Amer Hamidi-Sakr

Daniel Schiefer

Sangeetha Covindarassou

Laure Biniek

Michael Sommer

Martin Brinkmann

Highly oriented and crystalline films of a phenylsubstituted polythiophene prepared by epitaxy : structural model and influence of molecular weight

Abstract

The large majority of semiconducting polymers based on poly(alkylthiophene)s with either linear or branched alkyl side chains are reported to -stack in their crystalline phases. In regioregular poly(3-(2,5-dioctylphenyl)thiophene) (PDOPT), however,  interactions are absent due to the presence of the bulky 2,5-dioctylphenyl side groups.

In this work, high levels of crystallinity and orientation are created in thin films of PDOPT aligned on substrates of naphthalene by slow directional epitaxial crystallization of the side chains. Depending on molecular weight, both edge-on and flat-on lamellar crystals are obtained. As for poly(3-hexylthiophene) (P3HT), electron microscopy imaging reveals a transition from extended to folded chain crystallization in PDOPT for Mn  12.7 kDa. The high orientation and crystallinity result in high anisotropy in UV-vis absorption and photoluminescence with well defined vibronic structures. The singlecrystal-like electron diffraction patterns are further used to refine a structural model of PDOPT (a=29.09 Å, b=10.45 Å, c=7.72 Å , ===90° and space group P21/c). Uniquely, crystalline PDOPT features perfectly planarized chains despite the absence of -stacking between polythiophene backbones. The octyl side chains are interdigitated and crystallize in a dense subcell which is compared to that of other semiconducting polythiophenes e.g. form I poly(3-hexylthiophene) and poly(2,5-bis(3-dodecyl-2yl)thieno [3,2-b]thiophene) to draw a trend between overall crystallinity and density of the side chain packing. Conjugated polymers (CP) are of central importance in material science due to their unique optical and electronic properties that can be tuned by macromolecular engineering. [START_REF] Holliday | Advances in Charge Carrier Mobilities of Semiconducting Polymers Used in Organic Transistors[END_REF][START_REF] Klauk | Organic electronics : Materials, manufacturing and applications[END_REF] Moreover, CPs are easy to process from solution to yield large thin film surfaces at low cost. However, small changes in the side chain substituents modify not only their processing from solution but also their supramolecular packing which controls the final electronic properties in thin films. [START_REF] Biniek | Impact of the Alkyl Side Chains on the Optoelectronic Properties of a Series of Photovoltaic Low-Band-Gap Copolymers[END_REF][START_REF] Aasmundveit | Structural Ordering in Phenyl-Substituted Polythiophenes[END_REF][START_REF] Yiu | Side-Chain Tunability of Furan-Containing Low-Band-Gap Polymers Provides Control of Structural Order in Efficient Solar Cells[END_REF][START_REF] Kline | Critical Role of Side-Chain Attachment Density on the Order and Device Performance of Polythiophenes[END_REF][START_REF] Lee | Boosting the Ambipolar Performance of Solution-Processable Polymer Semiconductors via Hybrid Side-Chain Engineering[END_REF][START_REF] Kang | Record High Hole Mobility in Polymer Semiconductors via Side-Chain Engineering[END_REF] The role and impact of side chain regioregularity and ordering is essential as it determines the degree of planarization of the conjugated backbone, and the extent of -stacking upon crystallization. [START_REF] Pandey | Regioregularity vs regiodandomness : effect on photocarrier transport in poly(3hexylthiophene)[END_REF] The disordering of side chains, e.g. upon a crystal to liquid crystal phase transition, can induce a twisting of the backbone, i.e. a rupture of conjugation that may result in a characteristic thermochromism. [START_REF] Roux | Thermochromic Properties of Polythiophene Derivatives: Formation of Localized and Delocalized Conformational Defects[END_REF] As a general rule, disorder in the -stacking of semiconducting polymers such as P3HT and PBTTT determines the energetic disorder that controls charge mobility in layers of -stacked chains. [START_REF] Noriega | A general relationship between disorder, aggregation and charge transport in conjugated polymers[END_REF] At present, the influence of the side chain structure on charge transport is only poorly understood. One of the reasons might be that precise structural models of polythiophenes with different side chains are still relatively scarce. The inherent disorder of these semi-crystalline materials rests in the coexistence of crystalline and amorphous domains, and makes their structural refinement particularly challenging. Structure modeling based on grazing incidence X-ray diffraction (GIXD) or TEM data showed that alkyl side chains can be either interdigitated [START_REF] Rahimi | Controllable Processes for Generating Large Single Crystals of Poly(3-hexylthiophene)[END_REF][START_REF] Cho | Three-Dimensional Packing Structure and Electronic Properties of Biaxially Oriented Poly(2,5-bis(3-alkylthiophene-2-yl)thieno[3,2b]thiophene) Films[END_REF][14] as in PBTTT and form II of P3HT, or not interdigitated as in form I P3HT. [START_REF] Kayunkid | Structural Model of Regioregular Poly(3hexylthiophene) Obtained by Electron Diffraction Analysis[END_REF] Recently, structural models of a P3HT oligomer and an analogue with branched 2-ethylhexyl side chains were investigated. [START_REF] Himmerberger | Role of Side-Chain Branching on Thin-Film Structure and Electronic Properties of Polythiophenes[END_REF] Valuable structural data can be obtained from GIXD on non-oriented films and used to elaborate some structural models of CPs. However, limited structural data can impede a reliable space group determination which is essential in the structural modeling of CPs as it defines the symmetry relation between monomeric units in a unit cell. Space group identification can only be done properly by using single-crystal-like diffraction patterns obtained from highly oriented or, more rarely, from CP single crystals. [START_REF] Rahimi | Controllable Processes for Generating Large Single Crystals of Poly(3-hexylthiophene)[END_REF] For all these reasons, it is essential to develop means to grow polymer films with well defined orientation and high crystallinity that yield single-crystal-like diffraction patterns suitable for structural refinement. Different strategies have been used to reach such characteristics in CP films. To cite but a few: epitaxy and directional epitaxial crystallization (DEC) [START_REF] Brinkmann | Orientation of Regioregular Poly(3-hexylthiophene) by Directional Solidification: A Simple Method to Reveal the Semicrystalline Structure of a Conjugated Polymer[END_REF][START_REF] Brinkmann | Effect of Molecular Weight on the Structure and Morphology of Oriented Thin Films of Regioregular Poly(3-hexylthiophene) Grown by Directional Epitaxial Solidification[END_REF] , friction transfer 19 , controlled solvent vapor annealing (SVA) [START_REF] Crossland | Anisotropic Charge Transport in Spherulitic Poly(3-hexylthiophene) Films[END_REF][START_REF] Fischer | Highly Crystalline Films of PCPDTBT with Branched Side Chains by Solvent Vapor Crystallization: Influence on Opto-Electronic Properties[END_REF] and high temperature rubbing. [START_REF] Biniek | Large Scale Alignment and Charge Transport Anisotropy of pBTTT Films Oriented by High Temperature Rubbing[END_REF][START_REF] Biniek | High-Temperature Rubbing: A Versatile Method to Align π-Conjugated Polymers without Alignment Substrate[END_REF][START_REF] Hamidi Sakr | Precise Control of Lamellar Thickness in Highly Oriented Regioregular Poly(3-Hexylthiophene) Thin Films Prepared by High-Temperature Rubbing: Correlations with Optical Properties and Charge Transport[END_REF] Controlled crystallization through selfseeding helped to refine the structure of form II of P3HT from well defined single crystal domains. [START_REF] Rahimi | Controllable Processes for Generating Large Single Crystals of Poly(3-hexylthiophene)[END_REF] However, the preparation of such single crystals is usually tedious and therefore alternative approaches to prepare highly crystalline CP films are required.

