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Carotenoids are the precursors of important fragrance compounds in flowers of Osmanthus fragrans Lour. var. aurantiacus, which exhibit the highest diversity of carotenoid-derived volatiles among the flowering plants investigated. A cDNA encoding a carotenoid cleavage enzyme, OfCCD1, was identified from transcripts isolated from flowers of O. fragrans Lour. It is shown that the recombinant enzymes cleave carotenes to produce α-ionone and β-ionone in in vitro assays. It was also found that carotenoid content, volatile emissions, and OfCCD1 transcript levels are subjected to photorhythmic changes and principally increased during daylight hours. At the times when OfCCD1 transcript levels reached their maxima, the carotenoid content remained low or slightly decreased. The emission of ionones was also higher during the day; however, emissions decreased at a lower rate than the transcript levels. Moreover, carotenoid content increased from the first to the second day, whereas the volatile release decreased, and the OfCCD1 transcript levels displayed steady-state oscillations, suggesting that the substrate availability in the cellular compartments is changing or other regulatory factors are involved in volatile norisoprenoid formation. Furthermore, the sensory evaluation of the aroma of the model mixtures suggests that the proportionally higher contribution of α-ionone and β-ionone to total volatile emissions in the evening is probably the reason for the increased perception by humans of the scent emission of Osmanthus flowers.

Ethylene-Responsive Factor (ERF) is a key element found in the middle and lower reaches of the ethylene signal transduction pathway. It is widely distributed in plants and plays important roles in plant growth and development, hormone signal transduction, and various stress processes. Although there is research on AP/ERF family members, research on AP2/ERF in Osmanthus fragrans is lacking. Thus, in this work, AP2/ERF in O. fragrans was extensively and comprehensively analyzed. A total of 298 genes encoding OfAP2/ERF proteins with complete AP2/ERF domains were identified. Based on the number of AP2/ERF domains and the similarity among amino acid sequences between AP2/ERF proteins from A. thaliana and O. fragrans, the 298 putative OfAP2/ERF proteins were divided into four different families, including AP2 (45), ERF (247), RAV (5), and SOLOIST (1). In addition, the exon–intron structure characteristics of these putative OfAP2/ERF genes and the conserved protein motifs of their encoded OfAP2/ERF proteins were analyzed, and the results were found to be consistent with those of the population classification. A tissue-specific analysis showed the spatiotemporal expression of OfAP2/ERF in the stems and leaves of O. fragrans at different developmental stages. Specifically, 21 genes were not expressed in any tissue, while high levels of expression were found for 25 OfAP2/ERF genes in several tissues, 60 genes in the roots, 34 genes in the stems, 37 genes in young leaves, 34 genes in old leaves, 32 genes in the early flowering stage, 18 genes in the full flowering stage, and 37 genes in the late flowering stage. Quantitative RT-PCR experiments showed that OfERF110a and OfERF110b had the highest expression levels at the full-bloom stage (S4), and this gradually decreased with the senescence of petals. The expression of OfERF119c decreased first and then increased, while the expression levels of OfERF4c and OfERF5a increased constantly. This indicated that these genes may play roles in flower senescence and the ethylene response. In the subsequent subcellular localization experiments, we found that ERF1-4 was localized in the nucleus, indicating that it was expressed in the nucleus. In yeast self-activation experiments, we found that OfERF112, OfERF228, and OfERF23 had self-activation activity. Overall, these results suggest that OfERFs may have the function of regulating petal senescence in O. fragrans.

Background Flower size significantly influences the visitation of pollinators in angiosperms, and is also a crucial trait that affects commercial value of ornament plants. Sweet osmanthus ( Osmanthus fragrans ) is an ornamental species characterized by its small and aromatic flowers. GATA transcription factors have been reported to be involved in regulating plant development and growth. However, our understanding of the regulatory functions of GATA transcription factors concerning floral traits in ornamental plants such as sweet osmanthus remains limited. Results In this study, we functionally identified a member of the GATA family,, designated as OfGATA9, in sweet osmanthus. The expression of OfGATA9 was found to be transcriptionally up-regulated during the early stages of flower opening in sweet osmanthus. Furthermore, the expression pattern of OfGATA9 correlated with the flower size across various cultivars of sweet osmanthus. The nucleus-localized OfGATA9 harbors a conserved GATA-type zinc finger domain, and belongs to subfamily I of the GATA family. Ectopic over-expression of OfGATA9 in Nicotiana tabacum resulted in an increase in both flower size and epidermis cell dimensions. Comparative transcriptome analysis between control and transgenic N. tabacum showed that over-expressing OfGATA9 led to differential expression of genes including EXLB1 , XTHs , bHLHs , EFRs , Auxin/IAAs , SAURs , MYB-like , and MADS-box genes in flowers. Conclusions This study revealed the function of OfGATA9 in regulatory networks that influence flower size by promoting cell expansion and the expression of related genes. Our findings establish a theoretical foundation for breeding large-flowered sweet osmanthus.

