NORNICOTINE RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 62/595,983, filed December 7, 2017, the entire disclosure of which is incorporated herein by this reference.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy of the Sequence Listing, which was created on December 4, 2018, is named l3l77N-2l83US.txt and is 12 kilobytes in size.

TECHNICAL FIELD

[0003] The present invention relates to articles and methods for regulating conversion of nicotine to nomicotine. In particular, the presently-disclosed subject matter relates to transcription factors for regulating conversion of nicotine to nomicotine and methods of use thereof.

BACKGROUND

[0004] Nicotiana tabacum (common tobacco) is a natural allotetraploid originated about 200,000 years ago. The maternal S-genome is derived from ancestors of N. sylvestris and paternal T-genome from the relatives of N. tomentosiformis . Nicotine is the major alkaloid accumulated in most of the cultivated tobacco varieties. During the past decades, significant progress has been made in isolation and characterization structural genes in the nicotine biosynthetic pathway. Jasmonic acid (JA) is a major elicitor of nicotine biosynthesis. JA- responsive transcription factors (TFs) belong to two major families,

APETALA2/ETHYLENE RESPONSE FACTORS (AP2/ERFs) and the basic HELIX- LOOP-HELIX (bHLH), and are known to induce the expression of genes encoding key enzymes in the nicotine biosynthetic pathway.

[0005] Nicotine and other tropane alkaloids, such as hyoscyamine and scopolamine, are synthesized in roots, and transported through xylem to the leaves. A number transporters have been isolated and characterized for their role in transport and vacuolar sequestration of alkaloids in plants. In tobacco, a number of transporters belonging to the MULTIDRUG and TOXIC COMPOUND ETRUSION (MATE) family, including MATE 1/2 and Jasmonate- inducible Alkaloid Transporter (JAT1/2), are involved in transportation and vacuolar sequestration of nicotine. However, TFs involved in regulation of these transporter are not thoroughly studied.

[0006] In addition to nicotine, tobacco plants accumulate three other pyridine alkaloids namely, nomicotine, anabasine, and anatabine. Nomicotine is a demethylated nicotine (does not contain a methyl group) that is derived from nicotine by an enzymatic process. It is also a precursor to /V-nitrosonornicotine (NNN), which is produced during the curing and processing of tobacco materials. More specifically, during post-harvest processing, nomicotine chemically reacts with the nitrosating agents to form NNN. As NNN belongs to a class of smoking related carcinogens called tobacco specific nitrosamines (TSNA), it is highly desirable to reduce TSNA in tobacco products.

[0007] There are two possible ways to reduce TSNA. One is to reduce overall nicotine content; the other is to eliminate conversion of nicotine to nomicotine. Conversion of nicotine to nomicotine is catalyzed by nicotine N-demethylase (NND), a small family of cytochrome P450 enzymes. Three NND genes, CYP82E4vl (originated from N. tomentosiformis), CYP82E5v2 (originated from/V. tomentosiformis ), and CYP82E10 (originated from N.

sylvestris), have been identified in the conversion of nicotine to nomicotine in tobacco.

CYP82E4vl (E4) plays a major role in nicotine to nomicotine conversion in senescent leaves, while expression of CYP82E10 (E10) is reported to be in the roots and CYP82E5 (E5) functions in both roots and leaves. However, up to this point, transcription factors (TFs) involved in the regulation of nicotine to nomicotine conversion (i.e., transcriptional regulators of E4, 5, and 10 genes) have not been identified. Therefore, although significant progress has been made in biochemical and molecular characterization of these nicotine transporters and enzymes involved in nomicotine biosynthesis, the molecular mechanism underlying the regulation of these genes remains to be elucidated.

[0008] Accordingly, there is a need for articles and methods that regulate the conversion of nicotine to nomicotine.

SUMMARY

[0009] The presently-disclosed subject matter meets some or all of the above-identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.

[0010] This summary describes several embodiments of the presently-disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently-disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this summary does not list or suggest all possible combinations of such features.

