domain: sdv chim

text:
Moulay Sahaka

Eduardo Mateos-Diaz

Sawsan Amara

Jutarat Wattanakul

David Gray

Dominique Lafont

Brigitte Gontero

Hélène Launay

Frédéric Carrière
email: carriere@imm.cnrs.fr

In situ monitoring of galactolipid digestion by infrared spectroscopy in both model micelles and spinach chloroplasts

Keywords: Enzyme, galactolipase, lipid digestion, pancreatic lipase related protein 2, chloroplast, thin layer chromatography

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

Abbreviations: ALA, -linolenic acid; ATR, Attenuated Total Reflectance; C8-MGDG, monogalactosyl-dioctanoyl glycerol; CRF, chloroplast-rich fraction; DPPC, dipalmitoyl phosphatidylcholine; DW, dry weight; FA, fatty acid; FFA, free fatty acid; FTIR, Fouriertransform infrared spectroscopy; GPLRP2, guinea pig pancreatic lipase related protein 2; LC-MGDG, long chain monogalactosyl-diacyl glycerol; lyso-PC, lysophosphatidylcholine;

MGDG, monogalactosyl-diacyl glycerol; MGG, monogalactosyl glycerol; MGMG, monogalactosyl-monoacyl glycerol; NaGDC, sodium glycodeoxycholate; NaTC, sodium taurocholate; NaTDC,; sodium taurodeoxycholate; OA, octanoic acid; PA, palmitic acid; pD, pH in D2O; PLRP2, pancreatic lipase related protein 2; PUFA, polyunsaturated fatty acid; HDO, semiheavy water; TAG, triacylglycerol; TLC, thin-layer chromatography; TES, Ntris[hydroxymethyl]methyl-2-aminoethane-sulfonic acid;

Introduction

Fourier-transform infrared spectroscopy (FTIR) is an effective tool to investigate enzymatic lipolysis and its advantages have already been demonstrated by several studies. The first researchers to attempt to monitor lipolysis using transmission FTIR were [START_REF] Walde | A continuous assay for lipases in reverse micelles based on Fourier transform infrared spectroscopy[END_REF]. They succeeded in quantifying the substrate consumption and the fatty acids released during the hydrolysis of triacylglycerol (TAG) in reverse micelles, by exploiting the vibration bands of carbonyl (C=O) and C-O bond [START_REF] Walde | A continuous assay for lipases in reverse micelles based on Fourier transform infrared spectroscopy[END_REF]. Their method was then successfully applied by O'connor and Cleverly, in 1994, to analyse the activity of bile saltstimulated lipase on TAG [START_REF] O'connor | Fourier-transform infrared assay of bile salt-stimulated lipase activity in reversed micelles[END_REF]. Later, FTIR in the mode of Attenuated Total Reflectance (ATR-FTIR) was used to monitor enzymatic lipolysis of TAG films [START_REF] Snabe | Application of infrared spectroscopy (attenuated total reflection) for monitoring enzymatic activity on substrate films[END_REF] and oil-in-water emulsions [START_REF] Kaufhold | Novel μ-membrane module for online determination of the free fatty acid content in the dispersed phase of oil-in-water emulsions[END_REF][START_REF] Khaskheli | Monitoring the Rhizopus oryzae lipase catalyzed hydrolysis of castor oil by ATR-FTIR spectroscopy[END_REF][START_REF] Stöbener | In situ monitoring of the biocatalysed partial hydrolysis of cocoa butter and palm oil fraction[END_REF]. FTIR was also used to monitor other lipase-catalyzed reactions such as transesterification for the production of methyl ester [START_REF] Zagonel | Multivariate monitoring of soybean oil ethanolysis by FTIR[END_REF][START_REF] Müller | Online Monitoring of Biotransformations in High Viscous Multiphase Systems by Means of FT-IR and Chemometrics[END_REF][START_REF] Natalello | Enzymatic transesterification monitored by an easy-to-use Fourier transform infrared spectroscopy method[END_REF] or TAG [START_REF] Müller | Simultaneous Determination of Mono-, Di-, and Triglycerides in Multiphase Systems by Online Fourier Transform Infrared Spectroscopy[END_REF].

Previously, we studied the hydrolysis of dipalmitoylphosphatidylcholine (DPPC) by guinea pig pancreatic lipase-related protein 2 (GPLRP2) using transmission FTIR (Mateos-Diaz et al., 2018b). In this last study, we developed a method allowing the simultaneous quantification of residual DPPC and lipolysis products, lysophosphatidylcholine (lyso-PC) and free palmitic acid (PA), by fully exploiting the carbonyl band spectra using calibration curves made with reference standards.

In the present study, our objective was to further develop this approach to investigate the hydrolysis of galactolipids catalyzed by GPLRP2. Galactolipids are the main polar lipids from plant photosynthetic membranes but their digestion was overlooked until enzymes with galactolipase activity like PLRP2 were identified [START_REF] Sahaka | The digestion of galactolipids and its ubiquitous function in Nature for the uptake of the essential α-linolenic acid[END_REF]. In a first step, the substrate used was a synthetic monogalactosyl diacylglycerol with 8-carbon acyl chains . This is a model substrate that we have synthesized and used on several occasions to study the galactolipase activities of various enzymes [START_REF] Amara | Etude de l'activité galactolipase des lipases pancréatiques apparentées de type 2 (PLRP2)[END_REF][START_REF] Amara | Lipolysis of natural long chain and synthetic medium chain galactolipids by pancreatic lipase-related protein 2[END_REF][START_REF] Amara | Continuous measurement of galactolipid hydrolysis by pancreatic lipolytic enzymes using the pH-stat technique and a medium chain monogalactosyl diglyceride as substrate[END_REF][START_REF] Belhaj | Galactolipase activity of Talaromyces thermophilus lipase on galactolipid micelles, monomolecular films and UV-absorbing surfacecoated substrate[END_REF][START_REF] Sahaka | Quantitative monitoring of galactolipid hydrolysis by pancreatic lipase-related protein 2 using thin layer chromatography and thymol-sulfuric acid derivatization[END_REF]. The method was then tested with more complex substrates, including mixed micelles of a natural MGDG with long acyl chains (LC-MGDG), rich in polyunsaturated fatty acids (PUFA), and a chloroplast-rich fraction (CRF) isolated from spinach leaves, in which galactolipids are present in membranes.

Materials and methods

Reagents

MonoGalactosyl Dioctanoyl-Glycerol (C8-MGDG; Mw=506 g/mole) was synthetised as previously described [START_REF] Amara | Continuous measurement of galactolipid hydrolysis by pancreatic lipolytic enzymes using the pH-stat technique and a medium chain monogalactosyl diglyceride as substrate[END_REF][START_REF] Lafont | Syntheses of an α-d-Gal-(1→6)-βd-Gal diglyceride, as lipase substrate[END_REF][START_REF] Sias | Human Pancreatic Lipase-Related Protein 2 Is a Galactolipase †[END_REF]

Production and purification of GPLRP2

Recombinant GPLRP2 was produced in Aspergillus orizae and purified as previously described [START_REF] Hjorth | A structural domain (the lid) found in pancreatic lipases is absent in the guinea pig (phospho)lipase[END_REF]. Aliquots (50 μL) of GPLRP2 at 2.3 mg.mL -1 (or 50 µM) were freezedried, resuspended in the deuterated buffer solution used for the reactions, and finally incubated for 48 h at 4°C to reach H/D exchange equilibrium. The aliquots were then kept at -20°C until the reactions were performed. Before being added to the substrates, the aliquots were thawed and diluted to 500 or 1000 nM in D2O.

