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

Plant Physiology and Biochemistry xxx (2014) 1e7

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Research article

On the question of the light-harvesting role of b-carotene in photosystem II and photosystem I core complexes Kostas Stamatakis a, *, Merope Tsimilli-Michael b, George C. Papageorgiou a a b

Institute of Biosciences and Applications, National Center for Scentific Research Demokritos, Aghia Paraskevi, Attikis 15310, Greece Athanasiou Phylactou str. 3, Nicosia CY-1000, Cyprus

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 October 2013 Accepted 22 January 2014 Available online xxx

b-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 b-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 b-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 b-carotene absorption. In the present work we investigated the light harvesting role of b-carotenes in the cyanobacterium Synechococcus sp. PCC 7942 using selective b-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 b-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 b-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 b-Carabsorbed light can be attributed to the different excitation trapping efficiencies in the reaction centers of PSI and PSII. Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Chlorophyll fluorescence Cyanobacteria b-Carotene 77 K fluorescence spectra State transitions

1. Introduction In 1942, Emerson and Lewis (1942), reported that the quantum yield of photosynthesis in the cyanobacterium Chroococcus was about 70% lower for light absorbed by carotenes (450e520 nm) than for light absorbed by C-phycocyanin (CPC) and chlorophyll (Chl) a. Similar, but shallower, blue troughs in quantum yield spectra of photosynthesis were reported for the green alga Chlorella pyrenoidosa (Emerson and Lewis, 1943) and the red alga Porphyridium cruentum (Brody and Emerson, 1959), which contain accessory pigments (Chl b and phycoerythrin, respectively) that absorb in the same spectral region as carotenes. Early experiments of Goedheer (1961) (performed, however, after the discovery of the two light reactions (Emerson and Rabinowitch, 1960) and the formulation of

Abbreviations: APC, allophycocyanin; Chl, chlorophyll; CPC, C-phycocyanin; DPC, 1,5-diphenylcarbazide; DCMU, 3-(3,4-dichlorophenyl)-1,10 -dimethylurea; EE, excitation energy; PQ, plastoquinone; PS, photosystem; PSIRC, PSI reaction center complex; PSIIRC, PSII reaction center complex. * Corresponding author. Tel.: þ30 210 650 3518; fax: þ30 210 651 1767. E-mail address: [email protected] (K. Stamatakis).

the Z-scheme (Hill and Bendall, 1961) for oxygenic photosynthesis) with light petroleum-extracted cyanobacteria indicated that only in Photosystem I (PSI) do the b-carotenes (b-Cars) sensitize Chl a fluorescence and, hence, do harvest light for photosynthesis (for historical details, see Govindjee, 1999). In contrast, modern ultrafast spectroscopy did prove that singlet excitation energy (EE) transfer from b-Car to Chl a occurs in all photosystem I (PSI) (Kennis et al., 2001; De Weerd et al., 2003a; Holt et al., 2004; Hilbert et al., 2004; Wehling and Walla, 2005) and PSII holochromic complexes (De Weerd et al., 2003b; Holt et al., 2004), although with lower efficiency in PSII. EE transfer from carotenoids to Chl a was also reported recently by means of 77 K emission spectra (Mimuro et al., 2011). In cyanobacteria, the PSI core complex is trimeric, containing per monomer 96 Chls a and 22 b-Cars (Jordan et al., 2001). Twenty one b-Cars are in van der Waals contact with one or more Chl a headgroups (Wang et al., 2004) and several b-Cars show extensive pep electron stacking with them, thereby facilitating efficient bCar to Chl a EE transfer, as well as quenching of Chl a excited triplets. The PSII core complex exists as a dimer and contains 35 Chls a and 11 b-Cars per monomer, distributed in its holochromic

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Please cite this article in press as: Stamatakis, K., et al., On the question of the light-harvesting role of b-carotene in photosystem II and photosystem I core complexes, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.01.014

