Publications

A specialized metabolic network selectively modulates Arabidopsis root microbiota

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OpenPlant scientists Hans-Wilhelm Nützmann and Anne Osbourn demonstrate that model plant Arabidopsis thaliana produces a range of specialized triterpenes that direct the assembly and maintenance of a specific microbial community within and around its roots. Their work, which was part of a collaborative effort, was recently published in Science:

Ancheng C. Huang, Ting Jiang, Yong-Xin Liu, Yue-Chen Bai, James Reed, Baoyuan Qu, Alain Goossens, Hans-Wilhelm Nützmann, Yang Bai, Anne Osbourn

A specialized metabolic network selectively modulates Arabidopsis root microbiota

Science (2019) Vol. 364, Issue 6440, eaau6389
DOI: 10.1126/science.aau6389

https://science.sciencemag.org/content/364/6440/eaau6389

Abstract

Plant specialized metabolites have ecological functions, yet the presence of numerous uncharacterized biosynthetic genes in plant genomes suggests that many molecules remain unknown. We discovered a triterpene biosynthetic network in the roots of the small mustard plant Arabidopsis thaliana. Collectively, we have elucidated and reconstituted three divergent pathways for the biosynthesis of root triterpenes, namely thalianin (seven steps), thalianyl medium-chain fatty acid esters (three steps), and arabidin (five steps). A. thaliana mutants disrupted in the biosynthesis of these compounds have altered root microbiota. In vitro bioassays with purified compounds reveal selective growth modulation activities of pathway metabolites toward root microbiota members and their biochemical transformation and utilization by bacteria, supporting a role for this biosynthetic network in shaping an Arabidopsis-specific root microbial community.

Cas9-mediated mutagenesis of potato starch branching enzymes generates a range of tuber starch phenotypes

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OpenPlant scientists Aytug Tuncel, Nicola Patron and Alison Smith demonstrate that Cas9-mediated mutagenesis of starch-branching enzymes has the potential to generate new, potentially valuable starch properties.

Aytug Tuncel, Kendall R. Corbin, Jennifer Ahn‐Jarvis , Suzanne Harris, Erica Hawkins, Mark A. Smedley, Wendy Harwood, Frederick J. Warren, Nicola J. Patron, Alison M. Smith

Cas9-mediated mutagenesis of potato starch branching enzymes generates a range of tuber starch phenotypes

Plant Biotechnology Journal https://doi.org/10.1111/pbi.13137

Abstract

We investigated whether Cas9‐mediated mutagenesis of starch‐branching enzymes (SBEs) in tetraploid potatoes could generate tuber starches with a range of distinct properties. Constructs containing the Cas9 gene and sgRNAs targeting SBE1, SBE2 or both genes were introduced by Agrobacterium‐mediated transformation or by PEG‐mediated delivery into protoplasts. Outcomes included lines with mutations in all or only some of the homoeoalleles of SBE genes, and lines in which homoeoalleles carried several different mutations. DNA delivery into protoplasts resulted in mutants with no detectable Cas9 gene, suggesting the absence of foreign DNA. Selected mutants with starch granule abnormalities had reductions in tuber SBE1 and/or SBE2 protein that were broadly in line with expectations from genotype analysis. Strong reduction of both SBE isoforms created an extreme starch phenotype, as reported previously for low‐SBE potato tubers. HPLC‐SEC and 1H NMR revealed a decrease in short amylopectin chains, an increase in long chains and a large reduction in branching frequency relative to wild‐type starch. Mutants with strong reductions of SBE2 protein alone had near‐normal amylopectin chain length distributions and only small reductions in branching frequency. However, starch granule initiation was enormously increased: cells contained many granules of < 4 μm and granules with multiple hila. Thus large reductions in both SBEs reduce amylopectin branching during granule growth, whereas reduction of SBE2 alone primarily affects numbers of starch granule initiations. Our results demonstrate that Cas9‐mediated mutagenesis of SBE genes has the potential to generate new, potentially valuable starch properties without integration of foreign DNA into the genome.

The protosteryl and dammarenyl cation dichotomy in polycyclic triterpene biosynthesis revisited: has this ‘rule’ finally been broken?

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OpenPlant postdoc Michael Stephenson, and group leaders Rob Field and Anne Osbourn at the John Innes Centre in Norwich, reveal novel insights into triterpene biogenesis in their recent paper.

Stephenson MJ,  Field RA,  and  Osbourn A (2019) The protosteryl and dammarenyl cation dichotomy in polycyclic triterpene biosynthesis revisited: has this ‘rule’ finally been broken? Natural Product Reports, DOI: 10.1039/c8np00096d

Abstract

The triterpene alcohols represent an important and diverse class of natural products. This diversity is believed to originate from the differential enzymatically controlled cyclisation of 2,3-oxidosqualene. It is now a well-established presumption that all naturally occurring tetra- and penta-cyclic triterpene alcohols can be rationalised by the resolution of one of two intermediary tetracyclic cations, termed the protosteryl and dammarenyl cations. Here, a discussion of typical key triterpene structures and their proposed derivation from either of these progenitors is followed by comparison with a recently reported novel pentacyclic triterpene orysatinol which appears to correspond to an unprecedented divergence from this dichotomous protosteryl/dammarenyl view of triterpene biogenesis. Not only does this discovery widen the potential scope of triterpene scaffolds that could exist in nature, it could call into question the reliability of stereochemical assignments of some existing triterpene structures that are supported by only limited spectroscopic evidence. The discovery of orysatinol provides direct experimental evidence to support considering more flexibility in the stereochemical interpretation of the biogenic isoprene rule.

