Photosystem II reaction centres stay intact during low temperature photoinhibition

Photoinhibition of photosynthesis was studied in intact barley leaves at 5 and 20°C, to reveal if Photosystem II becomes predisposed to photoinhibition at low temperature by 1) creation of excessive excitation of Photosystem II or, 2) inhibition of the repair process of Photosystem II. The light and temperature dependence of the reduction state of Q_A was measured by modulated fluorescence. Photon flux densities giving 60% of Q_A in a reduced state at steady-state photosynthesis (300 mol m^–2s^–1 at 5°C and 1200 mol m^–2s^–1 at 20°C) resulted in a depression of the photochemical efficiency of Photosystem II (F_v/F_m) at both 5 and 20°C. Inhibition of F_v/F_m occurred with initially similar kinetics at the two temperatures. After 6h, F_v/F_m was inhibited by 30% and had reached steady-state at 20°C. However, at 5°C, F_v/F_m continued to decrease and after 10h, F_v/F_m was depressed to 55% of control. The light response of the reduction state of Q_A did not change during photoinhibition at 20°C, whereas after photoinhibition at 5°C, the proportion of closed reaction centres at a given photon flux density was 10–20% lower than before photoinhibition.Changes in the D1-content were measured by immunoblotting and by the atrazine binding capacity during photoinhibition at high and low temperatures, with and without the addition of chloramphenicol to block chloroplast encoded protein synthesis. At 20°C, there was a close correlation between the amount of D1-protein and the photochemical efficiency of photosystem II, both in the presence or in the absence of an active repair cycle. At 5°C, an accumulation of inactive reaction centres occurred, since the photochemical efficiency of Photosystem II was much more depressed than the loss of D1-protein. Furthermore, at 5°C the repair cycle was largely inhibited as concluded from the finding that blockage of chloroplast encoded protein synthesis did not enhance the susceptibility to photoinhibition at 5°C.It is concluded that, the kinetics of the initial decrease of F_v/F_m was determined by the reduction state of the primary electron acceptor Q_A, at both temperatures. However, the further suppression of F_v/F_m at 5°C after several hours of photoinhibition implies that the inhibited repair cycle started to have an effect in determining the photochemical efficiency of Photosystem II.Abbreviations CAP D-threochloramphenicol - F₀ and F ₀ fluorescence when all Photosystem II reaction centres are open in dark- and light-acclimated leaves, respectively - F_m and F _m fluorescence when all Photosystem II reaction centres are closed in dark- and light-acclimated leaves, respectively - F_s fluorescence at steady state - Q_A the primary, stable quinone acceptor of Photosystem II - q_N non-photochemical quenching of fluorescence - q_P photochemical quenching of fluorescence     

Rapid turnover of a component required for photosynthesis explains temperature dependence and kinetics of photoinhibition in a cyanobacterium,Synechococcus 6301

Illumination of a liquid culture of Synechococcus 6301 at high photon flux density (PFD) elicits a time-dependent first-order exponential decline in relative quantum yield of photosynthetic O₂ evolution to some steady-state value. Full photosynthetic activity is restored, also as a time-dependent first-order process, when the photoinhibited culture is transferred to lower PFD. Temperature and irradiation dependence of photoinhibition were measured under conditions which precluded simultaneous recovery from photoinhibition. Also the temperature and irradiation dependence of recovery from photoinhibition were determined under conditions which precluded simultaneous photoinhibition. Kinetics of photoinhibition were sensitive to PFD but relatively independent of temperature. Kinetics of recovery saturated at low PFD but were very temperature dependent at all PFDs. A general equation can be written to predict the change in photosynthetic activity versus time when a cell culture is placed at photoinhibitory PFD, assuming that first-order exponential photoinhibition and first-order exponential recovery from photoinhibition occur simultaneously. The equation can be made specific if the values of the kinetic constant for photoinhibition and for recovery from photoinhibition are known for the particular environmental conditions to which the cells are exposed. These values can be obtained by independently measuring the kinetics of photoinhibition without simultaneous recovery and the kinetics of recovery without simultaneous photoinhibition. The curve of photosynthetic activity versus time for cells placed at high PFD, which is predicted by this equation, precisely fits the experimentally determined kinetics of photoinhibition. This correlation remains valid over a wide range of temperatures and PFDs. Identical results were obtained with the marine cyanobacterium Synechococcus 7002. We conclude that the extent of net photoinhibition over a broad range of conditions represents a sum of individual rates of simultaneous photoinhibition and recovery from photoinhibition. The results support previous proposals that a protein required for photosystem II activity becomes functionally depleted during photoinhibition because protein synthesis or assembly into the membranes cannot keep up with the rate of its inactivation at excessively high PFDs. We also conclude that photoinhibition and light-dependent chilling sensitivity are manifestations of the same phenomenon.Abbreviations CAP chloramphenicol - Chl chlorophyll - PFD photon flux density - PSII photosystem II The authors thank Rockey Butler and Donna Scott for performing many of the preliminary experiments which led to this research. This work was supported by R.A. Welch and University Research Institute Grants to J.J.B.     

