The Biosynthesis Pathways of Agarwood Constituents

Agarwood formation can be related to the self-defense mechanism of Aquilaria trees in response to biotic and abiotic stresses (Gao et al., 2012b; Singh and Sharma, 2015). Stresses trigger the defense responses of Aquilaria species which in turn initiate the secondary metabolite biosynthesis and the accumulation of agarwood resin, forming of Agarwood Constituents.

Previously, we have mentioned that sesquiterpenes and PEC derivatives are the main constituents in agarwood. Hence, it is crucially important to understand the metabolic pathway for the regulation and biosynthesis of sesquiterpenes and chromone derivatives in Aquilaria species to effectively induce the agarwood formation.

In plants, the isoprenoid precursors for the biosynthesis of sesquiterpenes, triterpenes and sterols has generally been assumed to be provided from the mevalonic acid (MVA) pathway in cytosol. In plastids, the 1-deoxy-D-xylulose-5-phosphate (DXP) or known as methylerythritol phosphate (MEP) pathway provides precursors for the production of monoterpenes, diterpenes, and carotenoids (Rohmer, 1999; Dong et al., 2015; Singh and Sharma, 2015).

These two pathways biosynthesise C5 homoallylic isoprenoid precursor, that is isopentenyl pyrophosphate (IPP) and its electrophilic allylic isomer dimethylallyl pyrophosphate (DMAPP). An exchange of IPP and DMAPP was observed to happen in between plastids and cytosol even with the spatial partitioning of the two pathways (Dong et al., 2015).

The production of IPP and DMAPP precursors from pyruvate and acetyl-CoA involves a series of enzymes according to the respective pathway (Figure 4). The genes encode for these enzymes have been identified from Aquilaria species through transcriptome sequencing analysis (Xu et al., 2013; Ye et al., 2016).

These C5 isoprene units will later be channeled into the generation of C15 farnesyl pyrophosphate (FPP) by sequential condensation reactions in the presence of FPP synthase (FPS) (Rohmer, 1999; Yang et al., 2013; Ye et al., 2016). The FPS is one of the key-limiting enzymes responsible for the sesquiterpene biosynthesis (Gaffe et al., 2000; Yang et al., 2013; Liu X. al., 2017).

The genes encode for FPS have been cloned from Aquilaria microcarpa (Am-FaPS-1) (Kenmotsu et al., 2011) and Aquilaria sinensis (AsFPS1) (Yang et al., 2013). The transcript level of AsFPS1 was reported to be higher in stem and roots than the leaves, suggesting that sesquiterpene synthesis in Aquilaria species tends to be tissue-specific. Besides, the expression of Am-FaPS-1 was shown to be up-regulated upon exposure to methyl jasmonate (MeJA), yeast extract and Ca2+-ionophore A23187, indicating that the two former chemicals are effective to initiate the sesquiterpene biosynthesis pathway whereas Ca2+ can act as signaling molecule during the activation process (Kenmotsu et al., 2011). This provides clues for the artificial induction of agarwood formation via exogenous chemically induced approaches by triggering the sesquiterpene biosynthetic pathway in Aquilaria trees.


Schematic relationships between the wound-induced signal transduction mechanisms for the sesquiterpene biosynthesis and regulation in Aquilaria species for the agarwood production. External stimuli trigger the Ca2+ signaling pathway and induce the defense responses of Aquilaria species via hydrogen peroxide (H2O2) pathway, ethylene (ET) signals, Jasmonic acid (JA) signals, and salicylic acid (SA) signals.

MeJA treatment triggers H2O2 production that can induce programmed cell death (PCD) and increase the sesquiterpene synthesis. These signaling molecules activate the transcription factors such as MYB, MYC, and WRKY, which will bind to the cis-element on the promoter of terpenes biosynthesis genes in the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways and also the downstream terpene synthase genes (TPs). Direct and indirect interactions are shown as solid and dotted lines, respectively. AACT, acetyl-CoA C-acetyl transferase; HMGS, hydroxymethylglutaryl (HMG)-CoA synthase; HMGR, HMG-CoA reductase; MK, mevalonate kinase; MPK, phosphomevalonate kinase; MDD, mevalonate diphosphate decarboxylase; DXP, 1-deoxy-D-xylulose 5-phosphate; DXS, DXP synthase; DXR, DXP reductoisomerase; CMK, 4-(cytidine 50-diphospho)-2-C-methyl-D-erythritol kinase; MCS, 2-C-methyl-D-D-erythritol-2,4-cyclo diphosphate synthase; HDS, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase; IDS, isopentenyl diphosphate synthase; GPS, geranyl disphosphate synthase; GGPS, geranylgeranyl diphosphate synthase.

