTX on the UV-B-induced stomatal closure (Fig. 2A). Meanwhile, the Gα activator CTX induced increases of H2O2 (Fig. 5, A and B) and NO (Fig. 6, A and B) production in wild-type guard cells under light, which could be significantly inhibited or scavenged by either CAT and DPI (Supplemental Fig. S7, A and B) or c-PTIO and tungstate (Supplemental Fig. S8, A and B). Similarly, CTX-induced stomatal closure in the wild type under light was also blocked by either CAT and DPI (Supplemental Fig. S7C) or c-PTIO and tungstate (Supplemental Fig. S8C), but CTX failed to induce both H2O2 production (Fig. 5, A and B) and stomatal closure (Fig. 5C) in the single and double mutants of AtrbohD and ArrbohF or both NO production (Fig. 6, A and B) and stomatal closure (Fig. 6C) in the mutants Nia1-2 and Nia1-2/Nia2-5. These results strongly suggest that Gα mediates the UV-B-induced stomatal closure via modulating H2O2 and NO production in guard cells. Figure 5. Open in new tabDownload slide GPA1 mediates UV-B-induced stomatal closure via H2O2 production. A and B, Leaves of wild-type (WT) Col-0 with open stomata were incubated in MES buffer in the absence (Control) or presence of 400 ng mL−1 PTX under light alone (Light) or with 0.5 W m−2 UV-B (UV-B) for 3 h, or leaves of wild-type Col-0 and single mutants AtrbohD and AtrbohF or double mutant AtrbohD/AtrbohF with open stomata were incubated in MES buffer in the presence of 400 ng mL−1 CTX under light alone for 2 h, then fluorescence images (A) and pixel intensities (B) in guard cells preloaded with 50 µm H2DCFDA were recorded. C, Leaves of wild-type Col-0 and mutants AtrbohD, AtrbohF, and AtrbohD/AtrbohF were incubated in MES buffer in the absence or presence of 400 ng mL−1 CTX under light alone for 2 h, then stomatal apertures were measured in epidermal strips. D and E, Leaves of wild-type Ws, gpa1 mutants, the cGα1 line, and the cGα1 AtrbohD/AtrbohF construct were incubated in MES buffer under light alone or with 0.5 W m−2 UV-B for 3 h, then fluorescence images (D) and pixel intensities (E) in guard cells preloaded with 50 µm H2DCFDA were recorded. F, Leaves of wild-type Ws, gpa1 mutants, and the cGα1 AtrbohD/AtrbohF construct 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 stomatal apertures were measured in epidermal strips. Each assay was repeated at least three times. Data of stomatal aperture are displayed as means ± se (n = 150), and means with different letters are significantly different at P < 0.01. Data of fluorescence intensities are displayed as means ± se (n = 60), and means with different letters are significantly different at P < 0.05. Bars in A and D = 10 µm. [See online article for color version of this figure.] Figure 5. Open in new tabDownload slide GPA1 mediates UV-B-induced stomatal closure via H2O2 production. A and B, Leaves of wild-type (WT) Col-0 with open stomata were incubated in MES buffer in the absence (Control) or presence of 400 ng mL−1 PTX under light alone (Light) or with 0.5 W m−2 UV-B (UV-B) for 3 h, or leaves of wild-type Col-0 and single mutants AtrbohD and AtrbohF or double mutant AtrbohD/AtrbohF with open stomata were incubated in MES buffer in the presence of 400 ng mL−1 CTX under light alone for 2 h, then fluorescence images (A) and pixel intensities (B) in guard cells preloaded with 50 µm H2DCFDA were recorded. C, Leaves of wild-type Col-0 and mutants AtrbohD, AtrbohF, and AtrbohD/AtrbohF were incubated in MES buffer in the absence or presence of 400 ng mL−1 CTX under light alone for 2 h, then stomatal apertures were measured in epidermal strips. D and E, Leaves of wild-type Ws, gpa1 mutants, the cGα1 line, and the cGα1 AtrbohD/AtrbohF construct were incubated in MES buffer under light alone or with 0.5 W m−2 UV-B for 3 h, then fluorescence images (D) and pixel intensities (E) in guard cells preloaded with 50 µm H2DCFDA were recorded. F, Leaves of wild-type Ws, gpa1 mutants, and the cGα1 AtrbohD/AtrbohF construct 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 stomatal apertures were measured in epidermal strips. Each assay was repeated at least three times. Data of stomatal aperture are displayed as means ± se (n = 150), and means with different letters are significantly different at P < 0.01. Data of fluorescence intensities are displayed as means ± se (n = 60), and means with different letters are significantly different at P < 0.05. Bars in A and D = 10 µm. [See online article for color version of this figure.] Figure 6. Open in new tabDownload slide GPA1 regulates the UV-B-induced stomatal closure via NO generation. A and B, Leaves of wild-type (WT) Col-0 with open stomata were incubated in MES buffer in the absence (Control) or presence of 400 ng mL−1 PTX under light alone (Light) or with 0.5 W m−2 UV-B (UV-B) for 3 h, or leaves of wild-type Col-0, single mutant Nia1-2, and double mutant Nia1-2/Nia2-5 with open stomata were incubated in MES buffer in the presence of 400 ng mL−1 CTX under light alone for 2 h, then fluorescence images (A) and pixel intensities (B) in guard cells preloaded with 10 µm DAF-2DA were recorded. C, Leaves of wild-type Col-0 and mutants Nia1-2 and Nia1-2/Nia2-5 were incubated in MES buffer in the absence or presence of 400 ng mL−1 CTX under light alone for 2 h, then stomatal apertures were measured in epidermal strips. D and E, Leaves of wild-type Ws, gpa1 mutants, the cGα1 line, and the cGα1 Nia1-2 construct were incubated in MES buffer under light alone or with 0.5 W m−2 UV-B for 3 h, then fluorescence images (D) and pixel intensities (E) in guard cells preloaded with 10 µm DAF-2DA were recorded. F, Leaves of wild-type Ws, gpa1 mutants, and the cGα1 Nia1-2 construct were incubated in MES buffer in the absence or presence of 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. Each assay was repeated at least three times. Data of stomatal aperture are displayed as means ± se (n = 150), and means with different letters are significantly different at P < 0.01. Data of fluorescence intensities are displayed as means ± se (n = 60), and means with different letters are significantly different at P < 0.05. Bars in A and D = 10 µm. [See online article for color version of this figure.] Figure 6. Open in new tabDownload slide GPA1 regulates the UV-B-induced stomatal closure via NO generation. A and B, Leaves of wild-type (WT) Col-0 with open stomata were incubated in MES buffer in the absence (Control) or presence of 400 ng mL−1 PTX under light alone (Light) or with 0.5 W m−2 UV-B (UV-B) for 3 h, or leaves of wild-type Col-0, single mutant Nia1-2, and double mutant Nia1-2/Nia2-5 with open stomata were incubated in MES buffer in the presence of 400 ng mL−1 CTX under light alone for 2 h, then fluorescence images (A) and pixel intensities (B) in guard cells preloaded with 10 µm