Reduced sensitivity to photoinhibition following frost-hardening of winter rye is due to increased phosphate availability

The possibility of a role for phosphate metabolism in the photosynthetic regulation that occurs during frost hardening was investigated in winter rye (Secale cereale L. cv. Musketeer). Leaves of frost-hardened and non-hardened winter rye were studied during photosynthetic induction, and at steady state after being allowed to take up 20 mM orthophosphate through the transpiration stream for 3 h. At the growth irradiance (350 mol·m^-2·s^-1) frost-hardening increased the stationary rate of CO₂-dependent O₂ evolution by 57% and 25% when measured at 5 and 20° C, respectively. Frosthardening also reduced the lag phase to stationary photosynthesis by 40% at 5° C and decreased the susceptibility of leaves to oscillations during induction and after interruption of the actinic beam during steady-state photosynthesis. These responses are all indicative of increased phosphate availability in frost-hardened leaves. As reported previously by Öquist and Huner (1993, Planta 189, 150–156), frost-hardening also decreased the reduction state of Q_A, the primary, stable quinone acceptor of PSII, and decreased the sensitivity of winter rye to photoinhibition of photosynthesis. Non-hardened rye leaves fed orthophosphate also showed an increased photosynthetic capacity (25% at 20° C and light saturation), lower reduction state of Q_A, a reduced sensitivity to photoinhibition and lower susceptibility to oscillations resulting from a brief interruption of the actinic light. Thus, the data indicate that phosphate metabolism plays a key role in photosynthetic acclimation of winter rye to low temperatures.Abbreviations F_o and F_o minimal fluorescence when all PSII reaction centres are open in dark-and light-acclimated leaves, respectively - F_m and F_m maximal fluorescence when all PSII reaction centres are closed in dark-and light-acclimated leaves, respectively - F_v variable fluoresence (F_m -F_o) in dark-acclimated leaves - F_v variable fluorescence (F_m-F_o) in light-acclimated leaves - PCR photosynthetic carbon reduction - PPFD photosynthetic photon flux density - Q_A the primary, stable quinone acceptor of PSII - q_P photochemical quenching of fluorescence - q_N non-photochemical quenching of fluorescence This work was supported by the Swedish Natural Sciences Research Council. The authors are indebted to Dr. N. Huner, Department of Plant Sciences, UWO, London, Canada, for helpful discussions during the initiation of this work and for the gift of rye seeds.     

The site of photoinhibition in leaves of Cucumis sativus L. at low temperatures is photosystem I,not photosystem II

Maximum quantum yields (QY) of photosynthetic electron flows through PSI and PSII were separately assessed in thylakoid membranes isolated from leaves of Cucumis sativus L. (cucumber) that had been chilled in various ways. The QY(PSI) in the thylakoids prepared from the leaves treated at 4° C in moderate light at 220 mol quanta·m^–2·s^–1 (400–700 nm) for 5 h, was about 20–30% of that in the thylakoids prepared from untreated leaves, while QY(PSII) decreased, at most, by 20% in response to the same treatment. The decrease in QY(PSI) was observed only when the leaves were chilled at temperatures below 10° C, while such a marked temperature dependency was not observed for the decrease in QY(PSII). In the chilling treatment at 4° C for 5 h, the quantum flux density that was required to induce 50% loss of QY (PSI) was ca. 50 umol quanta·m^–2·s^–1. When the chilling treatment at 4° C in the light was conducted in an atmosphere of N₂, photoinhibition of PSI was largely suppressed, while the damage to PSII was somewhat enhanced. The ferricyanide-oxidised minus ascorbate-reduced difference spectra and the light-induced absorbance changes at 700 nm obtained with the thylakoid suspension, indicated the loss of P700 to extents that corresponded to the decreases in QY(PSI). Accordingly, the decreases in QY(PSI) can largely be attributed to destruction of the PSI reaction centre itself. These results clearly show that, at least in cucumber, a typical chillingsensitive plant, PSI is much more susceptible to aerobic photoinhibition than PSII.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - P700 primary electron donor of PSI - PPFD photosynthetically active photon flux density - QY quantum yield We are grateful to invaluable comments by Prof. S. Katoh, K. Hikosaka and the members of our laboratory. We also thank A. Aoyama for technical assistance. This work was partly supported by the grants from the Ministry of Education, Science, and Culture, Japan, to I. Terashima (#03740342 and #04640621).     

Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: effect of growth temperature on photoinhibition and recovery

Intact leaves of kiwifruit (Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson) from plants grown in a range of controlled temperatures from 15/10 to 30/25°C were exposed to a photon flux density (PFD) of 1500 μmol·m⁻²·s⁻¹ at leaf temperatures between 10 and 25°C. Photoinhibition and recovery were followed at the same temperatures and at a PFD of 20 μmol·m⁻²·s⁻¹, by measuring chlorophyll fluorescence at 77 K and 692 nm, by measuring the photon yield of photosynthetic O₂ evolution and light-saturated net photosynthetic CO₂ uptake. The growth of plants at low temperatures resulted in chronic photoinhibition as evident from reduced fluorescence and photon yields. However, low-temperature-grown plants apparently had a higher capacity to dissipate excess excitation energy than leaves from plants grown at high temperatures. Induced photoinhibition, from exposure to a PFD above that during growth, was less severe in low-temperature-grown plants, particularly at high exposure temperatures. Net changes in the instantaneous fluorescence,F ₀, indicated that little or no photoinhibition occurred when low-temperature-grown plants were exposed to high-light at high temperatures. In contrast, high-temperature-grown plants were highly susceptible to photoinhibitory damage at all exposure temperatures. These data indicate acclimation in photosynthesis and changes in the capacity to dissipate excess excitation energy occurred in kiwifruit leaves with changes in growth temperature. Both processes contributed to changes in susceptibility to photoinhibition at the different growth temperatures. However, growth temperature also affected the capacity for recovery, with leaves from plants grown at low temperatures having moderate rates of recovery at low temperatures compared with leaves from plants grown at high temperatures which had negligible recovery. This also contributed to the reduced susceptibility to photoinhibition in low-temperature-grown plants. However, extreme photoinhibition resulted in severe reductions in the efficiency and capacity for photosynthesis.     

Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: Recovery and its dependence on temperature

Recovery of photoinhibition in intact leaves of shade-grown kiwifruit was followed at temperatures between 10° and 35° C. Photoinhibition was initially induced by exposing the leaves for 240 min to a photon flux density (PFD) of 1 500 mol·m^-2·s^-1 at 20° C. In additional experiments to determine the effect of extent of photoinhibition on recovery, this period of exposure was varied between 90 and 400 min. The kinetics of recovery were followed by chlorophyll fluorescence at 77K. Recovery was rapid at temperatures of 25–35° and slow or negligible below 20° C. The results reinforce those from earlier studies that indicate chilling-sensitive species are particularly susceptible to photoinhibition at low temperatures because of the low rates of recovery. At all temperatures above 15° C, recovery followed pseudo first-order kinetics. The extent of photoinhibition affected the rate constant for recovery which declined in a linear fashion at all temperatures with increased photoinhibition. However, the extent of photoinhibition had little effect on the temperature-dependency of recovery. An analysis of the fluorescence characteristics indicated that a reduction in non-radiative energy dissipation and repair of damaged reaction centres contributed about equally to the apparent recovery though biochemical studies are needed to confirm this. From an interpretation of the kinetics of photoinhibition, we suggest that recovery occurring during photoinhibition is limited by factors different from those that affect post-photoinhibition recovery.Abbreviations and symbols F _o, F _m, F _v instantaneous, maximum, variable fluorescence - K _D, K _F, K _P, K _T rate constants for non-radiative energy dissipation, fluorescence, photochemistry, transfer to photosystem I - K(PI), k(R) rate constants for photoinhibition and recovery - PFD photon flux density - PSI, II photosystem I, II - _i photon yield of photosynthesis (incident light)     

Reversible photoinhibition of unhardened and cold-acclimated spinach leaves at chilling temperatures

The photoinhibition of photosynthesis at chilling temperatures was investigated in cold-acclimated and unhardened (acclimated to +18° C) spinach (Spinacia oleracea L.) leaves. In unhardened leaves, reversible photoinhibition caused by exposure to moderate light at +4° C was based on reduced activity of photosystem (PS) II. This is shown by determination of quantum yield and capacity of electron transport in thylakoids isolated subsequent to photoinhibition and recovery treatments. The activity of PSII declined to approximately the same extent as the quantum yield of photosynthesis of photoinhibited leaves whereas PSI activity was only marginally affected. Leaves from plants acclimated to cold either in the field or in a growth chamber (+1° C), were considerably less susceptible to the light treatment. Only relatively high light levels led to photoinhibition, characterized by quenching of variable chlorophyll a fluorescence (F_V) and slight inhibition of PSII-driven electron transport. Fluorescence data obtained at 77 K indicated that the photoinhibition of cold-acclimated leaves (like that of the unhardened ones) was related to increased thermal energy dissipation. But in contrast to the unhardened leaves, 77 K fluorescence of cold-acclimated leaves did not reveal a relative increase of PSI excitation. High-light-treated, cold-acclimated leaves showed increased rates of dark respiration and a higher light compensation point. The photoinhibitory fluorescence quenching was fully reversible in low light levels both at +18° C and +4° C; the recovery was much faster than in unhardened leaves. Reversible photoinhibition is discussed as a protective mechanism against excess light based on transformation of PSII reaction centers to fluorescence quenchers.Abbreviations F_O initial fluorescence - F_M maximal fluorescence - F_V devariable fluorescence (f_m-f_o) - PFD photon flux density - PS photosystem - SD standard deviation The authors thank the Deutsche Forschungsgemeinschaft and the Academy of Finland for financial support.     

Mechanism of photosystem-I photoinhibition in leaves ofCucumis sativus L.

