This research was funded by CSIC I+D 2020 grant No. 282, FVF 2017 grant No. 210, and PEDECIBA (Mar&#237;a Martha Sainz).

1. Plant growth and rhizobia inoculation
<ol>
	<li>To generate the required nodules, sow the soybean seeds of choice in the selected substrate in a growth chamber under controlled conditions. 
	NOTE: In this protocol, seeds were sown in a 0.5 L plastic bottle filled with a mix of sand:vermiculite (1:1). The growth chamber was set with a day/night cycle temperature of 28 &#176;C /20 &#176;C, respectively, and a light/darkness photoperiod of 16 h/8 h, respectively. The photosynthetically active radi...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+gapped-read+alignment+with+Bowtie+2.">Fast gapped-read alignment with Bowtie 2.</a> Nature Methods. 9 (4), 357-359 (2012).</li><li>Kim, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopHat2:+Accurate+alignment+of+transcriptomes+in+the+presence+of+insertions,+deletions+and+gene+fusions.">TopHat2: Accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions.</a> Genome Biology. 14 (4), 1-13 (2013).</li><li>Patro, R., Duggal, G., Love, M. I., Irizarry, R. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GEOexplorer:+A+webserver+for+gene+expression+analysis+and+visualisation.">GEOexplorer: A webserver for gene expression analysis and visualisation.</a> Nucleic Acids Research. , (2022).</li><li>Imbeaud, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+standardization+of+RNA+quality+assessment+using+user-independent+classifiers+of+microcapillary+electrophoresis+traces.">Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces.</a> Nucleic Acids Research. 33 (6), 1-12 (2005).</li><li>Di Paolo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDCD4+regulates+axonal+growth+by+translational+repression+of+neurite+growth-related+genes+and+is+modulated+during+nerve+injury+responses.">PDCD4 regulates axonal growth by translational repression of neurite growth-related genes and is modulated during nerve injury responses.</a> RNA. 26 (11), 1637-1653 (2020).</li><li>Smircich, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+profiling+reveals+translation+control+as+a+key+mechanism+generating+differential+gene+expression+in+Trypanosoma+cruzi.">Ribosome profiling reveals translation control as a key mechanism generating differential gene expression in Trypanosoma cruzi.</a> BMC Genomics. 16 (1), 443 (2015).</li><li>Eastman, G., Sharlow, E. 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The authors have no conflicts of interest.

Studying gene expression regulation at the translational level is critical to better comprehend different biological processes since the endpoint of cell gene expression is protein abundance13,14. This can be assessed by analyzing the translatome of the tissue or organism of interest for which the polysomal fraction should be purified and its associated mRNAs analyzed3,4,34</s...

