Is nitric oxide a critical key factor in ABA-induced stomatal closure?

Abstract

The role of nitric oxide (NO) in abscisic acid (ABA)-induced stomatal closure is a matter of debate. We conducted experiments in Vicia faba leaves using NO gas and sodium nitroprusside (SNP), a NO-donor compound, and compared their effects to those of ABA. In epidermal strips, stomatal closure was induced by ABA but not by NO, casting doubt on the role of NO in ABA-mediated stomatal closure. Leaf discs and intact leaves showed a dual dose response to NO: stomatal aperture widened at low dosage and narrowed at high dosage. Overcoming stomatal resistance by means of high CO₂ concentration ([CO₂]) restored photosynthesis in ABA-treated leaf discs but not in those exposed to NO. NO inhibited photosynthesis immediately, causing an instantaneous increase in intercellular [CO₂] (C_i), followed by stomatal closure. However, lowering C_i by using low ambient [CO₂] showed that it was not the main factor in NO-induced stomatal closure. In intact leaves, the rate of stomatal closure in response to NO was about one order of magnitude less than after ABA application. Because of the different kinetics of photosynthesis and stomatal closure that were observed, we conclude that NO is not likely to be the key factor in ABA-induced rapid stomatal closure, but that it fine-tunes stomatal aperture via different pathways.

Stomatal closure dynamics in Vicia faba following application of NO gas to epidermal strips, leaf discs, and intact leaves suggest that NO is not essential in ABA-induced stomatal closure, but that it acts independently of ABA in the presence of mesophyll.

Introduction

Stomata are the gateways for gas exchange between the inside of the leaf and the external atmosphere. In terrestrial plants, fine regulation of stomatal aperture is critical for balancing CO₂ uptake for photosynthesis whilst preventing excessive transpirational water loss. Evolution has resulted in a complex signalling network of pathways that trigger opening or closing of stomata in response to environmental cues. Abscisic acid (ABA) is considered to be the main phytohormone that induces stomatal closure, although an ABA-independent pathway for closure of stomata has also been proposed (Yoshida et al., 2006; Huang et al., 2009; Montillet et al., 2013; Roychoudhury et al., 2013).

Nitric oxide (NO) acts as important signalling molecule in diverse physiological processes, including adaptations to abiotic and biotic stresses (Wendehenne et al., 2004; Courtois et al., 2008; Neill et al., 2008; Jeandroz et al., 2013; Fancy et al., 2017) and it interacts with multiple plant hormones, including salicylic acid, ABA, and cytokinin (Freschi, 2013; Prakash et al., 2019). Its bioactivity is realized through redox-based post-translational protein modifications such as S-nitrosylation, which involves the addition of a NO group to a protein cysteine thiol to generate an S-nitrosothiol (Astier et al., 2011). Application of sodium nitroprusside (SNP), a NO donor, to detached leaves of Triticum aestivum, Tradescantia virginiana, and Poncirus trifoliata results in reduced water loss (García-Mata and Lamattina, 2001; Fan and Liu, 2012), whilst treatment of epidermal peels of Tradescantia species, Vicia faba, and Pisum sativum results in significant reductions in stomatal aperture (García-Mata and Lamattina, 2001; Neill et al., 2002). ABA promotes NO synthesis in guard cells, while the application of NO-scavengers prevents ABA-induced stomatal closure (García-Mata and Lamattina, 2001; Neill et al., 2002). Consequently, NO has repeatedly been suggested to be a key component of the ABA signalling pathway for stomatal closure, acting downstream of ABA (Desikan et al., 2002; Neill et al., 2002; Garcia-Mata et al., 2003; Bright et al., 2006; Murata et al., 2015).

However, other reports have raised doubts about this proposed role of NO. While ABA treatment reduces the water loss of detached leaves of Arabidopsis, application of SNP to leaves undergoing rapid dehydration does not result in pronounced stomatal closure compared with dehydrating control leaves, and scavenging of NO prevents neither the dehydration-induced nor the ABA-induced stomatal closure of the dehydrating leaves (Ribeiro et al., 2009). These findings suggest that in Arabidopsis, NO signalling is redundant in the ABA-induced dehydration response. Indeed, dehydrating leaves of both the Arabidopsis wild-type and the nia1 nia2 mutant with reduced NO biosynthesis show similar rates of stomatal closure following ABA treatment (Ribeiro et al., 2009). In addition, a critical role for NO downstream of ABA can also be questioned in non-dehydrating leaves, as stomata of the nia1 nia2 noa1-2 triple-mutant with impaired NO biosynthesis are hypersensitive to ABA and the plants show a strong resistance to drought (Lozano-Juste and León, 2010). Overall, this suggests that NO is not an indispensable component in the ABA signalling pathway.

Furthermore, stomata of the NO-overproducing Arabidopsis mutant gsnor1-3 are less sensitive to ABA (Wang et al., 2015b), and NO may function in a negative feedback mechanism to prevent over-activation of ABA signalling in guard cells by S-nitrosylation of Open Stomata 1 (OST1), a kinase that functions as a positive regulator for ABA-mediated stomatal closure. Laxalt et al. (2016) suggested that NO acts both in promoting and arresting the ABA-induced/phospholipid-mediated signals that trigger stomatal closure.

Thus, the role of NO in ABA-induced stomatal closure is far from clearly defined.

Epidermal peels are often used to study the interactions between NO and ABA; however, stomatal responses in peels can differ from those in intact leaf tissues, as shown for calcium (Atkinson et al., 1989), CO₂, and light (Mott et al., 2008; Mott, 2009; Fujita et al., 2013). Lee and Bowling (1992) found a stomatal response to light when an isolated epidermis was incubated in the presence of mesophyll cells or chloroplasts isolated from an illuminated leaf, but there was no response without the mesophyll or in the presence of chloroplasts isolated from dark-adapted leaves. They suggested that guard-cell responses are linked to a product of photosynthetic activity in the mesophyll via a diffusible factor. Lawson et al. (2014) suggested that stomatal movements are coordinated with mesophyll carbon assimilation via a complex interaction between ion channels, sugars, malate, and photosynthesis.

