Synergistic Effects of Melatonin and Gamma-Aminobutyric Acid on Protection of Photosynthesis System in Response to Multiple Abiotic Stressors

GABA (gamma-aminobutyric acid) and melatonin are endogenous compounds that enhance plant responses to abiotic stresses. The response of Vicia faba to different stressors (salinity (NaCl), poly ethylene glycol (PEG), and sulfur dioxide (SO2)) was studied after priming with sole application of GABA and melatonin or their co-application (GABA + melatonin). Both melatonin and GABA and their co-application increased leaf area, number of flowers, shoot dry and fresh weight, and total biomass. Plants treated with GABA, melatonin, and GABA + melatonin developed larger stomata with wider aperture compared to the stomata of control plants. The functionality of the photosynthetic system was improved in primed plants. To investigate the photosynthetic functionality in details, the leaf samples of primed plants were exposed to different stressors, including SO2, PEG, and NaCl. The maximum quantum yield of photosystem II (PS II) was higher in the leaf samples of primed plants, while the non-photochemical quenching (NPQ) of primed plants was decreased when leaf samples were exposed to the stressors. Correlation analysis showed the association of initial PIabs with post-stress FV/FM and NPQ. Stressors attenuated the association of initial PIabs with both FV/FM and NPQ, while priming plants with GABA, melatonin, or GABA + melatonin minimized the effect of stressors by attenuating these correlations. In conclusion, priming plants with both GABA and melatonin improved growth and photosynthetic performance of Vicia faba and mitigated the effects of abiotic stressors on the photosynthetic performance.


Introduction
Plants respond endogenously to abiotic stresses at the molecular and cellular levels. Understanding the underlying mechanism for stress tolerance is essential to develop stresstolerant crops) [1]. Experiencing single stressor enhances subsequently plant tolerance to diverse abiotic stresses, which is known as cross-stress tolerance [2,3]. Therefore, the mechanisms involved in response to diverse abiotic stresses have sharing similarities in some pathways or signaling molecules. Various approaches have been employed for enhancing plant tolerance to abiotic stresses; however, some of those approaches are tedious (e.g., conventional breeding), and some are controversial (e.g., plant genetic modification) [4]. Considering these limitations, attentions are directed toward priming responses [40]. An increasing number of studies focused on phytochemical and biochemical effects of melatonin and GABA on improving plant performance and alleviation of the stress impacts on plants, while the effect of melatonin and GABA on biophysical aspects of photosynthesis in normal and stress condition have been poorly investigated. Therefore, in this study, the possibility of using GABA (20 µM), melatonin (200 µM), and GABA + melatonin (GABA (20 µM) + melatonin (200 µM)) as priming agents for improving plants tolerance (by focus on photosynthesis functionality) to different stressors (PEG, salinity (NaCl), and sulfur dioxide (SO 2 ) was investigated using the faba bean as a model plant for stomata and photosynthesis studies. Although concerns regarding drought and salinity has been accentuated previously, increase in the concentration of SO 2 in the atmosphere due to the recent increase in rate of industrialization [41] and the adverse effects of SO 2 on plants has also emerged as a matter of concern [42,43]; thus, the possibility of alleviation of SO 2 stress on plants by melatonin and GABA, which has not been reported yet, was also targeted in the present study. This study can also be distinguished from previous studies since it targets both sole and combined effects of GABA and melatonin on plant growth and stress responses.

Plant Material and Growth Conditions
Vicia faba seeds were sown in pots with 15 cm diameters filled with mixture of cocopeat and perlite (2:1 v/v). Plants were grown under greenhouse conditions, and after seedling emergence, they were irrigated with modified Hoagland solution (half strength) three times per week. Ten days following seedling emergence, full-strength Hoagland solution was used for irrigation of the plants. GABA (20 µM), melatonin (200 µM), and GABA (20 µM) + melatonin (200 µM) were applied through irrigation once per week when the first true leaves emerged on the plants.

Measurements of Morphological Traits
Growth and morphological characteristics, such as root volume, fresh and dry weights, shoot fresh and dry weights, stem diameter, specific leaf area (SLA), number of leaves, and number of flowers were measured 45 days following seedling emergence. Flowering times were recorded when the flower primordia emerged on the plants. For measurement of SLA, first-leaf area was calculated by scanning of the detached leaves; then, their leaf area was calculated using Digimizer software (Digimizer V 4.1.1.0). To measure the dry weight, the samples were dried in an air-forced oven for 72 h at 75 • C. SLA was calculated by the ratio between leaf area and its dry weight. Root volume was measured based on the method described by [44].