DAF-2DA were recorded. C, Leaves of wild-type Col-0 and mutants Nia1-2 and Nia1-2/Nia2-5 were incubated in MES buffer in the absence or presence of 400 ng mL−1 CTX under light alone for 2 h, then stomatal apertures were measured in epidermal strips. D and E, Leaves of wild-type Ws, gpa1 mutants, the cGα1 line, and the cGα1 Nia1-2 construct were incubated in MES buffer under light alone or with 0.5 W m−2 UV-B for 3 h, then fluorescence images (D) and pixel intensities (E) in guard cells preloaded with 10 µm DAF-2DA were recorded. F, Leaves of wild-type Ws, gpa1 mutants, and the cGα1 Nia1-2 construct were incubated in MES buffer in the absence or presence of 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. Each assay was repeated at least three times. Data of stomatal aperture are displayed as means ± se (n = 150), and means with different letters are significantly different at P < 0.01. Data of fluorescence intensities are displayed as means ± se (n = 60), and means with different letters are significantly different at P < 0.05. Bars in A and D = 10 µm. [See online article for color version of this figure.] To confirm the role of Gα in the regulation of H2O2 and NO production in UV-B guard cell signaling, we next examined H2O2 and NO levels in guard cells of the gpa1 null mutants and of transgenic cGα lines (Okamoto et al., 2001). Levels of H2O2 (Supplemental Fig. S4) and NO (Supplemental Fig. S6) in the guard cells of two independent cGα lines were higher than that of the wild type (ecotype Ws) in response to UV-B (data from one cGα line are shown), while UV-B failed to induce H2O2 (Fig. 5, D and E; Supplemental Fig. S4) and NO (Fig. 6, D and E; Supplemental Fig. S6) accumulation in gpa1-1 and gpa1-2 mutants. These results clearly indicate that Gα is required for UV-B induction of H2O2 and NO generation. These data are consistent with the faster response of cGα stomata to UV-B (Fig. 2C) and the impaired stomatal closure of gpa1-1 and gpa1-2 with UV-B treatment (Fig. 2B; Supplemental Fig. S2). Furthermore, UV-B-triggered stomatal closure in cGα lines was blocked by either DPI and CAT (Supplemental Fig. S9A) or c-PTIO and tungstate (Supplemental Fig. S9B), and the defect of gpa1-1 and gpa1-2 in UV-B-induced stomatal closure was rescued not only by H2O2 (Fig. 5F) but also by the NO donor sodium nitroprusside (SNP; Fig. 6F), further supporting that H2O2 and NO act downstream of Gα in UV-B guard cell signaling. To further confirm the relationship between Gα and H2O2 or Gα and NO in UV-B guard cell signaling, we also crossed the cGα1 line (Okamoto et al., 2001) with the AtrbohD/AtrbohF or Noa1-2 mutant, respectively, and examined stomatal responses and the level of H2O2 or NO in guard cells of these constructs. As shown in Figure 5 and Supplemental Figures S2 and S4, UV-B failed to increase H2O2 level in guard cells of cGα1 AtrbohD/AtrbohF, and stomata of this construct also did not close when treated with UV-B. Similarly, UV-B also failed to induce NO production (Fig. 6, D and E; Supplemental Fig. S6) and subsequent stomatal closure (Fig. 6F; Supplemental Fig. S2) in the construct cGα1 Nia1-2. Taken together, our results clearly demonstrate that GPA1 G protein acts upstream of H2O2 and NO production to activate the UV-B-induced stomatal closure. Gα Regulation of NO Production in UV-B-Induced Stomatal Closure Depends on H2O2 Generation As both H2O2 and NO act downstream of Gα in UV-B-induced stomatal closure (Figs. 5 and 6) and reports from several groups imply that H2O2 induces NO generation in guard cells (He et al., 2005; Bright et al., 2006; Li et al., 2009), we finally investigated the relationship between H2O2 and NO in the Gα mediation of UV-B-induced stomatal closure. First, it is proposed that if NO acts downstream of H2O2, then the deficiency of H2O2 could be bypassed by NO, but the deficiency of NO could not be compensated by H2O2, and vice versa. Indeed, the defects of mutants AtrbohD, AtrbohF, and AtrbohD/AtrbohF in the UV-B-induced stomatal closure were significantly rescued by the NO donor SNP (Fig. 7A), but the defects of Nia1-2 and Nia1-2/Nia2-5 mutants in the UV-B-induced stomatal closure could not be compensated by H2O2 (Fig. 7D). Similarly, SNP could induce stomatal closure in mutants AtrbohD, AtrbohF, and AtrbohD/AtrbohF under light (Fig. 7A), but H2O2 failed to induce stomatal closure in mutants Nia1-2 and Nia1-2/Nia2-5 under light (Fig. 7D). These results suggest that NO acts downstream of H2O2 in the Gα mediation of UV-B-induced stomatal closure. To confirm this conclusion further, H2O2 or NO synthesis was next monitored in guard cells of the above mutants. As shown in Figure 7, B and C, UV-B failed to induce NO production in the mutants AtrbohD, AtrbohF, and AtrbohD/AtrbohF, but it could induce H2O2 generation in the mutants Nia1-2 and Nia1-2/Nia2-5 (Fig. 7, E and F). Together, the correlation between stomatal movement and change in H2O2 or NO level indicates an essential role of H2O2 generation in Gα-induced NO production during the UV-B-induced stomatal closure. Figure 7. Open in new tabDownload slide H2O2 generation is required for NO production in UV-B guard cell signaling. A to C, Leaves of wild-type (WT) Col-0 and mutants AtrbohD, AtrbohF, and AtrbohD/AtrbohF with open stomata were incubated in MES buffer in the absence or presence of 100 µm SNP and exposed to light alone (Light) or with 0.5 W m−2 UV-B (UV-B) for 3 h, then stomatal apertures were measured in epidermal strips (A) or fluorescence intensities (B) and images (C) of guard cells preloaded with 10 µm DAF-2DA were recorded. D to F, Leaves of wild-type Col-0, single mutant Nia1-2, or double mutant Nia1-2/Nia2-5 with open stomata were incubated in MES buffer in the absence or presence of 100 µm H2O2 and exposed to light alone or with 0.5 W m−2 UV-B for 3 h, then stomatal apertures were measured in epidermal strips (D) or fluorescence intensities (E) and images (F) of guard cells preloaded with 50 µm H2DCFDA were recorded. Each assay was repeated at least three times. The data of stomatal aperture are displayed as means ± se (n = 150), and means with different letters are significantly different at P < 0.01. Data of fluorescence intensities are displayed as means ± se (n = 60), and means with different letters are significantly different at P < 0.05. Bars in C and F = 10 µm. [See online article for color version of this figure.] Figure 7. Open in new tabDownload slide H2O2 generation is required for NO production in UV-B guard cell signaling. A to C, Leaves of wild-type (WT) Col-0 and mutants AtrbohD, AtrbohF, and AtrbohD/AtrbohF with open stomata were incubated in MES buffer in the absence or presence of 100 µm SNP and exposed to light alone (Light) or with 0.5 W m−2 UV-B (UV-B) for 3 h, then stomatal