Helena Isla

Nidal Saleh

Jiang-Kun Ou-Yang

Kais Dhbaibi

Marion Jean

Magdalena Dziurka

Ludovic Favereau

Nicolas Vanthuyne

Loïc Toupet

Bassem Jamoussi

Monika Srebro-Hooper

Jeanne Crassous

Bis-4-aza[6]helicene: a bis-helicenic 2,2'-bipyridine with chemically-triggered chiroptical switching activity

A novel enantiopure bis-helicenic 2,2'-bipyridine system was prepared using a Negishi coupling. Thanks to the bipyridine unit, the coordination with Zn II and protonation processes were studied revealing efficient tuning of photophysical (UV/Visible and emission) and chiroptical properties (electronic circular dichroism and circularly polarized emission) of the system. The coordination/decoordination and protonation/deprotonation processes appeared reversible thus constituting novel chiroptical switches.

INTRODUCTION

Chirality, ubiquitous in life and in chemical substances, has growing implications in diverse domains of materials science. 1 Indeed, there is a great demand in the development of novel chiral molecules and architectures with tunable electronic and optical properties, which may be used in molecular functional devices. In particular, contributions to the field of chiroptical switches 2 are stimulated by their potential applications as molecular memory elements or logic operators, in which data storage and processing are the ultimate goals. [START_REF] Balzani | Molecular Devices and Machines. Concepts and Perspectives for the Nanoworld[END_REF] Chiroptical switches have also found applications in sensing, especially in the detection of a multitude of different chiral analytes with a high level of sensitivity, 4 in asymmetric catalysis to offer switchable stereoselectivity, 5 and in molecular motors since chirality can provide unidirectionality, an important aspect in rotatory motors such as in the biological case of ATP-synthase and other original kinds of motions. [START_REF] Feringa | The Art of Building Small: From Molecular Switches to Molecular Motors[END_REF][START_REF] Balzani | Molecular Devices and Machines. Concepts and Perspectives for the Nanoworld[END_REF] Among chiroptical switching materials, helicenes play an increasing role due to their unprecedented inherent helical chirality accompanied by a -conjugated electronic structure. 6 The most appealing attributes of helicene derivatives are their large-magnitude chiroptical properties, i.e. optical rotation (OR), electronic circular dichroism (ECD), vibrational circular dichroism (VCD), and circularly polarized luminescence (CPL). [START_REF]Comprehensive Chiroptical Spectroscopy[END_REF] Despite remarkable progress that has been accomplished in helicene chemistry in the last decade, the development of straightforward and efficient strategies to diversify helicenes structure still remains a challenge for synthetic chemists. An attractive alternative to a stepwise organic approach to a construction of complex helicene-based architectures is coordination chemistry. [START_REF]Helicene and a Helical 1,10-Phenanthroline Ligand[END_REF]9 Indeed, metals are powerful templates for assembling -conjugated ligands into welldefined molecular structures. [START_REF] Anger | Rutheniumvinylhelicenes: remote metal-based tuning and redox switching of the chiroptical properties of a helicene core[END_REF] Furthermore, as the nature of the metal can strongly impact the chiroptical properties of the helical molecules, such connections give rise to chiral molecular systems combining novel topological features with enhanced photophysical and optical properties. In particular, one can take benefit from the inherent properties of a ligand and a metal ion to easily access novel helicene-based organometallic chiroptical switches 6e,f through the action of different stimuli such as a redox potential 10a-f or chemicals. 10g,h,11 In addition, the coordination process can induce strong conformational changes and intramolecular motions based on which new chiral switches and molecular motors can be envisioned. [START_REF] Isla | Conformational changes and chiroptical switching of enantiopure bishelicenic terpyridine upon Zn 2+ binding[END_REF] Among the helicenes bearing heteroatoms, azahelicenes [START_REF] Sato | Cyclophane chemistry fot the 21st century[END_REF] are especially interesting due to possibilities that nitrogen atoms offer. The involvement of the N lone-pair in coordination or protonation processes can induce a peculiar impact on the chiroptical properties of the helicene molecule. The bipyridine core, with the most widely used 2,2'-bipyridine unit [START_REF] Kaes | The Most Widely Used Ligand. A Review of Molecules Comprising at Least Two 2,2'-Bipyridine Units[END_REF] (Scheme 1), has received much attention due to its remarkable stability and exceptional coordination chemistry. Functionalized helical bipyridine systems 10d,h,13,14 exhibit an appealing set of properties: chirality, an extended -electron system, rich and high-affinity coordination chemistry with metals, good acid/base and alkylation reactivity, and redox activity. Complexation of such systems with a metallic ion (e.g. Zn II , Ru II , Os II , Re I ) may enhance their properties even further, based on strong optical activity, emission properties, and redox activity furnished by the metal. All this makes helicenicbipyridines promising candidates for unprecedented chiroptical switches.

In this article, we present the synthesis of a bis-azahelicenic system in which bipyridine core is integrated in the sophisticated aza[6]helicene structure (1, Scheme 1), and we address its chiroptical switching activity triggered chemically by (i) Zn II coordination/decoordination, and (ii) protonation/deprotonation processes. Read-out signals are observed using UV/Vis, ECD, OR, and luminescence techniques, and interpreted on the basis of first-principle calculations. As complexation/decomplexation process induces large conformational changes and therefore molecular motions around the zinc center, reversibly interconverting transoid and cisoid forms of 1 with different relative orientations of helicenes moieties, we also explore computationally unidirectionality of the motion and a possible application of 1 as a molecular hinge.

Scheme 1. Chemical structure of the bis-helicenic 2,2'-bipyridine 1 and reversible (i) coordination/decoordination process using Zn(OAc) 2 /TPEN chemical stimuli and (ii) protonation/deprotonation with triflic acid/NEt 3 as acid/base agents in dichloromethane.

RESULTS AND DISCUSSION

Synthesis and characterization of bis-helicene-bipyridine 1.

The synthetic strategy chosen for the construction of the helical architectures 1 is based on the Negishi coupling 15 between the bromoaza[6]helicene 5 with the zinc bromide derivative 6 (see Scheme 2). The 3-bromo-4-aza[6]helicene 5 was first prepared in two steps involving a Wittig reaction between 2-methyl-naphthalene-phosphonium bromide 2 [START_REF] Lightner | Absolute configuration[END_REF] and commercially available 2-bromo-pyridine-6-carboxaldehyde 3, yielding olefin 4 as a mixture of cis and trans isomers, followed by a photocyclization reaction with a 150 W mercury lamp with 2 equiv. of iodine and an excess of propylene oxide in toluene for 15 hrs. It is worth noting that the photo-irradiation conditions were compatible with the bromo-substituent functionality. Compound 5 was obtained in the racemic form and fully characterized by NMR spectroscopy and mass spectrometry (see Supporting Information, SI). For example, it displayed two unresolved triplets around 6.8 and 7.3 ppm that are typical of protons H 15 and H 14 , respectively. 11b Its structure was further ascertained by X-ray crystallographic analysis (see Figure 1 and SI). Racemic 5 crystallized in the P-1 space group. It displayed a helicity (dihedral angle between the two terminal rings) of 49.71° and a C-Br bond length of 1.917 Å which both correspond to regular values. Compound 5 can further be obtained as enantiopure P-(+)-and M-(-) samples by HPLC separation over a chiral stationary phase (see SI).

Scheme 2. Synthesis of 1 as a mixture of (P,P), (M,M), and (P,M) stereoisomers. i) n-BuLi, THF, rt, Ar, 5 hrs, 90%; ii) h, I 2 (2 equiv.), propylene oxide (49 equivs), toluene, rt, Ar, 15 hrs, 45%; iii) n-BuLi, ZnCl 2 , THF, rt, Ar, 5 hrs, 90%; iv) Pd(PPh 3 ) 4 , THF, reflux, Ar, 15 hrs; v) TPEN, CH 2 Cl 2 , 57% (two steps).

The bis-helicenic bipyridine system 1 was prepared by generating in situ the zinc bromide derivative 6 from 5 followed by coupling 5 and 6 together under the regular Negishi coupling conditions. Finally, decoordination of the Zn using an excess of N,N,N',N'-tetrakis (2-pyridylmethyl)ethane-1,2-diamine (TPEN) was necessary to obtain pure 1 with 57% yield. Under these conditions and starting from racemic (±)-5, a mixture of stereoisomers, namely chiral (P,P)-and (M,M)-1 and achiral meso compound (P,M)-1 was expected. This was indeed evidenced by chiral HPLC analysis, which showed three peaks in 34:34:24 respective proportions (see SI). Single crystals of (±)-1 were grown from pentane diffusion into a dichloromethane solution. X-ray crystallography revealed a mixture of (P,P)-and (M,M)-1 (Pccn orthorhombic centrosymmetric space group, see Figure 1 and SI). The structure showed i) the C 1 symmetry of the chiral compound 1, ii) the two nitrogen atoms placed trans to one another as common for 2,2'-bipyridines [START_REF] Kaes | The Most Widely Used Ligand. A Review of Molecules Comprising at Least Two 2,2'-Bipyridine Units[END_REF] with a dihedral angle between the different pyridine rings lower than 20°, and iii) typical helicity value of the azahelicene moieties (59.83°). [START_REF] Sato | Cyclophane chemistry fot the 21st century[END_REF] Compound (P*,P*)-1 17 was also characterized by NMR spectroscopy. For example, its 1 H NMR spectrum displayed the two typical unresolved triplets around 6.9 and 7.4 ppm corresponding to protons H 15 and H 14 , respectively. Note that the low quantities isolated and solubility issues did not allow to characterize the meso-1 compound by NMR spectroscopy. However, sufficient amounts of enantiopure (P,P)-and (M,M)-1 compounds were directly prepared under similar Negishi coupling conditions from enantiopure P-and M-5 samples.

Photophysical and chiroptical properties of (P,P)-and (M,M)-1

Photophysical and chiroptical properties of 1 were then examined and compared to those of the precursor 5. As seen in Figure 2a, the UV/Visible spectrum of (P*,P*)-1 [START_REF]P*)-1 means the racemic mixture of (P,P)-and[END_REF] shows a very strong absorption band centered around 275 nm (  7.6  10 4 M -1 cm -1 ) and a set of bands of gradually diminishing intensity that extends into the visible region with the longest absorption wavelength appearing at 420 nm ( ~ 10 4 M -1 cm -1 ). In comparison, the compound 5 displays overall less intense UV/Vis bands with the stronger one at 262 nm (  5.7  10 4 M -1 cm -1 ) and the weaker one at the longest wavelength of 405 nm ( ~ 10 3 M -1 cm -1 ). Furthermore, while 5 is not emissive, likely due to fluorescence quenching via internal heavy atom (Br) effect, 18 (P*,P*)-1 17 displays rather intense blue fluorescence in CH 2 Cl 2 at rt ( max = 420 nm,  = 22%, Table S1.1 in the SI, and Figure 4c). Its structured emission spectrum is characterized by a vibronic progression of ~ 1350 cm -1 with the maximum intensity being in the 0-0 band that is indicative of a rigid aromatic fluorophore. Note that emission behavior of 1 is almost identical to that previously observed for helicenic bipyridine and terpyridine systems. 10h,11 Linear-response time-dependent density functional theory (TDDFT) calculations, [START_REF] Autschbach | Computing chiroptical properties with first-principles theoretical methods: background and illustrative examples[END_REF] following the computational protocol that was established in Ref. 11 for a bis-helicenic terpyridine ligand and a corresponding Zn-based complex, reproduce experimental data well (see Figure 2a, inset, and Table S2.9 in the SI). Details regarding all the calculations presented in this article along with additional results not discussed herein in detail are provided in the SI. As expected, enhanced UV/Vis properties of 1 as compared to 5 can be linked to its extended -conjugation involving both azahelicene fragments. Indeed, the additional low-energy (> 350 nm) absorption observed for 1 originates from excitations (nos. 1 calculated at 372 nm and 3 at 359 nm) that may be predominantly considered as -* transitions across the whole molecule with some charge transfer (CT) components from helicene moieties to bipyridine fragment due to a partial spatial separation of the engaged MOs. For example, both excitations demonstrate high HOMO-LUMO contributions, with HOMO almost evenly extended over -electron system within the whole molecule and LUMO localized predominantly onto bipyridine fragment and its adjacent helicene rings (see Figure 3). The same was found for the frontier MOs at the optimized S 1 excited-state geometry that supports a predominantly bipyridine-centered LUMO-HOMO emission for 1.