Chlorophyll Index
A SPAD meter (Model: SPAD-502; Konica Minolta Sensing, Osaka, Japan) was used to directly estimate the chlorophyll content of the leaves. In each plot, the uppermost fully developed leaves were used for the SPAD readings. Since the sampling sensor area of the SPAD meter is small, the measurements were taken from three different spots of each leaf, for representative measurements, the average was used as the SPAD readings [45]. Finally, the SPAD meter readings were converted to actual chlorophyll content using the following conversion relationship where CC is chlorophyll content (µmol m −2 ), and SMR is the SPAD meter reading. CC = 10 (SMR 0.265 ) (1)

Stomatal Morphology and Conductivity
Stomatal morphology (stomatal length, stomatal width, pore aperture, stomatal index, and density) were measured on the young, fully developed leaves from each treatments according to the method described by [44]. A section of the young, fully developed leaf located midway between the tip and the base and equally distanced from each edge was used for microscopic analysis. To prepare the samples, the lower epidermis was coated with a thin layer of nail polish. After 10 min, a strip of transparent sticky tape was used to remove the dried polish. The dry polish sample along with the adhered sticky tape mounted on microscope slides and stomatal morphological details were investigated under a light microscope. Images were taken by an Omax top view software (version 3.5) and further analyzed using imageJ software (U.S. National Institutes of Health, Bethesda, MD, USA; http://imageJ.nih.gov/ij/ Version 1.53j 13 May 2021) to record stomatal length, stomatal width, pore length, aperture, and stomatal density. In total, 100 stomata from the leaves of each group were analyzed.
Stomatal conductance (g s ) was measured according to the method described by Fnourakis et al. (2013). In this calculation, stomatal pore depth was considered to be equal to the guard-cell width (i.e., stomatal width/2), assuming guard cells inflate to a circular cross-section. A 100× magnification was used to assess stomatal density [46,47]. Number of stomata was counted on three randomly chosen areas of the same leaf from which stomatal size measurements were taken. Analysis was done on 1 mm 2 of the middle of the leaf on both sides of the main vein [48]. Calculation of g s was done based on the following equation:

Chlorophyll Fluorescence Quenching Analysis
Samples were taken 45 days after growth from fully developed mature leaves. The samples were dark-adapted for 20 min after treating with PEG, NaCl, and SO 2 and were immediately used to measure slow induction of chlorophyll fluorescence with a FluorCam (FluorCam FC 1000-H, Photon Systems Instruments, PSI, Drasov, Czech Republic). A CCD camera and four fixed LED panels were included in the FluorCam. LED panels were used for supplying measuring pulses and for induction of saturating flash (https: //fluorcams.psi.cz/products/handy-fluorcam/#details). F V /F M was calculated using a custom-made protocol [48]. Measurements of chlorophyll fluorescence were started during exposure of the samples to short flashes in darkness and following a saturating pulse (3900 µmol m −2 s −1 Photosynthetic photon flux density (PPFD) at the end of the measurement to stop the electron transport as a result of reduction of quinone acceptors [49]. Based on the protocol, two sets of fluorescence data were recorded: one averaged over the time of short flashes in the darkness (F o ) and the other at the time of exposure to saturating flash (F M ).
For determination of NPQ, maximum fluorescence in light-adapted steady state (F M ) was recorded with that used for calculation of NPQ. The related data and calculations were done using version 7 (PSI, Drasov, Czech Republic) of FluorCam software [50].
The maximum quantum yield of PSII (F v /F M ) was calculated using the following equation: NPQ was calculated based on the following equation: The photochemical quenching or the amount of open PSII reaction centers at every consecutive pulse remains only influenced by the adaptation to the available light intensity and environmental conditions. Besides, two additional saturating pulses (F E " and F T " respectively) were given in darkness after completing the diurnal light cycle. This technique allows approximating the relaxation kinetics of the NPQ mechanisms, disentangling the energy-dependent quenching (qE), state-transition quenching (qT), and photo-inhibitory quenching (qI) based on their relaxation kinetics [51,52] according to the following equations:

Polyphasic Chlorophyll Fluorescence (OJIP) Transients
OJIP protocol was performed on fully developed, mature leaves of plants after 45 days of growth. The samples were dark-adapted for 20 min. A Fluorpen FP 100-MAX (Photon Systems Instruments, Drasov, Czech Republic) was used for measuring the OJIP transients. Different biophysical and phenomenological parameters related to PSII status [53] was investigated by JIP-test according to the protocol described by [20]. Parameters derived from the OJIP protocol provides information on the energy fluxes of light absorption (ABS) and trapping (TR) of the excitation energy and electron transport (ET O ) per reaction center (RC), which are described on the Supplementary Table S1.

Application of Abiotic Stresses SO 2 Stress
To impose SO 2 stress, plant samples were prepared based on the previously described protocols [44,48,54]. Leaf samples were cut from the petiole under water in order to prevent stress on the leaves during the analysis; each leaf was placed in 2 mL vials containing distilled water. The vials were placed into a sealed glass chamber (20 × 20 × 5 cm) with constant PPFD of 300 µmol m −2 s −1 at 25°C and incubated for 6 h with different concentrations of SO 2 (0, 0.5, 1, 2 ppm) before further analysis. Pure SO 2 was provided from a 5-L capsule with 99.99% purity. To calculate the inlet gas to the containers, two computational methods were used, including the ideal gas low [55] and Van der Waals equation [56]. All experimental procedures were conducted at 25°C and air pressure of 1 Pa.

Salinity and Osmotic Stresses
PEG 8000 (0 and −8 bar) and NaCl (0 and 100 mM) were used to impose salt and osmotic stresses on the leaf discs of primed (GABA, melatonin, and GABA+ melatonin) plants. Leaf disks were cut from 60-day-old faba bean plants. Seven leaf disks were put into distilled water (control), NaCl, and PEG solutions, then placed in a growth chamber with constant light (300 µmol m −2 s −1 PPFD) at 25°C and incubated for 6 h before chlorophyll fluorescence analysis [44,54].

