Signal Molecules Crosstalk and the Critical Role of Jasmonic Acid in Triterpenoid Synthesis Inducement of Aspergillus Niger Elicitor in Suspension Cultured Cyclocarya Paliurus Cells


 Background: Cyclocarya paliurus (C. paliurus) leaves contain multiple health benefical metabolites, and therefore are often used in functional foods and Chinese herbal medicines. However, breeding difficulties confines its utilization. Consequently, cell suspension culture was developed to produce the bioactive secondary metabolites of C. paliurus leaves, but the content was comparatively low. In the present paper, Aspergillus Niger Elicitor (ANE) was used to stimulate the synthesis of triterpenoids in the suspension cultured C. paliurus cells, and the signal molecules crosstalk involved in this elicitation was further studied to interpret the underlying mechanism. Results: Total triterpenoids accumulation of the cultured C. paliurus cells elicited by 200 µg/mL ANE was 9.17 times higher than that of the untreated cells. Nitric oxide (NO), hydrogen peroxide (H2O2) and jasmonic acid (JA) played important roles in the elicitation as signal molecules. Under the ANE stimulation, the concentrations of NO, H2O2 and JA all increased significantly, but presented different change profiles and peaked at different times. Based on series experiments of NO quenching by C-PTIO, H2O2 blocking by DMTU, and JA synthesis inhibition by IBU and NDGA, together with exogenous NO, H2O2 and JA addition experiments, it was deduced that ANE improved triterpenoids synthesis in the suspension cultured C. paliurus cells via a complex signal transduction network, in which three deduced and three hypothetical signal transduction pathways might be involved. JA was not only the junction of NO and H2O2 signal pathways, but also the critical point in the whole signal network. RNA-seq analysis showed that a total of 3 candidate JA synthesis pathway genes including 1 LOX and 2 OPR were found to be significantly up-regulated under the ANE stimulation, along with 5 down-regulated JAZs and 1 up-regulated JAR1 regulating to JA signal transduction.Conclusions: ANE can significantly increase the triterpenoids synthesis in the suspension cultured C. paliurus cells. RNA-seq analysis validated the pivotal role of JA in this ANE elicitation. Our results provided references for the further studies on triterpenoids synthesis in C. paliurus under ANE treatment.

A series of researches focused on the C. paliurus resources utilization had been carried out in our laboratory, such as metabolites identi cation and quanti cation [9,10], metabolites bioactivity [11][12][13], callus inducement and screening [14] and cell suspension culture [15,16]. Now, a stable cell suspension culture had been established to produce the main bioactive components of C. paliurus in our laboratory.
Triterpenoids of C. paliurus have been attracting the researcher's attention for its excellent bioactivities [17]. Eight triterpenoids, including arjunolic acid, betulinic acid, corosolic acid, maslinic acid, oleanolic acid, ursolic acid, β-amyrin and β-boswellic acid, were identi ed from the leaves of C. paliurus in our previous studies [10]. Among them, ve had been found in our suspension cultured C. paliurus cells [18]. To further improve the triterpenoids yield, sodium nitroprusside was applied into the cultured medium as an abiotic elicitor, and the results showed that total triterpenoids yield of the elicited cells was 2.14 times that of the control group [19].
Elicitation is one of the most effective ways to stimulate the secondary metabolite synthesis in the plant cell, and therefore is now widely employed to induce novel metabolites or enhance the accumulation of the target metabolites in the plant cell, tissue and organ cultures [20]. According to chemical property, the elicitors are normally classi ed into two types, including abiotic elicitors (e.g. methyl jasmonate, salicylic acid (SA), AgNO 3 and CaCl 2 ) and biotic elicitors (e.g. fungal extracts, bacterial extracts and chitin) [21,22]. These elicitors can trigger defense or stress-induced responses at a low concentration, which can further regulate the expression level of some biosynthesis pathway genes via complex signal transduction networks and nally promote the synthesis of secondary metabolites [20]. Fungal extract is one of the most effective and studied elicitors in the last three decades. Kümmritz et al [23] reported that adding fungal medium ltrates combined with sucrose feeding signi cantly enhanced triterpenes content of Salvia fruticosa cell suspension culture by approximately 140%, and the triterpenes yield almost reached the level of intact plants. According to Li et al., the glycyrrhetinic acid content of the cultured Glycyrrhiza uralensis adventitious roots raised 1.8 times with the addition of Aspergillus niger elicitors (ANE) [24]. However, up to now the mechanism of ANE elicitation is not well understood, especially the involved signal transduction network.
In the present paper, ANE was used as a biotic elicitor to increase the triterpenoids synthesis in the suspension cultured C. paliurus cells. To interpret the elicitation mechanism, the pro le changes of four signal molecules concentration were determined in detail after ANE inducing, meanwhile, the crosstalk among these signal molecules was further studied by a series of experiments of signal molecules blocking, as well as experiments of exogenous signal molecules addition. What's more, differentially expressed genes (DEGs) associated with the jasmonic acid (JA) synthesis and transduction were also analyzed to demonstrate the critical role of JA in this elicitation by RNA-sEq. To the best of our knowledge, at present there is no report on the signal molecules crosstalk involved in the ANE elicitation in the suspension cultured C. paliurus cells. Our results and analysis provided references for the further studies on the triterpenoids biosynthesis regulation of the cultured plant cells.

