Characterization of a 160 kD Photosystem II reaction center complex isolated from photoinhibited Dunaliella salina thylakoids

Photoinhibition in the green alga Dunaliella salina is accompanied by the formation of inactive Photosystem II reaction centers. In SDS-PAGE analysis, the latter appear as 160 kD complexes. These complexes are structurally stable, enough to withstand re-electrophoresis of excised gel slices from the 160 kD region. Western blot analyses with specific polyclonal antibodies raised against the D1 or D2 reaction center proteins provided evidence for the presence of both of these polypeptides in the re-electrophoresed 160 kD complex. Incubation of excised gel slices from the 160 kD region, under aerobic conditions at 4°C for a prolonged period of time, caused a break-up of the 160 kD complex into a 52 kD D1-containing and 80 and 26 kD D2-containing pieces. Western blot analysis with polyclonal antibodies raised against the apoproteins of CPI (reaction center proteins of PS I) did not show cross-reaction either with the 160 kD complex or with the 52, 80 and 26 kD pieces. The results show the presence of both D1 and D2 in the 160 kD complex and strengthen the notion of a higher molecular weight D1- and D2-containing complex that forms upon disassembly of photodamaged PS II units.Abbreviations Chl chlorophyll - PS II Photosystem II - D1 the 32 kD reaction center protein of PS II, encoded by the chloroplast psbA gene - D2 the 34 kD reaction center protein of PS II, encoded by the chloroplast psbD gene - CPI the 82 and 83 kD reaction center proteins of PS I, encoded by the chloroplast psaA and psaB genes - HL high light - LL low light This publication is dedicated to the memory of the late Professor Daniel Arnon, whom the first author will fondly remember for his many accounts of past scientific discovery and debate.     

Photoinhibition and repair in Dunaliella salina acclimated to different growth irradiances

The light-dependent rate of photosystem-II (PSII) damage and repair was measured in photoautotrophic cultures of Dunaliella salina Teod. grown at different irradiances in the range 50–3000 mol photons · m^–2· s^–1. Rates of cell growth increased in the range of 50–800 mol photons·m^–2·s^–1, remained constant at a maximum in the range of 800–1,500 mol photons·m^–2 ·s^–1, and declined due to photoinhibition in the range of 1500–3000 mol photons·m^–2·s^–1. Western blot analyses, upon addition of lincomycin to the cultures, revealed first-order kinetics for the loss of the PSII reaction-center protein (D1) from the 32-kDa position, occurring as a result of photodamage. The rate constant of this 32-kDa protein loss was a linear function of cell growth irradiance. In the presence of lincomycin, loss of the other PSII reaction-center protein (D2) from the 34-kDa position was also observed, occurring with kinetics similar to those of the 32-kDa form of D1. Increasing rates of photodamage as a function of irradiance were accompanied by an increase in the steady-state level of a higher-molecular-weight protein complex ( 160-kDa) that cross-reacted with D1 antibodies. The steady-state level of the 160-kDa complex in thylakoids was also a linear function of cell growth irradiance. These observations suggest that photodamage to D1 converts stoichiometric amounts of D1 and D2 (i.e., the D1/D2 heterodimer) into a 160-kDa complex. This complex may help to stabilize the reaction-center proteins until degradation and replacement of D1 can occur. The results indicated an intrinsic half-time of about 60 min for the repair of individual PSII units, supporting the idea that degradation of D1 after photodamage is the rate-limiting step in the PSII repair process.Abbreviations Chl chlorophyll - PSI photosystem I - PSII photosystem II - D1 the 32-kDa reaction-center protein of PSII, encoded by the chloroplast psbA gene - D2 the 34-kDa reactioncenter protein of PSII, encoded by the chloroplast psbD gene - Q_A primary electron-accepting plastoquinone of PSII The work was supported by grant 94-37100-7529 from the US Department of Agriculture, National Research Initiative Competitive Grants Program.     