Statistical Analysis
The results represented the average mean values of four replications. The data were analyzed using SAS software (version 9.0). The two-way analysis of variance (ANOVA) was used to find the significant differences (p ≤ 0.05), and then the Duncan multiple comparisons test was used to compare the means. For analyzing chlorophyll fluorescence parameters, obtained data were subjected to two-way ANOVA, and for mean comparison, the Tukey multiple comparison test was used. For stomatal characteristics, data obtained from one leaf were considered not independent, and for mean comparison, one-way ANOVA as well as Tukey multiple comparison test was used.

GABA and Melatonin Synergistically Improve Growth
The concentration of GABA and melatonin was selected according to the optimum concentration of GABA and melatonin that were reported to have a positive effect on plant abiotic stress tolerance [57][58][59]. Growth traits were significantly (p > 0.01) affected by priming treatments. The widest leaf area was obtained in plants treated with GABA + melatonin, and the lowest leaf area was observed in control plants. Plants treated with GABA + melatonin, GABA, and melatonin, respectively, showed 113%, 39%, and 38% increase in leaf area when compared to the leaf area of control plants ( Figure 1). Root volume was increased in plants treated with GABA + melatonin and melatonin; however, GABA did not significantly affect root volume of faba bean plants. The largest root volume was obtained in plants treated with GABA + melatonin, by 23% larger root volume than the control (Figure 2a). The maximum number of flowers was observed in GABA and GABA + melatonin, respectively, by 56% and 55% increase in comparison with the number of flowers in the control. This increase in the number of flowers by melatonin was 39% compared with control ( Figure 2b).
Plants treated with GABA + melatonin exhibited the highest fresh and dry weights of shoots, which showed 55% increase in shoot dry weight compared to the control. GABA and melatonin also led, respectively, to 30% and 34% increase in dry weight of shoot compared to the control (Figure 2c,d).
GABA+ melatonin treated plants also showed 37% increase in total biomass compared to the control. Total biomass of plants treated with either of GABA or melatonin was not significantly different from the control (Figure 2d).

Generation of Large Stomata and Improved Stomatal Conductance by GABA and Melatonin
Stomatal characteristics were significantly (p > 0.01) affected by the GABA and melatonin treatments (Figures 3a-d and 4). The highest stomatal length was obtained in plants treated with GABA + melatonin, which showed 7% longer stomata than the control. Moreover, GABA and melatonin caused 3% and 4% increase in stomatal length in comparison with the stomatal length of control ( Figure 3a). Plants treated with GABA + melatonin had the widest stomata, showing 3.5% increase in stomatal width than the control. The increase in width of stomata was 2.4% and 2.5% by sole application of GABA and melatonin, respectively ( Figure 3b). The maximum length of stomatal pore was also observed in plants treated with GABA + melatonin, which showed 9.5% longer stomatal pore than the control. GABA-and melatonin-treated plants showed, respectively, 5.6% and 4.7% increase in length of their stomatal pore in comparison with pore length of control plants (Figure 3c). Stomatal aperture (pore width) in plants treated with GABA + melatonin was three-fold wider than the stomata aperture of the control plants. GABA-and melatonin-treated plants also showed 10.1% and 4.5% wider aperture in comparison with stomatal aperture of the control (Figures 3d and 4). g s was also increased in primed plants compared to control. The g s in plants primed with GABA + melatonin, melatonin, and GABA was, respectively, 2.1-, 1.5-, and 1.3-fold higher than the g s of control plants (Figure 3e).

