Jasmonic acid is involved in the signaling pathway for fungal endophyte-induced volatile oil accumulation of Atractylodes lancea plantlets

Background Jasmonic acid (JA) is a well-characterized signaling molecule in plant defense responses. However, its relationships with other signal molecules in secondary metabolite production induced by endophytic fungus are largely unknown. Atractylodes lancea (Asteraceae) is a traditional Chinese medicinal plant that produces antimicrobial volatiles oils. We incubated plantlets of A. lancea with the fungus Gilmaniella sp. AL12. to research how JA interacted with other signal molecules in volatile oil production. Results Fungal inoculation increased JA generation and volatile oil accumulation. To investigate whether JA is required for volatile oil production, plantlets were treated with JA inhibitors ibuprofen (IBU) and nordihydroguaiaretic acid. The inhibitors suppressed both JA and volatile oil production, but fungal inoculation could still induce volatile oils. Plantlets were further treated with the nitric oxide (NO)-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO), the H2O2 inhibitors diphenylene iodonium (DPI) and catalase (CAT), and the salicylic acid (SA) biosynthesis inhibitors paclobutrazol and 2-aminoindan-2-phosphonic acid. With fungal inoculation, IBU did not inhibit NO production, and JA generation was significantly suppressed by cPTIO, showing that JA may act as a downstream signal of the NO pathway. Exogenous H2O2 could reverse the inhibitory effects of cPTIO on JA generation, indicating that NO mediates JA induction by the fungus through H2O2-dependent pathways. With fungal inoculation, the H2O2 scavenger DPI/CAT could inhibit JA generation, but IBU could not inhibit H2O2 production, implying that H2O2 directly mediated JA generation. Finally, JA generation was enhanced when SA production was suppressed, and vice versa. Conclusions Jasmonic acid acts as a downstream signaling molecule in NO- and H2O2-mediated volatile oil accumulation induced by endophytic fungus and has a complementary interaction with the SA signaling pathway.


Background
Atractylodes lancea, a member of the Compositae family, is a traditional Chinese medicinal plant [1,2]. Volatile oils from A. lancea show antimicrobial activities as well. These oils comprise active secondary metabolites, including the characteristic components atractylone, βeudesmol, hinesol, and atractylodin [3]. Secondary metabolites, such as terpenes, flavonoids, and alkaloids, are believed to be involved in plant responses to many biotic and abiotic stresses [4][5][6]. Another plant defense response is the activation of multiple signaling events [7,8]. For example, jasmonic acid (JA) biosynthesis by plants is induced by pathogen infection and elicitor treatment [9], and salicylic acid (SA) is involved in activating distinct sets of defense-related genes [10], such as those that encode pathogenesis-related (PR) proteins [11]. Also, many signaling molecules have been revealed to be involved in secondary metabolism [12][13][14].
Endophytes can coexist with their hosts and have great potential to affect the hosts' metabolism [15]; their effects on plant accumulation of medicinal components have received much attention recently [16,17]. Unlike pathogens, endophytic fungi do not cause strong hypersensitive reactions in the host. But long-term colonization can induce various kinds of metabolites to accrue in hosts [17,18]. How endophytic fungus-host interactions affect the accumulation of plant secondary metabolites is an intriguing issue.
Jasmonic acid is a well-characterized plant signaling molecule that mediates plant defense responses [19] by responding to microbial infection and elicitor treatment [20]. Kunkel et al.. [21] found that fungal elicitor caused rapid increases in JA production, secondary metabolite biosynthetic gene expression, and secondary metabolite accumulation in many plants. Exogenous JA application enhanced gene expression of secondary metabolite biosynthetic pathways, while the fungal elicitor-induced secondary metabolite accumulation could be abolished by JA synthesis inhibitors [13]. Most plant defense responses are regulated by many signal molecules, and "cross-talk" among multiple signaling pathways is important in plant cell signal transduction networks [21]. An increasing number of studies have shown that these signals do not function entirely independently; rather, they are influenced the magnitude or amplitude of various other signals [22].
Although interactions between SA-and JA-mediated signaling pathways have been reported to enhance the expression of plant defense-related genes, studies on interactions between JA and multiple signaling pathways (nitric oxide, hydrogen peroxide, and SA) in mediating plant secondary metabolite accumulation are rare. In this work, we report that JA acts as a downstream signal of nitric oxide (NO)-and hydrogen peroxide (H 2 O 2 )mediated volatile oil accumulation in A. lancea plantlets induced by endophytic fungus Gilmaniella sp. AL12. Furthermore, we reveal an unusual complementary relationship between JA and SA in mediating the biosynthesis of medicinal plant secondary metabolites.

