Under specific cultivation conditions, the sepal color of Hydrangea macrophylla (H. macrophylla) changes from red to blue due to the complexation of aluminum ions (Al³⁺), delphinidin 3-glucoside, and copigments. However, this phenomenon cannot occur in all cultivars despite the presence of sufficient Al³⁺ and copigments. To explore the mechanism of sepal bluing in H. macrophylla, there is an urgent need to study the molecular regulation of the anthocyanin biosynthesis pathway. However, the key structural genes, other than CHS, regulating anthocyanin biosynthesis in the sepals of H. macrophylla have not been identified. In this study, based on full-length transcriptome data from H.macrophylla ‘Bailmer’, the key structural genes regulating anthocyanin biosynthesis in the sepals of H. macrophylla were isolated and investigated. Ultimately, seven key structural genes, HmCHS1, HmCHI, HmF3H1, HmF3′H1, HmF3′5′H, HmDFR2, and HmANS3, were demonstrated to show high expression levels in colored sepals. The expression levels of these seven genes increased gradually with the development of sepals and were highest in the full-bloom stage. The trend of gene expression was consistent with the trend of anthocyanin contents. It was concluded that the seven selected genes were involved in anthocyanin biosynthesis in the sepals of H. macrophylla. The full-length sequence data have been deposited into the NCBI Sequence Read Archive (SRA) with accession number PRJNA849710. This study lays a good foundation for the further elucidation of the molecular mechanism of sepal coloration in H. macrophylla.

Hydrangea macrophylla is popular for its unique physiological characteristics and changeable colors. Previous studies have shown that the pH of the vacuoles of the sepal cells of hydrangea affects the color of the sepals. Located on the vacuolar membrane, NHX1 is an important H⁺ proton pump that drives the exchange of metal ions. This proton pump affects the physiological environment by controlling the accumulation of H⁺ in the vacuole. In hydrangea, the HmNHX1 gene has an open reading frame of 1626 bp and encodes a total of 541 amino acids. Bioinformatic analysis showed that HmNHX1, which encodes a Na⁺/H⁺ exchanger, is located on the vacuolar membrane. Tissue-specific expression analysis showed that the expression of this gene in the treatment group was higher than that in the control group. The ion flux in the vacuoles of colored hydrangea in the treatment group and the control group were measured, and the results showed that HmNHX1 was indeed a Na⁺/H⁺ exchanger. When the results of the HmNHX1 expression analysis and ion flux measurements are combined, it can be seen that HmNHX1 regulates the accumulation of H⁺ in the vacuole, ultimately affecting the color of the plant.