Mechanistic Insights on Salicylic Acid Mediated Enhancement of Photosystem II Function in Oregano Seedlings Subjected to Moderate Drought Stress

Dramatic climate change has led to an increase in the intensity and frequency of drought episodes and, together with the high light conditions of the Mediterranean area, detrimentally influences crop production. Salicylic acid (SA) has been shown to supress phototoxicity, offering photosystem II (PSII) photoprotection. In the current study, we attempted to reveal the mechanism by which SA is improving PSII efficiency in oregano seedlings under moderate drought stress (MoDS). Foliar application of SA decreased chlorophyll content under normal growth conditions, but under MoDS increased chlorophyll content, compared to H2O-sprayed oregano seedlings. SA improved the PSII efficiency of oregano seedlings under normal growth conditions at high light (HL), and under MoDS, at both low light (LL) and HL. The mechanism by which, under normal growth conditions and HL, SA sprayed oregano seedlings compared to H2O-sprayed exhibited a more efficient PSII photochemistry, was the increased (17%) fraction of open PSII reaction centers (qp), and the increased (7%) efficiency of these open reaction centers (Fv′/Fm′), which resulted in an enhanced (24%) electron transport rate (ETR). SA application under MoDS, by modulating chlorophyll content, resulted in optimized antenna size and enhanced effective quantum yield of PSII photochemistry (ΦPSII) under both LL (7%) and HL (25%), compared to non-SA-sprayed oregano seedlings. This increased effective quantum yield of PSII photochemistry (ΦPSII) was due to the enhanced efficiency of the oxygen evolving complex (OEC), and the increased fraction of open PSII reaction centers (qp), which resulted in an increased electron transport rate (ETR) and a lower amount of singlet oxygen (1O2) production with less excess excitation energy (EXC).


Introduction
Drought is the prevailing environmental factor affecting several physiological and biochemical processes of plants that detrimentally influences global crop production [1][2][3][4]. Drought stress (DS) episodes are expected to increase in frequency, intensity, and duration in plant stress defense against biotic or abiotic stresses [61,62]. An amplified SA production occurs through induction of plant defense responses with a simultaneous decrease in auxin biosynthesis, and their concurrent action orchestrates synchronized defense and plant growth responses [61,62]. SA has been reported to ameliorate the unfavorable consequences of DS and salinity, acting as a growth regulator and an antioxidant, improving the osmotic potential, transpiration rate, stomatal conductance, biochemical parameters, repairing membrane injury and restoring photosynthetic function and nutrient uptake [63][64][65].
Salicylic acid's impact on plants cannot be globalized, as the influence may vary not only with the concentration and the method of application, but also with the plant species and the exposure duration [60,66]. Foliar application of SA in tomato plants suppressed phototoxicity by decreasing chlorophyll content and offering photoprotection of PSII [60]. Thus, SA application was suggested to improve PSII function by reducing photoinhibition and photodamage [60,67]. Plant productivity is described by the photochemical efficacy of the absorbed amount of light energy [68]. Breeding for improved photosynthesis and light energy use in crops is a manageable and a useful shorter-term addition to genetic engineering to enhance crop potential [69].
Origanum vulgare L. is a perennial flowering species in the family Lamiaceae, native to the Mediterranean region and Central Asia and widely used both as a medicinal and culinary herb, especially in the Greek, Italian, Turkish, Mexican, Spanish, and French cuisine. The objectives of this study were to characterize the functional differences in photosystem II (PSII) of oregano (Origanum vulgare L.) seedlings, with or without foliar application of 1 mM salicylic acid (SA), grown under optimal conditions or under moderate drought stress (DS). In addition, we aimed to determine the molecular mechanisms in the allocation of the absorbed light energy in PSII of oregano seedlings sprayed with SA, under DS and low light (LL), or DS and high light (HL), and to elucidate the mechanism by which SA improves PSII efficiency under DS.

