Identification and characterization of two O-methyltransferases involved in biosynthesis of methylated 2-(2-phenethyl)chromones in agarwood.

The 2-(2-phenethyl)chromones (PECs) are the signature constituents responsible for the fragrance and pharmacological properties of agarwood. O-Methyltransferases (OMTs) are necessary for the biosynthesis of methylated PECs, but there is little known about OMTs in Aquilaria sinensis. In this study, we identified 29 OMT genes from the A. sinensis genome. Expression analysis showed they were differentially expressed in different tissues and responded to drill wounding. Comprehensive analysis of the gene expression and methylated PEC content revealed that AsOMT2, AsOMT8, AsOMT11, AsOMT16, and AsOMT28 could potentially be involved in methylated PECs biosynthesis. In vitro enzyme assays and functional analysis in Nicotiana benthamiana demonstrated that AsOMT11 and AsOMT16 could methylate 6-hydroxy-2-(2-phenylethyl)chromone to form 6-methoxy-2-(2-phenylethyl)chromone. A transient overexpression experiment in the variety 'Qi-Nan' revealed that AsOMT11 and AsOMT16 could significantly promote the accumulation of three major methylated PECs. Our results provide candidate genes for the mass production of methylated PECs using synthetic biology.

Pleistocene glaciation advances the cryptic speciation of Stellera chamaejasme L. in a major biodiversity hotspot.

The mountains of Southwest China comprise a significant large mountain range and biodiversity hotspot imperiled by global climate change. The high species diversity in this mountain system has long been attributed to a complex set of factors, and recent large-scale macroevolutionary investigations have placed a broad timeline on plant diversification that stretches from 10 million years ago (Mya) to the present. Despite our increasing understanding of the temporal mode of speciation, finer-scale population-level investigations are lacking to better refine these temporal trends and illuminate the abiotic and biotic influences of cryptic speciation. This is largely due to the dearth of organismal sampling among closely related species and populations, spanning the incredible size and topological heterogeneity of this region. Our study dives into these evolutionary dynamics of speciation using genomic and eco-morphological data of Stellera chamaejasme L. We identified four previously unrecognized cryptic species having indistinct morphological traits and large metapopulation of evolving lineages, suggesting a more recent diversification (~2.67-0.90 Mya), largely influenced by Pleistocene glaciation and biotic factors. These factors likely influenced allopatric speciation and advocated cyclical warming-cooling episodes along elevational gradients during the Pleistocene. The study refines the evolutionary timeline to be much younger than previously implicated and raises the concern that projected future warming may influence the alpine species diversity, necessitating increased conservation efforts.

Conservation Strategies for Aquilaria sinensis: Insights from DNA Barcoding and ISSR Markers.

The evergreen tree species Aquilaria sinensis holds significant economic importance due to its specific medicinal values and increasing market demand. However, the unrestricted illegal exploitation of its wild population poses a threat to its survival. This study aims to contribute to the conservation efforts of A. sinensis by constructing a library database of DNA barcodes, including two chloroplast genes (psbA-trnH and matK) and two nuclear genes (ITS and ITS2). Additionally, the genetic diversity and structure were estimated using inter-simple sequence repeats (ISSR) markers. Four barcodes of 57 collections gained 194 sequences, and 1371 polymorphic bands (98.63%) were observed using DNA ISSR fingerprinting. The Nei's gene diversity (H) of A. sinensis at the species level is 0.2132, while the Shannon information index (I) is 0.3128. The analysis of molecular variance revealed a large significant proportion of total genetic variations and differentiation among populations (Gst = 0.4219), despite a relatively gene flow (Nm = 0.6853) among populations, which were divided into two groups by cluster analysis. There was a close genetic relationship among populations with distances of 0.0845 to 0.5555. This study provides evidence of the efficacy and dependability of establishing a DNA barcode database and using ISSR markers to assess the extent of genetic diversity A. sinensis. Preserving the genetic resources through the conservation of existing populations offers a valuable proposition. The effective utilization of these resources will be further deliberated in subsequent breeding endeavors, with the potential to breed agarwood commercial lines.

