Synechococcus elongatus PCC7942: a cyanobacterium cell factory for producing useful chemicals and fuels under abiotic stress conditions.

Sucrose, a compatible osmolyte in cyanobacteria, functions both as an energy reserve and as osmoprotectant. Sugars are the most common substrates used by microorganisms to produce hydrogen (H2) by means of anaerobic dark fermentation. Cells of the unicellular, non-nitrogen fixing, freshwater cyanobacterium Synechococcus elongatus PCC7942 accumulate sucrose under salt stress. In the present work, we used this cyanobacterium and a genetically engineered strain of it (known as PAMCOD) to investigate the optimal conditions for (a) photosynthetic activity, (b) cell proliferation and (c) sucrose accumulation, which are necessary for H2 production via anaerobic dark fermentation of the accumulated sucrose. PAMCOD (Deshnium et al. in Plant Mol Biol 29:897-902, 1995) contains the gene codA that codes for choline oxidase, the enzyme which converts choline to the zwitterion glycine betaine. Glycine betaine is a compatible osmolyte which increases the salt tolerance of Synechococcus elongatus PCC7942. Furthermore, glycine betaine maintains cell proliferation under salt stress and results in increased sucrose accumulation. In the present study, we examine the environmental factors, such as the NaCl concentration, the culture medium pH, and the carbon dioxide content of the air bubbled through it. At optimal conditions, sucrose accumulated in the cyanobacteria cells up to 13.5 mol per mole Chl a. Overall, genetically engineered Synechococcus elongatus PCC7942 produces sucrose in sufficient quantities such that it may be a viable alternative (a) to sucrose synthesis, and (b) to H2 formation via anaerobic dark fermentation.

Eugene I. Rabinowitch: A prophet of photosynthesis and of peace in the world.

More than 45 years have passed since Eugene I. Rabinowitch died, on May 15, 1973, at the age of 75, but many still remember him as a photosynthesis giant, the author of a 2000-page "Bible" on photosynthesis, a great chemist and physicist, a discoverer of several basic photoreactions, one of the founders of modern biophysics, a peacemaker, a poet, an architect, an artist, a wonderful human being, and above all a great mentor. Sir John Rotblatt cited Eugene Rabinowitch, together with Bertrand Russell, for their key contributions that led to the Nobel Peace Prize awarded in 1995 jointly to Rotblatt and the Pugwash Conferences on Science and World Affairs "for their efforts to diminish the part played by nuclear arms in international politics and, in the longer run, to eliminate such arms." Already in 1965, Eugene Rabinowitch had received the prestigious Kalinga Prize from UNESCO "in recognition of his work to encourage international cooperation among scientists and to bring to light the potential dangers of science to the public."

Light-adaptive state transitions in the Ross Sea haptophyte Phaeocystis antarctica and in dinoflagellate cells hosting kleptoplasts derived from it.

Light state transitions (STs) is a reversible physiological process that oxygenic photosynthetic organisms use in order to minimize imbalances in the electronic excitation delivery to the reaction centers of Photosystems I and II, and thus to optimize photosynthesis. STs have been studied extensively in plants, green algae, red algae and cyanobacteria, but sparsely in algae with secondary red algal plastids, such as diatoms and haptophytes, despite their immense ecological significance. In the present work, we examine whether the haptophyte alga Phaeocystis antarctica, and dinoflagellate cells that host kleptoplasts derived from P. antarctica, both endemic in the Ross Sea, Antarctica, are capable of light adaptive STs. In these organisms, Chl a fluorescence can be excited either by direct light absorption, or indirectly by electronic excitation (EE) transfer from ultraviolet light absorbing mycosporine-like amino acids (MAAs) to Chl a (Stamatakis et al., Biochim. Biophys. Acta 1858 [2017] 189-195). Here we show that, on adaptation to PS II-selective light, dark-adapted P. antarctica cells shift from light state 1 (ST1; more EE ending up in PS II) to light state 2 (ST2; more EE ending up in PS I), as revealed by the spectral distribution of directly-excited Chl a fluorescence and by changes in the macro-organization of pigment-protein complexes evidenced by circular dichroism (CD) spectroscopy. In contrast, no STs are clearly detected in the case of the kleptoplast-hosting dinoflagellate cells, and in the case of indirectly excited Chls a, via MAAs, in P. antarctica cells.

