Investigating Auxin Metabolism in Arabidopsis thaliana Mutants With Altered Adventitious Rooting via High Throughput Indolealkanoic Acid Quantification

Auxins are a class of plant hormones, or phytohormones, that mediate the coordination of a number of important growth and behavioral processes in plants. The two widely distributed naturally occurring auxins are indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). Interestingly, IBA can be converted to IAA and IAA can serve as a precursor to IBA. These two events happen through two different pathways. IAA is converted into IBA in a two-step process that is only partially understood. This work aims to identify by genetic analysis the unknown enzyme involved in catalyzing the initial step in this biosynthetic conversion of IAA to IBA through the quantification of these auxins within lines of Arabidopsis thaliana mutated in genes that have been positively or negatively correlated with endogenous auxin levels and/or developmental events mediated by these hormones, such as adventitious root formation. Endogenous IAA and IBA were extracted via an automated high-throughput solid-phase extraction method and a modified post-extraction clean-up specifically designed to purify both IAA and IBA within the same sample. GC-SIM-MS analysis of samples utilized a novel IBA internal standard, [13C815N]-indole-3-butyric acid. This modified high-throughput method of multiple indolealkanoic acid quantification was applied to determine IAA and IBA concentrations in different ecotypes of Arabidopsis thaliana seedlings under different experimental growth conditions. Once optimal growth conditions for IBA production were identified, IBA and IAA levels in a series of insertional mutants revealed two gh3-6 mutants with an increased ratio of IAA to IBA concentrations suggesting that the GH3-6 protein is involved in maintaining endogenous levels of IAA and IBA