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.

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.

Functional characterization of polyphenol oxidase OfPPO2 supports its involvement in parallel biosynthetic pathways of acteoside.

Acteoside is a bioactive phenylethanoid glycoside widely distributed throughout the plant kingdom. Because of its two catechol moieties, acteoside displays a variety of beneficial activities. The biosynthetic pathway of acteoside has been largely elucidated, but the assembly logic of two catechol moieties in acteoside remains unclear. Here, we identified a novel polyphenol oxidase OfPPO2 from Osmanthus fragrans, which could hydroxylate various monophenolic substrates, including tyrosine, tyrosol, tyramine, 4-hydroxyphenylacetaldehyde, salidroside, and osmanthuside A, leading to the formation of corresponding catechol-containing intermediates for acteoside biosynthesis. OfPPO2 could also convert osmanthuside B into acteoside, creating catechol moieties directly via post-modification of the acteoside skeleton. The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis and subcellular localization assay further support the involvement of OfPPO2 in acteoside biosynthesis in planta. These findings suggest that the biosynthesis of acteoside in O. fragrans may follow "parallel routes" rather than the conventionally considered linear route. In support of this hypothesis, the glycosyltransferase OfUGT and the acyltransferase OfAT could direct the flux of diphenolic intermediates generated by OfPPO2 into acteoside. Significantly, OfPPO2 and its orthologs constitute a functionally conserved enzyme family that evolved independently from other known biosynthetic enzymes of acteoside, implying that the substrate promiscuity of this PPO family may offer acteoside-producing plants alternative ways to synthesize acteoside. Overall, this work expands our understanding of parallel pathways plants may employ to efficiently synthesize acteoside, a strategy that may contribute to plants' adaptation to environmental challenges.

Multiomics analyses provide insights into the genomic basis of differentiation among four sweet osmanthus groups.

Sweet osmanthus (Osmanthus fragrans) is famous in China for its flowers and contains four groups: Albus, Luteus, Aurantiacus, and Asiaticus. Understanding the relationships among these groups and the genetic mechanisms of flower color and aroma biosynthesis are of tremendous interest. In this study, we sequenced representative varieties from two of the four sweet osmanthus groups. Multiomics and phylogenetic analyses of varieties from each of the four groups showed that Asiaticus split first within the species, followed by Aurantiacus and the sister groups Albus and Luteus. We show that the difference in flower color between Aurantiacus and the other three groups was caused by a 4-bp deletion in the promoter region of carotenoid cleavage dioxygenase 4 (OfCCD4) that leads to expression decrease. In addition, we identified 44 gene pairs exhibiting significant structural differences between the multiseasonal flowering variety "Rixianggui" in the Asiaticus group and other autumn-flowering varieties. Through correlation analysis between intermediate products of aromatic components and gene expression, we identified eight genes associated with the linalool and α- and ß-ionone biosynthesis pathways. Overall, our study offers valuable genetic resources for sweet osmanthus, while also providing genetic clues for improving the flower color and multiseasonal flowering of osmanthus and other flowers.

Twelve newly assembled jasmine chloroplast genomes: unveiling genomic diversity, phylogenetic relationships and evolutionary patterns among Oleaceae and Jasminum species.

