- Split View
-
Views
-
Cite
Cite
Yan-Hong Xu, Yong-Cui Liao, Fei-Fei Lv, Zheng Zhang, Pei-Wen Sun, Zhi-Hui Gao, Ke-Ping Hu, Chun Sui, Yue Jin, Jian-He Wei, Transcription Factor AsMYC2 Controls the Jasmonate-Responsive Expression of ASS1 Regulating Sesquiterpene Biosynthesis in Aquilaria sinensis (Lour.) Gilg, Plant and Cell Physiology, Volume 58, Issue 11, November 2017, Pages 1924–1933, https://doi.org/10.1093/pcp/pcx122
- Share Icon Share
Abstract
Sesquiterpenes are one of the most important defensive secondary metabolite components of agarwood. Agarwood, which is a product of the Aquilaria sinensis response to external damage, is a fragrant and resinous wood that is widely used in traditional medicines, incense and perfume. We previously reported that jasmonic acid (JA) plays an important role in promoting agarwood sesquiterpene biosynthesis and induces expression of the sesquiterpene synthase ASS1, which is a key enzyme that is responsible for the biosynthesis of agarwood sesquiterpenes in A. sinensis. However, little is known about this molecular regulation mechanism. Here, we characterized a basic helix–loop–helix transcription factor, AsMYC2, from A. sinensis as an activator of ASS1 expression. AsMYC2 is an immediate-early jasmonate-responsive gene and is co-induced with ASS1. Using a combination of yeast one-hybrid assays and chromatin immunoprecipitation analyses, we showed that AsMYC2 bound the promoter of ASS1 containing a G-box motif. AsMYC2 activated expression of ASS1 in tobacco epidermis cells and up-regulated expression of sesquiterpene synthase genes (TPS21 and TPS11) in Arabidopsis, which was also promoted by methyl jasmonate. Our results suggest that AsMYC2 participates in the regulation of agarwood sesquiterpene biosynthesis in A. sinensis by controlling the expression of ASS1 through the JA signaling pathway.
Introduction
Agarwood is a non-timber dark resinous wood that is formed in the stems, branches and roots of Aquilaria and Gyrinops trees after they have been wounded (Ng 1997, Itoh et al. 2002, Pojanagaroon and Kaewrak 2005, Persoon, 2008). It is widely used in traditional medicines such as digestive, sedative and antiemetic drugs (China Pharmacopoeia Committee 2015), and is popular as incense and in perfumes in the Middle East, South Asia, Japan and China (Kumeta and Ito, 2010, Y.Y. Liu et al. 2013, Xu et al. 2013). In the international market, high-quality agarwood commands a higher price than gold. Aquilaria sinensis (Lour.) Gilg is one of the most important plant resources for producing agarwood in China, and it is also the only certified source of agarwood listed in the China Pharmacopoeia (China Pharmacopoeia Committee 2015). Sesquiterpenes are one of the major components in agarwood (Hashimoto et al. 1985, Chen et al. 2011, Chen et al. 2012), which is formed only when healthy Aquilaria trees are wounded. Like other sesquiterpene phytoalexins, agarwood sesquiterpenes are defensive compounds with antimicrobial and antidisease activity (Kumeta and Ito 2010, Chen et al. 2011) and only accumulate in response to elicitation or wound signals (Ito et al. 2005, Okudera and Ito 2009, Kumeta and Ito 2010, Chen et al. 2011, Xu et al. 2013).
Jasmonic acid (JA) and its derivates, collectively known as jasmonates (JAs), are essential signaling molecules that co-ordinate the plant defense response to biotic and abiotic challenges, in addition to multiple developmental processes (Wasternack 2007, Balbi and Devoto 2008, Browse and Howe 2008, Chico et al. 2008). Recent investigations hypothesized that, in the absence of JAs, jasmonate-ZIM domain proteins (JAZs) may repress expression of JA-responsive genes via their interactions with a series of transcription factors; when JA signaling is initiated, degradation of JAZs would disrupt these interactions, leading to activation of these transcription factors, which mediate various JA-regulated biological processes (Chini et al. 2007, Thines et al. 2007, Chico et al. 2008, Kazan and Manners 2008, Browse 2009, Pauwel et al, 2010). MYC2, a basic helix–loop–helix (bHLH)-type transcription factor, is a direct target of JAZs (Chini et al. 2007). As a master regulator in the JA signaling pathway, MYC2 both positively and negatively regulates diverse aspects of JA responses, including JA-mediated biosynthesis of secondary metabolites (for a review, see Kazan and Manners 2008, Kazan and Manners 2013).
