Essential Amino Acids in the Plant-Conserved and Class-Specific Regions of Cellulose Synthases

The Plant-Conserved Region (P-CR) and the Class-Specific Region (CSR) are two plant-unique sequences in the catalytic core of cellulose synthases (CESAs) for which specific functions have not been established. Here, we used site-directed mutagenesis to replace amino acids and motifs within these sequences predicted to be essential for assembly and function of CESAs. We developed an in vivo method to determine the ability of mutated CesA1 transgenes to complement an Arabidopsis (Arabidopsis thaliana) temperature-sensitive root-swelling1 (rsw1) mutant. Replacement of a Cys residue in the CSR, which blocks dimerization in vitro, rendered the AtCesA1 transgene unable to complement the rsw1 mutation. Examination of the CSR sequences from 33 diverse angiosperm species showed domains of high-sequence conservation in a class-specific manner but with variation in the degrees of disorder, indicating a nonredundant role of the CSR structures in different CESA isoform classes. The Cys residue essential for dimerization was not always located in domains of intrinsic disorder. Expression of AtCesA1 transgene constructs, in which Pro₄₁₇ and Arg₄₅₃ were substituted for Ala or Lys in the coiled-coil of the P-CR, were also unable to complement the rsw1 mutation. Despite an expected role for Arg₄₅₇ in trimerization of CESA proteins, AtCesA1 transgenes with Arg₄₅₇Ala mutations were able to fully restore the wild-type phenotype in rsw1. Our data support that Cys₆₆₂ within the CSR and Pro₄₁₇ and Arg₄₅₃ within the P-CR of Arabidopsis CESA1 are essential residues for functional synthase complex formation, but our data do not support a specific role for Arg₄₅₇ in trimerization in native CESA complexes.