JA-mediated transcriptional regulation of secondary metabolism in medicinal plants

Plants produce a wide spectrum of secondary metabolites that play critical roles in plant-environment interactions and against biotic and abiotic stresses. Moreover, many secondary metabolites have pharmaceutical efﬁcacy for a wide range of diseases (cancer, malaria, etc.). Controlled transcription of biosynthetic genes is one of the major mechanisms regulating secondary metabolism in plants. Several transcription factor families such as MYC, MYB, WRKY and AP2/ERF have been found to be involved in the regulation of secondary metabolism in different medicinal plants. In addition, the biosynthesis and proper accumulation of secondary metabolites are also induced by signaling molecule jasmonic acid (JA). This review provides an insight into JA signaling pathway and JA-mediated transcriptional regulation of secondary metabolism (vinblastine, nicotine, artemisinin, taxol and ginsenoside) in a range of medicinal plant species. ubiquitin ligase and form SCF COI1 complex, thereby recruiting the JAZ proteins, them for their degradation by the 26S proteasome and ultimately leading to the release of the TFs that can modulate JA-responsive gene expressions.


Introduction
Plants produce a variety of low molecular weight organic compounds that are usually ramified into two large classes: primary and secondary metabolites. Primary metabolites are essential for plant growth and development, while secondary metabolites act as defense molecules and protect plants in various adverse conditions. Many secondary metabolites have high pharmaceutical value and have been used for the treatment of wide range of diseases [1]. For example, vinblastine and nicotine are used for treatment of tumor [2] and asthma [3] diseases, respectively. Artemisinin, another essential secondary metabolite, is extensively employed as antimalarial drug [4]. Furthermore, two additional pharmaceutically important secondary metabolites, taxol and ginsenosides, are generally used for the treatment of breast, ovarian and non-small cell lung cancers [5,6]. Because of their beneficial effects to health, many researchers have been focusing on the comprehensive study of regulation and production of secondary metabolites. The biosynthesis and proper accumulation of secondary metabolites are strictly controlled in a spatial and temporal manner and influenced by a number of biotic and abiotic factors. The spatiotemporal transcriptional regulation of metabolic pathways is controlled by a complex network involving many regulatory proteins known as transcription factors (TFs). TFs are sequence-specific DNA-binding proteins that interact with the regulatory regions of the target genes and modulate the rate of transcriptional initiation by RNA polymerase [7]. Many TFs have been characterized for their roles in regulating biosynthetic pathways at the transcriptional level. TFs include a wide number of proteins that initiate and regulate the transcription of several genes. These proteins regulate gene transcription depending on tissue type and in response to external and internal signals [8]. Jasmonic acid (JA) that presents in the scent of jasmine flowers (Jasminum grandiflorum) is known as an essential signaling molecule which can coordinate a lot of cellular activities [9]. JA and its cyclic S. Afrin  precursors and derivatives (such as methyl jasmonate, MeJA) are collectively referred to as jasmonates (JAs). JAs act as ubiquitous and conserved elicitors for the production of secondary metabolites across the plant kingdom, from gymnosperms to angiosperms [10,11]. The promising depiction of transcriptional regulation of secondary metabolism suggests that, in response to phytohormones (JAs), TFs form dynamic regulatory networks that fine-tune the timing, amplitude and tissue-specific expression of pathway genes and the subsequent accumulation of secondary metabolites [8]. Here, we give an updated review on JA-responsive signaling pathway and speculate on JA-mediated transcriptional regulation of secondary metabolism (e.g., vinblastine, nicotine, artemisinin, taxol and ginsenoside) in a range of medicinal plant species.
2 Key players in the JA-mediated signaling pathway JAs are oxylipin-derived phytohormones that regulate a wide variety of physiological processes in plants including growth, development, reproduction and defense [12]. JAs are synthesized from the fatty acid a-linolenic acid via the octadecanoid pathway [13]. In general, the JAs signaling pathway is accomplished in two steps.

