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Silica Nanoparticles Enhance Disease Resistance in Arabidopsis Plants - RAW DATA
<p>These datasets are used to produce the figures/graphs published in our article</p> <p><strong>Silica Nanoparticles Enhance Disease Resistance in <em>Arabidopsis</em> Plants</strong></p> <p>in <em>Nat. Nanotechnol.</em> (2020). <a href="https://doi.org/10.1038/s41565-020-00812-0">https://doi.org/10.1038/s41565-020-00812-0</a></p> <p> </p><p><strong>Correspondence: </strong></p> <p></p> <p>fabienne.schwab@alumni.ethz.ch, Tel: +41 78 736 00 19;</p> <p>m.shetehy@uky.edu, Tel. +41 76 455 56 02</p> <p>Further raw data related to qPCR and microbiology are available upon reasonable request from M.H. El‑Shetehy.</p> <p>Further raw data related to the nanoparticles and plant microscopy are available upon reasonable request by F. Schwab.</p> <p> </p> <p><strong>Abstract</strong></p> <p>In plants, pathogen attack can induce an immune response known as systemic acquired resistance (SAR) that protects against a broad spectrum of pathogens. In the search for safer agrochemicals, silica nanoparticles (SiO<sub>2</sub>‑NPs, food additive E551) have recently been proposed as a new tool. However, initial results are controversial, and the molecular mechanisms of SiO<sub>2</sub>‑NP-induced disease resistance are unknown. Here, we show that SiO<sub>2</sub>‑NPs, as well as soluble orthosilicic acid (Si(OH)<sub>4</sub>), can induce SAR in a dose-dependent manner, that involves the defence hormone salicylic acid. Nanoparticle uptake and action occurred exclusively through stomata (leaf pores facilitating gas exchange) and involved extracellular adsorption in leaf air spaces of the spongy mesophyll. In contrast to treatment with SiO<sub>2</sub>‑NPs, induction of SAR by Si(OH)<sub>4 </sub>was problematic, since high concentrations caused stress. We conclude that SiO<sub>2</sub>‑NPs have the potential to serve as an inexpensive, highly efficient, safe, and sustainable alternative for plant disease protection.</p>
Prediction and analysis of phenotypes in the Arabidopsis clock mutant prr7prr9 using the Framework Model v2 (FMv2)
<p>This upload contains or links to the biological data, FMv2 model and simulations for the Chew et al. 2017 paper (bioRxiv <a href="https://doi.org/10.1101/105437">https://doi.org/10.1101/105437</a> ), updated 2022 as bioRxiv <a href="https://doi.org/10.1101/105437v2">https://doi.org/10.1101/105437v2</a>, mostly testing and simulating the effect of a slow circadian clock in the <em>prr7prr9 </em>double mutant compared to the Col wild type plants, with controls in <em>lsf1 </em>and <em>prr7 </em>single mutants. This is one of the outputs from the EU TiMet project, <a href="https://fairdomhub.org/projects/92">https://fairdomhub.org/projects/92</a>.</p> <p>Several data files contain results generated in the same studies, but not covered by the publication. For example, additional time points (18 or 21 days of growth), many additional metabolites, and additional genotypes including <em>pgm</em>, <em>lhy cca1, </em>and in one case, <em>toc1 </em>and <em>gi</em>.</p> <p>This data archive was updated during submisson to the journal _in Silico _Plants in 2022, and is formatted as a Research Object, generated by the Snapshot function of FairdomHub, based on <a href="https://fairdomhub.org/investigations/123">Investigation https://fairdomhub.org/investigations/123.</a> The same Snapshot is shared on FairdomHub and will be from the University of Edinburgh Datashare.</p> <p>We request that users gives appropriate credit to the authors of any data released here, as a norm of academic practice, including data released under CC-0 licence on the FairdomHub.</p>
Gene expression ATLAS of Arabidopsis thaliana (accession Columbia) across its lifecycle
<p><strong>Abstract: </strong>Arabidopsis thaliana (accession- Columbia) is an important model plant. RNA-Seq based study of 36 gene expression libraries was carried out to explore transcriptional programs operating in different plant parts (seedling, rosette, root, inflorescence, flower, fruit silique, and seed) and developmental stages (2-leaf stage, 6-leaf stage, 12-leaf stage, senescence stage, dry mature and imbibed seed stage). For each tissue type and developmental stage, three individual plants were used as biological replicates.</p> <div><strong><span>Organism part: </span></strong><span>inflorescence, whole plant, seed, root, silique fruit, flower, rosette</span></div> <div> </div> <div><span><strong>Developmental stage:</strong> </span><span>LP.02 two leaves visible stage, IL.00 inflorescence just visible stage, fruit size 30 to 50% stage, LP.12 twelve leaves visible stage, root development stage, fruit size 70% to final stage, LP.06 six leaves visible stage, dry seed stage, flowering stage, seed imbibition stage, sporophyte senescent stage, inflorescence development stage</span></div> <div> </div> <div> <div><strong><span>Organism: </span></strong><span>Arabidopsis thaliana</span></div> <div> </div> <div><span><strong>Ecotype:</strong> </span><span>Col-0</span></div> <div> </div> <div><strong><span>Genotype: </span></strong><span>wild type genotype</span></div> <div> </div> <div><span><strong>Age:</strong> Samples are from </span><span>20-day, 49-day, 39-day, 15-day, 21-day, 9-day, 22-day, 55-day, 26-day, 45-day</span></div> <div> </div> <div><span><strong><span>Experimental Designs: </span></strong><span>growth chamber study<a title="" href="https://www.ebi.ac.uk/ols4/ontologies/efo/terms?iri=http://purl.obolibrary.org/obo/EO_0007269" target="_blank" rel="noopener"> EFO</a></span>, <span>development or differentiation design<a title="" href="https://www.ebi.ac.uk/ols4/ontologies/efo/terms?iri=http://www.ebi.ac.uk/efo/EFO_0001746" target="_blank" rel="noopener"> EFO</a></span>, <span>organism part comparison design<a title="" href="https://www.ebi.ac.uk/ols4/ontologies/efo/terms?iri=http://www.ebi.ac.uk/efo/EFO_0001750" target="_blank" rel="noopener"> EFO</a></span></span></div> <div> </div> <div><span>For more description of the data and sample types see the file <a href="../api/records/11133989/draft/files/PRJEB24664_Sample_descriptors.xlsx/content" target="_blank" rel="noopener noreferrer">PRJEB24664_Sample_descriptors.xlsx or visit </a> or visit <a href="https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-6422/sdrf">https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-6422/sdrf</a></span></div> <div> </div> <div><span>Original data was submitted from </span></div> <div> <ul> <li><span>EMBL-EBI ArraExpress: <a href="https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-6422">https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-6422</a></span></li> <li><span>NCBI SRA: <a href="https://www.ncbi.nlm.nih.gov/bioproject/PRJEB24664">https://www.ncbi.nlm.nih.gov/bioproject/PRJEB24664</a></span></li> </ul> <p><strong><span>Protocol description:</span></strong></p> <table> <tbody><tr> <th>Name</th> <th>Type</th> <th>Description</th> <th>Hardware</th> </tr> </tbody><tbody> <tr> <td>P-MTAB-71349</td> <td><span>growth protocol<a title="" href="https://www.ebi.ac.uk/ols4/ontologies/efo/terms?iri=http://www.ebi.ac.uk/efo/EFO_0003789" target="_blank" rel="noopener"> EFO</a></span></td> <td>Seeds were planted in pots containing commercial potting mix with fertilizers. Pots were covered with clear perforated plastic wrap and kept at 4 degrees celsius for 3 days to break the dormancy. After 3 days plants were transferred to the Intellus Ultra growth chamber (Percival Scientific, IA, USA) which was set to temperature 22-23 degrees celsius, light intensity 120-150 micromol/m2sec under the cycle of 16h light and 8h dark. Soil was kept moist by gently spraying with water every 72 hours to maintain humidity to 50-60%. Sampling time point is given in days after germination.</td> <td> </td> </tr> <tr> <td>P-MTAB-71350</td> <td><span>nucleic acid extraction protocol<a title="" href="https://www.ebi.ac.uk/ols4/ontologies/efo/terms?iri=http://www.ebi.ac.uk/efo/EFO_0002944" target="_blank" rel="noopener"> EFO</a></span></td> <td>Total RNA from frozen samples was extracted as a method described in Filichkin et al., 2010. Total RNA was used to isolate large RNA as per manufacturer's protocol for miRNeasy Mini kits (Qiagen Inc., USA), and RNase-free DNase (Life Technologies Inc., USA).</td> <td> </td> </tr> <tr> <td>P-MTAB-71351</td> <td><span>nucleic acid library construction protocol<a title="" href="https://www.ebi.ac.uk/ols4/ontologies/efo/terms?iri=http://www.ebi.ac.uk/efo/EFO_0004184" target="_blank" rel="noopener"> EFO</a></span></td> <td>True-Seq kit (Illumina Inc.) was used to prepare RNA-seq libraries, according to the manufacturer’s protocol.</td> <td> </td> </tr> <tr> <td>P-MTAB-71352</td> <td><span>nucleic acid sequencing protocol<a title="" href="https://www.ebi.ac.uk/ols4/ontologies/efo/terms?iri=http://www.ebi.ac.uk/efo/EFO_0004170" target="_blank" rel="noopener"> EFO</a></span></td> <td>101bp paired-end sequencing of mRNA was performed by using the standard protocols on Illumina HiSeq 3000.</td> <td>Illumina HiSeq 3000</td> </tr> </tbody> </table> </div> </div>
Arabidopsis thaliana circadian mRNA-seq gene expression processed tables from Romanowski et al., TPJ 2020.
