Resin Embedding, Sectioning, and Immunocytochemical Analyses of Plant Cell Walls in Hard Tissues - Plant Cell Morphogenesis: Methods and Protocols - page 40

Chapter 3

Resin Embedding, Sectioning, and Immunocytochemical
Analyses of Plant Cell Walls in Hard Tissues

Kieran J. D. Lee and J. Paul Knox

Abstract

Plant cell walls are structurally diverse macromolecular composites. One of our best methodologies to
determine the temporal and spatial regulation of cell wall polysaccharides in relation to development are
monoclonal antibody (MAB) and carbohydrate-binding module (CBM) probes and their detection by
immunofl uorescence microscopy. Here we describe resin embedding, sectioning, and in situ chemical and
enzymatic cell wall disassembly and their use with immunocytochemical analyses as a means to unravel the
complexity of cell wall molecular architecture in hard tissues and seeds.

Key words Monoclonal antibody, Carbohydrate-binding module, Acrylic resin embedding, Cell wall
immunocytochemistry, Immunofl uorescence microscopy, Plant cell walls

1 Introduction

Plant cell walls are complex macromolecular composites comprised
of several interconnected networks. In primary cell walls cellulose
microfi brils are tethered by noncellulosic polysaccharides such as
xyloglucan, xylans, heteromannans, and mixed-linkage glucans,
which are often referred to as hemicelluloses; this fi brous network
is embedded in a gel-like matrix of pectic polysaccharides, glyco-
proteins, proteins, ions, and water. Pectic polysaccharides [ 1 ] are
structurally diverse and the constituent polymers are currently clas-
sifi ed as homogalacturonan (HG), rhamnogalacturonans I and II
(RG-I and RG-II), and xylogalacturonan (XGA). Noncellulosic
polysaccharides possess numerous structural elaborations that
impact on polymer properties and functions [ 2 ]. A current chal-
lenge is to understand how these structural variants infl uence
polymer-polymer interactions in individual cell wall architectures
in the context of tissues and organs.
Approaches to map cell wall architecture in relation to devel-
opment often make use of monoclonal antibody (MAB) and
carbohydrate-binding module (CBM) probes that recognize