Functional analysis of betalain biosynthesis genes in Portulaca umbraticola

Portulaca umbraticola blooms in summer and produces betalains as pigments.
Betalains are classified into yellow betaxanthin and reddish-purple betacyanin, and the flower color variation of P. umbraticola is caused by differences in the content of betaxanthin and betacyanin.
Betacyanins are mainly synthesized by three key enzyme proteins, cytochrome P450-α (CYP76AD-α), DOPA 4,5-dioxygenase (DOD), and cyclo-DOPA-5-O-glucosyltransferase (cDOPA5GT). In P. umbraticola, previous studies in our group have identified PuDOD1 and PuCYP76AD1, which encode DOD and CYP76AD-α protein, respectively.
In this study, we performed functional analysis of these genes to investigate their pigment biosynthetic activity.
The coding region of PuCYP76AD1 and PuDOD1 were amplified from a cDNA of P. umbraticola. For cDOPA5GT with known function, MjcDOPA5GT from Mirabilis jalapa was used because the gene encoding cDOPA5GT has not been identified in P. umbraticola. These genes were cloned into the binary vector pRI201-AN simultaneously.
The vector was transformed into Agrobacterium GV3101 strain.
Infiltration was performed by injecting the transgenic Agrobacterium into Nicotiana benthamiana leaves to transiently overexpress those genes.
Pigments were extracted from the leaves five days after infiltration and analyzed by HPLC. In N. benthamiana leaves overexpressed PuCYP76AD1, PuDOD1, and MjcDOPA5GT simultaneously, the inoculated area with Agrobacterium turned reddish.
HPLC analysis of the extracted pigment revealed that the peak corresponded to the retention time of betanin, a type of betacyanin, confirming that the accumulated pigment was betacyanin.
These results suggest that PuCYP76AD1 and PuDOD1 are responsible for betacyanin biosynthesis.

IV Asian Horticultural Congress – AHC2023

C. Sumi, M. Ishimori, M. Shibata, Y. Higuchi

betalains, betacyanin, gene function, pigments, transformation

https://doi.org/10.17660/ActaHortic.2024.1404.20