Epitaxial crystallization of CPs is an effective method to reach very high order and crystallinity as demonstrated recently. [START_REF] Brinkmann | Orientation of Regioregular Poly(3-hexylthiophene) by Directional Solidification: A Simple Method to Reveal the Semicrystalline Structure of a Conjugated Polymer[END_REF][START_REF] Brinkmann | Effect of Molecular Weight on the Structure and Morphology of Oriented Thin Films of Regioregular Poly(3-hexylthiophene) Grown by Directional Epitaxial Solidification[END_REF][START_REF] Fischer | Highly Crystalline Films of PCPDTBT with Branched Side Chains by Solvent Vapor Crystallization: Influence on Opto-Electronic Properties[END_REF][START_REF] Brinkmann | Highly Oriented and Nanotextured Films of Regioregular Poly(3-hexylthiophene) Grown by Epitaxy on the Nanostructured Surface of an Aromatic Substrate[END_REF][START_REF] Brinkmann | Understanding the Structure and Crystallization of Regioregular Poly (3-hexylthiophene) from the Perspective of Epitaxy[END_REF][START_REF] Brinkmann | Directional Epitaxial Crystallization and Tentative Crystal Structure of Poly(9,9'-di-n-octyl-2,7-fluorene)[END_REF][START_REF] Brinkmann | Segregated versus Mixed Interchain Stacking in Highly Oriented Films of Naphthalene Diimide Bithiophene Copolymers[END_REF] Directional epitaxial crystallization of P3HT and PFO was obtained with 1,3,5-trichlorobenzene (TCB) as a substrate. [START_REF] Brinkmann | Orientation of Regioregular Poly(3-hexylthiophene) by Directional Solidification: A Simple Method to Reveal the Semicrystalline Structure of a Conjugated Polymer[END_REF][START_REF] Brinkmann | Directional Epitaxial Crystallization and Tentative Crystal Structure of Poly(9,9'-di-n-octyl-2,7-fluorene)[END_REF] When using slow directional crystallization of TCB, it was possible to obtain very high orientation of both polymers over large surfaces. [START_REF] Brinkmann | Understanding the Structure and Crystallization of Regioregular Poly (3-hexylthiophene) from the Perspective of Epitaxy[END_REF] Such highly oriented systems are ideally suited for structural refinement using electron diffraction. [START_REF] Kayunkid | Structural Model of Regioregular Poly(3hexylthiophene) Obtained by Electron Diffraction Analysis[END_REF] In epitaxied PFO films, almost single-crystal like diffraction patterns were used to establish a structural model of the -polymorph. [START_REF] Brinkmann | Directional Epitaxial Crystallization and Tentative Crystal Structure of Poly(9,9'-di-n-octyl-2,7-fluorene)[END_REF] Herein we report that epitaxy on naphthalene can lead to high degrees of orientation and crystallinity of a phenyl-substituted poly(thiophene), poly(3-(2,5dioctylphenyl)thiophene) (PDOPT). PDOPT crystallizes well despite the presence of bulky 2,5-dioctylphenyl side chains (see Figure 1.a). [START_REF] Aasmundveit | Structural Ordering in Phenyl-Substituted Polythiophenes[END_REF] Sommer and coworkers reported on the first synthesis of highly regioregular PDOPT and the growth of micron-sized spherulites using isothermal crystallization, for which a high degree of regioregularity was crucial. 29,30 In the present contribution, thin films of highly oriented and crystalline PDOPT with single-crystal-like diffraction patterns are prepared by slow directional epitaxial crystallization on naphthalene for different samples with molecular weights Mn,SEC= 6-38 kDa. Liquid naphthalene is a solvent for PDOPT but becomes a substrate for epitaxy of PDOPT in its crystalline form. The influence of Mn on the film morphology has been investigated by TEM and electron diffraction. From single crystal-like diffraction patterns a structural model is determined which shows that the polythiophene backbone is highly planar despite the absence of π-π ineractions. Polarized UV-vis and fluorescence measurements are also used to investigate the impact of orientation and crystallinity on the thin film optical properties.

II. Experimental section a) Materials

Naphthalene with 99% purity was purchased from Sigma Aldrich. The synthesis of the PDOPT samples proceeds via Kumada catalyst transfer polycondensation and is described in detail elsewhere. 29 The study of the molecular weight of PDOPT on its structural characteristics was made on a set of six polymer batches with M n in the range 6.2-37.6 kDa. The relevant macromolecular parameters were obtained by size exclusion chromatography (SEC) and are summarized in Table 1 (Figure S1).

b) Thin film preparation

Epitaxied thin films are prepared by using a modified directional epitaxial crystallization method. In brief, it consists of using a crystallizable solvent (naphthalene) playing successively the role of (i) the solvent for PDOPT in its liquid form and (ii) once crystallized, the substrate for the epitaxial crystallization of the polymer. The process implies several steps described elsewhere. 31 In brief, a solution of PDOPT (

c) TEM analysis

Oriented areas are selected by optical microscopy (Leica DMR-X microscope) for TEM analysis. A thin carbon film is evaporated onto the PDOPT films using an Auto 306 evaporator (Edwards). The sample is removed from the glass substrate by floating the films on a 5 wt% aqueous HF solution and recovered on copper grids. TEM is performed in bright field, high resolution and diffraction modes using a CM12 Philips microscope equipped with a MVIII (Soft Imaging System) CCD camera. Specific conditions for low dose diffraction are given elsewhere. 32,33 Calibration of reticular distances is performed by using an oriented poly(tetrafluoroethylene) thin film.

Molecular modelling is performed on a Silicon Graphic station using the Cerius2 program (Accelrys). A trial-and-error procedure was used to refine the crystal structure of PDOPT, following the methodology used previously for P3HT 34 , PF8 35 and PCPDTBT 36 and conjugated co-oligomers 37 . For each step of the trial-and-error method, the molecular geometry is optimized using the "clean" procedure of the Cerius2 3.0 Program (additional information on this procedure is available in the User's guide on Modeling Environment). For the single crystal diffraction calculations, a crystal thickness of 30 nm was used. Energy minimization was performed using the minimizer module of the Cerius2 program and the United Force Field UFF1.1.

d) Polarized UV-vis and fluorescence spectroscopy

Polarized UV-vis absorption spectroscopy was performed using a Varian Carry 5000 UV-VIS-NIR spectrometer with polarized incident light. The photoluminescence of PDOPT films was measured with a Jobin -Yvon FluoroMax 4 spectrofluorimeter with a slit width of 3 nm for both the excitation and the detection. Excitation was performed at a wavelength of 540 nm.