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K. Stamatakis et al. / Plant Physiology and Biochemistry xxx (2014) 1e7

subcomplexes as follows: in CP43, 13 Chls a and 4 b-Cars; in CP47, 16 Chls a and 5 b-Cars; in D1D2Cytb559 (PSII reaction center, PSIIRC) 6 Chls a and 2 b-Cars (Umena et al., 2011). Since in cyanobacteria the stoichiometric core complex ratio of PSI to PSII ranges between 2 and 5, it is estimated that w80e95% of Chls a and w73e93% of bCars are located in PSI (Fujita et al., 1994). To optimize the quantum yield of oxygen evolution, photosynthetic organisms synchronize the photochemical turnover rates of PSIIRC and PSIRC by regulating the peripheral antennae sizes of PSI and PSII that supply excitation energy (EE) to them. The regulations are initiated in response to redox shifts in the plastoquinone pool (PQ-pool) in the membrane phase and can be post-translational rearrangements of peripheral antenna complexes (state transitions; shifts between state 1 and state 2; see recent reviews in Allen, 2003; Allen and Mullineaux, 2004; Mohanty et al., 2011; Papageorgiou and Govindjee, 2011), as well as transcriptional adjustments of the PSII/PSI stoichiometry (Allen et al., 2011; Puthiyaveetil et al., 2012). In the green algae, state transitions can be triggered also by shifts in the levels of ATP (adenosine triphosphate) and of NADPH (reduced nicotinamide adenine diphosphate) in the stroma phase (reviewed in Cardol et al., 2011). In state 1, the pigment antenna that supplies electronic excitation to PSIIRC is larger, and the pigment antenna that supplies excitation energy to PSIRC is smaller as compared to their respective sizes in state 2. In photosynthetic organisms that contain LHCIItype peripheral antenna complexes (i.e. Chl a-, Chl b- and xanthophyll-binding proteins) this occurs by shifting phosphorylated LHCII subunits (reviewed in Lemeille and Rochaix, 2010; Minagawa, 2011) and cytochrome b6f complexes (Vallon et al., 1991) from PSII-rich regions of the thylakoid membrane to PSIrich regions during the state 1 to state 2 transition; the opposite occurs during the state 2 to state 1 transition. The molecular mechanism is less clear in the case of cyanobacteria which instead of the membrane-intrinsic LHC proteins of plants and algae have extrinsic and non-covalently attached to the membrane light harvesting organelles, the phycobilisomes (PBS). Two distinct and mutually independent mechanisms of state transitions have been recognized in these organisms, both being initiated by redox shifts in the PQ-pool (McConnell et al., 2002): one focuses on the peripheral PBS and invokes structural rearrangements as causes for preferential excitation of PSIIRC or PSIRC in state transitions; the other focuses on the PSII core antenna complexes (Chl a and b-Car binding proteins) within the thylakoid membrane, which regulate the probability of EE transfer (spillover) from PSII Chl a to PSI Chl a. The two states can be recognized from the low temperature (at 77 K) fluorescence emission spectra of Synechococcus sp PCC 7942 cells, obtained by exciting Chl a. In these spectra, the emission bands of Chls a in PSI (at 717 nm; F717) and of Chls a in PSII (at 686 nm and 698 nm; F686 and F698, respectively) are clearly resolved: According to the definition of the two states, a high F698/ F717 ratio signals state 1, i.e. an oxidized PQ-pool, and a low ratio signals state 2, i.e. a reduced PQ-pool (Mullineaux and Allen, 1990; Mullineaux, 1992; Campbell et al., 1998). State transitions-related phenomenology, therefore, affords a powerful and convenient criterion to identify nondestructively redox shifts in the PQ-pool of intact photosynthetic cells. We applied this criterion in the present work aiming to investigate discrepancies in the literature about the light harvesting role of b-Car in cyanobacteria and to explain the reported poor efficiency of b-Car for photosynthetic O2 evolution. Specifically, we asked whether electronic excitation generated on the b-Cars of Synechococcus cells can drive the photoreduction of PQ-pool by electron donation by PSIIRC and its photo-oxidation by electron abstraction by PSIRC. We identified oxidized and reduced states of the PQ-pool state 1 and state 2, respectively by comparing steady-

