Photosynthetic Light Utilization Efficiency, Photosystem II Heterogeneity, and Fluorescence Quenching in Chlamydomonas reinhardtii during the Induction of the CO(2)-Concentrating Mechanism

The photosynthetic light-response curve, the relative amounts of the different photosystem II (PSII) units, and fluorescence quenching were altered in an adaptive manner when CO₂-enriched wild-type Chlamydomonas reinhardtii cells were transferred to low levels of CO₂. This treatment is known to result in the induction of an energy-dependent CO₂-concentrating mechanism (CCM) that increases the internal inorganic carbon concentration and thus the photosynthetic CO₂ utilization efficiency. After 3 to 6 h of low inorganic carbon treatment, several changes in the photosynthetic energy-transducing reactions appeared and proceeded for about 12 h. After this time, the fluorescence parameter variable/maximal fluorescence yield and the amounts of both PSIIα and PSIIβ (secondary quinone electron acceptor of PSII-reducing) centers had decreased, whereas the amount of PSIIβ (secondary quinone electron acceptor of PSII-nonreducing) centers had increased. The yield of noncyclic electron transport also decreased during the induction of the CCM, whereas both photochemical and nonphotochemical quenching of PSII fluorescence increased. Concurrent with these changes, the photosynthetic light-utilization efficiency also decreased significantly, largely attributed to a decline in the curvature parameter θ, the convexity of the photosynthetic light-response curve. Thus, it is concluded that the increased CO₂ utilization efficiency in algal cells possessing the CCM is maintained at the cost of a reduced light utilization efficiency, most probably due to the reduced energy flow through PSII.     

Activation of a Reserve Pool of Photosystem II in Chlamydomonas reinhardtii Counteracts Photoinhibition

The effect of strong irradiance (2000 micromole photons per square meter per second) on PSII heterogeneity in intact cells of Chlamydomonas reinhardtii was investigated. Low light (LL, 15 micromole photons per square meter per second) grown C. reinhardtii are photoinhibited upon exposure to strong irradiance, and the loss of photosynthetic functioning is due to damage to PSII. Under physiological growth conditions, PSII is distributed into two pools. The large antenna size (PSII_α) centers account for about 70% of all PSII in the thylakoid membrane and are responsible for plastoquinone reduction (Q_b-reducing centers). The smaller antenna (PSII_β) account for the remainder of PSII and exist in a state not yet able to photoreduce plastoquinone (Q_b-nonreducing centers). The exposure of C. reinhardtii cells to 60 minutes of strong irradiance disabled about half of the primary charge separation between P680 and pheophytin. The PSII_β content remained the same or slightly increased during strong-irradiance treatment, whereas the photochemical activity of PSII_α decreased by 80%. Analysis of fluorescence induction transients displayed by intact cells indicated that strong irradiance led to a conversion of PSII_β from a Q_b-nonreducing to a Q_b-reducing state. Parallel measurements of the rate of oxygen evolution revealed that photosynthetic electron transport was maintained at high rates, despite the loss of activity by a majority of PSII_α. The results suggest that PSII_β in C. reinhardtii may serve as a reserve pool of PSII that augments photosynthetic electron-transport rates during exposure to strong irradiance and partially compensates for the adverse effect of photoinhibition on PSII_α.     

Mutation of Residue Threonine-2 of the D2 Polypeptide and Its Effect on Photosystem II Function in Chlamydomonas reinhardtii

The D2 polypeptide of the photosystem II (PSII) complex in the green alga Chlamydomonas reinhardtii is thought to be reversibly phosphorylated. By analogy to higher plants, the phosphorylation site is likely to be at residue threonine-2 (Thr-2). We have investigated the role of D2 phosphorylation by constructing two mutants in which residue Thr-2 has been replaced by either alanine or serine. Both mutants grew photoautotrophically at wild-type rates, and noninvasive biophysical measurements, including the decay of chlorophyll fluorescence, the peak temperature of thermoluminescence bands, and rates of oxygen evolution, indicate little perturbation to electron transfer through the PSII complex. The susceptibility of mutant PSII to photoinactivation as measured by the light-induced loss of PSII activity in whole cells in the presence of the protein-synthesis inhibitors chloramphenicol or lincomycin was similar to that of wild type. These results indicate that phosphorylation at Thr-2 is not required for PSII function or for protection from photoinactivation. In control experiments the phosphorylation of D2 in wild-type C. reinhardtii was examined by ³²P labeling in vivo and in vitro. No evidence for the phosphorylation of D2 in the wild type could be obtained. [¹⁴C]Acetate-labeling experiments in the presence of an inhibitor of cytoplasmic protein synthesis also failed to identify phosphorylated (D2.1) and nonphosphorylated (D2.2) forms of D2 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results suggest that the existence of D2 phosphorylation in C. reinhardtii is still in question.     

