Arabidopsis Research Roundup: August 27th

The Arabdopsis Research Roundup broadens its remit this week. As well as including three original research papers, which look at casparian strip formation, light and hormone signaling, we also highlight an important viewpoint article that aims to set standards for synthetic biology parts. In addition we include a meeting report from a plant synthetic biology summer school and interviews with plant scientists at the JIC, Caroline Dean and Anne Osbourn.

Kamiya T, Borghi M, Wang P, Danku JM, Kalmbach L, Hosmani PS, Naseer S, Fujiwara T, Geldner N, Salt DE (2015) The MYB36 transcription factor orchestrates Casparian strip formation Proc Natl Acad Sci USA http://dx.doi.org/10.1073/pnas.1507691112 Open Access

GARNet Advisory Board Chairman David Salt (Aberdeen) leads this international collaboration that looks at the (relatively) poorly understood Casparian strip (CS), a lignin-based filter that lies in root endodermal cells. Formation of the CS is initiated by Casparian strip domain proteins (CASPs) that recruit other proteins, which begin the process of lignin deposition. In this study the authors look upstream this process and identify the transcription factor MYB36 that directly regulates expression of CASPs and is essential for CS formation. Ectopic expression of MYB36 in root cortical tissues is sufficient to stimulate expression of CASP1-GFP and subsequent deposit a CS-like structure in the cell wall of cortex cells. These results have implications for the design of future experiments that aim to control how nutrients are taken up by the plant as even though myb36 mutants have a ‘root-defect’, they also have changes to their leaf ionome.

Sadanandom A, Ádám É, Orosa B, Viczián A, Klose C, Zhang C, Josse EM, Kozma-Bognár L, Nagy F (2015) SUMOylation of phytochrome-B negatively regulates light-induced signaling in Arabidopsis thaliana Proc Natl Acad Sci USA http://dx.doi.org/10.1073/pnas.1415260112 Open Access

Ari Sadanandom (Durham) and Ferenc Nagy (Edinburgh) are the leaders of this study that investigates the precise function of the PhyB photoreceptor protein. PhyB interacts with a wide range of downstream signaling partners including the PHYTOCHROME INTERACTING FACTOR (PIF) transcription factors. The small ubiquitin-like modifier (SUMO) peptide is conjugated to larger proteins to bring about a variety of signaling outcomes. In this case the authors find that SUMO is preferentially attached to the C-term of PhyB under red light conditions, a relationship that occurs in a diurnal pattern. SUMOylation of PhyB prevents interaction with PIF5 whilst the OVERLY TOLERANT TO SALT 1 (OTS1) protein likely de-SUMOlyates PhyB in vivo. Altered levels of PhyB SUMOylation cause distinct light-responsive phenotypes and as such this paper adds another level of regulation to the already complex known network that controls light signaling.

Schuster C, Gaillochet C, Lohmann JU (2015) Arabidopsis HECATE genes function in phytohormone control during gynoecium development Development. http://dx.doi.org/10.1242/dev.120444 Open Access

Christopher Schuster who is now a postdoc based at the Sainsbury lab in Cambridge is the lead author on this investigation into the role of the HECATE (HEC) family of bHLH transcription factors on fruit development in Arabidopsis. During this process HEC proteins are involved in the response to both the phytohormones auxin and cytokinin, the authors proposing that HEC1 plays an essential role in Arabidopsis gynoecium formation.

Patron N et al (2015) Standards for plant synthetic biology: a common syntax for exchange of DNA parts New Phytologist http://dx.doi.org/10.1111/nph.13532 Open Access

Carmichael RE, Boyce A, Matthewman C Patron N (2015) An introduction to synthetic biology in plant systems New Phytologist http://dx.doi.org/10.1111/nph.13433 Open Access

Although not strictly based on Arabdopsis work, there are a couple of articles in New Phytologist that have broad relevance to plant scientists who are interested in plant synthetic biology. In the first of these Nicola Patron (The Sainsbury Laboratory) leads a wide consortium that aims to set parameters for the standardisation of parts in plant synthetic biology. It is hoped that as the principles of synbio are used more widley in the plant sciences that the proposals in this paper will serve as a useful guide to standidise part production. GARNet has recently written a blog post on this topic.
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The associated meeting report looks at the use of plant synthetic biology in a teaching context with a synopsis of the ERASynBio summer school hosted by John Innes Centre. In this event, young researchers from a range of backgrounds were introduced to the power and potential of plant synthetic biology through a diverse course of lectures, practical session and group projects.

 

Vicente C (2015) An interview with Caroline Dean Development http://dx.doi.org/10.1242/dev.127548 Open Access

An interview with Anne Osbourn (2015) New Phytologist http://dx.doi.org/10.1111/nph.13616 Open Access

These are interviews with eminent female plant molecular biologists who both work at the John Innes Centre. Caroline Dean’s lab focuses on the epigenetic mechanisms that regulate vernalisation whilst Anne Osbourn is interested in using synthetic biology approaches to engineer metabolic pathways for the production of novel compounds.

