Floral Scent Biosynthesis Research Articles

Research advances in regulation and genetic engineering of floral scents.

Floral scents play important ecological roles because they attract pollinators and seed-dispersers. Historically, humans have used plant volatiles, including floral scents, as food additives, cosmetic products, and medicines. Floral scent formation and emissions are sometimes considerably affected by environmental and climatic conditions. Both enzymes and genes involved in floral scent biosynthesis have been consistently identified, and have provided insights into the potential of metabolic engineering of floral scents. This review summarizes recent studies on various aspects of floral scent biosynthesis and emission, including biosynthetic enzymes and genetic engineering. The findings ultimately show that the metabolic pathways of floral volatiles may be regulated by a more complex system than previously thought.

Light Regulation of LoCOP1 and Its Role in Floral Scent Biosynthesis in Lilium 'Siberia'.

Light is an important environmental signal that governs plant growth, development, and metabolism. Constitutive photomorphogenic 1 (COP1) is a light signaling component that plays a vital role in plant light responses. We isolated the COP1 gene (LoCOP1) from the petals of Lilium 'Siberia' and investigated its function. The LoCOP1 protein was found to be the most similar to Apostasia shenzhenica COP1. LoCOP1 was found to be an important factor located in the nucleus and played a negative regulatory role in floral scent production and emission using the virus-induced gene silencing (VIGS) approach. The yeast two-hybrid, β-galactosidase, and bimolecular fluorescence complementation (BiFC) assays revealed that LoCOP1 interacts with LoMYB1 and LoMYB3. Furthermore, light modified both the subcellular distribution of LoCOP1 and its interactions with LoMYB1 and MYB3 in onion cells. The findings highlighted an important regulatory mechanism in the light signaling system that governs scent emission in Lilium 'Siberia' by the ubiquitination and degradation of transcription factors via the proteasome pathway.

Integrated metabolome and transcriptome analysis provides insights on the floral scent formation in Hydrangea arborescens.

Hydrangea (Hydrangea arborescens var. "Annabelle") flowers are composed of sweet aroma sepals rather than true petals and can change color. Floral volatiles plays important roles in plants, such as attracting pollinators, defending against herbivores, and signaling. However, the biosynthesis and regulatory mechanisms underlying fragrance formation in H. arborescens during flower development remain unknown. In this study, a combination of metabolite profiling and RNA sequencing (RNA-seq) was employed to identify genes associated with floral scent biosynthesis mechanisms in "Annabelle" flowers at three developmental stages (F1, F2, and F3). The floral volatile data revealed that the "Annabelle" volatile profile includes a total of 33 volatile organic compounds (VOCs), and VOCs were abundant during the F2 stage of flower development, followed by the F1 and F3 stages, respectively. Terpenoids and benzenoids/phenylpropanoids were abundant during the F2 and F1 stages, with the latter being the most abundant, whereas fatty acid derivatives and other compounds were found in large amounts during the F3 stage. According to ultra-performance liquid chromatography-tandem mass spectrometer analysis, benzene and substituted derivatives, carboxylic acids and derivatives, and fatty acyls play a significant role in the floral metabolite profile. The transcriptome data revealed a total of 17,461 differentially expressed genes (DEGs), with 7585, 12,795, and 9044 DEGs discovered between the F2 and F1, F3 and F1, and F2 and F3 stages, respectively. Several terpenoids and benzenoids/phenylpropanoids biosynthesis-related DEGs were identified, and GRAS/bHLH/MYB/AP2/WRKY were more abundant among transcription factors. Finally, DEGs interlinked with VOCs compounds were determined using Cytoscape and k-means analysis. Our results pave the way for the discovery of new genes, critical data for future genetic studies, and a platform for the metabolic engineering of genes involved in the production of Hydrangea's signature floral fragrance.

Biochemistry and Genetics of Floral Scent - A Historical Perspective.

