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Home , P700, Photophosphorylation, Photosystem II

Probing the electric field across membranes in

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 , , 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 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 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 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 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 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 /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 | | 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 , 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 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 . Today they for a photosystem II (PSII)-independent electron flow toward remain widespread primary photosynthetic producers and hold a (PSI). One is mediated by the NADPH:plastoqui- high biotechnological potential. In cyanobacteria, respiration and none (NDH) complexes (4, 5), with the other being photosynthesis are interconnected in a complex network of elec- an NDH-independent but -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- 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). After 100 ms, the homothetic decay in the signal of S. elongatus,exceptforaredshiftofabout10nmofthe 450- to 550-nm region is consistent with only 1 signal contributing broad positive feature in the 510- to 540-nm region. Most notably, to the long-lived absorption changes that we attribute to ECS. the difference between 480 to 485 nm and 500 to 505 nm was Indeed, after normalization on the (500 to 485 nm) difference (SI conserved. Again, the contribution of cyt f and cyt b redox signals Appendix,Fig.S3A), the spectra recorded at the different times was small in the presence of PMS, with the further addition of a were superimposed in the 450- to 550-nm region, except for a nonhomothetic negative signal present at 410 to 470 nm in the 5-ms small bleaching at 510 to 520 nm in the 19-ms recording. The recording. In the case of Synechocystis, minor spectral changes also identified ECS spectrum is very similar to the carotenoid band appeared between the forth and the back recordings (SI Appendix, shifts described in red and brown algae and in photosynthetic Fig. S4C) but, again, they did not affect the 480- to 505-nm region. bacteria (19–21), with peaks located at 30-nm intervals. To further The ECS gives a direct measure of the transmembrane electric remove the spectral contribution of cytochrome redox changes, we field irrespective of the molecular nature of the electrochromic used the redox catalyst PMS, which induces an artificial cyclic flow pigments and of their location in the membrane. Even so, in S. around PSI that bypasses the (26). In- elongatus and Synechocystis, the ECS spectra suggest that such deed, in its semireduced form, PMS is both an electron acceptor pigments are carotenoids. To also investigate their location, we and donor, and once fully reduced by PSI, it partitions into the recorded the ECS spectrum in a Synechocystis mutant lacking lumen and donates electrons to PSI, probably through plastocya- photosystem II (PSII, characterization in SI Appendix,Fig.S5)and nin (27), releasing protons. The spectra of flash-induced absorp- compared the long-lived absorption difference signals, when the tion changes were recorded in the presence of 15 μMPMSin redox spectral contributions have completely relaxed (i.e., >100 DCMU- and HA-treated S. elongatus cells. The addition of PMS ms), with those in the wild type. The 2 strains presented remarkably eliminated most of the contribution from the b haems and from c- similar spectra in the 470- to 510-nm region, both featuring the type cytochromes. Only a minor contribution of those signals carotenoid shift we described so far (Fig. 2A). We noted spectral remained 5 ms after the flash, which can be visualized in the 420- differences in the mutant in the blue and red sides of the spectrum to 440-nm region, and it relaxed completely within 100 ms (Fig. 1B (discussed in the SI Appendix,Fig.S6). We also looked whether and SI Appendix, Fig. S3B). We therefore consider that the spectra , although located at the thylakoid membrane

ACS. elongatus B S. elongatus PMS Synechocystis PMS 2000 2000 1000 1000 1000 1000

0 0 0 1000 500 500 6 6 -1000 6 -1000 -2000 x10 x10 0 0 x10 0 I/I I/I -2000 I/I -2000 -4000 ms ms ms 19 316 -1000 5 302 -500 5 302 -500 -3000 -3000 118 415 104 401 104 401 -6000 217 514 203 500 203 500 -2000 -4000 -1000 -4000 -1000 420 440 460 480 500 520 540 560 420 440 460 480 500 520 540 560 420 440 460 480 500 520 540 560 nm nm nm

Fig. 1. Absorption difference signals in S. elongatus and Synechocystis.(A) Spectra of flash-induced absorption changes in S. elongatus cells treated with DCMU and HA. (B and C) Spectra of flash-induced absorption changes in S. elongatus and Synechocystis cells, respectively, determined in presence of DCMU and HA and 15 μM PMS. Absorption changes were sampled at the indicated time intervals after the flash (in ms). Note that the y axis of the 410 to 450 nm region has a different scale for each spectrum.

