Formation of α- and β-Cembratriene-Diols in Tobacco (Nicotiana tabacum L.) Is Regulated by Jasmonate-Signaling Components via Manipulating Multiple Cembranoid Synthetic Genes

Cembranoids are a group of natural diterpenoid compounds with pharmaceutical potentials, and the cembratriene-diols produced by Nicotiana (tobacco) species display activities in anti-nicotine addiction and neuron protection. Although the enzymes catalyzing cembratriene-diols’ formation in tobacco have been investigated, the regulatory mechanism underlying this physiological process remains unknown. This study has investigated the roles of phytohormone jasmonic acid (JA) in regulating cembratriene-diol formation in N. tabacum cv. TN90 and found that JA and COI1, the receptor protein of the bioactive derivative of JA (i.e., JA-Ile), display critical roles in regulating cembratriene-diols’ formation and the expression of cembranoid synthetic genes CBTS, P450 and NtLTP1. Further studies showed that over-expressing either the gene encoding bHLH transcription factor MYC2a or that encoding MYB transcription factor MYB305 could upregulate the cembranoid synthetic genes and enhance the cembranoid production in plants with dysfunction of COI1. Further studies suggest that COI1 and its downstream regulators MYC2a and MYB305 also modulate the trichome secretion, which is correlated with cembranoid formation. Taken together, this study has demonstrated a critical role of JA-signaling components in governing the cembratriene-diol formation and the transcription of cembratriene-diol synthetic genes in tobacco. Findings in this study are of great importance to reveal the molecular regulatory mechanism underlying cembranoid synthesis.

