Essential oil is a kind of natural spice oil. The essential oil extracted from Yibin’s eucalyptus is mainly 1,8 eucalyptus oil  , which is widely used in many industries  . How to improve the production of essential oil, more effective, low cost and sustainable access to essential oil, has become an important content and new development direction of C. longepaniculatum resources and plant aromatic oil research. Endophytic fungus has a variety of species and roles     , which play an important role in the synthesis of volatile substances      . Calcium is a universal second messenger for plants in response to biotic and abiotic stresses. Studies have shown that calcium signaling pathways play an important role in the synthesis of volatile substances     .
In this study, the research object is endophytic fungi of the C. longepaniculatum, studies the relationship between the synthesis of the main volatile oils (1,8-eucalyptol, α-terpineol) and amount of Ca2+ by adding endophytic fungi elicitors, to reveal the signal transduction mechanism of endophytic fungal elicitors through Ca2+-mediated synthesis of volatile oil in C. longepaniculatum suspension cells, and is a signal for subsequent study of endophytic fungal elicitors to mediate monoterpenoid synthesis via calcium signaling pathway. The transduction mechanism provides a reference and also has a certain reference significance for the signal transduction mechanism of endophytic fungi through the synthesis of monoterpenoids by other signaling molecules.
2. Materials and Methods
The C. longepaniculatum was collected from the C. longepaniculatum base of Hongyan Mountain in Yibin, and an endophytic fungus 2J1 (Penicillium commune) was isolated from the C. longepaniculatum plant and identified in the early stage. It was preserved in PDA medium  .
2.2.1. Establishing the C. longepaniculatum Suspension Cell System
Collect fresh C. longepaniculatum leaves, and then disinfect them with washing powder water, running tap water, 75% alcohol, sterile water, mercury, and sterile water. The inoculated explants were light cultured at about 23˚C. After the callus induction was completed, subcultured twice. The well-grown and loosely-brown callus was inoculated into a 150 mL Erlenmeyer flask containing 50 mL of B5 medium at 25˚C, 120 r/min rotation speed, shading and shaking culture. 14 d subcultured once, followed by 2 times.
2.2.2. Preparation of Endophytic Fungus Elicitor
The strain was inoculated on potato medium and cultured at 28˚C for seven days. The activated endophytic fungi were inoculated into liquid PDA medium, and cultured at 28˚C, 130 r/min for 7 d. After the fermentation, the cells were separated from the fermentation broth by gauze. After crushing and homogenizing, it was mixed with the fermentation broth, suction filtered, and the filtrate was autoclaved at 121˚C for 20 min to prepare an endophytic fungus inducer. The content of the elicitor sugar was then determined by the fluorenone-sulfuric acid method.
2.2.3. Extraction and Determination of Essential Oil from Endophytic Fungi of C. longepaniculatum
The endophytic fungus of the C. longepaniculatum was removed from the culture flask under sterile conditions every 7 d and dried at 55˚C. Accurately weigh 0.3 g of endophytic fungus, add 4 times (1:4) of cyclohexane overnight cold soak, then ultrasonic extraction for 30 min, centrifugation at 5000 r/min at 25˚C for 4 min, then take the supernatant, the volume was adjusted to 5 mL with cyclohexane. The liquid was extracted with a syringe, and the filter was filtered into a sample bottle to determine the content of the essential oil, which was analyzed by GC-MS. Chromatographic conditions: column temperature 60˚C; HP-5MS column, 30 m × 0.250 mm; injection volume 1 μL; temperature programming: starting from 60˚C, rising to 190˚C at a heating rate of 10˚C/min and maintaining 2 min, then, it is raised to 210˚C for 2 min at a heating rate of 5˚C/min, and then raised to 220˚C for 8 min at a heating rate of 10˚C/min. After injection into GC-MS, the standard curve is obtained, that is, the essential oil content is obtained. The response value was used to calculate the essential oil content (1,8-eucalyptol: Y = 73,900X − 299,200, R2 = 0.999, α-terpineol Y = 51,620X + 162,200, R2 = 0.9992).
