Light increases resistance of thylakoid membranes to thermal inactivation

Light increases resistance of thylakoid membranes to thermal inactivation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Light increases resistance of thylakoid membranes to thermal inactivation Elena Lovyagina, Oksana Luneva, Aleksey Loktyushkin, Boris Semin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4019854/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Oct, 2024 Read the published version in Journal of Plant Research → Version 1 posted 4 You are reading this latest preprint version Abstract In the region of slightly acidic pH (рН 5.7), the manganese cluster in oxygen-evolving complex of photosystem II (PSII) is more resistant to exogenous reductants (Semin et al. 2015 ). The effect of such pH on the heat inactivation efficiency of the electron transport chain (O 2 evolution and 2,6-dichlorophenolindophenol reduction) in PSII membranes and thylakoid membranes was investigated. Under thylakoid membranes illumination accompanied by lumen acidification, their resistance to heat inactivation increases. In the presence of protonophores, the rate of heat inactivation increases, which seems to be associated not with the protonophore mechanism, but with structural and/or functional changes in membranes. In PSII membrane preparations, the efficiency of the oxygen evolution inhibition at pH 5.7 is also lower than at pH 6.5. The role of reactive oxygen species in thermal inactivation of photosynthetic membranes was investigated using a lipophilic cyclic hydroxylamine ESR spin probe. photosystem II protonophores oxygen-evolving complex calcium heat inactivation hydroxylamine spin probe Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction During the investigation of the mechanism of Fe(II) cations binding to Mn-binding sites in the oxygen-evolving complex (OEC), it was found highly efficient binding of these cations to the Mn-binding sites in Mn-depleted PSII membranes (Semin et al. 1995 ; Semin et al. 2002 ). Moreover, if OEC is available for exogenous factors (in the absence of extrinsic proteins PsbP and PsbQ) ferrous cations (strong reducing factor) are able to reach Mn cluster, interact with Mn cations and reduce part of Mn cations in OEC in the dark. As a result of this process, the reduced cations Mn(II) leave the binding site and the open Mn-binding sites are occupied by Fe(III) cations which leads to the formation in PSII chimeric clusters consisting of iron and manganese cations 2Mn2Fe (Semin and Seibert 2016 ) or 3Mn1Fe (Semin et al. 2018 ). Investigating this process, we have found that the substitution of Mn cations in OEC with Fe(II) cations depends on the pH of the buffer. At pH 6.5, Fe(II) cations replace 2 Mn cation, while at pH 5.7 only one Mn cation is replaced (Semin and Seibert 2016 ; Semin et al. 2018 ). A similar situation occurs in the case of other reducing agents - hydroquinone (H 2 Q) and hydrogen peroxide (Semin et al. 2015 ): at pH 6.5 these reductants extracted 3 Mn cations, whereas at pH 5.7 only 2 Mn cations were extracted. Thus, the redox potential of one of the Mn cations in the OEC strongly depends on the pH of the medium, which can increase the resistance of the manganese cluster to the action of reducing agents in the region of weakly acidic pH. A such effect may occur in the case of photoinhibition. It is known that the Mn cluster in the OEC of PSII can be effectively destroyed during the photoinhibition and, possibly, it is the first step in this process (Tyystjärvi 2008 ; Zavafer et al. 2015 ). Destruction of OEC manganese cluster may be the result of its exposure to some reactive oxygen species (ROS) O 2 ∙− and H 2 O 2 produced on the donor and acceptor sides of PSII during illumination (Pospišil 2012 ). Indeed, we found that at pH 5.7, the photoinactivation rate is less compared to pH 6.5 whereas in the Mn-depleted PSII membranes, photoinhibition does not depend on pH (Davletshina and Semin 2020 ). Moreover, the rate of photoinhibition of the thylakoid membranes (TM) was significantly increased in the presence of the uncouplers that changed the pH of lumen – the place of the OEC location. Yamashita et al. ( 2008 ) showed that ROS, apparently participate not only in photoinhibition, but also in the process of thermal inactivation of photosynthetic membranes. ROS are formed, apparently as a result of membrane lipids peroxidation initiated by heating and take part in heat inactivation of PSII membranes. Recently we found that that inactivation of electron transport (2,6-dichlorophenolindophenol [DCPIP] reduction) in membrane preparations of PSII without Ca 2+ in OEC (PSII(-Ca)) occurs at a slower rate at pH5.7 than at pH 6.5 (Lovyagina and Semin 2022 ). In the presented work, we examined the question of the relationship of the heat inactivation process with its pH dependence in more detail. We found that heat-induced inactivation of O 2 evolution reaction in PSII membranes occurs more slowly at pH 5.7 than at pH 6.5. In TM, light slows down the rate of heat inactivation as a result of lowering the pH of the lumen to slightly acidic values. Ca 2+ cations that increase the resistance of the Mn cluster to reductants also increase the resistance of PSII(-Ca) preparations to heat inactivation. ESR studies suggest a significant role of ROS in the process of heat inactivation. Materials and Methods Plant material We used fresh leaves of market spinach Spinacia oleracea L. without petioles and central veins. Sampling Preparation of PSII-enriched membranes PSII membranes (BBY type) were prepared according to Ghanotakis and Babcock ( 1983 ). Samples were stored at − 80°C in buffer A (50 mM 2-(N-morpholino)-ethanesulfonic acid at pH 6.5, 15 mM NaCl, and 400 mM sucrose) and thawed in the dark for ~ 1 h at 4°C before treatment or measurement. Preparation of thylakoid membranes Membranes were isolated from market spinach as described in the literature (McCauley and Melis 1986 ), suspended in the buffer B containing 50 mM Tricine, 400 mM sucrose, and 10 mM NaCl (pH 7.6) and stored at − 80°C. Preparation of Ca-depleted PSII membranes (PSII(-Ca)) PSII(-Ca) membranes without extrinsic proteins PsbP and PsbQ were prepared according to (Ono and Inoue 1990 ). PSII membranes (500 µg Chl /ml) were incubated in the buffer 2 M NaCl, 0.4 M sucrose, and 25 mM MES (pH 6.5) for 15 min at room temperature under low illumination (4–5 µE m − 2 s − 1 , room fluorescent light). The resulting material was washed twice and resuspended in a buffer A. Besides Ca 2+ cation PSII(-Ca/NaCl) membranes lack the PsbQ and PsbP extrinsic proteins, which prevent exogenous reducing agents from attacking the Mn/Ca cluster. Heat inactivation of thylakoid membranes and PSII particles Thermoincubation of TM and PSII preparations was carried out as follows. Samples with concentration of a chlorophyll 20 or 50 µg/ml were incubated at the definite temperature in buffer with pH 7.6 for 5 min (TM) or in buffer with pH 6.5 for 15 min (PSII and Ca-depleted PSII) (treatment time during which the effect of inactivation comes to the plateau) in previously warmed up buffer, then cooled up to 4 °С on ice for 3 min. All subsequent measurements were carried out at 25 °С temperature. Measurements of photosynthetic electron transport The photoreduction rate of the exogenous electron acceptor DCPIP Electron transport activity in PSII preparations was measured as the rate of the exogenous electron acceptor DCPIP photoreduction using a Specord UV-VIS spectrophotometer (Carl Zeiss Jena, Germany) in cuvettes with 1 cm optical path length. The XBDROY light diodes (Cree Inc., United States) with a maximum of 450 nm were used as the exciting light source to provide a saturating light intensity of 1800 µE m − 2 s − 1 . The cut-off excitation orange glass filter OS14 was installed in front of the photomultiplier tube of the spectrophotometer. Photoinduced changes in DCPIP optical density were recorded at wavelength of 600 nm and extinction coefficient for the deprotonated form of DCPIP ε = 21.8 mM − 1 ・cm − 1 (Armstrong 1964 ) was used for determination of the DCPIP reduction rate. Concentration of PSII during the measurement the rate of DCPIP photoreduction was 10 µg Chl/ml. Chlorophyll concentrations were determined in 80% acetone, according to the method of Porra et al. ( 1989 ). O 2 -evolving activity Kinetics of a photoinduced oxygen evolution by TM and PSII preparations were registered amperometrically using a closed Clark electrode. The measurements were carried out in a thermostatically controlled cell with a volume of 1 ml at 25 о С in the presence of 0.2 mM of an artificial electron acceptor 2,6-dichloro- p -benzoquinone (DCBQ). XBDROY light diodes were used as the excitation light source providing a saturating light intensity (1800 µE・m -2 ・s -1 ). The oxygen evolution rate was calculated using a linear part of a kinetic curve for the first 15 s after the illumination was turned on. Calibration of a diffusion current magnitude was carried out using the value of the oxygen concentration in water balanced with air (0,253 mM). The O 2 -evolving activity of the native PSII membranes ranged from 400 to 500 µmol O 2 ・mg Chl -1 ・h -1 , TM – from 195 to 220 µmol O 2 ・mg Chl -1 ・h -1 . The concentration of Chl in PSII preparations was 10 µg/ml, in TM preparations – 20 µg/ml. The data in the figures are presented as the arithmetic mean values obtained in independent experiments with at least 3 measurements in each experiment. ESR spin-trapping spectroscopy ESR method was used to detect the ROS formation during the heat inhibition of PSII membranes. To this end, a hydroxylamine spin probe (Dikalov et al. 2011 ; Kozuleva et al. 2011 ) was added to the preparation, and after incubation of the sample at the appropriate temperature, the ESR signal was measured. Lipophilic cyclic hydroxylamines N -(1-Hydroxy-2,2,6,6-tetramethylpiperidin-4-yl)-2-methylpropanamide (TMTH) was used as hydroxylamine spin probe. Hydroxylamines rapidly react with oxygen-centered free radicals, including superoxide (Dikalov et al. 2011 ), forming a nitroxide radicals which are measured by ESR equipment. Hydroxylamine spin probes invented by Rosen et al. ( 1982 ) successfully used by numerous researchers for superoxide measurement although the interaction of these probes with other types of ROS cannot be excluded and must be checked (Kozuleva et al. 2011 ). The PSII membranes (0.5 mg Chl/ml) were incubated with or without hydroxylamine spin probe