Intense mirror-image ECD spectra were then recorded for (M,M)-and (P,P)-1 enantiomers (Figure 2b), showing a strong negative band around 273 nm ( = -333 M -1 cm -1 ) and strong positive one at 332 nm ( = +309 M -1 cm -1 ) for the (P,P)-1. Weaker positive ECD bands are also present at longer wavelengths between 380 and 420 nm, i.e. at 382 ( = +70 M - 1 cm -1 ), 397 ( = +26 M -1 cm -1 ) and 417 nm ( = +23 M -1 cm - 1 ). For comparison, the P-5 displays a weaker ECD spectrum than (P,P)-1 and similar to those observed for other carbo[6]helicene derivatives, with a strong negative band around 258 nm ( = -200 M -1 cm -1 ), a strong positive one at 326 ( = +173 M -1 cm -1 ), and no ECD-active band above 380 nm. Note that some vibronic structure is roughly seen in both compounds 1 and 5. As seen in Figure 2b, inset, the calculated ECD spectral features of 1 and 5 agree well with the experiment. The MO analysis of dominant excitations calculated in the low-and medium-energy regions of the simulated ECD spectra of (P,P)-1 confirms that its main highly intense positive band originates predominantly from -* transitions within helicene moieties accompanied by helicene→bipyridine and bipyridine→helicene CTs. See for example, excitations nos. 2, 5, and 6 with sizable rotatory strengths calculated at respectively 368, 331, and 322 nm and their MO assignment in the SI. This clearly highlights the effect of elongated -electron system due to the presence of two azahelicene moieties in 1, resulting in a strong enhancement of its chiroptical properties as compared to 5. In line with the ECD spectra, high specific and molar optical rotation values were thus obtained for 1 with its molar rotation 1.7 times higher than for 5 ((P,P)-1: = +2600° cm 2 g -1 , = +17300° cm 2

[𝛼] 23

𝐷

[𝜙] 23

𝐷

dmol -1 (±5%, CH 2 Cl 2 , 4.2  10 -5 M) vs. P-5: = +2500°

[𝛼] 23 𝐷 cm 2 g -1 , = +10200° cm 2 dmol -1 (±5%, CH 2 Cl 2 , 6  10 -6

[𝜙] 23 𝐷 M).

Coordination-induced chiroptical switching activity and molecular motion in bis-helicenic bipyridine 1

The bipyridine system is a good scaffold for coordination to a variety of transition-metal centers yielding complexes with diversity of photophysical properties that may be appealing for device applications. [START_REF] Kaes | The Most Widely Used Ligand. A Review of Molecules Comprising at Least Two 2,2'-Bipyridine Units[END_REF] For example, Rebek, Jr. et al. have integrated crown ether functionalities within the 2,2'-bipyridine platform to study allosteric effects. 20 Furthermore, the molecular engineering of 2,2'-bipyridines with chiral units, introducing controlled steric congestion and chirality, enables to construct fascinating helicate structures, 21a and molecular motors displaying unidirectionality. Indeed, regarding the latter property, Haberhauer has designed a molecular hinge based on a 2,2'-bipyridine derivative grafted with a chiral peptidic clamp that shows a unidirectional open-close motion upon coordination to a Cu II chemical stimulus. 21b,c Yashima and coworkers have developed unprecedented molecular springs consisting of enantiomeric double-stranded spiroborate helicates bearing 2,2'-bipyridine and its N,N-dioxide linkages that demonstrate a unique allosterically regulated reversible extension-contraction motions coupled with unidirectional and helicity conservative twisting upon binding or release of protons and/or metal ions. 21d These examples open up the possibility for further design and development of molecular machines, both simpler or even more sophisticated ones. Based on this idea, we then explored how Zn II binding to a bis-helicenic 2,2'-bipyridine system could induce chemical switching of the UV/Vis absorption, ECD, and luminescence spectra via addition of i) Zn(OAc) 2 for coordination and ii) TPEN for decoordination (see Scheme 1).

Aliquots of Zn(OAc) 2 •2H 2 O (0.2 equiv. until saturation at 1.0-1.2 equivs) were thus added to 1 and the complexation process was followed by UV/Vis, ECD, and emission spectroscopy (Figure 4a-c). Several changes were observed in the UV/Vis spectrum of (P*,P*)-1 17 upon Zn II binding, as depicted in Figure 4a, with the presence of several isosbestic points, showing that the two species are in exchange. The main modification of the spectrum appears between 420 and 500 nm where an additional band emerges upon Zn complexation. Accordingly, a colour of the solution turned from pale yellow to dark yellow. Substantial modifications were also observed for chiroptical properties of (M,M)-1 after Zn(OAc) 2 •2H 2 O addition (see Figure 4b). For example, the strong positive ECD-active band at 273 nm decreases from +413 to +298 M -1 cm -1 , the strong negative band around 332 nm ( = -338 M -1 cm -1 ) evolves into a new one centered at 342 nm ( = -202 M -1 cm -1 ), and an additional negative band of medium intensity appears at 454 nm ( = -45 M -1 cm -1 ). Furthermore, magnitude of the molar rotation significantly increases for 1•Zn(OAc) 2 comparing to 1 (e.g. (M,M)-

1•Zn(OAc) 2 : = -25000° cm 2 dmol -1 , (M,M)-1: = - [𝜙] 23 𝐷 [𝜙] 23

𝐷

16700° cm 2 dmol -1 (±5%, CH 2 Cl 2 , 1.910 -4 M), see also SI).

Finally, as presented in Figure 4c, Zn II complexation led also to significant changes in the luminescence profile of 1, with the appearance of a structureless intense fluorescence at 520 nm ( = 44%) accompanied by a structured emission at 421 and 446 nm that resembles that from the ligand itself in line with the presence of equilibrium between 1 and 1•Zn(OAc) 2 . This was followed by a striking change in the emission colour from blue to green (see Figure 8d). Note that in order to obtain full transformation of the emission signal corresponding to full conversion to Zn complex, it was necessary to add an excess of Zn(OAc) 2 . Finally, having in hands chiral species displaying efficient fluorescence, it was appealing to examine their corresponding circularly polarized luminescence (CPL). 22,[START_REF] Aoki | Bis(dipyrrinato)zinc(II) Complex Chiroptical Wires: Exfoliation into Single Strands and Intensification of Circularly Polarized Luminescence[END_REF] The CPL responses for (P,P)-/(M,M)-1 and (P,P)-/(M,M)-1•Zn(OAc) 2 were thus measured in CH 2 Cl 2 at rt and the resulting spectra are depicted in Figure 6, showing maximum g lum = 2I/I values of -3.7 and +4.8  10 -3 for (M,M)-1 and (P,P)-1 at 425 nm, respectively, and of -1.5 and +1.8  10 -3 for (M,M)-1•Zn(OAc) 2 and (P,P)-1•Zn(OAc) 2 at 495 nm, respectively, which appear quite similar to values found for the previously examined helicene-bipyridine and bis-helicenic terpyridine systems (i.e. 3.4  10 -3 and 7  10 -3 for the bipyridine and terpyridine ligand, respectively, and 1  10 -3 for the terpyridine Zn complex, P enantiomers). 10h,11 Accordingly, based on the fact that 1 and its Zn complex show clearly distinct and good CPL responses, one may envision to use CPL-activity at one chosen fixed wavelength to read-out the coordination/decoordination switching process for 1.

Figure 6. CPL spectra of (P,P)-1 (solid black), its Zn complex (P,P)-1•Zn(OAc) 2 (solid red), and its fully protonated species (P,P)-1•2H + , 2OTf -along with the corresponding spectra of their mirror-images (dash lines) measured in CH 2 Cl 2 at rt (C ≈ 1 × 10 -5 M). Excitation wavelengths: λ ex at 350 nm for 1 and 1•Zn(OAc) 2 ; 460 nm for 1•2H + , 2OTf -. Maximum intensities have been normalized to 2.

Calculations (TDDFT LC-PBE0*/SV(P) with a continuum solvent model for CH 2 Cl 2 ) performed for bis-helicenic bipyridine complex of Zn II correctly reproduce the spectral trends observed experimentally upon binding of the metal fragment to 1. Due to a lack of X-ray crystal data for the 1•Zn(OAc) 2 compound, various conformations, differing in the spatial arrangement of acetate ligands around Zn II , were considered in geometry optimizations of this system. The lowest-energy structures with non-negligible populations, all comprising acetate ion(s) coordinated to the metal center in a bidentate fashion (see SI), reveal very similar overall UV/Vis and ECD spectral envelopes, and, as can be seen from Figure 5, their Boltzmann-averaged spectra agree well with the experiment. Contrary to the previously reported bis-helicenic terpyridine chiroptical switching system, [START_REF] Isla | Conformational changes and chiroptical switching of enantiopure bishelicenic terpyridine upon Zn 2+ binding[END_REF] the simulated UV/Vis and ECD spectra of the ligand 1 in the cis conformation of the bipyridine fragment clearly differ from those calculated for 1•Zn(OAc) 2 (see SI). This may indicate that the spectral modifications accompanying 1→1•Zn(OAc) 2 transition originate predominantly from electronic rather than geometric changes occurring in 1 upon complexation. Indeed, although no contributions of Zn d orbitals to the helicenic electron system can be noticed, as expected for closed-shell d 10 metal center, electronic interaction between Zn and 1 induces polarization of electron density and has a stabilizing effect on the orbital energies in 1•Zn(OAc) 2 vs. 1 (see Figure 3 and SI) that may rationalize differences in the spectral envelopes of 1 and 1•Zn(OAc) 2 . 11,10h In particular, a shift of the electronic density towards terminal rings of helicenes' moieties in HOMO and towards bipyridine fragment in LUMO, along with a decrease in the HOMO-LUMO gap due to a strong energetic stabilization of the LUMO level, observed for 1•Zn(OAc) 2 as compared to 1, activate intense low-energy helicenebipyridine CT excitation within the ligand -system (see excitation no. 1 calculated at ca. 390 nm of predominantly HOMO-LUMO character, Figure 5 and SI). The excitation is responsible for the appearance of the additional low-energy intensity in the UV/Vis and ECD spectra upon complexation. Furthermore, the calculations link the pronounced modification (a red-shift and a decrease in intensity) of the main band in the ECD spectrum for 1•Zn(OAc) 2 vs. 1 to the presence of two excitations with strong rotatory strength values of opposite signs (nos. 5 and 6 calculated at ca. 340 and 330 nm, respectively) which involve mainly helicenebipyridine CT transitions rather than classical helicene-centered -* ones (vide supra). The aforementioned activation of low-energy CT transitions in 1 due to Zn II coordination rationalizes also the experimentally observed emission switching process which is quite well reproduced by the computations: from 409 nm S 1 -S 0 fluorescence calculated for 1 to 440-470 nm (depending on the conformer) for 1•Zn(OAc) 2 . Note that strong interaction between Zn II and 1 in 1•Zn(OAc) 2 was indeed confirmed using the ETS-NOCV [START_REF] Mitoraj | A Combined Charge and Energy Decomposition Scheme for Bond Analysis[END_REF] charge and bonding-energy analysis, which showed one main NOCV-based contribution to the total deformation density corresponding to a formation of the Zn II -N -bonding associated with a charge transfer from lone-pair (LP) of bipyridine nitrogen atoms to the metal center (Figure 3).