BACKGROUND: Jasmine (Jasminum), renowned for its ornamental value and captivating fragrance, has given rise to numerous species and accessions. However, limited knowledge exists regarding the evolutionary relationships among various Jasminum species. RESULTS: In the present study, we sequenced seven distinct Jasminum species, resulting in the assembly of twelve high-quality complete chloroplast (cp) genomes. Our findings revealed that the size of the 12 cp genomes ranged from 159 to 165 kb and encoded 134-135 genes, including 86-88 protein-coding genes, 38-40 tRNA genes, and 8 rRNA genes. J. nudiflorum exhibited a larger genome size compared to other species, mainly attributed to the elevated number of forward repeats (FRs). Despite the typically conservative nature of chloroplasts, variations in the presence or absence of accD have been observed within J. sambac. The calculation of nucleotide diversity (Pi) values for 19 cp genomes indicated that potential mutation hotspots were more likely to be located in LSC regions than in other regions, particularly in genes ycf2, rbcL, atpE, ndhK, and ndhC (Pi > 0.2). Ka/Ks values revealed strong selection pressure on the genes rps2, atpA, rpoA, rpoC1, and rpl33 when comparing J. sambac with the three most closely related species (J. auriculatum, J. multiflorum, and J. dichotomum). Additionally, SNP identification, along with the results of Structure, PCA, and phylogenetic tree analyses, divided the Jasminum cp genomes into six groups. Notably, J. polyanthum showed gene flow signals from both the G5 group (J. nudiflorum) and the G3 group (J. tortuosum and J. fluminense). Phylogenetic tree analysis reflected that most species from the same genus clustered together with robust support in Oleaceae, strongly supporting the monophyletic nature of cp genomes within the genus Jasminum. CONCLUSION: Overall, this study provides comprehensive insights into the genomic composition, variation, and phylogenetic relationships among various Jasminum species. These findings enhance our understanding of the genetic diversity and evolutionary history of Jasminum.

The homomorphic self-incompatibility system in Oleaceae is controlled by a hemizygous genomic region expressing a gibberellin pathway gene.

In flowering plants, outcrossing is commonly ensured by self-incompatibility (SI) systems. These can be homomorphic (typically with many different allelic specificities) or can accompany flower heteromorphism (mostly with just two specificities and corresponding floral types). The SI system of the Oleaceae family is unusual, with the long-term maintenance of only two specificities but often without flower morphology differences. To elucidate the genomic architecture and molecular basis of this SI system, we obtained chromosome-scale genome assemblies of Phillyrea angustifolia individuals and related them to a genetic map. The S-locus region proved to have a segregating 543-kb indel unique to one specificity, suggesting a hemizygous region, as observed in all distylous systems so far studied at the genomic level. Only one of the predicted genes in this indel region is found in the olive tree, Olea europaea, genome, also within a segregating indel. We describe complete association between the presence/absence of this gene and the SI types determined for individuals of seven distantly related Oleaceae species. This gene is predicted to be involved in catabolism of the gibberellic acid (GA) hormone, and experimental manipulation of GA levels in developing buds modified the male and female SI responses of the two specificities in different ways. Our results provide a unique example of a homomorphic SI system, where a single conserved gibberellin-related gene in a hemizygous indel underlies the long-term maintenance of two groups of reproductive compatibility.

A hemizygous supergene controls homomorphic and heteromorphic self-incompatibility systems in Oleaceae.

Self-incompatibility (SI) has evolved independently multiple times and prevents self-fertilization in hermaphrodite angiosperms. Several groups of Oleaceae such as jasmines exhibit distylous flowers, with two compatibility groups each associated with a specific floral morph.1 Other Oleaceae species in the olive tribe have two compatibility groups without associated morphological variation.2,3,4,5 The genetic basis of both homomorphic and dimorphic SI systems in Oleaceae is unknown. By comparing genomic sequences of three olive subspecies (Olea europaea) belonging to the two compatibility groups, we first locate the genetic determinants of SI within a 700-kb hemizygous region present only in one compatibility group. We then demonstrate that the homologous hemizygous region also controls distyly in jasmine. Phylogenetic analyses support a common origin of both systems, following a segmental genomic duplication in a common ancestor. Examination of the gene content of the hemizygous region in different jasmine and olive species suggests that the mechanisms determining compatibility groups and floral phenotypes (whether homomorphic or dimorphic) in Oleaceae rely on the presence/absence of two genes involved in gibberellin and brassinosteroid regulation.

Genomic and Transcriptomic Analyses Illuminates Unique Traits of Elusive Night Flowering Jasmine Parijat (Nyctanthes arbor-tristis).

