F Gullo
email: gullo@laplace.univ-tlse.fr

T Christen

C Villeneuve-Faure

H Hillborg

C Laurent

S Le Roy

G Teyssedre

Field and charge distribution at semicon/polyethylene interfaces from combinations of probe force microscopy measurements

Keywords: HVDC insulation, space charge, semicon-contact, AFM, KPFM, polyethylene

Two modes of Atomic Force Microscopy, namely the Peak Force -Quantitative Nano Mechanics and the Kelvin Probe Force Microscopy, are used for the nanoscale characterization of semicon(SC)-polyethylene(PE) interfaces in a SC-PE-SC-sandwich. A combination of the two methods provides physical (local geometry, roughness) and electrical (electric field, space charge) properties.

Introduction

To understand electrical conduction mechanisms in polymer insulation is crucial for high voltage direct current (HVDC) applications, particularly for the prediction and control of the field distribution. Dielectric polymers exhibit properties which aggravate a sound physical understanding of bulk conductivity: amorphous or semi-crystalline structure, small intrinsic carrier density, lowmobility states and carrier traps, charge carrier injection etc.. In this contribution, we focus on the electrode contact, which is relevant because (1) the contact properties can strongly determine the electrical conductance behaviour of the insulation, e.g. due to injection of majority carriers in combination with the smallness of intrinsic carrier density, and (2) the calculation of electric fields based on drift-diffusion models requires realistic boundary conditions at the contacts [1]. In this work, we consider a typical prototype interface, as ubiquitous in HV equipment, between a carbon black (CB) filled host polymer ('semicon', SC) and the insulating pure host polymer (polyethylene, PE). The fundamental processes which govern the contact physics and their dependence on the microstructure properties in such systems are still poorly understood [2]. We report on results on the application of Peak Force -Quantitative Nano Mechanics (PF-QNM) and Kelvin Probe Force Microscopy (KPFM) techniques [3] to the SC/PE interface in a SC/PE/SC sandwich structure.

Results and Discussion

The geometrical interface was characterized by measuring the adhesion force with PF-QNM. This parameter shows a contrast between CB dots and the PE matrix, and provides the CB-induced interface roughness. Electrical properties were characterized by measuring the surface potential with KPFM. The determination of the charge distribution requires the solution of Poisson's equation, V= -free-pol, where the free and polarization charges need to be considered with special care on the nanoscale in this strongly nonuniform and anisotropic system. Also the spatial derivatives on experimental and thus noisy data needs a special treatment. We use the Savitzky-Golay method which fits locally a polynomial function such that the derivatives can be performed analytically. In figure 1, resulting E-field (first derivatives) and space charge (second derivatives) distributions are shown for a specific case. Charges appear close to the SC/PE interface (few microns) with negative charge in PE and positive charge in SC.

Conclusions

KPFM and PF-QNM measurements allows us to characterize the SC/PE interface roughness and the contact charge density. Our approach will allow to determine the boundary conditions for field simulations and to gain new insight into physical phenomena at the electrode interface.

Figure 1 :

 1 Figure 1: a) Electric field and b) charge density for a 50µm thick SC/PE/SC structure. Blue curve: profile along the dotted line.


Mazières Valentin
email: valentin.mazieres@laplace.univ-tlse.fr

Romain Pascaud

Olivier Pascal

Richard Clergereaux

Luc Stafford

Simon Dap

Laurent Liard

Spatio-temporal dynamics of a nanosecond pulsed microwave plasma ignited by time reversal

In the present paper, the first spatio temporal characterization of a time reversal microwave plasma generated in argon is provided using imaging. This novel source allows to ignite a plasma at a desired location in a reverberant cavity by focusing the electromagnetic energy in time and space. An important feature is the possibility to control the plasma position only by changing the input microwave waveform. The source is operated in a repetitive pulsed mode with very low duty cycle (typically 5×10 -2 %). Nanosecond pulses have rise time lower than one nanosecond. The generated plasmas have typical sizes in the millimeter range and are observed for dozens of nanoseconds. The plasma behavior is investigated for different pressures and repetition frequencies. A strong dependence is observed between each discharge pulse suggesting the existence of an important memory effect. The latter is probably due to argon metastable atoms and/or residual charges remaining in the post-discharge and allowing the next breakdown to occur at a moderate electric field.

Introduction

Microwaves discharges are intensively studied as they produce high plasma densities both in high and lowpressure conditions. Produced by travelling-waves as in the case of surfatron [1,2], by radiating microwave power with a slot antenna (SLAN) array [3,4] or by injecting the wave in closed resonant metallic cavities [5,6], microwave plasmas are controlled by the balance between [7]:

-the electromagnetic power absorption by the plasma: absorption process takes place through Joule heating of electrons directly controlled by the electric fields.

-the power losses in the plasma volume: losses involve electrons energy transfer to the neutrals and ions by elastic and inelastic collisions as well as gas heating and to the walls by heat conduction and convection.

While inelastic collisions are necessary for plasma ignition, gas and walls heating are limiting processes. Pulsed power can advantageously limit those losses. Indeed, time modulation of power injection has proven to offer different control capabilities of the plasma features [8]. The interruption of the power between repetitive short pulses allows, for example, to constrain the gas heating [9] or to control the chemical processes involved in the plasma volume and on the surfaces [START_REF] Martin | Handbook of deposition technologies for films and coatings: science, applications and technology[END_REF][START_REF] Hassouni | [END_REF]12,13,14]. With the electric field intensity, the pulse width w, the repetition rate f rep , the rise and fall times t r and t f , as well as the duty cycle D, defined as D = w.f rep are the main parameters that control plasma production and sustainability. A thorough investigation of pulsed microwave plasmas has been conducted on surface-wave-sustained plasmas. For example, Hubner et al. and Carbone et al. studied pulsed discharges with w down to 1 µs, t r down to a few tens of ns and D > 10 % [15,9].

A common feature of microwave discharges is the fact that plasma position is controlled and defined by the cavity size and the reactor design (position of the wave launcher, antennas, magnets, etc.). Recently, we have proposed a new kind of microwave plasma source -the so-called "plasma brush" -that allows to produce pulsed discharges with a control of their location independent of the reactor design [16]. In this case the idea is not to create a plasma filling the entire volume of a large plasma vessel, but rather to generate a localized plasma whose position is controlled in real time for local material processing of large objects. This source relies on the control of the waves inside the cavity by modifying the waveform of the input signal. It has been first demonstrated using time reversal (TR) [16] and recently using a more elaborated technique [17]. This requires two steps. In a TR experiment, an emitter first emits a short impulse and the information of the propagation of the waves (impulse response) is recorded using an array of sensors. This is a compulsory learning phase. Then, in order to produce a plasma discharge, this information is time reversed, amplified and re-emitted by the initial sensors. This leads to the reverse scenario: the waves, following the same path but backward, converge toward the initial location of the emission. This results in a spatiotemporal focusing of the electromagnetic energy and possibly a microwave breakdown.

This new plasma source possesses original features: the discharge is produced by focusing microwave energy on a small space (λ 0 /2 in diameter) isolated in the reverberant cavity during a nanosecond pulse width (w = 8 ns) in a repetitive mode (typically f rep = 6 kHz) corresponding to duty cycles lower than D = 5×10 -2 %. To the best of our knowledge, microwave plasma sources with such low rise time (in the order of the nanosecond) and such low duty cycle have never been studied. This paper aims to investigate the spatiotemporal behavior of the TR microwave plasmas. Some general features are presented related to the effect of the pressure (in the range 0.5 -4 Torr) and of the repetition rate. The signal s 1 (-t), i.e. the time reversed impulse response between the emitter located in the appendix and the receiver positioned randomly in the reverberant cavity, allows to focus a 8 ns peak near the receiver [16] and to reach plasma breakdown as observed by imaging through a faradized window.

Plasma source and diagnostic tools

The detailed description of the TR plasma source design and principle have been made in [16]. It is here briefly recalled for the sake of clarity.

Our experimental setup consists of a reverberant metallic cavity with dimensions 0.6 m × 0.6 m × 0.3 m, corresponding to 5λ 0 ×5λ 0 ×2.5λ 0 at the microwave carrier frequency f 0 = 2.55 GHz. As schematized in Figure 1, two coaxial probes acting as monopoles are used. The first one, located in an appendix at atmospheric pressure and connected to the main cavity through a glass window, is used as the emitter. The second one, located in the main cavity, is used as the receiver. The main cavity is pumped down using a primary pump. In these experiments, argon is used. It is injected at a constant flow rate controlled by a needle valve to a working pressure p ranging from 0.5 to 4 Torr. TR signal is defined following two steps [16]. First, a pulse of width w = 8 ns, modulated at a carrier frequency f 0 = 2.55 GHz, is applied on the receiver and the impulse response s 1 (t) between both monopoles is recorded. This signal is then time reversed s 1 (-t) and transmitted through the cavity by the emitter in the second step. This complex transmitted signal, shwon in Figure 1, generates a spatio-temporal focusing peak of w = 8 ns of dimension λ 0 /2 = 6 cm on the receiver (Figure 1). Note that, as demonstrated in [16], the location of the focusing inside the vacuum chamber is controlled by the waveform of the transmitted signal. As shown in Figure 1, the electrical signal received by the receiver is measured using an oscilloscope (Keysight MSO9254A). The power of the refocusing peak, defined as the square of the measured voltage over the input impedance of the oscilloscope (50 Ω), is proportional to the electric field near the receiving monopole at the focusing instant. However, due to the presence of a very diffusing environment between the monopoles (the reverberant cavity) and the use of broadband signals (bandwidth of 250 MHz), the relationship between this measured power and the value of the electric field is unknown and can not be easily estimated. Therefore, only relative measurements can be performed and power is given in arbitrary units.

