Date of Award
1-10-2013
Document Type
Dissertation
Degree Name
Molecular Biosciences, Ph.D.
First Advisor
Elizabeth Hood
Committee Members
Brett Savary; Gregory Phillips; Jianfeng Xu; Maureen Dolan
Call Number
LD 251 .A566d 2012 D32
Abstract
The cell wall, one of the most complex and interesting organelle in a plant cell, still holds a mystery to be solved by the research community. In maize (Zea mays L.), the primary cell wall contains several structural proteins, the most abundant and well-studied being hydroxyproline-rich glycoproteins (HRGPs). These structural proteins give strength, flexibility during cell expansion and participate in the process of cell wall formation and assembly. Extensin is the dominant member of the HRGP family is accumulated in silk and pericarp of maize. We hypothesized that extensin comprises a small gene family in maize and that a specific isoform is present in reproductive tissues such as silk and pericarp. Moreover, we predicted that the disruption of extensin expression in these tissues would affect corn reproduction, causing disruption in silk and pericarp structures which would decrease fertilization and cause low seed-set. In this study we discovered that the B73 extensin protein showed characteristics similar to extensins from other maize varieties, including the amount present in vegetative tissues and higher expression in silk and pericarp. The B73 protein size and migration analyzed in SDS-PAGE is altered by its rod-like shape, with an apparent size of 120 kDa. However, MALDI TOF MS analysis demonstrated that the actual molecular weight of B73 extensin is 44.2kDa for the glycosylated form. The deglycosylated form is 38.2kDa after deglycosylation. Thus, 13% of maize extensin molecular weight is represented by glycans. MALDI-TOF MS analysis of chymotryptic peptides confirmed that the extensin isolated from silk cell walls is a product of the gene on chromosome 2 of B73 corn. In order to address the question of extensin function in silk and pericarp, an antisense RNA strategy was employed to down-regulate extensin mRNA in the target tissues. A pericarp promoter was and we predicted it would also function in silk because they have the same maternal origin. Several transgenic plants with the antisense gene were generated and analyzed for protein and mRNA levels. An outcome of this study was the down-regulation of extensin in a maternal tissue (pericarp) in two events. This down-regulation could be possibly causing effects in seed set and reproduction in maize. The promoter used in this study was not effective in silk as hypothesized, thus conclusions about extensin in silk await a more specific promoter. Lastly, we conducted a detailed bioinformatics study of the extensin gene and native promoter from B73 corn. By comparing previously described extensin genes with the B73 genome, we concluded that one copy of the extensin gene is present in B73 maize and it is highly conserved through evolution when compared to other Zea spp. We also found that the extensin promoter has three conserved domains in its structure, but it seems that these domains alone do not control gene expression in silk or pericarp because transformed corn plants containing the full length promoter region with repetitive domains showed no expression of the visual marker gene in any studied tissue. This research has provided a series of tools for future research on maize extensin. Included among the tools developed are extensin mutants to study pericarp structure and thickness in functions like disease tolerance, biofuels, fresh corn and popcorn. Also, the extensin promoter can be used to study the mechanism of gene expression in reproductive tissues. As a single gene, extensin is a perfect model for breeding, gene silencing and other genetic studies.
Rights Management
This work is licensed under a Creative Commons Attribution 4.0 International License.
Recommended Citation
Dabul, Audrei Nisio, "Structural and functional studies of extensin, an hydroxyproline-rich glycoprotein in the reproductive tissues of B73 inbred maize (Zea mays L.)" (2013). Student Theses and Dissertations. 852.
https://arch.astate.edu/all-etd/852
