Next Article in Journal
New Evidence on a Distinction between Aβ40 and Aβ42 Amyloids: Thioflavin T Binding Modes, Clustering Tendency, Degradation Resistance, and Cross-Seeding
Next Article in Special Issue
BBX24 Interacts with DELLA to Regulate UV-B-Induced Photomorphogenesis in Arabidopsis thaliana
Previous Article in Journal
Behavioral Evidence for a Tau and HIV-gp120 Interaction
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 23
Issue 10
10.3390/ijms23105512
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

GCR1 Positively Regulates UV-B- and Ethylene-Induced Stomatal Closure via Activating GPA1-Dependent ROS and NO Production

by
Xue Li
†,
Qi Fu
†,
Fu-Xing Zhao
,
Yi-Qing Wu
,
Teng-Yue Zhang
,
Zhong-Qi Li
* and
Jun-Min He
*
School of Life Sciences, Shaanxi Normal University, Xi’an 710119, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2022, 23(10), 5512; https://doi.org/10.3390/ijms23105512
Submission received: 13 April 2022 / Revised: 7 May 2022 / Accepted: 12 May 2022 / Published: 15 May 2022
(This article belongs to the Special Issue UV-B Signaling and Its Molecular Control in Plant)
Browse Figures
Review Reports Versions Notes

Abstract

:
Heterotrimeric G proteins function as key players in guard cell signaling to many stimuli, including ultraviolet B (UV-B) and ethylene, but whether guard cell G protein signaling is activated by the only one potential G protein-coupled receptor, GCR1, is still unclear. Here, we found that gcr1 null mutants showed defects in UV-B- and ethylene-induced stomatal closure and production of reactive oxygen species (ROS) and nitric oxide (NO) in guard cells, but these defects could be rescued by the application of a Gα activator or overexpression of a constitutively active form of Gα subunit GPA1 (cGPA1). Moreover, the exogenous application of hydrogen peroxide (H2O2) or NO triggered stomatal closure in gcr1 mutants and cGPA1 transgenic plants in the absence or presence of UV-B or ethylene, but exogenous ethylene could not rescue the defect of gcr1 mutants in UV-B-induced stomatal closure, and gcr1 mutants did not affect UV-B-induced ethylene production in Arabidopsis leaves. These results indicate that GCR1 positively controls UV-B- and ethylene-induced stomatal closure by activating GPA1-dependent ROS and NO production in guard cells and that ethylene acts upstream of GCR1 to transduce UV-B guard cell signaling, which establishes the existence of a classic paradigm of G protein signaling in guard cell signaling to UV-B and ethylene.

1. Introduction

Ligand signaling through G protein-coupled receptors (GPCRs) is a widespread mechanism for extracellular signal perception in eukaryotic organisms. In the classic paradigm of G protein signaling, the GPCR associates with heterotrimeric G proteins composed of α, β, and γ subunits. Ligand binding to the GPCR causes Gα to exchange GDP for GTP, leading to the dissociation of the G protein complex into Gα-GTP and Gβγ dimers. These two functional subunits interact with a variety of downstream effectors to activate different signaling cascades. The intrinsic GTPase activity of Gα, which can be accelerated by the regulator of G protein signaling (RGS) proteins, returns the G protein to the inactive trimeric state [1].
Plant G protein signal transduction is modeled on the well-established animal paradigm, but many experimental pieces of evidence show that plant G protein signaling has taken a very different evolutionary path. Most importantly, while in animal systems G protein signaling is activated by thousands of identified GPCRs, only one canonical GPCR gene, named GCR1, was identified by homology searches of the EST databases in Arabidopsis thaliana [2,3]. Additional GPCR candidates were mined based on their transmembrane domains [4,5,6], with a list of up to 56 numbers, but of which GCR1 was bioinformatically considered to be the best candidate based on GPCR fold analysis [7]. Despite being the most studied potential candidate, the role of GCR1 as a GPCR remains biochemically unproven, due to the lack of an identified ligand that binds GCR1 and the lack of demonstration of guanine nucleotide exchange factors (GEFs) activity [5,8]. Therefore, it remains an unsettled controversy whether these GPCRs in animal models are present in plants [7,9]. Similarly, compared to the animal kingdom, where multiple Gα, Gβ, Gγ, and RGS genes exist, there is only one canonical G protein α-subunit1 (GPA1) [10], three extra-large GTP-binding proteins (XLG1, XLG2, and XLG3) [11], one Arabidopsis G protein β-subunit1 (AGB1) [12], three Arabidopsis G protein γ-subunit (AGG1, AGG2, and AGG3) [13,14], and one RGS (RGS1) [15] genes in Arabidopsis. However, consistent with that GCR1 functions as a GPCR, studies have shown that GCR1 physically interacts with GPA1 in a manner dependent on the intracellular domains of GCR1 [8], as has been shown for mammalian GPCRs [16]. Furthermore, gcr1 mutants exhibit a subset of the phenotypes seen in gpa1 mutants, such as on the cell cycle and the ABA sensitivity of seed germination [8,17,18,19,20]. In contrast, compared to animal Gαs, evidence has shown that GPA1 has a very low GTPase activity and spontaneously exchanges GDP for GTP without any involvement of GPCRs, suggesting that Gα subunits in plants are constitutively active [21]. Moreover, plant G proteins have been proven to physically interact with atypical receptors, such as the Arabidopsis RGS1 and some receptor-like kinases (RLKs) [15,22,23,24,25,26,27,28,29,30,31]. Thus, it has been proposed that, instead of the GPCR-dependent activation of G proteins employed in animals, plant G proteins are activated by phosphorylation or by signals inhibiting the deactivation of a constitutively active Gα [32,33]. Nevertheless, GCR1 satisfies the essential criteria for the identification of a protein as a GPCR. However, whether plant G protein signaling is also activated by GCR1 is still worthwhile to be researched.
Guard cells respond to many stimuli to regulate the stomatal aperture to an optimal level and thus optimize plant growth by controlling water loss, gas exchange, and innate immunity. Arabidopsis T-DNA insertional null mutants of the sole canonical Gα subunit, GPA1, are insensitive to stomata closing induced by sphingosine-1-P (S1P) [34,35], phosphatidic acid (PA) [36], extracellular calmodulin (ExtCaM) [37,38], extracellular ATP [39], ultraviolet B (UV-B) radiation [40], ethylene [41], brassinosteroid (BR) [42], and the pathogen-associated molecular pattern flg22 [43], as well as to abscisic acid (ABA) and S1P inhibition of stomatal opening [34,35,44,45], suggesting that plant Gα subunit GPA1 responds to various stimuli as a key regulator of stomatal signaling. Further studies indicate that GPA1 mediates several stimuli-regulated stomatal movements by inducing the production of reactive oxygen species (ROS) and nitric oxide (NO) and regulating the activities of inward K+ channels and slow anion channels in guard cells [37,38,39,40,41,42,44,45]. Despite the progress achieved in our understanding of the role of G proteins in stomatal signaling, it is still unclear how multiple stimuli activate G protein signaling and whether GCR1 is involved in the activation of G protein signaling in guard cells. To address this problem, Pandey and Assmann [8] compared the ABA and S1P responses between gcr1 and gpa1 mutant guard cells and found that the gcr1 mutants exhibited hypersensitivity in ABA and S1P inhibition of stomatal opening and promoting of stomatal closure, whereas the gpa1 mutants exhibited insensitivity in ABA and S1P inhibition of stomatal opening and normal response in ABA and S1P promoting of stomatal closure [8,34,44]. To reconcile these observations, the authors hypothesized that GCR1 negatively regulates ABA signaling via a mechanism dependent on its direct binding with and negatively regulating GPA1 or independent of its binding to GPA1 [8]. However, whether GCR1 is required for guard cell signaling to other stimuli is uncertain. If this is the case, the next question is whether and how it regulates G protein signaling in guard cells. All these problems are still unclear and worth studying.
In this study, we used Arabidopsis gcr1 null mutants and the transgenic lines overexpressing a constitutively active form of GPA1 (cGPA1) in wild-type and gcr1 mutants to show that GCR1 positively mediates ethylene- and UV-B-induced ROS and NO production in guard cells and subsequent stomatal closure in a GPA1-dependent manner. Our results establish that a classic paradigm of G protein signaling exists in the guard cell responses to ethylene and UV-B.