GABA and Melatonin Synergistically Improve Photosynthetic Functionality
Chlorophyll content in plants treated with GABA + melatonin, GABA, and melatonin showed, respectively, 72%, 61%, and 60% increase in comparison with the chlorophyll content in the leaves of control plants ( Figure 5).
The OJIP transient showed significant difference (p > 0.01) among treatments ( Table 1). The maximum value of F V /F M was obtained in plants treated with GABA + melatonin, which showed 4.8% increase compared to the F V /F M of control plants (Table 1). GABA-and melatonin-treated plants also exhibited, respectively, 3.7% and 3.3% increase in F V /F M in comparison with the control. Plants treated with GABA + melatonin, GABA, and melatonin had, respectively, 2.2-, 2-, and 1.8-fold higher PI abs than the control plants (Table 1).
ABS/RC was reduced by about 15% through priming treatments compared to ABS/RC of control plants. No significant difference was observed among ABS/RC of the primed plants (Table 1).
ET O /RC increased, respectively, by about 10%, 13%, and 6% in plants primed with GABA, GABA + melatonin, and melatonin when compared with the ET O /RC in the control plants (Table 1) Figure S1).    Exposing leaf samples to SO 2 resulted in significant difference (p > 0.01) among F V /F M of dark-adapted samples of plants that were primed with GABA, melatonin, and GABA + melatonin. Although exposure to SO 2 decreased F V /F M in control samples, this decrease was not detected or was smaller in primed samples (Figure 6a). Lowest F V /F M , among all samples was detected in those exposed to 2 ppm SO 2 . Following exposure to different concentrations of SO 2 , F V /F M in GABA + melatonin samples was higher than control. F V /F M in GABA + melatonin primed samples, in response to 0.5, 1, and 2 ppm SO 2 was 22%, 16%, and 22% higher than the F V /F M of control, respectively. The minimum value of F V /F M , regardless of SO 2 concentrations was associated with the control samples (Figures 6a and 7).  The value of NPQ in leaf discs of primed plants was significantly (p > 0.01) influenced by both priming (GABA, melatonin, and GABA + melatonin) and SO 2 concentration. In control samples, by exposure to 2 ppm SO 2 , a drastic reduction detected in NPQ compared to 0 ppm SO 2 , while in samples of plants primed with GABA + melatonin, a marked increase in NPQ was detected by exposure to 2 ppm SO 2 (Figures 6b and 8). Samples of plants primed with GABA also demonstrate the same reduction in NPQ as that of the control, and no significant difference was observed among samples of plants primed with melatonin in different concentrations of SO 2 . In general, the maximum NPQ was detected in samples of plants primed with GABA + melatonin when exposed to 2 ppm SO 2 , which showed 47% increase in comparison with the same priming samples exposed to 0 ppm SO2 (Figures 6b and 8, Supplementary Figure S2). The components of NPQ were also affected by SO 2 stress and priming agents. Highest qE was obtained in plants primed with melatonin, and priming with GABA + melatonin resulted in the highest qT and qI (Supplementary Figure S3).

Osmotic Stress
Exposing dark-adapted leaf discs of primed plants to PEG resulted in significant differences (p > 0.01) among F V /F M of primed samples (Figure 6c). Under non-osmotic stress condition, the maximum F V /F M was obtained in plants primed with GABA + melatonin and melatonin, showing, respectively, 8% and 7% higher F V /F M compared to F V /F M of control samples.
The maximum NPQ in samples that were exposed to PEG was detected in leaf samples of plants primed with melatonin and GABA, which showed 8% and 10% higher NPQ than the NPQ of control (Figures 6d and 9). In general, NPQ was increased in the samples of plants primed with GABA and melatonin, while in leaf samples of control plants, a drastic reduction in NPQ was detected (Figures 6d and 9; Supplementary Figure S2). The priming agent also affected NPQ components of plants under osmotic stress. Priming by melatonin reduced qE and qI components of NPQ, which resulted in higher qT than the other components in plants primed with GABA, qT was also highest among NPQ components (Supplementary Figure S3). For GABA + melatonin, all NPQ components contributed equally.

NaCl Stress
Exposing primed samples to 100 mM NaCl resulted in significant differences (p > 0.01) among F V /F M of dark-adapted samples (Figures 6e and 10). After exposing leaf samples to NaCl, the maximum F V /F M was related to samples of plants that were primed with GABA + melatonin, which showed 16% higher F V /F M than salt-exposed samples of control plants. After exposure to 100 mM NaCl, F V /F M of samples obtained from plants that were primed with sole GABA and melatonin was also higher than the F V /F M of control, which showed 15% higher F V /F M than the control samples. In general, under NaCl condition, primed plants with GABA, melatonin, and GABA + melatonin showed no significant difference with the F V /F M of their control, while NaCl exposure to control samples caused a significant decrease compared to the non-stressed control samples (Figures 6e and 10).
NPQ was increased in response to NaCl stress in leaf disks of plants primed with GABA and melatonin, while the value of NPQ in samples of control was reduced by exposure to 100 mM NaCl (Figures 6f and 10). The maximum value of NPQ was detected in samples of plants that were primed with GABA and exposed to 100 mM NaCl, which showed 24% higher NPQ compared to the control samples. Moreover, NPQ in leaf samples of plants primed with GABA and melatonin was, respectively, 24% and 14% higher than the control samples when exposed to salt stress (Figures 6f and 10, Supplementary Figure S2).
Exposing primed plants to NaCl stress caused more contribution of qT and qI to the NPQ than the qE in GABA-primed samples. In plants primed with melatonin, qI was lower than qE and qT. The components of NPQ showed almost the same value in plants primed with GABA + melatonin.

