Ethylene mediates brassinosteroid-induced stomatal closure via Gα protein-activated hydrogen peroxide and nitric oxide production in Arabidopsis.

The Plant Journal (2015) 82, 280–301

doi: 10.1111/tpj.12815

Ethylene mediates brassinosteroid-induced stomatal closure via Ga protein-activated hydrogen peroxide and nitric oxide production in Arabidopsis Chenyu Shi1,2, Cheng Qi1, Hongyan Ren1, Aixia Huang1, Shumei Hei1,3 and Xiaoping She1,* 1 School of Life Sciences, Shaanxi Normal University, Xi’an 710062, China, 2 School of Chemistry and Bioengineering, Hechi University, Yizhou 546300, China, and 3 School of Life Sciences, Yan’an University, Yan’an 716000, China Received 25 July 2014; revised 14 February 2015; accepted 23 February 2015; published online 6 March 2015. *For correspondence (e-mail [email protected])

SUMMARY Brassinosteroids (BRs) are essential for plant growth and development; however, whether and how they promote stomatal closure is not fully clear. In this study, we report that 24-epibrassinolide (EBR), a bioactive BR, induces stomatal closure in Arabidopsis (Arabidopsis thaliana) by triggering a signal transduction pathway including ethylene synthesis, the activation of Ga protein, and hydrogen peroxide (H2O2) and nitric oxide (NO) production. EBR initiated a marked rise in ethylene, H2O2 and NO levels, necessary for stomatal closure in the wild type. These effects were abolished in mutant bri1-301, and EBR failed to close the stomata of gpa1 mutants. Next, we found that both ethylene and Ga mediate the inductive effects of EBR on H2O2 and NO production. EBR-triggered H2O2 and NO accumulation were canceled in the etr1 and gpa1 mutants, but were strengthened in the eto1-1 mutant and the cGa line (constitutively overexpressing the G protein a-subunit AtGPA1). Exogenously applied H2O2 or sodium nitroprusside (SNP) rescued the defects of etr1-3 and gpa1 or etr1 and gpa1 mutants in EBR-induced stomatal closure, whereas the stomata of eto11/AtrbohF and cGa/AtrbohF or eto1-1/nia1-2 and cGa/nia1-2 constructs had an analogous response to H2O2 or SNP as those of AtrbohF or Nia1-2 mutants. Moreover, we provided evidence that Ga plays an important role in the responses of guard cells to ethylene. Ga activator CTX largely restored the lesion of the etr1-3 mutant, but ethylene precursor ACC failed to rescue the defects of gpa1 mutants in EBR-induced stomatal closure. Lastly, we demonstrated that Ga-activated H2O2 production is required for NO synthesis. EBR failed to induce NO synthesis in mutant AtrbohF, but it led to H2O2 production in mutant Nia1-2. Exogenously applied SNP rescued the defect of AtrbohF in EBR-induced stomatal closure, but H2O2 did not reverse the lesion of EBR-induced stomatal closure in Nia1-2. Together, our results strongly suggest a signaling pathway in which EBR induces ethylene synthesis, thereby activating Ga, and then promotes AtrbohF-dependent H2O2 production and subsequent Nia1-catalyzed NO accumulation, and finally closes stomata. Keywords: brassinosteroid, ethylene, Ga protein, hydrogen peroxide, nitric oxide, stomata.

INTRODUCTION Brassinosteroids (BRs) are a family of naturally occurring plant steroids that are important for a broad range of plant growth and development processes, including reproduction and senescence, leaf development, root growth, vascular differentiation and responses to light, as well as € ft and Harother environmental cues (Clouse, 2011; Wittho ter, 2011; Ye et al., 2011). As potent plant growth regulators, BRs have also been used to enhance plant growth and yield of important agricultural crops (Khripach et al., 2000). In plants, the BR signaling cascade involves the perception of the BR signal and further downstream relay of 280

events, leading to BR-induced gene expression. As detailed € ft and Harter, in previous reviews (Clouse, 2011; Wittho 2011; Yang et al., 2011), BR is perceived at the plasma membrane by its receptor, BRASSINOSTEROID INSENSITIVE 1 (BRI1). BRI1 contains a leucine-rich repeat (LRR) extracellular domain, a transmembrane domain and a cytoplasmic kinase domain with serine/threonine specificity (Li and Chory, 1997; Friedrichsen et al., 2000). The extracellular domain, especially the island and the neighboring C-terminal LRR repeat, has been confirmed to be responsible for perceiving BR (Kinoshita et al., 2005); however, like © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd

Ethylene mediates BR-induced stomatal closure 281 many bri1 strong alleles (Friedrichsen et al., 2000; Vert et al., 2005), bri1 weak alleles bri1-301 and bri1-8 have defects in their kinase domain, but exhibit a mild morphological phenotype (Vert et al., 2005; Xu et al., 2008), which suggests that the significance of the kinase activity in the biological function of BRI1 remains obscure. Stomata located on the aerial organs of terrestrial plants play an important role in controlling CO2 absorption, water transpiration and pathogen invasion. Stomatal movement is regulated by multiple plant hormones, such as abscisic acid (ABA; Pei et al., 2000; Dodd, 2003), ethylene (Desikan et al., 2006) and methyl jasmonate (MJ; Suhita et al., 2004). Although the involvement of the GSK3/SHAGGY-like kinase BRASSINOSTEROID INSENSITIVE 2 (BIN2) in BRmodulated stomatal development has been reported (Gudesblat et al., 2012; Kim et al., 2012), the role and the potential mechanism of BR in the regulation of stomatal movement have not been fully understood. Evidence indicated that the stomatal closure of the sax1, an Arabidopsis BR-deficient mutant, is hypersensitive to ABA (Ephritikhine et al., 1999). Conversely, brassinolide (BL), which is the most bioactive natural BR, not only induces stomatal closure in Vicia faba (broad bean), but also prevents the stomata opening (Haubrick et al., 2006). Surprisingly, a more recent investigation indicated that different concentrations of 24-epibrassinolide (EBR), a bioactive BR, have the opposite effect on stomatal behavior in Solanum lycopersicum (tomato; Xia et al., 2014). Low doses of EBR induce stomatal opening, whereas high concentrations of EBR promote closure. Low doses of EBR-induced opening are closely related to a transient H2O2 production in guard cells and the cellular redox status of glutathione, yet ABA and the prolonged increase in H2O2 level mediate the effect of high doses of EBR on stomatal closure. Plant hormone interactions play an important role in the regulation of plant growth, development and stress responses (Zhang et al., 2009; Wang and Irving, 2011; Vanstraelen and Benkov0 a, 2012; Denance et al., 2013). Stomatal movement is mediated by a complex network of signaling pathways, in which the major player ABA acts in concert with either one or several phytohormones (Acharya and Assmann, 2009; Daszkowska-Golec and Szarejko, 2013). The study indicated that ethylene activates the expression of BRP, a BR-repressed reporter gene, in darkgrown Arabidopsis seedlings (Gendron et al., 2008). BRs have also been shown to stimulate ethylene biosynthesis by inducing the expression of the ACC synthase (ACS) gene in mung bean (Vigna radiate) Yi et al., (1999) or increasing the stability of ACS protein in Arabidopsis seedlings (Hansen et al., 2009). Consistent with the inductive effect of BRs on ethylene biosynthesis, Shi et al. (2006) reported that ethylene, as a downstream signal of BR, promotes cotton fiber elongation. Although the role of ethylene in stomatal movement has been investigated for

decades, its effect on this process seems rather inconsistent. In some investigations, ethylene was shown to open stomata (Levitt et al., 1987) or to inhibit ABA-induced stomatal closure (Tanaka et al., 2005; Acharya and Assmann, 2009; Wilkinson and Davies, 2009; Beguerisse-Diaz et al., 2012; Chen et al., 2013; Watkins et al., 2014), or to reduce stomatal sensitivity to stresses (Benlloch-Gonzalez et al., 2010; Iqbal et al., 2011), whereas in other works it was reported to close stomata (Pallas and Kays, 1982; Gunderson and Taylor, 1991; Young et al., 2004; Desikan et al., 2006). Yet the role of ethylene in BR-regulated stomatal movement has not been studied. Heterotrimeric GTP-binding proteins (G proteins), composed of a-, b- and c-subunits, are signalling molecules found in a variety of eukaryotic organisms. Upon signal reception by G protein-coupled receptors (GPCRs), the G protein a-subunit (Ga) binds GTP, resulting in the separation of Ga from Gbc subunit pair. Ga and Gbc can both interact with downstream components of signaling pathways to propagate the signal (Oldham and Hamm, 2008). In contrast to humans, which have 21 Ga genes, five Gb genes and 12 Gc genes, Arabidopsis thaliana has only one Ga gene (GPA1), one Gb gene (AGB1), and three Gc genes (AGG1-AGG3) (Jones and Assmann, 2004; Chakravorty et al., 2011). The functions of plant G proteins can be evaluated by using G protein-deficient mutants, and such studies have suggested that G proteins are involved in multiple signaling pathways in Arabidopsis, including developmental processes, responses to abiotic and biotic environmental signals, and the action of phytohormones such as ABA, jasmonate, BR and ethylene (Okamoto et al., 2001, 2009; Ullah et al., 2002; Assmann, 2005; Wang et al., 2006; Temple and Jones, 2007; Warpeha et al., 2007; Nilson and Assmann, 2010; Tsugama et al., 2013). To date, the roles of G proteins and the relationship between them and ethylene in BR regulation of guard cell responses are still unknown. Both reactive oxygen species (ROS) and nitric oxide (NO) have been well established as signaling molecules, mediating a wide range of cellular responses. H2O2, a form of ROS, and NO have been confirmed to act as signaling intermediates in stomatal closure by several plant hormones and external stimuli, such as ABA (Pei et al., 2000; Desikan et al., 2002; Neill et al., 2002), MJ (Suhita et al., 2004; Liu et al., 2005), high CO2 concentration (Kolla and Raghavendra, 2007; Kolla et al., 2007) and light/dark transition (She et al., 2004). The findings that ABA promotes NADPH oxidase-dependent H2O2 synthesis (Kwak et al., 2003) and nitrate reductase (NR)-mediated NO generation (Desikan et al., 2002) in Arabidopsis guard cells are a significant discovery, which provide genetic evidence for the sources of ABA-triggered H2O2 and NO production in guard cells. It is worthwhile to note that both H2O2 generated from NADPH oxidase and NO sourced from NR

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 280–301

282 Chenyu Shi et al. activity participate in stomatal closure by ethylene (Desikan et al., 2006; Liu et al., 2010), and G proteins mediate extracellular calmodulin- and extracellular ATP-led stomatal closure via regulating H2O2 and/or NO production (Chen et al., 2004; Li et al., 2009; Hao et al., 2012). Nevertheless, the roles of H2O2 and NO, and the relationship between them and ethylene or G proteins in BR-induced stomatal closure remain unclear. In this study, we used pharmacological approach and the following Arabidopsis mutants: bri1-301, which shows a weak morphological phenotype and a reduced sensitivity to BR; eto1-1, in which ethylene is overproduced; gpa1-1 and gpa1-2, which are GPA1 null mutants; etr1-1 and etr1-3, which have a defect in the perception of ethylene; AtrbohD, AtrbohF and AtrbohD, AtrbohF, which exhibit the lesion in producing H2O2; and Nia1-2, Nia2-1 and Nia2-5 Nia1-2, in which NO synthesis is impaired. Our results not only show that ethylene, Ga protein, H2O2 and NO are involved in stomatal closure by BR in Arabidopsis, but also suggest a signaling transduction pathway, in which BR promotes ethylene synthesis, induces the activation of Ga protein, then stimulates H2O2 and subsequent NO production, and ultimately closes stomata.

(a)

(b)

RESULTS Ethylene is involved in stomatal closure by EBR To examine whether BR can close stomata, we used the Arabidopsis bri1-301 mutant containing a mutation of Gly989 to Iso in the BRI1 kinase domain, which leads to a reduced sensitivity to BR and weak morphological abnormalities, including round leaves, short petioles, slightly shorter plant height, prolonged lifespan and nearly normal fertility (Xu et al., 2008), and the wild-type ecotype Columbia (Col-0), and observed the effects of EBR, a bioactive BR, in a range of concentrations and exposure times, on stomatal aperture. Prior to EBR treatment, the stomatal aperture of bri1-301 was smaller than that of the wild-type plant (Figure 1). This might be because the inhibition of cell elongation resulted from the lesion of BR perception (Xu et al., 2008; Wang et al., 2009). Treatment with EBR closed the stomata of the wild type in dose- and timedependent manners; however, the effect of EBR on stomatal closure in the wild type was fully abolished in the bri1301 mutant (Figure 1a,b). Linked with the results that ABA also closes the stomata of bri1-301 (Figure 2a) and BR receptor-silenced plants (Xia et al., 2014), our data here clearly show that BR indeed has a specific effect on stomatal aperture in Arabidopsis, and a functional BRI1 protein is essential for BR to induce stomatal closure. Because the maximum biological response concentration and time of EBR are 5 lM and 3 h, respectively (Figure 1), we chose 5 lM of EBR exposure for 3 h for the following experiments.

Figure 1. 24-Epibrassinolide (EBR) induced stomatal closure in Arabidopsis leaves.(a) Leaves of wild-type ecotype (WT) Col-0 or mutant bri1-301 with open stomata were kept in MES buffer alone (0) or with the indicated concentrations of EBR for 3 h.(b) Leaves of wild-type Col-0 or mutant bri1-301 with open stomata were kept in MES buffer alone or with 5 lM EBR for the indicated times. After treatment, stomatal apertures were observed in epidermal fragments from abaxial surfaces of the treated leaves. The data are represented as means SEs of three replicates, each with 50 stomata. Means with different letters are significantly different at P < 0.01.

