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. 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. 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 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.

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.

Characterisation of Genetic Parts for Plant SynBio

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Published on: November 24, 2015

Paper Review: Quantitative characterization of genetic parts and circuits for plant synthetic biology. Nature Methods (2015) doi:10.1038/nmeth.3659

Professor June Medford andd Dr Ashok Prasad (Colorado State) have been in the vanguard of the development of tools for plant synthetic biology over the past few years. Here we highlight a recently published article in Nature Methods that presents their quantification of genetic parts and circuits designed for plant synthetic biology.

To date, quantitatively defined gene circuits have been almost exclusively characterised in unicellular organisms. Some of the initial challenges in transferring this characterisation to multi-cellular organisms includes use of orthogonally useful parts, that are active irrespective of developmental or organismal context, as well as developing methods for the quantification of input and output characteristics. This latter aim is hindered by the difficulties of plant transformation that adds significant levels of variation to this process. In this study the authors aimed to overcome some of these challenges by characterising parts using a medium-throughout method that analyses luciferase expression in protoplasts all within in a 96-well plant format.

This set of synthetic parts focused on the analysis of different repressive elements fused to a constitutive promotor region. These repressors were designed with orthogonal activity in mind and included both previously characterised and novel elements (Figure 1a). The output utilised a dual luciferase assay where one species of the Luc enzyme was constitutively expressed and compared to the expression level that was influenced by the designed repressor region (Figure 1b). This allowed tuneable expression that is dependent on the type of repressor elements that were present, all within the context of high levels of initial constitutive expression. In total, 128 different promotor combinations were tested in an Arabidopsis protoplast expression system. These expression levels were further varied by altering the levels of induction provided by two different compounds (DEX and OHT).


Figure 1 from Nature Methods

Initial experimentation generated significant ‘noise’, which is not ideal for generating predictable tools for use in synthetic biology. By analyzing a number of possible sources of experimental variation they found that the ‘batch effect’ was most significant, which described the differences between protoplast samples prepared on different days (attempts to resolve the precise aspect of the experimental technique that caused the variation were unsuccessful). Although it is somewhat harkening to know that the transformation efficiency or uptake of chemical inducers does not contribute as much to experimental variation, it is somewhat worrying that the fundamental preparation of protoplasts provided the highest amount of variation. However the authors were able to develop a mathematical model tat they used in their analysis that accounted for the ‘batch effect’. Ultimately the authors found that the repressor elements worked as expected but that some molecular motives acted more predictably in different pairings, the details of which can be found in the paper.

The authors then attempted to characterise a similar set of constructs in protoplasts generated from the monocot Sorghum. Although a slight alteration was made to the 5’ UTR region of the construct, they found that using the same normalization model to remove the batch effect generated a similar set of results as in the Arabidopsis protoplasts. This indicates that Arabidopsis can act as a good model for evaluating initial expression levels that might occur in other dicots or monocots.

Finally three different promotor-repressor genetic circuits were stably transformed into Arabidopsis and then multiple independent transgenic lines were isolated for each combination. Subsequently one transgenic line from each of three genetic combinations was chosen for comparison with the transient expression system. In a method outlined in Figure 2, protoplasts from the stably transformed plants were compared with those isolated from wildtype plaFigure2nts that were transiently transformed with the same construct. Although absolute expression was higher in the transiently transformed protoplasts, analysis of normalized data demonstrated that equivalent repressor elements performed similarly in the differently generated samples. This allowed the authors to suggest that use of the transient system was a reasonable proxy for that observed in stably transformed plants. Therefore they conclude that the transient system can be used as a more rapid screening procedure for newly developed synthetic elements.

This is an important piece of analysis that is absolutely necessary before the full potential of plant synthetic biology can be realised. This type of design, build, test procedure is common-place when using a unicellular chassis but has not been studied in this detail in a plant system.

However the amount of normalization that is required to ensure the data is directly comparable certainly highlights difficulties may lie ahead as researchers work toward the ultimate goal of developing synthetic genetic elements that are usable across many multi-cellular plant chassis. Although the GARNet blog has highlighted some excellent tools that have been recently developed for plant synthetic biology, this study acts as a cautionary tale for those entering this field and are unaware of the unpredictable nature of even the most commonly used experimental techniques.

