Arabidopsis Research Roundup: February 17th

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Published on: February 16, 2016

This weeks Arabidopsis Research Roundup features papers that build upon the history of research in each featured lab. Firstly Gareth Jenkins from Glasgow continues to investigate mechanisms of UV-B signaling whilst Laila Moubayidin, now at the JIC, is involved in work that investigates the multiple factors that control root meristem size. Finally we present a three protocol papers that are featured in a new colelction of articles that focus on protocols that can be used to assess different environmental responses.

Findlay KM, Jenkins GI (2016) Regulation of UVR8 photoreceptor dimer/monomer photo-equilibrium in Arabidopsis plants grown under photoperiodic conditions. Plant Cell Environment http://dx.doi.org/10.1111/pce.12724 Open Access
UVBmodel
The research group led by Gareth Jenkins (Glasgow) continues their work on the plant response to UV in this study that investigates the binding patterns of the UVR8 protein. UVR8 mediates the plant response to UV-B light and the protein either exists in a monomeric (active) or dimeric (inactive) form. This study shows that UVR8 maintains dimer/monomer photo-equilibrium through diurnal photoperiods and that the REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2 proteins are necessary for maintaining this equilibrium. Interestingly they show that the UVR8 balance is tipped toward the monomeric form in lower temperatures. This shows that the protein does not act as a simple switch to signal for changes in UV-B as its effect is influenced by environmental parameters outside of the light source.

Moubayidin L, Salvi E, Giustini L, Terpstra I, Heidstra R, Costantino P, Sabatini S (2016) A SCARECROW-based regulatory circuit controls Arabidopsis thaliana meristem size from the root endodermis Planta http://dx.doi.org/10.1007/s00425-016-2471-0 Open Access

Laila Moubayidin now works as a postdoc with Lars Ostergaard at the JIC but this work is the result of research conducted with Sabrina Sabatini in Rome. In this study they continue the labs investigation into the role of the SCARECROW (SCR) protein in the control of root meristem size. They show that SCR, from endodermal cells, sustains a gibberellic acid signal by regulating RGA REPRESSOR OF ga1-3 (RGA) protein stability. This in turn controls the activity of the cytokinin responsive transcription factor ARR1 at the root transition zone. This activity therefore maintains a balance of cell division and differentiation that maintains correct meristem size.

A new edition of ‘Methods in Molecular Biology’ focuses on ‘Environmental Responses in Plants and includes a number of papers featuring UK authors who work on Arabidopsis.

Hydrotropism: Analysis of the Root Response to a Moisture Gradient’ that features Malcolm Bennett from CPIB in Nottingham. http://dx.doi.org/10.1007/978-1-4939-3356-3_1

Monitoring Alternative Splicing Changes in Arabidopsis Circadian Clock Genes’ from the group of John Brown at the James Hutton in Dundee http://dx.doi.org/10.1007/978-1-4939-3356-3_11

Assessing the Impact of Photosynthetic Sugars on the Arabidopsis Circadian Clock’ from the lab of Alex Webb in Cambridge. http://dx.doi.org/10.1007/978-1-4939-3356-3_12

Arabidopsis Research Roundup: January 8th

For the inaugural Arabidopsis Research Roundup of 2016 we feature the final publications of UK researchers from 2015. Martin Howard kindly provides an audio description of a paper that looks at a fundamental aspect of transcriptional regulation, through the lense of the FLC gene, whilst his co-author Caroline Dean on that paper is an author on another manuscript that investigates RNA stability in the same FLC locus. Katja Graumann leads a paper that looks into gene expression at the periphery of the nucleus whilst Ian Colbeck looks at the effect of silver nanoparticles on plant growth. Ari Sadanandom is the UK lead of a study that investigates of SUMOylation and Ian Fricker looks at the role of a cytochrome P450 on the defence response. Finally Liam Dolan is involved in a comparative analysis of the genes involved in tip growth in the cells of plants and moss.

Wu Z, Ietswaart R, Liu F, Yang H, Howard M, Dean C (2015) Quantitative regulation of FLC via coordinated transcriptional initiation and elongation. Proc Natl Acad Sci U S A. http://dx.doi.org/10.1073/pnas.1518369112 Open Access

Martin Howard and Caroline Dean lead this study that comes out of the John Innes Centre and is the result of the same collaboration that featured in an ARR earlier in 2015. In this study they investigate the mechanisms that control the quantitative regulation of gene expression by focusing on the complex regulation of the FLOWERING LOCUS C (FLC). FLC expression is controlled by a chromatin silencing mechanism involving alternative polyadenylation of antisense transcripts. However they surprisingly show that the amount of RNA Polymerase II occupancy at FLC does not coincide very well with levels of FLC transcription. They used modeling to predict that there is a tight coordination between transcriptional initiation and elongation, which was validated by detailed measurements of the levels of FLC intronic RNA. Variation within initiation and elongation rates were significantly different and was coincident with changes in H3K36me3 and H3K4me2 levels in the FLC gene. The authors propose that chromatin state can influence transcriptional initiation and elongation rates and may be a general mechanism for quantitative gene regulation in a chromatin context.

Martin Howard kindly provides an audio description of this paper and wider aspects of transcriptional regulation.

Wu Z, Zhu D, Lin X, Miao J, Gu L, Deng X, Yang Q, Sun K, Zhu D, Cao X, Tsuge T, Dean C, Aoyama T, Gu H, Qu LJ (2015) RNA-binding proteins At RZ-1B and At RZ-1C play a critical role in regulation of pre-mRNA splicing and gene expression during Arabidopsis development Plant Cell http://dx.doi.org/10.1105/tpc.15.00949

This study investigates a set of previously mysterious RNA-binding proteins and is led by Chinese researchers with a UK contribution from Caroline Dean (JIC). They look at two Arabidopsis proteins, AtRZ-1B and At RZ-1C that have RNA-binding domains and are localised to the mysterious nuclear speckles. In addition these proteins physically interact with a range of serine/arginine-rich (SR) proteins and disrupting this binding causes a range of growth phenotypes that are similar to that observed in At rz-1b/At rz-1c double mutants. These include delayed seed germination, reduced stature, and serrated leaves and on the cellular level this is accompanied by defective splicing and global changes in gene expression. Interestingly AtRz-1C directly effects the expression of the floral repressor FLC, which links this work with other research in the Dean lab. Overall this highlights the important role of At RZ-1B/1C in RNA splicing and the link to many developmental phenotypes.

