Regulation of D1-protein degradation during photoinhibition of photosystem II in vivo: Phosphorylation of the D1 protein in various plant groups

Photoinhibition of PSII and turnover of the D1 reaction-centre protein in vivo were studied in pumpkin leaves (Cucurbita pepo L.) acclimated to different growth irradiances and in low-light-grown moss, (Ceratodon purpureus) (Hedw.) Brid. The low-light-acclimated pumpkins were most susceptible to photoinhibition. The production rate of photoinhibited PSII centres (k_PI), determined in the presence of a chloroplast-encoded protein-synthesis inhibitor, showed no marked difference between the high- and low-light-grown pumpkin leaves. On the other hand, the rate constant for the repair cycle (k_REC) of PSII was nearly three times higher in the high-light-grown pumpkin when compared to low-light-grown pumpkin. The slower degradation rate of the damaged D1 protein in the low-light-acclimated leaves, determined by pulsechase experiments with [³⁵S]methionine suggested that the degradation of the Dl protein retards the repair cycle of PSII under photoinhibitory light. Slow degradation of the D1 protein in low-light-grown pumpkin was accompanied by accumulation of a phosphorylated form of the D1 protein, which we postulate as being involved in the regulation of D1-protein degradation and therefore the whole PSII repair cycle. In spite of low growth irradiance the repair cycle of PSII in the moss Ceratodon was rapid under high irradiance. When compared to the high- or low-light-acclimated pumpkin leaves, Ceratodon had the highest rate of D1-protein degradation at 1000 mol photons m^–2 s^–1. In contrast to the higher plants, the D1 protein of Ceratodon was not phosphorylated either under high irradiance in vivo or under in-vitro conditions, which readily phosphorylate the D1 protein of higher plants. This is consistent with the rapid degradation of the D1 protein in Ceratodon. Screening experiments indicated that D1 protein can be phosphorylated in the thylakoid membranes of angiosperms and conifers but not in lower plants. The postulated regulation mechanism of D1-protein degradation involving phosphorylation and the role of thylakoid organization in the function of PSII repair cycle are discussed.Abbreviations Chl Chlorophyll - D1^* phosphorylated form of D1 protein - F_max and F_v maximal and variable fluorescence respectively - k_PJ and k_REC rate constants of photoinhibition and concurrent recovery respectively - LHCII lightharvesting chlorophyll a/bprotein of PSII - PFD photon flux density Dr. R. Barbato (Dipartimento di Biologia, Universita di Padova, Padova, Italy), Prof. P. Böger (Lehrstuhl fur Physiologie und Biochemie der Pflanzen, Universität Konstanz, Konstanz, Germany), Prof. A. Melis (Department of Plant Biology, University of California, Berkeley, USA), Prof. I. Ohad (Department of Biological Chemistry, Hebrew University, Jerusalem, Israel) and Mr. A. Soitamo (Department of Biology, University of Turku, Turku, Finland) are gratefully acknowledged for the D1-protein-specific antibodies. The authors thank Ms. Virpi Paakkarinen for excellent technical assistance. This work was supported by the Academy of Finland and the Foundation of the University of Turku.     

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 and light-dependent turnover of the D1 reaction-centre polypeptide of photosystem II are enhanced by mineral-stress conditions