Chlorophyll Content and Maximum Efficiency of Photosystem II under Normal Growth and Moderate Drought Stress
Leaves of oregano seedlings grown under optimal conditions were sprayed with 1 mM SA or double distilled H 2 O (control), and 72 h after spraying, the chlorophyll content was assessed. While chlorophyll content decreased significantly in the SA-sprayed oregano leaves under optimal growth conditions, compared to control (H 2 O-sprayed) (Figure 1a), the maximum efficiency of PSII photochemistry (Fv/Fm) remained unchanged after SA treatment ( Figure 1b). sequences of DS and salinity, acting as a growth regulator and an antioxidan the osmotic potential, transpiration rate, stomatal conductance, biochemica repairing membrane injury and restoring photosynthetic function and nu [63][64][65].
Salicylic acid's impact on plants cannot be globalized, as the influence only with the concentration and the method of application, but also with the and the exposure duration [60,66]. Foliar application of SA in tomato plant phototoxicity by decreasing chlorophyll content and offering photoprotectio Thus, SA application was suggested to improve PSII function by reducing ph and photodamage [60,67]. Plant productivity is described by the photochem of the absorbed amount of light energy [68]. Breeding for improved photo light energy use in crops is a manageable and a useful shorter-term additi engineering to enhance crop potential [69].
Origanum vulgare L. is a perennial flowering species in the family Lamia the Mediterranean region and Central Asia and widely used both as a medi inary herb, especially in the Greek, Italian, Turkish, Mexican, Spanish, and F The objectives of this study were to characterize the functional differences in II (PSII) of oregano (Origanum vulgare L.) seedlings, with or without foliar a 1 mM salicylic acid (SA), grown under optimal conditions or under mode stress (DS). In addition, we aimed to determine the molecular mechanisms tion of the absorbed light energy in PSII of oregano seedlings sprayed with and low light (LL), or DS and high light (HL), and to elucidate the mechan SA improves PSII efficiency under DS.

Chlorophyll Content and Maximum Efficiency of Photosystem II under Norma Moderate Drought Stress
Leaves of oregano seedlings grown under optimal conditions were sp mM SA or double distilled H2O (control), and 72 h after spraying, the chloro was assessed. While chlorophyll content decreased significantly in the SA gano leaves under optimal growth conditions, compared to control (H2O-spr 1a), the maximum efficiency of PSII photochemistry (Fv/Fm) remained unc SA treatment (Figure 1b).  Under moderate drought stress (MoDS) chlorophyll content, decreased significantly in both H 2 O-sprayed (−47%) and SA-sprayed leaves (−32%), compared to H 2 O-sprayed non-stressed leaves (control). Thus, chlorophyll content remained higher in SA-sprayed leaves compared to H 2 O-sprayed leaves (Figure 1a). Fv/Fm decreased significantly in MoDS H 2 O-sprayed oregano leaves compared to both non-stressed H 2 O-sprayed (−4%) and SA-sprayed leaves (−4%) (Figure 1b). Under MoDS, SA-sprayed leaves exhibited higher Fv/Fm values (2%) compared to H 2 O-sprayed MoDS leaves, but significantly lower values (−3%) compared to non-stressed SA-sprayed leaves.

Allocation of Absorbed Light Energy in Photosystem II under Normal Growth and Moderate Drought Stress
The light energy distribution to photochemistry (Φ PSII ), photoprotective heat dissipation (Φ NPQ ), and non-regulated energy loss (Φ NO ), was estimated under optimal growth conditions and MoDS, in both H 2 O-sprayed and SA-sprayed leaves.

Allocation of Absorbed Light Energy in Photosystem II under Normal Growth and Moderate Drought Stress
The light energy distribution to photochemistry (ΦPSII), photoprotective heat dissipation (ΦNPQ), and non-regulated energy loss (ΦNO), was estimated under optimal growth conditions and MoDS, in both H2O-sprayed and SA-sprayed leaves.
The effective quantum yield of PSII photochemistry (ΦPSII), under optimal growth conditions, did not differ between H2O-sprayed and SA-sprayed leaves at low light intensity ( The quantum yield of regulated non-photochemical energy loss (ΦNPQ), under normal growth conditions or MoDS, did not differ between H2O-sprayed and SA-sprayed leaves, at LL (Figure 2b). However, under HL, SA-sprayed leaves of oregano seedlings had significantly lower heat dissipation (ΦNPQ), under both optimal conditions (−11%) and under MoDS (−3%), compared to H2O-sprayed leaves ( Figure 2b).
The quantum yield of non-regulated energy loss (ΦNO), under optimal growth conditions, did not differ between H2O-sprayed and SA-sprayed leaves, at both LL and HL (Figure 3a), while under MoDS, SA-sprayed leaves displayed significantly lower ΦNO at both LL (−15%) and HL (−8%) compared to H2O-sprayed leaves (Figure 3a).

