Hydrangea spp. is renowned for its variety of color changes in its developmental stage and before and after aluminum treatment. We analyzed gene expression in hydrangeas sepals to study the causes of color change. The accuracy of quantitative RT-qPCR analysis depends on the reliability of reference genes. We selected reference genes for hydrangea of varying cultivars, at different developmental stages, and in aluminum treatment groups. We chose ‘Bailmer’ and ‘Duro’ as subject species. We selected eight candidate genes, all of which were ranked by geNorm, NormFinder, BestKeeper, and RefFinder. CCR, NHX1, and LODX were used to verify the exactitude of reference genes. According to the ranking result of RefFinder, the top-ranked reference genes in each group were different; the top four candidate reference genes in each group mostly included EF1-β, RPL34, GADPH, and RPL10. EF1-β and RPL34 ranked top in the ‘all materials’ group, and their expression trends, obtained from the analysis of CCR, NHX1, and LODX, were consistent. From the results, we gather that EF1-β and RPL34 can be used as reference genes to quantify target gene expression. In this study, we screened for reference genes in hydrangeas to provide a technical basis for hydrangea sepal formation and transformation for further experiments.

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.