Biochemistry

The growing plant cell wall is comprised of long, thin cellulose microfibrils embedded in a hydrated matrix of polysaccharides and glycoproteins. These components are typically constructed in layers (lamellae) on the inner surface of the cell wall, i.e., between the existing wall and the plasma membrane. The organization of these components is an important feature for plant cell growth and mechanics. To directly visualize the nano-scale structure of the newly-deposited surface of primary plant cell walls without dehydration or chemical extraction, a protocol of cell wall preparation for AFM imaging the most recently-synthesized cell wall surface in aqueous solutions was developed. Although the method was developed for onion scale epidermal peels, it can also be adapted to other organs, such as Arabidopsis hypocotyls, as well as ground samples of cell walls from the leaf petioles or hypocotyls of Arabidopsis and cucumber, maize coleoptiles and onion parenchyma. Potential artifacts of AFM imaging of plant cell walls are also discussed.

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.