state fluorescence spectra at 77 K of Synechococcus cells, which prior to freezing had been adapted to different light conditions. Our results show clearly that selectively excited b-Cars sensitize only the oxidation of the intersystem carriers via the PSI photoreaction but not their reduction via the PSII photoreaction. This means that the b-Cars of the PSI core complex have a light harvesting role, while those of the PSII core complex evidently do not. 2. Material and methods 2.1. Cell cultures and preparations Synechococcus sp. PCC 7942 cells were cultured in the BG11 medium (Rippka et al., 1979) which was buffered at pH 7.5 with 20 mM N-2-(2-hydroxylamine)-N0 -ethanesulfonic acid plus NaOH. The cultures were illuminated with white light from fluorescent lamps providing a photosynthetic active radiation (PAR) of 100 mmol photons m2 s1 and were aerated with 5% v/v CO2 in air. Cells were harvested during exponential growth phase (after 4 days) and were resuspended in buffered BG11 at 2 mg Chl a ml1 to be used for assays on the same day. Chl a concentration was determined in N,N-dimethylformamide extracts of cell pellets according to Moran (1982). Ion-permeable cyanobacteria cells (permeaplasts) were prepared by partial digestion of cell wall peptidoglycan with lysozyme (Papageorgiou, 1988). A Li-Cor Quantum Radiometer (Li-Cor, Lincoln, NE, USA) was used to measure light intensities. 2.2. Fluorescence spectrophotometry Assay samples for fluorometry were prepared by injecting 200 ml of a suspension into quartz capillary tubes (2.5 mm internal diameter). Cells were adapted at room temperature, either to darkness for 10 min or to light (at selected spectral bands) for 2 min. The 2-min light adaptations were sufficient for maximal effect as determined from the amplitude of the slow induction rise (SM) of Chl a fluorescence upon illumination of dark-adapted cyanobacterial cells (Stamatakis et al., 2007; Papageorgiou et al., 2007; Kana et al., 2012). Fluorescence was measured with a Hitachi F2500 spectrofluorometer (Hitachi High Technologies Corporation, Japan), which was equipped with liquid-nitrogen sample housing and a red-sensitive photomultiplier. Dark and light sample adaptations were carried out in the light-tight sample compartment of the spectrofluorometer just prior to freezing the samples to 77 K. Spectral bands for light-adaptation were selected from the exciting monochromator of the fluorometer as follows: lmax ¼ 484 nm, Dl ¼ 5 nm, 25 mmol photons m2 s1 (b-Car-selective light); lmax ¼ 710 nm, Dl ¼ 5 nm, 2.5 mmol photons m2 s1 (PSI-selective light). Dark- or light-adapted samples were frozen in the fluorometer sample holder by immersing them in liquid nitrogen. Sample fluorescence was excited either at 436 nm (Dl ¼ 10 nm; Chl a-selective excitation) or at 484 nm (Dl ¼ 10 nm; b-Car-selective excitation). Fluorescence spectra were scanned with a Dl ¼ 2.5 nm detection bandwidth; a Corning CS 2-60 was placed at the entrance slit of the measuring monochromator. Fluorescence ratios (F698/ F717) correspond to the respective peak emissions and are averages of the recorded 77 K spectra of four independent replicates. 2.3. Measurements of PSII and PSI electron transport activities Photoinduced electron transport rates were determined in Synechococcus permeaplasts at room temperature oxymetrically (for each Photosystem) with a Clark-type oxygen electrode (DW1; Oxygraph, Hansatech, King’s Lynn, U.K.). The instrument was fitted with a slide projector to provide actinic illumination to samples.

Please cite this article in press as: Stamatakis, K., et al., On the question of the light-harvesting role of b-carotene in photosystem II and photosystem I core complexes, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.01.014

K. Stamatakis et al. / Plant Physiology and Biochemistry xxx (2014) 1e7

Actinic light bands were selected with interference filters (IF) as follows: IF436 (lmax ¼ 436 nm, Dl ¼ 10 nm; 40 mmol photons m2 s1) for Chl a-selective excitation; IF484 (lmax ¼ 484; Dl ¼ 10 nm; 64 mmol photons m2 s1) for b-Car-selective excitation; and IF620 (lmax ¼ 620; Dl ¼ 10 nm; 150 mmol photons m2 s1) for CPC-selective excitation. PSI activity was determined by measuring the rate of oxygen uptake, in the presence of the post-PSII electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,10 -dimethylurea (DCMU), using Naascorbate/diaminodurene as electron donor to PSI and methyl viologen as post-PSI electron acceptor and mediator of oxygen uptake (after Trebst and Pistorius, 1965). The reaction mixture (1 ml, in buffered BG11) contained permeaplasts (5 mg Chl a), diaminodurene (1 mM), Na-ascorbate (2 mM), methyl viologen (0.15 mM) and DCMU (0.01 mM). PSII activity was determined by measuring the rate of oxygen evolution, with water as electron donor and p-benzoquinone as post-PSII electron acceptor. The reaction mixture (1 ml in buffered BG11) contained permeaplasts (5 mg Chl a ml1) and p-benzoquinone (1 mM). 3. Results Fig. 1 presents absorption spectra (relative values) of whole Synechococcus cells and of the constituent photosynthetic pigments CPC (Patil et al., 2006), APC (Gyzi and Zuber, 1974), Chl a and b-Car, together with the transmission spectrum of the interference filter IF484 (transmission peak at 484 nm, 12 nm full width at half maximum). The insert in Fig. 1 shows the inverted 2nd derivative absorption spectrum of whole cells, which exhibits two major