It was recently shown that the site of photoinhibition in leaves of Cucumis sativus L. at low temperatures is Photosystem I (PSI), not PSII (I. Terashima et al. 1994, Planta 193, 300–306). In the present study, the mechanisms of this PSI photoinhibition in vivo were examined. By lowering the photon flux density during the photoinhibitory treatment of leaves at 4°C for 5 h to less than 100 mol·m^–2s^–1, we were able to separate the steps of the destruction of the electron-transfer components. Although P-700, the reaction-center chlorophyll, was almost intact in this low-light treatment, the quantum yield of the electron transfer through PSI and photochemically induced absorption change at 701 nm were markedly inhibited. This, along with the results from the measurements of the light-induced absorption changes in the presence of various concentrations of methyl viologen, an artificial electron acceptor, indicates that the component on the acceptor side of the PSI, A₁ or F_x, is the first site of inactivation. When the photon flux density during the treatment was increased to 220 mol·m^–2s^–1, the destruction of P-700 itself was also observed. Furthermore, the partial degradation of the chlorophyll-binding large subunits was observed in photoinhibited leaves. This degradation of the subunits was not detected when the treatment was carried out under nitrogen atmosphere, the condition in which the electron transfer is not inhibited. Thus, the photoinhibitory processes in the reaction center of PSI go through three steps, the inactivation of the acceptor side, the destruction of the reaction-center chlorophyll and the degradation of the reaction center subunit(s). The similarities and the differences between the mechanisms of PSI photoinhibition and those of PSII photoinhibition are discussed.Abbreviations DAD 2,3,5,6-tetramethyl-p-phenylenediamine - LHCI, LHCII light-harvesting chlorophyll-a/b proteins associating with photosystems I and II, respectively - PFD photon flux density We are grateful to Dr. I. Enami (Department of Biology, Faculty of Science, Science University of Tokyo) and Drs. H. Matsubara and H. Oh-oka (Department of Biology, Faculty of Science, Osaka University) for generous gifts of antisera used in the present work. We also thank A. Aoyama for technical assistance. This work was partly supported by the grants from the Ministry of Education, Science and Culture, Japan.     

Freezing damage and frost tolerance of the photosynthetic apparatus studied with isolated mesophyll protoplasts of Valerianella locusta L.

Mesophyll protoplasts were isolated from unhardened and cold-acclimated leaves of Valerianella locusta L. and subjected to freeze-thaw treatment. To evaluate the extent and course of freezing injury, photosynthetic reactions of whole protoplasts and of free thylakoid membranes, liberated from protoplasts by osmotic lysis, were measured. In addition, the integrity of the protoplasts was determined by microscopy. The results reveal an increased frost tolerance of protoplasts isolated from acclimated leaves with respect to all parameters measured. CO₂-dependent O₂ evolution (representing net photosynthetic CO₂ fixation of protoplasts) was the most freezing-sensitive reaction; its inhibition due to freeze-thaw treatment of protoplasts was neither correlated with disintegration of the plasma membrane, nor was it initiated by inactivation of the thylakoid membranes. The frost-induced decline of protoplast integrity was not closely correlated to thylakoid damage either. Freezing injury of the thylakoid membranes was manifested by inhibition of photosynthetic electron transport and photophosphorylation. Both photosystems were affected by freezing and thawing with strongest inhibition occurring in the water-oxidation system or at the oxidizing site of photosystem II. Photophosphorylation responded more sensitively to freezing stress than electron transport, although uncoupling (increased permeability of the thylakoid membranes to protons) was not a conspicuous effect. The data are discussed in relation to freezing injury in leaves and seem to indicate that frost damage in vivo is initiated at multiple sites.Abbreviations Chl chlorphyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenylcarbazide - Hepes 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid - MES 2-(N-morpholino)-ethanesulfonic acid - PS I photosystem I - PS II photosystem II     

Nitrogen supply as a factor influencing photoinhibition and photosynthetic acclimation after transfer of shade-grown Solanum dulcamara to bright light

We have compared the ability of shadegrown clones of Solamum dulcamara L. from shade and sun habitats to acclimate to bright light, as a function of nitrogen nutrition before and after transfer to bright light. Leaves of S. dulcamara grown in the shade with 0.6 mM NO ₃ ^- have similar photosynthetic properties as leaves of plants grown with 12.0 mM NO ₃ ^- . When transferred to bright light for 1–2 d the leaves of these plants show substantial photoinhibition which is characterized by about 50% decrease in apparent quantum yield and a reduction in the rate of photosynthesis in air at light saturation. Photoinhibition of leaf photosynthesis is associated with reduction in the variable component of low-temperature fluorescence emission, and with loss of in-vitro electron transport, especially of photosystem II-dependent processes.We find no evidence for ecotypic differentiation in the potential for photosynthetic acclimation among shade and sun clones of S. dulcamara, or of differentiation with respect to nitrogen requirements for acclimation. Recovery from photoinhibition and subsequent acclimation of photosynthesis to bright light only occurs in leaves of plants provided with 12.0 mM NO ₃ ^- . In these, apparent quantum yield is fully restored after 14 d, and photosynthetic acclimation is shown by an increase in light-saturated photosynthesis in air, of light-and CO₂-saturated photosynthesis, and of the initial slope of the CO₂-response curve. The latter changes are highly correlated with changes in ribulose-bisphosphate-carboxylase activity in vitro. Plants supplied with 0.6 mM NO ₃ ^- show incomplete recovery of apparent quantum yield after 14 d, but CO₂-dependent leaf photosynthetic parameters return to control levels.Symbols and abbreviations F_o initial level of fluorescence at 77 K - F_m maximum level of fluorescence at 77 K - F_v variable components of fluorescence at 77 K (F_v=F_m-F_o) - PSI, PSII photosystem I and II, respectively - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase-oxygenase (EC 4.1.1.39)     

