The jasmonate-responsive transcription factor CbWRKY24

Journal of Genetics, Vol. 97, No. 5, December 2018, pp. 1379–1388 © Indian Academy of Scienceshttps://doi.org/10.1007/s12041-018-1026-5

RESEARCH ARTICLE

The jasmonate-responsive transcription factor CbWRKY24 regulatesterpenoid biosynthetic genes to promote saponin biosynthesis in Conyzablinii H. Lév.

WEN-JUN SUN, JUN-YI ZHAN, TIAN-RUN ZHENG, RONG SUN, TAO WANG, ZI-ZHONG TANG,TONG-LIANG BU, CHENG-LEI LI, QI WU and HUI CHEN∗

College of life Science, Sichuan Agricultural University, No. 46, XinKang Road, Ya’an 625014, Sichuan,People’s Republic of China*For correspondence. E-mail: [email protected].

Received 4 May 2018; revised 27 June 2018; accepted 27 July 2018; published online 14 November 2018

Abstract. Conyza blinii H. Lév., the most effective component is saponin, is a biennial medicinal material that needs to beoverwintered. WRKY transcription factors family is a large protein superfamily that plays a predominant role in plant secondarymetabolism, but their characteristics and functions have not been identified in C. blinii. The CbWRKY24 sequence was selectedfrom the transcriptome database of the C. blinii leaves constructed in our laboratory. Phylogenetic tree analysis revealed that it wasassociated withAaWRKY1which can regulate artemisinin synthesis inArtemisia annua. Expression analysis inC. blinii revealed thatCbWRKY24 was mainly induced by methyl jasmonate (MeJA) and cold treatments. Transcriptional activity assay showed that ithad an independent biological activity. Overexpression of CbWRKY24 in transient transformed C. blinii resulted in improved totalsaponins content, which was attributed to upregulate the expression level of keys genes frommevalonate (MVA) pathway in transienttransformed plants compared to wild type (WT) plants.Meanwhile, overexpression theCbWRKY24 in transient transformed tomatofruits showed that the transcript level of related genes in lycopene pathway decreased significantly when compared to WT tomatofruits. Additionally, the MeJA-response-element was found in the promoter regions of CbWRKY24 and the histochemical stainingexperiments showed that promoter had GUS activity in transiently transformed tobacco leaves. In summary, our results indicatedthat we may have found a transcription factor that can regulate the biosynthesis of terpenoids in C. blinii.

Keywords. CbWRKY24 gene; saponin; methyl jasmonate; Conyza blinii H. Lév.

Introduction

Conyza blinii H. Lév. is a biennial medicinal plantbelonging to compositae, which is distributed in southwestChina. Its dried aerial part is used to treat inflammatorydiseases such as gastroenteritis and chronic bronchitis infolk (Su et al.2000). It hasbeen found that its effective com-ponents are diterpenoids, triterpenoids and flavonoids (Suet al. 2003). Blinin is an important diterpenoid compound,which is a characteristic compound ofC. blinii (Yang et al.1989). According to previous studies, C. blinii saponinhas a strong protective effect on ethanol-induced acute

Electronic supplementary material: The online version of this article (https://doi.org/10.1007/s12041-018-1026-5) contains supplemen-tary material, which is available to authorized users.

gastric ulcer (MaandLiu 2014). Subsequently, the saponinfraction isolated from C. blinii was found to have excellentantitumour activity by inhibiting NF-KB signalling path-way (Ma et al. 2016). In addition, few studies showed thatC. blinii saponin can play an anti-cancer role in vivo and invitro in many ways (Ma et al. 2017).

