Identification of pectin methylesterase 3 as a basic pectin methylesterase isoform involved in adventitious rooting in Arabidopsis thaliana

New PhytologistVolume 192, Issue 1 p. 114-126 Full paperFree Access Identification of pectin methylesterase 3 as a basic pectin methylesterase isoform involved in adventitious rooting in Arabidopsis thaliana Stéphanie Guénin, Corresponding Author Stéphanie Guénin EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, France CRRBM – Bâtiment Serres Transfert, Université de Picardie Jules Verne, 33 Rue St Leu, F-80039 Amiens, FranceAuthor for correspondence: Jérôme Pelloux Tel: +33 322827038 Email: [email protected]Search for more papers by this authorAlain Mareck, Corresponding Author Alain Mareck Laboratoire 'Glycobiologie et Matrice Extracellulaire Végétale' UPRES-EA 4358, IFRMP 23, UFR des Sciences et Techniques, F-76821 Mont-Saint-Aignan, FranceAuthor for correspondence: Jérôme Pelloux Tel: +33 322827038 Email: [email protected]Search for more papers by this authorCatherine Rayon, Catherine Rayon EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this authorRomain Lamour, Romain Lamour Laboratoire 'Glycobiologie et Matrice Extracellulaire Végétale' UPRES-EA 4358, IFRMP 23, UFR des Sciences et Techniques, F-76821 Mont-Saint-Aignan, FranceSearch for more papers by this authorYves Assoumou Ndong, Yves Assoumou Ndong EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this authorJean-Marc Domon, Jean-Marc Domon EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this authorFabien Sénéchal, Fabien Sénéchal EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this authorFrançoise Fournet, Françoise Fournet EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this authorElisabeth Jamet, Elisabeth Jamet UPS, CNRS, UMR 5546 Surfaces Cellulaires et Signalisation chez les Végétaux, Université de Toulouse, BP42617, F-31326 Castanet-Tolosan, FranceSearch for more papers by this authorHervé Canut, Hervé Canut UPS, CNRS, UMR 5546 Surfaces Cellulaires et Signalisation chez les Végétaux, Université de Toulouse, BP42617, F-31326 Castanet-Tolosan, FranceSearch for more papers by this authorGiuseppe Percoco, Giuseppe Percoco Laboratoire 'Glycobiologie et Matrice Extracellulaire Végétale' UPRES-EA 4358, IFRMP 23, UFR des Sciences et Techniques, F-76821 Mont-Saint-Aignan, FranceSearch for more papers by this authorGrégory Mouille, Grégory Mouille Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, Bâtiment 2, INRA Centre de Versailles-Grignon, Route de St Cyr (RD 10), F-78026 Versailles Cedex FranceSearch for more papers by this authorAurélia Rolland, Aurélia Rolland Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, Bâtiment 2, INRA Centre de Versailles-Grignon, Route de St Cyr (RD 10), F-78026 Versailles Cedex FranceSearch for more papers by this authorChristine Rustérucci, Christine Rustérucci EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this authorFrançois Guerineau, François Guerineau EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this authorOlivier Van Wuytswinkel, Olivier Van Wuytswinkel EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this authorFrançoise Gillet, Françoise Gillet EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this authorAzeddine Driouich, Azeddine Driouich Laboratoire 'Glycobiologie et Matrice Extracellulaire Végétale' UPRES-EA 4358, IFRMP 23, UFR des Sciences et Techniques, F-76821 Mont-Saint-Aignan, FranceSearch for more papers by this authorPatrice Lerouge, Patrice Lerouge Laboratoire 'Glycobiologie et Matrice Extracellulaire Végétale' UPRES-EA 4358, IFRMP 23, UFR des Sciences et Techniques, F-76821 Mont-Saint-Aignan, FranceSearch for more papers by this authorLaurent Gutierrez, Laurent Gutierrez CRRBM – Bâtiment Serres Transfert, Université de Picardie Jules Verne, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this authorJérôme Pelloux, Jérôme