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. 2021 Sep 27;188(1):151–166. doi: 10.1093/plphys/kiab458

NtMYB305a binds to the jasmonate-responsive GAG region of NtPMT1a promoter to regulate nicotine biosynthesis

Shiquan Bian 1, Xueyi Sui 2, Jiahao Wang 1, Tian Tian 1, Chunkai Wang 1, Xue Zhao 1, Xiaofeng Liu 1, Ning Fang 1, Yu Zhang 1, Yanhua Liu 1, Yongmei Du 1, Bingwu Wang 2, Michael P Timko 3, Zhongfeng Zhang 1, Hongbo Zhang 1,✉,2
PMCID: PMC8774768  PMID: 34601578

Abstract

MYB transcription factors play essential roles in regulating plant secondary metabolism and jasmonate (JA) signaling. Putrescine N-methyltransferase is a key JA-regulated step in the biosynthesis of nicotine, an alkaloidal compound highly accumulated in Nicotiana spp. Here we report the identification of NtMYB305a in tobacco (Nicotiana tabacum) as a regulatory component of nicotine biosynthesis and demonstrate that it binds to the JA-responsive GAG region, which comprises a G-box, an AT-rich motif, and a GCC-box-like element, in the NtPMT1a promoter. Yeast one-hybrid analysis, electrophoretic mobility shift assay and chromatin immunoprecipitation assays showed that NtMYB305a binds to the GAG region in vitro and in vivo. Binding specifically occurs at the ∼30-bp AT-rich motif in a G/C-base-independent manner, thus defining the AT-rich motif as previously unknown MYB-binding element. NtMYB305a localized in the nucleus of tobacco cells where it is capable of activating the expression of a 4×GAG-driven GUS reporter in an AT-rich motif-dependent manner. NtMYB305a positively regulates nicotine biosynthesis and the expression of NtPMT and other nicotine pathway genes. NtMYB305a acts synergistically with NtMYC2a to regulate nicotine biosynthesis, but no interaction between these two proteins was detected. This identification of NtMYB305a provides insights into the regulation of nicotine biosynthesis and extends the roles played by MYB transcription factors in plant secondary metabolism.

NtMYB305a binds to the AT-rich motif of the jasmonate-responsive GAG region in the NtPMT1a promoter in a G/C-base-independent manner to regulate nicotine biosynthesis in tobacco.

Introduction

Plants produce numerous secondary metabolites throughout their developmental processes and in response to environmental changes (Wink, 2003; Wasternack and Strnad, 2019; Hatcher et al., 2020). Members of the Solanaceae synthesize a set of alkaloidal compounds as defense against insect herbivores (Bozorov et al., 2017; Zenkner et al., 2019). Nicotine is the most important and most abundant alkaloidal compound in Nicotiana spp., accounting for ˃90% of the total alkaloids in cultivated tobacco (Nicotiana tabacum L.; Saitoh et al., 1985; Dewey and Xie, 2013). In tobacco, nicotine is synthesized in the roots and transferred to the aerial organs through the xylem (Dawson 1941; Baldwin 1989). Production of nicotine is elicited by wounding, insect attack, and the phytohormone jasmonate (JA) (Imanishi et al., 1998; Shi et al., 2006; Bozorov et al., 2017; Li et al., 2018; Liu et al., 2020). The nicotine molecule is composed of a pyrrolidine ring and a pyridine ring. The pyrrolidine ring is formed from putrescine derived from ornithine under the catalyzation of ornithine decarboxylase (ODC) or from arginine catalyzed by arginine decarboxylase (ADC) (Chattopadhyay and Ghosh, 1998; Dalton et al., 2016). The conversion of putrescine to N-methylputrescine by putrescine N-methyltransferase (PMT) is the first committed step for nicotine biosynthesis (Hibi et al., 1994; Xu and Timko, 2004). N-methylputrescine is oxidized and cyclized to the l-methyl-Δ1-pyrrolinium cation in sequential steps, and then condensed with nicotinic acid derived from the pyridine nucleotide cycle under the catalyzation of quinolinate phosphoribosyltransferase (QPT) to form nicotine (Wagner et al., 1986; Sinclair et al., 2000). The NADPH-dependent PIP reductase A622 and the berberine bridging enzyme-like (BBL) proteins are supposed to catalyze the condensation of pyrrolidine and pyridine rings (Deboer et al., 2009; Kajikawa et al., 2009, 2011; Lewis et al., 2020). The majority of nicotine pathway genes, including ODC, PMT, QPT, A622, and BBL, are inducible by increase in endogenous JA content or the application of exogenous JA (Imanishi et al., 1998; Shoji et al., 2000; Deboer et al., 2009; Kajikawa et al., 2011; Dewey and Xie, 2013), which indicates that JA plays an important role in regulating nicotine biosynthesis.

In plants, bioactive JA-derivative jasmonoyl-isoleucine (JA-Ile) is perceived by a complex composed of CORONATINE INSENSITIVE 1 (COI1) and JA-ZIM domain (JAZ) proteins in cooperation with an inositol pentakisphosphate cofactor (Sheard et al., 2010; Wang et al., 2019). Basic helix–loop–helix (bHLH) transcription factors (e.g. AtMYC2/3) and R2R3-MYB proteins (e.g. AtMYB21/24) are two types of JA-signal transducers that interact with JAZ repressor proteins (Song et al., 2011; Qi et al., 2015; Liu et al., 2019). In the presence of JA-Ile, JAZ repressors are recruited to the proteasomal degradation system by the JA-Ile receptor, thus releasing the bHLH and MYB transcription activators and enhancing JA responses (Song et al., 2011; Qi et al., 2015). The bHLH transcription factors bind to G/E-box elements, and MYB transcription factors bind to MYB-core [C/T]NGTT[G/T] and AC-rich elements (Prouse and Campbell, 2012; Millard et al., 2019), which requires the presence of a certain number of G/C bases. MYB transcription factors are characterized by the conserved MYB DNA-binding domain and regulate the production of multiple secondary metabolites in plants (Millard et al., 2019). For instance, thale cress (Arabidopsis thaliana) AtMYB75 regulates JA-mediated anthocyanin accumulation (Qi et al., 2011), AtMYB12/111 controls flavonoid synthesis (Li and Zachgo, 2013), AtMYB51/122 mediates glucosinolate biosynthesis (Frerigmann and Gigolashvili, 2014; Mitreiter and Gigolashvili, 2021), and Vitis vinifera MYB14/15 regulates stilbene biosynthesis (Höll et al., 2013). MYB transcription factors are also involved in regulation of primary metabolism (Liu and Thornburg, 2012; Schubert et al., 2019), and some MYB transcription factors may cooperate with bHLHs by protein–protein interaction to regulate primary and secondary metabolism in plants (Frerigmann and Gigolashvili, 2014; Qi et al., 2015; Yang et al., 2020). In nicotine biosynthesis, tobacco NtMYC2s regulate this process under the control of JA-Ile receptor proteins (Shoji and Hashimoto, 2011; Zhang et al., 2012), but the involvement of MYB transcription factor in regulating nicotine biosynthesis remains unclear.