2. Results

2.1. GCR1 Positively Regulates UV-B- and Ethylene-Induced Stomatal Closure

In this study, based on the previous finding that heterotrimeric G protein α subunit GPA1 mediates stomatal closure in Arabidopsis leaves induced by 0.5 W/m2 UV-B radiation or 100 μmol/L 1-aminocyclopropane-1-carboxylic acid (ACC: the direct precursor of ethylene during its biosynthesis) [40,41], we used 0.5 W/m2 UV-B and 100 μmol/L ACC as UV-B and ethylene treatments, respectively, and further explored the role of the sole prototypical Arabidopsis GPCR gene, GCR1, in UV-B- and ethylene-induced stomatal closure in Arabidopsis. When freshly prepared Arabidopsis leaves with open stomata were exposed to light alone or with 0.5 W/m2 UV-B radiation for 3 h, UV-B induced stomatal closure in the wild-type (WT), but could not trigger the closing of stomata in the gcr1-1 and gcr1-2 mutants (Figure 1A). Similarly, when the leaves were treated with 100 μmol/L ACC for 3 h, ACC induced stomatal closure in the WT, but this effect of ACC was defective in the gcr1-1 and gcr1-2 mutants (Figure 1B). These genetic results indicate that GCR1 acting as a positive regulator mediates both UV-B- and ethylene-induced closing of stomata.
The size of stomatal pores controls water loss. Thus, we posited that UV-B or ACC treatment reduces water loss and that this effect is dependent on GCR1. When leaves of WT and gcr1 mutants treated with or without 0.5 W/m2 UV-B or 100 μmol/L ACC were placed under light without MES buffer for 2 h, the UV-B- and ACC-treated WT leaves lost less water than the untreated WT leaves, while UV-B and ACC treatments did not decrease the water loss from the gcr1 mutant leaves (Figure S1). These findings further confirmed the effect of UV-B and ethylene on stomatal aperture in WT and gcr1 leaves.
Our finding that GCR1 acts as a positive regulator in both UV-B- and ethylene-induced stomatal closure is contrary to the negative role of GCR1 in ABA and S1P inhibition of stomatal opening and promoting of stomatal closure [8]. To further confirm the role of GCR1 in ABA guard cell signaling, we repeated the experiments of Pandey and Assmann [8] in our experimental conditions. Consistent with the results of Pandey and Assmann [8] that gcr1-3 and gcr1-4 (two other allelic GCR1 mutants; Figure S2) exhibited hypersensitivity in ABA inhibition of stomatal opening and promoting of stomatal closure, our results showed that gcr1-1 and gcr1-2 were hypersensitive in both ABA responses (Figure S3A,B), which further confirms the results of Pandey and Assmann [8] and supports that GCR1 positively regulates UV-B and ethylene guard cell signaling but negatively controls ABA guard cell signaling.

2.2. GCR1 Mediates UV-B- and Ethylene-Induced Stomatal Closure via Activating Gα Subunit GPA1

In the classic paradigm of G protein signaling, heterotrimeric G proteins composed of α, β, and γ subunits link ligand perception by GPCRs with downstream effectors. Because GCR1 and Gα subunit GPA1 interact in plants [8], and both of them play positive roles in guard cell responses to UV-B and ethylene (Figure 1; [40,41]), we further explored whether GCR1 mediates guard cell UV-B and ethylene signaling by activating GPA1. Firstly, we investigated the stomatal responses of the WT Col-0 and mutants gcr1-1 and gcr1-2 to Gα activator cholera toxin (CTX) [37]. As shown in Figure 2A,B, CTX not only induced stomatal closure in the leaves of the WT and gcr1 mutants in the absence of UV-B or ACC but also rescued the defects of the gcr1-1 and gcr1-2 mutants in UV-B- and ACC-induced stomatal closure. These results suggest that GCR1 mediates both UV-B- and ethylene-induced stomatal closure via activating Gα. In Arabidopsis, GPA1 is the only prototypical Gα gene [46]. To further confirm the above pharmacological data, we generated the transgenic plants overexpressing a constitutively active form of GPA1 (cGPA1; GPA1 with a point mutation of Glu-222 to Leu, which locks GPA1 in the active state once activated) [47]. Their overexpression levels of GPA1 in the leaves of T2 transgenic lines used for further analysis were confirmed by quantitative real-time PCR (qPCR) and revealed no significant difference in the WT vs. gcr1-1 and gcr1-2 (Figure 2C). Under our plant growth condition, although the leaves of the cGPA1 transgenic lines were slightly smaller than those of the WT and gcr1 mutants, no other difference including stomatal density and development was detected between the control plants and the transgenic lines (data not shown). However, the cGPA1 transgenic lines in either the WT Col-0 or gcr1-1 and gcr1-2 mutant backgrounds not only exhibited smaller stomatal apertures compared with their corresponding WT and mutants in the absence of UV-B or ACC but also showed significant stomatal closure induced by UV-B and ACC, which is similar to the stomatal response of the WT to UV-B and ACC (Figure 2D). Together, our pharmacological and genetic results convincingly indicate that GCR1 mediates guard cell responses to both UV-B and ethylene by activating Gα subunit GPA1.

2.3. GCR1 Mediates UV-B- and Ethylene-Induced Stomatal Closure by Activating GPA1-Dependent ROS Production in Guard Cells

Having established that GCR1 mediates both UV-B- and ethylene-induced stomatal closure by activating Gα subunit GPA1 (Figure 2) and that GPA1 mediates both UV-B- and ethylene-induced stomatal closure via modulating ROS generation in guard cells [40,41], we further explored whether GCR1 mediates both UV-B and ethylene guard cell signaling by activating GPA1-dependent ROS generation. First, we used the ROS-sensitive fluorescent dye 2′,7′-dichlorofluorescin diacetate (H2DCFDA) to examine the ROS production in guard cells under the absence or presence of UV-B or ACC. Consistent with the impairment of the gcr1 mutants in UV-B- and ACC-induced stomatal closure (Figure 1A,B), both the UV-B- and ACC-induced ROS production in guard cells of the WT Col-0 were completely impaired in the guard cells of the gcr1 mutants (Figure 3), suggesting that GCR1 mediates both UV-B and ethylene stomatal responses by inducing ROS production in guard cells. Furthermore, the Gα activator CTX significantly induced ROS generation in guard cells of the WT Col-0 and gcr1 mutants in the absence or presence of UV-B or ACC (Figure 3). In addition, the overexpression of cGPA1 not only slightly but significantly induced ROS generation in guard cells of the WT Col-0 and gcr1 mutants in the absence of UV-B or ACC, but also rescued the defect of the gcr1-1 and gcr1-2 mutants in UV-B- and ACC-induced ROS production in guard cells (Figure 3). These results were consistent with the stomatal responses of the WT Col-0, gcr1 mutants, and cGPA1 transgenic lines to CTX, UV-B, and ACC (Figure 2), further suggesting that GCR1 mediates both UV-B- and ethylene-induced stomatal closure by activating Gα subunit GPA1-dependent ROS production in guard cells. To confirm this notion, we next investigated the stomatal response to exogenous hydrogen peroxide (H2O2). As expected, exogenous H2O2 not only induced stomatal closure in the leaves of the WT Col-0, gcr1 mutants, and cGPA1 transgenic lines in the absence of UV-B or ACC but also rescued the defects of the gcr1-1 and gcr1-2 mutants in UV-B- and ACC-induced stomatal closure (Figure 4). Together, our results firmly show that GCR1 mediates both UV-B and ethylene guard cell signaling by activating GPA1-dependent ROS production in guard cells.

