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

Osmanthus fragrans is widely used in gardening, but the short flowering period of O. fragrans affects its ornamental and economic value. ERF, as a plant ethylene response factor, is an important link in the regulation of plant senescence. In this study, we conducted a comprehensive analysis of the functional role of OfERF1-3 within the petals of O. fragrans. Specifically, the OfERF1-3 gene was cloned and subjected to rigorous sequence analysis. Subsequently, to evaluate its expression patterns and effects, gene overexpression experiments were carried out on both Nicotiana tabacum and O. fragrans. The results showed that OfERF1-3-overexpressing tobacco plants exhibited longer petal opening times compared with those of wild plants. Measurements of physiological parameters also showed that the flowers of overexpressed tobacco plants contained lower levels of malondialdehyde (MDA) and hydrogen peroxide (H2O2) than those of the wild type. There was a lower expression of senescence marker genes in overexpressed tobacco and O. fragrans. A yeast two-hybrid assay showed that OfERF1-3 interacted with OfSKIP14 in a manner related to the regulation of ABA. In summary, OfERF1-3 can play a delaying role in the petal senescence process in O. fragrans, and it interacts with OfSKIP14 to indirectly affect petal senescence by regulating the ABA pathway.

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.

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.

Osmanthus fragrans, belonging to the family Oleaceae, is listed as one of the most important traditional ornamental plant species in China. A new cultivar O. fragrans ‘Wucaigui’ has a very diversified form in terms of leaf colors, in which the leaf color changes from red to yellow-green and finally to dark green. To understand the mechanisms involved in leaf color changes, metabolome and transcriptome studies were performed on leaves at different developmental stages. A total of 79 metabolites, two chlorophyll, 26 carotenoids, and 51 anthocyanins, were detected in the 6 different developmental stages. An orthogonal partial least squares discriminant analysis identified key metabolites at different developmental stages, including lutein, pelargonidin-3-O-(6-O-p-coumaroyl)-glucoside, neoxanthin, and α-carotene. A total of 48,837 genes were obtained by transcriptome sequencing, including 3295 novel genes. Using a weighted gene co-expression network analysis to study the correlations between key metabolites and differentially expressed genes, we determined the characteristic modules having the highest correlations with key metabolites and selected associated candidate genes. Five genes (OfSHOU4L, OfATL1B, OfUGE5 OfEIF1AX, and OfUGE3) were finally identified as hub genes using real-time fluorescence quantitative PCR. In addition, we proposed a model based on the changes in key metabolite contents and the network regulatory map during the changes in O. fragrans ‘Wucaigui’ leaf color. The positive regulation of OfUGE3 led to an increase in the lutein content, which resulted in the leaves changing from grayish brown to moderate brown; during the change from moderate brown to dark greenish-yellow, the positive regulation of three genes (OfHOU4L, OfATL1B, and OfUGE5) increased the content of pelargonidin-3-O-(6-O-p-coumaroyl)-glucoside and the red color of the leaves gradually faded to dark greenish-yellow and then to strong yellow-green; the positive regulation of OfEIF1AX increased the content of neoxanthin; the stages in which the color changed from strong yellow-green to yellow-green and then to moderate olive-green were positively regulated by OfUGE3, which resulted in higher α-carotene content. These findings provided new insights into the mechanisms underlying the processes involved in O. fragrans ‘Wucaigui’ leaf color changes at the metabolic and transcriptional levels. This work seeks to contribute to the development of artificial regulate and control technology in the breeding and production of O. fragrans and other ornamental plants.

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-β-ionone, and trans-linalool oxide(furan) were key factors affecting the endophytic fungal community composition. Furthermore, dihydro-β-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.

