Sweet osmanthus (Osmanthus fragrans Lour.) is among the top ten most well-known flowers in China and is recognized as both an aromatic plant and ornamental flower. Here, manual sectioning, scanning electron microscopy, and transmission electron microscopy of sweet osmanthus petals revealed that large amounts of lipids are present inside the petal cells and on the cell surfaces. However, no secretory structures were observed. Instead, the petal cells protrude slightly outward, and the surfaces of the cells are adorned with highly regular brush-shaped hairs. The surfaces of the ‘Yingui’ petals possessed mostly curled and more numerous hairs, whereas the ‘Dangui’ petals possessed fewer brush-shaped and more sparsely arranged hairs. In addition, many granular substances were attached to the brush-shaped hairs, and the granules were denser on the hairs of the ‘Yingui’ petals compared to the hairs on the ‘Dangui’ petals. Furthermore, 35 aromatic components in the ‘Yingui’ petals and 30 aromatic components in the ‘Dangui’ petals were detected via GC-MS. The main aromatic component of the ‘Yingui’ petals was β-ionone, whereas that of the ‘Dangui’ petals was linalool and its oxides. Transcriptome sequencing and qRT-PCR indicated that the high β-ionone content in the ‘Yingui’ petals was due to the overexpression of CCD1 and CCD4 and that the high linalool content in the ‘Dangui’ petals was due to the overexpression of MECS, HDR, IDI1, and LIS1, which function upstream of the linalool synthetic pathway. In particular, the expression levels of CCD4 and LIS1 were upregulated by 5.5- and 5.1-fold in the ‘Yingui’ and ‘Dangui’ petals, respectively. One transcription factor (ERF61) was cloned and named, and the expression pattern of ERF61 in sweet osmanthus petals was found to be generally consistent with that of CCD4. Tobacco transformation experiments, yeast one-hybrid experiments, and electrophoretic mobility shift assays indicated that ERF61 binds to the CCD4 promoter and stimulates CCD4 expression, thereby regulating the synthesis of β-ionone in sweet osmanthus petals.

Linalool and ionone are two important aromatic components in sweet osmanthus petals, and the regulatory mechanisms that produce these two components remain unclear. In this study, we employed whole-genome methylation sequencing and ATAC-seq technology to analyze the genomic DNA methylation status and chromatin accessibility of the sweet osmanthus cultivars ‘Zaohuang’ and ‘Chenghong Dangui’. Results showed that the promoter region of TPS2, a key gene in the linalool synthesis pathway, was less methylated in ‘Chenghong Dangui’ than in ‘Zaohuang’. The chromatin was more accessible in ‘Chenghong Dangui’ than in ‘Zaohuang’, which resulted in a much stronger expression of this gene in ‘Chenghong Dangui’ than in ‘Zaohuang’. This eventually led to a high quantity of linalool and its oxides in the petals of ‘Chenghong Dangui’, but there were lower levels present in the petals of ‘Zaohuang’. These results suggest that DNA methylation and chromatin accessibility play major roles in linalool synthesis in sweet osmanthus. The methylation level of the promoter region of CCD4, a key gene for ionone synthesis, was higher in ‘Zaohuang’ than in ‘Chenghong Dangui’. The chromatin accessibility was lower in ‘Zaohuang’ than in ‘Chenghong Dangui’, although the expression of this gene was significantly higher in ‘Zaohuang’ than in ‘Chenghong Dangui’. ChIP-seq analysis and a series of experiments showed that the differential expression of CCD4 and CCD1 in the two cultivars may predominantly be the result of regulation by ERF2 and other transcription factors. However, a 183-bp deletion involving the CCD4 promoter region in ‘Chenghong Dangui’ may be the main reason for the low expression of this gene in its petals. This study provides an important theoretical basis for improving selective breeding of key floral fragrance components in sweet osmanthus.

Osmanthus fragrans Lour., one of the top 10 most popular flowers in China, is known for both its beauty and fragrance. It is rich in flavonoids, a class of secondary metabolites with significant neuroprotective, free-radical scavenging, and an...