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

Categories: Uncategorized
Tags: No Tags
Comments: No Comments
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.

Arabidopsis Research Roundup: July 20th

There is a bumper crop of publications in high quality journals in this weeks UK Arabidopsis Research Roundup, including manuscripts in PNAS, Nature Communications, PLoS Genetics , PloS One and Plant Physiology. Malcolm Bennett, Alex Webb and Anthony Hall lead a major collaborative effort that links the circadian clock with lateral root formation whilst Ottoline Leyser (SLCU) and Mike Bevan (JIC) participate in a similarly broad consortium in a study linking organ size and MAPK signaling. Liam Dolan’s group from Oxford looks at mechanisms of tip-growth across the plant kingdoms whilst elsewhere three members of faculty at the University of Birmingham are involved in two papers looking at the regulation of meiosis. Finally there are two US-led studies that include significant contributions from UK-based researchers, including Matthew Jones from the University of Essex.

 

Voß U, Wilson MH, Kenobi K, Gould PD, Robertson FC, Peer WA, Lucas M, Swarup K, Casimiro I, Holman TJ, Wells DM, Péret B, Goh T, Fukaki H, Hodgman TC, Laplaze L, Halliday KJ, Ljung K, Murphy AS, Hall AJ, Webb AA, Bennett MJ (2015) The circadian clock rephases during lateral root organ initiation in Arabidopsis thaliana Nature Communication 6:7641. http://dx.doi.org/10.1038/ncomms8641

Once again Malcolm Bennett (CPIB) leads a multi-Institute collaboration that includes Alex Webb (Cambridge) and current GARNet board member Anthony Hall (Liverpool). This is also an extremely international effect with groups from the UK, USA, Sweden, Japan, Spain and France. The science looks at lateral root stems cells and how the circadian clock is rephased during LR emergence. They show that the clock controls auxin levels and auxin-related genes. The conclusion is that the circadian clock acts to gate auxin signalling during LR development to facilitate organ emergence and adds to a growing portfolio of evidence that suggest the circadian clock might act in a cell autonomous manner. Anthony Hall, James Locke and Peter Gould currently have a grant that is looking at this phenomenon in Arabidopsis root cells.

 

Johnson KL, Ramm S, Kappel C, Ward S, Leyser O, Sakamoto T, Kurata T, Bevan MW, Lenhard M (2015) The Tinkerbell (Tink) Mutation Identifies the Dual-Specificity MAPK Phosphatase INDOLE-3-BUTYRIC ACID-RESPONSE5 (IBR5) as a Novel Regulator of Organ Size in Arabidopsis PLoS One.10(7):e0131103. http://dx.doi.org/10.1371/journal.pone.0131103

Ottoline Leyser, Sally Ward (Sainsbury lab, Cambridge) and Mike Bevan (JIC) are the UK contributors to this joint UK-German-Japanese-Australian collaboration. This study follows a screen for plants with reduced organ size and introduces a novel allele of the dual-specificity MAPK phosphatase INDOLE-3-BUTYRIC ACID-RESPONSE5 (IBR5), named Tinkerbell (tink). This mutation reveals that IBR5 is a novel regulator of organ size by changing the growth rate in petals and leaves although this occurs independent of the previously characterised KLU pathway. The authors use microarray data to suggest an additional role for TINK/IBR5 during male gametophyte development. Ultimately they conclude that IBR5 might influence organ size through auxin and TCP growth regulatory pathways.

 

Tam TH, Catarino B, Dolan L (2015) Conserved regulatory mechanism controls the development of cells with rooting functions in land plants Proc Natl Acad Sci U S A. http://dx.doi.org/10.1073/pnas.1416324112

Liam Dolan’s lab at the University of Oxford is a world leader in the study of root hair development. Previously it has been shown the group XI basic helix-loop-helix (bHLH) transcription factor (LOTUS JAPONICUS ROOTHAIRLESS1-LIKE (LRL) regulates root hair growth in Arabidopsis, Lotus or rice. This study investigates the equivalent proteins in the moss Phycomitrella patens and show that they are involved in an auxin signaling pathway that promotes cell outgrowth albeit via a different set of signaling intermediates. Overall the authors show that a core auxin network that supports cellular ‘tip-growth’ exists throughout land plant lineages even though the specificity of this signaling has diverged over the course of the ~420million years that separates angiosperms and mosses.

