An oligogalacturonide‐derived molecular probe demonstrates...

An oligogalacturonide‐derived molecular probe demonstrates the dynamics of calcium‐mediated pectin complexation in cell walls of tip‐growing structures - Mravec - 2017 - The Plant Journal - Wiley Online LibraryThe Plant JournalVolume 91, Issue 3 p. 534-546 Technical Advance Free Access An oligogalacturonide-derived molecular probe demonstrates the dynamics of calcium-mediated pectin complexation in cell walls of tip-growing structures Jozef Mravec, Corresponding Author mravec@plen.ku.dk Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg-C, DenmarkFor correspondence (e-mails mravec@plen.ku.dk; william.willats@newcastle.ac.uk).Search for more papers by this authorStjepan K. Kračun, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg-C, DenmarkSearch for more papers by this authorMaja G. Rydahl, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg-C, DenmarkSearch for more papers by this authorBjørge Westereng, Department of Chemistry, Biotechnology and Food Science (IKBM), Norwegian University of Life Sciences, Ås, NorwaySearch for more papers by this authorDaniela Pontiggia, Dipartimento di Biologia e Biotecnologie C. Darwin, Istituto Pasteur-Cenci Bolognetti, Università di Roma Sapienza, Piazzale A. Moro 5, 00185 Roma, ItalySearch for more papers by this authorGiulia De Lorenzo, Dipartimento di Biologia e Biotecnologie C. Darwin, Istituto Pasteur-Cenci Bolognetti, Università di Roma Sapienza, Piazzale A. Moro 5, 00185 Roma, ItalySearch for more papers by this authorDavid S. Domozych, Department of Biology and Skidmore Microscopy Imaging Center, Skidmore College, Saratoga Springs, NY, 12866 USASearch for more papers by this authorWilliam G. T. Willats, Corresponding Author william.willats@newcastle.ac.uk School of Agriculture, Food and Rural Development, Newcastle University, Newcastle upon Tyne, NE1 7RU UKFor correspondence (e-mails mravec@plen.ku.dk; william.willats@newcastle.ac.uk).Search for more papers by this author Jozef Mravec, Corresponding Author mravec@plen.ku.dk Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg-C, DenmarkFor correspondence (e-mails mravec@plen.ku.dk; william.willats@newcastle.ac.uk).Search for more papers by this authorStjepan K. Kračun, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg-C, DenmarkSearch for more papers by this authorMaja G. Rydahl, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg-C, DenmarkSearch for more papers by this authorBjørge Westereng, Department of Chemistry, Biotechnology and Food Science (IKBM), Norwegian University of Life Sciences, Ås, NorwaySearch for more papers by this authorDaniela Pontiggia, Dipartimento di Biologia e Biotecnologie C. Darwin, Istituto Pasteur-Cenci Bolognetti, Università di Roma Sapienza, Piazzale A. Moro 5, 00185 Roma, ItalySearch for more papers by this authorGiulia De Lorenzo, Dipartimento di Biologia e Biotecnologie C. Darwin, Istituto Pasteur-Cenci Bolognetti, Università di Roma Sapienza, Piazzale A. Moro 5, 00185 Roma, ItalySearch for more papers by this authorDavid S. Domozych, Department of Biology and Skidmore Microscopy Imaging Center, Skidmore College, Saratoga Springs, NY, 12866 USASearch for more papers by this authorWilliam G. T. Willats, Corresponding Author william.willats@newcastle.ac.uk School of Agriculture, Food and Rural Development, Newcastle University, Newcastle upon Tyne, NE1 7RU UKFor correspondence (e-mails mravec@plen.ku.dk; william.willats@newcastle.ac.uk).Search for more papers by this author 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 URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Summary Pectic homogalacturonan (HG) is one of the main constituents of plant cell walls. When processed to low degrees of esterification, HG can form complexes with divalent calcium ions. These macromolecular structures (also called egg boxes) play an important role in determining the biomechanics of cell walls and in mediating cell-to-cell adhesion. Current immunological methods enable only steady-state detection of egg box formation insitu. Here we present a tool for efficient real-time visualisation of available sites for HG crosslinking within cell wall microdomains. Our approach is based on calcium-mediated binding of fluorescently tagged long oligogalacturonides (OGs) with endogenous de-esterified HG. We established that more than seven galacturonic acid residues in the HG chain are required to form a stable complex with endogenous HG through calcium complexation insitu, confirming a recently suggested thermodynamic model. Using defined carbohydrate microarrays, we show that the long OG probe binds exclusively to HG that has a very low degree of esterification and in the presence of divalent ions. We used this probe to study real-time dynamics of HG during elongation of Arabidopsis pollen tubes and root hairs. Our results suggest a different spatial organisation of incorporation and processing of HG in the cell walls of these two tip-growing structures. Introduction The biological functions of plant cell walls are established not only by their composition per se but also by their precise molecular structures and intramolecular associations (Burton etal., 2010; Albersheim etal., 2011). Cell wall features can be fine-tuned according to local functional requirements, even within specific cell wall microdomains (Burton etal., 2010; Albersheim etal., 2011; Monniaux and Hay, 2016). One of the most dynamic cell wall components is the pectin homogalacturonan (HG), consisting of α-(1→4) linked d-galacturonic acid units. Post-synthetic processing of HG in muro underpins many cellular and developmental processes (Palin and Geitmann, 2012; Wolf and Greiner, 2012). It is generally accepted that HG is synthesised in the Golgi apparatus (Atmodjo etal., 2013; Anderson, 2016) and secreted in an esterified form and further processed by cell wall-located pectin methylesterases (PMEs) (Palin and Geitmann, 2012; Wolf and Greiner, 2012; Sénéchal etal., 2014; Levesque-Tremblay etal., 2015). These PMEs hydrolyse the methyl esters, producing polyanionic HG polymers than can form stable complexes (gels) with divalent cations, most commonly with calcium (Grant etal., 1973; Willats etal., 2001; Figure1a). These ‘egg box’ structures are abundant in the middle lamellae between adjacent plant cells and are critical for mediating cell-to-cell adhesion in plants (Willats etal., 2001; Bouton etal., 2002; Mouille etal., 2007; Palin and Geitmann, 2012; Daher and Braybrook, 2015). Schemeof pectin complexation and the principle of probe binding. (a) The model of pectin crosslinking in plant cell walls. Long stretches of de-esterified HG form stable complexes with calcium. These structures resemble egg boxes based on their schematic appearance. Side chains of rhamnogalacturonan I separate the stretches of adjacent HG modulating its crosslinking ability. (b) Schematic depicting the proposed principle of the binding interaction between the long oligogalacturonide (OG) probe and homogalacturonan (HG) resulting in egg box formation. A long OG conjugated to a fluorescent dye can form a complex with the endogenous HG in the presence of calcium. Approximately two calcium bridges are made per binding event. Homogalacturonan gels also largely determine the mechanical properties of cell walls. Spatiotemporally modulated de-esterification of HG has been shown to be instrumental in meristem formation (Peaucelle etal., 2008, 2011), hypocotyl elongation (Pelletier etal., 2010; Sénéchal etal., 2015) and border-like cell separation (Mravec etal., 2014). The gelling capacity of HG is not only determined by the degree of esterification (DE) but is also influenced by other structural features, such as the degree of