Characterizing new mutations of ARF6 and ARF8 in Arabidopsis thaliana

<p class="p1">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. <em>ARF6</em> and <em>ARF8</em> are activating transcription factors that promote flower maturation. Previous studies used random mutagenesis and T-DNA insertions to knock out <em>ARF6 </em>and <em>ARF8</em>. However, the T-DNA alleles often lead to transgene silencing. We investigated the phenotypic effects of new CRISPR-Cas9-generated indel mutations in <em>ARF6</em> and <em>ARF8</em> on flower phenotypes and fruit development. Using targeted guide RNAs, students in an undergraduate CURE course generated knock-out (N-terminal domain) and putative &ldquo;gain-of-function&rdquo; (C-terminal, PB1 domain) mutations within null backgrounds for the other gene (<em>arf6-</em><em>crispr arf8-8</em> and <em>arf6-2 arf8-crispr</em>). 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 (<em>arf6 arf8</em>) with knockout (N-terminal ARF domain) mutations had arrested growth, closed buds, and indehiscent anthers. Double mutants including an <em>arf6</em> PB1 domain mutation (C-terminal ARF domain) had weaker phenotypes. While we had hoped to identify a &ldquo;gain&rdquo; 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.