Two members of the DUF579 family are responsible for arabinogalactan methylation in Arabidopsis

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OpenPlant postdoc Henry Temple, in Prof Paul Dupree’s lab at the University of Cambridge, has published his discovery of a novel role for members of the DUF579 protein family in plant cell wall modification.

Temple H, Mortimer JC, Tryfona T, Yu X, Lopez‐Hernandez F, Sorieul M, Anders N, Dupree P (2019) Two members of the DUF579 family are responsible for arabinogalactan methylation in Arabidopsis. Plant Direct, https://doi.org/10.1002/pld3.117

Abstract

All members of the DUF579 family characterized so far have been described to affect the integrity of the hemicellulosic cell wall component xylan: GXMs are glucuronoxylan methyltransferases catalyzing 4‐O–methylation of glucuronic acid on xylan; IRX15 and IRX15L, although their enzymatic activity is unknown, are required for xylan biosynthesis and/or xylan deposition. Here we show that the DUF579 family members, AGM1 and AGM2, are required for 4‐O–methylation of glucuronic acid of a different plant cell wall component, the highly glycosylated arabinogalactan proteins (AGPs).

Two publications describe focus stacking setup developed through OpenPlant Fund

Dr Jennifer Deegan has built a Focus Stacking system that enables her to take close up photos of really small plant samples, in which the full sample is in focus. In her OpenPlant Fund project, she developed the system further, working with collaborators to try photography of new samples, and built up teaching tools to enable others to replicate the system. Read about her project here: https://www.biomaker.org/projects/focus-stacking-for-teaching-and-publication-in-plant-sciences and in the two publications below:

Part 1: Deegan, J. (2017). Photographing The Fern Gametophyte Developmental Series – The First Attempt. Pteridologist, 6 (4), 263-265. https://doi.org/10.17863/CAM.17067

Part 2: Deegan, J. I., & Deegan, T. (2018).  Macrophotography of Fern Gametophytes Using a Focus Stacking System. Pteridologist, 6 (5), 357-360. https://doi.org/10.17863/CAM.33541

miRNA-mediated regulation of synthetic gene circuits in the green alga Chlamydomonas reinhardtii

OpenPlant postdoc Francisco Navarro, in Prof David Baulcombe’s lab at the University of Cambridge, has published his work on regulation of synthetic gene circuits by miRNA in Chalmydomonas reinhardtii, in ACS Synthetic Biology. This work describes a new mechanism for regulation that can be used in in new synthetic biology applications in this green algae chassis.

Navarro F, Baulcombe DC (2019). miRNA-mediated regulation of synthetic gene circuits in the green alga Chlamydomonas reinhardtii. ACS Synthetic Biology, https://doi.org/10.1021/acssynbio.8b00393. [Epub ahead of print]

Abstract

microRNAs (miRNAs), small RNA molecules of 20-24 nts, have many features that make them useful tools for gene expression regulation - small size, flexible design, target predictability and action at a late stage of the gene expression pipeline. In addition, their role in fine-tuning gene expression can be harnessed to increase robustness of synthetic gene networks. In this work we apply a synthetic biology approach to characterize miRNA-mediated gene expression regulation in the unicellular green alga Chlamydomonas reinhardtii. This characterization is then used to build tools based on miRNAs, such as synthetic miRNAs, miRNA-responsive 3'UTRs, miRNA decoys and self-regulatory loops. These tools will facilitate the engineering of gene expression for new applications and improved traits in this alga.

OpenPlant researcher characterises pathway for active chemical in catnip

News article below has been copied with permission from the John Innes Centre website. The original article can be found here.

Benjamin R. Lichman, Mohamed O. Kamileen, Gabriel R. Titchiner, Gerhard Saalbach, Clare E. M. Stevenson, David M. Lawson & Sarah E. O’Connor. Uncoupled activation and cyclization in catmint reductive terpenoid biosynthesis. Nature Chemical Biologyvolume 15, pages71–79 (2019)


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Researchers at John Innes Centre have shed light on how catnip – also known as catmint – produces the chemical that sends cats into a state of wanton abandon.

The remarkable effect catnip has on cats is well known thanks to the scores on online videos showing pets enjoying its intoxicating highs.

The substance that triggers this state of feline ecstasy is called nepetalactone, a type of chemical called a terpene. This simple, small molecule is part of an unusual chain of events, not previously seen by chemists.

The researchers believe that understanding the production of these nepetalactones could help them recreate the way that plants synthesise other chemicals like vinblastine, which is used for chemotherapy. This could lead to the ability to create these useful medicines more efficiently and quickly than we are currently able to harvest them from nature.

Usually in plants, for example peppermint, terpenes are formed by a single enzyme. In their paper published online this week in Nature Chemical Biology, the researchers report that in catnip terpenes are formed in a two-step process; an enzyme activates a precursor compound which is then grabbed by a second enzyme to produce the substance of interest.

This two-step process has previously never been observed, and the researchers also expect something similar is occurring in the synthesis of anti-cancer drugs vincristine and vinblastine from Madagascan periwinkle, Catharanthus roseus, and elsewhere in olive and snapdragon.

In the publication, the team describe the process by which catmint produces nepetalactone in microscopic glands on the underside of its leaves. The study also identifies three new enzymes with unusual activity.

Dr Benjamin Lichman, who conducted the work while a post-doc at John Innes Centre and who is now a lecturer at the University of York, says: “We have made significant progress in understanding how catnip makes nepetalactones, the chemicals that sends cats crazy. Catnip is performing unusual and unique chemical processes, and we plan to use these to help us create useful compounds that can be used in treatment of diseases such as cancer. We are also working to understand the evolution of catnip to understand how it came to produce the cat-active chemicals.”

