[NaCl-induced photoinhibition and the recovery of the photosynthetic activity of katG- mutant of the cyanobacterium Synechocystis sp. PCC 6803].

The joint effects of 0.5 M NaCI and light of different intensities on the activity of the photosynthetic apparatus and ATP contents in cells of the katG- mutant of cyanobacterium Synechocystis sp. PCC 6803 have been studied. The mutant demonstrated a higher photoinhibition rate and a slower rate of recovery compared to the wild type, as it was shown by measurements of the CO2-dependent O2 production and delayed light emission of Chl a. The presence of 0.5 M NaCl in incubation medium induced the equal photoinhibition of the photosynthetic apparatus at I = 1200 microE m(-2) x (-1) in the mutant and wild-type cells. At I = 2400 microE m(-2) x c(-1), a stronger inhibition and a slower recovery of the photosynthetic apparatus activity in the kat- mutant than in wild-type cells was observed. The data obtained evidence an important role of catalase-peroxidase in the system of reparation of the photosynthetic apparatus damaged by high-intensty light, especially against the background of NaCI stress.

[Effect of oxidative stress inductors on the photosynthetic apparatus in cyanobacterium Synechocystis sp. PCC 6803 Prq20 mutant resistant to methyl viologen].

The damaging effect of oxidative stress inductors: methyl viologen, benzyl viologen, cumene hydroperoxide, H2O2, menadion, and high irradiance on the photosynthetic apparatus of cyanobacterium Synechocystis sp. PCC 6803 in cells of the wild type strain and the methyl viologen-resistant Prq20 mutant with the disrupted function of the regulatory gene prqR has been investigated by measuring the delayed fluorescence of chlorophyll a and the rate of CO2dependent -O2 gas exchange. It has been shown that the damage to the photosynthetic apparatus in the Prq20 mutant as compared with the wild type was less in the presence of methyl viologen and benzyl viologen. Reasons for the enhanced resistance of the photosynthetic apparatus in the mutant Prq20 to methyl viologen and benzyl viologen are discussed.

Photoinhibition and light-induced cyclic electron transport in ndhB(-) and psaE(-) mutants of Synechocystis sp. PCC 6803.

The ndhB(-) and psaE(-) mutants of the cyanobacterium Synechocystis sp. PCC 6803 are partly deficient in PSI-driven cyclic electron transport. We compared photoinhibition in these mutants to the wild type to test the hypothesis that PSI cyclic electron transport protects against photoinhibition. Photoinhibitory treatment greatly accelerated PSI cyclic electron transport in the wild type and also in both the mutants. The psaE(-) mutant showed rates of PSI cyclic electron transport similar to the wild type under all conditions tested. The ndhB(-) mutant showed much lower rates of PSI cyclic electron transport than the wild type following brief dark adaptation but exceeded wild type rates after exposure to photoinhibitory light. The wild type and both mutants showed similar rates of photoinhibition damage and photoinhibition repair at PSII. Photoinhibition at PSI was much slower than at PSII and was also similar between the wild type and both mutants, despite the known instability of PSI in the psaE(-) mutant. We conclude that photoinhibitory light induces sufficient PSI-driven cyclic electron transport in both the ndhB(-) and psaE(-) mutants to fulfill any role that cyclic electron transport plays in protection against photoinhibition.

New applications of photoacoustics to the study of photosynthesis.

Photoacoustic methods offer unique capabilities for photosynthesis research. Phenomena that are readily observed by photoacoustics include the storage of energy by electron transport, oxygen evolution by leaf tissue at microsecond time resolution, and the conformational changes of photosystems caused by charge separation. Despite these capabilities, photoacoustic methods have not been widely exploited in photosynthesis research. One factor that has contributed to their slow adoption is uncertainty in the interpretation of photoacoustic signals. Careful experimentation is resolving this uncertainty, however, and technical refinements of photoacoustic methods continue to be made. This review provides an overview of the application of photoacoustics to the study of photosynthesis with an emphasis on the resolution of uncertainties in the interpretation of photoacoustic signals. Recent developments in photoacoustic technology are also presented, including a microphotoacoustic spectrometer, gas permeable photoacoustic cells, the use of photoacoustics to monitor phytoplankton populations, and the use of photoacoustics to study protein dynamics.

Temperate Myxococcus xanthus phage Mx8 encodes a DNA adenine methylase, Mox.

