Substrate specificity of plant and fungi pectin methylesterases: Identification of novel inhibitors of PMEs

Accepted Manuscript Title: Substrate specificity of plant and fungi pectin methylesterases. Identification of novel inhibitors of PMEs Author: M´elanie L’Enfant Jean-Marc Domon Catherine Rayon Thierry Desnos Marie-Christine Ralet Estelle Bonnin J´erˆome Pelloux Corinne Pau-Roblot PII: DOI: Reference:

S0141-8130(15)00613-3 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.08.066 BIOMAC 5334

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

10-6-2015 27-8-2015 28-8-2015

Please cite this article as: M. L’Enfant, J.-M. Domon, C. Rayon, T. Desnos, M.-C. Ralet, E. Bonnin, J. Pelloux, C. Pau-Roblot, Substrate specificity of plant and fungi pectin methylesterases. Identification of novel inhibitors of PMEs, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.08.066 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Substrate specificity of plant and fungi pectin methylesterases. Identification of novel inhibitors of PMEs

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Mélanie L’Enfanta, Jean-Marc Domona, Catherine Rayona, Thierry Desnosb, Marie-Christine Raletc, Estelle Bonninc, Jérôme Pellouxa, Corinne Pau-Roblota

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CEA Cadarache, SBVME/LBDP, Bât 178, 13108 Saint-Paul-les-Durance cedex, France

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INRA, UR 1268 - Biopolymères - Interactions – Assemblages, BP 71627, 44316 Nantes cedex 03, France

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Unité de Biologie des Plantes et Innovation, EA3900, Université de Picardie Jules Verne, 33 rue Saint Leu, 80039 Amiens cedex, France

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Corresponding author : Dr. Corinne Pau-Roblot, e-mail: [email protected], Phone: +33-322-82-75-36, Fax: +33-3-22-82-74-36

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PME, pectin methylesterase; PMEI, pectin methylesterase inhibitor; EGCG, (-)-epigallocatechin-3-gallate; CsPME, PME from orange peel (Citrus sinensis); AtPME31, PME from Arabidopsis thaliana expressed in Escherichia coli, BcPME1, PME from Botrytis cinerea expressed in Pichia pastoris

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Keywords

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Pectin methylesterase; chemical inhibitors; chemical library; catechins; EGCG

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ABSTRACT

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Pectin methylesterases (PMEs) play a central role in pectin remodeling during plant development. They are also present in phytopathogens such as bacteria and fungi. We investigated the substrate specificity and pH dependence of plant and fungi PMEs using tailor-made pectic substrates. For this purpose, we used two plant PMEs (from orange peel: Citrus sinensis and from Arabidopsis thaliana) and one fungal PME (from Botrytis cinerea). We showed that plant and fungi PMEs differed in their substrate specificity and pH dependence, and that there were some differences between plant PMEs. We further investigated the inhibition of these enzyme activities using characterized polyphenols such as catechins and tannic acid. We showed that PMEs differed in their sensitivity to chemical compounds. In particular, fungal PME was not 1 Page 1 of 33

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sensitive to inhibition. Finally, we screened for novel chemical inhibitors of PMEs using a chemical library of ~3600 compounds. We identified a hundred new inhibitors of plant PMEs, but none had an effect on the fungal enzyme. This study sheds new light on the specificity of pectin methylesterases and provides new tools to modulate their activity.

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1. Introduction

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The plant cell wall is a complex matrix of polysaccharides and proteins, which includes notably cellulose, hemicellulose, and pectin as well as enzymes and structural proteins. It is the first external barrier and plays a key role in the interaction between plant and pathogens (fungi and bacteria). Pectin, which is the most abundant component of the plant primary cell wall in dicotyledonous species, represents up to 35% dry weight. It is composed of four distinct polysaccharides: homogalacturonan (HG), rhamnogalacturonans I (RG-I) and II (RG-II) and xylogalacturonan (XGA) [1]. HG, which represents ~65% of pectin, is a linear homopolymer of α-(1,4)-D-galacturonic acid [2,3]. It can be methylesterified at the C6 carboxyl and acetylated at O2 and/or O3 positions [4,5]. HG is the substrate of several pectin-remodeling enzymes, including pectin methylesterases (PME, E.C. 3.1.1.11), pectin acetylesterases (PAE, E.C. 3.1.1.6), polygalacturonases (PG, E.C. 3.2.1.15) and pectin/pectate lyases (PLL, E.C. 4.2.2.10/E.C. 4.2.2.2), all of which have been shown to play a key role in controlling the development of plants and their responses to stress [6]. In particular, in recent years, most attention has been paid to the role of PMEs in the fine-tuning of HG structure in developmental processes as diverse as pollen tube and hypocotyl elongation [7,8], primordia emergence at the shoot apical meristem [9], root growth [6], and adventitious root formation [10]. Although the diversity of the functional roles of PMEs is currently emerging, much remains to be discovered about their biochemical specificity. In all plant species sequenced to date, PMEs belong to a rather large multigenic family (>30 at least isoforms [6]), which obviously questions their potential substrate specificity, pH dependence and mode of action in the context of the cell wall. PMEs catalyze the specific hydrolysis of a methylester at C6 of galacturonic acid, thereby releasing methanol, protons and polygalacturonic acid (PGA) [11,12]. Their mode of action seems to be influenced by environmental factors, including the degree and pattern of methylesterification and the pH of the cell wall. For instance, at a given pH, some PMEs are more effective than others on highly methylated pectin. At acidic pH, some PMEs act randomly, thus promoting the formation of calcium bridges, whereas at alkaline pH, some PMEs have a processive action [13-15]. In addition to those present in plants, PMEs are produced by numerous organisms including bacteria, fungi and nematodes, particularly during their interaction with plants [16]. Plant/bacteria PMEs and fungal PMEs differ in their isoelectric pH (pI), mode of action and pH optimum. Plant PMEs and most bacterial PMEs present a neutral to alkaline pI, comprised between pH 7 and 8 [17, 18]. These PMEs act processively, generating blocks of galacturonic residues with free carboxyl groups [19,20]. The dynamics of the bacterial enzyme on the substrate have recently been resolved using molecular dynamics simulation, providing new insights into the mode of action of processive PMEs [21,22]. In contrast, fungal enzymes have been reported to act randomly on the methyl-esterified HG backbone, thus preferentially creating substrates for pectindegrading enzymes such as PGs and PLLs.

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One of the key points in the regulation of plant PME activity is their interaction with endogenous proteinaceous inhibitors, PMEIs (Pectin Methylesterase Inhibitors [23]). The inhibition is mediated by the formation of a 1:1 complex, and appears to be dependent on pH [24,25]. PMEIs are thus likely to be key components of the fine-tuning of HG structure as shown by recent reports [9,26,27]. Surprisingly, pathogen PMEs are not inhibited by plant PMEIs, suggesting that structural differences impair the interaction with inhibitors and that inhibition of pathogen PMEs, unlike the pathogen polygalacturonase-plant 2

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polygalacturonase inhibiting protein (PG-PGIP) interaction, is not a key component in preventing infection [24]. In addition to PMEIs, non-proteinaceous molecules such as catechins, phenolics extracted from green tea leaves, have been identified as inhibitors of plant PME activity [28]. These compounds were shown to be active on fungal enzymes, such as from Aspergillus, albeit at a much higher concentration [29]. Other chemicals, such as iodine and detergents (e.g., SDS), have also been shown to have an inhibitory effect on PMEs [30, 31]. The use of novel inhibitors of pectin methylesterases could be a means to target phytopathogens enzymes, thus reducing the use of synthetic pesticides.

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Using plant and fungal enzymes, as well as characterized tailor-made HG substrates, the aim of this study was to investigate the substrate specificity and pH dependence of PMEs. The enzymes were further used to study their sensitivity to previously reported inhibitors, as well as to novel chemical inhibitors, which were identified by screening a chemical library of ~3600 compounds. This study brings new insights into the specificity of these enigmatic enzymes, and the putative role of this specificity in the fine-tuning of the degree of methylesterification of pectins in planta and new tools to modulate the enzymes’ activities

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2. Materials and methods

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2.1. Source of enzymes

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Pectin methylesterase from orange peel (Citrus sinensis) was used as reference (Sigma, P5400). This protein was thereafter referred to as CsPME. Escherichia coli strain JM101, carrying the plasmid pREP4 [32] overexpressing PME-At3g29090, was used for the expression of the Arabidopsis protein. This protein was thereafter referred to as AtPME31. This strain was stored at -80 °C in glycerol. Pichia pastoris strain GS115, carrying the plasmid pPIC3.5 expressing Botrytis cinerea BcPME1, was obtained from Jan A. L. Van Kan and grown for 3 days at 30 °C in Yeast Extract Peptone Dextrose (YEPD) solid medium [33]. This protein was thereafter referred to as BcPME1.

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2.2. Expression, extraction and purification of recombinant proteins

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To express and produce recombinant AtPME31, a recombinant colony of E. coli was grown overnight at 30 °C in Lysogeny Broth (LB) solid medium containing 1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar and supplemented with ampicillin (100 µg.ml-1) and kanamycin (25 µg.ml-1). One colony of Escherichia coli was transferred into 5 ml liquid LB containing 1% glucose, ampicillin (100 µg.ml-1) and kanamycin (25 µg.ml-1) and grown at 30 °C and 250 rpm for 24 h. A volume of 200 ml of the same medium, to which 400 µl ampicillin (100 µg.ml-1), 50 µl kanamycin (25 µg.ml-1) and 4 ml glucose (1%) was added, was inoculated with 2 ml of the culture grown overnight. The culture was grown at 37 °C, 250 rpm. When the OD600nm reached 0.6, 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added and incubation was continued at 28 °C, 120 rpm for 4 h. Cells were harvested by centrifugation at 4,000 rpm for 10 min. The bacterial pellet was resuspended in 1 ml of PSB buffer (sodium-phosphate 50 mM, NaCl 300 mM; pH 8.0) containing lysozyme (1 mg.ml-1). After a 15 min incubation, the suspension was centrifuged at 4,000 rpm for 5 min and the pellet was resuspended in 1 ml of PSB buffer with imidazole (20 mM). Bacteria were lysed by 4x30 s sonication on ice and the lysate was centrifuged at 12,000 rpm for 15 min at 4 °C. The clarified culture supernatant was concentrated using a PelliconXL device with a Biomax10 membrane on a Labscale TFF pump (Millipore) and buffer exchanged to 0.3  M NaCl, 50  mM sodium phosphate, pH 7.5. The supernatant was applied to an AKTA prime FPLC fitted with a HisTrap FF column at a flow rate of 0.25 ml.min-1 and eluted in a linear gradient of 20 to 500  mM imidazole (0.5 ml.min-1) over 10  ml. Fractions containing pure AtPME31 were pooled and stored at -20 °C.

