Photoinhibition of photosystem II under environmental stress

Inhibition of the activity of photosystem II (PSII) under strong light is referred to as photoinhibition. This phenomenon is due to an imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. In the “classical” scheme for the mechanism of photoinhibition, strong light induces the production of reactive oxygen species (ROS), which directly inactivate the photochemical reaction center of PSII. By contrast, in a new scheme, we propose that photodamage is initiated by the direct effect of light on the oxygen-evolving complex and that ROS inhibit the repair of photodamaged PSII by suppressing primarily the synthesis of proteins de novo. The activity of PSII is restricted by a variety of environmental stresses. The effects of environmental stress on damage to and repair of PSII can be examined separately and it appears that environmental stresses, with the exception of strong light, act primarily by inhibiting the repair of PSII. Studies have demonstrated that repair-inhibitory stresses include CO₂ limitation, moderate heat, high concentrations of NaCl, and low temperature, each of which suppresses the synthesis of proteins de novo, which is required for the repair of PSII. We postulate that most types of environmental stress inhibit the fixation of CO₂ with the resultant generation of ROS, which, in turn, inhibit protein synthesis.     

Analysis of the slow phases of the in vivo chlorophyll fluorescence induction curve. Changes in the redox state of Photosystem II electron acceptors and fluorescence emission from Photosystems I and II

An analysis of the photo-induced decline in the in vivo chlorophyll a fluorescence emission (Kautsky phenomenon) from the bean leaf is presented. The redox state of PS II electron acceptors and the fluorescence emission from PS I and PS II were monitored during quenching of fluorescence from the maximum level at P to the steady state level at T. Simultaneous measurement of the kinetics of fluorescence emission associated with PS I and PS II indicated that the ratio of PS I/PS II emission changed in an antiparallel fashion to PS II emission throughout the induction curve. Estimation of the redox state of PS II electron acceptors at given points during P to T quenching was made by exposing the leaf to additional excitation irradiation and determining the amount of variable PS II fluorescence generated. An inverse relationship was found between the proportion of PS II electron acceptors in the oxidised state and PS II fluorescence emission. The interrelationships between the redox state of PS II electron acceptors and fluorescence emission from PS I and PS II remained similar when the shape of the induction curve from P to T was modified by increasing the excitation photon flux density. The contributions of photochemical and non-photochemical quenching to the in vivo fluorescence decline from P to T are discussed.     

Cooperation of photosystems II and I in leaves as analyzed by simultaneous measurements of chlorophyll fluorescence and transmittance at 800 nm

Parallel measurements of CO2 assimilation, Chl fluorescence and 800 nm transmittance were carried out on intact leaves of wild type and cytochrome b6/f deficient transgenic tobacco grown at two different light intensities and temperatures, with the aim to diagnose processes limiting quantum yield of photosynthesis and investigate their adaptations to growth conditions. Relative optical cross-sections of PSII and PSI antennae were calculated from measured gas exchange rates and fluorescence-related losses at PSII and P700 oxidation-related losses at PSI. In nonstress conditions (high light grown wild type and low light grown antisense type) optimal relative optical cross-section of PSII (aII) was 0.48-0.51 and that of PSI (aI) was 0.38-0.40, leaving a non-photosynthetic absorption cross-section (a0) of 0.09-0.14 for nitrite assimilation and absorption in PSII beta and other photosynthetically inactive pigments. Stress conditions (low light grown wild type and high light grown antisense type, elevated growth temperatures) tend to increase a0 and decrease PSII antenna cross-section more than that of PSI antenna, but this rule is reversed during senescence.     

The fluorescence decay kinetics of in vivo chlorophyll measured using low intensity excitation

We report fluorescence lifetimes for in vivo chlorophyll a using a time-correlated single-photon counting technique with tunable dye laser excitation. The fluorescence decay of dark-adapted chlorella is almost exponential with a lifetime of 490 ps, which is independent of excitation from 570 nm to 640 nm.Chloroplasts show a two-component decay of 410 ps and approximately 1.4 ns, the proportion of long component depending upon the fluorescence state of the chloroplasts. The fluorescence lifetime of Photosystem I was determined to be 110 ps from measurements on fragments enriched in Photosystem I prepared from chloroplasts with digitonin.     