Cold acclimation and photoinhibition of photosynthesis in Scots pine

Cold acclimation of Scots pine did not affect the susceptibility of photosynthesis to photoinhibition. Cold acclimation did however cause a suppression of the rate of CO₂ uptake, and at given light and temperature conditions a larger fraction of the photosystem II reaction centres were closed in cold-acclimated than in nonacclimated pine. Therefore, when assayed at the level of photosystem II reaction centres, i.e. in relation to the degree of photosystem closure, cold acclimation caused a significant increase in resistance to photoinhibition; at given levels of photosystem II closure the resistance to photoinhibition was higher after cold acclimation. This was particularly evident in measurements at 20° C. The amounts and activities of the majority of analyzed active oxygen scavengers were higher after cold acclimation. We suggest that this increase in protective enzymes and compounds, particularly Superoxide dismutase, ascorbate peroxidase, glutathione reductase and ascorbate of the chloroplasts, enables Scots pine to avoid excessive photoinhibition of photosynthesis despite partial suppression of photosynthesis upon cold acclimation. An increased capacity for light-induced de-epoxidation of violaxanthin to zeaxanthin upon cold acclimation may also be of significance.Abbreviations APX ascorbate peroxidase - DHA dehydroascorbate - DHAR dehydroascorbate reductase - Fm maximal fluorescence when all reaction centres are closed - Fv/Fm maximum photochemical yield of PSII - GR glutathione reductase - GSH reduced glutathione - Je rate of photosynthetic electron transport - MDAR monodehydroascorbate reductase - q_N nonphotochemical quenching of fluorescence - q_P photochemical quenching of fluorescence - SOD superoxide dismutase This work was supported by the Swedish Natural Science Research Council and the National Natural Science Foundation of China.     

Effect of long-term photoinhibition on growth and photosynthesis of cold-hardened spring and winter wheat

The effect of repeated exposure to high light (1200 mol · m^–2 · s^–1 photosynthetic photon flux density, PPFD) at 5° C was examined in attached leaves of cold-grown spring (cv. Katepwa) and winter (cv. Kharkov) wheat (Triticum aestivum L.) over an eight-week period. Under these conditions, Kharkov winter wheat exhibited a daily reduction of 24% in F_V/F_M (the ratio of variable to maximal fluorescence in the dark-adapted state), in contrast to 41% for cold-grown Katepwa spring wheat. Both cultivars were able to recover from this daily suppression of F_V/F_M such that the leaves exhibited an average morning F_V/F_M of 0.651 ± 0.004. Fluorescence measurements made under steady-state conditions as a function of irradiance from 60 to 2000 mol · m^–2 · s^–1 indicated that the yield of photosystem II (PSII) electron transport under light-saturating conditions was the same for photoinhibited and control cold-grown plants, regardless of cultivar. Repeated daily exposure to high light at low temperature did not increase resistance to short-term photoinhibition, although zeaxanthin levels increased by three- to fourfold. In addition, both cultivars increased the rate of dry-matter accumulation, relative to control plants maintained at 5° C and 250 mol · m^–2 · s^–1 PPFD (10% and 28% for Katepwa and Kharkov, respectively), despite exhibiting suppressed f_v/f_m and reduced photon yields for O₂ evolution following daily high-light treatments. Thus, although photosynthetic efficiency is suppressed by a longterm, photoinhibitory treatment, light-saturated rates of photosynthesis are sufficiently high during the high-light treatment to offset any reduction in photochemical efficiency of PSII. We suggest that in these cold-tolerant plants, photoinhibition of PSII may represent a longterm, stable, down-regulation of photochemistry to match the overall photosynthetic demand for ATP and reducing equivalents.Abbreviations and Symbols Chl chlorophyll - HL high light - PPFD photosynthetic photon flux density - F_O minimum fluorescence in the dark-adapted state - F_M maximum fluorescence in the dark-adapted state - F_V maximum variable fluorescence in the dark-adapted state (F_M-F_O) - F_V/F_V photosynthetic efficiency of the dark-adapted state - f_V/f_M photosynthetic efficiency of the light-adapted steady state - q_P photochemical quenching parameter - q_N non-photochemical quenching parameter - _e yield of electron transport and equals q_P · f_V/f_M - 1-q_O F_O quenching parameter - _app apparent photon yield. The assistance of Amy So is gratefully acknowledged. This research was supported by a Natural Sciences and Engineering Research Council of Canada (NSERCC) Operating Grant to N.P.A.H. G.Ö. was supported by an NSERCC International Exchange Award and the Swedish Natural Sciences Research Council.     