[Functional characterization of ent-kaurane-type diterpenoid synthases from Stellera chamaejasme].

Sequential catalysis by ent-copalyl diphosphate(CPS) and ent-kaurene synthase(KS) is a critical step for plants to initiate the biosynthesis of gibberellin with geranylgeranyl pyrophosphate(GGPP) as the substrate. This study mined the transcriptome data of Stellera chamaejasme and cloned two key diterpene synthase genes, SchCPS and SchKS, involved in the gibberellin pathway. The two genes had the complete open reading frames of 2 595 bp and 1 701 bp, encoding two hydrophilic proteins composed of 864 and 566 amino acid residues and with the relative molecular mass of 97.9 kDa and 64.6 kDa and the theoretical isoelectric points of 5.61 and 6.12, respectively. Sequence comparison and phylogenetic tree showed that SchCPS contained LHS, PNV, and DxDD motifs conserved in the CPS family and was categorized in the TPS-c subfamily, while SchKS contained DDxxD, NSE/DTE and PIx motifs conserved in the KS family and was categorized in the TPS-e subfamily. Functional validation showed that SchCPS catalyzed the protonation and cyclization of GGPP to ent-CPP, while SchKS acted on ent-CPP dephosphorylation and re-cyclization to ent-kaurene. In this study, the full-length sequences of SchCPS and SchKS were cloned and functionally verified for the first time, which not only enriched the existing CPS and KS gene libraries but also laid a foundation for the cloning and biosynthesis pathway analysis of more genes involved in the synthesis of active components in S. chamaejasme.

Aquilariperoxide A, a Sesquiterpene Dimer from Agarwood of Aquilaria sinensis with Dual Antitumor and Antimalarial Effects.

Aquilariperoxide A (1), an unprecedented sesquiterpene dimer characterized by a dioxepane ring connecting two sesquiterpene units via a C-C bond, was isolated from agarwood of Aquilaria sinensis-containing resins. The structure was elucidated by spectroscopic and computational methods. A bioassay revealed that 1 significantly inhibits cell proliferation and migration in human cancer cells. The mechanism of 1 against cancer cells was briefly discussed by analysis of RNA sequence data and epithelial-mesenchymal transition. Besides, the antimalarial activity of 1 was also evaluated.

Comprehensive Analysis of NAC Transcription Factors Reveals Their Evolution in Malvales and Functional Characterization of AsNAC019 and AsNAC098 in Aquilaria sinensis.

NAC is a class of plant-specific transcription factors that are widely involved in the growth, development and (a)biotic stress response of plants. However, their molecular evolution has not been extensively studied in Malvales, especially in Aquilaria sinensis, a commercial and horticultural crop that produces an aromatic resin named agarwood. In this study, 1502 members of the NAC gene family were identified from the genomes of nine species from Malvales and three model plants. The macroevolutionary analysis revealed that whole genome duplication (WGD) and dispersed duplication (DSD) have shaped the current architectural structure of NAC gene families in Malvales plants. Then, 111 NAC genes were systemically characterized in A. sinensis. The phylogenetic analysis suggests that NAC genes in A. sinensis can be classified into 16 known clusters and four new subfamilies, with each subfamily presenting similar gene structures and conserved motifs. RNA-seq analysis showed that AsNACs presents a broad transcriptional response to the agarwood inducer. The expression patterns of 15 AsNACs in A. sinensis after injury treatment indicated that AsNAC019 and AsNAC098 were positively correlated with the expression patterns of four polyketide synthase (PKS) genes. Additionally, AsNAC019 and AsNAC098 were also found to bind with the AsPKS07 promoter and activate its transcription. This comprehensive analysis provides valuable insights into the molecular evolution of the NAC gene family in Malvales plants and highlights the potential mechanisms of AsNACs for regulating secondary metabolite biosynthesis in A. sinensis, especially for the biosynthesis of 2-(2-phenyl) chromones in agarwood.

Revealing the Roles of the JAZ Family in Defense Signaling and the Agarwood Formation Process in Aquilaria sinensis.

