The role of antioxidant defense in freezing tolerance of resurrection plant Haberlea rhodopensis.

Haberlea rhodopensis Friv. is unique with its ability to survive two extreme environmental stresses-desiccation to air-dry state and subzero temperatures. In contrast to desiccation tolerance, the mechanisms of freezing tolerance of resurrection plants are scarcely investigated. In the present study, the role of antioxidant defense in the acquisition of cold acclimation and freezing tolerance in this resurrection plant was investigated comparing the results of two sets of experiments-short term freezing stress after cold acclimation in controlled conditions and long term freezing stress as a part of seasonal temperature fluctuations in an outdoor ex situ experiment. Significant enhancement in flavonoids and anthocyanin content was observed only as a result of freezing-induced desiccation. The total amount of polyphenols increased upon cold acclimation and it was similar to the control in post freezing stress and freezing-induced desiccation. The main role of phenylethanoid glucoside, myconoside and hispidulin 8-C-(2-O-syringoyl-b-glucopyranoside) in cold acclimation and freezing tolerance was elucidated. The treatments under controlled conditions in a growth chamber showed enhancement in antioxidant enzymes activity upon cold acclimation but it declined after subsequent exposure to -10 °C. Although it varied under ex situ conditions, the activity of antioxidant enzymes was high, indicating their important role in overcoming oxidative stress under all treatments. In addition, the activity of specific isoenzymes was upregulated as compared to the control plants, which could be more useful for stress counteraction compared to changes in the total enzyme activity, due to the action of these isoforms in the specific cellular compartments. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-00998-0.

Low temperature and high light dependent dynamic photoprotective strategies in Arabidopsis thaliana.

Arabidopsis thaliana has been recognized as a chilling tolerant species based on analysis of resistance to low temperature stress, however, the mechanisms involved in this tolerance are not yet clarified. The low temperature-induced effects are exacerbated when plants are exposed to low temperatures in the presence of high light irradiance but the experimental data on the impact of light intensity during cold stress and its influence during recovery from stress are rather limited. The main objective of this study was to re-examine the photosynthetic responses of A. thaliana plants to short term (6 days) low temperature stress (12/10°C) under optimal (150 µmol m-2 s-1 ) and high light (500 µmol m-2 s-1 ) intensity and the subsequent recovery from the stress. Simultaneous measurements of the in vivo and in vitro functional performance of both photosystem II (PSII) and photosystem I (PSI), as well as, net photosynthesis, low temperature (77 K) chlorophyll fluorescence and immunoblot analysis of the relative abundance of PSII and PSI reaction center proteins were used to evaluate the role of light in the development of possible protective mechanisms during low temperature stress and the consequent recovery from exposure to low temperature and different light intensities. The results presented clearly suggest that Arabidopsis plants can employ a number of highly dynamic photoprotective strategies depending on the light intensity. These strategies include one based on LHCII quenching and two other quenching mechanisms localized within the PSII and PSI reaction centers, which are all expressed to different extent depending on the severity of the photoinhibitory treatments under low temperature stress conditions.

Differential temperature effects on dissipation of excess light energy and energy partitioning in lut2 mutant of Arabidopsis thaliana under photoinhibitory conditions.

The high-light-induced alterations in photosynthetic performance of photosystem II (PSII) and photosystem I (PSI) as well as effectiveness of dissipation of excessive absorbed light during illumination for different periods of time at room (22 °C) and low (8-10 °C) temperature of leaves of Arabidopsis thaliana, wt and lut2, were followed with the aim of unraveling the role of lutein in the process of photoinhibition. Photosynthetic parameters of PSII and PSI were determined on whole leaves by PAM fluorometer and oxygen evolving activity-by a Clark-type electrode. In thylakoid membranes, isolated from non-illuminated and illuminated for 4.5 h leaves of wt and lut2 the photochemical activity of PSII and PSI and energy interaction between the main pigment-protein complexes was determined. Results indicate that in non-illuminated leaves of lut2 the maximum rate of oxygen evolution and energy utilization in PSII is lower, excitation pressure of PSII is higher and cyclic electron transport around PSI is faster than in wt leaves. Under high-light illumination, lut2 leaves are more sensitive in respect to PSII performance and the extent of increase of excitation pressure of PSII, ΦNO, and cyclic electron transport around PSI are higher than in wt leaves, especially when illumination is performed at low temperature. Significant part of the excessive light energy is dissipated via mechanism, not dependent on ∆pH and to functioning of xanthophyll cycle in LHCII, operating more intensively in lut2 leaves.

