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Tuning of Pectin Methylesterification

PECTIN METHYLESTERASE INHIBITOR 7 MODULATES THE PROCESSIVE ACTIVITY OF CO-EXPRESSED PECTIN METHYLESTERASE 3 IN A pH-DEPENDENT MANNER*
  • Fabien Sénéchal
    Affiliations
    EA3900-BIOPI, Biologie des Plantes et Innovation, Université de Picardie Jules Verne, 80039 Amiens, France
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  • Mélanie L'Enfant
    Affiliations
    EA3900-BIOPI, Biologie des Plantes et Innovation, Université de Picardie Jules Verne, 80039 Amiens, France
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  • Jean-Marc Domon
    Affiliations
    EA3900-BIOPI, Biologie des Plantes et Innovation, Université de Picardie Jules Verne, 80039 Amiens, France
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  • Emeline Rosiau
    Affiliations
    EA3900-BIOPI, Biologie des Plantes et Innovation, Université de Picardie Jules Verne, 80039 Amiens, France
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  • Marie-Jeanne Crépeau
    Affiliations
    INRA, UMR 1268, Biopolymères-Interactions-Assemblages, BP 71627, 44316 Nantes, France
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  • Ogier Surcouf
    Affiliations
    Laboratoire de Glycobiologie et Matrice Extracellulaire Végétale, UPRES EA 4358, Institut de Recherche et d'Innovation Biomédicale, Grand Réseau de Recherche-Végétal, Agronomie, Sol, Innovation, UFR des Sciences et Techniques, Normandie Université-Université de Rouen, 76821 Mont-Saint-Aignan Cedex 1, France
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  • Juan Esquivel-Rodriguez
    Affiliations
    Departments of Computer Sciences, Purdue University, West Lafayette, Indiana 47907
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  • Paulo Marcelo
    Affiliations
    Plateforme d'Ingénierie Cellulaire and Analyses des Protéines (ICAP), Université de Picardie Jules Verne, 80039 Amiens, France
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  • Alain Mareck
    Affiliations
    Laboratoire de Glycobiologie et Matrice Extracellulaire Végétale, UPRES EA 4358, Institut de Recherche et d'Innovation Biomédicale, Grand Réseau de Recherche-Végétal, Agronomie, Sol, Innovation, UFR des Sciences et Techniques, Normandie Université-Université de Rouen, 76821 Mont-Saint-Aignan Cedex 1, France
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  • François Guérineau
    Affiliations
    EA3900-BIOPI, Biologie des Plantes et Innovation, Université de Picardie Jules Verne, 80039 Amiens, France
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  • Hyung-Rae Kim
    Affiliations
    Biological Sciences, Purdue University, West Lafayette, Indiana 47907
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  • Jozef Mravec
    Affiliations
    Department of Plant and Environmental Sciences, University of Copenhagen, 1871 Frederiksberg, Denmark
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  • Estelle Bonnin
    Affiliations
    INRA, UMR 1268, Biopolymères-Interactions-Assemblages, BP 71627, 44316 Nantes, France
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  • Elisabeth Jamet
    Affiliations
    LRSV, UMR 5546 Université Toulouse 3/CNRS, 31326 Castanet-Tolosan, France
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  • Daisuke Kihara
    Footnotes
    Affiliations
    Departments of Computer Sciences, Purdue University, West Lafayette, Indiana 47907

    Biological Sciences, Purdue University, West Lafayette, Indiana 47907
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  • Patrice Lerouge
    Affiliations
    Laboratoire de Glycobiologie et Matrice Extracellulaire Végétale, UPRES EA 4358, Institut de Recherche et d'Innovation Biomédicale, Grand Réseau de Recherche-Végétal, Agronomie, Sol, Innovation, UFR des Sciences et Techniques, Normandie Université-Université de Rouen, 76821 Mont-Saint-Aignan Cedex 1, France
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  • Marie-Christine Ralet
    Affiliations
    INRA, UMR 1268, Biopolymères-Interactions-Assemblages, BP 71627, 44316 Nantes, France
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  • Jérôme Pelloux
    Footnotes
    Affiliations
    EA3900-BIOPI, Biologie des Plantes et Innovation, Université de Picardie Jules Verne, 80039 Amiens, France
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  • Catherine Rayon
    Correspondence
    To whom correspondence should be addressed: EA3900-BIOPI, Biologie des Plantes et Innovation Université de Picardie Jules Verne, 80039 Amiens, France. Tel.: 33-322-827-536.
