Adventures with Cyanobacteria: A Personal Perspective

Cyanobacteria, or the blue-green algae as they used to be called until 1974, are the oldest oxygenic photosynthesizers. We summarize here adventures with them since the early 1960s. This includes studies on light absorption by cyanobacteria, excitation energy transfer at room temperature down to liquid helium temperature, fluorescence (kinetics as well as spectra) and its relationship to photosynthesis, and afterglow (or thermoluminescence) from them. Further, we summarize experiments on their two-light reaction – two-pigment system, as well as the unique role of bicarbonate (hydrogen carbonate) on the electron-acceptor side of their photosystem II, PSII. This review, in addition, includes a discussion on the regulation of changes in phycobilins (mostly in PSII) and chlorophyll a (Chl a; mostly in photosystem I, PSI) under oscillating light, on the relationship of the slow fluorescence increase (the so-called S to M rise, especially in the presence of diuron) in minute time scale with the so-called state-changes, and on the possibility of limited oxygen evolution in mixotrophic PSI (minus) mutants, up to 30 min, in the presence of glucose. We end this review with a brief discussion on the position of cyanobacteria in the evolution of photosynthetic systems.

In this personal perspective, we will focus mainly on the adventures of one of us (G) that relate to his work on cyanobacteria with many graduate students, postdoctoral associates and researchers around the World. A perspective of this nature is essential to appreciate the Frontiers in Plant Physiology. We present here a summary of discoveries on specific cyanobacteria that will include new spectral forms of Chl a and P750 (in Anacystis (A.) nidulans), the two-light effect and the two-pigment system, excitation energy transfer that seems to follow Förster's Resonance Energy Transfer theory (from phycobilins to Chl a), the first observation of fast fluorescence changes, the first demonstration of the role of bicarbonate on the electron-acceptor side of PSII, the relationship of slow fluorescence changes with changes that are unrelated to the quencher "Q A ," but related to "conformational" changes and/or "state changes" (energy distribution between the two photosystems), and regulation of this energy distribution by various factors, and most recently, the observation of light-induced oxygen evolution in PSI-minus mutants in mixotrophic cultures and in the presence of glucose that demonstrates the flexibility of cyanobacteria.

cyanobacterIa have two-pIgment systems and two-lIght reactIons
Unlike anoxygenic photosynthetic bacteria (Blankenship et al., 1995;Hunter et al., 2009), cyanobacteria, like algae and plants, perform oxygenic photosynthesis . For that they use two-pigment systems and two-light reactions. Emerson and Chalmers (1958) discovered that, in A. nidulans, light absorbed in phycobilins enhanced the yield of oxygen evolution when added on top of light absorbed in Chl a. Thus, they proposed that in cyanobacteria, the "short-wave" photosystem is run by phycobilins and the "long-wave" photosystem is run by Chl a (also see Emerson and Rabinowitch, 1960). Kok (1959) showed that the long-wave (red light) system oxidized P700 (P700 + ; a special Chl a dimer; the reaction center, RC, of PSI), whereas the short-wave (orange light) system reduced P700 + and he related these results to Emerson's 2-light effect. Govindjee and Rabinowitch (1960a) found that a small peak for Chl a was present in the same system that phycobilins were. This solved the dilemma how phycobilins could run the primary photochemical reactions themselves since Duysens (1952) had found that, in cyanobacteria, phycobilins transferred energy very efficiently to Chl a. Cederstrand et al. (1966) discovered, using an integrating dodecahedron, at least two spectral absorption bands in the Chl a region: Chl a 670 and Chl a 680: the idea of several different Chl a spectral absorption bands was already known, from derivative absorption spectroscopy (see Brown and French, 1961).

FIgurE 1 | A schematic view of the changing concentration of atmospheric oxygen as a function of geological time in billions of years (ga).
Correlation between the estimated oxygen concentration changes and the major evolutionary events on Earth are based on data presented by Falkowski (2006;2011), Tomitani et al. (2006), Kump (2008), Blankenship (2002;2010), and Hohmann-Marriott and Blankenship (2011). The dates in the figure are not to scale and are approximations. Uncertainties of the selected events in the evolution of life are depicted with bars. A number of hypotheses exist as to when oxygenic photosynthesis was invented, by primitive cyanobacteria-like organisms. In this scheme, we have summarized currently available data and depict (by a black bar) that the first cyanobacteria could have evolved as early as 3.2 Ga or as late as 2.4 Ga ago. Therefore, an upper boundary of 3.2 Ga for the age of oxygenic photosynthesis and the initial rise of atmospheric oxygen in the atmosphere as indicated in this figure is still under discussion in the literature (see e.g., Tomitani et al., 2006;Allen and Martin, 2007;Blankenship et al., 2007;Buick, 2008). The figure is modified and adapted from .
For a historical perspective on the evolution of the Z-Scheme of electron transport in photosynthesis, see Govindjee and Björn (2011). In addition to the linear electron transport from water to NADP, there is also a cyclic electron transport around PSI. See Figure 4 for a current version of the Z-scheme of electron transport in cyanobacteria. Early measurements on ATP production in cyanobacteria, using the luciferin-luciferase assay, were made by Bedell and Govindjee (1973) in A. nidulans and by Lubberding and Schroten (1984) in Synechococcus sp.
The flexibility of cyanobacteria was demonstrated recently (see an abstract by Q. J. Wang, A. Singh, H. Li, L. Nedbal, L. A. Sherman, J. Whitmarsh and Govindjee, unpublished) when cells of a PSI-minus mutant of mixotrophically grown Synechocystis sp. PCC 6803 were shown to evolve oxygen (∼25% of that of wild type) for 30 min in the presence of glucose. Studies are under way to find the mechanism for this observation. Perhaps, it involves uphill electron transfer using ∆ pH made by PSII.

dIscovery of a new pIgment p750 In AnAcystis nidulAns
In addition to the Emerson enhancement effect, an unusual observation was that in several algae and in A. nidulans, far red light (720-760 nm range) caused inhibition (de-enhancement) of photosynthesis when added to short-wavelength light Govindjee et al., 1960;Rabinowitch et al., 1960). Soon thereafter, Owens and Hoch (1963) using 18 O-labeling and mass spectrometry showed that this de-enhancement was due to the effect of light on respiration (O 2 uptake).