Professor Sarah O’Connor, project leader at the John Innes Centre, says: “Nepetalactones have potential use in agriculture as they participate in certain plant-insect interactions. In future work we will explore the roles that these compounds have in plants.”

This research is funded by National Science Foundation Grant #1444499 and the UK Biotechnological and Biological Sciences Research Council (BBSRC) and Engineering and Physical Sciences Research Council (EPSRC) joint-funded OpenPlant Synthetic Biology Research Centre (BB/L014130/1).

Loop assembly: a simple and open system for recursive fabrication of DNA circuits

A new DNA assembly method has been developed by the Haseloff lab in Cambridge, in collaboration with the Patron lab at the Earlham Institute in Norwich and Fernan Federici in Chile. The method provides a simple solution for working with standardised DNA parts, e.g. those developed with the type IIS common syntax, and the system is openly shared under the OpenMTA.

Pollak B, Cerda A, Delmans M, Álamos S, Moyano T, West A, Gutiérrez RA, Patron N, Federici F, Haseloff J. Loop assembly: a simple and open system for recursive fabrication of DNA circuits. New Phytol. 2018 Dec 6. doi: 10.1111/nph.15625.

Abstract

High efficiency methods for DNA assembly have enabled routine assembly of synthetic DNAs of increased size and complexity. However, these techniques require customisation, elaborate vector sets or serial manipulations for the different stages of assembly. We have developed Loop assembly based on a recursive approach to DNA fabrication. The system makes use of two Type IIS restriction endonucleases and corresponding vector sets for efficient and parallel assembly of large DNA circuits. Standardised level 0 parts can be assembled into circuits containing 1, 4, 16 or more genes by looping between the two vector sets. The vectors also contain modular sites for hybrid assembly using sequence overlap methods. Loop assembly enables efficient and versatile DNA fabrication for plant transformation. We show construction of plasmids up to 16 genes and 38 Kb with high efficiency (>80%). We have characterized Loop assembly on over 200 different DNA constructs and validated the fidelity of the method by high-throughput Illumina plasmid sequencing. Our method provides a simple generalised solution for DNA construction with standardised parts. The cloning system is provided under an OpenMTA license for unrestricted sharing and open access.

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Speed breeding made accessible and democratic

Scientists at the John Innes Centre, Earlham Institute, and Quadram Institute in Norwich and the University of Queensland have improved the technique, known as speed breeding, adapting it to work in vast glasshouses and in scaled-down desktop growth chambers. The scaled-down chambers are the result of an OpenPlant Fund project to develop a “Bench-top Controlled Environment Growth Chamber for Speed-Breeding and Crop Transformation”.

Two papers have been published detailing the research on speed breeding and the protocols:

Watson A, Ghosh S, Williams MJ, Cuddy WS, Simmonds J, Rey MD, Asyraf Md Hatta M, Hinchliffe A, Steed A, Reynolds D, Adamski NM, Breakspear A, Korolev A, Rayner T, Dixon LE, Riaz A, Martin W, Ryan M, Edwards D, Batley J, Raman H, Carter J, Rogers C, Domoney C, Moore G, Harwood W, Nicholson P, Dieters MJ, DeLacy IH, Zhou J, Uauy C, Boden SA, Park RF, Wulff BBH, Hickey LT. Speed breeding is a powerful tool to accelerate crop research and breeding. Nat Plants. 2018 Jan;4(1):23-29. doi: 10.1038/s41477-017-0083-8.

Ghosh S, Watson A, Gonzalez-Navarro OE, Ramirez-Gonzalez RH, Yanes L, Mendoza-Suárez M, Simmonds J, Wells R, Rayner T, Green P, Hafeez A, Hayta S, Melton RE, Steed A, Sarkar A, Carter J, Perkins L, Lord J, Tester M, Osbourn A, Moscou MJ, Nicholson P, Harwood W, Martin C, Domoney C, Uauy C, Hazard B, Wulff BBH, Hickey LT. Speed breeding in growth chambers and glasshouses for crop breeding and model plant research. Nat Protoc. 2018 Dec;13(12):2944-2963. doi: 10.1038/s41596-018-0072-z.

There has been a lot of interest in the speed breeding technology and in the desktop speed breeding chamber, and the researchers highlighted the work in a piece on BBC Look East. The research is also described in a news article on the John Innes Centre website and through a series of videos.

Birth of a Photosynthetic Chassis: Microalga Chlamydomonas reinhardtii

An European collaboration that has included researchers in the OpenPlant labs of Prof. David Baulcombe, Prof. Alison Smith and Prof. Jim Haseloff has resulted in the publication of a key paper in ACS Synthetic Biology aiming to establish Chlamydomonas reinhardtii as a chassis for synthetic biology. As part of this effort, the authors have developed and characterised 119 openly distributed genetic parts.

Full Text

Crozet P, Navarro FJ, Willmund F, Mehrshahi P, Bakowski K, Lauersen KJ, Pérez-Pérez ME, Auroy P, Gorchs Rovira A, Sauret-Gueto S, Niemeyer J, Spaniol B, Theis J, Trösch R, Westrich LD, Vavitsas K, Baier T, Hübner W, de Carpentier F, Cassarini M, Danon A, Henri J, Marchand CH, de Mia M, Sarkissian K, Baulcombe DC, Peltier G, Crespo JL, Kruse O, Jensen PE, Schroda M, Smith AG, Lemaire SD. Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synth Biol. 2018 Sep 21;7(9):2074-2086. doi: 10.1021/acssynbio.8b00251.

