GARNet Research Roundup: November 22nd 2019

by Geraint
Categories: Cambridge University, Durham University, Glasgow University, Global, John Innes Centre, Lancaster Unversity, Nottingham University, SLCU, Uncategorized, University of Birmingham, University of Cambridge, University of Manchester, University of Nottingham, University of York
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Published on: November 22, 2019

This bumper edition of the GARNet Research Roundup begins with three papers that have a focus on the circadian clock. First is from Cambridge and looks at a novel role for TTG1 in control of the clock. The second paper also includes co-authors from Cambridge and looks at the clock Evening Complex. The final clock paper includes co-authors from York and looks at the new roles for EARLY FLOWERING 3 and GIGANTEA.

The next four papers include researchers from the John Innes Centre. Yiling Ding’s lab lead an exciting study into the role of RNA G-quadruplex to define liquid-liquid phase separations. Next David Seung and Alison Baker look at production of amylose starch across Arabidopsis accessions. The third JIC paper is from the Charpentier lab and looks at nuclear calcium signaling in the root. Finally Lars Ostergaard is a co-author on a paper that identifies a novel biostimulant that controls podshatter in Brassica.

The eighth paper is from Glasgow and describes the bioengineering of plants to express a novel antibiotic bacteriocin.

Next are three papers introduce exciting new research tools. 1. Weibei Yang in the Meyerowitz lab introduces a method for co-labeling of RNAs and protein 2. Researchers in Nottingham introduce RootNav2.0 for the automated measurement of root archtiectures 3. The Haydon Lab has developed a GAL4-GFP luciferase system for tissue-specific gene expression analysis.

Two Photosynthesis-based papers come next with firstly an analysis on the link between metabolism and the light response curve (from Manchester) and secondly a look at the role of aquaporins in control of CO2 conductance (Cambridge and Lancaster).

The fourteenth paper is from Durham and characterises an important protein regulator of the autophagy-dependent degradation pathway whilst the fifteenth is from Cambridge and uses cryo-SEM to analyse cell wall structures.

The penultimate paper is from Birmingham and looks at the role of redox signaling in aphid fecundity and the final paper includes co-authors from RHUL and looks at the interaction between the E2FB and RETINOBLASTOMA-RELATED proteins.

Airoldi CA, Hearn TJ, Brockington SF, Webb AAR, Glover BJ (2019) TTG1 proteins regulate circadian activity as well as epidermal cell fate and pigmentation. Nat Plants. doi: 10.1038/s41477-019-0544-3

This study from the University of Cambridge is led by Chiara Airoldi and introduces a new role for the TRANSPARENT TESTA GLABRA 1 (TTG1) WD-repeat (WDR) subfamily in the regulation of the circadian clock. TTG1 regulates epidermal cell differentiation and pigment production, while LIGHT-REGULATED WD1 and LIGHT-REGULATED WD2A are known to regulate the clock. The triple lwd1 lwd2 ttg1 mutant has no detectable circadian rhythym. This suggests that members of this protein family have undergone subfunctionalization to diverge from their core functions. This paper is of interest to those who research evolution of protein function as well as the to those interested in the control of the circadian clock.

https://www.nature.com/articles/s41477-019-0544-3

Tong M, Lee K, Ezer D, Cortijo S, Jung J, Charoensawan V, Box MS, Jaeger K, Takahashi N, Mas P, Wigge PA, Seo PJ (2019) The Evening Complex establishes repressive chromatin domains via H2A.Z deposition. Plant Physiol. doi: 10.1104/pp.19.00881

This collaboration between the UK and South Korea is led by Meixuezi Tong and investigates how the Evening Complex (EC) component of the circadian clock interacts with chromatin to control gene expression at dusk. This occurs through direct interaction with the SWI2/SNF2-RELATED complex and together they bind to the core clock genes PRR7 and PRR9, causing the deposition of H2A.Z at these loci subsequent to causing their repression at dusk.

Anwer MU, Davis A, Davis SJ, Quint M (2019) Photoperiod sensing of the circadian clock is controlled by EARLY FLOWERING 3 and GIGANTEA. Plant J. doi: 10.1111/tpj.14604

Amanda Davies and Seth Davies from the University of York are co-authors on this German-led study with Muhammad Anwer as both first and corresponding author. They look at the role of important circadian regulators ELF3 and GIGANTEA through generation of previously unanalysed elf3gi double mutants. In these plants the circadian oscillator fails to synchronize to light-dark cycles even under diurnal conditions, demonstrating that these genes act together to convey photoperiod sensing to the central oscillator.

Zhang Y, Yang M, Duncan S, Yang X, Abdelhamid MAS, Huang L, Zhang H, Benfey PN, Waller ZAE, Ding Y (2019) G-quadruplex structures trigger RNA phase separation. Nucleic Acids Res. doi: 10.1093/nar/gkz978
Open Access

Yueying Zhang is the first author of this study conducted in the lab of Yiliang Ding at the John Innes Centre, in collaboration with the Benfey lab in the USA. They reveal an exciting mode of regulating RNA activity through the formation of RNA G-quadruplex (GQ) complexes. They use the SHORTROOT mRNA as the model for this study, showing that GQ-mediated complex formation can bring liquid-liquid phase separation. This study is of fundamental importance as it provides the first evidence that RNA can adopt structural motifs to trigger and/or maintain the specificity of RNA-driven phase separation.

https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkz978/5624975?guestAccessKey=d3913912-fdbb-4f35-aa71-625442722842

Seung D, Echevarría-Poza A, Steuernagel B, Smith AM (2019) Natural polymorphisms in Arabidopsis result in wide variation or loss of the amylose component of starch. Plant Physiol. doi: 10.1104/pp.19.01062
Open Access

David Seung at the John Innes Centre the first and corresponding author of this study that used data from the Arabidopsis 1135 Genome project to investigate the prevelance of amylose production. Plants with amylose-free starch have no detrimental phenotypes so the function of this glucose-polymer, that accounts for up to 30% of all natural starch, is unknown. They looked at the polymorphisms within the GRANULE-BOUND STARCH SYNTHASE (GBSS) enzyme, identifying natural accessions that have no GBSS activity yet are viable within their natural environments. This study is a prelude to future research that will discover the adaptive significance of amylose.

Leitão N, Dangeville P, Carter R, Charpentier M (2019) Nuclear calcium signatures are associated with root development. Nat Commun. doi: 10.1038/s41467-019-12845-8
Open Access

Nuno Leitao is first author on this research from the Charpentier lab at the John Innes Centre. They looked at the role of nuclear Ca2+ signalling on primary root meristem development and auxin homeostasis through activity of the nuclear membrane localised ion channel DOES NOT MAKE INFECTIONS 1 (DMI1). This study discovers a previously unappreciated role for intracellular Ca2+ signalling during plant development.

Łangowski Ł, Goñi O, Quille P, Stephenson P, Carmody N, Feeney E, Barton D, Østergaard L, O’Connell S (2019 A plant biostimulant from the seaweed Ascophyllum nodosum (Sealicit) reduces podshatter and yield loss in oilseed rape through modulation of IND expression. Sci Rep. doi: 10.1038/s41598-019-52958-0
Open Access

Lars Ostergaard is a co-author on this Irish-study led by Lukasz Łangowski that investigates the factors that control pod shatter in oil seed rape. They show that the seaweed Ascophyllum nodosum-based biostimulant (Sealicit) is able to reduce podshatter by effecting the expression of the major regulator of pod shattering, INDEHISCENT. This has implications for the use of this compound by farmers wanting to reduce the amount of seed loss due to premature pod shatter.

Rooney WM, Grinter RW, Correia A, Parkhill J, Walker DC, Milner JJ (2019) Engineering bacteriocin-mediated resistance against the plant pathogen Pseudomonas syringae. Plant Biotechnol J. doi: 10.1111/pbi.13294
Open Access

William Rooney at the University of Glasgow is lead author on this study that attempts to combat Pseudomonas syringae infections through expression of a novel protein antibiotic bacteriocin, putidacin. They show that transgenic expression of this bacterial protein provides effective protection against Pseudomonas. This proof of concept opens the possibility for more widespread use of bacteriocins as an effective plant protection strategy.

https://onlinelibrary.wiley.com/doi/epdf/10.1111/pbi.13294

Yang W, Schuster C, Prunet N, Dong Q, Landrein B, Wightman R, Meyerowitz EM (2019) Visualization of Protein Coding, Long Non-coding and Nuclear RNAs by FISH in Sections of Shoot Apical Meristems and Developing Flowers. Plant Physiol. doi: 10.1104/pp.19.00980
This extended methods paper is led by Weibing Yang at the Sainsbury lab in Cambridge. They have adapted RNA fluorescence in situ hybridization (rnaFISH) to explore RNA localization in the shoot apical meristem of Arabidopsis. They are able to label mRNA as well as long ncRNAs and have developed double labeling to assay two separate RNAs in the same cell and to assess nucleo-cytoplasmic separation of RNA species. Finally they link rnaFISH with fluorescence immunocytochemistry for the simultaneous localization of a single genes mRNA and protein.

https://www.frontiersin.org/articles/10.3389/fpls.2019.01398/full

Yasrab R, Atkinson JA, Wells DM, French AP, Pridmore TP, Pound MP (2019) RootNav 2.0: Deep learning for automatic navigation of complex plant root architectures. Gigascience. doi: 10.1093/gigascience/giz123
Open Access

Robail Yasrab is lead author on this work from the University of Nottingham that introduces the RootNav2.0 software tool. This was developed by modern deep-learning approaches and allows the fully automated measurement of vertically growth root systems. RootNav2.0 was favourably compared with its semi-automated predecessor RootNav1.0 and can be used for measurement of root architectures from a range of different plant species.

Román Á, Golz JF, Webb AA, Graham IA, Haydon MJ (2019) Combining GAL4 GFP enhancer trap with split luciferase to measure spatiotemporal promoter activity in Arabidopsis. Plant J. doi: 10.1111/tpj.14603

This technical advance is led by Angela Roman, was in the Haydon lab during its time at the University of York. They have used the GAL4-GFP enhancer trap system, to develop a tissue-specific split luciferase assay for non-invasive detection of spatiotemporal gene expression in Arabidopsis. In this example they use the study to measure dynamics of circadian gene expression but is clearly applicable to answer many other experimental questions.

Herrmann HA, Schwartz JM, Johnson GN (2019) From empirical to theoretical models of light response curves – linking photosynthetic and metabolic acclimation. Photosynth Res. doi: 10.1007/s11120-019-00681-2
Open Access

Helena Herrmann is lead author on this work fro the University of Manchester. In this study they developed and then empirically tested a series of simple kinetic models that explains the metabolic changes that are required to alter light response curves (LRCs) across a range of temperatures. This allowed them to show how changes in NADPH and CO2 utilization respond to environmental changes. This provides useful information as to how a plant adapts its metabolic response to light depending on the growth temperature.