Formation of SCF COI1 complex and degradation of JAZ proteins by 26S proteasome
In JA signaling pathway, the F-box protein COR-ONATINE INSENSITIVE 1 (COI1) connects with Skp-Cullin-F-box-type E3 ubiquitin ligase and forms SCF COI1 complex to which it provides substrate specificity [10,12]. The targets of the SCF COI1 complex are the JA ZIM domain (JAZ) family of repressor proteins. JAZ is one of the key molecules in this pathway that serves as the on/off switch for JA signaling. JAZ and COI1 proteins directly interact in the presence of the bioactive JA-isoleucine (JA-Ile) conjugate to form a co-receptor complex. Although ubiquitination of the JAZ proteins also joins with SCF COI1 , this interaction ultimately triggers the degradation of the JAZ proteins by the 26S proteasome ( Fig. 1)

Transcriptional coordination and enhance physiological activity
In the presence of JA or its bioactive derivatives, JAZ proteins are degraded and freeing TFs for expression of specific sets of JA-responsive genes, thereby promoting physiological activity [9], such as produce specific sets of secondary metabolites (Fig. 1) . Based on these evidence, it is intriguing to recommend that many TFs are highly conserved and play a central role in the JA-mediated signaling pathway.

Transcriptional regulation of secondary metabolism in medicinal plants
Transcriptional regulation is the change in gene expression levels by altering transcription rates. Regulation of transcription is depending on the combined effects of structural properties and the interactions of TFs. The expression of genes encoding enzymes involved in one particular secondary metabolic pathway often displayed a marked concerted up-regulation after JA elicitation, leading to the recognition of so-called transcriptional regulons. Several genome-wide transcript profiling studies have demonstrated that JAs treatment triggers an extensive transcriptional reprogramming of metabolism [10]. Several TFs that can be elicited by JAs and regulate specific metabolic pathway are given in Table 1.

Vinblastine
The plant Madagascar periwinkle (C. roseus), a member of the Apocynaceae family, produces a large array of TIAs. TIAs can be characterized into two principle groups: monoterpene indole alkaloids (MIAs) and the bisindole alkaloids that composed of two joined MIAs. These TIAs, either natural or semisynthetic forms, have pharmaceutical applications. For example, the MIAs serpentine and ajmalicine are used as a tranquillizer and to reduce hypertension, respectively, and the bisindole alkaloids vincristine and vinblastine are potent as antitumor drugs [2,42]. Moreover, vinblastine also shows a strong antimicrobial activity [43]. The large interest in the anticancer compound vinblastine, which is derived from the coupling of catharanthine and vindoline, contrasts with the low amounts of these compounds found in the plants, making its extraction a very expensive process [44].  including the TGACG motifs, and suggests the involvement of novel TFs in this regulatory network [60, 61]. These findings demonstrate that the preferential expression of CrWRKY1 and its interaction with other TFs (including CrORCAs and CrMYCs) play a key role in determining the accumulation of vinblastine in C. roseus.