[0011] In some embodiments, the presently-disclosed subject matter includes a method of decreasing conversion of nicotine to nomicotine, the method comprising administering at least one basic region/leucine zipper (bZIP) type transcription factor inhibitor to an organism in need thereof. In some embodiments, the at least one bZIP transcription factor inhibitor is selected from the group consisting of group C bZIP transcription factor inhibitors, group S bZIP transcription factor inhibitors, and combinations thereof. In some embodiments, the at least one bZIP transcription factor inhibitor is selected from the group consisting of an NtbZIP la inhibitor, an NtbZIPlb inhibitor, an NtbZIP2a inhibitor, an NtbZIP2b inhibitor, and combinations thereof. In some embodiments, the at least one bZIP transcription factor inhibitor comprises an NtbZIP la inhibitor and an NtbZIPlb inhibitor. In some embodiments, the at least one bZIP transcription factor inhibitor comprises an NtbZIP2a inhibitor and an NtbZIP2b inhibitor. In some embodiments, the at least one bZIP transcription factor inhibitor comprises an NtbZIP la inhibitor, an NtbZIPlb inhibitor, an NtbZIP2a inhibitor, and an NtbZIP2b inhibitor.

[0012] In some embodiments, the at least one bZIP transcription factor inhibitor is selected from the group consisting of antisense oligonucleotides, miRNA, siRNA, locked nucleic acid (LNA) nucleotides, and combination thereof. In one embodiment, the at least one bZIP transcription factor inhibitor comprises an antisense oligonucleotide of a bZIP transcription factor selected from the group consisting of NtbZIP la, NtbZIPlb, NtbZIP2a, NtbZIP2b, and combinations thereof.

[0013] Also provided herein, in some embodiments, is a method of decreasing conversion of nicotine to nomicotine, the method comprising mutating a basic region/leucine zipper (bZIP) type transcription factor binding site on a promoter of a nicotine N-demethylase (NND). In some embodiments, the NND is selected from the group consisting of

CYP82E4vl , CYP82E5v2, and CYP82E10. In one embodiment, the NND is CYP82E4vl. In another embodiment, the bZIP binding site on the promoter of CYP82E4vl is an A/G box with a pre-mutated sequence of TACGTC. In a further embodiment, the mutated binding site has the sequence TGCGTC. In some embodiments, the mutated binding site is formed by site-directed mutagenesis. In some embodiments, the method also includes administering at least one bZIP type transcription factor inhibitor to an organism in need thereof. In one embodiment, the at least one bZIP transcription factor inhibitor is selected from the group consisting of an NtbZIPla inhibitor, an NtbZIPlb inhibitor, an NtbZIP2a inhibitor, an NtbZIP2b inhibitor, and combinations thereof.

[0014] Further provided herein, in some embodiments, is a method of decreasing conversion of nicotine to nomicotine, the method comprising mutating a plant genome to knockout at least one basic region/leucine zipper (bZIP) type transcription factor. In some embodiments, the at least one bZIP transcription factor is selected from the group consisting of group C bZIP transcription factor, group S bZIP transcription factor, and combinations thereof. In some embodiments, the at least one bZIP transcription factor is selected from the group consisting of NtbZIPla, NtbZIPlb, NtbZIP2a, NtbZIP2b, and combinations thereof. In some embodiments, the at least one bZIP transcription factor is selected from the group consisting of NtbZIPla and NtbZIP2a, NtbZIPlb and NtbZIP2b, and combinations thereof.

In some embodiments, the method also includes administering at least one bZIP type transcription factor inhibitor to an organism in need thereof. In one embodiment, the at least one bZIP transcription factor inhibitor is selected from the group consisting of an NtbZIPla inhibitor, an NtbZIPlb inhibitor, an NtbZIP2a inhibitor, an NtbZIP2b inhibitor, and combinations thereof.

[0015] Further features and advantages of the presently-disclosed subject matter will become evident to those of ordinary skill in the art after a study of the description, figures, and non-limiting examples in this document.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The presently-disclosed subject matter will be better understood, and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:

[0017] FIG. 1 shows a graph illustrating hierarchical cluster analysis of the transcriptome data of eight different tobacco tissues. Each tissue forms a distinct cluster based on the expression. [0018] FIG. 2 shows a graph illustrating distribution of different TF families in tobacco and its progenitors.

[0019] FIG. 3 shows a graph illustrating co-expression analysis of TF genes, and structural genes in nicotine biosynthetic pathway in different tissue. The TF and structural genes are grouped into 8 different modules (color-coded side bar) based on their expression. The black module contains majority of the structural gene nicotine biosynthetic pathway. YF, young flower; MF, mature flower; YL, young leaf; ML, mature leaf; SL, senesce leaf.

[0020] FIG. 4 shows a graph illustrating expression of NtbZIPl a/b and NNDs in different tobacco tissues.

[0021] FIG. 5 shows an image illustrating the bZIP family in tobacco. NtbZIPl a and NtbZIPlb are indicated by *.

[0022] FIG. 6 shows an image comparing the nucleotide and amino acid sequences of NtbZIPl a and lb.

