N Rea
email: rea@ieec.uab.es.

P Esposito

R Turolla

G L Israel

S Zane

L Stella

S Mereghetti

A Tiengo

D Götz

E Gögüs

C Kouveliotou

A low-magnetic-field Soft Gamma Repeater

Soft gamma repeaters and anomalous x-ray pulsars form a rapidly increasing group of x-ray sources exhibiting sporadic emission of short bursts. They are believed to be magnetars, i.e. neutron stars powered by extreme magnetic fields, B ∼ 10 14 -10 15 Gauss. We report on a soft gamma repeater with low magnetic field, SGR 0418+5729, recently detected after it emitted bursts similar to those of magnetars. X-ray observations show that its dipolar magnetic field cannot be greater than 7.5 × 10 12 Gauss, well in the range of ordinary radio pulsars, implying that a high surface dipolar magnetic field is not necessarily required for magnetar-like activity. The magnetar population may thus

include objects with a wider range of B-field strengths, ages and evolutionary stages than observed so far.

Magnetized, isolated rotating neutron stars are often detected as pulsating sources in the radio and x-ray bands, hence the name pulsars. Pulsars slow down with time as their rotational energy is lost via magnetic dipole radiation. The surface dipolar magnetic field (B) of a pulsar can be estimated using its spin period, P , and spin-down rate, Ṗ , as follows:

B = (3 I c 3 Ṗ P/8π 2 R 6 ) 1/2 ∼ 3.2 × 10 19 (P Ṗ ) 1/2 Gauss ( 1
)
where P is in seconds, Ṗ in seconds/second, and we assumed R ∼ 10 6 cm and I ∼ 10 45 g cm 2 , which are the neutron star radius and moment of inertia, respectively.

Although this expression was developed to estimate the magnetic fields of radio pulsars, usually ∼ 10 12 Gauss, it has been traditionally used also for magnetars, where the derived values of B reach ∼ 10 15 Gauss (1). To date only ∼ 16 of these ultra-magnetized neutron stars have been observed (5,6); their population includes soft gamma repeaters (SGRs) and anomalous x-ray pulsars (AXPs). All known magnetars are x-ray pulsars with luminosities of L X ∼ 10 32 -10 36 erg s -1 , usually much higher than the rate at which the star loses its rotational energy through spin-down. Their high luminosities together with the lack of evidence for accretion from a stellar companion (7,8), led to the conclusion that the energy reservoir fueling the SGR/AXP activity is their extreme magnetic field (3,9). Observationally, magnetars are characterized by stochastic outbursts (lasting from days to years) during which they emit very short x/γ-ray bursts; they have rotational periods in a narrow range (2-12 s) and, compared to other isolated neutron stars, large period derivatives of ∼ 10 -13 -10 -10 ß. Their large dipolar B-fields and relatively young characteristic ages are estimated to be over ∼ 5 × 10 13 Gauss, and t c = P/2 Ṗ ∼0.2 kyr -0.2 Myr [see (5) for a review].

In addition to the canonical SGRs and AXPs, two other sources are known to show magnetarlike activity: PSR J1846-0258 (10,11) and PSR 1622-4950 (12). The former is a 0.3 s, allegedly rotation-powered, x-ray pulsar, with a magnetic field of B ∼ 4.8 × 10 13 Gauss (in the lower end of the magnetar range), from which a typical magnetar outburst and short x-ray bursts were detected. In the latter, flaring radio emission with a rather flat spectrum (similar to those observed in the two transient radio magnetars; (13,14)) was detected from a 4.3 s radio pulsar with a magnetic field in the magnetar range (B ∼ 3 × 10 14 Gauss).

In all sources with magnetar-like activity, the dipolar field spans 5 × 10 13 G < B < 2 × 10 15 G, which is ∼ 10-1000 times the average value in radio pulsars and higher than the electron quantum field, B Q = m 2 e c 3 /eh ∼ 4.4×10 13 Gauss. The existence of radio pulsars with B > B Q and showing only normal behavior ( 15) is an indication that a magnetic field larger than the quantum electron field alone may not be a sufficient condition for the onset of magnetar-like activity. In contrast, so far the opposite always held: magnetar-like activity was observed only in sources with dipolar magnetic fields stronger than B Q .

SGR 0418+5729 was discovered on 5 June 2009 when the Fermi Gamma-ray Burst Monitor (GBM) observed two magnetar-like bursts (16). Follow-up observations with several x-ray satellites show that it has x-ray pulsations at ∼9.1 s, well within the range of periods of magnetar sources (17,18). Further studies show that SGR 0418+5729 exhibits all the typical characteristics of a magnetar: i) emission of short x-ray bursts, ii) enhanced persistent flux, iii) slow pulsations with a variable pulse profile, and iv) a x-ray spectrum characterized by a thermal plus non-thermal component, which softened as the outburst decayed.

What made this source distinctly different was the failure of detecting a period derivative in the first 160 days after the outburst onset, despite frequent observational coverage. Several

x-ray satellites (18) monitored the source almost weekly since its detection. This extensive observational campaign allowed the determination of an accurate ephemeris for the pulsar rota-tional period, but no sign of a spin down was detected. In the first 160 days after the outburst onset, the upper limit on the period derivative was 10 -13 ß (90% confidence level), which, according to equation ( 1), translates into a surface dipolar magnetic field B < 3 × 10 13 Gauss (18). This limit is quite low for a magnetar source, but not abnormally so, given the detection of a comparable magnetic field in the magnetar-like PSR J1846-0258 (10), or the case of AXP 1E 2259+586 with B ∼ 6 × 10 13 Gauss (19).

SGR 0418+5729 could not be monitored for a while after the first 160 days, because the Sun became too close to its position in the sky. On 2010 July 9th, soon after it became observable again, we started an extensive monitoring of the source with the Swift, Chandra and XMM-Newton X-ray satellites (Tab. S1). In particular, on 2010 July 23rd, we detected it with the Advanced CCD Imaging Spectrometer (ACIS) onboard Chandra at a flux of (1.2 ± 0.1) × 10 -13 erg s -1 cm -2 (0.5-10 keV), more than one order of magnitude fainter than in the previous available observation (18). The spectrum is well fit by an absorbed blackbody with a line of sight absorption N H = (1.5 ± 1.0) × 10 21 cm -2 and kT = 0.67 ± 0.11 keV (all quoted errors are at 90% confidence level). Pulsations were also clearly detected at the known magnetar period. On 2010 September 24th, we observed SGR 0418+5729 with the European Photon Imaging Camera (EPIC) onboard XMM-Newton, which detected it at a comparable flux, and could measure again the rotational period of the neutron star. We used our new Swift, Chandra and XMM-Newton observations, together with several other observations (Tab. S1) and phase-connected all the source data from 2009 June 5th till 2010 September 24th (Fig. 1 and Supporting on-line Material). We found a best fit period of 9.07838827(4) s referred to TJD (Truncated Julian Day) 14993.0 and to the Solar System barycenter. The phase evolution of SGR 0418+5729 is well described by a linear relation φ = φ 0 + 2π(t -t 0 )/P , and a quadratic term -2π Ṗ (t -t 0 ) 2 /2P 2 (which reflects the presence of a spin down), is not statistically required. This implies an upper limit on the period derivative of SGR 0418+5729 of Ṗ < 6.0 × 10 -15 ß (90% confidence level). This value is the smallest of all known SGRs/AXPs, of the two magnetar-like pulsars PSR J1846-0258 and PSR 1622-4950, and of the X-ray Dim Isolated Neutron Stars (XDINSs; 20) for which a measure of Ṗ is available (Fig. 2).

The corresponding limit on the surface dipolar magnetic field of SGR 0418+5729 is B < 7.5 × 10 12 Gauss, making it the magnetar with the lowest surface dipolar magnetic field yet.

The upper limit on the period derivative implies a characteristic age of the source t c > 24 Myr.

Despite the characteristic age is known to overestimate the true age of a neutron star in which magnetic field decay occurred (25), as is likely the case of SGR 0418+5729, the rather high Galactic latitude (b = 5.1 deg) and its position on the P -Ṗ plane [close to the death line for radio emission (26,27)], suggest that this system is quite older than the other SGRs/AXPs.

The existence of magnetar-like sources with low values of B has several consequences.

Among isolated pulsars, which are presumably rotation-powered, ∼18% have a dipolar magnetic field higher than the upper limit we derived for SGR 0418+5729 (Fig. 2). The discovery of PSR 1622-4950 (12) on the other hand, suggests that magnetar-like behavior may manifest itself mostly in the radio band. In this framework, our result indicates that a large number of apparently normal pulsars might turn on as magnetars at anytime, regardless of having a surface dipole magnetic field above the quantum limit or not. As a direct consequence, magnetar-like activity may occur in pulsars with a very wide range of magnetic fields and it may fill a continuum in the P -Ṗ diagram (Fig. 2).

So far we have been considering the relationship between the surface dipolar magnetic field and magnetar-like activity. However, it is likely that the magnetar activity is driven by the magnetic energy stored in the internal toroidal field (3,4); this component cannot be measured directly. If the magnetar model as it is currently understood is indeed valid, despite its low surface dipolar field, SGR 0418+5729 is expected to harbor a sufficiently intense internal toroidal component B tor in order to be able to undergo outbursts and emit bursts. This large internal field can stress the crust and ultimately deforms/cracks the star surface layers, periodically allowing magnetic helicity to be transferred to the external field, thus causing the (repeated) short x-ray bursts and the overall magnetar-like activity (3,[START_REF] Thompson | [END_REF]22).