Materials
Suspension cultured cell lines of C. paliurus was cultivated and maintained in Jiangxi Key Laboratory of Natural Products and Functional Food (Jiangxi Agricultural University, Nanchang, China).
Aspergillus niger elicitor preparation ANE (Aspergillus niger elicitor) was prepared according to the method of Xiong et al. [25].
Elicitation method of ANE on the cultured C.paliurus cells Elicitation was performed in accordance with the method described by Xiong et al. [25]. ANE at a concentration of 200 µg/mL was added into the culture medium on the 4 th day after cell inoculation to stimulate the synthesis of triterpenoids. The cultured cells were respectively sampled at 0 th , 20 th , 40 th , 60 th and 80 th hour after ANE elicitation for triterpenoids and signal molecules determinations.

Triterpenoids extraction and determination
Triterpenoids were extracted by ultrasound wave [18]. A total of 0.5 gram ne powder sample was dispersed in 6ml ethyl acetate and extracted in an ultrasonic extractor at 60℃ for 30min. After centrifugation at 4000 r/min for 10 min, the supernatant was taken out and volatilized to complete dryness at 60℃, then redissolved in methanol for triterpenoids determination. The triterpenoids determination was carried out according to the HPLC method of Yin et al. [9]. Total triterpenoids content was the sum content of maslinic acid, corosolic acid, betulinic acid, oleanolic acid and ursolic acid.

Signal molecules assay
A total of four signal molecules, including nitric oxide (NO), hydrogen peroxide (H 2 O 2 ), Jasmonic acid (JA) and salicylic acid (SA), were assayed by enzyme-linked immunosorbent assay method in the present paper. NO assay kit was purchased from Beyotime Bio-Technology Co., Ltd (Shanghai, China). H 2 O 2 , JA and SA assay kit were all provided by Yihan Bio-Technology Co., Ltd (Shanghai, China). All determinations were performed according to the instruction of assay kits.
Quenching of NO C-PTIO (Enzo life Sciences, Switzerland), a strong quencher of NO, was used to eliminate NO in the cell cultures [26]. 20 minutes before the addition of ANE, C-PTIO solution was put into the culture medium, and the nal C-PTIO concentration was controlled at 0.1, 1 and 100 μM respectively. The content of triterpenoids, H 2 O 2 , JA and SA in the cultured cells were respectively determined when the NO was quenched by the added C-PTIO.
Blocking of H 2 O 2 DMTU (Sigma-Aldrich, USA) was used as H 2 O 2 scavenger in this paper [27]. 20 minutes before the ANE elicitation, DMTU ( nal concentration of 0.5, 1 and 2 mM were set) was added into the culture medium. The changes of triterpenoids accumulation were tested as well as the concentration changes of NO, JA and SA content when H 2 O 2 were blocked by DMTU, Which were served to explore the role of H 2 O 2 in triterpenoid synthesis elicitation of ANE.