In order to reach plasma breakdown condition, the signal s 1 (-t) is amplified using a traveling wave tube (TWT) pulsed power amplifier (TMD PTC7353) before being transmitted to the cavity [16]. Note that, in the measurements reported in this paper, the input signal s 1 (-t) is injected with the same maximal power level of about 1 kW, unless otherwise specified. The power of the refocusing peak, which is proportional to the maximal input power, that is found in this condition is defined as P = 7 arb. units.

In addition to the electrical measurements, optical diagnostics are also performed. It consists in imaging using a camera (PI-MAX-512) that enables to measure the spatio-temporal evolution of the light intensity emitted by the plasma during the signal period. All of the camera measurements presented in this paper are performed without any filter. The camera is triggered by the control device of the amplifier, to synchronize the 4 ns gate time to the TR signal, with the adjusted delay. Each picture results from the accumulation of 20 different gates with a repetition rate of 10 Hz. red curve corresponding to a condition below the breakdown condition (i.e., at atmospheric pressure, where there is no evidence of plasma production at naked eyes) shows the TR refocusing peak at t = 300 ns with a duration of about 8 ns, similar, as expected, to the one of the initial pulse width w [18]. This peak is surrounded by side lobes before and after the refocusing peak as classically reported in TR experiments in reverberant cavities [19,20,18]. The "temporal peak-to-noise" ratio used to characterize the quality of a TR process [18] is estimated for our experimental setup between 4 and 5 [16]. This ratio is linked to the quantity of uncorrelated information and depends on the bandwidth of the initial pulse (here of about 250 MHz) and of the cavity characteristics (volume compared to the wavelength and losses).

Experimental results

Electrical measurements

For comparison, the signal measured with plasma (p = 1.5 Torr, f rep = 6 kHz) is reported in blue on Figure 2. The signal remains very similar until the refocusing peak at t = 300 ns. A relative comparison of levels before or after the TR refocusing peak and plasma ignition is meaningful. At first sight, one could hope to obtain data such as the absorbed energy by comparing those signals. However, the presence of the plasma near the monopole changes its sensitivity, i.e., the relation between the electric field applied to the monopole and the resulting voltage actually measured with the oscilloscope. Furthermore, depending on the plasma density, the waves may be more or less reflected by the plasma, leading to a lower level of the measured signal. Hence, the analysis cannot go further and absolute data cannot be obtained from that measurements limiting to a global observation of signal lowering and phase-shifting when the plasma is ignited. Although for pulsed plasma sources the classical maintain curves, i.e., the minimal sustaining power versus pressure is not so relevant, as a single plasma discharge depends on the previous one, it enables to define at least the operating domain.

Here, the following procedure is used to determine the maintain power. At a fixed gas pressure and repetition rate, the discharge is ignited by TR using a high power refocusing peak. Then, the power is smoothly decreased to the minimum value allowing to maintain the discharge. Below this threshold power, the plasma vanishes. These maintain curves are determined for two different repetition rates as plotted on Figure 3(a). The curves show a minimum maintain power near the condition p = 1.5 Torr. Considering the classical theory of power deposition in high frequency plasmas, as described e.g. in [START_REF] Moisan | Physics of Collisional Plasmas: Introduction to High-Frequency Discharges[END_REF], the power density absorbed by the plasma is given by:

P abs = θ e n e
where

θ e = e 2 m e E 2 0 ν m ν 2 m + ω 2 0 (1)
with θ e the power absorbed by an electron, n e , m e and e the electron density, mass, and charge, respectively, ν m the electron neutral momentum collision frequency, ω 0 the wave pulsation, and E 0 the electric field needed to maintain the plasma. The minimum of maintain power around p = 1.5 Torr means that the energy transfer is maximal around this pressure, i.e., that the ratio νm ν 2 m +ω 2 is maximum for ν m = ω 0 . Considering that the bandwidth of the microwave signal (250 MHz) is small compared to the carrier frequency f 0 , ω 0 can be approximated to be 2πf 0 ≈ 16 × 10 9 rad/s. We can deduce a ν m of about 16 GHz at 1.5 Torr in our experiments, in good agreement with data found in the literature in these conditions [START_REF] Lieberman | Principles of Plasma Discharges and Materials Processing[END_REF].

Regarding the repetition rates, one can expect that with duty cycles D < 5×10 -2 %, the influence of the previous pulse would disappear and that the maintain power is independent of the repetion rate. Nevertheless, Figure 3(a) shows that as the repetition frequency increases, the power needed to maintain the discharge decreases, as reported in [START_REF] Dawson | [END_REF]24]. These measurements clearly show some evidence of the influence of the previous pulse on the next one, as classically attributed to "memory effects".

To fully describe this phenomenon, the lowest repetition rate allowing to maintain the discharge is determined. A similar procedure is used to determine this lowest repetition frequency. Here, the discharge is ignited by TR using a high power refocusing peak with a high repetition rate (P = 25 a.u. and f rep = 6 kHz). For each gas pressure, the repetition rate is smoothly decreased to the minimum threshold value maintaining the discharge. Below this threshold, the plasma vanishes. The lowest repetition rate allowing to maintain the discharge is plotted in Figure 3(b): it shows that the plasma is maintained for repetition rate as low as 12 Hz for the condition of maximal energy transfer found around p = Torr. It means that the effect of the previous pulse is still present after a time off of approximately 100 ms.

To further investigate these different mechanisms, the dynamics of the discharge is analyzed by imaging.

Imaging measurements

The spatio-temporal evolution of a typical TR plasma discharge has been monitored using imaging. In agreement with the previous sections, experimental conditions were chosen as p = 1.5 Torr and f rep = 6 kHz. Figure 4 reports different snapshots recorded along the pulse injection. It shows that the discharge is ignited near the metallic tip of the monopole. It spreads over centimetric distances around the monopole with a quasi-spherical shape and does not move over time. The light intensity profiles along the x and y axes are reported for different instants on Figure 5. In contrast with the light intensity that presents a symmetrical shape along the vertical cut (for x = 0 cm), along a horizontal cut (for y = 0 cm) one can observe a dissymmetry attributed to the presence of the monopole, located from x = 0 cm to the positive x direction, with a diameter of about 0.1 cm on the y axis, symmetrically around x axis. In addition, one can note that the average plasma radius, of about R plasma = 5 mm, is smaller than half of the microwave wavelength (λ 0 /2 around 6 cm), which is the dimension of the spatial focusing by TR [18].

Temporal evolutions of the light intensity as well as of the apparent plasma surface are observed around the TR peak. The light intensity integrated across the entire image plotted together with the absolute value of the voltage on Figure 6(a) shows that the light intensity sharply increases when the TR signal is injected up to a maximum at around t = 308 ns, i.e., at the end of the TR signal.

After the main TR peak, the light intensity decreases following an exponential decay (scarcely heckled by the effect of the signal side lobes), as shown by the exponential fit reported as a dotted line in Figure 6(a) and called in the following the "early postdischarge". In the condition p = 1.5 Torr, f rep = 6 kHz, this decay time is estimated around 77 ns. Finally, an attentive observation highlights that the plasma is ignited before the TR signal injection (e.g. at t = 252 ns i.e. 50 ns before), in a so-called "late postdischarge". Indeed, between 220 and 300 ns, light emission is observed and clearly correlated with intense side lobes. The phenomena occuring during these two periods are discussed later in Section 4. Similar behaviors are observed on the plasma surface as plotted in Figure 6(b): the spatial spreading of the plasma is strongly correlated to the signal measured on the monopole and mainly due to the TR refocused peak. Nevertheless, the surface does not decrease at the end of the TR peak (308 ns) but few tens of ns after with some minor "rebounds" due to The volume is defined as the sum of the pixels with light intensity higher than the threshold taken just above the noise level and equal to 0.3.10 -3 a.u. (see Figure 4). signal side lobes.

Influence of the pressure

Figure 7 shows images of the plasma light intensity at the end of the TR signal (t = 308 ns) for a repetition rate fixed at f rep = 6 kHz and different gaz pressure. The pressure has a clear effect on the discharge: when the pressure increases, the plasma becomes more and more compressed near the tip of the monopole. This behavior is typical of diffusion controlled plasmas. As the pressure increases, the collision frequency between charged particles and neutrals increases limiting the diffusion process. It is thus reasonable to assume that a similar process occurs during such transient TR plasmas. As it is localized in front of the monopole tip where the electric field is the higher, the light intensity emitted by the plasma also increases. The temporal evolutions of the integrated light intensity as plotted in Figure 8 show a key role of the working pressure. First, it shows that together with a plasma spatial limitation with the pressure, the maximum of plasma light intensity decreases with the working pressure. The early post-discharge is always characterized by an exponential decay with a decay time, as reported in Figure 9, of few tens of ns. In addition, this shows that this decay time decreases with the pressure from 100 ns at 0.5 Torr down to 65 ns at 4 Torr, following a p -1 evolution. Finally, during the late post-discharge, light emission previously correlated with the signal side lobes appears in every condition.

