Epsilon Archive for Student Projects

Abstract

Auxins are one of the oldest phytohormones known to us, as studies on its physiological effects date back to the end of the nineteenth century. The cardinal auxin in plants is IAA, and attempts have been made to elucidate its biosynthesis and activity for over 70 years. IAA is known to be involved in a high multitude of developmental processes, and has a key role in many aspects of plant growth and development, such as root and shoot architecture, cell growth and division, response to environmental stimuli and stress response. Despite substantial progress made in the last two decades to understand the biosynthesis and catabolism of IAA, the metabolic pathways and regulatory mechanisms underlying its homeostasis in plant cells are still to be fully clarified.
This master dissertation pursued to clear up existing problems and gaps associated with IAA metabolism, aiming to identify novel genes directly or indirectly involved in IAA homeostasis, and resolve regulatory mechanisms behind auxin conjugation and degradation. For this purpose, Arabidopsis mutant lines were generated by ethyl methanesulfonate (EMS) treatment of auxin reporter lines. These were screened for their auxin metabolic profile by high-throughput liquid chromatography–tandem mass spectrometry (LC-MS/MS) profiling, and multivariate data analysis (MVDA) was used to identify candidate lines based on their diverging metabolic profiles from these in control lines. Selected lines were backcrossed and then analyzed by confocal imaging to study IAA distribution in the root tip.
A mapping-by-sequencing approach was performed for one particular line, DII365.3, which was identified as showing high levels of indole-3-acetaldoxime (IAOx). Nine candidate genes were identified carrying homozygous non-synonymous substitutions in their coding sequences, and T-DNA insertion lines carrying a disruption in these genes were ordered in an attempt to obtain stable knockout lines. Complementation tests and IAA metabolite profiling of the lines were initiated in this work and, together with establishing the knockout nature of the insertions, will be determinant in identifying the causal mutation.
Furthermore, to gain knowledge about regulatory mechanisms in the IAA conjugation/degradation pathways, different constructs were successfully generated carrying guide RNA (gRNA) sequences targeting members of the DIOXYGENASE FOR AUXIN OXIDATION (DAO) and UDP-glycosyl transferase (UGT) family of genes, by using a multiplex approach of the CRISPR-Cas9 technology. The different constructs were transformed into diverse Arabidopsis mutant backgrounds in order to faster and more efficiently generate different knockout combinations. Metabolic and transcriptional profiling of the lines generated here will be fundamental in modeling the regulation of the IAA inactivation pathways and their influence on IAA homeostasis.