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Hydrangea macrophylla (H. macrophylla), a species in the genus Hydrangea in the family Hydrangeaceae, is widely valued for its ornamental qualities in both domestic and international markets. Notably, H. macrophylla is known for its ability to accumulate aluminum (Al). Moreover, aluminum ions (Al3+) participate in sepal bluing. However, the underlying mechanisms of Al accumulation in the sepals remain unclear. In this study, we utilized transcriptome data from two cultivars to identify genes associated with Al accumulation. In total, 154 differentially expressed isoforms between the CK and Tr groups in the sepals of both cultivars were screened. Through gene enrichment analysis and similarity identification in the CDS (coding sequence) region, 43 differentially expressed genes were identified, including 30 upregulated and 13 downregulated genes, in the sepals of the Al treatment group. Further analysis revealed that seven of these upregulated genes are related to Al accumulation in sepals. Among the seven, the gene HmALS3.1 was identified as a potential key player in Al transport within the sepals of H. macrophylla. This study lays the groundwork for further exploration into the mechanisms by which HmALS3.1 regulates Al accumulation in H. macrophylla.

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.