Pelloux EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this author Stéphanie Guénin, Corresponding Author Stéphanie Guénin EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, France CRRBM – Bâtiment Serres Transfert, Université de Picardie Jules Verne, 33 Rue St Leu, F-80039 Amiens, FranceAuthor for correspondence: Jérôme Pelloux Tel: +33 322827038 Email: [email protected]Search for more papers by this authorAlain Mareck, Corresponding Author Alain Mareck Laboratoire 'Glycobiologie et Matrice Extracellulaire Végétale' UPRES-EA 4358, IFRMP 23, UFR des Sciences et Techniques, F-76821 Mont-Saint-Aignan, FranceAuthor for correspondence: Jérôme Pelloux Tel: +33 322827038 Email: [email protected]Search for more papers by this authorCatherine Rayon, Catherine Rayon EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this authorRomain Lamour, Romain Lamour Laboratoire 'Glycobiologie et Matrice Extracellulaire Végétale' UPRES-EA 4358, IFRMP 23, UFR des Sciences et Techniques, F-76821 Mont-Saint-Aignan, FranceSearch for more papers by this authorYves Assoumou Ndong, Yves Assoumou Ndong EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this authorJean-Marc Domon, Jean-Marc Domon EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this authorFabien Sénéchal, Fabien Sénéchal EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this authorFrançoise Fournet, Françoise Fournet EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this authorElisabeth Jamet, Elisabeth Jamet UPS, CNRS, UMR 5546 Surfaces Cellulaires et Signalisation chez les Végétaux, Université de Toulouse, BP42617, F-31326 Castanet-Tolosan, FranceSearch for more papers by this authorHervé Canut, Hervé Canut UPS, CNRS, UMR 5546 Surfaces Cellulaires et Signalisation chez les Végétaux, Université de Toulouse, BP42617, F-31326 Castanet-Tolosan, FranceSearch for more papers by this authorGiuseppe Percoco, Giuseppe Percoco Laboratoire 'Glycobiologie et Matrice Extracellulaire Végétale' UPRES-EA 4358, IFRMP 23, UFR des Sciences et Techniques, F-76821 Mont-Saint-Aignan, FranceSearch for more papers by this authorGrégory Mouille, Grégory Mouille Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, Bâtiment 2, INRA Centre de Versailles-Grignon, Route de St Cyr (RD 10), F-78026 Versailles Cedex FranceSearch for more papers by this authorAurélia Rolland, Aurélia Rolland Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, Bâtiment 2, INRA Centre de Versailles-Grignon, Route de St Cyr (RD 10), F-78026 Versailles Cedex FranceSearch for more papers by this authorChristine Rustérucci, Christine Rustérucci EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this authorFrançois Guerineau, François Guerineau EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this authorOlivier Van Wuytswinkel, Olivier Van Wuytswinkel EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this authorFrançoise Gillet, Françoise Gillet EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this authorAzeddine Driouich, Azeddine Driouich Laboratoire 'Glycobiologie et Matrice Extracellulaire Végétale' UPRES-EA 4358, IFRMP 23, UFR des Sciences et Techniques, F-76821 Mont-Saint-Aignan, FranceSearch for more papers by this authorPatrice Lerouge, Patrice Lerouge Laboratoire 'Glycobiologie et Matrice Extracellulaire Végétale' UPRES-EA 4358, IFRMP 23, UFR des Sciences et Techniques, F-76821 Mont-Saint-Aignan, FranceSearch for more papers by this authorLaurent Gutierrez, Laurent Gutierrez CRRBM – Bâtiment Serres Transfert, Université de Picardie Jules Verne, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this authorJérôme Pelloux, Jérôme Pelloux EA3900-BioPI Biologie des Plantes et Contrôle des Insectes Ravageurs, Université de Picardie, 33 Rue St Leu, F-80039 Amiens, FranceSearch for more papers by this author First published: 21 June 2011 https://doi.org/10.1111/j.1469-8137.2011.03797.xCitations: 51 These authors contributed equally to the work. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Summary • Here, we focused on the biochemical characterization of the Arabidopsis thaliana pectin methylesterase 3 gene (AtPME3; At3g14310) and its role in plant development. • A combination of biochemical, gene expression, Fourier transform-infrared (FT-IR) microspectroscopy and reverse genetics approaches were used. • We showed that AtPME3 is ubiquitously expressed in A. thaliana, particularly in vascular tissues. In cell wall-enriched fractions, only the mature part of the protein was identified, suggesting that it is processed before targeting the cell wall. In all the organs tested, PME activity was reduced in the atpme3-1 mutant compared with the wild type. This was related to the disappearance of an activity band corresponding to a pI of 9.6 revealed by a zymogram. Analysis of the cell wall composition showed that the degree of methylesterification (DM) of galacturonic acids was affected in the atpme3-1 mutant. A change in the number of adventitious roots was found in the mutant, which correlated with the expression of the gene in adventitious root primordia. • Our results enable the characterization of AtPME3 as a major basic PME isoform in A. thaliana and highlight its role in adventitious rooting. Introduction The growth of plant organs involves cell division and cell expansion. Cell expansion is driven by turgor pressure and relies on changes in the extensibility of the primary cell wall. In dicotyledonous species, such as the model plant Arabidopsis thaliana, the primary cell wall consists of a hydrogen-bonded network of cellulose microfibrils and xyloglucans embedded in a complex pectic and protein matrix (Carpita & Gibeaut, 1993). Cell growth is known to require creep between cellulose and xyloglucan, which is facilitated by the presence of expansins (Rose et al., 2002; Cosgrove, 2005). Very few data exist on the role of the pectin matrix in growth control. Pectins are complex polysaccharides rich in galacturonic acid (GalpA). They are organized around two molecular backbones and differ in the diversity of their side chains (Vincken et al., 2003; Caffall & Mohnen, 2009). Pectins contain four distinct domains: homogalacturonan (HG), rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II) and xylogalacturonan (XGA). HG, one of the main pectic constituents, is a linear homopolymer of α-(1-4) linked D galacturonic acids, which can be methylesterified at the C-6 carboxyl and/or carry acetyl groups at O-2 and O-3. HG is synthesized from nucleotide sugars in the Golgi apparatus, and then secreted in its fully methylesterified form into the cell wall (Sterling et al., 2001) where its structure can be modified by the activity of cell wall enzymes such as pectin methylesterases (PMEs; EC 3.1.1.11), which belong to a large multigene family in A. thaliana. PME activities are regulated by endogenous pectin methylesterase inhibitors (PMEIs) and control the degree of methylesterification (DM) of HG (Pelloux et al., 2007; Wolf et al., 2009a). The partially demethylesterified HG can either form Ca2+ bonds, which promote the formation of the so-called 'egg-box' structures that underlie the formation of pectin gels, or become a target for pectin-degrading enzymes such as polygalacturonases (PGs; EC 3.2.1.15) and pectate lyases (PLs; EC 4.2.2.10; EC 4.2.2.2). In either case, the demethylesterification of HG has dramatic consequences for the rheological properties of the cell wall, and is predicted to affect cell growth. The lack of experimental evidence for a role of pectin modification in growth control is partly a result of the recalcitrance of the PME gene family to functional analysis. However, pectin methylesterification status has recently been implicated in the control of cell elongation in the pollen tube (Jiang et al., 2005; Tian et