Floral Volatile Organic Compounds Change the Composition and Function of the Endophytic Fungal Community in the Flowers of Osmanthus fragrans.

Endophytic fungi in flowers influence plant health and reproduction. However, whether floral volatile organic compounds (VOCs) affect the composition and function of the endophytic fungal community remains unclear. Here, gas chromatography-mass spectrometry (GC-MS) and high-throughput sequencing were used to explore the relationship between floral VOCs and the endophytic fungal community during different flower development stages in Osmanthus fragrans 'Rixiang Gui'. The results showed that the composition of the endophytic fungal community and floral VOCs shifted along with flowering development. The highest and lowest α diversity of the endophytic fungal community occurred in the flower fading stage and full blooming stage, respectively. The dominant fungi, including Dothideomycetes (class), Pleosporales (order), and Neocladophialophora, Alternaria, and Setophoma (genera), were enriched in the flower fading stage and decreased in the full blooming stage, demonstrating the enrichment of the Pathotroph, Saprotroph, and Pathotroph-Saprotroph functions in the flower fading stage and their depletion in the full blooming stage. However, the total VOC and terpene contents were highest in the full blooming stage and lowest in the flower fading stage, which was opposite to the α diversity of the endophytic fungal community and the dominant fungi during flowering development. Linalool, dihydro-ß-ionone, and trans-linalool oxide(furan) were key factors affecting the endophytic fungal community composition. Furthermore, dihydro-ß-ionone played an extremely important role in inhibiting endophytic fungi in the full blooming stage. Based on the above results, it is believed that VOCs, especially terpenes, changed the endophytic fungal community composition in the flowers of O. fragrans 'Rixiang Gui'. These findings improve the understanding of the interaction between endophytic fungi and VOCs in flowers and provide new insight into the mechanism of flower development.

[Application of virus-induced gene silencing technology to investigate the phytochrome metabolism mechanism: a review].

Color is an important indicator for evaluating the ornamental traits of horticultural plants, and plant pigments is a key factor affecting the color phenotype of plants. Plant pigments and their metabolites play important roles in color formation of ornamental organs, regulation of plant growth and development, and response to adversity stress. It has therefore became a hot topic in the field of plant research. Virus-induced gene silencing (VIGS) is a vital genomics tool that specifically reduces host endogenous gene expression utilizing plant homology-dependent defense mechanisms. In addition, VIGS enables characterization of gene function by rapidly inducing the gene-silencing phenotypes in plants. It provides an efficient and feasible alternative for verifying gene function in plant species lacking genetic transformation systems. This paper reviews the current status of the application of VIGS technology in the biosynthesis, degradation and regulatory mechanisms of plant pigments. Moreover, this review discusses the potential and future prospects of VIGS technology in exploring the regulatory mechanisms of plant pigments, with the aim to further our understandings of the metabolic processes and regulatory mechanisms of different plant pigments as well as improving plant color traits.

DNA-Binding One Finger Transcription Factor PhDof28 Regulates Petal Size in Petunia.

Petal size is a key indicator of the ornamental value of plants, such as Petunia hybrida L., which is a popular ornamental species worldwide. Our previous study identified a flower-specific expression pattern of a DNA-binding one finger (Dof)-type transcription factor (TF) PhDof28, in the semi-flowering and full-flowering stages of petunia. In this study, subcellular localization and activation assays showed that PhDof28 was localized in the cell nucleus and could undergo in vitro self-activation. The expression levels of PhDof28 tended to be significantly up-regulated at the top parts of petals during petunia flower opening. Transgenic petunia 'W115' and tobacco plants overexpressing PhDof28 showed similar larger petal phenotypes. The cell sizes at the middle and top parts of transgenic petunia petals were significantly increased, along with higher levels of endogenous indole-3-acetic acid (IAA) hormone. Interestingly, the expression levels of two TFs, PhNAC100 and PhBPEp, which were reported as negative regulators for flower development, were dramatically increased, while the accumulation of jasmonic acid (JA), which induces PhBPEp expression, was also significantly enhanced in the transgenic petals. These results indicated that PhDof28 overexpression could increase petal size by enhancing the synthesis of endogenous IAA in petunias. Moreover, a JA-related feedback regulation mechanism was potentially activated to prevent overgrowth of petals in transgenic plants. This study will not only enhance our knowledge of the Dof TF family, but also provide crucial genetic resources for future improvements of plant ornamental traits.

