Methyl jasmonate induction of tanshinone biosynthesis in Salvia miltiorrhiza hairy roots is mediated by JASMONATE ZIM-DOMAIN repressor proteins

Jasmonic acid (JA) is an important plant hormone involved in regulation of many aspects of plant growth and development including secondary metabolism and JASMONATE ZIM-DOMAIN (JAZ) proteins are key components in JA signal processes. In this study, two new JAZ genes named SmJAZ3 and SmJAZ9 were cloned from S. miltiorrhiza hairy roots and characterized. Expression profiles under methyl jasmonate (MJ) treatment revealed that SmJAZ3 and SmJAZ9 were both MJ-responsive. Subcellular localization assay showed that SmJAZ3 was located in nucleus while SmJAZ9 was preferentially in nucleus. Expression of SmJAZ3 and SmJAZ9 in S. miltiorrhiza hairy roots differently affected the production of tanshinone. Over-expression of SmJAZ3 or SmJAZ9 in hairy roots produced lower level of tanshinone compared with the control, tanshinone production was as low as 0.077 mg/g DW in line SmJAZ3-3 and 0.266 mg/g DW in line SmJAZ9-22. Whereas, down-regulation of SmJAZs enhanced tanshione production, the content of tanshinone increased to 2.48 fold in anti-SmJAZ3-3 line, and 1.35-fold in anti-SmJAZ9-23 line. Our work indicated that SmJAZ3 and SmJAZ9 are involved in regulation of tanshinone biosynthesis and act as repressive transcriptional regulators in the JA signaling pathway, which paves the way to further dissect molecular mechanism in details in the future.

Scientific RepoRts | 6:20919 | DOI: 10.1038/srep20919 of JAZ repressors releases MYC2 transcription factor(s) through repression or activation the transcription of genes involved in plant defense responses 15,18 . The presence of bioactive JA-Ile, therefore, directly determines the duration of plant defense responses to biotic stresses that elicit JA signaling pathway.
JAZ family, interact with MYC2 transcription factor and repress its function in JA signaling, and act as repressors 17,19 . Twelve JAZ proteins are found in Arabidopsis, they share a conserved TIFY× G motif within the ZIM (or TIFY) domain in the N-terminus and a SLX2FX2KRX2RX5PY consensus motif within the Jas domain in the C-terminus. ZIM domains mediate the homo-and heteromeric interactions among the JAZ proteins as well as their interaction with other transcription factors [20][21][22] . In the presence of JA-Ile, JAZ proteins are recognized by Skp1-Cul1-F-box protein (SCF)-type E3-ubiquitin ligase complex, consequently got degraded by the 26 S proteasome. On the contrary, JAZ repressors bind to transcription factors (TFs) by recruiting the general co-repressors TOPLESS (TPL) and TOPLESS-Related (TPR) proteins through an interaction with the adaptor protein NINJA to prevent their activity. Up to now, JAZs have been testified to interact with some other TFs to mediate many process of development including trichome development and flavonoids synthesis.
Salvia miltiorrhiza Bunge, a well-known traditional Chinese herb, plays a crucial role in clinical treatment of cardiovascular and cerebrovascular diseases 23,24 . There are two kinds of active components in S. miltiorrhiza, one kind is the liposoluble tanshinone containing cryptotanshinone, tanshinone I, tanshinone IIA, dihydrotanshinone etc., which share a variety of biological activities including anti-oxidant, heart-protective, anti-ischemic, anti-bacteria and anti-tumor properties 23,[25][26][27][28] . The other kind is hydrosoluble phenolic acids including caffeic acid, lithospermic acid A, B, C and rosmarinic acid 24,26,27 . Diterpene tanshinone is mainly synthesized via the methylerythritol phosphate (MEP) pathway, with some degree of crosstalk between the MEP pathway and the mevalonate (MVA) pathway 23 . Enhanced accumulation of tanshinone elicited by addition of exogenous JA treatment in S. miltiorrhiza has been reported 28,29 , but the exact molecular regulation mechanism are still unclear. Recently, several tanshinone biosynthetic genes such as SmHMGR, SmDXR, SmHMGS, SmGGPPS and SmCYP76AH1 have been successfully cloned from S. miltiorrhiza 25,27,[30][31][32] , which provided the foundation to understand molecular mechanism of tanshinone biosynthesis induced by MJ in S. miltiorrhiza. As a key regulator in plant JA signal pathway reported recently, JAZ repression proteins should have important regulatory role in tanshinone biosynthesis in S. miltiorrhiza. However, little is known about exact role of JAZ on tanshinone biosynthesis in S. miltiorrhiza. Herein, two genes of JAZ family were isolated from S. miltiorrhiza and their roles in regulating the tanshinone biosynthetic pathway are functionally identified through genetic engineering strategy.

