Not much is known about the efficient trafficking of potentially toxic phytochemicals from their site of synthesis to their correct intracellular destinations. Anthocyanins, the colored products of the well characterized flavonoid pathway, were used as a convenient model system to understand the cellular, biochemical and molecular processes involved in its transport from the cytoplasmic face of the ER to the central vacuole. Cell biological observations of maize Black Mexican Sweet (BMS) cells in culture, constitutively expressing the MYB (C1) and bHLH (R) transcription factors, show these cells to accumulate anthocyanins in multiple vacuoles with anthocyanic vacuolar inclusions (AVIs). Exposure to light caused calli to darken. This was attributed to the fusion of the vacuoles and 'spread' of anthocyanins from the AVIs into the sap but not due to changes in transcripts of the enzymes or the accumulation of pigments. Formation of vacuoles from dynamic pigmented compartments was observed in maize tassel glumes. These observations indicated an alternate vesicular route of transport of anthocyanins to the central vacuole. In Arabidopsis , I exploited the transparent testa mutant tt5, deficient in the enzyme chalcone isomerase (CHI) and its anthocyanin complementation with naringenin to develop an easily manipulatable seedling model system. Naringenin-treated tt5 and wt seedlings accumulated a new anthocyanin peak identified as cyanidin 3-glucoside. Global transcriptome changes were monitored using microarrays to identify potential transferases and transporters involved in either the detoxification of naringenin or the transport of anthocyanins. The induction of signaling components, jasmonic acid biosynthetic genes and defense-related stress response genes, suggested additional roles of flavonoids in several cellular processes. Lastly, the need for the catalytic function of CHI was investigated in driving flux into the flavonoid pathway. The catalytic mutants Y104F, T46A and R34A were generated in maize CHI (ZmCHI). In vitro assays demonstrated ZmCHIY104F to retain 20% of the ZmCHIwt activity while ZmCHIT46A and ZmCHIR34A were catalytically inactive. Only ZmCHIY104F and, surprisingly, the in vitro catalytically inactive ZmCHIT46A complemented Arabidopsis tt5 mutants. These findings revealed additional roles of CHI in the flavonoid pathway. Taken together, these observations and results provided significant insights in understanding processes involved in phytochemical trafficking.
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