Inhibition of JA synthesis
Two inhibitors, named IBU and NDGA, were conjointly used to block JA synthesis in the cultured C. paliurus cells [28,29]. Three combinations of IBU and NDGA concentration were applied into the culture medium 20 minutes before the ANE elicitation in this paper, including 25 μM IBU and 25 μM NDGA, 50 μM IBU and 50 μM NDGA, and 100 μM IBU and 100 μM NDGA, which was served to investigate the role of JA in the triterpenoid synthesis stimulation of ANE.

Exogenous addition of signal molecules
Sodium nitroprusside (SNP, Sigma-Aldrich, USA, 99%) was used as exogenous NO donor to activate NO signal pathway [30]. Methyl jasmonate (MJA, Sigma-Aldrich, USA, 95%) was applied as JA donor to generate JA signal molecules [31]. SNP, MJA and H 2 O 2 (Xilong Chemical Co., Ltd. Guangdong, China, 30%) were respectively added into the C. paliurus cells culture medium at the same time of ANE elicitation as positive signal molecules control group, whose nal concentrations were set at 150 μM, 10 μM and 50 μM respectively RNA extraction and sequencing The cultured cells were harvested at 20 th hour after ANE elicitation and immediately frozen in liquid nitrogen and stored at -80 °C for the following total RNA extraction, as well as the untreated cells. Total RNA was extracted from the cultured C. paliurus cells using plant RNA puri cation reagent (Invitrogen, Carlsbad, CA, USA). The quantity and quality of total RNA were determined by Nanodrop 2000 (Thermo Fisher, America) and Agilent 2100. High-quality total RNA used for RNA-seq should have a concentration greater than 200 ng/μL, and the RNA integrity number (RIN) must reach 7.5, meanwhile OD 260 /280 should be between 1.8 and 2.2. Poly (A) mRNA was enriched from total RNA using Oligo (dT) magnetic beads. The enriched mRNA was randomly broken into short fragments and then was used to synthesize the rst-strand cDNA using reverse transcriptase and random primers, which was further served to synthesize the second-strand. These synthesized cDNA fragments were puri ed and dissolved in EB buffer for end repairing and adding of ploy (A), and then connected with sequencing adapters. The suitable fragments were selected for PCR ampli cation as templates to establish cDNA library. Finally the cDNA library was sequenced using an Illumina HiSeq 4000 platform with the 2×151 bp paired-end reads.

Reads alignment and De novo assembly
The raw reads, which were transformed from base calling le, were ltered out to generate clean reads by removing adaptor sequences, ambiguous reads and the low quality reads (quality value <20). The clean reads were assembled into transcript contigs using the short reads assembling program Trinity software (http://trinityrnaseq.sourceforge.net/, release-20140413), then the contigs were further connected until it could not be extended on either end, which were de ned as unigenes.

Function annotation and classi cation of unigenes
All assembled unigenes were annotated using BLASTx alignment (e-value<0.00001) against the following publicly available protein databases: NCBI non-redundant (NR), Swiss-Prot, Pfam, eukaryotic orthologous groups (COG) and KEGG. Combining NR annotation with the Blast2GO program [32], GO annotations of the unigenes were obtained. All GO functional classi cations were produced by WEGO software [33]. In the meantime, the pathway assignments were performed in conjunction with the KEGG database. When a con ict occurred among the database results, a priority order of alignments was Nr, Swiss-Port, KEGG, GO and COG.

Identi cation of differentially expressed genes
Normalized expression levels of the unigenes were calculated as fragment per kilobases of exon model per million mapped reads (FPKM) by the RSEM software [34]. The differential expression analysis of any two groups was analyzed using the EdgeR software and false discovery rate (FDR) method [35]. Afterward, the signi cantly differentially expressed genes (DEGs) were identi ed by the threshold of FDR < 0.05 and log2|Fold Change | ≥ 1. To further identify signi cantly over-represented metabolic pathways or signal transduction pathways, all DEGs were mapped to KEGG database with a threshold E-value of 10 −5 for pathway enrichment analysis.