Materials and Methods
Experimental materials. S. miltiorrhiza plants were grown in pots in the greenhouse under 25 °C with 16 h light/8 h dark period and relative air humidity of 60% or cultured on MS medium (pH5.8) containing 3% sugar and 0.8% agar as reported before 23,24,29 . Two-year-old flowering S. miltiorrhiza plants were obtained from the Plant Garden of Shanghai Normal University and used for the analysis of expression pattern of SmJAZs in taproot, fibril, leaf, petiole, stem, seed and flower. The seeds of N. benthamiana, plasmid pCAMBIA2300, pGBKT7 and pGADT7 were provided by Professor Kexuan Tang from Shanghai Jiaotong University. Escherichia coli DH5α , Agrobacterium C58C1 and GV3101 were kept in our own laboratory. The plasmid of pMON530 was gifted by Professor Zhongnan Yang from Shanghai Normal University. The pMD-18 T vector and reverse-transcription PCR Kit were purchased from TaKaRa Biotechnology Co., Ltd. Primers synthesis and DNA sequencing was performed by Shanghai Sangon Biotechnological Company, China. Standards of cryptotanshinone, tanshinone I, tanshinone IIA, dihydrotanshinone were purchased from Aladdin, China. All other chemicals used were of analytical grade.
Isolation of two JAZs from S. miltiorrhiza. The hairy roots of S. miltiorrhiza were dried and pulverized in liquid nitrogen with mortar and pestle for extraction of the total RNA. Total RNA was used to synthesize the first strand cDNA with primer AP (5′ -GGCCACGCGTCGACTAGTAC(T) 16 -3′ ) by using RT-PCR system (TaKaRa, Japan). A local S. miltiorrhiza transcription database was built up from public transcripts from NCBI as well as our own transcriptomic data sequenced by hairy roots. Conserved domains of ZIM and Jas were utilized to search our transcriptome database to identify putative JAZs from S. miltiorrhiza with Blastx and Blastn. ORF Finder was used to identify the length of the ORF. Among the candidate JAZ sequences, SmJAZ3 (lack of 3-terminal) was obtained with the specific forward primer SmJAZ3-F421 (5′ -GCCGTTGGAACCACTGATTTTAG-3′ ) together with the primer AUAP. Primer pairs (SmJAZ9-F: 5′ -ATGGAGAGAGATTTCATGGGGT-3′ , SmJAZ9-R: 5′ -TCAGTCATCCTTGCTGACGGA-3′ ) were synthesized to amplify the whole ORF of SmJAZ9. The PCR amplification conditions were as follows: initial denaturation at 95 °C for 8 min, followed by 35 cycles denaturation at 95 °C for 30 s, annealing at 58 °C for 1 min and extension at 72 °C for 1 min 30 s. Bioinformatics analysis of SmJAZs. Several bioinformatics softwares and websites were used for analysis of the two SmJAZs. The nucleotide sequence and open reading frame (ORF) were analyzed by ORF Finder and the sequence comparison was conducted through database search using BLAST (NCBI, http://www.ncbi.nlm. nih.gov). Alignment of nucleotide and deduced amino acid sequences containing the two SmJAZs and other JAZs such as AtJAZs and OsJAZs were aligned with the ClustalW with default parameters. MEGA5.1 combined with CLUSTAL W alignments were used to construct the phylogenetic tree.
