COP1 Mediates Dark-Induced Stomatal Closure by Suppressing FT, TSF and SOC1 Expression to Promote NO Accumulation in Arabidopsis Guard Cells

RING-finger-type ubiquitin E3 ligase Constitutively Photomorphogenic 1 (COP1) and floral integrators such as FLOWERING LOCUS T (FT), TWIN SISTER OF FT (TSF) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) have been identified as regulators of stomatal movement. However, little is known about their roles and relationship in dark-induced stomatal closure. Here, we demonstrated that COP1 is required for dark-induced stomatal closure using cop1 mutant. The cop1 mutant closed stomata in response to exogenous nitric oxide (NO) but not hydrogen peroxide (H2O2), and H2O2 but not NO accumulated in cop1 in darkness, further indicating that COP1 acts downstream of H2O2 and upstream of NO in dark-induced stomatal closure. Expression of FT, TSF and SOC1 in wild-type (WT) plants decreased significantly with dark duration time, but this process was blocked in cop1. Furthermore, ft, tsf, and soc1 mutants accumulated NO and closed stomata faster than WT plants in response to darkness. Altogether, our results indicate that COP1 transduces H2O2 signaling, promotes NO accumulation in guard cells by suppressing FT, TSF and SOC1 expression, and consequently leads to stomatal closure in darkness. These findings add new insights into the mechanisms of dark-induced stomatal closure.


Introduction
Plant guard cells open and close stomata to regulate CO 2 uptake for photosynthesis and control water loss from the plant. They are sensitive to multiple internal and external signals to set the appropriate stomatal aperture for the prevailing environments [1]. Light/dark is one of the most essential and well-studied environmental signals regulating stomatal movement [2][3][4]. Stomata open in response to light and close in the dark. Various components in light/dark-regulated stomatal movement, especially in light signaling, have been demonstrated, such as photoreceptors, plasma membrane H + -ATPase, ion channels, Constitutively Photomorphogenic 1 (COP1), CONSTANS (CO), hydrogen peroxide (H 2 O 2 ) and nitric oxide (NO) [2,[5][6][7].
COP1, an E3 ubiquitin ligase, has been extensively studied as a major negative regulator of photomorphogenesis [8]. Mao et al. [6] found that blue light-induced stomatal opening by cryptochromes (CRY) was mediated by negatively regulating COP1, first defining a new role of COP1 in guard cell signaling. After that, increasing evidence demonstrates that COP1 plays an essential role in modulating stomatal movement. Wang et al. [2] showed that COP1 acted downstream of Phytochrome B (PHYB) in regulating red light-induced stomatal opening. Several researchers further demonstrated that COP1 is not only involved in the light-induced stomatal opening but also plays a fundamental role in abscisic acid (ABA)- [9][10][11], dehydration- [12], and ultraviolet-B (UV-B)-induced stomatal closure [13].

COP1 Is Required for Dark-Induced Stomatal Closure
Previous studies have demonstrated that stomata of the cop1-4 mutant are constitutively open in darkness [6], indicating an important role of COP1 in dark-induced stomatal closure. To confirm the function of COP1, we compared the stomatal responses of cop1 mutants with that of the wild-type (WT) plants to continuous darkness for 4 h. Results showed that the stomatal aperture of WT plants decreased with the time of dark treatment. A significant decrease was observed at 2 h, and the minimum stomatal aperture appeared at 3 h of dark treatment. Two independent cop1 mutants showed a constitutive open-stomata phenotype in darkness ( Figure 1A), proving the essential role of COP1 in mediating dark-induced stomatal closure. COP1-mediated dark-induced stomatal closure, we compared stomatal response darkness of hy5 single mutants (hy5-1 in Ler background and hy5-ks50 in Ws backg and hy5hyh double mutant with that of the corresponding WT plants. Results show mutants for HY5 had the same stomatal responses to darkness as WT ( Figure 1B,C results, combined with the facts that HY5 is degraded by COP1 in darkness [8] a overexpression of HY5 results in constitutive closing of stomata under either light o ness [13], indicate that HY5 is not involved in dark-induced stomatal closure. Th COP1 mediates dark-induced stomatal closure in a way independent of HY5.