As with other magnetars, B tor can be estimated assuming that the magnetic energy stored in the internal toroidal field powers the quiescent emission of SGR 0418+5729 during its entire lifetime, B 2 tor ∼ 6L X t c /R 3 NS (3). Assuming a source distance of 2 kpc (16,18), and that the current luminosity L X ∼ 6.2 × 10 31 erg s -1 (the lowest measured so far for this source) corresponds to the quiescent luminosity, we obtain B tor ∼ 5 × 10 14 Gauss for a neutron star radius of R NS = 10 6 cm and a source characteristic age of t c ∼ 24 Myr. A value of the same order is obtained if the ratio of the toroidal to poloidal field strength is ∼ 50, as in the magneto-thermal evolution scenario (23,24). In this picture, SGR 0418+5729 may possess a high enough internal magnetic field to overcome the crustal yield and give rise to magnetar-like activity despite its low surface dipolar magnetic field. However, would the actual measurement of the surface dipolar B-field of SGR 0418+5729 turn out to be much smaller than the present upper limit, this may require to rethink some of the ingredients at the basis of the magnetar scenario.

SGR 0418+5729 may represent the tip of the iceberg of a large population of old and lowdipolar-field magnetars that are dissipating the last bits of their internal magnetic energy (29).

Indeed, a large fraction of the radio pulsar population may have magnetar-like internal fields not reflected in their normal dipolar component. The blue solid line marks the 90% upper limit for the dipolar magnetic field of SGR 0418+5729.

The value of the electron quantum magnetic field is also reported (dash-dotted grey line).

Fig. 1 . 12 Fig. 2 .

 1122 Fig. 1. Top panel: rotation phase versus time for the coherent timing solution for SGR 0418+5729 obtained using data taken with Rossi X-ray Timing Explorer (black circles), Swift (red triangles) XMM-Newton (blue squares), and Chandra (green stars). The solid line shows the best-fitting linear function (χ 2 = 1.8 for 18 degrees of freedom; root mean square ∼ 3%). Bottom panel: fit residuals.


Stefania Viola

Julien Sellés

Benjamin Bailleul

Pierre Joliot

Francis-André Wollman
email: wollman@ibpc.fr

In vivo electron donation from plastocyanin and cytochrome c 6 to PSI in Synechocystis sp. PCC6803

Keywords: Cyanobacteria, Photosystem I, Plastocyanin, Cytochrome c6, Respiration

 Both plastocyanin and cytochrome c6 form a pre-complex with photosystem I in Synechocystis  Fast electron donation to PSI occurs from both plastocyanin and cytochrome c6  The extent of formation of the pre-complex in the dark is not affected by respiration  Electron partitioning between respiration and photosynthesis is regulated in a similar way in presence of either of the two electron donors

Abstract

Many cyanobacteria species can use both plastocyanin and cytochrome c6 as lumenal electron carriers to shuttle electrons from the cytochrome b6f to either photosystem I or the respiratory cytochrome c oxidase. In Synechocystis sp. PCC6803 placed in darkness, about 60% of the active PSI centres are bound to a reduced electron donor which is responsible for the fast rereduction of P700 in vivo after a single charge separation. Here, we show that both cytochrome c6 and plastocyanin can bind to PSI in the dark and participate to the fast phase of P700 reduction, but the fraction of pre-bound PSI is smaller in the case of cytochrome c6 than with plastocyanin. Because of the inter-connection of respiration and photosynthesis in cyanobacteria, the inhibition of the cytochrome c oxidase results in the over-reduction of the photosynthetic electron transfer chain in the dark that translates into a lag in the kinetics of P700 oxidation at the onset of light. We show that this is true both with plastocyanin and cytochrome c6, indicating that the partitioning of electron transport between respiration and photosynthesis is regulated in the same way independently of which of the two lumenal electron carriers is present, although the mechanisms of such regulation are yet to be understood.

1-Introduction

In oxygenic photosynthesis, electrons extracted from water are transported from the photosystem II (PSII) donor side (thylakoid lumen) to the photosystem I (PSI) acceptor side (stroma / cytoplasm) via a third transmembrane complex, the cytochrome b6f (cyt b6f). Cyt b6f takes electrons from the membrane-soluble plastoquinone pool and passes them to an electron acceptor soluble in the lumenal space, which in turn reduces P700, the PSI chlorophyll that gets stably oxidised after charge separation. In higher plants this soluble electron carrier is, in most cases, a copper-containing protein named plastocyanin, whereas a c-type cytochrome named cytochrome c6 (cyt c6) can replace plastocyanin as a lumenal electron donor to PSI in many algae and cyanobacteria [START_REF] Díaz-Quintana | A comparative structural and functional analysis of cyanobacterial plastocyanin and cytochrome c6 as alternative electron donors to Photosystem I: Photosystem I reduction in cyanobacteria[END_REF]. It should be noted that cytochrome c6 is present also in plants, but its functional equivalence to plastocyanin has not yet been demonstrated [START_REF] Wastl | Higher plants contain a modified cytochrome c6[END_REF][START_REF] Gupta | Functional relationship of cytochrome c6 and plastocyanin in Arabidopsis[END_REF]. Both plastocyanin and cyt c6 are small (ca. 10 kDa) monomeric proteins with a redox potential of about +350 mV at pH 7. This redox potential is close to the one of cytochrome f (4), the cyt b6f subunit from which they accept electrons, but it is lower than the one of P700, that ranges between +400 and +470 mV depending on the organism [START_REF] Sétif | Chapter 14. Functional pattern of Photosystem I in oxygen evolving organisms[END_REF][START_REF] Nakamura | Species dependence of the redox potential of the primary electron donor P700 in photosystem I of oxygenic photosynthetic organisms revealed by spectroelectrochemistry[END_REF]. If plastocyanin from plants and algae are acidic proteins, plastocyanin and cytochrome c6 can have widely different isoelectric point values in cyanobacteria [START_REF] Díaz-Quintana | A comparative structural and functional analysis of cyanobacterial plastocyanin and cytochrome c6 as alternative electron donors to Photosystem I: Photosystem I reduction in cyanobacteria[END_REF].

The electron transfer kinetics between plastocyanin / cyt c6 and PSI have been investigated extensively in eukaryotes. In vitro studies have shown the existence, after a saturating light flash, of a fast (t½ of few μs) electron transfer phase responsible for the reduction of P700 in ~30-40% of isolated PSI particles from both plants [START_REF] Bottin | Interaction of plastocyanin with the photosystem I reaction center: a kinetic study by flash absorption spectroscopy[END_REF] and green algae [START_REF] Hippler | Fast electron transfer from cytochrome c6 and plastocyanin to photosystem I of Chlamydomonas reinhardtii requires PsaF[END_REF]. Such a fast phase also has been measured in intact plant chloroplasts and green algae cells [START_REF] Bottin | Interaction of plastocyanin with the photosystem I reaction center: a kinetic study by flash absorption spectroscopy[END_REF][START_REF] Haehnel | Electron transfer from plastocyanin to P700. Function of a subunit of photosystem I reaction center[END_REF][START_REF] Delosme | Electron transfer from cytochrome f to photosystem I in green algae[END_REF]. Thus, P700 re-reduction kinetics are bi-phasic, with the fast phase pointing to the fraction of PSI centres to which is bound, in the dark prior to the light flash, a reduced electron donor. The slower phase corresponds to the diffusion time of plastocyanin / cyt c6 in the lumenal space to PSI centres that do not have any pre-associated electron donor. In the case of the eukaryotic PSI, the PsaF subunit has been shown to carry the binding site for the transient electron carrier-PSI complex [START_REF] Hippler | Fast electron transfer from cytochrome c6 and plastocyanin to photosystem I of Chlamydomonas reinhardtii requires PsaF[END_REF][START_REF] Farah | Isolation of a psaFdeficient mutant of Chlamydomonas reinhardtii: efficient interaction of plastocyanin with the photosystem I reaction center is mediated by the PsaF subunit[END_REF], with a crucial role played by its lumenal N-terminal extension, rich in Lysine residues. These positive residues would electrostatically interact with the acidic surface of the electron donor, thus providing a high affinity binding site. In a Chlamydomonas reinhardtii mutant carrying a deletion of this N-terminal extension, the P700 reduction kinetics proved to be slower than in the wild type [START_REF] Hippler | The N-terminal domain of PsaF: Precise recognition site for binding and fast electron transfer from cytochrome c6 and plastocyanin to photosystem I of Chlamydomonas reinhardtii[END_REF]. In contrast to the chloroplast-located PsaF, the PsaF subunit of cyanobacterial PSI lacks the Lysin-rich lumenal extension. Together with the observation that some species have basic plastocyanin or cyt c6 isoforms, this sequence change in the cyanobacterial version of PsaF suggests that electrostatic interactions might not be the main factor determining their interaction with PSI in cyanobacteria. Rather, the presence of conserved Tryptophan residues on the lumenal side of the PsaA and PsaB PSI core subunits in both cyanobacteria [START_REF] Sun | Oxidizing side of the cyanobacterial photosystem I. Evidence for interaction between the electron donor proteins and a luminal surface helix of the PsaB subunit[END_REF] and algae [START_REF] Sommer | The luminal helix l of PsaB is essential for recognition of plastocyanin or cytochrome c6 and fast electron transfer to photosystem I in Chlamydomonas reinhardtii[END_REF][START_REF] Sommer | The hydrophobic recognition site formed by residues PsaA-Trp651 and PsaB-Trp627 of Photosystem I in Chlamydomonas reinhardtii confers distinct selectivity for binding of plastocyanin and cytochrome c6[END_REF] suggests an hydrophobic interaction mechanism, since these residues were shown to be pivotal for the binding of plastocyanin and cyt c6,.