2.4. GCR1 Mediates UV-B- and Ethylene-Induced Stomatal Closure by Activating GPA1-Dependent NO Production in Guard Cells

Because NO also mediates both UV-B- and ethylene-induced stomatal closure by working downstream of GPA1 and H2O2 [40,48], we further examined the relationship among NO, GCR1, and GPA1 in UV-B and ethylene stomatal signaling. We first measured NO levels in guard cells of the WT Col-0, gcr1 mutants, and cGPA1 transgenic lines using the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2DA) in the absence or presence of CTX, UV-B, and ACC. Similar to the effect of UV-B, ACC, and CTX on ROS production in guard cells of the WT Col-0, gcr1 mutants, and cGPA1 transgenic lines (Figure 3), both UV-B- and ACC-triggered NO generation in guard cells of the WT Col-0 was completely impaired in the gcr1 mutants; the activation of Gα by CTX could significantly induce NO production in guard cells of the WT Col-0 and gcr1 mutants in the either absence or presence of UV-B or ACC; the overexpression of cGPA1 slightly induced NO production in guard cells of the WT Col-0 and gcr1 mutants in the absence of UV-B or ACC and also rescued the defects of the gcr1 mutants in the UV-B- and ACC-induced NO generation in guard cells (Figure 5). These results were consistent with the stomatal responses of the WT Col-0, gcr1 mutants, and cGPA1 transgenic lines to CTX, UV-B, and ACC (Figure 2), indicating that GCR1 mediates both UV-B- and ethylene-induced stomatal closure through activating GPA1-dependent NO generation in guard cells. This notion was also supported by the finding that the exogenous application of NO donor sodium nitroprusside (SNP) not only induced stomatal closure in the WT Col-0, gcr1 mutants, and cGPA1 transgenic lines in the absence of UV-B or ACC but also rescued the defects of the gcr1 mutants in the UV-B- and ACC-induced stomatal closure (Figure 6).

2.5. Ethylene Functions Upstream of GCR1 to Transduce UV-B Signaling in Guard Cells

GCR1 mediates both UV-B- and ethylene-induced stomatal closure by working upstream of GPA1, ROS, and NO (Figure 1, Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6), while ethylene and GPA1 mediate UV-B-induced stomata closing as well as the production of ROS and NO in guard cells [40,49]. These findings suggest that ethylene mediates UV-B guard cell signaling via working upstream of GCR1. To test this suggestion, we examined the stomatal response of gcr1 mutants to ACC in the presence of UV-B, as well as the effect of UV-B on ethylene production in gcr1 mutants. As expected, the exogenous application of ACC could not reverse the defect of the gcr1 mutants in UV-B-induced stomatal closure (Figure 7A); UV-B-induced ethylene production in the WT Col-0 leaves was also not affected by the gcr1-1 and gcr1-2 mutants (Figure 7B). These results support that ethylene functions upstream of GCR1 to transduce UV-B signaling in guard cells.

3. Discussion

In animals, extracellular signals are often perceived by GPCRs and transduced through G proteins to downstream targets. In plants, G protein signaling plays essential roles in diverse biological processes, including guard cell signaling, but little is known about their upstream receptors [50]. In Arabidopsis, only GCR1 was identified as a canonical GPCR gene [2,3] and bioinformatically analyzed to be the best GPCR candidate [7]. Furthermore, GCR1 is a potential prototypical GPCR in plants, because it directly binds to the plant Gα subunit GPA1 [8] like that for animal GPCRs [16]. However, the unsettled controversy over the existence of or the need for the prototypical, animal model GPCRs in plants has overshadowed a more fundamental quest for the role of GCR1 in plant G protein signaling [32,33]. In this study, by genetic and pharmacological analyses, we provided convincing evidence that GCR1 positively mediates UV-B- and ethylene-induced stomatal closure by activating GPA1-dependent ROS and NO production in guard cells. Evidence is also provided to show that ethylene acts upstream of GCR1 to transduce UV-B signaling in guard cells. This study establishes the existence of a classic paradigm of G protein signaling in guard cell signaling to UV-B and ethylene.
In guard cell signaling, Gα subunit GPA1 mediates many factor-triggered stomatal movements by controlling the production of ROS and NO and regulating the activities of ion channels in guard cells [34,35,36,37,38,39,40,41,42,43,44]. However, by what mechanisms many stimuli activate G protein signaling and whether GCR1 participates in the activation of G protein signaling in guard cells is still unknown. In this report, we found that the Arabidopsis T-DNA insertional null mutants for GCR1 were insensitive to UV-B- and ethylene-induced stomatal closure as well as ROS and NO production in guard cells (Figure 1, Figure 3 and Figure 5), while exogenous H2O2 and NO induced stomatal closure in the gcr1 mutants in either the presence or absence of UV-B or ethylene (Figure 4 and Figure 6). This pharmacological and genetic evidence convincingly indicates that GCR1 mediates both UV-B- and ethylene-induced stomatal closure by regulating ROS and NO generation in the guard cells of Arabidopsis. This role of GCR1 is similar to the role of GPA1 in guard cell signaling triggered by UV-B and ethylene [40,41], suggesting that GCR1 and GPA1 function in the same pathway. This suggestion is consistent with the previous biochemical evidence that GCR1 physically interacts with GPA1 [8]. Moreover, the defect of gcr1 mutants in the UV-B- and ethylene-induced ROS and NO production in guard cells and subsequent stomatal closure can be rescued by the application of Gα activator CTX or overexpression of a constitutively active form of Gα subunit GPA1 (cGPA1) (Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6), further proving that GCR1 mediates both UV-B- and ethylene-induced ROS and NO production in guard cells and subsequent stomatal closure by the activation of Gα subunit GPA1. This finding not only indicates the role of GCR1 in guard cell signaling to UV-B and ethylene but also proves the existence of a classic paradigm of G protein signaling in plants. However, the experimental identification of a ligand for GCR1 and demonstration of GEF activity of GCR1 would unequivocally establish this classic paradigm of G protein signaling in UV-B or ethylene signaling. Further work is required in this direction.
Based on analysis of the stomatal response of GCR1 mutants gcr1-3 and gcr1-4, Pandey and Assmann [8] indicated that GCR1 has a negative role in ABA and S1P guard cell signaling, as these gcr1 mutants exhibit hypersensitivity in ABA and S1P inhibition of stomatal opening and promoting of stomatal closure. Here, our data of two other allelic GCR1 mutants, gcr1-1 and gcr1-2 (Figure 1 and Figure S1), not only confirm the results of Pandey and Assmann [8] but further indicate the positive role of GCR1 in UV-B and ethylene guard cell signaling. Because the ABA and S1P responses of gcr1 mutant guard cells are opposite to those of gpa1 mutants [8,34,44], it is hypothesized that GCR1 negatively regulates ABA and S1P signaling via a mechanism dependent on its binding to and negatively regulating GPA1 or independent of its binding to GPA1 [8]. However, the directly negative regulation of GPA1 by GCR1 is opposite to the classic paradigm of G protein signaling and lacks supporting biochemical and genetic evidence; thus, it is possible that GCR1 negatively regulates ABA and S1P guard cell signaling via a mechanism independent of GPA1. Combined with our results, it is clear that the role and relationship of GCR1 and GPA1 in UV-B and ethylene guard cell signaling are much different from those in ABA and S1P guard cell signaling. Previous studies have shown that in the absence of ABA, ethylene can induce stomatal closure [51], but in the presence of ABA, it inhibits ABA-induced stomatal closure [52]. Thus, whether these complex effects of ethylene on stomatal movement are caused by the different roles of GCR1 in ABA and ethylene guard cell signaling is an interesting question to be studied in the future.
Our previous studies have shown that both the UV-B-specific signaling pathway UV RESISTANCE LOCUS 8 (UVR8)-CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)-ELONGATED HYPOCOTYL5 (HY5)-dependent ethylene and GPA1 mediate UV-B-induced stomatal closing via functioning upstream of ROS and NO [40,49] and that GPA1 mediates ethylene-triggered stomatal closure via working downstream of the ethylene receptor ETHYLENE RESPONSE 1 (ETR1) and the ethylene signaling negative regulator CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1) and upstream of ROS and NO [41,42]. Here, we further show that GCR1 mediates both UV-B- and ethylene-induced stomatal closure by working upstream of GPA1, ROS, and NO (Figure 1, Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6). These results suggest that ethylene acts as a secondary messenger to mediate UV-B guard cell signaling via working downstream of the UVR8-COP1-HY5 signaling pathway and upstream of GCR1, which is further supported by the presented evidence that gcr1 mutants show insensitivity to ethylene induction of stomatal closure in the presence or absence of UV-B but sensitivity to UV-B induction of ethylene production (Figure 7).
In summary, the data presented here, combined with those of previous studies, provide compelling evidence for the essential role of GCR1, a potential prototypical GPCR in Arabidopsis, in UV-B and ethylene guard cell signaling and establish potential models of the UV-B- and ethylene-signaling pathways in guard cells (Figure 8). UV-B signaling in guard cells is initiated by the UV-B-specific signaling pathway UVR8-COP1-HY5 via the induction of ethylene production [48]. Ethylene is perceived by its receptor, ETR1, to inactivate the CTR1 kinase [40], releasing GCR1-dependent GPA1 activation (Figure 2). Then activated, GPA1 induces ROS and NO generation in guard cells and subsequent stomata closing (Figure 3, Figure 4, Figure 5, Figure 6 and Figure 8). However, the bacterial toxin CTX, which activates the Gα subunit as those of non-hydrolyzing GTP analogues that induce a conformational change in the Gα subunit that promotes the dissociation of the trimeric complex [37], could significantly induce ROS and NO production in guard cells and subsequent stomatal closure in WT and gcr1 mutants in either the absence or presence of UV-B or ACC (Figure 2, Figure 3 and Figure 5), while the overexpression of cGPA1, a potential constitutively active form of GPA1 that was shown to disable the GTPase activity of Gα once activated [53], only slightly induced ROS and NO generation and subsequent stomatal closure in the WT and gcr1 mutants in the absence of UV-B or ACC, but completely rescued the defects of the gcr1 mutants in UV-B- and ACC-induced ROS and NO production in guard cells and stomatal closure (Figure 2, Figure 3 and Figure 5). These different effects between CTX and cGPA1 suggest that the complete activation of this overexpressed cGPA1 still requires the presence of UV-B or ACC. It is possible that the active site of the cGPA1 is suppressed by the formation of heterotrimeric G protein complexes [47], and that the dissociation of cGPA1 from the trimeric complex, and thus its complete activation, requires other UV-B- or ethylene-triggered signals. Many studies have shown that plant G proteins can physically interact with atypical receptors such as the Arabidopsis RGS1 and some RLKs, which promote the dissociation of the heterotrimeric G protein complex, and thus Gα activation, by phosphorylation or by signals inhibiting the deactivation of a constitutively active Gα [15,22,23,24,25,26,27,28,29,30,31,32,33]. Combined together, it is proposed that both the classic GPCR-dependent and the atypical GPCR-independent G protein signaling may work cooperatively to control the stomatal response to UV-B and ethylene (Figure 8), but this suggestion should be studied in the future.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