apertures were measured in epidermal strips (A) or fluorescence intensities (B) and images (C) of guard cells preloaded with 10 µm DAF-2DA were recorded. D to F, Leaves of wild-type Col-0, single mutant Nia1-2, or double mutant Nia1-2/Nia2-5 with open stomata were incubated in MES buffer in the absence or presence of 100 µm H2O2 and exposed to light alone or with 0.5 W m−2 UV-B for 3 h, then stomatal apertures were measured in epidermal strips (D) or fluorescence intensities (E) and images (F) of guard cells preloaded with 50 µm H2DCFDA were recorded. Each assay was repeated at least three times. The data of stomatal aperture are displayed as means ± se (n = 150), and means with different letters are significantly different at P < 0.01. Data of fluorescence intensities are displayed as means ± se (n = 60), and means with different letters are significantly different at P < 0.05. Bars in C and F = 10 µm. [See online article for color version of this figure.] DISCUSSION In this report, we have provided convincing evidence for a linear signaling pathway controlling 0.5 W m−2 UV-B-induced stomatal closure in Arabidopsis. Our combined genetic and pharmacological analyses show that the activation of Gα of the heterotrimeric G protein, NADPH oxidase-dependent H2O2 generation, and Nia1-dependent NO production are all required for the UV-B-induced stomatal closure. Furthermore, we found that UV-B-triggered H2O2 and NO production are mediated by the activation of Gα. We also showed that the modulation of NO production by Gα requires NADPH oxidase-dependent H2O2 generation. Therefore, our results support a UV-B-activated guard cell signaling pathway that includes a cascade of UV-B, Gα, NADPH oxidase-dependent H2O2, and Nia1-dependent NO. Gα Plays an Important Role in UV-B Guard Cell Signaling In plants, heterotrimeric G proteins composed of Gα-, Gβ-, and Gγ-subunits function as important signaling agents mediating responses to a range of biotic and abiotic signals (Assmann, 2005; Temple and Jones, 2007; Warpeha et al., 2007; Okamoto et al., 2009; Nilson and Assmann, 2010). In animal cells, it has been shown that UV-B activates the Gα-Gβγ complex to release free Gα and Gβγ, both of which result in HB-EGF secretion and subsequent activation of the EGF signal transduction pathway (Seo et al., 2004, 2007; Seo and Juhnn, 2010), suggesting that both Gα and Gβγ are functional units to transmit UV-B signaling. However, until now, whether G proteins participate in the responses of plant cells to UV-B was not known. Our results in this report provide convincing evidence that Gα of the heterotrimeric G proteins in Arabidopsis plays an important role in the regulation of 0.5 W m−2 UV-B-induced stomatal closure. The Gα inhibitor PTX completely inhibited UV-B-induced stomatal closure, and the Gα activator CTX significantly induced stomatal closure under light, indicating that the activation of Gα is required for the UV-B-induced stomatal closure. The defect of null mutants for GPA1 in UV-B-induced stomatal closure and the faster stomatal closure of cGα lines further provide genetic evidence for the essential role of Gα in UV-B guard cell signaling (Fig. 2). Although our results here show an important role of Gα in UV-B guard cell signaling, whether Gβγ and Gα can act additively or synergically to mediate UV-B signaling in guard cells and other plant cells is still an interesting question to be addressed in the future. NADPH Oxidase- and Peroxidase-Dependent H2O2 Mediate Different Doses of UV-B-Induced Stomatal Closure H2O2 is one of the extensively studied signaling components involved in guard cell signaling (Neill et al., 2002; Wang and Song, 2008), and its origin in guard cells has been proposed to be multiple sources, including cell wall peroxidases (Khokon et al., 2010), NADPH oxidases (Kwak et al., 2003; Chen et al., 2004; Joo et al., 2005; Bright et al., 2006; Sagi and Fluhr, 2006; Li et al., 2009), and amine oxidase-type enzymes (An et al., 2008). With regard to the enzymatic source of H2O2 mediating UV-B-induced stomatal closure, our previous study has provided pharmacological evidence that 0.8 W m−2 UV-B-induced H2O2 generation in broad bean guard cells depends on peroxidases but not NADPH oxidases (He et al., 2011a). However, UV-B is reported to stimulate mRNA transcript levels and the activity of NADPH oxidases (Rao et al., 1996; A-H-Mackerness et al., 2001; Casati and Walbot, 2003), and mutants in the Arabidopsis AtrbohD and AtrbohF genes show reduced H2O2 accumulation in response to UV-B (Kalbina and Strid, 2006), suggesting that NADPH oxidases are also potential sources of H2O2 induced by UV-B. To explore whether different doses of UV-B can activate distinct sources of H2O2 to induce stomatal closure, we examined H2O2 sources during stomatal closure induced by 0.5 and 0.7 W m−2 UV-B. The data presented here clearly showed that 0.5 W m−2 UV-B-induced H2O2 production in guard cells and subsequent stomatal closure in wild-type Arabidopsis were significantly inhibited by the NADPH oxidase inhibitor DPI but not by the cell wall peroxidase inhibitor SHAM. Furthermore, the NADPH oxidase gene null mutants AtrbohD, AtrbohF, and AtrbohD/AtrbohF were also defective in this dose of UV-B-induced H2O2 generation and stomatal closure (Fig. 3; Supplemental Fig. S4). In contrast, consistent with our previous results in broad bean (He et al., 2011a), 0.7 W m−2 UV-B-induced H2O2 production and stomatal closure were observed not only in the wild type but also in the three NADPH oxidase mutants, and these effects were significantly reduced by SHAM but not by DPI (Supplemental Fig. S5). Clearly, our data provide convincing evidence that the low dose (0.5 W m−2) of UV-B induces H2O2 generation in guard cells and subsequent stomatal closure through NADPH oxidases and that both AtrbohD and AtrbohF NADPH oxidase genes are required for H2O2 generation, while the high dose (over 0.7 W m−2) of UV-B induces H2O2 production and subsequent stomatal closure mainly via cell wall peroxidases. A-H-Mackerness et al. (2001) have shown that the same dose of UV-B may activate diverse H2O2-generating pathways to produce the signals leading to different physiological processes. Our results here further show that the different doses of UV-B can also activate distinct sources of H2O2 to participate in the same physiological process. Kwak et al. (2003) observed that ABA-induced stomatal closing was partially impaired in AtrbohF and more strongly impaired in AtrbohD/AtrbohF, while the response of AtrbohD to ABA was the same as that of the wild type. So the authors suggest that there is some overlap in the functions of AtrbohD and AtrbohF. However, in our study, we observed that knockout of AtrbohD or AtrbohF both impaired the UV-B-induced