2of7 | www.pnas.org/cgi/doi/10.1073/pnas.1913099116 Viola et al. Downloaded by guest on October 2, 2021 surface, could contribute to the ECS signal. We measured the The latter should be proportional to the fraction of P700 that has spectra of the absorption changes in the PAL mutant (Fig. 2B), been rereduced between the 2 flashes, which we measured in lacking phycobilisomes (28, 29). The ECS signal proved much parallel by the absorption difference signals at 435 nm (Fig. 3). shorter-lived in this strain, which did not allow the extraction of a As described earlier, we performed these measurements in the pure ECS spectrum (note the important redox contributions at 425 presence of PMS (SI Appendix, SI Results and Discussion and Fig. to 430 nm and 555 to 560 nm). Nevertheless, 5 ms after a flash, the S7) to remove cyt b6f-related absorption changes at 435 nm and spectra were similar in the mutant and wild type in the 480- to 510- prevent back reactions within PSI. At 5 ms after the first flash, nm region. Taken together, these results are consistent with the ECS almost all P700 was reduced (see spectra in SI Appendix,Fig.S3B signal originating from carotenoids located within PSI cores (see also and C), and the ECS increase on a second flash was almost equal SI Appendix, SI Results and Discussion). It should be noted that to the first one (Fig. 3 A and B), indicating that only a small variations in the absolute amplitude of ECS signals due to variations fraction (≤10%) of PSI remained closed after 5 ms. This fraction in the content of the pigment or complexes har- likely corresponds to PSI centers that are not connected to sec- boring the probes (e.g., PSI) will not affect the measurements, pro- ondary donors, and thus remain oxidized 5 ms after the actinic vided that ECS signals are proportional to the electric field (see flash. Using this value at 5 ms to normalize the amount of PSI open centers (Materials and Methods), the kinetics measured with Flash-Induced ECS Kinetics: The a Phase and Its Use for Monitoring + the Activity of Photosystems and Fig. 3) and normalized to the ECS the ECS and the P700 absorption methods were superimposed signal generated by a single-turnover saturating flash, which corre- (Fig. 3D), demonstrating that the a phase of the ECS signal gives a sponds to 1 charge separation per active photosystem. direct measurement of the fraction of open centers. It also indi- Examination of the above spectra shows that the ECS can be cates that this ECS signal shows a linear response to the variation best measured by the difference between the absorption changes at of the transmembrane electric field, as it does in plants and green algae (11). The PSI reduction kinetics showed, in both cyanobac- 500 to 505 nm and at 480 to 485 nm in the 2 cyanobacteria; this ∼ μ ∼ difference is the difference with the largest amplitude, which teria, 2 phases completed in 100 sandin 3 ms, respectively corrects for the absorption changes associated with the oxidation/ (representative traces shown in Fig. 3C). The fast phase, having a half time of ∼7 μs, suggests the presence in vivo in the dark of a reduction of P700, c-type cytochromes, and b haems (25, 30) and is not affectedbythedistortionobservedinthegreenregionofthespec- fraction of PSI complexes (about 60% of the total) bound to re- trum. To further substantiate the attribution of the long-lived ab- duced plastocyanin or cyt c6. The half time for the electron transfer sorption difference to an ECS signal of appropriate lifetime and between the bound electron donor and P700 is close to that mea-

