1. Introduction
The
Cinnamomum genus comprises broad-leaved evergreen plant species belonging to the Lauraceae family; it is valued for its timber and aromatic properties and has been cultivated in China for at least 2000 years [
1]. Several commercially important EOs are derived from
Cinnamomum species, including
C. camphora,
C. kanehirae,
C. longipaniculatum, and
C. micranthum [
2,
3]. EOs are aromatic oily liquids extracted from a variety of plant materials, including leaves, roots, fruits, flowers, seeds, twigs, and bark [
4]. They are commercially significant, and are primarily used in pharmaceuticals, foodstuffs, biochemicals, flavors, and fragrances [
5]. The diverse chemical composition of plant EOs allows for their classification into several chemotypes, including those containing terpenes, aromatic compounds, and aliphatic compounds. For decades, numerous EO profiles and chemotypes have been identified in
Cinnamomum species, with terpenoids being the primary components [
6,
7]. However, the principal terpenoid components of the EOs of
Cinnamomum species display considerable intraspecific and interspecific variations in phytochemicals.
Cinnamomum plants are classified into various chemotypes, such as linalool-type, eucalyptol-type, camphor-type, borneol-type, and nerolidol-type chemotypes [
8,
9,
10]. Minor genetic and epigenetic alterations, which exert minimal or no influence on the plant’s morphology or anatomy, can nevertheless give rise to substantial alterations in the chemotype [
11]. In recent years, efforts have been made to explore the differences in the metabolic pathways of different chemotypes, with the objective of breeding high-quality varieties with specific chemical compositions.
Previous research on the biosynthesis and metabolic regulation of plant terpenoids have determined that the formation of terpenoids occurs through the condensation of isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP). IPP and DMAPP are synthesized in plastids by the 2-methylerythritol 4-phosphate pathway (MEP) and in the cytoplasm by the mevalonate (MVA) pathway [
12,
13,
14]. Terpene synthases (TPSs) catalyze the biosynthesis of sesquiterpene from MVA pathway precursors, and diterpenes and monoterpenes from MEP pathway precursors [
15,
16]. The development of RNA-sequencing (RNA-Seq) has enabled comprehensive transcriptomic investigations in species lacking a reference genome [
17]. The use of transcriptome sequencing has enabled the identification of genes involved in terpene biosynthesis in different chemotypes of
Cinnamomum [
9,
18,
19,
20,
21]. However, the lack of systematic and in-depth transcriptomic studies on terpenoids in various
Cinnamomum chemotypes has hindered the effective transformation and utilization of these plants. The recent publication of several
Cinnamomum whole-genome sequences provides reference data for an in-depth exploration of the molecular mechanisms underlying the formation of different
Cinnamomum chemotypes at the genomic level [
22,
23,
24,
25].
In this study, we extracted the EOs from the leaves of C. camphora var. linaloolifera, C. kanehirae, C. longipaniculatum, and C. micranthum and analyzed the main terpenoid constituents in the EOs using GC-MS to determine their chemotypes. We performed a comparative analysis of reference transcriptomes from the leaves of these four Cinnamomum species, using the published whole genome of C. camphora var. linaloolifera as the reference sequence. Following functional annotation and classification, the genes involved in the MEP/MVA pathways and terpene synthesis in different Cinnamomum species were identified and their expression levels were verified.
2. Materials and Methods
2.1. Plant Material
The study utilized four species of Cinnamomum. Two species, C. kanehirae and C. micranthum, were grown in the Fujian Forestry Science and Technology Experiment Center, Nanjing, Zhangzhou City, Fujian Province, China (127°19′ E, 24°30′ N). The two other species were represented by three-year-old C. longipaniculatum and two-year-old Nan’an 1, a new variety of C. camphora var. linaloolifera, which were cultivated in the Banlin state-owned forest farm in Anxi, Fujian Province (117°57′ E, 24°55′ N). No specific permits were required for this study. The plant material was identified by Prof. Shuangquan Zou (see author list). Healthy and pest-free fresh leaves were randomly collected from four directions (east, south, west, and north) from the plants for EO extraction and identification of the major components. In addition, a portion of fresh leaves was subjected to a thorough washing with sterile water and immediately frozen in liquid nitrogen for the purposes of transcriptome sequencing and qRT-PCR. Three relevant biological replicates were generated for each sample.
2.2. Essential Oil Extraction
EOs were extracted using a modified water vapor method in our laboratory. A 100 g sample of fresh leaves was collected and placed into an oven at 105 °C for 0.5 h. After the initial drying, the sample was transferred to an incubator set at 65 °C to complete the drying process, after which, the samples were weighed (W2). The dried sample was pulverized and placed into a 500 mL volatile oil extractor with 200 mL of ultrapure water and heated for 4 h. After distillation, the EO layer, which separates and floats on the surface, was collected, dried with anhydrous sodium sulfate, and weighed (W1). The distillation process was considered complete when two consecutive measurements showed no increase in the EO collected. Finally, the EO yield was calculated using the formula W1/W2 × 100.