In search of pigments, which absorb in this wavelength region, Govindjee et al. (1961) discovered a new pigment, in A. nidulans, that had an absorption band at 750 nm (also, see Govindjee, 1963a; FIgurE 2 | Structural organization of the antenna system of PSII for red algae and cyanobacteria (A) and energy transfer steps including charge separation (photochemical reaction) at the PSII rC (B) for cyanobacteria. The energy of absorbed photons is passed through a number of antenna molecules [phycoerythrin (absent in most cyanobacteria) → phycocyanin → allophycocyanin] until it reaches the RC Chl a (P680). The excited P680 donates its electron, which is in the excited state of the molecule, to an electron acceptor (A). The electron vacancy of the Chl a is filled by the electron from an electron donor (D). The wavelength numbers (nm) inside the circles represent pigments corresponding to the long wavelength absorption maxima of these pigments.

FIgurE 3 | Extrinsic proteins on the lumenal side of PSII in higher plants (A) and cyanobacteria (B).
PsbO (33 kDa), PsbQ (17 kDa) and PsbP (23 kDa) are the extrinsic proteins found in higher plants. In cyanobacteria, two of these proteins (PsbP and PsbQ) are replaced by PsbU (12 kDa) and PsbV (Cyt c550, 17 kDa). For a discussion of lipidated extrinsic proteins in cyanobacteria (CyanoP, CyanoQ, and Psb27), see recent review by Fagerlund and Eaton-Rye (2011). Both in higher plants and cyanobacteria, these proteins stabilize the Mn 4 CaO 5 cluster and optimize its water-splitting reactivity.
In the green alga Chlorella and in the diatom Navicula, Chl a 670 belonged to the same system as to where Chl b (or Chl c) belonged (Govindjee and Rabinowitch, 1960b). Amesz and Duysens (1962) provided experimental data on redox changes in the Cyt and NADP that supported the following picture of electron transfer path in cyanobacteria: Here, Q refers to a plastoquinone Q A , P700 to RC of PSI, and X to ferredoxin. however, Van Baalen (1965) found that it was involved in photooxidation of uric acid in A. nidulans;and Fischer and Metzner (1969) stated that P750 may be an open chain tetrapyrrole, but it not a bacteriopheophytin, and not a chlorin. Öquist (1974) found that iron-deficiency in A. nidulans increased fluorescence at 755 nm, perhaps, due to an increase in the concentration of P750. Goedheer and Hammans (1975) observed excitation energy transfer from several pigments to P750; and Hammans et al. (1977), however, established that P750 actually initiated oxygen uptake in A. nidulans. Further, Hammans (1978) showed that P750 sensitized photo-oxidation of several endogenous reductants in A. nidulans; however, these reductants did not serve as electron donors to PSI, nor were they oxidized in any Chl-sensitized reactions; there was, however, a hint that excitation of both P750 and Chl may cause the formation of singlet oxygen. Murata et al. (1981) showed that P750 was present in the cell envelope, not in the thylakoids, where there was also a small amount of pheophytin (Pheo)-like pigment with absorption maximum at 673 nm, and even some carotenoids. This observation explained why P750 had nothing to do with photosynthesis. Nultsch et al. (1983) found that P750 was absent in Anabaena and that it was not involved in phototaxis. Worcester et al. (1986) related an in vitro micellar system of aggregated Chls to P750; a correlation was suggested with the chlorosomes of the green bacteria. Later, Gombos et al. (1987) showed that P750, an aggregated form of Chl a, was formed during nitrogen starvation of cyanobacteria at ∼39°C, and that this process was reversible. Interestingly, however Shubin et al. (1991) found that, in Spirulina platensis, redox titration shows that F758 (fluorescence band at 758 nm) intensity has the same midpoint potential as P700; they concluded that the fluorescence quantum yield of F758 is proportional to the concentration of P700. Thus, in contrast, to other observations, they suggested a link of P750 to photosynthesis, at least in this organism. Further research is needed to understand fully the role of P750, i.e., where exactly it occurs in the cyanobacterial cells, what function or functions it performs, and possibly its importance in the evolutionary tree.

use of chlorophyll A fluorescence to understand photosynthetIc reactIons
Chl a fluorescence (FL) and its relation to photosynthesis has been reviewed by many (see chapters in Govindjee et al., 1986, andGovindjee, 2004). For historical perspectives on this subject, see Govindjee (1995) and Govindjee (2004). In order to fully appreciate this topic, we refer the readers to Wydrzynski and Satoh (2005) and Govindjee et al. (2010) to obtain a background on PSII since it is the system that has the most relationship with Chl a FL. For PSI, see Golbeck (2006).

excItatIon energy transfer In cyanobacterIa
A major use of FL has been in the use of detecting excitation energy transfer from one pigment (D for donor) to another (A for acceptor). If we excite D and all fluorescence appears from A, we know that there has been 100% energy transfer from D to A. However, if fluorescence appears from both D and A, we know that there has been a partial energy transfer (Duysens, 1952). This is the well-known "sensitized FL" method. Arnold and Oppenheimer (1950) were the first to show energy transfer in the cyanobacterium Chroococcus from phycocyanin to Chl a, and to provide a theory for excitation energy transfer. Duysens Figure 5). P750 had an emission band at 760 nm (Govindjee, 1963b) that was confirmed later by Goedheer and Hammans (1975). Unfortunately, no correlation of this band with photosynthesis was found (Gassner, 1962). This is an example of dead-end research as far as the laboratory of one of us (G) was concerned;  Govindjee et al., 1961). (B) Excitation spectra of fluorescence of P750 (measured at its red-most end, at 810 nm) of A. nidulans at 77 K (−196°C) and at 293 K (20°C). A portion of excitation spectrum of the green alga Chlorella is reproduced (open triangles) to show the absence of the excitation band at 750 nm. A curve for MgO is shown (open squares) to point out that the rising part of the curve beyond 760 nm is an artifact due to scattering (reproduced from Govindjee, 1963b).
