The plant cell wall is primarily composed of the polysaccharides cellulose, hemicellulose and pectin. The structural and compositional complexity of these components are important for determining cell wall function during plant growth. Moreover, cell wall structure defines a number of functional properties of plant-derived biomass, such as rheological properties of foods and feedstock suitability for the production of cellulosic biofuels. A typical characterization of cell wall chemistry in the molecular biology lab consists of a mild acid hydrolysis for the quantification of hemicellulose and pectin-derived monomers and a separate analysis of cellulose by the Updegraff method. We have adopted a streamlined ‘one-step two-step’ hydrolysis protocol that allows for the simultaneous determination of cellulose content, neutral sugars, and uronic acids by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of paired samples. In our work, this protocol has largely replaced Updegraff cellulose quantification and hydrolysis with 2 M TFA for the determination of matrix polysaccharide composition at the micro scale.

Cellulose is a main component of plant cell walls. Tools to analyze cellulose mainly rely on analytical chemistry, which yields information about cellulose amounts and structure, but cannot be applied to intact tissues. Moreover, these methods measure total cellulose and cannot be used to assay cellulose synthesis per se. Live cell imaging of the catalytic subunits of the cellulose synthesis complex (CSC) conjugated to fluorescent proteins is an important tool to understand the dynamics of the cellulose biosynthesis process (Paredez et al., 2006). This method can be used in various genetic backgrounds (Sorek et al., 2014) or with different chemical inhibitors (Brabham and Debolt, 2012). Here we describe in detail the procedure to visualize the movement of CSCs at the plasma membrane. As the movement of CSCs is likely caused by glucan synthesis and extrusion into the cell wall, live cell analysis of CSC velocity provides a method to directly measure cellulose synthesis in vivo.