Results
Stimulation effects of ANE on the triterpenoids synthesis in the cultured cells To further enhance the triterpenoids production, ANE, a widely used biotic elicitor, was applied to stimulate triterpenoids synthesis in the cultured cells. Our results (as showed in Fig.1) indicated that the accumulation of triterpenoids in the cultured C. paliurus cells enhanced signi cantly under the elicitation of ANE at a concentration of 200 µg/mL, while the biomass was unaffected. With the time increasing, the yield of total triterpenoids rose rst and fell later, and peaked at 60 hours after the ANE addition. The peak total triterpenoids yield of the elicited cells was 9.17 times higher than that of the untreated cells.
However, ANE's elicitation mechanism is still unclear. To better understand the mechanism, four signal molecules crosstalk was further investigated as well as the RNA-seq.

Pro le changes of four signal molecules concentration under the ANE elicitation
After the elicitation of 200 µg/mL ANE, the concentrations of NO, H 2 O 2 and JA all increased signi cantly in the cultured C. paliurus cells, but presented different change pro les and peaked at different time (as shown in Fig. 2(a-c)). While, SA concentration fell gradually at the beginning, then kept relatively stable and lower than that of the untreated cells until 80 hours post-elicitation ( Fig. 2(d)). Consequently, it was deduced that NO, H 2 O 2 and JA was involved in the signal transduction of ANE elicitation as important signal molecules.
NO concentration rise is a common physiological reaction in plant cells when stimulated by elicitors (Xu et al., 2005). Our results ( Fig. 2(a)) showed that NO concentration increased swiftly after the addition of ANE, and peaked at the 15 th minute, then decreased sharply and maintained at a similar level of the unelicited cells from 4 to 80 hours post-elicitation. Similarly, H 2 O 2 concentration also enhanced rapidly under the ANE stimulation, and also reached a high concentration at the 15 th minute ( Fig. 2(b)). Nevertheless, unlike NO, H 2 O 2 concentration always kept at a high level from 15 minutes to 80 hours, which was signi cantly higher than that in the untreated cells ( Fig. 2(b)). The concentration changes of JA lagged behind that of NO and H 2 O 2 ( Fig. 2(a-c)). Within 6 hours after ANE elicitation, JA concentration was almost the same as that in un-elicited cells. From the 7 th to 80 th hour post-elicitation, JA concentration was obviously higher than that of control group, with two peaks appearing at the 18 th and 42 nd hour after ANE addition respectively ( Fig. 2(c)). The highest concentration of JA occurred at the 42 nd post-elicitation. In view of the time sequence changes of these signal molecules, a series of experiments of NO quenching by C-PTIO, H 2 O 2 blocking by DMTU, and JA synthesis inhibition by IBU and NDGA were further performed to understand the crosstalk among these signal molecules, together with exogenous NO, H 2 O 2 and JA addition experiments.
Changes causing by NO quenching and exogenous addition As shown in Fig.3(a and b), NO concentration decreased gradually with the C-PTIO concentration increasing from 0.1 to 100 μmol/L, and fell to the level of the untreated cells at 100 μmol/L, but the C-PTIO addition and NO change had no in uence on the cell growth (biomass was given in Fig. 3(b)). When 100 μmol/L C-PTIO was applied to the cultured cells elicited by 200 μg/mL ANE, H 2 O 2 concentration was approximately the same as that in the elicited cells, while JA concentration declined signi cantly, but still obviously higher than that of control group (neither ANE nor C-PTIO was added) (Fig. 3(c and d)). With NO blocked by C-PTIO, triterpenoids yield of the ANE elicited cells reduced by 21.28% percent, but still markedly higher than that of the control group (Fig. 3(e)).
To further interpret the role of NO and its relationship with H 2 O 2 and JA, 150 μmol/L SNP (exgenous NO donor) was added into the culture medium of C. paliurus cells. Results (Fig. 3(f and g) indicated that exogenous NO showed no effect on H 2 O 2 concentration, while raised the concentration of JA signi cantly. However, JA concentration in the cells treated by exogenous NO was still lower than that in ANE elicited cells. Under the exogenous NO stimulation, triterpenoids yield was 4.86 times that of the control group, but signi cantly lower than that of the ANE treated cells (Fig. 3(h)).
Based on the above analysis, the following three conclusions were obtained: rstly, ANE elicitation promoted the synthesis of triterpenoids in C. paliurus cells partly through NO signal pathway; secondly, JA was involved in NO pathway in ANE elicitation, and located in downstream of NO pathway; thirdly, H 2 O 2 synthesis might be independent on the change of NO concentration.

