In vitro reconstitution of insertion and processing of cytochrome f in a homologous chloroplast translation system.

Using a homologous chloroplast translation system, we have reconstituted insertion and processing of the chloroplast-encoded thylakoid protein cytochrome f (pCytf). Cross-linking demonstrated that pCytf nascent chains when attached to the 70 S ribosome tightly interact with cpSecA, but this is strictly dependent on thylakoid membranes and a functional signal peptide. This indicates that cpSecA is only operative in pCytf biogenesis when it is bound to the membrane, most likely as part of the Sec translocon. No evidence for interaction between the 54-kDa subunit of the chloroplast signal recognition particle (cpSRP) and the pCytf nascent chain could be detected, suggesting that pCytf, in contrast to the polytopic D1 protein, does not require cpSRP for targeting. Insertion of pCytf occurred only co-translationally, resulting in processing and accumulation of both the processed signal peptide and the mature protein in the thylakoid. This co-translational membrane insertion and processing required a functional signal peptide and was inhibited by azide, demonstrating that cpSecA is essential for translocation of the soluble luminal domain. pCytf also associated post-translationally with thylakoids, but the soluble N-terminal domain could not be translocated into the lumen. This is the first study in which synthesis, targeting, and insertion of a chloroplast-encoded thylakoid membrane protein is reconstituted from exogenous transcripts and using the chloroplast translational machinery.

Complementation of bacterial SecE by a chloroplastic homologue.

The SecE protein is an essential component of the SecAYE-translocase, which mediates protein translocation across the cytoplasmic membrane in bacteria. In the thylakoid membranes of chloroplasts, a protein homologous to SecE, chloroplastic (cp) SecE, has been identified. However, the functional role of cpSecE has not been established experimentally. In this report we show that cpSecE in cells depleted for bacterial SecE (i) supports growth, (ii) stabilizes, just like bacterial SecE, the Sec-translocase core component SecY, and (iii) supports Sec-dependent protein translocation. This indicates that cpSecE can functionally replace bacterial SecE in vivo, and strongly suggests that the thylakoid membrane contains a SecAYE-like translocase with functional and structural similarities to the bacterial complex. This study further underscores the evolutionary link between chloroplasts and bacteria.

Identification of a 350-kDa ClpP protease complex with 10 different Clp isoforms in chloroplasts of Arabidopsis thaliana.

A 350-kDa ClpP protease complex with 10 different subunits was identified in chloroplast of Arabidopsis thaliana, using Blue-Native gel electrophoresis, followed by matrix-assisted laser desorption ionization time-of-flight and nano-electrospray tandem mass spectrometry. The complex was copurified with the thylakoid membranes, and all identified Clp subunits show chloroplast targeting signals, supporting that this complex is indeed localized in the chloroplast. The complex contains chloroplast-encoded pClpP and six nuclear-encoded proteins nCpP1-6, as well as two unassigned Clp homologues (nClpP7, nClpP8). An additional Clp protein was identified in this complex; it does not belong to any of the known Clp genes families and is here assigned ClpS1. Expression and accumulation of several of these Clp proteins have never been shown earlier. Sequence and phylogenetic tree analysis suggests that nClpP5, nClpP2, and nClpP8 are not catalytically active and form a new group of Clp higher plant proteins, orthologous to the cyanobacterial ClpR protein, and are renamed ClpR1, -2, and -3, respectively. We speculate that ClpR1, -2, and -3 are part of the heptameric rings, whereas ClpS1 is a regulatory subunit positioned at the axial opening of the ClpP/R core. Several truncations and errors in intron and exon prediction of the annotated Clp genes were corrected using mass spectrometry data and by matching genomic sequences with cDNA sequences. This strategy will be widely applicable for the much needed verification of protein prediction from genomic sequence. The extreme complexity of the chloroplast Clp complex is discussed.

Biogenesis of the chloroplast-encoded D1 protein: regulation of translation elongation, insertion, and assembly into photosystem II.