Changes in the Redox State of the Plastoquinone Pool, the Electron Transport Rate, and the Efficiency of Open Photosystem II Reaction Centers under Normal Growth and Moderate Drought Stress
The fraction of open PSII reaction centers (qp), representing the redox state of quinone A (QA) under optimal growth conditions at LL, did not differ between H2O-sprayed and SA-sprayed leaves; however, at HL, SA-sprayed leaves had a higher fraction of open PSII reaction centers (17%) (Figure 3b). Under MoDS, SA-sprayed leaves retained a higher fraction of open PSII reaction centers, at both LL (9%) and HL (23%) (Figure 3b).
The electron transport rate (ETR), under optimal growth conditions, did not differ between H2O-sprayed and SA-sprayed leaves at LL (Figure 4a), while under MoDS, SAsprayed leaves displayed a significantly higher ETR (7%) compared to H2O-sprayed leaves ( Figure 4a). Under HL, SA-sprayed leaves of oregano seedlings presented a significantly higher ETR, under both optimal conditions (24%) or under MoDS (25%), compared to H2O-sprayed leaves (Figure 4a).  Τhe efficiency of excitation energy capture by the open PSII rection centers (Fv'/Fm') under optimal growth conditions at LL did not differ in H2O-sprayed and SA-sprayed

Changes in the Redox State of the Plastoquinone Pool, the Electron Transport Rate, and the Efficiency of Open Photosystem II Reaction Centers under Normal Growth and Moderate Drought Stress
The fraction of open PSII reaction centers (qp), representing the redox state of quinone A (QA) under optimal growth conditions at LL, did not differ between H2O-sprayed and SA-sprayed leaves; however, at HL, SA-sprayed leaves had a higher fraction of open PSII reaction centers (17%) (Figure 3b). Under MoDS, SA-sprayed leaves retained a higher fraction of open PSII reaction centers, at both LL (9%) and HL (23%) (Figure 3b).

Changes in the Efficiency of the Oxygen Evolving Complex under Normal Growth and Moderate Drought Stress
Under optimal growth conditions, the efficiency of the oxygen evolving complex (OEC, Fv/Fo) did not differ in H2O-sprayed and SA-sprayed leaves ( Figure 5). However, under MoDS, SA-sprayed leaves showed enhanced efficiency (8%) of the OEC (Fv/Fo) ( Figure 5).

Changes in the Fraction of Closed Photosystem II Reaction Centers, and the Excess Excitation Energy in Photosystem II under Normal Growth and Moderate Drought Stress
The fraction of closed PSII reaction centers (1-qL), based on the "lake" model for the photosynthetic unit, under optimal growth conditions (control) did not differ in H2O-sprayed and SA-sprayed leaves at LL (Figure 6a); however, at HL, SA-sprayed leaves exhibited a smaller (−9%) fraction of closed PSII reaction centers (1-qL) (Figure 6a). Yet, under MoDS, SA-sprayed leaves had a smaller fraction of closed PSII reaction centers (1-qL) at both LL (−9%) and HL (−5%) (Figure 6a).

Changes in the Fraction of Closed Photosystem II Reaction Centers, and the Excess Excitation Energy in Photosystem II under Normal Growth and Moderate Drought Stress
The fraction of closed PSII reaction centers (1-qL), based on the "lake" model for the photosynthetic unit, under optimal growth conditions (control) did not differ in H 2 Osprayed and SA-sprayed leaves at LL (Figure 6a); however, at HL, SA-sprayed leaves exhibited a smaller (−9%) fraction of closed PSII reaction centers (1-qL) (Figure 6a). Yet, under MoDS, SA-sprayed leaves had a smaller fraction of closed PSII reaction centers (1-qL) at both LL (−9%) and HL (−5%) (Figure 6a).