The function of photosystem II (PSII) and the turnover of its D1 reaction-center protein were studied in spinach (Spinacia oleracea L.) plants set under mineral stress. The mineral deficiencies were induced either by supplying the plants with an acidic nutrient solution or by strongly reducing the supply of magnesium alone or together with sulfur. After exposure for 8–10 weeks to the different media, the plants were characterized by a loss of chlorophyll and an increase in starch content, indicating a disturbance in the allocation of assimilates. Depending on the severity of the mineral deficiencies the plants lost their ability to adapt even to moderate iradiances of 400 mol photons·m^–2·s^–1 and became photoinhibited, as indicated by the decrease in F_v/F_m (the ratio of yield of variable fluorescence to yield of maximal fluorescence when all reaction centers are closed). The loss of PSII function was induced by changes on the acceptor side of PSII. Fast fluorescence decay showed a loss of PSII centers with bound Q_B, the secondary quinone acceptor of PSII, and a fast reoxidation kinetic of q _a ^- , the primary quinone acceptor of PSII, in the photoinactivated plants. No appreciable change could be observed in the amount of PSII centers with unbound Q_B and in Q_B-nonreducing PSII centers. Immunological studies showed that the contents of the D1 and D2 proteins of the PSII reaction center and of the 33-kDa protein of the water-splitting complex were diminished in the photoinhibited plants, and the occurrance of a new polypetide of 14 kDa that reacted with an antibody against the C-termius of the D1 protein. As shown by pulse-labelling experiments with [¹⁴C]leucine both degradation and synthesis of the D1 protein were enhanced in the mineral-deficient plants when compared to non-deficient plants. A stimulation of D1-protein turnover was also observed in pH 3-grown plants, which were not inhibited at growth-light conditions. Obviously, stimulation of D1-protein turnover prevented photoinhibition in these plants. However, in the Mg- and Mg/S-deficient plants even a further stimulation of D1-protein turnover could not counteract the increased rate of photoinactivation.Abbreviations amp_(f,m,s) amplitude of the fast, (medium and slow) exponential component of fluorescence decay - F_m yield of maximum fluorescenc when all reaction centers are closed - F_o yield of intrinsic fluorescence at open PSII reaction centers in the dark - F_v yield of variable fluorescence, (difference between F_m and F_o) - LHC light-harvesting complex - PFD photon flux density - Q_A primary quinone acceptor of PSII - Q_B secondary quinone acceptor of PSII Dedicated to Professor Dr. Dres. hc. Achim Trebst on the occasion of his 65th birthdayThis work was supported by grants from the BMFT and the Ministerium für Umwelt, Raumordnung and Landwirtschaft, Nordrhein-Westfalen. The authors thank H. Wietoska and M. Bronzel for skilful technical assistance.     

Multiple strategies for a high light existence in a tropical marine macroalga

Acclimation to high light conditions on the top of coral reefs was examined in the coenocytic, filamentous green macroalga Chlorodesmis fastigiata (C. Ag.) Ducker. Despite having a pool of violaxanthin, high light does not induce formation of zeaxanthin in this macroalga. Exposure to 11 and 33% of surface irradiance resulted in parallel, reversible declines in F_v/F_mand in the number of functional PSII centers. The quantum requirement for PSII inactivation was calculated to be approx. 2×10⁷photons. Recovery of PSII activity after low photon exposures did not depend on protein synthesis, unlike at higher photon exposures, where recovery was inhibited by 50% in the presence of lincomycin. Accumulation of inactive, quenching PSII centers is proposed as a mechanism of energy dissipation; only some of these centers require protein synthesis for reactivation. In natural-sized populations, midday photoinhibition was greater in filament tips than in bases, but the number of inactive PSII centers within entire filaments did not significantly change over the course of the day. It is proposed that the higher chlorophyll concentration in the tips provides protective shading to chloroplasts in lower regions, and that cytoplasmic streaming of chloroplasts within this siphonous alga limits the cumulative exposure to high light, thereby providing another level of protection from high light stress.     

Light-induced increase in initial chlorophyll fluorescence Fo level and the reversible inactivation of PS II reaction centers in soybean leaves

With a portable PAM-2000 fluorometer it was observed that responses of initial chlorophyll fluorescence F_o level to strong light were different in various plant species examined. When the photochemical efficiency of Photosystem II, F_v/F_m, declined, F_o increased significantly in leaves of some plants such as soybean and cotton, while F_o decreased remarkably in other plants such as wheat and barley. In order to explore the mechanism of the increase in F_o in soybean leaves, the change in D1 protein amount and effects of lincomycin and far-red light on these fluorescence parameters were observed by SDS–PAGE combined with gel scanning and chlorophyll fluorescence analysis. The following results were obtained. (1) The amount of inactive PS II reaction centers increased under strong light and decreased during subsequent dark recovery [Hong and Xu (1997) Chinese Sci Bull 42(8): 684–689]. (2) No net loss of D1 protein occurred after strong light treatment. (3) Lincomycin taken up through petioles following strong light treatment had no significant effect on D1 protein level and the decay of F_o in the dark. (4) Far-red light applied after strong light treatment could largely attenuate the increase in F_o and accelerate F_o decay in the dark. Based on these results, it is deduced that the increase in F_o under strong light is mainly due to reversible inactivation of part of PS II reaction centers, rather than the net loss of D1 protein and that reversible inactivation of PS II is prevalent in some plants.     

