Metrics details. Many secondary metabolites of agarwood are known to have medicinal value to humans, including compounds that have been shown to elicit sedative effects and exhibit anti-cancer properties. However, little is known about the genome, transcriptome, and the biosynthetic pathways responsible for producing such secondary metabolites in agarwood. In this study, we present a draft genome and a putative pathway for cucurbitacin E and I, compounds with known medicinal value, from in vitro Aquilaria agallocha agarwood. The expression changes for cucurbitacin E and I are shown to be consistent with known responses of A.

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Metrics details. Many secondary metabolites of agarwood are known to have medicinal value to humans, including compounds that have been shown to elicit sedative effects and exhibit anti-cancer properties. However, little is known about the genome, transcriptome, and the biosynthetic pathways responsible for producing such secondary metabolites in agarwood. In this study, we present a draft genome and a putative pathway for cucurbitacin E and I, compounds with known medicinal value, from in vitro Aquilaria agallocha agarwood.

The expression changes for cucurbitacin E and I are shown to be consistent with known responses of A. This study is the first attempt to identify cucurbitacin E and I from in vitro agarwood and the first draft genome for any species of Aquilaria.

The results of this study will aid in future investigations of secondary metabolite pathways in Aquilaria and other non-model medicinal plants. Aquilaria agallocha is one of the largest producers of agarwood, a valuable product derived from Aquilaria and Gyrinops trees. Agarwood-producing tree species have become endangered due to the deforestation of tropical forests and the international trade of agarwood has come under strict control with a listing in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora.

The use of agarwood is prevalent in many cultures, particularly in the Middle East and Asia where it has been used for over a thousand years. In particular, the use of agarwood is prevalent in religious ceremonies, herbal medicine, and as fragrances for perfumes and aromatherapy.

The main compounds related to the medicinal properties of agarwood are terpenes and phenylethyl chromone derivatives [ 1 — 3 ] which can be highly variable in content and composition among different agarwood-producing tree species. Previous studies have focused on sesquiterpenes, the most abundant terpenes compound in agarwood [ 4 , 5 ]. However, little is known about triterpenoids in agarwood.

Terpenoid content is induced under biotic stress as an immune response to resist various pathogens and its derivatives have been shown to exhibit anti-microorganism and anti-tumour functions [ 6 , 7 ]. A specific compound of interest, cucurbitacin, is produced to combat fungal and bacterial pathogens [ 8 ].

Cucurbitacins have previously been isolated in Chinese medicinal herbs and have been shown to have pharmacological effects [ 6 ]. In particular, cucurbitacin I is known to repress cancer cell motility by perturbing actin dynamics and has also been known to exhibit cytotoxicity against MDA-MB human breast cancer cells from animal models and indirectly interrupt actin dynamics [ 9 ]. The study of anti-tumour compounds and related pathways is thus an important field in agarwood research.

In this study, we identified cucurbitacin E and I from in vitro A. In vitro materials were used to perform this study due to the lengthy growth period of resinous material in this species as well as to avoid contamination from microorganisms. Importantly, this process is applicable to plant factories for large-scale production in the future.

We inferred a putative pathway for cucurbitacin E and I from the genomic and transcriptomic data in order to better understand these important medicinal compounds in agarwood. The transcripts discovered to be related to the cucurbitacin pathway, were validated through qRT-PCR. To the best of our knowledge, this is the first draft genome for any species of Aquilaria as well as the first study to identify cucurbitacin E and I in agarwood from in vitro materials.

The strain of A. To the best of our knowledge, the cucurbitacin I content from in vitro A. Agarwood endogenous cucurbitacin E and I content. As mentioned, agarwood produces terpenoids under biotic stress [ 8 ].

To investigate whether pathogens induce cucurbitacin content, the in vitro material was treated with methyl jasmonate MJ , an important signal in pathogen related pathways. Various concentrations of MJ were applied to mimic different levels of biotic stress and the change in cucurbitacin content in response to different levels of biotic stress was measured. These results reveal that cucurbitacin pathway related genes were induced by MJ, indicating that agarwood may produce cucurbitacins and triterpenoids under biotic stress.