[0023] FIG. 7 shows a graph illustrating that transient overexpression of NtbZIPla in tobacco leaves induces the expression of CYP82E4vl, CYP82E5v2, and CYP82E10.

[0024] FIG. 8 shows a graph illustrating that NtbZIPla significantly activates

CYP82E4vl and CYP82E10 promoters in tobacco cells.

[0025] FIG. 9 shows a graph illustrating that NtbZIPl a activates CYP82E4vl promoter in tobacco cells by binding to the A/G box.

[0026] FIG. 10 shows a graph illustrating that NtbZIPla and b significantly activate CYP82E4vl promoter in tobacco cells.

[0027] FIG. 11 shows a graph illustrating that topping of tobacco plants downregulates the expression of NtbZIPs and NNDs.

[0028] FIGS. 12A-C show graphs and images illustrating overexpression of NtbZIPla in tobacco plants. (A) Genomic DNA PCR and cDNA PCR of control and three transgenic lines (line # 4, 5, and 9) confirming the integration and expression, respectively, of the antibiotic selection marker, neomycin phosphotransferase II (npt II; Kan). (B) Quantitative real-time (qRT-PCR) analysis showing the relative expression of NtbZIPl and E4 in control (EV) and transgenic lines (line # 4, 5, and 9). (C) Metabolic analysis showing conversion of nicotine to nomicotine in control and transgenic lines (To or first generation transgenic plants). [0029] FIG. 13 shows an image illustrating amino acid sequence alignment of NtbZIP2a and 2b.

[0030] FIG. 14 shows a graph illustrating that NtbZIP2a and b have similar expression patterns in tobacco tissues.

[0031] FIG. 15 shows a graph illustrating that NtbZIP2 acts synergistically with NtbZIPl to activate the E4 promoter in tobacco cells.

[0032] FIGS. 16A-B show images illustrating protein-protein interaction of NtbZIPl and NtbZIP2 using yeast two hybrid assay. (A) Yeast two hybrid assay showing protein-protein interaction between NtbZIPl and NtbZIP2. Colony growth on synthetic drop-out (SD) medium lacking leucine, tryptophan, histidine and adenine (-leu-trp-his-ade) indicates interaction between the proteins (bZIPs). (B) Schematic diagram of NtbZIPl and NtbZIP2. The bZIP domain is indicated by“shaded” rectangle. The numbers indicate the amino acid.

[0033] While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described below in detail. It should be understood, however, that the description of specific embodiments is not intended to limit the disclosure to cover all modifications, equivalents and alternatives falling within the spirit and scope of the disclosure as defined by the appended claims.

DEFINITIONS

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, including the methods and materials are described below.

[0035] Following long-standing patent law convention, the terms“a,”“an,” and“the” refer to“one or more” when used in this application, including the claims. Thus, for example, reference to“a cell” includes a plurality of cells, and so forth.

[0036] The terms“comprising,”“including,” and“having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0037] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term“about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

[0038] As used herein, the term“about,” when referring to a value or to an amount of mass, weight, time, volume, concentration, percentage, or the like is meant to encompass variations of in some embodiments ±50%, in some embodiments ±40%, in some

embodiments ±30%, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0039] As used herein, ranges can be expressed as from“about” one particular value, and/or to“about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. For example, if the value“10” is disclosed, then“about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0040] All combinations of method or process steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.

DETAILED DESCRIPTION

[0041] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.

[0042] The presently-disclosed subject matter relates to articles and methods for regulating conversion of nicotine to nomicotine. In some embodiments, the article includes one or more transcription factor (TF) inhibitors. In one embodiment, for example, the article includes one or more inhibitors of basic region/leucine zipper (bZIP) type transcription factors. In another embodiment, the bZIP type transcription factors are derived from tobacco. In a further embodiment, of the 133 bZIP type transcription factors identified in tobacco by the instant inventors, which are classified into ten sub-groups: A, B, C, D, E, F, G, H, and S, suitable bZIP type transcription factors include at least one of the 27 bZIPs in sub-group S, at least one of the 6 bZIPs in sub-group C, other bZIP 63 homologs, or a combination thereof.

In certain embodiments, the S sub-group bZIP transcription factor includes, but is not limited to, NtbZIPla (SEQ ID NOs: 1 and 2), NtbZIPlb (SEQ ID NOs: 3 and 4), or a combination thereof; and/or the C sub-group bZIP transcription factor includes, but is not limited to, NtbZIP2a (SEQ ID NO: 5), NtbZIP2b (SEQ ID NO: 6), or a combination thereof.