III. Results and discussion

Evidence for orientation (POM, UV-vis spectroscopy)

Directional epitaxial crystallization is an effective method to achieve high orientation and alignment of conjugated semiconducting polymers. 1,3,5-trichlorobenzene in its liquid form (T>63°C) is a perfect solvent for P3HT and PFO whereas when it crystallizes, it becomes the substrate for the epitaxial growth of the polymer. The effectivity of a crystallizable solvent such as TCB is related to the possibility for an epitaxial crystallization i.e. a registry between the substrate and the polymer crystal unit cell parameters in at least one direction (1D epitaxy). [START_REF] Brinkmann | Orientation of Regioregular Poly(3-hexylthiophene) by Directional Solidification: A Simple Method to Reveal the Semicrystalline Structure of a Conjugated Polymer[END_REF] Further evidence for the high alignment is supported by polarized UV-Vis and photoluminescence (PL). Figure 2 illustrates the high anisotropy observed in absorption and PL for PDOPT films with 6 kDa and 21 kDa oriented by slow-DEC on NA, resulting in dichroic ratios in the range 8-10 for absorption and PL. These values are higher than those observed by micro-photoluminescence on spherulites, presumably due to a higher level of in-plane orientation of crystalline domains in the epitaxied films whereas a splay of the lamellar orientations is common in spherulites. 30 Most interesting is the well defined vibronic structure in the PL of all PDOPT films. The PL spectra are well modeled with a single Franck-Condon progression (see Figure S2). The obtained Huang-Rhys factor S in the range 0.69 -0.77 indicate a small electron-phonon coupling. [START_REF] Pope | Electronic processes in organic crystals[END_REF] Fitting with a single F-C progression suggests that a single emitting species is involved in PDOPT films. In contrast, thin films of P3HT cooled from the melt have two H-type aggregate emitting species. [START_REF] Panzer | [END_REF] This difference might be related to the absence of  stacking in PDOPT for which inter-chain excitonic coupling might be very small as opposed to P3HT. However, the UV-vis absorption spectrum for Pol // cPDOPT (see Figure 2) is very similar to that of P3HT and cannot be accounted for by a single vibronic progression : it contains the signature of excitonic coupling in the crystal.

From previous analyses of the vibronic structure in crystalline P3HT, the predominance of the 0-0 vibronic component over the 0-1, is indicative of highly planarized chain segments in the crystaline domains. This is accordingly also the case for PDOPT as demonstrated further by TEM. The absorption spectra for Pol  cPDOPT are less structured and in general blue-shifted relative to Pol//cPDOPT. This results from the semi-crystalline lamellar morphology of the PDOPT films. For Pol  cPDOPT , the UV-vis absorption spectrum is dominated by the non structured absorption of amorphous, nonoriented PDOPT domains as also observed previously in oriented PBTTT and P3HT films. [START_REF] Biniek | Large Scale Alignment and Charge Transport Anisotropy of pBTTT Films Oriented by High Temperature Rubbing[END_REF][START_REF] Hamidi Sakr | Precise Control of Lamellar Thickness in Highly Oriented Regioregular Poly(3-Hexylthiophene) Thin Films Prepared by High-Temperature Rubbing: Correlations with Optical Properties and Charge Transport[END_REF] This result points to the semi-crystalline structure of PDOPT, that is again further evidenced by TEM. indicates the in-plane direction of the PDOPT chains.

Effect of molecular weight on structure, morphology and optical properties a) Lamellar periodicity

The morphology of the epitaxied films was observed in Bright field TEM (Figure 4a-d). Figure F.d features the evolution of the total lamellar period L as a function of Mn. [START_REF] Brinkmann | Orientation of Regioregular Poly(3-hexylthiophene) by Directional Solidification: A Simple Method to Reveal the Semicrystalline Structure of a Conjugated Polymer[END_REF] The regular lamellar structure is characteristic of semi-crystalline polymers and is similar to that observed previously for P3HT films oriented on TCB. [START_REF] Brinkmann | Orientation of Regioregular Poly(3-hexylthiophene) by Directional Solidification: A Simple Method to Reveal the Semicrystalline Structure of a Conjugated Polymer[END_REF] The contrast in the BF images is between amorphous interlamellar zones and crystalline lamellae of PDOPT. [START_REF] Brinkmann | Orientation of Regioregular Poly(3-hexylthiophene) by Directional Solidification: A Simple Method to Reveal the Semicrystalline Structure of a Conjugated Polymer[END_REF] In all PDOPT films, highly oriented lamellae are observed with edge-on orientation. PDOPT lamellae with very uniform thickness are formed for Mn=7.8 kDa whereas for Mn≥ 21 kDa strong fluctuations in the lamellar thickness result in peak broadening in the fast Fourier transforms (FFT) of the BF images. Molecular weight dispersity might be responsible for this behaviour, at least in part (see Table 1). 29 The lamellar periods L obtained from FFTs of the TEM images reveal a clear trend with Mn,

shown in Figure 3.e. For Mn≤12.7 kDa, L scales with Mn whereas for Mn≥21 kDa, L saturates at 29 nm. This trend is similar to that observed for epitaxied P3HT films (see A Gibbs-Thomson analysis was attempted using the melting temperatures determined by DSC 29 and the lamellar periods measured by TEM for PDOPT samples of different Mn.

The correlation between the melting temperature Tm and the lamellar period L of polymer crystals follows usually the equation [START_REF] Cheng | Phase transistions in polymers. The role of metastable states[END_REF] :

 T m  T m 0 1 2 e L H m         (1)
where is the equilibrium melting temperature of a PDOPT crystal with infinite size, ∆Hm is the equilibrium enthalpy of melting for a crystal of infinite size and e is the crystal surface free energy. As seen in

b) Electron diffraction

In our previous study on the impact of Mn on the structure and morphology of P3HT films prepared by slow DEC, the samples of lower Mn showed the most well defined ED patterns, indicating that crystallinity was most pronounced for shorter chains. Figure 5 shows the evolution of the ED patterns of oriented PDOPT films with increasing Mn. All the ED patterns show the same characteristic reflections, indicating that a single polymorph with one dominant contact plane is formed on naphthalene.