Overall there is little doubt that this is an exciting time for plant synthetic biology even though the generation of predictable, consistent gene expression across a variety of chassis will remain rather challenging in the immediate future.

Arabidopsis Research Roundup: November 13th.

This weeks Arabidopsis Research Roundup presents a wide range of topics from researchers across the UK. Firstly we highlight a study that documents the early stages of a potential biotechnological/synthetic biology approach to improve higher plant photosynthesis using algal components. Corresponding author Alistair McCormick also takes five minutes to discuss this work. Secondly a team based mostly at Bath introduces the function of the PAT14 gene, which is involved in S-palmitoylation. Thirdly is a study that successfully transfers SI components between evolutionary diverged plant species and the final paper documents research that adds additional complexity to the signalling pathway that responses to strigolactones.

Atkinson N, Feike D, Mackinder LC, Meyer MT, Griffiths H, Jonikas MC, Smith AM, McCormick AJ (2015) Introducing an algal carbon-concentrating mechanism into higher plants: location and incorporation of key components. Plant Biotechnol J. Open Access

This work results from a collaborative effort between the four groups that make up the Combining Algal and Plant Photosynthesis (CAPP) consortium and include Howard Griffiths (Cambridge), Martin Jonikas (Carnegie Institute for Science), Alison Smith (JIC) and Alistair McCormick (Edinburgh). Here they attempt to express in higher plants a range of algal proteins that are involved in carbon-concentrating mechanisms (CCM). They initially confirmed the intracellular locations of ten algal CCM components and showed that these locations were largely conserved when the proteins were expressed transiently in tobacco or stably in Arabidopsis. Although the expression of these CCMs components in Arabidopsis didn’t enhance growth, the authors suggest that stacking of multiple CCM proteins might be needed to confer an increase in productivity.

Alistair takes five minutes to discuss this paper here:

Li Y, Scott RJ, Doughty J, Grant M, Qi B (2015) Protein S-acyltransferase 14: a specific role for palmitoylation in leaf senescence in Arabidopsis. Plant Physiology Open Access

This Southwest-based study is led by Baoxiu Qi from the Plant-Lab at Bath University with input from Murray Grant (Exeter). They investigate Protein S-Acyl Transferase (PATs) protein, which are multi-pass transmembrane proteins that catalyze S-acylation (commonly known as S-palmitoylation). This process both confers correct protein localisation and is involved in signalling. These are 24 PATs in Arabidopsis and this study focuses on the novel PAT14, which they show has its predicted enzymatic role. Pat14 mutant plants show accelerated senescence that is associated with SA, but not JA or ABA-signaling. Therefore the authors suggest that AtPAT14 plays a pivotal role in regulating senescence via SA pathways and that this is the first published linkage between palmitoylation and leaf senescence.

Lin Z1, Eaves DJ1, Sanchez-Moran E1, Franklin FC1, Franklin-Tong VE1 (2015) The Papaver rhoeas S determinants confer self-incompatibility to Arabidopsis thaliana in planta Science 350(6261):684-7 http:/​/​dx.​doi.​org/​10.1126/science.aad2983

University of Birmingham researchers led by Noni Franklin- Tong publish this study in Science in which they transfer the elements that confer self-incompatibility (SI) in Papever rhoeas (Poppy) to Arabidopsis. They find that Arabidopsis pistils that express the self-determinant PrsS protein reject pollen that expresses the PrpS protein. This leads to a robust SI response in these plants, demonstrating that these two components are sufficient for the establishment of this interaction. Poppy and Arabidopsis are evolutionarily separated by 140million years so the authors suggest that the successful transfer of SI determinants between these divergent species will have potential utility in future crop production strategies.

Soundappan I, Bennett T, Morffy N, Liang Y, Stanga JP, Abbas A, Leyser O, Nelson DC (2015) SMAX1-LIKE/D53 Family Members Enable Distinct MAX2-Dependent Responses to Strigolactones and Karrikins in Arabidopsis The Plant Cell