H2O2 production in guard cells and subsequent stomatal closure (Fig. 3; Supplemental Figs. S2 and S4). These responses of both AtrbohD and AtrbohF to UV-B were similar to their responses to PA (Zhang et al., 2009) but also different from their responses to ethylene, as only AtrbohF appears essential for ethylene-induced H2O2 production and stomatal closure (Desikan et al., 2006). Combined together, we suggest that AtrbohD and AtrbohF may have different functions in mediating the responses of some stimuli such as ABA and ethylene but interact to mediate responses of other stimuli such as UV-B and PA. Nia1-Dependent NO Mediates UV-B-Induced Stomatal Closure Recently, the role of NO in the regulation of stomatal closure has been well documented, and a growing body of literature shows that the potential enzymatic sources of NO in guard cells are nitric oxide synthase (NOS) and NR (Neill et al., 2002, 2008; Qiao and Fan, 2008). However, so far, no genes or proteins with sequence homology to known mammalian-type NOS have been found in plants (Guo et al., 2003; Crawford, 2006). In Arabidopsis, NOS1/NOA1, which was identified originally as a potential NOS (Guo et al., 2003), was found to be unable to bind and oxidize Arg to NO and was later shown to be a circularly permuted GTPase (Moreau et al., 2008). Contrarily, there is a considerable body of genetic evidence to support a role for NR in NO production in plants (Crawford, 2006; Neill et al., 2008; Wilson et al., 2008). Our previous pharmacological evidence supports a role for the NR source of NO in mediating UV-B-induced stomatal closure in broad bean (He et al., 2011b). However, in Arabidopsis, two genes, Nia1 and Nia2, encoding NR have been cloned (Bright et al., 2006; Neill et al., 2008). So we would like to further know whether Nia1 and/or Nia2 mediate UV-B-activated NO generation in guard cells. In this report, our genetic evidence clearly shows that it is Nia1, not Nia2, that mediates 0.5 W m−2 UV-B-induced NO generation in guard cells and the subsequent stomatal closure in Arabidopsis. The UV-B-induced NO production in guard cells and the subsequent stomatal closure in the wild type were completely impaired in single mutant Nia1-2 and double mutant Nia1-2/Nia2-5, but the single mutant Nia2-1 responded normally to the UV-B radiation, as did the wild type (Fig. 4; Supplemental Figs. S2 and S6). Similar to our results, Bright et al. (2006) also reported that Nia1 is mainly responsible for the ABA-induced NO generation in guard cells. However, this is not the case in SA-induced NO production in guard cells, as NO production in guard cells and stomatal closure induced by SA in single mutants Nia1 and Nia2 were only partially suppressed in comparison with the double mutant Nia1/Nia2. Furthermore, the NO production and stomatal response of Nia1 are more sensitive to SA than those of Nia2 (Hao et al., 2010). These differences maybe suggest that Nia1 and Nia2 have differential mechanisms of activation and, thus, they respond differentially to different stimuli. A Linear Signaling Pathway Including Gα, H2O2, and NO Controls the UV-B-Induced Stomatal Closure Guard cells in gpa1 mutants were insensitive to ABA inhibition of inward K+ currents, activation of pH-independent anion channels, and inhibition of stomatal opening; thus, it is speculated that Gα regulates ion channels directly in guard cells (Wang et al., 2001). However, modulation of H2O2 by Gα has been found in ABA-, ExtCaM-, and extracellular ATP-regulated stomatal movement (Chen et al., 2004; Li et al., 2009; Zhang et al., 2011; Hao et al., 2012). In addition, regulation of NO by Gα has also been found in ExtCaM-induced stomatal closure (Li et al., 2009). Those reports indicate that Gα may play a central mediating role in guard cell signaling. However, it is not known whether Gα mediates UV-B-induced stomatal closure also via the modulation of H2O2 and NO. Our results here provide positive evidence for this speculation. gpa1 guard cells generated less H2O2 and NO and cGα lines generated higher levels of H2O2 and NO when irradiated with 0.5 W m−2 UV-B. Meanwhile, cGα AtrbohD/AtrbohF and cGα Nia1-2 constructs had similar responses to UV-B as AtrbohD/AtrbohF and Nia1-2, respectively. The defect of gpa1 mutants in the UV-B-induced stomatal closure was rescued by H2O2 and SNP (Figs. 5 and 6). These results convincingly confirm that Gα acts upstream of H2O2 and NO generation in UV-B-triggered guard cell signaling. Furthermore, our results also indicate that the UV-B-induced H2O2 synthesis is essential for NO production in Arabidopsis, since UV-B-induced NO production was greatly impaired in the guard cells of mutants AtrbohD, AtrbohF, and AtrbohD/AtrbohF, but the UV-B-induced H2O2 generation was not greatly impaired in the guard cells of Nia1-2 and Nia1-2/Nia2-5 (Fig. 7). This relationship between H2O2 and NO in stomatal movement is also supported by the findings of Bright et al. (2006) and Li et al. (2009) but somewhat different from our previous finding that an interrelationship between H2O2 and NO exists in 0.8 W m−2 UV-B-induced stomatal closure in broad bean epidermal strips (He et al., 2005), which discrepancy may be due to the different plant species or UV-B dosages used. In summary, this study has provided convincing evidence for a linear intracellular signaling pathway leading to 0.5 W m−2 UV-B-induced stomatal closure in Arabidopsis, which involves GPA1-dependent activation of H2O2 production by both AtrbohD and AtrbohF and subsequent Nia1-dependent NO accumulation. These findings not only extend the functions of G proteins but also provide clues to further understand the UV-B signal transduction mechanisms in plants. However, several remaining questions should be further studied in the future. First, although this study clearly shows that different doses of UV-B can activate distinct sources of H2O2 to induce stomatal closure, whether these results suggest that there is a switch of low-dosage UV-B-induced specific signaling to high-dosage UV-B-produced nonspecific stress response on stomata is still unclear. Previous studies have shown that different responses and signaling pathways are observed at different UV-B fluence rates. In general, exposure to high fluence rates of UV-B is likely to cause stress responses and possibly necrosis, in part through UV-B-nonspecific signaling pathways such as stress-signaling pathways. In contrast, low fluence rates of UV-B are sufficient to initiate regulatory responses via UV-B-specific signaling pathways (Heijde and Ulm, 2012; Jansen and Bornman, 2012). Recently, the UVR8 photoreceptor-mediated pathway was identified as a UV-B-specific signaling pathway that is crucial for UV-B acclimation and tolerance, but few of the molecular