Characterization of YABBY transcription factors in Osmanthus fragrans and functional analysis of OfYABBY12 in floral scent formation and leaf morphology.

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 ß-ionone biosynthesis. Correspondingly, a dual-luciferase assay showed that OfYABBY12 negatively regulated the expression of OfCCD4, which promotes ß-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 ß-ionone) in O. fragrans and may regulate leaf morphogenesis and lateral organs.

A model of prevention of mother-to-child transmission and health management team for improving adverse outcomes of pregnancy syphilis in Ningxia, China.

Regional variations exist in the implementation of Syphilis Mother-to-Child Transmission Prevention (PMTCT). Thus, it is crucial to assess the effectiveness of this model in the Ningxia region and explore the supplementary role of Health Management Teams (HMT). This study established the PMTCT + HMT model and examined its impact on adverse outcomes in pregnant women with syphilis infection. The majority of participants were urban residents, married, had a minimum high school education, and held public positions; 36.7% and 26.7% were from minority ethnic groups. The PMTCT + HMT model enhanced participants' knowledge, rates of voluntary counseling, and testing. The incidence of adverse pregnancy outcomes (miscarriages, preterm births, stillbirths) significantly decreased, and adverse neonatal outcomes (low birth weight, neonatal mortality, congenital syphilis) were notably reduced. Simultaneously, we identified factors associated with adverse outcomes, including non-residency, unmarried status, lower educational attainment, minority ethnicity, primary syphilis, and positive titers. Thus, HMT may be an effective intervention to enhance the effect of PMTCT for syphilis. The unique population structure in Ningxia is closely linked to adverse outcomes, highlighting the significance of providing equitable treatment for vulnerable populations.

Methylation Modification in Ornamental Plants: Impact on Floral Aroma and Color.

Methylation represents a crucial class of modification that orchestrates a spectrum of regulatory roles in plants, impacting ornamental characteristics, growth, development, and responses to abiotic stress. The establishment and maintenance of methylation involve the coordinated actions of multiple regulatory factors. Methyltransferases play a pivotal role by specifically recognizing and methylating targeted sites, which induces alterations in chromatin structure and gene expression, subsequently influencing the release of volatile aromatic substances and the accumulation of pigments in plant petals. In this paper, we review the regulatory mechanisms of methylation modification reactions and their effects on the changes in aromatic substances and pigments in plant petals. We also explore the potential of methylation modifications to unravel the regulatory mechanisms underlying aroma and color in plant petals. This aims to further elucidate the synthesis, metabolism, and regulatory mechanisms of various methylation modifications related to the aroma and color substances in plant petals, thereby providing a theoretical reference for improving the aroma and color of plant petals.

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.

Analysis of the Aging-Related AP2/ERF Transcription Factor Gene Family in Osmanthus fragrans.

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.

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 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.

Transcriptomic Analysis of the Candidate Genes Related to Aroma Formation in Osmanthus fragrans.

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.

Food additive salicylates inhibit human and rat placental 3ß-hydroxysteroid dehydrogenase: 3D-QSAR and in silico analysis.

The use of salicylates as flavoring agents in food and beverages is common, but their potential to disrupt the endocrine system remains unclear. Human placental 3ß-hydroxysteroid dehydrogenase 1 (h3ß-HSD1) plays a role in progesterone synthesis and is the potential target. This study evaluated the inhibition of 13 salicylates on h3ß-HSD1, structure-activity relationship (SAR) and compared with rat placental homolog r3ß-HSD4. Salicylates inhibited h3ß-HSD1, depending on carbon chain number in the alcohol moiety and the IC50 values for hexyl, ethylhexyl, homomenthyl, and menthyl salicylates were 53.27, 15.78, 2.35, and 2.31 µM, as mixed inhibitors, respectively, while methyl to benzyl salicylates were ineffective at 100 µM. Interestingly, only hexyl salicylate inhibited r3ß-HSD4 with IC50 of 31.05 µM. Bivariate analysis revealed a negative correlation between IC50 and hydrophobicity (LogP), molecular weight, heavy atoms, and carbon number in the alcohol moiety against h3ß-HSD1. Docking analysis demonstrated that these salicylates bind to cofactor binding sites or between the steroid and cofactor binding sites. Additionally, 3D-QSAR showed distinct binding via hydrogen bond donors and hydrophobic regions. In conclusion, the inhibition of h3ß-HSD1 by salicylates appears to be dependent on factors such as LogP, molecular weight, heavy atoms, and carbon-chain length and there is species-dependent inhibition sensitivity.

Insights into the bZIP gene family in Osmanthus fragrans and the role of OfbZIP98 in the callus and flower.

Osmanthus fragrans has important ornamental and economic value. As the callus re-differentiation process is difficult, a genetic transformation system has not been successfully established in O. fragrans. The basic leucine zipper (bZIP) transcription factors play an important role in plant growth and development. A total of 116 OfbZIP genes were identified in O. fragrans using bioinformatics methods. According to the evolutionary relationships, these genes were divided into 12 subfamilies and were found to be unevenly distributed on 23 chromosomes of O. fragrans. The quantitative real-time PCR results showed that the expression trend of OfbZIP98 was consistent with the proliferation of O. fragrans callus. The subcellular localization and yeast self-activation results showed that OfbZIP98 was localized in the nucleus and had self-activation activity. Further, phenotypic observation of over-expressed tobacco showed that compared with the wild type (WT), the transgenic strain formed callus 4 d earlier, and callus re-differentiation began on day 15. Analysis of the callus differentiation rate showed that after 20 d of culture, the differentiation rate of the leaf callus of the transgenic strain was about twice that of the WT plants. In addition, measurements of corolla diameter, corolla circumference, corolla area, and corolla tube diameter indicated that the petals of the transgenic strain were significantly larger than the WT plants. Our results showed that OfbZIP98 has the important function of promoting callus growth, re-differentiation, and flower organ enlargement. These findings provide new insights into the potential role of OfbZIP98 in the growth and development of O. fragrans.

[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.

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.

The Jasmine (Jasminum sambac) Genome Provides Insight into the Biosynthesis of Flower Fragrances and Jasmonates.

Jasminum sambac (jasmine flower), a world-renowned plant appreciated for its exceptional flower fragrance, is of cultural and economic importance. However, the genetic basis of its fragrance is largely unknown. Here, we present the first de novogenome assembly of J. sambac with 550.12 Mb (scaffold N50 = 40.10 Mb) assembled into 13 pseudochromosomes. Terpene synthase (TPS) genes associated with flower fragrance are considerably amplified in the form of gene clusters through tandem duplications in the genome. Gene clusters within the salicylic acid/benzoic acid/theobromine (SABATH) and benzylalcohol O-acetyltransferase/anthocyanin O-hydroxycinnamoyltransferases/anthranilate N-hydroxycinnamoyl/benzoyltransferase/deacetylvindoline 4-O-acetyltransferase (BAHD) superfamilies were identified to be related to the biosynthesis of phenylpropanoid/benzenoid compounds. Several key genes involved in jasmonate biosynthesis were duplicated, causing an increase in copy numbers. In addition, multi-omics analyses identified various aromatic compounds and many genes involved in fragrance biosynthesis pathways. Furthermore, the roles of JsTPS3 in ß-ocimene biosynthesis, as well as JsAOC1 and JsAOS in jasmonic acid biosynthesis, were functionally validated. The genome assembled in this study for J. sambac offers a basic genetic resource for studying floral scent and jasmonate biosynthesis, and provides a foundation for functional genomic research and variety improvements in Jasminum.

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.

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.

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.

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.