Studies have shown that JA-induced biosynthesis of nicotine in tobacco (Todd et al. 2010, Shoji and Hashimoto 2011), sesquiterpenes in Arabidopsis (Hong et al. 2012, Ran et al. 2014), alkaloids in Catharanthus roseus (Zhang et al. 2011) and sakuranetin, a flavonoid antifungal phytoalexin in rice (Ogawa et al. 2017), are all mediated by the transcription factor MYC2. In tobacco, NtMYC2 binds to the G-box sequence in the promoter of the putrescine N-methyltransferase (PMT) gene, regulating the expression of nicotine biosynthesis genes (Todd et al. 2010). Meanwhile, NtMYC2 also activates an AP2/ERF transcription factor gene, ERF189, that directly regulates several JA-inducible nicotine biosynthesis genes (Shoji and Hashimoto 2011). In C. roseus, methyl jasmonate (MeJA)-responsive expression of alkaloid biosynthesis genes is controlled by a transcription factor cascade; CrMYC2 binds to the promoter and activates expression of the AP2/ERF-domain transcription factors ORCA2 and ORCA3, which in turn regulate a subset of alkaloid biosynthesis genes (Zhang et al. 2011). In rice, OsMYC2 drastically enhances the activity of the OsNOMT promoter and is reinforced by OsMYL1 and OsMYL2 by interacting with each other, resulting in the inductive production of sakuranetin in the JA signaling pathway (Ogawa et al. 2017). MYC2 also positively regulates terpene synthase genes, such as AtTPS10 and NbTPS1, two whitefly defense-related genes (Ran et al. 2014) and two sesquiterpene synthase genes, TPS21 and TPS11, in Arabidopsis inflorescence (Hong et al. 2012) by directly binding to their promoters. In Aquilaria, the induction role played by JA has also been widely demonstrated in recent years. Exogenously applied MeJA in Aquilaria cell suspension cultures or calluses induced biosynthesis and accumulation of sesquiterpene compounds, especially δ-guaiene (Ito et al. 2005, Okudera and Ito 2009, Kumeta and Ito 2010, Xu et al. 2013, Xu et al. 2016). According to our latest results, JA is the most crucial endogenous signal molecule in the accumulation of biosynthesized sesquiterpenes in A. sinensis (Xu et al. 2016). However, the molecular mechanism by which JA regulates sesquiterpene biosynthesis is largely unknown.
The first committed step of sesquiterpene biosynthesis is the cyclization of farnesyl diphosphate, which is catalyzed by sesquiterpene synthases. The genes for two of these enzymes, AcC1 and ASS1, have been independently identified in Aquilaria by two groups (Kumeta and Ito 2010, Xu et al. 2013). Both genes encode enzymes that convert farnesyl diphosphate into δ-guaiene in addition to a few minor products such as α-guaiene and α-humulene. In healthy Aquilaria calluses or cell cultures, expression of the δ-guaiene synthase ASS1 was barely detectable, whereas in wounded tissues or MeJA-treated calluses, its expression increased significantly, and the sesquiterpene compound content correspondingly increased (Xu et al. 2013). These results demonstrate that ASS1 is the key enzyme that is responsible for the biosynthesis of agarwood sesquiterpene, whose expression is regulated at the transcriptional level by JA and wound signals.
In this study, to reveal the mechanism by which JA induces expression of ASS1 and agarwood sesquiterpene biosynthesis, we focused on the transcription factor AsMYC2, the master regulator in the JA signaling pathway in A. sinensis. Overexpression of AsMYC2 in Arabidopsis up-regulates expression of sesquiterpene synthase genes (TPS21 and TPS11) and partly rescues their expression and the MeJA responsiveness of the myc2-2 mutant. AsMYC2 binds the promoter of ASS1, which contains a G-box motif, and activates its expression in response to MeJA. These data suggest that AsMYC2 participates in the regulation of agarwood sesquiterpene biosynthesis by positively regulating ASS1 expression at the transcriptional level through a JA signaling pathway.