Frederick Yi-Tung Cho (1939-2011) : His PhD days in Biophysics, the Photosynthesis Lab, and his patents in engineering physics.

We present here a Tribute to Frederick Yi-Tung Cho (1939-2011), an innovative and ingenious biophysicist and an entrepreneur. He was one of the 4 earliest PhD students [see: Cederstrand (1965)-Carl Nelson Cederstrand; coadvisor: Eugene Rabinowitch; Papageorgiou (1968)-George C. Papageorgiou (coauthor of this paper); and Munday (1968)-John C. Munday Jr. (also a coauthor of this paper)] of one of us (Govindjee) in Biophysics at the University of Illinois at Urbana-Champaign (UIUC) during the late 1960s (1963-1968). Fred was best known, in the photosynthesis circle for his pioneering work on low temperature (down to liquid helium temperature, 4 K) absorption and fluorescence spectroscopy of photosynthetic systems; he showed temperature independence of excitation energy transfer from (i) chlorophyll (Chl) b to Chl a and (ii) from Chl a 670 to Chl a 678; and temperature dependence of energy transfer from the phycobilins to Chl a and from Chl a 678 to its suggested trap. After doing research in biophysics of photosynthesis, Fred shifted to do research in solid-state physics/engineering in the Government Electronics Division (Group) of the Motorola Company, Scottsdale, Arizona, from where he published research papers in that area and had several patents granted. We focus mainly on his days at the UIUC in context of the laboratory in which he worked. We also list some of his papers and most of his patents in engineering physics. His friends and colleagues have correctly described him as an innovator and an ingenious scientist of the highest order. On the personal side, he was a very easy-going and amiable individual.

The extraordinary longevity of kleptoplasts derived from the Ross Sea haptophyte Phaeocystis antarctica within dinoflagellate host cells relates to the diminished role of the oxygen-evolving Photosystem II and to supplementary light harvesting by mycosporine-like amino acid/s.

The haptophyte Phaeocystis antarctica and the novel Ross Sea dinoflagellate that hosts kleptoplasts derived from P. antarctica (RSD; R.J. Gast et al., 2006, J. Phycol. 42 233-242) were compared for photosynthetic light harvesting and for oxygen evolution activity. Both chloroplasts and kleptoplasts emit chlorophyll a (Chl a) fluorescence peaking at 683nm (F683) at 277K and at 689 (F689) at 77K. Second derivative analysis of the F689 band at 77K revealed two individual contributions centered at 683nm (Fi-683) and at 689 (Fi-689). Using the p-nitrothiophenol (p-NTP) treatment of Kobayashi et al. (Biochim. Biophys. Acta 423 (1976) 80-90) to differentiate between Photosystem (PS) II and I fluorescence emissions, we could identify PS II as the origin of Fi-683 and PS I as the origin of Fi-689. Both emissions could be excited not only by Chl a-selective light (436nm) but also by mycosporine-like amino acids (MAAs)-selective light (345nm). This suggests that a fraction of MAAs must be proximal to Chls a and, therefore, located within the plastids. On the basis of second derivative fluorescence spectra at 77K, of p-NTP resolved fluorescence spectra, as well as of PSII-driven oxygen evolution activities, PS II appears substantially less active (~1/5) in dinoflagellate kleptoplasts than in P. antarctica chloroplasts. We suggest that a diminished role of PS II, a known source of reactive oxygen species, and a diminished dependence on nucleus-encoded light-harvesting proteins, due to supplementary light-harvesting by MAAs, may account for the extraordinary longevity of RSD kleptoplasts.

Effects of exogenous ß-carotene, a chemical scavenger of singlet oxygen, on the millisecond rise of chlorophyll a fluorescence of cyanobacterium Synechococcus sp. PCC 7942.