Osmanthus fragrans ‘Yinbi Shuanghui’ is a colored leaf variety of O. fragrans. To study the mechanism of leaf color formation in O. fragrans ‘Yinbi Shuanghui’, we selected green and colored leaves with two different parts, namely yellow and green, as our research materials. We analyzed the expression changes related to leaf color in genes by performing qRT-PCR in the different leaf parts, finding that OfPAO was significantly up-regulated in the yellow part of colored leaves, and we initially determined that OfPAO was the key gene involved in the formation of colored leaves. Then, we constructed an OfPAO overexpression vector, before transforming it into tobacco through an Agrobacterium-mediated transformation to obtain transgenic plants. We found that the transgenic tobacco leaf color of OfPAO was lighter than that of the null carrier, the chlorophyll content in leaves decreased, and the expression of genes involved in the chlorophyll degradation pathway in OfPAO transgenic tobacco was up-regulated, suggesting that OfPAO regulates chlorophyll degradation, leading to changes in leaf color. According to the results of transcriptome sequencing and the genome data of O. fragrans ‘Rixianggui’, we cloned CDS and the promoter sequence of OfPAO, and the promoter regions 901-1307 of the OfPAO were sequenced through bisulfite genomic sequencing PCR (BSP), finding that the methylation level of CHH in the yellow part of colored leaves was lowest in colored and green leaves at 145 bp. The methylation of CHH in the promoter of OfPAO in O. fragrans ‘Yinbi Shuanghui’ was negatively correlated with the gene expression level, suggesting that the methylation of the promoter of OfPAO may regulate the expression of OfPAO, affecting chlorophyll degradation in the leaves.

Abstract 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.

Abstract 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.

Acer fabri is a widely distributed ornamental tree with colorful leaves and high ornamental value. Its young leaves change from red to red and green until turning fully green. To understand the mechanism of leaf color change, transcriptome sequencing and pigment content determination were performed in three stages during the leaf color change of A. fabri. In total, 53,550 genes, including 838 transcription factors (TFs), were identified by transcriptome sequencing. In addition, the results of orthogonal partial least squares-discriminant analysis (OPLS-DA) of three pigments in the three stages of leaf color development suggested that carotenoids played a major role in the process of leaf color change from red to red-green, whereas anthocyanins played an important role in the process of leaf color change from red to green. Based on weighted gene co-expression network analysis (WGCNA), Af0034384 (HSFB2A), Af0051627 (NMT1), and Af0052541 (THY-1) were selected as hub genes from characteristic modules with significant correlation between carotenoids and anthocyanins. The results of gene network regulation maps and real-time fluorescence quantitative PCR (qRT-PCR) showed that Af0010511 (NAC100) upregulated the expression of Af0034384 (HSFB2A), leading to an increase in carotenoid content and the gradual greening of leaves during the transition from red to green. However, during the transition from red to green leaves, Af0033232 (NAC83) and Af0049421 (WRKY24) downregulated the expression of Af0051627 (NMT1) and Af0052541 (THY-1), respectively, leading to a decrease in anthocyanin content and the complete greening of leaves. These results could provide new ideas for studying the molecular mechanism of leaf color change in A. fabri and other species.

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.

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.

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 β-ionone formation. In contrast, OfMYB1R201 decreases the synthesis of β-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.

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.

Genome sequences: Discovering a sweet secret A high-quality genome sequence for sweet olive, Osmanthus fragrans, has revealed which genes make the ornamental tree’s blossoms so fragrant. Lianggui Wang at Nanjing Forestry University in China and coworkers used cutting-edge approaches to produce a high-resolution picture of sweet olive’s genome. Analysis of the full sequence revealed that sweet olive has a large number of genes, more than 45,000. Further investigation showed that the entire genome had been duplicated, or accidentally copied, several million years ago. Thanks to the duplication, sweet olive has extra copies of the genes that produce its scent compound, resulting in its very strong scent. These results are only the beginning. Having this first sequence makes sequencing other varieties of sweet olive much easier, and future comparisons between genomes will help to pinpoint genes that control many other traits.

