Photoinactivation of functional photosystem II and D1-protein synthesis in vivo are independent of the modulation of the photosynthetic apparatus by growth irradiance

To investigate whether the in-vivo photoinhibition of photosystem II (PSII) function by excess light is an intrinsic property of PSII, the maximal photochemical efficiency of PSII (Fv/Fm) and the content of functional PSII (measured by repetitive flash yield of oxygen evolution) were determined in leaves of pea (Pisum sativum L.), grown in 50 (low light), 250 (medium light), and 650 (high light) mol photons·m^–2·s^–1. The modulation of PSII functionality in vivo was induced in 1.1% CO₂ by varying either (i) the duration (0–2 h) of light treatment (fixed at 1800 mol photons· m^–2·s^–1) or (ii) irradiance (0–3200 mol photons·m^–2·s^–1) at a fixed duration (1 h), after infiltration of leaves with water (control), lincomycin (an inhibitor of chloroplast-encoded protein synthesis), or a combination of lincomycin with nigericin (an uncoupler), through the cut petioles of leaves of 22-to 24-d-old plants. The reciprocity law of irradiance and duration of illumination for PSII function in vivo (Park et al. 1995, Planta 196: 401–411) holds in all differently light-grown peas, demonstrating that inactivation of functional PSII depends on photon exposure (mol photons·m^–2), not on the rate of photon absorption. In vivo, PSII acts as an intrinsic photon counter and at higher photon exposures is inactivated following absorption of about 3 × 10⁷ photons. There is a functional heterogeneity of PSII in vivo with 25% less-stable PSIIs that are inactivated at low photon exposure, compared to 75% more-stable PSIIs regardless of modulation of the photosynthetic apparatus. We suggest that the less-stable PSIIs represent monomers located in the nonappressed granal margins, while the more-stable PSIIs are dimers located in the appressed grana membrane cores. The capacity for D1-protein synthesis was the same in all the light-acclimated peas and saturated at low light, indicating that D1-protein repair is also an intrinsic property of PSII. This accounts for the low intensity required for recovery of photoinhibition in sun and shade plants which is independent of light-harvesting antennae size or PSII/PSI stoichiometries.Abbreviations D1-protein psbA gene product - D2 protein psbD gene product - Fo chlorophyll fluorescence corresponding to open PSII reaction centres - Fv, Fm variable and maximum fluorescence after dark incubation, respectively - PS photosystem - Q_B secondary quinone electron acceptor Financial support for this research by the Department of Employment, Education and Training/Australian Research Council International Research Fellowships Program (Korea) is gratefully acknowledged.     

Photoinhibition of photosynthesis represents a mechanism for the long-term regulation of photosystem II