The reversibility of the Zn II binding process was examined by using N,N,N',N'-tetrakis (2-pyridylmethyl)ethane-1,2-diamine (TPEN) as a competitive ligand. Indeed, upon addition of TPEN, the system returned to its original state, i.e. chiral (P,P)and (M,M)-1 non-coordinated species. Up to 9-10 steps could be performed upon successive additions of 1 equiv. of Zn(OAc) 2 and TPEN, following the ECD tuning at the selected wavelengths at which the changes are clearly visible (for example, 274 nm:  = 150 M -1 cm -1 , see Figure 4d). Accordingly, the bis-helicenic bipyridine system 1 constitutes another example of a chemically-triggered chiroptical switch with multi-output read-out including UV/Vis, ECD, and luminescence, for which the switching process is accompanied by nanomechanical molecular movements interconverting a trans conformation in the free ligand to a cis one in the Zncomplex. [START_REF] Isla | Conformational changes and chiroptical switching of enantiopure bishelicenic terpyridine upon Zn 2+ binding[END_REF] Having established that the Zn II coordination/decoordination effectively and reversibly interconverts transoid and cisoid forms of 1, the question arises as to whether 1 can act as a molecular hinge. Molecular hinges are systems that demonstrate a high relative amplitude of motion (> 90°) accompanied by controllable unidirectional 'open-close' movements. The first of these requirements can be in general easily fulfilled for 2,2'-bipyridine-based systems thanks to efficient metal-ion complexation / decomplexation processes that transform an 'open' non-complexed form (nitrogen atoms in trans configuration, NCC'N' dihedral angle of ~180°) to a 'closed' complexed one (nitrogen atoms in cis configuration, NCC'N' dihedral angle of ~0°) and vice versa. The second one (i.e. a control of the orientation of the movement between 'open' and 'close' states) is, however, more difficult to achieve as a rotation around the C-C' bipyridine bond is usually possible in both directions. The unidirectionality of the movement can be enforced for example by introducing a metabridge within the bipyridine part. 21a,b However, one could envision a similar effect in systems with spatially extended substituents attached to the bipyridine core (such as for example helicenes' moieties in 1) since such fragments, interacting with each other during a rotation around the C-C' axis, could make one of 'open'↔'closed' transition pathways preferable over the other providing a control over a movement' direction. Accordingly, the energy profile for the full rotation around the C-C' axis in 1 was calculated both at DFT and DFT-D3 (i.e. with inclusion of van der Waals (dispersive) forces via semi-empirical approach, see SI) level of theory and the corresponding results are shown in Figure 7. As can be seen, additional stabilizing dispersion interactions between helicenes' moieties located close to each other in the structure II (see also Figure S2.18 in the SI for overlays of the corresponding DFTand DFT-D3-optimized geometries) lower its energy compared to the structure IV in which helicenes' fragments are located apart (compare 7.0 kcal/mol vs. 8.9 kcal/mol for II and IV, respectively, relative to the lowest-energy structure I). This leads to asymmetry of the rotational profile for 1, which indicates that 'open': I → 'close': III transition may preferably occur via the pathway proceeding through structure II. However, even if we disregard a known tendency of DFT-D3 dispersion corrections to overestimate interactions between the aromatic rings of the helicene moieties, 25 this asymmetry seems to be too small to guarantee the preferential direction of rotation in 1. Accordingly, it is rather unlikely that this type of systems can act as molecular 'hinges'.

Chiroptical switching activity of 1 induced via protonation/deprotonation process

Finally, the acid-base switching for 1 was studied by using i) triflic acid and ii) triethylamine for protonation and deprotonation step, respectively (see Scheme 1). Upon adding aliquots of triflic acid (0.2 equiv.) to (M,M)-1 in CH 2 Cl 2 until saturation at 8 equivs, a full protonation of (M,M)-1 is expected with a formation of doubly protonated species (M,M)-1•2H + , 2OTf -. The process was followed by UV/Vis, ECD, and emission measurements, and the corresponding responses are presented in Figure 8. As can be seen, the UV/Vis spectrum of the protonated species (M,M)-1•2H + , 2OTf -significantly differs from that of the non-protonated system (M,M)-1 with 1) a slight increase in intensity of the band centered at 275 nm accompanied by its 5 nm bathochromic shift, 2) an intensity decrease of the bands at 325 and 383 nm, and 3) an appearance of strong broad band at longer wavelengths (420-550 nm, λ max = 475 nm, ε = 15000 M -1 cm -1 , Figure 8a). Similarly, the ECD spectrum of (M,M)-1 was substantially modified upon protonation showing a less intense positive band at 275 nm ( = +330 M -1 cm -1 ), a less intense negative band at 335 nm ( = -243 M -1 cm -1 ), and an additional moderate negative band at 460 nm ( = -32 M -1 cm -1 ) for the (M,M)-1•2H + , 2OTf - enantiomer (Figure 8b). Significant changes can also be observed for the luminescence profile of 1 for which, upon adding triflic acid, a structureless intense orange-red fluorescence emission appeared around 600 nm ( = 28%), while the structured blue emission at 421 and 446 nm, coming from the non-protonated system itself (vide supra), almost completely disappeared (Figures 8c and8d). This clearly confirms that under saturation conditions only protonated species are present in the solution. Finally, the CPL responses of (M,M)-and (P,P)-1•2H + , 2OTf -were also measured in CH 2 Cl 2 at rt (see Figure 6) and showed distinct signals centered around 605 nm with g lum maxima of the same order of magnitude as found for 1 and 1•Zn(OAc) 2 , namely -2.0 and +2.5  10 -3 for (M,M) and (P,P) enantiomer, respectively. Note that very similar g lum values were also obtained for protonated species of helicenic bipyridine system, although introduction of two helicene moieties onto the bipyridine core leads to much more red-shifted CPL signal (compare 643 nm for 1 vs. 590 nm with g lum = 3.2  10 -3 (P enantiomer) for helicene-bipyridine). 10h Summarizing, thanks to reversibility of the process (upon addition of aliquots of NEt 3 the UV/Vis and ECD, emission and CPL activity spectra of non-protonated species were recovered), the presented here bis-helicenic bipyridine system 1 can also act as an efficient pH-triggered (chir)optical switch. 26 To provide some insight into experimentally observed tuning of photophysical and chiroptical properties in 1 upon its protonation, (TD)DFT calculations were then performed for both singly (n = 1) and doubly (n = 2) protonated 1•nH + species, as they all are likely to be present in the reaction mixture below saturation limit. As expected, [START_REF] Alkorta | A theoretical study of the conformation, basicity and NMR properties of 2,2′-, 3,3′-and 4,4′bipyridines and their conjugated acids[END_REF] the monoprotonated system preferentially adopts a conformation with nitrogen atoms within bipyridine core placed in cis configuration (see structure 1•H + -I in Figure S2.9 in the SI), while for di-protonated species transoid arrangement is favoured (1•2H + -I and 1•2H + -II). Although the corresponding TDDFT calculations provide less satisfactory (than in the case of the neutral species) agreement with the experimental spectral envelopes for 1•2H + , 2OTf -, as a lowenergy band computed for 1•2H + appears too red-shifted, which is likely due to solvent and counter-ion(s) effects, 10h,28 the most important changes in photophysical and chiroptical properties of 1 observed experimentally upon its protonation are reproduced correctly. In particular, in line with the experiment, the computations for the protonated species demonstrate i) appearance of additional absorption and ECD intensity above 420 nm, ii) a significantly reduced intensity of the ECD band around 330 nm, and iii) a large red-shift (> 100 nm) of the emission signal. All these features can be linked to a strong impact of protonation on the -electronic system of 1 that manifests itself through a clear spatial separation of the frontier MOs of 1•nH + , with HOMO predominantly localized on the central and terminal parts of helicenic moieties and LUMO limited to the bipyridine core and the adjacent rings, accompanied by the strong stabilization of their energy levels (see Figure 3 and SI), especially pronounced for LUMO, resulting in a significant reduction of the HOMO-LUMO gap. Accordingly, photophysical and chiroptical properties of 1 upon its protonation appears to be dominated by low-energy CT-type transitions rather than classical helicene-centered -* ones (see SI). 10h For example, the new lowest-energy UV/Vis and ECD bands in 1•2H + correspond to a clear CT electronic transition between helicene -system and bipyridine core of the predominant HOMO-LUMO composition that further amplifies its CT character. Decreased (increased) contributions from helicene-centered -* (CT-type) transitions to the excitations calculated in the mediumenergy part of the ECD spectra are also responsible for a decrease in intensity of the ECD band around 330 nm. Similarly, very pronounced red-shift of the fluorescence signal for 1 observed upon protonation can be traced back to the predominant CT character of the corresponding S 1 excited state and the stabilization of the LUMO level. Note also that the calculations enable to shed some light on evolution of the emission spectra of 1 upon adding aliquots of triflic acid that involves i) a gradual diminishing of the signal centered at 420 and 440 nm accompanied by a gradual rise of emission intensity around 520 nm observed before reaching saturation, and ii) disappearance of the signal centered at 420 and 440 nm and appearance of separate intense band around 600 nm seen after saturation conditions are met. Based on the computed S 1 -S 0 fluorescence for the neutral system 1 (409 nm, see SI) and for its protonated species 1•nH + (495 nm for n = 1, and 660-690 nm (depending on the conformer) for n = 2), it appears therefore that below saturation limit neutral and mainly singly protonated species are present in the solution, while above it doubly protonated species clearly predominate.

CONCLUSIONS

In conclusion, a novel chiral 2,2'-bipyridine-based system endowed with two helicenic units has been synthesized in enantiopure forms from a 3-bromo-substituted 4aza [6]helicene. This compound displays strong photophysical and chiroptical properties including intense UV/Vis, ECD and emission spectra, and good CPL activity. The 2,2'-bipyridine chelating ability and basic properties were studied through the coordination and protonation processes with Zn(OAc) 2 and triflic acid, respectively. These reversible processes, as shown by quantum-chemical calculations, activate tuning between -*-type and chargetransfer-type excitations that leads to efficient changes of the spectral properties of 1. Accordingly, a new example of versatile molecular chiroptical switch emerges, behaving like a chiral dye. Finally, the 1 / 1•2H + , 2OTf switching system emits a circularly polarized luminescence in the red region, which may be of interest in the field of bioimaging.

EXPERIMENTAL SECTION

All experiments were performed under an atmosphere of dry argon using standard Schlenk techniques. Commercially available reagents were used as received without further purification. All solvents were dried according to standard procedures. Preparative separations were carried out by gravity column chromatography over silica gel (Merck Geduran 60, 0.063-0.200 mm) in 3.5-20 cm columns. Analytical thin layer chromatographies (TLC) were performed using aluminium-coated Merck Kieselgel 60 F254 plates. NMR spectra were recorded on a Bruker 300 Avance ( 1 H: 300 MHz; [START_REF] Kaes | The Most Widely Used Ligand. A Review of Molecules Comprising at Least Two 2,2'-Bipyridine Units[END_REF] 𝐷 are given in deg cm 2 dmol -1 . Circular dichroism (in M -1 cm - 1 ) was measured on a Jasco J-815 Circular Dichroism Spectrometer (IFR140 facility -Biosit -University of Rennes 1). For details on UV/Vis and non-polarised luminescence measurements see SI. Photoirradiation experiments were performed on a Heraeus TQ718 immersion Hg lamp (150 W).

3-Bromo-4-aza[6]helicene 5. Helicene derivative 5 was prepared in two steps, i.e. by a Wittig reaction followed by a photocyclization. 1) First, benzo [g]phenanthren-3-ylmethyl triphenylphosphonium bromide 2 was prepared according to the literature. [START_REF] Lightner | Absolute configuration[END_REF] A solution of 2 (400 mg, 0.686 mmol) in dry THF (16 mL) was cooled to -78º C and then n-BuLi (0.34 mL, 2.2 M in hexanes, 0.748 mmol) was added dropwise. The suspension turned orange and was left to stir at -78º C for 20 minutes, then the reaction mixture was warmed up to room temperature and kept stirring for 20 minutes more before cooling again (dark-red color). A solution of 6bromo-2-pyridinecarboxaldehyde 3 (116 mg, 0.624 mmol) in dry THF (5 mL) was added at -78º C. The reaction mixture was warmed up to room temperature and stirred for 5 hours. The orange suspension was filtered over Celite® and washed with THF. The solvent was stripped off and the olefin 4 was obtained as an E and Z isomers (230 mg, 90 %) which was used as such in the next step. 2) In a flask welladapted to the immersion Hg lamp, a solution of the olefinic precursor 4 (100 mg, 0.243 mmol) in degassed toluene (700 mL) was prepared. Iodine (123 mg, 0.486 mmol) and propylene oxide (0.85 mL, 12 mmol) were then added. The solution was strongly stirred overnight for 15 hours under the 150 W Hg lamp. The crude mixture was evaporated under vacuum and the product was purified by column chromatography (SiO 2 , heptane:Et 3 N 0.5% for packing, and the heptane:ethyl acetate 5-15%). The reaction was repeated five times (500 mg of precursor) to prepare (±)-5 as a yellow solid (222 mg, 45%).

