Introduction

Atractylodes lancea, a traditional Chinese medicinal herb, is a member of the Compositae family, also known as sword-like Atractylodes (Duan and others 2008; Yuan and others 2009). It is believed to act primarily on the digestive system. Volatile oils from A. lancea also show antimicrobial activities and include the characteristic atractylone, β-eudesmol, hinesol, and atractylodin (Wang and others 2009). Endophytes can coexist with their hosts for part of their life cycle without causing obvious symptoms of infection, have great potential to affect the metabolism of hosts (Suryanarayanana and others 2009), promote plant growth (Lewis 2004), and indirectly increase plant resistance to protect plants against environmental stress (Vega and others 2008; Hao and others 2010). Their effects on plant accumulation of medicinal components have received much attention recently (Saunders and Kohn 2009; Wang and others 2012). Unlike pathogens, endophytic fungi do not cause strong hypersensitive reactions in the host. However, long-term colonization can induce the accumulation of various kinds of metabolites in hosts (Li and Tao 2009; Saunders and Kohn 2009). How endophytic fungus–host interactions affect the accumulation of plant secondary metabolites is an intriguing issue.

More and more evidence indicates that nitric oxide (NO), identified as primarily a diffusible signal molecule in animals, plays roles in various physiological processes in plants, such as regulating plant growth, development, and defense responses (Neill and others 2003; Wendehenne and others 2004; Lamotte and others 2005). Gao and others (2012) reported that NO is a signalling molecule of the fungal endophyte Fusarium sp. E5 elicitor-induced isoeuphpekinensin accumulation in Euphorbia pekinensis suspension cells. NO also has been shown to be a key messenger in endophyte-induced volatile oil accumulation in A. lancea plantlets (Wang and others 2011). There are several potential sources of NO in plants, including the NOS-like enzyme nitrate reductase and nonenzymatic sources (Neill and others 2003; del Río and others 2004; Wendehenne and others 2004; Lamotte and others 2005). NO activity has been biochemically characterized in pea leaf peroxisomes; it is strictly dependent on CaM and requires Ca2+ (Corpas and others 2004). However, whether Ca2+–CaM is required for NO-induced secondary metabolism in plants, and, if so, what the regulatory relationship is remain to be determined.

Calcium is a universal second messenger in the responses of plant cells to biotic and abiotic stresses (Snedden and Fromm 2001; Zhang and Lu 2003; Bouche and others 2005). Increases in transient [Ca2+]cyt are sensed by several Ca2+ sensors such as calmodulin (CaM), calcium-dependent protein kinase, and calcineurin B-like protein (Snedden and Fromm 2001; Bouche and others 2005). As one of the most conserved Ca2+ receptors, CaM activates numerous downstream target proteins by binding Ca2+. It has been shown that Ca2+–CaM is involved in plant responses to environmental stimuli such as osmotic stress, temperature stress, hormones, and related microbes (Snedden and Fromm 2001; Yang and Poovaiah 2003). The activated Ca2+–CaM complex binds to target proteins and modulates their activities. Du and Poovaiah (2005) found that two brassinosteroid (BR) biosynthesis-related enzymes, DWF4 and CPD, were Ca2+–CaM binding proteins, and that binding regulates the physiological effects of BR in a wide-ranging manner. However, there is only limited evidence to date of these interactions between Ca2+–CaM and BR.

Using pharmacological and biochemical approaches, we investigated the signalling pathways between Ca2+–CaM, BR, and NO in endophyte-induced volatile oil accumulation in A. lancea plantlets. We found that Ca2+–CaM induced by the endophyte Gilmaniella sp. mediates NO generation and BL concentration and also functions downstream of BL signalling, resulting in the upregulation of volatile oil accumulation in A. lancea plantlets.

Materials and Methods

Plant Materials and Treatments

Meristem cultures of A. lancea (collected at Mao Mountain, Jiangsu Province, China) were established following the methods of Wang and others (2011). The explants were surface sterilized and grown in Murashige and Skoog medium (MS) (Murashige and Skoog 1962) supplemented with 0.3 mg/L naphthalene acetic acid (NAA), 2.0 mg/L 6-benzyladenine, 30 g/L sucrose, and 10 % agar. The rooting medium (1/2 MS) contained 0.25 mg/L NAA, 30 g/L sucrose, and 10 % agar. Explants were kept at 25/18 °C day/night, with a light intensity of 3,400 lm/m2 and a photoperiod of 12 h, and subcultured every 4 weeks. Thirty-day-old rooted plantlets were used for investigations.