Floral scent plays a crucial role in the reproduction process of many plants. Humans have been fascinated by floral scents throughout history, and have transported and traded floral scent products, for which they have found multiple uses as food additives, hygiene and perfume products, and medicines. Yet the scientific study of how plants synthesize floral scent compounds began later than studies on most other major plant metabolites, and the first report of the characterization of an enzyme responsible for the synthesis of a floral scent compound - linalool in Clarkia breweri, a California annual - appeared in 1994. In the almost 30 years since, enzymes and genes involved in the synthesis of hundreds of scent compounds from multiple plant species have been described. This review recapitulates this history and describes the major findings relating to the various aspects of floral scent biosynthesis and emission, including genes and enzymes and their evolution, storage and emission of scent volatiles, and the regulation of the biochemical processes.

Molecular and metabolic insights into floral scent biosynthesis during flowering in Dendrobium chrysotoxum.

Dendrobium chrysotoxum is considered as an important ornamental dendrobium because of its strong and long-lasting floral scent. Nevertheless, few information is known about the dynamic changes and related formation mechanism of dendrobium floral scent at different flowering stages. In this study, the characteristics and biosynthetic mechanism of floral scent in D. chrysotoxum during flowering was revealed by using widely-targeted volatilomics (WTV) combined with transcriptome analysis. Over 500 kinds of volatile organic compounds (VOCs) were detected in the floral scents of D. chrysotoxum, which improved the knowledge about floral scent components of dendrobium. A total of 153 differential VOCs and 4,487 differentially expressed genes (DEGs) were identified between flowers of different flowering stages, respectively. The results for both volatilomics and transcriptomics data indicated that terpenes and related genes played an important role in the formation of floral characteristics of D. chrysotoxum. But in general, the expression of genes showed an opposite trend to the accumulation of metabolites during flowering, suggesting that the regulation of floral scent biosynthesis might have started at the budding stage in D. chrysotoxum. Additionally, a transcriptional metabolic regulatory network consisting of terpenes, terpene synthases and candidate transcription factors was established. This research is the first systematic and comprehensive exploration of floral characteristics and related mechanisms during flowering in D. chrysotoxum. It provides basis for exploration of mechanisms on the floral scents and the breeding of aromatic dendrobium.

Tissue-Specific Transcriptomes Outline Halophyte Adaptive Strategies in the Gray Mangrove (Avicennia marina)

Avicennia marina forests fulfill essential blue carbon and ecosystem services, including halting coastal erosion and supporting fisheries. Genetic studies of A. marina tissues could yield insight into halophyte adaptive strategies, empowering saline agriculture research. We compare transcriptomes from A. marina pneumatophores, stems, leaves, flowers, seeds, and transcriptomes across four widely divergent environments in the Indo-Pacific (Red Sea, Arabian Gulf, Bay of Bengal, and Red River Delta) to decipher the shared and location-, tissue-, and condition-specific functions. On average, 4.8% of transcripts per tissue were uniquely expressed in that tissue, and 12.2% were shared in all five tissues. Flowers’ transcript expression was the most distinct, with domain-centric gene ontology analysis showing high enrichment for stimulus-responsive processes, as well as genes implicated in flowering (hydroxygeraniol dehydrogenase, TPM = 3687) and floral scent biosynthesis (e.g., benzoyl_coenzyme_A, 2497.2 TPM). Pneumatophores highly expressed antioxidant genes, such as glutathione S-transferase (GST, TPM = 4759) and thioredoxin (TRX, TPM = 936.2), as well as proteins in the GO term ‘Hydroquinone:oxygen oxidoreductase activity’ (enrichment Z = 7.69, FDR-corr. p = 0.000785). Tissue-specific metabolic pathway reconstruction revealed unique processes in the five tissues; for example, seeds showed the most complete expression of lipid biosynthetic and degradation pathways. The leaf transcriptome had the lowest functional diversity among the expressed genes in any tissue, but highly expressed a catalase (TPM = 4181) and was enriched for the GO term ‘transmembrane transporter activity’ (GO:0015238; Z = 11.83; FDR-corr. p = 1.58 × 10−9), underscoring the genes for salt exporters. Metallothioneins (MTs) were the highest-expressed genes in all tissues from the cultivars of all locations; the dominant expression of these metal-binding and oxidative-stress control genes indicates they are essential for A. marina in its natural habitats. Our study yields insight into how A. marina tissue-specific gene expression supports halotolerance and other coastal adaptative strategies in this halophytic angiosperm.

