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

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

Rapid turnover of the D1 reaction-center protein of photosystem II as a protection mechanism against photoinhibition in a moss,Ceratodon purpureus (Hedw.) Brid.

Susceptibility of a moss,Ceratodon purpureus (Hedw.) Brid., to photoinhibition and subsequent recovery of the photochemical efficiency of PSII was studied in the presence and absence of the chloroplast-encoded protein-synthesis inhibitor lincomycin.Ceratodon had a good capacity for repairing the damage to PSII centers induced by strong light. Tolerance against photoinhibition was associated with rapid turnover of the D1 protein, since blocking of D1 protein synthesis more than doubled the photoinhibition rate measured as the decline in the ratio of variable fluorescence to maximal fluorescence (F_v/F_max). Under exposure to strong light in the absence of lincomycin a net loss of D1 protein occurred, indicating that the degradation of damaged D1 protein inCeratodon was rapid and independent of the resynthesis of the polypeptide. The result suggests that synthesis is the limiting factor in the turnover of D1 protein during photoinhibition of the mossCeratodon. The level of initial fluorescence (F_o) correlated with the production of inactive PSII centers depleted of D1 protein. The higher the F_o level, the more severe was the loss of D1 protein seen in the samples during photoinhibition. Restoration of F_v/F_max at recovery light consisted of a fast and slow phase. The recovery of fluorescence yield in the presence of lincomycin, which was added at different times in the recovery, indicated that the chloroplast-encoded protein-synthesis-dependent repair of damaged PSII centers took place during the fast phase of recovery. Pulse-labelling experiments with [³⁵S]methionine supported the conclusion drawn from fluorescence measurements, since the rate of D1 protein synthesis after photoinhibition exceeded that of the control plants during the first hours under recovery conditions.     

D1 protein degradation during photoinhibition of intact leaves a modification of the D1 protein precedes degradation

Illumination of intact pumpkin leaves with high light led to severe photoinhibition of photosystem II with no net degradation of the D1 protein. Instead, however, a modified form of D1 protein with slightly slower electrophoretic mobility was induced with corresponding loss in the original form of the D1 protein. When the leaves were illuminated in the presence of chloramphenicol the modified form was degraded, which led to a decrease in the total amount of the D1 protein. Subfractionation of the thylakoid membranes further supported the conclusion that the novel form of the D1 protein was not a precursor but a high-light modified form that was subsequently degraded.     

Photoinactivation of photosystem II during photoinhibition in the cyanobacterium Microcystis aeruginosa

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

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.     

Stress-induced chlorosis and increase in D1-protein turnover precede photoinhibition in spinach suffering under magnesium/sulphur deficiency

To test wether chlorosis is induced by photoinhibitory damage to photosystem II (PSII), onset of chlorosis and loss of PSII function were compared in young spinach (Spinaciae oleracea L.) plants suffering under a combined magnesium and sulphur deficiency. Loss of chlorophyll already occurred after the first week of deficiency and preceded any permanent functional inhibition of the photosynthetic apparatus. Permanent disturbancies of photosynthetic electron transport measured in isolated thylakoids and of PSII function, determined via the ratio of variable fluorescence to maximal fluorescence, F_v/F_m, could be detected only after the second week of deficiency. After the third week, the plants had lost about 60% of their chlorophyll; even so, fluorescence data indicated that 85% of the existing PSII was still capable of initiating photosynthetic electron transport. However, quenching analysis of steady-state fluorescence showed an early increase in non-photochemical quenching and in down-regulated PSII centres with low steady-state quantum efficiency. Together with the down-regulation of PSII centres, a 1.4-fold increase in D1-protein synthesis, measured as incorporation of [¹⁴C]leucine, could be observed at the end of the first week before any loss of D1 protein, chlorophyll or photosynthetic activity could be detected. Immunological determiation by Western-blotting did not show a change in D1-protein content; thus, at this time, D1 protein was not only faster synthesised but was also faster degraded than before the imposition of mineral deficiency. The increased turnover was high enough to prevent any loss or functional inhibition of PSII. After 3 weeks, D1-protein synthesis on a chlorophyll basis was further stimulated by a factor of 2. However, this was not enough to prevent a net loss of D1 protein of about 70%, showing that the D1-protein was now degraded faster than it was synthesised. Immunological determination and electron-transport measurements showed that together with the loss of D1 protein the other polypetides of PSII were also degraded, resulting in a specific loss of PSII centres. The degradation of PSII centres prevented a large accumulation of damaged PSII centres. We assume that the decrease in PSII centres initiates the breakdown of the other thylakoid proteins.Abbreviations Fo yield of intrinsic fluorescence when all PSII centres are open in the dark - F_m yield of maximal fluorescence when all reaction centres are closed - F_m fluorescence yield when all reaction centres are closed under steady-state conditions - F_v yield of variable fluorescence, (difference between F_o and F_m) - F yield of variable fluorescence under steady-state conditions, difference between F_m and F_t, the fluorescence yield under steady-state conditions - PFD photon flux density - Q_A primary quinone acceptor of PSII - Q_B secondary quinone acceptor of PSII - q_p photochemical quenching - q_n non-photochemical quenching This work was supported by grants from the Bundesminister für Forschung und Technologie and the German Israeli Foundation. The authors thank Prof. I. Ohad (Department of Biological Chemistry, Hebrew University, Jerusalem, Israel) for fruitful discussions.     

