Short title Nicotine biosynthesis regulon in tobacco Name ... · 155 metabolism, a part of indole...

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Short title 1 Nicotine biosynthesis regulon in tobacco 2 3 Corresponding author details 4 Name; Tsubasa Shoji 5 Affiliation; Graduate School of Biological Sciences, Nara Institute of 6 Science and Technology 7 Address; Takayama-cho 8916-5, Ikoma, Nara 630-0101, Japan 8 Phone; +81-743-72-5521 9 Fax; +81-743-72-5529 10 e-mail;[email protected] 11 12 Article title 13 Genomic insights into the evolution of the nicotine biosynthesis pathway in 14 tobacco. 15 16 Author names and affiliations 17 Masataka Kajikawa1$, Nicolas Sierro2*$, Haruhiko Kawaguchi1, Nicolas 18 Bakaher2, Nikolai V. Ivanov2, Takashi Hashimoto1, and Tsubasa Shoji1* 19 20 1Graduate School of Biological Sciences, Nara Institute of Science and 21 Technology, Ikoma, Nara 630-0101, Japan 22 2Philip Morris International R&D, Philip Morris Products S.A., Quai 23 Jeanrenaud 5, 2000, Neuchâtel, Switzerland (Part of Philip Morris 24 International Group of Companies) 25 26 $ These authors have contributed equally to this work. 27 28 One sentence summary 29 A series of metabolic and transport genes involved in nicotine pathway 30 form a regulon under the control of jasmonate-responsive transcription 31 factors in tobacco. 32 33 Foot notes 34 List of author contributions 35 T.S. conceived the research plans. T.S., T.H., N.I. and N.S. supervised the 36

Plant Physiology Preview. Published on April 18, 2017, as DOI:10.1104/pp.17.00070

Copyright 2017 by the American Society of Plant Biologists

Plant Physiology Preview. Published on April 18, 2017, as DOI:10.1104/pp.17.00070

Copyright 2017 by the American Society of Plant Biologists

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experiments. M.K., H.K., T.S. and N.S performed the experiments; T.S, and 37 N.S analyzed the data with contributions from N.B.; T.S wrote the article 38 with contributions of N.S.; T.H. supervised and complemented the writing. 39 40 Funding information 41 This work was supported in part by the Japan Society for the Promotion of 42 Science (Grant-in-Aid for Scientific Research (C) no. 26440144 to T.S.) 43 and the Sumitomo Foundation (Grant for Basic Scientific Research to T.S.). 44 45 Conflicts of interest statement 46 N.S., N.B. and N.I. are employees of Philip Morris International R&D, 47 Philip Morris Products S.A. (part of Philip Morris International group of 48 companies). 49 50 Present address 51 Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan, 52 for M. K. 53



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Abstract 54 In Nicotiana tabacum (tobacco), nicotine is the predominant alkaloid. It is 55 produced in the roots and accumulated mainly in the leaves. Jasmonates 56 play a central signaling role in damage-induced nicotine formation. The 57 genome sequence of tobacco provides us an almost complete inventory of 58 structural and regulatory genes involved in nicotine pathway. Phylogenetic 59 and expression analyses revealed a series of structural genes of the nicotine 60 pathway, forming a regulon, under the control of jasmonate-responsive 61 ERF transcription factors. The duplication of NAD and polyamine 62 metabolic pathways and the subsequent recruitment of duplicated primary 63 metabolic genes into the nicotine biosynthesis regulon were suggested to be 64 the drivers for pyridine and pyrrolidine ring formation steps early in the 65 pathway. Transcriptional regulation by ERF and cooperatively acting 66 MYC2 transcription factors are corroborated by the frequent occurrence of 67 cognate cis-regulatory elements of the factors in the promoter regions of 68 the downstream structural genes. The allotetraploid tobacco has 69 homologous clusters of ERF genes on different chromosomes, which are 70 possibly derived from two ancestral diploids and include either 71 nicotine-controlling ERF189 or ERF199. A large chromosomal deletion 72 was found within one allele of the nicotine-controlling NICOTINE2 locus, 73 which is part of one of the ERF gene clusters, and which has been used to 74 breed tobacco cultivars with a low-nicotine content. 75 76 Key words: alkaloids, jasmonates, Nicotiana tabacum, nicotine, regulon 77



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Introduction 78 In plants, a large number of structurally diverse specialized metabolites are 79 produced through long, multi-step, and often branched pathways (Arimura 80 et al., 2017). The proper functioning of such pathways, allowing massive 81 metabolic flows leading to complex products from simple precursors, 82 largely relies on the concerted expression of a large set of metabolic and 83 transport genes, or structural genes, in different developmental and 84 environmental contexts. The transcription factors regulating these pathways, 85 play a critical role in such coordination, which often occurs at transcription 86 level. The regulatory transcription factors and downstream structural genes, 87 which form regulatory networks of multiple genes, or regulons, have begun 88 to be explored intensively through molecular and genomics studies (De 89 Geyter et al., 2012, Patra et al., 2013). 90

Tobacco is cultivated as an economically important crop around the 91 globe (Davis and Nielsen, 1999). The main cultivated species, Nicotiana 92 tabacum, hereafter called tobacco, is a natural allotetraploid possibly 93 derived through the hybridization between two ancestral diploids that are 94 closely related to current N. sylvestris and N. tomentosiformis (Murad et al., 95 2002, Sierro et al., 2013, Wang and Bennetzen, 2015). In tobacco, 96 nicotine is an abundant predominant alkaloid produced in the roots and 97 accumulating mainly in the leaves (Shoji and Hashimoto, 2011a, Dewey 98 and Xie, 2013). As a defense toxin, nicotine production is drastically 99 increased in response to damage caused by grazing herbivores (Baldwin, 100 1989), and jasmonates play a central signaling role in the damage-induced 101 nicotine biosynthesis (Baldwin et al., 1994, Shoji et al., 2000, Shoji et al., 102 2008). Nicotine has heterocyclic pyridine and pyrrolidine rings (Shoji and 103 Hashimoto, 2011a, Dewey and Xie, 2013); the pyridine ring is formed 104 through consecutive reactions catalyzed by ornithine decarboxylase (ODC; 105 Imanishi et al., 1998, De Boer et al., 2011a), putrescine 106 N-methyltransferase (PMT; Hibi et al., 1994), and N-methylputrescine 107 oxidase (MPO; Heim et al., 2007, Katoh et al., 2007), while enzymes 108 involved in early steps of NAD synthesis, aspartate oxidase (AO), 109 quinolinate synthase (QS), and quinolinate phosphoribosyl transferase 110 (QPT) are responsible for the formation of the pyrrolidine ring (Sinclair et 111 al., 2000, Katoh et al., 2006) (Fig. 1). It has been proposed that PMT and 112 MPO have evolved from spermidine synthase (SPDS) and diamine oxidase 113



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(DAO), two homologous enzymes with different catalytic activities, both of 114 which accept putrescine as a substrate and thus are involved in polyamine 115 metabolism (Hibi et al., 1994, Hashimoto et al., 1998, Junker et al., 2013, 116 Naconsie et al., 2014). Two orphan oxidoreductases of different families, 117 A622 (Hibi et al., 1994, De Boer et al., 2009, Kajikawa et al., 2009) and 118 berberine bridge enzyme-like (BBL) proteins (Kajikawa et al., 2011), are 119 required for later steps including coupling of the two rings (Fig. 1), but the 120 exact biochemical reactions catalyzed by these enzymes have yet to be 121



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determined. In tobacco roots, a pair of tonoplast-localized multidrug and 122 toxic compound extrusion (MATE)-family transporters, MATE1 and 123 MATE2 (Shoji et al., 2009) mediate vacuolar sequestration of nicotine, 124 whereas nicotine and vitamin B6, both with a pyridine ring, are imported 125 into the cells by a purine permease-like transporter, nicotine uptake 126 permease 1 (NUP1), localized at plasma membranes (Hildreth et al., 2011, 127 Kato et al., 2014). In addition to the transport function, NUP1 was 128 proposed to be involved in the regulation of root growth and nicotine 129 biosynthesis, and thus may have a regulatory role as well (Hildreth et al., 130 2011, Kato et al., 2015). 131