Cembranoids are synthesized in the glandular trichome, known as phytochemical factories, producing a large portion of plant metabolic compounds, such as polysaccharides, terpenes, phenylpropanoids and flavonoids [3][4][5]. Previously, the formation of cembranoids was investigated by multiple studies. The first step of cembranoid biosynthesis is catalyzed by cembratriene-ol synthase (CBTS) (Figure 1), which produces α-and β-cembratriene-ols from geranylgeranyl diphosphate (GGPP) [1]. The amount of cembratriene-ol production was increased by transient expressing of CBT2a [6], while the production of cembratriene-ol and cembratriene-diol was reduced by CBTS (NtCYC1) silencing in tobacco [7]. The second step of cembranoid biosynthesis is catalyzed by cytochrome P450 hydroxylase (P450) (Figure 1), which catalyzes the hydroxylation of cembratriene-ols to form cembratriene-diols [8]. Suppression of P450 caused a decrease in the content of cembratriene-diols, but an increase in the content of cembratriene-ols in tobacco [8]. Moreover, tobacco lipid transfer proteins (NtLTP1) play important roles in lipid secretion from glandular trichomes [9]. Over-expressing NtLTP1 in tobacco resulted in the increased secretion from glandular trichomes, while silencing of this gene led to reduced trichome secretion [9]. Although enzymes catalyzing multiple steps of cembranoid formation in tobacco have been identified by a number of studies [10,11], the regulatory mechanism underlying this physiological process remains largely unknown. Since glandular trichomes function as the factory producing cembranoids, this implies a critical role of trichome-developmental regulators in governing cembranoid synthesis. It was shown that the initiation, development and secretion of plant trichomes were regulated by several phytohormones, including jasmonate acid (JA), 6-benzylaminopurine (BAP) and gibberellin (GA) [12][13][14][15][16][17]. Studies in Artemisia annua indicated that JA and BAP positively regulate the density of glandular trichomes, but only JA could promote the secretion of glandular trichomes [16], suggesting a pivotal role of JA in regulating trichome secretion. JAs are a kind of fatty-acid-derived hormone functioning in plant responses to wounding and herbivore attacks and play important roles in regulating plant development, defense responses, secondary metabolism and other Since glandular trichomes function as the factory producing cembranoids, this implies a critical role of trichome-developmental regulators in governing cembranoid synthesis. It was shown that the initiation, development and secretion of plant trichomes were regulated by several phytohormones, including jasmonate acid (JA), 6-benzylaminopurine (BAP) and gibberellin (GA) [12][13][14][15][16][17]. Studies in Artemisia annua indicated that JA and BAP positively regulate the density of glandular trichomes, but only JA could promote the secretion of glandular trichomes [16], suggesting a pivotal role of JA in regulating trichome secretion. JAs are a kind of fatty-acid-derived hormone functioning in plant responses to wounding and herbivore attacks and play important roles in regulating plant development, defense responses, secondary metabolism and other physiological processes [18][19][20][21]. In Arabidopsis, the initiation and formation of trichomes can be greatly increased by herbivore attacks and JA [17,22], and proper JA perception is crucial for trichome induction [23]. Studies on Solanaceae plants showed that dysfunction of COI1 could suppress the development of glandular trichome in tobacco and notably reduce the secretion of glandular trichome in tobacco and tomato [24,25]. These facts suggested that JA, as well as COI1, the receptor protein of the bioactive derivative of JA (i.e., JA-Ile), display crucial roles in the development and secreting of trichomes.
Previous studies have established the molecular model of JA-signaling in plants. Jasmonoyl-isoleucine (JA-Ile), the bioactive derivative of JA, is perceived and transduced by the SCF COI1 (Skip/Cullin/F-box) complex [20,26], which results in the ubiquitination and subsequent degradation of JAZ (jasmonate ZIM domain) proteins, the repressors of JA-signaling, via the 26S proteasome pathway [27,28]. Then, the downstream bHLH (such as MYC2) and MYB transcription factors are released from the JAZ repressors to activate JA-signaling responses [27,29,30]. Studies in a number of plants have shown that the bHLH transcription factor MYC2 acts as a master regulator mediating multiple aspects of JA responses [31][32][33]. The R2R3 MYB transcription factor MYB305 from Antirrhinum was demonstrated to regulate the biosynthesis of flavonoid in flowers, and its homologue NtMYB305 from tobacco was shown to modulate the carbohydrate metabolism in both vegetative and reproductive organs [25,34,35]. Previously, cumulative studies in multiple plants have suggested that the WD40-bHLH-MYB complex displays a critical role in modulating the downstream JA responses, including trichome development, secondary metabolism and transcriptional regulations [14,15,[36][37][38][39], and a group of transcription factors are involved in this regulatory network [10].
Even though the roles of JA in regulating plant secondary metabolism have been intensively investigate, its function in cembranoid synthesis remain largely unknown. This study has analyzed the induction of cembranoid synthesis by JA in tobacco (N. tabacum cv. TN90) and investigated the mechanism of JA-signaling components including COI1, MYC2 and MYB305 in governing the cembranoid formation and modulating the transcription of cembranoid synthetic genes in tobacco. Findings of this study have provided important information for dissecting the molecular regulation of cembranoid synthesis in tobacco.

The Formation of Cembratriene-Diol Is JA-Inducible in Tobacco
To investigate the roles of JA in regulating cembranoid synthesis, the four-week-old tobacco seedlings were selected to determine the induction of cembratriene-diol formation by JA, for the seedlings at this stage had mid-sized leaves with much lower cembratriene-diol content than the aged plants ( Figure 2A Since cembranoids were secreted by the glandular trichomes on tobacco, the trichome secretion were observed under stereo-microscope after staining with I 2 /KI solution [1% (w/v) I 2 in 3% (w/v) KI (potassium iodide)]. The observations showed that the color of the trichome secretion droplets of the young plants was much lighter than those of the aged (eight-week-old) plants ( Figure 2E,F). While, the trichome secretion droplets of the MeJA-treated plants turned to a much darker color compared with that of the control, their color was still lighter than those of the eight-week-old plants ( Figure 2G). Further analysis indicated that the trichome secretion droplets of eight-week-old plants contained a higher soluble sugar content than those of the four-week-old plants; moreover, MeJA treatment could increase the soluble sugar content of the trichome secretions ( Figure 2H,I). These facts suggested that the cembratriene-diol formation in tobacco is regulated by JA, which may affect the composition of glandular trichome secretion.