Expression profiles of SmJAZ genes. To investigate the expression pattern of the SmJAZs in different tissues (taproot, fibril, leaf, petiole, stem, seed and flower) as well as its expression at 0 h, 0.5 h, 1.0 h, 2.0 h and 4.0 h after 100 μ M MeJA treatment, plant materials were collected accordingly and total RNA was isolated according to the method described before 23,24 . Reverse transcription (RT) reaction was carried out using the kit (TaKaRa, Japan) with 20 μ L volume consisting of 4 μ L 5× M-MLV buffer, 2 μ L 50 μ M primer AP, 1 μ L 10× dNTPs, 0.5 μ L 200 U/μ L RNase M-MLV and 0.5 μ L 40 U/μ L RNase inhibitor at 42 °C for 1.5 h, then inactivated at 70 °C for 15 min 24 . The qRT-PCR reaction was carried out using the Super Real PreMix kit (Tiangen, China) and performed on the Applied Biosystem StepOne Real Time PCR System (Applied Biosystems, USA) with an optional 48-well plate. The house-keeping gene (SmActinF: 5′ -AGCACCGAGCAGCATGAAGATT-3′ ; SmActinR: 5′ -AGCAAAGCAGCGAACGAAGAGT-3′ ) was performed as an internal control to estimate the expression level of the SmJAZs according to the relative quantitative analysis method (2 −∆∆CT ). Amplifications were performed under the following condition: 10 min denature at 95 °C, then 40 cycles of 15 s denature at 95 °C, 30 s annealing at 60 °C and 30 s extensions at 72 °C 24,33 . Subcellular localization of SmJAZs. BglII and KpnI site were added to the 5′ -and 3′ -of the ORF of SmJAZ3 and SmJAZ9 respectively via PCR. The PCR products were digested with BglII and KpnI and cloned into the vector pMON530. The constructed vectors were transferred into Agrobacterium strain ASE. Forty-day-old tobacco plants were used for the transient transformation experiment. After two days′ injection, localization of SmJAZs was observed using the confocal microscope (Carl Zeiss).

Construction of plant expression vectors and hairy root cultivation. The SmJAZ3/SmJAZ9
gene or anti-SmJAZ3/anti-SmJAZ9 was amplified via PCR as indicated above. The vectors pCAMBIA1304 and pCAMBIA2300 were double-digested with Hind III and EcoR I. The smaller DNA fragment containing GUS gene expression cassette from pCAMBIA1304 was ligated with the large pCAMBIA2300 fragment to obtain the modified vector pCAMBIA2300 + . The complete ORF of SmJAZs with restriction sites SpeI and BstEII were firstly cloned into pMD-18 T to generate pMD18T-SmJAZs. pCAMBIA2300 + and pMD18T-SmJAZs were then double-digested with SpeI and BstEII. The complete ORF of SmJAZs were then cloned into pCAMBIA2300 + to replace the GUS gene of the GUS gene expression cassette from pCAMBIA1304, under the control of CaMV35S promoter and the NOS terminator to generate pCAMBIA2300 + -SmJAZ3 and pCAMBIA2300 + -SmJAZ9. pCAM-BIA2300 + -anti-SmJAZ3 and pCAMBIA2300 + -anti-SmJAZ9 were constructed using similar strategy with opposite direction of ORF in place of GUS gene. These above constructed plastids were transformed into A. rhizogenes strain C58C1 respectively. The aseptic leaves of 30 day-old S. miltiorrhiza were cultured in 1/2 MS medium in darkness for two days and then immersed in the Agrobacterium suspension for 10 min, then co-cultivated on MS solid medium 23,29 . After 2 days, the leaves were transferred to 1/2 MS containing carbencillin (300 mg/L) and subcultured to 1/2 MS with reducing concentration of cefotaxime (300 mg/L, 100 mg/L) every two weeks. After 6 to 7 weeks, about 3-4 cm long hairy roots were excised from the leaves. The empty pCAMBIA2300 vector was used as a vector-only control. DNA isolation and PCR analysis procedure were carried out as we reported before 23,24,28 Positive colonies were cut down and moved to 100 mL 1/2 MS medium in 250 mL Erlenmeyer flasks shaking with the speed of 100 rpm in darkness at 25 °C and harvested after 60 days for RNA and tanshinone extraction.