Nicotine contents in tobacco plants is genetically controlled by two 132 distinct loci NICOTINE1 and NICOTINE2 (NIC1 and NIC2), and their 133 mutant alleles nic1 and nic2 have been used to breed a low-nicotine 134 tobacco cultivars (Legg and Collins, 1971, Chaplin, 1975, Hibi et al., 1994). 135 A small group of genes encoding closely related ETHYLENE RESPONSE 136 FACTOR (ERF) transcription factors, which are in a clade within group 137 IXa subfamily (Nakano et al., 2006), are clustered at NIC2 locus, and at 138 least seven such genes (ERF17, ERF104ΔC, ERF115, ERF168, ERF179, 139 ERF189, ERF221), called NIC2-locus ERFs, were found to be deleted in 140 the nic2 mutant (Shoji et al., 2010). In tobacco, all the NIC2-locus ERFs 141 and their homologs are induced by jasmonates (Shoji et al., 2010), while 142 salt stress induce the expression of most of the ERFs but not ERF189 and 143 its closest homolog ERF199 (Shoji and Hashimoto, 2015). As master 144 transcription factors regulating the pathway, jasmonate-inducible ERF189 145 and ERF199 directly up-regulate a nearly complete set of genes involved in 146 nicotine biosynthesis and transport (all the genes mentioned above except 147 NUP1) by recognizing GC-rich P box elements, which resemble, but differ 148 from a typical GCC box, in the promoters of the downstream genes (Shoji 149 et al., 2010, Shoji and Hashimoto, 2011b, 2011c, Shoji and Hashimoto, 150 2012, 2013, Shoji et al., 2013). Interestingly, ORCA3 from Catharanthus 151 roseus (van der Fits and Memelink, 2000) and JRE4/GAME9 from tomato 152 and potato (Cárdenas et al., 2016, Thagun et al., 2016) are homologs of 153 tobacco ERF189, and also regulate jasmonate-inducible defense 154 metabolism, a part of indole alkaloid pathway and a nearly complete 155 pathway for steroidal glycoalkaloid biosynthesis, respectively. Moreover, 156 JRE4/GAME9 is in a cluster of related ERF genes in tomato and potato 157



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genomes, just like the NIC2-locus genes in tobacco (Cárdenas et al., 2016, 158 Thagun et al., 2016). Through interaction with the nicotine-regulating 159 ERF factors, a bHLH-family transcription factor MYC2, a key component 160 in conserved jasmonate signaling (Goossens et al., 2016), positively 161 regulate the nicotine pathway genes by directly binding to G box elements 162 found in their promoters, as well as in way of the ERF genes (De Boer et 163 al., 2011b, Shoji and Hashimoto 2011c, Zhang et al., 2012). 164

Genomics has greatly facilitated our understandings on specialized 165 metabolism in plants. The genome sequence of tobacco (Sierro et al., 2014) 166 allows us to characterize a whole range of features of the entire suite of 167 genes involved in nicotine biosynthesis and related NAD and polyamine 168 metabolism, such as fine molecular phylogenies, genomic arrangements, 169 cis-element distributions in the promoters, and expression of individual 170 genes. Here, we discuss the evolution of this specialized metabolic 171 pathway specific to the Nicotiana linage, with particular focus on gene 172 regulatory aspects. 173 174 175



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Results 176 Genes in the tobacco genome involved in nicotine and related pathways 177 The genes encoding metabolic enzymes and transporters involved in 178 nicotine and related primary metabolism (Fig. 1) were retrieved from 179 tobacco genome sequence of TN90 cultivar (Sierro et al., 2014) using the 180 BLAST functionality at SOL Genomic Network 181 (https://solgenomics.net/tools/blast/) (Supplemental Table S1). To 182 investigate the phylogenetic relationships, the tobacco enzymes, except 183 A622 and BBL, were aligned with their homologs from N. sylvestris, N. 184 tomentosiformis, tomato, pepper, and Arabidopsis (Supplemental Fig. S1). 185 In the phylogenetic trees, most proteins have two copies in tobacco, which 186 group together with their counterparts in ancestral diploids, forming a 187 homeologous group of orthologs. It is possible to divide the proteins in 188 each tree into multiple such orthologous groups. According to the 189 groupings and ancestral origins of their products, the tobacco genes were 190 newly named in this study (Supplemental Table S1, Supplemental Fig. S1), 191 if not identical to already named ones. For A622 (Kajikawa et al., 2009), 192 MATE (Shoji et al., 2009), and NUP (Hildreth et al., 2011) genes, 193 orthologous genes (over 98% identity at the nucleotide level) were found in 194 the genome. ODC and PMT genes were given the names, due to no clear 195 matching to the reported ones (Riechers and Timko, 1999, Xu et al., 2004), 196 and so were BBLd.2 and BBLe and their relations to other Nicotiana BBLs 197 (Kajikawa et al., 2011) are shown (Supplemental Fig. S2). 198

Tobacco QPT1.2 and QPT2.2 of distinct groups, both from N. 199 tomentosiformis, are about 75 kb apart on Super Scaffold (SS) 1382, 200 suggesting a relatively recent duplication giving rise to these two genes 201 (Shoji and Hashimoto, 2011b). A two-gene cluster of non-homologous 202 A622L and MATE2 was found on chromosome 12; these genes from N. 203 tomentosiformis are away about 128 kb away from each other on SS753. 204 Other than those, no clustering was found at the SS level for the listed 205 genes (Supplemental Table S1). Because of their uncertain placements in 206 the genome, we could not confirm the genomic clustering of N. 207 sylvestris-derived counterparts of the clustered genes. 208 209 Expression of nicotine and related primary metabolic genes 210 Transcript levels were estimated by RNA-seq analysis, which yields 211



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expression levels in FPKM (fragments per kilobase of exon per million 212 mapped sequence reads), for each gene assigned on the genome. Based 213 on the FPKM values in various tobacco tissues, all of the metabolic and 214



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transport genes were clustered (Fig. 2A); QPT1.2 and QPT2.2 were 215 excluded because of apparently duplicate mapping of the reads. There is a 216 discrete cluster of 19 genes, including nearly all genes in the nicotine 217 biosynthesis regulon (Fig. 2A). The genes in this cluster are expressed 218 preferentially in the nicotine-producing roots. Such characteristic 219 expression largely restricted to the roots is not evident for genes that are 220 possibly involved in related primary pathway rather than the alkaloid 221 production and form other clusters (Fig. 2A). Though included in the 222 regulon, ODC2.1 and ODC2.2 with substantial expression also in stems 223 and flowers, and BBLc and BBLd.2 with very low FPKM values, are 224 exceptional in this clustering. 225

To complement the RNA-seq results and further address the 226 differential regulation, we examined the expression patterns with 227 quantitative reverse transcription (qRT)-PCR of the metabolic genes 228 involved in early parts of the pathways, which overlap or work in parallel 229 with related primary pathways (Fig. 1). Primer pairs were designed to 230 specifically amplify each gene or group of orthologs (Supplemental Table 231 S2). Transcript levels in organs from tobacco plants, including roots of 232 nic1nic2 mutant with a low-nicotine trait (Legg and Collins, 1971), and 233 cultured BY-2 cells elicited with methyl jasmonate (MeJA), were measured 234 and represented relative to those of a housekeeping gene EF1α (Fig. 2B). 235 As reflected in hierarchal clustering, the expression patterns were again 236 clearly distinguished into two major groups of genes, one possibly forming 237 nicotine biosynthesis regulon and one presumably devoted to parallel 238 primary pathways (Fig. 1). ODC1 clusters with the genes of the former 239 group, but only at a low level of statistical significance, which is likely to 240 be due to its very low expression levels. Thus ODC1 was assigned to the 241 latter group. The genes of the former, including ODC2, PMT, MPO1, 242 AO2, QS1, and QPT2, strongly or often nearly exclusively express in the 243 roots of wild-type tobacco and their levels are decreased to 18 to 68 % 244 levels in nic1nic2 mutant roots, reflecting the regulation by NIC loci and 245 therefore by NIC2-locus ERFs. The genes of this group are markedly 246 up-regulated by MeJA in the cultured cells, leading to alkaloid induction, 247 except for MPO1 as has been previously reported (Shoji and Hashimoto 248 2008),. In contrast to the genes involved in nicotine formation, expression 249 of ODC1, SPDS2, DAO1, AO1, and QPT1 are not restricted to nicotine 250



