Fructose Regulation of Stomatal Movement and its Effects on the Photosynthetic System in Vicia faba L.

Broad bean (Vicia faba L.) belongs to the legume family and are known for their high levels of protein, nutrient and bioactive compounds that are commonly used in food and feed (Dewangan et al., 2022). Sugar not only provides energy for plant growth, but also plays a role as a signaling molecule in the life cycle of plants (Rolland et al., 2006). ROS and NO are the core signals that regulate stomatal movement (Nazareno and Hernandez, 2017). Previous studies have proved that glucose and mannose can promote stomatal closure in broad beans through hydrogen peroxide (H₂O₂), NO and Ca²⁺-based signaling (Li et al., 2018). Sucrose can reduce stomatal opening and is easily decomposed by sucrase into glucose and fructose (Kelly et al., 2013). Studies have shown that fructose can also inhibit stomatal opening (Dittrich and Mayer, 1978), but details of the signaling mechanism and regulatory pathways are not well-understood.
       
Both the external environment and internal signaling molecules can regulate stomatal movement. NO can stimulate the release of Ca²⁺ from calcium pools, resulting in an increase in free cytoplasmic Ca²⁺ and inhibition of inward rectifying K⁺ channels. As a result, the plasma membrane localized anion channels in guard cells are activated, consequently leading to an anionic outflow. Simultaneously, it promotes the synthesis of phospholipid acid and enhances the ROS levels, causing stomatal closure (Sun et al., 2019). However, it is not known whether fructose induces ROS production in guard cells and information regarding its mechanism of production is also lacking.
       
Exogenous sugar can increase the photosynthesis-based substrate supply in plants, help the plants maintain a stable photosynthetic performance, enhance their tolerance under stress and reduce damage to the photosynthetic machinery (Sun et al., 2019). Previous studies have shown that glucose and sucrose can reduce stress-induced damage and inhibition and increase Pn and photosystem II (PSII)-based activity. PSII is highly susceptible to stress and is closely related to various photosynthesis-related physiological processes (Zhang et al., 2020a).
 
In this experiment, our objectives were to (1) demonstrate whether fructose may increase ROS production in guard cells inducing stomatal closure; (2) examine whether the exogenous application of fructose may regulate photosynthesis and chlorophyll fluorescence characteristics.

Plant materials and growth conditions
 
The seeds of the broad bean (Vicia faba L. cv. Da qing pi) were purchased from Shanghai NongLe Cultivation Co., Ltd., China. The experiment began in September 2022 and was carried out in the plant cultivation room and laboratories of Shaanxi University of Chinese Medicine. The seeds were sterilized in 75% ethanol for 30 min and then washed with water 5 to 6 times. Sterilized seeds were soaked in water for 48 h and then grown in a growth chamber at temperatures ranging from 20-25°C, under conditions of 70% relative humidity (RH), photosynthetically active radiation (PAR) of 300 µmol m^–2 s^–1 and a photoperiod of 14 h light/10 h dark, with daily watering schedule. Approximately 4 weeks after seeding, the completely expanded leaves were cut and the abaxial epidermis was peeled off.
 
Chemical reagents
 
Molecular probe 2’,7’ dichlorofiuorescin diacetate (H₂DCF-DA from Sigma-Aldrich, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO from Amresco, Solon, OH, USA) to produce a stock solution, which was aliquoted. Diphenylene iodonium chloride (DPI), salicylhydroxamic acid (SHAM), reduced glutathione (GSH), 2 (4 carboxyphenyl) 4, 4, 5, 5 tetramethylimidazoline 1 oxyl 3 oxide (c-PTIO), catalase (CAT, bovine liver), NG-nitro-L-arginine methyl ester (L-NAME) and 2 (N-morpholino) ethanesulfonic acid (MES) were all obtained from Sigma-Aldrich (St. Louis, MO, USA). Unless stated otherwise, the remaining chemicals were bought from companies based in China. All the chemicals used in this study were of the highest analytical grade available.
 
Stomatal bioassay
 
Stomatal bioassay experiments were performed as described with slight modifications. The details can be obtained from Gao et al., (2013). The strips were soaked in MES-KCl buffer solution (10 mM MES, 50 mM KCl, 100 mM KOH, pH= 6.1) for 2 h to induce stomatal opening and then transferred to fructose solution having different concentrations (0, 100, 125, 135 and 150 mM, respectively) for 30 min incubation in light. The stomatal aperture sizes were determined using digital images captured by a microscope (BX53, Olympus, Tokyo, Japan).
 
