We would like to thank Jean-Claude Walser of the Genetic Diversity Center Zurich for crucial expert advice in the early phase of this project. Work in the Vermeer lab was supported by an SNF Professorship grant (PP00P3_157524) and a R&#39;EQUIP equipment grant (316030_164086) from the Swiss National Science Foundation (SNSF) awarded to JEMV.

The authors have nothing to disclose.

Verification of RPL18 localization pattern 
Crucial to avoid misinterpretation of data from any TRAP experiment is the proper expression pattern of the tagged ribosomal subunit. Therefore, the incorporation of GFP as an epitope tag to RPL18 very elegantly allows verification of the desired expression pattern and consecutively, pulldown of the polysome fraction from the same tissue. More invasive approaches to assure proper promoter patterns are followed by Jiao and Mayerowitz 2010, which requires GU...

Driven by the increasing application of next-generation sequencing techniques, spatial resolution in developmental biology could be augmented. Contemporary studies aim at dissecting tissues down to specialized cell types, if not single-cell level1,2,3,4. To this end, a plethora of different methods has been devised over the last fifty years (see Figure 1A)5,6,7,8,9,10,11,12,13,14,15.
Many tools in plant science have been adaptations of techniques that were pioneered in animal research. This is not the case for the method we are introducing in detail here. In 2005, equipped with a strong background in protein translation, the Bailey-Serres Lab set out to engineer ribosomal proteins for subsequent affinity purification16. Thus, they could avoid time-consuming and labor-intensive polysome profiling, which is based on ultracentrifugation with a sucrose gradient and was used to assess translating ribosomes since the 1960s17,18. The method has since been referred to as translational ribosome affinity purification (TRAP)16. After successful translatome studies in plants, Heiman et al. adapted TRAP for animals19 and others extended its application to yeast20, Drosophila21, Xenopus22 and zebrafish23,24.
Although genetic modification of the model system is a prerequisite for TRAP, which limits its application to species amenable to genetic transformation, one can simultaneously harness this objection to target subsets of cells that are of special interest and otherwise extremely difficult to isolate from the intact tissue/organ25 (e.g., highly branched dendritic cells in a mouse brain or fungal hyphae in infected plant tissue). In plants, all cells are held in place via cell walls that form the basis of the hydrostatic skeleton26. To free a plant cell from this matrix, scientists have either physically cut the cell out of its surrounding tissue through laser capture microdissection (LCM)27 or performed enzymatic digestion of the cell walls28. Among the latter cells, so-called protoplasts, the population of interest is fluorescently labeled and can be separated via fluorescence-activated cell sorting (FACS)7. LCM usually requires a sample to be fixed and embedded in wax, which ultimately deteriorates the quality of its RNA29. FACS-based methods yield high-quality RNA, but the process of protoplasting itself introduces differences in gene expression30 and tissues with modified and thick secondary cell walls are notoriously difficult to treat. Moreover, many developmental processes in plants are assumed to rely on mechanically transmitted signals and therefore the integrity of the cell wall is of paramount importance31. Two methods, which use a shortcut to circumvent cell isolation by operating on the level of nucleii, are fluorescence-activated nuclear sorting (FANS) and isolation of nuclei tagged in specific cell types (INTACT). As in TRAP, they use cell type-specific promoters to mark nuclei, that subsequently get enriched via sorting or pull down, respectively8,15. A major challenge for all these approaches is to get sufficient RNA material from subsets of cells in a tissue. As TRAP captures only a fraction of the cellular RNAs, sample collection is a considerable bottleneck. Therefore, especially sensitive library preparation protocols are needed to produce high-quality data from low input amounts.
Since its establishment, TRAP has been either used in combination with DNA microarrays or, as sequencing costs dropped significantly in recent years, RNA-seq10,32,33. A multitude of research questions has already been elucidated as reviewed in Sablok et al.34. We are convinced that more reports will follow in coming years as the technique is very versatile when combining different promoters to target specific cell types. Eventually, this will be done even in an inducible way, and may be combined with probing the plant&#39;s reaction to many biotic and abiotic stress factors. Additionally, where stable transgenic lines are not available, hairy root expression systems have also been successfully used to perform TRAP in tomato and medicago35,36.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60919/60919fig1b.jpg" /> 