Enantiopure M-and P-5 samples were obtained by HPLC separation over a chiral stationary phase (see SI).

1 H NMR (300 MHz, CD 2 Cl 2 ) : 6.67 (1H, d, J = 9.0 Hz), 6.81 (1H, bt, J = 7.6 Hz), 7.32 (1H, bt, J = 7.6 Hz), 7.54 (1H, d, J = 8.6 Hz), 7.67 (1H, d, J = 9.0 Hz), 7.89 (1H, bd, J = 8.0 Hz), 7.96 (2H, bs), 8.00-8.11 (5H, m), 8.22 (1H, d, J = 9.0 Hz) ppm. [START_REF] Kaes | The Most Widely Used Ligand. A Review of Molecules Comprising at Least Two 2,2'-Bipyridine Units[END_REF] C{1H} NMR (75 MHz, CD 2 Cl 2 ) δ: 123. 8, 124.2, 124.9, 126.0, 126.6, 126.7, 127.65, 127.7, 127.8, 127.8, 128.2, 128.5, 128.6, 128.9, 129.7, 131.7, 131.9, 132.3, 132.8, 134.0, 137.8, 140.8, 148.3 3,3'-Bis-4-aza[6]helicene 1. Helicene derivative 1 was synthesized by means of a Negishi coupling of the enantiopure 3-bromo-4-aza[6]helicene 5. To a solution of n-BuLi (100 L, 0.168 mmol) in dry THF (0.4 mL) at -78º C, a solution of 3-bromo-4-aza[6]helicene 5 (36 mg, 0.088 mmol) in dry THF (0,4 mL) was added dropwise. A color of reaction mixture changed from colorless to wine red at the end of the addition. The solution was kept stirring for 30 minutes at -78º C and then ZnCl 2 (225 L, 1.0 M in diethyl ether) was added. The mixture changed from a red-wine solution to a dark-yellow suspension. The temperature was warmed up to 0º C for 1 hour and then to room temperature for 2 hours more. The reaction mixture was cooled again to 0º C and the 3-bromo-4-aza[6]helicene 5 (33 mg, 0.080 mmol) and catalyst Pd(PPh 3 ) 4 (3 mg) were then added in solution in dry THF (0.3 mL). After 1 hour at 0º C, the reaction mixture was warmed up to reflux. After stirring and refluxing for 15 hours, a bright-yellow precipitate was obtained. Dichloromethane (15 mL) and TPEN (36 mg, 0.0849 mmol) were then added and the mixture sonicated for 5 minutes. Final purification by column chromatography (SiO 2 , heptane:ethyl acetate 15%) yielded 1 (30 mg, 57%) as a bright-yellow solid. Note that around 10% of 4aza[6]helicene was obtained as a side product.

The same procedure was used to prepare enantiopure (M,M)-and (P,P)-1 by starting from M-or P-5, respectively. The meso-1 compound was not characterized due to low solubility and small mass quantity available.

(M,M)-1: 1 H NMR (400 MHz, CD 2 Cl 2 )  6.86 (2H, t, J = 7.6 Hz, H 15 ), 7.37 (2H, t, J = 7.6 Hz, H 14 ), 7.73 (2H, d, J = 8.5 Hz), 7.98 (2H, d, J = 7.9 Hz), 8.02 (8H, dd, J = 9.0, 3.0 Hz), 8.05-8.08 (7H, m), 8.08-8.16 (3H, m), 8.20 (2H, d, J = 8.9 Hz) ppm.. [START_REF] Kaes | The Most Widely Used Ligand. A Review of Molecules Comprising at Least Two 2,2'-Bipyridine Units[END_REF] C{1H} NMR (400 MHz, CD 2 Cl 2 ) : 154.7, 147.7, 136.1, 133.9, 132.8, 132.3, 132.0, 130.9, 130.2, 130.0, 128.9, 128.6, 128.3, 127.8, 127.6, 126.8, 126.4, 126.1, 126.0, 124.5, 117.4 1•Zn(OAc) 2 complex. The complexation was carried out by simple solvation of the ligand 1 (5 mg, 7.6 mmol) and Zn(OAc) 2 •2H 2 O (1.8 mg, 8.4 mmol, 1.1 equivalents) in 5 mL of dichloromethane. One minute of ultrasounds bath was used for total solvation of the complex. The colour intensity increased upon complexation, and once evaporated the solvent, only one set of signals was visible in 1 H NMR spectra.

The same procedure was used to prepare enantiopure (M,M)-and (P,P)-1•Zn(OAc) 2 by starting from (M,M)-or (P,P)-1, respectively.

(M,M)-1•Zn(OAc) 2 : 1 H NMR (400 MHz, CD 2 Cl 2 )  2.07 -2.00 (6H, s), 6.72 (2H, t, J = 7.6 Hz, H 15 ), 7.10 (2H, t, J = 7.6 Hz, H 14 ), 7.28 (2H, d, J = 9.0 Hz), 7.58 (2H, d, J = 8.5 Hz, H 16 ), 7.79 (2H, d, J = 8.0 Hz, H 13 ), 7.91-8.02 (4H, m), 8.11 (4H, bs) Acid-base switching of (M,M)-1. To a solution of (M,M)-1 (2.2  10 -5 M in CH 2 Cl 2 ) were added aliquots of a solution of CF 3 SO 3 H (1.14  10 -3 M in CH 2 Cl 2 ) until saturation of the UV/Vis and ECD signals (5 L added). Upon addition of aliquots of NEt 3 (1.4  10 -3 M in CH 2 Cl 2 ), the UV/Vis and ECD spectra of (M,M)-1 were recovered.

Figure 1 .

 1 Figure 1. X-ray crystallographic structures of compounds 1 and 5 (only one stereoisomer is shown).

  P)-, (M,M)-and (P,M)-

Figure 2 .

 2 Figure 2. Experimental UV/Vis spectra of 5 and (P*,P*)-1 17 (panel a) and ECD spectra of enantiopure M-and P-5 and (M,M)-and (P,P)-1 (panel b) in CH 2 Cl 2 (C ~ 3  10 -5 M). Insets: Corresponding TDDFT (LC-PBE0*/SV(P) with a continuum solvent model for CH 2 Cl 2 ) calculated data. No spectral shift has been applied. Numbered excitations (with oscillator strength f, and rotatory strength R, in 10 -40 cgs, values given in parentheses) are those analyzed in detail. See also SI.

Figure 3 .

 3 Figure 3. Left: Isosurfaces (±0.03 au) of frontier MOs of the bis-helicenic bipyridine ligand 1, of its corresponding Zn complex 1•Zn(OAc) 2 , and of its doubly protonated species 1•2H + . Right: Isosurface (±0.0015 au) of dominant ETS-NOCV deformation density channel depicting formation of the 1-Zn(OAc) 2 bonding. Only the most populated (lowest-energy) conformers' results have been shown; see SI for a full set of data.

Figure 4 .

 4 Figure 4. Evolution of UV/Vis spectrum of (P*,P*)-1 17 (panel a) and ECD spectrum of (M,M)-1 (panel b) upon addition of Zn(OAc) 2 •2H 2 O aliquots (0.2 equiv. until saturation at 1.0-1.2 equivs; C 3  10 -5 M, CH 2 Cl 2 , rt). Panel c: Evolution of fluorescence of (P*,P*)-1 17 ( ex = 350 nm, C 2.2  10 -5 M, CH 2 Cl 2 , rt). Panel d: Reversible ECD 274nm and ECD 454nm switching process.

Figure 5 .

 5 Figure 5. Comparison of the simulated UV/Vis (panel a) and ECD (panel b) spectra of the bis-helicenic bipyridine ligand 1 and of its corresponding Zn complex 1•Zn(OAc) 2 (Boltzmann-averaged spectrum at 25 ºC based on the most populated conformers found). No spectral shift has been applied. Calculated excitation energies along with the corresponding oscillator and rotatory strengths (for the lowest-energy conformer in the case of 1•Zn(OAc) 2 ) indicated as 'stick' spectra. Numbered excitations correspond to those analyzed in detail. See SI for a full set of data.

Figure 7 .

 7 Figure 7. Energy profiles for the rotation around CC' bond in bis-helicenic bipyridine 1 (panel a) along with molecular structures corresponding to their characteristic points (panel b). BP and BP-D3 SV(P) calculations with continuum solvent model for CH 2 Cl 2 . Values listed in panel b are relative energies ΔE (in kcal/mol) and NCC'N' dihedral angles (in deg) obtained with BP / BP-D3.

Figure 8 .

 8 Figure 8. Evolution of UV/Vis spectrum (panel a), ECD spectrum (panel b), and fluorescence spectrum (panel c) of (M,M)-1 upon addition of TfOH aliquots (0.2 equiv. until saturation at 8 equivs; C 2.2  10 -5 M, CH 2 Cl 2 , rt,  ex = 350 nm). Panel d: Picture of emissive solutions of 1, its Zn II complex, and its protonated species irradiated with 365 nm lamp.

  C: 75 MHz), a Bruker Avance 400 or a Bruker Avance 500 ( 1 H: 500 MHz; 13 C: 125 MHz) spectrometers at rt, unless stated otherwise, using partially deuterated solvents as internal standards. Coupling constants (J) are given in Hz and chemical shifts () in ppm. Multiplicities are denoted as follows: s = singlet, d = doublet, t = triplet, m = multiplet, while b = broad. Assignment of hydrogen atoms is based on 1H-1H COSY experiment. Assignment of carbon atoms is based on HMBC, HMQC and DEPT-135 experiments. Mass spectrometry was performed by the CRMPO, University of Rennes 1. Specific rotations ( in deg cm 2 g -1 ) were [𝛼] 20 𝐷 measured in a 1 dm thermostated quartz cell on a PerkinElmer Model 341 polarimeter. Molar rotations [𝜙] 20

  ppm. HR-MS (QTOF), ESI, m/z: calcd. for C 25 H 15 N 79 Br [M+H] + : 408.0382; found: 408.0383. Mp = 165-169 °C.

  ppm. HR-MS (QTOF), ESI, m/z: calcd. for C 50 H 28 N 2 Na [M+Na] + 679.2145 found (pos.) 679.2146. Mp = 170-175 °C.

  , 8.14 (2H, d, J = 8.1 Hz), 8.24 (2H, d, J = 8.1 Hz), 8.34 (2H, d, J = 9.0 Hz) 8.52 (2H, d, J = 9.0 Hz), 8.68 (2H, d, J = 9.0 Hz) ppm. 13 C: After experiment overnight in 500 MHz the signals were not large enough to give an accurate set of peaks. Yellowish solid; Mp = 162-167 °C.

The Journal of Organic Chemistry

ACKNOWLEDGMENTS

We acknowledge the Ministère de l'Education Nationale, de la Recherche et de la Technologie, the Centre National de la Recherche Scientifique (CNRS), the CNRS (Chirafun network), and the ANR (12-BS07-0004-METALHEL-01), for financial support. H. I. thanks the Région Bretagne for financial support. K. D. thanks The University of Gabès, the University of Rennes 1 and Campus France for financial support. M.S.-H. acknowledges the young researchers' Tsubsidy from the Ministry of Science and Higher Education in Poland.

ASSOCIATED CONTENT

Supporting Information 1 H, [START_REF] Kaes | The Most Widely Used Ligand. A Review of Molecules Comprising at Least Two 2,2'-Bipyridine Units[END_REF] C, and COSY spectra of compounds 5, (M,M)-1, and (M,M)-1•Zn(OAc) 2 . X-ray diffraction data of 1 and 5. HPLC conditions for 1 and 5. Photophysical and chiroptical properties of (M,M)-1, (M,M)-1•Zn(OAc) 2 , and (M,M)-1•2H + , 2OTf -. Computational details. Additional calculated results. This material is available free of charge on the ACS Publications website at DOI:

AUTHOR INFORMATION

Corresponding Authors * jeanne.crassous@univ-rennes1.fr * srebro@chemia.uj.edu.pl ORCID Jeanne Crassous: 0000-0002-4037-6067X Monika Srebro-Hooper: 0000-0003-4211-325X

Notes

The authors declare no competing financial interest.