 

Varas J, Sánchez-Morán E, Copenhaver GP, Santos JL, Pradillo M (2015) Analysis of the Relationships between DNA Double-Strand Breaks, Synaptonemal Complex and Crossovers Using the Atfas1-4 Mutant. PLoS Genet.11(7): e1005301. http://dx.doi.org/10.1371/journal.pgen.1005301

The work led by Monica Pradillo at the University of Madrid includes a contribution from Eugenio Sanchez-Moran from the University of Birmingham. This work focuses on the hetero-trimeric Chromatin Assembly Factor 1 (CAF-1), which is a histone chaperone that assembles acetylated histones H3/H4 onto newly synthesized DNA. In Arabidopsis the CAF1 complex is composed of the FAS1, FAS2 and MSI1 proteins. Atfas1 mutant plants are less fertility, have a higher number of double stranded breaks (DSB) and show a higher gene conversion frequency. The authors investigate how DSBs can influence meiotic recombination and synaptonemal complex (SC) formation by genetic analysis of Atfas1-containing double mutants. Ultimately their experiments provide new insights into the relationships between different recombinase proteins in Arabidopsis. Overall an increase in the number of DSBs does not translate to an increase in the number of crossovers (COs) but instead in a higher GC frequency. The authors provide different theories to explain this mechanism, including the possible existence of CO homeostasis in plants.

 

Lambing C, Osman K, Nuntasoontorn K, West A, Higgins JD, Copenhaver GP, Yang J, Armstrong SJ, Mechtler K, Roitinger E, Franklin FC (2015) Arabidopsis PCH2 Mediates Meiotic Chromosome Remodeling and Maturation of Crossovers PLoS Genetics 11(7):e1005372 http://dx.doi.org/10.1371/journal.pgen.1005372

The University of Birmingham is the lead Instiution in this study that also investigates regulation of meiosis. The groups of Chris Franklin and Sue Armstrong collaborate with US and Austrian partners to study the organization of meiotic chromosomes during prophase I. Using structured illumination microscopy (SIM) they show that dynamic changes in chromosome axis is coincident with synaptonemal complex (SC) formation and depletion of the ASY1 protein, which requires the function of the PCH2 ATPase. Using a pch2 mutant the authors are able to tease apart different aspects of ‘crossover’ (CO) biology and that the pch2 defect occurs precisely during CO maturation, not during designation. In addition, CO distribution is also affected in some chromosome regions showing that failure to deplete ASY1 can result in downstream events that include disruption of CO patterning.

 

Jones MA, Hu W, Litthauer S, Lagarias JC, Harmer S (2015) A Constitutively Active Allele of Phytochrome B Maintains Circadian Robustness in the Absence of Light Plant Physiology. http://dx.doi.org/pp.00782.2015

Matthew Jones (University of Essex) is the primary author of this work that comes from a collaboration from his time in the lab of Stacey Harmer in UC Davis. Since 2012 Matthew has been a lecturer at the University of Essex where he continues with work of this nature. In this study they introduce a constitutively active allele of the PHYB photoreceptor that is able to phenoopy red-light input into the circadian clock. In these mutants the pace of the clock is insensitive to light-intensity and this response is dependant on its PHYB nuclear localisation. Finally they show that fine tuning of PHYB signalling requires PHYC and overall they conclude that nuclear phytocrome signalling is necessary for sustaining clock function under red light.

 

Chakravorty D, Gookin TE, Milner M, Yu Y, Assmann SM (2015) Extra-Large G proteins (XLGs) expand the repertoire of subunits in Arabidopsis heterotrimeric G protein signalling Plant Physiol. http://dx.doi.org/10.1104/pp.15.00251

Sally Assman from Penn State University leads this study that includes a contribution from Matthew Milner who now works at NIAB. The number of proposed G protein subunits is greatly reduced in diploid plant genomes yet this study shows that a family of Arabidopsis GPA-related proteins (XLG1-3) can increase the repertoire of potential G proteins interactions by interacting with beta and gamma subunits. The authors propose they have uncovered a new plant-specific paradigm in cell signaling.