ANATOMICAL AND PHYSIOLOGICAL ACCLIMATION OF FATSIA JAPONICA LEAVES TO IRRADIANCE

Anatomical and physiological leaf characteristics and biomass production of Fatsia japonica plants were studied. Plants were grown in a growth chamber at 300 μmol m^-2 s^-1 (high light) and 50 μmol m^-2 s^-1 (low light) photosynthetic photon flux density. Plants grown under high light showed a net maximum photosynthetic rate 44% higher than plants grown under low light; the light compensation point and the light saturation point were also higher in high-light plants. Photosynthetic oxygen evolution in isolated chloroplasts was about 40% higher in high-light plants. However, chlorophyll content on a dry weight basis, on a leaf area basis, and per chloroplast was greater in plants grown under low light. Leaf thickness in high-light plants was 13% higher than in low-light plants. The number of chloroplasts was 30% higher in high-light leaves, while chloroplast size was only slightly higher. Chloroplast ultrastructure was also affected by light. Leaf dry weight, leaf area, and biomass production per plant were drastically reduced under low light. Thus, F. japonica is a plant that is able to acclimate to different photosynthetic photon flux density by altering its anatomical and physiological characteristics. However, low-light acclimation of this plant has a considerable limiting effect on biomass production.     

dr and spr/sr mutations of Chlamydomonas reinhardtii affecting D1 protein function and synthesis define two independent steps leading to chronic photoinhibition and confer differential fitness

The effects of introduced chloroplast gene mutations affecting D1 synthesis, turnover and function on photosynthesis, growth and competitive ability were examined in autotrophic cultures of Chlamydomonas reinhardtii (Chlorophyta) adapted to low or high irradiance. Few discernible effects were evident when the mutants were grown in low light (LL, 70 μmol m^?2 s^?1). The herbicide-resistant psbA mutation Ser²⁶⁴→ Ala (dr) slowed electron transfer and accelerated D1 degradation in cells grown under high light (HL, 600 μmol m^?2 s^?1). The maximum rate of light-and CO₂-saturated photosynthesis, cell growth rate and competitive ability in the dr mutant were reduced compared to wild type under HL. However, the wild-type rate of D1 synthesis in dr was adequate to compensate for accelerated D1 degradation. 16S rRNA mutations conferring resistance to streptomycin and spectinomycin (spr/sr) that altered chloroplast ribosome structure and assembly were used to inhibit chloroplast protein synthesis. In spr/sr cells grown under HL, D1 synthesis was reduced by 40–60% compared to wild type and D1 degradation was accelerated, leading to a 4-fold reduction in D1 pool size. The reduced D1 levels were accompanied by an elevation of F_o and a decline in F_v/F_m, quantum yield and maximum rate of CO₂-saturated photosynthesis. Chemostat experiments showed that the growth rate and competitive ability of spr/sr were reduced against both wild type and dr.     

Form II of D1 is important during transition from standard to high light intensity in Synechococcus sp. strain PCC 7942

In Synechococcus sp. strain PCC 7942 the D1 protein of Photosystem II is encoded by a multigene family; psbAI encodes Form I of D1 whereas both psbAII and psbAIII encode Form II. The psbA genes are differentially regulated in response to changes in light intensity, such that psbAI expression and Form I predominate at standard light intensity, whereas psbAII and psbAIII are induced at high light intensity, causing insertion of Form II into the thylakoids. The present study addressed whether high-light induced Form II is important for Synechococcus cells during adaptation to high light intensity. Wild-type Synechococcus, and mutants which produce only Form I (R2S2C3) or only Form II (R2K1), were co-cultured at standard light (130 E · m^–2 · s^–1) and then shifted to high light (750 E·m^–2·s^–1). Measurement of the proportion of each cell type at various time intervals revealed that the growth of R2S2C3, which has psbAII and psbAIII inactive, and thus lacks Form II, is transiently impaired upon shift to high light. Both mutants R2S2C3 and R2K1 maintained normal levels of psbA messages and D1 protein under standard and high light through an unknown mechanism that compensates for the inactive psbA genes. Thus, the impairment of R2S2C3 at high light is not due to a deficiency of D1 protein, but results from lack of Form II. We discounted the influence of possible secondary mutations by re-creating the psbA-inactivated mutants and testing the newly isolated strains. We conclude that Form II of D1 is intrinsically important for Synechococcus cells during a critical transition period after exposure to high light intensities.     