Isolation of CRF from spinach leaves

Spinach leaves, bought from local supermarket, were blanched in hot water at 85 °C for 3 min and then immediately cooled by immersion in an ice-water bath. Blanched spinach leaves were homogenised in a blender (Waring™) for 30 s with 0.3 M sucrose solution 1:6 (w/v). The homogenate was then filtered through a double-layered cheesecloth. The resulting juice was centrifuged at 10,000 rpm for 10 min at 4 °C. The CRF-containing pellet was freeze-dried for 3-5 days.

Preparation of micellar solutions of substrates (C8-MGDG, LC-MGDG) and fatty

acids (OA, ALA) and CRF suspensions.

Substrates were prepared in the form of mixed micelles with bile salts. Similarly, mixed micelles of free fatty acids (FFAs) were prepared to reproduce the environment of FFAs in the course of the lipolysis reaction in the presence of bile salts. To prepare solutions of C8-MGDG-NaTDC and OA-NaTDC micelles, C8-MGDG and OA were suspended in buffer solutions containing 65.5 mM NaTDC, 500 mM TES buffer, 100 mM NaCl and 5 mM CaCl2, in D2O, at pD 8. These buffer conditions were selected after screening various buffers and concentrations and their impact on the IR spectra, especially regarding fatty acid ionization and C8-MGDG phase transition. These optimization steps are described in the Supplementary Materials section with Figure S1 to S6. The suspensions were then heated to 65 °C for 5 min, vortexed and sonicated for 8 min to obtain micelles. C8-MGDG and OA concentrations were both 50 mM and NaTDC to C8-MGDG molar ratio was 1.3. For calibration experiments, a range of dilutions containing 0, 10, 20, 30, 40 and 50 mM of C8-MGDG and OA were prepared using the deuterated buffer solution as diluent.

Following a similar protocol, solutions of LC-MGDG-NaTDC and ALA-NaTDC micelles were prepared in buffer solutions containing 30 mM NaTDC, 200 mM TES buffer, 100 mM NaCl and 5 mM CaCl2, in D2O, at pD 8. LC-MGDG and ALA concentrations were 22.5 and 15 mM, respectively, and NaTDC/MGDG molar ratio was 1.3. For calibration experiments, a range of dilutions containing 0, 3, 6, 9, 12 and 15 mM of ALA was prepared using the deuterated buffer solution as diluent.

Freeze-dried CRF was suspended in the same deuterated buffer solution at a concentration of 100 g/L, in the absence and presence (30 mM) of NaTDC.

LC-MGDG-NaTDC micelles and CRF suspensions were also prepared in H2O for lipid extraction and analysis by TLC.

Analysis of MGDGs and CRF lipolysis by T-FTIR

Lipolysis experiments were carried out by adding 5µL of rGPLRP2 solution (500 or 1000 nM)

to 45 µL of substrate dispersions (C8-MGDG micelles, LC MGDG micelles or CRF suspensions) to obtain 50 or 100 nM final enzyme concentration. The reaction mixtures were placed and squeezed between two CaF2 transmission crystals with a 50 μm polytetrafluoroethylene (Teflon) spacer. IR-spectra were then recorded each 5 min during 2 h at 37 °C using a JASCO™ FT/IR-6100 Fourier-transform infrared spectrometer. The sample chamber was continuously purged with a flow of dry air to avoid the presence of water vapour and the formation of semiheavy water (HDO) molecules in the sample. All the spectra are an average of 128 scans, with 4 cm -1 of resolution, apodized with a cosine function.

Estimation of residual substrate and lipolysis product concentrations during C8-MGDG hydrolysis using FTIR absorbance

Upon lipolysis, a C8-MGDG molecule can be converted in monogalactosyl monooctanoylglycerol (C8-MGMG) and a free OA. To quantify these compounds from IR absorbance, we used equations (1), ( 2) and (3).

With Ai: absorbance at wavenumber i cm -1 , εx i : molar extinction coefficient (M -1 cm -1 ) of compound x at wavenumber i cm -1 , [x] : concentration (M) of compound x.

Estimation of free fatty acid (FFA) concentration during LC-MGDG and CRF hydrolysis using FTIR absorbance

To quantify FFA during lipolysis of LC-MGDG-NaTDC micelles and CRF suspensions (with and without NaTDC), we used equations (4) and (5).

[𝐹𝐹𝐴] = 𝐴 1555 𝜀 𝐴𝐿𝐴 1555 ⁄ (4)

[𝐹𝐹𝐴] = 𝐴 1407 𝜀 𝐴𝐿𝐴 1407 ⁄ (5)

Calibration of FTIR absorbance versus concentration of C8-MGDG, C8-MGMG, OA

and ALA.

For calibration, C8-MGDG-NaTDC, OA-NaTDC, and ALA-NaTDC micellar solutions were diluted with the buffer solutions in which they were prepared to obtain a concentrations range of 0, 10, 20, 30, 40 and 50 mM for C8-MGDG and OA and of 0, 3, 6, 9, 12 and 15 mM for ALA. FTIR spectra of these calibration solutions were then recorded under the same conditions as the lipolysis experiments.

For C8-MGMG calibration, no commercial or synthetic standard was available. To produce it, the mixed micelles of C8-MGDG-NaTDC were hydrolyzed by GPLRP2 (100 nM), at 37°C.

The reaction was stopped after 1 h of incubation to make sure that C8-MGDG disappeared completely and that there was enough accumulation of C8-MGMG. To stop the lipolysis, the reaction mixture was heated at 90 °C for 10 min to denature GPLRP2. The concentration of C8-MGMG in the hydrolysate (26 mM) was estimated by TLC-densitometry using the specific staining of galactose with thymol/sulfuric acid, as previously reported [START_REF] Sahaka | Quantitative monitoring of galactolipid hydrolysis by pancreatic lipase-related protein 2 using thin layer chromatography and thymol-sulfuric acid derivatization[END_REF]. A range of dilutions (1 to 5-fold) of the hydrolysate was prepared and their spectra were recorded at 37 °C. In these spectra, the carbonyl vibration band combines absorbance of C8-MGMG with that of free OA (Figure S7A). Some monogalactosyl glycerol (MGG) was formed that could not interfere with the vibration bands of carbonyl (C=O) bond. The carboxylate vibration band, on the other hand, is composed only of the absorbance of free OA (Figure S7A). This band allowed us to estimate free OA concentrations in the hydrolysate dilution range (14, 28, 42, 54, and 72 mM OA) with high accuracy, using equation (2). OA-NaTDC micellar solutions were then prepared at the same concentrations. Their spectra were recorded (Figure S7B) and subtracted from the spectra of the hydrolysate at the same dilution (Figure S7C). The latter were thus cleared of OA absorbances and used for calibration of C8-MGMG (Figure S7C).

All calibration curves are presented in Figure S7.

FTIR spectra treatment

All spectra presented in this study were submitted to baseline and water vapor corrections.

All "raw" spectra presented were subjected to subtraction of the buffer solutions spectra to present only the absorbance of lipids or CRFs.

Difference spectra were made by subtracting the initial spectrum from the reaction spectra at various times. In the case of C8-MGDG and LC-MGDG hydrolysis, this initial spectrum corresponds to the substrate spectrum without enzyme. In the case of CRF lipolysis, it corresponds to the spectrum recorded 5 min after the addition of enzyme. The spectrum of CRF without enzyme could not be used as initial spectrum because CRF spectra are not strictly reproducible in intensities because we were dealing with dispersions that form deposits in absence of continuous agitation.