To assess whether ethylene mediates BR signaling in guard cells, we measured the effect of EBR on ACS expression, ethylene production and stomatal aperture, and the expression of phyB activation-tagged suppressor 1 (BAS1), a sensitive marker gene for enhanced BR signaling (Wu et al., 2011), was also looked to corroborate the effect of EBR on ACS expression. EBR induced the expression of ACS5 and ACS9, known to be transcriptional BR target genes (Fridman et al., 2014), and BAS1, as well as ethylene production and closed stomata, in the wild type. These effects were highly repressed (ACS5 and BAS1) or nullified (ACS9, ethylene synthesis and stomatal closure) in the bri1-301 mutant (Figure 2a–c); however, ACC, an immediate precursor of ethylene, closed stomata in bri1-301, as in the wild type (Figure 2a). The data clearly indicate that ethylene acts as an intermediate signal molecule in BRinduced Arabidopsis stomatal closure, and a functional


Ethylene mediates BR-induced stomatal closure 283 Figure 2. Ethylene, H2O2 and NO are involved in 24-epibrassinolide (EBR)-induced stomatal closure. Leaves of wild-type (WT) Col-0 or mutant bri1-301 with open stomata were incubated in MES buffer alone (Control) or with 5 lM EBR, 100 lM ACC, 100 lM H2O2, 100 lM SNP, 5 lM ABA or 50 lM AOA for 3 h.(a) Stomatal apertures were measured in epidermal strips from the treated leaves.(b) The abundance of transcripts for ACS5, ACS9 and BAS1 was detected by RT-PCR of extracts of the treated leaves. Relative integrated density was shown below each band, where left first band in each row is set to 1.(c) The treated leaves were enclosed in a vial for another 3 h, the gas was gathered and ethylene was measured.(d–g) Fluorescence images (d) and pixel intensities (e) in guard cells pre-loaded with 50 lM H2DCFDA for 10 min, and fluorescence images (f) and pixel intensities (g) in guard cells pre-loaded with 10 lM DAF-2 DA for 30 min, in darkness, were recorded. Each assay was repeated at least three times. The blot of transcripts for ACS5, ACS9 and BAS1 shown is representative of three experiments. Data of stomatal aperture are displayed as means SES (n = 3), and means with different letters are significantly different at P < 0.01. Data of ethylene concentration are displayed as means SEs (n = 3), and means with different letters are significantly different at P < 0.05. Fluorescence pixel intensity data are displayed as means SEs (n = 3), and means with different letters are significantly different at P < 0.05. Scale bars: 10 lm.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

BRI1 protein mediates BR-induced ethylene production. Because the present study merely detected the effect of EBR on the expression of ACS5 and ACS9, we do not rule out the possibility that other ACS isozymes catalyze EBRinduced ethylene synthesis. Considering the fact that BR also increases the stability of ACS protein (Hansen et al., 2009), we postulate that the higher initial transcript levels of ACS5 and ACS9 in bri1-301 than in the wild type (Figure 2b) results from feedback upregulation caused by the loss of ACS stability following the lesion of BR perception. This supposition is supported by the effect of AOA, an ACS activity inhibitor, on ACS5 and ACS9 transcript levels in the wild type (Figure 2b). Conclusively determining whether this is the case will require further experimentation.

To further study whether ethylene is involved in BR-led stomatal closure, we used Arabidopsis ethylene-insensitive etr1-1 and etr1-3 mutants, in which the ethylene receptor ETR1 protein contains a Cys65Tyr and an Ala31Val mutation, respectively (Chang et al., 1993), and the wild type (Col-0). EBR-induced stomatal closure was completely abolished in etr1-1 and etr1-3, which is analogous to the response of the wild type pre-treated with ethylene perception inhibitors silver and 1-MCP, and ethylene synthesis inhibitor AOA and AVG, whereas the wild type responded normally to EBR (Figure 3a). Furthermore, we measured ethylene production in the leaves. As shown in Figure 3(b), EBR significantly induced ethylene production, AOA and AVG largely inhibited the effect of EBR, but silver and 1MCP inhibited EBR-induced ethylene production less, or


284 Chenyu Shi et al.

(a)

(b)

Figure 3. Ethylene is essential for 24-epibrassinolide (EBR)-induced stomatal closure. Arabidopsis wild-type (WT) Col-0 or etr1 mutant seedlings were pre-treated with 500 pl L1 1-MCP in a closed chamber, or not, and then leaves with open stomata were incubated in MES buffer without or with 50 lM AOA, 50 lM AVG or 10 lM Ag+ alone (Control), or with 5 lM EBR for 3 h.(a) Stomatal apertures were measured in epidermal strips from the abaxial surfaces of treated leaves.(b) Treated leaves were enclosed in a vial for another 3 h, and ethylene was then gathered and measured. Each assay was repeated at least three times. Data of stomatal aperture are displayed as means SEs (n = 3), and means with different letters are significantly different at P < 0.01. Data of ethylene concentration are displayed as means SEs (n = 3), and means with different letters are significantly different at P < 0.05.

not at all, in the wild type. In addition, EBR also largely stimulated ethylene synthesis in etr1-1 and etr1-3 mutants, as in the wild type (Figure 3b). The results further confirm the essential role of ethylene in the mediation of BRinduced stomatal closure. Incidentally, the effect of AOA, AVG, silver, 1-MCP or the ETR1 mutation on EBR-induced stomatal closure and ethylene synthesis is in accordance with their respective effect on ethylene synthesis or signaling. Both AtrbohF-dependent H2O2 and Nia1-catalyzed NO are required for stomatal closure by EBR To evaluate the potential role of H2O2 and NO in BRinduced stomatal closure, the effect of EBR on stomatal

aperture was measured, and EBR-induced H2O2 or NO synthesis in guard cells was monitored using confocal microscopy and the H2O2 fluorescent dye 20 ,70 -dichlorofluorescin diacetate (H2DCFDA), or the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2DA), in the wild type (Col-0) and bri1-301 mutant. EBR increased H2O2 and NO levels in guard cells and also closed stomata in the wild type; these effects were impaired in the bri1-301 mutant (Figure 2a,d–g). However, exogenously applied H2O2 or sodium nitroprusside (SNP), an NO donor, significantly closed stomata in bri1-301, as in the wild type (Figure 2a). These data clearly show that both H2O2 and NO are involved in BR-induced stomatal closure, and BRI1 protein is required for BR inducing H2O2 and NO generation in guard cells in Arabidopsis. To further study whether both H2O2 and NO participates in BR-induced stomatal closure, we then observed the effects of ascorbic acid (ASA), an important reducing substrate for H2O2 removal, catalase (CAT), an H2O2 scavenger enzyme, diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (c-PTIO), an NO scavenger and tungstate, an NR inhibitor, on EBR-induced stomatal closure and H2O2 or NO production in guard cells of the wild type (Col-0). ASA and CAT completely inhibited EBRinduced stomatal closure and H2O2 production in guard cells (Figure 4a–c). Similarly, EBR-induced stomatal closure and NO synthesis in guard cells were also fully prevented by c-PTIO (Figure 5a–c). These results suggest that both H2O2 and NO are essential for BR-induced stomatal closure. Furthermore, DPI or tungstate significantly repressed EBR-induced stomatal closure (Figures 4a and 5a), which is consistent with the effect of DPI or tungstate on H2O2 or NO production (Figures 4b,c and 5b,c). The data imply that both H2O2 sourced from NADPH oxidases and NO generated by NR are required for BR-led stomatal closure. To corroborate these pharmacological data, we further used Arabidopsis (Col-0) respiratory-burst oxidase homologue (Rboh) NADPH oxidases single and double atrbohD and atrbohF mutants (Kwak et al., 2003), and NR-deficient mutants Nia1-2, Nia2-1 and Nia2-5 Nia1-2 (Wilkinson and Crawford, 1993; Desikan et al., 2002). EBR closed the stomata of mutants AtrbohD and Nia2-1, but closed the stomata less or not at all in AtrbohF, AtrbohD AtrbohF, Nia1-2 and Nia2-5 Nia1-2 (Figures 4a and 5a). Meanwhile, EBR increased H2O2 or NO levels in the guard cells of mutant AtrbohD or Nia2-1, but failed to raise H2O2 or NO levels in the guard cells of mutants AtrbohF and AtrbohD AtrbohF or Nia1-2 and Nia2-5 Nia1-2 (Figures 4b,c and 5b,c). These responses of mutants AtrbohF and AtrbohD AtrbohF or Nia1-2 and Nia2-5 Nia1-2 are similar to those of the wild type pre-treated with DPI or tungstate (Figures 4a–c and 5a–c). The data not only confirm the essential role of H2O2 and NO in BR-induced stomatal closure in Arabidopsis, but


Ethylene mediates BR-induced stomatal closure 295 specific amino-acid residue in the kinase domain is indispensable for the function of BRI1 in BR-mediated stomatal regulation. Both ethylene and Ga are essential for stomatal closure by BR The cooperative effect between BR and other plant hormones, including ethylene, are crucial to a broad spectrum of plant physiological and developmental processes. BR induces ethylene biosynthesis through stimulating ACS and ACC oxidase activities (Hansen et al., 2009), BR signaling components also mediate ethylene-induced growth and defense responses (Bar et al., 2010; Deslauriers and Larsen, 2010; Cheung and Wu, 2011). G proteins play a vital role in the transduction of extracellular signals into intracellular responses, they are also shown to be a important regulator of guard cell responses to ABA, extracellular calmodulin and extracellular ATP (Wang et al., 2001; Chen et al., 2004; Li et al., 2009; Chakravorty et al., 2011; Zhang et al., 2011; Hao et al., 2012); however, until now, whether or not ethylene and G proteins mediate the response of stomatal guard cells to BR remains unknown. Our results in the present study provide convincing evidence that ethylene and Ga participate in the induction of stomatal closure by BR in Arabidopsis. Ethylene synthesis and perception inhibitors and Ga inhibitor fully prevented EBRled stomatal closure (Figures 3 and 6), and ethylene precursor and Ga activator, like EBR, significantly closed stomata (Figures 2 and 6), revealing that ethylene synthesis and perception and the activation of Ga are essential for BR to close stomata. The inability of EBR to close the stomata of etr1 and gpa1 mutants (Figures 3 and 6), and the more significant effect of EBR on stomatal closure in the eto1-1 mutant and cGa line than in the wild type (Figures 7–10), further provide genetic evidence for the essential role of ethylene and Ga in BR guard cell signaling. Because ABA is a main regulator of stomatal movements, the relationship between BR and ABA in the regulation of stomatal movement is an interesting question. A recent study has indicated that ABA acts downstream of H2O2 in the induction of stomatal closure by BR in tomato (Xia et al., 2014); however, our results here, along with the data from related studies, provide evidence that ABA does not

mediate the induction of stomatal closure by BR in Arabidopsis. The fact that EBR-induced stomatal closure is completely abolished in etr1 mutants (Figure 3) excludes the possibility that ABA also mediates the BR induction of stomatal closure. The finding that ABA closes the stomata of etr1 and gpa1 mutants (Wang et al., 2001; Desikan et al., 2006), but that BR does not (Figures 3 and 6), also supports the conclusion above. The difference of EBR response between tomato and Arabidopsis guard cells may be relative to the concentration of EBR used, the plant species, or both. The evidence indicates that ethylene can close stomata (Desikan et al., 2006). Ga was also known to be involved in ABA inhibition of stomatal opening and extracellular calmodulin- and extracellular ATP-induced stomatal closure (Wang et al., 2001; Chen et al., 2004; Li et al., 2009; Chakravorty et al., 2011; Zhang et al., 2011; Hao et al., 2012). Our results here further show that both ethylene and Ga are required for the BR induction of stomatal closure in Arabidopsis; however, the underlying mechanism by which BR induces ethylene synthesis and the roles of the other subunits of G proteins in BR guard cell signalling remain unclear. Both H2O2 and NO are required for the induction of stomatal closure by BR H2O2 and NO are extensively studied signaling molecules in plants. Numerous studies have shown that H2O2 and NO are involved in stomatal closure by multiple external stimuli or plant hormones, including ABA (Pei et al., 2000; Desikan et al., 2002; Neill et al., 2002). The ethylene inhibition of ABA-induced stomatal closure was also shown to be relative to an as-yet-unidentified antioxidant mechanism- and flavonol-induced reduction in the level of ROS, including H2O2, in guard cells (Beguerisse-Diaz et al., 2012; Watkins et al., 2014). Although H2O2 and NO are known to mediate BR-induced stress tolerance (Xia et al., 2009; Cui et al., 2011), the study of the role of H2O2 in BR-mediated stomatal movement is just beginning, and whether or not NO mediates the action of BR in stomatal behavior remains unknown. Our results clearly indicate that EBR-induced H2O2 and NO production and stomatal closure in wild-type Arabidopsis were fully abolished in the bri1-301 mutant (Figures 2a,c–f), but that H2O2 and SNP significantly closed

Figure 12. H2O2 production is necessary for NO synthesis in 24-epibrassinolide (EBR)-induced stomatal closure.(a) Leaves of wild-type Col-0 and mutants AtrbohD, AtrbohF and AtrbohD AtrbohF with open stomata incubated in MES buffer without or with 100 lM ASA, 100 units ml1 CAT or 10 lM DPI in the absence (Control) or presence of 5 lM EBR, 100 lM SNP or 5 lM EBR and 100 lM SNP for 3 h, and then stomatal apertures were measured in epidermal strips.(b, c) Leaves of wild-type Col-0 and mutants AtrbohD, AtrbohF and AtrbohD AtrbohF with open stomata were incubated in MES buffer without or with 100 lM ASA, 100 units ml1 CAT or 10 lM DPI in the absence (Control) or presence of 5 lM EBR for 3 h. Fluorescence intensities (b) and images (c) of guard cells pre-loaded with 10 lM DAF-2DA were recorded.(d) Leaves of wild-type Col-0 and mutants Nia1-2, Nia2-1 and Nia2-5 Nia1-2 with open stomata were incubated in MES buffer without or with 200 lM c-PTIO or 100 lM tungstate in the absence (Control) or presence of 5 lM EBR, 100 lM H2O2 or 5 lM EBR and 100 lM H2O2 for 3 h, then stomatal apertures were measured in epidermal strips.(e, f) Leaves of wild-type Col-0 and mutants Nia1-2, Nia2-1 and Nia2-5 Nia1-2 with open stomata were incubated in MES buffer without or with 200 lM c-PTIO or 100 lM tungstate in the absence (Control) or presence of 5 lM EBR for 3 h. Fluorescence intensities (e) and images (f) of guard cells pre-loaded with 50 lM H2DCFDA were recorded. Each assay was repeated at least three times. The data of stomatal aperture are displayed as means SEs (n = 3), and means with different letters are significantly different at P < 0.01. Data of fluorescence intensities are displayed as means SEs (n = 3), and means with different letters are significantly different at P < 0.05. Scale bars: 10 lm, for all images.