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To express and produce BcPME1, recombinant colonies of P. pastoris were grown overnight at 30 °C on Yeast Extract Peptone Dextrose (YPD) medium plate. One colony was transferred into 2 ml liquid Buffered Methanol-complex Medium Yeast (BMMY) maintained at 30 °C and shaken at 250 rpm for 15 h. A volume of 50 ml of the same medium was inoculated with 1 ml of preculture, at 30 °C, 250 rpm. When the OD600nm reached 1, methanol was added every 24 h to a final concentration of 0.5% (v/v). After 4 days of expression, yeasts were removed by centrifugation at 14,000 rpm for 5 min. The supernatant containing proteins was precipitated by ammonium sulfate at 90% for 6 h at 4 °C. Proteins were harvested by centrifugation at 12,000 rpm for 10 min at 4 °C and dialyzed in GEBAflex tubes (6-8 kDa) for 16 h at 4 °C; extracts were concentrated using 15 ml amicon tubes (10 kDa) for 30 min at 4 °C, 3500 rpm and then purified as follows: the material was applied twice to a DEAE-Sepharose FF column (GE Healthcare, Sweden). Protein was eluted with sodium-phosphate buffer (50 mM; pH 6.0). Fractions containing pure BcPME1 were pooled and stored at 4 °C.

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Pectin methylesterase from orange peel, CsPME, was diluted in sodium-phosphate buffer (pH 7.5; 50 mM) at a concentration of 0.26 µg.µl-1.

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The purity and molecular masses of PME were determined by SDS-PAGE [34] using gels prepared with 9% (resolving gel) and 3% (stacking gel) acrylamide. For each sample, 5 µl of protein extract was loaded in the wells in parallel with 5 µl of protein standard marker (Precision Plus Protein™ All Blue Standards#1610373, 10-250 kD, BioRad). Electrophoresis was run in a BioRad Mini protean III at 40 mM until the bromophenol blue reached the bottom of the gel. The proteins were subject to Coomassie blue G250 according to Scheler et al., 1998 [35].

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2.3. Mass spectroscopic sequencing

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To determine the amino acid sequence of the purified PME, each SDS–PAGE band was manually excised from the gels and hydrolyzed according to Shevchenko et al. (1996) [36]. All digested peptide mixtures were separated by on-line nano-LC and analyzed by nano-electrospray tandem mass spectrometry. The experiments were performed on an Ultimate 3000 RSLC system coupled with an LTQ-Orbitrap XL mass spectrometer (ThermoFisher Scientific). The digested products were analyzed as previously described [6].

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2.4. Determination of protein content

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The concentration of proteins was determined by the method described by Bradford (1976) [37] using the BioRad protein assay (5000-006) and bovine serum albumin (Sigma, 05479) as the standard in phosphate buffer (50 mM). The protein concentration was estimated by measuring the absorbance at 595 nm on a BioTeK PowerWaveXS2 spectrophotometer.

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2.5. Quantitative assay of PME activity

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PME activity was measured according to Anthon and Barrett (2004) [38]. A range of 0 to 20 µg.ml-1 of methanol was used as a standard. Incubation was performed at 30 °C for 30 min with 5 µl of purified protein (0.3 µg.µl-1), 5 µl of substrates (20 mg.ml-1, Table 1) and 85 µl of 50 mM sodium-phosphate buffer (pH 4, pH 6 or pH 7.5). The solution was then incubated for 10 min at 85 °C to inactivate the enzyme. The volume was divided into two equal volumes and lyophilized. 40 μl of cold water and 40 μl of 0.2 M NaOH was added to one of the lyophilized tubes and incubated for 1 h at 4 °C. 40 µl of 0.2 M HCl was then added to stop saponification. In parallel the second tube, not saponified, contained 40 µl of cold water, 40 μl of 0.2 M NaOH and 40 µl of 0.2 M HCl. In two new tubes, 100 µl of 200 mM Tris-HCl (pH 7.5), 40 µl of 3-Methyl-

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2-benzothiazolinone hydrazine (MBTH, 3 mg.ml-1), and 20 µl of 0.02 U.µl-1 alcohol oxidase were added to 50 µL of saponified and non-saponified solutions, respectively. These tubes were incubated at 30 °C for 20 min. 200 µl of solution containing 0.5% (FeNH4(SO4)2)·12H2O, 0.5% sulfamic acid was added and cooled to room temperature. 590 µl of water was added to stop the reaction. The absorbance was determined at 620 nm on a Uvikon spectrophotometer. PME activity was calculated as the difference in absorbance between saponified and non-saponified samples and expressed, using the standard curve, as nmol MeOH.min-1.µg-1 of proteins.

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Table 1 Location

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2.6. Quantitative assay of PME inhibition

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2.6.1. Inhibition by polyphenols

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Inhibition of CsPME and AtPME31 activity was determined using a colorimetric microassay adapted from Klavons and Bennett (1986) [39]. The reaction solution contained 5 µl of purified protein extract, 90% methylesterified Citrus pectin (Sigma P9561) at 1 mg.ml-1, 0.008 U of Pichia pastoris alcohol oxidase (Sigma A2404), 0.01 to 1 µg.µl-1 of inhibitor [Polyphenon 60 (PP60, Sigma, P1204, contains 60% catechin, which consists of 34% (-)-epigallocatechin-3-gallate, 16.7% (−)-epigallocatechin, 8.7% (−)-epicatechin-3gallate, 7.3% (−)-epicatechin, 2.8% (−)-gallocatechin gallate and 0.5% (−)-catechin gallate), tannic acid (Sigma 403040) or (-)-epigallocatechin-3-gallate (EGCG, E4143)] and 50 mM sodium-phosphate buffer (pH 7.5) to a final volume of 100 µl. The mixture was incubated at 28 °C for 30 min. A first OD420nm was read on a BioTeK PowerWaveXS2 spectrophotometer. 100 μl of developing solution (2 mM ammonium acetate, 0.02 M pentane-2,4-dione, 0.05 M glacial acetic acid) was added and the solution was incubated at 67 °C for 15 min. A second reading was taken at 420 nm on a BioTeK PowerWaveXS2 spectrophotometer. Standards from 0 to 10 nmol of methanol were included in each batch. Results were expressed as nmol MeOH.min1 .µg-1 of protein using the methanol standard curve.

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Considering the optimal pH activity of BcPME1 (pH 6) and the limitations of using the alcohol oxidase with regard to pH, the inhibition of BcPME1 activity was measured using the Anthon and Barrett [39] method described above.

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2.6.2. Screening of the LATCA chemical library

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Compounds of the LATCA chemical library (Library of AcTive Compounds on Arabidopsis, collected by Sean Cutler of University of California Riverside, http://cutlerlab.blogspot.com/2008/05/latca.html) were screened at a concentration of 75 µM using the colorimetric microassay adapted from Klavons and Bennett (1986) [39]. Following the first screen, 109 molecules were identified as inhibiting CsPME activity. These were further tested on AtPME31. Secondly, the dose dependence of the inhibition was tested for 9 molecules. Inhibition of PME activity was determined using a colorimetric method as described for polyphenols but the chemical molecules, given in Table 2, were dissolved in DMSO and used at concentrations in the range of 0 to 1500 µM. DMSO was used as a control.

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Table 2 location

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3. Results

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3.1. Production and purification of proteins

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In order to characterize their substrate specificity the three PMEs of interest (CsPME; AtPME31 from plants and BcPME1 from fungus) were first purified. The purity of the commercial PME from orange peel: Citrus sinensis (CsPME), used as a reference, was verified by SDS-PAGE (Fig. 1A).

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Fig. 1 Location

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A fraction containing a protein of the expected size (32 kDa) was observed. Fourteen single peptides were identified using nano-LC-ESI-MS/MS, corresponding to the mature part of the protein (Supp. data. Fig. S1). AtPME31 was produced in E. coli as previously reported [32] and purified by affinity binding on a His-Trap FF column. Considering the His-tag, this resulted in a fraction enriched in a protein of the expected size (40 kDa, Fig. 1B), with 13 single peptides identified (Supp data Fig. S1). Lastly, the PME from B. cinerea (BcPME1) was produced in P. pastoris and purified using a DEAE-Sepharose FF column. SDS-PAGE analysis of the purified fraction revealed a main band at a molecular weight above 37 kDa (Fig. 1C), corresponding to BcPME1, for which 8 single peptides were identified (Supp. data. Fig. S1).

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CsPME, AtPME31 and BcPME1 sequences were aligned using clustalX (Fig. 2).

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Fig. 2 Location

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Alignment revealed 30% of sequence identity between AtPME31 and CsPME. This weak identity between two plant PMEs could be related to the fact that CsPME is a group 2 PME while AtPME31 belongs to group 1 [6]. Overall, sequences alignment of the three PMEs showed that two stretches of amino acids of importance at the active site were conserved (i.e. DFIFG, LGRPW). The main differences between the plant and fungal PMEs lay in some substitution within the QAVAL and QDTL sequence stretches (CsPME sequence). The former sequence was slightly divergent from that of AtPME31 (QAVAI) with an I>L substitution and more divergent from that of BcPME1 (QNLAI) with substitution of three amino acids (A>N, V>L and L>I). The latter differed only for BcPME1 with an L>I substitution at the last position. Changes in the sequences could have an impact on the substrate/pH specificity and/or mode of action of these enzymes.

