Introduction

Atractylodis Rhizoma in Chinese pharmacopoeia is derived from the dry rhizome of Atractylodes lancea (Thunb.) DC. Atractylodis Rhizoma have several functions, namely, drying dampness and strengthening the spleen, eliminating pollution and turbidity, eliminating wind dampness, and clarifying the eyes in Traditional Chinese Medicine (TCM) applications1. Atractylodis Rhizoma is also one of the main materials in the prevention of COVID-19 infection2,3. The major volatile oils in Atractylodes lancea rhizome (AR) are the sesquiterpenoids hinesol, β-eudesmol, and atractylone, and the polyacetylene atractylodin, which are acknowledged as the four major medicinal components, as well as the marker compounds, of the quality evaluation of AR4. Thus, the higher the volatile oil content, the better the clinical efficacy of AR5.

Light is an essential factor that affects the growth, development, and metabolism of plants during their life cycles. Many studies have demonstrated that the accumulation of secondary metabolites can be affected by changes in light intensity. For example, the content of camptothecin in the leaves of Camptotheca acuminata seedlings increases as the degree of shading increases; however, it decreases in excessive shading6,7. Flavonoids accumulate in Anoectochilus roxburghii to a greater extent when the light intensity is low8. The synthesis of isorhamnetin can be induced when the light intensity is increased from low light above a certain threshold9. A. lancea varieties can be found in environments under a wide range of light conditions, according to a survey of wild resources10. However, previous studies have reported that bamboo-shaded or maize-shaded A. lancea contains more medicinal compounds than that grown in environments without the shade of other plants11,12. Shading is a key ecological factor that affects the accumulation of volatile oils in AR. Gu et al. showed that a shading value of 72.5% promotes the accumulation of volatile oils, but the physiological and biochemical indicators of plants are yet to be defined13. Sun et al. and Wang et al. detected the antioxidant enzymes and the photosynthetic parameters of A. lancea under different shading conditions, but the experimental period was very short and the results of volatile oils were not reported14,15. Presently, wild A. lancea is a rare resource, while cultivated A. lancea has become a major source of AR in production settings16. To promote the stable and sustainable production of high-quality AR in the future, establish a concrete shading value to guide the cultivation of high-quality A. lancea, and the corresponding mechanism of shading, which enhances the accumulation of volatile oils, also requires further investigation simultaneously.

Photosynthesis ensures life activities, and it is one of the most important metabolic processes. Photosynthetic efficiency is affected by various factors such as light, drought, and temperature, which in turn affect primary and secondary metabolic efficiency14,17,18. In many studies, photosynthetic efficiency is reflected by the intercellular CO2 concentration (Ci), transpiration rate (Tr), net photosynthetic rate (Pn), and stomatal conductance (Gs)19,20. More efficient photosynthesis can generate better plant qualities than those with poor physiological traits21. Phytohormones are the key endogenous signals of plant cells in response to changes in the external environment, and they are not only inducers of sesquiterpenoid biosynthesis22,23, but also the products of sesquiterpenoid biosynthesis such as abscisic acid (ABA). The expression of key enzymes in artemisinin biosynthesis can be regulated by jasmonic acid (JA), and this process requires light24. Therefore, to understand the mechanism of shading that regulates sesquiterpenoid accumulation, the effects of photosynthesis and the changes in plant hormone levels to light intensity are worthy of attention.

In this study, by testing six different light intensity conditions, including 100%, 80%, 60%, 40%, 20%, 7% of natural sunlight, we examined the effects of light intensity on the quality and the yield of AR, The reasons for the formation of high-quality AR were analyzed in terms of photosynthetic efficiency and phytohormone levels. The experiment was carried out in the field for one year, and the goals of this study were as follows: (1) to determine the shade value for the cultivation of high-quality A. lancea; (2) to understand the effects of different light intensities on photosynthetic efficiency and phytohormone levels in cultivated A. lancea; and (3) to determine the relationship between volatile oils, photosynthetic parameters, and phytohormones under different light intensities.