EFFECTS OF UV‐B RADIATION ON THE D1 PROTEIN REPAIR CYCLE OF NATURAL PHYTOPLANKTON COMMUNITIES FROM THREE LATITUDES (CANADA,BRAZIL, AND ARGENTINA)1

Ultraviolet radiation effects were examined in natural phytoplankton communities from Rimouski (Canada), Ubatuba (Brazil), and Ushuaia (Argentina). Outdoor pump‐mixed mesocosms were submitted to ambient solar radiation (NUVB) and ambient with additional UV‐B radiation (UVBR) from lamps (HUVB), corresponding to a local 60% ozone depletion scenario. At all sites, neither algal biomass nor dark‐adapted F_v/F_m were significantly affected by additional UVBR, suggesting the presence of active UV protection or repair mechanisms. To examine the role of D1 protein turnover, essential for PSII repair, short‐term surface incubations were performed in the presence or absence of lincomycin, a chloroplast protein synthesis inhibitor. Effects on PSII were determined using chl a in vivo fluorescence, whereas the D1 protein was detected immunochemically. In the absence of D1 repair, D1 pools and F_v/F_m decreased to a similar extent under both light treatments. In the presence of D1 repair, D1 pools suffered faster net degradation under HUVB compared with NUVB, whereas F_v/F_m was maintained for both light treatments, suggesting that HUVB exposure in field populations had more effect on D1 synthesis and PSII repair than on D1 degradation. The fewer undamaged reaction centers remaining in phytoplankton under HUVB were able to maintain F_v/F_m or actually recovered during the dark acclimation before F_v/F_m measurements. The D1 pools suffered faster net degradation at the tropical site where high irradiance drove faster D1 degradation and high water temperature enabled fast enzymatic activities. This study shows the crucial role of dynamic changes in D1 turnover in the photobiology of natural planktonic communities across a range of latitudes.     

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.     

Photoinhibition in outdoor Spirulina platensis cultures assessed by polyphasic chlorophyll fluorescence transients

Photoinhibition in outdoor cultures of Spirulina platensis was studied by measuring the polyphasic rise of chlorophyll fluorescence transients, which provide information on the primary photochemistry of PSII. The maximum efficiency of PSII photochemustry (F_v/F_m) declined in response to daily increasing irradiance and recovered as daily irradiance decreased. The greatest inhibition (15%) in F_v/F_m was observed at 12:00 hr which responded to the highest irradiance. The absorption flux, the trapping flux, and the electron transport flux per PSII reaction center increased in response to daily increasing irradiance and decreased as irradiance decreased. The daily change in the concentration of PSII reaction centers followed the same pattern as F_v/F_m. However, no significant changes in the probability of electron transport beyond Q_A (Ψ_o) were observed during the day. The results suggest that the decrease in F_v/F_m induced by photoinhibition in outdoor Spirulina cultures was a result of the inactivation of PSII reaction centers. The results also suggest that the measurement of polyphasic fluorescence transients is a powerful tool to study the mechanism of photoinhibition in outdoor Spirulina cultures and to screen strains for photoinhibition tolerance. This revised version was published online in June 2006 with corrections to the Cover Date.     