Previous studies on JA-induced gene expression of NtPMTs identified a composite regulatory region in the NtPMT1a promoter that comprised a G-box, an AT-rich motif, and a GCC-box-like element. This region has been termed the GAG region and has been shown to be required for JA induction of NtPMT1a (Xu and Timko, 2004; Sears et al., 2014). Transcriptional regulators binding to the GAG region at the G-box were identified as bHLH proteins. For example, Nicotiana benthamiana NbbHLH1/2 and N. tabacum NtMYC2s are G-box-binding bHLH proteins that positively regulate the expression of nicotine pathway genes (Todd et al., 2010; Shoji and Hashimoto, 2011; Zhang et al., 2012). Transcriptional regulators binding to the GCC-box-like element of the GAG region were identified as ethylene response factors (ERFs). For example, NbERF1, ORC1/JAP1, and NtERF32 bind to the GCC-box-like element of GAG and regulate nicotine biosynthesis (De Sutter et al., 2005; Todd et al., 2010; Sears et al., 2014; Liu et al., 2019). The NIC2 locus that controls nicotine biosynthesis was identified to be a cluster of ERFs (ERF189 and ERF221/ORC1) that bind to the GAG region at the GCC-box-like element (Shoji et al., 2010; Hayashi et al., 2020). However, the role of the AT-rich motif of the GAG region and its function in the regulation of NtPMT1a expression remains unclear.

In this study, we identified the R2R3-MYB transcription factor NtMYB305a as a transcriptional regulator that directly binds to the GAG region in the NtPMT1a promoter, the most highly JA-induced NtPMT homolog (Xu and Timko, 2004), in a yeast one-hybrid (Y1H) assay. Mutation assays showed that NtMYB305a interacts with the AT-rich motif of the GAG region in the NtPMT1a promoter and the presence of G/C bases is unnecessary for this interaction. NtMYB305a is a positive regulator of nicotine biosynthesis in tobacco and acts synergistically with NtMYC2a in regulating nicotine biosynthesis. The present findings provide important information for unraveling the regulatory mechanism underlying nicotine biosynthesis in tobacco.

Results

Isolation of NtMYB305a as GAG-binding factor by Y1H

We previously screened a number of cDNA libraries constructed from tobacco RNAs in yeast (Saccharomyces cerevisae) using Y1H assays, a powerful tool for isolating DNA-binding proteins, in order to isolate transcriptional regulators binding to the GAG region of NtPMT1a promoter. These studies only identified several ERFs with weak GAG-binding activity (Sears et al., 2014).

Recently, we constructed a yeast cDNA library with mixed mRNAs, including mRNAs from plants expressing NtMYB305a and NtMYC2a, whose Arabidopsis homologs AtMYB21/24 and AtMYC2 play important roles in controlling JA signaling (Thines et al., 2007; Song et al., 2011), to identify JA-responsive GAG-binding factors. The 4×GAG yeast reporter, constructed using yeast strain YM4271 and GAG tetramer-driven pHISi-1 and pLacZi vectors, was employed for the Y1H assays (Figure 1A). A total of 11 colonies were obtained in this screening that grew on the high stringency selection medium (SD/–His/–Ura/–Leu plus 45 mM 3-amino-1,2,4-triazole [3-AT]) and the subsequent assays revealed that they all contained a plasmid carrying the NtMYB305a sequence (Figure 1B). Further assays showed that the expression of AD-NtMYB305a, a fusion protein of GAL4 activation domain (AD) and NtMYB305a, allowed the 4×GAG yeast reporter to grow on the selection medium (SD/–His/–Ura/–Leu plus 45 mM 3-AT) and activated the expression of β-galactosidase (lacZ) (Figure 1C). In contrast, expression of only AD in the 4×GAG reporter or expression of AD-NtMYB305a in the control reporter (constructed with empty pHISi-1 and pLacZi vectors) did not enable growth of the 4×GAG yeast reporter. These results showed that NtMYB305a is a GAG-binding protein with strong GAG-binding activity in yeast, which was unexpected because the GAG region lacks a known MYB-binding element (Prouse and Campbell, 2012; Millard et al., 2019).

Figure 1.

Figure 1

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Isolation of the GAG-binding protein NtMYB305a by Y1H. A, Structures of the GAG tetramer-driven pHISi-1 and pLacZi vectors for Y1H reporter construction. B, Number of GAG-binding transcription factors obtained in yeast cDNA library screening by Y1H assays. C, Serial dilution growth assays of transformed yeast on the selection medium SD/-His/-Ura/-Leu with or without 45 mM 3-AT. Effector and reporter are indicated at left. Colonies grown on the selection medium without 3-AT were subjected to β-galactosidase activity determination by filter-lift assay (right).

Expression pattern and subcellular localization of NtMYB305a

By searching the genomic data accessible at the Sol Genomics Network and the GenBank of National Center for Biotechnology Information (NCBI), we identified four homologs of NtMYB305a (designated as NtMYB305a.1–2, and NtMYB305b.1–2) in the genome of N. tabacum cv TN90. These homologs were classified into two groups based on sequence similarity and exhibit 60% amino acid sequence identity to Arabidopsis AtMYB21/24 and tomato SlMYB2, and >90% sequence identity to ornamental tobacco LxS_MYB305 (Figure 2A;Supplemental Figures S1 and S2). In contrast, they show ˂40% identity to homologs in monocot species, such as maize ZmMYB305 and rice OsMYB305 (Figure 2A). Semi-quantitative reverse transcription-PCR (RT-PCR) analysis showed that the amplification products for NtMYB305a were much more abundant than those of NtMYB305b in all tissues analyzed from 16-week-old plants, and that each homolog was expressed at similar levels in the leaves, stems, and roots (Figure 2A). Further reverse transcription-quantitative PCR (RT-qPCR) analyses suggested that NtMYB305a/b are expressed at slightly higher levels in the stems than in the leaves and roots (Figure 2B). The expression level of NtMYB305a/b in tobacco roots increased ˃30-fold by exogenous methyl JA (MeJA) treatment and ˃10-fold by topping treatment, respectively, during a 24-h treatment period (Figure 2, C and D). To determine the subcellular localization of NtMYB305a/b proteins in tobacco, the NtMYB305a and NtMYB305b coding regions were cloned as in-frame with the yellow fluorescent protein (YFP) gene and the resulting constructs NtMYB305a-YFP and NtMYB305b-YFP were transiently expressed in tobacco leaves. The YFP protein gene was transiently expressed alone as a control. The fluorescent signals of the NtMYB305a-YFP and NtMYB305b-YFP fusion proteins were predominantly observed in the nucleus, whereas YFP signal was detected in the nucleus and cytoplasm, which suggested that NtMYB305a/b are nucleus-localized proteins (Figure 2E).

Figure 2.

Figure 2

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Gene expression and subcellular localization of NtMYB305a/b. A, Spatial expression pattern of NtMYB305a/b and the phylogenetic tree of their homologs. Gene expression was determined by RT-PCR. Scale bar in the phylogenetic tree represents the number of amino acid substitutions per site. Gene accessions at GenBank: NtMYB305a.1 (LOC107821652), NtMYB305a.2 (LOC107763989), NtMYB305b.1 (LOC107765438), NtMYB305b.2 (LOC107818761), AtMYB21 (At3g27810), AtMYB24 (At5g40350), LxS-MYB305 (EU111679), OsMYB305 (AK111807), and ZmMYB305 (EU960450). Gene accession at the Sol Genomics Network: SlMYB21 (Solyc02g067760). B, Quantification of the spatial expression level of NtMYB305a/b by RT-qPCR. Data are presented as means ± sd (n = 3). Expression in leaf was set as “1.” C and D, Relative expression of NtMYB305a/b in tobacco roots upon MeJA (C) and topping (D) treatment. Data are presented as means ± sd (n = 3). Expression in the sample at “0” time point of each treatment was set as “1.” E, Subcellular localization of NtMYB305a/b in tobacco. YFP is used as control. Scale bars indicate 50 μm.