sured in plant (∼4 μs) (31). The slower phase can be PLANT BIOLOGY characteristic spectrum, we examined the kinetic prediction of this attributed to the diffusion time of plastocyanin or cyt c molecules attribution. Indeed, the kinetics of an ECS signal after a single turn- 6 responsible for the reduction of the remaining fraction of PSI over flash display unique characteristics, being composed of 3 phases: complexes; its half time (∼200 μs) is also close to what was mea- afastrise(a phase, completed in <100 μs) corresponding to photo- sured in for the reaction between free plastocyanin and chemistry in PSII and PSI; a second rise (b phase, half-time ∼10 ms), P700 (31–33). The fast PSI reduction phase was present also in which corresponds to the electron transfer from PSII acceptors to PSI absence of PMS (SI Appendix,Fig.S8), indicating that it is not due donors, catalyzed by the cytochrome b6f; and a phase of ECS decay (c to a direct donation of electrons from PMS to PSI. Thus, a fast phase) reflecting the electric permeability of the thylakoid membrane donation of electrons from a prebound electron donor to P700 does (mostly the proton efflux catalyzed by the ATP synthase). occur in vivo in both S. elongatus and Synechocystis. The a phase, representing charge separations from active Flash-Induced ECS Kinetics: The a Phase and Its Use for Monitoring the photosystems, can also be used to assess the PSI/PSII ratio. Fig. 4A Activity of Photosystems. To test whether the fast rise in ECS that shows representative traces of the flash-induced ECS kinetics in S. follows a single turnover flash corresponds to the contribution elongatus in the control or when PSII is inhibited by the addition of of active photosystems, we measured the PSI open centers (in DCMU and HA. In the latter case, the a phase is solely due to PSI conditions of PSII inhibition, adding HA and DCMU), using 2 photochemistry, and the comparison with the control allowed us to independent methods. After a first saturating flash, generating a determine a PSI/PSII ratio of 2.3 ± 0.5 (5 biological replicates) in transmembrane electric field, a second flash is applied at in- S. elongatus, in line with previous reports (e.g., ref. 34). The same creasing time intervals and generates an additional electric field. experiment performed in Synechocystis (SI Appendix,Fig.S9C) also confirms the excess of PSI over PSII reaction centers.

A WT B WT Flash-Induced ECS Kinetics: b Phase and Decay. As mentioned 2 PSII- 2 PAL earlier, a flash-induced ECS kinetics should display, after the fast rise (a phase), a second rise called b phase, followed by a decay (c 0 phase) reflecting the dissipation of the transmembrane electric 0 field. We note that in S. elongatus PSII inhibition caused an overall -2 slowing down of the ECS decay (Fig. 4A), allowing the observation 2 of the slow electrogenic phase (maximal at 20 ms), due to the -2 1 -4 turnover of cyt b6f (13). This b phase was, as expected for a proper 1 ECS signal, a bona fide probe of electron transfer through the -6 -4 0 cytochrome b6f since it was sensitive to PMS (SI Appendix, Fig. norm. on (500 - 485 nm) norm. on (500 - 485 nm) 0 I/I I/I S7A), which bypasses this complex, and to the quinone analog -8 460 480 500 520 460 480 500 520 DBMIB, which inhibits its turnover (SI Appendix,Fig.S9A). The -6 420 440 460 480 500 520 540 560 420 440 460 480 500 520 540 560 flash-induced ECS kinetics in Synechocystis had an overall faster nm nm decay than those in S. elongatus, preventing the observation of the b phase (SI Appendix,Fig.S9B and C). Fig. 2. Spectra of flash-induced absorption changes in Synechocystis wild type Because the ECS decay depends on the permeability of the (WT) and mutants determined in presence of DCMU, HA, and PMS. The spectra are normalized on the 500- to 485-nm difference. (Insets) 450- to 530-nm re- thylakoid membrane, it reflects mostly the dissipation of the pro- gion on an expanded scale. (A) Comparison between WT and PSII- mutant ton gradient by the ATP synthase. Counter ion movements do also spectra taken at 100 ms after the flash. (B) Comparison between WT and PAL participate in modifying the membrane permeability (35), but their mutant at 5 ms after a flash. regulation remains largely unknown. On the contrary, the activity

Viola et al. PNAS Latest Articles | 3of7 Downloaded by guest on October 2, 2021 S. elongatus used to demonstrate that a permanent transthylakoid elec- ACFast phase Slow phase

).u.r(ISP/noitarap 1 Flash trochemical potential is maintained in the dark by the re-