Heat stress-induced effects of photosystem I: an overview of structural and functional responses.

Temperature is one of the main factors controlling the formation, development, and functional performance of the photosynthetic apparatus in all photoautotrophs (green plants, algae, and cyanobacteria) on Earth. The projected climate change scenarios predict increases in air temperature across Earth's biomes ranging from moderate (3-4 °C) to extreme (6-8 °C) by the year 2100 (IPCC in Climate change 2007: The physical science basis: summery for policymakers, IPCC WG1 Fourth Assessment Report 2007; Climate change 2014: Mitigation of Climate Change, IPCC WG3 Fifth Assessment Report 2014). In some areas, especially of the Northern hemisphere, even more extreme warm seasonal temperatures may occur, which possibly will cause significant negative effects on the development, growth, and yield of important agricultural crops. It is well documented that high temperatures can cause direct damages of the photosynthetic apparatus and photosystem II (PSII) is generally considered to be the primary target of heat-induced inactivation of photosynthesis. However, since photosystem I (PSI) is considered to determine the global amount of enthalpy in living systems (Nelson in Biochim Biophys Acta 1807:856-863, 2011; Photosynth Res 116:145-151, 2013), the effects of elevated temperatures on PSI might be of vital importance for regulating the photosynthetic response of all photoautotrophs in the changing environment. In this review, we summarize the experimental data that demonstrate the critical impact of heat-induced alterations on the structure, composition, and functional performance of PSI and their significant implications on photosynthesis under future climate change scenarios.

Effect of Low Light on Photosynthetic Performance of Tomato Plants-Ailsa Craig and Carotenoid Mutant Tangerine.

The effects of a five-day treatment with low light intensity on tomato plants-Ailsa Craig and tangerine mutant-at normal and low temperatures and after recovery for three days under control conditions were investigated. The tangerine tomato, which has orange fruits, yellowish young leaves, and pale blossoms, accumulates prolycopene rather than all-trans lycopene. We investigated the impact of low light at normal and low temperatures on the functioning and effectiveness of photosynthetic apparatuses of both plants. The photochemical activities of Photosystem I (PSI) and Photosystem II (PSII) were assessed, and the alterations in PSII antenna size were characterized by evaluating the abundance of PSII-associated proteins Lhcb1, Lhcb2, CP43, and CP47. Alterations in energy distribution and interaction of both photosystems were analyzed using 77K fluorescence. In Aisla Craig plants, an increase in thylakoid membrane fluidity was detected during treatment with low light at a low temperature, while for the tangerine mutant, no significant change was observed. The PSII activity of thylakoids from mutant tangerine was more strongly inhibited by treatment with low light at a low temperature while low light barely affected PSII in Aisla Craig. The obtained data indicated that the observed differences in the responses of photosynthetic apparatuses of Ailsa Craig and tangerine when exposed to low light intensity and suboptimal temperature were mainly related to the differences in sensitivity and antenna complexes of PSII.

Antioxidative Defense, Suppressed Nitric Oxide Accumulation, and Synthesis of Protective Proteins in Roots and Leaves Contribute to the Desiccation Tolerance of the Resurrection Plant Haberlea rhodopensis.

The desiccation tolerance of plants relies on defense mechanisms that enable the protection of macromolecules, biological structures, and metabolism. Although the defense of leaf tissues exposed to solar irradiation is challenging, mechanisms that protect the viability of the roots, yet largely unexplored, are equally important for survival. Although the photosynthetic apparatus in leaves contributes to the generation of oxidative stress under drought stress, we hypothesized that oxidative stress and thus antioxidative defense is also predominant in the roots. Thus, we aimed for a comparative analysis of the protective mechanisms in leaves and roots during the desiccation of Haberlea rhodopensis. Consequently, a high content of non-enzymatic antioxidants and high activity of antioxidant enzymes together with the activation of specific isoenzymes were found in both leaves and roots during the final stages of desiccation of H. rhodopensis. Among others, catalase and glutathione reductase activity showed a similar tendency of changes in roots and leaves, whereas, unlike that in the leaves, superoxide dismutase activity was enhanced under severe but not under medium desiccation in roots. Nitric oxide accumulation in the root tips was found to be sensitive to water restriction but suppressed under severe desiccation. In addition to the antioxidative defense, desiccation induced an enhanced abundance of dehydrins, ELIPs, and sHSP 17.7 in leaves, but this was significantly better in roots. In contrast to leaf cells, starch remained in the cells of the central cylinder of desiccated roots. Taken together, protective compounds and antioxidative defense mechanisms are equally important in protecting the roots to survive desiccation. Since drought-induced damage to the root system fundamentally affects the survival of plants, a better understanding of root desiccation tolerance mechanisms is essential to compensate for the challenges of prolonged dry periods.