GABA and Melatonin Affect the Correlation of Initial PI abs with F V /F M and NPQ after Exposure to Stress
To better understand the association between the initial photosynthesis efficiency of plants and their ability to tolerate abiotic stresses, a correlation analysis between the initial PI abs (before exposure to stressors) and quenching parameters of leaf samples of primed plants exposed to stressors was conducted. The level of correlation among those traits was differed in primed plants and was affected by stressors.
The initial PI abs and F V /F M showed −98% correlation in non-primed plants when the leaf samples were exposed to PEG stress. While in leaf samples of plants primed with GABA, the negative correlation was decreased to −19%, and leaf samples of plants primed with melatonin and GABA + melatonin showed positive correlations (57% and 31%, respectively). Under NaCl stress, leaf samples of plants had −70% correlation between initial PI abs and F V /F M , while in plants primed with GABA, melatonin, and GABA + melatonin, −16%, −15%, and 35% correlation, respectively, were detected. In absence of stressors, the correlation was −13%, 6%, 10%, and 28% in leaf samples of control, plants primed with GABA, melatonin, and GABA + melatonin, respectively. Under SO 2 stress, the correlation was −80% in control plants, while, when leaf discs of plants primed with GABA were exposed to SO 2 , the correlation was reduced to −18% (Figure 11).
Contrasting differences were detected in correlation of the initial PI abs and NPQ between primed and control plants under control or stress condition. The initial PI abs and NPQ of leaf samples exposed to PEG showed −68% correlation in control; however, those traits were negatively correlated in plants primed with GABA, melatonin, and GABA + melatonin, which showed −3%, 81%, and −49% correlations, respectively. When leaf samples of primed plants were exposed to NaCl stress, −80% correlation was detected in the control condition, while 79%, 36%, and 22% correlation was detected in plants primed with GABA, GABA + melatonin, and melatonin, respectively. In absence of stressors, PI abs and NPQ were weakly correlated. Under SO 2 stress, this correlation was −9% in the control condition, while more strong negative correlation was detected in leaf samples primed with GABA, melatonin, and GABA + melatonin, which showed −13%, −61%, and −83% correlation, respectively ( Figure 11). Figure 11. Changes in the correlation between initial PI abs (before exposure to stressors) and F V /F M or NPQ (the green and red semicircles related to the correlation between PI abs and F V /F M or NPQ, respectively) following exposure to PEG8000 (−8 bar), NaCl (100 mM), and SO 2 (2 ppm) stresses and control. Blue color represents positive correlation, whereas red color represents negative correlation. Color intensity and size of the circle are proportional to the correlation coefficients.

