Photoinhibitory Treatment Research Articles

Photoinhibition of Photosynthesis and its Recovery in Red Algae

AbstractIn different marine red algae (Chondrus crispus, Delesseria sanguinea, Membranoptera alata, Phycodrys rubens, Phyllophora truncata, Polyneura hilliae) photoinhibition of photosynthesis has been investigated by means of both fluorescence and oxygen measurements. Measurements of absolute oxygen production show that photoinhibition causes a decline in the initial slope and in the rate of bending of the fluence rate‐response curve (i.e. the photosynthetic efficiency at non‐saturating fluence rates), as well as a decline in the photosynthetic capacity (Pm) at saturating fluence rates. Fluorescence data (Fv/Fm) were consistent with the results of oxygen measurements. Under excessive light photoinhibition protects photosynthesis against photo‐damage in red algae. However, an increase in the initial fluorescence (Fo) after photoinhibitory treatment indicates that it could not prevent photodamage entirely.Action spectra of photoinhibition demonstrate that the main photoinhibition site in Polyneura hiliae is PS II, because far red light absorbed by PS I was ineffective. The strong increase of Fo in the blue wavelength range and the slight and partial recovery in weak blue light indicate that blue light especially causes photodamage.Recovery of photosynthesis requires dim white light conditions. Experiments with monochromatic light also show a wavelength dependence of recovery. Moreover, the recovery of photosynthesis after a photoinhibitory treatment is strongly temperature dependent, indicating participation of enzymatic processes. The comparison of fluorescence and oxygen measurement of the recovery shows different results in some species. The rate of oxygen production in red control light increased immediately after photoinhibited algae were exposed to weak light conditions. Surprisingly, the ratio of variable to maximum fluorescence (Fv/Fm) of Phyllophora truncata and the maximum fluorescence (Fm) of Polyneura hilliae show first a delay of the recovery under weak light conditions. Thus, in recovery experiments fluorescence and oxygen data are not quite consistent.

Recovery of photosynthesis and phtosystem II fluorescence in Chlamydomonas reinhardtii after exposure to three levels of high light

Recovery from 60 min of photoinhibitory treatment at photosynthetic photon flux densities of 500, 1400 and 2200 μMmol m−2 s− was followed in cells of the green alga Chlamydomonas reinhardtii grown at 125 μMmol m−2 s−1. These light treatments represent photoregulation, moderate photoinhibition and strong photoinhibition, respectively. Treatment in photoregulatory light resulted in an increased maximal rate of oxygen evolution (Pmax) and an increased quantum yield (Φ), but a 15% decrease in Fv/FM. Treatment at moderately photoinhibitory light resulted in a 30% decrease in Fv/FM and an approximately equal decrease in Φ. Recovery in dim light restored Fv/FM within 15 and 45 min after high light treatment at 500 and 1400 μMmol m−2 s−1, respectively. Convexity (Θ), a measure of the extent of co‐limitation between PS II turnover and whole‐chain electron transport, and Φ approached, but did not reach the control level during recovery after exposure to 1400 μMmol m−2 s−1, whereas Pmax increased above the control. Treatment at 2200 μMmol m−2 s−1 resulted in a strong reduction of the modeled parameters Φ, Θ and Pmax. Subsequent recovery was initially rapid but the rate decreased, and a complete recovery was not reached within 120 min. Based on the results, it is hypothesized that exposure to high light results in two phenomena. The first, expressed at all three light intensities, involves redistribution within the different aspects of PS II heterogeneity rather than a photoinhibitory destruction of PS II reaction centers. The second, most strongly expressed at 2200 μmol m−2 s−1, is a physical damage to PS II shown as an almost total loss of PS IIα and PS II QB‐reducing centers. Thus recovery displayed two phase, the first was rapid and the only visible phase in algae exposed to 500 and 1400 μmol m−2 s−1. The second phase was slow and visible only in the later part of recovery in cells exposed to 2200 μmol m−2 s−1.