Slow degradation of the d1 protein is related to the susceptibility of low-light-grown pumpkin plants to photoinhibition

Photoinhibition of photosystem II (PSII) electron transport and subsequent degradation of the D1 protein were studied in pumpkin (Cucurbita pepo L.) leaves developed under high (1000 μmol m⁻² s⁻¹) and low (80 μmol m⁻² s⁻¹) photon flux densities. The low-light leaves were more susceptible to high light. This difference was greatly diminished when illumination was performed in the presence of chloramphenicol, indicating that a poor capacity to repair photodamaged PSII centers is decisive in the susceptibility of low-light leaves to photoinhibition. In fact, the first phases of the repair cycle, degradation and removal of photodamaged D1 protein from the reaction center complex, occurred slowly in low-light leaves, whereas in high-light leaves the degradation of the D1 protein more readily followed photoinhibition of PSII electron transport. A modified form of the D1 protein, with slightly slower electrophoretic mobility than the original D1, accumulated in the appressed thylakoid membranes of low-light leaves during illumination and was subsequently degraded only slowly.     

Dynamics of photosystem II heterogeneity in Dunaliella salina (green algae)

Based on the electron-transport properties on the reducing side of the reaction center, photosystem II (PS II) in green plants and algae occurs in two distinct forms. Centers with efficient electron-transport from Q_A to plastoquinone (Q_B-reducing) account for 75% of the total PS II in the thylakoid membrane. Centers that are photochemically competent but unable to transfer electrons from Q_A to Q_B (Q_B-nonreducing) account for the remaining 25% of total PS II and do not participate in plastoquinone reduction. In Dunaliella salina, the pool size of Q_B-nonreducing centers changes transiently when the light regime is perturbed during cell growth. In cells grown under moderate illumination intensity (500 E m^-2s^-1), dark incubation induces an increase (half-time 45 min) in the Q_B-nonreducing pool size from 25% to 35% of the total PS II. Subsequent illumination of these cells restores the steady-state concentration of Q_B-nonreducing centers to 25%. In cells grown under low illumination intensity (30 µE m^–2s^–1), dark incubation elicits no change in the relative concentration of Q_B-nonreducing centers. However, a transfer of low-light grown cells to moderate light induces a rapid (half-time 10 min) decrease in the Q_B-nonreducing pool size and a concomitant increase in the Q_B-reducing pool size. These and other results are explained in terms of a pool of Q_B-nonreducing centers existing in a steady-state relationship with Q_B-reducing centers and with a photochemically silent form of PS II in the thylakoid membrane of D. salina. It is proposed that Q_B-nonreducing centers are an intermediate stage in the process of damage and repair of PS II. It is further proposed that cells regulate the inflow and outflow of centers from the Q_B-nonreducing pool to maintain a constant pool size of Q_B-nonreducing centers in the thylakoid membrane.Abbreviations Chl chlorophyll - PS photosystem - Q_A primary quinone electron acceptor of PS II - Q_B secondary quinone electron acceptor of PS II - LHC light harvesting complex - F_o non-variable fluorescence yield - F_pl intermediate fluorescence yield plateau level - F_max maximum fluorescence yield - F_i mitial fluorescence yield increase from F_o to F_pl(F_pl-F_o) - F_v total variable fluorescence yield (F_max-F_o) - DCMU dichlorophenyl-dimethylurea     

A 43 kD light-regulated chloroplast RNA-binding protein interacts with the psbA 5′ non-translated leader RNA