Figure 1: Translating ribosome affinity purification (TRAP) complements the &#34;omics&#34; analysis portfolio. A. Increasing levels of analytical precision, down to single-cell or even subcellular resolution can be achieved by a plethora of methods or combinations thereof. The scheme gives an overview of currently available tools in the plant and animal field. Tissue collection at cellular resolution can be achieved by protocols like LCM or FACS, which are then coupled to standard transcriptome or polysome profiling/translatome analysis. TRAP and INTACT integrate both tissue capture and RNA isolation as they are based on epitope-tagging. However, INTACT samples only cell nuclei and constitutes, therefore, a special case of transcriptome analysis. A small rabbit icon marks newly developed methods in the animal field: While SLAM-ITseq and Flura-seq rely on metabolic targetting of nascent RNAs with modified uracil bases in cells expressing the permissive enzyme, Slide-seq makes use of a coated glass slide with DNA barcodes that provide positional information in the cellular range. A proximity-labeling approach is followed in APEX-seq to sample RNAs in specific subcellular compartments. Notably, increased resolution often requires the generation of transgenic material (asterisks) and these methods are thus predominantly used for model species. TRAP is especially suited for plant science studies involving cell wall (CW) or mechanic signaling as well as cell species that are difficult to release from their CW matrix. B. Detailed wet-lab steps of the TRAP procedure: Seedlings expressing GFP-tagged ribosomal protein in distinct cell types (e.g. root endodermis) are grown on Petri dishes for seven days and root material harvested by snap freezing. A total RNA control sample is collected from the homogenized crude extract before pelleting the debris via centrifugation. Magnetic anti-GFP beads are added to the cleared extract to perform immunoprecipitation. After incubation and three wash steps, the polysome-associated RNA (TRAP/polysome RNA) is directly obtained via phenol-chloroform extraction. LCM: laser capture microdissection, FACS/FANS: fluorescence-activated cell/nuclear sorting, APEX-seq: method based on engineered ascorbate peroxidase, INTACT: isolation of nuclei tagged in specific cell types, SLAM-ITseq: thiol(SH)-linked alkylation for the metabolic sequencing of RNA in tissue, Flura-seq: fluorouracil-labeled RNA sequencing (Created with Biorender.com) <a href="https://www.jove.com/files/ftp_upload/60919/60919fig1blarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The goal of this article is to supply a detailed description of the TRAP method, to highlight critical steps and to provide guidance for a possible library preparation method.
A generic TRAP experiment will essentially consist of the following steps (see also Figure 1B): (1) Preparation of plant material including cloning of ribosome-tagging construct, transgenic line production and selection, growing and bulking up of seeds, sterilization and plating, and stress application/treatment (optional) and tissue harvesting; (2) immunopurification including tissue homogenization and clearing of the crude extract, bead wash and immunopurification, and wash steps; (3) RNA extraction and quality assessment; and (4) library preparation.
The Arabidopsis root has been a model system to study plant development ever since its introduction as a model plant37,38. Here, the application of TRAP is showcased in the context of plant lateral root development. In plants, the buildup of the entire root system relies on the execution of this program and is therefore very important for the survival of the organism39. In Arabidopsis, lateral roots originate from pericycle tissue that resides next to xylem vessels and therefore is termed xylem pole pericycle (XPP; see Figure 2C)40. Some XPP cells, which are located deep inside the root, acquire a founder cell identity and, upon a local hormonal trigger, start to proliferate by swelling and dividing anticlinally41. However, due to the presence of a rigid cell wall matrix, this process exerts mechanical stress on the surrounding tissues. In particular, the overlying endodermis is affected, as it is in the way of the lateral root growth axis42,43,44. Indeed, the newly forming primordium will have to grow through the overlying endodermis cell (Figure 2C2) whereas cortex and epidermis cells are just pushed aside for the primordium to finally emerge45,46. Recent work in our lab has shown that the endodermis is actively contributing to accommodate the proliferation in the pericycle. Targeted blocking of endodermal hormonal signaling is sufficient to inhibit even the very first division in the XPP cells47. Hence, pericycle-endodermis communication constitutes a very early checkpoint for lateral root development in Arabidopsis. It is, however, not known how this crosstalk is performed. To unravel this mystery, we chose the TRAP-seq approach to target XPP and endodermal cells. To enrich for cells in the lateral root program, we mimicked the hormonal trigger by exogenously applying an auxin analog (1-naphthaleneacetic acid, NAA)48, which at the same time allowed to temporally resolve the initial phase of lateral root formation.