The absorbance of HDO molecules was removed from the reaction spectra (raw and difference) by subtracting a model HDO spectrum from them. This model HDO spectrum was obtained by subtracting a pure D2O spectrum from a hydrated one. The hydration was induced either by adding a very small amount of water or by leaving the D2O in contact with the room air. Before being subtracted, the model HDO spectrum was multiplied by a coefficient α to match the hydration level of the spectrum to correct. This coefficient α was estimated using the equation ( 6).

𝐴 𝑠𝑝𝑒𝑐𝑡𝑟𝑢𝑚 𝑡𝑜 𝑐𝑜𝑟𝑟𝑒𝑐𝑡 1452 = 𝛼. 𝐴 𝐻𝐷𝑂 1452 (6)
With 𝐴 𝑠𝑝𝑒𝑐𝑡𝑟𝑢𝑚 𝑡𝑜 𝑐𝑜𝑟𝑟𝑒𝑐𝑡

1452

: Absorbance at 1452 cm -1 of the spectrum to correct, and 𝐴 𝐻𝐷𝑂 1452 : Absorbance of the HDO model spectrum at 1452 cm -1 .

All these treatments were performed using the software Spectra Manager® version V.2.07.

Analysis of C8-MGDG lipolysis by thin-later chromatography (TLC) and densitometry

A GPLRP2 aliquot (1000 nM) was added to C8-MGDG-NaTDC micelles to obtain a 100 nM enzyme concentration, in D2O, at 37 °C. The reaction volume was 200 μL. Aliquots of 25μL

were collected at different reaction times (0, 10, 20, 50, 90 and 120 min) and immediately mixed with 1 mL of a stop solution containing 200 mM HCl and 150 mM NaCl. Substrates and reaction products in aliquots were extracted and analysed using TLC-densitometry and thymol-sulfuric acid reagent, as previously described [START_REF] Sahaka | Quantitative monitoring of galactolipid hydrolysis by pancreatic lipase-related protein 2 using thin layer chromatography and thymol-sulfuric acid derivatization[END_REF], and a mixture of chloroform/methanol/water (25/15/1.25, v/v/v) for elution.

Analysis of LC-MGDG and CRF lipolysis by TLC

GPLRP2 aliquots (500 nM) were added to LC-MGDG-NaTDC micelles and CRF suspensions (with and without NaTDC) to obtain 50 nM enzyme concentration, in H2O, at 37 °C. Aliquots of 25μL were collected after 1h incubation and immediately mixed with 1 mL of stop solution.

Substrates and reaction products in aliquots were extracted and analysed using TLC, as previously described [START_REF] Sahaka | Quantitative monitoring of galactolipid hydrolysis by pancreatic lipase-related protein 2 using thin layer chromatography and thymol-sulfuric acid derivatization[END_REF], and a mixture of chloroform/methanol/water (47.5/10/1.25, v/v/v) for elution of long chain galactolipids [START_REF] Wattanakul | In vitro digestion of galactolipids from chloroplast-rich fraction (CRF) of postharvest, pea vine field residue (haulm) and spinach leaves[END_REF].

Results

FTIR spectra of C8-MGDG, LC MGDG, CRF, octanoic acid, palmitic acid and αlinolenic acid.

Figure 1A shows FTIR spectra of pure C8-MGDG, LC-chain MGDG, OA, ALA and PA, and CRFs, in the presence of NaTDC, at pD 8 and 37 °C.

In all spectra, the region between 3040 and 2813 cm -1 (Figure 1 B) is dominated by two large peaks attributed to symmetric (νsCH2) and asymmetric (νasCH2) methylene stretching vibrations, both peaks have a shoulder corresponding to symmetric (νsCH3) and asymmetric (νasCH3) vibrations of terminal CH3 groups, respectively [START_REF] Snyder | Carbon-hydrogen stretching modes and the structure of n-alkyl chains. 1. Long, disordered chains[END_REF]. In the same region, ALA, LC-MGDG and CRF spectra have in addition a peak corresponding to alkenyl C-H stretching vibration ʋCH (Figure 1 B) [START_REF] Vlachos | Applications of Fourier transform-infrared spectroscopy to edible oils[END_REF], indicating the presence of C=C double bonds.

Between 1800 and 1640 cm -1 all spectra present a band (Figure 1C) attributed to carbonyl stretching vibration νC=O [START_REF] Blume | Fourier transform infrared spectroscopy of 13C:O labeled phospholipids hydrogen bonding to carbonyl groups[END_REF][START_REF] Levin | Vibrational assignment of the sn-1 and sn-2 chain carbonyl stretching modes of membrane phospholipids[END_REF][START_REF] Lewis | Studies of mixed-chain diacyl phosphatidylcholines with highly asymmetric acyl chains: a Fourier transform infrared spectroscopic study of interfacial hydration and hydrocarbon chain packing in the mixed interdigitated gel phase[END_REF]Mateos-Diaz et al., 2018a;[START_REF] Yan | Conformation of the Acyl Chains in Diacylphospholipid Gels by IR Spectroscopy[END_REF]. In the case of the CRF spectrum, this carbonyl band is globally attributed to chloroplast membrane lipids.

Between 1600 cm -1 and 1350 cm -1 , ALA and OA spectra show two peaks (Figure 1D), attributed to the symmetric (νsCOO -) and asymmetric (νasCOO -) carboxylate stretching vibrations [START_REF] Mizuguchi | FT-IR study of the Ca 2+ -binding to bovine α-lactalbumin: Relationships between the type of coordination and characteristics of the bands due to the Asp COOgroups in the Ca 2+ -binding site[END_REF]. In the case of palmitic acid (PA), νsCOO -and νasCOO - occur as doublets (Figure 1D). The splitting of carboxylate vibrations into doublets has previously been reported in the spectra of carboxylate-calcium complexes and has been attributed to the coexistence of different structural organizations (Mielczarski et al., 1993a(Mielczarski et al., , 1993b)). Between its two doublets, PA present a band (Figure 1D) attributed to CH2 scissoring vibration (νsciCH2) [START_REF] Wieser | Infrared Band Progressions in the CH2 Scissoring Region and the Ring Puckering Vibration of Trimethylene Oxide and some Deuterated Analogs[END_REF].

Panel E of Figure 1 shows vibrations characteristic of proteins in the CRF spectrum. Between 1700 and 1600 cm -1 , there is a large band corresponding to deuterated amides I (amide I'). The shoulder between 1590 and 1540 cm -1 was attributed to the vibrations of non-deuterated amides II. The large band between 1500 and 1400 cm -1 corresponds to the vibrations of deuterated amides II (amide II') [START_REF] Goormaghtigh | Determination of Soluble and Membrane Protein Structure by Fourier Transform Infrared Spectroscopy[END_REF][START_REF] De Jongh | Amide-Proton Exchange of Water-Soluble Proteins of Different Structural Classes Studied at the Submolecular Level by Infrared Spectroscopy[END_REF][START_REF] Barth | What vibrations tell about proteins[END_REF].

Monitoring of C8-MGDG hydrolysis using FTIR spectroscopy

Hydrolysis of C8-MGDG-NaTDC micelles, by GPLRP2 (50 and 100 nM), in D2O was performed at pD 8 and 37° C in the transmission cell of the FTIR spectrophotometer. IR spectra were recorded every 5 minutes for 2 hours, in the presence and in absence (blank) of GPLRP2.