296 Chenyu Shi et al. the stomata in the bri1-301 mutant (Figure 2a). The data show that a functional BRI1 protein is vital for BR-induced H2O2 and NO productions and subsequent stomatal closure. Additionally, H2O2 or NO scavenger completely inhibited EBR-induced stomatal closure and H2O2 or NO accumulation in guard cells of the wild type (Figures 4a–c and 5a–c), which provides evidence that both H2O2 and NO, as intermediate signal molecules, act in stomatal closure by BR. The enzymatic sources of H2O2 in guard cells are known to be diverse, including NADPH oxidases (Kwak et al., 2003), cell wall peroxidases (Khokon et al., 2010) and amine oxidase (An et al., 2008). Nitric oxide synthase (NOS) and NR were also shown to be potential sources of NO in guard cells (Neill et al., 2002), although no genes or proteins with sequence homology to known mammaliantype NOS have been found in plants. So far, however, the origins of H2O2 and NO in BR-induced stomatal closure are incompletely clear. Our data here show that EBR-led stomatal closure in the wild type was largely abolished in the NADPH oxidase single mutant AtrbohF and double mutant AtrbohD AtrbohF, or in NR mutants Nia1-2 and Nia2-5 Nia1-2, in accordance with the effect of EBR on H2O2 or NO production in the guard cells of these plants (Figure 4a–c and 5a–c). Clearly the data provide convincing evidence for the important roles of AtrbohF-mediated H2O2 production and Nia1-dependent NO synthesis in BR-led stomatal closure in Arabidopsis. As aforementioned, H2O2 and NO are required for stomatal closure by multiple internal factors and abiotic or biotic environmental conditions (Pei et al., 2000; Desikan et al., 2002; Neill et al., 2002; She et al., 2004; Suhita et al., 2004; Liu et al., 2005; Melotto et al., 2006; Kolla and Raghavendra, 2007; Kolla et al., 2007). Our data here further show that both H2O2 sourced from AtrbohF and NO generated by Nia1 are crucial for stomatal closure by BR. Kwak et al. (2003) reported that ABA-induced stomatal closure is partially impaired in AtrbohF and more strongly impaired in AtrbohD AtrbohF, but AtrbohD, like the wild type, responds normally to ABA, which suggests that there is some overlap in the functions of AtrbohD and AtrbohF. Another study showed that only AtrbohF plays an essential role for ethylene-induced H2O2 production and stomatal closure (Desikan et al., 2006). Our results here indicate that the responses of both AtrbohD and AtrbohF to BR were similar to the responses to ethylene, but differed from the responses to ABA (Figure 4). The results suggest that AtrbohD and AtrbohF may have different roles in ABA- and ethylene- or BR-induced responses, and support that ethylene mediates the effect of BR on H2O2 production and the resulting stomatal closure (Figures 2 and 3). In addition, in this report our genetic evidence clearly shows that BR-induced NO production and subsequent stomatal closure were completely impaired in mutants Nia1-2 and Nia1-2 Nia2-5, but not in mutant Nia2-1, as in the wild type (Figure 5). Similar to our data, Bright et al.

(2006) proposed that Nia1 is mainly responsible for the ABA-induced NO generation in guard cells. The similar responses of Nia1-2 and Nia2-1 to BR and ABA might be related to the fact that both BR and ABA induction of NO production depend on H2O2 derived from NADPH oxidases (Figure 12; Bright et al., 2006). Both ethylene and Ga activate H2O2 and NO production in stomatal closure by BR Previous work indicated that BR can stimulate ethylene synthesis (Hansen et al., 2009), and that ethylene closes stomata by inducing H2O2 and NO production (Desikan et al., 2006; Liu et al., 2010). In addition, Ga-triggered H2O2 production has been known to be involved in ABA inhibition of stomatal opening and extracellular calmodulin- and extracellular ATP-led stomatal closure (Chen et al., 2004; Zhang et al., 2011; Hao et al., 2012), and the role of Ga in extracellular calmodulin-led stomatal closure is closely associated with its inductive effect on NO synthesis (Li et al., 2009). These studies suggest that ethylene and Ga may play an important mediating role in guard cell signaling; however, as yet it is unknown whether ethylene and Ga also mediate BR-induced stomatal closure via the activation of H2O2 and NO production. Our data here strongly support that ethylene- and Ga-activated H2O2 and NO production are involved in BR-led stomatal closure. The eto1-1 mutant and cGa line guard cells generated higher levels of H2O2 and NO than their respective wild type, and EBR failed to increase H2O2 and NO levels in the guard cells of etr1 and gpa1 mutants. Meanwhile, EBR-induced increases of H2O2 and NO in the eto1-1 mutant and cGa line were abolished in eto1-1 AtrbohF and cGa AtrbohF constructs, and in eto1-1 Nia1-2 and cGa Nia1-2 constructs, respectively. H2O2 rescued the defect of EBR-induced stomatal closure in etr1-3 and gpa1 mutants, whereas SNP recovered the lesions of etr1 and gpa1 mutants in EBR-induced stomatal closure (Figures 7–10). These data convincingly affirm that ethylene and Ga promote H2O2 and NO generation in BR-initiated guard cell signaling; however, how ethylene and Ga mediate the promoting effect of BR on H2O2 and NO production remains unclear. The G protein a-subunit acts downstream of ethylene in the BR induction of stomatal closure In plants, heterotrimeric G proteins function as important signaling molecules mediating responses to a range of abiotic and biotic signals, incuding plant hormones such as gibberellic acid (GA; Ullah et al., 2002), ABA (Pandey et al., 2006), BR (Ullah et al., 2001) and MJ (Trusov et al., 2006). As yet, whether G proteins are involved in the responses of plant guard cells to ethylene remains unclear. Both ethylene and Ga activate the AtrbohF-dependent H2O2 production and Nia1-catalyzed NO synthesis necessary for the stomatal closure induced by BR (Figures 4, 5, 7–10), which


Ethylene mediates BR-induced stomatal closure 297 implies that they may play a role in the same signaling pathway. Ga mediates the ethylene-induced triple response (Ullah et al., 2002) and inhibition of root elongation (Wang et al., 2006), although the detailed mechanisms have not been studied. This inspires us to assess its potential role in the response of the guard cell to ethylene. Our results in this report provide evidence that Ga plays an important role in the responses of Arabidopsis guard cells to ethylene. The Ga activator largely restored the lesion of EBR-induced stomatal closure, and also closed the stomata in the wild type treated with ethylene perception or synthesis inhibitor, and in the mutant etr1-3 (Figure 11c). On the contrary, the ethylene precursor failed to rescue the defect of EBR-induced stomatal closure, and also did not close the stomata in both the wild type treated with Ga inhibitor and the mutants gpa1 (Figure 11a). The data clearly show that Ga acts downstream of ethylene in BR guard cell signaling. The data indicating that EBR normally promoted ethylene production in the wild type treated with Ga inhibitor and in the mutant gpa1 (Figure 11b) also support this conclusion. In this context, the data that EBR has no promoting effect on the expression of GPA1 (Figure 11d) suggest that ethylene acts via increasing Ga activity in BR-induced stomatal closure. Although our results here show an important role of Ga in the responses of guard cells to ethylene, whether the other subunits of G proteins also acts to mediate the ethylene responses of guard cells and other plant cells is still an interesting question to be addressed in the future. In addition, the relationship between H2O2 and NO in BR-induced stomatal closure was also studied. EBR failed to induce NO synthesis in the guard cells of mutants AtrbohF and AtrbohD AtrbohF, but it induced H2O2 production in the guard cells of Nia1-2 and Nia2-5 Nia1-2 (Figure 12). The results indicate that BR-induced H2O2 is requisite for NO synthesis in Arabidopsis. This relationship between H2O2 and NO in stomatal movement is in agreement with the results of Bright et al. (2006), but differs somewhat from our previous study that suggested an interrelationship between H2O2 and NO in light/dark transition-induced stomatal closure in broad bean (She et al., 2004). A discrepancy that may reflect the difference between BR and the light/dark signal transduction mechanism in guard cells, or may result from the different plant species used. In summary, the data presented herein suggest a working model for BR action in the guard cell response of Arabidopsis (Figure 13). Binding of BR to its receptor BRI1 induces ACS expression and ethylene synthesis. Increased ethylene activates Ga. Active Ga stimulates AtrbohF-dependent H2O2 generation and subsequent Nia1-catalysed NO production, and finally closes the stomata. The findings confirm the effect of BR on stomatal movement, and elucidate the crucial role of ethylene and G proteins in the

guard cell BR signalling. The Arabidopsis guard cell offers an attractive tool with which to dissect novel aspects of BR signalling; however, several relevant questions need to be further studied in the future. First, although our study clearly shows that BRI1 protein mediates BR-induced ethylene production (Figure 2), how BRI1 mediates ethylene production remains unknown. Studies show that the MAPK cascade induces the expression and phosphorylation of ACS (Liu and Zhang, 2004; Li et al., 2012), hence resulting in higher cellular ACS activity and elevated ethylene. Recently, Meng et al. (2012) reported that ERECTA (ER), a receptor-like protein kinase (RLK), determines Arabidopsis inflorescence architecture by activiting an MAPK cascade. Furthermore, a more recent study provided evidence that MAPK cascade-activated ethylene and subsequent ROS production are essential for the salt sensitivity mediated by Salt Intolerance 1 (SIT1) in rice (Oryza sativa), a lectin receptor-like protein kinase (Li et al., 2014). The results, along with our data here (Figure 2), imply that BRI1, one of the best-studied RLKs, might directly or indirectly induce the expression and phosphorylation of ACS in the BR induction of stomatal closure; however, whether or not BRI1 acts in the manner discribed above remains to be studied. Second, how Ga activates NADPH oxidase remains to be elucidated. Previous studies indicate that one possible target of the heterotrimeric G proteins is an increase in cytosolic Ca2+ in the plant cell (Aharon et al., 1998), whereas NADPH oxidase is directly activated by Ca2+ and Ca2+dependent protein kinase (Kobayashi et al., 2007; Ogasawara et al., 2008). Two different GTPases, the small GTPase Rac, which is a homolog of the cytosolic regulator small GTPase Rac of mammalian NADPH oxidase in plants, and

Figure 13. Model showing the possible signaling pathway for 24-epibrassinolide (EBR)-induced stomatal closure. EBR induces the expression of ACS5 and ACS9 and ethylene synthesis via a BRI1-dependent mechanism, thereby activating G protein to induce H2O2 generation by AtrbohF, which increases Nia1-catalyzed NO production. NO subsequently closes the stomata.



(a)

(b)

(c)

(d)

(e)

(f)

Figure 7. Ethylene mediates 24-epibrassinolide (EBR)-induced stomatal closure via inducing H2O2 production.(a, b) Wild-type (WT) Col-0 seedlings were pretreated with 500 pl L1 1-MCP, or not, in a closed chamber, and then treated leaves with open stomata were incubated in MES buffer without or with 50 lM AOA, 50 lM AVG or 10 lM Ag+ alone (Control), or with 5 lM EBR for 3 h, or leaves of wild-type Col-0 or mutants AtrbohD, AtrbohF or AtrbohD AtrbohF with open stomata were incubated in MES buffer with 100 lM ACC for 3 h. Fluorescence images (a) and pixel intensities (b) in guard cells pre-loaded with 50 lM H2DCFDA were measured.(c) Leaves of wild-type Col-0 or mutants AtrbohD, AtrbohF or AtrbohD AtrbohF with open stomata were incubated in MES buffer alone (Control) or with 100 lM ACC for 3 h, then stomatal apertures were measured in epidermal strips.(d, e) Leaves of wild-type Col-0, etr1 and eto1-1 mutants, and the eto1-1 AtrbohF construct were incubated in MES buffer alone (Control) or with 5 lM EBR for 3 h, then fluorescence images (d) and pixel intensities (e) in guard cells pre-loaded with 50 lM H2DCFDA were recorded.(f) Leaves of wild-type Col-0, etr1 and eto1-1 mutants, and the eto1-1 AtrbohF construct, were incubated in MES buffer without (Control) or with 5 lM EBR, 100 lM H2O2, or 5 lM EBR and 100 lM H2O2 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 SEs (n = 3), and means with different letters are significantly different at P < 0.01. Data of fluorescence intensities are displayed as means SEs (n = 3); means with different letters are significantly different at P < 0.05. Scale bars: 10 lm, for all images.