substitution with other sugars (Willats etal., 1999, 2004; Morra etal., 2004; Figure1a). Detailed imaging of these processes in real time requires high-resolution probes with minimal processing of the living material. A large selection of carbohydrate-specific monoclonal antibodies (mAbs) commonly used to localise HG in fixed and sectioned samples already exists; however, immunohistological methods are cumbersome for use in real-time experiments and are prone to the masking effect caused by other polysaccharides which hinders the accessibility of the antibody to the epitope (Willats etal., 1999; Verhertbruggen etal., 2009; Hervé etal., 2011). Additionally, mAbs are developed under mammalian physiological conditions with a pH of 7–7.5, whereas the physiological pH of the apoplast is much lower. We have already described specialised reciprocal oligosaccharide-based probes for HG and de-acetylated chitin (chitosan) (Mravec etal., 2014). The positively charged chitosan oligosaccharide (COS) probes bind to de-esterified HG, and vice versa oligogalacturonide (OG) probes bind to chitosan. Although the COS probe localises to de-esterified HG it does not demonstrate the formation of a calcium-mediated complex or imply the ability of the target HG to do so per se. Binding of COS is dependent on ionic strength, so the binding might be influenced by other local environmental factors such as pH or the presence of other ions and polyionic molecules. Binding of COS to HG can also significantly affect cell wall homeostasis (Cabrera etal., 2010). Only one antibody (designated 2F4) has been reported to recognise HG in the egg box formation (Liners etal., 1989). Our approach was to use the complex-forming ability of HG itself as a base for producing invivo probes, and we developed a novel oligogalacturonide-based probe to visualise the sites of egg box formation insitu. We characterised the specificity of the probe using a variety of approaches and compared binding specificity with existing anti-HG probes in various plant systems. Using the new probe we were able to visualise and compare the dynamics of HG processing during pollen tube and root hair growth in real time and obtained new insight into the distinctive ways that the HG matrix operates in these tip-growing cellular structures. Synthesis of the probes and analysis of their binding ability on Arabidopsis stem sections Our aim was to use OGs long enough to form stable complexes with polygalacturonic chains in an environment containing calcium ions (Figure1b). First we tested OGs with a degree of polymerisation (DP) of 7, previously used as a probe against chitosan (Mravec etal., 2014; hereafter designated as OG7488). Co-incubation of OG7488 and 1mm calcium (CaCl2) with Arabidopsis stem sections did not yield any labelling (see below). We concluded that the DP7 is not long enough for OG to form a stable egg box complex. Hence we tried to use a longer OG preparation previously applied in physiological studies related to pathogen and hormonal signalling (Bellincampi etal., 2000; Savatin etal., 2014; Gravino etal., 2015; Gramegna etal., 2016). We precisely determined the DP distribution of this preparation and performed matrix assisted laser desorption ionisation-time of flight (MALDI-ToF) analysis, which showed that the sample is a mixture of oligomers with lengths between DP 7 and DP 13, with the most abundant DP being 10 (hereafter designated OG7-13; FigureS1 in the Supporting Information). This sample was modified at the reducing end with aminooxy derivatives of fluorophores Alexa Fluor 488 and 647, as previously described (Mravec etal., 2014), to generate probes OG7-13488 and OG7-13647, respectively. Contrary to the shorter OG7488, OG7-13488 was able to label fresh sections of Arabidopsis stem when co-incubated with calcium (1mm CaCl2) (FigureS2a–c). To determine if binding of OG7-13488 was indeed due to the formation of calcium complexes with endogenous de-esterified HG, we performed several control experiments. The lack of calcium in the labelling mixture resulted in no signal (FigureS2d), implying that exogenous calcium is required to stabilise new complexes. Furthermore, pre-treatment of sections with the HG-degrading enzymes polygalacturonase and pectate lyase diminished OG7-13488 labelling (FigureS2e–g). These initial insitu experiments showed that OGs with sufficient chain lengths can indeed associate with endogenous HG via calcium. We characterised the specificity of OG7-13488 binding by using carbohydrate microarrays populated with defined polysaccharide and proteoglycan cell wall components, as well as a set of pectins with various degrees and patterns of esterification (Pedersen etal., 2012). No binding could be observed when the arrays were incubated with OG7488 or OG7-13488 when no cation was present (Figures2 and S3). However, a highly specific recognition pattern was observed with OG7-13488 after co-incubation with divalent cations. OG7-13488 only bound to pectin samples on the arrays and the intensity of binding was inversely correlated with the DE (Figure2). The strongest binding was observed for lime pectins de-esterified to low levels by fungal (11% DE, random pattern) and plant PMEs (16% DE, block-wise pattern) with a gradual decrease of binding towards higher DEs. As expected, OG7-13488 also bound to polygalacturonic acid (Danisco) and commercial pectin (CPKelco). Some binding was observed for rhamnogalacturonan I (RGI) isolated from potato for all probes, suggesting the presence of de-esterified HG as part of the RG I in this preparation (FigureS3). The strength of binding correlated with the increasing concentration of calcium, with detectable binding starting at 0.5mm CaCl2 (Figures2 and S3). The divalent zinc cation (1mm ZnCl2) was able to mediate binding to the pectin samples, with more potency than 1mm Ca2+ and a lower stringency in distinguishing between the different DE values of pectin samples. For instance, Zn2+ promoted very similar binding to lime pectins, with DE 11% and DE 31% (random pattern of esterification). This observation supports recent thermodynamic models in which Zn2+ is a stronger HG binder (Assifaoui etal., 2015) and we suggest that one zinc atom alone is sufficient to complex one OG7-13 molecule to non-esterified HG. Determination of the specificity of the OG7-13488 using defined carbohydrate arrays. Anti-pectin probes were incubated with defined arrays containing a large selection of cell wall components. The intensity of the heatmap colour represents the strength of the binding and is directly correlated to the numerical value. The strongest signal was assigned value 100. The cut-off signal was set to 10. No binding could be seen for OG7488 or when no cation (calcium or zinc) was present. OG7-13488 bound only to pectins with a relatively low degree of esterification. The strength of the binding correlated with the concentration of CaCl2; 0.5mm but not 0.1mm CaCl2 was sufficient for binding. Incubation with 1mm ZnCl2 resulted in binding with higher strength than 1mm CaCl2 but with lower discrimination between the degree of esterification (DE). The binding of OG7-13488 was compared with other oligosaccharide probes COS488 and 2F4 monoclonal antibody. Note the binding to the same samples but with different binding intensities with respect to DE. RGI, rhamnogalacturonan I; AGP, arabinogalactan protein. Taken together the microarray data established that OG7-13488 binds to HG in a manner that is dependent on the presence of cations, and that is related to the DE of the HG. In comparison with 2F4 and COS488, OG7-13488 bound to a similar set of HG-containing samples on the arrays but only OG7-13488 binding is based on de novo formation of egg box structures with