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producing tissues; they occur more ubiquitously, although their expression 251 levels are generally being lower (except SPDS2), which possibly reflects 252 their contribution to primary pathways. 253 254 Cis-elements predicted in promoter regions 255 The availability of genomic sequences prompted us to predict 256 ERF189-binding P box and MYC2-binding G box elements in the promoter 257 regions of the genes involved in nicotine and related pathways. We 258 examined whether such elements were enriched in nicotine pathway genes. 259 Using weighted matrices (Shoji and Hashimoto 2011b, 2011c) representing 260 P and G boxes, we computationally searched for the binding elements with 261 cut-off scores of 5.0 for 10-mer P box and of 5.5 for 8-mer G box in 262 5’-flanking regions (-1,500 to -1; counted from the first ATG). The scores 263 of the predicted elements in each 100 bp bin along the entire promoter 264 sequences are visualized in a heat map (Fig. 3A). For both elements in 265 the primary metabolic genes, these average score for each bin are relatively 266 low (upper block), whereas for the nicotine pathway genes there are a 267 number of bins with high scores (lower two blocks). Bins scoring highly 268 for both P and G boxes (squared with white lines in the right column) are 269 present in 14 out of the 20 gene promoters of nicotine pathway, implying 270 that these elements are crucial to the expression of these genes. 271 To retrieve the cis-elements shared among the co-regulated genes in 272 a non-targeted way, MEME (Multiple EM for Motif Elicitation) analysis 273 (Bailey et al., 2006) was conducted using the 5’-flanking sequences (-1,500 274 to -1) of twenty genes included in the lower two blocks in Fig. 3A as 275 queries. Highly scoring sequences related to P box (rank; 2, e-value; 276 9.5e-004, log likelihood ratio; 203) and those to G box (rank; 4, e-value; 277 2.9e+0.00, log likelihood ratio; 188) were found in all 20 and 19 promoters, 278 respectively. Logo graphics (Crooks et al., 2004) representing conservation 279 among the retrieved sequences are shown (Fig. 3B). 280 281 Regulation of BBL gene promoter in tobacco 282 In concert with other genes involved in nicotine biosynthesis, BBL genes 283 are strongly expressed in the roots and induced by jasmonates in tobacco 284 (Kajikawa et al., 2011). In order to gain insight into the transcriptional 285 regulation of BBL genes, promoter reporter analyses were performed for 286



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NsBBLa from N. sylvestris (Supplemental Fig. S2). Since the genomic 287 sequence was not available when we began the experiment, the region from 288 -1.126 to -1bp (counted from the first ATG) of NsBBLa was obtained with 289 TAIL-PCR; the sequence was later confirmed 99 % identical to that in the 290



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genome database and 97 % to that of BBLa gene in tobacco genome. Four 291 P box (scores 5.8 to 6.9) and two G box (scores 9.1 and 5.5) elements were 292 predicted within the promoter sequence. To generate transgenic plants 293 and hairy roots, GUS reporter gene driven by the promoter was introduced 294 into tobacco via Agrobacterium-mediated transformation. 295 Expression patterns of the BBL promoter in transgenic seedlings 296 and hairy roots were analyzed by visualizing GUS activities 297 histochemically (Fig. 4). In transgenic seedlings at 2, 5, and 14 days after 298 germination, GUS staining was visible in the roots, but not in any aerial 299 tissues (Fig. 4A-C). Strong staining was also observed in transgenic hairy 300 roots (Fig. 4D-F). In apical regions of the hairy roots, promoter activity 301 was detected in differentiated cells as well as in actively dividing and 302 elongating cells, but was absent in the root cap and in epidermal cells (Fig. 303 4E and F). A cross section of the stained hairy roots in differentiated zone 304 was made to identify cell types associated with the activities; the promoter 305 was expressed most strongly in the outmost cortex layer and moderately in 306 the endodermis and parenchyma cells in the stele, but not in the epidermis 307 (Fig. 4 D). This expression pattern of the BBL promoter is quite similar to 308 those reported for other nicotine pathway genes, such as PMT (Shoji et al., 309 2000), QPT2 (Shoji and Hashimoto 2011a), A622 (Shoji et al., 2002), and 310 MATE1 (Shoji et al., 2009). 311

To examine the jasmonate-mediated response of the BBL promoter, 312 seven transgenic lines were treated with MeJA for 24 h, and the GUS 313 activities in crude root extracts were measured fluorometrically. In five 314 lines, significant increases of the activity (2.3 to 5.1 folds) relative to 315 mock-treated controls were observed (Supplemental Figure S3A). Indeed, 316 GUS staining intensities were increased in transgenic hairy roots treated 317 with MeJA, compared to the controls (Fig. 4E). To demonstrate the 318 regulation of the BBL promoter by NIC loci, multiple lines of transgenic 319 hairy roots with the promoter reporter were generated in wild-type and 320 nic1nic2 mutant genotypes. Higher GUS activities in the wild-type 321 background were clearly observed in histochemical (Fig. 4F) and 322 fluorometric (Supplemental Fig. S3B) assays. 323 324 Gene clusters of NIC2-locus ERFs and their homologs 325 Clustering of ERF genes at NIC2 locus was presumed in a previous study 326



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(Shoji et al., 2010). To understand the genomic organization of the 327 NIC2-locus gene cluster and its possible counterpart from another ancestral 328 diploid, NIC2-locus ERF genes and their homologs were retrieved from the 329



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tobacco genome sequence. 22 ERF genes were retrieved in total, 330 including four genes on unplaced SSs (Supplemental Table S3). Two 331 clusters of multiple ERF genes of the relevant group are found in the 332



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genome: one twelve-gene cluster (spanning about 660 kb, flanked with 333 genetic maker PT53353) from N. tomentosiformis on chromosome (chr.) 19 334 and one six-gene cluster (about 320 kb, flanked with PT51405 and 335