FIgurE 4 | A simplified Z-scheme representing the linear (black solid arrows) and a cyclic (black dashed arrow) electron transport in cyanobacteria. Straight yellow arrows are for excitation of RC Chl a molecules that leads to electrons in the ground state to be raised into higher (singlet) excited state (P680 in PSII and P700 in PSI) in response to the absorption of light quanta (wavy arrows) via the antenna system (see Figure 2). Other abbreviations: E m (eV), redox potential at pH 7 (in electron volts); e − , electron; PQ, plastoquinone; Cyt b 6 f, cytochrome b 6 f complex; Cyt c 553 , cytochrome c 553 ; PC, plastocyanin (for details on Cyt c 553 and PC, see Zhang et al., 1992); the numbers 680 and 700 are the wavelengths of the absorption maxima, in the first excited state, for the Chl a RC molecules in PSII and PSI, respectively. Modified and adapted from Govindjee et al. (2010) and  there are comparatively fewer RC molecules that are engaged in photosynthesis as compared to the number of antenna molecules that absorb the incoming light. Measurement of the action (or excitation) spectra of Chl FL is the method of choice to witness this phenomenon. By 1967, the following was known: (1) There is a "red drop" in FL yield, as was obtained through absorption spectra, and the application of the Stepanov relationship to A. nidulans (Szalay et al., 1967); data showed here that the red drop in FL began already around 640 nm in contrast to green algae where the red drop began at 685 nm. It confirmed the concept that in cyanobacteria most of Chl a is weakly fluorescent; (2) Spectral (FL) characteristics of various membrane fragments from Anacystis cells showed that Chl a of PSII was in a different fraction than Chl a of PSI (Shimony et al., 1967).
With this background, Govindjee's group was ready to make measurements on excitation energy transfer at different temperatures. Cho and Govindjee (1970) measured both the excitation and emission spectra as a function of temperature in A. nidulans down (1952) showed efficient energy transfer from phycobilins to Chl a in the cyanobacterium Oscillatoria. A powerful method to show energy migration among the same pigment is depolarization of FL, as was first used by Arnold and Meek (1956) for green algae.  showed that addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron; or DCMU), that blocks electron flow, led to increased depolarization of FL, thereby indicating increased energy migration in the antenna system. For a review on energy transfer in photosynthesis, see Goedheer (1972), Mimuro (2004), and Clegg et al. (2010). The general path of excitation energy transfer in most cyanobacteria is: A pathway of excitation energy transfer in cyanobacteria is shown in Figure 2B. Excitation energy migration and transfer, among the antenna molecules, is necessary for efficient photosynthesis since FIgurE 6 | Emission and excitation fluorescence spectra of Anacystis nidulans at low temperatures. (A) Emission spectra of fluorescence in the 4 K (liquid helium temperature) to 77 K (liquid nitrogen temperature) range in cells of A. nidulans; wavelength of excitation was at 620 nm (absorption by phycocyanin; see Figure 2B). For comparison, a room temperature (295 K) spectrum (black dashed curve) is shown. (B) Emission spectra of fluorescence in the 4-77 K range in cells of A. nidulans; wavelength of excitation was at 440 nm (absorption by Chl a; note that cyanobacteria have most of their Chl a in PSI). For comparison, a room temperature (295 K) spectrum is also shown. (C) Excitation (or action) spectra of fluorescence, as a function of temperature (4-77 K) in cells of A. nidulans, measured at 685 nm (mostly PSII fluorescence). (D) Excitation (or action) spectra of fluorescence as a function of temperature (4-79 K) in cells of A. nidulans, measured at 715 nm (mostly PSI fluorescence). All panels of the figure were modified and reproduced from Cho and Govindjee (1970). a rise to another maximum M and then a decline to a terminal steady state T within about a minute (see e.g., Govindjee and Papageorgiou, 1971;Papageorgiou, 1975;Papageorgiou et al., 2007). When this FL transient is plotted on a logarithmic time scale, two more inflections, called J and I, become obvious at about 2 and 30 ms, respectively (see Strasser et al., 1995;Strasser et al., 2004;Stirbet and Govindjee, 2011). The slow FL transient (minutes) in A. nidulans was first studied by Govindjee (1967, 1968), who showed that when phycobilins, but not Chl a, were excited, fluorescence rose from the S level to the M level, within a minute, in both normal and DCMUtreated A. nidulans cells (Figure 7). However, this change was severalfold higher in DCMU-treated cells; no change was observed at 77 K. Interestingly, carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP), which abolishes the pH difference across the thylakoids membrane, prevented the fluorescence increase only in normal, but not in DCMU-treated cells. Phlorizin, a phosphorylase inhibitor, had very little effect. These results suggested that these light-induced changes in Chl a FL yield may be related to the conformational changes which accompany photophosphorylation. Mohanty and Govindjee (1973a,b,c) were the first to study the fast, although of much reduced amplitude, FL transient in A. nidulans. They presented detailed studies with many uncouplers of phosphorylation, including salicylanilides, extending the concept that the slow FL changes were not related to "Q A " changes (reviewed in Mohanty and Govindjee, 1974). Recently, using state transitionblocked mutants, Kana et al. (2010) have shown that a large part of the slow S to M rise, in Synechocystis sp. PCC 6803, is due to state 2 to state 1 transition since it is absent in RpaC (minus) mutant that is locked in state 1 (Emlyn-Jones et al., 1999).
to 4 K (liquid helium temperature; Figure 6). They made several observations: (i) large proportions of Chl a 670, Chl a 678 and even Chl a 705 are present in PSI, and that PSII contains mainly phycocyanin, allophycocyanin and only some Chl a 670 and Chl a 678; (ii) phycocyanin (with absorption bands at 580, 625, and 634-637 nm) and allophycocyanin (at 650 nm) are responsible for the broad fluorescence band at 653-655 nm; Chl a 670 is responsible for fluorescence band at 680 nm (F680), and Chl a 678 is responsible for fluorescence band at 685 nm (F685); (iii) the ratio of FL band at 715 nm (F715) to those at 685 and 695 nm (F685 and F695) is greater in PSI than in PSII; (iv) the efficiency of excitation energy transfer from Chl a 670 to Chl a 678, that approaches 100%, even at 4 K, is independent of temperature suggesting strong coupling between the Chl a-protein complexes; perhaps, here an "exciton" migration occurs as opposed to "hopping" of excitation energy as suggested for energy transfer from phycocyanin and allophycocyanin to Chl a; and (v) transfer efficiency from phycocyanin to Chl a is temperature dependent; it is lower at 4 K than at 77 K suggesting that here Förster resonance energy transfer may be taking place.