Results

Mild shading facilitates sesquiterpenoid accumulation and growth in Atractylodes lancea rhizome

To determine a concrete shading value for the production of high-quality and high-yielding AR, we examined the major compounds, including the sesquiterpenoids hinesol (Hin), β-eudesmol (Edu), and atractylone (Atl), and the polyacetylene atractylodin (Atd), as well as the biomass of AR at different growth stages (Fig. 1A–C) under various light intensities. The sum of these four volatile oils as the total volatile oil content was subsequently analyzed. The results revealed that the accumulation of volatile oils was significantly different (p < 0.05) across seedling, expansion, and harvesting stages under different light intensities. The common features at the three life stages were that the total volatile oil content was the highest under 80% mild shading and the lowest under 7% low light intensity. At 80% mild shading, the total volatile oil content in seedling, expansion, and harvest stages increased by 58%, 52%, and 35%, respectively, compared to 100% strong light. Thus, excessive light inhibited the accumulation of volatile oils. Compared to 7% low light, the increased accumulation of volatile oils at 80% mild shading reached 144%, 178%, and 94% from seedling to harvesting stages. We found that the weaker the light intensity (20% and 7%), the lower the volatile oil accumulation. In addition, the biomass indicators of fresh and dry weights (Fig. 1G,H) indicated that the response to different light intensities yielded trends that were consistent with volatile oil accumulation. Thus, both quality and yield of AR can be achieved under 80% mild shading.

Figure 1
figure 1

Effects of different light intensities on the accumulation of total volatile compounds and each component (Hin, Edu, Atl, Atd) in the A. lancea rhizome in seedling (A,D), expansion (B,E), and harvest (C,F) stages. Effects of different light intensities on the biomass, namely, the fresh weight (FW) (G) and the dry weight (DW) (H) and the expression of upstream genes encoding key enzymes involved in sesquiterpenoid biosynthesis during the expansion stage (I). Lower-case letters represent significant differences (one-way ANOVA, p < 0.05).

Full size image

To further investigate the component that contributes the most to the total volatile oil accumulation due to changes in light intensity, individual compounds were analyzed at different life stages (Fig. 1D–F). The results revealed that each compound was significantly affected by changes in light intensity at different stages (Fig. 1D–F). Statistically, the sum of the difference contribution ratio (DCR) of the three sesquiterpenoids Hin, Edu, and Atl was greater than 90% of the total variation at any stage of growth, with less than 10% of Atd. Of these, Hin and Edu contributed approximately 70% to the total variation (Table S-1). Therefore, the effect of different light intensities on the accumulation of Hin and Edu was higher than that of Atl and Atd. Sesquiterpenoids, especially Hin and Edu, could reliably determine the variations in the total volatile oil content. Compared to 100% strong light, Hin increased by 60%, 52%, and 30% and Edu increased by 40%, 60%, and 45% at 80% mild shading, respectively, from seedling to harvesting stages. Compared to 7% low light, Hin increased by 111%, 185%, and 92%, and Edu increased by 182%, 224%, and 81% at 80% mild shading, respectively, from seedling to harvesting stages. In addition, we also measured the levels of the key enzyme genes of sesquiterpenoid biosynthesis during the expansion stage of AR. The expression levels of HMGR, DXR, and FPPS in the mevalonic acid (MVA) pathway and methylerythritol phosphate (MEP) pathway were measured by qRT-PCR. The results revealed that the expression of these genes under different light intensities significantly differed among the groups (Fig. 1I). The expression of each gene was the highest under 80% mild shading, that is, greater than 100% strong light, and the lowest under 7% low light. The differential expression patterns of these genes under different light intensities is consistent with sesquiterpenoid accumulation. Thus, the expression analysis further confirmed that light intensity affects sesquiterpenoid accumulation.

