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. 2024 Aug 13;10(16):e35874. doi: 10.1016/j.heliyon.2024.e35874

Comparative transcriptome analysis reveals the differences in wound-induced agarwood formation between Chi-Nan and ordinary germplasm of Aquilaria sinensis

Peiwen Sun a,1, Feifei Lv b,1, Yun Yang b, Wencheng Hou b, Mengjun Xiao a, Zhihui Gao a, Yanhong Xu a,⁎⁎, Jianhe Wei a,b,⁎
PMCID: PMC11388656  PMID: 39262957

Abstract

Agarwood is a rare and valuable heartwood derived from Aquilaria sinensis in China. Compared with ordinary germplasm, Chi-Nan, a special germplasm of A. sinensis, has a better agarwood-producing capacity. However, the mechanisms underlying their different qualities remain poorly characterized. Here, a comparative transcriptome analysis of Chi-Nan and ordinary A. sinensis was carried out to investigate the wound responses of both germplasms. A total of 198.19 Gb of clean data were obtained with an average of 6.61 Gb of clean reads for each sample. By comparing with their control groups, more differentially expressed genes (DEGs) were observed in Chi-Nan germplasm. Kyoto Encyclopedia of Genes and Genomes (KEGG) and expression profile analysis suggested that Chi-Nan possesses a stronger ability to respond to wounding. Furthermore, the enrichment of biosynthetic pathways related to sesquiterpenes and 2-(2-phenylethyl) chromones (PECs) were more significant in Chi-Nan than in ordinary germplasm, and related genes showed significantly higher up-regulation in Chi-Nan after wounding. Sixteen candidate genes presumably involved in biosynthesis of agarwood components were identified and found to exhibit higher up-regulation in Chi-Nan than in ordinary germplasm in response to wounding. Overall, these results are helpful in explaining reasons for the higher agarwood-producing properties of Chi-Nan, and contribute to a further understanding of the mechanism of agarwood formation in A. sinensis.

Keywords: Aquilaria sinensis, Chi-Nan, Transcriptome, Differentially expressed genes, Agarwood, Defense response

1. Introduction

Agarwood is a highly aromatic resinous heartwood of the Aquilaria trees which are a genus belonging to the Thymelaeaceae [1]. Agarwood has high medicinal values. For centuries, it has been widely used in traditional Chinese medicine to induce tranquility and reduce excitement [2,3]. Agarwood serves as a prized ingredient in the production of perfumes and fragrances, and is also used as an incense during Buddhist, Hindu, and Islam religious ceremonies. Owing to its high demand, high-quality agarwood is more expensive than gold in international markets. Agarwood cannot be naturally generated in healthy woody tissues of Aquilaria trees without external stimuli, such as mechanical wounding, insect gnawing, microbial infection or chemical stimulation [[4], [5], [6], [7], [8]]. As a result of extensive overexploitation and destructive harvesting, the wild Aquilaria are now critically endangered. The genus Aquilaria are mainly found in Southern China and countries in Southeast Asia, including Thailand, Malaysia, Vietnam, Indonesia and so on. There are 21 Aquilaria species recorded in the world, of which 13 species have been reported to produce agarwood and remaining eight species yet to be investigated. Among them, A. crassna, A. malaccensis and A. sinensis are the most studied species. A. crassna is mainly distributed in Indochina; A. malaccensis is found in Malaysia, Thailand and India; and A. sinensis is endemic in China [9,10]. To date, all species within this genus are listed in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) [11]. As an alternative, large-scale planting and cultivation of Aquilaria trees have provided a sustainable solution to the shortage of agarwood in the global market.

Aquilaria sinensis (Lour.) Gilg is the only certified medicinal resource of agarwood in China [12]. With decades of effort, it has a wide distribution in the Chinese southern provinces of Hainan, Guangdong, Yunnan, and Fujian [13]. However, despite human intervention, it still remains challenging to efficiently and rapidly produce large quantities of high-quality agarwood. In recent years, a special germplasm of A. sinensis called Chi-Nan has been cultivated in Guangdong and Hainan provinces. This germplasm possesses the easy agarwood-producing characteristics with high resin content compared to ordinary germplasm [14,15]. Sesquiterpenoids and 2-(2-phenylethyl) chromone (PEC) derivatives are the principal compounds in agarwood [1,[16], [17], [18]]. The total relative contents of PECs in Chi-Nan were significantly higher compared to ordinary A. sinensis, and the richness and total content of sesquiterpenes in Chi-Nan were also higher [14,15,19]. Based on these, Chi-Nan germplasm has been identified as a new chemotype of A. sinensis [15]. Consequently, investigating the mechanisms underlying different levels of agarwood production between the two germplasms is essential for sustainable utilization of agarwood resources and their industrial development.