Taken together, these findings show that NtMYB305a/b is JA- and topping-induced, nucleus-localized transcription factors in tobacco. Sequence alignment indicates that NtMYB305a and NtMYB305b have a high amino acid sequence identity within the DNA-binding domains and self-ADs identified in orthologous proteins of other species (Supplemental Figure S2), implying that these proteins may perform similar functions in tobacco. Given the significantly higher abundance of NtMYB305a transcripts in tobacco roots, we selected it for further study.

Interaction of NtMYB305a with the GAG region in tobacco

The in vivo interaction of NtMYB305a with the GAG region was investigated using chromatin immunoprecipitation (ChIP) and transcription activation assays. ChIP assays were performed using roots from transgenic plants expressing HA-tagged NtMYB305a (NtMYB305a-HA) and expression in tobacco roots was verified by western blotting (Supplemental Figure S3). The abundance of ChIP-enriched DNA fragments in the NtPMT1a promoter was assessed by means of a ChIP-qPCR assay, using amplification of ChIP-enriched NtActin as a control. A higher ratio (∼1%) of GAG-containing DNA was enriched from the NtMYB305a-HA expressing plants, whereas the DNA enrichment for regions upstream of the GAG region in the NtPMT1a promoter was ˂0.3% and for NtActin it was ∼0.1% (Figure 3A). These results provide further evidence of the in vivo interaction of NtMYB305a and the GAG-flanking region of the NtPMT1a promoter.

Figure 3.

Figure 3

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In vivo interaction between NtMYB305a and GAG composite. A, Amplification of ChIP-enriched DNAs of the NtPMT1a promoter. The positions of primers for ChIP-qPCR amplifications are indicated (top). The amplification of NtActin DNA was used as a control. B, Transactivation of NtMYB305a on the NtPMT1a promoter-driven GUS reporter. Left part shows the representative GUS staining of tobacco seedlings. Scale bars indicate 1 mm. Right part shows the quantification of GUS activity in the corresponding samples. C and D, Transactivation of NtMYB305a on the 4×GAG-driven GUS reporter. Top part of (C) shows the structure of reporter vectors (GAG-GUS, 4×GAG-driven GUS reporter; GG-GUS, 4×GG-driven GUS reporter; TATA, 35S TATA box). Bottom part of (C) shows the representative GUS staining of tobacco seedlings. Scale bars indicate 1 mm. Quantification of GUS activity in the corresponding samples is shown in (D). Data in (A, B, and D) are shown as means ± sd (n = 3). Asterisks indicate significant difference to the control (**P < 0.005, Student’s t test).

To determine the capability of NtMYB305a to transactivate the NtPMT1a promoter, the NtMYB305a-expressing vector and control effector vector were each co-transformed into 20-d-old tobacco seedlings via Agrobacterium-mediated infection along with a β-glucuronidase (GUS) reporter driven by the NtPMT1a promoter. GUS staining and GUS activity assays showed that Agrobacterium-mediated transient transformation of the NtMYB305a-expressing effector resulted in darker GUS staining and a three-fold higher GUS activity than the infection with that containing control effector (Figure 3B) confirming the capability of NtMYB305a to activate transcription of the NtPMT1a promoter-driven GUS reporter gene. To further determine the specific transactivity of NtMYB305a on the JA-responsive GAG region of the NtPMT1a promoter, we constructed a reporter vector (4×GAG-GUS) by cloning a tetramer of GAG upstream of the Cauliflower mosaic virus (CaMV) 35S TATA box (−46 to +8 bp) to drive GUS, and a control reporter (4×GG-GUS) using a tetramer of a GAG mutant with the AT-rich motif deleted (GG) (Figure 3, C and D). The 4×GAG-GUS and 4×GG-GUS reporters were, respectively, co-transformed with the NtMYB305a-expressing effector or the control effector into 20-d-old seedlings via Agrobacterium-mediated infection. Much darker GUS staining and a 10-fold higher GUS activity were observed in seedlings co-transformed with the 4×GAG-GUS reporter and the NtMYB305a-expressing effector, compared with seedlings co-transformed with the 4×GAG-GUS reporter and the control effector vector (Figure 3, C and D). No distinct increase in GUS activity was observed under co-transformation with the 4×GG-GUS reporter and the NtMYB305a-expressing effector, compared with that of the corresponding control (Figure 3, C and D). These results suggest that NtMYB305a activates the 4×GAG-driven GUS reporter in an AT-rich motif-dependent manner.

In vitro interaction between NtMYB305a and the AT-rich motif of the GAG region

To determine the specific NtMYB305a-binding site within the GAG region, an electrophoretic mobility shift assay (EMSA) was employed to analyze the in vitro interaction between GAG and 6×His-tagged NtMYB305a (NtMYB305a-His) expressed by Escherichia coli. In the EMSA with DIG-labeled GAG, a band shift was observed upon addition of the NtMYB305a-His protein and a stronger band shift was detected by addition of greater quantities of the protein (Figure 4, A and B), which provided evidence for NtMYB305a–GAG interaction in vitro. Next, the interaction of NtMYB305a-His with deletion mutants of GAG was analyzed. Deletion of the G-box or GCC-box-like element has no effect on the in vitro interaction, whereas deletion of the AT-rich motif (i.e. ΔATL, ΔATM, or ΔATR; including three bases at the junction of GCC-box-like element) abolished the interaction (Figure 4, A and C). In the competition assay, the unlabeled GAG mutant with deletion of the whole AT-rich motif has no effect on the NtMYB305a–GAG interaction, whereas partial deletion of the AT-rich motif (from either the left or right side) shows a suppression effect on this interaction, and the unlabeled native GAG could compete off most of the NtMYB305a-bound GAG at an amount 10-fold that of DIG-labeled GAG (Figure 4D). These findings suggest that the AT-rich motif is critical for NtMYB305a–GAG interaction.

Figure 4.

Figure 4

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In vitro interaction between NtMYB305a and the AT-rich motif of GAG. A, Sequences of GAG and its mutants. The region for deletion mutation is shown in pale gray, and the nucleotide for base replacement is shown in lowercase. Blue-dotted bases at the junction of GCC-box-like element are included in the mutation assays. B, EMSA for DIG-labeled GAG and different amounts of NtMYB305a-His. C, EMSA for NtMYB305a-His and GAG mutants with indicated deletion. D, Competition of NtMYB305a-GAG binding by GAG mutants with indicated deletion in EMSA. E, EMSA for NtMYB305a-His and GAG mutant with complementary base replacement. F, EMSA for NtMYB305a-His and GAG mutants with different G/C replacement. G, EMSA for NtMYB305a-His and the AT-rich mutants of mere A/T bases. The EMSA of NtMYB305a-His and native GAG probe served as a positive control in (C–G).