HVCfP Workshop on Synthetic Biology

Categories: Uncategorized
Tags: No Tags
Comments: No Comments
Published on: July 16, 2015

The High Value Chemicals from Plants (HVCfP) network is a BBSRC-funded consortium that aims to support the development of resources that will enable researchers to investigate different avenues to produce commercially relevant compounds from plants. These strategies might take advantage of naturally occurring novel biochemical pathways or use plant systems to produce heterologous products of commercial interest. This network is based at the University of York and led by Professor Ian Graham and by Professor Anna Osbourn (John Innes Centre). In their past research both these network-leaders have investigated the potential for increasing the production of high value chemicals, namely the anti-malarial compound Artesmisia or the defence-saponin Avenacin.

On July 13th and 14th the HVCfP network hosted a meeting in Dunston Hall, near Norwich that aimed to assess the potential of using synthetic biology (synbio) approaches in the production of high value chemicals. The use of synthetic biology is a key component of the growing bioeconomy, which aims to unlock the potential of biological systems for the production of useful compounds. Synbio was named as one of the governments ‘Eight great technologies’ and the subject of the RCUK ‘Synthetic Biology roadmap’. Principles of synbio encompass engineering molecular and metabolic systems for the economic and pastoral benefit of society. Although synthetic biology has largely been focused on single-celled experimental systems there is clear potential to transfer this technology to plants, as exemplified by the production of flu-vaccine in tobacco.

‘High value’ chemicals in plants are usually produced at low levels and/or in organisms that are currently not amenable to transgenic or synthetic biology procedures. Therefore at the start of this meeting it seemed that there was too much of a distance between principles of synbio and the production of high value chemicals for meaningful discussions to occur. However the meeting was structured in such a way to make it clear that there is not such the disconnect as initially thought, even though there is a long way to go until many of the goals are realized. The full meeting schedule can be obtained from Wendy Lawley.

The first session provided an overview to synthetic biology with Jim Haseloff (Cambridge) introducing general principles of the topic, including some work on the new ‘chassis’ that his lab are using for synbio, the liverwort Marchantia Polymorpha. Rowan McKibbin from the BBSRC then explained that the research councils continue to be committed to synbio as highlighted by a ‘refreshment’ of the Synthetic Biology Roadmap in coming months.

BBSRCsynbio

Latterly Joyce Tait (Edinburgh) provided an overview of the future ethical considerations that synthetic biologists might need to consider. Her overall message was that in terms of public acceptance of the technology, synthetic biology is currently in a strong position. Notably it differs from the debate with genetic modification in a number of critical ways 1. Most of the products from synbio are not food-stuffs and 2. The technology is not linked to multinational companies in the same way as, for example, GMO crops. However Kate Miller (Nottingham) made the important cautionary point that few members of the public have actually heard about synthetic biology at this point and so opinions might change….

Session Two focused on some of the technologies involved in synthetic biology. As Head of Synthetic Biology at The Sainsbury lab, Nicola Patron presented ideas about  standardization of molecular parts for use in synbio, highlighting a new paper in New Phytologist that attempts to develop a ‘common syntax for DNA parts in synthetic biology’ . The GARNet blog will have more on this paper in coming weeks but in short, it aims to bring together the community so that transfer of materials will be more routine in future.

Arguably the most striking presentation of the day was from Patrick Cai from the Edinburgh Genome Foundry who introduced approaches that have been developed in the multinational Synthetic Yeast 2.0 project. These include the construction of synthetic yeast chromosomes and attempts to SCRaMbLE the yeast genome, each of which have the grand goal of attempting to re-define what it takes to be a eukaryotic ‘genome’.

The session continued strongly with a classification-redefining presentation from Matias Zurbriggen (Freiburg) who demonstrated the feasibility of recapitulating auxin and light signaling in mammalian cell lines, with the aim of developing general molecular sensors. Marnix Medema (Wageningen) demonstrated the power of a bioinformatic approach to discover biosynthetic pathways in non-model organisms, which might be a critical technology in plants that produce important secondary metabolites.