).u.r(SC 1.2 2.0 2 Flashes versed activity of the ATP synthase, which hydrolyses the ATP 1.0 produced in the mitochondria and imported in the chloroplast 1.5 0.8 (16, 17, 39). When inhibiting mitochondrial respiration, the E

y 0.6

b transthylakoid electrochemical proton gradient decreases in

1.0 I

S 0.4 darkness and the ATP synthase switches to a partially inactive Pnep e

s 0.5 0.2 S. elongatus form. Because of the lower value of the electrochemical proton egrahC Synechocystis gradient in the dark, its subsequent increase during a saturating O 0.0 0.0 0501002000 4000 pulse of light is larger (16). In cyanobacteria, the thylakoid 0500 ms Time interval (µs) membranes house both the respiratory and the photosynthetic chains. Therefore, one can expect that a high transthylakoid of protons is maintained in the dark by BDSynechocystis the ongoing respiration. Respiration can be suppressed by a pro-

).u.r(ISP/noitar 1 Flash 1.0 longed (24 h or more) incubation in complete darkness, resulting 2.0 )u 2 Flashes . r( in the depletion of respiratory substrates (5). The prolonged dark 0.8 SC incubation also causes the oxidation of the 1.5 EybIS 0.6 and, presumably, of the cytosol. We tested the effects of dark 1.0 starvation on the membrane potential decay in S. elongatus (rep-

apesegrahC 0.4

P resentative traces in Fig. 4, average of 3 biological replicates in SI

0.5 n e S. elongatus Appendix,Fig.S10A and B). In light-grown cells briefly dark-

pO 0.2 Synechocystis adapted, the flash-induced increase in electric field was dissi- 0.0 0.0 pated in about 50 ms (Fig. 4B), a time shorter than that observed 0 500 ms 0.0 0.2 0.4 0.6 0.8 1.0 in green algae (13), suggesting a higher ATP synthase/counter ion + Open PSI by P700 (r.u.) exchange activity, and therefore, a high transmembrane electric + field preexisting in darkness and reducing conditions. Dark-starved Fig. 3. ComparisonofECSandP700 methods to measure PSI photochemistry in S. elongatus and Synechocystis. Experiments were performed in the presence cells showed a marked decrease in the decay rate of the ECS of DCMU, HA, and PMS. (A and B) ECS signals (absorption difference between signal. The flash-induced decay kinetics increased again when dark- 500 and 485 nm) in S. elongatus and Synechocystis, respectively, measured after starved cells were reincubated in the light for 2 h (recovery). The a single flash and after 2 flashes fired at a 5-ms time interval and normalized to effect of dark starvation was thus reversible when a pool of re- those generated by the single flash, corresponding to 1 charge separation/PSI (3 spiratory substrates (and therefore of reductants) was regenerated biological replicates ±SD). The value of the second flash with respect to the by photosynthetic activity. We note that starvation in darkness also ± ± single one was 1.89 0.07 for S. elongatus and 1,93 0.04 for Synechocystis. increased the amplitude of the electric field generated during a (C) Fraction of PSI open centers as a function of the time after the first flash, measured as the ECS increase on a second flash (Methods). (D) Comparison pulse of saturating light (Fig. 4C), while concomitantly slowing between PSI open centers measured with ECS (C) or through kinetics of re- down its decay. This demonstrates that the transmembrane electric + duction of P700 , measured as the absorption difference at 435 nm. The dashed field in darkness had collapsed on starvation, and oxidizing condi- lines represent the linear fit of the experimental points. tions were established. Again, recovery in the light increased the ECS decay rate. We verified that dark starvation also modified the decay kinetics of Synechocystis (SI Appendix,Fig.S10D). Addition of of the ATP synthase is regulated at various levels, one being the the ATP synthase inhibitor venturicidin to dark-starved cells further redox state of the thioredoxin system, which senses the redox state slowed down the ECS decay, whereas the subsequent addition of of the /cytosol. Oxidizing conditions thus result in the in- the CCCP reaccelerated it (Fig. 4D). This indicates that activation of the ATP synthase (36, 37). The ATP synthase is also dark starvation of S. elongatus leads to a partial down-regulation of regulated by the electrochemical proton gradient itself: when it the ATP synthase activity, but not to its complete inactivation. In stands below a “critical” threshold, the ATP synthase is partially dark-starved cells of S. elongatus treated with venturicidin, the ECS inactivated (38). In green algae, diatoms and plants, this has been increase generated by applying 100-μs light pulses given at short