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3.2. Substrate specificity and pH dependence of the enzymes

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First, the activity of the three proteins was tested using a commercial pectic substrate (Citrus pectin with a degree of methyl-esterification (DM) of 30%, 65% and 90%) at three different pHs (4, 6 and 7.5, Fig. 3).

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Fig. 3 Location

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Although the activities of CsPME and AtPME31 showed similar pH dependence, with a higher activity at pH 7.5 compared to pH 6, CsPME and AtPME31 activities were increased when substrates with increasing DM were used. In contrast, using similar substrates, BcPME1 showed the highest activity at acidic pH. In fact, the BcPME1 activity was ~4 times higher at pH 4 compared to pH 7.5 (Fig. 3C), which could be important for infection in the context of the acidic pH of the cell wall. BcPME1 appeared to have a strong affinity towards substrate with a DM>60%.

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Secondly, tailor-made HG substrates [40] were used to investigate further the substrate specificity of the three PMEs. The list of available substrates, with similar degrees of polymerization (DP), but different degrees of methylation (DM) and patterns of methylesterification, is shown in Table 1. Randomly methylesterified (B series) and blockwise methylesterified (P series) HGs were obtained by alkali or enzymatic treatment, respectively of the mother pectin (HG96). 6 Page 6 of 33

Fig. 4 Location

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At pH 7.5, CsPME activity increased with increasing DM, when both the B and P series were used. However, CsPME showed a preference for randomly methylesterified substrate, especially when a low DM (up to 56%) was considered (Fig. 4A). When substrates of relatively high DM (>60%) were used, no differences in CsPME activity could be measured between the B and P series. As a general feature, at pH 6, CsPME activity was lower than that measured at pH 7.5. However, these differences were not as marked for substrates of high DM (i.e. DM>60% for both the B and P series). At pH 4, a strong reduction in CsPME activity was observed for all the DM considered for the B series while no activity was measured for the P series. Similarly to that observed for CsPME, AtPME31 had an optimal activity at pH 7.5. Interestingly, at this pH, and in contrast to that observed for CsPME, AtPME31 did not have a strong specificity towards DM when the B series was considered (Fig. 4B). A slightly lower activity was observed with increasing DM. When the P series was used as substrates, AtPME31 activity increased with the DM, similarly to that observed for CsPME, reaching a maximum for HG96P64. At pH 6, an increase in AtPME31 activity occurred when a high DM of the B series was used, while the activity was very low for P series. At pH 4, a strong reduction in AtPME31 activity was seen whatever model of HG was used. Clearly, the pattern of methylation has a great influence on the activity of these enzymes. Therefore, although CsPME and AtPME31 are both plant enzymes, their substrate specificity appears different.

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In contrast to observations for the two plant PMEs, BcPME1 showed a different pH dependence and substrate specificity (Fig. 4C). BcPME1 activity was highest at acidic pH and when HG of high DM (>60 %) were used as substrate. For instance, at pH 4, up to a 4-fold increase in activity was observed when comparing HG96B20 and HG96B82, respectively. Similar results were obtained when substrates from the P series were used. Based on these results, BcPME1 does not appear to have a strong preference towards a pattern of methylesterification but is likely to target HG with a high DM.

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Overall, these results show that CsPME, AtPME31 and BcPME1 have distinct substrate specificities and pH dependences in vitro, which could be related to their putative roles in planta or during infection.

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3.3. Inhibition of PME by polyphenols

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These differences in substrate specificity and pH dependence prompted us to investigate whether the enzymes could be differentially inhibited. From previous studies it is known that CsPME can be inhibited by kiwi and Arabidopsis PMEI but AtPME31 cannot, due to structural differences in the binding interface ([32] and unpublished results). Fungal PME (BcPME1) was also not inhibited by any of the plant PMEIs tested (data not shown). Following a previous report showing an inhibition of PME activity by catechins extracted from green tea [35], we investigated whether CsPME, AtPME31 and BcPME1 could be differentially inhibited by chemical inhibitors. For this purpose, the effects of various chemicals, including Polyphenon 60 (PP60), (-)-epigallocatechin-3-gallate (EGCG) and tannic acid (TA), on enzyme activities were tested. The results are presented in Fig. 5 as the percentage of inhibition as a function of chemical concentration.

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Fig. 5 Location

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Inhibition of CsPME (Fig. 5A) increased rather linearly with increasing inhibitor concentration and was highest when tannic acid was used. For a chemical concentration of 0.5 µg.µl-1, CsPME activity was reduced by 50%, 74% and 88% for PP60, EGCG and tannic acid, respectively. The calculated IC50 were ~0.05 µg.µl1 , ~0.15 µg.µl-1 and ~0.5 µg.µl-1 for tannic acid, PP60 and EGCG, respectively. Thus, tannic acid had a strong inhibitory effect on CsPME, probably by cross-linking with the substrate.

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The effects of each of the chemical compounds on AtPME31 activity were very similar, with the three inhibition curves overlapping (Fig. 5B). For PP60, EGCG and TA, calculated IC50 were in the same range of 0.075 µg.µl-1 and the inhibition leveled off from 0.5 µg.µl-1 onwards. AtPME31 thus appeared more sensitive to PP60 and EGCG compared to CsPME.

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In contrast, BcPME1 showed a different response to the chemicals. Inhibition of BcPME1 was only observed when a high concentration (>0.7 µg.µl-1) of chemicals was used (Fig. 5C). This was particularly significant for TA where a ~90 % inhibition was reached when 1 µg.µl-1 was used.

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These results showed that green tea polyphenols could inhibit both A. thaliana and C. sinensis PMEs, as observed for other plant PMEs such as those from C. indivisa and C. pentagona [28]. Moreover, these compounds can also inhibit phytopathogen PME, but at much higher concentrations.

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As a means of identifying new chemical inhibitors of plant pectin methylesterases, the LATCA (Library of AcTive Compounds on Arabidopsis) chemical library [41] was screened. This is composed of ~3600 chemical compounds, most of which have been shown to be active in yeast, mammalian cells or plants. The library was first screened for compounds inhibiting CsPME and 109 molecules were identified (Table 2 and Supp. data. Table S2). For 77 of them, the inhibition was comprised between 10 and 50%. For the 32 remaining molecules, the inhibition was above 50 % and could reach up to 100%. Other compounds were either inactive or increased PME activity, which is beyond the focus of the current manuscript (data not shown). These 109 molecules were then tested for their inhibitory effect on AtPME31. It appeared that AtPME31 was less sensitive to inhibition and that the most effective compounds were not identical to those inhibiting CsPME (Table 2 and Supp. data Table S2).

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The dose dependence of inhibition for 9, commercially available chemical compounds (Table 3) was further investigated on CsPME, AtPME31 and BcPME1 (Fig. 6). In order to compare the sensitivity of the three enzymes to the chemical compounds and to avoid any pH-related effects on binding, all the experiments were realized in same conditions of pH (7.5, the optimal pH activity of CsPME and AtPME31) and with the same concentrations of inhibitors. Despite the lower activity of BcPME1 in these conditions, we believe that the strongest plant PMEs inhibitors (e.g. . 6-D6, 7-A7, 31-G8), should be efficient on this fungal enzyme.

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Table 3 location

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Fig. 6 Location

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Apart from two compounds (copper sulfate, 7-A7, and silver nitrate, 6-D6), all of these chemicals display a C6 ring with a structure similar to that of EGCG. CsPME was strongly inhibited by silver nitrate at low concentration (10 μM) and by 31-G8 (Na2-(3-chloro-2-fluorophenyl)-2,3-dihydroisothiazol-3-one) at 250 μM (Fig. 6A). For the other 7 compounds, the inhibition of CsPME was in the range of 10 to 50% depending on the concentration used. Interestingly, the response of AtPME31 to the chemicals differed from that of CsPME (Fig. 6B). For instance, the inhibition of AtPME31 by 31-G8 or 7-A7 was much higher compared to that measured for CsPME. In contrast, AtPME31 appeared less sensitive to other chemicals such as 31-H4, 31-H2 and 45-B8. Two molecules, DMSO and 45-B2, increased the activity of AtPME31, but this result, which could be of interest, was out of the scope of the present study where we focused only on compounds which had a clearly inhibitory effect. Although CsPME, AtPME3 and AtPME31 are enzymes

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from plants, their susceptibility to inhibitors appears different. Surprisingly, none of them was shown to inhibit the activity of the pathogen enzyme (BcPME1) (Fig. 6C).

323 4. Discussion

325 326 327 328 329 330 331 332 333 334 335 336 337 338

Pectin methylesterases are synthesized in higher plants, where they play a central role in the fine-tuning of the degree of methylesterification of pectins with dramatic consequences on cell wall rheology and growth [6,11,15]. In addition, several reports have shown that demethylesterification of homogalacturonan, which induces a change in the charge of the molecule and the production of methanol, and its consequences on calcium ion cross-linking, is an important mechanism in plant defense, notably against fungal pathogens [42, 43]. PMEs are also produced by bacteria or pathogenic fungi during their interaction with plants [44, 45], which questions the differences, in terms of substrate specificity and sensitivity to inhibitors, between plant and pathogen enzymes. It is known that protein inhibitors, called PMEIs, are effective against plant PMEs but ineffective against pathogen PMEs [46]. In order to identify new inhibitors of PMEs, the use of chemical inhibitors could be an alternative. For this purpose, three PMEs were used: CsPME, a commercial enzyme from Citrus cinensis, a PME from Arabidopsis thaliana (AtPME31) that was expressed in E. coli and a PME from Botrytis cinerea (BcPME1) that was expressed in Pichia pastoris. These enzymes were used to test their substrate specificity and pH-dependence using tailor-made HG substrates and then screened for novel inhibitors of PMEs using a chemical library.

