Molecular and functional characterization of arabidopsis OBP3 Responsive Gene 1
Restricted (Penn State Only)
- Author:
- Mu, Kemin
- Graduate Program:
- Plant Biology
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- October 18, 2024
- Committee Members:
- Teh-Hui Kao, Thesis Advisor/Co-Advisor
David Robert Huff, Committee Member
Teh-Hui Kao, Program Head/Chair
Carolee Bull, Committee Member - Keywords:
- ORG1
iron homeostasis
arabidopsis - Abstract:
- Iron homeostasis is critical to the normal growth of plants, and the proteins in the Ferric Reductase Oxidase (FRO) family play an essential role in this process. From the transcriptomic analyses of T-DNA insertion mutants of two FRO genes in Arabidopsis thaliana, an iron-deficiency induced gene, OBF-Binding Protein 3 Responsive Gene 1 (ORG1), was identified as significantly mis-regulated in response to iron deficiency. In this Thesis, I used multiple approaches of molecular biology, cellular biology, bioinformatics, and physiology to obtain clues as to the molecular and biological function of ORG1, a unique fusion protein with a kinase domain and a fibrillin domain. In plants, ORG1 orthologs are ubiquitous and the genes encoding them are highly syntenic. In A. thaliana, I confirmed that ORG1 has two distinct splice variants, named ORG1.1 and ORG1.2. Expression of ORG1, as indicated by a promoter-GFP system in vivo, was induced by iron deficiency, and its expression was restricted to vasculature tissues in roots and leaves. ORG1.1 protein, which contains a chloroplast transit peptide at the N-terminus, was shown to localize in the plastids of root cap cells and in the chloroplast of leaf stomata cells, while ORG1.2, which lacks the chloroplast transit peptide, was shown to localize in the cytosol of root cells. Co-IP/MS identification of ORG1.1-interacting proteins showed that ORG1.1 most likely interacted with proteins involved in photosynthesis in both iron deficient and iron sufficient conditions. To study the function of ORG1, I further obtained different genetic mutants of ORG1, including T-DNA insertion, CRISPR/Cas9 genome-editing, and artificial microRNA lines. Phenotypic analysis of these mutants showed that ORG1 is not likely to play a significant role in the whole plant level iron homeostasis, but may play a role in photosynthesis, seed germination, and regulation of flowering time. Overaccumulation of anthocyanin in the basal shoot was also observed in the two T-DNA insertion mutants, and this phenotype is related to specific up-regulation of early anthocyanin biosynthesis genes in these mutants. Taken together, this Thesis provides valuable genetic materials for future studies of this highly conserved ORG1 protein, and also sheds light on potential functions of this protein in different aspects of plant life.