Plant materials and treatments
Meristem cultures of Atractylodes lancea (collected in Maoshan, Jiangsu Province, China) were established according to Wang et al. [22]. The explants were surface sterilized and grown in MS medium [23] supplemented with 0.3 mg/L naphthaleneacetic acid (NAA), 2.0 mg/L 6-benzyladenine, 30 g/L sucrose, and 10% agar in 150 mL Erlenmeyer flasks. Rooting medium (1/2 MS) contained 0.25 mg/L NAA, 30 g/L sucrose, and 10% agar. All media were adjusted to a pH of 6.0 before being autoclaved. Cultures were maintained in a growth chamber (25/18°C day/night, with a light intensity of 3400 lm/ m 2 and a photoperiod of 12 h) and subcultured every four weeks. Thirty-day-old rooting plantlets were used for all treatments.

Fungal culture and treatments
The endophytic fungus AL12 (Gilmaniella sp.) was isolated from A. lancea, cultured on potato dextrose agar, and incubated at 28°C for five days [24]. Thirty-day-old plantlets were inoculated using 5-mm AL12 mycelial disks. An equal size of potato dextrose agar was used as a control. All treatments were conducted in a sterile environment and replicated at least three times to examine reproducibility.

Measurement of H 2 O 2 and NO
Thirty-day-old plants were incubated with fungal mycelia disks with or without inhibitors and were harvested 18 d later for determination of NO or H 2 O 2 . Inhibitors were 1.25 mmol L -1 cPTIO, 5.25 mKat L -1 CAT or 3 mmol L -1 DPI.
The generation of H 2 O 2 by A. lancea plantlets was measured by chemiluminescence in a ferricyanidecatalyzed oxidation of luminol according to Schwacke and Hager [25], with modification. Leaf samples (1 g) were ground with 5 ml double distilled water. The homogenate was centrifuged at 13,000 g for 10 min, then 100 μL supernatant, 50 μL luminol (5-amino-2,3-dihydro-l,4-phthalazinedione), and 800 μL phosphate-buffered saline were mixed in a cuvette. The reaction was initiated with 100 μL K 3 [Fe(CN) 6 ]. To compare independent experiments, we used H 2 O 2 as an internal standard. Fifty microliters of H 2 O 2 (1 μM, freshly prepared) was added to the assay mixture containing 750 μL potassium phosphate buffer. One unit of H 2 O 2 was defined as the chemiluminescence caused by the internal standard of 1 μM H 2 O 2 per gram fresh weight.
The generation of NO was monitored using a NO detection kit (Nanjing Jiancheng Bio-engineering Inst., Nanjing, China) according to 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.

Measurement of SA
Thirty-day-old plants were incubated with fungal mycelia disks with or without inhibitors and were harvested 18 d later for determination of SA. Inhibitors were 1 mmol L -1 PAC or 2.5 mmol L -1 AIP.
Salicylic acid was extracted followed the method of Verberne et al. [26], with some modifications. Five grams of whole plantlets was ground in liquid nitrogen and extracted in 2 ml methanol by sonication. After centrifugation at 14,000 g for 5 min, the supernatant was rotary evaporated, and the residue was resuspended in 250 μl of 5% trichloroacetic acid. The mixture was re-extracted with 800 μl acetic acid ester: cyclohexane (1:1 v/v). Finally, the organic phase was rotary evaporated until dry, dissolved with 600 μl HPLC mobile phase (methanol: 2% acetic acid: H 2 O, 50:40:10, v: v: v), and filtered with a 0.22-μm microporous membrane for determination.

Extraction and determination of volatile oils and JA
Thirty-day-old plantlets of Atractylodes lancea were incubated with 5-mm mycelial disks or PDA disks (control). Inhibitors (0.1 mmol L -1 IBU or NDGA) were added 1 d before fungal inoculation for JA determination.
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), according to Zhang et al. [27]. The volatile oils were dried with anhydrous sodium sulfate and stored in dark glass bottles at 4°C for gas chromatograph (GC) analysis.
Following Juergen et al. [28], JA was extracted by grinding plant material (1 g) frozen in liquid nitrogen and extracting with H 2 O: acetone (30:70, v:v). Samples were store in dark glass bottles at −21°C for GC analysis.
GC determination was carried out using an 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 according to the method of Fang et al. [29]; their retention times were 14.57, 15.24, 16.21, and 22.18 min, 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 the alHMGR genes to the absolute transcript level of EF1α was calculated for each sample of each treatment.

