FUNCTIONAL ANALYSIS OF THE CELLULOSE SYNTHASE CLASS SPECIFIC REGION IN PHYSCOMITRELLA PATENS

Abstract

Cellulose synthases are found in a wide range of organisms, from bacteria to land plants. However, the cellulose synthases found in land plants (CESAs) form large, multimeric, rosette-shaped cellulose synthase complexes (CSCs) and have three unique regions not found in other cellulose synthases; the N-terminal zinc-binding domain, the Plant Conserved Region (P-CR) and the Class Specific Region (CSR). The CSR, a portion of the large cytoplasmic region that contains the catalytic domain, has been predicted to be involved in CSC formation through in silico modeling. This project tested the hypothesis that the CSR is necessary for clade-specific CESA function. We have developed a complementation assay in the moss Physcomitrella patens based on the ppcesa5KO-2B line, which does not produce gametophores. In P. patens, CESAs in the A Clade (CESA3, 5 and 8) have similar CSR structures that are distinct from those in the B Clade (CESA4, 6, 7, 10). The ppcesa5KO-2B line is complemented by overexpression of PpCESA3 and PpCESA8, but not by the overexpression of CESAs in Clade B. An overexpression vector containing a Clade A CESA with a Clade B CSR was unable to rescue ppcesa5KO phenotype, indicating that the CSR is necessary for clade-specific CESA function. However, an overexpression vector containing a Clade B CESA with a Clade A CSR was also unable to rescue the ppcesa5KO phenotype. This signifies that the CSR is not the sole determinant of clade-specific CESA function. A vector containing a Clade A CESA and the C-terminus of a Clade B CESA was able to rescue the ppcesa5KO phenotype. This suggests that the functionality of the C-terminal region, containing six transmembrane helices, is not a determinant of clade-specific function. A vector containing a Clade A CESA with the N-terminus of a Clade B CESA was unable to rescue the ppcesa5KO phenotype. This suggests that CSR may be interacting with an N-terminal domain, possibly the P-CR or zinc-binding domain to confer clade-specific function.