<table><tbody><tr><td>&lt;strong&gt;Sterilization&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>bleach, 13%</td><td>Sigma</td><td>71696</td><td></td></tr><tr><td>beaker</td><td>VWR</td><td>214-1172/74/75</td><td></td></tr><tr><td>desiccator with porcelaine plate (DURAN)</td><td>Sigma/Merck</td><td>Z317454-1EA/Z317594-1EA</td><td></td></tr><tr><td>EtOH, p.a.</td><td>Honeywell</td><td>02860-1L</td><td></td></tr><tr><td>HCl, 37%</td><td>Roth</td><td>4625.1</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416</td><td></td></tr><tr><td>&lt;strong&gt;Plate growth + harvesting&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MS salts, basal salt mixture, incl. MES buffer</td><td>Duchefa</td><td>M0254</td><td></td></tr><tr><td>agar plant for cell culture</td><td>Applichem/Panreac</td><td>A2111.1000</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D4540</td><td></td></tr><tr><td>forcepts</td><td>Rubis Switzerland</td><td>5-SA model</td><td></td></tr><tr><td>KOH</td><td>Fluka</td><td>60370</td><td></td></tr><tr><td>micropore/surgical tape</td><td>3M</td><td>1530-0</td><td></td></tr><tr><td>NAA</td><td>Duchefa</td><td>N0903</td><td></td></tr><tr><td>petri dishes 120x120 mm</td><td>Greiner bio-one</td><td>688102</td><td></td></tr><tr><td>scalpel</td><td>VWR/Swann-Morton</td><td>233-5454</td><td></td></tr><tr><td>tissues, neutral, two-layered</td><td></td><td></td><td>any supplier of your choice</td></tr><tr><td>&lt;strong&gt;Immunoprecipitation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GFP-beads: gtma-100 GFP-Trap_MA</td><td>Chromotek</td><td>e.g. gtma-100</td><td></td></tr><tr><td>Brij-35</td><td>Sigma</td><td>P1254-500G</td><td></td></tr><tr><td>centrifuge tubes (in accordance with centrifuge)</td><td>Beckman Coulter</td><td>357001</td><td></td></tr><tr><td>Chloramphenicol</td><td>Applichem</td><td>C0378-25G</td><td></td></tr><tr><td>cotton gloves</td><td>VWR</td><td>113-7355</td><td></td></tr><tr><td>Cycloheximide, HPLC grade</td><td>Sigma</td><td>01810-1G</td><td></td></tr><tr><td>DEPC</td><td>VWR</td><td>E174</td><td>might have long delivery times</td></tr><tr><td>DTT</td><td>Fluka</td><td>43815</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>3054.3</td><td></td></tr><tr><td>homogenizers DUALL 23</td><td>KONTES GLASS CO (via VWR)</td><td>SCERSP885450-0023 (set)</td><td>SCERSP885451-0023 pestle only - SCERSP885452-0023 cylinder only; long delivery times</td></tr><tr><td>Igepal CA-360</td><td>Sigma</td><td>I3021-100ml</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>60130</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; hexahydrat</td><td>Roth</td><td>2189.2</td><td></td></tr><tr><td>mortar