players, such as COP1 and HY5, involved in this specific pathway are currently known (Rizzini et al., 2011; Christie et al., 2012; Heijde and Ulm, 2012), and whether this UVR8-dependent UV-B signaling pathway also mediates other responses, such as the guard cell response to UV-B, is still not clear. To further explore the new molecular players and better understand the functions of the UVR8-dependent UV-B signaling pathways in plants, future studies on the relationship between the UVR8- and Gα-dependent UV-B signaling pathways are still necessary. Second, it remains to be elucidated how UV-B can activate Gα. Since the UV-B guard cell response is clearly visible only after 3 h of radiation (Fig. 1B), which is clearly different from the faster stomatal responses to other signals such as ABA and light (Wang et al., 2001; Neill et al., 2008; Li et al., 2009), a UV-B indirect effect via modulating other signals to activate Gα may be plausible. Recently, it has been shown that some UV-B responses are ABA dependent in leaves of different plant species (Tossi et al., 2009; Berli et al., 2010). Meanwhile, a similar pathway to UV-B guard cell signaling appears to be responsible for the ABA regulation of stomatal movement (Wang et al., 2001; Kwak et al., 2003; Bright et al., 2006; Liu et al., 2007; Zhang et al., 2011). However, Wang et al. (2001) clearly showed that Gα is only involved in ABA inhibition of stomatal opening, not ABA-induced stomatal closure, which is obviously different from the role of Gα in UV-B guard cell signaling. Thus, in the UV-B-induced guard cell signaling pathway, UV-B may not activate Gα or Gα-coupled receptors (GCR) by ABA, but whether it activates Gα or GCR by UV-B photoreceptors such as UVR8 or other molecular players has yet to be studied. Another possible mechanism is that UV-B might activate GCR directly, as photons activate rhodopsins, a kind of GCR in the retina (Palczewski, 2006). Finally, although our study has well established that a linear intracellular signaling pathway including GPA1-dependent H2O2 production and subsequent NO accumulation controls the low dose of UV-B-induced stomatal closure, the mechanism by which UV-B-dependent NO induces stomatal closure is still unclear. The structure and dynamics of cytoskeleton, the main cellular players in stomatal movement (Huang et al., 2009; Eisinger et al., 2012a, 2012b) that can control ion channels in guard cells (Zhang et al., 2007), have been shown to be regulated by NO as well as H2O2 (Huang et al., 2009; Wilkins et al., 2011; Yemets et al., 2011). Recent evidence also suggests that NO mediates UV-B signaling in plant cells by modulating cytoskeleton (Yemets et al., 2011; Krasylenko et al., 2012). Thus, whether the UV-B-dependent NO induces stomatal closure via modulating cytoskeleton and the subsequent activation of ion channels is an interesting question to be addressed in the future. MATERIALS AND METHODS Plant Materials and Growth Conditions Seeds of the wild type and various mutants of Arabidopsis (Arabidopsis thaliana) were sown in potting mix and grown in plant growth chambers under a 16-h-light/8-h-dark cycle, a photon flux density of 0.1 mmol m−2 s−1 (provided by QML YZ26RR16/G fluorescent tubes with no emission below 330 nm [Chaomei Lighting Electric Appliance] and measured with a Skye RS232 meter equipped with a Quantum sensor [Skye Instruments]), and a day/night temperature cycle of 18°C/22°C. Seeds of cGα lines (background Ws) were obtained from Dr. L.G. Ma (Hebei Normal University). cGα plants were grown in the presence of 70 nm dexamethasone (Sigma) according to the method described by Okamoto et al. (2001). Seeds of the wild type and gpa1-1, gpa1-2, AtrbohD, AtrbohF, AtrbohD/AtrbohF, Nia1-2, Nia2-1, and Nia2-5/Nia1-2 mutants were obtained from the Nottingham Arabidopsis Stock Center. Genotypes of all mutants were confirmed by PCR analysis. Fully expanded leaves were harvested for immediate use at 4 to 6 weeks. Stomatal Bioassay Stomatal assays were performed on leaves essentially as described by Desikan et al. (2006). Briefly, freshly prepared leaves were first floated under the same light condition as that described above, with their abaxial surfaces facing up on MES-KCl buffer (10 mm MES, 50 mm KCl, and 0.1 mm CaCl2, pH 6.15) for 3 h to open the stomata, and then floated on the buffer alone or containing 400 ng mL−1 PTX, 400 ng mL−1 CTX, 100 µm ASA, 100 units mL−1 CAT, 10 µm DPI, 5 mm SHAM, 200 µm c-PTIO, 1 mm tungstate, 100 µm SNP, or 100 µm H2O2 for another 1 to 4 h under the same white light condition with or without supplementary UV-B irradiation. Control treatments involved the addition of buffer or appropriate solvents used with inhibitors. All chemicals were obtained from Sigma-Aldrich. After treatments, the epidermal strips were immediately peeled carefully from the abaxial surface of leaves, and stomatal apertures were then measured with a microscope. Fifty stomata were randomly selected for three independent repeats. The data are presented as means ± se (n = 150). UV-B Radiation UV-B irradiation was generated by filtered 40-W Q-panel UV 313 lamps (Largo; its maximum output is at 313 nm). The lamps were suspended above and perpendicular to the abaxial surface of leaves and filtered with 0.13-mm-thick cellulose diacetate (transmission down to 290 nm) for UV-B radiation or 0.13-nm polyester plastic films (which absorb all radiation below 320 nm) as a control. The desired radiation dose rate was obtained by adjusting the distance between the lamps and the leaves. The spectral irradiation from the lamps was determined with an Optronics Laboratories model 742 spectroradiometer and weighted with the generalized plant response action spectrum normalized to 300 nm. Measurement of Endogenous H2O2 and NO and Cell Viability by Confocal Laser Scanning Microscopy H2O2 and NO measurements in guard cells were performed with the fluorescent indicator dyes H2DCFDA and DAF-2DA, respectively, as described previously (He et al., 2005). Assays of guard cell viability were performed with the fluorescent indicator dyes FAD and PI (Jones and Senft, 1985). After the treatments described above, the epidermal strips were immediately peeled carefully from the abaxial surface of the treated leaves and placed into Tris-KCl buffer (10 mm Tris and 50 mm KCl, pH 7.2) containing 50 µm H2DCFDA, 10 µg mL−1 FAD, or 5 µg mL−1 PI for 10 min or containing 10 µm DAF-2DA for 30 min in the dark at 25°C. Excess dye was removed with fresh Tris-KCl buffer in the dark, and an examination of the peels was immediately performed with a TCS-SP2 confocal laser scanning microscope (Leica Lasertechnik) with the following settings: excitation at 488 nm and emission at 530 nm for H2DCFDA, DAF-2DA, and FAD or excitation at 536 nm and emission at 617 nm for PI. Images acquired from the confocal microscope were processed with Photoshop software and analyzed with Leica Image software. In each experiment, three epidermal strips were measured, each of which originated from a different plant. Each experiment was repeated three times. The selected confocal images represented the same results from approximately nine time measurements. Statistical Analysis Statistical analyses were performed using a one-way ANOVA followed by the lsd test. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers At2g26300 (GPA1), At5g47910 (rbohD), At1g64060 (rbohF), At1g77760 (nia1), and At1g37130 (nia2). Supplemental Data The following materials are available in the online version of this article. Supplemental Figure S1. Effects of UV-B radiation, PTX, and CTX on guard cell viability. Supplemental Figure S2. Time courses of stomatal responses of wild-type Ws and Col-0 and all the mutants used in this study to 0.5 W m−2 UV-B radiation. Supplemental Figure S3. Effects of PTX and CTX on the stomatal movement of gpa1 mutants gpa1-1 and gpa1-2. Supplemental Figure S4. Time courses of H2O2 production induced by 0.5 W m−2 UV-B in guard cells of wild-type Ws and Col-0 and all the mutants or constructs used in this study. Supplemental Figure S5. Effects of 0.7 W m−2 UV-B on stomatal aperture and H2O2 production in guard cells of wild-type Col-0 and Atrboh mutants. Supplemental Figure S6. Time courses of NO production induced by 0.5 W m−2 UV-B in guard cells of wild-type Ws and Col-0 and all the mutants or constructs used in this study. Supplemental Figure S7. CTX-induced H2O2 generation in guard cells and subsequent stomatal closure were prevented by CAT and DPI. Supplemental Figure S8. CTX-induced NO production in guard cells and subsequent stomatal closure were abolished by c-PTIO and tungstate. Supplemental Figure S9. UV-B-triggered stomatal closure in wild-type Ws and the cGα1 line was blocked by either DPI and CAT or c-PTIO and tungstate. ACKNOWLEDGMENTS We thank Dr. L.G. Ma and the Nottingham Arabidopsis Stock Center for providing Arabidopsis seeds. We also thank Drs. S. McCormick and G. Wu for their helpful discussion and critical reading of the manuscript. Glossary ABA abscisic acid PA phosphatidic acid ExtCaM extracellular calmodulin H2O2 hydrogen peroxide NO nitric oxide SA salicylic acid ROS reactive oxygen species PI propidium iodide PTX pertussis toxin CTX cholera toxin Ws Wassilewskija DPI diphenylene iodonium chloride SHAM salicylhydroxamic acid CAT catalase ASA ascorbic acid H2DCFDA 2′,7′-dichlorofluorescin diacetate Col-0 Columbia NR nitrate reductase c-PTIO 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide DAF-2DA 4,5-diaminofluorescein diacetate SNP sodium nitroprusside NOS nitric oxide synthase GCR Gα-coupled receptors FAD fluorescein diacetate LITERATURE CITED A-H-Mackerness S John CF Jordan B Thomas B ( 2001 ) Early signaling components in ultraviolet-B responses: distinct roles for different reactive oxygen species and nitric oxide . FEBS Lett 489 : 237 – 242 Google Scholar Crossref Search ADS PubMed WorldCat An Z Jing W Liu YL Zhang WH ( 2008 ) Hydrogen peroxide generated by copper amine oxidase is involved in abscisic acid-induced stomatal closure in Vicia faba . J Exp Bot 59 : 815 – 825 Google Scholar Crossref Search ADS PubMed WorldCat Assmann SM ( 2005 ) G proteins go green: a plant G protein signaling FAQ sheet . Science 310 : 71 – 73 Google Scholar Crossref Search ADS PubMed WorldCat Berli FJ Moreno D Piccoli P Hespanhol-Viana L Silva MF Bressan-Smith R Cavagnaro JB Bottini R ( 2010 ) Abscisic acid is involved in the response of grape (Vitis vinifera L.) cv. Malbec leaf tissues to ultraviolet-B radiation by enhancing ultraviolet-absorbing compounds, antioxidant enzymes and membrane sterols . Plant Cell Environ 33 : 1 – 10 Google Scholar PubMed OpenURL Placeholder Text WorldCat Bright J Desikan R Hancock JT Weir IS Neill SJ ( 2006 ) ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis . Plant J 45 : 113 – 122 Google Scholar Crossref Search ADS PubMed WorldCat Brown BA Jenkins GI ( 2008 ) UV-B signaling pathways with different fluence-rate response profiles are distinguished in mature Arabidopsis leaf tissue by requirement for UVR8, HY5, and HYH . Plant Physiol 146 : 576 – 588 Google Scholar Crossref Search ADS PubMed WorldCat Caldwell MM ( 1971 ) Solar UV-B irradiation and the growth and development of higher plant. In AC Giese, ed, Photophysiology, Vol 6. Academic Press, New York, pp 131–137 Casati P Walbot V ( 2003 ) Gene expression profiling in response to ultraviolet radiation in maize genotypes with varying flavonoid content . Plant Physiol 132 : 1739 – 1754 Google Scholar Crossref Search ADS PubMed WorldCat Chen YL Huang R Xiao YM Lü P Chen J Wang XC ( 2004 ) Extracellular calmodulin-induced stomatal closure is mediated by heterotrimeric G protein and H2O2 . Plant Physiol 136 : 4096 – 4103 Google Scholar Crossref Search ADS PubMed WorldCat Christie JM Arvai AS Baxter KJ Heilmann M Pratt AJ O’Hara A Kelly SM Hothorn M Smith BO Hitomi K et al. ( 2012 ) Plant UVR8 photoreceptor senses UV-B by tryptophan-mediated disruption of cross-dimer salt bridges . Science 335 : 1492 – 1496 Google Scholar Crossref Search ADS PubMed WorldCat Coursol S Fan LM Le Stunff H Spiegel S Gilroy S Assmann SM ( 2003 ) Sphingolipid signalling in Arabidopsis guard cells involves heterotrimeric G proteins . Nature 423 : 651 – 654 Google Scholar Crossref Search ADS PubMed WorldCat Coursol S Le Stunff H Lynch DV Gilroy S Assmann SM Spiegel S ( 2005 ) Arabidopsis sphingosine kinase and the effects of phytosphingosine-1-phosphate on stomatal aperture . Plant Physiol 137 : 724 – 737 Google Scholar Crossref Search ADS PubMed WorldCat Crawford NM ( 2006 ) Mechanisms for nitric oxide synthesis in plants . J Exp Bot 57 : 471 – 478 Google Scholar Crossref Search ADS PubMed WorldCat Desikan R Last K Harrett-Williams R Tagliavia C Harter K Hooley R Hancock JT Neill SJ ( 2006 ) Ethylene-induced stomatal closure in Arabidopsis occurs via AtrbohF-mediated hydrogen peroxide synthesis . Plant J 47 : 907 – 916 Google Scholar Crossref Search ADS PubMed WorldCat Eisinger W Ehrhardt D Briggs W ( 2012 a ) Microtubules are essential for guard-cell function in Vicia and Arabidopsis . Mol Plant 5 : 601 – 610 Google Scholar Crossref Search ADS PubMed WorldCat Eisinger WR Kirik V Lewis C Ehrhardt DW Briggs WR ( 2012 b ) Quantitative changes in microtubule distribution correlate with guard cell function in Arabidopsis . Mol Plant 5 : 716 – 725 Google Scholar Crossref Search ADS PubMed WorldCat Fan LM Zhang W Chen JG Taylor JP Jones AM Assmann SM ( 2008 ) Abscisic acid regulation of guard-cell K+ and anion channels in Gbeta- and RGS-deficient Arabidopsis lines . Proc