Discussion
Based on the finding of present study, melatonin and GABA increased leaf area of Vicia faba. The improving effect of GABA on leaf area has been also reported in Vinga mungo L. [60]. However, the negative effect of melatonin on leaf area has been reported [61,62]. Interestingly, when both GABA and melatonin were co-applied, the increase in leaf area was markedly higher than the increase obtained by sole application of either of GABA or melatonin. This result suggests an interactive effect for GABA and melatonin. In accordance, enhancing the effect of melatonin on GABA homeostasis through triggering GABA-shunt pathway has been reported [40].
Root volume was increased in plants treated with GABA + melatonin and melatonin; however, GABA did not significantly affect root volume of faba bean plants. Similarly, in muskmelon, GABA had no effect on root growth [63], which is in line with the findings of this study. The role of melatonin on de novo root formation in Hypericum perforatum L. [64], root elongation in rice [65], and promotion of adventitious and lateral root regeneration in barley has been also reported [66].
GABA application caused generation of more flowers in faba bean plants, which agrees with the previous reports on flowering enhancement in bitter gourd and white gourd [67,68]. The number of flowers of faba bean also increased in plants treated with melatonin, which can be related to the role of melatonin in the transition to flowering as reported in Chenopodium rubrum [39].
Dry and fresh weights of shoots were also increased in plants that were treated with GABA. The same finding was reported on shoot fresh weight of maize in response to exogenous GABA [9]. Moreover, the increase in dry weight of shoot obtained in this study is in line with the result of a previous study that showed melatonin-treated maize plants show enhanced fresh shoot weight under salt stress [69]. However, melatonin was shown to have no effect on dry weight of shoots in maize seedlings in normal environmental conditions [62].
In the present study, higher biomass production was obtained by co-application of GABA and melatonin compared to the biomass of the control. It was reported that GABA increased biomass of maize plants under cadmium stress [9]. In addition, increase in biomass production was reported in in vitro-regenerated plants of Prunella vulgaris L. in cultured media that were supplemented with melatonin [70] and in soybean plants developed from seeds that were primed with melatonin [17,71]. On the other hand, decreased biomass production due to melatonin application has been reported in cherry rootstock [71].
Despite of no significant effect of sole application of GABA or melatonin on biomass production in faba beans, co-application of GABA and melatonin resulted in the highest biomass production. This may be due to the accumulative effects of melatonin and GABA due to the involvement of melatonin in mediation of GABA-shunt [40] or inductive effect of melatonin on endogenous GABA [72].
Stomatal movements controls gas exchange, especially CO 2 uptake for photosynthesis and water loss through transpiration [73]. Thereby, stomata play a crucial role in water use efficiency and productivity of plants [74]. Discovering the first phytomelatonin receptor (CAND2/PMTR1) in Arabidopsis thaliana revealed that melatonin takes part in the receptor-dependent stomatal closure [75]. It was demonstrated that the effect of melatonin on stomatal aperture was concentration-dependent in both Arabidopsis thaliana and Vicia faba, and increase in concentration of melatonin from 0 to 20 µmol/L reduced the aperture of stomata, while increase in melatonin concentration from 20 to 200 µmol/L promoted stomatal opening [75]. Meng et al. (2014), also demonstrated that osmotic stress reduced stomatal aperture, while melatonin pretreatment resulted in wider and longer stomata with bigger aperture [76]. This is in line with the result of this study that both width and length of stomata and stomatal pore increased in plants that were treated with melatonin. Beside stomatal aperture, melatonin also improved g s , suggesting a role for melatonin in regulation of stomatal gas exchange in Vicia faba. The same effect of melatonin on g s was also reported in cucumber and wheat by exogenous application of melatonin [32,77]. Increased stomatal aperture has both advantage and disadvantage for plant stress tolerance. In one hand, it increases the water loss from the plant; if the lost water is not compensated by more water uptake, it would be harmful for the plant [73]; on the other hand, CO 2 restriction is a critical challenge for the plant photosynthesis under water stress circumstances. Therefore, stomatal opening would improve gas exchange efficiency; as a result, this improves stress tolerance by maintaining a steady state photosynthesis [77]. It has been reported that melatonin enhances osmotic stress tolerance by promoting the efficiency of CO 2 assimilation [78] and obstructing the inhibitory effects of osmotic stress on g s [79]. Melatonin takes part in increasing g s by maintaining cell turgor that facilitates the stomatal opening and conductance [76]. Increased stomatal conductance aids better water and CO 2 mobility and improves photosynthesis of melatonin-treated plants [77]. Moreover, melatonin down-regulates the key gene in ABA biosynthetic pathway (NCED3) and up-regulate the genes involved in degradation of ABA (CYP707A1 and CYP707A1), which improves the re-opening function of stomata and g s [80]. Moreover, it has been reported that regulation of water balance in Malus species is accompanied by up-regulation of melatonin synthesis genes (TDC1, AANAT2, T5H4, and ASMT1), suggesting a pivotal role of melatonin in plants' water balance under abiotic stresses [23,80].
Interestingly, in plants that were treated with GABA, open stomata were along with improved electron transport system of photosynthesis, suggesting that GABA increases stomatal aperture to facilitate CO 2 uptake to keep pace with improved light reactions of photosynthesis.
GABA exerted the same effect as melatonin on stomatal pore aperture. In plants primed with both GABA and melatonin, the stomatal aperture was also wider than the plants that were treated with either of GABA or melatonin, suggesting a synergistic effect of these two compounds on stomatal morphology. Mekonnen et al. (2016) indicated the role of GABA in stomatal aperture by studying GABA-depleted gad1/2 mutants of A. thaliana phenotype. The gad1/2 mutant showed wilting phenotype under drought stress, which was due to increased stomatal aperture and disturbance in stomatal closure [81]. A major pathway-mediating ion transfer during stomatal opening (ALMT9) is inhibited by GABA under osmotic stress to improve osmotic stress tolerance, while in wild-type plants under normal condition, stomatal aperture in plants treated with GABA was higher than the control, and the increase in stomatal aperture was contributed to higher g s [82], which agrees with the result of our study on improved g s in GABA-primed plants. Furthermore, our result is in line with the previous reports on the effect of GABA on increasing g s in maize and rice [80,83]. It can be postulated that GABA improves stress tolerance by maintaining cell turgor, which improves g s and further enhancement of photosynthesis [22].
Chlorophyll metabolism is largely affected by environmental cues, which influences plant growth and yields [51]. Increase in chlorophyll content is shown to be an early response to environmental fluctuations [84]. Exogenous melatonin was shown to play a protective role by maintaining higher levels of chlorophyll in Chara australis [21]. Application of melatonin maintained higher content of chlorophyll during leaf senescence in barley [85] and alleviated chlorophyll degradation during salt [29], cold [86], and heat [87] stresses. Our data confirmed this effect, as chlorophyll content was increased in plants treated with melatonin and GABA, which is in agreement with the results obtained in black cumin [88]. Moreover, chlorophyll content in plants that were treated with GABA + melatonin was more than that of plants treated with either of GABA or melatonin. This suggests an interactive effect between GABA and melatonin on chlorophyll homeostasis.
When plants are exposed to light, the energy of light is absorbed by chlorophyll molecules in leaves and converted to different levels of excitation. Most of the environmental factors exert direct or indirect effects on light reactions of photosynthesis, and abiotic stresses alter the bioenergic balance of photosynthesis [89]; this effect can be detected by fluorescence of chlorophyll molecules [9]. To further explore the behavior of PSII of faba beans in response to GABA and melatonin, in the present study, imaging of chlorophyll a fluorescence quenching and JIP-test were applied. F V /F M is a parameter derived from chlorophyll fluorescence data that reflects the maximum photochemical quantum yield of PSII and thus efficiency of photosynthesis [90]). In this research, both GABA and melatonin increased F V /F M in faba beans. The increase in F V /F M in response to exogenous GABA has been also reported in maize seedlings [9]. In addition, similar to our finding, melatonin also increased the value of F V /F M in Pisum sativum L. [26].