80% mild shading enhances photosynthetic efficiency in A. lancea, whose trend was affected by changes in light intensity, consistent with sesquiterpenoid accumulation

The intercellular CO2 concentration (Ci), transpiration rate (Tr), net photosynthetic rate (Pn), and stomatal conductance (GS) as gas exchange parameters were significantly different at the different light intensities. The Ci, Tr, Pn, and Gs values of 80% mild shading were the highest (Fig. 2), increasing by 30%, 33%, 27%, and 27%, respectively, compared to 100% strong light and increasing by 45%, 369%, 347%, and 81%, respectively, compared to 7% low light. The results showed that between 100% strong light and 80% mild shading, the differences of the four photosynthetic parameters are close to 30%. While between 7% low light and 80% mild shading, the changes of Tr, Pn are far higher than Ci, Gs. As the degree of shade increased, light utilization decreased starting at 80% light intensity. The results showed that mild shading promotes photosynthetic efficiency in A. lancea, and the trend of variation is consistent with sesquiterpenoid accumulation, which is affected by changes in light intensity.

Figure 2
figure 2

Effects of different light intensities on photosynthetic parameters, namely, the intercellular CO2 concentration (Ci), transpiration rate (Tr), net photosynthetic rate (Pn), and stomatal conductance (Gs) in A. lancea. Lower-case letters represent significant differences (one-way ANOVA, p < 0.05).

Full size image

80% mild shading significantly enhances the phytohormones ABA and GA3 in A. lancea, whose trend was affected by changes in light intensity, consistent with sesquiterpenoid accumulation

Phytohormones play important roles in signal transduction and secondary metabolism in plants. We measured the levels of common phytohormones in root (R) and leaf (L), as shown in Fig. 3. Data analysis found that the levels of JA, salicylic acid (SA), ABA, and gibberellin (GA3) under 80% mild shading were significantly (p > 0.05) higher than that of 100% and 7% light intensities, regardless of the plant part (Fig. 3). Furthermore, JA, SA, ABA, and GA3 levels in 80% mild shading were 1.7, 2.9, 3.1, and 2.2-fold higher than those in 100% strong light, and 4.2, 3.2, 22.9, 19.8-fold higher than those in 7% low light in root (Fig. 3A). And JA, SA, ABA, and GA3 levels in 80% mild shading were 4.2, 3.2, 21.2, and 19.8-fold higher than those in 100% strong light, and 7.8, 2.9, 11.8, 2.2-fold higher than those in 7% low light in leaves (Fig. 3B). The results showed that between 100% strong light and 80% mild shading, the phytohormone change folds of ABA and GA3 are higher than other phytohormones in leaves. While between 7% low light and mild shading, in root the phytohormone change folds of ABA and GA3 are higher than other phytohormones. Therefore, under strong light stress, ABA and GA3 in leaves were more responsive, while under weak light stress, ABA and GA3 in roots were more responsive. Other phytohormones content also fluctuated with different light intensity changes, but the fluctuation range was relatively small. In addition, all phytohormone levels gradually decreased with weakening light intensity starting at 80% light intensity (Fig. 3). The results indicated that the changes in the phytohormone levels are consistent with those of sesquiterpenoid accumulation.

Figure 3
figure 3

Effects of different light intensities on the phytohormones jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA), gibberellin (GA3) in A. lancea roots (A) and leaves (B). Lower-case letters represent significant differences (one-way ANOVA, p < 0.05).

Full size image

In addition, we compared the phytohormone response levels in root and leaf. When the phytohormone change fold (HCF) of roots to leaves was equal to 1, the hormone response level was similar in both root and leaf. When the HCF was higher than 1, the hormone response level was higher in root than in leaf, indicating that hormonal stress in roots is more sensitive to changes in light intensity. By contrast, when the HCF was lower than 1, the hormone response level was lower in root than in leaf, indicating that hormonal stress in leaves is more sensitive to changes in light intensity. Table 1 shows that JA was more sensitive to light intensity changes in leaves than in roots, while ABA was higher responseive to light intensity changes in roots than in leaves.