Smith S, Galinha C, Desset S, Tolmie F, Evans D, Tatout C, Graumann K (2015) Marker gene tethering by nucleoporins affects gene expression in plants. Nucleus. http://dx.doi.org/10.1080/19491034.2015.1126028

Expression of Seh1-LacI-YFP at the nuclear periphery. From http://dx.doi.org/10.1080/19491034.2015.1126028
Expression of Seh1-LacI-YFP at the nuclear periphery. From http://dx.doi.org/10.1080/19491034.2015.1126028

Katja Graumann and David Evans (Oxford Brookes) are the lead academics on this collaboration with the lab of Christophe Tatout from Clermont Ferrand in France. They are attempting to answer a long standing question in the field of the biology of the nucleus; whether genes that are located close to nuclear pore complexes have increased gene expression. They used the Lac Operator/ Lac Repressor (LacI-LacO) system to assess changes in gene expression when a loci is tethered to the NPC by attaching the LacI domain to the nucleoporins Seh1 or NUP50a. The Seh1 clones localised to the nuclear periphery and showed higher RNA and protein expression of Luc. When this interaction at the periphery was distributed, the higher levels of expression were abolished. The authors therefore show that association with the nuclear periphery is important for the regulation of gene expression.

Sosan A, Svistunenko D, Straltsova D, Tsiurkina K, Smolich I, Lawson T, Subramaniam S, Golovko V, Anderson D, Sokolik A, Colbeck I, Demidchik V (2015) Engineered silver nanoparticles are sensed at the plasma membrane and dramatically modify physiology of Arabidopsis thaliana plants. Plant Journal http://dx.doi.org/10.1111/tpj.13105

Ian Colbeck (Essex) is the UK lead on this study that involves a collaboration between researchers in New Zealand, Belarus and Russia and focuses on the effect of silver nanoparticles (Ag NPs) on the growth of Arabidopsis seedlings. This type of nanoparticle is used for many difference applications so worries exist about the safety of their use. This study looks at the effect of Ag NPs on Arabidopsis root elongation and leaf expansion, both of which were inhibited at over [300mg/l] Ag NPs. In addition there were reductions of photosynthetic efficiency and accumulation of silver in plant tissues. They also showed that these particles altered the influx and efflux of metal ions whilst, although they were unable to catalyse hydroxyl radical generation, they did directly oxidise the major plant antioxidant, L-ascorbic acid. Overall the authors show that silver nanoparticles induce classical stress signalling responses but also illicit specific detrimental effects at the plasma membrane. At the whole plant level this study provides a worrying example for the role of Ag NPs on whole plant growth, even though the concentrations used in food preparation might be lower.

Crozet P, Margalha L, Butowt R, Fernandes N, Elias A, Orosa B, Tomanov K, Teige M, Bachmair A, Sadanandom A, Baena-González E (2015) SUMOylation represses SnRK1 signaling in Arabidopsis. Plant Journal http://dx.doi.org/10.1111/tpj.13096

This pan-European study features researchers from Portugal, Austria and the UK’s Durham University, led by Ari Sadanandom. They investigate the role of the SnRK1 protein kinase, which is a key enzyme for modulating the plant stress response. This paper adds detail to the cellular mechanisms that regulate SnRK1 and they show that SnRK1 is SUMOylated by the SIZ1 E3 SUMO ligase. SnRK1 is ubiquitinated and degraded in a SIZ1-dependent manner that is lacking in siz1 mutants. Interestingly only active SnRK1 is degraded as the inactive SnRK1 protein is stable but can be easily degraded upon SUMOylation. Finally they show that SnRK1 is involved in a negative feedback loop wherein it controls its own SUMOylation and degradation that, in wildtype cells, prevents a potentially detrimental stress response.

Fuchs R, Kopischke M, Klapprodt C, Hause G, Meyer AJ, Schwarzländer M, Fricker MD, Lipka V (2015) Immobilized subpopulations of leaf epidermal mitochondria mediate PEN2-dependent pathogen entry control in Arabidopsis. Plant Cell http://dx.doi.org/10.1105/tpc.15.00887

Mark Fricker (Oxford) is the UK research lead on this study that investigates the role of the atypical myrosinase PEN2 in the response to pathogen attack. PEN2 is targeted to both peroxisomes and mitochondria and can also form homo-oligomer complexes. PEN2 localised to mitochondria are immobilized following fungal invasion and this accompanies mitochondrial arrest. The substrate for PEN2 is produced by the cytochrome P450 monooxygenase CYP81F2, which is localized to the ER and moves toward immobilized mitochondria. The critical function of PEN2 in that organelle was confirmed by the result that showed exclusive mitochondria targeting could rescue pen2 mutant phenotypes. The authors show by live-cell imaging that arrested mitochondria in domains of plant-microbe interaction exhibit a pathogen-induced redox imbalance that may lead to production of intracellular signals.

Ortiz-Ramírez C, Hernandez-Coronado M, Thamm A, Catarino B, Wang M, Dolan L, Feijó JA, Becker JD (2015) A transcriptome atlas of Physcomitrella patens provides insights into the evolution and development of land plants. Mol Plant. http://dx.doi.org/10.1016/j.molp.2015.12.002

Liam Dolan (Oxford) is an author on this study that is led from Portugal and is an investigation of the transcriptome of the model moss Physcomitrella patens throughout its life cycle. They also compare transcriptomes from P.patens and Arabidopsis, allowing the authors to identify transcription factors that are expressed in tip growing cells. Interestingly they identified differences in expression patterns that might account for the differences between tip growth in moss and the Arabidopsis root hairs, an area that is the expertise of the Dolan lab.