Helena explaining her research

Kromdijk J, Głowacka K, Long SP (2019) Photosynthetic efficiency and mesophyll conductance are unaffected in Arabidopsis thaliana aquaporin knock-out lines. J Exp Bot. doi: 10.1093/jxb/erz442

Open Access
Wanne Kromdijk leads this US-led research that includes contributions from the Universities of Cambridge and Lancaster. They looked at the potential role of membrane-bound aquaporins in the control of diffusion conductance for CO2 transfer from substomatal cavity to chloroplast stroma (gm). They tested three aquaporin mutants across a range of light and CO2 concentrations and surprisingly found that they appear to play no significant contribution to the control of gm. The reporting of this type of ‘negative’ result will prevent unnecessary replication of experiments and help to streamline the research process.

Wang P, Pleskot R, Zang J, Winkler J, Wang J, Yperman K, Zhang T, Wang K, Gong J, Guan Y, Richardson C, Duckney P, Vandorpe M, Mylle E, Fiserova J, Van Damme D, Hussey PJ (2019) Plant AtEH/Pan1 proteins drive autophagosome formation at ER-PM contact sites with actin and endocytic machinery. Nat Commun. doi: 10.1038/s41467-019-12782-6
Open Access

Pengwei Wang is first author in this research led from Durham University that incudes Chinese and Belgian collaborators. They show that the AtEH/Pan1 protein is involved with actin cytoskeleton regulated autophagy and recruits multiple other components to autophagosomes during this process. In addition they show vesicle bound-AtEH/Pan1 interact with VAP27-1 at the ER-PM. This demonstrates that AtEH/Pan1 is a key component of the autophagy-dependent degradation pathway.

Lyczakowski JJ, Bourdon M, Terrett OM, Helariutta Y, Wightman R, Dupree P (2019) Structural Imaging of Native Cryo-Preserved Secondary Cell Walls Reveals the Presence of Macrofibrils and Their Formation Requires Normal Cellulose, Lignin and Xylan Biosynthesis. Front Plant Sci. doi: 10.3389/fpls.2019.01398
Open Access

Jan Lyczakowski from the Dupree lab at the University of Cambridge is first author on this study that has adapted low temperature scanning electron microscopy (cryo-SEM) to visualize the cell walls of both angiosperm and gymnosperms. They have used Arabidopsis mutants to reveal that cell wall macrofibrils at composed of cellulose, xylan, and lignin. They demonstrate that cryo-SEM is a useful tool for native nanoscale cell wall architectures.

https://www.frontiersin.org/articles/10.3389/fpls.2019.01398/full

Rasool B, Karpinska B, Pascual J, Kangasjärvi S, Foyer CH (2019) Catalase, glutathione and protein phosphatase 2A-dependent organellar redox signalling regulate aphid fecundity under moderate and high irradiance. Plant Cell Environ. doi: 10.1111/pce.13669
Brwa Rasool is first author on this collaboration between the Universities of Birmingham and Helsinki that looks at how aphids respond to redox changes in Arabidopsis thaliana grown under different light conditions. They also identified defence-related transcription factors differentially upregulated by aphid predation in different light conditions. Overall they show aphid fecundity is in part determined by the plants cellular redox signaling.

Őszi E, Papdi C, Mohammed B, Pettkó-Szandtner A, Vaskó-Leviczky T, Molnár E, Ampudia CG, Khan S, Lopez-Juez E, Horváth B, Bögre L, Magyar Z (2019) E2FB interacts with RETINOBLASTOMA RELATED and regulates cell proliferation during leaf development. Plant Physiol. doi: 10.1104/pp.19.00212
Erika Oszi is first author of this Hungarian-led research that includes co-authors from Royal Holloway University of London. This research looks at the interaction between the transcription factors E2FB and RETINOBLASTOMA-RELATED (RBR) and how this contributes to cell proliferation during organ development in Arabidopsis leaves. The relationship between these proteins changes throughout the stages of leaf development and is critical to determine final leaf cell number.

http://www.plantphysiol.org/content/early/2019/11/06/pp.19.00212.long

GARNet Research Roundup: November 1st 2019

by Geraint
Categories: Cantebury University, Global, John Innes Centre, NIAB, Rothamsted Research, Uncategorized, University of Cambridge, University of Leicester, Warwick University
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Published on: November 1, 2019

This edition of the GARNet Research Roundup begins with a pan-UK study that has identified a gene involved in starch granule formation in polyploid wheat. Second is a study from Canterbury that identifies Arabidopsis QTLs involved in alternative splicing. Third is research from Cambridge that investigates the role of the nuclear circadian oscillator on sub-cellular calcium fluctuations. The fourth paper describes the development of a computer-vision tool designed for automated measurements of wheat spikes in the field. The fifth paper is a Korean-led study that has identified a transcription factor involved in pollen development and includes co-authors from Leicester. Last is a study from the University of Warwick that has looked into light-regulated gene expression during bulb initiation in onion.

Chia T, Chirico M, King R, Ramirez-Gonzalez R, Saccomanno B, Seung D, Simmonds J, Trick M, Uauy C, Verhoeven T, Trafford K (2019) A carbohydrate-binding protein, B-GRANULE CONTENT 1, influences starch granule size distribution in a dose-dependent manner in polyploid wheat. J Exp Bot. doi: 10.1093/jxb/erz405
Open Access

Tansy Chia is lead author on this study that brings together three of the UKs major plant breeding research centres; NIAB, Rothamsted and the JIC. They take advantage of the new genomic tools and mutant populations available in wheat to characterize the complex role of the BGC1 (B-GRANULE CONTENT 1) gene during formation of B-type starch granules.

https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/erz405/5601560

Khokhar W, Hassan MA, Reddy ASN, Chaudhary S, Jabre I, Byrne LJ, Syed NH (2019) Genome-Wide Identification of Splicing Quantitative Trait Loci (sQTLs) in Diverse Ecotypes of Arabidopsis thaliana Front Plant Sci. doi: 10.3389/fpls.2019.01160
Open Access

This work from Canterbury Christ Church University is led by Waqas Khokhar and Naeem Syed. They analysed 666 diverse Arabidopsis ecotypes to look for splicing quantitative trait loci (sQTLs)] that alter rates of alternative splicing. They identified a number of trans-sQTLs hotspots that align with known functional SNPs. This study provides the first sQTL resource across diverse ecotypes that can be used to compliment other available genome and transcriptome datasets.

Martí Ruiz MC, Jung HJ, Webb AAR (2019) Circadian gating of dark-induced increases in chloroplast- and cytosolic-free calcium in Arabidopsis. New Phytol. doi: 10.1111/nph.16280

María Carmen Martí Ruiz is lead author on this research undertaken in Alex Webb’s lab in Cambridge. They have looked at the role of the circadian clock in the control of calcium fluctuations in both cytoplasm and chloroplast stroma. They show the extent these changes are dependent on a nuclear-encoded circadian oscillator, adding a new role in sub-cellular Ca2+ signaling to the circadian machinery.

Sadeghi-Tehran P, Virlet N, Ampe EM, Reyns P, Hawkesford MJ (2019) DeepCount: In-Field Automatic Quantification of Wheat Spikes Using Simple Linear Iterative Clustering and Deep Convolutional Neural Networks. Front Plant Sci. doi: 10.3389/fpls.2019.01176
Open Access

Pouria Sadeghi-Tehran leads this theorectical study from Rothamsted Research that has developed an automated ‘DeepCount’ system for quantifying wheat spikes in the field. They use a deep convolutional neural network to test their program on field images and compare this method to other automated systems based on edge detection techniques and morphological analysis. Overall they show that this method has potential toward development of a portable and smartphone-assisted wheat-ear counting systems, that will have the associated benefits of counting accuracy and reduced labour.

https://www.frontiersin.org/articles/10.3389/fpls.2019.01176/full

Oh SA, Hoai TNT, Park HJ, Zhao M, Twell D, Honys D, Park SK (2019) MYB81, a microspore-specific GAMYB transcription factor, promotes pollen mitosis I and cell lineage formation in Arabidopsis. Plant J. doi: 10.1111/tpj.14564

Mingmin Zhao and David Twell are co-authors on this project led by Sung‐Aeong Oh and Korean colleagues. After screening pollen cell patterning mutants they have identified a role for the GAMYB transcription factor MYB81 during a narrow window prior to pollen mitosis I. They demonstrate that this protein is essential for establishing the male cell lineage in Arabidopsis pollen.

Rashid MHA, Cheng W, Thomas B (2019) Temporal and Spatial Expression of Arabidopsis Gene Homologs Control Daylength Adaptation and Bulb Formation in Onion (Allium cepa L.). Sci Rep. doi: 10.1038/s41598-019-51262-1 Open Access

This collaboration between the University of Warwick and Bangladesh Agricultural University is led by Harun Ar Rashid. They look at genetic regulation of light-dependent onion bulb initiation by growing plants under short and long days and testing the expression of known regulators of flowering time; AcFT, Ac LFY and AcGA3ox1. They also performed tissue-specific analysis to demonstrate differences in expression patterns that begin to suggest how these genes are involved in bulb initiation.

https://www.nature.com/articles/s41598-019-51262-1

GARNet Research Roundup: October 17th 2019

by Geraint
Categories: Bristol University, Cambridge University, Durham University, Edinburgh University, Global, James Hutton Institute, John Innes Centre, Nottingham University, Rothamsted Research, SLCU, Uncategorized, University of Bristol, University of Cambridge, University of Dundee, University of Edinburgh, University of Nottingham, University of Sheffield
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Published on: October 17, 2019

This edition of the GARNet Research Roundup includes a superb selection of papers by scientists from across the UK. First is work from the Spoel lab in Edinburgh that characterizes the fine-tuning of NPR1 activity during the plant immune response. Second is work from SLCU and the University of Helsinki that is an extensive investigation into the molecular basis of cambial development. Next is research from Nottingham that looks at the importance of soil macro-structures during the growth of wheat roots.

Fourth are three papers that highlight the breadth of research occurring at the John Innes Centre. The first paper is from Enrico Coen’s lab that applies their expertise in computational modeling to leaf development in the carnivorous plant Utricularia gibba. Second is work from Saskia Hogenhout’s lab that looks at immunity to infection by Phytoplasma pathogens. Last is work from Lars Ostergaard’s lab that characterises the role of Auxin Binding promoter elements in floral development.

The seventh paper from Bristol and Glasgow looks at shade avoidance signaling via PIF5, COP1 and UVR8 whilst the eighth paper, which is from Rothamsted, demonstrates how metabolic engineering in Arabidopsis seeds can result in a high proportion of human milk fat substitute. The next paper is from the University of Durham and investigates how the composition of the Arabidopsis cell wall impacts freezing tolerance. The first author of this paper, Dr Paige Panter discusses the paper on the GARNet community podcast.