<p>This dataset is an add-on for Romanowski et al., TPJ 2020 (https://doi.org/10.1111/tpj.14776) containing processed files for the circadian RNAseq data in tab delimited txt format.</p> <p><br> Here, you can the raw counts file, the normalized CPM values, and the full JTK result (without recalculated circadian phases, just the original ones). All genes with a read density > 0.05 in at least one timepoint were considered expressed. The read density is calculated as the amount of reads divided by the effective length of a gene (total reads / length). Genes rd file is also included.</p> <p>Some useful notes:<br> 1) Counts were assigned using ASpli and the AtRTDv2 annotation (34,212 genes).<br> 2) After filtering by rd we had a total of 18,503 expressed genes.<br> 3) 13,256 genes passed the QL F-tests.<br> 4) 9,127 genes were rhythmic according to JTK_cycle. </p> <p>For detailed protocols, please see Romanowski et al., TPJ 2020 (https://doi.org/10.1111/tpj.14776)</p> <p>The RNA-seq raw data supporting the conclusions of this article have been deposited in ArrayExpress (Kolesnikov et al., 2015) at EMBL-EBI (www.ebi.ac.uk/arrayexpress), under accession numbers E-MTAB-7933.</p> <p>All relevant custom r scripts are available at https://github.com/aromanowski/Circadian_rhythms_and_alternative_splicing</p>
Arabidopsis thaliana images used in the paper entitled "Led Color Gradient As A New Screening Tool For Rapid Phenotyping Of Plant Responses To Light Quality" by Pierre LEJEUNE et al.
<p><em>Arabidopsis thaliana</em> images used in the paper entitled "Led Color Gradient As A New Screening Tool For Rapid Phenotyping Of Plant Responses To Light Quality" by Pierre LEJEUNE, Anthony FRATAMICO, Frédéric BOUCHÉ, Samuel HUERGA-FERNÁNDEZ, Pierre TOCQUIN, Claire PÉRILLEUX</p>
The proximity interactome of the peach-potato aphid (Myzus persicae) cathepsin B in Arabidopsis thaliana
<p><strong>Introduction</strong></p> <p>In agriculture, the peach-potato aphid <em>Myzus persicae</em> (Sulzer) has one of the broadest host ranges among insects and cause devastating crop losses worldwide (CABI, 2022). They are highly adaptable, displaying a wide range of plastic responses to environmental cues, including the ability to develop as either winged or wingless forms and to reproduce through either asexual or sexual means (Brisson, 2010; Ogawa and Miura, 2014; Grantham and Brisson, 2018). Remarkably, <em>M. persicae </em>differentially regulate the transcription of certain gene clusters to facilitate colonization of diverse plant species (Mathers et al., 2017; Chen et al., 2020). Among these gene clusters are members of the cysteine protease family, cathepsin B (CathB).</p> <p>Host responsive CathB genes are organized in tandemly repeated clusters in the <em>M. persicae</em> genome and belong to a recently expanded clade in phylogeny (Mathers et al., 2017). They are upregulated when aphids feed on <em>Arabidopsis thaliana </em>and <em>Brassica rapa</em> and knock down of their expression using RNA interference reduces aphid reproduction on <em>A. thaliana </em>(Chen et al., 2020). Intriguingly, peptides corresponding to CathB proteins are detected in <em>M. persicae</em> oral secretion (OS), indicating that at least some CathB proteins are directly delivered into plant cells during aphid feeding (Guo et al., 2020; Liu et al., 2024).</p> <p>Among <em>M. persicae</em> CathB proteins, CathB6 is most highly expressed in aphids on <em>A. thaliana</em> (Chen et al., 2020) and most abundant in <em>M. persicae</em> OS (Liu et al., 2024). To identify the potential plant targets of <em>M. persicae</em> CathB, we optimized the TurboID-based proximity labelling and MS (PL-MS) protocol (Fig. 1).</p> <p>As a first step, we generated stable transgenic <em>A. thaliana</em> lines producing GFP or CathB6 as C-terminal TurboID-3×FLAG fusions (GFP-TurboID or CathB6-TurboID). Seedlings of these plants were treated with biotin followed by affinity capture with streptavidin beads (Fig. 2A). Enrichment of biotinylated proteins was confirmed by western blotting (Fig. 2B), followed by nanoLC-MS/MS analyses.</p> <p>Principal component analysis (PCA) of the MS data showed that the three CathB6-TurboID samples were grouped together, separately from three GFP-TurboID samples (Fig. 2C). Furthermore, MA plot confirmed that the CathB6-TurboID and GFP-TurboID samples are distinct (Fig. 2D). From the complete dataset, 267 <em>A. thaliana</em> proteins exhibited statistically significant enrichment (<em>p</em>-value < 0.05) of more than 2-fold and were consistently identified in at least two replicates of the CathB6-TurboID samples compared to the GFP-TurboID controls (Fig. 2E, Table 1). This compares to 223 proteins in the GFP-TurboID samples versus CathB6-TurboID samples (Fig. 2E, Table 1). Additionally, we identified 20 unique peptides corresponding to CathB6 in the CathB6-TurboID samples and 19 unique peptides corresponding to GFP in the GFP-TurboID samples (Table 1). These data suggest that this PL-MS protocol worked dnd identified genuine interactors of CathB6.</p> <p>Together, this dataset identifies 267 potential plant interactors of aphid CathB6, which may contribute to CathB6 modulation of <em>A. thaliana</em> plant for colonization. Further mechanistic studies should be done to characterize if these potential interactors are involved and how the relevant pathways are affected after CathB delivery through aphid feeding.</p> <p> </p> <p><strong>Materials and Methods</strong></p> <p><em>Plasmid construction</em></p> <p>For the construction of plasmids producing CathB6-TurboID-3×FLAG, the coding sequences corresponding to the catalytic domain (without signal peptide and prodomain regions) of CathB6 (Arg61-Asn338) and TurboID-3×FLAG were separately amplified. Then, the two fragments were connected using overlap PCR (Nelson and Fitch, 2011). After cloning of the sequence corresponding to the CathB6-TurboID-3×FLAG fragment into the pJET vector and sequencing, CathB6-TurboID-3×FLAG was amplified with primers containing <em>attB</em> extensions and cloned into the pDONOR207 vector, followed by the ligation to Gateway destination vector pB7WG2 containing a 35S promoter. Similar cloning methods were used for construction of GFP-TurboID-3×FLAG.</p> <p><em>Plant transformation</em></p> <p>The constructed plasmids were introduced into <em>Agrobacterium tumefaciens</em> strain GV3101, and the cultures were grown on plates at 28 °C for 24–48 hrs. Then, positive colonies were identified via PCR using plasmids extracted from overnight liquid cultures and gene-specific primers. Positive colonies were grown at 28 °C in liquid cultures and transformed into <em>A. thaliana</em> Col-0 plants using the floral dipping method (Bechtold, 1993). Transgenic seeds were harvested and selected on Murashige and Skoog (MS) medium supplemented with 20 μg/mL phosphinothricin (BASTA) and screened for ratio of 3:1 alive/dead segregation. After screening for two or three generations, transgenic plants were deemed to harbor single homozygous transgenes and were used for proximity labeling once germinated seeds achieved a 100% survival rate.