2.2.4. Determination of Ca2+ Concentration in Suspension Cells of C. longepaniculatum
The C. longepaniculatum cells were dried at 70˚C, ground into a powder, and weigh 0.2 g into a Nitrifying tube. 10 ml of concentrated sulfuric acid-perchloric acid (5:1) was added to the mixture. Set overnight, digested in the fume hood the next day. The standard solution of Ca2+ was set, and then 2 mL of 5% LaCl3 solution was added separately, and the volume was made up with ion-free water. Take 2 - 10 ml of the solution to be tested and blank into a 100 ml volumetric flask, add 2 mL of 5% LaCl3 solution, and make up to volume with ion-free water. The concentration of Ca2+ was measured using a flame photometer   .
2.2.5. Method for Adding Exogenous Ca2+
The C. longepaniculatum suspension cells cultured for 14 days were added to a Ca2+ solution with a concentration of 10 mmol/l after sterilization.
2.2.6. Addition Method of Ca2+ Channel Blocker LaCl3
The C. longepaniculatum suspension cells cultured for 14 days were used, and the Ca2+ channel blocker LaCl3 was added 20 min before the endogenous fungal inducer or exogenous Ca2+ was added, and the concentration was 20 mmol/L  .
3.1. Endophytic Fungal Elicitors Induce Accumulation of Ca2+ and Essential Oil in Suspension Cells of C. longepaniculatum
40 mg/L of endophytic fungus 2J1 crude elicitor was added to the C. longepaniculatum suspension cell culture until the 7th day, and an equal amount of PDA medium was added as a control. The effects of endophytic fungus 2J1 crude elicitor on Ca2+ concentration and essential oil content in C. longepaniculatum suspension cells within 24 h were shown in Figures 1-3, respectively. The results shown in the figure are the average of 3 independent experiments.
It can be seen from Figure 1 that the C. longepaniculatum suspension cells treated by the 2J1 crude elicitor can produce a change in Ca2+ concentration. The maximum intracellular Ca2+ concentration is 21 h after the elicitor treatment, and the Ca2+ concentration in the treated group at this time is about 1.66 times that of the control group. As shown in Figure 2 and Figure 3, the amount of essential oil produced by the C. longepaniculatum cell suspension cells after the inducer treatment is significantly different from that of the control group, and the content of the cells grows with the growth of the cells, and the cells grow for 21 days, (14 d after treatment), the yield peaked. At this time, the cells treated with the inducer 2J1 produced 1,8-eucalyptus oil in an amount of 11.725 mg/L, and the produced α-terpineol was 4.577 Mg/L. The amount of volatile oil that is subsequently produced gradually decreases.
3.2. Effects of Different Concentrations of Endophytic Fungi Elicitors on Ca2+ Concentration Changes and Essential Oil Accumulation in Suspension Culture Cells
After 7 days of culture of suspension culture of C. longepaniculatum, 2J1 crude elicitors of endophytic fungi with different concentrations (0, 20, 40, 60, 80 mg/L) were added to detect the change of Ca2+ concentration and accumulation of volatile oil in suspension cells of C. longepaniculatum. The amount is shown in Figures 4-6. The results shown in the figure are the average of 3 experiments.
Figure 4 shows that the change in Ca2+ concentration in the suspension cells of C. longepaniculatum is dependent on the concentration of endophytic 2J1 elicitor. When the concentration of elicitor was 0 - 40 mg/L, the concentration of Ca2+ in the suspension cells of C. longepaniculatum increased gradually. When the concentration of inducer was 40 mg/L, the concentration of Ca2+ in the suspension cells of C. longepaniculatum peaked. When the concentration is greater than 40 mg/L, the intracellular Ca2+ concentration gradually decreases. This phenomenon is similar to the dependence of the accumulation of volatile oil in the C. longepaniculatum suspension cells on the endophytic fungal 2J1 elicitor concentration as shown in Figure 5 and Figure 6. This suggests that the Ca2+ signaling pathway may be one of the signaling pathways that mediate endophytic fungal elicitors to promote the accumulation of volatile oil in C. longepaniculatum suspension cells.