Tetiana Kalachova

Martin Janda

Vladimír Šašek

Jitka Ortmannová

Pavla Nováková

Petre I Dobrev

Volodymyr Kravets

Anne Guivarc'h

Deborah Moura

Lenka Burketová

Kalachova + Tetiana

Janda + Martin

Šašek Vladimír

Ortmannová Jitka

Nováková Pavla

Dobrev I Petre

Kravets Volodymyr

Moura Deborah

Burketová Lenka

Olga Valentová

Ruelland Eric
email: eric.ruelland@upmc.fr

Identification of salicylic acid-independent responses in an Arabidopsis 3 phosphatidylinositol 4-kinase beta double mutant

Keywords: pi4kβ1β2 / PI4Ks, callose, salicylic acid, phytohormones, isochorismate synthase 1, biotic stress, pathogens, Arabidopsis thaliana

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Salicylic acid (SA) is a phytohormone playing a role in many plant physiological processes although its role is mainly documented in plant response to biotic stress when SA accumulates within tissues, both at the site of attack and in a systemic manner [START_REF] Vlot | Salicylic Acid, a Multifaceted Hormone to Combat Disease[END_REF][START_REF] Janda | Magical mystery tour: salicylic acid signalling[END_REF]. In plants, SA is biosynthesized via two pathways. One is dependent on phenylalanine ammonia-lyase (PAL; EC 4.3.1.24) which catalyses the conversion of phenylalanine to transcinnamic acid. In the other pathway, the key enzyme is ISOCHORISMATE SYNTHASE (ICS; EC 5.4.4.2) which catalyses the isomerization of chorismate into isochorismate (Dempsey et al. 2011). The ICS-dependent pathway was shown to be responsible for most SA accumulation upon pathogen attack. In A. thaliana, two ICS isoforms exist, but the major role in SA biosynthesis is played by ICS1. The ICS1 mutant is known as sid2 for salicylic acid induction deficient 2 (Wildermuth et al. 2001;Wagner et al. 2013;[START_REF] Cui | A core function of EDS1 with PAD4 is to protect the salicylic acid defense sector in Arabidopsis immunity[END_REF]. When SA levels increase, downstream signalling events are triggered, and the best described molecular pathway is dependent on NONEXPRESSOR OF PATHOGENESIS RELATED 1 (NPR1).

Upon SA action, homo-oligomeric NPR1 protein undergoes dissociation by reduction and the resulting monomers move into the nucleus where they interact with TGA-transcription factors to induce the expression of SA responsive genes. A NPR1 independent pathway also exists in response to SA [START_REF] Janda | Magical mystery tour: salicylic acid signalling[END_REF]. The activation of SA signalling pathways leads to robust changes in the plant transcriptome, including defence related genes [START_REF] Seyfferth | Salicylic acid signal transduction: the initiation of biosynthesis, perception and transcriptional reprogramming[END_REF]). Among the immune responses affected by changes in SA levels or by SA treatments is the accumulation of callose (Antignani et al. 2015;[START_REF] Dong | Expression of callose synthase genes and its connection with Npr1 signaling pathway during pathogen infection[END_REF][START_REF] Kohler | Benzothiadiazole-induced priming for potentiated responses to pathogen infection, wounding, and infiltration of water into leaves requires the NPR1/NIM1 gene in Arabidopsis[END_REF] ,a (1,3)-β-Glucan occurring in plant cell walls.

The signaling pathways triggered by SA are still the subject of current research. We have shown that phosphoinositides, the phosphorylated derivatives of phosphatidylinositol (PI), are involved in SA transduction. Indeed, PI can be phosphorylated at the D4 position of the inositol ring by phosphatidylinositol-4-kinases (PI4K) thus leading to phosphatidylinositol 4phosphate (PI4P); which can be phosphorylated further into phosphatidylinositol-4,5bisphosphate (PI-4,5-P2). There are two types of PI4Ks according to their primary sequences and pharmacological sensitivities. Type II PI4Ks are inhibited by adenosine while type III PI4Ks are inhibited by micromolar concentrations of wortmannin, a steroid metabolite produced by the fungus Penicillium funiculosum [START_REF] Nakanishi | A wortmannin-sensitive phosphatidylinositol 4-kinase that regulates hormone-sensitive pools of inositolphospholipids[END_REF][START_REF] Balla | Imaging and manipulating phosphoinositides in living cells[END_REF][START_REF] Krinke | Phosphatidylinositol 4-Kinase activation is an early response to salicylic acid in Arabidopsis suspension cells[END_REF]). From the A. thaliana genome, twelve putative PI4K isoforms have been identified.

Eight belong to type-II (AtPI4Kγ1-8), and four belong to type-III (AtPI4Kα1 and α2, AtPI4Kβ1 and β2) [START_REF] Delage | Arabidopsis type-III phosphatidylinositol 4-kinases beta1 and beta2 are upstream of the phospholipase C pathway triggered by cold exposure[END_REF]Janda et al. 2013). We have shown that type III-PI4Ks are activated when A. thaliana suspension cells respond to SA, thus leading to an increase in PI4P and PI-4,5-P2 [START_REF] Krinke | Phosphatidylinositol 4-Kinase activation is an early response to salicylic acid in Arabidopsis suspension cells[END_REF]). PI-4,5-P2 can act as a cofactor to some phospholipase Ds [START_REF] Pappan | Substrate selectivities and lipid modulation of plant phospholipase D alpha, -beta, and -gamma[END_REF]. Interestingly, there is an overlap between SA responsive genes controlled by PI4Ks and those controlled by PLDs, leading to the working model that in response to SA, PI4P and PI-4,5-P2 are produced with PI-4,5-P2 acting as a cofactor for a PLD, whose product, PA, will trigger a signalling cascade [START_REF] Krinke | Phospholipase D activation is an early component of the salicylic acid signaling pathway in Arabidopsis cell suspensions[END_REF][START_REF] Kalachova | The inhibition of basal phosphoinositide-dependent phospholipase C activity in Arabidopsis suspension cells by abscisic or salicylic acid acts as a signalling hub accounting for an important overlap in transcriptome remodelling induced by these hormones[END_REF].

In order to better characterize the role of PI4Ks in the response to SA, we have used A. thaliana mutants altered in type III PI4Ks. We previously worked on a double mutant defective in the two PI4K genes. Surprisingly, pi4kβ1β2 exhibited a constitutively high SA level that resulted in constitutive high transcription of SA responsive genes such as PR-1 (PATHOGENESIS RELATED 1). Therefore, PI4Ks are not only involved in SA transduction but they can also impact SA concentration. Furthermore, this double mutant exhibited dwarfism and was more resistant to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326 [START_REF] Sasek | Constitutive salicylic acid accumulation in pi4kIIIbeta1beta2 Arabidopsis plants stunts rosette but not root growth[END_REF]. The pi4kβ1β2 plant was crossed with mutants impaired in components of SA synthesis (sid2, impaired in ICS1 expression; eds1), SA transduction (npr1) or a mutant expressing bacterial SA-hydroxylase (NahG) that degrades SA to catechol. The resulting triple mutants allowed us to conclude that the dwarf phenotype of pi4kβ1β2 plants was dependent on SA accumulation and its transduction via the NPR1 pathway [START_REF] Janda | The Arabidopsis pi4kIIIbeta1beta2 double mutant is salicylic acid-overaccumulating: a new example of salicylic acid influence on plant stature[END_REF][START_REF] Sasek | Constitutive salicylic acid accumulation in pi4kIIIbeta1beta2 Arabidopsis plants stunts rosette but not root growth[END_REF]).

In the present study, our aim was to identify amongst the cellular and physiological processes affected by the deficiency of PI4Kβ1β2, those that were either SA-dependent or SAindependent. To achieve this, we used the sid2pi4k12 triple mutant that does not accumulate SA and exhibits wild-type sized rosettes [START_REF] Sasek | Constitutive salicylic acid accumulation in pi4kIIIbeta1beta2 Arabidopsis plants stunts rosette but not root growth[END_REF]. In this mutant, the effects of PI4Kß1 and PI4Kß2 mutations would not be masked by high SA levels. We showed that hormonal levels and pathogen resistance were mainly dependent on SA. However, we could show that sid2pi4k12 plants accumulated higher amounts of callose in response to flg22 and wounding. Interestingly, this SA-independent callose accumulation was also observed during early stages of interactions with Blumeria graminis when penetration was observed. Our data suggest that PI4Ks are involved in plant immune responses not only through SA accumulation but also via SA-independent processes.

MATERIALS AND METHODS

Plant material, growth conditions

In this study, we used the following genotypes of A. thaliana: Columbia-0 (WT), sid2-3 [START_REF] Gross | A plant locus essential for phylloquinone (vitamin K1) biosynthesis originated from a fusion of four eubacterial genes[END_REF]), npr1-1 [START_REF] Cao | The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats[END_REF]) NahG [START_REF] Delaney | A central role of salicylic Acid in plant disease resistance[END_REF], pi4kβ1β2

(SALK_040479 / SALK_09069; [START_REF] Preuss | A role for the RabA4b effector protein PI-4Kbeta1 in polarized expansion of root hair cells in Arabidopsis thaliana[END_REF]), sid2pi4kβ1β2, NahGpi4kβ1β2, and npr1pi4kβ1β2 mutants previously described [START_REF] Sasek | Constitutive salicylic acid accumulation in pi4kIIIbeta1beta2 Arabidopsis plants stunts rosette but not root growth[END_REF].

All plants were cultivated in Jiffy 7 peat pellets at 22°C with 70% relative humidity. All plants were watered without additional fertilizers. As for the light regimes, plants were routinely cultivated in daily cycles of 10 h light (100-130 μE m -2 s -1 ) and 14 h dark. Plants that would be used for hormonal analysis or transcription analysis were cultivated under 16 h light (130-150 μE m -2 s -1 ) and 8 h dark.

Pathogen inoculation

Two-week-old plants grown at high density in one pot were sprayed with H. arabidopsidis NoCo2 spores (~100 spores/μl). The infected plants were cultivated in closed transparent plastic boxes in high humidity for 6 days under 16 h light/8 h dark (100-130 μE m -2 s -1 ) at 19°C. For analysis, leaves collected from one pot were considered as one sample (for each genotype, 11 samples were analysed). Spores were counted under a microscope using a Bürker chamber and expressed as relative spore number in [%], where relative spore number for a given control genotype (WT or sid2) was set to 100%. The spores were counted as spores per mg of tissue fresh weight. The experiments [WT vs pi4kβ1β2] and [sid2 vs sid2pi4kβ1β2] were conducted independently.

Inoculation with Pseudomonas syringae was performed according to [START_REF] Katagiri | The Arabidopsis thaliana-pseudomonas syringae interaction[END_REF] with modifications. Bacteria were cultivated overnight on King's B medium plates containing rifampicin (50 µg/µl). Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) and Pseudomonas syringae pv. tomato DC3000 AvrRpt2 (Pst DC3000 AvrRpt2) were taken from the respective plate and resuspended in 10mM MgCl2 to give an OD600=0.001. Four-week-old plants were infiltrated with this suspension.

One disc (6 mm) from one leaf, three leaves at a similar developmental stage from one plant and three plants were collected as one sample of one genotype at 0 dpi and 3 dpi (3 dpi only for Pst DC3000; 2 dpi for Pst DC3000 AvrRpt2). Leaf discs were ground in 10mM MgCl2 and decimal dilutions were performed. Colony forming units were counted.

Four-week-old A. thaliana plants were treated with 6 µl drops containing Botrytis cinerea BMM spores (5.10 4 spores/ml) by applying a single drop to each leaf, with three leaves at a similar developmental stage inoculated for each plant. Treated plants were placed into closed plastic boxes and kept in low light (16h light/8h dark, 21°C; 10-20 μE m -2 s -1 ) for 56 hpi.