Jasmonate ZIM-domain family proteins (JAZs) are repressors in the signaling cascades triggered by jasmonates (JAs). It has been proposed that JAs play essential roles in the sesquiterpene induction and agarwood formation processes in Aquilaria sinensis. However, the specific roles of JAZs in A. sinensis remain elusive. This study employed various methods, including phylogenetic analysis, real-time quantitative PCR, transcriptomic sequencing, yeast two-hybrid assay, and pull-down assay, to characterize A. sinensis JAZ family members and explore their correlations with WRKY transcription factors. The bioinformatic analysis revealed twelve putative AsJAZ proteins in five groups and sixty-four putative AsWRKY transcription factors in three groups. The AsJAZ and AsWRKY genes exhibited various tissue-specific or hormone-induced expression patterns. Some AsJAZ and AsWRKY genes were highly expressed in agarwood or significantly induced by methyl jasmonate in suspension cells. Potential relationships were proposed between AsJAZ4 and several AsWRKY transcription factors. The interaction between AsJAZ4 and AsWRKY75n was confirmed by yeast two-hybrid and pull-down assays. This study characterized the JAZ family members in A. sinensis and proposed a model of the function of the AsJAZ4/WRKY75n complex. This will advance our understanding of the roles of the AsJAZ proteins and their regulatory pathways.

[Characterization and phylogenetic analysis of complete chloroplast genome of cultivated Qinan agarwood].

"Tangjie" leaves of cultivated Qinan agarwood were used to obtain the complete chloroplast genome using high-throughput sequencing technology. Combined with 12 chloroplast genomes of Aquilaria species downloaded from NCBI, bioinformatics method was employed to determine the chloroplast genome characteristics and phylogenetic relationships. The results showed that the chloroplast genome sequence length of cultivated Qinan agarwood "Tangjie" leaves was 174 909 bp with a GC content of 36.7%. A total of 136 genes were annotated, including 90 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. Sequence repeat analysis detected 80 simple sequence repeats(SSRs) and 124 long sequence repeats, with most SSRs composed of A and T bases. Codon preference analysis revealed that AUU was the most frequently used codon, and codons with A and U endings were preferred. Comparative analysis of Aquilaria chloroplast genomes showed relative conservation of the IR region boundaries and identified five highly variable regions: trnD-trnY, trnT-trnL, trnF-ndhJ, petA-cemA, and rpl32, which could serve as potential DNA barcodes specific to the Aquilaria genus. Selection pressure analysis indicated positive selection in the rbcL, rps11, and rpl32 genes. Phylogenetic analysis revealed that cultivated Qinan agarwood "Tangjie" and Aquilaria agallocha clustered together(100% support), supporting the Chinese origin of Qinan agarwood from Aquilaria agallocha. The chloroplast genome data obtained in this study provide a foundation for studying the genetic diversity of cultivated Qinan agarwood and molecular identification of the Aquilaria genus.

Comparative transcriptome analysis reveals different defence responses during the early stage of wounding stress in Chi-Nan germplasm and ordinary Aquilaria sinensis.

BACKGROUND: Agarwood is a valuable Chinese medicinal herb and spice that is produced from wounded Aquilaria spp., is widely used in Southeast Asia and is highly traded on the market. The lack of highly responsive Aquilaria lines has seriously restricted agarwood yield and the development of its industry. In this article, a comparative transcriptome analysis was carried out between ordinary A. sinensis and Chi-Nan germplasm, which is a kind of A. sinensis tree with high agarwood-producing capacity in response to wounding stress, to elucidate the molecular mechanism underlying wounding stress in different A. sinensis germplasm resources and to help identify and breed high agarwood-producing strains. RESULTS: A total of 2427 and 1153 differentially expressed genes (DEGs) were detected in wounded ordinary A. sinensis and Chi-Nan germplasm compared with the control groups, respectively. KEGG enrichment analysis revealed that genes participating in starch metabolism, secondary metabolism and plant hormone signal transduction might play major roles in the early regulation of wound stress. 86 DEGs related to oxygen metabolism, JA pathway and sesquiterpene biosynthesis were identified. The majority of the expression of these genes was differentially induced between two germplasm resources under wounding stress. 13 candidate genes related to defence and sesquiterpene biosynthesis were obtained by WGCNA. Furthermore, the expression pattern of genes were verified by qRT-PCR. The candidate genes expression levels were higher in Chi-Nan germplasm than that in ordinary A. sinensis during early stage of wounding stress, which may play important roles in regulating high agarwood-producing capacity in Chi-Nan germplasm. CONCLUSIONS: Compared with A. sinensis, Chi-Nan germplasm invoked different biological processes in response to wounding stress. The genes related to defence signals and sesquiterepene biosynthesis pathway were induced to expression differentially between two germplasm resources. A total of 13 candidate genes were identified, which may correlate with high agarwood-producting capacity in Chi-Nan germplasm during the early stage of wounding stress. These genes will contribute to the development of functional molecular markers and the rapid breeding highly of responsive Aquilaria lines.