Down regulation of photosynthesis in Artabotrys hexapetatus by high light

Using variable to maximum fluorescence (F_v/F_m) as the criterion, the down regulation of photosynthesis by high light stress was characterized in the detached leaves of Artabotrys hexapetatus. The decrease in F_v/F_m was corelated with the decrease in oxygen evolution by thylakoids isolated from high light exposed leaves. The decrease in F_v/F_m was linear with increasing time of exposure to high light. A comparison of recovery measured as F_v/F_m, in low light versus dark, revealed that the recovery in darkness was as significant as in low light. Since the relaxation of fluorescence was a rapid response after exposure to high light and the fact that the recovery occurs in total darkness, it is concluded that photoinhibition and down regulation of photosynthesis by high light are independent events.Abbreviation Fpl- initial plateau - F_m- maximum fluorescence - F_o- prompt fluorescence - F_v- variable fluorescence - PFD- photon flux density - PS I (II)- Photosystem I (II)     

Chlamydomonas raudensis (UWO 241), Chlorophyceae,exhibits the capacity for rapid D1 repair in response to chronic photoinhibition at low temperature1

Maximum photosynthetic capacity indicates that the Antarctic psychrophile Chlamydomonas raudensis H. Ettl UWO 241 is photosynthetically adapted to low temperature. Despite this finding, C. raudensis UWO 241 exhibited greater sensitivity to low‐temperature photoinhibition of PSII than the mesophile Chlamydomonas reinhardtii P. A. Dang. However, in contrast with results for C. reinhardtii, the quantum requirement to induce 50% photoinhibition of PSII in C. raudensis UWO 241 (50 μmol photons) was comparable at either 8°C or 29°C. To our knowledge, this is the first report of a photoautotroph whose susceptibility to photoinhibition is temperature independent. In contrast, the capacity of the psychrophile to recover from photoinhibition of PSII was sensitive to temperature and inhibited at 29°C. The maximum rate of recovery from photoinhibition of the psychrophile at 8°C was comparable to the maximum rate of recovery of the mesophile at 29°C. We provide evidence that photoinhibition in C. raudensis UWO 241 is chronic rather than dynamic. The photoinhibition‐induced decrease in the D1 content in C. raudensis recovered within 30 min at 8°C. Both the recovery of the D1 content as well as the initial fast phase of the recovery of F_v/F_m at 8°C were inhibited by lincomycin, a chloroplast protein synthesis inhibitor. We conclude that the susceptibility of C. raudensis UWO 241 to low‐temperature photoinhibition reflects its adaptation to low growth irradiance, whereas the unusually rapid rate of recovery at low temperature exhibited by this psychrophile is due to a novel D1 repair cycle that is adapted to and is maximally operative at low temperature.     

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)     

Changes in D1-protein turnover and recovery of photosystem II activity precede accumulation of chlorophyll in plants after release from mineral stress

After seven weeks of a combined magnesium and sulphur deficiency, spinach (Spinacea oleracea L.) plants showed a substantial accumulation of inactivated photosystem II (PSII) centres as indicated by a 40% decrease of the chlorophyll (Chl) fluorescence parameter F_v/F_m (F_v being the yield of variable fluorescence and F_m the yield of maximal fluorescence when all reaction centres are closed) together with a severe loss of leaf Chl content of 75%. The responses of the photosynthetic apparatus were examined when the deficient plants were transferred back to a rich nutrient medium. During the first 24 h of the recovery phase, thylakoid protein synthesis measured as incorporation of [¹⁴C]leucine per unit of Chl increased substantially. The synthesis rate of the D1 reaction-centre polypeptide of PSII, which in the deficient plants was reduced to 50% of the non-deficient control, was stimulated eight- to ninefold. D1-protein content, which in the deficient plants was reduced to 40% of the non-deficient control, started to increase 2 d later. Thus, D1-protein degradation was also enhanced. The increased D1-protein turnover led to a rapid repair of the existing PSII centres as indicated by the rise of F_v/F_m. It was completed at day 7 of the recovery phase. At day 2 of the recovery phase, the synthesis of other thylakoid proteins such as the D2 protein, cytochrome b ₅₅₉, CP 47 and the 33-kDa polypeptide of the water-splitting system, became stimulated. This process resulted in an accumulation of new PSII centres. During the first week, formation of new PSII centres was not associated with an increase in leaf Chl content. The Chl content of the recovering leaves only started to increase when the ratio of PSII polypeptides versus LHCII (light-harvesting complex of PSII), which was substantially diminished in the deficient plants, became comparable to that of the control. The recovery process was accompanied by substantial changes in thylakoid protein phosphorylation. Their relevance to thylakoid protein turnover and stability is discussed.Abbreviations Chl chlorophyll - cyt cytochrome - F_o yield of intrinsic fluorescence when all PSII centres are open in the dark - F_m yield of maximal fluorescence when all reaction centres are closed - F_m fluorescence yield when all reaction centres are closed (after a saturating flash) under steady-state conditions - F_v yield of variable fluorescence, (difference between F_oand F_m) - F yield of variable fluorescence under steady state conditions - LHC light-harvesting complex - PQ plastoquinone - Q_A primary quinone acceptor of PSII - Q_B secondary quinone acceptor of PSII - q_P photochemical quenching - q_n non-photochemical quenching The authors like to thank Dipl. Biol. Britta Untereiser for determining the chlorophyll fluorescence quenching factors. This work was supported by grants from the Bundesminister für Forschung und Technologie, the Project Europäisches Forschungszentrum and the German Israeli Foundation in cooperation with Prof. I. Ohad, Hebrew University, Jerusalem, Israel.     