Tanshinone extraction and HPLC analysis. After 2 month culture, the hairy roots were dried at 50 °C in an oven and then ground into powder, 100 mg powder was placed in 16 mL solvent of methanol/dichloromethane (3:1, v/v) for tanshinone extraction, and then kept in dark for 24 hours after 1 hour′ s ultrasonic process with frequency of 40000 Hz at room temperature (Kunshan HC-2002 S, China). The substance was dried under vacuum and re-dissolved in 2 ml methanol. The solution was filtered and used for HPLC on Agilent 1260 apparatus equipped with a Waters reversed-phase C18 symmetry column. Acetonitrile-water (65:35, v/v) worked as the mobile phase at a flow rate of 1 mL/min with the detection wavelength at 270 nm 5,7 . Chromatographic peaks were integrated and compared with the respective standard curves to calculate the individual and total amount of tanshinone 23,24 . The sum of HT, CT, T1 and T2A were calculated as total tanshinone (TT) in this paper.
Yeast two-hybrid assays. To assess possible protein interactions, the corresponding plasmids (pGB-KT7-SmJAZs and pGADT7-AtMYC2) were co-transformed into Saccharomyces cerevisiae AH109 cells following standard protocols 5,19 . Both the SmJAZ3 and SmJAZ9 cDNAs were cloned into pGBKT7 vector and the cDNA of AtMYC2 was inserted into pGADT7. Plasmids pGBKT7-AtJAZ9 and pGADT7-AtMYC2 were co-transformed into AH109 as the positive control. The empty vector pGADT7 and pGBKT7 constructs were co-transformed as the negative control. The indicated construct pairs were transformed into yeast AH109 and screened on yeast synthetic drop-out-Trp-Leu at 30 °C. Three days after transformation, yeast colonies were grown in selective liquid media for about 7 h, and the cell density was adjusted to OD 600 = 1. A volume of 10 μ L sample of the cell suspensions was plated out on yeast synthetic drop-out (SD/-Trp-Leu-His-Ade) to test protein interaction. Plates were incubated at 30 °C for 2-4 days.

Statistical analysis.
All the experiments including culture of hairy root clones, PCR identification, qRT-PCR and HPLC analysis were repeated three times. Results of tanshinone content were presented as mean values ± SD. The error bars were from biological triplicates. The statistical significant difference was analyzed by one sample T test, and the difference of various hairy root lines were used in the one-way analysis of variance (ANOVA) using SPSS 11.5 software (SPSS, Inc.).

Results
Isolation and bioinformatics analysis of JAZs from S. miltiorrhiza. To isolate S. miltiorrhiza JAZ genes which are induced by MJ and functionally identify their roles in tanshinone biosynthesis, conserved domains of ZIM and Jas were used to blast S. miltiorrhiza transcriptome database. Two JAZ fragments were found, one lacked 3′ terminal (partial JAZ3) and the other contained complete 921 bp ORF (later named JAZ9). Based on sequences of JAZ domains, a primer F421 was designed to amplify 3′ terminal cDNA of the JAZ3 together with the primer AUAP (Fig. S1) to get the complete coding sequence of JAZ3. The SmJAZ3 gene described here encodes a 336-amino acid protein. Sequence alignment (Fig. 1) revealed that the two JAZs share high amino acid sequence identity with JAZs from Arabidopsis thaliana. Phylogenetic analysis of selected JAZ proteins showed the one SmJAZ was highly homologous to AtJAZ3, thus we named it SmJAZ3 (Fig. 2). And the other SmJAZ contains a 921 bp ORF which encodes 306 amino acid residues (Fig. S1) showing the highest homology with AtJAZ9, accordingly, this SmJAZ was named as SmJAZ9. Both SmJAZ3 and SmJAZ9 processed two conserved domains, the TIFY domain located near the N-terminus consists of 28 amino acids and the Jas motif in the C-terminal.
Tissue expression patterns of SmJAZ3 and SmJAZ9. The expression profile of SmJAZ3 and SmJAZ9 in various S.miltiorrhiza tissues including taproot, fibril, leaf, petiole, stem, seed and flower was examined using qRT-PCR. High expression of SmJAZ3 was detected in mature leaf and petiole, while low expression presented in taproot and fibril, and SmJAZ3 expression was hardly observed in stem and reproductive organs, such as seed and flower (Fig. 3A). The expression of SmJAZ9 was detected in almost all the tissues, highest SmJAZ9 expression was found in mature leaf, less SmJAZ9 expression was observed in petiole and fibril (Fig. 3A).  , 1 h, 2 h, 4 h). The expression of SmJAZ3 increased from 0.5 h and reached to the highest level at 1 h after MJ elicitation, which is nearly 6-fold higher than the control (Fig. 3B). This indicated that the SmJAZ3 was responsive to MJ induction. The expression profile of SmJAZ9 was similar to SmJAZ3, also increased to the highest level at 1 h after MJ treatment, which was 8 times higher than the control (Fig. 3B).