Arabidopsis Research Roundup: August 21st.

There are a wide array of topics included in this weeks Arabidopsis Research Roundup, ranging from studies on stomatal density, thylakoid transport, metabolic flux analysis, mutant detection and root development. We feature unlinked studies from three researchers from the University of Oxford Plant Science (Paul Jarvis, Lee Sweetlove and Nick Harberd), whilst the papers from Julie Gray and Brian Forde share the broad theme that investigates different mechanisms that might be used to improve nitrogen uptake, either by modifying the expression of a single gene involved in root development or by altering stomatal density.

Hepworth C, Doheny-Adams T, Hunt L, Cameron DD, Gray JE (2015) Manipulating stomatal density enhances drought tolerance without deleterious effect on nutrient uptake New Phytol. http://dx.doi.org/10.1111/nph.13598

Julie Gray (University of Sheffield) is an expert on both stomatal biology and on the potential for manipulating stomatal density to improve crop production. In this study drought tolerance and soil water retention were measured in four Arabidopsis mutants with defects in epidermal patterning and stomatal density. Nutrient uptake was measured by mass flow of 15N. Plants with less stomata had reduced transpiration and were drought-tolerant yet interestingly showed little reduction in shoot N concentrations, especially when water availability is restricted. In contrast, plants with extra stomata could take up more N except when access to water was reduced. Therefore the authors show that by altering stomatal density they can generate plants that are drought resistance yet maintain nutrient uptake or generate plants with enhancing nutrient uptake is conditions with plentiful water.

Trösch R, Töpel M, Flores-Pérez Ú, Jarvis P (2015) Genetic and Physical Interaction Studies Reveal Functional Similarities between ALB3 and ALB4 in Arabidopsis. Plant Physiol. http://dx.doi.org/10.1104/pp.15.00376

This German, Swedish and UK collaboration is led by Paul Jarvis at the University of Oxford and broadly investigates thylakoid protein targeting. The ALB3 complex has previously been shown to target light harvesting complex proteins (LHCP) to the thylakoid. A related Arabidopsis protein, ALB4, had been proposed to interact not the LHCPs but rather with the ATP synthase complex. However this study shows that ALB3 and ALB4 have some overlapping roles in addition to their specific functions and that they can engage with a similar set of interactor proteins to bring their substrates to the thylakoid membrane.

Cheung CY, Ratcliffe RG, Sweetlove LJ (2015) A method of accounting for enzyme costs in flux balance analysis reveals alternative pathways and metabolite stores in an illuminated Arabidopsis leaf Plant Physiol. http://dx.doi.org/10.1104/pp.15.00880

Lee Sweetlove (Oxford University) leads this study that looks at the Flux Balance Analysis (FBA) of plant metabolism across several metabolic pathways by attaching ‘flux weighting factors’ to allow for the variable intrinsic cost of supporting each flux. This model has been applied to the Arabidopsis leaf exposed to different light regimes to explore the flexibility of the network in meeting its metabolic requirements. The authors discover interesting trade-offs between use of different carbon storage forms and in the variable consumption of ATP and NADPH by different metabolic pathways.

Belfield EJ, Brown C, Gan X, Jiang C, Baban D, Mithani A, Mott R, Ragoussis J, Harberd NP (2014) Microarray-based optimization to detect genomic deletion mutations Genom Data Dec;2:53-54 http://dx.doi.org/10.1016/j.gdata.2014.04.005

GARNet Advisory Board member Nick Harberd (Oxford University) leads this short communication that highlights the development of a tool for detection of genomic deletion mutants in Arabidopsis. Using a NimbleGen whole genome custom tiling array they successfully identify five mutants with deletion ranging from 4bp to 5kb and therefore introduce a powerful tool for analysing this type of genetic lesion in Arabidopsis and other plant species with well-constructed genomes.

Yu C, Liu Y, Zhang A, Su S, Yan A, Huang L, Ali I, Liu Y, Forde BG, Gan Y (2015) MADS-box Transcription Factor OsMADS25 Regulates Root Development through Affection of Nitrate Accumulation in Rice PLoS One http://dx.doi.org/10.1371/journal.pone.0135196

Brian Forde (Lancaster University) is the UK lead on this Chinese collaboration that focuses on nitrate accumulation and how it regulates root development in rice. This occurs via a MADS-box transcription factor OsMADS25 that, when overexpressed in Arabidopsis, promotes primary and lateral root development. Altered expression of this gene also affects root development in transgenic rice and includes significant changes in nitrate accumulation. Therefore this gene might prove to be an important target for future attempts to improve plant growth in regions with altered nitrate concentrations.

Arabidopsis Research Roundup: August 12th

The UK Arabidopsis Research Roundup this week includes a couple of EVO-DEVO-type studies that compare processes within different organisms (Physcomitrella and Cardamine) to those occurring in Arabidopsis. These include the evolution of both hormone signaling and leaf development. Elsewhere a cell-biological focused study looks at the factors that control formation of plasmodesmata whilst another manuscript investigates the details of a plants mechanism to avoid photoinhibition.

