Transcription Factor OpWRKY3 Is Involved in the Development and Biosynthesis of Camptothecin and Its Precursors in Ophiorrhiza pumila Hairy Roots

The plant Ophiorrhiza pumila produces camptothecin (CPT), a kind of terpene indole alkaloid (TIAs) that has been widely used in treatment of cancer. Tryptophan-arginine-lysine-tyrosine (WRKY) transcription factors have been reported to play important roles in plant metabolism and development. In this study, a novel WRKY transcription factor named OpWRKY3 was isolated from O. pumila, with full-length open reading frame (ORF) of 1128 bp, encoding 375 amino acids. Phylogenetic tree analysis revealed that OpWRKY3 shared the highest homology with VvWRKY30, and it is a significant feature belonging to group III. OpWRKY3 was responsive to various treatments, including gibberellin (GA3), methyl jasmonate (MJ), acetylsalicylic acid (ASA), salicylic acid (SA), and abscisic acid (ABA). Besides, OpWRKY3 is expressed predominantly in stems. Subcellular localization analysis showed that OpWRKY3 localized in the nucleus. The biomass of OpWRKY3-SRDX transgenic hairy roots (S line) was visibly suppressed, while there were slight changes between overexpression of the OpWRKY3 line (OE line) and the control. In addition, the concentration and total production of camptothecin precursors including loganin and secologanin were significantly changed in both OE and S lines while total production of CPT was significantly changed in most transgenic lines. Thus, the present work revealed that OpWRKY3 may act as a regulator in the growth and development of O. pumila, and in production of camptothecin and its precursors.


Introduction
Camptothecin (CPT) is a well-known terpene indole alkaloid (TIA), which was isolated for the first time by M. E. Wall and M. C. Wani in 1966 from Camptotheca acuminata [1]. CPT has been found in some distantly related plants, including C. acuminata, Ervatamia heyneana, Merrilliodendron megacarpum, Nothapodytes foetida, N. nimmoniana, and several Ophiorrhiza species [2][3][4][5]. CPT exhibits excellent anti-tumor activity by inhibiting the activity of topoisomerase I [1,6]. Its two semisynthetic water-soluble derivatives, irinothecan and topothecan, were approved by the US Food and Drug Administration (FDA) in 1994 and are used extensively for the treatment of ovarian cancer, lung cancer, metastatic colorectal cancer, and cervical cancer throughout the world [7][8][9]. Like many TIAs, CPT is derived from the shikimate pathway and iridoids biosynthesis pathway, which supply the precursor's tryptamine and secologanin for CPT synthesis [5,10]. The terpene part of CPT is received from the Here, in this study, a novel gene OpWRKY3 encoding for WRKY transcription factor was isolated from O. pumila and functionally characterized. OpWRKY3 shared high homology with VvWRKY30, VqWRKY52, PtWRKY30, AcWRKY41, and AtWRKY30. OpWRKY3 possessed one WRKY domain and a C2HC motif and was classified into group III. The biomass of repression OpWRKY3 transgenic hairy roots was significantly reduced by suppressing the growth and development of root tip. While there were slight changes observed in OpWRKY3-overexpressing lines. In addition, overexpression of OpWRKY3 in hairy roots resulted in increased concentration and total production (hairy roots and medium) of loganin and decrease of secologanin contentration and production in hairy roots, while repression of OpWRKY3 resulted in opposite effect compared with the overexpressed lines. The overexpression of OpWRKY3 resulted in increased the total production of CPT while repression of OpWRKY3 suppressed significantly the CPT production. Thus, the findings of this study will serve as an accentuating reference for future research on the regulators of alkaloid production in various medicinal plants.