In the absence of enzyme (Figure 2 A), no change with time is visually noticeable in the raw spectra of C8-MGDG. However, by subtracting the initial spectrum (t = 0 min) from the other spectra/times, an increase in absorbance at 1451 cm -1 (Figure 2A') is revealed, that can be attributed to the scissoring vibration of HDO molecules (δHDO) [START_REF] Ceccaldi | Le spectre d'absorption des mélanges H2O-D2O à l'état liquide entre 0,8 μ et 30 μ[END_REF]. The appearance of HDO results from the hydration of liquid D2O by the atmospheric H2O vapor, according to equation ( 7). This phenomenon occurs despite a continuous purge of the sample with dry air.

H2Ovap + D2Oliq ⇌ 2 HDOliq (7)

In the presence of enzyme, important changes in the raw and difference spectra of C8-MGDG are observed over time (Figure 2 B and B'). Changes related to HDO production were suppressed in these spectra in order to present only the evolutions induced by the enzyme. The hydrolysis was manifested in raw spectra by a progressive disappearance of the νC=O band of C8-MGDG (at around 1744 cm -1 ) giving rise to νC=O bands of C8-MGMG and protonated free OA (Figure 2 B). We also observe the appearance of two imposing peaks corresponding to the νasCOO -and νsCOO -vibrations of ionized free OA carboxylate (Figure 2 B). The difference spectra shows a decrease in absorbance at 1744 cm -1 and an increase in absorbance at 1682, 1553 and 1408 cm -1 attributed to the disappearance of νC=O band of C8-MGDG, and the appearance of νC=O, νasCOO -and νsCOO -bands of OA, respectively (Figure 2 B').

To monitor quantitatively the reaction products using νC=O vibrations, absorbances at 1744 cm -1 , 1722 cm -1 , and 1682 cm -1 were extracted from the raw spectra. These wavenumbers correspond to the absorption maxima of C8-MGDG (Figure 3A), C8-MGMG (Figure 3B) and free OA (Figure 3C) carbonyl bands, respectively. For each reaction time, C8-MGDG, C8-MGMG and OA concentrations were estimated from these absorbances using the matrix system

(1) with 3 equations and 3 unknowns adapted from the study on phospholipid hydrolysis by Mateos-Diaz et al. (Mateos-Diaz et al., 2018b).

To monitor quantitatively the reaction products using carboxylate vibrations, we extracted absorbances at 1553 cm -1 and 1408 cm -1 , corresponding to maxima of νasCOO -(Figure 3E) and νsCOO -(Figure 3F) from the difference spectra. Free OA concentrations were then estimated from these absorbances using equations ( 2) and (3).

The resolution of equations ( 1), ( 2) and (3) requires to determine the molar extinction coefficients of C8-MGDG, C8-MGMG and OA. For this purpose, carbonyl and carboxylate band spectra of C8-MGDG (Figure 3A), C8-MGMG (Figure 3B) and OA (Figure 3C, E andF)

were recorded at different concentrations, under the same conditions as the lipolysis reactions.

These spectra were used to draw calibration curves at the wavenumbers relevant for reaction monitoring (Figure S2 A, B, C and D). Table 1 shows the molar extinction coefficients estimated from these calibration curves. C8-MGMG hydrolysis indicates the production of MGG (Figure 9). However, as no band was assigned to MGG in the FTIR spectra, its quantification was not possible. MGG concentrations shown in Figure 4 A and C were deduced from those of C8-MGDG, C8-MGMG and OA, using equation ( 8).

[

𝑀𝐺𝐺] = [𝐶8 ̵ 𝑀𝐺𝐷𝐺] 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 -[𝐶8 ̵ 𝑀𝐺𝐷𝐺] -[𝑀𝐺𝑀𝐺] (8)
With [C8-MGDG] initial: concentration of C8-MGDG at 0 minute incubation time.

Monitoring of C8-MGDG hydrolysis using TLC and densitometry

To validate the results obtained by FTIR spectroscopy, we also analysed the lipolysis of C8-MGDG-NaTDC micelles, by 100 nM GPLRP2, using TLC-densitometry and thymol-sulfuric acid derivatization (Figure 4B). Thymol-sulphuric acid reagent allowed to quantify only galactosylated compounds (C8-MGDG, C8-MGMG and MGG). OA concentrations were calculated from C8-MGMG and MGG concentrations, using equation ( 9).

[𝑂𝐴] = [𝐶8 ̵ 𝑀𝐺𝑀𝐺] + 2.

[𝑀𝐺𝐺] (9)

Concentrations and kinetic curves obtained from TLC analysis (Figure 4B) are very similar with those obtained using FTIR spectroscopy and the same concentration of enzyme (Figure 4 A). The specific activities of GPLRP2 estimated from both methods using 100 nM GPLRP2 are very close (FTIR: 981 ± 97 U/mg, TLC-densitometry: 727 ± 67 U/mg; with 1U = 1 µmole of FFA released per min; see Table 2). However, the specific activity is reduced to 439 ± 125 U/mg using 50 nM GPLRP2, which indicates that the assay conditions are not fully optimized to measure a constant specific activity when varying the enzyme amount.

Monitoring of LC-MGDG hydrolysis using FTIR spectroscopy

Lipolysis of LC-MGDG-NaTDC micelles, by 50 nM GPLRP2, in D2O, at pD 8 and 37° C, was studied using IR spectroscopy. Spectra were recorded every 5 minutes for 2 hours, in presence and in absence of GPLRP2.

In the absence of enzyme, only the production of HDO molecules was observed in the difference spectra (spectra not shown), as previously observed during the incubation of C8-MGDG without enzyme (Figure 2 A').

In the presence of enzyme, the raw and difference spectra (Figure 2 C and C') show the same changes as those observed during the hydrolysis of C8-MGDG (Figure 2 B and B').

Carboxylate vibrations (νasCOO -and νsCOO -) of released fatty acids appear as two peaks as in the spectrum of ALA-NaTDC (Figure 1D), this is consistent with the fact that ALA is the main fatty acid in LC-MGDG (60 % w/w of total FA) [START_REF] Amara | Lipolysis of natural long chain and synthetic medium chain galactolipids by pancreatic lipase-related protein 2[END_REF].

FFA concentration in the reaction medium over time was estimated from a calibration established with ALA. Carbonyl and carboxylate band spectra of ALA (Figure 3G andH) were recorded at different concentrations, under the same conditions as the lipolysis reactions. These spectra were used to draw calibration curves at the wavenumbers relevant for reaction monitoring: 1555 cm -1 for νasCOO -and 1408 cm -1 for νsCOO -(Figure S2 E). FFA concentrations were estimated from absorbances at these wavenumbers using equations ( 4) and

(5) and the molar extinction coefficients estimated from the calibration curves (Table 1).

Figure 4 D shows FFA concentrations over time estimated by this method. The concentrations estimated from absorbances at 1555 cm -1 and 1407 cm -1 were found to be identical. The FFA concentration reached after 120 min (27 mM; Figure 4D) corresponds to a lipolysis level of 60%, taking into account a substrate concentration of 22.5 mM and the possibility to release two fatty acid molecules from LC-MGDG. For the sake of comparison, the FFA concentration reached after 120 min with C8-MGDG and the same enzyme concentration (50 nM) was 2-fold higher (61 mM; Figure 4C) than the concentration reached upon LC-MGDG lipolysis.

However, the initial concentration in the C8-MGDG substrate was also higher (50 mM) and this level of FFA corresponds to a lipolysis level of 61 % that is the same as with LC-MGDG.

Similarly, the specific activity estimated for GPLRP2 acting on LC MGDG (492 ± 30 U/mg) is in the same range as the specific activity measured with C8-MGDG (439 ± 125 U/mg; see Table 2), at the same enzyme concentration (50 nM).