Abstract

Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows. To this end we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii. It is based on Golden Gate cloning with standard syntax, and comprises 119 openly distributed genetic parts, most of which have been functionally validated in several strains. It contains promoters, UTRs, terminators, tags, reporters, antibiotic resistance genes, and introns cloned in various positions to allow maximum modularity. The toolkit enables rapid building of engineered cells for both fundamental research and algal biotechnology. This work will make Chlamydomonas the next chassis for sustainable synthetic biology.

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Visual method for triterpene production in Nicotiana benthamiana

OpenPlant post-doc Michael Stephenson in Prof. Anne Osbourn’s lab at the John Innes Centre, has starred in a video supporting a protocols paper in the Journal of Visual Experimentation:


Abstract

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

Stephenson MJ, Reed J, Brouwer B, Osbourn A. Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale. J Vis Exp. 2018 Aug 16;(138). doi: 10.3791/58169.

Control of cell size and division driven through the circadian clock in cyanobacteria

OpenPlant postdoc Bruno Martins, in Dr James Locke’s lab in the Sainsbury Laboratory, Cambridge, has published and article in PNAS identifying the control that the circadian clock exerts on cell size and division. This could be a first step towards engineering the "rules” of size control in this single-celled organism.

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When and at what size to divide are critical decisions, requiring cells to integrate internal and external cues. While it is known that the 24-h circadian clock and the environment modulate division timings across organisms, how these signals combine to set the size at which cells divide is not understood.

Iterating between modeling and experiments, the authors show that, in both constant and light−dark conditions, the cyanobacterial clock produces distinctly sized and timed subpopulations. These arise from continuous coupling of the clock to the cell cycle, which, in light−dark cycles, steers cell divisions away from dawn and dusk. Stochastic modeling allows them to predict how these effects emerge from the complex interactions between the environment, clock, and cell size control.

Abstract

How cells maintain their size has been extensively studied under constant conditions. In the wild, however, cells rarely experience constant environments. Here, we examine how the 24-h circadian clock and environmental cycles modulate cell size control and division timings in the cyanobacterium Synechococcus elongatus using single-cell time-lapse microscopy. Under constant light, wild-type cells follow an apparent sizer-like principle. Closer inspection reveals that the clock generates two subpopulations, with cells born in the subjective day following different division rules from cells born in subjective night. A stochastic model explains how this behavior emerges from the interaction of cell size control with the clock. We demonstrate that the clock continuously modulates the probability of cell division throughout day and night, rather than solely applying an on−off gate to division, as previously proposed. Iterating between modeling and experiments, we go on to identify an effective coupling of the division rate to time of day through the combined effects of the environment and the clock on cell division. Under naturally graded light−dark cycles, this coupling narrows the time window of cell divisions and shifts divisions away from when light levels are low and cell growth is reduced. Our analysis allows us to disentangle, and predict the effects of, the complex interactions between the environment, clock, and cell size control.

Martins BMC, Tooke AK, Thomas P, Locke JCW. Cell size control driven by the circadian clock and environment in cyanobacteria. PNAS November 27, 2018 115 (48) E11415-E11424; published ahead of print November 8, 2018 https://doi.org/10.1073/pnas.1811309115

Sticking to it: phytopathogen effector molecules may converge on evolutionarily conserved host targets in green plants

OpenPlant postdoc Phillip Carella, from the lab of Dr Sebastian Schornack in the Sainsbury Laboratory, Cambridge, has published his research on the targets of effector proteins in green plants.

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Abstract

Plant-associated microbes secrete effector proteins that subvert host cellular machinery to facilitate the colonization of plant tissues and cells. Accumulating data suggests that independently evolved effectors from bacterial, fungal, and oomycete pathogens may converge on a similar set of host proteins in certain angiosperm models, however, whether this concept is relevant throughout the green plant lineage is unknown. Here, we explore the idea that pathogen effector molecules target host proteins present across evolutionarily distant land plant lineages to promote disease. We discuss that host proteins targeted by phytopathogens or integrated into angiosperm immune receptors are likely found across green plant genomes, from early diverging non-vascular lineages (bryophytes) to flowering plants (angiosperms). This would suggest that independently evolved pathogens might manipulate their hosts by targeting `vulnerability’ hubs that are present across land plants. Future work focusing on accessible early divergent land plant model systems may therefore provide an insightful evolutionary backdrop for effector–target research.

Find the full article at

Carella P, Evangelisti E, Schornack S. Sticking to it: phytopathogen effector molecules may converge on evolutionarily conserved host targets in green plants. Current Opinion in Plant Biology 44, August 2018, Pages 175-180; https://doi.org/10.1016/j.pbi.2018.04.019

Opening up Global Biotech Innovation: Publication of OpenMTA

The OpenMTA was launched with a commentary published in the journal Nature Biotechnology in October 2018. It provides a new way to exchange materials commonly used in biological research and engineering, complementing existing, more restrictive arrangements. The OpenMTA also promotes access for researchers and individuals working in less privileged institutions and world regions.

Download: OpenMTA Commentary. “Opening options for material transfer”. Linda Kahl, Jennifer Molloy, Nicola Patron, Colette Matthewman, Jim Haseloff, David Grewal, Richard Johnson & Drew Endy. Nature Biotechnology 36:923–927 (2018). https://doi.org/10.1038/nbt.4263


Abstract

Material-transfer agreements (MTAs) underlie the legal frameworks within which biotechnology practitioners define the terms and conditions for sharing biomaterials ranging, for example, from plasmid DNA to patient samples. If MTAs are easy to use and well adapted to the needs of individual researchers, institutions, and broader communities, then more sharing, innovation, and translation can occur. However, the MTA frameworks currently in place were developed in the 1990s—before widespread adoption of the World Wide Web, genome sequencing, and gene synthesis—and are not always well adapted for contemporary research and translation practices or aligned with social objectives.

