Yoshihiko Fujita

Short-term and Long-term Adaptation of the Photosynthetic Apparatus: Homeostatic Properties of Thylakoids

Light-energy conversion in thylakoids is accomplished by cooperative interactions between two photoreactions. The balance between these two photoreactions determines the efficiency of energy conversion. The efficiency of energy conversion is maintained at a high level by at least two regulatory mechanisms: short-term adaptation and long-term adaptation. Short-term adaptation, i.e. the state transition, is a regulatory mechanism that controls the distribution of excitation energy transfer from the light-harvesting antenna complexes, the phycobilisomes (PBS), to the two photosystems. The transfer of energy trapped by PBS to the Chl a of Photosystem I (PS I) or Photosystem II (PS II) is regulated either at the transfer point from the PBS to the two photosystems or at a transfer point between the Chl a of PS II and PS I. Energy transfer from the PBS to PS I increases, and to PS II decreases, when most of PS II centers are closed, and the opposite occurs upon a shift to conditions under which most PS I centers are closed. This regulation occurs in response to the state of balance between the two photoreactions through monitoring of the redox status of electron transport between the two photosystems or through monitoring the electrochemical potential of the membrane around the two photosystem complexes. This regulatory process is called’ short-term adaptation’ because this regulation can occur rather rapidly (usually completed within several minutes or less). ‘Long-term adaptation’ refers to a regulated change in the stoichiometry of PS I and PS II in the thylakoids. The PS LPS II ratio becomes greater-values of 2.0 to 3.0 are typical-when cells are grown under conditions where most PS II centers are closed (e.g., green-rich light). The ratio becomes small, approximately 1.0 , upon a shift to conditions where most of PS I centers are closed (e.g., growth in red-rich light). The PS LPS II ratio is also regulated in response to the redox state of electron transport between two photosystems. Regulation of PS I synthesis appears to be the general pattern in cyanobacteria. Synthesis of PS I complexes apparently is controlled at the assembly level but not at the level of apoprotein synthesis. This regulation seems to occur by controlling Chl a synthesis or transport to the site for PS I assembly. The regulation of PS I synthesis is as rapid as state transition. However, the PS I:PS II ratio changes more slowly over a period of hours or days, and hence this process is referred to as ‘long-term adaptation.’ These short-term and long-term adaptation regulatory responses operate together to produce fine-and coarse-tuning adjustments, respectively, and to balance the activities of the two photoreactions in response to changing photosynthetic environments. The phenomena associated with these responses as well as possible mechanisms for their regulation are discussed.

EXCITATION ENERGY TRANSFER IN THE LIGHT HARVESTING ANTENNA SYSTEM OF THE RED ALGA Porphyridium cruentum AND THE BLUE‐GREEN ALGA Anacystis nidulans: ANALYSIS OF TIME‐RESOLVED FLUORESCENCE SPECTRA

Abstract— Time‐resolved fluorescence spectra of intact cells of red and blue‐green algae Porphyridium cruentum and Anacystis nidulans were measured by means of a ps laser and a time‐correlated photon counting system. Fluorescence spectra were observed successively from various pigments in the light harvesting system in the order of phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC) and chlorophyll a (Chl a). The spectrum changes with time in the range of0–400 ps in P. cruentum and of0–1000 ps in A. nidulans. The time‐resolved spectra were analyzed into components to obtain the rise and decay curve of each fluorescence component. Overall time behaviors of the sequential fluorescence emissions from various pigments can be interpreted with a decay kinetics ofexp(–2kt½). The rate constants of the energy transfer show that the energy transfer takes place much faster in the red alga P. cruentum than in the blue‐green alga A. nidulans, particularly in the step PCAPC. Results also indicated that a special form of APC, far‐emitting APC, exists in the pigment system of A. nidulans, but it does not mediate a main energy transfer from phycobilisome to Chl a.

Chromatic regulation inChlamydomonas reinhardtii alters photosystem stoichiometry and improves the quantum efficiency of photosynthesis

The work addressed the adjustment of the photosystem ratio in the green algaChlamydomonas reinhardtii. It is shown that green algae, much like cyanophytes and higher plants, adjust and optimize the ratio of the two photosystems in chloroplasts in response to the quality of irradiance during growth. Such adjustments are compensation reactions and helpC. reinhardtii to retain a quantum efficiency of oxygen evolution near the theoretical maximum. Results show variable amounts of PS I and a fairly constant amount of PS II in chloroplasts and suggest that photosystem stoichiometry adjustments, occurring in response to the quality of irradiance during plant growth, are mainly an adjustment in the concentration of PS I. The work delineates chromatic effects on chlorophyll accumulation in the chloroplast ofC. reinhardtii from those pertaining to the regulation of the PS I/PS II ratio. The detection of the operation of a molecular feedback mechanism for the PS I/PS II ratio adjustment in green algae strengthens the notion of the highly conserved nature of this mechanism among probably all oxygen evolving photosynthetic organisms. Findings in this work are expected to serve as the basis of future biochemical and mutagenesis experiments for the elucidation of the photosystem ratio adjustment in oxygenic photosynthesis.