al., 2006) and the hypocotyl (Derbyshire et al., 2007; Pelletier et al., 2010). Other major roles of PMEs include cambial cell differentiation and fiber length determination in trees (Micheli et al., 2000; Siedlecka et al., 2008), microsporogenesis (Lacoux et al., 2003; Francis et al., 2006) and fleshy fruit maturation (Eriksson et al., 2004; Deytieux-Belleau et al., 2008). In addition, the control of pectin demethylesterification via the PME–PMEI balance has recently been shown to be involved in organ formation and phyllotaxis in the A. thaliana meristem (Peaucelle et al., 2008). These results indicate a role for PME-mediated HG changes in plant development. In addition, numerous studies have reported a role for pectin methylesterification status in plant defense responses. In particular, in A. thaliana and strawberry (Fragaria vesca), the PME-mediated demethylesterification of pectins has been implicated in the response to Botrytis (Lionetti et al., 2007; Osorio et al., 2008). In A. thaliana, this could involve the regulation of AtPME3 activity by AtPMEI-1 and AtPMEI-2, which were shown to interact with AtPME3 (Lionetti et al., 2007). AtPME3 was further shown to interact with a cellulose-binding protein (CBP) of the parasitic nematode Heterodora schachtii (Hewezi et al., 2008) and to act as a susceptibility factor that is required for the initial colonization of the host (Raiola et al., 2011). AtPME3 (At3g14310) is a group 2 PME where the active domain (the PME domain; PF01095) is preceded by an N-terminal extension (the PRO region) which exhibits similarity with the PME inhibitor (the PMEI domain; PF04043) (Pelloux et al., 2007). It has been shown that the PRO region mediates the retention of unprocessed group 2 PMEs in the Golgi apparatus, thus regulating PME activity through a post-translational mechanism (Wolf et al., 2009b). To investigate the roles of AtPME3 in plant development, we first analyzed the processing of the protein and the pattern of expression of the corresponding gene. A T-DNA mutant line for AtPME3 (atpme3-1) was characterized. We showed that the reduced PME activity measured in several organs was related to the disappearance of a basic PME isoform. The reduction in PME activity had an impact on the DM of pectins. Moreover, the changes in pectic structure had consequences for plant growth and development, as revealed by changes in adventitious rooting. Materials and Methods Plant material and growth conditions The Arabidopsis thaliana (L.) Heynh atpme3-1 mutant was isolated from the Versailles T-DNA insertion collection (FLAG585E02; WS ecotype). The left flanking sequence of the T-DNA insertion site was amplified on genomic DNA by PCR using the T-DNA specific primer Tag3 (5′-CTGATACCAGACGTTGCCCGCATAA-3′) and AtPME3 specific primers KOF (5′-GGAGATCTTCCACCGTTTCA-3′) and KOR (5′-CGTAGCTTTGCTCTTCGTAGC-3′). PCR products were sequenced and the site of insertion confirmed as being localized in the first exon. The reference gene APT1 (Adenosine Phosphoribosyl Transferase 1)/At1g27450 (APT1F 5′-CGGGGATTTTAAGTGGAACA-3′ and APT1R 5′-GAGACATTTTGCGTGGGATT-3′) was used as a control. N506425 and N62907 mutants, in At3g43270 and At3g29090, respectively, were isolated from the Salk T-DNA collection using specific gene primers (5′-TGATTCAGCAGAAGAAAAATCTG-3′/5′-CGTCGACGCTATAACAAAAGC-3′ and 5′-ACAAAAGAATTCATCCACCATT-3′/5′-TTGCGAAACCCTAACCATTC-3′, respectively). Plants were grown on soil in a glasshouse (16-h photoperiod at 200 μmol m−2 s−1 and a day : night temperature of 22 : 20°C) or on solid media on plates in a phytotronic chamber (16-h photoperiod at 120 μmol m−2 s−1 and at 20°C), as described previously (Estelle & Somerville, 1987), without sucrose. Seeds were cold-treated for 48 h to synchronize germination. For dark growth conditions, seeds were exposed to fluorescent white light (120 μmol m−2s−1) for 4 h to induce germination, after