Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp.
(A) Agroinfiltrated N. benthamiana leaf with the AmRosea1:AmDelila:MtANR:MtLAR multigenic construct showing purple pigmentation. (B) Infiltrated leaf with DsRed (control). (C) Non-infiltrated wild-type leaf. (D) Expression levels of the AmRosea1, AmDelila, MtANR and MtLAR transgenes in agroinfiltrated leaves of N. benthamiana. NbACT8 is used as positive control. PCR results were obteined after 30 amplification cycles for all analized genes. (E) RT-PCR analysis of the anthocyanin biosynthetic pathway genes in agroinfiltrated leaves of N. benthamiana. The PCR results were obteined after 30 amplification cycles for all genes and 25 cycles for the housekeeping NbACT8 gene. (F) PA levels in agroinfiltrated leaf extracts of N. benthamiana obteined by the dimethylaminocinnamaldehyde (DMACA) colorimetric reaction. The DMACA reaction showed an increase of the PA’s content in the agroinfiltrated leaf area compared with the non-infiltrated WT leaves. The maximum PA levels were found in the plants infiltrated with the multigenic construct. Statistical T-Test values for (-) catechin (n = 3, t = 11.46, df = 4, p = 0.0003) and (-) epicatechin (n = 3, t = 11.46, df = 4, p = 0.0003).