In the final stage of sesquiterpenes production, the enzymes accountable for the diversification of sesquiterpene mainly come from the classes of sesquiterpene synthases (SesTPs) and cytochrome P450 dependent mono-oxygenases (P450s). The SesTP enzymes are responsible to catalyze the formation of multicyclic scaffold complexes from FPP, followed by oxidative functionalization of the resulting scaffolds by cytochrome P450 enzymes. The added hydroxyl groups by P450s can serve as molecular handles for further modifications, such as alkylations, esterifications and the addition of sugar residues ().

In addition, the P450 enzymes which carry out stereospecific hydroxylation on the hydrocarbon backbones, that is important for the novel chiralities and further modifications of the sesquiterpene molecules, have never been reported from Aquilaria species thus far. Similarly, the NADPH-dependent cytochrome P450 oxidoreductases (POR) in Aquilaria that act as redox partners of P450s catalysis activity are basically unexplored. Several studies have been reported to isolate genes encode for SesTPs from Aquilaria, which can be considered as the early attempts to study SesTPs involved in the agarwood formation (; ).

Structure of cytochrome P450

In the study of , five genes encode for sesquiterpene synthases which shared highly similar amino acid sequences have been isolated from Aquilaria crassna. Three out of these genes have been successfully expressed in Escherichia coli and enzymatically converted FPP into δ-guaiene as their major product. Besides, three other sesquiterpene synthase genes (ASS1, ASS2, and ASS3) identified from A. sinensis via transcriptome sequencing have been revealed to encode enzymes that produce δ-guaiene as well (). The isolation of SesTP genes was also described in A. malaccensis where the temporal and spatial expression of the two SesTPs reported in the study, i.e., the guaiene (AmGuaiS1) and sesquiterpene synthase (AmSesTPS1), was elucidated ().

The AmSesTPS1 was found to be highly expressed after 6 h of wounding while AmGuaiS1 was induced after 2 h of wounding at a magnitude of 18- and 5.5-fold higher than unwounded control, respectively. Recently, a novel sesquiterpene synthase gene (As-sesTPS) was isolated from A. sinensis where the recombinant As-sesTPS catalyzed FPP into nerolidol (). Expression analysis showed that the transcript level of As-sesTPS was much higher in agarwood than the healthy wood, implying that the gene can be participated in the agarwood formation. Despite the fact that many sesquiterpene compounds have been discovered from the agarwood, the corresponding SesTPs responsible for their production have yet to be reported from Aquilaria. For that reason, the sesquiterpene biosynthesis pathways involve in the functionalization of terpenes in Aquilaria is urged for further clarification.

On the other hand, chromones are a large group of secondary metabolites with wide-ranging potential therapeutic indications toward immunomodulation, inflammation, cancer, diabetes, neurological conditions, bacterial and viral infections (; ; ). Chromone is derived from a polycyclic organic compound namely benzopyran ring, with a keto group substitution on its oxime ring. It is generally believed that derivations of chromones take place as a consequence of the convergence of multiple secondary metabolite biosynthetic pathways involving pentaketide pathway, shikimic acid pathway and the addition of nitrogenous moiety from amino acids or other sources (). Owing to the extensive pharmacological properties associated with its bicyclic ring structure, chromones have been used as the privileged scaffold in the development of new drugs ().

The PECs are small class of chromones, which hold a phenylethyl substituent at the C2 of benzopyran ring of the chromone that happened to be structurally unique in the family (). Until now, the PECs have only been found to be present in a few species of plants for example Bothriochloa ischaemum (), Imperata cylindrical (), Cucumis melo L. (), Gyrinops salicifolia (), and Aquilaria species (; ). Recently, a hypothetical scheme for the biosynthetic pathway of PECs was proposed by based on in-dept analysis of agarwood chemical constituents using GC-EL-MS and UPLC-ESI-MS/MS methods.