Bioinformatics and Molecular Characteristics of OpWRKY3
In this study, OpWRKY3 was cloned and functionally characterized from O. pumila. It owned a full-length open reading frame (ORF) of 1128 bp, encoding a protein with 375 amino acid residues, calculated molecular weight of 41.95 kDa, and isoelectric point of 5.77. BLASTp search (NCBI website) revealed that OpWRKY3 shared 55.53%, 54.85%, 53.77%, and 53.51% identity with PtWRKY30, AcWRKY41, VqWRKY52, and VvWRKY30, respectively. Based on the OpWRKY3 and other species' WRKYs, a phylogenetic tree was constructed and it showed that they could be classified into three major groups, and OpWRKY3, together with AtWRKY30 and AtWRKY41, belongs to group III ( Figure 1A). Multiple sequence alignment indicted that OpWRKY3 possesses a highly conserved WRKY domain (WRKYGQK) and a C2H2-type zinc-finger structure ( Figure 1B).

Tissue and Induction Expression Profiles of OpWRKY3
To investigate the tissue expression pattern of OpWRKY3, roots, stems, petioles, leaves, and hairy roots were analyzed. Figure 2A reveals that high expression of OpWRKY3 was found in stems only, and the others have little expression.
To investigate whether OpWRKY3 responded to exogenous elicitor treatment, 30-day old hairy roots were treated with different inducers, including GA 3 , MJ, ASA, SA, and ABA. The hairy roots were harvested at different time points. The results showed that the exogenous GA 3 , MJ, or ASA significantly induced OpWRKY3 expression, reaching a peak at 1 h ( Figure 2B-D), and thereafter declined rapidly. In addition, the transcript level of OpWRKY3 was raised rapidly after SA or ABA induction, with a maximum peak at 0.5 h, followed by a gradual decrease ( Figure 2E,F). These results indicate that OpWRKY3 can respond to exogenous elicitors.

Subcellular Localization of OpWRKY3
To experimentally confirm the subcellular localization of OpWRKY3, OpWRKY3 was cloned into the pMON530 vector (the binary vector) and fused with the green fluorescent protein (GFP) reporter gene to generate vector pMON530-OpWRKY3-GFP. Then, the constructed vector and the pMON530 (used as the control) was transformed into the ASE strain and expressed in tobacco leaves, respectively. In the leaves of the transformed control vector plant, the fluorescence of the GFP was detected in the cytoplasm and nucleus, whereas the fluorescence of the OpWRKY3/GFP fusion protein was exclusively found in the nucleus (Figure 3). The expression pattern was consistent with the character of OpWRKY3 as a TF. OpWRKY3 is highlighted with a red triangle. The phylogenetic tree was constructed with the neighbor-joining test using MEGA 7.0. (B) Multiple alignment of OpWRKY3 with other WRKY members. The following proteins were used for analysis: VqWRKY52 (AQS95512.1), VvWRKY30 (ALM9663.1), AcWRKY41 (PSS28797.1), PtWRKY30 (AZQ19316.1), AtWRKY41 (AAL35289.1), and AtWRKY30 (NP_568439.1). Identical and similar amino acids are shaded in black and red, respectively. The WRKY domains and the zinc-finger structures are boxed and marked by squares, respectively. roots were treated with different inducers, including GA3, MJ, ASA, SA, and ABA. The hairy roots were harvested at different time points. The results showed that the exogenous GA3, MJ, or ASA significantly induced OpWRKY3 expression, reaching a peak at 1 h ( Figure 2B-D), and thereafter declined rapidly. In addition, the transcript level of OpWRKY3 was raised rapidly after SA or ABA induction, with a maximum peak at 0.5 h, followed by a gradual decrease ( Figure 2E,F). These results indicate that OpWRKY3 can respond to exogenous elicitors.

Subcellular Localization of OpWRKY3
To experimentally confirm the subcellular localization of OpWRKY3, OpWRKY3 was cloned into the pMON530 vector (the binary vector) and fused with the green fluorescent protein (GFP) reporter gene to generate vector pMON530-OpWRKY3-GFP. Then, the constructed vector and the pMON530 (used as the control) was transformed into the ASE strain and expressed in tobacco leaves, respectively. In the leaves of the transformed control vector plant, the fluorescence of the GFP was detected in the cytoplasm and nucleus, whereas the fluorescence of the OpWRKY3/GFP fusion protein was exclusively found in the nucleus ( Figure 3). The expression pattern was consistent with the character of OpWRKY3 as a TF.