Nicotine
Nicotiana, a member of the Solanaceae family, is one of the most important research model plants and of high economic value worldwide. The main secondary metabolites found in Nicotiana species are tobacco alkaloids [62]. The biosynthesis of nicotine involves pyrrolidine ring formation, pyridine ring formation and the coupling of both rings. Several enzymes and corresponding genes are involved in the major steps of the nicotine synthetic pathway [63]. In N. tabacum, arginine decarboxylase (ADC), ornithine decarboxylase (ODC), putrescine N-methyltransferase (PMT) and N-methyl-putrescine oxidase (MPO) are involved in the formation of the pyrrolidine ring; on the other hand, aspartate oxidase (AO), quinolinate synthase (QS) and quinolinic acid phosphoribosyltransferase (QPT) are responsible for the biosynthesis of the pyridine ring [64]. Furthermore, a PIP (pinoresinol-lariciresinol reductase, isoflavone reductase and phenylcoumaran benzylic ether reductase) family isoflavone reductase-like protein A622 and berberine bridge enzymelike protein (BBL) are required for nicotine ring coupling [62]. However, it remains unclear how the two rings are joined to form nicotine. Data from a range of studies revealed that the complex regulation of nicotine biosynthesis involves a number of TFs and JAs is a positive regulator for nicotine biosynthesis [62, 64,65]. In response to JA, the JA receptor NtCOI1 mediates degradation of the NtJAZs protein, a transcriptional repressor, resulting in inducible expression of PMT (Fig. 2) [36]. In N. benthamiana, two bHLH TFs bHLH1 and bHLH2, which are homologs of MYC2, bind to the PMT promoter to provide positive regulation of nicotine synthesis. A series of ERF TFs that regulate nicotine levels play a role in regulating nicotine content [22]. In N. tabacum, 7 AP2/ERF TFs were found in the NIC2 locus [36].The JA-inducible ORC1 (also known as ERF221) and ERF189 TFs cluster together in the NIC2 locus and are up-regulated by JAs elicitation. ORC1 and ERF189 have overlapping but non-redundant roles in regulating nicotine biosynthetic genes [36,66]. ORC1 recognition of the GCC-box of the PMT promoter was necessary but not sufficient for activation. ORC1 is a close homolog of C. roseus ORCA3, and its overexpression stimulated alkaloid biosynthesis [66]. NtMYC2 directly regulates the expression of selected nicotine biosynthetic genes and also regulates nicotine biosynthesis through regulation of the NIC2 AP2/ERF TFs [20,67]. In N. benthamiana, MYC2 homolog NbbHLH1 functions in combination with ORC1, in binding to the G and GCC-boxes of PMT1 and quinolinate phosphoribosyl transferase (QPRT2) promoters, respectively; both TFs are necessary for optimal PMT1 and QPRT2 activation [66]. Future work is needed to determine the roles of the other five NIC2 locus ERFs specifically play in regulating nicotine biosynthesis. In addition, JA-induced phosphorylation cascade has also been shown to play a crucial role in nicotine biosynthesis; for example, the activity of both ORC1 and the MYC-type bHLH proteins can be posttranslationally up-regulated by a JA-modulated phosphorylation cascade. By contrast, in tobacco, the JA-inducible MYBJS1 (a R2R3-MYB TF) was shown to induce phenylpropanoid biosynthetic genes, and the accumulation of phenylpropanoid polyamine conjugates during stress [35]. A related MYB, MYB8, was recently found to control inducible phenolamide levels in Nicotiana attenuata [37]. Collectively, these findings indicate the intricate interaction between different TFs in the regulation of nicotine and other secondary metabolites in Nicotiana species.

Artemisinin
Artemisinin, a sesquiterpene lactone endoperoxide that isolated from Artemisia annua (family Asteraceae), has been widely employed as antimalarial drugs [4,30]. Artemisinin is effective against chloroquine-and quinineresistant Plasmodium falciparum as well as other malariacausing parasites [68]. Besides their antimalarial activity, recently artemisinins have also been reported to possess antiviral [31], anticancer [32] and antischistosomal activities [69]. To date, A. annua is the only natural source of artemisinin. The relatively low yield (0.1 %-0.8 % by dry weight) of artemisinin in A. annua is a major limitation to the commercialization of the drug, so the enhanced production of artemisinin is highly desirable [70]. Caretto et al. [71] have recognized that the plant A. annua are able to produce high content of artemisinin in respond to the elicitor effect of JAs. So, controlled transcription of biosynthetic genes by JA-responsive TFs is an alternative way to produce high artemisinin from A. annua. Recently, the artemisinin biosynthetic pathway has been almost completely revealed by several groups [72]. In artemisinin biosynthesis pathway, amorpha-4,11-diene synthase (ADS) catalyzes the conversion of farnesyl diphosphate into amorpha-4,11-diene, which is the first committed step in A. annua [73]. In the second step, amorpha-4,11-diene is hydroxylated to yield artemisinic alcohol, which is subsequently oxidized to form artemisinic aldehyde. The two steps are catalyzed by a bifunctional cytochrome P450dependent hydroxylase (CYP71AV1) together with the NADPH: cytochrome P450 oxidoreductase (CPR) [74]. Most of the genes involved in the artemisinin biosynthetic pathway are likely to be trichome specific and to be controlled by one or several TFs [75]. Two jasmonate-responsive AP2/ERF TFs AaERF1 and AaERF2 can regulate the transcription of ADS and CYP sesquiterpene oxidase (CYP71AV1) and to activate expression of both genes. Overexpression of AaERF1 or AaERF2 led to increased accumulation of artemisinin and artemisinic acids [30]. The promoters of ADS contain two reverse-oriented TTGACC W-box cis-acting elements, which are the proposed binding sites of WRKY TFs. Ma et al. [4] first reported about WRKY1 TF in A. annua by isolating fulllength cDNA of AaWRKY1. In A. annua, JA-responsive WRKY1 can transactivate the promoter of the ADS gene and might participate in the regulation of artemisinin biosynthesis. Majority of the artemisinin biosynthetic gene expressions were activated by transient expression of AaWRKY1 cDNA [4,76]. Recently, the AP2/ERF TF AaORA1 has been shown to regulate accumulation of artemisinin [77]. With some exception, AaORA1 regulates the same enzymes as AaWRKY1 that directing the pathway toward artemisinin formation. Moreover, Ji et al. [70] report about the isolation and characterization of novel bHLH TF (AabHLH1) from A. annua. AabHLH1 can positively regulate the transcription of ADS and CYP71AV1 by binding to the E boxes in the promoters, and transient expression of the AabHLH1 TF in leaves of A. annua resulted in increased levels of ADS and CYP71AV1. The previous study indicates that JA-responsive expression of alkaloid biosynthesis genes in C. roseus is controlled by a TF cascade consisting of the bHLH protein, CrMYC2, which regulates the gene expression AP2/ERF-domain transcription factor ORCA (ORCA2 and ORCA3) [54]. It should be interesting to investigate in the future whether there is a TF cascade consisting of AabHLHs regulating AP2/ERF-domain TFs (AaERF1 and AaERF2) in artemisinin biosynthesis in A. annua.