Regulation of translation elongation, membrane insertion, and assembly of the chloroplast-encoded D1 protein of photosystem II (PSII) was studied using a chloroplast translation system in organello. Translation elongation of D1 protein was found to be regulated by (1) a redox component that can be activated not only by photosynthetic electron transfer but also by reduction with DTT; (2) the trans-thylakoid proton gradient, which is absolutely required for elongation of D1 nascent chains on the thylakoid membrane; and (3) the thiol reactants N-ethylmaleimide (NEM) and iodosobenzoic acid (IBZ), which inhibit translation elongation with concomitant accumulation of distinct D1 pausing intermediates. These results demonstrate that D1 translation elongation and membrane insertion are tightly coupled and highly regulated processes in that proper insertion is a prerequisite for translation elongation of D1. Cotranslational and post-translational assembly steps of D1 into PSII reaction center and core complexes occurred independently of photosynthetic electron transfer or trans-thylakoid proton gradient but were strongly affected by the thiol reactants DTT, NEM, and IBZ. These compounds reduced the stability of the early PSII assembly intermediates, hampered the C-terminal processing of the precursor of D1, and prevented the post-translational reassociation of CP43, indicating a strong dependence of the D1 assembly steps on proper redox conditions and the formation of disulfide bonds.

Proteomics of the chloroplast: systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins.

The soluble and peripheral proteins in the thylakoids of pea were systematically analyzed by using two-dimensional electrophoresis, mass spectrometry, and N-terminal Edman sequencing, followed by database searching. After correcting to eliminate possible isoforms and post-translational modifications, we estimated that there are at least 200 to 230 different lumenal and peripheral proteins. Sixty-one proteins were identified; for 33 of these proteins, a clear function or functional domain could be identified, whereas for 10 proteins, no function could be assigned. For 18 proteins, no expressed sequence tag or full-length gene could be identified in the databases, despite experimental determination of a significant amount of amino acid sequence. Nine previously unidentified proteins with lumenal transit peptides are presented along with their full-length genes; seven of these proteins possess the twin arginine motif that is characteristic for substrates of the TAT pathway. Logoplots were used to provide a detailed analysis of the lumenal targeting signals, and all nuclear-encoded proteins identified on the two-dimensional gels were used to test predictions for chloroplast localization and transit peptides made by the software programs ChloroP, PSORT, and SignalP. A combination of these three programs was found to provide a useful tool for evaluating chloroplast localization and transit peptides and also could reveal possible alternative processing sites and dual targeting. The potential of proteomics for plant biology and homology-based searching with mass spectrometry data is discussed.

Co-translational assembly of the D1 protein into photosystem II.

Assembly of multi-subunit membrane protein complexes is poorly understood. In this study, we present direct evidence that the D1 protein, a multiple membrane spanning protein, assembles co-translationally into the large membrane-bound complex, photosystem II. During pulse-chase studies in intact chloroplasts, incorporation of the D1 protein occurred without transient accumulation of free labeled protein in the thylakoid membrane, and photosystem II subcomplexes contained nascent D1 intermediates of 17, 22, and 25 kDa. These N-terminal D1 intermediates could be co-immunoprecipitated with antiserum directed against the D2 protein, suggesting co-translational assembly of the D1 protein into PS II complexes. Further evidence for a co-translational assembly of the D1 protein into photosystem II was obtained by analyzing ribosome nascent chain complexes liberated from the thylakoid membrane after a short pulse labeling. Radiolabeled D1 intermediates could be immunoprecipitated under nondenaturing conditions with antisera raised against the D1 and D2 protein as well as CP47. However, when the ribosome pellets were solubilized with SDS, the interaction of these intermediates with CP47 was completely lost, but strong interaction of a 25-kDa D1 intermediate with the D2 protein still remained. Taken together, our results indicate that during the repair of photosystem II, the assembly of the newly synthesized D1 protein into photosystem II occurs co-translationally involving direct interaction of the nascent D1 chains with the D2 protein.

Interactions of ribosome nascent chain complexes of the chloroplast-encoded D1 thylakoid membrane protein with cpSRP54.

The mechanisms of targeting, insertion and assembly of the chloroplast-encoded thylakoid membrane proteins are unknown. In this study, we investigated these mechanisms for the chloroplast-encoded polytopic D1 thylakoid membrane protein, using a homologous translation system isolated from tobacco chloroplasts. Truncated forms of the psbA gene were translated and stable ribosome nascent chain complexes were purified. To probe the interactions with the soluble components of the targeting machinery, we used UV-activatable cross-linkers incorporated at specific positions in the nascent chains, as well as conventional sulfhydryl cross-linkers. With both cross-linking approaches, the D1 ribosome nascent chain was photocross-linked to cpSRP54. cpSRP54 was shown to interact only when the D1 nascent chain was still attached to the ribosome. The interaction was strongly dependent on the length of the nascent chain that emerged from the ribosome, as well as the cross-link position. No interactions with soluble SecA or cpSRP43 were found. These results imply a role for cpSRP54 in D1 biogenesis.