In plants, there are two biosynthetic pathways of sesquiterpenoids, namely the mevalonic acid (MVA) pathway and the methylerythritol phosphate (MEP) pathway [20,21]. Numerous enzymes in sesquiterpenoid biosynthetic pathway have been reported and identified [6,22,23]. In A. sinensis, many terpene synthases (TPSs) responsible for catalyzing the generation of sesquiterpenes have been isolated [[24], [25], [26], [27]]. And genes encoding these synthases exhibit inducible characteristics, which are closely related to stimulation caused by wounding [28,29]. However, the biosynthesis of PECs in A. sinensis is not well understood. Four types PECs, including simple 2-(2-phenylethyl) chromone, oxy-heterocyclic 2-(2-phenylethyl) chromone, tetrahydro 2-(2-phenylethyl) chromone and 2-(2-phenylethyl) chromone polymers were found in agarwood [15,30,31]. The core structure of PECs consists of a phenylpropanoid unit, which is speculated to be derived from the phenylpropanoid pathway [32]. However, the downstream synthetic pathway is not presently clear. The type III polyketide synthases (PKSs) play important roles in the biosynthesis of most plant polyketides [[32], [33], [34]]. These enzymes catalyze the decarboxylative condensation of acyl-CoAs of varying length (C2 to C20) as starter substrates with extender CoA esters (e.g., malonyl-CoA units) to produce a large variety of metabolites [32,[35], [36], [37], [38], [39]]. It has been reported that the diarylpentanoid-producing polyketide synthase (PECPS) can catalyze the generation of the precursor C6–C5–C6 skeleton of PECs by condensing malonyl-CoA with dihydro-4-coumaroyl-CoA to form a diketone intermediate, which then undergoes a second condensation with benzoyl-CoA in A. sinensis [40]. Thus, further identification and study of PKSs in A. sinensis are necessary and urgent. Transcriptomic comparisons between two germplasms of A. sinensis offer a promising approach to discover the key genes involved in the biosynthesis of sesquiterpenes and PECs.

To clarify the mechanism of agarwood formation in A. sinensis and to understand the underlying mechanism for better ability of agarwood-producing in Chi-Nan, a comparative transcriptome analysis between Chi-Nan and ordinary A. sinensis at different time points after wounding was carried out. It will help to uncover and identify candidate genes related to agarwood formation, and provide insights for further studies on the formation of secondary metabolites in A. sinensis specifically and in other plants.

2. Materials and methods

2.1. Plant materials and treatments

The ordinary A. sinensis and Chi-Nan germplasms were grown in the greenhouse at the Hainan Branch of the Institute of Medicinal Plant Development in Haikou City, Hainan Province, China. The formal identification of the plant was undertaken by the professor JH Wei. Stems from three-year-old ordinary A. sinensis and Chi-Nan trees were used for RNA sequencing. The diameter of the selected stems was 1.0 ± 0.2 cm. The stems were treated by drilling. In each stem, there were 10 holes drilled (0.2 cm diameter spaced 2.0 cm apart). Wounded stems were collected separately at each timepoint (1, 5, 15 and 30 d) after drilling, and then were frozen in liquid nitrogen immediately and stored at −80 °C. Healthy stems (0 h) served as control and were collected and frozen using the same methods. Three biological replicates were used in each experiment.

2.2. Detection of chromones in stems

The wounded stems at 15 d and healthy stems of the two germplasms were used to detect the agarwood chromones. According to the literature [14,15], the acetone extracts of the samples were analyzed by GC-MS instrument (Agilent 7890B–5975C). The chromatographic separation was performed on an Agilent DB-5MS column. (+)-Aromadendrene was used as a standard at the concentration of 0.1 g/mL, and acetone served as the control. The initial temperature was 50 °C for 1 min, then rose to 140 °C at a rate of 15 °C/min and kept for 8 min. Subsequently, the temperature was increased to 155 °C by a gradual rate of 1 °C/min and held for 8 min, then rapidly increased to 175 °C at a rate of 10 °C/min and sustained for 7.5 min. Later, the temperature was up to 200 °C at a rate of 5 °C/min and maintained for 9.5 min, and then continued to rise to 260 °C at a rate of 20 °C/min, and kept at 260 °C for 5 min. The inlet temperature was 250 °C and the ultrapure helium (99.9995 %) was used as the carrier gas with a flow rate at 1 mL/min. The injection was splitless, and the detection range was m/z 30–550. The area of each peak identified as a chromone in the chromatogram was used to calculate its relative content.