COP1 Acts Downstream of H2O2 in Dark-Induced Stomatal Closure
H2O2 is a common signal for dark-, ABA-and UV-B-induced stomatal [7,13,22]. To explore the signaling pathway in dark-induced stomatal closure, we vestigated the stomatal responses of cop1 mutant to exogenous H2O2 treatment. T mutant showed a similar stomatal aperture to WT plants under a light. Exogenou significantly closed the stomata of WT but not that of the cop1 mutant. When tran to the dark for 3 h, the stomata of the cop1 mutant are still kept open and exogenou cannot affect this constitutive open-stomata phenotype (Figure 2A). These results i that COP1 may function downstream of H2O2 during dark-induced stomatal clos further confirm this, we detected the H2O2 content in guard cells of cop1 mutant Leaves of WT Col-0, Ler and Ws, and the cop1-4, cop1-6, hy5-1, hy5-ks50 and hy5hyh mutants were incubated in MES buffer under darkness for 3-4 h. The stomatal apertures of epidermal peels taken from the leaf abaxial surfaces were examined at the indicated time. Data are means ± standard error (SE, n = 3). Different small letters indicate significant differences (p < 0.01).
We previously reported that COP1 mediated UV-B-induced stomatal closure through an HY5-dependent pathway [13]. To determine whether HY5 is also involved in COP1mediated dark-induced stomatal closure, we compared stomatal responses to the darkness of hy5 single mutants (hy5-1 in Ler background and hy5-ks50 in Ws background) and hy5hyh double mutant with that of the corresponding WT plants. Results showed that mutants for HY5 had the same stomatal responses to darkness as WT ( Figure 1B,C). These results, combined with the facts that HY5 is degraded by COP1 in darkness [8] and that overexpression of HY5 results in constitutive closing of stomata under either light or darkness [13], indicate that HY5 is not involved in dark-induced stomatal closure. Therefore, COP1 mediates dark-induced stomatal closure in a way independent of HY5.  Figure 2B,C). Clear mutation of COP1 did not affect dark-induced H2O2 accumulation; however, in contr to WT plants, the H2O2 accumulation in the cop1 mutant did not lead to stomatal closu This result further confirms that both H2O2 and COP1 are involved in dark signaling guard cells, and COP1 is required for transducing the H2O2 signal.

COP1 Acts Upstream of NO in Dark-Induced Stomatal Closure
NO is another important signal molecule, and NO generation in guard cells duri stomatal closure is usually mediated by H2O2 [19,20,23]. Therefore, both NO and CO function downstream of H2O2. To further define the relationship between NO and CO in dark-induced stomatal closure, we investigated the effect of exogenous NO, applied the form of the NO donor sodium nitroprusside (SNP), on stomatal apertures of cop1 m tant. SNP treatment significantly reduced the stomatal aperture of WT plants under lig conditions and further decreased it under dark treatment ( Figure 3A). Meanwhile, SN treatment not only induced stomatal closure of the cop1 mutant under light conditions b also significantly rescued the defect of dark-induced stomatal closure in the cop1 muta ( Figure 3A). These results suggest that COP1 may induce stomatal closure upstream NO in darkness. Using NO-specific fluorescent dye 4,5-diaminofluorescein diacet (DAF-2DA), we found that both dark and exogenous H2O2 treatment significantly e hanced NO accumulation in WT guard cells but did not do that in cop1 guard cells (Figu 3B,C). This result indicates that dark-and H2O2-induced NO accumulation are blocked cop1 mutant, further confirming that NO accumulation is downstream of COP1 in t dark. However, the stomatal aperture in the cop1 mutant was significantly larger than th in WT plants under SNP treatment ( Figure 3A), indicating that COP1 also mediates da induced stomatal closure through alternative pathways.