Expression of a dominant negative form of cpSRP54 inhibits chloroplast biogenesis in Arabidopsis.

The chloroplast homolog of the 54 kDa subunit of signal recognition particle is required for the in vitro targeting of chlorophyll a/b binding proteins (LHCP) to the thylakoid membrane. To explore the function of cpSRP54 in vivo, plants that are mutated in cpSRP54 function were generated. Dominant negative forms of cpSRP54 altered in single amino acids within the conserved guanine nucleotide binding domain were expressed in Arabidopsis. Transformed plants contained less than 30% of the wild-type level of cpSRP54 protein. As a consequence of the reduced cpSRP54 protein content, the first emerging leaves were yellow and contained immature chloroplasts. Although the chlorophyll (chl) content of the leaves was reduced by 75%, the chl a/b ratio was unaffected, indicating a role of cpSRP54 in the biogenesis of proteins besides LHCP. Many chloroplast proteins were less abundant in the first emerging leaves, including non-pigmented proteins, thylakoid proteins known to be targeted by alternative pathways, and soluble proteins. These observations indicate that the cpSRP54 mutation also has a pleiotropic effect on chloroplast biogenesis. Whereas the level of cpSRP54 remained low as the plants aged, leaves emerging subsequently had a wild-type appearance, suggesting that the adult plants compensated for the reduction in cpSRP54 protein.

Transcriptional and translational adjustments of psbA gene expression in mature chloroplasts during photoinhibition and subsequent repair of photosystem II.

The D1 reaction centre protein of photosystem II (PSII), encoded by the plastid psbA gene, has the highest turnover rate of all thylakoid proteins, due to light-induced damage to D1. The expression of the psbA gene was studied in chloroplasts of fully developed pea (Pisum sativum L.) leaves during high-light photoinhibitory treatment and subsequent restoration of PSII function at low light. psbA transcript levels were determined and the translational activity was followed by in vivo pulse-labelling, by in vitro translations with intact chloroplasts, and by run-off translations on isolated thylakoid membranes. PSII photochemical efficiency was determined in vivo by monitoring the ratio of variable fluorescence to maximal fluorescence (F(V)/F(M)). Enhanced D1 synthesis in pea leaves, upon a shift first from darkness to growth light and subsequently to high light, was accompanied by a substantial increase in the total number of pshA transcripts and by the accumulation of psbA mRNA x initiation complexes on thylakoid membrane. This suggested that high-light illumination increased the transcriptional activity of the psbA gene in mature leaves, and that enhanced translational initiation of psbA mRNA was followed by docking of the initiation complexes to the thylakoid membrane. The high-light-induced increase in the number of thylakoid-associated psbA mRNA x initiation complexes, occurred in parallel with enhanced in vivo D1 synthesis. This, however, did not result in an enhanced accumulation of D1 translation products in in vitro run-off translations when pea leaves were shifted from growth light to high light. This may suggest that at high light only a portion of thylakoid-associated psbA mRNA can be under translational elongation at a given moment. When the leaves were shifted from high light to low light to allow repair of PSII, thylakoid-associated psbA mRNA was rapidly released from the membrane and the high-light-induced pool of psbA transcripts was degraded. The synthesis of the D1 protein decreased on the same time scale. However, the restoration of PSII photochemical function, measured as F(V)/F(M), took a substantially longer time. It is concluded that during changing light conditions, mature leaves are able to adjust psbA gene expression both at the transcriptional and at the translational level.

Synthesis and assembly of the D1 protein into photosystem II: processing of the C-terminus and identification of the initial assembly partners and complexes during photosystem II repair.