Genome-wide investigation and expression analysis of the AP2/ERF family for selection of agarwood-related genes in Aquilaria sinensis (Lour.) Gilg.

Aquilaria sinensis is an important non-timber tree species for producing high-value agarwood, which is widely used as a traditional medicine and incense. Agarwood is the product of Aquilaria trees in response to injury and fungal infection. The APETALA2/ethylene responsive factor (AP2/ERF) transcription factors (TFs) play important roles in plant stress responses and metabolite biosynthesis. In this study, 119 AsAP2/ERF genes were identified from the A. sinensis genome and divided into ERF, AP2, RAV, and Soloist subfamilies. Their conserved motif, gene structure, chromosomal localization, and subcellular localization were characterized. A stress/defense-related ERF-associated amphiphilic repression (EAR) motif and an EDLL motif were identified. Moreover, 11 genes that were highly expressed in the agarwood layer in response to whole-tree agarwood induction technique (Agar-Wit) treatment were chosen, and their expression levels in response to methyl jasmonate (MeJA), salicylic acid (SA), or salt treatment were further analyzed using the quantitative real time PCR (qRT-PCR). Among the 11 genes, eight belonged to subgroup B-3. All 11 genes were significantly upregulated under salt treatment, while eight genes were significantly induced by both MeJA and SA. In addition, the gene clusters containing these upregulated genes on chromosomes were observed. The results obtained from this research not only provide useful information for understanding the functions of AP2/ERF genes in A. sinensis but also identify candidate genes and gene clusters to dissect their regulatory roles in agarwood formation for future research.

A tetraploidization event shaped the Aquilaria sinensis genome and contributed to the ability of sesquiterpenes synthesis.

BACKGROUND: Agarwood, generated from the Aquilaria sinensis, has high economic and medicinal value. Although its genome has been sequenced, the ploidy of A. sinensis paleopolyploid remains unclear. Moreover, the expression changes of genes associated with agarwood formation were not analyzed either. RESULTS: In the present work, we reanalyzed the genome of A. sinensis and found that it experienced a recent tetraploidization event ~ 63-71 million years ago (Mya). The results also demonstrated that the A. sinensis genome had suffered extensive gene deletion or relocation after the tetraploidization event, and exhibited accelerated evolutionary rates. At the same time, an alignment of homologous genes related to different events of polyploidization and speciation were generated as well, which provides an important comparative genomics resource for Thymelaeaceae and related families. Interestingly, the expression changes of genes related to sesquiterpene synthesis in wounded stems of A. sinensis were also observed. Further analysis demonstrated that polyploidization promotes the functional differentiation of the key genes in the sesquiterpene synthesis pathway. CONCLUSIONS: By reanalyzing its genome, we found that the tetraploidization event shaped the A. sinensis genome and contributed to the ability of sesquiterpenes synthesis. We hope that these results will facilitate our understanding of the evolution of A. sinensis and the function of genes involved in agarwood formation.

WRKY44 represses expression of the wound-induced sesquiterpene biosynthetic gene ASS1 in Aquilaria sinensis.

