Sweet osmanthus (Osmanthus fragrans) is a very popular ornamental tree species throughout Southeast Asia and USA particularly for its extremely fragrant aroma. We constructed a chromosome-level reference genome of O. fragrans to assist in studies of the evolution, genetic diversity, and molecular mechanism of aroma development. A total of over 118 Gb of polished reads was produced from HiSeq (45.1 Gb) and PacBio Sequel (73.35 Gb), giving 100× depth coverage for long reads. The combination of Illumina-short reads, PacBio-long reads, and Hi-C data produced the final chromosome quality genome of O. fragrans with a genome size of 727 Mb and a heterozygosity of 1.45 %. The genome was annotated using de novo and homology comparison and further refined with transcriptome data. The genome of O. fragrans was predicted to have 45,542 genes, of which 95.68 % were functionally annotated. Genome annotation found 49.35 % as the repetitive sequences, with long terminal repeats (LTR) being the richest (28.94 %). Genome evolution analysis indicated the evidence of whole-genome duplication 15 million years ago, which contributed to the current content of 45,242 genes. Metabolic analysis revealed that linalool, a monoterpene is the main aroma compound. Based on the genome and transcriptome, we further demonstrated the direct connection between terpene synthases (TPSs) and the rich aromatic molecules in O. fragrans. We identified three new flower-specific TPS genes, of which the expression coincided with the production of linalool. Our results suggest that the high number of TPS genes and the flower tissue- and stage-specific TPS genes expressions might drive the strong unique aroma production of O. fragrans.

Lycoris longituba, belonging to the Amaryllidaceae family, is a perennial bulb bearing flowers with diverse colors and fragrance. Selection of cultivars with excellent colored and scented flowers has always been the breeding aim for ornamental plants. However, the molecular mechanisms underlying color fading and aroma production during flower expansion in L. longituba remain unclear. Therefore, to systematically investigate these important biological phenomena, the tepals of L. longituba from different developmental stages were used to screen and analyze the metabolic components and relevant genes. Utilizing the Illumina platform, a total of 144,922 unigenes were obtained from the RNA-Seq libraries. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the phenylpropanoid biosynthesis and flavonoid biosynthesis pathways might play important roles during color and aroma changes. Metabolomic analysis identified 29 volatile organic components (VOCs) from different developmental stages of L. longituba tepals, and orthogonal partial least-squares discriminate analysis (OPLS-DA) revealed that trans-β-ocimene—a terpene—was the most important aroma compound. Meanwhile, we found the content of anthocyanin was significantly reduced during the tepal color fading process. Then, we identified two dihydroflavonol-4-reductase (DFR) and three terpene synthase (TPS) genes, for which expression changes coincided with the production patterns of anthocyanins and trans-β-ocimene, respectively. Furthermore, a number of MYB and bHLH transcription factors (TFs) which might be involved in color- and aroma-formation were also identified in L. longituba tepal transcriptomes. Taken together, this is the first comprehensive report of the color and fragrance in tepals of L. longituba and these results could be helpful in understanding these characteristics and their regulation networks.

The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway is responsible for the biosynthesis of many crucial secondary metabolites, such as carotenoids, monoterpenes, plastoquinone, and tocopherols. In this study, we isolated and identified 10 MEP pathway genes in the important aromatic plant sweet osmanthus (Osmanthus fragrans). Multiple sequence alignments revealed that 10 MEP pathway genes shared high identities with other reported proteins. The genes showed distinctive expression profiles in various tissues, or at different flower stages and diel time points. The qRT-PCR results demonstrated that these genes were highly expressed in inflorescences, which suggested a tissue-specific transcript pattern. Our results also showed that OfDXS1, OfDXS2, and OfHDR1 had a clear diurnal oscillation pattern. The isolation and expression analysis provides a strong foundation for further research on the MEP pathway involved in gene function and molecular evolution, and improves our understanding of the molecular mechanism underlying this pathway in plants.