and pestle</td><td>VWR</td><td>470148-960 &amp;amp; 470019-978</td><td></td></tr><tr><td>PMSF</td><td>Roche</td><td>10 837 091 001</td><td></td></tr><tr><td>Polyoxyethylene-(10)-tridecylether/PTE</td><td>Sigma</td><td>P2393-500G</td><td></td></tr><tr><td>RNase-free water</td><td>Roth</td><td>T143.3</td><td></td></tr><tr><td>RNAZap</td><td>Thermo Fisher</td><td>AM9780/AM9782</td><td>for cleaning surfaces</td></tr><tr><td>Tris, &gt;99.3%</td><td>Roth</td><td>AE15.3</td><td></td></tr><tr><td>Triton X-100</td><td>Fluka</td><td>T8787-250ml</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416-100ml</td><td></td></tr><tr><td>&lt;strong&gt;RNA extraction&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2-Propanol, p.a.</td><td>Sigma</td><td>33539-1L-GL-R</td><td></td></tr><tr><td>Chloroform, HPLC grade</td><td>Scharlau</td><td>CL02181000</td><td></td></tr><tr><td>EtOH, p.a.</td><td>Honeywell</td><td>02860-1L</td><td></td></tr><tr><td>low-retention microcentrifuge tubes, 1.5 ml</td><td>Eppendorf/Sigma</td><td>Z666548-250EA</td><td>LoBind</td></tr><tr><td>RNase-free DNase set</td><td>Qiagen</td><td>79254</td><td></td></tr><tr><td>RNeasy MiniElute Cleanup Kit</td><td>Qiagen</td><td>74204</td><td></td></tr><tr><td>TRIzol reagent</td><td>ThermoFisher/Ambion</td><td>15596018</td><td></td></tr><tr><td>&lt;strong&gt;Library preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>15/50 mL Tube Magnetic Separator</td><td>Abraxis</td><td>PN 472250</td><td></td></tr><tr><td>AMPure beads</td><td>Beckman Coulter</td><td>A63881</td><td></td></tr><tr><td>Index Kit A</td><td>Illumina</td><td>FC-131-2001</td><td></td></tr><tr><td>Index Kit D</td><td>Illumina</td><td>FC-131-2004</td><td></td></tr><tr><td>neodymium magnets</td><td>Amazon/other</td><td>6 x 1.5 mm range: N42 (NdFeB)</td><td></td></tr><tr><td>Nextera XT kit</td><td>Illumina</td><td>FC-131-1024/1096</td><td>&lt;a href=&quot;https://emea.support.illumina.com/sequencing/sequencing_kits/nextera_xt_dna_kit/documentation.html&quot; target=&quot;_blank&quot;&gt;https://emea.support.illumina.com/&lt;/a&gt;</td></tr><tr><td>PCR strips</td><td>ThermoScientific</td><td>AB-0266</td><td></td></tr><tr><td>SMARTer v4 kit</td><td>Takara Bioscience</td><td>634892</td><td>&lt;a href=&quot;https://www.takarabio.com/products/next-generation-sequencing/rna-seq/smart-seq-v4-for-mrna-seq&quot; target=&quot;_blank&quot;&gt;https://www.takarabio.com/&lt;/a&gt;</td></tr><tr><td>Bioanalyzer</td><td>Agilent</td><td>2100 Bioanalyzer Instrument</td><td>specialized equipment for RNA/DNA quality