NO-donor compounds such as sodium nitroprusside (SNP) are often used to investigate stomatal responses to NO. This has several disadvantages. NO donors produce decomposition products in addition to NO that can themselves affect cells (Feelisch, 1998). For example, SNP can release iron complexes, disulphides, and cyanide (CN^–) together with NO (Feelisch, 1998; Wang et al., 2002). Interactions between CN^–, NO, and ABA are known in seed dormancy (Bethke et al., 2006) and CN^– will affect photosynthesis (Wodala et al., 2008). Because of these possible side-effects, the results of experiments that use SNP and other NO donors to study stomatal responses should be interpreted carefully. Moreover, precise control of NO dosage (time × concentration) is difficult when NO donors are used.

In light of this, in addition to using SNP (the most common NO donor) in the current study, we also used NO in its pure gas form when investigating stomatal responses to NO in epidermal peels, leaf discs, and intact leaves of Vicia faba. Adding gaseous NO to flow-through gas-mixing systems enables instantaneous administration and removal of NO in order to observe the dynamics of stomatal closure as well as photosynthesis responses. Despite these advantages, to our knowledge there are no studies of NO as an ABA-signalling intermediate in stomatal closure in which it has been supplied as a gas rather than via a donor. Our results showed that in intact leaves the rate of stomatal closure after NO application was about one order of magnitude less than after ABA application. Experiments with epidermal peels were largely affected by factors other than NO, most likely CO₂.

Materials and methods

Plant material and growth conditions

Fava bean (Vicia faba L. cv Longpod) plants were cultivated in a growth chamber at 20±1 °C and 60±5% relative humidity (RH) with a 12-h photoperiod (300 μmol m^–2 s^–1). Plants were grown in soil in pots and were regularly watered with nutrient solution (for details see Aliniaeifard et al., 2014). Fully developed leaves (fourth and fifth in acropetal order) were used for measurements.

Stomatal responses to NO gas, SNP, and ABA in epidermis peels

Epidermal strips were isolated from the abaxial surfaces of fully developed leaves in order to investigate stomatal responses to NO gas (5000 µmol mol ^–1 in N₂; Linde Gas Benelux BV, Schiedam, The Netherlands), sodium nitroprusside (SNP, Na₂[Fe(CN)₅NO].2H₂O; Sigma), and ABA (Sigma). The strips were pre-incubated for 1 h in a stomatal-opening medium (50 mM KCl, 10 mM MES-KOH, pH 6.15, 50 µM CaCl₂ in de-gassed distilled water; Aliniaeifard and van Meeteren, 2014) in Petri dishes placed in a test chamber at 20–25 °C and 30 µmol m^–2 s^–1 photosynthetically active radiation (PAR, 400–700 nm; spectrum given in Supplementary Fig S1A at JXB online). The strips were then transferred to freshly de-gassed stomatal-opening solutions with different concentrations of SNP (50, 200, and 1000 µM) and ABA (100 µM), or to fresh medium alone (control) and kept for 1 h in the test chamber. Images were taken for the measurement of stomatal apertures.

To study the effects of NO gas, a self-built gas-mixing system was connected to a desiccator, into which the Petri dishes with the epidermal strips were placed (12 µmol m^–2 s^–1 PAR inside the desiccator; spectrum as above). The strips were pre-incubated for 1 h in stomatal-opening medium and kept in an atmosphere with a lowered O₂ concentration (20 mmol mol^–1 O₂, 380 µmol mol^–1 CO₂, remainder N₂) in order to diminish the oxidation of NO gas in the next step. Before exposure to NO gas, some strips were taken out of the desiccator as controls and images were taken immediately for determination of stomatal apertures. The gas-mixing system was adjusted to obtain different concentrations of NO in the same O₂ and CO₂ concentrations as during pre-incubation, and the strips were treated for 1 h.

In each experiment, 10–12 strips were taken from two leaves of the same plant (fourth and fifth: see above). For each treatment, three strips were randomly selected and an image was taken of each, from which the pore widths of 10 randomly selected stomata were measured. Various concentrations of NO gas and SNP were tested in separate experiments, each of which included a control (for NO) or control and ABA (for SNP). All experiments were repeated three times.

Images (500 × magnification) were taken using a DXM-1200 digital camera (Nikon) attached to an Aristoplan microscope (Leica). Pore widths were measured using the ImageJ software.

Stomatal responses to NO gas, SNP, and ABA in leaf discs

Leaf discs were cut with a 1.5-cm diameter cork-borer, using the middle of a leaf between the main vein and leaf margin. In each experiment three discs per treatment from leaves of one plant were used. The discs were incubated with their adaxial side upwards for 1 h in Petri dishes filled with the stomata-opening medium and then transferred to dishes with either opening medium alone (control and NO gas treatments) or with different concentrations of SNP (50, 200, and 1000 µM) or ABA (100 µM). To achieve fast, uniform uptake of the solutions, the dishes were twice subjected to a vacuum for 30 s (Filippou et al., 2012). After 90 min, the dishes were placed in a flow-through cuvette under a system for chlorophyll fluorescence imaging (see below) and different concentrations of NO gas were applied. Different concentrations of SNP were also tested in separate experiments. Each experiment included a control (for NO) or control and ABA (for SNP). All experiments were repeated three times.