Reagents used as specific scavengers or inhibitors, including 10 mM ethylene glycol tetraacetic acid (EGTA), 5 mM LaCl3, 100 μM trifluoperazine (TFP), 100 μM W7 [N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide hydrochloride], 1.25 mM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO), and 50 μM verapamil, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Brassinazole (BRz) (100 μM) was purchased from TCI (Tokyo Kasei Kogyo Co., Ltd., Japan). Exogenous signalling molecules were 1–10 mM CaCl2 and 10 nM brassinolide (TCI). All exogenous signalling molecules and inhibitors were filtered before use through 0.22-μm-diameter microporous membranes. Unless otherwise stated, inhibitors were applied 1 day before the application of signalling molecules or fungal inoculation and sprayed directly on plant leaves and roots.

Fungal Culture and In Vitro Inoculation

The endophytic fungus AL12 (Gilmaniella sp.) was isolated from A. lancea, cultured on potato dextrose agar, and incubated at 28 °C for 5 days. Young plantlets were inoculated with 5-mm AL12 mycelial disks. All treatments were conducted in a sterile environment and performed in triplicate to examine the reproducibility.

Measurement of [Ca2+]cyt and CaM Levels

Atractylodes lancea mesophyll protoplasts were isolated using the method described by Shang and others (2005). Leaf sections were digested in enzyme solution containing 1 % cellulose R-10 (Yakult Honsha, Tokyo, Japan) and 0.4 % macerozyme R-10 (Yakult Honsha, Tokyo, Japan). The isolated protoplasts were washed twice with washing and incubation solution (0.6 M mannitol, 4 mM MES, and 20 mM KCl, pH 5.7). The mesophyll protoplasts were incubated with Fluo-3/AM ester (Molecular Probes, Eugene, OR, USA) at 4 °C for 1 h and then incubated at 25 °C for 1 h in the dark. The incubation solution contained 10 μM Fluo-3/AM ester, 0.4 M mannitol, 5 mM MES (pH 5.7), and 20 mM KCl. Pictures were taken by scanning three times every 30 s using confocal laser scanning microscopy, excited with a 488-nm laser, and fluorescent emissions were filtered by a 515-nm filter to eliminate the autofluorescence of chlorophyll. The fluorescence intensities of these pictures were measured by fluorescence microscopy after establishing a stable baseline.

For isolation of CaM, the A. lancea plantlets were ground in liquid N2 and then homogenized in buffer solution (1:1 w/v): 50 mM Tris–HCl (pH 8.0), 1 mM EGTA, 0.5 mM PMSF, 20 mM NaHSO4, and 0.15 M NaCl. The homogenates were sonicated for 2 min, incubated at 90 °C for 2 min, and then centrifuged at 10,000×g and 4 °C for 30 min. Supernatants were used to measure protein levels and CaM concentration. CaM concentration was determined by enzyme-linked immunosorbent assay (ELISA) following Sun and others (1995).

Determination of Brassinolide Levels

The endogenous brassinolide of A. lancea was extracted following Jana and others (2007). Freeze-dried plant tissues were ground to a fine powder under liquid nitrogen and extracted twice in ice-cold 80 % (v/v) methanol in an ultrasonic bath, 30 min each time (10 mL/g FW). The mixture was centrifuged at 14,000×g and 4 °C for 10 min, then the supernatant was evaporated to 200 μl under a vacuum and stored at −20 °C for analysis. The generation of brassinolide was monitored using a Plant Brassinolide ELISA Kit (Sunred Biological Technology, Shanghai, China) following the manufacturer’s instructions. No fewer than 15 plantlets were used at each time point. All treatments were performed in triplicate.