JA Displays a Role in Regulating the Transcription of Cembratriene-Diol Synthetic Genes in Tobacco
To figure out the molecular mechanism underlying JA-regulated cembratriene-diol formation in tobacco, we carried out a transcriptional assay to determine whether the cembratriene-diol synthetic genes are regulated by JA treatment. The transcription levels of a set of cembratriene-diol synthetic genes, including LTP1 (lipid transfer protein), CBTS (cembratriene-ol synthase) and P450 (cytochrome P450 hydroxylase) [10,11], were analyzed in the qRT-PCR assays. The results showed that the expression level of LTP1 was increased to five folds of that in the control after three days of JA-treatment and to 17 folds of that in the control after five days of treatment ( Figure 3A); CBTS was upregulated by four-fold after seven days of JA treatment ( Figure 3B); and P450 was increased by two-fold after five days of treatment ( Figure 3C). These findings suggest that JA functions as a regulator of the cembratriene-diol synthetic genes.

JA Displays a Role in Regulating the Transcription of Cembratriene-Diol Synthetic Genes in Tobacco
To figure out the molecular mechanism underlying JA-regulated cembratriene-diol formation in tobacco, we carried out a transcriptional assay to determine whether the cembratriene-diol synthetic genes are regulated by JA treatment. The transcription levels of a set of cembratriene-diol synthetic genes, including LTP1 (lipid transfer protein), CBTS (cembratriene-ol synthase) and P450 (cytochrome P450 hydroxylase) [10,11], were analyzed in the qRT-PCR assays. The results showed that the expression level of LTP1 was increased to five folds of that in the control after three days of JA-treatment and to 17 folds of that in the control after five days of treatment ( Figure 3A); CBTS was upregulated by four-fold after seven days of JA treatment ( Figure 3B); and P450 was increased by two-fold after five days of treatment ( Figure 3C). These findings suggest that JA functions as a regulator of the cembratriene-diol synthetic genes.

COI1 Is Required for the Formation of Cembratriene-Diols in Tobacco
In order to uncover the mechanism of the JA-signaling pathway in regulating cembratriene-diol formation, tobacco plants with dysfunction of COI1 [25] were adopted for further analyses. Our previous studies showed that the trichome secretion of these plants was decreased to an extremely lower level ( Figure 4A,B) [25]. The cembratriene-diol determination with the eight-week-old plants showed that the α-and β-cembratriene-diol contents of plants with dysfunction of COI1 were decreased to an undetectable level, while those of the control plants were 5.68 and 3.96 mg/g FW, respectively ( Figure 4D,E). The transcriptional analyses revealed that dysfunction of COI1 greatly attenuated the expression LTP1, CBTS and P450 genes ( Figure 4F). Additionally, the plants with dysfunction of COI1 possessed significantly decreased soluble sugar content of the trichome secretions ( Figure 4C). These results further demonstrated the importance of proper JA-signaling for cembratriene-diol synthesis in tobacco.

COI1 Is Required for the Formation of Cembratriene-Diols in Tobacco
In order to uncover the mechanism of the JA-signaling pathway in regulating cembratriene-diol formation, tobacco plants with dysfunction of COI1 [25] were adopted for further analyses. Our previous studies showed that the trichome secretion of these plants was decreased to an extremely lower level ( Figure 4A,B) [25]. The cembratriene-diol determination with the eight-week-old plants showed that the αand β-cembratriene-diol contents of plants with dysfunction of COI1 were decreased to an undetectable level, while those of the control plants were 5.68 and 3.96 mg/g FW, respectively ( Figure 4D,E). The transcriptional analyses revealed that dysfunction of COI1 greatly attenuated the expression LTP1, CBTS and P450 genes ( Figure 4F). Additionally, the plants with dysfunction of COI1 possessed significantly decreased soluble sugar content of the trichome secretions ( Figure 4C). These results further demonstrated the importance of proper JA-signaling for cembratriene-diol synthesis in tobacco.