This same polymorph is present in films prepared by doctor-blading and isothermal crystallization. The main reflections are indexed with an orthorhombic unit cell (a=29.1Å, b=10.5 Å and c=7.72Å, ===90°). All ED patterns correspond to the [1 0 0] zone with very intense 0 0 ±2, 0 ±2 1 and 0 2 ±1 reflections forming a hexagonal-like pattern (d002 = 3.86 Å and d021 = 4.33 Å). As shown hereafter, this pseudo-hexagonal pattern is similar to that of polyethylene single crystals and is a fingerprint of the regular packing of the n-octyl side chains. [START_REF] Dorset | Crystallography of the polymethylene chain : an inquiry into the structure of waxes[END_REF] The pattern dominates the ED of PDOPT films because the orientation of crystalline lamellae is such that the electron beam is parallel to the n-octyl side chain axis, with the polythiophene backbones lying in the plane of the substrate (see Figure 5.e). This contact plane is also observed in the doctor bladed films and the spherulites after isothermal crystallization on SiO2 substrate. With increasing Mn, the presence of higher order reflections with dhkl up to 1.3 Å -1 indicates a progressive improvement of crystalline order. This trend is a consequence of the increased total lamellar period. Larger values of L imply larger crystalline domains in the chain axis (c axis) direction, hence, improved diffraction of crystallites. PDOPT behaves therefore differently from P3HT whose crystallinity in epitaxied films was observed to decrease with increasing Mn. [START_REF] Brinkmann | Effect of Molecular Weight on the Structure and Morphology of Oriented Thin Films of Regioregular Poly(3-hexylthiophene) Grown by Directional Epitaxial Solidification[END_REF] This comparison points at the beneficial role of the 2,5-dioctylphenyl groups on the crystallization of higher molecular weight PDOPT.

For PDOPT with Mn= 6.2 kDa, the majority of crystals have a (1 0 0) contact plane on naphthalene i.e. like other molecular weights. However in some parts of the samples (essentially the thinnest parts of the films), additional and different ED patterns corresponding to a minority fraction of PDOPT crystals with two different contact planes on naphthalene are identified. For flat-on crystals, the quantized changes in grey levels reveal multilamellar morphologies. The presence of the PDOPT crystals with standing chains for the 6.2kDa sample reminds the case of P3HT films epitaxied on TCB. [START_REF] Brinkmann | Effect of Molecular Weight on the Structure and Morphology of Oriented Thin Films of Regioregular Poly(3-hexylthiophene) Grown by Directional Epitaxial Solidification[END_REF][START_REF] Brinkmann | Understanding the Structure and Crystallization of Regioregular Poly (3-hexylthiophene) from the Perspective of Epitaxy[END_REF] Indeed, it has been shown that P3HT crystals with standing chains are also observed for epitaxied films with low molecular weight (7.3 kDa). [START_REF] Brinkmann | Effect of Molecular Weight on the Structure and Morphology of Oriented Thin Films of Regioregular Poly(3-hexylthiophene) Grown by Directional Epitaxial Solidification[END_REF] In the case of P3HT, it was suggested that a mechanism of homoepitaxy could explain the growth of flat-on lamellar crystals on edge-on crystals. Such a mechanism is only possible for polymers crystallizing with extended chains i.e.

when the surface of the lamellar crystals is not made of folded i.e. disordered chains. In other words, the presence of PDOPT crystals with standing chain for 6.2 kDa is a further evidence for extended chain crystallization for such low Mn.

c) Epitaxy

The relative orientation of PDOPT on NA corresponds to : (1 0 0)PDOPT // (0 0 1)NA and cPDOPT // aNA (the growth direction of naphthalene using a Bridgman type of growth corresponds to the (1 0 0)NA direction 38 ). To determine if epitaxy is a possible mechanism for the observed orientation of PDOPT chains, it is necessary to compare the unit cell parameters of the PDOPT overlayer and the naphthalene substrate. The possibility for epitaxy is usually determined by the mismatch factors between the unit cell parameters of the overlayer and the substrate. 44 These parameters can be evaluated for PDOPT on NA :

-along the chain direction : cPDOPT -aNA/cPDOPT  4 % -perpendicular to the chain direction : bPDOPT -2bNA/bPDOPT  12%.

Only the mismatch parameter along the chain direction of PDOPT is below 10% which is commonly considered as the upper limit for epitaxial growth. Accordingly, it seems that the epitaxy of PDOPT on NA involves essentially a 1D epitaxial orientation mechanism.

This situation is close to that observed for P3HT epitaxy on TCB for which cP3HT//cTCB with a mismatch cP3HT-2*cTCB/cP3HT  2.5%. [START_REF] Brinkmann | Orientation of Regioregular Poly(3-hexylthiophene) by Directional Solidification: A Simple Method to Reveal the Semicrystalline Structure of a Conjugated Polymer[END_REF] As an illustration, Figure 7

Structural model of PDOPT

The control of orientation and crystallinity in epitaxied films of PDOPT opens the route to a structural refinement of this polymer. The trial-and-error approach followed herein is similar to that used in our previous studies on form I P3HT [START_REF] Kayunkid | Structural Model of Regioregular Poly(3hexylthiophene) Obtained by Electron Diffraction Analysis[END_REF] , PFO [START_REF] Brinkmann | Directional Epitaxial Crystallization and Tentative Crystal Structure of Poly(9,9'-di-n-octyl-2,7-fluorene)[END_REF] and PCPDTBT/F-PCPDTBT. [START_REF] Fischer | Highly Crystalline Films of PCPDTBT with Branched Side Chains by Solvent Vapor Crystallization: Influence on Opto-Electronic Properties[END_REF] First, the different diffraction patterns obtained for different zone axes are used to determine the selection rules of the observed reflections. The 35 -40 independent reflections obtained from the collected ED patterns are indexed using an orthorhombic unit cell with parameters a=29.1Å, b=10.5 Å and c=7.72Å, ===90°, i.e.

unit cell dimensions essentially similar to those proposed by Aasmundveit et al. on the basis of powder diffraction data. [START_REF] Aasmundveit | Structural Ordering in Phenyl-Substituted Polythiophenes[END_REF] In their case, the absence of diffraction data along the chain direction hampered the space group identification. This uncertainty is presently aleviated. The following selection rules are identified and point to the P21/c space group [START_REF]Supporting informations Highly oriented and crystalline films of a phenylsubstituted polythiophene prepared by epitaxy : structural model and influence of molecular weight Amer Hamidi-Sakr 1 , Daniel Schiefer 2 , Sangeetha Covindarassou 1[END_REF] :

(h 0 0) with h=2n (k 0 0) with k=2n (0 0 l) with l=2n and (h k 0) with h+k=2n

Accordingly, the unit cell contains four monomer units related by the symmetry conditions (x, y, z), (-x+1/2, -y+1/2, z+1/2), (-x, -y, -z) and (x+1/2, y+1/2, -z+1/2). As expected for a regioregular PDOPT chain, successive monomers in a chain are related by a 21 screw axis.