Singlet-excited oxygen (1O 2* ) has been recognized as the most destructive member of the reactive oxygen species (ROS) which are formed during oxygenic photosynthesis by plants, algae, and cyanobacteria. ROS and 1O 2* are known to damage protein and phospholipid structures and to impair photosynthetic electron transport and de novo protein synthesis. Partial protection is afforded to photosynthetic organism by the ß-carotene (ß-Car) molecules which accompany chlorophyll (Chl) a in the pigment-protein complexes of Photosystem II (PS II). In this paper, we studied the effects of exogenously added ß-Car on the initial kinetic rise of Chl a fluorescence (10-1000 µs, the OJ segment) from the unicellular cyanobacterium Synechococcus sp. PCC7942. We show that the added ß-Car enhances Chl a fluorescence when it is excited at an intensity of 3000 µmol photons m-2 s-1 but not when excited at 1000 µmol photons m-2 s-1. Since ß-Car is an efficient scavenger of 1O 2* , as well as a quencher of 3Chl a * (precursor of 1O 2* ), both of which are more abundant at higher excitations, we assume that the higher Chl a fluorescence in its presence signifies a protective effect against photo-oxidative damages of Chl proteins. The protective effect of added ß-Car is not observed in O2-depleted cell suspensions. Lastly, in contrast to ß-Car, a water-insoluble molecule, a water-soluble scavenger of 1O 2* , histidine, provides no protection to Chl proteins during the same time period (10-1000 µs).

ΔpH-dependent non-photochemical quenching (qE) of excited chlorophylls in the photosystem II core complex of the freshwater cyanobacterium Synechococcus sp PCC 7942.

Light-induced and lumen acidity-dependent quenching (qE) of excited chlorophylls (Chl) in vivo has been amply documented in plants and algae, but not in cyanobacteria, using primarily the saturation pulse method of quenching analysis which is applied to continuously illuminated samples. This method is unsuitable for cyanobacteria because the background illumination elicits in them a very large Chl a fluorescence signal, due to a state 2 to state 1 transition, which masks fluorescence changes due to other causes. We investigated the qE problem in the cyanobacterium Synechococcus sp. PCC 7942 using a kinetic method (Chl a fluorescence induction) with which qE can be examined before the onset of the state 2 to state 1 transition and the attendant rise of Chl a fluorescence. Our results confirm the existence of a qE mechanism that operates on excited Chls a in Photosystem II core complexes of cyanobacteria.

On the question of the light-harvesting role of ß-carotene in photosystem II and photosystem I core complexes.

ß-Carotene is the only carotenoid present in the core complexes of Photosystems I and II. Its proximity to chlorophyll a molecules enables intermolecular electronic interactions, including ß-carotene to chlorophyll a electronic excitation transfers. However, it has been well documented that, compared to chlorophylls and to phycobilins, the light harvesting efficiency of ß-carotenes for photosynthetic O2 evolution is poor. This is more evident in cyanobacteria than in plants and algae because they lack accessory light harvesting pigments with absorptions that overlap the ß-carotene absorption. In the present work we investigated the light harvesting role of ß-carotenes in the cyanobacterium Synechococcus sp. PCC 7942 using selective ß-carotene excitation and selective Photosystem detection of photo-induced electron transport to and from the intersystem plastoquinones (the plastoquinone pool). We report that, although selectively excited ß-carotenes transfer electronic excitation to the chlorophyll a of both photosystems, they enable only the oxidation of the plastoquinone pool by Photosystem I but not its reduction by Photosystem II. This may suggest a light harvesting role for the ß-carotenes of the Photosystem I core complex but not for those of the Photosystem II core complex. According to the present investigation, performed with whole cyanobacterial cells, the lower photosynthesis yields measured with ß-Car-absorbed light can be attributed to the different excitation trapping efficiencies in the reaction centers of PSI and PSII.

The slow S to M fluorescence rise in cyanobacteria is due to a state 2 to state 1 transition.