An anther-specific GRP gene, regulated by PhMYC2 , causes a significant reduction of male fertility when overexpressed in petunia, and its promoter is efficient in genetic engineering of male-sterile lines. Glycine-rich proteins (GRPs) play important roles in plant anther development; however, the underlying mechanisms and related regulatory networks are poorly understood. In this study, a novel glycine-rich family gene designated as PhGRP was isolated from Petunia hybrida 'Fantasy Red'. The qRT-PCR analysis showed that it expressed specifically in anthers, and its expression peaked earlier than those well-known tapetum-specific genes, such as TA29, and several genes with the classic cis-regulatory element 'anther-box' in petunia during its anther development. The male fertility was significantly reduced in PhGRP overexpression lines, due to the abnormal formation of pollen wall. The PhGRP promoter (pPhGRP) could drive the GUS genes expressing specifically in the anthers of the transgenic Arabidopsis plants, indicating that the anther-specific characteristic of this promoter was conserved. In addition, when pPhGRP was used to drive the expression of BARNASE, complete male-sterile petunia lines were created without changes in vegetative organs and floral parts other than anthers. Finally, when pPhGRP was used as the bait to screen a yeast-one-hybrid (Y1H) library, a transcription factor (PhMYC2) belonging to the bHLH family was successfully selected, and the binding between pPhGRP and PhMYC2 was validated both by Y1H and dual-luciferase reporter assay. Overall, these results suggest that PhGRP, which is a male fertility-related gene that expresses specifically in anthers, is regulated by PhMYC2 and whose promoter can be used as an effective tool in the creation of male-sterile lines.

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.

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, or “RiXiangGui”, is an ornamental, woody, evergreen plant that is cultivated widely because it blooms recurrently and emits a strong fragrance. Recently, the germplasm resources, classification, and aroma compositions of O. fragrans have been investigated. However, the molecular mechanisms of the floral scent formation and regulation have remained largely unknown. To obtain a global perspective on the molecular mechanism of the aroma formation during blooming, nine RNA Sequencing (RNA-Seq) libraries were constructed from three flowering stages: The initial, full, and final flowering stage. In short, a total of 523,961,310 high-quality clean reads were assembled into 136,611unigenes, with an average sequence length of 792 bp. About 47.43% of the unigenes (64,795) could be annotated in the NCBI non-redundant protein database. A number of candidate genes were identified in the terpenoid metabolic pathways and 1327 transcription factors (TFs), which showed differential expression patterns among the floral scent formation stages, were also identified, especially OfMYB1, OfMYB6, OfWRKY1, and OfWRKY3, which could play critical roles in the floral scent formation. These results indicated that the floral scent formation of O. fragrans was a very complex process which involved a large number of TFs. This study provides reliable resources for further studies of the O.fragrans floral scent formation.

Lycoris, which is known as the ‘Chinese tulip,’ has diverse flower colors and shapes, and some species have a delicate fragrance. However, limited studies have reported the volatile organic compounds (VOCs) of Lycoris. In this study, headspace solid-phase microextraction combined with gas chromatography-mass spectrometry was used to analyze the floral VOCs of six typical Lycoris taxa. Thirty-two VOCs were identified, including terpenoids, alcohols, esters, aldehydes, ketones, and phenols. The aldehyde and terpenoid contents in Lycoris aurea were higher than in the other taxa, and the ester and alcohol contents in L. sprengeri were the highest compared to all taxa tested. Compared with other species and cultivars, L. longituba and L. longituba var. flava were the two most scented taxa and the VOCs were dominated by terpenoids and esters. L. radiate and L. chinensis were two unscented taxa and, accordingly, the VOC content was weak. A partial least squares discriminate analysis of the floral VOCs among the six Lycoris taxa showed that the six taxa could be successfully separated. Moreover, the VOCs of L. longituba and L. longituba var. flava clustered together. β-Ocimene was verified as the most important aroma compound, as determined via the calculation of the variable importance in projection values and significance analysis. β-Ocimene and its trans isomer, trans-β-ocimene, had a high relative content in L. longituba, L. longituba var. flava, L. aurea, and L. chinensis but were not detected in L. sprengeri and L. radiata. These results indicate that floral VOCs might be selected during the evolutional processes of Lycoris, and β-ocimene could be the most typical VOC among the different Lycoris taxa.