Ethylene mediates BR-induced stomatal closure 289

(a)

(b)

(c)

(d)

(e)

(f)

Figure 8. Ethylene mediates 24-epibrassinolide (EBR)-induced stomatal closure via initiating NO generation.(a, b) Wild-type (WT) Col-0 seedlings were pre-treated with 500 pl L1 1-MCP, or not, in a closed chamber, and then treated leaves with open stomata were incubated in MES buffer without or with 50 lM AOA, 50 lM AVG or 10 lM Ag+ alone (Control), or with 5 lM EBR for 3 h, or leaves of wild-type Col-0 or mutants Nia1-2, Nia2-1 or Nia2-5 Nia1-2 with open stomata were incubated in MES buffer with 100 lM ACC for 3 h. Fluorescence images (a) and pixel intensities (b) in guard cells pre-loaded with 10 lM DAF-2DA were recorded.(c) Leaves of wild-type Col-0 or mutants Nia1-2, Nia2-1 or Nia2-5 Nia1-2 with open stomata were incubated in MES buffer alone (Control) or with 100 lM ACC for 3 h, and then stomatal apertures were measured in epidermal strips.(d, e) Leaves of wild-type Col-0, etr1 and eto1-1 mutants or the eto1-1Nia1-2 construct were incubated in MES buffer alone (Control) or with 5 lM EBR for 3 h, then fluorescence images (d) and pixel intensities (e) in guard cells pre-loaded with 10 lM DAF-2DA were recorded.(f) Leaves of wild-type Col-0, etr1 and eto1-1 mutants, and the eto1-1Nia1-2 construct were incubated in MES buffer without (Control) or with 5 lM EBR, 100 lM SNP or 5 lM EBR and 100 lM SNP 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 SEs (n = 3); means with different letters are significantly different at P < 0.01. Data of fluorescence intensities are displayed as means SEs (n = 3); means with different letters are significantly different at P < 0.05. Scale bars: 10 lm, for all images.



(a)

(d)

(b)

(e)

(c)

(f)

Figure 9. GPA1 mediates 24-epibrassinolide (EBR)-induced stomatal closure via activating H2O2 production.(a, b) Leaves of wild-type (WT) Col-0 with open stomata were incubated in MES buffer without or with 400 ng ml1 PTX alone (Control), or with 5 lM EBR for 3 h, or leaves of wild-type Col-0 and mutants AtrbohD, AtrbohF or AtrbohD AtrbohF with open stomata were incubated in MES buffer with 400 ng ml1 CTX for 3 h, and then fluorescence images (a) and pixel intensities (b) in guard cells pre-loaded with 50 lM 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 (Control) or presence of 400 ng ml1 CTX for 3 h, and then stomatal apertures were measured in epidermal strips.(d, e) Leaves of wild-type Ws, the gpa1 mutant, the cGa1 line, and the cGa1 AtrbohF construct were incubated in MES buffer without (Control) or with 5 lM EBR for 3 h, and then fluorescence images (d) and pixel intensities (e) in guard cells pre-loaded with 50 lM H2DCFDA were recorded.(f) Leaves of wild-type Ws, the gpa1 mutant, the cGa1 line, and the cGa1 AtrbohF construct were incubated in MES buffer without (Control) or with 5 lM EBR, 100 lM H2O2, 5 lM EBR and 100 lM H2O2 for 3 h, and then stomatal apertures were measured in epidermal strips. Each assay was repeated at least three times. Data of stomatal aperture are displayed as means SEs (n = 3); means with different letters are significantly different at P < 0.01. Data of fluorescence intensities are displayed as means SEs (n = 3), and means with different letters are significantly different at P < 0.05. Scale bars: 10 lm, for all images.

was stronger in the cGa line than in the wild type (Ws), whereas EBR-led increases of H2O2 (Figure 9d,e) and NO (Figure 10d,e) fluorescence intensity in the guard cells of the wild type were fully prevented in the guard cells of gpa1 mutants. Together with the more marked effect of EBR on stomatal closure in the cGa line than in the wild type (Figures 6c, 9f, 10f), and the defect of gpa1 mutants in EBR-led stomatal closure (Figure 6b, 9f, 10f), the effects of EBR on H2O2 and NO production in gpa1 mutants and the cGa line provide genetic evidence that Ga, as a regulatory

molecule of H2O2 and NO synthesis, mediates BR-led stomatal closure. Additionally, both H2O2 (Figure 9f) and NO donor SNP (Figure 10f) largely redeemed the deficiencies of gpa1 mutants in EBR-led stomatal closure, and the data also offer support for Ga promoting H2O2 and NO production in BR-stimulated stomatal closure. To further provide genetic evidence for Ga promoting H2O2 and NO production in BR-led stomatal closure, we next crossed the cGa1 line with the AtrbohF or Nia1-2 mutants, respectively, and then observed the stomatal


Ethylene mediates BR-induced stomatal closure 291

(a)

(d)

(b)

(e)

(c)

(f)

Figure 10. GPA1 mediates 24-epibrassinolide (EBR)-induced stomatal closure via regulating NO production.(a, b) Leaves of wild-type (WT) Col-0 with open stomata were incubated in MES buffer without or with 400 ng ml1 PTX alone (Control), or with 5 lM EBR for 3 h, or leaves of wild-type Col-0 and mutants Nia1-2, Nia2-1 or Nia2-5 Nia1-2 with open stomata were incubated in MES buffer with 400 ng ml1 CTX for 3 h, and then fluorescence images (a) and pixel intensities (b) in guard cells pre-loaded with 10 lM DAF-2DA were recorded.(c) Leaves of wild-type Col-0 and mutants Nia1-2, Nia2-1 or Nia2-5 Nia1-2 were incubated in MES buffer in the absence (Control) or presence of 400 ng ml1 CTX for 3 h, and then stomatal apertures were measured in epidermal strips.(d, e) Leaves of wild-type Ws, the gpa1 mutant, the cGa1 line, and the cGa1 AtrbohF construct were incubated in MES buffer without (Control) or with 5 lM EBR for 3 h, and then fluorescence images (d) and pixel intensities (e) in guard cells pre-loaded with 10 lM DAF-2DA were recorded.(f) Leaves of wild-type Ws, the gpa1 mutant, the cGa1 line, and the cGa1 Nia1-2 construct were incubated in MES buffer without (Control) or with 5 lM EBR, 100 lM SNP or 5 lM EBR and 100 lM SNP for 3 h, and then stomatal apertures were measured in epidermal strips. Each assay was repeated at least three times. Data for stomatal aperture are displayed as means SEs (n = 3), and means with different letters are significantly different at P < 0.01. Data of fluorescence intensities are displayed as means SEs (n = 3); means with different letters are significantly different at P < 0.05. Scale bars: 10 lm, for all images.

behavior and measured the H2O2 and NO levels in the guard cells of these constructs. As shown in Figure 9, the cGa AtrbohF construct had an obvious defect in EBRinduced H2O2 production in guard cells (Figure 9d,e) and stomatal closure (Figure 9f). Likewise, EBR-induced NO synthesis in the guard cells (Figure 10d,e) and stomatal closure (Figure 10f) in the wild type (Ws) were fully abolished in cGa Nia1-2. The data also support that H2O2 and NO production, as downstream events of Ga action, mediate BR-induced stomatal closure.

Overall, our data described above provide convincing evidence that G protein a-subunit acts upstream of H2O2 and NO production to mediate BR-induced Arabidopsis stomatal closure. Ga is essential for the action of ethylene in stomatal closure by EBR Both ethylene and Ga activation of AtrbohF-dependent H2O2 and Nia1-catalyzed NO production (Figures 7–10) implies that they act in the same pathway. To investigate


292 Chenyu Shi et al. the interrelationship between ethylene and Ga, we used pharmacological and genetic approaches. The Ga activator CTX largely restored the lesion of EBR-induced stomatal closure in the wild type (Col-0) treated with ethylene perception and synthesis inhibitors silver, 1-MCP, AOA and AVG, and in the mutant etr1-3 (Figure 11c), but ethylene precursor ACC had no rescue effect on the defect of EBRinduced stomatal closure in the wild type (Ws) treated with Ga inhibitor PTX and in the mutant gpa1 (Figure 11a). The results show that Ga mediates the action of ethylene in BR-induced stomatal closure. To confirm this verdict, the effect of EBR on ethylene production in the wild type (Ws) treated with PTX and in the mutant gpa1 was monitored. As in the wild type, EBR largely promoted ethylene production in the wild type treated with PTX and in the mutant gpa1 (Figure 11b), which supports the conclusion that Ga

acts downstream of ethylene in BR-induced stomatal closure. We noticed that CTX failed to reverse the defect of EBR-induced stomatal closure in mutant etr1-1 (Figure 11c), which is similar to the effect of H2O2 (Figure 7f). This coherence between the effects of CTX and H2O2 suggests that the role of Ga is associated with the induction of H2O2 production in EBR-led stomatal closure. Because etr1-1 has the defect in both ethylene-induced H2O2 synthesis and H2O2-induced stomatal closure (Desikan et al., 2005, 2006), the inability of CTX to rescue the fault of EBRinduced stomatal closure in etr1-1 (Figure 11c) is not incompatible with ethylene acting upstream of Ga in BRled stomatal closure. Afterwards, the effect of EBR on GPA1 expression was detected. gpa1-1 and gpa1-2 plants failed to express GPA1 transcripts (Figure 11d), as expected. Although EBR

(a)

(b)

(c)

(d)

Figure 11. GPA1 mediates the action of ethylene in 24-epibrassinolide (EBR)-induced stomatal closure.(a) Leaves of wild-type Ws and the gpa1 mutant with open stomata were kept in MES buffer without or with 400 ng ml1 PTX in the absence (Control) or presence of 5 lM EBR, 100 lM ACC or 5 lM EBR and 100 lM ACC for 3 h. Stomatal apertures were measured in epidermal strips.(b) Leaves of wild-type Ws and the gpa1 mutant with open stomata were incubated in MES buffer without or with 400 ng ml1 PTX in the absence (Control) or presence of 5 lM EBR for 3 h. Treated leaves were enclosed in a vial for another 3 h, the gas was gathered and the ethylene was measured.(c) Wild-type Col-0 seedlings were pre-treated with 500 pl L1 1-MCP, or not, in a closed chamber, and then the treated leaves with open stomata were floated in MES buffer without or with 50 lM AOA, 50 lM AVG or 10 lM Ag+ in the absence (Control) or presence of 5 lM EBR, 400 ng ml1 CTX or 5 lM EBR and 400 ng ml1 CTX for 3 h. Stomatal apertures were measured in epidermal strips.(d) Leaves of wild-type Ws and the gpa1 mutant with open stomata were kept in MES buffer without or with 5 lM EBR for 3 h, the abundance of transcripts for GPA1 and BAS1 was detected by RT-PCR of extracts of the treated leaves. The relative integrated density was shown below each band where the left first band in each row is set to 1. Each assay was repeated at least three times. Data of stomatal aperture are displayed as means SEs (n = 3); means with different letters are significantly different at P < 0.01. Data of ethylene concentration are displayed as means SEs (n = 3); means with different letters are significantly different at P < 0.05. The blot of transcripts for GPA1 and BAS1 shown is representative of three experiments.


Ethylene mediates BR-induced stomatal closure 293 significantly increased the expression of BAS1 in the wild type and in gpa1 mutants, it did not elevate the transcript levels of GPA1 in the wild type (Figure 11d). The data show that EBR has no inductive effect on GPA1 expression. Linked with the conclusion above that Ga acts downstream of ethylene to mediate stomatal closure by EBR, and the failure of ACC to rescue the defect of EBR-induced stomatal closure in the wild type treated with Ga inhibitor PTX, and the reverse effect of Ga activator CTX on the lesion of EBR-induced stomatal closure in the wild type treated with inhibitors of ethylene synthesis and perception, and in the etr1-3 mutant (Figure 11a–c), the inability of EBR to promote the expression of GPA1 (Figure 11d) indicates that EBR-induced ethylene stimulates the increase of Ga activity, and hence closes stomata. NO synthesis depends on H2O2 production in EBR-induced stomatal closure Having established that Ga acts downstream of ethylene to induce H2O2 and NO production and stomatal closure (Figures 7–11), and the data from several laboratories show that H2O2-induced NO generation has an important role in light/dark transition and ABA-led stomatal closure (She et al., 2004; Bright et al., 2006), we want to elucidate the relationship between H2O2 and NO in BR-induced stomatal closure. SNP significantly rescued the defects in mutants AtrbohF and AtrbohD AtrbohF, and in the wild type treated with ASA, CAT or DPI (Figure 12a), but H2O2 did not restore the deficiencies of Nia1-2 and Nia2-5 Nia1-2 mutants and the wild type treated with c-PTIO or tungstate in EBR-induced stomatal closure (Figure 12d). Analogously, SNP closed the stomata in mutants AtrbohF and AtrbohD AtrbohF, and in the wild type treated with ASA, CAT or DPI (Figure 12a), but H2O2 did not close or reduce the closure of stomata in mutants Nia1-2 and Nia2-5 Nia12, and in the wild type treated with c-PTIO or tungstate (Figure 12d). These data clearly show that H2O2 induces NO synthesis in BR-led stomatal closure. To support this conclusion, we measured H2O2 or NO synthesis in the guard cells of the above plants. EBR did not stimulate NO synthesis in mutants AtrbohF and AtrbohD AtrbohF, and in the wild type treated with ASA, CAT or DPI (Figure 12b,c), but it triggered H2O2 production in mutants Nia1-2 and Nia2-5 Nia1-2, and in the wild type treated with c-PTIO or tungstate (Figure 12e,f). The relevance between stomatal movement and the alteration in H2O2 or NO level also provides evidence that H2O2 production is required by NO synthesis during the BR-led stomatal closure. DISCUSSION BR can close stomata in Arabidopsis The stomata embedded in the epidermis of terrestrial plants consist of a pair of guard cells; they provide a route