Arabidopsis thaliana seeds were obtained from the Nottingham Arabidopsis Stock Center (Nottingham, UK). Ecotype Col-0 was used as the WT control, mutants of gcr1-1 (N6539) and gcr1-2 (N6540) are in the Col-0 ecotype background, and their genotypes were confirmed by PCR. The seeds were sterilized, sown in a potting mix of soil: vermiculite: perlite (3:1:1, v/v/v), and grown in plant growth chambers under a 16 h photoperiod (0.1 mmol/m2/s) at a day/night temperature cycle of 22/18 °C and relative humidity of 80%. Fully expanded rosette leaves of 4-week-old plants were harvested and used for analysis.

4.2. UV-B Treatments

In this paper, 0.5 W/m2 UV-B radiation was used for all UV-B treatments and carried out, as previously described [40,54].

4.3. Stomatal Bioassays

Freshly detached leaves with their abaxial surfaces facing up were first incubated in MES-KCl buffer (10 mmol/L MES, 50 mmol/L KCl, and pH 6.15) under white light (0.1 mmol/m2/s) for 2 h to open stomata and then transferred to the MES-KCl buffer with or without 100 μmol/L ACC, 10 μmol/L ABA, 400 ng/mL CTX, 100 μmol/L H2O2, or 100 μmol/L SNP under white light alone or supplemented with 0.5 W/m2 UV-B radiation for another 3 h. The pH of the MES-KCl buffer containing ACC was adjusted to 6.15 with KOH to avoid any effect of the pH. After the treatments, the abaxial epidermis was immediately peeled from the treated leaves, and the stomatal apertures were recorded using a calibrated light microscope.

4.4. Measurement of Leaf Water Loss Rate

For the measurement of leaf water loss, ten freshly detached leaves with open stomata were incubated in MES-KCl buffer alone or containing 100 μmol/L ACC under white light (0.1 mmol/m2/s) alone or supplemented with 0.5 W/m2 UV-B radiation for 3 h, weighed and labeled as W0. The treated leaves were then placed under the same light conditions without MES-KCl buffer for 2 h, weighed and labeled as W1. The leaf water loss rate was calculated as W0–W1/W0 × 100.

4.5. Measurement of ROS and NO Levels in Guard Cells

The fluorescent probes H2DCFDA and DAF-2DA were used to detect levels of ROS and NO in guard cells, respectively, as previously mentioned [48,54]. Briefly, the freshly prepared epidermal strips peeled from the abaxial surfaces of leaves were incubated in Tris-KCl loading buffer (10 mmol/L Tris and 50 mmol/L KCl; pH 7.2) in the presence of 50 μmol/L H2DCF-DA for 10 min or 10 μmol/L DAF-2 DA for 30 min under darkness. After washing in fresh Tris-KCl buffer under darkness, an inverted fluorescence microscope (Eclipse TE 200; Nikon) with an excitation filter (450–490 nm) and an emission filter (520–560 nm) was used to detect H2DCF and DAF-2 fluorescence in guard cells. In each experiment, five epidermal strips taken from the leaves of different plants were examined, and each experiment was repeated at least three times. The fluorescence pixel intensity was analyzed in the whole stomata areas of the fluorescence microscope images with Leica Image software (Leica Microsystems), and the data are presented as means ± SE of three independent experiments, each with 20 stomata. The presented images are representative of the three or more independent experiments performed.

4.6. Generation of Transgenic Plants Overexpressing cGPA1

The generation of transgenic lines overexpressing cGPA1 was based on the protocol described [47], with some modifications. A cDNA library was generated from RNA isolated from seedlings of Arabidopsis ecotype Col-0. The wild-type GPA1 cDNA was amplified by PCR using this cDNA library as a template with gene-specific primers GPA1 KpnI+ (5′-GGTACCATGGGCTTACTCTGCAGTAGAA-3′) and GPA1 SmaI- (5′-TCCCCCGGGTAAAAGGCCAGCCTCCAGTAA-3′). The cGPA1 was generated by changing Glu-222 to Leu by PCR–based mutagenesis using primers M1 (5-TGACGTGGGTGGACTGAGAAATGAGAGGAGG-3) and M2 (5-CCTCCTCTCATTTCTCAGTCCACCCACGTCA-3) in combination with the primers GPA1 KpnI+ and GPA1 SmaI-. These fragments were inserted into SmaI and KpnI sites in the pCambia1305 vector, resulting in 35S:cGPA1, and transferred into A. tumefaciens strain GV3101 cells, which were then used to transform the Col-0, gcr1-1, or gcr1-2 plants by using the floral dip method. Seedlings were selected on 1/2 Murashige and Skoog solid medium containing 40 μg/mL hygromycin-B, and T3 transgenic plants were used for the experiments.

4.7. RNA Extraction and qPCR

Total RNA was extracted from the treated leaves using MagzolTM reagent (Trizol reagent, Magen). An amount of 1 μg of total RNA was used to synthesize cDNA using the Prime Script™ RT Master Mix (Takara Bio), according to the manufacturer’s instructions. qPCR analysis was performed with SYBR Green TaqMix (Takara Bio) on the IQ5 real-time system (Bio-Rad). Each qPCR result was the average of three independent biological repeats. The relative expression level is shown as a value relative to the WT Col-0 sample after normalization to that of ACTIN 2. The sequences of the primers for qPCR are GCR1-F (5′-TCTGCGTTGCTTCGTTCTTGT-3′), GCR1-R (5′-TTTCGCTTCGCTGTTGTTGTT-3′), ACTIN2-F (5′-CAAGGCCGAGTATGATGAGG-3′), and ACTIN2-R (5′-GAAACGCAGACGTAAGTAAAAAC-3′).

4.8. Ethylene Measurement

After the treatments, the leaves were enclosed in a vial. After 3 h, the ethylene levels in the vial were analyzed, as previously mentioned [49]. For each treatment, five leaves from different plants were measured, and the treatment was repeated at least four times. Data are shown as means ± SE of three independent experiments.