Here, we introduce a new MTA, the Open Material Transfer Agreement (OpenMTA), that relaxes restrictions on the redistribution and commercial use of biomaterials while maintaining aspects of standard MTAs that support widespread adoption (for example, incorporation into semiautomated administration systems). In developing the OpenMTA, our motivation was to realize a simple, standardized legal tool for sharing biological materials as broadly as possible without undue restrictions, while respecting the rights of creators and promoting safe practices and responsible research. Importantly, we wanted the tool to work within the practical realities of technology transfer and to be sufficiently flexible to accommodate the needs of many groups globally (for example, providing support for international transfers and compatibility with public and philanthropic funding policies).

Colour bio-factories: anthocyanin production in plant cell cultures

Bioreactors with engineered tobacco (left) and wild-type grape (right) cell cultures.

Bioreactors with engineered tobacco (left) and wild-type grape (right) cell cultures.

OpenPlant Postdoc, Ingo Appelhagen, in Prof Cathie Martin's lab in the John Innes Centre has recently published an article in the journal Metabolic Engineering about his research to develop a system for production of high-levels of anthocyanins in plant cell cultures.

Anthocyanins give many fruits and flowers their red, purple or blue colouration. The martin lab are interested in the beneficial effects of anthocyanins in our diets and their use as natural colourants in the food and cosmetic industries.

 

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Appelhagen I, Wulff-Vester AK, Wendell M, Hvoslef-Eide AK, Russell J, Oertel A, Martens S, Mock HP, Martin C, Matros A (2018). Colour bio-factories: Towards scale-up production of anthocyanins in plant cell cultures. Metabolic Engineering. Doi: https://doi.org/10.1016/j.ymben.2018.06.004

Abstract

Anthocyanins are widely distributed, glycosylated, water-soluble plant pigments, which give many fruits and flowers their red, purple or blue colouration. Their beneficial effects in a dietary context have encouraged increasing use of anthocyanins as natural colourants in the food and cosmetic industries. However, the limited availability and diversity of anthocyanins commercially have initiated searches for alternative sources of these natural colourants. In plants, high-level production of secondary metabolites, such as anthocyanins, can be achieved by engineering of regulatory genes as well as genes encoding biosynthetic enzymes. We have used tobacco lines which constitutively produce high levels of cyanidin 3-O-rutinoside, delphinidin 3-O-rutinoside or a novel anthocyanin, acylated cyanidin 3-O-(coumaroyl) rutinoside to generate cell suspension cultures. The cell lines are stable in their production rates and superior to conventional plant cell cultures. Scale-up of anthocyanin production in small scale fermenters has been demonstrated. The cell cultures have also proven to be a suitable system for production of 13C-labelled anthocyanins. Our method for anthocyanin production is transferable to other plant species, such as Arabidopsis thaliana, demonstrating the potential of this approach for making a wide range of highly-decorated anthocyanins. The tobacco cell cultures represent a customisable and sustainable alternative to conventional anthocyanin production platforms and have considerable potential for use in industrial and medical applications of anthocyanins.

Engineering plant production systems to synthesise terpenoids

Nicotiana benthamiana by Aymeric Leveau (John Innes Centre);  NRP-103

Nicotiana benthamiana by Aymeric Leveau (John Innes Centre); NRP-103

Researchers at the John Innes Centre, including OpenPlant PI Prof Anne Osbourn have recently published a review describing strategies developed in the lab to engineer terpenoid biosynthesis in plant-based production systems.

Terpenoids are the most structurally diverse class of plant natural products with a huge range of commercial and medical applications. Exploiting this enormous potential has historically been hindered due to low levels of these compounds in their natural sources, making isolation difficult, while their structural complexity frequently makes synthetic chemistry approaches uneconomical.

Reed, J. & Osbourn A. (2018), Engineering terpenoid production through transient expression in Nicotiana benthamiana. Plant Cell Rep. https://doi.org/10.1007/s00299-018-2296-3

Abstract

Terpenoids are the most structurally diverse class of plant natural products with a huge range of commercial and medical applications. Exploiting this enormous potential has historically been hindered due to low levels of these compounds in their natural sources, making isolation difficult, while their structural complexity frequently makes synthetic chemistry approaches uneconomical. Engineering terpenoid biosynthesis in heterologous host production platforms provides a means to overcome these obstacles. In particular, plant-based production systems are attractive as they provide the compartmentalisation and cofactors necessary for the transfer of functional pathways from other plants. Nicotiana benthamiana, a wild relative of tobacco, has become increasingly popular as a heterologous expression platform for reconstituting plant natural product pathways, because it is amenable to Agrobacterium-mediated transient expression, a scalable and highly flexible process that enables rapid expression of genes and enzymes from other plant species. Here, we review recent work describing terpene production in N. benthamiana. We examine various strategies taken to engineer this host for increased production of the target metabolite. We also look at how transient expression can be utilised for rapid generation of molecular diversity, including new-to-nature products. Finally, we highlight current issues surrounding this expression platform and discuss the future directions and developments which will be needed to fully realise the potential of this system.

OpenPlant Fund project publishes on droplet-based microfluidics for rapid phenotyping of plant systems

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Researchers from the University of Cambridge have this month published the results of a collaborative project, supported by the OpenPlant Fund, to develop droplet-based microfluidics systems for rapid prototyping in plant systems. The paper describes how they have achieved on-chip encapsulation and analysis of protoplasts isolated from the emergent plant model Marchantia polymorpha. We caught up with the team to find out more about their OpenPlant Fund project. Read on to find out more...