    Affiliations
    EA3900-BIOPI, Biologie des Plantes et Innovation, Université de Picardie Jules Verne, 80039 Amiens, France
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  • Author Footnotes
    * This work was supported by Agence Nationale de la Recherche Grants ANR-09-BLANC-0007-01 GROWPEC and ANR-12-BSV5-0001 GALAPAGOS and by the Conseil Régional de Picardie through a Ph.D. studentship (to F. S.). Work was also supported in part by the “Trans Channel Wallnet” project, which was selected by the INTERREG IVA program France (Channel)-England European cross-border cooperation program and National Institutes of Health Grant R01GM097528 (to D. K.). The authors declare that they have no conflicts of interest with the contents of this article.
    1 Supported in part by National Science Foundation Grants IIS1319551, DBI1262189, and IOS1127027 and National Research Foundation of Korea Grant NRF-2011-220-C00004.
    2 Supported by the Institut Universitaire de France.
Open AccessPublished:July 16, 2015DOI:https://doi.org/10.1074/jbc.M115.639534
      Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.

      Introduction

      The primary cell wall of dicots consists of cellulose primarily cross-linked by xyloglucans and embedded in a complex matrix of pectic polysaccharides (
      • Carpita N.C.
      • Gibeaut D.M.
      Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth.
      ). Pectins are important structural polysaccharides in cell walls, representing up to one-third of primary wall dry mass. They are complex polysaccharides, rich in galacturonic acids (GalA),
      The abbreviations used are: GalA
      galacturonic acid(s)
      HG
      homogalacturonan
      PME
      pectin methylesterase
      PMEI
      DM, degree(s) of methylesterification
      DP
      degree(s) of polymerization
      DB
      degree of blockiness
      DBMe
      DB of highly methylesterified zones
      PMEI
      pectin methylesterase inhibitor
      TEV
      tobacco etch virus
      Ni-NTA
      nickel-nitrilotriacetic acid
      ESI
      electrospray ionization
      MST
      microscale thermophoresis
      PDB
      Protein Data Bank
      ANOVA
      analysis of variance.
      which comprise three main domains: homogalacturonan (HG), rhamnogalacturonan-I, and minor amounts of rhamnogalacturonan-II (
      • Caffall K.H.
      • Mohnen D.
      The structure, function, and biosynthesis of plant cell wall pectic polysaccharides.
      ). HG is composed of α-1,4-linked-d-galacturonic acid units. It can be methylesterified at the C-6 carboxyl and/or acetylated at the O-2 or O-3 residues (
      • Ralet M.-C.
      • Cabrera J.C.
      • Bonnin E.
      • Quéméner B.
      • Hellìn P.
      • Thibault J.-F.
      Mapping sugar beet pectin acetylation pattern.
      ). HG is synthesized from nucleotide sugars in the Golgi apparatus and then secreted as a fully methylesterified (up to 80%) form into the cell wall (
      • Sterling J.D.
      • Quigley H.F.
      • Orellana A.
      • Mohnen D.
      The catalytic site of the pectin biosynthetic enzyme α-1,4-galacturonosyltransferase is located in the lumen of the Golgi.
      ), where it can be de-esterified by cell wall enzymes, pectin methylesterases (PMEs; EC 3.1.1.11).
      PMEs are encoded by a large multigene family of 66 members in Arabidopsis (
      • Pelloux J.
      • Rustérucci C.
      • Mellerowicz E.J.
      New insights into pectin methylesterase structure and function.
      ). Based on their structure, plant PMEs have been classified into two groups. Both group 1 and group 2 PMEs possess a conserved PME domain (Pfam 01095). Group 2 PMEs contain an N-terminal extension called the PRO region, which shares similarity with the PME inhibitor domain (Pfam 04043 (
      • Pelloux J.
      • Rustérucci C.
      • Mellerowicz E.J.
      New insights into pectin methylesterase structure and function.
      )). It has been shown that the PRO region mediates the retention of unprocessed group 2 PMEs in the Golgi apparatus, thus regulating PME enzyme activity through a post-translational mechanism (
      • Wolf S.
      • Rausch T.
      • Greiner S.
      The N-terminal pro region mediates retention of unprocessed type-I PME in the Golgi apparatus.
      ). PME isoforms are either constitutively or differentially expressed in plant tissues at specific developmental stages or in response to biotic and abiotic stresses (
      • Louvet R.
      • Cavel E.
      • Gutierrez L.
      • Guénin S.
      • Roger D.
      • Gillet F.
      • Guerineau F.
      • Pelloux J.