Natl Acad Sci USA 105 : 8476 – 8481 Google Scholar Crossref Search ADS PubMed WorldCat Frohnmeyer H Staiger D ( 2003 ) Ultraviolet-B radiation-mediated responses in plants: balancing damage and protection . Plant Physiol 133 : 1420 – 1428 Google Scholar Crossref Search ADS PubMed WorldCat Guo FQ Okamoto M Crawford NM ( 2003 ) Identification of a plant nitric oxide synthase gene involved in hormonal signaling . Science 302 : 100 – 103 Google Scholar Crossref Search ADS PubMed WorldCat Hao F Zhao S Dong H Zhang H Sun L Miao C ( 2010 ) Nia1 and Nia2 are involved in exogenous salicylic acid-induced nitric oxide generation and stomatal closure in Arabidopsis . J Integr Plant Biol 52 : 298 – 307 Google Scholar Crossref Search ADS PubMed WorldCat Hao L-H Wang W-X Chen C Wang Y-F Liu T Li X Shang Z-L ( 2012 ) Extracellular ATP promotes stomatal opening of Arabidopsis thaliana through heterotrimeric G protein α subunit and reactive oxygen species. Mol Plant 5 : 852 – 864 He J-M She X-P Liu C Zhao W-M ( 2004 ) [Stomatal and nonstomatal limitations of photosynthesis in mung bean leaves under the combination of enhanced UV-B radiation and NaCl stress] . Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao 30 : 53 – 58 Google Scholar PubMed OpenURL Placeholder Text WorldCat He J-M Xu H She X-P Song X-G Zhao W-M ( 2005 ) The role and the interrelationship of hydrogen peroxide and nitric oxide in the UV-B induced stomatal closure in broad bean . Funct Plant Biol 32 : 237 – 247 Google Scholar Crossref Search ADS PubMed WorldCat He J-M Yue X-Z Wang R-B Zhang Y ( 2011 a ) Ethylene mediates UV-B-induced stomatal closure via peroxidase-dependent hydrogen peroxide synthesis in Vicia faba L . J Exp Bot 62 : 2657 – 2666 Google Scholar Crossref Search ADS PubMed WorldCat He J-M Zhang Z Wang R-B Chen Y-P ( 2011 b ) UV-B-induced stomatal closure via ethylene-dependent NO generation in Vicia faba . Funct Plant Biol 38 : 293 – 302 Google Scholar Crossref Search ADS PubMed WorldCat Heijde M Ulm R ( 2012 ) UV-B photoreceptor-mediated signalling in plants . Trends Plant Sci 17 : 230 – 237 Google Scholar Crossref Search ADS PubMed WorldCat Huang A-X She X-P Cao B Zhang B Mu J Zhang S-J ( 2009 ) Nitric oxide, actin reorganization and vacuoles change are involved in PEG 6000-induced stomatal closure in Vicia faba . Physiol Plant 136 : 45 – 56 Google Scholar Crossref Search ADS PubMed WorldCat Jansen MAK Bornman JF ( 2012 ) UV-B radiation: from generic stressor to specific regulator . Physiol Plant 145 : 501 – 504 Google Scholar Crossref Search ADS PubMed WorldCat Jansen MAK Noort REVD ( 2000 ) Ultraviolet-B radiation induces complex alterations in stomatal behaviour . Physiol Plant 110 : 189 – 194 Google Scholar Crossref Search ADS WorldCat Jones KH Senft JA ( 1985 ) An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide . J Histochem Cytochem 33 : 77 – 79 Google Scholar Crossref Search ADS PubMed WorldCat Joo JH Wang S Chen JG Jones AM Fedoroff NV ( 2005 ) Different signaling and cell death roles of heterotrimeric G protein α and β subunits in the Arabidopsis oxidative stress response to ozone . Plant Cell 17 : 957 – 970 Google Scholar Crossref Search ADS PubMed WorldCat Kalbina I Strid A ( 2006 ) The role of NADPH oxidase and MAP kinase phosphatase in UV-B-dependent gene expression in Arabidopsis . Plant Cell Environ 29 : 1783 – 1793 Google Scholar Crossref Search ADS PubMed WorldCat Khokon MAR Hossain MA Munemasa S Uraji M Nakamura Y Mori IC Murata Y ( 2010 ) Yeast elicitor-induced stomatal closure and peroxidase-mediated ROS production in Arabidopsis . Plant Cell Physiol 51 : 1915 – 1921 Google Scholar Crossref Search ADS PubMed WorldCat Krasylenko YA Yemets AI Sheremet YA Blume YB ( 2012 ) Nitric oxide as a critical factor for perception of UV-B irradiation by microtubules in Arabidopsis . Physiol Plant 145 : 505 – 515 Google Scholar Crossref Search ADS PubMed WorldCat Kwak JM Mori IC Pei ZM Leonhardt N Torres MA Dangl JL Bloom RE Bodde S Jones JDG Schroeder JI ( 2003 ) NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis . EMBO J 22 : 2623 – 2633 Google Scholar Crossref Search ADS PubMed WorldCat Li JH Liu YQ Lü P Lin HF Bai Y Wang XC Chen YL ( 2009 ) A signaling pathway linking nitric oxide production to heterotrimeric G protein and hydrogen peroxide regulates extracellular calmodulin induction of stomatal closure in Arabidopsis . Plant Physiol 150 : 114 – 124 Google Scholar Crossref Search ADS PubMed WorldCat Liu X Yue Y Li B Nie Y Li W Wu WH Ma L ( 2007 ) A G protein-coupled receptor is a plasma membrane receptor for the plant hormone abscisic acid . Science 315 : 1712 – 1716 Google Scholar Crossref Search ADS PubMed WorldCat Mishra G Zhang W Deng F Zhao J Wang X ( 2006 ) A bifurcating pathway directs abscisic acid effects on stomatal closure and opening in Arabidopsis . Science 312 : 264 – 266 Google Scholar Crossref Search ADS PubMed WorldCat Moreau M Lee GI Wang Y Crane BR Klessig DF ( 2008 ) AtNOS/AtNOA1 is a functional Arabidopsis thaliana cGTPase and not a nitric-oxide synthase. J Biol Chem 283: 32957–32967 Musil CF Wand SJE ( 1993 ) Responses of sclerophyllous Ericaceae to enhanced level of ultraviolet-B radiation . Environ Exp Bot 33 : 233 – 242 Google Scholar Crossref Search ADS WorldCat Neill S Barros R Bright J Desikan R Hancock J Harrison J Morris P Ribeiro D Wilson I ( 2008 ) Nitric oxide, stomatal closure, and abiotic stress . J Exp Bot 59 : 165 – 176 Google Scholar Crossref Search ADS PubMed WorldCat Neill SJ Desikan R Clarke A Hurst RD Hancock JT ( 2002 ) Hydrogen peroxide and nitric oxide as signalling molecules in plants . J Exp Bot 53 : 1237 – 1247 Google Scholar Crossref Search ADS PubMed WorldCat Nilson SE Assmann SM ( 2010 ) The α-subunit of the Arabidopsis heterotrimeric G protein, GPA1, is a regulator of transpiration efficiency . Plant Physiol 152 : 2067 – 2077 Google Scholar Crossref Search ADS PubMed WorldCat Nogués S Allen DJ Morison JIL Baker NR ( 1999 ) Characterization of stomatal closure caused by ultraviolet-B radiation . Plant Physiol 121 : 489 – 496 Google Scholar Crossref Search ADS PubMed WorldCat Okamoto H Göbel C Capper RG Saunders N Feussner I Knight MR ( 2009 ) The α-subunit of the heterotrimeric G-protein affects jasmonate responses in Arabidopsis thaliana . J Exp Bot 60 : 1991 – 2003 Google Scholar Crossref Search ADS PubMed WorldCat Okamoto H Matsui M Deng XW ( 2001 ) Overexpression of the heterotrimeric G-protein α-subunit enhances phytochrome-mediated inhibition of hypocotyl elongation in Arabidopsis . Plant Cell 13 : 1639 – 1652 Google Scholar PubMed OpenURL Placeholder Text WorldCat Oldham WM Hamm HE ( 2008 ) Heterotrimeric G protein activation by G-protein-coupled receptors . Nat Rev Mol Cell Biol 9 : 60 – 71 Google Scholar Crossref Search ADS PubMed