Leguminous plants, such as soybean (Glycine max), can establish symbiosis with specific soil bacteria called rhizobia. This mutualistic relationship elicits the formation of novel organs, the symbiotic nodules, on the plant roots. The nodules are the plant organs hosting the bacteria and consist of host cells whose cytoplasm is colonized with a specialized form of rhizobia called bacteroids. These bacteroids catalyze the reduction of atmospheric nitrogen (N2) into ammonia, which is transferred to the plant in return for carbohydrates1,2.
Although this nitrogen-fixing symbiosis is one of the most well-studied plant-microbe symbioses, many aspects remain to be better understood, such as how plants subjected to different abiotic stress conditions modulate their interaction with their symbiotic partner and how this affects nodule metabolism. These processes could be better understood by analyzing the nodule translatome (i.e., the subset of messenger RNAs [mRNAs] actively translated). Polyribosomes or polysomes are complexes of multiple ribosomes associated with mRNA, commonly used to study translation3. The polysome profiling method consists of the analysis of the mRNAs associated with polysomes and has been successfully used to study the posttranscriptional mechanisms controlling gene expression that occurs in diverse biological processes4,5.
Historically, genome expression analysis has focused primarily on determining mRNA abundance6,7,8,9. However, there is a lack of correlation between transcript and protein levels due to the different stages of posttranscriptional regulation of gene expression, particularly translation10,11,12. Moreover, no dependence has been observed between the changes at the level of the transcriptome and those that occur at the level of the translatome13. The direct analysis of the set of mRNAs that are being translated allows a more accurate and complete measurement of the cell gene expression (whose endpoint is protein abundance) than the one obtained when only mRNA levels are analyzed14,15,16.
This protocol describes how plant-derived polysomes are purified from intact soybean nodules by differential centrifugation through a two-layer sucrose cushion (Figure 1). However, since bacteroid-derived ribosomes are also present in the nodules, a mix of ribosomes and RNA species are purified, even though the eukaryotic ones represent the main fraction (90%-95%). The subsequent RNA isolation, quantification, and quality control are also described (Figure 1). This protocol, in combination with RNA-seq, should provide experimental results on the translational regulation of eukaryotic mRNAs in a complex tissue such as the symbiotic nodule.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64269/64269fig01.jpg" /> 
Figure 1: Schematic overview of the proposed methodology for eukaryotic polysome purification from symbiotic nodules. The scheme gives an overview of the steps followed in the protocol from (1) plant growth and (2) nodule harvest to (3) preparation of the cytosolic extracts, (3) obtaining TOTAL samples and (4) PAR samples, and (5) RNA extraction and quality control. Abbreviations: PEB = polysome extraction buffer; RB = resuspension buffer; TOTAL = total RNA; PAR = polysome-associated mRNA. <a href="https://www.jove.com/files/ftp_upload/64269/64269fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Plant growth and rhizobia inoculation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Daihan Scientific</td>
			<td></td>
			<td>Model SHO-1D</td>
		</tr>
		<tr>
			<td>YEM-medium</td>
			<td>Amresco</td>
			<td>J850 (yeast extract) 0122 (mannitol)</td>
			<td></td>
		</tr>
		<tr>
			<td>Water deficit treatment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KNO3</td>
			<td>Merck</td>
			<td>221295</td>
			<td></td>
		</tr>
		<tr>
			<td>Porometer</td>
			<td>Decagon Device</td>
			<td></td>
			<td>Model SC-1</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Preparation of cytosolic extracts</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brij L23</td>
			<td>Sigma-Aldrich</td>
			<td>P1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sigma</td>
			<td></td>
			<td>Model 2K15</td>
		</tr>
		<tr>
			<td>Chloranphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C0378</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma-Aldrich</td>
			<td>C7698</td>
			<td></td>
		</tr>
		<tr>
			<td>DOC</td>
			<td>Sigma-Aldrich</td>
			<td>30970</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Igepal CA 360</td>
			<td>Sigma-Aldrich</td>
			<td>I8896</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Merck</td>
			<td>1.04936</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic tissue grinder</td>
			<td>Fisher Scientific</td>
			<td>12649595</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr>
			<td>PTE</td>
			<td>Sigma-Aldrich</td>
			<td>P2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Invitrogen</td>
			<td>15504-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing dish&#160;</td>
			<td>Deltalab</td>
			<td>1911103</td>
			<td></td>
		</tr>
		<tr>
			<td>Preparation of sucrose cushions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Invitrogen</td>
			<td>15503022</td>
			<td></td>
		</tr>
		<tr>
			<td>SW 40 Ti rotor</td>
			<td>Beckman-Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman-Coulter</td>
			<td></td>
			<td>Optima L-100K</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes</td>
			<td>Beckman-Coulter</td>
			<td>344059</td>
			<td>13.2 mL tubes</td>
		</tr>
		<tr>
			<td>RNA extraction and quality control</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Thermo scientific</td>
			<td>R0492</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioanalyzer</td>
			<td>Agilent</td>
			<td></td>
			<td>Model 2100. Eukaryote total RNA nano assay</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>DI</td>
			<td>41191</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Dorwil</td>
			<td>UN1170</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Mallinckrodt</td>
			<td>3032-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Sigma</td>
			<td>10814-010</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol LS</td>
			<td>Ambion</td>
			<td>102960028</td>
			<td></td>
		</tr>
		<tr>
			<td>Miscellaneous</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes 15 mL</td>
			<td>Biologix</td>
			<td>10-0152</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips 10 &#181;L</td>
			<td>BioPointe Scientific</td>
			<td>321-4050</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips 1000 &#181;L</td>
			<td>BioPointe Scientific</td>
			<td>361-1050</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips 20 &#181;L</td>
			<td>BioPointe Scientific</td>
			<td>341-4050</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips 200 &#181;L</td>
			<td>Tarsons</td>
			<td>528104</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 1.5 mL</td>
			<td>Tarsons</td>
			<td>500010-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 2.0 mL</td>
			<td>Tarsons</td>
			<td>500020-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencing company</td>
			<td>Macrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 250 mL flask</td>
			<td>Marienfeld</td>
			<td>4110207</td>
			<td></td>
		</tr>
	</tbody>
</table>