The structural refinement was performed in two steps. First, the best agreement between calculated and experimental ED pattern was sought by a simple trial-and-error approach. In a second step the lattice energy of this unit cell was minimized. The experimental and the calculated ED patterns for the [1 0 0] and the [0 0 1] zones before and after energy minimization are given in Figure 8. The corresponding projections of the structural models are shown in Figure 9. After energy minimization, a final stable structure that is very similar to the initial model (see Figure 9) was obtained. The calculated ED patterns for both models are similar, indicating that the trial-and-error approach leads to structural models that are relatively accurate first order solutions even though the exact packing of the n-octyl side chains could only be obtained after energy minimization. Closer inspection of the calculated ED patterns show some differences because the energy minimization results in a small reorientation of the noctyl side chains in the unit cell. In the initial model obtained by trial-and-error approach, n-octyl side chains are set parallel to the a axis, as in the model of Aasmundveit et al. [START_REF] Aasmundveit | Structural Ordering in Phenyl-Substituted Polythiophenes[END_REF] However, in this structure, the packing of n-octyl side chains was not optimized. Only for the second model i.e. after energy minimization is a satisfactory packing of the side chains obtained at the expense of a small tilt of the n-octyl side chains to the a axis. The optimized model yields a substantial improvement of the [0 0 1] zone, in particular the low intensity of the ±1 1 0 reflection is well reproduced while in the initial model it was strongly overestimated. A contrario, the strong 0 ±1 2 reflection is lost after energy minimization in the calculated [1 0 0] zone.

The refined model of PDOPT is close to that proposed earlier by Aasmundveit et al. [START_REF] Aasmundveit | Structural Ordering in Phenyl-Substituted Polythiophenes[END_REF] The major improvement lies in the clearcut determination of the space group given the highly defined ED patterns obtained for the [1 0 0] and [0 0 1] zones. In particular, all other symmetry relations between monomers in the unit cell proposed as alternative models by Aasmundveit et al. can be discarded because they lead to some unobserved reflections in the diffraction patterns, e.g. 0 1 0 and 0 0 1 reflections. [START_REF] Aasmundveit | Structural Ordering in Phenyl-Substituted Polythiophenes[END_REF] PDOPT chains have a perfectly planarized backbone, in agreement with the conclusions drawn from optical properties. Contrary to most semi-conducting polymers such as P3HT or PBTTT, the backbone planarization of PDOPT is not enforced by - stacking. The 2,5dioctylphenyl side groups are oriented in a plane perpendicular to the chain axis and the n-octyl side chains form a strongly interdigitated sublattice. The plane of the phenyls is almost perpendicular to the plane of the polythiophene backbone. Successive phenyl groups along the b axis are however not in a situation of favorable  stacking. The top view of the n-octyl side chain sublattice is shown in Figure 10.g, its parameters are a0=bPDOPT=10.5Å and b0=cPDOPT=7.72Å. Such a sublattice can be compared to that of regular polymethylenes e.g. the M, T and O sublattices. [START_REF] Dorset | Crystallography of the polymethylene chain : an inquiry into the structure of waxes[END_REF] As a matter of fact, the n-octyl sublattice in PDOPT is twice a classical O subcell with slightly larger unit cell parameters (typically a0=4.96Å and b0=7.40Å for O subcell). [START_REF] Dorset | Crystallography of the polymethylene chain : an inquiry into the structure of waxes[END_REF] As a consequence, the average molecular area per n-octyl stem (20.17 Å 2 ) is close to that observed for the classical O polymethylene subcell (18.4 Å 2 ). [START_REF] Dorset | Crystallography of the polymethylene chain : an inquiry into the structure of waxes[END_REF] The doubling of the subcell area is due to the fact that two n-octyl side chains are linked to the phenyl group as illustrated in Figure 10.

IV. Discussion

At present, only a limited number of structures of CPs have been determined

precisely by using X-ray diffraction, electron diffraction or molecular mechanics methods. [START_REF] Cho | Three-Dimensional Packing Structure and Electronic Properties of Biaxially Oriented Poly(2,5-bis(3-alkylthiophene-2-yl)thieno[3,2b]thiophene) Films[END_REF][START_REF] Kayunkid | Structural Model of Regioregular Poly(3hexylthiophene) Obtained by Electron Diffraction Analysis[END_REF] However, to correlate properties such as charge transport to the packing of the chains in a crystal, it is important to determine the type of sublattice formed by the alkyl side chains, especially because the side chain ordering/disordering can impact the planarization of the conjugated backbone and hence electronic properties of the CPs. In this perspective, it is instructive to compare the sublattices formed by alkyl side chains in a number of poly(thiophene)s for which structural models have been recently

proposed e.g. PDOPT, form I P3HT and C12-PBTTT. Figure 10 compares the projections of the polymethylene subcells along the alkyl chain direction for P3HT, PDOPT and C12-PBTTT. The two former subcells are extracted from a structural refinement whereas for C12-PBTTT it was obtained by molecular mechanics calculations. [START_REF] Cho | Three-Dimensional Packing Structure and Electronic Properties of Biaxially Oriented Poly(2,5-bis(3-alkylthiophene-2-yl)thieno[3,2b]thiophene) Films[END_REF] For all three structures, the alkyl side chains form a different sublattice. [START_REF] Dorset | Crystallography of the polymethylene chain : an inquiry into the structure of waxes[END_REF] Following the nomenclature of polymethylene lattices by Kitaigorodski, form I P3HT and PDOPT show an O-type sublattice (a0=4.96Å, b0=4.4Å, 0=90°) whereas PBTTT has an M-type sublattice (a0=4.2Å, b0=4.4Å, 0=111°). For these sublattices, the density of the alkyl side chain (area per stem) follows the progression : PDOPT (20.17Å 2 ), PBTTT (24.5 Å 2 ) and form I P3HT (27 Å 2 ). As a matter of fact, neither P3HT nor PBTTT did show such high levels of crystallinity as observed here in PDOPT. As an example, Figure S5 shows one of the most well defined ED patterns of an oriented P3HT film crystallized by high-T rubbing at 180°C. The ED pattern of highly oriented and crystalline P3HT always shows fuzzy reflections, indicative of disorder in the side chain packing. The same is true for C12-PBTTT which shows only a few mixed indices reflections in thin films. [START_REF] Biniek | Large Scale Alignment and Charge Transport Anisotropy of pBTTT Films Oriented by High Temperature Rubbing[END_REF] In PDOPT, neither streaking, nor fuzzy reflections, two common signatures of structural disorder, are observed in the epitaxied films. This comparison points at a highly ordered n-octyl side chain sublattice in PDOPT compared to loose and defective side chain packing of non interdigitated n-hexyl side chains in form I P3HT. [START_REF] Kayunkid | Structural Model of Regioregular Poly(3hexylthiophene) Obtained by Electron Diffraction Analysis[END_REF] The comparison between the three polymers suggests that the overall crystallinity of the CPs is correlated, at least in part, to the density of the side chain sublattice: the higher the density of side chain packing, the larger the overall crystallinity of the CP.

