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 significantly 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 first 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.
Profile changes of four signal molecules concentration under the ANE elicitation
After the elicitation of 200 µg/mL ANE, the concentrations of NO, H2O2 and JA all increased significantly in the cultured C. paliurus cells, but presented different change profiles 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, H2O2 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 15th minute, then decreased sharply and maintained at a similar level of the un-elicited cells from 4 to 80 hours post-elicitation. Similarly, H2O2 concentration also enhanced rapidly under the ANE stimulation, and also reached a high concentration at the 15th minute (Fig. 2(b)). Nevertheless, unlike NO, H2O2 concentration always kept at a high level from 15 minutes to 80 hours, which was significantly higher than that in the untreated cells (Fig. 2(b)). The concentration changes of JA lagged behind that of NO and H2O2 (Fig. 2(a-c)). Within 6 hours after ANE elicitation, JA concentration was almost the same as that in un-elicited cells. From the 7th to 80th hour post-elicitation, JA concentration was obviously higher than that of control group, with two peaks appearing at the 18th and 42nd hour after ANE addition respectively (Fig. 2(c)). The highest concentration of JA occurred at the 42nd post-elicitation. In view of the time sequence changes of these signal molecules, a series of experiments of NO quenching by C-PTIO, H2O2 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, H2O2 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 influence 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, H2O2 concentration was approximately the same as that in the elicited cells, while JA concentration declined significantly, 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 H2O2 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 H2O2 concentration, while raised the concentration of JA significantly. 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 significantly lower than that of the ANE treated cells (Fig. 3(h)).
Based on the above analysis, the following three conclusions were obtained: firstly, 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, H2O2 synthesis might be independent on the change of NO concentration.
Changes causing by H2O2 blocking and exogenous addition
As DMTU concentration rose from 0.5 to 2 mmol/L, the elimination rate of H2O2, increased in the C. paliurus cell cultures accordingly (Fig. 4(a)). Under the scavenging of 2 mmol/L DMTU, H2O2 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 significant 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 H2O2 was added into the culture medium of C. paliurus cells, JA concentration increased by 11.31% percent correspondingly, but was still significantly lower than that of the ANE elicited cells (Fig. 4(e)). Stimulated by 50μmol/L H2O2, 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 H2O2 signal pathway, and the change of H2O2 concentration could cause the change of JA concentration and finally led to a fluctuation 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 significantly 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 H2O2 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 H2O2 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.
Summary of Signal molecules crosstalk and deduced signal transduction pathways involved in ANE elicitation
Our signal molecules blocking and exogenous addition experiments suggested that NO, H2O2 and JA were all involved in the responses of ANE elicitation in the suspension cultured C. paliurus cells; the blocking of any one of NO, H2O2 and JA led to triterpenoids accumulation decline in the ANE elicited cells. Conversely, exogenous addition of any one of NO, H2O2 and JA resulted in triterpenoids synthesis rising, but their induced increments were all lower than that of ANE; both NO and H2O2 had apparent impacts on JA synthesis; the change of NO concentration showed no effect on the H2O2 synthesis; the influence of JA on triterpenoids synthesis was relatively more significant than that of NO and H2O2. Based on the above analyses, three signal transduction pathways were deduced, which were shown in Fig. 6 and described as following: (1) ANE → receptor → nitric oxide synthase (NOS) → NO → jasmonic acid synthesis enzymes → JA → TFs → triterpenoids synthesis enzymes → triterpenoids; (2) ANE → receptor → NADPH oxidase → H2O2 → jasmonic acid synthesis enzymes → JA → TFs → triterpenoids synthesis enzymes → triterpenoids; (3) ANE → receptor →NADPH oxidase → H2O2 → nitric oxide synthase (NOS) → NO → jasmonic acid synthesis enzymes → JA→ TFs → triterpenoids synthesis enzymes → triterpenoids. In addition, there might be another three hypothetical signal transduction pathways involved in ANE elicitation, which need to be further validated: (1) ANE → receptor → nitric oxide synthase (NOS) → NO → TFs → triterpenoids synthesis enzymes → triterpenoids; (2) ANE → receptor → NADPH oxidase → H2O2 → TFs → triterpenoids synthesis enzymes → triterpenoids; (3) ANE → receptor → other pathways independent of NO, H2O2 and JA → TFs → triterpenoids synthesis enzymes → triterpenoids. In summary, ANE elicitation improved the triterpenoids accumulation in the suspension cultured C. paliurus cells via a complex signal transduction network, in which NO, H2O2 and JA were involved, and JA was the critical signal molecule.
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 . 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 filtering 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 fine quality, and therefore suitable for further annotation.
Function annotations and classifications 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 first 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 classification by possible function. Overall, a total of 7,694 unigenes (11.44%) were classified 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 classified as “Extracellular structures” and “Nuclear structure”, which need to be further studied.
According to the annotation against Gene Ontology (GO), altogether 19398 unigenes were classified into three main categories: “cellular component” (42,253 unigenes, 32.91%), “molecular function” (24,703 unigenes, 19.24%), and “biological process” (61440 unigenes, 47.85%), which were further divided into 65 sub-categories. (Fig.S3 (b)).Within the cellular component category, cell sub-category (8,221 unigenes, 19.46%) and cell part sub-category (8,221 unigenes, 19.46%) were the two most significant representation. Among the molecular function unigenes, the majority were classified into “binding sub-category” (11,100 unigenes, 44.93%) and “catalytic activity sub-category” (10,098 unigenes, 40.88%).
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 five 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 significant role in the growth and metabolism of the cultured cells (Table S4), such as phenylpropanoid biosynthesis (179 unigenes), terpenoid backbone biosynthesis (75 unigenes) and flavonoid 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 up-regulated DEGs and 12,089 down-regulated DEGs. Among these DEGs, 774 genes were significantly up-regulated, while 876 were significantly down-regulated. Similarly, between the un-elicited cells and elicited cells treated for 60 hours, there were 340 significantly up-regulated genes and 129 significantly down-regulated genes. Furthermore, cells elicited for 20 hours showed different gene expression profile by comparison with that elicited for 60 hours. Between these two treated groups, 406 significant up-regulation and 224 significant down-regulation genes were identified. The three groups of DEGs were further analyzed. Among them, 1650, 469 and 630 genes were significantly 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 significantly up-regulated (Table 1). Within the 6 up-regulated OPR unigenes, 2 were found to be significantly up-regulated. Meanwhile, the expression of 2 AOC and 1 AOS unigenes increased, but not significantly (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 finally 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 20th hours after ANE elicitation (Table 2). Consequently, it was speculated that the down-regulation 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.