| Summary: | The taste of foods is perceived as one or a combination of five sensations which are sweet, sour, bitter, salty and umami. The umami taste is best described as a savoury sensation (Ikeda, 2002) and it is of central importance in food flavour. The mammalian taste receptors TIRs form two heteromeric G-protein-coupled receptor complexes and taste receptors T 1 R 1 and T 1 R3 function together as an umami taste detector on the tongue (Nelson et al., 2002; Li et al., 2002; Zhao et al., 2003). It is already well established that monosodium glutamate and L-aspartate evoke the umami sensation and are highly detected by these taste receptors (Li et aI., 2002). Yamaguchi (1991) and Ninomiya (1998) reported that umami flavour is enhanced in the presence of inosine monophosphate (IMP). This project aims to develop an in vitro assay that could mimic human perception of umami so that it will be possible to screen extracts of the tomato introgression lines (ILs) for novelumami compounds. A functional umami calcium assay based on G-protein coupled receptor signaling was used to test pure compounds of monosodium glutamate (MSG), inosine-5'monophosphate (IMP) and the ctude tomato IL extracts. The calcium assay was not suitable for use on ctude tomato extracts because the crude samples elicited non specific responses. This led to the development of another high-throughput assay based on a different principle known as the bioluminescence resonance energy transfer (BRET2). This assay was designed to study interactions between proteins through resonance energy transfer between the donor molecule (Rluc) and the acceptor molecule (GFp2). Umami receptors were cloned into an expression vector with the reporter constmcts and then transfected into Flp-In T -Rex cells. Stably transformed cells were challenged with MSG, IMP and crude tomato IL extracts. Additionally transgenic plants were generated with altered expression of GAD and AMP deaminase to modulate the levels of umami compounds in tomato. The BRET2 assay demonstrated the ability of this system to respond to different levels of MSG and IMP through reproducible dose response curves and also established a good correlation with the in vivo sensory panel results. Tests on the ctude tomato IL lines generated umami responses. Lines with low levels of glutamic acid showed high BRET2 response values which suggest the presence of enhancers in the tomato extracts that contributed to the umami taste intensity. The knockout of the GAD gene to increase glutamate levels in plants was unsuccessful. However, overexpression of AMP deaminase produced plants at To generation, which will be the subject of future analysis. Future work will involve the optimization of the newly developed umami BRET2 assay to improve robustness and reproducibility. This assay then can be utilized as a universal umami screen for complex samples, including those from the AMP deaminase plants.
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