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.
SynBioWorkshopPic
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 <a href="http://dx.doi acheter cialis.org/10.1111/nph.13616″ onclick=”_gaq.push([‘_trackEvent’, ‘outbound-article’, ‘http://dx.doi.org/10.1111/nph.13616’, ‘http://dx.doi.org/10.1111/nph.13616 ‘]);” target=”_blank”>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 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 (<a href="http://www2.warwick cialis professional 20 mg.ac.uk/fac/sci/lifesci/people/lfrigerio/” onclick=”_gaq.push([‘_trackEvent’, ‘outbound-article’, ‘http://www2.warwick.ac.uk/fac/sci/lifesci/people/lfrigerio/’, ‘Frigerio’]);” target=”_blank”>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

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.

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.

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 Research Roundup

This week roundup features a wide range of research topics from two current members of GARNet Advisory board as well as two papers featuring work from the lab of Laszlo Bogre at Royal Hollaway. The studies range from an investigation into the similarity between the barley and Arabidopsis circadian clocks, the role of MYR3R during regulation of organ growth, documenting a novel interaction of a MAPK protein and the development of new fluorescent probes for study of cysteine proteases.

 

Kusakina J, Rutterford Z, Cotter S, Martí MC, Laurie DA, Greenland AJ, Hall A, Webb AA (2015) Barley Hv CIRCADIAN CLOCK ASSOCIATED 1 and Hv PHOTOPERIOD H1 Are Circadian Regulators That Can Affect Circadian Rhythms in Arabidopsis. PLoS One. 10(6):e0127449. http://dx.doi.org/10.1371/journal.pone.0127449

This publication is the result of a multi-site collaboration between the Alex Webb at Cambridge, GARNet Advisory board member Anthony Hall at Liverpool, Andy Greenland at NIAB and David Laurie at the JIC. The focus of this study are the barley CIRCADIAN CLOCK ASSOCIATED 1 and PHOTOPERIODH1 genes, which are involved in regulation of the circadian clock. The authors investigated the circadian rhythms in barley whilst using heterologous expression in Arabidopsis to show that the barley CCA1 is functionally equivalent to AtCCA1 and that barley PHOTOPERIODH1 functions similar to AtPRR7.

 

Kobayashi K, Suzuki T, Iwata E, Nakamichi N, Suzuki T, Chen P, Ohtani M, Ishida T, Hosoya H, Müller S, Leviczky T, Pettkó-Szandtner A, Darula Z, Iwamoto A, Nomoto M, Tada Y, Higashiyama T, Demura T, Doonan JH, Hauser MT, Sugimoto K, Umeda M, Magyar Z, Bögre L, Ito M (2015) Transcriptional repression by MYB3R proteins regulates plant organ growth. EMBO J. http://dx.doi.org/10.15252/embj.201490899

GARNet advisory board member John Doonan and Royal Hollaway-based Laszlo Bogre are collaborators on this multi-nation publication that looked at the role of three MYB2R3 proteins in cell cycle control. Arabidopsis plants that have mutations in three repressor type-myb3r genes display enlarged organs. In addition, MYB3R3 binds to G2/M-specific genes and associates with the repressor-type E2F and RBR proteins. The authors perform a range of pair-wise interaction studies to identify components of multiprotein complexes, that have also been identified in other organisms. Ultimately they show that these MYC3R genes are important for periodic expression during the cell cycle and for establishing a post-mitotic quiescent state that determines organ size.

 

Kohoutová L1, Kourová H1, Nagy SK2, Volc J1, Halada P1, Mészáros T2,3, Meskiene I4,5, Bögre L6, Binarová P1 (2015) The Arabidopsis mitogen-activated protein kinase 6 is associated with γ-tubulin on microtubules, phosphorylates EB1c and maintains spindle orientation under nitrosative stress New Phytologist. http://dx.doi.org/10.1111/nph.13501

Laszlo Bogre also features as a collaborator in this East European-led study that investigated the interaction of the MAPK-protein MPK6 with microtubules. Immunoprecitations showed that the active form of MPK6 interacted with γ-tubulin, sedimenting with in vitro polymerised microtubules. In addition they identified a novel substrate for MPK6, the microtubule plus-end protein, EB1c. Overall the authors propose that MPK6 plays a significant role in maintaining regular planes of cell division, particularly during stress conditions.