Expression of the chloroplast psbA gene coding for the D1 protein of Photosystem II is subject to regulation at different levels in higher plants, including control of mRNA accumulation and translation. In dicots, the conserved 5 non-translated leader (5-UTR) of the psbA mRNA is sufficient to direct the light-dependent translation of the D1 protein. In this report we show that the psbA mRNA 5-UTR forms a stem-loop structure and binds a 43 kD chloroplast protein (43RNP). Binding of the 43RNP is sensitive to competition with poly(U), but insensitive to high concentrations of tRNA, the RNA homopolymers poly(A), poly(G), poly(C), or poly(A):poly(U) as a double-strand RNA. The 43RNP does not bind efficiently to the psbA mRNA 3 non-translated region, although the RNA sequence is U-rich and folds into a stem-loop. A deletion mutant of the psbA 5-UTR RNA in which 5 sequences of the stem-loop are removed does not affect 43RNP binding. Together, these properties suggest that the 43RNP binds most effectively to a specific single-strand U-rich sequence preceding the AUG start codon in the psbA mRNA. Binding of the 43RNP is not detectable in plastid protein extracts from 5-day-old dark-grown seedlings, but is detectable in light-grown seedlings as well as mature plants in the light and after shifted to the dark. The 43RNP is therefore a candidate for a regulatory RNA-binding protein that may control the accumulation and/or translation of the psbA mRNA during light-dependent seedling development.Abbreviations DMS dimethylsulfate - psb Photosystem II genes - RNP ribonucleoprotein - UTR non-translated leader - UV crosslinking ultra-violet light crosslinking     

Over-production of the D1 protein of photosystem II reaction centre in the cyanobacterium Synechococcus sp. PCC 7942

The unicellular cyanobacterium Synechococcus sp. PCC 7942 has three psbA genes encoding two different forms of the photosystem II reaction centre protein D1 (D1:1 and D1:2). The level of expression of these psbA genes and the synthesis of D1:1 and D1:2 are strongly regulated under varying light conditions. In order to better understand the regulatory mechanisms underlying these processes, we have constructed a strain of Synechococcus sp. PCC 7942 capable of over-producing psbA mRNA and D1 protein. In this study, we describe the over-expression of D1:1 using a tac-hybrid promoter in front of the psbAI gene in combination with lacI ^Q repressor system. Over-production of D1:1 was induced by growing cells for 12 h at 50 mol photons m^-2 s^-1 in the presence of 40 or 80 g/ml IPTG. The amount of psbAI mRNA and that of D1:1 protein in cells grown with IPTG was three times and two times higher, respectively. A higher concentration of IPTG (i.e., 150 g/ml) did not further increase the production of the psbAI message or D1:1. The over-production of D1:1 caused a decrease in the level of D1:2 synthesised, resulting in most PSII reaction centres containing D1:1. However, the over-production of D1:1 had no effect on the pigment composition (chlorophyll a or phycocyanin/number of cells) or the light-saturated rate of photosynthesis. This and the fact that the total amounts of D1 and D2 proteins were not affected by IPTG suggest that the number of PSII centres within the membranes remained unchanged. From these results, we conclude that expression of psbAI can be regulated by using the tac promoter and lacI ^Q system. However, the accumulation of D1:1 protein into the membrane is regulated by the number of PSII centres.     

Phosphatase activities in spinach thylakoid membranes-effectors, regulation and location

The dephosphorylation of seven phosphoproteins associated with Photosystem II or its chlorophyll a/b antenna in spinach thylakoids, was characterised. The rates were found to fall into two distinct groups. One, rapidly dephosphorylated, consisted of the two subunits (25 and 27 kD) of the major light harvesting complex of Photosystem II (LHC II) and a 12 kD polypeptide of unknown identity. A marked correlation between the dephosphorylation of these three phosphoproteins, strongly suggested that they were all dephosphorylated by the same enzyme. Within this group, the 25 kD subunit was consistently dephosphorylated most rapidly, probably reflecting its exclusive location in the peripheral pool of LHC II. The other group, only slowly dephosphorylated, included several PS II proteins such as the D1 and D2 reaction centre proteins, the chlorophyll-a binding protein CP43 and the 9 kD PS II-H phosphoprotein. No dephosphorylation was observed in either of the two groups in the absence of Mg²⁺-ions. Dephosphorylation of the two LHC II subunits took place in both grana and stroma-exposed regions of the thylakoid membrane. However, deposphorylation in the latter region was significantly more rapid, indicating a preferential dephosphorylation of the peripheral (or mobile) LHC II. Dephosphorylation of LHC II was found to be markedly affected by the redox state of thiol-groups, which may suggest a possible regulation of LHC II dephosphorylation involving the ferredoxin-thioredoxin system.Abbreviations CP 43 43 kD chlorophyll a- binding protein - D1 and D2 reaction centre proteins of PS II - LHC II light-harvesting complex of PS II - LHC II-25 25 kD subunit of LHC II - LHC II-27 27 kD subunit of LHC II - NEM N-ethylmaleimide - PP2C protein phosphatase 2C - PS II-H psb H gene product     