COP1 Acts Upstream of NO in Dark-Induced Stomatal Closure
NO is another important signal molecule, and NO generation in guard cells during stomatal closure is usually mediated by H 2 O 2 [19,20,23]. Therefore, both NO and COP1 function downstream of H 2 O 2 . To further define the relationship between NO and COP1 in dark-induced stomatal closure, we investigated the effect of exogenous NO, applied in the form of the NO donor sodium nitroprusside (SNP), on stomatal apertures of cop1 mutant. SNP treatment significantly reduced the stomatal aperture of WT plants under light conditions and further decreased it under dark treatment ( Figure 3A). Meanwhile, SNP treatment not only induced stomatal closure of the cop1 mutant under light conditions but also significantly rescued the defect of dark-induced stomatal closure in the cop1 mutant ( Figure 3A). These results suggest that COP1 may induce stomatal closure upstream of NO in darkness. Using NO-specific fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2DA), we found that both dark and exogenous H 2 O 2 treatment significantly enhanced NO accumulation in WT guard cells but did not do that in cop1 guard cells ( Figure 3B,C). This result indicates that dark-and H 2 O 2 -induced NO accumulation are blocked in cop1 mutant, further confirming that NO accumulation is downstream of COP1 in the dark. However, the stomatal aperture in the cop1 mutant was significantly larger than that in WT plants under SNP treatment ( Figure 3A), indicating that COP1 also mediates dark-induced stomatal closure through alternative pathways.

Expression of FT, TSF and SOC1 Is Downregulated by Darkness via the COP1-Dependen Manner
The floral integrators FT, TSF and SOC1, have been shown to express in guard ce and positively regulate light-induced stomatal opening [5,16,17]. We performed both R PCR and qRT-PCR analysis, and they consistently showed that gene expression of FT, T and SOC1 in epidermal peels of WT Arabidopsis leaves was downregulated in darkne and the longer the dark duration time, the lower transcription of these genes was observ during the whole experiment period ( Figure 4A,B). Changes in their expression were agreement with the stomatal responses of WT plants to dark treatment, suggesting a po itive correlation between dark-induced stomatal closure and the decreased gene expr sion of these floral integrators.

Expression of FT, TSF and SOC1 Is Downregulated by Darkness via the COP1-Dependent Manner
The floral integrators FT, TSF and SOC1, have been shown to express in guard cells and positively regulate light-induced stomatal opening [5,16,17]. We performed both RT-PCR and qRT-PCR analysis, and they consistently showed that gene expression of FT, TSF and SOC1 in epidermal peels of WT Arabidopsis leaves was downregulated in darkness, and the longer the dark duration time, the lower transcription of these genes was observed during the whole experiment period ( Figure 4A

Expression of FT, TSF and SOC1 Is Downregulated by Darkness via the COP1-Dependent Manner
The floral integrators FT, TSF and SOC1, have been shown to express in guard cells and positively regulate light-induced stomatal opening [5,16,17]. We performed both RT-PCR and qRT-PCR analysis, and they consistently showed that gene expression of FT, TSF and SOC1 in epidermal peels of WT Arabidopsis leaves was downregulated in darkness, and the longer the dark duration time, the lower transcription of these genes was observed during the whole experiment period ( Figure 4A,B). Changes in their expression were in agreement with the stomatal responses of WT plants to dark treatment, suggesting a positive correlation between dark-induced stomatal closure and the decreased gene expression of these floral integrators.  To test whether COP1 functions in downregulating the expression of FT, TSF and SOC1 in guard cells in darkness, we investigated the effects of COP1 mutation on the expression of FT, TSF and SOC1 in epidermal peels. Results showed that mutation of COP1 totally inhibited dark-triggered downregulation of these floral integrator genes ( Figure 4C,D). These results indicate that the decrease in FT, TSF and SOC1 expression are mainly dependent on COP1, and they, in turn, transduce COP1-mediated guard cell signaling in darkness.