Arabidopsis Research Roundup: December 18th

The final Arabidopsis Research Roundup of 2015 contains a bumper crop of papers that again highlights the diversity of research occuring in UK plant science. Justin Goodrich from the University of Edinburgh kindly provides an audio description of work that identifies a novel role for a member of a transposon gene family. Elsewhere are studies about a specific aspect of the biochemistry of crytochromes as well as confirmation of a role for DNA gyrases in Arabidopsis. Paul Dupree (Cambridge) leads a study into the mechanism of ascorbic acid production while Heather Knight is the UK representative in a study about cell wall composition. We also present an investigation into the mechanism and subsequent expression changes that occur following infection with different isolates of the Turnip Mosaic Potyvirus. Finally are two short studies from Ive de Smet (Nottingham) and Matt Jones (Essex).

Liang SC, Hartwig B, Perera P, Mora-García S, de Leau E, Thornton H, de Alves FL, Rapsilber J, Yang S, James GV, Schneeberger K, Finnegan EJ, Turck F, Goodrich J (2015) Kicking against the PRCs – A Domesticated Transposase Antagonises Silencing Mediated by Polycomb Group Proteins and Is an Accessory Component of Polycomb Repressive Complex 2. PLoS Genet. 11 e1005660. http://dx.doi.org/10.1371/journal.pgen.1005660 Open Access

Justin Goodrich (Edinburgh) is the lead of this collaborative study between UK, German and Australian researchers that investigates the role of the evolutionarily conserved Polycomb group (PcG) and trithorax group (trxG) genes during plant development. These homeotic genes influence gene expression by causing epigenetic chromatin changes, usually in the form of histone methylation. Previously the ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN1 (ALP1) gene was found to act as a genetic suppressor the Arabidopsis PcG gene, LIKE HETEROCHROMATIN PROTEIN1 (LHP1). In this study ALP1 is shown to genetically interact with members of these two gene families and its activity is necessary for the activation of several floral homeotic genes. Surprisingly the ALP1 gene is shown to encode for a transposase of the PIF/Harbinger class, which is conserved throughout land plants. The authors suspect that the transposase activity has been lost in the angiosperm lineage, where the gene obtained a novel function. Interestingly ALP1 can interact with the core PrC complex, which most notably participates in H3K27me3 methylation and therefore appears to act, along with other proteins such as EMBRYONIC FLOWER 1 (EMF1), as a plant-specific accessory component that controls histone modification. The authors speculate that this novel function might have arisen as a “means for the cognate transposon to evade host surveillance or for the host to exploit features of the transposition machinery beneficial for epigenetic regulation of gene activity”. Over the coming years it will be interesting to discover if other transposon-encoded genes share novel functions and this study represents an important lesson for researchers not to ignore transposon sequences as ‘junk’ DNA that they might feel can clutter up their analysis!

Justin Goodrich kindly provides an audio summary of this paper:

van Wilderen LJ, Silkstone G, Mason M, van Thor JJ, Wilson MT (2015) Kinetic studies on the oxidation of semiquinone and hydroquinone forms of Arabidopsis cryptochrome by molecular oxygen FEBS Open Bio. 5:885-892 http://dx.doi.org/10.1016/j.fob.2015.10.007 Open Access

This study is a collaborative effort between researchers from Imperial College and the University of Essex, led by emeritus biochemistry Professor Michael Wilson and is an in vitro analysis of the oxidation of the Arabidopsis cryptochrome (CRY) photoreceptor in the presence and absence of an external electron donor. They show that a more complex model than previously thought is required to explain the mechanism by which the CRY-associated flavin molecule is oxidised. The authors propose that the final steps in this reaction require cooperative interaction between partners in a CRY homodimer or between separate CRY molecules.

Evans-Roberts KM, Mitchenall LA, Wall MK, Leroux J, Mylne JS, Maxwell A (2015) DNA Gyrase is the Target for the Quinolone Drug Ciprofloxacin in Arabidopsis thaliana. J Biol Chem. http://dx.doi.org/10.1074/jbc.M115.689554 Open Access

Antony Maxwell from the Biological Chemistry department from the John Innes Centre is the UK academic lead on this UK-Australian study. This group has previously shown that Arabidopsis contains three proteins thought to function as DNA Gyrases (AtGYRA, ATGYRB1, ATGYRB2) although they could not provide direct evidence that are were involved in DNA supercoiling. This study moves the work on by identifying mutant plants that are resistant to the drug ciprofloxacin and contain a point mutation in AtGYRA. Furthermore ATGYRA heterologously expressed in insect cells has supercoiling activity. Therefore the authors unequivocally show that plants encode an organellar-targeted DNA gyrase that, like bacterial gyrases, is a  target for ciprofloxacin. This work has important consequences for our understanding of plant physiology and in the future development of novel herbicides.

Sawake S, Tajima N, Mortimer JC, Lao J, Ishikawa T, Yu X, Yamanashi Y, Yoshimi Y, Kawai-Yamada M, Dupree P, Tsumuraya Y, Kotake T (2015) KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis The Plant Cell http://dx.doi.org/10.1105/tpc.15.00379

Paul Dupree (Cambridge) is the British lead on the UK-Japanese collaboration that investigates the role of the GDP-mannose pyrophosphorylase (GMPP), VITAMIN C DEFECTIVE1 (VTC1) enzyme in catalysis of the rate-limiting step in the production of ascorbic acid (AsA). They identify two novel pyrophosphorylase-like proteins, KONJAC1 (KJC1) and KJC2 that stimulate VTC1. Mutant analysis showed that these proteins are necessary for normal growth that coincides with control of AsA production via stimulating GMPP activity. Yeast 2 Hybrid  analysis is indicative of a direct interactin between KJC and VTC1 proteins. In future, it will be interesting to investigate the role of these proteins in plants that are more relevant to human consumption of AsA.