control</td></tr><tr><td>Tapestation</td><td>Agilent</td><td>4200 Tapestation Instrument</td><td>specialized equipment for RNA/DNA quality control</td></tr><tr><td>Fragment Analyzer</td><td>Agilent</td><td>5400 Fragment Analyzer System</td><td>specialized equipment for RNA/DNA quality control (high throughput)</td></tr><tr><td>LabChip</td><td>PerkinElmer</td><td>LabChip GX Touch Nucleic Acid Analyzer</td><td>specialized equipment for RNA/DNA quality control (high throughput)</td></tr><tr><td>Qubit 4 Fluorometer</td><td>ThermoFisher</td><td>&lt;a href=&quot;https://www.thermofisher.com/order/catalog/product/Q33239&quot; target=&quot;_blank&quot;&gt;Q33239&lt;/a&gt;</td><td>specialized equipment for RNA/DNA concentration determination</td></tr><tr><td>&lt;strong&gt;qRT-PCR&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GATA23</td><td>Microsynth</td><td></td><td>fwd: AGTGAGAATGAA&lt;br/&gt; AGAAGAGAAGGG;&lt;br/&gt; rev: GTGGCTGCGAAT&lt;br/&gt; AATATGAATACC</td></tr><tr><td>GH3.3</td><td>Microsynth</td><td></td><td>fwd: CAAACCAATCCT&lt;br/&gt; CCAAATGAC;&lt;br/&gt; rev: ACTTATCCGCAA&lt;br/&gt; CCCGACT</td></tr><tr><td>LBD29</td><td>Microsynth</td><td></td><td>fwd: TCTCCAACAACA&lt;br/&gt; GGTTGTGAAT;&lt;br/&gt; rev: AAGGAGCCTTAG&lt;br/&gt; TAGTGTCTCCA</td></tr><tr><td>UBC21</td><td>Microsynth</td><td></td><td>fwd: TGCGACTCAGGG&lt;br/&gt; AATCTTCT;&lt;br/&gt; rev: TCATCCTTTCTT&lt;br/&gt; AGGCATAGCG</td></tr><tr><td>SsoAdvanced Universal SYBR Green</td><td>Bio-Rad</td><td>#172-5270</td><td></td></tr><tr><td>iScript Adv cDNA Kit</td><td>Bio-Rad</td><td>#172-5038</td><td></td></tr><tr><td>&lt;strong&gt;miscellaneous&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Falcon tubes 15 ml, Cellstar</td><td>Greiner bio-one</td><td>188261</td><td></td></tr><tr><td>Falcon tubes 50 ml, Cellstar</td><td>Greiner bio-one</td><td>210261</td><td></td></tr><tr><td>filter tips 1 ml</td><td>Axygen</td><td>TF-1000-R-S</td><td></td></tr><tr><td>filter tips 10 &amp;micro;l</td><td>Axygen</td><td>TF-10-R-S</td><td></td></tr><tr><td>filter tips 100 &amp;micro;l</td><td>Axygen</td><td>TF-100-R-S</td><td></td></tr><tr><td>filter tips 20 &amp;micro;l</td><td>Axygen</td><td>TF-20-R-S</td><td></td></tr><tr><td>filter tips 200 &amp;micro;l</td><td>Axygen</td><td>TF-200-R-S</td><td></td></tr><tr><td>microcentrifuge tubes 1.5 ml</td><td>SARSTEDT</td><td>72.690.001</td><td></td></tr><tr><td>Propidium iodide</td><td>Sigma</td><td>P4170-100MG</td><td></td></tr><tr><td>sequencing company</td><td>Novogene</td><td></td><td>&lt;a href=&quot;http://en.novogene.com&quot; target=&quot;_blank&quot;&gt;en.novogene.com&lt;/a&gt;</td></tr></tbody></table>