In dark-adapted plants and algae, chlorophyll a fluorescence induction peaks within 1s after irradiation due to well documented photochemical and non-photochemical processes. Here we show that the much slower fluorescence rise in cyanobacteria (the so-called "S to M rise" in tens of seconds) is due to state 2 to state 1 transition. This has been demonstrated in particular for Synechocystis PCC6803, using its RpaC(-) mutant (locked in state 1) and its wild-type cells kept in hyperosmotic suspension (locked in state 2). In both cases, the inhibition of state changes correlates with the disappearance of the S to M fluorescence rise, confirming its assignment to the state 2 to state 1 transition. The general physiological relevance of the SM rise is supported by its occurrence in several cyanobacterial strains: Synechococcus (PCC 7942, WH 5701) and diazotrophic single cell cyanobacterium (Cyanothece sp. ATCC 51142). We also show here that the SM fluorescence rise, and also the state transition changes are less prominent in filamentous diazotrophic cyanobacterium Nostoc sp. (PCC 7120) and absent in phycobilisome-less cyanobacterium Prochlorococcus marinus PCC 9511. Surprisingly, it is also absent in the phycobiliprotein rod containing Acaryochloris marina (MBIC 11017). All these results show that the S to M fluorescence rise reflects state 2 to state 1 transition in cyanobacteria with phycobilisomes formed by rods and core parts. We show that the pronounced SM fluorescence rise may reflect a protective mechanism for excess energy dissipation in those cyanobacteria (e.g. in Synechococcus PCC 7942) that are less efficient in other protective mechanisms, such as blue light induced non-photochemical quenching. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Photosystem II fluorescence: slow changes--scaling from the past.

With the advent of photoelectric devices (photocells, photomultipliers) in the 1930s, fluorometry of chlorophyll (Chl) a in vivo emerged as a major method in the science of photosynthesis. Early researchers employed fluorometry primarily for two tasks: to elucidate the role in photosynthesis, if any, of other plant pigments, such as Chl b, Chl c, carotenoids and phycobilins; and to use it as a convenient inverse measure of photosynthetic activity. In pursuing the latter task, it became apparent that Chl a fluorescence emission is influenced (i) by redox active Chl a molecules in the reaction center of photosystem (PS) II (photochemical quenching); (ii) by an electrochemical imbalance across the thylakoid membrane (high energy quenching); and (iii) by the size of the peripheral antennae of weakly fluorescent PSI and strongly fluorescent PSII in response to changes in the ambient light (state transitions). In this perspective we trace the historical evolution of our awareness of these concepts, particularly of the so-called 'State Transitions'.

Spectral characteristic of fluorescence induction in a model cyanobacterium, Synechococcus sp. (PCC 7942).

We present here three-dimensional time-wavelength-intensity displays of changes in variable fluorescence, during the O(JI)PSMT transient, observed in cyanobacterium at room temperature. We were able to measure contributions of individual chromophores to fluorescence spectra at various times of fluorescence induction (FI). The method was applied to a freshwater cyanobacterium, Synechococcus sp. (PCC 7942). Analysis of our experimental results provides the following new conclusions: (i) the main chlorophyll (Chl) a emission band at approximately 685 nm that originates in Photosystem (PS) II exhibits typical fast (OPS) and slow (SMT) FI kinetics with both orange (622 nm) and blue (464 nm) excitation. (ii) Similar kinetics are exhibited for its far-red emission satellite band centered at approximately 745 nm, where the PS II contribution predominates. (iii) A significant OPS-SMT-type kinetics of C-phycocyanin emission at approximately 650 nm are observed with the blue light excitation, but not with orange light excitation where the signal rose only slightly to a maximum. The induction of F650 was not caused by an admixture of the F685 fluorescence and thus our data show light-inducible and dark-reversible changes of phycobilin fluorescence in vivo. We discuss possible interpretations of this new observation.

Dark-to-light transition in Synechococcus sp. PCC 7942 cells studied by fluorescence kinetics assesses plastoquinone redox poise in the dark and photosystem II fluorescence component and dynamics during state 2 to state 1 transition.

We investigated the dark-to-light transition in Synechococcus sp. PCC 7942 cells by a detailed analysis of fluorescence transients induced by strong red light. The transients, recorded with high data-acquisition, revealed all the steps of the fast (OJIP; 10(-5)-1 s) and slow phase (PSM(T); 1-10(3) s), kinetically distinguished with precision. Focusing on the OJIP-rise, we show, for the first time, how the variable to initial fluorescence ratio and the relative height of J-level can serve as indexes of the plastoquinone redox poise and the established state in the dark; hence, differences among cyanobacteria can be recognised in a simple way. Applying intermittent illumination (20-s light pulses separated by 10-s dark intervals) to induce dark-to-light transition and analysing the individual transients, we establish a method by which we determine the fluorescence component not originating from photosystem (PS) II and we assess PSII dynamics during state 2 to state 1 transition. The development of photochemical and non-photochemical quenching is also discussed, as well as evidences favouring the mobile antenna model.