Determination of the PS I content of PS II core preparations using selective emission: A new emission of PS II at 780 nm

Routinely prepared PS II core samples are often contaminated by a significant (~ 1–5%) fraction of PS I, as well as related proteins. This contamination is of little importance in many experiments, but masks the optical behaviour of the deep red state in PS II, which absorbs in the same spectral range (700–730 nm) as PS I (Hughes et al. 2006). When contamination levels are less than ~ 1%, it becomes difficult to quantify the PS I related components by gel-based, chromatographic, circular dichroism or EPR techniques. We have developed a fluorescence-based technique, taking advantage of the distinctively different low-temperature emission characteristics of PS II and PS I when excited near 700 nm. The approach has the advantage of providing the relative concentration of the two photosystems in a single spectral measurement. A sensitivity limit of 0.01% PS I (or better) can be achieved. The procedure is applied to PS II core preparations from spinach and Thermosynechococcus vulcanus. Measurements made of T. vulcanus PS II preparations prepared by re-dissolving crystallised material indicate a low but measurable PS I related content. The analysis provides strong evidence for a previously unreported fluorescence of PS II cores peaking near 780 nm. The excitation dependence of this emission as well as its appearance in both low PS I cyanobacterial and plant based PS II core preparations suggests its association with the deep red state of PS II.     

Role of chloroplast photosystems II and I in apoptosis of pea guard cells

We investigated the CN^–-induced apoptosis of guard cells in epidermal peels isolated from pea (Pisum sativum L.) leaves. This process was considerably stimulated by illumination and suppressed by the herbicides DCMU (an inhibitor of the electron transfer between quinones Q_A and Q_B in PS II) and methyl viologen (an electron acceptor from PS I). These data favor the conclusion drawn by us earlier that chloroplasts are involved in the apoptosis of guard cells. Pea mutants with impaired PS I (Chl-5), PS II (Chl-I), and PS II + PS I (Xa-17) were tested. Their lesions were confirmed by the ESR spectra of Signal I (oxidized PS I reaction centers) and Signal II (oxidized tyrosine residue Y_D in PS II). Destruction of nuclei (a symptom of apoptosis) and their consecutive disappearance in guard cells were brought about by CN^– in all the three mutants and in the normal pea plants. These results indicate that the light-induced enhancement of apoptosis of guard cells and its removal by DCMU are associated with PS II function. The effect of methyl viologen preventing CN^–-induced apoptosis in wild-type plants was removed or considerably decreased upon the impairment of the PS II and/or PS I activity.     

Light-induced quenching of chlorophyll fluorescence at 77 K in leaves,chloroplasts and Photosystem II particles

The light-induced chlorophyll (Chl) fluorescence decline at 77 K was investigated in segments of leaves, isolated thylakoids or Photosystem (PS) II particles. The intensity of chlorophyll fluorescence declines by about 40% upon 16 min of irradiation with 1000 μmol m⁻² s⁻¹ of white light. The decline follows biphasic kinetics, which can be fitted by two exponentials with amplitudes of approximately 20 and 22% and decay times of 0.42 and 4.6 min, respectively. The decline is stable at 77 K, however, it is reversed by warming of samples up to 270 K. This proves that the decline is caused by quenching of fluorescence and not by pigment photodegradation. The quantum yield for the induction of the fluorescence decline is by four to five orders lower than the quantum yield of Q_A reduction. Fluorescence quenching is only slightly affected by addition of ferricyanide or dithionite which are known to prevent or stimulate the light-induced accumulation of reduced pheophytin (Pheo). The normalised spectrum of the fluorescence quenching has two maxima at 685 and 695 nm for PS II emission and a plateau for PS I emission showing that the major quenching occurs within PS II. ‘Light-minus-dark’ difference absorbance spectra in the blue spectral region show an electrochromic shift for all samples. No absorbance change indicating Chl oxidation or Pheo reduction is observed in the blue (410–600 nm) and near infrared (730–900 nm) spectral regions. Absorbance change in the red spectral region shows a broad-band decrease at approximately 680 nm for thylakoids or two narrow bands at 677 and 670–672 nm for PS II particles, likely resulting also from electrochromism. These absorbance changes follow the slow component of the fluorescence decline. No absorbance changes corresponding to the fast component are found between 410 and 900 nm. This proves that the two components of the fluorescence decline reflect the formation of two different quenchers. The slow component of the light-induced fluorescence decline at 77 K is related to charge accumulation on a non-pigment molecule of the PS II complex. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photoinhibition of Photosystem I electron transfer activity in isolated Photosystem I preparations with different chlorophyll contents

