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Iridoid compounds have been reported to be accumulated in the dried fruits of Gardenia jasminoides (Rubiaceae), which are used in traditional Chinese medicine. These compounds exhibit obvious pharmacological activities, including effective protective effects on the liver and therapeutic efficacy for cardiovascular diseases. However, the biosynthetic pathway of iridoid remains uninvestigated. In this study, 12 transcriptomes from four tissues (lower leaves, top leaves, flowers, and fruits) were analyzed to characterize the accumulation of active constituents and to identify the corresponding genes involved in iridoid biosynthesis. Almost all genes in the mevalonate (MVA) pathway and the 2-C-methyl- d -erythritol-4-phosphate (MEP) pathway were found in our transcriptome database; these pathways comprise upstream pathways of iridoid compounds (monoterpene derivatives), most of which are abundant in flowers and fruits. Geraniol synthase (GES), which is involved in the first branch step in the iridoid biosynthetic pathway, was cloned, and quantitative real-time PCR (qRT-PCR) revealed that GjGES showed a similar expression pattern to that in the transcriptome in terms of reads per kilobase per million (RPKM) mapped reads. The recombinant GjGES protein efficiently converted geranyl diphosphate (GPP) to geraniol. Subsequently, the metabolic profiling of seven iridoids and iridoid glycosides, namely geniposide, gardenoside, genipin, geniposidic acid, genipin-1-β-gentiobioside, shanzhiside, and shanzhiside methyl ester, revealed that these compounds were highly accumulated in fruits. Consequently, the metabolites and speculated unigenes involved in iridoid metabolic pathways enabled the identification of glucosyltransferase, hydroxylase, and O-methyltransferase candidate genes responsible for the biosynthesis of iridoid and iridoid glycosides. The results of the present study can serve as a reference for the functional characterization of enzyme-coding genes and are beneficial for the engineering of biosynthetic pathways of iridoids and iridoid glycosides in the future.

Chrysanthemum indicum has long been used in traditional Chinese medicine for its health-promoting benefits. Studies on C. indicum have mainly focused on the flowers. Terpenoid distribution in various parts of the plant and characterization of terpene synthases remain unclear. In this study, volatile metabolic profiling was performed to compare the composition and quantity of terpenoids distributed in the root, stem, leaf, flower bud and flower of C. indicum. The potential for extracting active ingredients from the root, stem, and leaf was also examined. In total, 17 monoterpenoids and 27 sesquiterpenoids were identified. Transcriptome data were used to clone two monoterpene synthases and two sesquiterpene synthases highly expressed in the root. The recombinant proteins of full-length and truncated versions of C. indicum terpene synthase (CiTPS1) produced α-pinene, but the truncated one was catalytically more efficient than the full-length version. No product could be detected when full-length version of CiTPS2 was used for catalyzing GPP, but the truncated one can produce a minor amount of α-pinene. CiTPS3 contributed to the production of three sesquiterpenoids, namely β-farnesene, petasitene, and α-bisabolene. CiTPS4 acted as a difunctional enzyme, contributing to the production of four monoterpenoids and three sesquiterpenoids, including petasitene. The evidence suggests that petasitene and the genes responsible for its biosynthesis were first found in the genus Chrysanthemum. The present findings provide insights into the composition, formation, and regulation of these bioactive compounds.

The JASMONATE ZIM DOMAIN (JAZ) proteins act as negative regulators in the jasmonic acid (JA) signaling pathways of plants, and these proteins have been reported to play key roles in plant secondary metabolism mediated by JA. In this study, we firstly isolated one JAZ from P. cablin, PatJAZ6, which was characterized and revealed based on multiple alignments and a phylogenic tree analysis. The result of subcellular localization indicated that the PatJAZ6 protein was located in the nucleus of plant protoplasts. The expression level of PatJAZ6 was significantly induced by the methyl jasmonate (MeJA). Furthermore, by means of yeast two-hybrid screening, we identified two transcription factors that interact with the PatJAZ6, the PatMYC2b1 and PatMYC2b2. Virus-induced gene silencing (VIGS) of PatJAZ6 caused a decrease in expression abundance, resulting in a significant increase in the accumulation of patchouli alcohol. Moreover, we overexpressed PatJAZ6 in P. cablin, which down-regulated the patchoulol synthase expression, and then suppressed the biosynthesis of patchouli alcohol. The results demonstrate that PatJAZ6 probably acts as a repressor in the regulation of patchouli alcohol biosynthesis, contributed to a model proposed for the potential JA signaling pathway in P. cablin.