In regular polymethylene sublattices, well defined offsets along the polymethylene chain direction are observed. [START_REF] Dorset | Crystallography of the polymethylene chain : an inquiry into the structure of waxes[END_REF] The analysis of the three refined polythiophenes indicates that the grafted extremity of the side chains to the polythiophene backbone imposes a sterical limit that impedes the alkyl side chains to adopt the most favorable packing along their long axis. In other words, the polymethylene sublattice might be optimized with respect to the packing of the chains in the plane perpendicular to the polymethylene chain direction, but not in the direction parallel to it.

The results on PDOPT further suggest that planarization of conjugated backbones can be enforced in systems in which  stacking is absent, provided that the alkyl side chains can form a dense sublattice. The phenyl unit between the solubilizing alkyl chains and the conjugated backbone is particularly beneficial because it helps reducing the direct impact of the packing constraints imposed by the ordering of the alkyl side chains on the polythiophene backbone conformation, a prevailing situation observed in poly(alkylthiophene)s such as P3HT and C12-PBTTT for which the alkyl side chains are directly linked to the conjugated backbone. In addition, in the case of P3HT, Figure S4 features the evolution of the UV-vis absorption spectrum and max versus molecular weight of PDOPT. Regarding, the UV-vis absorption, one observes a 10 nm red shift of the 0-0 peak position for the higher Mn of 21 kDa. The position of the 0-0 component in epitaxied P3HT films showed a similar red-shift with increasing Mn. 31 From the evolution of lamellar period Lp verus Mn, this trend is possibly related to the impact of reduced crystal size along the chain direction on the position of the 0-0 peak position. Higher Mn PDOPT show longer planarized chain segments in the crystalline domains, hence a larger conjugation length which should translate to a red-shift of the 0-0 peak.

Figure

  Figure for Title of content

38 Figure 1

 381 Figure1shows the polarized optical microscopy images of two highly oriented

Figure 1 .

 1 Figure 1. (a) Chemical formula of PDOPT. (b) Setup used for the growth of oriented

Figure 2 .

 2 Figure 2. Comparison of the polarized absorption and photoluminescence of highly

Figure 3 .

 3 Figure 3. Comparison of the bright field TEM images and corresponding diffraction

Figure 4 .

 4 Figure 4.e). It indicates that PDOPT chains with Mn≤ 12.7 kDa tend to crystallize with

Figure 4 .

 4 Figure 4. a) -d) : Evolution of the semicrystalline lamellar morphology in oriented

  Figure 4.f, a very good agreement is obtained when the experimental values of Tm measured by DSC are plotted versus 1/L, yielding a value =127.6°C±1°C. It must be stressed that this relation seems to hold true even if the melting temperatures are measured for PDOPT powders and not epitaxied PDOPT films, suggesting that the lamellar periods observed in the powders of the different PDOPT samples should be comparable to those observed in the films grown by epitaxy. At present, the value of ∆Hm for a 100% crystalline PDOPT sample remains unknown, making the extraction of e from equation (1) difficult. Additional efforts are currently in progress to determine ∆Hm of 100% crystalline PDOPT.

Figure 5 .

 5 Figure 5. Comparison of the ED patterns for oriented films of PDOPT prepared by

Figure 6 ,

 6 Figure 6e anf 6f). In both cases, the lamellae show up as light grey areas in the

Figure 6 .

 6 Figure 6. Selected area diffraction patterns and associated morphologies in 6.2kDa

  features the section profile of the PDOPT/NA interface. It shows that ordering of the n-octyl side chains by 2D epitaxy on naphthalene is clearly the driving force for the alignment of PDOPT since the polythiophene backbones are approx. 10 Å away from the NA contact plane.

Figure 7 .

 7 Figure 7. Structural model showing the section profile of the (1 0 0)PDOPT/(0 0 1)NA

Figure 8 .

 8 Figure 8. Comparison of the experimental electron diffraction patterns of highly

Figure 9 .

 9 Figure 9. Projections of the refined structure of PDOPT along the c, b and a unit cell axes

  regioregularity defects imped a tight packing of side chains and can therefore ruptur chain conjugation by twisting of the backbone. More generally, packing defects of the side chains can cause unwanted twisting of the backbone, shortening conjugation lengths and reducing intra-chain transport. It can be anticipated that the ordering in phenyl substituted polythiophenes might be further improved by the use of symmetric 3,5-dialkylphenyls.

Figure 10 .

 10 Figure 10. Comparison of the packing of n-alkyl side chains in polythiophene

Figure S5 .

 S5 Figure S5. Electron diffraction pattern of a highly crystalline and oriented P3HT film obtained by high-temperature rubbing at 180°C. The two arrows point at fuzzy reflections indicative of structural disorder in the packing of n-hexyl side chains in form I P3HT.

Table 1 .

 1 Macromolecular data of the PDOPT samples.

Mn a [kDa] Mw a [kDa] PDI a DPn

	6.2	6.7	1.08	16.2
	7.8	8.7	1.12	20.4
	9.2	12.1	1.31	24.1
	12.7	19.8	1.56	33.2
	21.0	36.1	1.72	55.0
	37.6	74.4	1.97	98.4
	a Polydispersity (PDI)index and degree of polymerization (DPn) obtained via SEC in
	THF with polystyrene standard.

Acknowledgments

Support from the DFG (IRTG 1642 and project SO 1213/5-1) is gratefully acknowledged. M.B. acknowledges also the support from the interreg IV-A Rhin Solar program (project C25). B. Lotz is acknowledged for fruitfull discussions. M. Schmutz and C. Blanck are acknowledged for technical support in TEM. We are grateful to Patrick Brocorens for supplying the structural model of C12-PBTTT used in the comparative study of this work.


M Tissut•

G Mrlina

E Genies

J P Calmon

p-NITROACETOPHENOXIME N-METHYLCARBAMATE, A NEW ELECTRON ACCEPTOR IN ISOLATED THYLAKOIDS

Keywords: N-methylcarbamate of p, nitroacetophenoxime, chloroplasts, electron transfer, methylviologen, thylakoids, spinach

p-Nitroacetophenoxime N-methylcarbamate (MCPNA) is a rather potent inhibitor of the electron transfer in spinach class A chloroplasts. In isolated thylakoids, MCPNA is an electron acceptor at the level of photosystem 1 (PS 1). lt inhibits 0 1 evolution in the presence of NADP and ferredoxin but not the reduction of ferricyanide. MCPNA is active as an acceptor between 3 µM and 100 µM. At concentrations higher than 300 µM, inhibition of photosystem Il (PS Il) occurs. MCPNA has no uncoupling effect on photophosphorylation. Reduction of MCPNA by thylakoids in the presence of light is in accordance with the E� of this compound (:>-0.57 V) and is followed by an electron transfer to 0 1 , This reaction probably explains the inhibitory effect of MCPNA on class A chloro plasts.

Introduction

The aim of this investigation was to study the action of MCPNA on isolated thylakoids. An oxime function is present in this com pound as in the well-known insecticide aldicarb. MCPNA is related to phenylcarba mate and phenylurea herbicides (Scheme II). The synthesis of MCPNA has already been described [ 1 ] .