Blumeria graminis f. sp. hordei (Bgh) was cultivated continuously on fresh barley (Golden promise) grown under short day conditions (19°C, 10/14 h, 50% humidity, at a light intensity of 70 μmol m -2 s -1 ). Plants, approximately 4 weeks old, were inoculated by spreading spores from infected barley onto the adaxial side of their leaves (from leaf to leaf). The 5th -6th leaves were cut off at selected hpi and cleared with 96% ethanol or chloral hydrate. For penetration rate, fungal structures were stained with 250 mg/ml trypan blue in a lactophenol/ethanol solution [START_REF] Vogel | Isolation and characterization of powdery mildew-resistant Arabidopsis mutants[END_REF]. Stained leaves were observed by classical epifluorescence microscopy or bright-field microscopy using a Zeiss AxioImager ApoTome2 (objective 100x).

Callose deposition

Four-week-old A. thaliana plants were treated for 24 h with 100 nM flg22 or infiltrated with Pst DC3000. Distilled water infiltration was used as a control (mock) treatment. Infiltrated leaves were decolored in ethanol:glacial acetic acid (3:1 v/v). The leaves were then rehydrated in successive baths of 70% ethanol (at least 1 h), 50% ethanol (at least 1 h), 30% ethanol (at least 1 h) and water (at least 2h). Leaves were stained for 4 h with 0.01 % aniline blue in 150 mM K2HPO4, pH 9.5. Callose deposition was observed by fluorescence microscopy using a Zeiss AxioImager ApoTome2 (objective 10x). In the case of Bgh infection analysis, we calculated only callose spots using the high circularity function of the measurement settings at an interval of 0.5-1 which allowed us to distinguish only the cells with the size exclusion limit for spots corresponding to either encased haustoria or enormous papilla. Callose deposition was observed by fluorescence microscopy using a Zeiss AxioImager ApoTome2 (objective 10x). Images were processed with ImageJ software. At least four leaves from three independent plants were analysed for each variant.

RNA extraction and qPCR analysis

Plant tissues were homogenised in 2-ml screw-cap tubes containing 1 g of 1.3-mm diameter silica beads using a FastPrep-24 instrument (MP Biomedicals, Santa Ana, CA, USA). Total RNA was isolated using a Spectrum Plant Total RNA kit (Sigma-Aldrich, USA) and treated with a DNA-free kit (Ambion, USA). Subsequently, 1 μg of RNA was converted to cDNA with M-MLV RNase H-Point Mutant reverse transcriptase (Promega Corp., USA) and an anchored oligo dT21 primer (Metabion, Germany). Gene transcription was quantified by qPCR using a LightCycler 480 SYBR Green I Master kit and a LightCycler 480 (Roche, Switzerland). The PCR conditions employed were 95°C for 10 min followed by 45 cycles of 95°C for 10 s, 55°C for 20 s, and 72°C for 20 s. Melting curve analyses were then carried out.

Relative transcription was normalized to the housekeeping genes SAND or TIP41 [START_REF] Czechowski | Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis[END_REF]. Primers were designed using PerlPrimer v1.1.21 (Marshall, 2004).

The primers used were CalS1_FP, AAGAGCGGAGGGTCACTTTG;

CalS1_RP, GGCGACACGAATAGACGGAT; CalS12_FP, TTCACTCCGTTTTCCCGAGG; CalS12_RP, GGAGAGAGACGCATCTGAGC.

Analysis of plant hormones

Plant hormones were extracted from 100 mg of frozen tissues and their concentrations were determined as previously described [START_REF] Dobrev | Quantification of abscisic Acid, cytokinin, and auxin content in salt-stressed plant tissues[END_REF][START_REF] Dobrev | Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed-mode solid-phase extraction[END_REF] after the addition of appropriate internal standards. Hormone analysis was carried out on four samples, each taken from three plants. Briefly, samples were homogenized in tubes with 1.3 mm silica beads using a FastPrep-24 instrument (MP Biomedicals, USA). Samples were then extracted with a methanol/H2O/formic acid (15:4:1, v:v:v) mixture, which was supplemented with stable isotope-labeled phytohormone internal standards (10 pmol per sample) in order to check recovery during purification and to validate the quantification. The clarified supernatants were subjected to solid phase extraction using Oasis MCX cartridges (Waters Co., USA). The eluates were evaporated to dryness and the generated solids were dissolved in 30 μl of 15% (v/v) acetonitrile in water. Hormones were separated and quantified by Ultimate 3000 high-performance liquid chromatography (Dionex, Bannockburn, IL, USA) coupled to a 3200 Q TRAP hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems, Foster City, CA, USA) as described by [START_REF] Dobrev | Analytical Determination of Auxins and Cytokinins[END_REF]. Metabolite levels were expressed in pmol/g fresh weight (FW).

Statistical analysis

At least three independent biological replicates were performed for all experiments. Statistical analysis was conducted by paired t-test or ANOVA with Tukey honestly significant difference (HSD) multiple mean comparison post hoc test. The number of analysed samples was specified for each condition. The correlation matrix for hormonal levels was prepared using R-software Hmisc and corrplot packages based on the Pearson correlation [START_REF] Team | R: A language and environment for statistical computing[END_REF].

RESULTS

The pi4k12 double mutant has altered phytohormonal levels

Our goal was to identify SA-dependent and SA-independent processes triggered by the double pi4kβ1β2 mutation. To do so, we used a sid2pi4kβ1β2 triple mutant. If a process triggered by the double pi4kβ1β2 mutation is SA-dependent, then it should disappear in the sid2pi4kβ1β2 triple mutant. On the other hand, if a process triggered by the double pi4kβ1β2 mutation is SA-independent, then it should still be observed in the sid2pi4kβ1β2 triple mutant.

Since the main effect of the double pi4kβ1β2 mutation was on the level of SA, we decided to quantify a broad spectrum of phytohormones in the sid2pi4kβ1β2 triple mutant. The hormone levels obtained were compared to those of pi4k12, sid2 and WT plants. A first look allowed us to establish that the pi4k12 double mutation does not impact only SA levels. Many hormone-related metabolites showed significantly different levels in pi4k12 plants when compared to WT plants while most of them remained at WT levels in sid2pi4kβ1β2

(supplemental Table S1). In our previous study, we created an additional triple mutant by crossing pi4kβ1β2 with a NahG mutant impaired in SA accumulation: NahGpi4kβ1β2 [START_REF] Sasek | Constitutive salicylic acid accumulation in pi4kIIIbeta1beta2 Arabidopsis plants stunts rosette but not root growth[END_REF]. To confirm and to strengthen the results obtained with sid2pi4kβ1β2, we also quantified phytohormones in NahGpi4kβ1β2, together with their corresponding single mutants (Supplemental table S1). From these data, we built a correlation matrix (Fig. 1).

Many hormone levels were correlated to higher SA levels (Pearson correlation higher than 0.7) as seen for the abscisic acid (ABA) derivatives such as 9-hydroxy-ABA (9OH-ABA), phaseic acid (PA) and dihydrophaseic acid (DPA). However, the high levels of DPA, PA and 9OH-ABA observed in pi4kβ1β2 versus WT were no longer seen in the triple mutants with low SA; therefore, these metabolite levels were controlled by SA in the pi4kβ1β2 double mutant (Fig. S1A).

Since the main genetic pathway of SA response is controlled by NPR1, we investigated whether the hormonal control of SA was NPR1-dependent. This was achieved using a previously generated npr1pi4kβ1β2 triple mutant where the SA level was 30-fold that of WT and 4-fold that of pi4kβ1β2. Interestingly, levels of DPA, PA and 9-OH were still high in npr1pi4kβ1β2, showing that the SA effect on these metabolites was only partially NPR1dependent. It should be noted that ABA levels did not correlate with SA (Pearson correlation 0.3), thus indicating that the action of SA on ABA-derivatives is probably not directly connected with the biosynthesis of ABA, but with its metabolism.

Other hormones with high Pearson correlations to SA were the Asp conjugated form of indole-3-acetic acid (Asp-IAA) and some cytokinins. The increased level of IAA-Asp observed in pi4kβ1β2 was no longer observed in the triple mutants with low SA. However, it was still visible in the npr1pi4kβ1β2 triple mutant thus suggesting that this metabolite is controlled by SA but it is only partially NPR1-independent (Fig. S1B). The pattern of IAA was different from that of Asp-IAA, suggesting that SA control of Asp-IAA is on aspartate conjugation. As for cytokinins, the increase in cis-zeatin (cZ), cis-zeatin-riboside (cZR), ciszeatin-7-N-glucoside (cZ7G), cis-zeatin-riboside-O-glucoside (cZROG) and trans-zeatin-Oglucoside (tZOG) and the decrease of trans-zeatin-7-N-glucoside (tZ7G) and trans-zeatin -9-N-glucoside (tZ9G) in the pi4k12 double mutant was SA-driven. In contrast to the other hormones tested, SA action on tZROG appeared to be partially independent of NPR1 (Fig. S1C).

We identified one hormone for which its level was altered in pi4kβ1β2 plants independently of SA. Indeed, the increase of oxIAA-GE observed in pi4kβ1β2 was still visible in the triple mutants with low SA. It should be noted that oxIAA did not follow the same pattern (Fig. S2).

Pathogen resistance, including a necrotroph, of pi4kβ1β2 plants is SA-dependent

At the hormonal level our data show that the major change induced by the pi4k12 double mutation was an increase in SA, with this change determining the levels of many other hormones. Since a major role of SA is related to biotic stress responses, we reasoned that processes related to biotic stress, dependent or not on SA, might also be altered in the double mutant.

Whether the PI4K double mutation per se was accompanied by an enhanced resistance to pathogens was investigated. Comparing resistance in sid2pi4k12 plants to that in pi4k12

or sid2 would allow us to distinguish between the effectiveness of SA-dependent and independent responses. Therefore, different pathogens with different lifestyles (biotrophs, hemibiotrophs and necrotrophs) were tested [START_REF] Glazebrook | Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens[END_REF]. The pi4k12 double mutant plants were more resistant to the biotroph H. arabidopsidis NoCo2 compared to WT.

However, sid2pi4kβ1β2 resistance was similar to that of sid2 plants (Fig. 2A). We then studied resistance to the hemibiotroph Pseudomonas syringae pv. tomato DC3000 in its wild type (Pst DC3000) or AvrRpt2 expressing form (Pst DC3000 AvrRpt2). Pst DC3000

AvrRpt2 leads to a strong effector triggered immunity (ETI) response compared to Pst DC3000. With both forms, pathogen development was reduced in pi4k12 plants compared to the WT while sid2pi4kβ1β2 resistance was comparable to that of sid2 and lower than WT plants (Fig. 2B, Fig. S3). Unexpectedly, the double mutant also showed an increased resistance to the necrotroph B. cinerea which was also SA-dependent since sid2pi4kβ1β2

resistance was similar to that of sid2 and WT plants (Fig. 2C). For each pathogenic assay, triple mutant resistance was similar to sid2, indicating that SA-dependent pathways were dominant in the immune response. Putative mechanisms regulated by PI4K activity alone were not sufficient to establish pathogen resistance.

The enhanced level of basal callose deposition in pi4kβ1β2 is mainly SA-dependent while stress-induced callose accumulation is not SA levels modulate numerous processes associated with immune responses including the strengthening of leaf tissues and particularly cell walls around the infection site by lignification and callose accumulation (Voigt 2014). Interestingly, SA pre-treatment also has a positive effect on flagellin-induced callose accumulation (Yi et al. 2014). We therefore studied callose levels, accumulated in leaf tissues in response to treatment with the flagellin epitope (flg22). At first, we studied callose accumulation in the absence of flg22 treatment (control; Fig. 3A). The pi4kβ1β2 double mutant exhibited a constitutively high level of callose deposition, as previously shown (Antignani et al. 2015). We were able to show that a spatial pattern in callose accumulation existed, since the examination of different regions of interest (ROI) indicated a higher accumulation in the upper part of the leaf edges (Fig. 3B).