Taxol
Taxol (generic name paclitaxel), a complex diterpene found in the gymnosperm genus Taxus, is one of the most promising anticancer agents and widely used for the treatment of breast, ovarian and non-small cell lung cancers as well as AIDS-related Kaposi's sarcoma [5]. Paclitaxel is also being investigated to use in the treatment of neurological disorders and in postsurgery heart patients. Production of taxol from yew trees remains a challenging problem due to the limited resources of Taxus sp. Moreover, paclitaxel found small quantities in plants (0.01 %-0.03 % of the dry weight of the bark of Taxus), and total biosynthesis comprises several steps and is therefore low yielding [39]. The biosynthetic pathway is believed to contain 19 steps; however, there are certain transformations which have yet to be fully characterized [78]. Like other secondary metabolites, JAs can significantly induce the production of taxol and related taxanes in Taxus sp. [39,79]. The time-course analysis revealed that there are two regulatory steps existing in the paclitaxel biosynthesis: (i) the taxane ring formation step that is up-regulated by JAs and (ii) the acylation step at the C-13 position. The enzyme 10-deacetylbaccatin III-10 b-o-acetyl transferase (DBAT) is a key rate-limiting step in the biosynthesis of taxol [80]. Li et al. [81] identified a WRKY TF, TcWRKY1, with the yeast one-hybrid system from Taxus chinensis cells, as a regulator of DBAT. The gene expression of TcWRKY1 in T. chinensis suspension cells was specifically induced by JAs. Biochemical analysis indicated that TcWRKY1 protein specifically interacts with the two W-box (TGAC) cis-elements among the important regulatory elements. Overexpression of TcWRKY1 enhanced DBAT expression in T. chinensis suspension cells, and RNA interference (RNAi) reduced the transcript levels of DBAT. These results suggest that TcWRKY1 participates in regulation of taxol biosynthesis in T. chinensis cells and DBAT is a target gene of this WRKY TF. Recently, Lenka et al.
[39] identified three JA-inducible MYC TFs TcJAMYC1, TcJAMYC2 and TcJAMYC4 from Taxus cuspidata. The MYC TFs from Taxus sp. exhibit high similarity to Arabidopsis MYC2. The similarity in sequence and expression between TcJAMYC(s) and the well-characterized AtMYC2 suggests a conserved response to JAs despite significant divergence between the gymnosperm and angiosperm lineages. The results suggest that TcJAMYC(s) are regulators of JA-mediated expression of taxane biosynthetic genes in Taxus, but that they function as negative rather than positive regulators of these genes. Further analysis of other TFs will be helpful to understand the regulation patterns upon JAs elicitation and the molecular mechanisms of JA-mediated taxane biosynthesis in Taxus sp.

