Time-resolved tracking of cellulose biosynthesis and assembly during cell wall regeneration in live protoplasts.

Cellulose, the most abundant polysaccharide on earth composing plant cell walls, is synthesized by coordinated action of multiple enzymes in cellulose synthase complexes embedded within the plasma membrane. Multiple chains of cellulose fibrils form intertwined extracellular matrix networks. It remains largely unknown how newly synthesized cellulose is assembled into an intricate fibril network on cell surfaces. Here, we have established an in vivo time-resolved imaging platform to continuously visualize cellulose biosynthesis and fibril network assembly on protoplast surfaces as the primary cell wall regenerates. Our observations provide the basis for a model of cellulose fibril network development in protoplasts driven by an interplay of multiscale dynamics that includes rapid diffusion and coalescence of nascent cellulose fibrils, processive elongation of single fibrils, and cellulose fibrillar network rearrangement during maturation. This study provides fresh insights into the dynamic and mechanistic aspects of cell wall synthesis at the single-cell level.