Table 1 Hormonal changes folds (HCF) in roots compared to leaves under the influence of different light intensities.
Full size table

Ci, Tr, and Pn in photosynthesis; GA3 and ABA in root; and JA in leaf were strongly correlated with the accumulation of each sesquiterpenoid

The relationships among various physiological and biochemical factors and volatile oils were assessed by Pearson correlation coefficients under different light intensities. As shown in Table S-2, most indexes were significantly correlated with the volatile oil content (p < 0.01). The correlation coefficient represents the strength of the correlation between the two indexes. Correlation coefficients greater than 0.85 were used as the screening criteria. As shown in Fig. 4, the values of Ci, Tr and Pn were strongly correlated with the sesquiterpenoids hinesol and β-eudesmol (r > 0.85). From the correlation network, we found that R-ABA, R-GA3 and L-JA showed the highest correlation with the accumulation of sesquiterpenoids. Based on the results of photosynthetic efficiency test, phytohormone test and Pearson correlation analysis, Tr and Pn in photosynthesis, GA3 and ABA in root, and JA in leaf that are not only significantly responsive to light intensity changes but also significantly related to sesquiterpenes are screened out.

Figure 4
figure 4

Pearson correlation network diagram between volatile oils and physiological and biochemical indexes. Different colors of nodes represent volatile oils (purple), photosynthetic parameters (pink), phytohormones in leaves (green), and phytohormones in roots (orange). The size of each node indicates the amount of the correlated index. The width of each edge represents the correlation. As the width of each edge increases, the correlation coefficient is greater (r > 0.85 and p< 0.05). The connectivity degree is indicated by the size of the symbols.

Full size image

Promoter regions of HMGR, DXRs, and FPPS contain both light and phytohormone cis-acting regulatory elements

HMGR, DXR, and FPPS have been reported as key enzyme genes involved in biosynthesis of sesquiterpenoids. Therefore, we analyzed the cis-acting elements within the upstream promoter regions (2000 bp) of the three genes which had been selected for qRT-PCR analysis. The relevant sequences are shown in Fig. S-1. The results indicated that HMGR, DXR, and FPPS promoter regions included several light and phytohormone regulatory elements (Table 2), confirming that sesquiterpenoid genes may co-regulated by light and phytohormones.

Table 2 The cis-acting elements related light and phytohormone were contained in the promoter upstream 2000 bp region of HMGR,DXR,FPPS.
Full size table

Discussion

Mild shading facilitates the accumulation of volatile oils and growth in A. lancea

Synthesize multiple results showed that Tr and Pn may be the key extrinsic factors affecting the synthesis and the accumulation of the sesquiterpenoids hinesol and β-eudesmol. We know that photosynthesis is an important indicator of growth and development as well as stress resistance in plants36,37. This study showed that Pn, which reflects plant organic matter accumulation, was the highest under 80% mild shading. In addition, AR also accumulates more biomass under this condition. The substances produced by photosynthesis far exceeded the substances consumed by the plant for its own life activities, indicating that it grew well. The 100% strong light provided more light energy than 80% mild shading, but the biomass of 100% strong light was lower than that under 80% mild shading. This can be explained by the fact that high light and high temperature in the summer cause severe leaf burns, and photosynthetic structures are irreversibly damaged14, leading to an imbalance of Tr and a decline of Pn. According to Farquhar and Sharkey, who commented on the reduced photosynthetic rate, when Pn and Gs decrease, if Ci also decreases, this indicates that the reduction of Gs is the main reason for the reduced photosynthetic rate38. Meanwhile, under the long-term regulation of shaded environments, stomatal density can decrease, leading to the decline of Gs39. Furthermore, Gs is the main factor of Tr intensity and Pn efficiency. Combined with the results of this study, under conditions of < 80% mild shading, with the increase of shading degree, photosynthetic efficiency was mainly affected by stomatal limitations. Studies have demonstrated that low light can reduce photosynthesis by inhibiting Ci and Gs to suppress secondary metabolic processes such as phycocyanin synthesis and yield in crops such as wheat40,41. Therefore, under 80% mild shading, A. lancea maintained the best photosynthesis state, and it was more capable of resisting the fluctuations of the external environment to produce more sesquiterpene secondary metabolites in a long-time environment. In addition to having better resistance to the environment at 80% mild shading, more organic substances accumulated in AR, thereby providing the elements needed for energy production such as acetyl coenzyme A, malonyl coenzyme A, and ATP42 using for sesquiterpenoid biosynthesis.