Agarwood is derived from wounds in Aquilaria trees and is widely used in traditional medicine, incense, and perfume. Sesquiterpenes are one of the main active components in agarwood and are known to be induced by wounding or injury; However, the molecular mechanisms by which wounding leads to sesquiterpene formation remain largely unknown. Agarwood sesquiterpene synthase 1 (ASS1) is one of key enzymes responsible for the biosynthesis of sesquiterpenes and is a crucial jasmonate (JA)-responsive wound-inducible synthase. However, it is not known why ASS1 is not expressed in healthy trees and how its expression is induced as a result of wounding. Here, we report that ASS1 is a wound-induced gene with a promoter in which a 242-bp region (-973 to -731bp) is identified as the core sequence for responding to wound signals. AsWRKY44 binds directly to this region and represses ASS1 promoter activity. Down-regulation or disruption of AsWRKY44 can relieve the inhibition and activate ASS1 expression. In addition, AsWRKY44 is degraded and the expression of ASS1 is significantly up-regulated in response to exogenous application of methyl jasmonate. Thus, AsWRKY44 is a crucial negative regulator of wound-induced ASS1 transcription, and is central to the mechanism of sesquiterpene biosynthesis in agarwood.

[Analysis of bacteria distribution characteristics in different layers of agarwood based on Hiseq sequencing].

To explore the diversity of bacterial community structure between different layers of agarwood, Hiseq(high-throughput sequencing) was used to analyze the bacterial community structure of samples from different layers of agarwood. Our results showed that 1 150 096 optimized sequences and 9 690 OTUs were obtained from 15 samples of 5 layers of agarwood, which belonged to 28 bacterial phyla, 61 classes, 110 orders, 212 families and 384 genera. Further analysis revealed that the normal layer(NL) had the lowest bacterial species richness and the smallest number of OTUs. And the total number of OTUs of the agarwood layer(AL) and NL was zero, which was quite different.At the same time, there were significant differences in bacterial community structure and species diversity between NL and the other four layers. While there were some common dominant bacterial genera in both transition layer(TL) and NL. The similarity of bacterial distribution in 4 non-NL layers was relatively high, which had four common genera, such as Acidibacter, Bradyrhizobium, Acidothemus and Sphingomonas. While Acidibacter, Bradyrhizobium and Acidothemus were the dominant bacterial genus of DA and AL, and all of these layers contained volatile oil. In addition, the Bradyrhizobium was the most abundant in agarwood layer. Our results showed that bacterial community diversity and abundance were decreasing from DL to AL, and different layers showed significant differences in bacterial enrichment. It provided the clues to investigate how bacteria participate in the formation of agarwood.

Genetic, evolutionary and phylogenetic aspects of the plastome of annatto (Bixa orellana L.), the Amazonian commercial species of natural dyes.

MAIN CONCLUSION: The plastome of B. orellana reveals specific evolutionary features, unique RNA editing sites, molecular markers and the position of Bixaceae within Malvales. Annatto (Bixa orellana L.) is a native species of tropical Americas with center of origin in Brazilian Amazonia. Its seeds accumulate the apocarotenoids, bixin and norbixin, which are only found in high content in this species. The seeds of B. orellana are commercially valued by the food industry because its dyes replace synthetic ones from the market due to potential carcinogenic risks. The increasing consumption of B. orellana seeds for dye extraction makes necessary the increase of productivity, which is possible accessing the genetic basis and searching for elite genotypes. The identification and characterization of molecular markers are essential to analyse the genetic diversity of natural populations and to establish suitable strategies for conservation, domestication, germplasm characterization and genetic breeding. Therefore, we sequenced and characterized in detail the plastome of B. orellana. The plastome of B. orellana is a circular DNA molecule of 159,708 bp with a typical quadripartite structure and 112 unique genes. Additionally, a total of 312 SSR loci were identified in the plastome of B. orellana. Moreover, we predicted in 23 genes a total of 57 RNA-editing sites of which 11 are unique for B. orellana. Furthermore, our plastid phylogenomic analyses, using the plastome sequences available in the plastid database belonging to species of order Malvales, indicate a closed relationship between Bixaceae and Malvaceae, which formed a sister group to Thymelaeaceae. Finally, our study provided useful data to be employed in several genetic and biotechnological approaches in B. orellana and related species of the family Bixaceae.

iTRAQ-Based Quantitative Proteomic Analysis of Chemically Induced Aquilaria sinensis Provides Insights into Agarwood Formation Mechanism.

