1. Introduction
Paclitaxel is an important anticancer drug, which is mainly produced by chemical semi-synthesis by using paclitaxel, which is biosynthesized in Pacific yew. Due to its complex molecular structure, its raw materials have been dependent on
Taxus biomass, and its biosynthesis and regulation have been studied [
1]. The phytohormone jasmonic acid can significantly induce the biosynthesis of paclitaxel in
Taxus, but the molecular mechanism has not yet been resolved [
2,
3,
4].
The biosynthetic pathway of paclitaxel is very complex, involving at least 19 steps of enzyme-catalysed reactions, and the related enzymes and synthetic pathways have not been fully resolved. The synthetic pathway can be divided into the following four steps: taxane ring synthesis; side hydroxylation, acetylation and phenylacetylation modification; epoxidation modification at C5 position and oxidation at C9; and side chain at C13 position addition [
5].
The precursor of taxadiene ring biosynthesis is derived from isopentenyl pyrophosphate (IPP) and dimethyl propylene pyrophosphate (DMAPP), the synthesis, mainly through the plastids of
Taxus cells [
5,
6,
7]. After ring formation, the biosynthesis process of paclitaxel includes various modifications to the sites on the ring, including hydroxylation, acetylation and benzoylation modification, epoxidation at the C5 position, and oxidative modification at the C9 position [
7].
The known modification process is first hydroxylation of the taxane-4(5), 11(12)-diene at the 5α position. This is the rate-limiting step for paclitaxel synthesis [
5]. Subsequently, there is hydroxylation of 1β(1βOH), 2α(2αOH), 7β(7βOH), 9α(9αOH), 10β(10βOH), 13α(13αOH), acetylation of 5α(TBT) and 10β(TAT), and phenylacetylation of 2α(DBAT). In addition to the 1β and 9α position hydroxylases, the genes of other enzymes have been confirmed [
5]. Oxidation at the 9α position and epoxidation between C4, C5 and C20 occur after the desired modification, resulting in the formation of baccatin III (see
Figure 1), but the genes in both steps have not been isolated [
5]. At least 11 enzymes are involved in the various modification processes, but the exact order has not been determined. Finally, baccatin III binds to the side chain under the catalysis of the enzyme, and undergoes hydroxylation of the opposite side chain and phenylacetylation to form the final product, paclitaxel. Phenylalanine oxidase (PAM), phenylpropionyl-CoA transferase (BAPT) at position C13, and phenylacetylase (DBTNBT) in the side chain have been identified in this process [
5,
8].
The biosynthesis step of paclitaxel is very complicated, and it is very difficult to directly increase the expression of the biosynthetic pathway gene to increase the paclitaxel content. As the analysis of all synthetic pathways is not complete, the metabolic pathways cannot be precisely regulated to control the metabolic flux. In this case, for such a complex metabolic pathway, a better choice for regulating genes in the synthetic pathway is to regulate upstream transcription factors, thereby comprehensively regulating the expression of genes in the synthetic pathway [
9]. It is important to analyse the transcription factors that regulate the expression of paclitaxel synthesis pathway genes.
Transcription factors regulating Taxol biosynthesis in
Taxus are being closely studied [
2,
3,
4]. Previous studies have used two methods to isolate related transcription factors: one is to first isolate the promoter of the key gene of the Taxol synthesis pathway, and then to study the transcription factor that can be combined with the promoter; the other is to study jasmonic acid-induced transcription factors in order to analyse whether it can regulate gene expression of paclitaxel.
In the transcription factor-related studies in Taxus, no relevant research results of jasmonic acid signaling pathways in model plants have been used. There is also no systematic and in-depth discussion of the signaling pathway of jasmonic acid in Taxus. Li et al. used high-throughput sequencing to study the transcriptome of jasmonic acid treatment for 16 h, and obtained an AtJAZ-like gene induced by jasmonic acid, and a similar AtMYC2 gene, but no in-depth study [
10]. In the transcriptome sequencing study published by the Sun et al., the related gene expression after eight days of jasmonic acid treatment was involved, but no jasmine similar to that in the model plants induced in the early stage of jasmonic acid treatment was involved in the ketoacid signalling pathway transcription factor [
11]. Lenka et al. published article on AtMYC2-like genes [
8]; except for the TcMYC2 gene, which is known to have a full-length gene, other transcription factors were not found to be similar to AtMYC2 in model plants. The AtJAZ-binding region also does not contain active regions that activate transcription. Maybe due to the shortcomings of the adopted technical methods, the conclusions obtained were also different from those of similar genes in model organisms [
12].
