Effects of Cellulose Synthase (cesa) Mutations on Catalytic Activity, Cell Wall Structure, and Isoform Expression Profile

Open Access
Author:
Russo, Daniel Alexander
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Ming Tien, Thesis Supervisor
  • Craig Eugene Cameron, Honors Advisor
  • Scott Brian Selleck, Faculty Reader
Keywords:
  • Site-directed Mutagenesis
  • Cellulose synthase
  • CesA
  • Cellulose
  • Molecular Biology
  • GUS
  • Gene Expression
Abstract:
In higher plants, cellulose is thought to be produced by cellulose synthase (CesA)-containing complexes. In Arabidopsis thaliana, there are 10 CesA isoforms. Six of these isoforms (1, 3, 4, 6, 7, and 8) have been observed within one of two complexes: the primary cell wall CSC (1, 3, 6) and the secondary cell wall CSC (4, 7, 8), however the CesA stoichiometry within these complexes is currently unknown. This study seeks to determine the individual contribution of CesA 4, 7, & 8 catalytic activity on the structure of the secondary cell wall. Catalytically inactivated CesAs were developed using site-directed mutagenesis of a key aspartate residue in each. These mutant genes were then used to complement CesA knockout plants. Spectroscopic analysis of the resultant cell wall structures should yield insight into the number and arrangement of CesA 4, 7, & 8 in the secondary cell wall cellulose synthesis complex. Additionally, this research plans to address the effect of various CesA mutations on the expression patterns of the remaining isoforms. The CesA promoters were each cloned upstream of the beta-glucuronidase (GUS) gene to create a reporter system. These constructs are then to be introduced into a range of different mutant Arabidopsis lines. GUS staining of these lines will reveal any modulation in CesA expression caused by each background mutation, leading to better understanding of the role of each of the 10 CesA isoforms. Currently, the CesA4 and CesA7 reporters are ready for plant transformation and the other reporters are currently in construction.