In previous studies [van Wijk, K. J., Bingsmark, S., Aro, E.-M., & Andersson, B. (1995) J. Biol. Chem. 270, 25685-25695; van Wijk, K. J., Andersson, B., & Aro, E.-M. (1996) J. Biol. Chem 271, 9627-9636], we have demonstrated that D1 protein synthesized in isolated chloroplasts and thylakoids is incorporated into the photosystem II (PSII) core complex. By pulse-chase experiments in these in vitro systems, followed by sucrose gradient fractionation of solubilized thylakoid membranes, it was shown that this assembly proceeded stepwise; first the D1 protein was incorporated to form a PSII reaction center complex (PSII rc), and through additional assembly steps the PSII core complex was formed. In this study, we have analyzed this assembly process in more detail, with special emphasis on the initial events, through further purification and analysis of the assembly intermediates by nondenaturing Deriphat-PAGE and by flatbed isoelectric focusing. The D2 protein was found to be the dominant PSII reaction center protein initially associating with the new D1 protein. This strongly suggests that the D2 protein is the primary "receptor" or stabilizing component during or directly after synthesis of the D1 protein. After formation of the D1-D2 heterodimer, cyt b559 became attached, whereas the psbI gene product was assembled as a subsequent step, thereby forming a PSII reaction center complex. Subsequent formation of the PSII core occurred by binding of CP47 and then CP43 to the PSII rc. The rapid radiolabeling of a minor population of a PSII core subcomplex without CP43 indicated that an association of newly synthesized D1 protein with a preexisting complex consisting of D2/cyt b55q/psbI gene product/CP47 was possibly occurring, in parallel to the predominant sequential assembly pathway. The kinetics of synthesis and processing of the precursor D1 protein were followed in isolated chloroplasts and were compared with its incorporation into PSII assembly intermediates. No precursor D1 protein was found in PSII core complexes, indicating either that incorporation into the PSII core complex facilitates the cleavage of the C-terminus or, more likely, that processing is more rapid than the assembly into the PSII core.

Light is required for efficient translation elongation and subsequent integration of the D1-protein into photosystem II.

The light dependence of translation and successive assembly of the D1 reaction center protein into Photosystem II subcomplexes was followed in fully developed chloroplasts isolated from the dark phase of diurnally grown spinach. The incorporation of synthesized D1 protein into Photosystem II (PSII) was analyzed by fractionation of radiolabeled unassembled protein and PSII (sub)complexes on sucrose density gradients. The ribosomes with attached nascent chains were recovered as pellets in the same gradients, and nascent chains of the D1 protein were immunoprecipitated. The analysis showed that absence of light during translation leads to an increased accumulation of polysome-bound D1 translation intermediates, indicating that light is required for efficient elongation of the D1 protein. The accumulation of the D1 protein and CP43 decreased three-fold in darkness, whereas accumulation of the D2 reaction center protein was not affected by light. In addition, light was also required for efficient incorporation of the D1 protein into the PSII core complex. In darkness, the newly synthesized D1 protein accumulated predominantly as unassembled protein or in PSII subcomplexes smaller than 100 kDa.

Kinetic resolution of the incorporation of the D1 protein into photosystem II and localization of assembly intermediates in thylakoid membranes of spinach chloroplasts.

The chloroplast-encoded D1 protein of photosystem II (PSII) has a much higher turnover rate than the other subunits of the PSII complex as a consequence of photodamage and subsequent repair of its reaction center. The replacement of the D1 protein in existing PSII complexes was followed in two in vitro translation systems consisting of isolated chloroplasts or isolated thylakoid membranes with attached ribosomes. By application of pulse-chase translation experiments, we followed translation elongation, release of proteins from the ribosomes, and subsequent incorporation of newly synthesized products into PSII (sub)complexes. The time course of incorporation of newly synthesized proteins into the different PSII (sub)complexes was analyzed by sucrose density gradient centrifugation. Immediately after termination of translation, the D1 protein was found both unassembled in the membrane as well as already incorporated into PSII reaction center complexes, possibly due to a cotranslational association of the D1 protein with other PSII reaction center components. Later steps in the reassembly of PSII were clearly post-translational and sequential. Different rate-limiting steps in the assembly process were found to be related to the depletion of nuclear encoded and stromal components as well as the lateral migration of subcomplexes within the heterogeneous thylakoid membrane. The slow processing of precursor D1 in the thylakoid translation system revealed that processing was not required for the assembly of the D1 protein into a PSII (sub)complex and that processing of the unassembled precursor could take place. The limited incorporation into PSII subcomplexes of three other PSII core proteins (D2 protein, CP43, and CP47) was clearly post-translational in both translation systems. Radiolabeled assembly intermediates smaller than the PSII core complex were found to be located in the stroma-exposed thylakoid membranes, the site of protein synthesis. Larger PSII assembly intermediates were almost exclusively located in the appressed regions of the membranes.

In vitro synthesis and assembly of photosystem II core proteins. The D1 protein can be incorporated into photosystem II in isolated chloroplasts and thylakoids.