Acquisition of OpWRKY3 Transgenic Hairy Roots
To further investigate the function of OpWRKY3 in O. pumila, the Agrobacterium rhizogenes strain C58C1 harboring OpWRKY3 or OpWRKY3-SRDX fusion gene were used to infect O. pumila explants ( Figure S1). The empty vector was used as a control. The positive lines carrying the OpWRKY3 gene were verified by PCR. The expression of OpWRKY3 in transformed hairy roots was determined by qRT-PCR. The gene expression levels of OpWRKY3 in overexpression transgenic hairy root lines were found to be 15 to 360 times higher than in the empty vector control-transformed lines ( Figure  S2A). Five high-expression lines, including OE-1, 8, 11, 13, and 14, were chosen for further analysis. In OpWRKY3-SRDX overexpression lines, OpWRKY3 was expressed 15 to 110 times higher

Acquisition of OpWRKY3 Transgenic Hairy Roots
To further investigate the function of OpWRKY3 in O. pumila, the Agrobacterium rhizogenes strain C58C1 harboring OpWRKY3 or OpWRKY3-SRDX fusion gene were used to infect O. pumila explants ( Figure S1). The empty vector was used as a control. The positive lines carrying the OpWRKY3 gene were verified by PCR. The expression of OpWRKY3 in transformed hairy roots was determined by qRT-PCR. The gene expression levels of OpWRKY3 in overexpression transgenic hairy root lines were found to be 15 to 360 times higher than in the empty vector control-transformed lines ( Figure  S2A). Five high-expression lines, including OE-1, 8, 11, 13, and 14, were chosen for further analysis. In OpWRKY3-SRDX overexpression lines, OpWRKY3 was expressed 15 to 110 times higher compared to the empty vector (control) transformed lines ( Figure S2B). The five high expression lines, including S-3, 10, 11, 13, and 15, were chosen for further analysis.

Autofluorescent Analysis of Camptothecin in Transgenic Hairy Roots
Camptothecin can be autofluorescent, which provides a direct way to observe the distribution of CPT in hairy roots. Besides, relative concentrations of CPT in different hairy roots can be examined by comparing the size of the fluorescence area and the intensity of fluorescence. The selected root tips were collected from OE-14, S-10, and EV (transformed empty vector) transgenic hairy roots. The area of fluorescence at the root tip was significantly smaller in OpWRKY3-SRDX overexpression hairy roots compared with the control (Figure 4). The root tips of the S-10 line were extremely shortened with a decreasing number of branches in the root-hair zone. However, the fluorescence pattern showed no significant difference between OE-14 and EV hairy root tips. These results indicated that OpWRKY3 may be involved in the regulation of hairy root growth and root tip morphogenesis.

Metabolism of Camptothecin and Intermediate Compound in Hairy Roots Overexpressing or Repressing OpWRKY3
Based on the expression level of OpWRKY3, the biomass of chosen hairy roots lines was analyzed. The fresh weight (1.278 g) and dry weight (0.042 g) were significantly decreased in