which plates were wrapped in three layers of aluminum foil. For the phenotyping presented in Table 1, depending on the experiment, 15–30 seedlings/plants were analyzed for each of the three biological replicates. Data were statistically analyzed using Student's t-test (Statisca; Statsoft, Maison-Alfort, France). Table 1. Phenotypic traits associated with the Arabidopsis thaliana pectin methylesterase 3-1 (atpme3-1) mutant WS atpme3-1 n Seedling roots (cm) 1.96 ± 0.05 2.03 ± 0.05 90 Secondary roots density (nb cm−1) 0.31 ± 0.03 0.28 ± 0.02 55 Dark-grown hypocotyls (cm) 1.06 ± 0.04 1.14 ± 0.04* 60 Rosette diameter (cm) 5.52 ± 0.08 4.99 ± 0.10*** 80 Inflorescence stem (cm) 25.86 ± 0.62 23.83 ± 0.56* 80 Measurements were carried out either on seedlings grown on agar plates (root and hypocotyl lengths, and number of secondary roots) or on 4-wk-old Arabidopsis thaliana plants grown on soil (rosette diameter and stem length). Results are expressed in cm. Data represent the mean ± SE of three independent experiments. Data were analyzed statistically using Student's t-test (Statisca; Statsoft, Maison-Alfort, France). *, P < 0.05; ***, P < 0.001. n, number of measurements. For counting of adventitious roots (ARs), seeds were sterilized and sown in vitro as previously described (Sorin et al., 2005). After 48 h of incubation at 4°C, plates were transferred to the light for 6–8 h to induce germination and were then wrapped in three layers of aluminum foil. They were kept in the dark until the seedling hypocotyls reached 6 mm in length (c. 48 h). They were then transferred to the light for the induction of AR formation. Adventitious roots were counted 7 d after transfer to the light. For each biological replicate, at least 30 seedlings were analyzed, and each experiment was repeated three times. A one-way ANOVA combined with a Tukey's multiple comparison test was performed to analyze the differences between the mean and variance of the genotypes using the software GraphPad Prism version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Identification of AtPME3 as a cell wall-associated protein Etiolated hypocotyls grown on plates were collected at 5 and 11 d (Irshad et al., 2008). Hypocotyls 1.0 to 1.8 cm in length were used for the isolation of cell walls, as previously reported (Feiz et al., 2006). The cell wall fraction obtained was ground in liquid nitrogen in a mortar with a pestle prior to lyophilization. Proteins were extracted from the cell wall preparation by successive steps of CaCl2 extraction followed by LiCl extraction. The salt-extracted proteins were separated by cation exchange chromatography. The column fractions were separated by SDS-PAGE (Laemmli, 1970), stained as previously described (Irshad et al., 2008). Colored bands were excised from gels and digested with trypsin (Boudart et al., 2005; Borderies et al., 2003). Sample preparation for all mass spectrometry (MS) analyses was performed as previously described (Borderies et al., 2003). Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS analyses were performed using a Voyager-DE STR mass spectrometer (Applied Biosystems/MDS, Foster City, CA, USA). Spectra were acquired in reflectron mode as previously reported (Borderies et al., 2003; Boudart et al., 2005). Peptides were identified at least once out of seven analyses. Fourier transform-infrared (FT-IR) microspectroscopy analysis Four-day-old dark-grown seedlings grown on plates were squashed between two BaF2 windows and thoroughly rinsed in distilled water for 2 min. The samples were then dried on the windows at 37°C for 20 min. For each mutant, 20 spectra were collected for individual hypocotyls from seedlings from four independent cultures (five seedlings from each culture), as described by Mouille et al. (2003). Normalization of the data and the discriminant variable selection method were performed as described by