In their study, the PECs was found to be the major agarwood resin constituents, which is comprised mostly of flindersia-type 2-(2-phenylethyl) chromones (FTPECs). The formation of FTPECs is further elucidated to be possibly catalyzed by type III polyketide synthase (PKs) through condensation of dihydro-cinnamoyl-CoA analogs and malonyl-CoA with 2-hydroxy-benzoyl-CoA to produce PEC scaffold that will subsequently be catalyzed by hydroxylases or O-methyltransferases (OMTs) to form structurally diverse FTPECs (). Recent study showed that salinity stress could induce the biosynthesis of PECs in A. sinensis calli (). Transcriptomic analysis of these salt-induced A. sinensis calli have identified several upregulated candidate genes potentially involved in the biosynthesis of PECs, including three OMT-encoding genes (flavonol-OMT 1, flavonol-3-OMT and caffeoyl-CoA-OMT) and a type III polyketide synthase gene encodes for chalcone synthase 1 (AsCHS1).

In spite of the recent progress made on the understandings of PECs biosynthesis, a tremendous effort is necessary to experimentally determine the missing steps in this complex PEC biosynthetic pathway. Even with the inadequate knowledge on the detailed PEC biosynthetic pathway, artificial synthesis of chromones and some of its derivatives is nevertheless feasible due to the advancement of chemical processes (; ). Agarwood is a rich source of PEC derivatives which deserves further investigation to uncover the structure of new chromone compounds and improve the understanding toward its biosynthetic mechanism at the molecular level.

Signaling and Regulation Mechanism of Agarwood Formation

In nature, the probability of getting agarwood-containing Aquilaria trees are extremely low (1–2%), where can only be found on pathogenically infected or wounded trees (Cui et al., 2013; Chhipa and Kaushik, 2017). Therefore, it is sensible to assume that there is a wound-inducible signal transduction process causing the expression of sesquiterpene synthases prior to agarwood formation. In order to clarify the relationship of wound signal transduction and regulation of agarwood formation, high-throughput studies on agarwood formation have recently gained attention in researches (Table 2). A schematic diagram of the proposed signal transduction mechanism of sesquiterpene biosynthesis and regulation in Aquilaria species is provided in Figure 4.

2012 Identification of conserved and novel microRNAs in Aquilaria sinensis based on small RNA sequencing and transcriptome sequence data Gao Z. H., Wei J. H., Yang Y., Zhang Z., Zhao W. T. (2012b). Selection and validation of reference genes for studying stress-related agarwood formation of Aquilaria sinensis. Plant Cell Rep. 31 1759–1768. 10.1007/s00299-012-1289-x. [PubMed] [CrossRef] [Google Scholar]


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The mitogen-activated protein kinase (MAPK) signaling pathway has been proposed as wound-induced signaling mechanism for the agarwood formation in A. sinensis, which phosphorylates downstream transcription factors (TFs) like MYB or WRKY that eventually lead to the expression of sesquiterpene synthase genes (ASSs) (Xu et al., 2013). The MAPK signaling cascade consists of three sequentially activated components [MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs], which is highly conserved signaling mechanism in eukaryotes in mediating extracellular signals to downstream responsive genes (Sinha et al., 2011; Xu C. et al., 2017). Xu et al. (2013) has reported that a total of 41 unigenes from the transcriptome analysis of wounded A. sinensis are annotated as being related to MAPK signaling pathway and 25 to calcium signaling pathways which may play roles in wound-induced agarwood formation.