Materials and Methods

Isolation and purification of chloroplasts

Spinach leaves were eut into small pieces directly within the ice-cold chloroplast extraction medium (330 mM mannitol, 2 mM EDT A, 0 .2 g • r 1 bovine serum albumin, 30 mM sodium pyrophosphate (pH 7.9); tissue/medium ratio 1: 3.3). Leaves were homogenized using a Waring Blendor for 3 X 2 s. The brei was fi!tered through 8 layers of muslin and intact chloroplasts were pre pared by differential centrifugation according to Nakatani and Barber [2]. The chloroplasts thus obtained ( chlorophyll concentration: 7 to 8 mg • mr 1 ) were purified by isopycnic centrifugation in Percoll, a non-toxic silica sol gradient (Pharmacia); isoosmotic condi tions were thus maintained throughout the isolation procedure [ 3]. The intact purified chloroplasts (class A chloroplasts) were diluted with the extraction medium (1: 10, v/v) and recovered as a pellet after centrifuga tion (5500 X g, 90 s, SS 34 rotor, Sorvall RC5).

Chlorophyll concentration

Chlorophyll concentration was measured according to Arnon [ 4] .

Photosynthesis assay

Percentage of broken chloroplasts

Broken chloroplasts were determined from ferricyanide-dependent 0 2 evolution in the presence of NH 4 Cl (2 mM) before and after an osmotic shock [ 5] and under reduced irradiance (700 W • m• 2 ). Thylakoids (class C chloroplasts) were obtained by an osmotic shock (10 s in distilled water) and diluted in the reaction medium. Reduction of NADP was fol!owed by 0 2 evolution at pH 7 .9 in a medium containing 1 mM NADP, 10 µM ferredoxin. concentration increased from 10-, to 4.10-2 mg• mi-1 , the 1 50 for MCPNA in creased only from 4 µM to 6 µM. With more purified class A chloroplast suspensions (95% class A obtained on Percoll gradients) 0 2 evolution was not as sensitive to MCPNA and 20 µM were needed to induce a 50% inhibition at a 10-2 mg • mr 1 chlorophyll concentration. The chloroplasts were insen sitive to the presence of small arnounts of Percoll which originated from the gradient: up to 2%, Percoll had no effect on the elec tron transfer in thylakoids and on the inhibi tory action of MCPNA.

Determination of E�. Cyclic voltametry

PS I electron transfer was studied using ascorbate-reduced N-tetrarnethyl-p-phenylene diarnine (TMPD) as electron donor and methylviologen (MV) as electron accepter. Although 300 µM MCPNA markedly inhibited the electron transfer between H 2 0 and MV, no major inhibition appeared at the level of PS !: 1 mM MCPNA only induced a 14% decrease in the electron flow in PSI (Table [). PS II electron transfer was then investigated between H 2 0 and 100 µM p-phenylenedi arnine in the presence of 5 mM potassium ferricyanide and after addition of 3 µM 2,5dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) as a plastoquinone antagonist [8][9][10]. Under these conditions, 500 µM MCPNA gave rise to an 80% inhibition of the electron flow (Table !). Then, the electron transfer was studied between reduced duro quinone [11) and MV, this test allowing the analysis of inhibitions located in the plasto quinone region. MCPNA was completely ineffective in this test, even at a 200 µM concentration.

An electron transfer between H 2 0 as donor and NADP as accepter was then studied in the presence of ferredoxin. This transfer took place in the light with freshly I). MCPNA at a concentration close to 25 µM fully inhibited 0 evolution in these conditions. Further more, 100 µM MCPNA induced a very marked decrease of the 0 2 concentration in the reaction vesse[, showing a significant light dependent 0 2 consumption which was never seen in the presence of classic inhibitors such as DBMIB or DCMU. Addition at this stage of small amounts of ferricyanide (500 µM) was followed by a transient 0 2 evolution which stopped when ferricyanide was fully reduced.

As suggested from the preceding experi ment, MCPNA atone (10 µM) induced an intense 0 2 consumption (150 µmol 0 2 • h-1 • mg• 1 chlorophyll) by class C chloroplasts in the presence of light (Table I). This consump tion was inhibited by lindophenol (DPIP). The 0 2 consumption appeared when MCPNA concentration reached 2 µ M and its intensity increased thereafter for concentrations between 2 µM and 10 µM. Between 10 µM and 100 µM, a constant value of 150 µM 0 2 • h-1 , mg• 1 chlorophyll was obtained, At concentrations greater than 150 µM, the intensity of the electron transfer gradually decreased (Fig. 2).

A comparative study of the redox systems of MCPNA and MV has been carried out through cyclic voltametry (Fig. 3 ). The voltamogram of MV (curve A, Fig, 3) indi cates that it is a perfect monoelectronic, fast and reversible electrochemical system [START_REF] Baizer | Organic Electrochemistry, an Introduction and a Guide[END_REF] . In this case, the potential difference t>E P between the cathodic and anodic peaks at 20 mV • s• 1 is almost 60 mV and the E� of MV is -0.53 ± 0.01 V. For MCPNA ( curve B, Fig. 3) the electrochemical reaction is much more complex. The cathodic and corresponding anodic peaks of the voltamo-10 100 �M MCPNA gram are of -0.62 and +0.27 Vat 200 mV • s-1, respectively. The t.E P is 890 mV. This strongly suggests a slow overall transfer of several particles. According to the structure, it must be 2e-and 2H•. In such a case, it is probably an ECEC mechanism [START_REF] Baizer | Organic Electrochemistry, an Introduction and a Guide[END_REF] where a proton transfer is a determining step. In this situation, it is not possible to give a quasi thermodynamic standard potential value E� for MCPNA. We can give an E�-value for the reduction of MCPNA ox to MCPNA red which is a maximum value: E� = -0.57 V ± 0.03 ox ➔ red. In homogeneous oxido reduction transfer we probably have to take into account a lower value. In the inverse direction red ➔ ox, the potential E� is about +0.20 ± 0.03 V. In homogeneous phase with different intermediates, owing to proton acceptor or to donor effect, the potential that we have to take into account can be between -0.57 and +0.2 V.

The E� for MCPNA ox ➔ MCPNA red is in accordance with the acceptance of e generated by PS I.

Discussion

The 0 2 uptake induced by MCPNA indi cates that MCPNA from 2 to 100 µM is able to accept electrons emitted by PS I in the light: when the two photo systems are opera ting, the electron transfer to MCPNA is inhibited by DCMU and restored by addition of ascorbate-DPIP. As can be predicted when considering the E� of MCPNA, ascorbate and DPIP, these two latter compounds are not able to transfer e-directly to MCPNA without thylakoids acting in the presence of light.