TMTH (0.5 mM) in buffer A at a certain temperature for 15 minutes and put into a flat-type quartz ESR cell (70 µl). The ESR settings were: microwave power 20 mW, time constant 0.1 s, sweep time 100 s. All experiments were performed at 25°C. ESR spectra were plotted as the first derivative of the radio frequency absorption. Each characteristic spectrum presented in the figures is an average of 5 replicates. Results and Discussion Comparison of heat inactivation effect on the oxygen evolution in PSII membranes at different pH. We have previously shown (Lovyagina and Semin 2022 ) that the heat treatment of PSII(Ca) membranes is accompanied by inactivation of the water oxidation process in OEC and electron transport from OEC to the electron acceptor DCPIP on the acceptor side of Ca-depleted PSII. The effectiveness of temperature influence depends on the pH of the medium. In the area of slightly acidic pH (pH 5.7), heating of the sample affects the electron transport in PSII(-Ca) less effectively than at pH 6.5 (Lovyagina and Semin 2022 ). It should be noted that in this work we used PSII particles without calcium, in which manganese catalytic center cannot oxidize water molecules to molecular oxygen. However, Ca-depleted PSII particles have a high electron transport speed (about 70% of the control (Semin et al. 2008 )). The reduction of the DCPIP artificial acceptor is determined by electrons formed as a result of incomplete oxidation of water to hydrogen peroxide (Semin et al. 2013 ). In this regard, in the experiment described below, we investigated the effect of pH on the heat stress in native membrane preparations PSII. The results are shown in Fig. 1 . The obtained data show that heat inactivation at pH 5.7 develops more slowly than at pH 6.5. The effect found is most pronounced in region of moderate heat stress (about 40 о С). The difference between the data obtained at pH 5.7 and pH 6.5 is small (about 10%) and corresponds to the value of a similar difference in the case of photoinhibition (Davletshina and Semin 2020 ). Heat inactivation of thylakoid membranes. Effect of light and protonophores. In our previous work, we conducted a comparative study of the effect of the medium with pH 5.7 and 6.5 on photoinhibition of PSII membranes (Davletshina and Semin 2020 ). In addition to PSII membranes, in which, as well as in the present work, the anti-destructive effect of slightly acidic pH was found, we also used TM. The medium in TM lumen has a neutral pH, but under illumination it is acidified due to the operation of OEC to pH in the region of 5.7 (Kramer et al. 1999 ; Takizawa et al. 2007 ). We compared the rate of photoinhibition of OEC in TM under normal conditions and in the presence of protonophores, which remove the resulting proton gradient, causing alkalization of lumen. As a result, we found that protonophores increase the photoinhibition rate of TM (Davletshina and Semin 2020 ). We conducted similar experiments investigating the influence of light and protonophores on the heat inactivation of TM. Results are shown in Figs. 2 , 3 and Tables 1 , 2 . Figure 2 shows the kinetics of heat inactivation of the oxygen-evolving activity of TM and PSII membranes. From the presented results it can be seen that TM are more sensitive to heat stress than PSII particles. Perhaps this is due to the large pH value of the external medium for TM (pH 7.6), at which OEC is inactivated. Sequence of events of heat inactivation of TM can be as follows. Under the heat effect, the TM begin to break down and become permeable to the medium. As a result, lumen begins to alkalize and inactivation of OEC accelerates. Table 1 Effect of protonophores on heat inactivation of thylakoid membranes in the dark and under illumination. Sample a Rate of О 2 evolution in µmol/mg Chl • h (%) Dark Light Thylakoid membranes (25 о С) 217 ± 10 (100% ± 4.6%) 216 ± 13 (100% ± 6.0%) Thylakoid membranes (40 о С) 122 ± 9 (56% ± 4.2%) 168 ± 6 (78% ± 2.8%) + 2 µМ nigericin (40 о С) 56 ± 4 (26% ± 1.8%) 46 ± 5 (21% ± 2.3%) + 2 мМ NH 4 Cl (40 о С) 87 ± 6 (40% ± 2.8%) 83 ± 7 (38% ± 3.2%) a TM (20 µg Ch/ml) were incubated at a given temperature and stirring for 5 minutes. Then, rapidly cooled on ice for 3 min and the rate of oxygen evolution at 25 o C was measured in buffer containing 50 мМ Tricine, 400 мМ sucrose, 10 мМ NaCl (рН 7,6). Light intensity at thermal incubation − 30% of the saturating light intensity in the Soret band. Table 2 Effect of protonophores on the PSII membranes heat inactivation. Sample a Rate of О 2 evolution (%) 5 min incubation at 40 о С 15 min incubation at 40 о С PSII membranes 100% ± 5.0% 100% ± 5.1% + 2 µМ nigericin 79% ± 4.1% 68% ± 3.4% + 2 мМ NH 4 Cl 95% ± 5.3% 77% ± 3.9% a PSII membrane preparations (20 µg Ch/mL) were incubated at a given temperature for 5 or 15 minutes. Then, rapidly cooled in ice for 3 minutes, diluted with cold buffer A to C = 10 µg Ch/ml, and the rate of oxygen evolution at 25°C was measured. 100% - oxygen evolution activity 360 ± 18 µmol/mg Chl • h after 5 min incubation and 295 ± 15 µmol/mg Chl • h after 15 min incubation. Figure 3 shows the temperature dependencies of heat inactivation of TM oxygen-evolving activity in the dark and in the light. The results clearly demonstrate that when illuminated, the resistance to heat is increased. Taking into account presented above photoinhibition results, one can assume a significant role of the lumen acidification process in this effect resulted due to the illumination. Next, we decided to test the effect of protonophores on the observed effect of light. The obtained results showed that in the presence of protonophores (nigericin or NH 4 Cl), the rate of heat inactivation under illumination increases (Table 1 ), as in the case of photoinhibition (Davletshina and Semin 2020 ). However, this effect is not related to the protonophore action of the uncouplers (i.e., the reduction of the proton gradient), but is determined by more complex reasons not related to pH. This is evidenced by the following facts. First, in the dark, uncouplers also increase the effect of thermal inactivation (Table 1 ) and this fact suggests that the mechanism of sensitization of TM by uncouplers to heat is not related to their ability to influence the proton gradient. Secondly, it is confirmed by the data of an experiment in which the effect of uncouplers on heat inactivation of PSII membranes was investigated (Table 2 ). The results showed that in membrane preparations of PSII, where a proton gradient is not formed, uncouplers also stimulate thermal inactivation. Possibly uncouplers directly interact with membrane components involved in thermal denaturation, thereby providing catalysis of this process. In fact, it is well known about the possibility of interaction of uncouplers with membrane proteins (Kotova and Antonenko 2022 ). Effect of Ca 2+ and Fe cations on the thermal inactivation of PSII membranes at pH 5.7 and 6.5. The obtained results show that the pH-induced changes in the manganese cluster of OEC at pH 5.7, leading to a change in the redox potential of one or more manganese cations, is accompanied by an increase in resistance not only to photoinhibition (Davletshina and Semin 2020 ), but also to thermal inactivation (the above results). It should be noted that similar changes, accompanied by an increase in the resistance of the manganese cation to the action of reductants, take place and in the case of Ca 2+ cations additions. Thus, for example, Fe(II) cations replace in PSII OEC at pH 5.7 and pH 6.5 respectively 1 and 2 cations, whereas in the presence of 10 mM Ca 2+ these digits are less by one – 0 and 1 Mn cation (Semin et al. 2021 ), i.e. in the presence of Сa 2+ reducing agent Fe(II) restores in a manganese cluster at 1 manganese cation less. In this respect, the action of Ca 2+ cation is similar to the effect of pH. In this connection we tested the effect of Ca 2+ cations on thermal inactivation (Table 3 ). We found that in the presence of Ca 2+ (10 mM), resistance of electron transport (reduction of DCPIP) to heat stress increases significantly especially at pH 5.7. This effect is particularly noticeable in the region of high temperature 50°C (Table 3 ), in which the disassembly of the Mn cluster occurs (Pospišil et al. 2003 ). Recently, Yang et al. ( 2015 ) found that calcium nitrate protects peanut seedlings from elevated temperature and photoinhibition. In line with the results of their study, they suggested that under heat and high irradiation stress, the Ca 2+ signal transduction pathway can alleviate the photoinhibition and heat stress through regulating the protein repair process besides an enhanced capacity for scavenging ROS. Since we used mainly in our experiments PSII particles without a molecular apparatus for protein reparation, it can be assumed that the influence of Ca 2+ on heat stress is carried out in a more complex way, including the effect of Ca 2+ cation directly on manganese cations. Table 3 Effect of Ca 2+ cations on heat inactivation of electron transport (reduction of DCPIP) in membrane preparations of Ca-depleted PSII, containing Mn cluster of 4 cations and membrane preparations of Ca-depleted PSII with chimeric Mn/Fe cluster (3Mn/1Fe and 2Mn/2Fe) Sample a Rate DCPIP reduction, % Thermal incubation at рН 6,5 Thermal incubation at рН 5,7 Ca-depleted PSII 45 о С 50 о С 45 о С 50 о С 58,7% ± 6.3% 5,5% ± 4.1% 41,7% ± 6.0% 10,9% ± 3.5% Ca-depleted PSII + 10 mМ Са 73,1% ± 7.1% 16,7% ± 5.2% 71,55% ± 7.4% 40,7% ± 5.9% b Ca-depleted PSII (100 µg Chl/ml) after incubation with Fe(II) (20 µМ) for 120 min at pH 6.5 without thermal incubation 48.8% ± 5.9% Ca-depleted PSII (100 µg Chl/ml) after incubation with Fe(II) (20 µМ) for 120 min at pH 6.5 followed by centrifugation and thermal incubation (40 о С) Thermal incubation at рН 6,5 27.2% ± 5.6% Thermal incubation at рН 5,7 12,4% ± 4.4% c Ca-depleted PSII (100 µg Chl/ml) after incubation with Fe(II) (20 µМ) and Ca 2+ (10 mM) for 120 min at pH 6.5 without thermal incubation 92% ± 8.1% c Ca-depleted PSII (100 µg Chl/ml) after incubation with Fe(II) (20 µМ) and Ca 2+ (10 mM) for 120 min followed by centrifugation and thermal incubation (40 о С) Thermal incubation at рН 6,5 57.1% ± 5.7% Thermal incubation at рН 5,7 50.2% ± 4.9% a Ca-depleted PSII preparations (50 µg Ch/ml) were incubated at a given temperature for 15 minutes at pH 6.5 or 5.7 in the dark. After incubation, the samples were quickly cooled on ice for 3 minutes and centrifuged. The reduction of DCPIP (40 µM) was measured at C = 10 µg Chl/ml in kinetic mode at pH 6.5. The activity of PSII(-Ca) preparations at pH 6.5 without incubation at a given temperature was taken as 100%. b Sample contains chimeric cluster 2Mn/2Fe in OEC (Semin and Seibert 2016 ). c Sample contains chimeric cluster 3Mn/1Fe in OEC, since the substitution was carried out in the presence of Ca (Semin et al. 2021 ). In addition to Ca 2+ , iron cations also affect the resistance to reducing agents, although their mechanism of action is different from the mechanism of action of calcium cations. Stabilizing effect of iron cations is manifested only in case of their binding to Mn cluster during replacement of extracted manganese cations by them. So, for example, substitution of 1 or 2 Mn cations with Fe cations in OEC significantly increases the resistance to extraction by the reductant (H 2 Q) of the remaining manganese cations (Semin et al. 2018 ). So, H 2 Q extracts all Mn cations from native membranes PSII, containing 4 Mn cations, or from membranes, containing stable Mn dimer, except for one cation. At the same time, H 2 Q cannot extract any Mn cation from chimeric clusters of PSII(2Mn,2Fe) and PSII(3Mn,1Fe) (Semin et al. 2018 ). In this regard, we tested the resistance of PSII membranes with a chimeric cluster to temperature shock. Results are presented in Table 3 . PSII particles with chimeric cluster 2Mn/2Fe were obtained by incubating Ca-depleted PSII membranes with Fe(II) cations at pH 6.5 for 120 min at room temperature, which ensures substitution of 2 Mn cations in OEC with iron cations (Semin and Seibert 2016 ). It should be noted that there was no effect of substitution on the resistance of PSII particles to heat inactivation (Table 3 ) which can be explained by the influence of iron cations. It should be noted that electron transport in PSII with chimeric cluster 2Mn/2Fe has a very low rate, which nevertheless demonstrates the possibility of water molecules oxidation by the chimeric cluster to hydrogen peroxide (Semin et al. 2013 ). Substitution of Mn cations with Fe(II) cations in the presence of Ca 2+ cations is significantly different from the case of substitution without Ca 2+ (Table 3 ). This difference is that PSII particles with a chimeric cluster have significantly greater electron transport activity before heat inactivation, and after. The following mechanism can be the cause of such effect. In our previous studies (Semin et al. 2021 ), we have found that in the presence of Ca 2+ , the Fe cation substitutes at pH 6.5 for 1 cation Mn less than at pH 6.5 without Ca. I.e. in this case, we have a substitution at pH 6.5 only 1 manganese cation and formation of chimeric cluster not 2Mn2Fe but 3Mn1Fe. This may be the reason for the increased activity of chimeric clusters, obtained in the presence of Ca 2+ . On the other hand, the reason for the increase of electron-transport activity may be the binding of Ca 2+ to the chimeric cluster during its formation. Production of reactive oxygen species in PSII membranes during thermal incubation. ESR study with cyclic hydroxylamine probe. Heat inactivation of PSII is a complex process, many aspects of which are quite well investigated (Mathur et al. 2014 ; Allakhverdiev et al. 2008 ). Characteristic features of this process are the dissociation of extrinsic proteins PsbO, PsbP and PsbQ in the case of plant PSII (Nash et al. 1985 ; Yamashita et al. 2008 ) and release of Mn cations from its binding sites of OEC (Enami et al. 1994 ; Pospíšil et al. 2003; Yamashita et al. 2008 ). А prominent role in heat inactivation belongs to the ROS (Pospíšil et al. 2007 ; Yamashita et al. 2008 ; Pospíšil 2016 ) and lipid peroxidation products (Pospíšil and Yamamoto 2017 ). Considering the possible role of the redox process in the destruction of the manganese cluster and the possibility of the involvement of ROS (some of which are reductant agents) in thermal inactivation, we investigated the possibility of ROS generation during the heat stress. To do this, we used hydroxylamine spin probe TMTH, which is often used to detect superoxide generation (Dikalov et al. 2011 ; Kozuleva et al. 2011 ). However, it should be noted that many reagents for O 2 ∙− are non-specific and react with other ROS, especially with HO ∙ and 1 O 2 , giving the same or hardly distinguishable products (Kozuleva et al. 2015 ). This property can be clearly seen from the data presented in Fig. 4 . First figure ( a ) shows the ESR spectrum of the product of the TMTH probe reaction with superoxide generated in the O 2 ∙− generation system (adrenalin in CAPS buffer, pH 10.5), which does not contain superoxide dismutase (SOD) (1) and contains SOD (2). These data clearly demonstrate that in the presence of superoxide, the ESR signal of nitroxide radical (the oxidation product of TMTH with superoxide) appears. A similar spectrum appears in the singlet oxygen (4 b ) generation system, but, importantly, the efficiency of suppressing the ESR signal by the SOD enzyme is significantly less, which can be used to identify the ROS product. Figure 4 c also demonstrates the appearance of a similar signal in the hydroxyl radical generation system. The results presented in Fig. 5 were obtained during an ESR study of a TMTH probe in the presence of PSII membranes. Control curves 1 (probe in buffer A exposed to 4°C for 15 min) and 2 (probe in buffer А exposed to 40°C for 15 min) show absence of ESR signal when heating pure probe solution. Control curve 3 demonstrates the absence of ESR signal in PSII membranes in buffer А exposed to 40°C for 15 min without probe. However, during incubation of the membrane preparation at 40°C in the presence of the TMTH probe, an intense ESR signal is observed (Fig. 5 b, curve 2). In the case of incubation at 4 °С ESR signal is absent, which indicates the appearance of ROS only during thermal inactivation of PSII. In the case of temperature incubation of the sample together with the SOD, there was practically no effect of the enzyme on the signal intensity. This means that thermal inactivation of PSII does not produce superoxide and oxidation of the probe occurs due to other forms of ROS. Abbreviations CAPS N -cyclohexyl-3-aminopropanesulfonic acid Chl chlorophyll DCPIP 2,6-dichlorophenolindophenol H 2 Q hydroquinone MES 2-( N -morpholino)-ethanesulfonic acid OEC oxygen-evolving complex PSII photosystem II PSII(-Ca) Ca 2+ -depleted PSII membranes PSII(-Mn) Mn-depleted PSII membranes ROS reactive oxygen species SOD superoxide dismutase TM thylakoid membrane TMTH N -(1-Hydroxy-2,2,6,6-tetramethylpiperidin-4-yl)-2-methylpropanamide Declarations Acknowledgments The research was carried out as part of the Scientific Project of the State Order of the Government of Russian Federation to Lomonosov Moscow State University No. 121032500058-7 Conflicts of interest The authors declare that they have no conflicts of interest. References Allakhverdiev SI, Kreslavski VD, Klimov VV, Los DA, Carpentier R, Mohanty P (2008) Heat stress: an overview of molecular responses in photosynthesis. 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Biochim Biophys Acta 975:384−394 https://doi.org/10.1016/S0005-2728(89)80347-0 Pospišil P, Haumann M, Dittmer J, Solé VA, Dau H (2003) Stepwise transition of the tetra-manganese complex of photosystem II to a binuclear Mn 2 (μ-O) 2 complex in response to a temperature jump: a time-resolved structural investigation employing X-ray absorption spectroscopy. Biophys J 84:1370–1386 https://doi.org/10.1016/S0006-3495(03)74952-2 Pospíšil P, Šnyrychová I, Nauš J (2007) Dark production of reactive oxygen species in photosystem II membrane particles at elevated temperature: EPR spin-trapping study. Biochim Biophys Acta 1767:854–859 https://doi.org/10.1016/j.bbabio.2007.02.011 Pospišil P (2012) Molecular mechanisms of production and scavenging of reactive oxygen species by photosystem II. Biochim Biophys Acta 1817:218–231 https://doi.org/10.1016/j.bbabio.2011.05.017 Pospíšil P (2016) Production of reactive oxygen species by photosystem II as a response to light and temperature stress. Fronts Plant Sci 7:1950 https://doi.org/10.3389/fpls.2016.01950 Pospíšil P, Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products. Biochim Biophys Acta 1861:457–466 https://doi.org/10.1016/j.bbagen.2016.10.005 Rosen GM, Finkelstein E, Rauckman EJ (1982) A method for the detection of superoxide in biological systems. Arch Biochem Biophys 215:367–378 https://doi.org/10.1016/0003-9861(82)90097-2 Semin BK, Ivanov II, Rubin AB, Parak F (1995) High-specific binding of Fe(II) at the Mn-binding site in Mn-depleted PSII membranes from spinach. FEBS Lett 375:223−226 https://doi.org/10.1016/0014-5793(95)01215-Z Semin BK, Ghirardi ML, Seibert M (2002) Blocking of electron donation by Mn(II) to Y Z · following incubation of Mn-depleted photosystem II membranes with Fe(II) in the light. Biochemistry 41:5854−5864 https://doi.org/10.1021/bi0200054 Semin BK, Davletshina LN, Ivanov II, Rubin AB, Seibert M (2008) Decoupling of the processes of molecular oxygen synthesis and electron transport in Ca 2+ -depleted PSII membranes. Photosynth Res 98:235−249 https://doi.org/10.1007/s11120-008-9347-5 Semin BK, Davletshina LN, Timofeev KN, Ivanov II, Rubin AB, Seibert M (2013) Production of reactive oxygen species in decoupled, Ca 2+ -depleted PSII and their use in assigning a function to chloride on both sides of PSII. Photosynth Res. 117(1):385−399. https://doi.org/10.1007/s11120-013-9870-x Semin BK, Davletshina LN, Rubin AB (2015) Correlation between pH dependence of O 2 evolution and sensitivity of Mn cations in the oxygen-evolving complex to exogenous reductants. Photosynth Res 125:95−103 https://doi.org/10.1007/s11120-015-0155-4 Semin BK, Seibert M (2016) Substituting Fe for two of the four Mn ions in photosystem II—effects on water-oxidation. J Bioenerg Biomembr 48:227−240 https://doi.org/10.1007/s10863-016-9651-2 Semin BK, Davletshina LN, Seibert M, Rubin AB (2018) Creation of a 3Mn/1Fe cluster in the oxygen-evolving complex of photosystem II and investigation of its functional activity. J Photochem Photobiol B 178:192–200 https://doi.org/10.1016/j.jphotobiol.2017.11.016 Semin BK, Davletshina LN, Goryachev SN, Seibert M (2021) Ca 2+ effects on Fe(II) interactions with Mn-binding sites in Mn-depleted oxygen-evolving complexes of photosystem II and on Fe replacement of Mn in Mn-containing, Ca-depleted complexes. Photosynth Res 147(2):229−237. https://doi.org/10.1007/s11120-020-00813-z Takizawa K, Cruz