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PT50089) from N. sylvestris on chr. 7 (Fig. 5) (Bindler et al., 2011). A 336 phylogenetic tree of the retrieved ERFs and their homologs can be found in 337 Supplemental Fig. S4. While most ERFs retrieved are practically 338 identical (over 98% at nucleotide level) to the queries, ERF91Ls, ERF17Ls, 339 and JRE5Ls were newly defined according to their phylogenies 340 (Supplemental Fig. S4), the ERF91L genes differ by an N-terminal 64 341 amino acid extensions, but are nearly identical to ERF91 (not in the tree). 342 Also the ERF17L genes are not orthologous to ERF17 or JRE4 from 343 tomato, the nearest neighbor in the tree. Some ERFs are truncated and do 344 not including full-length DNA-binding domains, and therefore may be 345 non-functional as transcription factors; these are denoted with ΔN or ΔC. 346 As reflected in disproportional gene numbers between the clusters, only 347 five out of ten functional NIC2-locus ERFs in the T-genome have clear 348 counterparts in the S-genome; at least four pairs of such ERFs in the 349 clusters (Fig. 5). 350 To delimit the genomic region deleted in nic2 mutant, genomic PCR 351 analysis (Supplemental Fig. S5) was performed with primers designed to 352 detect the sequences at various positions around the NIC2-locus gene 353 cluster (Supplemental Table S4). Genomic DNAs from various NIC 354 genotypes in two cultivars, Burley 21 and NC95, were used for the analysis. 355 Some of the amplified fragments were sequenced to confirm the specific 356 amplifications (Supplemental Fig. S5), which are sometime unsuccessful 357 possibly due to highly repetitive nature of tobacco genome (Sierro et al., 358 2014). A large chromosomal region (about 650 kb) including ten out of 359 twelve clustered ERF genes is missing in nic2 genetic background, while 360 the remaining two genes, ERF163 and ERF91L1, are retained in the 361 genotype (Fig. 5, Supplemental Fig. S5), in agreement with the previous 362 data (Shoji et al., 2010); ERF91L1 was considered an equivalent of former 363 ERF91. These results were also in line with expression data in nic2 mutant 364 of the clustered ERFs and a gene that encodes COP9 signalosome subunit 7 365 and resides between ERF91L1 and the putative breakpoint of the deletion; 366 expression levels of the genes within the deleted region are very low, or 367 below reliable detection limits, in nic2 mutant (Supplemental Fig. S6). As 368 expression levels of ERF168L1ΔC and ERF104ΔC are also below the 369 detection limits (Supplemental Fig. S6), these genes on unplaced SS3881 370 were assumed to reside somewhere in the deleted part of the cluster. 371



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Since genes were predicted with transcriptomics data, only a small 372 fraction of the high expressed ERF genes are found in the current database 373 (Supplemental Table S3) and can be assigned FPKM data; eight ERF genes 374 were annotated, including only one orthologous pair of functional genes, 375 ERF189 and ERF199. FPKM data indicate that ERF189 and ERF199 are 376 expressed almost exclusively in the roots, while a similar root-specific 377 expression (albeit at a lower level) was observed for ERF16 and 378 non-functional ERFs, ERF17L3ΔC and ERF168L1ΔC (Fig. 4C). 379 Expression of JRE5L2 and ERF91L1 is not restricted to the roots, but is 380 also apparent in flowers and other tissues (Fig. 4C). 381 382 Responses of ERF189 and its close homologs to jasmonates and salt 383 stress 384 The availability of the genome sequence allowed us to detect individual 385 ERF genes with qRT-PCR by using carefully designed primers 386 (Supplemental Table S2). We analyzed the responses to jasmonates and 387 salt stress of ERF189 and ERF199, which were proposed to regulate 388 nicotine pathway (Shoji et al., 2010, Shoji and Hashimoto, 2015), and their 389 close homologs, ERF16, ERF29, ERF115, and ERF221, which had been 390 detected collectively in the previous studies, in tobacco hairy roots; the 391 cultured roots were treated with MeJA at 100 μM and NaCl at 300 mM. 392 A pair of ERF189 and ERF199 are gradually induced by MeJA, while NaCl 393 treatment has negative impact on their expression (Fig. 6), as reported 394 previously (Shoji et al., 2010, Shoji and Hashimoto 2015). ERF16 is from 395 N. sylvestris and has no obvious counterpart from N. tomentosiformis 396 (Supplemental Fig. S4). ERF16 is induced by both MeJA and NaCl (Fig. 6) 397 and has the highest expression levels among the examined genes 398 (according to qRT-PCR data) (Fig. 5C, Fig. 6); it boasts a 7-fold induction 399 at 0.5 h by MeJA and 5-fold induction at 5 h by NaCl. The expression 400 levels of ERF29, ERF115, and ERF221, which are highly similar to each 401 other as the sequence level (Supplemental Fig. S4), have very low 402 expression relative to ERF16, ERF189, and ERF199 (Fig. 6), and thus may 403 be not annotated as genes in the database. MeJA treatment caused rapid 404 (reaching maximums within 0.5 to 1.5 h) and substantial induction (15 to 405 33 times) of ERF29, ERF115, and ERF221, while their expression also 406 rapidly rose and reached 59- to 226-fold levels after 5 h upon NaCl 407



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treatment (Fig. 6); such large fold changes may reflect their low basal 408 expression. 409 410 Genomic deletions found in nic1nic2 mutant of Burley 21 cultivar 411 In order to identify the genomic deletions in nic1nic2 double mutant other 412 than the ERF gene cluster (Fig. 5), genomic PCR analysis was carried out 413 for the genes strongly down-regulated in the low-nicotine mutant 414



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(Supplemental Fig. S7); the genes highly ranked in a microarray-based 415 screen (Shoji et al., 2010) along with ERF189 (rank; 8, signal ratio of 416 nic1nic2 mutant to wild type; 0.025), were analyzed. We found that 13 417 genes have been deleted (rank: 1 to 38, ratio; 0.013 to 0.133), including 418 presumed genes residing on the same SSs with the confirmed ones, on six 419 SSs associated with at least four regions on three chromosomes in the 420 nic1nic2 double mutant of cv. Burley 21, or cv. LA Burley 21 (Legg and 421 Collins, 1971) (Supplemental Table S5). The presumptive deletions 422 encompass a relatively large chromosomal region, i.e. about 1,730 kb 423 deletion at a one end of chr. 17 (Supplemental Table S5). The deletions 424 were found only in LA Burley 21 but not in other lines, such as nic1 and 425 nic2 single mutant of the same Burley 21 cultivar and nic1nic2 double 426 mutant of cv. NC95 (Chaplin, 1975) (Supplemental Fig. S7). As these are 427 specific to a single line of LA Burley 21, the correlations of these deletions 428 with NIC genotypes and thus nicotine biosynthesis are unlikely. The 429 deletions found at two distinct positions on chr. 19 are far away and thus 430 are apparently different from the deletion at NIC2 locus (Fig. 5A). 431 432 433



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Discussion 434 435 A regulon for nicotine biosynthesis pathway 436 Concerted and substantial expression of structural genes involved in certain 437 pathways is required for massive metabolic flows allowing production and 438 accumulation of specialized metabolites. Such coordination of multiple 439 genes is often extended into preceding primary pathways that supply 440 precursors to downstream metabolism (van der Fits and Memelink, 2000, 441 Cárdenas et al., 2016, Thagun et al., 2016, Moerkercke et al., 2015). In 442 tobacco, a series of metabolic and transport genes involved in a pathway 443 leading to nicotine from ornithine and aspartate express nearly exclusively 444 in the roots and jasmonate-elicited cultured cells (Fig. 2) (Shoji and 445 Hashimoto, 2011a). Such co-expression of these genes largely depends 446 on pathway-controlling ERF transcription factors (Shoji and Hashimoto, 447 2013). The organization of these genes into such a regulon allows the 448 coordination of distinct portions of the pathway from early ring formation 449 steps to late steps including ring coupling and nicotine transport (Fig. 1). 450

Detailed phylogenies (Supplemental Fig. S1) and expression 451 profiling (Fig. 2) clearly distinguished the genes involved in the regulon, as 452 shown in Fig. 1. Genes involved in each step specific to nicotine pathway, 453 such as PMT, MPO, A622, BBL, and MATE, which generally belong to a 454 single orthologous group (Supplemental Fig. S1, Supplemental Fig. S2), 455 are commonly regulated as members of the regulon (Fig. 2). In contrast to 456 the nicotine-specific genes, ODC, AO, QS and QPT genes are involved in 457 early steps that overlap with polyamine or NAD pathway (Fig. 1). To 458 satisfy metabolic demands of different downstream pathways, two types of 459 genes (genes from two groups of orthologs), are present for every 460 overlapping step, except QS (Fig. 1, Supplemental Fig. S1); ODC2, AO2, 461 and QPT2 are involved in the nicotine biosynthesis regulon, while ODC1, 462 AO1, and QPT1 are not subjected to the regulation by the ERFs and 463 jasmonates and are possibly devoted to the primary housekeeping pathways 464 (Fig. 1, Fig. 2). Of course, since such functional differentiations among 465 the genes were presumed from their biased, but not mutually exclusive, 466 expression patterns, transgenic or mutational approaches may be required 467 to validate the contributions of each gene to specific metabolic pathways. 468 Although no differences other than the transcriptional regulation between 469