The above studies were followed by measurements on the lifetime of FL both at room temperature and at 77 K . In contrast to green alga Chlorella, Anacystis was calculated to have two times the rate constant of heat loss and had a lower efficiency of energy trapping than in algae. Further, the quantum yield of Chl a FL in Anacystis was about half of that in Chlorella. These observations are in agreement with what is known about photosynthesis measurements in these organisms. Further, both photosynthesis and Chl a FL are not static, but affected by dynamic changes in the environment. Nedbal et al. (2003) discovered a negative feedback regulation of the energetic coupling between phycobilisomes and PSII in the cyanobacterium Synechocystis sp. PCC 6803; they found that dynamic changes in the coupling of the phycobilisomes to PSII are not accompanied by corresponding antiparallel changes in the PSI excitation, suggesting a regulation limited to PSII. On the other hand, Kana et al. (2009) have shown changes in emission spectra, in Synechococcus sp., during the FL transient (for earlier observations in A. nidulans, see Govindjee, 1967, 1968). Further, heating of A. nidulans at 60°C for 10 min leads to several changes (Singhal et al., 1981): loss of absorption by phycobilins, and changes in excitation energy transfer from the leftover phycobilins to Chl a of PSI and or conversion of Chl a of PSI to Chl a of PSII. Maksimov et al. (2011) have recently shown that the effective fluorescence cross section decreases from a value of ∼ 900 Å 2 in the wild type Synechococcus sp. PCC 6803 to ∼200 Å 2 (in phycocyaninless mutant) or to ∼80 Å 2 (in phycobilisome-less mutant). These mutants are expected to provide further information on excitation energy mechanisms in cyanobacteria.

chlorophyll A fluorescence transIents: fast (seconds) and slow (mInutes) and theIr relatIon to photosynthetIc reactIons
We know that when green algae and higher plant leaves are illuminated with strong (high-intensity) light, Chl a FL rises from a minimum original low level (O, the origin) to a peak P within 1 s, and then declines to a semi-steady state S within about 10 s; this is followed by  Govindjee and Papageorgiou, 1971). This fluorescence transient (or fluorescence induction) is quite different from that observed in green algae and higher plants, where the "OPS" transient dominates. Cyanobacteria in darkness are usually in what is called "state 2" (low fluorescence state) and it takes a long time (almost 100 s) to be transferred to "state 1" (high fluorescence state). For a recent historical review on "state changes, " see Papageorgiou and Govindjee (2011).
• Based on several measurements including TL, Meetam et al. (1999) concluded that the PsbY protein in Synechocystis sp. PCC 6803 is not essential for oxygenic photosynthesis and does not provide an important binding site for manganese in the OEC of PSII. • In a mutant of S. platensis, where electron transport from plastoquinol (PQH 2 ) to Cyt b 6 f was slowed, Ruengjitchatchawalya et al. (2005) suggested, from TL measurements, the presence of a reversed electron flow via PQ -a decrease in reoxidation of PQH 2 seems to explain TL and other data. We note that a TL band at 45°C was present in this mutant, and, that this band was enhanced by trans-thylakoid pH and eliminated by an uncoupler FCCP. Further research is necessary to understand how cyanobacteria -like changes were converted to plant-like changes (see the section above). • Cser and Vass (2007)

afterglow (thermolumInescence): back reactIons of photosystem II
When plants, algae, and cyanobacteria are illuminated and then quickly frozen to very low temperatures (e.g., 77 K) in darkness, and then warmed in dark, they emit light, and, this light emission is the afterglow, called thermoluminescence (TL). Arnold and Sherwood (1957) were the first to observe this phenomenon in dried chloroplasts. TL has been used extensively to monitor the water oxidation cycle (referred to as the S-state or the Kok cycle) as well as the redox status of the primary and secondary electron acceptors (Q A and Q B , respectively) of PSII since TL involves recombination reactions of the electron donor and acceptor sides of PSII. The so-called "B" bands of TL are due to recombination of S 2 /S 3 states with reduced Q B , whereas the "Q" band is due to recombination of the S 2 state with reduced Q A (Sane and Rutherford, 1986;Demeter and Govindjee, 1989;Vass and Govindjee, 1996;Vass, 2005). Excited Chl a (Chl*), which is needed to give light, is produced when electrons on reduced Q A and Q B move up by thermal energy, and positive charges on the S-states provide the "holes" to the ground state of Chl a, with which electrons can recombine. In their theory for TL, DeVault et al. (1983) and DeVault and  included temperature-dependent equilibrium between the various steps in the electron transport pathways and provided a generalized theory to explain the major TL bands in the photosynthetic systems. For TL bands, due to reduced Q B , Rose et al. (2008) did show that the theory of DeVault and  is essential for explaining all the TL bands, although, for TL band due to reduced Q A , simpler models may be sufficient (Rappaport and Lavergne, 2009). Many investigators have observed TL from cyanobacteria; these have provided important information about their photosynthesis characteristics. Some examples are: • Govindjee et al. (1985) observed that, in a thermophilic cyanobacterium Synechococcus vulcanus, TL bands were at higher temperatures (Figure 8): The B band was shifted from 25-30°C (in mesophilic organisms) to 50-55°C; and the Q (or D) band from 0-10°C to 35°C; further, the deactivation of the S 2 /S 3 states was observed to slow down from 20-30 to 100-200 s range in this thermophilic cyanobacterium.   1, 2, 3, and 4, respectively). The dashed curve 5 is the spectrum obtained in the presence of DCMU that blocks electron transport from Q A − to Q B . Insert shows flash number dependence of the 53°C TL band, the B band, due to S 2 /S 3 and Q B − recombination (closed circles), and that of oxygen evolution (closed triangles). The "Q" band, due to S 2 and Q A − recombination (curve 5) was obtained at 35°C in this organism. The figure was modified and reproduced from Govindjee et al. (1985).