The first plant image of the workshop appeared courtesy of Sarah O’Connor (JIC) who presented the Madagascar Periwinkle, which produces important monoterpene indole alkaloids.One of the strengths of this meeting was the breadth of ‘experimental chassis’ that were discussed. These ranged from conventional model organisms such a Saccharomyces cerevisae or Arabidopsis thaliana through the less widely used tobacco, microalgae or Dictyostelium and ultimately to plants such as Madagascar periwinkle or Dioscorea (Yam), whose potential is now being investigated due to the novel compounds they are able to produce.

The second day of talks had a more practical edge as Session Three looked at ‘Production Platforms’ currently in place including use of microalgae, tobacco or E.coli. Anil Day (University of Manchester) introduced the benefits of chloroplast expression, either of individual genes or as fusions to the large subunit of Rubisco. This technology appears to have great potential yet it is striking that no transplastomically-transformed plants have been licensed for growth. Dr Day thought that this is due to regulatory and not technological problems. However it was conceded that one potential problem with this approach is the current inability to transform monocots.

The final session of the workshop focused on ‘Metabolic Engineering’ where examples were given for the output and potential of different systems. Jon Marles-Wright (Edinburgh) discussed engineering bacterial nano- and micro-compartments for different applications. He highlighted three potential benefits: 1. They can protect enzymes from high-temperatures or non-physiological conditions, 2. They can safely hold toxins within cells and 3. They can carry heavy metals. However the challenge is now to go from basic research to develop an ‘Encapsulation Platform’. Watch this space…

Arguably the most ‘complete’ story was presented by Olga Sayanova who described a decade-long journey to move production of fish oils from algae into camelina. This has been a cause-celebre in recent years and an exciting exemplar of what is possible with GM technology. Olga did have some cautionary words about the potential of synthetic biology as ‘one-module’ might not fit all. The heterologous expression of algal genes worked fine in Camelina (just a ‘big Arabidopsis’) but did not work in Flax/Linseed. This highlights that each experimental ‘chassis’ might require their own set of molecular tools to enable expression of your product of interest.

Overall the meeting seemed to be a great success with researchers across a range of disciplines learning plenty outside of their comfort zones. It might be a many years until we are able to produce Galanthamine (or similarly complex molecules) in E.coli but the potential is there to move in that direction….

In addition to discussions of HVCfP, approximately 30 attendees were able to take advantage of a Science-Art-Writing workshop over dinner. This JIC-based initiative is led by Jenni Rant and Anne Osbourn with the aim of linking science and art to enthuse schoolchildren about scientific topics. After general discussions about the topic, the attendees were encouraged to write an original ‘ode or haiku’ about synthetic biology and then to blindly draw their table-mates (two representations of your author are below…), which led to great hilarity all around.

However the important message was that seemingly-disparate topics can be brought together to aid education. Hopefully the workshop attendees were given food for thought concerning their next pubic outreach engagement.

I will finish with my indifferently-received ‘Ode to Plant Synthetic Biology’:

O GREEN MACHINE.

GPpic

Arabidopsis Research Roundup: July 11th

A couple of weeks since the last update as it’s been quiet for UK Arabidopsis Research publications. However we now see a variety of publications that address some important questions in different signaling pathways. Firstly a multinational collaboration performs a genome-wide analysis of DELLA binding, followed by two studies looking different aspects of light signaling, specifically the link with the production of protective carotenoids and also with the tight control of protein degradation. Elsewhere there is the description of a systems biology approach developed to aid the definition of signaling pathways in non-model organisms and finally a commentary piece about some work on Arabidopsis Arenosa.