JA, Kanazawa A, Kramer DM (2007) The thylakoid proton motive force in vivo. Quantitative, non-invasive probes, energetics, and regulatory consequences of light-induced PMF. Biochim Biophys Acta 1767:1233–1244 https://doi.org/10.1016/j.bbabio.2007.07.006 Tyystjärvi E (2008) Photoinhibition of photosystem II and photodamage of the oxygen evolving manganese cluster. Coord Chem Rev 252:361–376 https://doi.org/10.1016/j.ccr.2007.08.021 Zavafer A, Cheah MH, Hillier W, Chow WS, Takahashi S (2015) Photodamage to the oxygen evolving complex of photosystem II by visible light. Sci Rep 5:16363 https://doi.org/10.1038/srep16363 Yamashita A, Nijo M, Pospišil P, Morita N, Takenaka D, Aminaka R, Yamamoto Yo, Yamamoto Ya (2008) Quality control of photosystem II. Reactive oxygen species are responsible for the damage to photosystem II under moderate heat stress. J Biol Chem 283:28380–28391 https://doi.org/10.1074/jbc.M710465200 Yang S, Wang F, Guo F, Meng JJ, Li X., Wan S. (2015) Calcium contributes to photoprotection and repair of photosystem II in peanut leaves during heat and high irradiance. J Integr Plant Biol, 57(5):486–495 https://doi.org/10.1111/jipb.12249 Cite Share Download PDF Status: Published Journal Publication published 02 Oct, 2024 Read the published version in Journal of Plant Research → Version 1 posted Reviewers agreed at journal 07 Apr, 2024 Reviewers invited by journal 25 Mar, 2024 Editor assigned by journal 07 Mar, 2024 First submitted to journal 05 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4019854","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":283818405,"identity":"c7725e23-e020-45fb-bade-98770ab45493","order_by":0,"name":"Elena Lovyagina","email":"","orcid":"","institution":"Lomonosov Moscow State University: Moskovskij gosudarstvennyj universitet imeni M V Lomonosova","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"","lastName":"Lovyagina","suffix":""},{"id":283818406,"identity":"784f02b7-dfe7-4fd2-9856-daea2492c9c6","order_by":1,"name":"Oksana Luneva","email":"","orcid":"","institution":"Lomonosov Moscow State University: Moskovskij gosudarstvennyj universitet imeni M V Lomonosova","correspondingAuthor":false,"prefix":"","firstName":"Oksana","middleName":"","lastName":"Luneva","suffix":""},{"id":283818407,"identity":"e942e1fa-737c-4242-bc53-4a212e8ab80c","order_by":2,"name":"Aleksey Loktyushkin","email":"","orcid":"","institution":"Lomonosov Moscow State University: Moskovskij gosudarstvennyj universitet imeni M V Lomonosova","correspondingAuthor":false,"prefix":"","firstName":"Aleksey","middleName":"","lastName":"Loktyushkin","suffix":""},{"id":283818408,"identity":"cf55c4e4-5785-4b15-8296-f8e697c4bca7","order_by":3,"name":"Boris Semin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArklEQVRIiWNgGAWjYJCCA4wNCXIMDIyNB0jSYgzU0kC8FqDihMQGkF6iVMu3dyceLtyRlr62/XDDAcY9hwlrMThzdsPhmWdycredSQQ67BkxWiRyNxzmbavI3XYApOUAEVrk578Fa0k3O/+QSC0MN3hBWnISzG4Qa4vBGZDDzqQZbrsBtCXhQDoRDms/u/kz745kebPz6Q8ffDhgTYTDUEACqRpGwSgYBaNgFGAHAOSRSJcrW08SAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-4173-2809","institution":"Lomonosov Moscow State University: Moskovskij gosudarstvennyj universitet imeni M V Lomonosova","correspondingAuthor":true,"prefix":"","firstName":"Boris","middleName":"","lastName":"Semin","suffix":""}],"badges":[],"createdAt":"2024-03-06 07:47:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4019854/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4019854/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10265-024-01584-7","type":"published","date":"2024-10-02T15:56:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53631537,"identity":"a53a3f74-d710-42db-b526-51b9c19e1297","added_by":"auto","created_at":"2024-03-28 10:00:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11115,"visible":true,"origin":"","legend":"\u003cp\u003eTemperature dependence of PSII oxygen-evolving activity at pH 6.5 and pH 5.7. А suspension of PSII membranes in buffer A (20µg Chl/ml) was incubated at a definite temperature and intensive stirring. Then the preparations were cooled rapidly in ice for 3 minutes, diluted 2 times and the rate of oxygen evolution measured at 25 °C. The 100% preparations activity – the activity of PSII membranes after 15 min incubation at 25 \u003csup\u003eo\u003c/sup\u003eC at the corresponding pH. Measurements were made at pH 6.5 and pH 5.7 according to incubation pH.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4019854/v1/46eed82d1094d61080412841.png"},{"id":53631538,"identity":"04c2fd13-1454-4179-8861-15fa5e5bd0d7","added_by":"auto","created_at":"2024-03-28 10:00:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12034,"visible":true,"origin":"","legend":"\u003cp\u003eTime dependence of heat inactivation of thylakoid and PSII membranes at 45 \u003csup\u003eо\u003c/sup\u003eС. А suspension of thylakoid and PSII membranes in buffer A or in buffer B (20 µg Chl/ml) were incubated at a definite temperature and intensive stirring. Then the preparations were cooled rapidly in ice for 3 minutes, and the rate of oxygen evolution measured at 25 °C. The concentration of Chl in PSII preparations was 10 µg/ml, in thylakoid membranes preparations – 20 µg/ml.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4019854/v1/b3cb4b2de522207336597676.png"},{"id":53631539,"identity":"70c8d8c5-b5ad-4d31-874d-6599d0b25325","added_by":"auto","created_at":"2024-03-28 10:00:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":15464,"visible":true,"origin":"","legend":"\u003cp\u003eRate of oxygen evolution by thylakoid membranes after incubation at a given temperature in the dark or in the light. Аsuspension of thylakoid membranes (20 µg Chl/ml) in buffer B was incubated for 5 minutes at a definite temperature and intensive stirring in dark or under illumination (light intensity was 30% of the intensity of the saturating light in the Soret band). Then the preparations were cooled rapidly in ice for 3 minutes and the rate of oxygen evolution measured at 25 °C. Insert: kinetic curves of oxygen evolution by thylakoid membranes after incubation at 40 °C in the dark and in the light.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4019854/v1/3b15ac5c5c1de836ce56a8e0.png"},{"id":53631513,"identity":"76af82a0-c67d-428b-9033-5deed15e1fc6","added_by":"auto","created_at":"2024-03-28 10:00:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":45812,"visible":true,"origin":"","legend":"\u003cp\u003eESR detection of the nitroxides formed from the spin probe TMTH in: \u003cstrong\u003ea\u003c/strong\u003e, O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e•-\u003c/sup\u003e – generating system (0.2 mM adrenalin in 10 mM CAPS buffer, pH 10.5), 1 – without SOD, 2 – with 100 u/ml SOD; \u003cstrong\u003eb\u003c/strong\u003e, \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e - generating system (red light illumination of 10 µM methylene blue), 1 – without SOD, 2 – with 100 u/ml SOD; \u003cstrong\u003ec\u003c/strong\u003e, OH\u003csup\u003e•\u003c/sup\u003e - generating system (1 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/1 mM FeSO\u003csub\u003e4\u003c/sub\u003e), 1 – without ethanol, 2 – with 0.2 M ethanol. Model ESR spectrum was obtained using 0.5 mM TMTH. The incubation time for all generating systems was 5 minutes.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4019854/v1/e640e45c6f929cd5d9780881.png"},{"id":53631514,"identity":"4ccd8d36-a857-451a-8f45-b84a9ac55da0","added_by":"auto","created_at":"2024-03-28 10:00:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":37834,"visible":true,"origin":"","legend":"\u003cp\u003eDark ТМТН adduct ESR spectra measured in PSII membrane particles exposed to 40 °C for 15 min. \u003cstrong\u003ea\u003c/strong\u003e, control experiments: 1 – probe in buffer A exposed to 4 °C for 15 min, 2 – probe in buffer А exposed to 40 °C for 15 min, 3 – PSII preparation in buffer А exposed to 40 °C for 15 min without probe. \u003cstrong\u003eb\u003c/strong\u003e, 1 – PSII preparation in buffer А exposed to 4 °C for 15 min with probe, 2 – PSII preparation in buffer А exposed to 40 °C for 15 min with probe, 3 – PSII preparation in buffer А exposed to 40 °C for 15 min with probe and SOD (100 u/ml). TMTH adduct ESR spectra were obtained in the presence of 0,5 mM TMTH, 500 μg of Chl ml\u003csup\u003e−1\u003c/sup\u003e in buffer A (pH 6.5).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4019854/v1/b1ce9e0e57541b4f9a17884a.png"},{"id":66096669,"identity":"3fa01913-77ca-408c-8ecb-cc907d8f7340","added_by":"auto","created_at":"2024-10-07 16:06:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":805725,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4019854/v1/fbc13451-6f13-40dd-8cb8-1d677a7e65c8.pdf"}],"financialInterests":"","formattedTitle":"Light increases resistance of thylakoid membranes to thermal inactivation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDuring the investigation of the mechanism of Fe(II) cations binding to Mn-binding sites in the oxygen-evolving complex (OEC), it was found highly efficient binding of these cations to the Mn-binding sites in Mn-depleted PSII membranes (Semin et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Semin et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Moreover, if OEC is available for exogenous factors (in the absence of extrinsic proteins PsbP and PsbQ) ferrous cations (strong reducing factor) are able to reach Mn cluster, interact with Mn cations and reduce part of Mn cations in OEC in the dark. As a result of this process, the reduced cations Mn(II) leave the binding site and the open Mn-binding sites are occupied by Fe(III) cations which leads to the formation in PSII chimeric clusters consisting of iron and manganese cations 2Mn2Fe (Semin and Seibert \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) or 3Mn1Fe (Semin et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Investigating this process, we have found that the substitution of Mn cations in OEC with Fe(II) cations depends on the pH of the buffer. At pH 6.5, Fe(II) cations replace 2 Mn cation, while at pH 5.7 only one Mn cation is replaced (Semin and Seibert \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Semin et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). A similar situation occurs in the case of other reducing agents - hydroquinone (H\u003csub\u003e2\u003c/sub\u003eQ) and hydrogen peroxide (Semin et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e): at pH 6.5 these reductants extracted 3 Mn cations, whereas at pH 5.7 only 2 Mn cations were extracted. Thus, the redox potential of one of the Mn cations in the OEC strongly depends on the pH of the medium, which can increase the resistance of the manganese cluster to the action of reducing agents in the region of weakly acidic pH.