title: Proteomics and SSH Analyses of ALA-Promoted Fruit Coloration and Evidence for the Involvement of a MADS-Box Gene,MdMADS1.
abstract: Skin color is a key quality attribute of fruits and how to improve fruit coloration has long been a major concern. 5-Aminolevulinic acid (ALA), a natural plant growth regulator, can significantly increase anthocyanin accumulation in fruit skin and therefore effectively improve coloration of many fruits, including apple. However, the molecular mechanism how ALA stimulates anthocyanin accumulation in fruit skin remains unknown. Here, we investigated the impact of ALA on apple skin at the protein and mRNA levels. A total of 85 differentially expressed proteins in apple skins between ALA and water treatment (control) were identified by complementary gel-based and gel-free separation techniques. Most of these differentially expressed proteins were up-regulated by ALA. Function analysis suggested that 87.06% of the ALA-responsive proteins were associated with fruit ripening. To further screen ALA-responsive regulators, we constructed a subtracted cDNA library (tester: ALA treatment; driver: control) and obtained 104 differentially expressed unigenes, of which 38 unigenes were indicators for the fruit ripening-related genes. The differentially changed proteins and transcripts did not correspond well at an individual level, but showed similar regulated direction in function at the pathway level. Among the identified fruit ripening-related genes, the expression ofMdMADS1, a developmental transcription regulator of fruit ripening, was positively correlated with expression of anthocyanin biosynthetic genes (MdCHS, MdDFR, MdLDOX, and MdUFGT) in apple skin under ALA treatment. Moreover, overexpression ofMdMADS1enhanced anthocyanin content in transformed apple calli, which was further enhanced by ALA. The anthocyanin content inMdMADS1-silenced calli was less than that in the control with ALA treatment, but higher than that without ALA treatment. These results indicated thatMdMADS1is involved in ALA-induced anthocyanin accumulation. In addition, anthocyanin-related verification in apple calli suggested that the regulation ofMdMADS1on anthocyanin biosynthesis was partially independent of fruit ripening process. Taken together, our findings provide insight into the mechanism how ALA regulates anthocyanin accumulation and add new information on transcriptase regulators of fruit coloration.
authors.affiliation: College of Horticulture, Nanjing Agricultural University Nanjing, China.
authors.forename: Xinxin
authors.initials: X
authors.lastname: Feng
authors.affiliation: College of Horticulture, Nanjing Agricultural University Nanjing, China.
authors.forename: Yuyan
authors.initials: Y
authors.lastname: An
authors.affiliation: College of Horticulture, Nanjing Agricultural University Nanjing, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zheng
authors.affiliation: College of Horticulture, Nanjing Agricultural University Nanjing, China.
authors.forename: Miao
authors.initials: M
authors.lastname: Sun
authors.affiliation: College of Horticulture, Nanjing Agricultural University Nanjing, China.
authors.forename: Liangju
authors.initials: L
authors.lastname: Wang
keywords: 5-aminolevulinic acid (ALA); MdMADS1; anthocyanin; apple; proteomics; suppression subtractive hybridization (SSH)