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Functional Genomics Studies of Core Arabidopsis F-box (CAF) Genes

Yapa, Madhura Mihiranga
http://orcid.org/0000-0003-1165-4619
http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1724325748688028

Abstract Details

2024, Doctor of Philosophy (PhD), Ohio University, Plant Biology (Arts and Sciences).
Plants encounter various biotic and abiotic stresses daily and have developed defense mechanisms to overcome these challenges. One key system involved in these defense mechanisms is the ubiquitin (Ub)-26S proteasome system (UPS), which targets malfunctioning proteins for degradation through Ub-tagged proteasomal pathways. The E3 ligases, specifically S-phase kinase-associated protein 1 (SKP1), Cullin 1 (CUL1), and F-box (SCF) complexes, play crucial roles in this process by recognizing and tagging specific protein substrates. Arabidopsis thaliana, with over 700 F-box proteins, has the largest group of E3 ligases, yet only 5% have been functionally characterized. Phylogenetic relationships among 111 plant species have identified four clusters of F-box genes, including a cluster with more conserved F-boxes, referred to as core Arabidopsis F-box (CAF) genes. Given that CAF genes have more known functions compared to other clusters, this dissertation hypothesizes significant potential for discovering new functions among the uncharacterized F-boxes within this group. Considering the evolutionary conservation of most CAFs, I adopted a genetic approach to investigate the roles of CAFs during seed germination and seed development. To address the challenges posed by functional redundancy of duplicated CAF genes and the lethality associated with constitutive F-box overexpression in transgenic plants, I created a library of inducible overexpression lines for 40 CAF genes, many of which lacked known biological functions. By systematically examining the effects of conditional overexpression of these 40 CAFs, I found that CAF overexpression during seed germination and seed development can positively or negatively regulate radicle rupture growth, thus controlling the germination process. Specifically, I identified 24 CAFs that enhance radicle rupture and two that inhibited it by interfering with abscisic acid (ABA)-mediated germination suppression. Induction of CAFs during seed developmental stages successfully enhanced germination under ABA treatment, with five genes enhancing and 12 inhibiting radicle rupture, indicating that CAFs play a development-dependent role in seed germination. Overall, I successfully transferred 40 CAFs into plants using an inducible overexpression method and categorized their impact on seed germination and seed development under ABA treatment. Next, I focused on studying the F-box substrate MULTIPLE ORGANELLAR RNA EDITING FACTOR 2 (MORF2) in plant growth and development. Initially recognized as a plastidial RNA-editing factor involved in retrograde signaling (RS), recent interactions of MORF2 with GENOMES UNCOUPLED 1 (GUN1) and the unviable phenotypes of morf2 mutants suggested that MORF2 might have dosage dependent functions or roles beyond RNA editing. Using an inducible Clustered Interspaced Short Palindromic Repeat interference (iCRISPRi) approach, we successfully reduced MORF2 transcripts in a controlled manner. This reduction led to MORF2-dosage dependent RNA-editing errors and stimulated the expression of stress-responsive genes, triggered plastidial retrograde signaling, repressed ethylene signaling and skotomorphogenesis, and increased the accumulation of hydrogen peroxide. These findings, along with previous discoveries, suggest that MORF2 is a crucial regulator involved in plastidial metabolic pathways, and its reduction can activate multiple retrograde signaling molecules, possibly involving reactive oxygen species, to adjust plant growth.
Zhihua Hua (Advisor)
Yang Li (Committee Member)
John Schenk (Committee Member)
Morgan Vis (Committee Member)
240 p.
Biology; Genetics; Molecular Biology; Plant Biology; Plant Sciences
Arabidopsis thaliana; Ubiquitylation; F-box; Core Arabidopsis F-box (CAF); Inducible overexpression; Seed germination; MULTIPLE ORGANELLAR RNA EDITING FACTOR 2 (MORF2); RNA editing; Inducible CRISPR interference (iCRISPRi); Skotomorphogenesis, Stress response

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Citations

  • Yapa, M. M. (2024). Functional Genomics Studies of Core Arabidopsis F-box (CAF) Genes [Doctoral dissertation, Ohio University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1724325748688028

    APA Style (7th edition)

  • Yapa, Madhura. Functional Genomics Studies of Core Arabidopsis F-box (CAF) Genes. 2024. Ohio University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1724325748688028.

    MLA Style (8th edition)

  • Yapa, Madhura. "Functional Genomics Studies of Core Arabidopsis F-box (CAF) Genes." Doctoral dissertation, Ohio University, 2024. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1724325748688028

    Chicago Manual of Style (17th edition)

ohiou1724325748688028
© 2024, all rights reserved.
This open access ETD is published by Ohio University and OhioLINK.
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