 

Yu Z, Boehm CR, Hibberd JM, Abell C, Haseloff J, Burgess SJ, et al. (2018) Droplet-based microfluidic analysis and screening of single plant cells. PLoS ONE 13(5): e0196810.

 

Publication abstract

Droplet-based microfluidics has been used to facilitate high-throughput analysis of individual prokaryote and mammalian cells. However, there is a scarcity of similar workflows applicable to rapid phenotyping of plant systems where phenotyping analyses typically are time-consuming and low-throughput. We report on-chip encapsulation and analysis of protoplasts isolated from the emergent plant model Marchantia polymorpha at processing rates of >100,000 cells per hour. We use our microfluidic system to quantify the stochastic properties of a heat-inducible promoter across a population of transgenic protoplasts to demonstrate its potential for assessing gene expression activity in response to environmental conditions. We further demonstrate on-chip sorting of droplets containing YFP-expressing protoplasts from wild type cells using dielectrophoresis force. This work opens the door to droplet-based microfluidic analysis of plant cells for applications ranging from high-throughput characterisation of DNA parts to single-cell genomics to selection of rare plant phenotypes.

 

Interview with the OpenPlant Fund team

A brief overview of the project

A current limitation for plant synthetic biology involves high-throughput screening of genetic parts in plants. Current approaches require testing circuits in individual plants, through transient or stable transgenics. Applying these techniques to hundreds of different circuits is not feasible at a laboratory scale. In this project, we use droplet based microfluidics to isolate and characterise both gene expression activity and chlorophyll content on single plant protoplasts at a high throughput scale. Our device can potentially analyse protoplasts at a processing rate of > 100,000 cells per hour.  We use this system to quantify the stochastic properties of a heat-inducible promoter across a population of transgenic Marchantia polymorpha protoplasts to demonstrate its potential for assessing gene expression activity in response to environmental conditions. In addition, we managed to sort droplets containing YFP-expressing protoplasts from wild type cells using dielectrophoresis force.  This work opens the door to droplet-based microfluidic analysis of plant cells for applications ranging from high-throughput characterisation of DNA parts to single-cell genomics to selection of rare plant phenotypes.

 

A schematic of the droplet based microfluidics setup

A schematic of the droplet based microfluidics setup

 

What inspired the project?

Part of our research is focused on identifying DNA regulatory elements that could be used for designing synthetic promoters in plants. To achieve this, we would normally create a reporter construct containing the element of interest and then test its activity on individual plants through transient or stable genetic transformation. This approach can be very time consuming and impedes the researcher to test more than a handful of constructs at the time. Based on this we consider there was a need to develop methods that could accelerate the process. We knew Droplet-based microfluidics has been used to facilitate high throughput analysis of individual prokaryote and mammalian cells so we thought implementing this method in plants will be very useful.  

How did this project develop links between Cambridge and Norwich?

During the early stages of the project we teamed up with Oleg Raitskin from the Patron lab at the Earlham Institute. Oleg was very kind to share his experiences with protoplast isolation and also he showed us his method for protoplast transformation in tobacco leaves.

 

Microscopy images of protoplasts captured in droplets and sorted by fluorescence

Microscopy images of protoplasts captured in droplets and sorted by fluorescence

 

What was your favourite aspect of the project?

We really enjoyed learning more about each other’s area of expertise. This project spans the disciplines of physical chemistry optomechatronics and biology so it gave us an opportunity to approach disciplines in which we were not very familiar.

Sorting of M. polymorpha protoplasts: Microscopy images of microdroplets sorted into positive and negative channels based on their fluorescence intensity.

Sorting of M. polymorpha protoplasts: Microscopy images of microdroplets sorted into positive and negative channels based on their fluorescence intensity.

What is the biggest challenge the team faced?

Preparing high quality protoplast preparations from Marchantia was one of the greater challenges we encountered.

Is there something that came out of the project that you never expected at the beginning?

Being able to perform the sorting was unexpected at the beginning. Protoplasts are very sensitive cells that can burst spontaneously. The fact that we manage not just to isolate and measure but also sort opens a lot of possibilities for further applications.

How did the OpenPlant Fund enable the development of the project?

We could have never done this kind of project without the OpenPlant Fund. Apart of the funding which played a primal role in the development of the project, the OpenPlant fund offered great support across the whole process. For instance, our collaboration was established during a fund mixer organized by the synbio fund. Thanks to the Open Plant initiative we showed our project in various Open Plant meetings which resulted in very useful comments from various colleagues. Finally the costs derived from publishing the paper and making it open access were also covered by the grant.

What are the future opportunities to take this project forward?

Now that the system is set up, the next step could be to expand it to on-chip protoplast transformation (as has been done for other cell types). Protoplast transformation currently requires large amounts of materials (cells and DNA) and is low throughput, so this would be a big plus.

Researchers find first land plants were parasitised by microbes

The following blog was first published on the website of The Sainsbury Laboratory, Cambridge, and has been reproduced with the permission. The original can be found here.

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Sainsbury Laboratory researchers have found that the relationship between plants and filamentous microbes not only dates back millions of years, but that modern plants have maintained this ancient mechanism to accommodate and respond to microbial invaders.

Why do some plants welcome some microbes with open arms while giving others the cold-shoulder? Like most relationships, it’s complicated, and it all goes back a long way.