\u003c/p\u003e \u003cp\u003eA such effect may occur in the case of photoinhibition. It is known that the Mn cluster in the OEC of PSII can be effectively destroyed during the photoinhibition and, possibly, it is the first step in this process (Tyystj\u0026auml;rvi \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zavafer et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Destruction of OEC manganese cluster may be the result of its exposure to some reactive oxygen species (ROS) O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e∙\u0026minus;\u003c/sup\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e produced on the donor and acceptor sides of PSII during illumination (Pospišil \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Indeed, we found that at pH 5.7, the photoinactivation rate is less compared to pH 6.5 whereas in the Mn-depleted PSII membranes, photoinhibition does not depend on pH (Davletshina and Semin \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, the rate of photoinhibition of the thylakoid membranes (TM) was significantly increased in the presence of the uncouplers that changed the pH of lumen \u0026ndash; the place of the OEC location. Yamashita et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) showed that ROS, apparently participate not only in photoinhibition, but also in the process of thermal inactivation of photosynthetic membranes. ROS are formed, apparently as a result of membrane lipids peroxidation initiated by heating and take part in heat inactivation of PSII membranes. Recently we found that that inactivation of electron transport (2,6-dichlorophenolindophenol [DCPIP] reduction) in membrane preparations of PSII without Ca\u003csup\u003e2+\u003c/sup\u003e in OEC (PSII(-Ca)) occurs at a slower rate at pH5.7 than at pH 6.5 (Lovyagina and Semin \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the presented work, we examined the question of the relationship of the heat inactivation process with its pH dependence in more detail. We found that heat-induced inactivation of O\u003csub\u003e2\u003c/sub\u003e evolution reaction in PSII membranes occurs more slowly at pH 5.7 than at pH 6.5. In TM, light slows down the rate of heat inactivation as a result of lowering the pH of the lumen to slightly acidic values. Ca\u003csup\u003e2+\u003c/sup\u003e cations that increase the resistance of the Mn cluster to reductants also increase the resistance of PSII(-Ca) preparations to heat inactivation. ESR studies suggest a significant role of ROS in the process of heat inactivation.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material\u003c/h2\u003e \u003cp\u003eWe used fresh leaves of market spinach \u003cem\u003eSpinacia oleracea\u003c/em\u003e L. without petioles and central veins.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSampling\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003ePreparation of PSII-enriched membranes\u003c/h2\u003e \u003cp\u003ePSII membranes (BBY type) were prepared according to Ghanotakis and Babcock (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). Samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C in buffer A (50 mM 2-(N-morpholino)-ethanesulfonic acid at pH 6.5, 15 mM NaCl, and 400 mM sucrose) and thawed in the dark for ~\u0026thinsp;1 h at 4\u0026deg;C before treatment or measurement.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of thylakoid membranes\u003c/h2\u003e \u003cp\u003eMembranes were isolated from market spinach as described in the literature (McCauley and Melis \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1986\u003c/span\u003e), suspended in the buffer B containing 50 mM Tricine, 400 mM sucrose, and 10 mM NaCl (pH 7.6) and stored at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of Ca-depleted PSII membranes (PSII(-Ca))\u003c/h2\u003e \u003cp\u003ePSII(-Ca) membranes without extrinsic proteins PsbP and PsbQ were prepared according to (Ono and Inoue \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). PSII membranes (500 \u0026micro;g Chl /ml) were incubated in the buffer 2 M NaCl, 0.4 M sucrose, and 25 mM MES (pH 6.5) for 15 min at room temperature under low illumination (4\u0026ndash;5 \u0026micro;E m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, room fluorescent light). The resulting material was washed twice and resuspended in a buffer A. Besides Ca\u003csup\u003e2+\u003c/sup\u003e cation PSII(-Ca/NaCl) membranes lack the PsbQ and PsbP extrinsic proteins, which prevent exogenous reducing agents from attacking the Mn/Ca cluster.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHeat inactivation of thylakoid membranes and PSII particles\u003c/h2\u003e \u003cp\u003eThermoincubation of TM and PSII preparations was carried out as follows. Samples with concentration of a chlorophyll 20 or 50 \u0026micro;g/ml were incubated at the definite temperature in buffer with pH 7.6 for 5 min (TM) or in buffer with pH 6.5 for 15 min (PSII and Ca-depleted PSII) (treatment time during which the effect of inactivation comes to the plateau) in previously warmed up buffer, then cooled up to 4 \u0026deg;С on ice for 3 min. All subsequent measurements were carried out at 25 \u0026deg;С temperature.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMeasurements of photosynthetic electron transport\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eThe photoreduction rate of the exogenous electron acceptor DCPIP\u003c/h2\u003e \u003cp\u003eElectron transport activity in PSII preparations was measured as the rate of the exogenous electron acceptor DCPIP photoreduction using a Specord UV-VIS spectrophotometer (Carl Zeiss Jena, Germany) in cuvettes with 1 cm optical path length. The XBDROY light diodes (Cree Inc., United States) with a maximum of 450 nm were used as the exciting light source to provide a saturating light intensity of 1800 \u0026micro;E m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The cut-off excitation orange glass filter OS14 was installed in front of the photomultiplier tube of the spectrophotometer. Photoinduced changes in DCPIP optical density were recorded at wavelength of 600 nm and extinction coefficient for the deprotonated form of DCPIP ε\u0026thinsp;=\u0026thinsp;21.8 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e・cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Armstrong \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1964\u003c/span\u003e) was used for determination of the DCPIP reduction rate. Concentration of PSII during the measurement the rate of DCPIP photoreduction was 10 \u0026micro;g Chl/ml. Chlorophyll concentrations were determined in 80% acetone, according to the method of Porra et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1989\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eO\u003csub\u003e2\u003c/sub\u003e-evolving activity\u003c/h2\u003e \u003cp\u003eKinetics of a photoinduced oxygen evolution by TM and PSII preparations were registered amperometrically using a closed Clark electrode. The measurements were carried out in a thermostatically controlled cell with a volume of 1 ml at 25 \u003csup\u003eо\u003c/sup\u003eС in the presence of 0.2 mM of an artificial electron acceptor 2,6-dichloro-\u003cem\u003ep\u003c/em\u003e-benzoquinone (DCBQ). XBDROY light diodes were used as the excitation light source providing a saturating light intensity (1800 \u0026micro;E・m\u003csup\u003e-2\u003c/sup\u003e・s\u003csup\u003e-1\u003c/sup\u003e). The oxygen evolution rate was calculated using a linear part of a kinetic curve for the first 15 s after the illumination was turned on. Calibration of a diffusion current magnitude was carried out using the value of the oxygen concentration in water balanced with air (0,253 mM). The O\u003csub\u003e2\u003c/sub\u003e-evolving activity of the native PSII membranes ranged from 400 to 500 \u0026micro;mol O\u003csub\u003e2\u003c/sub\u003e・mg Chl\u003csup\u003e-1\u003c/sup\u003e・h\u003csup\u003e-1\u003c/sup\u003e, TM \u0026ndash; from 195 to 220 \u0026micro;mol O\u003csub\u003e2\u003c/sub\u003e・mg Chl\u003csup\u003e-1\u003c/sup\u003e・h\u003csup\u003e-1\u003c/sup\u003e. The concentration of Chl in PSII preparations was 10 \u0026micro;g/ml, in TM preparations \u0026ndash; 20 \u0026micro;g/ml.\u003c/p\u003e \u003cp\u003eThe data in the figures are presented as the arithmetic mean values obtained in independent experiments with at least 3 measurements in each experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eESR spin-trapping spectroscopy\u003c/h2\u003e \u003cp\u003eESR method was used to detect the ROS formation during the heat inhibition of PSII membranes. To this end, a hydroxylamine spin probe (Dikalov et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kozuleva et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) was added to the preparation, and after incubation of the sample at the appropriate temperature, the ESR signal was measured. Lipophilic cyclic hydroxylamines \u003cem\u003eN\u003c/em\u003e-(1-Hydroxy-2,2,6,6-tetramethylpiperidin-4-yl)-2-methylpropanamide (TMTH) was used as hydroxylamine spin probe. Hydroxylamines rapidly react with oxygen-centered free radicals, including superoxide (Dikalov et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), forming a nitroxide radicals which are measured by ESR equipment. Hydroxylamine spin probes invented by Rosen et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1982\u003c/span\u003e) successfully used by numerous researchers for superoxide measurement although the interaction of these probes with other types of ROS cannot be excluded and must be checked (Kozuleva et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The PSII membranes (0.5 mg Chl/ml) were incubated with or without hydroxylamine spin probe TMTH (0.5 mM) in buffer A at a certain temperature for 15 minutes and put into a flat-type quartz ESR cell (70 \u0026micro;l). The ESR settings were: microwave power 20 mW, time constant 0.1 s, sweep time 100 s. All experiments were performed at 25\u0026deg;C. ESR spectra were plotted as the first derivative of the radio frequency absorption. Each characteristic spectrum presented in the figures is an average of 5 replicates.