Quantitative confocal imaging method for analyzing cellulose dynamics during cell wall regeneration in Arabidopsis mesophyll protoplasts

The network structure of cellulose fibrils provides mechanical properties to the primary cell wall, thereby determining the shapes and growth patterns of plant cells. Despite intensive studies, the construction process of the network structure in muro remains largely unknown, mainly due to the lack of a robust, straightforward technique to evaluate network configuration. Here, we developed a quantitative confocal imaging method for general use in the study of cell wall dynamics in protoplasts derived from Arabidopsis leaf mesophyll cells. Confocal imaging of regenerating cell walls in protoplasts stained with Calcofluor allowed us to visualize the cellulose network, comprising strings of bundled cellulosic fibrils. Using image analysis techniques, we measured several metrics including total length, which is a measure of the spread of the cellulose network. The total length increased during cell wall regeneration. In a proof-of-concept experiment using microtubule-modifying agents, oryzalin, an inhibitor of microtubule polymerization, inhibited the increase in total length and caused abnormal orientation of the network, as shown by the decrease in the average angle of the cellulose with respect to the cell long axis. Taxol, a microtubule stabilizer, stimulated the bundling of cellulose fibrils, as shown by the increase in skewness in the fluorescence intensity distribution of Calcofluor, and inhibited the increase in total length. These results demonstrate the validity of this method for quantitative imaging of the cellulose network, providing an opportunity to gain insight into the dynamic aspects of cell wall regeneration.