C. blinii is a unique Chinese herbal medicine with greatmedicinal and economic value in southwest China. How-ever, the low content of terpenes seriously hampers theclinical application of C. blinii. Many studies have beendevoted to elucidating the terpenoid synthesis pathwaysofC. blinii, so as to improve terpenoid contents by playing

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the role of key enzyme genes (Sun et al. 2017). Themevalonate (MVA) pathway in C. blinii formed the triter-penoid compounds represented by oleanane type of triter-penoid saponins, and the methylerythritol phosphate(MEP) pathway formed the diterpenoid compounds rep-resented by blinin (Sykora and Legut 2015; Sun et al.2017). Studies have shown that the content of secondarymetabolites in plants can be improved by overexpress-ing the terpene synthase gene (Yin et al. 2017; Bansalet al. 2018), but sometimes the regulation effect of a singleenzyme gene is minimal (Carleton et al. 2017). Transcrip-tional factors can regulate the whole metabolic pathway,thus in recent years, it has attracted wide attention in theregulation of the growth, development and the synthesis ofeffective components in plants (Dai et al. 2009; Zhu et al.2012; Huang et al. 2016; Zhao and Liu 2017).

At present, the transcription factors that regulateterpenoid pathways are mainly found in the AP2-ERF,WRKY, bZIP and bHLH families. The WRKY proteincannot only participate in plant growth regulation, butalso play a necessary role in regulate plant metabolic path-ways (Li et al. 2012; Tang et al. 2014). In recent years,many WRKY transcription factors that regulate effectivecomponents of medicinal plants have been isolated andidentified (Ma et al. 2009; Chen et al. 2017).

However, there are few studies on WRKY transcrip-tion factors regulating terpenoid pathways in C. blini.Therefore, it is necessary to isolate and identify thesetranscription factors from the C. blinii. In this study,based on the transcriptome database of C. blinii, a novelWRKY transcription factor (designated as CbWRKY24)that responds toMeJAwas isolatedandcharacterized.Ourresults indicated that CbWRKY24 may improve the con-tent of total saponins through upregulating the expressionlevel of multiple MVA pathway genes.

Materials and methods

Plant materials and stress conditions

C. blinii known as ‘jin long dan cao’, was grown in agreenhouse at 25◦C. Tobacco and tomatoes plants werealso grown in 25◦C chamber with 16 h light/8 h dark.Seedlings ofC. bliniiwere treatedwith the following condi-tions: 100μMmethyl jasmonate (MeJA), 100μMabscisicacid (ABA), 100μM gibberellin (GA), and 4◦C (Xionget al. 2013; Shen et al. 2016). The control group was nottreated with these hormones. The samples were collectedat 0 h, 1 h, 3 h, 6 h, 12 h, 24 h, 36 h and 48 h, and quicklyfrozen in liquid nitrogen and stored at −80◦C for furtheranalysis.

Isolation and phylogenetic analysis of CbWRKY24

Extraction of total RNA with an RNAout kit andreversed into cDNA with PrimeScript RT reagent kit

(Takara, China). A novel WRKY gene (designated asCbWRKY24) was isolated from C. blinii. The completeamino acid sequences of CbWRKY24 and 30 WRKYproteins from other plants were aligned by Clustalx1.81program and then a neighbour-joining phylogenetic treewas constructed using MEGA 5 with 10,000-bootstrapreplicates (Stracke et al. 2001). The gene sequence ofCbWRKY24 was analysed via the NCBI database andthe amino acid sequence alignments of CbWRKY24 andAaWRKY1 were performed with DNAMAN.

Transcriptional assay

The coding region of CbWRKY24 was constructedon the pBridge vector by specific primers to formthe pBridge-CbWRKY24 constructs, and then trans-criptional activity was analysed. The empty vector,the positive control pBridge-GmMYBJ6, and thepBridge-CbWRKY24 plasmids were transformed intothe yeast strain AH109 respectively. The transformedcells in SD/H-Trp medium were validated by colonyPCR. The galactosidase filter lift assay was performedto evaluate its transcriptional activation. The primers arelisted in table 1 in electronic supplementary material athttp://www.ias.ac.in/jgenet/.