The existence of a fast phase of electron transfer to PSI in cyanobacteria has been a matter of debate. In vitro studies using Synechocystis sp. PCC6803, showed that electron donation from either plastocyanin or cyt c6 to the endogenous PSI displays monophasic kinetics, with a halftime intermediate between those from the two phases described in chloroplasts [START_REF] Hervás | Laser flash kinetic analysis of Synechocystis PCC 6803 cytochrome c6 and plastocyanin oxidation by Photosystem I[END_REF]. Consistent with these data, plastocyanin and cyt c6 from Anabaena sp. PCC7119, as well as plastocyanin isolated from Synechocystis sp. PCC6803, cannot quickly donate electrons to spinach PSI.

However the reduction of P700 in PSI particles from Anabaena displays a fast phase with cyt c6, but not with plastocyanin, from the same organism [START_REF] Hervás | Laser-flash kinetic analysis of the fast electron transfer from plastocyanin and cytochrome c6 to photosystem I. Experimental evidence on the evolution of the reaction mechanism[END_REF]. P700 reduction kinetics comprising a fast phase also have been observed in vivo in conditions where only cyt c6 is used as PSI electron donor: this is the case in the cyanobacteria Plectonema boryanum [START_REF] Hiyama | Laser-induced reactions of P700 and cytochrome f in a bluegreen alga, Plectonema boryanum[END_REF], in a thermophilic Synechococcus sp. [START_REF] Nanba | Reaction kinetics of P-700, cytochrome c-553 and cytochrome f in the cyanobacterium, Synechococcus sp[END_REF][START_REF] Nanba | Electron transport from cytochrome b6f complexes to Photosystem I reaction center complexes in Synechococcus sp. Is cytochrome c-553 a mobile electron carrier?[END_REF] and in Synechococcus sp. PCC 7002 [START_REF] Baymann | Rapid electron transfer to photosystem I and unusual spectral features of cytochrome c6 in Synechococcus sp. PCC 7002 in vivo[END_REF]. Recently we have shown the existence in vivo of such a fast phase in Synechococcus elongatus sp. PCC7942 and Synechocystis sp. PCC6803 [START_REF] Viola | Probing the electric field across thylakoid membranes in cyanobacteria[END_REF], grown in BG11 medium containing 0,5 µM Cu, in conditions which favour expression of plastocyanin [START_REF] Zhang | Copper-mediated regulation of cytochrome c553 and plastocyanin in the cyanobacterium Synechocystis 6803[END_REF][START_REF] Clarke | Inactivation of the petE gene for plastocyanin lowers photosynthetic capacity and exacerbates chilling-induced photoinhibition in the cyanobacterium Synechococcus[END_REF]. In these cyanobacteria, which harbour both the genes coding for plastocyanin and cyt c6, the expression of the two electron donors is indeed regulated according to the availability of the metals used as cofactors, iron and copper. Using Synechocystis sp. PCC6803 wild type grown in presence of different metal concentrations and in mutant strains for either plastocyanin or cyt c6, the two proteins have been shown to be functional electron carriers in both the photosynthetic and the respiratory chains [START_REF] Zhang | Copper-mediated regulation of cytochrome c553 and plastocyanin in the cyanobacterium Synechocystis 6803[END_REF][START_REF] Durán | The efficient functioning of photosynthesis and respiration in Synechocystis sp. PCC 6803 strictly requires the presence of either cytochrome c6 or plastocyanin[END_REF][START_REF] Ardelean | Unexpected changes in photosystem I function in a cytochrome c6-deficient mutant of the cyanobacterium Synechocystis PCC 6803[END_REF], which are located and interconnected in the same cyanobacterial thylakoids (reviewed in [START_REF] Clarke | Inactivation of the petE gene for plastocyanin lowers photosynthetic capacity and exacerbates chilling-induced photoinhibition in the cyanobacterium Synechococcus[END_REF][START_REF] Durán | The efficient functioning of photosynthesis and respiration in Synechocystis sp. PCC 6803 strictly requires the presence of either cytochrome c6 or plastocyanin[END_REF]. In these organisms, once reduced by the cyt b6f, plastocyanin and cyt c6 can donate electrons to both PSI and Cytochrome c Oxidase (COX) [START_REF] Howitt | Quinol and cytochrome oxidases in the cyanobacterium Synechocystis sp. PCC 6803[END_REF][START_REF] Dj | Thylakoid terminal oxidases are essential for the cyanobacterium Synechocystis sp. PCC 6803 to survive rapidly changing light intensities[END_REF]. Cyanobacteria possess an aa3-type COX enzyme, using two haems and three copper atoms as cofactors [START_REF] Peschek | Characterization of cytochrome-c oxidase in isolated and purified plasma and thylakoid membranes from cyanobacteria[END_REF]. The most oxidising cofactors of COX (CuB, +412 mV at pH 6.5 in Paracoccus denitrificans [START_REF] Gorbikova | Redox titration of all electron carriers of cytochrome c oxidase by Fourier transform infrared spectroscopy[END_REF]) and of PSI (P700, between +400 and +470 mV) have similar redox potentials, providing similar driving forces for electron donation from plastocyanin and cyt c6. However, the determinants of the in vivo kinetic competition between the two pathways are still unknown, limiting our understanding of the competition between the respiratory and photosynthetic flows. Here, we present a detailed in vivo analysis of the electron donation kinetics from either plastocyanin or cyt c6 to PSI, in the wild type strain Synechocystis sp. PCC6803 (hereafter referred to as Synechocystis) and in a mutant devoid of plastocyanin and expressing cyt c6 instead. We also address the possible influence of the respiratory activity on such kinetics.

2-Materials and Methods

2.1-Cyanobacteria Cultures

Synechocystis sp. PCC6803 cells were grown under white light (30-35 µmol photons/m 2 /s) at 25°C in BG11 medium (containing 0.5 μM Cu and 20 μM Fe, pH 8.0) with constant shaking. For the generation of the Synechocystis ΔPC mutant, all DNA techniques such as plasmid isolation, restriction and ligation were performed according to standard protocols [START_REF] Sambrook | Molecular Cloning[END_REF]. All the enzymes used here were purchased from New England Biolabs. A 1405 bp long region encompassing the petE gene (SYNPCCP_2261) was amplified from Synechocystis sp. PCC6803 genomic DNA using the primers SacI-petE FW (5'-aatataGAGCTCAACCACCGTTATCAGCAGAAC, SacI restriction site underlined, nongenomic sequence in small letters) and EcoRI-petE RV (5'-aatataGAATTCCTACTCTATTTAACCATCTGTATATCGAGG, EcoRI restriction site underlined, non-genomic sequence in small letters). The fragment was gel-purified and digested with the restriction enzymes SacI and EcoRI and subsequently ligated into the plasmid pSBA2 [START_REF] Lagarde | Increased production of zeaxanthin and other pigments by application of genetic engineering techniques to Synechocystis sp. strain PCC 6803[END_REF] previously digested with the same enzymes. The plasmid was then linearized with the enzymes SmaI and HpaI and ligated with a blunt-ended kanamycin resistance cassette (Kan R ). The resulting plasmid in which part of the petE gene was replaced by the resistance cassette was sequenced and introduced into the wild-type Synechocystis strain, and transformants were selected on increasing concentrations of kanamycin. Completely segregated ΔPC mutant clones were obtained and verified by PCR using the primer pairs SacI-petE FW + petE del RV (5'-TTTAGACATACTTCTTGGCGATTG, specific for the wild-type allele) and SacI-petE FW + KanR RV (5'-ATGTAAGCCCACTGCAAGCTAC, specific for the Kan R -containing allele). The ΔPC mutant was maintained in presence of 100 µg/ml kanamycin. When used for measurements, cells were grown in absence of kanamycin, harvested by centrifugation in the mid exponential phase of growth, resuspended in their own supernatant to the appropriate concentration and incubated in the same conditions as for growth for at least one hour before starting the experiments.

2.2-Inhibitors and redox mediators

3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was dissolved in ethanol; hydroxylamine (HA), 5-Methylphenazinium methyl sulphate (PMS), potassium cyanide (KCN) and methyl viologen (MV) were dissolved in water; rotenone (Rot) was dissolved in DMSO. PSII inhibition was achieved using 20 µM DCMU and 500 µM HA. PMS was used at a final concentration of 15 µM, KCN at 50 µM, MV at 300 µM and Rot at 50 µM.

2.3-Whole-cell absorption spectra

These were determined with a Cary 300 UV-VIS spectrophotometer (Varian). Spectra were recorded from 400 to 750 nm and normalized to the absorption at 630 nm.

