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Probing the Electric Field Across Thylakoid Membranes in Cyanobacteria

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Probing the Electric Field Across Thylakoid Membranes in Cyanobacteria Probing the electric field across thylakoid membranes in cyanobacteria Stefania Violaa,b, Benjamin Bailleula, Jianfeng Yub, Peter Nixonb, Julien Sellésa, Pierre Joliota, and Francis-André Wollmana,1 aLaboratoire de Biologie du chloroplaste et perception de la lumière chez les micro-algues-UMR7141, Institut de Biologie Physico-Chimique, CNRS-Sorbonne Université, 75005 Paris, France; and bDepartment of Life Sciences, Imperial College, SW7 2AZ London, United Kingdom Edited by Krishna K. Niyogi, University of California, Berkeley, CA, and approved September 16, 2019 (received for review August 9, 2019) In plants, algae, and some photosynthetic bacteria, the ElectroChromic the photosynthetic activity. Upon illumination, each of the com- Shift (ECS) of photosynthetic pigments, which senses the electric ponents of the photosynthetic chain participates in either the for- field across photosynthetic membranes, is widely used to quantify mation (PSII, cytochrome b6f,PSI)orthedissipation(ATP the activity of the photosynthetic chain. In cyanobacteria, ECS synthase) of a proton motive force, which has an electric and an signals have never been used for physiological studies, although osmotic component. Light-induced ECS signals can thus be used to they can provide a unique tool to study the architecture and assess the activities and/or relative amounts of each of the photo- function of the respiratory and photosynthetic electron transfer synthetic complexes (13). ECS measurements can also be used to chains, entangled in the thylakoid membranes. Here, we identified calculate the turnover rates of the photosystems during illumina- bona fide ECS signals, likely corresponding to carotenoid band shifts, tion (14, 15), and to determine the levels of the preexisting elec- in the model cyanobacteria Synechococcus elongatus PCC7942 and trochemical proton gradient in the dark (16, 17). Synechocystis sp. PCC6803. These band shifts, most likely originating In chlorophytes, the electrochromic signals in the 450- to 600-nm from pigments located in photosystem I, have highly similar spectra region of the spectrum are attributed to a combination of ca- in the 2 species and can be best measured as the difference between rotenoid and chlorophyll b band shifts (12, 18). ECS signals the absorption changes at 500 to 505 nm and the ones at 480 to corresponding, in the same spectral region, only to carotenoid 485 nm. These signals respond linearly to the electric field and band shifts have been described in red and brown algae (19), as display the basic kinetic features of ECS as characterized in other well as in microalgae (20) and photosynthetic bacteria (21). In PLANT BIOLOGY organisms. We demonstrate that these probes are an ideal tool to the case of cyanobacteria, earlier studies concluded that the study photosynthetic physiology in vivo, e.g., the fraction of PSI absence of light-induced signals that could be ascribed to ca- centers that are prebound by plastocyanin/cytochrome c6 in dark- rotenoid band shifts (19, 20), in contrast with subsequent reports ness (about 60% in both cyanobacteria, in our experiments), the of putative ECS signals in intact cells (22, 23) and isolated PSI conductivity of the thylakoid membrane (largely reflecting the complexes (24) from different species. Still no ECS-based mea- activity of the ATP synthase), or the steady-state rates of the pho- surements have ever been used for physiological studies. tosynthetic electron transport pathways. Here, we identified bona fide ECS signals in the model cya- nobacteria Synechococcus elongatus PCC7942 and Synechocystis sp. cyanobacteria | photosynthesis | ElectroChromic Shift | electron fluxes PCC6803. We show that these signals are consistent with carot- enoid band shifts, do not originate from PSII, are linearly pro- bout 2.8 billion years ago began the great oxygenation event portional to the light-induced transmembrane electric field, and Acaused by cyanobacteria, the first organisms to perform display all the kinetic properties of ECS. We provide conclusive oxygenic photosynthesis using 2 photosystems linked in series and evidence for a rapid phase in the kinetics of rereduction of P700, water as an electron donor (1, 2). In cyanobacteria, the photo- synthetic and the respiratory electron transfer chains are both lo- Significance cated in the thylakoid membranes, and they share several redox components (3), giving rise to a complex network of electron Cyanobacteria were the first organisms to develop oxygenic fluxes. Moreover, cyanobacteria are proposed to house 2 pathways photosynthesis using water as a source of electrons. Today they for a photosystem II (PSII)-independent electron flow toward remain widespread primary photosynthetic producers and hold a photosystem I (PSI). One is mediated by the NADPH:plastoqui- high biotechnological potential. In cyanobacteria, respiration and none oxidoreductase (NDH) complexes (4, 5), with the other being