Phosphorylation and disassembly of the photosystem II core as an early stage of photoinhibition

The presence of heterogeneity in phosphorylated PSII core populations in grana membranes of spinach (Spinacia oleracea L.) was previously demonstrated (Giardi et al., 1991, Biochem. Biophys. Res. Commun. 176, 1298–1304). The effect of photoinhibitory conditions on the distribution of these phosphorylated PSII core populations in thylakoids and PSII particles has been investigated. The sensitivity of the PSII core to strong illumination depended on the phosphorylation state of D₁ and D₂ proteins as well as on the content of the 9-kDa PsbH phosphoprotein. When D₁ and D₂ proteins are under-phosphorylated, the 9-kDa phosphoprotein is tightly bound to the PSII core; thus, a partial protection from photoinhibition is observed. Of the different PSII core populations isolated from membranes photoinhibited for 10 min, the highly phosphorylated populations lack internal antennae CP43 and CP47; perhaps these migrate out to the non-appressed regions of thylakoids. The degradation of the D₁ protein seems to follow the disassembly of the PSII core.     

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.     

Application of low temperatures during photoinhibition allows characterization of individual steps in photodamage and the repair of photosystem II

Recent investigations of photoinhibition have revealed that photodamage to photosystem II (PSII) involves two temporally separated steps: the first is the inactivation of the oxygen-evolving complex by light that has been absorbed by the manganese cluster and the second is the impairment of the photochemical reaction center by light that has been absorbed by chlorophyll. Our studies of photoinhibition in Synechocystis sp. PCC 6803 at various temperatures demonstrated that the first step in photodamage is not completed at low temperatures, such as 10°C. Further investigations suggested that an intermediate state, which is stabilized at low temperatures, might exist at the first stage of photodamage. The repair of PSII involves many steps, including degradation and removal of the D1 protein, synthesis de novo of the precursor to the D1 protein, assembly of the PSII complex, and processing of the precursor to the D1 protein. Detailed analysis of photodamage and repair at various temperatures has demonstrated that, among these steps, only the synthesis of the precursor to D1 appears to proceed at low temperatures. Investigations of photoinhibition at low temperatures have also indicated that prolonged exposure of cyanobacterial cells or plant leaves to strong light diminishes their ability to repair PSII. Such non-repairable photoinhibition is caused by inhibition of the processing of the precursor to the D1 protein after prolonged illumination with strong light at low temperatures.     