Real-Time Quantitative PCR Analysis

Total RNA was extracted from plantlet leaves as described by Dong and Beer (2000). First-strand cDNA was synthesized from 1 μg of total RNA (PrimeScript RT Reagent Kit, Takara, Dalian, China). Real-time qPCR was performed using the DNA Engine Opticon 2 Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) and SYBR® Green probe (SYBR Premix Ex Taq system, Takara). The constitutively expressed gene EF1α was used as an internal positive control. The gene-specific primers used to amplify EF1α were 5′-CAGGCTGATTGTGCTGTTCTTA-3′ and 5′-TGGTGGCATCCATCTTGT-3′ (241-bp product), and the primers for CaM1 genes were 5′-ACTTCTTCATCCGTCAGC-3′ and 5′-GGAATGGGACTATTGATTT-3′ (141 bp). The GenBank accession numbers of the CaM1 and EF1α genes are EF090602.1 and GR724649.1, respectively. The thermocycler program was as follows: 90 s at 95 °C; 40 cycles of 30 s at 95 °C, 30 s at 57 °C, and 30 s at 72 °C; and 5 min at 72 °C. To standardize the data, the ratio of the absolute transcript level of CaM1 genes to the absolute transcript level of EF1α was calculated for each sample of each treatment.

Measurement of NO

The generation of NO was monitored using a NO detection kit (Nanjing Jiancheng Bioengineering Inst., Nanjing, China) following the manufacturer’s instructions. Leaf samples (1 g) were ground with 5 mL of 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.2) and the homogenate was centrifuged at 14,000×g for 10 min. The supernatant was used for the NO assays. One unit of NO was defined as the absorbance variation caused by the internal standard of 1 μM NO per gram fresh weight. At least 15 plantlets were assayed for each time point, and all treatments were performed in triplicate.

Extraction and Determination of Volatile Oils

Volatile oils were extracted from whole plantlets of A. lancea, including leaves and rhizomes (0.8–1.6 % oil content in leaves, 2.2–3.4 % in rhizomes) following Zhang and others (2009). The volatile oils were dried with anhydrous sodium sulfate and stored in dark glass bottles at 4 °C for gas chromatography (GC) analysis. GC determination was carried out using a 1890 series GC (Hewlett-Packard, Palo Alto, CA) equipped with a flame ionization detector. A DB-5 ms (30 m × 0.25 mm × 0.25 μm) column (Agilent, Santa Clara, CA, USA) was used with the following temperature program: column held at 60 °C for 1 min after injection, increased by 10 °C/min to 190 °C, held for 2 min, increased by 5 °C/min to 210 °C, held for 2 min, increased by 10 °C/min to 220 °C, and held for 8 min. Nitrogen was used as carrier and the flow rate was 4 mL/min. Four main components of the volatile oils, atractylone, hinesol, β-eudesmol, and atractylodin, were quantitatively analyzed following the method of Fang and others (2009); their retention times were 14.57, 15.24, 16.21, and 22.18 min, respectively.

Statistical Analyses

Data were compiled using Microsoft Excel (Redmond, WA, USA). Values are presented as mean ± SD (standard deviation) of three replicates for each treatment. One-way ANOVA and Duncan’s multiple-range test were used to identify significant differences (SPSS ver. 13.0, SPSS Inc., Chicago, IL, USA).

Results

Ca2+–CaM is Required for Fungus-Induced Volatile Oil Production

To investigate the possible role of Ca2+–CaM in fungal endophyte-induced volatile oil accumulation, A. lancea plantlets were inoculated with Gilmaniella sp. AL12. When protoplasts in the mesophyll cells from A. lancea leaves were isolated and loaded with Fluo-3/AM. The relative fluorescence intensity increased significantly 4 days after endophyte inoculation (Fig. 1a) , indicating that the fungus may elevate [Ca2+]cyt concentration in A. lancea cells. Concurrently, the levels of CaM also began increasing significantly 4 days after fungus inoculation (Fig. 1c). To investigate whether Ca2+–CaM was involved in the fungus-induced volatile oil accumulation, the Ca2+ chelator EGTA, channel blockers La3+ and verapamil, and CaM antagonists W7 and TFP were applied individually or in combination. Compared with the fungus inoculation group, application of La3+ and verapamil and EGTA significantly reduced the fungus-induced [Ca2+]cyt concentration and the fungus-triggered volatile oil production, but none of the La3+, verapamil, or EGTA alone could affect the volatile oil production without fungus inoculation (Fig. 1b). Compared with the fungus inoculation group, W7 and TFP significantly suppressed the fungus-induced CaM level and the fungus-triggered volatile oil production, but it could not affect the volatile oil production when they were applied alone (Fig. 1d). These results suggested that Ca2+–CaM is important for fungus-induced volatile-oil synthesis in A. lancea plantlets.