FT, TSF and SOC1 Participate in Dark-Induced Stomatal Closure
To confirm the role of FT, TSF and SOC1 in dark-induced stomatal closure, we observed the stomatal responses of ft, tsf and soc1 mutants to dark treatment. Under light condition (0 h), stomatal apertures of ft-1, ft-2, tsf-1, tsf-2, soc1-11 and soc1-12 mutants were all significantly smaller than that of WT controls ( Figure 5), confirming light-induced stomatal opening is suppressed in these mutants [5,16,17]. Under darkness for 1 h, the stomatal aperture in WT plants showed a slight but not significant decrease, while it significantly decreased in ft, tsf and soc1 mutants. Under darkness for 2 h, both WT and mutants closed their stomata further. However, by comparing stomatal apertures, we found that ft, tsf and soc1 mutants maximumly closed their stomata at 2 h ( Figure 5), while WT did it at 3 h (Figures 1 and 5). These results demonstrate the hypersensitive stomatal responses of ft, tsf and soc1 mutants to darkness, indicating that FT, TSF and SOC1 not only positively regulate light-induced stomatal opening but also negatively modulate dark-induced stomatal closing. To test whether COP1 functions in downregulating the expression of FT, TSF and SOC1 in guard cells in darkness, we investigated the effects of COP1 mutation on the expression of FT, TSF and SOC1 in epidermal peels. Results showed that mutation of COP1 totally inhibited dark-triggered downregulation of these floral integrator genes ( Figure  4C,D). These results indicate that the decrease in FT, TSF and SOC1 expression are mainly dependent on COP1, and they, in turn, transduce COP1-mediated guard cell signaling in darkness.

FT, TSF and SOC1 Participate in Dark-Induced Stomatal Closure
To confirm the role of FT, TSF and SOC1 in dark-induced stomatal closure, we observed the stomatal responses of ft, tsf and soc1 mutants to dark treatment. Under light condition (0 h), stomatal apertures of ft-1, ft-2, tsf-1, tsf-2, soc1-11 and soc1-12 mutants were all significantly smaller than that of WT controls ( Figure 5), confirming light-induced stomatal opening is suppressed in these mutants [5,16,17]. Under darkness for 1 h, the stomatal aperture in WT plants showed a slight but not significant decrease, while it significantly decreased in ft, tsf and soc1 mutants. Under darkness for 2 h, both WT and mutants closed their stomata further. However, by comparing stomatal apertures, we found that ft, tsf and soc1 mutants maximumly closed their stomata at 2 h ( Figure 5), while WT did it at 3 h (Figures 1 and 5). These results demonstrate the hypersensitive stomatal responses of ft, tsf and soc1 mutants to darkness, indicating that FT, TSF and SOC1 not only positively regulate light-induced stomatal opening but also negatively modulate dark-induced stomatal closing.  ft-2, (B)), soc1 (soc1-11 and soc1-12, (C)) and tsf (tsf-1 and tsf-2, (D)) mutants were incubated in MES buffer in darkness for 0-3 h. The stomata apertures in epidermal peels were examined at 1-h intervals. Data are means ± standard error (SE, n = 3). Different small letters indicate significant differences (p < 0.01).  ft-2, (B)), soc1 (soc1-11 and soc1-12, (C)) and tsf (tsf-1 and tsf-2, (D)) mutants were incubated in MES buffer in darkness for 0-3 h. The stomata apertures in epidermal peels were examined at 1-h intervals. Data are means ± standard error (SE, n = 3). Different small letters indicate significant differences (p < 0.01).

Mutation of FT, TSF and SOC1 Accelerates Dark-Induced NO but Not H 2 O 2 Accumulation in Guard Cells
Given that FT, TSF and SOC1, as well as H 2 O 2 and NO, all participate in dark-induced stomatal closure, we further clarified the relationship of H 2 O 2 or NO with the floral integrators FT, TSF and SOC1 by monitoring H 2 O 2 and NO levels in guard cells of ft, tsf and soc1 mutants. We found that ft, tsf and soc1 mutants showed the same level of H 2 O 2 as the WT plants under light or during the dark treatment for 1-3 h (Figure 6), indicating that mutation of FT, TSF and SOC1 do not affect dark-induced H 2 O 2 accumulation.