The tenth paper is from Julia Davies’s lab at the University of Cambridge and introduces an uncharacterised response to extracellular ATP signals in Arabidopsis roots. The next paper is from Mike Blatt’s group at University of Glasgow and characterises a new interaction between vesicular transport and ion channels. The penultimate entry includes co-authors from the JIC on a Chinese-led study that demonstrates improved seed vigour in wheat through overexpression of a NAC transcription factor. Finally are two methods papers taken from a special journal issue on ‘Plant Meiosis’.

Skelly MJ, Furniss JJ, Grey HL, Wong KW, Spoel SH (2019) Dynamic ubiquitination determines transcriptional activity of the plant immune coactivator NPR1. Elife. doi: 10.7554/eLife.47005
Open Access

https://elifesciences.org/articles/47005

Michael Skelly is lead author on this paper from the lab of current GARNet chair Steven Spoel. In it they investigate the mechanisms that fine-tune the function of NPR1, a key player in the plant immune response. Progressive ubiquitination of NPR1 by an E3 ligase causes both its interaction with target genes and its subsequent degradation by an E4 ligase. This latter occurrence is opposed by the deubiquitinase activity of UBP6/7, setting up a complex regulatory environment that allows the plant to rapidly response to pathogen attack.

Zhang J, Eswaran G, Alonso-Serra J, Kucukoglu M, Xiang J, Yang W, Elo A, Nieminen K, Damén T, Joung JG, Yun JY, Lee JH, Ragni L, Barbier de Reuille P, Ahnert SE, Lee JY, Mähönen AP, Helariutta Y (2019) Transcriptional regulatory framework for vascular cambium development in Arabidopsis roots. Nat Plants. doi: 10.1038/s41477-019-0522-9

This pan-European-Korean collaboration has Jing Zhang from the University of Helsinki and the Sainsbury Laboratory, University of Cambridge as first author. They use cambium cell-specific transcript profiling and follow-on network analysis to discover 62 new transcription factors involved in cambial development in Arabidopsis. This information was used to engineer plants with increased radial growth through ectopic cambial activity as well as to generate plants with no cambial activity. This understanding provides a platform for possible future improvements in production of woody biomass.

Atkinson JA, Hawkesford MJ, Whalley WR, Zhou H, Mooney SJ (2019) Soil strength influences wheat root interactions with soil macropores. Plant Cell Environ. doi: 10.1111/pce.13659
This work is led from the University of Nottingham by John Atkinson and Sacha Mooney. They use X-ray Computed Tomography to investigate a trait called trematotropism, which applies to the ability of deep rooting plants to search out macropores and avoid densely packed soil. They show root colonisation of macropores is an important adaptive trait and that strategies should be put in place to increase these structures within the natural soil environment.

Lee KJI, Bushell C, Koide Y, Fozard JA, Piao C, Yu M, Newman J, Whitewoods C, Avondo J, Kennaway R, Marée AFM, Cui M, Coen E (2019) Shaping of a three-dimensional carnivorous trap through modulation of a planar growth mechanism. PLoS Biol. doi: 10.1371/journal.pbio.3000427
Open Access

https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3000427

Karen Lee, Claire Bushell and Yohei Koide are co-first authors on this work led by Enrico Coen at the John Innes Centre and Minlong Cui at the Zhejiang Agriculture and Forestry University in China. This study uses 3D imaging, cellular and clonal analysis, combined with computational modelling to analyse the development of cup-shaped traps of the carnivorous plant Utricularia gibba. They identify growth ansiotrophies that result in the final leave shape that develops from an initial near-spherical form. These processes have some similarities to the polar growth seen in Arabidopsis leaves. Overall they show that ‘simple modulations of a common growth framework underlie the shaping of a diverse range of morphologies’.

Pecher P, Moro G, Canale MC, Capdevielle S, Singh A, MacLean A, Sugio A, Kuo CH, Lopes JRS, Hogenhout SA (2019) Phytoplasma SAP11 effector destabilization of TCP transcription factors differentially impact development and defence of Arabidopsis versus maize. PLoS Pathog. doi: 10.1371/journal.ppat.1008035
Open Access

This work from Saskia Hogenhout’s lab at the John Innes Centre is led by Pascal Pecher and Gabriele Moro. They look at the effect of SAP11 effectors from Phytoplasma species that infect either Arabidopsis or maize. They demonstrate that although both related versions of SAP11 destabilise plant TCP transcription factors, their modes of action have significant differences. Please look out for Saskia discussing this paper on the GARNet Community podcast next week.

Kuhn A, Runciman B, Tasker-Brown W, Østergaard L 92019) Two Auxin Response Elements Fine-Tune PINOID Expression During Gynoecium Development in Arabidopsis thaliana. Biomolecules. doi: 10.3390/biom9100526
Open Access

Andre Kuhn is first author of this research from Lars Østergaard’s lab at the John Innes Centre. They functional characterise two Auxin-responsive Elements (AuxRE) within the promotor of the PINOID gene, which are bound by the ETITIN/ARF3 Auxin Response Factor. Alteration of this AuxRE causes phenotypic changes during flower development demonstrating that even with a complex regulatory environment, small changes to cis-elements can have significant developmental consequences.

Sharma A, Sharma B, Hayes S, Kerner K, Hoecker U, Jenkins GI, Franklin KA (2019) UVR8 disrupts stabilisation of PIF5 by COP1 to inhibit plant stem elongation in sunlight. Nat Commun. doi: 10.1038/s41467-019-12369-1
Open Access

https://www.nature.com/articles/s41467-019-12369-1

Ashutosh Sharma is first author of this UK-Spanish-Germany collaboration led by Keara Franklin at University of Bristol. They have characterised the interaction between three significant molecular players that function during the shade avoidance response in Arabidopsis; PIF5, UVR8 and COP1. In shaded conditions, UVR8 indirectly promotes rapid degradation of PIF5 through their interactions with the E3 ubiquitin ligase COP1.

van Erp H, Bryant FM, Martin-Moreno J, Michaelson LV, Bhutada G, Eastmond PJ (2019) Engineering the stereoisomeric structure of seed oil to mimic human milk fat. Proc Natl Acad Sci U S A. doi: 10.1073/pnas.1907915116

Open Access

https://www.pnas.org/content/early/2019/09/24/1907915116.long

Harrie Van Arp and Peter Eastmond at Rothamsted Research lead this extremely translational study in which they have modified the metabolic pathway for triacylglycerol (TAG) biosynthesis. By modifying the location of one biosynthesis enzyme and removing the activity of another, the fats produced in these Arabidopsis seeds are enriched to contain TAGs that are similar to those found in human milk. They propose that this technology could be used to develop a source of plant-derived human milk fat substitute.

Panter PE, Kent O, Dale M, Smith SJ, Skipsey M, Thorlby G, Cummins I, Ramsay N, Begum RA, Sanhueza D, Fry SC, Knight MR, Knight H (2019) MUR1-mediated cell-wall fucosylation is required for freezing tolerance in Arabidopsis thaliana. New Phytol. doi: 10.1111/nph.16209

Paige Panter led this work as part of her PhD at the University of Durham in the lab of Heather Knight. They characterise the role of the MUR1 protein in the control of cell wall fucosylation and how this contributes to plant freezing tolerance. Paige discusses this paper and the long history of MUR1 on the GARNet Community podcast. Please check it out!

Wang L, Stacey G, Leblanc-Fournier N, Legué V, Moulia B, Davies JM (2019) Early Extracellular ATP Signaling in Arabidopsis Root Epidermis: A Multi-Conductance Process. Front Plant Sci. doi: 10.3389/fpls.2019.01064.

Open Access

The UK-French collaboration is led by Limin Wang from Julia Davies’s lab in Cambridge. They use patch clamp electrophysiology to identify previously uncharacterized channel conductances that respond to extracellular ATP across the root elongation zone epidermal plasma membrane.

Waghmare S, Lefoulon C, Zhang B, Lileikyte E, Donald NA, Blatt MR (2019) K+ channel-SEC11 binding exchange regulates SNARE assembly for secretory traffic. Plant Physiol. doi: 10.1104/pp.19.00919

Open Access

This work from Mike Blatt’s lab in Glasgow is led by Sakharam Waghmare. They look at the interaction between SNARE proteins, which are involved in vesicular fusion and K+ channels, which help control turgor pressure during cell expansion. Through combining analysis of protein-protein interactions and electrophysiological measurement they have found that this interaction requires the activity of the regulatory protein SEC11.

Li W, He X, Chen Y, Jing Y, Shen C, Yang J, Teng W, Zhao X, Hu W, Hu M, Li H, Miller AJ, Tong Y (2019) A wheat transcription factor positively sets seed vigour by regulating the grain nitrate signal. New Phytol. doi: 10.1111/nph.16234
Wenjing Li is first author of this Chinese study that includes Yi Chen and Anthony Miller from the John Innes Centre as co-authors. This research shows that seed vigour and nitrate accumulation in wheat is regulated by the TaNAC2 transcriptions factor through its control of the TaNRT2.5 nitrate transporter. The authors suggest that both genes could be used as potential future targets to increase grain yield and nitrogen use efficiency.

The Special Issue of Methods in Molecular Biology on Plant Meiosis includes papers from the University of Cambridge, led by Christophe Lambing and the James Hutton Institute, led by Benoit Darrier.

Lambing C, Choi K, Blackwell AR, Henderson IR (2019) Chromatin Immunoprecipitation of Meiotically Expressed Proteins from Arabidopsis thaliana Flowers. Methods Mol Biol. doi: 10.1007/978-1-4939-9818-0_16
Darrier B, Arrieta M, Mittmann SU, Sourdille P, Ramsay L, Waugh R, Colas I (2019) Following the Formation of Synaptonemal Complex Formation in Wheat and Barley by High-Resolution Microscopy. Methods Mol Biol. doi: 10.1007/978-1-4939-9818-0_15

GARNet Research Roundup: September 24th 2019

by Geraint
Categories: Global, James Hutton Institute, John Innes Centre, Lancaster University, Nottingham University, Oxford Brookes University, Oxford University, Rothamsted Research, Uncategorized, University of Bath, University of Birmingham, University of Bristol, University of Cambridge, University of Dundee, University of Edinburgh, University of Leeds, University of Oxford, University of York, Warwick University
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Published on: September 24, 2019

Due to a significant delay this GARNet Research Roundup is a bumper overview of recent(ish) publications across discovery-led plant science, which have at least one contributor from a UK institution.

These can be (very) loosely separated into the following categories:

Circadian Clock: Greenwood et al, PloS Biology. Belbin et al, Nature Communications.

Environmental responses: Rodríguez-Celma et al, PNAS. Walker and Bennett, Nature Plants. Conn et al, PLoS Comput Biology. de Jong et al,PLoS Genetics. Molina-Contreras et al,The Plant Cell.

Defence signaling: Van de Weyer et al, Cell.Hurst et al, Scientific Reports. Xiao et al, Nature. Wong et al, PNAS.