HG. Arabidopsis stems are widely used as a model to study specialised cell walls of differentiated cell types. First, we studied the binding of oligosaccharide-based probes on fresh stem sections obtained from 1-month-old Arabidopsis plants from the region 1–2cm above the leaf rosette. FigureS4(a,b) shows phluoroglucinol and toluidine blue staining of the sections with the indication of the different cell types. By using OG7-13488 on the same sections, labelling was visible in all cells with primary cell walls, with no detectable signal in the cells with secondary cell walls of xylem and interfascicular tissue (FigureS4c). Contrary to OG7-13488, COS488 labelling could also be observed in interfascicular tissues; however, COS488 staining was generally weaker in the phloem and cortex cell walls compared with OG7-13488 (FigureS4d). These not entirely identical binding patterns of OG- and COS-based probes point to potentially different penetrative abilities or slightly different affinities of the two probes to HG as observed on microarrays. A closer examination of pith parenchyma revealed the highest binding of both OG7-13488 and the far-red variant OG7-13647 to triangular junctions with enhanced binding to cell corners (FigureS4e,f). By scanning with higher photomultiplier (PMT) gain settings, we could also detect a more delocalised signal distributed throughout the cell walls (FigureS4f,g). Consistent with the results from defined microarrays, the binding of OG7-13488 insitu was also strongly dependent on calcium concentration: a CaCl2 concentration of at least 0.5mm was required for binding, and labelling was restricted to the corners of the cell (FigureS5). Co-labelling with Calcofluor White, a dye specific for β-glucans, and JIM7 mAb, which recognises HG with a high DE, revealed distinct cell wall microdomains of HG esterification levels in muro (Figure3). The JIM7 signal was present in the cell wall region closer to the plasma membrane, while the OG7-13488 signal was localised to the lining of intercellular spaces (Figure3). Co-localisation analysis of OG7-13488 and JIM7 monoclonal antibody (mAb) in cells of Arabidopsis pith parenchyma. (a) OG7-13488 labelling of the pith parenchyma cell walls in the region of three-cell connection (cells are marked as c1–c3 and intercellular space as ‘is’). The labelling can be seen in the walls forming triangular junctions (open arrowhead) with particularly strong binding to cell corners (closed arrowhead) and weaker staining of the middle lamella (arrow). (b) Signal from the labelling with JIM7 mAb (and secondary anti-rat antibody conjugated to Alexa Fluor 555) marks plasma membrane in the proximal part of the cell walls. (c) Calcofluor White (CW) labelling of cell wall β-(1,4)-glucans. (d) Overlay of the three channels demonstrates distinct cell wall microdomains with homogalacturonan with different levels of esterification. Scale bar=5μm. Subsequently, we studied the properties of OG7-13488-binding sites in higher resolution on semi-thin sections of resin-embedded Arabidopsis stems (for morphological analyses see Figure4a,b) and performed several types of pre-treatments of the sections before labelling (Figure4c–h): (i) polygalacturonase treatment completely removed the OG7-13488 binding sites, whereas (ii) pre-treatment with the calcium chelating agent 1,2-diaminocyclohexanetetraacetic acid (CDTA) destroyed endogenous egg boxes, thus exposing more binding sites for OG7-13488. This could be observed as a stronger signal in triangular intercellular junctions and a new signal in middle lamellae (Figure4f). (iii) Pre-treatment with PME generated binding sites from the hitherto esterified HG and, as expected, OG7-13488 binding was observed throughout cell walls. (iv) De-esterification by chemical means using 100mm NaOH had the same effect as PME treatment. These experiments demonstrate that OG7-13488 does not bind to existing egg box sites, which can be destroyed by calcium chelation; they also show that there is a considerable amount of HG that is not available for binding to OG7-13488, due to the presence of ester groups. OGs can be therefore used in conjunction with different treatments to study pre-existing egg boxes, readily available sites and the esterified stretches of HG. OG7-13488 binding sites in resin-embedded Arabidopsis stem. (a, b) Toluidine blue staining of 1μm thick sections of the Arabidopsis stem. (a) The region of the stem encompassing the different tissue: xy, xylem; co, cortex; ep, epidermis; ss, starch sheath’ ph, phloem; phc, phloem cap; pp, pith parenchyma. (b) Close-up of the region analysed. Note the secondary cell walls of interfascicular fibres in blue stemming from lignin and in violet from the more acidic components of primary cell walls. (c–h). Analysis of the OG7-13488 binding sites on the resin-embedded sections. The pictures are an overlay of OG7-13488 signal (green) and counterstain with Calcofluor White (blue). Upper panels show the region from the cortex through interfascicular fibres to parenchyma; lower panels, the pith parenchyma in the centre of the stem. (c) Control labelling with no probe used. (d) Labelling with OG7-13488. Note the labelling of triangular junction/cell corners in all cell types (arrowheads). (e) Pre-treatment with polygalacturonase diminishes the OG7-13488 binding sites. (f) Pre-treatment with chelating agent 1,2-diaminocyclohexanetetraacetic acid (CDTA) exposes new binding sites. Note the enhanced signal in the triangular junction and middle lamella (arrowhead). (g), (h) Pre-treatment with (g) orange peel pectin methylesterase (PME) and (h) NaOH creates new binding sites from esterified homogalacturonan which could be seen throughout the cell wall. Scale bars=20μm. 2F4 mAb differs from OG7-13488 in labelling of cell wall microdomains in stem sections We compared OG7-13488 labelling with that by 2F4 mAb, reported to recognise the HG-Ca2+ complex (Liners etal., 1989). The labelling obtained with 2F4 was generally weak and confined to the tissues also stained by OG7-13488, that is the epidermis, cortex and pith parenchyma (FigureS6). Pre-treatment with polygalacturonase completely removed the signal from primary cell walls, showing the specificity of 2F4 towards HG (FigureS6). Closer examination at higher magnification showed some labelling of cell wall microdomains but this was not well defined in the triangular junctions. Thus, although giving very similar recognition pattern in microarray analysis, the 2F4 antibody and OG7-13488 show a different insitu labelling pattern, which could be due to a difficulty of the antibody with penetrating cell wall matrices or due to masking of the epitopes by other polysaccharides. We also tested OG7-13488 as an anti-HG probe invivo on whole organisms. The synthesis of HG originated early in the evolution of the Streptophyta, i.e. the group of extant green plants that includes the charophycean green algae and land plants (Sørensen etal., 2011). Many unicellular species of the charophycean group Zygnematophyceae possess cell walls that are rich in HG complexes that form highly distinctive structures on their outer wall surfaces. The topology of these structures and the dynamic events that occur during their formation can be monitored using live cell labelling protocols (Domozych etal., 2014). Previous methods used to visualise these structures have employed anti-HG mAbs and COS probes (Sørensen etal., 2011; Domozych etal., 2014; Mravec etal., 2014). In this study, we tested the new probe invivo on three species of unicellular charophytes. Labelling with OG7-13488 revealed in high resolution the HG-rich cell wall structures that are typical for the particular species (Figure5): the lattice-like assembly of the outermost layer