Regulatory Roles of NtMYC2a in COI1-Mediated Cembratriene-Diol Formation in Tobacco
To investigate the regulation of cembratriene-diol formation by the regulators functioning downstream of the JA-Ile receptor, the plants with dysfunction of COI1 were employed for further assays. The tobacco homologue of Arabidopsis MYC2, i.e., bHLH transcription factor NtMYC2a, was overexpressed in the tobacco with dysfunction of COI1. Trichome staining with I2/KI showed that overexpression of NtMYC2a had no obvious effect on the volume of trichome secretion droplets, but turned the secretion droplets to a darker color and increased the soluble sugar content of the trichome secretions, as well ( Figure 5A-C). Following cembratriene-diol determination revealed that overexpression of NtMYC2a notably enhanced the cembratriene-diol production of plants with dysfunction of COI1 ( Figure 5D,E). The transcriptional analyses suggested that overexpression of NtMYC2 accentuated the transcription levels of LTP1, CBTS and P450 genes in the plants with dysfunction of COI1 ( Figure 5F). This evidence suggested the involvement of NtMYC2 in regulating COI1-mediated cembratriene-diol formation in tobacco.

Regulatory Roles of NtMYC2a in COI1-Mediated Cembratriene-Diol Formation in Tobacco
To investigate the regulation of cembratriene-diol formation by the regulators functioning downstream of the JA-Ile receptor, the plants with dysfunction of COI1 were employed for further assays. The tobacco homologue of Arabidopsis MYC2, i.e., bHLH transcription factor NtMYC2a, was overexpressed in the tobacco with dysfunction of COI1. Trichome staining with I 2 /KI showed that overexpression of NtMYC2a had no obvious effect on the volume of trichome secretion droplets, but turned the secretion droplets to a darker color and increased the soluble sugar content of the trichome secretions, as well ( Figure 5A-C). Following cembratriene-diol determination revealed that overexpression of NtMYC2a notably enhanced the cembratriene-diol production of plants with dysfunction of COI1 ( Figure 5D,E). The transcriptional analyses suggested that overexpression of NtMYC2 accentuated the transcription levels of LTP1, CBTS and P450 genes in the plants with dysfunction of COI1 ( Figure 5F). This evidence suggested the involvement of NtMYC2 in regulating COI1-mediated cembratriene-diol formation in tobacco.

Regulatory Roles of NtMYB305 in COI1-Mediated Cembratriene-diol Formation in Tobacco
Furthermore, the tobacco homologue of Arabidopsis MYB24, i.e., MYB transcription factor NtMYB305, was also overexpressed in the tobacco with dysfunction of COI1. Staining with I2/KI showed that overexpression of NtMYB305 resulted in a pronounced increase in the volume of trichome secretion droplets of plants with dysfunction of COI1, and subsequent analysis also revealed an increase in the soluble sugar content of the trichome secretions ( Figure 6A-C); whereas no obvious color change in the trichome secretion droplets was observed ( Figure 6A,B). Further determination showed that the contents of cembratriene-diol of the trichome secretions was obviously increased by overexpression of NtMYB305 in tobacco with dysfunction of COI1 ( Figure  6D,E). The transcription levels of LTP1, CBTS and P450 genes were also accentuated by overexpression of NtMYB305 ( Figure 6F). This evidence suggested the involvement of NtMYB305 in regulating COI1-mediated cembratriene-diol formation in tobacco. Interestingly, an unidentified compound observed in the above results was abolished by overexpressing NtMYB305 in the plant with dysfunction of COI1, but a novel unidentified compound appeared with the retention time shifted to 5 min from 6.8 min.