2.3. RNA extraction and sequencing

Total RNAs were extracted from stem samples at the different timepoints using the Trizol reagent (Invitrogen, Waltham, MA, USA). RNA concentration and purity were assessed using NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE). RNA integrity was assessed using the RNA Nano 6000 Assay Kit with the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). For the RNA sample preparations, 1 μg RNA of per sample was used as input material. Sequencing libraries were generated using the NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA), and index codes were added to attribute sequences for each sample. The mRNA molecules were purified and then fragmented into small pieces for cDNA synthesis. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, adaptors with hairpin loop structure were ligated to prepare for hybridization. To preferentially select cDNA fragments of 370–420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers and Index (X) Primer. Finally, the cDNA library quality was assessed on the Agilent Bioanalyzer 2100 system. The prepared libraries were sequenced on an Illumina Hiseq platform.

2.4. Transcriptome data assembly

After removing readers with adapter, ploy-N and low-quality reads from the raw data, clean reads were obtained and assembled. To assess and ensure the high quality of the data, the Q20, Q30, GC content and sequence duplication levels of the clean data was calculated. Then, the clean reads were aligned with the reference genome of A. sinensis (BioProject ID: PRJNA524272) by software Hisat2 (v2.0.5). Gene expression levels were estimated by fragments per kilobase of transcript per million fragments (FPKM) mapped. Pearson correlation analysis based on gene expression was carried out to assess the reliability of biological replicates at the same timepoint in each group [41].

2.5. Differential expression and functional analysis

Differential gene expression analysis was performed using DESeq2 [42]. The resulting P-values were adjusted using Benjamini and Hochberg's approach to control the false discovery rate. Genes with an adjusted P-value <0.05 and Fold Change (FC) ≥2, determined using DESeq2, were assigned as differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG), a database resource for understanding high-level functions and utilities of the biological system, was carried out online (http://www.genome.jp/kegg/) [43]. Cluster profiler R package was used to test the statistical enrichment of differential expression genes in KEGG pathways. The heatmap were drawn based on the normalized values (log2(FPKM)) from RNA-seq data. And it was normalized using the Z-score standardization method. Weighted correlation network analysis (WGCNA) was performed. Module identification was implemented after merging modules in which expression profiles were similar. The minModuleSize parameter was set to 30, and the merge CutHeight was set to 0.25. WGCNA analysis were performed using BMKCloud (www.biocloud.net).

2.6. Quantitative real-time PCR validation

The samples for quantitative real-time (qRT)-PCR were collected at the same time as those for the RNA sequence analysis, and the samples of the two analyses were consistent. Total RNA was extracted using a total RNA extraction kit (Aidlab, China), supplemented with on-column DNA digestion, according to the manufacturer's instructions. RNA samples were reverse-transcribed into cDNA using a c-DNA synthesis Supermix kit (Transgene, China), and analyzed using LightCycler 480 SYBR Green I Mastermix, and a LightCycler®96 (Roche, Switzerland). The quantification of transcripts was estimated using the 2−ΔΔCTmethod. The gene GADPH was used as internal control. The primers used for real-time PCR were designed using Primer Premier 5.0 and listed in Supplementary Table S1.