Results
Isolation of AsMYC2 from A. sinensis
MYC2, a key downstream component of the JA signaling pathway, has been reported as a master that regulates most JA-responsive genes (Kazan and Manners 2008, Kazan and Manners 2013). The importance of JA signaling in regulating sesquiterpene biosynthesis and ASS1 expression in Aquilaria spp. (Ito et al. 2005, Okudera and Ito 2009, Kumeta and Ito 2010, Xu et al. 2013, Xu et al. 2016) prompted us to investigate the function of MYC2 in A. sinensis. Based on the partial sequences of unigenes in A. sinensis transcriptome data (Xu et al. 2013), the full-length cDNA of AsMYC2 was obtained using the 5′/3′-RACE (rapid amplification of cDNA ends) method (accession No. KP677282). It was found to encode a 673 amino acid protein with a predicted molecular mass of 73.5 kDa and a pI of 5.53 (Fig. 1A). The search for conserved domains in the AsMYC2 protein against the NCBI Conserved Domain Database showed that AsMYC2 contains an HLH domain in its carboxyl region and a bHLH_MYC_N domain (Fig. 1A). By aligning multiple amino acid sequences, an unrooted Neighbor–Joining tree was constructed, showing that AsMYC2 is most homologous (73%) with the Theobroma cacao bHLH protein (Fig. 1B).
Analysis of AsMYC2 characteristics
A putative nuclear localization signal (NLS) in AsMYC2 is indicated in Fig. 1A. It was previously reported that C-terminal NLSs are responsible for targeting to the nucleus (Hedhili et al. 2010). To confirm that AsMYC2 is a nuclear protein in A. sinensis, the open reading frame (ORF) of the AsMYC2 gene was fused to the N-terminus of a green fluorescent protein (GFP) reporter gene under the control of the Cauliflower mosaic virus (CaMV) 35 S promoter. The recombinant constructs of the AsMYC2–GFP fusion and GFP alone were individually introduced into onion epidermal cells by gold particle bombardment. As shown in Fig. 2A, the AsMYC2–GFP fusion protein was specifically localized in the nucleus, whereas GFP alone was ubiquitously distributed in both the nucleus and the cytoplasm.
Next, we preliminarily elucidated the function of AsMYC2 by analyzing its expression patterns in the roots, stems, leaves and branches of 4-year-old A. sinensis trees. Quantitatiuve real-time PCR (RT-PCR) experiments showed that AsMYC2 was expressed in all the analyzed tissues (Fig. 2B), with the highest transcript level, approximately eight times higher than that in leaves, occurring in stems; roots expressed the second highest amount, four times higher than that in leaves (Fig. 2B). These data indicated that AsMYC2 is mainly accumulated and functions in stems and roots during the development of Aquilaria trees. Coincidentally or not, agarwood is mainly formed in stems and roots of wounded Aquilaria trees, implying that there may be a positive correlation in different tissues between AsMYC2 expression and the active principle content of agarwood.
JA signaling is regulated by some protein complexes. MYC2, as a master regulator in JA signaling, is recruited by JAZ repressors to form COI1–JAZ–MYC2 complexes and inhibit MYC2-targeted gene transcripts (Thines et al. 2007, Chico et al. 2008, Kazan et al. 2008). To demonstrate whether there is an interaction between AsJAZ1 and AsMYC2 proteins in A. sinensis, we performed pull-down experiments using recombinant purified glutathione S-transferase (GST)–AsMYC2 and His-AsJAZ1 fusion proteins. As shown in Fig. 2C, AsJAZ1 could be pulled down by the GST–AsMYC2 fusion protein (lane 1), and AsMYC2 could also be pulled down by His-AsJAZ1 fusion protein (lane 3), demonstrating that there is an interaction between AsJAZ1 and AsMYC2 proteins.