Characterization of Photosystem II Activity and Heterogeneity during the Cell Cycle of the Green Alga Scenedesmus quadricauda

The photosynthetic activity of the green alga Scenedesmus quadricauda was investigated during synchronous growth in light/dark cycles. The rate of O₂ evolution increased 2-fold during the first 3 to 4 h of the light period, remained high for the next 3 to 4 h, and then declined during the last half of the light period. During cell division, which occurred at the beginning of the dark period, the ability of the cells to evolve O₂ was at a minimum. To determine if photosystem II (PSII) controls the photosynthetic capacity of the cells during the cell cycle we measured PSII activity and heterogeneity. Measurements of electron-transport activity revealed two populations of PSII, active centers that contribute to carbon reduction and inactive centers that do not. Measurements of PSII antenna sizes also revealed two populations, PSII_α and PSII_β, which differ from one another by their antenna size. During the early light period the photosynthetic capacity of the cells doubled, the O₂-evolving capacity of PSII was nearly constant, the proportion of PSII_β centers decreased to nearly zero, and the proportion of inactive PSII centers remained constant. During the period of minimum photosynthetic activity 30% of the PSII centers were insensitive to the inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, which may be related to reorganization of the thylakoid membrane. We conclude from these results that PSII does not limit the photosynthetic activity of the cells during the first half of the light period. However, the decline in photosynthetic activity observed during the last half of the light period can be accounted for by limited PSII activity.     

Comparison of Photosynthetic Performance in Triazine-Resistant and Susceptible Biotypes of Amaranthus hybridus

The rate of CO₂ reduction in the S-triazine-resistant biotype of smooth pigweed (Amaranthus hybridus L.) was lower at all levels of irradiance than the rate of CO₂ reduction in the susceptible biotype. The intent of this study was to determine whether or not the lower rates of CO₂ reduction are a direct consequence of the same factors which confer triazine resistance. The quantum yield of CO₂ reduction was 23 ± 2% lower in the resistant biotype of pigweed and the resistant biotype of pigweed had about 25% fewer active photosystem II centers on both a chlorophyll and leaf area basis. This quantum inefficiency of the resistant biotype can be accounted for by a decrease in the equilibrium constant between the primary and secondary quinone acceptors of the photosystem II reaction centers which in turn would lead to a higher average level of reduced primary quinone acceptor in the resistant biotype. Thus, the photosystem II quantum inefficiency of the resistant biotype appears to be a direct consequence of those factors responsible for triazine resistance but a caveat to this conclusion is discussed. The effects of the quantum inefficiency of photosystem II on CO₂ reduction should be overcome at high light and therefore cannot account for the lower light-saturated rate of CO₂ reduction in the resistant biotype. Chloroplast lamellar membranes isolated from both triazine-resistant and triazine-susceptible pigweed support equivalent rates of whole chain electron transfer and these rates are sufficient to account for the rate of light-saturated CO₂ reduction. This observation shows that the slower transfer of electrons from the primary to the secondary quinone acceptor of photosystem II, a trait which is characteristic of the resistant biotype, is nevertheless still more rapid than subsequent reactions of photosynthetic CO₂ reduction. Thus, it appears that the lower rate of light-saturated CO₂ reduction of the resistant biotype is not limited by electron transfer capacity and therefore is not a direct consequence of those factors which confer triazine resistance.     

