Hormetic Responses of Photosystem II in Tomato to Botrytis cinerea

Botrytis cinerea, a fungal pathogen that causes gray mold, is damaging more than 200 plant species, and especially tomato. Photosystem II (PSII) responses in tomato (Solanum lycopersicum L.) leaves to Botrytis cinerea spore suspension application were evaluated by chlorophyll fluorescence imaging analysis. Hydrogen peroxide (H2O2) that was detected 30 min after Botrytis application with an increasing trend up to 240 min, is possibly convening tolerance against B. cinerea at short-time exposure, but when increasing at relative longer exposure, is becoming a damaging molecule. In accordance, an enhanced photosystem II (PSII) functionality was observed 30 min after application of B. cinerea, with a higher fraction of absorbed light energy to be directed to photochemistry (ΦPSΙΙ). The concomitant increase in the photoprotective mechanism of non-photochemical quenching of photosynthesis (NPQ) resulted in a significant decrease in the dissipated non-regulated energy (ΦNO), indicating a possible decreased singlet oxygen (1O2) formation, thus specifying a modified reactive oxygen species (ROS) homeostasis. Therefore, 30 min after application of Botrytis spore suspension, before any visual symptoms appeared, defense response mechanisms were triggered, with PSII photochemistry to be adjusted by NPQ in a such way that PSII functionality to be enhanced, but being fully inhibited at the application spot and the adjacent area, after longer exposure (240 min). Hence, the response of tomato PSII to B. cinerea, indicates a hormetic temporal response in terms of “stress defense response” and “toxicity”, expanding the features of hormesis to biotic factors also. The enhanced PSII functionality 30 min after Botrytis application can possible be related with the need of an increased sugar production that is associated with a stronger plant defense potential through the induction of defense genes.


Introduction
Botrytis cinerea, a fungal pathogen that causes gray mold, is damaging more than 200 plant species, especially tomato, cucumber, strawberry, potato, and ornamental plants [1,2]. B. cinerea releases ethylene to accelerate leaf wither, decreasing photosynthetic efficiency and affecting plant production resulting in huge economic losses [2,3]. This pathogen kills plant tissues previous to feeding on them, and uses a variety of toxic molecules to decompose the host cells [4,5]. Symptoms initial develop on infected tissues as limited lesions that subsequently become necrotic and spread to other tissues [5]. Thus, lesion development induced by the necrotrophic fungus B. cinerea influences growth performance gain an insight into the mechanisms that play a role in plant defense response to the fungal pathogen.

Visible Symptoms of Botrytis cinerea Spore Application on Tomato
Visual damage on tomato leaflets could be detected only after 24 h of the 20 μL spore suspension application (10 5 -10 7 spores/mL) (Figure 1a), with the apparent hyphae formation to be also observed ( Figure 1b). Leaflet damage appeared as a discoloration of the leaf epidermis (white spot) (arrow in Figure 1a).

Hydrogen Peroxide Detection after Botrytis cinerea Spore Application
At 30 min after B. cinerea spore suspension application, H2O2 production was detected at the drop's application area only (arrowhead in Figure 1c), but with time, H2O2 production gradually increased and spread out ( Figure 1d). Thus, after 240 min, DCF-DA signal intensified in the application area (arrowhead in Figure 1d) and H2O2 detection expanded to the leaf veins as well (arrow in Figure 1d).

Hydrogen Peroxide Detection after Botrytis cinerea Spore Application
At 30 min after B. cinerea spore suspension application, H 2 O 2 production was detected at the drop's application area only (arrowhead in Figure 1c), but with time, H 2 O 2 production gradually increased and spread out ( Figure 1d). Thus, after 240 min, DCF-DA signal intensified in the application area (arrowhead in Figure 1d) and H 2 O 2 detection expanded to the leaf veins as well (arrow in Figure 1d).