Temperate bacteriophage Mx8 of Myxococcus xanthus encapsidates terminally repetitious DNA, packaged as circular permutations of its 49-kbp genome. During both lytic and lysogenic development, Mx8 expresses a nonessential DNA methylase, Mox, which modifies adenine residues in occurrences of XhoI and PstI recognition sites, CTCGAG and CTGCAG, respectively, on both phage DNA and the host chromosome. The mox gene is necessary for methylase activity in vivo, because an amber mutation in the mox gene abolishes activity. The mox gene is the only phage gene required for methylase activity in vivo, because ectopic expression of mox as part of the M. xanthus mglBA operon results in partial methylation of the host chromosome. The predicted amino acid sequence of Mox is related most closely to that of the methylase involved in the cell cycle control of Caulobacter crescentus. We speculate that Mox acts to protect Mx8 phage DNA against restriction upon infection of a subset of natural M. xanthus hosts. One natural isolate of M. xanthus, the lysogenic source of related phage Mx81, produces a restriction endonuclease with the cleavage specificity of endonuclease BstBI.

Light adaptation of cyclic electron transport through Photosystem I in the cyanobacterium Synechococcus sp. PCC 7942.

Photosystem I-driven cyclic electron transport was measured in intact cells of Synechococcus sp PCC 7942 grown under different light intensities using photoacoustic and spectroscopic methods. The light-saturated capacity for PS I cyclic electron transport increased relative to chlorophyll concentration, PS I concentration, and linear electron transport capacity as growth light intensity was raised. In cells grown under moderate to high light intensity, PS I cyclic electron transport was nearly insensitive to methyl viologen, indicating that the cyclic electron supply to PS I derived almost exclusively from a thylakoid dehydrogenase. In cells grown under low light intensity, PS I cyclic electron transport was partially inhibited by methyl viologen, indicating that part of the cyclic electron supply to PS I derived directly from ferredoxin. It is proposed that the increased PSI cyclic electron transport observed in cells grown under high light intensity is a response to chronic photoinhibition.

Acclimation of the Photosynthetic Apparatus to Growth Irradiance in a Mutant Strain of Synechococcus Lacking Iron Superoxide Dismutase.

The acclimation of the photosynthetic apparatus to growth irradiance in a mutant strain of Synechococcus sp. PCC 7942 lacking detectable iron superoxide dismutase activity was studied. The growth of the mutant was inhibited at concentrations of methyl viologen 4 orders of magnitude smaller than those required to inhibit the growth of the wild-type strain. An increased sensitivity of photosynthetic electron transport near photosystem I (PSI) toward photooxidative stress was also observed in the mutant strain. In the absence of methyl viologen, the mutant exhibited similar growth rates compared with those of the wild type, even at high growth irradiance (350 [mu]E m-2 s-1) where chronic inhibition of photosystem II (PSII) was observed in both strains. Under high growth irradiance, the ratios of PSII to PSI and of [alpha]-phycocyanin to chlorophyll a were less than one-third of the values for the wild type. In both strains, cellular contents of chlorophyll a, [alpha]-phycocyanin, and [beta]-carotene, as well as the length of the phycobilisome rods, declined with increasing growth irradiance. Only the cellular content of the carotenoid zeaxanthin seemed to be independent of growth irradiance. These results suggest an altered acclimation to growth irradiance in the sodB mutant in which the stoichiometry between PSI and PSII is adjusted to compensate for the loss of PSI efficiency occurring under high growth irradiance. Similar shortening of the phycobilisome rods in the sodB mutant and wild-type strain suggest that phycobilisome rod length is regulated independently of photosystem stoichiometry.

Changes in the cyanobacterial photosynthetic apparatus during acclimation to macronutrient deprivation.