Figure 1. Endophytic fungal elicitors induce changes in Ca2+ concentration in suspension cells of C. longepaniculatum.
Figure 2. Effects of endophytic fungal elicitors on the production of 1,8-eucalyptus in suspension cells of C. longepaniculatum.
Figure 3. Effects of endophytic fungal elicitors on the production of α-terpineol in suspension cells of C. longepaniculatum.
Figure 4. Effects of different elicitor concentrations on Ca2+ in suspension cells of C. longepaniculatum.
Figure 5. Effects of different elicitor concentrations on the production of 1,8-eucalyptus in suspension cells of C. longepaniculatum.
Figure 6. Effects of different elicitor concentrations on the production of α-terpineol in suspension cells of C. longepaniculatum.
3.3. Role of Ca2+ Endophytic Fungal Elicitor in Promoting Volatile Oil Synthesis from C. longepaniculatum Suspension Cells
In order to further study the role of Ca2+ in the endophytic fungus 2J1 crude elicitor in the synthesis of volatile oil from the suspension of C. longepaniculatum, the concentration of Ca2+ in the suspension of C. longepaniculatum in 5 groups was detected after 7 days of culture. The situation (Figure 7) and the synthesis of volatile oil (Figure 8), wherein the endophytic fungi 2J1 crude elicitor, exogenous Ca2+, LaCl3 were added at a concentration of 40 mg/L, 10 mmol/L, 20 mmol/L.
Figure 7. The changes of Ca2+ concentration in 5 groups.
Figure 8. Synthesis of volatile oil in 5 groups.
As can be seen from Figure 7 and Figure 8, in the control group without the inducer added, the concentration of Ca2+ in the suspension cells of the C. longepaniculatum was low, and the amount of 1,8-eucalyptus oil that can be detected was only 4.43 mg/L. Adding the endophytic fungus 2J1 crude elicitor can increase the concentration of Ca2+ in the suspension cells of C. longepaniculatum (compared with A and B in Figure 7); it can also promote the accumulation of volatile oil, and the 1,8-eucalyptol under the action of 2J1 elicitor, the amount of synthetic oil was 13.41 mg/L, which was 3.03 times that of the control group (compared with A and B in Figure 8). The addition of exogenous Ca2+ promoted the increase of Ca2+ concentration in the suspension of C. longepaniculatum (compared with A and C in Figure 7); it also promoted the accumulation of volatile oil (compared with A and C in Figure 8), but The promotion effect was inferior to the addition of the endophytic fungus 2J1 crude elicitor experimental group (comparison of A, B and C in Figure 8). Adding endophytic fungus 2J1 crude elicitor and exogenous Ca2+ promoted the increase of Ca2+ concentration in the suspension of C. longepaniculatum, and the Ca2+ concentration reached the maximum (compared with A and D in Figure 7), and also promoted the synthesis of volatile oil (compared with A and D in Figure 8). The concentration of Ca2+ in the suspension cells was increased after add exogenous Ca2+ and LaCl3 compared with the control group, but the content of volatile oil decreased slightly (compared with A and E in Figure 7, A and in Figure 8). This indicates that LaCl3 can inhibit the promotion of volatile oil synthesis by endophytic fungal elicitors and exogenous Ca2+.
