“Indole-3-Glycerol Phosphate Synthase Characterization in Maize Plants”
Project Summary
In plants, indole-3-glycerol phosphate synthase (IGPS) proteins control the biosynthesis of precursors for the production of important metabolites, including tryptophan, benzoxazinoids, and volatile indole. The maize genome contains three predicted IGPS genes. In our research, we aimed to characterize these genes by investigating their activity in the production of defense compounds, as well as their localization within maize cells.
To determine the functional activity of IGPS genes, we prepared cDNA constructs from each gene and transformed them into E.coli trpC9800, which has a tryptophan auxotrophy due to lack of IGPS. Thereby, a tryptophan complementation assay was used to confirm the activity of each maize IGPS gene. To examine IGPS activity in planta, we obtained maize knockout lines with mutations in each individual IGPS gene, and measured benzoxazinoid and tryptophan content by liquid chromatography-mass spectrometry, as well as volatile indole production by gas chromatography-mass spectrometry.
To localize the IGPS proteins in maize cells we used the Gateway cloning technique to fuse each gene with a yellow fluorescent protein (YFP) marker. IGPS-YFP in the plant cells was visualized by fluorescence microscopy to identify the subcellular location of the fluorescence, which is indicative of the location of the IGPS protein. This research is important for understanding how plants defend against herbivory. By understanding the function of IGPS genes in maize plants, scientists will be able to obtain a more complete picture of the plant, contributing important knowledge and technology for maize production with improved food safety, high quality, and yield.
My Experience:
My summer at BTI has been invaluable. Over the past six weeks, I have learned what it is like to propose, design and perform a research project. I have learned a variety of lab techniques ranging from PCR and RNA extraction to plasmid preparation and Gateway gene cloning. One of the most valuable skills that I have learned through this program is how to think critically and how to face setbacks when experiments do not work. This internship taught me that science is not one dimensional, and that I should approach questions and analyze results with an open mind. I know that these skills will definitely follow me in my future research endeavors. I would like to thank my mentor Annett Richter, Georg Jander, the Jander Lab, and BTI for providing me with this incredible opportunity.