When the cyanobacterium Synechococcus sp. Strain PCC 7942 is deprived of an essential macronutrient such as nitrogen, sulfur or phosphorus, cellular phycobiliprotein and chlorophyll contents decline. The level of ß-carotene declines proportionately to chlorophyll, but the level of zeaxanthin increases relative to chlorophyll. In nitrogen- or sulfur-deprived cells there is a net degradation of phycobiliproteins. Otherwise, the declines in cellular pigmentation are due largely to the diluting effect of continued cell division after new pigment synthesis ceases and not to net pigment degradation. There was also a rapid decrease in O2 evolution when Synechococcus sp. Strain PCC 7942 was deprived of macronutrients. The rate of O2 evolution declined by more than 90% in nitrogen- or sulfur-deprived cells, and by approximately 40% in phosphorus-deprived cells. In addition, in all three cases the fluorescence emissions from Photosystem II and its antennae were reduced relative to that of Photosystem I and the remaining phycobilisomes. Furthermore, state transitions were not observed in cells deprived of sulfur or nitrogen and were greatly reduced in cells deprived of phosphorus. Photoacoustic measurements of the energy storage capacity of photosynthesis also showed that Photosystem II activity declined in nutrient-deprived cells. In contrast, energy storage by Photosystem I was unaffected, suggesting that Photosystem I-driven cyclic electron flow persisted in nutrient-deprived cells. These results indicate that in the modified photosynthetic apparatus of nutrient-deprived cells, a much larger fraction of the photosynthetic activity is driven by Photosystem I than in nutrient-replete cells.

Electron transport and photophosphorylation by Photosystem I in vivo in plants and cyanobacteria.

Recently, a number of techniques, some of them relatively new and many often used in combination, have given a clearer picture of the dynamic role of electron transport in Photosystem I of photosynthesis and of coupled cyclic photophosphorylation. For example, the photoacoustic technique has detected cyclic electron transport in vivo in all the major algal groups and in leaves of higher plants. Spectroscopic measurements of the Photosystem I reaction center and of the changes in light scattering associated with thylakoid membrane energization also indicate that cyclic photophosphorylation occurs in living plants and cyanobacteria, particularly under stressful conditions.In cyanobacteria, the path of cyclic electron transport has recently been proposed to include an NAD(P)H dehydrogenase, a complex that may also participate in respiratory electron transport. Photosynthesis and respiration may share common electron carriers in eukaryotes also. Chlororespiration, the uptake of O2 in the dark by chloroplasts, is inhibited by excitation of Photosystem I, which diverts electrons away from the chlororespiratory chain into the photosynthetic electron transport chain. Chlororespiration in N-starved Chlamydomonas increases ten fold over that of the control, perhaps because carbohydrates and NAD(P)H are oxidized and ATP produced by this process.The regulation of energy distribution to the photosystems and of cyclic and non-cyclic phosphorylation via state 1 to state 2 transitions may involve the cytochrome b 6-f complex. An increased demand for ATP lowers the transthylakoid pH gradient, activates the b 6-f complex, stimulates phosphorylation of the light-harvesting chlorophyll-protein complex of Photosystem II and decreases energy input to Photosystem II upon induction of state 2. The resulting increase in the absorption by Photosystem I favors cyclic electron flow and ATP production over linear electron flow to NADP and 'poises' the system by slowing down the flow of electrons originating in Photosystem II.Cyclic electron transport may function to prevent photoinhibition to the photosynthetic apparatus as well as to provide ATP. Thus, under high light intensities where CO2 can limit photosynthesis, especially when stomates are closed as a result of water stress, the proton gradient established by coupled cyclic electron transport can prevent over-reduction of the electron transport system by increasing thermal de-excitation in Photosystem II (Weis and Berry 1987). Increased cyclic photophosphorylation may also serve to drive ion uptake in nutrient-deprived cells or ion export in salt-stressed cells.There is evidence in some plants for a specialization of Photosystem I. For example, in the red alga Porphyra about one third of the total Photosystem I units are engaged in linear electron transfer from Photosystem II and the remaining two thirds of the Photosystem I units are specialized for cyclic electron flow. Other organisms show evidence of similar specialization.Improved understanding of the biological role of cyclic photophosphorylation will depend on experiments made on living cells and measurements of cyclic photophosphorylation in vivo.

Characterization of damage to photosystems I and II in a cyanobacterium lacking detectable iron superoxide dismutase activity.