 

Genome Wide Binding Site Analysis Reveals Transcriptional Coactivation of Cytokinin-Responsive Genes by DELLA Proteins (2015) Marín-de la Rosa N, Pfeiffer A, Hill K, Locascio A, Bhalerao RP, Miskolczi P, Grønlund AL, Wanchoo-Kohli A, Thomas SG, Bennett MJ, Lohmann JU, Blázquez MA, Alabadí D PLoS Genet. 11(7):e1005337. http://dx.doi.org/10.1371/journal.pgen.1005337

The Centre for Integrative Biology in Nottingham and Rothamstead Plant Science partner with groups from Sweden, Germany, Spain and Saudi Arabia in this truly international collaboration. They investigate the role of DELLA proteins in the relay of environmental cues to multiple transcriptional circuits. The primary experimentation in this study uses ChIP-Seq to analyse the DNA-binding sites of one DELLA protein. Perhaps as expected the DELLA protein binds multiple promotor regions yet with a particular enrichment in regions upstream of cytokinin-regulated genes, where they interact with type-B ARABIDOPSIS RESPONSE REGULATOR (ARR) proteins. The biological relevance of this mechanism is underpinned by the requirement for both DELLAs and B-type ARRs in the control of root growth and photomorphogenesis.

 

Regulation of carotenoid biosynthesis by shade relies on specific subsets of antagonistic transcription factors and co-factors (2015) Bou-Torrent J, Toledo-Ortiz G, Ortiz-Alcaide M, Cifuentes-Esquivel N, Halliday KJ, Martinez-Garcia JF, Rodriguez-Concepcion M Plant Physiol.

Karen Halliday at the University of Edinburgh is part of this UK-Spanish team that studied the regulation of carotenoid biosynthesis via a light signaling module formed by PIF1 and HY5. In shade conditions, PIF proteins signal for a decrease in carotenoid accumulation, thus saving the plant unneeded energy consumption. The PIF1 response focusses on the phytoene synthase (PSY) biosynthetic gene and is antagonised by the PAR1 transcriptional co-factor. However this is not a universal response carried out by known antagonisers of PIF1 function, demonstrating that carotenoid biosynthesis is finely regulated by a precise subset of regulatory proteins.

 

High-level expression and phosphorylation of phytochrome B modulates flowering time in Arabidopsis (2015) Hajdu A, Ádám É, Sheerin DJ, Dobos O, Bernula P, Hiltbrunner A,, Kozma-Bognár L, Nagy F Plant Journal http://dx.doi.org/10.1111/tpj.12926

Professor Ferenc Nagy has dual appointments in Edinburgh and in Hungary and this output results from work performed in Hungary. This study looks at control of flowering via phytochrome B signalling, which has been previously shown to rely on the degradation of the CONSTANS (CO) protein that in turn delays flowering by attenuating FLOWERING LOCUS T (FT) expression. Therefore phyB mutants show accelerated flowering, yet this is unexpectedly also true following PHYB overexpression. The novelty of this study comes from showing that PHYB overexpression induces FT without affecting CO transcription but rather acts by causing accumulation of the CO protein, due to an affect on a COP1-ubiquitin ligase complex. This article adds further detail to the already complex relationship between light signaling, the circadian clock, protein degradation and de novo transcription in the control of flowering in Arabidopsis.

 

Inferring orthologous gene regulatory networks using interspecies data fusion (2015) Penfold CA, Millar JB, Wild DL. Bioinformatics. 31(12):i97-i105. http://dx.doi.org/10.1093/bioinformatics/btv267

This study was led by David Wild from Warwick Systems Biology Centre. The authors have used two related Bayesian approaches to network inference that allow Gene Regulatory Networks (GRN) to be jointly inferred in, or leveraged between, several related species, for example between Arabidopsis and related crop species. Inferring gene function is achieved with more accuracy when GRNs are compared between species rather than attempting to use stand alone inference. The manuscript uses data from the yeast S.pombe but the broader principles could be applied to other experimental systems.

 

The High Life: Alpine Dwarfism in Arabidopsis (2015) Bomblies K Plant Physiol. 168(3):767. http://dx.doi.org/10.1104/pp.15.00745

This commentary piece about high altitude growth of Arabidopsis aernosa is the first published work from Kristen Bomblies since she moved her lab to the John Innes Centre from Havard (together with the lab of Levi Yant). Having these two talented young researchers relocate to the UK is be great for UK plant science so I sure everyone in the community wishes them all the best. Watch Kristen talk about her work at a New Phytologist conference from 2014.

Levi Yant also has two postdoctoral posts currently available in his lab.