The Role of Alternative Electron Pathways for Effectiveness of Photosynthetic Performance of Arabidopsis thaliana, Wt and Lut2, under Low Temperature and High Light Intensity.

A recent investigation has suggested that the enhanced capacity for PSI-dependent cyclic electron flow (CEF) and PSI-dependent energy quenching that is related to chloroplast structural changes may explain the lower susceptibility of lut2 to combined stresses-a low temperature and a high light intensity. The possible involvement of alternative electron transport pathways, proton gradient regulator 5 (PGR5)-dependent CEF and plastid terminal oxidase (PTOX)-mediated electron transfer to oxygen in the response of Arabidopsis plants-wild type (wt) and lut2-to treatment with these two stressors was assessed by using specific electron transport inhibitors. Re-reduction kinetics of P700+ indicated that the capacity for CEF was higher in lut2 when this was compared to wt. Exposure of wt plants to the stress conditions caused increased CEF and was accompanied by a substantial raise in PGR5 and PTOX quantities. In contrast, both PGR5 and PTOX levels decreased under the same stress conditions in lut2, and inhibiting PGR5-dependent pathway by AntA did not exhibit any significant effects on CEF during the stress treatment and recovery period. Electron microscopy observations demonstrated that under control conditions the degree of grana stacking was much lower in lut2, and it almost disappeared under the combined stresses, compared to wt. The role of differential responses of alternative electron transport pathways in the acclimation to the stress conditions that are studied is discussed.

Reactivation of the Photosynthetic Apparatus of Resurrection Plant Haberlea rhodopensis during the Early Phase of Recovery from Drought- and Freezing-Induced Desiccation.

Haberlea rhodopensis is a unique desiccation-tolerant angiosperm that also survives winter frost. As, upon freezing temperatures, H. rhodopensis desiccates, the taxon is proposed to survive low temperature stress using its desiccation tolerance mechanisms. To reveal the validity of this hypothesis, we analyzed the structural alterations and organization of photosynthetic apparatus during the first hours of recovery after drought- and freezing-induced desiccation. The dynamics of the ultrastructure remodeling in the mesophyll cells and the restoration of the thylakoid membranes shared similarities independent of the reason for desiccation. Among the most obvious changes in thylakoid complexes, the proportion of the PSI-LHCII complex strongly increased around 70% relative water content (RWC), whereas the proportion of Lhc monomers decreased from the beginning of rehydration. We identified enhanced levels of cyt b6f complex proteins that contributed to the enhanced electron flow. The high abundance of proteins related to excitation energy dissipation, PsbS, Lhcb5, Lhcb6 and ELIPs, together with the increased content of dehydrins contributed to the preservation of cellular integrity. ELIP expression was maintained at high levels up to 9 h into recovery. Although the recovery processes from drought- and freezing-induced desiccation were found to be similar in progress and time scale, slight variations indicate that they are not identical.

Response of isolated thylakoid membranes with altered fluidity to short term heat stress.

The effect of alterations of lipid phase order of thylakoid membranes on the thermosensitivity of photosystem I (PS I) and photosystem II (PS II) was studied. Plant sterols stigmasterol and cholesterol were applied to decrease the fluidity in isolated membranes. After sterol treatment, a decrease of the temperature of 50 % inhibition of PSII activity was observed. Heat stress-induced stimulation of PSI-mediated electron transport rate was registered for control, but not for sterol-treated membranes. Effect of altered lipid order on oxygen evolving complex was evaluated by means of flash oxygen yields revealing changes in the stoichiometry of PSIIα and PSIIß centers. The effect of sterol incorporation on the changes in the thermotropic behavior of the main pigment-protein complexes was studied by differential scanning calorimetry (DSC). DSC traces of control thylakoids in the temperature range 20-98 °C exhibited several irreversible endothermic transitions. Incorporation of cholesterol and stigmasterol results in superimposition of the transitions and only two main bands could be resolved. While high temperature band peaks at the same temperature after treatment with both sterols, the band that combines low temperature transitions shows different melting temperature (Tm): 70 °C for stigmasterol- and 65 °C for cholesterol-treated membranes. The data presented here emphasise the crucial role of lipid order for the response of thylakoids to high temperatures, mediated not only by changes in the fluidity of bulk lipid phase as result of sterol incorporation but also by changes in the thermotropic properties of pigment-protein complexes.