3. Results and discussion

3.1. Differences in agarwood formation between Chi-Nan and the ordinary germplasm

Agarwood is a kind of resinous wood produced in the stems, branches and roots of A. sinensis after wounding stress [6]. As shown in Fig. 1A, in healthy samples of Chi-Nan and the ordinary A. sinensis, the color of stems was yellowish-white. However, after wounding, the apparent discoloration of the stems around the holes was observed in both germplasms. Over the same time for the wounding treatments, the dark accumulation in Chi-Nan was significantly greater than that in the ordinary A. sinensis. Additionally, the range of apparent discoloration in the stems around the holes in Chi-Nan was significantly wider compared to that in ordinary A. sinensis. 2-(2-phenylethyl) chromone and 2-[2-(4′-methoxyphenyl) ethyl] chromone are two representative compounds of PECs in agarwood [15,44]. The relative amounts of these two PECs were 9.8 and 32.2 times higher, respectively, in Chi-Nan than in ordinary germplasm after wounding (Fig. 1B). Furthermore, unlike ordinary germplasm, Chi-Nan could synthesize trace amounts of PECs even under heathy conditions. These results confirmed that the differences between Chi-Nan and ordinary A. sinensis were marked in the agarwood production, and Chi-Nan has a stronger ability to produce agarwood.

Fig. 1.

Fig. 1

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Effect of wounding stress on agarwood formation and content of PECs in two germplasms of A. sinensis. A The differences of apparent discoloration between Chi-Nan and the ordinary type after wounding. B Relative amount of 2-(2-phenylethyl) chromone and 2-[2-(4′-methoxyphenyl) ethyl] chromone after wounding. CN, Chi-Nan germplasm; AS, the ordinary A. sinensis; H, the healthy samples with wounding at 0 h; W, the treatment samples with wounding at 15 d.

3.2. Transcriptome sequencing and DEGs analysis

To clarify the underlying mechanism of the differences in agarwood formation between Chi-Nan and ordinary A. sinensis germplasm, transcriptomic analyses were performed on their stem tissues at 0 h (control), and 1, 5, 15, and 30 d after mechanical wounding. There were 198.19 Gb of clean data in total were obtained, averaging 6.61 Gb of clean reads for each sample. The GC content of the reads was in the range of 45.91 %–47.81 %, and the percentages of Q30 ranged from 92.91 %–93.92 %. The percentage of clean reads of all samples mapped to the A. sinensis reference genome sequence (BioProject ID: PRJNA524272) was 88.72 %–94.88 %, and the uniquely mapped rate was 82.04 %–91.37 % (Table S2). As shown in Fig. S1, Chi-Nan and ordinary germplasm could be separated clearly, and the expression values of three biological replicates in each timepoint were highly correlated, indicating that the quality of the RNA-seq data were reliable.

To determine the wound-induced genes in Chi-Nan germplasm and ordinary type of A. sinensis, DEGs were identified by comparing their expression values to those of respective control libraries (0 h). Genes with adjusted P-value <0.05 and FC ≥ 2 were considered as DEGs. The total number of DEGs detected in Chi-Nan (8628) was more than that in ordinary germplasm (6370). Among the eight pairwise comparison groups, the wounded stems of ordinary A. sinensis in 15 d vs. 0 h comparison generated the smallest number of DEGs (2149), whereas the maximum of DEGs (5556) was observed in Chi-Nan at 5 d vs. 0 h (Fig. 2A). In both germplasms, the number of DEGs significantly increased within 5 d after wounding, indicating the strong reactions of the plant to the wounding. Subsequently, the number of DEGs decreased at 15 d, and then increased again at 30 d, suggesting the complexity of the responses to the wounding. Besides, to compare the distribution of DEGs in different comparisons, UpSet plots were graphed (Fig. 2B & C). Totals of 512 genes were differentially expressed in all comparisons of ordinary A. sinensis, while 1156 DEGs were found in all comparisons of Chi-Nan germplasm. It was found that more genes in ordinary A. sinensis were only differentially expresses in single comparison, whereas Chi-Nan possesses more co-expressed genes in multiple comparisons. Overall, these results clearly stated that the differences in transcriptional levels between two germplasms were obvious. The number of wound response genes in Chi-Nan was obviously more than that in ordinary germplasm of A. sinensis, suggesting that more genes in Chi-Nan might be involved in the wound response.

Fig. 2.

Fig. 2

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The statistical analyses of differentially expressed genes (DEGs) in Chi-Nan and ordinary A. sinensis germplasm. A The number of DEGs in different comparisons. We judged genes with P-value <0.05 and FC ≥ 2 as DEGs. B The UpSet plot shows the distribution of DEGs in different pairwise comparisons in Chi-Nan. C The UpSet plot shows the distribution of DEGs in different pairwise comparisons in ordinary A. sinensis. The set size indicates the total number of DEGs in each comparison. The intersect size refers to the number of DEGs detected in each type of event shown in the dotted line. CN, Chi-Nan germplasm; AS, ordinary A. sinensis.