Metabolite and Transcriptome Profiling Analysis Revealed That Melatonin Positively Regulates Floral Scent Production in Hedychium coronarium.

Melatonin is a pleiotropic molecule that regulates a variety of developmental processes. Floral volatiles are important features of flowers that facilitate flower–visitor interactions by attracting pollinators, structure flower–visitor communities, and play defensive roles against plant and flower antagonists. Aside from their role in plants, floral volatiles are an essential ingredient in cosmetics, perfumes, pharmaceuticals, and flavorings. Herein, integrated metabolomic and transcriptomic approaches were carried out to analyze the changes triggered by melatonin exposure during the Hedychium coronarium flower development stages. Quantitative analysis of the volatiles of H. coronarium flowers revealed that volatile organic compound emission was significantly enhanced after melatonin exposure during the half bloom (HS), full bloom (FB) and fade stage (FS). Under the melatonin treatment, the emission of volatile contents was highest during the full bloom stage of the flower. Variable importance in projection (VIP) analysis and partial least-squares discriminant analysis (PLS-DA) identified 15 volatile compounds with VIP > 1 that were prominently altered by the melatonin treatments. According to the transcriptome sequencing data of the HS, FB, and FS of the flowers, 1,372, 1,510, and 1,488 differentially expressed genes were identified between CK-HS and 100MT-HS, CK-FB and 100MT-FB, and CK-FS and 100MT-FS, respectively. Among the significant differentially expressed genes (DEGs), 76 were significantly upregulated and directly involved in the floral scent biosynthesis process. In addition, certain volatile organic compounds were substantially linked with various DEGs after combining the metabolome and transcriptome datasets. Moreover, some transcription factors, such as MYB and bHLH, were also significantly upregulated in the comparison, which might be related to the floral aroma mechanism. Our results suggested that melatonin increased floral aroma production in H. coronarium flowers by modifying the expression level of genes involved in the floral scent biosynthesis pathway. These findings serve as a foundation for future research into the molecular mechanisms underlying the dynamic changes in volatile contents induced by melatonin treatment in H. coronarium.

Integration of Transcriptome and Methylome Analyses Provides Insight Into the Pathway of Floral Scent Biosynthesis in Prunus mume.

DNA methylation is a common epigenetic modification involved in regulating many biological processes. However, the epigenetic mechanisms involved in the formation of floral scent have rarely been reported within a famous traditional ornamental plant Prunus mume emitting pleasant fragrance in China. By combining whole-genome bisulfite sequencing and RNA-seq, we determined the global change in DNA methylation and expression levels of genes involved in the biosynthesis of floral scent in four different flowering stages of P. mume. During flowering, the methylation status in the “CHH” sequence context (with H representing A, T, or C) in the promoter regions of genes showed the most significant change. Enrichment analysis showed that the differentially methylated genes (DMGs) were widely involved in eight pathways known to be related to floral scent biosynthesis. As the key biosynthesis pathway of the dominant volatile fragrance of P. mume, the phenylpropane biosynthesis pathway contained the most differentially expressed genes (DEGs) and DMGs. We detected 97 DMGs participated in the most biosynthetic steps of the phenylpropane biosynthesis pathway. Furthermore, among the previously identified genes encoding key enzymes in the biosynthesis of the floral scent of P. mume, 47 candidate genes showed an expression pattern matching the release of floral fragrances and 22 of them were differentially methylated during flowering. Some of these DMGs may or have already been proven to play an important role in biosynthesis of the key floral scent components of P. mume, such as PmCFAT1a/1c, PmBEAT36/37, PmPAL2, PmPAAS3, PmBAR8/9/10, and PmCNL1/3/5/6/14/17/20. In conclusion, our results for the first time revealed that DNA methylation is widely involved in the biosynthesis of floral scent and may play critical roles in regulating the floral scent biosynthesis of P. mume. This study provided insights into floral scent metabolism for molecular breeding.