Addition of Jess than 10 µM MCPNA is su fficient to obtain complete oxygen con sumption in our conditions. This suggests that MCPNA does not accumulate in the reduced form. The fact that the e-transfer to this compound is followed by an oxygen consumption, and that MCPNA does not accumulate in the reduced form during the course of water photolysis in the thylakoids, shows that an electron transfer takes place between the reduced form of MCPNA and 0 2 , in a way which can probably be compared to that affecting 'Y ,'Y' dipyridyf salts and low potential quinones such as anthraquinone sulfonates [13] (Scheme I).

H,O x X• x MCPNAred x Q 2 -2 1 0 2 + 2H'+2e• x- MCPNAo, H 0 2 2
The oxido-reduction activity of MCPNA appears to be associated with the presence of a =NO-structure, as suggested by Scheme II.

CH,

MCPNAred

We have studied elsewhere the effects of amides and phenylcarbamates [START_REF] Tissut | [END_REF]16] on photosynthesis. Such compounds, the struc tures of which are close to that of MCPNA, are unable to transfer e-to 0 2 in the presence of thylakoids.

In the case of MCPNA itself, the addition of an electron withdrawing region on the phenyl ring, which is represented by a p-N0 2 , seems to increase the reactivity of the oxime function.

At concentrations higher than 300 µM, MCPNA inhibits the electron transfer in the thylakoids. This inhibition can be seen with the PS II acting alone, with PD and FeCNf as acceptors, after addition of DBMIB. PS II inhibition by MCPNA could be related to the occurence of the carbaomyl function -CO-NH-CH 3 and could thus be compared to that by DCMU [ 1 7] . An s-triazine-like structure might also account for the effect of MCPNA on PS IL It should be noted that the inhibitory effect of MCPNA on PS II is low, as in the cas e of comparable structures of the phenylamide or phenylcarbamate fam:Jies [START_REF] Tissut | [END_REF]16]. For this action, p-substitution by NO 2 on the phenyl ring is not favourable.

Addition of MCPNA to thylakoids does not prevent the acceleration of the electron transfer induced by NH,Cl (result not shown). MCPNA is therefore devoid of uncoupling activity. The inhibitory effect of MCPNA at low concentration, on the whole photosyn thetic mechanism in class A chloroplasts can be explained neither by the inhibition of the electron transfer in the thylakoids, nor by an uncoupling effect. On the other hand, the MCPNA concentration needed for electron transfer to 0 2 and for the inhibitory action on class A chloroplasts are ahnost the same. This suggests that MCPNA is able to enter into the class A chloroplasts and compete with electron transfer from PS I to NADP.

Nevertheless, we have to note that the presence of small amounts of class C chloro plasts in class A suspensions increases the 0 2 consumption associated with MCPNA action and leads to a reduction of the oxygen concentration in the medium. This explains why the apparent inhibitory effect of MCPNA is greater on preparations of A plastids containing a small amount of thylakoids than on very pure class A preparations.

The demonstration of the redox proper ties of the -Ç =N--0-CO-NH-CH, chain of MCPNA and of its correlative effect on photosynthesis leads to further studies in the chemical family of the systemic insecti cide aldicarb in which the same chain is also present.

  Abbreviations: DBMIB, 2,5-dibromo-3-methyl-6isopropyl-p-benzoquinone; Diuron (DCMU), 3-(3,4dichlorophenyl)-l,1-dimethylurea; DPIP, dichloro phenolindophenol; l 50 , concentration of the product giving a 50% inhibition; MCPNA, p-nitroacetopheno xime N-methylcarbamate; MOPS, morpholinopropane sulfonic acid; MV, methylviologen; PS 1, photosystem !; PS II, photosystem Il; TMPD, N•tetramethyl-p phenylenediamine.

0 2

 2 Photosynthesis assay 0 2 evolution was followed polarographi cally at 25 °C using a Clark-type electrode system. The reaction medium contained: 330 mM mannitol, 50 mM morpholinopro pane su!fonic acid (MOPS)-NaOH (pH 7 .6), 2 mM EDTA, 5 mM NaHCO,, 100 µM Na, HPO 4 and chloroplasts in an amount equiva lent to 70-80 µg of chlorophyll in a total volume ôf 1 ml. The reaction medium was gassed with argon before chloroplast addition and equilibrated with the 0 2 electrode at 25 °C. The light source was a 150 W xenon arc lamp giving an irradiance of 1700 W • m•• at the surface of the vesse!.

Fig. 1 .

 1 Fig. 1. Effect of MCPNA on light-dependent O, evolution by Spinach class A chloroplasts in the presence of NaHCO,, 1 mM, Na 2 HPQ 4 , 0.1 mM. (A) chlorophyll concentration reaches îO µg , mI-1 in the reaction medium; (B) chlorophyll concen• tration is 40 µg , m1-1 ,

Table 1 .

 1 Intensity of the photosynthetic electron flow in class C chloroplasts with different electron donors and acceptors when increasing MCPNA concentration. Columns 1-6: percentage of the value obtained without MCPNA. Columns 7-8: percentage of the maximum value of the oxygen consumption when MCPNA was elec tron acceptor. Experimental conditions: ( 1) H20--+ NADP: ox ygen ev olution measured in the presence of 1 mM NADP and 10 µM Fd with freshly broken chloroplasts (class C). (2) H20 _,. MV: oxygen consumption measured in the presence of 100 µM MV and 10 mM NH4Cl. (3) Ase+ DPIP _,. MV: oxygen consumption measured in the presence of 100 µM MV, 10 mM NH,Cl, 10 µM DCMU, 500 µM ascorbate and 100 µM DPIP. (4) Dq -Mv: oxygen consumption measured în the presence of 100 µM reduced duroquinone, 100 µM MV and 10 mM NH 4 Cl. (5) H 2 Q-,. FeCN!-: oxygen evolution measured in the presence of 5 mM FeCN!and 10 mM NH 4 Cl. (6) H 1 0 -PD: oxygen evolution measured in the presence of 5 mM FeCN!-, 3 µM DBMIB, 100 µM PD and 10 mM NHjCI. (7) H,O -MCPNA: oxygen consumption measured in the presence of MCPNA as electron acceptor, with 10 mM NH4Cl. (8) Ase+ DPIP _,. MCPNA: oxygen consumption measured in the presence of 10 mM NH4Cl, 10 µM DCMU, 500 µM ascorbate, 100 µM DPIP and MCPNA. For tests 1-6 see Refs. 12,13. Ase, ascorbate; Dq, reduced duroquinone; FeCN!-, potassium ferricyanide; PD, p-phenylene-diamine.

  5 µM 3-(3,4-dichloro phenyl)-1,l-dimethylurea (Diuron, DCMU) and was thereafter restored by addition of ascorbate reduced TMPD or dichloropheno-

Fig. 2 .Fig. 3 .

 23 Fig. 2. Relation between the oxygen consumption rate with MCPNA as electron acceptor and MCPNA concen• tration. One hundred percent refers to 160 µ,mol O i consumed • h-1 • mg-1 chlorophyll.