In plants, calcium ions (Ca2+) are important intracellular secondary messenger molecules to regulate many signal transduction pathways reacting to the external stimuli (Tuteja and Mahajan, 2007). Earlier studies in other plants have shown that TFs are crucial regulators in stress-responsive signaling pathways to transmit signals to different cellular centers to activate plant adaptation/defense mechanisms against adverse environments, including TFs like bZIP, ERF, EIN3, MYB, MYC, and WRKY (Ambawat et al., 2013; Phukan et al., 2016; Schmiesing et al., 2016). Overexpression of AaWRKY1 in Artemisia annua was found to have positively regulated the expression of amorpha-4,11-diene synthase gene (ADS) and significantly increased the production of artemisinin (Ma et al., 2009). The expression of cotton TF GaWRKY was also shown to upregulate the sesquiterpene synthase genes for the biosynthesis of (+)-δ-cadinene and gossypol sesquiterpene (Xu et al., 2004).

Besides mechanical wounding, the MeJA is an effective elicitor to increase the sesquiterpenes content in Aquilaria (Xu et al., 2013, 2016; Xu Y. H. et al., 2017). Previous studies have shown that heat shock can increase the expression of genes involved in the Jasmonic acid (JA) biosynthesis, including allene oxide cyclase (AOC), allene oxide synthase (AOS), lipoxygenase (LOX) and 12-oxophytodienoate reductase 3 (OPR3) genes, which subsequently lead to the production of JA and the accumulation of sesquiterpene in the A. sinensis suspension cell culture (Xu et al., 2016). The A. sinensis coronatine-insensitive protein 1 (AsCOI1), acts as a receptor in MeJA signaling pathway, has been cloned and characterized (Liao et al., 2015). Expression study of AsCOI1 has demonstrated that the gene was expressed in a tissue-specific pattern which is highest in stem, followed by root and leaves.

The findings imply that resin production due to external stimuli may be more responsive in stem of Aquilaria. The AsCOI1 gene is responsive toward early treatment of MeJA, mechanical wounding and heat stress. The application of MeJA in A. sinensis has increased the expression level of 17 wound signaling-related genes, including TFs WRKY4 and MYB4, protein kinases CAPKs, MAPKs and MAPKK, NADPH oxidase noxB and some regulators related to signal molecules MeJA, ethylene and hydrogen peroxide (Xu et al., 2013). Interestingly, the hydrogen peroxide (H2O2) producing NADPH oxidase noxB was found to be significantly up-regulated by the MeJA treatment (Xu et al., 2013; Gong et al., 2017), which is consistent with the findings that MeJA triggers H2O2 production in plants (Orozco-Cardenas et al., 2001; Hung et al., 2006). A study on suspension cultures of A. sinensis revealed that H2O2 can induce the programmed cell death (PCD) and sesquiterpene synthesis by the elevated expression of ASS genes due to the endogenous accumulation of salicylic acid (SA) (Liu et al., 2015).

Meanwhile, the expression of jasmonate-responsive key sesquiterpene synthase ASS1 in A. sinensis was described to be regulated by a TF AsMYC2 (Xu Y. H. et al., 2017). As an immediate-early responsive gene toward MeJA treatment, AsMYC2 binds to the ASS1 promoter containing a G-box motif to initiate the expression of ASS1. Similarly, the homolog of AsMYC2 in Arabidopsis (MYC2) was shown to be MeJA-responsive and up-regulated the expression of two sesquiterpene synthases (TPS11 and TPS21) upon treatment (Hong et al., 2012). In addition, MeJA treatment has successfully induced the synthesis of 3 sesquiterpenes, that are α-guaiene, α-humulene and δ-guaiene in A. crassna cell culture (Ito et al., 2005; Kumeta and Ito, 2010).

In contrary to the sesquiterpene biosynthesis pathway, the biosynthesis and regulation of PECs remains almost unknown. In the study of Wang et al. (2016), forty-one PECs have been produced in A. sinensis calli by salt treatment. With the help of high-throughput transcriptome analysis, a total of 18,069 differentially expressed transcripts between the control and the NaCl-treated A. sinensis calli induced by 24 or 120 h salinity stress were obtained. Many differentially expressed genes are reported to be involved in the hormone signal transduction; including genes encode for MAPK cascades, receptor-like kinases, Ca2+ signal transduction and transcription factors (Wang et al., 2016).