Yasumura Y1, Pierik R2, Kelly S3, Sakuta M4, Voesenek LA5, Harberd NP (2015) An Ancestral Role for Constitutive Triple Response 1 (CTR1) Proteins in Both Ethylene and Abscisic Acid Signaling Plant Physiology http://dx.doi.org/10.1104/pp.15.00233

GARNet Advisory Board Member Nick Harberd leads this study that investigates the evolution of the CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1) protein, which has known to be involved in ethylene signalling for two decades. CTR1 is compared between mosses, lycophytes and angiosperms, showing that PpCTR1 from moss Physcomitrella patens has the same function and the Arabidopsis equivalent, indicating that this signaling pathway predates the land plant lineage. However PpCTR1 is also involved in ABA signaling, which is not the case with AtCTR1 and may be explained by the presence of an AtCTR1 homolog in angiosperms. The authors state that this work provides new insights into the molecular events that contributed to the adaptive evolution of regulatory mechanisms across plant species

Kirsten Knox, Pengwei Wang, Verena Kriechbaumer, Jens Tilsner, Lorenzo Frigerio, Imogen Sparkes, Chris Hawes, Karl Oparka (2015) Putting the Squeeze on Plasmodesmata: A Role for Reticulons in Primary Plasmodesmata Formation Plant Physiology http://dx.doi.org/10.1104/pp.15.00668

This study is led by Karl Oparka (Edinburgh) and Chris Hawes (Oxford Brookes) as well as including PIs from Exeter (Sparkes), Warwick (Frigerio) and St Andrews (Tilsner). The manuscript investigates formation of plasmodesmata (PD), which are known to form from endoplasmic reticulum (ER) via an intermediant termed the desmotubule. Members of the Reticulon (RTNLB) family of ER-tubulating proteins are found in the PD proteome are are associated with developing PD following cell division. The authors use super-resolution imaging to show that RTNLB6 colocalises with desmotubules. The mobility of these RTNLB proteins was show, using FRAP, to vary dependent on their positioning within a developing cell plate. Mutant studies show that RTNLB proteins act as important regulators of the formation of PDs and the authors discuss the wider potential roles of these proteins in this process.

Ware MA, Giovagnetti V, Belgio E, Ruban AV (2015) PsbS protein modulates non-photochemical chlorophyll fluorescence quenching in membranes depleted of photosystems J Photochem Photobiol B http://dx.doi.org/10.1016/j.jphotobiol.2015.07.016

Alexander Ruban (QMUL) continues a fine run of recent publications with this study that investigates plants that express increased levels of the photosynthetic PsbS protein, in the context of a subsequent increase in levels of non-photochemical fluorescence quenching (NPQ). In these PsbS overexpressors, there is increased amplitude of the irreversible NPQ component, qI, which likely results from aggregation of the LHCII antenna complex. Use of freeze-fracture electron microscopy show that quenched thylakoids have 3x more aggregated LHCII particles compared to those that are dark-adapted. Overall, these results demonstrate the importance of this LHCII aggregation in the NPQ mechanism whilst showing that structure of the PSII supercomplex plays no role in formation in process of quenching.

Cartolano M, Pieper B, Lempe J, Tattersall A, Huijser P, Tresch A, Darrah PR, Hay A, Tsiantis M (2015) Heterochrony underpins natural variation in Cardamine hirsuta leaf form Proc Natl Acad Sci U S A. 2015 Aug 4. http://dx.doi.org/10.1073/pnas.1419791112

The study is a continuation of many years of work led by Miltos Tsiantis (who maintains links with Oxford University), aimed at increasing the understanding of how different morphological patterns develop. They compare leaf patterning in Arabidopsis (which has a simple leaf) and in the related plant, Cardamine (that has a complex leaf). They have identified a novel QTL from Cardamine that shows that age-dependent progression of leaf form underlies variation in this trait within species. Interestingly the QTL mapped to a cis-acting region controlling expression of the floral regulator FLC. Genotypes expressing low levels of FLC show early flowering and accelerated changes in leaf form, including faster leaflet production. These findings link reproductive timing with leaf development and the authors speculate that this may help to optimize resource allocation to the next generation.

Arabidopsis Research Roundup: August 5th

This weeks Arabidopsis Research Roundup bucks the recent trend of featuring large consortium-led studies as it contains four studies each from a single UK Institution. Matthew Jones (Essex) looks at the link between photosynthesis, the circadian clock and blue-light signaling whilst Miriam Gifford (Warwick) uses cell sorting to more precisely define the plants response to an oomycete pathogen. Elsewhere Peter Eastmond (Rothamstead) looks at lipid metabolism and Keith Lindsey (Durham) leads a theorectical study on the effectiveness of methods for modelling hormone crosstalk in the root.