The enzyme superoxide dismutase is ubiquitous in aerobic organisms where it plays a major role in alleviating oxygen-radical toxicity. An insertion mutation introduced into the iron superoxide dismutase locus (designated sodB) of the cyanobacterium Synechococcus sp. PCC 7942 created a mutant strain devoid of detectable iron superoxide dismutase activity. Both wild-type and mutant strains exhibited similar photosynthetic activity and viability when grown with 17 mumol.m-2.s-1 illumination in liquid culture supplemented with 3% carbon dioxide. In contrast, the sodB mutant exhibited significantly greater damage to its photosynthetic system than the wild-type strain when grown under increased oxygen tension or with methyl viologen. Although damage occurs at both photosystems I and II, it is primarily localized at photosystem I in the sodB mutant. Growth in 100% molecular oxygen for 24 hr decreased photoacoustically measured energy storage in 3-(3,4-dichlorophenyl)-1,1-dimethylurea and abolished the fluorescence state 2 to state 1 transition in the sodB mutant, indicating interruption of cyclic electron flow around photosystem I. Analysis of the flash-induced absorption transient at 705 nm indicated that the interruption of cyclic electron flow occurred in the return part of the cycle, between the two [4 Fe-4 S] centers of photosystem I, FA and FB, and cytochrome f. Even though the sodB mutant was more sensitive to damage by active oxygen than wild-type cells, both strains were equally sensitive to the photoinhibition of photosystem II caused by exposure to strong light.

Light Energy Distribution in the Brown Alga Macrocystis pyrifera (Giant Kelp).

The brown alga Macrocystis pyrifera (giant kelp) was studied by a combination of fluorescence spectroscopy at 77 kelvin, room temperature modulated fluorimetry, and photoacoustic techniques to determine how light energy is partitioned between photosystems I and II in states 1 and 2. Preillumination with farred light induced the high fluorescence state (state 1) as determined by fluorescence emission spectra measured at 77K and preillumination with green light produced a low fluorescence state (state 2). Upon transition from state 1 to state 2, there was an almost parallel decrease of all of the fluorescence bands at 693, 705, and 750 nanometers and not the expected decrease of fluorescence of photosystem II and increase of fluorescence in photosystem I. The momentary level of room temperature fluorescence (fluorescence in the steady state, F(s)), as well as the fluorescence levels corresponding to all closed (F(m)) or all open (F(o)) reaction-center states were measured following the kinetics of the transition between states 1 and 2. Calculation of the distribution of light 2 (540 nanometers) between the two photosystems was done assuming both the ;separate package' and ;spill-over' models. Unlike green plants, red algae, and cyanobacteria, the changes here of the light distribution were rather small in Macrocystis so that there was approximately an even distribution of the photosystem II light at 540 nanometers to photosystem I and photosystem II in both states 1 and 2. Photoacoustic measurements confirmed the conclusions reached as a result of fluorescence measurements, i.e. an almost equal distribution of light-2 quanta to both photosystems in each state. This conclusion was reached by analyzing the enhancement phenomenon by light 2 of the energy storage measured in far red light. The effect of light 1 in decreasing the energy storage measured in light 2 is also consistent with this conclusion. The photoacoustic experiments showed that there was a significant energy storage in light 1 which could be explained by cyclic electron transport around photosystem I. From a quantitative analysis of the enhancement effect of background light 2 (maximum enhancement of 1.4-1.5) it was shown that around 70% of light 1 was distributed to this cyclic photosystem I transport.

A gas-permeable photoacoustic cell.

A photoacoustic cell assembly is described that is permeable to CO2 and other gases but not water vapor. As a replacement for the usually employed solid cover, this cell uses a cover containing a small fritted glass disk that holds a small piece of 6.4 µm Teflon film against the sample.With the above arrangement it was possible to increase the rate of O2 evolution measured photoacoustically about 3 times in Zea mays leaves and about 1.7 times in Phaseolus vulgaris leaves upon adding CO2 to the gas stream. The extent of energy storage was also enhanced with supplemental CO2 in Zea and Ulva but less so in Phaseolus. The maximum improvements of photosynthetic activities were obtained when the gas stream contained 2.5-5% CO2. These high concentrations were presumably necessary as the result of a high resistance to diffusion through the gas-permeable cover.

Photoacoustic measurements in vivo of energy storage by cyclic electron flow in algae and higher plants.

Energy storage by cyclic electron flow through photosystem I (PSI) was measured in vivo using the photoacoustic technique. A wide variety of photosynthetic organisms were considered and all showed measurable energy storage by PSI-cyclic electron flow except for higher plants using the C-3 carbon fixation pathway. The capacity for energy storage by PSI-cyclic electron flow alone was found to be small in comparison to that of linear and cyclic electron flows combined but may be significant, nonetheless, under conditions when photosystem II is damaged, particularly in cyanobacteria. Light-induced dynamics of energy storage by PSI-cyclic electron flow were evident, demonstrating regulation under changing environmental conditions.