Ginsenoside
Ginseng (Panax ginseng C.A. Meyer) belonging to the family Araliaceae has been used as a traditional medicine since the ancient times. The major active component of ginseng roots is ginsenoside, a triterpenoid saponin. Saponins have been shown to have many beneficial bioactive effects on human health, such as antitumor, antihyperglycemic, antiinflammatory, antistress, antiaging and improving immune function [82,83]. To date, more than 40 ginsenosides have been characterized, which can be divided into three categories according to the position of side chains on the shared a glycine skeleton. These three types are the protopanaxadiol (PPD), protopanaxatriol (PPT) and oleanolic acid [83]. Due to their broad pharmaceutical properties, special attention has been paid to genes involved in ginsenoside biosynthesis for large-scale production. Ginsenosides are synthesized by the cyclization of 2,3-oxidosqualene via the mevalonate pathway. This cyclization occurs at the branching point leading to oleanane-and dammarene-type ginsenosides. Dammarenetype triterpenes undergo further modifications such as oxidation/hydroxylation, substitution and glycosylation leading to PPD-and PPT-type ginsenosides. Despite commercial interest in ginsenosides, very little is known about the genes involved in the biosynthetic pathway of triterpenoid saponins [83,84].
Various elicitation strategies have been developed to stimulate ginsenoside biosynthesis of ginseng plants. For example, JAs treatment can increase the production of ginsenosides. The previous study indicates that JAs (especially MeJA) have a great impact on ginsenoside production in ginseng hairy root and adventitious root cultures. The stimulation of ginsenosides production with JAs may be mediated by the up-regulation of the genes involved in the biosynthesis and accumulation of saponins [85][86][87]. Recently, Sun et al.
[41] isolated one JA-responsive WRKY family TF (PqWRKY1) that positively regulate triterpene ginsenoside biosynthesis in P. quinquefolius. In our work, one MYB TF (PgMYB1) has been isolated from P. ginseng that exhibit negative transcript effect by JAs [88]. However, transcriptional regulation of ginsenoside is little known, and the research in this field will be emerged in near future.