3.3. Functional enrichment analyses of DEGs in Chi-Nan and ordinary A. sinensis

To gain a comprehensive understanding of the functions and associated pathways in response to wounding in both germplasms, DEGs were mapped in the KEGG database. In total, there were 127 pathways annotated in ordinary A. sinensis and 128 pathways in Chi-Nan germplasms after wounding. The top 20 pathways in each comparison of two germplasms were shown in Fig. 3 and Table S3, and padj <0.05 was set as the cut-off value to judge significant enrichment for pathways. The analysis revealed that the pathways enriched in each comparison were dynamic. All DEGs enriched in these pathways were up-regulated in both germplasms.

Fig. 3.

Fig. 3

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KEGG enrichment analysis of DEGs in different pairwise comparisons in Chi-Nan and the ordinary A. sinensis. CN, Chi-Nan germplasm; AS, the ordinary A. sinensis.

Sesquiterpenes and PECs, which are the principal compounds of agarwood, have antioxidant and antibacterial activities, and they are synthesized to subject to various stresses in A. sinensis [[45], [46], [47]]. Among the top20 enriched pathways, the phenylpropanoid biosynthesis, flavonoid biosynthesis and sesquiterpenoid and triterpenoid biosynthesis pathway were enriched in both germplasms. And phenylpropanoid biosynthesis and flavonoid biosynthesis were significantly enriched in 5 d–30 d after wounding in both germplasms. In Chi-Nan, they were significantly enriched in the comparison of 1d vs. 0 h. However, these two pathways were not enriched in ordinary A. sinensis in 1d vs. 0 h. These results indicated that these biosynthesis pathways had been activated after 1d of wounding in Chi-Nan. Sesquiterpenoid and triterpenoid biosynthesis pathway was significantly enriched from 5d to 30d after wounding in Chi-Nan, whereas it was obviously enriched in the comparisons of 5 d vs. 0 h and 15 d vs. 0 h in ordinary A. sinensis. It was suggested that compared to ordinary germplasm, the pathway was enriched consistently for a longer time after it was activated in Chi-Nan.

It was suggested that the starch and sucrose metabolism is closely related to the agarwood formation [48]. Notably, we observed that the starch and sucrose metabolism were markedly enriched in Chi-Nan from 5 d to 30 d after wounding. However, in ordinary germplasm, the metabolism pathway was only enriched in the comparison 15 d vs. 0 h. Among the top 20 enriched pathways in each group, special attention was also given to the pathways associated with defense responses. Obviously, in Chi-Nan, MAPK signaling pathway, plant hormone signal transduction and plant-pathogen interaction were significantly enriched. However, the enrichment of these pathways was not so significant in ordinary germplasm. MAPK activation is one of the earliest signaling events when the plant senses wounding [49]. MAPK cascades are involved in signaling multiple defense responses, including the plant hormone signal transduction and plant-pathogen interaction [[50], [51], [52]]. Previously, it has been suggested that Jasmonate (JA), Salicylic acid (SA) signaling were closely related to agarwood formation [26,28,29]. We found that alpha-Linolenic acid metabolism and Brassinosteroid (BR) biosynthesis, which were related to JA and BR signaling [53,54], were also enriched. These results indicated that upon sensing the wounding, many pathways linked with defense responses were enriched, then these signaling caused the enrichment of pathways associated with agarwood formation. And according to Fig. 3, the enrichment of these pathways was more obviously in Chi-Nan than that in ordinary A. sinensis, suggesting that Chi-Nan is more sensitive in response to wounding in comparison to the ordinary germplasm.

3.4. Expression patterns of genes in the agarwood biosynthesis pathway

To explore the expression patterns of genes involved in agarwood formation in Chi-Nan and ordinary A. sinensis, genes associated with biosynthesis of sesquiterpenes and PECs were screened based on the results of KEGG enrichment. The expression profiles of the genes in Chi-Nan and ordinary A. sinensis were clustered according to the FPKM values (Fig. 4 and Table S4).

Fig. 4.

Fig. 4

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Heat maps of genes involved in the agarwood formation. A Expression profile of genes related to sesquiterpene biosynthesis. B Expression profile of genes related to PEC biosynthesis. The color scale is displayed on the right. The high expression levels were in red while low expression levels were shown in blue. CN, Chi-Nan germplasm; AS, the ordinary A. sinensis.