The obligate shade plant, Tradescantia albiflora Kunth grown at 50 mol photons · m^–2 s^–1 and Pisum sativum L. acclimated to two photon fluence rates, 50 and 300 mol · m^–2 · s^–1, were exposed to photoinhibitory light conditions of 1700 mol · m^–2 · s^–1 for 4 h at 22° C. Photosynthesis was assayed by measurement of CO₂-saturated O₂ evolution, and photosystem II (PSII) was assayed using modulated chlorophyll fluorescence and flash-yield determinations of functional reaction centres. Tradescantia was most sensitive to photoinhibition, while pea grown at 300 mol · m^–2 · s^–1 was most resistant, with pea grown at 50 mol · m^–2 · s^–1 showing an intermediate sensitivity. A very good correlation was found between the decrease of functional PSII reaction centres and both the inhibition of photosynthesis and PSII photochemistry. Photoinhibition caused a decline in the maximum quantum yield for PSII electron transport as determined by the product of photochemical quenching (q_p) and the yield of open PSII reaction centres as given by the steady-state fluorescence ratio, F_vF_m, according to Genty et al. (1989, Biochim. Biophys. Acta 990, 81–92). The decrease in the quantum yield for PSII electron transport was fully accounted for by a decrease in F_vF_m, since q_p at a given photon fluence rate was similar for photoinhibited and noninhibited plants. Under lightsaturating conditions, the quantum yield of PSII electron transport was similar in photoinhibited and noninhibited plants. The data give support for the view that photoinhibition of the reaction centres of PSII represents a stable, long-term, down-regulation of photochemistry, which occurs in plants under sustained high-light conditions, and replaces part of the regulation usually exerted by the transthylakoid pH gradient. Furthermore, by investigating the susceptibility of differently lightacclimated sun and shade species to photoinhibition in relation to q_p, i.e. the fraction of open-to-closed PSII reaction centres, we also show that irrespective of light acclimation, plants become susceptible to photoinhibition when the majority of their PSII reaction centres are still open (i.e. primary quinone acceptor oxidized). Photoinhibition appears to be an unavoidable consequence of PSII function when light causes sustained closure of more than 40% of PSII reaction centres.Abbreviations F_o and F_o minimal fluorescence when all PSII reaction centres are open in darkness and steady-state light, respectively - F_m and F_m maximal fluorescence when all PSII reaction centres are closed in darkand light-acclimated leaves, respectively - F_v variable fluorescence - (F_m-F_o) under steady-state light con-ditions - F_s steady-state fluorescence in light - Q_A the primary,stable quinone acceptor of PSII - q_Ne non-photochemical quench-ing of fluorescence due to high energy state - (pH); q_Ni non-photochemical quenching of fluorescence due to photoinhibition - q_p photochemical quenching of fluorescence To whom correspondence should be addressedThis work was supported by the Swedish Natural Science Research Council (G.Ö.) and the award of a National Research Fellowship to J.M.A and W.S.C. We thank Dr. Paul Kriedemann, Division of Forestry and Forest Products, CSIRO, Canberra, Australia, for helpful discussions.     

Photoinhibition and repair in Dunaliella salina acclimated to different growth irradiances

The light-dependent rate of photosystem-II (PSII) damage and repair was measured in photoautotrophic cultures of Dunaliella salina Teod. grown at different irradiances in the range 50–3000 mol photons · m^–2· s^–1. Rates of cell growth increased in the range of 50–800 mol photons·m^–2·s^–1, remained constant at a maximum in the range of 800–1,500 mol photons·m^–2 ·s^–1, and declined due to photoinhibition in the range of 1500–3000 mol photons·m^–2·s^–1. Western blot analyses, upon addition of lincomycin to the cultures, revealed first-order kinetics for the loss of the PSII reaction-center protein (D1) from the 32-kDa position, occurring as a result of photodamage. The rate constant of this 32-kDa protein loss was a linear function of cell growth irradiance. In the presence of lincomycin, loss of the other PSII reaction-center protein (D2) from the 34-kDa position was also observed, occurring with kinetics similar to those of the 32-kDa form of D1. Increasing rates of photodamage as a function of irradiance were accompanied by an increase in the steady-state level of a higher-molecular-weight protein complex ( 160-kDa) that cross-reacted with D1 antibodies. The steady-state level of the 160-kDa complex in thylakoids was also a linear function of cell growth irradiance. These observations suggest that photodamage to D1 converts stoichiometric amounts of D1 and D2 (i.e., the D1/D2 heterodimer) into a 160-kDa complex. This complex may help to stabilize the reaction-center proteins until degradation and replacement of D1 can occur. The results indicated an intrinsic half-time of about 60 min for the repair of individual PSII units, supporting the idea that degradation of D1 after photodamage is the rate-limiting step in the PSII repair process.Abbreviations Chl chlorophyll - PSI photosystem I - PSII photosystem II - D1 the 32-kDa reaction-center protein of PSII, encoded by the chloroplast psbA gene - D2 the 34-kDa reactioncenter protein of PSII, encoded by the chloroplast psbD gene - Q_A primary electron-accepting plastoquinone of PSII The work was supported by grant 94-37100-7529 from the US Department of Agriculture, National Research Initiative Competitive Grants Program.     

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: 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.     