Interestingly, almost complete replacement of the AT-rich motif with complementary bases had no obvious impact on NtMYB305a-binding activity (Figure 4E). This result was consistent with the previous finding that replacement of the AT-rich motif with complementary bases did not alter the responses to JA (Sears et al., 2014). Subsequently, a more precise mutagenesis of the AT-rich motif was performed to identify the crucial bases determining the NtMYB305a–GAG interaction. We observed that replacement of single or multiple G/C bases with A/T bases in the AT-rich motif had no obvious effect on the NtMYB305a binding activity (Figure 4F). These results imply that NtMYB305a may bind to DNA fragments composed entirely of A/T bases. To verify this hypothesis, we determined the interaction of NtMYB305a with 31-bp and 19-bp AT-rich fragments derived from the GAG region and used a 31-bp complementary poly(A)/poly(T) fragment as a control (Figure 4, A and G). The results show that NtMYB305a could bind to the 31-bp AT-rich fragment, but not the 19-bp fragment or the 31-bp complementary poly(A)/poly(T) fragment (Figure 4, A and G). These findings suggest that NtMYB305a interacts with the GAG region at the AT-rich motif and may bind to ∼30-bp AT-rich motif lacking G/C bases, which differs from the currently known MYB-binding elements (Prouse and Campbell, 2012; Millard et al., 2019).

Regulation of nicotine biosynthesis by NtMYB305a

The role of NtMYB305a in regulating nicotine biosynthesis was investigated by measuring the nicotine content in the leaves of NtMYB305a-overexpression plants (NtMYB305a-HA) generated previously (Sui et al., 2018) and in RNAi-mediated gene-silenced plants (NtMYB305a-RI) exhibiting over 70% suppression of NtMYB305a transcription (Figure 5A). Consistent with the findings on Arabidopsis AtMYB21/24 (Song et al., 2011), the NtMYB305a-RI plants are sterile and must be propagated by crossing with wild-type plants. The NtMYB305a-RI seedlings are less sensitive to MeJA (5 μM) treatment than control seedlings in a root elongation assay, whereas NtMYB305a-HA seedlings showed sensitivity to MeJA like that of the control (Figure 5B). In accordance with the overexpression of NtMYB305a (Figure 5A), nicotine content in the leaves of NtMYB305a-HA plants before topping treatment was increased by almost 40% compared with that of control plants (transformed with empty vector; Figure 5C). These results indicate that NtMYB305a plays a positive role in regulating nicotine biosynthesis. Silencing of NtMYB305a resulted in a ˃50% decrease in nicotine content before topping treatment (Figure 5C). We subsequently determined the nicotine contents in NtMYB305a-HA, NtMYB305a-RI, and control plants 2 weeks after topping treatment. We observed an ∼30% increase in the nicotine content in NtMYB305a-HA plants and an ∼40% decrease in NtMYB305a-RI plants compared to the control plant levels (Figure 5C). The determination of NtMYB305b expression level in NtMYB305a-RI plants showed no significant decrease compared with that of control plants (Supplemental Figure S4), even though it is highly similar to NtMYB305b in sequence (Supplemental Figure S1). Presumably, this has resulted from the low abundance of NtMYB305b in the plant (Figure 2A). Taken together, these findings suggest that NtMYB305a plays a positive role in regulating nicotine biosynthesis in tobacco.

Figure 5.

Figure 5

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Regulation of nicotine biosynthesis by NtMYB305a. A, Expression levels of NtMYB305a in NtMYB305a-HA (OE), NtMYB305a-RI (RI), and control (Ctrl) plants. B, MeJA sensitivity of NtMYB305a-HA, NtMYB305a-RI, and control plant seedlings. C, Nicotine contents in the leaves of NtMYB305a-HA (OE), NtMYB305a-RI (RI), and control (Ctrl) plants before topping and 2 weeks after topping. Data in (A and C) are shown as means ± sd (n = 3). Asterisks indicate significant difference (*P < 0.05; Student’s t test) from that of control.

Regulation of nicotine pathway genes by NtMYB305a

To determine the effect of NtMYB305a on genes in the nicotine biosynthetic pathway, RT-qPCR was carried out to determine the relative expression levels of NtPMT, NtODC, NtADC, NtQPT, NtA662, and NtBBL in NtMYB305a-HA, NtMYB305a-RI, and control plants. In the roots of 5-week-old tobacco seedlings, NtPMT expression was enhanced by overexpression of NtMYB305a and attenuated by suppression of NtMYB305a expression (Figure 6A). MeJA treatment resulted in an over three-fold increase in NtPMT expression in NtMYB305a-HA plants, whereas the JA induction of NtPMT was abolished in NtMYB305a-RI plants (Figure 6A). These results support that NtMYB305a is a positive regulator of NtPMT expression. The expression of NtODC, NtADC, and NtQPT was not affected by NtMYB305a overexpression but was suppressed by silencing of NtMYB305a in the roots of 5-week-old tobacco seedlings before or after MeJA treatment (Figure 6A). The expression patterns of NtA622 and NtBBL are similar to that of NtPMT except that no change in the expression of NtA622 was observed in NtMYB305a-RI plants without MeJA treatment (Figure 6A). These results suggested that NtMYB305a positively regulates JA-mediated expression of genes functioning at different steps of nicotine biosynthesis. In the roots of 16-week-old tobacco plants before topping treatment, the expression of NtPMT, NtODC, NtA622, and NtBBL, but not NtQPT and NtADC, is increased in NtMYB305a-HA plants, while these genes are all suppressed in NtMYB305a-RI plants (Supplemental Figure S5). The expression of nicotine pathway genes is differentially enhanced in NtMYB305a-HA and NtMYB305a-RI plants after 6 h or 12 h of topping treatment, whereas the expression of NtPMT, NtA622, and NtBBL is enhanced to a higher level in NtMYB305a-HA plants after 12 h of topping treatment (Supplemental Figure S5). Yet, most of these genes are at lower expression levels in NtMYB305a-RI plants than in control (Supplemental Figure S5). These results further support that NtMYB305a is a positive regulator of nicotine biosynthesis.

Figure 6.

Figure 6

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Regulation of nicotine pathway genes by NtMYB305a. A, Relative expression of nicotine pathway genes in the roots of 5-week-old NtMYB305a-HA (OE), NtMYB305a-RI (RI), and control (Ctrl) plant seedlings with or without MeJA treatment. The expression of each gene in the untreated control was set as “1.” Data are shown as means ± sd (n = 3). Asterisks indicate significant difference from control of the same treatment (*P < 0.05, **P < 0.005; Student’s t test). B–E, Interaction assay of NtMYB305a and AT-rich motifs in the promoters of indicated nicotine pathway genes by EMSA (C) and ChIP-qPCR (D). Locations of NtMYB305a-binding AT-rich motifs (orange blocks) and the ChIP-qPCR primers are indicated in (B), and the relative locations of other AT-rich motifs are indicated with gray blocks. Probes of AT-rich motifs in EMSA are indicated by the gene names. EMSA of NtMYB305-His and the AT-rich motif of GAG region in the NtPMT1a promoter served as a positive control. The sequence logo of NtMYB305a-binding AT-rich motifs is shown in (E). Data of ChIP-qPCR are shown as means ± sd (n = 3). Asterisks indicate significant difference from the control amplification of NtActin (P < 0.005, Student’s t test).