 

Lu H, Chandrasekar B, Oeljeklaus J, Misas-Villamil JC, Wang Z, Shindo T, Bogyo M, Kaiser M, van der Hoorn RA (2015) Subfamily-specific Fluorescent Probes for Cys proteases Display Dynamic Protease Activities During Seed Germination. Plant Physiology http://dx.doi.org/10.1104/pp.114.254466

Renier Van De Hoorn who works in the Department of Plant Chemetics at the University of Oxford, leads this study that investigates the activity of plant cysteine proteases. They developed a novel set of fluorescent probes that specifically target different subfamilies of Cys proteases. In order to test these probes they used Arabidopsis mutant lines alongside transient expression studies in tobacco. In addition they show that these probes have broad applicable across 8 plant species. Finally they use these new tools to reveal the dynamic properties of different protease sub-families during remobilization of seed storage proteins in Arabidopsis.

Genome Resequencing for Mutant Identification

As most biologists will be aware, the cost of DNA sequencing has been falling well in advance of the costs predicted by Moores law (although argued by Neil Hall a few years ago, this might not have been the best thing to happen, intellectually at least).

Instead of simply sequencing many genomes for the sake of it, this also offers opportunities for researchers to use this technology to ‘do-science’ that might previously have been prohibitively laborious or expensive. One such area where this is true is in the identification of novel mutations in plants, especially in Arabidopsis.

Classic approaches to identity the location of an EMS mutation involved mutant identification, backcrossing, selection, rough mapping by PCR or CAPS markers, probably more crossing and then a little guesswork toward the end..…..before using Sanger sequencing to identify what you hope is the causative mutation. Even with a strong following wind this process could take upwards of a year……. many a 1990s PhD thesis was written off the back of mutant identification. In contrast it is now relatively cheap to resequence the Arabidopsis genome so a lot of time can be taken out of this process. In addition, resequencing can remove some of the difficulty involved with selective of mutants that have a subtle phenotypes wherein inaccurate selection of putative mutants would significantly set back the process.

Back in 20111, Anthony Hall’s group in Liverpool University used resequencing in parallel with classic genetics to identify the lesion in the novel early bird1 gene (ebi1), which has a defect in function of the circadian clock. In this case ebi1, which was generated using EMS, was backcrossed 4 times to reduce the number of EMS-induced SNPs not associated with phenotype, and then sequenced alongside the original wildtype plant (from the WS ecotype). The critical part of the protocol came in the power of the software they used to detect homozygous SNPs in the ebi1 line. Indeed the researchers ran into some difficulties due to a high number of SNPs they initially identified. However, when they combined altering the stringency of SNP-calling together with classical rough mapping they were left with approximately 30 SNPs to finally assess. Using a priori knowledge of proposed gene function and by investigating expression changes in these candidates they ultimately identified a novel mutant. Although this process was ultimately successful, it took some extra time due to the difficulty of mutant selection, optimization of the SNP-calling software and subsequent analysis of gene expression.

A recent paper from the lab of Lucia Strader at Washington University in St Louis shows how powerful resequencing can be if you are using a robust method of mutant selection. In their case they isolated mutants with a defect in the root growth response to ABA, which is an unequivocal phenotype to score. They backcrossed their initial mutants, selected for ABA resistance in F2 generation before resequencing these resistant plants. Using this process the authors report that they narrowed their search to between 3-10 candidate genes and that they have subsequently identified novel (unpublished) genes using this method. In addition, as an exemplar of their protocol they used it to isolate novel alleles of known ABA-resistant mutants.

Schematic for mutant identification using NGS. Reproduced from Taylor and Francis PSB http://dx.doi.org/10.1080/15592324.2014.1000167
Schematic for mutant identification using NGS. Reproduced from Taylor and Francis PSB http://dx.doi.org/10.1080/15592324.2014.1000167

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

In parallel they used a similar protocol to the Hall lab where they resequenced non-backcrossed plants and then selected SNPs that only lay within exons.Using this approach they identified between 100-200 homozygous SNPs, a potentially fifty-fold increase compared to their other method. Therefore when you are working with a strong robust phenotype it is probably worth the extra time to obtain a back-crossed population in order to have greater confidence you are isolated your mutant of interest.

The authors importantly note that one limitation of this protocol is that by only selecting for exonic mutations, they are removing the possibility of identifying mutants with splicing or non-coding defects, which may in turn rule out a number of candidate genes.

 

For me the take-home message from this second study is that if you have a robust phenotype to select for and are confident that your mutation is novel then use of ever-improving NGS is now a time and cost effective way of mutant identification.

In fact this technology might inspire a return to the forward genetic screens of the 80s and 90s , with the aim of identifying novel genes involved in well characterised signaling pathways……..except that PhD students might now have to characterise 10 novel genes prior to graduation….

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