Spare quinones in the QB cavity of crystallized photosystem II from Thermosynechococcus elongatus

The recent crystallographic structure at 3.0 Å resolution of PSII from Thermosynechococcus elongatus has revealed a cavity in the protein which connects the membrane phase to the binding pocket of the secondary plastoquinone Q_B. The cavity may serve as a quinone diffusion pathway. By fluorescence methods, electron transfer at the donor and acceptor sides was investigated in the same membrane-free PSII core particle preparation from T. elongatus prior to and after crystallization; PSII membrane fragments from spinach were studied as a reference. The data suggest selective enrichment of those PSII centers in the crystal that are intact with respect to O₂ evolution at the manganese-calcium complex of water oxidation and with respect to the integrity of the quinone binding site. One and more functional quinone molecules (per PSII monomer) besides of Q_A and Q_B were found in the crystallized PSII. We propose that the extra quinones are located in the Q_B cavity and serve as a PSII intrinsic pool of electron acceptors.     

The Intracellular Localization of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Chlamydomonas reinhardtii

The pyrenoid is a proteinaceous structure found in the chloroplast of most unicellular algae. Various studies indicate that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is present in the pyrenoid, although the fraction of Rubisco localized there remains controversial. Estimates of the amount of Rubisco in the pyrenoid of Chlamydomonas reinhardtii range from 5% to nearly 100%. Using immunolocalization, the amount of Rubisco localized to the pyrenoid or to the chloroplast stroma was estimated for C. reinhardtii cells grown under different conditions. It was observed that the amount of Rubisco in the pyrenoid varied with growth condition; about 40% was in the pyrenoid when the cells were grown under elevated CO₂ and about 90% with ambient CO₂. In addition, it is likely that pyrenoidal Rubisco is active in CO₂ fixation because in vitro activity measurements showed that most of the Rubisco must be active to account for CO₂-fixation rates observed in whole cells. These results are consistent with the idea that the pyrenoid is the site of CO₂ fixation in C. reinhardtii and other unicellular algae containing CO₂-concentrating mechanisms.     

IMPACT OF IRON LIMITATION ON THE PHOTOSYNTHETIC APPARATUS OF THE DIATOM CHAETOCEROS MUELLERI (BACILLARIOPHYCEAE)

Iron starvation induced marked increases in flavodoxin abundance and decreases in light-saturated and light-limited photosynthesis rates in the diatom Chaetoceros muelleri. Consistent with the substitution of flavodoxin for ferredoxin as an early response to iron starvation, increases of flavodoxin abundance were observed before declines of cell division rate or chl a specific photosynthesis rates. Changes in the abundance of flavodoxin after the addition of iron to iron-starved cells indicated that flavodoxin was not actively degraded under iron-replete conditions. Greater declines in light-saturated oxygen evolution rates than dark oxygen consumption rates indicated that the mitochondrial electron transfer chain was not affected as greatly by iron starvation as the photosynthetic electron transfer chain. The carbon:nitrogen ratio was unaffected by iron starvation, suggesting that photosynthetic electron transfer was a primary target of iron starvation and that reductions in nitrate assimilation were due to energy limitation (the C:N ratio would be expected to rise under nitrogen-limited but energy-replete conditions). Parallel changes were observed in the maximum light-saturated photosynthesis rate and the light-limited initial slope of the photosynthesis-light curve during iron starvation and recovery. The lowest photosynthesis rates were observed in iron-starved cells and the highest values in iron-replete cells. The light saturation parameter, I_k, was not affected by iron starvation, nor was the chl-to-C ratio markedly reduced. These observations were consistent with iron starvation having a similar or greater effect on photochemical charge separation in PSII than on downstream electron transfer steps. Declines of the ratio of variable to maximum fluorescence in iron-starved cells were consistent with PSII being a primary target of iron starvation. The functional cross-section of PSII was affected only marginally (<20%) by iron starvation, with the largest values observed in iron-starved cells. The rate constant for electron transfer calculated from fast repetition rate fluorescence was found to covary with the light-saturated photosynthesis rate; it was lowest in the most severely starved cells.     