4.9. Statistical Analysis

The samples were arranged in completely randomized designs with three replications. The data were presented as the mean value ± standard errors (SE). Results from different treatments were compared using one-way ANOVA (analysis of variance). Following ANOVA, post hoc comparisons of means were made using Mann–Whitney multiple comparisons. Statistical significance was determined at p < 0.01 or p < 0.05, as indicated in the figure legends. Statistical analyses were carried out using SPSS16.0.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms23105512/s1.

Author Contributions

Conceptualization, J.-M.H.; methodology, Z.-Q.L.; validation, X.L., Q.F., F.-X.Z., Y.-Q.W. and T.-Y.Z.; writing—original draft preparation, X.L. and Q.F.; writing—review and editing, J.-M.H.; funding acquisition, J.-M.H. and Z.-Q.L.. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (grant number 31870375), the Fundamental Research Funds for the Central Universities (grant number SY20210003), and the Excellent Doctoral Dissertation Project of Shaanxi Normal University (grant number 2019TS077).

Institutional Review Board Statement

This study only involved model plant experiments with no human or animal material, and therefore, ethical approval was not required.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data will be made available upon request.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Oldham, W.M.; Hamm, H.E. Heterotrimeric G protein activation by G-protein-coupled receptors. Nat. Rev. Mol. Cell Biol. 2008, 9, 60–71. [Google Scholar] [CrossRef]
  2. Josefsson, L.-G.; Rask, L. Cloning of a putative G-protein-coupled receptor from Arabidopsis thaliana. Eur. J. Biochem. 1997, 249, 415–420. [Google Scholar] [CrossRef] [Green Version]
  3. Plakidou-Dymock, S.; Dymock, D.; Hooley, R. A higher plant seven-transmembrane receptor that influences sensitivity to cytokinins. Curr. Biol. 1998, 8, 315–324. [Google Scholar] [CrossRef] [Green Version]
  4. Moriyama, E.N.; Strope, P.K.; Opiyo, S.O.; Chen, Z.; Jones, A.M. Mining the Arabidopsis thaliana genome for highly-divergent seven transmembrane receptors. Genome Biol. 2006, 7, R96. [Google Scholar] [CrossRef] [Green Version]
  5. Gookin, T.E.; Kim, J.; Assmann, S.M. Whole proteome identification of plant candidate G-protein coupled receptors in Arabidopsis, rice, and poplar: Computational prediction and in-vivo protein coupling. Genome Biol. 2008, 9, R120. [Google Scholar] [CrossRef] [Green Version]
  6. Pandey, S.; Nelson, D.C.; Assmann, S.M. Two novel GPCR-type G proteins are abscisic acid receptors in Arabidopsis. Cell 2009, 136, 136–148. [Google Scholar] [CrossRef] [Green Version]
  7. Taddese, B.; Upton, G.J.; Bailey, G.R.; Jordan, S.R.; Abdulla, N.Y.; Reeves, P.J.; Reynolds, C.A. Do plants contain G protein-coupled receptors? Plant Physiol. 2014, 164, 287–307. [Google Scholar] [CrossRef] [Green Version]
  8. Pandey, S.; Assmann, S.M. The Arabidopsis putative G protein-coupled receptor GCR1 interacts with the G protein alpha subunit GPA1 and regulates abscisic acid signaling. Plant Cell 2004, 16, 1616–1632. [Google Scholar] [CrossRef] [Green Version]
  9. Urano, D.; Jones, A.M. “Round up the usual suspects”: A comment on nonexistent plant G protein-coupled receptors. Plant Physiol. 2013, 161, 1097–1102. [Google Scholar] [CrossRef] [Green Version]
  10. Ma, H.; Yanofsky, M.F.; Meyerowitz, E.M. Molecular cloning and characterization of GPA1, a G protein alpha subunit gene from Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 1990, 87, 3821–3825. [Google Scholar] [CrossRef] [Green Version]
  11. Heo, J.B.; Sung, S.; Assmann, S.M. Ca2+-dependent GTPase, extra-large G protein 2 (XLG2), promotes activation of DNA-binding protein related to vernalization 1 (RTV1), leading to activation of floral integrator genes and early flowering in Arabidopsis. J. Biol. Chem. 2012, 287, 8242–8253. [Google Scholar] [CrossRef] [Green Version]
  12. Weiss, C.A.; Garnaat, C.W.; Mukai, K.; Hu, Y.; Ma, H. Isolation of cDNAs encoding guanine nucleotide-binding protein betasubunit homologues from maize (ZGB1) and Arabidopsis (AGB1). Proc. Natl. Acad. Sci. USA 1994, 91, 9554–9558. [Google Scholar] [CrossRef] [Green Version]
  13. Mason, M.G.; Botella, J.R. Completing the heterotrimer: Isolation and characterization of an Arabidopsis thaliana G protein γ-subunit cDNA. Proc. Natl. Acad. Sci. USA 2000, 97, 14784–14788. [Google Scholar] [CrossRef] [Green Version]
  14. Mason, M.G.; Botella, J.R. Isolation of a novel G-protein γ-subunit from Arabidopsis thaliana and its interaction with Gβ. Biochim. Biophys. Acta 2001, 1520, 147–153. [Google Scholar] [CrossRef]
  15. Chen, J.G.; Willard, F.S.; Huang, J.; Liang, J.; Chasse, S.A.; Jones, A.M.; Siderovski, D.P. A seven-transmembrane RGS protein that modulates plant cell proliferation. Science 2003, 30, 1728–1731. [Google Scholar] [CrossRef] [Green Version]
  16. Wess, J. Molecular basis of receptor/G-protein-coupling selectivity. Pharmacol. Ther. 1998, 80, 231–264. [Google Scholar] [CrossRef]
  17. Ullah, H.; Chen, J.G.; Young, J.C.; Im, K.H.; Sussman, M.R.; Jones, A.M. Modulation of cell proliferation by heterotrimeric G protein in Arabidopsis. Science 2001, 292, 2066–2069. [Google Scholar] [CrossRef] [Green Version]
  18. Ullah, H.; Chen, J.G.; Wang, S.; Jones, A.M. Role of a heterotrimeric G protein in regulation of Arabidopsis seed germination. Plant Physiol. 2002, 129, 897–907. [Google Scholar] [CrossRef] [Green Version]
  19. Colucci, G.; Apone, F.; Alyeshmerni, N.; Chalmers, D.; Chrispeels, M.J. GCR1, the putative Arabidopsis G protein-coupled receptor gene is cell cycle-regulated, and its overexpression abolishes seed dormancy and shortens time to flowering. Proc. Natl. Acad. Sci. USA 2002, 99, 4736–4741. [Google Scholar] [CrossRef] [Green Version]
  20. Apone, F.; Alyeshmerni, N.; Wiens, K.; Chalmers, D.; Chrispeels, M.J.; Colucci, G. The G-protein-coupled receptor GCR1 regulates DNA synthesis through activation of phosphatidylinositol specific phospholipase C. Plant Physiol. 2003, 133, 571–579. [Google Scholar] [CrossRef] [Green Version]
  21. Jones, J.C.; Jones, A.M.; Temple, B.R.S.; Dohlman, H.G. Differences in intradomain and interdomain motion confer distinct activation properties to structurally similar Gα proteins. Proc. Natl. Acad. Sci. USA 2012, 109, 7275–7279. [Google Scholar] [CrossRef] [Green Version]
  22. Jones, J.C.; Temple, B.R.S.; Jones, A.M.; Dohlman, H.G. Functional reconstitution of an atypical G Protein heterotrimer and regulator of G Protein signaling protein (RGS1) from Arabidopsis thaliana. J. Biol. Chem. 2011, 286, 13143–13150. [Google Scholar] [CrossRef] [Green Version]
  23. Bommert, P.; Il Je, B.; Goldshmidt, A.; Jackson, D. The maize Galpha gene COMPACT PLANT2 functions in CLAVATA signalling to control shoot meristem size. Nature 2013, 502, 555–558. [Google Scholar] [CrossRef]
  24. Choudhury, S.R.; Pandey, S. Specific subunits of heterotrimeric G proteins play important roles during nodulation in Soybean. Plant Physiol. 2013, 162, 522–533. [Google Scholar] [CrossRef] [Green Version]
  25. Aranda-Sicilia, M.N.; Trusov, Y.; Maruta, N.; Chakravorty, D.; Zhang, Y.L.; Botella, J.R. Heterotrimeric G proteins interact with defense-related receptor-like kinases in Arabidopsis. J. Plant Physiol. 2015, 188, 44–48. [Google Scholar] [CrossRef]
  26. Liang, X.; Ding, P.; Lian, K.; Wang, J.; Ma, M.; Li, L.; Li, L.; Li, M.; Zhang, X.; Chen, S.; et al. Arabidopsis heterotrimeric G proteins regulate immunity by directly coupling to the FLS2 receptor. eLife 2016, 5, e13568. [Google Scholar] [CrossRef]
  27. Liang, X.; Ma, M.; Zhou, Z.; Wang, J.; Yang, X.; Rao, S.; Bi, G.; Li, L.; Zhang, X.; Chai, J.; et al. Ligand-triggered de-repression of Arabidopsis heterotrimeric G proteins coupled to immune receptor kinases. Cell Res. 2018, 28, 529–543. [Google Scholar] [CrossRef] [Green Version]
  28. Li, B.; Tunc-Ozdemir, M.; Urano, D.; Jia, H.; Werth, E.G.; Mowrey, D.D.; Hicks, L.M.; Dokholyan, N.V.; Torres, M.P.; Jones, A.M. Tyrosine phosphorylation switching of a G protein. J. Biol. Chem. 2018, 293, 4752–4766. [Google Scholar] [CrossRef] [Green Version]
  29. Peng, Y.; Chen, L.; Li, S.; Zhang, Y.; Xu, R.; Liu, Z.; Liu, W.; Kong, J.; Huang, X.; Wang, Y.; et al. BRI1 and BAK1 interact with G proteins and regulate sugar-responsive growth and development in Arabidopsis. Nat. Commun. 2018, 9, 1522. [Google Scholar] [CrossRef] [Green Version]
  30. Yu, Y.; Chakravorty, D.; Assmann, S.M. The G protein β-subunit, AGB1, interacts with FERONIA in RALF1-regulated stomatal movement. Plant Physiol. 2018, 176, 2426–2440. [Google Scholar] [CrossRef] [Green Version]
  31. Xue, J.; Gong, B.-Q.; Yao, X.; Huang, X.; Li, J.-F. BAK1-mediated phosphorylation of canonical G protein alpha during flagellin signaling in Arabidopsis. J. Integr. Plant Biol. 2020, 62, 690–701. [Google Scholar] [CrossRef]