UV-B response of greening barley seedlings.

The relationship between the greening stage of barley seedlings and their response to UV-B irradiation was studied. Etiolated barley seedlings ( Hordeum vulgare L., cv. Alfa) greened 12, 24 and 48 h were exposed to UV-B irradiation (312 nm) for 5 h. As a result of UV-B treatment the rate of CO(2) fixation and chlorophyll contents decreased but flavonoids, UV-B-induced compounds and carotenoids increased. The inhibition of photosynthesis in green plants was lower in comparison to greening ones. The 12 h greening plants were more sensitive to UV-B treatment than the plants greening 24 h and particularly 48 h, estimated by the quantum efficiency of PSII photochemistry and the oxygen production rate. The levels of flavonoids and UV-B induced compounds enhanced with increasing the greening time. Activity of antioxidant enzymes catalase, peroxidase and superoxide dismutase increased during the seedlings greening and as a result of UV-B irradiation, but the pattern of isoforms remained similar to those found in the controls. UV-B preferentially induced Cu,Zn-superoxide dismutase. Increase of UVB induced synthesis of antioxidant enzymes is in line with their important role in the plant response to UV-B stress. Data presented show that the response of barley seedlings to UV-B irradiation is related to the development stage of photosynthetic apparatus.

Acquired tolerance of the photosynthetic apparatus to photoinhibition as a result of growing Solanum lycopersicum at moderately higher temperature and light intensity.

The kinetics of photoinhibition in detached leaves from tomato plants (Solanium lycopersicum L. cv. M82) grown for 6 days under different combinations of optimal and moderately high temperature and optimal and high light intensity were studied. The inhibition of PSII was evaluated by changes in maximal quantum yield, the coefficient of photochemical quenching and the quantum yield of PSII. The changes of PSI activity was estimated by the redox state of P700. The involvement of different possible protective processes was checked by determination of nonphotochemical quenching and cyclic electron flow around PSI. To evaluate to what extent the photosynthetic apparatus and its response to high light treatment was affected by growth conditions, the kinetics of photoinhibition in isolated thylakoid membranes were also studied. The photochemical activities of both photosystems and changes in the energy distribution and interactions between them were evaluated by means of a Clark electrode and 77 K fluorescence analysis. The data showed an increased tolerance to photoinhibition in plants grown under a combination of moderately high temperature and light intensity, which was related to the stimulation of cyclic electron flow, PSI activity and rearrangements of pigment-protein complexes, leading to a decrease in the excitation energy delivered to PSII.

Quality control of Photosystem II: cleavage and aggregation of heat-damaged D1 protein in spinach thylakoids.

Moderate heat stress (40 degrees C, 30 min) on spinach thylakoids induced cleavage of the D1 protein, producing an N-terminal 23-kDa fragment, a C-terminal 9-kDa fragment, and aggregation of the D1 protein. A homologue of Arabidopsis FtsH2 protease, which is responsible for degradation of the damaged D1 protein, was abundant in the stroma thylakoids. Two processes occurred in the thylakoids in response to heat stress: dephosphorylation of the D1 protein in the stroma thylakoids, and aggregation of the phosphorylated D1 protein in the grana. Heat stress also induced the release of the extrinsic PsbO, P and Q proteins from Photosystem II, which affected D1 degradation and aggregation significantly. The cleavage and aggregation of the D1 protein appear to be two alternative processes influenced by protein phosphorylation/dephosphorylation, distribution of FtsH, and intactness of the thylakoids.

Valorization of a plant ß-amylase: Immobilization and dataset on the kinetic process.

The data presented in this article are related to the research article titled "Immobilization nd topochemical mechanism of a new ß-amylase extracted from Pergularia tomentosa" (Lahmar et al., 2017) [1]. This article documented information on the determination of the molecular weight of the ß-amylase, the method of its immobilization and a comparison of the kinetic mechanism between the free and the immobilized forms by a mathematical method. Fresh Pergularia tomentosa was collected from Tunisia and a special method for ß-amylase extraction was followed (Yotova et al., 2000) [2]. Public dissemination of this dataset will allow further analyses of the data.

