Chlorophyll fluorescence in the resurrection plant Selaginella lepidophylla (Hook. & Grev.) Spring during high-light and desiccation stress,and evidence for zeaxanthin-associated photoprotection

The function of photosystem (PS)II during desiccation and exposure to high photon flux density (PFD) was investigated via analysis of chlorophyll fluorescence in the desert resurrection plant Selaginella lepidophylla (Hook. and Grev.) Spring. Exposure of hydrated, physiologically competent stems to 2000 mol · m^–2 · s^–1 PFD caused significant reductions in both intrinsic fluorescence yield (F_O) and photochemical efficiency of PSII (F_V/F_M) but recovery to pre-exposure values was rapid under low PFD. Desiccation under low PFD also affected fluorescence characteristics. Both F_V/F_M and photochemical fluorescence quenching remained high until about 40% relative water content and both then decreased rapidly as plants approached 0% relative water content. In contrast, the maximum fluorescence yield (F_M) decreased and non-photochemical fluorescence quenching increased early during desiccation. In plants dried at high PFD, the decrease in F_V/F_M was accentuated and F_O was reduced, however, fluorescence characteristics returned to near pre-exposure values after 24-h of rehydration and recovery at low PFD. Pretreatment of stems with dithiothreitol, an inhibitor of zeaxanthin synthesis, accelerated the decline in F_V/F_M and significantly increased F_O relative to controls at 925 mol · m^–2 · s^–1 PFD, and the differences persisted over a 3-h low-PFD recovery period. Pretreatment with dithiothreitol also significantly decreased non-photochemical fluorescence quenching, increased the reduction state of Q_A, the primary electron acceptor of PSII, and prevented the synthesis of zeaxanthin relative to controls when stems were exposed to PFDs in excess of 250 mol · m^–2 · s^–1. These results indicate that a zeaxanthin-associated mechanism of photoprotection exists in this desert pteridophyte that may help to prevent photoinhibitory damage in the fully hydrated state and which may play an additional role in protecting PSII as thylakoid membranes undergo water loss.Abbreviations and Symbols DTT dithiothreitol - EPS epoxidation state - F_O yield of instantaneous fluorescence at open PSII centers - F_M maximum yield of fluorescence at closed PSII centers induced by saturating light - F_M F_M determined during actinic illumination - F_V yield of variable fluorescence (F_M-F_O) - F_V/F_M photochemical efficiency of PSII - q_P photochemical fluorescence quenching - q_NP non-photochemical fluorescence quenching of Schreiber et al. (1986) - NPQ non-photochemical fluorescence quenching from the Stern-Volmer equation - PFD photon flux density - RWC relative water content This paper is based on research done while W.G.E. was on leave of absence at Duke University during the fall of 1990. We would like to thank Dan Yakir, John Skillman, Steve Grace, and Suchandra Balachandran and many others at Duke University for their help and input with this research. Dr. Barbara Demmig-Adams provided zeaxanthin for standard-curve purposes.     

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     

Analysis of oxygen evolution during photosynthetic induction and in multiple-turnover flashes in sunflower leaves

Exchange of CO₂ and O₂ and chlorophyll fluorescence were measured in the presence of 360 1 · 1^–1 CO₂ in nitrogen in Helianthus annuss L. leaves which had been preconditioned in the dark or at a photon flux density (PFD) of 24 mol · m^–2 · s^–1 either in 21 or 0% O₂. An initial light-dependent O₂ outburst of 6 mol · m^–2 was measured after aerobic dark incubation. It was attributed to the reduction of electron carriers, predominantly plastoquinone. The maximum initial rate of O₂ evolution at PFD 8000 mol · m^–2 · s^–1 was 170 mol · m^–2 · s^–2 or about four times the steady CO₂-and light-saturated rate of photosynthesis. Fluorescence measurements showed that the rate was still acceptor-limited. Fast O₂ evolution ceased after electron carriers were reduced in the dark-adapted leaf, but continued for a short time at the lower rate of 62 mol · m^–2 · s^–1 in the light-adapted leaf. The data are interpreted to show that enzymes involved in 3-phosphoglycerate reduction are dark-inhibited, but were fully active in low light. In a dark-adapted leaf, respiratory CO₂ evolution continued under nitrogen; it was partially inhibited by illumination. Prolonged exposure of a leaf to anaerobic conditions caused reducing equivalents to accumulate. This was shown by a slowly increasing chlorophyll fluorescence yield which indicated the reduction of the PSII acceptor Q_A in the dark. When the leaf was illuminated, no O₂ evolution was detected from short light pulses, although transient O₂ production was appreciable during longer light pulses. This indicates that an electron donor (pool size about 2–3 e/PSII reaction center) became reduced in the dark and the first photons were used to oxidise this donor instead of water.Abbreviations Chl chlorophyll - CRC carbon reduction cycle - GAPDH NADP-glyceraldehyde-phosphate dehydrogenase - PFD photon flux density - PGA 3-phosphoglycerate - RuBP ribulose bisphosphate - TCA tricarboxylic acid cycle To whom correspondence should be addressedThis work received support by the Estonian Academy of Sciences, the Gottfried-Wilhelm-Leibniz Program of the Deutsche For-schungsgemeinschaft and the Sonderforschungsbereich 251 of the University of Würzburg.     