polysome purification from soybean symbiotic nodules

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper describes methods optimized to isolate plant-derived polyribosomes and their associated mRNAs to be analyzed using RNA-sequencing. First, cytoplasmic lysates are obtained through homogenization in polysome- and RNA-preserving conditions from whole, frozen soybean nodules. Then, lysates are cleared by low-speed centrifugation, and 15% of the supernatant is used for total RNA (TOTAL) isolation. The remaining cleared lysate is used to isolate polysomes by ultracentrifugation through a two-layer sucrose cushion (12% and 33.5%). Polysome-associated mRNA (PAR) is purified from polysomal pellets after resuspension. Both TOTAL and PAR are evaluated by highly sensitive capillary electrophoresis to meet the quality standards of sequencing libraries for RNA-seq. As an example of a downstream application, after sequencing, standard pipelines for gene expression analysis can be used to obtain differentially expressed genes at the transcriptome and translatome levels. In summary, this method, in combination with RNA-seq, allows the study of the translational regulation of eukaryotic mRNAs in a complex tissue such as the symbiotic nodule.

The quantity and quality assessment of the TOTAL and PAR fractions purified with the abovementioned procedure is key to determining its success, since for most downstream applications, such as RNA sequencing, high-quality samples are fundamental for library preparation and sequencing. Moreover, the integrity of the RNA molecules allows the capture of a snapshot of the gene expression profile at the moment of sample collection18. In this context, an RNA integrity number (RIN) is obtained when perfo...

Watch this Scientific Journal Video about Polysome Purification from Soybean Symbiotic Nodules at JoVE.com

Polysome Purification from Soybean Symbiotic Nodules

This protocol describes a method for eukaryotic polysome purification from intact soybean nodules. After sequencing, standard pipelines for gene expression analysis can be used to identify differentially expressed genes at the transcriptome and translatome levels.

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper ...

