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 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.
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.
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. Please click here to view a larger version of this figure.
1. Plant growth and rhizobia inoculation
2. Water deficit treatment (optional)
NOTE: This protocol outlines the water deficit treatment of the soybean plants. This part can be changed or omitted entirely depending on the experimental question at hand.
3. Nodule harvest
4. Preparation of cytosolic extracts
NOTE: The final aim of this protocol is to obtain high-quality total RNA (TOTAL) and polysome-associated RNA (PAR). Therefore, work under conditions that prevent RNA degradation, always keeping the samples at 4 °C and using RNase-free laboratory equipment and solutions. Unless specified, all the solutions are prepared with sterile ultrapure water.
5. Preparation of sucrose cushions
NOTE: This protocol uses a two-layer sucrose cushion (12% and 33.5%) in 13.2 mL ultracentrifuge tubes (see the Table of Materials). All solutions are prepared with sterile ultrapure water.
6. Polysome purification
7. RNA extraction and quality control
NOTE: This step is performed for TOTAL (step 4.8) and PAR samples (step 6.4).
8. RNA precipitation
9. Standard pipeline for gene expression analysis
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 performing these measurements using a Bioanalyzer. The RIN is used for assigning integrity values in a robust, reliable, and user-independent way, ranging from 10 (intact) to 1 (totally degraded)18.
Figure 2A,B show the standard output of the high-sensitivity capillary electrophoresis system (Bioanalyzer) for several TOTAL and PAR sample fractions. Very good quality is observed for all the samples since sharp ribosomal RNA (rRNA) bands can be visualized 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. Nevertheless, as previously mentioned, there is no visual sign of sample degradation, as can be seen in Figure 2C. Figure 2D shows an example of a bioanalyzer result of an almost entirely degraded sample for comparison. The equipment also failed to identify the 18S and 25S peaks, as seen in the electropherograms both for TOTAL and PAR samples (Figure 2B). Consequently, the proportion of the ribosomal bands (25S:18S; another indicator of RNA integrity) could not be calculated. High-quality RNA usually exhibits a 25S:18S 2:1 ratio33.
Figure 2: Total RNA and polysome-associated mRNA quantity and quality assessment. (A) Representative virtual gel reconstruction of high-sensitivity capillary electrophoresis (Bioanalyzer) results from 12 samples with their respective RIN and concentration values. Lane 1 to lane 6 correspond to total RNA (TOTAL) fractions of six nodule samples, and lane 7 to lane 12 correspond to polysome-associated mRNA (PAR) fractions of the same samples. Green line: lower marker present in all lanes. (B) Electropherogram representation is shown for sample 4 (as an example of TOTAL) and sample 10 (as an example of PAR). 18S and 25S peaks are indicated. Peaks possibly corresponding to 16S and 23S (prokaryotic rRNAs) are indicated with *. Zoom-ins on these peaks are shown (B1 and B2). The Eukaryote Total RNA Nano assay was used in the Bioanalyzer. (C) Representative agarose gel electrophoresis results from six samples (left): 10 µL of TOTAL and PAR fractions were loaded on a 2% agarose gel and separated (80 V for 35 min). On the right, there is another gel of sample 2 only, where the prokaryotic rRNAs can be visualized more clearly. Eukaryotic rRNA (18S and 25S) are indicated by black arrowheads and prokaryotic rRNAs (16S and 23S) by grey arrowheads.(D) Example of a Bioanalyzer result of a degraded sample. L: standard ladder. Green line: lower marker present in all lanes. Abbreviations: RIN = RNA integrity number; PAR = polysome-associated mRNA; nt = nucleotides; L = standard ladder. Please click here to view a larger version of this figure.
Since these samples are from symbiotic nodules where eukaryotic and prokaryotic RNAs coexist, the purified RNA is a mix of both species (although the eukaryotic ones are the majority). Hence, it is expected to observe peaks corresponding to 16S and 23S rRNA in the electropherograms (Figure 2B and zoom-in B1 and B2). These "extra" peaks could explain the lower RIN values obtained.
The equipment also calculates sample concentrations considering the standard ladder in lane 1 as a reference. As expected, the TOTAL samples present higher concentration values than the PAR samples. Still, even for these, there is enough RNA to proceed with library preparation and RNA-seq since sample quantity requirements typically recommend at least 1.0 µg.