Changes causing by H 2 O 2 blocking and exogenous addition
As DMTU concentration rose from 0.5 to 2 mmol/L, the elimination rate of H 2 O 2 , increased in the C.
paliurus cell cultures accordingly ( Fig. 4(a)). Under the scavenging of 2 mmol/L DMTU, H 2 O 2 concentration in the ANE elicitation cells fell to the level of the un-elicited cells. Meanwhile JA concentration reduced by 14.53% percent, but still obviously higher than that in the control (Fig. 4(c)). 2 mmol/L DMTU showed no signi cant impact on cell growth (( Fig. 4(b))), but decreasing the triterpenoids yield of the ANE treated cells by 13.76% percent (Fig. 4(d)). Although triterpenoids accumulation in ANE elicited cells was reduced by 2 mmol/L DMTU, but still higher than that in the control group ( Fig. 4(d)).
When exogenous 50 μmol/L H 2 O 2 was added into the culture medium of C. paliurus cells, JA concentration increased by 11.31% percent correspondingly, but was still signi cantly lower than that of the ANE elicited cells (Fig. 4(e)). Stimulated by 50μmol/L H 2 O 2 , triterpenoids accumulation rose by 78.35 % percent, however, it was notably lower than that in the ANE treated cells (Fig. 4(f)).
In conclusion, ANE elicitation improved triterpenoids synthesis in C. paliurus cells partly through H 2 O 2 signal pathway, and the change of H 2 O 2 concentration could cause the change of JA concentration and nally led to a uctuation of triterpenoids yield accordingly.
Changes causing by JA synthesis inhibition and exogenous JA addition The combined application of IBU and NDGA could effectively as inhibit JA synthesis in the cultured C. paliurus cells. With the addition of 100 μmol/L IBU and NDGA, JA concentration in the ANE elicited cells declined to the level of the un-elicited cells (Fig.5(a)), while the cell growth was unaffected (Fig.5(b)). Under the inhibition of 100 μmol/L IBU and NDGA, triterpenoids yield reduced to 16.69 mg/40mL, which was 59.41% percent of the ANE treated cells, but still signi cantly higher than that of the control (Fig.5(c)). When 10 μmol/L MJA was used as JA donor in the culture medium, triterpenoids yield improved to 24.33mg/40mL, which was 4.92 times that of the control (Fig.5(d)).
Comparatively speaking, JA blocking resulted in the biggest decline of triterpenoids yield in the ANE elicited C. paliurus cells (Fig.5(c)), followed by NO and H 2 O 2 blocking (Fig.3(e) and Fig.4(d)). Similarly, exogenous JA addition led to the highest increase of triterpenoids yield in the cultured C. paliurus cells (Fig.5(d)), followed by NO and H 2 O 2 addition (Fig.3(h) and Fig.4(f)).
Consequently, it was deduced that JA was the critical signal molecule for triterpenoids yield promotion in the ANE elicitation, and the change of JA concentration could cause a notable change of triterpenoids accumulation in the culture C. paliurus cells. RNA-seq and de novo assembly Transcriptome sequencing technology, also known as RNA-seq, is now widely used to explore the mechanism of biological phenomena from the perspective of gene expression difference [36]. In the present paper, three cDNA libraries from the cultured C. paliurus cells, named CK (control), 20h (cells elicited for 20 h), 60h (cells elicited for 60 h) respectively, were sequenced using Illumina Hiseq 4000 platform, which generated 65,074,242, 66,016,592 and 53,951,932 raw reads separately (Table S1). After data ltering and stringent quality evaluation, clean reads were obtained. Subsequently, using Trinity program, these clean reads were assembled into 88,144 transcripts with an N50 of 1,590 bp and the average length of 869 bp, which were then joined into 67230 unigenes with an N50 of 1375 bp and the average length of 744 bp (Table S2). Furthermore, the length and number of transcripts and unigenes were statistically analyzed. As shown in Fig.S1, the length of transcripts and unigenes ranged from 201 to 17,098 bp and the majority distributed in 201-400bp, accounting for 45.56% and 52.99% respectively. The above-mentioned data indicated that the generated unigenes in our experiments were of ne quality, and therefore suitable for further annotation.