[0043] The one or more transcription factor inhibitors include, but are not limited to, antisense oligonucleotides, miRNA, siRNA, locked nucleic acid (LNA) nucleotides, or a combination thereof. In some embodiments, the inhibitors provide RNAi-mediated knock down/silencing of the bZIP type transcription factors. As will be appreciated by those skilled in the art, the specific sequence/structure of the transcription factor inhibitors is based upon the sequence of the specific transcription factor. Accordingly, as will also be appreciated by those skilled in the art, the antisense oligonucleotides and/or LNAs may be formed by any suitable method using the bZIP type transcription factor sequences provided herein. For example, in one embodiment, the inhibitor includes an antisense oligonucleotide having 100% sequence homology with the complementary bZIP type transcription factor. In another embodiment, the transcription factor inhibitor includes an antisense oligonucleotide having 100% sequence homology with a bZIP type transcription factor complementary to NtbZIPla, NtbZIPlb, NtbZIP2a, and/or NtbZIP2b. In such embodiments, the transcription factor inhibitor(s) provide RNAi-mediated knock-do wn/silencing of NtbZIPla, NtbZIPlb, NtbZIP2a, and/or NtbZIP2b expression in tobacco.

[0044] Also provided herein, in some embodiments, is a method of regulating the conversion of nicotine to nomicotine in a tobacco plant or other nicotine containing organism. In one embodiment, the method includes administering one or more of the bZIP inhibitors disclosed herein to a nicotine containing organism. Administration of these one or more bZIP inhibitors decreases or eliminates conversion of the nicotine to nomicotine. The one or more inhibitors may be administered for a single type of bZIP transcription factor, or for a combination of bZIP transcription factors. For example, in one embodiment, the method includes administering one or more inhibitors for S bZIP type transcription factors or C bZIP type transcription factors. In another embodiment, the method includes administering one or more inhibitors for S bZIP type transcription factors and one or more transcription factors for C bZIP type transcription factors. In a further embodiment, the method includes

administering one or more inhibitors ofNtbZIPla, NtbZIPlb, NtbZIP2a, and/or NtbZIP2b to the organism. In certain embodiments, inhibiting both S bZIP type transcription factors and C bZIP type transcription factors has a synergistic effect on the reduction or elimination of nicotine conversion to nomicotine.

[0045] The methods disclosed herein include administering a single type of inhibitor or any suitable combination of inhibitors, which may be the same or different for each bZIP transcription factor being inhibited. For example, in one embodiment, the method includes administering antisense oligonucleotides ofNtbZIPla, NtbZIPlb, NtbZIP2a, and/or NtbZIP2b. In another embodiment, the method includes administering antisense

oligonucleotides of one bZIP transcription factor, such as NtbZIPla, and LNA nucleotides of another bZIP transcription factor, such as NtbZIPlb. As will be appreciated by those skilled in the art, although discussed above with regard to certain combinations of bZIP transcription factors and transcription factor inhibitors, the disclosure is not so limited and may include any other suitable combination of TFs and TF inhibitors.

[0046] Additionally or alternatively, the method may include bZIP type transcription factor knockout and/or mutation of a bZIP type transcription factor binding site on the promoter of the nicotine N-demethylase (NND). For example, in one embodiment, the method includes editing the plant genome to knock-out NtbZIPla, NtbZIPlb, NtbZIP2a, and/or NtbZIP2b. The genome editing may be performed through any suitable process, such as, but not limited to, CRISPR/Cas9-mediated genome editing. In another embodiment, the method includes mutating the bZIP binding element in the E4 promoter, called A/G box (TACGTC), to TGCGTC by site-directed mutagenesis. Although discussed above with regard to a specific mutation in the E4 promoter, as will be appreciated by those skilled in the art, the disclosure is not so limited and includes any other mutation in the E4, E5, and/or E10 promoter to reduce or eliminate activation of the respective NND by the bZIP type transcription factor.

[0047] The administration of the TF inhibitors, the TF knockout, and/or the binding site mutation disclosed herein reduces or eliminates activation of the NND by the bZIP type transcription factor, which decreases or eliminates conversion of nicotine to nomicotine. As opposed to existing articles that include E4, E5, and E10 mutants, the articles disclosed herein control the expression of E4, E5, and E10 to reduce or eliminate the conversion of nicotine to nomicotine. By reducing or eliminating the conversion of nicotine to nomicotine the articles and methods disclosed herein decrease the harmful effects of products which typically contain the carcinogenic nomicotine, such as, but not limited to, tobacco products.