Changes in the Efficiency of the Oxygen Evolving Complex under Normal Growth and Moderate Drought Stress
Under optimal growth conditions, the efficiency of the oxygen evolving complex (OEC, Fv/Fo) did not differ in H2O-sprayed and SA-sprayed leaves ( Figure 5). However, under MoDS, SA-sprayed leaves showed enhanced efficiency (8%) of the OEC (Fv/Fo) ( Figure 5).

Changes in the Fraction of Closed Photosystem II Reaction Centers, and the Excess Excitation Energy in Photosystem II under Normal Growth and Moderate Drought Stress
The fraction of closed PSII reaction centers (1-qL), based on the "lake" model for the photosynthetic unit, under optimal growth conditions (control) did not differ in H2O-sprayed and SA-sprayed leaves at LL (Figure 6a); however, at HL, SA-sprayed leaves exhibited a smaller (−9%) fraction of closed PSII reaction centers (1-qL) (Figure 6a). Yet, under MoDS, SA-sprayed leaves had a smaller fraction of closed PSII reaction centers (1-qL) at both LL (−9%) and HL (−5%) (Figure 6a). The excess excitation energy (EXC), calculated as (Fv/Fm − Φ PSII )/Fv/Fm, under optimal growth conditions (control), did not differ in H 2 O-sprayed and SA-sprayed leaves at LL (Figure 6b); however, at HL, SA-sprayed leaves exhibited significantly less (−10%) EXC (Figure 6b). Moreover, under MoDS, SA-sprayed leaves presented significantly less EXC at both LL (−5%), and HL (−5%) (Figure 6b).