Genome-Wide Analysis of ZAT Gene Family in Osmanthus fragrans and the Function Exploration of OfZAT35 in Cold Stress.

Osmanthus fragrans is a popular ornamental and odorant plant with high commercial value, but its cultivation and exploitation are limited by low temperature. The ZAT (zinc finger of Arabidopsis thaliana) genes as a subclass of the C2H2-type zinc finger proteins (C2H2-ZFP) family play essential roles in various abiotic stresses. However, their roles in cold stress response in O. fragrans remain unclear. This study identified 38 OfZATs, which could be divided into 5 subgroups based on the phylogenetic tree, with OfZATs in the same subgroup harboring similar gene structures and motif patterns. In addition, 49 segmental and 5 tandem duplication events were detected among OfZAT genes, while some OfZAT genes exhibited specific expression patterns in different tissues. Furthermore, two OfZATs were induced in salt stress and eight OfZATs responded to cold stress. Interestingly, OfZAT35 showed a continuously increasing expression trend under cold stress, while its protein showed nucleus localization with no transcriptional activation activity. Transiently transformed tobacco overexpressing OfZAT35 exhibited a significantly higher relative electrolyte leakage (REL) level and increased activities of superoxide dismutase (SOD), peroxidase (POD), and Ascorbate peroxidase (APX), while there was significantly decreased activity of catalase (CAT). Moreover, CAT, DREB3, and LEA5, which are associated with cold stress, were dramatically decreased after cold treatment in transiently transformed tobacco, suggesting that overexpression of OfZAT35 negatively regulated cold stress. This study provides a basis for exploring the roles of ZAT genes and contributes to uncovering the mechanism of ZAT-mediated cold stress response in O. fragrans.

Insights into the Cytochrome P450 Monooxygenase Superfamily in Osmanthus fragrans and the Role of OfCYP142 in Linalool Synthesis.

Osmanthus fragrans flowers have long been used as raw materials in food, tea, beverage, and perfume industries due to their attractive and strong fragrance. The P450 superfamily proteins have been reported to widely participate in the synthesis of plant floral volatile organic compounds (VOCs). To investigate the potential functions of P450 superfamily proteins in the fragrance synthesis of O. fragrans, we investigated the P450 superfamily genome wide. A total of 276 P450 genes were identified belonging to 40 families. The RNA-seq data suggested that many OfCYP genes were preferentially expressed in the flower or other organs, and some were also induced by multiple abiotic stresses. The expression patterns of seven flower-preferentially expressed OfCYPs during the five different flower aroma content stages were further explored using quantitative real-time PCR, showing that the CYP94C subfamily member OfCYP142 had the highest positive correlation with linalool synthesis gene OfTPS2. The transient expression of OfCYP142 in O. fragrans petals suggested that OfCYP142 can increase the content of linalool, an important VOC of the O. fragrans floral aroma, and a similar result was also obtained in flowers of OfCYP142 transgenic tobacco. Combined with RNA-seq data of the transiently transformed O. fragrans petals, we found that the biosynthesis pathway of secondary metabolites was significantly enriched, and many 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway genes were also upregulated. This evidence indicated that the OfCYP proteins may play critical roles in the flower development and abiotic response of O. fragrans, and that OfCYP142 can participate in linalool synthesis. This study provides valuable information about the functions of P450 genes and a valuable guide for studying further functions of OfCYPs in promoting fragrance biosynthesis of ornamental plants.

Integrated transcriptome and endogenous hormone analysis provides new insights into callus proliferation in Osmanthus fragrans.