  32. Stateczny, D.; Oppenheimer, J.; Bommert, P. G protein signaling in plants: Minus times minus equals plus. Curr. Opin. Plant Biol. 2016, 34, 127–135. [Google Scholar] [CrossRef]
  33. Trusov, Y.; Botella, J.R. Plant G-proteins come of age: Breaking the bond with animal models. Front. Chem. 2016, 4, 24. [Google Scholar] [CrossRef] [Green Version]
  34. Coursol, S.; Fan, L.M.; Le Stunff, H.; Spiegel, S.; Gilroy, S.; Assmann, S.M. Sphingolipid signalling in Arabidopsis guard cells involves heterotrimeric G proteins. Nature 2003, 423, 651–654. [Google Scholar] [CrossRef]
  35. Coursol, S.; Le Stunff, H.; Lynch, D.V.; Gilroy, S.; Assmann, S.M.; Spiegel, S. Arabidopsis sphingosine kinase and the effects of phytosphingosine-1-phosphate on stomatal aperture. Plant Physiol. 2005, 137, 724–737. [Google Scholar] [CrossRef] [Green Version]
  36. Mishra, G.; Zhang, W.; Deng, F.; Zhao, J.; Wang, X. A bifurcating pathway directs abscisic acid effects on stomatal closure and opening in Arabidopsis. Science 2006, 312, 264–266. [Google Scholar] [CrossRef] [Green Version]
  37. Chen, Y.L.; Huang, R.; Xiao, Y.M.; Lü, P.; Chen, J.; Wang, X.C. Extracellular calmodulin-induced stomatal closure is mediated by heterotrimeric G protein and H2O2. Plant Physiol. 2004, 136, 4096–4103. [Google Scholar] [CrossRef] [Green Version]
  38. Li, J.H.; Liu, Y.Q.; Lü, P.; Lin, H.F.; Bai, Y.; Wang, X.C.; Chen, Y.L. 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. 2009, 150, 114–124. [Google Scholar] [CrossRef] [Green Version]
  39. Hao, L.H.; Wang, W.X.; Chen, C.; Wang, Y.F.; Liu, T.; Li, X.; Shang, Z.L. Extracellular ATP promotes stomatal opening of Arabidopsis thaliana through heterotrimeric G protein a subunit and reactive oxygen species. Mol. Plant 2012, 5, 852–864. [Google Scholar] [CrossRef] [Green Version]
  40. He, J.M.; Ma, X.G.; Zhang, Y.; Sun, T.F.; Xu, F.F.; Chen, Y.P.; Liu, X.; Yue, M. Role and interrelationship of Ga protein, hydrogen peroxide, and nitric oxide in ultraviolet B-induced stomatal closure in Arabidopsis leaves. Plant Physiol. 2013, 161, 1570–1583. [Google Scholar] [CrossRef] [Green Version]
  41. Ge, X.M.; Cai, H.L.; Lei, X.; Zhou, X.; Yue, M.; He, J.M. Heterotrimeric G protein mediates ethylene-induced stomatal closure via hydrogen peroxide synthesis in Arabidopsis. Plant J. 2015, 82, 138–150. [Google Scholar] [CrossRef]
  42. Shi, C.; Qi, C.; Ren, H.; Huang, A.; Hei, S.; She, X. Ethylene mediates brassinosteroid-induced stomatal closure via Galpha protein-activated hydrogen peroxide and nitric oxide production in Arabidopsis. Plant J. 2015, 82, 280–301. [Google Scholar] [CrossRef]
  43. Zhang, W.; He, S.Y.; Assmann, S.M. The plant innate immunity response in stomatal guard cells invokes G-protein-dependent ion channel regulation. Plant J. 2008, 56, 984–996. [Google Scholar] [CrossRef] [Green Version]
  44. Wang, X.Q.; Ullah, H.; Jones, A.M.; Assmann, S.M. G protein regulation of ion channels and abscisic acid signaling in Arabidopsis guard cells. Science 2001, 292, 2070–2072. [Google Scholar] [CrossRef] [Green Version]
  45. Zhang, W.; Jeon, B.W.; Assmann, S.M. Heterotrimeric G-protein regulation of ROS signalling and calcium currents in Arabidopsis guard cells. J. Exp. Bot. 2011, 62, 2371–2379. [Google Scholar] [CrossRef] [Green Version]
  46. Temple, B.R.S.; Jones, A.M. The plant heterotrimeric G-protein complex. Annu. Rev. Plant Biol. 2007, 58, 249–266. [Google Scholar] [CrossRef] [Green Version]
  47. Okamoto, H.; Matsui, M.; Deng, X.W. Overexpression of the heterotrimeric G-protein α-subunit enhances phytochrome-mediated inhibition of hypocotyl elongation in Arabidopsis. Plant Cell 2001, 13, 1639–1652. [Google Scholar]
  48. Zhang, T.Y.; Li, Z.Q.; Zhao, Y.D.; Shen, W.J.; Chen, M.S.; Gao, H.Q.; Ge, X.M.; Wang, H.Q.; Li, X.; He, J.M. Ethylene-induced stomatal closure is mediated via MKK1/3-MPK3/6 cascade to EIN2 and EIN3. J. Integr. Plant Biol. 2021, 63, 1324–1340. [Google Scholar] [CrossRef]
  49. Ge, X.M.; Hu, X.; Zhang, J.; Huang, Q.M.; Gao, Y.; Li, Z.Q.; Li, S.; He, J.M. UV resistance LOCUS8 mediates ultraviolet-B-induced stomatal closure in an ethylene-dependent manner. Plant Sci. 2020, 301, e110679. [Google Scholar] [CrossRef]
  50. Urano, D.; Chen, J.-G.; Botella, J.R.; Jones, A.M. Heterotrimeric G protein signalling in the plant kingdom. Open Biol 2013, 3, 120186. [Google Scholar] [CrossRef] [Green Version]
  51. Desikan, R.; Last, K.; Harrett-Williams, R.; Tagliavia, C.; Harter, K.; Hooley, R.; Hancock, J.T.; Neill, S.J. Ethylene-induced stomatal closure in Arabidopsis occurs via AtrbohF-mediated hydrogen peroxide synthesis. Plant J. 2006, 47, 907–916. [Google Scholar] [CrossRef]
  52. Tanaka, Y.; Sano, T.; Tamaoki, M.; Nakajima, N.; Kondo, N.; Hasezawa, S. Ethylene inhibits abscisic acid-induced stomatal closure in Arabidopsis. Plant Physiol. 2005, 138, 2337–2343. [Google Scholar] [CrossRef] [Green Version]
  53. Aharon, G.; Gelli, A.; Snedden, W.A.; Blumwald, E. Activation of a plant plasma membrane Ca2+ channel by TGα1, a heterotrimeric G protein α-subunit homologue. FEBS Lett. 1998, 424, 17–21. [Google Scholar] [CrossRef] [Green Version]
  54. Li, F.C.; Wang, J.; Wu, M.M.; Fan, C.M.; Li, X.; He, J.M. Mitogen-activated protein kinase phosphatases affect UV-B-induced stomatal closure via controlling NO in guard cells. Plant Physiol. 2017, 173, 760–770. [Google Scholar] [CrossRef] [Green Version]
Ijms 23 05512 g001 550
Figure 1. GCR1 plays a positive role in UV-B and ethylene induction of stomatal closure. (A) Leaves of the wild-type Col-0 (WT) and gcr1 mutants with open stomata were exposed to light alone (Control) or with 0.5 W/m2 UV-B (UV-B) for 3 h. (B) Leaves of the WT and gcr1 mutants with open stomata were incubated in MES buffer alone (Control) or containing 100 μmol/L ACC (ACC) for 3 h under the light. After treatments, stomatal apertures were measured in epidermal strips taken from the abaxial surfaces of the treated leaves. Data are presented as means ± SE of three independent experiments, each with 30 stomata. Means with different letters are significantly different at p < 0.01.
Figure 1. GCR1 plays a positive role in UV-B and ethylene induction of stomatal closure. (A) Leaves of the wild-type Col-0 (WT) and gcr1 mutants with open stomata were exposed to light alone (Control) or with 0.5 W/m2 UV-B (UV-B) for 3 h. (B) Leaves of the WT and gcr1 mutants with open stomata were incubated in MES buffer alone (Control) or containing 100 μmol/L ACC (ACC) for 3 h under the light. After treatments, stomatal apertures were measured in epidermal strips taken from the abaxial surfaces of the treated leaves. Data are presented as means ± SE of three independent experiments, each with 30 stomata. Means with different letters are significantly different at p < 0.01.
Ijms 23 05512 g001
Ijms 23 05512 g002 550
Figure 2. GCR1 mediates UV-B- and ethylene-induced stomatal closure via activating Gα subunit GPA1. (A) Leaves of the wild-type Col-0 (WT) and gcr1 mutants with open stomata were floated on MES buffer under light alone (Control) or with 0.5 W/m2 UV-B (UV-B) or the MES buffer containing 400 ng/mL CTX (CTX) for 3 h. (B) Leaves of the WT and gcr1 mutants with open stomata were incubated in MES buffer in the absence (Control) or presence of 100 μmol/L ACC (ACC) or 400 ng/mL CTX (CTX) for 3 h under the light. (C) qPCR to assess GPA1 relative expression level in the leaves of T2 transgenic lines overexpressing cGPA1 in the background of wild-type Col-0 and the gcr1-1 and gcr1-2 mutants. The transcript level of GPA1 in Col-0 was set to one. Data are shown as means ± SE of three independent biological determinations, and means with different letters are significantly different at p < 0.01. (D) Leaves of the WT Col-0, gcr1 mutants, and transgenic lines cGPA1/Col-0/gcr1-1/gcr1-2 with open stomata were floated on MES buffer under light alone (Control) or with 0.5 W/m2 UV-B (UV-B) or the MES buffer containing 100 μmol/L ACC (ACC) for 3 h. For (A), (B), and (D), stomatal apertures were measured in epidermal strips taken from the abaxial surfaces of the treated leaves. Data are shown as means ± SE (n = 3), each with 30 stomata. Means with different letters are significantly different at p < 0.01.
Figure 2. GCR1 mediates UV-B- and ethylene-induced stomatal closure via activating Gα subunit GPA1. (A) Leaves of the wild-type Col-0 (WT) and gcr1 mutants with open stomata were floated on MES buffer under light alone (Control) or with 0.5 W/m2 UV-B (UV-B) or the MES buffer containing 400 ng/mL CTX (CTX) for 3 h. (B) Leaves of the WT and gcr1 mutants with open stomata were incubated in MES buffer in the absence (Control) or presence of 100 μmol/L ACC (ACC) or 400 ng/mL CTX (CTX) for 3 h under the light. (C) qPCR to assess GPA1 relative expression level in the leaves of T2 transgenic lines overexpressing cGPA1 in the background of wild-type Col-0 and the gcr1-1 and gcr1-2 mutants. The transcript level of GPA1 in Col-0 was set to one. Data are shown as means ± SE of three independent biological determinations, and means with different letters are significantly different at p < 0.01. (D) Leaves of the WT Col-0, gcr1 mutants, and transgenic lines cGPA1/Col-0/gcr1-1/gcr1-2 with open stomata were floated on MES buffer under light alone (Control) or with 0.5 W/m2 UV-B (UV-B) or the MES buffer containing 100 μmol/L ACC (ACC) for 3 h. For (A), (B), and (D), stomatal apertures were measured in epidermal strips taken from the abaxial surfaces of the treated leaves. Data are shown as means ± SE (n = 3), each with 30 stomata. Means with different letters are significantly different at p < 0.01.
