Changes in photosynthesis and pigmentation in an agp deletion mutant of the cyanobacterium Synechocystis sp

The agp gene encoding ADP-glucose pyrophosphorylase is involved in cyanobacterial glycogen synthesis. By in vitro DNA recombination technology, agp deletion mutant (agp ^–) of cyanobacterium Synechocystis sp. PCC 6803 was constructed. This mutation led to a complete absence of glycogen biosynthesis. As compared with WT (wild type), a 60% decrease in ratio of the c-phycocyanine/chlorophyll a and no significant change in the carotenoid/chlorophyll a were observed in agp ^– cells. The agp ^– mutant had 38% less photosynthetic capacity when grown in light over 600 mol m^–2 s^–1. Under lower light intensity, the final biomass of the mutant strain was only 1.1 times of that of the WT strain under mixotrophic condition after 6 d culture. Under higher light intensity, however, the final biomass of the WT strain under mixotrophic conditions was 3 times that of the mutant strain after 6 d culture and 1.5 times under photoautotrophic conditions. The results indicate that there is a minimum requirement for glycogen synthesis for normal growth and development in cyanobacteria.     

The lumenal loop connecting transmembrane helices I and II of the D1 polypeptide is important for assembly of the photosystem two complex

Current structural models indicate that the D1 and D2 polypeptides of the Photosystem two reaction center complex (PS II RC) each span the thylakoid membrane five times. In order to assess the importance of the lumenal extrinsic loop that connects transmembrane helices I and II of D1 we have constructed five deletion mutants and two double mutants in the cyanobaterium Synechocystic sp. PCC 6803. Four of the deletion mutants (59–65, 69–74, 79–86 and 109–110) are obligate photoheterotrophs unable to accumulate D1 in the membrane as assayed by immunoblotting experiments or pulse-labelling experiments using [³⁵S]-methionine. In contrast deletion mutant 100 which lacks A100 behaved very similarly to the WT control strain in terms of photoautotrophic growth rate, saturated rates of oxygen evolution, flash-induced oxygen evolution, fluorescence induction and decay, and thermoluminescence. 100 is the first example of an internal deletion on the lumenal side of the D1 polypeptide that is benign to photosystem two function. Double mutant D103G/E104A also behaves similarly to the WT control strain leading to the conclusion that residues D103 and E104 are unlikely to be involved in ligating the metal ions Mn or Ca²⁺, which are needed for photosynthetic oxygen evolution. Double mutant, G109A/G110A, was constructed to assess the significance of this GlyGly motif which is also conserved in the L subunit of purple bacterial reaction centres. The G109A/G110A mutant is able to evolve oxygen at approximately 50–70% of WT rates but is unable to grow phatoautotrophically apparently because of an enhanced sensitivity to photoinactivation than the WT control strain. A photoautotropic revertant was isolated from this strain and shown to result from a mutation that restored the WT codon at position 109. Pulse-chase experiments in cells using [³⁵S]-methionine showed that resistance to photoinhibition in the revertant correlated with an enhanced rate of incorporation of D1 into the membrane compared to mutant G109A/G110A. The sensitivity to photoinhibition shown by the G109A/G110A mutant is therefore consistent with a perturbation to the D1 repair cycle possibly at the level of D1 synthesis or incorporation of D1 into the PS II complex.Abbreviations DCMU- 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Hepes- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes- 4-morpholineethanesulfonic acid - PCR- polymerase chain reaction - PS II- Photosystem II - TL- thermoluminescence - PQ- plastoquinone - PS II^–- absence of PS II activity - PS^–- incapable of photoautotrophic growth - Q_A- primary plastoquinone electron acceptor - Q_B- secondary plastoquinone electron acceptor - SDS- sodium dodecyl sulphate     

On the functional significance of the polypeptide PsbY for photosynthetic water oxidation in the cyanobacterium<Emphasis Type="Italic"> Synechocystis</Emphasis> sp. strain PCC 6803