Statistical analyses
Data were compiled using Microsoft Excel (Redmond, WA, USA). The values were represented as mean ± SD of three replicates for each treatment. Student's t-test, one-way ANOVA, and Duncan's multiple range test were used to identify significant differences (SPSS ver. 13.0, SPSS Inc., Chicago, IL, USA).

Dependence of JA in fungus-induced volatile oil accumulation
The JA contents of the plantlets increased significantly after endophytic fungus inoculation ( Figure 1A), indicating that the fungus may trigger JA biosynthesis in the plantlets. Concurrently, the total amount of volatile oils increased significantly (Table 1). Both IBU and NDGA are inhibitors of the octadecanoid pathway that synthesizes JA and are usually applied in plant systems as JAspecific inhibitors [13]. To investigate whether JA was involved in the fungus-induced volatile oil accumulation, IBU and NDGA were applied; as shown in Figure 1B, both inhibitors suppressed not only the fungus-induced JA generation, but also the fungus-triggered volatile oil production. The results suggested that JA was important for fungus-induced volatile-oil synthesis in A. lancea plantlets. However, volatile oils in the A. lancea plantlets treated with both fungus and JA inhibitors could still accumulate, compared with the control, even though JA generation was lower than control ( Figure 1B), implying that fungus-induced volatile oil synthesis is not solely dependent on the JA signaling pathway.

JA acts as a downstream signal of NO and H 2 O 2 pathway
Previous results showed that JA is not the sole signaling pathway involved in fungus-induced volatile oil synthesis; NO, H 2 O 2 , and SA are also known to mediate this process in A. lancea plantlets [22]. To investigate a possible relationships between JA and one or more of these other pathways, A. lancea plantlets were treated with the NO-specific scavenger cPTIO, the membrane NADPH oxidase inhibitor DPI/CAT, the SA inhibitor PAC/AIP, IBU, and fungal inoculation. The NO scavenger cPTIO could inhibit JA production in inoculated plantlets, but IBU could not inhibit NO production (Figure 2A), showing that JA may act as a downstream signal of NO. Exogenous H 2 O 2 could reverse JA suppression, implying that JA is mediated by NO though H 2 O 2 in endophyte-induced volatile-oil accumulation. In  Thirty-day-old plantlets were incubated with 5-mm mycelia disks or with an equal size of potato dextrose agar (control). Data are mean ± standard deviation (SD) of triplicate samples. Within each row, values followed by different lower-case letters were significantly different (one-way ANOVA, Duncan's multiple range test, P <0.05).
addition, the H 2 O 2 inhibitor DPI/CAT could inhibit JA production, but IBU could not inhibit H 2 O 2 production with inoculation ( Figure 2A). The one-way dependence of JA on H 2 O 2 confirmed that H 2 O 2 was the intermediary factor between JA and NO.
Paclobutrazol is an effective SA biosynthesis-related benzoic acid hydroxylase (BA2H) inhibitor [31] that also inhibits gibberellin biosynthesis [32]. Therefore, we also used AIP, a specific SA biosynthesis-related phenylalanine ammonialyase (PAL) inhibitor [33,34], to confirm that SA generation was suppressed. Interestingly, PAC and AIP could abolish the suppression of JA by DPI/ CAT with fungus inoculation ( Figure 2B). This result implied that the SA and JA signaling pathways were closely linked in endophyte-induced volatile-oil accumulation in A. lancea plantlets.