Two sites of primary degradation of the D1-protein induced by acceptor or donor side photo-inhibition in photosystem II core complexes

Depending on experimental conditions we have found that photo-inhibitory treatment of photosystem II (PSII) core complexes, isolated from wheat, can generate two fragments of about 23–24 kDa that contain either the C-terminal or N-terminal regions of the D1-protein. A 24 kDa C-terminal fragment appears when the water splitting reaction is not functional and an electron acceptor is present. This ‘donor’-side inhibition also generates an N-terminal fragment of about 10 kDa and is suggested to be due to the cleavage of a peptide bond in the region connecting transmembrane segments I and II of the D1-protein. In contrast, an N-terminal 23 kDa D1-protein fragment is detected when the water splitting reactions of the isolated complex are active, and occurs in the absence of an added electron acceptor. This ‘acceptor’-side photo-inhibition also generates a C-terminal fragment of about 10 kDa.

Effect of High-Irradiance Stress on Primary Photochemistry and Light Regulated Enzymes of Photosynthetic Carbon Metabolism

The photosynthetic electron transport, chlorophyll fluorescence and light regulated CO 2 reduction cycle enzymes of chloroplasts isolated from photoinhibited Sorghum bicolor L. (R 109A) were determined. The leaves were exposed to a range of irradiance (850-2500 µtmol m -2 s -1 ) at 5 °C and 20 °C. The photosynthetic electron transport and chlorophyll fluorescence (F v /Fm ratio) decreased with increasing irradiance. This decrease was more at 5 °C than at 20 °C. Exposure of seedlings to higher irradiance caused damage to the oxidizing side of PS II while PS I remained comparatively unaffected. The activities of light-regulated CO 2 reduction enzymes, NADP-malate dehydrogenase, PEP-carboxylase and phosphoribulokinase in leaves increased upon exposure to higher irradiance. Increase in the activities of enzymes was temperature dependent. Extending the photoinhibitory treatment for longer than 2 hours showed little further increase in the enzyme activities. The results show that photoinhibition of leaves affects the photosynthetic electron transport of their chloroplasts with primary damage to the oxidizing side of PS II, and also effects the CO 2 fixation properties of the chloroplasts.

Effects of photoinhibition on the PS II acceptor side including the endogenous high spin Fe2+ in thylakoids, PS II-membrane fragments and PS II core complexes

Effects of photoinhibition at 0 °C on the PS II acceptor side have been analyzed by comparative studies in isolated thylakoids, PS II membrane fragments and PS II core complexes from spinach under conditions where degradation of polypeptide(s) D1(D2) is highly retarded. The following results were obtained by measurements of the transient fluorescence quantum and oxygen yield, respectively, induced by a train of short flashes in dark-adapted samples: (a) in the control the decay of the fluorescence quantum yield is very rapid after the first flash, if the dark incubation was performed in the presence of 300 μM K3[Fe(CN)6]; whereas, a characteristic binary oscillation was observed in the presence of 100 μM phenyl-p-benzoquinone with a very fast relaxation after the even flashes (2nd, 4th. . . ) of the sequence; (b) illumination of the samples in the presence of K3[Fe(CN)6] for only 5 min with white light (180 W m(-2)) largely eliminates the very fast fluorescence decay after the first flash due to QA (-) reoxidation by preoxidized endogenous non-heme Fe(3+), while a smaller effect arises on the relaxation kinetics of the fluorescence transients induced by the subsequent flashes; (c) the extent of the normalized variable fluorescence due to the second (and subsequent) flash(es) declines in all sample types with a biphasic time dependence at longer illumination. The decay times of the fast (6-9 min) and the slow degradation component (60-75 min) are practically independent of the absence or presence of K3[Fe(CN)6] and of anaerobic and aerobic conditions during the photo-inhibitory treatment, while the relative extent of the fast decay component is higher under anaerobic conditions. (d) The relaxation kinetics of the variable fluorescence induced by the second (and subsequent) flash(es) become retarded due to photoinhibition, and (e) the oscillation pattern of the oxygen yield caused by a flash train is not drastically changed due to photoinhibition.Based on these findings, it is concluded that photoinhibition modifies the reaction pattern of the PS II acceptor side prior to protein degradation. The endogenous high spin Fe(2+) located between QA and QB is shown to become highly susceptible to modification by photoinhibition in the presence of K3[Fe(CN)6] (and other exogenous acceptors), while the rate constant of QA (-) reoxidation by QB(QB (-)) and other acceptors (except the special reaction via Fe(3+)) is markedly less affected by a short photoinhibition. The equilibrium constant between QA (-) and QB(QB (-)) is not drastically changed as reflected by the damping parameters of the oscillation pattern of oxygen evolution.