Van Klimov (2005), andMcConnell et al. (2011). For the absence of bicarbonate effect in the photosystems of anoxygenic photosynthetic bacteria, see Shopes et al. (1989), and Wang et al. (1992). HC has been suggested to function both on the electron acceptor as well as on the electron donor side of PSII; there is no effect on PSI, however (Khanna et al., 1977;Eaton-Rye and Govindjee, 1984). Data on higher plant thylakoids suggest that the diffusing species is CO 2 and the binding species is HCO CO 3 3 2 − − / (Sarojini and Govindjee, 1981;Blubaugh and Govindjee, 1986). Using thylakoids, the HC effect has been located on the electron-acceptor side, close to the herbicide-binding side, particularly on the electron transport and protonation reactions at the secondary quinone Q B and near the non-heme iron (Govindjee et al., 1976(Govindjee et al., , 1991aJursinic et al., 1976;Khanna et al., 1980Khanna et al., , 1981Van Rensen and Vermaas, 1981; Eaton-Rye and Govindjee, 1988a,b). Several studies have established that the HC effect can be observed in intact cells and in leaves (Garab et al., 1988;. To study the HC effect, very often formate (HCO 2 − ) is used to obtain HCO CO 3 2 − / -free system to which HCO 3 − anions are added to measure the HC effect ( Figure 9A). Nugent et al. (1988) showed clear effects of removal of HC (by formate addition) directly on the Q A -Fe-Q B signals in the PSII of the cyanobacterium Phormidium laminosum (Figure 9B). Both the ESR bands related to Q A and Q B (in the g = 1.8 and g = 1.6 regions) as well as to non-heme region (g = 6 region; shown on spinach samples; Figure 9C) were clearly affected upon the addition of formate (100 mM). The release of CO 2 by formate addition was shown first by Govindjee et al. (1991b; using mass spectrometry and infrared spectroscopy; this has been recently confirmed by Shevela et al. (2008a,b).
From the early work of Blubaugh and Govindjee (1988), we have known that there are at least two binding sites of HC. Thus, besides the site at non-heme iron between Q A and Q B , there has to • From TL and other measurements, Cser et al. (2008) showed that in Acaryochloris marina, which contains predominantly Chl d, instead of Chl a, the charge recombination energetics is modified: the E m of Q A and Q B is increased, and the redox gap between RCIIChl* and RCIIChl + Pheo − is decreased. Further, the free energy span between RCIIChl and RCIIChl* is decreased indicating the involvement of Chl d in primary electron donor activity. Interestingly, in spite of these differences in the energetics on the electron-acceptor side of PSII, the redox potentials and kinetics within the OEC of the Chl d-containing cyanobacterium A. marina closely resembles that of Chl a-containing cyanobacteria and higher plants (see Shevela et al., 2006 and references therein). Therefore, it appears, that the replacement of a large number of Chl a by Chl d in PSII of oxygen-evolving organisms has virtually no effect on the properties of the OEC. Although the redox potentials of Q A and Pheo were found to be different in Chl d-containing A. marina and in Chl a-containing Synechocystis sp. PCC 6803, Allakhverdiev et al. (2011) found that the energetics in PSII is conserved, i.e., the difference in the potentials between Q A and Pheo is the same.

FIgurE 9 | Fluorescence and EPr data representing the effects of HC removal on the electron-acceptor side of PSII. (A) Fluorescence measurements reflecting the decay of Q A − , upon illumination of thylakoids of
Synechocystis 6803 after the third flash (repetitive rate of 1 Hz) at pH 7.5 (modified and reproduced from Cao and Govindjee, 1988). The fluorescence decay was measured in control (i.e., HC-containing) samples (closed squares), HC-depleted samples (open triangles), and HC-depleted samples after the re-addition of 2.5 mM HCO 3 − . (B) EPR spectra of the ironsemiquinone in the dark-adapted PSII samples from thermophilic cyanobacterium P. laminosum upon 5 min illumination at 77 K in the absence (spectrum 1) and the presence of 100 mM formate (spectrum 2). The used concentration of formate (100 mM) has been shown to be sufficient for the removal all HC bound to PSII (Govindjee et al., 1991b;Shevela et al., 2008b). (C) EPR spectra of the g = 6 non-heme iron region of the dark-adapted PSII samples from spinach at pH 7.5. The spectra were obtained in the absence (spectrum 1) and the presence of 100 mM formate (spectrum 2). For EPR settings, see Nugent et al. (1988). Abbreviations: g, is the electronic splitting factor (in case of free electron g-factor, it has a value of 2.0023); mT (millitesla), values for the magnetic field. (B,C) were modified and reproduced from Nugent et al. (1988). in the following order: S264A-F255L > S264A = N266T > N266T-S264A > F255L = N266D = WT > > N266T-A251V = A251V. On the basis of the absence of additivity of the effects between single and double mutants, Vernotte et al. (1995) concluded that the couples S264 and N266, N266 and A251, and S264 and F255 interact with each other in formate binding. These results are best explained to be due to the modification, to some extent, of the "overall conformation of the D1 protein which, in turn, modified the HCO /HCO − and their recombination (Maenpaa et al., 1995;Mulo et al., 1997). Maenpaa et al. (1995) found that formate which inhibits the Q A to Q B reaction was several-fold less effective in the CA1 mutant (where three glutamates at 242, 243, and 244 positions were deleted and glutamine 241 was changed to His); on the other hand D1-E229D and D1-E243K mutants were like the wild type Synechocystis sp. PCC 6803. Mulo et al. (1997) demonstrated, also in Synechocystis sp. PCC 6803, that deletion of either the PEST like sequence (∆R225-F239) or the putative cleavage region (∆G240-V249, ∆R225-V249) of the D1 protein resulted in severe perturbations on the function of the Q B electron acceptor of PSII. Xiong et al. (1997), considering all the results available till then, presented a model of PSII, based on homology with the bacterial system, and introduced the hypothesis of how HCO 3 − may function in PSII. Obviously, only the basic points are known: HC was suggested in the literature to bind to the non-heme iron that sits between Q A and Q B . To accommodate other experiments, particularly those on Chlamydomonas mutants (Hutchison et al., 1996;Xiong et al., 1997), specific arginines were implicated as well as conformational changes. What is known from the work of Eaton-Rye and Govindjee (1988a,b) on spinach thylakoids is that HC functions not only in the reduction of Q B , but also in its protonation. Recent studies have suggested that D2-K264 (Cox et al., 2009), but more likely D1-Y246 and D2-Y244 (Takahashi et al., 2009) are directly involved in HC binding. Figure 10 shows the binding of HCO 3 − to the electron-acceptor side of PSII from the 1.9 Å resolution structure of PSII . Here the importance of D1-Y246 and D2-Y244 in the HC effect is clearly obvious. Komenda et al. (2002) discovered that a mutant of Synechocystis sp. PCC 6803, that lacked PsbH protein, had a much weakened binding of HC on the PSII acceptor side; this observation needs to be understood in light of the recent PSII crystal structure .