Leaf structure and patterns of photoinhibition in two neotropical palms in clearings and forest understory during the dry season

We studied the leaf structural, water status, and fast fluorescence responses of two palms, Socratea exorrhiza and Scheelea zonensis, under natural dry season conditions in a clearing (high light [HL] palms) and the forest understory (low light [LL] palms) on Barro Colorado Island, Panama. HL-Socratea leaves were more shade-adapted, less xeromorphic, and more strongly affected by drought than HL-Scheelea. F_v/F_m (the ratio of variable to maximum chlorophyll fluorescence) and t_½ (the half-rise time of F_m) was lower in HL-leaves of both species, indicating photoinhibition. In HL-Scheelea, the light-induced reduction of F_v/F_m was much less than in HL-Socratea, and F_v/F_m recovered completely overnight. Patterns of relative water content, specific leaf dry weight, stable carbon isotope composition, and leaf conductance suggest that increased drought resistance in Scheelea reduces susceptibility to photoinhibition. An increase in F_o indicated the inactivation of PSII reaction centers in HL-Socratea. The very low chlorophyll a/b ratio and alterations in chloroplast ultrastructure in HL-Socratea are consistent with photoinhibition. Under LL, the species showed no appreciable interspecific differences in chlorophyll fluorescence. Excess light leads to low values of F_v/F_m in HL-plants relative to LL-plants on both leaf surfaces, particularly on the lower surface, due to a decrease of F_m in both surfaces and an increase in F., of lower surface. For both species, F_o for the lower surfaces of HL-plants was higher and t_½ was markedly lower than for the upper surface, as is typical for shade-adapted leaves. Xeromorphic leaf structure may reduce susceptibility to photoinhibition during the dry season. Drought-enhanced photoinhibition could limit the ability of some species to exploit treefall gaps.     

Recovery from Photoinhibition in Peas (Pisum sativum L.) Acclimated to Varying Growth Irradiances (Role of D1 Protein Turnover)