Discussion
Climate change's impacts on agriculture and the increasing world population both threaten global food security [70]. Drought is the main global threat that affects agricultural production [71]. Photosynthesis is the main process in plants that can be intensely disturbed by environmental parameters [72]. Thus, the challenge of improving crop performance by increasing the photosynthetic efficiency of crop plants is a crucial and significant research issue [56,67]. Enhanced photosynthetic efficiency can be accomplished via improved distribution of the absorbed light energy [12]. Absorbed light energy can be used via photochemistry or dissipated via various thermal processes at the light reactions of photosynthesis; these comprise a set of redox reactions which are the basis of energy production in plant cells [23,29,73,74]. When the absorbed light energy exceeds the amount that can be used for photochemistry, increased formation of reactive oxygen species (ROS), such as hydrogen peroxide (H 2 O 2 ), superoxide anion radical (O 2 •− ), and singlet oxygen ( 1 O 2 ), occurs [24,31,[75][76][77]. Later, 1 O 2 , is produced from the triplet chlorophyll excited-state ( 3 Chl*) which is formed through an intersystem crossing of the singlet excited-state chlorophyl ( 1 Chl*) [2,20,31]. Under DS, there is an overexcitation of PSII, because the absorbed light energy exceeds chloroplasts' capabilities to use it, and the excess photons increase the amount of 1 Chl* and thus the probability of 3 Chl* and 1 O 2 formation, prompting subsequent photoinhibition [31,38,42,78]. Chlorophyll molecules are the key pigments for capturing light energy and transferring it to the reaction centres and the consequential electron transport in light reactions [20,[79][80][81].
The decline in chlorophyll content under MoDS in oregano seedlings (Figure 1a) might be attributed to the possible oxidation of chlorophyll molecules [82,83]. However, this reduction in the chlorophyll content under MoDS was partially reversed by the foliar application of SA, which is known to ameliorate oxidative stress and serve as an antioxidant [60,84]. It seems that under MoDS, the application of SA, which acted as an antioxidant, decreased the oxidation of chlorophyll molecules and modulated the chlorophyll content, resulted in improving antenna size. Optimizing antenna size can maximize photosynthetic efficiency [55]. Thus, in SA-sprayed oregano seedlings, the improved antenna size under MoDS growth conditions was followed by an enhancement of PSII photochemistry under both LL and HL. This was evident in the increased Φ PSII (Figure 2a), the increased qp (Figure 3b), the increased ETR (Figure 4a), but also the decreased Φ NO (Figure 3a) and the decreased EXC (Figure 6b). Using Φ NO , the probability of 3 Chl* and 1 O 2 formation can be calculated [60,85]. Thus, a decreased Φ NO reflects the ability of a plant to protect itself against excess light energy that leads to photoinhibition and photodamage [60,[86][87][88].
The decreased chlorophyll content in oregano leaves under MoDS, compared to no stress, results in the downsizing of their light-harvesting capacity to prevent photooxidative stress [53,55,89]. The modulation of antenna size, through foliar application of SA that decreased chlorophyll content (Figure 1a) and enhanced photosynthetic efficiency, was verified under non-stressed conditions and HL. Foliar application of SA, under nonstressed conditions and HL, increased Φ PSII (Figure 2a), qp (Figure 3b), ETR (Figure 4a), and Fv /Fm (Figure 4b), and also resulted in less EXC (Figure 6b), a smaller fraction of closed PSII reaction centers (1-qL) based on the "lake" model for the photosynthetic unit (Figure 6a), and a significantly lower heat dissipation (Φ NPQ ) (Figure 2b). The significantly lower Φ NPQ , under non-stressed conditions and HL, after SA application, indicates the photoprotective quality of SA in oregano seedlings against damage by excess illumination [60]. Reducing the size of the light-harvesting antenna has been recognised as an effective approach to mitigate photosynthetic inadequacy related to over-absorption of light energy [90,91].
Limitation of photoprotection under DS subsequently leads to photooxidative damage, indicated by an increase in Φ NO as well as a decrease in the maximum quantum efficiency of PSII (Fv/Fm) [12,39,42,92,93]. Chlorophyll a fluorescence analysis revealed a higher value of minimum fluorescence (Fo) (data not shown), and a significant decrease in Fv/Fm (Figure 1b) in both H 2 O-sprayed and SA-sprayed oregano leaves under MoDS. Thus, a higher fraction of absorbed light energy was lost as fluorescence under MoDS compared to optimal growth conditions. Yet, lower Fv/Fm values under MoDS (Figure 1b) indicate a higher degree of photoinhibition [94,95]. Nevertheless, SA-sprayed oregano leaves under MoDS had a higher Fv/Fm ratio compared to the H 2 O-sprayed leaves (Figure 1b).
PSII photodamage can appear through photooxidative stress, either at the acceptor side through 3 Chl*, which by exchanging energy and spinning with O 2 in the triplet state (molecular oxygen) results in 1 O 2 formation, or at the donor side through inactivation of the oxygen-evolving complex (OEC) [60,[96][97][98]. The chlorophyll fluorescence parameter 1−q L [101] has been shown to act as a signal to stomatal guard cells [102]. Accordingly, the lower fraction of closed reaction centres, or alternatively, the more oxidized Q A pool in SA-sprayed leaves under MoDS (Figure 6a), corresponds to a lower stomatal opening, which was accompanied by a lower EXC (Figure 6b), indicating improved PSII efficiency. The fraction of open PSII reaction centers (q p ) decreases during DS, and this leads to decreases in Φ PSII and increases in Φ NPQ [12,92,93,103,104]. However, in SA-sprayed leaves, compared to H 2 O-sprayed, under HL and normal growth conditions or HL and MoDS, the captured light energy was preferentially converted into photochemical energy (Φ PSII ) (Figure 2a), rather than dissipated as heat (Φ NPQ ) (Figure 2b). The enhanced ETR in SA-sprayed leaves compared to H 2 O-sprayed, under MoDS at both LL and HL (Figure 4a) was due to an increased qp ( Figure 3b) and an increased Fv /Fm ( Figure 4b). However, SA has been shown to slow down ETR in tobacco [66] but enhance ETR in tomatoes at both LL and HL [60]. In Hordeum vulgare, SA triggered a concentration-related decreased efficiency of the OEC, resulting also in a decreased fraction of open PSII centres [105]. It appears that SA's mode of action depends considerably on several characteristics, such as the plant species, exposure duration, the concentration used, and the environmental conditions [60,61,106,107]. Thus, data on the effects of SA on plant physiological processes under stressed or non-stressed conditions remain debatable [106], but generally it can be recognized that SA has a positive effect on plant responses to many abiotic stresses such as heat, chilling, salinity, drought, and heavy metal toxicity [60,84,[108][109][110][111][112][113][114][115][116]. The diverse impact of SA on different plant species may be due to the diversification of the SA signaling and biosynthesis pathways in plants [117].