339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362

The mode of action of PMEs has been described in the literature, indicating that this is likely to differ depending on the pH and ionic environment, substrate specificity and origin of PMEs [14,47]. In our study, we showed that CsPME preferred randomly methyl esterified substrates with a DM of 69% at basic pH. This is likely to be one of the major substrate in planta, HG being synthesized in the Golgi in a relatively highly methyl-esterified form, before being excreted towards the cell wall [48]. Surprisingly, despite being a plant PME, AtPME31 showed distinct substrate specificity and pH dependence when compared to CsPME. In particular, AtPME31 had a strong preference for substrates with a low DM and randomly distributed methylester at pH 7.5. In contrast, at pH 6, AtPME31 preferred substrates with high levels of randomly distributed methylesters. Considering the fact that PMEs are encoded by rather large multigenic families, this further highlights the diversity of plant PMEs, and their potential differences in pH dependence and substrate specificity, as previously reported when studying the mode of action of apple PME at various pHs [14]. To our knowledge, this is the first report of the substrate specificity of BcPME1. In contrast to plant PMEs, BcPME1 from Botrytis cinerea was more active at acidic pH (4.5-6). This pH dependence has previously been observed for several fungal PMEs including Aspergillus flavus [29]. Interestingly, AtPME31, which was previously characterized as atypical with regard to the lack of inhibition by PMEI [32], showed features of both plant and pathogen enzymes. For instance, while AtPME31 was active at pH 7.5 like CsPME, its substrate specificity overlapped that of BcPME1 at pH 6. This could be related to the specific structural features of AtPME31, which was predicted to be a soluble protein (no signal peptide or transmembrane domain) and which showed only 30% identity with other Arabidopsis PMEs [32]. Although the role of AtPME31 remains to be determined, it could have a dual function, which has previously been shown for other Arabidopsis PMEs (AtPME18, At1g11580) [49]. The pH dependence and substrate specificity of BcPME1 is likely to be a key determinant of its pathogenicity [33]. BcPME1 was indeed active over a wide range of acidic pHs, which is the local pH conditions at the cell wall, and could target both randomly and blockwise methylesterified substrates with similar affinities.

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Previous studies have shown that plant PMEs can be inhibited by endogenous PMEIs while fungal PMEs cannot [46]. One of the key controls of fungal enzymes is related rather to the inhibition of their polygalacturonases by plant PGIPs [50,51]. In an attempt to uncover putative chemical inhibitors of plant and fungal PMEs, catechins extracted from green tea were used [28], and a chemical library of ~3600 compounds was tested. It has been reported that some non-proteinaceous compounds, such as iodine, detergent, tannins, phenolic acids, glycerol and epigallocatechin-3-gallate (EGCG) [16,28,29], can inhibit PMEs. Our results showed that the chemical compound used (PP60, EGCG and tannic acid) had different inhibitory capacities towards plant and fungal PMEs. For instance, 0.05 µg.µl-1 (109 µM) EGCG induced a 40% inhibition of AtPME31 and CsPME activities. In contrast, when considering BcPME1, only a weak inhibition (20%) was measured, at higher concentrations (from 1 µg.µl-1, or 2.18 mM). Plant PMEs were therefore more sensitive to this chemical compound compared to fungal enzymes. Recent results showed that the PME from Aspergillus could be inhibited by EGCG, but at concentrations in the range of 20 mg.ml-1 [29]. Overall, this confirms that fungal enzymes are likely to be equally sensitive to EGCG and that structural features of proteins might explain the differences in inhibitor sensitivity between plant and fungal enzymes. We hypothesized that the gallate group could be responsible for this inhibition since it is the most common one in the tested molecules (PP60, EGCG, TA). EGCG was shown to be a competitive inhibitor of Aspergillus PME [29]. The docking of EGCG onto Aspergillus and plant PMEs showed that gallate esters are likely to be key components of the inhibition of PME activity [28, 29]. Structural modelling demonstrated that the position of the gallate group could impair the entry of the substrate. The differences in the sensitivity of plant and fungal PMEs to EGCG might therefore reflect different binding affinities.

383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402

To investigate further the inhibition of PME by chemical compounds, the LATCA chemical library was used. This has previously been used to unravel some potential chemical tools to investigate plant development [41,52]. In addition, the use of a chemical library of compounds to identify novel enzyme inhibitors has proved useful on several enzymes, including a chitinase synthase from B. cinerea [53] and cyclopropane fatty acid synthase from E. coli [54]. Using this strategy, we identified more than one hundred compounds that showed inhibitory capacity towards plant PMEs. The molecules could be clustered into different families, including sulfonamides, naphthoquinones, amines, molecules with azole groups, aromatic rings and halogen atoms. Some of the compounds targeted both PMEs (i.e. 1-(3,4-dihydroxyphenyl)-2-{[1(1-naphthyl)-1H-tetrazol-5-yl]thio}, see Table 2) while others appeared specific for a given isoform (i.e. 2(3-chloro-2-fluorophenyl)-2,3-dihydroisothiazol-3-one for AtPME31). Overall, AtPME31 was less inhibited by the identified chemicals compared to CsPME. These results are in line with those observed using proteinaceous inhibitors, and shed new light on the putative specificity of the chemical inhibition depending on protein structure. Using the above-mentioned classification, it was found that CsPME was more specifically inhibited by hydrazones with carbothioamide groups and urea-type molecules. In contrast, for AtPME31, isothiazole groups and nitrile appeared specific for the inhibition. Thus, enzymes were diversely sensitive to the different compounds and ultimate 6-D6 was the strongest inhibitor. Further experiments, which could include docking analysis, would be needed to relate the difference in the compound’s structure to their inhibitory activities. Moreover, experiments with lower concentrations of 6-D6 and 31-G8 could be required to determine the limit concentration for an inhibition. Surprisingly, none of the chemical compounds active on plant PMEs appeared to inhibit fungal enzymes at this concentration.

403 404 405 406 407

In conclusion, the use of purified proteins from both plants (Arabidopsis thaliana and Citrus sinensis) and pathogenic fungi Botrytis cinerea has further highlighted the pH dependence and substrate specificity of PMEs. Our results shed new light on the diversity of these enzymes and their putative preferred substrates. This is of the utmost importance to understand how the pectin DM could be fine-tuned during plant development and in response to stress such as pathogens. Using a chemical library screen, we identified a

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number of novel inhibitors of PME activity that could be used for further analysis of these enzymes. This study brings new insight into the modulation of pectin methylesterification in planta and a new biotechnological application to regulate PME activity.

411 5. Acknowledgments

413 414 415

This work was supported by a grant from the Conseil Régional de Picardie, France and the European Regional Development Fund (PECTINHIB project) through a studentship awarded to ML’ and from the Agence National de la Recherche (GALAPAGOS project, ANR-12-BSV5-0001).

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[24] A. Raiola, L. Camardella, A. Giovane, B. Mattei, G. De Lorenzo, F. Cervone, Bellincampi D. Two Arabidopsis thaliana genes encode functional pectin methylesterase inhibitors, FEBS Lett. 557 (2004) 199– 203.

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[25] A.D. Matteo, A. Giovane, A. Raiola, L. Camardella, D. Bonivento, G.D. Lorenzo, F. Cervone, D. Bellincampi, D. Tsernoglou, Structural basis for the interaction between pectin methylesterase and a specific inhibitor protein, Plant Cell 17 (2005) 849–858.

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[28] K.C. Lewis, T. Selzer, C. Shahar, Y. Udi, D. Tworowski, I. Sagi Inhibition of pectin methyl esterase activity by green tea catechins, Phytochem. 69 (2008) 2586–2592.

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[29] X. Jiang, Q. Jia, L. Chen, Q. Chen, Q. Yang, Recombinant expression and inhibition mechanism analysis of pectin methylesterase from Aspergillus flavus, FEMS Microbiol. Lett. 355 (2014) 12–19.

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[30] J.A.E. Benen, G.J.W.M. Van Alebeek, A.G.J. Voragen Handbook of food enzymology. New York: Basel, 2003.

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[31] C.H Chen, M.C. Wu, C.Y. Hou, C.M. Jiang, C.M. Huang, Y.T. Wang, Effect of phenolic acid on antioxidant activity of wine and inhibition of pectin methyl esterase, J. Instit. Brew. 115 (2009) 328–333.

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[32] S. Dedeurwaerder, L. Menu-Bouaouiche, A. Mareck, P. Lerouge, F. Guerineau, Activity of an atypical Arabidopsis thaliana pectin methylesterase, Planta 229 (2009) 311–321.

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[33] I. Kars, M. McCalman, L. Wagemakers, J.A.L. van Kan Functional analysis of Botrytis cinerea pectin methylesterase genes by PCR-based targeted mutagenesis: Bcpme1 and Bcpme2 are dispensable for virulence of strain B05.10, Mol. Plant Pathol. 6 (2005) 641–652.

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[39] J.A. Klavons, R.D. Bennett, Determination of methanol using alcohol oxidase and its application to methyl ester content of pectins. J. Agric. Food Chem. 34 (1986) 597–599.

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[40] A. Tanhatan-Nasseri, M.J. Crépeau, J.F. Thibault, M.C. Ralet, Isolation and characterization of model homogalacturonans of tailored methylesterification patterns, Carbohydr. Polym. 86 (2011) 1236–1243.

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[41] Y. Zhao, T.F. Chow, R.S. Puckrin, S.E. Alfred, A.K. Korir, C.K. Larive, S.R. Cutler, Chemical genetic interrogation of natural variation uncovers a molecule that is glycoactived, Nat. Chem. Biol. 3 (2007) 716721.

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[51] F. Spinelli, L. Mariotti, B. Mattei, G. Salvi, F. Cervone, C. Caprari, Three aspartic acid residues of polygalacturonase-inhibiting protein (PGIP) from Phaseolus vulgaris are critical for inhibition of Fusarium phyllophilum PG, Plant Biol. 11 (2009) 738–743.