Subcellular localization of SmJAZ3 and SmJAZ9. To determine the Subcellular localization of SmJAZ3
and SmJAZ9, the ORF of SmJAZ3 and SmJAZ9 were fused with the green fluorescent protein (GFP) in vector pMON530. Then the constructs were introduced into Agrobacterium strain ASE, respectively. Subcellular localization results showed that SmJAZ3 was only localized to the nucleus while SmJAZ9 was mainly in the nucleus but with some signal in the membrane in one-month-old tobacco (Fig. 4). This indicates that SmJAZ3 and SmJAZ9 are preferentially nuclear-localized, implying that they may function as transcriptional regulators.  PCR analysis of genetically engineered hairy roots. Genomic DNA was isolated from all the hairy root lines individually and was used for PCR analysis using gene-specific primers to amplify the CaMV35S promoter and partial SmJAZ3 or SmJAZ9 gene. Plastid pCAMBIA2300-SmJAZ3 and pCAMBIA2300-SmJAZ9 were used as positive control. The blank vector pCAMBIA2300 + -transformed lines and water were set as the negative controls. Representative results of PCR analysis of transgenic hairy roots were showed in Fig. S3. The PCR-positive rates of hairy root lines from JAZ3, JAZ9, anti-SmJAZ3 and anti-SmJAZ9 lines were 21/38 (55.3%), 14/28 (50.0%), 16/36 (44.4%) and 16/36 (44.4%) (Fig. S3). Our results suggested that SmJAZ3 and SmJAZ9 were successfully transformed into the genome of S.miltiorrhiza. Here we selected 5 SmJAZ3, 5 SmJAZ9, 3 anti-SmJAZ3 and 3 anti-SmJAZ9 of PCR-positive hairy root lines for further experiments.

Effect on tanshinone biosynthetic genes by expression of SmJAZ3 or SmJAZ9 in transgenic hairy roots.
To determine whether the SmJAZ3 or SmJAZ9 can affect tanshinone biosynthetic genes, expression profiles of several key tanshinone biosynthetic genes such as SmGGPPS, SmDXS2 and SmKSL were examined, respectively. In the SmJAZ3 overexpressing S. miltiorrhiza hairy root lines, SmJAZ3 expression was up-regulated by 5-40 times in comparison to the control lines, while the transcripts of SmGGPPS, SmDXS2 and SmKSL were all reduced with different level in the SmJAZ3 overexpressing S. miltiorrhiza hairy root lines (Fig. 5). Among them, SmKSL showed the most significant decrease, nearly 20-fold lower in SmJAZ3-38 line than the control (Fig. 5). Meanwhile, the key MEP pathway gene SmDXS2 decreased to a lesser extent (Fig. 5). On the contrary, the expression of a few genes including SmCPS increased by 2-6 folds than the empty vector control, furthermore SmCYP76AH1 did not show obvious change. In the SmJAZ9-overpressing hairy roots lines, the level of SmJAZ9 were elevated by 5 times to the most, whereas the expression profiles of SmGGPPS, SmDXS2 and SmKSL didn't show similar expression pattern as those in SmJAZ3, indicated that SmJAZ9 may have much weaker effect to repress biosynthesis of tanshinone when compared to SmJAZ3. Effect on tanshinone accumulation by expression of SmJAZ3 or SmJAZ9 in transgenic hairy roots. Tanshinone is synthesized from universal precursor IPP that derived through two different pathways in separate cellular compartments, the MVA pathway occurring in the cytosol and MEP pathway occurring in the plastids, while the MEP plays a more important role in tanshinone biosynthesis 23 . The contents of tanshinone including dihydrotanshinone (HT), cryptotanshinone (CT), tanshinone I (T1) and tanshinone IIA (T2A) in S. miltiorrhiza hairy root lines were determined by HPLC, and the results were shown in Fig. 6. Consistent with the gene expression profile, the accumulation of tanshinone in SmJAZ3-overexpressing lines appeared to decrease. Over-expression of SmJAZ3 in hairy root produced evidently lower level of tanshinone ranging from 0.077 to 0.94 mg/g DW compared with the control (1.37 mg/g DW), tanshinone production in line SmJAZ3-3 was as low as 0.077 mg/g DW, a 94% decrease. Transgenic hairy roots lines harboring SmJAZ9 exhibited a slightly suppressive effect on the tanshinone biosynthesis, and the content of tanshinone decreased to 0.266 mg/g DW in SmJAZ9-22 line. Whereas, the content of tanshinone increased to 4.78 mg/g DW in anti-SmJAZ3-3 lines which was about 2.48-fold higher than the control, and it was about 3.22 mg/g in anti-SmJAZ9-23 lines with 1.35-fold higher than the control. The above results inferred that the inhibitory effect of SmJAZ3 on tanshinone biosynthesis is much stronger than SmJAZ9.