Litthauer S, Battle M, Lawson T, Jones MA (2015) Phototropins Maintain Robust Circadian Oscillation of PSII Operating Efficiency Under Blue Light Plant J. http://dx.doi.org/10.1111/tpj.12947

Matt Jones is a Leuverhulme Research Fellow at the University of Essex and this study is his first output as a group leader. It investigates the affect of the circadian clock on the operating efficiency of photosystem II (PSII). Previous this efficiency had been shown to be controlled by transcriptional feedback loops within the nucleus. However this study shows that in blue light it is maintained by phototropin receptors, which do not influence the nucleus. The novel imaging methodology used in this study highlight differences between the modulation of circadian outputs in distinct subcellular compartments.

Coker TL, Cevik V, Beynon JL, Gifford ML (2015) Spatial dissection of the Arabidopsis thaliana transcriptional response to downy mildew using Fluorescence Activated Cell Sorting Front Plant Sci. http://dx.doi.org/10.3389/fpls.2015.00527

Miriam Gifford leads this study from the University of Warwick that looks at the transcriptional response of Arabidopsis to downy mildew infection. The Gifford lab are experts in analysis of transcriptional data from microarrays. This study uses FACS-sorted cells infected with the biotrophic oomycete pathogen Hyaloperonospora arabidopsidis in an attempt to focus-in on infected cells without the diluting effects of non-infected cells within the same tissue. Almost 300 transcripts were differentially expressed between haustoriated and non-haustoriated cells and this technique uncovered novel genes that had previously not been implicated in playing a role in this pathogen response.

Craddock CP, Adams N, Bryant FM, Kurup S, Eastmond PJ (2015) Regulation of endomembrane biogenesis in Arabidopsis by PHOSPATIDIC ACID HYDROLASE Plant Signal Behav. http://dx.doi.org/10.1080/15592324.2015.1065367

This study was wholly undertaken at Rothamsted Research led by Peter Eastmond. They investigate the coordination of lipid biosynthesis by focussing on the activity of two different enzymes, PHOSPHATIDIC ACID PHOSPHOHYDROLASE (PAH) and PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE (CCT). These enzymes participate in a feedback loop to control the biosynthesis of phosphaticylcholine (PC) and phosphatidic acid (PA), which is linked to biogenesis of the endoplasmic reticulum. This work offers a clue that PAH activity may require phosphorylation even though this data is not yet clear.

Simon Moore, Xiaoxian Zhang, Junli Liu & Keith Lindsey (2015) Some fundamental aspects of modelling auxin patterning in the context of auxin-ethylene-cytokinin crosstalk Plant Signalling and Behaviour http://dx.doi.org/10.1080/15592324.2015.1056424

In this manuscript Keith Lindsey and colleagues from Durham University use the paradigm of root-tip auxin-ethylene-cytokinin signaling to discuss the effectiveness of linking experimental data, reaction kinetics and spatiotemporal modelling to dissect hormonal crosstalk. The authors agree that the integration of kinetic equations with spatial root structure can produce powerful models for assessing the crosstalk of multiple hormone interactions in a spatiotemporal manner. Finally the authors come up with key recommendations to be considered when developing models for spatiotemporal hormonal crosstalk in the Arabidopsis root

Introducing OpenPlant.

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Published on: August 3, 2015

OpenPlant is one of the BBSRC Synthetic Biology hubs and is a collaboration between researchers at the Cambridge University and in the John Innes Centre and The Sainsbury lab in Norwich. Last week the inaugural public OpenPlant meeting took place in the Department of Plant Science in Cambridge and had the broad remit to introduce the ideals of the program whilst highlighting the excellent science that is ongoing in the labs affiliated with the project. GARNet were very pleased to be able to attend the event which involved being part of the judging panel for the OpenPlant Fund as well as being able to interact with many significant members of the world synthetic biology research.

Day One: OpenPlant Fund

One of the key aims of OpenPlant is to support innovative synthetic biology research in any and all possible areas. To that end, the OpenPlant grant has the facility to fund small (£4K+1K) grants to Cambridge and Norwich researchers who come up with novel collaborative ideas. Throughout the day each of the 17 applicants gave a five-minute pitch to explain and sell their ideas. This is the full list of pitches and as advertised, they covered all aspects of what could be considered synthetic biology, from projects that built on already established research to those that were the result of real blue-sky thinking.

More conventional lab-based projects included the development of codon-optimisation tools in Chlamydomonas or investigating LysM receptor-like kinases in legumes and cereals. The proposed in silico projects included development of an ‘Open-Pi image’, which is low-cost system for image analysis and a project proposed by TGAC PI Rob Davey to look into setting up a hackathon to develop software for synbio literature mining. The more ‘unconventional’ synbio projects included two that are linked to the development of synbio resources in Africa, in collaboration with the organisation TrendinAfrica. Undoubtedly the most enthusiastic presentation came from Paolo Bombelli, whose vision is to set up the ‘Big-Algal Open Experiment’ that aims to provide low-cost algal growth incubators to involve school groups across the UK in the study of algal growth.