The night-flowering Jasmine, Nyctanthes arbor-tristis also known as Parijat, is a perennial woody shrub belonging to the family of Oleaceae. It is popular for its fragrant flowers that bloom in the night and is a potent source of secondary metabolites. However, knowledge about its genome and the expression of genes regulating flowering or secondary metabolite accumulation is lacking. In this study, we generated whole genome sequencing data to assemble the first de novo assembly of Parijat and use it for comparative genomics and demographic history reconstruction. The temporal dynamics of effective population size (Ne ) experienced a positive influence of colder climates suggesting the switch to night flowering may have provided an evolutionary advantage. We employed multi-tissue transcriptome sequencing of floral stages/parts to obtain insights into the transcriptional regulation of nocturnal flower development and the production of volatiles/metabolites. Tissue-specific transcripts for mature flowers revealed key players in circadian regulation and flower development, including the auxin pathway and cell wall modifying genes. Furthermore, we identified tissue-specific transcripts responsible for producing numerous secondary metabolites, mainly terpenoids and carotenoids. The diversity and specificity of Terpene Synthase (TPS) and CCDs (Carotenoid Cleavage Deoxygenases) mediate the bio-synthesis of specialised metabolites in Parijat. Our study establishes Parijat as a novel non-model species to understand the molecular mechanisms of nocturnal blooming and secondary metabolite production.

Male fertility advantage within and between seasons in the perennial androdioecious plant Phillyrea angustifolia.

BACKGROUND AND AIMS: Androdioecy, the co-occurrence of males and hermaphrodites, is a rare reproductive system. Males can be maintained if they benefit from a higher male fitness than hermaphrodites, referred to as male advantage. Male advantage can emerge from increased fertility owing to resource reallocation. However, empirical studies usually compare sexual phenotypes over a single flowering season, thus ignoring potential cumulative effects over successive seasons in perennials. In this study, we quantify various components of male fertility advantage, both within and between seasons, in the long-lived perennial shrub Phillyrea angustifolia (Oleaceae). Although, owing to a peculiar diallelic self-incompatibility system and female sterility mutation strictly associated with a breakdown of incompatibility, males do not need fertility advantage to persist in this species, this advantage remains an important determinant of their equilibrium frequency. METHODS: A survey of >1000 full-sib plants allowed us to compare males and hermaphrodites for several components of male fertility. Individuals were characterized for proxies of pollen production and vegetative growth. By analysing maternal progeny, we compared the siring success of males and hermaphrodites. Finally, using a multistate capture-recapture model we assessed, for each sexual morph, how the intensity of flowering in one year impacts next-year growth and reproduction. KEY RESULTS: Males benefitted from a greater vegetative growth and flowering intensity. Within one season, males sired twice as many seeds as equidistant, compatible hermaphroditic competitors. In addition, males more often maintained intense flowering over successive years. Finally, investment in male reproductive function appeared to differ between the two incompatibility groups of hermaphrodites. CONCLUSION: Males, by sparing the cost of female reproduction, have a higher flowering frequency and vegetative growth, both of which contribute to male advantage over an individual lifetime. This suggests that studies analysing sexual phenotypes during only single reproductive periods are likely to provide inadequate estimates of male advantage in perennials.

Argopistes sexvittatus and Argopistes capensis (Chrysomelidae: Alticini): Mitogenomics and Phylogeny of Two Flea Beetles Affecting Olive Trees.

The genus Argopistes (Chrysomelidae: Alticini) is the only group of flea beetles specialized in plant hosts in the family Oleaceae. In southern Africa, Argopistes are often found feeding on African Wild Olive (Olea europaea subsp. cuspidata) and European cultivated olive (O. e. subsp. europaea), and heavy infestations can be devastating to mature trees and compromise the development of young trees. Despite their negative agricultural impact, African Argopistes are an understudied group for which no genetic data were available. We assessed the species diversity of olive flea beetles in the Western Cape province of South Africa, the largest olive-producing region in sub-Saharan Africa, by collecting adult specimens on wild and cultivated olive trees between 2015 and 2017. Argopistes sexvittatus Bryant, 1922 (n = 289) dominated at all sampling sites, and Argopistes capensis Bryant, 1944 (n = 2) was found only once. Argopistes oleae Bryant, 1922, a third species previously reported in the region, was not found. The complete mitogenomes of one A. capensis and two A. sexvittatus (striped and black morphotypes) individuals were sequenced for phylogenetic reconstruction in the context of other 64 species. The two olive flea beetle species form a monophyletic clade with other Argopistes, supporting the hypothesis that the exclusive feeding habit on Oleaceae is an evolutionary adaptation in this genus.

