Dataset Information

Amino acids essential for function in the Plant-Conserved and Class-Specific Regions of plant cellulose synthase

ABSTRACT: The Plant-Conserved Region (P-CR) and the Class-Specific Region (CSR) are two unique sequences inserted into the catalytic core of plant cellulose synthases. Either or both of these domains might promote assembly of the synthase complex. From structural studies of these domains, we have identified candidate amino acids and motifs that might be essential for assembly of the complex or catalytic function. We used site-directed mutagenesis to replace them in the CSR and P-CR in an Arabidopsis AtCesA1 and tested the ability of the mutated CesA1 transgenes to complement an Arabidopsis temperature-sensitive root-swelling1 (rsw1) mutant and restore the wild-type phenotype. Elimination of conserved basic and acidic motifs characteristic of the CSR in AtCesA1 constructs were able to complement wild-type growth and cellulose synthesis in rsw1. However, transgenes that replaced Cys662 with Ala failed to complement, indicating an essential function for this residue. Replacement of the cognate Cys662 with Ala in a recombinant rice CESA8 catalytic domain eliminated the ability of the recombinant protein to dimerize. Expression of transgene constructs in which of Pro417 and Arg453 were substituted for Ala or Lys in the coiled-coil of the P-CR also were unable to complement the rsw1 mutation. However, mutation of Arg457, residue predicted to function in trimerization of reconstituted poplar CESA8, fully restored the wild-type phenotype in transgenic rsw1. Taken together, these results indicate that Cys residues are essential for CesA function, most likely through their role in dimerization of CSR domains. Although evidence supports roles for both the CSR and P-CR in oligomerization of CesAs, our data do not support a role for Arg457 in trimerization in the native CesA complex. Examination of the 3-dimensional structure of a poplar CESA8 revealed other amino acids that might stabilize trimer structures in the absence of Arg457.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Arabidopsis Thaliana (ncbitaxon:3702)

SUBMITTER: Nicholas Carpita