This protocol is significant because it is, to our knowledge, the first one to describe a new centrifuge based metal for kinetic polysome purification from soybean symbiotic nodules. The big advantage of this technique is that in combination with RNA-Seq, allows the study of the relation and organized in a complex tissue such as the symbiotic nodule. To begin, weigh approximately 0.2 grams of intact nodules in a weighing dish, and transfer them to a pre-cooled two milliliter tube.Then add 1.2 milliliters of polysome extraction buffer, let the sample thaw for two minutes, and homogenize with a tissue grinder until complete disruption and homogenization of the nodules. Next, incubate the samples on ice with gentle agitation for 10 minutes or until all samples have been processed and centrifuge at 16, 000 times G for 15 minutes at four degrees Celsius to pellet the debris. Then, recover the supernatant, and repeat the centrifuge step.After carefully recovering the clarified cytosolic extract, transfer a 200 microliter aliquot to a clean 1.5 milliliter micro centrifuge tube for total isolation. Then, pour 4.5 milliliters of the 33.5%sucrose layer into the centrifuge tubes, and add 4.5 milliliters of the 12%layer carefully and slowly with a P-1000 micropipette. Next, put the tubes on ice until the addition of the clarified cytosolic extract.Load one milliliter of the clarified cytosolic extract on top of the sucrose cushion by carefully pipetting onto the side wall of the tube. Then, transfer the ultra centrifuge tubes to pre-cooled buckets, and centrifuge at 217, 874 G for two hours at four degrees Celsius. After discarding the remaining cytosolic extract and sucrose cushion, re-suspend the polysomal pellet with 200 microliters of pre-cooled re-suspension buffer.Next, incubate for 30 minutes on ice, and then transfer the polysomal re-suspension to 1.5 milliliter pre-cooled tubes to proceed with the RNA extraction. After homogenizing the samples with 750 microliters of the RNA isolation reagent incubate for five minutes at room temperature. Then, add 200 microliters of cold chloroform, and shake the tubes vigorously for 15 seconds.Next, incubate at room temperature for 10 minutes. Centrifuge at 12, 000 times G for 15 minutes at four degrees Celsius for phase separation, and transfer 500 microliters of the upper aqueous phase to a clean tube without disturbing the pink organic phase. Then, add 375 microliters of cold isopropanol and 0.5 microliters of RNAs free glycogen.Afterward, mix thoroughly by pipetting up and down. Incubate the mix for 10 minutes at four degrees Celsius, and centrifuge at 12, 000 times G for 15 minutes. Then, discard the supernatant, and wash the RNA precipitate with one milliliter of cold, 75%ethanol.Mix by brief vortexing. Next, centrifuge at 7, 500 G for five minutes at four degrees Celsius. Discard the supernatant, and air dry the RNA pellet.Then, dissolve the pellet in 50 microliters of RNAs free water, and incubate at 65 degrees Celsius for five minutes. Assess the RNA concentration and integrity with highly sensitive capillary electrophoresis and, or electrophoresis on a 2%RNAs free agarose gel. After estimating the sample volume, add 0.1 volumes of three molar sodium acetate, three volumes of cold ethanol, and 0.5 microliters of RNAs, free glycogen.Then, mix them thoroughly. Capillary electrophoresis was performed for several total and polysomal associated RNA sample fractions, which demonstrated sharp ribosomal RNA bands, without any smeared appearance. However, this is not reflected in the RIN values, as they range between 5.9 and 7.5, which corresponds to non-intact samples.The bioanalyzer failed to identify the 18S and 25S peaks as seen in the electropherograms, both for total and polysome associated RNA samples. There was no visual sign of sample degradation. However, a sample was analyzed to visualize the result of almost entirely degraded sample, for comparison.The most important thing is to work in polysome and RNA preserving conditions during the whole protocol. After sequencing the total and par RNA fractions standard patterns for the gene expression analyzed can be used to identify differential express genes at the transcription and the translation level.

polysome-purification-from-soybean-symbiotic-nodules

Research

JoVE Journal

Developmental Biology

1.4K Görüntüleme.  Universidad de la República. Bu protokol önemlidir, çünkü bildiğimiz kadarıyla, soya fasulyesi simbiyotik nodüllerinden kinetik polizom saflaştırması için yeni bir santrifüj bazlı metali tanımlayan ilk protokoldür. Bu tekniğin en büyük avantajı, RNA-Seq ile kombinasyon halinde, ilişkinin incelenmesine izin vermesi ve simbiyotik nodül gibi karmaşık bir dokuda organize olmasıdır. Başlamak için, bir tartım kabında yaklaşık 0,2 gram bozulmamış nodül tartın ve bunları önceden soğutulmuş ...