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 of 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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Summary TT19 orthologs, essential for the final step in anthocyanidin synthesis, which appear to have been repeatedly and entirely lost. Additional late‐stage flavonoid pathway genes upstream ofTT19 also 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.Free, publicly-accessible full text available January 1, 2025 -
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Summary This work revisits a publication by Bean
et 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 Bean
et 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.Multiple
in vivo assays, in bothSaccharomyces cerevisiae andNicotiana benthamiana , did not result in visible DODA activity in BvDODAα2‐mut3, with betalain production always 10‐fold below BvDODAα1.In vitro assays 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 the
in vivo analyses of Beanet al. (2018 ), and our quantitativein vivo andin vitro analyses 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 ).Free, publicly-accessible full text available September 1, 2024 -
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.more » « less
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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.more » « less
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l-Tyrosine is an essential amino acid for protein synthesis and is also used in plants to synthesize diverse natural products. Plants primarily synthesize tyrosine via TyrA arogenate dehydrogenase (TyrAa or ADH), which are typically strongly feedback inhibited by tyrosine. However, two plant lineages, Fabaceae (legumes) and Caryophyllales, have TyrA enzymes that exhibit relaxed sensitivity to tyrosine inhibition and are associated with elevated production of tyrosine-derived compounds, such as betalain pigments uniquely produced in core Caryophyllales. Although we previously showed that a single D222N substitution is primarily responsible for the deregulation of legume TyrAs, it is unknown when and how the deregulated Caryophyllales TyrA emerged. Here, through phylogeny-guided TyrA structure–function analysis, we found that functionally deregulated TyrAs evolved early in the core Caryophyllales before the origin of betalains, where the E208D amino acid substitution in the active site, which is at a different and opposite location from D222N found in legume TyrAs, played a key role in the TyrA functionalization. Unlike legumes, however, additional substitutions on non-active site residues further contributed to the deregulation of TyrAs in Caryophyllales. The introduction of a mutation analogous to E208D partially deregulated tyrosine-sensitive TyrAs, such as Arabidopsis TyrA2 (AtTyrA2). Moreover, the combined introduction of D222N and E208D additively deregulated AtTyrA2, for which the expression in Nicotiana benthamiana led to highly elevated accumulation of tyrosine in planta. The present study demonstrates that phylogeny-guided characterization of key residues underlying primary metabolic innovations can provide powerful tools to boost the production of essential plant natural products.more » « less
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Summary The evolution of
l ‐DOPA 4,5‐dioxygenase activity, encoded by the geneDODA l ‐DOPA 4,5‐dioxygenase activity evolved via a single Caryophyllales‐specific neofunctionalisation event within theDODA DODA To address this, we functionally characterised 23 distinct
DODA proteins forl ‐DOPA 4,5‐dioxygenase activity, from four betalain‐pigmented and five anthocyanin‐pigmented species, representing key evolutionary transitions across Caryophyllales. By mapping these functional data to an updatedDODA phylogeny, we then explored the evolution ofl ‐DOPA 4,5‐dioxygenase activity.We find that low
l ‐DOPA 4,5‐dioxygenase activity is distributed across theDODA DODA l ‐DOPA 4,5‐dioxygenase activity, accompanied by convergent shifts in key functional residues and distinct genomic patterns of micro‐synteny.In the context of an updated organismal phylogeny and newly inferred pigment reconstructions, we argue that repeated convergent acquisition of elevated
l ‐DOPA 4,5‐dioxygenase activity is consistent with recurrent specialisation to betalain synthesis in Caryophyllales. -
Green plants (Viridiplantae) include around 450,000–500,000 species of great diversity and have important roles in terrestrial and aquatic ecosystems. Here, as part of the One Thousand Plant Transcriptomes Initiative, we sequenced the vegetative transcriptomes of 1,124 species that span the diversity of plants in a broad sense (Archaeplastida), including green plants (Viridiplantae), glaucophytes (Glaucophyta) and red algae (Rhodophyta). Our analysis provides a robust phylogenomic framework for examining the evolution of green plants. Most inferred species relationships are well supported across multiple species tree and supermatrix analyses, but discordance among plastid and nuclear gene trees at a few important nodes highlights the complexity of plant genome evolution, including polyploidy, periods of rapid speciation, and extinction. Incomplete sorting of ancestral variation, polyploidization and massive expansions of gene families punctuate the evolutionary history of green plants. Notably, we find that large expansions of gene families preceded the origins of green plants, land plants and vascular plants, whereas whole-genome duplications are inferred to have occurred repeatedly throughout the evolution of flowering plants and ferns. The increasing availability of high-quality plant genome sequences and advances in functional genomics are enabling research on genome evolution across the green tree of life.more » « less