Fig. 1. Absorption spectra of Synechococcus sp. PCC 7942 whole cells and of constituent photosynthetic pigments, as follows: whole cells (S7942; open circles); chlorophyll a (Chla, in diethyl ether, obtained from omlc.ogi.edu/spectra/PhotochemCAD/html; closed circles); b-Carotene (b-Car, in n-hexane; closed squares); C-phycocyanin (CPC, in water, purified from Spirulina platensis (Patil et al., 2006); closed diamonds); allophycocyanin (APC, in water, purified from Mastigocladus laminosus Cohn (Gyzi and Zuber, 1974); closed triangles). The transmission spectrum of the 484 nm interference filter (IF484) is also shown (long-dashed line, gray-shaded area). Absorption (A) and transmission (T) are given by relative values (Rel.) after normalization between 0 (set at 700 nm) and 1. Insert: Inverted 2nd derivative of the absorption spectrum of whole cells in the 400e550 nm spectral range, revealing a Chl a band (peak at 436 nm) and a b-Car band (peak at 489 nm).

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absorption components in the 400e560 nm range, peaking at 436 nm and 489 nm, which correspond to Chl a and b-Car absorptions, respectively. According to Fig. 1, the transmission band of IF484 overlaps substantially with the absorption band of b-Car and almost negligibly with the absorption bands of Chl a and phycobilins. Therefore, we can well consider the 484 nm transmission band to be b-Car-selective. According to the state transitions phenomenology (Allen, 2003; Mullineaux, 2008), as applied to cyanobacteria, adaptation to light absorbed preferentially by PSII (mainly by phycobilins) induces a transition from state 1 to state 2, while light absorbed preferentially by PSI (by Chls a) induces the transition from state 2 to state 1. It is generally postulated that dark adaptation brings cyanobacteria to state 2, due to PQ-pool reduction by the respiratory electron transport (Dominy and Williams, 1987). This is true also for Synechococcus sp. PCC 7942, although probably not for all cyanobacteria (Tsimilli-Michael et al., 2009). Hence, it is expected that adaptation of dark adapted cells to PSI-light would drive them from state 2 to state 1, while PSII-light would have no effect on them. In Fig. 2 experiment, cells were first adapted to darkness, or to 484 nm light (b-Car-selective), or to 710 nm light (PSI Chl a-selective) and then they were immediately frozen to 77 K with liquid nitrogen. Fluorescence spectra were recorded by exciting the frozen samples with 436 nm light which is absorbed mainly by the Chls a of both photosystems and negligibly by phycobilins and b-Car. These spectra are displayed in Fig. 2 after normalization to equal intensities at 717 nm. They clearly show enhanced PSII fluorescence (F686 and F698) for cells adapted to b-Car-selective light (484 nm), as well as for cells adapted to PSI Chl a-selective light (710 nm), compared to dark-adapted cells. This means that selective excitation of b-Cars of both photosystems, drives the oxidation of PQpool, just as PSI Chl a-selective excitation does, thereby demonstrating that, in vivo, b-Cars transfer, within the PSI core complex, EE to Chl a to sensitize, concomitantly, PSI photochemistry.

Fig. 2. Fluorescence emission spectra of Synechococcus cells recorded at 77 K upon excitation with 436 nm (Dl ¼ 10 nm)excitation wavelength (lexc). Before freezing, samples were adapted to darkness (open circles), or to 484 nm light (Dl ¼ 5 nm, 25 mmol photons m2 s1; black circles), or to 710 nm light (Dl ¼ 5 nm, 5 mmol photons m2 s1; gray circles). The emission maxima of PSII Chl a (F686 and F698, at 686 and 698 nm, respectively) and of PSI Chl a (F717; at 717 nm), as well as the shoulder at 660 nm (F660) attributed to APC fluorescence emission, are marked. The spectra are normalized to equal intensities at 717 nm (equal to F717 of the darkadapted sample).