Biosynthesis and emission of methyl hexanoate, the major constituent of floral scent of a night-blooming water lily Victoriacruziana

Among the factors that have made flowering plants the most species-rich lineage of land plants is the interaction between flower and insect pollinators, for which floral scent plays a pivotal role. Water lilies belong to the ANA (Amborellales, Nymphaeales, and Austrobaileyales) grade of basal flowering plants. In this study, Victoria cruziana was investigated as a model night-blooming water lily for floral scent biosynthesis. Four volatile compounds, including three benzenoids and one fatty acid methyl ester methyl hexanoate, were detected from the flowers of V. cruziana during their first bloom, with methyl hexanoate accounting for 45 % of total floral volatile emission. Emission rates were largely constant before significant drop starting at the end of second bloom. To understand the molecular basis of floral scent biosynthesis in V. cruziana, particularly methyl hexanoate, a transcriptome from the whole flowers at the full-bloom stage was created and analyzed. Methyl hexanoate was hypothesized to be biosynthesized by SABATH methyltransferases. From the transcriptome, three full-length SABATH genes designated VcSABATH1-3 were identified. A full-length cDNA for each of the three VcSABATH genes was expressed in Escherichia coli to produce recombinant proteins. When tested in in vitro methyltransferase enzyme assays with different fatty acids, both VcSABATH1 and VcSABATH3 exhibited highest levels of activity with hexanoic acid to produce methyl hexanoate, with the specific activity of VcSABATH1 being about 15 % of that for VcSABATH3. VcSABATH1 and VcSABATH3 showed the highest levels of expression in stamen and pistil, respectively. In phylogenetic analysis, three VcSABATH genes clustered with other water lily SABATH methyltransferase genes including the one known for making other fatty acid methyl esters, implying both a common evolutionary origin and functional divergence. Fatty acid methyl esters are not frequent constituents of floral scents of mesangiosperms, pointing to the importance for the evolution of novel fatty acid methyltransferase for making fatty acid methyl esters in the pollination biology of water lilies.

Genome-Wide Analysis Reveals the Potential Role of MYB Transcription Factors in Floral Scent Formation in Hedychium coronarium.

The MYB gene family is one of the largest groups of transcription factors (TFs) playing diverse roles in several biological processes. Hedychium coronarium (white ginger lily) is a renowned ornamental plant both in tropical and subtropical regions due to its flower shape and strong floral scent mainly composed of terpenes and benzenoids. However, there is no information available regarding the role of the MYB gene family in H. coronarium. In the current study, the MYB gene family was identified and extensively analyzed. The identified 253 HcMYB genes were unevenly mapped on 17 chromosomes at a different density. Promoter sequence analysis showed numerous phytohormones related to cis-regulatory elements. The majority of HcMYB genes contain two to three introns and motif composition analysis showed their functional conservation. Phylogenetic analysis revealed that HcMYBs could be classified into 15 distinct clades, and the segmental duplication events played an essential role in the expansion of the HcMYB gene family. Tissue-specific expression patterns of HcMYB genes displayed spatial and temporal expression. Furthermore, seven HcMYB (HcMYB7/8/75/79/145/238/248) were selected for further investigation. Through RT-qPCR, the response of candidates HcMYB genes toward jasmonic acid methyl ester (MeJA), abscisic acid (ABA), ethylene, and auxin was examined. Yeast one-hybrid (Y1H) assays revealed that candidate genes directly bind to the promoter of bottom structural volatile synthesis genes (HcTPS1, HcTPS3, HcTPS10, and HcBSMT2). Moreover, yeast two-hybrid (Y2H) assay showed that HcMYB7/8/75/145/248 interact with HcJAZ1 protein. In HcMYB7/8/79/145/248-silenced flowers, the floral volatile contents were decreased and downregulated the expression of key structural genes, suggesting that these genes might play crucial roles in floral scent formation in H. coronarium by regulating the expression of floral scent biosynthesis genes. Collectively, these findings indicate that HcMYB genes might be involved in the regulatory mechanism of terpenoids and benzenoid biosynthesis in H. coronarium.