Recent investigations have revealed that the cyanobacterial photosystem II complex contains more than 26 polypeptides. The functions of most of the low-molecular-mass polypeptides, including PsbY, have remained elusive. Here we present a comparative characterization of the wild-type Synechocystis sp. strain PCC 6803 and a PsbY-free mutant derived from it. The results show that growth of the PsbY-free mutant was comparable to that of the wild-type when cells were cultivated in complete BG11 medium or under initial manganese or chloride limitation, and when illuminated at 20 or 200 E m^–2 s^–1. However, while growth rates of both the wild-type and the PsbY-free mutant were reduced when cells were cultivated in BG11 medium in the absence of calcium, the reduction was significantly greater in the case of the PsbY-free mutant. This differential effect on growth of the mutant relative to the wild-type in CaCl₂ deficient medium was detected when the cells were illuminated with high-intensity light (200 E m^–2 s^–1) but not when light levels were lower (20 E m^–2 s^–1). The differential effect on growth was associated with lower O₂ evolving activity in the mutant compared to wild-type cells. The mutant was also found to be more sensitive to photoinhibition, and showed an altered pattern of fluorescence emission at 77 K. In addition, mass spectrometric analysis revealed that PsbY-free cells cultivated in CaCl₂ sufficient medium (in which no growth reduction was observed) had a significantly higher O₂ evolution from hydrogen peroxide and a lower O₂ evolution from water under flash light illumination than wild-type cells. These results imply that photosystem II is slightly impaired in the PsbY-free mutant, and that the mutant is less capable of coping with low levels of Ca²⁺ than the wild-type.Communicated by R. G. Herrmann     

Characteristics of mixotrophic growth of Synechocystis sp. in an enclosed photobioreactor

Synechocystis sp. PCC 6803 was grown in a 2.5 l enclosed photobioreactor on medium with or without glucose. The incident light intensities ranged from 1.5 klux to 7 klux. The highest average specific growth rates of mixotrophic culture and photoautotrophic culture were, respectively, 1.3 h^–1 at a light intensity of 7 klux on 3.2 g l^–1 glucose and 0.3 h^–1 at both light intensities of 5 klux and 7 klux. The highest cell density 2.5 g l ^–1 was obtained at both of light intensities 5 klux and 7 klux on 3.2 g glucose l^–1. Glucose consumption decreased with decreasing light intensity. The energy yields of mixotrophic cultures were 4 to 6 times higher than that of photoautotrophic cultures. Light favored mixotrophic growth of Synechocystis sp. PCC 6803, especially at higher light intensities (5–7 klux).     

Investigation of a requirement for the PsbP-like protein in Synechocystis sp. PCC 6803

The sll1418 gene encodes a PsbP-like protein in Synechocystis sp. PCC 6803. Expression of sll1418 was similar in BG-11 and in Cl⁻- or Ca²⁺-limiting media, and inactivation of sll1418 did not prevent photoautotrophic growth in normal or nutrient-limiting conditions. Also the wild-type and ΔPsbP strains exhibited similar oxygen evolution and assembly of Photosystem II (PS II) centers. Inactivation of sll1418 in the ΔPsbO: ΔPsbP, ΔPsbQ:ΔPsbP, ΔPsbU:ΔPsbP and ΔPsbV:ΔPsbP mutants did not prevent photoautotrophy or alter PS II assembly and oxygen evolution in these strains. Moreover, the absence of PsbP did not affect the ability of alkaline pH to restore photoautotrophic growth in the ΔPsbO:ΔPsbU strain. The PsbO, PsbU and PsbV proteins are required for thermostability of PS II and thermal acclimation in Synechocystis sp. PCC 6803 [Kimura et al. (2002) Plant Cell Physiol 43: 932–938]. However, thermostability and thermal acclimation in ΔPsbP cells were similar to wild type. These results are consistent with the conclusion that PsbP is associated with ∼3 of PS II centers, and may play a regulatory role in PS II [Thornton et al. (2004) Plant Cell 16: 2164–2175].     

