Transcription factor MdNAC33 is involved in ALA-induced anthocyanin accumulation in apples

5-Aminolevulinic acid (ALA), as a new natural plant growth regulator, has a significant function in promoting anthocyanin accumulation in many species of fruits. However, the mechanisms underlying remain obscure. In a transcriptome study of our group, it was found that many transcription factors (TFs) including NACs responsive to ALA treatment during anthocyanin accumulation. In the present study, we found a NAC of apple, MdNAC33 was coordinatively expressed with anthocyanin accumulation after ALA treatment in the apple fruits and leaves, suggesting that this TF may be involved in anthocyanin accumulation induced by ALA. We found that the MdNAC33 protein was localized in the nucleus and exhibited strong transcriptional activity in both yeast cells and plants, where its C-terminal contributed to the transcriptional activity. Functional analysis showed that overexpression of MdNAC33 promoted the accumulation of anthocyanin, while the silencing vector of MdNAC33 (RNAi) significantly impaired the anthocyanin accumulation induced by ALA. Yeast one-hybrid (Y1H), luciferase assay and electrophoretic mobility shift assay (EMSA) indicated that MdNAC33 could bind to promoters of MdbHLH3, MdDFR and MdANS to activate the gene expressions. In addition, MdNAC33 specifically interacts with MdMYB1, a positive regulator of anthocyanin biosynthesis, which was then in turn binding to its target genes MdUFGT and MdGSTF12, to promote anthocyanin accumulation in apples. Taken together, our data indicate that MdNAC33 plays multiple roles in ALA-induced anthocyanin biosynthesis. It provides new insights into the mechanisms of anthocyanin accumulation induced by ALA.