Metabolism of Camptothecin and Intermediate Compound in Hairy Roots Overexpressing or Repressing OpWRKY3
Based on the expression level of OpWRKY3, the biomass of chosen hairy roots lines was analyzed. The fresh weight (1.278 g) and dry weight (0.042 g) were significantly decreased in OpWRKY3 repression transgenic hairy roots, compared to the control (5.5 g, 0.30g) ( Figure S3). The fresh weight (6.148 g) of OpWRKY3 overexpression transgenic hairy roots was significantly increased whereas dry weight (0.312 g) was not significantly different compared to the control. Among them, dry weight of OE-8, 13, 14 lines was slight increase. The results indicated that repression of OpWRKY3 inhibited the growth of hairy roots, and OpWRKY3 may be a transcription factor involved in growth and development of hairy roots.
To study whether OpWRKY3 participated in the regulation of camptothecin biosynthesis, the concentration and total production of camptothecin and three intermediate compounds (tryptamine, loganin and secologanin) in OpWRKY3 transgenic hairy roots and medium were investigated. Compared with OE lines an opposite accumulation pattern was observed for each compound in S lines. The overexpression of OpWRKY3 resulted in increased significantly lognin concentration in transgenic hairy roots while repression of OpWRKY3 decreased the lognin concentration, compared to control lines ( Figure S4B). In contrast, the secologanin concentration was down-regulated significantly by 70% in OE lines, whereas it was up-regulated significantly by 1.7-fold in S lines ( Figure S4C). The tryptamine concentration was slight change in OE lines while it was decreased significantly in S lines ( Figure S4A). In addition, the overexpression or repression of OpWRKY3 showed no significant change in the concentration of camptothecin, compared to the control ( Figure S4D).
The total production (hairy roots and medium) of tryptamine, loginin and CPT were up-regulated significantly by 1.28, 1.59, 1.37-fold in OE lines while the total production of tryptamine, loginin and CPT were down-regulated significantly by 86%, 81%, and 77% in S lines, compared to control lines ( Figure 5). Among them, the CPT production was increased significantly in line OE-8, 13, and 14, while CPT production was slightly high in line OE-1 and 4 ( Figure 5D). However, the overexpression of OpWRKY3 resulted in decreased significantly secologanin production in transgenic hairy roots while repression of OpWRKY3 increased significantly the secologanin production ( Figure 5C). These results suggested that OpWRKY3 may be related to the biosynthesis of tryptamine, loganin, secologanin and camptothecin.

Expression of Camptothecin Biosynthetic Genes in OpWRKY3 Transgenic Hairy Roots
To identify the camptothecin biosynthetic genes regulated by OpWRKY3 in O. pumila, transcript levels of eight genes (OpTSB, OpTDC, OpG10H, Op10HGO, OpSLS, OpCPR, OpSTR and OpSGD) were analyzed by qRT-PCR ( Figure S5). Compared with the empty vector (control), the OpTDC and OpCPR genes were up-regulated in OpWRKY3 overexpressing hairy roots, which were approximately 5-fold and 2.5-fold more respectively as compared with the control (Figure 6). However, OpG10H, Op10HGO, OpSLS, OpSTR and OpSGD genes were down-regulated in the five independent OpWRKY3 overexpression hairy roots. In contrast, OpTDC and OpCPR genes were significantly decreased in the five independent OpWRKY3-SRDX lines, while OpG10H, Op10HGO, OpSLS, OpSTR and OpSGD genes were significantly increased compared with control. In addition, the OpTSB gene expression level of transgenic hairy roots was no significant change. All these results suggested that OpWRKY3 may be involved in the regulation of camptothecin biosynthesis. compared to control lines ( Figure 5). Among them, the CPT production was increased significantly in line OE-8, 13, and 14, while CPT production was slightly high in line OE-1 and 4 ( Figure 5D). However, the overexpression of OpWRKY3 resulted in decreased significantly secologanin production in transgenic hairy roots while repression of OpWRKY3 increased significantly the secologanin production ( Figure 5C). These results suggested that OpWRKY3 may be related to the biosynthesis of tryptamine, loganin, secologanin and camptothecin. The total production of tryptamine in OpWRKY3 transgenic hairy roots and medium by high performance liquid chromatography (HPLC); (B) The total production of loganin in OpWRKY3 transgenic hairy roots and medium by HPLC; (C) The total production of secologanin in OpWRKY3 transgenic hairy roots by HPLC; (D) The total production of CPT in OpWRKY3 transgenic hairy roots and medium by HPLC. Values are means ± standard deviation of triplicate analyses. * , significant at P < 0.05, * * , highly significant at P < 0.01.