in Guard Cells
Given that FT, TSF and SOC1, as well as H2O2 and NO, all participate in dark-induced stomatal closure, we further clarified the relationship of H2O2 or NO with the floral integrators FT, TSF and SOC1 by monitoring H2O2 and NO levels in guard cells of ft, tsf and soc1 mutants. We found that ft, tsf and soc1 mutants showed the same level of H2O2 as the WT plants under light or during the dark treatment for 1-3 h (Figure 6), indicating that mutation of FT, TSF and SOC1 do not affect dark-induced H2O2 accumulation.   In contrast, the NO level in the guard cells of these mutants was significantly higher than that of WT plants under light conditions and increased faster than that of WT in response to dark treatment (Figure 7). These results indicate that mutation of FT, TSF and SOC1 accelerates guard cell NO accumulation under either light or darkness. Obviously, changes in NO level have a significantly negative correlation with the stomatal apertures in these mutants under light and dark treatment (r = −0.849, p = 0.008 for ft-2; r = −0.891, p = 0.003 for tsf-1; and r = −0.890, p = 0.003 for soc1-11), suggesting that downregulation of FT, TSF and SOC1 expression by darkness facilitates NO accumulation in guard cells and therefore contributes to dark-induced stomatal closure.

in Guard Cells
Given that FT, TSF and SOC1, as well as H2O2 and NO, all participate in dark-induced stomatal closure, we further clarified the relationship of H2O2 or NO with the floral integrators FT, TSF and SOC1 by monitoring H2O2 and NO levels in guard cells of ft, tsf and soc1 mutants. We found that ft, tsf and soc1 mutants showed the same level of H2O2 as the WT plants under light or during the dark treatment for 1-3 h (Figure 6), indicating that mutation of FT, TSF and SOC1 do not affect dark-induced H2O2 accumulation.

COP1 Mediates Dark-Induced Stomatal Closure through Promotion of NO Accumulation
The E3 ubiquitin ligase COP1 has been well-documented as a central, negative determinant for plant photomorphogenesis [8]. It directs the degradation of diverse light-regulated transcription factors by the 26S proteasome [8,24]. Additionally, it also acts as a negative regulator of stomatal development [25]. Recently, increasing evidence also suggests a crucial role of COP1 in regulating stomatal movement [2,[9][10][11][12][13]. These studies demonstrate that COP1 participates in both light-induced stomatal opening and ABA-and UV-B-induced stomatal closure. In addition, COP1 has also been indicated to play a role in dark-induced stomatal closing [6]. Here, the constitutive open-stomata phenotype of both cop1-4 and cop1-6 mutants provides further evidence that COP1 is required for dark-induced stomatal closure.
HY5 is one of the most studied signaling transcription factors, which sometimes acts redundantly with its closely related homolog HYH (HY5 HOMOLOG) [20,26]. The interaction between HY5 and COP1 is involved in many developmental processes [26]. In photomorphogenesis, COP1 acts as a negative factor to promote the degradation of HY5 [26]. However, it has been reported that, in UV-B signaling, COP1 positively regulates HY5, and HY5 then mediates UV-B-induced stomatal closure [13,20]. In the present study, neither hy5 single mutants nor hy5hyh double mutants affected stomatal responses to dark treatment (Figure 1), indicating HY5 is not involved in the dark-signaling of guard cells. Our result indicates that, in contrast to UV-B signaling, COP1 acts in a way independent of HY5 during dark-induced stomatal closure.
H 2 O 2 and NO are famous signaling molecules mediating various physiological processes in plants, including stomatal movement [7,[18][19][20][21][22][23]27]. Zhang et al. [21] provided convincing evidence that H 2 O 2 and NO participate in dark-induced stomatal closure, in which H 2 O 2 acts upstream of NO. Here, after confirming the essential role of COP1, we further revealed the relationships among COP1, H 2 O 2 and NO in dark-induced stomatal closure. Pharmacological and confocal studies strongly support that COP1 functions downstream of H 2 O 2 and upstream of NO in dark-induced stomatal closure (Figures 2 and 3). Similar relationships have also been reported for COP1, H 2 O 2 and NO in UV-B-induced stomatal closure [13]. Further studies are needed to uncover how COP1 is regulated by H 2 O 2 and then mediates NO production in UV-B-and dark-induced stomatal closure. Except for COP1, the MEK1-MPK6 cascade also functions in dark-induced stomatal closure by mediating H 2 O 2 -induced NO accumulation [21]. Whether and how COP1 has crosstalk with MEK1-MPK6 cascade is worth to be further studied. AtMYB61, an R2R3-MYB transcription factor specifically expressed in guard cells, plays a major role in dark-induced stomatal closure [28]. Moazzam-Jazi et al. [12] reported that COP1 expression in darkness is required for the expression of AtMYB61. A study on the relationship between AtMYB61 and NO will also provide a better understanding of COP1 signaling in darkness.