Two mechanisms of recovery from photoinhibition in vivo: Reactivation of photosystem II related and unrelated to D1-protein turnover

Recovery (at 20° C) of spinach (Spinacia oleracea L.) leaf sections from photoinhibition of photosynthesis was monitored by means of the fluorescence parameter F_V/F_M of intact leaf tissue and of PSII-driven electron-transport activity of isolated thylakoids. Different degrees of photoinactivation of PSII were obtained by preillumination in ambient air (at 4 or 20° C), CO₂-free air or at low and high O₂ levels (2 or 41 %) in N₂. The kinetics of recovery exhibited two distinct phases. The first phase usually was completed within about 20-60 min and was most pronounced after preillumination in low O₂. The slow phase proceeded for several hours leading to almost complete reactivation of PSII. Preincubation of the leaves with streptomycin (SM), which inhibits chloroplast-encoded protein synthesis, inhibited the slow recovery phase only, indicating the dependence of this phase on resynthesis of the reaction-centre protein, D1. The fast recovery phase remained largely unaffected by SM. Both phases were strongly but not totally dependent on irradiation of the leaf with low light. When SM was absent, net degradation of the D1 protein could neither be detected upon photoinhibitory irradiation nor during following incubation of the leaf sections in low light or darkness. In the presence of SM, net D1 degradation was seen and tended to increase with O₂ concentration during photoinhibition treatment. Based on these data, we suggest that photoinactivation of PSII in vivo occurs in at least two steps. From the first step, reactivation appears possible in low light without D1 turnover (fast recovery phase). Action of oxygen then may lead to a second step, in which the D1 protein is affected and reactivation requires its removal and replacement (slow phase).Abbreviations Chl chlorophyll - F₀, F_M and F_V initial, maximum total and maximum variable chlorophyll fluorescence yield, respectively - PFD photon flux density - SM streptomycin We thank Professor P. Böger (Department of Plant Physiology and Biochemistry, University of Konstanz, Germany) for a gift of D1-specific antibodies. The paper contains part of the thesis work of J.L. The study was supported by the Deutsche Forschungs-gemeinschaft (SFB 189).     

Photoinhibition at chilling temperature

The effects of moderate light at chilling temperature on the photosynthesis of unhardened (acclimated to +18° C) and hardened (cold-acclimated) spinach (Spinacea oleracea L.) leaves were studied by means of fluorescence-induction measurements at 20° C and 77K and by determination of quantum yield of O₂ evolution. Exposure to 550 mol photons·m^-2·s^-1 at +4° C induced a strong photoinhibition in the unhardened leaves within a few hours. Photoinhibition manifested by a decline in quantum yield was characterized by an increase in initial fluorescence (F _o) and a decrease in variable fluorescence (F _v) and in the ratio of variable to maximum fluorescence (F _V/F _M), both at 77K and 20° C. The decline in quantum yield was more closely related to the decrease in the F _V/F _M ratio measured at 20° C, as compared with F _V/F _M at 77K. Quenching of the variable fluorescence of photosystem II was accompanied by a decline in photosystem-I fluorescence at 77K, indicating increased thermal de-excitation of pigments as the main consequence of the light treatment. All these changes detected in fluorescence parameters as well as in the quantum yield of O₂ evolution were fully reversible within 1–3 h at a higher temperature in low light. The fast recovery led us to the view that this photoinhibition represents a regulatory mechanism protecting the photosynthetic apparatus from the adverse effects of excess light by increasing thermal energy dissipation. Long-term cold acclimation probably enforces other protective mechanisms, as the hardened leaves were insensitive to the same light treatment that induced strong inhibition of photosynthesis in unhardened leaves.Abbreviations F ₀ initial fluorescence - F _M maximum fluorescence - F _V variable fluorescence (F _M-F ₀ - PFD photon flux density - PS photosystem     

Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: Effect of temperature

Photoinhibition of photosynthesis was induced in attached leaves of kiwifruit grown in natural light not exceeding a photon flux density (PFD) of 300 mol·m^-2·s^-1, by exposing them to a PFD of 1500 mol·m^-2·s^-1. The temperature was held constant, between 5 and 35° C, during the exposure to high light. The kinetics of photoinhibition were measured by chlorophyll fluorescence at 77K and the photon yield of photosynthetic O₂ evolution. Photoinhibition occurred at all temperatures but was greatest at low temperatures. Photoinhibition followed pseudo first-order kinetics, as determined by the variable fluorescence (F _v) and photon yield, with the long-term steady-state of photoinhibition strongly dependent on temperature wheareas the observed rate constant was only weakly temperature-dependent. Temperature had little effect on the decrease in the maximum fluorescence (F _m) but the increase in the instantaneous fluorescence (F _o) was significantly affected by low temperatures in particular. These changes in fluorescence indicate that kiwifruit leaves have some capacity to dissipate excessive excitation energy by increasing the rate constant for non-radiative (thermal) energy dissipation although temperature apparently had little effect on this. Direct photoinhibitory damage to the photosystem II reaction centres was evident by the increases in F _o and extreme, irreversible damage occurred at the lower temperatures. This indicates that kiwifruit leaves were most susceptible to photoinhibition at low temperatures because direct damage to the reaction centres was greatest at these temperatures. The results also imply that mechanisms to dissipate excess energy were inadequate to afford any protection from photoinhibition over a wide temperature range in these shade-grown leaves.Abbreviations and symbols fluorescence yield correction coefficient - F _o, F _m, F _v instantaneous, maximum, variable fluorescence - K _D, K _F, K _P, K _T rate constants for non-radiative energy dissipation, fluorescence, photochemistry, energy transfer to photosystem I - PFD photon flux density - PSI, II photosystem I, II - _i photon yield of photosynthesis (incident light)     

Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: Changes in susceptibility to photoinhibition and recovery during the growth season

Kiwifruit (Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson) plants grown in an outdoor enclosure were exposed to the natural conditions of temperature and photon flux density (PFD) over the growing season (October to May). Temperatures ranged from 14 to 21° C while the mean monthly maximum PFD varied from 1000 to 1700 mol · m^–2 · s^–1, although the peak PFDs exceeded 2100 mol · m^–2 · s^–1. At intervals, the daily variation in chlorophyll fluorescence at 692 nm and 77K and the photon yield of O₂ evolution in attached leaves was monitored. Similarly, the susceptibility of intact leaves to a standard photoinhibitory treatment of 20° C and a PFD of 2000 mol · m^–2 · s^–1 and the ability to recover at 25° C and 20 mol · m^–2 · s^–2 was followed through the season. On a few occasions, plants were transferred either to or from a shade enclosure to assess the suceptibility to natural photoinhibition and the capacity for recovery. There were minor though significant changes in early-morning fluorescence emission and photon yield throughout the growing season. The initial fluorescence, F_o, and the maximum fluorescence, F_m, were, however, significantly and persistently different from that in shade-grown kiwifruit leaves, indicative of chronic photoinhibition occurring in the sun leaves. In spring and autumn, kiwifruit leaves were photoinhibited through the day whereas in summer, when the PFDs were highest, no photoinhibition occurred. However, there was apparently no non-radiative energy dissipation occurring then also, indicating that the kiwifruit leaves appeared to fully utilize the available excitation energy. Nevertheless, the propensity for kiwifruit leaves to be susceptible to photoinhibition remained high throughout the season. The cause of a discrepancy between the severe photoinhibition under controlled conditions and the lack of photoinhibition under comparable, natural conditions remains uncertain. Recovery from photoinhibition, by contrast, varied over the season and was maximal in summer and declined markedly in autumn. Transfer of shade-grown plants to full sun had a catastrophic effect on the fluorescence characteristics of the leaf and photon yield. Within 3 d the variable fluorescence, F_v, and the photon yield were reduced by 80 and 40%, respectively, and this effect persisted for at least 20 d. The restoration of fluorescence characteristics on transfer of sun leaves to shade, however, was very slow and not complete within 15 d.Abbreviations and Symbols F_o, F_m, F_v initial, maximum, variable fluorescence - F_i F_v at t = 0 - F F_v at t = - PFD photon flux density - PSII photosystem II - leaf absorptance ratio - (_a photon yield of O₂ evolution (absorbed basis) - _{i a} at t = 0 - _a at t = We thank Miss Linda Muir and Amanda Yeates for their technical assistance in this study.     

Photoinhibition of photosynthesis: effect on chlorophyll fluorescence at 77K in intact leaves and in chloroplast membranes of Nerium oleander

The effect of exposing intact leaves and isolated chloroplast membranes of Nerium oleander L. to excessive light levels under otherwise favorable conditions was followed by measuring photosynthetic CO₂ uptake, electron transport and low-temperature (77K=-196°C) fluorescence kinetics. Photoinhibition, as manifested by a reduced rate and photon (quantum) yield of photosynthesis and a reduced electron transport rate, was accompanied by marked changes in fluorescence characteristics of the exposed upper leaf surface while there was little effect on the shaded lower surface. The most prominent effect of photoinhibitory treatment of leaves and chloroplasts was a strong quenching of the variable fluorescence emission at 692 nm (F_v,692) while the instantaneous fluorescence (F_o,692) was slightly increased. The maximum and the variable fluorescence at 734 nm were also reduced but not as much as F_M,692 and F_v,692. The results support the view that photoinhibition involves an inactivation of the primary photochemistry of photosystem II by damaging the reaction-center complex. In intact leaves photoinhibition increased with increased light level, increased exposure time, and with decreased temperature. Increased CO₂ pressure or decreased O₂ pressure provided no protection against photoinhibition. With isolated chloroplasts, inhibition of photosystem II occurred even under essentially anaerobic conditions. Measurements of fluorescence characteristics at 77K provides a simple, rapid, sensitive and reproducible method for assessing photoinhibitory injury to leaves. The method should prove especially useful in studies of the occurrence of photoinhibition in nature and of interactive effects between high light levels and major environmental stress factors.Abbreviations and symbols PFD photon flux area density - PSI, PSII photosystem I, II - F_M, F_O, F_V maximum, instantaneous, variable fluorescence emission C.I.W.-D.P.B. Publication No. 773     