Osmanthus fragrans is an important evergreen species with both medicinal and ornamental value in China. Given the low efficiency of callus proliferation and the difficulty of adventitious bud differentiation, tissue culture and regeneration systems have not been successfully established for this species. To understand the mechanism of callus proliferation, transcriptome sequencing and endogenous hormone content determination were performed from the initial growth stages to the early stages of senescence on O. fragrans calli. In total, 47,340 genes were identified by transcriptome sequencing, including 1798 previously unidentified genes specifically involved in callus development. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes (DEGs) was significantly enriched in plant hormone signal transduction pathways. Furthermore, our results from the orthogonal projections to latent structures discrimination analysis (OPLS-DA) of six typical hormones in five development stages of O. fragrans calli showed jasmonic acid (JA) could play important role in the initial stages of calli growth, whereas JA and auxin (IAA) were dominant in the early stages of calli senescence. Based on the weighted gene co-expression network analysis, OfSRC2, OfPP2CD5 and OfARR1, OfPYL3, OfEIL3b were selected as hub genes from the modules with the significant relevance to JA and IAA respectively. The gene regulation network and quantitative real-time PCR implied that during the initial stages of callus growth, the transcription factors (TFs) OfERF4 and OfMYC2a could down-regulate the expression of hub genes OfSRC2 and OfPP2CD5, resulting in decreased JA content and rapid callus growth; during the late stage of callus growth, the TFs OfERF4, OfMYC2a and OfTGA21c, OfHSFA1 could positively regulate the expression of hub genes OfSRC2, OfPP2CD5 and OfARR1, OfPYL3, OfEIL3b, respectively, leading to increased JA and IAA contents and inducing the senescence of O. fragrans calli. Hopefully, our results could provide new insights into the molecular mechanism of the proliferation of O. fragrans calli.

Insights into the trihelix transcription factor responses to salt and other stresses in Osmanthus fragrans.

BACKGROUND: Osmanthus fragrans is an evergreen plant with high ornamental and economic values. However, they are easily injured by salt stress, which severely limits their use in high salinity areas. The trihelix transcription factor (TF) family, as one of the earliest discovered TF families in plants, plays an essential part in responses to different abiotic stresses, and it has potential functions in improving the salt-tolerance capability of O. fragrans. RESULTS: In this study, 56 trihelix genes (OfGTs) were first identified in O. fragrans and then divided into five subfamilies in accordance with a phylogenetic tree analysis. The OfGTs were found to be located randomly on the 20 O. fragrans chromosomes, and an analysis of gene replication events indicated that the OfGT gene family underwent strong purification selection during the evolutionary process. The analysis of conserved motifs and gene structures implied that the OfGT members in the same subfamily have similar conserved motifs and gene structures. A promoter cis-elements analysis showed that all the OfGT genes contained multiple abiotic and hormonal stress-related cis-elements. The RNA-seq data suggested that the OfGTs have specific expression patterns in different tissues, and some were induced by salt stress. The qRT-PCR analysis of 12 selected OfGTs confirmed that OfGT1/3/21/33/42/45/46/52 were induced, with OfGT3/42/46 being the most highly expressed. In addition, OfGT42/OfGT46 had a co-expression pattern under salt-stress conditions. OfGT3/42/46 were mainly localized in the nuclei and exhibited no transcriptional activities based on the analysis of the subcellular localization and transcriptional activity assay. Furthermore, the expression levels of most of the selected OfGTs were induced by multiple abiotic and hormonal stresses, and the expression patterns of some OfGTs were also highly correlated with gibberellic acid and methyl jasmonate levels. Remarkably, the transient transformation results showed lower MDA content and increased expression of ROS-related genes NbAPX in transgenic plants, which implying OfGT3/42/46 may improve the salt tolerance of tobacco. CONCLUSIONS: The results implied that the OfGT genes were related to abiotic and hormonal stress responses in O. fragrans, and that the OfGT3/42/46 genes in particular might play crucial roles in responses to salt stress. This study made a comprehensive summary of the OfGT gene family, including functions and co-expression patterns in response to salt and other stresses, as well as an evolutionary perspective. Consequently, it lays a foundation for further functional characterizations of these genes.

Insights Into the MYB-Related Transcription Factors Involved in Regulating Floral Aroma Synthesis in Sweet Osmanthus.