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the gene types had been suggested to date, slight differences in the protein 470 sequences (Supplemental Fig. S1) could give rise to functional 471 differentiations, thus allowing for distinct metabolic roles (Schenck et al., 472 2015). 473

As reflected by the existence of two types of the genes for the 474 overlapping steps, pyridine and pyrrolidine formation branches of nicotine 475 pathway are presumed to have evolved from universal polyamine and NAD 476 pathways through gene duplication followed with subfunctionalization or 477 neofunctionalization of the duplicates (Fig. 1) (Shoji and Hashimoto, 2011b, 478 Naconsie et al., 2014). The ornithine-derived pyridine ring is utilized as a 479 common building block for formation of tropane, nortropane, and nicotine 480 alkaloids in a number of species of Solanaceae and other families (Shoji 481 and Hashimoto, 2011a). The pyridine formation branch composed with 482 ODC, PMT, and MPO, may have arisen before the diversification of the 483 plants producing alkaloids containing the ornithine-derived moiety. The 484 assumption is supported with the fact that tomato and pepper have at least 485 one gene of ODC2, PMT, and MPO1 (Supplemental Fig. S1) (Stenzel et al., 486 2006). While ODC2, which acts in parallel with ODC1, has retained its 487 original catalytic activity, PMT and MPO1, which have been derived from 488 SPDS and DAO respectively, have acquired novel activities through 489 neofunctionalization (Junker et al., 2013, Naconsie et al., 2014), thus 490 contributing to the innovation of the pyridine-forming extension (Fig. 1). 491 In contrast to the relatively ancient diversification of the pyridine branch 492 from polyamine metabolism, establishment of paralleled routes including 493 AO, QS, and QPT for pyridine formation (Fig. 1), which supply the ring to 494 NAD and nicotine production in tobacco, is presumed to have occurred 495 around the time of the diversification of the Nicotiana lineage, given the 496 specificity of AO2 and QPT2 to the lineage (Supplemental Fig. S1) (Shoji 497 and Hashimoto 2011b, Ryan et al., 2012). This scenario is also in 498 agreement with existence of a gene cluster of QPT1.1 and QPT2.1 in the 499 tobacco genome (Supplemental Table S1) that may have arisen by a 500 relatively recent gene duplication event. It remains to be addressed how 501 and when the ERF factors, which play a central regulatory role in the 502 nicotine biosynthesis regulon in present day tobacco, have evolved to 503 regulate distinct portions of the pathways that may have developed 504 independently at different points in time. 505



23

Clusters of genes encoding non-homologous proteins involved in 506 specialized pathways has been widely recognized in plants (Nützmann and 507 Osborn, 2014), and alkaloid pathways follow suit (Winzer et al., 2012). In 508 the tobacco genome, a cluster of A622L and MATE2 genes, which encode 509 enzymes (De Boer et al., 2009, Kajikawa et al., 2009) and transporters 510 (Shoji et al., 2009), and which are both involved in late steps of the 511 pathway, is situated on chromosome 12 (Supplemental Fig. S1). 512 Localization of the cluster opens up the possibility of identifying genes for 513 as yet undefined late steps based on their proximities to the cluster. Apart 514 from this cluster, we could not find any clustering of the other 515 non-homologous genes; of course, before concluding, exact placements of 516 unplaced SSs should be determined (Supplemental Fig. S1). The 517 dispersion of these structural genes throughout the genome (Supplemental 518 Table S1) implies that regulation of this functionality is dependent on 519 promoter-binding transcription factors, rather than the chromatin-level 520 regulation proposed for clustered genes (Wegel et al., 2009). 521

A number of P and G box elements, which are targeted by ERF189 522 and MYC2 transcription factors, respectively, were computationally 523 predicted in the promoter regions of the genes (Fig. 3A). Occurrences of 524 the predicted elements are more frequent amongst nicotine pathway genes 525 regulated by those transcription factors, than they are amongst primary 526 metabolic genes (Fig. 3A). This finding was complemented by the 527 identification of P and G boxes within the regulated promoters (Fig. 3B). 528 These results support the notion that downstream structural genes have 529 been recruited into the regulons under the control of the transcription 530 factors through generating cognate cis-elements in their promoters (Shoji 531 and Hashimoto, 2011b, Moghe and Last, 2015). Proximities of the two 532 distinct boxes within the promoters (Fig. 3A) (Shoji et al., 2010, Shoji and 533 Hashimoto, 2011b, 2011c), imply the importance of such arrangements of 534 functional cis-elements that possibly support cooperative action of the two 535 transcription factors. As observed previously, both elements are likely to 536 be present in similarly situated proximal promoter regions (Shoji et al., 537 2010, Shoji and Hashimoto, 2011b, Thagun et al., 2016); this is observed 538 for genes such as PMT, QPT2, BBL, and MATE (Fig. 3A),. However, this 539 seems not always to be the case; the likely elements, though none of them 540 have been validated experimentally, are also present in relatively distal 541



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promoter regions of some genes (Fig. 3A) (Xu et al., 2004). It remains to 542 be addressed whether such different placements of cis-elements in the 543 promoters account for slight but significant differences in the expression 544 patterns of the generally co-regulated genes involved in the regulon, such 545 as no elicitation of MPO1 in cultured cells and relatively relaxed 546 suppression of ODC2 and MPO1 in nic1nic2 mutant (Fig. 3A) (Shoji and 547 Hashimoto, 2008). 548

Promoter of the BBLa gene follow the pattern of those of other 549 genes involved in the regulon (Shoji et al., 2000, Shoji et al., 2002, Shoji et 550 al., 2009, Shoji and Hashimoto 2011b) in terms of cell-type specificity (Fig. 551 4), response to jasmonates (Fig. 4E, Supplemental Fig. S3A), and 552 suppression in the nic1nic2 mutant (Fig. 4F, Supplemental Fig. S3B). By 553 recognizing resident P box elements, pathway-controlling ERF factors with 554 the help of MYC2, may mainly contribute to transcriptional regulation 555 underlying such common characteristic expression. The functional 556 importance of P and G box elements for regulation has been demonstrated 557 by loss-of-function experiments with mutated promoters (Shoji et al., 2010, 558 Shoji and Hashimoto, 2011b, 2011c). As yet, no gain-of-function analyses 559 has been performed for these elements (Shoji et al., 2000, Xu and Timko, 560 2004). Compared to regulation by jasmonates and NIC genotypes, it has 561 remained less clear how much the ERF factors contribute to the 562 tissue-specific expression of the promoters. In tomato, expression of 563 JRE4 is well correlated with those of downstream structural genes during 564 developmental progressions (Cárdenas et al., 2016, Thagun et al., 2016). 565 Root-specific expression of ERF189 and ERF199 indicated by RNA-seq 566 (Fig. 5C) gives a first important cue to this issue. Cell-type specificity, 567 in addition to root specificity, is considered characteristic to the regulon, as 568 reflected by the epidermal expression of NUP1 promoter contrasted with 569 those of the ERF-regulated promoters (Kato et al., 2014). 570 571 Evolution of clustered transcription factor genes 572 The genetic locus NIC2 controlling nicotine content in tobacco (Legg and 573 Collins, 1971, Hibi et al., 1994, Shoji et al., 2010) was finally elucidated; 574 complete sequence of the tobacco genome (Sierro et al., 2014) allowed us 575 to figure out the structure of ERF gene clusters at NIC2 locus originated 576 from N. tomentosiformis and its counterpart from N. sylvestris (Fig. 5A and 577