2.3. Gas Chromatography–Mass Spectrometry (GC-MS) Analysis
The composition of the EOs was determined by GC-MS, performed using a Shimadzu QP2020 GC-MS instrument (chromatographic column: SH-RXI-5SILMS, 30 m × 0.25 mm × 0.25 µm). The GC-MS procedure entailed maintaining the EO samples initially at 80 °C for 2 min, subsequently elevating the temperature to 160 °C at a rate of 8 °C/min, and then further increasing the temperature to 250 °C at a rate of 8 °C/min, where it was maintained for 2 min. The injection volume was 1.0 µL, and a split ratio of 20:1 was employed. The temperature of the injection port was 280 °C, the EI ion source was 230 °C, and the connection line was 200 °C. The MS scan range (m/z) was 50–650. For the analysis, the essential oils were dissolved in alcohol (30 mg/mL) and directly injected.
2.4. Transcriptome Sequencing and Assembly
High-quality RNA was extracted from healthy mature leaves of Nan’an 1 (
C. camphora var.
linaloolifera),
C. kanehirae,
C. longipaniculatum, and
C. micranthum using the RNeasy Plant Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. The mass and concentration of the extracted RNA were quantified using 1% agarose gel electrophoresis and UV spectrophotometry. The quality-checked RNAs were enriched with oligo-dTs, randomly interrupted, and double-stranded cDNAs were synthesized. After the DNA was amplified by PCR with specific primers, the single-stranded DNA was circularized with a bridge primer to obtain a single-stranded circular DNA library. Afterwards, it was sequenced on the Novaseq 6000 platform (Illumina, San Diego, CA, USA). SOAPnuke v1.4.0 [
26] and Trimmomatic v0.36 [
27] were used to count and filter the raw data. The clean data were assembled using Bowtie2 v2.2.5 [
28], with the Nan’an 1 genome as the reference genome.
2.5. Sequencing Data Analyses
RSEM v1.2.8 [
29] was used to obtain the FPKM (expected number of fragments per kilobase of transcript sequence per million base pairs sequenced) values. DESeq2 [
30] was used to conduct the analysis on the differentially expressed genes (DEGs). Genes with |log2(FC)| ≥ 2 and a Q value ≤ 0.05 were considered to be significantly differentially expressed. The ‘ggVolcano’ package in R was used to perform the volcano mapping, and the ‘ggVennDiagram’ and the ‘UpsetR’ packages were used to perform the Upset mapping. Based on Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), significantly different DEGs were functionally annotated using Goseq R package. The FPKM values of candidate genes for EO synthesis in different tissues were visualized by using TBtools-II v2.101 [
31].
2.6. Quantitative Real-Time PCR Evaluation
A total of 24 key genes for EO synthesis were selected for qRT-PCR analysis to verify the accuracy of the transcriptome data and the actual expression levels of the selected genes. Leaf RNA was extracted using a total plant RNA kit (Polysaccharide Polyphenol Plant Total RNA Extraction Kit, developed by Hangzhou Bo Ri Technology Co., Ltd, (Hangzhou, China) for qRT-PCR validation. Primer Premier 5.0 software was used to design the primers, and all the primer information are shown in
Table A1. CcEF1a was used as an internal reference gene to calculate the relative expression of each target gene within a sample. The qualified RNA was employed as the template to construct an RT reaction solution in accordance with the TransScript
® One-Step gDNA Removal and cDNA Synthesis SuperMix instructions. The final whole reaction system volume was 20 µL, which was inactivated at 85 °C for 5 s to produce cDNA. The reaction conditions were as follows: 94 °C for 30 s, 40 cycles of 94 °C for 5 s and 60 °C for 30 s, 72 °C for 10 s, 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 1 s. Each reaction was completed for three technical replicates, and each biological replicate included three technical replications. The relative expression levels of all genes were determined using the 2
−ΔΔCt method [
32].
4. Discussion
Cinnamomum species are rich in EOs, which are employed globally as fragrances, spices, and traditional herbal remedies. Our comparative analysis of the fresh leaf EO yields from
C. camphora var.
linaloolifera,
C. kanehirae,
C. longipaniculatum, and
C. micranthum revealed that
C. camphora var.
linaloolifera exhibited the highest EO yield (5.3%), notably higher than those of other
Cinnamomum species that were previously reported in the literature [
33]. Chemotypic diversity is prevalent among
Cinnamomum species. Our chemical compositional analysis showed that the acyclic monoterpene linalool was detected in the EOs of all four
Cinnamomum species. The principal component of the EOs of
C. camphora var.
linaloolifera and
C. kanehirae was linalool, comprising 88.3% and 29.99% of the EO, respectively. In this study, the higher EO yield and linalool content from
C. camphora var.
linaloolifera can be attributed to the use of the high-yield cultivar Nan’an 1 that was selected by our research group [
34]. Eucalyptol, which is commonly added to flavorings, spices, and cosmetics, was identified in our study as the main component of the
C. longipaniculatum EO, with a content of 39.55%. This contrasts with previous findings by Zhao et al. [
21] who only detected seven terpenoids in
C. longipaniculatum EOs and did not find eucalyptol. The discrepancy is likely due to the different chemotypes of
C. longipaniculatum used in the two studies. Although a significant number of aliphatic aldehydes were detected in the EO of
C. micranthum leaves, its primary constituent was the bicyclic sesquiterpene β-caryophyllene (18.94%). This compound is known for its strong medical value, including anti-inflammatory and analgesic properties [
35]. Collectively, while the main constituents of the EOs from the four
Cinnamomum species were terpenoids, the specific terpenoid content varied significantly among species.