Background: Osmanthus fragrans is an important ornamental tree and has been widely planted in China because of its pleasant aroma, which is mainly due to terpenes. The monoterpenoid and sesquiterpenoid metabolic pathways of sweet osmanthus have been well studied. However, these studies were mainly focused on volatile small molecule compounds. The molecular regulation mechanism of synthesis of large molecule compounds (triterpenoids) remains unclear. Squalene synthase (SQS), squalene epoxidase (SQE), and beta-amyrin synthase (BETA-AS) are three critical enzymes of the triterpenoid biosynthesis pathway. Results: In this study, the full-length cDNA and gDNA sequences of OfSQS, OfSQE, and OfBETA-AS were isolated from sweet osmanthus. Phylogenetic analysis suggested that OfSQS and OfSQE had the closest relationship with Sesamum indicum, and OfBETA-AS sequence shared the highest similarity of 99% with that of Olea europaea. The qRT-PCR analysis revealed that the three genes were highly expressed in flowers, especially OfSQE and OfBETA-AS, which were predominantly expressed in the flowers of both “Boye” and “Rixiang” cultivars, suggesting that they might play important roles in the accumulation of triterpenoids in flowers of O. fragrans. Furthermore, the expression of OfBETA-AS in the two cultivars was significantly different during all the five flowering stages; this suggested that OfBETA-AS may be the critical gene for the differences in the accumulation of triterpenoids. Conclusion: The evidence indicates that OfBETA-AS could be the key gene in the triterpenoid synthesis pathway, and it could also be used as a critical gene resource in the synthesis of essential oils by using bioengineered bacteria.How to cite: Yang X, Ding W, Yue Y, et al. Cloning and expression analysis of three critical triterpenoids pathway genes in Osmanthus fragrans. Electron J Biotechnol 2018;36. https://doi.org/10.1016/j.ejbt.2018.08.007. Keywords: Aroma, Essential oils, Flower gene expression, Ornamental tree, Osmanthus fragrans, Terpenoid metabolic pathway, Squalene epoxidaes, Squalene synthase, Terpenes, Triterpenoid biosynthesis

Sweet osmanthus (Osmanthus fragrans) is an evergreen woody plant that emits a floral aroma and is widely used in the landscape and fragrance industries. However, its application and cultivation regions are limited by cold stress. Heat-shock transcription factor (HSF) family members are widely present in plants and participate in, and regulate, the defense processes of plants under various abiotic stress conditions, but now, the role of this family in the responses of O. fragrans to cold stress is still not clear. Here, 46 OfHSF members were identified in the O. fragrans genome and divided into three subfamilies on the basis of a phylogenetic analysis. The promoter regions of most OfHSFs contained many cis-acting elements involved in multiple hormonal and abiotic stresses. RNA-seq data revealed that most of OfHSF genes were differentially expressed in various tissues, and some OfHSF members were induced by cold stress. The qRT-PCR analysis identified four OfHSFs that were induced by both cold and heat stresses, in which OfHSF11 and OfHSF43 had contrary expression trends under cold stress conditions and their expression patterns both showed recovery tendencies after the cold stress. OfHSF11 and OfHSF43 localized to the nuclei and their expression patterns were also induced under multiple abiotic stresses and hormonal treatments, indicating that they play critical roles in responses to multiple stresses. Furthermore, after a cold treatment, transient expression revealed that the malondialdehyde (MDA) content of OfHSF11-transformed tobacco significantly increased, and the expression levels of cold-response regulatory gene NbDREB3, cold response gene NbLEA5 and ROS detoxification gene NbCAT were significantly inhibited, implying that OfHSF11 is a negative regulator of cold responses in O. fragrans. Our study contributes to the further functional characterization of OfHSFs and will be useful in developing improved cold-tolerant cultivars of O. fragrans.

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.

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.

Auxins have long been implicated in many aspects of plant growth and development. Auxin response factors (ARFs) are important proteins in auxin-mediated pathways and they play key roles in plant physiological and biochemical processes, including flower development. Endogenous indoleacetic acid (IAA) levels were measured and ARFs were studied in the flowers during the developmental stages in order to further elucidate the role of auxin in flower development of Osmanthus fragrans. A systematic analysis of OfARFs was conducted by carrying out a genome-wide search of ARFs. A total of 50 ARF genes (OfARFs) were detected and validated from the Osmanthus fragrans genome. Furthermore, a comprehensive overview of the OfARFs was undertaken, including phylogenetic relationship, gene structures, conserved domains, motifs, promoters, chromosome locations, gene duplications, and subcellular locations of the gene product. Finally, expression profiling, while using transcriptome sequencing from a previous study and quantitative real-time PCR (qRT-PCR), revealed that many OfARF genes have different expression levels in various tissues and flower developmental stages. By comparing the expression profiles among the flower developmental stages, and the relationship between ARFs and endogenous IAA levels, it can be supposed that OfARFs function in flower development of O. fragrans in an auxin-mediated pathway.

