NSFDEB-NERC; Collaborative Resource; A phytochemical "tug-of-war" and its impact on organismal diversification and niche occupancy in Caryophyllales
Lead Research Organisation:
University of Cambridge
Department Name: Plant Sciences
Abstract
Our proposed work seeks to understand the step-wise evolution of complex specialized metabolic traits in
flowering plants, and to explore how the evolution of such traits influence subsequent adaptation and
diversification in flowering plants. To address these questions, we will focus on the flowering plant order
Caryophyllales, which is well recognized for extraordinary adaptations to extreme environments and
unusually high diversity of metabolites derived from the amino-acid Tyrosine. Our central hypothesis is
that changes in availability and abundance of Tyrosine in Caryophyllales has led to the evolution of
numerous lineage-specific tyrosine-derived metabolites that in turn has profoundly influenced the
adaptation and diversification of species within Caryophyllales. To test this hypothesis, we will build an
evolutionary framework for Caryophyllales, that integrates transcriptomic, genomic, and metabolic
datasets, with patterns of trait evolution, on a macroevolutionary scale. Specifically, we will: 1) perform
an extensive survey to establish the occurrence and distribution of tyrosine-derived metabolic traits across
Caryophyllales; 2) examine the association of these tyrosine-derived metabolic traits with organismal
adaptation and diversification patterns; and 3) determine the evolutionary genetic mechanisms responsible
for the biosynthesis of these metabolites. On completion of the proposed work, we expect to have
comprehensively described the extent of tyrosine-enriched metabolism in Caryophyllales, to have defined
the degree to which they are associated with organismal diversification patterns across Caryophyllales,
and to have resolved the stepwise evolutionary assembly of the genetic pathways underlying complex
tyrosine-derived metabolic traits.
Understanding the evolution of complex traits is a fundamental challenge for biologists, as the stepwise
fashion by which such traits have evolved is not always readily apparent. Furthermore, the connections
between the various stages of complex trait assembly (e.g. genetic, biochemical, and morphological) and
subsequent organismal diversification patterns are not well explored, with methodological approaches
still in their infancy. Recent advances in phylogenetics, with the integration of -omic scale data, now
provide timely opportunities to marry the assembly of complex traits with lineage-specific and nichespecific
organismal diversification. Specialized metabolites are chemicals that confer adaptive advantages
in certain ecological and evolutionary contexts. The stepwise nature of the biosynthetic pathways
underlying complex specialized metabolites ensure that they are especially tractable for reconstructing
stepwise evolution of complexity. While the phylogenetically restricted distributions of specialized
metabolites are fundamental to resolving the influence of complex traits on niche-specific and lineagespecific
organismal adaptation and diversification. Our approach, using the tyrosine-enriched specialized
metabolism in Caryophyllales as a model system, therefore has the potential to lead to new and
fundamental insights into the causes and consequences of the evolutionary assembly of complex traits at a
variety of evolutionary scales.
flowering plants, and to explore how the evolution of such traits influence subsequent adaptation and
diversification in flowering plants. To address these questions, we will focus on the flowering plant order
Caryophyllales, which is well recognized for extraordinary adaptations to extreme environments and
unusually high diversity of metabolites derived from the amino-acid Tyrosine. Our central hypothesis is
that changes in availability and abundance of Tyrosine in Caryophyllales has led to the evolution of
numerous lineage-specific tyrosine-derived metabolites that in turn has profoundly influenced the
adaptation and diversification of species within Caryophyllales. To test this hypothesis, we will build an
evolutionary framework for Caryophyllales, that integrates transcriptomic, genomic, and metabolic
datasets, with patterns of trait evolution, on a macroevolutionary scale. Specifically, we will: 1) perform
an extensive survey to establish the occurrence and distribution of tyrosine-derived metabolic traits across
Caryophyllales; 2) examine the association of these tyrosine-derived metabolic traits with organismal
adaptation and diversification patterns; and 3) determine the evolutionary genetic mechanisms responsible
for the biosynthesis of these metabolites. On completion of the proposed work, we expect to have
comprehensively described the extent of tyrosine-enriched metabolism in Caryophyllales, to have defined
the degree to which they are associated with organismal diversification patterns across Caryophyllales,
and to have resolved the stepwise evolutionary assembly of the genetic pathways underlying complex
tyrosine-derived metabolic traits.