Ijms 23 05512 g002
Ijms 23 05512 g003 550
Figure 3. GCR1 mediates UV-B- and ethylene-induced ROS production in guard cells via activating Gα subunit GPA1. Leaves of the wild-type Col-0, gcr1 mutants, and transgenic plants cGPA1/Col-0/gcr1-1/gcr1-2 were floated on MES buffer in the absence or presence of 400 ng/mL CTX or 100 μmol/L ACC (ACC) and exposed to light alone (Control) or with 0.5 W/m2 UV-B (UV-B) for 3 h. Then the images (A) and fluorescence intensities (B) of guard cells preloaded with H2DCFDA were recorded. For (A), scale bar = 10 μm. For (B), data are shown as means ± SE (n = 3), each with 20 stomata. Means with different letters are significantly different at p < 0.05.
Figure 3. GCR1 mediates UV-B- and ethylene-induced ROS production in guard cells via activating Gα subunit GPA1. Leaves of the wild-type Col-0, gcr1 mutants, and transgenic plants cGPA1/Col-0/gcr1-1/gcr1-2 were floated on MES buffer in the absence or presence of 400 ng/mL CTX or 100 μmol/L ACC (ACC) and exposed to light alone (Control) or with 0.5 W/m2 UV-B (UV-B) for 3 h. Then the images (A) and fluorescence intensities (B) of guard cells preloaded with H2DCFDA were recorded. For (A), scale bar = 10 μm. For (B), data are shown as means ± SE (n = 3), each with 20 stomata. Means with different letters are significantly different at p < 0.05.
Ijms 23 05512 g003
Ijms 23 05512 g004 550
Figure 4. Exogenous H2O2 induced stomatal closure in the gcr1 mutants and cGPA1 transgenic plants in the absence or presence of UV-B or ACC. Leaves of the wild-type Col-0, gcr1 mutants, and transgenic plants cGPA1/Col-0/gcr1-1/gcr1-2 with open stomata were floated on MES buffer in the absence or presence of 100 μmol/L H2O2 (H2O2) under light alone (Control) or with 0.5 W/m2 UV-B (UV-B) (A) or the MES buffer containing 100 μmol/L ACC (ACC) (B) for 3 h. Then stomatal apertures were measured in epidermal strips. Data are shown as means ± SE (n = 3), each with 30 stomata. Means with different letters are significantly different at p < 0.01.
Figure 4. Exogenous H2O2 induced stomatal closure in the gcr1 mutants and cGPA1 transgenic plants in the absence or presence of UV-B or ACC. Leaves of the wild-type Col-0, gcr1 mutants, and transgenic plants cGPA1/Col-0/gcr1-1/gcr1-2 with open stomata were floated on MES buffer in the absence or presence of 100 μmol/L H2O2 (H2O2) under light alone (Control) or with 0.5 W/m2 UV-B (UV-B) (A) or the MES buffer containing 100 μmol/L ACC (ACC) (B) for 3 h. Then stomatal apertures were measured in epidermal strips. Data are shown as means ± SE (n = 3), each with 30 stomata. Means with different letters are significantly different at p < 0.01.
Ijms 23 05512 g004
Ijms 23 05512 g005 550
Figure 5. GCR1 mediated UV-B- and ethylene-induced NO generation in guard cells via activating Gα subunit GPA1. Leaves of the wild-type Col-0, gcr1 mutants, and transgenic plants cGPA1/Col-0/gcr1-1/gcr1-2 were floated on MES buffer in the absence or presence of 400 ng/mL CTX (CTX) or 100 μmol/L ACC (ACC) and exposed to light alone (Control) or with 0.5 W/m2 UV-B (UV-B) for 3 h. Then the images (A) and fluorescence intensities (B) of guard cells preloaded with DAF-2DA were recorded. For (A), scale bar = 10 μm. For (B), data are shown as means ± SE (n = 3), each with 20 stomata. Means with different letters are significantly different at p < 0.05.
Figure 5. GCR1 mediated UV-B- and ethylene-induced NO generation in guard cells via activating Gα subunit GPA1. Leaves of the wild-type Col-0, gcr1 mutants, and transgenic plants cGPA1/Col-0/gcr1-1/gcr1-2 were floated on MES buffer in the absence or presence of 400 ng/mL CTX (CTX) or 100 μmol/L ACC (ACC) and exposed to light alone (Control) or with 0.5 W/m2 UV-B (UV-B) for 3 h. Then the images (A) and fluorescence intensities (B) of guard cells preloaded with DAF-2DA were recorded. For (A), scale bar = 10 μm. For (B), data are shown as means ± SE (n = 3), each with 20 stomata. Means with different letters are significantly different at p < 0.05.
Ijms 23 05512 g005
Ijms 23 05512 g006 550
Figure 6. Exogenous NO induced stomatal closure in the gcr1 mutants and cGPA1 transgenic plants in the absence or presence of UV-B or ACC. Leaves of the wild-type Col-0, gcr1 mutants, and transgenic plants cGPA1/Col-0/gcr1-1/gcr1-2 with open stomata were floated on MES buffer in the absence or presence of 100 μmol/L SNP (SNP) and exposed to light alone (Control) or with 0.5 W/m2 UV-B (UV-B) (A) or the MES buffer containing with 100 μmol/L ACC (ACC) (B) for 3 h. Then stomatal apertures were measured in epidermal strips. Data are shown as means ± SE (n = 3), each with 30 stomata. Means with different letters are significantly different at p < 0.01.
Figure 6. Exogenous NO induced stomatal closure in the gcr1 mutants and cGPA1 transgenic plants in the absence or presence of UV-B or ACC. Leaves of the wild-type Col-0, gcr1 mutants, and transgenic plants cGPA1/Col-0/gcr1-1/gcr1-2 with open stomata were floated on MES buffer in the absence or presence of 100 μmol/L SNP (SNP) and exposed to light alone (Control) or with 0.5 W/m2 UV-B (UV-B) (A) or the MES buffer containing with 100 μmol/L ACC (ACC) (B) for 3 h. Then stomatal apertures were measured in epidermal strips. Data are shown as means ± SE (n = 3), each with 30 stomata. Means with different letters are significantly different at p < 0.01.
Ijms 23 05512 g006
Ijms 23 05512 g007 550
Figure 7. Ethylene acted upstream of GCR1 in UV-B guard cell signaling. Leaves of the wild-type Col-0 and gcr1 mutants were floated on MES buffer in the absence or presence of 100 μmol/L ACC (ACC) and exposed to light alone (Control) or with 0.5 W/m2 UV-B (UV-B) for 3 h. Then the stomatal apertures were measured in the epidermal strips (A), or ethylene production rate was analyzed in the treated leaves by gas chromatography (B). Data are shown as means ± SE (n = 3), and means with different letters are significantly different at p < 0.01.
Figure 7. Ethylene acted upstream of GCR1 in UV-B guard cell signaling. Leaves of the wild-type Col-0 and gcr1 mutants were floated on MES buffer in the absence or presence of 100 μmol/L ACC (ACC) and exposed to light alone (Control) or with 0.5 W/m2 UV-B (UV-B) for 3 h. Then the stomatal apertures were measured in the epidermal strips (A), or ethylene production rate was analyzed in the treated leaves by gas chromatography (B). Data are shown as means ± SE (n = 3), and means with different letters are significantly different at p < 0.01.
Ijms 23 05512 g007
Ijms 23 05512 g008 550
Figure 8. Modules showing the possible signaling pathways for UV-B- and ethylene-induced stomata closing. UV-B signaling in guard cells is initiated by the UV-B-specific signaling pathway UVR8-COP1-HY5 via induction of ethylene production. Ethylene is perceived by its receptor, ETR1, to inactivate the CTR1 kinase, releasing GCR1-dependent GPA1 activation. Then activated, GPA1 induces ROS and NO generation in guard cells and subsequent stomata closing. UV-B and ethylene maybe also activate GPA1 by inducing receptor-like kinases (RLKs)-phosphorylated GPA1 or by inhibiting RGS1-dependent deactivation of GPA1. Arrows and bars indicate induction and inhibition, respectively. The dashed line indicates a hypothetical cell response.
Figure 8. Modules showing the possible signaling pathways for UV-B- and ethylene-induced stomata closing. UV-B signaling in guard cells is initiated by the UV-B-specific signaling pathway UVR8-COP1-HY5 via induction of ethylene production. Ethylene is perceived by its receptor, ETR1, to inactivate the CTR1 kinase, releasing GCR1-dependent GPA1 activation. Then activated, GPA1 induces ROS and NO generation in guard cells and subsequent stomata closing. UV-B and ethylene maybe also activate GPA1 by inducing receptor-like kinases (RLKs)-phosphorylated GPA1 or by inhibiting RGS1-dependent deactivation of GPA1. Arrows and bars indicate induction and inhibition, respectively. The dashed line indicates a hypothetical cell response.
Ijms 23 05512 g008
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Li, X.; Fu, Q.; Zhao, F.-X.; Wu, Y.-Q.; Zhang, T.-Y.; Li, Z.-Q.; He, J.-M. GCR1 Positively Regulates UV-B- and Ethylene-Induced Stomatal Closure via Activating GPA1-Dependent ROS and NO Production. Int. J. Mol. Sci. 2022, 23, 5512. https://doi.org/10.3390/ijms23105512