Cell Biology: Miller et al, The Plant Cell. Coudert et al, Current Biology. Burgess et al,The Plant Cell. Harrington et al, BMC Plant Biology.

Metabolism: Jia et al, J Biol Chem. Perdomo et al, Biochem J. Gurrieri et al, Frontiers in Plant Science. Mucha et al, The Plant Cell. Atkinson et al, JXBot.

Cell Wall Composition: Wightman et al, Micron. Milhinhos et al, PNAS.

Signaling: Hartman et al, Nature Communications. Dittrich et al, Nature Plants. Villaécija-Aguilar et al, PLoS Genetics

Greenwood M, Domijan M, Gould PD, Hall AJW, Locke JCW (2019) Coordinated circadian timing through the integration of local inputs in Arabidopsis thaliana. PLoS Biol. 17(8):e3000407. doi: 10.1371/journal.pbio.300040 Open Access

https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3000407

Lead author is Mark Greenwood. UK contribution from The Sainsbury lab University of Cambridge, University of Liverpool and Earlham Institute. Using a mixture of experimental and modeling this paper shows that individual organs have circadian clocks that runs at different speeds.

Belbin FE, Hall GJ, Jackson AB, Schanschieff FE, Archibald G, Formstone C, Dodd AN (2019) Plant circadian rhythms regulate the effectiveness of a glyphosate-based herbicide. Nat Commun. 2019 Aug 16;10(1):3704. doi: 10.1038/s41467-019-11709-5 Open Access

Lead author is Fiona Belbin. UK contribution from University of Bristol and Syngenta Jealott’s Hill. Activity of the circadian clock determines that the plant response to the herbicide glyphosate is lessened at dusk, promoting the idea of agricultural chronotherapy. Fiona discusses this paper on the GARNet Community Podcast.

Rodríguez-Celma J, Connorton JM, Kruse I, Green RT, Franceschetti M, Chen YT, Cui Y, Ling HQ, Yeh KC, Balk J (2019) Arabidopsis BRUTUS-LIKE E3 ligases negatively regulate iron uptake by targeting transcription factor FIT for recycling. Proc Natl Acad Sci U S A. doi: 10.1073/pnas.1907971116 Open Access

https://www.pnas.org/content/early/2019/08/13/1907971116.long

Lead author is Jorge Rodríguez-Celma. UK contribution from John Innes Centre, University of East Anglia.The Arabidopsis E3 ubiquitin ligases, BRUTUS-LIKE1 (BTSL1) and BTSL2 target the FIT transcription factor for degradation, altering the plant response to harmful level of iron.

Walker CH, Bennett T (2019) A distributive ‘50% rule’ determines floral initiation rates in the Brassicaceae. Nat Plants. doi: 10.1038/s41477-019-0503-z
Lead author Catriona Walker. UK contribution from the University of Leeds. The authors introduce the 50%-rule that defines the relationshop between the total number of flowers the number of secondary inflorescences

Conn A, Chandrasekhar A, Rongen MV, Leyser O, Chory J, Navlakha S (2019) Network trade-offs and homeostasis in Arabidopsis shoot architectures. PLoS Comput Biol. doi: 10.1371/journal.pcbi.100732 Open Access

Lead author is Adam Conn. UK contribution from Sainsbury Laboratory, University of Cambridge. This study performed 3D scanning of 152 Arabidopsis shoot architectures to investigate how plants make trade-offs between competing objectives.

de Jong M, Tavares H, Pasam RK, Butler R, Ward S, George G, Melnyk CW, Challis R, Kover PX, Leyser O (2019) Natural variation in Arabidopsis shoot branching plasticity in response to nitrate supply affects fitness. PLoS Genet. doi: 10.1371/journal.pgen.1008366 Open Access

Lead author is Maaike de Jong. UK contribution from the Sainsbury Laboratory, University of Cambridge, the University of York and the University of Bath. This study looks at phenotypic plasticity of shoot branching in Arabidopsis diversity panels grown until different nitrate concentrations.

Molina-Contreras MJ, Paulišić S, Then C, Moreno-Romero J, Pastor-Andreu P, Morelli L, Roig-Villanova I, Jenkins H, Hallab A, Gan X, Gómez-Cadenas A, Tsiantis M, Rodriguez-Concepcion M, Martinez-Garcia JF (2019) Photoreceptor Activity Contributes to Contrasting Responses to Shade in Cardamine and Arabidopsis Seedlings. Plant Cell. doi: 10.1105/tpc.19.00275 Open Access

Lead author is Maria Jose Molina-Contreras. UK contribution from the University of Oxford. The authors looks at the response to different light conditions and how they contribute to phenotypic determination in Cardamine and Arabidopsis seedlings.

Van de Weyer AL, Monteiro F, Furzer OJ, Nishimura MT, Cevik V, Witek K, Jones JDG, Dangl JL, Weigel D, Bemm F (2019) A Species-Wide Inventory of NLR Genes and Alleles in Arabidopsis thaliana. Cell. doi: 10.1016/j.cell.2019.07.038 Open Access

https://www.sciencedirect.com/science/article/pii/S0092867419308372

Lead author is Anna-Lena Van de Weyer. UK contribution from The Sainsbury Laboratory, Norwich. Using sequence enrichment and long-read sequencing the authors present the pan-NLRome constructed from 40 Arabidopsis accessions.

Hurst CH, Wright KM, Turnbull D, Leslie K, Jones S, Hemsley PA (2019) Juxta-membrane S-acylation of plant receptor-like kinases is likely fortuitous and does not necessarily impact upon function. Sci Rep. doi: 10.1038/s41598-019-49302-x Open Access

Lead author is Charlotte Hurst. UK contribution from the James Hutton Institute and the University of Dundee. They look at the functional role of post-translational modification S-acylation with a focus on the plant pathogen perceiving receptor-like kinase FLS2.

Xiao Y, Stegmann M, Han Z, DeFalco TA, Parys K, Xu L, Belkhadir Y, Zipfel C, Chai J (2019) Mechanisms of RALF peptide perception by a heterotypic receptor complex. Nature. doi: 10.1038/s41586-019-1409-7
Lead author is Yu Xiao. UK contribution from The Sainsbury Laboratory, Norwich. This study investigates how RAPID ALKALINIZATION FACTOR (RALF) peptides induce receptor complex formation to regulate immune signaling.

Wong JEMM, Nadzieja M, Madsen LH, Bücherl CA, Dam S, Sandal NN, Couto D, Derbyshire P, Uldum-Berentsen M, Schroeder S, Schwämmle V, Nogueira FCS, Asmussen MH, Thirup S, Radutoiu S, Blaise M, Andersen KR, Menke FLH, Zipfel C, Stougaard J (2019). A Lotus japonicus cytoplasmic kinase connects Nod factor perception by the NFR5 LysM receptor to nodulation. Proc Natl Acad Sci U S A. doi: 10.1073/pnas.1815425116
Open Access

Lead author is Jaslyn Wong. UK contribution from The Sainsbury Laboratory, University of East Anglia. This work was conducted in the legume Lotus and after a proteomic screen, the authors identified NFR5-interacting cytoplasmic kinase 4 that is involved in control of Nod factor perception.

Miller C, Wells R, McKenzie N, Trick M, Ball J, Fatihi A, Dubreucq B, Chardot T, Lepiniec L, Bevan MW (2019) Variation in expression of the HECT E3 ligase UPL3 modulates LEC2 levels, seed size and crop yield in Brassica napus. Plant Cell. doi: 10.1105/tpc.18.00577
Open Access

Lead author in Charlotte Miller. UK contribution from the John Innes Centre. Activity of the Brassica napus HECT E3 ligase gene BnaUPL3 controls seed weight per pod through degradation of LEC2, a master transcriptional regulator of seed maturation and reveals a potential target for crop improvement

Coudert Y, Novák O, Harrison CJ (2019) A KNOX-Cytokinin Regulatory Module Predates the Origin of Indeterminate Vascular Plants. Curr Biol. 2019 Aug 19;29(16):2743-2750.e5. doi: 10.1016/j.cub.2019.06.083

https://www.cell.com/current-biology/fulltext/S0960-9822%2819%2930843-7

Lead author is Yoan Coudert. UK contribution from the University of Cambridge and University of Bristol. Class I KNOX gene activity is shown to be necessary for axis extension from an intercalary region of determinate moss shoots, in part through promotion of cytokinin biosynthesis.

Burgess SJ, Reyna-Llorens I, Stevenson SR, Singh P, Jaeger K, Hibberd JM (2019) Genome-wide transcription factor binding in leaves from C3 and C4 grasses Plant Cell.  doi: 10.1105/tpc.19.00078 Open Access

Lead author is Steven Burgess. UK contribution from University of Cambridge, The Sainsbury lab University of Cambridge, University of Leeds The authors use DNaseI-SEQ to assess the similarities and differences in transcription factor binding sites in the leaves across a set of four C3 and C4 grasses.

Harrington SA, Overend LE, Cobo N, Borrill P, Uauy C (2019) Conserved residues in the wheat (Triticum aestivum) NAM-A1 NAC domain are required for protein binding and when mutated lead to delayed peduncle and flag leaf senescence. BMC Plant Biol. doi: 10.1186/s12870-019-2022-
Lead author is Sophie Harrington. UK contributions from the John Innes Centre and University of Birmingham. The authors used a wheat TILLING resource to investigate mutrant allele with the NAC domain of the NAM-A1 transcription factor and their contribution to phenotypes in lab and field.

Jia Y, Burbidge CA, Sweetman C, Schutz E, Soole K, Jenkins C, Hancock RD, Bruning JB, Ford CM (2019) An aldo-keto reductase with 2-keto- L-gulonate reductase activity functions in L-tartaric acid biosynthesis from vitamin C in Vitis vinifera. J Biol Chem. doi: 10.1074/jbc.RA119.010196 Open Access

Lead author Yong Jia. UK contribution from the James Hutton Institute. This work conducted in grape reveals the mechanism by which an aldo-keto reductase functions in tartaric acid biosynthesis.

Perdomo JA, Degen GE, Worrall D, Carmo-Silva E (2019) Rubisco activation by wheat Rubisco activase isoform 2β is insensitive to inhibition by ADP. Biochem J. doi: 10.1042/BCJ2019011 Open Access

Lead author is Juan Alejandro Perdomo. UK contribution from Lancaster University. They show through analysis of site-directed mutations across three isoforms of wheat Rubisco activase that these isoforms have different sensitivities to ADP.

Gurrieri L, Distefano L, Pirone C, Horrer D, Seung D, Zaffagnini M, Rouhier N, Trost P, Santelia D, Sparla F (2019) The Thioredoxin-Regulated α-Amylase 3 of Arabidopsis thaliana Is a Target of S-Glutathionylation. Front Plant Sci. doi: 10.3389/fpls.2019.00993 Open Access

Lead author is Libero Gurrieri. UK contribution from John Innes Centre. The chloroplastic α-Amylases, AtAMY3 is post-translationally modified by S-glutathionylation in response to oxidative stress.