Movement and compartmentation of abscisic acid in guard cells of Valerianella locusta: Effects of osmotic stress,external H+-concentration and fusicoccin

Epidermal peels of Valerianella locusta were acid-treated for 1 h at pH 3.9 to kill all cells other than guard cells. These guard-cell preparations were used to explore the steady-state one-way fluxes and the cytoplasmic and vacuolar contents of abscisic acid (ABA). The method of compartmental analysis has been applied. The intracellular ABA concentrations were surprisingly high. At an external pH of 5.8 the cytoplasm contained 1.28 mmol·dm^-3 of ABA, twice of the amount which accumulated in the vacuoles (0.57 mmol·dm^-3). The fluxes of ABA at the plasmalemma (_oc=_oc=0.43 fmol · cell ^–1 · h ^–1) were higher than those at the tonoplast (_cv=_vc=0.12 fmol · cell ^–1 · h ^–1). Moderate stress (0.1 and 0.3 mol·dm^-3 sorbitol in the medium) caused a change in the kinetics of ABA movement. The rate constants of the fluxes from the cytoplasm into the vacuole (_cv) and into the apoplast (_co) were increased while the rate constant of the flux from the vacuoles into the cytoplasm (_vc) was decreased. As a consequence the amount of ABA sequestered in the vacuole remained unchanged; the cytoplasmic ABA content, however, was reduced to only 20% of that found in the control treatments (no sorbitol in the medium). Under moderate stress, one Valerianella guard cell released rapidly about 0.36 fmol·cell^-1 to its direct cell-wall space. This surprising result is discussed in regard to rapid stomatal closure under reduced water supply.Abbreviations ABA abscisic acid - FC fusicoccin     

Photoinactivation of photosystem II during photoinhibition in the cyanobacterium Microcystis aeruginosa

Sites of photoinhibition and photo-oxidative damage to the photosynthetic electrontransport system of the unicellular cyanobacterium Microcystis aeruginosa were identified by studies of the kinetics of chlorophyll fluorescence induction by whole cells at room temperature and from partial photosynthetic electron-transport reactions in vitro in thylakoid preparations. Chlorophyll fluorescence intensity decreased following photoinhibitory light treatment. This was attributed to decreases both in the activity of photosystem II and in electron flow through the primary electron acceptor, Q. This inhibition was only partially reversed over a 50-min dark recovery period. Partial photosynthetic electron-transport experiments in vitro demonstrated that photosystem I was not affected by the photoinhibitory treatment. Light damage was associated exclusively with the light reactions, of photosystem II, at a site close to the reaction centre, between the site where diphenylcarbazide can donate electrons and the site where silicomolybdate can accept electrons. This damage presumably reduced production of ATP by noncyclic photophosphorylation and production of NADPH by photosystem I, decreasing the availability of these co-factors for reducing CO₂ in the dark reactions of photosynthesis. The importance of these findings is discussed.Abbreviations Chl chlorophyll - DCPIP 2,6-dichlorophenolindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DPC diphenylcarbazide - PSI photosystem I - PSH photosystem II     

Dithiothreitol,an inhibitor of violaxanthin de-epoxidation,increases the susceptibility of leaves ofNerium oleander L. to photoinhibition of photosynthesis

Leaves ofNerium oleander L. plants, which had been previously kept in a shaded glasshouse for at least two months, were fed 1 mM dithiothreitol (DTT) through their petioles, either for 12h in darkness (overnight) or for 2h in low light (28 μmol photons·m⁻²·s⁻¹), in each case followed by a 3-h exposure to high light (1260 μmol photons·m⁻²·s⁻¹). During exposure to high light, violaxanthin became converted to zeaxanthin in control leaves, to which water had been fed, whereas zeaxanthin did not accumulate in leaves treated with DTT. Total carbon gain was not reduced by DTT during the photoinhibitory treatment. Exposure to high light led to a decrease in the photochemical efficiency of photosystem II, measured as the ratio of variable over maximum fluorescence emission,F _v/F _M, at both 298 K and 77K. The decrease was much more pronounced in the presence of DTT, mainly owing to a sustained increase in the instantaneous fluorescence,F _o. By contrast, in the control leaves,F _o determined immediately after the high-light treatment showed a transient decrease below theF _o value obtained before the onset of the photoinhibitory treatment (i.e. after 12 h dark adaptation), followed by a rapid return (within seconds) to this original level ofF _o during the following recovery period in darkness. Incubation of leaves with DTT led to large, sustained decreases in the photon-use efficiency of photosynthetic O₂ evolution by bright light, whilst the capacity of photosynthetic O₂ evolution at light and CO₂ saturation was less affected. In the control leaves, only small reductions in the photon yield and in the photosynthetic capacity were observed. These findings are consistent with previous suggestions that zeaxanthin, formed in the xanthophyll cycle by de-epoxidation of violaxanthin, is involved in protecting the photosynthetic apparatus against the adverse effects of excessive light.