Floral Integrators FT, TSF and SOC1 Negatively Regulate Dark-Induced Stomatal Closure
Although FT, TSF and SOC1 were originally identified and further well-studied as crucial flowering regulators, their roles in other physiological processes, such as fruit set and vegetative growth, have also received a lot of attention [14,15]. These researches suggest that they are general growth regulators in plants. In the past 11 years, a novel function of these proteins as regulators of stomatal movement was defined successively [5,16,17,29]. However, these studies focused on light-induced stomatal opening, in which FT, TSF and SOC1 expressed highly and functioned as positive regulators. Here, we not only revealed that expressions of FT, TSF and SOC1 all decreased with dark duration time (Figure 4) but also provided genetic evidence that these floral integrators are negative regulators in dark-induced stomatal closure ( Figure 5). These findings add new information on the role of FT, TSF and SOC1 as stomatal movement regulators. However, the partially closed phenotypes of ft, tsf and soc1 mutants under light suggest that alternative signaling pathway(s) independent of FT, TSF and SOC1 also exist in dark-induced stomatal closure, which should be further studied.

COP1 Promotes NO Accumulation by Suppressing FT, TSF and SOC1 Expression
The decreased expression levels of FT, TSF and SOC1, were positively correlated with the gradually reduced stomatal aperture during dark treatment ( Figure 4A). This result is consistent with previous results that stomatal opening was accompanied by the upregulation of FT, TSF and SOC1 expression [5,16,17]. Together, these results demonstrate that the regulation of dark-induced stomatal closure by FT, TSF and SOC1 is due to the downregulation of their expressions. Moreover, COP1 mutation not only blocked the downregulation of FT, TSF and SOC1 expression in darkness ( Figure 4B) but also impaired dark-induced stomatal closure (Figure 1). These results indicate that COP1 mediates darkinduced stomatal closure by suppressing FT, TSF and SOC1 expression. CO, a major regulator of photoperiodic flowering response, is also involved in stomatal opening [5]. It is well-known that FT and TSF are targets of CO protein, and SOC1 expression is mediated by FT [5,29]. Besides, we also know that COP1 targets CO for proteasome-mediated degradation in the dark [8,24]. Therefore, COP1 mediates the down-regulation of FT, TSF and SOC1 expression in darkness by promoting the degradation of CO, the upstream regulator of FT, TSF and SOC1. Further studies are needed to test this hypothesis.
To date, little information is available on the relationship of signaling molecules H 2 O 2 and NO with floral integrators FT, TSF and SOC1, especially in guard cells. In the present study, we showed that H 2 O 2 accumulation in guard cells of ft, tsf and soc1 mutants is similar to that of WT plants (Figure 6), while NO in guard cells of ft, tsf and soc1 mutants accumulated faster and higher than that of WT plants under dark treatment (Figure 7). This result, together with the stomatal phenotypes, indicates that decreased expression of FT, TSF and SOC1 does not affect H 2 O 2 but promotes NO accumulation in guard cells and consequently facilitates dark-induced stomatal closure. Our findings enrich mechanisms of guard cell signaling in darkness and also pose an interesting challenge in elucidating the molecular mechanisms underlying FT-, TSF-, and SOC1-mediated stomatal movement.
It has been recognized that an appropriately functioning circadian system confers an adaptive advantage to plants [30]. Wang et al. [31] demonstrated that manipulating H + -ATPase in guard cells to make stomata open wider in the daytime and still close at night enhanced plant growth while making the stomata constantly open, even in the dark, did not. As major regulatory components were mediating the onset of flowering, expression of the floral integrators (FT, TSF and SOC1) will be activated prior to flowering time [15]. These photoperiodic flowering components positively affect light-regulated stomatal opening, probably resulting in the acceleration of photosynthesis to prepare for flowering [5]. The improved growth may be offset by increased water loss if stomata are still open in the dark [30]. Therefore, our finding that downregulation of FT, TSF and SOC1 by COP1 to allow stomata to close normally in the dark may be an important mechanism facilitating plant growth, especially in the period prior to the flowering time when the floral integrators are highly expressed.
In summary, the data presented herein demonstrate that COP1 acts downstream of H 2 O 2 to mediate dark-induced NO accumulation in guard cells and subsequent stomatal closure by suppressing expressions of the floral integrator genes FT, TSF and SOC1. Based on our findings, we put forward a possible model by which COP1 mediates darkinduced stomatal closure ( Figure 8). The novel role of FT, TSF and SOC1 as negative regulators of dark-induced stomatal closure and the interrelationships among H 2 O 2 , COP1, FT/TSF/SOC1, and NO would no doubt provide clues directing a further study on guard cell signal transduction.
Seeds of wild types and mutants were surface sterilized with bleaching power (20%, w/v) for 10 min, washed with sterilized water three times, then germinated and grown on vermiculite. Seedlings were cultured as previously described [21]. Fully expanded leaves of four-five-week-old healthy plants were harvested and immediately used for different treatments.