Fig. 1
figure 1

The influence of [Ca2+]cyt and CaM on endophytic fungus-induced volatile oil accumulation of A. lancea. a Changes in relative fluorescence intensity of [Ca2+]cyt. The mesophyll protoplasts loaded with Fluo-3/AM were observed by LSCM. The fluorescence intensity was measured by fluorescence microscopy and calculated as the average value of those obtained by scanning over 100 protoplasts from five different experiments. b Effects of the Ca2+ chelator and channel blockers on volatile oil accumulation after 18 days. The Ca2+ chelator EGTA (10 mM) and channel blockers (1 mM LaCl3, 50 μM verapamil) were added 1 day before fungal inoculation. c Changes in the content of CaM. d Effects of CaM antagonists on volatile oil accumulation after 18 days. CaM antagonists W7 100 μM and TFP 100 μM were added 1 day before fungal inoculation. Thirty-day-old plantlets of Atractylodes lancea were incubated with 5-mm mycelial disks or PDA disks (control). Values are the mean of three independent experiments. Bars with different lower-case letters were significantly different (one-way ANOVA, Duncan’s multiple range test, P < 0.05)

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Brassinolide is Required for Fungus-Induced Volatile Oil Accumulation

To investigate whether brassinolide was involved in fungus-induced volatile oil accumulation, A. lancea plantlets were inoculated with Gilmaniella sp. AL12. Compared with the control, brassinolide levels significantly fluctuated in the plantlets inoculated with the fungus (Fig. 2a). The brassinolide content in the plantlets began to increase 6 days after fungus infection. The maximum concentration of brassinolide in plant tissues induced by the fungus was approximately eight times that in the control. When pretreated with the brassinosteroid biosynthesis-specific inhibitor brassinazole, the volatile oil accumulation induced by fungus was significantly suppressed, to 74.48 % of that in the fungus treatment, whereas brassinolide generation was completely blocked (Fig. 2b). Jana and others (2007) successfully applied the ELISA method to the measurement of BR; the highest cross-reactivity with nontarget BR analogs is 1.3 %. Therefore, the above data indicate that brassinolide may be involved in fungus-induced volatile oil accumulation.

Fig. 2
figure 2

The influence of brassinolide on endophytic fungus-induced volatile oil accumulation of A. lancea. a Brassinolide content at 2-day intervals. b Effects of brassinazole (BRz) volatile oil accumulation after 18 days. An inhibitor (100 μM BRz) was added 1 day before fungal inoculation. Thirty-day-old plantlets of Atractylodes lancea were incubated with 5-mm mycelial disks or PDA disks (control). Values are the mean of three independent experiments. Bars with different lower-case letters were significantly different (one-way ANOVA, Duncan’s multiple range test, P < 0.05)

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Ca2+–CaM Functions Both Upstream and Downstream of Brassinolide Production Induced by Fungus

Because both Ca2+–CaM and brassinolide signalling may be involved in volatile oil accumulation induced by the endophyte, interactions between Ca2+–CaM and brassinolide were further investigated. When treated with 10 mM CaCl2, brassinolide levels increased steadily in plantlets and peaked at 48 h (Fig. 3a. The CaCl2-induced increase was also dose-dependent in the concentration range of 1–10 mM CaCl2 compared with the control (Fig. 3b). Pretreatments with the CaM antagonists W7 and TFP substantially reduced the brassinolide accumulation induced by CaCl2 treatment (Fig. 3b). These results clearly indicate that Ca2+ can induce increased production of brassinolide in A. lancea. To investigate whether brassinolide also affects the levels of Ca2+–CaM, exogenous brassinolide was used. Compared with the control, we observed an increase in the Ca2+-sensitive fluorescence of the protoplasts, which were isolated from A. lancea leaves, treated with brassinolide, and loaded with Fluo-3/AM, a Ca2+-sensitive fluorescent probe (Zhang and others 1998), and the fluorescence intensity was similar to fungus inoculation (Fig. 4a). In addition, pretreatments with BRz were not able to reduce the increase in fluorescence intensity induced by fungus treatment (Fig. 4a). Changes in CaM content and CaM1 gene expression under the same conditions are consistent with [Ca2+]cyt (Fig. 4b, c). These results suggest that Ca2+–CaM functions both upstream and downstream of brassinolide production in A. lancea plantlets induced by the endophyte.