Selective photobleaching of chlorophylls and carotenoids in photosystem I particles under high-light treatment.

Photosystem I particles (PSI-200) isolated from spinach leaves were studied by means of absorbance, 77K fluorescence and resonance Raman (RR) spectroscopy. The aim was to obtain better insight into the changes of the pigment spectral properties in those particles during prolonged exposure to high-light intensities and to reveal the involvement of these pigments in the photoprotection of the PSI. During prolonged exposure to high-light intensities of spinach PSI particles, a loss of a significant amount of photosynthetic pigments was observed. It was shown that various pigments exhibited different susceptibility to photodamage. In addition to bleaching of chlorophyll a (Chl a), bleaching of carotenoids was also clearly observed. RR technique allowed us to recognize the type and conformation of photobleached carotenoid molecules. Raman data revealed a nearly full photobleaching of the long-wavelength lutein molecules. The observed similar bleaching rate of the lutein molecules and the most-red shifted long-wavelength Chl a, located in the antenna membrane protein Lhca4, suggested that these molecules are located closely. Our results showed that the photobleached antenna pigments and especially luteins and the most long-wavelength absorbing chlorophylls are involved in photoprotection of PSI core complex.

Effect of membrane fluidity on photosynthetic oxygen production reactions.

The effect of changes of membrane fluidity on the oxygen evolving capability of isolated thylakoids was investigated. Alteration of the lipid phase fluidity was achieved by incorporation of the plant sterol stigmasterol. Incorporation of stigmasterol in the lipid bilayer of thylakoid membranes results in rigidization of the hydrophobic phase of thylakoid membranes and decreases the degree of packing of the lipid head groups. These changes of lipid order are accompanied by a reduction of oxygen evolution, measured with 1,4-benzoquinone as an electron acceptor, and by a more pronounced inhibition of PSI-mediated electron transport. By analysis of the parameters of oxygen flash yields and oxygen burst under continuous illumination it was shown that after treatment with stigmasterol: 1.) the number of active oxygen-evolving centres decreased; 2.) the remaining active oxygen-evolving centres were not affected in respect to the oscillation pattern; 3.) the contribution of the slow oxygen-evolving centres in oxygen burst yield was increased. The effect of stigmasterol was compared with the well-studied effect of cholesterol. Results were discussed in terms of determining the role of lipid order for the organization and functioning of the photosynthetic machinery.

The Lack of Lutein Accelerates the Extent of Light-induced Bleaching of Photosynthetic Pigments in Thylakoid Membranes of Arabidopsis thaliana.

The high light-induced bleaching of photosynthetic pigments and the degradation of proteins of light-harvesting complexes of PSI and PSII were investigated in isolated thylakoid membranes of Arabidopsis thaliana, wt and lutein-deficient mutant lut2, with the aim of unraveling the role of lutein for the degree of bleaching and degradation. By the means of absorption spectroscopy and western blot analysis, we show that the lack of lutein leads to a higher extent of pigment photobleaching and protein degradation in mutant thylakoid membranes in comparison with wt. The highest extent of bleaching is suffered by chlorophyll a and carotenoids, while chlorophyll b is bleached in lut2 thylakoids during long periods at high illumination. The high light-induced degradation of Lhca1, Lhcb2 proteins and PsbS was followed and it is shown that Lhca1 is more damaged than Lhcb2. The degradation of analyzed proteins is more pronounced in lut2 mutant thylakoid membranes. The lack of lutein influences the high light-induced alterations in organization of pigment-protein complexes as revealed by 77 K fluorescence.

Tomato plants acclimate better to elevated temperature and high light than to treatment with each factor separately.

The influence of two factors - high temperature and high light intensity, acting separately or simultaneously on the pigment composition, fluorescent characteristics, membrane integrity and synthesis of protective substances was investigated in tomato plants (Solanum lycopersicum cv. M 82). Moderate elevated temperatures (38/29 °C) were applied under optimum or high light intensity for 2 and 6 days and after that the plants are allowed to recover for 5 days at optimum conditions. Parameters of chlorophyll fluorescence were used to evaluate the alterations of photosystem I and photosystem II activity and malondialdehyde content was determined as a measure of stress-induced peroxidation of membrane lipids. The response of treated plants to high light and elevated temperature was estimated by analyzing the accumulation of anthocyanins. Both stress factors exhibit different impact on studied parameters - high light intensity influences considerably quantum yield of photosystem II and photochemical quenching that is compensated to some extent when applied at elevated temperature. High temperature reduces strongly non-photochemical quenching. Data obtained show that after two days under particular conditions, the plants tend to acclimate, but this is achieved after longer treatment - 6 days. During the recovery period the activity of photosystem I and the quantum yield of photosystem II recover almost completely, while the values of non-photochemical quenching although slightly higher, did not reach the levels at the beginning of treatment.

