Screening of Genes Important for Artificial Accumulation of 6′-Deoxychalcone in <i>Nicotiana benthamiana</i>

6′-Deoxychalcone (isoliquiritigenin and butein) is a yellow flavonoid pigment that is promising target for molecular breeding of yellow flowers. In our previous study, it was suggested that co-overexpression of CaMYBA, a positive anthocyanin MYB transcription factor in pepper (Capsicum annuum) with a dahlia (Dahlia variabilis) aldo-keto reductase (AKR) DvAKR1 and snapdragon (Antirrhinum majus) chalcone 4′-O-glucosyltransferase (4′CGT) Am4′CGT was sufficient to accumulate isoliquiritigenin. However, CaMYBA expression induced not only accumulation of 6′-deoxychalcone, but also that of delphinidin-based anthocyanin. Since this anthocyanin accumulation is unnecessary for molecular breeding of yellow flowers, it is necessary to identify genes important for the accumulation of only 6′-deoxychalcone. In this study, we conducted comparative RNA-seq analysis between co-overexpressing CaMYBA, DvAKR1, and Am4′CGT (referred as the CaMYBA combination) and co-overexpressing NtAN2, DvAKR1, and Am4′CGT (referred as the NtAN2 combination) Nicotiana benthamiana leaves. The CaMYBA combination successfully induced isoliquiritigenin, while the NtAN2 combination failed to do so. Because the NbCHS2 gene was detected as a differentially expressed gene, CHS genes from N. benthamiana, pepper, and dahlia were co-overexpressed with DvAKR1 and Am4′CGT. Our results reconfirm that CHS plays an important role in the accumulation of isoliquiritigenin, but the detected peak of isoliquiritigenin was small, also indicating that other genes are required for the abundant accumulation of isoliquiritigenin. We also analyzed NbPAL4, which was more highly expressed in the CaMYBA combination than in the NtAN2 combination, but our results indicated that NbPAL4 is not essential for isoliquiritigenin accumulation. Finally, we investigated why the NtAN2 combination did not work for isoliquiritigenin and anthocyanin accumulation. RNA-seq analysis indicated one bHLH transcription factor was down-regulated in the NtAN2 combination, so we co-overexpressed a dahlia bHLH transcription factor DvIVS with the NtAN2 combination. Isoliquiritigenin accumulation was successfully detected suggesting that the failure of isoliquiritigenin accumulation in the NtAN2 combination is due to the weak ability of NtAN2 to activate bHLH transcription factors.