Function annotations and classi cations of unigenes
A total of 33,470 unigenes (accounting for 49.78% of all unigenes) were successfully annotated against seven public databases such as NR, Swiss-Prot, KOG KEGG, GO, COG (Table S3). The results of unigenes homology searches against the NR database were shown in Fig.S2, including the similar, E-value and species distribution. As seen in Fig.S2(c), 3972 annotated unigenes of C. paliurus had the rst top matches with sequences from Vitis vinifera, followed by the Theobroma cacao (3417 unigenes), Prunus persica (2702 unigenes), Prunus mume (2548 unigenes) and Morus notabilis (1807 unigenes).
All C. paliurus unigenes were aligned to the COG database for prediction and classi cation by possible function. Overall, a total of 7,694 unigenes (11.44%) were classi ed into 25 COG functional categories (Fig S3(a)).Our results indicated that 1,008 annotated unigenes (13.10%) fell into "General function prediction only" group, which was the largest among the 25 COG functional categories, followed by "Signal transduction mechanisms" (966, 10.11%). It should be noted that no unigenes was classi ed as "Extracellular structures" and "Nuclear structure", which need to be further studied.
To better understand the biological functions of these unigenes in the cultured C. paliurus cells, a total of 13,634 unigenes (20.28%) were mapped to 348 KEGG pathways and divided into ve branches including Metabolism; Genetic Information Processing, Environmental Information Processing, Environmental Information Processing, Cellular the Processes and Organismal Systems (Fig.S3 (c)). The most represented pathways were Metabolic pathways (2778 unigenes, 18.74%), followed by biosynthesis of secondary metabolites (1336 unigenes, 9.80%). In addition, 29 metabolic pathways play a signi cant role in the growth and metabolism of the cultured cells (Table S4), such as phenylpropanoid biosynthesis (179 unigenes), terpenoid backbone biosynthesis (75 unigenes) and avonoid biosynthesis (42 unigenes). Our annotation against KEGG provided lots of useful information to interpret the metabolic characteristics of the cultured C. paliurus cells.

Overall analysis of differentially expressed genes
From the perspective of differentially expressed genes (DEGs), ANE elicitation caused great changes in metabolism related genes expression (Fig. S4), which might further result in markedly changes of the metabolites synthesis and accumulation in the cultured C. paliurus cells. A total of 24,788 DEGs were found between the un-elicited cells and elicited cells treated for 20 hours, which consisted of 12,699 upregulated DEGs and 12,089 down-regulated DEGs. Among these DEGs, 774 genes were signi cantly upregulated, while 876 were signi cantly down-regulated. Similarly, between the un-elicited cells and elicited cells treated for 60 hours, there were 340 signi cantly up-regulated genes and 129 signi cantly downregulated genes. Furthermore, cells elicited for 20 hours showed different gene expression pro le by comparison with that elicited for 60 hours. Between these two treated groups, 406 signi cant upregulation and 224 signi cant down-regulation genes were identi ed. The three groups of DEGs were further analyzed. Among them, 1650, 469 and 630 genes were signi cantly differentially expressed, and 35 genes belonged to the differentially expressed genes in three samples. The vene diagram is shown in Fig. S5.