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[52] H. Abdel-Hamid, K. Chin, W. Moeder, K Yoshioka, High throughput chemical screening supports the involvement of Ca2+ in cyclic nucleotide-gated ion channel-mediated programmed cell death in Arabidopsis, Plant Signal. Behav. 6 (2011) 1817–1819.

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[53] H. Magellan, M. Boccara, T. Drujon, M.C. Soulié, C. Guillou, J. Dubois, H.F. Becker, Discovery of two new inhibitors of Botrytis cinerea chitin synthase by a chemical library screening, Bioorg. Med. Chem. 21 (2013) 4997–5003.

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[54] D. Guianvarc'h, E. Guangqi, T. Drujon, C. Rey, Q. Wang, O. Ploux, Identification of inhibitors of the E. coli cyclopropane fatty acid synthase from the screening of a chemical library: in vitro and in vivo studies. Biochim. Biophys. Acta 1784 (2008) 1652–1658

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14 Page 14 of 33

550

551

552

562

563

564

565

566

567

568

569

ip t

96

B series HG96B20

20

HG96B39

39

HG96B56

56

HG96B69

69

HG96B82

82

P series

cr

561

HG96

us

560

DM

an

559

HG

M

558

HG96P14

14

HG96P36

36

HG96P56

56

HG96P64

64

HG96P75

75

d

557

Table 1: HG models used for characterizing the substrate specificity of CsPME, AtPME31 and BcPME1. Randomly demethylesterified HG substrate (B series) were obtained following NaOH treatment of the mother pectin (HG96). Blockwise demethylesterified substrates (P series) were obtained following treatment with a plant PME [40]. DM: degree of methyl-esterification.

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570 Table 2: List of newly identified chemical inhibitors of PME activity. The 3650 chemical compounds of the LATCA chemical library were screened to identify new inhibitors of PME activity. DMSO was used as a control, and 109 compounds were identified as inhibitors of CsPME (25 compounds were presented in this table. For other, see supplementary data). Their inhibitory capacity was further screened on AtPME31. Results are the means of three replicates. % Inhibition CsPME

% Inhibition AtPME31

Silver nitrate

7761-88-8

100

100

7-A7

Copper sulfate

7758-98-7

7.9

31-F2

N-(1,4-dibenzoyl-5-phenyl-4,5-dihydro-1H-1,2,4triazol-3-yl)-benzamide

882282-61-3

58.1

28-H4

1-(3,6-dichloro-9H-carbazoyl-9-yl)-3morpholinopropan-2-ol

253448-97-4

56.0

34.7

31-H2

2-chloro-3-(2-methylphenoxy)-1,4-dihydronaphthalene-1,4-dione

134380-08-8

55.4

39.1

31-G8

2-(3-chloro-2-fluorophenyl)-2,3-dihydroisothiazol-3one

31-H4

9(10H)-Phenanthrenone, 10-(hydroxymethylene)-

45-B8

30.8

40.8

75.3

864162-30-1

33.9

40.9

Benzene, 1-(2-nitroethenyl)-4-(2-propenyloxy)-, (E)(9CI)

34209-93-3

32.9

41.5

45-B2

3H-Pyrazol-3-one, 2-[(4-chlorophenyl)sulfonyl]-2,4dihydro-4-(2-propen-1-yl)-5-propyl-

883014-35-5

30.4

40.2

18-C5

1-(3,4-dihydroxyphenyl)-2-{[1-(1-naphthyl)-1Htetrazol-5-yl]thio}

713111-08-1

71.0

58.9

41-E2

Hydrazine carbothioamide,2-(3,4-dihydro-1(2H)naphthalenylidene

3689-17-6

63.4

35.5

43-D4

N-(3,5-dichlorophenyl)-N’-[3-(2-furyl)-1H-pyrazol-5yl] urea

1030313-57-5

61.8

33.1

43-E3

4-(3-hydroxy-3-methylbut-1-ynyl)benzaldehyde-1phenylhydrazone

882308-14-7

61.1

30.6

3-D2

Phenylmercuric acetate

62-38-4

60.7

85.6

39-H10

2-methoxyacridin-9-amine

3407-99-6

60.3

31.7

46-B9

4-[1-(fur-2-oyl)pyrazol-5-yl]-5-methyl-1phenylpyrazole

95333-13-0

59.4

41.5

43-F2

N4-(2-furylmethyl)-2-(2,3-dihydro-1,4-benzodioxin2-yl)-1,3-thiazole-4-carboxamide

848782-30-9

59.2

26.2

46-H2

2-{2-[4-(trifluoromethyl)phenyl]-hydrazono}malonitrile

7089-17-0

58.5

38.1

d

M

220862-89-5

77.1

cr

us

6-D6

an

Name of molecule

ip t

CAS number Code

Ac ce pt e

571 572 573 574 575

16 Page 16 of 33

malonitrile N-(4-chloro-2-nitrophenyl)-N’-phenylurea

883022-11-5

57.0

42.2

43-E2

O5-(2-chlorobenzoyl)2,1,3-benzoxadiazole-5carbohydroximamide

882288-85-9

56.4

36.9

43-D3

N’1-[2-(tert-butyl)-5-(trifluoromethyl)-pyrazolo[1,5α]-pyrimidin-7-yl]-4-chlorobenzen-1-carbohydrazide

882306-79-8

56.1

34.5

45-G9

N’6-[3,5-di-(trifluoromethyl)phenyl]-5-oxo-2,3dihydro-5H-pyrimido-[2,1-b][1,3]thiazole-6carbohydrazide

254749-22-9

55.6

30.7

45-H9

N1-[3-(trifluoromethyl)phenyl]-3-(2-thienylthio)propanamide

255395-53-0

55.5

43-D6

[5-(4-chlorophenyl)-3-thienyl]-(piperidino)methanone

883018-92-6

55.2

41-D3

N-(4-methyl-2-thienyl)-N’-[4(trifluoromethyl)phenyl]-urea

676594-50-6

55.2

ip t

43-E6

us

cr

37.7

27.3

an

576

29.8

Ac ce pt e

d

M

577

17 Page 17 of 33

577 Table 3: Structure of the nine inhibitors and solvent (DMSO) screened for the dose-dependence inhibition of CsPME, AtPME31 and BcPME1. 31-F2 = N-(1,4dibenzoyl-5-phenyl-4,5dihydro-1H-1,2,4-triazol3-yl)-benzamide (Ambinter, France)

31-G8= 2-(3-chloro-2fluorophenyl)-2,3dihydroisothiazol-3-one (Maybridge, Great Britain)

O S

N N N

N

F

Cl

cr

O

O

ip t

DMSO = dimethylsulfoxide (Sigma, France)

NH

7-A7 = copper sulfate (Sigma, France)

Cu2+

d

M

an

us

O

45-B2 = 3H-Pyrazol-3-one, 2-[(4-chlorophenyl) sulfonyl]-2,4-dihydro-4-(2propen-1-yl)-5-propyl(Maybridge, Great Britain)

Ac ce pt e

578 579

O O

S

28-H4 = 1-(3,6-dichloro9H-carbazol-9-yl)-3morpholinopropan-2-ol (Maybridge, Great Britain)

O

Cl

O

O

O Cl

N S

N

31-H4 = 10-(hydroxymethylene) phenanthren-9(10H)-one (Maybridge, Great Britain)

O N

Cl

31-H2 = 2-chloro-3-(2methylphenoxy)-1,4dihydronaphthalene-1,4dione (Maybridge, Great Britain)

OH

O

N

45-B8 = 1-(allyloxy)-4(2-nitrovinyl)benzene (Maybridge, Great Britain)

O OH

O

O

N

O

O

Cl O

O

18 Page 18 of 33

6-D6 = silver nitrate (Sigma, France)

Ag+

O

N

O

O

ip t

580

Ac ce pt e

d

M

an

us

cr

581

19 Page 19 of 33

581 Figure captions

583 584 585 586 587 588 589

Fig. 1. Purification of pectin methylesterases (PMEs) from various organisms. (A) CsPME from Citrus sinensis was obtained commercially and further purified using ammonium sulfate precipitation. (B) AtPME31 was heterologously expressed in Escherichia coli and purified using Ni-NTA columns. (C) BcPME1 was expressed in Pichia pastoris as a c-myc tagged version and purified from the culture medium using ammonium sulfate precipitation. The purified proteins were resolved by SDS-PAGE and stained using Coomassie blue. Molecular mass marker proteins are in lane 1, unpurified PME in lane 2, purified PME in lane 3. The identity of the protein was confirmed using nano-LC/MS (see Fig. S1).

590 591 592 593

Fig. 2. Amino-acid sequence alignment of CsPME, AtPME31 and BcPME1. For CsPME, only the putative mature part (minus the PRO part) was used. For AtPME31, as it is predicted to be soluble, the full-length sequence was used. For BcPME1, the sequence minus the putative signal peptide was used. Sequences were aligned using ClustalW. Amino-acids involved in the catalytic site are box-shaded in gray.

594 595 596 597

Fig. 3. pH dependence of purified PMEs using commercial pectic substrates of various DMs. (A) CsPME, (B) AtPME31 and (C) BcPME1. Purified PME activities were assayed at three pHs ( : pH 4.0; : pH 6.0; : pH 7.5) using commercial substrates of various DMs (30%, 65% and 90%). Data represent mean values ± SD from 3 samples. Assays were performed according to method adapted from Anthon & Barett [38].

598 599 600 601 602 603 604

Fig. 4. Substrats specificity and pH dependence of the three PMEs using tailor-made HG substrates of different DMs and patterns of methylesterification. (A) CsPME, (B) AtPME31 and (C) BcPME1. PME activities were assayed at three pHs ( : pH4.0; : pH6.0; : pH7.5) on HGs with different patterns and degrees of methylation. HG96, the mother pectin, was chemically (B-series) or enzymatically (P-series) demethylesterified to reach a specific degree of methylesterification (DM). The characteristics of the various substrates are shown in Table 1. Data represent mean values ± SD from 3 samples. Assays were performed according to method adapted from Anthon and Barett [38].