D1 protein turnover and restoration of the photochemical efficiency of photosystem II (PSII) after photoinhibition of pea leaves (Pisum sativum L. cv Greenfeast) acclimated to different light intensities were investigated. All peas acclimated to different light intensities were able to recover from photoinhibition, at least partially, at light intensities far above their growth light irradiance. However, the capacity of pea leaves to recover from photoinhibition under increasing high irradiances was strictly dependent on the light acclimation of the leaves; i.e. the higher the irradiance during growth, the better the capacity of pea leaves to recover from photoinhibition at moderate and high light. In our experimental conditions, mainly D1 protein turnover-dependent recovery was monitored, since in the presence of an inhibitor of chloroplast-encoded protein synthesis, lincomycin, only negligible recovery took place. In darkness, neither the restoration of PSII photochemical efficiency nor any notable degradation of damaged D1 protein took place. In low light, however, good recovery of PSII occurred in all peas acclimated to different light intensities and was accompanied by fast degradation of the D1 protein. The rate of degradation of the D1 protein was estimated to be 3 to 4 times faster in photoinhibited leaves than in nonphotoinhibited leaves under the recovery conditions of 50 [mu]mol of photons m-2 s-1. In moderate light of 400 [mu]mol of photons m-2 s-1, the photoinhibited low-light peas were not able to increase further the rate of D1 protein degradation above that observed in nonphotoinhibited leaves, nor was the restoration of PSII function possible. On the other hand, photoinhibited high-light leaves were able to increase the rate of D1 protein degradation above that of nonphotoinhibited leaves even in moderate and high light, ensuring at least partial restoration of PSII function. We conclude that the capacity of photoinhibited leaves to restore PSII function at different irradiances was directly related to the capacity of the leaves to degrade damaged D1 protein under the recovery conditions.     

Seasonal changes in photosystem II organisation and pigment composition in Pinus sylvestris

Conifers of the boreal zone encounter considerable combined stress of low temperature and high light during winter, when photosynthetic consumption of excitation energy is blocked. In the evergreen Pinus sylvestris L. these stresses coincided with major seasonal changes in photosystem II (PSII) organisation and pigment composition. The earliest changes occurred in September, before any freezing stress, with initial losses of chlorophyll, the D1-protein of the PSII reaction centre and of PSII light-harvesting-complex (LHC II) proteins. In October there was a transient increase in F₀, resulting from detachment of the light-harvesting antennae as reaction centres lost D1. The D1-protein content eventually decreased to 90%, reaching a minimum by December, but PSII photochemical efficiency [variable fluorescence (F_v)/maximum fluorescence (F_m)] did not reach the winter minimum until mid-February. The carotenoid composition varied seasonally with a twofold increase in lutein and the carotenoids of the xanthophyll cycle during winter, while the epoxidation state of the xanthophylls decreased from 0.9 to 0.1 from October to January. The loss of chlorophyll was complete by October and during winter much of the remaining chlorophyll was reorganised in aggregates of specific polypeptide composition, which apparently efficiently quench excitation energy through non-radiative dissipation. The timing of the autumn and winter changes indicated that xanthophyll de-epoxidation correlates with winter quenching of chlorophyll fluorescence while the drop in photochemical efficiency relates more to loss of D1-protein. In April and May recovery of the photochemistry of PSII, protein synthesis, pigment rearrangements and zeaxanthin epoxidation occurred concomitantly. Indoor recovery of photosynthesis in winter-stressed branches under favourable conditions was completed within 3 d, with rapid increases in F₀, the epoxidation state of the xanthophylls and in light-harvesting polypeptides, followed by recovery of D1-protein content and F_v/F_m, all without net increase in chlorophyll. The fall and winter reorganisation allow Pinus sylvestris to maintain a large stock of chlorophyll in a quenched, photoprotected state, allowing rapid recovery of photosynthesis in spring.Abbreviations Elips early light-induced proteins - EPS epoxidation state - F₀ instantaneous fluorescence - F_m maximum fluorescence - F_v variable fluorescence - LHC II light-harvesting complex of PSII - LiDS lithium dodecyl sulfate This research was supported by the Swedish Natural Science Research Council. We wish to thank Dr. Adrian Clarke¹ (Department of Plant Physiology, University of Umeå, Sweden) for advice on electrophoresis, valuable discussion and providing antibodies. Dr. Stefan Jansson¹ and Dr. Torill Hundal (Department for Biochemistry, University of Stockholm, Sweden) provided antibodies. Jan Karlsson¹ helped with the HPLC, Dr. Marianna Krol gave advice on green gels and Dr. Vaughan Hurry (Cooperative Research Centre for Plant Sciences, Australian National University, Canberra, Australia) provided valuable discussion.     

