Background Dextrorotatory borneol (D-borneol), a cyclic monoterpene, is widely used in traditional Chinese medicine as an efficient topical analgesic drug. Fresh leaves of Cinnamomum trees, e.g., C. burmannii and C. camphor, are the main sources from which D-borneol is extracted by steam distillation, yet with low yields. Insufficient supply of D-borneol has hampered its clinical use and production of patent remedies for a long time. Biological synthesis of D-borneol offers an additional approach; however, mechanisms of D-borneol biosynthesis remain mostly unresolved. Hence, it is important and necessary to elucidate the biosynthetic pathway of D-borneol. Results Comparative analysis on the gene expression patterns of different D-borneol production C. burmannii samples facilitates elucidation on the underlying biosynthetic pathway of D-borneol. Herein, we collected three different chemotypes of C. burmannii, which harbor different contents of D-borneol.A total of 100,218 unigenes with an N50 of 1,128 bp were assembled de novo using Trinity from a total of 21.21 Gb clean bases. We used BLASTx analysis against several public databases to annotate 45,485 unigenes (45.38%) to at least one database, among which 82 unigenes were assigned to terpenoid biosynthesis pathways by KEGG annotation. In addition, we defined 8,860 unigenes as differentially expressed genes (DEGs), among which 13 DEGs were associated with terpenoid biosynthesis pathways. One 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and two monoterpene synthase, designated as CbDXS9, CbTPS2 and CbTPS3, were up-regulated in the high-borneol group compared to the low-borneol and borneol-free groups, and might be vital to biosynthesis of D-borneol in C. burmannii. In addition, we identified one WRKY, two BHLH, one AP2/ERF and three MYB candidate genes, which exhibited the same expression patterns as CbTPS2 and CbTPS3, suggesting that these transcription factors might potentially regulate D-borneol biosynthesis. Finally, quantitative real-time PCR was conducted to detect the actual expression level of those candidate genes related to the D-borneol biosynthesis pathway, and the result showed that the expression patterns of the candidate genes related to D-borneol biosynthesis were basically consistent with those revealed by transcriptome analysis. Conclusions We used transcriptome sequencing to analyze three different chemotypes of C. burmannii, identifying three candidate structural genes (one DXS, two monoterpene synthases) and seven potential transcription factor candidates (one WRKY, two BHLH, one AP2/ERF and three MYB) involved in D-borneol biosynthesis. These results provide new insight into our understanding of the production and accumulation of D-borneol in C. burmannii.

Background The large genus Ficus comprises approximately 800 species, most of which possess high ornamental and ecological values. However, its evolutionary history remains largely unknown. Plastome (chloroplast genome) analysis had become an essential tool for species identification and for unveiling evolutionary relationships between species, genus and other rank groups. In this work we present the plastomes of ten Ficus species. Results The complete chloroplast (CP) genomes of eleven Ficus specimens belonging to ten species were determined and analysed. The full length of the Ficus plastome was nearly 160 kbp with a similar overall GC content, ranging from 35.88 to 36.02%. A total of 114 unique genes, distributed in 80 protein-coding genes, 30 tRNAs, and 4 rRNAs, were annotated in each of the Ficus CP genome. In addition, these CP genomes showed variation in their inverted repeat regions (IR). Tandem repeats and mononucleotide simple sequence repeat (SSR) are widely distributed across the Ficus CP genome. Comparative genome analysis showed low sequence variability. In addition, eight variable regions to be used as potential molecular markers were proposed for future Ficus species identification. According to the phylogenetic analysis, these ten Ficus species were clustered together and further divided into three clades based on different subgenera. Simultaneously, it also showed the relatedness between Ficus and Morus. Conclusion The chloroplast genome structure of 10 Ficus species was similar to that of other angiosperms, with a typical four-part structure. Chloroplast genome sizes vary slightly due to expansion and contraction of the IR region. And the variation of noncoding regions of the chloroplast genome is larger than that of coding regions. Phylogenetic analysis showed that these eleven sampled CP genomes were divided into three clades, clustered with species from subgenus Urostigma, Sycomorus, and Ficus, respectively. These results support the Berg classification system, in which the subgenus Ficus was further decomposed into the subgenus Sycomorus. In general, the sequencing and analysis of Ficus plastomes, especially the ones of species with no or limited sequences available yet, contribute to the study of genetic diversity and species evolution of Ficus, while providing useful information for taxonomic and phylogenetic studies of Ficus.