The chromosome-level wintersweet (Chimonanthus praecox) genome provides insights into floral scent biosynthesis and flowering in winter

BackgroundWintersweet (Chimonanthus praecox), an important ornamental plant, has evolved unique fragrant aroma and winter-flowering properties, which are critical for its successful sexual reproduction. However, the molecular mechanisms underlying these traits are largely unknown in this species. In addition, wintersweet is also a typical representative species of the magnoliids, where the phylogenetic position of which relative to eudicots and monocots has not been conclusively resolved.ResultsHere, we present a chromosome-level wintersweet genome assembly with a total size of 695.36 Mb and a draft genome assembly of Calycanthus chinensis. Phylogenetic analyses of 17 representative angiosperm genomes suggest that Magnoliids and eudicots are sister to monocots. Whole-genome duplication signatures reveal two major duplication events in the evolutionary history of the wintersweet genome, with an ancient one shared by Laurales, and a more recent one shared by the Calycantaceae. Whole-genome duplication and tandem duplication events have significant impacts on copy numbers of genes related to terpene and benzenoid/phenylpropanoid (the main floral scent volatiles) biosynthesis, which may contribute to the characteristic aroma formation. An integrative analysis combining cytology with genomic and transcriptomic data reveals biological characteristics of wintersweet, such as floral transition in spring, floral organ specification, low temperature-mediated floral bud break, early blooming in winter, and strong cold tolerance.ConclusionsThese findings provide insights into the evolutionary history of wintersweet and the relationships among the Magnoliids, monocots, and eudicots; the molecular basis underlying floral scent biosynthesis; and winter flowering, and highlight the utility of multi-omics data in deciphering important ornamental traits in wintersweet.

Identification and Molecular Characterization of Geranyl Diphosphate Synthase (GPPS) Genes in Wintersweet Flower.

Geranyl diphosphate synthase (GPPS) is a plastid localized enzyme that catalyzes the biosynthesis of Geranyl diphosphate (GPP), which is a universal precursor of monoterpenes. Wintersweet (Chimonanthus praecox L.), a famous deciduous flowering shrub with a strong floral scent character, could have GPPS-like homologs that are involved in monoterpenes biosynthesis, but it remains unclear. In the present study, five full-length GPPS and geranylgeranyl diphosphate synthases (GGPPS) genes were identified in the wintersweet transcriptome database. The isolated cDNAs showed high protein sequence similarity with the other plants GPPS and GGPPS. The phylogenetic analysis further classified these cDNAs into four distinct clades, representing heterodimeric GPPS small subunits (SSU1 and SSU2), homodimeric GPPS, and GGPPS. Analysis of temporal expression revealed that all genes have the highest transcript level at the full-open flower stage. From tissue-specific expression analysis, CpGPPS.SSU1 and CpGGPPS1 were predominantly expressed in petal and flower, whereas CpGPPS.SSU2, GPPS, and GGPPS2 showed a constitutive expression. Additionally, the subcellular localization assay identified the chloroplast localization of SSUs and GGPPSs proteins, and the yeast two-hybrid assay showed that both CpGPPS.SSU1 and CpGPPS.SSU2 can interact with the GGPPS proteins. Taken together, these preliminary results suggest that the heterodimeric GPPS can regulate floral scent biosynthesis in wintersweet flower.

Composition and Biosynthesis of Scent Compounds from Sterile Flowers of an Ornamental Plant Clematis florida cv. 'Kaiser'.

Clematisflorida is a popular ornamental vine species known for diverse colors and shapes of its flowers but not for scent. Here we investigated the composition and biosynthesis of floral scent in ‘Kaiser’, a fragrant cultivar of C. florida that has sterile flowers. Volatile profiling revealed that flowers of ‘Kaiser’ emit more than 20 compounds, with monoterpenes being most abundant. Among the three floral organs, namely sepals, transformed-petals, and ovaries, ovaries had the highest rates of total volatile emission. To determine the molecular mechanism underlying floral scent biosynthesis in ‘Kaiser’, we sequenced a flower transcriptome and searched the transcriptome for terpene synthase genes (TPSs), which are key genes for terpene biosynthesis. Among the TPS genes identified, three were putative intact full-length genes and were designated CfTPS1, CfTPS2, and CfTPS3. Phylogenetic analysis placed CfTPS1, CfTPS2, and CfTPS3 to the TPS-g, TPS-b, and TPS-a subfamily, respectively. Through in vitro enzyme assays with Escherichia coli-expressed recombinant proteins, both CfTPS1 and CfTPS2 were demonstrated to catalyze the conversion of geranyl diphosphate to linalool, the most abundant constituent of C. florida floral scent. In addition, CfTPS1 and CfTPS2 produced the sesquiterpene nerolidol from (E,E)-farnesyl diphosphate. CfTPS3 showed sesquiterpene synthase activity and produced multiple products in vitro. All three CfTPS genes showed higher levels of expression in sepals than those in transformed-petals and ovaries. Our results show that despite being sterile, the flowers of ‘Kaiser’ have normal mechanisms for floral scent biosynthesis that make the flowers fragrant.