[0048] The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data that are representative of data gathered at various times during the course of

development and experimentation related to the presently-disclosed subject matter.

EXAMPLES

[0049] Example 1

[0050] This Example describes the analysis of transcriptome data sets of different tobacco tissues, including leaf (young, mature, and senesce leaf), root, stem, flower (young and mature flower), and capsule to generate a co-expression network. First, a hierarchical cluster analysis was performed, which revealed that each individual tissue type exhibits a unique expression pattern (FIG. 1). Next, the genes encoding all major TF families in tobacco and its progenitors were identified. The tobacco genome contains more TF genes than its progenitors and the number of TFs belonging to MYB, AP2/ERFs, and bHLH families are significantly higher than other families (FIG. 2). In view thereof, the TFs and structural genes in the nicotine biosynthetic pathway were then grouped into 8 different modules (color-coded side bar) based on their expression pattern in different tissues (FIG. 3). The“black” module is particularly interesting as majority of the nicotine biosynthetic genes were found in this module along with a number of TF genes. Many of these TFs belong to MYB, bHLH, bZIP, and ERF families. As discussed in Examples 2-4 below, the role of these TFs in nicotine biosynthesis in tobacco is established through isolation and functional characterization thereof.

[0051] Example 2

[0052] This Example describes two bZIP type transcription factors from tobacco (FIG. 4), which regulate the conversion of nicotine to nomicotine, can be used for reduction of smoking related carcinogen, tobacco specific nitrosamines (TSNA).

[0053] bZIP TFs are characterized by a conserved leucine zipper motif that mediates dimer formation for DNA binding. In plants, bZIP TFs regulate processes including pathogen defense, light and stress signaling, seed maturation, and flower development. Many bZIP factors, especially those in tobacco, are not well characterized. By co-expression and clustering analyses, two bZIP TF genes that co-express with E4, E5, and E10 were identified herein. These tobacco bZIP TFs have been termed NtbZIPla and NtbZIPlb.

[0054] NtbZIPl a/b exhibit similar expression patterns as compared to CYP82E4vl, the major NND enzyme involved in nicotine to nomi cotine conversion, and are highly expressed in flowers and senescent leaves (FIG. 4). As illustrated in FIG. 5, 133 bZIPs were identified in tobacco and classified into ten sub-groups: A, B, C, D, E, F, G, H, I, and S, with NtbZIPl a/b belonging to sub-group S. In maize, expression of group-S bZIPs are induced by wounding, cold, and drought stress. Referring to FIG. 6, it was also found that NtbZIPla and b are more than 97% identical at nucleotide and amino acid level. Without wishing to be bound by theory, it is believed that the two homologous bZIPs are derived from two progenitors of tobacco, N. sylvestris and N. tometosiformis .

[0055] After identifying the two bZIP TFs, whether overexpression of NtbZIPla leads to upregulation of E4, 5, and 10 was tested. NtbZIPla was cloned into pCAMBIA2300 (binary vector) under the control of the CaMV 35 S promoter and rbcS terminator. The binary vectors (empty control and NtbZIPla) were mobilized into Agrobacterium, and tobacco leaves were infiltrated using the transformed Agrobacterium. Total RNA isolated from Agrobacterium- infiltrated leaf discs were used for cDNA synthesis and real-time quantitative PCR (qRT- PCR) was used to detect the transcript levels of NtbZIPla , NtbZIPlb, E4, 5, and 10. An ubiquitously expressed house-keeping gene, tubulin, was used as internal control in qRT- PCR. The results showed that, when NtbZIPla was highly expressed transiently, the endogenous NtbZIPlb, E4, 5, and 10 were upregulated (approx. 3-10 fold), indicating that NtbZIPla induces the expression of NtbZIPlb, E4, 5, and 10, hence a possible transcriptional activator for these genes (FIG. 7).

[0056] Next, whether NtbZIPla can bind to the promoters of its potential target genes was tested. The promoters (approximately l.Okb fragment of the 5’ untranslated region of each coding gene) of E4, 5, and 10 were isolated, and the individual promoters were fused to a firefly luciferase reporter gene. The NtbZIPla gene was cloned into the pBlueScript (pBS) vector under the control of the CaMV 35S promoter and rbcS terminator. The plasmids were electroporated into tobacco protoplasts. NtbZIPla significantly induced the luciferase gene expression controlled by the E4 and E10 promoters, but not E5 promoter, suggesting that NtbZIPla can directly activate E4 and E10 genes, likely by binding to their promoters (FIG. 8). NtbZIPla did not appear to bind to the l.Okb promoter region of E5, used for the activation experiment. However, as mentioned above, overexpression of NtbZIPla led to upregulation of E5, together with E4 and 10. The promoter activation experiment indicates two possible NtbZIPla regulatory relationships with E5 gene: (1) NtbZIPla binds to a site outside of the l.Okb promoter fragment, or (2) NtbZIPla indirectly activates E5, through another unidentified activator in tobacco (e.g., NtbZIPla activates another activator, which in turn activates E5).