Cold-hardening-induced resistance to photoinhibition of photosynthesis in winter rye is dependent upon an increased capacity for photosynthesis

Analyses of chlorophyll fluorescence and photosynthetic oxygen evolution were conducted to understand why cold-hardened winter rye (Secale cereale L.) is more resistant to photoinhibition of photosynthesis than is non-hardened winter rye. Under similar light and temperature conditions, leaves of cold-hardened rye were able to keep a larger fraction of the PS II reaction centres in an open configuration, i.e. a higher ratio of oxidized to reduced Q_A (the primary, stable quinone acceptor of PSII), than leaves of non-hardened rye. Three fold-higher photon fluence rates were required for cold-hardened leaves than for non-hardened leaves in order to establish the same proportion of oxidized to reduced Q_A. This ability of cold-hardened rye fully accounted for its higher resistance to photoinhibition; under similar redox states of q_a cold-hardened and non-hardened leaves of winter rye exhibited similar sensitivities to photoinhibition. Under given light and temperature conditions, it was the higher capacity for light-saturated photosynthesis in cold-hardened than in non-hardened leaves, which was responsible for maintaining a higher proportion of oxidized to reduced Q_A. This higher capacity for photosynthesis of cold-hardened leaves also explained the increased resistance of photosynthesis to photoinhibition upon cold-hardening.Abbreviations F_m and F'_m fluorescence when all PSII reaction centres are closed in dark- and light-acclimated leaves, respectively - F_o and F'_o fluorescence when all PSII reaction centres are open in darkness and steady-state light, respectively - F_v variable fluorescence (F'_m-F'_o) under steady-state light conditions - F_v/F_m the ratio of variable to maximum fluorescence as an expression of the maximum photochemical yield of PSII in dark-acclimated leaves - Q_A the primary, stable, quinone electron acceptor of PSII - q_N non-photochemical quenching of fluorescence due to high energy state (pH) - q_p photochemical quenching of fluorescence - RH cold-hardened rye - RNH non-hardened rye This work 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 by the Swedish Natural Science Research Council.     

Chlorophyll fluorescence and photoinhibition in a tropical rainforest understory plant

The data presented here deal with the effects of high-light exposure on the 77 K fluorescence characteristics of Elatostema repens. It is shown that the decrease of the variable fluorescence during the treatment is biphasic. The reactions responsible for the first phase of fluorescence quenching are saturated under 700 mol photon m^-2 s^-1 and insensitive to streptomycin, whereas those responsible for the second phase are not yet saturated under 700 mol photon m^-2 s^-1 and sensitive to streptomycin. It is concluded that only the second phase of fluorescence quenching is associated with photoinhibitory processes. Rate and amplitude of recovery from photoinhibition are maximum under very low light (3.5 mol photon m^-2 s^-1), and very small at a moderate light (160 mol photon m^-2 s^-1) which does not cause photoinhibition. It is concluded that recovery processes are inhibited during photoinhibition. It is suggested that they could be associated with damage occuring on the oxidizing side of PSII.Abbreviations F_o, F_v, F_m initial, variable and maximum fluorescence, respectively - PFD photon flux density - PS II photosystem II     

Two components of onset and recovery during photoinhibition of Ulva rotundata

Short-term (up to 5 h) transfers of shade-adapted (100 mol · m^–2 · s^–1) clonal tissue of the marine macroalga Ulva rotundata Blid. (Chlorophyta) to higher irradiances (1700, 850, and 350 mol · m^–2 · s^–1) led to photoinhibition of room-temperature chlorophyll fluorescence and O₂ evolution. The ratio of variable to maximum (F_v/F_m) and variable (F_v) fluorescence, and quantum yield () declined with increasing irradiance and duration of exposure. This decline could be resolved into two components, consistent with the separation of photoinhibition into energy-dissipative processes (photoprotection) and damage to photosystem II (PSII) by excess excitation. The first component, a rapid decrease in F_v/F_m and in F_v, corresponds to an increase in initial (F_o) fluorescence and is highly sensitive to 1 mM chloramphenicol. This component is rapidly reversible under dim (40 mol · m^–2 · s^–1) light, but is less reversible with increasing duration of exposure, and may reflect damage to PSII. The second (after 1 h exposure) component, a slower decline in F_v/F_m and F_v with declining F_o, appears to be associated with the photoprotective interconversion of violaxanthin to zeaxanthin and is sensitive to dithiothreitol. The accumulation of zeaxanthin in U. rotundata is very slow, and may account for the predominance of increases in F_o at high irradiances.Abbreviations and Symbols CAP chloramphenicol - DTT dithiothreitol - F_o, F_m, F_v initial, maximum, and variable fluorescence - quantum yield - PFD photon flux density - PSII photosystem II To whom correspondence should be addressedWe are grateful to O. Björkman and S. Thayer, Carnegie Institution of Washington, Stanford, Cal., USA, for analysis of xanthophyll pigments reported here. This research was supported by National Science Foundation grant OCE-8812157 to C.B.O. and J.R. Support for G.L. was provided by a NSF-CNRS (Centre National de la Recherche Scientifique) exchange fellowship.     