Analysis of DEGs involved in JA synthesis pathway under ANE treatment
To further validate the key role of JA in the responses of ANE elicitation in C. paliurus cells, the expression levels of JA synthesis-related genes such as LOX, AOS, AOC, and OPR were detected using comparative transcriptome sequencing. A total of 7 annotated candidate LOX unigenes were up-regulated, among which 1were signi cantly up-regulated (Table 1). Within the 6 up-regulated OPR unigenes, 2 were found to be signi cantly up-regulated. Meanwhile, the expression of 2 AOC and 1 AOS unigenes increased, but not signi cantly ( Table 1). The above DEGs analysis associated with JA synthesis was in consistence with the determination data of JA concentration in the elicited cells. Our results indicated that ANE elicitation activated the expression of JA synthesis pathway genes, which nally enhanced the triterpenoids accumulation in the culture cells.

Analysis of DEGs involved in JA signal transduction pathway under ANE treatment
Researches showed that JAZ and JAR1 were key transcription factors in JA signal transduction pathway, and played critical roles in the synthesis of secondary metabolites [37,38]. In the present paper, a total of 7 JAZ candidate unigenes were annotated, among which 5 were found to be down-regulated, and 1 kept almost unchanged, while 1 was up-regulated (Table 2). In addition, one JAR1 candidate unigene (c39500_g1) was annotated and observed to be up-regulated, whose FPKM increased from 48.614 to 77.295 at the 20 th hours after ANE elicitation (Table 2). Consequently, it was speculated that the downregulation of JAZs together with the up-regulation of JAR1 mediated the ANE elicitation signal transduction, and led to the rising of triterpenoids synthesis in the cultured cells, which further validated the above deduced JA signal pathway and the critical role of JA in ANE elicitation.