Allocation of Absorbed Light Energy at PSII before and after Spore Application
We estimated the fraction of the absorbed light energy that is used for photochemistry (Φ PSII ), is lost by regulated heat dissipation (Φ NPQ ), and that of non-regulated energy loss (Φ NO ), that add up to unity [35]. The effective quantum yield of PSII photochemistry (Φ PSII ) 30 min after spore suspension application increased significantly compared to control, while 120 min after spore suspension application it did not differ compared to control, at both LL and HL treatments. Φ PSII decreased significantly compared to control, 240 min after spore suspension application, at both LL and HL ( Figure 2). The quantum yield of regulated non-photochemical energy loss in PSII (Φ NPQ ) increased significantly up to 120 min after spore suspension application, but later on decreased to control level at LL (Figure 3a), while at HL treatment there was no significant difference compared to control at all measurements after spore suspension application (Figure 3b).

Allocation of Absorbed Light Energy at PSII before and after Spore Application
We estimated the fraction of the absorbed light energy that is used for photochemistry (ΦPSΙΙ), is lost by regulated heat dissipation (ΦNPQ), and that of non-regulated energy loss (ΦNO), that add up to unity [35]. The effective quantum yield of PSII photochemistry (ΦPSII) 30 min after spore suspension application increased significantly compared to control, while 120 min after spore suspension application it did not differ compared to control, at both LL and HL treatments. ΦPSII decreased significantly compared to control, 240 min after spore suspension application, at both LL and HL ( Figure 2). The quantum yield of regulated non-photochemical energy loss in PSII (ΦNPQ) increased significantly up to 120 min after spore suspension application, but later on decreased to control level at LL (Figure 3a), while at HL treatment there was no significant difference compared to control at all measurements after spore suspension application (Figure 3b).
Due to the increase of ΦNPQ at 30 and 120 min after spore suspension application, the quantum yield of non-regulated energy loss in PSII (ΦNO) decreased compared to control, but increased significantly 240 min after spore suspension application at LL (Figure 4a). At HL, the pattern was similar to LL, with the exception that of 120 min after spore suspension application, where ΦNO was at the same level with control plants (Figure 4b).

Allocation of Absorbed Light Energy at PSII before and after Spore Application
We estimated the fraction of the absorbed light energy that is used for photochemistry (ΦPSΙΙ), is lost by regulated heat dissipation (ΦNPQ), and that of non-regulated energy loss (ΦNO), that add up to unity [35]. The effective quantum yield of PSII photochemistry (ΦPSII) 30 min after spore suspension application increased significantly compared to control, while 120 min after spore suspension application it did not differ compared to control, at both LL and HL treatments. ΦPSII decreased significantly compared to control, 240 min after spore suspension application, at both LL and HL ( Figure 2). The quantum yield of regulated non-photochemical energy loss in PSII (ΦNPQ) increased significantly up to 120 min after spore suspension application, but later on decreased to control level at LL (Figure 3a), while at HL treatment there was no significant difference compared to control at all measurements after spore suspension application (Figure 3b).
Due to the increase of ΦNPQ at 30 and 120 min after spore suspension application, the quantum yield of non-regulated energy loss in PSII (ΦNO) decreased compared to control, but increased significantly 240 min after spore suspension application at LL (Figure 4a). At HL, the pattern was similar to LL, with the exception that of 120 min after spore suspension application, where ΦNO was at the same level with control plants (Figure 4b).   Due to the increase of Φ NPQ at 30 and 120 min after spore suspension application, the quantum yield of non-regulated energy loss in PSII (Φ NO ) decreased compared to control, but increased significantly 240 min after spore suspension application at LL (Figure 4a). At HL, the pattern was similar to LL, with the exception that of 120 min after spore suspension application, where ΦNO was at the same level with control plants (Figure 4b).

Photoprotective Dissipation of Excitation Energy as Heat (NPQ)
The non-photochemical chlorophyll fluorescence quenching (NPQ) increased significantly up to 120 min after spore application, but later on decreased to control level at LL (Figure 5a). At HL treatment, there was no significant difference compared to control, at 30 and 120 min after spore application, while at 240 min after spore application, NPQ was significantly lower than control (Figure 5b).

Chlorophyll a Fluorescence Images
At 30 min after application of B. cinerea suspension, ΦPSII decreased in the direct vicinity of the spore application area, compared to control values (arrow in Figure 6). At the same time at the surrounding area and the rest of the leaflet, an increased ΦPSII was observed, having as a result a higher ΦPSII value at the whole leaflet compared to control. At 240 min after spore application, the effective quantum yield of PSII photochemistry was severely affected at the whole leaflet area, being totally interrupted (ΦPSII = 0) at the appli-

Photoprotective Dissipation of Excitation Energy as Heat (NPQ)
The non-photochemical chlorophyll fluorescence quenching (NPQ) increased significantly up to 120 min after spore application, but later on decreased to control level at LL (Figure 5a). At HL treatment, there was no significant difference compared to control, at 30 and 120 min after spore application, while at 240 min after spore application, NPQ was significantly lower than control ( Figure 5b).

Photoprotective Dissipation of Excitation Energy as Heat (NPQ)
The non-photochemical chlorophyll fluorescence quenching (NPQ) increased significantly up to 120 min after spore application, but later on decreased to control level at LL (Figure 5a). At HL treatment, there was no significant difference compared to control, at 30 and 120 min after spore application, while at 240 min after spore application, NPQ was significantly lower than control (Figure 5b).

Chlorophyll a Fluorescence Images
At 30 min after application of B. cinerea suspension, ΦPSII decreased in the direct vicinity of the spore application area, compared to control values (arrow in Figure 6). At the same time at the surrounding area and the rest of the leaflet, an increased ΦPSII was observed, having as a result a higher ΦPSII value at the whole leaflet compared to control. At 240 min after spore application, the effective quantum yield of PSII photochemistry was

Chlorophyll a Fluorescence Images
At 30 min after application of B. cinerea suspension, Φ PSII decreased in the direct vicinity of the spore application area, compared to control values (arrow in Figure 6). At the same time at the surrounding area and the rest of the leaflet, an increased Φ PSII was observed, having as a result a higher Φ PSII value at the whole leaflet compared to control. At 240 min after spore application, the effective quantum yield of PSII photochemistry was severely affected at the whole leaflet area, being totally interrupted (Φ PSII = 0) at the application spot and the adjacent area, and on most of the leaflet, with a high photosynthetic heterogeneity to be observed ( Figure 6).

Discussion
Plant pathogens are divided into biotrophics, that attack the host cells conserving host viability and gaining nutrients from living cells, and necrotrophics, that get their nutrients by killing the host plant [8]. Botrytis cinerea is a necrotrophic plant pathogen causing gray mold disease on several crop plant species, producing enormous damage in crop  (Figure 7). In accordance to Φ PSII pattern, at 240 min after spore application, most reaction centers at the application spot and the adjacent area, were completely closed (q P = 0), and also on most of the leaflet, with only a 5% of reaction centers to remain open at the whole leaflet ( Figure 7). Following the pattern of ΦPSII, the redox state of the plastoquinone pool, that is a measure of the number of open PSII reaction centers (qP), 30 min after spore suspension application increased at the whole leaflet (54% open), compared to control (44% open) (Figure 7). In accordance to ΦPSII pattern, at 240 min after spore application, most reaction centers at the application spot and the adjacent area, were completely closed (qP = 0), and also on most of the leaflet, with only a 5% of reaction centers to remain open at the whole leaflet ( Figure 7).

Discussion
Plant pathogens are divided into biotrophics, that attack the host cells conserving host viability and gaining nutrients from living cells, and necrotrophics, that get their nutrients by killing the host plant [8]. Botrytis cinerea is a necrotrophic plant pathogen causing gray mold disease on several crop plant species, producing enormous damage in crop

Discussion
Plant pathogens are divided into biotrophics, that attack the host cells conserving host viability and gaining nutrients from living cells, and necrotrophics, that get their nutrients by killing the host plant [8]. Botrytis cinerea is a necrotrophic plant pathogen causing gray mold disease on several crop plant species, producing enormous damage in crop production [4]. Thus, there is increasing interest in the mechanism(s) utilized by plants to counteract infection by this fungus [8].
The necrotrophic pathogen B. cinerea is able to induce ROS generation, especially H 2 O 2 , in a plethora of plant species [50,51], either directly or indirectly (e.g., via the toxin botrydial) [52]. In our experimental set up, H 2 O 2 was detected 30 min after spore suspension application and gradually increased (Figure 1). The question that arises is on the role of this H 2 O 2 production. One possible explanation proposed from various researchers was that the H 2 O 2 produced could directly fight off the fungi [51]. However, this hypothesis was abandoned since H 2 O 2 production does not affect fungi development, given that B. cinerea poses effective ROS-detoxification systems, while at the same time B. cinerea can even contribute to ROS increase by producing its own ROS [50,51]. The rapid accumulation of H 2 O 2 observed (Figure 1), is a widespread defense mechanism of higher plants against pathogen attack [50].
Non-photochemical chlorophyll fluorescence quenching (NPQ), the key photoprotective process in plants that dissipates excess light energy as heat and protects photosynthesis [11,12,[53][54][55][56], increased significantly up to 120 min after spore suspension application, but later on decreased to control level (Figure 5a). Similarly, NPQ increased in ice leaves in the surrounding area of the infection by B. cinerea [24]. After 240 min of B. cinerea spore suspension application, NPQ at HL was significantly lower than the control (Figure 5b). This indicates that when Botrytis application was combined with HL, the photoprotective mechanism was no longer buffering the excess light stress levels, indicating an imbalance between energy supply and demand [57][58][59][60]. This, results in increased 1 O 2 and H 2 O 2 production [61] as we observed (Figures 1b and 4b) respectively. Non-photochemical quenching is a major component of the systemic acquired acclimation and systemic acquired resistance which is tightly related to ROS [10,56]. Chloroplasts through the operation of photosynthesis play an important role as redox sensors of environmental conditions and elicit acclimatory or stress defense responses [62,63]. Tomato leaflets after B. cinerea spore suspensions application show an increased capacity to keep quinone (QA) oxidized, thus, to have a higher fraction of open PSII reaction centers (q P ) compared to controls (Figure 7), indicating an enhanced PSII functionality.
At 30 min after spore suspensions application, a decreased Φ PSII in the direct vicinity of the application spot compared to control values was detected (Figure 6), while at the surrounding area and the rest of the leaflet, an increased Φ PSII was observed, specifying that the biotic stress was signaled to the rest of the leaflet regions, distant from the spore application area, having as a result a higher Φ PSII value at the whole leaflet compared to control. It has been frequently shown that H 2 O 2 diffuses through leaf veins to act as a long-distance molecule, triggering the stress defense response in plants [9,12,14,16,23]. An inhibition of photosynthesis by decreased Φ PSII values in the direct vicinity of the B. cinerea infection sites of tomato leaflets was recorded [64], but, with no alterations in the primary metabolism in the rest of the leaf tissue [64]. Decays in the effective quantum yield of PSII photochemistry (Φ PSII ) and increases in NPQ values before any visual symptoms appeared were observed in cashew seedlings inoculated with Lasiodiplodia theobromae [65].
The enhancement of Φ PSII 30 min after B. cinerea spore application ( Figure 2) and the concurrent increase of the regulated non-photochemical energy loss in PSII (Φ NPQ ) (Figure 3), resulted in a significant decrease in the quantum yield of non-regulated energy loss in PSII (Φ NO ) (Figure 4). Φ NO comprises of chlorophyll fluorescence internal conversions and intersystem crossing, that results to 1 O 2 formation via the triplet state of chlorophyll ( 3 chl*) [53,[66][67][68]. Thus, after application of B. cinerea on tomato leaflets, the decreased Φ NO indicates decreased 1 O 2 , that is considered as a highly damaging ROS produced by PSII [14,[69][70][71][72]. However, 240 min after B. cinerea application, the increased Φ NO suggests high levels of 1 O 2 (Figure 4) that could act synergistically with the high H 2 O 2 level (Figure 1d), in chloroplast damage [15,73]. Botrytis cinerea is a necrotrophic fungus that produces constantly toxic compounds which ultimately cause cell death [4]. Then, the fungus feeds on the dead tissue, thus, causing noticeable necrotic lesions [4].
While 1 O 2 creation via 3 chl* decreased 30 min after spore application compared to control (Figure 4a), H 2 O 2 was detected to appear 30 min after spore application (Figure 1c), and gradually to increase (Figure 1d). Hydrogen peroxide levels were positively correlated with disease severity [74], and low hydrogen peroxide levels appear to be the best indicator for leaf resistance to B. cinerea in strawberry leaves [74], while, chloroplast-generated ROS play a major role in B. cinerea -induced leaf damage [73], that eventually results in cell death (necrosis) [4]. As ROS formation by energy transfer ( 1 O 2 ), and electron transport (H 2 O 2 ) is simultaneous, it seems likely that their signaling pathways occasionally antagonize each other [13]. A low increase in ROS level is considered as favorable for activating defense responses [13,50,75,76], but an excessive ROS level is damaging to PSII functionality [9,13], which seems to be the case in our experimentation.
Botrytis cinerea that has been placed second in rank order of the top 10 fungal plant pathogens [77] can develop microscopic infections in epidermal cells of leaves that remain hidden for a certain period but after sporulation they convert in sources of primary inoculum and spread to other tissues [74].
The enhanced PSII functionality 30 min after Botrytis application can possible be related with the need of an increased sugar production that is associated with a stronger plant defense potential through the induction of defense genes [7]. However, increased levels of soluble sugars have been shown to support B. cinerea growth in tomato leaves [6]. In Rosa chinensis leaves, exogenous jasmonate (JA) spraying was shown to be essential for inducing defense response against B. cinerea [5]. Plant defenses can be activated by ROS [50], or by elicitor molecules such as chitosan [78], and γ-aminobutyric acid (GABA) [79], that are able to induce resistance mechanisms against B. cinerea.
Photosystem II responses of tomato leaflets to B. cinerea inoculation can be described as a time-dependent hormetic response (Figure 8) in terms of "stress defense response" and "toxicity" [76,80]. Activation by a low-dose effect or short-time exposure is a common phenomenon that is associated with the term "hormesis", typically indicating a positive biological response [13,44,55,[81][82][83][84][85][86]. There are published studies reporting a time-dependent hormesis [55,82], but it is only recently that analysis has been made regarding the features of hormesis as a function of time [13,81,82], and according to our data, under biotic stress. While 1 O2 creation via 3 chl* decreased 30 min after spore application compared to control (Figure 4a), H2O2 was detected to appear 30 min after spore application ( Figure  1c), and gradually to increase (Figure 1d). Hydrogen peroxide levels were positively correlated with disease severity [74], and low hydrogen peroxide levels appear to be the best indicator for leaf resistance to B. cinerea in strawberry leaves [74], while, chloroplast-generated ROS play a major role in B. cinerea -induced leaf damage [73], that eventually results in cell death (necrosis) [4]. As ROS formation by energy transfer ( 1 O2), and electron transport (H2O2) is simultaneous, it seems likely that their signaling pathways occasionally antagonize each other [13]. A low increase in ROS level is considered as favorable for activating defense responses [13,50,75,76], but an excessive ROS level is damaging to PSII functionality [9,13], which seems to be the case in our experimentation.
Botrytis cinerea that has been placed second in rank order of the top 10 fungal plant pathogens [77] can develop microscopic infections in epidermal cells of leaves that remain hidden for a certain period but after sporulation they convert in sources of primary inoculum and spread to other tissues [74].
The enhanced PSII functionality 30 min after Botrytis application can possible be related with the need of an increased sugar production that is associated with a stronger plant defense potential through the induction of defense genes [7]. However, increased levels of soluble sugars have been shown to support B. cinerea growth in tomato leaves [6]. In Rosa chinensis leaves, exogenous jasmonate (JA) spraying was shown to be essential for inducing defense response against B. cinerea [5]. Plant defenses can be activated by ROS [50], or by elicitor molecules such as chitosan [78], and γ-aminobutyric acid (GABA) [79], that are able to induce resistance mechanisms against B. cinerea.
Photosystem II responses of tomato leaflets to B. cinerea inoculation can be described as a time-dependent hormetic response (Figure 8) in terms of "stress defense response" and "toxicity" [76,80]. Activation by a low-dose effect or short-time exposure is a common phenomenon that is associated with the term "hormesis", typically indicating a positive biological response [13,44,55,[81][82][83][84][85][86]. There are published studies reporting a time-dependent hormesis [55,82], but it is only recently that analysis has been made regarding the features of hormesis as a function of time [13,81,82], and according to our data, under biotic stress. Figure 8. Overview of the hormetic response of photosystem II in tomato (at moderate light intensity), to B. cinerea spore application. The hormetic effect is defined by an inverse J-shaped biphasic curve with a short exposure time to have a stimulatory effect but at longer exposure time severe toxic effects to be apparent. Figure 8. Overview of the hormetic response of photosystem II in tomato (at moderate light intensity), to B. cinerea spore application. The hormetic effect is defined by an inverse J-shaped biphasic curve with a short exposure time to have a stimulatory effect but at longer exposure time severe toxic effects to be apparent.