[0057] To support the possibility of NtbZIPla directly binding to the E4 promoter to regulate transactivation the bZIP binding element in the E4 promoter, called A/G box (TACGTC), was mutated to TGCGTC by site-directed mutagenesis. The mutated promoter was fused to the luciferase reporter gene as described above. A transactivation experiment was then performed using the mutant reporter plasmid and the NtbZIPla expression vector, as described above. The result showed that NtbZIPla is unable to activate the luciferase gene expression under the control of the mutant promoter (FIG. 9). This experiment demonstrated that the E4 promoter is activated through an A/G-box binding factor, most likely NtbZIPla.

[0058] Referring to FIG. 10, the transactivation experiment was also performed using a NtbZIPlb expression vector and the E4 promoter-luciferase reporter plasmid, as described for NtbZIPla. NtbZIPlb also activated the E4 promoter at the similar level as NtbZIPla.

[0059] Finally, as it has been established that the agronomic practice of tobacco topping (removal of the axillary shoots) induces nicotine production, the instant inventors analyzed the transcriptome data from tobacco plants that were topped or un-topped. More specifically, leaf samples were collected after 24 hours from the control (un-topped) and topped plants. RNA isolated from un-topped and topped leaves were used for cDNA synthesis and qRT- PCR analyses. The results showed that topping resulted in decreased expression of bZIPl a/b, as well as E4, 5, and 10, by approximately 70-90%, compared to the un-topped plants, suggesting that topping negatively regulates the expression of bZIPl a/b and NNDs in tobacco (FIG. 11). The result also indicates that bZIPl a/b and NNDs are coordinately expressed in tobacco, as gene regulators and their target genes usually do.

[0060] Based upon the Example above, NtbZIPla and lb are believed to be involved in the regulation of the three NND genes as activators. Reduction or inactivation of NtbZIPla/b may lead to reduction of nomicotine. [0061] Example 3

[0062] This Example describes the formation of transgenic lines overexpressing

NtbZIPla and the effect of these transgenic lines on endogenous E4 expression.

[0063] To form the transgenic lines, the pCAMBIA2300 (binary vector), containing NtbZIPla under the control of the CaMV 35S promoter and rbcS terminator, was mobilized into Agrobacterium, and tobacco leaf discs were infected with the transformed

Agrobacterium. More than 20 transgenic lines overexpressing NtbZIPla were generated from Agrobacterium- infected leaf discs. Genomic DNA isolated from control and three transgenic lines were used to verify the transgenic status of the plants by PCR amplification of the antibiotic selection marker, neomycin phophotransferase II ( nptll ; kan). Total RNA isolated from leaves of control and three transgenic lines were used for cDNA synthesis. RT-PCR was used to verify the expression of nptll (kan) gene in the transgenic plants (FIG. 12A). Real time quantitative PCR (qRT-PCR) was used to detect the transcript levels of NtbZIPla, and E4 (FIG. 12B). An ubiquitously expressed house-keeping gene, tubulin, was used as internal control in qRT-PCR.

[0064] The results of this Example showed that NtbZIPla expression was significantly higher in the transgenic plants compared with control. When NtbZIPla was highly expressed the endogenous E4 expression was upregulated (approx. 6-8 fold), indicating that NtbZIPla induces the expression of E4 and therefore is a possible transcriptional activator for E4 gene. Additionally, metabolic analysis shows that nicotine to nomicotine conversion is higher in transgenic tobacco leaves as compared with a control (FIG. 12C). The formula used for calculating the conversion of nicotine to nomicotine was:

Nomicotine

Nicotine conversion = — - — - x 100

Nicotine + Nomicotine

Two of the three lines analyzes showed higher nicotine to nomicotine conversion. Because the metabolic analysis was performed with independent To (first generation transgenic plants) segregating population, the metabolic outcomes can vary.

[0065] Example 4

[0066] As described in Example 2 above, the instant inventors have characterized NtbZIPla and b, two NtbZIP belonging to group S bZIP factors. In Arabidopsis , group S bZIP factors are known to interact with certain group C factors to regulate gene expression.