In this study, high-throughput sequencing was used to obtain transcription factors after an early induction with jasmonic acid. In our previous study, the sequence of several genes related to the paclitaxel synthesis pathway and jasmonic acid signaling pathway were obtained by sequencing analysis of the transcriptome and expression profile of Taxus. Based on this, the full-length sequence of jasmonic acid signalling pathway-related transcription factors in T. media was obtained by sequence comparison analysis and RACE PCR. It includes sequences similar to the important genes of the jasmonic acid signalling pathways such as AtCOI1, AtMYC2, AtJAZ and AtMED25 (At, Arabidopsis thaliana, if the source of the gene is not clearly prefixed, it is from the T. media). The interaction between JAZ and MYC transcription factors and other genes was studied, and the jasmonic acid signalling pathway in Taxus was confirmed. The above work further studied the regulation of the promoter of the paclitaxel synthesis pathway gene by the MYC transcription factor, and elucidated the regulation mechanism of the transcription factor of the jasmonic acid signaling pathway on the biosynthesis pathway of paclitaxel.
We also found that some of the genes involved in the paclitaxel synthesis pathway were highly expressed by jasmonic acid at 0.5 h, similar to the transcription factors of the jasmonic acid signalling pathway and the expression of self-synthesis-related genes, but faster. Expression of anthocyanin synthesis-related genes regulated by the jasmonic acid signalling pathway was noted at three hours [
13]. This indicated that the jasmonic acid signalling pathway plays an important role in regulating the gene expression of the paclitaxel synthesis pathway.
To analyse the mechanism of jasmonic acid signalling pathway specifically regulating the paclitaxel synthesis pathway gene, 14 promoters of paclitaxel synthesis pathway gene were isolated, and five MYC transcription factors were verified by fluorescence activity assay in Arabidopsis protoplasts. Regulation of the promoter of the paclitaxel biosynthetic pathway gene and the transcription factor mutants with application value were designed, which lays the foundation for the future construction of a high-yield paclitaxel-based Taxus cell line.
3. Discussion
3.1. Taxus Has a Similar Mechanism of Signal Accepting and Conduction of Jasmonic Acid
As an important phytohormone, jasmonic acid is an important component of plant stress response. It has been proven to be ubiquitous in plants, not only in the model plant
Arabidopsis thaliana [
14,
18,
19,
20,
21]. Similar acceptance and transmission pathways for jasmonic acid signals should also be present in the Taxus. Prior to this study, a small number of genes for the jasmonic acid signalling pathway have been isolated by high-throughput sequencing [
10], but have not conducted in-depth studies.
The jasmonic acid signalling pathway in Taxus was confirmed by high-throughput sequencing and identification of related genes and their function. Twenty-one isolated genes of the jasmonic acid signalling pathways were similar to those found in the model plants, considering characteristic protein domains and protein–protein interactions. It is shown that a similar jasmonic acid signalling pathway may exist in Taxus and play an important role.
Firstly, 12 JAZ interacted with two COI1 in the presence of coronatine, and the interaction intensity of some JAZ and COI1 increased with the increase of coronin concentration, indicating that these are probably active molecules of jasmonic acid signalling in Taxus. Jasmonyl isoleucine also mediates the interaction between the JAZ and COI1. However, JAZ8 and JAZ9 also have strong interaction with COI1 in the absence of coronatine, which is different from that in model plants. This indicates that the signalling pathways in Taxus may be different. The relevant biological significance requires in-depth research.