ET o /RC, TR o /RC, and ABS/RC, which are parameters of specific energy fluxes, changed in response to the priming treatments. TR o /RC and ABS/RC decreased in primed plants, while ET o /RC was higher in primed plants compared to the control. Our result demonstrated that melatonin decreased ABS/RC. In line with our finding, reduction in ABS/RC has been reported in Chara australis following melatonin application [21]. Influence of GABA on specific energy fluxes in tomato, and consequently, improved ET o /RC has been reported [91].
PI abs is a delicate parameter of JIP-test that indicates the photochemical performance of plants. This parameter is a multi-parametric expression of light energy absorption, excitation energy trapping, and conversion of excitation energy to electron transport that depicts photosynthetic activity through a reaction center complex of PSII [92]. It has been found that exogenous melatonin increases the value of PI abs . Since PI abs is a function of ABS/RC and other chlorophyll fluorescence parameters, variation in the value of PI abs reversely corresponds with ABS/RC. The increase in PI abs in response to exogenous melatonin was also reported in Chara australis [21].
Photosynthetic performance is a central indicator of plants' fitness to their growth conditions. Most stresses, including osmotic stress, affect chlorophyll fluorescence of plants.
Tracking and analyzing the chlorophyll fluorescence provides a reliable, rapid, and noninvasive indicator to investigate photosynthetic function under different environments [93].
Plants need to regulate the absorbed light energy to abate the damage imposed to photosynthesis aperture. If the excess light energy is absorbed by plants, not quenched by photoprotective mechanisms or photochemistry, it can result in formation of damaging reactive-oxygen species, singlet oxygen, or superoxide [94,95]. Therefore, to avoid cellular damage and to maintain photosynthesis, plants optimize the absorbed light by directing its energy path from photosynthesis to photoprotective mechanisms. The efficiency of photoprotective mechanisms can be also quantified by chlorophyll fluorescence. For instance, F V /F M can be used as an indicator of photoinhibition, and NPQ quantifies non-photochemical energy dissipation [93].
A marked decline in the ratio of F V /F M was observed in control plants following exposure to SO 2 stress, while in plants treated with GABA, melatonin, and GABA + melatonin, the ratio of F V /F M was higher than that of the control, which further explains the reduction in negative correlation between initial PI abs and F V /F M of plants primed with GABA compared to the control. In agreement with the results obtained from an experiments on S. oblate [42], our results also indicate that the capability of plants to use photon energy was not impaired in plants primed with melatonin or GABA + melatonin when exposed to SO 2 stress. The same results were obtained by application of melatonin on tomato seedlings that were exposed to sulfuric acid-simulated acid rain, which depicted that melatonin treatment alleviated the acid-rain stress injury by increasing photosynthetic capacity [96]. Under SO 2 stress, the NPQ was higher in primed plants compared to the control. In plants treated with GABA, the qT component of NPQ was higher than qE and equal to qI. In plants treated with melatonin, qE mainly contributed in improving NPQ, suggesting that GABA favors energy dissipation through state transition more than energy quenching through xanthophyll cycle, while melatonin improves energy quenching more than the other NPQ components under SO 2 stress.
The higher correlation between PI abs and NPQ in primed plants is due to higher initial PI abs and NPQ compared with control, suggesting the conclusion that primed plants characterized with higher PI abs dissipate the excess energy better than plants with lower photosynthetic efficiency, confirmed by improved F V /F M in primed plants.
Osmotic stress is one of the most important abiotic stresses limiting plant growth [32] through dehydration; this restricts photosynthesis by pigment degradation and exerts irreversible damage to photosynthetic apparatus [97]. F V /F M drastically decreased in leaf samples of control plants exposed to PEG, while sole application of GABA and melatonin or their co-application increased F V /F M to obviously higher levels than non-primed plants under PEG stress. In agreement, in cucumber and soybean seedlings, osmotic stress significantly affected photosynthesis by decreasing F V /F M , and melatonin minimized the adverse effects of PEG [32,98].
PI abs is one of the most sensitive parameters of JIP-test, which is considered as a generic response suitable to quantify responses to various abiotic stresses, such as drought, osmotic stress, salinity, air pollutants, etc. [99]. Under PEG stress, F V /F M was negatively correlated with initial PI abs (−98%), which may be explained by severe reduction of F V /F M in response to PEG. GABA reduced this negative correlation to −19%, and melatonin and GABA + melatonin made the correlation positive and reached to 57% and 31%, respectively, which is due to the effect of GABA and melatonin in maintaining F V /F M under PEG stress. The same finding has been reported by [32].
NPQ is a photoprotective mechanism acting through a set of biochemical processes that control light harvesting [100]. NPQ reflects the quenching of thermal energy through the xanthophyll cycle and other energy quenching pathways that are induced by the concentration of proton inside thylakoids [101]. Thermal energy dissipation at PSII was estimated by investigating the NPQ in leaf samples of plants treated with either or both melatonin and GABA. NPQ was increased when exposed to PEG stress, indicating that GABA and melatonin directed the excess excitation energy through non-radiative dissipative processes, which agrees with the results of previous study on the role of melatonin in alleviating PEG stress [32].
The correlation between initial PI abs with NPQ was smaller and negative in control compared with plants primed with GABA, melatonin, and GABA + melatonin in both presence and absence of PEG stress. It can be concluded that the stronger correlation in primed plants compared with the control was due to higher initial PI abs parallel with increase in NPQ under PEG stress. In this regard, GABA has been reported to enhance PEG-induced stress tolerance in Piper nigrum through increasing PSII activity [102], and melatonin showed to improve PI abs in oat plants exposed to PEG stress, which minimizes the inhibitory effects of PEG [78].
Salt stress adversely affects crop productivity and survival by exerting osmotic and ionic stresses on plants [103]. GABA accumulation is an endogenous response of plants to salinity stress [28,104], suggesting a role for GABA in alleviating salt stress [20]. Our results showed that priming with GABA and melatonin minimized the adverse effect of NaCl by preventing the reduction of F V /F M . These findings agrees with those of the earlier studies, where enhanced F V /F M was detected in GABA-treated plants under salt stress condition [104].
Melatonin improves the efficiency of light energy absorption and electron transport in PSII during salt stress [22]. The protective effect of melatonin on photosynthesis is due to alleviation of negative impact of NaCl on F V /F M [29], which is in line with our results, where melatonin priming maintained F V /F M in leaf samples that were exposed to NaCl stress. The same improving effect of melatonin has been reported in Avena sativa [105] and grapevine [106] plants. Considering the correlation between PI abs and F V /F M under NaCl stress, priming with GABA and melatonin increased the correlation compared to the control, which can be seen by higher F V /F M in primed plants compared to control. Further, it is indicative of the protective role of GABA and melatonin on photosynthesis machinery, which leads to improved efficiency of the light reaction process in plants exposed to salinity stress.
When leaf discs of primed plants were exposed to NaCl, the value of NPQ was higher in plants primed with GABA and melatonin compared to control. The similar result has been reported when wheat plants were exposed to NaCl and GABA [23]. However, NPQ in leaf samples primed with melatonin was initially lower than the control before exposure to NaCl stress. The higher NPQ in NaCl-exposed samples indicated that the excitation energy was higher than the capacity of electron transport; as a result, it dissipates as heat that enables plants to withstand stressful condition [107]. Among NPQ components, qT and qI was higher than qE in plants primed with melatonin while in GABA-primed plants qE and qT was of the same level and higher than qI, suggesting that melatonin orients energy dissipation through state transition and favors the excitation energy distribution through PSII and PSI. At the same time, GABA causes the same level of energy dissipation through state transition and xanthophyll cycle.
The correlation between PI abs before stress with NPQ after stress was negative in control plants, while in primed plants, this correlation was positive, indicating higher capacity of primed plants for dissipating excess energy, resulting in maintenance of PI abs under stress condition by priming treatments. The protective role of melatonin on PI abs under salt stress has been also reported in Avena sativa [105].