Previous investigations into terpenoid biosynthesis in different chemotypes of
Cinnamomum species relied on transcriptome data [
9,
19,
20]. Numerous terpenoid synthases have been isolated and characterized from various plant species [
36,
37]. Transcriptome sequencing allows for the acquisition of a large number of transcripts, thereby providing a time-efficient and cost-effective approach for the rapid identification of functional genes. However, due to the temporal and spatial limitations of gene transcription, transcriptome sequencing is only capable of providing transient gene transcription information during RNA extraction, which may result in the omission of time-specific active functional genes. Whole-genome sequencing addresses this limitation by providing a comprehensive genetic resource. Recently, several
C. camphora reference genomes have been released, including a new variety of
C. camphora var.
linaloolifera (Nan’an 1), which has a high linalool content [
22,
23,
24,
25]. In this study, we integrated genomic and transcriptomic data to elucidate the molecular regulatory mechanisms underlying the synthesis of the primary terpenoid components in
C. camphora var.
linaloolifera,
C. kanehirae,
C. longipaniculatum, and
C. micranthum.
Six sets of DEGs were analyzed, revealing that Cl vs. Cm exhibited the highest number of DEGs and shared DEGs, whereas Cc vs. Ck showed the lowest number. This variation may be attributed to the distinct main terpenoid components in these species:
C. longipaniculatum primarily produces the monoterpene eucalyptol, while
C. micranthum produces the sesquiterpene β-caryophyllene. Conversely, both the
C. camphora var.
linaloolifera and
C. kanehirae leaf EOs predominantly contained the cyclic monoterpene linalool. The functional enrichment analysis further revealed that the DEGs were significantly enriched in terpene-related pathways, consistent with the results of Zhao et al. [
21]. Terpenoids are synthesized via the MVP pathway in plastids and the MVA pathway in the cytosol [
38]. A total of 42 DEGs were found to be associated with the MVA and MEP pathways, indicating that the EOs in
Cinnamomum species are synthesized via both pathways. The expression levels of the candidate genes in the MEP pathway were higher than those in the MVA pathway in both
C. camphora var.
linaloolifera and
C. kanehirae, indicating a higher abundance of monoterpenes.
TPSs are the rate-limiting enzymes responsible for the production of various terpenoids [
14,
39]. Phylogenetic analyses have divided the TPS gene family into several subfamilies, of which, the TPS-b/g subfamily typically encodes monoterpene synthases, with TPS-g specifically encoding acyclic monoterpene synthases, while the TPS-a subfamily is involved in the biosynthesis of sesquiterpenes [
40,
41]. The expansion of the TPS family (especially the TPS-b subfamily) in the genomes of
C. camphora and
C. kanehirae partially explains the diversity of terpenoids in these
Cinnamomum species [
7,
22,
23,
24]. In total, 24 TPS candidate genes were identified, with 19 belonging to the TPS-b subfamily. The expression validation indicated that only one TPS-b gene is involved in the biosynthesis of the monoterpene eucalyptol.
5. Conclusions
In this study, we systematically and comparatively analyzed the principal components of the leaf EOs of four Cinnamomum species: C. camphora var. linaloolifera, C. kanehirae, C. longipaniculatum, and C. micranthum. Terpenoids were identified as the primary constituents of the EOs of all four species. Specifically, linalool, an acyclic monoterpene, was the predominant constituent in the EOs of C. camphora var. linaloolifera and C. kanehirae, the monoterpene eucalyptol was the main constituent in the C. longipaniculatum EO, while the sesquiterpene β-caryophyllene was the main constituent in the C. micranthum EO. Notably, the leaf EO yield and linalool content of C. camphora var. linaloolifera were found to be higher than those of other Cinnamomum plants reported in the literature, suggesting that the leaves of C. camphora var. linaloolifera are an ideal sample for studying linalool biosynthesis and are a high-quality source of naturally occurring linalool for extraction. A total of 12 samples of mature leaves from four Cinnamomum species were subjected to transcriptome sequencing and assembly, using the C. camphora var. linaloolifera genome as the reference. We identified 66 candidate genes that are involved in the synthesis of the major EO components (linalool, eucalyptol, and β-caryophyllene) across the four species, including 42 structural genes belonging to the MEP and MVA pathways and 24 TPS genes. Furthermore, the qRT-PCR data revealed that the expression patterns of these genes varied among the different Cinnamomum species, correlating with the different principal components and their contents in the EOs. In conclusion, these results provide a theoretical basis for further exploration of the biosynthesis of the major components of EOs from different chemotypes of Cinnamomum.