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B). A similar cluster of five ERF genes is present in tomato genome 578 (Cárdenas et al., 2016, Thagun et al., 2016). While ERF and COP9 579 signalosome genes conserved between distantly related tomato and tobacco 580 are similarly situated around the peripheral area of the clusters, central 581 regions are occupied by ERF genes specific to each lineage, such as 582 ERF189 and JRE4 (Fig. 5B, Supplemental Fig. S4, Supplemental Fig. S6). 583 The structural arrangement of the clusters suggests more dynamic 584 rearrangement of the divergent central area, which may favor formation of 585 the ERFs devoted to linage-specific alkaloid regulation. Tobacco has two 586 sequences with similarities to JRE6 from tomato (not shown in 587 Supplemental Table S3 and Supplemental Fig. S4 because of incomplete 588 reading frames). In tomato, apart from the ERF gene cluster on chr. 1, 589 JRE6 is present as a singleton on chr. 5 (Thagun et al. 2016). The tobacco 590 JRE6-like sequences may be situated somewhere around clusters, since 591 they reside on unplaced SSs associated with same chromosomes as the 592 clusters. The possible association of the JRE6-like sequences with the 593 clusters in tobacco implies the involvement of JRE6 orthologs in ancient 594 clusters and consequent relocation of them to other chromosomes during 595 formation of the tomato cluster. 596

There is considerable structural divergence even between the two 597 clusters originating from distinct Nicotiana diploids in tobacco (Fig. 5), 598 suggesting relatively frequent loss and gain of ERF genes and their 599 replacements within the clusters even after diversification of the two 600 ancestral diploids. Tandem repeats of homologous sequences may have 601 contributed to highly frequent unequal crossing-over events leading to such 602 chromosomal changes around the regions. A number of genes encoding 603 truncated ERFs (Fig. 5, Supplemental Table S3) may be vestiges of 604 recombination events which have occurred in the intragenic regions. 605 Phylogenetic analysis revealed the existence of only very few homeologous 606 pairs of functional ERF genes in tobacco genome as well as a considerable 607 number of remaining unpaired or possibly non-functional genes 608 (Supplemental Fig. S4). For these, the biological relevance is unclear, 609 considering evolutionary proximity of N. tomentosiformis and N. sylvestris. 610 Phylogenetic (Supplemental Fig. S4) and gene expression (Fig. 5C, Fig. 6) 611 evidence point to the functional importance of a pair of ERF189 and 612 ERF199, one of the three Nicotiana-specific pairs (Fig. 5B, Supplemental 613



26

Fig. S4). These have been previously proposed to function as a regulator of 614 the nicotine pathway (Shoji et al., 2010, Shoji and Hashimoto, 2015). A 615 pair of genes, ERF221 and ERF29, which is the nearest functional pair to 616 ERF189 and ERF199 in the phylogenetic tree (Supplemental Fig. S4), is 617 expressed basally at low levels and is induced not only by MeJA but also 618 by NaCl (Fig. 5C, Fig. 6). It is intriguing to uncover how only a certain 619 ERF gene (or a certain pair of genes in tobacco case) among the many 620 clustered genes, has become the predominant regulatory gene (Fig. 5C, Fig. 621 6) (Thagun et al., 2016) and functionally important for a specialized 622 pathway in each plant lineage. 623

624 Genomic deletions in low-nicotine mutants 625 Molecular basis of nic2 mutation was described; the mutant allele has a 626 large chromosomal deletion that encompasses ten ERF genes in the cluster 627 at NIC2 locus on chr. 19, including ERF189 (Fig. 5B, Supplemental Fig. 628 S5). Repetitive sequences present throughout the tobacco genome (Sierro 629 et al., 2014) prevented further delimitation of the deleted region; regions of 630 about 13 and 20 kb in size remain to be elucidated (Supplemental Fig. S5). 631 Whole-genome or targeted re-sequencing and cytogenetic analyses may 632 help to precisely determine breakpoints of the deletion and to detect 633 possible structural changes at chromosomal level, respectively. Absence 634 of a major deletion in the counterpart locus on chr. 7 has yet to be 635 confirmed in nic1 genetic background (unpublished data). 636

We found a number of genomic deletions other than that at NIC2 637 locus in nic1nic2 mutant of Burley 21 cultivar, or cv. LA Burley 21, at least 638 at four positions on three chromosomes (Supplemental Table S5, 639 Supplemental Fig. S7). A lot of genes, the expression of which is severely 640 suppressed in nic1nic2 mutant (signal ratio of nic1nic2 to wild type; < 641 0.113) (Shoji et al., 2010), are missing in the mutant (Supplemental Table 642 S5, Supplemental Fig. S7). Since these deletions are specific to LA 643 Burley 21 and not found in other varieties, and thus may not be associated 644 with the NIC genotypes and thus with nicotine biosynthesis. Note that the 645 nic1nic2 double mutant was not generated by crossing the corresponding 646 single mutants, but rather derived from an original Cuban cigar cultivar as 647 its genotype (Legg and Collins 1971). The presence of multiple deletions 648 specific to a certain line implies that the nature of the tobacco genome is 649



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highly prone to structural variations. 650 651 Materials and Methods 652 Plant growth and treatment 653 Sterilized seeds of Nicotiana tabacum (tobacco), N. sylvestris, and N. 654 tomentosiformis were germinated and grown to seedlings on half-strength 655 Gamborg B5 medium solidified with 0.3% (w/v) gellan gam and 656 supplemented with 2% (w/v) sucrose. The wild-type, nic1, nic2, and 657 nic1nic2 (registered as cv. LA Burley 21) genotypes in the Burley 21 658 cultivar (Legg and Collins, 1971) were obtained from USDA, the wild-type 659 and nic1nic2 (registered as LAFC53) genotypes in the NC95 cultivar 660 (Chaplin, 1975) from John Hamill (Deakin University), and N. tabacum cv 661 Petit Havana line SR1, N. sylvestris, and N. tomentosiformis from Japan 662 Tobacco. Two-week-old seedlings were transferred onto soil in pots and 663 grown to maturity in the green house. Tobacco BY-2 cells were cultured 664 as described (Nagata et al., 1992). To induce nicotine biosynthesis, 665 4-d-old BY-2 cells were rinsed five times to remove auxin, transferred to 666 auxin-free medium supplemented with MeJA at 100 μM, and cultured for 667 24 h. 668 669 RNA-seq analysis 670 Sequencing data for RNA from dry capsule (SRX495517, SRX495518, 671 SRX495519), senescent flower (SRX495530, SRX495531), mature flower 672 (SRX495520, SRX495521, SRX495522), immature flower (SRX495602, 673 SRX495603, SRX495605), senescent leaf (SRX495532, SRX495534, 674 SRX495535), mature leaf (SRX495523, SRX495524, SRX495525), young 675 leaf (SRX495606, SRX495607, SRX495608), stem (SRX495598, 676 SRX495600, SRX495601) and root (SRX495526, SRX495527, 677 SRX495529) of Nicotiana tabacum TN90 were obtained from BioProject 678 PRJNA208209 (Sierro et al., 2014). 679

Reads were mapped to the genome of N. tabacum TN90 680 (ftp://ftp.solgenomics.net/genomes/Nicotiana_tabacum/assembly/Ntab-TN681 90_AYMY-SS.fa.gz) using hisat2 (v2.0.5) (Kim et al., 2015) and filtered 682 using samtools (v1.3.1) (Li et al., 2009) to retain properly paired read pairs 683 not annotated as secondary, QC failed, duplicate or supplementary. 684

For each N. tabacum TN90 gene model 685



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(ftp://ftp.solgenomics.net/genomes/Nicotiana_tabacum/annotation/Ntab-T686 N90_AYMY-SS_NGS_rnaseq.gff3), expression was calculated from the 687 filtered mapped reads using stringtie (v1.3.1c) (Pertea et al., 2016). Gene 688 level expression values were obtained by summing the FPKM of the gene's 689 isoforms. 690 691 Computational analyses 692 Full-length protein sequences were aligned with ClustalW (Thompson et al., 693 1994), and using the alignments, phylogenetic trees were generated with 694 MEGA6 software (Tamura et al., 2013) with the neighbor-joining 695 algorithm. 696