Callose accumulation was then assessed in either mock-infiltrated or flg22-treated plants (Fig. 4). In this case, callose accumulation was much higher in pi4kβ1β2 when compared to WT plants (Fig. 4A,B). The sid2pi4kβ1β2 triple mutant was used to investigate whether high callose deposition in the double mutant depended on its high SA level. For mock treatments, callose deposition was much lower in sid2pi4kβ1β2 compared to pi4kβ1β2 plants. This provides arguments for a SA-dependent higher basal callose deposition. This was confirmed by the response to flg22. In WT plants, flg22 induced 20-times more callose compared to control mock infiltrations. Again, the callose level in pi4kβ1β2 was higher (2-fold) compared to WT plants. This increase was reduced when the sid2 mutation was introduced into the pi4kβ1β2 double mutant as the level in the triple mutant was circa 2-fold lower than in the double mutant and in the same range as the WT level. This indicated that SA was a major inducer of callose accumulation in the pi4kβ1β2 genotype context. Yet, the sid2pi4kβ1β2 triple mutant exhibited a higher callose deposition than sid2 plants. Therefore, the pi4kβ1β2 double mutation per se had a role in the high callose accumulation observed in the pi4kβ1β2 double mutant.

We also studied callose accumulation in leaf tissues in response to mechanical wounding (Fig. S4). Again, in response to wounding, the PI4K double mutation enhanced callose accumulation via a SA-dependent pathway.

Whether callose overaccumulation correlated with the transcription of callose synthases (CalSs) was then investigated. Among 12 callose synthases, CalS1, CalS12 have been shown

to be related to SA and/or biotic stresses [START_REF] Dong | Expression of callose synthase genes and its connection with Npr1 signaling pathway during pathogen infection[END_REF]. The transcript levels of CalS1 and CalS12 were tested by qPCR in WT, pi4kβ1β2, sid2 and sid2pi4kβ1β2 plants treated or not with flg22. No correlation was observed between CalS1 and CalS12 transcript levels and callose accumulation (Fig. S5).

pi4kβ1β2 has altered non-host resistance that is SA-independent

The establishment of non-host resistance is based on different mechanisms, involving vesicular secretion as well as callose accumulation [START_REF] Collins | SNARE-protein-mediated disease resistance at the plant cell wall[END_REF][START_REF] Assaad | The PEN1 syntaxin defines a novel cellular compartment upon fungal attack and is required for the timely assembly of papillae[END_REF][START_REF] Takemoto | Re-organization of the cytoskeleton and endoplasmic reticulum in the Arabidopsis pen1-1 mutant inoculated with the nonadapted powdery mildew pathogen, Blumeria graminis f. sp. hordei[END_REF], Bohlenius et al. 2010[START_REF] Lee | Current understandings of plant Nonhost Resistance[END_REF]. To study the role of PI4K in such responses, we tested penetration success and callose production in response to the nonhost pathogen Blumeria graminis f. sp. hordei (Bgh). In Bgh/A. thaliana interactions, callose accumulates in defensive papillae and haustorial encasements or around dead cells [START_REF] Assaad | The PEN1 syntaxin defines a novel cellular compartment upon fungal attack and is required for the timely assembly of papillae[END_REF], Ellinger et al. 2013[START_REF] Jacobs | An Arabidopsis callose synthase, GSL5, is required for wound and papillary callose formation[END_REF]. The enhanced number of plant cells with developed haustoria or dead cells reflects the penetration success of fungal hyphae (Fig. 5A).

In our experiments a higher penetration correlated with greater callose accumulation in the plant tissue. In particular the pi4kβ1β2 double mutant showed an enhanced successful penetration of Bgh 24 h post inoculation (hpi), as seen by the enhanced number of haustoria and dead cells. A similar defect in penetration resistance was seen in sid2/pi4kβ1β2, thus revealing the SA-independent character of this phenomenon (Fig. 5B). Both, pi4kβ1β2 and sid2/pi4kβ1β2 accumulated more callose (Fig. 5C) and over larger areas compared to WT plants (Fig. 5C,D). Thus, the lower penetration resistance accompanied by callose accumulation in pi4kβ1β2 was independent of the SA pathway.

DISCUSSION

The aim of this study was to investigate SA-dependent and -independent processes caused by the pi4k12 double mutation. As previously shown [START_REF] Sasek | Constitutive salicylic acid accumulation in pi4kIIIbeta1beta2 Arabidopsis plants stunts rosette but not root growth[END_REF], this mutant accumulates a constitutively high level of SA. In pi4k12, SA biosynthesis is dependent on ISOCHORISMATE SYNTHASE 1 (ICS1), demonstrated by an absence of SA accumulation in the sid2pi4kβ1β2 triple mutant with impaired ICS1 transcription [START_REF] Sasek | Constitutive salicylic acid accumulation in pi4kIIIbeta1beta2 Arabidopsis plants stunts rosette but not root growth[END_REF]. This was confirmed with the hormone analysis described in the present study. The sid2pi4kβ1β2 triple mutant lacking high SA was used as a tool to distinguish between SA-dependent and SA-independent effects caused by the double mutation in pi4k12.

We first measured phytohormonal levels in fully developed leaves of four-week-old A.

thaliana WT, pi4k12, sid2 and sid2pi4kβ1β2 plants. According to our knowledge, this is the broadest phytohormonal study carried out with a SA over-accumulating mutant (in our case pi4k12) and its comparison with a plant having the same background but with an impaired SA pathway (sid2pi4kβ1β2). The level of 15 hormone derivatives (excluding SA)

was altered in pi4k12 compared to WT leaves. For 13 of these, this was SA-dependent. We could identify two metabolites, cis-zeatin-riboside-5'-monophosphate and glucosylesters of oxindole-3-acetic acid, for which the pi4k12 double mutation effect was SA-independent.

The same conclusions were also reached with another triple mutant, NahGpi4kβ1β2. Amongst the hormones controlled by SA were ABA derivatives such as DPA, PA and 9OH-ABA. As ABA levels did not correlate with SA, the action of SA on ABA-derivatives did not appear to act on ABA biosynthesis but on its metabolism. Such an effect of SA on ABA catabolism is poorly described. A slight induction of ABA 8'-hydroxylase expression was observed after a 24 h SA treatment of rice seedlings [START_REF] Mega | Sustained low abscisic acid levels increase seedling vigor under cold stress in rice (Oryza sativa L.)[END_REF]. In an A. thaliana cpr22 mutant (constitutive expressor of PR genes 22), the increase of SA and ABA levels due to a high-low humidity shift was also followed by the SA-dependent expression of genes encoding ABA metabolising enzymes [START_REF] Mosher | The lesion-mimic mutant cpr22 shows alterations in abscisic acid signaling and abscisic acid insensitivity in a salicylic aciddependent manner[END_REF].

Other hormones displaying a high correlation to SA were IAA-Asp and some cytokinins (SA positively controlled cZROG and tZOG and negatively controlled tZ7G and tZ9G). The pattern of IAA was different from that of IAA-ASP, suggesting that SA control of IAA-Asp was on aspartate conjugation. Aspartate conjugation is catalysed by GH3.2-GH3.6 (Normanly 2010), with our transcriptome data obtained with in vitro grown pi4k12 seedlings (Sasek et al. 2014) indicating that GH3.3 (At2g23170) was overexpressed in the double mutant. It would be interesting to investigate whether it was responsible for the IAA-Asp/SA correlation in our double mutant.

Our results demonstrate the major role of hormonal cross-talk between SA and other hormones but only a minor role of impairment of PI4Kβ1/β2 per se. Since a major role of SA is related to responses to biotic stresses, we reasoned that other processes related to biotic stress, whether SA-dependent or not, could also be altered in the double mutant. We tested the resistance of WT, pi4k12, sid2 and sid2pi4k12 plants against representative biotrophic (oomycete H. arabidopsidis NoCo2), hemibiotrophic (bacteria Pst DC3000) and necrotrophic (fungus B. cinerea) pathogens. Results clearly showed that resistance to these pathogens was dependent on a high SA content. Resistance to H. arabidopsidis NoCo2 and Pst DC3000 was perhaps not surprising as resistance to such pathogens is generally associated with SA signalling [START_REF] Glazebrook | Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens[END_REF]. On the other hand, the role of SA in regulating resistance to necrotrophs is rather uncommon. Indeed, plant defence against necrotrophs is commonly associated with jasmonic acid signalling [START_REF] Glazebrook | Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens[END_REF][START_REF] Ferrari | Arabidopsis local resistance to Botrytis cinerea involves salicylic acid and camalexin and requires EDS4 and PAD2, but not SID2, EDS5 or PAD4[END_REF]. However, [START_REF] Ferrari | Arabidopsis local resistance to Botrytis cinerea involves salicylic acid and camalexin and requires EDS4 and PAD2, but not SID2, EDS5 or PAD4[END_REF] showed that resistance to B. cinerea could be dependent on high SA levels, in accordance with our observations. A similar finding was reported for defence response to the necrotroph Sclerotinia sclerotiorum [START_REF] Novakova | Plant hormones in defense response of Brassica napus to Sclerotinia sclerotiorum -reassessing the role of salicylic acid in the interaction with a necrotroph[END_REF]. Moreover, we tested A. thaliana ETI by using a bacterial strain highly expressing the AvrRpt2 effector (Pst DC3000 AvrRpt2

). An ETI response can induce the expression of genes commonly associated with SA, such as PR-1, in a SA-independent manner in A. thaliana [START_REF] Tsuda | Dual regulation of gene expression mediated by extended MAPK activation and salicylic acid contributes to robust innate immunity in Arabidopsis thaliana[END_REF]). Yet, we found that the higher resistance of pi4k12 plants against Pst DC3000 AvrRpt2 was SAdependent. In conclusion, the higher resistance of pi4k12 mutant against all the host pathogens assayed was strongly SA-dependent.

Non-host resistance, efficient against non-adapted pathogens, does not rely fully on the SA pathway. Here we show that pi4kβ1β2 exhibited a SA-independent defective resistance towards penetration of the non-host pathogen Bgh.

Callose is a linear polysaccharide (1,3-β-glucan) occurring in plant cells where it is important for many plant physiological processes such as cytokinesis [START_REF] Chen | Callose synthesis in higher plants[END_REF]. Callose accumulation is triggered in response to pathogens and is used as a common test of PTI upon treatment with typical PAMPs such as flg22, the epitope of flagellin [START_REF] Luna | Callose deposition: a multifaceted plant defense response[END_REF]. In mock inoculated pi4k12, callose deposition was greater than in WT leaves, thus confirming findings of Antignani et al. (2015). Interestingly, this was also true for the sid2pi4kβ1β2 triple mutant when compared to sid2 plants, thus indicating a SA-independent phenomenon.

Following inoculation with flg22, an increase in callose deposition was observed in all genotypes tested, but this was still higher in pi4k12 compared to WT leaves, and callose deposition was higher in sid2pi4kβ1β2 with respect to sid2 plants. Therefore, it seems that the pi4k12 double mutation per se enabled higher callose deposition under biotic stress conditions.

The biosynthesis of callose occurs outside of the cell [START_REF] Ellinger | Callose biosynthesis in Arabidopsis with a focus on pathogen response: what we have learned within the last decade[END_REF].

Accumulation can be regulated at different levels: transcriptional, translational, or during enzyme transport to the plasma membrane and out of the cell via vesicular trafficking.