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 in intact bean leaves: role of light and temperature,and requirement for chloroplast-protein synthesis during recovery

Photoinhibition of photosynthesis was induced in intact leaves of Phaseolus vulgaris L. grown at a photon flux density (PFD; photon fluence rate) of 300 mol·m^-2·s^-1, by exposure to a PFD of 1400 mol·m^-2·s^-1. Subsequent recovery from photoinhibition was followed at temperatures ranging from 5 to 35°C and at a PFD of either 20 or 140 mol·m^-2·s^-1 or in complete darkness. Photoinhibition and recovery were monitored mainly by chlorophyll fluorescence emission at 77K but also by photosynthetic O₂ evolution. The effects of the protein-synthesis inhibitors, cycloheximide and chloramphenicol, on photoinhibition and recovery were also determined. The results demonstrate that recovery was temperature-dependent with rates slow below 15°C and optimal at 30°C. Light was required for maximum recovery but the process was light-saturated at a PFD of 20 mol·m^-2·s^-1. Chloramphenicol, but not cycloheximide, inactivated the repair process, indicating that recovery involved the synthesis of one or more chloroplast-encoded proteins. With chloramphenicol, it was shown that photoinhibition and recovery occurred concomitantly. The temperature-dependency of the photoinhibition process was, therefore, in part determined by the effect of temperature on the recovery process. Consequently, photoinhibition is the net difference between the rate of damage and the rate of repair. The susceptibility of chilling-sensitive plant species to photoinhibition at low temperatures is proposed to result from the low rates of recovery in this temperature range.Abbreviations and symbols Da Dalton - Fo, Fm, Fv instantaneous, maximum, variable fluorescence emission - PFD photon flux density - PSII photosystem II - photon yield C.I.W.-D.P.B. Publication No. 871     

Photoinhibition of photosynthesis under natural conditions in ivy (Hedera helix L.) growing in an understory of deciduous trees

We investigated to what extent south-exposed leaves (E-leaves) of the evergreen ivy (Hedera helix L.) growing in the shadow of two deciduous trees suffered from photoinhibition of photosynthesis when leaf-shedding started in autumn. Since air temperatures drop concomitantly with increase in light levels, changes in photosynthetic parameters (apparent quantum yield, _i and maximal photosynthetic capacity of O₂ evolution, P_max; chlorophyll-a fluorescence at room temperature) as well as pigment composition were compared with those in north-exposed leaves of the same clone (N-leaves; photosynthetic photon flux density PPFD< 100 mol · m^–2 · s^–2) and phenotypic sun leaves (S-leaves; PPFD up to 2000 mol · m^–2 · s^–1).In leaves exposed to drastic light changes during winter (E-leaves) strong photoinhibition of photosynthesis could be observed as soon as the incident PPFD increased in autumn. In contrast, in N-leaves the ratio of variable fluorescence to maximum fluorescence (F_V/F_Mm) and _i did not decline appreciably prior to severe frosts (up to -12° C) in January. At this time, _i was reduced to a similar extent in all leaves, from about 0.073 mol O₂ · mol^–1 photons before stress to about 0.020. Changes in _i were linearly correlated with changes in f_v/f_m (r = 0.955). The strong reduction in F_V/F_M on exposure to stress was caused by quenching in F_M. The initial fluorescence (F₀), however, was also quenched in all leaves. The diminished fluorescence yield was accompanied by an increase in zeaxanthin content. These effects indicate that winter stress in ivy primarily induces an increase in non-radiative energy-dissipation followed by photoinhibitory damage of PSII. Although a pronounced photooxidative bleaching of chloroplast pigments occurred in January (especially in E-leaves), photosynthetic parameters recovered completely in spring. Thus, the reduction in potential photosynthetic yield in winter may be up to three times greater in leaves subjected to increasing light levels than in leaves not exposed to a changing light environment.Abbreviations and Symbols F₀, F_M initial and maximal fluorescence yield when all PSII centres are open and closed - F_V variable fluorescence (F_M-F₀) - P_max maximal photosynthetic capacity at 1000 umol · m^–2 · s^–1 PPFD and CO₂ saturation - PPFD photosynthetic photon flux density - _i apparent quantum yield of photosynthetic O₂ evolution - E-leaves, N-leaves shade leaves exposed, not exposed to drastic light changes during winter - S-leaves sun leaves from an open ivy stand Dedicated to Professor Otto Härtel on the occasion of his 80th birthdayThis work was supported by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung.     