As an important member of the MYB transcription factor (TF) family, the MYB-related TFs play multiple roles in regulating the synthesis of secondary metabolites and developmental processes, as well as in response to numerous biotic and abiotic stressors in plants. However, little is known regarding their roles in regulating the formation of floral volatile organic compounds (VOCs). In this study, we conducted a genome-wide analysis of MYB-related proteins in sweet osmanthus; 212 OfMYB-related TFs were divided into three distinct subgroups based on the phylogenetic analysis. Additionally, we found that the expansion of the OfMYB-related genes occurred primarily through segmental duplication events, and purifying selection occurred in all duplicated gene pairs. RNA-seq data revealed that the OfMYB-related genes were widely expressed in different organs of sweet osmanthus, and some showed flower organ/development stage-preferential expression patterns. Here, three OfMYB-related genes (OfMYB1R70/114/201), which were expressed nuclearly in floral organs, were found to be significantly involved in regulating the synthesis of floral VOCs. Only, OfMYB1R201 had transcriptional activity, thus implying that this gene participates in regulating the expression of VOC synthesis related genes. Remarkably, the transient expression results suggested that OfMYB1R70, OfMYB1R114, and OfMYB1R201 are involved in the regulation of VOC synthesis; OfMYB1R114 and OfMYB1R70 are involved in accelerating ß-ionone formation. In contrast, OfMYB1R201 decreases the synthesis of ß-ionone. Our results deepen our knowledge of the functions of MYB-related TFs and provide critical candidate genes for the floral aroma breeding of sweet osmanthus in the future.

Exploration of the Potential Transcriptional Regulatory Mechanisms of DNA Methyltransferases and MBD Genes in Petunia Anther Development and Multi-Stress Responses.

Cytosine-5 DNA methyltransferases (C5-MTases) and methyl-CpG-binding-domain (MBD) genes can be co-expressed. They directly control target gene expression by enhancing their DNA methylation levels in humans; however, the presence of this kind of cooperative relationship in plants has not been determined. A popular garden plant worldwide, petunia (Petunia hybrida) is also a model plant in molecular biology. In this study, 9 PhC5-MTase and 11 PhMBD proteins were identified in petunia, and they were categorized into four and six subgroups, respectively, on the basis of phylogenetic analyses. An expression correlation analysis was performed to explore the co-expression relationships between PhC5-MTases and PhMBDs using RNA-seq data, and 11 PhC5-MTase/PhMBD pairs preferentially expressed in anthers were identified as having the most significant correlations (Pearson's correlation coefficients > 0.9). Remarkably, the stability levels of the PhC5-MTase and PhMBD pairs significantly decreased in different tissues and organs compared with that in anthers, and most of the selected PhC5-MTases and PhMBDs responded to the abiotic and hormonal stresses. However, highly correlated expression relationships between most pairs were not observed under different stress conditions, indicating that anther developmental processes are preferentially influenced by the co-expression of PhC5-MTases and PhMBDs. Interestingly, the nuclear localization genes PhDRM2 and PhMBD2 still had higher correlations under GA treatment conditions, implying that they play important roles in the GA-mediated development of petunia. Collectively, our study suggests a regulatory role for DNA methylation by C5-MTase and MBD genes in petunia anther maturation processes and multi-stress responses, and it provides a framework for the functional characterization of C5-MTases and MBDs in the future.

Genome-Wide Identification, Classification, and Expression Profiling Reveals R2R3-MYB Transcription Factors Related to Monoterpenoid Biosynthesis in Osmanthus fragrans.

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.

Biochemical and Comparative Transcriptome Analyses Reveal Key Genes Involved in Major Metabolic Regulation Related to Colored Leaf Formation in Osmanthus fragrans 'Yinbi Shuanghui' during Development.

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.

Genome-wide investigation of WRKY transcription factors in sweet osmanthus and their potential regulation of aroma synthesis.

WRKY transcription factors, one of the largest transcription factor families, play important roles in regulating the synthesis of secondary metabolites. In sweet osmanthus (Osmanthus fragrans), the monoterpenes have been demonstrated as the most important volatile compounds, and the W-box, which is the cognate binding site of WRKY transcription factors, could be identified in most of the terpene-synthesis-related genes' promoters. However, the role of the WRKY family in terpene synthesis in sweet osmanthus has rarely been examined. In this study, 154 WRKY genes with conserved WRKY domain were identified and classified into three groups. The group II was further divided into five subgroups, and almost all members of IId contained a plant zinc cluster domain. Eight OfWRKYs (OfWRKY7/19/36/38/42/84/95/139) were screened from 20 OfWRKYs for their flower-specific expression patterns in different tissues. Simultaneously, the expression patterns of OfWRKYs and emission patterns of volatile compounds during the flowering process were determined and gas chromatography-mass spectrometry results showed that monoterpenes, such as linalool and ocimene, accounted for the highest proportion, contributing to the floral scent of sweet osmanthus in two cultivars. In addition, correlation analysis revealed the expression patterns of OfWRKYs (OfWRKY7/19/36/139) were each correlated with distinct monoterpenes (linalool, linalool derivatives, ocimene and ocimene derivatives). Subcellular localization analysis showed that p35S::GFP-OfWRKY7/38/95/139 were localized in the nucleus and OfWRKY139 had very strong transactivation activity. Collectively, the results indicated potential roles of OfWRKY139 and OfWRKYs with plant zinc cluster domain in regulating synthesis of aromatic compounds in sweet osmanthus, laying the foundation for use of OfWRKYs to improve the aroma of ornamental plants.