of the cell wall of Penium margaritaceum, the outer fringes of the pore complexes found in the secondary cell wall of Closterium acerosum and the punctate pore coverings of the cell wall of Micrasterias sp. These experiments demonstrate the diversity of the calcium-crosslinked pectin gel matrix, which very likely gives these structures mechanical stability, and might also support the role of these pectin polymers in the evolution of the middle lamella (Domozych etal., 2014). They also show that OG-based probes represent a rapid and effective tool in the study of the evolutionary aspects of HG function in plant multicellularity. In vivo labelling of unicellular charophytes with OG7-13488. Labelling of (a) Penium margaritaceum, (b) Closterium acerosum and (c) Micrasterias sp. with OG7-13488. In (a) and (b) left panels show the scan of the entire alga and right panels show a close-up of the surface structures. Note the fine resolution of the intricate cellular structures present on the surface of these algae. Scale bars=5μm. Some of the best models for studying rapid cell wall formation in land plants are tip-growing cellular structures: pollen tubes and root hairs (Gu and Nielsen, 2013; Hepler etal., 2013; Rounds and Bezanilla, 2013; Grierson etal., 2014; Larson etal., 2014). We utilised Arabidopsis pollen tubes and root hairs to test the versatility of the probes further through real-time analysis of HG dynamics. It is known that the cell walls of pollen tubes are primarily constructed of pectins with relatively low amounts of cellulose (Lehner etal., 2010; Chebli etal., 2012). Analyses of pollen-specific PME and PMEI trafficking and immunolocalisation of HG epitopes suggest that methyl-esterified HG is secreted at the tip and is then de-esterified behind the tip by the PMEs released from inhibition by PMEIs (Röckel etal., 2008; Dardelle etal., 2010). Immunohistological experiments showed that the newly synthesised highly esterified HG recognised by JIM7 mAb was present at the tip whereas the partially de-methyl-esterified HG recognised by JIM5 mAb was located in the subapical region, suggesting a ‘fountain-like’ progression of HG secretion and subsequent de-esterification (Dardelle etal., 2010; Lehner etal., 2010; Sanati Nezhad etal., 2014). We performed co-labelling experiments of the pollen tubes with OG7-13488 and JIM7 to corroborate this theory, and we could indeed observe tip-specific JIM7 labelling that was progressively replaced by the OG7-13488 signal present at the shank but completely absent at the tip of the pollen tube (Figures6a and S7). Although co-labelling with JIM5 was not successful, most likely because of partially shared epitopes, we could confirm the absence of JIM5 binding to the tip of the pollen tube (Figure6b). In situ and real-time study of homogalacturonan (HG) dynamics in Arabidopsis pollen tubes. (a) Co-labelling of fixed pollen tubes with OG7-13488, JIM7 monoclonal antibody (mAb) recognising HG with a high degree of esterification (DE) and Calcofluor White (CW) specific for cell wall β-glucans. Note the tip-localised JIM7 but shank-localised OG7-13488 labelling. (b) Labelling of pollen tube with JIM5 mAb recognising HG with a low DE. Note the absence of the signal at the tip of the pollen tube. (c) Schemeof the setup used for the invivo pollen tube staining experiment. Pollen tubes were germinated in the chambered cover slip in the germination medium containing the probes and recorded using an inverted confocal microscope. (d) Time course of real-time imaging of a pollen tube growing in a medium containing the OG7-13488 probe over the 32-min long interval. The OG7-13488 is continuously incorporated at the shank of the pollen tube. Scale bars=5μm. Next, we monitored the incorporation of OG7-13488 in pollen tubes in real time. First, we investigated whether the OG had any inhibitory effect on germination or tube elongation. OG7-13 at a concentration of 10μgml−1 in the medium had some negligible effect on the rate of germination (FigureS8). To accomplish real-time visualisation, we let the pollen germinate and grow directly inside chambered coverslips filled with germination media supplemented with the probe (1:500 dilution) and scanned by an inverted laser scanning confocal microscope (LSCM) (Figure6b). Since the standard pollen germination medium (Rodriguez-Enriquez etal., 2013) already contained 1mm CaCl2 there was no requirement for any further modification or supplementation. Sequential scans of the growing pollen tube indeed showed that the probe is being continuously incorporated in the main body of the pollen tube over the course of its elongation (Figure6c, Movie S1). The main body is conclusively the site of the HG crosslinking required for integrity of the entire cellular structure (Fayant etal., 2010). These experiments demonstrate the potential for real-time imaging of HG post-synthetic processing using an OG probe and strongly support the proposed model of pectin matrix layout in pollen tubes. A root hair is an elongated tube-like cellular structure derived from a root trichoblast that develops via a polar, tip-growing mechanism (Gu and Nielsen, 2013; Grierson etal., 2014). We used Arabidopsis root hairs for developmental analysis of the pectin dynamics involved in polar growth and for comparison with the pollen tube. Unlike in the pollen tube, invivo labelling of the root hairs with OG7-13488 showed a rather even distribution of the signal along the root hairs from the point of their initiation to the apex (Figure7a–f). This could be quantified as a ratio between the signal present at the tip and the main body at an approximate distance of 20μm from the apex (Figure7g). Moreover, when grown in the presence of the PME inhibitor (–)-epigallocatechin gallate (Lewis etal., 2008; Wolf etal., 2012) or pre-digested with polygalacturonase, the OG7-13488 signal was strongly reduced along the entire root hair (FigureS9a–c). These experiments indicated that, unlike in the pollen tube, de-esterified HG available for crosslinking is present throughout the root hair cell wall, including its tip. In order to further elaborate on this observation, we performed, in a similar manner as for the pollen tube, real-time imaging of the growing root hair in the MS medium. Movie S2 shows the young root hair after emergence scanned over a period of 45min during which it elongated by approximately 1–1.5μm. The signal of bound OG7-13488 significantly increased over this period in the entire cell wall but without any specific pattern. Dynamics of homogalacturonan (HG) secretion and de-esterification in Arabidopsis root hairs. (a) In vivo labelling of the root hairs with OG7-13488 (green). Left panel: Early stage of root hair emergence. Right panel: elongated root hair was approximately 100μm in length. The labelling could be seen equally distributed along the root hairs. (b–f) Close-up of the root hair tip co-labelled with (b) OG7-13488 and (c) Calcofluor White (CW). (d) Overlay of the two channels with marked regions used for signal quantification in (g). (e) Plasma membrane staining with FM4-64. Note the endocytosis event (arrowhead) which is not visible in the OG7-13488 channel. (f) Differential contrast channel (DIC). (g) Quantification of the signal ratio between the tip and shaft region (marked by numbers in d) of CW labelling and OG7-13488 labelling. In both cases the ratio is close to 1, suggesting equal distribution of the labelled epitopes (n=11; error bars represent SEM). (h) Quantification of the sequential labelling experiment. Seedlings were first labelled with the OG7-13647 probe (initial labelling), washed, left to grow for 4h and relabelled with the OG7-13488 probe (final labelling marking the new available epitopes). The