Sorek N, Szemenyei H, Sorek H, Landers A, Knight H, Bauer S, Wemmer DE, Somerville CR (2015) Identification of MEDIATOR16 as the Arabidopsis COBRA suppressor MONGOOSE1. PNAS http://dx.doi.org/10.1073/pnas.1521675112

Heather Knight (Durham) is the sole UK representative on this manuscript that is led by the lab of Chris Somerville from the University of California. In this work the authors identified suppressors of the Arabidopsis cobra mutant, which have defects in cellulose formation. The appropriately named mongoose (mon1) mutant partially restored cellulose levels and also restored the esterification ratio of pectin to wild-type levels. MON1 was cloned to the MEDIATOR16 (MED16)/ SENSITIVE TO FREEZING6 (SFR6) locus and single mon1 mutants are resistant to cellulose biosynthesis inhibitors. Concomitantly, transcriptome analysis demonstrated that a set of ‘cell wall’ genes are misregulated in mon1/med16/sfr6, including two encoding pectin methylesterase inhibitors. Overall the authors suggest that cellulose biosynthesis is closely linked to esterification levels of pectin and offer a number of possible explanations for this functional relationship.

Sánchez F, Manrique P, Mansilla C, Lunello P, Wang X, Rodrigo G, López-González S, Jenner C, González-Melendi P, Elena SF, Walsh J, Ponz F (2015) Viral Strain-Specific Differential Alterations in Arabidopsis Developmental Patterns Mol Plant Microbe Interact. http://dx.doi.org/10.1094/MPMI-05-15-0111-R

The UK contributor to this Spanish-led study is Carol Jenner, who at the time was a research fellow at the University of Warwick. This study highlights the morphological changes that occur in Arabidopsis plants infected by different isolates of Turnip mosaic virus (TuMW). The UK1 and JPN1 versions of TuMW were shown to have highest levels of sequence divergence in the P3 cistron and following the generation and use of viral chimeras, it is this region that was identified as the major viral determinant of plant developmental changes. However when the P3 gene was constitutively expressed in Arabidopsis it did not cause any development effects, which highlights the importance of performing infection studies in a whole-plant context. Latterly the authors performed transcriptomic and interactomic analysis, showing that infection with the most severe UK1 strain primarily causes changes, perhaps unsurprisingly, in genes involved in transport and in the stress response.

Czyzewicz N, De Smet I (2015) The Arabidopsis thaliana CLAVATA3/EMBRYO-SURROUNDING REGION 26 (CLE26) peptide is able to alter root architecture of Solanum lycopersicum and Brassica napus. Plant Signal Behav http://dx.doi.org/10.1080/15592324.2015.1118598

This work was performed in the lab of Ive De Smet, who is a BBSRC research fellow at the University of Nottingham. In this short communication they show that overexpression of the Arabidopsis AtCLE26 peptide is able to induce architectural change in the agriculturally important crops, Brassica napus and Solanum lycopersicum. Having previously shown that AtCLE26 is similarly active in Arabidopsis, Brachypodium and Triticum, these experiments further demonstrate that small peptide signaling plays an important role in root development across plant lineages.

Litthauer S1, Battle MW1, Jones MA (2015) Phototropins do not alter accumulation of evening-phased circadian transcripts under blue light. Plant Signal Behav. http://dx.doi.org/10.1080/15592324.2015.1126029

Matt Jones (Essex) leads this accompanying study to the more substantial project previously published in Plant Journal. This manuscript reports that phototropin photoreceptors are not involved in the nuclear accumulation of evening-phased circadian transcripts. In addition they show that even in phototropin mutants, the rhythms of nuclear clock transcript accumulation are maintained under fluctuating light regimes.

Arabidopsis Research Roundup: December 9th.

This December 9th Arabidopsis Research Roundup includes four rather different studies. Firstly we include an excellent audio description from David Salt about a new type of GWAS analysis that his lab was involved in developing. This allowed identification of new genetic loci involved in molybdenum signalling. Secondly Isabelle Carre’s group from Warwick presents a study into the interactions that define the functioning of the circadian clock. Thirdly Mike Blatt leads a study that models stomatal opening and finally we include an investigation of the DOG1 gene, that includes a contribution from Fuquan Liu.

Forsberg SK, Andreatta ME, Huang XY, Danku J, Salt DE, Carlborg Ö (2015) The Multi-allelic Genetic Architecture of a Variance-Heterogeneity Locus for Molybdenum Concentration in Leaves Acts as a Source of Unexplained Additive Genetic Variance PLoS Genet. e1005648. http://dx.doi.org/10.1371/journal.pgen.1005648 Open Access.

Current GARNet Chairman David Salt (Aberdeen) is the UK lead on this collaboration with the lab of Orjan Carlborg from Uppsala in Sweden. The novelty of this paper is in the development of a new technique to measure Genome-Wide Association using the variance in SNP differences instead of using the mean. Professor Salt explained this vGWA technique in the attached audio-file, which is especially useful for people not so familiar with GWAS. Using this vGWA technique the authors were able to re-analyse an old dataset to gain additional understanding of how certain genetic loci are regulated to explain differences in the production of the essential nutrient molybdenum. Overall this paper introduces an analysis technique that can hopefully be used by other members of the community to analyse/re-analyse their data with increased rigour.

This is the 10minute audio file where David explains the paper:

Adams S, Manfield I, Stockley P, Carré IA (2015) Revised Morning Loops of the Arabidopsis Circadian Clock Based on Analyses of Direct Regulatory Interactions. PLoS One.10(12):e0143943. http://dx.doi.org/ 10.1371/journal.pone.0143943 Open Access

This collaboration between the Universities of Warwick and Leeds is led by Isabelle Carré and investigates the Arabidopsis circadian clock. They analysed the in vivo interactions of the LATE ELONGATED HYPOCOTYL (LHY) protein with promotors of other clock components. This uncovered a novel regulatory loop between LHY and the CIRCADIAN CLOCK ASSOCIATED-1 (CCA1) gene. Furthermore they show LHY acts as a repressor of all other clock components, clearly placing this protein as a key regulatory component of the Arabidopsis clock.