Please cite this article in press as: Stamatakis, K., et al., On the question of the light-harvesting role of b-carotene in photosystem II and photosystem I core complexes, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.01.014

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K. Stamatakis et al. / Plant Physiology and Biochemistry xxx (2014) 1e7

The finding that the b-Car-absorbed light plays the same role as the PSI-absorbed light does not necessarily mean that b-Cars fail to sensitize photochemistry in the PSII core complex. It is also possible that the observed net oxidation of the PQ-pool is due to the higher preponderance of b-Cars in PSI relative to PSII, resulting in an oxidative PSI photoreaction that overpowers the reductive PSII photoreaction. It must be taken into consideration, also, that the samples were dark-adapted prior to light-adaptation and hence their PQ-pools were already reduced. The Fig. 3 experiment is similar to the Fig. 2 experiment, with the exception that the fluorescence of the dark- or light-adapted and subsequently frozen cell samples was now excited at 484 nm, i.e. within the b-Car absorption band. In our knowledge this is the first time that such emission spectra, i.e. obtained with lexc ¼ 484 nm at 77 K, were used to recognize the state (1 or 2) to which the cells were brought prior to freezing. It is worth noting that the peak fluorescence intensities were lower than in the spectra of Fig. 2 (note the different scales). This is expected since the concentration of b-Car is lower than that of Chl a and, also, that the efficiency of EE transfer from b-Car to Chl a is lower than unity. The first to observe in Fig. 3 is that, independent of the type of adaptation, the 484 nm excitation gave rise to all three low temperature bands of Chl a fluorescence (F686, F698, F717), thereby demonstrating that b-Car-to-Chl a EE transfer occurs within both PSI and PSII core complexes. However, the F698 peaks (and correspondingly the F698/F717 ratios) are lower in Fig. 3 (484 nm excitation) than in Fig. 2 (436 nm excitation) indicating a lower EE transfer efficiency within PSII than within PSI, in agreement with reports of detailed investigations in purified photosynthetic complexes with ultrafast pump-probe spectroscopic methods (Kennis et al., 2001; De Weerd et al., 2003a, 2003b; Holt et al., 2004). We also observe, in all spectra of Fig. 3, a low fluorescence band at w670 nm, corresponding to APC fluorescence emission, which indicates that a fraction of the 484 nm excitation light is absorbed by CPC and APC of the Synechococcus phycobilisome. A relatively smaller but distinct band at w670 nm is also seen in the emission spectra obtained with 436 nm excitation (Fig. 2). The ratio F698/F717 of the peak intensities of the PSII and PSI Chl a fluorescence bands at 77 K has often been used to quantify the

extent of state transitions of oxygenic photosynthetic cells (e.g. McConnell et al., 2002). The upper extreme of this ratio indicates nearly a fully oxidized PQ-pool (state 1) while the lower extreme nearly a fully reduced PQ-pool (state 2). Since state shifts are continuous, the F698/F717 ratio can take any value between the high and the low extremes, depending on the conditions used to light-adapt the cells at room temperature. In Fig. 4, we plot the F698/F717 ratio against the wavelength of the light band the cells were adapted to prior to freezing, together with the absorption spectrum of whole Synechococcus sp. PCC 7942 cells. The resulting action spectrum of state transitions in this cyanobacterium shows that the maximum shift towards state 1 occurred at 498 nm (upward arrow) and the maximum shift toward state 2 at 608 nm (downward arrow). The 498 nm spectral band of the action spectrum lies primarily within the b-Car absorption, while the 608 nm band is in a region where CPC and APC absorb strongly, Chl a minimally and b-Car has no absorption at all (see Fig. 1). Characteristically, the value of F698/F717 after dark adaptation, which is known to shift cyanobacteria to state 2 (Dominy and Williams, 1987; Tsimilli-Michael et al., 2009), was measured at 0.72. This value lies below the F698/F717 trace in Fig. 4, suggesting that a fuller reduction of the PQ-pool in this cyanobacterium is achieved after dark-adaptation than after a light-induced state 1 to state 2 transition. The data on Fig. 4 demonstrate that, among light-adapted cells, the fullest net oxidation of the PQ-pool in Synechococcus is attained with b-Car-selective excitation (at w498 nm), while the fullest net reduction occurs in a spectral region where b-Car does not absorb (at w608 nm) and, besides a minor direct absorption by Chl a, it is mainly the phycobilin-sensitized Chls a of PSII that are functional. Chls a and b-Cars occur both in PSII and in PSI and, as deduced from Fig. 3, EE of b-Car is transferred to Chl a in both photosystems. The question then arises why the EE that is generated by light absorption on b-Cars, and then is transferred to Chls a, does oxidize the PQ-pool more effectively than the EE which is directly generated on Chls a. Considering that, potentially, upon excitation of Chls a or b-Cars the photoreactions of both photosystems would occur, but the PSI oxidative photoreaction overpowers the reductive PSII photoreaction and results in a net oxidation of the PQ-pool, we may

Fig. 3. Fluorescence emission spectra of Synechococcus cells recorded at 77 K upon excitation with lexc ¼ 484 nm. Before freezing, the samples were adapted as indicated. Other details, as in the legend of Fig. 2.