root hairs were scanned and the signal ratios between the tip and the shaft calculated as in (g) for both signals. The ratios for both probes are close to 1 (n=8; error bars represent the SEM). (i) Whole mount root hair immunolocalisation of highly esterified HG with the JIM7 monoclonal antibody (mAb). Note the punctate pattern marked by the arrowhead. (j), (k) Scan of the root hairs labelled with JIM7 mAb (red) and CW (blue). (j) The scan of the surface of the root hair approximately 30μm from the apex. Note the potential secretion events marked by the arrowhead. (k) Scan of the longitudinal section of the resin embedded root hair labelled with JIM7. Note the labelled intracellular compartments marked by the arrowhead. (l), (m) Scan of the cellular compartment marker lines: (l) wave 18R marking the Golgi apparatus and (m) secreted RFP (secRFP). In both cases the compartments containing RFP are not exclusively accumulated in the tip region but are also positioned along the wall of the root hair (arrowheads). (n) brefeldin A (BFA) treatment aggregates the secretory vesicles marked by secRFP construct in the BFA compartment (arrowhead) which is distant from the tip. Scale bars=10μm. Longer real-time scanning of entire root hairs is very difficult due to constant root movement and stress inflicted upon the root hairs. In order to monitor the formation of OG7-13-binding sites over a longer period of time, we performed sequential labelling experiments using two fluorescent variants of the probe (Figures7h and S9d–h). Firstly, we labelled the seedlings using the red variant OG7-13647, washed them and let them grow in the MS medium for an additional 4h. The seedlings were re-labelled with the green variant OG7-13488 and observed. In theory, this experiment should visualise initial OG7-13-binding sites and those freshly formed over the 4-h growth period. The scans and overlays of the two channels showed a large overlap with no clear distinction (FigureS9g), which again pointed to the possibility that the formation of OG7-13 epitopes has no elaborate spatial pattern as is the case for pollen tubes. To confirm these observations we labelled fixed whole mount root hairs with the JIM5 and JIM7 mAbs (Figures7i,j and S9i,j). No signal could be observed for JIM5, which is in line with previous reports on live non-sectioned material (Larson etal., 2014). On the other hand labelling with JIM7 produced a specific signal which could be observed along the root hair cell wall but often appeared in a punctate pattern, suggesting distinct periodic secretion events (Figure7i,j). Interestingly, a similar pattern was also noted in the visualisation of pectin post-synthetic delivery using click chemistry (Anderson etal., 2012). In addition, in longitudinal sections of the resin-embedded root hairs, the JIM7 signal was observed inside cellular compartments (Figure7k). To relate the observed pattern to the general secretion machinery that operates in the root hair, we also analysed the mRFP Golgi marker line, wave 18R (Geldner etal., 2009), and a line expressing mRFP fused with a secretion signal (secRFP; Faso etal., 2009). Golgi compartments marked with mRFP signal in wave 18R were detectable alongside the root hair (Figure7l). Similarly, secRFP-marked compartments were equally distributed below the wall along the root hair and did not exclusively accumulate at the tip (Figure7m). These compartments showed aggregation in the presence of the exocytosis inhibitor brefeldin A (BFA) (Klausner etal., 1992; Geldner etal., 2001). The aggregates (‘BFA bodies’) were found in the centre of the hair at a considerable distance from the tip (Figure7n). These results strongly suggested that the spatial distribution of the secretion machinery is not exclusively restricted to the apical region. We demonstrated the potential for real-time imaging of calcium-mediated crosslinking of HG in muro using fluorescently tagged long oligogalacturonides (OG7-13). This oligosaccharide-based type of probe has similar but not identical specificity towards HGs with low DE as the COS probe reported earlier (Mravec etal., 2014). The main advantage resides in direct one-step labelling in a buffer with a pH value close to the apoplastic pH or directly in the growth medium. The ease of conjugation of OGs to other molecules using reducing ends offers high versatility in the choice of the detection tags (fluorescent, nanogold or enzymatic). Different fluorescent variants enable studies on the progression of egg-box-site formation during the growth of plant organs and can also be used in multispectral co-labelling experiments. The advantage of the OG7-13 probe over the COS probe is that it does not rely solely on the ionic interaction which can occur with other polycationic molecules. Moreover, it utilises an endogenously occurring phenomenon that most likely does not extensively affect cell wall homeostasis of the studied object at a low concentration of the probe. Additionally, we propose that the de-esterified HG regions that bind to the COS probe due to the presence of oligo/polyanionic blocks that are long enough for ionic interactions are different from the regions bound by the novel OG7-13 probe, which probably requires fewer calcium-mediated interactions. This provides additional and not necessarily equivalent information about the relation of the local degree of de-esterification of the HG and the ability of HG to form calcium complexes as two similar but not necessarily coinciding properties as indicated by microarray analysis. On the other hand OGs are also damage-associated molecular patterns (DAMPs; Vallarino and Osorio, 2012; Ferrari etal., 2013), and invivo labelling might trigger defence-related signalling. This could be overcome by using the lowest possible concentration of the probe and short labelling times. The concentration of the OG7-13 used in this study is one order of magnitude lower than that used for studies of the DAMP effect and showed no significant retardation of the growth when tested on pollen. Another drawback associated with the OG7-13 probe is that OGs are relatively long and charged molecules which might make them unsuitable for invivo labelling of plant organs covered with a cuticle. Here, we utilised an OG7-13 probe to study HG secretion and de-esterification dynamics on two tip-growing cellular structures. Both root hairs and pollen tubes elongate in a unidirectional fashion, but there are important differences in their function, size and the velocity of their elongation. For instance the pollen tube can elongate at a much higher rate (up to 1μmsec−1) than a root hair (up to 1μmmin−1) (Chebli etal., 2012; Grierson etal., 2014). These physiological differences are clearly reflected in the specific mechanisms of build-up of their nascent cell walls. Based on our results we postulate a hypothesis that the pectin components of root hair cell walls are formed by a uniformly deposited pectin matrix rather than an elaborate tip secretion/shank de-esterification pattern that is typical of the pollen tube. Although not fully proven, this idea provides an interesting base for future research on the biology of root hair cell walls. The presented probe can also be used in thermodynamic analysis of egg box formation. By comparing the complex-forming ability of OGs of different lengths (DP7 compared with DP7-13) we also support the recently proposed molecular dynamics model of insitu egg boxes (Grant etal., 1973; Braccini and Pérez, 2001; Assifaoui etal., 2015). The model, based on isothermal titration calorimetry (ITC) experiments and consequent calculations, proposes that for a stable crosslink-forming complex to form, one calcium ion needs to mediate an interaction between at least every