Agarwood is a precious traditional Chinese medicine with a variety of pharmacological effects. Although efforts have been made in elucidating the mechanism of agarwood formation, little progress is obtained till now. Therefore, the molecular mechanism of agarwood formation needs to be further explored using different biological approaches. In this study, the quantitative proteomic analysis using iTRAQ technology combined with transcriptomic and metabolomic analyses on chemically induced Aquilaria sinensis is performed to elucidate the agarwood formation mechanism by formic acid stimulus. Data are available via ProteomeXchange with identifier PXD007586; 1884 proteins are detected, 504 differential proteins that show at least twofold differences in their expression levels are selected based on GO annotations, KEGG, STRING analysis, and quantitative RT-PCR analysis. The results indicate that sesquiterpene synthase, germin-like protein, pathogenesis-related protein, 6-phosphogluconate dehydrogenase, lipoyl synthase, and superoxide dismutase play important roles in the agarwood formation, suggesting that the proteins related to the plant defensive response, the removal of peroxide, the disease-resistance, the biosythesis of glycan, fatty acids, and sesquiterpene are crucial for agarwood formation.

Plastome sequences and exploration of tree-space help to resolve the phylogeny of riceflowers (Thymelaeaceae: Pimelea).

Data sets comprising small numbers of genetic markers are not always able to resolve phylogenetic relationships. This has frequently been the case in molecular systematic studies of plants, with many analyses being based on sequence data from only two or three chloroplast genes. An example of this comes from the riceflowers Pimelea Banks & Sol. ex Gaertn. (Thymelaeaceae), a large genus of flowering plants predominantly distributed in Australia. Despite the considerable morphological variation in the genus, low sequence divergence in chloroplast markers has led to the phylogeny of Pimelea remaining largely uncertain. In this study, we resolve the backbone of the phylogeny of Pimelea in comprehensive Bayesian and maximum-likelihood analyses of plastome sequences from 41 taxa. However, some relationships received only moderate to poor support, and the Pimelea clade contained extremely short internal branches. By using topology-clustering analyses, we demonstrate that conflicting phylogenetic signals can be found across the trees estimated from individual chloroplast protein-coding genes. A relaxed-clock dating analysis reveals that Pimelea arose in the mid-Miocene, with most divergences within the genus occurring during a subsequent rapid diversification. Our new phylogenetic estimate offers better resolution and is more strongly supported than previous estimates, providing a platform for future taxonomic revisions of both Pimelea and the broader subfamily. Our study has demonstrated the substantial improvements in phylogenetic resolution that can be achieved using plastome-scale data sets in plant molecular systematics.

Evolutionary analysis and structural characterization of Aquilaria sinensis sesquiterpene synthase in agarwood formation: A computational study.

Agarwood originating from Aquilaria sinensis contains sesquiterpenoids that have tremendous commercial value in the pharmaceutical and fragrance industries. Aquilaria sinensis sesquiterpene synthase (AsSTS) is the key enzyme in the agarwood biosynthesis pathway, and its activity directly affects the chemical composition of agarwood; however, its role in species evolution remains unclear. In this study, we performed an evolutionary analysis based on 68 plant sesquiterpene synthase (STS) genes and further structural characterization of the gene encoding AsSTS to explore its molecular evolution. The phylogenetic tree indicated that these STS genes included three subfamilies. Additionally, 23 positively selected sites were detected, and no influence of recombination was found. Furthermore, the protein structure of AsSTS was characterized using primary sequence and structural analyses as having a functional active site lid domain, a substrate binding site, two post-translational modification sites and four conserved motifs. Finally, most virtual mutations of positively selected sites could be stabilized against thermal denaturation by a decrease in free energy, and three virtual mutations (D403R, G470Q and S538K) were shown to play important roles in the function and stability of AsSTS. The molecular evolutionary analysis of plant STSs provides essential clues for further experimental site-directed mutagenesis and molecular modification of AsSTS.