Expression of Camptothecin Biosynthetic Genes in OpWRKY3 Transgenic Hairy Roots
To identify the camptothecin biosynthetic genes regulated by OpWRKY3 in O. pumila, transcript levels of eight genes (OpTSB, OpTDC, OpG10H, Op10HGO, OpSLS, OpCPR, OpSTR and OpSGD) were analyzed by qRT-PCR ( Figure S5). Compared with the empty vector (control), the OpTDC and OpCPR genes were up-regulated in OpWRKY3 overexpressing hairy roots, which were approximately 5-fold and 2.5-fold more respectively as compared with the control (Figure 6).
However, OpG10H, Op10HGO, OpSLS, OpSTR and OpSGD genes were down-regulated in the five independent OpWRKY3 overexpression hairy roots. In contrast, OpTDC and OpCPR genes were

Activation of the Expression of OpCPR by OpWRKY3
Expression profiles showed that the overexpression of OpWRKY3 resulted in increased the expression of OpTDC and OpCPR. By analyzing the promoter sequence of OpTDC and OpCPR genes, we found that there was no W-box on the OpTDC promoter and OpCPR promoter contained two W-boxes (W-box 1, 2). The W-box 1 and W-box 2 of OpCPR promoter were located between

Activation of the Expression of OpCPR by OpWRKY3
Expression profiles showed that the overexpression of OpWRKY3 resulted in increased the expression of OpTDC and OpCPR. By analyzing the promoter sequence of OpTDC and OpCPR genes, we found that there was no W-box on the OpTDC promoter and OpCPR promoter contained two W-boxes (W-box 1, 2). The W-box 1 and W-box 2 of OpCPR promoter were located between positions -80 and -86, -433 and 439, respectively ( Figure 7A). Y1H assay showed that OpWRKY3 was bound to the W-box 1 of OpCPR promoter ( Figure 7B). These results indicated that OpWRKY3 increased the transcription of OpCPR by binding to the W-box 1 of its promoter.

Discussion
In O. pumila, WRKY3 transcription factor shared high identity with AtWRKY30, AtWRKY41, and VvWRKY30. Furthermore, OpWRKY3 possesses a highly conserved WRKYGQK motif and a C2H2-type zinc-finger structure, belonging to the group III category. The group III WRKYs, such as Arabidopsis thaliana WRKY75 and WRKY41, have been shown previously and played essential roles in regulating plant growth and development [30,31]. In addition, knockout of the AtWRKY75 mutant increased the number and length of root hairs [30]. AtWRKY46, AtWRKY54, and AtWRKY70 are involved in both brassinosteroid-regulated plant growth and drought response [36,37]. In the present study, the results of OpWRKY3 overexpression and repression revealed that the expression level of OpWRKY3 was linear with the biomass of transgenic hairy roots. Repression of OpWRKY3 inhibited the growth and development of the root tip and ultimately decreased the biomass of transgenic hairy roots. This is consistent with the WRKY transcription factor gene CbNN1 increased rooting ability, promoting adventitious root formation [38]. These results indicated that OpWRKY3 is involved in the growth and development of hairy roots. In addition, previous reports shown that group III WRKYs possessed the function of regulating secondary metabolism [39,40]. In another study has been shown that overexpression of CrWRKY1-SRDX decrease cellular tryptophan decarboxylase (TDC) activity and tryptamine accumulation in transgenic hairy roots [40]. However, their characteristics and functions have not been identified in O. pumila. Therefore, the function of WRKYs in O. pumila requires in-depth research, especially OpWRKY3.
Furthermore, overexpression and repression of OpWRKY3 affected the expression level of genes involved in biosynthesis of camptothecin, tryptamine, loganin, and secologanin in transgenic hairy roots. The TDC gene expression and loganin concentration were increased significantly while