fifth pair of galacturonic acid units (Assifaoui etal., 2015). By extrapolating these data, we can approximate that the binding event occurs only with OG probes that are long enough to allow at least two calcium ion bridges with HG necessary to stabilise the complex sufficiently for binding to be detectable. We also support the notion that zinc has a different capacity as a mediator of the complex and possibly binds to galacturonic acid as to a monodentate ligand (Assifaoui etal., 2015). On the basis of that information and our data, we propose that most likely only one zinc cation is required to mediate binding of OG7-13 to HG. This supports our hypothesis that when OG7-13 probes bind to a stretch of de-esterified HG, they need approximately two calcium ions to allow binding at two binding sites, whereas COS probes probably depend on more binding sites mediated by the interaction of positively charged protonated amino groups and negatively charged deprotonated carboxyl groups. In conclusion, this type of small oligosaccharide-based molecular probe is an important addition to the repertoire of cell wall imaging tools. The demonstrated versatility of the long OG probe that depends on calcium complexation for binding adds another dimension to COS probes, whose interaction with HG is based on ionic interaction. We believe that future application of this probe will greatly facilitate studies of the challenging aspects of HG dynamics, such as in meristem formation and tissue patterning, as well as provide a highly efficient and flexible tool for real-time invivo imaging. We used the following lines of Arabidopsis thaliana: wild-type Col-0, secRFP (Faso etal., 2009) and wave 18R (Geldner etal., 2009). The adult plants were grown in the greenhouse under long day at 20–23°C. Arabidopsis seedlings were grown vertically on solid MS medium (Duchefa, M0221) with 1% sucrose for 3days before analyses. The flowers of 4- to 5-week-old plants were used for pollen germination studies which were performed on the pads made of medium covered by cellophane in a wet chamber as described (Rodriguez-Enriquez etal., 2013). The charophytes Penium margaritaceum, Closterium acerosum and Micrasterias sp. were grown in sterile liquid Woods Hole medium at 18°C under a 16-h light, 8-h dark photocycle. Cells were collected during log phase growth (i.e. 10–14days after the start of a subculture) by gentle centrifugation (500g for 1min), and were washed three times with fresh medium before the labelling experiments. Sample of OGs used to generate the conjugates to fluorescent tags were prepared as previously described (Pontiggia etal., 2015). The OGs were conjugated to aminoxy derivatives of the Alexa Fluor dyes 488 and 647 (A30629 and A30632, Invitrogen, http://www.invitrogen.com/) using the method described for OG7 (Mravec etal., 2014). The concentration of the synthesised probes is approximately 1mgml−1. The Arabidopsis stem sections for vibratome sectioning were embedded in 8% agarose and 100μm thick sections were generated using a Leica VT1000S vibratome (http://www.leicabiosystems.com/). The samples were incubated in the solution in the microsieves. Calcofluor white (Fluorescent Brightener 28; F3543, Sigma, http://www.sigmaaldrich.com/) was used as a counterstain from (10mgml−1) stock solution in water at 1:100 final dilution. The FM4-64 (T-3166, Invitrogen, http://www.invitrogen.com/) was used as 1mm stock solution in DMSO and a final concentration of 1μm. For embedding in LR White resin (62661, Sigma) the specimens were fixed in 4% formaldehyde in PBS washed and dehydrated in series of methanol:water mixtures up to 100% methanol. The methanol was substituted by 1:1 mixure methanol:LR White resin for at least 8h and samples were finally embedded in pure resin overnight. The polymerisation was initiated at 60°C overnight. The blocks were cut on a Leica EM-UC7 ultramicrotome and the 1μm thick sections were mounted on SuperFrost slides. Staining was then performed on the slides using the same conditions as for fresh samples. The pollen was germinated on the cellophane pads which were removed after 4h and fixed with 4% formaldehyde (Sigma) in a 24-well plate. The 4-day-old seedlings were removed from the MS plates to microsieves and fixed with 4% formaldehyde. The fixation was followed by two washes with PBS. The whole mount samples and resin sections adhered to SuperFrost slides were blocked with 5% milk powder in PBS for 15min and probed with primary antibody. Probing with JIM5 and JIM7 mAbs (PlantProbes, http://www.plantprobes.net/) was done using 1:10 diluted mAbs in 5% milk powder/PBS. Probing with 2F4 mAb (Plant Probes) was done using 1:100 diluted mAb in T/Ca/S buffer [20mm 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS)-HCl, 0.5mm CaCl2 and 150mm NaCl, pH 8.2] with 5% milk powder. After 1h incubation the probing solution was removed and samples were washed three times with PBS and probed for 1h with anti-rat secondary antibody conjugated to Alexa Fluor 555 (A-21434, Invitrogen) in the case of JIM5 and JIM7 or anti-mouse antibody conjugated to Alexa Fluor 488 (115-545-044, Jackson Immunoresearch, https://www.jacksonimmuno.com/) diluted 1:300 in 3% BSA in PBS. The samples were washed three times with PBS and probed with OG7-13488 and Calcofluor White as other samples. After repeated washings and collection by centrifugation (500g for 1min), the algal cell pellets were resuspended in Woods Hole medium containing 25mm 2-(N-morpholine)-ethanesulphonic acid) (MES) and supplemented with 1.5mm CaCl2. The pH was adjusted to 5.7 with KOH. After 10min the cells were centrifuged and the pellet resuspended in a solution of 1μlml−1 of OG7-13488 in the aforementioned pH 5.7 buffer. The cells were incubated in the dark with gentle shaking for 90min. The cells were then collected by centrifugation and the pellets were washed three times with Woods Hole medium. Microscopic observation occurred within 4h of labelling. For the enzymatic treatment of the sections we used the endo-polygalacturonase (E-PGALUSP, Megazyme, https://www.megazyme.com/) in 100mm sodium acetate buffer pH 4.2 at 5Uml−1, pectate lyase (E-PECLY, Megazyme) in 100mm TRIS-HCl, pH 8.0 at 0.5Uml−1 and orange peel PME (P5400, Sigma) in PBS pH 7.6. at 1Uml−1. Incubations were done at room temperature (22–24°C). After the treatment, the samples were washed twice with 25mm MES buffer, pH 5.7 and probed like the other samples. Then 50mm CDTA was used for a short wash (10min) to remove endogenous calcium. Epigallocatechin gallate (E4143, Sigma) stock was prepared at 10mm in water and used at a final concentration of 50μm. The BFA (B7651, Sigma) stock was prepared in DMSO and used at a concentration of 50μm for 1h following washout and probing. The OG probing was performed in 25mm MES buffer, pH 5.7, with 1mm CaCl2 (if not indicated otherwise) for 30min, washed twice and co-labelled with other dyes. The samples were mounted in 10% glycerol in MES buffer. The labelled samples were scanned using a Leica SP5 or Olympus Fluoview 1200 confocal laser scanning microscope equipped with UV diode (405nm), argon (488nm) and helium–neon (543nm) lasers. All images presented were recorded on root hairs shorter than 100μm (except for Figure7n) and for all experiments considerable numbers of root hairs (usually at least three from five independent roots) below 20μm were also inspected. Pollen tubes were analysed 4h after germination. Real-time imaging of pollen tube growth was performed in liquid pollen germination medium (Rodriguez-Enriquez etal., 2013) in four-chambered cover slips (Thermo-Fisher, 155383) with a probe dilution of 1:500. The real-time imaging of root hairs was performed in MS medium supplemented with 1mm CaCl2 and OG7-13488 