The above findings prompted us to explore the potential NtMYB305a-binding AT-rich motifs in the promoters of nicotine pathway genes other than NtPMT. By analyzing the promoter sequences of NtODC, NtADC1, NtQPT1, NtA662, and NtBBLa, regions that show similarity to the AT-rich motif of GAG region (i.e. ∼30-nt length with >27 A/T bases) were selected within the 1,000-bp region upstream of the ATG codons for protein–DNA binding assays, except for NtADC1 whose 5′-untranslated region is over 400 bp. Approximately 2–6 this type of AT-rich regions were found in the promoter of each nicotine pathway gene, and those could be bound by NtMYB305a were determined in both EMSA and ChIP-qPCR assays (Figure 6, B–D). In each of these promoters, one AT-rich motif was observed to be bound by NtMYB305a in EMSA assay (Figure 6, B and C; Supplemental Table S1) and had the flanking DNA enriched at a proportion ∼0.7% in ChIP-qPCR assay (Figure 6D). In comparison, the DNA enrichment for NtActin is ˂0.1% (Figure 6D). These results suggest an interaction between NtMYB305a and the promoters of nicotine pathway genes other than NtPMT. Using the MEME Suite (https://meme-suite.org), the consensus NtMYB305a-binding sequence was characterized by sequence analysis of these NtMYB305a-binding AT-rich motifs (Figure 6E), which indicates an essential contribution of the intermediate A bases and the two-side flanking T bases to the interaction with NtMYB305a. These findings suggest that NtMYB305a could directly regulate the expression of these genes.

NtMYB305a acts synergistically with NtMYC2a in regulating nicotine biosynthesis

NtMYB305a is a homolog of Arabidopsis AtMYB21/24, which interacts with the bHLH transcription factor AtMYC2 to regulate JA-mediated responses (Song et al., 2011; Qi et al., 2015). We, therefore, explored the involvement of the bHLH transcription factor NtMYC2a in NtMYB305a-mediated nicotine biosynthesis by comparing the gene expression and nicotine accumulation in plants expressing NtMYB305a, NtMYC2a, or both proteins. Transcription assay indicates that the overexpression levels of NtMYB305a and NtMYC2a in the hybrid plants are similar to that in plants overexpressing either of them (Figure 7A). In the gene expression assay, a synergistic effect on the expression of nicotine synthetics genes NtPMT, NtADC, NtQPT, NtA622, and NtBBL was observed by co-expression of NtMYB305a and NtMYC2a compared with the effect of their individual expression, but this effect was not observed on the expression of NtODC (Figure 7B). In the plants without topping treatment, the nicotine content is ∼12 μg/mg in control plants, ∼16 μg/mg in the plants expressing either NtMYB305a-HA or NtMYC2a-HA, and ∼22 μg/mg in the plants expressing both proteins (Figure 7C). Thus, the co-expression of NtMYC2a-HA with NtMYB305a-HA resulted in a ∼38% increase in nicotine content compared with that of plants expressing either protein, supporting a synergistic action on the nicotine biosynthesis. After topping treatment, the nicotine content is ∼19 μg/mg in the control plants, ∼28 μg/mg in the plants expressing either NtMYB305a-HA or NtMYC2a-HA, and ∼32 μg/mg in the plants expressing both proteins (Figure 7C), showing a weaker synergistic effect than that before topping treatment. Presumably, an unknown feedback mechanism may operate.

Figure 7.

Figure 7

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Synergistic effect of NtMYB305a and NtMYC2a on nicotine biosynthesis. A, Expression level of NtMYB305a- and NtMYC2a-transgene in plants expressing NtMYB305a-HA, NtMYC2a-HA, or both proteins (Hybrid). B, Relative expression of nicotine pathway genes in the roots of 5-week-old plant seedlings expressing NtMYB305a-HA, NtMYC2a-HA, or both proteins. The expression of each gene in the control was set as “1.” C, Nicotine contents in the leaves of plants expressing NtMYB305a-HA, NtMYC2a-HA, or both proteins with and without topping treatment. Data in (A–C) are shown as means ± sd (n = 3), and the asterisks indicate significant difference both from plants expressing NtMYB305a-HA and those expressing NtMYC2a-HA (*P < 0.05, **P < 0.005; Student’s t test). D and E, Protein interaction analyses of NtMYB305a and NtMYC2a by Y2H (D) and BiFC assays (E). SD/-2 and SD/-4 in Y2H assay indicate SD/–Leu/–Trp medium and SD/–Ade/–His/–Leu/–Trp medium (with X-α-Gal), respectively. The expression of NtMYB305a and NtMYC2a proteins in yeast was determined by western blotting (bottom part of (D); M indicates protein marker). The protein interaction assays for NtMYC2a and NtJAZ1 serve as a positive control. The negative controls of BiFC assay are not shown. The scale bars in (E) indicate 50 μm.

The potential interaction between NtMYB305a and NtMYC2a was assessed by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays using the interaction of NtMYC2a and NtJAZ1 as a positive control. In the Y2H assay, the self-AD deletion variants of NtMYB305a and NtMYC2a were used as bait due to their self-activation activity (Supplemental Figure S6), and their full-length proteins were used as prey. The Y2H assays showed no interaction between NtMYB305a and NtMYC2a, although their protein could be successfully expressed in yeast (Figure 7D). We also did not detect a physical interaction between the two proteins using BiFC assays (Figure 7E). These results suggest that the regulatory pattern in tobacco may be different from that observed in Arabidopsis.

Discussion

In plants, MYB transcription factors play important roles in regulating the biosynthesis of secondary metabolites, such as flavonoids, terpenoids, and stilbenes, and some MYB family members also interact with JAZ proteins and bHLH transcription factors to mediate JA signaling critical in plant secondary metabolism (Wasternack and Strnad, 2019). In this study, we reveal that NtMYB305a is capable of binding to the JA-responsive GAG region of the NtPMT1a promoter and regulates nicotine biosynthesis in tobacco, extending our understanding of MYB-mediated regulation of plant secondary metabolism.

Prior studies have established that MYB transcription factors physically interact with specific promoter elements in various functional genes to regulate plant secondary metabolism (Millard et al., 2019). For example, pea MYB26 binds to the P-box element (GTTAGGTT) to regulate flavonoid biosynthesis (Uimari and Strommer, 1997), the poplar R2R3-MYB transcription factor MYB134/6 binds to the AC element (ACCTACC or ACCTNNC) to mediate proanthocyanidin formation (Mellway, et al., 2009; Wang et al., 2019), Freesia hybrida MYB21 binds to the AC element (CAACCG) to control terpene metabolism (Yang et al., 2020), ornamental tobacco MYB305 binds to the AC element (CACCTAA) to modulate primary metabolism (Liu and Thornburg, 2012), chrysanthemum CmMYB15 binds to AC elements to regulate lignin synthesis (An et al., 2019), and citrus CiMYB42 binds to the MYB-core (TTGTTG) element to regulate limonoid formation (Zhang et al., 2020). In summary, previous studies indicate that plant R2R3-MYB transcription factors bind to MYB-core [C/T]NGTT[G/T] and AC-rich elements, which contain a certain number of G/C bases, to regulate the metabolism in plants (Prouse and Campbell, 2012; Millard et al., 2019). The present work shows that NtMYB305a regulates nicotine biosynthesis and binds to the JA-responsive GAG region in the NtPMT1a promoter. Analysis of the interaction between NtMYB305a and the GAG region indicates that NtMYB305a directly binds to the ∼30-bp AT-rich motif of the GAG region in a G/C-bases-independent manner. These results have revealed an additional DNA-binding pattern among MYB transcription factors.