Influence of the repetition rate

The effect of the repetition rate on the discharge appears to be quite strong as illustrated in Figure 10 for two different repetition frequencies and three different pressures. The dotted lines correspond to the previous case with a repetition frequency of 6 kHz whereas the plain lines correspond to a repetition frequency of 60 kHz. For a given pressure, the integrated light emission increases when increasing the frequency. Moreover, it is more pronounced for higher pressure. In addition, the decay time of the light intensity after the main peak remains almost identical in the 6 -60 kHz range as summarized in Figure 9. The latter thus seems to be mainly related to the pressure. Finally, the early light emission before the TR peak is also tremendously affected by the increase of the frequency. At 60 kHz, its level can reach up to 40 % of the maximum light emission in good agreement with a memory effect between two TR refocusing peaks.

Discussion

To describe and analyse TR plasma production, the pulsed discharge will be studied starting from the main TR refocusing peak (t = 300 ns) until the next one.

Refocusing peak

As it was already noticed, the duration of the refocusing peak is quite unusual for microwave plasma sources. Indeed, thanks to TR a high microwave power level is injected around the monopole during w = 8 ns with a rise time lower than t r = 1 ns. With the TR technique, the electromagnetic energy injected in the cavity is focused in time and space. This main peak (at 300 ns on the plot), spatially centered on the monopole, triggers an important light emission due to the formation of a plasma discharge. Over the range of experimental conditions investigated, the electron density remains unknown. The only available experimental information is the difference observed in the voltage collected by the monopole after the main peak with and without plasma. This suggests that the electron density changes either the sensitivity of the monopole, i.e., the relationship between the electromagnetic waves at the monopole and the voltage collected, or the propagation of electromagnetic waves at the vicinity of the monopole, or both.

Early post-discharge

When the main peak has ended, the emission intensity decreases exponentially, only slightly disturbed by the side lobes of the microwave signal. On Figure 9, this decay time is inversely proportional to the pressure. Assuming that light emission predominantly comes from the spontaneous decay of Ar 2p i states and that these levels are mostly excited by electron-impact excitation of ground-state Ar atoms, the total emission intensity can be written as:

I T otal = i hc λ i f (λ i )k i (T e )n e n Ar ( 2
)
where h is the Planck constant, c is the speed of light, λ i is the wavelength of the Ar 2p i -1s j transitions, f (λ i ) is the optical response of the detection system at wavelength λ i , k i (T e ) is the reaction rate for electronimpact excitation of the Ar 2p i state from ground state Ar atoms (a function of the electron temperature, T e ), n e is the electron density, and n Ar is the number density of ground state Ar atoms. Based on Equation 2, the decay of the total emission intensity in the early post-discharge for a given pressure could be related to a decrease of either the electron density or electron temperature. We now look closer at these different possible causes.

In diffusion-controlled plasmas, charged particles losses are ruled by either free or ambipolar diffusion depending on the absolute number of the electron density (free diffusion in weakly ionized discharges and ambipolar diffusion in more strongly ionized plasmas). In such conditions, the electron density decay time is linked to the characteristic diffusion times that can be estimated using the following equation:

τ dif f = Λ 2 D dif f ( 3
)
where Λ is the diffusion length and D dif f is the free or ambipolar diffusion coefficient. In spherical geometry, the diffusion length writes Λ = L/π , where L is the characteristic size of the plasma vessel. In our case, there is no correlation between the typical size of the plasma and the vessel dimension. Consequently, L is set equal to a typical plasma radius, i.e. L = 5 mm. Considering an electron temperature T e of about 2 eV in a 1.5 Torr Ar plasma, the characteristic time is tens of nanoseconds for the free diffusion regime and tens of microseconds in the ambipolar regime. Note that in this last case, electron-ion recombination can shorten the electron density decay time as shown by Hubner et al. [15]. In any case, as the working pressure increases, the characteristic diffusion coefficient decreases such that the electron density decay time (Equation 3) increases, which is inconsistent with the set of data presented in Figure 9.

Electron energy relaxation following the extinction of the microwave signal can also induced a decrease of the total emission intensity in the early postdischarge. Neglecting power deposition and heat transport phenomena, the energy balance equation for electrons can be written as [START_REF] Lieberman | Principles of Plasma Discharges and Materials Processing[END_REF] :

dT e dt = -n n   j k j (T e ) j   (4)
where n n is the neutral density, k j (T e ) is the reaction rate for electron-atom collisions (elastic and inelastic collisions), and j is electron energy loss per electronatom collision.

Here, the summation takes into account electron momentum transfer collisions as well as electron-impact excitation and ionization processes [START_REF] Lieberman | Principles of Plasma Discharges and Materials Processing[END_REF]. From the corresponding set of reaction rates, this non-linear differential equation can be numerically resolved using the number density of ground state Ar atoms and the initial value of the electron temperature T e0 as the only adjustable parameters. It is worth highlighting that for argon plasmas with electron temperatures of a few eV, electron energy relaxation is dominated by excitation and ionization processes. Considering an initial electron temperature of 2 eV in a 1.5 Torr Ar plasma, T e (t) and thus I T otal (t) can thus be calculated using Equations 4 and 2, respectively. In such conditions, using the set of cross sections described in [25] the characteristic decay time of the total emission intensity is around 100 ns, which is comparable to the experimental data. Moreover, Equation 4 reveals that the electron energy relaxation time is inversely proportional to the number density of ground state Ar atoms, which is in very good agreement with the set of data presented in Figure 9. Therefore, over the range of experimental conditions investigated, the observed exponential decay time of the total emission intensity in the early post-discharge is mostly linked to the electron energy relaxation as the electron density decay time is much longer.

Late post-discharge

Between two pulses, once T e has relaxed as discussed in the previous section, one can assume a classical postdischarge evolution, with densities of charged species and metastable states evolving together. Experimental measurements reported in the previous sections clearly show the existence of a memory effect resulting from previous discharges and influencing the development of the following ones. This effect often occurs in discharges working in repetitive mode. It is related to the persistence of species such as electrons, ions, metastable states or radicals, from one discharge to the following one. Various mechanisms can be involved in the memory effects, their occurrence being strongly determined by the operating conditions such as the gas composition, pressure, discharge type... It can promote the ignition of the next discharge [26,9,27,28] e.g. because of the presence of seed electrons, or on the contrary perturb the next discharge e.g. if the remaining ion density is sufficient to disturb the electric field [29,30].

Assuming an electron temperature of 300 K, a new estimation of the characteristic diffusion times of charged particles can be provided. For this very low electron temperature, an approximated collision rate of 2 × 10 -15 m 3 s -1 is used [START_REF] Lieberman | Principles of Plasma Discharges and Materials Processing[END_REF]. The resulting characteristic diffusion times for a pressure of 1 Torr are 20 ns and 500 µs for the free and ambipolar diffusion respectively. Thus, if the plasma is sufficiently dense for the ambipolar process to rule the diffusion of free charges, a non-negligible amount of charges can remain in the medium until the next pulse. Electron-ion recombination can also play a role as previously mentioned. The dissociative recombination (DR) process Ar + 2 + e → Ar * + Ar generally plays a major role at the very beginning of the post-discharge whereas the collisional-radiative (CR) recombination Ar + + e + e → Ar * + e dominates at larger time scale when the electron temperatures becomes very low [31]. Their respective coefficients can be found in [32] for (DR) recombination and [START_REF] Biberman | Kinetics of Nonequilibrium Low-Temperature Plasmas[END_REF] for (CR) recombination. Depending on the plasma density, simple calculations indicate that electrons can remain in the medium for dozens of ms after the pulse. Nevertheless, it is interesting to note that these processes do not only result in charged particles losses but also in the production of metastable argon atoms. Metastable atoms coming either from the discharge or from recombination processes can play an important role in the memory effect as shown e.g. by Carbone et al. [9]. Based on [START_REF] Stefanović | [END_REF], the lifetime of the Ar( 3 P 2 , 1s 5 ) metastable state is essentially determined by the rate of two-and three-body collisional deexcitation with neutral argon. Indeed, the low electron temperature makes unlikely any process involving energetic electrons such as excitation towards upper levels or mixing of the metastable and resonant 1s argon state [31]. It results in an Ar( 3 P 2 , 1s 5 ) lifetime at 1.5 Torr around 10 ms. Thus, for f rep ≥ 100 Hz, metastables can greatly facilitate the breakdown before the next TR peak through step-wise processes. The presence of residual charges and metastable atoms in the late post-discharge are thus probably responsible for the observed memory effect. It explains the early light emission observed during the first side lobes of the voltage magnitude and the strong dependence of the breakdown behavior with respect to the operating conditions such as the repetition frequency. This behavior can be referred as ionization in moderate electric field.