AsMYC2 is co-induced with ASS1 by MeJA
Because ASS1 transcription could be activated by mechanical wounding and MeJA (Xu et al. 2013), we would expect the inducible expression of AsMYC2 if it regulates ASS1. Therefore, we analyzed its expression in response to MeJA exposure. Examination by RT-PCR revealed that AsMYC2 was significantly up-regulated by MeJA treatment (Fig. 3), showing two peaks within a 24 h window, with the first peak at 0.5 h and the second peak at 4 h; this second peak was followed by a slow decline and finally returned to normal at 24 h. Comparatively, the sesquiterpene biosynthesis gene ASS1 showed a slower rate of induction, its transcriptional level was not significantly elevated until approximately 1 h post treatment and it reached its peak expression at 6 h (Fig. 3). Thus, the transcript level of AsMYC2 increased rapidly and transiently upon elicitation, preceding ASS1 induction, which suggested that AsMYC2 is an immediate-early jasmonate-responsive gene in A. sinensis and is co-induced with ASS1.
Overexpression of AsMYC2 up-regulates expression of sesquiterpene synthase genes TPS21 and TPS11 in Arabidopsis
To determine further whether AsMYC2 is a regulator of sesquiterpene biosynthesis genes, we transformed the AsMYC2 gene into wild-type Arabidopsis (marked as 35 S-AsMYC2) and AtMYC2 knockout mutant myc2-2 Arabidopsis (marked as 35 S-AsMYC2-myc2-2) and then analyzed the expression of the sesquiterpene synthase genes TPS21 and TPS11, which are responsible for the formation of virtually all Arabidopsis floral volatile sesquiterpenes (Tholl et al. 2006, Hong et al. 2012). As shown in Fig. 4, in the myc2-2 mutant, the knocking out of AtMYC2 down-regulates the expression levels of TPS11 and TPS21, and the mutant showed a MeJA-insensitive phenotype in the MeJA-induced promotion of TPS11 and TPS21 expression; induction by MeJA was almost lost compared with the response in the wild type, demonstrating that AtMYC2 is essential for MeJA-responsive expression of TPS11 and TPS21. Interestingly, overexpression of AsMYC2 partially rescued the expression of TPS11 and TPS21 to wild-type levels and restored their inducibility by MeJA. Meanwhile, in the 35 S-AsMYC2 transgenic lines, AsMYC2 increased the transcription levels of TPS11 and TPS21 2-fold, and MeJA treatment significantly induced their expression (Fig. 4). These data provided genetic evidence that the function of AsMYC2 is similar to that of AtMYC2 in Arabidopsis: specifically, it regulates the expression of sesquiterpene synthase genes through a JA signaling pathway (Hong et al. 2012).
AsMYC2 binds the promoter of ASS1 containing a G-box motif and activates its expression
To test whether AsMYC2 binds to the ASS1 promoter in vitro, a yeast one-hybrid assay was performed. It was shown that AsMYC2 interacts with the promoter of ASS1, as the cells grow well on SD medium lacking tryptophan, leucine and histidine (Fig. 5B). Previous in vitro and in vivo binding assays showed that MYC2 binds directly to the G-box cis-element (CACGTG) and G-box-like motifs (AACGTG or CATGTG or CACATG) (Boter et al. 2004, Dombrecht et al. 2007, Hou et al. 2010, Hong et al. 2012, Li et al. 2014). In a broader sense, E-box elements (CANNTG) serve as binding sites of bHLH transcription factors (Chaudhary and Skinner 1999). Coincidentally, six E-box (CANNTG) motifs were found in the promoter of ASS1 (PASS1) and thus may be the binding site of AsMYC2. To investigate the binding sites of AsMYC2 in the ASS1 promoter, a chromatin immunoprecipitation (ChIP) assay was performed, followed by RT-PCR. In the ChIP assay assessing AsMYC2–PASS1 interactions, three domains in the ASS1 promoter were isolated (Supplementary Table S1): domain I (–973 to –731) covers two E-boxes (CACCTG and CAAGTG), domain II (–707 to –436) covers one E-box (CATTTG) and domain III (–256 to –47) covers three E-boxes (CACGTG, CAAGTG and CAGGTG). Our results demonstrated that AsMYC2 binds domain III (Fig. 5C, D), as the experiment showed that ChIP with either anti-acetyl-histone 3 or anti-AsMYC2 antibody detected the ASS1 promoter amplification products (Fig. 5C, lanes 2 and 4). However, almost no products were observed in ChIP assays with anti-rabbit IgG (Fig. 5C, lane 1). Samples of DNA that was not immunoprecipitated displayed a bright lane (Fig. 5C, lane 3) and thus served as a control to determine ChIP efficiency. ChIP-qPCR analysis also illustrated that AsMYC2 strongly binds to the ASS1 promoter that encompasses the G-box motifs; in contrast, no interaction was observed between AsMYC2 and the ASS1 promoter without the G-box motifs (Fig. 5D). These results suggested that AsMYC2 binds to ASS1 promoter that contains the G-box motifs.