Complementary interactions between JA and SA in fungus-induced volatile-oil accumulation
To further investigate the relationship between JA and SA, gradient concentrations of the JA-inhibiter IBU and the SA-inhibitors PAC and AIP were applied. As shown in Figure 3, the fungus-induced JA level of the plantlets decreased gradually as IBU concentration increased, but both SA accumulation and volatile oil content were enhanced as well, although the amounts did not exceed those obtained with fungal inoculation alone. Similarly, SA levels in plantlets were inhibited by 0.1, 1, and 2.5 mmol L -1 AIP and by 3 mmol L -1 PAC, whereas JA was enhanced significantly ( Figure 3A). Volatile oil accumulation was enhanced by 2.5 mmol L -1 AIP and 3 mmol L -1 PAC ( Figure 3B). The results suggested that JA may have a complementary interaction with SA to mediate fungal endophyte-induced volatile-oil accumulation. However, combining IBU and paclobutrazol could not completely inhibit volatile oil synthesis. We added the H 2 O 2 -inhibitor DPI/CAT to IBU and paclobutrazol, which reduced volatile-oil accumulation to the level of the control. The results suggested that H 2 O 2 , SA, and JA may work simultaneously in fungus-induced volatile-oil synthesis in A. lancea plantlets.

Dependence of fungus-induced sesquiterpenoid production on JA production
The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate, which is the key step in the terpenoid biosynthesis pathway in plants [35,36]. We further investigated the possible mediating role of JA on HMGR gene expression. The results showed that exogenous JA could strongly stimulate HMGR gene expression ( Figure 4A). Three sesquiterpenoid components of A. lancea volatile oils, atractylone, β-eudesmol, and hinesol, were all induced by JA and suppressed by IBU with fungal inoculation ( Figure 4B).

Discussion
Secondary metabolite accumulation is a common plant response to biotic or abiotic environmental stress, and secondary messengers are widely employed to mediate the accumulation of plant secondary metabolites. This work demonstrated that the fungus Gilmaniella sp. can induce JA production and promote the accumulation of volatile oils in host plantlets. As an important signal molecule, JA plays key roles in regulating the induction of volatile oils by the endophytic fungus. The specific inhibitors IBU and NDGA could block the JA signaling pathway and reduce the accumulation of related metabolites. Our previous study showed that NO, H 2 O 2 , and SA acted as signal molecules to mediate the accumulation of volatile oils in suspension cells of A. lancea caused by endophytic fungal elicitor [22]. Thus, the possible relationships between JA and other known signaling pathways in the accumulation of secondary metabolites were further investigated.
Cross-talk between different signal transduction pathways, as opposed to single signaling pathways, mediates gene expression and the production of secondary metabolites during plant defense responses [37,38]. Hydrogen peroxide has been reported to be a possible upstream signal for NO production in mung bean plantlets [39]. Nitric oxide also can mediated fungal elicitor-induced taxol biosynthesis in Taxus chinensis suspension cells through reactive oxygen signaling pathways, stimulate SA accumulation in tobacco cell cultures, and induce PAL expression via an SA independent pathway [31,40,41]. Moreover, our previous work demonstrated that NO mediates volatile oil accumulation induced by the fungus through SA-and H 2 O 2 -dependent pathways. Hydrogen peroxide can enhance SA production but does not act as upstream signal molecule [22]. The present work showed that endophytic fungus-induced JA was directly mediated by H 2 O 2 and acted as a downstream signal molecule for both H 2 O 2 and NO pathways.
In our study, JA had an unusual complementary interaction with the SA signaling pathway. Jasmonic acid is commonly postulated to act antagonistically on the SA signaling pathway and on the expression of SA-dependent genes [42,43]. Other studies have shown that SA is a potent suppressor of JA signaling pathways and JAdependent defense gene expression in various pharmacological and genetic experiments [44,45]. In addition, both JA and SA are important signaling molecules in plant defense responses, such as the activation of distinct sets of defense-related genes and the development of systemic acquired resistance [21,46]. Our results showed that when JA biosynthesis was suppressed by the inhibitor IBU, accumulation of SA was enhanced to compensate for the loss of JA-mediating function in fungus-triggered volatile-oil production. Similarly, JA production/signaling could substitute for the SA pathway when SA accumulation was impaired.

Conclusions
The value of medicinal herbs relies mainly on the accumulation of active pharmaceutical ingredients; low yield is the main challenge to producing high-quality herbs. In this work, we demonstrated that JA acts as a downstream signaling molecule in NO-and H 2 O 2 -mediated volatile oil accumulation induced by endophytic fungus and has a complementary interaction with the SA signaling pathway and clarified that HMGR gene expression was significantly stimulated by JA along with increasing sesquiterpenoid components. This information will help to better understand the relationships between fungal endophytes and their host plants. Furthermore, it also suggests strategies to improve the quality of medicinal herbs.