Monitoring of CRF lipolysis using FTIR spectroscopy

We then studied whether GPLRP2 was able to get access to galactolipids and hydrolyze them when they are present in the photosynthetic membranes of the chloroplast. Lipolysis of CRF was performed using 50 nM GPLRP2 in D2O, at pD 8 and 37 °C, without and with bile salts (NaTDC) to reproduce digestive conditions in the gastrointestinal tract. FTIR spectra of CRF suspension were recorded every 5 min during two hours.

Changes in the CRF spectra over time were too weak to be visible on the raw spectra (see Figure S9). They were revealed and analysed by establishing the difference spectra (Figure 5).

In absence of enzyme and presence of NaTDC, we observed an important decrease in absorbance that extends from 1700 to 1350 cm -1 (Figure 5 A). This decrease is composed of two large bands, that we attributed to amide I' (between 1700 and 1600 cm -1 ) and amide II'

(between 1500 and 1350 cm -1 ) vibrations, and a third less important band that could correspond to amide II vibrations (between 1600 and 1500 cm -1 ). This phenomenon was not observed without NaTDC (Figure 5 A') and was therefore related to the presence of this surfactant.

Surfactants are known to bind to protein complexes in chloroplast membranes and solubilise them [START_REF] Bril | Fractionation of spinach chloroplasts with sodium deoxycholate[END_REF][START_REF] Yu | Triton X-100 as an effective surfactant for the isolation and purification of photosystem I from Arthrospira platensis[END_REF]. Therefore, the decrease in amide absorbances could correspond to protein solubilisation, more precisely to the formation of proteins-NaTDC complexes. To investigate this hypothesis, we incubated CRF with other surfactants: sodium glycodeoxycholate (NaGDC), sodium taurocholate (NaTC) and the non-ionic surfactant Triton X-100, whose ability to disturb chloroplast membranes has already been observed and established [START_REF] Yu | Triton X-100 as an effective surfactant for the isolation and purification of photosystem I from Arthrospira platensis[END_REF]. Similar decreases in absorbance were observed with all surfactants tested, with differences in intensity (Figure S10). This reinforces the hypothesis of changes in CRF membranes by NaTDC. We also observed that the presence of NaTDC shifted the asymmetric vibrations of the C-H bonds (νasCH2 and νasCH3) towards higher wavenumbers (Figure 6A). This shows that the presence of NaTDC increases the acyl chain mobility in CRF membranes. However, the membrane disturbance is probably limited because the microscope observation of CRF dispersed with NaTDC showed non-disintegrated chloroplasts (Figure 6B), which appear similar to those observed in intact cells from spinach leaves (Figure 6C).

In the presence of NaTDC and GPLRP2 (Figure 5B), we observe the same decrease in protein absorbance as with NaTDC alone but additional changes resulting from the lipolysis by GPLRP2 also appeared. To isolate these last ones, the spectra in absence of enzyme (Figure 5 A) were subtracted from those in presence of enzyme (Figure 5B). The differential spectra obtained (Figure 5C) are very similar to those recorded in the course of LC-MGDG hydrolysis (Figure 2 C'). They show a decrease in absorbance of lipids νC=O vibrations at 1741 cm -1 and the appearance of carboxylate peaks (νsCOO -and νasCOO -) of released fatty acids at 1556 and 1405 cm -1 . We also observed a decrease in absorbance between 1700 and 1600 cm -1 (minimum at 1646 cm -1 ; Figure 5 C) which did not appear during the hydrolysis of LC MGDG. We attributed it to the vibrations of amides I'. This could be due to an increase in proteins-NaTDC interaction caused by the disappearance of galactolipids linked to proteins, or to novel interactions of lipolysis products with proteins. Indeed, lyso-galactolipids like MGMG and DGMG are also surfactants.

The lipolysis reaction was monitored by quantifying the released fatty acids. The concentration of FFA was estimated from the absorbances at 1556 and 1405 cm -1 , corresponding to νsCOO - and νasCOO -peaks, using equations ( 4) and ( 5). After 2 h of reaction, a concentration of 5.3 mM FFA (ALA equivalent) was reached, which corresponds to the release of 71% of the fatty acids of chloroplast galactolipids (Figure 7 A). This percentage has been estimated by considering that (1) there are 28.59 mg and 25.81 mg of MGDG and DGDG per g of spinach CRF (DW) [START_REF] Wattanakul | In vitro digestion of galactolipids from chloroplast-rich fraction (CRF) of postharvest, pea vine field residue (haulm) and spinach leaves[END_REF] and ( 2) the MGDG and DGDG of spinach leaves contain 71% and 58% (w/w) of FA [START_REF] Amara | Lipolysis of natural long chain and synthetic medium chain galactolipids by pancreatic lipase-related protein 2[END_REF]. We could estimate a specific activity of 89 ± 7 U/mg that is about two times less than the one obtained with LC-MGDG (166 ± 9 U/mg) in the presence of NaTDC (see Table 2).

Given the effects of bile salts on CRF, we wondered whether their action could favour the activity of GPLRP2 on the membrane galactolipids. The hydrolysis of CRF by 50 nM GPLRP2 was therefore tested in the absence of NaTDC.

The raw spectra of CRF were larger in absence (Figure S9 A' and B') than in the presence of NaTDC (Figure S9 A and B). This is consistent with our hypothesis that NaTDC causes a decrease in the absorbance of membrane protein bands. However, as before in the presence of NaTDC, changes with time on the raw spectra were difficult to observe because of their low intensity . Again, the spectra evolutions could be revealed and analysed using the difference spectra (Figure 5).

In absence of NaTDC and GPLRP2 (Figure 5 A'), the difference spectra did not show the large decrease in absorbance due to NaTDC and observed in Figure 5A. There were however rather small decrease and increase in absorbance at 1530 and 1442 cm -1 , which we attributed to the disappearance of amides II and the appearance of amides II', respectively. The increase in absorbance at 1442 cm -1 may also be due in part to the appearance of HDO molecules.

In presence of GPLRP2 (Figure 5 B'), we observed the same variations as in the absence of GPLRP2, mixed with the classical variations attributed to lipolysis (disappearance of galactolipid carbonyls at 1741 cm -1 and appearance of FFA carboxylates at 1560 and 1405 cm - 1 ). We also observed bands appearing between 1700 and 1600 cm -1 which we attributed to amide I' vibrations (Figure 5 B'). Four peaks are observed at 1704, 1683, 1648 and 1617 cm -1 .

The peaks at 1704 and 1683 cm -1 may correspond more precisely to β turn structures and the peaks at 1648 and 1617 cm -1 to random coil and β sheet structures of proteins [START_REF] Kong | Fourier Transform Infrared Spectroscopic Analysis of Protein Secondary Structures[END_REF]. The peak at 1683 cm -1 may also be partly composed of the νC=O vibrations of FFA as observed in the difference spectra of C8-MGDG and LC-MGDG hydrolysis (Figure 2 B' and C'). The increase in absorbance of amides I' bands may have been caused by the disappearance of proteins-galactolipids interactions and/or the appearance of interactions between proteins and other compounds, such as the proteins themselves or the lipolysis products (lysogalactolipids and FFA).