A viewpoint: why chlorophyll a?

Chlorophyll a (Chl a) serves a dual role in oxygenic photosynthesis: in light harvesting as well as in converting energy of absorbed photons to chemical energy. No other Chl is as omnipresent in oxygenic photosynthesis as is Chl a, and this is particularly true if we include Chl a(2), (=[8-vinyl]-Chl a), which occurs in Prochlorococcus, as a type of Chl a. One exception to this near universal pattern is Chl d, which is found in some cyanobacteria that live in filtered light that is enriched in wavelengths >700 nm. They trap the long wavelength electronic excitation, and convert it into chemical energy. In this Viewpoint, we have traced the possible reasons for the near ubiquity of Chl a for its use in the primary photochemistry of Photosystem II (PS II) that leads to water oxidation and of Photosystem I (PS I) that leads to ferredoxin reduction. Chl a appears to be unique and irreplaceable, particularly if global scale oxygenic photosynthesis is considered. Its uniqueness is determined by its physicochemical properties, but there is more. Other contributing factors include specially tailored protein environments, and functional compatibility with neighboring electron transporting cofactors. Thus, the same molecule, Chl a in vivo, is capable of generating a radical cation at +1 V or higher (in PS II), a radical anion at -1 V or lower (in PS I), or of being completely redox silent (in antenna holochromes).

The fast and slow kinetics of chlorophyll a fluorescence induction in plants, algae and cyanobacteria: a viewpoint.

The light-induced/dark-reversible changes in the chlorophyll (Chl) a fluorescence of photosynthetic cells and membranes in the mus-to-several min time window (fluorescence induction, FI; or Kautsky transient) reflect quantum yield changes (quenching/de-quenching) as well as changes in the number of Chls a in photosystem II (PS II; state transitions). Both relate to excitation trapping in PS II and the ensuing photosynthetic electron transport (PSET), and to secondary PSET effects, such as ion translocation across thylakoid membranes and filling or depletion of post-PS II and post-PS I pools of metabolites. In addition, high actinic light doses may depress Chl a fluorescence irreversibly (photoinhibitory lowering; q(I)). FI has been studied quite extensively in plants an algae (less so in cyanobacteria) as it affords a low resolution panoramic view of the photosynthesis process. Total FI comprises two transients, a fast initial (OPS; for Origin, Peak, Steady state) and a second slower transient (SMT; for Steady state, Maximum, Terminal state), whose details are characteristically different in eukaryotic (plants and algae) and prokaryotic (cyanobacteria) oxygenic photosynthetic organisms. In the former, maximal fluorescence output occurs at peak P, with peak M lying much lower or being absent, in which case the PSMT phases are replaced by a monotonous PT fluorescence decay. In contrast, in phycobilisome (PBS)-containing cyanobacteria maximal fluorescence occurs at M which lies much higher than peak P. It will be argued that this difference is caused by a fluorescence lowering trend (state 1 --> 2 transition) that dominates the FI pattern of plants and algae, and correspondingly by a fluorescence increasing trend (state 2 --> 1 transition) that dominates the FI of PBS-containing cyanobacteria. Characteristically, however, the FI pattern of the PBS-minus cyanobacterium Acaryochloris marina resembles the FI patterns of algae and plants and not of the PBS-containing cyanobacteria.

Fluorescence induction in the phycobilisome-containing cyanobacterium Synechococcus sp PCC 7942: analysis of the slow fluorescence transient.