Fig. 4. Action spectrum (open circles; left vertical axis) of the F698/F717 ratio (indicating the extent of state transition) calculated from emission spectra obtained at 77 K by exciting differently adapted samples with 436 nm light; the ratio is plotted vs. the wavelength of the light (Dl ¼ 5 nm) to which Synechococcus cells were adapted for 2 min before freezing (adaptation light). The upward and downward arrows indicate the maximum and the minimum of the action spectrum, respectively. The dotted line presents the value of the F698/F717 ratio (0.72; left vertical axis) in dark adapted cells (D-adp). The relative absorption spectrum of Synechococcus sp. PCC 7942 whole cells (as in Fig. 1) is also plotted, for comparison (closed circles; right vertical axis).

Please cite this article in press as: Stamatakis, K., et al., On the question of the light-harvesting role of b-carotene in photosystem II and photosystem I core complexes, Plant Physiology and Biochemistry (2014), http://dx.doi.org/10.1016/j.plaphy.2014.01.014

K. Stamatakis et al. / Plant Physiology and Biochemistry xxx (2014) 1e7

consider two possibilities: (a) because of the lower efficiency of EE transfer from b-Car to Chl a in PSII than in PSI (as deduced by the comparison of Figs. 2 and 3), the PQ-pool is shifted more to the oxidized state when the Chls a receive EE from b-Car than when they are directly excited by absorption; or (b) selective excitation of PSII b-Car excitation fails to drive PSII photochemistry. To resolve this dilemma, we employed selective b-Car excitation in conjunction with selective detection of the PSII (Vernon and Shaw, 1969) and the PSI electron transporting activities at room temperature using Synechococcus permeaplasts (Papageorgiou et al., 1998). Using Synechococcus permeaplasts, we measured oxymetrically electron transport activities across both PSII (electron donor: water; post-PSII electron acceptor: p-benzoquinone) and PSI (postPSII inhibitor: DCMU; post-PSII electron donor: diaminodurene plus ascorbate; post-PSI electron acceptor: methyl viologen). The results, displayed on Table 1, show that the electron transport activities of PSII and PSI depend on the spectral band of the exciting light. Particularly revealing are the displayed relative quantum efficiencies of the three pigment-selective excitations, which were calculated from the fractional absorptions (1 e transmittance) at 436, 484 and 620 nm (last two columns). Thus, for both the PSII and the PSI photoreactions, the relative quantum efficiencies were maximal upon CPC excitation (620 nm), and about 40% as much upon Chl a excitation (436 nm). On the other hand, upon b-Carselective excitation (484 nm), the relative quantum efficiency of the PSII photoreaction was about zero, while that of the PSI photoreaction was as high as that with CPC-selective excitation. We conclude from these results that the observed oxidation of the PQ-pool after selective excitation of the b-Cars does not reflect an outperformed PSII but, most likely, the inability of PSII to reduce the PQ-pool. 4. Discussion Carotenoids, which co-exist with Chls in all major protein complexes of PSI and PSII, have been recognized to play essential roles in light harvesting for photosynthesis (see e.g. reviews by Frank et al., 1999; Faller et al., 2005; Govindjee et al., 2010) as well as in the structure dynamics of pigmenteprotein complexes that regulate the non-photochemical dissipation of excess Chl a excitation (see reviews by Pogson et al., 2005; Horton, 2012; DemmigAdams et al., 2012; Kirilovsky and Kerfeld, 2012, and citations therein). The light harvesting function of carotenoids was first recognized at about the middle of the 20th century (see historical reviews by Dutton, 1997; Govindjee, 1999) and was subsequently investigated intensively, particularly after the advent of ultrafast spectroscopic techniques (reviewed e.g. by Berrera et al., 2009; Renger and Schlodder, 2011). The main evidence for photosynthetic light harvesting by carotenoids are demonstrations of EE transfer to Chl a and the concomitant emission of Chl a fluorescence. EE transfer from b-Car to Chl a has been investigated in detail in purified photosynthetic