Over-production of the D1:2 protein makes Synechococcus cells more tolerant to photoinhibition of photosystem II

Over-expression of the psbAIII gene encoding for the D1 protein (form II; D1:2) of the photosystem II reaction centre in the Synechococcus sp. PCC 7942 was studied using a tac promoter and the lacI ^Q system. Over-expression was induced with 40 g/ml IPTG in the growth medium for either 6 or 12 h at growth irradiance (50 mol photons m^-2 s^-1). This treatment doubled the amount of psbAII/III mRNA and the D1:2 protein in membranes but decreased the amount of psbAI messages and the D1:1 protein. The total amount of both heterodimeric reaction centre proteins, D1 and D2, remained constant under growth light conditions, indicating that the number of PSII centres in the membranes was not affected, only the form of the D1 protein was changed from D1:1 to D1:2 in most centres. When the cells were photoinhibited either at 500 or 1000 mol photons m^-2 s^-1, in the presence or absence of the protein synthesis inhibitor lincomycin, the D1:2 protein remained at a higher level in cells in which over-expression had been induced by IPTG. These cells were also less prone to photoinhibition of PSII. It is suggested that the tolerance of cells to photoinhibition increases when most PSII reaction centres contain the D1:2 protein at the beginning of high irradiance. This tolerance is further strengthened by maintaining psbAIII gene over-expression during the photoinhibitory treatment.     

Photoinhibition of photosystem II in vivo is preceded by down-regulation through light-induced acidification of the lumen: Consequences for the mechanism of photoinhibition in vivo

The mechanism of photoinhibition of photosystem II (PSII) was studied in intact leaf discs of Spinacia oleracea L. and detached leaves of Vigna unguiculata L. The leaf material was exposed to different photon flux densities (PFDs) for 100 min, while non-photochemical (q_N) and photochemical quenching (q_p) of chlorophyll fluorescence were monitored. The ‘energy’ and redox state of PSII were manipulated quite independently of the PFD by application of different temperatures (5–20° C), [CO₂] and [O₂] at different PFDs. A linear or curvilinear relationship between q_p and photoinhibition of PSII was observed. When [CO₂] and [O₂] were both low (30 μl · l^?1 and 2%, respectively), PSII was less susceptible at a given q_p than at ambient or higher [CO₂] and photoinhibition became only substantial when q_p decreased below 0.3. When high levels of energy-dependent quenching (q_E) (between 0.6 and 0.8) were reached, a further increase of the PFD or a further decrease of the metabolic demand for ATP and NADPH led to a shift from q_E to photoinhibitory quenching (q_I). This shift indicated that photoinhibition was preceded by down-regulation through light-induced acidification of the lumen. We propose that photoinhibition took place in the centers down-regulated by q_E. The shift from q_E to q_I occurred concomitant with q_P decreasing to zero. The results clearly show that photoinhibition does not primarily depend on the photon density in the antenna, but that photoinhibition depends on the energy state of the membrane in combination with the redox balance of PSII. The results are discussed with regard to the mechanism of photoinhibition of PSII, considering, in particular, effects of light-induced acidification on the donor side of PSII. Interestingly, cold-acclimation of spinach leaves did not significantly affect the relationship between q_P, q_E and photoinhibition of PSII at low temperature.     

A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II

Inhibition of the activity of photosystem II (PSII) under strong light is referred to as photoinhibition. This phenomenon is due to the imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. Photodamage is initiated by the direct effects of light on the oxygen-evolving complex and, thus, photodamage to PSII is unavoidable. Studies of the effects of oxidative stress on photodamage and subsequent repair have revealed that reactive oxygen species (ROS) act primarily by inhibiting the repair of photodamaged PSII and not by damaging PSII directly. Thus, strong light has two distinct effects on PSII; it damages PSII directly and it inhibits the repair of PSII via production of ROS. Investigations of the ROS-induced inhibition of repair have demonstrated that ROS suppress the synthesis de novo of proteins and, in particular, of the D1 protein, that are required for the repair of PSII. Moreover, a primary target for inhibition by ROS appears to be the elongation step of translation. Inhibition of the repair of PSII by ROS is accelerated by the deceleration of the Calvin cycle that occurs when the availability of CO₂ is limited. In this review, we present a new paradigm for the action of ROS in photoinhibition.     