Confocal laser-scanning microscopy

To verify its presence in guard cells, a fluorescent NO-indicator dye, 4,5-diaminofluorescein diacetate (DAF-2DA; Sigma), was used (Bright et al., 2006). The DAF-2DA solution was prepared in darkness in DMSO on the day of the experiment. Epidermal strips were pre-incubated for 1 h in the stomatal opening medium and 15 µM DAF-2DA was added for 20 min. The epidermal strips were then rinsed with opening medium for 20 min followed by incubation in opening medium alone (control) or with NO gas, SNP, or ABA before imaging using a Zeiss LSM 510-META confocal laser-scanning microscope (excitation 488 nm, emission 515–560 nm). The fluorescence intensity was assessed using ImageJ and for each stoma it was corrected as described by Gavet and Pines (2010):

Corrected fluorescence = Guard cells fluorescence – (Guard cells area × Mean fluorescence background)/1000

where the background reading corresponds to the pixel intensity of areas outside the stoma.

Mapping of PSII photochemical efficiency using chlorophyll fluorescence

Imaging of chlorophyll fluorescence and calculation of PSII efficiency (Φ _PSII) was done as described by Rezaei Nejad et al. (2006), with slight modifications. A gas-mixing system was attached to a flow-through cuvette that was placed in the imaging area of a chlorophyll fluorescence imaging system (FluorCam 700MF, Photon System Instruments, Brno, Czech Republic) and Petri dishes containing leaf discs were placed in the cuvette. Light intensity in the cuvette was 75 µmol m^–2 s^–1 PAR (see Supplementary Fig S1B for spectrum), the temperature was 22±1 °C and RH was 40±3%. Measurements in the cuvette were conducted under 20 mmol mol^–1 O₂, 380 µmol mol^–1 CO₂, and the remainder N₂; these non-photorespiratory conditions were deemed appropriate because, by means of Φ _PSII, they allowed areas where the stomata might be closed to be identified. NO gas was applied as described above. Preliminary experiments showed that 10 min light exposure was sufficient to reach the steady state of Φ _PSII. Hence, images were taken after 10 min and values for F_t and F_m^´ were determined and then averaged over all pixels per leaf disc. Φ _PSII was then calculated as (F_m^´–F_t)/F_m^´ (Rezaei Nejad et al., 2006).

To determine whether changes in Φ _PSII were caused by a decrease in stomatal conductance or by a direct effect of the treatments on photosynthetic activity, images of chlorophyll fluorescence were taken after 5 min exposure to a high CO₂ concentration (50 000 µmol mol ^–1 CO₂, other gases unchanged) following the measurements at 380 µmol mol^–1 CO₂.

Stomatal impressions

After chlorophyll fluorescence imaging, the stomatal apertures of the leaf discs were determined using a silicon rubber impression technique. Silicone (elite HD+ light body; Zhermack, Badia Polesine, Italy) was applied on the abaxial leaf surface and removed after hardening. For positive replicas of the impressions, the surface of the silicone was painted with a clear nail polish, which was removed after hardening. The stomatal apertures of the positive replicas were determined using light microscopy (Rezaei Nejad et al., 2006) for 10 randomly selected stomata per disc.

Gas exchange measurements

Net photosynthetic rate (P_n), leaf intercellular CO₂ concentration (C_i), and stomatal conductance (g_s)were measured on one leaf per plant using a LI-6400 photosynthesis system (LI-COR). Light intensity in the cuvette was 300 μmol m^–2 s^–1 with a 90/10% red/blue mixture (see Supplementary Fig S1C for spectrum), except when testing for the effects of the PAR spectrum on NO-induced stomatal closure, in which case 100% red light was used. RH was set to 60%, the block temperature was 23 °C, and the CO₂ concentration was set at either 50 μmol mol ^–1 or 380 μmol mol^–1. A gas-mixing system was used for the NO treatment. Leaf samples were initially exposed to an atmosphere containing 20 mmol mol^–1 O₂, 380 µmol mol^–1 CO₂, and remainder N₂. The NO treatment was started after the leaves had reached a steady state for g_s. For the ABA treatment, the leaf petioles were cut under water and placed in 10-ml vials containing water before the leaves were positioned in the cuvette of the gas analyser. After reaching a steady state for g_s, ABA was added to the vial to a final concentration of 100 µM.

Statistical analysis

Data were subjected to ANOVA, except for Φ _PSII. Data from one plant were treated as a block in a randomized block design. Treatment means were compared using Fischer’s least-significant difference (LSD) post hoc test. Genstat 17th Edition (VSN International, Hemel Hempstead, UK) was used for statistical analysis. Because the Φ _PSII data showed non-homogeneous variances, they were analysed as a complete randomized design using Welch’s ANOVA followed by the Games–Howel post hoc test, using IBM-SPSS v. 23. In all cases, P<0.05 was considered statistically significant.

Results

Stomata in epidermal peels do not close in response to NO

In a first series of experiments, stomatal responses to NO gas, SNP, and ABA were investigated in epidermal peels. Fumigation with different concentrations of NO (1–100 µmol mol^–1) did not close the stomata (Table 1), and neither did exposure to different SNP concentrations (50–1000 µM) (Table 2). When the epidermal peels were placed in an ABA solution the stomatal apertures were reduced by ~50% (Table 2), indicating that the stomata had not lost their ability to close.

Table 1.

Stomatal aperture (µm) in epidermal strips of Vicia faba after 1 h fumigation with NO gas

Control	NO gas
	1 µmol mo –1	10 µmol mol–1	100 µmol mol–1
9.39 a	10. 06 a
8.64 a		8.45 a
8.54 a			8.15 a

Different concentrations of NO gas were tested in separate experiments (each including a control treatment). Each experiment was repeated three times and in each one the apertures of 30 stomata from three epidermal strips (10 per strip) were measured for each treatment. All strips in each experiment were obtained from the same plant. Means followed by different letters within a row differ significantly (P<0.05) as determined by Fisher’s LSD test.

Table 2.