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complexes by means of ultrafast pump-probe spectroscopic methods. In all PSI (Kennis et al., 2001; De Weerd et al., 2003a; Holt et al., 2004) and PSII (De Weerd et al., 2003b; Holt et al., 2004) core complexes, b-Cars transfer singlet excitation from their S2 state to Chls a with efficiencies of w33% in CP43 and CP47, w26% in D1D2Cytb559 and w57% in PSI (Holt et al., 2004). Electronic excitation has been shown to equilibrate at room temperature faster (within w1e3 ps, de Weerd et al., 2002; Pawlowicz et al., 2007; Casazza et al., 2010) in the PSII core antenna complexes CP43 and CP47 than between antenna complexes and the reaction center complex (within w20e50 ps, Pawlowicz et al., 2007; van der Weij-de Wit et al., 2011) as expected in view of the larger inter pigment distances in the latter case (Raszewski and Renger, 2008). On the other hand, the increased functional connectivity of the denser pigment network in the PSI core complex allows for an even faster equilibration of EE (Melkozernov et al., 2006) which was measured to occur at w2 ps (Müller et al., 2003). Finally, primary charge separation has been shown to occur faster in PSIRC (w100 fs; Shelaev et al., 2010) compared to PSIIRC (0.9e3 ps; Holzwarth et al., 2006; Shelaev et al., 2011). Thus, the EE of b-Car is transferred faster and more efficiently to PSIRC than to PSIIRC. Despite the amply established ability of b-Cars to donate singlet excitation to Chl a, both in the PSII and in the PSI core complex, there had been no demonstrations in the literature of primary charge separations sensitized by b-Car-selective excitation, in vivo. This information is, however, absolutely necessary for assigning light harvesting roles to the b-Cars of PSII and of PSI, in the sense not only of collecting light but also of driving primary photochemistry. Our experiments with whole Synechococcus sp. PCC 7942 cells show that light selectively absorbed by b-Car gives rise to the three characteristic emission bands of Chl a in vivo, i.e. F686 (originating mainly from CP43), F698 (originating from CP47) and F717 (originating from PSI; see Fig. 3), and therefore prove the transfer of EE transfer from b-Cars to Chls a within both the PSI and PSII core complexes, although with a lower efficiency in PSII than in PSI (comparison of fluorescence intensities in Figs. 2 and 3). It was also demonstrated that, upon receiving EE from b-Cars (adaptation to 484 nm light), the Chls a of PSI succeed in oxidizing the PQ-pool, while those of PSII fail to reduce it (Figs. 2 and 3). On the other hand, when Chls a are excited directly by absorbing light, or indirectly by receiving EE from phycobilins, they can sensitize, respectively, a PSI-driven oxidation (Figs. 2 and 3) or a PSII-driven reduction of the PQ-pool (Fig. 4). These conclusions are deduced from the comparison of low temperature (77 K) fluorescence emission spectra of cells that had been pre-adapted at room temperature to different light conditions (dark, or light of different wavelengths selectively absorbed by Chl a, of b-Car, or CPC). This comparison permits us to recognize whether the cells have been adapted to state I or to state II, and from this to identify whether, respectively, the PQ-pool has been oxidized or reduced.

Table 1 Photosystem II and I electron transport activities measured with Synechococcus sp. PCC 7942 permeaplastsa after selective excitation of chlorophyll a (436 nm), b-carotene (484 nm) and C-phycocyanin (620 nm). Electron transport activityc

Pigment-selective actinic light nm

Incident

436 484 620

40 64 150

a b c d

b

b

Relative quantum efficiencyd

Absorbed

PSII

PSI

PSII

PSI

11.5 9.2 20.5

9.0  1.0 w0 40.2  1.2

142.4  2.3 284.0  4.4 633.6  14.5

0.78 (w40) w0 (w0) 1.96 (100)

12.43 (w40) 30.79 (w100) 30.86 (100)

Oxymetric assays, as described in Materials and Methods. In mmol photons m2 s1. In mmol O2 mgChl1 h1. In (electron transport activity) (mmol absorbed photons m2 s1)1; data are means  s.e. (n ¼ 3).

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K. Stamatakis et al. / Plant Physiology and Biochemistry xxx (2014) 1e7