Transient transformation of CbWRKY24 into C. blinii andtomato fruits

The codon region of CbWRKY24 was PCR-amplifiedfrom C. blinii using the specific primers in table 1 inelectronic supplementary material and the fragment wascloned into the vector pCHF3-YFP. The recombinantplasmid was transiently transformed into the leaves ofC. blinii and tomato fruits by agrobacterium tumefa-ciens mediated transformation. The PCR was performedto identify whether the recombinant plasmid was tran-siently transformed into C. blinii and tomato fruits. TheqRT-PCR was used to detect the expression of terpenoidrelated genes in transiently transformed, empty trans-formed, and wild type C. blinii. The expression level ofkey genes from lycopene pathway in transient transformedtomato fruits were determined according to the samemethod. The content of blinin and total saponinswere alsodetermined.

Cloning of the CbWRKY24 promoter and GUS histochemistry oftransient transfection of tobacco

The isolated of CbWRKY24 promoter from C. bliniigenome was performed according to Genome Walking(TaKaRa, Japan) with nested specific primers sp1, sp2, sp3andAP primers provided in the kit. The nested PCRprod-uct was cloned into the pMD19-T vector and sequenced.Analysis the cloned promoter sequence of CbWRKY24 inthe PlantCARE database.

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The Jasmonate-responsive transcription factor 1381

The obtained promoter sequence was designated asCbWRKY24P and constructed on the plant expressionvector pBI101-GUS. The empty vector, the recombinantplasmid CbWRKY24P-GUS and the positive cauliflowermosaic virus (CaMV) 35S-GUS were transiently trans-fected into tobacco leaves using agrobacterium tumefa-ciens strain GV3101. Histochemical staining was per-formed in transiently transformed tobacco leaves. Theprimers are listed table 1 in electronic supplementarymate-rial.

Relative expression analysis by quantitative real-time PCR

The relative expression level of CbWRKY24 andterpenoid pathway related genes in C. blinii were anal-ysed by qRT-PCR. The primers for qRT-PCR weredesigned with Primer Premier 5 software according tothe CbWRKY24 cDNA sequences. The qRT-PCR wasperformed with a SYBR Premix EX Taq kit (TaKaRa,China). The housekeeping genes GAPDH (KF027475)in C. blinii and UBI3 (X58253.1) in Solanum lycoper-sicum were used as the internal standards (Mascia et al.2010; Sykora and Legut 2015). The experimental data wasprocessed by 2−��CT method and each qRT-PCR experi-mentwasperformedwithat least threebiological replicates(Livak and Schmittgen 2012).

Statistical analysis

The experimental data were analysed by using theStudent’s t-test and it was considered to be significant ifP< 0.05. The experimental data was plotted with Origin8.0 software.

Results

Cloning and characterizations analysis of CbWRKY24

To identify putative WRKY genes that may be involvedin the terpene synthesis, the WRKY gene CbWRKY24was screened from the C. blinii transcriptome databaseconstructed by our laboratory. The CbWRKY24 pro-tein was clustered together with the transcription factorsinvolved in artemisinin biosynthesis in A. annua (Zhanget al. 2009) (figure 1 in electronic supplementary mate-rial at http://www.ias.ac.in/jgenet/). TheCbWRKY24 geneencodes a protein of 342 amino acid residues with a pre-dicted molecular mass of 38.44 kDa and a calculated pIof 5.51. The CbWRKY24 has an 1029-bp open read-ing frame (GenBank accession no. MG148350). WRKYtranscription factors of different structures perform dif-ferent functions. The multiple sequence alignment resultshowed that CbWRKY24 has a single WRKYGQKdomain and has a C2H2-type zinc-finger motif at the C-terminal which belongs to group II (figure 2 in electronicsupplementary material). The WRKY family belongs togroup II is not only involved in environmental stresses,but also involved in the development of trichome andsecondary metabolism of plants (Eulgem et al. 2000).Understanding the structure of WRKY family transcrip-tion factors can lay a foundation for functional verification(Dai et al. 2016; Schluttenhofer et al. 2014; Cao et al.2018).

