The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes

Abstract

MdMYB16 forms homodimers and directly inhibits anthocyanin synthesis via its C-terminal EAR repressor. It weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis when overexpressing MdbHLH33 in callus overexpressing MdMYB16. MdMYB16 could interact with MdbHLH33. Anthocyanins are strong antioxidants that play a key role in the prevention of cardiovascular disease, cancer, and diabetes. The germplasm of Malus sieversii f. neidzwetzkyana is important for the study of anthocyanin metabolism. To date, only limited studies have examined the negative regulatory mechanisms underlying anthocyanin synthesis in apple. Here, we analyzed the relationship between anthocyanin levels and MdMYB16 expression in mature Red Crisp 1-5 apple (M. domestica) fruit, generated an evolutionary tree, and identified an EAR suppression sequence and a bHLH binding motif of the MdMYB16 protein using protein sequence analyses. Overexpression of MdMYB16 or MdMYB16 without bHLH binding sequence (LBSMdMYB16) in red-fleshed callus inhibited MdUFGT and MdANS expression and anthocyanin synthesis. However, overexpression of MdMYB16 without the EAR sequence (LESMdMYB16) in red-fleshed callus had no inhibitory effect on anthocyanin. The yeast one-hybrid assay showed that MdMYB16 and LESMdMYB16 interacted the promoters of MdANS and MdUFGT, respectively. Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays showed that MdMYB16 formed homodimers and interacted with MdbHLH33, however, the LBSMdMYB16 could not interact with MdbHLH33. We overexpressed MdbHLH33 in callus overexpressing MdMYB16 and found that it weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis. Together, these results suggested that MdMYB16 and MdbHLH33 may be important part of the regulatory network controlling the anthocyanin biosynthetic pathway.

R2R3‐MYB transcription factor MdMYB23 is involved in the cold tolerance and proanthocyanidin accumulation in apple

“…In Arabidopsis, AtPAP1, AtPAP2, AtMYB113, AtMYB114, and AtMYBL2 act as crucial modulators of flavonoid biosynthesis (Borevitz et al, 2000;Gonzalez et al, 2008;Matsui et al, 2008;Rowan et al, 2009). In apple, MdMYB1/10/A, MdMYB3, MdMYB12, MdMYB16, and MdMYB22 positively affect flavonoid biosynthesis (Takos et al, 2006;Ban et al, 2007;Espley et al, 2007;Vimolmangkang et al, 2013;Xu et al, 2017;Wang et al, 2017). Decreased ROS accumulation in MdMYB23-overexpressing apple calli and Arabidopsis in response to cold stress suggested that MdMYB23 might be involved in the modulation of flavonoid accumulation.…”

PbMYB120 Negatively Regulates Anthocyanin Accumulation in Pear

“…For example, apple (Malus × domestica) MYB16 directly binds MdANS and MdUFGT, and inhibits their expression through the C-terminal EAR motif. AtMYB3 inhibits the biosynthesis of sinapoyl malate and anthocyanin directly by targeting and repressing AtC4H [29,41,42].…”

“…The expression of UFGT has been inhibited in anthocyanin MYB repressor-overexpression lines. However, only a few MYB repressors, apple MdMYB16 and peach PpMYB18, for instance, can target the promoter of UFGT or inhibit the promoter activity of UFGT directly [30,41,42]. The actions of lignin and flavonol MYB regulators usually do not require the bHLH cofactor, while the actions of anthocyanin and proanthocyanidin MYB regulators generally do require the bHLH cofactor [30].…”

Phytochemical profiles, antioxidant, and antiproliferative activities of four red‐fleshed apple varieties in China

“…Over the past decade, our group has constructed a series of hybrid populations of M. sieversii f. niedzwetzkyana (Luntai National Fruit Germplasm Resources Garden, China) crossed with two excellent white‐fleshed varieties (Fuji & Gala). So far, these studies have focused mainly on their genetic structure (Zhang, Chen, He, Liu, & Tao, ), tissue cryopreservation in vitro (Wu et al., ), breeding of functional apples (Chen et al., ), the mechanism of anthocyanin and flavonoid formation (Wang et al., ; Wang et al., ; Xu et al., ), and processing technology (Zuo et al., ). However, the phytochemical characteristics of new red‐fleshed varieties in China, such as free phenolics, bound phenolics, antioxidant activity especially in cellular antioxidant activity (CAA) and antiproliferative activity, have not been systematically evaluated.…”