Answer to the HC problem lies in future studies on the mutants of these and some other amino acids. So, we would say that a new hope has arisen and we can hardly wait to get the final answers!

remarks on evolutIon
We end this review with remarks on evolution (for recent reviews, see Buick, 2008;Blankenship, 2010;Hohmann-Marriott and Blankenship, 2011). Figure 11 shows a simplified model of the origin and evolution of life (tree of life) based on small-subunit RNA analysis. Despite the simple representation of the origin of the first oxygenic photosynthesizers in this Figure, the earliest steps in the origin of photosynthesis are not known, and are the subject of vivid discussions in the literature (Blankenship et al., 2007;Buick, 2008 and references therein). be another HCO 3 − -binding site, which, however, was not seen by the recent crystallographic studies of PSII (Guskov et al., 2010;Umena et al., 2011). Most probably, at the second binding site, HCO 3 − may be loosely bound to PSII, and may have escaped detection due possibly to its loss caused by X-ray irradiation as well as by the treatments required during the X-ray measurement.
In view of a large body of experimental evidence for HC effect on the PSII acceptor side, and the latest PSII crystal structure at the resolution of 1.9 Å that shows the clear presence of HC on the non-heme iron between Q A and Q B in a thermophilic cyanobacterium Thermosynechococcus vulcanus , there is a renewed interest in understanding the mechanism of HC effect on the PSII acceptor side. There are clear evidences that HC is not a tightly bound constituent of the OEC of PSII (Aoyama et al., 2008;Shevela et al., 2008b;Ulas et al., 2008;Umena et al., 2011); however, there are numerous indications for the action of HC on the watersplitting side of PSII. For the HC effects on the donor side of PSII, see reviews by Stemler (2002) and Van Rensen and Klimov (2005). Govindjee (1988, 1990) were the first to show the effect of HC on PSII in transformable Synechocystis sp. PCC 6803.  confirmed this effect in other cyanobacterial cells. Through Chl a FL measurements, the effect was located on the electron-acceptor side of PSII. In view of the fact that in chloroplasts, HC effect was suggested to be near the herbicide-binding site,  examined herbicide-resistant D1 mutants of Synechocystis 6714. Chl a FL and O 2 evolution measurements revealed a differential sensitivity to HCO 3 − -reversible formate inhibition; resistance to formate treatment followed the order (highest to lowest): [double mutant] A251V/F211S (AZ V) > [single mutant] F211S (AZ 1) > wild type > [single mutant] S264A (DCMU II-A). These results are in agreement with the concept that there may be overlapping herbicide and bicarbonate niches in cyanobacteria as well.
Soon thereafter, the concept arose that even D2 protein may be involved with the HC effect, particularly its specific arginines (Cao et al., 1991). Measurements of oxygen evolution showed that the D2 mutants R233Q (arginine-233 ∼ glutamine) and R251S (arginine-251 ∼ serine) were 10-fold more sensitive to formate than the wild type of Synechocystis sp. PCC 6803, suggesting their possible role in HC binding. However, further, studies on D1-S264 and D1-F255 mutants of Synechococcus sp. PCC 6942 led to the conclusion that the equilibrium dissociation constant for HC is increased in the mutants, while that of the formate remains unchanged. The hierarchy of the equilibrium dissociation constant for HC (highest to lowest, ±2 mM) was: D1-F255L/S264A (46 mM) > D1-F255Y/ S264A (31 mM) -D1-S264A (34 mM) -D1-F255Y (33 mM) > wild type (25 mM). These data suggested the importance of D1-S264 and D1-F255 in the HCO 3 − -binding niche. A possible involvement of HC and these two residues in the protonation of Q B − , the reduced secondary plastoquinone of PSII, in the D1 protein was discussed .
A picture began to emerge that we may not be dealing with specific binding site of HC that is involved in the reactions, but that conformation changes may be responsible for some of our observations in cyanobacteria. This became rather obvious when Vernotte et al. (1995) used several single and double mutants in the d-e interhelical loop of D1 protein in Synechocystis sp. PCC 6714. They showed a differential sensitivity of formate inhibition on the acceptor side of PSII How did the first cyanobacteria contrive to do it? The following evolutionary developments are thought to have preceded the successful water-splitting by the first O 2 -producing organisms: • Origin of RCs and the development of the two types (PSII and PSI) of functionally linked RCs ending up in the same organism was undoubtedly, the key evolutionary step toward the ability by the first cyanobacteria to extract electrons from water. While cyanobacteria (as well as green algae and higher plants) contain two types of RCs (PSII and PSI; Figure 4), all anoxygenic bacteria have only one of the two types: RC1 (pheophytin-quinone type) or RC2 (iron-sulfur type; Figure 12). Therefore, it is generally accepted that the origin of the oxygenic photosynthetic organisms begins with the origin of the linked RCs in a common ancestor of O 2 -evolving cyanobacteria (for details, see, Xiong and Bauer, 2002;Olson and Blankenship, 2004). Currently two models for the origin the fIrst cyanobacterIa and the advent of water oxIdatIon The first appearance of the atmosphere and the oceans on our Earth is dated to about 4.5 Ga ago. It happened soon after the Earth completed its formation stage (Nisbet and Sleep, 2001). Based on the carbon isotope data, the earliest forms of life are thought to have emerged about 3.7-3.8 Ga ago (Mojzsis et al., 1996;Falkowski, 2006). Although photosynthesis was, probably, not the earliest metabolic system on Earth, it, however, arose very early in the history of our planet. It is generally accepted that simple anoxygenic organisms carried out the first photosynthetic activity. These first photobacteria were dependent on the availability of electron donors (reductants) such as H 2 , H 2 S, and/or Fe(OH) + which they used as substrates (Olson, 2006;Blankenship, 2010;Hohmann-Marriott and Blankenship, 2011). However, these substances were much less abundant and thus limited compared to the huge (almost unlimited) H 2 O pool on the surface of the planet. During the period dated between 3.2 and 2.4 Ga ago, some cyanobacteria-like organisms managed to utilize H 2 O as a substrate (i.e., as a source of electrons and protons) for the reduction of CO 2 using the energy of sunlight to drive this reaction (Oparin, 1965;Xiong and Bauer, 2002;Tomitani et al., 2006;Allen and Martin, 2007;Blankenship et al., 2007; see also Figure 1 and its legend).