Inactivation of the water-oxidizing enzyme in manganese stabilizing protein-free mutant cells of the cyanobacteria Synechococcus PCC7942 and Synechocystic PCC6803 during dark incubation and conditions leading to photoactivation

The previously constructed MSP (manganese stabilizing protein-psbO gene product)-free mutant of Synechococcus PCC7942 (Bockholt R, Masepohl B and Pistorius E K (1991) FEBS Lett 294: 59–63) and a newly constructed MSP-free mutant of Synechocystis PCC6803 were investigated with respect to the inactivation of the water-oxidizing enzyme during dark incubation. O₂ evolution in the MSP-free mutant cells, when measured with a sequence of short saturating light flashes, was practically zero after an extended dark adaptation, while O₂ evolution in the corresponding wild type cells remained nearly constant. It could be shown that this inactivation could be reversed by photoactivation. With isolated thylakoid membranes from the MSP-free mutant of PCC7942, it could be demonstrated that photoactivation required illumination in the presence of Mn²⁺ and Ca²⁺, while Cl^– addition was not required under our experimental conditions. Moreover, an extended analysis of the kinetic properties of the water-oxidizing enzyme (kinetics of the S₃(S₄)S₀ transition, S-state distribution, deactivation kinetics) in wild type and mutant cells of Synechococcus PCC7942 and Synechocystis PCC6803 was performed, and the events possibly leading to the reversible inactivation of the water-oxidizing enzyme in the mutant cells are discussed. We could also show that the water-oxidizing enzyme in the MSP-free mutant cells is more sensitive to inhibition by added NH₄Cl-suggesting that NH₃ might be a physiological inhibitor of the water oxidizing enzyme in the absence of MSP.Abbreviations Chl chlorophyll - DCBQ 2,6-Dichloro-p-benzoquinone - MSP manganese stabilizing protein (psbO gene product) - PS II Photosystem II - WOE water oxidizing enzyme - WT wild type This paper is dedicated to Prof. Dr. Bernard Axelrod on the occasion of his 80th birthday     

Decrease of polypeptides in the PS I antenna complex with increasing growth irradiance in the red alga Porphyridium cruentum

Thylakoids isolated from cells of the red alga Porphyridium cruentum exhibit an increased PS I activity on a chlorophyll basis with increasing growth irradiance, even though the stoichiometry of Photosystems I and II in such cells shows little change (Cunningham et al. (1989) Plant Physiol 91: 1179–1187). PS I activity was 26% greater in thylakoids of cells acclimated at 280 mol photons · m^–2 · s^–1 (VHL) than in cells acclimated at 10 mol photons · m^–2 · s^–1 (LL), indicating a change in the light absorbance capacity of PS I. Upon isolating PS I holocomplexes from VHL cells it was found that they contained 132±9 Chl/P700 while those obtained from LL cells had 165±4 Chl/P700. Examination of the polypeptide composition of PS I holocomplexes on SDS-PAGE showed a notable decrease of three polypeptides (19.5, 21.0 and 22 kDa) in VHL-complexes relative to LL-complexes. These polypeptides belong to a novel LHC I complex, recently discovered in red algae (Wolfe et al. (1994a) Nature 367: 566–568), that lacks Chl b and includes at least six different polypeptides. We suggest that the decrease in PS I Chl antenna size observed with increasing irradiance is attributable to changes occurring in the LHC I-antenna complex. Evidence for a Chl-binding antenna complex associated with PS II core complexes is lacking at this point. LHC II-type polypeptides were not observed in functionally active PS II preparations (Wolfe et al. (1994b) Biochimica Biophysica Acta 1188: 357–366), nor did we detect polypeptides that showed immunocross-reactivity with LHC II specific antisera (made to Chlamydomonas and Euglena LHC II).Abbreviations Bis-Tris bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane - DCPIP 2,6-dichlorophenol indophenol - -dm dodecyl--d-maltoside - HL high light of 150 mol photons · m^–2 · s^–1 - LGB lower green band - LHC I light-harvesting complex of PS I - LHC II light-harvesting complex of PS II - LL low light of 10 mol photons · m^–2 · s^–1 - ML medium light of 50 mol photons · m^–2 · s^–1 - MES 2-(N-morpholino) ethanesulfonic acid - P700 reaction center of PS I - PFD photon flux density - Trizma tris(hydroxymethyl)aminomethane - UGB upper green band - VHL very high light of 280 mol photons · m^–2 · s^–1     

