Among naturally occurring pigments, carotenoids are importantly involved in the photosynthesis of plants and responsible for the coloration of petals and fruits. Osmanthus fragrans Lour., a famous ornamental plant, has many cultivars with different flower color. Petal coloration in O. fragrans mainly depends on the kinds of carotenoids and their contents. To investigate the mechanism of flower coloration in different cultivars, an analysis of phenotypic classification, phytochemistry, as well as the expression of carotenoid metabolism genes based on different groups was performed in the present study. Two main clusters including the orange-red cluster containing Aurantiacus cultivars and the yellowish-white cluster containing the other three cultivar groups were classified using the CIEL∗a∗b∗ system. No significant differences in flavonoid contents were observed between these two clusters. However, carotenoids, especially α-carotene and β-carotene, were found to have crucial roles in the diversity of floral coloration among the different cultivars. Carotenoid compositions in the petals of cultivars from both clusters consisted of α-carotene, β-carotene, α-cryptoxanthin, β-cryptoxanthin, lutein, and zeaxanthin, but carotenoid accumulation patterns during the flowering process were different. The petals of the yellowish-white cultivars exhibited high contents of β-carotene, lutein and α-carotene, whereas the petals of the orange-red cultivars mainly contained β-carotene and α-carotene. The profound diversity in the total carotenoid concentrations in the two clusters was determined by the transcript levels of OfCCD4. Furthermore, the accumulation of upstream products with orange color in orange-red cultivars was partially due to the low expression of OfCHYB, whereas the relatively higher OfCHYB expression in the petals of the yellowish-white cultivars led to higher proportions of lutein, which is yellow. We also found that downregulation of OfLCYE, which encodes 𝜀-ring cyclase, indicated that the carotenoid flux of most cultivars mainly resulted in more β, β-branched products. Additionally, carotenoid biosynthesis in green tissues and petals was compared, revealing the tissue specificity of carotenoid accumulation in O. fragrans. Therefore, the effects of multiple genes on carotenoid accumulation give rise to the colorful O. fragrans.

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Quantitative real-time PCR (RT-qPCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. Several studies examining the selection of reference genes have been performed in ornamental plants but none in sweet osmanthus (Osmanthus fragrans Lour.). Based on transcriptomic sequencing data from O. fragrans buds at four developmental stages, six reference genes (OfACT, OfEF1α, OfIDH, OfRAN1, OfTUB, and OfUBC2) with stable expression (0.5 to 2 fold change in expression levels between any two developmental stages), as well as the commonly used reference gene Of18S, were selected as candidates for gene expression normalization in the RT-qPCR analysis of O. fragrans. For the normalization of RT-qPCR with two dyes, SYBR Green and EvaGreen, the expressional stability of seven candidate reference genes in 43 O. fragrans samples was analyzed using geNorm, NormFinder and BestKeeper. For RT-qPCR using SYBR Green, OfRAN1 and OfUBC2 were the optimal reference genes for all samples and different cultivars, OfACT and OfEF1α were suitable for different floral developmental stages, and OfACT was the optimal reference gene for different temperature treatments. The geometric mean values of the optimal reference gene pairs for the normalization of RT-qPCR are recommended to be used for all samples, different cultivars and different floral developmental stages in O. fragrans. For RT-qPCR using EvaGreen, OfUBC2 was the optimal reference gene for all samples and different cultivars, and OfACT was the optimal reference gene for different floral developmental stages and different temperature treatments. As the worst reference gene, Of18S should not be used as a reference gene in O. fragrans in the future. Our results provide a reference gene application guideline for O. fragrans gene expression characterization using RT-qPCR.

Floral scent is an important part of volatile organic compounds (VOCs) emitted from plants, and is influenced by many environmental and endogenous factors. To investigate the influence of temperature on the emission of the floral scent of Osmanthus fragrans, the number of chemical compounds and their relative release amounts from four cultivars of O. fragrans under different temperature treatments, were identified using the solid-phase microextraction (SPME) technique and gas chromatography-mass spectrometry (GC-MS) in this study. Results revealed that the numbers and release amounts of floral scent components were significantly influenced by different temperatures, and depend on different cultivars and different types of compounds. Overall, most cultivars had the largest number of chemical compounds in 19 °C and the numbers of chemical compounds decreased with the increase or decrease in the temperature. Alcohols and ketones were the two main kinds of compounds responding to temperature change. The response of a specific chemical compound to temperature change was different in four cultivars. Generally, linalool, α-ionone, β-ionone, and γ-decalactone accounted for the highest proportion in the nine main compounds, and changes of these four chemical compounds to different temperatures had obvious contributions to the floral scent of O. fragrans. The results obtained provide evidence that temperatures can greatly influence the emission of floral scent.

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Several unigenes encoding ACS and ERF involved in ethylene biosynthesis and signal transduction were greatly down-regulated in the petal transcriptome of cut tree peony 'Luoyang Hong' with glucose treatment. Glucose also repressed stress-related transcription factor genes DREB, CBF, NAC, WRKY and bHLH. Tree peony (Paeonia suffruticosa Andrews) is a famous traditional flower in China. Glucose supply prolonging vase life of cut tree peony flowers is associated with its role in the suppression of sensitivity to ethylene and ethylene production, but the regulation mechanism of sugar on ethylene biosynthesis and signaling is unclear. In the present work, a normalized cDNA pool was constructed as the reference transcriptome from mixed petals of different developmental cut tree peony 'Luoyang Hong' and sequenced using the Illumina HiSeq™ 2000 platform. We obtained 33,117 unigenes annotated with public protein databases. In addition, the transcriptome change in petals of cut tree peony with glucose supply and the control treatment was investigated. With non-redundant annotation, 173 differentially expressed genes were identified, with 41 up-regulated genes and 132 down-regulated genes. According to RNA-Seq data and real-time quantitative polymerase chain reaction validation, one unigene encoding ACS, a key ethylene synthetic enzyme, and four unigenes encoding ERF, which is involved in ethylene signal transduction was greatly down-regulated with glucose treatment. Furthermore, stress-related transcription factor genes DREB, CBF, NAC, WRKY and bHLH were also repressed with glucose supply, as well as several other stress-responsive and stress-tolerance genes, indicating that glucose supply probably releases the effects induced by various environmental stress. All the results and analysis are valuable resources for better understanding of the beneficial influence of exogenous sugars on cut tree peony.