It is generally believed that the signal transduction process of endophytic fungal inducer synthesis of secondary metabolites in plant cells is: endophytic fungal elicitor as an extracellular stimulator first recognizes and binds to specific receptors on plant cell membranes, thereby promoting the cells produce specific intracellular messenger substances and regulate the expression of related genes in the nucleus through corresponding signal transduction pathways  , ultimately activating the defensive secondary metabolic system in the cells to promote secondary metabolite synthesis. Therefore, according to this theory, it can be speculated that the change of Ca2+ concentration in cells after 2J1 crude elicitor treatment may be the pre-reaction, and calcium as a common second messenger in plant cells, after a series of process reactions, eventually leading to the accumulation of volatile oil. Under stress conditions such as elicitors, plant cells can sense and transduce external stress signals through a variety of signaling molecules and signaling pathways  . Studies have shown that the calcium signaling pathway is located upstream of the NO signaling molecule  . The calcium signaling pathway is thought to be an important pathway for fungal elicitors to induce plant cell defense responses  . The results of this study indicated that the concentration of Ca2+ reached the peak at 21 h after culture of the endophytic fungus 2J1 crude elicitor. At the same time, the endophytic fungus 2J1 crude elicitor was used to treat the suspension of C. longepaniculatum. After data analysis, it was found that the concentration of Ca2+ and the accumulation of volatile oil in the suspension of C. longepaniculatum showed a similar trend with the change of concentration; further study the role of Ca2+ on the synthesis of volatile oil in the suspension of C. longepaniculatum by 2J1 crude elicitor. The C. longepaniculatum suspension cells may exist a signal pathway regulating the synthesis of volatile oil: calcium signaling pathway. And this pathway has different effects in regulating the different components of synthetic volatile oil. Adding endophytic fungus 2J1 crude elicitor can promote the synthesis of α-terpineol to 3.31 times of the control group, while 1,8-eucalyptol can only increase by 1.90 times; addition of exogenous Ca2+ can also promote the increase of volatile oil, but the effect is not as good as after the addition of inducer. Moreover, the three experimental groups added with LaCl3 showed that the synthesis of volatile oil was significantly inhibited.
Although this study has further studied the regulation mechanism of Ca2+-mediated endophytic fungal elicitors on the synthesis of oil volatile oil, there are two aspects that can be further studied: the endophytic fungus crude inducer is used in this study. The filtrate of the mycelium is inactivated, and the endophytic fungal inducers are divided into four categories: oligosaccharides, glycoproteins, proteins, and unsaturated fatty acids. The components are complex, and it is presumed to be oligosaccharides in this experiment. Oligosaccharides are one of the more common inducers  . However, in order to fully exert the inducer effect, it is necessary to further isolate and purify the endophytic fungus 2J1 crude elicitor, and explore a better method for preparing elicitors  . The addition of LaCl3 could not completely inhibit the accumulation of volatile oil from the endophytic fungus 2J1 crude elicitor on the suspension of C. longepaniculatum, indicating that endophytic fungal elicitors can also cause accumulation of volatile oil by other means, or it may be calcium signal. With multiple calcium signaling pathways, LaCl3 may only inhibit one of the signaling pathways. Therefore, the synthetic pathway of plant secondary metabolites is very complicated, especially the involvement of calcium signaling pathways in secondary metabolic regulation is particularly complicated, and further research is needed.
In this study, the induction time, induced concentration of endophytic fungal elicitors and the addition of exogenous Ca2+ and LaCl3 were investigated. The endophytic fungal elicitors were induced by Ca2+ to mediate the accumulation of volatile oil in the suspension cells. The results indicated that there may be a signal pathway for the regulation of volatile oil synthesis by Ca2+ as a signaling molecule in C. longepaniculatum suspension cells. The addition of exogenous Ca2+ promotes the synthesis of volatile oils in C. longepaniculatum cells. LaCl3 inhibits the accumulation of some Ca2+ in the cells and leads to a decrease in the yield of volatile oil. However, the addition of LaCl3 did not completely inhibit the accumulation of volatile oil in C. longepaniculatum cells caused by endophytic fungal elicitors, suggesting that endophytic fungal elicitors may not completely inhibit all pathways of calcium signaling, and may also through other signal transduction pathways to promote the synthesis of volatile oil in C. longepaniculatum suspension cells, so this aspect needs further study.
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