This work was supported by the French National Research Agency (ANR-14-CE02-0010). We thank Dr Benjamin Field and Dr. Elodie Lanet for critical reading of the manuscript. We thank Mr. Michel Terese for his help with video editing.

1. Preparation of 20 to 50% (w/v) Sucrose Gradients
Note: The gradients are made of 4 layers of sucrose (50%, 35% and 2 layers of 20%) in a 13.2 ml ultracentrifuge tube. In our experience, pouring the 20% sucrose in two separate layers greatly improves the quality of polysome preparations.
<ol>
	<li>Prepare the stock solutions. Ensure that all solutions are RNAse and DNAse free.
	<ol>
		<li>Prepare 10X Salt Solution: 400 mM Tris-HCl pH 8.4, 200 mM KCl and 100 mM MgCl2.</li>
		<li>Prepare 2 M Sucrose Solution: For 200 ml, dissolve 137 g of sucrose in 1X Salt Solution.</li>
	</ol>
	</li>
	<li>Dilute the Sucrose Solution in Salt Solution, as described in Table 1, to prepare the gradients. The volumes given are for six gradients.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Final sucrose concentration</td>
			<td>Sucrose 2M (ml)</td>
			<td>Salt solution 1X (ml)</td>
			<td>Final Vol. (ml)</td>
		</tr>
		<tr>
			<td>50%</td>
			<td>8.8</td>
			<td>3.2</td>
			<td>12</td>
		</tr>
		<tr>
			<td>35%</td>
			<td>12.9</td>
			<td>12.1</td>
			<td>25</td>
		</tr>
		<tr>
			<td>20%</td>
			<td>7.4</td>
			<td>17.6</td>
			<td>25</td>
		</tr>
		<tr>
			<td>20%</td>
			<td>5.8</td>
			<td>14.2</td>
			<td>20</td>
		</tr>
	</tbody>
</table>
Table 1. Dilutions of the Sucrose Solution to prepare six gradients.
<ol start="3">
	<li>Pour the layers as per Table 2.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sucrose layer</td>
			<td>50%</td>
			<td>35%</td>
			<td>20%</td>
			<td>20%</td>
		</tr>
		<tr>
			<td>Vol. (ml)</td>
			<td>1.85</td>
			<td>3.65</td>
			<td>3.65</td>
			<td>1.35</td>
		</tr>
	</tbody>
</table>
Table 2. Volume of sucrose solution per layer.
<ol start="4">
	<li>After pouring each layer, keep the tubes in a -40 &#176;C or -80 &#176;C freezer until complete freezing before adding the next sucrose layer. Freezing is usually achieved after 2 hr at -40 &#176;C, but waiting about 6 hr before pouring the next layer is recommended. The last 20% layer can be frozen or added freshly on the day of the experiment. 
	Note: Freezing each layer before adding the next one prevents from any mixing or disturbance of the layers. Gradients can be kept in a -40 &#176;C or -80 &#176;C freezer for at least six months.</li>
	<li>If it has not already been done, add the last 20% layer to the gradients on the day of the experiment. Then, let the gradients thaw out in a cold room or a fridge.</li>
</ol>
2. Preparation of Cytosolic Extracts
Note: We recommend using two gradients per biological sample. 300 mg is the optimum amount of plant material to prepare two gradients when working with 6 days old Arabidopsis thaliana seedlings. When working with less translationally active tissues, the amount of plant material can be increased up to 600 mg.
<ol>
	<li>Harvest six day old seedlings grown on &#189; Murashige and Skoog8 medium supplemented with 1% sucrose or equivalent media by quick-freezing in liquid nitrogen</li>
	<li>Grind plant material in a precooled mortar and pestle with liquid nitrogen.</li>
	<li>Weigh 300 mg of powdered material in a precooled weighing dish. Perform this step quickly to avoid thawing of the sample. 
	Note: Keep the frozen samples for no more than one week in a -80 &#176;C freezer.</li>