photosynthesis are interconnected in a complex network of elec- an NDH-independent but ferredoxin-dependent pathway. The tron fluxes. The study of cyanobacterial physiology is hampered molecular nature of the latter requires further elucidation, after by the lack of techniques, allowing a direct measurement of the the characterization of a mutant showing some altered electron transmembrane electric field that develops across their photo- flow (6). No accurate estimation of the physiological turnover rates synthetic/respiratory membranes. Here, we characterized a probe of these pathways is available so far. of the transmembrane electric field, based on the ElectroChromic The study of cyanobacterial photosynthetic fluxes is further Shifts of carotenoids, thus opening unprecedented avenues to complicated by the fact that current methods such as PSII fluo- bioenergetics studies of these major photosynthetic organisms. rescence (7) and PSI absorption spectroscopy (8) measure distinct variables that cannot be quantitatively compared without potential Author contributions: S.V., B.B., P.N., J.S., P.J., and F.-A.W. designed research; S.V., B.B., biases. Moreover, the quantitative interpretation of fluorescence J.Y., J.S., and P.J. performed research; S.V., B.B., J.S., and P.J. analyzed data; and S.V., B.B., parameters in cyanobacteria is hampered by a number of pitfalls (9, J.S., P.J., and F.-A.W. wrote the paper. 10). A powerful tool to measure photosynthetic activity, extensively The authors declare no competing interest. used in plants, algae, and several photosynthetic bacteria, is the This article is a PNAS Direct Submission. ElectroChromic Shift (ECS) of photosynthetic pigments (11, 12), Published under the PNAS license. which is based on a physical phenomenon known as the Stark ef- 1To whom correspondence may be addressed. Email: [email protected]. fect. This method relies on the shift of the absorption spectrum of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. some photosynthetic pigments in the thylakoid membrane in re- 1073/pnas.1913099116/-/DCSupplemental. sponse to changes in the transmembrane electric field generated by www.pnas.org/cgi/doi/10.1073/pnas.1913099116 PNAS Latest Articles | 1of7 Downloaded by guest on October 2, 2021 the special pair of PSI, in vivo in both cyanobacteria. We also measured after 100 ms are pure ECS spectra, which present demonstrate that the identified electrochromic probes can be minima at 430, 455, 485, and 560 nm; maxima at 450, 470, and used to study the changes in membrane permeability and the 500 nm; and a broad positive region at 510 to 540 nm. We also electron flow rates. note that the addition of PMS caused a progressive decrease in the ECS amplitude in the green region of the spectrum (510 to 540 Results and Discussion nm) in S. elongatus. This is caused by the appearance of a negative Flash-Induced Carotenoid Band Shifts in S. elongatus and Synechocystis. signal peaking around 515 nm that superimposed itself to ECS We recorded the spectra of the absorption changes induced by during the time course of the experiment (about 2 h between the single-turnover saturating flashes in S. elongatus. Cells were placed forth and the back recordings in anaerobic conditions). As shown in anaerobic conditions in the presence of the PSII inhibitors 3- in SI Appendix,Fig.S4A and B and further discussed in the SI (3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and hydrox- Appendix, the progressive bleaching was present also when re- ylamine (HA), to eliminate the absorption changes produced by cording spectra in the absence of PMS, although to a much lesser PSII turnovers. We recorded these spectra 19 ms after the flash, extent. We do not have direct evidence of the nature of such a followed by 5 detecting flashes given 100 ms apart, to minimize the signal, although the spectrum of the difference between the forth contribution of redox-dependent absorption changes that mostly and the back recordings may also be due to a change in the band relax before 19 ms (SI Appendix,Fig.S2). As shown in Fig. 1A,the shift of a carotenoid. These spectral changes did not affect the decay of the absorption changes was uniform in the 450- to 550-nm amplitudes of the negative peak at 480 to 485 nm and the positive region: all spectra show 2 minima peaking around 455 and 485 nm; peak at 500 to 505 nm. 3 maxima at 450, 470, and 500 nm; and a broad featureless positive We investigated the presence of ECS in another commonly region between 510 and 540 nm. On the edges of the recorded used cyanobacterium, Synechocystis sp. PCC6803. Fig. 1C and SI spectral region was a transient bleaching at 420 to 425 nm and Appendix,Fig.S3C show the spectra of flash-induced absorption 554 nm that we attribute to the oxidation of the c-type haem of changes recorded in presence of 15 μMPMSinDCMUandHA- cytochrome f (and possibly of cyt c6), and an absorbance increase treated cells. The spectra showed a predominant component with at 435 nm and 560 nm, which we attribute to the reduction of the homothetic decay and a shape highly similar to the electrochromic bH haem of cyt b (25).
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