Stomatal aperture (µm) in epidermal strips of Vicia faba in response to the NO-donor SNP (sodium nitroprusside) and ABA

Control	SNP			ABA (100 µM)
	50 µM	200 µM	1000 µM
7.37 b	7.60 b			4.09 a
7.56 b		7.65 b		4.01 a
7.19 b			7.78 b	3.25 a

Different concentrations of SNP were tested in separate experiments (each including a control and ABA treatment). All other details are as described in Table 1. Means followed by different letters within a row differ significantly (P<0.05) as determined by Fisher’s LSD test.

In light of the unexpected absence of SNP- and NO-induced stomatal closure, a cell-permeable fluorescent detector of NO (DAF-2DA) was applied to the epidermal strips together with the treatments. The resulting fluorescence indicated that application of NO gas, SNP, and ABA all resulted in significant accumulation of NO in guard cells (Fig. 1), while only stomata in the ABA treatment were (partly) closed. Exposure to 10 µmol mol^–1 NO gas resulted in an increase in fluorescence that was comparable to application of 100 µM ABA. Exposure to 100 µmol mol^–1 NO gas and treatment of the strips with 1 mM SNP solution resulted in fluorescence levels that were at least four times higher than those in the ABA treatment.

Fig. 1.

NO is accumulated in guard cells of Vicia faba after exposure of epidermal strips to ABA, NO gas, or the NO-donor sodium nitroprusside (SNP). Epidermal strips were loaded with the fluorescent NO detector DAF-2DA and treated with ABA (100 µM), nitric oxide in its pure gas form (NO; 10 µmol mol^–1 or 100 µmol mol^-1), or SNP (1000 µM). The presence of NO in the guard cells is measured as fluorescence. Data are means (±SE) of 10–15 replicates. Different letters indicate significant differences as determined by Fisher’s LSD test (P<0.05).

These results indicated that even when NO was clearly present, it was not a critical component in ABA-induced stomatal closure in isolated epidermal tissues of Vicia faba.

Stomata in leaf discs respond differently to NO gas, SNP, and ABA

To test whether the lack of stomatal response to NO in epidermal peels was due to the absence of mesophyll tissue, the efficiency of photosystem II (Φ _PSII) and stomatal pore width were examined in leaf discs. Under the non-photorespiratory conditions at which it was measured (20 mmol mol^–1 O₂), Φ _PSII relates to stomatal conductance (g_s) if CO₂ availability is the only factor limiting photosynthesis. To determine the contribution of g_s to the changes in Φ _PSII under the different treatments, measurements of Φ _PSII were taken at physiological (380 µmol mol^–1) and at extremely high (50 000 µmol mol^–1) CO₂ concentrations: at the latter concentration, g_s does not limit photosynthesis.

NO gas (10 µmol mol^–1 and 100 µmol mol^–1), SNP, and ABA all caused significant reductions in Φ _PSII in leaf discs (Fig. 2), and the higher SNP concentrations (200 µM and 1000 µM) resulted in similar or larger decreases in Φ _PSII as observed with ABA (Fig. 2B, C). Exposure to high CO₂ recovered Φ _PSII in ABA-treated discs (91–93% of control) but did not recover it under NO- or SNP-treatments. This indicated that ABA decreased Φ _PSII mainly because of stomatal closure, while in the case of NO gas and SNP linear photosynthetic electron transport was inhibited by non-stomatal factors. Exposure to control air (0 µmol mol^–1 NO and 380 µmol mol^–1 CO₂) for 10–15 min after NO gas treatment recovered Φ _PSII to ~90% of its initial value (‘R’ in Fig. 2A).

Fig. 2.

Efficiency of photosystem II (Φ _PSII) of Vicia faba leaf discs in response to treatment with NO gas, sodium nitroprusside (SNP), or ABA. Measurements were made with 75 µmol m^–2 s^–1 PAR, and under 20 mmol mol^–1 O₂ plus the gases indicated and the remainder was N₂. Data are means (±SE). Different NO and different SNP concentrations were tested in separate experiments. Each experiment was repeated three times with three replicates per treatment in each one. (A) Φ _PSII after exposure to various concentrations NO gas for 10–15 min. Measurements were made under 380 µmol mol^–1 CO₂ except for the NO+CO₂ treatment (high CO₂) where 50 000 µmol mol^–1 CO₂ was used. Following the NO+CO₂ treatment, the discs were exposed to 380 µmol mol^–1 CO₂ without NO for a 10–15-min recovery period, R. Different letters indicate significant differences as determined by Fisher’s LSD test (P<0.05). (B) Representative images of Φ _PSII in the leaf discs as represented by false colours. (C) Φ _PSII after treatment with different concentrations of SNP or 100 µM ABA. Measurements were made under 380 µmol mol^–1 CO₂ (open columns) or under 50 000 µmol mol^–1 CO₂ (filled columns). Different letters indicate significant differences as determined by Welch’s ANOVA followed by the Games–Howell post hoc test (P<0.05).

Given that the effects of NO and SNP on Φ _PSII were not related to the stomata, impressions of the leaf discs were made to measure stomatal apertures directly. Some small, concentration-dependent effects on stomatal aperture were observed as a result of exposure to the three concentrations of NO gas (Table 3). At 1 µmol mol^–1, the apertures were significantly wider compared with the control, at 10 µmol mol^–1 there was a small, but significant reduction in aperture, and at 100 µmol mol^–1 there was no significant effect of NO gas. In contrast with epidermal strips, in leaf discs the stomatal aperture was decreased by SNP by ~50% for all three concentrations tested (Table 3). ABA treatment resulted in a reduction in aperture of ~90% compared with the control, which was much more than the ~50% reduction seen in epidermal strips (Table 2) and a significantly larger reduction compared to the SNP treatments of the discs (Table 3). In addition to the effect on mean aperture, the frequency distributions of pore sizes were also different between ABA and SNP concentrations (Supplementary Fig. S2). ABA treatment resulted in a skewed distribution towards smaller apertures with a median pore size (0.64 µm) of about half of the mean (1.20 µm), while the three SNP treatments had distributions that were about normal (median values 6.3, 4.9, 5.3 and means 6.3, 4.9, 5.8 µm for 50, 200, and 1000 µM, respectively).