</p> <p><em>Proximity labelling</em></p> <p>Seeds of <em>A. thaliana</em> plants stably expressing GFP-TurboID-3×FLAG or CathB6-TurboID-3×FLAG were sowed on ½ MS plates containing 1.0% sucrose and 0.3% phytagel and placed under long-day condition (16 h light/8 h dark) at 22 °C. After 10 days, 2.5 g seedlings were collected and submerged in 50 µM biotin solution for 4 hrs at RT. Afterwards, seedlings were rinsed with ice-cold MilliQ water for 5 times. After removing excess liquid with paper towel, seedings were ground with pestle, mortar and nitrogen to a fine powder. Protein extraction was performed in 5 mL of extraction buffer [150 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% Glycerol, 10 mM DTT, 0.4% Nonidet-40, 0.1% (w/v) Deoxycholic acid, 2% (w/v) PVPP, 1 tablet of cOmplete protease Inhibitor cocktail (Roche, Catalog number 10697498001)] and incubation on a rotor wheel at 4 °C for 30 min, followed by centrifugation of the tubes at 5000 g for 15 min to remove the cell debris. The upper soluble fraction was then run through the Zeba Spin Desalting Column (Thermo Fisher Scientific, Catalog number 89893) to remove excess biotin from the lysates. Fifty (50) µL of desalted lystate was used as input for western blot analysis, while the rest of the desalted lysate was incubated with High Capacity Streptavidin Agarose Resin (Thermo Fisher Scientific, Catalog number 20361) on a rotor wheel at 4 °C overnight. The next day, Streptavidin beads were sequentially washed once in 1 mL Buffer 1 (2% SDS in water), once in 1 mL Buffer 2 [150 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.1% (w/v) Deoxycholic acid (w/v), 1% Triton X-100], once in buffer 3 [10 mM Tris-HCl (pH 7.4), 250 mM LiCl, 1 mM EDTA, 0.1% (w/v) Deoxycholic acid, 1% (v/v) NP40], twice in Buffer 4 [50mM Tris-HCl (pH 7.5)], and six times in Buffer 5 (50mM ammonium bicarbonate, pH 8.0). Finally, the streptavidin beads were resuspended in 200 µL of 50 mM ammonium bicarbonate. For quality control of the TurboID immunoprecipitation, 10% (20 µL) of the suspension was taken out for Western blot analysis, and the remaining bead suspension was flash-frozen in liquid nitrogen and stored at -80 °C and submitted to nano LC-MS/MS analysis.</p> <p>For western blot analysis, 20 µL of suspended streptavidin beads in washing buffer 5 were added to 10 µL of 4× LDS Sample Loading Buffer, 10 mM DTT and 2 mM biotin, and boiled for 10 min. Samples were loaded onto 12% SDS-PAGE gels (Invitrogen) and transferred to 0.22 μm PVDF membranes using the Bio-Rad mini-PROTEAN Electrophoresis system. Membranes were hybridized with Streptavidin-HRP.</p> <p><em>NanoLC-MS/MS</em></p> <p><em> </em>Biotinylated proteins enriched with streptavidin beads were processed with trypsin via on bead digestion. The beads were washed in water and resuspended in of 1.5% sodium deoxycholate (SDC; Merck) in 0.2 M EPPS-buffer (Merck) to 50% bead slurry vol/vol, pH 8.5 and vortexed under heating. Cysteine residues were reduced with dithiothreitol, alkylated with iodoacetamide, and the proteins digested with trypsin in the SDC buffer according to standard procedures for 8 hrs. The beads were then pelleted by centrifugation and the supernatant was collected for SDC precipitation by adding trifluoroacetic acid (TFA) to a final concentration of 0.2%. The clear supernatant was subjected to C18 SPE using home-made stage tips with C18 Reprosil_pur 120, 5 µm (Dr. Maisch GmbH, Germany). Aliquots were analyzed by nano LC-MS/MS on an Orbitrap Eclipse™ Tribrid™ mass spectrometer equipped with a FAIMS Pro Duo interface coupled to an UltiMate® 3000 RSLC nano LC system (Thermo Fisher Scientific, Hemel Hempstead, UK). The samples were loaded onto a trap cartridge (PepMap™ Neo Trap Cartridge, C18, 5um, 0.3x5mm, Thermo) with 0.1% TFA at 15 µl min-1 for 3 min. The trap column was then switched in-line with the analytical column (Aurora Frontier TS, 60 cm nanoflow UHPLC column, ID 75 µm, reversed phase C18, 1.7 µm, 120 Å; IonOpticks, Fitzroy, Australia) for separation at 55°C using the following gradient of solvents: A (water, 0.1% formic acid) and B (80% acetonitrile, 0.1% formic acid) at a flow rate of 0.26 µl min-1 : 0-3 min 1% B (parallel to trapping); 3-10 min increase B (curve 4) to 8%; 10-102 min linear increase B to 48; followed by a ramp to 99% B and re-equilibration to 0% B. Total runtime was 140 min.</p> <p>Mass spectrometry data were acquired between 10 and 110 min with the FAIMS device set to three compensation voltages (-35V, -50V, -65V) at standard resolution for 1.0 s each with the following MS settings in positive ion mode: OT resolution 120K, profile mode, mass range m/z 300-1600, normalized AGC target 100%, max inject time 50 ms; MS2 in IT Turbo mode: quadrupole isolation window 1 Da, charge states 2-5, threshold 1e4, HCD CE = 30, AGC target standard, max. injection time dynamic, dynamic exclusion 1 count for 15 s with mass tolerance of ±10 ppm, one charge state per precursor only.</p> <p>The mass spectrometry raw data were processed and quantified in Proteome Discoverer 3.1 (PD3.1) (Thermo) using the search engine CHIMERYS (MSAID, Munich, Germany); all mentioned tools of the following workflow are nodes of the proprietary Proteome Discoverer (PD) software. The <em>A. thaliana</em> protein sequence database (TAIR10, 35,386 entries, from 14/12/2010), the two sequences of the used TurboID constructs, and the MaxQuant contaminants database (240812, 246 entries) were imported into PD adding a reversed sequence database for decoy searches.</p> <p>The database search was performed using the search engine CHIMERYS (MSAID, Munich, Germany). The processing workflow started with spectrum recalibration, Minora Feature Detection with min. trace length 5, S/N 2.5, PSM confidence high, and Top N Peak Filter with 20 peaks per 100 Da. For CHIMERYS, the inferys_3.0.0_fragmentation prediction model with FDR targets 0.01 (strict) and 0.05 (relaxed), a fragment tolerance of 0.3 Da, enzyme trypsin with 2 missed cleavages, variable modification oxidation (M), fixed modification carbamidomethyl (C) were used.</p> <p>The consensus workflow in the PD3.1 software was used to evaluate the peptide identifications and to measure the abundances of the peptides based on the LC-peak intensities. For chromatographic alignment and feature mapping, a retention time tolerance of 2 min, a mass tolerance of 1 ppm, and an S/N threshold of 5 were used. For quantification, three replicates per condition were measured. In PD3.1, the following parameters were used for ratio calculation: normalization on total peptide abundances, protein abundance-based ratio calculation using the Top3 most abundant peptides, missing values imputation by low abundance resampling, hypothesis testing by t-test (background based), adjusted <em>p</em>-value calculation by BH-method. The results were exported into a Microsoft Excel table including data for protein abundances, ratios, <em>p</em>-values, number of peptides, protein coverage, the CHIMERYS identification score and other important values.</p> <p> </p> <p><strong>Data availability statement</strong></p> <p>The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier with the dataset identifier PXD057789 and 10.6019/PXD057789.</p> <p> </p> <p><strong>Acknowledgements</strong></p> <p><strong> </strong>This research was funded by UK Research and Innovation (UKRI) Biotechnology and Biological Sciences Research Council (BBSRC) grants to SAH (BB/V008544/1 and BB/R009481/1). Additional Support is provided by the BBSRC Institute Strategy Programmes (BBS/E/J/000PR9797 and BBS/E/JI/230001B) awarded to the JIC. The JIC is grant-aided by the John Innes Foundation.</p> <p> </p> <p><strong>Conflicts of Interest</strong></p> <p><strong> </strong>The authors declare that no conflicts of interest exist.</p> <p> </p> <p><strong>Legends of figures and tables</strong></p> <p><strong>Figure 1. Principle of CathB6-TurboID based proximity labelling with MS (PL-MS). </strong>The TurboID biotin ligase (TurboID) is fused to C-terminus of CathB6. Exogenous addition of biotin (yellow stars) biotinylates proteins in the proximity of CathB6-TurboID fusion protein, whereas distal proteins are not biotinylated. The biotinylated proteins are captured by incubating total proteins extracts with streptavidin beads. Peptides derived from biotinylated proteins, most of which are in the proximity of CathB6-TurboID, are detected by nanoLC-MS.