AMA Style

Li X, Fu Q, Zhao F-X, Wu Y-Q, Zhang T-Y, Li Z-Q, He J-M. GCR1 Positively Regulates UV-B- and Ethylene-Induced Stomatal Closure via Activating GPA1-Dependent ROS and NO Production. International Journal of Molecular Sciences. 2022; 23(10):5512. https://doi.org/10.3390/ijms23105512

Chicago/Turabian Style

Li, Xue, Qi Fu, Fu-Xing Zhao, Yi-Qing Wu, Teng-Yue Zhang, Zhong-Qi Li, and Jun-Min He. 2022. "GCR1 Positively Regulates UV-B- and Ethylene-Induced Stomatal Closure via Activating GPA1-Dependent ROS and NO Production" International Journal of Molecular Sciences 23, no. 10: 5512. https://doi.org/10.3390/ijms23105512

APA Style

Li, X., Fu, Q., Zhao, F. -X., Wu, Y. -Q., Zhang, T. -Y., Li, Z. -Q., & He, J. -M. (2022). GCR1 Positively Regulates UV-B- and Ethylene-Induced Stomatal Closure via Activating GPA1-Dependent ROS and NO Production. International Journal of Molecular Sciences, 23(10), 5512. https://doi.org/10.3390/ijms23105512

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop