International Journal of Molecular Sciences

Article Salicylic Acid Protects II by Alleviating Photoinhibition in under High Light

1, , 1, 1, 1 1 Yang-Er Chen * † , Hao-Tian Mao †, Nan Wu †, Atta Mohi Ud Din , Ahsin Khan , Huai-Yu Zhang 1 and Shu Yuan 2 1 College of Life Sciences, Sichuan Agricultural University, Ya’an 625014, China; [email protected] (H.-T.M.); [email protected] (N.W.); [email protected] (A.M.U.D.); [email protected] (A.K.); [email protected] (H.-Y.Z.) 2 College of Resources Science and Technology, Sichuan Agricultural University, Chengdu 611130, China; [email protected] * Correspondence: [email protected]; Tel.: +86-835-2886653 These authors contributed equally to this study. †  Received: 19 January 2020; Accepted: 9 February 2020; Published: 12 February 2020 

Abstract: Salicylic acid (SA) is considered to play an important role in responses to environmental stresses. However, the detailed protective mechanisms in are still unclear. We therefore explored the protective roles of SA in photosystem II (PSII) in Arabidopsis thaliana under high light. The results demonstrated that 3 h of high light exposure resulted in a decline in photochemical efficiency and the dissipation of excess excitation energy. However, SA application significantly improved the photosynthetic capacity and the dissipation of excitation energy under high light. Western blot analysis revealed that SA application alleviated the decrease in the levels of D1 and D2 protein and increased the amount of Lhcb5 and PsbS protein under high light. Results from photoinhibition highlighted that SA application could accelerate the repair of D1 protein. Furthermore, the phosphorylated levels of D1 and D2 proteins were significantly increased under high light in the presence of SA. In addition, we found that SA application significantly alleviated the disassembly of PSII-LHCII super complexes and LHCII under high light for 3 h. Overall, our findings demonstrated that SA may efficiently alleviate photoinhibition and improve by dissipating excess excitation energy, enhancing the phosphorylation of PSII reaction center proteins, and preventing the disassembly of PSII super complexes.

Keywords: salicylic acid; fluorescence; photosystem; Arabidopsis thaliana

1. Introduction In higher , (PSI) and photosystem II (PSII) are two large multiple protein super complexes in the membranes of photosynthetic organisms [1]. To cooperate with other complexes, carry out photosynthetic electron transfer from water to NADP+; however, ever-changing environments often cause them to become imbalanced. The reaction center of PSII has been shown to be the key site of damage from different environmental stresses in the photosynthetic apparatus of plants [2,3]. Plants have evolved to develop several protective mechanisms against excess light including nonphotochemical quenching (NPQ), the defense system, and state transitions [4–7]. NPQ facilitates the dissipation of excess light energy in the form of heat, thereby preventing the overreduction of PSII [4,5]. (ROS) induced by excessive light cause oxidative damage to proteins and lipids in the thylakoid membrane, which can be ameliorated by antioxidant defense systems including enzymes and [6]. State transitions occur to balance

Int. J. Mol. Sci. 2020, 21, 1229; doi:10.3390/ijms21041229 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 1229 2 of 17 absorbed light energy between the two photosystems for efficient photosynthesis under changing light conditions by relocating light-harvesting complex II proteins [7]. It is, therefore, very important to explore how to protect PSII against excess light. High light is one of the most fiercely fluctuating environmental factors that can lead to the degradation of pigments, decline in photosynthetic efficiency [8], stomatal closure [9], inactivation of PSII reaction center, and eventually, decrease in plant yield [10]. Although light is necessary for photosynthesis, exposure to strong light can result in severe inhibition in the activity of PSII due to photo-oxidative damage to photosynthetic machinery [11]. This phenomenon is referred to as photoinhibition, which is usually unavoidable in photosynthetic organisms. It is well known that PSII is highly prone to high light, and the activity of PSII decreases more rapidly than many other physiological activities [12,13]. Many studies have suggested that photodamage of PSII is accelerated under environmental stress [8,14]. The degree of photoinhibition mainly depends on the balance between the repair of PSII damage and photodamage to PSII. The PSII repair cycle maintains photodamage of the PSII reaction center, the subsequent degradation of D1, de novo synthesis of D1, and cotranslational assembly [10,15]. Under all light intensities, the PSII reaction center, and the D1 protein in particular, is very sensitive to damage; this protein not only provides a binding site for many cofactors, but also maintains conformational stability of PSII in the PSII repair cycle [11]. Furthermore, D1 protein can be used as a good indicator to measure the extent of photoinhibition [12,13]. However, plants have developed some repair mechanisms to prevent the accumulation of damaged PSII and the photoinhibition caused under stressful conditions [16,17]. Salicylic acid (SA) is a small phenolic compound and well-known phytohormone involved in many physiological and biochemical processes of plants, such as seed germination, seedling growth, stomatal aperture, respiration, and senescence [18–22]. Moreover, it has been reported to play an important regulatory role in enhancing plant tolerance against various environmental stresses including osmotic stress, salt stress, heavy metal, and chilling, and as a signaling molecule at moderate concentrations [23–26]. Much evidence has also indicated that SA markedly alleviates damage to plants under high light by activating the antioxidant system and protecting photosynthesis [24,26–28]. However, the detailed regulatory mechanisms of SA in protecting PSII are less well understood under high light, particularly for PSII proteins and thylakoid protein phosphorylation. Thylakoid can achieve optimum photosynthetic performance rapidly by changing its structure and functions as a key part of photosynthetic machinery under external environmental conditions [29]. In the present study, our aim was to elucidate the protective roles of SA on PSII by investigating the changes in thylakoid membrane complexes, PSII reaction center damage, protein phosphorylation, and energy dissipation in Arabidopsis thaliana under high light. Our findings demonstrated that high light caused severe photodamage to PSII, and that SA application can alleviate such adverse effects through dissipating excess excitation energy and accelerating the repair of D1.

2. Results

2.1. Chl Content and Carotenoid Content To investigate the effects of SA on photosynthetic capacity, pigment contents of Arabidopsis plants were determined under high light in the presence or absence of exogenous SA application (Figure1). The results showed that there were no significant di fferences in the total chlorophyll (Chl) content, Chl a/b, and carotenoid content between the control and SA-pretreated plants under normal conditions. Although Chl a/b had no significant changes under all conditions, 3 h of high light resulted in a remarkable decrease in the contents of Chl and carotenoid in the presence or absence of SA. Furthermore, we found that SA application increased the contents of Chl and carotenoid when compared with non-SA-pretreated plants under high light. Int. J. Mol. Sci. 2020, 21, 1229 3 of 17 Int. J. Mol. Sci. 2020, 21, 1229 3 of 17

FigureFigure 1. E ff1.ects Effects of SA of on SA total on total chlorophyll chlorophyll content content (A), ( chlorophyllA), chlorophylla/b ratioa/b ratio (B), (B and), and carotenoid carotenoid content (C) incontentArabidopsis (C) in thalianaArabidopsisunder thaliana high under light. high The light. data The represent data represent means meansSD (standard ± SD (standard deviations) deviations) from four independent biological replicates (n = 4). Different lower± ‐case letters indicate from four independent biological replicates (n = 4). Different lower-case letters indicate significant significant differences (p < 0.05) according to Duncan’s multiplication range test. HL, high light. SA + differences (p < 0.05) according to Duncan’s multiplication range test. HL, high light. SA + HL, high HL, high light after SA pretreatment for 3 d. 0–3 h, high light for 0 h, 1 h, and 3 h in the presence or light after SA pretreatment for 3 d. 0–3 h, high light for 0 h, 1 h, and 3 h in the presence or absence of absence of SA pretreatment, respectively. SA pretreatment, respectively. 2.2. SA Improved Photosynthetic Efficiency under High Light 2.2. SA Improved Photosynthetic Efficiency under High Light To further test the effects of SA on photosynthetic efficiency, PSI photochemistry was measured Toin Arabidopsis further test plants the eexposedffects of to SA high on light photosynthetic in the presence effi orciency, absence PSI of photochemistry SA. As shown in wasFigure measured S1, in Arabidopsisthe effects plantsof high exposedlight on PSI to highphotochemistry light in the were presence minor. orFour absence representative of SA. AsPSI shownparameters in Figure (the S1, the effphotochemicalects of high lightquantum on PSI yield photochemistry of PSI (ΦPSI), oxidation were minor.status of Four PSI donor representative side (ΦND), PSIreduction parameters status (the photochemicalof PSI acceptor quantum side (Φ yieldNA), and of PSImaximal (ΦPSI P700), oxidation change (P statusm)) did of not PSI significantly donor side decrease (ΦND), reduction under high status of PSIlight acceptor for 1 h, side compared (ΦNA), with and the maximal control, P700 in the change absence (P ofm)) SA. did However, not significantly only 3 h of decreasehigh light under led to high lighta for remarkable 1 h, compared decline with in Φ theNA control,and Pm. in Although the absence four of representative SA. However, PSI only parameters 3 h of high showed light no led to a observed differences between the control and SA‐pretreated plants, SA pretreatments significantly remarkable decline in ΦNA and Pm. Although four representative PSI parameters showed no observed improved the value of ΦNA and Pm under high light for 3 h. These results suggested that exogenous differences between the control and SA-pretreated plants, SA pretreatments significantly improved SA could protect PSI against the damage from high light in Arabidopsis thaliana. P the valueNext, of Φ NAPSIIand photochemistrym under high was determined light for 3 h.using These a modulated results suggested imaging fluorometer that exogenous under high SA could protectlight PSI in againstthe presence the damage or absence from of SA. high As lightshown in inArabidopsis Figure 2, 3 h thaliana of high. light resulted in a significant Next,decline PSII in maximum photochemistry efficiency was of determined PSII photochemistry using a modulated(Fv/Fm), effective imaging quantum fluorometer yield of under PSII high lightelectro in the presencetransport or (Φ absencePSII), and of photochemical SA. As shown quenching in Figure2 ,(qP), 3 h of and high a light remarkable resulted increase in a significant in declinenonphotochemical in maximum e ffiquenchingciency of (NPQ) PSII photochemistry compared with the (Fv control/Fm), einff theective absence quantum of SA. yield SA application of PSII electro transportalleviated (ΦPSII the), anddecrease photochemical in the Fv/Fm, quenching ΦPSII, and (qP),qP and and stimulated a remarkable the increase increase in inNPQ nonphotochemical under high quenchinglight for (NPQ) 3 h relative compared to the withcontrol. the This control suggests in the that absence SA could of SA. effectively SA application maintain alleviatedthe photosynthetic the decrease capacity in Arabidopsis plants under high light. In addition, we found that SA treatments significantly in the Fv/Fm, ΦPSII, and qP and stimulated the increase in NPQ under high light for 3 h relative to the increased the values of quantum yield of nonregulated energy dissipation (Y(NO)) compared to non‐ control. This suggests that SA could effectively maintain the photosynthetic capacity in Arabidopsis SA treated plants. plants under high light. In addition, we found that SA treatments significantly increased the values of quantum yield of nonregulated energy dissipation (Y(NO)) compared to non-SA treated plants. NPQ is related to nonphotochemical quenching, and is regarded as the key protective mechanism against excess light energy in PSII. As shown in Figure3, the NPQ induction was more pronounced with the increase of high light treatment. The NPQ induction in SA-pretreated plants were faster and reached a higher amplitude compared to the non-SA treated plants under the same high light treatment. The kinetics of dark relaxation were still faster than in non-SA treated plants under 0 and 3 h of high light. State transitions are a good way to indicate the rate of energy dissipation by monitoring changes in chlorophyll fluorescence characteristics [30]. As shown in Figure S2, the levels of Chl a fluorescence in non-SA treated plant was greater than SA-treated plants. However, there was no obvious difference between the non-SA treated and SA-treated plants. In contrast, a transitory increase in fluorescence under 3 h of high light was presented when the far-red light was turned off in SA-treated plant. These results suggested that SA could participate in regulating the dissipation of excess light energy under high light.

Figure 2. Effects of SA on chlorophyll fluorescence parameters in Arabidopsis thaliana under high light.

Fv/Fm, maximum efficiency of PSII photochemistry; ΦPSII, effective quantum yield of PSII electro Int. J. Mol. Sci. 2020, 21, 1229 3 of 17

Figure 1. Effects of SA on total chlorophyll content (A), chlorophyll a/b ratio (B), and carotenoid content (C) in Arabidopsis thaliana under high light. The data represent means ± SD (standard deviations) from four independent biological replicates (n = 4). Different lower‐case letters indicate significant differences (p < 0.05) according to Duncan’s multiplication range test. HL, high light. SA + HL, high light after SA pretreatment for 3 d. 0–3 h, high light for 0 h, 1 h, and 3 h in the presence or absence of SA pretreatment, respectively.

2.2. SA Improved Photosynthetic Efficiency under High Light To further test the effects of SA on photosynthetic efficiency, PSI photochemistry was measured in Arabidopsis plants exposed to high light in the presence or absence of SA. As shown in Figure S1, the effects of high light on PSI photochemistry were minor. Four representative PSI parameters (the photochemical quantum yield of PSI (ΦPSI), oxidation status of PSI donor side (ΦND), reduction status of PSI acceptor side (ΦNA), and maximal P700 change (Pm)) did not significantly decrease under high light for 1 h, compared with the control, in the absence of SA. However, only 3 h of high light led to a remarkable decline in ΦNA and Pm. Although four representative PSI parameters showed no observed differences between the control and SA‐pretreated plants, SA pretreatments significantly improved the value of ΦNA and Pm under high light for 3 h. These results suggested that exogenous SA could protect PSI against the damage from high light in Arabidopsis thaliana. Next, PSII photochemistry was determined using a modulated imaging fluorometer under high light in the presence or absence of SA. As shown in Figure 2, 3 h of high light resulted in a significant decline in maximum efficiency of PSII photochemistry (Fv/Fm), effective quantum yield of PSII electro transport (ΦPSII), and photochemical quenching (qP), and a remarkable increase in nonphotochemical quenching (NPQ) compared with the control in the absence of SA. SA application alleviated the decrease in the Fv/Fm, ΦPSII, and qP and stimulated the increase in NPQ under high light for 3 h relative to the control. This suggests that SA could effectively maintain the photosynthetic capacity in Arabidopsis plants under high light. In addition, we found that SA treatments significantly Int. J.increased Mol. Sci. 2020 the, 21 values, 1229 of quantum yield of nonregulated energy dissipation (Y(NO)) compared to non‐4 of 17 SA treated plants.

Int. J. Mol. Sci. 2020, 21, 1229 4 of 17

transport; NPQ, nonphotochemical quenching; qP, photochemical quenching; Y(NO), quantum yield of nonregulated energy dissipation. The individual fluorescence images with quantitative values (± SD) are presented. HL, high light. SA + HL, high light after SA pretreatment for 3 d. 0–3 h, high light for 0 h, 1 h, and 3 h in the presence or absence of SA pretreatment, respectively.

NPQ is related to nonphotochemical quenching, and is regarded as the key protective mechanism against excess light energy in PSII. As shown in Figure 3, the NPQ induction was more pronounced with the increase of high light treatment. The NPQ induction in SA‐pretreated plants were faster and reached a higher amplitude compared to the non‐SA treated plants under the same high light treatment. The kinetics of dark relaxation were still faster than in non‐SA treated plants under 0 and 3 h of high light. State transitions are a good way to indicate the rate of energy dissipation byFigure monitoringFigure 2. E ff2.ects Effects changes of SAof SA on in on chlorophyll chlorophyll chlorophyll fluorescence fluorescencefluorescence parameters parameters characteristics in inArabidopsisArabidopsis [30]. As thaliana shown thaliana under inunder Figurehigh light. high S2, light. the levelsFv/Fm,Fv/Fm, of maximum Chl maximum a fluorescence effi efficiencyciency in of nonof PSII PSII‐SA photochemistry; photochemistry; treated plant was ΦΦPSIIPSII greater, effective, effective than quantum quantumSA‐treated yield yield ofplants. PSII of PSIIelectro However, electro theretransport; was NPQ,no obvious nonphotochemical difference between quenching; the qP,non photochemical‐SA treated and quenching; SA‐treated Y(NO), plants. quantum In contrast, yield ofa transitorynonregulated increase energy in dissipation.fluorescence The under individual 3 h of high fluorescence light was images presented with when quantitative the far‐red values light ( wasSD) ± turnedare presented. off in SA HL,‐treated high light. plant. SA These+ HL, results high light suggested after SA that pretreatment SA could participate for 3 d. 0–3 in h, regulating high light forthe 0 dissipationh, 1 h, and 3of h excess in the presencelight energy or absence under high of SA light. pretreatment, respectively.

FigureFigure 3. NPQ 3. NPQ kinetics kinetics of ofArabidopsis Arabidopsis thaliana thaliana under high high light. light. Bars Bars on ontop, top, white white bar (light bar (light on) and on) and blackblack bar (dark).bar (dark). The The data data represent represent means means ± SDSD from from four four independent independent biological biological replicates replicates (n = 4). (n = 4). ± HL, highHL, high light. light. SA SA+ HL, + HL, high high light light after after SASA pretreatment for for 3 3d. d. 0–3 0–3 h, high h, high light light for 0 for h, 1 0 h, h, and 1 h, 3 and h 3 h in thein presence the presence or absenceor absence of of SA SA pretreatment, pretreatment, respectively.respectively.

To furtherTo further test test the the roles roles of of SA SA in in improving improving the photosynthetic photosynthetic capacities capacities under under environmental environmental stress,stress, four four gas exchangegas exchange parameters parameters including including the the net net photosynthetic photosynthetic rate rate ((PPn),n), transpirationtranspiration rate rate (Tr), (Tr), intercellular CO2 concentration (Ci), and stomatal conductance (Gs) were determined in intercellular CO concentration (Ci), and stomatal conductance (Gs) were determined in Arabidopsis Arabidopsis plants2 exposed to high light in the presence or absence of SA pretreatment (Figure S3). plantsCompared exposed with to high the lightcontrol, in theSA pretreatments presence or absence significantly of SA decreased pretreatment four gas (Figure exchange S3). parameters Compared with the control,under nonstressful SA pretreatments conditions. significantly At the same decreased time, high four light gas resulted exchange in a parameters remarkable underdecline nonstressful in Pn, conditions.Tr, and G Ats in the the same control time, and high SA‐treated light resulted plants. However, in a remarkable we found decline that SA in treatmentsPn, Tr, and alleviatedGs in the the control and SA-treateddecrease in plants.Pn, Ci, However,Tr, and G wes. These found results that SA further treatments demonstrated alleviated that the SA decrease could improve in Pn, C i, Tr, and Gphotosynthetics. These results efficiency further under demonstrated high light. that SA could improve photosynthetic efficiency under high light. 2.3. PSII Photoinhibition Analysis 2.3. PSII PhotoinhibitionWe tested the sensitivity Analysis of PSII to photoinhibition with or without lincomycin in Arabidopsis plantsWe tested in the the presence sensitivity or absence of PSII of SA to photoinhibitionbecause chlorophyll with fluorescence or without analysis lincomycin revealed in thatArabidopsis SA could alleviate the photodamage to PSII under high light. Lincomycin was used to block PSII repair plants in the presence or absence of SA because chlorophyll fluorescence analysis revealed that SA by inhibiting protein synthesis [31]. The control and SA‐treated plants were illuminated couldwith alleviate heterochromatic the photodamage light for 4 toh, after PSII which under the high samples light. were Lincomycin recovered was at low used intensity to block (10 μ PSIImol repair by inhibitingphotons m chloroplast−2∙s−1) for 21 h. protein Changes synthesis in Fv/Fm [31 and]. Thethe levels control of two and thylakoid SA-treated proteins plants (D1 were and illuminated PsaD) Int. J. Mol. Sci. 2020, 21, 1229 5 of 17 with heterochromatic light for 4 h, after which the samples were recovered at low intensity (10 µmol 2 1 photonsInt. J. m Mol. Sci.s 2020) for, 21 21, 1229 h. Changes in Fv/Fm and the levels of two thylakoid proteins (D1 and5 of 17 PsaD) − · − are presented in Figure4. During 4 h of high light, the values of Fv /Fm for SA-treated plants were significantlyare presented higher in than Figure that 4. During for the 4 control h of high in light, the absence the values and of presence Fv/Fm for of SA lincomycin‐treated plants (Figure were4 A), suggestingsignificantly that thehigher protective than that e forffect the of control the SA in the application absence and was presence on the of concurrent lincomycin (Figure recovery 4A), from suggesting that the protective effect of the SA application was on the concurrent recovery from photoinhibition. Furthermore, the rates of recovery from photoinhibition were similar in control and photoinhibition. Furthermore, the rates of recovery from photoinhibition were similar in control and SA-treatedSA‐treated plants plants (Figure (Figure4B). 4B). Therefore, Therefore, the the greater greater decline decline in Fv/Fm Fv /Fm may may be be attributed attributed to elevated to elevated photosensitivityphotosensitivity in the in control.the control.

FigureFigure 4. PSII 4. PSII photosensitivity photosensitivity of ofArabidopsis Arabidopsis thaliana thaliana in the presence presence or or absence absence of SA of SA pretreatment pretreatment duringduring high high light light illumination. illumination. (A ()A Untreated) Untreated ( (−)) andand lincomycin-treatedlincomycin‐treated (+) ( +detached) detached leaves leaves were were exposed to high‐light (HL) conditions (1000 μ−mol photons m−22∙s−1)1 for 4 h. (B) The photoinhibited exposed to high-light (HL) conditions (1000 µmol photons m− s− ) for 4 h. (B) The photoinhibited detached leaves were recovered at low light (LL) intensity (10 μmol· photons m−2∙s2−1) up1 to 24 h with detached leaves were recovered at low light (LL) intensity (10 µmol photons m− s− ) up to 24 h with regular measurement of Fv/Fm. Values are means ± SD from three independent biological· replicates regular measurement of Fv/Fm. Values are means SD from three independent biological replicates (n (n = 3). (C) Immunoblot analysis of thylakoid ±proteins obtained from Arabidopsis thaliana in the = 3). (C) Immunoblot analysis of thylakoid proteins obtained from Arabidopsis thaliana in the presence presence or absence of SA pretreatment with D1 and PsaD antibodies before (−) and after (+) or absence of SA pretreatment with D1 and PsaD antibodies before ( ) and after (+) photoinhibition photoinhibition using a light intensity of 1000 μmol photons m−2∙s−1 for− 3 h. PsaD was as a loading using a light intensity of 1000 µmol photons m 2 s 1 for 3 h. PsaD was as a loading control. control. − · −

To furtherTo further identify identify these these results results from from photoinhibition, photoinhibition, we we analyzed analyzed the the accumulation accumulation of PSII of PSII reactionreaction center center D1 proteinD1 protein in leavesin leaves from from the the control control andand SA SA-treated‐treated plants plants under under high high light light in the in the presencepresence or absence or absence of lincomycin.of lincomycin. As As shown in in Figure Figure 4C,4C, the the abundance abundance of D1 of protein D1 protein showed showed no no obviousobvious di differencesfferences between between the control and and SA SA-treated‐treated plants plants under under nonstress nonstress condition condition in the in the absenceabsence and presenceand presence of lincomycin.of lincomycin. However, However, afterafter 3 h of of illumination, illumination, the the levels levels of D1 of D1declined declined significantlysignificantly in the in controlthe control and and moderately moderately in in SA-treated SA‐treated plants in in the the absence absence lincomycin. lincomycin. In the In the presence of lincomycin, this effect was more pronounced. In addition, the amount of PSI protein PsaD presence of lincomycin, this effect was more pronounced. In addition, the amount of PSI protein PsaD Int. J. Mol. Sci. 2020, 21, 1229 6 of 17

showedInt. J. no Mol. obvious Sci. 2020, 21 changes, 1229 in all samples (Figure4C and Figure S4). These results indicated6 of 17 that SA could effectively enhance the repair of D1 protein, and thus alleviate the sensitivity of PSII to photoinhibitionshowed no obvious under high changes light. in all samples (Figures 4C and S4). These results indicated that SA could effectively enhance the repair of D1 protein, and thus alleviate the sensitivity of PSII to 2.4. Changesphotoinhibition in Thylakoid under Proteins high light. and Phosphorylation under High Light

To2.4. further Changes verify in Thylakoid the eff Proteinsects of exogenous and Phosphorylation SA on photosynthesis, under High Light immunoblot analysis of thylakoid membrane proteins was carried out in Arabidopsis plants under high light in the presence or absence of To further verify the effects of exogenous SA on photosynthesis, immunoblot analysis of SA (Figure5 and Figure S5). The levels of several PSII proteins changed under high light. Compared thylakoid membrane proteins was carried out in Arabidopsis plants under high light in the presence with theor absence control, of high SA (Figures light resulted 5 and S5). in the The significant levels of several reduction PSII inproteins the amount changed of under D1 and high D2 light. proteins. However,Compared the content with the of control, PsbS was high significantly light resulted increasedin the significant under reduction high light in comparedthe amount toof D1 the and control, especiallyD2 proteins. in SA-treated However, plants. the content The of amount PsbS was of significantly D1 and D2 increased showed diunderfferences high light between compared SA-treated to plantsthe and control, the control especially under in SA high‐treated light, plants. suggesting The amount that SA of couldD1 and e ffD2ectively showed alleviate differences the between degradation of PSIISA reaction‐treated centerplants and proteins the control and increase under high the light, accumulation suggesting of that PsbS SA under could effectively high light. alleviate Although the high light diddegradation not lead of to PSII changes reaction in center the level proteins of Lhcb5 and increase in the absence the accumulation of SA, SA of applicationPsbS under high increased light. the contentAlthough of Lhcb5 high protein light did under not lead high to light changes compared in the level with of Lhcb5 the control. in the absence of SA, SA application increased the content of Lhcb5 protein under high light compared with the control.

FigureFigure 5. Immunoblot 5. Immunoblot analyses analyses of thylakoid of thylakoid proteins proteins obtained obtained from fromArabidopsis Arabidopsis thaliana thalianaunder under highhigh light in thelight presence in the or presence absence or of absence SA pretreatment. of SA pretreatment. (A) Immunoblotting (A) Immunoblotting were done were using done specific using specific antibodies againstantibodies representative against PSIrepresentative and PSII proteins. PSI and PSII (B) Quantitativeproteins. (B) Quantitative data for D1, data D2, for Lhcb5, D1, D2, and Lhcb5, PsbS and proteins in ArabidopsisPsbS proteins thaliana in Arabidopsisunder high thaliana light withunder or high without light SAwith pretreatment. or without SA Results pretreatment. are presented Results are relative presented relative to the amount of the respective control (HL 0h, 100%). Significantly different values to the amount of the respective control (HL 0 h, 100%). Significantly different values are marked with are marked with an asterisk (*) at p < 0.05 level (n = 4). HL, high light. SA + HL, high light after SA an asterisk (*) at p < 0.05 level (n = 4). HL, high light. SA + HL, high light after SA pretreatment for 3 d. pretreatment for 3 d. 0–3 h, high light for 0 h, 1 h, and 3 h in the presence or absence of SA 0–3 h, high light for 0 h, 1 h, and 3 h in the presence or absence of SA pretreatment, respectively. pretreatment, respectively. The levels of thylakoid membrane protein phosphorylation were further analyzed under high light in the presence or absence of SA. As presented in Figure6 and Figure S6, the phosphorylation Int. J.Int. Mol. J. Sci.Mol.2020 Sci. 2020, 21,, 122921, 1229 7 of 17 7 of 17

The levels of thylakoid membrane protein phosphorylation were further analyzed under high levellight of CP43 in the and presence LHCII or showed absence of no SA. obvious As presented differences in Figures under 6 and high S6, light the phosphorylation in the presence level or absence of of SACP43 compared and LHCII to that showed of the no control. obvious However,differences theunder significant high light accumulation in the presence of or phosphorylated-D1 absence of SA (P-D1)compared and P-D2 to that was of observedthe control. under However, high the light significant in the accumulation absences of of SA phosphorylated compared with‐D1 the(P‐D1) control. Furthermore,and P‐D2 SA was pretreatments observed under remarkably high light upregulated in the absences the levels of SA of compared P-D1 and P-D2with underthe control. high light for 3Furthermore, h (Figure6A SA and pretreatments Figure S6). remarkably upregulated the levels of P‐D1 and P‐D2 under high light for 3 h (Figures 6A and S6).

FigureFigure 6. Thylakoid 6. Thylakoid protein protein phosphorylation phosphorylation of ofArabidopsis Arabidopsis thaliana underunder high high light light in in the the presence presence or absenceor absence of SA. of (A SA.) Immunoblotting (A) Immunoblotting of of thylakoid thylakoid membrane membrane proteins proteins analysis analysis was was performed performed using using antiphosphothreonine antibodies. (B) Coomassie blue staining (CBS) of SDS‐PAGE was presented in antiphosphothreonine antibodies. (B) Coomassie blue staining (CBS) of SDS-PAGE was presented in the bottom panel. HL, high light. SA + HL, high light after SA pretreatment for 3 d. 0–3 h, high light the bottom panel. HL, high light. SA + HL, high light after SA pretreatment for 3 d. 0–3 h, high light for 0 h, 1 h, and 3 h in the presence or absence of SA pretreatment, respectively. for 0 h, 1 h, and 3 h in the presence or absence of SA pretreatment, respectively. Photoinhibition leads to photosynthetic imbalance through the photosynthetic electron transportPhotoinhibition chain, which, leads in to turn, photosynthetic causes excess imbalance reactive oxygen through species the photosynthetic (ROS) formation. electron Direct color transport chain,staining which, with in turn, NBT causes (blue) excessand DAB reactive (orange) oxygen were species used to (ROS) detect formation. the oxidative Direct stress color of detached staining with NBTleaves (blue) in and the DAB process (orange) of photoinhibition were used to detect(Figure the S7). oxidative NBT and stress DAB of staining detached indicated leaves inthat the the process of photoinhibitiondetached leaves (Figure under high S7). light NBT accumulated and DAB staining more O2ˉ indicatedand H2O2 compared that the detached to the control, leaves while under SA high – lightapplication accumulated alleviated more Othe2 productionand H2O2 ofcompared ROS under to high the light. control, while SA application alleviated the production of ROS under high light. 2.5. Alterations in Oxygen Evolution, Thylakoid Membrane Complexes, and Chloroplast Organization 2.5. AlterationsNext, we in Oxygeninvestigated Evolution, the activity Thylakoid and integrity Membrane of thylakoid Complexes, by andmeasuring Chloroplast of oxygen Organization evolution ratesNext, and we investigatedthylakoid membrane the activity complexes and integrity (Figure of 7). thylakoid When compared by measuring to the of control oxygen plants, evolution SA rates pretreatments did not result in the significant changes in the rates of oxygen evolution under and thylakoid membrane complexes (Figure7). When compared to the control plants, SA pretreatments nonstressed conditions. However, high light significantly decreased the rate of oxygen evolution in did not result in the significant changes in the rates of oxygen evolution under nonstressed conditions. the presence and absence of SA relative to the control (Figure 7A). Compared with SA‐pretreated However,plants, high non‐ lightSA‐pretreated significantly plants decreased presented the a lower rate of rate oxygen of oxygen evolution evolution, in the suggesting presence that and SA absence of SAapplication relative to could the enhance control the (Figure activity7A). of Comparedthylakoid membrane with SA-pretreated under high light. plants, In addition, non-SA-pretreated changes plantsin presentedthe organization a lower of thylakoid rate of oxygen membrane evolution, complexes suggesting were also analyzed that SA application by BN‐PAGE could in Arabidopsis enhance the activityplants of thylakoidunder high membrane light in the presence under high or absence light. In of addition,SA. As shown changes in Figure in the 7B, organization SA pretreatment of thylakoid did membranenot result complexes in the obvious were changes also analyzed in thylakoid by BN-PAGE complexes in relativeArabidopsis to the plantscontrol under under highnonstressed light in the presencecondition. or absence Compared of SA. with As the shown control, in only Figure high7 B,light SA for pretreatment 3 h significantly did reduced not result the in amount the obvious of changesPSII‐ inLHCII thylakoid super complexes, complexes LHCII relative assembly, to the and control LHCII under trimer nonstressed in the absence condition. of SA, while Compared a marked with increase in LHCII monomer was observed under 3 h of high light. However, SA application the control, only high light for 3 h significantly reduced the amount of PSII-LHCII super complexes, significantly alleviated the decline in the amount of PSII‐LHCII super complexes, LHCII assembly, LHCIIand assembly, LHCII trimer and in LHCII Arabidopsis trimer plants in the exposed absence to ofhigh SA, light while for 3 a h marked (Figure increase7C). in LHCII monomer was observed under 3 h of high light. However, SA application significantly alleviated the decline in the amount of PSII-LHCII super complexes, LHCII assembly, and LHCII trimer in Arabidopsis plants exposed to high light for 3 h (Figure7C). Int. J. Mol. Sci. 2020, 21, 1229 8 of 17 Int. J. Mol. Sci. 2020, 21, 1229 8 of 17

FigureFigure 7. Oxygen 7. Oxygen evolution evolution and thylakoidand thylakoid membrane membrane complexes complexes analysis analysis of thylakoid of thylakoid proteins proteins obtained from obtainedArabidopsis from thaliana Arabidopsisunder thaliana high light under in high the presence light in the or absencepresence ofor SA.absence (A) Oxygenof SA. (A evolution) Oxygen rates evolution rates of thylakoid membranes were measured at 20 °C with 0.5 mM phenyl‐p‐benzoquinone of thylakoid membranes were measured at 20 ◦C with 0.5 mM phenyl-p-benzoquinone under saturating light intensities.under saturating The data light represent intensities. means The dataSD represent (standard means deviations) ± SD (standard from four deviations) independent from biological four independent biological replicates (n = 4).± Different lower‐case letters indicate significant differences replicates (n = 4). Different lower-case letters indicate significant differences (p < 0.05) according to (p < 0.05) according to Duncan’s multiplication range test. (B) BN‐PAGE of thylakoid membranes was Duncan’s multiplication range test. (B) BN-PAGE of thylakoid membranes was performed using performed using 5–12.5% acrylamide after solubilization using 1% (w/v) DM. NDH, NAD(P)H 5–12.5% acrylamide after solubilization using 1% (w/v) DM. NDH, NAD(P)H dehydrogenase; PS, dehydrogenase; PS, photosystem; LHC, light‐harvesting complex; Cyt b6/f, b6/f; mc, photosystem;megacomplex; LHC, sc, light-harvesting super complex. complex;(C) Quantitative Cyt b6 /fdata, cytochrome for PSII‐LHCIIb6/f ; mc,super megacomplex; complexes, LHCII sc, super complex.trimer, (C LHCII) Quantitative assembly, dataand LHCII for PSII-LHCII monomer in super Arabidopsis complexes, thaliana LHCII under trimer,high light LHCII with or assembly, without and LHCIISA monomer pretreatment. in Arabidopsis Results are shown thaliana relativeunder to high the amount light with of the or respective without SAcontrol pretreatment. (HL 0h, 100%). Results are shownSignificantly relative different to the values amount are ofmarked the respective with an asterisk control (*) at (HL p < 00.05 h, 100%).level (n = Significantly 4). HL, high light. different valuesSA are + HL, marked high light with after an SA asterisk pretreatment (*) at p for< 0.053 d. 0–3 level h, high (n = light4). HL,for 0 highh, 1 h, light. and 3 SAh in+ theHL, presence high light afteror SA absence pretreatment of SA pretreatment, for 3 d. 0–3 respectively. h, high light for 0 h, 1 h, and 3 h in the presence or absence of SA pretreatment, respectively. To further study the roles of SA in protecting thylakoid structures under environmental stresses, Tothe further chloroplast study ultrastructure the roles of was SA analyzed in protecting with transmission thylakoid structures electron microscopy under environmental under high light stresses, for 3 h in the presence or absence of SA. Relative to the control plants, SA pretreatment did not lead the chloroplast ultrastructure was analyzed with transmission electron microscopy under high light to obvious changes in the thylakoid structures under nonstressed condition (Figure 8), while a for 3 h in the presence or absence of SA. Relative to the control plants, SA pretreatment did not lead to significant increase in the number of starch granules was identified under 3 h of high light in the obviousabsence changes of SA. in However, the thylakoid SA application structures reduced under nonstressedthe number of condition starch granules (Figure in8 Arabidopsis), while a significantplants increaseunder in high the number light for 3 of h starch compared granules with non was‐SA identified‐treated plants under (Figure 3 h of S8). high light in the absence of SA. However, SA application reduced the number of starch granules in Arabidopsis plants under high light for 3 h compared with non-SA-treated plants (Figure S8). Int. J. Mol. Sci. 2020, 21, 1229 9 of 17 Int. J. Mol. Sci. 2020, 21, 1229 9 of 17

FigureFigure 8. Transmission 8. Transmission electron electron microscope microscope analysis analysis of of chloroplasts in inArabidopsisArabidopsis thaliana thaliana underunder high high lightlight in the in presence the presence or absenceor absence of of SA. SA. HL HL 0 0h, h, highhigh light for for 0 0 h h without without SA SA pretreatment. pretreatment. SA+HL SA+ 0h,HL 0 h, highhigh light light for 0 for h with0 h with SA SA pretreatment. pretreatment. HL HL 3 3 h, h, high high light light for 3 h without without SA SA pretreatment. pretreatment. SA SA + HL+ HL 3 3 h, high light for 3 h with SA pretreatment. Scale bar represents 1 μm in each figure. h, high light for 3 h with SA pretreatment. Scale bar represents 1 µm in each figure.

3. Discussion3. Discussion It is widely known that high light is one of the key environmental stresses in the natural It is widely known that high light is one of the key environmental stresses in the natural environment, and that it influences many aspects of physiological and metabolic processes, including environment, and that it influences many aspects of physiological and metabolic processes, including antioxidant response, gene induction, and photosynthesis [32,33]. Although PSI may be damaged by antioxidanthigh light, response, potential gene PSI activity induction, has a and high photosynthesis tolerance to extreme [32, 33light]. Although intensity relative PSI may to PSII be damaged [32,34]. by highThe light, alterations potential in PSI PSII activity activity has were a highmainly tolerance investigated to extreme in Arabidopsis light intensity thaliana under relative high to light. PSII In [32 ,34]. The alterationsplants, SA is in an PSII important activity phytohormone were mainly and investigated may regulate in manyArabidopsis key physiological thaliana underand biochemical high light. In plants,processes, SA is an especially important in phytohormone response to many and abiotic may regulate stresses many such keyas salinity physiological and drought and biochemical stress processes,[13,35,36]. especially A previous in response study indicated to many that abiotic SA stressescould play such an as essential salinity role and in drought the acclimatization stress [13,35 ,36]. A previousprocesses study and indicatedin the redox that homeostasis SA could playunder an high essential light [33]. role In in the the present acclimatization study, we investigated processes and in the redoxthe regulatory homeostasis roles under of exogenous high light SA [in33 ].photoprotection In the present of study, PSII in we Arabidopsis investigated thaliana the regulatoryunder high roles light. of exogenous SA in photoprotection of PSII in Arabidopsis thaliana under high light. Photosynthetic pigments are among the most important indicators of responses to Photosynthetic pigments are among the most important indicators of responses to environmental environmental stresses [9,33,37,38]. In the present study, although Chl a/b showed no obvious stresseschanges [9,33 ,under37,38 ].high In the light, present total Chl study, contents although significantly Chl a/b showeddeclined nounder obvious high light. changes The underreason high may light, totalbe Chl that contents the biosynthesis significantly of chlorophyll declined was under inhibited high light. under The environmental reason may stresses. be that Previous the biosynthesis studies of chlorophyllreported was that inhibited SA application under environmental can protect the stresses. degradation Previous of studiesphotosynthetic reported pigments that SA applicationunder can protectenvironmental the degradation stresses [25,39]. of photosynthetic In agreement with pigments these findings, under our environmental results indicated stresses that exogenous [25,39 ]. In agreementSA can with minimize these the findings, degradation our results of pigments indicated under that high exogenous light. SA can minimize the degradation of pigmentsA previous under high study light. indicated that PSI is less susceptible to damage than PSII, and PSI gets Adamaged previous only study when indicated electron flow that from PSI is PSII less exceeds susceptible the processing to damage capacity than of PSII, PSI and[40]. PSI In agreement gets damaged with this report, the present experiment showed that the significant decline in PSI activity (ΦND, ΦNA, only when electron flow from PSII exceeds the processing capacity of PSI [40]. In agreement with and Pm) only was found under high light for 3 h. In the present experiment, the high ΦND and ΦNA this report, the present experiment showed that the significant decline in PSI activity (Φ , Φ , observed under high light for 3 h in the presence of SA was probably because SA may regulateND the NA and Pphotooxidationm) only was found rate and under the reduction high light rate for of 3 P700 h. In under the present severe environmental experiment, the stress. high InΦ addition,ND and ΦNA observedthe marked under decrease high light in photo for 3‐oxidizable h in the presence PSI (Pm) ofwas SA probably was probably the result because of a permanent SA may reduction regulate the photooxidationof the PSI acceptor rate and side the under reduction 3 h of rate high of light. P700 Here, under we severe found environmentalthat SA can effectively stress. Inprotect addition, PSI the markedagainst decrease the damage in photo-oxidizable from extreme light PSI conditions. (Pm) was probably the result of a permanent reduction of the PSI acceptorMany side studies under including 3 h of high our light. recent Here, works we found have thatdemonstrated SA can eff ectivelythat high protect irradiance PSI against may the damagesignificantly from extreme decrease light PSII conditions. activities such as Fv/Fm, ΦPSII, and qP in some plants [11,32,41]. Previous Manyreports studies have indicated including that our SA recent application works havecan protect demonstrated the function that highof PSII irradiance under environmental may significantly decrease PSII activities such as Fv/Fm, ΦPSII, and qP in some plants [11,32,41]. Previous reports have indicated that SA application can protect the function of PSII under environmental stresses [25,36]. Consistent with these findings, our results showed that SA treatments obviously upregulated the levels Int. J. Mol. Sci. 2020, 21, 1229 10 of 17

of Fv/Fm, ΦPSII, and qP under 3 h of high light. Under high light combined with SA application, the slight reduction in Fv/Fm and ΦPSII was probably because exogenous SA may protect the PSII reaction center under environmental stresses. The low decline in qP under high light in the presence of SA could be because SA alleviated the photodamage to the organization of the thylakoid protein complex. In the present study, these results were further demonstrated by the data from NPQ and Y(NO). NPQ, which is nonphotochemical quenching, is regarded to be the key protective mechanism against excess light energy in PSII [42]. The high value of NPQ observed under high light combined with SA was probably because exogenous SA participates in regulating dissipating light energy, and thus, enhances a suboptimal capacity of photoprotective reaction under high light. Y(NO) is the quantum yield of nonregulated energy dissipation in PSII. The increase of Y(NO) in SA-pretreated plants was probably because exogenous SA could participate as an uncoupler in regulating the dissipation of the nonregulated light energy [43]. Therefore, our results suggest that exogenous SA may maintain the photosynthetic activities by protecting the PSII reaction center or dissipating excessive light energy under high light. The ability of SA-pretreated and non-SA-pretreated plants to undergo nonphotochemical quenching upon exposure to high light was further confirmed. In the present study, we found that under 3 h of high light, the dark recovery became slower, suggesting that long-term high light could severely damage the protective mechanism of PSII in Arabidopsis plants. However, we noticed that exogenous SA application significantly amplified the dark recovery under 3 h of high light, indicating that SA application has a regulatory role in dissipating excess light energy, thereby reducing the damage of photoinhibition to PSII in Arabidopsis under high light. In addition, state transition is also an important light-adaptation mechanism in an imbalance of excitation energy between PSI and PSII, depending on the reduction status of under environmental stresses [44]. In the present experiment, our results showed that exogenous SA application increased fluorescence under 3 h of high light when far-red light was turned off. The reason for this was probably because that SA could effectively regulate the state of LHCII between unquenched and quenched through protein reversible phosphorylation under high light. It is well known that photosynthesis is influenced by stomatal opening under environmental stresses. Many studies have indicated that the application of SA can improve the net photosynthetic rate transpiration rate, and stomatal conductance in many plants under abiotic stress [19,36,45]. Consistent with these reports, our results indicated that exogenously applied SA increased the net photosynthetic rate, intercellular CO2 concentration, transpiration rate, and stomatal conductance under high light. The reason for this may be that SA plays an important role in regulating the stomatal status, and thereby, improves photosynthetic capacity as a regulator of photosynthesis under environmental stresses. Photoinhibition is the result of an imbalance between the rate of PSII damage and its repair [17]. It has been reported that the increase of photoinhibition is more likely caused by the suppression of PSII repair than by the enhanced photodamage to PSII under high light [16,17]. A previous study indicated that SA plays an essential role in light acclimation processes [33]. In the present study, we found that Fv/Fm in SA-treated plants were significantly higher than that of the control in the absence and presence of lincomycin during high light for 4 h, suggesting that SA alleviated the photoinhibition of PSII. The reason for this was probably because SA could regulate the dissipation of excess light, and thus protect PSII reaction center from the attack of ROS (reactive oxygen species) which accumulate under high light. In addition, photoinhibition is accompanied with oxidative damage to D1 protein that is necessary for the PSII repair cycle [8]. It was also reported that exogenous SA could maintain relative contents of D1 protein and induce Deg1 protease in Satsuma mandarin leaves under strong light-induced photodamage [46]. Deg1 protease participates in PSII protein complex assembly by interacting with the PSII reaction center D2 protein [47]. In the present experiment, our results showed that SA application alleviated the decline in the level of D1 protein in the absence or presence of lincomycin under high light, which was consistent with the changes in Fv/Fm. This may be because Int. J. Mol. Sci. 2020, 21, 1229 11 of 17

SA may effectively enhance the repair of D1 protein and promote the assembly of the PSII reaction center under high light. Other works have indicated that the PSII reaction center proteins are the main targets that are hampered by ROS under environmental stresses including high light, thereby leading to a decrease in PSII activity [32,48,49]. In accordance with these findings, our results showed that the contents of D1 and D2 were significantly decreased under high light for 3 h. This decline may because high light impaired PSII reaction center. However, we found that the application of exogenous SA alleviated the decline in the levels of PSII reaction center proteins under high light, indicating that SA could retard the degradation of PSII core proteins under high light, thereby possibly contributing to high tolerance to photoinhibition in Arabidopsis plants. Our results were consistent with a previous study, in which application of 0.3 mM SA alleviated the reduction of D1 protein in wheat plants under heat and high light stress [50]. This is mainly through accelerating the turnover of D1 protein in PSII by inducing PsbA gene transcription, as PsbA is responsible for the regeneration and replacement of D1 protein that has been injured under stress. Lhcb5 is a component of the light-harvesting antenna and plays a role in energy dissipation [51]. In the present study, exogenous SA application increased the amount of Lhcb5 under high light, implying that SA may reduce the damage of photoinhibition to PSII by enhancing the components related to heat dissipation. PsbS is a small subunit of PSII and play a key role in qE, which is the ∆pH-dependent component of NPQ [52]. Our previous work indicated that long-term drought stress upregulated the levels of PsbS protein in Arabidopsis [9]. In accordance with this finding, our results showed that the amount of PsbS was significantly increased under high light. However, SA application lowered the increase in the content of PsbS under high light, suggesting that SA probably plays an important role in regulating excitation energy dissipation by adjusting the level of PsbS. Phosphorylation of thylakoid membrane proteins including D1, D2, CP43, and LHCII plays important regulatory roles in the PSII repair cycle and the energy balance between PSI and PSII in response to different environmental stresses [32,53–55]. A previous study demonstrated that D1 and D2 phosphorylation is related to the degradation of damaged proteins of PSII reaction center with insertion of new synthetic proteins into PSII [56]. Consistent with these findings, our results showed that high light significantly increased the levels of phosphorylated D1 and D2. Furthermore, we found that SA application maintained higher phosphorylation of D1 and D2 proteins under high light, implying that SA could promote the phosphorylation of PSII reaction center proteins, and then maintain the content of D1 and D2 protein under environmental stress. In addition, our work has indicated that a different disassembly of PSII-LHCII super complexes and the LHCII assembly occurred in response to different environmental stresses [9,32,57]. In the present experiment, we found that PSII-LHCII super complexes, LHCII assembly, and LHCII trimer were obviously disassembled in Arabidopsis under 3 h of high light, suggesting that these thylakoid membrane complexes are sensitive to long-term high light. However, SA application alleviated the reduction of these thylakoid complexes under high light for 3 h, indicating that SA may maintain the structures of PSII complexes effectively under high light. These findings were further demonstrated by the results obtained from oxygen evolution rate and thylakoid structure. Starch is a photosynthetic product synthesized in chloroplasts in many higher plants. Evidence has shown that the accumulation of starch granules might contribute to thylakoid stability and normal photosynthetic phosphorylation by maintaining a high sugar concentration near the [58]. In the present study, SA treatment increased the accumulation of starch granules under normal growth conditions, which may be more conducive to maintaining the stability of the photosynthetic apparatus. However, the accumulation of photosynthetic products may inhibit the photosynthesis rate, resulting in the repression of photosynthesis [59], which could explain the decrease in the net photosynthetic rate caused by SA treatment under normal growth conditions. Int. J. Mol. Sci. 2020, 21, 1229 12 of 17

4. Material and Methods

4.1. Plant Materials and Stress Treatments Arabidopsis thaliana ecotype Columbia (Col-0) plants were grown in a growth chamber with 8 h dark/16 h light cycles, irradiance of 120 µmol photons m 2 s 1, relative humidity of 70% and a − · − temperature of 23 1 C. After four weeks, the Arabidopsis plants were subjected to different treatments. ± ◦ Six treatments consisting of two SA concentrations (0 and 0.3 mM) and three different times (0, 1, and 3 h) under high light (1000 µmol photons m 2 s 1) were conducted as follows: CK (distilled water); − · − SA (distilled water with 0.3 mM SA); HL 1 h (high light for 1 h); SA + HL 1 h (high light for 1 h in the presence of 0.3 mM SA pretreatment); HL 3 h (high light for 3 h); SA + HL 3 h (high light for 3 h in the presence of 0.3 mM SA pretreatment). The SA was dissolved in ethanol with 0.1% Tween-20, and then 0.3 mM SA was obtained using double-distilled water. The SA solution was sprayed on the Arabidopsis leaves twice daily for three days. The effective concentration of SA and way of application were selected based on our previous work [9,60]. After three days, high light treatments were imposed on Arabidopsis thaliana. For each assay, three to five pots of rosette leaves were used for each treatment.

4.2. Measurements of Chlorophyll and Carotenoid Contents Chlorophyll (Chl) and carotenoid contents were determined according to a previous method [61]. Briefly, 0.5 g of fresh samples was taken and ground using 80% acetone in combination with 0.1% SiO2 and 0.1% CaCO3 at room temperature. After grinding, the homogenate was filtered through the filter paper, and then the absorbance of the filter solution was recorded at 470, 645, and 663 nm with a spectrophotometer (Hitachi-U2000, Hitachi, Ltd., Tokyo, Japan).

4.3. Measurements of P700 Parameters and Chl Fluorescence Measurements of P700 redox state was performed with a Dual PAM-100 fluorometer (Heinz-Walz Instruments, Effeltrich, Germany) according to the manufacturer’s instructions. Before the measurements, Arabidopsis plants were adapted for 30 min in the dark. P700 parameters, including the photochemical quantum yield of PSI (ΦPSI), oxidation status of PSI donor side (ΦND), and reduction status of PSI acceptor side (ΦNA), were calculated based on the method described by Klughammer and Schreiber [62]. The PSII photochemistry was measured using modulated imaging PAM M-series chlorophyll fluorescence system (Heinz-Walz Instruments, Effeltrich, Germany) according to our methods published previously [55]. Prior to the measurements, plants were dark adapted for at least 30 min. The actinic light was at 145 µmol m 2 s 1, and the saturating pulse intensity was at 8000 µmol m 2 s 1. The − · − − − maximum efficiency of PSII photochemistry (Fv/Fm), quantum yield of PSII electron transport (ΦPSII), nonphotochemical quenching (NPQ), photochemical quenching (qP), and the quantum yield of nonregulated energy dissipation (Y(NO)) were obtained based on the previous methods [63,64]. Measurements of NPQ kinetic and state transition were carried out with whole plants using a Dual PAM-100 fluorometer (Heinz-Walz Instruments, Effeltrich, Germany) following established protocols [55]. Prior to measurements, Arabidopsis plants were kept in the dark for 1 h. The Fm value measured from an untreated plant was used to calculate the NPQ kinetics. Fm level in State I (Fm’) and State II (Fm”) was obtained by the application of the saturating light pulse at the end of each state transition cycle.

