Anthocyanins are red and violet pigments that color flowers, fruits and epidermal tissues in virtually all flowering plants. A single order, Caryophyllales, contains families in which an unrelated family of pigments, the betalains, color tissues normally pigmented by anthocyanins. Here we show that CYP76AD1 encoding a novel cytochrome P450 is required to produce the red betacyanin pigments in beets. Gene silencing of CYP76AD1 results in loss of red pigment and production of only yellow betaxanthin pigment. Yellow betalain mutants are complemented by transgenic expression of CYP76AD1, and an insertion in CYP76AD1 maps to the R locus that is responsible for yellow versus red pigmentation. Finally, expression of CYP76AD1 in yeast verifies its position in the betalain biosynthetic pathway. Thus, this cytochrome P450 performs the biosynthetic step that provides the cyclo-DOPA moiety of all red betacyanins. This discovery will contribute to our ability to engineer this simple, nutritionally valuable pathway into heterologous species.

Nearly all flowering plants produce red/violet anthocyanin pigments. Caryophyllales is the only order containing families that replace anthocyanins with unrelated red and yellow betalain pigments. Close biological correlation of pigmentation patterns suggested that betalains might be regulated by a conserved anthocyanin-regulating transcription factor complex consisting of a MYB, a bHLH and a WD repeat-containing protein (the MBW complex). Here we show that a previously uncharacterized anthocyanin MYB-like protein, Beta vulgaris MYB1 (BvMYB1), regulates the betalain pathway in beets. Silencing BvMYB1 downregulates betalain biosynthetic genes and pigmentation, and overexpressing BvMYB1 upregulates them. However, unlike anthocyanin MYBs, BvMYB1 will not interact with bHLH members of heterologous anthocyanin MBW complexes because of identified nonconserved residues. BvMYB1 resides at the historic beet pigment-patterning locus, Y, required for red-fleshed beets. We show that Y and y express different levels of BvMYB1 transcripts. The co-option of a transcription factor regulating anthocyanin biosynthesis would be an important evolutionary event allowing betalains to largely functionally replace anthocyanins.

The key enzymatic step in betalain biosynthesis involves conversion of l-3,4-dihydroxyphenylalanine (l-DOPA) to betalamic acid. One class of enzymes capable of this is 3,4-dihydroxyphenylalanine 4,5-dioxygenase (DODA). In betalain-producing species, multiple paralogs of this gene are maintained. This study demonstrates which paralogs function in the betalain pathway and determines the residue changes required to evolve a betalain-nonfunctional DODA into a betalain-functional DODA. Functionalities of two pairs of DODAs were tested by expression in beets, Arabidopsis and yeast, and gene silencing was performed by virus-induced gene silencing. Site-directed mutagenesis identified amino acid residues essential for betalamic acid production. Beta vulgaris and Mirabilis jalapa both possess a DODA1 lineage that functions in the betalain pathway and at least one other lineage, DODA2, that does not. Site-directed mutagenesis resulted in betalain biosynthesis by a previously nonfunctional DODA, revealing key residues required for evolution of the betalain pathway. Divergent functionality of DODA paralogs, one clade involved in betalain biosynthesis but others not, is present in various Caryophyllales species. A minimum of seven amino acid residue changes conferred betalain enzymatic activity to a betalain-nonfunctional DODA paralog, providing insight into the evolution of the betalain pigment pathway in plants.

Yellow and red-violet betalain plant pigments are restricted to several families in the order Caryophyllales, where betacyanins play analogous biological roles to anthocyanins. The initial step in betalain biosynthesis is the hydroxylation of tyrosine to form L-DOPA. Using gene expression experiments in beets, yeast, and Arabidopsis, along with HPLC/MS analysis, the present study shows that two novel cytochrome P450 (CYP450) enzymes, CYP76AD6 and CYP76AD5, and the previously described CYP76AD1 can perform this initial step. Co-expressing these CYP450s with DOPA 4,5-dioxygenase in yeast, and overexpression of these CYP450s in yellow beets show that CYP76AD1 efficiently uses L-DOPA leading to red betacyanins while CYP76AD6 and CYP76AD5 lack this activity. Furthermore, CYP76AD1 can complement yellow beetroots to red while CYP76AD6 and CYP76AD5 cannot. Therefore CYP76AD1 uniquely performs the beet R locus function and beets appear to be genetically redundant for tyrosine hydroxylation. These new functional data and ancestral character state reconstructions indicate that tyrosine hydroxylation alone was the most likely ancestral function of the CYP76AD alpha and beta groups and the ability to convert L-DOPA to cyclo-DOPA evolved later in the alpha group.

Introduction: We describe the development and performance of a new sample-to-report targeted sequencing solution for testing solid tissue cancers using the Genexus Integrated Sequencing System and accompanying software. The assay is designed for research applications from either formalin fixed paraffin embedded (FFPE) solid tumor samples or cell-free total nucleic acid (cfTNA) from liquid biopsy samples. The Genexus Integrated Sequencer is a fully automated system requiring minimal touch points and hands on time allowing a novice user to go from nucleic acid to variant calls for somatic variant testing across multiple cancer types in less than two days. Methods: The Oncomine Precision Assay is a new amplicon-based assay targeting specific somatic variants in 50 genes with coverage for multiple cancer types. The assay uses AmpliSeq HD chemistry capable of distinguishing true sample biological variants from errors generated during library preparation, templating, and sequencing through incorporation of molecular tags during target amplification. With about 15 minutes of hands on time, a run is set-up using pre-filled reagent strips for a fully automated run that includes library prep, templating, sequencing, variant calling, and a final report if desired. Results: Reported here are the results generated from an early external test site along with development data. The Oncomine Precision Assay is designed to detect somatic variants in 50 unique genes testing all major variant types important in the oncology research. The content was selected based on published accounts of target actionability and prevalence across multiple cancer types. All major variant types are targeted including SNVs, insertions, deletions, copy number variation, fusion transcripts and alternate splice forms. Data is shared from an external test lab using the Oncomine Precision Assay on the Genexus Integrated Sequencer with control and research samples. Results from both multiplexed FFPE and liquid biopsy runs are presented. Data demonstrates use of a single assay and system to effectively call variants from both FFPE and liquid biopsy sample types with a turn-around time of less than 30 hours. Conclusion: The Oncomine Precision Assay and Genexus Integrated Sequencer enable detection of key oncology variants in 50 genes using either solid tissue or liquid biopsy samples as input. This fully automated solution for oncology research generates variant calls from nucleic acid input in less than 2 days with minimal hands-on time and touch-points from the user. Many features of the automated system increase success rates by ensuring the appropriate reagents are properly installed before a run. The user friendly, highly automated, and fully optimized sample to answer system described here has great potential for targeted oncology sequencing in the research setting. Citation Format: Jian Gu, Ru Cao, Jeff Schageman, Kris Lea, Priyanka Kshatriya, Amir Marcovitz, Paul Williams, Rasika Sunnadeniya, Varun Bagai, Khalid Hanif, Jose L. Costa, Kelli Bramlett. Demonstration of the genexus integrated sequencing system with the oncomine precision assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 213.