Background Temperature is involved in the regulation of carotenoid accumulation in many plants. The floral color of sweet osmanthus ( Osmanthus fragrans Lour.) which is mainly contributed by carotenoid content, is affected by temperature in autumn. However, the mechanism remains unknown. Here, to reveal how temperature regulates the floral color of sweet osmanthus, potted sweet osmanthus ‘Jinqiu Gui’ were treated by different temperatures (15 °C, 19 °C or 32 °C). The floral color, carotenoid content, and the expression level of carotenoid-related genes in petals of sweet osmanthus ‘Jinqiu Gui’ under different temperature treatments were investigated. Results Compared to the control (19 °C), high temperature (32 °C) changed the floral color from yellow to yellowish-white with higher lightness ( L *) value and lower redness ( a *) value, while low temperature (15 °C) turned the floral color from yellow to pale orange with decreased L * value and increased a * value. Total carotenoid content and the content of individual carotenoids (α-carotene, β-carotene, α-cryptoxanthin, β-cryptoxanthin, lutein and zeaxanthin) were inhibited by high temperature, but were enhanced by low temperature. Lower carotenoid accumulation under high temperature was probably attributed to transcriptional down-regulation of the biosynthesis gene OfPSY1 , OfZ-ISO1 and OfLCYB1 , and up-regulation of degradation genes OfNCED3 , OfCCD1-1 , OfCCD1-2 , and OfCCD4-1 . Up-regulation of OfLCYB1 , and down-regulation of OfNCED3 and OfCCD4-1 were predicted to be involved in low-temperature-regulated carotenoid accumulation. Luciferase assays showed that the promoter activity of OfLCYB1 was activated by low temperature, and repressed by high temperature. However, the promoter activity of OfCCD4-1 was repressed by low temperature, and activated by high temperature. Conclusions Our study revealed that high temperature suppressed the floral coloration by repressing the expression of carotenoid biosynthesis genes, and activating the expression of carotenoid degradation genes. However, the relative low temperature had opposite effects on floral coloration and carotenoid biosynthesis in sweet osmanthus. These results will help reveal the regulatory mechanism of temperature on carotenoid accumulation in the petals of sweet osmanthus.

Background The plant-specific YABBY transcription factor family plays important roles in plant growth and development, particularly leaf growth, floral organ formation, and secondary metabolite synthesis. Results Here, we identified a total of 13 OfYABBY genes from the Osmanthus fragrans genome. These 13 OfYABBY genes were divided into five subfamilies through phylogenetic analysis, and genes in the same subfamily showed similar gene structures and conserved protein motifs. Gene duplication promoted the expansion of the OfYABBY family in O . fragrans . Tissue-specific expression analysis showed that the OfYABBY family was mainly expressed in O . fragrans leaves and floral organs. To better understand the role of OfYABBY genes in plant growth and development, OfYABBY12 was selected for heterologous stable overexpression in tobacco, and OfYABBY12 -overexpressing tobacco leaves released significantly fewer volatile organic compounds than wild-type tobacco leaves. Overexpression of OfYABBY12 led to the downregulation of NtCCD1/4 and decreased β-ionone biosynthesis. Correspondingly, a dual-luciferase assay showed that OfYABBY12 negatively regulated the expression of OfCCD4 , which promotes β-ionone synthesis. Furthermore, tobacco leaves overexpressing OfYABBY12 were curled and wrinkled and had significantly reduced leaf thickness and leaf inclusions and significantly extended flower pistils (styles). Conclusion Overall, the results suggest that the OfYABBY gene family may influence the biosynthesis of the floral scent (especially β-ionone) in O . fragrans and may regulate leaf morphogenesis and lateral organs.

Carotenoids are important natural pigments that are responsible for the fruit and flower colors of many plants. The composition and content of carotenoid can greatly influence the color phenotype of plants. However, the regulatory mechanism underling the divergent behaviors of carotenoid accumulation, especially in flower, remains unclear. In this study, a new cultivar Osmanthus fragrans ‘Yanzhi Hong’ was used to study the regulation of carotenoid pigmentation in flower. Liquid chromatograph–mass spectrometer (LC-MS) analysis showed that β-carotene, phytoene, lycopene, γ-carotene, and lutein were the top five pigments enriched in the petals of ‘Yanzhi Hong’. Through transcriptome analysis, we found that the expression of the structural genes in carotenoid pathway was imbalanced: most of the structural genes responsible for lycopene biosynthesis were highly expressed throughout the flower developmental stages, while those for lycopene metabolism kept at a relatively lower level. The downregulation of LYCE, especially at the late developmental stages, suppressed the conversion from lycopene to α-carotene but promoted the accumulation of β-carotene, which had great effect on the carotenoid composition of ‘Yanzhi Hong’. Ethylene response factor (ERF), WRKY, basic helix-loop-helix (bHLH), v-myb avian myeloblastosis viral oncogene homolog (MYB), N-Acetylcysteine (NAC), auxin response factor (ARF), and other transcription factors (TFs) have participated in the flower color regulation of ‘Yanzhi Hong’, which formed co-expression networks with the structural genes and functioned in multiple links of the carotenoid pathway. The results suggested that the cyclization of lycopene is a key link in determining flower color. The modification of the related TFs will break the expression balance between the upstream and downstream genes and greatly influence the carotenoid profile in flowers, which can be further used for creating colorful plant germplasms.