The carboxyl-terminal processing of precursor D1 protein of the photosystem II reaction center

The D1 protein, a key subunit of photosystem II reaction center, is synthesized as a precursor form with a carboxyl-terminal extension, in oxygenic photosynthetic organisms with some exceptions. This part of the protein is removed by the action of an endopeptidase, and the proteolytic processing is indispensable for the manifestation of oxygen-evolving activity in photosynthesis. The carboxyl-terminus of mature D1 protein, which appears upon the cleavage, has recently been demonstrated to be a ligand for a manganese atom in the Mn₄Ca-cluster, which is responsible for the water oxidation chemistry in photosystem II, based on the isotope-edited Fourier transform infrared spectroscopy and the X-ray crystallography. On the other hand, the structure of a peptidase involved in the cleavage of precursor D1 protein has been resolved at a higher resolution, and the enzyme–substrate interactions have extensively been analyzed both in vivo and in vitro. The present article briefly summarizes the history of research and the present state of our knowledge on the carboxyl-terminal processing of precursor D1 protein in the photosystem II reaction center.     

Core protein phosphorylation facilitates the repair of photodamaged photosystem II at high light

Phosphorylation of photosystem II (PSII) reaction center protein D1 has been hypothesised to function as a signal for the migration of photodamaged PSII core complex from grana membranes to stroma lamellae for concerted degradation and replacement of the photodamaged D1 protein. Here, by using the mutants with impaired capacity (stn8) or complete lack (stn7 stn8) in phosphorylation of PSII core proteins, the role of phosphorylation in PSII photodamage and repair was investigated. We show that the lack of PSII core protein phosphorylation disturbs the disassembly of PSII supercomplexes at high light, which is a prerequisite for efficient migration of damaged PSII complexes from grana to stroma lamellae for repair. This results in accumulation of photodamaged PSII complexes, which in turn results, upon prolonged exposure to high light (HL), in general oxidative damage of photosynthetic proteins in the thylakoid membrane.     

Studies on the mechanism of photosystem II photoinhibition I. A two-step degradation of D1-protein

The role of D1-protein in photoinhibition was examined. Photoinhibition of spinach thylakoids at 20°C caused considerable degradation of D1-protein and a parallel loss of variable fluorescence, Q_B-independent electron flow and Q_B-dependent electron flow. The breakdown of D1-protein as well as the loss of variable fluorescence and Q_B-independent electron flow were largely prevented when thylakoids were photoinhibited at 0°C. The Q_B-dependent electron flow markedly decreased under the same conditions. This inactivation may represent the primary event in photoinhibition and could be the result of some modification at the Q_B-site of D1-protein. Evidence for this comes from fluorescence relaxation kinetics following photoinhibition at 0°C which indicate a partial inactivation of Q_A ^--reoxidation. These results support the idea of D1-protein breakdown during photoinhibition as a two step process consisting of an initial inactivation at the Q_B-site of the protein followed by its degradation. The latter is accompanied by the loss of PS II-reaction centre function.Abbreviations Asc ascorbate - p-BQ 1, 4-benzoquinone - DAD diaminodurene - DPC diphenylcarbazide - DQH₂ duroquinole - Fecy ferricyanide - MV methylviologen - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - SiMo silicomolybdate     

A Psb27 homologue in Arabidopsis thaliana is required for efficient repair of photodamaged photosystem II

Psb27 has been identified as a lumenal protein associated with photosystem II (PSII). To gain insight into the function of Psb27, we isolated a mutant Arabidopsis plant with a loss of psb27 function. The quantity of PSII complexes and electron transfer within PSII remained largely unaffected in the psb27 mutant. Our results also showed that under high-light-illumination, PSII activity and the content of the PSII reaction center protein D1 decreased more significantly in the psb27 mutant than in wild-type (WT) plant. Treatment of leaves with a chloroplast protein synthesis inhibitor resulted in similar light-induced PSII inactivation levels and D1 protein degradation rates in the WT and psb27 mutant plants. Recovery of PSII activity after photoinhibition was delayed in the psb27 mutant, suggesting that Psb27 is required for efficient recovery of the photodamaged PSII complex. Overall, these results demonstrated that Psb27 in Arabidopsis is not essential for oxygenic photosynthesis and PSII formation. Instead, our results provide evidence for the involvement of this lumenal protein in the recovery process of PSII. Hua Chen and Dongyuan Zhang contribute equally to this work.     