Mucha S, Heinzlmeir S, Kriechbaumer V, Strickland B, Kirchhelle C, Choudhary M, Kowalski N, Eichmann R, Hueckelhoven R, Grill E, Kuster B, Glawischnig E (2019) The formation of a camalexin-biosynthetic metabolon. Plant Cell. doi: 10.1105/tpc.19.00403 Open Access

Lead author is Stefanie Mucha. UK contribution from Oxford Brookes University and University of Warwick. The authors performed two independent untargeted co-immunoprecipitations to identify components involved in biosynthesis of the antifungal phytoalexin camalexin.

Atkinson N, Velanis CN, Wunder T, Clarke DJ, Mueller-Cajar O, McCormick AJ (2019) The pyrenoidal linker protein EPYC1 phase separates with hybrid Arabidopsis-Chlamydomonas Rubisco through interactions with the algal Rubisco small subunit. J Exp Bot. doi: 10.1093/jxb/erz275
Open Access

Lead author is Nicky Atkinson. UK contribution from the University of Edinburgh. This study uses Arabidopsis-Chlamydomonas to investigate the protein-protein interaction between Rubisco and essential pyrenoid component 1 (EPYC1).

Wightman R, Busse-Wicher M, Dupree P (2019) Correlative FLIM-confocal-Raman mapping applied to plant lignin composition and autofluorescence. Micron. doi: 10.1016/j.micron.2019.102733
Lead author Raymond Wightman. UK contribution from the Sainsbury Laboratory, University of Cambridge and the University of Cambridge. This study uses applies a novelmethod of correlative FLIM-confocal-Raman imaging to analyse lignin composition in Arabidopsis stems.

Milhinhos A, Vera-Sirera F, Blanco-Touriñán N, Mari-Carmona C, Carrió-Seguí À, Forment J, Champion C, Thamm A, Urbez C, Prescott H, Agustí J (2019) SOBIR1/EVR prevents precocious initiation of fiber differentiation during wood development through a mechanism involving BP and ERECTA. Proc Natl Acad Sci U S A. doi: 10.1073/pnas.1807863116
Lead author is Ana Milhinho. UK contribution from the University of Oxford. The authors used GWAS in Arabidopsis to identify the SOBIR1/EVR as an important regulator of the control of secondary growth in xylem fibers.

Hartman S, Liu Z, van Veen H, Vicente J, Reinen E, Martopawiro S, Zhang H, van Dongen N, Bosman F, Bassel GW, Visser EJW, Bailey-Serres J, Theodoulou FL, Hebelstrup KH, Gibbs DJ, Holdsworth MJ, Sasidharan R, Voesenek LACJ (2019) Ethylene-mediated nitric oxide depletion pre-adapts plants to hypoxia stress. Nat Commun. doi: 10.1038/s41467-019-12045-4 Open Access

https://www.nature.com/articles/s41467-019-12045-4

Lead author is Sjon Hartman. UK contribution from the University of Nottingham, Rothamsted Research and the University of Birmingham. This multinational collaboration looks into the relationship of how ethylene mediated nitric-oxide signaling responds to environmental signals.

Dittrich M, Mueller HM, Bauer H, Peirats-Llobet M, Rodriguez PL, Geilfus CM, Carpentier SC, Al Rasheid KAS, Kollist H, Merilo E, Herrmann J, Müller T, Ache P, Hetherington AM, Hedrich R (2019) The role of Arabidopsis ABA receptors from the PYR/PYL/RCAR family in stomatal acclimation and closure signal integration. Nat Plants. doi: 10.1038/s41477-019-0490-0
Lead author Marcus Dittrich. UK contribution from the University of Bristol. This work looks at the role of ABA signaling in stomatal responses and that the multiple ABA receptors can be modulated differentially in a stimulus-specific manner.

Villaécija-Aguilar JA, Hamon-Josse M, Carbonnel S, Kretschmar A, Schmid C, Dawid C, Bennett T, Gutjahr C (2019). SMAX1/SMXL2 regulate root and root hair development downstream of KAI2-mediated signalling in Arabidopsis. PLoS Genet. doi: 10.1371/journal.pgen.1008327 Open Access

Lead author Jose Antonio Villaécija-Aguilar. UK contribution from the University of Leeds and The Sainsbury lab, University of Cambridge. This demonstrates that KAI2 signalling through SMAX1/SMXL2 , is an important new regulator of root hair and root development in Arabidopsis.

https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1008327

GARNet Research Roundup: August 16th 2019

by Geraint
Categories: Bristol University, Cambridge University, Edinburgh University, Global, John Innes Centre, Lancaster University, Nottingham University, Oxford University, Rothamsted Research, Uncategorized, University of Bristol, University of Cambridge, University of Oxford, University of York, Warwick University
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Published on: August 16, 2019

This holiday-time edition of the GARNet research roundup begins with two papers that include the late Ian Moore from the University of Oxford as a co-author. The first looks at the role of RAB-A5c in the control of cellular growth anisotropy whilst the second characterises the Transport Protein Particle II (TRAPPII) complex.

The third paper is a UK-wide collaboration that assesses the role of UVA signaling on stomatal development. Next is a paper from Cambridge and the JIC that has identified the TAF4b protein as a novel regulator of meiotic crossovers.

The fifth paper is from the University of York and characterizes a role for cis-12-oxo-phytodienoic acid (OPDA) during seed germination.

The next three papers feature scientists from the University of Leeds in research that investigates 1, a peroxisomal ABC transporter; 2, the role of LRR-RLKs in plasmodesmata development and 3, the cell wall characteristics of banana and mango fruit.

The ninth paper is from the University of Edinburgh and investigates the role of S-nitrosylation in the control of SUMO conjugation.

The next two papers include Steve Penfield at the JIC as a corresponding author; the first looks at the role of endosperm-expressed transcriptional factors during seed dormancy and the second, in collaboration with researchers at the University of Warwick, identifies novel QTLs involved in seed dormancy.

The penultimate study is from Lancaster and presents a surprising outcome resulting from the overexpression of the wheat CA1Pase gene. The final paper includes Alison Tidy and Zoe Wilson from University of Nottingham as co-authors on a study that looks at male fertility in Arabidopsis.

Kirchhelle C, Garcia-Gonzalez D, Irani NG, Jérusalem A, Moore I (2019) Two mechanisms regulate directional cell growth in Arabidopsis lateral roots. Elife. pii: e47988. doi: 10.7554/eLife.47988

Open Access

Charlotte Kirchhelle leads this work that was conducted in the lab of the late Ian Moore at the University of Oxford. She investigates the role of the plant-specific small GTPase RAB-A5c during growth anisotropy in lateral roots, which involves coordinated orientations of cellulose microfibrils (CMFs) and by cortical microtubules (CMTs). They identify RAB-A5c dependent and independent mechanisms to control cellular growth anisotropy in this growing tissue.

From https://elifesciences.org/articles/47988

Kalde M, Elliott L, Ravikumar R, Rybak K, Altmann M, Klaeger S, Wiese C, Abele M, Al B, Kalbfuß N, Qi X, Steiner A, Meng C, Zheng H, Kuster B, Falter-Braun P, Ludwig C, Moore I, Assaad FF (2019) Interactions between Transport Protein Particle (TRAPP) complexes and Rab GTPases in Arabidopsis. Plant J. doi: 10.1111/tpj.14442

This German-led study includes Monika Kalde from the University of Oxford as first author as well Ian Moore as co-author. They characterize the components and function of the Transport Protein Particle II (TRAPPII) complex. TRAPPII plays multiple roles in intra-cellular transport and this study identified 13 subunits, including several that were previously uncharacterised.

Isner JC, Olteanu VA, Hetherington AJ, Coupel-Ledru A, Sun P, Pridgeon AJ, Jones GS, Oates M, Williams TA, Maathuis FJM, Kift R, Webb AR, Gough J, Franklin KA, Hetherington AM (2019). Short- and Long-Term Effects of UVA on Arabidopsis Are Mediated by a Novel cGMP Phosphodiesterase. Curr Biol.29(15):2580-2585.e4. doi: 10.1016/j.cub.2019.06.071

Open Access

Jean-Charles Isner is the first author on this collaboration between labs in Bristol, York, Oxford and Cambridge. They show that UVA radiation (which represents 95% of the UV radiation reaching earth) inhibits stomatal opening through a process that involves a reduction in the cytosolic level of cGMP. The AtCN-PDE1 gene (a cGMP-activated phosphodiesterase) is needed to decrease cGMP levels in Arabidopsis. This response is present across the tree of life except in metazoans. They show AtCN-PDE1 is needed for the UVA response and that prolonged UVA exposure causes increased growth yet reduced water use efficiency.

From https://www.cell.com/current-biology/fulltext/S0960-9822(19)30798-5

Lawrence EJ, Gao H, Tock AJ, Lambing C, Blackwell AR, Feng X, Henderson IR (2019) Natural Variation in TBP-ASSOCIATED FACTOR 4b Controls Meiotic Crossover and Germline Transcription in Arabidopsis. Curr Biol. pii: S0960-9822(19)30844-9. doi: 10.1016/j.cub.2019.06.084

Open Access

This work from Ian Henderson’s lab in Cambridge and Xiaoqi Feng’s lab at the JIC is led by Emma Lawrence and isolates a novel modifier of meiotic crossover frequency, TBP-ASSOCIATED FACTOR 4b (TAF4b), which encodes a subunit of the RNA polymerase II general transcription factor TFIID. They show TAF4b expression is enriched in meiocytes, compared to the more general expression of its paralog TAF4. Ultimately they reveal TAF4b drives a novel mode of meiotic recombination control through its activity as a general transcription factor.

From https://www.cell.com/current-biology/fulltext/S0960-9822(19)30844-9

Barros-Galvão T, Dave A, Cole A, Harvey D, Langer S, Larson TR, Vaistij FE, Graham IA (2019) cis-12-oxo-phytodienoic acid represses Arabidopsis thaliana seed germination in shade light conditions. J Exp Bot. pii: erz337. doi: 10.1093/jxb/erz337

Open Access

Thiago Barros-Galvão is first author on this study from Ian Graham’s lab at the University of York. They investigate how the jasmonic acid pre-cursor cis-12-oxo-phytodienoic acid (OPDA) contributes to control of seed germination, particularly under shade conditions. OPDA acts through the activity of the transcription factor MOTHER-OF-FT-AND-TFL1 (MFT).