Conclusions
In the present study, priming with GABA and melatonin improved growth, stomatal conductance, and photosynthetic performance of Vicia faba. Priming caused higher initial photosynthetic performance of plants before being exposed to stressors, as indicated by higher initial PI abs . GABA and melatonin conferred to better photosynthetic performance under different abiotic stresses. Considering higher NPQ in primed plants when exposed to stresses, a role for priming agents in improving energy dissipation as a protective mechanism is conclusive. Not only was the quantity of energy dissipation affected by treatments but also the process of energy dissipation affected by both priming agents and stressors. The findings of the present study suggest a synergistic effect of GABA and melatonin since the growth parameters and photosynthetic performances was higher in plants primed with both GABA and melatonin compared to sole application of them. The use of chemical agents for priming to enhance plant tolerance against diverse abiotic stresses and discovering the underlying mechanisms of the action of priming substances is promising. The findings of the present study could provide a prompt approach for crop stress management as an alternative for other tedious techniques of breeding for improving stress tolerance of plants.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/cells10071631/s1: Table S1. Measured parameters related to JIP test; Figure  (a) when exposed to SO 2 stress (2 ppm) (b), PEG stress (−8 bar) (c), and NaCl stress (100 mM) (d); Figure S3: NPQ partitioning kinetics (qE, qT, and qI) of Vicia faba plants primed with GABA (20 µM), melatonin (200 µM), and GABA (20 µM) + melatonin (200 µM) (a) when exposed to SO 2 stress (2 ppm) (b), PEG stress (−8 bar) (c), and NaCl stress (100 mM) (d); Figure S4: Graphical representation of a correlation matrix of initial PI abs with F V /F M and NPQ in control and leaf discs of plants primed with GABA (G), melatonin (M), or both of them (GM). Red color represents positive correlation, whereas black represents negative correlation. Color intensity is proportional to the correlation, which depicted in the legend at the bottom; Figure S5: Graphical representation of a correlation matrix of initial PI abs with F V /F M and NPQ in control and leaf discs of plants primed with GABA (G), melatonin (M), or both of them (GM) when exposed to PEG stress. Red color represents positive correlation whereas black represents negative correlation. Color intensity is proportional to the correlation, which depicted in the legend at the bottom; Figure S6: Graphical representation of a correlation matrix of initial PI abs with F V /F M and NPQ in control and leaf discs of plants primed with GABA (G), melatonin (M), or both of them (GM) when exposed to NaCl stress. Red color represents positive correlation whereas black represents negative correlation. Color intensity is proportional to the correlation, which depicted in the legend at the bottom; Figure S7: Graphical representation of a correlation matrix of initial PI abs with F V /F M and NPQ in control and leaf discs of plants primed with GABA (G), melatonin (M), or both of them (GM) when exposed to SO 2 stress. Red color represents positive correlation whereas black color represents negative correlation. Color intensity is proportional to the correlation, which depicted in the legend at the bottom.  Data Availability Statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest:
Authors have no conflict of interest.