Phylogenetic Identification and Substrate Uptake Patterns of Sulfate-Reducing Bacteria Inhabiting an Oxic-Anoxic Sewer Biofilm Determined by Combining Microautoradiography and Fluorescent In Situ Hybridization

We simultaneously determined the phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria (SRB) inhabiting a sewer biofilm with oxygen, nitrate, or sulfate as an electron acceptor by combining microautoradiography and fluorescent in situ hybridization (MAR-FISH) with family- and genus-specific 16S rRNA probes. The MAR-FISH analysis revealed that Desulfobulbus hybridized with probe 660 was a dominant SRB subgroup in this sewer biofilm, accounting for 23% of the total SRB. Approximately 9 and 27% of Desulfobulbus cells detected with probe 660 could take up [¹⁴C]propionate with oxygen and nitrate, respectively, as an electron acceptor, which might explain the high abundance of this species in various oxic environments. Furthermore, more than 40% of Desulfobulbus cells incorporated acetate under anoxic conditions. SRB were also numerically important members of H₂-utilizing and ¹⁴CO₂-fixing microbial populations in this sewer biofilm, accounting for roughly 42% of total H₂-utilizing bacteria hybridized with probe EUB338. A comparative 16S ribosomal DNA analysis revealed that two SRB populations, related to the Desulfomicrobium hypogeium and the Desulfovibrio desulfuricans MB lineages, were found to be important H₂ utilizers in this biofilm. The substrate uptake characteristics of different phylogenetic SRB subgroups were compared with the characteristics described to date. These results provide further insight into the correlation between the 16S rRNA phylogenetic diversity and the physiological diversity of SRB populations inhabiting sewer biofilms.     

PHOTOPHYSIOLOGICAL RESPONSES OF FRAGILARIOPSIS CYLINDRUS (BACILLARIOPHYCEAE) TO NITROGEN DEPLETION AT TWO TEMPERATURES1

The photosynthetic efficiency and photoprotective capacity of the sea‐ice diatom, Fragilariopsis cylindrus (Grunow) W. Krieg., grown in a matrix of nitrogen repletion and depletion at two different temperatures (?1°C and +6°C) was investigated. Temperature showed no significant effect on photosynthetic efficiency or photoprotection in F. cylindrus. Cultures under nitrogen depletion showed enhanced photoprotective capacity with an increase in nonphotochemical quenching (NPQ) when compared with nitrogen‐replete cultures. This phenomenon was achieved at no apparent cost to the photosynthetic efficiency of PSII (F_V/F_M). Nitrogen depletion yielded a partially reduced electron transport chain in which maximum fluorescence (F_M) could only be obtained by adding 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU). reoxidation curves showed the presence of Q_B nonreducing PSII centers under nitrogen depletion. Fast induction curves (FICs) and electron transport rates (ETRs) revealed slowing of the electrons transferred from the primary (Q_A) to the secondary (Q_B) quinone electron acceptors of PSII. The data presented show that nitrogen depletion in F. cylindrus leads to the formation of Q_B nonreducing PSII centers within the photosystem. On a physiological level, the formation of Q_B nonreducing PSII centers in F. cylindrus provides the cell with protection against photoinhibition by facilitating the rapid induction of NPQ. This strategy provides an important ecological advantage, especially during the Antarctic spring, maintaining photosynthetic efficiency under high light and nutrient‐limiting conditions.     

Recovery of photosystem I and II activities during re-hydration of lichen<Emphasis Type="Italic"> Hypogymnia physodes</Emphasis> thalli