Phosphorylation and direct translocation of callose synthase is crucial in the regulation of biosynthesis, whereas transcriptional control might have only a minor role [START_REF] Ellinger | Callose biosynthesis in Arabidopsis with a focus on pathogen response: what we have learned within the last decade[END_REF]. Our data on the transcription levels of CALS1 and CALS12 indicate that in pi4k12 a transcriptional effect is not involved in the observed over accumulation of callose. So how is it possible to explain the action on callose of the pi4k12 double mutation per se? A number of reports indicate that PI4Ks can impact trafficking. In A. thaliana, PI4K1 was shown to be recruited by the GTP bound Rab4b GTPAse. Both RabA4b and PI4Kβ1 localize to budding secretory vesicles in the trans-golgi network (TGN) and to secretory vesicles en route to the cell surface. A pi4k12 double mutant produces secretory vesicles of highly variable sizes (Antignani et al. 2015;[START_REF] Preuss | A role for the RabA4b effector protein PI-4Kbeta1 in polarized expansion of root hair cells in Arabidopsis thaliana[END_REF][START_REF] Kang | Electron tomography of RabA4b-and PI-4Kbeta1-labeled trans Golgi network compartments in Arabidopsis[END_REF]. The product of PI4Ks activity, PI4P massively accumulates at the plasma membrane and at early endosomes / TGN and Golgi (Platre and Jaillais 2016; Noack and Jaillais 2017). Therefore PI4Kβ1/β2 are important in vesicle trafficking. Interestingly, inhibiting PI4K with phenylarsine oxide (PAO)

suppressed the salt-induced endocytosis of plasma membrane intrinsic protein 2;1 (Ueda et al., 2016). Similarly, inhibiting PI4K led to the internalization of CELLULOSE SYNTHASE3 from the plasma membrane [START_REF] Fujimoto | Phosphatidylinositol 3kinase and 4-kinase have distinct roles in intracellular trafficking of cellulose synthase complexes in Arabidopsis thaliana[END_REF]. Can the impact of PI4K betas on trafficking explain the increased callose accumulation? Callose biosynthesis and accumulation have been shown to be affected by vesicle trafficking [START_REF] Ellinger | Callose biosynthesis in Arabidopsis with a focus on pathogen response: what we have learned within the last decade[END_REF]. PI4Ks has been shown to play an important role in cytokinesis [START_REF] Lin | A dual role for cell plate-associated PI4Kβ in endocytosis and phragmoplast dynamics during plant somatic cytokinesis[END_REF], especially in the correct organization of the vesicles at the cell division plane and further formation of a cell plate. During phragmoplast formation, PI4Kβ1 likely interacts with MPK4, a member of the MAP65 protein family that regulates microtubule organization [START_REF] Lin | A dual role for cell plate-associated PI4Kβ in endocytosis and phragmoplast dynamics during plant somatic cytokinesis[END_REF]. Callose is also essential for cytokinesis [START_REF] Thiele | The timely deposition of callose is essential for cytokinesis in Arabidopsis[END_REF]. We can therefore only speculate whether the effects of PI4Ks on callose, cytokinesis and trafficking are interconnected. Interestingly, the role of SA in these processes has not been tested. Since

PI4Kβ1/β2 can impact the secretory pathway, they could also impact the translocation of callose synthases. Furthermore, PMR4 (CALS12) binds to small RabA4c GTPase at TGN and PI4Kβ1 binds to RabA4b GTPase, the most similar small GTPase to RabA4c, at TGN [START_REF] Böhlenius | The multivesicular body-localized GTPase ARFA1b/1c is important for callose deposition and ROR2 syntaxin-dependent preinvasive basal defense in barley[END_REF]. It should also be noted that the impact of PI4K betas on trafficking could also explain our non-host resistance data. The syp121 mutant altered in a SNARE protein involved in trafficking has been reported to accumulate SA and also display defective non-host resistance [START_REF] Collins | SNARE-protein-mediated disease resistance at the plant cell wall[END_REF].

In conclusion (Fig. 6), the pi4k12 double mutant constitutively accumulated a high SA level via ICS1/SID2 and this had a big impact on other hormone levels and it was associated with an increased resistance to several plant pathogens (P. syringae, H. arabidopsidis, B. cinerea). The pi4k12 double mutation also affected pathogen-related processes in a high SA-independent manner as seen by differences in callose accumulation in response to flg22, to Bgh infection, to wounding, and the higher penetration success of Bgh. The identification of such processes directly affected by the mutation on PI4Ks will now allow us to better investigate the role of these enzymes, in relation to signalling or trafficking events.

This work was supported by the Czech Science Foundation [grant number. 17-05151S].

Collaboration of Ukrainian and French teams was partially supported by a "Projet international de coopération scientifique" from the "Centre National de la Recherche

Scientifique" (2015)(2016)(2017)(2018) For each treatment, statistical differences between the genotypes were assessed using a oneway ANOVA, with a Tukey honestly significant difference (HSD) multiple mean comparison post hoc test. Different letters indicate a significant difference, Tukey HSD, P < 0.01, n=4. For each hormone, statistical differences between the genotypes were assessed using a one-way ANOVA, with a Tukey honestly significant difference (HSD) multiple mean comparison post hoc test. Different letters indicate a significant difference, Tukey HSD, P < 0.05. n=4.

FIGURE CAPTIONS Figure 1 .

 CAPTIONS1 FIGURE CAPTIONS

Figure 2 .

 2 Figure 2. Resistance to biotic stresses of pi4kβ1β2 and sid2pi4kβ1β2 mutants. A. Resistance

Figure 3 .

 3 Figure 3. Pattern of callose accumulation in pi4kβ1β2 leaves. A. Aniline blue staining, and

Figure 4 .

 4 Figure 4. Callose deposition in response to flagellin. A. Representative images of callose

Figure 5 .

 5 Figure 5. Resistance of pi4kβ1β2 to penetration by the non-host pathogen Blumeria graminis

Figure 6 .

 6 Figure 6. Schematic representation of the effects of the pi4kβ1β2 double mutation on

Fig. S3 .

 S3 Fig. S3. Resistance to P. syringae pv. tomato DC3000 AvrRpt2. 4-week-old A. thaliana

Fig. S4 .

 S4 Fig. S4. Callose deposition in response to wounding. A. Representative images of callose

Fig. S5 .

 S5 Fig. S5. Relative transcription of some CalS genes in untreated rosette leaves or 24h after

ACKNOWLEDGEMENTS

We thank Lucie Lamparová and Romana Pospíchalová for their technical support and Michael Hodges for language editing.

LITERATURE CITED

Antignani V, Klocko AL, Bak G, Chandrasekaran SD, Dunivin T, Nielsen E. 2015.

Funding information


P Mozer

Moreau-Gaudry

J 1 Troccaz

Framework for 3D TransRectal Ultrasound (TRUS) Image-Based Tracking -Example of Use -Evaluation of 2D TRUS Prostate Biopsies Mapping

Introduction and Objective:

Prostate biopsies are mainly performed under 2D TransRectal UltraSound (TRUS) control by sampling the prostate according to a predefined pattern. In case of first biopsies, this pattern follows a random systematic plan. Sometimes, repeat biopsies can be needed to target regions unsampled by previous biopsies or resample critical regions (for example in case of cancer expectant management or previous prostatic intraepithelial neoplasia findings). From a clinical point of view, it could be useful to control the 3D spatial distribution of theses biopsies inside the prostate. Modern 3D-TRUS probes allow acquiring high-quality volumes of the prostate in few seconds. We developed a framework to track the prostate in 3D TRUS images. It means that if one acquires a reference volume at the beginning of the session and another during each biopsy, it is possible to determine the relationship between the prostate in the reference and the others volumes by aligning images. We used this tool to evaluate the ability of a single operator (a young urologist assistant professor) to perform a pattern of 12 biopsies under 2D TRUS guidance.

Methods:

After approval by ethical committee, the operator performed 12-core TRUS biopsies on 32 patients according to a classical pattern (hence 12 identical square coronal sectors -Figure 1) using a 2D-3D TRUS probe (RIC5-9 on a Voluson-i, both from General Electric Medical). Prostate volume was on average 45ml (min: 20ml; max: 100ml).

• During the biopsy session:

o Before the first biopsy, a 3D reference volume is acquired. o For biopsy targeting, the probe is switched to 2D mode. o After each biopsy gun shot, the needle is left inside the prostate, on average during 5 seconds, and a 3D TRUS volume is acquired. During this acquisition the operator took care to apply a minimal force on the probe to minimize deformation on the prostate.

• After the biopsy session:

The needle is manually selected in each 3D volume and is fused automatically into the reference volume with our rigid image-based registration algorithm [START_REF]Towards 3D ultrasound image based soft tissue tracking: a transrectal ultrasound prostate image alignment system[END_REF]. Registration success was determined visually. To validate registration, we segmented clearly visible point-like fiducials (e.g. calcifications) in the volumes. The distances between corresponding fiducials after application of the registration transformation were used as gold standard for accuracy evaluation.

After registration, biopsies can be represented into the reference volume (Figure 2 and Figure 3) allowing an analysis of their spatial distribution. To perform a quantitative analysis, the 3D volume is reformatted in the coronal plane and the preoperative targets are created (Figure 4). For each target we computed the percentage of planned biopsies hitting the target and the average biopsy length inside the target. Nevertheless, as the apex lateral target area was often small on the preoperative planning, we fused it with the apex parasagital target for the analysis. Results:

• Registration: The registration method was validated on 237 3D images acquired during biopsy of 14 different patients with an average error < 1.44 mm and a max error 3.84mm. Registration between each volume was computed in 6 seconds. The success rate of all the registration was 96.7% (371 good registrations on 384 volumes).

• Operator accuracy and learning curve: Table 1 and Figure 5 show the ratio of biopsies reaching their target and their inner length. On average, the operator reached the target in 67% of all cases. The ratio decreases as the planning approaches the boundaries of the prostate. If we split this data in two equal sets in a chronological order, the success rate is 60% on the first 16 patients and 72% on the last 16 patients. It seems that a learning curve can be highlighted (test for independence of all factors: Chi2 = 5.89, p-value = 0.01523). Registration:

Target

# of biopsies % (#) of biopsies inside the target

In this study, the registration process does not take into account the deformation induced by the TRUS probe. Nevertheless, this system can be used for quality control, with an accuracy of few millimeters, if the operator is well aware of the deformations. Moreover, this framework can be used to register data acquired at different time to check if a previous suspicious target has been hit (Figure 6).

Operator accuracy and learning curve:

The low percentage of reached targets and the low inner lengths of sampled tissue inside the targets tend to prove the inadequacy between a theoretical planning pattern and the actual constrained transrectal access. At lateral base and apex sides the lowest ratios may be explained by the difficulty to target and by the low prostate presence in those sectors.

For the first time, at our knowledge, this study allows to quantify a learning curve based on spatial distribution of 2D TRUS prostate biopsies.

Future:

Work is in progress to estimate deformations during the procedure. Some preliminary results show that we can estimate theses deformations but the accuracy of such approach is still unknown (see Figure 6). With 6 seconds per registration the algorithm should be fast enough to achieve framerates of 5Hz when making advantage of the massive parallelization capacities provided by modern high-end computers. Then, this method seems robust enough to allow continuous tracking. It could be possible to select a target in the reference volume and guide the clinician to reach it. Work is in progress to register MRI and 3D TRUS reference volumes. This tool should allow to select the target in MRI images and tracking it by this framework. Note that this approach does not rely on any external optical or magnetic tracking system, which means that no cumbersome additional hardware is required.

A good way to enhance the accuracy of this framework could be to hold the TRUS probe with a robot as show on Figure 7.

Conclusion:

This study shows that it is difficult to accurately reach targets in the prostate using a 2D TRUS probe. On average, the operator reached the target in 67% of all cases and it seems that a learning curve can be highlighted.

Figure 1 :

 1 Figure 1: (a) illustrates the 12-core systematic standard protocol, which is defined on a schematic coronal plane of the prostate. (b) Coronal sector definition for the accuracy study (B=Base, M=Mid-Gland, A=Apex, L=Lateral, P=Parasagittal).

Figure 2 :

 2 Figure 2: 3D biopsy distribution in a reference volume.

Figure 3 :

 3 Figure 3: Examples of biopsies distribution in the coronal plan for 8 patients.

Figure 4 :

 4 Figure 4: Biopsies and reference volume

Figure

  Figure 5: Targeting accuracy evaluation result

Figure 6 :Figure

 6 Figure 6: 2 sets of biopsies in one reference volume (with deformation)

/Average 371 67% (248) 14Table 1 :

 1 Targeting accuracy evaluation result

					Biopsy
					length
					inside the
					target
					(mm)
	Base	Right	33	70% (23)	14
	Lateral (BL)	Left	31	55% (17)	12
	Base	Right	31	65% (20)	15
	Sagital (BS)	Left	32	66% (21)	14
	Mid	Right	32	81% (26)	15
	Lateral (ML)	Left	30	77% (23)	15
	Mid	Right	32	100% (32)	15
	Sagital (MS)	Left	31	90% (31)	16
	Apex	Right	60	52% (31)	12
	(AL+AS) Left	59	46% (27)	13
	Sum

Currently, this research framework can be used as a biopsy quality control tool. Moreover, different biopsies sessions can be register to verify that a previous suspicious area has been sampled. Finally, use of a robot in the loop could allow to increase the accuracy and reproducibility.