Although transcriptome assembly has become commonplace, the majority of transcripts obtained from transcriptome assembly are partial transcripts. Genome assembly may potentially allow for more complete transcript information to be obtained when compared to a de novo transcriptome procedure, thus easing primer design. These factors allow for deeper studies of gene expression mechanisms.

The size of the haploid A. Five DNA libraries were constructed for a total of For the A. This resulted in Transposable elements are generally non-coding DNA sequences that can change its location within a genome and can play an important role in development and evolution [ 13 ].

RNA-seq data was aligned to the repeat-masked genome achieving a mapping rate of Ab initio gene prediction combined with protein alignment resulted in annotations for 40, protein-coding genes, among which 3, genes encoded for multiple isoforms, representing 44, transcripts in total. On average, the predicted gene-models consisted of transcript lengths of 3, A total of The transcriptome sequence data was able to be mapped to the draft genome at a mapping rate of As well, a total of A de novo assembled transcriptome was also aligned to the draft genome, where Functional classification of annotated proteins.

The gene count for various protein classes as determined by Gene Ontology for the set of annotated proteins in A. We compared our gene set to those from a recent study by Xu et al. In their study, 38, transcripts were annotated with an average assembled sequence length of As well, 35, transcripts in our gene set contained both start and stop codons, which is another indicator for completeness.

For gene discovery, assembling a draft genome clearly aids in the number of genes discovered as well as in the completeness of the assembled sequence.

The short reads from RNA-seq data were aligned to the set of annotated transcripts and the gene expression for each treatment condition was quantified, resulting in 4, differentially expressed genes Additional file 2 : Table S1 with at least a two-fold change in expression, of which 2, genes were up-regulated and 2, genes were down-regulated.

After treatment with 0. Functional classification of differentially expressed transcripts. Functional classifications of the set of differentially expressed transcripts through Gene Ontology, separated into up and down modes of regulation. It can be seen that metabolic processes and catalytic activity were enriched with up-regulated genes in the presence of biotic stress. Although the importance of medicinal compounds in A.

Thus, an assay of the cucurbitacin bio-synthetic pathway is currently not available. To investigate the cucurbitacin pathway in A. The annotated transcripts were then used to infer a putative cucurbitacin pathway in A. Cucurbitacin E and I pathways in A.

A schematic illustration showing cucurbitacin E and I bio-synthesis, including isoprenoid precursors via MEP and Mevalonate pathways. These gene expression results are consistent with the knowledge that cucurbitacins are synthesized to resist pathogens [ 8 ]. We also investigated the vascular plant one-zinc-finger transcription factor VOZ , described as follows.

VOZ s are transcription factors that are highly conserved in land plant evolution [ 18 , 19 ]. VOZ s have been observed to be both positive and negative transcription factors of biotic and abiotic stress-response pathways, respectively, in A. Although the voz1voz mutant did not notably change in endogenous ABA content, the voz1voz2 double mutant was inable to combat pathogens Pseudomonas syringae and Colletotrichum higginsianum due to low gene expression from defense-response genes [ 18 ].

Therefore, we speculated that VOZ plays a postive role in the cucurbitacin pathway. A transcript from our gene set homologous to VOZ in A. This provides evidence that VOZ transcription factors are a positive regulator that play a role, directly or indirectly, in the cucurbitacin pathway and biotic stress-response related genes. CYPs are one of the largest gene families in plants and catalyzes most oxidation steps in secondary metabolism such as in the biosynthesis of defense compounds, pigment, and antioxidants [ 21 , 22 ].

Putatively, CYPs may catalyze the conversion of cucurbitadienol. SAM-Mtases may also act on cucurbitadienol by catalyzing methylation, as it is known that many compounds with anti-microorganism functions have cucurbitadienol backbones activated by methylation [ 23 ]. These genes can be considered candidate genes that are possibly involved in the cucurbitacin pathway. As well, we identified a small number of SAM-Mtases that contained the VOZ cis -element, though their expression was not observed to be significantly up-regulated.