605 606 607 608

Fig. 5. Inhibition of PME activities by polyphenosl (š: PP60; +: EGCG; ¯: TA). (A) CsPME, (B) AtPME31 and (C) BcPME1. PME activities assayed on pectic substrate (DE 90%) at pH 7.5 for CsPME and AtPME31 and at pH 6.0 for BcPME1. Data represent mean values ± SD from 3 samples. Assays were performed according to method adapted from Klavons and Bennett [39].

609 610 611 612 613 614

Fig. 6. Inhibition of PME activities by newly identified chemical inhibitors at different concentrations. ( -0 µM; -10 µM; -50 µM; -100 µM; -250 µM; -500 µM; -1000 µM; -1500 µM). (A) CsPME, (B) AtPME31, (C) BcPME1. Purified PME activities assayed on pectic substrate (DE 90%) at pH 7.5 for CsPME and AtPME31 and pH 6 for BcPME1 with or without chemical compounds. DMSO was used as a control. 31-H4, 31-H2 and 45-B8 were not tested for BcPME1. Data represent mean values ± SD from 3 samples. Assays were performed according to from Klavons and Bennett [39].

Ac ce pt e

d

M

an

us

cr

ip t

582

615

616

617

Highlights

618

• Characterization of PME from Citrus sinensis, Arabidopsis thaliana, Botrytis cinerea.

619

• Substrate specificity and pH dependence using tailor-made pectic substrates. 20 Page 20 of 33

620

• Inhibition of PME by characterized polyphenols

621

• Identification of novel inhibitors using a chemical library

622

623 >CsPME

625 626 627 628

QATTVVPDVTVAADGSGNYLTVAAAVAAAPEGSSRRYIIRIKAGEYRENVEVPKKKINLMFIGDGRTTTIITGSRNVVDGSTTFNSATV AVVGDGFLARDITFQNTAGPSKHQAVALRVGSDLSAFYRCDMLAYQDTLYVHSLRQFYTSCIIAGTVDFIFGNAAAVFQNCDIHARRP NPNQRNMVTAQGRDDPNQNTGIVIQKCRIGATSDLLAVKGSFETYLGRPWKRYSRTVVMQSDISDVINPAGWYEWSGNFALDTLFY AEYQNTGAGADTSNRVKWSTFKVITSAAEAQTYTAANFIAGSTWLGSTGFPFSLGL

ip t

624

629 >AtPME31

631 632 633 634

MATTRMVRVSQDGSGDYCSVQDAIDSVPLGNTCRTVIRLSPGIYRQPVYVPKRKNFITFAGISPEITVLTWNNTASKIEHHQASRVIGT GTFGCGSVIVEGEDFIAENITFENSAPEGSGQAVAIRVTADRCAFYNCRFLGWQDTLYLHHGKQYLKDCYIEGSVDFIFGNSTALLEH CHIHCKSQGFITAQSRKSSQESTGYVFLRCVITGNGQSGYMYLGRPWGPFGRVVLAYTYMDACIRNVGWHNWGNAENERSACFY EYRCFGPGSCSSERVPWSRELMDDEAGHFVHHSFVDPEQDRPWLCLRMGVKTPYSA

us

cr

630

an

635 >BcPME1

637 638 639 640

MYSLIPILSFAATLLGAVSAAPLEARAVSRTSAPSGAVIVDATGKTAGSYTTFQKGVNALSTTTTTPQYLFIYPGTYTEQVYVPALNSNL TIQGYTTDASTYAGNQVTLTYNLALKDTTSDDLTATLRQWNKNTKVYNLIIQNTFGHISSNGQNLAISAHTTNQGYYATQFIGYQDTIL ANTGTQLYAKCLVVGAIDFIFGQTAQAWFENNDIRTIAAGSITASGRADDSNPSWYVINNSNIQNINSSVATGNNYLGRPWRNYARVV FQNSYLGNNIKAAGWSVWSSSTANTDHVVFEEYGNTGPGSNSSGVQRATFSSGISKAIPITTVLGSAYLNEWWVDSSYL

643 644

Supplementary data figure 1 : Amino-acid sequence of CsPME, AtPME31 and BcPME1. Underlined sequences correspond to peptides identified after proteomics sequencing.

645

646

Ac ce pt e

642

d

641

M

636

21 Page 21 of 33

646 Supplementary Table S2: List of newly identified chemical inhibitors of PME activity. The 3650 chemical compounds of the LATCA chemical library were screened for identifying new inhibitors of PME activity. DMSO was used as a control, and 109 compounds were identified as inhibitors of CsPME. Their inhibitory capacity was further screened on AtPME31. Results are the means of three replicates. % Inhibition CsPME

% Inhibition AtPME31

Silver nitrate

7761-88-8

100.0

100.0

7-A7

Copper sulfate

7758-98-7

7.9

31-F2

N-(1,4-dibenzoyl-5-phenyl-4,5-dihydro-1H-1,2,4triazol-3-yl)-benzamide

882282-61-3

58.1

28-H4

1-(3,6-dichloro-9H-carbazoyl-9-yl)-3morpholinopropan-2-ol

253448-97-4

56.0

31-H2

2-chloro-3-(2-methylphenoxy)-1,4-dihydronaphthalene-1,4-dione

134380-08-8

55.4

39.1

31-G8

2-(3-chloro-2-fluorophenyl)-2,3-dihydroisothiazol-3one

220862-89-5

40.8

75.3

31-H4

9(10H)-Phenanthrenone, 10-(hydroxymethylene)-

864162-30-1

33.9

40.9

45-B8

Benzene, 1-(2-nitroethenyl)-4-(2-propenyloxy)-, (E)(9CI)

34209-93-3

32.9

41.5

45-B2

3H-Pyrazol-3-one, 2-[(4-chlorophenyl)sulfonyl]-2,4dihydro-4-(2-propen-1-yl)-5-propyl-

883014-35-5

30.4

40.2

18-C5

1-(3,4-dihydroxyphenyl)-2-{[1-(1-naphthyl)-1H-tetrazol5-yl]thio}

713111-08-1

70.9

58.9

41-E2

Hydrazine carbothioamide, 2-(3,4-dihydro-1(2H)naphthalenylidene

3689-17-6

63.4

35.5

43-D4

N-(3,5-dichlorophenyl)-N’-[3-(2-furyl)-1H-pyrazol-5-yl] urea

1030313-57-5

61.8

33.1

43-E3

4-(3-hydroxy-3-methylbut-1-ynyl)benzaldehyde-1phenylhydrazone

882308-14-7

61.1

30.6

3-D2

Phenylmercuric acetate

62-38-4

60.7

85.6

39-H10

2-methoxyacridin-9-amine

3407-99-6

60.3

31.7

46-B9

4-[1-(fur-2-oyl)pyrazol-5-yl]-5-methyl-1-phenylpyrazole

95333-13-0

59.4

41.5

43-F2

N4-(2-furylmethyl)-2-(2,3-dihydro-1,4-benzodioxin-2yl)-1,3-thiazole-4-carboxamide

848782-30-9

59.2

26.2

46-H2

2-{2-[4-(trifluoromethyl)phenyl]-hydrazono}-malonitrile

7089-17-0

58.5

38.1

43-E6

N-(4-chloro-2-nitrophenyl)-N’-phenylurea

883022-11-5

57.0

42.2

43-E2

O5-(2-chlorobenzoyl)2,1,3-benzoxadiazole-5carbohydroximamide

882288-85-9

56.4

36.9

77.1

d

30.8

cr

us

an

6-D6

M

Name of molecule

ip t

CAS number

Code

Ac ce pt e

647 648 649 650 651

34.7

22 Page 22 of 33

N’1-[2-(tert-butyl)-5-(trifluoromethyl)-pyrazolo[1,5-α]pyrimidin-7-yl]-4-chlorobenzen-1-carbohydrazide

882306-79-8

56.1

34.5

45-G9

N’6-[3,5-di-(trifluoromethyl)phenyl]-5-oxo-2,3-dihydro5H-pyrimido-[2,1-b][1,3]thiazole-6-carbohydrazide

254749-22-9

55.6

30.7

45-H9

N1-[3-(trifluoromethyl)phenyl]-3-(2-thienylthio)propanamide

255395-53-0

55.5

29.8

43-D6

[5-(4-chlorophenyl)-3-thienyl]-(piperidino)-methanone

883018-92-6

55.2

37.7

41-D3

N-(4-methyl-2-thienyl)-N’-[4-(trifluoromethyl)phenyl]urea

676594-50-6

55.2

27.3

18-H10

1-(1,3-benzodioxol-5-yl)-3-(pyridine-2-yl-methyl)-urea

708215-45-6

54.7

31.5

28-H3

N1-[3-(2,3-diphenyl-1H-inden-1-yliden)-prop-1-enyl]N1-methyl-aniline

2473-61-2

53.4

28-F6

2,5-dibromo-3,6-dimethylbenzo-1,4-quinone

28293-38-1

53.1

28-F4

1,3,6-tribromo-9H-carbazole

55119-10-3

53.1

28-H11

N1-(2,4-difluorophenyl)-2,3,4,5,6-pentamethylbenzene -1-sulfonamide

661475-79-2

52.6

46.9

35-G5

N-benzoyl-N’-{4-[2-(2-chlorophenyl)-diaz-1-enyl]-3cyclopropyl-1H-pyrazol-5-yl-}-thiourée