Pathogen Culture and Spore Suspension Application
Botrytis cinerea ATHUM 4850 obtained from the ATHUM Culture Collection of Fungi of the National and Kapodistrian University of Athens Mycetotheca was used for tomato leaf spore application and was cultured on a solid nutrient medium (Potato Dextrose Agar, PDA, BD Difco, Oxford, UK) containing 0.5 mL L −1 lactic acid 1N at 23 • C until sporulation. Sporulated cultures were transferred in 100 mL sterilized distilled water and gently vortexed for spore release.
Tomato 5th leaflets were detached and laid on Petri dishes containing two sterilized blotting filter papers (Whatman, Schleicher and Schuell, Ottawa, Canada), wetted with sterile water. A drop (20 µL) of B. cinerea aqueous spore suspension (10 5 -10 7 spores/mL) was applied on tomato leaflets with a micropipette [87] as shown on Figure 1a, while leaves treated with a drop (20 µL) of sterile water (without B. cinera spores) were considered as controls.

Hydrogen Peroxide Imaging Detection
H 2 O 2 imaging detection, in control, and at 30, 120, and 240 min after spore suspension application, was performed as described earlier [12]. Briefly, inoculated and non-inoculated tomato leaflets were incubated with 25 µM 2 , 7 -dichlorofluorescein diacetate (DCF-DA, Sigma-Aldrich, Chemie GmbH, Schnelldorf, Germany) for 30 min in the dark, to visualize H 2 O 2 production.many Inoculated and control tomato leaflets were observed under a Zeiss Axioplan epifluoresence microscope equipped with an AxioCam MRc 5 digital camera. Photographs were obtained with the ZEN 2 version software according to the manufacturer's instructions.

Chlorophyll Fluorescence Imaging Analysis
An Imaging PAM M-Series system (Heinz Walz Instruments, Effeltrich, Germany) was used for the chlorophyll fluorescence measurements of the dark adapted (15 min) control and infected tomato leaflets as described in detail previously [39]. Measurements were conducted in leaves treated with a drop (20 µL) of sterile water (without B. cinera spores, control, 0 min) and after 30, 120 and 240 min of B. cinerea aquatic spore suspension application. The light intensities that were used for the photosynthetic efficiency measurements were 230 µmol photons m −2 s −1 , a low light (LL) intensity similar to the growth light, a moderate light (ML) intensity at 640 µmol photons m −2 s −1 , and a high light (HL) one, at 900 µmol photons m −2 s −1 . Representative areas of interest (AOIs) were selected in each leaflet so as to cover the whole leaflet area. The chlorophyll fluorescence parameters that were measured together with their definitions are shown in Table 1. Color-coded images of the effective quantum yield of PSII photochemistry (Φ PSII ) and the redox state of the plastoquinone pool (q P ), obtained at ML intensity at 640 µmol photons m −2 s −1 , are also presented.

Statistics
Statistically significant differences among the means were determined, from three independent treatments with each treatment consisting of three leaflets from three different tomato plants, using two-way ANOVA. Means (± SD) were considered statistically different at a level of p < 0.05.

Conclusions
In the present study, 30 min after application of Botrytis spore suspension in tomato leaflets, before any visual symptoms appeared, defense response mechanisms were triggered, with light energy use to be adjusted by NPQ in a such way that PSII functionality to be enhanced. The underlying mechanism involved was possible activated by H 2 O 2 , that was detected 30 min after Botrytis application with an increasing trend up to 240 min. This, is possibly convening tolerance against B. cinerea at short-time exposure, but at relatively longer exposure time when H 2 O 2 is increasing, it is becoming a damaging molecule. Hence, the response of tomato PSII to B. cinerea, indicates a hormetic temporal response, in terms of "stress defense response" and "toxicity", expanding the features of hormesis to biotic factors also.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.