Click chemistry-based tracking reveals putative cell wall-located auxin binding sites in expanding cells

Abstract Auxin is a key plant regulatory molecule, which acts upon a plethora of cellular processes, including those related to cell differentiation and elongation. Despite the stunning progress in all disciplines of auxin research, the mechanisms of auxin-mediated rapid promotion of cell expansion and underlying rearrangement of cell wall components are poorly understood. This is partly due to the limitations of current methodologies for probing auxin. Here we describe a click chemistry-based approach, using an azido derivative of indole-3-propionic acid. This compound is as an active auxin analogue, which can be tagged in situ. Using this new tool, we demonstrate the existence of putative auxin binding sites in the cell walls of expanding/elongating cells. These binding sites are of protein nature but are distinct from those provided by the extensively studied AUXIN BINDING PROTEIN 1 (ABP1). Using immunohistochemistry, we have shown the apoplastic presence of endogenous auxin epitopes recognised by an anti-IAA antibody. Our results are intriguingly in line with previous observations suggesting some transcription-independent (non-genomic) activity of auxin in cell elongation

High throughput screening of starch structures using carbohydrate microarrays

In this study we introduce the starch-recognising carbohydrate binding module family 20 (CBM20) from Aspergillus niger for screening biological variations in starch molecular structure using high throughput carbohydrate microarray technology. Defined linear, branched and phosphorylated maltooligosaccharides, pure starch samples including a variety of different structures with variations in the amylopectin branching pattern, amylose content and phosphate content, enzymatically modified starches and glycogen were included. Using this technique, different important structures, including amylose content and branching degrees could be differentiated in a high throughput fashion. The screening method was validated using transgenic barley grain analysed during development and subjected to germination. Typically, extreme branching or linearity were detected less than normal starch structures. The method offers the potential for rapidly analysing resistant and slowly digested dietary starches

Characterization of the LM5 pectic galactan epitope with synthetic analogues of β-1,4-d-galactotetraose

Plant cell wall glycans are important polymers that are crucial to plant development and serve as an important source of sustainable biomass. The study of polysaccharides in the plant cell wall relies heavily on monoclonal antibodies for localization and visualization of glycans, using e.g. Immunofluorescent microscopy. Here, we describe the detailed epitope mapping of the mab LM5 that is shown to bind to a minimum of three sugar residues at the non-reducing end of linear beta-1,4-linked galactan. The study uses de novo synthetic analogs of galactans combined with carbohydrate microarray and competitive inhibition ELISA for analysis of antibody-carbohydrate interactions

An oligogalacturonide-derived molecular probe demonstrates the dynamics of calcium-mediated pectin complexation in cell walls of tip-growing structures

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 cell wall biomechanics and in mediating cell-to-cell adhesion. Current immunological methods enable only steady-state detection of egg box formation in situ. 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 in situ, 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 different spatial organisation of HG incorporation and processing in cell walls of these two tip-growing structures

Production and fine characterisation of new monoclonal antibodies against rhamnogalacturonan I

National audienceThe functional role of the complex pectic rhamnogalacturonane I (RGI) in the context of cell biology is still unclear. RGI is mainly composed of a repeating disaccharide unit [,2)-a-L-rhamnosep-(1,4)-a-D-galacturonic acidp-(1,]n (RU)n decorated primarily with arabinan and (arabino)-galactan side-chains. Monoclonal antibodies (mAbs) are useful to probe pectin structural domains in situ. Several mAbs to the RGI backbone and to arabinan and (arabino)-galactan side-chains have been developed but up to now, there is no mAb against rhamnogalacturonan stretches bearing arabinose or galactose units. Here, we report on the production and fine characterisation of new mAbs against RGI side chains connected to the rhamnogalacturonan backbone. The study first focused on the generation of adequate antigens. A pool of low-branched RU oligosaccharides was prepared and purified from potato pulp. mAbs were raised against low-branched RU oligosaccharides-ovalbumin conjugates. Promising clones producing mAbs recognizing low-branched RU oligosaccharides but recognizing neither unbranched RU oligosaccharides nor galactan oligosaccharides were recovered. The combination of glycan microarray and sub-fractionation of the low-branched RU oligosaccharides pool by anion exchange chromatography prior to competitive ELISA studies allowed fine mAbs characterisation. The use of these new mAbs in immunocytochemistry is expected to give a better understanding of RGI role in planta

A biology-driven approach for producing monoclonal antibodies against green algal cell wall components

A biology-driven approach for producing monoclonal antibodies against green algal cell wall components. 14th cell wall meetin

Production and fine characterization of new antibodies against rhamnogalacturonan I

Production and fine characterization of new antibodies against rhamnogalacturonan I. WallTraC Symposiu

Biology-driven, microarray-assisted selection of cell wall directed antibodies

Biology-driven, microarray-assisted selection of cell wall directed antibodies. Gordon Research Conference on Plant Cell Wall