Year: 2025
Type: dissertation
Abstract: Auxin is a key phytohormone that regulates various developmental and maturation processes in plants. Intracellular auxin induces ARF (Auxin Response Factor) activation through the turnover of Aux/IAA (Auxin/Indole-3-Acetic Acid) repressors that otherwise interact with the C-terminal PB1 domain of ARFs. ARF6 and ARF8 are activating transcription factors that promote flower maturation. Previous studies used random mutagenesis and T-DNA insertions to knock out ARF6 and ARF8. However, the T-DNA alleles often lead to transgene silencing. We investigated the phenotypic effects of new CRISPR-Cas9-generated indel mutations in ARF6 and ARF8 on flower phenotypes and fruit development. Using targeted guide RNAs, students in an undergraduate CURE course generated knock-out (N-terminal domain) and putative “gain-of-function” (C-terminal, PB1 domain) mutations within null backgrounds for the other gene (arf6-crispr arf8-8 and arf6-2 arf8-crispr). We genetically characterized these mutations and performed flower organ length, seedling growth, fruit growth after emasculation, and low light stress assays of the mutants. Double mutants (arf6 arf8) with knockout (N-terminal ARF domain) mutations had arrested growth, closed buds, and indehiscent anthers. Double mutants including an arf6 PB1 domain mutation (C-terminal ARF domain) had weaker phenotypes. While we had hoped to identify a “gain” in ARF function to unmask Aux/IAA repressor effects, these mutants resemble partial loss-of-function mutants in light stress, hypocotyl growth, and emasculation assays conducted thus far. Through this investigation, we can uncover effects of Aux/IAA proteins on ARF function and may develop tools to manipulate reproductive fitness in flowers and fruits and providing applications in managing crop yield.
DOI: 10.17615/dwm7-m026
Author: Ramanujam, Rashmi
Institution: University of North Carolina at Chapel Hill
Language: English
Subfield: Plant Science
Domain: Life Sciences
SDG: Zero hunger
Open Access status: green
Funder: National Science Foundation
OpenAlex ID: W6906791948
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