24-Epibrassinolide enhances 5-ALA-induced anthocyanin and flavonol accumulation in calli of 'Fuji' apple flesh

Color is a key factor for fruit commercial value. 5-Aminolevulinic acid (5-ALA), as an eco-friendly plant growth regulator, shows an attractively promotive effect on plant secondary metabolism, especially for fruit coloration. Brassinosteroids (BRs) can also improve plant flavonoid biosynthesis. No information is now available on the relationship between 5-ALA and BR. Here, we found that 1.5 mg L-1 24-epibrassinolide (24-EBL) promoted 50 mg L-1 5-ALA-induced anthocyanin accumulation, while, brassinazole (Brz) significantly inhibited the 5-ALA-induced flavonoid accumulation. HPLC analysis further showed that the inductive effects of 5-ALA on the accumulation of cyanidin-3-galactoside, quercetin-3-galactoside, quercetin and kaempferol were elevated by 24-EBL, but impaired by Brz. These results suggest that brassinolide biosynthesis might involve in 5-ALA-induced flavonoid accumulation. Gene expression analysis showed that 5-ALA and 5-ALA + 24-EBL induced the expression of regulatory genes MdMYB10, MdMYB9, MdbHLH3 and MdbHLH33. These two treatments also up-regulated the structural gene expressions of anthocyanin biosynthesis and transportation, including MdCHS, MdF3'H, MdDFR, MdANS, MdUFGT, MdGST and MdMATE, as well as flavonol biosynthetic gene MdFLS. But Brz decreased 5-ALA-induced up-regulation of these genes. In addition, 5-ALA also induced the expression of MdBRI1, MdBAK1 and MdBZR1, which are involved in brassinolide signal transduction. These results indicate that 24-EBL enhances 5-ALA-promoted expression of genes related to flavonoid biosynthesis and brassinolide signal transduction, while Brz exhibits the opposite effects. Taken together, we propose that 24-EBL is involved in 5-ALA-induced anthocyanin and flavonol accumulation in calli of apples. Our results provide new insights into 5-ALA-induced fruit coloration.