By studying liverworts – which diverged from other land plants early in the history of plant evolution – researchers from the Sainsbury Laboratory at the University of Cambridge have found that the relationship between plants and filamentous microbes not only dates back millions of years, but that modern plants have maintained this ancient mechanism to accommodate and respond to microbial invaders.

Liverworts

Close up of a liverwort.

Close up of a liverwort.

Liverworts are small green plants that don’t have roots, stems, leaves or flowers. They belong to a group of plants called Bryophytes, which also includes mosses and hornworts. Bryophytes diverged from other plant lineages early in the evolution of plants and are thought to be similar to some of the earliest diverging land plant lineages. Liverworts are found all over the world and are often seen growing as a weed in the cracks of paving or on the soil of potted plants. Marchantia polymorpha, which is also known as the common liverwort or umbrella liverwort, was used in this research.

Published today in the journal Proceedings of the National Academy of Sciences, a new study shows that aggressive filamentous microbial (fungi-like) pathogens can invade liverworts and that some elements of the liverwort’s response are shared with distantly related plants. The first author of the paper, Dr Philip Carella, said the research showed that liverworts could be infected by the common and devastating microorganism Phytophthora: “We know a great deal about microbial infections of modern flowering plants, but until now we haven’t known how distantly related plant lineages dealt with an invasion by an aggressive microbe. To test this, we first wanted to see if Phytophthora could infect and complete its life cycle in a liverwort."

 

A healthy Marchantia polymorpha liverwort (left) and one that has been infected by Phytophthora palmivora (right).

A healthy Marchantia polymorpha liverwort (left) and one that has been infected by Phytophthora palmivora (right).

We found that Phytophthora palmivora can colonise the photosynthetic tissues of the liverwort Marchantia polymorpha by invading living cells. Marchantia responds to this by deploying proteins around the invading Phytophthora hyphal structures. These proteins are similar to those that are produced in flowering plants such as tobacco, legumes or Arabidopsis in response to infections by both symbiont and pathogenic microbes.”

 

Microscopy image of a cross-section of a Marchantia polymorpha thallus showing the Phytophthora infection (red) in the upper photosynthetic layer of the liverwort plant.

Microscopy image of a cross-section of a Marchantia polymorpha thallus showing the Phytophthora infection (red) in the upper photosynthetic layer of the liverwort plant.

These lineages share a common ancestor that lived over 400 million years ago, and fossils from this time period show evidence that plants were already forming beneficial relationships with filamentous microbes. Dr Carella added: “These findings raise interesting questions about how plants and microbes have interacted and evolved pathogenic and symbiotic relationships. Which mechanisms evolved early in a common ancestor before the plant groups diverged and which evolved independently?”

Phytophthora

Phytophthora  growing on  Marchantia thallus

Phytophthora growing on Marchantia thallus

Phytophthora is a water mould. Although it looks like it, it is not a fungus at all. Instead it belongs to the oomycetes and is a type of filamentous microbe. Phytophthora pathogens are best known for devastating crops, such as causing the Irish potato famine through potato late blight disease as well as many tropical diseases. This research used the tropical species, Phytophthora palmivora, which causes diseases in cocoa, oil palms, coconut palms and rubber trees.

Dr Sebastian Schornack, who led the research team, says the study indicates that early land plants were already genetically equipped to respond to microbial infections: “This discovery reveals that certain response mechanisms were already in place very early on in plant evolution.”

“Finding that pathogenic filamentous microbes can invade living liverwort cells and that liverworts respond using similar proteins as in flowering plants suggests that the relationship between filamentous pathogens and plants can be considered ancient. We will continue to study whether pathogens are exploiting mechanisms evolved to support symbionts and, hopefully, this will allow us to establish future crop plants that both benefit from symbionts whilst remaining more resistant to pathogens.

“Liverworts are showing great promise as a model plant system and this discovery that they can be colonised by pathogens of flowering plants makes them a valuable model plant to continue research into plant-microbe interactions.”

Read the full paper online.

This research was funded by the Gatsby Charitable Foundation, the Royal Society, the BBSRC OpenPlant initiative and the Natural Environment Research Council.

 

Photo Credits: Images by Philip Carella.

Collaboration including OpenPlant researchers discovers that C4 photosynthesis has co-opted an ancient C3 regulatory code

C4Maize_Ninghui Shi_CC BY-SA 3.0.jpg

A new publication in Molecular Biology and Evolution has resulted from a collaboration of OpenPlant PI Prof. Julian Hibberd and researcher Dr Ivan Reyna-Llorens with colleagues in Portugal at the Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, and the Instituto de Biologia Experimental e Tecnológica in Portugal. The paper shows that C4 photosynthesis has co-opted an ancient C3 regulatory code:

Borba AR, Serra TS, Górska A, Gouveia P, Cordeiro AM, Reyna-Llorens I, Kneřová J, Barros PM, Abreu IA, Oliveira MM, Hibberd JM, Saibo NJM (2018). Synergistic binding of bHLH transcription factors to the promoter of the maize NADP-ME gene used in C4 photosynthesis is based on an ancient code found in the ancestral C3 state. Molecular Biology and Evolution, msy060, https://doi.org/10.1093/molbev/msy060