Trans-isopentenyl diphosphate synthases (TIDSs) genes are known to be important determinants for terpene diversity and the accumulation of terpenoids. The essential oil of Cinnamomum camphora, which is rich in monoterpenes, sesquiterpenes, and other aromatic compounds, has a wide range of pharmacological activities and has therefore attracted considerable interest. However, the TIDS gene family, and its relationship to the camphor tree (C. camphora L. Presl.), has not yet been characterized. In this study, we identified 10 TIDS genes in the genome of the C. camphora borneol chemotype that were unevenly distributed on chromosomes. Synteny analysis revealed that the TIDS gene family in this species likely expanded through segmental duplication events. Furthermore, cis-element analyses demonstrated that C. camphora TIDS (CcTIDS) genes can respond to multiple abiotic stresses. Finally, functional characterization of eight putative short-chain TIDS proteins revealed that CcTIDS3 and CcTIDS9 exhibit farnesyl diphosphate synthase (FPPS) activity, while CcTIDS1 and CcTIDS2 encode geranylgeranyl diphosphate synthases (GGPPS). Although, CcTIDS8 and CcTIDS10 were found to be catalytically inactive alone, they were able to bind to each other to form a heterodimeric functional geranyl diphosphate synthase (GPPS) in vitro, and this interaction was confirmed using a yeast two-hybrid assay. Furthermore, transcriptome analysis revealed that the CcTIDS3, CcTIDS8, CcTIDS9, and CcTIDS10 genes were found to be more active in C. camphora roots as compared to stems and leaves, which were verified by quantitative real-time PCR (qRT-PCR). These novel results provide a foundation for further exploration of the role of the TIDS gene family in camphor trees, and also provide a potential mechanism by which the production of camphor tree essential oil could be increased for pharmacological purposes through metabolic engineering.

Background The borneol chemotype of Cinnamomum camphora (BCC), a monoterpene-rich woody plant species, is the sole source prescribed by the Chinese Pharmacopoeia for the production of natural D-borneol, a major monoterpene in BCC used for millennia as a topical analgesic in China. Nevertheless, the possible gene-regulatory roles of transcription factors (TFs) in BCC’s monoterpenoid biosynthesis remained unknown. Here, a joint analysis of the transcriptome and terpenoid metabolome of BCC induced by mechanical damage (MD) was used to comprehensively explore the interaction between TFs and terpene synthase (TPS) unigenes that might participate in monoterpene biosynthesis in BCC. Results Gas chromatography–mass spectrometry analysis detected 14 monoterpenes and seven sesquiterpenes. All but two monoterpenes underwent a significantly increased accumulation after the MD treatment. RNA sequencing data revealed that 10 TPS, 82 MYB, 70 AP2/ERF, 38 BHLH, 31 WRKY, and 29 bZIP unigenes responded to the MD treatment. A correlation analysis revealed that three monoterpene synthase genes (CcTPS1, CcTPS3, CcTPS4) highly correlated with multiple monoterpenes, namely D-borneol, camphor, and bornyl acetate, which could be responsible for monoterpenoid biosynthesis in BCC. Furthermore, five WRKY, 15 MYB, 10 ERF/AP2, five bZIP, and two BHLH genes had strong, positive correlations with CcTPS1 or CcTPS4, judging by their high coefficient values (R2 > 0.8). The bioinformatics results were verified by quantitative real-time PCR. Conclusion This study provides insight into the genes involved in the biosynthesis and regulation of monoterpene in BCC and thus provides a pool of candidate genes for future mechanistic analyses of how monoterpenes accumulate in BCC.

Ardisia Sw. (Primulaceae) is naturally distributed in tropical and subtropical areas. Most of them possess edible and medicinal values and are popular in clinical and daily use in China. However, ambiguous species delineation and genetic information limit the development and utilization of this genus. In this study, the chloroplast genomes of four Ardisia species, namely A. gigantifolia Stapf, A. crenata Sims, A. villosa Roxb. and A. mamillata Hance, were sequenced, annotated, and analyzed comparatively. All the four chloroplast genomes possess a typical quadripartite structure, and each of the genomes is about 156 Kb in size. The structure and gene content of the Ardisia plastomes were conservative and showed low sequence divergence. Furthermore, we identified five mutation hotspots as candidate DNA barcodes for Ardisia, namely, trnT-psbD, ndhF-rpl32, rpl32-ccsA, ccsA-ndhD and ycf1. Phylogenetic analysis based on the whole-chloroplast genomes data showed that Ardisia was sister to Tapeinosperma Hook. f. In addition, the results revealed a great topological profile of Ardisia’s with strong support values, which matches their geographical distribution patterns. Summarily, our results provide useful information for investigations on taxonomic differences, molecular identification, and phylogenetic relationships of Ardisia plants.