ABCDControl Light Light Dark DCMU+HA Dark Dark Dark+Vent 2000 Dark+Rec Dark+Vent+CCCP ) Dark+Rec 3 ) . 2000 . u u .r . mn mn084-305

r( 3 PSI+PSII ( la 1500 la 0 i i 84-30 t 1500 2 tn net e PSI to 2 1000 o 5 p 1000 penarb 6 6 enarbmeM 01x 01x 1 1 500 500 I/I I/I m eM 0 0 0 0 0255075 0 100 200 0 100 200 300 0 100 200 300 ms ms ms ms

Fig. 4. ECS kinetics in light-grown and dark-starved S. elongatus cells. (A) Representative kinetics of flash-induced ECS signals (absorption difference between 503 and 480 nm) in S. elongatus cells without (Control) and with the addition of PSII inhibitors (DCMU and HA). (B) Representative kinetics of flash-induced ECS signals measured in light-grown cells (Light), after 24 h of dark starvation (Dark) and in dark-starved cells after 2 h of recovery in the light (Dark+Rec). (C) Membrane potential kinetics measured with ECS (normalized on the single-turnover flash) during saturating light pulses in the same samples as in B. The pulse duration was adjusted to reach the maximal potential levels; the time 0 is at the pulse offset. (D) Membrane potential kinetics measured with ECS (normalized on the single- turnover flash) during saturating light pulses in dark-starved cells (same as C) and in dark-starved cells in presence of venturicidin without or with the further addition of the uncoupler CCCP.

4of7 | www.pnas.org/cgi/doi/10.1073/pnas.1913099116 Viola et al. Downloaded by guest on October 2, 2021 time intervals could be integrated in a linear manner, owing to its A S. elongatus B Synechocystis very slow decay (SI Appendix,Fig.S10C), further demonstrating 1 1 DCMU+HA 1 1 DCMU+HA that the ECS (measured as the difference between 500 to 505 and ) 2 2 DCMU+HA )IS 2 2 DCMU+HA ISP 480 to 485 nm absorption changes) depends linearly upon the 3 3 DCMU+HA 3 3 DCMU+HA P/ /s 25 35 s/

electric field. It is of note that the integration properties and / e e(e

( 30 the opposite effects of ATP synthase inhibitors and ionophores on 20 et tarl 25 the decay phase further confirmed that we measure a bona fide arla 15 20 ECS signal that probes the actual electric field across the thylakoid a cim membranes. Clearly, we cannot exclude that membrane perme- cimehcotohP 10 15

e 10 ability changes induced by dark starvation also depend on 5 hc 5 o changes in counter ion exchanges. Since it provides a direct tohP 0 0 measurement of the transthylakoid potential, ECS is ideally 0 200 400 600 0 200 400 600 suited to study further the in vivo activity of ion channels in Light intensity (µmol photons/m2/s) Light intensity (µmol photons/m2/s) thylakoid membranes of cyanobacteria. Fig. 5. Total (solid symbols) and PSII-independent (in presence of DCMU and ECS-Based Measurements of Steady-State Electron Flows in S. HA, open symbols) electron flow capacities in S. elongatus (A)andSynecho- elongatus and Synechocystis. In chloroplasts, Cyclic Electron cystis (B) cells. Electron flows were measured at increasing intensities of con- Flow (CEF) around PSI is considered a major way to generate a stant light using the DIRK(ECS) method. Rates are normalized on the single- turnover flash in presence of DCMU and HA (i.e., per PSI) and correspond to proton motive force without the net production of NADPH and, 3 independent biological replicates. therefore, to ensure the correct ATP:NADPH ratio for carbon assimilation, which cannot be provided by the sole Linear Electron Flow (LEF) (40, 41). This constraint may be different in cyano- segregation of NDH complexes may contribute to the low level of bacteria, where the interconnection of the photosynthetic and re- PSII-independent EF that we measured. spiratory electron transport pathways and other alternative electron fluxes (3, 42) could balance the production ratio of Conclusions ATP and NADPH. Here, instead of using the concepts of “linear” We have shown that ECS signals, likely corresponding to carot- and “cyclic” electron flows, we rather distinguished between total enoid band shifts originating from the PSI cores, are present in