2.4-Protein analyses

All steps of protein extraction were performed at 4° C. Cells in exponential growth phase were harvested by centrifugation, washed once in ice-cold PBS buffer, re-suspended in 1/400 volumes of ice-cold PBS containing protease inhibitors (cOmplete EDTA-free, Roche) and mixed with 2/3 volumes of glass beads (425-600 µm, acid-washed, Sigma). Cells were broken by subsequent cycles of freezing/thawing and vortexing. Glass beads were sedimented by a brief slow-speed centrifugation and the total cell extract (T) recovered in the supernatant. Half volume of the total cell extract was centrifuged at 16000 rcf for 30 minutes and the supernatant was recovered as the soluble fraction (S), whereas the pellet, corresponding to the membrane fraction (M) was washed once in PBS and finally resuspended in the same PBS volume as the soluble fraction. The chlorophyll concentration of the whole cell extract and membrane fraction was calculated according to [START_REF] Porra | Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy[END_REF]. Whole cell extracts and membrane protein samples were adjusted to the same chlorophyll concentration, whereas soluble protein samples were adjusted on a volume basis (same as the corresponding membrane sample). Proteins were denatured by boiling in presence of 3% SDS, 100 mM DTT and 100 mM Na2CO3 and then loaded (3 µg Chl a per lane) on a 4-20% SDS-PAGE gel (TGX Mini gel, BioRad). After electrophoresis, proteins were transferred by semi-dry blotting on a nitrocellulose membrane (Amersham Protran 0.1 µm NC, GE Helthcare). For c-type haem detection, the membrane was rinsed in PBS and immediately incubated with ECL (Clarity Western ECL Blotting Substrate, BioRad). For immunodetection, the membrane was saturated with 5% milk proteins in PBS-T and immunodecorated with primary antibodies, diluted 10 000-fold (PsbA and plastocyanin, Agrisera), then revealed by horseradish peroxidase-conjugated antibodies against rabbit IgG (Promega). Signals were detected by ECL using the ChemiDoc Imager (BioRad).

2.5-Spectroscopy measurements

For all spectroscopic measurements, several experimental traces recorded on the same biological material were averaged until a satisfying signal-to-noise ratio was achieved. All the data shown in this work are therefore averages of several experimental traces, the number depending on the signal quality of each individual trace (typically around 10).

A home-made Optical Parametric Oscillator (OPO)-based spectrophotometer [START_REF] Joliot | A new high-sensitivity 10-ns time-resolution spectrophotometric technique adapted to in vivo analysis of the photosynthetic apparatus[END_REF] with a time resolution of 10 ns and a spectral resolution of 2 nm was used for the pump-probe and pumppump-probe measurements of section 3.2. Cells were injected in two closed horizontal cuvettes, one used as a reference (without actinic light) and the other used for measurement with actinic light. In this setup, detecting flashes are split between the two cuvettes and monitored carefully in order to avoid actinic effects, whereas the actinic light illuminates only one of the two. The cells were incubated in the cuvettes in presence of specified inhibitors in the dark for at least 30 minutes before the experiments, in anaerobic conditions. The actinic flashes were provided by one or two Nd:YAG lasers at 532 nm which pumped an OPO producing monochromatic saturating flashes at 690 nm. The absorption changes induced by these actinic flashes were sampled at discrete times by short detecting flashes generated by another OPO. The time delay between the laser delivering the actinic flashes and the laser delivering the detecting flashes was controlled by a digital delay/pulse generator (DG645, Stanford Research). The lightdetecting photodiodes were protected from transmitted and scattered actinic light and fluorescence of the samples by BG39 Schott (Mainz, Germany) filters. The pump-pump-probe measurements of PSI re-reduction kinetics were performed as described in [START_REF] Viola | Probing the electric field across thylakoid membranes in cyanobacteria[END_REF]. In this setup, the maximal P700 + levels were measured as the ΔI/I 435 nm at 200 ns after a single-turnover flash in presence of the PSII inhibitors DCMU and HA.

A LED-based spectrophotometer (JTS10, Biologic, France) was used for measurements of absorbance change kinetics in the >100 µs time range. Cells were injected in a closed horizontal cuvette in presence of the specified inhibitors and measurements were performed in presence and absence of actinic illumination on each biological sample. The absorption changes induced by the measuring light alone (actinic effects or changes in the LED output intensity due to heating) were then subtracted from those recorded in presence of actinic illumination. In this setup the cells were kept in aerobic conditions by manual re-oxygenation every few minutes. Actinic flashes were provided by a dye laser at 690 nm, whereas multi-turnover light pulses were provided by a red LED array. For measurements in the 540 -573 nm region, measuring pulses were provided by a converted green LED (Unicolor Converted Green OSTAR Projection Cube, OSRAM Opto Semiconductor) using appropriate interference filters (10 nm full width at half-maximum, Edmund Optics). P700 redox changes were measured through the difference of the absorbance changes at 705 and 730 nm using measuring pulses provided by a combination of red LEDs and appropriate interference filters. The light-detecting diodes were protected from scattered actinic light by a BG39 filter (Schott) for measurements in the 540 -573 nm region and by a RG695 (Schott) filter for P700 measurements. In this setup, the maximal P700 + levels were measured as the maximal ΔI/I 705 -730 nm obtained during a multi-turnover saturating pulse in presence of the PSII inhibitor DCMU and of methyl viologen.

3-Results

3.1-Generation and characterization of the Synechocystis ΔPC mutant

We generated the Synechocystis plastocyanin deficient strain by partially replacing the endogenous petE gene (from position -158 to position +180 with respect to the start of the coding sequence) with a kanamycin resistance cassette, as shown in Fig. 1A (see section 2.1 for details). The complete segregation of the ΔPC mutant clones was verified by PCR (Fig. 1B). Clone number 3 (from now on indicated as ΔPC) was used for further analyses. The whole cell absorption spectra (Fig. 1C) revealed only minor differences between the wild type and the ΔPC mutant, with a slightly lower Chl a (peaks at 440 and 682 nm) to phycocyanin (peak at 630 nm) ratio in the latter, as previously reported [START_REF] Wang | Inactivation of the petE gene encoding plastocyanin causes different photosynthetic responses in cyanobacterium Synechocystis PCC 6803 under light-dark photoperiod and continuous light conditions[END_REF]. Both the wild-type and ΔPC strains were grown photoautotrophically in unmodified BG11 medium, containing 0,5 µM Cu: at this Cu concentration Synechocystis wild-type cells express mostly the copper-based plastocyanin with only low levels of the alternative electron transporter cyt c6 [START_REF] Zhang | Copper-mediated regulation of cytochrome c553 and plastocyanin in the cyanobacterium Synechocystis 6803[END_REF]. Indeed, when performing protein analyses (Fig. 1D) the wild type displayed a large amount of plastocyanin but little cyt c6 (compare its haem signal to that of cyt f in the total cell extract). It must be noted that we cannot exclude that the faint signal present in the wild type that we attribute to cyt c6 could rather correspond to another c-type cytochrome of comparable size, as for example the cytochrome cM. This cytochrome has been detected in Synechocystis cells grown at 30°C [START_REF] Cho | Cytochrome cM from Synechocystis 6803. detection in cells, expression in Escherichia Coli, purification and physical characterization[END_REF] and its expression has been shown to increase when cells are grown at room temperature [START_REF] Malakhov | Balanced regulation of expression of the gene for cytochrome c(M) and that of genes for plastocyanin and cytochrome c6 in Synechocystis[END_REF], as in the present work. Since the identity of this cytochrome is not particularly relevant for our study, in the following we will refer to it as cyt c6 for economy. In contrast, the ΔPC strain expressed no plastocyanin but much higher amounts of cyt c6 than the wild type. Both proteins were mostly found in soluble fractions and their low amounts in the membrane fractions can be attributed to a minor contamination from soluble proteins, evident also in the distribution of the phycobiliproteins. Although the relative changes in the amounts of each of the two transporters are clear when comparing the two strains, we could not quantify their absolute amounts per cell, owing to the lack of protein standards. The plastocyanin deficient mutant did not display any significant differences in the accumulation of either PSI or cyt b6f with respect to the wild type, as also confirmed by Clear Native-PAGE analysis of the membrane fractions (Fig. S1C).