SmJAZ9 protein interacts with AtMYC2. It has been reported that JAZ repressors interact with some transcription factors such as MYC2 which play an important role in JA-activated response. In order to test whether there is interaction between SmJAZs and AtMYC2 (MYC2-like proteins were not identified from S. miltiorrhiza so far), we fused the SmJAZ3, SmJAZ9 to the pGBKT7 vector (BD) and AtMYC2 to the pGADT7 vector (AD), followed by co-introduction of the above vectors into yeast cells AH109 for Y2H. Plasmids pGB-KT7-AtJAZ9 and pGADT7-AtMYC2 were co-transformed into AH109 as the positive control and the empty vector pGADT7 was co-transformed with pGBKT7 constructs as a negative control (Fig. 7). Our results showed only SmJAZ9 can interact with AtMYC2 whereas SmJAZ3 couldn't, which implied some degree of interaction of JAZ and MYC2 possibly existed in S. miltiorrhiza to regulate biosynthesis.

Discussion
Being one kind of diterpenes with good pharmaceutical activities, tanshinone was essentially derived from two common precursors, isopentenl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) 29,34,35 . The two   precursors were synthesized via two different pathways in separate cellular compartments, the MVA pathway occurring in the cytosol and MEP pathway in the plastids 23 . Metabolic engineering approach has been considered as an effective approach to increase the valuable metabolites in some medical plants [36][37] . Based on the successful isolation of several tanshinone biosynthetic genes such as SmHMGR, SmDXR, SmDXS and SmGGPPS from S. miltiorrhiza, the attempts to increase the tanshinone production in S. miltiorrhiza via metabolic engineering approach and combination with elicitor treatments have been reported 23,24,28 . However, due to lack of genome sequences for S. miltiorrhiza, several catalytic enzymes in the late steps of tanshinone synthesis still remain unknown. The rapid development of next-generation sequencing technology has greatly pushed the advance in transcription profile and new gene pools. Accumulations of tanshinone could be induced by various elicitors including plant hormone MJ, whereas little is known about molecular regulatory mechanism in tanshinone biosynthetic pathway. In this study, two JAZ genes SmJAZ3 and SmJAZ9 were cloned from S. miltiorrhiza and functionally identified as repressors involved in tanshinone biosynthesis. Bioinformatics analysis revealed that the two cloned SmJAZ genes showed high homology with JAZs from Arabidopsis and contained typical ZIM and Jas domains, indicating those functional domains are conserved for plant JAZ family during evolution. The expression profiles under methyl jasmonate (MJ) elicitation revealed that expression of SmJAZ3 and SmJAZ9 was quickly MJ-responsive, which suggested that SmJAZ3 and SmJAZ9 may be involved in MJ signal pathway and its related activated responses, in good agreement with those JAZs previously reported in model plant A. thaliana 17 . It has been reported that methyl jasmonate could promote SCF COI1 and JAZ to generate protein complex and then 26 S proteomes were recruited to degrade JAZ proteins to liberate positive regulators such as MYC2 whose transcriptional activation was repressed by JAZs under normal condition 5,7,17 . It may be hypothesized that MJ may promote SCF COI1 -like protein interact with JAZs and make JAZ proteins degraded by 26 S proteomes in S. miltiorrhiza, and then some positive transcription regulators such as MYC2 were released from repression by JAZs to transcriptionally activate the expression of some tanshinone biosynthetic genes and correspondingly modulate tanshinone biosynthesis. SmJAZ9 could interact with AtMYC2, implied that the above hypothesis may exist in S. miltiorrhiza, which needed to be investigated further in the future. Over-expression of SmJAZ3 and SmJAZ9 in S. miltiorrhiza hairy roots resulted in reduced accumulation of tanshinone while down-regulation of SmJAZ3 and SmJAZ9 effectively improved the production of tanshiones. These indicated that SmJAZ3 and SmJAZ9 play important roles in tanshinone biosynthesis and act as repressive components in the JA signal pathway for tanshinone production, which paves a way to dissect molecular mechanism in details in the future.
