Fig 1.
Identification of candidate genes for tyrosine hydroxylation.
A. Biosynthetic scheme for betacyanin and betaxanthin production. Tyrosine hydroxylation is step 1. B. LOGO analysis of the three CYP76AD subfamily clades. C. qRT-PCR analysis of the beet CYP76AD1, CYP76AD5, and CYP76AD6 genes in non-transgenic beets. Bull’s Blood is a very dark red table beet and Albina Vereduna is a white table beet. Bull’s Blood is YY, expressing the dominant, high-pigment Y allele of BvMYB1 while Albina Vereduna is yy.
Fig 2.
Overexpression of CYP76AD1, CYP76AD6, or CYP76AD5 results in increased L-DOPA content in beet.
A. and B. CYP76AD1 complemented, but CYP76AD5 did not complement the rr (CYP76AD1) beet mutant (Golden Globe) from yellow to red. C. qRT-PCR verified that CYP76AD1 and CYP76AD5 were overexpressed in transgenic hairy root cultures. D, E, F, and G. Mass spectrometry analysis of L-DOPA in Golden Globe beet roots overexpressing CYP76AD1, CYP76AD6, CYP76AD5, and GUS, respectively. Error bars are s.d. Each experiment was replicated three times. Scale bars are 3.5 mm.
Fig 3.
All tested CYP76AD proteins perform tyrosine hydroxylation in yeast.
A. HPLC/ms analysis of yeast expressing CYP76AD1, CYP76AD6, and CYP76AD5 individually shows that all will hydroxylate tyrosine to L-DOPA. Inset shows expanded view of lower peaks. Yeast containing empty vector either have weak tyrosine hydroxylation activity or produce a compound that co-migrates with L-DOPA. B to F. Same yeast strains as in A with the addition of PgDODA. B. images of the cultures fed L-DOPA, tyrosine, or no added substrate with the tyrosine-fed panel corresponding to C through F. C and D. HPLC/MS analysis of the red betanidin pigment with the lower peaks for CYP76AD6, CYP76AD5, and empty vector expanded in D. E. and F. HPLC/MS analysis of the yellow tyrosine-betaxanthin pigment with the lower peaks for CYP76AD6, CYP76AD1, and empty vector expanded in F. Note that CYP76AD1 produces large amounts of betanidin and small amounts of betaxanthin. G. Yeast cultures expressing PgDODA alone and PgDODA with CYP76AD3 or CYP76AD15 from Mirabilis jalapa and fed tyrosine. CYP76AD3 has CYP76AD1-like activity producing red pigment and CYP76AD15 has CYP76AD6/5-like activity producing yellow pigment.
Fig 4.
Arabidopsis expressing betalain biosynthetic genes produce visible pigment phenotypes.
All Arabidopsis are ttg1-1 mutant except the wild type Ler ecotype in panel E. A. left, 35S:CYP76AD5 alone, right 35S:CYP76AD5 and 35S:PgDODA. B. left two flowers and right two flowers the same as in A. C and D are images of the same two petals. Left and right, the same as in A and B. C. white light, D. excitation with UV irradiation showing fluorescence of betaxanthins. E. mature siliques with seeds; top-35S:CYP76AD5 in ttg1-1 alone, middle-35S:PgDODA in ttg1-1 alone, bottom-wild type Ler, no transgene. F. seedlings, left to right- 35S:CYP76AD1 and 35S:PgDODA unfed; 35S:CYP76AD1 and 35S:PgDODA fed 1.5 mM tyrosine; 35S:CYP76AD5 and 35S:PgDODA fed 1.5 mM tyrosine; 35S:CYP76AD5 and 35S:PgDODA unfed. G. Thin Layer Chromatography of betalain pigments: top-untransformed red beet; middle-Arabidopsis transformed with 35S:CYP76AD1 and 35S:PgDODA fed 1.5 mM tyrosine; bottom-Arabidopsis transformed with 35S:CYP76AD5 and 35S:PgDODA fed 1.5 mM tyrosine. Asterisks mark the presence of red betacyanins.