      Comprehensive expression profiling of the pectin methylesterase gene family during silique development in Arabidopsis thaliana.
      ,
      • Sexton T.R.
      • Henry R.J.
      • Harwood C.E.
      • Thomas D.S.
      • McManus L.J.
      • Raymond C.
      • Henson M.
      • Shepherd M.
      Pectin methylesterase genes influence solid wood properties of Eucalyptus pilularis.
      ,
      • Lionetti V.
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      • Bellincampi D.
      Methyl esterification of pectin plays a role during plant-pathogen interactions and affects plant resistance to diseases.
      ,
      • Weber M.
      • Deinlein U.
      • Fischer S.
      • Rogowski M.
      • Geimer S.
      • Tenhaken R.
      • Clemens S.
      A mutation in the Arabidopsis thaliana cell wall biosynthesis gene pectin methylesterase 3 as well as its aberrant expression cause hypersensitivity specifically to Zn.
      ,
      • Futamura N.
      • Mori H.
      • Kouchi H.
      • Shinohara K.
      Male flower-specific expression of genes for polygalacturonase, pectin methylesterase and β-1,3-glucanase in a dioecious willow (Salix gilgiana Seemen).
      ).
      The mechanism of action of PMEs consists of the hydrolysis of the methyl ester bond at the C-6 position of GalA of HG. This releases methanol and provides a free carboxyl group on the pectin backbone, thus lowering the degree of methylesterification (DM). As a result, the gelling properties and calcium reactivity of the pectic polymer are modified (
      • Yoo S.-H.
      • Fishman M.L.
      • Hotchkiss Jr., A.T.
      • Lee H.G.
      Viscometric behavior of high-methoxy and low-methoxy pectin solutions.
      ,
      • Agoda-Tandjawa G.
      • Durand S.
      • Gaillard C.
      • Garnier C.
      • Doublier J.L.
      Properties of cellulose/pectins composites: implication for structural and mechanical properties of cell wall.
      ). The enzyme activity of PMEs is regulated by pH (
      • Denès J.M.
      • Baron A.
      • Renard C.M.
      • Péan C.
      • Drilleau J.F.
      Different action patterns for apple pectin methylesterase at pH 7.0 and 4.5.
      ,
      • Duvetter T.
      • Fraeye I.
      • Sila D.N.
      • Verlent I.
      • Smout C.
      • Hendrickx M.
      • Van Loey A.
      Mode of de-esterification of alkaline and acidic pectin methyl esterases at different pH conditions.
      ,
      • Thonar C.
      • Liners F.
      • Van Cutsem P.
      Polymorphism and modulation of cell wall esterase enzyme activities in the chicory root during the growing season.
      ,
      • Cameron R.G.
      • Luzio G.A.
      • Goodner K.
      • Williams M.A.K.
      Demethylation of a model homogalacturonan with a salt-independent pectin methylesterase from citrus: I. Effect of pH on demethylated block size, block number and enzyme mode of action.
      ). It is generally assumed that PMEs with an alkaline pI remove methyl ester in a blockwise manner, leading to the formation of demethylated stretches (
      • Denès J.M.
      • Baron A.
      • Renard C.M.
      • Péan C.
      • Drilleau J.F.
      Different action patterns for apple pectin methylesterase at pH 7.0 and 4.5.
      ,
      • Ralet M.C.
      • Dronnet V.
      • Buchholt H.C.
      • Thibault J.F.
      Enzymatically and chemically de-esterified lime pectins: characterisation, polyelectrolyte behaviour and calcium binding properties.
      ,
      • van Alebeek G.-J.W.M.
      • van Scherpenzeel K.
      • Beldman G.
      • Schols H.A.
      • Voragen A.G.J.
      Partially esterified oligogalacturonides are the preferred substrates for pectin methylesterase of Aspergillus niger.
      ), whereas acidic isoform activity results in a random-like distribution of the non-methylated GalA residues (
      • Daas P.J.
      • Voragen A.G.
      • Schols H.A.
      Study of the methyl ester distribution in pectin with endo-polygalacturonase and high-performance size-exclusion chromatography.
      ). The activity of PMEs is also regulated by PMEIs (
      • Jolie R.P.
      • Duvetter T.
      • Van Loey A.M.
      • Hendrickx M.E.
      Pectin methylesterase and its proteinaceous inhibitor: a review.
      ). In Arabidopsis, 76 genes have been annotated as encoding putative PMEIs, and some of them have been characterized at a biochemical (
      • Raiola A.
      • Camardella L.
      • Giovane A.
      • Mattei B.