In order to get a semi-quantitative estimation of the levels of cyt c6 expressed in the ΔPC mutant, we spectroscopically determined the ratio between the c-type cytochromes and P700. To ensure a full oxidation of all c-type cytochromes and P700, we used a saturating light pulse (high oxidation rate) in presence of the PSII inhibitors 3-(3,4-dichlorophenyl)-1,1dimethylurea (DCMU) and hydroxylamine (HA) to prevent their reduction by electrons extracted from PSII. The time-resolved absorption changes were recorded between 545 and 573 nm (Fig. 2), where stand the α bands of all cytochromes [START_REF] Baymann | Rapid electron transfer to photosystem I and unusual spectral features of cytochrome c6 in Synechococcus sp. PCC 7002 in vivo[END_REF]. These absorption changes were measured during a 1.5 s illumination with a strong (1000 µmol photons/m 2 /s) red light and they were normalized to the maximal P700 + signal (ΔI/I 705-730 nm) measured in the same conditions. A linear regression between the ΔI/I at 545 and at 573 nm was set as baseline. It must be noted that in these measurements, performed using the JTS10 spectrophotometer, the wavelengths of the measuring pulses are selected using interference filters (10 nm full width at half-maximum), so the spectral resolution is lower than the one obtained when using the OPObased spectrophotometer, as in section 3.2. As shown in Fig. 2, the amplitude of the bleaching corresponding to the oxidation of cytochromes was about three times larger in the ΔPC mutant than in the wild type after 1.5 s illumination. In the latter, the signal corresponded predominantly to the oxidation of cytochrome f from the cyt b6f complex, whereas the bleaching corresponded to the oxidation of both cyt f and cyt c6 in the ΔPC mutant. Indeed, the peak in the absorption changes in the latter was blue shifted by two nm -554 nm versus 556 nm-in agreement with the spectral differences previously reported for the two cytochromes [START_REF] Baymann | Rapid electron transfer to photosystem I and unusual spectral features of cytochrome c6 in Synechococcus sp. PCC 7002 in vivo[END_REF]. By measuring in different cultures the maximal bleach corresponding to c-type cytochrome oxidation with respect to the maximal bleach at 700 nm due to P700 oxidation, we determined that the ΔPC mutant accumulates 3.2±0.3 (3 replicates) times more c-type cytochromes per PSI than the wild type. Since we observed no significant differences in the cyt f/PSI ratio at the protein level between the two strains (Fig 1 ), the additional bleaching measured in the mutant must be ascribed to the accumulation of oxidized cyt c6. Taken together, these observations point to the presence of about two cyt c6 molecules per cyt b6f monomer in the ΔPC mutant. The spectroscopic detection of cyt c6 accumulation levels were similar in the ΔPC clones 1 and 3, as shown in Fig. S1. It must be noted that the spectra recorded in the wild type also presented an unusual broadening towards the shortest wavelengths with respect to a pure cytochrome f spectrum. This is in line with the biochemical observation that small amounts of cyt c6 are present in addition to plastocyanin and thus contribute to the overall bleach.

3.2-Flash-induced in vivo kinetics of plastocyanin/cyt c6-dependent electron donation to PSI

We then investigated kinetics of the redox changes on these c-type cytochromes after a single turnover saturating flash. These were measured in presence of DCMU and HA in the α-band region of c-type cytochromes (Fig. 3A andB, traces recorded in biological replicates are shown in Fig. S2A andB). These measurements were performed using the OPO-based spectrophotometer, having a spectral resolution of 2 nm. The spectra were normalized on the total P700 + signals, and the baseline calculation was done as described above for Fig. 2. In the wild type, the bleaching appearing with a peak at 556 nm corresponds for the major part to the oxidation of cyt f. Again, a small shoulder around 550-552 nm could be observed, corresponding to the small amounts of cyt c6 present in this strain. The relative amplitude of this shoulder is lower in the spectra in Fig. S2A with respect to those in Fig. 3A, suggesting variable cyt c6 amounts in the different cultures. In the ΔPC mutant the bleaching peaking at 553 nm has a maximal amplitude twice as large as in the wild type. This is because the charge is shared by cyt c6 and cyt f in the mutant, whereas in the wild type it is shared between cyt f and plastocyanin that does not contribute to the absorption changes in this region. The kinetics of the absorption changes in the 550-558 nm region are shown in Fig. 3C andD (and Fig. S2C and D): in the wild type the kinetics were very similar at all wavelengths, as they are dominated by the absorption changes due to cytochrome f, which gets oxidised with a t½ of ~200 µs. A minor faster bleach is visible in the 100 µs time range at 550-552 nm: this likely corresponds to the fast oxidation of the small amounts of cyt c6 present. In the ΔPC mutant the kinetics were more complex, clearly showing a faster bleaching at 550-554 nm and a slower one at 556-558 nm. Therefore, the oxidation of cyt c6, mostly responsible for the ΔI/I 550-554 nm, preceded the one of cyt f, that has a major contribution in the ΔI/I 556-558 nm. From the above kinetics we estimate that cyt c6 oxidation occurs with a t½ of ~20 µs, indicative of a fast electron donation to PSI. In a previous study [START_REF] Viola | Probing the electric field across thylakoid membranes in cyanobacteria[END_REF] we have shown that the re-reduction of oxidized P700 after a singleturnover saturating flash is bi-phasic in vivo in the wild type strain of Synechocystis: it displays a fast phase that corresponds to electron donation by a pre-fixed plastocyanin (or possibly cyt c6, in a small fraction of PSI centres) and a slow phase which accounts for the diffusion time of additional electron donors in the thylakoid lumen. We thus investigated the kinetics of electron donation from cyt c6 to PSI in the ΔPC mutant, following the same approach as in [START_REF] Viola | Probing the electric field across thylakoid membranes in cyanobacteria[END_REF] which is based on ElectroChromic Shift (ECS) measurements. The light-induced ECS signal that can be measured as ΔI/I (500-485nm) in Synechocystis, is proportional to the electric field generated by charge separations within each photosystem. The amplitude of the ECS signal measured within 1 ms after a single-turnover saturating flash given in presence of PSII inhibitors (DCMU and HA) corresponds to one charge separation per active PSI. During a few milliseconds after the flash, the positive charge originally present on P700 + is stabilized on secondary electron donors on the lumenal side of PSI, thus maintaining the trans-membrane electric field but allowing another charge separation within the PSI reaction centres in which P700 has been re-reduced. It follows that the fraction of PSI centres whose P700 has been reduced by a soluble donor at a given time after the first flash can be measured through the increase in ECS generated by a second flash. In our experimental conditions, where the cells were incubated in anaerobiosis and darkness for at least one hour before starting the measurements, the transmembrane electric field generated by the single flash remained quite stable in the 5 ms time range and any decay was taken into account (see Fig. S3 for further explanations). The advantage of using ECS instead of P700 + absorption spectroscopy is that the evolution between the two flashes allows us to discriminate between charge recombination and electron transfer from plastocyanin / cyt c6. Both charge recombination and electron transfer from secondary donors will translate into a reduction of P700. The ECS discriminates the two processes: charge recombination will suppress the electric field generated by the first flash whereas electron transfer from plastocyanin / cyt c6 to P700 will keep the electric field constant. Therefore, we measured the in vivo kinetics of P700 reduction by secondary donors in the wild type and in the ΔPC strains as described above (Fig. 4 A, B andC). The fitted experimental traces are shown individually in Fig. S4. As expected, the kinetics in the wild type were bi-phasic, with about 60% of P700 being re-reduced in less than 100 μs (t½ ~10 μs). A fast phase with similar kinetics (t½ ~15 μs) was observed also in the case of the ΔPC mutant, although the fraction of P700 reduced in less than 100 μs was only about 30% of the total. The fast re-reduction kinetics of P700 in the ΔPC strains correspond very well to the cyt c6 oxidation kinetics measured in the same conditions (Fig. 3D). In both strains the fast P700 reduction phase was followed by a considerably slower phase, with about 20 and 40 % of the total P700 still remaining oxidized 5 ms after the first flash in the wild type and the mutant, respectively. In order to promote the full re-reduction of P700 in the time during which the electric field generated by the first charge separation is preserved (few ms), we used the redox mediator PMS. PMS accepts electrons from PSI on the stromal/cytoplasmic side and when it gets doubly-reduced and protonated it partitions inside the lumen and donates electrons to P700, likely via plastocyanin or cyt c6 (40), while releasing protons. In our previous work [START_REF] Viola | Probing the electric field across thylakoid membranes in cyanobacteria[END_REF] we demonstrated that the presence of PMS did not affect the fraction of PSI centres in which P700 was reduced during the fast (100 μs) phase but it shortened the slow phase of reduction. This suggests that PMS should not donate electrons directly to PSI fast enough to outcompete electron donation from the pre-fixed plastocyanin or cytochrome c6. Indeed, the addition of PMS did not significantly change the kinetics of the fast phase in either of the two strains (Table S1), while it considerably accelerated the slow reduction phase, leading to the full re-reduction of P700 within 5 ms. These conditions allowed us to confirm that the ECS signal in the ΔPC mutant is linearly proportional to the electric field, as previously demonstrate for the wild type [START_REF] Viola | Probing the electric field across thylakoid membranes in cyanobacteria[END_REF]. These results indicate that a secondary donor is pre-bound in the dark to a fraction of PSI in both the wild-type and the mutant strain. This fast-donating electron carrier is either plastocyanin (plus traces of cyt c6) in the wild type or cytochrome c6 in the ΔPC mutant, the latter case involving a much smaller fraction of the PSI centres.