Colored leaves, a notable horticultural trait, have high research and ornamental value. The evergreen sweet olive ( Osmanthus fragrans ), one of the top ten traditional flowers in China, has been cultivated for more than two thousand years. However, in recent years, an increasing number of O. fragrans cultivars with colored leaves have been cultivated for their ornamental value. To study the molecular mechanism underlying the observed changes in leaf color, we selected O. fragrans ‘Yinbi Shuanghui’ (Y), which has yellow-white leaves, and O. fragrans ‘Sijigui’ (S), which has green leaves, as materials. Pigment content measurement showed that the chlorophyll, carotenoid and anthocyanin contents in Y were lower than in S. According to the SWATH-MS sequencing results, a total of 3,959 proteins were quantitatively identified, 1,300 of which were differentially expressed proteins (DEPs), including 782 up-regulated and 518 down-regulated proteins in Y compared to S. Functional enrichment analysis of DEPs revealed that down-regulated expression of photosynthesis related proteins may lead to the inhibition of chlorophyll synthesis in Y, this may be the main cause of leaf color change. Moreover, a protein interaction prediction model also showed that proteins such as PetC, PsbO, PsbP, and PsbQ were key proteins in the interaction network, and the up-regulated proteins participating in the anthocyanin and carotenoid pathways may be related to the formation of yellow-white leaves. Taken together, our findings represent the first SWATH-MS-based proteomic report on colored leaf O. fragrans and reveal that chlorophyll synthesis and secondary metabolism pathways contribute to the changes in leaf color.

Osmanthus fragrans, a native to China, is renowned as a highly popular gardening plant. However, this plant faces significant challenges from drought stress, which can adversely affect its flowering. In this study, we found that the plasma membrane-localized gene OfPIP2 exhibited a substantial upregulation during the flowering stages and in response to drought stress. GUS staining has illustrated that the OfPIP2 promoter can drive GUS activity under drought conditions. The overexpression of OfPIP2 was found to enhance petal size by modulating epidermal cell dimensions in Petunia and tobacco. Moreover, this overexpression also bolstered drought tolerance, as evidenced by a reduction in stomatal aperture in both species. Furthermore, yeast one-hybrid (Y1H) and dual-luciferase (Dual-LUC) assays have indicated that the transcription factor OfMYB28 directly binds to the OfPIP2 promoter, thereby regulating its expression. Together, we speculated that a module of OfMYB28-OfPIP2 was not only involved in the enhancement of petal size but also conferred the improvement of drought tolerance in O. fragrans. These results contribute valuable insights into the molecular function of the OfPIP2 gene and lay a foundation for molecular breeding strategies in O. fragrans.

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 is widely grown for the purpose of urban greening and the pleasant aroma emitted from its flowers. The floral scent is determined by several monoterpenoid volatiles, such as linalool and its oxides, which are a few of the most common volatiles and the main components of the essential oils in most sweet osmanthus cultivars. In addition, the relative contents of cis- and trans-linalool oxide (furan) may affect the aromas and quality of the essential oils. MYB proteins represent the largest family of transcription factors in plants and participate in regulating secondary metabolites. Several cis-elements, especially AC-rich regions, are known to be bound by 2R-MYBs and could be found in the promoter of the enzyme genes in the terpenoid metabolic pathway. However, there has to date been no investigation into the 2R-MYB family genes involved in regulating terpenoid biosynthesis in O. fragrans. Here, 243 non-redundant 2R-MYB proteins were grouped into 33 clusters based on the phylogeny and exon-intron distribution. These genes were unevenly distributed on 23 chromosomes. Ka/Ks analysis showed that the major mode of 2R-MYB gene evolution was purifying selection. Expression analysis indicated that 2R-MYB genes in O. fragrans exhibited varied expression patterns. A total of 35 OfMYBs representing the highest per kilobase per million mapped reads in the flower were selected for quantitative real-time PCR analysis. The correlation analysis between the expression level and the contents of fragrant compounds at different flowering stages suggested that OfMYB19/20 exhibited remarkably positive correlation with the accumulation of cis-linalool oxides. OfMYB51/65/88/121/137/144 showed significantly negative correlations with one or more linalool oxides. Characterization of these proteins revealed that OfMYB19 and OfMYB137 were localized in the nuclei, but did not show transcriptional activation in the yeast system, which suggested that they may be bound to other transcription factors to exert regulatory functions. These findings provide useful information for further functional investigation of the 2R-MYBs and offer a foundation for clarifying the 2R-MYB transcription factors involved in the molecular mechanism of the regulation of monoterpenoid biosynthesis in Osmanthus fragrans.