In view of this interaction, the instant inventors identified the tobacco homologs of Arabidopsis bZIP 63, a group C member that interacts with group S factors. In tobacco, there are two bZIP 63 homologs, termed here as NtbZIP2a and b, that share greater than 95% in amino acid identity (FIG. 13). Without wishing to be bound by theory, it is believed that NtbZIP2a and b are originated from the two tobacco progenitors, N. sylvestris and N.

tometosiformis , and functionally redundant.

[0067] According to transcriptomic analysis, NtbZIP2a and b have similar expression patterns in tobacco flowers, leaves, stems, and roots (FIG. 14). Based upon the foregoing discussion, the instant inventors hypothesized that NtbZIP2a and b also regulate E4I5I10, individually and/or cooperatively with NtbZIPla and b. Thus, NtbZIP2a was tested for transactivation of the E4 promoter, individually or in combination with NtbZIPla. More specifically, the E4 promoter was fused to the firefly luciferase reporter gene and the bZIP TFs were cloned into pBS vector under the control of the CaMV 35S promoter and rbcs terminator. The results showed that, individually, both NtbZIPla and NtbZIP2a activate the E4 promoter; however, when both NtbZIPla and 2a were co-expressed, transactivation of the E4 promoter was significantly increased, compared to that was induced by each factor alone (FIG. 15)

[0068] Next, to determine whether NtbZIPla/b interact with NtbZIP2a, the inventors performed yeast hybrid assay. The growth of the yeast cells on synthetic drop-out (SD) medium lacking leucine, trptophan, histidine, and adenine (SD-leu-trp-his-ade) suggests that NtbZIPla/b interact with NtbZIP2a (FIG. 16A; 1 and 2). In addition, NtbZIP2 interacts with itself to form a homo-dimer (FIG. 16A; 3). The bZIP domains of NtbZIPl and NtbZIP2 are illustrated in FIG. 16B.

[0069] Although the instant Example only tested NtbZIP2a activity on the E4 promoter, but not the E5 or E10 promoters, it is believed that both NtbZIP2 factors are activators of E4I5I10 genes. That is, this Example suggests that the group C NtbZIP2a and b are two previously uncharacterized regulators of E4I5I10 genes. In addition, this Example shows that NtbZIPl and NtbZIP2 are synergistic in activation of the E4 (possibly E5 and 10) promoter. In particular, without wishing to be bound by theory, it is believed that NtbZIP2a and/or b interact with NtbZIPla and/or b to enhance DNA binding ability and significantly increase activation of the E4I5I10 promoters, as compared to NtbZIPl or NtbZIP2 alone.

[0070] In summary, two group S and two group C NtbZIP factors that are positive regulators of E4I5I10 genes were characterized herein. The transactivation activities of NtbZIPla and 2a on the E4 promoter are additive. Therefore, without wishing to be bound by theory, it is believed that knockout approaches to inactivate one or all of these genes reduces

E4I5I10 gene expression.

REFERENCES

[1] Gavilano, L.B., and Siminszky, B. (2007). Isolation and characterization of the cytochrome P450 gene CYP82E5v2 that mediates nicotine to nomicotine conversion in the green leaves of tobacco. Plant & cell physiology 48, 1567-1574.

[2] Higo, K., Ugawa, Y., Iwamoto, M., and Higo, H. (1998). PLACE: a database of plant cis-acting regulatory DNA elements. Nucleic acids research 26, 358-359.

[3] Leitch, I.J., Hanson, L., Lim, K.Y., Kovarik, A., Chase, M.W., Clarkson, J.J., and Leitch, A.R. (2008). The ups and downs of genome size evolution in polyploid species of Nicotiana (Solanaceae). Annals of botany 101, 805-814.

[4] Lescot, M., Dehais, P., Thijs, G., Marchal, K., Moreau, Y., Van de Peer, Y., Rouze, P., and Rombauts, S. (2002). PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic acids research 30, 325-327.

[5] Lewis, R.S., Bowen, S.W., Keogh, M.R., and Dewey, R.E. (2010). Three nicotine demethylase genes mediate nomicotine biosynthesis in Nicotiana tabacum L.:

functional characterization of the CYP82E10 gene. Phytochemistry 71, 1988-1998.

[6] Lim, K.Y., Matyasek, R., Kovarik, A., and Leitch, A.R. (2004). Genome evolution in allotetraploid Nicotiana. Biol J. Linn Soc 82, 599-606.