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.

Identification of miRNAs and Target Genes at Key Stages of Sexual Differentiation in Androdioecious Osmanthus fragrans.

Androdioecy is the crucial transition state in the evolutionary direction of hermaphroditism to dioecy, however, the molecular mechanisms underlying the formation of this sex system remain unclear. While popular in China for its ornamental and cultural value, Osmanthus fragrans has an extremely rare androdioecy breeding system, meaning that there are both male and hermaphroditic plants in a population. To unravel the mechanisms underlying the formation of androdioecy, we performed small RNA sequencing studies on male and hermaphroditic O. fragrans. A total of 334 miRNAs were identified, of which 59 were differentially expressed. Functional categorization revealed that the target genes of differentially expressed miRNAs were mainly involved in the biological processes of reproductive development and the hormone signal transduction pathway. We speculated that the miRNA160, miRNA167, miRNA393 and miRNA396 families may influence the sex differentiation in O. fragrans. Overall, our study is the first exploration of miRNAs in the growth and development process of O. fragrans, and is also the first study of androdioecious plants from the miRNA sequencing perspective. The analysis of miRNAs and target genes that may be involved in the sex differentiation process lay a foundation for the ultimate discovery of the androdioecious molecular mechanism in O. fragrans.

A chromosome-level genome of Syringa oblata provides new insights into chromosome formation in Oleaceae and evolutionary history of lilacs.

Lilacs (Syringa L.), a group of well-known ornamental and aromatic woody plants, have long been used for gardening, essential oils and medicine purposes in East Asia and Europe. The lack of knowledge about the complete genome of Syringa not only hampers effort to better understand its evolutionary history, but also prevents genome-based functional gene mining that can help in the variety improvement and medicine development. Here, a chromosome-level genome of Syringa oblata is presented, which has a size of 1.12 Gb including 53 944 protein coding genes. Synteny analysis revealed that a recent duplication event and parallel evolution of two subgenomes formed the current karyotype. Evolutionary analysis, transcriptomics and metabolic profiling showed that segment and tandem duplications contributed to scent formation in the woody aromatic species. Moreover, phylogenetic analysis indicated that S. oblata shared a common ancestor with Osmanthus fragrans and Olea europaea approximately 27.61 million years ago (Mya). Biogeographic reconstruction based on a resequenced data set of 26 species suggested that Syringa originated in the northern part of East Asia during the Miocene (approximately 14.73 Mya) and that the five Syringa groups initially formed before the Late Miocene (approximately 9.97 Mya). Furthermore, multidirectional dispersals accompanied by gene introgression among Syringa species from Northern China during the Miocene were detected by biogeographic reconstruction. Taken together, the results showed that complex gene introgression, which occurred during speciation history, greatly contributed to Syringa diversity.

Temperature regulation of carotenoid accumulation in the petals of sweet osmanthus via modulating expression of carotenoid biosynthesis and degradation genes.

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

Cloning, Expression, and Tobacco Overexpression Analyses of a PISTILLATA/GLOBOSA-like (OfGLO1) Gene from Osmanthus fragrans.

GLOBOSA (GLO), a B-class MADS-box gene, is involved in floral organ determination but has rarely been studied in Osmanthus fragrans, which is a very popular ornamental tree species in China. Here, the full-length cDNA of a homologous GLO1 gene (named OfGLO1) was cloned from a flower bud of O. fragrans using the RACE technique. The OfGLO1 has a 645 bp open reading frame, encoding 214 amino acids. Similar to other PI/GLO proteins, OfGLO1 has two conserved domains, MADS MEF2-like and K-box, and a 16-amino-acid PI motif in the C terminal region. Our phylogeny analysis classified OfGLO1 as a PI-type member of the B-class MADS-box gene family. The qRT-PCR assay showed that the expression of OfGLO1 in O. fragrans was continuously upregulated from the tight bud stage to the full flowering stage but barely expressed in the pistils, sepals, and non-floral organs, such as root, leaf, and stem. The genetic effect of OfGLO1 was assayed by ectopic expression in tobacco plants. Compared with the wild-type, OfGLO1 transformants showed reduced plant size, earlier flowering, shorter stamens, and lower seed setting rates. Furthermore, some stamens were changed into petal-like structures. These findings indicate that OfGLO1 plays an important role in the regulation of flower development. This study improved our understanding of class B gene function in woody plants.