Conclusions

Time reversal plasmas are pulsed discharges produced by refocusing microwave power. This paper brings the first insight on the microwave TR plasma properties. Indeed, TR allows to generate microwave plasmas as short as w = 8 ns with repetition rate down to f rep = 12 Hz resulting, to the best of our knowledge, in the very lowest duty cycle pulsed discharge with D < 10 -2 %. It works at low pressure with a condition of maximal energy transfer in argon around 1.5 Torr. The size of the generated plasmas was found with centimetric dimension, i.e., lower than the microwave wavelength. The latter is an important aspect of this source as the plasma and the cavity dimensions are uncorrelated making the mechanisms involved in ignition and afterglow phases unusual.

From the spatio-temporal dynamics of TR plasmas, three different steps are identified. First, when the microwave power is focused around the monopole, plasma breakdown condition is reached as observed by the sharp increase of the light intensity. The power deposition leading to the plasma ignition is followed by a post-discharge. Early, the light intensity was found to decrease exponentially with a characteristic decay time of about tens of nanoseconds inversely proportional to the pressure and independent on the repetition rate. This behavior was attributed to electron energy dissipation due to collisions with the background gas. In addition, a late post-discharge mechanism plays also a role in the discharge dynamics. It is probably related to the presence of energetic species and/or residual charges, which submitted to moderate electric field, i.e., of the side lobes -inherent to TR process and observed 100 ns before the main TR peak -allow the ionization of the gaz, especially in low pressure and high repetition frequency conditions. This strong dependence regarding experimental conditions confirms the importance of a memory effect. Future work will be devoted to a deeper understanding of its fundamental parameters.

Figure 1 .

 1 Figure1. Schematic of the TR plasma source. The signal s 1 (-t), i.e. the time reversed impulse response between the emitter located in the appendix and the receiver positioned randomly in the reverberant cavity, allows to focus a 8 ns peak near the receiver[16] and to reach plasma breakdown as observed by imaging through a faradized window.

  Two typical signals measured on the receiver during TR experiments are plotted on Figure2. The

Figure 2 .

 2 Figure 2. Electrical signals measured on the receiver during a TR experiment. The red curve corresponds to the TR signal at atmospheric pressure, i.e. in a condition below the plasma breakdown and the blue one in an Ar plasma (p = 1.5 Torr and frep = 6 kHz).

Figure 3 .

 3 Figure 3. (a) Minimum microwave power and (b) minimum repetition frequency of the TR peak needed to maintain a plasma as a function of the pressure. In both cases, these values were determined from the disappearance of the discharge at naked eyes.

Figure 4 .

 4 Figure 4. Images of plasma light intensity collected by imaging at p = 1.5 Torr and frep = 6 kHz for different times around the TR injection time (t = 300 ns). The light intensity is represented as a color map. White parts correspond to the region below a threshold level corresponding to the noise level of the camera measurements and equal to 0.3.10 -3 a.u.. The monopole outline is represented with dashed lines on the image at 500 ns.

Figure 5 .

 5 Figure 5. (a) Horizontal cut, meaning along the x axis for y = 0 cm, and (b) vertical cut, meaning along the y axis for x = 0 cm, for different times including the times presented in Figure 4.

Figure 6 .

 6 Figure 6. Temporal evolution of (a) the integrated light intensity and of (b) the plasma volume along with the absolute value of the voltage. The volume is defined as the sum of the pixels with light intensity higher than the threshold taken just above the noise level and equal to 0.3.10 -3 a.u. (see Figure4).

Figure 7 .

 7 Figure 7. Images of plasma light intensity collected by imaging after the TR peak instant (t = 308 ns) for different pressures.

Figure 8 .Figure 9 .

 89 Figure 8. Temporal evolution of the integrated light intensity.

Figure 10 .

 10 Figure 10. Evolution of the integrated temporal intensity for two different repetition rates, frep = 6 kHz in dotted line and frep = 60 kHz in plain line, and for three pressures p = 0.5 Torr (blue), p = 1.5 Torr (red) and p = 4 Torr (green).

Acknowledgments

The authors would like to acknowledge the DGA/AID for their funding support and the CEA-Gramat for the material support.


Marine Messant

Ginga Shimakawa

François Perreau

Chikahiro Miyake

Anja Krieger-Liszkay
email: anja.krieger-liszkay@cea.fr

Anja Krieger

Evolutive differentiation between alga-and plant-type plastid terminal oxidase: study of plastid terminal oxidase PTOX isoforms in Marchantia polymorpha

Keywords: plastid terminal oxidase, Marchantia polymorpha, plastoquinone pool, chlororphyll fluorescence, P700 absorption High lights - AOX, Alternative Oxidase, DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, NDH, Proton Gradient Regulation 5, PGRL1, PRG5-like protein, PIFT, Post-Illumination Fluorescence Transient, PQH2, plastoquinol, PQ, plastoquinone, PS, Photosystem, PTOX, Plastid Terminal Oxidase

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

One of the most important events in the evolution of photosynthetic organisms has been the colonization of lands. Alboresi et al. [START_REF] Alboresi | Balancing protection and efficiency in the regulation of photosynthetic electron transport across plant evolution[END_REF] have highlighted the great diversity of protection mechanisms that allow photosynthetic organisms to face environmental conditions that could induce oxidative stress and destruction of the photosynthetic apparatus (photoinhibition) when more light is absorbed than needed for photosynthesis. All oxygenic phototrophs ranging from algae to plants have the following regulation mechanisms in common: photosynthetic control limiting electron transport at the level of the cytochrome b6f complex when the pH in the lumen decreases below a certain threshold value [START_REF] Foyer | The mechanisms contributing to photosynthetic control of electron transport by carbon assimilation in leaves[END_REF]; mechanisms to dissipate excess energy in the antenna system, the so-called non-photochemical quenching (NPQ) [START_REF] Müller | Non-photochemical quenching. A response to excess light energy[END_REF]; the proton gradient regulation 5 (PGR5) and PGR5-like 1 (PGRL1) proteins having a supposed role in the cyclic electron flow [START_REF] Munekage | PGR5 is involved in cyclic electron flow around photosystem I and is essential for photoprotection in Arabidopsis[END_REF][START_REF] Suorsa | PRG5-PGRL1-dependant cyclic electron transport modulates linear electron transport rate in Arabidopsis thaliana[END_REF]; the chloroplast NADPH dehydrogenase (NDH) complex involved in cyclic electron flow and in chlororespiration [START_REF] Bennoun | Evidence for a respiratory chain in the chloroplast[END_REF][START_REF] Peltier | NDH-1 and NDH-2 plastoquinone reductases in oxygenic photosynthesis[END_REF] as well as the so-called Mehler reaction (the waterwater cycle) that occurs when photosystem I (PSI) reduces O2 to the superoxide anion radical which is then converted into O2 and H2O by detoxification enzymes [START_REF] Asada | The water-water cycle as alternative photon and electron sinks[END_REF]. Others, such as flavodiiron proteins (FLV) that reduce O2 to H2O at the acceptor side of PSI [START_REF] Helman | Genes encoding A-type flavoproteins are essential for photoreduction of O2 in cyanobacteria[END_REF][START_REF] Alboresi | Role and regulation of class-C flavodiiron proteins in photosynthetic organisms[END_REF] have disappeared in angiosperms, but are present from cyanobacteria up to gymnosperms. Finally, some proteins have evolved over the time into separate subclasses such as the plastid terminal oxidase (PTOX).