Arabidopsis arenosa02

The cautionary tale of ABP1.

Categories: Uncategorized
Tags: No Tags
Comments: No Comments
Published on: June 29, 2015

The plant hormone auxin has long been known to play a significant role in plant growth, even featuring in Charles Darwin’s book ‘The Power and Movement of Plants’.

However until the mid-2000s the identity of any receptor for the hormone remained a mystery. Until that time, the site of auxin reception was somewhat of an enigma with the main candidate being a protein named ‘Auxin Binding Protein (ABP)’ that, in many biochemical studies, was shown to bind auxin at physiological concentrations (for review of ABP1 work1). This protein is present in Arabidopsis but investigations into its in vivo function were somewhat stalled by a 2001 publication that demonstrated that an abp1 T-DNA insertion mutant was embryo lethal2. This did not overly surprise the research community given the fundamental and wide-ranging role of auxin in plant development.

Meanwhile researchers at York University in the UK and at Indiana University in the US were working on a novel hypothesis that proposed the auxin receptor was linked to the degradation of AuxIAA proteins, which are negative regulators of the auxin response. Ultimately after years of painstaking research the labs of Ottoline Leyser and Mark Estelle demonstrated that the F-box protein TIR1, when in complex with an AuxIAA protein, was also able to bind auxin3-4. This finding nicely pieced together an elegant pathway of control that involved tightly regulated protein degradation controlling both positive and negative feedback regulation of the hormone signal.

At this time the work on ABP1 had mostly retreated into the long weeds as research in the community revolved around the TIR1-AuxIAA receptor paradigm. However toward the end of the 2000s ABP1 had a renaissance as molecular techniques such as RNAi and cellular immunisation allowed identification of transgenic plants with reduced expression of ABP1. Plants with a deficiency in ABP1 exhibited robust auxin-deficient phenotypes including cell-cycle arrest in tobacco BY2 cells5 and changes in cell-wall composition during cell expansion6. Initially these effects were thought to occur independent of TIR1 but more recently ABP1 was shown to lie genetically upstream of the TIR1-receptor pathway7. In keeping with its initially proposed role in the rapid auxin response, ABP1 was found, by use of a weak abp1-5 allele, to have a defect in auxin-induced transcriptionally-independent clathrin-mediated endocytosis of PIN proteins8 as well as playing a critical role in the activation of ROP proteins that control epidermal cell interdigitation9. Therefore reducing the expression of ABP1 had demonstrated, as would be predicted, that the protein plays a critical and wide-ranging role in cell division and expansion.

Therefore at this time the functional relationship between ABP1 and TIR1 appeared to satisfy researchers…… that was until a paper was published at the start of 2015 that greatly altered perceptions within this research community10.

The labs of Yunde Zhao and Mark Estelle at UC-San Diego initially used a CRISPR-based strategy to generate a null abp1 mutant allele, with the full expectation that the resulting plant would be unable to survive. Surprisingly they discovered that this abp1-c1 allele exhibited a completely wildtype phenotype. This prompted the isolation of a new T-DNA insertion mutant that was an abp1 null mutant. These plants also showed wildtype phenotypes across a range of assays that were thought to be ABP1-dependent. Ultimately the authors concluded that ‘ABP1 is not a key component in auxin signaling or Arabidopsis development’.

The discrepancy between these findings and the previous 15 years of data is striking and inevitably leads to questions about the previous work. In a recent calm editorial comment in the Journal of Integrative Plant Biology, Professor Chun-Ming Liu called for a careful reexamination of the previous data and for experimental materials to be exchanged in order to get to the bottom of this growing controversy11.

Even more recently the lab of Lucia Strader at Washington University in St Louis published a short paper in Plant Cell that questioned the abp1-5 mutant12. As stated above this allele has been used in a number of studies where its many interesting phenotypes include long hypocotyl growth in red light9. The Strader lab were interested in this response but ran into difficulties when characterising the genetic basis of this phenotype. Ultimately they decided to sequence the abp1-5 genome and surprisingly found that it contained significant portions from the Arabidopsis ecotype Wassilewskija, which is known to display mild resistance to auxin. Perhaps most worrying was that they discovered abp1-5 contained a null mutation in phyB, which is likely the causative effect of the long hypocotyl phenotype. The authors conclude by warning that their findings ‘provide a cautionary tale illustrating the need to use multiple alleles or complementation lines when attributing roles to a gene product’.