The JA-signaling pathway is indispensable in the regulation of nicotine biosynthesis and controls the expression of crucial nicotine pathway genes, such as ODC, PMT, QPT, A622, and BBL, whose expression is inducible by increase in endogenous JA content or application of exogenous JA (Imanishi et al., 1998; Shoji et al., 2000; Deboer et al., 2009; Kajikawa et al., 2011). PMT is the rate-limiting enzyme of nicotine biosynthesis (Hibi et al., 1994; Dewey and Xie, 2013). Previous studies of PMT expression identified the JA-responsive GAG region in NtPMT promoters, which comprises a G-box, an AT-rich motif, and a GCC-box-like element that are all essential for JA-mediated induction (Xu and Timko, 2004; Sears et al., 2014). bHLH transcription factors, such as NbbHLH1/2 from N. benthamiana and NtMYC2s from N. tabacum, are G-box-binding regulators of nicotine biosynthesis (Todd et al., 2010; Shoji and Hashimoto, 2011; Zhang et al., 2012), and ERF transcription factors, including NbERF1, ORC1/JAP1, NtERF32, and NIC2-locus regulators (ERF189 and ERF221/ORC1), are GCC-box-like-binding regulators of nicotine biosynthesis (De Sutter et al., 2005; Todd et al., 2010; Sears et al., 2014; Paul et al., 2020; Shoji and Yuan, 2021). This study identifies NtMYB305a as a regulator of nicotine biosynthesis that binds to the AT-rich motif in the GAG region of the NtPMT1a promoter, and provides a further clue to unraveling the molecular modulation of the GAG composite and JA-mediated regulation of nicotine biosynthesis. NtMYB305a has a strong GAG-binding activity in Y1H assay, which implies that NtMYB305a may have a higher biological affinity to bind to the GAG region in vivo and may play a critical role in manipulating the nicotine biosynthesis. This finding also helps establish a regulatory module with similarity to Arabidopsis MYB transcription factors that cooperates with bHLHs to regulate plant metabolic processes (Qi et al., 2011; Frerigmann and Gigolashvili, 2014; Yang et al., 2020). The ChIP-qPCR, EMSA, and transactivation assays all show that NtMYB305a not only binds to the AT-rich motif of GAG in NtPMT1a promoter but also binds to AT-rich motifs in the promoters of other genes in the nicotine biosynthetic pathway, supporting a direct role for NtMYB305a in the regulation of multiple nicotine pathway genes in tobacco.

In Arabidopsis, AtMYB21/24 (homologs of NtMYB305a) interacts with and cooperates with bHLH transcription factors to mediate JA responses (Song et al., 2011; Liu and Thornburg, 2012; Wang et al., 2014; Qi et al., 2015; Wasternack and Strnad, 2019). A similar modulator has been observed in other plant species. For example, Gerbera hybrida GMYB10 interacts with bHLH factor GMYC1 to control the expression of anthocyanin biosynthetic genes (Elomaa et al., 2003), Arabidopsis AtMYB21 interacts with AtMYC2 to regulate the expression of terpene synthase genes in flowers (Yang et al., 2020), Arabidopsis MYB51 interacts with bHLH05 to modulate glucosinolate biosynthesis (Frerigmann and Gigolashvili, 2014), and Arabidopsis MYB75 interacts with bHLHs to regulate JA-mediated anthocyanin accumulation (Qi et al., 2011). The current results indicate that NtMYB305a is an activator of the expression of nicotine pathway genes and acts synergistically with NtMYC2a in the regulation of nicotine biosynthesis. The transcription assays showed that NtMYB305a positively regulates the expression of nicotine pathway genes including NtPMT, NtA622, NtBBL, NtODC, NtADC, and NtQPT, and its co-expression with NtMYC2a exerted differential synergistic effects on the expression of these genes. However, no interaction between these two proteins was detected in Y2H or BiFC assays. These results imply that the function pattern of NtMYB305a and NtMYC2a in tobacco may differ from that of AtMYC2 and AtMYB21/24 in Arabidopsis, or that an unknown factor may be involved in their synergistic regulation of nicotine biosynthesis. Moreover, the change of nicotine accumulation by overexpression or silencing of NtMYB305a is in a limited extent, and this may imply the requirement of other strong activators (e.g. bHLHs and AP2/ERFs) in cooperating with NtMYB305a to control the nicotine biosynthesis in tobacco.

Taken together, the present results provide evidence that NtMYB305a is a regulator of nicotine biosynthesis in tobacco. A molecular connection between NtMYB305a and the JA-responsive GAG composite region of the NtPMT1a promoter involving specific interaction with the AT-rich motif was revealed. In addition, NtMYB305a binds to the AT-rich motifs in the promoters of other nicotine pathway genes and cooperates with NtMYC2a to regulate nicotine biosynthesis in tobacco.

Materials and methods

Plant materials and growth conditions

Tobacco (N. tabacum L. cv TN90) was used for gene expression assays and transgenic plant development. The binary vectors and plants expressing HA-tagged NtMYB305a (NtMYB305a-HA) and HA-tagged NtMYC2a (NtMYC2a-HA) were generated previously (Sui et al., 2018). These plants were used to produce hybrid plants expressing both NtMYB305a-HA and NtMYC2a-HA. To construct the vector for RNAi-mediated gene silencing of NtMYB305a, ∼300-bp coding sequence of NtMYB305a (Supplemental Figure S1) was amplified with primer 5′-TGATAATGGAACTGCATGCTAA-3′ and 5′-ATCGCCGTTAAGCAATTGCAT-3′, cloned into the pENTR-D-TOPO vector (Invitrogen, Carlsbad, CA, USA), and integrated into a Gateway-compatible plant RNAi vector modified by replacing the gene expression module of pBin19-attR-YFP (Subramanian et al., 2006) with the RNAi cassette from pHZPRi–Hyg (Zhang et al., 2012) using Gateway cloning method. The vector carrying the NtPMT1a promoter-driven β-glucuronidase (GUS) reporter was constructed by cloning the sequence of the NtPMT1a promoter upstream of the GUS gene in pBT10–GUS vector (Sprenger-Haussels and Weisshaar, 2000). The obtained binary vectors were introduced into Agrobacterium tumefaciens LBA4404 and then used to transform tobacco plants as described previously (Wang et al., 2014). NtMYB305a-RI plants were propagated by crossing wild-type tobacco plants with pollen grains from the dehisced anthers of NtMYB305a-RI plant (the naturally abscised anthers were incubated to dehiscence in covered petri dishes).

Tobacco plants were grown in an indoor growth room at 23°C with a 14-h light/10-h dark photoperiod. For transcriptional analyses with MeJA treatment, tobacco seeds after sterilization were germinated on 1/2 Murashige and Skoog (MS) medium with the desired antibiotics for 1  week, then cultured in liquid 1/2 MS medium (refreshed each week) for 4 more weeks, and then treated with fresh liquid 1/2 MS medium containing 100 μM MeJA for the specified period to collect root samples. The root elongation assay to estimate JA sensitivity was performed as previously described (Wang et al., 2014).

For measurement of nicotine contents, tobacco plants were grown in soil in pots to flowering stage (about 16-week-old). Leaves at the middle internodes were collected before or after topping treatment for nicotine extraction. Root samples were collected at the same time to determine the expression of nicotine pathway genes.