Stomatal Bioassay
The stomatal bioassays were performed according to Zhang et al. [21] using the rosette leaves of four-five-week-old WT plants or tested mutants with slight modifications. Freshly detached leaves were incubated in MES-KCl buffer (50 mmol/L KCl, 10 mmol/L MES-KOH, pH 6.15) under white light (PPFD 0.1 mmol/m 2 /s) for 2 h to open the stomata, and then incubated in the buffer alone or with 100 µmol/L H 2 O 2 or 50 µmol/L SNP under the same white light or darkness for 1-4 h. After treatments, abaxial epidermal strips were peeled from the leaves, and stomatal apertures were measured with a calibrated light microscope. Stomatal bioassays were always started at the same time of the day to avoid the potential rhythmic effects on stomatal aperture. Data are displayed as means ± standard errors (SE) of three biological replicates, each with 40 stomata.

Measurement of Endogenous H 2 O 2 and NO in Guard Cells
Endogenous H 2 O 2 and NO in guard cells were measured according to a previous study [21] with fluorescent dye H 2 DCFDA and DAF-2DA, respectively. After being incubated with the fluorescent dye and removing the excess dye in the dark, fluorescence of the epidermal strips was immediately observed using a confocal laser scanning microscopy (Leica TCS-SP8 Lasertechnik GmbH, Wien, Austria, excitation 488 nm, emission 515 ± 15 nm, frame 1024 × 1024). Images were then processed with Photoshop software, and the whole stomata areas were analyzed with Leica Image software to quantify the fluorescence intensity. All presented images represented similar results from three independent experiments, and the data of fluorescence intensity are presented as means ± SE of three independent experiments, each with 20 stomata.

Gene Expression Analysis
Total RNAs were extracted from the epidermal peels with E.Z.N.A.™ Plant RNA Kit (OMEGA, La Chaux-de-Fonds, Switzerland), and first-strand cDNAs were synthesized using the PrimeScript™ RT Master Mix (TAKARA, Shiga, Japan) following the manufacturer's instructions. TUBELIN BETA CHAIN2 (TUB2) was used as the control. Twenty-five cycles of PCR were performed for FT, TSF, SOC1 and TUB2 in RT-PCR. Primer sequences for RT-PCR analysis of these genes were reported previously [17]. Quantitative gene expression was analyzed by qRT-PCR with SYBR Green PCR Master Mix as previously described [32]. The primers that have been successfully used for FT, SOC1, TUB2 [29] and TSF [33] were used in this study for qRT-PCR analysis.

Statistical Analysis
All experiments were repeated three times with similar results. Statistical analysis was performed using a one-way ANOVA to discriminate significant differences followed by least significant difference test (LSD).

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

Informed Consent Statement: Not applicable.
Data Availability Statement: Data will be made available upon request.