Photoinhibition of photosynthesis in vivo: The role of protein turnover in photosystem II

Divergent theories on the mechanism behind, and the nature of, photoinhibition are discussed, especially in relation to observations made in higher plant leaves. Comparisons are made with 'lower' plant groups and results of in vivo and in vitro experiments are considered. Irradiance-induced mechanisms involved in the regulation of PSII function and structure are discussed in connection with turnover of the DI protein. A model is presented in which a structural change in DI protein facilitates the formation of a population of dissipative PSII centres that do not participate in linear electron transport to PSI. We suggest a sophisticated regulatory mechanism whereby this variable PSII function is controlled with respect to both incident light and biochemical demand, a control which relies on feedback from both light and dark reactions.     

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.     

Molecular analysis of the Chlamydomonas nuclear gene encoding PsbW and demonstration that PsbW is a subunit of photosystem II,but not photosystem I

PsbW is a nuclear-encoded protein located in the thylakoid membrane of the chloroplast. Studies in higher plants have provided substantial evidence that PsbW is a core component of photosystem II. However, recent data have been presented to suggest that PsbW is also a subunit of photosystem I. Such a sharing of subunits between the two photosystems would represent a novel phenomenon. To investigate this, we have cloned and characterized the psbW gene from the green alga Chlamydomonas reinhardtii. The gene is split by five introns and encodes a polypeptide of 115 residues comprising the 6.1 kDa mature PsbW protein preceded by a 59 amino acid bipartite transit sequence. Using antibodies raised to PsbW we have examined: (1) C. reinhardtii mutants lacking either photosystem and (2) purified photosystem preparations. We find that PsbW is a subunit of photosystem II, but not photosystem I.     

Analysis of the genes of the OEE1 and OEE3 proteins of the photosystem II complex from Chlamydomonas reinhardtii

The sequences of the nuclear genes of the 33 kDa (OEE1) and the 16 kDa (OEE3) polypeptides of the oxygen evolving complex of Chlamydomonas reinhardtii have been established. Comparison between the OEE1 protein sequences of C. reinhardtii and higher plants and cyanobacteria reveals 67 and 47% homology. In contrast, C. reinhardtii and higher plants have only 28% overall homology for OEE3 which is mostly limited to the central portion of the protein. The transit peptides of the C. reinhardtii proteins consist of 52 (OEE1) and, most likely, 51 (OEE1) amino acids. They have a basic amino terminal region and, at least in the case of OEE1, a hydrophobic segment at their carboxy terminal end typical of thylakoid lumen proteins. Comparison of the genomic and cDNA clones indicates that the OEE1 and OEE3 genes contain five and four introns, respectively, some of which are located within the coding sequences of the transit peptides.     

The carboxy-terminal extension of the D1-precursor protein is dispensable for a functional photosystem II complex in Chlamydomonas reinhardtii

The D1-precursor protein of the photosystem II reaction centre contains a carboxy-terminal extension whose proteolytic removal is necessary for oxygen-evolving activity. To address the question of the role of the carboxy-terminal extension in the green alga Chlamydomonas reinhardtii, we truncated D1 by converting codon Ser345 of the psbA gene into a stop codon. Particle gun transformation of an in vitro modified psbA gene fragment also carrying mutations conferring herbicide resistance yielded a homoplasmic transformant containing the stop codon. Since oxygen evolution capacity is not affected in this mutant as compared with herbicide-resistant control cells, the carboxy-terminal extension is dispensable for a functional photosystem II complex under normal growth conditions.     

Changes in antioxidant enzyme activity in the fine roots of black poplar (Populus nigra L.) and cottonwood (Populus deltoides Bartr. ex Marsch) in a heavy-metal-polluted environment

The effect of heavy metal deposition onto soil from a copper smelter on lipid peroxidation and antioxidant enzyme activity in the fine roots of two poplars (Populus nigra L. and Populus deltoides Bartr. ex Marsch) was analyzed. The subjects were mature trees growing in real environments. In both analyzed species, heavy metals stimulated the overproduction of free radicals in fine roots (measured as malondialdehyde level), which was directly proportional to advancing senescence. In young fine roots, heavy metals caused a decrease in guaiacol peroxidase activity and presumably disturbed the lignification process. Catalase was highly sensitive to the presence of heavy metals in the soil. In contrast, ascorbate peroxidase and glutathione reductase activities were unaffected by heavy metals. In the case of superoxide dismutase, a clear increase in enzyme activity was observed only in P. nigra under drought conditions, whereas it was inhibited in polluted stands.     

