The study of Tryptophan-Dependent Indole-3-Acetic Acid Biosynthesis pathways in Maize Endosperm

University of Minnesota Ph.D. dissertation. August 2017. Major: Plant Biological Sciences. Advisor: Adrian Hegeman. 1 computer file (PDF); x, 196 pages.Auxin as the most important plant hormone regulates almost all aspects of plant growth and development. Indole-3-acetic acid (IAA), the most abundant form of auxin, plays essential roles in the housekeeping functions. IAA can be de novo synthesized via tryptophan-dependent pathways or tryptophan-independent pathways. The findings on the most studied tryptophan-dependent IAA biosynthesis suggested that there may be 4 routes involving 6 key intermediates contribute to the IAA production from tryptophan in maize. Due to the genetic redundancies of the IAA biosynthesis pathways and the low activities of the exogenously expressed enzymes involved in the pathways, the classic genetics and biochemical approaches have proven challenging. The work in chapter 2 demonstrated that maize endosperm in vitro system retained a stable and high enzyme activities in the reaction of converting tryptophan to IAA, and converting indole-3-pyruvic acid (IPyA) to IAA enzymatically. Thus, maize endosperm in vitro system provides an excellent platform to study tryptophan-dependent IAA biosynthesis. The enzymatic parameters of the IAA biosynthesis reaction using the maize endosperm system were also characterized in chapter 2. The work in chapter 3 is to investigate the contribution of TAA1/YUCCA pathway to the overall tryptophan-dependent IAA biosynthesis in maize endosperm system. This chapter deployed two methodological approaches, the isotopic labeling dilution assay, and the oxygen depletion and 18O2 labeling experiments, to address this question. The results from both methods strongly indicated that TAA1/YUCCA is the main tryptophan dependent pathway. Furthermore, the 18O2 labeling experiments determined that TAA1/YUCCA pathway contributes at least 80% of the tryptophan-dependent IAA biosynthesis pathways. In order to get around the obstacles presented by classic genetics methods, the work in chapter 4 aimed to find effective inhibitors that can potentially be used to selectively and temporarily block enzymes in certain IAA biosynthesis routes, at different development stages, under various environmental conditions. Four groups of compounds, including indole derivative inhibitors, tryptophan transaminase inhibitors, kynurenine pathway related inhibitors, and yucasin has been tested. The data suggested that yucasin, very likely targeting at YUCCA, is a very potent inhibitor in the maize endosperm in vitro system. AOPP, which is very likely targeting at TAA1, and MPP+ iodide are also good inhibitors in tryptophan-dependent IAA biosynthesis pathways