SSR marker development in Clerodendrum trichotomum using transcriptome sequencing.

Clerodendrum trichotomum, a member of the Lamiaceae (Verbenaceae) family, is an ornamental plant widely distributed in South Asia. Previous studies have focused primarily on its growth characteristics, stress resistance, and pharmacological applications; however, molecular investigations remain limited. Considering germplasm conservation and the extensive applications of this plant, it is necessary to explore transcriptome resources and SSR makers for C. trichotomum. In the present study, RNA sequencing was used to determine the transcriptome of C. trichotomum. Subsequently, unigene annotations and classifications were obtained, and SSRs were mined with MIcroSAtellite. Finally, primer pairs designed with Oligo 6.0 were selected for polymorphism validation. In total, 127,325,666 high-quality reads were obtained, and 58,345 non-redundant unigenes were generated, of which 36,900 (63.24%) were annotated. Among the annotated unigenes, 35,980 (97.51%) had significant similarity to 607 species in Nr databases. In addition, a total of 6,444 SSRs were identified in 5,530 unigenes, and 200 random primer pairs were designed for polymorphism validation. Furthermore, after primary polymorphism identification, 30 polymorphic primer pairs were selected for the further polymorphism screening, and 200 alleles were identified, 197 of which showed polymorphism. In this work, a large number of unigenes were generated, and numerous SSRs were detected. These findings should be beneficial for further investigations into germplasm conservation and various applications of C. trichotomum. These results should also provide a solid foundation for future molecular biology studies in C. trichotomum.

Exploration of Floral Volatile Organic Compounds in Six Typical Lycoris taxa by GC-MS.

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. ß-Ocimene was verified as the most important aroma compound, as determined via the calculation of the variable importance in projection values and significance analysis. ß-Ocimene and its trans isomer, trans-ß-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 ß-ocimene could be the most typical VOC among the different Lycoris taxa.

Erratum: Author Correction: The chromosome-level quality genome provides insights into the evolution of the biosynthesis genes for aroma compounds of Osmanthus fragrans.

[This corrects the article DOI: 10.1038/s41438-018-0108-0.].

Integrating Transcriptomic and GC-MS Metabolomic Analysis to Characterize Color and Aroma Formation during Tepal Development in Lycoris longituba.

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-ß-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-ß-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.

Preparation and evaluation of dummy-template molecularly imprinted polymer as a potential sorbent for solid phase extraction of imidazole fungicides from river water.

Dummy molecularly imprinted polymer (DMIP) for imidazole fungicides was prepared for the first time using alpha-(2,4-Dichlorophenyl)-1H-imidazole-1-ethanol (DCE) as the fragment template. The imprinting selectivity of DCE-DMIP was evaluated for climbazole (CBZ), clotrimazole (CMZ) and miconazole (MNZ) by liquid chromatography, imprinting factors of 10.9, 10.8 and >10.7 were achieved, respectively. Heterogeneous binding sites were found in the DCE-DMIP, the corresponding saturation capacity and dissociation constant for the high affinity binding sites were 13.05 µmol g-1 and 0.4701 mmol L-1. High efficient method based on dummy molecularly imprinted solid phase extraction (DMISPE) coupled with HPLC was established for the selective enrichment of CBZ, CMZ and MNZ in river water using DCE-DMIP as sorbent. DMISPE conditions including sample loading pH/volume, selective washing and elution solvents were carefully optimized. The developed method showed good recoveries (84.2-95.0%) and precision (RSDs 1.7-5.0%, n = 5) for samples spiked at two different concentration levels (0.5 and 2.5 µg L-1). The detection limits were ranged from 0.023 to 0.031 µg L-1. The results demonstrated good potential of this method for sample pretreatment of azole fungicides in environmental water samples.

The chromosome-level quality genome provides insights into the evolution of the biosynthesis genes for aroma compounds of Osmanthus fragrans.

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.