To observe only the changes caused by lipolysis, the spectra in absence of enzyme were subtracted from those in presence of enzyme. This greatly reduced the changes due to amide II' appearance and amide II disappearance and made the carboxylates peaks more quantitative (Figure 5 C'). The quantitative analysis of the reaction consisted of monitoring the concentration of fatty acid released. FFA concentration was estimated from the two carboxylate peaks (νsCOO -and νasCOO -) using equations ( 4) and ( 5). The FFA concentrations (Figure 7 B) and the specific activity obtained (133 ± 29 U/mg; Table 2) was more important than in the presence of NaTDC (89 ± 7 U/mg, see Table 2). After 2 h of reaction, the estimated amount of FFA was equivalent to the total amount of fatty acids present in chloroplast galactolipids (Figure 7 B). It is worth noticing, however, that the calibration curve for ALA measurement was performed with ALA-NaTDC micelles, as in the previous experiment performed with NaTDC. Since the interaction of ALA with NaTDC may change the molar extinction coefficient of ALA, it is possible that we have overestimated or underestimated the amounts of ALA equivalents released during the lipolysis of CRF in the absence of NaTDC. We had however no obvious alternative because ALA is insoluble in water and NaTDC micelles allow dispersing it.

Analysis of LC-MGDG and CRF lipolysis using TLC

To validate FTIR analysis of galactolipids and CRF lipolysis by GPLRP2, lipolysis reactions were reproduced in water instead of D2O and the lipolysis products were extracted and analyzed by thin-layer chromatography coupled to densitometry after staining of galactolipids with thymol/sulfuric acid reagent. Figure 8A shows the TLC separation of the lipolysis products generated upon hydrolysis of LC-MGDG by GPLRP2 in the presence of NaTDC. In the absence of enzyme, only the LC MGDG band is observed in the form of a doublet. After 1 h of incubation with GPLRP2, the latter has almost disappeared and novel bands appear in the organic phase resulting from the extraction: a lower red band that can be attributed to MGMG [START_REF] Sahaka | Quantitative monitoring of galactolipid hydrolysis by pancreatic lipase-related protein 2 using thin layer chromatography and thymol-sulfuric acid derivatization[END_REF] and two upper bands corresponding to less polar compounds that can be attributed to FFA. When compared to the ALA standard loaded on the same plate, on can see that the yellow band corresponds to free ALA. This is the first time that we observed this ALA yellow staining by the thymol-sulphuric acid reagent. The separate analysis of the aqueous phase resulting from the extraction shows the appearance of MGG, also stained in red by the thymol-sulphuric acid reagent. Therefore, all the lipolysis products identified by FTIR can be seen by TLC analysis.

The action of GPLRP2 on CRF dispersions, in the presence and absence of NaTDC, was also analysed using TLC (Figure 8B). Without the enzyme, CRF showed two large red bands attributed to MGDG and DGDG [START_REF] Sahaka | Quantitative monitoring of galactolipid hydrolysis by pancreatic lipase-related protein 2 using thin layer chromatography and thymol-sulfuric acid derivatization[END_REF][START_REF] Wattanakul | In vitro digestion of galactolipids from chloroplast-rich fraction (CRF) of postharvest, pea vine field residue (haulm) and spinach leaves[END_REF]. After 1 h incubation with GPLRP2 at 37 °C, in the presence and absence of NaTDC, MGDG and DGDG bands have almost disappeared and MGMG and free ALA bands appeared (Figure 8). The weakness of MGMG band and the absence of DGMG band indicates that these intermediate lipolysis products have also been hydrolysed to a large extent, which is in agreement with the FTIR analysis. Qualitatively the reaction appears to be more advanced in the presence of NaTDC than in its absence.

Discussion

Monitoring galactolipid lipolysis by FTIR

Previously, we monitored the lipolysis of DPPC-NaTDC micelles by GPLRP2, in D2O, using T-FTIR. We used the composite carbonyl band to estimate the concentration of DPPC, lysoPC and palmitic acid (PA) throughout the reaction (Mateos-Diaz et al., 2018b). In the present study, we used the same method to monitor the hydrolysis of a medium chain galactolipid, C8-MGDG, also presented in the form of mixed micelles with NaTDC. We quantified the residual substrate (C8-MGDG) and reaction products (C8-MGMG and OA) by exploiting the carbonyl band, as previously. The final reaction product, MGG, was estimated from the contents in residual MGDG, MGMG, OA and the reaction stoichiometry. The C8-MGMG standard used for calibration was produced by hydrolysing C8-MGDG with GPLRP2. This allowed to obtain a perfect match between the concentration of C8-MGMG estimated and that expected from C8-MGDG disappearance and OA release. We also used the carboxylate bands to quantify FFA (OA), what was not attempted before. The concentrations obtained with these peaks were the same as those obtained with the carbonyl band. Then, in order to validate the FTIR results, the reaction, carried out in D2O, was also monitored with TLC-densitometry analysis of lipolysis products. This allowed to estimate similar reaction rates and product concentrations with both methods.

Substrate specificity and specific activity of GPLRP2

During lipolysis of C8-MGDG and NaTDC mixed micelles, the disappearance of the intermediate lipolysis product C8-MGMG and the production of MGG deduced from FFA release shows that GPLRP2 can hydrolyse the two ester functions in C8-MGDG and confirms our previous studies using TLC analysis [START_REF] Sahaka | Quantitative monitoring of galactolipid hydrolysis by pancreatic lipase-related protein 2 using thin layer chromatography and thymol-sulfuric acid derivatization[END_REF]. This means that GPLRP2 can attack at both sn-1 and sn-2 positions of C8-MGDG, or, that after the hydrolysis of sn-1 position, an intramolecular migration of the remaining acyl chain from the sn-2 to sn-1 position occurs, followed by a second hydrolysis at this position (Figure 9). Since GPLRP2 has been described in several studies as an enzyme preferring the sn-1 position [START_REF] Carrière | Structural basis for the substrate selectivity of pancreatic lipases and some related proteins[END_REF][START_REF] Fauvel | Purification of two lipases with high phospholipase A1 activity from guinea-pig pancreas[END_REF]Mateos-Diaz et al., 2018b;[START_REF] Withers-Martinez | A pancreatic lipase with a phospholipase A1 activity: crystal structure of a chimeric pancreatic lipase-related protein 2 from guinea pig[END_REF], the latter hypothesis is the one we consider most likely. However, in a study using synthetic glycerophosphatidylcholines containing -eleostearic acid, either at the sn-1 position or at the sn-2 position and a nonhydrolyzable ether bond at the other position to prevent acyl chain migration during lipolysis, it was shown that GPLRP2 could release -eleostearic acid from the sn-2 position (phospholipase A2 activity) at a rate that was 10-fold lower than the rate of eleostearic acid release from the sn-1 position (phospholipase A1 activity) [START_REF] El Alaoui | A continuous spectrophotometric assay that distinguishes between phospholipase A 1 and A 2 activities[END_REF]. Therefore, some of the FFA released from galactolipids by GPLRP2 may also be generated by a direct attack of the sn-2 position.

T-FTIR was also used to monitor the hydrolysis of a natural LC-MGDG, solubilized in mixed micelles with NaTDC, by GPLRP2. The spectra evolved in the same way as those of C8-MGDG, showing that this technique is applicable to natural lipids having a heterogeneous fatty acid composition. However, due to the lack of MGMG standard for calibration, we could not quantitatively exploit the carbonyl band and we only quantified the FFA from the carboxylate bands using ALA as a reference standard (Figure 4D).

The specific activities of GPLRP2 obtained with LC-MGDG and C8-MGDG were similar (Table 2). These data confirm the previous study by [START_REF] Amara | Lipolysis of natural long chain and synthetic medium chain galactolipids by pancreatic lipase-related protein 2[END_REF] showing that the galactolipase activity of PLRP2 is poorly affected by acyl chain length and/or degree of unsaturation. Assay conditions were however quite different with FFA estimated from continuous pH-stat titration under strong mechanical stirring of the substrate micellar dispersion. Higher specific activities reaching 5000 U/mg were recorded under these conditions. Nevertheless, FTIR analysis can be performed in a much smaller volume (50 µL versus 5 to 15 mL) which allows using low amounts of rare natural substrates.