Understanding the evolution of complex traits is a fundamental challenge for biologists, as the stepwise
fashion by which such traits have evolved is not always readily apparent. Furthermore, the connections
between the various stages of complex trait assembly (e.g. genetic, biochemical, and morphological) and
subsequent organismal diversification patterns are not well explored, with methodological approaches
still in their infancy. Recent advances in phylogenetics, with the integration of -omic scale data, now
provide timely opportunities to marry the assembly of complex traits with lineage-specific and nichespecific
organismal diversification. Specialized metabolites are chemicals that confer adaptive advantages
in certain ecological and evolutionary contexts. The stepwise nature of the biosynthetic pathways
underlying complex specialized metabolites ensure that they are especially tractable for reconstructing
stepwise evolution of complexity. While the phylogenetically restricted distributions of specialized
metabolites are fundamental to resolving the influence of complex traits on niche-specific and lineagespecific
organismal adaptation and diversification. Our approach, using the tyrosine-enriched specialized
metabolism in Caryophyllales as a model system, therefore has the potential to lead to new and
fundamental insights into the causes and consequences of the evolutionary assembly of complex traits at a
variety of evolutionary scales.
Planned Impact
The proposed work sits at the interface of phylogenetics, evolutionary biology, and specialized plant
metabolism, and consequently will influence a number of frontiers in scientific discovery. Numerous
tyrosine-derived metabolites are important in human health and nutrition. Our work will reveal genetic
components that help improve industrial production of betalain pigments and further help to elucidate
biosynthesis pathways of a number of plant-derived alkaloids with pharmaceutical values, such as
catecholamines, isoquinoline, and benzylisoquinoline alkaloids. The international collaboration among
the Minnesota, Wisconsin, Michigan, and Cambridge (UK) teams will provide multidisciplinary training
opportunities for students and postdocs in addressing broader evolutionary questions by applying various
omics and biochemical methods in a phylogenetic framework. All data and analytical tools generated by
the project will be made freely available within six months via NCBI SRA and genome databases, after
quality control and processing. Phylogenomics workshops at professional society meetings, a K-12
summer camp module, and multiple public outreach modules are planned for both the US and UK
institutions, which will bring these concepts to the public, and highlight the importance of biodiversity
with respect to high-value plant chemicals.
metabolism, and consequently will influence a number of frontiers in scientific discovery. Numerous
tyrosine-derived metabolites are important in human health and nutrition. Our work will reveal genetic
components that help improve industrial production of betalain pigments and further help to elucidate
biosynthesis pathways of a number of plant-derived alkaloids with pharmaceutical values, such as
catecholamines, isoquinoline, and benzylisoquinoline alkaloids. The international collaboration among
the Minnesota, Wisconsin, Michigan, and Cambridge (UK) teams will provide multidisciplinary training
opportunities for students and postdocs in addressing broader evolutionary questions by applying various
omics and biochemical methods in a phylogenetic framework. All data and analytical tools generated by
the project will be made freely available within six months via NCBI SRA and genome databases, after
quality control and processing. Phylogenomics workshops at professional society meetings, a K-12
summer camp module, and multiple public outreach modules are planned for both the US and UK
institutions, which will bring these concepts to the public, and highlight the importance of biodiversity
with respect to high-value plant chemicals.
People |
ORCID iD |
| Samuel Brockington (Principal Investigator) |
Publications
Feng K
(2024)
The link between ancient whole-genome duplications and cold adaptations in the Caryophyllaceae
in American Journal of Botany
Feng T
(2023)
The genome of the glasshouse plant noble rhubarb (Rheum nobile) provides a window into alpine adaptation
in Communications Biology
Lopez-Nieves S
(2022)
Two independently evolved natural mutations additively deregulate TyrA enzymes and boost tyrosine production in planta.
in The Plant journal : for cell and molecular biology
Montero H
(2021)
A mycorrhiza-associated receptor-like kinase with an ancient origin in the green lineage.
in Proceedings of the National Academy of Sciences of the United States of America
Pucker B
(2022)
The evidence for anthocyanins in the betalain-pigmented genus Hylocereus is weak.