It was encouraging that many PhD students and postdocs took the opportunity to come up with innovative ideas that had the support of their relevant PIs. Some of the presentations were a little ‘flabby’ which may have been a consequence of knowing there was funding for 20 projects, with ‘only’ 17 presentations. Ultimately OpenPlant was happy to fund each of the proposals although in a few cases the judges asked for clarifications and amendments prior to that final agreement. Overall it was a very strong start to the Open Plant Fund and with another 4 years of funding, hopefully these projects will encourage other researchers to apply in coming years. As a community-facing organisation, GARNet would hope to see researchers outside of the OpenPlant sites getting involved in the development of collaborative ideas that might be submitted to future funding rounds.

Days 2-3: OpenPlant Forum

The meat of the meeting took place over two days and involved formal presentations across a range of topics important to the ideas and emphasis of OpenPlant. The section headings were: ‘Frameworks for Open Innovation’, ‘Open DNA parts and assembly’,Foundational Systems’,Plant-based Bioproduction’ and ‘OpenPlant and Open technologies’ that followed opening presentations from Cambridge project lead Jim Haseloff, who outlined the aims of OpenPlant and the keynote lecture from Tom Knight, founder of the recently minted Ginkgo Bioworks. Tom has been involved since the beginnings of synthetic biology and gave an interesting talk about both the history of the field and its future opportunities.

The first session entitled ‘Frameworks for Open Innovation’ touched upon one of the key tenants of OpenPlant, which is to encourage the free and open exchange of technology. The ultimate aim of this is in the development of an OpenMTA that would allow free transfer of materials between labs, in the hope of aiding innovation and collaboration. Linda Kahl from the Biobricks foundation outlined the history of Material Transfer Agreements and the challenges of moving toward an oMTA. Perhaps the most alarming statistic presented was that with the current MTA system, ~50% of academics were discouraged from continuing with their experiments due to difficulties obtaining samples.

Throughout the meeting it was very encouraging that the plant science community appears to have a more enlightened opinion about the potential uptake of the oMTA. Although it is a great idea to establish this oMTA, researchers will require a mechanism to give future (non-financial) attribution for the work they have put in. We will watch this space with interest. This topic was the subject of the ‘Intellectual Property Working Group’ meeting that following the OpenPlant Forum and GARNet was honoured to be able to sit in on the early discussions where experts in this area discussed what might be possible with an oMTA system. Look out for updates on the OpenPlant website for updates as this challenging process moves forward.

Prior to the first session Randy Rettberg from the iGEM organization announced the exciting news that there will be a specific ‘plant –track’ in the iGEM 2016 competition. This will hopefully inspire more teams to use a plant chassis in their projects. GARNet will certainly look to encourage plant academics to support teams in 2016. In the 2015 competition there are 14 teams from the UK so hopefully these numbers will rise in coming years.

It was clear throughout this meeting both in formal presentations/ questiontime and during informal discussions that the ‘DNA of iGEM runs through the worldwide synthetic biology community’ as many of those who are involved with iGEM are advocates of the competition for their entire careers. If you don’t know much about the competition then take a look through their extensive catalogue of information on the iGEM website.

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The second session was led off by Nicola Patron from The Sainsbury Lab who explained the foundation and rationale behind developing a common syntax for DNA parts, which has been discussed elsewhere on the GARNet blog. Later Christian Rogers explained how the Engineering Nitrogen Synbiosis for Africa (ENSA) project had embraced the need for a common set of DNA parts across their many projects. In addition he presented that there is the beginnings of a consensus across each of the large Gates Foundation funded projects (ENSA, C4 rice and RIPE) to be more efficient in the way they carry out tasks, which includes creation of DNA tools. It was reassuring to know that there are academic researchers who are attempting solve some of the coming ‘big problems for humanity’ in part using synthetic biology techniques.

Between Nicola and Christian’s talks, Fernan Federici introduced his project that looked at low cost gene assembly systems by showing a stunning set of images. In addition Markus Gershater, who used to work as a plant scientist but is now the co-founder of Syntace introduced their Antha software, which is their answer to dealing with biological complexity that aims to ease the automation of synthetic biological process. No doubt this will be software that is implemented in many synthetic biology labs in years to come.

AnthaSessions 3 and 4 treaded more familiar ground for those of use with a background of plant molecular cell biology as the talks focused either on the potential for using ‘newer’ experimental organisms (cyanobacteria, algae, marchantia) and on successful bioproduction in plant systems. Arguably most striking was the work from Cathie Martin’s group by Yang Zhang that documented their successful production of many medicinal compounds in tomato fruit. In one particularly exciting instance, they used CRISPR technology to knockdown one enzyme in a biosynthetic pathway so that an intermediate was ‘shunted’ into the production of Genistein.