Ottoline Leyser (SLCU) is the UK lead on this US-UK collaboration that investigates the plant response to butenolide signals, namely the plant hormone strigolactones and smoke-derived karrikins. It is known that these molecules are perceived by the F-box protein MORE AXILLARY GROWTH2 (MAX2) and that the Arabidopsis SUPPRESSOR OF MAX2 1 (SMAX1) protein acts downstream of this perception. This study documents an extensive genetic study that shows that the activity of the SMAX1-LIKE genes, SMXL6, SMXL7, and SMXL8 promote shoot branching. smxl6,7,8 mutant plants suppress several strigolactone-related phenotypes in max2, that focus on the response to auxin but not on germination or hypocotyl elongation responses, which are only suppressed in smax1 mutants. On a molecular level these responses are controlled by the MAX2-dependant degradation of the SMAX1/SMXL proteins, which result in changes in gene expression. Therefore this shows that the diversity of SMAX1/SMXL proteins allows the signaling pathway that responses to butenolide signals to bifurcate downstream of the initial perception.

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 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 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. Open Access

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

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

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.

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.

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.

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.

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.

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.

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.


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

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.


Provart et al (2015) 50 years of Arabidopsis research: highlights and future directions New Phytol.

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.

Variation in Agroinflitration

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Published on: October 19, 2015

Agroinflitration of Nicotiana benthamiana is an increasingly important technique that many plant scientists may use for transient expression of their gene of interest. These experiments might include localisation or interaction studies, monitoring of pathogen effects or simply for protein production where, in extreme cases, up to 50% of the total soluble protein will be expressed by the transgene.

As a previous user of this technique, I know that many aspects of early test experiments can be undertaken haphazardly, with little understanding or expectation regarding the optimum agrobacterium concentration, best leaf segment or inoculation timings to use. There also seems to be significant variation within the same inoculated leaves, between leafs on the same plant and especially so between leaves from different plants. Therefore it is satisfying to read a recent paper published in Plant Methods from the lab of Teemu Teeri (University of Helsinki) that attempts to provide some understanding as to the sources of variation in these infiltration experiments [1].

They conduct a simple set of experiments whereby they use an intron-containing luciferase gene as a reporter to test the factors that most influenced the infiltration efficiency. They inoculated the upper leaves of 6-week old tobacco plants with either luciferase driven by the constitutive 35S promoter or by an estradiol-inducible promoter. They showed that estradiol induced an eight-fold increase in enzyme production and that these induced levels were similar to those observed with the constitutive promoter.
In order to assess variation within experiments they compared samples from the same leaf, between leaves from the same plant and between leaves from different plants. Importantly they used a nested experimental design so that they could compare the amount of variation generated between hierarchical levels of infiltration, sampling or enzyme activity.

Somewhat surprisingly they found that the largest component of variation in these experiments was between disks on the same leaf, whereas use of different leaves or different plants generated a 2.5x lower level of variation. Within disks from a single leaf, the amount of bacteria and protein showed little alteration, even though the luciferase enzyme activity was greatly different. The reason for this difference is unknown.

Therefore based on their results the authors make the following recommendations:

  1. Ensure tested plants are in the same physiological state.
  2. Combine the expression values from several sample disks on the same leaf, do not rely on one disk as a representative amount of expression on that leaf.
  3. Inflitrate more plants but less leaves on those plants. Sample more disks on each of the leaves that are used.
  4. Technical replicates (for luciferase assays) are not as important since these measurements are more reliable.

Good luck with your experiments!

1- Bashandy et al (2015) Plant Methods11:47 DOI: 10.1186/s13007-015-0091-5

Arabidopsis Research Roundup: October 12th

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Published on: October 11, 2015

The Arabidopsis Research Roundup is ‘defense-focused’ this week. We present three papers that highlight different aspects of plant immunity, two of which result from UK-US-China collaborations. Firstly a team from the Sainsbury Lab, Norwich looks at two molecular mechanisms that control stomatal closure. There are then two studies that involve University of Exeter researchers that investigate either the role of plant hormones in the response to bacterial pathogens or the role that the physical barrier of the cell wall plays in the prevention of infection. Next a group of JIC researchers present a Large Scale Biology investigation of microtubule interacting proteins. Finally a study from QMUL looks at the interaction between NPQ and photoinhibition in controlling the activity of Photosystem II.

Gou M, Zhang Z, Zhang N, Huang Q, Monaghan J, Yang H, Shi Z, Zipfel C, Hua J (2015) Opposing effects on two phases of defense responses from concerted actions of HSC70 and BON1 in Arabidopsis. Plant Physiol.