Patchoulol, the major active ingredient in Pogostemon cablin (Blanco) Benth., has considerable clinical and industrial value. A number of genes that participate in the patchoulol biosynthesis pathway have been identified; however, little is known about the transcription factors involved in regulating patchoulol synthesis. In this study, PatSWC4, a homologous protein of Arabidopsis SWC4 belonging to the MYB (v-myb avian myeloblastosis viral oncogene homolog)-related transcription factor family, was identified and characterized. Subcellular localization showed that PatSWC4 protein localized in the nucleus. The highest expression level of PatSWC4 gene was found in old leaves of P. cablin and it was significantly induced by methyl jasmonate (MeJA). Additionally, yeast one-hybrid (Y1H) and dual-luciferase (dual-LUC) assays revealed that PatSWC4 could bind to the promoter of PatPTS gene to increase its transcriptional activity. Yeast two-hybrid (Y2H) assays also confirmed the interaction between PatSWC4 and PatJAZ4 proteins, indicating that PatSWC4 might function in the JA response network. Moreover, transient overexpression of PatSWC4 gene in P. cablin leaves markedly increased the production of patchoulol, and qRT-PCR analysis further revealed that genes in the patchoulol biosynthesis pathway were significantly upregulated at the transcriptional level. Ultimately, we proposed a work model depicting how JA signaling regulates PatPTS via the interaction between PatSWC4 and PatJAZ4, thereby regulating the biosynthesis of patchoulol. Our findings not only help to elucidate the regulatory mechanism governing JA-induced patchoulol biosynthesis, but also lay a foundation for future studies on improving patchoulol production through genetic and metabolic engineering.

Ilex asprella, a plant widely used as a folk herbal drug in southern China, produces and stores a large amount of triterpenoid saponins, most of which are of the α-amyrin type. In this study, two oxidosqualene cyclase (OSC) cDNAs, IaAS1 and IaAS2, were cloned from the I. asprella root. Functional characterisation was performed by heterologous expression in the yeast Saccharomyces cerevisiae. Analysis of the resulting products by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) showed that both genes encode a mixed amyrin synthase, producing α-amyrin and β-amyrin at different ratios. IaAS1, which mainly produces α-amyrin, is the second triterpene synthase so far identified in which the level of α-amyrin produced is ≥80% of total amyrin production. By contrast, IaAS2 mainly synthesises β-amyrin, with a yield of 95%. Gene expression patterns of these two amyrin synthases in roots and leaves of I. asprella were found to be consistent with the content patterns of total saponins. Finally, phylogenetic analysis and multiple sequence alignment of the two amyrin synthases against several known OSCs from other plants were conducted to further elucidate their evolutionary relationship.

Native to Southern China, Ilex pubescens and Ilex asprella are frequently used in traditional Chinese medicine. Both of them produce a large variety of ursane-type triterpenoid saponins, which have been demonstrated to have different pharmacological effects. However, little is known about their biosynthesis. In this study, transcriptomic analysis of I. pubescens and comparison with its closely related specie I. asprella were carried out to identify potential genes involved in triterpenoid saponin biosynthesis. Through RNA sequencing (RNA-seq) and de novo transcriptome assembly of I. pubescens, a total of 68,688 UniGene clusters are obtained, of which 32,184 (46.86%) are successfully annotated by comparison with the sequences in major public databases (NCBI, Swiss-Prot, and KEGG). It includes 128 UniGenes related to triterpenoid backbone biosynthesis, 11 OSCs (oxidosqualene cyclases), 233 CYPs (cytochrome P450), and 269 UGTs (UDP-glycosyltransferases). By homology-based blast and phylogenetic analysis with well-characterized genes involved in triterpenoid saponin biosynthesis, 5 OSCs, 14 CYPs, and 1 UGT are further proposed as the most promising candidate genes. Transcriptomic comparison between two Ilex species using blastp and OrthoMCL method reveals high sequence similarity. All OSCs and UGTs as well as most CYPs are classified as orthologous genes, while only 5 CYPs in I. pubescens and 3 CYPs in I. asprella are species-specific. One of OSC candidates, named as IpAS1, was successfully cloned and expressed in Saccharomyces cerevisiae INVSc1. Analysis of the yeast extract by gas chromatography (GC) and gas chromatography–mass spectrometry (GC-MS) shows IpAS1 is a mixed amyrin synthase, producing α-amyrin and β-amyrin at ratio of 5:1, which is similar to its ortholog IaAS1 from I. asprella. This study is the first exploration to profile the transcriptome of I. pubescens, the generated data and gene models will facilitate further molecular studies on the physiology and metabolism in this plant. By comparative transcriptomic analysis, a series of candidate genes involved in the biosynthetic pathway of triterpenoid saponins are identified, providing new insight into their biosynthesis at transcriptome level.