Y1H assay

To construct the yeast reporter for isolating GAG-binding factors, a GAG tetramer was synthesized (Supplemental Table S2) and cloned into the pHISi-1 and pLacZi vectors (Clontech, San Jose, CA, USA), respectively. The obtained pHISi-1-GAG and pLacZi-GAG vectors were integrated into yeast strain YM4271 (Clontech, Mountain View, CA, USA) and cultured on SD/–His/–Ura selection medium at 30°C to generate the yeast GAG reporter, which was then used to screen yeast cDNA libraries constructed with tobacco mRNAs using a BD Matchmaker Library Construction & Screening Kit (Clontech, Mountain View, CA, USA) in accordance with the manufacture’s instruction. The selective medium SD/–His/–Ura/–Leu supplemented with 45 mM 3-AT was used for cDNA library screening.

To evaluate the specific interaction between NtMYB305a and the GAG region, a control yeast reporter was constructed with pHISi-1 and pLacZi empty vectors. The coding sequence of NtMYB305a was amplified with gene-specific primers (Supplemental Table S2) and cloned into pGADT7-Rec2 vector (Clontech, Mountain View, CA, USA) for the Y1H assay, with the empty vector used as a control. The NtMYB305a-expressing vector and pGADT7-Rec2 empty vector were separately transformed into the yeast GAG reporter or the control reporter. Yeast transformants were cultured on SD/–His/–Ura/–Leu selection medium and the positive colonies were cultured in the liquid SD/–His/–Ura/–Leu medium before serial dilution and spotting onto the selection medium SD/–His/–Ura/–Leu supplemented with the specified amount of 3-AT. The colonies that developed on the selection medium SD/–His/–Ura/–Leu were subjected to a filter-lift assay for determination of β-galactosidase activity, which was imaged after 30 min of incubation at 30°C.

Sequence alignment

The tobacco homologs of NtMYB305a were retrieved from both the GenBank of National Center for Biotechnology Information (NCBI) and the Sol Genomics Network (https://solgenomics.net) by performing a BLAST search using the protein sequence of the previously cloned NtMYB305a (GenBank accession: KC792284) as the query. The homologs of NtMYB305a in Arabidopsis, ornamental tobacco, rice, and maize were retrieved from the GenBank of NCBI, and that in tomato was retrieved from the Sol Genomics Network. A phylogenetic tree was constructed with the MEGA version 7.0 software using the deduced protein sequences of NtMYB305a and other homologous genes. A multiple DNA sequence alignment for NtMYB305a homologs from tobacco was generated with Clone Manager version 8.0. The multiple sequence alignment for NtMYB305a and other homologous proteins was generated with DNAMAN version 6.0.

Gene expression analyses

Total RNAs were extracted from samples for gene expression analyses with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). The RNAs were reverse-transcribed into cDNAs using the PrimeScript 1st Strand cDNA Synthesis Kit (Takara, Shiga, Japan) in accordance with the manufacturer’s instructions. Semi-quantitative RT-PCR reactions were performed (with 30 cycles for NtMYB305a/b) using cDNAs transcribed from the same amount of total RNAs and separated in 1.0% (w/v) agarose gel. NtActin was amplified (with 26 cycles) as an internal control. RT-qPCR was performed with the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) on a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). NtActin was amplified as an internal control (Zhang et al., 2012). The relative expression level was calibrated using the 2−ΔΔCt algorithm as described previously (Zhang et al., 2012). All experiments were performed with three independent biological replicates. Data in the figures are presented as the mean ± sd. Statistical significance was evaluated using one-way analysis of variance followed by a two-tailed Student’s t test. Differences considered statistically significant are indicted by *P < 0.05, **P < 0.005. The RT-qPCR primers used for each nicotine pathway gene were designed based on regions highly conserved among the homologs. Accessions of genes subjected to transcription assays and the corresponding primers used in the RT-PCR and RT-qPCR assays are listed in Supplemental Table S3. The overexpression of NtMYB305a and NtMYC2a in the transgenic plants was amplified with primer 5′-AGCATTCTACTTCTATTGCAGC-3′ that is specific to a transcribable vector region and gene-specific primers 5′-CCCACTTAGCATGCAGTTCCA-3′ for NtMYB305a and 5′-TCCACGTCTCACGAGCACCA-3′ for NtMYC2a.

Subcellular localization assay

To determine the subcellular localization of NtMYB305a and NtMYB305b, vectors for expressing the YFP-tagged fusion proteins (NtMYB305a-YFP and NtMYB305b-YFP) were constructed, respectively. The coding sequence of NtMYB305a was amplified with primers 5′-AAACCGCGGATGGATAAAAAACCATGCAAC-3′ and 5′-ATCGCCGTTAAGCAATTGCAT-3′, cloned into the pENTR-D-TOPO vector, and then integrated into the 2 × 35S-promoter carrying binary vector pBin19-attR-YFP (Subramanian et al., 2006) using the Gateway cloning method as described above. The coding sequence of NtMYB305b was amplified with primers 5′-AAACCGCGGATGGATAAAAGAACATGCAA-3′ and 5′-GTTGGTTGCATCATTAAGCA-3′, and cloned into pBin19-attR-YFP using the same method. A vector for expressing YFP alone was modified from the pBin19-attR-YFP vector to serve as a control. The obtained binary vectors were introduced into A. tumefaciens strain GV3101, cultured in yeast extract beef (YEB) medium at 28°C to OD600 = 1.0 (the optical density at 600 nm), and then resuspended in infiltration buffer (10 mM MES-KOH pH 5.5, 10 mM MgCl2, 100 μM acetosyringone) to infiltrate the N. tabacum leaves with a needleless syringe. The agro-infiltrated plants were cultured in an indoor growth room for 48 h. Fluorescence was observed under a Leica TCS-SP8 confocal microscope (Leica Microsystems, Germany) with excitation at 488 nm (solid-state laser) and emission in the range 520–540 nm for YFP, and with excitation at 552 nm (solid-state laser) and emission in the range 650–760 nm for chlorophyll.

ChIP assay

ChIP assays were performed as previously described (Liu et al., 2019). Root samples (∼1.5 g) from 20-d-old NtMYB305a-HA expressing plants were collected and fixed in 1% (v/v) formaldehyde for 10 min under vacuum. Chromatin preparation, shearing, and pre-clearing with single-stranded DNA (ssDNA)/protein G agarose were performed as previously described with necessary modification. The chromatin was immunoprecipitated with anti-HA antibody (Roche, Mannheim, Germany) and ssDNA/protein G agarose. The DNA enriched by immunoprecipitation was used to amplify the specified DNA regions by qPCR. A fragment of NtActin was amplified as an internal control. The ChIP-qPCR primers used to amplify the different promoter regions and the control DNA are listed in Supplemental Table S1.

Transactivation assay

To determine the capacity of NtMYB305a to transactivate the NtPMT1a promoter, the reporter vector was constructed by cloning the DNA of the NtPMT1a promoter upstream of the GUS gene in pBT10–GUS vector (Sprenger-Haussels and Weisshaar, 2000). The effector vector was generated by removing the kanamycin resistance gene from the backbone of pBin19-NtMYB305a-HA to generate a vector only conferring resistance to chloramphenicol in the bacterium. An empty vector modified in the same manner was used as control effector vector. These effector vectors were paired with the reporter vector to co-transform A. tumefaciens LBA4404 in a method similar to Parkhi et al. (2005). Culture of the obtained Agrobacterium (resistant to kanamycin and chloramphenicol) was resuspended with liquid 1/2 MS medium (containing 100 μM acetosyringone) to OD600 = 1.0 to infect the 20-d-old seedlings of wild-type tobacco. The transfected seedlings were co-cultivated on solid MS medium with 100 μM acetosyringone for 2 d in dark and changed onto solid MS medium with 250 mg/L cefotaxime for another 3 d cultivation. Subsequently, the tobacco seedlings were subjected to GUS staining and GUS activity quantification as described previously (Jefferson et al., 1987). The GUS activity was calculated according to a calibration curve prepared using 4-methylumbelliferone (MU) standards and was expressed as picomoles MU/minute/milligram protein.