Nonetheless, the pace of research did not just stop at searching for effective inducers and their underlying signal transduction process, but also extended to their post-transcriptional regulation mechanism. By deep sequencing of sRNAs from healthy control and wounded samples of A. sinensis, Gao et al. (2012a) have identified ten stress-responsive miRNAs from 74 putative conserved miRNAs and their hairpin forming precursors were also confirmed. Expression pattern revealed that six of these stress-responsive miRNAs were up-regulated, including miR159, miR168, miR171, miR396, miR397, and miR408, whereas miR160 and miR398 were down-regulated and continued their reduced level at 2 day point (Gao et al., 2012a). The different responses of miRNAs reacted toward treatment and the effect lasted for various time lengths reflecting the diversity of their positions in the post-transcriptional regulation of wound response in A. sinensis. Among the identified miRNAs, the down-regulated miR398 is of interest where it was demonstrated to negatively regulate the pathogen-associated molecular pattern (PAMP)-triggered callose deposition and plant innate immunity against bacteria (Li et al., 2010). The oppositely reacted miR160 and miR398 in A. sinensis suggested that they might be important regulators and play a more distinct role on agarwood formation.

Further study on miRNAs profiling of wounded A. sinensis showed that some of the most conserved miRNAs such as miR159 and miR396 families elevated and subsided quickly in early period of treatment implying their function at the upstream of wound responses (Gao Z. H. et al., 2014). The down-regulated miR396b2 in wounded tissues of A. sinensis was suggested to involve in the biosynthesis and accumulation of agarwood constituents. The target of miR396b2, which has glutamyl-tRNA reductase activity, was believed to bind NADP and produce NADPH. The NADPH is the cofactor for the two key enzymes in terpene biosynthesis, i.e., 1-deoxy-D-xylulose 5 phosphate (DXR) and 3-hydroxy-3-methylglutaryl-CoA (HMGR) (Nagegowda, 2010). Furthermore, plant P450s that oxidatively functionalize the terpene scaffolds also require reducing agents for its catalytic activity, which is commonly provided by NADH or NADPH. Despite in the situation of lacking sequenced genome, high-throughput transcriptome analysis provides a feasible approach to examine the overall changes of gene expression on Aquilaria species responding to a variety of stresses. Further investigation of the functions of the identified regulator sequences would help to reveal the regulation mechanism of agarwood formation.

Future Prospects of Agarwood Induction Technology

Previous studies have shown that agarwood formation can be influenced by many factors. Together with the exceedingly complex agarwood resin composition, it is believed that agarwood formation is an intricate process which involved a variety of physiological changes occurs on Aquilaria trees to cope with the external stimuli either in the form of biotic or abiotic. This whole agarwood formation process is in any case inseparable from the gene-expression response of the trees toward the triggering factors. Consequently, future improvement of agarwood induction technology should emphasize on two aspects that are to further improve the induction efficiency and to screen more responsive lines of Aquilaria for resin production under breeding program.

In order to improve the induction efficiency, an induction technique plays a decisive role. The concept of currently available induction approaches can be summarized as either to provide external stimuli to activate the production of plant signaling molecules that eventually lead to the resin biosynthesis, or to bypass the external stimuli via direct introduction of signaling molecules to the plants. In any case, the overall concern is to increase agarwood yield and quality as well to reduce human intervention (e.g., holing process) during the induction process. Since the aforementioned physical wounding and biological induction method have their inevitable drawbacks of inconsistent agarwood quality and requiring intensive workforce, the chemical induction method can be regarded as a promising approach for further optimization (Table 1). Comprehensive understanding of agarwood formation at the molecular level via high-throughput using omics approach such as trancriptomic and metabolomic appears to be advantageous for more targeted and directional improvement of the induction formulation rather than the trial- and error-based experimentations.

By coupling with omics approaches such as single molecule real time sequencing technology (SMRT) which offers longer read lengths and highly contiguous de novo assemblies (Rhoads and Au, 2015), thus it tends to be particularly useful for unsolved problems in genome and transcriptome of non-model Aquilaria species where their genomes are not available. With longer reads, the highly repetitive non-coding regulatory sequence of genes controlling the agarwood resin production can be easily obtained. Sequence similarity/identity-based integration can be used to establish the cross-reference data sets between the query sequences and their homologous references from various public databases and genetic resources, where genomic sequence structures, domain features, promoter regions and gene ontology for motifs can be assigned (Mochida and Shinozaki, 2011).