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 μmol · m(-2) · s(-1), although the peak PFDs exceeded 2100 μmol · 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 μmol · m(-2) · s(-1) and the ability to recover at 25° C and 20 μmol · 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, Fo, and the maximum fluorescence, Fm, 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, Fv, 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.

The effects of low temperature acclimation and photoinhibitory treatments on Photosystem 2 studied by thermoluminescence and fluorescence decay kinetics

The effects of low temperature acclimation and photoinhibitory treatment on Photosystem 2 (PS 2) have been studied by thermoluminescence and chlorophyll fluorescence decay kinetics after a single turnover saturating flash. A comparison of unhardened and hardened leaves showed that, in the hardened case, a decrease in overall and B-band thermoluminescence emissions occurred, indicating the presence of fewer active PS 2 reaction centers. A modification in the form of the B-band emission was also observed and is attributed to a decrease in the apparent activation energy of recombination in the hardened leaves. The acclimated leaves also produced slower QA (-) reoxidation kinetics as judged from the chlorophyll fluorescence decay kinetics. This change was mainly seen in an increased lifetime of the slow reoxidation component with only a small increase in its amplitude. Similar changes in both thermoluminescence and fluorescence decay kinetics were observed when unhardened leaves were given a high light photoinhibitory treatment at 4°C, whereas the hardened leaves were affected to a much lesser extent by a similar treatment. These results suggest that the acclimated plants undergo photoinhibition at 4°C even at low light intensities and that a subsequent high light treatment produces only a small additive photoinhibitory effect. Furthermore, it can be seen that photoinhibition eventually gives rise to PS 2 reaction centers which are no longer functional and which do not produce thermoluminescence or variable chlorophyll fluorescence.

Light as a hazard for the desiccation‐resistant ‘resurrection’ fern Polypodium polypodioides

Abstract. Using fluorescence measurements at 77 K, the susceptibility to photoinhibition was determined for variously dehydrated leaves of the desiccation–resistant fern Polypodium polypodioides. As expected, water loss increased the potential danger of light‐induced damage. However, the long‐term consequences of that damage differed depending on the hydration state of the leaves during the light treatment, suggesting that different targets were affected. This became evident when the rehydrated leaves were subjected to conditions conducive to recovery from typical photoinhibition. After an exposure to high photon flux densities, full restoration of normal photochemical efficiencies occurred only when the photoinhibitory treatment had occurred while the leaves were fully hydrated, or when the dehydration had reached a stage at which only the primary charge separation and storage were operative. On the other hand, lesions caused by high light intensities in slightly dehydrated leaves which had retained some electron transport activity between the two photosystems, or in desiccated leaves incapable of any measureable photosynthetic functions, were very inefficiently or not at all repaired. One unexpected hazard of high light intensities for dehydrated leaves turned out to be further desiccation which could reach a threshold at which extensive cellular damage began to ensue. In nature, P. polypodioides avoids light–induced damage by rolling its leaves during dehydration so that only the reflectant abaxial leaf surface is exposed to ambient light, and by occupying shaded habitats.