To determine the transactivity of NtMYB305a on the JA-responsive GAG region of the NtPMT1a promoter, the reporter vectors were constructed by cloning the synthesized tetramers of GAG and its mutant GG (with the AT-rich motif deleted) upstream of the CaMV 35S TATA box (–46 to +8 bp) followed by the GUS gene in pBT10–GUS vector, respectively. These reporter vectors were paired with the effector vectors as specified to co-transform A. tumefaciens LBA4404 and to infect the 20-d-old seedlings of wild-type tobacco for GUS activity assay.

Electrophoretic mobility shift assay

The coding region of NtMYB305a was amplified with the primers 5′-AAACCATGGATAAAAAACCATGCAA-3′ and 5′-AAACTCGAGATCGCCGTTAAGCAATTGCA-3′, and cloned into the pET28b vector (Novagen Madison, WI, USA) via the Nco I and Xho I restriction sites for prokaryotic expression 6×His-tagged NtMYB305a (NtMYB305a-His) in E. coli strain BL21. The E. coli cells were cultured at 16°C, and the protein expression was induced by addition of 0.5 mM IPTG. The expressed proteins were purified using an affinity column made with Ni-NTA Sefinose Resin as described previously (Zhang et al., 2012). The probes used for EMSA were synthesized and labeled with digoxin (DIG) as desired. The EMSA reaction was made of 2 μg purified NtMYB305a-His protein and 2 ng DIG-labeled DNA probes in 20 μL binding buffer (25 mM HEPES–KOH, pH 7.4, 100 mM KCl, 0.1 mM EDTA, 8% (v/v) glycerol, 1 mM dithiothreitol (DTT), and 100 ng of poly(dA-dT)), and separated in 6% (w/v) polyacrylamide gel in 0.5×TBE (Tris-borate-EDTA) buffer. For the competition assay, the indicated amount of unlabeled probe DNA was included in the reactions. The separations were blotted onto a Hybond-N+ membrane and detected using the DIG High Prime DNA Labeling and Detection Starter Kit (Roche Basel, Switzerland). The probes used in the EMSA are listed in Supplemental Table S4.

Nicotine measurement

The nicotine content of tobacco leaves was measured as previously described with minor modification (Zhang et al., 2012; Ma et al., 2016). Briefly, 0.1 g tobacco leaf sample was dried and ground into fine powder. After soaking in 1 mL of 10% (w/v) NaOH for 20 min, the sample was extracted in the equivalent volume of dichloromethane with vortexing. The nicotine content was determined using an Agilent Technologies 7890A Chromatograph equipped with a DB 5 MS column, using nicotine from Sigma-Aldrich as a standard control. The data for each sample were determined from three independent biological replicates.

Protein interaction assays

To test the protein interaction by means of Y2H assays, the bait vectors for expressing the self-AD deleted NtMYB305a and NtMYC2a were constructed by cloning the corresponding sequences into pGBK-T7 vector (Takara Bio, San Jose, CA USA), and the prey vectors for expressing NtMYB305a and NtMYC2a were constructed by cloning the full coding sequences into pGAD-T7 vector (Takara Bio, USA). The generated constructs were transformed into yeast (S. cerevisae) strain AH109 (Takara Bio, USA) as indicated pairs and cultured on SD/–Leu/–Trp medium at 30°C to select the desired colonies. The colonies were dropped onto SD/–Ade/–His/–Leu/–Trp medium supplemented with 20 mg/L X-α-Gal and cultured at 30°C for 3 d to test the protein interaction. Three individual colonies were tested for each transformation and representative results were presented. The expression of NtMYB305a and NtMYC2a proteins in yeast was determined by western blotting with anti-AD primary antibody (Takara Bio, USA) and an HRP-labeled goat anti-mouse secondary antibody and imaged using the electrochemiluminescence method. NtJAZ1 was cloned into pGBK-T7 and co-transformed with NtMYC2a-expressing pGAD-T7 to serve as a positive control. The cloning primers are listed in Supplemental Table S5.

To investigate the interaction between NtMYB305a and NtMYC2a in plants by BiFC assay, the coding sequences of NtMYB305a and NtMYC2a were cloned into the pDOE-01 vector of the pDOE mVenus210 BiFC system (Gookin and Assmann, 2014) to co-express NmVen210-tagged NtMYB305a and CVen210-tagged NtMYC2a. The derived vector was introduced into A. tumefaciens strain GV3101 (pSoup-P19) and Agro-infiltrated into the leaves of 4-week-old N. benthamiana. After 2 d of growth in the indoor growth room, the leaves were imaged under the Leica TCS-SP8 confocal microscope with above-described excitation and emission wavelengths. A vector for co-expressing NmVen201-tagged NtJAZ1 and CVen201-tagged NtMYC2a was constructed to serve as a positive control. The cloning primers are listed in Supplemental Table S5.

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1. Sequence alignment of NtMYB305a homologs in tobacco.

Supplemental Figure S2. Alignment of NtMYB305a/b and their homologous proteins in other plant species.

Supplemental Figure S3. Western blotting of NtMYB305a-HA in tobacco roots.

Supplemental Figure S4. Expression of NtMYB305b in NtMYB305a-RI plants.

Supplemental Figure S5. Relative expression of nicotine pathway genes in the roots of NtMYB305a-HA and NtMYB305a-RI plants before or after indicated topping treatment.

Supplemental Figure S6. Self-activation tests of NtMYB305a, NtMYC2a, and their self-AD deleted variants in yeast.

Supplemental Table S1. Primers for ChIP-qPCR.

Supplemental Table S2. Nucleotide sequences and gene cloning primers for Y1H assays.

Supplemental Table S3. Primers used for RT-qPCR.

Supplemental Table S4. Nucleotide sequences of EMSA probes.

Supplemental Table S5. Gene cloning primers for protein interaction assays.

Supplementary Material

kiab458_Supplementary_Data
Click here for additional data file. (1.4MB, pdf)

Acknowledgments

We gratefully thank Prof. Wei Zeng at Zhejiang A&F University for sharing the pDOE mVenus210 BiFC system.

Funding

Funding for this project was provided by the Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences (Elite youth program to H.Z., ASTIP-TRIC05), Yunnan Tobacco Company (2018530000241001 and 2019530000241005), the National Natural Science Foundation of China (32101643), and Sichuan Tobacco Company (SCYC202014). M.P.T. was supported in part by grants from Altria Client Services (GI15099).

Conflict of interest statement. None declared.

 

H.Z. and S.B. conceived this study and wrote the manuscript. S.B., X.S., J.W., T.T, C.W., X.Z., X.L., N.F., and Y.Z. performed the experiments. Y.L., Y.D., B.W., M.P.T., and Z.Z. provided assistance in data analysis and manuscript preparation.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is Hongbo Zhang (zhanghongbo@caas.cn).

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