From https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/erz337/5536641

Carrier DJ, van Roermund CWT, Schaedler TA, Rong HL, IJlst L, Wanders RJA, Baldwin SA, Waterham HR, Theodoulou FL, Baker A (2019) Mutagenesis separates ATPase and thioesterase activities of the peroxisomal ABC transporter, Comatose. Sci Rep. 9(1):10502. doi: 10.1038/s41598-019-46685-9

Open Access

Alison Baker at the University of Leeds is the corresponding author of this UK, Dutch collaboration that includes David Carrier as first author. They characterise the peroxisomal ABC transporter, Comatose (CTS) through mutagenesis of key residues responsible for the proteins intrinsic acyl-CoA thioesterase (ACOT) activity. Ultimately they show that ACOT activity depends of endogenous ATPase activity but that these activities could be functional separated by mutagenesis of key residues.

Grison M, Kirk P, Brault M, Wu XN, Schulze WX, Benitez-Alfonso Y, Immel F, Bayer EMF (2019). Plasma membrane-associated receptor like kinases relocalize to plasmodesmata in response to osmotic stress. Plant Physiol. pii: pp.00473.2019. doi: 10.1104/pp.19.00473

Open Access

GARNet advisory committee member Yoselin Benitez-Alfonso and members of her research group are co-authors on the next two studies. This work is led by Magali Grison in Emmanuelle Bayer’s lab in Bordeaux. They show that the PM-localised Leucine-Rich-Repeat Receptor-Like-Kinases (LRR-RLKs), QSK1 and IMK2 relocate and cluster to the plasmodesmata under osmotic stress conditions. Through a variety of assays that focuses on QSK1 the authors show that reorganisation of RLKs can be important for the regulation of callose deposition at plasmodesmata and under osmotic stress this can have a functional effect on lateral root development.

Rongkaumpan G, Amsbury S, Andablo-Reyes E, Linford H, Connell S, Knox JP, Sarkar A, Benitez-Alfonso Y, Orfila C (2019) Cell Wall Polymer Composition and Spatial Distribution in Ripe Banana and Mango Fruit: Implications for Cell Adhesion and Texture Perception. Front Plant Sci. 10:858. doi: 10.3389/fpls.2019.00858

Open Access

Ganittha Rongkaumpan is first author on this interdisciplinary collaborative research from multiple departments at the University of Leeds. They characterise the composition of the cell wall in two fruits, banana and mango, which soften during ripening. The authors compared structural information, obtained using Atomic Force Microscopy and biochemical analysis, with data from rheology and tribology assays to understand why these fruits feel different in the mouth during ingestion.

From https://www.frontiersin.org/articles/10.3389/fpls.2019.00858/full

Skelly MJ, Malik SI, Le Bihan T, Bo Y, Jiang J, Spoel SH, Loake GJ (2019) A role for S-nitrosylation of the SUMO-conjugating enzyme SCE1 in plant immunity Proc Natl Acad Sci U S A. pii: 201900052. doi: 10.1073/pnas.1900052116

Michael Skelly from the University of Edinburgh is the lead author of this study from the labs of Gary Loake and GARNet chairman Steven Spoel. They investigate the mechanism through which nitric oxide signaling after pathogen recognition stimulates inhibitory S-nitrosylation of the Arabidopsis SUMO E2 enzyme, SCE1. S-nitrosylation occurs on the evolutionary conserved Cys139 of SCE1 and they investigate the wider significant of this residue in the control of immune responses across eukaryotes.

MacGregor DR, Zhang N, Iwasaki M, Chen M, Dave A, Lopez-Molina L, Penfield S (2019) ICE1 and ZOU determine the depth of primary seed dormancy in Arabidopsis independently of their role in endosperm development. Plant J. 98(2):277-290. doi: 10.1111/tpj.14211

Open Access

Dana MacGregor (now at Rothamsted Research) leads this work from the lab of Steve Penfield at the JIC that investigates the extent of control on depth of primary dormancy that is mediated by the endosperm-expressed transcription factors ZHOUPI (ZOU) and INDUCER OF CBF EXPRESSION1 (ICE1). These effects are additive and independent of their role in endosperm development since the dormancy defect in ice1 and zou mutants can be ameliorated without altering seed morphology. They show that ICE1 acts primarily through control of ABA INSENSITIVE 3 (ABI3).

From https://onlinelibrary.wiley.com/doi/full/10.1111/tpj.14211

Footitt S, Walley PG, Lynn JR, Hambidge AJ, Penfield S, Finch-Savage WE (2019) Trait analysis reveals DOG1 determines initial depth of seed dormancy, but not changes during dormancy cycling that result in seedling emergence timing. New Phytol. doi: 10.1111/nph.16081

This research is a collaboration between the John Innes Centre and the Universities Liverpool and Warwick, from which Steven Footitt is first author. They used two Arabidopsis ecotypes that have differences in the timing of seedling emergence to identify new QTLs involved in depth of seed dormancy and Seedling Emergence Timing (SET). They revealed that DOG1 is important for determining depth of dormancy. In addition they identified three new SET QTLs, which are each physically close to DOG1, that play a role in the control of SET in the field.

Lobo AKM, Orr D, Gutierrez MO, Andralojc J, Sparks C, Parry MAJ, Carmo-Silva E (2019) Overexpression of ca1pase decreases Rubisco abundance and grain yield in wheat. Plant Physiol. pii: pp.00693.2019. doi: 10.1104/pp.19.00693

Open Access

This research from Lancaster Environmental Centre and their Brazilian collaborators is led by Ana Karla Lobo and demonstrates that overexpression of 2-carboxy-D-arabinitol-1-phosphate phosphatase (CA1Pase) in wheat causes a reduction in above ground biomass and compromises wheat grain yields. As CA1Pase is involved in removing inhibitors of Rubisco activity this result is contrary to the anticipated outcome. This suggests that Rubisco inhibitors might actually protect enzyme activity, thus maintaining the number of active sites that the enzyme is able to support.

From http://www.plantphysiol.org/content/early/2019/07/31/pp.19.00693.long

Zhao SQ, Li WC, Zhang Y, Tidy AC, Wilson ZA (2019) Knockdown of Arabidopsis ROOT UVB SENSITIVE4 Disrupts Anther Dehiscence by Suppressing Secondary Thickening in the Endothecium. Plant Cell Physiol. doi: 10.1093/pcp/pcz127

Shu-Qing Zhao is the lead author on this China-UK collaboration that includes Alison Tidy and Zoe Wilson from the University of Nottingham. They show that using an artificial microRNA to reduce levels of the RUS4 gene in Arabidopsis causes a decline in male fertility. They perform a detailed analysis of the RUS4 expression module and how it impacts fertility.

GARNet Research Roundup: July 26th 2019

by Geraint
Categories: Aberystwyth University, Brassica, Cambridge University, Cardiff University, Edinburgh University, Global, John Innes Centre, Nottingham University, Oxford University, Uncategorized, University of Bath, University of Cambridge, University of Edinburgh, University of Nottingham, University of Oxford, University of Sheffield, University of Southampton, University of York
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Published on: July 26, 2019

This summer-time-reading bumper edition of the GARNet Research Roundup begins with two papers from the University of Sheffield that each use advanced imaging techniques. Firstly Andrew Fleming’s group leads a study on the link between stomatal function and mesophyll space morphology. Second is a study from Matthew Johnson’s group that looks at the dynamic arrangement of thylakoid stacks.

Next are two papers that include Alison Smith from the JIC as a corresponding author. The first also includes Vasilios Andriotis from the University of Newcastle and looks at the role of the plastidial pentose phosphate pathway during post-germination growth. Second uses a gene-editing strategy to generate potatoes with altered starch morphologies.

The fifth paper also looks at starch; researchers from Cambridge and Norwich are involved in a study that characterises the role of the LIKE SEX4 1 protein in starch degradation.

The sixth paper is from Aberystwyth University and identifies a transcription factor that alters secondary cell wall composition in Brachypodium and maize. Next is research from the University of Bath that looks at the role of a protein S-acyl transferase during seed germination.

The eighth and ninth papers are led by Spanish research groups and include contributions from UK-based co-authors in Cambridge and Nottingham, working on photoperiod perception or phosphate signaling respectively.

The tenth paper features work from Cardiff University and looks at the role of heterologous expression of the Arabidopsis WEE1 protein. The Bancroft lab from the University of York leads the next paper that investigates glucosinolate signaling in Brassica napus.

The final three manuscripts are methods papers. The first from Edinburgh introduces a new NanoLUC reporter whilst the other two include techniques involved in the investigation of light-regulated growth processes.

Lundgren MR, Mathers A, Baillie AL, Dunn J, Wilson MJ, Hunt L, Pajor R, Fradera-Soler M, Rolfe S, Osborne CP, Sturrock CJ, Gray JE, Mooney SJ, Fleming AJ (2019) Mesophyll porosity is modulated by the presence of functional stomata. Nat Commun. doi: 10.1038/s41467-019-10826-5

Open Access

This UK-wide study is led from Andrew Fleming’s lab in Sheffield and includes Marjorie Lundgren as first author (now working in Lancaster). They use microCT imaging alongside more traditional measurements linked to analysis of gas exchange to show that mesophyll airspace formation is linked to stomatal function in both Arabidopsis and wheat. This allows the authors to propose that coordination of stomata and mesophyll airspace pattern underpins water use efficiency in crops.

https://www.nature.com/articles/s41467-019-10826-5

Wood WH, Barnett SFH, Flannery S, Hunter CN, Johnson MP (2019) Dynamic thylakoid stacking is regulated by LHCII phosphorylation but not its interaction with photosystem I. Plant Physiol. doi: 10.1104/pp.19.00503

Open Access

William Wood is the first author on this study from the University of Sheffield that uses 3D structured illumination microscopy (3D-SIM) to look at the dynamics of thylakoid stacking in both Arabidopsis and spinach. They show that the processes they observe are dependent on light harvesting complex II phosphorylation.

http://www.plantphysiol.org/content/early/2019/06/11/pp.19.00503.long

Andriotis VME, Smith AM (2019) The plastidial pentose phosphate pathway is essential for postglobular embryo development in Arabidopsis. Proc Natl Acad Sci U S A. doi: 10.1073/pnas.1908556116

Open Access

Vasilios Andriotis (now at the University of Newcastle) is the lead author of this work performed in Alison Smith’s lab at the JIC. They look at the role of the plastidial oxidative pentose phosphate pathway (OPPP) during embryo development. This involved demonstrating that production of ribose-5-phosphate (R5P), which in turn leads to synthesis of purine nucleotides, is a critical function of the OPPP.

Tuncel A, Corbin KR, Ahn-Jarvis J, Harris S, Hawkins E, Smedley MA, Harwood W, Warren FJ, Patron NJ, Smith AM (2019) Cas9-mediated mutagenesis of potato starch-branching enzymes generates a range of tuber starch phenotypes. Plant Biotechnol J. doi: 10.1111/pbi.13137

Open Access

Alison Smith and Nicola Patron who work in Norwich Research Park are corresponding authors of this study that includes Aytug Tuncel as first author. They have used Cas9-mediated gene editing to generate potato plants that have a range of different tuber starch structures. This shows that gene-editing techniques allows the transgene-free alteration to generate potentially healthier crops.

