Overexpression of the Citrus Derived R2R3-MYB Gene (Ruby) Strongly Upregulates the MYB-bHLH-WD40 (MBW) Complex Associated Genes and Results in Enhanced Anthocyanin Production in Transgenic Citrus

Commercially cultivated sweet oranges and grapefruits do not produce anthocyanins under warm tropical and subtropical conditions. The blood oranges that do produce anthocyanins require an extended exposure to cold during fruit maturity for activation of an R2R3-MYB transcriptional factor (Ruby) which regulates the subsequent anthocyanin biosynthesis in the fruit. In efforts to develop novel blood orange like citrus cultivars, the Ruby transgene was constitutively overexpressed in ‘Mexican’ lime (Citrus aurantifolia Swingle), a citrus cultivar that does not naturally produce anthocyanin and thrives in the warm subtropical citrus production belt. Ruby overexpression resulted in anthocyanin production as indicated by characteristic pigmentation in the leaves, stems, flowers, and fruit. Transcriptome analysis of the juice vesicles indicated that the MYB-bHLH-WD40 (MBW) complex associated genes were highly upregulated only in the transgenic ‘Mexican’ lime. This resulted in overexpression of the flavonoid biosynthesis associated genes such as chalcone and stilbene synthase family protein (CHI), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), L-phenylalanine ammonia lyase (PAL), UDP-glucosyl transferase (UGT), and Transparent Testa (TT). In the control ‘Mexican’ lime fruit, negative regulator of anthocyanin biosynthesis such as the squmosa promoter binding protein-9 (SPL-9) and S-like Ribonuclease (RNS1) transcripts were highly upregulated. Hierarchical clustering (HCA) and principal component analysis (PCA) demonstrated that the transgenic ‘Mexican’ lime has distant gene expression profiling with the non transgenic ‘Mexican’ lime. Our results suggest that overexpression of Ruby promotes anthocyanin production by triggering the expression of the MBW complex genes.