Discussion
Fungal elicitor, as a highly effective stimulator, can trigger rapid defense responses when applied to in vitro plant cell, tissue and organ cultures, and thus stimulate the secondary metabolites synthesis by activating the expression of speci c genes [39,40]. Our elicitation experiment suggested that ANE can signi cantly improve the triterpenoids yield of the cultured C. paliurus cells, and therefore has a promising application prospect in the production of plant-derived triterpenoids. However, more investigations are still needed to better understand the elicitation mechanism. Our previous report showed that ANE elicitation up-regulated the expression of triterpenoid synthesis pathway genes, and therefore enhanced the triterpenoid accumulation in the cultured C. paliurus cells [41], but the involved signal mechanism causing the triterpenoid synthesis genes up-regulation hasn't been revealed yet.
Researches indicated that there are four common signal molecules, including NO, H 2 O 2 , JA and SA, which mediate signal transduction when plants face various environmental stresses, such as fungal invasion, insect attack, drought, osmotic pressure [42][43][44]. Usually, these signal molecules play their roles in the complicated signal transduction network harmoniously, and result in differential expression of some related key genes, which further causes various physiological responses in the plants [45,46].
Signal transduction is a very complex biological process, and therefore has been attracting many researchers' attention in recent decades. Domingos et al. [47]  The research of Xu and Dong [49] suggested that nitric oxide mediated the fungal elicitor-induced taxol biosynthesis in the T. chinensis suspension cultured cells through both reactive oxygen speciesdependent and -independent signal pathways. As shown in their report, both NO quenching and reactive oxygen species (ROS, including H 2 O 2 ) blocking inhibited the elicitor-induced taxol production, which was in accordance with the results of our experiment performed in the suspension cultured C. paliurus cells. Their data also showed that NO quenching suppressed the H 2 O 2 synthesis, and exogenous NO addition increased the H 2 O 2 accumulation, however according to our results, the change of NO concentration had no effect on the H 2 O 2 synthesis. According to the schematic representation of action mode of elicitor in plant cell given by Halder et al. [20], NO synthesis was the downstream event of the ROS burst under elicitation, which coincided with our conclusion. These different phenomena and results may be caused by plant species differences or other unknown reasons, and further studies are still needed.
Both SA and JA are important signal molecules associated with plant defense responses [50]. Our previous studies showed that any one of SA and JA inducing could stimulate the synthesis of secondary metabolites [51]. However, according to Li et al. [52], there was antagonism between SA and JA pathways in A. thaliana in response to elicitation, and WRKY70 acted as an activator of SA-induced genes and a repressor of JA-responsive genes to integrate signals from these mutually antagonistic pathways. Similar results were reported by Xu et al. [53]. In our ANE elicitation experiments, JA concentration increased signi cantly after the ANE addition, and played a critical role in stimulating triterpenoids synthesis, while SA concentration was lower than that of the untreated cells. Our results also con rmed the antagonism between SA and JA pathways.
In this study, JA was found to be not only the junction of NO and H 2 O 2 signal pathways but also the critical point in the whole signal network of ANE elicitation. Researches indicated that JA, as an important endogenous phytohormone, was synthesized in plant using α-linolenic acid as starting compound [54], and a series of cascade enzymes of its synthesis pathways were involved, including LOX, AOS, AOC, and OPR [55,56], which were also identi ed in our elicited C. paliurus cells (Table 1). Our RNA-seq analysis showed that a total of 3 candidate JA synthesis pathway genes including 1 LOX and 2 OPR were found to be signi cantly up-regulated under the ANE stimulation,, as well as 5 down-regulated JAZs and 1 upregulated JAR1 relating to JA signal transduction, which further validated the key role of JA in response to ANE elicitation. JAs (jasmonates) usually refer to a category of organic compounds derived from JA, including jasmonic acid (JA), methyl jasmonate (MeJA), and jasmonic acid isoleucine (JA-Ile), which acted as ubiquitous and conserved regulators for the production of secondary metabolites in the most majority of plants, from gymnosperms to angiosperms [37,57]. It is generally recognized that JA shows its effects through a bioactive form -JA-Ile, which is synthesized from JA and isoleucine under the catalysis of JA amino acid synthetase (JAR1) [37,38]. Normally, JA-Ile concentration is very low, and MYC2 is bound by jasmonate ZIM-domain protein (JAZ) and presents in an inactive form; when the JA-Ile concentration increases to a certain level under stimulation, the COI1 protein of SCF COI1 complex interacts physically with JAZ protein and make the latter to be labeled with ubiquitin, which causes MYC2 to be freed from JAZ and JAZ to be degraded by 26S-proteasome; the free MYC2 released from JAZ activates the expression of the early jasmonate-response genes and nally improved the synthesis of secondary metabolites to adapt to the environment changes [37,[57][58][59][60]. Our results also indicated that JA mediated the signal transduction of ANE elicitation, in which TFs like JAZ and JAR played important roles.
Meanwhile, MYC2 were also annotated in our ANE treated cells, but its expression declined slightly at the 20th hour after elicitation (Table 2). However, further studies are still needed to interpret the underlying mechanism of these TFs associated with JA signal transduction pathways. Conclusion ANE elicitation signi cantly enhanced the triterpenoids accumulation in the suspension cultured C. paliurus cells. NO, H 2 O 2 and JA were all involved in this elicitation as important signal molecules. Three related signal pathways were deduced based on a series of experiments of signal molecules blocking and exogenous addition, together with three hypothetical signal transduction pathways involved in ANE elicitation. JA was not only the junction of NO and H 2 O 2 signal pathways but also the critical point in the whole signal network of ANE elicitation. RNA-seq analysis further validated the pivotal role of JA in ANE elicitation. As far as we know, up to now this paper is the rst report on the signal molecules crosstalk involved in the ANE elicitation in the suspension cultured C. paliurus cells. Superscript symbol "*" indicates that this unigene expression changed signi cantly.

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