We tested whether AsMYC2 regulates the promoter activity of the ASS1 gene using an in vivo system that involved co-transforming tobacco leaves with AsMYC2 and the ASS1 promoter linked to GFP (PASS1–GFP). The result showed that AsMYC2 also interacts with the ASS1 promoter in vivo and activates its expression, as reported by the GFP fluorescence. GFP fluorescence from tobacco leaves that had been infiltrated by both 35 S:AsMYC2 and PASS1-GFP recombinant plasmids was stronger than the fluorescence of leaves with PASS1–GFP alone (Fig. 6). In addition, applications of exogenous MeJA specifically promoted their interaction, as epidermal cells of tobacco leaves with these applications showed the strongest fluorescence (Fig. 6).
Discussion
Agarwood, a type of precious and rare traditional Chinese medicinal material and a highly valued worldwide resource, is a resinous heartwood produced by wounded Aquilaria spp. trees, and sesquiterpenes are one of its major components (Hashimoto et al. 1985, Chen et al. 2011, Chen et al. 2012). It has been widely demonstrated that JA plays important roles in agarwood sesquiterpene biosynthesis in A. sinensis (Okudera and Ito 2009, Kumeta and Ito 2010, Xu et al. 2013), but whether there is a direct relationship between JA and this process is unknown, as is the exact regulatory mechanism of the JA signaling pathway that is involved in agarwood sesquiterpene biosynthesis.
As extensively reported, primary signal transduction processes following JA perception converge on related bHLH transcription factors. Of these, the best characterized and most multifunctional is MYC2. Considering the important role of MYC2 in JA signaling and plant secondary metabolism (for a review, see Kazan and Manners 2013), as well as the positive role of JA in agarwood production (Ito et al. 2005, Okudera and Ito 2009, Kumeta and Ito 2010, Xu et al. 2013, Xu et al. 2016), it is interesting to ask whether the transcription factor MYC2 regulates the expression of sesquiterpene synthase in A. sinensis.
In this study, we first isolated and characterized the transcription factor AsMYC2 from A. sinensis. We provided several lines of evidence that AsMYC2 is a positive regulator of ASS1 in the JA signaling pathway. First, AsMYC2 is an immediate JA-responsive gene that is co-induced with ASS1 and is mainly expressed in the stems and roots of A. sinensis, where agarwood is usually formed (Figs. 2B, 3), demonstrating that it is likely to function mostly in these parts and might be a regulator of ASS1. Secondly, the expression of sesquiterpene synthase genes TPS11 and TPS21 in Arabidopsis is up-regulated by overexpression of AsMYC2 in the myc2-2 mutant and partly restores their response to MeJA (Fig. 4), which provides genetic evidence that AsMYC2 has a function similar to that of AtMYC2 in regulating sesquiterpene synthase genes through the JA signaling pathway. Thirdly, yeast one-hybrid and ChIP assays showed that AsMYC2 directly binds the ASS1 promoter, which contains a G-box motif (Fig. 5). Lastly, in tobacco leaves co-transformed with both 35 S-AsMYC2 and ASS1 native promoter–GFP constructs, we observed that AsMYC2 specifically activates expression of ASS1 in vivo and MeJA promoted this process (Fig. 6). Thus, the present experiments allow us to identify AsMYC2 as a positive regulator that targets ASS1 and promotes its expression in A. sinensis.
Additionally, pull-down assays indicated that there exists an interaction between AsJAZ1 and AsMYC2 in vitro (Fig. 2C), which is consistent with the previously described working model in which in the absence of JA, MYC2 is recruited by JAZ repressors to form COI1–JAZ–MYC2 complexes and inhibit MYC2-targeted gene transcripts (Thines et al. 2007, Chico et al. 2008, Kazan et al. 2008). As ASS1 is the enzyme most responsible for the biosynthesis of agarwood sesquiterpenes and its expression is regulated at the transcriptional level by JA (Xu et al. 2013), we speculate a model, based on the data here, in which AsMYC2 regulates ASS1 expression (Fig. 7): in healthy A. sinensis, AsMYC2 is repressed by the AsJAZ1 protein without targeting ASS1, but the expression of ASS1 itself is very low and it may not even be expressed, resulting in a lack of synthesis of agarwood sesquiterpenes; in wounded A. sinensis, with the biosynthesis of endogenous JA, AsMYC2 is released and directly targets and activates ASS1 expression, leading to biosynthesis of agarwood sesquiterpenes.