MYB1 transcription factor regulation through floral scent in Cymbidium cultivar ‘Sael Bit’

Cymbidium belongs to Orchidaceae, one of the most abundant angiosperm families. Cymbidium cultivars have a high aesthetic value and commercially valued characteristics such as a pleasant scent and a variety of flower colors. Cymbidium cultivar ‘Sael Bit’, which was developed by National Institute of Horticultural & Herbal Science (NIHHS), is highly fragrant with small green and light yellow flowers. An analysis of ‘Sael Bit’ floral scent emissions by an electronic nose showed that the petal and initial flowering stage emitted a strong scent. In an headspace-solid phase micro extraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) ‘Sael Bit’ study, we identified alkenes and benzenoids, such as tridecane, benzene, and Z-2-octenal, as major volatile organic compounds. V-myb myeloblastosis viral oncogene homolog (MYB) transcription factors (TFs) play a positive role in floral scent regulation. However, little is known about the MYB transcriptional regulation of floral scent biosynthesis. Due to the importance of the plant’s biology, we need to study the floral scent MYB TFs in this plant. In the present work, we isolated and cloned the full-length cDNA of Cymbidium cultivar ‘Sael Bit’ MYB1 from fully open flower. CsMYB1 encodes an 840-bp open reading frame and 280 amino acids by functional characterization. In q-RT-PCR expression analysis, CsMYB1 TF showed the highest expression in petals and columns compared to sepals and labella. Moreover, this MYB gene expressed various flower developmental stages and was highly expressed at the fully open flower stage. The phenylpropanoid/benzenoid and ester compounds responsible genes expressed through CsMYB1 TF regulation in q-RT-PCR analysis. According to these results, we suggest that CsMYB1 might be involved in the regulation of phenylpropanoid/benzenoid genes in floral scent profile. In future studies, we will focus on develop regulation models of floral scent in Cymbidium cultivar ‘Sael Bit’.

Morphological, Physiological and Ultrastructural Changes in Flowers Explain the Spatio-Temporal Emission of Scent Volatiles in Polianthes tuberosa L.

Tuberose or Polianthes tuberosa L. is a horticultural crop of tropical origin, widely cultivated for its pleasant and intense floral fragrance in the evening. Here an investigation was made into the physiological and cell biological aspects of floral scent biosynthesis, tissue localization and emission that have not previously been examined. Volatiles collected from floral headspace were analyzed by gas chromatography-mass spectrometry (GC-MS) for identification of individual compounds and elucidation of emission patterns. Transcript accumulation and the amount of active enzyme were measured to understand the enzymatic route of scent volatile biosynthesis. Localization of scent volatiles was investigated by histochemical and ultrastructural studies. Scent emission was found to be rhythmic and nocturnal under normal day-night influence, peaking at night. Enhanced enzyme activities and transcript accumulation were recorded just prior to maximum emission. Through scanning electron microscopy (SEM) analysis, the presence of a large number of floral stomata on the adaxial surface of the tepal was revealed which might have bearing on tissue-specific emission. Guard cells of stomata responded significantly to histochemical tests, which also indicated that epidermal tissues are mostly involved in scent emission. High metabolic activity was found in epidermal layers during anthesis as shown by transmission electron microscopy (TEM) analysis. Further, new insight into the localization of scent compounds, the plausible tissue involved in their release along with the preceding ultrastructural changes at the cellular levels is presented. Finally, ultrastructural analysis of the tepal surface has been able to fill a major gap in knowledge of stomatal involvement during scent emission.

Transcriptome Sequencing Analysis Reveals a Difference in Monoterpene Biosynthesis between Scented Lilium 'Siberia' and Unscented Lilium 'Novano'.