4.4. Measurements of Gas Exchange Parameters and Oxygen Evolution Gas exchange parameters of leaves were determined immediately after treatments in steady-state conditions using a portable photosynthesis system (PP systems TPS-1, Hitchin, UK). The conditions during the measurements were set to 120 µmol photons m 2 s 1, 360 µmol mol 1 CO content, and − · − − 2 60–80% relative humidity in the assimilation chamber at leaf temperature 25 ◦C[3]. Int. J. Mol. Sci. 2020, 21, 1229 13 of 17

Oxygen evolving activities of thylakoid membranes from different treatments were determined using a Clark-type electrode (Hansatech, Norfolk, United Kingdom) at 20 ◦C under saturating light based on the method of Chen et al. [55]. The assay media was 25 mM HEPES (pH 7.6, KOH) buffer containing 0.2 M sucrose, 10 mM NaCl, 5 mM CaCl2, and a final concentration of 0.5 mM phenyl-p-benzoquinone (PpBQ) as the artificial electron acceptor.

4.5. Analysis of Photoinhibition and Recovery The sensitivity of PSII to high light, measured in terms of changes in Fv/Fm, was analyzed using the intact leaves of the control and 3 h of high light plants in the presence or absence of lincomycin [55]. In order to determine PSII recovery under prevailing photoinactivation, the leaves were subjected 2 1 to polychromatic light conditions (1000 µmol photons m− s− ) for 4 h followed by light transition 2 1 · from high to low (10 µmol photons m− s− ) for 24 h. Fv/Fm was measured using the Dual PAM-100 fluorometer. To evaluate the loss of D1 during photoinhibition, thylakoid proteins extracted from the detached leaves were separated by SDS-PAGE and then immunoblotted with specific D1 antibody. DAB and NBT were used to indicate the accumulation of H2O2 and superoxide anions respectively in detached leaves with different treatments [55]. Each measurement was done at least three times.

4.6. Immunoblotting Analysis Thylakoid membrane proteins were isolated from Arabidopsis leaves with 10 mM NaF under dim light following the method of Chen et al. [65]. After measuring Chl concentrations from thylakoid membrane extracts, thylakoid proteins containing equal chlorophyll were separated by 15% SDS-PAGE with 6 M urea and then transferred to polyvinylidene difluoride (PVDF) membrane (Immobilone, Millipore, Darmstadt, Germany). Some PSI and PSII proteins were detected by specific antibodies against Lhca1-3, PsaD, CP43, D1, D2, Lhcb1-6, and PsbS, which were purchased from Agrisera (Umea, Sweden). In addition, phosphorylation of thylakoid membrane proteins was analyzed using an antiphosphothreonine antibody (Cell Signaling, Ipswich, MA, USA). For the detection of the immunoblots, horseradish peroxidase-conjugated secondary antibody (Agrisera, Umea, Sweden) and the ECL reagent (GE Healthcare Buckinghamshire, UK) were used. Quantification of the immunoblots was done using quantity one software (Bio-Rad Comp. Hercules, CA, USA).

4.7. Blue Native PAGE Blue native polyacrylamide gel electrophoresis (BN-PAGE) of thylakoid complexes was done according to the method of Chen et al. [65]. Thylakoid membranes (20 µg Chl) were solubilized with 1% (w/v) n-dodecyl-β-D-maltoside with continuous gentle mixing for 10 min on the ice in the dark. After centrifugation (18,000 g for 20 min) at 4 ◦C, the samples were separated on a gradient of 5–12.5% acrylamide in the separation gel. BN-PAGE was carried out with a gradually increasing voltage (from 75 to 200 V) for 3–4 h at 4 ◦C. Quantitative analysis of the thylakoid membrane complexes was performed using quantity one software (Bio-Rad Comp. Hercules, CA, USA).

4.8. Electron Microscopy Arabidopsis leaves were fixed in glutaraldehyde (2.5%, v/v) for overnight. Post fixation was done for 1–2 h by soaking the samples in 1% (v/v) osmium tetroxide. Then, the samples were dehydrated in series acetone and subsequently embedded in Epon 812. Thin sections were cut using the ultramicrotome (Ultracut F-701704, Reichert-Jung, Reichert, Austria) and negatively stained with uranyl acetate (2%) on glow discharged carbon-coated copper grids. The ultrastructures of chloroplast were observed with the TEM H-9500 electron microscope (Itachi, Tokyo, Japan) at 75 kV. A quantitative analysis of the thylakoid membrane complexes was performed using the Quantity One software (Bio-Rad Comp. Hercules, CA, USA). Analysis for the area of chloroplast and starch granule was performed using ImageJ [66]. Int. J. Mol. Sci. 2020, 21, 1229 14 of 17

4.9. Statistical Analysis At least three independent replicates were carried out and values were expressed as means ± standard deviation (SD). ANOVA analysis was done with the SPSS Statistics 19.0 software (IBM, Chicago, IL, USA). Duncan’s multiplication range test was used to compare differences between the means among treatments when p < 0.05.

5. Conclusions In the present study, our results suggest that the application of SA significantly alleviated the photodamage to PSII by enhancing the repair of D1, accelerating the phosphorylation of PSII reaction center proteins, and efficiently dissipating excess excitation energy under high light. Therefore, we propose that SA plays an important regulatory role in the photoprotection of PSII and in improving photosynthetic efficiency under environmental stresses.

Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/21/4/1229/s1, Figure S1. Effects of SA on PSI photochemistry in Arabidopsis thaliana under high light. Figure S2. Assays of state transitions in Arabidopsis thaliana under high light in the presence or absence of SA pretreatment. Figure S3. Effects of SA on gas exchange parameters in Arabidopsis thaliana under high light. Figure S4. Quantitative data for D1 and PsaD of detached leaves in the presence or absence of SA pretreatment before untreated ( ) and after 2 1 − lincomycin-treated (+) photoinhibition using a light intensity of 1000 µmol photons m− s− for 3 h. Figure S5. The SDS-PAGE results after Coomassie blue staining (CBS). Figure S6. Quantitative data· for thylakoid protein phosphorylation in Arabidopsis thaliana under high light in the presence or absence of SA pretreatment. Figure S7. Oxidative stress analysis of Arabidopsis thaliana in the presence or absence of SA pretreatment before untreated 2 1 ( ) and after lincomycin-treated (+) photoinhibition using a light intensity of 1000 µmol photons m− s− for 3 h. Figure− S8. Quantitative data for transmission electron microscope analysis of chloroplasts in Arabidopsis· thaliana under high light in the presence or absence of SA. Author Contributions: Y.-E.C. conceived and designed this experiment. Y.-E.C., H.-T.M. and N.W. performed the experiments. H.-Y.Z. and S.Y. helped to analyze the data. Y.-E.C., A.M.U.D. and A.K. wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Sichuan Province Academic and Technical Leaders Fund: num; Xichang Municipal Science and Technology Program: 18JSYJ09; Sichuan Science and Technology Program: 2018HH0129; Sichuan Science and Technology Program: 2019ZHFP0128. Acknowledgments: We thank Shozeb Haider (University College London) for critical reading and language polishing during the revision of this manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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