Measurement of ROS production in guard cells
 
The details can be obtained from Gao et al., (2013). The intensity of fluorescence was quantified using the Image Pro Plus 6.0 software (Media Cybernetics, Silver Springs, MD, USA). In each treatment, we recorded the measurements from three epidermal strips originating from different source plants. The experiment was repeated three times. The data represent the means of 120 measurements ± SE.
 
Gas exchange measurements
 
The 4-week-old plants were sprayed with 125 mM fructose dissolved in water and the controls were sprayed with water. At 3 h, 24 h and 48 h after treatment, leaf gas exchange was measured using a portable photosynthetic apparatus CIRAS-3 (PP Systems, UK) (Li et al., 2018). The carboxylation efficiency (CE) was calculated as Pn/Ci (Teramura et al., 1990). The stomatal limit value LS was calculated as 1 Pn/ca. The experiment was repeated four times (n= 4).
 
Chlorophyll fluorescence measurements
 
The chlorophyll fluorescence parameters were determined using a Pocket PEA fluorimeter (Plant Efficiency Analyzer PE 321JL, Hansatech Instruments Ltd., King’s Lynn Norfolk, UK) (Guha et al., 2013). The 4-week-old plants were sprayed with 120 mM fructose dissolved in water and the controls were sprayed with water. Gas exchange estimations were measured on the same leaves for which the chlorophyll fluorescence induction (OJIP) parameters were conducted. Leaves used for chlorophyll fluorescence assay were collected at 3 h, 24 h and 48 h after treatment. After full dark adaptation for 30 min, the indices were determined. Leaves at the same stages of photosynthesis were selected for the estimation. The chlorophyll fluorescence parameters were: average absorbed photon flux per PSII reaction center (ABS/RC), dissipated energy flux per PSII (DI_O/RC), maximum trapped excitation flux per PSII (TR_O/RC), electron transport flux from Q_A to Q_B (ET_O/RC), electron transport flux until PSI acceptors per PSII (RE_O/RC), performance index for energy conservation from photons absorbed by PSII antenna to the reduction of PSI acceptors (PI_total), performance index for energy conservation from photons absorbed by PSII antenna to the reduction of Q_B (PI_ABS), maximum quantum yield of primary PSII photochemistry (φ_PO), probability of electron transport beyond QA– (Ψ_EO), quantum yield of the electron transport flux from Q_A to Q_B (φ_EO), efficiency with which an electron from Q_B is transferred to PSI acceptors (δ_RO) and quantum yield of the electron transport flux till the electron reaches the PSI electron acceptors (φ_RO), respectively. The experiment was repeated six times (n = 6).
 
Statistical analysis
 
Standard error (SE) for each treatment was calculated. Differences among treatments were compared using least significant difference (LSD, P<0.05) test. All statistical analyses were performed using the SPSS 26.0 package (SPSS, Chicago, USA).

Fructose-induced stomatal closure in epidermal strips
 
Fructose is a soluble sugar found in higher plants, which has high superoxide scavenging capacity (Bogdanović et al., 2008). In this study, after full illumination, the stomata opened and their apertures increased to approximately 10.09 µm in the control group (Fig 1A). After the application of 100 mM, 125 mM and 135 mM fructose, respectively, for 30 min under light, stomatal apertures were reduced by 9.79% (P<0.01), 30.27% (P<0.01) and 3.58% (P>0.05), respectively, compared to the control (Fig 1A). However, stomatal aperture increased by 6.28% (P<0.05) under 150 mM fructose treatment (Fig 1A). With the increase of fructose concentration, stomatal opening decreased first and then increased. The results showed that fructose-induced stomatal closure in the epidermal strips of the broad bean was concentration-dependent. The effects of fructose at higher concentrations (135 and 150 mM) were less, which rules out an osmotic role of sugars in mediating stomatal closure (Li et al., 2016). The time course of stomatal closure in broad beans is shown in Fig 1B. Stomatal aperture decreased rapidly with the treatment time and reached the minimum value at 30 min, showing a significant reduction by 23.62% (P<0.05) compared with that in the control. Our results suggested that fructose induce stomatal closure in a dose- and time-dependent manner.
 