</p> <p><strong>Fig. 2. Sample preparation and quantification for CathB6-TurboID interactome in <em>A. thaliana</em>. </strong>(<strong>A</strong>) Sample preparation working flow for TurboID-based proximity labeling. GFP-TurboID and CathB6-TurboID seedlings were treated with 50 µM biotin for 4 hrs at room temperature. (<strong>B</strong>) Visualization on western blots of biotinylated proteins detected after desalting step (input) and 12 wash steps of Streptavidin beads (Streptavidin IP) as per workflow shown in (A). (<strong>C</strong>) Principal component analysis (PCA) of three replicates of GFP-TurboID and CathB6-TurboID samples. (<strong>D</strong>) MA plot of three replicates of GFP-TurboID and CathB6-TurboID samples. (<strong>E</strong>) Venn diagrams showing the overlap of proteins identified in three biological replicates of GFP-TurboID (left) and CathB6-TurboID (right) upon a fold-change of CathB6-TurboID/GFP-TurboID > 2, n = 267. </p> <p><strong>Table 1. Full list of proteins detected from CathB6-TurboID PL-MS.</strong></p> <p> </p> <p> </p> <p><strong>References</strong></p> <p><strong>Bechtold, N.</strong> (1993). In planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. CR Acad. Sci. Paris, Life Sci. <strong>316, </strong>1194-1199.</p> <p><strong>Brisson, J.A.</strong> (2010). Aphid wing dimorphisms: linking environmental and genetic control of trait variation. Philos Trans R Soc Lond B Biol Sci <strong>365, </strong>605-616.</p> <p><strong>CABI, C.f.A.a.B.I.</strong> (2022). Myzus persicae (green peach aphid). CABI Compendium.</p> <p><strong>Chen, Y., Singh, A., Kaithakottil, G.G., Mathers, T.C., Gravino, M., Mugford, S.T., van Oosterhout, C., Swarbreck, D., and Hogenhout, S.A.</strong> (2020). An aphid RNA transcript migrates systemically within plants and is a virulence factor. Proc Natl Acad Sci U S A <strong>117, </strong>12763-12771.</p> <p><strong>Grantham, M.E., and Brisson, J.A.</strong> (2018). Extensive Differential Splicing Underlies Phenotypically Plastic Aphid Morphs. Mol Biol Evol <strong>35, </strong>1934-1946.</p> <p><strong>Guo, H., Zhang, Y., Tong, J., Ge, P., Wang, Q., Zhao, Z., Zhu-Salzman, K., Hogenhout, S.A., Ge, F., and Sun, Y.</strong> (2020). An Aphid-Secreted Salivary Protease Activates Plant Defense in Phloem. Curr Biol <strong>30, </strong>4826-4836.e4827.</p> <p><strong>Liu, Q., Goldberg, J.K., Mugford, S.T., Saalbach, G., Martins, C., Singh, A., Kaithakotti, G.G., Swarbreck, D., and Hogenhout, S.A.</strong> (2024). The salivary proteome of the green peach aphid/peach-potato aphid (Myzus persicae) (Sulzer, 1776) (Hemiptera, Aphididae) (Zenodo).</p> <p><strong>Mathers, T.C., Chen, Y., Kaithakottil, G., Legeai, F., Mugford, S.T., Baa-Puyoulet, P., Bretaudeau, A., Clavijo, B., Colella, S., Collin, O., Dalmay, T., Derrien, T., Feng, H., Gabaldón, T., Jordan, A., Julca, I., Kettles, G.J., Kowitwanich, K., Lavenier, D., Lenzi, P., Lopez-Gomollon, S., Loska, D., Mapleson, D., Maumus, F., Moxon, S., Price, D.R., Sugio, A., van Munster, M., Uzest, M., Waite, D., Jander, G., Tagu, D., Wilson, A.C., van Oosterhout, C., Swarbreck, D., and Hogenhout, S.A.</strong>(2017). Rapid transcriptional plasticity of duplicated gene clusters enables a clonally reproducing aphid to colonise diverse plant species. Genome Biol <strong>18, </strong>27.</p> <p><strong>Nelson, M.D., and Fitch, D.H.</strong> (2011). Overlap extension PCR: an efficient method for transgene construction. Methods Mol Biol <strong>772, </strong>459-470.</p> <p><strong>Ogawa, K., and Miura, T.</strong> (2014). Aphid polyphenisms: trans-generational developmental regulation through viviparity. Front Physiol <strong>5, </strong>1.</p>
Isoprenoid Gene Network in Arabidopsis Thaliana
<p>To gain more insight into the cross-talk between both pathways at the transcriptional level, gene-expression patterns were monitored under various experimental conditions using 118 GeneChip (Affymetrix) microarrays. To construct a genetic regulatory network in Wille et al. (2004), the interventional dataset focusses on 40 genes, 16 of which were assigned to the cytosolic pathway, 19 to the plastidal pathway and five encode proteins located in the mitochondrion.</p> <p> </p> <p><strong>Task: </strong>The dataset can be used to study causal discovery algorithms.</p> <p> </p> <p><strong>Summary: </strong></p> <ul> <li><strong>Size of dataset</strong>: 118 x 41</li> <li><strong>Task:</strong> Causal Discovery Problem</li> <li><strong>Data Type:</strong> Continuous Data</li> <li><strong>Dataset Scope:</strong> Standalone Dataset</li> <li><strong>Ground Truth:</strong> Known Graph</li> <li><strong>Temporal Structure:</strong> Static Data</li> <li><strong>License:</strong> CC BY 2.0 (see https://genomebiology.biomedcentral.com/articles/10.1186/gb-2004-5-11-r92#MOESM1)</li> <li><strong>Missing Values:</strong> No Missing Values</li> </ul> <p> </p> <p><strong>Missingness Statement: </strong>There are no missing values.<strong><br></strong></p> <p> </p> <p><strong>Samples:</strong></p> <p>Experimental procedures involved seedlings, leaves and roots. For experiments involving seedlings or leaves, plants were grown in growth chambers at 70% humidity and daily cycles of 16 h light at 21◦C and 8 h darkness at 21◦C. Plant material from three independent experiments (replications not in parallel) for each experiment group respectively was pooled prior to RNA extraction. The 118 different experimental conditions are denoted by c1-c118:</p> <ul> <li><strong>c1-c2: </strong>Experiment with wild-type and era mutant seedlings. (growth stage 1.0; tissue: whole seedlings)</li> <li><strong>c3-c5:</strong> Arabidopsis tissue culture, leaf and seedling in a baseline experiment (growth stage: - ; tissue: tissue culture, seedling, adult leaf).</li> <li><strong>c6-c14: </strong>RNA was extracted from seedlings and adult leaves of wild-type and prenylation mutant plants grown under standard conditions (growth stage 1.0; tissue: whole seedlings and adult leaves).</li> <li><strong>c15-c22: </strong>RNA was extracted from wild-type and several transgenic seedlings (growth stage: 1.0; tissue: whole seedlings).</li> <li><strong>c23-c30:</strong> RNA was extracted from a root inducible system exposed to hormonal treatments. (growth stage: 1.0; tissue: lateral roots).</li> <li><strong>c31-c56: </strong>Arabidopsis seedlings were exposed to light and dark conditions in a time-course experiment (0, 10 min, 1h, 5h, 2d, 5d). (growth stage: 1.0; tissue: whole seedlings).</li> <li><strong>c70-c92:</strong> Experiment to assess the effect of inhibitors of the MVA pathway (lovastatin) and the MVA-independent pathway (fosmidomycin) on the expression of genes involved in isoprenoid biosynthesis. (growth stage: 1.0 and 3.90; tissues: whole seedlings and adult leaves).</li> <li><strong>c93-c118:</strong> Arabidopsis seedlings and adult leaves were exposed to ozone for several periods of time. (growth stage: 1.0 and 3.90; tissues: whole seedlings, cauline leaves and adult leaves).</li> </ul> <p> </p> <p><strong>Features: </strong></p> <ul> <li><strong>c[...]: </strong>This row indicates the experiment as descried above</li> <li> <p><strong>AACT1, AACT2, CMK, DPPS1, ...:</strong> Gene expressions level. The column names indicate the gene name.</p> </li> </ul> <div> <p> </p> <p><strong>Note: </strong>Although the gene UPPS2 was analyzed in Willie et al. (2004), it is not provided in its supporting material and in this dataset.</p> </div>
Locally adaptive temperature response of vegetative growth in Arabidopsis thaliana