Regulatory Roles of NtMYB305 in COI1-Mediated Cembratriene-Diol Formation in Tobacco
Furthermore, the tobacco homologue of Arabidopsis MYB24, i.e., MYB transcription factor NtMYB305, was also overexpressed in the tobacco with dysfunction of COI1. Staining with I 2 /KI showed that overexpression of NtMYB305 resulted in a pronounced increase in the volume of trichome secretion droplets of plants with dysfunction of COI1, and subsequent analysis also revealed an increase in the soluble sugar content of the trichome secretions ( Figure 6A-C); whereas no obvious color change in the trichome secretion droplets was observed ( Figure 6A,B). Further determination showed that the contents of cembratriene-diol of the trichome secretions was obviously increased by overexpression of NtMYB305 in tobacco with dysfunction of COI1 ( Figure 6D,E). The transcription levels of LTP1, CBTS and P450 genes were also accentuated by overexpression of NtMYB305 ( Figure 6F). This evidence suggested the involvement of NtMYB305 in regulating COI1-mediated cembratriene-diol formation in tobacco. Interestingly, an unidentified compound observed in the above results was abolished by overexpressing NtMYB305 in the plant with dysfunction of COI1, but a novel unidentified compound appeared with the retention time shifted to 5 min from 6.8 min.

Discussion
The therapeutic activities of cembranoids in cancer and inflammation treatment, as well as their fascinating architectures are attractive to pharmaceutical developers [1,2]. Currently, the majority of cembranoids from plants were identified from conifers and tobacco [1], and tobacco is the most feasible plant that could be applied to dissect the molecular mechanism of cembranoids' synthesis in plants. Thus, the studies of JA-induced cembranoid formation in this study are of great importance for the utilization of plant-derived cembranoids.
Many studies have proven that JAs display important roles in regulating the secondary metabolism in plants [18][19][20][21] and also act as regulators in controlling the initiation and secondary metabolism of glandular trichomes, which are the factories producing cembranoids [4,17,22,23,40,41]. In this study, treatment of tobacco seedlings with MeJA could not only increase the secretion level of glandular trichome, but also promote the formation of cembratriene-diols, which evidenced the regulatory function of JA in cembranoid formation. Previous studies identified several genes involved in cembranoid synthesis, CBTS (cembratrienol synthase), P450 (cytochrome P450 hydroxylase) and LTP1 (lipid transfer protein) [1,8,9], and the qRT-PCR analyses carried out in this study demonstrated that MeJA treatment could significantly enhance the transcription levels of CBTS, P450 and NtLTP1 genes in tobacco. This finding implied that JA functions as positive regulator of cembratriene-diol formation via upregulating the cembranoid synthetic genes.
COI1 is a critical component of the SCF COI1 receptor complex of the JA-signaling pathway, which displays diverse roles in the regulation of JA-mediated plant growth and development, defense response and secondary metabolism [20,[26][27][28]. In this study, we analyzed the roles of tobacco COI1 in regulating cembranoid synthesis using previously developed transgenic plants [25], and the results showed that dysfunction of COI1 decreased the cembratriene-diol content of tobacco leaves to an undetectable level. Subsequent research found that the transcription levels of CBTS, P450 and

