Nicotine, the signature alkaloid of Nicotiana species responsible for the addictive properties of human tobacco smoking, functions as a defensive neurotoxin against attacking herbivores. However, the evolution of the genetic features that contributed to the assembly of the nicotine biosynthetic pathway remains unknown. We sequenced and assembled genomes of two wild tobaccos, Nicotiana attenuata (2.5 Gb) and Nicotiana obtusifolia (1.5 Gb), two ecological models for investigating adaptive traits in nature. We show that after the Solanaceae whole-genome triplication event, a repertoire of rapidly expanding transposable elements (TEs) bloated these Nicotiana genomes, promoted expression divergences among duplicated genes, and contributed to the evolution of herbivoryinduced signaling and defenses, including nicotine biosynthesis. The biosynthetic machinery that allows for nicotine synthesis in the roots evolved from the stepwise duplications of two ancient primary metabolic pathways: the polyamine and nicotinamide adenine dinucleotide (NAD) pathways. In contrast to the duplication of the polyamine pathway that is shared among several solanaceous genera producing polyamine-derived tropane alkaloids, we found that lineage-specific duplications within the NAD pathway and the evolution of rootspecific expression of the duplicated Solanaceae-specific ethylene response factor that activates the expression of all nicotine biosynthetic genes resulted in the innovative and efficient production of nicotine in the genus Nicotiana. Transcription factor binding motifs derived from TEs may have contributed to the coexpression of nicotine biosynthetic pathway genes and coordinated the metabolic flux. Together, these results provide evidence that TEs and gene duplications facilitated the emergence of a key metabolic innovation relevant to plant fitness.Nicotiana genomes | genome-wide multiplications | transposable elements | nicotine biosynthesis | expression divergence

Background Nicotiana attenuata (coyote tobacco) is an ecological model for studying plant-environment interactions and plant gene function under real-world conditions. During the last decade, large amounts of genomic, transcriptomic and metabolomic data have been generated with this plant which has provided new insights into how native plants interact with herbivores, pollinators and microbes. However, an integrative and open access platform that allows for the efficient mining of these -omics data remained unavailable until now.DescriptionWe present the Nicotiana attenuata Data Hub (NaDH) as a centralized platform for integrating and visualizing genomic, phylogenomic, transcriptomic and metabolomic data in N. attenuata. The NaDH currently hosts collections of predicted protein coding sequences of 11 plant species, including two recently sequenced Nicotiana species, and their functional annotations, 222 microarray datasets from 10 different experiments, a transcriptomic atlas based on 20 RNA-seq expression profiles and a metabolomic atlas based on 895 metabolite spectra analyzed by mass spectrometry. We implemented several visualization tools, including a modified version of the Electronic Fluorescent Pictograph (eFP) browser, co-expression networks and the Interactive Tree Of Life (iTOL) for studying gene expression divergence among duplicated homologous. In addition, the NaDH allows researchers to query phylogenetic trees of 16,305 gene families and provides tools for analyzing their evolutionary history. Furthermore, we also implemented tools to identify co-expressed genes and metabolites, which can be used for predicting the functions of genes. Using the transcription factor NaMYB8 as an example, we illustrate that the tools and data in NaDH can facilitate identification of candidate genes involved in the biosynthesis of specialized metabolites.ConclusionThe NaDH provides interactive visualization and data analysis tools that integrate the expression and evolutionary history of genes in Nicotiana, which can facilitate rapid gene discovery and comparative genomic analysis. Because N. attenuata shares many genome-wide features with other Nicotiana species including cultivated tobacco, and hence NaDH can be a resource for exploring the function and evolution of genes in Nicotiana species in general. The NaDH can be accessed at: http://nadh.ice.mpg.de/.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3465-9) contains supplementary material, which is available to authorized users.

Herbivore-induced defenses are widespread, rapidly evolving and relevant for plant fitness. Such induced defenses are often mediated by early defense signaling (EDS) rapidly activated by the perception of herbivore associated elicitors (HAE) that includes transient accumulations of jasmonic acid (JA). Analyzing 60 HAE-induced leaf transcriptomes from closely-related Nicotiana species revealed a key gene co-expression network (M4 module) which is co-activated with the HAE-induced JA accumulations but is elicited independently of JA, as revealed in plants silenced in JA signaling. Functional annotations of the M4 module were consistent with roles in EDS and a newly identified hub gene of the M4 module (NaLRRK1) mediates a negative feedback loop with JA signaling. Phylogenomic analysis revealed preferential gene retention after genome-wide duplications shaped the evolution of HAE-induced EDS in Nicotiana. These results highlight the importance of genome-wide duplications in the evolution of adaptive traits in plants.DOI: http://dx.doi.org/10.7554/eLife.19531.001

Alternative pre-mRNA splicing (AS) is prevalent in plants and is involved in many interactions between plants and environmental stresses. However, the patterns and underlying mechanisms of AS evolution in plants remain unclear. By analyzing the transcriptomes of four eudicot species, we revealed that the divergence of AS is largely due to the gains and losses of AS events among orthologous genes. Furthermore, based on a subset of AS, in which AS can be directly associated with specific transcripts, we found that AS that generates transcripts containing premature termination codons (PTC), are likely more conserved than those that generate non-PTC containing transcripts. This suggests that AS coupled with nonsense-mediated decay (NMD) might play an important role in affecting mRNA levels post-transcriptionally. To understand the mechanisms underlying the divergence of AS, we analyzed the key determinants of AS using a machine learning approach. We found that the presence/absence of alternative splice site (SS) within the junction, the distance between the authentic SS and the nearest alternative SS, the size of exon–exon junctions were the major determinants for both alternative 5′ donor site and 3′ acceptor site among the studied species, suggesting a relatively conserved AS mechanism. The comparative analysis further demonstrated that variations of the identified AS determinants significantly contributed to the AS divergence among closely related species in both Solanaceae and Brassicaceae taxa. Together, these results provide detailed insights into the evolution of AS in plants.

Alternative pre-mRNA splicing (AS) is prevalent among all plants and is involved in many interactions with environmental stresses. However, the evolutionary patterns and underlying mechanisms of AS in plants remain unclear. By analyzing the transcriptomes of six plant species, we revealed that AS diverged rapidly among closely related species, largely due to the gains and losses of AS events among orthologous genes. Furthermore, AS that generates transcripts containing premature termination codons (PTC), although only representing a small fraction of the total AS, are more conserved than those that generate non-PTC containing transcripts, suggesting that AS coupled with nonsense-mediated decay (NMD) might play an important role in regulating mRNA levels post-transcriptionally. With a machine learning approach we analyzed the key determinants of AS to understand the mechanisms underlying its rapid divergence. Among the studied species, the presence/absence of alternative splicing site (SS) within the junction, the distance between the authentic SS and the nearest alternative SS, the size of exon-exon junctions were the major determinants for both alternative donor site and acceptor site, suggesting a relatively conserved AS mechanism. Comparative analysis further demonstrated that variations of the identified AS determinants, mostly are located in introns, significantly contributed to the AS turnover among closely related species in both Solanaceae and Brassicaceae taxa. These new mechanistic insights into the evolution of AS in plants highlight the importance of post-transcriptional regulation in mediating plant-environment interactions