INVOLVEMENT OF THYLAKOID MEMBRANE‐DEPENDENT PHOTOSENSITIZATION IN PHOTOINHIBITION OF THE CALVIN CYCLE ACTIVITY IN SPINACH CHLOROPLASTS

AbstractPhotoinhibition of the light‐regulated key enzymes of the photosynthetic carbon reduction (PCR) cycle was investigated using chloroplasts isolated from spinach leaves. Light quality dependence of the light‐induced activity change (activation or inactivation) of key PCR enzymes in situ demonstrated that, while light activation is promoted mainly by red light (Λ.> 600 nm), inactivation takes place largely in the region of blue light (Λ < 500 nm). Inactivation was suppressed by a lipid soluble singlet oxygen (1O2,1Δg) quencher. When “stromal protein” was subjected to a severe photoinhibitory treatment, no significant loss of activity was observed for any PCR enzyme assayed. However, the inclusion of thylakoids in the photolysis system resulted in a substantial inactivation of the enzymes; this inactivation was significantly diminished in the presence of imidazole and enhanced to some extent by a partial deuteration of medium. In contrast, superoxide dismutase did not exert any effect. The blue light‐induced inactivation of the enzymes was remarkably decreased in the presence of thylakoids whose Fe‐S centers were destroyed. The results obtained in this study suggest that photoinactivation of the PCR enzymes in situ is mediated mainly by 1O2, which is photoproduced primarily by the Fe‐S centers of thylakoids and diffuses into the stroma.

Resistance to Low Temperature Photoinhibition Is Not Associated with Isolated Thylakoid Membranes of Winter Rye

In vivo measurements of chlorophyll a fluorescence indicate that cold-hardened winter rye (Secale cereale L. cv Musketeer) develops a resistance to low temperature-induced photoinhibition compared with nonhardened rye. After 7.2 hours at 5 degrees C and 1550 micromoles per square meter per second, the ratio of variable fluorescence/maximum fluorescence was depressed by only 23% in cold-hardened rye compared with 46% in nonhardened rye. We have tested the hypothesis that the principal site of this resistance to photoinhibition resides at the level of rye thylakoid membranes. Thylakoids were isolated from cold-hardened and nonhardened rye and exposed to high irradiance (1000-2600 micromoles per square meter per second) at either 5 or 20 degrees C. The photoinhibitory response measured by room temperature fluorescence induction, photosystem II electron transport, photoacoustic spectroscopy, or [(14)C]atrazine binding indicates that the differential resistance to low temperature-induced photoinhibition in vivo is not observed in isolated thylakoids. Similar results were obtained whether isolated rye thylakoids were photoinhibited or thylakoids were isolated from rye leaves preexposed to a photoinhibitory treatment. Thus, we conclude that increased resistance to low temperature-induced photoinhibition is not a property of thylakoid membranes but is associated with a higher level of cellular organization.

Interaction of Salt Stress and Photoinhibition on Photosynthesis in Barley and Sorghum

Summary The effect of salinity (short term), high light and interaction of the two was investigated in leaves and isolated chloroplasts from these leaves of barley (C3) and sorghum (C4) plants. The light saturated rate of CO 2 uptake and maximum quantum yield decreased with increasing salt concentrations. Stomatal conductance was more sensitive to salt stress than photoinhibition. Photoinhibition treatment of salt stressed plants resulted in a further decrease of the CO 2 assimilation rate and quantum efficiency of photosynthesis. The recovery of the CO 2 assimilation rate was considerably lower in salt and high light stressed plants compared to plants which were given a photoinhibition treatment only (no salt). It is concluded that salt stress itself does not exert adverse effects on the primary photosynthesis process since the F v /F m ratio and electron transport activity did not decrease with higher salt concentrations. However, salinity stress enhanced substantially the susceptibility to photoinhibition; this may be due to the predisposing of the photosynthetic apparatus to damage under salt stress, resulting in lower light saturated rates of photosynthesis a decreased ability to recover from photodamage.