SWATH-MS based quantitive proteomics reveal regulatory metabolism and networks of androdioecy breeding system in Osmanthus fragrans.

BACKGROUND: The fragrant flower plant Osmanthus fragrans has an extremely rare androdioecious breeding system displaying the occurrence of males and hermaphrodites in a single population, which occupies a crucial intermediate stage in the evolutionary transition between hermaphroditism and dioecy. However, the molecular mechanism of androdioecy plant is very limited and still largely unknown. RESULTS: Here, we used SWATH-MS-based quantitative approach to study the proteome changes between male and hermaphroditic O. fragrans pistils. A total of 428 proteins of diverse functions were determined to show significant abundance changes including 210 up-regulated and 218 down-regulated proteins in male compared to hermaphroditic pistils. Functional categorization revealed that the differentially expressed proteins (DEPs) primarily distributed in the carbohydrate metabolism, secondary metabolism as well as signaling cascades. Further experimental analysis showed the substantial carbohydrates accumulation associated with promoted net photosynthetic rate and water use efficiency were observed in purplish red pedicel of hermaphroditic flower compared with green pedicel of male flower, implicating glucose metabolism serves as nutritional modulator for the differentiation of male and hermaphroditic flower. Meanwhile, the entire upregulation of secondary metabolism including flavonoids, isoprenoids and lignins seem to protect and maintain the male function in male flowers, well explaining important feature of androdioecy that aborted pistil of a male flower still has a male function. Furthermore, nine selected DEPs were validated via gene expression analysis, suggesting an extra layer of post-transcriptional regulation occurs during O. fragrans floral development. CONCLUSION: Taken together, our findings represent the first SWATH-MS-based proteomic report in androdioecy plant O. fragrans, which reveal carbohydrate metabolism, secondary metabolism and post-transcriptional regulation contributing to the androdioecy breeding system and ultimately extend our understanding on genetic basis as well as the industrialization development of O. fragrans.

Genome-wide miRNA analysis and integrated network for flavonoid biosynthesis in Osmanthus fragrans.

BACKGROUND: Osmanthus fragrans is an important economical plant containing multiple secondary metabolites including flavonoids and anthocyanins. During the past years, the roles of miRNAs in regulating the biosynthesis of secondary metabolites in plants have been widely investigated. However, few studies on miRNA expression profiles and the potential roles in regulating flavonoid biosynthesis have been reported in O. fragrans. RESULTS: In this study, we used high-throughput sequencing technology to analyze the expression profiles of miRNAs in leaf and flower tissues of O. fragrans. As a result, 106 conserved miRNAs distributed in 47 families and 88 novel miRNAs were identified. Further analysis showed there were 133 miRNAs differentially expressed in leaves and flowers. Additionally, the potential target genes of miRNAs as well as the related metabolic pathways were predicted. In the end, flavonoid content was measured in flower and leaf tissues and potential role of miR858 in regulating flavonoid synthesis was illustrated in O. fragrans. CONCLUSIONS: This study not only provided the genome-wide miRNA profiles in the flower and leaf tissue of O. fragrans, but also investigated the potential regulatory role of miR858a in flavonoid synthesis in O. fragrans. The results specifically indicated the connection of miRNAs to the regulation of secondary metabolite biosynthesis in non-model economical plant.

Resolving the Phylogeny of the Olive Family (Oleaceae): Confronting Information from Organellar and Nuclear Genomes.