Abstract

C4 photosynthesis has evolved repeatedly from the ancestral C3 state to generate a carbon concentrating mechanism that increases photosynthetic efficiency. This specialised form of photosynthesis is particularly common in the PACMAD clade of grasses, and is used by many of the world’s most productive crops. The C4 cycle is accomplished through cell-type specific accumulation of enzymes but cis-elements and transcription factors controlling C4 photosynthesis remain largely unknown. Using the NADP-Malic Enzyme (NADP-ME) gene as a model we tested whether mechanisms impacting on transcription in C4 plants evolved from ancestral components found in C3 species. Two basic Helix-Loop-Helix (bHLH) transcription factors, ZmbHLH128 and ZmbHLH129, were shown to bind the C4NADP-ME promoter from maize. These proteins form heterodimers and ZmbHLH129 impairs trans-activation by ZmbHLH128. Electrophoretic mobility shift assays indicate that a pair of cis-elements separated by a seven base pair spacer synergistically bind either ZmbHLH128 or ZmbHLH129. This pair of cis-elements is found in both C3 and C4 Panicoid grass species of the PACMAD clade. Our analysis is consistent with this cis-element pair originating from a single motif present in the ancestral C3 state. We conclude that C4 photosynthesis has co-opted an ancient C3 regulatory code built on G-box recognition by bHLH to regulate the NADP-ME gene. More broadly, our findings also contribute to the understanding of gene regulatory networks controlling C4 photosynthesis.


Image by Ninghui Shi: Cross section of a C4 plant. Specifically of a maize leaf. Vascular bundles shown. Drawing based on microscopic images courtesy of Cambridge University Plant Sciences Department. Image is shared under licence CC BY-SA 3.0

Bio-solar panel developed by researchers at University of Cambridge and Imperial College London

A two-in-one solar bio-battery and solar panel has been created by researchers who printed living cyanobacteria and circuitry onto paper.

Cyanobacteria are photosynthetic micro-organisms that have been on Earth for billions of years. They are thought to be the primary reason why the Earth’s atmosphere is oxygen rich. Several synthetic biology groups in Cambridge are working on these useful organisams, including OpenPlant PI Prof Chris Howe and OpenPlant Fund grantee Dr Paolo Bombelli (both Department of Biochemistry).

Together with researchers from Imperial College London and Central Saint Martins, they demonstrated that cyanobacteria could be used as an ink and printed from an inkjet printer in precise patterns onto electrically conductive carbon nanotubes, which were also inkjet-printed onto the piece of paper. The team showed that the cyanobacteria survived the printing process and were able to perform photosynthesis so that small amounts of electrical energy could be harvested over a period of 100 hours.

A bio-solar panel made in this way, the approximate size of an iPad, could power a simple digital clock, and in separate experiments, a small LED light bulb.

The team suggest their breakthrough could lead to new types of electrical devices that are made from paper and printed photosynthetic bacteria. These could include disposable power supplies integrated into paper-based sensors for monitoring patients with diabetes or devices that resemble wallpaper but are in fact environmental sensors for monitoring air quality in the home.

Dr Marin Sawa, a co-author from the Department of Chemical Engineering at Imperial College London, said: “We think our technology could have a range of applications such as acting as a sensor in the environment. Imagine a paper-based, disposable environmental sensor disguised as wallpaper, which could monitor air quality in the home. When it has done its job it could be removed and left to biodegrade in the garden without any impact on the environment.” 

New type of renewable energy

The solar bio-battery pushes forward research into a new type of renewable energy technology currently being developed by scientists globally called microbial biophotoltaics (BPV). It exploits the ability of cyanobacteria and other algae that use photosynthesis to convert light energy into an electrical current using water as the source of electrons.

One of the advantages of using BPVs to harvest energy from cells like cyanobacteria is that they can produce small amounts electricity in daylight and carry on producing it even in the dark from molecules produced in the light.

Some of the current limitations that scientists have previously faced when developing BPVs are that they are expensive to make, have low power output, and a short lifespan. All these drawbacks have prevented scientists from being able to scale up the technology to an industrial level.

The team says their approach of using an off-the-shelf inkjet printer to construct BPVs demonstrates a potential method for easily scaling up the technology, which may pave the way for its wider use.

Dr Andrea Fantuzzi, a co-author of the study from Department of Life Sciences at Imperial College London, said: “Paper-based BPVs are not meant to replace conventional solar cell technology for large-scale power production, but instead, could be used to construct power supplies that are both disposable and biodegradable. Their low power output means they are more suited to devices and applications that require a small and finite amount of energy, such as environmental sensing and biosensors.”

New types of paper-based sensors

The researchers suggest BPVs could be used in new forms of sensors built entirely from paper, which would mean that they are cheaper and more cost effective to make with less impact on resources and the environment.

Another example for BPVs, suggest the team, is in the healthcare industry.

Dr Andrea Fantuzzi said: “Paper-based BPVs integrated with printed electronics and biosensor technology could usher in an age of disposable paper-based sensors that monitor health indicators such as blood glucose levels in patients with diabetes. Once a measurement is taken, the device could be easily disposed of with low environmental impact and its ease of use could facilitate its direct employment by the patients. Furthermore, this approach has the potential to be very cost-effective, which could also pave the way for its use in developing countries with limited healthcare budgets and strains on resources.”

Next steps

The current paper-based BPV unit is a palm size. The next step will see the team scale up their proof-of-concept to A4 size to determine the electrical output on a larger scale.

Professor Christopher Howe, a co-author from the Department of Biochemistry at the University of Cambridge, added: “This is an exciting proof-of-concept. The challenge now is to make panels that are more powerful, long-lasting and robust.”

Publication

Sawa, Marin, Andrea Fantuzzi, Paolo Bombelli, Christopher J. Howe, Klaus Hellgardt, and Peter J. Nixon. "Electricity generation from digitally printed cyanobacteria." Nature Communications 8, no. 1 (2017): 1327.

Press release text is from Imperial College London and is available under an Attribution-NonCommercial-ShareAlike Creative Commons license.

Image credit: From publication, licensed under CC-BY 4.0