<p>We investigated early vegetative growth of natural <em>Arabidopsis thaliana</em> accessions in cold, non-freezing temperatures, similar to temperatures these plants naturally encounter in fall at northern latitudes.</p> <p>Dataset includes:<br> - rosette area measurements over 3 weeks in a 16ºC and a 6ºC treatment. First phenoptying time point is at 14 days after stratification. Measurements were take twice per day.<br> These data are in file <a href="https://zenodo.org/api/files/54fc7139-99a8-4e7f-af87-d6754031f5d9/rawdata_combined_annotation.txt?versionId=7b707f81-723f-4059-b72b-9dfb9f5ddd2e">rawdata_combined_annotation.txt</a> and go together with <a href="https://zenodo.org/api/files/54fc7139-99a8-4e7f-af87-d6754031f5d9/outliers.csv?versionId=7287c919-1ed1-4b65-8e25-a75bb312c8fa">outliers.csv</a>, which contains outlying datapoints.</p> <p>- Seed Size measurements.<br> These data are in file <a href="https://zenodo.org/api/files/54fc7139-99a8-4e7f-af87-d6754031f5d9/seed_size_swedes_lab_updated.csv?versionId=fb739477-862b-45cb-8074-7a1d8e1650bb">seed_size_swedes_lab_updated.csv </a><br> </p> <p>The remainnig files are required to rerun the analyses and recreate figures.<br> Scripts to do so can be found in https://github.com/picla/growth_16C_6C/</p> <p><a href="https://zenodo.org/api/files/54fc7139-99a8-4e7f-af87-d6754031f5d9/1001genomes-accessions.csv?versionId=ee605038-bd9e-448f-9c96-1a8e980c1755">1001genomes-accessions.csv</a>: lists all accession from the 1001genomes project and their respective subpopulations.</p> <p><a href="https://zenodo.org/api/files/54fc7139-99a8-4e7f-af87-d6754031f5d9/2029_modified_MN_SH_wc2.0_30s_bilinear.csv?versionId=73c6c2bf-97bd-425f-bf7e-14b5a7cb162f">2029_modified_MN_SH_wc2.0_30s_bilinear.csv</a>: contains climate data for each accession, downloaded and prcocessed from www.worldclim.org</p> <p><a href="https://zenodo.org/api/files/54fc7139-99a8-4e7f-af87-d6754031f5d9/metabolic_distance.csv?versionId=8456f998-d0dc-4a80-b96f-c0c66c1c9731">metabolic_distance.csv</a>: contains the metabolic distance as calculated in Weiszmann et al. (https://www.biorxiv.org/content/10.1101/2020.09.24.311092v1)</p> <p><a href="https://zenodo.org/api/files/54fc7139-99a8-4e7f-af87-d6754031f5d9/RNAseq_samples.txt?versionId=6ae1518b-1a70-440d-b0bd-0ccdcb66665e">RNAseq_samples.txt</a>: sample description of the RNA-seq samples (data is downloadable from <a href="http://www.ncbi.nlm.nih.gov/bioproject/807069">http://www.ncbi.nlm.nih.gov/bioproject/807069)</a></p> <p><a href="https://zenodo.org/api/files/54fc7139-99a8-4e7f-af87-d6754031f5d9/ZAT12_downregulated_table10.csv?versionId=c6f7aa54-cb07-4378-a5a0-de12c6979b9b">ZAT12_downregulated_table10.csv</a>, <a href="https://zenodo.org/api/files/54fc7139-99a8-4e7f-af87-d6754031f5d9/ZAT12_upregulated_table9.csv?versionId=911a2aa2-f08f-4a20-85de-cfa7c58b73a8">ZAT12_upregulated_table9.csv</a>, <a href="https://zenodo.org/api/files/54fc7139-99a8-4e7f-af87-d6754031f5d9/CBF_regulon_DOWN_ParkEtAl2015.txt">CBF_regulon_DOWN_ParkEtAl2015.txt</a>, <a href="https://zenodo.org/api/files/54fc7139-99a8-4e7f-af87-d6754031f5d9/CBF_regulon_UP_ParkEtAl2015.txt?versionId=f7cacbda-eea6-4ac9-8f71-5ba74e3a67c4">CBF_regulon_UP_ParkEtAl2015.txt, </a><a href="https://zenodo.org/api/files/54fc7139-99a8-4e7f-af87-d6754031f5d9/CBF2_downregulated_table8.csv">CBF2_downregulated_table8.csv, </a><a href="https://zenodo.org/api/files/54fc7139-99a8-4e7f-af87-d6754031f5d9/CBF2_upregulated_table7.csv">CBF2_upregulated_table7.csv, </a><a href="https://zenodo.org/api/files/54fc7139-99a8-4e7f-af87-d6754031f5d9/HSFC1_regulon_ParkEtAl2015.txt">HSFC1_regulon_ParkEtAl2015.txt</a>: these files list genes that are involve din cold acclimation as described by Park et al. (https://onlinelibrary.wiley.com/doi/10.1111/tpj.12796), and Vogel et al.(https://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2004.02288.x).</p> <p><strong>Material and Methods</strong></p> <p><em><strong>Rosette growth</strong></em></p> <p>Seeds of 249 natural accessions (Suppl. Data 1) of <em>Arabidopsis thaliana</em> described in the 1001 genomes project <a href="https://paperpile.com/c/UDgV3V/DUBI">(1001 Genomes Consortium 2016)</a> were sown on sieved (6 mm) substrate (Einheitserde ED63). Pots were filled with 71.5 g ±1.5 g of soil to assure homogenous packing. The prepared pots were all covered with blue mats <a href="https://paperpile.com/c/UDgV3V/1WUv">(Junker et al. 2014)</a> to enable a robust performance of the high-throughput image analysis algorithm. Seeds were stratified (4 days at 4ºC in darkness) after which they germinated and left to grow for 2 weeks at 21ºC (relative humidity: 55 %; light intensity: 160 µmol m-2 s-1; 14 h light). The temperature treatments were started by transferring the seedlings to either 6 °C or 16 °C. To simulate natural conditions temperatures fluctuated diurnally between 16-21 °C, 0.5-6 °C and 8-16 °C for the 21 °C initial growth conditions and the 6 °C and 16 °C treatments, respectively (<a href="https://docs.google.com/document/d/1Bmr7p24ZMh4yPFVV5oPeH2-T5S41TOFDS3au8JhtwsU/edit#fig_design">Fig.2</a>). Light intensity was kept constant at 160 µmol m-2 s-1 throughout the experiment. Relative humidity was set at 55% but in colder temperatures it rose uncontrollably to maximum 95%. Daylength was 9h during the 16°C and 6°C treatments.</p> <p>Each temperature treatment was repeated in three independent experiments. Five replicate plants were grown for every genotype per experiment. Plants were randomly distributed across the growth chamber with an independent randomisation pattern for each experiment. During the temperature treatments (14 DAS – 35 DAS), plants were photographed twice a day (1 hour. after/before lights switched on/off), using an RGB camera (IDS uEye UI-548xRE-C; 5MP) mounted to a robotic arm. At 35 DAS, whole rosettes were harvested, immediately frozen in liquid nitrogen and stored at -80 °C until further analysis. Rosette areas were extracted from the plant images using Lemnatec OS (LemnaTec GmbH, Aachen, Germany) software.</p> <p><em><strong>Seed size</strong></em></p> <p>We used the seeds produced by <a href="https://paperpile.com/c/UDgV3V/Jqsd">(Kerdaffrec et al. 2016)</a> and limited our measurements to the set of 123 Swedish accessions that overlapped with our growth dataset. After seed stratification for four days at 4ºC in darkness, mother plants were grown for 8 weeks at 4ºC under long-day conditions (16h light; 8h dark) to ensure proper vernalization. Temperature was raised to 21ºC (light) and 16ºC (dark) for flowering and seed ripening. Seeds were kept in darkness at 16ºC and 30% relative humidity, from the harvest until seed size measurements. For each genotype three replicates were pooled and about 200-300 seeds were sprinkled on 12 x 12 cm square, transparent Petri dishes. Image acquisition was performed as described in <a href="https://paperpile.com/c/UDgV3V/WH1e">(Exposito-Alonso et al. 2018)</a> by scanning dishes on a cluster of eight Epson V600 scanners. The resulting 1200 dpi .tiff images were analyzed in the Fiji software. Images were converted to 8-bit binary images and thresholded with the <em>setAutoThreshold("Defaultdark”) </em>command, and seed area was measured in squared mm by running the <em>Analyse Particles</em> command (inclusion parameters: size=0.04-0.25 circularity=0.70-1.00).</p> <p> </p> <p> </p> <p> </p> <p> </p>
Multi-omic analysis of the Arabidopsis clock activator mutant rve 4 6 8 reveals connections to carbohydrate metabolism and proteasome regulation
<p>Plants are able to sense changes in their light environments, such as the onset of day and night, as well as anticipate these changes in order to adapt and survive. Central to this ability is the plant circadian clock, a molecular circuit that precisely orchestrates plant cell processes over the course of a day. REVEILLE proteins (RVEs) are recently discovered members of the plant circadian circuitry that activate the evening complex and PRR genes to maintain regular circadian oscillation. The RVE 8 protein and its two homologs, RVE 4 and 6, have been shown to limit the length of the circadian period, with rve 4 6 8 triple-knockout plants possessing an elongated period along with increased leaf surface area, biomass, cell size and delayed flowering relative to wild-type Col-0 plants. Here, using a multi-omics approach consisting of phenomics, transcriptomics, proteomics, and metabolomics we draw novel connections between RVE8-like proteins and a number of core plant cell processes. In particular, we reveal that loss of RVE8-like proteins results in altered carbohydrate, organic acid and lipid metabolism, including a starch excess phenotype at dawn. We further demonstrate that rve 4 6 8 plants have lower levels of 20S proteasome subunits and possess significantly reduced proteasome activity, potentially explaining the increase in cell-size observed in RVE8-like mutants. Overall, this robust, multi-omic dataset, provides substantial new insights into the far reaching impact RVE8-like proteins have on the diel plant cell environment.<br> <br> This dataset has the raw search outputs for the mass-spec analysis for this manuscript. </p>