The D1 reaction center protein of the membrane-bound photosystem II complex (PSII) has a much higher turnover rate than the other PSII proteins. Thus, the D1 protein has to be replaced while the other PSII components are not newly synthesized. In this study, this D1 protein replacement into PSII complexes was followed in two in vitro translation systems: isolated chloroplasts and a homologous run-off translation system consisting primarily of isolated thylakoids with attached ribosomes. The incorporation of newly synthesized radiolabeled products into different (sub)complexes was analyzed by sucrose density gradient centrifugation of n-dodecyl beta -D-maltoside-solubilized thylakoid membranes. This analysis allowed us to follow the release of the nascent polypeptide chains from the ribosomes and identification of at least four assembly steps of the PSII complex, as shown below. (i) Both in isolated chloroplasts and in thylakoids, newly synthesized D1 protein is predominantly incorporated into existing PSII subcomplexes, indicating that synthesis and import of nuclear-encoded factors is not needed for D1 protein replacement. (ii) In chloroplasts, D1 protein incorporation into PSII core complexes is more efficient than during translation in isolated thylakoids. In the thylakoid translation system, a large percentage of radiolabeled D1 protein is found in smaller PSII subcomplexes, like PSII reaction center particles, and as unassembled protein in the membrane. This indicates that stromal factors are required in the replacement process of the D1 protein. (iii) Both in isolated chloroplasts and in thylakoids, the other PSII core proteins D2, CP43, and CP47 are also synthesized and released from the membrane-bound ribosomes, but incorporation into PSII complexes occurs to a much smaller extent than the D1 protein. Instead they accumulate predominantly as unassembled proteins in the thylakoid membrane. (iv) In chloroplasts, synthesis of the D1 protein seems to be adjusted according to the possibilities of incorporation into PSII complexes, while synthesis of the D2 protein, CP43, and CP47 is less regulated and their accumulation as unassembled protein in the membrane is abundant.

Evidence for SecA- and delta pH-independent insertion of D1 into thylakoids.

Many nuclear-encoded proteins are targeted into chloroplast thylakoids by an azide sensitive Sec-related mechanism or by a delta pH-driven mechanism. In this report, the requirements for the integration of chloroplast-encoded thylakoid proteins have been analysed in pulse-labeled intact chloroplasts. We show that the integration of the photosystem II reaction centre protein, D1, continues in the absence of a delta pH and in the presence of azide. A range of other proteins are similarly targeted to thylakoids in the presence of azide, suggesting that the SecA-related mechanism is not widely used for the targeting of chloroplast-encoded proteins.

Synthesis of reaction center proteins and reactivation of redox components during repair of photosystem II after light-induced inactivation.

The D1 reaction center protein in photosystem II (PSII) has a high turnover rate due to light-induced inactivation of the redox components. We have studied the reactivation kinetics of the redox components of PSII after strong illumination and compared these kinetics with the turnover of the D1 protein and translation kinetics of the plastid-encoded PSII core proteins in Chlamydomonas reinhardtii cells. Repair of PSII was to a large extent dependent on protein translation. During the first hours of repair, D1 translation was highly accelerated as compared to the other PSII core proteins. By addition of protein synthesis inhibitors during the recovery process, it was found that the time from protein synthesis to full reassembly and reactivation of the individual PSII complexes was about 55 +/- 10 min. Inactivation and reactivation of the redox components in PSII were followed by electron spin resonance and electron transport measurements. Combining the data shows that reactivation of the individual components proceeded together or shortly after one another. Thus, no accumulation of any partially active reactivation intermediate occurred. We conclude that the rate-limiting step of the repair cycle of PSII lies in the degradation and synthesis of the PSII reaction center proteins. Once stable synthesis of the PSII core proteins is achieved, reactivation of the redox components occurs very quickly.

Spectroscopic characterization of tyrosine-Z in histidine 190 mutants of the D1 protein in photosystem II (PSII) in Chlamydomonas reinhardtii. Implications for the structural model of the donor side of PSII.