Fig. 3
figure 3

Brassinolide production in response to CaCl2 treatment in plantlets of A. lancea. a Time course for CaCl2-induced brassinolide production in A. lancea. The plantlets were treated with 10 mM CaCl2 for various times. b Dose dependence for CaCl2-induced brassinolide production and effects of pretreatments with CaM antagonists on fungus-induced brassinolide production. Inhibitors (100 μM BRz, 100 μM W7, 100 μM TFP) were added 1 day before fungal inoculation or CaCl2 treatment. Brassinolide content was detected 18 days after various treatments. Values are the mean of three independent experiments. Bars with different lower-case letters were significantly different (one-way ANOVA, Duncan’s multiple range test, P < 0.05)

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Fig. 4
figure 4

Effects of pretreatments with brassinolide and brassinolide inhibitor on Ca2+, CaM, and CaM1 gene expression in A. lancea plantlets inoculated with endophytic fungus. a Changes in [Ca2+]cyt concentration. The mesophyll protoplasts loaded with Fluo-3/AM were observed by LSCM. b Changes in CaM content. c Expression levels of CaM1 genes by real-time PCR analysis. Leaves were used for RNA isolation. Relative expression is based on the expression of EF1α. Changes in [Ca2+]cyt, CaM, and CaM1 gene expression were investigated 4 days after various treatments. Values are the mean of three independent experiments. Bars with different lower-case letters were significantly different (one-way ANOVA, Duncan’s multiple range test, P < 0.05)

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NO is Involved in Ca2+–CaM Pathway and has no Interaction with the BL Pathway

Previous work has shown that NO is a key upstream messenger in the signalling pathway regulating endophyte-induced volatile oil accumulation in A. lancea plantlets (Wang and others 2011; Ren and Dai 2012). To investigate whether NO is involved in the Ca2+–CaM and brassinolide interaction, NO generation was measured in A. lancea plantlets pretreated with CaCl2, exogenous brassinolide, Ca2+ channel blockers (LaCl3 and verapamil), CaM antagonists (W7 and TFP), and a brassinolide biosynthesis inhibitor (BRz). NO production was continually enhanced by CaCl2 within 12 days and maintained at a concentration higher than that in the control (Fig. 5a). The Ca2+ channel blockers were able to inhibit NO production in inoculated plantlets, but the NO scavenger cPTIO was not able to inhibit Ca2+ production in inoculated plantlets (Fig. 5b), showing that NO may act as a downstream signal of Ca2+. In addition, the brassinolide inhibitor BRz was not able to inhibit NO production, and cPTIO was also unable to inhibit brassinolide production with fungus inoculation (Fig. 5c). Although NO levels could be enhanced by exogenous brassinolide treatment, the enhancement was blocked by the CaM antagonist TFP combined with the Ca2+ channel blockers verapamil and EGTA (Fig. 5a). These results imply that NO may act as a downstream signal of Ca2+–CaM and may not direct regulation on the brassinolide pathway.

Fig. 5
figure 5

Interactions between NO and Ca2+–CaM or brassinolide signaling pathways induced by endophytic fungus. Endogenous NO, [Ca2+]cyt, and brassinolide were measured at 12, 4 and 18 days, respectively, after different treatments. a, b Interactions between NO and [Ca2+]cyt pathways. Inhibitors were 1.25 mM cPTIO, 1 mM LaCl3, and 50 μM verapamil. c Interactions between NO and brassinolide pathways. Inhibitor was 1.25 mM cPTIO or 100 μM BRz. All inhibitors were added 1 day before fungus inoculation. Values are means of three independent experiments. Bars with different lower-case letters were significantly different (one-way ANOVA, Duncan’s multiple range test, P < 0.05)

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Discussion

Secondary metabolite accumulation is a common plant response to biotic or abiotic environmental stresses, and secondary messengers are widely employed to mediate the accumulation of plant secondary metabolites (Reymond and Farmer 1998; John and Jeffery 2000; Hahlbrock and others 2003; Mur and others 2006). Our study demonstrates that the fungus Gilmaniella sp. can induce calcium–calmodulin and brassinolide production (Figs. 1a and 2a), and promote the accumulation of volatile oils in host plantlets (Table 1). Acting as important signalling molecules, calcium–calmodulin and brassinolide play important roles in regulating the volatile oil production induced by the fungus. By using relevant scavengers and inhibitors, the links between signalling molecules are blocked and, in turn, the accumulation of related secondary metabolites is altered.