	<li>Add 2.4 ml of precooled polysome buffer (Salt Solution 4X, 5.26 mM EGTA, 0.5% (v/v) Octylphenoxy poly(ethyleneoxy)ethanol, branched, 50 &#181;g.ml-1 cycloheximide, 50 &#181;g.ml1 chloramphenicol) and homogenize by mixing with the pipette tip. Transfer into two 1.5 ml tubes. Work quickly to prevent the samples from warming.</li>
	<li>Centrifuge at 16,000 x g for 15 min at 4 &#176;C in a microcentrifuge to pellet debris. 
	Note: To prevent RNA degradation, always keep the samples at 4&#176;C, use RNase/DNase free solutions and work in RNase/DNase free conditions. Heparin can be added to the polysome buffer, to a final concentration of 300 &#181;g.ml-1, to enhance RNA protection. However, since heparin can interfere with downstream analysis, it will have to be removed during the RNA precipitation step by performing lithium chloride precipitation instead of ethanol precipitation (cf. note 4.7).</li>
</ol>
3. Polysome Profiling
<ol>
	<li>Carefully pipette the supernatant without disturbing the pellet. If plant fragments have been pipetted, repeat step 2.5. Load the supernatant on top of the gradient by gently pipetting onto the sidewall of the tube in a constant stream. Use one gradient for each 1.5 ml tube.</li>
	<li>Transfer to precooled buckets and centrifuge at 175,000 x g for 2 hr 45 min at 4 &#176;C, in an ultracentrifuge (e.g. 32,000 rpm when using a SW41 rotor).</li>
	<li>Set up the gradient collection system as described in Figure 1. The UV cuvette has a 1 mm pathlength.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/54231/54231fig1.jpg" /> 
Figure 1.&#160;Gradient collection system.&#160;The UV cuvette is connected by polyvinyl chloride tubing to a glass capillary tube that descends to the bottom of the gradient. The gradient progresses through the system thanks to a peristaltic pump. OD260 is continuously read and 2 ml fractions are collected. <a href="https://www.jove.com/files/ftp_upload/54231/54231fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Adjust the fraction collector to RT, and set the carousel at a speed that will allow the collection of 2 ml fractions. Use pre-cooled collection tubes.</li>
	<li>Collect the fractions from bottom to top using a glass capillary tube connected by polyvinyl chloride tubing to the gradient collection system. Use a plastic paraffin film to secure the connection between the polyvinyl chloride tubing and the glass capillary tube.</li>
	<li>Read the absorbance at 260 nm from the bottom to the top of the gradient. When the entire gradient is collected, place the collection tubes on ice before RNA extraction.</li>
</ol>
4. RNA Extraction
<ol>
	<li>Pool the fractions collected from two gradients, in 50 ml capped centrifuge tubes, as follows: 
	Polysomes: fractions 1 to 3 (12 ml) 
	Monosomes: fraction 4 (4 ml) 
	Supernatant: fractions 5 and 6 (8 ml)</li>
	<li>To each fraction, add 1 vol. 8M guanidine hydrochloride, 50 &#181;g acryl carrier and 1.5 vol. isopropanol. 
	Note: Acryl carrier is linear acrylamide, used as a coprecipitant to improve the recovery of nucleic acids during alcohol precipitation.</li>
	<li>Mix by inverting the tubes and precipitate O/N&#160;at -20 &#176;C.</li>
	<li>Centrifuge at 175,000 x g for 1 hr at 4 &#176;C, in an ultracentrifuge (32,000 rpm in a SW32 rotor). If the precipitation step is performed in a tube which is not compatible with ultracentrifugation, transfer to a suitable tube.</li>