There is also an interaction between the protein of MYC transcription factor and the JAZ protein in
Taxus, which is an important way for JAZ to inhibit the transcriptional activities of MYC2, MYC3 and MYC4. JAM1, lacking the JID domain, does not bind to most JAZ, indicating that the mutual binding is based on the JID domain in the MYC-like transcription factor. JAZ8, which contains two EAR domains and has a long length and JAS domain, is not combined with MYC3 and MYC4, but can be combined with MYC2 and the JAM1 without the JID domain. It may play a special role in the jasmonic acid signalling pathway of
Taxus. Subsequent structural analysis of the complexes formed by AtMYC3 and AtJAZ1 of
A. thaliana resulted in mutations in MYC2 and MYC3, and mutations in the JID domain resulted in their inability to bind to JAZ. This also suggests that the jasmonic acid signalling pathway in
Taxus is similar to that in
Arabidopsis [
16,
22,
23,
24]. Interactions exist between JAZ proteins, especially between any combinations of any two of the six JAZs from JAZ7 to JAZ12, but there is less interaction between any combinations of any two of the six JAZs from JAZ1 to JAZ6, indicating that JAZ7–JAZ12 plays an important role in the formation of inhibitory protein complexes [
22].
There are also interactions between MYC transcription factors, MYC2 and MYC3, and JAM2 interacts with itself. This differs from the broad interaction between inhibitory MYC transcription factors between activated MYC-like transcription factors in model plants.
The final MYC-type transcription factor recruits RNA polymerase by binding to MED25 to activate transcription of downstream genes. MYC2, MYC3 and MYC4 in Taxus have the same function. JAM1 without activation activity could not bind to MED25. However, JAM2, which has no apparent transcriptional activity, binds to MED25, indicating that it may bind to MED25 competitively, thereby inhibiting jasmonic acid signalling.
3.2. Jasmonic Acid Signalling Pathway Transcription Factor Regulates Paclitaxel Biosynthesis Pathway Gene Expression
The transcription factors MYC2, MYC3 and MYC4 of the jasmonic acid signalling pathway play an important role in the paclitaxel biosynthesis pathway. In the promoter region of most paclitaxel synthesis pathway genes, there are G-boxes and similar cis-elements that bind to MYC-type transcription factors, including the promoters of TAT, TBT, DBTNBT, 2αOH, 7βOH, 5αOH, 13αOH, DBAT, 10βOH and PAM, TS. The promoter of BAPT, GGPPS has fewer cis-elements, while the promoter of 14βOH has no corresponding cis-elements.
The activity of the three transcription factors also showed transcriptional activation activity against the promoters of DBTNBT, 2αOH, 7βOH, 5αOH, 13αOH, DBAT, 10βOH, PAM, GGPPS and TS, which contain cis-elements. However, there was no strong transcriptional activation activity for BAPT and 14βOH. It is shown that the G-box cis-element is important for the activity of the above three transcription factors. However, the promoters of TAT and TBT, which contain a large number of cis-like elements similar to G-box, suggest that MYC transcription factors should bind to the transcription factors of the two genes. However, in the Arabidopsis protoplasts, they are not strongly activated by MYC transcription factors; in particular, MYC3 and MYC4 have a weak regulation effect on them. Whether the excessive cis-elements in the promoters of TAT and TBT lead to inhibition requires further analysis.
JAM2 has a significant inhibitory effect on all paclitaxel synthesis pathway genes, and its expression is induced by jasmonic acid signalling. It is likely to be a feedback inhibition signal of the jasmonic acid signalling pathway in Taxus. The inhibitory effect of JAM1 is weaker than JAM2. JAM2 can be combined with MED25. JAM1 and MYC4 almost have the same sequence. Besides the above functions, whether they are different from model plants, these two transcription factors have other unknown functions and need further research.