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e \u003cb\u003eComparison of heat inactivation effect on the oxygen evolution in PSII membranes at different pH.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe have previously shown (Lovyagina and Semin \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) that the heat treatment of PSII(Ca) membranes is accompanied by inactivation of the water oxidation process in OEC and electron transport from OEC to the electron acceptor DCPIP on the acceptor side of Ca-depleted PSII. The effectiveness of temperature influence depends on the pH of the medium. In the area of slightly acidic pH (pH 5.7), heating of the sample affects the electron transport in PSII(-Ca) less effectively than at pH 6.5 (Lovyagina and Semin \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It should be noted that in this work we used PSII particles without calcium, in which manganese catalytic center cannot oxidize water molecules to molecular oxygen. However, Ca-depleted PSII particles have a high electron transport speed (about 70% of the control (Semin et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2008\u003c/span\u003e)). The reduction of the DCPIP artificial acceptor is determined by electrons formed as a result of incomplete oxidation of water to hydrogen peroxide (Semin et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In this regard, in the experiment described below, we investigated the effect of pH on the heat stress in native membrane preparations PSII. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The obtained data show that heat inactivation at pH 5.7 develops more slowly than at pH 6.5. The effect found is most pronounced in region of moderate heat stress (about 40 \u003csup\u003eо\u003c/sup\u003eС). The difference between the data obtained at pH 5.7 and pH 6.5 is small (about 10%) and corresponds to the value of a similar difference in the case of photoinhibition (Davletshina and Semin \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHeat inactivation of thylakoid membranes. Effect of light and protonophores.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn our previous work, we conducted a comparative study of the effect of the medium with pH 5.7 and 6.5 on photoinhibition of PSII membranes (Davletshina and Semin \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition to PSII membranes, in which, as well as in the present work, the anti-destructive effect of slightly acidic pH was found, we also used TM. The medium in TM lumen has a neutral pH, but under illumination it is acidified due to the operation of OEC to pH in the region of 5.7 (Kramer et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Takizawa et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). We compared the rate of photoinhibition of OEC in TM under normal conditions and in the presence of protonophores, which remove the resulting proton gradient, causing alkalization of lumen. As a result, we found that protonophores increase the photoinhibition rate of TM (Davletshina and Semin \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). We conducted similar experiments investigating the influence of light and protonophores on the heat inactivation of TM. Results are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the kinetics of heat inactivation of the oxygen-evolving activity of TM and PSII membranes. From the presented results it can be seen that TM are more sensitive to heat stress than PSII particles. Perhaps this is due to the large pH value of the external medium for TM (pH 7.6), at which OEC is inactivated. Sequence of events of heat inactivation of TM can be as follows. Under the heat effect, the TM begin to break down and become permeable to the medium. As a result, lumen begins to alkalize and inactivation of OEC accelerates.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of protonophores on heat inactivation of thylakoid membranes in the dark and under illumination.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eRate of О\u003csub\u003e2\u003c/sub\u003e evolution in \u0026micro;mol/mg Chl \u0026bull; h (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDark\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLight\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThylakoid membranes (25 \u003csup\u003eо\u003c/sup\u003eС)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e217\u0026thinsp;\u0026plusmn;\u0026thinsp;10 (100% \u0026plusmn; 4.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e216\u0026thinsp;\u0026plusmn;\u0026thinsp;13 (100% \u0026plusmn; 6.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThylakoid membranes (40 \u003csup\u003eо\u003c/sup\u003eС)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e122\u0026thinsp;\u0026plusmn;\u0026thinsp;9 (56% \u0026plusmn; 4.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e168\u0026thinsp;\u0026plusmn;\u0026thinsp;6 (78% \u0026plusmn; 2.8%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u0026thinsp;2 \u0026micro;М nigericin (40 \u003csup\u003eо\u003c/sup\u003eС)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e56\u0026thinsp;\u0026plusmn;\u0026thinsp;4 (26% \u0026plusmn; 1.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e46\u0026thinsp;\u0026plusmn;\u0026thinsp;5 (21% \u0026plusmn; 2.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u0026thinsp;2 мМ NH\u003csub\u003e4\u003c/sub\u003eCl (40 \u003csup\u003eо\u003c/sup\u003eС)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e87\u0026thinsp;\u0026plusmn;\u0026thinsp;6 (40% \u0026plusmn; 2.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e83\u0026thinsp;\u0026plusmn;\u0026thinsp;7 (38% \u0026plusmn; 3.2%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e TM (20 \u0026micro;g Ch/ml) were incubated at a given temperature and stirring for 5 minutes. Then, rapidly cooled on ice for 3 min and the rate of oxygen evolution at 25 \u003csup\u003eo\u003c/sup\u003eC was measured in buffer containing 50 мМ Tricine, 400 мМ sucrose, 10 мМ NaCl (рН 7,6). Light intensity at thermal incubation \u0026minus;\u0026thinsp;30% of the saturating light intensity in the Soret band.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of protonophores on the PSII membranes heat inactivation.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eRate of О\u003csub\u003e2\u003c/sub\u003e evolution (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 min incubation at 40 \u003csup\u003eо\u003c/sup\u003eС\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 min incubation at 40 \u003csup\u003eо\u003c/sup\u003eС\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePSII membranes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e100% \u0026plusmn; 5.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e100% \u0026plusmn; 5.1%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u0026thinsp;2 \u0026micro;М nigericin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e79% \u0026plusmn; 4.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e68% \u0026plusmn; 3.4%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e+\u0026thinsp;2 мМ NH\u003csub\u003e4\u003c/sub\u003eCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e95% \u0026plusmn; 5.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e77% \u0026plusmn; 3.9%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003ea\u003c/sup\u003ePSII membrane preparations (20 \u0026micro;g Ch/mL) were incubated at a given temperature for 5 or 15 minutes. Then, rapidly cooled in ice for 3 minutes, diluted with cold buffer A to C\u0026thinsp;=\u0026thinsp;10 \u0026micro;g Ch/ml, and the rate of oxygen evolution at 25\u0026deg;C was measured. 100% - oxygen evolution activity 360\u0026thinsp;\u0026plusmn;\u0026thinsp;18 \u0026micro;mol/mg Chl \u0026bull; h after 5 min incubation and 295\u0026thinsp;\u0026plusmn;\u0026thinsp;15 \u0026micro;mol/mg Chl \u0026bull; h after 15 min incubation.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the temperature dependencies of heat inactivation of TM oxygen-evolving activity in the dark and in the light. The results clearly demonstrate that when illuminated, the resistance to heat is increased. Taking into account presented above photoinhibition results, one can assume a significant role of the lumen acidification process in this effect resulted due to the illumination. Next, we decided to test the effect of protonophores on the observed effect of light. The obtained results showed that in the presence of protonophores (nigericin or NH\u003csub\u003e4\u003c/sub\u003eCl), the rate of heat inactivation under illumination increases (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), as in the case of photoinhibition (Davletshina and Semin \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, this effect is not related to the protonophore action of the uncouplers (i.e., the reduction of the proton gradient), but is determined by more complex reasons not related to pH. This is evidenced by the following facts. First, in the dark, uncouplers also increase the effect of thermal inactivation (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and this fact suggests that the mechanism of sensitization of TM by uncouplers to heat is not related to their ability to influence the proton gradient. Secondly, it is confirmed by the data of an experiment in which the effect of uncouplers on heat inactivation of PSII membranes was investigated (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The results showed that in membrane preparations of PSII, where a proton gradient is not formed, uncouplers also stimulate thermal inactivation. Possibly uncouplers directly interact with membrane components involved in thermal denaturation, thereby providing catalysis of this process. In fact, it is well known about the possibility of interaction of uncouplers with membrane proteins (Kotova and Antonenko \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of Ca\u003c/b\u003e \u003csup\u003e \u003cb\u003e2+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eand Fe cations on the thermal inactivation of PSII membranes at pH 5.7 and 6.5.