TPM - Tailoring Plant Metabolism - Work package 2 (WP2) - Designer Willows: high value phenolic glycosides for health and industry

Lead Research Organisation: Rothamsted Research
Department Name: Unlisted

Abstract

Willow contains high levels of natural phenolic glycosides which are probably best known for their role in the development of Aspirin but which also offer an alternative route to petroleum-derived chemicals. Application of chemical fingerprinting to Rothamsted's willow germplasm collections has revealed novel products with new pharmacologies, offering a timely and exciting opportunity to exploit willow chemistry and genes for the development of new medicines. In addition, further chemical diversity, present in abundance in some members of the willow family, offers green chemistry routes to BTX-derived phenolics for the chemical industry. Furthermore, low-input perennial cropping systems, such as short rotation coppice willow offer opportunities to achieve carbon-neutral production of materials, whilst also providing energy to drive industrial processes for the recovery of those materials, via the established technology of willow chip combustion for power generation. WP2 will combine natural product chemistry, genetics and genomics to design willow varieties producing novel high-value phenolic glycosides for multiple markets. Through a "plug and play" breeding approach, diverse substrates will be united with species-specific biosynthetic modules. Chemical space will be defined via metabolomics screening of the national willow collection. Metabolomic-transcriptomic time course experiments and mQTL analysis of mapping populations will elucidate the salicinoid biosynthetic pathway(s) and their regulation. Candidate genes will be characterised in microbial systems and/or RNAi and genome editing of the corresponding genes in the closely related poplar. Models of metabolite flux in willow will be derived from stable isotope labelling data and genetic factors influencing metabolism in hybridised genomes will be revealed through 'omics analysis of judicious crosses. Finally, product yields and extraction processes will be optimised for onward exploitation.
 
Description The project addresses the challenges involved in the development of fast growing willow biomass into a dual-purpose chemical and bioenergy crop. It is based on the propensity of willow to accumulate phenolic glycosides, useful natural products with potential in a number of industries including pharmaceuticals, polymers and resins. The work is arranged around four over-arching objectives, each containing several sub-work packages. Key findings so far:-

Objective 1. Map the extent of phenolic diversity across unique genetic resources

• Application of the high throughput metabolomics screen to a large number of willow species, using nuclear magnetic resonance (NMR) and mass spectrometric (MS) analysis that was developed in the previous ISP programme 'Cropping Carbon', has allowed us to build a comprehensive chemical map that spans the National Willow Collection (NWC) and the parents and progeny of a number of genetic mapping populations. Visualisation of the chemical data as a network has allowed us to quickly construct an interactive 'star-map' where constellations of genetically and chemically related species are easily identified. The visualisation of data in this format has allowed rapid selection of future breeding material for specific chemicals.

• Many novel structures have been determined by isolation of pure components and full NMR and MS characterisation. New molecules with pharmaceutical interest have been discovered. These include salicin-7-sulfate, an analogue of aspirin that has implications in the safety and efficacy of commercial herbal Salix products (paper published 2018; doi: 10.1016/j.fitote.2018.02.009). In year 2 we have completed our initial work on structural determination, and testing of an anti-cancer compound of a completely novel type from willow, and have filed a patent (Jan 2019). A further two research papers describing novel willow components (sulfated flavonoids and phenylalkanoid disaccharides) have been submitted (Feb 2019). The potential of plant derived polyphenols, including those from willow, in neuroscience has been developed through a collaboration with Daniel Otzen at the Interdisciplinary Nanoscience Center, Aarhus University that has resulted in 2 publications (Jan 2019) on reduction a-synuclein fibrillation by plant natural products, including a possible connection between plant longevity and anti-fibrillation activity.

• Field trials have been established and harvesting at different growth stages commenced, for 8 lines bearing different chemistries, to determine the optimum time (in a three year growth cycle) and highest yielding plant part (e.g. top, middle or bottom stems) for compounds of interest. These include the anti-cancer salicinoid patented above as well as chemicals with neuro-protective properties. Comprehensive phenotyping and metabolomic data has been collected for the early timepoints. In year 2, the data collection from the year 1 harvest has been completed and analysed and tissues from the second season growth have been prepared for analysis. Year 1 results have allowed an estimation of chemical yield at a per hectare scale.