Photoinhibition of the light-induced Photosystem I (PS I) electron transfer activity from the reduced dichlorophenol indophenol to methyl viologen was studied. PS I preparations with Chl/P700 ratios of about 180 (PS I-180), 100 (PS I-100) and 40 (PS I(HA)-40) were isolated from spinach thylakoid membranes by the treatments with Triton X-100, followed by sucrose density gradient centrifugation and hydroxylapatite column chromatography. White light irradiation (1.1 × 10⁴E m^–2 s^–1) of PS I-180 for 2 hours bleached 50% of the chlorophyll and caused a 58% decrease in the electron transfer activity with virtually no loss of the primary donor, P700. The flash-induced absorbance change showed the decay phase with a half time of about 10 s that was attributed to the P700 triplet, suggesting that the photoinhibitory light treatment caused the destruction of the PS I acceptor(s), F_x and possibly A₁. PS I-100 was similarly photobleached by the irradiation and the electron transfer activity decreased. There was, however, no apparent photoinhibition of the electron transport activity in PS I(HA)-40. Photoinhibition similar to that seen in PS I-180 also occurred in membrane fragments that were isolated without any detergent from a PS II-deficient mutant strain of the cyanobacterium Synechocystis sp. PCC 6803. PS I-180 was not photoinhibited under anaerobic conditions. The production of superoxide and fatty acid hydroperoxide during white light irradiation was significantly greater in PS I-180 than in PS I(HA)-40. The mechanism of photoinhibition in PS I preparations is discussed in relation to the formation of toxic oxygen molecules.Abbreviations A₀,A₁ primary and secondary electron acceptors of PS I - CD circular dichroism - DCPIP 2,6-dichlorophenol indophenol - F_A, F_B, F_X iron-sulfur centers A, B, X - HA hydroxylapatite - LHCI lightharvesting complex of PS I - MDA malondialdehyde - MV methyl viologen - Na-Asc sodium L-ascorbate - P700 primary electron donor of PS I - PFD photon flux density - PS I-A and PS I-B psaA and psaB gene products - TBA thiobarbituric acid     

Photoinhibition in cork-oak leaves under stress: influence of the bark-stripping on the chlorophyll fluorescence emission inQuercus suber L.

Quercus suber is the primary source for industrial cork and becomes bark-stripped every 9–10 years. Recurring cork extraction is a major stress factor and the large water loss from the stripped trunk surface may affect the water balance and tree productivity. To evaluate the effect of bark-stripping, fluorescence emission and stomatal conductance of leaves were determined in groups of bark-stripped and control trees. Fv/Fm ratio was found to be significantly lower in bark-stripped trees indicating a reduced photosynthetic efficiency of PSII. Photosynthesis was not found to be stomata limited. The reduction in Fv/Fm resulted from a decline in maximum and variable fluorescence while the initial fluorescence of the dark-adapted state (Fo) remained constant. A general decline in photosynthetic efficiency of PSII was found in all trees during the summer, probably reflecting the prolonged environmental stresses during a hot and dry season. Additional stress caused by the bark-stripping seems to enhance the susceptibility to photoinhibition of the trees.     