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe obtained results show that the pH-induced changes in the manganese cluster of OEC at pH 5.7, leading to a change in the redox potential of one or more manganese cations, is accompanied by an increase in resistance not only to photoinhibition (Davletshina and Semin \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), but also to thermal inactivation (the above results). It should be noted that similar changes, accompanied by an increase in the resistance of the manganese cation to the action of reductants, take place and in the case of Ca\u003csup\u003e2+\u003c/sup\u003e cations additions. Thus, for example, Fe(II) cations replace in PSII OEC at pH 5.7 and pH 6.5 respectively 1 and 2 cations, whereas in the presence of 10 mM Ca\u003csup\u003e2+\u003c/sup\u003e these digits are less by one \u0026ndash; 0 and 1 Mn cation (Semin et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), i.e. in the presence of Сa\u003csup\u003e2+\u003c/sup\u003e reducing agent Fe(II) restores in a manganese cluster at 1 manganese cation less. In this respect, the action of Ca\u003csup\u003e2+\u003c/sup\u003e cation is similar to the effect of pH. In this connection we tested the effect of Ca\u003csup\u003e2+\u003c/sup\u003e cations on thermal inactivation (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). We found that in the presence of Ca\u003csup\u003e2+\u003c/sup\u003e (10 mM), resistance of electron transport (reduction of DCPIP) to heat stress increases significantly especially at pH 5.7. This effect is particularly noticeable in the region of high temperature 50\u0026deg;C (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), in which the disassembly of the Mn cluster occurs (Pospišil et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Recently, Yang et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) found that calcium nitrate protects peanut seedlings from elevated temperature and photoinhibition. In line with the results of their study, they suggested that under heat and high irradiation stress, the Ca\u003csup\u003e2+\u003c/sup\u003e signal transduction pathway can alleviate the photoinhibition and heat stress through regulating the protein repair process besides an enhanced capacity for scavenging ROS. Since we used mainly in our experiments PSII particles without a molecular apparatus for protein reparation, it can be assumed that the influence of Ca\u003csup\u003e2+\u003c/sup\u003e on heat stress is carried out in a more complex way, including the effect of Ca\u003csup\u003e2+\u003c/sup\u003e cation directly on manganese cations.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of Ca\u003csup\u003e2+\u003c/sup\u003e cations on heat inactivation of electron transport (reduction of DCPIP) in membrane preparations of Ca-depleted PSII, containing Mn cluster of 4 cations and membrane preparations of Ca-depleted PSII with chimeric Mn/Fe cluster (3Mn/1Fe and 2Mn/2Fe)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eRate DCPIP reduction, %\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eThermal incubation at рН 6,5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eThermal incubation at рН 5,7\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCa-depleted PSII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45 \u003csup\u003eо\u003c/sup\u003eС\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50 \u003csup\u003eо\u003c/sup\u003eС\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45 \u003csup\u003eо\u003c/sup\u003eС\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50 \u003csup\u003eо\u003c/sup\u003eС\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e58,7% \u0026plusmn; 6.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5,5% \u0026plusmn; 4.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41,7% \u0026plusmn; 6.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10,9% \u0026plusmn; 3.5%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa-depleted PSII\u0026thinsp;+\u0026thinsp;10 mМ Са\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73,1% \u0026plusmn; 7.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16,7% \u0026plusmn; 5.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e71,55% \u0026plusmn; 7.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40,7% \u0026plusmn; 5.9%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003csup\u003eb\u003c/sup\u003eCa-depleted PSII (100 \u0026micro;g Chl/ml) after incubation with Fe(II) (20 \u0026micro;М) for 120 min at pH 6.5 without thermal incubation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003e48.8% \u0026plusmn; 5.9%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa-depleted PSII (100 \u0026micro;g Chl/ml) after incubation with Fe(II) (20 \u0026micro;М) for 120 min at pH 6.5 followed by centrifugation and thermal incubation (40 \u003csup\u003eо\u003c/sup\u003eС)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eThermal incubation at рН 6,5\u003c/p\u003e \u003cp\u003e27.2% \u0026plusmn; 5.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eThermal incubation at рН 5,7\u003c/p\u003e \u003cp\u003e12,4% \u0026plusmn; 4.4%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003csup\u003ec\u003c/sup\u003eCa-depleted PSII (100 \u0026micro;g Chl/ml) after incubation with Fe(II) (20 \u0026micro;М) and Ca\u003csup\u003e2+\u003c/sup\u003e (10 mM) for 120 min at pH 6.5 without thermal incubation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003e92% \u0026plusmn; 8.1%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003csup\u003ec\u003c/sup\u003eCa-depleted PSII (100 \u0026micro;g Chl/ml) after incubation with Fe(II) (20 \u0026micro;М) and Ca\u003csup\u003e2+\u003c/sup\u003e (10 mM) for 120 min followed by centrifugation and thermal incubation (40 \u003csup\u003eо\u003c/sup\u003eС)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eThermal incubation at рН 6,5\u003c/p\u003e \u003cp\u003e57.1% \u0026plusmn; 5.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eThermal incubation at рН 5,7\u003c/p\u003e \u003cp\u003e50.2% \u0026plusmn; 4.9%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003ea\u003c/sup\u003e Ca-depleted PSII preparations (50 \u0026micro;g Ch/ml) were incubated at a given temperature for 15 minutes at pH 6.5 or 5.7 in the dark. After incubation, the samples were quickly cooled on ice for 3 minutes and centrifuged. The reduction of DCPIP (40 \u0026micro;M) was measured at C\u0026thinsp;=\u0026thinsp;10 \u0026micro;g Chl/ml in kinetic mode at pH 6.5. The activity of PSII(-Ca) preparations at pH 6.5 without incubation at a given temperature was taken as 100%.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003eb\u003c/sup\u003e Sample contains chimeric cluster 2Mn/2Fe in OEC (Semin and Seibert \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003ec\u003c/sup\u003eSample contains chimeric cluster 3Mn/1Fe in OEC, since the substitution was carried out in the presence of Ca (Semin et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn addition to Ca\u003csup\u003e2+\u003c/sup\u003e, iron cations also affect the resistance to reducing agents, although their mechanism of action is different from the mechanism of action of calcium cations. Stabilizing effect of iron cations is manifested only in case of their binding to Mn cluster during replacement of extracted manganese cations by them. So, for example, substitution of 1 or 2 Mn cations with Fe cations in OEC significantly increases the resistance to extraction by the reductant (H\u003csub\u003e2\u003c/sub\u003eQ) of the remaining manganese cations (Semin et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). So, H\u003csub\u003e2\u003c/sub\u003eQ extracts all Mn cations from native membranes PSII, containing 4 Mn cations, or from membranes, containing stable Mn dimer, except for one cation. At the same time, H\u003csub\u003e2\u003c/sub\u003eQ cannot extract any Mn cation from chimeric clusters of PSII(2Mn,2Fe) and PSII(3Mn,1Fe) (Semin et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In this regard, we tested the resistance of PSII membranes with a chimeric cluster to temperature shock. Results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. PSII particles with chimeric cluster 2Mn/2Fe were obtained by incubating Ca-depleted PSII membranes with Fe(II) cations at pH 6.5 for 120 min at room temperature, which ensures substitution of 2 Mn cations in OEC with iron cations (Semin and Seibert \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). It should be noted that there was no effect of substitution on the resistance of PSII particles to heat inactivation (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) which can be explained by the influence of iron cations. It should be noted that electron transport in PSII with chimeric cluster 2Mn/2Fe has a very low rate, which nevertheless demonstrates the possibility of water molecules oxidation by the chimeric cluster to hydrogen peroxide (Semin et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Substitution of Mn cations with Fe(II) cations in the presence of Ca\u003csup\u003e2+\u003c/sup\u003e cations is significantly different from the case of substitution without Ca\u003csup\u003e2+\u003c/sup\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This difference is that PSII particles with a chimeric cluster have significantly greater electron transport activity before heat inactivation, and after. The following mechanism can be the cause of such effect. In our previous studies (Semin et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), we have found that in the presence of Ca\u003csup\u003e2+\u003c/sup\u003e, the Fe cation substitutes at pH 6.5 for 1 cation Mn less than at pH 6.5 without Ca. I.e. in this case, we have a substitution at pH 6.5 only 1 manganese cation and formation of chimeric cluster not 2Mn2Fe but 3Mn1Fe. This may be the reason for the increased activity of chimeric clusters, obtained in the presence of Ca\u003csup\u003e2+\u003c/sup\u003e. On the other hand, the reason for the increase of electron-transport activity may be the binding of Ca\u003csup\u003e2+\u003c/sup\u003e to the chimeric cluster during its formation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProduction of reactive oxygen species in PSII membranes during thermal incubation. ESR study with cyclic hydroxylamine probe.