Objective 2. Define the phenolic glycoside pathways, their regulation and species-specific reaction mechanisms

• A large-scale combined-metabolomic-transcriptomics 60 day time-course experiment for six key lines expressing different branches of a putative biosynthetic grid has been completed. The data has been mined for associations between metabolites and genes, using as anchors two genes of proven involvement in salicinoid biosynthesis. One of these 'known' genes, a cytochrome P450 oxidase was previously isolated by us via a mQTL and map-based cloning approach, and functionally characterised by RNAi knockdown in transgenic poplar via metabolomic analysis. A list of further candidate genes (enzymes) for the pathway has been developed from the transcriptomics data and passed into our poplar transformation pipeline for determination of gene function via metabolomics of knockdowns. The small list of ~20 genes contains further P450s, glycosyltransferases, epoxide lyase and reductases. One the glycosyl transferases (UGTs) has been cloned and expressed in E.coli and shown to accept a range of salicyl derivatives, confirming a potential role in the pathway. A transient expression system utilising young plantlets of poplar has been developed and will be used initially to further characterise this UGT in vivo. Transgenic poplar with RNAi/overexpressing glycosyl transferase have also been generated and will soon emerge from the pipeline for metabolomic analysis. A second cytochrome P450 from the list is under investigation by RNAi knockdown in poplar and metabolomics of the transgenic plants has demonstrated a central role in the pathway, apparently upstream of the first P450. Similar timecourses (3) have been set up, harvested and analysed by metabolomics in readiness for generation of transcriptome data aimed at elucidation of regulatory switches. We have successfully developed an in-house genome editing protocol for Populus with our first 'proof of concept' experiment indicating successful knockout of a phytoene destaurase (albino phenotype) gene. This approach will now be used where appropriate to elucidate pathways.

• Mapping of metabolites across populations mpF (n=394) and mpG (n=593) grown in the field has commenced to try and determine a QTL for early versus late expression of salicortin production, to further enhance our knowledge of the genes of the pathway, as well as to inform on potential regulatory genes that will be important in engineering higher yield varieties. Metabolomics data for leaf and stem tissue from all members of both populations is in hand and mQTL mapping is now in progress. Already, a novel QTL for this trait has been identified on chromosome II in mpG which initially appears to co-locate with an important phenology controlling locus. Fine mapping is underway to confirm this. Bioinformatic tools for high-throughput, genome-wide, genotyping assay design in these populations have also now been completed.
Establishment of a hydroponic growth protocol for willow has been achieved and studies using lead genotypes perturbed with either heavy metals and aromatic chemical pollutants have been initiated. The effect on these stressors on secondary metabolism can now be investigated.

Objective 3. Novel designer willows and products

• Having screened through the pure species in the NWC we have used the chemotype data to predict the composition of existing hybrids (originally produced to enhance biomass) where new substrates are brought together with alternate genes to form 'designer molecules' that increase the number of analogues for biological testing. Novel structural variants have been obtained in the hybrid progeny serving as a proof of concept. These include variant forms of our lead anti-cancer molecule, thus demonstrating applicability in the approach. • We are now seeking to optimise levels and create new chemistry through the generation of novel hybrids. Based on chemistry, stock for the breeding of new hybrids has now been identified and entered into the local breeding programme. Lines for the determination of the genetic factors influencing metabolism in hybridised Salix genomes have been selected from the chemistry and genetic profile data. Cuttings for these large scale experiments have been collected for growth in the coming year.

•New, dedicated intraspecific, interspecific and polyploid crosses for studying genetic factors influencing metabolism in hybridised Salix genomes have been created and validated by molecular markers. Bespoke, bioinformatics tools for designing a high throughput, genome-wide, sequence-based genotyping assay have been created and cuttings made for scheduled multiomic screening and analysis.

In-house screening of a Salix viminalis association mapping population with a new set of genome-wide molecular (GBS) markers was completed (136K markers aligned to our draft reference genome of S. viminalis). This enabled analyses resulting in a rich database of novel, genome-wide, marker-metabolite associations (over 6K new associations at P < 10-5, roughly half of which reach Bonferroni-adjusted genome-wide significance). These data have also been used to develop and demonstrate the potential of multiomic prediction and Mendelian Randomisation (MR) approaches for inferring causative links between metabolites and adaptive phenotypic traits.

Objective 4. Prototypes and scale-up for exploitation

• Field demonstration trials (80 plants x 16 lines) have been planted and are established in order to provide a demonstration and quantitation of chemical product versus biomass in a conventional coppicing regime. This is vital information for forward crop improvement. Chipped material has been obtained for experimentation after the first year growth. A set of experiments, designed to inform on the best storage and down-stream processing regimes for optimum product yield from wood chips has been carried out. Results from this one year experiment have informed on compound integrity following storage or oven drying. Chipped material from the experiment has been stored and provides a resource for the development of larger scale extraction protocols.