Chimaeric CP47 mutants of the cyanobacterium Synechocystis sp. PCC 6803 carrying spinach sequences: Construction and function

Chimaeric mutants of the cyanobacterium Synechocystis sp. PCC 6803 have been generated carrying part or all of the spinach psbB gene, encoding CP47 (one of the chlorophyll-binding core antenna proteins in Photosystem II). The mutant in which the entire psbB gene had been replaced by the homologous gene from spinach was an obligate photoheterotroph and lacked Photosystem II complexes in its thylakoid membranes. However, this strain could be transformed with plasmids carrying selected regions of Synechocystis psbB to give rise to photoautotrophs with a chimaeric spinach/cyanobacterial CP47 protein. This process involved heterologous recombination in the cyanobacterium between psbB sequences from spinach and Synechocystis 6803; which was found to be reasonably effective in Synechocystis. Also other approaches were used that can produce a broad spectrum of chimaeric mutants in a single experiment. Functional characterization of the chimaeric photoautotrophic mutants indicated that if a decrease in the photoautotrophic growth rates was observed, this was correlated with a decrease in the number of Photosystem II reaction centers (on a chlorophyll basis) in the thylakoid membrane and with a decrease in oxygen evolution rates. Remaining Photosystem II reaction centers in these chimaeric mutants appeared to function rather normally, but thermoluminescence and chlorophyll a fluorescence measurements provided evidence for a destabilization of Q_B ^–. This illustrates the sensitivity of the functional properties of the PS II reaction center to mild perturbations in a neighboring protein.Abbreviations diuron 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_v variable chlorophyll a fluorescence - HEPES N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) - (k)bp (kilo)base pairs - PS II Photosystem II - Q_A primary electron-accepting plastoquinone in Photosystem II - Q_B secondary electron-accepting plastoquinone in Photosystem II - SDS sodium dodecyl sulfate     

A New Type of Adaptation of the Cyanobacterium Spirulina platensis to Illumination Conditions

Incubation of cells of the cyanobacterium Spirulina platensis under conditions of exposure to low-intensity (2–3 E m^–2 s^–1) red light, which was predominantly absorbed by photosystem I (PS I), caused atypical adaptation changes. Invariable pigment composition and stoichiometry of the photosystems was observed in the cells incubated under these conditions against the background of a decrease in the rate of photosynthetic fixation of ₂ (by one-half) and a 1.5-fold increase in the rate of dark respiration relative to cells incubated under conditions of exposure to green light. Comparison of these data with a high rate of dark relaxation of P700⁺ in the presence of diuron suggests that deficiency of reduced equivalents on the donor side of PS I in Spirulina cells exposed to red light is compensated by electron supply from the respiratory chain NAD(P)H dehydrogenase complex.     

Characterization of a Synechococcus sp. strain PCC 7002 mutant lacking Photosystem I. Protein assembly and energy distribution in the absence of the Photosystem I reaction center core complex

A Synechococcus sp. strain PCC 7002 psaAB::cat mutant has been constructed by deletional interposon mutagenesis of the psaA and psaB genes through selection and segregation under low-light conditions. This strain can grow photoheterotrophically with glycerol as carbon source with a doubling time of 25 h at low light intensity (10 E m^–2 s^–1). No Photosystem I (PS I)-associated chlorophyll fluorescence emission peak was detected in the psaAB::cat mutant. The chlorophyll content of the psaAB::cat mutant was approximately 20% that of the wild-type strain on a per cell basis. In the absence of the PsaA and PsaB proteins, several other PS I proteins do not accumulate to normal levels. Assembly of the peripheral PS I proteins PsaC,PsaD, PsaE, and PsaL is dependent on the presence of the PsaA and PsaB heterodimer core. The precursor form of PsaF may be inserted into the thylakoid membrane but is not processed to its mature form in the absence of PsaA and PsaB. The absence of PS I reaction centers has no apparent effect on Photosystem II (PS II) assembly and activity. Although the mutant exhibited somewhat greater fluorescence emission from phycocyanin, most of the light energy absorbed by phycobilisomes was efficiently transferred to the PS II reaction centers in the absence of the PS I. No light state transition could be detected in the psaAB::cat strain; in the absence of PS I, cells remain in state 1. Development of this relatively light-tolerant strain lacking PS I provides an important new tool for the genetic manipulation of PS I and further demonstrates the utility of Synechococcus sp. PCC 7002 for structural and functional analyses of the PS I reaction center.Abbreviations ATCC American type culture collection - Chl chlorophyll - DCMU 3-(3,4-dichlorophyl)-1,1-dimethylurea - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid] - PCC Pasteur culture collection - PS I Photosystem I - PS II Photosystem II - SDS sodium dodecyl sulfate     