Saturating Irradiance-Induced Photoinhibition without Monomerisation of Photosystem 2 Dimer in Soybean Leaves

The oligomeric state of photosystem 2 (PS2) complex in soybean leaves treated with saturating irradiance was studied by non-denaturing polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography. PS2 dimers resolved by non-denaturing PAGE accounted for about 75 % of total PS2 complex and there was no significant difference in the ratio of PS2 dimer to monomer between samples from saturating irradiance-treated and fully dark-adapted leaves. Furthermore, BBY particles were resolved into four chlorophyll-enriched fractions by gel filtration chromatography. From their molecular masses and protein components, these fractions were deduced to be PS2 dimer, PS2 monomer, oligomeric light-harvesting complex 2 (LHC2), and monomeric LHC2. Also, no change in the proportion of PS2 dimer in total PS2 was observed in the granal region of thylakoid membranes from soybean leaves after saturating irradiation. Hence the dimer is the predominant natural form of PS2 in vivo and no monomerisation of PS2 dimer occurs during saturating irradiance-induced photoinhibition in soybean leaves.     

Chromatic regulation inChlamydomonas reinhardtii alters photosystem stoichiometry and improves the quantum efficiency of photosynthesis

The work addressed the adjustment of the photosystem ratio in the green algaChlamydomonas reinhardtii. It is shown that green algae, much like cyanophytes and higher plants, adjust and optimize the ratio of the two photosystems in chloroplasts in response to the quality of irradiance during growth. Such adjustments are compensation reactions and helpC. reinhardtii to retain a quantum efficiency of oxygen evolution near the theoretical maximum. Results show variable amounts of PS I and a fairly constant amount of PS II in chloroplasts and suggest that photosystem stoichiometry adjustments, occurring in response to the quality of irradiance during plant growth, are mainly an adjustment in the concentration of PS I. The work delineates chromatic effects on chlorophyll accumulation in the chloroplast ofC. reinhardtii from those pertaining to the regulation of the PS I/PS II ratio. The detection of the operation of a molecular feedback mechanism for the PS I/PS II ratio adjustment in green algae strengthens the notion of the highly conserved nature of this mechanism among probably all oxygen evolving photosynthetic organisms. Findings in this work are expected to serve as the basis of future biochemical and mutagenesis experiments for the elucidation of the photosystem ratio adjustment in oxygenic photosynthesis.     

Application of genetic markers to the discrimination of European Black Poplar (Populus nigra) from American Black Poplar (P. deltoides) and Hybrid Poplars (P. x canadensis) in Switzerland

European Black Poplar (Populus nigra) is considered a rare and endangered tree species because of severe reduction of its natural riverine habitat and potential hybridisation with the related non-indigenous taxa P. deltoides and P. x canadensis. As it is difficult to distinguish these taxa solely based on their morphology, we applied a PCR-based assay with an easy-to-use and robust molecular marker set (cpDNA trnL-trnF/RsaI RFLP, nDNA win3 and nDNA POPX/MspI RFLP) in order to identify pure P. nigra. Different plant tissues could be used for fast and standardised DNA extraction. The application of the three marker types was tested on a number of different Populus taxa, and they were also used for the verification of pure P. nigra in a sample of 304 putative P. nigra individuals from Switzerland. Cross-checking of the DNA data with those using a traditional allozyme approach resulted in complete agreement. The availability of molecular identification methods is an important prerequisite for the conservation of European Black Poplar, because pure, non-introgressed plant material can then be used in restoration projects of European floodplains.     