Table 1 Accumulation of volatile oils by Atractylodes lancea over time
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BRs are a class of plant steroid hormone involved in the regulation of growth, development, and various physiological responses (Bajguz 2007). In addition, BRs play important roles in inducing plant tolerance to various abiotic stresses (Kagale and others 2007; Xia and others 2009). Brz is a specific inhibitor for DWF4, a cytochrome P450 monooxygenase of the BR biosynthetic pathway (Asami and others 2001; Bajguz and Asami 2005), and 5-μM Brz treatment can alter the endogenous BR levels (Xia and others 2009). Pretreatment with Brz suppressed the endogenous level of brassinolide, and the degree of volatile oil accumulation induced by endophyte treatment (Fig. 2b). CaM (calmodulin) transduces cytosolic Ca2+ changes into cellular responses by changing its conformation in the presence of Ca2+ and concomitantly binding and altering the activities of a series of target proteins (Bouche and others 2005; DeFalco and others 2009). The effects of CaM antagonists such as TFP and W7 have suggested a role for the Ca2+–CaM system in the responses to hormones (Schroeder and others 2001), light (Frohnmeyer and others 1999), abiotic stresses (Gong and others 1998), and microbial elicitors (Blume and others 2000). Pretreatment with TFP or W7 suppressed endogenous brassinolide production induced by endophytic fungus inoculation, and 1–10 mM CaCl2 was able to elevate endogenous brassinolide production (Fig. 3b). Plant defense reactions, including enzyme activation and the production of secondary metabolites, are not regulated by individual signalling pathways but by a collaborative regulation between multiple processes (Xu and Dong 2006). Exogenous brassinolide treatment was able to induce [Ca2+]cyt concentration and CaM1 gene expression and increase CaM levels. However, 100-mM Brz treatment, which was effective in suppressing endogenous brassinolide production, had almost no effect on Ca2+ concentration, CaM1 gene expression, or CaM levels (Fig. 4). Moreover, combining Ca2+–CaM pathway inhibitors with BRz was more effective in suppressing fungus-induced volatile oil accumulation than BRz treatment alone (Fig. 2b). These results indicate that Ca2+–CaM induced by Gilmaniella sp. may mediate BL concentration and also functions downstream of BL signalling, resulting in upregulation of volatile oil accumulation in A. lancea plantlets.

Our previous work demonstrated that NO acts through SA- and H2O2-dependent pathways as an upstream signal in mediating volatile oil accumulation induced by the fungus in A. lancea plantlets (Ren and Dai 2012). In the study by Gao and others (2012), NO seems to function as an important signalling molecule of the endophyte Fusarium sp. E5 elicitor, which induces isoeuphpekinensin accumulation in E. pekinensis suspension cells. We thus used effective inhibitors of NO, Ca2+–CaM, and brassinolide pathways to explore the possible relationships between NO, Ca2+–CaM, and brassinolide. We found that BRz and cPTIO failed to suppress the production of other signalling molecules (Fig. 5c), which could indicate that the two pathways may not be directly related. The enhancement of brassinolide on NO production was almost completely blocked by Ca2+–CaM inhibitors (TFP+VERA+EGTA) (Fig. 5a), confirming that NO is regulated by Ca2+, independent of the brassinolide pathway. Moreover, the enhancement between Ca2+–CaM and brassinolide may indicate a regulation loop (Fig. 6).

Fig. 6
figure 6

Proposed cross-talk between signaling pathways for volatile oil accumulation induced by endophytic fungus in A. lancea plantlets

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The complex relationships between the diverse signal transduction pathways imply their key roles in regulating the accumulation of volatile oil induced by the fungus. However, it seemed that the stimulating effects on the synthesis of each component of volatile oil by the fungus were not of the same strength. The four detectable components, atractylone, β-eudesmol, hinesol, and atractylodin, were enhanced 3.78-fold, 0.44-fold, 1.82-fold, and 0.75-fold, respectively, in their maximum amount by the endophyte Gilmaniella sp. (Table 1). The selective initiation of specific secondary metabolite accumulation may be due to the unique signalling pathway launched by the endophyte or to the diversity of different secondary metabolites.

Volatile oil is the pharmaceutically active ingredient of A. lancea, and the low yield of active pharmaceutical ingredients is one of the greatest challenges for medicinal plant culture. Our study showed the potential signal transduction pathways in which the endophytic fungus Gilmaniella sp. affects the accumulation of volatile oil in plantlets of A. lancea. This information will aid in the understanding of the relationships between fungal endophytes and their host plants.