Analysis of xanthophyll cycle carotenoids and chlorophyll fluorescence in light intensity-dependent chlorophyll-deficient mutants of wheat and barley

Three light intensity-dependent Chl b-deficient mutants, two in wheat and one in barley, were analyzed for their xanthophyll cycle carotenoids and Chl fluorescence characteristics under two different growth PFDs (30 versus 600 mol photons·m^–2 s^–1 incident light). Mutants grown under low light possessed lower levels of total Chls and carotenoids per unit leaf area compared to wild type plants, but the relative proportions of the two did not vary markedly between strains. In contrast, mutants grown under high light had much lower levels of Chl, leading to markedly greater carotenoid to Chl ratios in the mutants when compared to wild type. Under low light conditions the carotenoids of the xanthophyll cycle comprised approximately 15% of the total carotenoids in all strains; under high light the xanthophyll cycle pool increased to over 30% of the total carotenoids in wild type plants and to over 50% of the total carotenoids in the three mutant strains. Whereas the xanthophyll cycle remained fairly epoxidized in all plants grown under low light, plants grown under high light exhibited a considerable degree of conversion of the xanthophyll cycle into antheraxanthin and zeaxanthin during the diurnal cycle, with almost complete conversion (over 90%) occurring only in the mutants. 50 to 95% of the xanthophyll cycle was retained as antheraxanthin and zeaxanthin overnight in these mutants which also exhibited sustained depressions in PS II photochemical efficiency (F_v/F_m), which may have resulted from a sustained high level of photoprotective energy dissipation activity. The relatively larger xanthophyll cycle pool in the Chl b-deficient mutant could result in part from the reported concentration of the xanthophyll cycle in the inner antenna complexes, given that the Chl b-deficient mutants are deficient in the peripheral LHC-II complexes.Abbreviations A antheraxanthin - Chl chlorophyll - F_o and F_m minimal yield (at open PS II reaction centers) and maximal yield (at closed centers) of chlorophyll fluorescence in darkness - F level of fluorescence during illumination with photosynthetically active radiation - F_m maximal yield (at closed centers) of chlorophyll fluorescence during illumination with photosynthetically active radiation - (F_m–F)/F_m actual efficiency of PS II during illumination with photosynthetically active radiation - F_v/F_m+(F_m–F_o)/F_m intrinsic efficiency of PS II in darkness - LHC_II light-harvesting chlorophyll-protein complex of Photosystem II - PFD photon flux density (between 400 and 700 nm) - PS I Photosystem I - PS II Photosystem II - V violaxanthin - Z zeaxanthin     

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

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

Changes in Photosystem II fluorescence in Chlamydomonas reinhardtii exposed to increasing levels of irradiance in relationship to the photosynthetic response to light