To further verify the characterization of CbWRKY24as a transcription factor, the transcriptional activity ofCbWRKY24 were analysed using a yeast assay system.Yeast cells containing pBridge-GmMYBJ6 and pBridge-CbWRKY24 grew well in SD/-Trp/-His medium, whereas

Figure 1. Transcriptional activation activity verification of CbWRKY24 in yeast. (a) The transformed cells were screened onSD/-His-Trpmedium. (b) After the galactosidase filter lift assay. Negative control 1 (NC1), empty pBridge plasmid; negative control 2(NC2), AH109 cells; C24, pBridge-WRKY24; positive control (PC), pBridge-GmMYBJ6. Fusion proteins of pBridge-CbWRKY24,pBridge- GmMYBJ6 and pBridge were expressed in the yeast strain AH109.

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Figure 2. Tissue-specific genes expression of CbWRKY24 andkey genes in terpenoid pathway. (a) Represents the expressionlevel of CbWRKY24 and genes in MVA pathway in differenttissues. (b) Represents the expression level of CbWRKY24 andgenes in MEP pathway in different tissues. The mRNA expres-sion patterns of genes in root, stem, leaf, and flower tissues wereexamined by qPCR assay. The 2−��CT method was used todetermine the relative expression, and the expression level ofgenes in flower tissuewere set to ‘1’.GAPDHwas used as a house-keeping gene.All experiments were performed using at least threebiological replicates and error bars indicated standard deviations(±SD). Statistical significance was determined by Student’s t-test(*P< 0.05; **P< 0.01). Different genes were represented by dif-ferent colours in columns, and the relative expression of the samegene in root, stem and leave were significantly analysed withwhich in flower.

cells containing empty vector did not grow (figure 1a).After treatment withX-gal, the pBridge-CbWRKY24 andthe positive control showed a significant blue reaction,while thenegative control hadnocolour change (figure1b).These results indicate thatCbWRKY24has transcriptionalactivity.

Expression analysis of CbWRKY24 and key genes in terpenoidpathway under different phytohormones and cold treatmentconditions

The expression of tissue-specific genes is supposed to beclosely related to some growth and development functions.

Previous experiments proved that blinin and saponin weremainly found in C. blinii leaves (figure 3 in electronic sup-plementary material). The qRT-PCR experimental resultsindicated that CbWRKY24 had the highest expressionlevel in leaves, which was similar to CbHMGR, CbβAS,CbSQE, CbSQS in MVA pathway and CbDXR, CbG-GPPS, CbMCS in MEP pathway (figure 2). From tis-sue specific expression experiments, it is speculated thatCbWRKY24 probably regulates the terpenoid synthesis inC. blinii.The relative expression level of CbWRKY24 and

terpenoid related genes were measured under stress treat-ments. From the result, it is shown that CbWRKY24almost responded to every stress treatment, but the tran-script level changed most obviously under MeJA andcold treatments (figure 3). The expression pattern ofCbWRKY24 reached the maximum at 12 h and was subse-quently declinedunderMeJA treatment,whichwas similarto CbFPPS, CbHMGR, CbBAS in MVA pathway, andCbDXS in theMEP pathways (figure 4). Under cold treat-ment, the expression profile of CbWRKY24 achieved thehighest at 6 h and was dropped, which was similar toCbSQE, CbBAS in MVA pathway, and CbDXR, CbDXSand CbGGPPS in the MEP pathways (figure 5).

Determination of blinin and saponin under differentphytohormones and cold treatments

The contents of blinin and total saponins in C. blinii werechanged under hormone and cold treatments (figure 4 inelectronic supplementary material). It was suggested fromfigure 4 in electronic supplementary material that in thefirst 24 h the change of the blinin content had the sametrend under these three hormone treatments.In brief, hormones and cold treatments have a great

influence on the content of blinin and saponin in C. blinii.It can be speculated from the experimental results thatCbWRKY24 may regulate the expression of terpenoidgenes and thus affect the changes of blinin and totalsaponin content.