1239978-22-3

52.6

37.4

41-F11

2-(2,3-dihydro-1,4-benzodioxin-6-yl)-6-methylimidazol[1,2-a]-pyridine

702636-92-8

43-B4

3-phenoxythiophene-2-carbaldehyde-2-(4chlorophenyl)-hydrazone

43-B5

N-[3-chloro-2-isopropylthio)-phenyl]-N’-phenylurea

43-D5

ip t

43-D3

49.6

an

us

cr

47.6

32.0

33.0

882314-76-3

51.8

27.3

8830-50-5

51.8

26.8

N-(3,5-dichlorophenyl)-N’-{2-[(2-furyl-methyl)thio]ethyl}-urea

883025-44-3

50.9

32.5

29-H6

7-(4-chlorobenzylidène)-3-(4-chlorophenyl)3,3a,4,5,6,7-hexahydro-2H-indazole

99160-04-0

49.7

39.5

42-C8

Benzenepropanenitrile, α-[bis(methylthio)methylene]2-chloro-β-oxo-

175137-51-6

36.2

53.1

43-F4

3-(dimethylamino)-1-[4-(2-phenyleth-1-ynyl)phenyl] prop-2-en-1-one

883002-66-2

49.4

36.4

45-H5

2,6-Piperidinedione, 4-(2,6-dichlorophenyl)-1-[3(methylthio)phenyl]

883014-94-6

48.9

39.5

46-A9

1H-1,2,4-Triazole, 3-[3,5-bis(trifluoromethyl)phenyl]-5(methylthio)-

261511-26-6

48.5

33.7

31-F11

2,1,3-Benzoxadiazole, 4-[[(4-methoxyphenyl) methyl]thio]-7-nitro-

882289-31-8

48.3

38.0

46-C9

1H-1,2,4-Triazole, 3-(3,5-dichlorophenyl)-5-[[2-nitro-4(trifluoromethyl)phenyl]thio]-

261714-89-0

48.1

32.1

14-C2

1-Piperidinecarbothioamide, N-(4-chloro-2methylphenyl)-4-(hydroxydiphenylmethyl)-

516459-85-1

46.7

28.2

22-D7

9H-Indeno[1,2-d][1,2,4]triazolo[1,5-a]pyrimidin-9-one, 10-(4-methoxyphenyl)-

500268-01-9

46.3

24.6

Ac ce pt e

d

M

51.9

23 Page 23 of 33

10-(4-methoxyphenyl)2-Furancarboxylic acid, 5-nitro-, 2-(2,6-dichlorophenyl) hydrazide

261626-73-7

46.3

33.4

14-C4

3,3,6,6-tetramethyl-9-(3-nitrophenyl)-3,4,5,6,7,9hexahydro-1H-xanthene-1,8(2H)-dione

40588-50-9

45.9

27.0

28-E4

1,2-Propanediol, 3-(3,6-dibromo-9H-carbazol-9-yl)-

173157-92-1

45.4

28.7

43-C4

Urea, N-(3,5-dichlorophenyl)-N'-[5-(2-thienyl)-1Hpyrazol-3-yl]-

1030313-55-3

45.3

23.8

43-C3

2-Furancarbothioic acid, 5-[(4-bromo-3,5-dimethyl-1Hpyrazol-1-yl)methyl]-, S-[5-(trifluoromethyl)-2-pyridinyl] ester

957332-27-3

44.4

34.8

31-E11

2,1,3-Benzoxadiazole, 4-[(2,4-dichlorophenyl)thio]-7nitro-

882289-29-4

44.1

28-H10

1H-2-Benzothiopyran-4(3H)-one, O-[2,6-dinitro-4(trifluoromethyl)phenyl]oxime

658039-99-7

43.9

41-E3

Benzaldehyde, 2-chloro-, 2-(5-methylthieno[2,3d]pyrimidin-4-yl)hydrazone

676997-25-4

43.5

26.6

45-A7

4-Thiazolidinone, 2,3-bis[4-(trifluoromethyl)phenyl]-

883030-39-5

43.4

39.7

45-H6

4-Thiazolidinone, 3-(2,4-difluorophenyl)-5-methyl-2-(3pyridinyl)-

75741-54-7

43.4

41.3

29-G5

Thiourea, N-(3-chloro-4-methylphenyl)-N'-(4-chloro-2nitrophenyl)-

671754-94-2

43.3

42.6

47-F4

Hydrazinecarbothioamide, 2-[1-[4-[(3,5-dichloro-4pyridinyl)oxy]phenyl]ethylidene]-

583059-54-5

43.1

32.7

14-A9

Benzeneacetic acid, α-hydroxy-α-methyl-, 2-[(2hydroxy-1-naphthalenyl)methylene]hydrazide

413617-69-3

43.0

29.6

28-G4

1,2-Propanediol, 3-(1,3,6-tribromo-9H-carbazol-9-yl)-

173157-93-2

42.4

37.0

46-E9

Benzoic acid, 3,5-dichloro-, 2-(4-iodophenyl)hydrazide

261626-74-8

41.3

30.8

31-H9

Urea, N-(4-chlorophenyl)-N'-[(3-chloro-4,5,6,7tetrahydro-5,5-dimethyl-7-oxobenzo[c]thien-1-yl) methyl-λ4-sulfanylidene]-

1005958-07-5

41.3

43.2

45-B4

Methanediamine, 1-phenyl-N,N'-bis(phenylmethylene) -

92-29-5

41.2

39.5

14-G5

Benzenesulfonamide, N-(4-bromo-3-methylphenyl)-4methyl-3-nitro-

367960-27-8

40.8

32.1

45-A3

1,3,4-Thiadiazole-2(3H)-thione, 5-[(4-iodophenyl) amino]-

50796-24-2

40.1

32.7

42-B8

Acetonitrile, 2-[2-(2-chlorophenyl)hydrazinylidene]-2[(4-chlorophenyl)sulfonyl]-

882278-42-4

40.0

34.2

29-F5

Thiazole, 5-[[2,6-dinitro-4-(trifluoromethyl)phenyl]thio]4-[(4-methylphenyl)sulfonyl]-

671754-87-3

39.7

37.2

31-H8

Thiocyanic acid, 2-(4-chlorophenyl)-2-oxoethyl ester

19339-59-4

39.3

37.8

ip t

46-D9

cr

us

an

M

d

Ac ce pt e

41.6

39.7

24 Page 24 of 33

3H-1,2,4-Triazol-3-one, 2,4-dihydro-4-(2-methylbutyl)5-[(5-nitro-2-thiazolyl)thio]-

883061-26-5

39.1

35.6

22-C7

9H-Indeno[1,2-d][1,2,4]triazolo[1,5-a]pyrimidin-9-one, 10-(4-fluorophenyl)-

488716-08-1

38.0

23.1

29-H5

Urea, N-(4-chlorophenyl)-N'-(5-nitro-3-thienyl)-

672286-02-1

37.7

42.9

45-A2

Hydrazinecarbothioamide, 2-[(2-hydroxyphenyl) methylene]-N-(2-methoxyphenyl)-

76628-63-2

36.5

43.5

41-F3

Benzenesulfonamide, N-[4-(4-fluorophenoxy)phenyl]4-methyl-

677276-96-9

35.8

32.7

45-A8

2H-1,3,5-Thiadiazine-2-thione, tetrahydro-3,5-bis[(4methoxyphenyl)methyl]-

58078-78-7

33.9

38.5

47-H7

Carbamic acid, (3,5-dichlorophenyl)-, 1-methylethyl ester (9CI)

2150-29-0

33.8

42-E4

Benzothiazole, 2-[[(2-nitrophenyl)methyl]sulfonyl]-

882282-96-4

32.5

37.5

29-G3

3-Thiophenesulfonamide, 2,5-dichloro-N-[2-(4chlorophenyl)ethyl]-

667399-49-7

32.5

33.2

26-H2

Acetic acid, 2-[2-[[2-(1,2-dihydro-1-methyl-2-oxo-3Hindol-3-ylidene)hydrazinylidene]methyl]phenoxy]-

304652-47-9

32.5

31.3

26-G2

9,10-Anthracenedione, 1,2,3-trichloro-4(dimethylamino)-

15160-47-1

32.4

21.6

47-D6

Acetamide, 2-[2-(4-chloro-2-methoxyphenyl) hydrazinylidene -2-cyano-

883050-59-7

32.3

42.3

43-B6

4-Quinolinamine, 7-chloro-N-(3-fluoro-4methylphenyl)-

883016-87-3

31.8

28.7

22-E8

Acetamide, 2-[[4-(4-methoxyphenyl)-5-phenyl-4H1,2,4-triazol-3-yl]thio]-N-(phenylmethyl)-

675194-48-6

31.8

23.7

19-F6

1H-Benzimidazol-5-amine, 1-cyclohexyl-N-(2pyridinylmethyl)-, hydrochloride (1:2)

1049719-36-9

31.7

23.2

42-D8

Benzenepropanenitrile, α-[bis(methylthio)methylene]3-methoxy-β-oxo-

882280-92-4

30.5

39.5

22-G8

Acetamide, 2-[[4-(4-fluorophenyl)-5-phenyl-4H-1,2,4triazol-3-yl]thio]-N-[(4-methoxyphenyl)methyl]-

702649-31-8

30.0

23.5

43-C6

Methanone, [4-(7-chloro-4-quinolinyl)-1-piperazinyl]-2naphthalenyl-

883017-00-3

29.9

32.5

29-H3

3(2H)-Thiophenone, dihydro-, O-[[(4chlorophenyl)amino]carbonyl]oxime (9CI)

667866-23-1

29.6

36.0

19-A6

Acetamide, 2-[2-methyl-4[(propylamino)sulfonyl]phenoxy]-N-(2-pyridinylmethyl)-

878436-19-2

28.9

15.2

19-G6

N-[(benzyloxy)carbonyl]-N-(2-pyridinylmethyl) phenylalaninamide

1009244-43-2

28.9

18.6

22-A6

Acetamide, 2-[[5-(3-chlorophenyl)-4-phenyl-4H-1,2,4triazol-3-yl]thio]-N-(phenylmethyl)-

305376-22-1

27.5

12.0

42-E6

4-(2-pyridin-2-yldiazenyl)benzene-1,3-diol

1141-59-9

27.1

40.2

ip t

47-F5

cr

us

an

M

d

Ac ce pt e

36.9

25 Page 25 of 33

2-Thiophenecarboxylic acid, 4-(1,3-dithiolan-2-yl) phenyl ester

261928-91-0

26.6

37.0

22-D8

Acetamide, 2-[[5-(4-chlorophenyl)-4-phenyl-4H-1,2,4triazol-3-yl]thio]-N-(phenylmethyl)-