      • De Lorenzo G.
      • Cervone F.
      • Bellincampi D.
      Two Arabidopsis thaliana genes encode functional pectin methylesterase inhibitors.
      ,
      • Wolf S.
      • Grsic-Rausch S.
      • Rausch T.
      • Greiner S.
      Identification of pollen-expressed pectin methylesterase inhibitors in Arabidopsis.
      ) or functional level (
      • Peaucelle A.
      • Louvet R.
      • Johansen J.N.
      • Höfte H.
      • Laufs P.
      • Pelloux J.
      • Mouille G.
      Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins.
      ,
      • Pelletier S.
      • Van Orden J.
      • Wolf S.
      • Vissenberg K.
      • Delacourt J.
      • Ndong Y.A.
      • Pelloux J.
      • Bischoff V.
      • Urbain A.
      • Mouille G.
      • Lemonnier G.
      • Renou J.-P.
      • Höfte H.
      A role for pectin de-methylesterification in a developmentally regulated growth acceleration in dark-grown Arabidopsis hypocotyls.
      ,
      • Müller K.
      • Levesque-Tremblay G.
      • Bartels S.
      • Weitbrecht K.
      • Wormit A.
      • Usadel B.
      • Haughn G.
      • Kermode A.R.
      Demethylesterification of cell wall pectins in Arabidopsis plays a role in seed germination.
      ,
      • Saez-Aguayo S.
      • Ralet M.C.
      • Berger A.
      • Botran L.
      • Ropartz D.
      • Marion-Poll A.
      • North H.M.
      PECTIN METHYLESTERASE INHIBITOR6 promotes Arabidopsis mucilage release by limiting methylesterification of homogalacturonan in seed coat epidermal cells.
      ). PMEI inhibits plant PME through interaction in a complex of 1:1 stoichiometry, in which PMEI covers the pectin-binding cleft of PME and hides its putative catalytic site, thereby impairing access to the substrate (
      • Hothorn M.
      • Wolf S.
      • Aloy P.
      • Greiner S.
      • Scheffzek K.
      Structural insights into the target specificity of plant invertase and pectin methylesterase inhibitory proteins.
      ,
      • Di Matteo A.
      • Giovane A.
      • Raiola A.
      • Camardella L.
      • Bonivento D.
      • De Lorenzo G.
      • Cervone F.
      • Bellincampi D.
      • Tsernoglou D.
      Structural basis for the interaction between pectin methylesterase and a specific inhibitor protein.
      ,
      • Hothorn M.
      • Van den Ende W.
      • Lammens W.
      • Rybin V.
      • Scheffzek K.
      Structural insights into the pH-controlled targeting of plant cell-wall invertase by a specific inhibitor protein.
      ).
      Considering the sizes of the PME and PMEI gene families, the determination of the specificity of the interactions between the proteins is a key issue for understanding the fine tuning of pectin methylesterification and its effects on plant development and defense mechanisms. In this report, we have identified AtPME3 (At3g14310) and AtPMEI7 (At4g25260) as being two of the major PME and PMEI isoforms expressed in Arabidopsis dark-grown hypocotyls, both at the transcript and protein levels. This co-expression suggested that AtPME3 (thereafter PME3) and AtPMEI7 (thereafter PMEI7) could interact in vivo. Both proteins were overexpressed in heterologous systems and purified by affinity chromatography. We showed that purified PME3 has an optimal enzyme activity at slightly alkaline pH and exhibits strong substrate specificity toward HG with a DM between 60 and 80% and a random distribution of methyl esters. The inhibition of recombinant PME3 by purified PMEI7 was shown to be regulated by pH. Using structural modeling, the docking of PMEI7 into PME3 revealed a strong conservation of the interaction. Altogether, this study brings new insights into the interactions between PMEs and PMEIs and provides new tools for the identification of PME-PMEI pairs in vitro and in muro.

      Discussion

      In this study, we characterized PME3 and its inhibition by PMEI7. On the basis of gene expression analysis, PME3 and PMEI7 were identified, among other genes encoding HG-modifying enzymes (
      • Pelletier S.
      • Van Orden J.
      • Wolf S.
      • Vissenberg K.
      • Delacourt J.
      • Ndong Y.A.
      • Pelloux J.
      • Bischoff V.
      • Urbain A.
      • Mouille G.
      • Lemonnier G.
      • Renou J.-P.
      • Höfte H.
      A role for pectin de-methylesterification in a developmentally regulated growth acceleration in dark-grown Arabidopsis hypocotyls.