PTOX oxidizes plastoquinol (PQH2) and reduces O2 to H2O. It may play an important physiological role since the redox state of the plastoquinone (PQ) pool is a key point of photosynthesis regulation in many physiological conditions. In angiosperms, PTOX is often regarded in the literature as a "safety valve", allowing to avoid the over-reduction of PQ pool in conditions such as for example high light [START_REF] Laureau | Plastid Terminal Oxidase (PTOX) has the potential to act as a safety valve for excess excitation energy in the alpine plant species Ranunculus glacialis L[END_REF] or salt stress [START_REF] Stepien | Plastid terminal oxidase requires translocation to the grana stacks to act as a sink for electron transport[END_REF]. An increase of PTOX expression levels and of protein amounts have been observed upon exposure of plants to abiotic stress conditions like low or high temperature, drought etc. [START_REF] Feilke | Effect of Chlamydomonas plastid terminal oxidase 1 expressed in tobacco on photosynthetic electron transfer[END_REF][START_REF] Johnson | Plastid terminal oxidase as a route to improving plant stress tolerance: Known knowns and known unknowns[END_REF] Furthermore, PTOX is involved in carotenoid biosynthesis by maintaining PQ in its oxidized form [START_REF] Wetzel | Nuclear-organelle interactions: the immutans variegation mutant of Arabidopsis is plastid autonomous and impaired in carotenoid biosynthesis[END_REF][START_REF] Carol | Mutations in the Arabidopsis gene IMMUTANS cause a variegated phenotype by inactivating a chloroplast terminal oxidase associated with phytoene desaturation[END_REF]. In addition, PTOX acts as a terminal oxidase during chlororespiration [17] and in non-green plastids in etiorespiration [START_REF] Kambakam | PTOX mediates novel pathways of electron transport in etioplasts of Arabidopsis[END_REF] and chromorespiration [START_REF] Renato | Tomato fruit chromoplasts behave as respiratory bioenergetic organelles during ripening[END_REF][START_REF] Grabsztunowicz | Electron transport pathways in isolated chromoplasts from Narcissus pseudonarcissus[END_REF]. Finally, PTOX is important in the first steps of chloroplast biogenesis, based on the publication by [START_REF] Wetzel | Nuclear-organelle interactions: the immutans variegation mutant of Arabidopsis is plastid autonomous and impaired in carotenoid biosynthesis[END_REF] [START_REF] Wetzel | Nuclear-organelle interactions: the immutans variegation mutant of Arabidopsis is plastid autonomous and impaired in carotenoid biosynthesis[END_REF], highlighting that mutants have heteroplastidic cells. Phylogenetic trees on PTOX evolution have been published previously focussing on amino acid sequences comparison with other oxidases [START_REF] Mcdonald | Prokaryotic orthologues of mitochondrial alternative oxidase and plastid terminal oxidase[END_REF][START_REF] Finnegan | Prokaryotic origins for the mitochondrial alternative oxidase and plastid terminal oxidase nuclear genes[END_REF]. Nawrocki et al. [START_REF] Nawrocki | The plastid terminal oxidase: its elusive function points to multiple contributions to plastid physiology[END_REF] has investigated the evolutionary relationship among PTOX isoforms in various photosynthetic organisms, which suggested that PTOX in angiosperms has been evolved along with the evolution from green algae to plants with several independent duplication events. The appearance of the ancestral protein is not clearly identified. Atteia and coworkers [START_REF] Atteia | Identification of prokaryotic homologues indicates an endosymbiotic origin for the alternative oxidases of mitochondria (AOX) and chloroplasts (PTOX)[END_REF] hypothesized that the origin of PTOX predates primary endosymbiotic events, justifying its presence in the progeny of chlorophytes. Nevertheless, most articles agree that PTOX appears to diverge from its analogue, the mitochondrial alternative oxidase (AOX) that catalyzes the oxidation of ubiquinol by reducing O2 to H2O. AOXs are non-heme diiron carboxylate enzymes with six well conserved iron binding sites. It has been shown that this catalytic site including the ligands is conserved in PTOX [START_REF] Fu | Sequences required for the activity of PTOX (IMMUTANS), a plastid terminal oxidase[END_REF] but PTOX diverges from AOX by insertion of amino acids conserved in all PTOX-containing photosynthetic organisms.

Characterization of PTOX activity in vivo has been mainly conducted in angiosperms by the characterization of immutans and ghost mutants in Arabidopsis thaliana [START_REF] Wetzel | Nuclear-organelle interactions: the immutans variegation mutant of Arabidopsis is plastid autonomous and impaired in carotenoid biosynthesis[END_REF][START_REF] Meehan | Lhcb transcription is coordinated with cell size and chlorophyll accumulation (Studies on fluorescence-activated, cell-shorterpurified single cells from wild-type and immutans Arabidopsis thaliana)[END_REF][START_REF] Wetzel | Regulation of phytoene desaturase expression is independent of leaf pigment content in Arabidopsis thaliana[END_REF][START_REF] Wu | The IMMUTANs variegation locus of Arabidopsis defines a mitochondrial alternative oxidase homolog that functions during early chloroplast biogenesis[END_REF] and Solanum lycopersicum [START_REF] Barr | The GHOST terminal oxidase regulated developmental programming in tomato fruit[END_REF], respectively. These mutants are known to have a variegated phenotype which is more important when exposed to increasing light intensities [30] . In addition, PTOX activity has been characterized in vivo in the green alga Chlamydomonas reinhartii using a mutant affected in the isoform PTOX2 (Crptox2). Houilles-Vernes et al. [START_REF] Houilles-Vernes | Plastid terminal oxidase 2 (PTOX2) is the major oxidase involved in chlororespiration in Chlamydomonas[END_REF] have highlighted its involvement in chlororespiration. Crptox2 shows an over-reduction of PQ pool and is also affected in redistributing light excitation energy between photosystem II (PSII) and PSI (the socalled state transition). Using a single mutant of the cytochrome b6f complex as well as a double mutant of both cytochrome b6f and PTOX2, they concluded that PTOX2 is the major oxidase used to reoxidize PQ pool compared to PTOX1 in C. reinhartii. Crptox2 did not exhibit a defect in carotenoid contents and did not show a variegation phenotype such as immutans or ghost.

Houille-Vernes and coworkers concluded that PTOX1 is sufficient for regenerating oxidized PQ for phytoene desaturase while being less active than PTOX2 in controlling the redox poise of PQ pool during photosynthesis. No PTOX1 mutant of C. reinhartii has been characterized so far.

Liverworts can be regarded as the oldest living land plants or at least the land plants which have kept the most ancestral characters, among them Marchantia polymorpha has been used as a model with the sequencing of its genome and the development of genetic tools in order to better understand the evolution of land plants [START_REF] Shimamura | Marchantia polymorpha: Taxonomy, phylogeny and morphology of a model system[END_REF][START_REF] Bowman | Insights into land plant evolution garnered from the Marchantia polymorpha genome[END_REF]. Here we demonstrate that the two M. polymorpha PTOX isoforms are phylogenetically significantly different and representative of algal-and angiosperm-type of PTOX respectively, and if they differ in their activity by using mutants affected in one of the isoforms. First, we focussed on PTOX evolution through the green lineage using in silico approaches. Then, mutation of both proteins independently allowed us to highlight the partial redundancy of these proteins in vivo by performing mainly chlorophyll fluorescence analyses.

Materials and Methods

Phylogenetic analysis

31 amino acid sequences of PTOX and AOX in oxygenic phototrophs were aligned using Muscle [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF], and then the extra N-and C-terminal regions were removed based on the comparison to cyanobacterial PTOX sequences. The evolutionary history was inferred by using the Maximum Likelihood method [START_REF] Le | An improved general amino acid replacement matrix[END_REF].

The tree with the highest log likelihood (-8225.18) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms [36] to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA X [START_REF] Kumar | MEGA X: Molecular evolutionary genetics analysis across computing platforms[END_REF].

Material

A male accession of Marchantia polymorpha, Takaragaike (Tak)-1 and the mutants were asexually cultured on one-half-strength Gamborg's B5 agar medium [START_REF] Gamborg | Nutrient requirements of suspension cultures of soybean root cells[END_REF] under a light-dark cycle (16 h-light, 22°C, 120 µmol photons m -2 s -1 , white fluorescent lamp/8 h-dark, 20°C). For biochemical and physiological measurements, 3-4 week-old thalli were used.

Genome editing by CRISPR/Cas9 system

The PTOX mutants of M. polymorpha were prepared by CRISPR/Cas9-based targeted mutagenesis [START_REF] Sugano | Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha[END_REF]. The target regions (ptoxa.1, TTACACATGTACGAGAGCTT; ptoxb.1, CCAGGTTGTGAGTCCCAACC; ptoxb.2, AAGCACCTTGGTGGATTCT) were introduced into the Gateway entry vector pMpGE_En03, and finally included in the destination vector pMpGE010 and pMpGE011. Thalli of M. polymorpha were infected with Rhizobium radiobactor C58C1 (GV2260) transformed with the resulting plasmids, and the transformants were selected on half-strength Gamborg's B5 agar medium supplemented with hygromycin or chlorsulfuron [40] . The genome DNA was extracted from the thalli of mutants, and the mutations were confirmed by DNA sequencing (Supplementary Material Fig. S2). The mutants Δptoxa.1

and Δptoxb.1 were mainly used in this study as Δptoxa and Δptoxb. We confirmed the same phenotype of Δptoxb.2 as Δptoxb.1 by chlorophyll fluorescence measurements (Supplementry Material Fig. S3).

Pigment analysis

Liquid chromatography-UV-absorption analysis was conducted using a HPLC column (Uptisphere Strategy C18-HQ 250 × 3 mm 3 µm, Interchim, Montluçon, France) with a liquid chromatography chain HPLC/UV (Shimadzu, Tokyo, Japan), consisting of two pumps (LC-20AD), a sample manager (SIL-20AC HT), a column oven (CTO-20A) and an UV diode array detector (UVSPD-M20A) with a flow of 0.5 mL min -1 . The mobile phase A was composed of 10% water and 90% acetonitrile with 0.5% acetic acid and the mobile phase B was ethyl acetate with 0.5% acetic acid. The elution gradient was as follows: initially 10% B, 1 min 10% B; 25 min 95% B. Then the column was equilibrated to initial conditions for 15 min. Absorbance was monitored at 450 nm. Peak areas were normalized by known absorption factors for each compound at 450 nm, and amounts of each pigment were calculated by normalization to chlorophyll a.