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHeat inactivation of PSII is a complex process, many aspects of which are quite well investigated (Mathur et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Allakhverdiev et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Characteristic features of this process are the dissociation of extrinsic proteins PsbO, PsbP and PsbQ in the case of plant PSII (Nash et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Yamashita et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) and release of Mn cations from its binding sites of OEC (Enami et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Posp\u0026iacute;šil et al. 2003; Yamashita et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). А prominent role in heat inactivation belongs to the ROS (Posp\u0026iacute;šil et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Yamashita et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Posp\u0026iacute;šil \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and lipid peroxidation products (Posp\u0026iacute;šil and Yamamoto \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Considering the possible role of the redox process in the destruction of the manganese cluster and the possibility of the involvement of ROS (some of which are reductant agents) in thermal inactivation, we investigated the possibility of ROS generation during the heat stress. To do this, we used hydroxylamine spin probe TMTH, which is often used to detect superoxide generation (Dikalov et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kozuleva et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). However, it should be noted that many reagents for O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e∙\u0026minus;\u003c/sup\u003e are non-specific and react with other ROS, especially with HO\u003csup\u003e∙\u003c/sup\u003e and \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e, giving the same or hardly distinguishable products (Kozuleva et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis property can be clearly seen from the data presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. First figure (\u003cb\u003ea\u003c/b\u003e) shows the ESR spectrum of the product of the TMTH probe reaction with superoxide generated in the O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e∙\u0026minus;\u003c/sup\u003e generation system (adrenalin in CAPS buffer, pH 10.5), which does not contain superoxide dismutase (SOD) (1) and contains SOD (2). These data clearly demonstrate that in the presence of superoxide, the ESR signal of nitroxide radical (the oxidation product of TMTH with superoxide) appears. A similar spectrum appears in the singlet oxygen (4\u003cb\u003eb\u003c/b\u003e) generation system, but, importantly, the efficiency of suppressing the ESR signal by the SOD enzyme is significantly less, which can be used to identify the ROS product. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec also demonstrates the appearance of a similar signal in the hydroxyl radical generation system.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e were obtained during an ESR study of a TMTH probe in the presence of PSII membranes. Control curves 1 (probe in buffer A exposed to 4\u0026deg;C for 15 min) and 2 (probe in buffer А exposed to 40\u0026deg;C for 15 min) show absence of ESR signal when heating pure probe solution. Control curve 3 demonstrates the absence of ESR signal in PSII membranes in buffer А exposed to 40\u0026deg;C for 15 min without probe. However, during incubation of the membrane preparation at 40\u0026deg;C in the presence of the TMTH probe, an intense ESR signal is observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, curve 2). In the case of incubation at 4 \u0026deg;С ESR signal is absent, which indicates the appearance of ROS only during thermal inactivation of PSII. In the case of temperature incubation of the sample together with the SOD, there was practically no effect of the enzyme on the signal intensity. This means that thermal inactivation of PSII does not produce superoxide and oxidation of the probe occurs due to other forms of ROS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eCAPS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003cem\u003eN\u003c/em\u003e-cyclohexyl-3-aminopropanesulfonic acid\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChl\u003c/strong\u003e \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;chlorophyll\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDCPIP\u003c/strong\u003e \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;2,6-dichlorophenolindophenol\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u003csub\u003e2\u003c/sub\u003eQ\u003c/strong\u003e \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;hydroquinone\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMES\u003c/strong\u003e \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;2-(\u003cem\u003eN\u003c/em\u003e-morpholino)-ethanesulfonic acid\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOEC\u003c/strong\u003e \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;oxygen-evolving complex\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePSII\u003c/strong\u003e \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;photosystem II\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePSII(-Ca)\u003c/strong\u003e \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Ca\u003csup\u003e2+\u003c/sup\u003e-depleted PSII membranes\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePSII(-Mn)\u003c/strong\u003e \u0026nbsp; \u0026nbsp; \u0026nbsp;Mn-depleted PSII membranes\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eROS\u003c/strong\u003e \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;reactive oxygen species\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSOD\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;superoxide dismutase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTM\u003c/strong\u003e \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;thylakoid membrane\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTMTH\u003c/strong\u003e \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003cem\u003eN\u003c/em\u003e-(1-Hydroxy-2,2,6,6-tetramethylpiperidin-4-yl)-2-methylpropanamide\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003eThe research was carried out as part of the Scientific Project of the State Order of the Government of Russian Federation to Lomonosov Moscow State University No. 121032500058-7\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e The authors declare that they have no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAllakhverdiev SI, Kreslavski VD, Klimov VV, Los DA, Carpentier R, Mohanty P (2008) Heat stress: an overview of molecular responses in photosynthesis. 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Coord Chem Rev 252:361\u0026ndash;376 https://doi.org/10.1016/j.ccr.2007.08.021 \u003c/li\u003e\n \u003cli\u003eZavafer A, Cheah MH, Hillier W, Chow WS, Takahashi S (2015) Photodamage to the oxygen evolving complex of photosystem II by visible light. Sci Rep 5:16363 https://doi.org/10.1038/srep16363\u003c/li\u003e\n \u003cli\u003eYamashita A, Nijo M, Pospi\u0026scaron;il P, Morita N, Takenaka D, Aminaka R, Yamamoto Yo, Yamamoto Ya (2008) Quality control of photosystem II. Reactive oxygen species are responsible for the damage to photosystem II under moderate heat stress. J Biol Chem 283:28380\u0026ndash;28391 https://doi.org/10.1074/jbc.M710465200\u003c/li\u003e\n \u003cli\u003eYang S, Wang F, Guo F, Meng JJ, Li X., Wan S. (2015) Calcium contributes to photoprotection and repair of photosystem II in peanut leaves during heat and high irradiance. J Integr Plant Biol, 57(5):486\u0026ndash;495 https://doi.org/10.1111/jipb.12249\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpre","sideBox":"Learn more about [Journal of Plant Research](http://link.springer.com/journal/10265)","snPcode":"10265","submissionUrl":"https://www.editorialmanager.com/jpre/default2.aspx","title":"Journal of Plant Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"photosystem II, protonophores, oxygen-evolving complex, calcium, heat inactivation, hydroxylamine spin probe","lastPublishedDoi":"10.21203/rs.3.rs-4019854/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4019854/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn the region of slightly acidic pH (рН 5.7), the manganese cluster in oxygen-evolving complex of photosystem II (PSII) is more resistant to exogenous reductants (Semin et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The effect of such pH on the heat inactivation efficiency of the electron transport chain (O\u003csub\u003e2\u003c/sub\u003e evolution and 2,6-dichlorophenolindophenol reduction) in PSII membranes and thylakoid membranes was investigated. Under thylakoid membranes illumination accompanied by lumen acidification, their resistance to heat inactivation increases. In the presence of protonophores, the rate of heat inactivation increases, which seems to be associated not with the protonophore mechanism, but with structural and/or functional changes in membranes. In PSII membrane preparations, the efficiency of the oxygen evolution inhibition at pH 5.7 is also lower than at pH 6.5. The role of reactive oxygen species in thermal inactivation of photosynthetic membranes was investigated using a lipophilic cyclic hydroxylamine ESR spin probe.\u003c/p\u003e","manuscriptTitle":"Light increases resistance of thylakoid membranes to thermal inactivation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-28 10:00:04","doi":"10.21203/rs.3.rs-4019854/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-04-07T07:21:23+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-25T23:49:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-07T07:09:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Plant Research","date":"2024-03-06T02:47:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpre","sideBox":"Learn more about [Journal of Plant Research](http://link.springer.com/journal/10265)","snPcode":"10265","submissionUrl":"https://www.editorialmanager.com/jpre/default2.aspx","title":"Journal of Plant Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"83fdef6b-6b87-4eaf-85cf-bf070dc142a7","owner":[],"postedDate":"March 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-07T15:58:53+00:00","versionOfRecord":{"articleIdentity":"rs-4019854","link":"https://doi.org/10.1007/s10265-024-01584-7","journal":{"identity":"journal-of-plant-research","isVorOnly":false,"title":"Journal of Plant Research"},"publishedOn":"2024-10-02 15:56:54","publishedOnDateReadable":"October 2nd, 2024"},"versionCreatedAt":"2024-03-28 10:00:04","video":"","vorDoi":"10.1007/s10265-024-01584-7","vorDoiUrl":"https://doi.org/10.1007/s10265-024-01584-7","workflowStages":[]},"version":"v1","identity":"rs-4019854","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4019854","identity":"rs-4019854","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}