Genomic Insights into the Evolution of the Nicotine Biosynthesis Pathway in Tobacco

Abstract

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

Figure 1.

Pathways of nicotine, NAD, and polyamine metabolism in tobacco. Each defined enzymatic step is represented by an arrow and enzyme name, whereas undefined single or multiple step processes are represented by broken arrows. Boxes denote enzymes hypothesized to be involved predominantly in the nicotine biosynthesis pathway, whereas enzymes predominantly associated with related primary metabolic pathways are not framed. QS is underlined as it contributes to both nicotine and NAD pathways.

Figure 2.

Heat map visualization of expression data of metabolic genes involved in nicotine and related primary metabolism in tobacco. A, Heat map visualizing FKPM values obtained by RNA-seq analysis. B, Heat map visualizing transcript levels estimated by qRT-PCR analysis. Transcript levels in various organs of tobacco plants and cultured BY-2 cells elicited or not with MeJA were analyzed. Using average values of three biological replicates, the levels are calculated relative to those of EF1α, and are shown relative to the highest one (set to 100) in the data set. Clustering was done with Cluster 3.0 (http://bonsai.hgc.jp/∼mdehoon/software/cluster/software.htm) and heat maps with a tree were drawn with Java TreeView (http://jtreeview.sourceforge.net/).

Figure 3.

ERF189-binding P box and MYC2-binding G box elements predicted in 5′-flanking regions from −1,500 to −1 (numbered from the first ATG) of metabolic and transport genes involved in nicotine and related primary metabolism. A, Heat map visualizing distributions of the elements predicted with Regulatory Sequence Analysis Tools (http://rsat.ulb.ac.be/rsat/); elements with scores greater than 5.5 for P box and 5.0 for G box were included. Colors reflect scores of the elements (or sums of those when multiples are predicted) in each bin. At borders between bins, elements were assigned into those proximal to the first ATG. Sums of scores for both boxes are in the right column (merge), where the bins including both P box and G box are squared with white lines. The values are averaged for a gene set in each block. Genomic sequence of a region from −1,500 to −1,200 is not available for MPO1.1 and BBLd.2. PMT3 and BBLc were excluded, because 5′-flanking sequences available were too short (<200 bp). B, Sequence logos representing conservations of sets of sequences related to P box (left) and G box (right) retrieved by MEME analysis from the promoter regions of the genes included in the lower two blocks of (A). NA, Not available.

Figure 4.

Histochemical GUS staining of tobacco seedlings and hairy roots transformed with the reporter gene driven by NsBBLa promoter. The GUS reporter gene was driven by a promoter region from −1,162 to −1 (numbered from first ATG) of NsBBLa gene from N. sylvestris. Two- (A), five- (B), and fourteen- (C) day-old seedlings. D, A cross section of a transgenic hairy root in the differentiation zone. E, Hairy roots (wild-type line no. 8) were treated with 100 μm MeJA for 24 h. F, Hairy roots of wild type (line no. 13) and of nic1nic2 mutant (line no. 6). Bars = 0.5 mm in A, 2 mm in B and C, and 0.1 mm in D. co, Cortex; en, endodermis; ep, epidermis; WT, wild type.

Figure 5.

Clusters of NIC2-locus ERF genes and their homologs on tobacco chromosomes. A, Positions of clustered ERFs and flanking genetic markers on chromosomes. Positions of ERFs at ends of the clusters and the nearest genetic markers anchored on genome sequence are shown. Positions of genomic deletions on chr. 19 found in cv LA Burley 21 (Supplemental Table S5) are shown with gray arrowheads and corresponding SS numbers. B, Graphic views of the gene clusters. A large chromosomal region (∼650 kb) indicated was found to be deleted in nic2 mutant (Supplemental Fig. S5). Arrowheads indicate positions and orientations of predicted open reading frames of ERFs. The ERF genes, denoted with Δ and shown in gray, encode possibly nonfunctional transcription factors without full-length DNA-binding domains. Functional ERFs of same ortholog groups (Supplemental Fig. S4) are paired with dotted lines. C, Heat map visualizing FPKM values of ERF genes. ERF genes on unplaced SSs (but associated with chr. 7 or chr. 19; Supplemental Fig. S2) are marked with asterisks.

Figure 6.

Response of ERF189 and related genes to jasmonate and salt stress in tobacco hairy roots. Transcript levels were analyzed with qRT-PCR in tobacco hairy roots treated with 100 μm MeJA (A) or 300 mm NaCl (B) for 0, 0.5, 1.5, 5, or 24 h. The error bars indicate sds over three biological replicates. The levels are expressed relative to those of EF1α. Note that different vertical scales are adapted for ERF16 and for others.