Schreier TB, Umhang M, Lee SK, Lue WL, Shen Z, Silver D, Graf A, Müller A, Eicke S, Stadler M, Seung D, Bischof S, Briggs SP, Kötting O, Moorhead GB, Chen J, Zeeman SC (2019) LIKE SEX4 1 acts as a β-amylase-binding scaffold on starch granules during starch degradation. Plant Cell. doi: 10.1105/tpc.19.00089

Open Access

Tina Schreier from the University of Cambridge is the first author on this international study led from Switzerland that also includes Alexander Graf and David Seung from the JIC as co-authors. This study defines a precise role for the LIKE SEX FOUR 1 (LSF1) protein that binds starch and is required for normal starch degradation. Through a variety of experiments they show that the glucan binding, rather than phosphatase activity, is required for LSF1 function during starch degradation.

http://www.plantcell.org/content/early/2019/07/02/tpc.19.00089.long

Bhatia R, Dalton S, Roberts LA, Moron-Garcia OM, Iacono R, Kosik O, Gallagher JA, Bosch M (2019) Modified expression of ZmMYB167 in Brachypodium distachyon and Zea mays leads to increased cell wall lignin and phenolic content. Sci Rep. doi: 10.1038/s41598-019-45225-9

Open Access

Rakesh Bhatia is the first author on this work from the lab of Maurice Bosch at Aberystwyth University. They overexpress the maize MYB transcription factor ZmMYB167 in both Brachypodium and maize. Both species show increased lignin content with Brachypodium but not maize showing a biomass deficit. This indicates that ZmMYB167 could be a useful molecular tool for the alteration of secondary cell wall biosynthesis.

https://www.nature.com/articles/s41598-019-45225-9

Li Y, Xu J, Li G, Wan S, Batistic O, Sun M, Zhang Y, Scott R, Qi B (2019) Protein S-acyl Transferase 15 is Involved in Seed Triacylglycerol Catabolism during Early Seedling Growth in Arabidopsis (2019) J Exp Bot. doi: 10.1093/jxb/erz282

First author on this UK-Chinese collaboration is Yaxiao Li who works with Baoxiu Qi at the University of Bath. The authors characterise the function of Arabidopsis Protein Acyl Transferase 15, AtPAT15. This protein is involved in essential β-oxidation of triacylglycerols during post-germination growth.

Ramos-Sánchez JM, Triozzi PM, Alique D, Geng F, Gao M, Jaeger KE, Wigge PA, Allona I, Perales M (2019) LHY2 Integrates Night-Length Information to Determine Timing of Poplar Photoperiodic Growth. Curr Biol. doi: 10.1016/j.cub.2019.06.003

Open Access

This Spanish-led study includes co-authors from the Sainsbury Laboratory in Cambridge and attempts to define the factors that control photoperiod perception in trees, using poplar as a model system. FLOWERING LOCUS T2 (FT2) has been previously shown to be involved in this process and this study builds on that work to show that night-length information is transmitted by the clock gene LATE ELONGATED HYPOCOTYL 2 (LHY2) and is able to control FT2 expression.

https://www.cell.com/current-biology/fulltext/S0960-9822(19)30696-7?

Silva-Navas J, Conesa CM, Saez A, Navarro-Neila S, Garcia-Mina JM, Zamarreño AM, Baigorri R, Swarup R, Del Pozo JC (2019) Role of cis-zeatin in root responses to phosphate starvation. New Phytol. doi: 10.1111/nph.16020

Ranjan Swarup from the University of Nottingham is a co-author on this Spanish-led study that has Javier Silva-Navas as first author. Through analysis of dark-grown seedlings they have identified a set of new genes involved in root phosphate signaling. In addition they provide evidence of a links between cytokinin and phosphate signaling through modulation of the cell cycle.

Siciliano I, Lentz Grønlund A, Ševčíková H, Spadafora ND, Rafiei G, Francis D, Herbert RJ, Bitonti MB, Rogers HJ, Lipavská H (2019) Expression of Arabidopsis WEE1 in tobacco induces unexpected morphological and developmental changes. Sci Rep. 2019 Jun 18;9(1):8695. doi: 10.1038/s41598-019-45015-3

Open Access

Ilario Siciliano leads this work that includes colleagues from Hilary Rogers’ lab at Cardiff University. The WEE1 protein regulates the cell cycle across eukaryote lineages. In this work they show that overexpression of AtWEE1 in tobacco causes precocious flowering and increased shoot morphogenesis of stem explants whilst in cell culture this WEE1 OX causes smaller cell sizes.

Kittipol V, He Z, Wang L, Doheny-Adams T, Langer S, Bancroft I (2019) Genetic architecture of glucosinolate variation in Brassica napus. J Plant Physiol. doi: 10.1016/j.jplph.2019.06.001

Open Access

This study from the Bancroft lab at the University of York is led by Varanya Kittipol. Through use of Associative Transcriptomics (AT) across a diversity panel of 288 Brassica napus genotypes they are able to identify a set of genes involved in synthesis of glucosinate hydrolysis products.

Urquiza-García U, Millar AJ (2019). Expanding the bioluminescent reporter toolkit for plant science with NanoLUC. Plant Methods. doi: 10.1186/s13007-019-0454-4

Open Access

This study from the University of Edinburgh introduces NanoLUC, a new more stable luciferase-based reporter for use by the plant community.

The final two papers are methods papers that focus on different aspects of light-regulated growth. These are from the University of Southampton and University of York.

https://plantmethods.biomedcentral.com/articles/10.1186/s13007-019-0454-4

Terry MJ, Kacprzak SM (2019) A Simple Method for Quantification of Protochlorophyllide in Etiolated Arabidopsis Seedlings. Methods Mol Biol. doi: 10.1007/978-1-4939-9612-4_14

Oakenfull RJ, Ronald J, Davis SJ (2019) Measuring Phytochrome-Dependent Light Input to the Plant Circadian Clock. Methods Mol Biol. doi: 10.1007/978-1-4939-9612-4_15

John Innes Centre Gene Editing Workshop.

by Geraint
Categories: John Innes Centre, Uncategorized
Tags: No Tags
Comments: No Comments
Published on: July 22, 2019

Professor Wendy Harwood leads a team of researchers and technicians at the John Innes Centre who have expertise in tissue-culture based plant transformation techniques (BRACT- Biotechnology Resources for Arable Crop Transformation).

            Transformation is often considered a dark-art of plant science, as history informs that it requires an established set of protocols that have been well-tested for local experimental conditions. It takes significant time and expertise to set up an effective and reliable transformation pipeline, which is why few places around the UK undertake this type of work. These challenges can provide a significant bottleneck in the establishment of high-throughout transformation platforms.

            The BRACT expertise in transformation was aligned with gene editing technology to successfully gain UKRI-BBSRC Bioinformatics and Biological Resources Funding (BBR) funding in 2016 with a grant entitled ‘Targeted gene knockouts in crops using RNA-guided Cas9 nucleases’. Professor Harwood shares Co-I status on this grant with Dr Nicola Patron who is an expert on synthetic biology and genome editing based at the Earlham Institute.

Professor Harwood introduces the workshop. Photo @SadiyeHayta

Expanding UK capability in gene editing of crops

            This BBR funding is absolutely community-facing and included the provision for 50 genes, selected from open applications, to be targeted by CRISPR-Cas9 gene editing. Initially successful applicants would benefit from BRACT expertise in guide RNA (gRNA) design after which the optimised gRNAs and Cas9 would then be introduced into either barley or Brassica oleracea. However, the success of the BRACT service meant that over 60 genes were ultimately selected for targeted knock-out and in the second round of applications the available crops were expanded to include wheat, Brassica napus and tomato.

            This grant allowed users throughout the UK to take advantage of expertise in gene editing and transformation, which is beyond the capabilities of most individual research departments. The grant also included a requirement to provide training for new users and a training event was successfully delivered between July 10-12th at the JIC. On days 1 and 3, 12 successful applicants worked with the BRACT team to learn how to design CRISPR guides, assemble constructs, screen edited plants and perform the basics of plant transformation. In between these two days was a day of scientific talks that included talks from successful community applicants as well as updates on the latest technical developments at BRACT. The day 2 workshop was attended by 62 participants from across the UK.  The demand for training was significantly higher than the number of spaces available, with the course being 3x oversubscribed. Hopefully this may encourage the course organisers to consider running similar training events again.   

Demonstrating a clear community need.

During the two rounds of applications, researchers from 43 UK research groups across 26 institutions applied to have one or more genes of interest targeted by this resource. Due to high demand ultimately 61 genes were selected for targeting from a total of 126 requested genes. Multiple guide RNAs were designed and primary transformation conducted at BRACT before transgenic and potentially edited plants were sent out to users. The users then screen these plants for Non-homologous end joining (NHEJ)-editing events and, if needed, identified transgene-free germplasm. BRACT provided help with screening and growing on the selected plants as required.

Success Stories

            Mikhaela Neequaye presented the most complete study of those that have used the BRACT facility through this BBR funding. She works with Richard Mithen, formally from the Quadram Institute and Lars Ostergaard at the JIC on the Brassica oleracea MYB28 transcription factor, which is involved in glucosinolate biosynthesis. In collaboration with BRACT she has identified plants with edited MYB28 that show altered glucosinate levels and has even successfully gone through the onerous process of applying to DEFRA to conduct a field trial with these edited plants. An impressive output from a PhD project!

The DEFRA application

            Kate Henbest is a PhD student who works with Lorraine Williams at the University of Southampton on the challenge of developing plants with altered capacity to transport zinc. She targeting two loci encoding bZIP transcription factors in barley and had successfully identified the predicted double ‘CRISPR mutants’. In addition she also found a surprising triple mutant that also contained a mutation in a related bZIP family member. These multi-mutants were identified from a relatively small number of edited lines, which so far is a very satisfying result.

            However the appearance of the triple mutant highlights that we have an incomplete understanding of the editing events that will occur after NHEJ and demonstrate the importance of users feeding back this information to BRACT. This will allow them to build a valuable database about the particular editing events observed by users. The handful of users who presented their research at the workshop documented both small deletions as well as single nucleotide substitutions and deletions.

Growing plants can be surprisingly challenging!

            Early user feedback indicates that obtaining full-value from this BBR grant might rely on the extent of post-editing support that the BRACT team is able to provide (and how this might be financially supported). Some applicants had previously only worked with Arabidopsis, so after a successful application they will be confronted with the challenge of growing much larger (transgenic) plants that will require more space and different growth conditions. This might seem like a trivial problem but in order for the BRACT team to show that their project has been successful (in terms of follow-on publications or future grants) then this relies on end-users being able to correctly grow plants in order to obtain useful data. The BRACT team has been able to provide some advice on these issues without impinging on the time they must devote to other projects, but as is often the case, these additional requirements are often hidden time constraints that aren’t always factored into grant funding.