	<li>Discard supernatant and dissolve the pellet in 200 &#181;l RNAse-free TE buffer (10 mM Tris-HCl pH 7.5 - 1 mM EDTA). Transfer to a fresh 1.5 ml tube.</li>
	<li>Extract RNA by adding 1 vol. water saturated phenol (pH 6.6) and 1 vol. chloroform:isoamyl alcohol (24:1). Mix vigorously. Centrifuge at 15,000 x g for 20 min at 4 &#176;C. Transfer the aqueous phase to a new 1.5 ml tube. 
	Note: Acid phenol (pH 4.5) can be used in order to minimize DNA contamination.</li>
	<li>Precipitate RNA by adding 1/10 vol. 3 M sodium acetate and 3 vol. 100% ethanol. Allow precipitation at -80 &#176;C for 20 min or O/N at -20 &#176;C. Centrifuge at 15,000 x g for 15 min at 4 &#176;C. 
	Note: If using heparin in the polysome buffer (cf. note 2.5), perform a lithium chloride (LiCl) precipitation instead of ethanol precipitation to properly remove any trace of heparin that could interfere with downstream analysis9. After extraction, add LiCl to a final concentration of 2.5 M. Allow precipitation for 30 min at -20 &#176;C or O/N at 4 &#176;C. Centrifuge at 15,000 x g for 15 min at 4 &#176;C.</li>
	<li>Wash the pellet with 500 &#181;l 75% ethanol. Air dry the pellet and dissolve in 30 &#181;l TE buffer. Assess RNA quality by electrophoresis on an RNAse free 1.2% agarose gel (100V, 15 min) or by capillary electrophoresis methods.</li>
</ol>
5. Data Analysis
<ol>
	<li>Export raw data to a graphic and data analysis software. 
	Note: As shown in Figure 2A and B, the raw profiles give qualitative information: the number of polysome peaks that can be seen, the height of the monosome peak, and the presence of a shoulder corresponding to the free ribosome subunits.</li>
	<li>Merge profiles to allow comparison of the profiles between samples. Use the screen reader tool to determine the X value of the monosome peak for each sample and align the monosome peaks by removing extra data points at the beginning of the curves. Identify the lowest point of the curve and determine its Y value (using the screen reader tool). Set the lowest point Y value to 0 by using the &#34;set columns value&#34; function.</li>
	<li>Determine the polysomes/monosomes ratio by integrating the area under the curve from raw profiles (Figure 3C). Use the data selector tool to define the border of the area to be integrated. Then use the integrate function (Analysis-Mathematics).</li>
	<li>Normalize the curves to the monosome peak by dividing all data points of a curve by the Y value of the summit of the monosome peak (determined using the screen reader tool). Select the column, then under Analysis-Mathematics, select Normalize and choose the &#34;divided by a specified value&#34; methods. This step allows comparison of the relative level of polysomes (Figure 3D).</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/54231/54231fig2.jpg" /> 
Figure 2. Representative polysome profiles. Arabidopsis thaliana wild-type (wt, ecotype Col-0) and mutant seedlings were grown for six days on &#189; Murashige and Skoog medium under long day photoperiods (16 hr light, 8 hr dark). A: raw polysome profiles from wt seedlings. B: raw polysome profiles from mutant seedlings. C: Determination of the percentage of polysomes and monosomes by integration of the area under the curve D:Polysome profiles normalized to the monosome peak. <a href="https://www.jove.com/files/ftp_upload/54231/54231fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have nothing to disclose