The olive family, Oleaceae, is a group of woody plants comprising 28 genera and ca. 700 species, distributed on all continents (except Antarctica) in both temperate and tropical environments. It includes several genera of major economic and ecological importance such as olives, ash trees, jasmines, forsythias, osmanthuses, privets and lilacs. The natural history of the group is not completely understood yet, but its diversification seems to be associated with polyploidisation events and the evolution of various reproductive and dispersal strategies. In addition, some taxonomical issues still need to be resolved, particularly in the paleopolyploid tribe Oleeae. Reconstructing a robust phylogenetic hypothesis is thus an important step toward a better comprehension of Oleaceae's diversity. Here, we reconstructed phylogenies of the olive family using 80 plastid coding sequences, 37 mitochondrial genes, the complete nuclear ribosomal cluster and a small multigene family encoding phytochromes (phyB and phyE) of 61 representative species. Tribes and subtribes were strongly supported by all phylogenetic reconstructions, while a few Oleeae genera are still polyphyletic (Chionanthus, Olea, Osmanthus, Nestegis) or paraphyletic (Schrebera, Syringa). Some phylogenetic relationships among tribes remain poorly resolved with conflicts between topologies reconstructed from different genomic regions. The use of nuclear data remains an important challenge especially in a group with ploidy changes (both paleo- and neo-polyploids). This work provides new genomic datasets that will assist the study of the biogeography and taxonomy of the whole Oleaceae.

Transcriptomic analysis of flower opening response to relatively low temperatures in Osmanthus fragrans.

BACKGROUND: Sweet osmanthus (Osmanthus fragrans Lour.) is one of the top ten traditional ornamental flowers in China. The flowering time of once-flowering cultivars in O. fragrans is greatly affected by the relatively low temperature, but there are few reports on its molecular mechanism to date. A hypothesis had been raised that genes related with flower opening might be up-regulated in response to relatively low temperature in O. fragrans. Thus, our work was aimed to explore the underlying molecular mechanism of flower opening regulated by relatively low temperature in O. fragrans. RESULTS: The cell size of adaxial and abaxial petal epidermal cells and ultrastructural morphology of petal cells at different developmental stages were observed. The cell size of adaxial and abaxial petal epidermal cells increased gradually with the process of flower opening. Then the transcriptomic sequencing was employed to analyze the differentially expressed genes (DEGs) under different number of days' treatments with relatively low temperatures (19 °C) or 23 °C. Analysis of DEGs in Gene Ontology analysis showed that "metabolic process", "cellular process", "binding", "catalytic activity", "cell", "cell part", "membrane", "membrane part", "single-organism process", and "organelle" were highly enriched. In KEGG analysis, "metabolic pathways", "biosynthesis of secondary metabolites", "plant-pathogen interaction", "starch and sucrose metabolism", and "plant hormone signal transduction" were the top five pathways containing the greatest number of DEGs. The DEGs involved in cell wall metabolism, phytohormone signal transduction pathways, and eight kinds of transcription factors were analyzed in depth. CONCLUSIONS: Several unigenes involved in cell wall metabolism, phytohormone signal transduction pathway, and transcription factors with highly variable expression levels between different temperature treatments may be involved in petal cell expansion during flower opening process in response to the relatively low temperature. These results could improve our understanding of the molecular mechanism of relatively-low-temperature-regulated flower opening of O. fragrans, provide practical information for the prediction and regulation of flowering time in O. fragrans, and ultimately pave the way for genetic modification in O. fragrans.

Virus-Induced Gene Silencing in Olive Tree (Oleaceae).

Research on gene functions in non-model tree species is hampered by a number of difficulties such as time-consuming genetic transformation protocols and extended period for the production of healthy transformed offspring, among others. Virus-induced gene silencing (VIGS) is an alternative approach to transiently knock out an endogenous gene of interest (GOI) by the introduction of viral sequences encompassing a fragment of the GOI and to exploit the posttranscriptional gene silencing (PTGS) mechanism of the plant, thus triggering silencing of the GOI. Here we describe the successful application of Tobacco rattle virus (TRV)-mediated VIGS through agroinoculation of olive plantlets. This methodology is expected to serve as a fast tracking and powerful tool enabling researchers from diversified fields to perform functional genomic analyses in the olive tree.