Materials and Methods
Plant materials and growth conditions
Arabidopsis thaliana ecotype Columbia (Col-0) and the myc2-2 mutant were used in the generation of transgenic plants. The plants were grown in a growth chamber at 19–20°C on Murashige and Skoog (MS) medium (Sigma) at approximatley 80 µmol photons m–2 s–1 or in compost soil at approximately 120 µmol photons m–2 s–1 over a 16 h photoperiod. The tobacco Nicotiana benthamiana was cultivated in a greenhouse at 22°C and 1 µmol photons m–2 s–1 over a 16 h photoperiod. The A. sinensis calli were grown in MS medium at 25 °C in the dark.
AsMYC2 cloning with the RACE method
Total RNA was isolated from A. sinensis calluses using the Total RNA Purification Kit (Aidlab), supplemented with on-column DNA digestion according to the manufacturer’s instructions. Single-stranded cDNA was synthesized from total RNA according to the SMART™ cDNA Library Construction Kit (Clontech) protocols. Using this cDNA as a template and based on the unigenes of 454 data (Xu et al. 2013), 5′-RACE and 3′-RACE were performed with AsMYC2-specific primers, following the manufacturer’s instructions. The primers 3′-RACE CDS Primer A (3′-PA) and 5′-RACE CDS Primer A (5′-PA) were used as the primers to synthesize the 3′ and 5′ first-strand cDNAs, respectively. Gene-specific primers targeting AsMYC2 were designed based on the transcriptome sequence. Antisense primers AsMYC2-5′GSP1 (5′-GGGTCGTTCTCGCCCTGATCCGGATTC-3′) and AsMYC2-5′GSP2 (5′-CAGGAACG CCTGACCCGGTAATCCCG-3′) were synthesized for 5′-RACE, and sense primers AsMYC2-3′GSP1 (5′-CGCCTTGCGTGCTGTGGTCCCAAATGTG-3′) and AsMYC2-3′GSP2 (5′-CCCGGCCGCAAGACTCATGGCTGCATTG-3′) were synthesized for 3′-RACE. These two types of primers were both paired with 10 × Universal Primer A Mix (UPM) to amplify the 5′ and 3′ cDNA ends. The Nested Universal Primer A (NUP) was used as the nested primer. The PCR procedure employed Touchdown-PCRs: 9°C for 4 min and then 95°C for 30 s, 70°C for 30 s and 72°C for 90 s for the first cycle; the annealing temperature decreased by 1°C per cycle. After 10 cycles, the conditions were changed to 94°C for 30 s, 60°C for 30 s and 72°C for 90 s for 20 cycles. The duration of the 72°C elongation step was 10 min. The PCR product was cloned into the pGM-T vector (TIANGEN) and then sequenced.
Real-time PCR analysis
Total RNA was isolated using a Total RNA Rapid Extraction kit (BioTeke), then treated with RNase-free DNase I (TAKARA) at 37°C for 30 min to degrade genomic DNA, and purified using an RNA Purification kit (BioTeke). A 2 µg aliquot of RNA was subjected to first-strand cDNA synthesis using Moloney murine leukemia virus reverse transcriptase (Promega) and an oligo(dT)18 primer. Real-time PCRs were performed with specific primers: for AsMYC2, forward 5′-ATGCATGCCATCGTCCAATGG-3′ and reverse 5′-CCAGAAGCCGATGCGATTC-3′; for ASS1, forward 5′-CAGACATACAAGGCTGAAGAAAAG-3′ and reverse 5′-TTCTATCTTTGGTCACACCTTGG-3′; for TPS11, forward 5′-CACTTTGGGACAACGACAGA-3′ and reverse 5′-CTTGGAAGTAATGAAGCTGCAAG-3′; and for TPS21, forward 5′-TCGCCTTGGTGTCT CCTATCAC-3′ and reverse 5′-CTTTGAACTTCCCATTTTCGTCC-3′. The A. sinensis Tubulin gene (forward 5′-GCCAAGTGACACAAGCGTAGGT-3′ and reverse 5′-TCCTTGCCAGAAATAAGTTGCTC-3′) was used as an internal control. Analysis was performed using a Bio-Rad Real-Time System CFX96TM C1000 Thermal Cycler (Singapore).