The putative ERF189 and MYC2-binding elements in the 697 5’-flanking regions from -1,500 to -1 bp (numbered from the first ATG) of 698 metabolic and transport genes (Supplemental Table S1) were searched and 699 scored with Regulatory Sequence Analysis Tools (RSAT; Turatsinze et al., 700 2008, http://rsat.ulb.ac.be/rsat/), using weigh matrices for P box (Shoji et al., 701 2011a) and G box (Shoji et al., 2011b). 702

MEME analysis (v. 4.,11.2) (Bailey et al., 2006) was performed by 703 selecting an option of zero or one occurrence per sequence and setting 704 minimum width to 6 and maximum width to 15 and 12 when retrieving P 705 box- and G box-related sequences, respectively. 706 707 Plant transformation 708 The 5’-flanking region of NsBBLa was cloned from N. sylvestris genomic 709 DNA by a TAIL-PCR method (Liu et al., 1995). A promoter region from 710 -1,126 to -1 (numbered from the first ATG) of NsBBLa was placed 711 upstream of GUS coding sequence on pGBW3 using a Gateway 712 Technology (Thermo Fisher Scientific). To generate transgenic tobacco 713 plants and hairy roots, leaf discs from N. tabacum cv. NC95 of wild type 714 and nic1nic2 mutant were infected with Agrobacterium tumefaciens strain 715 EHA105 and A. rhizogenes strain ATCC15834 harboring the GUS reporter 716 construct, respectively (Horsch et al., 1985). Wild-type tobacco hairy 717 roots used in Fig. 6 were induced in a similar fashion, but with N. tabacum 718 cv. Petit Havana line SR1 and the A. rhizogenes strain without the binary 719 vector. The hairy root lines were subcultured every week in 125 ml glass 720 flasks filled with 30 ml of liquid B5 medium supplemented with 2% (w/v) 721



29

sucrose with shaking at 100 r.p.m. in the dark. MeJA and NaCl were 722 directly added to 4-day-old cultures to final concentrations of 100 μM and 723 300 mM, respectively. 724 725 GUS reporter assays 726 The GUS activity was detected histochemically and cross sections of the 727 hairy roots were prepared as described (Shoji et al., 2000). Images of the 728 stained tissues and sections were captured with SZX12 (Olympus) or 729 Eclipse E-1000 (Nikon) microscopes, both equipped with a digital camera 730 DP-70 (Olympus). 731

Frozen hairy roots were homogenized in 50 mM potassium 732 phosphate buffer (pH 7.0), 10 mM EDTA (pH 8.0), 0.1% (v/v) Triton 733 X-100, 0.1% (w/v) Sarcosyl. After centrifugation, the supernatants were 734 desalted through NAP-5 columns (GE health care) with phosphate buffered 735 saline. Protein concentrations were determined with Coomassie Protein 736 Assay Reagent (Thermo Fisher Scientific). The protein solutions was 737 supplemented with 4-methylumbelliferyl-β-D-glucuronide at 150 μM and 738 incubated at 37 C for the reactions. The amount of 4-methylumbeliferone 739 formed was measured by using the fluorescence spectrophotometer F-4500 740 (Hitachi). 741 742 Genomic PCR analysis 743 Genomic DNA was isolated using the CTAB method (Murray and 744 Thompson, 1980) and used for PCR with Ex Taq DNA polymerase 745 (Takara). The thermal program details for each primer pair are available 746 upon request. 747 748 Accession Number 749 The promoter sequence of NsBBLa genes from N. sylvestris can be found in 750 the GenBank/EMBL/DDBJ database under the accession number 751 LC201808. 752 753 Acknowledgments 754 We thank Drs. Tsuyoshi Nakagawa (Shimane University) and John Hamill 755 (Deakin University) for providing pGBW3 vector and seeds of the NC95 756 cultivar, respectively. We are grateful to Drs. James Battey for David 757



30

Page critically reading and correcting the manuscript. 758 759 Supplemental Data 760 Supplemental Figure S1. Phylogenetic trees of enzyme proteins involved 761 in nicotine and related primary metabolism. 762 Supplemental Figure S2. A phylogenetic tree of BBL proteins from 763 tobacco and its ancestral Nicotiana diploids. 764 Supplemental Figure S3. GUS activities in tobacco hairy roots 765 transformed with the reporter gene driven by NsBBLa promoter. 766 Supplemental Figure S4. A phylogenetic tree of NIC2-locus ERFs and 767 related proteins. 768 Supplemental Figure S5. A chromosomal region deleted in nic2 mutant 769 was delimitated with genomic PCR analysis. 770 Supplemental Figure S6. Transcript levels of genes around NIC2 locus in 771 the roots of wild-type and nic2 mutant tobacco were analyzed by qRT-PCR. 772 Supplemental Figure S7. Genes deleted in LA Burley 21 of nic1nic2 773 genotype were found by genomic PCR analysis. 774 Supplemental Table S1. Metabolic enzyme and transporter genes involved 775 in nicotine and related metabolism in tobacco. 776 Supplemental Table S2. Primer sequences for qRT-PCR analysis. 777 Supplemental Table S3. NIC2-locus ERF genes and their homologs in 778 tobacco. 779 Supplemental Table S4. Primer sequences for genomic PCR analysis to 780 detect the chromosomal delation around NIC2 locus in the mutants. 781 Supplemental Table S5. Gene found to be deleted in LA Burley21. 782 Supplemental Table S6. Primer sequences for genomic PCR analysis to 783 detect genes delated in LA Burley21. 784 785 Figure legends 786 787 Figure 1. Pathways of nicotine, NAD, and polyamine metabolism in 788 tobacco. Each defined enzymatic step is represented by an arrow and 789 enzyme name, while undefined single or multiple step processes are 790 represented by broken arrows. Boxes denote enzymes hypothesized to be 791 involved predominantly in the nicotine biosynthesis pathway, while 792 enzymes predominantly associated with related primary metabolic 793



31

pathways are not framed. Quinolinate synthase (QS) is underlined as it 794 contributes to both nicotine and NAD pathways. AO, aspartate oxidase; QS, 795 quinolinate synthase; QPT, quinolinate phosphoribosyl transferase; ODC, 796 ornithine decarboxylase; PMT, putrescine N-methyltransferase; DAO, 797 diamine oxidase; MPO, N-methylputrescine oxidase; BBL, berberine 798 bridge enzyme-like protein. 799

800

Figure 2. Heat map visualization of expression data of metabolic genes 801 involved in nicotine and related primary metabolism in tobacco. A, Heat 802 map visualizing FKPM values obtained by RNA-seq analysis. B, Heat 803 map visualizing transcript levels estimated by qRT-PCR analysis. 804 Transcript levels in various organs of tobacco plants and cultured BY-2 805 cells elicited or not with MeJA were analyzed. Using average values of 806 three biological replicates, the levels are calculated relative to those of 807 EF1α, and are shown relative to the highest one (set to 100, marked with 808 white asterisk) in the dataset. Clustering was done with Cluster 3.0 and 809 heat maps with a tree was drawn with Java TreeView. 810 811 Figure 3. ERF189-binding P box and MYC2-binding G box elements 812 predicted in 5’-flanking regions from -1,500 to -1 (numbered from the first 813 ATG) of metabolic and transport genes involved in nicotine and related 814 primary metabolism. A, Heat map visualizing distributions of the elements 815 predicted with RSAT software; elements with scores over 5.5 for P box and 816 5.0 for G box included. Colors reflect scores of the elements (or sums of 817 those when multiples are predicted) in each bin. At borders between bins, 818 elements were assigned into ones proximal to the first ATG. Sums of 819 scores for the both boxes are in the right column (merge), where the bins 820 including both P box and G box are squared with white lines. The values 821 are averaged for a gene set in each block. Genomic sequence of a region 822 from -1,500 to -1,200 is not available (NA) for BBLd. PMT3 and BBLc 823 were excluded, since 5’-flanking sequences available were too short (less 824 than 200 bp). B, Sequence logos representing conservations of sets of 825 sequences related to P box (left) and G box (right) retrieved by MEME 826 analysis from the promoter regions of the genes included in lower two 827 blocks of A. 828