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 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.     

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 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)     

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)     

Effect of daily photon receipt on the susceptibility of dwarf bean (Phaseolus vulgaris L.) leaves to photoinhibition of photosynthesis

Bean (Phaseolus vulgaris L.) plants were grown at two light periods of 8 and 13 h with a similar photon flux density (PFD) giving a daily photon receipt (DPR) of 17.9 and 38.2 mol · m–2, respectively. Shoot growth and leaf area development were followed at regular intervals and diurnal whole-plant photosynthesis measured. Single mature trifoliate leaves were exposed to photoinhibitory treatments at PFDs of 800 and 1400 mol · m–2 · s–1 and at temperatures of 12 and 20°C. Chlorophyll fluorescence and photon yields were measured at regular intervals throughout each treatment. Plants grown in 13 h had significantly greater leaf areas than those grown in 8 h. There were no differences in maximum rates of photosynthesis, photon yields and only minor but significant differences in F_v/F_m for plants in the two treatments, showing photosynthetic characteristics were dependent on PFD but not DPR. A significant decline in photosynthesis and F_v/F_m occurred over the 13-h but little change in photosynthesis for plants in the 8 h, indicating some feedback inhibition of photosynthesis was occurring. Plants grown in 8 h were consistently more susceptible to photoinhibition of photosynthesis at all treatments than 13-h plants. Nevertheless, photoinhibition was exacerbated by increases in PFD, and by decreases in temperature for leaves from both treatments. However, for plants from the 8-h day, exposing leaves to 12°C and 1400 mol · m–2 · s–1 caused photo-oxidation and severe bleaching but no visible damage on leaves from 13-h-grown plants. Closure of the photosystem II reaction-centre pool was partially correlated with increasing extents of photoinhibition but the relationship was similar for plants from both treatments. There remains no clear explanation for their wide differences in susceptibility to photoinhibition.Abbreviations and Symbols DPR daily photon receipt - F₀ and F_m initial and maximal fluorescence - F_v/F_m fluorescence ratio in dark-treated leaves - F/F_m intrinsic efficiency of PSII during illumination - PFD photon flux density - _i photon yield (incident basis) - _psi quantum yield of PSII electron transport - P_max maximum rate of photosynthesis - q_N non-photochemical quenching coefficient - q_P photochemical quenching coefficient Many thanks to my colleague William Laing who spent a considerable effort in developing the programme to run the photosynthesis apparatus. I am also indebted to one reviewer with whom I corresponded to resolve some issues in the paper. This project was funded by the New Zealand Foundation for Research, Science and Technology.     

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.     

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)     

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.     

Concurrent measurements of oxygen- and carbon-dioxide exchange during lightflecks inAlocasia macrorrhiza (L.) G. Don

Oxygen and CO₂ exchange were measured concurrently in leaves of shade-grownAlocasia macrorrhiza (L.) G. Don during lightflecks consisting of short periods of high photon flux density (PFD) superimposed on a low-PFD background illumination. Oxygen exchange was measured with a zirconium-oxide ceramic cell in an atmosphere containing 1 600 bar O₂ and 350 bar CO₂. Following an increase in PFD from 10 to 500 mol photons·m^-2·s^-1, O₂ evolution immediately increased to a maximum rate that was about twice as high as the highest CO₂-exchange rates that were observed. Oxygen evolution then decreased over the next 5–10 s to rates equal to the much more slowly increasing rates of CO₂ uptake. When the PFD was decreased at the end of a lightfleck, O₂ evolution decreased nearly instantaneously to the low-PFD rate while CO₂ fixation continued at an elevated rate for about 20 s. When PFD during the lightfleck was at a level that was limiting for steady-state CO₂ exchange, then the O₂-evolution rate was constant during the lightfleck. This observed pattern of O₂ evolution during lightflecks indicated that the maximum rate of electron transport exceeded the maximum rate of CO₂ fixation in these leaves. In noninduced leaves, rates of O₂ evolution for the first fraction of a second were about as high as rates in fully induced leaves, indicating that O₂ evolution and the electron-transport chain are not directly affected by the leaf's induction state. Severalfold differences between induced and noninduced leaves in O₂ evolution during a lightfleck were seen for lightflecks longer than a few seconds where the rate of O₂ evolution appeared to be limited by the utilization of reducing power in CO₂ fixation.Abbreviation PFD photon flux density (of photosynthetically active radiation)     