at 1:500 dilution. All imaging was performed at room temperature. The light microscopy images were taken on an Olympus BX41 equipped with a ColourView I camera. The signal intensities were calculated using ImageJ (https://imagej.nih.gov/ij/). The images were processed using GIMP2 software for contrast and brightness enhancement and images for signal comparison were treated equally. The defined carbohydrate microarrays were prepared by printing carbohydrate solutions on the nitrocellulose membrane using microarray printers as described (Pedersen etal., 2012; Mravec etal., 2014). The arrays were blocked in 5% milk/PBS for 30min and then incubated in 25mm MES, pH 5.7, containing the OG conjugates at a 1:500 dilution for 1h at 4°C with various concentrations of CaCl2 or ZnCl2. The arrays were washed with MES buffer twice, dried and scanned by GenePix scanner using the 488nm laser. The signals on the scans were quantified using ProScanArray Express software (Perkin Elmer, http://www.perkinelmer.com/) by quantifying the colour saturation of spot signals. The values for all the signals were then normalised to a scale of values from 0 to 100 and transformed into a heatmap. The values for 2F4 mAb staining were normalised separately as that staining was performed differently because of indirect immunodetection, as described in the section Immunohistochemistry. JM, SKK and WGTW designed the study, DP prepared the oligogalacturonides under the supervision of GDL, SKK synthesised the probes, JM and DD performed the insitu experiments, SKK, MGR and BW performed the biochemical analysis, JM, SKK, GDL, DSD and WGTW wrote the manuscript. This work was supported by EU FP7 Marie Curie IEF project CeWalDyn under contract 329830 (JM), the B21st project from the Danish Advanced Technology Foundation (JM and WW), the BioValue SPIR project, funded by The Innovation Fund Denmark (case no. 0603-00522B), the Villum Foundation PLANET project (JM, SKK and WW) and the National Science Foundation (USA) grants NSF-MCB-RUI 1517345 and NSF-MRI 1337280 (DSD). tpj13574-sup-0001-FigS1.pdfPDF document, 193 KB FigureS1. The matrix assisted laser desorption ionisation-time of flight analysis of the oligogalacturonide sample used to prepare the probes to show the distribution of the length of the galacturonic acid residues. tpj13574-sup-0002-FigS2.pdfPDF document, 339.1 KB FigureS2. Specificity of OG7-13488 binding studied on the stem sections of Arabidopsis stem. tpj13574-sup-0003-FigS3.pdfPDF document, 251.2 KB FigureS3. The comprehensive microarray binding data of the anti-homogalacturonan probes including all carbohydrates present on the defined arrays. tpj13574-sup-0004-FigS4.pdfPDF document, 396.2 KB FigureS4. Comparative analysis of the oligogalacturonide- and chitosan oligosaccharide-derived probes labelling pattern on fresh sections through the Arabidopsis stems. tpj13574-sup-0005-FigS5.pdfPDF document, 278.6 KB FigureS5. Analysis of calcium dependence of OG7-13488 binding insitu. tpj13574-sup-0006-FigS6.pdfPDF document, 461.8 KB FigureS6. Analysis of 2F4 mAb labelling pattern of fresh Arabidopsis stem sections. tpj13574-sup-0007-FigS7.pdfPDF document, 263.6 KB FigureS7. Control analysis of OG7-13488 pollen tube labelling. tpj13574-sup-0008-FigS8.pdfPDF document, 332.6 KB FigureS8. Analysis of the physiological effect of oligogalacturonides on pollen germination and pollen tube elongation. tpj13574-sup-0009-FigS9.pdfPDF document, 352.9 KB FigureS9. Additional analysis of the homogalacturonan dynamics in root hairs including control treatments, sequential labelling, and immunolocalisation on fixed whole mount root hairs using JIM5 and JIM7 mAbs. tpj13574-sup-0010-MovieS1.mp4MPEG-4 video, 8.6 MB MovieS1. Real-time scanning of an elongating pollen tube labelled with OG7-13488 present directly in the pollen germination medium over the period of 40min. tpj13574-sup-0011-MovieS2.mp4MPEG-4 video, 8.7 MB MovieS2. Real-time scanning of an elongating root hair with OG7-13488 present directly in the MS medium over a period of 45min. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Albersheim, P., Darvill, A., Roberts, K., Sederoff, R. and Staehelin, A. (2011) Plant Cell Walls. New York, USA: Garland Science. Anderson, C.T. (2016) We be jammin’: an update on pectin biosynthesis, trafficking and dynamics. J. Exp. Bot. 67, 495– 502. Anderson, C.T., Wallace, I.S. and Somerville, C.R. (2012) Metabolic click-labeling with a fucose analog reveals pectin delivery, architecture, and dynamics in Arabidopsis cell walls. Proc. Natl Acad. Sci. USA, 109, 1329– 1334. Assifaoui, A., Lerbret, A., Uyen, H.T.D., Neiers, F., Chambin, O., Loupiac, C. and Cousin, F. (2015) Structural behaviour differences in low methoxy pectin solutions in the presence of divalent cations (Ca2+ and Zn2+): a process driven by the binding mechanism of the cation with the galacturonate unit. Soft Matter, 11, 551– 560. Atmodjo, M.A., Hao, Z. and Mohnen, D. (2013) Evolving views of pectin biosynthesis. Annu. Rev. Plant Biol. m64, 747– 779. Bellincampi, D., Dipierro, N., Salvi, G., Cervone, F. and De Lorenzo, G. (2000) Extracellular H(2)O(2) induced by oligogalacturonides is not involved in the inhibition of the auxin-regulated rolB gene expression in tobacco leaf explants. Plant Physiol. 122, 1379– 1385. Bouton, S., Leboeuf, E., Mouille, G., Leydecker, M.T., Talbotec, J., Granier, F., Lahaye, M., Höfte, H. and Truong, H.N. (2002) QUASIMODO1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion in Arabidopsis. Plant Cell, 14, 2577– 2590. Braccini, I. and Pérez, S. (2001) Molecular basis of Ca2+-induced gelation in alginates and pectins: the egg-box model revisited. Biomacromolecules, 4, 1089– 1096. Burton, R.A., Gidley, M.J. and Fincher, G.B. (2010) Heterogeneity in the chemistry, structure and function of plant cell walls. Nat. Chem. Biol. 6, 724– 732. Cabrera, J.C., Boland, A., Cambier, P., Frettinger, P. and Van Cutsem, P. (2010) Chitosan oligosaccharides modulate the supramolecular conformation and the biological activity of oligogalacturonides in Arabidopsis. Glycobiology, 20, 775– 786. Chebli, Y., Kaneda, M., Zerzour, R. and Geitmann, A. (2012) The cell wall of the Arabidopsis pollen tube – spatial distribution, recycling, and network formation of polysaccharides. Plant Physiol. 160, 1940– 1955. Daher, F.B. and Braybrook, S.A. (2015) How to let go: pectin and plant cell adhesion. Front. Plant Sci. 14, 523. Dardelle, F., Lehner, A., Ramdani, Y., Bardor, M., Lerouge, P., Driouich, A. and Mollet, J.C. (2010) Biochemical and immunocytological characterizations of Arabidopsis pollen tube cell wall. Plant Physiol. 153, 1563– 1576. Domozych, D.S., Sørensen, I., Popper, Z.A. etal. (2014) Pectin metabolism and assembly in the cell wall of the charophyte green alga Penium margaritaceum. Plant Physiol. 