JAZ proteins belong to a larger group of ZIM-domain proteins, named after the putative transcription factor Zinc-finger inflorescence meristem (ZIM). ZIM family members were assigned to this group according to a conserved 28 amino acid ZIM motif. In addition to ZIM, JAZ family members also have the highly conserved Jas motif of 26 amino located near the C-terminus, it is now well established that Jas motif participates in protein-protein interaction with both transcription factors like the member of bHLH family (MYC2) and COI1. In the present study, it was found that SmJAZ9 protein can interact with the AtMYC2 by yeast two hybrids (Y2H), which was consistent with former reports that the JAZ family has the character to interact with MYC2 38,39 . We didn't detect SmJAZ3 protein's interaction with the AtMYC2 by Y2H, which may be attributed to genes from different plant species with uncertain complexity. The above data suggested that there may be a potential MYC2-like transcription factor in S. miltiorrhiza, which may be the target of JAZ proteins and play an important role in accumulation of tanshinone as positive regulator. Whether the SmJAZs have an interaction with the potential SmMYC2 needed to be further investigated. Thus work towards isolation and identification of MYC2-like transcription factor from S. miltiorrhiza may further decipher this action mechanism.
In A. thaliana and some other model plants, several reports have revealed the direct involvement of JA and JAZ proteins in growth and development processes such as secondary growth 40 , phytochrome, anthocyanin accumulation, trichrome initiation 41 , stamen development 42 , and defense response against biotic or abiotic stresses [43][44][45] . Our work revealed that over-expression of SmJAZ3 in S. miltiorrhiza hairy roots down-regulated several tanshinone biosynthesis key genes such as SmDXS2, SmKSL and SmGGPPS in comparison to the control lines. At the same time, the production of tanshinone was decreased to 0.077 mg/g DW in JAZ3 over-expressing line JAZ3-3 in comparison to the control line (1.37 mg/g DW). While down regulated expression of JAZ3 in anti-JAZ3-3 line increased the content of tanshinone to 4.78 mg/g DW, which was 2.48-fold higher than the control. In transgenic SmJAZ9 line, average production of tanshinone was 1.32 mg/g DW, whereas, the content of tanshinone increased to 3.22 mg/g DW in anti-JAZ9-23 lines, a 1.35-fold increase as the control. These results reflect that the SmJAZ3 has much stronger suppressive effect on tanshinone biosynthesis than the SmJAZ9. Furthermore it was found that SmJAZ3 and SmJAZ9 acted as repressors in tanshinone biosynthesis. The above data testified the role of JA in regulating tanshinone biosynthesis genes and thereby affecting the production of tanshinone in S. miltiorrhiza. However, further investigations are needed to figure out whether SmJAZs interact with SmMYC2 or other transcription factors, how and which SmJAZ they interact with. We also need to further elucidate how the JA signaling functions or whether it cross talks with other signaling pathways in S. miltiorrhiza. Overall, this study extends our understanding in the molecular regulation of tanshinone biosynthesis induced by MJ signal. Our work indicated JAZs such as SmJAZ3 and SmJAZ9 play important role in regulation of tanshinone biosynthesis, it may be involved in other secondary metabolism. The isolation and functional characterization of the two JAZs from S. miltiorrhiza paves a way to further dissect molecular mechanism and manipulate tanshinone production in plant.