 

TomatoEva Thuenemann from the lab of George Lomonossoff presented the latest developments from the HyperTrans project, which uses transient expression in tobacco leaves to produce valuable bioactive compounds (such a flu vaccine). She detailed the steps that are involved to ensure that your gene of interest is expressed in the right amounts and that the product correctly interacts with co-expressed proteins. It was clearly stated that it’s not just a case of transforming your clone and having guaranteed reliable expression! Excitingly Eva mentioned the opening of a new high-throughout facility for HyperTrans expression on the JIC campus in late 2016. In addition they are currently searching for researchers to suggest clones for ‘proof of concept’ experiments at the facility. More on this in the coming months….

The final session entitled ‘OpenPlant and Open Technologies’ took a wider view about what it means to be ‘open’. Chas Bountra gave an inspirational talk about the progress his organisation is making in reducing the time and money that is wasted within the pharmaceutical industry, while attempting to streamline the process of getting a novel drug to market. Rob Mullins (University of Cambridge) then told the story of how and why the incredibly successful Raspberry Pi computer was developed and how they were able to bring this technology into schools and to the community, all without becoming a big business and succumbing to the pressures of the profit margin.

Finally Jim Ajioka gave the cautionary tale of their attempts to obtain regulatory support for the Arsenic sensor that was developed following the work of a previous iGEM team. It was a sadly frustrating tale of having an important tool available to use that gets incredibly held up in European red tape before it is either able to get to the market or more importantly to the people who would benefit from it.

EuCycleOverall the meeting was a great start to the OpenPlant era and it will be especially interesting to keep up with the developments of the ‘OpenPlant Fund’ed projects in the coming year. Next years meeting has already been advertised as happening in Norwich July 26-27th 2016 so if you are intrigued as to the progress of the research that is being funded by OpenPlant, then put the dates in your diary!! After all the meetings are ‘Open’.

TGAC iPlant workshop: Big Data Analysis

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Published on: July 31, 2015

Guest post by Adam Talbot and Elspeth Ransom who work at the University of Warwick with GARNet Advisory Board member Dr Katherine Denby
iPlantpicBig data is an ever-increasing part of scientific research. With the expanding use of high throughput genomics, biologists of the future will have to not only possess laboratory skills, but will also need to become specialists in advanced computing. The sheer volume of data is increasing at an alarming and/or encouraging rate, depending on your point of view. This makes it more difficult for biologists to manage, process, manipulate and analyse their datasets.

The iPlant Collaborative is an US-based NSF-funded initiative that offers an easy-to-use, standardised and high performance platform for biologists across all areas to perform complex analyses from initial data evaluation to final visualization. It provides a computing cluster available for all academic biologists and a platform for safely storing and sharing data. The iPlant interface is easy to navigate, providing a large selection of software programs in a manner which makes them straightforward to use, both for those with little prior knowledge of big data analysis whilst also offering multiple running options for those with more experience.

In July 2015 we attended the iPlant Tools and Services Workshop at The Genome Analysis Centre, Norwich. It provided a great grounding in how to utilise the iPlant platform and tools that it offers, from simply storing and exchanging data to RNA-Sequencing and GWAS analysis. The course was well structured and presented, suitable for both iPlant novices and for those who wished to learn how to use iPlant to analyse data in different ways.

The course began with the basic functions of iPlant. We were shown how to manage and import data into the iPlant Discovery environment and how to then create public links and sharing folders to enable the sharing of data with colleagues, collaborators and other iPlant users. This included how to use iDrop, which allows users to directly import data from their hard drives and also iCommands, which allows interaction with the iPlant Data Store. The session also included how to properly annotate data within iPlant using their metadata and how to quickly and easily extract data using these metadata fields.

The workshop then moved to describe how to navigate the Discovery Environment – one of the main portals for exploring iPlant. The Discovery Environment allows you to visualise file storage and perform analysis through a web browser on the cluster. Here we were given a taste of the uses of iPlant by performing a large sequence alignment using MUSCLE and analysing RNA-Seq data using the Tuxedo pipeline (Tophat, Cufflinks and CummeRbund).

The final part of the course focussed on Atmosphere cloud computing. This interface is designed for a user to start and save a virtual computer running on the iPlant high-performance computers, allowing performance of many tasks. You can outsource some computing power to these machines, install software you can’t or don’t want to use on your own machine, and use the interface to set up an external server to view from your computer. We used Atmosphere to visualize our RNA-Seq results within a web browser using the software JBrowse.

This workshop gave a broad introduction to computational analysis using iPlant, as well as introducing us to the concept of traceable, reproducible computing. The iPlant platform was easy to use and fully supports the research analysis requirements of today’s life scientists. A must use resource for all biologists!

The BBSRC has just funded an iPlant UK node so look out on all the usual forums for the announcements of future iPlant tutorial workshops run by iPlant-UK in collaboration with GARNet.
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Arabidopsis Research Roundup: July 30th

Two broad topics dominant the studies featured in this weeks Arabidopsis Research Roundup. Environmental and hormonal factors that control different types of ‘dormancy’ are presented in studies from the labs of Caroline Dean (JIC) and Ian Graham (York). Elsewhere two Sainsbury lab (Norwich) led studies investigate different aspects of the interaction between plants and bacterial pathogens. Finally Colin Turnbull from Imperial College is involved in an interesting assessment of cytokinin concentrations across the root tip.