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     

Development of photoautotrophy and photoinhibition of Gardenia jasminoides plantlets during micropropagation

This paper reports on the fast fluorescence responses of Gardenia jasminoides Ellis plantlets, at two successive stages (shoot multiplication and root induction) of culture in vitro. We test whether plantlets in vitro suffer photoinhibition during culture and whether the degree of photoautotrophy of these mixotrophic plantlets has any effect on the extent of photoinhibitory impairment. In this regard the effects of different sucrose levels in the medium and PPFD during growth on the development of photoautotrophy and the extent of photoinhibition were evaluated. Plantlets were grown under low, intermediate, and high (50, 100, and 300 mol m^-2 s^-1) PPFD, and at 3 different sucrose concentrations (0.5, 1.5, and 3.0%, w/v) in the medium, during shoot multiplication. During root induction the same growth conditions were assayed except for the high PPFD. The development of photoautotrophy was assessed via the difference between the stable carbon isotope composition of sucrose used as heterotrophic carbon source and that of leaflets grown in vitro. Plantlets from root induction showed more developed photoautotrophy than those from shoot multiplication. For both stages the low-sucrose medium stimulated the photoautotrophy of plantlets in vitro. In addition, intermediate PPFD induced photoautotrophy during shoot multiplication. For plantlets of both culture stages at the lowest PPFD no photoinhibition occurred irrespective of the sucrose concentration in media. However, during the shoot multiplication stage chlorophyll fluorescence measurements showed a decrease in F _v /F _m and in t _1/2 as growing PPFD increased, indicating photoinhibitory damage. The decline of F _v /F _m was caused mostly by an increase in F _o , indicating the inactivation of PSII reaction centers. However plantlets growing under low sucrose showed reduced susceptibility to photoinhibition. During root induction, only plantlets cultured with high sucrose showed a decrease in F _v /F _m as PPFD increased, although t _1/2 remained unchanged. In this case, the decline of F _v /F _m was mostly due to a decrease in F _m , which indicates increased photoprotection rather than occurrence of photodamage. Therefore, growth in low-sucrose media had a protective effect on the resistance of PSII to light stress. In addition, plantlets were more resistant to photoinhibition during root induction than during shoot multiplication. Results suggest that increased photoautotrophy of plantlets reduces susceptibility to photoinhibition during gardenia culture in vitro.Abbreviations AP apparent photosynthesis - Chl total chlorophyll content - Chl a/b chlorophyll a-to-b ratio - Chl/Car total chlorophyll-to-carotenoids ratio - ¹³C ratio of ¹³C/¹²C relative to PeeDee belemnite standard - F _m maximum chlorophyll fluorescence - F _o fluorescence emission when all reaction centres are open and the photochemical quenching is minimal - F _v variable chlorophyll fluorescence (F _m -F _o ) - F _v /F _m the ratio of variable to maximum chlorophyll fluorescence, indicator photochemical efficiency of PSII - MS medium Murashige and Skoog (1962) medium - PPFD photosynthetic photon flux density - Rd dark respiration, t _1/2 the half-time of the increase from F _o to F _m - IAA indole butyric acid