Chlorophyll fluorescence analysis

Chlorophyll a fluorescence was measured in 5 min dark-adapted thalli using an Imaging-PAM (Fig. 2 and Supplementary Material Fig. S3A-C, and S4) or a Dual-PAM-100 fluorometer (Fig. 3 and4, and Supplementary Material Fig. S3D) (Walz, Effeltrich, Germany).

Fluorescence parameters during illumination with blue actinic light (230 µmol photons m -2 s -1 ) and recovery curves were measured after the determination of Fo and Fm parameters. Fm and Fmʹ were determined using saturating flashes (10,000 µmol photons m -2 s -1 , duration 300 ms): Fo, minimum and Fm, maximum fluorescence in a darkadapted sample; Fmʹ, maximum fluorescence and Fʹ, fluorescence emission from a light-adapted sample [START_REF] Baker | Chlorophyll fluorescence: a probe of photosynthesis in vivo[END_REF]. Post-Illumination Fluorescence Transient (PIFT) was monitored in the dark after exposure to actinic red light (400 µmol photons m -2 s -1 ) for 10 min. When indicated, far-red light was given. Quantum yield of photochemical energy conversion in PSII, Y(II), and NPQ were determined as (Fmʹ-Fʹ)/Fmʹ and (Fm-Fmʹ)/Fmʹ, respectively. Electron transport rate through PSII was calculated as the product of Y(II) multiplied with photon flux density of red actinic light, the sample absorptance, and the ratio of the absorbance by PSII to that by both PSI and PSII [START_REF] Krall | Relationship between photosystem II activity and CO2 fixation in leaves[END_REF]; we used 0.8 and 0.5, respectively, as these absorptance values which had been determined for leaves.

P700 measurement

P700 absorbance was measured using a Dual-PAM-100 fluorometer [START_REF] Klughammer | An improved method, using saturating light pulses, for the determination of photosystem I quantum yield via P700 + -absorbance changes at 830 nm[END_REF] (Walz, Effeltrich, Germany). Near-infrared measuring lights (830 and 870 nm) were applied to measure the transmittance of oxidized P700 (P700 + ). Five minutes dark-adapted thalli were exposed to actinic light and far-red light for 15s. Then, a saturating flash was given in the presence of farred light after switching off the light. First, this experiment was done on intact thalli which were then immersed for 5 min in 100 µM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or in a combination of DCMU and 500 µM methylviologen.

Immunoblots

Thalli were grinded in 50 mM HEPES pH 7.6. The extract was filtered through cheese, centrifuged at 4000g, the pellet was resuspended in 50 mM HEPES pH 7.6, the chlorophyll concentration was determined and this membrane fraction was used for analysis by SDS-PAGE (10% acrylamide) and immunoblotting. Proteins were blotted onto nitrocellulose membrane.

Labelling of the membranes with anti-PGR5, anti-NDH-B and anti-RbcL (Agrisera, Vännäs, Sweden) was carried out at room temperature in 1x PBS (50 mM Tris-HCl pH 7.6, 150 mM NaCl), 0.1% Tween 20 and 1% non-fat powder milk). After washing, bound antibodies were revealed with a peroxidase-linked secondary anti-rabbit antibody (Agrisera, Vännäs, Sweden) and visualized by enhanced chemiluminescence. PTOX antibodies raised against the proteins from Arabidopsis (gift from Marcel Kuntz, CEA Grenoble) and Chlamydomonas reinhardtii (gift from Xenie Johnson, CEA Cadarache) did not reveal bands in Marchantia polymorpha.

Results

Marchantia polymorpha contains two PTOX isoforms (MpPTOXs) that differ significantly in their sequences. The study of PTOX evolution has so far been limited to the comparison of their protein sequences with those of AOXs. However, the more recent evolution of different PTOX isoforms is just as interesting. Sequence comparison shows that MpPTOXa is closely related to PTOX of angiosperms that usually contain only one isoform, while MpPTOXb is more similar to PTOX isoforms found in the green alga Chlamydomonas reinhardtii (CrPTOXs). Since the PTOX isoform of Arabidopsis thaliana has been highlighted first, the isoforms resembling to it were defined here as "PTOXa" and the other isoforms as "PTOXb". MpPTOXa shows 96% identity with PTOX in Arabidopsis thaliana (AtPTOX), while MpPTOXa and MpPTOXb share only 53% identity. Since the two MpPTOX isoforms are so different in respect to their sequence homology, it can be hypothesized that these proteins have different physiological functions. A closer look at the sequences revealed that MpPTOXb and CrPTOXs (hereafter defined as "algatype PTOX") are larger than MpPTOXa and AtPTOX (hereafter defined as "plant-type PTOX"; Fig. 1A). CrPTOX1 and CrPTOX2 are composed of 471 and 416 amino acid residues.

MpPTOXb has 497 amino acid residues while MpPTOXa and AtPTOX contain only 376 and 352 ones (Supplementary Material, Figure S1).

31 amino-acid sequences of PTOX isoforms were investigated in a variety of photosynthetic organisms, including cyanobacteria, algae, and land plants. Although the Cterminal parts of the proteins are not particularly well conserved between these isoforms, the longer N-terminal part is mostly recognized in alga-type PTOX. Plant-type PTOX seems to have lost a part of the sequence between the transit peptide and the conserved motif containing the catalytic site (Fig. 1A), resulting in the shorter primary structures, although there are some exceptions such as Zea mays and Nannochloropsis oceanica. A phylogenetic tree including cyanobacterial PTOX, alga-type PTOX, plant-type PTOX, and AOX was built (Fig. 1B) based on the amino acid sequence alignment in the largely conserved region containing the catalytic site (the part "Fe-Fe" in Fig. 1A) among PTOX isoforms in a variety of photosynthetic organisms, clearly showing the three different PTOX groups. Several amino acid residues around the catalytic site are highly conserved in alga-and plant-type PTOX (Supplementary Material Fig. S1). The green alga Coccomyxa subellipsoidea has one PTOX belonging to the alga-type. Chlamydomonas reinhardtii and Volvox carteriv have two PTOX isoforms, both belonging to the alga-type. The other green alga Ostreococcus tauri has both alga-and planttype PTOX isoforms. Seed plants are proposed to be evolved from green algae through charophytes, bryophytes (liverworts, mosses, and hornworts), and pteridophytes (ferns), termed as photosynthetic green lineage [START_REF] Falkowski | The evolution of modern eukaryotic phytoplankton[END_REF]. Like in Ostreococcus tauri both alga-and plant-type PTOX isoforms are conserved in the charophyte alga Klebsormidium nitens, the liverwort Marchantia polymorpha, and the fern Azolla filiculoides, but alga-type one is lost in the fern Salvinia cucullata and in all angiosperms (Fig. 1B). In contrast to the green lineage, photosynthetic red lineage is defined as having evolved from red algae to secondary algae such as eustigmatophytes and diatoms [44] . The red alga Cyanidioschyzon merolae has two PTOX isoforms, both of which are closer to the alga-type ones, but the eustigmatophyte alga Nannochloropsis oceanica and two diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana have both, alga-and plant-type PTOX (Fig. 1B).

Since the two MpPTOX isoforms are so different in respect to their sequence homology, it can be hypothesized that these proteins have different physiological functions. To characterize the physiological role of the two PTOX isoforms in vivo, M. polymorpha has been chosen since it has one PTOX belonging to the plant-type and another one of the alga-type enzyme, and its position in the evolution of the green line is intermediate between green algae and plants [START_REF] Bowman | Insights into land plant evolution garnered from the Marchantia polymorpha genome[END_REF]. The mutants deficient in MpPTOXa and MpPTOXb, named hereafter Δptoxa and Δptoxb, were constructed using CRISPR/Cas9-genome editing system [START_REF] Sugano | Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha[END_REF]. The specific regions to MpPTOXa and MpPTOXb were determined as the target site for CRISPR/Cas9. The Marchantia transformants were generated by the infection of Agrobacterium containing binary vectors with young thalli in the presence of antibiotics (see "Materials and Methods"). The gemma from the transformants were cultured on agar plates with the antibiotics, and finally the frameshift mutation in the target regions were confirmed by DNA sequencing (Supplementary Material Fig. S2). Concerning that the mutation occurred at the later part of the gene in the mutant Δptoxb (Δptoxb.1), we constructed another line (Δptoxb.2) to confirm that the phenotype was due to the mutation of PTOXb (Supplementary Material Fig. S2 andS3). Similar to Crptox2, no variegation phenotype such as immutans or ghost was seen in both Δptoxa and Δptoxb mutants.

In order to characterize the role of PTOX on photosynthetic electron transport, measurements of chlorophyll fluorescence upon illumination and dark recovery were carried out. As shown in Fig. 2, upon illumination with actinic light Δptoxb showed a lower fluorescence level during saturating pulses while Δptoxa was very similar to Tak-1 (wild type).

When the actinic light was switched off, fluorescence levels in mutants decreased below the Fo level, more significantly for Δptoxb than for Δptoxa. This can be either caused by a higher level of NPQ leading to a quenching of Fo or chlororespiratory activity resulting in a higher reduction state of PQ pool, QB and QA in the dark giving rise to a higher fluorescence level. In this case, dark adaptation of the material prior to the fluorescence measurement does not assure that all reaction centers are in the "open state".