Cytochrome c-553 is not required for photosynthetic activity in the cyanobacterium Synechococcus.

In cyanobacteria, the water-soluble cytochrome c-553 functions as a mobile carrier of electrons between the membrane-bound cytochrome b6-f complex and P-700 reaction centers of Photosystem I. The structural gene for cytochrome c-553 (designated cytA) of the cyanobacterium Synechococcus sp. PCC 7942 was cloned, and the deduced amino acid sequence was shown to be similar to known cyanobacterial cytochrome c-553 proteins. A deletion mutant was constructed that had no detectable cytochrome c-553 based on spectral analyses and tetramethylbenzidine-hydrogen peroxide staining of proteins resolved by polyacrylamide gel electrophoresis. The mutant strain was not impaired in overall photosynthetic activity. However, this mutant exhibited a decreased efficiency of cytochrome f oxidation. These results indicate that cytochrome c-553 is not an absolute requirement for reducing Photosystem I reaction centers in Synechococcus sp. PCC 7942.

Photoinhibition Resistance in the Red Alga Porphyra perforata: The Role of Photoinhibition Repair.

Photoinhibition resistance exhibited by the high intertidal red alga Porphyra perforata relative to its subtidal congener Porphyra nereocystis was examined using the protein synthesis inhibitor chloramphenicol to separate the damage and repair components of photoinhibition. Under photoinhibitory conditions, the rates of both damage to and replacement of photoinhibition-sensitive proteins was much higher in P. nereocystis than in P. perforata. Thus, photoinhibition resistance in P. perforata appears to be due to a reduced rate of photoinhibition damage rather than to an accelerated rate of photoinhibition repair. Reduction of photoinhibition damage in P. perforata may be by means of biophysical processes which increase the radiationless decay of excitation to heat in photosystem II. Alternatively, the photoinhibition-sensitive proteins in P. perforata may have slight structural alterations that improve their stability or they may be protected by enzyme systems that quench radicals formed by overexcitation of photosystem II. Reduction of the damage component of photoinhibition is a reasonable way to limit photoinhibition in P. perforata during the severe desiccation and exposure to full sun that occur simultaneously during daily low tides, conditions under which the protein synthesis required for photoinhibition repair could not occur.

Fluorescence characteristics of photoinhibition and recovery in a sun and a shade species of the red algal genus porphyra.

The effects of light treatment (2000 micromole photons per square meter per second) for varying periods (up to 60 minutes) on chlorophyll fluorescence characteristics and light-limited rates of O(2) evolution were examined in two Porphyra species. Brief light exposures (5-60 seconds) produced a large decrease in variable fluorescence which was not accompained by photoinhibition of light-limited O(2) evolution rates. This rapid decrease in variable fluorescence was suppressed by carbonylcyanide m-chlorophenylhydrazone, indicating that it was related to formation of a proton gradient across the thylakiod membranes. A second phase of fluorescence quenching started after 5 minutes of illumination in the case of the shade species, Porphyra nereocystis Anderson, and after 30 minutes of illumination in the case of the sun species, Porphyra perforata J. Agardh. The rate of fluorescence quenching in the second phase was similar to the rate of photoinhibition of light-limited O(2) evolution in both cases. The dark recovery of variable fluorescence in light-treated plants was also biphasic consisting of a rapid first phase and a slower second phase in both the Porphyra species. Recovery of P. perforata was more complete than that of P. nereocystis over the same recovery period. This greater capacity for recovery could represent a mechanism by which P. perforata is more resistant to photoinhibition than P. nereocystis.

Radiationless transitions as a protection mechanism against photoinhibition in higher plants and a red alga.

Exposure of the red alga Porphyra perforata or leaves of Phytolacca americana and Echinodorus sp. to white light equivalent to full sunlight for short periods induced large decreases of variable fluorescence measured at 695 nm at 77K. This change was not produced by photoinhibition but rather appeared to result from an inorease in the rate constant of radiationless transition in the reaction centers of photosystem II. It is proposed that this increase is related to the formation of the high energy state which serves as a photoprotective mechanism in plants.