in BMC genomics
| Description | As a result of this award we have been able show how to couple the betalain synthesis pathway to arbuscula mycorrhizal promoters that report arbusclar Mycorrhiza infection in crop plants as a system for real-time reporting of this beneficial symbiosis that is important for productivity of agricultural crops. We have also discovered the genetic mechanisms underpinning the loss of anthocyanin pigments in beta lain pigmented lineages including the loss of an anthocyanin trasnporter, and parallel deterioration of a regulatory transcription factor complex. We have shown that the key betalain pigment enzyme DODA has evolved multiple times, and has yield different enzymatic isoforms with different catalytic abilities or relevance to biotechnology. We have also shown that L-DOPA and betalains, as neurotransmitters, have an impact on herbivory rates etc. We have shown that we can split the DODA protein to make a promising technological application using natural pigments to report on protein-preotin interaction. |
| Exploitation Route | Other researchers could use this approach to further understand the dynamics of important fungal symbiosis on crop productivity. This information could be used to inform researchers seeking to engineer the production anthocyanin pigments in beta lain pigmented lineages. The discovery of the deterrence effects of betalains and L-DOPA has practical implications for crop protection. |
| Sectors | Agriculture Food and Drink Environment |
| Description | Our research is beginning to leverage the phenomenon of repeated evolution of metabolic pathway, to identify alternative high performance enzymes of value to the bioengineering of improved pipelines for the production of specialised plant metabolites (2021). We have also used our understanding of betalain enzymes to create split-enzyme visible dye reporters that can be used as biological reporters for protein-portion interactions, and to detect drug and pollution substrates. |
| First Year Of Impact | 2021 |
| Sector | Agriculture, Food and Drink,Environment,Pharmaceuticals and Medical Biotechnology |
| Impact Types | Societal Economic |
| Title | Betalain reporter of AM fungal colonisation |
| Description | This was a collaboration with the Schornack Lab, which coupled our expertise in betalain biosynthesis with the goal of implementing a real-time reporter of AM symbiosis. In addition to the reporter system we created a rhizotron system that allowed AM colonisation to be visualised in planta and in real-time The system we created has huge potential to help track and understand the progression and ecology of AM colonisation, and to understand how it is affected by the application of agricultural fertilizers and pesticides. By accurately reporting colonisation it also opens up avenues to further uncover unknown elements of the pathway through RNAseq approaches. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2021 |
| Provided To Others? | Yes |
| Impact | The article was extensively covered in the press in over 110 new outlets, with an altmetric score of 971, and highlighted in PloS Biology Year in Review. |
| Title | split betalain protein |
| Description | GFP- and luciferase-based protein-fragment complementation assays (PCAs) are widely used in protein-protein interaction (PPI) studies, biosensor development, and drug discovery. We are developing a plant pigment-based PCA that offers significant advantages in signal observation and stability. This approach leverages the vivid pigmentation and simple biosynthesis of betalains, a group of plant pigments that provide a robust and visually distinct readout. Betalain biosynthesis begins with the conversion of tyrosine to L-DOPA by CYP76AD5/6, or to L-DOPA and cyclo-DOPA by CYP76AD1. L-DOPA is further oxidized by dioxygenase (DODA) to form betalamic acid, which reacts with amines (e.g., amino acids) to produce yellow betaxanthins or with cyclo-DOPA to form red betacyanins. This spontaneous pigment formation makes betalains an ideal candidate for developing a PCA system with clear and stable visual outputs. The key challenge in developing a betalain-based PCA is determining whether any of these CYP76ADs or DODA enzymes can be split into fragments that can later be reconstituted. We have successfully split DODA and applied it to test different PPIs, and our current focus is on splitting CYP76ADs to expand the capabilities of this approach. Through this project, we have identified a conserved split site in CYP76AD1 and CYP76AD6 and successfully split and reconstituted these enzymes. We found that the enzyme fragments do not automatically reconstitute unless each fragment is fused to another protein that interacts with its partner. To test this, we used the well-known Jun-Fos protein interaction system, fusing Fos to the N-terminal fragment (CYP76ADn) and Jun to the C-terminal fragment (CYP76ADc). We then co-transformed these constructs into plant leaves along with DODA using agroinfiltration. If reconstitution occurred, pigmentation was expected to appear within two days. Our results confirmed that both split CYP76AD1 and CYP76AD6 can be functionally reconstituted. The C-terminal fragment (CYP76ADc) is responsible for pigment synthesis but is only active when recruited by the N-terminal fragment (CYP76ADn) through the interaction of Jun and Fos. Furthermore, we found that split fragments between CYP76AD1 and CYP76AD6 can be swapped, demonstrating the versatility of this approach. These findings indicate that split CYP76ADs can be effectively used to test PPIs, paving the way for a new generation of plant pigment-based PCA systems. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2025 |