Minguet-Parramona C, Wang Y, Hills A, Vialet-Chabrand S, Griffiths H, Rogers S, Lawson T, Lew V, Blatt MR (2015) An optimal frequency in Ca2+ oscillations for stomatal closure is an emergent property of ion transport in guard cells. Plant Physiol. http://dx.doi.org/10.1104/pp.15.01607 Open Access

Mike Blatt is the corresponding author for this collaboration between Glasgow, Cambridge and Essex Universities. There are a good number of UK researchers who investigate the factors that regulate stomatal opening and this study looks at the role of calcium oscillations in this process. They have used the Arabidopsis OnGuard model that faithfully reproduces the optimum 10minute period of Ca2+ oscillation in guard cells. They used experimentally derived kinetics to describe the activity of ion transporters in the plasma membrane and tonoplast. Overall they discovered that the calcium oscillations are actually a by-product of the ion transport that determines stomatal aperature and not the overall controlling factor.

Cyrek M, Fedak H, Ciesielski A, Guo Y, Śliwa A, Brzeźniak L, Krzyczmonik K, Pietras Z, Liu F, Kaczanowski S, Swiezewski S (2015) Seed dormancy in Arabidopsis thaliana is controlled by alternative polyadenylation of DOG1 Plant Physiol. http://dx.doi.org/10.1104/pp.15.01483

Fuquan Liu (Queens, Belfast) is the UK contributor to this Polish-led study focused on the DOG1 gene, which is a key regulator of Arabidopsis seed dormancy. Previously it had been shown that the C-terminus of DOG1 is not conserved in many other plant species. The DOG1 transcript is alternatively polyadenylated and the authors show that Arabidopsis mutants that lack current 3’ RNA processing also show defects in seed dormancy. The shorter version of DOG1 is able to rescue the dog1 phenotype, which allows the authors to propose that DOG1 is a key regulator of seed dormancy and that the phenotypes of RNA processing mutants are linked to the incorrect processing of this specific mRNA species.

Arabidopsis Research Roundup: November 25th

This weeks Arabidopsis Research Roundup contains four papers each with a different focus. Firstly is a large-scale investigation that attempts to define the transcriptional changes that occur in response to bacterial infection. Second is a study that investigates a newly proposed role for the chloroplast chaperone Hsp93. Thirdly is another piece of work that also involves University of Oxford researchers and investigates the genetic networks that control leaf morphology. Finally is an updated plant-specific protocol for the commonly used technique of Chromatin Immunoprecipitation.

Lewis LA, Polanski K, de Torres-Zabala M, Jayaraman S, Bowden L, Moore J, Penfold CA, Jenkins DJ, Hill C, Baxter L, Kulasekaran S, Truman W, Littlejohn G, Prusinska J, Mead A, Steinbrenner J, Hickman R, Rand D, Wild DL, Ott S, Buchanan-Wollaston V, Smirnoff N, Beynon J, Denby K, Grant M (2015) Transcriptional Dynamics Driving MAMP-Triggered Immunity and Pathogen Effector-Mediated Immunosuppression in Arabidopsis Leaves Following Infection with Pseudomonas syringae pv tomato DC3000 Plant Cell. http://dx.doi.org/10.1105/tpc.15.00471 Open Access

This ‘Large Scale Biology’ publication is a collaboration between the Universities of Exeter and Warwick, led by Murray Grant and current GARNet Advisory board member Katherine Denby. This study investigates the transcriptional changes that occur over a long time course in response to infection by the pathogen Pseudomonas syringae pv tomato DC3000. The authors aim to differentiate between the changes associated with endogenous microbial-associated molecular pattern (MAMP)-triggered immunity (MTI) and those orchestrated by pathogen effectors. The responses to pathogenic and non-pathogenic P.syringae were compared and using novel computational analysis, it was shown that the majority of gene expression changes that contribute to disease or defense responses occurred within 6hour post-infection, well before pathogen multiplication. Broadly it was found that chloroplast-associated genes are suppressed by a MAMP-triggered response, presumably to restrict nutrient availability. Ultimately this manuscript identified specific promotor elements that are involved in either the MTI response or utilised by the infecting bacteria.

Corresponding author Professor Murray Grant kindly takes ten minutes to discuss the finding of this paper and the community resource that it represents. He also discusses another paper involving the Jasmonate response that resulted from this dataset and was recently highlighted in the Research Roundup. Interview end at 11m10s.

Flores-Pérez Ú1, Bédard J1, Tanabe N2, Lymperopoulos P2, Clarke AK3, Jarvis P (2015) Functional analysis of the Hsp93/ClpC chaperone at the chloroplast envelope Plant Physiology. http://dx.doi.org/10.1104/pp.15.01538 Open Access

Paul Jarvis (Oxford) is the corresponding author on this study in which his lab collaborates with Swedish researchers to investigate the role of the Hsp93/ClpC chaperone protein in protein import into the chloroplast. This recently postulated role for this protein has not yet been experimental tested so they generated a hsp93[P-] mutant that lacked a functional ClpP-binding motif (PBM), which confers the already determined role for Hsp93 in proteolysis that occurs in the chloroplast stroma. The hsp93[P-] mutant localises to the chloroplast envelope and associates with TIC transport machinery but was unable to complement the phenotypes of a hsp93 null mutant. This showed that the PBM domain was essential for its function. Expression of the Hsp93[P-] mutant in the hsp93 null background did not improve protein import so the authors concluded that these results do not confirm this newly postulated role for the protein and they suggest that its functional role occurs immediately after its substrate had been transported into the chloroplast.