Resonance Raman spectroscopy of carotenoids in Photosystem I particles.

Low-temperature resonance Raman (RR) spectroscopy was used for the first time to study the spectral properties, binding sites and composition of major carotenoids in spinach Photosystem I (PSI) particles. Excitation was provided by an argon ion laser at 457.9, 476.5, 488, 496.5, 502 and 514.5 nm. Raman spectra contained the four known groups of bands characteristic for carotenoids (called from nu(1) to nu4). Upon 514.5, 496.5 and 476.5 nm excitations, the nu(1)-nu(3) frequencies coincided with those established for lutein. Spectrum upon 502-nm excitation could be assigned to originate from violaxanthin, at 488 nm to 9-cis neoxanthin, and at 457.9 nm to beta-carotene and 9-cis neoxanthin. The overall configuration and composition of these bound carotenoid molecules in Photosystem I particles were compared with the composition of pigment extracts from the same PSI particles dissolved in pyridine, as well as to configuration in the main chlorophyll a/b light-harvesting protein complex of photosystem II. The absorption transitions for lutein, violaxanthin and 9-cis neoxanthin in spinach photosystem I particles are characterized, and the binding sites of lutein and neoxanthin are discussed. Resonance Raman data suggest that beta-carotene molecules are also present in all-trans and, probably, in 9-cis configurations.

High light-induced changes of 77 K fluorescence emission of pea thylakoid membranes with altered membrane fluidity.

The effect of lipid phase order of isolated thylakoid membranes on fluorescent characteristics of both photosystems during illumination with high light intensity at 22 degrees C and 4 degrees C was investigated. For artificial modification of membrane fluidity two membrane perturbing agents were applied-cholesterol and benzyl alcohol. 77 K fluorescence emission and excitation spectra of control, cholesterol- and benzyl alcohol-treated thylakoid membranes were analysed in order to determine the high light-induced changes of emission bands attributed to different chlorophyll-protein complexes-F 735, emitted by photosystem I-light-harvesting complex I; and F 685 and F 695, emitted by photosystem II-light-harvesting complex II. Analysis of emission bands showed that high light treatment leads to a decrease of the area of band at 695 nm and a concomitant increase of intensity of the band at 735 nm. The involvement of different pigment pools (chlorophyll a and chlorophyll b) in the energy supply of both photosystems before and after photoinhibitory treatment was estimated on the basis of excitation fluorescence spectra. The dependence of the ratios F 735/F 685 and the band areas at 685 and 695 nm on the illumination time was studied at both temperatures. Data presented indicate that cholesterol incorporation stabilized the intersystem structure in respect to light-induced changes of fluorescence emission of PSI and PSII. It was shown that the effect of fluid properties of thylakoid membranes on the 77 K fluorescence characteristics of main pigment protein complexes of pea thyalkoid membranes depends on the temperature during high light treatment.

Different kinetics of photoinactivation of photosystem I-mediated electron transport and P700 in isolated thylakoid membranes.

Photoinactivation kinetics of photosystem I (PSI)-mediated electron transport rate was compared to that of P700 content at room (22 degrees C) and low (4 degrees C) temperatures in isolated spinach thylakoid membranes. The high light treatment was carried out under aerobic and anaerobic conditions. At 22 degrees C the decrease of electron transport rate showed first order exponential kinetics. The amount of P700 decreased linearly, being less affected in the first hours of illumination. During photoinhibition at 4 degrees C in the presence of oxygen, the kinetics of inactivation of PSI photochemical activity and the content of P700 were different. It was found that 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) had different protective effect on the electron transport rate and on P700 content at both temperatures. Treatment with high light intensity under N(2) atmosphere had no effect on the electron transport rate or P700 content. The possible degradation of PSI reaction centre proteins was determined using immunoblot methods. In the presence of linear electron transport at 22 degrees C correlation between formation of toxic hydroxyl radicals and inhibition of oxygen uptake was observed.