The development of sequencing technology has made the future research on the whole genome sequencing of Aquilaria species to be easier to fill in the lack of genome-wide information in the current situation. Data integration based on genome sequence is important to allow analysis of global changes of transcriptome through whole genome microarrays. The gene expression analysis of induced Aquilaria at its entirety can be examined in broad coordinated trends by this approach, which is indiscernible by individual assays. The expression profiles obtained in this way will help to identify potential agarwood-producing biomarker genes that are important indicators for downstream applications of agarwood induction.

The integration of systems biology and omics approaches, covering genomics, transcriptomics, proteomics, metabolomics and functional analysis; provide a potential solution to comprehend the multigenic nature of resin biosynthesis in Aquilaria. On the basis of large number of previously conducted agarwood induction experiments, transcriptomic and metabolites studies (Naef, 2011; Gao X. et al., 2014; Ye et al., 2016; Wu et al., 2017), integrated omics analysis can indeed serve as a platform to build a more comprehensive picture of agarwood resin biosynthetic mechanism that involve different omics layers. The development of high-throughput mass spectrometry (MS), microarray and sequencing technologies (DNA and RNA) have made it possible to integrate such data into a system biological framework via integrated-omics which would help to predict gene-gene interactions, identify driver genes and the molecular signatures of agarwood formation (Figure 5).

Likewise, potential signaling molecules for agarwood formation can be predicted in a wake of this dramatically increased availability of data. Experimentally validated effective signaling molecules can be added into the existing recipe of chemical inducer to further enhance the induction performance. Moreover, predetermination of resin composition or the agarwood quality is conceivable with deep understanding of the key quality indicators and the specific pathways involved for agarwood production by integrated elucidation of different metabolite and transcript profiles under different induction methods. Future improvement of agarwood induction efficiency should accompany with the development of monitoring system for early detection of non-responsive trees which could avoid cutting down of Aquilaria trees that are unsuccessfully been induced. This can be achieved by monitoring the expression of a set of genes involved in agarwood resin biosynthesis.

Schematic representation of application of omic approaches in the development of agarwood inducers. PacBio, Pacific Biosciences; Illumina, Illumina sequencing; Pyroseq, pyrosequencing; RNAseq, RNA sequencing; 2D-PAGE, 2-dimensional polyacrylamide gel electrophoresis; DIGE, differential gel electrophoresis; ChIP seq, chromatin immunoprecipitation sequencing; GC-MS, gas chromatography-mass spectrometry; LC-MS, liquid chromatography-mass spectrometry; NMR, nuclear magnetic resonance.

Aside of the effective inducer, the responsiveness of Aquilaria trees toward stimulation is another determining factor for the production of agarwood. By knowing that the degree of plant response on stimuli is largely dependent on their genetic makeup, the utilization of highly responsive Aquilaria line as induction target is expected to further increase the agarwood yield rather than optimizing the inducer recipe alone.

Conventionally, selective breeding based on phenotypic selection have been adopted to develop new plant lines with desirable traits. Current technology associates these beneficial traits of plants to genetic (DNA/RNA variations) or biochemical (signature metabolites) markers to allow marker-assisted selection (MAS). The approach of MAS offers a great promise for the selection of elite Aquilaria lines as these biomarkers can be applied to predict the phenotypic characteristics before these features develop into more noticeable. Such biomarkers can also be used for the development of fast and targeted diagnostic assays that will assist the selection program. As an alternative to obtain a high-yield line, a combined approach of genetic engineering (e.g., CRISPR-Cas9 genome editing technology) with tissue culture could pose a possibility to manipulate the key regulator genes of Aquilaria involved in the agarwood production which will help to fine tune or redirect the metabolic flux toward desired metabolic pathways.

On the whole, an integrated and high-throughput strategy will provide sufficient information to continually improve the agarwood induction methods, which is superior compare to the traditional way of induction method establishment that rely on the visual observation and personal experience. A deeper insight into the essential compounds and the biosynthesis mechanism of agarwood resin would greatly ease to control the stability of agarwood yield, quality and its price in the future.


Source: Scientific report on Agarwood by the authors:

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