Discussion
The therapeutic activities of cembranoids in cancer and inflammation treatment, as well as their fascinating architectures are attractive to pharmaceutical developers [1,2]. Currently, the majority of cembranoids from plants were identified from conifers and tobacco [1], and tobacco is the most feasible plant that could be applied to dissect the molecular mechanism of cembranoids' synthesis in plants. Thus, the studies of JA-induced cembranoid formation in this study are of great importance for the utilization of plant-derived cembranoids.
Many studies have proven that JAs display important roles in regulating the secondary metabolism in plants [18][19][20][21] and also act as regulators in controlling the initiation and secondary metabolism of glandular trichomes, which are the factories producing cembranoids [4,17,22,23,40,41]. In this study, treatment of tobacco seedlings with MeJA could not only increase the secretion level of glandular trichome, but also promote the formation of cembratriene-diols, which evidenced the regulatory function of JA in cembranoid formation. Previous studies identified several genes involved in cembranoid synthesis, CBTS (cembratrienol synthase), P450 (cytochrome P450 hydroxylase) and LTP1 (lipid transfer protein) [1,8,9], and the qRT-PCR analyses carried out in this study demonstrated that MeJA treatment could significantly enhance the transcription levels of CBTS, P450 and NtLTP1 genes in tobacco. This finding implied that JA functions as positive regulator of cembratriene-diol formation via upregulating the cembranoid synthetic genes.
COI1 is a critical component of the SCF COI1 receptor complex of the JA-signaling pathway, which displays diverse roles in the regulation of JA-mediated plant growth and development, defense response and secondary metabolism [20,[26][27][28]. In this study, we analyzed the roles of tobacco COI1 in regulating cembranoid synthesis using previously developed transgenic plants [25], and the results showed that dysfunction of COI1 decreased the cembratriene-diol content of tobacco leaves to an undetectable level. Subsequent research found that the transcription levels of CBTS, P450 and NtLTP1 genes were significantly decreased in plants with dysfunction of COI1 compared to that in the control plants. These results evidenced the involvement of COI1 in regulating the formation of cembratriene-diols and the transcription of cembranoid synthetic genes including CBTS, P450 and NtLTP1. Since the COI1-mediated JA-signaling network was well established [28,[42][43][44][45], the involvement of the JA perception complex in cembranoid synthesis will provide important clues to uncover the regulatory mechanism underlying cembranoid formation.
Previous studies established that COI1 and JAZ proteins are the key components of the JA-Ile receptor complex and play pivotal roles in governing the JA responses in plants [20,[26][27][28]. The WD40-bHLH-MYB complex acts as a critical modulator in controlling the downstream JA responses, including secondary metabolism and transcriptional regulations [10,14,15,[36][37][38][39], in which the bHLH (such as MYC2) and MYB transcription factors are the direct targets of JAZ proteins and exert important functions in mediating JA responses [27,29,30,46]. Thus, it is of great importance to study the functions of JA-responsive bHLH and MYB transcription factors in regulating cembranoid synthesis, which may provide direct evidence of the molecular regulation of cembranoid synthesis. To analyze the roles of NtMYC2a and NtMYB305 in regulating cembranoid synthesis, NtMYC2a and NtMYB305 were overexpressed in the tobacco with dysfunction of COI1, respectively. The studies showed that MYC2-overexpression could notably enhance the cembratriene-diol production of plants with dysfunction of COI1, but with no obvious effects on the trichome secretion. MYB305-overexpression not only increased the cembratriene-diol production, but also increased the quantity of the trichome secretion of plants with dysfunction of COI1. Moreover, the transcriptional assays revealed that overexpression of MYC2 or MYB305 could increase the transcriptional levels of cembranoid synthetic genes including CBTS, P450 and NtLTP1 in the plants with dysfunction of COI1. Thus, these findings demonstrated that MYC2 and MYB305 function as positive regulators of cembratriene-diols synthesis, but these two kinds of regulators worked in distinctive patterns. As mentioned above, bHLH transcription factor MYC2 and MYB transcription factor MYB305, the tobacco homologues of Arabidopsis MYB21 and MYB24 [47], are the direct targets of JAZ proteins and function downstream of COI1 to regulate secondary metabolism in plants [31,34,[46][47][48][49][50][51]. The enhancement of cembratriene-diol production, as well as the upregulation of cembranoid synthetic genes in plants with dysfunction of COI1 by overexpression of MYC2 or MYB305 evidenced that MYC2 and MYB305 both function downstream of COI1 in regulating cembratriene-diol synthesis in tobacco, which is consistent with their function patterns in regulating other JA responses [10,14,15,27,29,30,[36][37][38][39]46].

Plant Materials
Tobacco cultivar Nicotiana tabacum L. cv. TN90 was used in this study. The NtCOI1-silenced tobacco plants and the empty-vector-transformed control plants were developed in our previous study [25].
The hybrid plant of NtCOI1-silenced and NtMYC2a-overexpression was generated by crossing of the NtCOI1-silenced plants with pollens from NtMYC2a-overexpressing plants. The hybrid of NtCOI1-silenced and NtMYB305-overexpression was generated in a similar manner.