Ilex asprella, which contains abundant α-amyrin type triterpenoid saponins, is an anti-influenza herbal drug widely used in south China. In this work, we first analysed the transcriptome of the I. asprella root using RNA-Seq, which provided a dataset for functional gene mining. mRNA was isolated from the total RNA of the I. asprella root and reverse-transcribed into cDNA. Then, the cDNA library was sequenced using an Illumina HiSeq™ 2000, which generated 55,028,452 clean reads. De novo assembly of these reads generated 51,865 unigenes, in which 39,269 unigenes were annotated (75.71% yield). According to the structures of the triterpenoid saponins of I. asprella, a putative biosynthetic pathway downstream of 2,3-oxidosqualene was proposed and candidate unigenes in the transcriptome data that were potentially involved in the pathway were screened using homology-based BLAST and phylogenetic analysis. Further amplification and functional analysis of these putative unigenes will provide insight into the biosynthesis of Ilex triterpenoid saponins.

Exogenous hormone application is an effective and feasible method to regulate production of economic plant and synthesis of high-value natural medical products. Jasmonate and ethylene are two crucial plant hormones regulate growth and development, secondary metabolism, and stress responses in Pogostemon cablin. However, the regulatory mechanism of these phytohormones in this important medicinal plant has not been elucidated to date. In the present study, alterations of secondary metabolites and photosynthesis of P. cablin leaves treated with exogenous methyl jasmonate (MeJA), ethrel (ETH), and simultaneously with both MeJA and ETH, respectively, were explored. To uncover the potential protein functional network involved in the hormone-induction mechanism, we performed a proteomic analysis of differently treated leaves using isobaric tags for relative and absolute quantification (iTRAQ). In P. cablin leaves before and after three different treatments, 254, 229 and 400 differentially expressed proteins (DEPs) involving a wide range of physiological processes were clearly identified respectively. The quantitative data were further validated by parallel reaction monitoring (PRM) and qRT-PCR. Functional enrichment analysis indicated photosynthesis-related proteins showed a significant increase expression in P. cablin leaves after MeJA treatment, while proteins involved in secondary metabolite biosynthesis and thiamine metabolism were significantly down-regulated. Exogenous ETH mainly repressed the expression of proteins related to carbohydrate and energy metabolism in P. cablin leaves while markedly raised protein abundance in photosynthesis and protein processing in the endoplasmic reticulum. Regarding P. cablin leaves treated with both MeJA and ETH, expression of proteins related to carbohydrate and energy metabolism, secondary metabolite biosynthesis, tyrosine metabolism, thiamine metabolism and lipid metabolism were significantly down-regulated, while that of proteins involved in photosynthesis, phenylpropanoid biosynthesis and nitrogen metabolism were considerably up-regulated. This is the first study using iTRAQ-based proteomics approach to comprehensively elucidate exogenous hormone response of P. cablin, providing important pathways/proteins involved in secondary metabolism regulation, which will contribute to potential genetic improvement of medical constituents.

Amomum villosum Lour. (officinal name Fructus Amomi) is one of the most well-known and authentic herbs in South China. Monoterpenes in its volatile oil are the main medicinal compounds. The condensation of pyruvate with D-glyceraldhyde-3-phosphate to form 1-deoxy-D-xylulose-5-phosphate (DXP) catalyzed by DXP synthase (DXS, EC: 2.2.1.7) and the conversion of DXP to 2-C-methyl-D-erythritol-4-phosphate (MEP), catalyzed by DXP reductoisomerase (DXR, EC:1.1.1.267), are the initial steps of the MEP pathway for isoprenoid biosynthesis. A DXR gene, AvDXR (GenBank accession no. FJ459894), and a DXS gene, AvDXS (FJ455512), were isolated from the leaves of Amomum villosum. The 1749-bp full-length cDNA of AvDXR contained a 1416-bp open reading frame (ORF) encoding a peptide of 472 amino acids, and the 2347-bp full-length cDNA of AvDXS contained a 2148-bp ORF encoding a peptide of 715 amino acids. The deduced amino acid sequences of the AvDXR and AvDXS proteins shared high homology with DXRs and DXSs from other plant species, respectively, and the AvDXS belonged to class 1 plant DXS. Chloroplast transit peptides were found in the N-terminal region of AvDXR and AvDXS. A proline-rich region, two highly conserved NADPH-binding domains and two substrate-binding domains consistent with the enzymatic functions of DXR were found in AvDXR. AvDXS contained a TPP-binding domain and a DRAG domain related to the catalysis of DXS. The functional color assay in Escherichia coli with pAC-BETA implied that AvDXR and AvDXS encoded functional proteins that manipulate the biosynthesis of isoprenoid precursors. Both AvDXR and AvDXS were expressed extensively in the leaves, stems, roots, pericarps and seeds of A. villosum. AvDXS expression was similar in all tissues investigated, whereas higher levels of AvDXR were observed in the fruits, the main part for the accumulation of volatile oil in A. villosum, which suggested that AvDXR might play a more influential role in monoterpene precursor biosynthesis than AvDXS. The role of AvDXS in other isoprenoid biosynthesis, especially the primary isoprenoid biosynthesis remained to be investigated.