JA-responsive transcriptional networks and elicitation of secondary metabolites
Secondary metabolites play diverse roles in plants.
Biosynthesis of defensive secondary metabolites is often induced by the following attack of plant. Primary signals specifying attack which are recognized by the plant are called elicitors. These elicitors activate signal transduction pathways that generate secondary signals produced by plants [11]. Three major secondary signaling molecules are JAs, ethylene and salicylic acid [89,90]. Production of these hormones generates a signal transduction network that leads to a cascade of events responsible for the physiological adaptation of the plant cell to the external stress [91]. Among these three key regulatory signals, by far the most important molecules for induction of secondary metabolism are the JAs. JAs play key roles in defense against insects and certain pathogens, among others by controlling the biosynthesis of protective secondary metabolites [92]. Coordinate transcriptional control of biosynthetic genes emerges as a major mechanism dictating the final levels of secondary metabolites in plant cells. This regulation of biosynthetic pathways is achieved by specific TFs. TFs form useful tools for engineering the production of valuable secondary metabolites [7]. Studies related to the regulation of secondary metabolite pathways have been focused mainly on characterization of TFs regulating the structural genes. However, only a few have provided mechanistic insight about regulation. The bHLH TF MYC2 forms a very valuable tool, since it occupies the highest position in the regulatory hierarchy and it controls the expression of both the ORCA2 and ORCA3 genes and possibly of other genes encoding TFs involved in alkaloid biosynthesis in C. roseus. Activation of CrMYC2 involves JA-induced degradation of JAZ proteins that forms a repressor complex with CrMYC2 in the absence of JA [19]. Similarly, biosynthesis of nicotine is also induced by JA in a COI1-JAZ-dependent manner. Zhang et al. [21] have shown that tobacco contains multiple MYC2 genes (NtMYC2a, NtMYC2b and NtMYC2c) involved in nicotine biosynthesis. The similarities between the components involved in regulation of JA-responsive alkaloid metabolism in tobacco and in C. roseus suggest that the overall regulatory circuits are highly similar (Fig. 2). The above finding indicates that JAZ proteins are conserved components of JAs signaling in plants. However, JA-mediated WRKY TF also acts as a powerful tool for medicinal plants secondary metabolism. In C. roseus, CrWRKY1 interacts with other TFs and helps to accumulate important secondary metabolites [93]. AaWRKY1 and TcWRKY1 also participate in regulation of artemisinin and taxol biosynthesis in A. annua and T. chinensis, respectively [4,82].
Recently, Sun et al.
[41] reported about PqWRKY1, which positively regulates triterpene ginsenoside biosynthesis in P. quinquefolius. These evidences suggest that the concerted transcriptional activation of whole pathways is not necessarily activated by the action of a single TF; quite the opposite, a combinatorial role for several TFs in the regulation of different enzymes or suites of enzymes seems currently more reasonable to clarification for the control of biosynthetic pathways by JAs. Combinatorial exploit of TFs has already been confirmed in non-medicinal secondary metabolite flavonoid biosynthesis [94]. In case of medicinal plants, the JA-mediated elicitation of tobacco nicotine biosynthesis also involves the concerted action of AP2/ERF and bHLH TFs [67]. It implies that combinatorial interactions of TF families are proficient to biosynthesis various important secondary metabolites. JAs have an evolutionarily conserved role in the reprogramming of plant secondary metabolism in response to various environmental or developmental stimuli, and JAmediated induction of secondary metabolism is strongly and rapidly influenced by defense responses [95]. When the plant is under attack, the JA signal needs to persist or even to intensify for the induction of secondary metabolite accumulation. As a result, plants have developed a positive response system with loops at various control points, termed as autoregulatory JA loop [96]. The mechanisms involved in the autoregulatory JA loop have mainly been studied in Arabidopsis, but the phenomenon is conserved among plants. Positive and negative feedback loops might be increased the system of secondary metabolism but not at all costs. In case of positive loops, a control point is situated at the genes encoding JAs biosynthesis enzymes that are all JAs inducible and controlled by JA-responsive TFs (such as MYC2), that increase bioactive JAs synthesis [10,96]. After that, many genes encoding TFs involved in the primary JAs response are rapidly induced by the same signal and can modulate their own expression [10, 17]. Finally, some other sets of regulators and a number of auxin response factors (ARF6 and ARF8) can positively influence JA biosynthesis in a developmental context [97]. In addition to positive loops, negative feedback is also needed to shut down plant defense and stress responses to avoid energy being wasted. Multiple regulatory mechanisms have been developed to maintain energy consumption. As a result, negative regulators are key components in the control of stress-related gene expression. ERF-associated amphiphilic repression (EAR)-domain-containing proteins have been identified as characteristic elements of transcriptional repression of gene expression in plants [18]. Some EAR proteins have been shown to be active in metabolic pathways. In C. roseus, three members of the EAR-domain-containing TF IIIA-type zinc-finger protein family, ZCT1, ZCT2 and ZCT3, were reported to bind the promoters of the STR and TDC genes and therefore repress their expression [29]. Such information can potentially be used to engineer metabolic pathways to enhance the accumulation of important secondary compounds in medicinal plants.

Conclusions and perspectives
The plant secondary metabolites play important roles in plant-environment interactions. Also they have been proven to be beneficial for human health, and many secondary metabolites are highly valuable in the pharmacological application. However, direct extraction of these metabolites is usually complicated and inefficient due to their low concentrations in plant tissues. Biosynthesis and regulation of secondary metabolites is multilayer network that requires common signaling pathways and key molecules such as JAs. JAs are instrumental in controlling plant insect defense, wounding responses, development and secondary metabolite biosynthesis. TFs play a paramount role in regulating genes involved in likely all aspects of plant growth and development, including secondary metabolism. In the last decades, an increasing number of TFs and the underlying mechanisms of secondary metabolism have been elucidated. Although the basic JA signaling pathway that releases many TFs is much clearer, the binding sites of TFs and other details need to be further clarified. Meanwhile, JA-responsive transcriptional regulations have been identified mostly in model plants, but the cases in medicinal plants remain elusive. JAs have an evolutionarily conserved role in reprogramming plant secondary metabolism. An important aspect herein is the concerted transcriptional activation of the genes encoding the enzymes that catalyze the secondary metabolic reactions. Often, JAs simultaneously induce all known biosynthetic genes from a particular pathway, as illustrated by the vinblastine, nicotine, artemisinin, taxol and ginsenoside pathways in C. roseus, N. tabacum, A. annua, Taxus sp. and P. ginseng, respectively. Increasing secondary metabolites by engineering TFs is a promising method in near future.