Transient transformation of C. blinii and tomato fruits

To further verify the function of CbWRKY24, transienttransfected C. blinii plants and tomato fruits withCbWRKY24were generated. The PCR verification resultsshowed that CbWRKY24 was expressed in all of the tran-sient transfectedC. blinii plants and tomato fruits but notin WT and empty vector transient transfected plants (fig-ure 5 in electronic supplementary material). Transientlytransformed plants were used for the following experi-ments.


Figure 3. Expression patterns of CbWRKY24 and key genes in terpenoid pathway under stress treatments. Expression patterns ofCbWRKY24 and key genes in terpenoid pathway under MeJA (100μM), gibberellin (GA, 100μM), abscisic acid (ABA, 100μM)and 4◦C treatments after 48 h. The mRNA expression patterns ofCbWRKY24 and key genes inC. bliniiwere examined by qRT-PCRassay. The 2−��CT method was used to determine the relative expression. GAPDH was used as a housekeeping gene. The diagramwas obtained by the heat map making tool HemL.

CbWRKY24 positively regulates the saponin pathway in C. bliniiand negatively regulates lycopene pathway in tomato

To confirm whether CbWRKY24 was involved inbiosynthesis of terpenoids, the expression patterns of keygenes from terpenoid pathways in transiently transformedplants with CbWRKY24, empty vector and WT plantswere measured. From the results, we can see that thetranscript level of CbβAS in transiently transformed C.blinii with CbWRKY24 was ∼4.6-fold compared to thatin WT plants, meanwhile the content of total saponinsalso increased (figures 6 and 7). However, the expressionlevel ofCbDXS,CbDXR andCbGGPPS inMEP pathwaydecreased, as well as the content of blinin. Conclusively,it showed that CbWRKY24 could increase the content oftotal saponins in C. blinii in some degree.

To further verify the function of CbWRKY24, it wastransiently transformed into tomato fruits. It can be seenfrom the results that the expression level of related genesfrom lycopene pathway in transient transformed tomatofruits with CbWRKY24 dropped rapidly compared toWT plants, andCbWRKY24may negatively regulated theexpression of related genes in lycopene pathway (figure 8).

Isolation and sequence analysis of the CbWRKY24P

A 2025-bp promoter fragments upstream of the startcodon of CbWRKY24 was cloned. Analysing its sequenceon plant cis-acting regulatory elements (CARE), it isfound that CbWRKY24P contained multiple cis-actingelements involved in abiotic and biotic stress responses.For example, there are heat shock response element(HSE), elicitor response element (W box) and droughtstress response element (MBS) in CbWRKY24P. In addi-tion, the cis-acting elements in CbWRKY24P also con-tains components which are involved in regulating theabiotic stress genes, including MeJA response element(TGACG-motif), abscisic acid response element (ABRE),gibberellin response element (GARE) and so on (table 2in electronic supplementary material).The CbWRKY24P: GUS fusion constructs was

transient transformed into tobacco leaves to detect theactivity. The CaMV 35S promoter transformed plants aspositive controls and nontransformed plants were used asnegative controls. Experimental results show that the tran-siently converted tobaccowithCbWRKY24Pandpositivecontrol stained different degrees of blue (figure 9).