Our results are consistent with the expectation of cucurbitacin pathway genes being up-regulated in response to biotic stress. However, this is likely due to differences in platform. The qRT-PCR analysis results showing relative change in expression for each gene involved in the cucurbitacin pathway, as well as the VOZ transcription factor and the internal control AcHistone , after treatment with MJ.

Cucurbitacin, a compound of interest with medicinal value which exhibits anti-microorganism and anti-tumour properties was identified and observed to be abundant in in vitro A. To better understand the genes and pathways related to cucurbitacin, a draft genome was assembled, from which, many genes and protein functions were able to be annotated and analyzed. The expression change for cucurbitacins was shown to be consistent with known responses of A. The VOZ transcription factor, a positive regulator of biotic stress-response pathways was identified as well as a putative regulation target among the genes related to the cucurbitacin pathway, HMGR , in which the VOZ cis -element was present in its promoter.

The draft genome from this study can provide a resource for the investigation of secondary metabolite pathways not only in Aquilaria trees, but also in other non-model medicinal plants. The confirmation that cucurbitacins can be obtained from in vitro materials with a relatively high yield has positive implications with regards to plant factories to save on costs and time, as well as limiting the effects of research on this endangered species in the wild.

A plant regeneration system from shoot tips into in vitro plants was created using a tissue culture process similar to the processes described by He et al. Separation was performed with reversed-phase liquid chromatography on a BEH C8 column 2. The flow rate was set to 0.

The A. First, SGA was used to assemble the paired-end reads kmer length of 41 for error correction, 65 and 79 for the minimum overlap and overlap lengths respectively into contigs. Following scaffolding, gap closing was performed using GapFiller [ 26 ]. RNA-seq reads were aligned to the repeat masked genome and potential transcripts were assembled using TopHat followed by Cufflinks [ 29 ]. The RNA-seq alignment results and assembled transcript sequences were used to generate extrinsic data for the gene prediction tool Augustus [ 30 ], which was used to predict gene models and transcripts from the draft genome assembly.

The quality of the gene prediction was checked by performing both reference based transcript assembly with the draft genome using TopHat and Cufflinks as well as a de novo assembly using Velvet [ 31 ] followed by Oases [ 32 ].

For the reference based assembly, RNA-seq reads were aligned to the draft genome using TopHat, after which Cufflinks was used to assemble transcripts. The Cufflinks assembled transcripts were checked against the predicted transcripts from Augustus using blastn. The Velvet assembled transcripts were aligned to the draft genome using TopHat and blat.

The regions in the draft genome which were able to be mapped by Velvet assembled transcripts were overlapped with Augustus predicted exon regions in order to determine how many exon locations were successfully predicted by Augustus.


Associated Content

Agarwood , aloeswood, eaglewood, or gharuwood is a fragrant dark resinous wood used in incense, perfume, and small carvings. It is formed in the heartwood of aquilaria trees when they become infected with a type of mold Phialophora parasitica. Prior to infection, the heartwood is odourless, relatively light and pale coloured; however, as the infection progresses, the tree produces a dark aromatic resin, called aloes not to be confused with Aloe ferox , the succulent known as bitter aloes or agar not to be confused with the edible, algae-derived agar as well as gaharu , jinko , oud , or oodh aguru not to be confused with bukhoor , in response to the attack, which results in a very dense, dark, resin-embedded heartwood. The resin-embedded wood is valued in Indian - North Eastern culture for its distinctive fragrance, and thus is used for incense and perfumes. The aromatic qualities of agarwood are influenced by the species, geographic location, its branch, trunk and root origin, length of time since infection, and methods of harvesting and processing. One of the main reasons for the relative rarity and high cost of agarwood is the depletion of the wild resource. The odour of agarwood is complex and pleasing, [7] with few or no similar natural analogues.



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