In summary, the jasmonic acid signalling pathway in Taxus is summarized in
Figure 9. JAR1 catalyzes the binding of JA and isoleucine to form a biologically active jasmonoyl isoleucine (JA-Ile). It can mediate the Coronatine Insensitive 1-E3 ubiquitin ligase complex (SCFCOI1) binding to the jasmonic acid-inducing protein (JAZ) containing the ZIM domain. SCFCOI1 then labels ubiquitin on the JAZ protein. Furthermore, it promotes the degradation of JAZ protein bound to MYC by protease, stops the inhibion of MYC’s activity caused by JAZ’s binding. Eventually, activate the activity of transcription factors such as MYC2, cooperate with different transcription factors, recruit transcriptional complexes including Mediator25 (MED25), and activate genes expression in the downstream pathway. MYC transcription factors play a central role in the
T. media signaling pathway, and include three MYC factors. The three MYC factors have an upregulation effect on the regulation of genes in each step of the paclitaxel synthesis pathway. In addition to MYC3 and MYC4 having an inhibitory effect of BAPT and 14βOH, JAM1 and JAM2 are MYC transcription factors in
T. media. They inhibit every step’s gene expression in the downstream pathway.
To confirm the function of the above jasmonic acid signalling pathway-related genes, the construction of related variants is the most important evidence, and the construction of the transgenic method of Taxus cell line is a worldwide problem, which is also related to this study. The study will validate the function of related genes and realize the construction of high-yield Taxus cell lines in the future.
4. Materials and Methods
4.1. Cell Culture, Jasmonate Treatment, and RNA Isolation
The
T. media cell line Zike used in this study was induced from sapling leaf purchased in Beijing. The cells were subcultured using modified SSS medium. For jasmonate treatment, 25 μL 100 mM methyl jasmonate (MeJA) dissolved in ethanol was added to 2 g cells that were suspension cultured in 50 mL of medium 14 days after subculture. Samples were collected after 0, 0.5, 3, and 24 h MeJA treatment using a Büchner funnel. Three biologically independent experiments were performed and a total of 12 samples were obtained. Total RNA was isolated from each of the samples as described before [
25]. Generally, the cells were frozen in liquid nitrogen and ground to powder. Every 2 g of cell powder was lysed in 10 mL GTC buffer (62.5 mM Tris-HCl, 12.5 mM EDTA, and 5 M guanidinium isothiocyanate, pH 8.0), then incubated on ice for 15 min and centrifuged at 15,000×
g for 15 min. The supernatant was added to an equal volume of isopropanol to precipitate the total RNA. The total RNA was purified using an RNeasy Mini kit (Qiagen, City, Germany) according to the manufacturer’s instructions.
4.2. RACE PCR
The full length sequence of the 5′ and 3′ ends of the gene was obtained using the GeneRacer Kit with SuperScript III RT kit based on Invitrogen’s RACE PCR technology (CA, USA) using high quality RNA extraction. First, the extracted RNA is dephosphorylated, and then the enzyme is used to remove the 5′ end cap of the RNA, and then the known RNA linker sequence is ligated. Using this as a template, the first strand of the cDNA is synthesized by the oligo(T) used as a locking primer to bind the the 3′ end ploy(A) tail of mRNA. Design specific primers for nested PCR at the 5′ and 3′ ends, respectively, clone the obtained DNA fragments, analyse the sequence of the coding region after sequencing, and after splicing, both stop codons and reach the longest coding region to determine the full length sequence at both ends.
4.3. Yeast Two-Hybrid
Based on Clontech’s MATCHMAKER LexA Two-Hybrid System, JAZ and other related genes are ligated into a pLexA vector containing a LexA promoter binding domain (BD), and another gene for detecting interaction is linked to transcriptional activation domain(AD) of the pB42AD vector. The above two plasmids were then transferred to the lacZ-expressing active EGY48 yeast strain containing LexA control using the Frozen-EZ Yeast Transformation Kit of Zymo Research. After overnight culture in SD/–His/–Trp/–Ura liquid medium, transfer 5–20 μL of yeast culture solution to SD/Gal/Raf/–His/–Trp/–Ura/ for activity detection. In X-gal + BU salts solid medium, the appearance of blue spots was observed after four days of culture to evaluate the interaction between lacZ activity and protein.