Light-hormone interactions regulate key enzyme gene expression in sesquiterpenoid synthesis and further influence sesquiterpenoid accumulation

As physiological and biochemical indicators, phytohormones reflect the intrinsic expression of plant life activities. The Pearson correlation results showed that the accumulation of sesquiterpenoids is strongly related to the changes of multiple hormones at different light intensities. Our findings are consistent with the results that the promoters of three key enzyme genes in the sesquiterpenoid synthesis pathway contain multiple cis-acting elements of phytohormones. Chuanchao and colleagues demonstrated that JA can up-regulate the expression of sesquiterpenoid genes through fungal induction; it also has functional interactions with SA in plant defense43. On the other hand, ABA and GA3 up-regulate DXS and HMGR genes under bacterial induction and promote the accumulation of volatile compounds44,45. It was also found that blocking the production of just one hormone did not completely inhibit sesquiterpenoid production43, suggesting that sesquiterpenoid synthesis is not dependent on a single signaling event and that multiple phytohormones work together to regulate their synthesis. These findings reveal that multiple phytohormones showed good correlation with the accumulation of sesquiterpenoids. A previous molecular study demonstrated a relationship between light and several hormone signaling pathways via signaling integrators (PIFs, HY5)46. It has also been demonstrated that the function of JA in artemisinin is dependent on the presence of light47. Therefore, light may be the basic condition required to induce changes in phytohormone levels. However, A. lancea maintains different phytohormone levels, and their responses are the same at different light intensities. The levels were higher in mild shading, and lower in low light shading. This may be related to the growth inhibitory mechanism used by A. lancea under the stress of strong light or weak light for a long time to maintain a longer lifespan. In summary, the results of this study suggest that mild shade induces changes in the phytohormones ABA, and GA3, JA, which regulate the expression levels of key enzyme genes for sesquiterpenoid synthesis and ultimately influence the changes in bioactive components.

Our findings revealed that ABA decreases under strong light, which is inconsistent with the results of a previous study that reported ABA unaffected by light signals48. Under long-term high light conditions, the leaves of A. lancea showed burning and yellowing as well as poor growth. Abscisic acid is a hormone that triggers leaf abscission, so ABA can be used as an indicator of the abscission of injured leaves, indicating that ABA levels change with plant growth and development. In addition, it was found that the content of ABA in roots was significantly higher than that in leaves. Currently, ABA is synthesized at several sites, such as roots and leaves, and translocated to its sites of action, which includes guard cells49. Given that plants have multiple ABA transporters, ABA transport is dynamically regulated under various growth conditions, which regulates root system expansion50,51.

In addition, we found that the promoters of key enzyme genes that regulate the synthesis of sesquiterpenoids contain numerous light response elements. Several studies have demonstrated that light can directly regulate the synthesis of isoprene, the precursor of sesquiterpenoids52. Therefore, in the future, studies should focus on the significance of light, which directly regulates the synthesis of sesquiterpenoids via the downstream factors of the light response.

Conclusion

In conclusion, 80% mild shading is a proper shade condition suitable for guiding stable and sustainable production of high-quality AR. It promotes the accumulation of total volatile oils mainly by regulating the biosynthesis of the sesquiterpenoids hinesol and β-eudesmol. Mild shading promotes the synthesis and accumulation of the medicinal compound in A. lancea via integration and regulation of photosynthesis and phytohormones. In addition, light and phytohormones are important internal and external factors affecting plant growth, development, and metabolism, although the direction of molecular research of A. lancea has been slow. Thus, this study provides a solid foundation upon which future studies can be designed. In the future, the relationship between photosynthesis (Tr, Ci, Pn), multiple hormones (ABA, GA3, JA), and sesquiterpenoids (hinesol, β-eudesmol) at the molecular level should be studied, laying the foundation for the regulation and the improvement of AR quality.

Materials and methods

All plant experiments were performed in accordance with relevant guidelines and regulations.

