The photosynthesis of individual algal cells during the cell cycle of Scenedesmus quadricauda studied by chlorophyll fluorescence kinetic microscopy.

A microscope for imaging of chlorophyll fluorescence kinetics was equipped with a chamber that allows the growth of an immobilised population of algae and their study under well-defined conditions. Single cells of the chlorococcal alga Scenedesmus quadricauda were grown and recorded for periods of whole cell cycles (up to 48 h) displaying a normal course of cell development. Heterogeneity in fluorescence yield among individual coenobia in the population and among different cells in one coenobium were analysed. Differences were observed both in the shape of Kautsky transients and in the modulation of fluorescence parameter values during the progress of the cell cycle. The extent of heterogeneity in fluorescence parameters was cell cycle dependent - in some phases of the cycle, the population was almost homogeneous, while distinct heterogeneity was observed, in particular between the protoplast division and the release of the daughter coenobia. The heterogeneity was not random but reflected developmental processes.

Synechocystis 6803 mutants expressing distinct forms of the Photosystem II D1 protein from Synechococcus 7942: relationship between the psbA coding region and sensitivity to visible and UV-B radiation.

Synechocystis PCC 6803 mutants expressing either the "low light" (D1:1) or the "high light" (D1:2) form of the Photosystem II (PSII) D1 protein from Synechococcus PCC 7942 were constructed and characterized with respect to properties of PSII and sensitivity to visible and UV-B radiation. The AI and AIII mutants (containing only the D1:1 and D1:2 forms, respectively) exhibited very similar PSII characteristics as the control strain and they differed only in the accelerated decay kinetics of flash-induced variable fluorescence measured in the presence of DCMU. However, the mutants showed increased sensitivity to photodamage induced by visible and UV-B radiation, with higher loss of PSII activity in the AI than in the AIII strain. Thus, the difference between strains containing D1:1 and D1:2 found previously in Synechococcus 7942 is maintained after transfer of corresponding psbA genes into Synechocystis 6803 and is directly related to the coding region of these genes. The higher light sensitivity of the AI mutant is caused partly by the higher rate of photodamage and partly by the less efficient PSII repair.

Nitrogen deprivation strongly affects photosystem II but not phycoerythrin level in the divinyl-chlorophyll b-containing cyanobacterium Prochlorococcus marinus.

Effects of nitrogen limitation on Photosystem II (PSII) activities and on phycoerythrin were studied in batch cultures of the marine oxyphotobacterium Prochlorococcus marinus. Dramatic decreases in photochemical quantum yields (F(V)/F(M)), the amplitude of thermoluminescence (TL) B-band, and the rate of Q(A) reoxidation were observed within 12 h of growth in nitrogen-limited conditions. The decline in F(V)/F(M) paralleled changes in the TL B-band amplitude, indicative of losses in PSII activities and formation of non-functional PSII centers. These changes were accompanied by a continuous reduction in D1 protein content. In contrast, nitrogen deprivation did not cause any significant reduction in phycoerythrin content. Our results refute phycoerythrin as a nitrogen storage complex in Prochlorococcus. Regulation of phycoerythrin gene expression in Prochlorococcus is different from that in typical phycobilisome-containing cyanobacteria and eukaryotic algae investigated so far.

Characterization of processes responsible for the distinct effect of herbicides DCMU and BNT on Photosystem II photoinactivation in cells of the cyanobacterium Synechococcus sp. PCC 7942.

Light-induced modification of Photosystem II (PS II) complex was characterized in the cyanobacterium Synechococcus sp. PCC 7942 treated with either DCMU (a phenylurea PS II inhibitor) or BNT (a phenolic PS II inhibitor). The irradiance response of photoinactivation of PS II oxygen evolution indicated a BNT-specific photoinhibition that saturated at relatively low intensity of light. This BNT-specific process was slowed down under anaerobiosis, was accompanied by the oxygen-dependent formation of a 39 kDa D1 protein adduct, and was not related to stable Q(A) reduction or the ADRY effect. In the BNT-treated cells, the light-induced, oxygen-independent initial drop of PS II electron flow was not affected by formate, an anion modifying properties of the PS II non-heme iron. For DCMU-treated cells, anaerobiosis did not significantly affect PS II photoinactivation, the D1 adduct was not observed and addition of formate induced similar initial decrease of PS II electron flow as in the BNT-treated cells. Our results indicate that reactive oxygen species (most likely singlet oxygen) and modification of the PS II acceptor side are responsible for the fast BNT-induced photoinactivation of PS II.