Molecular Identification of Three Aquilaria (Thymelaeaceae) Species through DNA Barcoding.

Aquilaria LAM. is an endangered tropical tree that produces agarwood, a common ingredient in medicine, perfumes and incense. The species endemic to China, Aquilaria yunnanensis, is often misidentified as the two valuable species, Aquilaria sinensis and Aquilaria crassna. In present study, three DNA barcodes (internal transcribed spacer (ITS), maturase K gene (matK) and trnL-trnF) were used to evaluate whether these genes can be used to discriminate the three species, and evaluate the phylogenetic relationship between the three Aquilaria species. For accurate identification of the three Aquilaria species, a total of 26 nucleotide variations were detected when comparing the three DNA barcodes. We found that A. sinensis is closely related to A. crassna based on combination of nuclear and chloroplast DNA barcodes, and is closely related to A. yunnanensis based on chloroplast DNA barcodes. Taken together, we suggest that the combination of ITS+matK and ITS+trnL-trnF are suitable for identifying these three Aquilaria species.

Transcription Factor AsMYC2 Controls the Jasmonate-Responsive Expression of ASS1 Regulating Sesquiterpene Biosynthesis in Aquilaria sinensis (Lour.) Gilg.

Sesquiterpenes are one of the most important defensive secondary metabolite components of agarwood. Agarwood, which is a product of the Aquilaria sinensis response to external damage, is a fragrant and resinous wood that is widely used in traditional medicines, incense and perfume. We previously reported that jasmonic acid (JA) plays an important role in promoting agarwood sesquiterpene biosynthesis and induces expression of the sesquiterpene synthase ASS1, which is a key enzyme that is responsible for the biosynthesis of agarwood sesquiterpenes in A. sinensis. However, little is known about this molecular regulation mechanism. Here, we characterized a basic helix-loop-helix transcription factor, AsMYC2, from A. sinensis as an activator of ASS1 expression. AsMYC2 is an immediate-early jasmonate-responsive gene and is co-induced with ASS1. Using a combination of yeast one-hybrid assays and chromatin immunoprecipitation analyses, we showed that AsMYC2 bound the promoter of ASS1 containing a G-box motif. AsMYC2 activated expression of ASS1 in tobacco epidermis cells and up-regulated expression of sesquiterpene synthase genes (TPS21 and TPS11) in Arabidopsis, which was also promoted by methyl jasmonate. Our results suggest that AsMYC2 participates in the regulation of agarwood sesquiterpene biosynthesis in A. sinensis by controlling the expression of ASS1 through the JA signaling pathway.

[Cloning and expression analysis of transcription factor AsMYB1 and AsMYB2 from Aquilaria sinensis].

The MYB gene family comprises one of the richest groups of transcription factors in plants. The full length of two MYB genes were isolated through heterologous screening of Aquilaria sinensis calli transcriptome data, and the reverse transcription PCR was performed to obstain the corrected MYB clones, named AsMYB1, AsMYB2. The MYB transmembrane domain and phylogenetic analysis were predicted by different software to analyze the bioinformatics of MYB proteins. The transcript level of AsMYB1, AsMYB2 was performed by real-time quantitative RT-PCR in different tissues and in responds to abiotic stresses including salt, cold, metal and drought stress, and hormone treatments including abscisic acid (ABA), salicylic acid (SA), gibberellins (GA3) and methyl jasmonate (MeJA) treatment. The AsMYB1 cDNA sequence had an ORF of 1 063 nucleotides, encoding a protein of 353 amino acids. The largest AsMYB2 ORF was 1 081 nucleotides, and its predicted translation products consisted of 359 amino acids. Two MYB genes had a tissues-specific pattern in A. sinensis. Moreover, the expression level of AsMYB1 and AsMYB2 was regulated by different abiotic stresses and hormone treatments, suggesting the transcription factors AsMYB1 and AsMYB2 play an important role in plant defense and hormone signal transduction in A. sinensis.