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.     

The effects of photoinhibition on the photosynthetic light-response curve of green plant cells (Chlamydomonas reinhardtii)

The photosynthetic response to light can be accurately defined in terms of (1) the initial slope (quantum yield); (2) the asymptote (light-saturated rate); (3) the convexity (rate of bending); and (4) the intercept (dark respiration). The effects of photoinhibition [which damages the reaction centre of photosystem II (PSII)] on these four parameters were measured in optically thin cultures of green plant cells (Chlamydomonas reinhardtii). The convexity of the light-response curve decreased steadily from a value of 0.98 (indicating a sharply bending response) to zero (indicating Michaelis-Menten kinetics) in response to increasing photoinhibition. Photoinhibition was quantified from the quantum yield of inhibited cells relative to that of control cells. The quantum yield was estimated by applying linear regression to low-light data or by fitting a non-rectangular hyperbola. Assuming the initial slope is linear allowed comparison with earlier work. However, as the convexity was lowered this assumption resulted in a significant underestimate of the true quantum yield. Thus, the apparent level of photoinhibition required for a zero convexity and the initial decrease in light-saturated photosynthesis depended upon how the quantum yield was estimated. If the initial slope of the light response was assumed to be linear the critical level of inhibition was 60%. If the linear assumption was not made, the critical level was 40%. At the level of inhibition where the convexity reached zero, the light-saturated rate of photosynthesis also began to decrease, indicating that this level of inhibition caused photosynthesis to be limited at all light intensities by the rate of PSII electron transport. At this level of inhibition the F_m-F_i signal (where F_m is maximal chlorophyll fluorescence and F_i is intermediate chlorophyll fluorescence of dark adapted cells; Briantais et al. 1988) from the fluorescence induction curve was zero and the F_i-F_o signal (where F_o is initial chlorophyll fluorescence of dark adapted cells) was 30% of the control, indicating dramatic reduction or complete elimination of one type of PSII. These data do not contradict published mathematical models showing that the ratio of the maximum speed of electron transport in PSII relative to the maximum speed of plastoquinone electron transport can determine the convexity of the photosynthetic response to light.Abbreviations and Symbols Chl chlorophyll content - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_o, F_i, F_m initial, intermediate, and maximal Chl fluorescence of dark adapted cells - P rate of net photosynthesis per unit chlorophyll (mol-(mg Chl)^–1 · s^–1) - PSII photosystem II - PQ plastoquinone - initial slope to the light-response curve - convexity (rate of bending) of the light-response curve of photosynthesis - Q photosynthetically active photon flux density (400–700 nm, mol · m^–2 · ^–1) The present investigation was supported by the Swedish Council for Forestry and Agricultural Research, the Swedish Environmental Protection Board, and the Swedish Natural Science Research Council. We thank Dr. Deborah D. Kaska (Department of Biological Sciences, University of California, Santa Barbara, Calif., USA) for giving us Chlamydomonas algae. We thank Professor G. Öquist (Department of Plant Physiology, University of Umea, Umea, Sweden) for his encouragement, valuable comments and discussion.     

Protective systems against active oxygen species in spinach: response to cold acclimation in excess light

Spinach (Spinacia oleracea L.) plants were acclimated to 1° C or maintained at 18° C under the same light regime (260–300 mol photons·m^–2·s^–1). The cold acclimation led to several metabolic and biochemical changes that apparently include improved protection of the photosynthetic apparatus against active oxygen species. In particular, cold-acclimated leaves exhibited a considerably higher ascorbate content and significantly increased activities of superoxide dismutase, ascorbate peroxidase, and monodehydroascorbate reductase in the chloroplasts. The level of dehydroascorbate reductase did not alter. Catalase activity decreased. The photosynthetic pigment composition of cold-acclimated spinach was characterized by increased levels of the xanthophylls lutein + zeaxanthin and violaxanthin. The observed changes are discussed in terms of their possible relevance for plant resistance to photoinhibition at chilling temperatures.Abbreviations DHA dehydroascorbate - GSH reduced glutathione - MDA monodehydroascorbate - SOD superoxide dismutase The authors thank the Deutsche Forschungsgemeinschaft for financial support of this study.     