One way to determine chlororespiratory activity is to follow the so-called Post-Illumination Transient Fluorescence (PIFT) rise. Illumination with actinic light for a given time leads to the production of NADPH and ATP that can be consumed in the dark leading to a transient reduction of the PQ pool, QB and QA. Figure 3 shows after previous illumination in the dark a strong increase in the fluorescence level (PIFT) in both mutants detected using low intensity measuring light. Applying far-red light removed the PIFT, confirming the high reduction state of the PQ pool in both mutants.

If one calculated Fv/Fm values using dark-adapted material and without taking into account that the Fo may be not correct, one obtains values for Fv/Fm being 0.56 ± 0.04 for Δptoxa and 0.61 ± 0.04 for Δptoxb (n = 6). If these values were determined after far-red light illumination, 0.69 ± 0.01 and 0.71 ± 0.01 were obtained for Δptoxa and Δptoxb (n = 6), similar to those measured for Tak-1. In the literature, the Fv/Fm value in Tak-1 was determined to be approximately 0.8 [START_REF] Shimakawa | The liverwort, Marchantia, drives alternative electron flow using a flavodiiron protein to protect PSI[END_REF], but, when determined with an Imaging-PAM as done for the data shown in Fig. 2, a lower value was determined even when higher plants like Arabidopsis thaliana were used. This lower value is caused by a too high intensity of the measuring light in the Imaging-PAM system.

Next, we measured NPQ and the electron transport rate as a function of actinic light intensity (Fig. 4). Δptoxb showed a lower electron transport rate and a higher NPQ while Δptoxa behaved like Tak-1. Pigment analysis showed that the amounts of certain carotenoids such as neoxanthin and violaxanthin were slightly larger in Δptoxb (Fig. 5). The increase in pigment content of the violaxanthin pool may explain the stronger NPQ found in this genotype. It may also indicate that Δptoxb is more stressed than Δptoxa or Tak-1, leading to a lower electron transport rate. However, no higher susceptibility to photoinhibition was observed in Δptoxb (Supplementary Material Fig. S4).

When the PQ pool is more oxidized in the dark, it is expected that P700 + is accumulating to a higher level in response to the illumination at a given light intensity. As can be seen in Figure 6, more P700 + accumulated in Tak-1 upon illumination with either actinic or far-red light. Both mutants showed the same lower level of P700 oxidation suggesting that PQ pool was more reduced, if we consider that there was no PSI acceptor side limitation thanks to the presence of FLV [START_REF] Shimakawa | The liverwort, Marchantia, drives alternative electron flow using a flavodiiron protein to protect PSI[END_REF]. There are nevertheless striking differences between Δptoxa and Δptoxb.

Upon illumination with actinic light that excites both photosystems and favours linear electron flow, the oxidation of P700 was delayed in Δptoxa compared with Δptoxb, especially at the initial rise. This seems to indicate that the PQ pool was more reduced in Δptoxa, suggesting a higher activity of this enzyme. However, when illuminated with far-red light that excites preferentially PSI and favours cyclic electron flow, P700 was faster oxidized in Δptoxa than in Δptoxb. To show the electron donation to P700 and the reduction state of the PQ pool in the absence of electron input from PSII, we added DCMU, an inhibitor of PSII blocking the QBbinding pocket. The re-reduction of P700 + after far-red illumination was much faster in Δptoxa than in Δptoxb and Tak-1, with Δptoxb being slightly faster than Tak-1 (Fig. 7). This indicates again a higher reduction state of the PQ pool in Δptoxa. In the absence of inhibitors, the rereduction of P700 + after far-red illumination was slightly faster in both mutants than in Tak-1.

As expected, addition of the electron acceptor methylviologen together with DCMU abolished the differences between the mutants and Tak-1 (Supplementary Material Fig. S5). Immunoblots probing for NDH and PGR5 showed an increase in the amount of NDH in Δptoxa and of PGR5 in both, Δptoxa and Δptoxb.

Discussion

Here, we categorized PTOX isoforms in oxygenic phototrophs into three different groups (Fig. 1): cyanobacterial, alga-type, and plant-type PTOXs. Based on the present phylogenetic analysis, two different types of PTOX isoforms had been inherited to basal land plants from green algae like Ostreococcus tauri that is often described as one of the ancient members of the photosynthetic green lineage [START_REF] Derelle | Genome analysis of the smallest free-living eukaryote Ostreococcus tauri unveils many unique features[END_REF]. Both alga-and plant-type PTOXs are conserved in most basal land plants, while angiosperms have lost the alga-type PTOX. Also, the fern Salvinia cucullata has only a plant-type PTOX (Fig. 1B), and another fern, Azolla filiculoides, lost a conserved ligand of the non-heme diiron center, most probably leading to the loss or alteration in the catalytic activity in its alga-type PTOX (Supplementary Material Fig. S1). These facts suggest that alga-type PTOX are already not functional in some pteridophytes. Both plant-type and alga-type PTOX are also found in secondary algae that are supposed to be derived from red algae in photosynthetic red lineage (Fig. 1B). In this study, we show that the red alga Cyanidioschyzon merolae has only two isoforms of alga-type PTOX. This may not be the case for all red algae and improved open genome sources of red algae may uncover the red algae species having both alga-and plant-type PTOX isoforms in near future. The appearance of the ancestral protein for PTOX has not been identified to date. However, many studies based on genomic sequence analysis seem to favor the hypothesis of PTOX acquisition during the primary endosymbiotic event, suggesting that this protein appeared much earlier than the eukaryotic lineage [START_REF] Mcdonald | Prokaryotic orthologues of mitochondrial alternative oxidase and plastid terminal oxidase[END_REF][START_REF] Finnegan | Prokaryotic origins for the mitochondrial alternative oxidase and plastid terminal oxidase nuclear genes[END_REF][START_REF] Atteia | Identification of prokaryotic homologues indicates an endosymbiotic origin for the alternative oxidases of mitochondria (AOX) and chloroplasts (PTOX)[END_REF][START_REF] Mcdonald | Flexibility in photosynthetic electron transport: the physiological role of plastoquinol terminal oxidase (PTOX)[END_REF]. The question is when these alga-and plant-type PTOXs had been separated in the evolutionary history of photosynthetic organisms. It can be speculated that a single protein has been duplicated in eukaryotic algae, finally resulting in alga-and planttype PTOXs, if we consider that cyanobacterial PTOX is closer to plant-type PTOX but can be clearly distinguished from both alga-and plant-type PTOXs. Interestingly, most of alga-type PTOX isoforms show longer N-terminal regions, resulting in bigger size of proteins (Fig. 1A and Supplementary Material Fig. S1). In addition, some amino acid residues are specifically conserved in each type of PTOX even in the motif containing catalytic sites (Supplementary Material Fig. S1), reflected also in the phylogenetic tree (Fig. 1B). Differences in enzymatic activities of these different types of PTOX remain to be characterized in future, possibly uncovering why angiosperms had lost alga-type PTOX during the evolutionary history.

The results shown here (Figs. 2, 3, 6, 7) indicate that both MpPTOX isoforms have redundant functions and are both important for controlling the redox state of the PQ pool.

Especially the PIFT showing the slow electron donation to the PQ pool via the NDH complex during chlororespiration is similar in both mutants. However, there are small differences when comparing Δptoxa and Δptoxb. Δptoxa shows no difference in electron transport rate and NPQ compared with Tak1, whereas in Δptoxb the electron transport rate is lower while NPQ is higher. The lower electron transport rate and the higher NPQ and violaxanthin amount in Δptoxb suggest that this mutant is slightly stressed although no obvious phenotype was visible.

Crptox2 mutant showed a lower fitness when cells were grown under phototrophic conditions and was more susceptible to photoinhibition [START_REF] Houilles-Vernes | Plastid terminal oxidase 2 (PTOX2) is the major oxidase involved in chlororespiration in Chlamydomonas[END_REF]. In addition, Crptox2 was affected in the state transition being preferentially in state 2. In contrast, Δptoxb in M. polymorpha showed neither signs of a defect in the state transition nor a higher susceptibility to high light (Supplementary Material Fig. S4) suggesting that the plant-type MpPTOXa could compensate better for the absence of MpPTOXb than it was the case in C. reinhardtii possessing only two alga-type isoforms. In respect to carotenoid synthesis MpPTOXb seems to replace perfectly MpPTOXa since the absence of MpPTOXa did not alter the carotenoid composition neither induced a variegated phenotype as described in angiosperms for immutans and ghost.

