Fibrous matrix component of cell wall in the giant-celled green alga Valonia utricularis observed by atomic force microscopy in liquid

The cell walls of the giant-celled green alga Valonia exhibit the “crossed-fibril” arrangement of cellulose microfibrils (CMFs). The existence of fibrous matrix components coiling around the CMFs was recently reported using atomic force microscopy (AFM). To understand the biological role of this fibrous wall component, we attempted to reveal the detailed morphology of the fibrous structure of the cell walls isolated from V. utricularis specimens in artificial seawater (ASW), by using an improved method of AFM. We also investigated the effect of incubation in an acidic environment that had been known to make the cell walls liable to separate into layers, on the morphology of the fibrous structures. Thin, fibrous structures were found to entangle around a single CMF or multiple adjacent CMFs, the thickness of which was 0.85 nm on average. Incubation in acidic ASW (pH 4), greatly modified the morphology of the fibrous structures in quality and quantity, compared with those incubated in the original ASW (pH 8). Thickness of the fibrous structures was increased to 4.63 nm on average, remarkable deformations were observed, and the density of the structures was reduced to less than half by incubation in the acidic ASW. In addition, same fibrous structures extended over CMFs and forming aggregates were observed on the surface of the cell wall layers separated artificially. Fluorescence microscopy of cell walls treated with Vicia villosa lectin conjugated with fluorescein isothiocyanate showed specific labelling of cell wall regions where the fibrous structures were present abundantly as observed by AFM. From these observations, it can be assumed that the fibrous structures of V. utricularis are the matrix component of the cell wall containing N-acetylgalactosamine, involved in the maintenance of the cell wall integrity through bonding the neighboring cell wall layers, and their morphology and function of V. utricularis are sensitive to acidic pH.