• Scale-up of chromatographic isolation has allowed us to obtain a relatively large quantity (>100mg) of a lead anti-cancer compound for onward testing.
Exploitation Route The findings represent the most comprehensive study of willow (and poplar) secondary chemistry and biochemistry ever, and builds a platform for many future research projects, including this project which is still very active. As well as the current area of focus (high value chemicals and dual purpose crops) the knowledge of the pathway genes and metabolite network will underpin many projects, basic and applied, in areas of tree and biomass crop biology such as agronomy, pest resilience, phytoremediation, carbon cycling and climate science. Novel molecules are being processed through a KEC platform through patenting and commercial pipelines.
Sectors Agriculture, Food and Drink,Chemicals,Energy,Pharmaceuticals and Medical Biotechnology

 
Description Pathways to impact for the designer willows project are enriched by the historic platform of willow usage in the agro-economy. From the general public's perspective, the growing of willow for basket making and the discovery of aspirin are well known historically. In addition, the principles behind the breeding and utilisation of fast growing willow varieties for the production of biomass, for heat and power generation, are also well understood. This project aims to take the next step and add value to the willow biomass chain, via the extraction of chemical and pharmaceutical products, before use of the wood chips for energy generation. This logical extension of the industry has the potential for impact across multiple sectors - agro-industry, chemicals, bio-energy and pharmaceuticals. Scientific impact arises from the use of state-of-the-art metabolomic and genomic technologies to understand a biochemical pathway that has not yet been characterised. The discovery of new medically bioactive entities and the breeding of willow to produce high yields and panels of analogues for pharmaceutical exploitation broadens the commercial attractiveness of the project. This aspect of the work was highlighted during the project launch and picked up by a scientific journalist and led to a published feature article in Chemistry World. in addition to the medical aspects, the use of non-food crops to replace petrochemicals as source of carbon-based building blocks for chemical and polymer industries has green credentials in that the process has the potential to be carbon neutral in that greenhouse gas emissions are negated by carbon dioxide uptake by the crop. Also in year one of the project we have taken the following steps to disseminate our research:- The normal academic routes of published papers and presentations at conferences are listed in the engagement section of this form. This year, working with a film maker at the University of Hertfordshire, members of the TPM team have constructed a video entitled 'Willow: Then and Now' which tells the history of the use of willow for the past 100years. The film is centered around the National Willow Collection and laboratory facilities at Rothamsted, and contains interview footage from members of the team talking about the TPM project. The film has been released on the University of Hertfordshire web-site and subsequently on the Rothamsted web-site. In year 2 of the project a further video 'willow 2.0' was released by Rothamsted via Youtube and this describes the research of TPM. Also in 2018 the project was prominent in the Festival of Ideas, a major 3 day open Rothamsted event celebrating the 175th anniversary of the Institute.
First Year Of Impact 2017
Sector Agriculture, Food and Drink,Education,Energy,Culture, Heritage, Museums and Collections
Impact Types Cultural,Policy & public services

 
Description Collaboration with Prof. Daniel Otzen, Interdisciplinary Nanoscience Center, Aarhus Univeristy 
Organisation Aarhus University
Department Interdisciplinary Nanoscience Center (iNano)
Country Denmark 
Sector Academic/University 
PI Contribution Methodologies for natural product screening and fractionation of plant extracts, developed with the Tailoring Plant Metabolism ISP, have been applied to a range of plants to identify inhibitors of protein fibrillation. Annotation of natural products within the extracts has been carried out as has the characterisation of purified fractions. A number of compounds have been suggested to act as inhibitors of alpha-synuclein fibrillation.
Collaborator Contribution Partners, supplied crude plant extracts from a number of plants for analysis by the RRes team. They also conducted the biological assays associated with fibrillation testing. The partners have also surveyed a panel of willow extracts and have provided activity data suggesting that there are compounds present in willow that inhibit alpha-synuclein and thus may be promising targets for new neuroprotective drugs.
Impact The collaboration has resulted in two publications so far. In addition we have bioactivity data in hand indicating willow species that contain potential new neuroprotectives.
Start Year 2014
 
Description Chemistry world article (Ward) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Phone interview (Jane Ward) given to science journalist for article in Royal Society of Chemistry's magazine, Chemistry World. Article title: Metabolomic Wind in the Willows (published 31 May 2017) https://www.chemistryworld.com/news/willow-promises-new-medicines/3007491.article
Year(s) Of Engagement Activity 2017
URL https://www.chemistryworld.com/news/willow-promises-new-medicines/3007491.article
 
Description Cultivation and Use of Basket Willows 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This guidebook was published by the Basketmakers Association, a trade group and is a revision of a pervious booklet distributed by the trade. Project scientists were involve in updating and drafting this new version. Reference:- Macalpine, W. J., Burns, H., Hammerin, A., Shield, I. F., Butcher, M., Davies, O. and Bertram, G. 2018. Cultivation and Use of Basket Willows - a guide to growing basket willows. The Basketmakers Association.
Year(s) Of Engagement Activity 2018
 