Lilium is a world famous fragrant bulb flower with high ornamental and economic values, and significant differences in fragrance are found among different Lilium genotypes. In order to explore the mechanism underlying the different fragrances, the floral scents of Lilium ‘Sibeia’, with a strong fragrance, and Lilium ‘Novano’, with a very faint fragrance, were collected in vivo using a dynamic headspace technique. These scents were identified using automated thermal desorption—gas chromatography/mass spectrometry (ATD-GC/MS) at different flowering stages. We used RNA-Seq technique to determine the petal transcriptome at the full-bloom stage and analyzed differentially expressed genes (DEGs) to investigate the molecular mechanism of floral scent biosynthesis. The results showed that a significantly higher amount of Lilium ‘Siberia’ floral scent was released compared with Lilium ‘Novano’. Moreover, monoterpenes played a dominant role in the floral scent of Lilium ‘Siberia’; therefore, it is believed that the different emissions of monoterpenes mainly contributed to the difference in the floral scent between the two Lilium genotypes. Transcriptome sequencing analysis indicated that ~29.24 Gb of raw data were generated and assembled into 124,233 unigenes, of which 35,749 unigenes were annotated. Through a comparison of gene expression between these two Lilium genotypes, 6,496 DEGs were identified. The genes in the terpenoid backbone biosynthesis pathway showed significantly different expression levels. The gene expressions of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), 4-hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), isopentenyl diphosphate isomerase (IDI), and geranyl diphosphate synthase (GPS/GGPS), were upregulated in Lilium ‘Siberia’ compared to Lilium ‘Novano’, and two monoterpene synthase genes, ocimene synthase gene (OCS) and myrcene synthase gene (MYS), were also expressed at higher levels in the tepals of Lilium ‘Siberia’, which was consistent with the monoterpene release amounts. We demonstrated that the high activation levels of the pathways contributed to monoterpene biosynthesis in Lilium ‘Siberia’ resulting in high accumulations and emissions of monoterpenes, which led to the difference in fragrance between these two Lilium genotypes.

Integrated metabolome and transcriptome analysis of Magnolia champaca identifies biosynthetic pathways for floral volatile organic compounds

BackgroundMagnolia champaca, commonly known as champak is a well-known tree due to its highly fragrant flowers. Champak floral scent is attributed to a complex mix of volatile organic compounds (VOCs). These aromatic flowers are widely used in flavors and fragrances industry. Despite its commercial importance, the VOC biosynthesis pathways in these flowers are largely unknown. Here, we combine metabolite and RNA sequencing (RNA-seq) analyses of fully opened champak flowers to discover the active VOC biosynthesis pathways as well as floral scent-related genes.ResultsVolatile collection by headspace method and analysis by gas chromatography-mass spectrometry (GC-MS) identified a total of 43 VOCs from fully opened champak flowers, of which 46.9% were terpenoids, 38.9% were volatile esters and 5.2% belonged to phenylpropanoids/benzenoids. Sequencing and de novo assembly of champak flower transcriptome yielded 47,688 non-redundant unigenes. Transcriptome assembly was validated using standard polymerase chain reaction (PCR) based approach for randomly selected unigenes. The detailed profiles of VOCs led to the discovery of pathways and genes involved in floral scent biosynthesis from RNA-seq data. Analysis of expression levels of many floral-scent biosynthesis-related unigenes in flowers and leaves showed that most of them were expressed higher in flowers than in leaf tissues. Moreover, our metabolite-guided transcriptomics, in vitro and in vivo enzyme assays and transgenic studies identified (R)-linalool synthase that is essential for the production of major VOCs of champak flowers, (R)-linalool and linalool oxides.ConclusionAs our study is the first report on transcriptome analysis of Magnolia champaca, this transcriptome dataset that serves as an important public information for functional genomics will not only facilitate better understanding of ecological functions of champak floral VOCs, but also provide biotechnological targets for sustainable production of champak floral scent.

Transcriptomic analysis of flower development in wintersweet (Chimonanthus praecox).