GARNet Advisory Board Member Cyril Zipfel is the UK lead on this US-China-UK collaboration that looks at two aspects of the plant immune response that are regulated by the same proteins, albeit in an antagonistic way. The heat shock protein HSC70 and the calcium binding protein BON1 both are involved in stomatal closure, the formers effect mediated by the SNC1 protein and the latter (BON1) via the activitation of SGT1 that in turn inhibits HSC70. These new functions demonstrate opposing roles for HSC80 and BON1 in the immune response and further highlight the complexity of the signaling pathways that ultimately feed into the gross phenotypic change of stomatal closure.

de Torres Zabala M, Zhai B, Jayaraman S, Eleftheriadou G, Winsbury R, Yang R, Truman W, Tang S, Smirnoff N, Grant M (2015) Novel JAZ co-operativity and unexpected JA dynamics underpin Arabidopsis defence responses to Pseudomonas syringae infection New Phytol.

This is another UK-USA-China collaboration led by Murray Grant at the University of Exeter, in which the role of the plant hormones is assessed in the response to bacterial pathogens. The defence response is mediated by both the hormones salicylic acid (SA) and jasmonic acid (JA) which antagnise many of each others activity. Pathogens have been shown to produce a JA-mimic cornatine (COR) in order to stall SA-mediated effects. In this study the authors use a systems-biology based approach that involved targeted hormone profiling, high-temporal-resolution micro-array analysis, reverse genetics and mRNA-seq to introduce a complex network of regulation that involves JAZ proteins, which are repressors of the JA signal. In short they show that JAZ5 and JAZ10 specifically co-operate to inhibit pathogen growth by restricting COR cytotoxicity by novel mechanisms, which do not involve previously well-defined signaling proteins.

Marcos R, Izquierdo Y, Vellosillo T, Kulasekaran S, Cascón T, Hamberg M, Castresana C (2015) 9-Lipoxygenase-derived oxylipins activate brassinosteroid signaling to promote cell wall-based defense and limit pathogen infection Plant Physiol.

This work was performed in Madrid under the supervision of Carmen Castresana but includes the work of Satish Kulasekaran who is now at the Exeter. The focus of the work is the oxylipins, which are oxygenated lipid derivatives that regulate plant development and immunity. Using a variety of noxy mutants (non-responding to oxylipins) they show that the effect of the oxylipins is mediated via changes in the cell wall and this is signalled via the Brassinosteriod response pathway. Suspectibility to bacterial and fungal infection was enhanced in noxy mutants but plants were resistance when BR signalling was switched on. Therefore this manuscript introduces an important interaction between the oxylipins and BR signalling and helps to clarify their role in modulating plant defense.

Derbyshire P, Ménard D, Green P, Saalbach G, Buschmann H, Lloyd CW, Pesquet E (2015) Proteomic Analysis of Microtubule Interacting Proteins over the Course of Xylem Tracheary Element Formation in Arabidopsis Plant Cell.

The experiments in this Large Scale Biology paper were performed in the lab of Clive Lloyd (John Innes Centre) which included the work of Eduoard Pesquet, who now has his own lab in Sweden. They looked the microtubule patterning that defines the nature of tracheary element (TEs) thickening in plant vascular tissues. They used Arabidopsis cell suspension culture to isolate microtubule interacting proteins present during TE differentiation. One protein of interest was CELLULOSE SYNTHASE-INTERACTING PROTEIN1, associated with primary wall synthesis, which was enriched during secondary cell wall formation of TEs. The authors knocked-down the expression of some of their identified proteins and indeed showed that they were important for this differentiation. A take-home message is that the proteins that interact with microtubules and link them to different metabolic compartments do indeed specifically vary during TE differentiation, regulating different aspects of cell wall patterning.

Giovagnetti V, Ruban AV (2015) Discerning the effects of photoinhibition and photoprotection on the rate of oxygen evolution in Arabidopsis leaves J Photochem Photobiol B.

Arabidopsis Research Roundup Regular Alexandre Ruban (QMUL) again looks at the mechanisms that lessen the amount of photoinhibition (when photosystem II is damaged by being exposed to too much light). An opposing response is Non-photochemical quenching (NPQ) of chlorophyll a fluorescence which serves to protect PSII from high light conditions. In this study they confirm that a recently devised procedure that aims to discern between the effect of NPQ and photoinhibition works well as a measurement for the efficiency of PSII activity.

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