for CO2 absorption, water transpiration and pathogen entry into the plant (Melotto et al., 2006). Thus, the regulation of stomatal movement is extremely important for controlling water loss, preventing microbial invasion and maintaining the ability to conduct gas exchange essential for photosynthesis. Stomatal aperture is regulated by numerous internal factors (e.g. plant hormone ABA), and abiotic (e.g. light) or biotic (e.g. bacterial pathogen) environmental conditions; however, the role and the underlying mechanism of BR in regulating stomatal behavior are incompletely clear. Previous studies indicated that BR has either a positive (Haubrick et al., 2006) or a negative (Ephritikhine et al., 1999) effect on stomatal closure. A recent investigation reported that the different doses of EBR have opposite effects on stomatal movement (Xia et al., 2014). Low doses of EBR open stomata, whereas high doses close them. Stomatal opening with low doses of EBR is strongly coupled with the transient production of H2O2 in guard cells and the cellular redox status of glutathione, whereas ABA synthesis and the prolonged increase of H2O2 level mediate the effect of high doses of EBR inducing stomatal closure. Interestingly, stomatal closure was shown to be part of the innate immune response of Arabidopsis to flg22, a 22-amino acid peptide derived from bacterial flagellin (Melotto et al., 2006). BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) functions as a co-receptor of BR-induced BRI1 signaling (Li et al., 2002; Nam and Li, 2002), and flg22-induced receptor kinases flagellin sensitive 2 (FLS2) signaling (Chinchilla et al., 2007). Our data here clearly indicate that BR does close stomata in Arabidopsis. The EBR induction of stomatal closure in the wild type (Figure 1) suggests that BR has a promoting effect on stomatal closure, which is coincident with the previous results in broad bean and tomato (Haubrick et al., 2006; Xia et al., 2014). Furthermore, the inability of EBR to close the stomata of BR receptor mutant bri1-301 (Figures 1 and 2) provides genetic evidence for the BR induction of stomatal closure. To date, many bri1 alleles have been isolated, and many of them have a defect in the BRI1 kinase domain (Friedrichsen et al., 2000; Vert et al., 2005; Xu et al., 2008). Previous work reported that the bri1-301 mutant has undetectable kinase activity, yet displays weak morphological abnormalities (Xu et al., 2008). Our data here indicate that EBR failed to close the stomata of the bri1-301 mutant (Figures 1 and 2). The results suggest that the BRI1 kinase activity has a more important role for the function of BRI1 in BR-induced stomatal closure than in the mediation of plant growth and development. This might reflect the difference between the regulatory mechanisms of plant growth and development and stomatal movement. In addition, the present study only addressed the stomatal response of the bri1-301 mutant to BR, and further investigation of more bri1 mutants that have mutations in the kinase domain will help us to understand whether the BRI1 kinase domain or the



(a)

(b)

(c)

(d)

(e)

(f)


Ethylene mediates BR-induced stomatal closure 295 specific amino-acid residue in the kinase domain is indispensable for the function of BRI1 in BR-mediated stomatal regulation. Both ethylene and Ga are essential for stomatal closure by BR The cooperative effect between BR and other plant hormones, including ethylene, are crucial to a broad spectrum of plant physiological and developmental processes. BR induces ethylene biosynthesis through stimulating ACS and ACC oxidase activities (Hansen et al., 2009), BR signaling components also mediate ethylene-induced growth and defense responses (Bar et al., 2010; Deslauriers and Larsen, 2010; Cheung and Wu, 2011). G proteins play a vital role in the transduction of extracellular signals into intracellular responses, they are also shown to be a important regulator of guard cell responses to ABA, extracellular calmodulin and extracellular ATP (Wang et al., 2001; Chen et al., 2004; Li et al., 2009; Chakravorty et al., 2011; Zhang et al., 2011; Hao et al., 2012); however, until now, whether or not ethylene and G proteins mediate the response of stomatal guard cells to BR remains unknown. Our results in the present study provide convincing evidence that ethylene and Ga participate in the induction of stomatal closure by BR in Arabidopsis. Ethylene synthesis and perception inhibitors and Ga inhibitor fully prevented EBRled stomatal closure (Figures 3 and 6), and ethylene precursor and Ga activator, like EBR, significantly closed stomata (Figures 2 and 6), revealing that ethylene synthesis and perception and the activation of Ga are essential for BR to close stomata. The inability of EBR to close the stomata of etr1 and gpa1 mutants (Figures 3 and 6), and the more significant effect of EBR on stomatal closure in the eto1-1 mutant and cGa line than in the wild type (Figures 7–10), further provide genetic evidence for the essential role of ethylene and Ga in BR guard cell signaling. Because ABA is a main regulator of stomatal movements, the relationship between BR and ABA in the regulation of stomatal movement is an interesting question. A recent study has indicated that ABA acts downstream of H2O2 in the induction of stomatal closure by BR in tomato (Xia et al., 2014); however, our results here, along with the data from related studies, provide evidence that ABA does not

mediate the induction of stomatal closure by BR in Arabidopsis. The fact that EBR-induced stomatal closure is completely abolished in etr1 mutants (Figure 3) excludes the possibility that ABA also mediates the BR induction of stomatal closure. The finding that ABA closes the stomata of etr1 and gpa1 mutants (Wang et al., 2001; Desikan et al., 2006), but that BR does not (Figures 3 and 6), also supports the conclusion above. The difference of EBR response between tomato and Arabidopsis guard cells may be relative to the concentration of EBR used, the plant species, or both. The evidence indicates that ethylene can close stomata (Desikan et al., 2006). Ga was also known to be involved in ABA inhibition of stomatal opening and extracellular calmodulin- and extracellular ATP-induced stomatal closure (Wang et al., 2001; Chen et al., 2004; Li et al., 2009; Chakravorty et al., 2011; Zhang et al., 2011; Hao et al., 2012). Our results here further show that both ethylene and Ga are required for the BR induction of stomatal closure in Arabidopsis; however, the underlying mechanism by which BR induces ethylene synthesis and the roles of the other subunits of G proteins in BR guard cell signalling remain unclear. Both H2O2 and NO are required for the induction of stomatal closure by BR H2O2 and NO are extensively studied signaling molecules in plants. Numerous studies have shown that H2O2 and NO are involved in stomatal closure by multiple external stimuli or plant hormones, including ABA (Pei et al., 2000; Desikan et al., 2002; Neill et al., 2002). The ethylene inhibition of ABA-induced stomatal closure was also shown to be relative to an as-yet-unidentified antioxidant mechanism- and flavonol-induced reduction in the level of ROS, including H2O2, in guard cells (Beguerisse-Diaz et al., 2012; Watkins et al., 2014). Although H2O2 and NO are known to mediate BR-induced stress tolerance (Xia et al., 2009; Cui et al., 2011), the study of the role of H2O2 in BR-mediated stomatal movement is just beginning, and whether or not NO mediates the action of BR in stomatal behavior remains unknown. Our results clearly indicate that EBR-induced H2O2 and NO production and stomatal closure in wild-type Arabidopsis were fully abolished in the bri1-301 mutant (Figures 2a,c–f), but that H2O2 and SNP significantly closed

Figure 12. H2O2 production is necessary for NO synthesis in 24-epibrassinolide (EBR)-induced stomatal closure.(a) Leaves of wild-type Col-0 and mutants AtrbohD, AtrbohF and AtrbohD AtrbohF with open stomata incubated in MES buffer without or with 100 lM ASA, 100 units ml1 CAT or 10 lM DPI in the absence (Control) or presence of 5 lM EBR, 100 lM SNP or 5 lM EBR and 100 lM SNP for 3 h, and then stomatal apertures were measured in epidermal strips.(b, c) Leaves of wild-type Col-0 and mutants AtrbohD, AtrbohF and AtrbohD AtrbohF with open stomata were incubated in MES buffer without or with 100 lM ASA, 100 units ml1 CAT or 10 lM DPI in the absence (Control) or presence of 5 lM EBR for 3 h. Fluorescence intensities (b) and images (c) of guard cells pre-loaded with 10 lM DAF-2DA were recorded.(d) Leaves of wild-type Col-0 and mutants Nia1-2, Nia2-1 and Nia2-5 Nia1-2 with open stomata were incubated in MES buffer without or with 200 lM c-PTIO or 100 lM tungstate in the absence (Control) or presence of 5 lM EBR, 100 lM H2O2 or 5 lM EBR and 100 lM H2O2 for 3 h, then stomatal apertures were measured in epidermal strips.(e, f) Leaves of wild-type Col-0 and mutants Nia1-2, Nia2-1 and Nia2-5 Nia1-2 with open stomata were incubated in MES buffer without or with 200 lM c-PTIO or 100 lM tungstate in the absence (Control) or presence of 5 lM EBR for 3 h. Fluorescence intensities (e) and images (f) of guard cells pre-loaded with 50 lM H2DCFDA were recorded. Each assay was repeated at least three times. The data of stomatal aperture are displayed as means SEs (n = 3), and means with different letters are significantly different at P < 0.01. Data of fluorescence intensities are displayed as means SEs (n = 3), and means with different letters are significantly different at P < 0.05. Scale bars: 10 lm, for all images.


296 Chenyu Shi et al. the stomata in the bri1-301 mutant (Figure 2a). The data show that a functional BRI1 protein is vital for BR-induced H2O2 and NO productions and subsequent stomatal closure. Additionally, H2O2 or NO scavenger completely inhibited EBR-induced stomatal closure and H2O2 or NO accumulation in guard cells of the wild type (Figures 4a–c and 5a–c), which provides evidence that both H2O2 and NO, as intermediate signal molecules, act in stomatal closure by BR. The enzymatic sources of H2O2 in guard cells are known to be diverse, including NADPH oxidases (Kwak et al., 2003), cell wall peroxidases (Khokon et al., 2010) and amine oxidase (An et al., 2008). Nitric oxide synthase (NOS) and NR were also shown to be potential sources of NO in guard cells (Neill et al., 2002), although no genes or proteins with sequence homology to known mammaliantype NOS have been found in plants. So far, however, the origins of H2O2 and NO in BR-induced stomatal closure are incompletely clear. Our data here show that EBR-led stomatal closure in the wild type was largely abolished in the NADPH oxidase single mutant AtrbohF and double mutant AtrbohD AtrbohF, or in NR mutants Nia1-2 and Nia2-5 Nia1-2, in accordance with the effect of EBR on H2O2 or NO production in the guard cells of these plants (Figure 4a–c and 5a–c). Clearly the data provide convincing evidence for the important roles of AtrbohF-mediated H2O2 production and Nia1-dependent NO synthesis in BR-led stomatal closure in Arabidopsis. As aforementioned, H2O2 and NO are required for stomatal closure by multiple internal factors and abiotic or biotic environmental conditions (Pei et al., 2000; Desikan et al., 2002; Neill et al., 2002; She et al., 2004; Suhita et al., 2004; Liu et al., 2005; Melotto et al., 2006; Kolla and Raghavendra, 2007; Kolla et al., 2007). Our data here further show that both H2O2 sourced from AtrbohF and NO generated by Nia1 are crucial for stomatal closure by BR. Kwak et al. (2003) reported that ABA-induced stomatal closure is partially impaired in AtrbohF and more strongly impaired in AtrbohD AtrbohF, but AtrbohD, like the wild type, responds normally to ABA, which suggests that there is some overlap in the functions of AtrbohD and AtrbohF. Another study showed that only AtrbohF plays an essential role for ethylene-induced H2O2 production and stomatal closure (Desikan et al., 2006). Our results here indicate that the responses of both AtrbohD and AtrbohF to BR were similar to the responses to ethylene, but differed from the responses to ABA (Figure 4). The results suggest that AtrbohD and AtrbohF may have different roles in ABA- and ethylene- or BR-induced responses, and support that ethylene mediates the effect of BR on H2O2 production and the resulting stomatal closure (Figures 2 and 3). In addition, in this report our genetic evidence clearly shows that BR-induced NO production and subsequent stomatal closure were completely impaired in mutants Nia1-2 and Nia1-2 Nia2-5, but not in mutant Nia2-1, as in the wild type (Figure 5). Similar to our data, Bright et al.

(2006) proposed that Nia1 is mainly responsible for the ABA-induced NO generation in guard cells. The similar responses of Nia1-2 and Nia2-1 to BR and ABA might be related to the fact that both BR and ABA induction of NO production depend on H2O2 derived from NADPH oxidases (Figure 12; Bright et al., 2006). Both ethylene and Ga activate H2O2 and NO production in stomatal closure by BR Previous work indicated that BR can stimulate ethylene synthesis (Hansen et al., 2009), and that ethylene closes stomata by inducing H2O2 and NO production (Desikan et al., 2006; Liu et al., 2010). In addition, Ga-triggered H2O2 production has been known to be involved in ABA inhibition of stomatal opening and extracellular calmodulin- and extracellular ATP-led stomatal closure (Chen et al., 2004; Zhang et al., 2011; Hao et al., 2012), and the role of Ga in extracellular calmodulin-led stomatal closure is closely associated with its inductive effect on NO synthesis (Li et al., 2009). These studies suggest that ethylene and Ga may play an important mediating role in guard cell signaling; however, as yet it is unknown whether ethylene and Ga also mediate BR-induced stomatal closure via the activation of H2O2 and NO production. Our data here strongly support that ethylene- and Ga-activated H2O2 and NO production are involved in BR-led stomatal closure. The eto1-1 mutant and cGa line guard cells generated higher levels of H2O2 and NO than their respective wild type, and EBR failed to increase H2O2 and NO levels in the guard cells of etr1 and gpa1 mutants. Meanwhile, EBR-induced increases of H2O2 and NO in the eto1-1 mutant and cGa line were abolished in eto1-1 AtrbohF and cGa AtrbohF constructs, and in eto1-1 Nia1-2 and cGa Nia1-2 constructs, respectively. H2O2 rescued the defect of EBR-induced stomatal closure in etr1-3 and gpa1 mutants, whereas SNP recovered the lesions of etr1 and gpa1 mutants in EBR-induced stomatal closure (Figures 7–10). These data convincingly affirm that ethylene and Ga promote H2O2 and NO generation in BR-initiated guard cell signaling; however, how ethylene and Ga mediate the promoting effect of BR on H2O2 and NO production remains unclear. The G protein a-subunit acts downstream of ethylene in the BR induction of stomatal closure In plants, heterotrimeric G proteins function as important signaling molecules mediating responses to a range of abiotic and biotic signals, incuding plant hormones such as gibberellic acid (GA; Ullah et al., 2002), ABA (Pandey et al., 2006), BR (Ullah et al., 2001) and MJ (Trusov et al., 2006). As yet, whether G proteins are involved in the responses of plant guard cells to ethylene remains unclear. Both ethylene and Ga activate the AtrbohF-dependent H2O2 production and Nia1-catalyzed NO synthesis necessary for the stomatal closure induced by BR (Figures 4, 5, 7–10), which