Water is a very stable compound. If one considers oxidation of water to O 2 and protons, the E m for such a process would be +0.82 V at pH 7 (Atkins and De Paula, 2006;Messinger and Renger, 2008).  Van Rensen et al., 1999). The model shown here provides detailed information that will allow future experimenters to find the molecular mechanism of the HC effect on the PSII.

FIgurE 11 | The origin and evolutionary tree of life that is based on
small-subunit rNA. The branches that perform oxygenic photosynthesis are labeled with 'O 2 '. The black arrow indicates the plastid endosymbiotic event that resulted in the origin of eukaryotic photosynthesis from cyanobacteria-like organisms, which ultimately became chloroplasts in algae and later in plants. However, while chloroplasts of the higher plants, glaucophytes, green and red algae are thought to be the result of the plastid (primary) endosymbiosis, all other groups of algae are assumed to have arisen due to the algal (secondary and tertiary) endosymbiosis (not shown), in which one eukaryotic alga was incorporated into another eukaryote. For details, see Olson and Blankenship (2004), Blankenship et al. (2007), Hohmann-Marriott and Blankenship (2011), Kim et al. (2011), Garcia-Mendoza et al. (2011. Only some branches of bacteria, eukarya, and archaea are displayed. Modified and adapted from Blankenship (2002) and Blankenship et al. (2007).
• The evolutionary development of photosynthetic pigments (Chls) and of a strongly oxidizing RC (PSII) most probably preceded or evolved simultaneously with the invention of the OEC (Blankenship and Hartman, 1998;Blankenship, 2010). A strong oxidant with redox potential greater than +0.82 V was needed to split water into O 2 and four protons. We do not know the midpoint redox potential of the RC photoactive pigment in ancient cyanobacteria. In modern water-splitting organisms, the E m value of the RC photoactive Chl a (P680 •+ ) is about +1.25 V (Rappaport et al., 2002). Such a high value must be due to a special protein environment (Renger and Holzwarth, 2005). This oxidizing power is about half a volt above the special bacteriochlorophyll (BChl) pair (BChl-L-BChl-M) in the RCs of all anoxygenic bacteria that contain various BChl forms: a, b, or g (Blankenship et al., 1995;Figure 12). BChl a has been suggested to have evolved before Chl a (see e.g., Burke et al., 1993;Raymond et al., 2003;Olson and Blankenship, 2004). In the common ancestor of cyanobacteria, Fe(OH) + and later H 2 O 2 may have been the electron donors for photosynthetic CO 2 fixation until BChl a was replaced by Chl a in the RCs and the OEC was invented (Rutherford and Nitschke, 1996;Blankenship and Hartman, 1998;Borda et al., 2001;Olson, 2006;Raymond and Blankenship, 2008). However, Ohashi et al. (2010) have proposed that the oxygensensitive BChl g could be the precursor for Chl a. For many years, Chl a had been thought to be unique and be the essential pigment among the Chl species in photosynthetic O 2 -evolving organisms. However, with the discovery by Miyashita et al. (1996) of the Chl d-containing cyanobacterium A. marina, the role of Chl a versus Chl d began to be discussed (Akimoto et al., 2006;Kobayashi et al., 2007;Tomo et al., 2007;Cser et al., 2008; of RCs are discussed (for reviews, see Vermaas, 2002;Olson and Blankenship, 2004). According to the selective loss model, the two types of RCs (RC2 and RC1) developed separately in the same organism and later become functionally linked. In this model, the anoxygenic photosynthetic organisms are derived from a primitive cyanobacterium by a loss of either RC1 or RC2. In the fusion model, the RC2 and RC1 developed separately in distinct organisms and later ended up together in one organism by a large-scale lateral gene transfer. For details on the models, see Blankenship (1992), Xiong and Bauer (2002), Blankenship et al. (2007), Hohmann-Marriott and Blankenship (2011). However, whatever model is true, in order to oxidize water to molecular oxygen, two more major changes from the more primitive non-O 2 -evolving RCs were also required: addition of a charge-accumulating system (i.e., OEC) and that of a RC pigment with a very high oxidizing potential. • The development of the catalytic site of water oxidation (MnCaprotein complex/OEC) that is capable of collecting and storing positive charges was, undoubtedly, a central stage in the transition from anoxygenic bacteria to oxygenic cyanobacteria (Allen and Martin, 2007;Raymond and Blankenship, 2008). The evolutionary origin of the OEC is obscure. Currently, it is thought to have been derived either from Mn catalase enzymes (Blankenship and Hartman, 1998), Mn-HC complexes (Dismukes et al., 2001;Khorobrykh et al., 2008), or Mn(Ca)-containing minerals (Sauer and Yachandra, 2002;Najafpour et al., 2010). Dash et al. (2011) speculated that Cyt c in anoxygenic photosynthetic bacteria might have been replaced by the addition of a Mn-Ca entity by assimilating Mn-Ca minerals like hollandite.  (2002) and Blankenship (2010).