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

The landscape application of sweet osmanthus (Osmanthus fragrans) with flower fragrance and high ornamental value is severely limited by salinity stress. Gamma irradiation applied to seeds enhanced their tolerance to salinity stress as reported in other plants. In this study, O. fragrans ‘Huangchuang Jingui’ seeds were pretreated with different doses of gamma irradiation, and tolerance of the seedlings germinated from the irradiated seeds to salinity stress and the changes of reactive oxygen species (ROS) production and ROS scavenging systems induced by gamma irradiation were observed. The results showed that seed pretreatment with different doses of gamma irradiation enhanced the tolerance of sweet osmanthus seedlings to salinity stress, and the positive effect induced by gamma irradiation was more remarkable with the increase of radiation dose (50−150 Gy). The pretreatment with high-dose irradiation decreased O2− production under salinity stress and mitigated the oxidative damage marked by a lower malondialdehyde (MDA) level, which could be related to the significant increase of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities in the seedlings germinated from the irradiated seeds compared to the corresponding control seedlings. In addition, the accumulation of proline in the irradiated seedlings may contribute to enhancing their tolerance to salt stress by the osmotic adjustment. The study demonstrated the importance of regulating plant ROS balance under salt stress and provided a potential approach to improve the tolerance of sweet osmanthus to salt stress.

Since the publication of this article, the authors have noticed that the NCBI accession number is missing from article.

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

Different mulches have variable effects on soil physical properties and plant growth. This study aimed to compare the effects of mulching with inorganic (round gravel, RG), organic (wood chips, WC), and living (manila turf grass, MG) materials on soil properties at 0-5-cm and 5-10-cm depths, as well as on the growth and physiological features of Osmanthus fragrans L. 'Rixianggui' plants. Soil samples were collected at three different time points from field plots of O. fragrans plants treated with the different mulching treatments. Moisture at both soil depths was significantly higher after mulching with RG and WC than that in the unmulched control (CK) treatment. Mulching did not affect soil bulk density, pH, or total nitrogen content, but consistently improved soil organic matter. The available nitrogen in the soil increased after RG and WC treatments, but decreased after MG treatment during the experimental period. Mulching improved plant growth by increasing root activity, soluble sugar, and chlorophyll a content, as well as by providing suitable moisture conditions and nutrients in the root zone. Plant height and trunk diameter were remarkably increased after mulching, especially with RG and WC. However, while MG improved plant growth at the beginning of the treatment, the 'Rixianggui' plants later showed no improvement in growth. This was probably because MG competed with the plants for water and available nitrogen in the soil. Thus, our findings suggest that RG and WC, but not MG, improved the soil environment and the growth of 'Rixianggui' plants. Considering the effect of mulching on soil properties and plant growth and physiology, round gravel and wood chips appear to be a better choice than manila turf grass in 'Rixianggui' nurseries. Further studies are required to determine the effects of mulch quality and mulch-layer thickness on shoot and root growths.

Ethylene phytic acid and unsaturated itaconic acid could be copolymerized insitu and cross-linked on the cotton fabrics to enhance flame-retardant and crease-proofing properties. Natural phytic acid was modified with glycidyl methacrylate and characterized by IR, 1H NMR, and TG. The IR indicated that new characteristic absorption peaks appear near 1720 c/m, 1470 c/m, 1380 c/m, and 1002 c/m for the molar ratios phytic acid: glycidyl methacrylate of 1:3 or 1:6, the peaks correspond to the bending vibration of C=O, C-H, CH3, and C=O-O respectively in the glycidyl methacrylate. The 1H NMR results further confirmed the reaction between glycidyl methacrylate and phytic acid, and the TG results revealed the thermal properties of phytic acid. [ABSTRACT FROM AUTHOR]