Photoinhibition, 77K chlorophyll fluorescence quenching and phosphorylation of the light-harvesting chlorophyll-protein complex of photosystem II in soybean leaves

When the capacity of leaves for orderly dissipation of excitation energy in photosynthesis is exceeded, one mechanism by which the excess energy appears to be dissipated is through a nonradiative decay process. This process is observed as a reversible quenching of chlorophyll fluorescence emission (77K) from both photosystem II and photosystem I which persists in darkness (Demmig and Björkman 1987, Planta 171, 171–184). Fluorescence quenching was induced in soybean (Glycine max (L.) Merr.) leaves by two methods: 1) changing the composition of the gas surrounding the leaf from normal air to 2% O₂, 0% CO₂ at a low, constant photon flux density (PFD=photon fluence rate), and 2) increasing the PFD in the presence of normal air. In either case the quenching was fully reversible after return to the original condition (low PFD, normal air). The half-time of the relaxation of the quenching was in the order of 30 min. Both treatments resulted in reversible dephosphorylation of the light-harvesting chlorophyll-protein complex of photosystem II (LHC-II). Treatment under photoinhibitory conditions (high PFD plus chloramphenicol) also caused dephosphorylation of LHC-II. Therefore, phosphorylation of LHC-II cannot account for the observed fluorescence quenching. In addition, our results indicate that in vivo a factor other than the redox state of the plastoquinone pool controls LHC-II phosphorylation. This factor may be pH, the pH gradient across the thylakoid membranes.Abbreviations and symbols CAP chloramphenicol - F_o, F_M, F_v instantaneous, maximumr variable fluorescence emission - LHC-II light-haryesting chlorophyll-protein complex of PSII - kDa kilodalton - pH pH gradient across the thylakoid membrane - PFD photon flux density (photon fluence rate) - PQ plastoquinone - PSI, PSII photosystem I, II - Q acceptor of PSII C.I.W.-D.P.B. Publication No. 926     

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.     

Estimation of the light distribution between photosystems I and II in intact wheat leaves by fluorescence and photoacoustic measurements

Usisng intact leaves, the extent of the decrease in chlorophyll a fluorescenece caused by the addition of continuous 710 nm light superimposed on modulated (20 Hz) 550 nm light was used to determine the distribution of this absorbed light between photosystems I () and II (). The F_o and F_m levels, which defined the total variable fluorescenece, were taken as equal to those obtained with excess 710 nm light and with saturating blue-green light, respectively.An analogous procedure was used with a photoacoustic detector, saturating white light defining a base line for oxygen yield, the levels with an without 710 nm light being used to define and respectively.The two methods gave similar values for the distribution of light between the two photosystems for the experimental conditions used, averaging 0.55 for a range of Triticum genotypes and Brachypodium sylvaticum grown in high or low light.     

Effects of enhanced solar irradiation on chlorophyll fluorescence and photosynthetic oxygen production of five species of phytoplankton

Abstract The effects of solar irradiation on chlorophyll a fluorescence and photosynthetic oxygen production of three Cryptomonas species, Euglena gracilis and Scenedesmus cf. quadricauda were investigated in comparative field studies in Erlangen (280 m above sea level) and at Zugspitze (2957 m above sea level). The experiments showed that the decrease of fluorescence and the inhibition of photosynthetic oxygen production occurred after shorter times of exposure to solar radiation at Zugspitze compared to Erlangen in all tested organisms. Cryptomonas maculata was more sensitive towards radiation than the other organisms: fluorescence decreased earlier, while Scenedesmus cf. quadricauda seemed to be much less sensitive since fluorescence and photosynthetic oxygen production decreased later and to a smaller extent compared to the other organisms. The results of the present study indicate that increased solar radiation (with an increased level of UV-B radiation) at higher geographical altitudes may have significant effects on phytoplankton populations.     