| Provided To Others? | No |
| Impact | non as yet |
| Title | Pitaya transcriptome assemblies and investigation of transcript abundances |
| Description | A reanalysis of co-expression datasets in different coloured coloured fruits of Pitaya |
| Type Of Material | Database/Collection of data |
| Year Produced | 2020 |
| Provided To Others? | Yes |
| Impact | It forms the basis of an upcoming rebuttal for a published dataset with problems, relevant to the grant |
| URL | https://doi.org/10.4119/unibi/2946374 |
| Description | Elucidating the biosynthesis of mesembrine for medicine |
| Organisation | University of Stellenbosch |
| Country | South Africa |
| Sector | Academic/University |
| PI Contribution | This is a new collaboration between a South African research group of Professor Nox Makunga and the UK research group of Professor Samuel Brockington. Collaboration is essential to the success of this proposal, leveraging the strengths and resources of both teams. The UK team's experience in genomic technologies and data analysis would complement the South African team's expertise with S. tortuosa and South African natural product development, initiating an exploration of the biosynthetic pathways involved in mesembrine production. The research group of Sam Brockington offers expertise in genomic and transcriptomic analyses, with the capability for the high-throughput sequencing, bioinformatics, and synthetic biology aspects of the project. The Brockington research group offer world-leading expertise in the metabolism within Caryophyllales, the order to which S. tortuosum belongs, and have a track-record in solving metabolic pathways closely related to mesembrine synthesis, via a similar combination of genomics, transcriptomics and synthetic biology approaches. |
| Collaborator Contribution | The research group of Nox Makunga has deep-rooted expertise in ethnobotany and access to native Sceletium tortuosum populations, and will on lead the collection, cultivation, initial phenotypic characterization, and RNA extraction and sequencing. The Makunga research group offer knowledge of the plant's traditional uses, in-vitro cultivation and quantitative analysis of mesembrine, understanding of the biotechnological landscape in South Africa, and have preliminary and published data on mesembrine accumulation in S. tortuosum. The collaboration would be sustained through a carefully choreographed research programme. The South African group would conduct the tissue sampling, metabolite analysis and RNAseq. A South African researcher would then travel to Cambridge, for training in genomic assembly and annotation, which would be done in collaboration with an existing PDRA in the UK research group. Transcriptomic analyses will similarly be performed by the SA researcher with support by the UK-based PDRA. Functional analyses would be performed by an existing UK-based PDRA, with metabolite analyses performed by the SA research group. Collaboration and communication would be supported by joint lab meetings attended by the all the members of both research groups to discuss research, data, ideas and for trouble shooting. |
| Impact | non as yet, just started |
| Start Year | 2025 |
| Description | Regulation of betalain transcription |
| Organisation | University of Cologne |
| Country | Germany |
| Sector | Academic/University |
| PI Contribution | We are collaborating to understand the transcriptional regulation of betalains pigments in different evolutionary origins. We are developing transgenic resources in order to do this. |
| Collaborator Contribution | The partners are developing Amaranth's tricolour as a crop system to study transcriptional regulation in complement to our transgenic approaches. |
| Impact | None as yet |
| Start Year | 2023 |
| Description | Festival of Plant Exhibition |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Public/other audiences |
| Results and Impact | The research team created and manned a stall at the Cambridge University Botanic Garden Festival of Plants, and communicated our research to the public through talks, demonstrations, and child-focussed games. |
| Year(s) Of Engagement Activity | 2021 |
| Description | Festival of Plants in Cambridge |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Public/other audiences |
| Results and Impact | The research team created and manned a stall at the Cambridge University Botanic Garden Festival of Plants, and communicated our research to the public through talks, demonstrations, and child-focussed games. |
| Year(s) Of Engagement Activity | 2024 |