P700 measurements show that the isoform MpPTOXa is more efficient in keeping the PQ pool oxidized since the oxidation of P700 + in actinic light was retarded in Δptoxa compared with Δptoxb (Fig. 6). However, under illumination with far-red light the opposite was observed with slower P700 oxidation in Δptoxb than in Δptoxa. Actinic light drives mainly reduction of the PQ pool by linear electron transport, while far-red light oxidizes the PQ pool and may also favour PQ reduction by cyclic electron flow. This leads us to hypothesize that PTOXa is more active in linear electron transport and PTOXb more in cyclic electron transport. This different behaviour of the PTOX isoforms at the different light qualities can be rationalized by different enzymatic properties, with MpPTOXb having a higher affinity (low Km-value) for PQH2 but a lower Vmax than MpPTOXa. This would make MpPTOXa the more efficient enzyme keeping the PQ pool oxidized under conditions favouring linear electron flow. The proposition of a high activity of MpPTOXa is supported by the fact that, in the presence of DCMU, the reduction of P700 + is accelerated in Δptoxa (Fig. 7). However, the amount of the NDH complex is also increased in Δptoxa (Fig. 8), and a higher chlororespiratory activity may also explain the higher reduction state of the PQ pool in this mutant. Since cyclic electron flow in far-red light is expected to be relatively low, the reduction state of the PQ pool may also be relatively low and MpPTOXb may become under these conditions highly efficient to keep the PQ pool oxidized.

Alternatively, the two PTOX isoforms may be heterogeneously distributed within the thylakoid membrane. MpPTOXb may be preferentially associated with supercomplexes involved in cyclic flow composed of PSI, ferredoxin, cytochrome b6f and a putative ferredoxin quinone reductase possibly linked to PGR5 and PGRL1 [START_REF] Iwai | Isolation of the elusive supercomplex that rives cyclic electron flow in photosynthesis[END_REF][START_REF] Steinbeck | Structure of a PSI-LHCI-cyt b6f supercomplex in Chlamydomonas reinhardtii promoting cyclic electron flow under anaerobic conditions[END_REF] while MpPTOXa may be preferentially associated with components of linear electron flow. A third possibility to explain the different behaviour of the two mutants may simply be a difference in the protein level of the two isoforms.

Both mutants have a higher amount of PGR5 (Fig. 8), indicating that the loss of a part of PTOX activity triggers the accumulation of higher amounts PGR5 and likely increases the capacity for cyclic electron transport. Cyclic electron transport may be important in these mutants to compensate for a reduction of the PTOX-dependent chlororespiratory activity and to keep the size of the proton motive force.

PTOX activity may also be regulated as a function of the physiological state of the chloroplast. Stepien and Johnson [START_REF] Stepien | Plastid terminal oxidase requires translocation to the grana stacks to act as a sink for electron transport[END_REF] have demonstrated that plant PTOX is localized under control conditions at the stroma lamellae while it is localized at the grana stacks under salt stress in the halophile Eutrema salsugineum. Furthermore, it has been shown that AtPTOX could be found partially in a soluble protein fraction and in a membrane-associated fraction. The strength of the membrane association of AtPTOX was shown to depend on the proton motive force [START_REF] Feilke | Effect of Chlamydomonas plastid terminal oxidase 1 expressed in tobacco on photosynthetic electron transfer[END_REF][START_REF] Bolte | Dynamics of the localization of the plastid terminal oxidase PTOX inside the chloroplast[END_REF]. A difference between plant-type and alga-type PTOX may lie in the strength of the membrane association. Nawrocki and co-workers [START_REF] Nawrocki | The plastid terminal oxidase: its elusive function points to multiple contributions to plastid physiology[END_REF] carried out a modelling of the structure of CrPTOX2 based on the x-ray structure of AOX from Trypanosoma brucei (TbAOX) [START_REF] Shiba | Structure of the trypanosome cyanide-insensitive alternative oxidase[END_REF].

Shiba and coworkers [START_REF] Shiba | Structure of the trypanosome cyanide-insensitive alternative oxidase[END_REF] proposed that two alpha helices upstream of the catalytic site of TbAOX are involved in the anchoring to the inner mitochondrial membrane. According to the calculation by Nawrocki et al. [START_REF] Nawrocki | The plastid terminal oxidase: its elusive function points to multiple contributions to plastid physiology[END_REF] the amphipathic helices of CrPTOX dock to a depth of approximately 8Å into the membrane. They suggest that the N-terminal extension in CrPTOX contributes to the interaction of the two monomers of the protein. We propose here that the increase in length may as well strengthen the association of the protein to the upper membrane leaflet. Further studies are needed to investigate the difference in plant-type and alga-type PTOX regarding their attachment to the membrane and differences in the regulation of the activity of these isoforms.

Fig. 1 .

 1 Fig. 1. Phylogenetic analysis of plastid terminal oxidase (PTOX) and alternative oxidase (AOX) isoforms in oxygenic phototrophs. (A) Typical primary structures of plant-and alga-type PTOX. TP, transit peptide; Fe-Fe, highly conserved region containing the catalytic site; C, Cterminal region; X, an extended region often observed in alga-type PTOX. (B) Phylogenetic tree of the evolutionary relationship between plastid terminal oxidase (PTOX) in oxygenic phototrophs. Each highly conserved region (Fe-Fe) was used for the alignment analysis to build the phylogenetic tree (Supplementary Material Fig. S2). Branch lengths correspond to the evolutionary distances. Organisms included in this phylogenetic analysis are cyanobacteria (Gloeobacter violaceus, Anabaena cylindrica, and Nostoc sp.), green algae (Coccomyxa subellipsoidea, Ostreococcus tauri, Chlamydomonas reinhardtii, and Volvox carteriv), the charophyte alga Klebsormidium nitens, the liverwort Marchantia polymorpha, ferns (Azolla filiculoides and Salvinia cucullata), angiosperms (Arabidopsis thaliana, Oryza sativa, Zostera marina, and Zea mays), the eustigmatophyte alga Nannochloropsis oceanica, diatoms (Phaeodactylum tricornutum and Thalassiosira pseudonana), and the red alga Cyanidioschyzon merolae. Alternative oxidase (AOX) in Chlamydomonas reinhardtii (AOX1) and Arabidopsis thaliana (AOX1A) are also included in the analysis.

Fig. 2 .

 2 Fig. 2. Response of chlorophyll fluorescence of wild type and mutants deficient in plastid terminal oxidase to illumination by blue actinic light (230 µmol photons m -2 s -1 ). A: M. polymorpha wild type (Tak-1), B: Δptoxa, C: Δptoxb). Before the measurements, the plants were dark-adapted for 5 min. Dashed lines indicate the chlorophyll fluorescence levels before the first saturation flash. Blue bar indicates the duration of the actinic light. Measurements were conducted independently three times (biological replicates), and representative traces are shown.

Fig. 3 .

 3 Fig. 3. Typical trace of chlorophyll fluorescence change after switching off the actinic red light (400 µmol photons m -2 s -1 , 10 min) at 0 min in wild type (Tak-1; black) and the mutants deficient in plastid terminal oxidases a (Δptoxa; red) and b (Δptoxb; blue). Measurements were conducted independently three times (biological replicates), and representative traces are

Fig. 4 .

 4 Fig. 4. Dependence of electron transport rate through PSII (A) and non-photochemical quenching (NPQ; B) on light intensity at steady-state photosynthesis (4 min after onset of actinic light) in M. polymorpha wild type (Tak-1; black) and the mutants deficient in plastid terminal oxidases a (Δptoxa; red) and b (Δptoxb; blue). Data are represented as the mean ± SD, n = 3 (biological replicates).

Fig. 5 .

 5 Fig. 5. Pigment content in thalli of M. polymorpha wild type (Tak-1; black) and the mutants deficient in plastid terminal oxidases a (Δptoxa; red) and b (Δptoxb; blue) determined by HPLC-UV measurement. Data are represented as the mean ± SD, n = 3 (technical replicates). Statistically significant differences (*) in Δptoxb compared to Tak-1 were determined by Student's t-test (p < 0.05).

Fig. 6 .

 6 Fig. 6. Oxidation of P700 in response to light in M. polymorpha wild type (Tak-1; black) and the mutants deficient in plastid terminal oxidases a (Δptoxa; red) and b (Δptoxb; blue). Red actinic light (500 µmol photons m -2 s -1 ) and far-red light were given as indicated by red and white bars. Experiments were conducted independently three times (biological replicates), and the representative traces are shown. The maximum oxidation amplitude of P700 was determined by a saturation flash in at the end of the far-red light at 40 s.

Fig. 7 .

 7 Fig. 7. P700 + re-reduction after illumination with far-red light in DCMU-treated thalli. Representative traces of M. polymorpha wild type (Tak-1, black) and the mutants deficient in plastid terminal oxidases a (Δptoxa; red) and b (Δptoxb; blue) are shown.

Fig. 8 .

 8 Fig. 8. PGR5 and NDH-B content in membranes fractions of M. polymorpha wild type (Tak-1) and the mutants deficient in plastid terminal oxidases a (Δptoxa) and b (Δptoxb) analyzed by SDS-PAGE and immunoblotting using specific antibodies. As loading controls, the signals

Figure 6 29

 6 Figure 6

Figure 7 Figure 8 Δptoxb

 78 Figure 7

Acknowledgements

We thank Dr. Kimitsune Ishizaki (Kobe University, Japan) for kindly giving us the Tak-1 strain. The I2BC and the IJPB benefit from the support of Saclay Plant Sciences-SPS (ANR-17-EUR-0007) and the I2BC from the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05. This work has benefited from the support of IJPB's Plant Observatory technological platforms. G.S. is supported by a JSPS oversea research fellowship (201860126).