Since neither the b-Car selective excitation nor the b-Car to Chl a EE transfer are photosystem selective, the shift of the PQ-pool towards oxidation after direct or indirect excitation of Chl a in vivo could reflect the preponderance of Chl a (w80e95%) and of b-Cars (w73e93%) in the PSI of cyanobacteria (vide infra) as well as the faster and more efficient transfer of EE to the reaction center in the PSI core complex (Demmig-Adams et al., 2012; Kirilovsky and Kerfeld, 2012), compared to the PSII core complex (Dutton, 1997; Berrera et al., 2009). Does, indeed, the observed oxidation of the PQ-pool after selective b-Car excitation reflects a PSII outperformed by PSI, or does it reflect a completely failed PSII? Based on the results of Table 1 we favor the second alternative. According to Table 1, the relative quantum efficiency of the PSII photoreaction was maximal for CPC-absorbed light (620 nm), intermediate for Chl a-absorbed light (436 nm) and about zero for b-Car-absorbed light (484 nm). A photosynthetically silent PSII, after selective excitation of its bCars, may also explain why, according to Fig. 4, the plastoquinone pool is oxidized more completely by b-Car-sensitized Chls a than by directly excited Chls a, namely why the F698/F717 ratio is greater at 498 nm, where b-Car absorbs, than at 440 nm, where Chl a absorbs (see Fig. 4). The disparity is even more dramatic when the cells are excited at w670 nm, a spectral band absorbed by Chl a alone. In contrast, the fullest reduction of the plastoquinone pool is achieved by phycobilin-excited Chls a of PSII (at w608 nm). In conclusion, our results suggest that the b-Cars of PSI core complex have a clear light harvesting role, while those of the PSII core complex evidently do not. References Allen, J.F., 2003. State transitions e a question of balance. Science 299, 1530e1532. Allen, J.F., Mullineaux, C.W., 2004. Probing the mechanisms of state transitions in oxygenic photosynthesis by chlorophyll fluorescence spectroscopy, kinetics and imaging. In: Papageorgiou, G.C., Govindjee (Eds.), Chlorophyll a Fluorescence: a Signature of Photosynthesis, Advances in Photosynthesis and Respiration, vol. 19. Springer, Dordrecht, pp. 447e461. Allen, J.F., Santabarbara, S., Allen, C.A., Puthiyaveetil, S., 2011. Discreet redox signaling pathways regulate light-harvesting and chloroplast gene transcription. PLoS One 6, e26372. Berrera, R., van Grondelle, R., Kennis, J.T.M., 2009. Ultrafast light absorption spectroscopy: principles and application to photosynthetic systems. Photosynth. Res. 101, 115e118. Brody, M., Emerson, R., 1959. The quantum yield of photosynthesis in Porphyridium cruentum and the role of chlorophyll a in the photosynthesis of red algae. J. Gen. Physiol. 43, 251e264. Campbell, D., Hurry, V., Clarke, A.K., Gustafsson, P., Öquist, G., 1998. Chlorophyll fluorescence analysis of cyanobacterial photosynthesis and acclimation. Microbiol. Mol. Biol. Rev. 62, 667e683. Cardol, P., Forti, G., Finazzi, G., 2011. Regulation of electron transport in microalgae. Biochim. Biophys. Acta 1807, 912e918. Casazza, A.P., Szczepaniak, M., Muller, M.G., Zucchelli, G., Holzwarth, A.R., 2010. Energy transfer in isolated core antenna complexes CP43 and CP47 of Photosystem II. Biochim. Biophys. Acta 1797, 1606e1616. Demmig-Adams, B., Cohu, C.M., Muller, O., Adams III, W.W., 2012. Modulation of photo-synthetic energy conversion efficiency in nature: from seconds to seasons. Photosynth. Res. 113, 75e88. De Weerd, F.L., van Stokkum, I.H.M., van Amerongen, H., Dekker, J.P., van Grondelle, R., 2002. Pathways for energy transfer in the core light-harvesting complexes CP43 and CP47 of Photosystem II. Biophys. J. 82, 1586e1597. De Weerd, F.L., Kennis, J.T.M., Dekker, J.P., van Grondelle, R., 2003a. b-Carotene to chlorophyll singlet energy transfer in the Photosystem I core of Synechococcus elongatus proceeds via the b-carotene S2 and S1 states. J. Phys. Chem. B 107, 5995e6002. De Weerd, F.L., Dekker, J.P., van Grondelle, R., 2003b. Dynamics of b-carotene-tochlorophyll singlet energy transfer in the core of Photosystem II. Phys. Chem. B 107, 6214e6220. Dominy, P.J., Williams, W.P., 1987. The role of respiratory electron flow in the control of excitation energy distribution in blue-green algae. Biochim. Biophys. Acta 892, 264e274. Dutton, H.J., 1997. Carotenoid-sensitized photosynthesis: quantum efficiency, fluorescence and energy transfer. Photosynth. Res. 52, 175e185. Emerson, R., Lewis, C.M., 1942. The photosynthetic efficiency of phycocyanin in Chroococcus and the problem of carotenoid participation in photosynthesis. J. Gen. Physiol. 25, 579e595.

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