need to learn how to transform the unlimited supply of sunlight into a storable and transportable form of energy, such as biomass, or hydrogen that is ecologically a "clean" fuel. This would help us in solving the rising energy demand, taking into consideration the necessity for the supply of "clean" energy. We ask: Can cyanobacteria help us in this regard? And, what can we learn from them? The answer to the first question is "Yes," but the details of what can we learn from them is being debated around the World. It is a common knowledge that a large number of cyanobacteria (as well as green algae and plants) use light as the driving force to extract electrons from water in order to generate strong reductants, such as NADPH and reduced ferredoxin, which can, in principle, be utilized as substrates for molecular hydrogen production by either hydrogenases and nitrogenases (for recent reviews, see Lubitz et al., 2008;Ghirardi et al., 2009). Figure 13 shows a hydrogenase-dependent pathway for molecular hydrogen production in cyanobacteria. However, several biochemical barriers exist for the sustained and sufficient H 2 production in cyanobacteria. The most important are: (i) the irreversible inhibition of most hydrogenases by O 2 ; (ii) the consumption of photobiologically produced H 2 by the bidirectional hydrogenases; (iii) the competition of the Calvin-Benson cycle and H 2 production for NADPH (Tamagnini et al., 2007). Because of these limitations, photobiological H 2 production occurs only under special conditions and with low yields in the wild type cyanobacteria. Mutagenesis, metabolic engineering, as well as other approaches are being employed to overcome these restrictions (Esper et al., 2006;Ghirardi et al., 2007Ghirardi et al., , 2009Lubitz et al., 2008), but this unique and complex system, which evolved ∼ 3 Ga ago, has not been successfully used by humanity thus far.
In this respect, Francis Crick was absolutely right, saying that "Evolution is cleverer than you are" (Dennett, 1984). Undoubtedly, in the future, the efforts of the numerous cyanobacteria research groups hold a promise for us all. The adventure with cyanobacteria continues… Allakhverdiev et al., 2011). Blankenship and Hartman (1998) proposed that Chl d may have been a transitional evolutionary pigment between BChl a-containing RCs of anoxygenic bacteria and Chl a-containing RCs of the first oxygenic cyanobacteria (Blankenship et al., 2007). However, alternatively, it could also well be a more recent adaptation to a particular light environment : the Acaryochloris-like organisms live in environments rich in near-IR light, but show features of adaptation to strong light Miller et al., 2005). The latter option is supported by the data of Schliep et al. (2010) indicating that Chl a is the ancestor of Chl d.
• Finally, the coupling of HCO 3 − ions to PSII in the first oxygenic photosynthesizers to facilitate quinone reduction could be speculated to be an additional and the latest evolutionary step from anoxygenic/low-efficient-oxygenic to oxygenic/highly efficient oxygenic photosynthesis. The presence of HC bound between Q A and Q B is unique as it exists only in O 2 -evolving organisms and is absent in anoxygenic photosynthesizers (Shopes et al., 1989;Wang et al., 1992).

consequences of the orIgIn of cyanobacterIa
Already ∼2.3 Ga ago, photosynthetic H 2 O-splitting cyanobacteria started to be the dominant photosynthetic organisms. Geological and geochemical evidence clearly indicates that around this time, free molecular oxygen started to accumulate in significant amounts in the atmosphere (Figure 1; Bekker et al., 2004). Mass spectrometric evidence for a high driving force of O 2 evolution by PSII in cyanobacteria and higher plants has been provided by , confirming earlier indications (Haumann et al., 2008;Kolling et al., 2009). All together, the data obtained clearly indicate that elevated O 2 levels (up to 20-50 bars) do not suppress photosynthetic oxygen production. Therefore, it is obvious, that the evolution of oxygenic photosynthesis was not influenced and restricted by the rise of the O 2 content in the atmosphere in the past. The rapid development of prokaryotic oxygenic photosynthesis by the first cyanobacteria dramatically changed the Earth by creating an aerobic atmosphere. In turn, this event forced the anaerobic forms of life either to find O 2 -free niches or to adapt to the high O 2 levels by developing protective mechanisms. Those organisms that did not adapt to the presence of O 2 faced extinction (Sleep and Bird, 2008). Subsequent evolution of eukaryotic oxygenic photosynthesis due to plastid (primary) endosymbiosis (see Figures 1 and 11), and later due to algal (secondary) endosymbiosis (see Chan and Bhattacharya, 2010;Chan et al., 2011;Hohmann-Marriott and Blankenship, 2011) led to the rise of [O 2 ] to the present day level (∼ 20%). Moreover, the appearance of oxygen levels in high amounts in the atmosphere also led to the formation of the protective ozone layer that absorbs a large part of the UV radiation from the Sun. These new conditions ultimately permitted the development of an aerobic metabolism and more-advanced forms of live. All fossil fuels that we use at this moment are derived from past photosynthesis.
perspectIves for the future Today, the natural process of oxygenic photosynthesis may serve as a blueprint for developing artificial photosynthesis for human needs Najafpour and Govindjee, 2011). We FIgurE 13 | A schematic representation of H 2 photoproduction pathway catalyzed by a hydrogenase in cyanobacteria. As in green algae, photosynthetic electron transport delivers electrons after light-induced water-splitting at the OEC to the hydrogenases (H 2 -ase), leading to photophosphorylation and H 2 production. In addition to hydrogenases, nitrogenases are also known to produce H 2 in oxygenic cyanobacteria and in anoxygenic purple bacteria. Abbreviations: PQ, plastoquinone; Fd, ferredoxin; FNR, ferredoxin-NADP reductase; ATPase, adenosine triphosphate. The figure is modified and adapted from  time-line on photosynthesis (Govindjee and Krogmann, 2004), to a web article "Photosynthesis Online" (Orr and Govindjee, 2011): <http://www.life.illinois.edu/govindjee/photoweb/>; and to a conversation between Donald R. Ort and one of us (G): <http:// ensemble.atlas.uiuc.edu/app/sites/JZ64_U8FmEmx7Liwjl36Mw. aspx?webSiteID=JZ64> acknowledgments We thank all the co-authors of our past research, as without their participation this perspective of cyanobacterial photosynthesis would not have been possible. Jian-Ren Shen is gratefully acknowledged for providing the figure of his recent PSII crystal structure. We also take this opportunity to refer the readers to a