Duncan S, Holm S, Questa J, Irwin J, Grant A, Dean C (2015) Seasonal shift in timing of vernalization as an adaptation to extreme winter Elife. http://dx.doi.org/10.7554/eLife.06620

Caroline Dean (JIC) again publishes in the open access journal eLife as her lab continues to investigate the precise detail of the vernalisation response. This response shows natural variation that is dependent on the geographic distribution of Arabidopsis ecotypes. Plants collected from northern latitudes showed maximum vernalisaton at 8oC, both at the level of flowering time and FLC chromatin silencing. The vernalisation response was measured both in controlled and field conditions and all Northern ecotypes were importantly shown to vernalise prior to snowfall, which would allow flowering immediately after thawing. These findings have important implications for models aimed at predicting the affect of climate change on flowering time.

Ibarra SE1, Tognacca RS1, Dave A2, Graham IA2, Sánchez RA1, Botto JF (2015) Molecular mechanisms underlying the entrance in secondary dormancy of Arabidopsis seeds Plant Cell Environ http://dx.doi.org/10.1111/pce.12607

Ian Graham is the leader of the Centre for Novel Agricultural Products (CNAP) at the University of York and contributes to this Argentinian-led study that looks into the molecular factors that underlie secondary dormancy in Arabidopsis seeds. They show that this process involves changes in the content and sensitivity to GA (but not ABA) that requires the activity of the RGL2 protein acting through ABI5. A wide geographical study then perhaps unsurprisingly showed that temperature is also an important variable influencing the induction of secondary dormancy

Lee D, Bourdais G, Yu G, Robatzek S, Coaker G (2015) Phosphorylation of the Plant Immune Regulator RPM1-INTERACTING PROTEIN4 Enhances Plant Plasma Membrane H+-ATPase Activity and Inhibits Flagellin-Triggered Immune Responses in Arabidopsis Plant Cell http://dx.doi.org/10.1105/tpc.114.132308

Silke Robatek (TSL) is the UK lead on this collaboration with UC-Davis that looks at phosphorylation of RPM1-INTERACTING PROTEIN4 (RIN4) in a range of Arabidopsis genotypes that are suspectible to infection. Flexibility of the RIN4 protein is affected by phosphorylation and this causes enhanced suspectibility coincident with increasing plasma membrane H+-ATPase activity. The expression of the AHA1 ATPase is high in guard cells and therefore linked to stomatal opening. As such bacterial infection works to phosphorylate RIN4 that in turn increases the chance of bacterial entry.

Pfeilmeier S, Saur IM, Rathjen JP, Zipfel C, Malone JG (2015) High levels of cyclic-di-GMP in plant-associated Pseudomonas correlate with evasion of plant immunity Mol Plant Pathology http://dx.doi.org/10.1111/mpp.12297

GARNet Advisory Board Member Cyril Zipfel (TSL) and Jacob Malone (JIC) investigate the response to pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) by the plant innate immune system. The resulting pattern-triggered immunity (PTI) fends off pathogen attack by recognition of bacterial flagellin by, amongst others, the FLAGELLIN SENSING2 (FLS2) protein. In this study the authors focus on the bacterial side of the response and show that cyclic-di-GMP is involved in the evasion of PTI, although this also reduces virulence, likely due to reduced flagellar motility. This results in a trade off for the bacteria in which it is not recognised as readily by plant yet isn’t as virulent.

Antoniadi I, Plačková L, Simonovik B, Doležal K, Turnbull C, Ljung K, Novák O (2015) Cell-Type-Specific Cytokinin Distribution within the Arabidopsis Primary Root Apex Plant Cell http://dx.doi.org/10.1105/tpc.15.00176

Colin Turnbull (Imperial College) is a contributor to this Swedish-Czech collaboration that measures cytokinin concentrations in root cell files isolated by FACS and analysed by MS. The authors show a gradient of cytokinin across the root tip with maximum concentrations in the lateral root cap, columnella and QC cells. As these are also areas of high auxin concentration, the authors suggest that this implies that interactions between the two hormone groups are cell type specific.

Standardising DNA parts for plant Synthetic Biology

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Published on: July 22, 2015

The understanding of synthetic biology is slowing percolating through the plant science community. Whereas the microbial research community started to adopt this technology a decade ago, it is only over the past five years that plant scientists are becoming aware of the possibilities that might exist when using the principles of synthetic biology in their research. In 2012 New Phytologist held a synthetic biology workshop whilst in 2013 GARNet hosted a plant synthetic biology meeting that aimed to set the agenda for the coming decade. In addition and perhaps more importantly, the BBSRC chose to fund the joint Cambridge-Norwich OpenPlant initiative as one of its centres of synthetic biology.