A total of 36 DEGs mapped in the sesquiterpenoid biosynthesis pathway were detected for investigation. The expression levels of these DEGs were obviously up-regulated in wounding groups compared with those in the control (0 h) (Fig. 4A). There were 13 TPS genes with higher expression levels in Chi-Nan after wounding. Among them, SCA128967.3, ASN14699, ASN13036, SCA130759.32, SCA104463.27, SCA121315.43 and SCA121315.44 exhibited more obvious up-regulation. Meanwhile, the expression of two TPS genes, SCA16155.51 and SCA131879.18, were higher in ordinary germplasm than in Chi-Nan. In the biosynthetic pathway of sesquiterpenoids in A. sinensis, C5 isoprene units are formed via MVA and MEP pathways and are later converted to C15 farnesyl pyrophosphate (FPP) [55]. In the upstream pathways of sesquiterpenes, the expression of these detected genes was obviously up-regulated in both germplasms. Furthermore, most of them were expressed at higher levels in Chi-Nan. Interestingly, gene encoding 1-deoxy-D-xylulose-5-phosphate synthase (DXS) was much more highly expressed in ordinary germplasm, while genes encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) were more highly expressed in Chi-Nan. These results indicated that the expression patterns of genes involved in sesquiterpene biosynthesis were differed in the two germplasms, and more genes in Chi-Nan had higher expression levels in response to wounding.

At present, the biosynthetic pathway of PECs in A. sinensis is not well understood. Based on reports related to PECs synthesis [44,46], KEGG enrichment and the structural features of PECs, phenylpropanoid biosynthesis and flavonoid biosynthesis were considered to be correlated with PECs biosynthesis. Here, 45 DEGs related to the biosynthesis of PECs were identified. The expression patterns of these genes exhibit wound-inducible properties (Fig. 4B). Almost all genes detected were more up-regulated in Chi-Nan. Specifically, 10 of the 12 PKSs showed higher expression in Chi-Nan, while only one had a relatively higher expression in ordinary germplasm. Among the 10 highly expressed DEGs in Chi-Nan, SCA114857.4, SCA85417.12, SCA85417.14, SCA29571.30, SCA132661.6, and SCA142139.27 were particularly prominent. Moreover, genes identified in the phenylpropanoid biosynthesis pathway, such as genes encoding phenylalanine ammonia-lyase (PAL) and 4-coumarate-CoA ligase (4CL), were significantly up-regulated after wounding, and showed obviously higher expressions in Chi-Nan. And genes involved in the flavonoid biosynthesis pathways, such as genes encoding O-methyltransferase (OMT), Chalcone-flavanone isomerase (CHI) and cytochrome P450(P450) [17,56,57], also exhibited relatively higher expressions in Chi-Nan after wounding. Thus, the higher content of PECs in Chi-Nan than in ordinary germplasm of A. sinensis may result from the higher expression profiles of these related DEGs in Chi-Nan germplasm. Studies on these genes will provide insights to clarify the biosynthesis pathway of PECs in A. sinensis.

3.5. Expression changes of genes related to defense response and starch and sucrose metabolism

In this study, the mechanism of agarwood formation in response to mechanical wounding in two germplasms of A. sinensis were conducted. Previous studies have shown that mechanical stress induced JA, SA, BR, ethylene (ET), abscisic acid (ABA) and hydrogen peroxide accumulation to mediate multiple phytohormone signaling pathways in plants [58]. In A. sinensis, these signaling pathways triggered the biosynthesis of agarwood sesquiterpenes and PECs which were considered crucial defensive metabolites [59,60]. We detected the expression profiles of genes enriched in the defense response pathways including MAPK signaling pathway, plant hormone signal transduction and plant-pathogen interaction in both germplasms (Fig. S2A and Table S4). These genes were markedly up-regulated after wounding in two germplasms of A. sinensis. Obviously, we found that genes in Chi-Nan were expressed at higher levels than in ordinary A. sinensis after wounding. Previously, it was reported that compared with ordinary A. sinensis, Chi-Nan exhibited higher levels of hormones, such as JA and ET, as well as higher antioxidant enzyme activities after wounding [44]. These results suggested that Chi-Nan was more sensitive to wounding and exhibited a better capacity to cope with wounding.