Ethylene mediates BR-induced stomatal closure 297 implies that they may play a role in the same signaling pathway. Ga mediates the ethylene-induced triple response (Ullah et al., 2002) and inhibition of root elongation (Wang et al., 2006), although the detailed mechanisms have not been studied. This inspires us to assess its potential role in the response of the guard cell to ethylene. Our results in this report provide evidence that Ga plays an important role in the responses of Arabidopsis guard cells to ethylene. The Ga activator largely restored the lesion of EBR-induced stomatal closure, and also closed the stomata in the wild type treated with ethylene perception or synthesis inhibitor, and in the mutant etr1-3 (Figure 11c). On the contrary, the ethylene precursor failed to rescue the defect of EBR-induced stomatal closure, and also did not close the stomata in both the wild type treated with Ga inhibitor and the mutants gpa1 (Figure 11a). The data clearly show that Ga acts downstream of ethylene in BR guard cell signaling. The data indicating that EBR normally promoted ethylene production in the wild type treated with Ga inhibitor and in the mutant gpa1 (Figure 11b) also support this conclusion. In this context, the data that EBR has no promoting effect on the expression of GPA1 (Figure 11d) suggest that ethylene acts via increasing Ga activity in BR-induced stomatal closure. Although our results here show an important role of Ga in the responses of guard cells to ethylene, whether the other subunits of G proteins also acts to mediate the ethylene responses of guard cells and other plant cells is still an interesting question to be addressed in the future. In addition, the relationship between H2O2 and NO in BR-induced stomatal closure was also studied. EBR failed to induce NO synthesis in the guard cells of mutants AtrbohF and AtrbohD AtrbohF, but it induced H2O2 production in the guard cells of Nia1-2 and Nia2-5 Nia1-2 (Figure 12). The results indicate that BR-induced H2O2 is requisite for NO synthesis in Arabidopsis. This relationship between H2O2 and NO in stomatal movement is in agreement with the results of Bright et al. (2006), but differs somewhat from our previous study that suggested an interrelationship between H2O2 and NO in light/dark transition-induced stomatal closure in broad bean (She et al., 2004). A discrepancy that may reflect the difference between BR and the light/dark signal transduction mechanism in guard cells, or may result from the different plant species used. In summary, the data presented herein suggest a working model for BR action in the guard cell response of Arabidopsis (Figure 13). Binding of BR to its receptor BRI1 induces ACS expression and ethylene synthesis. Increased ethylene activates Ga. Active Ga stimulates AtrbohF-dependent H2O2 generation and subsequent Nia1-catalysed NO production, and finally closes the stomata. The findings confirm the effect of BR on stomatal movement, and elucidate the crucial role of ethylene and G proteins in the

guard cell BR signalling. The Arabidopsis guard cell offers an attractive tool with which to dissect novel aspects of BR signalling; however, several relevant questions need to be further studied in the future. First, although our study clearly shows that BRI1 protein mediates BR-induced ethylene production (Figure 2), how BRI1 mediates ethylene production remains unknown. Studies show that the MAPK cascade induces the expression and phosphorylation of ACS (Liu and Zhang, 2004; Li et al., 2012), hence resulting in higher cellular ACS activity and elevated ethylene. Recently, Meng et al. (2012) reported that ERECTA (ER), a receptor-like protein kinase (RLK), determines Arabidopsis inflorescence architecture by activiting an MAPK cascade. Furthermore, a more recent study provided evidence that MAPK cascade-activated ethylene and subsequent ROS production are essential for the salt sensitivity mediated by Salt Intolerance 1 (SIT1) in rice (Oryza sativa), a lectin receptor-like protein kinase (Li et al., 2014). The results, along with our data here (Figure 2), imply that BRI1, one of the best-studied RLKs, might directly or indirectly induce the expression and phosphorylation of ACS in the BR induction of stomatal closure; however, whether or not BRI1 acts in the manner discribed above remains to be studied. Second, how Ga activates NADPH oxidase remains to be elucidated. Previous studies indicate that one possible target of the heterotrimeric G proteins is an increase in cytosolic Ca2+ in the plant cell (Aharon et al., 1998), whereas NADPH oxidase is directly activated by Ca2+ and Ca2+dependent protein kinase (Kobayashi et al., 2007; Ogasawara et al., 2008). Two different GTPases, the small GTPase Rac, which is a homolog of the cytosolic regulator small GTPase Rac of mammalian NADPH oxidase in plants, and

Figure 13. Model showing the possible signaling pathway for 24-epibrassinolide (EBR)-induced stomatal closure. EBR induces the expression of ACS5 and ACS9 and ethylene synthesis via a BRI1-dependent mechanism, thereby activating G protein to induce H2O2 generation by AtrbohF, which increases Nia1-catalyzed NO production. NO subsequently closes the stomata.


298 Chenyu Shi et al. the heterotrimeric G proteins are also shown to induce the activation of NADPH oxidase (Kawasaki et al., 1999; Joo et al., 2005; Moeder et al., 2005). Furthermore, Suharsono et al. (2002) reported that the small GTPase Rac acts downstream of Ga to activate ROS production in disease resistance of rice. Whether or not the GTPases and other upstream modulators of NADPH oxidase activity have a similar role in the BR induction of H2O2 production and subsequent stomatal closure remains unclear, however, and how they operate mechanistically should be studied further. Finally, the linkage between ethylene and G proteins is also an important problem. In mammals, different ligands activate G proteins via stimulating the G protein-coupled receptor (GPCR); however, the activation of plant G proteins arises from the inhibition of the regulator of G protein signalling (RGS) protein, which is a G protein inhibitor, whereas the inhibition of RGS depends on its phosphorylation by signal-triggered kinases activity and resulting endocytosis (Urano et al., 2013). Because Arabidopsis has more than 600 RLKs, and some of them have genetic links with G proteins (Lease et al., 2001; Llorente et al., 2005), it is possible that RLKs transmit signals to G proteins through the phosphorylation and endocytosis of RGS. Whether or not different RLKs transmit different signals, including the plant hormone ethylene, to G proteins, and thereby result in a specific response, remains unknown. The findings that ethylene induces the expression of bacterial flagellin flg22 receptor FLS2, one of RLKs in Arabidopsis (Mersmann et al., 2010), and that the inhibitory effect of flg22 on stomatal opening is blocked in mutant gpa1 (Zhang et al., 2008) seem to support the speculation above. Our data here show that Ga is involved in the action of ethylene in the BR induction of stomatal closure (Figure 11). Whether or not RLKs mediate the activating effect of ethylene on Ga in BR-induced stomatal closure awaits exploration.

Plant material. Seeds of the wild type and various mutants of Arabidopsis (A. thaliana) were sown in potting mix and grown in plant growth chambers under a 16-h light/8-h dark cycle, with a photon flux density of 0.1 mmol m2 sec1 (provided by QMX YZ28RR16/G fluorescent tubes with no emission below 330 nm; Philips, http://www.philips.com) and measured with a Skye RS232 meter equipped with a Quantum sensor; Skye Instruments, http:// www.skyeinstruments.com), and a day/night temperature cycle of 22°C/18°C. Seeds of the bri1-301 mutant were obtained from Dr G Wu (Shaanxi Normal University). Seeds of cGa lines (background Ws) were obtained from Dr LG Ma (Hebei Normal University). cGa plants were grown in the presence of 70 nM dexamethasone according to the method described by Okamoto et al. (2001). Seeds of etr1-1 and eto1-1 mutants were obtained from the Arabidopsis Biological Resource Center. Seeds of the wild type and etr1-3, 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 Centre (uNASC, http://arabidopsis.info). Genotypes of all mutants were confirmed by PCR analysis. Fully expanded rosette leaves were harvested for immediate use at 4–6 weeks.

Stomatal bioassay. The stomatal assay was performed as described by Desikan et al. (2006). In brief, rosette leaves from the wild type and various mutant seedlings were incubated in MESKCl buffer (50 mM KCl, 10 mM MES-KOH, pH 6.15) for 3 h in the same light conditions as that described above, at a temperature of 22°C. Once the stomata were fully open, leaves were incubated in MES-KCl buffer alone or containing various compounds or inhibitors, with or without EBR, which is a bioactive BR, for another 3 h under light conditions. Control treatments involved the addition of buffer or appropriate solvents used with various compounds or inhibitors. After treatments, the epidermal strips were immediately peeled carefully from the abaxial surface of leaves, and stomatal apertures were recorded with a light microscope and an eyepiece graticule previously calibrated with a stage micrometer. To avoid any potential rhythmic effects on stomatal aperture, experiments were always started at the same time each day. The data are represented as means SEs of three replicates, each with 50 stomata.

Chemicals. Molecular probes 20 ,70 -dichlorofluorescein diacetate

Treatment with 1-methylcyclopropene (1-MCP). For treatment with 1-MCP, 4.8 mg of 1-MCP was dissolved with 73 ll of distilled water in a tube, and plants that were 4–6 weeks of age were laid with the tube in a closed chamber for 12 h. The final concentration of 1-MCP in the gas phase was expected to be 500 pl L1. After treatment, the rosette leaves were incubated in MES-KCl buffer under light, to open the stomata, before being treated as described above and used for subsequent experiments.

(H2DCF-DA; Biotium, http://biotium.com) or 4,5-diaminofluorescein diacetate (DAF-2 DA; Sigma-Aldrich, http://www.sigmaaldrich.com) were dissolved in dimethyl sulphoxide (DMSO; Amresco, http:// www.amresco-inc.com) to produce a stock solution, which was then aliquoted. 2-(N-morpholino) ethanesulphonic acid (MES), 24-epibrassinolide (EBR), abscissic acid (ABA), 1-aminocyclopropane-1-carboxylic acid (ACC), aminoethoxyvinyl glycine (AVG), aminoxyacetic acid (AOA), 1-methylcyclopropene (1-MCP), pertussis toxin (PTX), cholera toxin (CTX), ascorbic acid (ASA), catalase (CAT, from bovine liver), diphenylene iodonium (DPI), sodium nitroprusside (SNP) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) were obtained from Sigma-Aldrich. Unless stated otherwise, the remaining chemicals were of the highest analytical grade available from various Chinese suppliers.

RNA isolation and transcript analysis by RT-PCR. Total RNA was isolated from the treated leaves using Trizol (Life Technologies, http://www.lifetechnologies.com) according to the manufacturer’s instructions. Each RNA sample was quantified with a spectrophotometeric method. The A260/A280 values of the RNA samples were all greater than 1.7. To ensure the quality of RNA for RT-PCR analysis, the RNA samples were also visualized on agarose gels following ethidium bromide staining (data not shown). First-strand cDNA was synthesized from 1 lg of total RNA with QuantiTectâ Reverse Transcription kit (Qiagen #205311, http://www.qiagen.com), following the manufacturer’s instructions, in a final volume of 20 ll of reaction mixture containing

EXPERIMENTAL PROCEDURES Materials and methods


Ethylene mediates BR-induced stomatal closure 299 gDNA Wipeout Buffer for the effective elimination of genomic DNA contamination from starting RNA samples. The cDNA was then amplified by PCR with gene-specific primers for ACS5, ACS9, GPA1, BAS1 and ACTIN2 at an annealing temperature of 56°C. Primers used for RT-PCR with cycles are as follows: 50 GCTATGGCTGAGTTTATGG-30 and 50 -TCATTTCGTCGTTGGAGT-30 for ACS5 with 39 cycles, 50 -GGGATGGGAAGAATACGA-30 and 50 ACATTGTGCCAAGAGGGT-30 for ACS9 with 39 cycles, 50 -CACAGGCTGCTGAAATCG-30 and 50 -CTCCCACAGGGCTGAACT-30 for GPA1 with 32 cycles, 50 -GCTCTCCTTTTTGTGTTTTCTCTCT-30 and 50 -AGTCCGGAACAAATTTTTGACCGTT-30 for BAS1 with 32 cycles, 50 -TTCCGCTCTTTCTTTCCAAGCTCA-30 and 50 -AAGAGGCATCAATT CGATCACTCA-30 for ACTIN2 with 32 cycles. Bands in gels from RT-PCR experiments were measured on images with the BIODOC-IT imaging system (UVP, http://uvp.com), followed by validation with IMAGEJ. The integrated density was calculated for each band. The relative integrated density was calculated as a ratio of each measurement to a common data point of each gene, and was shown below each band in the gel.

Ethylene measurement. After the aforementioned treatments, the rosette leaves were enclosed in a vial containing filter paper moistened by MES-KCl buffer with the corresponding compounds or inhibitors, under light conditions. After 3 h the gas in the vial was gathered with a syringe, and then the concentration of ethylene was measured with an Agilent 6890 N gas chromatogram system (Agilent Technologies, Hhttp://www.agilent.com), equipped with a flame ionization detector on an HP-5 capillary column (Agilent Technologies). In each treatment, six leaves from different plants were measured, and the treatment was repeated at least nine times. Data are presented as means SEs. Measurement of endogenous H2O2 and NO. The H2O2 and NO levels in guard cells were monitored by using the fluorescent indicator dyes H2DCF-DA and DAF-2 DA, respectively, as previously described (Allan and Fluhr, 1997; Kojima et al., 1998), with minor changes. After the aforementioned treatments, the epidermal strips were carefully peeled from the abaxial surface of the treated leaves and immediately loaded with 50 lM H2DCF-DA for 10 min or 10 lM DAF-2 DA for 30 min in Tris-KCl loading buffer (Tris 10 mM and KCl 50 mM, pH 7.2) in darkness at 25°C. Excess dye was removed with fresh Tris-KCl buffer in the darkness, and an examination of the peels was immediately performed with a TCS-SP5 confocal laser scanning microscope (Leica, http:// www.leica-microsystems.com), with the following settings: excitation at 488 nm and emission at 530 nm. Images acquired from the confocal microscope were processed with PHOTOSHOP and analyzed with Leica IMAGE. In each treatment, three epidermal strips originated from different plants were measured, and the treatment 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 lowed by the least significant difference test.