Table 3.

Stomatal aperture (µm) in leaf discs of Vicia faba in response to NO gas (10 min fumigation), SNP, and ABA

Control	NO gas			SNP			ABA (100 µM)
	1 µmol mol–1	10 µmol mol–1	100 µmol mol–1	50 µM	200 µM	1000 µM
11.71 c	12.69 d			6.27 b			1.52 a
11.53 d		10.87 c			4.91 b		1.32 a
10.84 c			11.05 c			5.84 b	0.76 a

Different concentrations of NO and SNP were tested in separate experiments (each including a control and ABA treatment). All other details are as described in Table 1. Means followed by different letters within a row differ significantly (P<0.05) as determined by Fisher’s LSD test.

These results indicated that stomata in leaf discs responded differently to the various treatments than stomata in epidermal peels. The effects between NO gas, SNP, and ABA were also different.

Is photosynthesis involved in the stomatal response to NO?

To examine whether the mesophyll tissue in the leaf discs influenced the stomatal response to NO through photosynthetic activity, chlorophyll fluorescence and gas exchange were measured simultaneously in intact leaves subjected to NO or ABA treatment. During 1 h of exposure to 10 µmol mol^–1 NO, stomatal conductance to water vapour (g_s) gradually decreased by 30%, and then slowly recovered after the NO was flushed out (Fig. 3A). With ABA feeding (100 µM), g_s had decreased by 37% within 15 min, and it further decreased by 67% after 60 min. The dynamic behaviour of net photosynthetic rate (P_n) and intercellular CO₂ concentration (C_i) differed from that of g_s when the leaves were exposed to NO (Fig. 3B, C). P_n dropped sharply to a very low level within minutes of exposure and then remained low for the rest of the exposure time. With ABA feeding, P_n showed a logistic pattern of decrease: it declined slowly during the first 15 min, followed by a faster decrease in the next 15 min. When NO was flushed out, P_n recovered to 65, 87, and 93% of its original level after 3, 30, and 60 min of exposure to NO-free air, respectively. C_i increased sharply within 5 min of exposure to NO gas, remained at the same high level during the rest of the exposure period, and decreased quickly to its original level after removal of the gas (Fig. 3C). The time course of C_i in response to ABA treatment was similar to that of P_n.

Fig. 3.

Effects of NO gas and ABA on gas-exchange parameters in leaves of Vicia faba. Time-courses of (A) stomatal conductance for water vapour (g_s), (B) net photosynthetic rate (P_n), and (C) intercellular CO₂ concentration (C_i) of leaves in response to 10 µmol mol^–1 NO gas or 100 µM ABA After 60 min, the NO gas was flushed out with NO-free air (arrows). Measurements were performed in an atmosphere containing 20 mmol mol^–1 O₂, 380 µmol mol^–1 CO₂, and the remainder N₂. Data are means (±SE), n=14.

These results indicated that ABA induced closure of stomata that resulted in a decrease in photosynthesis due to restricted influx of CO₂, whilst NO directly inhibited photosynthesis (confirming the Φ _PSII measurements in leaf discs), which resulted in an instantaneous increase in C_i that was followed by stomatal closure.

To investigate a possible effect of NO on g_s via C_i, we decreased C_i experimentally by lowering the CO₂ concentration (in combination with NO) in the ambient gas flow. At a [CO₂] of 380 µmol mol^–1, g_s increased during the first 9–15 min of NO application (Fig. 4A, C, Phase 1a), whilst photosynthesis sharply declined immediately after NO application (Fig. 4B). C_i initially increased, as would be expected from the inhibition of photosynthesis. After about 15 min of NO application, the stomata started to close (Fig. 4A, C, Phase 1b). The rate of closure had the form of an exponential time curve; g_s changed at a mean rate of –0.10 mmol m^–2 s^–2 during the period 15–30 min (Fig. 4A, C, Phase 1b), followed by a rate of –0.02 mmol m^–2 s^–2 during the following 2-h period (Phase 1c). The external [CO₂] was then lowered (20 µmol mol^–1, Phase 2), and in response C_i decreased sharply to 23–25 µmol mol^–1 CO₂ (Fig. 4B) whilst stomatal aperture increased very slowly (at a rate of 0.005 mmol m^–2 s^–2). The NO gas was then removed (Phase 3), and in response the rate of stomatal opening increased (Fig. 4A, C) while P_n remained at a low level due to the low [CO₂] (Fig. 4B). These results indicated that high C_i had a significant but only small negative effect on stomatal aperture, while removal of NO resulted in much faster stomatal opening compared to lowering C_i alone. For comparison, g_s decreased from ~5–30 min after the start of ABA application (Fig. 4D) at a rate of about –0.30 mmol m^–2 s^–2 during the linear part of the logistic time curve, and thus stomata closed much faster after ABA treatment than after NO treatment (compare Fig. 4A and 4D).

Fig. 4.