Discussion
In O. pumila, WRKY3 transcription factor shared high identity with AtWRKY30, AtWRKY41, and VvWRKY30. Furthermore, OpWRKY3 possesses a highly conserved WRKYGQK motif and a C2H2-type zinc-finger structure, belonging to the group III category. The group III WRKYs, such as Arabidopsis thaliana WRKY75 and WRKY41, have been shown previously and played essential roles in regulating plant growth and development [30,31]. In addition, knockout of the AtWRKY75 mutant increased the number and length of root hairs [30]. AtWRKY46, AtWRKY54, and AtWRKY70 are involved in both brassinosteroid-regulated plant growth and drought response [36,37]. In the present study, the results of OpWRKY3 overexpression and repression revealed that the expression level of OpWRKY3 was linear with the biomass of transgenic hairy roots. Repression of OpWRKY3 inhibited the growth and development of the root tip and ultimately decreased the biomass of transgenic hairy roots. This is consistent with the WRKY transcription factor gene CbNN1 increased rooting ability, promoting adventitious root formation [38]. These results indicated that OpWRKY3 is involved in the growth and development of hairy roots. In addition, previous reports shown that group III WRKYs possessed the function of regulating secondary metabolism [39,40]. In another study has been shown that overexpression of CrWRKY1-SRDX decrease cellular tryptophan decarboxylase (TDC) activity and tryptamine accumulation in transgenic hairy roots [40]. However, their characteristics and functions have not been identified in O. pumila. Therefore, the function of WRKYs in O. pumila requires in-depth research, especially OpWRKY3.
Furthermore, overexpression and repression of OpWRKY3 affected the expression level of genes involved in biosynthesis of camptothecin, tryptamine, loganin, and secologanin in transgenic hairy roots. The TDC gene expression and loganin concentration were increased significantly while the SLS gene expression and secologanin concentration were decreased significantly in OpWRKY3 overexpression hairy roots. This is consistent with a previous report that the accumulation levels of secologanin exhibited a strong negative correlation with the expression level of TDC, and that of loganin exhibited a negative correlation with the expression level of SLS [17]. These results suggested that OpWRKY3 is involved in biosynthesis of tryptamine, loganin and secologanin, which as CPT biosynthetic precursors. Numerous reports showed that WRKY TFs regulate their target genes by binding to the W-box elements containing the TTGAC(C/T) motif [41]. Simultaneously, previous reports indicated that overexpression of CrWRKY1-SRDX repressed the transcription of TDC by binding to the W-box elements of its promoter [40]. SmWRKY2 increased accumulation of tanshinones by binding to the W-box of the SmCPS [42]. The yeast one-hybrid experiments implied that OpWRKY3 regulates the biosynthesis of camptothecin and its precursors by binding to the W-box elements of the OpCPR gene promoter.
The total production (hairy roots and medium) of CPT has been increased significantly in OE-8, 13, 14 lines while it was significantly decreased in the S lines. However, the CPT concentrations in transgenic hairy roots were not significantly changed in OE and S lines, compared to control. It may be due to overexpression of OpWRKY3 slightly increased the biomass of hairy roots while repression of OpWRKY3 down-regulated the biomass of hairy roots. These results indicated that OpWRKY3 influenced the production of CPT by regulating the growth and development of hairy roots.
We found that OpWRKY3-SRDX decreased CPT production as quantified through a confocal microscope and high performance liquid chromatography (HPLC) method. The previous report has been reported that camptothecin was mainly accumulated in the root tip of O. pumila roots [43]. The camptothecin can emit blue auto-fluorescence under 360 nm UV light [43,44]. According to this feature, it was found that the camptothecin was mainly accumulated in glandular trichomes and secretory canals of the stem and leaf in C. acuminate [45,46]. Therefore, it requires direct and effective methods to further examine the concentration of camptothecin by confocal microscope observation. These results can accurately identify the content of camptothecin, which is helpful to explore the transport and accumulation of camptothecin in plants.

Plant Materials and Elicitor Treatments
The aseptic O. pumila plants were cultured as per the previously reported method [18]. Roots, stems, leaves, and petioles from O. pumila seedlings and hairy roots were gathered to detect OpWRKY3 expression patterns. For the analysis of OpWRKY3 expression patterns by different elicitors including 50 µM gibberellin (GA3), 200 µM methyl jasmonate (MJ), 50 µM acetylsalicylic acid (ASA), 50 µM salicylic acid (SA), and 100 µM abscisic acid (ABA) were used and water or ethanol was used as controls. Elicitor treatments were conducted on O. pumila hairy roots sub-cultured for 30 days infected with modified A. tumefaciens strain C58C1. The treated samples were harvested separately at different time points, including 0, 0.5, 1, 3, 6, 9, and 12 hour. All the treated materials were immediately frozen in liquid nitrogen and stored at −80 • C in a refrigerator.