ANOVA

fol-

ACKNOWLEDGEMENTS We thank Dr G Wu (Shaanxi Normal University) for helpful comments on the article. This work was supported by the Fundamental Research Funds for the Central Universities (grant no. GK201401005) and the National Spark Program Project of China (grant no. 2012GA850002).

REFERENCES Acharya, B.R. and Assmann, S.M. (2009) Hormone interactions in stomatal function. Plant Mol. Biol. 69, 451–462. Aharon, G.S., Gelli, A., Snedden, W.A. and Blumwald, E. (1998) Activation of a plant plasma membrane Ca2+ channel by TGa1, a heterotrimeric G proteina-subunit homologue. FEBS Lett. 424, 17–21. Allan, A.C. and Fluhr, R. (1997) Two distinct sources of elicited reactive oxygen species in tobacco epidermal cells. Plant Cell, 9, 1559–1572. An, Z.F., Jing, W., Liu, Y.L. and Zhang, W.H. (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. Assmann, S.M. (2005) G proteins go green: a plant G protein signaling FAQ sheet. Science, 310, 71–73. Bar, M., Sharfman, M., Ron, M. and Avni, A. (2010) BAK1 is required for the attenuation of ethylene-inducing xylanase (Eix)-induced defense responses by the decoy receptor LeEix1. Plant J. 63, 791–800. Beguerisse-Diaz, M., Hern0 andez-G0 omez, M.C., Lizzul, A.M., Barahona, M. and Desikan, R. (2012) Compound stress response in stomatal closure: a mathematical model of ABA and ethylene interaction in guard cells. BMC Syst. Biol. 6, 146. Benlloch-Gonzalez, M., Romera, J., Cristescu, S., Harren, F., Fournier, J.M. and Benlloch, M. (2010) K+ starvation inhibits water-stress induced stomatal closure via ethylene synthesis in sunflower plants. J. Exp. Bot. 61, 1139–1145. Bright, J., Desikan, R., Hancock, J.T., Weir, I.S. and Neill, S.J. (2006) ABAinduced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis. Plant J. 45, 113–122. Chakravorty, D., Trusov, Y., Zhang, W., Acharya, B.R., Sheahan, M.B., McCurdy, D.W., Assmann, S.M. and Botella, J.R. (2011) An atypical heterotrimeric G-protein c-subunit is involved in guard cell K+-channel regulation and morphological development in Arabidopsis thaliana. Plant J. 67, 840–851. Chang, C., Kwok, S.F., Bleecker, A.B. and Meyerowitz, E.M. (1993) Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators. Science, 262, 539–544. Chen, Y.L., Huang, R., Xiao, Y.M., L€u, P., Chen, J. and Wang, X.C. (2004) Extracellular calmodulin-induced stomatal closure is mediated by heterotrimeric G protein and H2O2. Plant Physiol. 136, 4096–4103. Chen, L., Dodd, I.C., Davies, W.J. and Wilkinson, S. (2013) Ethylene limits abscisic acid- or soil drying-induced stomatal closure in aged wheat leaves. Plant, Cell Environ. 36, 1850–1859. Cheung, A.Y. and Wu, H.M. (2011) THESEUS 1, FERONIA and relatives: a family of cell wall-sensing receptor kinases? Curr. Opin. Plant Biol. 14, 632–641. Chinchilla, D., Zipfel, C., Robatzek, S., Kemmerling, B., Nurnberger, T., Jones, J.D., Felix, G. and Boller, T. (2007) A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature, 448, 497– 500. Clouse, S.D. (2011) Brassinosteroid signal transduction: from receptor kinase activation to transcriptional networks regulating plant development. Plant Cell, 23, 1219–1230. Cui, J.X., Zhou, Y.H., Ding, J.G., Xia, X.J., Shi, K., Chen, S.C., Asami, T., Chen, Z.X. and Yu, J.Q. (2011) Role of nitric oxide in hydrogen peroxidedependent induction of abiotic stress tolerance by brassinosteroids in cucumber. Plant, Cell Environ. 34, 347–358. Daszkowska-Golec, A. and Szarejko, I. (2013) Open or close the gate – stomata action under the control of phytohormones in drought stress conditions. Front. Plant Sci. 4, 138. Denance, N., Sanchez-Vallet, A., Goffner, D. and Antonio, M. (2013) Disease resistance or growth: the role of plant hormones in balancing immune responses and fitness costs. Front. Plant Sci. 4, 155. Desikan, R., Griffiths, R., Hancock, J.T. and Neill, S.J. (2002) A new role for an old enzyme: nitrate reductase-mediated nitric oxide generation is required for abscisic acid-induced stomatal closure in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA, 99, 16314–16318. Desikan, R., Hancock, J.T., Bright, J., Harrison, J., Weir, I., Hooley, R. and Neill, S.J. (2005) A role for ETR1 in hydrogen peroxide signaling in stomatal guard cells. Plant Physiol. 137, 831–834. Desikan, R., Last, K., Harrett-Williams, R., Tagliavia, C., Harter, K., Hooley, R., Hancock, J.T. and Neill, S.J. (2006) Ethylene-induced stomatal closure


300 Chenyu Shi et al. in Arabidopsis occurs via AtrbohF-mediated hydrogen peroxide synthesis. Plant J. 47, 907–916. Deslauriers, S.D. and Larsen, P.B. (2010) FERONIA is a key modulator of brassinosteroid and ethylene responsiveness in Arabidopsis hypocotyls. Mol. Plant 3, 626–640. Dodd, I.C. (2003) Hormonal interactions and stomatal responses. J. Plant Growth Regul. 22, 32–46. Ephritikhine, G., Fellner, M., Vannini, C., Lapous, D. and Barbier-Brygoo, H. (1999) The sax1 dwarf mutant of Arabidopsis thaliana shows altered sensitivity of growth responses to abscisic acid, auxin, gibberellins and ethylene and is partially rescued by exogenous brassinosteroid. Plant J. 18, 303–314. Fridman, Y., Elkouby, L., Holland, N., Vragovic, K., Elbaum, R. and SavaldiGoldstein, S. (2014) Root growth is modulated by differential hormonal sensitivity in neighboring cells. Genes Dev. 28, 912–920. Friedrichsen, D.M., Joazeiro, C.A., Li, J., Hunter, T. and Chory, J. (2000) Brassinosteroid-insensitive-1 is a ubiquitously expressed leucine-rich repeat receptor Serine/Threonine kinase. Plant Physiol. 123, 1247–1256. Gendron, J., Haque, A., Gendron, N., Chang, T., Asami, T. and Wang, Z. (2008) Chemical genetic dissection of brassinosteroid-ethylene interaction. Mol. Plant, 1, 368–379. Gudesblat, G.E., Schneider-Pizon0 , J., Betti, C. et al. (2012) SPEECHLESS integrates brassinosteroid and stomata signalling pathways. Nat. Cell Biol. 14, 548–554. Gunderson, C.A. and Taylor, G.E. (1991) Ethylene directly inhibits foliar gas exchange in Glycine max. Plant Physiol. 95, 337–339. Hansen, M., Chae, H.S. and Kieber, J.J. (2009) Regulation of ACS protein stability by cytokinin and brassinosteroid. Plant J. 57, 606–614. Hao, L.H., Wang, W.X., Chen, C., Wang, Y.F., Liu, T., Li, X. and Shang, Z.L. (2012) Extracellular ATP promotes stomatal opening of Arabidopsis thaliana through heterotrimeric G protein a subunit and reactive oxygen species. Mol. Plant, 5, 852–864. Haubrick, L.L., Torsethaugen, G. and Assmann, S.M. (2006) Effect of brassinolide, alone and in concert with abscisic acid, on control of stomatal aperture and potassium currents of Vicia faba guard cell protoplasts. Physiol. Plant. 128, 134–143. Iqbal, N., Nazar, R., Syeed, S., Masood, A. and Khan, N.A. (2011) Exogenously sourced ethylene increases stomatal conductance, photosynthesis, and growth under optimal and deficient nitrogen fertilization in mustard. J. Exp. Bot. 62, 4955–4963. Jones, A.M. and Assmann, S.M. (2004) Plants: the latest model system for G-protein research. EMBO Rep. 5, 572–578. Joo, J.H., Wang, S.Y., Chen, J.G., Jones, A.M. and Fedoroff, N.V. (2005) Different signaling and cell death roles of heterotrimeric G protein a- and bsubunits in the Arabidopsis oxidative stress response to ozone. Plant Cell, 17, 957–970. Kawasaki, T., Henmi, K., Ono, E., Hatakeyama, S., Iwano, M., Satoh, H. and Shimamoto, K. (1999) The small GTP-binding protein Rac is a regulator of cell death in plants. Proc. Natl Acad. Sci. USA, 96, 10922–10926. Khokon, M.A., Hossain, M.A., Munemasa, S., Uraji, M., Nakamura, Y., Mori, I.C. and Murata, Y. (2010) Yeast elicitor-induced stomatal closure and peroxidase-mediated ROS production in Arabidopsis. Plant Cell Physiol. 51, 1915–1921. Khripach, V.A., Zhabinskii, V.N. and Groot, A.D. (2000) Twenty years of brassinosteroids: steroidal plant hormones warrant better crops for the XXI century. Ann. Bot. 86, 441–447. Kim, T.W., Michniewicz, M., Bergmann, D.C. and Wang, Z.Y. (2012) Brassinosteroid regulates stomatal development by GSK3-mediated inhibition of a MAPK pathway. Nature, 482, 419–422. Kinoshita, T., Cano-Delgado, A., Seto, H., Hiranuma, S., Fujioka, S., Yoshida, S. and Chory, J. (2005) Binding of brassinosteroids to the extracellular domain of plant receptor kinase BRI1. Nature, 433, 167–171. Kobayashi, M., Ohura, I., Kawakita, K., Yokota, N., Fujiwara, M., Shimamoto, K., Doke, N. and Yoshioka, H. (2007) Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase. Plant Cell, 19, 1065–1080. Kojima, H., Nakatsubo, N., Kikuchi, K., Kawahara, S., Kirino, Y., Nagoshi, H., Hirata, Y. and Nagano, T. (1998) Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal. Chem. 70, 2446–2453.

Kolla, V.A. and Raghavendra, A.S. (2007) Nitric oxide is a signaling intermediate during bicarbonate-induced stomatal closure in Pisum sativum. Physiol. Plant, 130, 91–98. Kolla, V.A., Vavasseur, A. and Raghavendra, A.S. (2007) Hydrogen peroxide production is an early event during bicarbonate induced stomatal closure in abaxial epidermis of Arabidopsis. Planta, 225, 1421–1429. Kwak, J.M., Mori, I.C., Pei, Z.-M., Leonhardt, N., Torres, M.A., Dangl, J.L., Bloom, R.E., Bodde, S., Jones, J.D.G. and Schroeder, J.I. (2003) NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis. EMBO J. 22, 2623–2633. Lease, K.A., Wen, J., Li, J., Doke, J.T., Liscum, E. and Walker, J.C. (2001) A mutant Arabidopsis heterotrimeric G-protein b-subunit affects leaf, flower, and fruit development. Plant Cell, 13, 2631–2641. Levitt, L.K., Stein, D.B. and Rubinstein, B. (1987) Promotion of stomatal opening by indoleacetic acid and ethrel in epidermal strips of Vicia faba L. Plant Physiol. 85, 318–322. Li, J.M. and Chory, J. (1997) A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell, 90, 929–938. Li, J., Wen, J.Q., Lease, K.A., Doke, J.T., Tax, F.E. and Walker, J.C. (2002) BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signalling. Cell, 110, 213–222. Li, J.H., Liu, Y.Q., L€ u, P., Lin, H.F., Bai, Y., Wang, X.C. and Chen, Y.L. (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. Li, G., Meng, X., Wang, R., Mao, G., Han, L., Liu, Y. and Zhang, S. (2012) Dual-level regulation of ACC synthase activity by MPK3/MPK6 cascade and its downstream WRKY transcription factor during ethylene induction in Arabidopsis. PLoS Genet. 8, e1002767. Li, C.H., Wang, G., Zhao, J.L., Zhang, L.Q., Ai, L.F., Han, Y.F., Sun, D.Y., Zhang, S.W. and Sun, Y. (2014) The receptor-like kinase SIT1 mediates salt sensitivity by activating MAPK3/6 and regulating ethylene Homeostasis in rice. Plant Cell, 26, 2538–2553. Liu, Y. and Zhang, S. (2004) Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces ethylene biosynthesis in Arabidopsis. Plant Cell, 16, 3386–3399. Liu, X., Shi, W.L., Zhang, S.Q. and Lou, C.H. (2005) Nitric oxide involved in signal transduction of jasmonic acid-induced stomatal closure of Vicia faba L. Chin. Sci. Bull. 50, 520–525. Liu, J., Liu, G.H., Hou, L.X. and Liu, X. (2010) Ethylene-induced nitric oxide production and stomatal closure in Arabidopsis thaliana depend on changes in cytosolic pH. Chin. Sci. Bull. 55, 2403–2409. Llorente, F., Alonso-Blanco, C., Sanchez-Rodriguez, C., Jorda, L. and Molina, A. (2005) ERECTA receptor-like kinase and heterotrimeric G protein from Arabidopsis are required for resistance to the necrotrophic fungus Plectosphaerella cucumerina. Plant J. 43, 165–180. Melotto, M., Underwood, W., Koczan, J., Nomura, K. and He, S.Y. (2006) Plant stomata function in innate immunity against bacterial invasion. Cell, 126, 969–980. Meng, X.Z., Wang, H.C., He, Y.X., Liu, Y.D., John, C.W., Keiko, U.T. and Zhang, S.Q. (2012) A MAPK cascade downstream of ERECTA receptorlike protein kinase regulates Arabidopsis nflorescence architecture by promoting localized cell proliferation. Plant Cell, 24, 4948–4960. Mersmann, S., Bourdais, G., Rietz, S. and Robatzek, S. (2010) Ethylene signaling regulates accumulation of the FLS2 receptor and is required for the oxidative burst contributing to plant immunity. Plant Physiol. 154, 391–400. Moeder, W., Yoshioka, K. and Klessig, D.F. (2005) Involvement of the small GTPase Rac in the defense responses of tobacco to pathogens. Mol. Plant Microbe Interact. 18, 116–124. Nam, K.H. and Li, J.M. (2002) BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling. Cell, 110, 203–212. Neill, S.J., Desikan, R., Clarke, A., Hurst, R.D. and Hancock, J.T. (2002) Hydrogen peroxide and nitric oxide as signalling molecules in plants. J. Exp. Bot. 53, 1237–1247. Nilson, S.E. and Assmann, S.M. (2010) The a-subunit of the Arabidopsis heterotrimeric G protein, GPA1, is a regulator of transpiration efficiency. Plant Physiol. 152, 2067–2077.