The principles of synthetic biology put great importance on the ability to easily switch between the DNA parts used in different experiments. Widespread uptake of synthetic biology is dependent on a significant amount of front-end design and planning to prepare and define universal cloning strategies and then provide the DNA parts that might be used by a downstream researcher. To that end a number of cloning strategies are available that might fit this bill, each which their own benefits and drawbacks. These include ligation-based strategies such as Goldengate/MoClo/Goldenbraid or overlap based methods such as Gibson Assembly (reviewed here).

Therefore with this backdrop, an important Viewpoint article was recently published in New Phytologist where a collaborative of ~50 plant scientists define: ‘Standards for plant synthetic biology: a common syntax for the exchange of DNA parts. This work is led by members of the OpenPlant collaboration, namely Dr Jim Haseloff at Cambridge University and Dr Nicola Patron who is Head of Synthetic Biology at The Sainsbury Laboratory in Norwich. In this article the authors introduce the Type IIS assembly method (otherwise known as MoClo assembly) as a common mode of production for DNA parts for plant synthetic biology.

Importantly the article sets out the aims and future principles of plant synthetic biology as having analogy with mechanical or electrical engineering, wherein (DNA) parts made by multiple manufacturers (researchers) are immediate interchangeable, which facilitates and enables technological innovation.

The power of MoClo/GoldenGate cloning comes in the use of the BsaI enzyme, whose 6bp non-palindromic recognition sequence is physically separated from a 4bp restriction site (whose sequence is unrelated to the recognition site). The design of these 4bp ‘fusion sites’ is critical for the modular assembly of DNA parts in MoClo, where multiple parts can be fused within a single reaction using a range of fusion sites across the entire transcriptional unit (TU) (see Figure 1 from Patron et al (2015)).

This Viewpoint article describes 12 ‘parts’ that span a TU from 5’ promoter regions through to 3’ termination sequences. However previous articles have introduced many more DNA parts that are already available to researchers interested in this technology, for example, the 96-well MoClo Tool kit (see figure 2 below) or the items available on the GoldenBraid 2.0 website. In these collections each DNA part is held within an Universal Acceptor Plasmid (UAP) and can be mixed and matched to create many functional units as long as the ‘fusion units’ match up along the length of the clone.

MoCloToolkitFigure 2: The MoClo 96-well Toolkit

 

However across the grand scheme of things, the specific nature of these parts is mostly irrelevant. What is most important is that DNA parts that lie in a similar position within any TU contain the same fusion sites so that they can correctly fit into an amalgamated clone at the functionally relevant location. If this is correct then a TU can be made up of any number of available parts designed for a specific regulatory function.

Therefore one future aim of this technology might be to generate a plasmid library that includes the entirety of Arabidopsis promoter sequences, which would all contain the same directional fusion sites so that each of them could be used to drive expression of any particular gene of interest.

Maximising the potential of synthetic biology resources will come as the availability of parts increases. Of course having the DNA parts available is one thing but understanding how they work in different biological contexts is an entirely different question. As researchers looks to use a range of experimental ‘chassis’ such as Arabidopsis, Tobacco or Marchantia then it might be necessary to provide DNA parts that have been optimised for each ‘chassis’.

 

So what does a lab researcher with their ‘clone of interest’ need to do to take advantage of this type of synthetic biology resource?

Firstly their clone needs to be ‘domesticated’ which, in its most simple terms, involves removal of any internal BsaI sites. As with any cloning reaction, this may not be trivial since it involves two overlapping PCR reactions to incorporate a single mismatch designed to destroy the internal BsaI site. As an aid, the GB2.0 website contains an optimisation tool to help this process.

The researchers clone of interest is fully ‘domesticated’ into the UAP by addition of the appropriate 4bp fusion site that will allow cloning into the correct location of any designed TU. The power of this system truly emerges when considering the vast range of possible regulatory regions that can be attached to the clone, all of which are held within an appropriate binary vector for plant transformation*.

With this Viewpoint article the UK plant synthetic biology community proposes that, given its ease of use, any future DNA parts repository might be best based around MoClo-type technology. However this does not prevent the use of other useful assembly technologies and in time, it might be possible to link seemingly disparate cloning strategies to provide a truly universal mode of DNA-part preparation.

Ultimately the effectiveness of any centralized parts repository is dependent on a number of important factors:

  1. Stimulating community involvement to assess which DNA parts would be most useful for the largest number of researchers
  2. Providing incentives for academic researchers to develop parts for the community and ensure they get appropriate credit for doing so.
  3. Investment in the hardware and software infrastructure to support the physical location of a repository and the upkeep of that facility once it is established.
  4. Continued support for wider synthetic biology so that the DNA parts can be used to fully develop the technological capability of the burgeoning bioeconomy.

Finally it is worth noting that although all the synthetic biology tools can be provided for researchers to use, if the individual researcher is unable to amplify their (cursed?) gene of interest then it might all be for nothing!!! Therefore it is vital to keep supporting basic skills in research so that a fundamental knowledge of molecular biology is not lost to following generations.

*- A discussion of transformation efficiency into both model or non-model plants is beyond the scope of this blog, even though it remains a potential bottleneck in many organisms.

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