The Gene Clone and Bioinformatics Analyses of OpWRKY3
Based on the sequence information of the WRKY transcription factor in O. pumila transcriptome databases, the full length of OpWRKY3 was amplified using PCR. The amplified primers of the full-length coding sequence (CDS) of OpWRKY3 are listed in Table S1. The physicochemical properties of the amino acids were determined with the ExPASy server (http://web.expasy.org/compute_pi/). Homologous proteins were searched using the BLASTp program against a non-redundant (nr) protein sequence. Phylogenetic analysis of OpWRKY3 with the Arabidopsis thaliana and other species WRKYs was performed using the neighbor-joining method and 1000 bootstrap replicates in MEGA 7.0. Multiple sequences alignment analyses were performed between OpWRKY3 with six selected WRKYs from the same clade from the phylogenetic analysis using DNAMAN version 5.0.

Subcellular Localization of OpWRKY3
To investigate the subcellular localization of OpWRKY3, its full length of OpWRKY3 was amplified and inserted into vector pMON530-GFP located at the restriction site of Bgl II and Kpn I to generate pMON530-OpWRKY3-GFP. The constructed expression vector was transferred into Agrobacterium strain ASE and injected into 60-day old tobacco leaves. GFP fluorescence was observed using the confocal microscope after 48 h of cultivation [47]. In total, 1 µg mL −1 4, 6-diamidino-2-phenylindole (DAPI) was injected into tobacco leaves for 3 h before observation. A corresponding empty vector was considered as the control.

Construction of Plant Expression Vectors
To investigate the role OpWRKY3 played in camptothecin biosynthesis, transgenic plants overexpress and repress vectors of the OpWRKY3 gene were constructed. For the construction of the OpWRKY3 overexpress vectors, the full length of OpWRKY3 was cloned from the cDNA library and inserted into the modified vector pCAMBIA2300 sm located at the restriction site of Spe I and BstE II to generate pCAMBIA2300 sm -OpWRKY3 ( Figure S6) [33]. For the construction of the OpWRKY3 repress vectors, the OpWRKY3 gene was linked to an SRDX domain for gene expression repression based on the Chimeric REpressor gene-Silencing Technology (CRES-T) to make a chimeric gene (OpWRKY3-SRDX) construct, with the SRDX domain linked to the C-terminal end of OpWRKY3 [48]. The OpWRKY3-SRDX was amplified by PCR with gene-specific primers pCAMBIA 2300 sm -OpWRKY3-SRDX-KF-SpeI and 2300 sm -OpWRKY3-SRDX-KR-BstEII and cloned into the pCAMBIA2300 sm vector (Table S1). A corresponding empty vector was considered as the control.

Acquisition of Transgenic Hairy Root Lines
Aseptic stems were separated from the O. pumila plants and cultured on B5 solid medium in the darkness for 2 days. The A. tumefaciens strain C58C1 containing the constructed vectors were used to infect the isolated stems. The transformation procedure was conducted according to a previous report [18]. The positive transgenic hairy roots were chosen for further study. For acquisition of positive transgenic lines, individual hairy root samples were collected for isolation of genomic DNA using the cetyltrimethylammonium ammonium bromide (CTAB) method [49]. Positive colonies were identified by PCR with the specific primers. All the primers used in this article are listed in Table S1. Fragments of the positive hairy roots were cut down and inoculated into 50 mL B5 liquid media and cultured for about 45 days under dark conditions with the rate of 120 rpm at 25 • C [18].

Quantitative Real-Time PCR
Total RNA was isolated from all collected samples by using the RNAprep pure Tissue Kit according to the instructions (TIANGEN, Beijing, China). Total RNA of 2 µg was used to perform the reverse transcription analysis using the RNaseH-Kit and Cloned Ribonuclease Inhibitor Kit (TransGen Biotech, Beijing, China). Real-time quantitative PCR analysis was carried out to detect the expression pattern of OpWRKY3 and eight genes involved in the camptothecin biosynthesis pathway ( Figure S3). The expression level was normalized to the internal reference gene Opactin. The amplification reaction using was performed in Applied Biosystem Step One with an optional 48-well plate (Applied Biosystem) by the relative quantitative analysis method (2 −∆∆Ct ) [50]. The amplification profile was 95 • C for 10 min, followed by 40 cycles of 95 • C for 15 s, 60 • C for 30 s, and 72 • C for 30 s. All the primers used in this article are listed in Table S1.