560126-48-9

26.4

19.1

19-C3

Benzo[b]thiophene-3-carboxylic acid, 4,5,6,7tetrahydro-2-[[2-oxo-2-[(2-pyridinylmethyl) amino]acetyl]amino]-, ethyl ester

839685-14-2

26.1

18.7

22-C8

Acetamide, 2-[[5-(4-chlorophenyl)-4-(3,4dimethylphenyl -4H-1,2,4-triazol-3-yl]thio]-N(phenylmethyl)-

669697-20-5

25.8

25.5

19-H5

9H-Carbazole-3-methanamine, 9-ethyl-N-(2pyridinylmethyl)-, hydrochloride (1:1)

1030021-40-9

24.4

12.3

42-F6

2-Propenamide, N-(3-chlorophenyl)-3-(4chlorophenyl)-, (2E)-

959154-31-5

22.3

19-H7

Acetamide, 2-[4-(4-morpholinylsulfonyl)phenoxy]-N-(2pyridinylmethyl)-

831242-58-1

22.1

17-H11

4(3H)-Pyrimidinone, 2-[[2-[3,5-bis(1,1-dimethylethyl)4-hydroxyphenyl]-2-oxoethyl]thio]-6-methyl-

700851-15-6

20.4

12.3

41-G3

1H-Pyrazole-4-carboxylic acid, 1-(4-nitrophenyl)-5(trifluoromethyl)-, [(4-chlorophenyl)iminomethyl]azanyl ester

677277-19-9

18.6

36.0

19-F5

Benzenemethanol, α-[1-[(2-pyridinylmethyl) amino]ethyl]-, hydrochloride (1:1)

1049713-17-8

17.6

20.6

22-A7

Acetamide, 2-[[5-(3-methoxyphenyl)-4-phenyl-4H1,2,4-triazol-3-yl]thio]-N-(1-phenylethyl)-

345988-64-9

17.1

9.8

19-H9

Pyrazolo[1,5-a]pyrimidine-2-carboxamide, 4,5,6,7tetrahydro-5-phenyl-N-(2-pyridinylmethyl)-7(trifluoromethyl)-

830349-36-5

11.9

14.7

16-H6

3-Pyridinecarbonitrile, 2-[[2-(3,4-dihydroxyphenyl)-2oxoethyl]thio]-4-(2-furanyl)-6-methyl-

337500-83-1

11.7

21.5

17-C6

Acetamide, N-[(5-benzoyl-2-hydroxyphenyl)methyl]-2[[4-methyl-5-(phenylmethyl)-4H-1,2,4-triazol-3-yl]thio]-

690651-65-1

10.5

10.7

17-E6

2-Propen-1-one, 3-(2-fluorophenyl)-1-(2-hydroxy-4methoxyphenyl)-

488852-01-3

10.4

20.5

19-E3

Benzamide, 4-(acetylamino)-5-chloro-2-methoxy-N-(2pyridinylmethyl)-

876883-95-3

10.3

14.5

47-F8

Benzenesulfonamide, N-[4-(3-chloro-4-methylphenyl)5-methyl-2-thiazolyl]-4-methyl-

883050-73-5

9.6

32.3

47-E5

1H-Pyrazole-3-carboxamide, N-(4-chlorophenyl)-1(1,1-dimethylethyl)-5-methyl-

353504-90-2

9.1

34.6

19-G5

3-Pyrrolidinecarboxamide, 1-[2-(4-fluorophenyl)ethyl]5-oxo-N-(2-pyridinylmethyl)-

879597-98-5

3.3

23.4

47-F7

4-(2-methylbutyl)-5-[(5-nitro-1,3-thiazol-2-yl)thio]-4H1,2,4-triazol-3-ol

883061-26-5

1.3

39.9

ip t

46-G9

cr

us

an

M

d

Ac ce pt e

35.8

14.0

652 26 Page 26 of 33

653

654

Ac ce pt e

d

M

an

us

cr

ip t

655

27 Page 27 of 33

Ac

ce

pt

ed

M

an

us

cr

i

Figure 1

Page 28 of 33

Figure 2

----------QATTVVPDVTVAADGSGNYLTVAAAVAAAPEGSSRRYIIRIKAGEYRENV 50 ------------MATTRMVRVSQDGSGDYCSVQDAIDSVPLGNTCRTVIRLSPGIYRQPV 48 PLEARAVSRTSAPSGAVIVDATGKTAGSYTTFQKGVNALSTTTTTPQYLFIYPGTYTEQV 60 : . * .: . :*.* :. .: : . .: : : .* * : *

CsPME1 AtPME31 BcPME1

EVPKKKINLMFIGDGRTTTIITGS----------RNVVDGSTTFNSATVAVVGDGFLARD 100 YVPKRKNFITFAGISPEITVLTWNNTASKIEHHQASRVIGTGTFGCGSVIVEGEDFIAEN 108 YVPALNSNLTIQGYTTDASTYAGNQVTLTYN---LALKDTTSDDLTATLRQWNKNTKVYN 117 ** : : : * : : . : .:: ... . :

CsPME1 AtPME31 BcPME1

ITFQNTAG--PSKHQAVALRVGSDLSAFYRCDMLAYQDTLYVHSLRQFYTSCIIAGTVDF 158 ITFENSAP--EGSGQAVAIRVTADRCAFYNCRFLGWQDTLYLHHGKQYLKDCYIEGSVDF 166 LIIQNTFGHISSNGQNLAISAHTTNQGYYATQFIGYQDTILANTGTQLYAKCLVVGAIDF 177 : ::*: .. * :*: . : .:* ::.:***: : * .* : *::**

CsPME1 AtPME31 BcPME1

IFGN-AAAVFQNCDIHARRPNPNQRNMVTAQGRDDPNQNTGIVIQKCRIGATSDLLAVKG 217 IFGN-STALLEHCHIHCK-----SQGFITAQSRKSSQESTGYVFLRCVITGN------GQ 214 IFGQTAQAWFENNDIRTI-----AAGSITASGRADDSNPSWYVINNSNIQNIN---SSVA 229 ***: : * ::: .*: . :**..* . .: : *: .. *

CsPME1 AtPME31 BcPME1

SFETYLGRPWKRYSRTVVMQSDISDVINPAGWYEWSGNFA-LDTLFYAEYQNTGAGADTS 276 SGYMYLGRPWGPFGRVVLAYTYMDACIRNVGWHNWGNAEN-ERSACFYEYRCFGPGSCSS 273 TGNNYLGRPWRNYARVVFQNSYLGNNIKAAGWSVWSSSTANTDHVVFEEYGNTGPGSNSS 289 : ****** :.*.*. : :. *. .** *.. : ** *.*: :*

CsPME1 AtPME31 BcPME1

NRVKWSTFKVITSAAEAQTYTAANFIAGSTWLGS-TGFPFSLGL 319 ERVPWSRELMDDEAGHFVHHSFVDPEQDRPWLCLRMGVKTPYSA 317 G-VQRATFSSGISKAIPITTVLGSAYLNEWWVDSSYL------- 325

Ac

ce pt

ed

M

an

us

cr

ip t

CsPME1 AtPME31 BcPME1

Page 29 of 33

Figure 3

5

A

4 3 2

ip t

nmol MeOH.min-1.µg-1 of proteins

6

1 0

2

us

2.5

B

an

1.5 1 0.5 0 DM 30%

DM 65% Pectic substrates

DM 90%

d

1

C

te

0.8 0.6 0.4

Ac ce p

nmol MeOH.min-1.µg-1 of proteins

DM 90%

cr

DM 65% Pectic substrates

M

nmol MeOH.min-1.µg-1 of proteins

DM 30%

0.2 0

DM 30%

DM 65%

DM 90%

Pectic substrates

Page 30 of 33

6

A

5 4 3

1 0

1.2

B

us

1.4

1 0.8 0.6 0.4

an

nmol MeOH.min-1.µg-1 of proteins

Homogalacturonan substrates

ip t

2

cr

nmol MeOH.min-1.µg -1 of proteins

Figure 4

0.2

M

0

Homogalacturonan substrates

nmol MeOH.min-1.µg -1 of proteins

0.6

C

0.3 0.2

Ac ce p

0.1

te

0.4

d

0.5

0

Homogalacturonan substrates

Page 31 of 33

Figure 5

A

80

60 40 20

ip t

Inhibition (%)

100

0

B

80

us

60

40 20

an

Inhibition (%)

100

0.01 0.05 0.1 0.2 0.5 0.7 1 Inhibitor concentration (µg.µl -1)

cr

0

0 0

1

M

C

80 60

d

40

20 0

0.01 0.05 0.1 0.2 0.5 0.7 Inhibitor concentration (µg.µl-1)

1

Ac ce p

0

te

Inhibition (%)

100

0.01 0.05 0.1 0.2 0.5 0.7 Inhibitor concentration (µg.µl-1)

Page 32 of 33

3.5

A

3 2.5 2

ip t

1.5 1 0.5

cr

nmol MeOH.min-1.µg-1 of proteins

Figure 6

0 None DMSO 31-F2 31-G8

7-A7

45-B2 28-H4 31-H4 31-H2 45-B8

B

1.2

an

1 0.8 0.6 0.4 0.2 0 None DMSO 31-F2 31-G8

M

nmol MeOH.min-1.µg-1 of proteins

1.4

us

Chemical compounds

6-D6

7-A7

45-B2 28-H4 31-H4 31-H2 45-B8

6-D6

0.12 0.1

d

C

te

0.14

Ac ce p

nmol MeOH.min-1.µg-1 of proteins

Chemical compounds

0.08 0.06 0.04 0.02 0

None DMSO 31-F2 31-G8 7-A7 45-B2 28-H4 31-H4 31-H2 45-B8 Chemical compounds

6-D6

Page 33 of 33