Figure 4. Expression analysis of CbWRKY24 and key genes interpenoid pathway under MeJA (100μM) treatments after 48 h.(a) Represents the expression level of CbWRKY24 and genes inMVA pathway after MeJA treatment. (b) Represents the expres-sion level ofCbWRKY24 and genes inMEP pathway afterMeJAtreatment. The mRNA expression patterns of CbWRKY24 andkey genes in C. blinii were examined by qRT-PCR assay. The2−��CT method was used to determine the relative expression.GAPDH was used as a housekeeping gene. All experiments wereperformed using at least three biological replicates and error barsindicated standard deviations (±SD). Statistical significance wasdetermined by Student’s t-test (*P< 0.05; **P< 0.01). Differentgenes were represented by different colors in columns, and therelative expression of the same gene was significantly analysedwith which at 0 h.

Discussion

The WRKY transcription factors not only play animportant role in plant biotic and abiotic stresses, but alsoregulate the synthesis of secondary metabolites (Phukanet al. 2016). It owns a long domain with the length of 60amino acid, along with a highly conserved WRKYGQKstructure at the N-terminus and a zinc-finger structureat the C-terminus. There are three groups of WRKYtranscription factors according to the amount of WRKY

Figure 5. Expression analysis of CbWRKY24 and key genesin terpenoid pathway under cold stress treatment after 48 h. (a)Represents the expression level ofCbWRKY24andgenes inMVApathway after cold treatment. (b) Represents the expression levelofCbWRKY24 and genes inMEP pathway after cold treatment.The mRNA expression patterns of these genes in C. blinii wereexamined by qRT-PCR assay. The 2−��CT method was used todetermine the relative expression. GAPDH was used as a house-keeping gene. All experiments were performed using at least threebiological replicates and error bars indicated standard deviations(±SD). Statistical significance was determined by Student’s t-test(*P < 0.05; **P < 0.01). Different genes were represented bydifferent colours in columns, and the relative expression of thesame gene was significantly analysed with which at 0 h.

domains and the typeof zinc-finger structure (Eulgem et al.2000). Contrary to group I WRKY genes which own twoWRKYdomains, there is only one domain in group II andIII WRKY genes. It was found that there is a zinc-fingermotif with a C2H2-type structure (C–X4–5–C–X22–23–H–X1–H) in group I and II at the C-terminal, while groupIII have a C2HC zinc-finger motif C–X7–C–X23–H–X–C. Understanding the classification of WRKY family canlay a foundation for the isolation and identification oftranscription factors. Several plant WRKY factors that


Figure 6. The transcript expression of key enzymes genes fromterpenoid biosynthesis pathway in of C. blinii lines transientlytransfected with CbWRKY24. (a) Represents the transcriptionallevel of genes from MVA pathway in transient transfectedC. blinii with CbWRKY24. (b) Represents the transcriptionallevel of genes from MEP pathway in transient transfectedC. blinii with CbWRKY24. The MVA pathway formed thetriterpenoid compounds represented by saponin and the MEPpathway formed the diterpenoid compounds represented byblinin. All experiments were performed using at least three bio-logical replicates and error bars indicated standard deviations(±SD). Statistical significance was determined by Student’s t-test(*P < 0.05; **P < 0.01). Different genes were represented bydifferent colours in columns, and the relative expression of thesame gene was significantly analysed with which in WT plants.

have been functionally verified and identified are involvedin biosynthesis of terpenoids, such as AaWRKY1 (Maet al. 2009; Chen et al. 2017), TcWRKY1 (Li et al. 2013),pqWRKY1 (Sun et al. 2013), AtWRKY1 (Schluttenhofer

Figure 7. Percentage content of blinin and total saponins con-tent analysis in of C. blinii lines transiently transfected withCbWRKY24. (a) Respresents percentage of blinin in tran-sient transfected of C. blinii with CbWRKY24. (b) Respresentstotal saponins content in transient transfected of C. blinii withCbWRKY24. The content of blinin was determined by highperformance liquid chromatography, and the content of totalsaponins was determined by spectrophotometry. All experimentswere performed using at least three biological replicates and errorbars indicated standard deviations (±SD). Statistical significancewas determined by Student’s t-test (*P < 0.05; **P < 0.01).

et al. 2014) and GaWRKY1 (Xu et al. 2004). AaWRKY1,which is found inA. annua, can regulate the biosynthesis ofartemisinin. The paclitaxel owns the excellent antitumouractivity of Taxus chinensis. Now the related transcriptionfactors on the synthesis of taxol were also found in T. chi-nensis such as TcWRKY1.