Implication of D1 degradation in phosphorylation-induced state transitions

State transitions and lateral migration of phosphorylated mobile-LHC II upon thylakoid unstacking have been reported as being interdependent. However, now the thyakoid unstacking event can be separated from the thyakoid phosphorylation and the associated F730/F685 enhancement by using the serine-type-protease inhibitor benzamidine. Thus, lateral migration appears not be necessary, and it can be shown that LHC II-rich fragments, originating in peripheral granal membranes, can be released by digitonin although in reduced amounts. On the other hand, phosphorylation of thylakoid proteins greatly stimulates the light-induced D1 degradation, which is observed in chloroplasts phosphorylated even at very low light (15 µmol m^–2s^–1). Thylakoid pretreatment with FSBA (the PS II protein-kinase inhibitor) blocks the light-induced and ATP-stimulated D1 degradation, and the F730/F685 ratio increase; this suggests that the dissociation of the PS II unit, resulting from the introduction of repulsive negative charges ( ATP groups) into LHC II and PS II core proteins, leads to D1 degradation. In chloroplast samples transferred to darkness following short-time phosphorylation, the D1 level is recovered. The results suggest that disassembly of PS II and D1 degradation occur parallel to State transitions. The removal of outer phospho-LHC II from PS II and its association with PS I at the periphery of grana may allow D1 degradation and increased light utilization by PS I, while net de novo synthesis of D1, stimulated by ATP, may lead to the assembly of new PS II units which could bind dephosphorylated LHC II in the dark, resulting in increased light utilization by PS II.     

Light acclimation in leaves of the juvenile and adult life phases of ivy (Hedera helix)

Hoflacher, H. and Bauer, H. 1982. Light acclimation in leaves of the juvenile and adult life phases of ivy (Hedera helix). – Physiol. Plant. 56: 177–182. Light acclimation was investigated during the juvenile and adult life phases of the whole-plant-development in Hedera helix L. For this purpose, cuttings of the juvenile and adult parts of one single parent plant were grown under low-light (PAR 30–50 μmol photons m^?2 s^?1) and high-light (PAR 300–500 μmol m^?2 s^?1) conditions: CO₂ exchange, chloroplast functions, and specific anatomy of fully developed leaves differentiated under these conditions were determined. In juvenile plants the leaves formed under low and high light had light-saturated rates of net photosynthesis of 6.5 and 11.1 mg CO₂ (dm leaf area)^?2 h^?1, respectively. In adult plants the rates were 9.4 and 22.2 mg dm^?2 h^?1, indicating a more pronounced capacity for acclimation to strong light in the adult life phase. Higher photosynthetic capacities were accompanied by higher conductances for the CO₂ transfer through the stomata, leading to almost the same CO₂ concentration in the intercellular spaces. Thus, stomatal conductances were not primarily responsible for the different photo-synthetic capacities. The higher rates in adult and high-light grown leaves were mainly the result of formation of thicker leaves with more chloroplasts per unit leaf area. Expressed per chloroplast, the photosynthetic capacity, the Hill reaction, and the activity of ribulose bisphosphate carboxylase were almost identical in plants grown in low-light and high-light. Measurements of photosynthetic capacity and thickness of leaves of Hedera sampled from field habitats with contrasting light regimes confirm the results of growth chamber studies. It is, therefore, concluded that both life phases of Hedera are capable of acclimating to strong light, but that during the juvenile phase this capacity is not fully developed.     

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.     

