The mechanism through which Hydrangea macrophylla are able to change color has been the focus of investigation for many studies. However, the molecular mechanism involved in the complexation of aluminum ions and anthocyanins to regulate the color change remains unclear. Here, the color-changing mechanism was investigated in Hydrangea macrophylla plants under aluminum stress using proteome and transcriptome sequencing methods. Catalase, peroxidase, superoxide dismutase, 3-O-delphinidin and Al³⁺ contents in sepal were significantly upregulated upon Al³⁺ treatment. Moreover, 1628 differentially expressed genes and 448 differentially expressed proteins were identified between the treated and untreated conditions. GO and KEGG enrichment analyses revealed that the differentially expressed genes and differentially expressed proteins were enriched in categories related to the cell wall, peroxidase activity, and peroxisome pathways. Importantly, eight genes involved in anthocyanin biosynthesis were significantly downregulated at the transcript and protein levels under aluminum stress. These results suggest that aluminum ions induce expression changes of related key genes, which regulate the hydrangea’s flower color. Overall, this study provides a valuable reference for the molecular mechanism relating to the color change and adaptation of Hydrangea macrophylla in response to aluminum stress.

Hydrangea macrophylla is an ornamental plant with varied calyx colors. Interestingly, from red, to purple, to blue, the colors of all Hydrangea macrophylla are formed by unique delphinidin-3-O-glucoside and aluminum ions (Al³⁺) and 5-O-p-coumaroylquinic acid. The sepals of ‘Blue Mama’ changed from pink to blue, and the contents of delphinidin-3-O-glucoside and aluminum ions increased under 3 g/L aluminum sulfate treatment. However, the mechanism of the effect of aluminum ions on the synthesis and metabolism of anthocyanins in Hydrangea macrophylla is still unclear. In this project, transcriptome sequencing and anthocyanin metabolome analysis were performed on the sepals of ‘Blue Mama’ during flower development at the bud stage (S1), discoloration stage (S2) and full-bloom stage (S3) under aluminum treatment. It was found that delphinidin, delphinidin-3-O-glucoside and delphinidin-3-O-galactoside were the main differential metabolites. The structural genes CHS, F3H, ANS, DFR and BZI in the anthocyanin synthesis pathway were up-regulated with the deepening in sepal color. There was no significant difference between the aluminum treatment and the non-aluminum treatment groups. However, seven transcription factors were up-regulated and expressed to regulate anthocyanin synthesis genes CHS, F3H, BZI and 4CL, promoting the sepals to turn blue. The KEGG enrichment pathway analysis of differentially expressed genes showed that the glutathione metabolism and the ABC transporter pathway were closely related to anthocyanin synthesis and aluminum-ion transport. GST (Hma1.2p1_0158F.1_g069560.gene) may be involved in the vacuolar transport of anthocyanins. The expression of anthocyanin transporter genes ABCC1 (Hma1.2p1_0021F.1_g014400.gene), ABCC2 (Hma1.2p1_0491F.1_g164450.gene) and aluminum transporter gene ALS3 (Hma1.2p1_0111F.1_g053440.gene) were significantly up-regulated in the aluminum treatment group, which may be an important reason for promoting the transport of anthocyanin and aluminum ions to vacuoles and making the sepals blue. These results preliminarily clarified the mechanism of aluminum ion in the synthesis and transport of anthocyanin in Hydrangea macrophylla, laying a foundation for the further study of the formation mechanism of ‘blue complex’ in Hydrangea macrophylla.