Search

Sweet osmanthus (Osmanthus fragrans) is an evergreen aromatic woody tree widely used in landscaping. However, O. fragrans is very sensitive to cold stress, which seriously limits its cultivation and application in regions of low temperature. The NAC transcription factor gene family which has been characterized in many plant species could participate in multiple processes and play critical roles in plant response to external stress. However, little is known about this family in O. fragrans, an important woody ornamental plant. Here, the structural characteristics, chromosomal location, evolutionary relationship, cold stress expression patterns, and subcellular localization analysis of NAC family genes in O. fragrans were analyzed on a genome-wide scale. A total of 119 O. fragrans NAC genes were identified, and gene structure analysis showed that most OfNACs contained two introns, suggesting that structural diversity of the O. fragrans NAC family is relatively low. The phylogeny evolutionary tree showed that the NACs of O. fragrans were classified into 13 branches. The transcriptome data suggested that each branch members had different expression patterns in various tissues and a number of OfNACs showed tissue-specific expression profiles. The quantitative real-time PCR and subcellular localization results indicated that expression patterns of most branch ‘5B’ members were significantly induced by cold stress and they were mainly located in the nucleus, especially OfNAC49 and 59, indicating that branch ‘5B’ members could have important roles in cold tolerance in O. fragrans. Remarkably, expression levels of many branch ‘5B’ genes were also significantly changed under waterlogging stress, suggesting that this branch might be widely involved in tolerance to various abiotic stresses. Our study should supply useful gene resources for the cold resistance breeding of O. fragrans.

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.

Osmanthus fragrans &lsquo, Yinbi Shuanghui&rsquo, 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 &lsquo, 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 &lsquo, leaves at the transcriptional level.

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 a widely used evergreen aromatic ornamental tree with poor tolerance to low temperatures. The basic helix-loop-helix (bHLH) transcription factor protein family, which is the second largest protein family in plants, has been found to play essential roles in the response to multiple abiotic and biotic stresses, especially cold stress. However, the bHLH gene family in sweet osmanthus has not yet been analyzed. In this study, 206 OfbHLH genes, which were classified into 25 subfamilies, were successfully identified from recently released whole-genome sequence data. Segmental duplication events had contributed mainly to the expansion of the OfbHLH gene family and all these duplicated gene pairs had experienced purifying selection. The transcriptome data exhibited different expression levels of OfbHLH genes in various tissues. Quantitative real-time PCR results indicated that the expression patterns of most IIIb, III(d + e), IVc, IX, and XII subfamilies could be up- or down-regulated under cold stress, and, among them, IIIb, III(d + e), and part of the XII subfamily participated in the response to multiple abiotic stresses. Six candidate OfbHLH genes (OfbHLH120/128/133/163/164/191), which were significantly up- or down-regulated during the cold stress process, were identified. Further studies indicated that OfbHLH120/128/163 had transcriptional activation activity and were located in the nucleus, implying that these genes are involved in the regulation of expression of cold tolerance genes. This study provides a solid foundation for the functional analysis of the OfbHLH gene family members involved in cold stress response and could be useful in the breeding of sweet osmanthus with improved cold tolerance.

Osmanthus fragrans is widely grown for the purpose of urban greening and the pleasant aroma emitted from its flowers. The floral scent is determined by several monoterpenoid volatiles, such as linalool and its oxides, which are a few of the most common volatiles and the main components of the essential oils in most sweet osmanthus cultivars. In addition, the relative contents of cis- and trans-linalool oxide (furan) may affect the aromas and quality of the essential oils. MYB proteins represent the largest family of transcription factors in plants and participate in regulating secondary metabolites. Several cis-elements, especially AC-rich regions, are known to be bound by 2R-MYBs and could be found in the promoter of the enzyme genes in the terpenoid metabolic pathway. However, there has to date been no investigation into the 2R-MYB family genes involved in regulating terpenoid biosynthesis in O. fragrans. Here, 243 non-redundant 2R-MYB proteins were grouped into 33 clusters based on the phylogeny and exon-intron distribution. These genes were unevenly distributed on 23 chromosomes. Ka/Ks analysis showed that the major mode of 2R-MYB gene evolution was purifying selection. Expression analysis indicated that 2R-MYB genes in O. fragrans exhibited varied expression patterns. A total of 35 OfMYBs representing the highest per kilobase per million mapped reads in the flower were selected for quantitative real-time PCR analysis. The correlation analysis between the expression level and the contents of fragrant compounds at different flowering stages suggested that OfMYB19/20 exhibited remarkably positive correlation with the accumulation of cis-linalool oxides. OfMYB51/65/88/121/137/144 showed significantly negative correlations with one or more linalool oxides. Characterization of these proteins revealed that OfMYB19 and OfMYB137 were localized in the nuclei, but did not show transcriptional activation in the yeast system, which suggested that they may be bound to other transcription factors to exert regulatory functions. These findings provide useful information for further functional investigation of the 2R-MYBs and offer a foundation for clarifying the 2R-MYB transcription factors involved in the molecular mechanism of the regulation of monoterpenoid biosynthesis in Osmanthus fragrans.

Osmanthus fragrans, or &ldquo, RiXiangGui&rdquo, 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.

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