Investigation of cell wall uptake, trafficking, and organization in Arabidopsis thaliana using a metabolically incorporated click reporter

open_access
Open Access
Author:
Price, Emma Caroline
Area of Honors:
Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
Dr. Charles T Anderson, Thesis Supervisor
Dr. Stephen Wade Schaeffer, Thesis Honors Advisor
Keywords:
arabidopsis thaliana
pectin
click chemistry
CuAAC
SPAAC
fluorescence
microscopy
plant cell wall
fucose alkyne
Abstract:
Limited tools for imaging cell walls in plants have made it difficult to visualize cell wall synthesis and dynamics. It has been reported that the sugar analog, fucose alkyne (FucAl), may be incorporated into the matrix polysaccharide, rhamnogalacturonan I (RGI), and can be labeled using a copper-catalyzed click reaction. In order to study the uptake, processing, trafficking, and organization of labeled cell wall components, mutants in various steps in the molecular pathways thought to be involved in fucose metabolism and wall synthesis were assessed for defects in the amount (mean fluorescence intensity, MFI) and organization (anisotropy) of FucAl-related fluorescence, using spinning disk confocal microscopy. We found that mutations in genes related to the uptake and processing of sugars have differing effects on the incorporation of FucAl. Because the copper-catalyzed click labeling reaction is toxic to cells, a copper-free strain-promoted click reaction was used to incorporate another sugar analog, Kdo azide (KdoAz), to develop a tool to study the molecular pathways involved in matrix polysaccharide synthesis and dynamics in vivo. Recently developed and improved cyclic alkyne reporters provide opportunities to optimize this labeling technique. The utility of these metabolic biochemical labeling tools could have far-reaching applications in the study of the plant cell wall, increasing our understanding of cell wall formation and dynamics.