The biosynthesis of the primary auxin-type plant hormone, indole-3-acetic acid (IAA), has remained an active area of study for more than half a century and numerous different pathways have been predicted but few have been shown to be critical for normal plant development. The biosynthesis of IAA during late kernel development in maize is important for the accumulation of relatively large quantities of stored conjugated IAA and has been shown to originate from the amino acid tryptophan. The physiologically relevant production of high levels of IAA during a short time period in endosperm development makes this a unique system for analysis of IAA biosynthesis pathways. Using two complementary approaches to identify the enzymes responsible for the different conversions in the pathway as well as the identification of the intermediate compounds, it was shown that previously reported pathways could not account for the measured IAA production. A metabolite screen for potential intermediates revealed the majority of the radioactivity that was not either tryptophan or IAA was associated with a higher molecular weight fraction. Gel electrophoresis and size exclusion HPLC indicated radioactivity was associated with proteins in the size range of 10-20 kDa, where tryptophan was linked apparently via a thio-ester bond. The focus of this project is to utilize biochemical analyses to isolate, characterize and microsequence proteins, which become bound to tryptophan, for subsequent cloning of corresponding genes. The ability to produce the tryptophyl-proteins in larger amounts and at higher purities will help resolve their role(s) in IAA biosynthesis.
Broader impacts: This project will train a postdoctoral scientist in both research and in the grant writing process. Results obtained in this project will be used as part of a separately funded effort to train and mentor minority undergraduate students. In addition, the laboratory will host visiting students, postdocs and other scientists from universities and 4-year colleges to learn techniques related to the project goals.