Plant Cultivation and Phytohormone Treatment
All tobacco plants were grown at 23 • C in a greenhouse with a photoperiod of 14 h light/10 h dark. For phytohormone treatment with jasmonate (JA), the 4-week-old seedlings of wild type TN90 were sprayed daily with MeJA aqueous solution (100 µM) for seven days, while the seedlings of control treatment were sprayed with distilled water simultaneously. The leaf samples were collected at indicated time points for further analyses.

Visualization of Trichome Secretion Droplets by Iodine Staining
To visualize the trichome secretion droplets, tobacco leaves were stained by I 2 /KI solution (1% (w/v) I 2 in 3% (w/v) KI), as previously described [25]. The stained leaves were observed under the stereoscopic microscope and photographed. A darker color indicates higher soluble sugar content of the tested samples.

Soluble Sugar Content Determination
The soluble sugar content of the trichome secretion was determined using the method for total soluble sugar measurement with anthrone reagent as previously described [53]. Briefly, the soluble sugar of glandular trichome secretion was extracted with anhydrous alcohol by washing the leaf sample, and then, 1/4 V (volume) distilled water was added into the extraction for a dilution. Following that, 0.5 mL of extraction were mixed with 2.5 mL of ice-cold anthrone solution (0.2% (w/v) anthrone in 72% (v/v) H 2 SO 4 ) and incubated in the water-bath of 100 • C for exactly 11 min. Then, the reactions were transferred onto ice immediately to stop the reaction. The absorbances of the reactions were measured at 630 nm, and the soluble sugar content of each sample was calculated as previously described [53].

Measurement of Cembratriene-Diols
The cembratriene-diol content of tobacco glandular trichome secretion was determined using the UPLC (ultra performance liquid chromatography) system. In brief, the cembratriene-diol of glandular trichome secretion was extracted by washing tobacco leaves with ethyl acetate and dried in the nitrogen flow. Then, the extraction was dissolved in the solvent of 80% acetonitrile in water (v/v) and filtrated through filters with a membrane with a 0.22-µm pore size for injection (10 µL). The chromatography assay was performed on an ACQUITY UPLC system (Waters, Milford, CT, USA) under the following optimized conditions: BEH C18 column (1.7 µm, 2.1 mm × 100 mm) with the column temperature of 35 • C, a mobile phase of gradient acetonitrile (Table 1) at the flow rate of 0.3 mL min −1 and a UV detector for the detection of cembratriene-diols at 200 nm. Cembratriene-diol purified from tobacco leaves with preparative LC (liquid chromatography) system and verified by the MS spectrum was used as the standard. Standard curves were made for quantification by serial dilutions of the cembratriene-diol stock solutions (1 mg/mL). 4.6. Quantitative RT-PCR (qRT-PCR) Samples for total RNA extraction were collected from fully-expanded leaves of the indicated plants. Total RNAs were extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. First-strand cDNAs were synthesized using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China) and used as templates for qRT-PCR. Reactions were performed using an ABI 7500 real-time PCR system with GoTaq ® qPCR Master Mix (Promega, Madison, USA).

Statistical Analysis
Data in the figures are presented as the mean ± SE. Student's t-test was carried out for the statistical analyses of cembratriene-diol and soluble sugar contents and for the qRT-PCR data. Differences in the tests were considered statistically significant at a p-value < 0.05 (*) or <0.005 (**).

Conclusions
This study has demonstrated a critical role of JA, as well as COI1, the receptor protein of the bioactive derivative of JA (i.e., JA-Ile), in governing cembratriene-diol synthesis in tobacco. Findings of this study suggested that JA-signaling pathways may control the cembratriene-diol formation in tobacco via manipulating not only the formation of trichome secretion, but also its composition, which involves the function of downstream bHLH and MYB transcription factors. However, NtMYC2a and NtMYB305 exhibit distinctive roles in regulating the cembratriene-diol formation and trichome secretion in tobacco.