Relationship Between Photosystem 2 Electron Transport and Photosynthetic CO2 Assimilation Responses to Irradiance in Young Apple Tree Leaves

The responses to irradiance of photosynthetic CO₂ assimilation and photosystem 2 (PS2) electron transport were simultaneously studied by gas exchange and chlorophyll (Chl) fluorescence measurement in two-year-old apple tree leaves (Malus pumila Mill. cv. Tengmu No.1/Malus hupehensis Rehd). Net photosynthetic rate (P _N) was saturated at photosynthetic photon flux density (PPFD) 600-1 100 (mol m^-2 s^-1, while the PS2 non-cyclic electron transport (P-rate) showed a maximum at PPFD 800 mol m^-2 s^-1. With PPFD increasing, either leaf potential photosynthetic CO₂ assimilation activity (F_d/F_s) and PS2 maximal photochemical activity (F_v/F_m) decreased or the ratio of the inactive PS2 reaction centres (RC) [(F_i – F_o)/(F_m – F_o)] and the slow relaxing non-photochemical Chl fluorescence quenching (q_s) increased from PPFD 1 200 mol m^-2 s^-1, but cyclic electron transport around photosystem 1 (RFp), irradiance induced PS2 RC closure [(F_s – F_o)/F_m – F_o)], and the fast and medium relaxing non-photochemical Chl fluorescence quenching (q_f and q_m) increased remarkably from PPFD 900 (mol m^-2 s^-1. Hence leaf photosynthesis of young apple leaves saturated at PPFD 800 mol m^-2 s^-1 and photoinhibition occurred above PPFD 900 mol m^-2 s^-1. During the photoinhibition at different irradiances, young apple tree leaves could dissipate excess photons mainly by energy quenching and state transition mechanisms at PPFD 900-1 100 mol m^-2 s^-1, but photosynthetic apparatus damage was unavoidable from PPFD 1 200 mol m^-2 s^-1. We propose that Chl fluorescence parameter P-rate is superior to the gas exchange parameter P _N and the Chl fluorescence parameter F_v/F_m as a definition of saturation irradiance and photoinhibition of plant leaves.     

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.     

Comparison of the effect of excessive light on chlorophyll fluorescence (77K) and photon yield of O2 evolution in leaves of higher plants

High-light treatments (1750–2000 mol photons m^–2 · s^–1) of leaves from a number of higher-plant species invariably resulted in quenching of the maximum 77K chlorophyll fluorescence at both 692 and 734 nm (F _M, 692 and F _M, 734). The response of instantaneous fluorescence at 692 nm (F _O, 692) was complex. In leaves of some species F _O, 692 increased dramatically in others it was quenched, and in others yet it showed no marked, consistent change. Regardless of the response of F _O, 692 an apparently linear relationship was obtained between the ratio of variable to maximum fluorescence (F _V/F _M, 692) and the photon yield of O₂ evolution, indicating that photoinhibition affects these two variables to approximately the same extent. Treatment of leaves in a CO₂–free gas stream containing 2% O₂ and 98% N₂ under weak light (100 mol · m^–2 · s^–1) resulted in a general and fully reversible quenching of 77K fluorescence at 692 and 734 nm. In this case both F _O, 692 and F _M, 692 were invariably quenched, indicating that the quenching was caused by an increased non-radiative energy dissipation in the pigment bed. We propose that high-light treatments can have at least two different, concurrent effects on 77K fluorescence in leaves. One results from damage to the photosystem II (PSII) reaction-center complex and leads to a rise in F _O, 692; the other results from an increased non-radiative energy dissipation and leads to quenching of both F _O, 692 and F _M, 692 This general quenching had a much longer relaxation time than reported for pH-dependent quenching in algae and chloroplasts. Sun leaves, whose F _V/F _M, 692 ratios were little affected by high-light exposure in normal air, suffered pronounced photoinhibition when the exposure was made under conditions that prevent photosynthetic gas exchange (2% O₂, 0% CO₂). However, they were still less susceptible than shade leaves, indicating that the higher capacity for energy dissipation via photosynthesis is not the only cause of their lower susceptibility. The rate constant for recovery from photoinhibition was much higher in mature sun leaves than in mature shade leaves, indicating that differences in the capacity for continuous repair may in part account for the difference in their susceptibility to photoinhibition.Abbreviations and symbols kDa kilodalton - LHC-II light-harvesting chlorophyll-protein complex - PFD photon flux density (photon fluence rate) - PSI, PSII photosystem I, II - F _O, F _M, F _V instantaneous, maximum, variable fluorescence emission - absorptance - _a photon yield of O₂ evolution (absorbed light) C.I.W.-D.P.B. Publication No. 925     