High frequency direct plant regeneration from leaf, internode, and root segments of Eastern Cottonwood (Populus deltoides)

Simple, reproducible, high frequency, improved plant regeneration protocol in Eastern Cottonwood (Populus deltoides) clones, WIMCO199 and L34, has been reported. Initially, aseptic cultures established from axillary buds of nodal segments from mature plus trees on MS liquid medium supplemented with 0.25 mg l⁻¹ KIN and 0.25 mg l⁻¹ IAA. Nodal and internodal segments were found to be extra-prolific over shoot apices during course of aseptic culture establishment, while 0.25 mg l⁻¹ KIN concentration played a stimulatory role in high frequency plant regeneration. Diverse explants, such as various leaf segments, internodes, and roots from in vitro raised cultures, were employed. Direct plant regeneration was at high frequency of 92% in internodes, 88% in leaf segments, and 43% in root segments. This led to the formation of multiple shoot clusters on established culture media with rapid proliferation rates. Many-fold enhanced shoot elongation and growth of the clusters could be achieved on liquid MS medium supplemented with borosilicate glass beads, which offer physical support for proliferating shoots leading to faster growth in comparison to semi-solid agar or direct liquid medium. SEM examination of initial cultures confirmed direct plant regeneration events without intervening calli. In vitro regenerated plants induced roots on half-strength MS medium with 0.15 mg l⁻¹ IAA. Rooted 5- to 6-week-old in vitro regenerated plants were transferred into a transgenic greenhouse in pots containing 1:1 mixture of vermicompost and soil at 27 ± 2°C for hardening and acclimatization. 14- to 15-week-old well-established hardened plants were transplanted to the field and grown to maturity. The mature in vitro raised poplar trees exhibited a high survival rate of 85%; 4-year-old healthy trees attained an average height of 8 m and an average trunk diameter of 25 cm and have performed well under field conditions. The regeneration protocol presented here will be very useful for undertaking genetic manipulation, providing a value addition to Eastern Cottonwood propagation in future.     

Changes of amino acid sequence in PEST-like area and QEEET motif affect degradation rate of D1 polypeptide in photosystem II

The degradation rate of the D1 polypeptide was measured in threeSynechocystis PCC 6803 mutantsin vivo. Mutations were introduced into a putative cleavage area of the D1 polypeptide (QEEET motif) and into the PEST-like area. PEST sequences are often found in proteins with a high turnover rate. The QEEET-motif mutants are CA1 [(E242-E244);Q241H] and E243K, and the third mutation, E229D, was directed to the PEST-like area. During high-light illumination (1500 mol photons m^-2s^-1) that induced photoinhibition of photosystem II (PSII), the half-life time of the D1 polypeptide in mutant E229D (t _1/2=35 min) was about twice as long as in AR (control strain) cells (t _1/2=19 min). In growth light (40 mol photons m^-2s^-1), the degradation rate of the D1 polypeptide in E229D and AR strains was the same (t _1/25 h). In growth light the D1 polypeptide was degraded faster in both QEEET-motif mutants than in the AR strain, but in photoinhibitory light the degradation rates were similar. According to these results, the highly conservative QEEET motif as such is not required for the proteolytic cut of the D1 polypeptide, but it does affect the rate of degradation. No simple correlation existed between the degradation rate of the D1 polypeptide and the susceptibility of PSII to photoinhibition in mutant and AR cells under our experimental conditions.     

Polymorphism and Mendelian inheritance of photosystem II 23-kilodalton polypeptide

