Molecular analysis of genes involved in the synthesis of proanthocyanidins in Theobroma cacao

The flavonoids catechin and epicatechin, and their polymerized oligomers, the proanthocyanidins (PAs, also called condensed tannins), accumulate to levels of up to 15% of the total weight of dry seeds of Theobroma cacao L. These compounds have been associated with several health benefits in humans including antioxidant activity, improvement of cardiovascular health and reduction of cholesterol levels. They also play important roles in pest and disease defense throughout the plant. This research focuses on molecularly dissecting the proanthocyanidin biosynthetic pathway of Theobroma cacao. To this end, I first isolated candidate genes from T.cacao (Tc) encoding key structural enzymes of this pathway which include, anthocyanidin reductase (ANR), leucoanthocyanidin dioxygenase (LDOX, also called anthocyanidin synthase, ANS) and leucoanthocyanidin reductase (LAR). I performed gene expression profiling of candidate TcANR, TcANS and TcLAR in various tissues through different developmental stages and also evaluated PA accumulation levels in those tissues. My results suggested that all PA candidate genes are co-regulated and positively correlated with PA synthesis. To functionally analyze the candidate genes, I used the model plants Arabidopsis and tobacco as expression platforms. Results from Arabidopsis mutant complementation tests and transgenic tobacco plants constitutively overexpressing cacao genes demonstrate that the candidate structural genes isolated from cacao are true ANS, ANR and LAR genes and all actively involved in PA synthesis in cacao. To further explore the transcriptional regulation of the PA synthesis pathway, I then isolated and characterized an R2R3 type MYB transcription factor TcMYBPA from cacao. I examined the spatial and temporal gene expression patterns of TcMYBPA in cacao and found it to be developmentally expressed in a manner consistent with its involvement in PAs as well as anthocyanin synthesis. Complementation test of TcMYBPA in Arabidopsis tt2 mutant suggested that TcMYBPA could functionally substitute Arabidopsis TT2 gene. Interestingly, except PA accumulation in seeds, I also observed an obvious increase of anthocyanidin accumulation in hypocotyls of transgenic Arabidopsis plants. This is consistent with gene expression analysis which showed that the entire PA pathway could be induced by overexpression of TcMYBPA gene, including DFR, LDOX (ANS) and BAN (ANR). Therefore I concluded that the isolated TcMYBPA gene encodes an R2R3 type MYB transcription factor and is involved in the regulation of both anthocyanin and PA synthesis in cacao. This research will not only offer us the knowledge of secondary metabolites production in cacao, but also provides molecular tools for breeding of cacao varieties with improved disease resistance and enhanced flavonoid profiles for nutritional and pharmaceutical applications.