Photochemical efficiencies of photosystem I (PSI) and photosystem II (PSII) were studied in dry thalli of the lichen Hypogymnia physodes and during their re-hydration. In dry thalli, PSII reaction centers are photochemically inactive, as evidenced by the absence of variable chlorophyll (Chl) fluorescence, whereas the primary electron donor of PSI, P700, exhibits irreversible oxidation under continuous light. Upon application of multiple- and, particularly, single-turnover pulses in dry lichen, P700 oxidation partially reversed, which indicated recombination between P700⁺ and the reduced acceptor F_X of PSI. Re-wetting of air-dried H. physodes initiated the gradual restoration of reversible light-induced redox reactions in both PSII and PSI, but the recovery was faster in PSI. Two slow components of P700⁺ reduction occurred after irradiation of partially and completely hydrated thalli with strong white light. In contrast, no slow component was found in the kinetics of re-oxidation of Q_A^–, the reduced primary acceptor of PSII, after exposure of such thalli to white light. This finding indicated the inability of PSII in H. physodes to provide the reduction of the plastoquinone pool to significant levels. It is concluded that slow alternative electron transport routes may contribute to the energetics of photosynthesis to a larger extent in H. physodes than in higher plants.Abbreviations A₀ and A₁ Primary acceptor chlorophyll and secondary electron acceptor phylloquinone - Chl a Chlorophyll a - F_m Maximal level of chlorophyll fluorescence when all PSII centers are closed - F_o Minimal level of fluorescence when all PSII centers are open after dark adaptation - FR Far-red - F_v Variable fluorescence (=F_m–F_o) - F_X, F_A, and F_B Iron–sulfur centers - MT pulse Multiple-turnover pulse - PS Photosystem - P700 Reaction center chlorophyll of PSI - Q_A Primary quinone acceptor of PSII - Q_B Secondary quinone acceptor of PSII - ST pulse Single-turnover pulse     

Estimating the Excess Investment in Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Leaves of Spring Wheat Grown under Elevated CO2

Wheat (Triticum aestivum L.) was grown under CO₂ partial pressures of 36 and 70 Pa with two N-application regimes. Responses of photosynthesis to varying CO₂ partial pressure were fitted to estimate the maximal carboxylation rate and the nonphotorespiratory respiration rate in flag and preceding leaves. The maximal carboxylation rate was proportional to ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) content, and the light-saturated photosynthetic rate at 70 Pa CO₂ was proportional to the thylakoid ATP-synthase content. Potential photosynthetic rates at 70 Pa CO₂ were calculated and compared with the observed values to estimate excess investment in Rubisco. The excess was greater in leaves grown with high N application than in those grown with low N application and declined as the leaves senesced. The fraction of Rubisco that was estimated to be in excess was strongly dependent on leaf N content, increasing from approximately 5% in leaves with 1 g N m⁻² to approximately 40% in leaves with 2 g N m⁻². Growth at elevated CO₂ usually decreased the excess somewhat but only as a consequence of a general reduction in leaf N, since relationships between the amount of components and N content were unaffected by CO₂. We conclude that there is scope for improving the N-use efficiency of C₃ crop species under elevated CO₂ conditions.     

Compensatory Alterations in the Photochemical Apparatus of a Photoregulatory, Chlorophyll b-Deficient Mutant of Maize

Characterization of the functional organization of the photochemical apparatus in the light sensitive chlorophyll b-deficient oil yellow-yellow green (OY-YG) mutant of maize (Zea mays) is presented. Spectrophotometric and kinetic analysis revealed substantially lower amounts of the light harvesting complex of photosystem II (LHCII-peripheral) in high light-grown OY-YG thylakoids. However, accumulation of a tightly bound LHCII appears unaffected by the lesion. Changes in photosystem (PS) stoichiometry include lower amounts of PSII with characteristic fast kinetics (PSII_α) and a substantial accumulation of PSII centers with characteristic slow kinetics (PSII_β) in the thylakoid membrane of the OY-YG mutant. Thus, PSII_β is the dominant photosystem in the mutant chloroplasts. In contrast to wild type, roughly 80% of the mutant PSII_β centers are functionally coupled to the plastoquinone pool and are probably localized in the appressed regions of the thylakoid membrane. These centers, designated PSII_β-Q_B-reducing (Q_B being the secondary electron quinone acceptor of PSII), are clearly distinct from the typical PSII_β-Q_B-nonreducing centers found in the stroma lamellae of wild-type chloroplasts. It is concluded that the observed changes in the stoichiometry of electron-transport complexes reflect the existence of a regulatory mechanism for the adjustment of photosystem stoichiometry in chloroplasts designed to correct any imbalance in light absorption by the two photosystems.     