      ,
      • Guénin S.
      • Mareck A.
      • Rayon C.
      • Lamour R.
      • Assoumou Ndong Y.
      • Domon J.-M.
      • Sénéchal F.
      • Fournet F.
      • Jamet E.
      • Canut H.
      • Percoco G.
      • Mouille G.
      • Rolland A.
      • Rustérucci C.
      • Guerineau F.
      • Van Wuytswinkel O.
      • Gillet F.
      • Driouich A.
      • Lerouge P.
      • Gutierrez L.
      • Pelloux J.
      Identification of pectin methylesterase 3 as a basic pectin methylesterase isoform involved in adventitious rooting in Arabidopsis thaliana.
      ), as being expressed during dark-grown hypocotyl development. The proteins were identified in cell wall-enriched fractions of such hypocotyls using both nano-LC-ESI-MS/MS and MALDI-TOF MS analysis. PME3 was only present in a processed form, which is in accordance with the processing of group 2 PME isoforms at specific sites (
      • Wolf S.
      • Rausch T.
      • Greiner S.
      The N-terminal pro region mediates retention of unprocessed type-I PME in the Golgi apparatus.
      ) and the recent identification of PME3 processing (
      • Weber M.
      • Deinlein U.
      • Fischer S.
      • Rogowski M.
      • Geimer S.
      • Tenhaken R.
      • Clemens S.
      A mutation in the Arabidopsis thaliana cell wall biosynthesis gene pectin methylesterase 3 as well as its aberrant expression cause hypersensitivity specifically to Zn.
      ). Transcript level and proteome analyses strongly suggested that PME3 and PMEI7 were two major isoforms present in the cell wall of developing dark-grown hypocotyls and that these proteins could interact in muro. PME3 was previously shown to be a putative interacting protein of AtPMEI1 and AtPMEI2, regulating PME activity in response to plant pathogens, such as Botrytis (
      • Lionetti V.
      • Raiola A.
      • Camardella L.
      • Giovane A.
      • Obel N.
      • Pauly M.
      • Favaron F.
      • Cervone F.
      • Bellincampi D.
      Overexpression of pectin methylesterase inhibitors in Arabidopsis restricts fungal infection by Botrytis cinerea.
      ). However, the existence of PME3-PMEI1 or PME3-PMEI2 pairs in vivo is highly questionable because it is known that whereas the two PMEI genes are specifically expressed in pollen, PME3 is widely expressed except in pollen (
      • Louvet R.
      • Cavel E.
      • Gutierrez L.
      • Guénin S.
      • Roger D.
      • Gillet F.
      • Guerineau F.
      • Pelloux J.
      Comprehensive expression profiling of the pectin methylesterase gene family during silique development in Arabidopsis thaliana.
      ,
      • Raiola A.
      • Camardella L.
      • Giovane A.
      • Mattei B.
      • De Lorenzo G.
      • Cervone F.
      • Bellincampi D.
      Two Arabidopsis thaliana genes encode functional pectin methylesterase inhibitors.
      ,
      • Wolf S.
      • Grsic-Rausch S.
      • Rausch T.
      • Greiner S.
      Identification of pollen-expressed pectin methylesterase inhibitors in Arabidopsis.
      ). The occurrence of specific spatial and temporal PME-PMEI interactions is likely to be a key determinant of the fine tuning of HG-methylesterification status, as shown previously in the pollen tube (
      • Röckel N.
      • Wolf S.
      • Kost B.
      • Rausch T.
      • Greiner S.
      Elaborate spatial patterning of cell-wall PME and PMEI at the pollen tube tip involves PMEI endocytosis, and reflects the distribution of esterified and de-esterified pectins.
      ).
      The purification of the recombinant PME3-His6 protein enabled the characterization of its substrate specificity using dedicated HG models. To our knowledge, this is the first report of such a characterization for a PME from Arabidopsis. PME3-His6 activity was optimal at an alkaline pH (pH 7.5), which is similar to that reported for apple, banana, and green pepper PMEs (
      • Denès J.M.
      • Baron A.
      • Renard C.M.
      • Péan C.
      • Drilleau J.F.
      Different action patterns for apple pectin methylesterase at pH 7.0 and 4.5.
      ,
      • Grsic-Rausch S.
      • Rausch T.
      A coupled spectrophotometric enzyme assay for the determination of pectin methylesterase activity and its inhibition by proteinaceous inhibitors.
      ,
      • Castro S.M.
      • Van Loey A.
      • Saraiva J.A.
      • Smout C.
      • Hendrickx M.