In order to further confirm that the fast P700 reduction phase corresponds to the electron donation from a lumenal donor to PSI and is not due to a direct donation from PMS, we measured in the same conditions as above the kinetics of the absorption changes at 416 nm. At this wavelength, light-induced absorption changes measured in presence of PSII inhibitors contain a P700 + contribution mixed with an ECS contribution and with the absorption peaks of oxidized cytochromes in the Soret region [START_REF] Baymann | Rapid electron transfer to photosystem I and unusual spectral features of cytochrome c6 in Synechococcus sp. PCC 7002 in vivo[END_REF]. We have previously shown [START_REF] Viola | Probing the electric field across thylakoid membranes in cyanobacteria[END_REF] that PMS strongly suppresses the oxidation of cytochrome f induced by a single-turnover flash, thus eliminating its contribution from the flash-induced absorption changes at 416 nm. Consequently, the wild type incubated with PMS should display only a minor contribution from cyt c6 oxidation on top of P700 redox changes and ECS, whereas in the case of the ΔPC mutant, one would expect an additional contribution coming from cyt c6 oxidation, unless PMS by-passes it and re-reduces P700 directly. As shown in Fig. 4D, the absorption kinetics at 416 nm in the ΔPC mutant indeed were very different from those in the wild type: in the latter the bleaching was maximal at 200 ns after the flash (corresponding to the maximal P700 oxidation and to the rapid rise phase of ECS) and it relaxed steadily (see inset Fig. 4D), whereas in the mutant, owing to cyt c6 oxidation, the bleaching further increased during the first 100 μs, before relaxing. This confirms that PMS does not by-pass cyt c6 during the fast phase, which thus gets oxidized by PSI before getting re-reduced. Although we do not have experimental data showing the oxidation of plastocyanin in the wild type under the same circumstances, we assume that it occurs in the same way, given the very similar redox potentials of the two lumenal electron carriers.

3.3-Partitioning of the intersystem electron fluxes in presence of plastocyanin or cyt c6

In cyanobacteria, as already mentioned in the introduction, plastocyanin and cyt c6 can donate electrons to both PSI and the respiratory cytochrome oxidase complex, COX. The partitioning between the photosynthetic and respiratory flows at this intersection should depend on the affinity of the plastocyanin or cytochrome c6 for PSI or COX but the modalities of this partitioning are still unresolved in cyanobacteria. This aspect was irrelevant to the previous experiments since the kinetics of electron donation from plastocyanin/cyt c6 to PSI after a single-turnover flash reported above were measured in anaerobic conditions. In absence of oxygen, COX is not functional and therefore cannot compete for electrons with PSI. In the following, we maintained the cells in aerobic conditions by frequent mixing and reinjection in the sample cuvette to investigate whether the in vivo partitioning of electrons between PSI and COX depends on the molecular nature of the electron donor in Synechocystis. To this aim, we measured the P700 oxidation-reduction kinetics upon single or multiple turnover illumination in the presence of DCMU -to prevent re-reduction of P700 by PSII -and of various combinations of: i) potassium cyanide (KCN), an inhibitor of COX [START_REF] Howitt | Quinol and cytochrome oxidases in the cyanobacterium Synechocystis sp. PCC 6803[END_REF]; ii) methyl viologen, serving as artificial PSI acceptor (41); and iii) rotenone (Rot), that inhibits the activity of the NADPH dehydrogenase complex (NDH), thus also blocking one of the pathways of electron injection in the intersystem electron carriers [START_REF] Mi | NAD(P)H dehydrogenasedependent cyclic electron flow around photosystem-I in the cyanobacterium Synechocystis PCC-6803 -A Study of dark-starved cells and spheroplasts[END_REF]. All P700 traces were normalized to the total PSI amounts present in each sample, measured as the maximal ΔI/I (705 -730) nm obtained at the end of a multi-turnover light pulse in presence of DCMU and methyl viologen (MV). First, we investigated if the fraction of pre-bound PSI secondary donors is affected by COX activity, using single turnover flashes (Fig. 5). In the kinetics of P700 reduction, the first point was recorded 100 μs after the flash: at this time, about 60% of P700 in the wild type and 20% in the ΔPC mutant were already re-reduced, no matter which inhibitors or mediators were present.

Fig. 5. P 700 + kinetics (ΔI/I 705-730 nm) measured in Synechocystis WT (A) and ΔPC (B) cells measured after a single-turnover saturating flash (first measuring time 100 μs after the flash) in presence of DCMU and various combinations of: methyl viologen (MV), Rotenone (Rot) and potassium cyanide (KCN). All traces are normalized to the total PSI present in each sample (maximal ΔI/I 705-730 nm measured during a 75 ms light pulse in presence of DCMU and MV). The flash was applied at time-point 0. This is consistent with the ECS-based kinetics of P700 reduction reported above in anaerobic conditions, which suggests that the fraction of pre-bound electron donor in the dark depends neither on the redox state of the intersystem electron carriers nor on the activity of the COX. The inhibition of COX by KCN did not modify the fraction of rapidly reduced P700, further confirming that the pre-bound fraction does not depend on a competition between the affinity of plastocyanin / cyt c6 for PSI and for COX, in the dark. This also demonstrates that in our experimental conditions KCN did not lead to Cu removal from plastocyanin, as had been previously reported in broken chloroplasts, at a much higher KCN concentrations than the 50 µM used here [START_REF] Berg | Mechanism of KCN inhibition of photosystem I[END_REF]. To investigate the electron partitioning between PSI and COX in the light, we used instead multi turnover pulses, again in presence of DCMU and aerobic conditions. The addition of the COX inhibitor KCN strongly prevented the full oxidation of P700 during a short (75 ms) saturating pulse, in a similar way in both strains (Fig. 6A andC). When applying longer pulses of lower intensity light (Fig. 6B andD showing traces recorded in independent cultures with respect to those used for panels A and C), an almost complete oxidation of P700 could be reached even in presence of KCN, although the oxidation kinetics remained much slower than in its absence. In addition, the P700 reduction kinetics at the light offset (time-point 0) were faster in presence of KCN+DCMU than with DCMU alone with both short and long pulses. Since in presence of DCMU no electrons coming from PSII are injected in the intersystem electron transport chain, the lag in P700 oxidation during illumination and the faster reduction at the light offset induced by KCN suggest that the inhibition of the COX results in: i) a transitory higher rate of cyclic electron flow (CEF), ii) a transient acceptor side limitation of PSI resulting in charge recombination, or iii) a combination of both [START_REF] Nawrocki | Maximal cyclic electron flow rate is independent of PGRL1 in Chlamydomonas[END_REF]. Independently of the process behind the lag in P700 oxidation in presence of KCN, the inhibition of COX led to an accumulation of reduced PSI electron donors and/or acceptors prior to and during illumination. In dark-adapted Arabidopsis leaves the P700 + decay at the offset of a saturating light pulse comprises a fast phase which is completed in 1 ms and is attributed to P700 + A1 -charge recombination within PSI [START_REF] Joliot | Cyclic electron flow under saturating excitation of dark-adapted Arabidopsis leaves[END_REF]. Since the P700 reduction kinetics that we measured in Synechocystis did not show a faster reduction phase completed in 1 ms (Fig. 6), it is tempting to suggest that the KCN-induced faster reduction of P700 is due to a faster cyclic electron flow around PSI. However, we cannot fully exclude a role of charge recombination involving the reduced terminal PSI iron-sulphur clusters FA and FB: as reviewed in [START_REF] Brettel | Electron transfer and arrangement of the redox cofactors in photosystem I[END_REF], charge recombination between P700 + and (FA/FB) -has been shown to occur over tens of milliseconds in isolated plant PSI particles. Such recombination may occur in vivo in absence of available ferredoxin, were it detached from PSI centres or over-reduced upon inhibition of the respiratory chain in presence of KCN. Charge recombination could especially contribute to the strong lag in P700 oxidation that we observed during the first tens of ms of the multiple turnover pulses (Fig. 6A, B, C andD) in presence of KCN. The fact that P700 + absorption spectroscopy cannot easily distinguish between cyclic electron flow and charge recombination has been recently discussed in experiments with C. reinhardtii, where up to half of the PSI charge separations measured with this method during the first hundreds of milliseconds of illumination in darkadapted cells were attributed to charge recombination rather than to an actual electron flow [START_REF] Nawrocki | Maximal cyclic electron flow rate is independent of PGRL1 in Chlamydomonas[END_REF]. However, there is one situation where the contribution of charge recombination can be neglected compared to CEF. This is at the end of the long pulse (Fig. 6 B and D), when P700 is almost fully oxidized: the acceptor side limitation is absent in these conditions. The rereduction rates of P700 are still faster in the presence of KCN than in its absence which indicates that CEF is promoted by the inhibition of the COX. KCN could lead to PSI over-reduction not only via the inhibition of COX but also by inhibiting the Calvin-Benson cycle, as shown for example in [START_REF] Kobayashi | Rates of vectorial proton transport supported by cyclic electron flow during oxygen reduction by illuminated intact chloroplasts[END_REF]. Such an inhibition could result in both PSI acceptor side limitation and an increase in CEF. However, a COX-specific effect of KCN was revealed by the effect of an addition of 300 µM methyl viologen. This addition counteracted the KCN-induced acceleration of the P700 reduction kinetics at the end of the long pulse (Fig. 6 B andD), which is easily interpreted by the competition of MV for the electrons used in the CEF promoted by the presence of KCN. Besides competing for electrons with CEF, MV has also been shown to efficiently prevent charge recombination in PSI [START_REF] Sétif | Electron-transfer kinetics in cyanobacterial cells: Methyl viologen is a poor inhibitor of linear electron flow[END_REF]. Despite these two effects, its addition did not fully prevent the lag in P700 oxidation induced by KCN during a short pulse and during the initial phase of illumination when applying a longer pulse (see Fig. S5 for details). This can be explained by the accumulation of electrons on the donor side of PSI prior to illumination because of the inhibition of COX. It must be noted that KCN also inhibits the cyanobacterial plastoquinone terminal oxidase Cyd. However at a 50 µM concentration, as used here, COX is fully inhibited whereas part of Cyd remains active [START_REF] Pils | Characterization of three bioenergetically active respiratory terminal oxidases in the cyanobacterium Synechocystis sp. strain PCC 6803[END_REF]. Nonetheless, a partial inhibition of Cyd could contribute to the accumulation of reduced PSI donors/acceptors that we observe in presence of KCN. In accordance with this, a retardation in P700 oxidation upon illumination and its faster rereduction at the light offset have been previously reported in a Synechocystis mutant devoid of COX and even more markedly in a mutant devoid of both COX and Cyd [START_REF] Ermakova | Distinguishing the roles of thylakoid respiratory terminal oxidases in the cyanobacterium Synechocystis sp. PCC 6803[END_REF]. In cyanobacteria, one of the entry points of electrons in the intersystem chain is represented by the NDH complex, which accepts electrons from stromal reductants and re-injects them upstream of PSI at the level of the plastoquinone pool. This pathway can be inhibited by rotenone [START_REF] Mi | NAD(P)H dehydrogenasedependent cyclic electron flow around photosystem-I in the cyanobacterium Synechocystis PCC-6803 -A Study of dark-starved cells and spheroplasts[END_REF]. Addition of this inhibitor partially relieved the P700 over-reduction induced by KCN during illumination and it did not significantly slow down the fast PSI re-reduction at the offset of the light pulse (Figure 6). The effect of rotenone, although limited, points to the availability, in presence of KCN, of a higher pool of electron donors to PSI in part maintained by the activity of the NDH. The fact that rotenone counteracted only partly the effect of KCN on the redox changes of P700 during the pulses can be explained by the fact that it does not prevent charge recombination and by the presence of a NDH-independent cyclic pathway in cyanobacteria [START_REF] Yeremenko | Open reading frame ssr2016 is required for antimycin Asensitive photosystem I-driven cyclic electron flow in the cyanobacterium Synechocystis sp. PCC 6803[END_REF]. In conclusion, the response of the oxidation and reduction kinetics of P700 to the inhibitors and mediator used here were similar in the wild type and ΔPC strains.