Mouille et al. (2003). Cell wall preparation, neutral sugar analysis and determination of the degree of methylesterification of GalUA Isolation of cell walls was carried out according to Carpita et al. (2001). Frozen plant material was ground into a fine powder with stainless steel balls in a grinding jar using a Retschmill (30 s; 25 Hz, Retsch France, Eragny sur Oise, France). Frozen powder (100 mg) was heated to 70°C in 95% ethanol for 15 min. The extract was centrifuged at 17 000 g for 5 min, and another ethanol extraction was performed. The pellet was homogenized in 1% (w/v) SDS in 50 mM Tris-HCl, pH 7.2, and heated at 70°C for 30 min. After cooling, the suspension was filtered through a 2.5-cm-diameter 41-μm nylon cloth disk placed in a Millipore-type manifold, and then washed sequentially with water, ethanol, acetone and water. Finally, the material was freeze-dried. Dry cell wall material (DCW; 10 mg) was digested with amylase according to Fleischer et al. (1999). Briefly, the cell wall was suspended in 100 mM potassium phosphate, pH 6.8 (500 μl), and digested for 24 h at room temperature with α-amylase (0.51 U α-amylase from Bacillus subtilis (Sigma, Saint-Quentin Fallavier, France) mg−1 DCW). After digestion, the cell wall was centrifuged at 17 000 g for 5 min and washed three times in 1 ml of water and freeze-dried. The destarched cell wall material (c. 5 mg) was hydrolyzed in 1 ml of 2 M TFA (TriFluoro Acetic Acid) containing 20 μg of myoinositol (internal standard) at 120°C for 90 min. After cooling, the mixture was centrifuged and the supernatant was evaporated at 45°C in a stream of nitrogen. The sugars were reduced with NaBH4, and alditol acetates were prepared as previously described (Gibeaut & Carpita, 1991). Derivatives were separated by gas liquid chromatography on a 0.25 mm × 30 cm column of TR-FAME (Thermo, Strasbourg, France) according to the method described previously (Peña & Carpita, 2004). The flame ionization detector was integrated and the amount of neutral sugar calculated relative to the myoinositol internal standard. Uronic acid content and the DM of the samples were determined as described elsewhere (Blumenkrantz & Asboe-Hansen, 1973; Klavons & Bennett, 1986). Analyses of neutral sugar composition, uronic acid content and the DM of the samples were performed in triplicate. Data were analyzed statistically using a Mann–Whitney test (Statisca; Statsoft). Promoter amplification and GUS staining One kb of the promoter of At3g14310 was amplified using AmpliTaq polymerase (Applied Biosystems, Courtaboeuf, France) with the following primers: PrF (5′-GTAGGATCCTTCGACACTAGTGAGTGATG-3′) and PrR (5′-TCAAAGCTTTGACAGGCTTGCAGCCATCG-3′). The PCR product was cloned into the pGEM-T Easy vector (Promega, Charbonnières-les-Bains, France), sequenced and subcloned into the binary vector pBI101.3 (Clontech, Saint-Germain-en-Laye, France) upstream of the GUS coding sequence using BamHI/HindIII restriction sites. Plant transformation, using Agrobacterium tumefaciens strain LBA4404, was performed according to the floral dip method (Clough & Bent, 1998). Transformants were selected on kanamycin at 80 μg ml−1 and β-glucuronidase staining was carried out as described previously (Sessions et al., 1999), with 10 mM K3Fe(CN)6 and 10 mM K4Fe(CN)6, to limit stain diffusion. Plant samples were destained in 75% ethanol and digital pictures were taken with a Nikon Coolpix 995 camera (Nikon France, Champigny sur Marne, France). Production of Anti-LuPME3 antibodies and western blot analysis Two peptide sequences were chosen in the LuPME3 protein (AF188895), QSVKGSFGT and DAEAAGFTPGR, being specific for this flax PME. These peptides were coupled to KLH for immunization. Peptide synthesis and the immunization procedure were carried out by Eurogentec (Angers, France). After collecting the pre-immune serum, a rabbit was immunized using 1 mg of the