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.

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).

Figure1

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. http://dx.doi.org/10.1111/pbi.12497 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 http://dx.doi.org/10.1104/pp.15.00448 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 http://dx.doi.org/10.1105/tpc.15.00562

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

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.

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