Light and the maintenance of photosynthetic competence in leaves of Populus balsamifera L. during short-term exposures to high concentrations of sulfur dioxide

Leaves of Populus balsamifera grown under full natural sunlight were treated with 0, 1, or 2 l SO₂·1^-1 air under one of four different photon flux densities (PFD). When the SO₂ exposures took place in darkness or at 300 mol photons·m^-2·s^-1, sulfate accumulated to the levels predicted by measurements of stomatal conductance during SO₂ exposure. Under conditions of higher PFD (750 and 1550 mol·m^-2·s^-1), however, the predicted levels of accumulated sulfate were substantially higher than those obtained from anion chromatography of the leaf extracts. Light-and CO₂-saturated capacity as well as the photon yield of photosynthetic O₂ evolution were reduced with increasing concentration of SO₂. At 2 l SO₂·1^-1 air, the greatest reductions in both photosynthetic, capacity and photon yield occurred when the leaves were exposed to SO₂ in the dark, and increasingly smaller reductions in each occurred with increasing PFD during SO₂ exposure. This indicates that the inhibition of photosynthesis resulting from SO₂ exposure was reduced when the exposure occurred under conditions of higher light. The ratio F _v/F _M (variable/maximum fluorescence emission) for photosyntem II (PSII), a measure of the photochemical efficiency of PSII, remained unaffected by exposure of leaves to SO₂ in the dark and exhibited only moderate reductions with increasing PFD during the exposure, indicating that PSII was not a primary site of damage by SO₂. Pretreatment of leaves with SO₂ in the dark, however, increased the susceptibility of PSII to photoinhibition, as such pretreated leaves exhibited much greater reductions inF _V/F _M when transferred to moderate or high light in air than comparable control leaves.Abbreviations and symbols A₁₂₀₀ photosynthetic capacity (CO₂-saturated rate of O₂ evolution at 1200 mol photons·m^-2·s^-1) - F_o instantaneous fluorescence emission - F_M maximum fluorescence emission - F_V variable fluorescence emission - PFD photon flux density (400–700 nm) - PSII photosystem II     

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.     

Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: Effect of light during growth on photoinhibition and recovery

Photoinhibition of photosynthesis was induced in intact kiwifruit (Actinidia deliciosa (A. Chev.) C. F. Liang et A. R. Ferguson) leaves grown at two photon flux densities (PFDs) of 700 and 1300 mol·m^-2·s^-1 in a controlled environment, by exposing the leaves to PFD between 1000 and 2000 mol·m^-2·s^-1 at temperatures between 10 and 25°C; recovery from photoinhibition was followed at the same range of temperatures and at a PFD between 0 and 500 mol·m^-2·s^-1. In either case the time-courses of photoinhibition and recovery were followed by measuring chlorophyll fluorescence at 692 nm and 77K and by measuring the photon yield of photosynthetic O₂ evolution. The initial rate of photoinhibition was lower in the high-light-grown plants but the long-term extent of photoinhibition was not different from that in low-light-grown plants. The rate constants for recovery after photoinhibition for the plants grown at 700 and 1300 mol·m^-2·s^-1 or for those grown in shade were similar, indicating that differences between sun and shade leaves in their susceptibility to photoinhibition could not be accounted for by differences in capacity for recovery during photoinhibition. Recovery following photoinhibition was increasingly suppressed by an increasing PFD above 20 mol·m^-2·s^-1, indicating that recovery in photoinhibitory conditions would, in any case, be very slow. Differences in photosynthetic capacity and in the capacity for dissipation of non-radiative energy seemed more likely to contribute to differences in susceptibility to photoinhibition between sun and shade leaves of kiwifruit.Abbreviations and symbols F _o , F _m , F _v instantaneous, maximum, variable fluorescence - F _v /F _m fluorescence ratio - F _i =F _v at t=0 - F F _v at t= - K _D rate constant for photochemistry - k(F _p ) first-order rate constant for photoinhibition - k(F _r ) first-order rate constant for recovery - PFD photon flux density - PSII photosystem II - _i photon yield of O₂ evolution (incident light)     