32

829 Figure 4. Histochemical GUS staining of tobacco seedlings and hairy 830 roots transformed with the reporter gene driven by NsBBLa promoter. 831 The GUS reporter gene was driven by a promoter region from -1,162 to -1 832 (numbered from first ATG) of NsBBLa gene from N. sylvestris. Two (A), 833 five (B), and fourteen (C) day-olds seedlings. D, A cross section of a 834 transgenic hairy root in the differentiation zone. E, Hairy roots (wild-type 835 line no. 8) were treated with 100μM MeJA for 24 h. F, Hairy roots of wild 836 type (WT) (line no. 13) and of nic1nic2 mutant (line no. 6). Scale bars; 0.5 837 mm in A, 2 mm in B and C, 0.1 mm in D. co; cortex, en; endodermis, ep; 838 epidermis. 839 840 Figure 5 Clusters of NIC2-locus ERF genes and their homologs on tobacco 841 chromosomes. A, Positions of clustered ERFs and flanking genetic markers 842 on chromosomes. Positions of ERFs at ends of the clusters and the nearest 843 genetic markers anchored on genome sequence are shown. Positions of 844 genomic deletions on chr. 19 found in cv. LA Burley 21 (Supplemental 845 Table S5) are shown with gray arrowheads and corresponding SS numbers. 846 B, Graphic views of the gene clusters. A large chromosomal region (ca. 847 650 kb) indicated was found to be deleted in nic2 mutant (Supplemental 848 Figure S5). Arrowheads indicate positions and orientations of predicted 849 open reading frames of ERFs. ERF genes denoted with Δ and shown in 850 gray, encode possibly non-functional transcription factors without 851 full-length DNA-binding domains. Functional ERFs of same ortholog 852 groups (Supplemental Figure S4) are paired with dotted lines. C, Heat 853 map visualizing FPKM values of ERF genes. ERF genes on unplaced SSs 854 (but associated with chr. 7 or chr. 19) (Supplemental Fig. S2) are marked 855 with asterisks. 856

857

Figure 6. Response of ERF189 and related genes to jasmonate and salt 858 stress in tobacco hairy roots. Transcript levels were analyzed with 859 qRT-PCR in tobacco hairy roots treated with 100 μM MeJA (A) or 300 mM 860 NaCl (B) for 0, 0.5, 1.5, 5, or 24 h. The error bars indicate standard 861 deviations over three biological replicates. The levels are expressed 862



33

relative to those of EF1α. Note that different vertical scales are adapted 863 for ERF16 and for others. 864 865 866



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http://www.ncbi.nlm.nih.gov/pubmed?term=Kim D%2C Langmead B%2C Salzberg SL %282015%29 HISAT%3A a fast spliced aligner with low memory requirements%2E Nature Methods 12%3A 357%2D360&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Legg PG%2C Collins GB %281971%29 Inheritance of percent total alkaloids in Nicotiana tabacum L%2E II%2E Genetic effects of two loci in Burley 21 x LA Burley 21 populations%2E Can J Genet Cytol 13%3A 287%2D291&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Li H%2C Handsaker B%2C Wysoker A%2C Fennell T%2C Ruan J%2C Homer N%2C Marth G%2C Abecasis G%2C Durbin R and 1000 Genome Project Data Processing Subgroup %282009%29 The Sequence alignment%2Fmap %28SAM%29 format and SAMtools%2E Bioinformatics 25%3A 2078%2D2079&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Liu YG%2C Mitsukawa N%2C Oosumi T%2C Whittier RF %281995%29 Efficient isolation and mapping of Arabidopsis thaliana T%2DDNA insert junction by thermal asymmetric interlaced PCR%2E Plant J 8%3A 457%2D463&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Moerkercke AV%2C Steensma P%2C Schweizer F%2C Pollier J%2C Gariboldi I%2C Payne R%2C Bossche RV%2C Miettinen K%2C Espoz J%2C Purnama PC et al %282015%29 The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus%2E Proc Natl Acad Sci USA 112%3A 8130%2D8135&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Moghe GD%2C Last RL %282015%29 Something old%2C something new%3A conserved enzyme and the evolution of novelty in plant specialized metabolism%2E Plant Physiol 169%3A 1512%2D1523&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Murad L%2C Lim KY%2C Christopodulou V%2C Matyasek R%2C Lichtenstein C%2C Kovarik A%2C Leitch A %282002%29 The origin of tobacco%27s T genome is traced to a particular linage within Nicotiana tomentosiformis %28Solanaceae%29%2E Am J Bot 89%3A 921%2D928&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Murray MG%2C Thompson WF %281980%29 Rapid isolation of high molecular weight plant DNA%2E Nucleic Acids Res 8%3A 4321%2D4325&dopt=abstract

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Wegel E, Koumproglou R, Shaw P, Osbourn A (2009) Cell type-specific chromatin decondensation of a metabolic gene cluster inoats. Plant Cell 21: 3926-3936


Winzer T, Gazda V, He Z, Kaminski F, Kern M, Larson TR, Li Y, Meade F, Teodor R, Vaistij FE et al (2012) A Papaver somniferum 10-gene cluster for synthesis of the anticancer alkaloid noscapine. Science 336: 1704-1708


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Xu B, Timko MP (2004) Methyl jasmonate induced expression of the tobacco putrescine N-methyltransferase genes requires bothG-box and GCC-motif elements. Plant Mol Biol 55: 743-761


Zhang HB, Bokowiec MT, Rushton PJ, Han SC, Timko MP (2012) Tobacco transcription factors NtMYC2a and NtMYC2b formnuclear complexes with the NtJAZ1 repressor and regulate multiple jasmonate-inducible steps in nicotine biosynthesis. Mol Plant5: 73-84



http://www.ncbi.nlm.nih.gov/pubmed?term=Wegel E%2C Koumproglou R%2C Shaw P%2C Osbourn A %282009%29 Cell type%2Dspecific chromatin decondensation of a metabolic gene cluster in oats%2E Plant Cell 21%3A 3926%2D3936&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Winzer T%2C Gazda V%2C He Z%2C Kaminski F%2C Kern M%2C Larson TR%2C Li Y%2C Meade F%2C Teodor R%2C Vaistij FE et al %282012%29 A Papaver somniferum 10%2Dgene cluster for synthesis of the anticancer alkaloid noscapine%2E Science 336%3A 1704%2D1708&dopt=abstract

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http://scholar.google.com/scholar?as_q=Methyl jasmonate induced expression of the tobacco putrescine N-methyltransferase genes requires both G-box and GCC-motif elements&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Xu&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Zhang HB%2C Bokowiec MT%2C Rushton PJ%2C Han SC%2C Timko MP %282012%29 Tobacco transcription factors NtMYC2a and NtMYC2b form nuclear complexes with the NtJAZ1 repressor and regulate multiple jasmonate%2Dinducible steps in nicotine biosynthesis%2E Mol Plant 5%3A 73%2D84&dopt=abstract

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http://scholar.google.com/scholar?as_q=Tobacco transcription factors NtMYC2a and NtMYC2b form nuclear complexes with the NtJAZ1 repressor and regulate multiple jasmonate-inducible steps in nicotine biosynthesis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhang&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1


Short title Nicotine biosynthesis regulon in tobacco Name ... · 155 metabolism, a part of indole...

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