Confocal Microscope Analysis
In order to observe the distribution of camptothecin in hairy roots, transgenic hairy root tips from cultures in B5 solid medium in the darkness for 20 days were used for temporary slide specimens. CPT fluorescence in all of samples was observed and photographed under a confocal microscopy with 360 nm UV excitation [43]. A corresponding empty vector was considered as the control.

Camptothecin Content Measurement
Positive transgenic hairy roots cultured for 45 days were collected for the determination of camptothecin content and production. The hairy roots were dried in an oven to a constant weight and ground to a powder and weighted. An aliquot of 0.1 g powder was weighed for extraction of camptothecin according to a previous report [18]. In order to detect camptothecin excreted into the medium, medium was extracted by equal volumes of chloroform and methanol (4:1) three times after that organic layer was separated and evaporated the organic solvent. Residue was dissolved in 2 mL of methanol for HPLC analysis. Different concentration of camptothecin standard (100, 83.3, 66.7, 50, 33.3 and 16.7 µg/mL) was used to describe the standard curve ( Figure S7). The detection parameters were the same as that described previously [18]. CPT production per flask = CPT content in hairy roots × hairy roots biomass per flask + CPT content in medium × medium volume per flask.

Extraction and Analysis of Tryptamine, Loganin and Secologanin
Extraction method of trypamine and loganin was the same as camptothecin, while the secologanin was extracted from hairy roots with distilled water. The HPLC parameters of trypamine were as follows: The mobile phase consisted of acetonitrile, methanol, distilled water, and glacial acetic acid with the ratio of 43:30:26:1. The detection wavelength was 254 nm. The HPLC parameters of loganin and secologanin were as follows: The mobile phase consisted of acetonitrile and distilled water at a ratio of 1:3 (v/v). The detection wavelength was 236 nm. The flow rate was 1.0 mL/min with an injection volume of 10 µL. The standard curves of tryptamine, logan and secologanin were described using different concentrations of its standards ( Figure S7). The production of tryptamine and loganin were calculated according to the formula of CPT production. The secologanin in medium was undetectable, so secologanin production per flask = Secologanin content in hairy roots × hairy roots biomass per flask.

Yeast-One Hybrid Assay
Yeast one-hybrid assay was performed according to previous report [50]. The coding sequence of full-length OpWRKY3 was inserted into the pB42AD vector. Meanwhile, the OpCPR promoter sequences (from −80 bp to −86 bp and -433 bp to -439 bp, relative to translation start site) were constructed into a pLacZ plasmid, respectively. The resulting recombinant plasmids were co-transformed into yeast strain EGY48a. The transformed reporter strains were cultivated on SD/-Ura/-Trp medium for 48 h, and tested on SD/-Ura/-Trp medium with 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal) for 24 h. The empty vectors pB42AD and pLacZ were used as negative controls.

Statistical Analysis
All experiments, including identification of positive hairy roots lines, qRT-PCR, HPLC analysis of camptothecin, and its intermediate compound, were performed three times and the results are shown as the mean values of three independent repeats ± standard deviation. Statistical significance was determined by the one sample t-test and one-way analysis of variance using SPSS 11.5 software (SPSS).

Supplementary Materials:
The following are available online at http://www.mdpi.com/1422-0067/20/16/3996/s1, Figure S1: Transformation of O. pumila explants for hairy roots lines. Figure S2: Expression pattern of OpWRKY3 in transgenic hairy roots. Figure S3: The fresh weight (FW) and dry weight (DW) of OpWRKY3 transgenic hairy roots after shake-flask culture. Figure S4: The content of CPT and its precursors in OpWRKY3 transgenic hairy roots by HPLC. Figure S5: Camptothecin biosynthetic pathway in O. pumila. Figure S6: Expression vectors construction for transformation. Figure S7: Analysis of Tryptamine, Loganin, Secologanin and CPT standard by HPLC. Table S1: Oligonucleotide primers used in this study.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.