Effects of constitutive expression of nitrate reductase in transgenic Nicotiana plumbaginifolia L. in response to varying nitrogen supply

Transformed Nicotiana plumbaginifolia plants with constitutive expression of nitrate reductase (NR) activity were grown at different levels of nitrogen nutrition. The gradients in foliar NO ₃ ^– content and maximum extractable NR activity observed with leaf order on the shoot, from base to apex, were much decreased as a result of N-deficiency in both the transformed plants and wild type controls grown under identical conditions. Constitutive expression of NR did not influence the foliar protein and chlorophyll contents under any circumstances. A reciprocal relationship between the observed maximal extractable NR activity of the leaves and their NO ₃ ^– content was observed in plants grown in nitrogen replete conditions at low irradiance (170 mol photons·m^–2 ·s^–1). This relationship disappeared at higher irradiance (450 mol photons·m^–2·S^–1) because the maximal extractable NR activity in the leaves of the wild type plants in these conditions increased to a level that was similar to, or greater than that found in constitutive NR-expressors. Much more NO ₃ ^– accumulated in the leaves of plants grown at 450 mol photons·m^–2·s^–1 than in those grown at 170 mol photons·m^–2·s^–1 in N-replete conditions. The foliar NO ₃ ^– level and maximal NR activity decreased with the imposition of N-deficiency in all plant types such that after prolonged exposure to nitrogen depletion very little NO ₃ ^– was found in the leaves and NR activity had decreased to almost zero. The activity of NR decreased under conditions of nitrogen deficiency. This regulation is multifactoral since there is no regulation of NR gene expression by NO ₃ ^– in the constitutive NR-expressors. We conclude that the NR protein is specifically targetted for destruction under nitrogen deficiency. Consequently, constitutive expression of NR activity does not benefit the plant in terms of increased biomass production in conditions of limiting nitrogen.Abbreviations Chl chlorophyll - N nitrogen - NR NADH-nitrate reductase - WT wild type     

Reduced photoinhibition with stem curling in the resurrection plant Selaginella lepidophylla

Summary Selaginella lepidophylla, the resurrection plant, curls dramatically during desiccation and the hypothesis that curling may help limit bright light-induced damage during desiccation and rehydration was tested under laboratory conditions. Restraint of curling during desiccation at 25° C and a constant irradiance of 2000 mol m^–2 s^]t-1 significantly decreased PSII and whole-chain electron transport and the F_v/F_m fluorescence yield ratio following rehydration relative to unrestrained plants. Normal curling during desiccation at 37.5°C and 200 mol m^–2 s^–1 irradiance did not fully protect against photoinhibition or chlorophyll photooxidation indicating that some light-induced damage occurred early in the desiccation process before substantial curling. Photosystem I electron transport was less inhibited by high-temperature, high-irradiance desiccation than either PSII or whole-chain electron transport and PSI was not significantly affected by restraint of curling during desiccation at 25°C and high irradiance. Previous curling also helped prevent photoinhibition of PSII electron transport and loss of whole-plant photosynthetic capacity as the plants uncurled during rehydration at high light. These results demonstrate that high-temperature desiccation exacerbated photoinhibition, PSI was less photoinhibited than PSII or whole-chain electron transport, and stem curling ameliorated bright light-induced damage helping to make rapid recovery of photosynthetic competence possible when the plants are next wetted.     