So what has happened in these experiments? In the Gao et al paper10 the authors took the simple approach of testing abp1 mutants for phenotypes and didn’t find anything different from the wildtype. It would be difficult to imagine where any errors would have occurred in these experiments especially given the nature of the genetic lesion in these new abp1 mutants. Gao et al suggest that the previous work based on ABP1 knock-down lines might have in fact been the consequence of off-target transgenic effects. Given the varied role that auxin plays in Arabidopsis development this is not an impossible conclusion to draw as numerous genes are involved in some aspects of this hormone response.

Most concerning is the initial characteristic of the original null abp1 mutant lines that was found to be embryo-lethal. This work is the cornerstone of the subsequent work that aimed to create plants with reduced ABP1 expression. The characterised abp1 mutant lines in Chen et al and Gao et al have T-DNA insertions close together at the 5’ end of the coding sequence so it is unclear why there are such differing results. It was unusual that in the first study only a single abp1 mutant allele was characterised, especially when the authors claimed embryo-lethality2. It would have been helpful for Gao et al (2015) to reexamine the original abp1 mutant line assessed in Chen et al (2001) but perhaps that might have appeared rather too confrontational.

At it stands the future direction of research into ABP1 is in flux with the onus now on those researchers with a vested interest, of which there are plenty, to try to understand the role of this protein.

In many ways this story is an excellent example of how science should work, where claims are independently tested to ensure that earlier experiments have been conducted or interpreted correctly. However this can be difficult as there is usually little value in retroactive testing of published claims. The plant science community awaits the resolution of the mystery of ABP1 whilst commiserating with those who have been negatively affected by this new development in this story.

 

1- Sauer and Klein-Vehn (2011) Plant Cell 23, 2033-2043

2- Chen et al (2001) Genes and Development 15, 902-911

3- Dharmasiri et al (2005) 435, 441-445

4- Kepinski and Leyser (2005) 435 446-451

5- David et al (2007) Plant Journal 50, 197-206

6- Paque et al (2014) Plant Cell 26, 280-295

7- Tromas et al (2013) Nature Communications 4, 2496

8- Robert et al (2010) Cell 143 111-121

9- Xu et al (2010) Cell 143, 99-110

10- Gao et al (2015) PNAS 112, 2275-2280

11- Liu (2015) JIPB 57, 234-235

12- Enders et al (2015) The Plant Cell Epub

COPO 2015 Meeting

Tags: No Tags
Comments: No Comments
Published on: June 25, 2015

COPO has big plans…. and if it is to be truly successful and benefit the plant community there needs to be a cultural change! That’s the simple if revolutionary message that came out of the recent COPO meeting held at TGAC on June 23rd-24th.

So the uninitiated will be asking: ‘What is COPO?‘ The answer is the Collaborative Open Plant Omics group which was funded by a BBSRC BBR grant in 2014. This is a >£1m collaboration between The Genome Analysis Centre (TGAC), University of Oxford, the European Bioinformatics Institute (EMBL) and the University of Warwick.

This workshop was to introduce the aims of COPO to a range of stakeholders, from curators of available data repositories to experimentalists who are generating large datasets. By the end of the 2-day session it was hoped that everyone would gain an understanding of what COPO can offer the community with regard facilitating the sharing of large datasets.

White Board Discussions

The workshop was led by Rob Davey (TGAC) and Ruth Bastow (GPC, GARNet, Warwick). Rob kicked off the meeting by describing the aims of COPO which included asking ‘What are the barriers for you and your data and how can COPO facilitate access to the workflows used to analyse data’.