Adjustment of thylakoid plastoquinone content and Photosystem I electron donor pool size in response to growth temperature and growth irradiance in winter rye (Secale cereale L.)

Winter rye (Secale cereale L. cv Musketeer) grown at 5 °C/250 µmol photons m^–2 s^–1 exhibited a relative reduction state of PS II comparable to that of rye grown at 20 °C but high light (800 µmol photons m^–2 s^–1) (1-q_P = 0.32) whereas winter rye grown at 20 °C/250 µmol photons m^–2 s^–1 exhibited values of 1-q_P ( 0.15) comparable to plants grown at 5 °C but low light (50 µmol photons m^–2 s^–1). The apparent size of the electron donor pool to PS I, estimated either in vivo or in vitro in the presence of methylviologen by A₈₂₀ was positively correlated with the relative reduction state of PS II under the steady-state growth conditions. Immunoblotting of rye thylakoid polypeptides indicated that the relative contents of Lhcb1, Lhcb2, D1, Cyt f, PC, PsaA/PsaB heterodimer and the -subunit of ATPase complex exhibited minimal changes on a Chl basis. In contrast, a 2-fold increase in plastoquinone A content was associated with increasing growth irradiance at growth temperatures of either 5 or 20 °C. We suggest that the increases in the apparent size of the electron donor pool to PS I associated with rye grown at either 5 °C/250 µmol photons m^–2 s^–1or 20 °C/800 µmol photons m^–2 s^–1 may be explained by an increased thylakoid plastoquinone A content, coupled with possible enhanced PS I cyclic electron transport and/or increased capacity for electron donation from the stroma to the intersystem electron transport chain. The results are discussed with respect to photosynthetic adjustment to changes in PS II excitation pressure in winter rye.     

Mutation of the Chlamydomonas reinhardtii analogue of residue M210 of the Rhodobacter sphaeroides reaction center slows primary electron transfer in Photosystem II

Primary charge separation within Photosystem II (PS II) is much slower (time constant 21 ps) than the equivalent step in the related reaction center (RC) found in purple bacteria ( 3 ps). In the case of the bacterial RC, replacement of a specific tyrosine residue within the M subunit (at position 210 in Rhodobacter sphaeroides), by a leucine residue slows down charge separation to 20 ps. Significantly the analogous residue in PS II, within the D2 polypeptide, is a leucine not a tyrosine (at position D2-205, Chlamydomonas reinhardtii numbering). Consequently, it has been postulated [Hastings et al. (1992) Biochemistry 31: 7638–7647] that the rate of electron transfer could be increased in PS II by replacing this leucine residue with tyrosine. We have tested this hypothesis by constructing the D2-Leu205Tyr mutant in the green alga, Chlamydomonas reinhardtii, through transformation of the chloroplast genome. Primary charge separation was examined in isolated PS II RCs by time-resolved optical spectroscopy and was found to occur with a time constant of 40 ps. We conclude that mutation of D2-Leu205 to Tyr does not increase the rate of charge separation in PS II. The slower kinetics of primary charge separation in wild type PS II are probably not due to a specific difference in primary structure compared with the bacterial RC but rather a consequence of the P680 singlet excited state being a shallower trap for excitation energy within the reaction center.     

A Fourier transform infrared spectroscopic study of the effects of hydrogen peroxide and high light on protein conformations of Photosystem II

Degradation of the reaction center-binding protein D1 of Photosystem II (PS II) during photoinhibition is dependent on the action of active oxygen species and/or D1-specific proteases. Protein conformational changes may be involved in the process of D1 degradation. In the present study, we determined the effect of H₂O₂ on spinach PS II-enriched membranes and core complexes with respect to electron transport, Mn content and protein secondary structural changes as measured by Fourier transform infrared (FTIR) spectroscopy. H₂O₂ is effective in removing catalytic Mn in PS II, especially in PS II core complexes depleted of OEC18 and OEC24, impairing the donor-side. By quantitative analysis of the amide I band (1600 – 1700 cm^-1) with both aqueous and dehydrated PS II samples, we found that no significant secondary structural changes are associated with H₂O₂ treatment in the dark, even though there is some cleavage of the D1 protein by H₂O₂ treatment as determined by Western analysis with specific antibodies. In contrast, a large decrease in the -helices in the PS II core occurs, with or without H₂O₂ treatment, after 20 min strong illumination and there is more extensive degradation of the D1 protein. Our results suggest that high light enhances the cleavage of the D1 protein which is reflected in the large protein secondary structural changes in PS II detected by FTIR measurements.     