 
H₂O₂ and NO participate in fructose induced stomatal closure in guard cells
 
H₂O₂ exists in cells and tissues as a molecule form of ROS (Govindaraj et al., 2017). As shown in Fig 2, when compared to the 125 mM fructose treatment, the CAT, DPI, GSH and SHAM-based treatments increased stomatal aperture by 66.64%, 46.90%, 27.19% and 27.03%, respectively. The implication is that H₂O₂ is involved in fructose-induced stomatal closure. In this study, CAT was found to completely reverse fructose-induced stomatal closure. CAT is membrane-impermeable with efficient catalytic and regulatory properties and can degrade H₂O₂ into water and oxygen (Sharma and Ahmad, 2014), implying that sugar-induced ROS production occurs exclusively outside the plasma membrane of guard cells (Li et al., 2016). Additionally, ROS production can be inhibited by both DPI and SHAM to similar extents, which suggested that both NADPH oxidase and peroxidase activity can be responsible for fructose-triggered oxidative burst (Hemetsberger et al., 2012).
 

       
NO is important components of complex signals transmitted during the process of stomatal movement (Ajaykumar et al., 2023; Li et al., 2018). Meanwhile, NO protects plants from damage caused by oxidative stress (Dadasoglu et al., 2021). Fig 2 also showed that fructose-induced stomatal closure can be inhibited by 10 µM NO scavenger (c-PTIO), 25 µM NOS inhibitor (L-NAME) and 25 µM NR inhibitor (NaN₃), with stomatal opening being restored up to 93.99%, 101.92% and 101.28%, respectively, of that in the control group. These data suggested that fructose-mediated NO production is generated via NOS and NR-mediated mechanisms (Melotto et al., 2006).
       
The fluorescence intensity of H₂DCF-DA in guard cells of broad beans increased significantly by 61.60% (P<0.05) and 126.94% (P<0.05) after 100 mM and 125 mM fructose treatment, respectively, when compared to the control, while there was no significant change when treated with other concentrations of fructose (Fig 3B; P>0.05). In contrast to the 125 mM fructose treatment, H2DCF-DA fluorescence intensity of the guard cells of broad beans decreased significantly by 75.20% (P<0.05), 65.48% (P<0.05), 45.62% (P<0.05) and 45.22% (P<0.05), respectively, after CAT, DPI, GSH and SHAM treatment (Fig 3A). These results indicate that fructose can induce an increase in the H₂O₂ content in the guard cells and the H₂O₂ catalyzed by NADPH oxidase and peroxidase is involved in fructose-induced stomatal closure. This observation is in agreement with the stomatal response noted in Fig 2, further suggesting that fructose-induced stomatal closure is mainly mediated by ROS via DPI-sensitive plasma membrane NADPH oxidases and not via SHAM-sensitive peroxidases.
 

 
Ca²⁺ and aquaporin are involved in fructose induced stomatal closure
 
2 mM calcium chelating agent ethylene glycol tetra acetic acid (EGTA) and 1 mM calcium antagonist lanthanum chloride (LaCl₃) significantly inhibited fructose-induced stomatal closure (P<0.05; Fig 2). Treatment regimens with EGTA and LaCl3, respectively, after treatment with 125 mM fructose, allowed stomatal aperture recovery to 85.70% (P<0.05) and 115.54% (P<0.05) of that in the control. Fructose-induced stomatal closure was inhibited by 50 µM water channel blocker mercuric chloride (HgCl₂), with the stomatal aperture of 125 mM fructose-treated epidermis being restored to 97.26% of that in the control. β-Thiol (β-ME) prevented binding of HgCl2 to aquaporin. The effect of HgC_l2 on stomatal movement was neutralized by β-ME (Fig 2). These results suggest that Ca²⁺ and aquaporin are involved in fructose induced stomatal closure.
 