Description International Poplar Symposium (Buenos Aires)(Beale,Ward, MacAlpine, Noleto-Dias) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Four members of the research team participated the 7th International Polar Symposium: New bioeconomies: exploring the potential role of Salicaceae. held in Buenos Aires. Invited talks were give by Prof Beale, Dr Ward and Mr MacAlpine, and a poster presented by Dr Clarice Noleto-Dias. The symposium also included a field trip and discussions with scientists at the INTA institute and commercial willow plantations in the Parana delta region, where Salix are grown for feedstock to a local paper mill.
Year(s) Of Engagement Activity 2018
 
Description Invited presentation (Hanley) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation by Steve Hanley at Sustainable bioenergy crops conference, Oxford, Sept 2017 entitled 'dissecting complex traits in Salix: progress in gene discovery'
Year(s) Of Engagement Activity 2017
 
Description Invited presentation (Slavov): Bioenergy Genomics 17 Oxford Conference. Integrated genomic prediction in bioenergy crops. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Generated discussions and outlined future research with peers working in bioenergy crops.
Year(s) Of Engagement Activity 2017
URL http://www.watbio.eu/bioenergy-genomics-17-oxford-conference/
 
Description Joint worksop with JIC Molecules from Nature ISP (Beale, Ward, Hanley) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact A joint 2 day workshop between this ISP and the John Innes centre ISP 'Molecules from Nature' was held in Norwich, November 2018. The discussions allowed us to exchange details of the two programmes and look for synergies and new ideas and technologies in the area of high value products from plants.
Year(s) Of Engagement Activity 2018
 
Description Public release video (Willow 2.0) describing project 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Willow 2.0 is a professionally made video, part of a portfolio made by Rothamsted for publicity and educational purposes. It describes in detail the context and aspirations of the TPM project and has now been made available on youtube
Year(s) Of Engagement Activity 2018
URL https://www.youtube.com/watch?v=tXV7I6vqMAE
 
Description Slavov (cross-institute BBSRC workshop on predictive breeding) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Explored potential for developing a cross-institute (Roslin, Earlham, JIC, NIAB, RRes, IBERS ...) ISP on predictive breeding. A report was submitted to BBSRC outlining next steps / key questions.
Year(s) Of Engagement Activity 2018
 
Description Slavov (invited presentation) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Multi-omic dissection and prediction of complex traits in plants. XIV International Symposium on Integrative Bioinformatics, Harpenden, UK, June 14, 2018
Year(s) Of Engagement Activity 2018
URL https://www.rothamsted.ac.uk/events/14th-international-symposium-integrative-bioinformatics
 
Description Special interest group visit (macalpine) 
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 A local natural history group were given a tour of the National Willow Collection. Aspects of the history of willow growing and uses in the UK, the agronomy and breeding of willow for biomass use, research in the programme towards dual use willows and the biodiversity benefits of willow plantations were discussed.
Year(s) Of Engagement Activity 2017
 
Description Willow video 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Working with film makers from the University of Hertfordshire, under the banner of an initiative called 'everyday lives in war', a video was constructed that begins with the history of willow cultivation and uses in the first world war and ends with a overview of the current project on biomass for energy and for industrial chemicals and medicines. The film includes current project members, speaking about the work, and footage from the field trials established by the project. The film will shortly be released on the University of Herts website, and will also be placed on Rothamsted's web-site.
Year(s) Of Engagement Activity 2017,2018
URL https://everydaylivesinwar.herts.ac.uk/?page_id=2848
 
Description Willow works (Rothamsted Festival of Ideas) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact 'Willow works' consisted of an interactive, walk through of field, chemistry and genetics manned pop-up labs leading onto visits to NMR and Mass spectroscopy suites, and an optional tour of the National Willow Collection. The event, which formed one of a number of exhibits/tours that were staged during the 3 day Rothamsted Festival of Ideas, in June 2018 (celebrating the 175th anniversary of Rothamsted) was visited by many thousands of members of the public. All members of the research team were involved and both formal and informal feedback received was extremely positive.
Year(s) Of Engagement Activity 2018
 
Description Workshop - Committee on Climate Change 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact William MacAlpine was invited and attended 'Steps to scaling up UK sustainable bioenergy supply workshop', held by the Committee on Climate Change. The outcome was a document released to the public https://www.theccc.org.uk/wp-content/uploads/2018/11/Biomass-in-a-low-carbon-economy-CCC-2018.pdf
Year(s) Of Engagement Activity 2018
URL https://www.theccc.org.uk/wp-content/uploads/2018/11/Biomass-in-a-low-carbon-economy-CCC-2018.pdf
 
Description nottingham students visit (Ward and Macalpine) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact Two talks presented by team members (Ward and Macalpine) to a large party of bioscience undergraduate and masters students from the University of Nottingham. The presentations covered - 'Harnessing chemical diversity, genetics and willow breeding.
Year(s) Of Engagement Activity 2018