Cyanobacterial ycf27 gene products regulate energy transfer from phycobilisomes to photosystems I and II

Two open reading frames (slr0115 and slr0947) in the genome of the cyanobacterium Synechocystis sp. PCC 6803 are shown to be involved in the regulation of the coupling of phycobilisomes to photosynthetic reaction centres. Homologues of these genes, called ycf27, have been found in a range of phycobilin-containing organisms. The slr0115 and slr0947 gene products are OmpR-type DNA-binding response regulator proteins. Deletion of slr0115 results in increased efficiency of energy transfer from phycobilisomes to photosystem II relative to photosystem I. Reduction of the copy number of slr0947 has the opposite phenotypic effect. We have given the slr0115 and slr0947 genes the designations rpaA and rpaB respectively.     

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

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

A quantitative determination of photochemical and non-photochemical quenching during the slow phase of the chlorophyll fluorescence induction curve of bean leaves

Estimations of the changes in the reduction-oxidation state of Photosystem II electron acceptors in Phaseolus vulgaris leaves were made during the slow decline in chlorophyll fluorescence emission from the maximal level at P to the steady-state level at T. The relative contributions of photochemical and non-photochemical processes to the fluorescence quenching were determined from these data. At a low photon flux density of 100 μmol · m^?2 · s^?1, non-photochemical quenching was the major contributor to the fluorescence decline from P to T, although large charges were observed in photochemical quenching immediately after P. On increasing the light intensity 10-fold, the contribution of photochemical processes to fluorescence quenching was markedly diminished, with nearly all the P-to-T fluorescence decline being attributable to changes in non-photochemical quenching. The possible factors responsible for changes in non-photochemical quenching within the leaves are discussed.     

Two temperature-sensitive photosystem II mutants of pea

Two nuclear gene mutants of pea, chlorotica-887 and chlorina-5756, are temperature-sensitive in the development of photosystem II activity. Low temperature flourescence emission spectra of leaves show that the peak at 697 nm from the reaction center of photosystem II is present when the mutants have been grown at 18°C, but absent when they have been grown at 30°C. For leaves of chlorina-5756 grown at 18°C the relative size of the peak at 697 nm is reduced compared to that of leaves of the wild type or chlorotica-887 grown at this temperature. Flourescence induction curves of leaves from wild type plants and chlorotica-887 grown at 18°C possess two steps, while those of leaves from chlorina-5756 grown at 18°C or 30°C and chlorotica-887 grown at 30°C show at fast rise to the maximal level of fluorescence. Measurements on chloroplasts isolated from the mutants indicated that the photosystem I activity per g leaf material is comparable for plants grown at 18°C and plants grown at 30°C. In contrast, no photosystem II activity was detected when the mutants had been grown at 30°C. It is suggested that these mutants are affected in a component required for the assembly of functional photosystem II complexes.     

Photoinhibition and its wavelength dependence in the cyanobacterium Anabaena variabilis

In the cyanobacterium Anabaena variabilis the dependence of photoinhibition on fluence rate, duration and wavelength of irradiation were studied by measurements of oxygen production and fluorescence emission spectra. The analysis of the photosynthetic activity revealed that photoinhibition affects exclusively photosystem II (PS II), whereas photosystem I (PS I) remained largely unimpaired. Furthermore, PS II fluorescence emission decreased much faster in bleached than in unbleached controls.Studying the wavelength dependence of photoinhibition it was found that only radiation between 520 and 680 nm causes photoinhibition. This is about the same range of wavelengths which causes photobleaching. Fluorescence emission spectra of samples exposed to high fluence rates of 582 and 662 nm, respectively, essentially agree with those samples exposed to high fluence rates of white light, whereas the fluorescence emission spectra of samples exposed to blue light resemble those exposed to dim white light.NaN₃, a substance which prevents photobleaching, inhibits the photosynthetic O₂ production of Anabaena and, hence, enhances the photoinhibitory effect.