Rast-Somssich MI, Broholm S, Jenkins H, Canales C, Vlad D, Kwantes M, Bilsborough G, Dello Ioio R, Ewing RM, Laufs P, Huijser P, Ohno C, Heisler MG, Hay A, Tsiantis M (2015) Alternate wiring of a KNOXI genetic network underlies differences in leaf development of A. thaliana and C. hirsuta Genes Dev. 29(22):2391-404 http://dx.doi.org/10.1101/gad.269050.115 Open Access

The study includes researchers from Oxford and Southampton Universities in collaboration with those from Italy, France and Germany in work that is led by Angela Hay and Miltos Tsiantis, who were both previously based in Oxford. This is familiar territory for this group as they compare leaf development between Arabidopsis, which has simple leaves, and the related , Cardamine hirsuta, which has dissected leaves. In this new work they transfer the SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP) homeobox genes between the two species and investigate their ability to modify leaf form. In Cardamine, expression of BP is controlled by crosstalk between the microRNA164A (MIR164A)/ChCUP-SHAPED COTYLEDON (ChCUC) module and ChASYMMETRIC LEAVES1 (ChAS1) gene. However this regulatory network does not function in Arabidopsis and therefore leads to the establishment of differing regulatory networks that the authors propose are responsible for the alterations in organ geometry.

Posé D, Yant L (2016) DNA-Binding Factor Target Identification by Chromatin Immunoprecipitation (ChIP) in Plants Methods Mol Biol. 1363:25-35. http://dx.doi.org/10.1007/978-1-4939-3115-6_3

Levi Yant is a new member of faculty at the John Innes Centre and is the lead author on this paper that introduces an updated protocol for Chromatin Immunoprecipitation in Plants (ChIP). They have used this technique in his lab to identify target genes for a number of transcriptional regulators that are involved in Arabidopsis floral development.

Arabidopsis Research Roundup: November 5th

Academics from the John Innes Centre lead two of the papers featured in this week Arabidopsis Research Roundup. Firstly Veronica Grieneisen leads a study that combines modeling and experimental work to assess the factors that establish the root auxin maximum and secondly the structural biologist David Lawson heads up an investigation into the plastid-localised enzyme, DPE1. Seemingly a common theme in UK-Arabidopsis research focuses on the factors that control the dynamics of stomatal opening and this week Mike Blatt from Glasgow heads a team that investigates the role of potassium and nitric oxide in this process. Finally we present a paper that investigates proteins that interact within the ER.

El-Showk S, Help-Rinta-Rahko H, Blomster T, Siligato R, Marée AF, Mähönen AP, Grieneisen VA (2015) Parsimonious Model of Vascular Patterning Links Transverse Hormone Fluxes to Lateral Root Initiation: Auxin Leads the Way, while Cytokinin Levels Out PLoS Comput Biol. e1004450Picture

http://dx.doi.org/10.1371/journal.pcbi.1004450 Open Access

Veronica Grieneisen (JIC) is the UK-based leader of this work that was performed with her Finnish collaborators. They work on the modeling the processes that define the auxin maximum in the root meristem. This patterning is defined by the activity of the PIN-formed auxin efflux transport proteins and the AHP6 protein, an inhibitor of cytokinin signaling. The authors implement a parsimonious computational model of auxin transport that considers hormonal regulation of the auxin transporters within a spatial context, explicitly taking into account cell shape and polarity and the presence of cell walls. They initially find that variation in cytokinin signaling, mediated by diffusion of the hormone is insufficient for patterning but rather it is an auxin-dependent modification of the cytokinin signal that can define the auxin maximum. Although the role that the PIN proteins play in root vascular patterning is well established, the authors experimentally verify a role for the AUX/LAX auxin influx carrier family of proteins. They also show that polar PIN localisation generates a flux of auxin flow that ultimately causes its own accumulation in the pericycle cells that signal for lateral root initiation. Finally their model confirms the supposition that these pericycle cells compete for auxin accumulation, therefore ensuring that lateral roots develop in the correct localisation. The associated figure is from this paper.

O’Neill EC, Stevenson CE, Tantanarat K, Latousakis D, Donaldson MI, Rejzek M, Nepogodiev SA, Limpaseni T, Field RA, Lawson DM (2015) Structural Dissection of the Maltodextrin Disproportionation Cycle of the Arabidopsis Plastidial Enzyme DPE1. Journal of Biological Chemistry http://dx.doi.org/10.1074/jbc.M115.682245 Open Access

This is another paper led by JIC researchers, this time in collaboration with Thai partners. This focuses on determining the structure of the Arabidopsis Plastidial Disproportionating Enzyme 1 (DPE1) that acts to convert two maltotriose molecules to a molecule of maltopentaose and a molecule of glucose, which, for different reasons, are both more functional useful molecules for the plant. They have used ligand soaking techniques to trap the DPE1 in a different set of conformational states and have found that it exists as a homodimer with a variety of interesting features. This includes a dynamic ‘gate’ loop that may play a role in substrate capture, subtle changes in which could alter the efficacy of the active site. The structural insights provided by this study allow the authors to confidently delineate the complete AtDPE1 disproportionation cycle

Chen ZH, Wang Y, Wang JW, Babla M, Zhao C, García-Mata C, Sani E, Differ C, Mak M, Hills A, Amtmann A, Blatt MR (2015) Nitrate reductase mutation alters potassium nutrition as well as nitric oxide-mediated control of guard cell ion channels in Arabidopsis New Phytol.http://dx.doi.org/10.1111/nph.13714 Open Access

<a href="http://www.gla cialis vente en france.ac.uk/researchinstitutes/biology/staff/michaelblatt/” onclick=”_gaq.push([‘_trackEvent’, ‘outbound-article’, ‘http://www.gla.ac.uk/researchinstitutes/biology/staff/michaelblatt/’, ‘Mike Blatt’]);” target=”_blank”>Mike Blatt (Glasgow) is the lead on this UK-Sino-Australino-Argentine collaboration that investigates the role of nitrate reductase enzyme in potassium flux in guard cells. This flux is necessary for a plants adaption to the environment and is controlled by the activity of ABA via the activity of H2O2 and Nitric Oxide (NO). The authors showed that multiple responses to ABA were impaired in nia1nia2 nitrate reductase mutants, which includes the K+ IN current in guard cells, required for stomatal closure. This response was rescued by exogenous NO and allowed the authors to demonstrate that there exists a complex interaction involving ABA, NO, potassium nutrition and nitrogen metabolism that is necessary to ensure correct stomatal responses.