and PSII-independent electron flows. We investigated the steady- both S. elongatus and Synechocystis. These ECS signals respond PLANT BIOLOGY state Total Electron Flow (TEF, when both photosystems are ac- linearly to the electric field and can be best measured in these tive) and the PSII-independent Electron Flow (PSII-independent cyanobacteria in the 480- to 505-nm region. Even though the ab- EF, in presence of DCMU and HA) rates in S. elongatus and sorption changes in this region include other contributions, the Synechocystis. We used the ECS signals to perform Dark Interval difference between absorption changes at 500 to 505 nm and 480 to Relaxation Kinetics (DIRK[ECS]) measurements (15), as detailed 485 nm provide measures of ECS cleared from those contributions. in the SI Appendix, Supplementary Materials and Methods.Sta- The ECS signals measured this way display classical flash-induced tionary photochemical rates were established by adapting the cells kinetics: a fast rise reflecting photosystem activity (sensitive to in- for several minutes to increasing intensities of constant actinic hibitors of PSII), a slower rise dependent on cytochrome b6f activity light. Fig. 5 A and B show the dependency on the actinic light (sensitive to PMS and DBMIB), and a decay phase reflecting the intensity of the electron flows measured in the 2 cyanobacteria. In electric permeability of the membrane (sensitive to uncouplers and both cases, the TEF rates showed a dependency on light intensity ATPase inhibitors). − ∼ μ that saturated at 20 to 35 e /s/PSI (light intensity of 350 mol Although an extensive investigation of the physiology of the photons/m2/s), depending on the experiment. On the contrary, the − various electron transport pathways is beyond the scope of the PSII-independent EF did not exceed 2 to 7 e /s/PSI at any light present study, we showed here that a direct measure of the electric intensity. Accordingly, the PSII and PSI yields measured in the field resulting from the photosynthetic activity can provide unique same conditions as the DIRK(ECS) also showed a strong de- information to understand cyanobacterial bioenergetics. Through pendency on light intensity in the absence of inhibitors, with the the development of oxygenic photosynthesis, cyanobacteria greatly PSI yields collapsing in the presence of DCMU and HA, which is influenced the evolution of life on Earth and are organisms of high consistent with the low PSII-independent EF (SI Appendix,Fig. ecological and biotechnological relevance. For these reasons, the S11). It is of note that these slow PSI turnover rates in absence of development of ECS-based physiological measurements should be PSII electron donation were not due to a strong derivation of instrumental in a better understanding of the complex reductants toward highly active cyanobacterial respiratory oxi- of these organisms. dases, since their inhibition with KCN did not cause an increase in the PSII-independent EF rates measured (SI Appendix,Fig.S12). Materials and Methods This low electron flow, similar to what is observed in the same Cyanobacteria Cultures. Synechococcus elongatus PCC7942 and Synechocystis experimental conditions in eukaryotic photosynthesis (43), does sp. PCC6803 (respectively S. elongatus and Synechocystis in the following) not qualify cyanobacterial NDH, despite its high affinity for re- strains were kindly provided by Diana Kirilovsky, CNRS, Commissariat à duced ferredoxin presumably produced by PSI (44), as a PSII l’Énergie Atomique et aux Énergies Alternatives, Paris-Saclay, France. bypassing specifically dedicated to cyclic electron flow in The Synechocystis PAL mutant was kindly provided by Ghada Ajlani, prokaryotic photosynthesis. CNRS, Commissariat à l’Énergie Atomique et aux Énergies Alternatives, Our observation is consistent with a previous report of low Paris-Saclay, France; the Synechocystis PSII- mutant was generated and PSI turnover rates measured in cyanobacteria in the presence characterized as described in the SI Appendix. Cells were grown under + white light (30 to 35 μmol photons/m2/s) in BG11 medium (supplemented of DCMU through P700 absorption spectroscopy (45). We note, however, that much higher rates, attributed to CEF, have been with 5 mM glucose in the case of the PAL and PSII- mutants) with constant shaking. reported in chloroplasts in transient situations (14), and more sustained PSI-only electron flow may occur when PSII is active. In Inhibitors. The 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,5- addition, in S. elongatus, DCMU was reported to prevent an even dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB), and carbonyl cya- distribution of the NDH-1 complex in the thylakoid membranes on nide m-chlorophenyl hydrazone (CCCP) were dissolved in ethanol; HA, moderate illumination, maintaining it aggregated in distinct spots 5-methylphenazinium methyl sulfate (PMS), venturicidin, and potassium as in the dark (46). Thus, one should also consider that a spatial cyanide (KCN) were dissolved in water. Final concentrations used: 20 μM