Generation of AsMYC2-overexpressing plants
To generate a 35 S:AsMYC2 construct, the coding sequence of the AsMYC2 gene was amplified with the primer pair (AsMYC2-LF, 5′-gcTCTAGAATGACCGATTACAGGCTCCC-3′; and AsMYC2-LR, 5′-cgGGATCCTTACCTTG CATCCCCCACTTTG-3′) and inserted into a plant binary vector pBI121 at the restriction sites XbaΙ/BamHΙ. The recombined plasmids were then introduced into Agrobacterium tumefaciens (LBA4404) and then transformed into A. thaliana plants using the floral dip method (Clough and Bent 1998). Arabidopsis thaliana ecotype Col-0 and the AtMYC2 knockout mutant myc2-2 were used in the generation of transgenic plants. The homozygous T3 seeds of the transgenic plants were used for analysis.
In vitro pull-down assays
Coding sequences of full-length AsMYC2 and AsJAZ1 genes were PCR-amplified from A. sinensis cDNA templates, cloned into pGM-T and recombined in pGEX-4 T-1 or PET-28a to obtain GST–AsMYC2 and His-AsJAZ1 fusion proteins. Recombinant plasmids were expressed in Escherichia coli BL21 (DE3), and the fused proteins were purified according to standard protocols. For GST pull-down experiments, 200 µg of GST–AsMYC2 fusion protein or GST protein was added to 1 mg of His-AsJAZ1 fusion protein and incubated for 12 h at 4°C with rotation. After the samples were washed and denaturalized, the proteins were loaded on 10% SDS–polyacrylamide gels, transferred to nitrocellulose membranes and incubated with anti-His [HRP] mouse polyclonal antibody (Beijing ComWin Biotech Co., Ltd.). Similarly, for His pull-down experiments, 50 µg of His-AsJAZ1 fusion protein or His protein was added to 1 mg of GST–AsMYC2 fusion protein and incubated for 12 h at 4°C with rotation. After the samples were washed and denaturalized, they were loaded on 8% SDS–polyacrylamide gels, transferred to nitrocellulose membranes and incubated with anti-GST [HRP] mouse polyclonal antibody (Beijing ComWin Biotech Co., Ltd.).
AsMYC2–ASS1 promoter interaction tested with yeast one-hybrid assay
Yeast one-hybrid assays were performed as described (R. Liu et al. 2013). The ASS1 promoter was amplified by PCR using the following primer pairs: forward primer 5′-ATTGTCCGCCGCACCTGAG-3′ and reverse primer 5′-TCGGCGGCTAACATCTTCG-3′. The promoter DNA fragment was subcloned into the EcoRI/MluI sites of a pHIS2 vector. The one-hybrid assays were performed using the Y187 yeast strain according to the manufacturer’s instructions. Yeast cells were co-transformed with a pHIS2 bait vector harboring the promoter of ASS1 and a pGADT7 prey vector harboring the ORF of AsMYC2. As negative controls, the yeast cells were co-transformed with the combination of pGADT7-MYC2 and an empty pHIS2 vector, an empty pGADT7 vector and pHIS2 harboring the ASS1 promoter, or two empty vectors, pGADT7 and pHIS2. Transformed yeast cells were first grown in SD–Trp–Leu medium to ensure that the yeast cells were successfully co-transformed, and the co-transformed yeast cells were then grown on SD–Trp–Leu–His medium plates. The SD–Trp–Leu and SD–Trp–Leu–His media were supplemented with 25 mM 3-amino-1,2,4-triazole (Sigma). The plates were then incubated for 3 d at 30 °C.
