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  1. Abstract Premise

    Better understanding of the relationship between plant specialized metabolism and traditional medicine has the potential to aid in bioprospecting and untangling of cross‐cultural use patterns. However, given the limited information available for metabolites in most plant species, understanding medicinal use–metabolite relationships can be difficult. The order Caryophyllales has a unique pattern of lineages of tyrosine‐ or phenylalanine‐dominated specialized metabolism, represented by mutually exclusive anthocyanin and betalain pigments, making Caryophyllales a compelling system to explore the relationship between medicine and metabolites by using pigment as a proxy for dominant metabolism.

    Methods

    We compiled a list of medicinal species in select tyrosine‐ or phenylalanine‐dominant families of Caryophyllales (Nepenthaceae, Polygonaceae, Simmondsiaceae, Microteaceae, Caryophyllaceae, Amaranthaceae, Limeaceae, Molluginaceae, Portulacaceae, Cactaceae, and Nyctaginaceae) by searching scientific literature until no new uses were recovered. We then tested for phylogenetic clustering of uses using a “hot nodes” approach. To test potential non‐metabolite drivers of medicinal use, like how often humans encounter a species (apparency), we repeated the analysis using only North American species across the entire order and performed phylogenetic generalized least squares regression (PGLS) with occurrence data from the Global Biodiversity Information Facility (GBIF).

    Results

    We hypothesized families with tyrosine‐enriched metabolism would show clustering of different types of medicinal use compared to phenylalanine‐enriched metabolism. Instead, wide‐ranging, apparent clades in Polygonaceae and Amaranthaceae are overrepresented across nearly all types of medicinal use.

    Conclusions

    Our results suggest that apparency is a better predictor of medicinal use than metabolism, although metabolism type may still be a contributing factor.

     
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    Free, publicly-accessible full text available April 1, 2025
  2. Summary

    In this study, we investigate the genetic mechanisms responsible for the loss of anthocyanins in betalain‐pigmented Caryophyllales, considering our hypothesis of multiple transitions to betalain pigmentation.

    Utilizing transcriptomic and genomic datasets across 357 species and 31 families, we scrutinize 18 flavonoid pathway genes and six regulatory genes spanning four transitions to betalain pigmentation. We examined evidence for hypotheses of wholesale gene loss, modified gene function, altered gene expression, and degeneration of the MBW (MYB‐bHLH‐WD40) trasnscription factor complex, within betalain‐pigmented lineages.

    Our analyses reveal that most flavonoid synthesis genes remain conserved in betalain‐pigmented lineages, with the notable exception ofTT19orthologs, essential for the final step in anthocyanidin synthesis, which appear to have been repeatedly and entirely lost. Additional late‐stage flavonoid pathway genes upstream ofTT19also manifest strikingly reduced expression in betalain‐pigmented species. Additionally, we find repeated loss and alteration in the MBW transcription complex essential for canonical anthocyanin synthesis.

    Consequently, the loss and exclusion of anthocyanins in betalain‐pigmented species appear to be orchestrated through several mechanisms: loss of a key enzyme, downregulation of synthesis genes, and degeneration of regulatory complexes. These changes have occurred iteratively in Caryophyllales, often coinciding with evolutionary transitions to betalain pigmentation.

     
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    Free, publicly-accessible full text available January 1, 2025
  3. Summary

    This work revisits a publication by Beanet al.(2018) that reports seven amino acid substitutions are essential for the evolution ofl‐DOPA 4,5‐dioxygenase (DODA) activity in Caryophyllales. In this study, we explore several concerns which led us to replicate the analyses of Beanet al.(2018).

    Our comparative analyses, with structural modelling, implicate numerous residues additional to those identified by Beanet al.(2018), with many of these additional residues occurring around the active site of BvDODAα1. We therefore replicated the analyses of Beanet al.(2018) to re‐observe the effect of their original seven residue substitutions in a BvDODAα2 background, that is the BvDODAα2‐mut3 variant.

    Multiplein vivoassays, in bothSaccharomyces cerevisiaeandNicotiana benthamiana, did not result in visible DODA activity in BvDODAα2‐mut3, with betalain production always 10‐fold below BvDODAα1.In vitroassays also revealed substantial differences in both catalytic activity and pH optima between BvDODAα1, BvDODAα2 and BvDODAα2‐mut3 proteins, explaining their differing performancein vivo.

    In summary, we were unable to replicate thein vivoanalyses of Beanet al.(2018), and our quantitativein vivoandin vitroanalyses suggest a minimal effect of these seven residues in altering catalytic activity of BvDODAα2. We conclude that the evolutionary pathway to high DODA activity is substantially more complex than implied by Beanet al.(2018).

     
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    Free, publicly-accessible full text available September 1, 2024
  4. Abstract Here we respond to Zhou (BMC Genomics 21:734, 2020) “Combined Transcriptome and Metabolome analysis of Pitaya fruit unveiled the mechanisms underlying peel and pulp color formation” published in BMC Genomics. Given the evolutionary conserved anthocyanin biosynthesis pathway in betalain-pigmented species, we are open to the idea that species with both anthocyanins and betalains might exist. However, in absence of LC-MS/MS spectra, apparent lack of biological replicates, and no comparison to authentic standards, the findings of Zhou (BMC Genomics 21:734, 2020) are not a strong basis to propose the presence of anthocyanins in betalain-pigmented pitaya. In addition, our re-analysis of the datasets indicates the misidentification of important genes and the omission of key flavonoid and anthocyanin synthesis genes ANS and DFR. Finally, our re-analysis of the RNA-Seq dataset reveals no correlation between anthocyanin biosynthesis gene expression and pigment status. 
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  5. Abstract Here we respond to the paper entitled “ Contribution of anthocyanin pathways to fruit flesh coloration in pitayas ” (Fan et al., BMC Plant Biol 20:361, 2020). In this paper Fan et al. 2020 propose that the anthocyanins can be detected in the betalain-pigmented genus Hylocereus , and suggest they are responsible for the colouration of the fruit flesh. We are open to the idea that, given the evolutionary maintenance of fully functional anthocyanin synthesis genes in betalain-pigmented species, anthocyanin pigmentation might co-occur with betalain pigments, as yet undetected, in some species. However, in absence of the LC-MS/MS spectra and co-elution/fragmentation of the authentic standard comparison, the findings of Fan et al. 2020 are not credible. Furthermore, our close examination of the paper, and re-analysis of datasets that have been made available, indicate numerous additional problems. Namely, the failure to detect betalains in an untargeted metabolite analysis, accumulation of reported anthocyanins that does not correlate with the colour of the fruit, absence of key anthocyanin synthesis genes from qPCR data, likely mis-identification of key anthocyanin genes, unreproducible patterns of correlated RNAseq data, lack of gene expression correlation with pigmentation accumulation, and putative transcription factors that are weak candidates for transcriptional up-regulation of the anthocyanin pathway. 
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