The soluble sugars play crucial roles in maintaining the cellular homeostasis and enhancing the plant resistance [61]. After wounding, starch grains undergo a series of changes and are gradually converted into agarwood resin in A. sinensis [48]. In this study, the expression patterns of genes enriched in the pathway of the starch and sucrose metabolism were detected. As shown in Fig. S2B and Table S4, these genes had a higher expression level in Chi-Nan than in ordinary A. sinensis after wounding. And the fold-change of the expressions is more obvious in Chi-Nan than in ordinary germplasm, indicating genes in this metabolism were more active in Chi-Nan germplasm after wounding. Therefore, it can be inferred that the ability to convert starch into carbon sources for agarwood is stronger in Chi-Nan than in ordinary A. sinensis. Combined with the higher expression profiles of DEGs in the biosynthesis pathways related to agarwood (Fig. 4), these results demonstrated that upon external wounding, Chi-Nan germplasm has a greater ability to activate the defense signaling pathways, a stronger ability to convert starch, and a higher level of biosynthesis genes, facilitating a better ability in response to wounding and agarwood production.

3.6. Identification of hub modules between two A. sinensis germplasms after wounding

In order to reveal the key factors contributing to the changes observed in wound-induced responses in Chi-Nan germplasm, WGCNA analysis was carried out. A total of 5241 genes were screened out for the analysis between the two germplasms. As shown in Fig. 5A, 12 modules were finalized, each containing 74 to 644 DEGs. The module-trait relationships analysis showed that three co-expression modules (MEplum2, MEdarkolivegreen, and MEcyan) were considered to be hub modules (Fig. 5B). The results of their gene expression pattern analysis indicated that DEGs in these three modules had a higher expression level in Chi-Nan germplasm than in ordinary germplasm (Fig. 5C). DEGs of MEplum2 and MEdarkolivegreen may primarily be involved in early responses, and MEcyan may be related to the defense reactions in the late stages after wounding. Subsequently, we performed KEGG enrichment analysis of DEGs in these modules (Figs. S3–S5). The results demonstrated that pathways of plant hormone signal transduction and plant-pathogen interaction were significantly enriched in the three modules, showing that they were involved in the defense response. Notably, the enrichment of agarwood sesquiterpene and PEC biosynthesis was critical and evident in these modules. In MEcyan, there was notable enrichment in pathways related to biosynthesis of agarwood compounds. In MEdarkolivegreen, the gene annotations were focused on phenylpropanoid biosynthesis, starch and sucrose metabolism and sesquiterpenoid and triterpenoid biosynthesis. In MEplum2, the eigengene expressions were prominent in Chi-Nan at 1 d, and the pathways were mainly associated with the synthesis and metabolism of primary substances, such as amino acids biosynthesis and carbon metabolism. Furthermore, pathways including phenylpropanoid biosynthesis and terpenoid backbone biosynthesis were obviously enriched. These findings indicated that significant changes in these metabolic pathways may contribute to the higher agarwood formation capacity of Chi-Nan.

Fig. 5.

Fig. 5

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WGCNA analysis of Chi-Nan and ordinary A. sinensis at different timepoints after wounding. A Eigengene-trait correlation analysis B Module-trait relationships. The modules in red rectangular boxes were judged to be hub modules. C Expression patterns of the co-expressed genes in hub modules. Numbers on the heatmap represent correlation coefficient values and P_values. CN, Chi-Nan germplasm; AS, the ordinary A. sinensis.