Light acclimation potential and xanthophyll cycle pigments in photoautotrophic suspension cells of Chenopodium rubrum

The present study investigates the light acclimation potential of photoautotrophic suspension culture cells of Chenopodium rubrum L. grown in 16 h light/8 h dark cycles. Typical features of sun/shade acclimation could be demonstrated in cultures grown at photon flux densities of 30 and 150 μmol m−2 s−1. Low light grown cells had lower chlorophyll a/b ratios, lower respiration rates and lower light compensation points than high light grown cells. Maximum photosynthetic rate per cell dry weight was highest in low light conditions, indicating that the cells did not enlarge their photosynthetic machinery upon exposure to high light. Transfer of cultures to 800 μmol m−2 s−1 caused photoinhibition as indicated by a decrease in photosynthetic efficiency and by the occurrence of a slowly reversible quenching of variable chlorophyll fluorescence. Extension of the photoinhibitory treatment over six light dark cycles did not result in further dramatic changes of these parameters, whereas the chlorophyll content per dry weight and the chlorophyll a/b ratio decreased. Measurements of photochemical quenching showed that the capability of the cells to dissipate excessive energy had increased during the acclimation process. The presence of the xanthophyll cycle pigments and the operation of the cycle could be demonstrated. In agreement with the putative photoprotective function of antheraxanthin and zeaxanthin these pigments could only be detected under photoinhibitory conditions. Prolonged photoinhibitory treatment resulted in increases in the xanthophyll pigment concentration but not of the potential to deepoxidate violaxanthin. The limited potential of the cells to accumulate zeaxanthin and antheraxanthin might indicate that the xanthophyll cycle is not the main factor determining their resistance to high light stress.

Temperature-dependent changes in Photosystem II heterogeneity support a cycle of Photosystem II during photoinhibition

High light treatments were given to attached leaves of pumpkin (Cucurbita pepo L.) at room temperature and at 1°C where the diffusion- and enzyme-dependent repair processes of Photosystem II are at a minimum. After treatments, electron transfer activities and fluorescence induction were measured from thylakoids isolated from the treated leaves. When the photoinhibition treatment was given at 1°C, the Photosystem II electron transfer assays that were designed to require electron transfer to the plastoquinone pool showed greater inhibition than electron transfer from H2O to paraphenyl-benzoquinone, which measures all PS II centers. When the light treatment was given at room temperature, electron transfer from H2O to paraphenyl-benzoquinone was inhibited more than whole-chain electron transfer. Variable fluorescence measured in the presence of ferricyanide decreased only during room-temperature treatments. These results suggest that reaction centers of one pool of Photosystem II, non-QB-PS II, replace photoinhibited reaction centers at room temperature, while no replacement occurs at 1°C. A simulation of photoinhibition at 1°C supports this conclusion.

Protection from photoinhibition by low temperature in Synechocystis 6714 and in Chlamydomonas reinhardtii: detection of an intermediary state

Photoinhibition was induced in a cyanobacterium strain, Synechocystis 6714, and a green alga, Chlamydomonas reinhardtii, by exposing them to light intensities from 1000 to 4000 microE/(m2.s) at various temperatures. The photoinhibition process was followed by measurements of chlorophyll fluorescence and oxygen evolution. During exposure to high light, fluorescent active reaction centers II became low fluorescent inactive centers. This process involved several reversible and irreversible steps. The pathway from the active state to the inactive low fluorescent state was different in Synechocystis and Chlamydomonas. In the latter there was a reversible intermediary step characterized by an increase of F0. This state was stable at 5 degrees C and slowly reversible at room temperature. The high F0 fluorescence level corresponded to a state of photosystem II centers that were inactive for oxygen evolution. An F0 decrease occurred in the dark in the absence of protein synthesis and was correlated to a restoration of oxygen evolution. Further experiments suggested that the existence of the intermediate fluorescent state is due to modified closed centers in which the reduced primary acceptor is less accessible to reoxidation. In cyanobacteria this reversible state was not detected. In both organisms, the decrease of Fmax reflected an irreversible damage of photosystem II centers. These centers need replacement of proteins in order to be active again. The quenching of Fmax and the irreversible inhibition of oxygen evolution were slowed down in both organisms by decreasing the temperature of the photoinhibitory treatment from 34 to 5 degrees C. We conclude that low temperature protected the reaction center II from irreversible photodamage.

Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes, fluorescence and photosynthesis in leaves of Hedera canariensis

The role of the xanthophyll cycle in regulating the energy flow to the PS II reaction centers and therefore in photoprotection was studied by measurements of light-induced absorbance changes, Chl fluorescence, and photosynthetic O2 evolution in sun and shade leaves of Hedera canariensis. The light-induced absorbance change at 510 nm (ΔA510) was used for continuous monitoring of zeaxanthin formation by de-epoxidation of violaxanthin. Non-radiative energy dissipation (NRD) was estimated from non-photochemical fluorescence quenching (NPQ).High capacity for zeaxanthin formation in sun leaves was accompanied by large NRD in the pigment bed at high PFDs as indicated by a very strong NPQ both when all PS II centers are closed (F'm) and when all centers are open (F'o). Such Fo quenching, although present, was less pronounced in shade leaves which have a much smaller xanthophyll cycle pool.Dithiothreitol (DTT) provided through the cut petiole completely blocked zeaxanthin formation. DTT had no detectable effect on photosynthetic O2 evolution or the photochemical yield of PS II in the short term but fully inhibited the quenching of Fo and 75% of the quenching of Fm, indicating that NRD in the antenna was largely blocked. This inhibition of quenching was accompanied by an increased closure of the PS II reaction centers.In the presence of DTT a photoinhibitory treatment at a PFD of 200 μmol m(-2) s(-1), followed by a 45 min recovery period at a low PFD, caused a 35% decrease in the photon yield of O2 evolution, compared to a decrease of less than 5% in the absence of DTT. The Fv/Fm ratio, measured in darkness showed a much greater decrease in the presence than in the absence of DTT. In the presence of DTT Fo rose by 15-20% whereas no change was detected in control leaves.The results support the conclusion that the xanthophyll cycle has a central role in regulating the energy flow to the PS II reaction centers and also provide direct evidence that zeaxanthin protects against photoinhibitory injury to the photosynthetic system.

In vitro studies on light-induced inhibition of Photosystem II and D 1-protein degradation at low temperatures

In order to get information on the molecular background behind the aggrevated photodamage to photosynthesis at low temperatures and to investigate the general mechanism of D 1-protein degradation, isolated spinach thylakoids were subjected to photoinhibitory treatment at various temperatures. The results reveal that: (i) the Photosystem II electron transport per se is less sensitive to high light at low temperatures in contrast to the overall photosynthetic process; (ii) the degradation of D 1-protein is severely retarded below 7°C; (iii) inhibition of Photosystem II electron transport and D 1-protein degradation are separate events the two reactions could be completely separated in time; (iv) D 1-protein is degraded by enzymatic proteolysis and not by a direct photocleavage reaction; (v) degradation of the D 1-protein readily proceeds in the dark but its triggering for the proteolytic attack requires light; (vi) strong illumination at low temperature does not induce any lateral rearrangement in the location of Photosystem II; and (vii) D 1-protein fragments can be identified in vitro and be used to verify the specificity of D 1-protein degradation under various experimental conditions.

Estimation of the effect of photoinhibition on the carbon gain in leaves of a willow canopy

The occurrence of photoinhibition of photosynthesis in leaves of a willow canopy was examined by measuring the chlorophyll-a fluorescence ratio of F V/F M (FM is the maximum fluorescence level of the induction curve, and FV is the variable fluorescence, F V=F M-F 0, where F0 is the minimal fluorescence). The majority of the leaves situated on the upper parts of peripheral shoots showed an afternoon inhibition of this ratio on clear days. This was the consequence of both a decrease in F M and a rise in F O. In the same leaves the diurnal variation in intercepted photosynthetic photon flux density (PPFD) was monitored using leaf-mounted sensors. Using the multivariate method, partial least squares in latent variables, it is shown that the dose of PPFD, integrated and linearly weighted over the last 6-h period, best predicts photoinhibition. Photoinhibition occurred even among leaves that did not intercept PPFDs above 1000 μmol·m(-2)·s(-1). Exposure of leaves to a standard photoinhibitory treatment demonstrated that the depression in the F V/F M ratio was paralleled by an equal depression in the maximal quantum yield of CO2 uptake and a nearly equal depression in the rate of bending (convexity) of the light-response curve of CO2 uptake. As a result, the rate of net photosynthesis is depressed over the whole natural range of PPFD. By simulating the daily course in the rate of net photosynthesis, it is estimated that in the order of one-tenth of the potential carbon gain of peripheral willow shoots is lost on clear days as a result of photoinhibition. This applies to conditions of optimal temperatures. Photoinhibition is even more pronounced at air temperatures below 23° C, as judged from measurements of the FV/FM ratio on clear days: the afternoon inhibition of this ratio increased in a curvilinear manner from 15% to 25% with a temperature decrease from 23° to 14° C.