Figure 8. The transcript expression of key enzymes genes fromlycopene biosynthesis pathway in of tomato fruits transientlytransfectedwithCbWRKY24. ThemRNAexpressionpatterns ofthese genes inSolanum lycopersicumwere examined by qRT-PCRassay. The 2−��CT method was used to determine the relativeexpression. UBI3 was used as a housekeeping gene. All experi-ments were performed using at least three biological replicatesand error bars indicated standard deviations (±SD). Statisti-cal significance was determined by Student’s t-test (*P < 0.05;**P < 0.01). Different genes were represented by different col-ors in columns, and the relative expression of the same gene wassignificantly analysed with which in WT plants.

In this study, MeJA-inducible CbWRKY24 wasidentified in C. blinii. It has a single WRKYGQK domainand has a C2H2-type zinc-finger motif at the C-terminal,which belongs to group II (figure 2 in electronic supple-mentary material). Transcriptional activity assay showsthat it is transcriptional activator (figure 1). The phytohor-mones GA3, ABA andMeJA play an indispensable role inregulating plant development and enhancing biosynthesisof secondary metabolites, including terpenoids (Mansouriand Asrar 2012; Wild et al. 2012; Yu et al. 2012). How-ever, low temperature is also the main environmentalstress that influences plant cell survival and physiological

activities (Chinnusamy et al. 2007). In our experiments,the CbWRKY24 has different response to each treatment,and the response are most intense under MeJA and coldtreatment (figures 3 and 4). Transcription factors are usu-ally induced rapidly under abiotic stress in the early stage,reach the maximum induction level after a few hours, andthen the expression level decreased (Dai et al. 2007). Forinstance, the transcript level of AaMYC2 reached its peakat 6 h after MeJA treatment inA. annua (Shen et al. 2016).However, the expression level of OsEBP2 which comesfrom rice accumulated quicklywithin 4 h afterMeJA treat-ment and then begun to downregulate compared withthat of control (Lin et al. 2007). The transcript level ofCbWRKY24 in our experiment begun to accumulate afterMeJA treatment for 6 h, and reached themaximum level at12 h, then the transcript level begun to decrease graduallycompared with that of control (figure 3). It is necessary toexperiment a low temperature environment in the growthcycle of C. blinii. However, the expression of CbWRKY24upregulated to 11.96-fold after cold treatment for 6 h, indi-cating thatCbWRKY24 should response to the cold stressin C. blinii (figure 4). Meanwhile, the expression patternsof CbWRKY24 and key genes in terpenoid pathway ofC. blinii were similar under MeJA and cold treatments. Itis possible to infer that CbWRKY24 may play an impor-tant role in terpene biosynthesis. To test this hypothesis,we transiently transfected CbWRKY24 to the leaves of C.blinii and tomato fruits.To verify the role of CbWRKY24 in terpenoid

biosynthesis ofC. blinii, multiple terpenoid pathway geneswere analysed by qRT-PCR in transiently transfectedplants with CbWRKY24 and WT plants. The results indi-cated that several genes, including CbFPPS, CbSQE,CbHMGR,CbBAS andCbMCS were significantly upreg-ulated in transiently transfectedC. bliniiwithCbWRKY24compared to that in WT plants; while the expressionlevel of CbGGPPS, CbDXS and CbDXR were downreg-ulated (figure 6). These genes including CbBAS, CbH-MGR, CbSQE and CbFPPS belong to MVA pathway,which can generate oleanane type of triterpenoid saponinsfrom C. blinii. Thus, the higher expression of thesegenes may improve the quality of C. blinii. Besides, the

Figure 9. Histochemical analysis of GUS in transient transformed tobacco leaves with the CbWRKY24 promoter. (a) Nontrans-formed plants as negative controls; (b) Transformed plants with the CaMV 35S promoter as a positive control; (c–e) positive lineswith CbWRKY24 promoter.