Calculation of leaf photosynthetic parameters from light-response curves for ecophysiological applications

Measurement of the light response of photosynthetic CO₂ uptake is often used as an implement in ecophysiological studies. A method is described to calculate photosynthetic parameters, such as the maximum rate of whole electron transport and dissimilative respiration in the light, from the light response of CO₂ uptake. Examples of the light-response curves of flag leaves and ears of wheat (Triticum aestivum cv. ARKAS) are shown.Abbreviations and symbols A net photosynthesis rate - D ¹ rate of dissimilative respiration occurring in the light - f loss factor - I incident PPFD - I effective absorbed PPFD - J rate of whole electron transport - J _m maximum rate of whole electron transport - p ^c intercellular CO₂ partial pressure - PPFD photosynthetic photon flux density - q effectivity factor for the use of light (electrons/quanta) - absorption coefficient - I ^* CO₂ compensation point in the absence of dissimilative respiration (bar) - II conversion factor for calculation of CO₂ uptake from the rate of whole electron transport - convexity factor Gas-exchange rates relate to the projective area and are given in mol·m^-2·s^-1. Electron-transport rates are given in mol electrons·m^-2·s^-1; PPFD is given in mol quanta·m^-2·s^-1.     

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.     

Carotenoid composition and photon-use efficiency of photosynthesis inGossypium hirsutum L. grown under conditions of slightly suboptimum leaf temperatures and high levels of irradiance

Summary Cotton (Gossypium hirsutum L. var. DP 61) was grown at different temperatures during 12-h light periods, with either 1800–2000 mol photons m^–2 s^–1 (high photon flux density, PFD) or 1000–1100 mol m^–2 s^–1 (medium PFD) incident on the plants. Night temperature was 25°C in all experiments. Growth was less when leaf temperatures were below 30°C during illumination, the effect being greater in plants grown with high PFD (Winter and Königer 1991). Leaf pigment composition and the photon-use efficiency of photosynthesis were analysed to assess whether plants grown with high PFD and suboptimal temperatures experienced a higher degree of high irradiance stress during development than those grown with medium PFD. The chlorophyll content per unit area was 3–4 times less, and the content of total carotenoids about 2 times less, with the proportion of the three xanthophylls zeaxanthin + antheraxanthin + violaxanthin being greater in leaves grown at 20–21°C than in leaves grown at 33–34°C. In leaves from plants grown at 21°C and 1800–2000 mol photons m^–2 s^–1, zeaxanthin accounted for as much as 34% of total carotenoids in the middle of the photoperiod, the highest level recorded in this study. This finding is consistent with a protective role of zeaxanthin under conditions of excess light. At the lower temperatures, the photochemical efficiency of photosystem II, measured as the ratio of variable to maximum fluorescence yield (F _V/F _M) after 12-h dark adaptation, was 0.76 in medium PFD plants and 0.75 in high PFD plants compared with 0.83 and 0.79, respectively, at the higher temperatures. The photon-use efficiency of O₂ evolution () based on absorbed light between 630 and 700nm, decreased with decrease in temperature from 0.102 to 0.07 under conditions of high PFD, but remained above 0.1 at medium PFD. Owing to compensatory reactions in these long-term growth experiments, sustained differences inF _V/F _M and were much less pronounced than the differences in chlorophyll content and dry matter, particularly in plants which had developed at high PFD and low temperature. In fact, in these plants, which exhibited pronounced photobleaching, a largely functional photosynthetic apparatus was still maintained in cells adjacent to the lower leaf surfaces. This was indicated by measurements of photon use efficiencies of photosynthetic O₂ evolution with leaves illuminated first at the upper, and then at the lower surface.Abbreviations F _O yield of dark level fluorescence - F _M maximum yield of fluorescence, induced in a pulse of saturating light - F _V yield of variable fluorescence (=F _M-F _o) - PFD photon flux density - _iw photon use efficiency of O₂ evolution based on white (400–700 nm) incident light - _ir photon use efficiency based on red (630–700 nm) incident light - _aw photon use efficiency based on white absorbed light - _ar photon use efficiency based on red absorbed light     

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