On the Mechanism of Photoinhibition in Chloroplasts. Relationship Between Changes in Fluorescence and Activity of Photosystem II

Spinach ( Spinacia oleracea L.) leaves were subjected to photoinhibitory treatments. Thylakoid membranes isolated from such leaves showed inhibition of the optimal quantum yield and of the capacity of photosystem (PS)II-dependent electron transport to the same extent. The decline in PS II capacity was linearly related to the decrease in the ratio of variable to maximum fluorescence (F v /F M ) of the leaves. Chlorophyll fluorescence properties (in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea, DCMU) and PS II activity were studied in thylakoid membranes released from photoinhibited spinach chloroplasts (or mesophyll protoplasts of Valerianella locusta L.). PS II electron transport capacity was linearly related to F v /F M and to the DCMU binding capacity. The maximum complementary area above the fluorescence induction curve (A max ) declined with progressing photoinhibition to the same extent as F v . Area growth analysis indicated a predominant photoinhibition of PS IIα, but no transformation of a to β units. The results support the view that photoinhibition is based on transformation of active reaction centers to photochemically inactive fluorescence quenchers, which convert excitation energy to heat. Indications for increased thermal deactivation in the antenna system of PS II were not observed.

Induction kinetics of delayed fluorescence of sun and shade leaves ofFagus sylvatica in the ms-range

Induction kinetics of luminescence (= delayed chlorophyll fluorescence or delayed light emission) were measured with sun and shade leaves of a tall beech tree (Fagus sylvatica 'pendula', weeping beech). The kinetics detected in the ms-range are contrasted for the upper and the lower leaf side. The influence of the following parameters is demonstrated: time of dark-adaptation prior to the measurement, intensity of the excitation light and photoinhibitory treatment. The effects are discussed with respect to chlorophyll concentration, absorption of the excitation light, reabsorption of the luminescence and photosynthetic activity of the leaf tissue. It is shown that the luminescence signal and its kinetic are determined mainly by the properties of the mesophyll parenchyma facing the detector. Thus the more densely packed palisade parenchyma at the upper leaf side exhibits a lower luminescence and a slower kinetic than the spongy parenchyma at the lower leaf side, which is characterized by many aerial interspaces. Our study shows that luminescence kinetics can be applied to interpret the physiological state of a specific leaf tissue. They may serve as an indicator of disorders in the photosynthetic function.

Studies on the mechanism of photosystem II photoinhibition II. The involvement of toxic oxygen species

In a previous paper it was shown that photoinhibition of reaction centre II of spinach thylakoids was predominantly caused by the degradation of D1-protein. An initial inactivation step at the QB-site was distinguished from its breakdown. The present paper deals with the question as to whether this loss of QB-function is caused by oxygen radical attack. For this purpose the photoinhibition of thylakoids was induced at 20°C in the presence of either superoxide dismutase and catalase or the antioxidants glutathione and ascorbic acid. This resulted in comparable though not total protection of D1-protein, photochemistry and fluorescence from photoinhibition. The combined action of both the enzymatic and the non-enzymatic radical scavenging systems brought about an even more pronounced protective effect against photoinhibition than did either of the two systems singularly at saturating concentrations. The results signify a major contribution of activated oxygen species to the degradation process of D1-protein and the related phenomena of photoinhibition. Thylakoids treated with hydroxyl radicals generated through a Fenton reaction showed a loss of atrazine binding sites, electron transport capacity and variable fluorescence in a similar manner, though not to the same extent, as usually observed following photoinhibitory treatment.