Light inactivation of functional photosystem II in leaves of peas grown in moderate light depends on photon exposure

To determine the dependence of in vivo photosystem (PS) II function on photon exposure and to assign the relative importance of some photoprotective strategies of PSII against excess light, the maximal photochemical efficiency of PSII (Fv/Fm) and the content of functional PSII complexes (measured by repetitive flash yield of oxygen evolution) were determined in leaves of pea (Pisum satlvum L.) grown in moderate light. The modulation of PSII functionality in vivo was induced by varying either the duration (from 0 to 3 h) of light treatment (fixed at 1200 or 1800 mol photons · m^-2 · s^-1) or irradiance (from 0 to 3000 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), nigericin (an uncoupler), or dithiothreitol (an inhibitor of the xanthophyll cycle) through the cut petioles of leaves of 22 to 24-day-old plants. We observed a reciprocity of irradiance and duration of illumination for PSII function, demonstrating that inactivation of functional PSII depends on the total number of photons absorbed, not on the rate of photon absorption. The Fv/Fm ratios from photoinhibitory light-treated leaves, with or without inhibitors, declined pseudo-linearly with photon exposure. The number of functional PSII complexes declined multiphasically with increasing photon exposure, in the following decreasing order of inhibitor effect: lincomycin > nigericin > DTT, indicating the central role of D1 protein turnover. While functional PSII and Fv/Fm ratio showed a linear relationship under high photon exposure conditions, in inhibitor-treated leaves the Fv/Fm ratio failed to reveal the loss of up to 25% of the total functional PSII under low photon exposure. The loss of this 25% of less-stable functional PSII was accompanied by a decrease of excitation-energy trapping capacity at the reaction centre of PSII (revealed by the fluorescence parameter, 1/Fo-1/Fm, where Fo and Fm stand for chlorophyll fluorescence when PSII reaction centres are open and closed, respectively), but not by a loss of excitation energy at the antenna (revealed by the fluorescence parameter, 1/Fm). We conclude that (i) PSII is an intrinsic photon counter under photoinhibitory conditions, (ii) PSII functionality is mainly regulated by D1 protein turnover, and to a lesser extent, by events mediated via the transthylakoid pH gradient, and (iii) peas exhibit PSII heterogeneity in terms of functional stability during photon exposure.Abbreviations D1 protein psbA gene product - DTT dithiothreitol - Fo chlorophyll fluorescence corresponding to open PSII reaction centres - Fv, Fm variable and maximum fluorescence after dark incubation, respectively - Fs, Fm steady-state and maximum fluorescence during illumination, respectively - P680 reactioncentre chlorophyll and primary electron donor of PSII - PS photosystem Financial support of this work by Department of Employment, Education and Training/Australian Research Council International Research Fellowships Program (Korea) is gratefully acknowledged.     

Light response of D1 turnover and photosystem II efficiency in the seagrassZostera capricorni

Biochemical and biophysical parameters, including D1-protein turnover, chlorophyll fluorescence, oxygen evolution activity and zeaxanthin formation were measured in the marine seagrassZostera capricorni (Aschers) in response to limiting (100 mol·m^–2·^–1), saturating (350 mol·m^–2·s^–1) or photoinhibitory (1100 mol·m^–2·s^–1) irradiances. Synthesis of D1 was maximal at 350 mol·m^–2·s^–1 which was also the irradiance at which the rate of photosynthetic O₂ evolution was maximal. Degradation of D1 was saturated at 350 mol·m^–2·s^–1. The rate of D1 synthesis at 1100 mol·m^–2·s^–1 was very similar to that at 350 mol·m^–2·s^–1 for the first 90 min but then declined. At limiting or saturating irradiance little change was observed in the ratio of variable to maximal fluorescence (Fv/Fm) measured after dark adaptation of the leaves, while significant photoinhibition occurred at 1100 mol·m^–2·s^–1. The proportion of zeaxanthin in the total xanthophyll pool increased with increasing irradiance, indicative of the presence of a photoprotective xanthophyll cycle in this seagrass. These results are consistent with a high level of regulatory D1 turnover inZostera under non-photoinhibitory irradiance conditions, as has been found previously for terrestrial plants.We would like to thank Professor Peter Böger (Department of Plant Biochemistry, University of Konstanz, Germany) for the kind gift of D1 antibodies. This work was partly supported by a University of Queensland Enabling Grant to CC.