New insights into the genetic manipulation of the R2R3-MYB and CHI gene families on anthocyanin pigmentation in Petunia hybrida.

Several R2R3-MYB genes control anthocyanin pigmentation in petunia, and ANTHOCYANIN-2 (AN2) is treated as the main player in petal limbs. However, the actual roles of R2R3-MYBs in the coloration of different floral tissues in the so called "darkly-veined" petunias are still not clear. The genetic background and expression of AN2 paralogs from various petunias with different color patterns were identified. All "darkly-veined" genotypes have the identical mutation in the AN2 gene, but express a different functional paralog - ANTHOCYANIN-4 (AN4) - abundantly in flowers. Constitutive overexpression of PhAN4 in this petunia resulted not only in a fully colored flower but also in a clearly visible pigmentation in the green tissue and roots, which can be rapidly increased by stress conditions. Suppression of AN4 gene resulted in discolored petals and whitish anthers. Interestingly, when a similar white flower phenotype was achieved by knockout of an essential structural gene of anthocyanin biosynthesis - CHALCONE ISOMERASE-A (CHI-A) - the plant responded directly by upregulating of another paralogs - DEEP PURPLE (DPL) and PURPLE HAZE (PHZ). Moreover, we also found that CHI-B can partially substitute for CHI-A in anthers, but not in vegetative tissues. Further, no significant effects on the longevity of white or enhanced colored flowers were observed compared with the wild type. We concluded that endogenous up-regulation of AN4 leads to the restoration of petal color in the "darkly-veined" phenotypes as a result of the breeding process under human selection, and CHI-B is a backup for CHI-A acitvity in some floral tissues.