165, 105– 118. Faso, C., Chen, Y.N., Tamura, K. etal. (2009) A missense mutation in the Arabidopsis COPII coat protein Sec24A induces the formation of clusters of the endoplasmic reticulum and Golgi apparatus. Plant Cell, 21, 3655– 3671. Fayant, P., Girlanda, O., Chebli, Y., Aubin, C.E., Villemure, I. and Geitmann, A. (2010) Finite element model of polar growth in pollen tubes. Plant Cell, 22, 2579– 2593. Ferrari, S., Savatin, D.V., Sicilia, F., Gramegna, G., Cervone, F. and Lorenzo, G.D. (2013) Oligogalacturonides: plant damage-associated molecular patterns and regulators of growth and development. Front. Plant Sci. 4, 49. Geldner, N., Friml, J., Stierhof, Y.D., Jürgens, G. and Palme, K. (2001) Auxin transport inhibitors block PIN1 cycling and vesicle trafficking. Nature, 413, 425– 428. Geldner, N., Dénervaud-Tendon, V., Hyman, D.L., Mayer, U., Stierhof, Y.D. and Chory, J. (2009) Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set. Plant J. 59, 169– 178. Wiley Online Library Gramegna, G., Modesti, V., Savatin, D.V., Sicilia, F., Cervone, F. and De Lorenzo, G. (2016) GRP-3 and KAPP, encoding interactors of WAK1, negatively affect defense responses induced by oligogalacturonides and local response to wounding. J. Exp. Bot. 67, 1715– 1729. Grant, G.T., Morris, E.R., Rees, D.A., Smith, P.J.C. and Thom, D. (1973) Biological interactions between polysaccharides and divalent cations – egg-box model. FEBS Lett. 32, 195– 198. Wiley Online Library Gravino, M., Savatin, D.V., Macone, A. and De Lorenzo, G. (2015) Ethylene production in Botrytis cinerea- and oligogalacturonide-induced immunity requires calcium-dependent protein kinases. Plant J. 84, 1073– 1086. Wiley Online Library Grierson, C., Nielsen, E., Ketelaarc, T. and Schiefelbein, J. (2014) Root hairs. Arabidopsis Book, 12, e0172. Gu, F. and Nielsen, E.J. (2013) Targeting and regulation of cell wall synthesis during tip growth in plants. J. Integr. Plant Biol. 55, 835– 846. Wiley Online Library Hepler, P.K., Rounds, C.M. and Winship, L.J. (2013) Control of cell wall extensibility during pollen tube growth. Mol. Plant. 6, 998– 1017. Hervé, C., Marcus, S.E. and Knox, J.P. (2011) Monoclonal antibodies, carbohydrate-binding modules, and the detection of polysaccharides in plant cell walls. Methods Mol. Biol. 715, 103– 113. Klausner, R.D., Donaldson, J.G. and Lippincott-Schwartz, J. (1992) Brefeldin A: insights into the control of membrane traffic and organelle structure. J. Cell Biol. 116, 1071– 1080. Larson, E.R., Tierney, M.L., Tinaz, B. and Domozych, D.S. (2014) Using monoclonal antibodies to label living root hairs: a novel tool for studying cell wall microarchitecture and dynamics in Arabidopsis. Plant Methods, 10, 30. Lehner, A., Dardelle, F., Soret-Morvan, O., Lerouge, P., Driouich, A. and Mollet, J.C. (2010) Pectins in the cell wall of Arabidopsis thaliana pollen tube and pistil. Plant Signal. Behav. 5, 282– 1285. Levesque-Tremblay, G., Pelloux, J., Braybrook, S.A. and Müller, K. (2015) Tuning of pectin methylesterification: consequences for cell wall biomechanics and development. Planta, 242, 791– 811. Lewis, K.C., Selzer, T., Shahar, C., Udi, Y., Tworowski, D. and Sagi, I. (2008) Inhibition of pectin methyl esterase activity by green tea catechins. Phytochemistry, 69, 2586– 2592. Liners, F., Letesson, J.J., Didembourg, C. and Van Cutsem, P. (1989) Monoclonal antibodies against pectin: recognition of a conformation induced by calcium. Plant Physiol. 91, 1419– 1424. Monniaux, M. and Hay, A. (2016) Cells, walls, and endless forms. Curr. Opin. Plant Biol. 34, 114– 121. Morra, M., Cassinelli, C., Cascardo, G. etal. (2004) Effects on interfacial properties and cell adhesion of surface modification by pectic hairy regions. Biomacromolecules, 5, 2094– 2104. Mouille, G., Ralet, M.C., Cavelier, C. etal. (2007) Homogalacturonan synthesis in Arabidopsis thaliana requires a Golgi-localized protein with a putative methyltransferase domain. Plant J. 50, 605– 614. Wiley Online Library Mravec, J., Kračun, S.K., Rydahl, M.G. etal. (2014) Tracking developmentally regulated post-synthetic processing of homogalacturonan and chitin using reciprocal oligosaccharide probes. Development, 141, 4841– 4850. Palin, R. and Geitmann, A. (2012) The role of pectin in plant morphogenesis. Biosystems, 109, 397– 402. Peaucelle, A., Louvet, R., Johansen, J.N., Höfte, H., Laufs, P., Pelloux, J. and Mouille, G. (2008) Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins. Curr. Biol. 18, 1943– 1948. Peaucelle, A., Braybrook, S.A., Le Guillou, L., Bron, E., Kuhlemeier, C. and Höfte, H. (2011) Pectin-induced changes in cell wall mechanics underlie organ initiation in Arabidopsis. Curr. Biol. 21, 1720– 1726. Pedersen, H.L., Fangel, J.U., McCleary, B. etal. (2012) Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research. J. Biol. Chem. 287, 39429– 39438. Pelletier, S., Van Orden, J. and Wolf, S. (2010) A role for pectin de-methylesterification in a developmentally regulated growth acceleration in dark-grown Arabidopsis hypocotyls. New Phytol. 188, 726– 739. Wiley Online Library Pontiggia, D., Ciarcianelli, J., Salvi, G., Cervone, F., De Lorenzo, G. and Mattei, B. (2015) Sensitive detection and measurement of oligogalacturonides in Arabidopsis. Front. Plant Sci. 6, 258. Röckel, N., Wolf, S., Kost, B., Rausch, T. and Greiner, S. (2008) 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. Plant J. 53, 133– 143. Wiley Online Library Rodriguez-Enriquez, M.J., Mehdi, S., Dickinson, H.G. and Grant-Downton, R.T. (2013) A novel method for efficient invitro germination and tube growth of Arabidopsis thaliana pollen. New Phytol. 197, 668– 679. Wiley Online Library Rounds, C.M. and Bezanilla, M. (2013) Growth mechanisms in tip-growing plant cells. Annu. Rev. Plant Biol. 64, 243– 265. Sanati Nezhad, A., Packirisamy, M. and Geitmann, A. (2014) Dynamic, high precision targeting of growth modulating agents is able to trigger pollen tube growth reorientation. Plant J. 80, 185– 195. Wiley Online Library Savatin, D.V., Bisceglia, N.G., Marti, L., Fabbri, C., Cervone, F. and De Lorenzo, G. (2014) The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein kinases are required for elicitor-induced oxidative burst and immunity. Plant Physiol. 165, 1188– 1202. Sénéchal, F., Wattier, C., Rustérucci, C. and Pelloux, J. (2014) Homogalacturonan-modifying enzymes: structure, expression, and roles in plants. J. Exp. Bot. 65, 5125– 5160. Sénéchal, F., L\'Enfant, M., Domon, J.M. etal. (2015) Tuning of Pectin Methylesterification: PECTIN METHYLESTERASE INHIBITOR 7 MODULATES THE PROCESSIVE ACTIVITY OF CO-EXPRESSED PECTIN METHYLESTERASE 3 IN A pH-DEPENDENT MANNER. J. Biol. Chem. 290, 23320– 23335. Sørensen, I., Pettolino, F.A., Bacic, A., Ralph, J., Lu, F., O\'Neill, M.A., Fei, Z., Rose, J.K., Domozych, D.S. and Willats, W.G.T. (2011) The charophycean green algae provide insights into the early origins of plant cell walls. Plant J. 68, 201– 211. Wiley Online Library Vallarino, J.G. and Osorio, S. (2012) Signaling role of oligogalacturonides derived during cell wall degradation. Plant Signal. Behav. 7, 1447– 1449. Verhertbruggen, Y., Marcus, S.E., Haeger, A., Ordaz-Ortiz, J.J. and Knox, J.P. (2009) An extended set of monoclonal antibodies to pectic homogalacturonan. Carbohydr. Res. 344, 1858– 1862. Willats, W.G.T., Steele-King, C.G., Marcus, S.E. and Knox, J.P. (1999) Side chains of pectic polysaccharides are regulated in relation to cell proliferation and cell differentiation. Plant J. 20, 619– 628. Wiley Online Library Willats, W.G., Orfila, C. and Limberg, G. (2001) Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls. Implications for pectin methyl esterase action, matrix properties, and cell adhesion. J. Biol. Chem. 276, 19404– 19413. Willats, W.G., McCartney, L. and Steele-King, C.G. (2004) A xylogalacturonan epitope is specifically associated with plant cell detachment. Planta, 218, 673– 681. Wolf, S. and Greiner, S. (2012) Growth control by cell wall pectins. Protoplasma, 249, S169– S175. Wolf, S., Mravec, J., Greiner, S., Mouille, G. and Höfte, H. (2012) Plant cell wall homeostasis is mediated by brassinosteroid feedback signaling. Curr. Biol. 22, 1732– 1737. The full text of this article hosted at iucr.org is unavailable due to technical difficulties. Please check your email for instructions on resetting your password. If you do not receive an email within 10 minutes, your email address may not be registered, and you may need to create a new Wiley Online Library account. Can\'t sign in? Forgot your username? Enter your email address below and we will send you your username If the address matches an existing account you will receive an email with instructions to retrieve your username