[7] Morita, M., Shitan, N., Sawada, K., Van Montagu, M.C., Inze, D., Rischer, H., Goossens, A., Oksman-Caldentey, K.M., Moriyama, Y., and Yazaki, K. (2009). Vacuolar transport of nicotine is mediated by a multi drug and toxic compound extrusion (MATE) transporter in Nicotiana tabacum. Proceedings of the National Academy of Sciences of the United States of America 106, 2447-2452.

[8] Pattanaik, S., Werkman, J.R., Kong, Q., and Yuan, L. (20l0a). Site-directed mutagenesis and saturation mutagenesis for the functional study of transcription factors involved in plant secondary metabolite biosynthesis. Methods in molecular biology 643, 47-57. [9] Pattanaik, S., Kong, Q., Zaitlin, D., Werkman, J.R., Xie, C.H., Patra, B., and Yuan, L. (20l0b). Isolation and functional characterization of a floral tissue-specific R2R3 MYB regulator from tobacco. Planta 231, 1061-1076.

[10] Shoji, T., and Hashimoto, T. (2011). Tobacco MYC2 regulates jasmonate-inducible nicotine biosynthesis genes directly and by way of the NIC2-locus ERF genes. Plant & cell physiology 52, 1117-1130.

[11] Shoji, T., Kajikawa, M., and Hashimoto, T. (2010). Clustered transcription factor genes regulate nicotine biosynthesis in tobacco. The Plant cell 22, 3390-3409.

[12] Shoji, T., Inai, K., Yazaki, Y., Sato, Y., Takase, H., Shitan, N., Yazaki, K., Goto, Y., Toyooka, K., Matsuoka, K., and Hashimoto, T. (2009). Multi drug and toxic compound extrusion-type transporters implicated in vacuolar sequestration of nicotine in tobacco roots. Plant physiology 149, 708-718.

[13] Sierro, N., van Oeveren, J., van Eijk, M.J., Martin, F., Stormo, K.E., Peitsch, M.C., and Ivanov, N.V. (2013a). Whole genome profiling physical map and ancestral annotation of tobacco Hicks Broadleaf. The Plant journal : for cell and molecular biology 75, 880-889.

[14] Sierro, N., Battey, J.N., Ouadi, S., Bovet, L., Goepfert, S., Bakaher, N., Peitsch, M.C., and Ivanov, N.V. (2013b). Reference genomes and transcriptomes of

Nicotiana sylvestris and Nicotiana tomentosiformis. Genome biology 14, R60.

[15] Sierro, N., Battey, J.N., Ouadi, S., Bakaher, N., Bovet, L., Willig, A., Goepfert, S., Peitsch, M.C., and Ivanov, N.V. (2014). The tobacco genome sequence and its comparison with those of tomato and potato. Nature communications 5, 3833.

[16] Siminszky, B., Gavilano, L., Bowen, S.W., and Dewey, R.E. (2005). Conversion of nicotine to nomicotine in Nicotiana tabacum is mediated by CYP82E4, a cytochrome P450 monooxygenase. Proceedings of the National Academy of Sciences of the United States of America 102, 14919-14924.

[17] Singh, S.K., Wu, Y., Ghosh, J.S., Pattanaik, S., Fisher, C., Wang, Y., Lawson, D., and Yuan, L. (2015). RNA-sequencing Reveals Global Transcriptomic Changes in Nicotiana tabacum Responding to Topping and Treatment of Axillary-shoot Control Chemicals. Scientific reports 5, 18148. [18] Van Moerkercke, A., Steensma, P., Schweizer, F., Pollier, J., Gariboldi, I., Payne, R., Vanden Bossche, R., Miettinen, K., Espoz, J., Purnama, P.C., Kellner, F., Seppanen-Laakso, T., O'Connor, S.E., Rischer, H., Memelink, J., and Goossens,

A. (2015). The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus. Proceedings of the National Academy of Sciences of the United States of America 112, 8130-8135.

[19] Yu, F., and De Luca, V. (2013). ATP-binding cassette transporter controls leaf

surface secretion of anticancer drug components in Catharanthus roseus. Proceedings of the National Academy of Sciences of the United States of America 110, 15830- 15835.

[20] Ehlert, A., et al. (2006) Two-hybrid protein-protein interaction analysis in

Arabidopsis protoplasts: establishment of a heterodimerization map of group C and group S bZIP transcription factors. The Plant Journal, 46: 890-900.

[0071] While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described below in detail. It should be understood, however, that the description of specific embodiments is not intended to limit the disclosure to cover all modifications, equivalents and alternatives falling within the spirit and scope of the disclosure as defined by the appended claims.