Specific activity of GPLRP2 in D2O versus H2O

The specific activity of GPLRP2 on C8-MGDG in D2O was estimated from the release of octanoic acid during the first 10 min of the reaction and expressed in μmoles of fatty acid released per min and per mg of enzyme (or U/mg). The values obtained were 727 ± 67 U/mg by FTIR and 981 ± 97 U/mg by TLC analysis (Table 2). These activities were similar but 2 to 2.7-fold lower than the activity (1945 ± 116 U/mg) obtained when studying the same reaction in H2O under similar conditions and the same enzyme concentration [START_REF] Sahaka | Quantitative monitoring of galactolipid hydrolysis by pancreatic lipase-related protein 2 using thin layer chromatography and thymol-sulfuric acid derivatization[END_REF]. This finding is in line with our previous study on DPPC lipolysis by GPLRP2, in which we also found a lower rate of lipolysis in D2O compared to H2O (Mateos-Diaz et al., 2018b). A difference was also observed between the hydrolysis of C8-MGMG in D2O and that in H2O. In water, the hydrolysis started as soon as C8-MGMG appeared [START_REF] Sahaka | Quantitative monitoring of galactolipid hydrolysis by pancreatic lipase-related protein 2 using thin layer chromatography and thymol-sulfuric acid derivatization[END_REF], whereas in D2O it only started when the amount of C8-MGDG reached 4 times that of the residual C8-MGDG, independently of the amount of enzyme present (Figure 4). Therefore, the lower hydrolytic activity of GPLRP2 in D2O compared to water can be generalized. Moreover, the nature of the solvent may also influence the substrate preference of GPLRP2.

Monitoring galactolipid lipolysis by FTIR in the complex environment of the chloroplast

T-FTIR was used to monitor the action of GPLRP2 on CRF. Although this natural material presents a low mass concentration of galactolipid (54 mg/g CRF (DW) [START_REF] Wattanakul | In vitro digestion of galactolipids from chloroplast-rich fraction (CRF) of postharvest, pea vine field residue (haulm) and spinach leaves[END_REF]), it was possible to observe the hydrolysis of chloroplast membrane lipids in differential spectra. The previous data obtained with model galactolipid substrates allowed identifying the spectral region of interest, and the lipolysis of CRF was monitored by quantifying FFA over time using the carboxylate peaks. Bile salts (NaTDC) were initially added to favour the action of GPLRP2 on membrane galactolipids since the optimum activity of GPLRP2 on purified galactolipids was observed in the presence of bile salts [START_REF] Amara | Lipolysis of natural long chain and synthetic medium chain galactolipids by pancreatic lipase-related protein 2[END_REF][START_REF] Amara | Continuous measurement of galactolipid hydrolysis by pancreatic lipolytic enzymes using the pH-stat technique and a medium chain monogalactosyl diglyceride as substrate[END_REF]. About 70% of the CRF galactolipid fatty acids were released after 2 h of incubation under these conditions (Figure 7A). It was hypothesized that the presence of bile salts could lead to membrane disruption by these surfactants and galactolipid substrate dispersion in water in the form of mixed micelles. Indeed, we have shown that NaTDC has an impact on the CRF membrane with an increased fluidity of acyl chains (Figure 6) and presumably an interaction with membranes proteins suggested by a decrease in absorbance of amide bands (Figures 5 andS4). We hypothesized that this decrease in absorbance could reflect the interaction between NaTDC and proteins and the formation of protein/NaTDC/lipid aggregates.

Nevertheless, the activity of GPLRP2 on CRF galactolipids was also shown in the absence of bile salts (7B), which demonstrated that GPLRP2 can access its galactolipid substrate in CRF membranes without the need of an interaction with bile salts. Moreover, the action of GPLRP2 on CRF in the absence of bile salts was characterized by a higher specific activity (Table 2) and the percent of galactolipid fatty acid released reached 100% (Figure 7B).

This implies that fatty acids from both sn-1 and sn-2 positions of galactolipids were released as observed during lipolysis of the model C8-MGDG micelles. Thus, PLRP2 is tailored for the total release of fatty acids from dietary galactolipids present in the chloroplast of vegetables.

PLRP2 is present in human pancreatic juice (HPJ), what certainly explains the efficient digestion of galactolipids of CRF from spinach leaves and pea vine field residue observed in a previous study [START_REF] Wattanakul | In vitro digestion of galactolipids from chloroplast-rich fraction (CRF) of postharvest, pea vine field residue (haulm) and spinach leaves[END_REF]. Further studies with whole plant leaves are now required for a better understanding of galactolipid digestibility.

The direct action of GPLRP2 on CRF galactolipids was not expected from the previous characterization of this enzyme. Indeed, studies with galactolipid and phospholipid monomolecular films [START_REF] Hjorth | A structural domain (the lid) found in pancreatic lipases is absent in the guinea pig (phospho)lipase[END_REF] have shown the dependency of enzyme activity on surface pressure and optimum activities at low surface pressure. It was thus likely that GPLRP2, but also human PLRP2, could not hydrolyze galactolipids and phospholipids at the lateral surface pressure of natural membranes (30 mN/m) [START_REF] Amara | The galactolipase activity of some microbial lipases and pancreatic enzymes[END_REF]. Moreover, we have shown that GPLRP2 does not interact with and does not hydrolyze liposome of DPPC, while it is active on mixed micelles of the same phospholipids (Mateos-Diaz et al., 2018a).

CRF lipolysis by GPLRP2 in the absence of NaTDC, was accompanied by an increase in absorbance of the amide I' band, more precisely amide I' of β turn, random coil and β sheet structures. This increase in absorbance may have been caused by the disappearance of proteingalactolipid interactions and/or the appearance of interactions between proteins and other compounds, such as the proteins themselves or the lipolysis products (lyso-galactolipids and FFA) generated by GPLRP2. Since lyso-galactolipids are polar, it would not be surprising if they bound to proteins in the same way as galactolipids. FFAs are also known to interact with chloroplast proteins and modify their tertiary structures [START_REF] Molotkovsky | The influence of heating on the morphology and photochemical activity of isolated chloroplasts[END_REF].

TLC analysis of CRF lipolysis confirmed the ability of GPLRP2 to hydrolyze chloroplasts without the presence of NaTDC. However, lipolysis seems less advanced in the absence than in the presence of NaTDC (Figure 8B), suggesting an overestimation by FTIR of the amount of FFA released during lipolysis of CRF in the absence of NaTDC. This may result from the fact that the FFA calibration curve was established with ALA in the presence of NaTDC.

Indeed this later was required to ensure ALA dispersion in water.