      Activity and process stability of purified green pepper (Capsicum annuum) pectin methylesterase.
      ,
      • Ly Nguyen B.
      • Van Loey A.
      • Fachin D.
      • Verlent I.
      • Indrawati
      • Hendrickx M.
      • Hendrickx I.M.
      Purification, characterization, thermal, and high-pressure inactivation of pectin methylesterase from bananas (cv. Cavendish).
      ) and contrasts with that reported for fungal PMEs (
      • Christgau S.
      • Kofod L.V.
      • Halkier T.
      • Andersen L.N.
      • Hockauf M.
      • Dörreich K.
      • Dalbøge H.
      • Kauppinen S.
      Pectin methyl esterase from Aspergillus aculeatus: expression cloning in yeast and characterization of the recombinant enzyme.
      ,
      • Gonzalez S.L.
      • Rosso N.D.
      Determination of pectin methylesterase activity in commercial pectinases and study of the inactivation kinetics through two potentiometric procedures.
      ). Interestingly, at an optimal pH, PME3-His6 activity was dependent on the DM and DP values and methylesterification distribution within HG. Using HG models, PME3-His6 showed a strong activity toward substrates, with a DM of 60–80%, a long chain of HG (DP >37%), and a random distribution of methyl ester groups, as reflected by the determination of apparent Km values. The apparent Km values obtained with the best substrates (HG98B69, HG96B77P63, and HG96B82) were lower but still in the same range as those found with LuPME5, a flax ortholog of PME3 (
      • Al-Qsous S.
      • Carpentier E.
      • Klein-Eude D.
      • Burel C.
      • Mareck A.
      • Dauchel H.L.N.
      • Gomord V.
      • Balangé A.P.
      Identification and isolation of a pectin methylesterase isoform that could be involved in flax cell wall stiffening.
      ). The difference could be related to the types of substrates used. In fact, we worked on pure HG and not on commercial pectin (DM 64), which was used for LuPME5. Surprisingly, the apparent Km value obtained with HG96B20 showed a stronger affinity of PME3-His6 for HG96B20 than for HG96B82. At pH 7.5, PME3 (pI ∼9.6) is positively charged and therefore interacts strongly with the many free carboxylic groups of HG96B20. Although plant PME enzymatic activity was previously shown to be optimal using a broad spectrum of pectic substrates, in the DM range of 62–94% (
      • Denès J.M.
      • Baron A.
      • Renard C.M.
      • Péan C.
      • Drilleau J.F.
      Different action patterns for apple pectin methylesterase at pH 7.0 and 4.5.
      ,
      • Duvetter T.
      • Fraeye I.
      • Sila D.N.
      • Verlent I.
      • Smout C.
      • Hendrickx M.
      • Van Loey A.
      Mode of de-esterification of alkaline and acidic pectin methyl esterases at different pH conditions.
      ,
      • Cameron R.G.
      • Luzio G.A.
      • Goodner K.
      • Williams M.A.K.
      Demethylation of a model homogalacturonan with a salt-independent pectin methylesterase from citrus: I. Effect of pH on demethylated block size, block number and enzyme mode of action.
      ,
      • Jolie R.P.
      • Duvetter T.
      • Houben K.
      • Clynen E.
      • Sila D.N.
      • Van Loey A.M.
      • Hendrickx M.E.
      Carrot pectin methylesterase and its inhibitor from kiwi fruit: study of activity, stability and inhibition.
      ), our study brings new insight into the close relationship between HG structure (DP, DM, and pattern of methylesterification) and enzyme activity. This probably reflects, at the level of methylesterification, the preferential substrates that PME3 could target within the cell wall. The action of PME3 on HG96B82 (B-series) produces a methyl distribution pattern bearing a high level of blockwise non-methylesterified GalA zones and short methylesterified stretches. Our study could not assess the intermolecular heterogeneity of methyl ester group distribution. However, the identification of long demethylesterified blocks after the action of PME3-His6 on HG96B82 indicates that it is a processive enzyme in vitro and that the PME mode of action could be a single chain mechanism. We did not characterize the number and the size of the demethylesterified blocks per HG molecule. That could be investigated in the future and thus allow the determination of either a single chain mechanism or multiple attack mode of action of PME3 as described previously with apple PME and citrus PME (
      • Denès J.M.
      • Baron A.
      • Renard C.M.
      • Péan C.
      • Drilleau J.F.
      Different action patterns for apple pectin methylesterase at pH 7.0 and 4.5.
      ,
      • Cameron R.G.
      • Luzio G.A.