Data and code for the publication "DNA methylation underpins the epigenomic landscape regulating genome transcription in Arabidopsis"
<p>The zipped file of this repository contains code and data to reproduce the results of the publication:</p> <p>Zhao et al, DNA methylation underpins the epigenomic landscape regulating genome transcription in Arabidopsis. Genome Biology (2022). </p> <p>All sequence data have been deposited in NCBI GEO accession codes GSE183987 and GSE169497.</p> <p> </p> <p>Please see the README document for detailed:</p> <p>- Descriptions of the code and data provided</p> <p>- Lists of the required dependencies</p>
Intraspecific variation in reproductive barriers between two closely-related Arabidopsis sister species
<p>Reproductive isolation (RI) is a critical component of speciation and varies strongly in timing and strength among different sister taxa, depending e.g.<em>,</em> on the geography of speciation and divergence time. However, these factors may also produce variation in timing and strength among populations within species. Here we tested for variation in the expression of RI among replicate population pairs between the sister taxa <em>Arabidopsis lyrata</em> subsp. <em>lyrata</em> and <em>A. arenicola</em>. While the former is predominantly outcrossing, the latter is predominantly selfing<em>.</em> We focused on intrinsic prezygotic and postzygotic RI as both species occur largely in allopatry. We assessed RI by performing within-population crosses and interspecific between-population crosses, and by raising offspring. RI was generally high between all interspecific population pairs, but it varied in timing and strength depending on population history. Prezygotic isolation was strongest between the closest-related population pair, while early postzygotic isolation was high for all other population pairs. Furthermore, the timing and strength of RI depended strongly on cross direction. Our study provides empirical support that reproductive barriers between species are highly variable among population pairs and asymmetric within population pairs, and this variation seems to follow patterns typically described across species pairs.</p>
In-depth Tissue-Level Dimerization Analysis of AtLEA proteins from Arabidopsis thaliana - Replicate 2
<p>Post 48-hour infiltration, leaves were examined utilizing a 3I Mariana Spinning Disc Confocal microscope equipped with a Zeiss Observer Z.1 Inverted base and a fluorescence spinning disk. In each experimental run, four plants were analyzed, each expressing one of three specific genetic constructs fused to complementary fragments of Yellow Fluorescent Protein: pYFN-4-/5pYFC-4-5 (representing the complete AtLEA4-5 protein), pYFN-4-51-77/pYFC-4-51-77 (encompassing the N-terminal region of AtLEA4-5), pYFN-4-578-158/pYFC-4-578-158 (comprising the C-terminal region of AtLEA4-5),pYFN-4-2/pYFC-4-2 (full length AtLEA4-2 protein), or pYFN-pYFC (serving as the control condition). Observations were conducted using a 20X—/0.8 NA air lens from Zeiss, under illumination provided by a pre-centered fiber illuminator from Intelligent Imaging Innovations, Inc. The detection of YFP fluorescence was facilitated by a 515 nm excitation laser and a 542/27 nm emission filter. Images were captured using an Andor Ixon 3 EMCCD camera (Model: DU-897E-CS0-#BV), adhering to protocols defined in SlideBook software version 6. Images were captured at specified intervals with exposure times ranging from 400 to 700 milliseconds. Each image consisted of a plane with a pixel size of 0.8 microns, spaced at 2 micrometer intervals. A total of 16 planes, correlating to an estimated depth of 32 micrometers per z-plane, were scrutinized. From each plant, up to three leaves were examined. The analysis included over 50 volumetric scenes per plant, with each scene encompassing a volume of 100 x 100 x 25 mm³. Image stacks were exported as *.tif </p> <p>Notation:</p> <p><strong> </strong></p> <p>Exp2_PYF: Replicate2 expressing one of four specific genetic constructs fused to complementary fragments of Yellow Fluorescent Protein.</p> <p>45: Fused to the full length AtLEA4-5 protein (pYFN-4-/5pYFC-4-5).</p> <p>4H: Fused to the N-terminal region of AtLEA4-5 (pYFN-4-51-77/pYFC-4-51-77).</p> <p>RC: Fused to the C-terminal region of AtLEA4-5 (pYFN-4-578-158/pYFC-4-578-158).</p> <p>control: The control condition (pYFN-pYFC).</p> <p>42: Fused to the full length AtLEA4-2 protein (pYFN-4-2/pYFC-4-2).</p> <p> </p>
In-depth Tissue-Level Dimerization Analysis of AtLEA proteins from Arabidopsis thaliana - Replicate 1
<p>Post 48-hour infiltration, leaves were examined utilizing a 3I Mariana Spinning Disc Confocal microscope equipped with a Zeiss Observer Z.1 Inverted base and a fluorescence spinning disk. In each experimental run, four plants were analyzed, each expressing one of three specific genetic constructs fused to complementary fragments of Yellow Fluorescent Protein: pYFN-4-/5pYFC-4-5 (representing the complete AtLEA4-5 protein), pYFN-4-51-77/pYFC-4-51-77 (encompassing the N-terminal region of AtLEA4-5), pYFN-4-578-158/pYFC-4-578-158 (comprising the C-terminal region of AtLEA4-5),pYFN-4-2/pYFC-4-2 (full length AtLEA4-2 protein), or pYFN-pYFC (serving as the control condition). Observations were conducted using a 20X—/0.8 NA air lens from Zeiss, under illumination provided by a pre-centered fiber illuminator from Intelligent Imaging Innovations, Inc. The detection of YFP fluorescence was facilitated by a 515 nm excitation laser and a 542/27 nm emission filter. Images were captured using an Andor Ixon 3 EMCCD camera (Model: DU-897E-CS0-#BV), adhering to protocols defined in SlideBook software version 6. Images were captured at specified intervals with exposure times ranging from 400 to 700 milliseconds. Each image consisted of a plane with a pixel size of 0.8 microns, spaced at 2 micrometer intervals. A total of 16 planes, correlating to an estimated depth of 32 micrometers per z-plane, were scrutinized. From each plant, up to three leaves were examined. The analysis included over 50 volumetric scenes per plant, with each scene encompassing a volume of 100 x 100 x 25 mm³. Image stacks were exported as *.tif </p> <p><strong> </strong>Notation:</p> <p>Exp1_PYF: Replicate1 expressing one of four specific genetic constructs fused to complementary fragments of Yellow Fluorescent Protein.</p> <p>45: Fused to the full length AtLEA4-5 protein (pYFN-4-/5pYFC-4-5).</p> <p>4H: Fused to the N-terminal region of AtLEA4-5 (pYFN-4-51-77/pYFC-4-51-77).</p> <p>RC: Fused to the C-terminal region of AtLEA4-5 (pYFN-4-578-158/pYFC-4-578-158).</p> <p>control: The control condition (pYFN-pYFC).</p> <p>42: Fused to the full length AtLEA4-2 protein (pYFN-4-2/pYFC-4-2).</p> <p> </p>
In-depth Tissue-Level Dimerization Analysis of AtLEA proteins from Arabidopsis thaliana - Replicate 4
