Identification and functional analysis of betalain pathway genes
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Betalains, comprised of red betacyanins and yellow betaxanthins, are found in the single order, Caryophyllales, where most other flowering plants produce anthocyanins. They are derived from tyrosine via three enzymatic steps: 1. tyrosine is converted to L-DOPA; 2. L-DOPA is converted to the yellow betalamic acid (BA) intermediate; and 3. L-DOPA is also converted to the cyclo-DOPA intermediate. BA spontaneously condenses with amines or amino acid, to make yellow betaxanthins, and with cyclo-DOPA to make red betacyanins. Before the work reported here, the only step for which a pigment ring biosynthetic gene had been cloned was DOPA 4, 5-dioxygenase (DODA) functioning to produce BA. The work described here identified a novel cytochrome P450, CYP76AD1, and showed that CYP76AD1 is absolutely required to produce red pigments in beets. Expression in yeast verifies that it converts L-DOPA to cyclo-DOPA. Transcriptome data was generated on white and red beet varieties by next generation sequencing. In an attempt to find gene(s) responsible for the tyrosine hydroxylation step, two new CYP450 genes, CYP76AD6 and CYP76AD5 were identified. Expression in yeast showed that CYP76AD6, CYP76AD5, and CYP76AD1 are responsible for tyrosine hydroxylation of the betalain biosynthetic pathway. However, unlike CYP76AD1, CYP76AD6 and CYP76AD5 show only very slight activity on L-DOPA to produce cyclo-DOPA and cannot complement yellow beet roots to red. This thesis also studied the functional differences of the two DODA homologs, DODA1, known to act in the betalain pathway, and DODA2, which is more similar to DODA-like proteins in anthocyanin producing non–Caryophyllales species. Expression in beets, Arabidopsis, and in yeast shows that BvDODA1 functions in the betalain pathway and BvDODA2 does not. The conserved amino acids in the two DODA homologs were identified and mutated proteins were expressed in yeast to test whether they are responsible for the functional differences of the two homologs. Identifying members of this pathway represents an important contribution toward understanding the evolutionary replacement of anthocyanins by betalains within a single order, and fills a lack of knowledge by identifying the genes functioning at the two uncharacterized steps in the synthesis of betalain ring structure.