Cellulose synthase 'class specific regions' are intrinsically disordered and functionally undifferentiated.

Cellulose synthases (CESAs) are glycosyltransferases that catalyze formation of cellulose microfibrils in plant cell walls. Seed plant CESA isoforms cluster in six phylogenetic clades, whose non-interchangeable members play distinct roles within cellulose synthesis complexes (CSCs). A 'class specific region' (CSR), with higher sequence similarity within versus between functional CESA classes, has been suggested to contribute to specific activities or interactions of different isoforms. We investigated CESA isoform specificity in the moss, Physcomitrella patens (Hedw.) B. S. G. to gain evolutionary insights into CESA structure/function relationships. Like seed plants, P. patens has oligomeric rosette-type CSCs, but the PpCESAs diverged independently and form a separate CESA clade. We showed that P. patens has two functionally distinct CESAs classes, based on the ability to complement the gametophore-negative phenotype of a ppcesa5 knockout line. Thus, non-interchangeable CESA classes evolved separately in mosses and seed plants. However, testing of chimeric moss CESA genes for complementation demonstrated that functional class-specificity is not determined by the CSR. Sequence analysis and computational modeling showed that the CSR is intrinsically disordered and contains predicted molecular recognition features, consistent with a possible role in CESA oligomerization and explaining the evolution of class-specific sequences without selection for class-specific function.