ChIP assay
For ChIP analyses, leaves (1.5 g) were cross-linked with 37 ml of formaldehyde in a vacuum for 20 min. A 2.5 ml aliquot of 2 M glycine was added to stop the cross-linking. The leaves were rinsed with water and then ground to powder with liquid nitrogen, and chromatin was extracted. The samples were then sonicated four times for 10 s at 20% power and kept on ice for 1 min during each interval to achieve an average fragment size ranging from 0.2 to 1.0 kb. Before precipitation, 1 : 10 dilutions of the supernatant were reserved as the ‘input fraction’. Appropriate antibodies were added to each diluted sample, and each sample was then incubated overnight at 4°C with gentle rotation. The polyclonal antibody against MYC2 was mouse anti-MYC2 serum, and anti-acetyl-histone 3 and normal mouse IgG were used as positive and negative controls, respectively. Protein A–agarose beads (Beyotime Biotechnology) were added to the immune complex and vortexed to mix. This step was followed by standing the samples on a magnetic stand for 1 h, after which the supernatant was discarded. The beads were washed for 3–5 min each time with gentle rotation at 4°C and then centrifuged so that the supernatants could be discarded. The complexes were then eluted from the beads and centrifuged at 4°C. The elution step was repeated once, and the elution products were combined. Subsequently, 5 M NaCl was added, and the combined sample was incubated at 65°C overnight to reverse cross-linking. RNase was then added, and the sample was incubated at 37°C for 15 min. The protein was digested, and the DNA was purified using a DNA fragment purification kit (Genview). Finally, the DNA fragment concentration was determined by semi-quantitative PCR and quantitative RT-PCR using primers specifically to amplify sequences that contained the G-box (from –256 to –47) in the ASS1 gene promoter (forward, 5′-ACAGCCCACGTGGTCATACAAG-3′; reverse, 5′-CAAGTTTGCTGTTTTGAGCGATG-3′) and the sequences outside the G-box (from –731 to –568) (forward, 5′-TGATGCGTATTTGTTCTTTCTTTTC-3′; reverse, 5′-TTGAATGGTATGAGAACCCGAAG-3′). The sequences of the three domains that were used in the ChIP assay are listed in Supplementary Table S1.
Transient expression assays in tobacco
Transient expression analysis was conducted by agroinfiltration of tobacco leaves according to the method of Yang et al. (2000). Six-week-old tobacco plants were used for infiltration. The recombinant plasmids containing GFP1 and AsMYC2 were individually transformed into the A. tumefaciens strain GV3101. Agrobacterium tumefaciens cells at an appropriate concentration (OD600 = 0.6) were collected and then resuspended in tobacco infiltration buffer (100 mM MES, 100 mM MgCl2 and 10 mM acetosyringone, pH 5.7). In total, 100 µl of each bacterial suspension was infiltrated into the intercellular spaces of intact leaves using a 1 ml needleless syringe. After infiltration, tobacco plants were first grown in darkness for 12 h and then maintained in a growth chamber at 25°C for 60 h. The epidermis of the agroinfiltrated tobacco leaves was used for fluorescence observation via confocal microscopy (OLYMPUS V-TV0.5XC-3 n).
Supplementary data
Supplementary data are available at PCP online.
Abbreviations
- bHLH
basic helix–loop–helix
- ChIP
chromatin immunoprecipitation
- GFP
green fluorescent protein
- GST
glutathione S-transferase
- JA
jasmonic acid
- JAZ
jasmonate-ZIM domain protein
- MeJA
methyl jasmonate
- MS
Murashige and Skoog
- ORF
open reading frame
- RACE
rapid amplification of cDNA ends
- RT-PCR
real-time PCR
Acknowledgments
We are grateful to Professor Hong-Wei Guo (Peking University) for providing the myc2-2 mutant, and to Dr Fu-Qing Wu (Chinese Academy of Agriculture Science) for providing the GFP vector pAN580. Y.X. participated in the design of the study, performed the experiments, analyzed the data and drafted the manuscript. Y.L. and F.L. performed the experiments, and participated in the design of the study and analysis of the data. Z.Z. helped to analyze the data. Y.J. and P.S. prepared the A sinensis cell suspension. K.H. helped in making the monoclonal antibody of AsMYC2 against mice. Z.G. and C.S. participated in the discussion of the results and helped to revise the manuscript. J.W. initiated the project, helped conceive the study and revised the manuscript.
Funding
This work was supported by the National Natural Science Foundation of China [81573525 and 81173539] and the Major Science and Technology Project of Hainan Province [ZDKJ2016004].
Disclosures
The authors have no conflicts of interest to declare.