Viola et al. PNAS Latest Articles | 5of7 Downloaded by guest on October 2, 2021 DCMU, 500 μMHA,40μM venturicidin, 100 μM CCCP, 15 μMPMS,40μM centers that reopened between the 2 flashes. This value was normalized by the DBMIB, 50 μMKCN. maximal value obtained when the 2 actinic flashes were separated by 5 ms. A LED-based spectrophotometer (JTS10, Biologic, France) was used for Spectroscopy Measurements. Two experimental setups were used in this work. measurements of fluorescence and absorbance kinetics. In this setup, in the Full details on the light sources and filters used are given in the SI Appendix. case of absorption changes, all measurements were performed in the pres- A homemade Optical Parametric Oscillator-based spectrophotometer (47) ence and absence (measuring light only) of actinic illumination on each bi- having a time resolution of 10 ns was used for fast (<50 μs) measurements ological sample. The absorption changes induced by the measuring light and for spectra recording in the 410 to 570 nm region. Cells were injected in 2 alone (actinic effects or changes in the LED output intensity due to heating) closed horizontal cuvettes: 1 used on the reference and 1 on the measuring were then subtracted from those recorded in presence of actinic illumination. photodiodes. In this setup, the measuring beam is split between the 2 cuvettes, Representative traces are shown in SI Appendix, Fig. S1. For single-turnover whereas the actinic light illuminates only 1 of the 2. The absorption changes flash and saturating light pulse measurements, cells were injected in a closed induced by the measuring light alone are automatically subtracted from those horizontal cuvette in the presence of the specified inhibitors and manually measured with actinic illumination to correct for actinic effects of the mea- reoxygenated every few minutes. For longer measurements in continuous suring light. The cells were incubated in the cuvettes in presence of specified light, cell samples were deposited in the cavity of a horizontal cuvette over- inhibitors in the dark for 30 min before the experiments, in anaerobic condi- laid with a semipermeable membrane separating them from a second cavity, tions. The spectra were recorded back and forth from 570 nm to 410 nm, and where a constant flow of air was pumped. PSII and PSI photosynthetic yields the back and forth recordings were averaged to correct for sedimentation or were calculated as in refs. 7 and 8, respectively. Photochemical rates mea- physiological changes occurring during the experiment. A total of 8 actinic surements were performed using DIRK(ECS), as described in the SI Appendix. flashes were integrated for each wavelength. For the measurement of open PSI (Fig. 3) at time t after a saturating flash, the fraction of reduced P was 700 ACKNOWLEDGMENTS. This work was supported by UMR7141, CNRS/ measured as the difference between the ΔI/I435 nm at time t and the one at Sorbonne Université and by the Agence Nationale de la Recherche Δ 5 ms, normalized by the difference between the I/I 435 nm at 200 ns (fully (ReCyFuel: ANR-16-CE05-0026-01). B.B. also acknowledges the support by oxidized PSI) and the one at 5 ms. In parallel, we measured the ECS increase the European Research Council (ERC) PhotoPHYTOMIX project (grant agree- (difference in ΔI/I at 500 and 485 nm) 5 ms after 2 single turnover flashes ment No. 715579). We thank Diana Kirilovsky and Pierre Sétif for fruitful separated by a time interval t. The difference between the ECS increase after 2 discussions and Stefano Santabarbara for his critical reading of the manu- flashes and the one after a single flash is equal to the contribution to ECS of PSI script. We thank Ghada Ajlani for the Synechocystis PAL mutant.

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