Osmanthus fragrans ‘Yinbi Shuanghui’ not only has a beautiful shape and fresh floral fragrance, but also rich leaf colors that change, making the tree useful for landscaping. In order to study the mechanisms of color formation in O. fragrans ‘Yinbi Shuanghui’ leaves, we analyzed the colored and green leaves at different developmental stages in terms of leaf pigment content, cell structure, and transcriptome data. We found that the chlorophyll content in the colored leaves was lower than that of green leaves throughout development. By analyzing the structure of chloroplasts, the colored leaves demonstrated more stromal lamellae and low numbers of granum thylakoid. However, there was a large number of plastoglobuli. Using transcriptome sequencing, we demonstrated that the expression of differentially expressed genes (DEGs) involved in chlorophyll degradation was upregulated, i.e., heme oxygennase-1 (HO1), pheophorbide a oxidase (PAO), and chlorophyllase-2 (CLH2), affecting the synthesis of chlorophyll in colored leaves. The stay-green gene (SGR) was upregulated in colored leaves. Genes involved in carotenoid synthesis, i.e., phytoene synthase 1 (PSY1) and 1-Deoxyxylulose-5-phosphate synthase (DXS), were downregulated in colored leaves, impeding the synthesis of carotenoids. In the later stage of leaf development, the downregulated expression of Golden2-Like (GLK) inhibited chloroplast development in colored leaves. Using weighted gene co-expression network analysis (WGCNA) to investigate the correlation between physiological indicators and DEGs, we chose the modules with the highest degree of relevance to chlorophyll degradation and carotenoid metabolism. A total of five genes (HSFA2, NFYC9, TCP20, WRKY3, and WRKY4) were identified as hub genes. These analyses provide new insights into color formation mechanisms in O. fragrans ‘Yinbi Shuanghui’ leaves at the transcriptional level.

Osmanthus fragrans ‘Yinbi Shuanghui’ is a colored leaf variety of O. fragrans. To study the mechanism of leaf color formation in O. fragrans ‘Yinbi Shuanghui’, we selected green and colored leaves with two different parts, namely yellow and green, as our research materials. We analyzed the expression changes related to leaf color in genes by performing qRT-PCR in the different leaf parts, finding that OfPAO was significantly up-regulated in the yellow part of colored leaves, and we initially determined that OfPAO was the key gene involved in the formation of colored leaves. Then, we constructed an OfPAO overexpression vector, before transforming it into tobacco through an Agrobacterium-mediated transformation to obtain transgenic plants. We found that the transgenic tobacco leaf color of OfPAO was lighter than that of the null carrier, the chlorophyll content in leaves decreased, and the expression of genes involved in the chlorophyll degradation pathway in OfPAO transgenic tobacco was up-regulated, suggesting that OfPAO regulates chlorophyll degradation, leading to changes in leaf color. According to the results of transcriptome sequencing and the genome data of O. fragrans ‘Rixianggui’, we cloned CDS and the promoter sequence of OfPAO, and the promoter regions 901-1307 of the OfPAO were sequenced through bisulfite genomic sequencing PCR (BSP), finding that the methylation level of CHH in the yellow part of colored leaves was lowest in colored and green leaves at 145 bp. The methylation of CHH in the promoter of OfPAO in O. fragrans ‘Yinbi Shuanghui’ was negatively correlated with the gene expression level, suggesting that the methylation of the promoter of OfPAO may regulate the expression of OfPAO, affecting chlorophyll degradation in the leaves.