Effects of fructose on stomatal opening and gas exchange parameters
 
As noted in Fig 4A, Pn increased by 20% (P<0.001) and 18.11% (P<0.001), respectively, at 3 h and 48 h after fructose treatment compared to that in the control. The Ci at 3 h after fructose treatment increased by 11.55% (P<0.001) compared to the control (Fig 4D). Compared with the control, Gs and Tr increased at 3 h and 48 h, respectively, after fructose treatment and decreased significantly at 24 h after fructose treatment (Fig 4B, C). Meanwhile, exogenous application of fructose significantly increased the carboxylation rate (CE) at 24 and 48 h (Fig 4E) (P<0.001). LS reached its maximum value at 24 h after fructose treatment (Fig 4F) (P<0.001). The saturated vapor pressure difference (VPD) was significantly lower than that of the control at 48 h after fructose treatment (P<0.001) and there was no significant difference at other time points (Fig 4G). WUE was significantly increased at 24 h after fructose treatment (P<0.001) and there was no significant difference at other time points (Fig 4H). Compared to the control, stomatal aperture decreased by 22.77% (P<0.001) and 5.27% (P<0.05) at 3 h and 24 h, respectively, after spraying of fructose (Fig 4I). Therefore, Pn did not differ significantly between the treated group and the control at 24 h.
 

 
Effects of fructose on rapid chlorophyll fluorescence induction kinetic curve
 
The rapid chlorophyll fluorescence induction kinetic curve of plants contains a lot of information about the initial photochemical reaction of the PSII reaction center. The non-destructive analysis of polyphasic fast chlorophyll a fluorescence (ChlF) has been widely used to reflect a plant’s photosynthetic performance (Živčák et al.,  2015; Hajihashemi et al., 2020). Exogenous fructose reduced the rapid-induction curve between J and I phase, but the difference was not significant. OJIP from fructose-treated plants gave a lower fluorescence yield at J-I phase (Fig 5).
 

 
Effects of fructose on PSII reaction center activity
 
Table 1 shows that the light energy absorbed by the chloroplast unit reaction center (ABS/RC) of broad bean leaves after exogenous fructose treatment decreased by 13.9% (P<0.05) and 14.0% (P<0.05) at 3 h and 48 h, respectively, compared with the control. ABS/RC, TR_O/RC, ET_O/RC and DI_O/RC on PSII receptor side belong to the specific activity parameters of PSII in unit reaction center and the decrease in ABS/RC, TR_O/RC and ET_O/RC indicates that the electron transport capacity has increased (Papazi et al., 2008). The maximum photochemical efficiency (φ_PO) and the energy dissipated per unit reaction center (DI_O/RC and RE_O/RC) in the fructose treated groups were not statistically different compared with the control group (P>0.05). The light energy captured per unit reaction center (TRO/RC) and the energy used for electron transport per unit reaction center (ET_O/RC) were significantly reduced at 3 h and 24 h, respectively, after fructose treatment. The probability of electron transport outside QA (ψ_EO), photosynthetic performance index (PI_ABS: the energy conservation from photons absorbed by PSII antenna, to the reduction of Q_B) and the total fluorescence basis (PI_total: the energy conservation from photons absorbed by PSII antenna, until the reduction of PSI acceptors) used for electron transport at 48 h increased by 7.27% (P<0.05), 33.99% (P<0.05) and 41.45% (P<0.05), respectively, compared with the control. The PI_ABS is a more sensitive parameter than F_v/F_m. Fructose significantly improved PI_ABS and PSI end acceptors (PI_total) at 48 h. The increase in PI_ABS indicated that the energy conservation ability and photosynthetic apparatus activity were improved (Hajihashemi et al., 2020). Our results indicated that the trans-thylakoid sub-gradient was increased (Baghbani et al., 2019) and light chemical efficiency was high after fructose treatment. The increase in ψ_EO reflected the increase in the quantum yield of captured light energy used for electron transport downstream of Q_A.
 

In this study, fructose-induced stomatal closure was found to be dose- and time-dependent, with maximum effects being observed at 125 mM and 30 min. NO, Ca²⁺, aquaporin and ROS were all involved in fructose-induced stomatal closure. Exogenous application of fructose can improve photochemical efficiency, Pn and WUE. This result serves as a reference for further investigation into the impact of sugar on plant physiology.

This research was supported by the National Natural Science Foundation of China (Grant No.31600320), Natural Science Basic Research Program of Shaanxi (2022 JM 455) and Subject Innovation Team of Quality Control and Resources Development of “Qin medicine” of Shaanxi University of Chinese Medicine (2019 QN01).

All authors declared that there is no conflict of interest.