CbDXS,CbDXR,CbMCS andCbGGPPSbelong toMEPpathway, which can generate diterpenoid blinin from C.blinii. In brief, the CbWRKY24 may regulate biosynthe-sis of terpenoids from C. blinii. To further verify theeffect of CbWRKY24 on terpenoid synthesis, it was tran-siently transformed into tomato fruits. Lycopene, a fourterpenoid, is a natural fat soluble carotenoid. The MVApathway of synthetic lycopene is relatively clear. The tran-sient transformed tomato fruits with CbWRKY24 werefound that the transcript level of SlPSY, SlPDS, SlZDSin lycopene pathway decreased sharply compared to thatinWT fruits (figure 8). It can be seen from the above resultsthatCbWRKY24 can regulate terpenoid biosynthesis, butthe regulation effects of different terpenoid pathways werequite different.Studies have shown that MeJA can increase the expres-

sion of genes including ADS, CYP71AV1 and DBR2 inartemisinin biosynthesis, thereby increasing the densityof glandular trichomes and resulting in the accumula-tion of sesquiterpenes (Maes et al. 2011). In addition,the expression of AaWRKY1 which is an A. annua tran-scription factor that regulates artemisinin biosynthesiswas also strongly induced under MeJA treatment (Zhanget al. 2009). Similar to previous studies, the CbWRKY24in our experiment showed strong induction under MeJAtreatment, meanwhile the expression of upstream genesCbHMGR, CbFPPS, and downstream genes CbSQE,CbβAS in MVA pathway and upstream gene CbDXS inMEP pathway also increased (figure 4). The expressionof the CbWRKY24 is rapidly induced by MeJA, whichimplies that CbWRKY24 either self-regulation its owngene expression level or regulated byone ormore upstreamtranscription factors. It has been reported that the tran-scriptional cascade of different signalling pathways canregulate plant stress response (Chinnusamy et al. 2003).Under the MeJA treatment, the accumulation time of keygenes expression were earlier than that of CbWRKY24,whileCbWRKY24 response to cold stress was earlier thanthat of key genes. Therefore, we are bold to speculatethat CbWRKY24 is MeJA activated transcription factor,and may also participate in jasmonic acid signal transduc-tion pathway through interaction with cold stress. To testthis hypothesis, we cloned the upstream promoter regionof CbWRKY24, and analysed the cis-acting elements ofthe CbWRKY24P, we found many biotic and abioticstress responsive elements including MeJA response ele-ment (JRE), but no cold responsive element was found.Many of the MeJA responsive elements have been identi-fied from different plant promoters (Brown et al. 2003; Xuand Timko 2004). Meanwhile, it was found that GUS his-tochemistry test of transient transformed tobacco leaveswithCbWRKY24P appeared blue (figure 9). Based on thisstudy, the CbWRKY24 promoter can be transformed intoArabidopsis thaliana, and GUS activity can be determinedunder MeJA and other stress treatments to further verifythe function of CbWRKY24.

In summary, a jasmonate-responsive gene,CbWRKY24,was isolated from C. blinii and functions as an importantTF in changing terpenoid content. The overexpression ofCbWRKY24 upregulate the transcript level of terpenoidrelated genes in transient transformed C. blinii leaves; butdownregulated the expression of lycopene related genesin transiently transfected tomato fruits. In conclusion, theexperimental results can be used to lay the foundation forfurther study on improving the quality ofmedicinal plants.

Acknowledgement

We thank all people who participated in this study for their sup-port.

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Corresponding editor: H. A. Ranganath

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The jasmonate-responsive transcription factor CbWRKY24