Subsequently a range of stakeholders introduced the fantastic tools that are out there for repositing data of many different types. These included David Salt (University of Aberdeen, Ionomics), Elizabeth Arnaud (Montpellier, CropOntology), David Marshall (James Hutton Institute, Germinate: Plant Genetic Resources), Esther Kabore (INRA, Wheat Data Repository) Reza Salek (EMBL, Metabolights), and finally Tomasz Zielinski (Edinburgh, BioDare) who made the telling observation that ‘data Management is a user interface/user experience problem NOT a software engineering/ data modelling problem’. Many researchers are often reluctant to take the time and effort required to submit their data to an appropriate repository in a reasonable manner, for any number of opaque reasons. However the take-home message from the early talks was very positive as there are a large number of data platforms available for people to use and benefit from. One of the challenges for COPO is to not only to help convince people to use these resources but encourage them to share data in a standardised manner.

Following a useful coffee break it was time for researchers to explain the data they are producing and the challenges for their analysis. Miriam Gifford (Warwick) discussed her generation of transcriptomic data, Christine Sambles (Exeter) talked about developing a workflow for metabolomics data and TGAC group leader Ksenia Krasileva introduced her work on wheat functional genomics. Ksenia also highlighted a new portal for communication between data generators and data users called Grassroot Genomics.

The final three talks of the day highlighted the amount of data that can be produced in different sets of biological experiments. Ji Zhou (TGAC) and Chris Rawlings (Rothamstead) introduced cutting-edge field phenotyping technologies that use large imagers to capture visible and spectral aspects of plant growth. Workshop attendee Professor Peter Murray-Rust summed it up with a tweet: ‘Blown away by the crop monitoring equipment at Rothamstead’. On an opposite end of the spectrum Jim Murray (Cardiff) showed a single fluorescent image of a zebrafish taken on a light-sheet microscope that weighed in at an impressive 23Tb of data. Overall these talks served to highlight the vast amounts of data that can be produced and provided the second take-home message of the day that ‘Getting data is NOT the issue, making any sense of it IS the challenge’……

 

The task of second day discussions was to make sense of what had been presented the previous day and identify the best opportunities for COPO to impact on the process of data sharing. A lively first hour of debate included Dr Philippe Rocca-Serra (COPO Co-PI from Oxford) presenting a somewhat sobering eight slides of ‘Pain Points’ that he had taken out of the previous days presentations! However it was refreshing to observe that the challenge of the task was not underestimated and being tackled with realistic planning.

Pain Points!

Later in the morning discussions turned more specific with a white-board brainstorming session that was divided into ‘Data Collection’.Data Storage’ and ‘Data Analysis’ sections. Most progress was made in the first two sections with a long list of storage repositories identified that bridged the breath of biological data and with which COPO could potentially interact.

It was felt that successful interactions would be predicated on some level of data standardisation so perhaps the most effective initial use of the COPO resources would be to develop a workflow for standard data collection. This would hopefully make experimentalists think about the format of their data submission as they are planning and generating the data. The consensus was that attaching these standards to legacy-data might be a difficult task but that for future data generation, COPO could influence data sharing at this level.

IMG_8241

Ultimately it is clear that plant science has the same generic problems as many other disciplines and the greatest challenge is to change the ‘culture’ of sharing data. The most obvious and direct way to promote this change will be via the funders and publishers. Some progress has been made in this arena with a recent shift towards open access publication in the REF process, and it would only take another small additional step to make it a requirement to share data in any REF-returnable publications. So I hope that those with greater power and influence than me, are reading the GARNet blog!

Regardless of the pace of cultural change, the feeling in the meeting was that the COPO mandate is to encourage data sharing whilst moving to a position to effectively interact with the data that is shared. There is plenty of work to do but at the end of this exploratory workshop the COPO organisers had plenty to think about regarding the direction of the project. Watch their space!

Storify of tweets from the meeting

GARNet-OpenPlant CRISPR workshop

Categories: Uncategorized
Tags: No Tags
Comments: No Comments
Published on: June 23, 2015

Please see program in the poster below and register for the meeting here. For more information please contact geraint@garnetcommunity.org.uk

CRISPR Poster

«page 1 of 32

Follow Me
TwitterRSS
GARNetweets
July 2015
M T W T F S S
« Jun    
 12345
6789101112
13141516171819
20212223242526
2728293031  

Welcome , today is Friday, July 31, 2015