BoMYB2 plays a key role in anthocyanin accumulation in tobacco plants overexpressing BoMYB2 , BoTT8 and BoTTG1 transcription factors from purple cauliflower

Purple cauliflower exhibits substantial anthocyanin accumulation in the curd and young tissues. The accumulation of anthocyanin was mainly regulated independently by MYB, bHLH and WD40 transcription factors, or the MBW complex formed by them, whether these three transcription factors independently or synergistically affect anthocyanin accumulation remains unclear in the purple cauliflower. Herein, BoMYB2 (MYB), BoTT8 (bHLH) and BoTTG1 (WD40) elements were isolated from the purple cauliflower variety ‘Graffiti’ and heterologously expressed in tobacco to investigate the effect of these three transcription factors on anthocyanin accumulation. Among the transgenic plants, anthocyanin accumulated in the callus, leaves, stems, buds, flowers, capsules and immature seeds of 35S:MYB2 , 35S:MYB2TTG1 , 35S:MYB2TT8 and 35S:MYB2TT8TTG1, but not 35S:TT8 , 35S:TTG1 and 35S:TT8TTG1 . The structural gene NtDFR for anthocyanin synthesis was upregulated in the young leaves and buds of 35S:MYB2 , 35S:MYB2TTG1 and 35S:MYB2TT8 . More anthocyanin accumulation were induced by additional BoTT8 co-expression in 35S:MYB2TT8 , when compared with 35S:MYB2 and 35S:MYB2TTG1 transgenic plants, and further anthocyanin accumulation was induced by co-expression of BoMYB2 , BoTTG1 and BoTT8 in 35S:MYB2TT8TTG1 . The transgenic plants of 35S:MYB2TT8TTG1 with higher anthocyanin contents showed weaker growth and vitality. These results demonstrated that BoMYB2 was the key factor in regulating anthocyanin accumulation at the seedling, vegetative and floral stages. BoTT8 and BoTTG1 did not induce anthocyanin pigment independently, but they may cooperate with BoMYB2 to enhance anthocyanin accumulation and slow the rate of vegetative growth.