Field experiment, sampling, and sample preparation

The field experiment was conducted from November 2016 to November 2017 at the GAP production base of Jiufang Pharmaceutical Company, Yuexi County, Anhui Province, China, and A. lancea seedlings were developed from the buds of the second-year rhizome by vegetative propagation. The germplasm identification of the seedlings was confirmed by Professor Lanping Guo from the National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences. Healthy and uniformly sized A. lancea seedlings were planted in an experimental field located on a flat plot at an altitude of 870 m with a row spacing of 0.5 m and a plant spacing of 0.3 m. Different levels of shade above the seedlings were created in the greenhouse using materials of different densities such as white gauze and sunshade net. There were six experimental groups with five replicates in each group. A TR-72U light quantum instrument (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the light intensity of the different shading treatments every 10 min from 8:00 to 17:00 on sunny days, and the full daylight intensity without any shade was measured, which served as a control. The percentage of light intensity in the other groups relative to the full light intensity was used as an indicator of the light intensity in the growth environment. The experimental groups were as follows: 100% (full light group), 80% light intensity group (two layers of white gauze), 60% light intensity group (four layers of white gauze), 40% light intensity group (one layer of sunshade net), 20% (one layer of white gauze and one of sunshade net, respectively), and 7% (two layers of sunshade net). An area up to 30 cm above the ground surrounding the greenhouse was enclosed to ensure the smooth flow of air.

Extraction and determination of volatile compounds at different developmental stages

The concentrations of volatile oils in the rhizome of A. lancea were determined at different developmental stages, including seedling, expansion (period of rapid growth of rhizome), and harvest. First, the fresh rhizome samples were dried in an oven at 40 °C to a constant weight and weighed. The dried samples were crushed into a powder with a ball mill, passed through a 60-mesh sieve, and used to determine the chemical composition of A. lancea. Approximately 500 mg of each powdered sample was obtained and placed in a 50-mL centrifuge tube. After adding 25 mL of n-hexane (purity ≥ 95%, Beijing Chemical Works, Beijing, China), ultrasonic extraction was conducted at 40 kHz for 30 min. The samples were centrifuged at 3000 r min− 1 for 10 min, and the supernatants were collected. The pellet residues were suspended, and 25 mL of n-hexane was continuously added and extracted. This process was repeated, and after two rounds of centrifugation, the supernatants were combined, mixed well, and finally diluted with n-hexane to 50 mL. After filtration through an organic membrane, the samples were analyzed by gas chromatography–mass spectrometry (GC–MS)53. A TRACE 1310 GC instrument coupled to a TSQ 8000 mass spectrometer (Thermo Fisher Scientific) was used. An Agilent DB-5MS capillary column (0.25 mm × 30 m, 0.25 μm) was used; the carrier gas was helium gas, and the flow rate was 1 mL min− 1. The injection mode was split flow (ratio 50:1), the injection port temperature was 240 ℃, and the injection volume was 1 μL. The temperature was increased to 120 °C for the first 2 min, and then to 240 °C at a rate of 5 °C min-1, and then maintained at 240 °C for 5 min. The other parameters were as follows: EI (ionization voltage: 70 V, ion source 230 ℃, quadrupole temperature: 150 ℃) and MSD data acquisition mode (scanning ion range m/z 40–500). Figure 5 shows the GC–MS chromatograms of the four major volatile compounds in A. lancea 53.

Figure 5
figure 5

Typical GC–MS chromatogram of the classical volatile oils present in A. lancea.

Full size image

Expression of genes encoding key enzymes involved in the biosynthesis of sesquiterpenoids in the expansion stage

The fresh rhizome samples of the expansion period, a mixture of roots, and leaves of A. lancea were stored in a small liquid nitrogen tank in the field and used for RNA extraction. Approximately 50 mg of leaves and roots were mixed and RNA was extracted with the TransZol UP Plus RNA Kit (TransGen Biotech Co., Beijing, China). The RNA was reverse-transcribed to obtain cDNA using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen Biotech Co.). Primers of genes encoding key enzymes involved in the sesquiterpenoid biosynthesis pathway (DXR, HMGR, and FPPS) were designed based on our transcriptome database (Table S-3). Real-time quantitative PCR (qRT-PCR) was conducted according to the instructions of the TransStart Top Green qPCR SuperMix Kit (TransGen Biotech Co.), and EF-1α served as the internal reference gene.