Two sites of photoinhibition of the electron transfer in oxygen evolving and Tris-treated PS II membrane fragments from spinach

Photoinhibition was analyzed in O₂-evolving and in Tris-treated PS II membrane fragments by measuring flash-induced absorption changes at 830 nm reflecting the transient P680⁺ formation and oxygen evolution. Irradiation by visible light affects the PS II electron transfer at two different sites: a) photoinhibition of site I eliminates the capability to perform a stable charge separation between P680⁺ and Q_A ^- within the reaction center (RC) and b) photoinhibition of site II blocks the electron transfer from Y_Z to P680⁺. The quantum yield of site I photoinhibition (2–3×10^-7 inhibited RC/quantum) is independent of the functional integrity of the water oxidizing system. In contrast, the quantum yield of photoinhibition at site II depends strongly on the oxygen evolution capacity. In O₂-evolving samples, the quantum yield of site II photoinhibition is about 10^-7 inhibited RC/quantum. After selective elimination of the O₂-evolving capacity by Tris-treatment, the quantum yield of photoinhibition at site II depends on the light intensity. At low intensity (<3 W/m²), the quantum yield is 10^-4 inhibited RC/quantum (about 1000 times higher than in oxygen evolving samples). Based on these results it is inferred that the dominating deleterious effect of photoinhibition cannot be ascribed to an unique target site or a single mechanism because it depends on different experimental conditions (e.g., light intensity) and the functional status of the PS II complex.Abbreviations A₈₃₀ absorption change at 830 nm - P680 primary electron donor of PS II - PS II photosystem II - Mes 2(N-morpholino)ethansulfonic acid - Q_A, Q_B primary and secondary acceptors of PS II - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenylcarbohydrazide - FWHM fullwidth at half maximum - Ph-p-BQ phenyl-p-benzoquinone - PFR photon fluence rate - Pheo pheophytin - RC reaction center     

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.     

Inversion of gravitropism by symmetric blue light on the clinostat

Gravitropic stimulation of maize (Zea mays L.) seedlings resulted in a continuous curvature of the coleoptiles in a direction opposing the vector of gravity when the seedlings were rotated on a horizontal clinostat. The orientation of this response, however, was reversed when the gravitropic stimulation was preceeded by symmetric preirradiation with blue light (12.7 mol photons·m^–2). The fluence-response curve of this blue light exhibited a lower threshold at 0.5 mol·m^–2, and could be separated into two parts: fluences exceeding 5 mol·m^–2 reversed the direction of the gravitropic response, whereas for a range between the threshold and 4 mol·m^–2 a split population was obtained. In all cases a very strong curvature resulted either in the direction of gravity or in the opposite orientation. A minor fraction of seedlings, however, curved towards the caryopsis. Furthermore, the capacity of blue light to reverse the direction of the gravitropic response disappeared with the duration of gravitropic stimulation and it depended on the delay time between both stimulations. Thistonic blue-light influence appears to be transient, which is in contrast to the stability observed fortropistic blue-light effects.This work was supported by the Deutsche Forschungsgemeinschaft.     

Light-dependent enhanced metabolism of chlorotoluron in a substituted urea herbicide-resistant biotype ofLolium rigidum Gaud.

A population ofLolium rigidum Gaud. displays resistance to the herbicide chlorotoluron endowed by enhanced metabolism of this herbicide. The level of resistance in intact plants of this population is light dependent. Resistance is about 4-fold at 110 mol photons·m^–2·s^–1, but increases to 11-fold at 600 mol photons·m^–2·s^–1. For seedlings grown in the dark, the rate of chlorotoluron metabolism is identical between biotypes; however, seedlings of the resistant biotype grown in the light display enhanced chlorotoluron metabolism compared to the susceptible biotype. Specifically, light with blue wavelengths induces chlorotoluron metabolism in the resistant biotype. An analysis of the metabolites produced indicates that two routes of chlorotoluron metabolism occur inL. rigidum. These are characterised by initial reactions leading to ringmethyl hydroxylation orN-demethylation of the herbicide. The ring-methyl hydroxylation pathway is increased greatly in light-grown resistant seedlings compared to susceptible seedlings, whereas theN-demethylation pathway is only slightly increased. The differential induction of these two pathways in resistantL. rigidum by light suggests that enhanced activity of two different enzymes may be involved in chlorotoluron resistance.Abbreviations ABT 1-aminobenzotriazole - LD₅₀ dose giving 50% mortality - LSS liquid scintillation spectroscopy