The protocol we present here is an easy and cheap method for generating polysome profiles and isolating mRNAs associated with polysomes, single ribosomes or free of ribosomes. A wide range of different polysome fractionation methods is described in the literature. The method we have described here has been optimized to keep only the necessary compounds and has been adapted for plant material. In particular, we reduced the amount of detergent11 and added chloramphenicol to the buffer to fix the chloroplastic ri...

Protein synthesis is an essential and energetically costly process in all cells 1. First of all, cells must invest energy in the production of the translation machinery, the ribosomes. For example an actively dividing yeast cell produces as much as 2,000 ribosomes per minute. Such a production requires up to 60% of the total transcriptional activity and up to 90% of the total splicing activity of the cell 2. In addition, energy is required for the synthesis of amino acids, aminoacyl-tRNA and peptide bonds. In plants, adding one amino acid to a peptide chain costs from 4.5 to 5.9 molecules of ATP 3. Therefore, it is not surprising that the translation of mRNA to protein is a major site of regulation, particularly when it comes to dealing with changing environmental conditions.
The initiation step of translation, that is the association of a mRNA with the ribosome, is the main target of the regulation of translation4. As a consequence of the regulation of translation as well as other post-transcriptional regulatory steps, only 40% of the variations in protein concentration can be explained by mRNA abundance 5,6. Thus, the study of total mRNA gives relatively poor information about protein abundance. On the other hand, the association of mRNA with ribosomes gives better insight into protein abundance by giving access to those mRNAs involved in translation. Actively translated mRNAs are associated with several ribosomes in structures called polysomes. Conversely, poorly translated mRNAs will be associated with only one ribosome (monosome). Consequently, the translational status of an mRNA can be evaluated by monitoring its association with ribosomes7.
This protocol describes the isolation of polysomes from six days old Arabidopsis thaliana seedlings, the subsequent isolation of RNA, and the analysis of the results. Polysomes and monosomes are separated through a sucrose density gradient. Gradients are collected into six fractions. Some of the fractions are pooled to obtain three well separated fractions: polysomes, monosomes and the light fraction (hereafter called supernatant), which contains the free 60S and 40S ribosomal subunits and mRNAs that are not associated with ribosomes. Global translation activity can be estimated by generating a polysome/monosome ratio, which is determined by integration of the area under the curve, and by comparing the polysomes profiles. mRNAs and proteins are then extracted from the different fractions and used for analysis by RT-PCR, qRT-PCR, Northern blot, microarray, western blot or proteomics. This protocol has been validated for other plants and tissues.
The equipment required to perform this protocol are commonly found in most laboratories: There is no need for a gradient maker. Freezing each layer before adding the next one prevents from any mix or disturbance of the layers. No tube piercer is used for gradient collection which can be achieved by immersion of a glass capillary tube in the gradient. Therefore, the costly ultracentrifuge tubes remain undamaged and can be re-used many times. Collectively, this makes the present protocol an easy and cheap method for polysome profiling.

<table border="0" cellpadding="0" cellspacing="0" width="735">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge tube, thinwall, polyallomer - 13.2 ml</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">331372</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge tube, thinwall, polyallomer - 38.5 ml</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">326823</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Glass capillary tube</td>
			<td style="width:109px;">Drummond Scientific</td>
			<td style="width:132px;">1-000-1000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge&#160;</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">Optima series</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge Rotor SW41</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">331362</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge Rotor SW32</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">369650</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Peristaltic pump</td>
			<td style="width:109px;">Any</td>
			<td style="width:132px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Tygon R3607 polyvinyl chloride tubing&#160;</td>
			<td>Fisher Scientific</td>
			<td>070534-22</td>
			<td>Polyvinyl chloride tubing, 2.29 mm&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Fraction collector Model 2110</td>
			<td style="width:109px;">Bio-Rad</td>
			<td style="width:132px;">731-8120</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">UV cuvette</td>
			<td style="width:109px;">Hellma</td>
			<td style="width:132px;">170.700-QS</td>
			<td>Quartz flow-through cuvette</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">UV Spectrophotometer</td>
			<td style="width:109px;">Varian</td>
			<td style="width:132px;">Cary50</td>
			<td>Read every 0.0125 sec</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">All chemicals</td>
			<td style="width:109px;">Any</td>
			<td style="width:132px;"></td>
			<td>Use only Molecular Biology Grade</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Murashige and Skoog Basal Salt Mixture (MS)</td>
			<td>Sigma-Aldrich</td>
			<td>M5524</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Rnase-Free water</td>
			<td style="width:109px;">Any</td>
			<td style="width:132px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>10083251&#160;</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:233px;">Octylphenoxy poly(ethyleneoxy)ethanol, branched (Nonidet P40)</td>
			<td style="width:109px;">Euromedex</td>
			<td style="width:132px;">UN3500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Linear acrylamide (acryl carrier)</td>
			<td style="width:109px;">ThermoFischer scientific</td>
			<td style="width:132px;">AM9520</td>
			<td>RNA precipitation carrier</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">OriginPro 8</td>
			<td style="width:109px;">OriginLab</td>
			<td style="width:132px;"></td>
			<td>Analysis software</td>
		</tr>
	</tbody>
</table>

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.

In the literature, polysome profiles are often shown from the light fraction to the heavy fraction as a result of the way the gradients are collected, i.e. from the top to the bottom. Since in the protocol described here the gradients are collected from the bottom to the top, the profiles we show start with the heavy fraction (the polysomes) and go to the light fraction (free ribosome subunits and RNAs) (Figure 2A). We then collect each gradient in six 2 ml fractions, but...

Watch this Scientific Journal Video about An Easy Method for Plant Polysome Profiling at JoVE.com

An Easy Method for Plant Polysome Profiling

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 ...

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12.4K Views.  Aix-Marseille Université. 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.

Video: An Easy Method for Plant Polysome Profiling

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.