Developing Arabidopsis thaliana seed coat specific promoter as a tool for basic and applied research

• Main Author: Esfandiari, Elahe
• Language: English
• Published: University of British Columbia 2012
• Online Access: http://hdl.handle.net/2429/43122

During differentiation of Arabidopsis thaliana seed coat epidermal cells, dramatic changes occur highlighted by the synthesis and secretion of large amounts of pectinaceous mucilage. This cell type, therefore, provides an excellent molecular-genetic model to study the biosynthesis, secretion and modification of plant cell wall polysaccharides. Here, I describe the development of an experimental tool to aid in studying cell wall components found in mucilage. I sought to identify a promoter that drives gene expression specifically in the mucilage secretory cells to investigate the effect of manipulating different cell wall polymers in the mucilage without detrimental effects to the rest of the plant. The search for such a promoter was initiated by analyzing seed coat microarray data, followed by investigating expression pattern of the candidate genes by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). By fusing the regulatory region of these candidate genes to beta-glucuronidase gene (GUS), one promoter was identified, that of the DIRIGENT PROTEIN1 (DP1) gene, that was able to drive expression specifically in the epidermis and palisade layer of Arabidopsis and Brassica napus seed coats. These results were confirmed using Citrine Yellow Fluorescent Protein (YFP). To verify the potential ability of the DP1 promoter (DP1Pro) to express enzyme-encoding genes and determine putative role(s) of homogalacturonan in mucilage, the ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2) encoding a polygalacturonase was expressed under the control of DP1Pro and targeted to the apoplast. Although RT-PCR results showed that the DP1Pro drives expression of this gene in the seed coat, no significant difference was found between transgenic and wild type mucilage or the seed coat epidermal cell morphology. MUCILAGE-MODIFIED4 (MUM4) gene was found to encode a putative UDP-L-rhamnose synthase, required for synthesis of mucilage. While the transcript of MUM4 is found throughout the plant, it is specifically up-regulated during mucilage biosynthesis. To identify the sequences responsible for this up-regulation, a deletion analysis of the MUM4 transcription regulatory region was undertaken. Dissection of the MUM4 promoter region led to identification of two functional domains: one conferring higher level of gene expression in root, cotyledon and silique walls, and one that promotes expression specifically in the seed coat.