The MIR-Domain of PbbHLH2 Is Involved in Regulation of the Anthocyanin Biosynthetic Pathway in ”Red Zaosu” (PyrusBretschneideri Rehd.) Pear Fruit

Abstract

The N-terminal of Myc-like basic helix-loop-helix transcription factors (bHLH TFs) contains an interaction domain, namely the MYB-interacting region (MIR), which interacts with the R2R3-MYB proteins to regulate genes involved in the anthocyanin biosynthetic pathway. However, the functions of MIR-domain bHLHs in this pathway are not fully understood. In this study, PbbHLH2 containing the MIR-domain was identified and its function investigated. The overexpression of PbbHLH2 in ”Zaosu” pear peel increased the anthocyanin content and the expression levels of late biosynthetic genes. Bimolecular fluorescence complementation showed that PbbHLH2 interacted with R2R3-MYB TFs PbMYB9, 10, and 10b in onion epidermal cells and confirmed that MIR-domain plays important roles in the interaction between the MIR-domain bHLH and R2R3-MYB TFs. Moreover, PbbHLH2 bound and activated the dihydroflavonol reductase promoter in yeast one-hybrid (Y1H) and dual-luciferase assays. Taken together these results suggested that the MIR domain of PbbHLH2 regulated anthocyanin biosynthesis in pear fruit peel.

DhMYB2 and DhbHLH1 regulates anthocyanin accumulation via activation of late biosynthesis genes in Phalaenopsis-type Dendrobium

“…Direct interactions of MYB or bHLH alone with promoters of structural genes has not been previously shown. Rather, they regulate ABP structural genes by forming MBW protein complexes ( Li et al., 2021b ; Yan et al., 2021 ). Similarly, we found DhMYB2 and DhbHLH1 alone are not sufficient to bind and activate expression of DhDFR and DhANS , respectively, in PD.…”

“…MYB binding elements involved in flavonoid biosynthesis h a v e b e e n i d e n t i fi e d i n m a n y r e p o r t s , i n c l u d i n g AGMOTIFNTMYB2, MYB26PS, MYBPZM, MYB core, and MYBPLANT (Singh et al, 2015;Yan et al, 2021). Likewise, Ebox cis-acting elements are the most common binding targets of bHLH, of which G-box is the most common type (Li et al, 2021b;Qian et al, 2021). In this study, several putative ciselements recognized by MYB or bHLH were identified in the promoter regions of DhF3'H1, DhF3'5'H2, DhDFR, DhANS, and DhGT4 (Figure 6).…”

ZeMYB9 regulates cyanidin synthesis by activating the expression of flavonoid 3′-hydroxylase gene in Zinnia elegans

“…AtGL3 and AtEGL3 can also coordinate with MYB transcription factors to regulate the synthesis of anthocyanin but not proanthocyanidins ( Payne et al., 2000 ; Morohashi et al., 2007 ). PbbHLH2 from Pyrus bretschneideri , interacts with MYB through the N-terminal MIR domain to promote anthocyanin synthesis ( Li et al., 2021 ). Evolutionary analysis showed that ZeGL3 belonged to the GL3 clade and there was a MIR domain region present in its N-terminal ( Figure S5 ), suggesting that this protein may possess positive regulation on anthocyanin synthesis.…”

The Basic Helix-Loop-Helix Transcription Factor SmbHLH1 Represses Anthocyanin Biosynthesis in Eggplant

“…The bHLH, R2R3-MYB, and WD repeat proteins can form a ternary MBW complex to modulate the expression of anthocyanin biosynthesis-related genes. Previous studies have revealed that several bHLH TFs, including AtTT8, AtGL3, AtEGL3, PhAN1, PhJAF13, GhMYC1, SlAN1, MdbHLH3, and PbbHLH2, interact with their respective MYB partners AtPAP1, AtTT2, PhAN2, GhMYB10, SlAN2, MdMYB1, and PbMYB9/10/10b to regulate anthocyanin or proanthocyanidin biosynthesis in Arabidopsis , Petunia hybrida , Gerbera hybrida , S. lycopersicum , M. domestica , and Pyrus bretschneideri ( Elomaa et al, 2003 ; Koes et al, 2005 ; Ramsay and Glover, 2005 ; Xie et al, 2012 ; Sun et al, 2020a ; Li et al, 2021b ). Xie et al (2012) reported that two regions (amino acids 1–24 and 186–228) at the N terminus of MdbHLH3 were crucial for the interaction between MdbHLH3 and MdMYB1.…”