4-Discussion

We have shown that in Synechocystis wild type grown in normal BG11 the electron carrier plastocyanin is expressed preferentially, although some cytochrome c6 is also present, in line with previous literature (see section 3.1). Upon deletion of the gene encoding plastocyanin the cells accumulate cytochrome c6 instead.

The in vivo reduction kinetics of P700 after a single-turnover flash is bi-phasic with either plastocyanin or cytochrome c6 as electron donor (Fig. 4). Both these donors can thus pre-bind in the dark to PSI and can reduce the P700 + formed upon illumination with comparable kinetics (t½ ~10-15 µs). Very convincingly, the kinetics of cyt c6 oxidation after a flash (t½ slightly higher than 10 µs, see Fig 3B) is similar to the fast phase of the kinetics of P700 reduction in the ΔPC mutant. We showed that even in presence of PMS the fast P700 reduction phase after a flash represents an electron transfer from a pre-bound plastocyanin / cyt c6 to PSI. In our results, though, the slower phase of P700 reduction was PMS-sensitive and we cannot exclude that PMS could compete with lumenal electron donors for a direct P700 reduction in the hundreds of µs to ms time scale. In presence of PMS we could indeed observe a fast flash-induced oxidation of cyt c6 in the ΔPC mutant, completed in 100 µs, corresponding to the fast electron donation to PSI centres forming a pre-complex, followed by a re-reduction having t½ ~1.5 ms (Fig 4D). Such re-reduction kinetics are well suited to account for the slow P700 + reduction phase observed in the same conditions (Fig. 4B), which could suggest that cyt c6 does mediate electron donation from PMS to PSI. Nonetheless, PMS could at the same time donate electrons directly to PSI, as it is known to do in vitro when no other soluble electron carrier is present (see for example [START_REF] Schlodder | P700 + -and 3 P700-induced quenching of the fluorescence at 760 nm in trimeric Photosystem I complexes from the cyanobacterium Arthrospira platensis[END_REF]). This could especially be true if the diffusible lumenal electron carriers were present in sub-stoichiometric amounts compared to PSI, as discussed below.

The fraction of PSI centres having a pre-bound electron donor is lower in the ΔPC mutant than in the wild type (~30% and ~60% of the total, respectively). Using spectroscopy, we estimated the presence of about two cytochrome c6 molecules per cytochrome b6f complex in the mutant (Fig. 2) but we know neither the concentration of cyt c6, nor that of plastocyanin in the wild type, relatively to PSI. The difference in the fraction of PSI centres having a pre-bound electron donor observed in the two strains could reflect a difference in the total amount of lumenal donors, with the cyt c6/PSI ratio being lower in the ΔPC mutant than the plastocyanin/PSI ratio in the wild type. However, it is of note that the correlation between the amount of the sole plastocyanin relative to PSI and the amplitude of the fast P700 reduction phase varies in plant chloroplasts. Indeed, in spinach chloroplast a plastocyanin/PSI ratio of ~3 resulted in the reduction of almost all P700 + in tens of µs [START_REF] Haehnel | Lateral distribution and diffusion of plastocyanin in chloroplast thylakoids[END_REF], whereas in Arabidopsis leaves the amount of P700 reduced in 100 µs was reported to be ~60% even in overexpressor lines accumulating a large excess of plastocyanin [START_REF] Pesaresi | Mutants, overexpressors, and interactors of Arabidopsis plastocyanin isoforms: Revised roles of plastocyanin in photosynthetic electron flow and thylakoid redox state[END_REF]. Another comparative in vitro study performed with isolated particles from plants, green algae and cyanobacteria (17) described a fast P700 reduction phase, the amplitude of which depended on species and on the molecular nature of the electron donor (plastocyanin or cyt c6). Therefore, the differences in the fraction of P700 reduced in 100 µs in the wild type and ΔPC mutant could also be in part due to changes in the affinity of the electron donor to the PSI binding site.

In isolated PSI particles from Chlamydomonas the amplitude, but not the kinetics, of the fast P700 reduction phase has been shown to be dependent on the concentration of the soluble electron donor, were it plastocyanin or cytochrome c6 [START_REF] Hippler | Fast electron transfer from cytochrome c6 and plastocyanin to photosystem I of Chlamydomonas reinhardtii requires PsaF[END_REF]. Our results could therefore be accounted for if cytochrome c6 has the same properties of electron donation to PSI as plastocyanin, but that it remains sub-stoichiometric with respect to PSI in the ΔPC strain. Indeed, in the flash-induced kinetics of cytochrome oxidation in absence of PMS, we observed that the oxidation of cytochrome c6 is mostly completed in 100 µs, when only 30% of P700 is reduced (Fig. 3 and Fig. S2). This suggests that most of the available cyt c6 is oxidised during this phase. Therefore, we suggest that the stoichiometry of lumenal donor per PSI would be of ~0.6 for plastocyanin versus ~0.3 for cytochrome c6. This is consistent with the higher proportion of P700 remaining oxidised in the ΔPC mutant, 5 ms after a single-turnover flash, when no PMS is present (Fig. 4B). A plastocyanin/PSI ratio of ~1.3 has been recently measured in Synechocystis cells by near-infrared spectroscopy [START_REF] Theune | In-vivo quantification of electron flow through photosystem I -Cyclic electron transport makes up about 35% in a cyanobacterium[END_REF]. In our study the cells are grown at lower temperature and lower light intensity than in [START_REF] Theune | In-vivo quantification of electron flow through photosystem I -Cyclic electron transport makes up about 35% in a cyanobacterium[END_REF], which could explain the discrepancy in the estimated ratios since the expression of the petE gene has been shown to be affected by both factors [START_REF] Malakhov | Balanced regulation of expression of the gene for cytochrome c(M) and that of genes for plastocyanin and cytochrome c6 in Synechocystis[END_REF]. Sub-stoichiometric amounts of soluble cytochromes relative to PSI have been previously reported in a thermophilic Synechococcus sp. in which plastocyanin was absent [START_REF] Aoki | Contents of cytochromes, quinones and reaction centers of photosystems I and II in a cyanobacterium Synechococcus sp[END_REF]. Based on spectroscopy results (Fig. 2) we estimated that in the ΔPC mutant the cyt b6f amounts are about half of the cyt c6 ones, which would result in a cyt b6f per PSI stoichiometry of ~0.15. These quantitative estimates, that are not meant to be precise, are not in disagreement with the literature, where cyt b6f to PSI ratios as low as 0.2 have been reported, varying in function of the cyanobacterial species analysed and the growth conditions [START_REF] Murakami | Changes in photosystem stoichiometry in response to environmental conditions for cell growth observed with the cyanophyte Synechocystis PCC 6714[END_REF][START_REF] Zorz | The RUBISCO to photosystem II ratio limits the maximum photosynthetic rate in picocyanobacteria[END_REF][START_REF] Casella | Dissecting the native architecture and dynamics of cyanobacterial photosynthetic machinery[END_REF].

In both the wild type and the ΔPC mutant the percentage of PSI centres having in the dark a pre-bound electron donor was comparable whether the cytochrome c oxidase is active or inhibited by KCN in aerobic conditions and it did not change either when switching to anaerobic conditions (compare Fig. 4A and B and Fig. 5). Therefore, despite the fact that COX, as PSI, relies on plastocyanin and cytochrome c6 as electron donors, their oxidation in the dark via respiration does not affect the fraction that is pre-bound to the lumenal side of PSI, as if the two donor systems were not in competition.