Salycilic Acid Treatment
Oregano seedlings under normal growth conditions were sprayed with 1 mM salicylic acid (SA) or double distilled H 2 O, and after 72h the chlorophyll content and PSII function were evaluated [60]. In addition, chlorophyll content and PSII function were evaluated in oregano seedlings that were sprayed with 1 mM SA or double distilled H 2 O, and exposed to moderate drought stress (MoDS). Each plant received 10 mL of 1 mM SA or double distilled H 2 O, applied by a hand sprayer only once during the experiment at 72 h before the measurements. All treatments were performed with four independent biological replicates.

Drought Stress Treatment and Soil Water Status
Moderate drought stress (MoDS) was induced by withholding irrigation of oregano seedlings until a 60% soil volumetric H 2 O content (SWC) was maintained in the control seedlings. SWC was measured with ProCheck device coupled with the soil moisture sensor 5TE (Decagon Devices, Pullman, WA, USA), as described previously [118].

Chlorophyll Fluorescence Analysis
Chlorophyll fluorescence analysis of dark-adapted oregano plants was performed as described in detail previously [120], using an Imaging-PAM Fluorometer M-Series MINI-Version (Heinz Walz GmbH, Effeltrich, Germany). The minimum (Fo) and the maximum (Fm) chlorophyll a fluorescence in the dark was measured after 20 min dark adaptation. The maximum chlorophyll a fluorescence in the light (Fm ) was measured after a saturation pulse, while the minimum chlorophyll a fluorescence in the light (Fo ) was computed by Win software (Heinz Walz GmbH, Effeltrich, Germany) as Fo = Fo/(Fv/Fm + Fo/Fm ) [121]. Steady-state photosynthesis (Fs) was measured after 5 min of illumination time with either 205 µmol photons m −2 s −1 , actinic light (AL) low light intensity (LL), which corresponds to the growth light intensity, or with 1000 µmol photons m −2 s −1 , high light intensity (HL). The following chlorophyll fluorescence parameters (Table 1) were estimated by Win software (Heinz Walz GmbH, Effeltrich, Germany).

Statistics
All data were tested for normality with a Shapiro-Wilk test, and for homogeneity of variance with Levene's test prior to statistical analysis [122]. The populations of variances were not equal, so we performed a Welch's ANOVA to compare the four treatments, followed by a post hoc analysis with a Games-Howell test [60]. All the analyses were performed in SPSS version 28.0 (IBM, Chicago, IL, United States) for Windows. The data are presented as means ± SD (n ≥ 4).

Conclusions
Salicylic acid application increased the effective quantum yield of PSII photochemistry (Φ PSII ) by enhancing the efficiency of the oxygen evolving complex (OEC) and increasing the fraction of open PSII reaction centers (qp), which resulted in an increased electron transport rate (ETR). We can conclude that SA application may reduce the excess excitation energy by reducing 1 O 2 formation, and may also enhance the photosynthetic function of oregano seedlings to challenge DS; thus, SA can be regarded as a promising tool for improving the ability of crop plants to face drought episodes in combination with the high light conditions of the Mediterranean area that influence crop production detrimentally. However, since the impact of SA application on different crop plants is diverse, possibly due to the diversification of the SA signaling and biosynthesis pathways in plants, more experiments must be executed in different crop species to establish the large-scale use of SA in agriculture in order to achieve sustainable crop production to confront the challenge of climate change.