Kriechbaumer V, Botchway SW, Slade SE, Knox K, Frigerio L, Oparka K, Hawes C (2015) Reticulomics: Protein-Protein Interaction Studies with Two Plasmodesmata-Localized Reticulon Family Proteins Identify Binding Partners Enriched at Plasmodesmata, Endoplasmic Reticulum, and the Plasma Membrane Plant Physiol. 169(3):1933-45 http://dx.doi.org/10.1104/pp.15.01153

This proteomic analysis of endoplasmic reticulum components is a collaboration between the Central Laser Facility at Didcot, Warwick, Edinburgh and Oxford Brookes Universities, led by Professor Chris Hawes. Plant Reticulon proteins (RTNLB) specifically populate and tubulate the ER, mediated by their varied multi-meric interactions. In addition, certain RTNLB are also present in plasmodesmata (PD) and two of these proteins, RTNLB3 and RTNLB6 were GFP-tagged, Co-IPed and interacting proteins were analysed by MS. This identified a range of known PD-localised proteins, and these interactions were experimentally verified in tobacco cells using FRET-microscopy. The authors suggest that this data shows that RTNLB proteins may play important roles in linking the cortical ER to the plasma membrane. This paper is the ‘sister’ to another manuscript in Plant Physiology that was highlighted in a recent Arabidopsis Research Roundup.

Arabidopsis Research Roundup: Oct 28th

This latest Arabidopsis Research Roundup is rather GARNet-focused as members of the current Advisory Board lead three of the featured papers. Firstly we present a study into mechanisms that control meiotic recombination, which also includes a short audio-description from the lead author Dr Ian Henderson. Secondly we introduce a paper that identifies the function of a novel gene in the control of male fertility and thirdly, a study of a translation control-factor that is involved in regulation of cell size and ovule development. In addition we introduce some highly collaborative work that looks into the role of SUMO proteases in SA signaling. Finally there is a methods paper that presents a new protocol for measurement of cellulose content in Arabidopsis stems.

Yelina N, Lambing C, Hardcastle T, Zhao X, Santos B, Henderson I (2015) DNA methylation epigenetically silences crossover hot spots and controls chromosomal domains of meiotic recombination in Arabidopsis Genes & Dev. 29: 2183-2202 http://dx.doi.org/10.1101/gad.270876.115

GARNet advisory board member Ian Henderson leads this study that assesses how methylation state influences the chromosomal regions that undergo meiotic recombination. It was previously known that highly-methylated regions, such as centromeres, do not often undergo recombination. This work naturally extends that knowledge by using RNA-directed DNA methylation to show that methylation of local euchromatic regions also have reduced recombination levels. Equally they show that global reductions in CG methylation, such as in met1 mutants, cause wide-scale alterations in recombination remodeling. Use of recombination mutants shows that these changes are due to the redistribution of interfering crossovers. Overall they confirm that DNA methylation is critical in establishing domains of meiotic recombination.

In this short audio file, Dr Henderson explains the main features of this paper.

Visscher AM, Belfield EJ, Vlad D, Irani N, Moore I, Harberd NP (2015) Overexpressing the Multiple-Stress Responsive Gene At1g74450 Reduces Plant Height and Male Fertility in Arabidopsis thaliana. PLoS One.;10(10):e0140368. http://dx.doi.org/10.1371/journal.pone.0140368

Ian Moore and Nick Harberd (Oxford), who is also on the GARNet Advisory Board,  present this investigation of five unknown genes that had been previously identified from global expression studies as playing a role in multiple stress-responses. These are somewhat unimaginatively identified by their ‘At’ numbers and even though they are each responsive to multiple stresses, mutants with a T-DNA insertion in any of these genes have no change in phenotype compared to wildtype plants. In contrast, overexpression of At1g74450, but no other of the tested genes, resulted in stunted growth and reduced male fertility. As the stress-response is often manifested by alterations in male gametophyte development, this work introduces the function of a gene that may provide an important link between multiple environmental factors, fertility and plant growth. In future the authors hope to provide further insight into the function of At1g74450.

Bush M, Crowe N, Zheng T, Doonan J (2015) The RNA helicase, eIF4A-1, is required for ovule development and cell size homeostasis in Arabidopsis Plant J. http://dx.doi.org/10.1111/tpj.13062

John Doonan, another GARNet board member, leads this collaborative work between Aberystwyth and Norwich. They investigate the function of the RNA helicase/ATPase eIF4A-1 that is involved in the initiation of mRNA translation. Arabidopsis contains two isoforms of this genes and the knockdown eif4a-1 mutant displays a range of altered phenotypes that includes a reduction in the amount of mitotic cells in the root meristem. This change skews the relationship between cell size and cell cycle progression. Concomitantly several cell cycle-regulated genes have reduced expression in this mutant. Each of the eIF4A isoforms plays an important role in plant fertility as although single eif4a-1 mutants display some defects in ovule development, double eif4a1eif4a2 mutants cannot be isolated.