The effects of a 60 min exposure to photosynthetic photon flux densities ranging from 300 to 2200 mol m^–2s^–1 on the photosynthetic light response curve and on PS II heterogeneity as reflected in chlorophyll a fluorescence were investigated using the unicellular green alga Chlamydomonas reinhardtii. It was established that exposure to high light acts at three different regulatory or inhibitory levels; 1) regulation occurs from 300 to 780 mol m^–2s^–1 where total amount of PS II centers and the shape of the light response curve is not significantly changed, 2) a first photoinhibitory range above 780 up to 1600 mol m^–2s^–1 where a progressive inhibition of the quantum yield and the rate of bending (convexity) of the light response curve can be related to the loss of Q_B-reducing centers and 3) a second photoinhibitory range above 1600 mol m^–2s^–1 where the rate of light saturated photosynthesis also decreases and convexity reaches zero. This was related to a particularly large decrease in PS II centers and a large increase in spill-over in energy to PS I.Abbreviations Chl chlorophyll - DCMU 3,(3,4-dichlorophenyl)-1,1-dimethylurea - F_M maximal fluorescence yield - F_pl intermediate fluorescence yield plateau level - F₀ non-variable fluorescence yield - F_v total variable fluorescence yield (F_M-F₀) - initial slope to the light response curve, used as an estimate of initial quantum yield - convexity (rate of bending) of the light response curve of photosynthesis - LHC light-harvesting complex - P_max maximum rate of photosynthesis - PQ plastoquinone - Q photosynthetically active photon flux density (400–700 nm, mol m^–2s^–1) - PS photosystem - Q_A and Q_B primary and secondary quinone electron acceptor of PS II     

Optimization of growth and fatty acid composition of a unicellular marine picoplankton,Nannochloropsis sp., with enriched carbon sources

A unicellular marine picoplankton, Nannochloropsis sp., was grown under CO₂-enriched photoautotrophic or/and acetate-added mixotrophic conditions. Photoautotrophic conditions with enriched CO₂ of 2800 l CO₂ l^–1 and aeration gave the highest biomass yield (634 mg dry wt l^–1), the highest total lipid content (9% of dry wt), total fatty acids (64 mg g^–1 dry wt), polyunsaturated fatty acids (35% total fatty acids) and eicosapentaenoic acid (EPA, 20:53) (16 mg g^–1 dry wt or 25% of total fatty acids). Mixotrophic cultures gave a greater protein content but less carbohydrates. Adding sodium acetate (2 mM) decreased the amounts of the total fatty acids and EPA. Elevation of CO₂ in photoautotrophic culture thus enhances growth and raises the production of EPA in Nannochloropsis sp.     

Very high light resistant mutants of Chlamydomonas reinhardtii: Responses of Photosystem II,nonphotochemical quenching and xanthophyll pigments to light and CO2

We have isolated very high light resistant nuclear mutants (VHL ^R) in Chlamydomonas reinhardtii, that grow in 1500–2000 mol photons m^–2 s^–1 (VHL) lethal to wildtype. Four nonallelic mutants have been characterized in terms of Photosystem II (PS II) function, nonphotochemical quenching (NPQ) and xanthophyll pigments in relation to acclimation and survival under light stress. In one class of VHL ^R mutants isolated from wild type (S4 and S9), VHL resistance was accompanied by slower PS II electron transfer, reduced connectivity between PS II centers and decreased PS II efficiency. These lesions in PS II function were already present in the herbicide resistant D1 mutant A251L (L ^*) from which another class of VHL ^R mutants (L4 and L30) were isolated, confirming that optimal PS II function was not critical for survival in very high light. Survival of all four VHL ^R mutants was independent of CO₂ availability, whereas photoprotective processes were not. The de-epoxidation state (DPS) of the xanthophyll cycle pigments in high light (HL, 600 mol photons m^–2 s^–1) was strongly depressed when all genotypes were grown in 5% CO₂. In S4 and S9 grown in air under HL and VHL, high DPS was well correlated with high NPQ. However when the same genotypes were grown in 5% CO₂, high DPS did not result in high NPQ, probably because high photosynthetic rates decreased thylakoid pH. Although high NPQ lowered the reduction state of PS II in air compared to 5% CO₂ at HL in wildtype, S4 and S9, this did not occur during growth of S4 and S9 in VHL. L ^* and VHL ^R mutants L4 and L30, also showed high DPS with low NPQ when grown air or 5% CO₂, possibly because they were unable to maintain sufficiently high pH due to constitutively impaired PS II electron transport. Although dissipation of excess photon energy through NPQ may contribute to VHL resistance, there is little evidence that the different genes conferring the VHL ^R phenotype affect this form of photoprotection. Rather, the decline of chlorophyll per biomass in all VHL ^R mutants grown under VHL suggests these genes may be involved in regulating antenna components and photosystem stoichiometries.This revised version was published online in October 2005 with corrections to the Cover Date.     