4.4. Bimolecular Fluorescence Complementation
The sequences of the two genes detecting the interaction were ligated into p35SnYFP and p35ScYFP, respectively, and the fusion protein was formed with the C-terminus and the N-terminus of the yellow fluorescent protein YFP, respectively. The assembled plasmids were then transferred to GV3101 Agrobaterium tumefaciens by electroporation. After induction with acetosyringone for two days, the two plasmids were simultaneously transferred into the tobacco leaves of the 4-week culture by injection, and then the fluorescence signal of the injection site on the leaves was observed using a laser confocal microscope.
4.5. Genome Walker PCR Separation Promoter Sequence
To obtain the promoter of the paclitaxel biosynthetic pathway gene, the genome of T. media was extracted using the Plant Genomic DNA Purification Kit (Tiangen, Beijing, China). Based on Clontech’s Universal Genome Walker Kit (Clontech, Takara, Japan), genomic DNA was digested with four different blunt-end restriction enzymes DraI, EcoRV, PvuII, and StuI, followed by ligation to the linker. Primers were designed for two rounds of nested PCR by known coding region sequences and linker sequences, and the obtained DNA bands were cloned, sequenced, spliced and analysed to obtain the promoter sequences of the corresponding genes.
4.6. Detection of Transcription Factor Activity in Protoplasts
To analyse the transcriptional activity, the transcription factor was transferred to a plasmid 35SGAL4DB containing a GAL4DB domain in a plant cell to form a plasmid expressing a fusion of the GAL4DB domain and a transcription factor. Based on the mature
Arabidopsis protoplast transformation method [
26], the
Arabidopsis protoplasts were transferred by a PEG transformation method, and a plasmid containing a GAL4 promoter-controlled transcription factor was simultaneously transformed. The plasmid also contained the reporter gene for the LUC fluorescent protein in the plasmid and the plasmid for the continuous expression of the REN fluorescent protein internal reference. The fluorescence intensity changes of the two fluorescent proteins were detected using the Promega Dual Luciferase Reporter Assay system kit (Cat. E1910) (Madison, WI, USA), and the activity of the transcription factors in the plant cells was analysed by comparing the changes in the fluorescence intensity ratio.
4.7. Detection of Transcription Factor Regulation in Protoplasts
The promoter of the paclitaxel biosynthetic pathway gene was inserted into the coding region of the LUC fluorescent protein in the 0800 plasmid to construct a plasmid, which contained the reporter gene with promoter-controlled LUC fluorescent protein and the continuous expression of REN fluorescent protein as internal reference. At the same time, the transcription factor to be detected is inserted into another plasmid 62sk, and the transcription factor can be continuously expressed in the plant by 35S regulation. The two plasmids were co-transformed into protoplasts by PEG transformation. The fluorescence intensity of the two fluorescent proteins was simultaneously detected using the Promega Dual Luciferase Reporter Assay system (Cat. E1910) kit, and the regulation of the transcription factor on the paclitaxel biosynthesis pathway promoter was analyzed.
5. Conclusions
In this study, based on the jasmonic acid signalling pathway of the Arabidopsis model, the jasmonic acid signalling pathway of T. media was isolated and identified. The interaction between related proteins was analysed, and the molecular mechanism of the jasmonic acid signalling pathway in Taxus was constructed. Further analysis of the activation or inhibition of the promoter of paclitaxel biosynthesis pathway gene by transcription factors such as MYC2 confirmed the regulation of MYC2 and other transcription factors on the biosynthesis pathway of paclitaxel. Based on this design, the transcriptional activity of MYC2 and MYC3 was not affected by the JAZ inhibition function, which laid a foundation for the construction of a high-yield paclitaxel variant Taxus cell line.
Taxus has a corresponding jasmonic acid signalling pathway, and it directly regulates the expression of paclitaxel biosynthesis pathway genes through MYC transcription factors, and JAZ and non-transcriptionally active MYC transcription factors comprehensively regulate the expression of related genes. Based on the signalling pathway, a variant MYC transcription factor was constructed to activate transcription without inhibition by JAZ protein, and was the basis for constructing a taxol-producing Taxus cell line.