Ethylene mediates BR-induced stomatal closure 301 Ogasawara, Y., Kaya, H., Hiraoka, G. et al. (2008) Synergistic activation of the Arabidopsis NADPH oxidase AtrbohD by Ca2+ and phosphorylation. J. Biol. Chem. 283, 8885–8892. Okamoto, H., Matsui, M. and Deng, X.W. (2001) Overexpression of the heterotrimeric G-protein a-subunit enhances phytochrome-mediated inhibition of hypocotyl elongation in Arabidopsis. Plant Cell, 13, 1639–1652. € bel, C., Capper, R.G., Saunders, N., Feussner, I. and Okamoto, H., Go Knight, M.R. (2009) The a-subunit of the heterotrimeric G-protein affects jasmonate responses in Arabidopsis thaliana. J. Exp. Bot. 60, 1991–2003. Oldham, W.M. and Hamm, H.E. (2008) Heterotrimeric G protein activation by G-protein-coupled receptors. Nat. Rev. Mol. Cell Biol. 9, 60–71. Pallas, J.E. and Kays, S.J. (1982) Inhibition of photosynthesis by ethylene — a stomatal effect. Plant Physiol. 70, 598–601. Pandey, S., Chen, J.G., Jones, A.M. and Assmann, S.M. (2006) G-protein complex mutants are hypersensitive to abscisic acid regulation of germination and postgermination development. Plant Physiol. 141, 243– 256. Pei, Z.M., Murata, Y., Benning, G., Thomine, S., Klusener, B., Allen, G.J., Grill, E. and Schroeder, J.I. (2000) Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells. Nature, 406, 731–734. She, X.P., Song, X.G. and He, J.M. (2004) Role and relationship of nitric oxide and hydrogen peroxide in light/dark-regulated stomatal movement in Vicia faba. Acta Bot. Sin. 46, 1292–1300. Shi, Y.H., Zhu, S.W., Mao, X.Z., Feng, J.X., Qin, Y.M., Zhang, L., Cheng, J., Wei, L.P., Wang, Z.Y. and Zhu, Y.X. (2006) Transcriptome profiling, molecular biological and physiological studies reveal a major role for ethylene in cotton fiber cell elongation. Plant Cell, 18, 651–664. Suharsono, U., Fujisawa, Y., Kawasaki, T., Iwasaki, Y., Satoh, H. and Shimamoto, K. (2002) The heterotrimeric G protein a subunit acts upstream of the small GTPase Rac in disease resistance of rice. Proc. Natl Acad. Sci. USA, 99, 13307–13312. Suhita, D., Raghavendra, A.S., Kwak, J.M. and Vavasseur, A. (2004) Cytoplasmic alkalization precedes reactive oxygen species production during methyl jasmonate- and abscisic acid-induced stomatal closure. Plant Physiol. 134, 1536–1545. Tanaka, Y., Sano, T., Tamaoki, M., Nakajima, N., Kondo, N. and Hasezawa, S. (2005) Ethylene inhibits abscisic acid-induced stomatal closure in Arabidopsis. Plant Physiol. 138, 2337–2343. Temple, B.R.S. and Jones, A.M. (2007) The plant heterotrimeric G-protein complex. Annu. Rev. Plant Biol. 58, 249–266. Trusov, Y., Rookes, J.E., Chakravorty, D., Armour, D., Schenk, P.M. and Botella, J.R. (2006) Heterotrimeric G proteins facilitate Arabidopsis resistance to necrotrophic pathogens and are involved in jasmonate signaling. Plant Physiol. 140, 210–220. Tsugama, D., Liu, S. and Takano, T. (2013) Arabidopsis heterotrimeric G protein b subunit, AGB1, regulates brassinosteroid signalling independently of BZR1. J. Exp. Bot. 64, 3213–3223. Ullah, H., Chen, J.G., Young, J., Im, K.H., Sussman, M.R. and Jones, A.M. (2001) Modulation of cell proliferation by heterotrimeric G protein in Arabidopsis. Science, 292, 2066–2069. Ullah, H., Chen, J.G., Wang, S. and Jones, A.M. (2002) Role of a heterotrimeric G protein in regulation of Arabidopsis seed germination. Plant Physiol. 129, 897–907. Urano, D., Chen, J.G., Botella, J.R. and Jones, A.M. (2013) Heterotrimeric G protein signalling in the plant kingdom. Open Biol. 3, 120186. Vanstraelen, M. and Benkov0 a, E. (2012) Hormonal interactions in the regulation of plant development. Annu. Rev. Cell Dev. Biol. 28, 463–487. Vert, G., Nemhauser, J.L., Geldner, N., Hong, F.X. and Chory, J. (2005) Molecular mechanisms of steroid hormone signaling in plants. Annu. Rev. Cell Dev. Biol. 21, 177–201. Wang, Y.H. and Irving, H.R. (2011) Developing a model of plant hormone interactions. Plant Signal. Behav. 6, 494–500.

Wang, X.Q., Ullah, H., Jones, A.M. and Assmann, S.M. (2001) G protein regulation of ion channels and abscisic acid signaling in Arabidopsis guard cells. Science, 292, 2070–2072. Wang, K.L.C., Yoshida, H., Lurin, C. and Ecker, J.R. (2004) Regulation of ethylene gas biosynthesis by the Arabidopsis ETO1 protein. Nature, 428, 945–950. Wang, L., Xu, Y.Y., Ma, Q.B., Li, D., Xu, Z.H. and Chong, K. (2006) Heterotrimeric G protein a subunit is involved in rice brassinosteroid response. Cell Res. 16, 916–922. Wang, H., Zhu, Y.Y., Fujioka, S., Asami, T., Li, J.Y. and Li, J.M. (2009) Regulation of Arabidopsis brassinosteroid signaling by atypical basic helixloop-helix proteins. Plant Cell, 21, 3781–3791. Warpeha, K.M., Upadhyay, S., Yeh, J., Adamiak, J., Hawkins, S.I., Lapik, Y.R., Anderson, M.B. and Kaufman, L.S. (2007) The GCR1, GPA1, PRN1, NF-Y signal chain mediates both blue light and abscisic acid responses in Arabidopsis. Plant Physiol. 143, 1590–1600. Watkins, J.M., Hechler, P.J. and Muday, G.K. (2014) Ethylene-induced flavonol accumulation in guard cells suppresses reactive oxygen species and moderates stomatal aperture. Plant Physiol. 164, 1707–1717. Wilkinson, J.Q. and Crawford, N.M. (1993) Identification and characterization of a chlorate resistant mutant of Arabidopsis with mutations in both NIA1 and NIA2 nitrate reductase structural genes. Mol. Gen. Genet. 239, 289–297. Wilkinson, S. and Davies, W.J. (2009) Ozone suppresses soil drying- and abscisic acid (ABA)-induced stomatal closure via an ethylene dependent mechanism. Plant, Cell Environ. 32, 949–959. € ft, J. and Harter, K. (2011) Latest news on Arabidopsis brassinosterWittho oid perception and signaling. Front. Plant Sci. 2, 58–61. Wu, G., Wang, X.L., Li, X.B., Kamiya, Y.J., Otegui, M.S. and Chory, J. (2011) Methylation of a phosphatase specifies dephosphorylation and degradation of activated brassinosteroid receptors. Sci. Signal. 4, ra29. doi: 10.1126/scisignal.2001258. Xia, X.J., Wang, Y.J., Zhou, Y.H., Tao, Y., Mao, W.H., Shi, K., Asami, T., Chen, Z.X. and Yu, J.Q. (2009) Reactive oxygen species are involved in brassinosteroid-induced stress tolerance in cucumber. Plant Physiol. 150, 801–814. Xia, X.J., Gao, C.J., Song, L.X., Zhou, Y.H., Shi, K. and Yu, J.Q. (2014) Role of H2O2 dynamics in brassinosteroid-induced stomatal closure and opening in Solanum lycopersicum. Plant, Cell Environ. 37, 2036–2050. Xu, W.H., Huang, J., Li, B.H., Li, J.Y. and Wang, Y.H. (2008) Is kinase activity essential for biological functions of BRI1? Cell Res. 18, 472–478. Yang, C.J., Zhang, C., Lu, Y.N., Jin, J.Q. and Wang, X.L. (2011) The mechanisms of brassinosteroids’ action: from signal transduction to plant development. Mol. Plant, 4, 588–600. Ye, H.X., Li, L. and Yin, Y.H. (2011) Recent advances in the regulation of brassinosteroid signaling and biosynthesis pathways. J. Integr. Plant Biol. 53, 455–468. Yi, H.C., Joo, S., Nam, K.H., Lee, J.S., Kang, B.G. and Kim, W.T. (1999) Auxin and brassinosteroid differentially regulate the expression of the three members of the 1-aminocyclopropane-1-carboxylate synthase family in mung bean (Vigna radiata L.). Plant Mol. Biol. 41, 443–454. Young, T.E., Meeley, R.B. and Gallie, D.R. (2004) ACC synthase expression regulates leaf performance and drought tolerance in maize. Plant J. 40, 813–825. Zhang, W., He, S.Y. and Assmann, S.M. (2008) The plant innate immunity response in stomatal guard cells invokes G-protein-dependent ion channel regulation. Plant J. 56, 984–996. Zhang, S.S., Wei, Y., Lu, Y.N. and Wang, X. (2009) Mechanisms of brassinosteroids interacting with multiple hormones. Plant Signal. Behav. 4, 1117–1120. Zhang, W., Jeon, B.W. and Assmann, S.M. (2011) Heterotrimeric G-protein regulation of ROS signalling and calcium currents in Arabidopsis guard cells. J. Exp. Bot. 62, 2371–2379.


Heterotrimeric G protein mediates ethylene-induced stomatal closure via hydrogen peroxide synthesis in Arabidopsis.

Reactive Oxygen Species-Dependent Nitric Oxide Production Contributes to Hydrogen-Promoted Stomatal Closure in Arabidopsis.

3 complex-mediated actin dynamics is required for hydrogen peroxide-induced stomatal closure in Arabidopsis.

8-Mercapto-Cyclic GMP Mediates Hydrogen Sulfide-Induced Stomatal Closure in Arabidopsis.

Nitric Oxide and Hydrogen Peroxide Production are Involved in Systemic Drought Tolerance Induced by 2R,3R-Butanediol in Arabidopsis thaliana.

Hydrogen peroxide acts upstream of nitric oxide in the heat shock pathway in Arabidopsis seedlings.

Aerobic exercise training protects against endothelial dysfunction by increasing nitric oxide and hydrogen peroxide production in LDL receptor-deficient mice.

Ethylene Oxide and Hydrogen Peroxide Gas Plasma Sterilization: Precautionary Practices in U.S. Hospitals.

Nitric Oxide, Ethylene, and Auxin Cross Talk Mediates Greening and Plastid Development in Deetiolating Tomato Seedlings.

Hydrogen Peroxide- and Nitric Oxide-mediated Disease Control of Bacterial Wilt in Tomato Plants.

calmodulin-dependent formation of hydrogen peroxide by brain nitric oxide synthase.

NO), a class of hydrogen peroxide inducible nitric oxide (NO) donors.

Arabidopsis CaM1 and CaM4 Promote Nitric Oxide Production and Salt Resistance by Inhibiting S-Nitrosoglutathione Reductase via Direct Binding.

Nitric oxide as a secondary messenger during stomatal closure as a part of plant immunity response against pathogens.

Nitric oxide mediates iron release from ferritin.

Detection of Hydrogen Peroxide by DAB Staining in Arabidopsis Leaves.

Hydrogen peroxide production during experimental protein glycation.

Mobile gene silencing in Arabidopsis is regulated by hydrogen peroxide.

Exenatide induces aortic vasodilation increasing hydrogen sulphide, carbon monoxide and nitric oxide production.

Nitric oxide mediates bleomycin-induced angiogenesis and pulmonary fibrosis via regulation of VEGF.

Hydrogen peroxide, nitric oxide and UV RESISTANCE LOCUS8 interact to mediate UV-B-induced anthocyanin biosynthesis in radish sprouts.

Interaction of Polyamines, Abscisic Acid, Nitric Oxide, and Hydrogen Peroxide under Chilling Stress in Tomato (Lycopersicon esculentum Mill.) Seedlings.

Copper amine oxidase 8 regulates arginine-dependent nitric oxide production in Arabidopsis thaliana.

Catalase influence in the regulation of coronary resistance by estrogen: joint action of nitric oxide and hydrogen peroxide.