WorldCat Palczewski K ( 2006 ) G protein-coupled receptor rhodopsin . Annu Rev Biochem 75 : 743 – 767 Google Scholar Crossref Search ADS PubMed WorldCat Pandey S Assmann SM ( 2004 ) The Arabidopsis putative G protein-coupled receptor GCR1 interacts with the G protein α subunit GPA1 and regulates abscisic acid signaling . Plant Cell 16 : 1616 – 1632 Google Scholar Crossref Search ADS PubMed WorldCat Qiao W Fan LM ( 2008 ) Nitric oxide signaling in plant responses to abiotic stresses . J Integr Plant Biol 50 : 1238 – 1246 Google Scholar Crossref Search ADS PubMed WorldCat Rao MV Paliyath G Ormrod DP ( 1996 ) Ultraviolet-B- and ozone-induced biochemical changes in antioxidant enzymes of Arabidopsis thaliana . Plant Physiol 110 : 125 – 136 Google Scholar Crossref Search ADS PubMed WorldCat Rizzini L Favory J-J Cloix C Faggionato D O’Hara A Kaiserli E Baumeister R Schäfer E Nagy F Jenkins GI et al. ( 2011 ) Perception of UV-B by the Arabidopsis UVR8 protein . Science 332 : 103 – 106 Google Scholar Crossref Search ADS PubMed WorldCat Sagi M Fluhr R ( 2006 ) Production of reactive oxygen species by plant NADPH oxidases . Plant Physiol 141 : 336 – 340 Google Scholar Crossref Search ADS PubMed WorldCat Seo M Cho CH Lee YI Shin EY Park D Bae CD Lee JW Lee ES Juhnn YS ( 2004 ) Cdc42-dependent mediation of UV-induced p38 activation by G protein betagamma subunits . J Biol Chem 279 : 17366 – 17375 Google Scholar Crossref Search ADS PubMed WorldCat Seo M Juhnn YS ( 2010 ) Gq protein mediates UVB-induced cyclooxygenase-2 expression by stimulating HB-EGF secretion from HaCaT human keratinocytes . Biochem Biophys Res Commun 393 : 190 – 195 Google Scholar Crossref Search ADS PubMed WorldCat Seo M Lee MJ Heo JH Lee YI Kim Y Kim SY Lee ES Juhnn YS ( 2007 ) G protein betagamma subunits augment UVB-induced apoptosis by stimulating the release of soluble heparin-binding epidermal growth factor from human keratinocytes . J Biol Chem 282 : 24720 – 24730 Google Scholar Crossref Search ADS PubMed WorldCat Suharsono U Fujisawa Y Kawasaki T Iwasaki Y Satoh H Shimamoto K ( 2002 ) The heterotrimeric G protein α subunit acts upstream of the small GTPase Rac in disease resistance of rice . Proc Natl Acad Sci USA 99 : 13307 – 13312 Google Scholar Crossref Search ADS PubMed WorldCat Temple BRS Jones AM ( 2007 ) The plant heterotrimeric G-protein complex . Annu Rev Plant Biol 58 : 249 – 266 Google Scholar Crossref Search ADS PubMed WorldCat Tossi V Lamattina L Cassia R ( 2009 ) An increase in the concentration of abscisic acid is critical for nitric oxide-mediated plant adaptive responses to UV-B irradiation . New Phytol 181 : 871 – 879 Google Scholar Crossref Search ADS PubMed WorldCat Wang P Song C-P ( 2008 ) Guard-cell signalling for hydrogen peroxide and abscisic acid . New Phytol 178 : 703 – 718 Google Scholar Crossref Search ADS PubMed WorldCat Wang XQ Ullah H Jones AM Assmann SM ( 2001 ) G protein regulation of ion channels and abscisic acid signaling in Arabidopsis guard cells . Science 292 : 2070 – 2072 Google Scholar Crossref Search ADS PubMed WorldCat Warpeha KM Upadhyay S Yeh J Adamiak J Hawkins SI Lapik YR Anderson MB Kaufman LS ( 2007 ) The GCR1, GPA1, PRN1, NF-Y signal chain mediates both blue light and abscisic acid responses in Arabidopsis . Plant Physiol 143 : 1590 – 1600 Google Scholar Crossref Search ADS PubMed WorldCat Wei Q Zhou W Hu G Wei J Yang H Huang J ( 2008 ) Heterotrimeric G-protein is involved in phytochrome A-mediated cell death of Arabidopsis hypocotyls . Cell Res 18 : 949 – 960 Google Scholar Crossref Search ADS PubMed WorldCat Wilkins KA Bancroft J Bosch M Ings J Smirnoff N Franklin-Tong VE ( 2011 ) Reactive oxygen species and nitric oxide mediate actin reorganization and programmed cell death in the self-incompatibility response of papaver . Plant Physiol 156 : 404 – 416 Google Scholar Crossref Search ADS PubMed WorldCat Wilson ID Neill SJ Hancock JT ( 2008 ) Nitric oxide synthesis and signalling in plants . Plant Cell Environ 31 : 622 – 631 Google Scholar Crossref Search ADS PubMed WorldCat Yemets AI Krasylenko YA Lytvyn DI Sheremet YA Blume YB ( 2011 ) Nitric oxide signalling via cytoskeleton in plants . Plant Sci 181 : 545 – 554 Google Scholar Crossref Search ADS PubMed WorldCat Zhang W Fan LM Wu WH ( 2007 ) Osmo-sensitive and stretch-activated calcium-permeable channels in Vicia faba guard cells are regulated by actin dynamics . Plant Physiol 143 : 1140 – 1151 Google Scholar Crossref Search ADS PubMed WorldCat Zhang W He SY Assmann SM ( 2008 ) The plant innate immunity response in stomatal guard cells invokes G-protein-dependent ion channel regulation . Plant J 56 : 984 – 996 Google Scholar Crossref Search ADS PubMed WorldCat Zhang W Jeon BW Assmann SM ( 2011 ) Heterotrimeric G-protein regulation of ROS signalling and calcium currents in Arabidopsis guard cells . J Exp Bot 62 : 2371 – 2379 Google Scholar Crossref Search ADS PubMed WorldCat Zhang Y Zhu H Zhang Q Li M Yan M Wang R Wang L Welti R Zhang W Wang X ( 2009 ) Phospholipase dalpha1 and phosphatidic acid regulate NADPH oxidase activity and production of reactive oxygen species in ABA-mediated stomatal closure in Arabidopsis . Plant Cell 21 : 2357 – 2377 Google Scholar Crossref Search ADS PubMed WorldCat Zhao Z Stanley BA Zhang W Assmann SM ( 2010 ) ABA-regulated G protein signaling in Arabidopsis guard cells: a proteomic perspective . J Proteome Res 9 : 1637 – 1647 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported by the National Science Foundation of China (grant no. 31170370 to J.-M.H., X.-G.M., and Y.Z.), the Fundamental Research Funds for the Central Universities (grant no. GK200901013 to J.-M.H.), the Science Foundation of the State Key Laboratory of Loess and Quaternary Geology, Institute of Earth Environment, Chinese Academy of Sciences (grant no. SKLLQG1004 to J.-M.H.), and the Undergraduate Students Innovative Experimental Projects of Shaanxi Normal University (grant no. CX11073 to T.-F.S. and F.-F.X.). * Corresponding author; e-mail hejm@snnu.edu.cn. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Jun-Min He (hejm@snnu.edu.cn). [C] Some figures in this article are displayed in color online but in black and white in the print edition. [W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.112.211623 © 2013 American Society of Plant Biologists. All Rights Reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Role and Interrelationship of Gα Protein, Hydrogen Peroxide, and Nitric Oxide in Ultraviolet B-Induced Stomatal Closure in Arabidopsis Leaves    
JF - Plant Physiology
DO - 10.1104/pp.112.211623
DA - 2013-02-28
UR - https://www.deepdyve.com/lp/oxford-university-press/role-and-interrelationship-of-g-protein-hydrogen-peroxide-and-nitric-uEn9prlaNT
SP - 1570
EP - 1583
VL - 161
IS - 3
DP - DeepDyve
ER -