The role of chloroplast-encoded protein biosynthesis on the rate of D1 protein degradation in Dunaliella salina

Mechanistic aspects of the Photosystem II (PS II) damage and repair cycle in Dunaliella salina were investigated. The work addressed the role of chloroplast-encoded protein biosynthesis on the rate of the D1 protein (chloroplast psbA gene product) degradation, following photoinhibition of PS II under in vivo conditions. Cells were grown under different light-intensities and the rate of D1 photodamage and degradation was measured via pulse-chase measurements with (³⁵S)sulfate. It is shown that no detectable difference exists in the rate of D1 degradation in D. salina, measured in the presence or absence of lincomycin, a chloroplast protein biosynthesis inhibitor. The results suggest that de novo D1 biosynthesis does not play a role in the regulation of D1 degradation. In low-light (100 mol photons m^–2 s^–1) grown cells, the rate of photodamage to D1 did not exceed the rate of its degradation and replacement. In high-light (2200 mol photons m^–1 s^–1) grown cells, the rate of D1 photodamage was faster than the rate of its degradation, resulting in a significant accumulation of photoinactivated PS II centers in the chloroplast thylakoids (chronic photoinhibition). The latter was coincident with the appearance of a 160 kD complex that contained photodamaged D1. Electron micrographs of D. salina thylakoids revealed extensive grana stacks in the thylakoid membrane of low-light grown cells. Only rudimentary appressions consisting of simple membrane pairings were found in the high-light grown cells. The results are discussed in terms of the regulation of D1 degradation in chloroplasts under in vivo conditions.Abbreviations Chl chlorophyll - 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 - HL high light - LL low light - Linc lincomycin     

Oxidizing side of the cyanobacterial Photosystem I: Mutational analysis of the luminal H loop of the PsaB subunit

PS I core proteins are expected to interact with the electron donor proteins plastocyanin or cytochrome c ₆. To investigate the role of the luminal H loop of PsaB in the assembly and function of the PS I complex, we generated 15 deletion and repetition mutations in the H loop of the PsaB protein from Synechocystis sp. PCC 6803. The mutant strains differed in their photoautotrophic growth. The PS I proteins could not be detected in the membranes of mutants in which the N438–E448, I453–T464, or S500–G512 region was deleted from the PsaB protein, indicating the essential role of these segments in proper folding of the PsaB protein. Mutants with partial or complete deletion of the L469–D496 segment contained the PS I proteins. These results indicate that the regions near the transmembrane helices are more important for the assembly of PsaB than the middle region of the H loop. The L469-D496 segment in the H loop of PsaB is dispensable in the interaction between the PS I complex and the soluble donor proteins. These results suggested that sections of the H loop of PsaB are crucial for the structural integrity of the PsaB protein.     

Towards efficient hydrogen production: the impact of antenna size and external factors on electron transport dynamics in <Emphasis Type="Italic">Synechocystis</Emphasis> PCC 6803

Three Synechocystis PCC 6803 strains with different levels of phycobilisome antenna-deficiency have been investigated for their impact on photosynthetic electron transport and response to environmental factors (i.e. light-quality, -quantity and composition of growth media). Oxygen yield and P₇₀₀ reduction kinetic measurements showed enhanced linear electron transport rates—especially under photoautotrophic conditions—with impaired antenna-size, starting from wild type (WT) (full antenna) over ΔapcE- (phycobilisomes functionally dissociated) and Olive (lacking phycocyanin) up to the PAL mutant (lacking the whole phycobilisome). In contrast to mixotrophic conditions (up to 80% contribution), cyclic electron transport plays only a minor role (below 10%) under photoautotrophic conditions for all the strains, while linear electron transport increased up to 5.5-fold from WT to PAL mutant. The minor contribution of the cyclic electron transport was proportionally increased with the linear one in the ΔapcE and Olive mutant, but was not altered in the PAL mutant, indicating that upregulation of the linear route does not have to be correlated with downregulation of the cyclic electron transport. Antenna-deficiency involves higher linear electron transport rates by tuning the PS2/PS1 ratio from 1:5 in WT up to 1:1 in the PAL mutant. While state transitions were observed only in the WT and Olive mutant, a further ~30% increase in the PS2/PS1 ratio was achieved in all the strains by long-term adaptation to far red light (720 nm). These results are discussed in the context of using these cells for future H₂ production in direct combination with the photosynthetic electron transport and suggest both Olive and PAL as potential candidates for future manipulations toward this goal. In conclusion, the highest rates can be expected if mutants deficient in phycobilisome antennas are grown under photoautotrophic conditions in combination with uncoupling of electron transport and an illumination which excites preferably PS1.     