At room temperature, the chlorophyll (Chl) a fluorescence induction (FI) kinetics of plants, algae and cyanobacteria go through two maxima, P at approximately 0.2-1 and M at approximately 100-500 s, with a minimum S at approximately 2-10 s in between. Thus, the whole FI kinetic pattern comprises a fast OPS transient (with O denoting origin) and a slower SMT transient (with T denoting terminal state). Here, we examined the phenomenology and the etiology of the SMT transient of the phycobilisome (PBS)-containing cyanobacterium Synechococcus sp PCC 7942 by modifying PBS-->Photosystem (PS) II excitation transfer indirectly, either by blocking or by maximizing the PBS-->PS I excitation transfer. Blocking the PBS-->PS I excitation transfer route with N-ethyl-maleimide [NEM; A. N. Glazer, Y. Gindt, C. F. Chan, and K.Sauer, Photosynth. Research 40 (1994) 167-173] increases both the PBS excitation share of PS II and Chl a fluorescence. Maximizing it, on the other hand, by suspending cyanobacterial cells in hyper-osmotic media [G. C. Papageorgiou, A. Alygizaki-Zorba, Biochim. Biophys. Acta 1335 (1997) 1-4] diminishes both the PBS excitation share of PS II and Chl a fluorescence. Here, we show for the first time that, in either case, the slow SMT transient of FI disappears and is replaced by continuous P-->T fluorescence decay, reminiscent of the typical P-->T fluorescence decay of higher plants and algae. A similar P-->T decay was also displayed by DCMU-treated Synechococcus cells at 2 degrees C. To interpret this phenomenology, we assume that after dark adaptation cyanobacteria exist in a low fluorescence state (state 2) and transit to a high fluorescence state (state 1) when, upon light acclimation, PS I is forced to run faster than PS II. In these organisms, a state 2-->1 fluorescence increase plus electron transport-dependent dequenching processes dominate the SM rise and maximal fluorescence output is at M which lies above the P maximum of the fast FI transient. In contrast, dark-adapted plants and algae exist in state 1 and upon illumination they display an extended P-->T decay that sometimes is interrupted by a shallow SMT transient, with M below P. This decay is dominated by a state 1-->2 fluorescence lowering, as well as by electron transport-dependent quenching processes. When the regulation of the PBS-->PS I electronic excitation transfer is eliminated (as for example in hyper-osmotic suspensions, after NEM treatment and at low temperature), the FI pattern of Synechococcus becomes plant-like.

Facilitated water transport in cyanobacterium Synechococcus sp. PCC 7942 studied by phycobilisome-sensitized chlorophyll a fluorescence.

Water transport across plant cell membranes is difficult to measure. We present here a model assay, based on chlorophyll (Chl) a fluorometry, with which net water transport across the cell membrane of freshwater cyanobacterium Synechococcus sp. PCC7942 (S7942) can be followed kinetically with millisecond-time resolution. In cyanobacteria, the phycobilisome (PBS)-sensitized Chl a fluorescence increases when cells expand (e.g., in hypo-osmotic suspension) and decreases when cells contract (e.g., in hyper-osmotic suspension). The osmotically-induced Chl a fluorescence changes are proportional to the reciprocal of the suspension osmolality (DeltaF proportional, variant Osm-1; Papageorgiou GC and Alygizaki-Zorba A (1997) Biochim Biophys Acta 1335: 1-4). In our model assay, S7942 cells were loaded with NaCl (passively penetrating solute) and shrunk in hyper-osmotic glycine betaine (nonpenetrating solute). Upon injecting these cells into hypo-osmotic medium, the PBS-sensitized Chl a fluorescence rose to a maximum due to the osmotically-driven water uptake. The rise of Chl a fluorescence (water uptake) was partially inhibited by HgCl2, at micromolar concentrations. Arrhenius plots of the water uptake rates gave activation energies of EA=4.9 kcal mol-1, in the absence of HgCl2, and EA=11.9 kcal mol-1 in its presence. These results satisfy the usual criteria for facilitated water transport through protein water pores of plasma membranes (aquaporins), namely sensitivity to Hg2+ ions and low activation energy.

Photosynthesis research in Greece: a historical snapshot (1960-2001).

The origin of photosynthesis research in Greece can be traced to the early 1960s, and the first dedicated laboratory was established by George Akoyunoglou in the Nuclear Reseach Center (now National Center for Scientific Research) Demokritos, in Athens. More photosynthesis groups subsequently emerged, in Demokritos and in the universities. Research in Greece benefited greatly from the links of Greek scientists with laboratories and personalities, primarily in the USA and western Europe. The local research output is a proportional part of global research and, more or less, in tune with the shifting priorities of the latter. The list of references provided includes only a sample of publications: it is not inclusive.