Either of the three following hypothesis could account for these observations : i) the concentration ratio of COX to PSI in Synechocystis thylakoids is so low that the activity of COX as a negligible effect on the amount of electron donors available for PSI; ii) PSI and COX are connected to two physically separated pools of cytochrome b6f and lumenal donors; or iii) PSI and COX are connected to the same pool of electron donors, but in the dark the affinity of reduced plastocyanin or cyt c6 for PSI is so high that none of the pre-bound electron donors are available as COX substrates. Although there are no reports of an actual quantification of the COX/PSI ratio in the literature, the localization of COX in the thylakoids membranes of cyanobacteria is well documented [START_REF] Peschek | Characterization of cytochrome-c oxidase in isolated and purified plasma and thylakoid membranes from cyanobacteria[END_REF][START_REF] Peschek | Characterization of the cytochrome c oxidase in isolated and purified plasma membranes from the cyanobacterium Anacystis nidulans[END_REF][START_REF] Peschek | Correlation between immuno-gold labels and activities of the cytochrome-c oxidase (aa3-type) in membranes of salt stressed cyanobactria[END_REF], as are the effects of its inhibition [START_REF] Cooley | Succinate dehydrogenase and other respiratory pathways in thylakoid membranes of Synechocystis sp. strain PCC 6803: Capacity comparisons and physiological function[END_REF] or absence [START_REF] Dj | Thylakoid terminal oxidases are essential for the cyanobacterium Synechocystis sp. PCC 6803 to survive rapidly changing light intensities[END_REF][START_REF] Pils | Characterization of three bioenergetically active respiratory terminal oxidases in the cyanobacterium Synechocystis sp. strain PCC 6803[END_REF] on the photosynthetic electron transport. In our experiments, the inhibition of COX with KCN resulted in a marked lag in P700 oxidation during short saturating light pulses and at the onset of longer pulses (Fig. 6). These results rule out hypothesis i), as they imply that in our experimental samples COX was present in sufficient amounts to maintain, in absence of KCN, the electron transport chain upstream of PSI in a more oxidised state than in presence of the inhibitor.

The inhibition of COX thus results in a larger pool of PSI electron donors available prior to illumination, but it does not increase the proportion of PSI centres that have a pre-bound donor. This observation can be equally well accounted for by hypotheses ii) or iii). If there were distinct PSI and COX domains, still there would be diffusion-limited electron transfer from cyt b6f complexes closely associated to either PSI or COX. The inhibition of COX would strongly affect the P700 oxidation kinetics upon illumination, because the two electron transfer chains would still be connected through the plastoquinone pool, that would get over-reduced in presence of KCN. A spatial segregation of COX in cyanobacteria thylakoids has not been experimentally demonstrated, but such a segregation was reported for the respiratory complexes NDH and succinate dehydrogenase [START_REF] Liu | Control of electron transport routes through redox-regulated redistribution of respiratory complexes[END_REF]. Alternatively, if PSI and COX rely on a shared pool of electron donors but with a much higher affinity of PSI for the electron donors in darkness, these would partition differently in the dark and under illumination: in the dark the partitioning of donors between PSI and COX mainly depends on their relative affinity, whereas upon illumination the availability of luminal donors becomes diffusion-limited. It also has been suggested that the light-induced changes in the ionic strength of the lumen could favour association of plastocyanin or cyt c6 with COX, while their association with PSI would be favoured in the dark [START_REF] Nicholls | Cytochrome c and c-554 oxidation by membranous Anacystis nidulans cytochrome oxidase[END_REF]. However, such a switch mechanism has not yet been demonstrated in vivo.

Independently of the underlying model, though, we have shown that the in vivo partitioning of electrons between PSI and COX upon multi-turnover illumination is rather insensitive to whether the lumenal electron carrier is plastocyanin or cyt c6. Indeed, in both cases the inhibition of COX activity results in the accumulation of electrons in the photosynthetic chain, resulting in a lag in the oxidation of P700 at the dark-to-light transition. Our results could not discriminate between the role of cyclic electron flow and of charge recombination in inducing the lag, although it is very likely that both processes were involved. Indeed, the transient overreduction of the intersystem chain induced by the inhibition of COX activity would also result in the over-reduction of the cytoplasmic pool of PSI acceptors (ferredoxin, FNR and NADP + ). Reduced ferredoxin is the electron donor of the NDH complex [START_REF] Schuller | Structural adaptations of photosynthetic complex I enable ferredoxin-dependent electron transfer[END_REF], which in turn injects electrons in the plastoquinone pool [START_REF] Mi | NAD(P)H dehydrogenasedependent cyclic electron flow around photosystem-I in the cyanobacterium Synechocystis PCC-6803 -A Study of dark-starved cells and spheroplasts[END_REF][START_REF] Mi | Electron donation from cyclic and respiratory flows to the photosynthetic intersystem chain is mediated by pyridine nucleotide dehydrogenase in the cyanobacterium Synechocystis PCC 6803[END_REF]. In both our strains the inhibition of NDH with rotenone partially relieved the P700 over-reduction induced by KCN during light pulses (Fig. 6), thus confirming that the NDH activity was in part responsible for the dark reduction of the photosynthetic chain. This indicates that the lag in P700 oxidation induced by the inhibition of COX was not solely due to charge recombination. The NDH has been shown to play a role, together with the flavodiiron (Flv) proteins, in regulating the redox homeostasis of the photosynthetic chain [START_REF] Nikkanen | Functional redundancy between flavodiiron proteins and NDH-1 in Synechocystis sp. PCC 6803[END_REF]. Nonetheless, we show here that when the electron flux towards oxygen mediated by COX is inhibited, the NDH and Flv activities are not sufficient to prevent a transient over-reduction of P700 during the first tens of milliseconds of the dark to high light transition. This observation confirms the role of respiratory oxidases to prevent photodamage, as it has been previously shown in Synechocystis cultures subjected to a growth regime characterized by rapid dark-to-high light transitions [START_REF] Dj | Thylakoid terminal oxidases are essential for the cyanobacterium Synechocystis sp. PCC 6803 to survive rapidly changing light intensities[END_REF].

The present study provides further experimental evidence that plastocyanin and cyt c6 are functionally equivalent in Synechocystis in vivo, with respect the photosynthetic and respiratory chains. The functional interaction between these two metabolic chains is far from being understood, but further time-resolved analysis of the partitioning of electron transport between respiration and photosynthesis in light-dark cycles should provide key information in that respect.

Fig. 1 .

 1 Fig. 1. Generation and characterization of the Synechocystis ΔPC mutant. (A) Schematic representation of the partial replacement of the petE gene with the kanamycin resistance cassette (kan R ) in the ΔPC mutant strain. FW (SacI-petE FW), RP (petE del RV) and RK (KanR RV) are the primers used for genotyping. (B) PCR-based genotyping, using the primers depicted in (A), of two independent ΔPC

Fig. 2 .

 2 Fig. 2. Time-resolved accumulation of oxidized c-type cytochromes in Synechocystis WT (A) and ΔPC (B) cells. Absorption changes were measured in presence of DCMU and HA at the indicated times (in ms) after the onset of a 1.5 s illumination with a strong red actinic light and normalized on total P 700 + (maximal ΔI/I 705-730 nm measured at the end of the light pulse). The linear regression between ΔI/I 545 and ΔI/I 573 nm was set as baseline for each recording time. The dotted vertical lines mark the 556 nm wavelength.

Fig. 3 .

 3 Fig. 3. Flash-induced oxidation and reduction kinetics of c-type cytochromes in Synechocystis WT (A and C) and ΔPC (B and D) cells. (A) and (B) Absorption changes were measured in presence of DCMU and HA at the indicated times (in μs) after a single-turnover saturating flash and normalized on total PSI (ΔI/I 435 nm measured at 200 ns after the flash), and the linear regression between ΔI/I 545 and ΔI/I 573 nm was set as baseline for each recording time. (C) and (D) Kinetics of the absorption changes between 550 and 580 nm from panels A and B.

Fig. 4 .

 4 Fig. 4. Electron transfer kinetics between secondary electron donors and PSI in Synechocystis. (A) and (B) P 700 + re-reduction kinetics measured in WT and ΔPC cells, respectively, in presence of DCMU and

Fig. 6 .

 6 Fig. 6. P 700 + kinetics (ΔI/I 705-730 nm) measured in Synechocystis WT (A and B) and ΔPC (C and D) cells in presence of DCMU and various combinations of: methyl viologen (MV), Rotenone (Rot) and potassium cyanide (KCN). All traces are normalized to the total PSI present in each sample (maximal ΔI/I 705-730 nm measured at the end of a light pulse in presence of DCMU and MV). (A) and (C) P 700 + kinetics measured during short (75 ms) pulses of saturating light (2500 µmol photons/m 2 /s). The dashed lines indicate a change of scale in the time (ms) axis. (B) and (D) P 700 + kinetics measured during long (1.5 s) pulses of strong light (1200 µmol photons/m 2 /s). In all panels, the time-point 0 is at the pulse offset. Cells used in panels (A) and (C) were the same as those used in Fig. 5, while cells used in panels (B) and (D) are from independent cultures.

Acknowledgments

This work was supported by UMR7141, CNRS/ Sorbonne Université, by the Agence Nationale