Soluble proteins from leaves of Nicotiana glauca Grah., N. langsdorffii Weinm., their reciprocal hybrids and amphiploid hybrid (N. glaucaxN. langsdorffii) were resolved by two-dimensional gel electrophoresis. Among a group of well-resolved polypeptides, in the isoelectric-point range of 5–5.5 and relative-molecular-mass (M_r) range of 18–23 kilodaltons (kDa), species-specific variation was observed. Polypeptides designated L and l are specific to N. langsdorffii, and G and g to N. glauca, while C is common to both species. Polypeptides L, G and C are localized in the chloroplasts and associated with thylakoid membranes. Polypeptide L is more acidic than polypeptide G, and both polypeptides have an M_r of 23 kDa. They were isolated from two-dimensional gels and their first 13 N-terminal amino-acid sequences were determined. These were found to be identical to the 13N-terminal amino acids of the photosystem II (PSII) 23-kDa polypeptide from spinach (T. Jansen et al. (1987) FEBS Lett. 216, 234–240) and, except for one change, to those from pea (R. Wales et al. (1989) Plant Molec. Biol., in press). Polypeptides G and L cross-react with antiserum against the PSII 23-kDa polypeptide from pea. Therefore, polypeptides G and L are extrinsic PSII 23-kDa polypeptides. They appear jointly and in equal amounts in the reciprocal hybrids. Since chloroplasts in Nicotiana are maternally inherited, these results demonstrate that polypeptides G and L are encoded by nuclear genes, are polymorphic variants of the PSII 23-kDa polypeptide, and are inherited in a Mendelian manner.Abbreviations kDa kilodalton - LS large subunit of Rubisco - M_r relative molecular mass - NEPHGE non-equilibrium pH gradient gel electrophoresis - PSII photosystem II - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - SS small subunit of Rubisco     

Stoichiometric determination of pheophytin in photosystem II of oxygenic photosynthesis

Pheophytin and chlorophyll extracted from oxygen-evolving photosystem II particles, chloroplast thylakoids and cyanobacterial cells were separated by column chromatography with DEAE-Toyopearl, and quantitatively determined by spectrophotometry. The molecular ratio of chlorophyll a+b to pheophytin a was about 100 in spinach photosystem II particles and about 140 in spinach thylakoids. Using flash spectrophotometry of P680 and measurement of flash-induced oxygen yield, the molecular ratio of the chlorophyll to the photochemical reaction center II was determined to be about 200 in the photosystem II particles. These findings suggest that the stoichiometry in photosystem II particles is one reaction center II and two pheophytin a molecules per about 200 chlorophyll molecules. The same stoichiometry for pheophytin to the reaction center II was obtained in the cyanobacteria, Anacystis nidulans and Synechocystis PCC 6714. A quantitative determination of pheophytin a and the electron donor P700 in stroma thylakoids from pokeweed suggests that photosystem I does not contain pheophytin.Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Directed disruption of the Chlamydomonas chloroplast psbK gene destabilizes the photosystem II reaction center complex

Using particle gun-mediated chloroplast transformation we have disrupted the psbK gene of Chlamydomonas reihardtii with an aadA expression cassette that confers resistance to spectinomycin. The transformants are unable to grow photoautotrophically, but they grow normally in acetate-containing medium. They are deficient in photosystem II activity as measured by fluorescence transients and O₂ evolution and they accumulate less than 10% of wild-type levels of photosystem II as measured by immunochemical means. Pulse-labeling experiments indicate that the photosystem II complex is synthesized normally in the transformants. These results differ from those obtained previously with similar cyanobacterial psbK mutants that were still capable of photoautotrophic growth (Ikeuchi et al., J. Biol. Chem. 266 (1991) 1111–1115). In C. reinhardtii the psbK product is required for the stable assembly and/or stability of the photosystem II complex and essential for photoautotrophic growth. The data also suggest that the stability requirements of the photosynthetic complexes differ considerably between C. reinhardtii and cyanobacteria.     

Diurnal and seasonal patterns of net photosynthesis by irrigated Chrysothamnus nauseosus under field conditions

Photosystem II particles from spinach and barley contained 2.5 and 4.2 Cu per 300 chlorophylls respectively. This Cu was resistant to removal by EDTA. A large percentage of the PSII Cu in both plants is associated with the light-harvesting chlorophyll a/b protein, LHCII; 46% in barley and 76% in spinach. Several experiments have been performed to rule out the possibility that the Cu was introduced during the isolation procedures and to ensure that the Cu is associated with PSII. Since the PSII Cu is mainly associated with LHCII, it is unlikely that it is involved in O₂ evolution.Abbreviations BBY PSII particles prepared as in [4] with modifications - LHCII Light Harvesting Chlorophyll a/b protein - MMN 50mM Mes-NaOH, 5 mM MgCl₂, 15 mM NaCl - SOD Superoxide Dismutase - TEMED N,N,N,N-tetramethylethylenediamine Some of the results in this paper appeared in preliminary form in the Proceedings of the VIIth International Congress on Photosynthesis [24].     