Wintersweet (Chimonanthus praecox) is familiar as a garden plant and woody ornamental flower. On account of its unique flowering time and strong fragrance, it has a high ornamental and economic value. Despite a long history of human cultivation, our understanding of wintersweet genetics and molecular biology remains scant, reflecting a lack of basic genomic and transcriptomic data. In this study, we assembled three cDNA libraries, from three successive stages in flower development, designated as the flower bud with displayed petal, open flower and senescing flower stages. Using the Illumina RNA-Seq method, we obtained 21,412,928, 26,950,404, 24,912,954 qualified Illumina reads, respectively, for the three successive stages. The pooled reads from all three libraries were then assembled into 106,995 transcripts, 51,793 of which were annotated in the NCBI non-redundant protein database. Of these annotated sequences, 32,649 and 21,893 transcripts were assigned to gene ontology categories and clusters of orthologous groups, respectively. We could map 15,587 transcripts onto 312 pathways using the Kyoto Encyclopedia of Genes and Genomes pathway database. Based on these transcriptomic data, we obtained a large number of candidate genes that were differentially expressed at the open flower and senescing flower stages. An analysis of differentially expressed genes involved in plant hormone signal transduction pathways indicated that although flower opening and senescence may be independent of the ethylene signaling pathway in wintersweet, salicylic acid may be involved in the regulation of flower senescence. We also succeeded in isolating key genes of floral scent biosynthesis and proposed a biosynthetic pathway for monoterpenes and sesquiterpenes in wintersweet flowers, based on the annotated sequences. This comprehensive transcriptomic analysis presents fundamental information on the genes and pathways which are involved in flower development in wintersweet. And our data provided a useful database for further research of wintersweet and other Calycanthaceae family plants.

Floral Scent in Wisteria: Chemical Composition, Emission Pattern, and Regulation

Volatile chemicals emitted from the flowers of chinese wisteria (Wisteria sinenesis) and japanese wisteria (W. floribunda) were collected using a dynamic headspace technique and identified using gas chromatography–mass spectrometry; 28 and 22 compounds were detected from chinese wisteria and japanese wisteria flowers, respectively. These chemicals can be classified into four major classes, including fatty acid derivatives, benzenoids/phenylpropanoids, terpenoids, and nitrogen-containing compounds. Two monoterpenes, (E)-β-ocimene and linalool, belonging to the class of terpenoids, were the most abundant compounds emitted from both species. Despite strong similarity, the floral volatile profiles of the two species displayed variations in both quality and quantity. Chinese wisteria was selected as a model for further study of volatile emission from different parts of flowers, emission dynamics, and regulation of floral scent production. Although floral volatiles were detected from all flower parts, petals emitted the most. The emission of floral volatiles displayed a diurnal pattern with the maximal emissions occurring during the daytime. This rhythmic pattern was determined to be light-dependent. Regulation of floral volatile emission by exogenous chemicals, including silver thiosulphate (an ethylene inhibitor), salicylic acid, and jasmonic acid, also was analyzed. Generally, jasmonic acid promoted the emission of floral volatiles. In contrast, neither silver thiosulphate nor salicylic acid showed a significant effect on floral volatile emission. The results presented in this article suggest that wisteria can serve as a useful system for exploring novel biochemistry of floral scent biosynthesis. They also build a foundation for the study of the biological/ecological significance of floral volatiles on the reproductive biology of wisteria species.

A putative PhODO1 homologous MYB transcription factor gene, MdMYBB, is not involved in the regulation of aroma volatile biosynthesis in apple

To get deeper insight on the molecular mechanism underlying production of volatile compounds in apple (Malus domestica Borkh.), we performed the isolation and expression analysis of one R2R3-type MYB gene named MdMYBB. The amino acid sequence and the structural features of MdMYBB highly resembled those of PhODO1, which is a key regulator for floral scent biosynthesis in petunia. The expression of MdMYBB was repressed concomitantly with the inhibition of ethylene production, which regulates the volatile synthesis in apple. However, MdMYBB expression was not detected in the flesh from nearly ripened apple fruits, although the detection of exogenous volatiles had actually occurred in the same portion. In addition, overexpression of MdMYBB did not cause any induction of the volatile compounds in the transgenic tobacco lines. Thus, the features of MdMYBB were not in accordance with the aroma volatile emission, unlike the case of PhODO1, suggesting that MdMYBB may not be involved in the regulation of the biosynthesis for apple aroma volatiles. On the basis of the specific expression patterns, we discussed possible physiological roles of MdMYBB in apple.