(A) Time-courses of stomatal conductance (g_s) for three individual Vicia faba leaves from different plants (shown in different colours). After g_s reached a steady-state under 380 µmol mol^–1 CO₂ (time 0), NO was applied at 10 µmol mol^–1 (+NO +380 CO₂; Phase 1a–c). After ~150 min (indicated by a solid arrow for each leaf), the CO₂ concentration was lowered to 20 µmol mol^–1 (+NO +20 CO₂; Phase 2). At ~190–210 min (indicated by an open arrow for each leaf), NO was flushed out (–NO +20 CO₂; Phase 3). (B) Typical time-courses of net photosynthetic rate (P_n; solid line) and intercellular CO₂ concentration (C_i; dashed line) for one of the leaves in (A). All measurements were performed in an atmosphere with 20 mmol mol^–1 O₂ and the remainder N₂. (C) Mean changes in g_s during the phases indicated in (A). The changes were calculated as the slope of linear regression for g_s versus time during each phase. Because g_s initially increased, followed by an exponential decrease during NO exposure under normal CO₂, Phase 1 was sub-divided into 1a, 1b, and 1c. Data are the means (±SE) of the three leaves. (D) Time-courses of g_s for three individual leaves (shown in different colours) after application of 100 µM ABA. ABA application to the cut petioles started at time 0.

Confirming that stomatal closure during NO treatment was not caused by increased C_i, photosynthesis was reduced by low CO₂ before the NO treatment began. At an ambient [CO₂] of 50 µmol mol^–1, C_i was ~40 µmol mol^–1 and photosynthesis was decreased to ~2 µmol m^-2 s^-1 (Fig. 5). Stomata were opened wide (g_s=0.46 mol m^–2 s^–1), probably because of the low C_i. When NO was added, photosynthesis dropped to ~0 µmol m^–2 s^–1, and C_i increased to 50 µmol mol^–1. About 10 min from the start of the NO treatment, g_s started to decrease quickly and continued to do so at the same rate as seen previously (about –0.13 mmol m^–2 s^–2; Phase 1b in Fig. 4A, C) for the next ~60 min.

Fig. 5.

Time-courses of stomatal conductance for water vapour (g_s), net photosynthetic rate (P_n), and intercellular CO₂ concentration (C_i) of a Vicia faba leaf kept under 50 µmol mol^–1 CO₂, 20 mmol mol^–1 O₂, and the remainder N₂. Logging was started when g_s reached a steady state (after ~90 min in the apparatus), and 10 µmol mol^–1 NO was added after 2 min.

These results indicated that NO by itself induced a decrease in stomatal aperture, while an increase of [CO₂] in the sub-stomatal cavities had only a limited effect on aperture.

Discussion

NO and mesophyll-driven signals for stomatal closure: sub-stomatal [CO₂] is not the main factor

In our experiments, application of ABA to epidermal peels induced stomatal closure, whereas NO (gaseous or via the NO-donor SNP) did not (Tables 1, 2). However, exposing intact leaves to NO resulted in a gradual decrease of stomatal conductance over a period of 1–2 h (Figs 3–5), suggesting that NO interacted with mesophyll-driven signals for stomatal closure. Feedback of photosynthetic activity on stomatal aperture is of central importance for plants. Light acts on stomatal opening through two parallel signalling pathways: blue light specifically acts via phototropins in the guard cells, whereas red light induces opening via photosynthesis (Shimazaki et al., 2007). Coordination between mesophyll photosynthesis and stomatal conductance is a well-known phenomenon (Lawson and Blatt, 2014). When leaves (intact or as discs) were exposed to NO, photosynthesis dropped instantaneously (Figs. 2A, 3B, 4B) and was not recovered by adding a very high concentration of CO₂. Thus, the reduction was not due to stomatal closure (as in the case of ABA treatment). After removal of the NO gas, photosynthesis quickly reverted to its original rates (Figs. 2A, 3B). Decreases in Φ _PSII and photosynthesis as a result of fumigation with NO have been reported previously (Clyde Hill and Bennett, 1970; Procházková et al., 2013), and it reversibly inhibits photophosphorylation and ATP synthesis in thylakoid membranes of spinach chloroplasts (Takahashi and Yamasaki, 2002). Concurrently with the sharp decline in P_n in response to NO, there was a steep increase in C_i (Figs. 3, 4B), probably because CO₂ was no longer consumed by photosynthesis. In contrast to the instantaneous changes in P_n and C_i, g_s gradually decreased over 1–2 h of NO exposure. When the NO gas was removed, P_n and C_i quickly recovered to their original levels, and this was followed by a gradual increase of g_s (Figs. 3, 4). These results at first suggested that stomatal closure by NO may have been induced by increased sub-stomatal [CO₂], as closing of stomata due to high C_i is a well-known phenomenon (Mansfield, 1965; Allaway and Mansfield, 1967).

However, when C_i was additionally lowered by a low ambient [CO₂] during the NO treatment, only minor effects on stomata were observed (Fig. 4), indicating that an increase in C_i caused by suppression of photosynthesis via NO was not the main factor. This was confirmed by decreases of g_s after NO treatment when C_i was already low before the treatment started due to low ambient [CO₂] (Fig. 5). Therefore, a factor other than high sub-stomatal [CO₂] must be involved in mesophyll-driven signals of stomatal closure that interact with NO. This could be further examined using transformants with reduced Rubisco (Quick et al., 1991): because such plants have low P_n whilst g_s is still at normal levels, NO would not cause strong increases in C_i. Stomatal closure induced by elevated CO₂ in tomato has been shown to be dependent on NO production in guard cells in an ABA-independent manner (Shi et al., 2015).