Measurements of variable chlorophyll fluorescence using fast repetition rate techniques: defining methodology and experimental protocols

We present a methodology, called fast repetition rate (FRR) fluorescence, that measures the functional absorption cross-section (sigmaPS II) of Photosystem II (PS II), energy transfer between PS II units (p), photochemical and nonphotochemical quenching of chlorophyll fluorescence, and the kinetics of electron transfer on the acceptor side of PS II. The FRR fluorescence technique applies a sequence of subsaturating excitation pulses ('flashlets') at microsecond intervals to induce fluorescence transients. This approach is extremely flexible and allows the generation of both single-turnover (ST) and multiple-turnover (MT) flashes. Using a combination of ST and MT flashes, we investigated the effect of excitation protocols on the measured fluorescence parameters. The maximum fluorescence yield induced by an ST flash applied shortly (10 &mgr;s to 5 ms) following an MT flash increased to a level comparable to that of an MT flash, while the functional absorption cross-section decreased by about 40%. We interpret this phenomenon as evidence that an MT flash induces an increase in the fluorescence-rate constant, concomitant with a decrease in the photosynthetic-rate constant in PS II reaction centers. The simultaneous measurements of sigmaPS II, p, and the kinetics of Q-A reoxidation, which can be derived only from a combination of ST and MT flash fluorescence transients, permits robust characterization of the processes of photosynthetic energy-conversion.

Biophysical, biochemical, and physiological characterization of Chlamydomonas reinhardtii mutants with amino acid substitutions at the Ala251 residue in the D1 protein that result in varying levels of photosynthetic competence.

The QB binding site of the D1 reaction center protein, located within a stromal loop between transmembrane helices IV and V formed by residues Ile219 to Leu272, is essential for photosynthetic electron transport through photosystem II (PSII). We have examined the function of the highly conserved Ala251 D1 residue in this domain in chloroplast transformants of Chlamydomonas reinhardtii and found that Arg, Asp, Gln, Glu, and His substitutions are nonphotosynthetic, whereas Cys, Ser, Pro, Gly, Ile, Val, and Leu substitutions show various alterations in D1 turnover, photosynthesis, and photoautotrophic growth. The latter mutations reduce the rate of QA to QB electron transfer, but this is not necessarily rate-limiting for photoautotrophic growth. The Cys mutant divides and evolves O2 at wild type rates, although it has slightly higher rates of D1 synthesis and turnover and reduced electron transfer between QA and QB. O2 evolution, D1 synthesis, and accumulation in the Ser, Pro, and Gly mutants in high light is reduced, but photoautotrophic growth rate is not affected. In contrast, the Ile, Val, and Leu mutants are impaired in photoautotrophic growth and photosynthesis in both low and high light and have elevated rates of D1 synthesis and degradation, but D1 accumulation is normal. While rates of synthesis/degradation of the D1 protein are not necessarily correlated with alterations in specific parameters of PSII function in these mutants, bulkiness of the substituted amino acids is highly correlated with the dissociation constant for QB in the seven mutants examined. These observations imply that the Ala251 residue plays a key role in D1 protein.

Cyclic electron flow around Photosystem II in vivo.

The oxygen flash yield (YO2) and photochemical yield of PS II (ΦPS II) were simultaneously detected in intact Chlorella cells on a bare platinum oxygen rate electrode. The two yields were measured as a function of background irradiance in the steady-state and following a transition from light to darkness. During steady-state illumination at moderate irradiance levels, YO2 and ΦPS II followed each other, suggesting a close coupling between the oxidation of water and QA reduction (Falkowski et al. (1988) Biochim. Biophys. Acta 933: 432-443). Following a light-to-dark transition, however, the relationship between QA reduction and the fraction of PS II reaction centers capable of evolving O2 became temporarily uncoupled. ΦPS II recovered to the preillumination levels within 5-10 s, while the YO2 required up to 60 s to recover under aerobic conditions. The recovery of YO2 was independent of the redox state of QA, but was accompanied by a 30% increase in the functional absorption cross-section of PS II (σPS II). The hysteresis between YO2 and the reduction of QA during the light-to-dark transition was dependent upon the reduction level of the plastoquinone pool and does not appear to be due to a direct radiative charge back-reaction, but rather is a consequence of a transient cyclic electron flow around PS II. The cycle is engaged in vivo only when the plastoquinone pool is reduced. Hence, the plastoquinone pool can act as a clutch that disconnects the oxygen evolution from photochemical charge separation in PS II.

