A role of DAO1 in oxidation of IAA amino acid conjugates revealed through metabolite, high throughput transcript and protein level analysis

After analysing auxin metabolism in auxin dependent tobacco BY-2 cell line grown in presence or absence of synthetic auxin 2,4-D we found that both conditions were similarly characterized by very low levels of endogenous indole-3-acetic acid (IAA) and its metabolites. However, metabolic profiling after exogenous application of IAA uncovered that the concentration of N-(2-oxindole-3-acetyl)-L-aspartic acid (oxIAA-Asp), the most abundantly formed auxin metabolite in the control culture, dramatically decreased in auxin-starved conditions. To describe the molecular mechanism behind this regulation, we analysed transcriptome and proteome changes caused by auxin starvation. While no changes in the expression of auxin biosynthetic machinery were observed, many genes related to auxin conjugation and degradation showed differential expression. Selected putative auxin glycosylating enzymes as well as members of the Gretchen Hagen 3 gene family involved in auxin amino acid conjugation showed both up- and down-regulation. Contrarily to that, all tobacco homologs of Arabidopsis thaliana DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1), known to be responsible for the formation of oxIAA from IAA, showed significant downregulation at both transcript and protein levels. Overall design: Seven biological replicates of Nicotiana tabacum L. cv. Bright Yellow (BY-2) cell culture samples cultured at auxin supplemented conditions (ctrl) and three replicates of BY-2 cell culture samples cultured at auxin-starved (AF) conditions. Samples collected 48 hours after culture inoculation.