NO- and mesophyll-driven signals for stomatal closure: no relationship with CO₂ assimilation

Lawson et al. (2014) argued that mesophyll-driven signals coordinate stomatal movements with mesophyll carbon assimilation via a complex interaction between ion channels and metabolites derived from photosynthesis. However, our results showed that NO also affected stomatal aperture when CO₂ assimilation was around zero due to low ambient CO₂ (Figs. 4, 5). A current hypothesis is that, rather than C_i, it is the redox state of the chloroplastic plastoquinone (Q_A) pool that is the photosynthetic signal that interacts with the stomatal guard cells to balance g_s with P_n (Busch, 2014; Głowacka et al., 2018; Kromdijk et al., 2019). Limitation of CO₂ supply to the Calvin–Benson cycle affects the photosynthetic electron sink capacity in the chloroplasts and the Q_A pool may become more reduced (Quick et al., 1991), resulting in an increase of g_s. NO reversibly blocks linear electron flow between PSII and PSI (Takahashi and Yamasaki, 2002), which means that there is no plastoquinone reduction; this will result in a more oxidized Q_A pool (Busch, 2014) and probably in a decrease of g_s. Transgenic plants with differing Q_A redox states resulting from differing expression levels of Photosystem II Subunit S (Głowacka et al., 2018) could be used to provide insights into the role of mesophyll-driven signals for NO signalling in stomatal closure and the inter-relationship of ABA and NO.

Dual dose–response relationship between NO and stomatal aperture

NO gas showed a dual dose–response effect on stomatal aperture. After 10 min exposure of leaf discs to 1 µmol mol^–1 NO the stomatal aperture was increased, whilst exposure to 10 µmol mol^–1 resulted in smaller apertures than the control (Table 3). An increase in g_s was observed during the first 9–15 min of exposure of leaves to 10 µmol mol^–1 NO, followed by a decrease thereafter (Fig. 4). This dual dose-dependent effect may be the result of two opposite effects of NO on g_s: an increase due to S-nitrosylation of OST1, as reported by Wang et al. (2015b), and a decrease because of a more oxidized Q_A pool, as discussed above. It would therefore be expected that the positive effect of NO on g_s at low doses would be absent in ost1 loss-of-function mutants (Mustilli et al., 2002).

The effects of NO on stomatal closure in epidermal peels are controversial

The lack of stomatal closure that we observed when exposing epidermal peels to SNP or NO gas (Tables 1, 2) contradicts previous results for epidermal fragments of Pisum sativum, Salpichroa organifolia, Tradescantia species, and Vicia faba (García-Mata and Lamattina, 2001; Desikan et al., 2002; Neill et al., 2002; She et al., 2004), whilst the response to ABA in our peels was similar or greater to what has previously been reported in V. faba (Iwai et al., 2003; Yan et al., 2007; Arve et al., 2014). Hettenhausen et al. (2012) also observed no reduction in stomatal aperture when applying 100 μM SNP in Nicotiana attenuate, and Sakihama et al. (2003) even found an increase in aperture in V. faba leaf peels treated with the NO-donor S-Nitroso-N-Acetyl-D,L-Penicillamine (SNAP). These contradictions could be related to variations in the procedures of the different experiments: together with some other studies, we used a degassed (CO₂-free) MES/KCl stomata-opening medium as the incubation solution, while other studies did not degas the medium, or subjected it to shaking. Furthermore, the exposure time to SNP/SNAP before the measurement of stomatal aperture varied from 20–60 min in our experiments and those of Sakihama et al. (2003) and Hettenhausen et al. (2012), and from 1–3 h in the experiments of García-Mata and Lamattina (2001, Desikan et al. (2002), and She et al. (2004). Wang et al. (2015a) showed that elevated CO₂ concentrations can induce stomatal closure in epidermal strips within about 2 h. It is likely that removing dissolved CO₂ from the opening medium by degassing and/or the length of the period in which CO₂ can be produced by epidermal tissue or re-dissolve from the surrounding air into the medium are factors that will influence stomatal aperture. To determine the importance of the time between degassing the bathing solution and the observations of the epidermal peels, we performed an additional experiment in which stomatal apertures were measured 1 h and 3 h after degassing, with ABA (100 µM) and SNP (200 µM) being applied to the strips immediately after degassing (Supplementary Fig. S3A). Over the 2 h-period between the measurements, the apertures decreased in the control (12%) and the SNP (19%) treatments. In the ABA treatment, the mean aperture was already small after 1 h (4.0 µm) and did not decrease over the next 2 h. In addition, the shape of the frequency distributions of stomatal apertures differed between the ABA and SNP treatments (Supplementary Fig S3C). When the bathing solutions were not degassed there was no decrease in stomatal aperture for the control, and the SNP treatment actually showed a small but significant opening of the stomata between 1–3 h (Supplementary Fig S3B).These results demonstrated the importance of taking precautions to prevent differences and/or changes in [CO₂] in the bathing solution of epidermal peels.

Different outcomes between NO gas and SNP treatments of leaf discs

When leaf discs were used, NO gas showed a concentration-dependent effect on stomatal aperture, while the three SNP concentrations all resulted in a decrease in stomatal aperture of ~50% compared to the control discs (Table 3). There are several possible explanations for these different outcomes between treatment with NO gas and SNP, as follows.

First, SNP-induced stomatal closure is the result of other products in addition to the NO that is released, including CN⁻, as has been demonstrated for some other physiological processes. For example, gases generated by SNP have been shown to increase Arabidopsis seed germination from 0% in controls (exposed to water vapour) to 90%, while germination after exposure to NO gas is ~30% (Libourel et al., 2006). Wodala et al. (2010) demonstrated that application of an NO-scavenger (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide) only partially restored SNP-induced changes in Φ _PSII, which suggests that CN⁻ and NO are both responsible for the effect of SNP on Φ _PSII. It has previously been reported that CN⁻ affects Φ _PSII (Jones et al., 1999), and Zelitch (1965) mentions that CN⁻ inhibits stomatal opening in the light and prevents closure in the dark. These results indicate that SNP is an unsuitable NO donor for studies on stomatal closure.

Second, in our study NO treatment was performed for 10 min while SNP was applied for 90 min before the stomatal apertures were measured. The strong inhibition of Φ _PSII after 10 min of NO treatment demonstrated that penetration of the gas into the leaf discs was not a limiting factor during this time. However, as shown in Fig. 4, g_s increased during the first 9–15 min of NO application to leaves, and stomata only started to close around the end of this period. So, the fact that stomatal closure was not observed in the leaf discs after treatment with NO was very likely due to the short exposure time.