KLH-coupled synthetic peptides and boosted twice every 2 wk, then twice every month for 3 months. Proteins from total crude extracts of A. thaliana plants were separated by 15% SDS-PAGE and either stained with Coomassie blue R-250 or electrotransferred onto polyvinylidene difluoride membranes (Roche Diagnostics Corporation, Indianapolis, IN, USA) and blocked at 4°C overnight with 5% skimmed milk and 0.1% Tween-20 in Tris-buffered saline (TBS-T). The membranes were then rinsed twice with 2% skimmed milk and 0.1% Tween-20 in TBS-T for 10 min at room temperature. Immunoblotting was performed by incubation with anti-LuPME3 antibody (1/200) for 4 h at room temperature and membranes were rinsed twice with 2% skimmed milk and 0.1% Tween-20 in TBS-T for 10 min at room temperature. A horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (H + L) (Bio-Rad, Hercules, CA, USA) was used as a secondary antibody for membranes treated with the heavy chain-specific antibody. For detection of HRP activity, the membranes were revealed by incubation for 10–15 min in 100 ml of a TBS buffer containing 30 mg of 4-chloro-1-naphthol (Sigma, Saint-Quentin Fallavier, France) and 150 μl of hydrogen peroxide (Sigma). Immunofluorescence staining with anti-PME3 antibodies Six-day-old seedlings were fixed in 4% paraformaldehyde, 50 mM 1,4-piperazinediethanesulfonic acid and 1 mM CaCl2 for 2 h and washed 3 times for 15 min each in phosphate-buffered saline (PBS). The samples were then dehydrated in a graded aqueous ethanol series, and embedded in butyl-methyl methacrylate resin as described by Baskin & Wilson (1997). Thin cross-sections (2.5 μm) were immunostained and observed as described by Andème-Onzighi et al. (2002). The antibodies were used at 1 : 1 dilution for anti-PME3 (primary antibody) and 1 : 50 for fluorescein isothiocyanate conjugate secondary antibody. PME activity and zymograms Protein extraction was adapted from Pilling et al. (2000). Frozen material (roots, stems, leaves or hypocotyls) was ground with a pestle and mortar in liquid nitrogen to obtain a fine powder. Proteins were extracted in 20 mM sodium phosphate buffer, pH 7.5, containing 1 M NaCl, 0.2% PVPP (PolyVinylPolyPyrrolidone) and 0.01% Tween 20 for 1 h at 4°C under shaking. Cellular fragments were discarded by centrifugation at 12 000 g for 15 min. Proteins from the supernatant were quantified by the Bradford method (Bradford, 1976) with a Protein Assay kit (Bio-Rad, Marnes-la-Coquette, France). The protein extract was assayed for PME activity using the alcohol oxidase coupled assay (Klavons & Bennett, 1986). Data are the means of four to six independent replicates. Data were analyzed statistically using a Mann–Whitney test (Statisca; Statsoft). After extensive dialysis, active fractions were submitted to IEF (IsoElectricFocusing) (Pharmalyte, pH 8–10.5; Pharmacia, Pharmacia France, St Quentin Yvelines, France) on a 0.5-mm-thick polyacrylamide gel, according to the supplier's recommendations. The pH gradient was estimated with a contact electrode (pH Inlab 426; VWR International, Fontenay sous Bois, France), along a central gel sample strip. After IEF, PME activities were visualized using the citrus pectin–agar method using pectins of DM 85% (Bertheau et al., 1984). The demethoxylation of pectins was detected by staining with ruthenium red. Assessment of AtPME3 and AUXIN RESPONSE FACTOR (ARF) gene expression by RT-PCR The absence of PME3 transcripts was assessed in the atpme3-1 mutant and PME3 transcripts were quantified in the arf6-3, arf8-7, MIR160c-OX (plants over-expressing the MIR160c-OX (overexpressor) gene) and ARF17-OX AR mutants as described in Gutierrez et al. (2009). To check for the absence of AtPME3 c-DNA and for the possible presence of a transcript of T-DNA fused to cDNA in the atpme3-1 mutant, RT-PCR was carried out on 4-d-old dark-grown hypocotyls using already described KO