EPR spectra attributed to the redox active tyrosine residues on the oxidizing side of photosystem II (TyrZ and TyrD) have almost identical line shapes, although the tyrosyl radicals differ in stability and redox characteristics. Strongly modified spectra of oxidized TyrD in site-directed mutants in a histidine residue, H189 on the D2 reaction center protein in the cyanobacterium Synechocystis 6803, support a structural model where H189 interacts closely, probably via a hydrogen bond, to TyrD (Tommos, C., Davidsson, L., Svensson, B., Madsen, C., Vermass, W., and Styring, S. (1993) Biochemistry 32, 5436-5441). To determine whether TyrZ and the corresponding histidine on the D1 protein (D1-H190) interacts similarly, we have generated His-Phe (H190F) and His-Tyr (H190Y) mutations in the C2 symmetry related H190 residue on the D1 reaction center protein by site-directed mutagenesis in Chlamydomonas reinhardtii. The H190F and H190Y mutants assemble photosystem II reaction centers capable of primary photochemistry but unable to oxidize water. We have obtained kinetic spectra of a flash-induced transient EPR signal that we assign to oxidized TyrZ in the D1-H190 mutants. The spectra are identical in line width (18-20 G) and hyperfine structure to the wild-type spectrum from oxidized TyrZ and exhibit decay kinetics (t 1/2 approximately 500 ms) typical for the TyrZ radical in managenese-depleted photosystem II membranes. However, both TyrZ and TyrD were oxidized with reduced (10-15%) quantum yield in these mutants, indicating that the kinetics of electron donation to P+680 were significantly modified as a result of the mutation. Thus, the altered kinetics of TyrZ in the mutants suggest that there is an interaction between TyrZ and His-190 on the D1 protein. However, unlike the situation on the D2 side, the presence of a hydrogen bond between TyrZ and H190 on the D1 protein is improbable.

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 (qN) and photochemical quenching (qp) 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 qp 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 qp than at ambient or higher [CO2] and photoinhibition became only substantial when qp decreased below 0.3. When high levels of energy-dependent quenching (qE) (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 qE to photoinhibitory quenching (qI). 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 qE. The shift from qE to qI occurred concomitant with qP 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 qP, qE and photoinhibition of PSII at low temperature.

Oxygen dependence of photoinhibition at low temperature in intact protoplasts of Valerianella locusta L.

Photoinhibition of photosynthesis in vivo is shown to be considerably promoted by O2 under circumstances where energy turnover by photorespiration and photosynthetic carbon metabolism are low. Intact protoplasts of Valerianella locusta L. were photoinhibited by 30 min irradiation with 3000 µmol photons · m(-2) · s(-1) at 4° C in saturating [CO2] at different oxygen concentrations, corresponding to 2-40% O2 in air. The photoinhibition of light-limited CO2-dependent photosynthetic O2 evolution increased with increasing oxygen concentration. The uncoupled photochemical activity of photosystem II, measured in the presence of the electron acceptor 1,4-benzoquinone, and maximum variable fluorescence, Fv, were strongly affected and this inhibition was closely correlated to the O2 concentration. The effect of O2 did not saturate at the highest concentrations applied. An increase in photoinhibitory fluorescence quenching with [O2], although less pronounced than in protoplasts, was also observed with intact leaves irradiated at 4° C in air. Initial fluorescence, Fo, was slightly (about 10%) increased by the inhibitory treatments but not influenced by [O2]. A long-term cold acclimation of the plants did not substantially alter the O2-sensitivity of the protoplasts under the high-light treatment. From these experiments we conclude that oxygen is involved in the photoinactivation of photosystem II by excess light in vivo.

The quantum efficiency of photosystem II and its relation to non-photochemical quenching of chlorophyll fluorescence; the effect of measuring-and growth temperature.

The relation between the quantum yield of oxygen evolution of open photosystem II reactions centers (Φp), calculated according to Weis and Berry (1987), and non-photochemical quenching of chlorophyll fluorescence of plants grown at 19°C and 7°C was measured at 19°C and 7°C. The relation was linear when measured at 19°C, but when measured at 7°C a deviation from linearity was observed at high values of non-photochemical quenching. In plants grown at 7°C this deviation occurred at higher values of non-photochemical quenching than in plants grown at 19°C. The deviations at high light intensity and low temperature are ascribed to an increase in an inhibition-related, non-photochemical quenching component (qI).The relation between the quantum yield of excitation capture of open photosystem II reaction centers (Φexe), calculated according to Genty et al. (1989), and non-photochemical quenching of chlorophyll fluorescence was found to be non-linear and was neither influenced by growth temperature nor by measuring temperature.At high PFD the efficiency of overall steady state electron transport measured by oxygen-evolution, correlated well with the product of q N and the efficiency of excitation capture (Φexe) but it deviated at low PFD. The deviations at low light intensity are attributed to the different populations of chloroplasts measured by gas exchange and chlorophyll fluorescence and to the light gradient within the leaf.