Survival of Enterococcus faecalis in an Oligotrophic Microcosm: Changes in Morphology, Development of General Stress Resistance, and Analysis of Protein Synthesis

The ability of Enterococcus faecalis to metabolically adapt to an oligotrophic environment has been analyzed. E. faecalis is able to survive for prolonged periods under conditions of complete starvation established by incubation in tap water. During incubation in this microcosm, cells developed a rippled cell surface with irregular shapes. Exponentially growing cells survived to the same extent as cells starved for glucose prior to exposure to the multiple nutrient deficient stress. Chloramphenicol treatment during incubation in tap water led to a rapid decline in plate counts for exponentially growing cells but showed progressively reduced influence on stationary-phase cells harvested after different times of glucose starvation. During incubation in the oligotrophic environment, cells from the exponential-growth phase and early-stationary phase became progressively more resistant to other environmental stresses (heat [62°C], acid [pH 3.3], UV_{254 nm} light [180 J/m²], and sodium hypochlorite [0.05%]) until they reached a maximum of survival characteristic for each treatment. In contrast, cells starved of glucose for 24 h did not become more resistant to the different treatments during incubation in tap water. Our combined data suggest that energy starvation induces a response similar to that triggered by oligotrophy. Analysis of protein synthesis by two-dimensional gel electrophoresis revealed the enhanced synthesis of 51 proteins which were induced in the oligotrophic environment. A comparison of these oligotrophy-inducible proteins with the 42 glucose starvation-induced polypeptides (J. C. Giard, A. Hartke, S. Flahaut, P. Boutibonnes, and Y. Auffray, Res. Microbiol. 148:27–35, 1997) showed that 16 are common between the two different starvation conditions. These proteins and the corresponding genes seem to play a key role in the observed phenomena of long-term survival and development of general stress resistance of starved cultures of E. faecalis.     

Acetate in mixotrophic growth medium affects photosystem II in Chlamydomonas reinhardtii and protects against photoinhibition

Chlamydomonas reinhardtii is a photoautotrophic green alga, which can be grown mixotrophically in acetate-supplemented media (Tris–acetate–phosphate). We show that acetate has a direct effect on photosystem II (PSII). As a consequence, Tris–acetate–phosphate-grown mixotrophic C. reinhardtii cultures are less susceptible to photoinhibition than photoautotrophic cultures when subjected to high light. Spin-trapping electron paramagnetic resonance spectroscopy showed that thylakoids from mixotrophic C. reinhardtii produced less ¹O₂ than those from photoautotrophic cultures. The same was observed in vivo by measuring DanePy oxalate fluorescence quenching. Photoinhibition can be induced by the production of ¹O₂ originating from charge recombination events in photosystem II, which are governed by the midpoint potentials (E_m) of the quinone electron acceptors. Thermoluminescence indicated that the E_m of the primary quinone acceptor (Q_A/Q_A⁻) of mixotrophic cells was stabilised while the E_m of the secondary quinone acceptor (Q_B/Q_B⁻) was destabilised, therefore favouring direct non-radiative charge recombination events that do not lead to ¹O₂ production. Acetate treatment of photosystem II-enriched membrane fragments from spinach led to the same thermoluminescence shifts as observed in C. reinhardtii, showing that acetate exhibits a direct effect on photosystem II independent from the metabolic state of a cell. A change in the environment of the non-heme iron of acetate-treated photosystem II particles was detected by low temperature electron paramagnetic resonance spectroscopy. We hypothesise that acetate replaces the bicarbonate associated to the non-heme iron and changes the environment of Q_A and Q_B affecting photosystem II charge recombination events and photoinhibition.     

Photoacclimation in the Red Alga Porphyridium cruentum: Changes in Photosynthetic Enzymes, Electron Carriers, and Light-Saturated Rate of Photosynthesis as a Function of Irradiance and Spectral Quality