THE EFFECT OF IRON NUTRITION ON PHOTOSYNTHESIS AND NITROGEN FIXATION IN CULTURES OF TRICHODESMIUM (CYANOPHYCEAE)1

Cultures of Trichodesmium NIBB 1067 were grown in the synthetic medium AQUIL with a range of iron added from none to 5 × 10−7 M Fe for 15 days. Chlorophyll‐a, cell counts, and total cell volume were two or three times higher in medium with 10−7 M Fe than with no added Fe. Oxygen production rate per chlorophyll‐a was over 60% higher with higher iron. Increased iron stimulated photosynthesis at all irradiances from about 12–250 μE · m−2· s−1. Nitrogen fixation rate, estimated from acetylene reduction, for 10−7 and 10−8 M Fe cultures was approximately twice that of the cultures with no added Fe. The range of rates of O2 production and N2 fixation in cultures at the iron concentrations we used were similar to the rates from natural samples of Trichodesmium from both the Atlantic, and the Pacific oceans. This similarity may allow this clone to be used, with some caution, for future physiological ecology studies. This study demonstrates the importance of iron to photosynthesis and nitrogen fixation and suggests that Trichodesmium plays a central role in the biogeochemical cycles of iron, carbon and nitrogen.

Regulation of nitrogen-fixation by different nitrogen sources in the marine non-heterocystous cyanobacterium Trichodesmium sp. NIBB1067

The effect of various nitrogen sources on the synthesis and activity of nitrogenase was studied in the marine, non-heterocystous cyanobacterium Trichodesmium sp. NIBB1067 grown under defined culture conditions. Cells grown with N2 as the sole inorganic nitrogen source showed light-dependent nitrogenase activity (acetylene reduction). Nitrogenase activity in cells grown on N2 was not suppressed after 7 h incubation with 2 mM NaNO3 or 0.02 mM NH4Cl. However, after 3 h of exposure to 0.5 mM of urea, nitrogenase was inactivated. Cells grown in medium containing 2 mM NaNO3, 0.5 mM urea or 0.02 mM NH4Cl completely lacked the ability to reduce acetylene. Western immunoblots tested with polyclonal antisera against the Fe-protein and the Mo−Fe protein, revealed the following: (1) both the Fe-protein and the Mo−Fe protein were synthesized in cells grown with N2 as well as in cells grown with NaNO3 or low concentration of NH4Cl; (2) two bands (apparent molecular mass of 38 000 and 40 000) which cross-reacted with the antiserum to the Fe-protein, were found in nitrogen-fixing cells; (3) only one protein band, corresponding to the high molecular mass form of the Fe-protein, was found in cells grown with NaNO3 or low concentration of NH4Cl; (4) neither the Fe-protein nor the Mo−Fe protein was found in cells grown with urea; (5) the apparent molecular mass of the Fe-protein of Trichodesmium sp. NIBB1067 was about 5000 dalton higher than that of the heterocystous cyanobacterium, Anabaena cylindrica IAM-M1.

Aerobic nitrogenase activity measured as acetylene reduction in the marine non-heterocystous cyanobacterium Trichodesmium spp. grown under artificial conditions

Aerobic nitrogenase activity in the marine non-heterocystous cyanobacterium Trichodesmium spp. NIBB 1067, isolated off the Izu Peninsula, Japan in 1983 and grown under artificial conditions, was assayed by the acetylene reduction method. This strain exhibited acetylene reduction activity under aerobic conditions when cells had been grown in the medium free of combined nitrogen. Activity was markedly enhanced by light, and dependent on the growth phase being higher during the exponential growth phase and lower during the late linear and stationary growth phases. Since typical colony formation occurred during the last growth phase, the present results contradict the idea that N2-fixation depends on colony formation. The photosynthesis inhibitor DCMU at 10-6M inhibited light-dependent acetylene reduction completely. Acetylene reduction by Trichodesmium spp. was tolerant of O2 as strongly as that in the heterocystous cyanobacteria. Even at a partial pressure of oxygen (pO2) of ∼3 atm, the activity still remained as high as half of the maximum. It was almost under anaerobic conditions. Maximum activity was obtained at pO2 of ca. 0.1 atm.

Cultures of the pelagic cyanophytes Trichodesmium erythraeum and T. thiebautii in synthetic medium

Trials for determination of culture conditions for the marine cyanophytes of Trichodesmium erythraeum and T. thiebautii were made with use of a synthetic medium. The “Aquil” medium, either with or without combined nitrogen, brought about stable growth of the two strains, T. erythraeum and T. thiebautii. However, they failed to grow in an ASP7 medium. The failure was found to be due to the toxic effect of Tris-aminomethane, the pH-buffer in this medium. Two important chemical conditions for the stable growth of Trichodesmium spp. were revealed. (1) Stable growth was supported by Ca2+ at high concentrations; in a concentration lower than 0.9 mM, cell-lysis promptly occurred, while the cells could grow without cell-lysis at Ca2+ concentrations higher than 7.5 mM even at a salinity as low as 19‰ S. Ca2+ is probably essential for the osmotic regulation in this organism. (2) Phosphate-toxicity at high concentrations was at least partly due to heavy metal(s) contaminating the reagent of inorganic phosphate. After treatment with a Chelex-100 column, phosphate concentration could be increased up to four times the previous concentrations without toxicity.