<p> Post 48-hour infiltration, leaves were examined utilizing a 3I Mariana Spinning Disc Confocal microscope equipped with a Zeiss Observer Z.1 Inverted base and a fluorescence spinning disk. In each experimental run, four plants were analyzed, each expressing one of three specific genetic constructs fused to complementary fragments of Yellow Fluorescent Protein: pYFN-4-/5pYFC-4-5 (representing the complete AtLEA4-5 protein), pYFN-4-51-77/pYFC-4-51-77 (encompassing the N-terminal region of AtLEA4-5), pYFN-4-578-158/pYFC-4-578-158 (comprising the C-terminal region of AtLEA4-5),pYFN-4-2/pYFC-4-2 (full length AtLEA4-2 protein), or pYFN-pYFC (serving as the control condition). Observations were conducted using a 20X/0.8 NA air lens from Zeiss, under illumination provided by a pre-centered fiber illuminator from Intelligent Imaging Innovations, Inc. The detection of YFP fluorescence was facilitated by a 515 nm excitation laser and a 542/27 nm emission filter. Images were captured using an Andor Ixon 3 EMCCD camera (Model: DU-897E-CS0-#BV), adhering to protocols defined in SlideBook software version 6. Images were captured at specified intervals with exposure times ranging from 400 to 700 milliseconds. Each image consisted of a plane with a pixel size of 0.8 microns, spaced at 2 micrometer intervals. A total of 16 planes, correlating to an estimated depth of 32 micrometers per z-plane, were scrutinized. From each plant, up to three leaves were examined. The analysis included over 50 volumetric scenes per plant, with each scene encompassing a volume of 100 x 100 x 25 mm³. Image stacks were exported as *.tif </p> <p>Notation:</p> <p>Exp1_PYF: Replicate 4 expressing one of four specific genetic constructs fused to complementary fragments of Yellow Fluorescent Protein.</p> <p>45: Fused to the full length AtLEA4-5 protein (pYFN-4-/5pYFC-4-5).</p> <p>4H: Fused to the N-terminal region of AtLEA4-5 (pYFN-4-51-77/pYFC-4-51-77).</p> <p>RC: Fused to the C-terminal region of AtLEA4-5 (pYFN-4-578-158/pYFC-4-578-158).</p> <p>control: The control condition (pYFN-pYFC).</p> <p>42: Fused to the full length AtLEA4-2 protein (pYFN-4-2/pYFC-4-2).</p>
In-depth Tissue-Level Dimerization Analysis of AtLEA proteins from Arabidopsis thaliana - Replicate 3
<p>Post 48-hour infiltration, leaves were examined utilizing a 3I Mariana Spinning Disc Confocal microscope equipped with a Zeiss Observer Z.1 Inverted base and a fluorescence spinning disk. In each experimental run, four plants were analyzed, each expressing one of three specific genetic constructs fused to complementary fragments of Yellow Fluorescent Protein: pYFN-4-/5pYFC-4-5 (representing the complete AtLEA4-5 protein), pYFN-4-51-77/pYFC-4-51-77 (encompassing the N-terminal region of AtLEA4-5), pYFN-4-578-158/pYFC-4-578-158 (comprising the C-terminal region of AtLEA4-5),pYFN-4-2/pYFC-4-2 (full length AtLEA4-2 protein), or pYFN-pYFC (serving as the control condition). Observations were conducted using a 20X—/0.8 NA air lens from Zeiss, under illumination provided by a pre-centered fiber illuminator from Intelligent Imaging Innovations, Inc. The detection of YFP fluorescence was facilitated by a 515 nm excitation laser and a 542/27 nm emission filter. Images were captured using an Andor Ixon 3 EMCCD camera (Model: DU-897E-CS0-#BV), adhering to protocols defined in SlideBook software version 6. Images were captured at specified intervals with exposure times ranging from 400 to 700 milliseconds. Each image consisted of a plane with a pixel size of 0.8 microns, spaced at 2 micrometer intervals. A total of 16 planes, correlating to an estimated depth of 32 micrometers per z-plane, were scrutinized. From each plant, up to three leaves were examined. The analysis included over 50 volumetric scenes per plant, with each scene encompassing a volume of 100 x 100 x 25 mm³. Image stacks were exported as *.tif </p> <p> </p> <p>Notation:</p> <p> </p> <p>Exp3_PYF: Replicate 3 expressing one of four specific genetic constructs fused to complementary fragments of Yellow Fluorescent Protein.</p> <p>45: Fused to the full length AtLEA4-5 protein (pYFN-4-/5pYFC-4-5).</p> <p>4H: Fused to the N-terminal region of AtLEA4-5 (pYFN-4-51-77/pYFC-4-51-77).</p> <p>RC: Fused to the C-terminal region of AtLEA4-5 (pYFN-4-578-158/pYFC-4-578-158).</p> <p>control: The control condition (pYFN-pYFC).</p> <p>42: Fused to the full length AtLEA4-2 protein (pYFN-4-2/pYFC-4-2).</p> <p> </p> <p> </p>
Simulated Arabidopsis thaliana sequencing datasets for chloroplast assembler benchmarking
<p><strong>Changes</strong></p> <ul> <li>Fixed non-circular sampling from chloroplast and mitochondrion in version 1.1.0</li> <li>Fixed off-by-one error in reverse read in version 1.0.0</li> </ul> <p><strong>Purpose and Documentation</strong></p> <p>See: <a href="https://github.com/chloroExtractorTeam/benchmark">github.com/chloroExtractorTeam/benchmark</a></p> <p><strong>Original data</strong><br> The original <em>Arabidopsis thaliana </em>sequences were downloaded from TAIR: </p> <p>The Arabidopsis Information Resource (<a>TAIR</a>) on www.arabidopsis.org, Mar 22, 2019 available under the <a href="http://www.arabidopsis.org/doc/about/tair_terms_of_use/417">TAIR Terms of Use</a> </p> <p><em>Tanya Z. Berardini, Leonore Reiser, Donghui Li, Yarik Mezheritsky, Robert Muller, Emily Strait and Eva Huala. "The Arabidopsis Information Resource: Making and mining the "gold standard" annotated reference plant genome." genesis 2015 <a href="https://doi.org/10.1002/dvg.22877">doi:10.1002/dvg.22877</a></em></p> <p><strong>Programs used to generate this data</strong><br> - <a href="https://github.com/shenwei356/seqkit">seqkit</a> (v0.10.1): Shen W, Le S, Li Y, Hu F (2016) "SeqKit: A Cross-Platform and Ultrafast Toolkit for FASTA/Q File Manipulation." PLOS ONE 11(10): e0163962. <a href="https://doi.org/10.1371/journal.pone.0163962">doi:10.1371/journal.pone.0163962</a></p> <p> </p>
Microclimate predicts frost-hardiness of alpine Arabidopsis thaliana populations better than elevation
<p>In mountain regions, topological differences on the micro-scale can strongly affect microclimate and may counteract the average effects of elevation, such as decreasing temperatures. While these interactions are well understood, their effect on plant adaptation is understudied.</p> <p> </p> <p>We investigated winter frost hardiness of Arabidopsis thaliana accessions originating from 13 sites along altitudinal gradients in the Southern Alps during three winters on an experimental field station on the Swabian Jura and compared levels of frost damage with the observed number of frost days and the lowest temperature in eight collection sites.</p> <p> </p> <p>We found that frost-hardiness increased with elevation in a log-linear fashion. This is consistent with adaptation to a higher frequency of frost conditions, but also indicates a decreasing rate of change in frost hardiness with increasing elevation. Moreover, the number of frost days measured with temperature loggers at the collection sites correlated much better with frost-hardiness than the elevation of collection sites, suggesting that populations were adapted to their local microclimate. Notably, the variance in frost days across sites increased exponentially with elevation. Together, our results suggest that strong microclimate heterogeneity of high alpine environments can preserve functional genetic diversity among small populations.</p> <p> </p> <p>Synthesis. Here we tested how plant populations differed in their adaptation to frost exposure along an elevation gradient and whether microsite temperatures improve the prediction of frost hardiness. We found that local temperatures, particularly the number of frost days, is a better predictor of the frost hardiness of plants than elevation. This reflects a substantial variance in frost frequency between sites at similar high elevations. We conclude that high mountain regions harbor microsites that differ in their local microclimate and thereby can preserve a high functional genetic diversity among them. Therefore, high mountain regions have the potential to function as a refugium in times of global change.</p>
Generation of transcriptional novelty by transposable element insertions in Arabidopsis, Genome Sequencing and eccDNA Data