A robust protocol for site-directed mutagenesis of the D1 protein inChlamydomonas reinhardtii: A PCR-splicedpsbA gene in a plasmid conferring spectinomycin resistance was introduced into apsbA deletion strain

In this paper, we describe a protocol to obtain a site-directed mutants in thepsbA gene ofChlamydomonas reinhardtii, which overcomes several drawbacks of previous protocols, and makes it possible to generate a mutant within a month. Since the large size of the gene, and the presence of four large introns has made molecular genetics of thepsbA gene rather unwieldy, we have spliced all of the exons of thepsbA gene by PCR to facilitate genetic manipulation and sequencing of the gene. The resultant construct (plasmid pBA153, with several unique restriction sites introduced at exon boundaries) carried 1.2 and 1.8 kb intact sequences from the 5- and 3-flanking regions, respectively. The plasmid was used to transform a D1-deletion mutant and was found to complement the deletion and restore photosynthetic activity. In addition, a bacterialaadA gene conferring spectinomycin resistance (spe ^r) was inserted downstream of the intron-freepsbA gene, to give construct pBA155. This allowed selection of mutant strains deficient in photosynthesis by using spectinomycin resistance, and eliminated the possibility of selection for revertant strains which is a consequence of having to use photosynthetic activity as a selection pressure. Finally, pBA155 was used to construct pBA157, in which additional restriction sites were inserted to facilitate cassette mutagenesis for generation of mutations in spans thought to be involved in donor-side interactions. AllpsbA deletion strains transformed with intron-freepsbA-aadA constructs encoding the wild-type D1 sequence, and screened on spectinomycin plates for thespe ^r phenotype, were able to grow photosynthetically, and all showed identical kinetics for electron transfer from primary (Q_A) to secondary quinone (Q_B) in Photosystem II, as assayed by the decay of the high fluorescence yield on oxidation of the reduced primary acceptor (Q_A ^–).     

Wavelength-dependent photodamage to <Emphasis Type="Italic">Chlorella</Emphasis> investigated with a new type of multi-color PAM chlorophyll fluorometer

A new type of multi-color PAM chlorophyll fluorometer (Schreiber et al. 2012) was applied for measurements of photodamage to photosystem II (PS II) in optically thin suspensions of Chlorella (200 μg Chl l^?1) in the presence of 1 mM lincomycin. An action spectrum of the relative decrease of F _v/F _m in the 440–625 nm range was measured, which not only showed the expected high activity in the blue, but at a lower level also substantial activity above 540 nm. With the same dilute suspension, a PS II absorption spectrum was derived via measurements of the O-I₁ rise kinetics induced by differently colored strong light at defined incident quantum flux densities. After normalization of the two spectra at 625 nm, the relative extent of photodamage at 440–480 nm proved substantially higher than absorption by PS II, whereas the two spectra were close to identical in the 540–625 nm range. Hence, overall photodamage to PS II appears to consist of two components, one of which is due to light absorbed by PS II pigments, whereas the other one is likely to involve direct light absorption by Mn in the oxygen-evolving complex (Hakala et al. Biochim Biophys Acta 1706:68–80, 2005). Based on this rationale, an action spectrum of the Mn mechanism of photodamage was deconvoluted from the overall action spectrum, declining steeply above 480 nm. An almost identical Mn-spectrum was derived by another approach with the PAR of the various colors being adjusted to give identical rates of PS II turnover, PAR _II. The tentative, basic assumption of negligibly small contribution of the Mn mechanism to photodamage above 540 nm was supported by supplementary measurements using an external 665 nm lamp. 665 nm not only gave about two times PS II turnover as compared to 625 nm, but also about two times photodamage.