A Comprehensive Toolkit of Plant Cell Wall Glycan-Directed Monoclonal Antibodies    

Abstract

A collection of 130 new plant cell wall glycan-directed monoclonal antibodies (mAbs) was generated with the aim of facilitating in-depth analysis of cell wall glycans. An enzyme-linked immunosorbent assay-based screen against a diverse panel of 54 plant polysaccharides was used to characterize the binding patterns of these new mAbs, together with 50 other previously generated mAbs, against plant cell wall glycans. Hierarchical clustering analysis was used to group these mAbs based on the polysaccharide recognition patterns observed. The mAb groupings in the resulting cladogram were further verified by immunolocalization studies in Arabidopsis (Arabidopsis thaliana) stems. The mAbs could be resolved into 19 clades of antibodies that recognize distinct epitopes present on all major classes of plant cell wall glycans, including arabinogalactans (both protein-and polysaccharide-linked), pectins (homogalacturonan, rhamnogalacturonan I), xyloglucans, xylans, mannans, and glucans. In most cases, multiple subclades of antibodies were observed to bind to each glycan class, suggesting that the mAbs in these subgroups recognize distinct epitopes present on the cell wall glycans. The epitopes recognized by many of the mAbs in the toolkit, particularly those recognizing arabinose-and/or galactose-containing structures, are present on more than one glycan class, consistent with the known structural diversity and complexity of plant cell wall glycans. Thus, these cell wall glycan-directed mAbs should be viewed and utilized as epitope-specific, rather than polymer-specific, probes. The current world-wide toolkit of approximately 180 glycan-directed antibodies from various laboratories provides a large and diverse set of probes for studies of plant cell wall structure, function, dynamics, and biosynthesis.

SALT-OVERLY SENSITIVE5 Mediates Arabidopsis Seed Coat Mucilage Adherence and Organization through Pectins

“…JIM5 is specific for partially methylesterified HG (up to 40%; Vandenbosch et al, 1989;Knox et al, 1990;Willats et al, 2000), whereas CCRC-M36 is specific for the unsubstituted RG I backbone that is found in Arabidopsis seed mucilage (Young et al, 2008;Arsovski et al, 2009;Pattathil et al, 2010). CCRC-M36 strongly labeled the periphery of the mucilage halo in wild-type seeds ( Fig.…”

“…Wild-type, cesa5-1, and sos5-2 seeds were labeled with JIM13 [epitope: Knox et al, 1991;Yates and Knox, 1994;Yates et al, 1996;Majewska-Sawka and Nothnagel, 2000], MAC207 [epitope: a-GlcA-(1,3)-a-GalA-(1,2)-a-Rha; Bradley et al, 1988;Pennell et al, 1989;Yates and Knox, 1994;Yates et al, 1996;Pattathil et al, 2010], JIM8 (epitope: arabinogalactan; Pennell et al, 1991;Majewska-Sawka and Nothnagel, 2000), and CCRC-M7 (epitope: recognizes 6-linked b-D-Gal oligomers that contain arabinose; Puhlmann et al, 1994;Steffan et al, 1995;Pattathil et al, 2010) antibodies (Supplemental Figs. S7 and S8).…”

GALACTURONOSYLTRANSFERASE-LIKE5 Is Involved in the Production of Arabidopsis Seed Coat Mucilage

“…Monoclonal antibodies were obtained as hybridoma cell culture supernatants from laboratory stocks (CCRC series and JIM series antibodies are available from CarboSource [http://www.carbosource/net]). Detailed information about all monoclonal antibodies included in this study can be found at WallMabDB (http://www.wallmabdb.net), an online database that provides information about the immunogens used to develop the antibodies, their isotype, the cell wall polysaccharide class they primarily recognize, and detailed epitope information where available (Pattathil et al, 2010). Whole-seed immunolabeling was conducted as described (Sullivan et al, 2011).…”

“…Immunogold labeling, using CCRC-M38 (a nonmethylesterified HG-directed monoclonal antibody; Pattathil et al, 2010), was employed to visualize the localization of HG epitopes in seed coat epidermal cells. Nonmethylesterified HG is evenly distributed in the middle lamella and cell corners of seed coat epidermal cells (Fig.…”

Reduction of Plant-Specific Arabinogalactan-TypeO-Glycosylation by Treating Tobacco Plants with Ferrous Chelator 2,2′-Dipyridyl

“…The antibodies used were PN16.4B4 and MAC204 with a respective dilution of 1:100, 15) and the antigen-antibody complex on the membrane was detected by using ECL Plex goat--mouse IgG-Cy5 (GE Healthcare) or Alexa fluor 488 goat--rat IgM (Invitrogen) at a respective dilution of 1:2500, the signals being detected by a Typhoon 9640 image analyzer. As shown on the left of Fig.…”