      • Goodner K.
      • Williams M.A.K.
      Demethylation of a model homogalacturonan with a salt-independent pectin methylesterase from citrus: I. Effect of pH on demethylated block size, block number and enzyme mode of action.
      ). Plant PMEs with basic pI have been shown to produce order distributions of methylated stretches within HG (
      • Ralet M.-C.
      • Cabrera J.C.
      • Bonnin E.
      • Quéméner B.
      • Hellìn P.
      • Thibault J.-F.
      Mapping sugar beet pectin acetylation pattern.
      ,
      • Cameron R.G.
      • Luzio G.A.
      • Goodner K.
      • Williams M.A.K.
      Demethylation of a model homogalacturonan with a salt-independent pectin methylesterase from citrus: I. Effect of pH on demethylated block size, block number and enzyme mode of action.
      ,
      • Ralet M.-C.
      • Williams M.A.K.
      • Tanhatan-Nasseri A.
      • Ropartz D.
      • Quéméner B.
      • Bonnin E.
      Innovative enzymatic approach to resolve homogalacturonans based on their methylesterification pattern.
      ).
      Understanding the specificity of the interactions between PME and PMEI is likely to be a key point in our understanding of how PME enzymatic activities could be fine tuned within the cell wall. So far, purified PMEIs from Arabidopsis have only been tested for their inhibitory capacity on either cell wall-enriched protein extracts or commercially available tomato and orange PMEs (
      • Raiola A.
      • Camardella L.
      • Giovane A.
      • Mattei B.
      • De Lorenzo G.
      • Cervone F.
      • Bellincampi D.
      Two Arabidopsis thaliana genes encode functional pectin methylesterase inhibitors.
      ,
      • Wolf S.
      • Grsic-Rausch S.
      • Rausch T.
      • Greiner S.
      Identification of pollen-expressed pectin methylesterase inhibitors in Arabidopsis.
      ). In our study, we expressed and purified PMEI7-His6 and showed, using MST experiments, that PME3-His6 and PMEI7-His6 formed a complex with higher affinity at acidic pH. At pH 7.5, the affinity of PMEI7-His6 for PME3-His6 was markedly decreased, as assessed by MST analysis, mainly due to a faster dissociation of the complex, as observed with the kaki PME·kiwi PMEI complex (
      • Ciardiello M.A.
      • Tamburrini M.
      • Tuppo L.
      • Carratore V.
      • Giovane A.
      • Mattei B.
      • Camardella L.
      Pectin methylesterase from kiwi and kaki fruits: purification, characterization, and role of pH in the enzyme regulation and interaction with the kiwi proteinaceous inhibitor.
      ). The inhibition of PME3-His6 by PMEI7-His6 is pH-dependent, with an optimal inhibition at acidic pH (pH 5.0) and a stoichiometric ratio of 1:1. This suggests that, within the cell wall, at this pH, the majority of PME3 would be complexed with PMEI7 in a 1:1 molecular ratio. The pH dependence of the inhibition of PME by PMEI was previously reported for Arabidopsis PMEI·tomato PME complexes, but the optimal inhibition was at pH 6.5 (
      • Raiola A.
      • Camardella L.
      • Giovane A.
      • Mattei B.
      • De Lorenzo G.
      • Cervone F.
      • Bellincampi D.
      Two Arabidopsis thaliana genes encode functional pectin methylesterase inhibitors.
      ). When using kiwi PMEI, distinct pH dependence of the inhibition was observed. In response to changes in pH, the inhibitory capacity of PMEI7-His6 was more similar to that observed with kiwi PMEI. This suggests that, depending on the presence of specific PME isoforms and the local changes in pH in the cell wall, distinct PME-PMEI pairs might occur. This hypothesis is in accordance with the observed differences in the inhibitory capacity of PMEI7-His6 toward cell wall-enriched extracts of various organs and with previous results (
      • Ciardiello M.A.
      • Tamburrini M.
      • Tuppo L.
      • Carratore V.
      • Giovane A.
      • Mattei B.
      • Camardella L.
      Pectin methylesterase from kiwi and kaki fruits: purification, characterization, and role of pH in the enzyme regulation and interaction with the kiwi proteinaceous inhibitor.
      ,
      • Balestrieri C.
      • Castaldo D.
      • Giovane A.
      • Quagliuolo L.
      • Servillo L.
      A glycoprotein inhibitor of pectin methylesterase in kiwi fruit (Actinidia chinensis).