Third, during the treatment with NO the leaf discs in the flow-through cuvette of the chlorophyll fluorescence imaging system were exposed to red light, while during the SNP treatments the discs were exposed to light from white fluorescent tubes (spectra in Supplementary Fig. S1B and S1A, respectively). It is known that stomata open in response to weak blue light under a background of strong red light and that ABA inhibits blue light-dependent opening (Shimazaki et al., 2007). It is also known that NO inhibits stomatal opening specific to blue light (Zhang et al., 2007). The g_s time-courses shown in Figs 3 and 4 were obtained under a spectrum of 90% red plus 10% blue light. The possibility cannot be excluded that NO may be less effective in stomatal closure under 100% red light.

To get a better understanding of the limited effect of NO in stomatal closure that was found in leaf discs (Table 3), additional measurements were done in which the discs were exposed to NO gas for 2 h under an irradiance of 90% red plus 10% blue. At the end of this treatment, mean aperture was decreased by 50% compared with the control (Supplementary Fig. S4A; the bathing solution was not degassed at start to prevent an increase of [CO₂] by ambient CO₂). Without NO, the aperture increased under the same light and CO₂ conditions over the same time period (Supplementary Fig. S4B). The effect of NO after 2 h resembled the effect of SNP on leaf discs (Table 3). In addition, the dynamics of g_s of intact leaves exposed to NO were recorded under 100% red or 90% red plus 10% blue light. This demonstrated that the spectral composition interacted with the NO effect on g_s (Supplementary Fig. S5): when blue light was added to the red light, the start of stomatal closing was delayed by 6 min, but the rate of closure was about twice as fast.

Taken together, it is likely that the main reason for the limited and dual effects of NO on stomatal aperture (Table 3) were due to the relatively short exposure time of the leaf discs.

Is NO a critical key factor in ABA-induced stomatal closure?

A critical role for NO in ABA-induced stomatal closure has been proposed downstream of ABA (García-Mata and Lamattina, 2001; Neill et al., 2002); however, multiple conflicting results for this role have been published (see Introduction). Most data on NO production and function rely on experimental approaches based on the application of NO donors and scavengers. Inhibition of ABA-induced stomatal closure by treatment with 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) as a NO scavenger has often used as an argument for a key role of NO in ABA-induced stomatal closure. Although c-PTIO reacts rapidly with NO, its effects are diverse and its specificity has been questioned (Pfeiffer et al., 1997). Bethke et al. (2006) demonstrated that c-PTIO prevents both cyanide- and NO-stimulated seed germination in Arabidopsis. In our experiments, the effects on stomatal closure as well as their dynamics differed between ABA and NO gas treatments. The effect of ABA was fast and was seen in epidermal peels as well as in intact leaves, while the effect of NO gas was slow (delayed and with a low rate) and was affected by the type of tissue sampled (epidermis peel or with mesophyll present) and by exposure time. In epidermis peels, none of the concentrations of NO gas that we used (1, 10, and 100 µmol mol^–1) resulted in stomatal closure, while floating the peels in an ABA solution decreased the aperture to ~50% of that in the controls. As discussed above, the effects of SNP in epidermal peels in previous studies could be (partly) related to an increase of [CO₂] in the bathing solution during long exposure times. When mesophyll was present (leaf discs or intact leaves), NO had a dual dose–response effect with low concentrations or short exposures increasing stomatal aperture. Upon exposure to 10 µmol mol^–1 NO, stomatal conductance started to decrease after 10–15 min (depending on the spectral composition of the light; Supplementary Fig. S5), with a rate of ~20% of that of ABA-induced stomatal closure. On the other hand, after application of ABA via cut leaf petioles, stomatal conductance decreased after ~5 min and reached its minimal value after ~30 min. These results indicate that NO affects stomatal aperture but that it is not the critical key factor in rapid responses of stomata to ABA.

Conclusions

Despite NO being ubiquitously produced in plants, many details regarding its generation and signalling pathways are still unclear (Domingos et al., 2015). As a reactive oxygen species, NO is an important signalling molecule under various abiotic stresses. Our results suggest that NO is not the critical key factor in ABA-induced rapid stomatal closure but that it acts independently of ABA, thus implicating NO as an effector of stomatal closure in its own right, especially in the presence of mesophyll tissue. This points to an alternative mechanism that plants use to reduce stomatal aperture to restrict water loss under stressful conditions.

Supplementary data

Supplementary data are available at JXB online.

Fig. S1. Spectra of the various light sources used in the different experiments.

Fig. S2. Frequency distribution of stomatal apertures in leaf discs as affected by NO gas, SNP, and ABA.

Fig. S3. Interactions between degassing the bathing solution, SNP treatment, and time since degassing on stomatal apertures in epidermal peels.

Fig. S4. Stomatal apertures in leaf discs after 2 h treatment with 10 µmol mol^–1 NO gas.

Fig. S5. Interaction of spectral composition and NO on stomatal conductance and photosynthesis.

Glossary

Abbreviations:

ABA

abscisic acid

C_i

leaf intercellular CO₂ concentration

CN^–

cyanide

c-PTIO

2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide

DAF-2DA

4,5-diaminofluorescein diacetate

F_m´

maximal fluorescence level in light-adapted leaves

F_t

steady-state yield of fluorescence in the light

ΦPSII

photochemical efficiency of PSII

g_s

stomatal conductance

NO

nitric oxide

OST1

Open Stomata 1

P_n

net photosynthesis rate

QA

chloroplastic plastoquinone

SNAP

S-Nitroso-N-Acetyl-D,L-Penicillamine

SNP

sodium nitroprusside