Photosynthetic characteristics of photomixotrophic and photoautotrophic cell suspension cultures ofChenopodium rubrum

Summary Cell suspension cultures ofChenopodium rubrum have been grown for more than 2 years photoautotrophically with CO₂ as sole carbon source. Average increase in fresh weight is appr. 600% within 14 days. The chlorophyll content of photoautotrophic cells (200 g/g fresh weight) is much higher than of photomixotrophic cells (50 g/g fresh weight). The photosynthetic activity of the cells (190 moles CO₂×mg^–1 chlorophyllXh^–1) is comparable to the values found with intact leaves. As shown by short-term¹⁴CO₂ photosynthesis, both, the photomixotrophic and the photoautotrophic cell suspension cultures assimilate CO₂ predominantly via the Calvin pathway.Major differences were found with cells from either exponential or stationary phase of growth with regard to differential labelling of 3-phosphoglyceric acid, malate, sucrose and glucose/fructose.In vitro measurements of carboxylation reactions only partially corroborate our findings with¹⁴CO₂ incorporation. The ratio of ribulosebisphosphate to phosphoenolpyruvate carboxylase activity is 4.7 for leaves of C.rubrum, 1.2 for photoautotrophic cells during stationary growth and 0.5 for cells during exponential growth phase, however, 0.18 was found for photomixotrophic cells. Though the¹⁴CO₂ incorporation into 3-phosphoglyceric acid is clearly higher than into malate, thein vitro activity of phosphoenolpyruvatecarboxylase is 2–6 fold higher than that of ribulosebisphosphate carboxylase. We postulate that anaplerotic reactions of the tricarboxylic acid cycle are involved in the regulation of phosphoenolpyruvate carboxylase.Abbreviations 2,4-D didilorophenoxyacetic acid - EDTA ethylene-diamine-tetraacetic acid - fr. w. fresh weight - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PGA 3-phosphoglyceric acid - PPO 2,5-diphenyloxazole - PEP phosphoenolpyruvate - RuBP nbulosebisphosphate     

Regulating photoprotection improves photosynthetic growth and biomass production in Q<Subscript>C</Subscript>-site mutant cells of the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

We characterized the photosynthetic growth of wild-type (WT) and QC-site mutant cells of the cyanobacterium Synechocystis sp. PCC 6803 grown in a photobioreactor under medium-intensity [~70 μmol(photon) m^–2 s^–1] and high-intensity [~200 μmol(photon) m^–2 s^–1] light conditions. Photosynthetic growth rate (the exponential phase) increased about 1.1–1.2 fold for the A16FJ, S28Aβ, and V32Fβ mutant compared with WT cells under medium-intensity light and about 1.2–1.3 fold under high-intensity light. Biomass production increased about 17–20% for A16FJ and S28Aβ mutant cells as compared with WT cells under medium-intensity light and about 14–17% for A16FJ and V32Fβ mutant cells under high-intensity light. The greater photosynthetic growth rate and biomass production of these Q_C-site mutant cells could be attributed to the increased photosynthesis efficiency and decreased dissipation of wasteful energy from phycobilisomes in mutants vs. WT cells. Our results support that manipulation of photoprotection may improve photosynthesis and biomass production of photosynthetic organisms.