<p><strong>Raw Illumina sequencing data from the Manuscript entitled "Generation of transcriptional novelty by transposable element insertions in Arabidopsis"</strong></p> <p><strong>A. Illumina genome sequencing reads of Arabidopsis control and hcLines that contain novel transposable element insertions.</strong></p> <p>To identify the genomic position of the new <em>ONSEN</em> insertions, the extracted DNA of the 11 selected lines (nine lines with new insertions and two control lines) was sent to BGI, Hong-Kong for Illumina paired-end 150 bp sequencing, aiming for a minimum of 20X sequencing coverage. Quality control of the raw reads was done using FastQC (Andrews S. (2010). FastQC: a quality control tool for high throughput sequence data. Available online at: <a href="http://www.bioinformatics.babraham.ac.uk/projects/fastqc">http://www.bioinformatics.babraham.ac.uk/projects/fastqc</a>) and trimming/clipping was done using Trimmomatic with parameters ILLUMINACLIP: TruSeq3:2:30:10 LEADING:20 TRAILING:20 SLIDINGWINDOW:4:20 and MINLEN:36. Quality of the reads was deemed excellent and no further actions were taken.</p> <p>Samples identifications: genome_hcLineX with "_1" indicating the forward and "_2" the reverse reads.</p> <p><strong>B. Illumina eccDNA sequencing of Arabidopsis control and hcLines following stress treatments</strong></p> <p>Extrachromosomal circular DNA was prepared and sequenced as follows: twenty plants from each petri dish were pooled separately and DNA was extracted using the CTAB method (<a href="https://dx.doi.org/10.17504/protocols.io.quidwue">dx.doi.org/10.17504/protocols.io.quidwue</a>). Following the mobilome-seq method described in (Lanciano et al., 2017), for all samples, we digested linear DNA from 2 µg of total DNA for 17 hours at 37<sup>o</sup>C using 10 U of PlasmidSafe (<em>LubioScience cat# E3101K</em>), followed by enzyme denaturation (30 mins at 70<sup>o</sup>C). Digested DNA was precipitated with isopropanol supplemented with 1 µg of GlycoBlue coprecipitant (<em>Fisher Scientific cat# 10391565</em>). Circular DNA was then amplified through rolling circle amplification (RCA) with the Illustra TempliPhi kit (<em>GE Healthcare cat# 25-6400-10</em>), following the manufacturer recommendation and leaving the reaction for 16h at 30<sup>o</sup>C. DNA was once again precipitated with isopropanol and sent for Illumina paired end 150 bp sequencing at BGI, Hong Kong. </p> <p>Samples identification: </p> <p>eccDNA_A.thaliana_ctrl: control reads</p> <p>eccDNA_A.thaliana_HS: heat stressed plants reads</p> <p>eccDNA_A.thaliana_AZ_HS: reads of alpha-amanitin, zebularine and heat-stressed plants</p> <p>"R1" indicates forward and "R2" reverse reads.</p> <p> </p>
Unraveling the secrets of plant roots: Simplified method for large scale root exudate sampling and analysis in Arabidopsis thaliana
<p>Plants exude a plethora of compounds, both above- and below ground, to communicate with their environment. Although much is known about this communication above ground, we are only beginning to fathom the identity, level of variation, and role of below-ground chemical signals in a plant’s life. There have been many challenges associated with establishing a standardized methodology for studying root-exuded compounds, thus their role in plant-environment communication is still not well described.</p> <p>Here, we develop an interdisciplinary workflow to explore the natural variation in root exudate chemical composition of the model plant <em>Arabidopsis thaliana</em>. We highlight key challenges associated with sampling strategies and develop a framework for analyzing narrow and broad-scale patterns in root exudate composition of a large sample set of natural <em>A. thaliana</em> accessions.</p> <p>Our method consists of <em>in vitro</em> cultivation of individual seedlings inside a plastic mesh followed by a short hydroponic sampling period in micro quantities of ultrapure water. The mesh eases handling individual plants of varying sizes, thus making this setup advantageous for large-scale characterization of root exudates of individual plants in axenic conditions, and can be easily extended for prolonged temporal exudate collection experiments. Furthermore, a short sampling duration minimizes the experiment duration from days to mere hours, and is validated by yielding sufficient signal even with the small volume of sampling solution. An untargeted metabolic profiling analytical approach using ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-QTOF-MS), followed by compound identification using open access software MZMine3 and SIRIUS 5, was used to capture a broad picture of the root exudate composition of <em>A. thaliana</em> accessions. This methodology can be broadly applied for investigating the role of root exudates as signals involved in plant belowground interactions. We report the first findings from the analysis here with results from Columbia genotype.</p> <p>We include here</p> <p> </p> <p>In <strong>Raw datafiles</strong>: The raw datasets obtained from MZmine 3 analysis, which contains aligned features of Columbia genotypes (Sheet1) as well as control samples (Sheet2). The dataset consists of <em>feature (row)ID, average (mass-to-charge ratios) m/z</em> and <em>retention times (RT)</em> across samples for individual features. It also includes sample-specific information including <em>feature status, name, m/z, RT, feature peak height,</em> and <em>area</em>. We also include a filtered datasheet excluding the features obtained in control as well as samples (Sheet3). Sheet 4 contains the phenotypic data on the number of leaves and rosette size of the 28 replicates at the time of sampling, along with the total peak area for each sample from MZmine data.</p> <p>In <strong>Supplementary tables</strong>: The 354 metabolites obtained after filtering out control features are listed with their <em>mass-to-charge ratios</em>, <em>retention times, and the mean, variance and coefficient of variation of the peak areas </em>(Supplementary Table 1). Supplementary Table 2 details the features identified by SIRIUS 5 with their <em>mass-to-charge ratios, retention times, chemical formula, chemical annotations,</em> and corresponding <em>probabilities scored by SIRIUS</em></p> <p>In <strong>Extended data analysis:</strong> Contains supporting data analysis for reproducibility and validity of our method for root exudate collection and analysis in <em>Arabidopsis thaliana.</em> These extended data analyses enhance the understanding of the relationship between plant phenotypic traits, peak area, and variation in compound abundance</p>
Laser Ablation Electrospray Ionization Mass Spectrometry Imaging (LAESI MSI) of Arabidopsis thaliana leaf
<p>Mass spectrometry imaging (MSI) data set in imzML format, obtained from the 5th leaf of an Arabidopsis thaliana wildtype plant using Laser Ablation Electrospray Ionization. Laser ablation took place with 20 pulses per pixel at an energy of 58.4 µJ/pulse. The ROI measures 9 mm by 5 mm and was sampled with a step size of 200 µm.</p>
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Allen Brain Atlas
Allen Brain Atlas is an Allen Institute collection of brain map atlases, datasets, APIs, and analysis tools covering mouse, human, and non-human primate brain resources.
Annotated Behaviour and Observability Dataset (ABODe)
ABODe is a University of Edinburgh DataShare dataset for behavior classification in group-housed mice using home-cage video, identities, bounding boxes, ground-plate positions, and annotator labels.
DANDI Archive for NWB datasets
DANDI is a BRAIN Initiative archive for publishing and sharing neurophysiology data, including electrophysiology, optophysiology, and behavioral data packaged as NWB and related standards.
International Brain Laboratory public data
The International Brain Laboratory public data releases expose standardized mouse decision-making experiments, including Neuropixels recordings, widefield calcium imaging, behavior, and session metadata accessed through the ONE API.
OpenNeuro
OpenNeuro is a free, open platform for sharing neuroimaging datasets, with public search, dataset pages, and download paths for web, S3, DataLad, and the OpenNeuro CLI.