Regulation of nitrogenase activity in relation to the light-dark regime in the filamentous non-heterocystous cyanobacterium Trichodesmium sp. NIBB 1067

SUMMARY: A periodicity in nitrogen fixation potential with respect to the light-dark regime was studied in the filamentous non-heterocystous cyanobacterium Trichodesmium sp. NIBB 1067. During a 12 h light/12 h dark cycle, potential nitrogenase activity measured by acetylene reduction in the light was insignificant in the dark period, but developed after illumination for 1 to 3 h. Maximum nitrogenase activity was found at the middle of the light period, and activity decreased near the end of the light period. Manipulation of the length of the light and dark periods, and use of the glutamine synthetase inhibitor L-methionine sulphoximine, led to the conclusion that (1) the periodicity in activity was not attributable to an endogenous rhythm, (2) development and maintenance of nitrogenase activity in Trichodesmium was regulated by the light period, and (3) the decrease in activity at the end of the light period was due to the accumulation of an intermediate(s) in nitrogen metabolism. The nitrogenase Fe- and MoFe-proteins were always present despite the changes in nitrogenase activity associated with the light-dark cycle. However, a change in apparent molecular mass of the Fe-protein on SDS-PAGE correlated with the change in nitrogenase activity. The results indicate that changes of nitrogenase activity in Trichodesmium under a light-dark regime can be attributed to activation and deactivation of the Fe-protein, and that the activation of the protein depends on light.

Effect of redox state on the dynamics Photosystem II during steady-state photosynthesis in eucaryotic algae

Abstract Using a pump and probe-flash technique we simultaneously measured changes in fluorescence yields (ΔΦ) and the yield of oxygen produced by the pump flash ( Y ) during steady-state photosynthesis in continuous background light in two species of eucaryotic algae, Chlorella pyrenoidosa , a chlorophyte, and Chaetoceros gracilis , a diatom. By varying the pump-flash intensity, we examined the flash-intensity saturation curves for ΔΦ and Y . When a small fraction of Photosystem (PS) II traps is closed by a blue-green background light the flash-intensity saturation curves for ΔΨ and Y closely followed a cumulative one-hit Poisson function. However, in cells exposed to a far-red background light (more than 720 nm) the flash-intensity saturation curve for ΔΦ (but not Y ) rose faster than predicted by a one-hit Poisson model. These results suggest that energy transfer between PS II reaction centers is moderated by the spectral quality of a continuous photon flux. Using saturating pump flashes we calculated the relative fraction of open/closed PS II reaction centers as perceived from the donor and acceptor sides as the background irradiance was changed. The relative changes in ΔΦ and Y were highly correlated at low and moderate levels of continuous background light, regardless of spectral quality. At high continuous photon fluxes, however, Y declined faster with increasing background irradiance than ΔΦ. Based on kinetic data is appears that the uncoupling between Y and ΔΦ at high photon flux densities is largely due to cyclic electron flow around PS II, which is negligible at subsaturating background irradiance levels. Cyclic electron flow around PS II accounted for 15–28% of the linear electron flow and may reduce damage to PS II reaction centers at supraoptimal irradiance levels.

OCCURRENCE OF A TEMPERATE CYANOPHAGE LYSOGENIZING THE MARINE CYANOPHYTE PHORMIDIUM PERSICINUM1

A temperate cyanophage was found to lysogenize the marine cyanophyte Phormidium persicinum (Reinke) Com. (Provasoli strain). The lytic cycle was induced by the addition of mitomycin C or by brief illumination with ultraviolet light. The lytic process observed under the electron microscope showed that phage particles appeared in a nucleoplasm region 15 to 24 h after the addition of mitomycin C. The induction of the lytic process occurred simultaneously in almost all cells of every trichome. Matured phage particles were released to the medium 30 to 50 h after the addition of mitomycin C. Phage particles isolated from algal lysates had a polyhedral head (about 40 nm in diameter) with a long (about 300 nm) and noncontractile tail. The most abundant protein, presumably a structural protein, had an apparent molecular mass of about 38 kDa. The genome size estimated from restriction analysis was about 50 kbp. Phage DNA was digested with several restriction endonucleases including Sau3AI and DpnI. However, MboI failed to digest the phage DNA, suggesting that the phage DNA is highly methylated. Southern blot analysis suggested that some part of the phage was in the lytic cycle in algal cells growing under normal conditions. A possible role of temperate cyanophages in the regulation of cyanophyte populations in the marine environment is discussed.