Measurement of the biomass of A. lancea at different developmental stages

Rhizome biomass, including the fresh weight (FW) and dry weight (DW) of A. lancea at seedling, expansion, and harvest stages was measured. The aerial parts and roots of the harvested A. lancea samples were removed, and the weight of the underground rhizomes was measured. There were ten replicates for each treatment group.

Determination of the photosynthetic parameters of A. lancea leaves at the expansion stage

The leaf photosynthetic parameters of A. lancea, including the intercellular CO2 concentration (Ci), transpiration rate (Tr), net photosynthetic rate (Pn), and stomatal conductance (Gs), were determined under different light intensities using a Li-6400 portable photosynthesis instrument on a sunny day from 9:00 to 11:00 a.m. and 2:00 to 4:00 p.m. when plants were in the expansion stage. A. lancea leaves fully exposed to light and at the same positions were randomly selected. For each group, five plants were selected, and measurements were taken from two leaves on each plant. Each leaf was measured three times.

Determination of the endogenous hormone content in roots and leaves of A. lancea at the expansion stage

The contents of JA, ABA, SA, and GA3 were determined in the roots and leaves of A. lancea53, and each group had three replicates. After removing the surface dirt, the roots and leaves were immediately ground in a liquid nitrogen dry ice bath, weighed, and added to a 2-mL centrifuge tube. Next, 0.5 mL of isopropyl alcohol:water:HCl (2:1:2) extract was added to the centrifuge tube and mixed (4 ℃, 100 r min−1) for 30 min, and 1 mL of dichloromethane was then added to the centrifuge tube and mixed for 30 min. The samples were centrifuged (4 ℃, 13,000 r min−1) for 5 min, and 0.9 mL of the organic phase layer (middle layer) was absorbed and blow-dried with nitrogen. Next, 200 µL of a 50% methanol–water solution was added, followed by centrifugation (4 °C, 13,000 r min-1) for 15 min. The supernatant was then extracted for UPLC-MS/MS analysis. Chromatography was conducted with a Waters ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 µm). The flow phase was 0.05% formic acid water–acetonitrile (Thermo Fisher Scientific). The linear gradient elution was as follows: 0–0.3 min, 10% acetonitrile; 0.3–3 min, 10–60% acetonitrile; 3–6 min, 60–95% acetonitrile; and 6–6.2 min, 10% acetonitrile. The flow rate was 0.6 mL min-1, and the column temperature was 4 ℃. The sample tray temperature was 4 ℃, the injection volume was 10 µL, and the analysis time was 7 min. The mass spectrometry conditions were as follows: the ionization mode was electrospray ion source (ESI), the scanning mode was negative ion scanning, the monitoring method was multi-response monitoring (MRM), the pressure of the air curtain was 30 psi, the ionization voltage was − 4500 V, the temperature of the ion source was 550 ℃, the spray gas pressure was 50 psi, and the auxiliary heating gas pressure was 50 psi. Data processing was conducted with Multi Quant Software.

Analysis of cis-acting elements in the predicted promoter regions of HMGR, DXR, FPPS genes that regulate sesquiterpenoid biosynthesis

Using the genomic data from this experimental platform, the upstream predicted promoter regions of HMGR, DXR, and FPPS was obtained by TBtools, and the potential cis-acting elements were predicted using Plantcare Software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Data analysis

Microsoft Excel 2016 and SPSS v26.0 Software (IBM, Armonk, NY, USA) were used for statistical and correlation analyses. The results were expressed as means ± standard deviations (SD). One-way analysis of variance (ANOVA), followed by the Fisher’s protected least significance difference test, was performed to determine the main effects. The figures in the manuscript were created with GraphPad Prism Software and Adobe Illustrator CS6. The graphs for Pearson correlation network diagram were prepared with Cytoscape Software.