Acclimation of the photosynthetic apparatus to changes in the light environment was studied in the unicellular red alga Porphyridium cruentum (American Type Culture Collection No. 50161). Absolute or relative amounts of four photosynthetic enzymes and electron carriers were measured, and the data were compared with earlier observations on light-harvesting components (F.X. Cunningham, Jr., R.J. Dennenberg, L. Mustárdy, P.A. Jursinic, E. Gantt [1989] Plant Physiol 91: 1179-1187; F.X. Cunningham, Jr., R.J. Dennenberg, P.A. Jursinic, E. Gantt [1990] Plant Physiol 93: 888-895) and with measurements of photosynthetic capacity. P_max, the light-saturated rate of photosynthesis on a chlorophyll (Chl) basis, increased more than 4-fold with increase in growth irradiance from 6 to 280 μeinsteins·m⁻²·s⁻¹. Amounts of ferredoxin-NADP⁺ reductase, ribulose-1,5-bisphosphate carboxylase, and cytochrome f increased in parallel with P_max, whereas numbers of the light-harvesting complexes (photosystem [PS] I, PSII, and phycobilisomes) changed little, and ATP synthase increased 7-fold relative to Chl. The calculated minimal turnover time for PSII under the highest irradiance, 5 ms, was thus about 4-fold faster than that calculated for cultures grown under the lowest irradiance (19 ms). A change in the spectral composition of the growth light (irradiance kept constant at 15 μeinsteins·m⁻²·s⁻¹) from green (absorbed predominantly by the phycobilisome antenna of PSII) to red (absorbed primarily by the Chl antenna of PSI) had little effect on the amounts of ribulose-1,5-bisphosphate carboxylase, ATP synthase, and phycobilisomes on a Chl, protein, or thylakoid area basis. However, the number of PSI centers declined by 40%, cytochrome f increased by 40%, and both PSII and ferredoxin-NADP⁺ reductase increased approximately 3-fold on a thylakoid area basis. The substantial increase in ferredoxin-NADP⁺ reductase under PSI light is inconsistent with a PSI-mediated reduction of NADP as the sole function of this enzyme. Our results demonstrate a high degree of plasticity in content and composition of thylakoid membranes of P. cruentum.     

Evaluation of photosynthetic activities in thylakoid membranes by means of Fourier transform infrared spectroscopy

Light-induced Fourier transformed infrared (FTIR) difference spectroscopy is a powerful method to study the structures and reactions of redox cofactors involved in the photosynthetic electron transport chain. So far, most of the FTIR studies of the reactions of oxygenic photosynthesis have been performed using isolated photosystem I (PSI) and photosystem II (PSII) preparations, which, however, could be modified during isolation procedures. In this study, we developed a methodology to evaluate the photosynthetic activities of thylakoids using FTIR spectroscopy. FTIR difference spectra upon successive flashes using thylakoids from spinach exhibited signals typical of the S-state cycle at the Mn₄CaO₅ cluster and Q_B reactions in PSII with period-four and -two oscillations, respectively. Similar measurement in the presence of an artificial quinone as an exogenous electron acceptor showed features specific to the S-state cycle. Simulations of the oscillation patterns provided the quantum efficiencies of the S-state cycle and electron transfer in PSII. Moreover, FTIR measurement under continuous illumination on thylakoids in the presence of DCMU showed signals due to Q_A reduction and P700 oxidation simultaneously. From the relative amplitudes of marker bands of Q_A^? and P700⁺, the molar ratio of photoactive PSII and PSI centers in thylakoids was estimated. FTIR analyses of the photo-reactions in thylakoids, which are more intact than isolated photosystems, will be useful in investigations of the photosynthetic mechanism especially by genetic modification of photosystem proteins.     

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.     

Pigment mutants of the green microalga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>: Morphological properties and photosynthetic performance

Pigment mutants CC-124y-1, СС-124у-2, and СС-124р-2 of the green microalga Chlamydomonas reinhardtii Dangeard were obtained by means of UV-induced mutagenesis. Morphological properties and photosynthetic activity of these mutants were examined. The mutants displayed a lowered content of chlorophyll b and carotenoids. Analysis of fluorescence induction curves revealed the decreased rates of electron transport in photosystem II (PSII), the increase in the fraction of Q_B-nonreducing centers, and the enhancement of nonphotochemical fluorescence quenching. The mutations had no direct impact on oxidation of pigment P₇₀₀ in PSI and on the decline of delayed fluorescence. The parameters of induction curves of prompt and delayed fluorescence are proposed for use in early diagnostics of UV-induced mutagenesis. The pigment mutants obtained can be applied in biomonitoring studies.