Molecular studies of cellulose synthase supercomplex from cotton fiber reveal its unique biochemical properties

Cotton fiber is a highly elongated and thickened single cell that produces large quantities of cellulose, which is synthesized and assembled into cell wall microfibrils by the cellulose synthase complex (CSC). In this study, we report that in cotton (Gossypium hirsutum) fibers harvested during secondary cell wall (SCW) synthesis, GhCesA 4, 7, and 8 assembled into heteromers in a previously uncharacterized 36-mer-like cellulose synthase supercomplex (CSS). This super CSC was observed in samples prepared using cotton fiber cells harvested during the SCW synthesis period but not from cotton stem tissue or any samples obtained from Arabidopsis. Knock-out of any of GhCesA 4, 7, and 8 resulted in the disappearance of the CSS and the production of fiber cells with no SCW thickening. Cotton fiber CSS showed significantly higher enzyme activity than samples prepared from knock-out cotton lines. We found that the microfibrils from the SCW of wild-type cotton fibers may contain 72 glucan chains in a bundle, unlike other plant materials studied. GhCesA4, 7, and 8 restored both the dwarf and reduced vascular bundle phenotypes of their orthologous Arabidopsis mutants, potentially by reforming the CSC hexamers. Genetic complementation was not observed when non-orthologous CesA genes were used, indicating that each of the three subunits is indispensable for CSC formation and for full cellulose synthase function. Characterization of cotton CSS will increase our understanding of the regulation of SCW biosynthesis.