      ). The methylesterification pattern of HG was determined at pH 6.0, to evaluate the DBMe, in the presence of the PME3-His6·PMEI7-His6 complex. That condition provided a partial inhibition of PME3-His6 by PMEI7-His6 as expected, which means that the proteins alternately interact and dissociate. In order to gain insight into the specificity of the PME3-PMEI7 interaction, three-dimensional homology modeling of both proteins PME3 and PMEI7 was carried out and showed strong structural similarities with plant PME and plant PMEI structures (
      • Di Matteo A.
      • Giovane A.
      • Raiola A.
      • Camardella L.
      • Bonivento D.
      • De Lorenzo G.
      • Cervone F.
      • Bellincampi D.
      • Tsernoglou D.
      Structural basis for the interaction between pectin methylesterase and a specific inhibitor protein.
      ,
      • Johansson K.
      • El-Ahmad M.
      • Friemann R.
      • Jörnvall H.
      • Markovic O.
      • Eklund H.
      Crystal structure of plant pectin methylesterase.
      ,
      • Ciardiello M.A.
      • D'Avino R.
      • Amoresano A.
      • Tuppo L.
      • Carpentieri A.
      • Carratore V.
      • Tamburrini M.
      • Giovane A.
      • Pucci P.
      • Camardella L.
      The peculiar structural features of kiwi fruit pectin methylesterase: amino acid sequence, oligosaccharides structure, and modeling of the interaction with its natural proteinaceous inhibitor.
      ). Docking analysis showed that inhibition most probably occurs through the interaction of PMEI7 with the ligand-binding cleft structure within PME3 as described previously for the crystallized structure of the tomato or kiwi PME·kiwi PMEI complex (
      • Di Matteo A.
      • Giovane A.
      • Raiola A.
      • Camardella L.
      • Bonivento D.
      • De Lorenzo G.
      • Cervone F.
      • Bellincampi D.
      • Tsernoglou D.
      Structural basis for the interaction between pectin methylesterase and a specific inhibitor protein.
      ,
      • Ciardiello M.A.
      • D'Avino R.
      • Amoresano A.
      • Tuppo L.
      • Carpentieri A.
      • Carratore V.
      • Tamburrini M.
      • Giovane A.
      • Pucci P.
      • Camardella L.
      The peculiar structural features of kiwi fruit pectin methylesterase: amino acid sequence, oligosaccharides structure, and modeling of the interaction with its natural proteinaceous inhibitor.
      ). Amino acid sequence alignment between kiwi PMEI and PMEI7 displayed important common amino acid residues for the interaction. Among them, Asp-137 found in PMEI7 is conserved in kiwi PMEI. However, docking analysis indicated that Asp-137 is unlikely to be involved in the interaction. This might reveal structural differences among plant PMEIs. The discrepancy observed between tomato PME-kiwi PMEI and our PME3-PMEI7 model could also be due to species specificities. The determination of the crystallographic structure of PME3 and PMEI7 will help in deciding which hypothesis is the most likely.
      From our in vitro experiments and the fact that genes are co-expressed, we assume that PME3-PMEI7 is one of the pairs that indeed fine tune the degree of methylesterification of pectins in the cell wall in dark-grown hypocotyls. However, considering the size of the PME and PMEI gene families, it is likely that distinct PMEIs could target PME3 and conversely that each PMEI could target several PMEs. For instance, PME32, which is as abundant as PME3 in dark-grown hypcotyl, based on proteomics data, could be another candidate for PMEI7. Our data should shed new light on the key role of PME-PMEI interactions in the regulation of PME activity in muro and the consequences on HG remodeling and development. Further studies are required to understand how PME-PMEI pairs could spatially and temporally control the DM of HG and its consequences for cell wall rheology.

      Author Contributions

      F. S. performed transcriptomic expression of PME3 and PMEI7, GUS assays, subcellular localization, and MST experiments. E. R and C. R. designed and cloned PME3. M. L., J.-M. D., and C. R. led PME3 purification. M. L. and C. R. performed PME3 enzyme activity assay experiments. O. S., A. M., and C. R. performed kinetic assay studies, and P. L. assisted in interpretations. P. M. and E. J. performed proteomic experiments. J. M., F. G. and F. S designed, cloned, and purified PMEI7. F. S. performed gel diffusion assays experiments, and J. P. assisted in interpretations. M.-C. R. and E. B. led studies of degree of blockiness (DB) with M.-J. C. J. E.-S., H. R. K., C. R., and D. K. led studies of protein modeling and docking. J. P. and C. R. designed the experiments. F. S., J. P., and C. R. wrote the manuscript.

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