Conclusion

FTIR transmission spectroscopy allowed quantitative measurement of the lipolysis of galactolipids by PLRP2, in situ and in real time. Using mixed micelles of a medium chain galactolipid (C8-MGDG) and bile salts (NaTDC) in D2O as a model system, the concentrations of the residual substrate and lipolysis products were estimated from the carbonyl and carboxylate vibration bands after calibration with reference standards. The results were validated by TLC analysis after lipid extraction. In situ FTIR measurement performed in a 50-µL liquid cell allowed monitoring the lipolysis reaction with a low amount of substrate, no sampling and no lipid extraction step. With more complex substrates like natural MGDG with long chain unsaturated FAs and intact chloroplasts isolated from spinach leaves, it was not possible to quantify all lipolysis products by FTIR. Nevertheless, the carboxylate vibration band allowed quantifying FFA released by PLRP2 using a calibration curve established with ALA dispersed in mixed micelles with bile salts. It was thus possible to estimate the specific activity of PLRP2 on all the substrates tested. Interestingly, all specific activities were in the same order of magnitude with values ranging from 133 ± 29 U/mg on CRF galactolipids to 492 ± 30 U/mg on LC-MGDG and 727 ± 67 U/mg on C8-MGDG. The highest values were obtained with galactolipids in mixed micelles with bile salts, in agreement with previous studies on the galactolipase activity of PLRP2 [START_REF] Amara | Lipolysis of natural long chain and synthetic medium chain galactolipids by pancreatic lipase-related protein 2[END_REF]. However, we have shown for the first time the direct action of PLRP2 on the galactolipids of chloroplast membranes without the need for bile salts. PLRP2 can therefore access to galactolipids present in bilayers and not only into Lipolysis was catalyzed by 50 nM GPLRP2, at 37°C and pD 8. NaTDC. The activities were calculated using the amounts of fatty acid released during 10 minutes from the start of the reaction (0-10 min) or from the fifth minute of reaction (5-15 min).

The reactions were performed at 37 °C and pD 8.

  In situ monitoring of galactolipid digestion by infrared spectroscopy in both model micelles and spinach chloroplasts Moulay Sahaka, Eduardo Mateos-Diaz, Sawsan Amara, Jutarat Wattanakul, David Gray, Dominique Lafont, Brigitte Gontero, Hélène Launay, Frédéric Carrière To cite this version: Moulay Sahaka, Eduardo Mateos-Diaz, Sawsan Amara, Jutarat Wattanakul, David Gray, et al.. In situ monitoring of galactolipid digestion by infrared spectroscopy in both model micelles and spinach chloroplasts. Chemistry and Physics of Lipids, 2023, pp.105291. 10.1016/j.chemphyslip.2023.105291. hal-04028212

Figure 4

 4 Figure4shows the evolution with time of the lipolysis reaction of C8-MGDG in the presence

Figure legends Figure 1 .

 legends1 Figure legends

Figure 4 .

 4 Figure 4. Time-course variations in the concentrations of lipolysis products and residual

Figure 5 .

 5 Figure 5. Difference spectra of carbonyl, carboxylate and amide vibrations during the lipolysis

Figure 6 .

 6 Figure 6. Spectra of CRF acyl chain vibrations, in the absence (green line) and presence (red

Figure 8 .

 8 Figure 8. TLC analysis of CRF hydrolysis, in the presence and in absence of NaTDC. The

Table 1 .

 1 Molar extinction coefficients (ε, M -1 cm -1 ) of C8-MGDG, C8-MGMG, OA and ALA from IR absorbance. The ε-values represent the slope of the standard calibration curves of IR absorbance of C8-MGDG, C8-MGMG, OA and ALA at different wavenumbers, at pD 8 (see FigureS2). All IR measurements were performed in D2O, in the presence of NaTDC, at pD 8

	and 37 °C.
	Compound	Vibration band Wavenumber	ε	R 2
			1744 cm -1	478.1	0.998
	C8-MGDG	ʋC=O	1722 cm -1	454.4	0.999
			1682 cm -1	38.6	0.929
			1744 cm -1	134.8	0.988
	C8-MGMG	ʋC=O	1722 cm -1	302.7	0.998
			1682 cm -1	33.3	0.957
			1744 cm -1	22.0	0.928
		ʋC=O	1722 cm -1	33.5	0.946
	OA		1682 cm -1	89.2	0.996
		ʋasCOO -	1553 cm -1	751.8	0.997
		ʋsCOO -	1408 cm -1	227.6	0.996
		ʋasCOO -	1555 cm -1	595	0.967
	ALA
		ʋsCOO -	1407 cm -1	204.2	0.988

Table 2 :

 2 Specific activities (U/mg or µmole FFA per min per mg of enzyme) of GPLRP2 on C8-MGDG-NaTDC micelles, LC-MGDG-NaTDC micelles and spinach CRF with and without

Acknowledgements

The PhD thesis of Moulay Sahaka was supported by a fellowship from Aix Marseille Université, France. The PhD thesis of Miss Jutarat Wattanakul was supported by Thai Royal Government, Thailand.

Funding

This work received the financial support of Agence Nationale de la Recherche (France) in the framework of the GALACTOLIPASE project (ANR-09-CP2D-06-01).

micelles. Moreover, an almost complete release of FA from chloroplast galactolipids was observed, what fits with the ability of PLRP2 to release the two FAs present in C8-MGDG.

These findings provide new insights into the substrate specificity of PLRP2 and further support its major contribution to galactolipid digestion in mammalian species.

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Author contribution

Moulay Sahaka was involved in the conception and design of the study, in acquisition, analysis and interpretation of data, in drafting the and revising it critically for important intellectual content. Eduardo Mateos-Diaz, Sawsan Amara and Jutarat Wattanakul were involved in acquisition, analysis and interpretation of data, and revising the article critically for important intellectual content. Dominique Lafont was involved in the chemical synthesis of C8-MGDG. David Gray, Brigitte Gontero, Hélène Launay were involved in revising the article critically for important intellectual content. Frédéric Carrière was involved in the conception and design of the study, in analysis and interpretation of data, in drafting the article and revising it critically for important intellectual content. All authors were involved in the final approval of the version to be submitted. NaTDC concentration was 56 mM for C8-MGDG, C8-MGMG and OA, and 30 mM for ALA.

IR spectra were recorded at 37 °C and pD 8.


Sara Abada
email: abada@ifpen@fr

Amandine Martin Petit

Amandine Lecocq

Guy Marlair

Valérie Sauvant-Moynot

François Huet

Ah Purepower He

Ah Lifebatt

Accelerated rate calorimetry dedicated to the modelling of Li-ion batteries thermal runaway of fresh and aged cells

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.

• An original experimental/modeling approach was developed to understand the effect of aging on Li-ion batteries safety

•

•

  the coupled thermal runaway model was validated at the cell level on BTC tests at INERIS testing plateform • the validated model was used in order to discuss why aged cells appear safer than fresh ones • Further work has to be done to take into account cycling aging: • Study of safety degradation linked to cycling aging (Li plating…) • Study of high energy cells with up-to-date Li-ion technologies Prada et al, VPPC 2010 [1] S. Abada et al., "Safety focused modeling of lithium-ion batteries: A review," J. Power Sources, vol. 306, pp. 178-192, 2016. • Choice of 3 relevant technologies • 2 LFP/C cells • 1 NMC/C cell • Calendar aging of cells • Accelerated aging @ high State of Charge (SOC): 100% @ high Temperature : 60 °C • In order to reach 2 aging targets: o 10 % of capacity loss o 30% of capacity losses • Abusive tests • Heat-Wait and Search on Battery Testing Calorimeter (BTC) device for model calibration and validation • Coupling of 3 submodels • 3D thermal model • Static aging submodel Taking into account the formation of a SEI layer on the negative electrode • 0D cell chemical degradation model Based on the four main degradation mechanisms Evidence of 3 key temperatures on BTC tests for all cells: o T 1 : First exothermal reaction / end of safe phase o T 2 : Fusion of the separator o T 3 : Thermal runaway A123s LifeBatt • Using of modeling to further discuss the experiments: • First exothermal reaction posponed in aged cells o Passivation effect of the SEI layer formed during aging o The second degradation reaction is slower Fresh A123s cell Aged A123s cell (30% capacity fade) CONCLUSIONS AND PERSPECTIVES Develop and validate physical and 3D model of Li-ion battery electro-thermal behavior nearby thermal runaway conditions, for a better understanding of phenomena as basis for improved engineering.