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.

MdMYB110a, directly and indirectly, activates the structural genes for the ALA-induced accumulation of anthocyanin in apple.

5-Aminolevulinic acid (ALA), an essential biosynthetic precursor of tetrapyrrole compounds, promotes the anthocyanin accumulation in many plant species. However, the underlying mechanism of ALA-induced accumulation is not yet fully understood. In this study, we identified an important regulator of the anthocyanin accumulation, MdMYB110a, which plays an important role in the ALA-induced anthocyanin accumulation. MdMYB110a activated the expression of MdGSTF12 by binding to its promoter. Additionally, two interacting MdMYB110a proteins, MdWD40-280 and MdHsfB3a, were isolated and confirmed as positive regulators of the ALA-induced anthocyanin accumulation. Both MdWD40-280 and MdHsfB3a enhanced the ability of MdMYB110a to transcribe MdGSTF12. A yeast one-hybrid assay revealed that MdWD40-280 did not bind to most structural genes in the anthocyanin biosynthetic and transport pathways, thus promoting anthocyanin accumulation by MdWD40-280 to depend on MdMYB110a. However, MdHsfB3a could bind to both the MdDFR and MdANS promoters, thereby directly regulating anthocyanin biosynthesis. Collectively, these results provide new insight into the mechanism of ALA-induced anthocyanin accumulation.

The Transcription Factor MdERF78 Is Involved in ALA-Induced Anthocyanin Accumulation in Apples.

As a friendly plant growth regulator to the environment, 5-aminolevulinic acid (ALA) has been widely used in plant production, such as fruit coloration, stress resistance, and so on. Previous studies have identified some genes that have a function in the anthocyanin accumulation induced by ALA. However, the regulatory mechanism has not been well revealed. In the current study, we proposed that an ALA-responsive transcription factor, MdERF78, regulated anthocyanin accumulation. MdERF78, overexpressed in apple peels or calli, resulted in a significant increase of anthocyanins, while MdERF78 interference had an opposite trend. Furthermore, the anthocyanin accumulation induced by MdERF78 overexpression was enhanced by exogenous ALA treatment, suggesting that MdERF78 was involved in the ALA-induced anthocyanin accumulation. Yeast one-hybrid and dual luciferase reporter assays revealed that MdERF78 bound to the promoters of MdF3H and MdANS directly and activated their expressions. Additionally, MdERF78 interacted with MdMYB1 and enhanced the transcriptional activity of MdMYB1 to its target gene promoters. Based on these, it can be concluded that MdERF78 has a positive function in ALA-induced anthocyanin accumulation via the MdERF78-MdF3Hpro/MdANSpro and MdERF78-MdMYB1-MdDFRpro/MdUFGTpro/MdGSTF12pro regulatory network. These findings provide new insights into the regulatory mechanism of ALA-promoted anthocyanin accumulation.

MdSCL8 as a Negative Regulator Participates in ALA-Induced FLS1 to Promote Flavonol Accumulation in Apples.

Apples (Malus domestica) are rich in flavonols, and 5-aminolevulinic acid (ALA) plays an important role in the regulation of plant flavonoid metabolism. To date, the underlying mechanism of ALA promoting flavonol accumulation is unclear. Flavonol synthase (FLS) is a key enzyme in flavonol biosynthesis. In this study, we found that ALA could enhance the promoter activity of MdFLS1 in the 'Fuji' apple and improve its expression. With MdFLS1 as bait, we screened a novel transcription factor MdSCL8 by the Yeast One-Hybrid (Y1H) system from the apple cDNA library which we previously constructed. Using luciferase reporter assay and transient GUS activity assay, we verified that MdSCL8 inhibits the activity of MdFLS1 promoter and hinders MdFLS1 expression, thus reducing flavonol accumulation in apple. ALA significantly inhibited MdSCL8 expression. Therefore, ALA promoted the expression of MdFLS1 and the consequent flavonol accumulation probably by down-regulating MdSCL8. We also found that ALA significantly enhanced the gene expression of MdMYB22 and MdHY5, two positive regulators of MdFLS. We further demonstrated that MdMYB22 interacts with MdHY5, but neither of them interacts with MdSCL8. Taken together, our data suggest MdSCL8 as a novel regulator of MdFLS1 and provide important insights into mechanisms of ALA-induced flavonol accumulation in apples.