Bailey M, Srivastava A, Conti L, Nelis S, Zhang C, Florance H, Love A, Milner J, Napier R, Grant M, Sadanandom A (2015) Stability of small ubiquitin-like modifier (SUMO) proteases OVERLY TOLERANT TO SALT1 and -2 modulates salicylic acid signalling and SUMO1/2 conjugation in Arabidopsis thaliana J Exp Bot. http://dx.doi.org/10.1093/jxb/erv468

This study of the SUMO proteases OVERLY TOLERANT TO SALT1 and -2 (OTS) is a real pan-UK collaboration that features researchers from six institutions, led by Ari Sadanandom at Durham. The OTS proteins have been previously linked to salicylic acid (SA) signaling and this manuscript shows that in addition to containing higher level of SA, ots1ots2 double mutants are more resistant to virulent Pseudomonas syringae. This is in part linked to an upregulation of the SA biosynthetic gene ICS1. In wildtype plants SA promotes degradation of OTS1/2, which indicates that these proteins are involved in a positive feedback loop that ensures a higher SA response, which increases the efficacy of certain defence responses. However de novo synthesis of OTS1/2 will be antagonistic to SA biosynthesis and provides a brake to prevent the over-accumulation of SA-responses.

Kumar M, Turner S (2015) Protocol: a medium-throughput method for determination of cellulose content from single stem pieces of Arabidopsis thaliana Plant Methods. 11:46. http://dx.doi.org/10.1186/s13007-015-0090-6

Simon Turner (Manchester) is the lead author of this paper that presents a new method for determining cellulose content from Arabidopsis stems. This protocol is an adaptation of a previous method and uses aspiration rather than centrifugation for recovery of liquids throughout the procedure. This increases the throughout of the method and improves its potential usage as a screening protocol to identify mutants with altered cell wall composition.

Arabidopsis Research Roundup: October 20th

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Published on: October 20, 2015

There are just three research papers in this weeks Arabidopsis Roundup but they each represent important projects from established groups. Firstly is a significant output from the Edinburgh SynthSys Centre that documents their analysis of the Arabidopsis circadian clock. Secondly an international collaborative effort looks into the molecular signaling pathways that control the physiological response to increasing CO2 levels and thirdly a paper that uncovers a novel plant-specific molecular mechanism that controls the biogenesis of certain siRNAs. Finally we highlight a major review concerning the importance of Arabidopsis research over the past 50 years.

Flis A, Fernández AP, Zielinski T, Mengin V, Sulpice R, Stratford K, Hume A, Pokhilko A, Southern MM, Seaton DD, McWatters HG, Stitt M, Halliday KJ, Millar AJ (2015) Defining the robust behaviour of the plant clock gene circuit with absolute RNA timeseries and open infrastructure. Open Biol. 5(10). pii: 150042. http://dx.doi.org/10.1098/rsob.150042

This study of the Arabidopsis circadian clock, impressive in its breadth, is led by faculty members from the University of Edinburgh SynthSys Synthetic Biology Centre. The team measured RNA profiles of clock genes in plants grown with or without exogenous sucrose or from wildtype or mutant soil growth plants. They found surprisingly robust patterns of expression together with some novel genetic behaviours. In addition they discovered major differences in the absolute expression of certain clock genes, ranging from 50 up to 1500 copies/ cell. Importantly this information is freely-available within the BioDare repository and it is hoped that this will benefit future attempts at modeling the circadian clock.

Chater C, Peng K, Movahedi M, Dunn JA, Walker HJ, Liang YK, McLachlan DH, Casson S, Isner JC, Wilson I, Neill SJ, Hedrich R, Gray JE, Hetherington AM (2015) Elevated CO2-Induced Responses in Stomata Require ABA and ABA Signaling Curr Biol. pii: S0960-9822(15)01092-1. http://dx.doi.org/10.1016/j.cub.2015.09.013

This broad collaboration between UK, German and Chinese researchers is led by Alistair Hetherington (Bristol) and Julie Gray (Sheffield) and looks into the molecular events that respond to changing levels of CO2, specifically in guard cells. The new findings in this manuscript show that reduction in stomatal density in response to higher [CO2] relies on the production of reactive oxygen species (ROS), adding a new element to this signaling pathway. In addition they show that the ABA response pathway is also involved in this process and that, following genetic analysis, the CO2 response is mediated via this hormone pathway. However it is unclear whether this is due to ABA increasing CO2 sensitivity in this system or whether CO2 acts specifically in guard cells to increase ABA biosynthesis. A plants response to CO2 is ancestral in evolutionary terms so the authors suggest that this link with ABA signaling is similarly ancient.

CO2Pic

Zhai J, Bischof S, Wang H, Feng S, Lee TF, Teng C, Chen X, Park SY, Liu L, Gallego-Bartolome J, Liu W, Henderson IR, Meyers BC, Ausin I, Jacobsen SE (2015) A One Precursor One siRNA Model for Pol IV-Dependent siRNA Biogenesis. Cell. 163(2):445-55 http://dx.doi.org/10.1016/j.cell.2015.09.032

GARNet Advisory Board member Ian Henderson is an author in this rare plant-focused paper in Cell, in work that results from his post-doc with Steve Jacobson at UCLA. The manuscript describes a novel mode of action surrounding the plant-specific RNA Polymerase IV (Pol IV). RNAs generated from the activity of Pol IV play an important role in RNA-directed DNA methylation. Intriguingly the authors found that Pol IV transcripts are surprisingly short, just 30 to 40 nt and are similarly adundant to the siRNAs that they subsequently form. The Pol IV RNAs exhibit transcriptional start points similar to those generated by Pol II, which might indicate there are similar mechanisms that control their activity. In addition they find that methylated DNA plays a role in locally reinforcing the silencing reaction. Overall this indicates that the transcripts produced by Pol IV go through a unique “one precursor, one siRNA” model, although the physiological significance of this remains opaque. Another paper on this topic is presented in ELife by the lab of Craig Pikaard.

PolIVpic

Provart et al (2015) 50 years of Arabidopsis research: highlights and future directions New Phytol. http://dx.doi.org/10.1111/nph.13687

Also worth noting this week is a Tansley Review in New Phytologist, which coincides with 50 years since the inaugural Arabidopsis Conference held in 1965. This review has been written by a number of senior Arabidopsis researchers, although no-one from the UK, to discuss the many important findings that have resulted from work on our favourite organism.

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