Photoinhibition of photosynthesis in willow leaves under field conditions

Chlorophyll fluorescence of leaves of a willow (Salix sp.) stand grown in the field in northern Sweden was measured on several occasions during the growing season of 1987. For leaves that received mostly full daylight, the F _V/F _P ratio declined roughtly 15% in the afternoon on cloudless days in July (F _P is the fluorescence at the peak of the induction curve obtained at the prevailing air temperature after 45 min of dark adaptation, and F _V is variable fluoresence, F _V=F _P-F _O, where F _O is minimal fluorescence). There was no decrease in the F _V/F _P ratio on cloudy days, while the effect was intermediate on changeable days. In view of this light dependence, together with the fact that the decline in the F _V/F _P ratio was paralleled with an equal decline in the corresponding fluorescence ratio F _V/F _M at 77K, and a similar decline in the maximum quantum yield of O₂ evolution, it is suggested that the decline in the F _V/F _P ratio represents a damage in photosyntem II attributable to photoinhibition. Recovery of the F _V/F _P ratio in dim light following a decline on a cloudless day took 7–16 h to go to completion; the F _V/F _P ratio was fully restored the following morning. When all active leaves of a peripheral shoot were compared, the F _V/F _P ratio in the afternoon of a day of bright light varied greatly from leaf to leaf, though the majority of leaves showed a decline. This variation was matched by a pronounced variation in intercepted photon flux density. When leaves developed in the shade were exposed to full sunlight by trimming of the stand an increased sensitivity to photoinhibition was observed as compared to peripheral leaves. The present study indicates that peripheral willow shoots experienced in the order of 10–20% photoinhibition during an appreciable part of their life. This occurred even though the environmental conditions were within the optimal range of photosynthesis and growth.Abbreviations and symbols F _O minimum fluorescence - F _P fluorescence at the peak of the induction curve obtained at normal ambient temperatures - F _V variable fluorescence - F _M maximum fluorescence obtained at 77K - PPFD photosynthetic photon flux density     

Characterization of the photosystem II 32 kDa protein in Synechococcus PCC7942

A rapidly labeled photosynthetic membrane protein was identified in the cyanobacterium Synechococcus PCC7942 R2 as the 32 kDA protein that is involved in electron transport and quinone binding in the photosystem II complex. Partial proteolysis of the membrane-bound protein indicates that the internal architecture and the topology of the Synechococcus 32 kDa protein resembles the analogous protein of higher plants. In addition to the R2 wild-type strain, we characterized three psbA-inactivated Synechococcus strains, in which two of the three endogenous psbA genes were inactivated. In all strains, a 32 kDa protein cross-reacts with an antiserum that was raised against a higher-plant 32 kDa protein and displays in vivo light-dependent turnover. In Synechococcus, the herbicide DCMU inhibits the 32 kDa protein turnover at similar concentration ranges as in higher plants; however, a fraction of the molecules always displays a DCMU-insensitive degradation.     

Degradation andde novo synthesis of D1 protein andpsb A transcript inreinhardtii during UV-B inactivation of photosynthesis

UV-B induces intensity and time dependent inhibition of photosynthetic O2 evolution and PS II electron transport activity in Chlamydomonas reinhardtii. The D1 and D2 proteins of chloroplast membranes are rapidly and specifically degraded in the course of irradiation of cells to UV-B. Continuous synthesis of the two proteins was essential for the repair of damaged PS II as chloramphenicol accelerated UV-B inactivation of photosynthesis and prevented photoreactivation. Northern analysis revealed that UV-B also affected the expression of psbA gene coding for the D1 protein. Cells showing 72% inhibition of PS II activity, revealed a modest net loss of 25% in the level of D1 protein. This shows that synthesis of D1 protein is not the only component involved in the recovery process. Our results indicate that besides affecting the synthesis of the D1 protein UV-B may impair certain post-translational events, which in turn may limit the repair of damaged PS II.