3.7. Validation of candidate genes related to agarwood formation by qRT-PCR

To further validate and confirm the expression characteristics of genes linked to agarwood formation in both germplasms after wounding, 16 DEGs from hub modules in the relevant biosynthetic pathways were screened (Fig. 6). A total of four genes were involved in sesquiterpene biosynthesis, including three TPSs and one HMGR. Twelve candidate DEGs related to biosynthesis of PECs were determined, including those encoding PKS, CHI, caffeoyl-CoA O-methyltransferase (CCoAOMT), caffeic acid 3-O-methyltransferase (COMT), and cytochrome P450. The qRT-PCR results were consistent with those of the RNA-seq determination. As shown, 16 candidate genes were all up-regulated after wounding in both germplasms, indicating that these genes were obviously induced by wounding. The expression of the three TPS genes showed significantly higher up-regulation in Chi-Nan than in ordinary A. sinensis. In particular, TPS6(SCA121315.43) showed a more than 10-fold higher expression in Chi-Nan than that in ordinary A. sinensis after wounding. The expression of SCA50665.126, which encodes HMGR, was also obviously higher in Chi-Nan. In addition, the eight PKS genes were significantly more highly expressed in Chi-Nan compared with the ordinary germplasm. Specifically, PKS1(SCA142139.27) and PKS7(SCA17299.2) were observed to be highly expressed after wounding and showed 10-fold higher expression in Chi-Nan than in ordinary A. sinensis. The other six PKS genes exhibited a more than two-fold higher expression in Chi-Nan compared with that in ordinary germplasm. Previously, it was reported that PKS7 and PKS11 (SCA85417.14) could catalyze the generation of 1-methyl-2-phenethylquinolin-4(1H)-one whose structure is very similar to that of PECs [34], suggesting that these two PKSs may be related to the agarwood PECs synthesis. From this, it is suggested that the study of PKSs is a breakthrough in understanding PEC biosynthesis, and the key to investigate the differences of agarwood PEC content between the two germplasms. Meanwhile, we found that four DEGs from the phenylpropanoid biosynthesis and flavonoid biosynthesis were also significantly highly expressed in Chi-Nan. Among them, COMT(SCA19487.9) and CHI(SCA136519.52) exhibited obvious differences between the two germplasms after wounding, with more than 10-fold up-regulation in Chi-Nan than in ordinary A. sinensis after wounding. Overall, these candidate genes were involved in the biosynthesis of sesquiterpenes and PECs. Owning to their obvious differences of expression in response to wounding, these genes might be used as molecular markers to identify the two germplasms and contribute to clarifying the differences in agarwood production between two germplasms of A. sinensis. In the future, we will focus on the functions of these candidate genes in agarwood formation, and further investigate the differences of these two germplasms of A. sinensis in combination with proteome and metabolome analysis.

Fig. 6.

Fig. 6

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The relative expression levels of 16 candidate genes associated with biosynthesis of sesquiterpenes and PECs under wounding treatments. The stems of Chi-Nan and the ordinary germplasm of A. sinensis were treated by drilling for different times (0 h, 1d, 5d, 15d and 30 d). The expression levels of genes at different times were compared with those at 0 h, and GADPH was used as an internal control. Data are means (±SE) of three independent biological replicates. CN, Chi-Nan germplasm; AS, the ordinary A. sinensis.

4. Conclusions

This study demonstrated that there were significant differences in the transcriptional levels between Chi-Nan and ordinary A. sinensis after wounding. The biosynthetic pathways of phenylpropanoids, flavonoids and sesquiterpenoids may play crucial roles in agarwood formation. Chi-Nan germplasm exhibits a more effective wounding response compared to ordinary germplasm. The expression profiles of genes involved in the biosynthesis of agarwood sesquiterpenes and PECs were obviously more up-regulated after wounding in Chi-Nan than in ordinary germplasm. The screened candidate genes related to agarwood formation showed significantly higher expression levels in Chi-Nan than in ordinary A. sinensis, which might be used for molecular markers to distinguish the two germplasms. These finding also contributes to a deeper understanding of plant defense systems in general.

Funding

This research was funded by the National Natural Science Foundation of China (Grant Nos. 82104332, 82104335), and CAMS Innovation Fund for Medical Sciences (CIFMS) (2021-I2M-1–032).

Date availability statement

Data will be available on request.

CRediT authorship contribution statement

Peiwen Sun: Writing – original draft, Investigation, Formal analysis. Feifei Lv: Writing – review & editing, Investigation, Formal analysis. Yun Yang: Writing – review & editing, Resources. Wencheng Hou: Writing – review & editing, Investigation. Mengjun Xiao: Writing – review & editing, Investigation. Zhihui Gao: Writing – review & editing, Investigation. Yanhong Xu: Supervision, Investigation. Jianhe Wei: Supervision, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2024.e35874.

Contributor Information

Yanhong Xu, Email: yhxu@implad.ac.cn.

Jianhe Wei, Email: jhwei@implad.ac.cn.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

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mmc2.xlsx (11.1KB, xlsx)
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mmc3.xlsx (25.1KB, xlsx)
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mmc4.xlsx (30.9KB, xlsx)
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mmc5.docx (1.1MB, docx)

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