translating ribosome affinity purification (trap) to investigate arabidopsis thaliana root development at a cell type-specific scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

For quality assessment, the above-mentioned procedure should be probed at several intermediate steps: expression pattern validation in planta, quality control of the isolated polysomal RNA as well as of the final libraries. qRT-PCR using known marker genes can, in addition, be performed to confirm the response to the treatment condition or to fine-tune the experimental conditions.
Confocal analysis of GFP signal distribu...

Watch this Scientific Journal Video about Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale at JoVE.com

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

translating-ribosome-affinity-purification-trap-to-investigate

Research

JoVE Journal

Developmental Biology

11.9K Views.  University of Zurich. Met TRAP kunnen we ontwikkelingsverwerking onderzoeken op cellulaire resolutie. We gebruiken cel-type-specifieke promotors om een gelabelde ribosomale sub-eenheid te besturen, en van daaruit kunnen we polysome RNA isoleren. Dit protocol levert RNA van hoge kwaliteit op, maar in zeer beperkte hoeveelheden zijn daarom methoden nodig die gespecialiseerd zijn voor ultralage invoer om RNA-bibliotheken van hoge kwaliteit betrouwbaar te produceren.TRAP is een ideaal hulpmiddel voor plantenond...

Video: Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

Biochemistry

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Bioengineering

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Biology

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, rhizosphere colonization by plant growth-promoting bacteria (PGPB) can increase the health or the development of host plants relative to uncolonized plants. However, in field settings, bacterial treatments with PGPB often do not provide substantial benefits to crops. One explanation is that this may be due to loss of the PGPB during interactions with endogenous soil microbes over the lifespan of the plant. This possibility has been difficult to confirm, since most studies focus on the initial colonization rather than maintenance of PGPB within rhizosphere communities. It is hypothesized here that the assembly, coexistence, and maintenance of bacterial communities are shaped by deterministic features of the rhizosphere microenvironment, and that these interactions may impact PGPB survival in native settings. To study these behaviors, a hydroponic plant-growth assay is optimized using Arabidopsis thaliana to quantify and visualize the spatial distribution of bacteria during initial colonization of plant roots and after transfer to different growth environments. This system&#8217;s reproducibility and utility are then validated with the well-studied PGPB Pseudomonas simiae. To investigate how the presence of multiple bacterial species may affect colonization and maintenance dynamics on the plant root, a model community from three bacterial strains (an Arthrobacter, Curtobacterium, and Microbacterium species) originally isolated from the A. thaliana rhizosphere is constructed. It is shown that the presence of these diverse bacterial species can be measured using this hydroponic plant-maintanence assay, which provides an alternative to sequencing-based bacterial community studies. Future studies using this system may improve the understanding of bacterial behavior in multispecies plant microbiomes over time and in changing environmental conditions.

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, ...

Immunology and Infection

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given point in time. The protocol dubbed Translating Ribosome Affinity Purification (TRAP) is able to capture this mRNA translation process in a cell-type-specific manner. Based on the affinity purification of polysomes carrying a tagged ribosomal subunit, TRAP can be applied to translatome analyses in individual cells, making it possible to compare cell types during the course of developmental processes or to track disease development progress and the impact of potential therapies at molecular level. Here we report an optimized version of the TRAP protocol, called TRAP-rc (rare cells), dedicated to identifying engaged-in-translation RNAs from rare cell populations. TRAP-rc was validated using the Gal4/UAS targeting system in a restricted population of muscle cells in Drosophila embryos. This novel protocol allows the recovery of cell-type-specific RNA in sufficient quantities for global gene expression analytics such as microarrays or RNA-seq. The robustness of the protocol and the large collections of Gal4 drivers make TRAP-rc a highly versatile approach with potential applications in cell-specific genome-wide studies.

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Translating Ribosome Affinity Purification (TRAP) is able to capture cell-type-specific translation of mRNA. Here we report the first TRAP protocol dedicated to isolation of mRNA in rare cell populations of Drosophila embryos.

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

A Simple Protocol for Mapping the Plant Root System Architecture Traits

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar tolerance to environmental challenges. An experimental protocol is presented for setting up the hydroponic system, plantlet growth, RSA spreading, and imaging. The approach used a magenta box-based hydroponic system containing polypropylene mesh supported by polycarbonate wedges. Experimental settings are exemplified by assessing the RSA of the plantlets under varying nutrient (phosphate [Pi]) supply. The system was established to examine the RSA of Arabidopsis, but it is readily adaptable to study other plants like Medicago sativa (Alfalfa). Arabidopsis thaliana (Col-0) plantlets are used in this investigation as an example to understand the plant RSA. Seeds are surface sterilized by treating ethanol and diluted commercial bleach, and kept at 4 &#176;C for stratification. The seeds are germinated and grown on a liquid half-MS medium on a polypropylene mesh supported by polycarbonate wedges. The plantlets are grown under standard growth conditions for the desired number days, gently picked out from the mesh, and submersed in water-containing agar plates. Each root system of the plantlets is spread gently on the water-filled plate with the help of a round art brush. These Petri plates are photographed or scanned at high resolution to document the RSA traits. The root traits, such as primary root, lateral roots, and branching zone, are measured using the freely available ImageJ software. This study provides techniques for measuring plant root characteristics in controlled environmental settings. We discuss how to (1) grow the plantlets, and collect and spread root samples, (2) obtain pictures of spread RSA samples, (3) capture the images, and (4) use image analysis software to quantify root attributes. The advantage of the present method is the versatile, easy, and efficient measurement of the RSA traits.

We use simple laboratory tools to examine the root system architecture (RSA) of Arabidopsis and Medicago. The plantlets are grown hydroponically over mesh and spread using an art brush to reveal the RSA. Images are taken using scanning or a high-resolution camera, then analyzed with ImageJ to map traits.

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar ...

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

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