Role of plastoquinol oxidoreduction in regulation of photochemical reaction center IID1 protein turnover in vivo.

The light-induced turnover of the D1 protein subunit of reaction center II (RCII) was investigated in Chlamydomonas reinhardtii y-1 (control) and D6, AC208, and B4 mutants lacking cytochrome b6/f, plastocyanin or photosystem I activity, respectively, and, thus, impaired in light-dependent plastoquinol (PQH2) oxidation. Charge recombination assayed by thermoluminescence measurements indicated similar RCII properties in control and mutant cells. The D1 protein is not degraded in the mutants during photoinactivation; however, RCII-D1 is irreversibly altered, and the protein is degraded when the cells are incubated in low light permitting slow reoxidation of the PQH2 pool. Photoinactivation precedes D1 degradation also in the control cells. Thus, in vivo under physiological conditions photoinactivation and "tagging" of RCII-D1 are resolved from the degradation process. RCII activity in photoinactivated cells may be recovered only following D1 degradation and replacement. Recovery may occur either in the light or dark in the absence of de novo chlorophyll synthesis. The degradation of the photoinactivated RCII-D1 protein is a prerequisite for the synthesis and stable integration of new D1 indicating that tagged D1 is still assembled in the inactive reaction centers. The physiological implication of these results is that oxidation of the PQH2 pool in photoinactivated cells affects RCII-D1 protein degradation and replacement, and, thus, D1 turnover in vivo is regulated by the turnover of PQ at the binding site of the secondary stable electron acceptor quinone of RCII.

Inhibition of PS II photochemistry by PAR and UV radiation in natural phytoplankton communities.

The effects of PAR and UV radiation on PS II photochemistry were examined in natural phytoplankton communities from coastal waters off Rhode Island (USA) and the subtropical Pacific. The photochemical energy conversion efficiency, the functional absorption cross section and the kinetics of electron transfer on the acceptor side of PS II were derived from variable fluorescence parameters using both pump and probe and fast repetition rate techniques. In both environments, the natural phytoplankton communities displayed marked decreases in PS II photochemical energy conversion efficiency that were correlated with increased PAR. In the coastal waters, the changes in photochemical energy conversion efficiency were not statistically different for samples treated with supplementary UV-B radiation or screened to exclude ambient UV-B. Moreover, no significant light-dependent changes in the functional absorption cross section of PS II were observed. The rate of electron transfer between QA (-) and QB was, however, slightly reduced in photodamaged cells, indicative of damage on the acceptor side. In the subtropical Pacific, the decrease in photochemical energy conversion efficiency was significantly greater for samples exposed to natural levels of UV-A and/or UV-B compared with those exposed to PAR alone. The cells displayed large diurnal changes in the functional absorption cross section of PS II, indicative of non-photochemical quenching in the antenna. The changes in the functional absorption cross section were highly correlated with PAR but independent of UV radiation. The time course of changes in photochemical efficiency reveals that the photoinhibited reaction centers rapidly recover (within an hour or two) to their preillumination values. Thus, while we found definitive evidence for photoinhibition of PS II photochemistry in both coastal and open ocean phytoplankton communities, we did not find any effect of UV-B on the former, but a clear effect on the latter. The results of this study indicate that the effects of UV-B radiation on phytoplankton photosynthesis are as dependent on the radiative transfer properties of the water body and the mixing rate, as on the wavelength and energy distribution of the radiation and the absorption cross sections of the biophysical targets.

Three types of Photosystem II photoinactivation : 2. Slow processes.

Oxygen evolving Photosystem II particles were exposed for up to 10 h to 100 W m(-2) white light at 20°C under aerobic, low oxygen, strictly anaerobic and strongly reducing conditions. The fast and slow photoinactivation processes described earlier (Setlík et al. 1989) were observed during the first 120 min. The third and by far the slowest process impaired the primary charge separation P680(+)-Pheo(-). Its half-time was about 2.5 h under aerobic and strongly reducing conditions and about 4 h under anaerobic and low oxygen conditions. In these time intervals there were no changes in the chlorophyll-protein and polypeptide composition of the particles irradiated under anaerobic, low oxygen or strongly reducing conditions while a dramatic degradation of chlorophyll-proteins and polypeptides occurred under aerobic conditions.