            Somewhat related to these issues was a talk from Gustaf Degen from Lancaster University who had received Brassica oleracea plants with edits within a gene involved in photosynthesis. His progress in this project has been delayed as all of the edited plants died during a particularly (surprisingly) hot summer in Lancaster! Fortunately, the plants were saved by returning to the original tissue-culture and although this was a fluke event, it does rather highlight that users need to be able to grow the plants that they intend to work with!

Penny Hundleby overseeing the Brassica transformation practical Photo @MarkSmedley15

Overcoming the challenges of HDR.

            Tufan Oz had recently joined the Patron lab at the Earlham Institute but gave a talk about his previous research conducted in Florida. He worked with sugarcane and had successfully targeted the acetolactate synthase (ALS) gene using homology-directed repair (HDR). He explained that sugarcane provides an excellent model for HDR as the tissue-culture transformation frequency is very high.

            One of the BRACT CRISPR-experts is Tom Lawrenson and he gave an informative update about his work aiming to undertake HDR in barley. HDR uses a ‘conventional’ gRNA/CRISPR system along with an additional DNA template with homology arms to a specific pre-selected region within the genome. Although HDR results in the insertion of a ‘foreign’ gene unlike conventional GMOs that rely on random insertion, HDR allows targeted insertion to a chosen place in the genome.

            A particular challenge of using HDR is ensuring that there is a sufficiently high quantity of the DNA template in the vicinity of the Cas9 cut site. Poor transformation frequencies will also be an impediment to this work as with fewer transformations there is less chance of obtaining a line with sufficient high levels of the template. Tom reported that overall this means that successful HDR occurs 10-100x times less efficiently than NHEJ.

            A major strength of this BBR grant is that it complements other funded work [wh(1] aiming to troubleshoot challenging protocols (such as HDR) precluding the need for replicated efforts in labs around the country. In these HDR experiments Tom is attempting to directly knock-in a N-terminal mCherry fusion and his primary advice is to ensure that you use very efficient gRNAs. Due to the low efficiency of HDR in order to obtain a correctly edited plant a researcher has the choice to either a) look at more primary transformants, b) to use a viral vector to improve expression or c) to screen a lower number of transformants over multiple generations. Tom has experimented with viral-derived components but found that this wasn’t necessary for successful HDR. As with NHEJ, the requirements for efficient HDR will vary on a plant-to-plant basis but the BRACT team are working out the basics so that the entire community will benefit from using this powerful technique in the future.

Future technologies

            The latter stages of the workshop focused on future technologies that could be of use to the community. The BRACT team discussed their recent work on:

1. Use of Cpf as an alternative to Cas9 (it doesn’t work as well in their hands)

2. On their testing of base-editing and epigenome editing techniques (work in progress)

3. Strategies to obtain multiple knock-outs from a single transformation (use different promotors to ensure even expression of all gRNAs in a single construct).

            Workshop participants were invited to suggest what they needed to improve their research using CRISPR-based technologies. The consensus amongst those few people I talked to was that more hands-on training would be very useful and that nothing is too simple to be taught. This type of training requires a significant commitment in time and space so there needs to be a community discussion around the mechanisms that could be used to enable these opportunities. CRISPR-based gene editing techniques are clearly game-changing technologies but, as with anything, without correct use they can take a lot of time for little reward.

            During discussions Professor Harwood gave a comment on the ‘Freedom To Operate’ study that they have commissioned as part of this BBR grant. This suggests that they will have no difficulty using CRISPR-based technology for projects working with academic applicants. Interestingly it was positive to learn that whereas there may be complications when they work with for-profit companies, it should be possible for those companies to use the CRISPR technology without having to pay a prohibitive licensing fee. However as the legal battles over the CRISPR-patent have not yet been resolved any opinion given in this area might be liable to change.

Relaxing after the meeting. Photo @SadiyeHayta

            The day of presentations ended in traditional John Innes Centre fashion with some locally brewed beer and wine on the terrace in the summer sun! The three-day workshop / training course then concluded for the trainees with the third day allowing hands-on experience of tissue-culture transformation techniques. Hopefully the training course participants will take this knowledge back to their home institutions in order to allow more researchers to master one of the dark-arts of plant science!

GARNet Research Roundup: July 5th 2019

by Geraint
Categories: Cambridge University, Glasgow University, Global, James Hutton Institute, John Innes Centre, Nottingham University, Oxford Brookes University, Uncategorized, University of Dundee, University of Leicester, University of Nottingham
Tags: No Tags
Comments: No Comments
Published on: July 5, 2019

This edition of the GARNet research roundup begins with a study from the University Leicester that investigates the rate of selection of genes expressed in Arabidopsis pollen.

The second and third papers focus on the function of members of the AP2 family of transcription factors. Sarah McKim’s lab in Dundee characterizes the role of APETALA2 during barley stem elongation whilst the other paper investigates the function of the Arabidopsis PUCHI gene and includes co-authors from the University of Nottingham.

The fourth paper is from Lars Ostergaard’s lab at the John Innes Centre and demonstrates the benefit of using models to understand developmental processes in crop plants. The next paper from the University of Glasgow investigates the plant response to low fluence rates of UV-B light.

The penultimate paper features authors from Oxford Brookes University and characterizes a novel LINC-KASH protein in maize whilst the final paper is from the University of Cambridge and investigates the novel function of two members of DUF579 family in methylation of glucuronic acid residues.

Harrison MC, Mallon EB, Twell D, Hammond RL (2019) Deleterious mutation accumulation in Arabidopsis thaliana pollen genes: a role for a recent relaxation of selection. Genome Biol Evol. doi: 10.1093/gbe/evz127

Open Access

This research from Hammond and Twell lab’s at the University of Leicester uses Arabidopsis to investigate the hypothesis that pollen genes evolve faster than sporophytic genes. This study is challenging to perform in Arabidopsis as for the past million years the plant has been self-compatible, which causes reduction in pollen competition, increased homozygosity and a dilution of masking in diploid expressed, sporophytic genes. This study has two main findings: firstly prior to becoming self-compatible pollen genes evolved faster than sporophytic genes. Secondly, since becoming self-compatible selection has relaxed causing higher polymorphism levels and a higher build-up of deleterious mutations.

https://academic.oup.com/gbe/advance-article/doi/10.1093/gbe/evz127/5519772

Patil V, McDermott HI, McAllister T, Cummins M, Silva JC, Mollison E, Meikle R, Morris J, Hedley PE, Waugh R, Dockter C, Hansson M, McKim SM (2019) APETALA2 control of barley internode elongation. Development. doi: 10.1242/dev.170373

Open Access

Vrushali Patil leads his study from the lab of current GARNet committee member Sarah McKim at the James Hutton Institute in Dundee. They show that the APETALA2 (AP2) transcription factor is necessary for stem elongation in Barley. In addition they demonstrate that AP2 expression is controlled by the activity of the microRNA mi172 as well as jasmonate signaling.

https://dev.biologists.org/content/146/11/dev170373.long

Trinh DC, Lavenus J, Goh T, Boutté Y, Drogue Q, Vaissayre V, Tellier F, Lucas M, Voß U, Gantet P, Faure JD, Dussert S, Fukaki H, Bennett MJ, Laplaze L, Guyomarc’h S (2019) PUCHI regulates very long chain fatty acid biosynthesis during lateral root and callus formation. Proc Natl Acad Sci U S A. doi: 10.1073/pnas.1906300116

Julien Lavenus, Ute Voß and Malcolm Bennett from University of Nottingham are co-authors on this French-led study that investigates the mechanism by which the AP2 family transcription factor PUCHI controls lateral root development. By performing a transcriptional analysis of developing lateral root cells they show that genes involved in very long chain fatty acid (VLCFA) biosynthesis enzymes are induced in a PUCHI dependent manner. Concomitantly they show puchi-1 mutant roots have reduced VLCFA content when compared with wildtype roots. They conclude that PUCHI regulates VLCFA biosynthesis as part of a pathway controlling cell proliferation during lateral root formation.

Stephenson P, Stacey N, Brüser M, Pullen N, Ilyas M, O’Neill C, Wells R, Østergaard L (2019) The power of model-to-crop translation illustrated by reducing seed loss from pod shatter in oilseed rape. Plant Reprod. doi: 10.1007/s00497-019-00374-9

Open Access

Pauline Stephenson and Lars Østergaard at the John Innes Centre lead this study in which they demonstrate that lessons learnt from understanding the genes involved in fruit ripening in Arabidopsis lead to an ability to adjust the pod-opening process in oilseed rape. They have combined two mutant alleles, first characterized in Arabidopsis, to develop OSR plants that have significantly increased yield. In addition they present a new software tool for the analysis of pod shatter data in other crops plants.

O’Hara A, Headland LR, Díaz-Ramos LA, Morales LO, Strid Å, Jenkins GI (2019) Regulation of Arabidopsis gene expression by low fluence rate UV-B independently of UVR8 and stress signaling. Photochem Photobiol Sci. doi: 10.1039/c9pp00151d

Open Access

This UK-Swedish collaboration is led by Andrew O’Hara from the Jenkins lab in the University of Glasgow. They continue the lab focus on the UV-B receptor UVR8, in this case performing a transcriptomic analysis of wildtype and uvr8 mutants grown under low UV-B fluence rates. They analyse one differentially expressed gene in more detail, the transcription factor ARABIDOPSIS NAC DOMAIN CONTAINING PROTEIN 13 (ANAC13), which was induced by UV-B but by the activity of any other photoreceptor.

Gumber HK, McKenna JF, Tolmie AF, Jalovec AM, Kartick AC, Graumann K, Bass HW (2019) MLKS2 is an ARM domain and F-actin-associated KASH protein that functions in stomatal complex development and meiotic chromosome segregation Nucleus. doi: 10.1080/19491034.2019.1629795

Open Access

Hardeep Gumber is first author on this US-led study that includes Joe KcKenna, Andrea Tolmie and Katja Graumann from Oxford Brookes as co-authors. They characterise the Maize LINC KASH AtSINE-like2 protein, MLKS2, whose targeting to the nuclear periphery requires its N-terminal armadillo repeats. Mutant mlks2 plants have pleiotropic plant phenotypes and on a nuclear level show defects in chromosome segregation and positioning. These findings support a model in which cytoplasmic actin is linked to chromatin through the LINC-KASH nuclear envelope network.

https://www.tandfonline.com/doi/full/10.1080/19491034.2019.1629795

Temple H, Mortimer JC, Tryfona T, Yu X, Lopez-Hernandez F, Sorieul M, Anders N, Dupree P (2019) Two members of the DUF579 family are responsible for arabinogalactan methylation in Arabidopsis. Plant Direct. doi: 10.1002/pld3.117

Open Access

Henry Temple is first author on this work from the University of Cambridge that characterizes two members of the DUF579 family (AGM1 and AGM2). These proteins are required for 4-O-methylation of glucuronic acid within highly glycosylated arabinogalactan proteins (AGPs).

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