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BackgroundBenzylisoquinoline alkaloids (BIAs) represent a diverse class of plant specialized metabolites sharing a common biosynthetic origin beginning with tyrosine. Many BIAs have potent pharmacological activities, and plants accumulating them boast long histories of use in traditional medicine and cultural practices. The decades-long focus on a select number of plant species as model systems has allowed near or full elucidation of major BIA pathways, including those of morphine, sanguinarine and berberine. However, this focus has created a dearth of knowledge surrounding non-model species, which also are known to accumulate a wide-range of BIAs but whose biosynthesis is thus far entirely unexplored. Further, these non-model species represent a rich source of catalyst diversity valuable to plant biochemists and emerging synthetic biology efforts.ResultsIn order to access the genetic diversity of non-model plants accumulating BIAs, we selected 20 species representing 4 families within the Ranunculales. RNA extracted from each species was processed for analysis by both 1) Roche GS-FLX Titanium and 2) Illumina GA/HiSeq platforms, generating a total of 40 deep-sequencing transcriptome libraries. De novo assembly, annotation and subsequent full-length coding sequence (CDS) predictions indicated greater success for most species using the Illumina-based platform. Assembled data for each transcriptome were deposited into an established web-based BLAST portal (www.phytometasyn.ca) to allow public access. Homology-based mining of libraries using BIA-biosynthetic enzymes as queries yielded ~850 gene candidates potentially involved in alkaloid biosynthesis. Expression analysis of these candidates was performed using inter-library FPKM normalization methods. These expression data provide a basis for the rational selection of gene candidates, and suggest possible metabolic bottlenecks within BIA metabolism. Phylogenetic analysis was performed for each of 15 different enzyme/protein groupings, highlighting many novel genes with potential involvement in the formation of one or more alkaloid types, including morphinan, aporphine, and phthalideisoquinoline alkaloids. Transcriptome resources were used to design and execute a case study of candidate N-methyltransferases (NMTs) from Glaucium flavum, which revealed predicted and novel enzyme activities.ConclusionsThis study establishes an essential resource for the isolation and discovery of 1) functional homologues and 2) entirely novel catalysts within BIA metabolism. Functional analysis of G. flavum NMTs demonstrated the utility of this resource and underscored the importance of empirical determination of proposed enzymatic function. Publically accessible, fully annotated, BLAST-accessible transcriptomes were not previously available for most species included in this report, despite the rich repertoire of bioactive alkaloids found in these plants and their importance to traditional medicine. The results presented herein provide essential sequence information and inform experiment...

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Lifetime reproductive success of males is often dependent upon the ability to physically compete for mates. However, species variation in social structure leads to differences in the relative importance of intraspecific aggression. Here, we present a large comparative dataset on sexual dimorphism in skeletal shape in Carnivora to test the hypotheses that carnivorans exhibit sexual dimorphism in skeletal anatomy that is reflective of greater specialization for physical aggression in males relative to females and that this dimorphism is associated with the intensity of sexual selection. We tested these hypotheses using a set of functional indices predicted to improve aggressive performance. Our results indicate that skeletal shape dimorphism is widespread within our sample. Functional traits thought to enhance aggressive performance are more pronounced in males. Phylogenetic model selection suggests that the evolution of this dimorphism is driven by sexual selection, with the best-fitting model indicating greater dimorphism in polygynous versus nonpolygynous species. Skeletal shape dimorphism is correlated with body size dimorphism, a common indicator of the intensity of male-male competition, but not with mean body size. These results represent the first evidence of sexual dimorphism in the primary locomotor system of a large sample of mammals.

O - and N -methylations are ubiquitous and recurring features in the biosynthesis of many specialized metabolites. Accordingly, the methyltransferase (MT) enzymes catalyzing these modifications are directly responsible for a substantial fraction of the vast chemodiversity observed in plants. Enabled by DNA sequencing and synthesizing technologies, recent studies have revealed and experimentally validated the trajectories of molecular evolution through which MTs, such as those biosynthesizing caffeine, emerge and shape plant chemistry. Despite these advances, the evolutionary origins of many other alkaloid MTs are still unclear. Focusing on benzylisoquinoline alkaloid (BIA)-producing plants such as opium poppy, we review the functional breadth of BIA N - and O -MT enzymes and their relationship with the chemical diversity of their host species. Drawing on recent structural studies, we discuss newfound insight regarding the molecular determinants of BIA MT function and highlight key hypotheses to be tested. We explore what is known and suspected concerning the evolutionary histories of BIA MTs and show that substantial advances in this domain are within reach. This new knowledge is expected to greatly enhance our conceptual understanding of the evolutionary origins of specialized metabolism.

Aggressive behaviour is important in the life history of many animals. In grey wolves (Canis lupus), territory defence through direct competition with conspecifics is severe and often lethal. Thus, performance in aggressive encounters may be under strong selection. Additionally, grey wolves frequently kill large dangerous prey species. Because both sexes actively participate in aggressive activities and prey capture, wolves are expected to exhibit a low level of musculoskeletal sexual dimorphism. However, male wolves more often lead in agonistic encounters with conspecifics and must provision the nursing female during the pup-rearing period of the breeding season. These behaviours may select for males that exhibit a higher degree of morphological adaptation associated with aggression and prey capture performance. To test this prediction, we assessed skeletal sexual dimorphism in three subspecies of grey wolves using functional indices reflecting morphological specialization for aggression. As expected, sexual dimorphism in skeletal shape was limited. However, in two of three subspecies, we found sexually dimorphic traits in the skull, forelimbs and hindlimbs that are consistent with the hypothesis that males are more specialized for aggression. These characters may also be associated with selection for improved prey capture performance by males. Thus, the sexually dimorphic functional traits identified by our analysis may be adaptive in the contexts of both natural and sexual selection. Several of these traits may conflict with locomotor economy, indicating the importance of aggression in the life history of male grey wolves. The presence of functional specialization for aggression in a generally monogamous species indicates that sexual dimorphism in specific musculoskeletal traits may be widespread among mammals.

Benzylisoquinoline alkaloids are a large group of plant-specialized metabolites displaying an array of biological and pharmacological properties associated with numerous structural scaffolds and diverse functional group modification. N-Methylation is one of the most common tailoring reactions, yielding tertiary and quaternary pathway intermediates and products. Two N-methyltransferases accepting (i) early 1-benzylisoquinoline intermediates possessing a secondary amine and leading to the key branch-point intermediate (S)-reticuline and (ii) downstream protoberberines containing a tertiary amine and forming quaternary intermediates destined for phthalideisoquinolines and antimicrobial benzo[c]phenanthridines were previously characterized. We report the isolation and characterization of a phylogenetically related yet functionally distinct N-methyltransferase (NMT) from opium poppy (Papaver somniferum) that primarily accepts 1-benzylisoquinoline and aporphine substrates possessing a tertiary amine. The preferred substrates were the R and S conformers of reticuline and the aporphine (S)-corytuberine, which are proposed intermediates in the biosynthesis of magnoflorine, a quaternary aporphine alkaloid common in plants. Suppression of the gene encoding reticuline N-methyltransferase (RNMT) using virus-induced gene silencing in opium poppy resulted in a significant decrease in magnoflorine accumulation and a concomitant increase in corytuberine levels in roots. RNMT transcript levels were also most abundant in roots, in contrast to the distribution of transcripts encoding other NMTs, which occur predominantly in aerial plant organs. The characterization of a third functionally unique NMT involved in benzylisoquinoline alkaloid metabolism will facilitate the establishment of structure-function relationships among a large group of related enzymes.