Autoclavable solutions to be stored at room temperature |
2 M Tris-HCl pH 9.0 |
2 M KCl |
0.5 M EGTA pH 8.0 (adjusted with 10 M NaOH) |
1 M MgCl2 |
20% (v/v) PTE (shake bottle before pipetting the solution) |
10% DOC |
20% Detergent mix: 20% (w/v) Brij L23, 20% (v/v) Tritón X-100, Igepal CA 360, 20% (v/v) Tween 20 (dissolve while heating at 60 °C) |
Filter-sterilizable solutions to be frozen at -20 °C |
0.5 M DTT |
0.5 M PMSF |
50 mg.mL-1 cycloheximide (CHX) (in EtOH) |
50 mg.mL-1 chloranphenicol (CHL) (in EtOH) |
Stock solution | Volume (µL) for 10 mL PEB | Volume (µL) for 2 mL RB |
2 M Tris, pH 9.0 | 1,000 | 200 |
2 M KCl | 1,000 | 200 |
0.5 M EGTA pH 8.0 | 500 | 100 |
1 M MgCl2 | 356 | 70 |
20% (v/v) PTE | 500 | – |
10% DOC | 1,000 | – |
20% Detergent mix | 500 | – |
0.5 M DTT | 100 | 20 |
0.5 M PMSF | 20 | – |
50 mg.mL-1 CHX | 10 | 2 |
50 mg.mL-1 CHL | 10 | 2 |
H2O | 5,004 | 1,406 |
2 M sucrose solution (68.5%): 68.5 g of sucrose in water. Keep at room temperature (RT). | ||
10x salt solution: 0.4 M Tris-HCl pH 8.4, 0.2 M KCl, 0.1 M MgCl2; autoclave for 15 min, freeze at -20 °C. |
Table 1: Buffers and solutions used in this protocol. The volumes given are for six samples. Abbreviations: EGTA = ethylene glycol-bis(2-aminoethylether)-N,N,N´,N´-tetraacetic acid; PTE = polyoxyethylene (10) tridecyl ether; DOC = sodium deoxycholate; DTT = dithiothreitol; PMSF = phenylmethanesulfonyl fluoride.
Sucrose layer (%) | Sucrose 2 M (mL) | Salt solution 10x (mL) | CHX 50 mg.mL-1 (µL) | CHL 50 mg.mL-1 (µL) | H2O (mL) |
12 | 5.25 | 3 | 3 | 3 | 21.75 |
33.5 | 14.7 | 3 | 3 | 3 | 12.3 |
Table 2: Composition of the two sucrose layers (12% and 33.5%). The volumes given are for the preparation of six cushions.
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 analyzed3,4,34,35,36.
A method is presented here to purify soybean symbiotic nodule polysomes through sucrose cushions, followed by TOTAL and PAR extraction. Obtaining both RNA fractions is one relevant point of this protocol since RNA-seq and standard pipelines for gene expression could be implemented afterward. Thus, the transcriptome and the translatome of the nodule can be studied.
An alternative method for polysome purification is the translating ribosome affinity purification (TRAP) method, in which the expression of an epitope-tagged version of ribosomal protein L18 allows for the immunoprecipitation of ribosomes37,38. However, this method requires the generation of transgenic plants, which can be challenging for some species such as the soybean. Moreover, the ribosome profiling or Ribo-seq method15,39,40,41, which involves the deep sequencing of ribosome-protected mRNA fragments after polysome purification, is an alternative for studying the translatome with higher resolution than polysome profiling, since the exact number and position of ribosomes on individual mRNAs can be assessed. Nevertheless, this approach is more laborious, requires higher amounts of sample, and is computationally intensive, since small RNA fragments are recovered.
There are several critical experimental aspects to consider, such as the quality of the sample from which the polysomes will be purified, especially if frozen tissue is used, as is the case in this protocol. Intact nodules should be detached from the root and immediately frozen in liquid nitrogen before storage at −80 °C. In our experience, polysomes and their associated mRNAs can be successfully purified from nodules within a few months of storage. Furthermore, the whole protocol must be implemented under conditions that prevent RNA degradation to obtain high-quality TOTAL and PAR, which is critical for downstream applications such as cDNA synthesis and high-throughput sequencing.
A major limitation of the method is the ultracentrifugation step, as ultracentrifuges are not widely available in many laboratories. Also, it is relevant to mention that the pellet obtained after the centrifugation step might contain, besides polysomes, other ribonucleoproteins complexes that are not translationally active. Moreover, one consideration if performing the protocol presented here with another soybean tissue is that it will probably require optimizing the amount of sample, the sample:PEB ratio, and the homogenization procedure.
Considering that the sample used in this protocol is an organ formed due to a symbiotic interaction between a plant root cell and a bacteria, performing a Dual RNA-seq analysis42 (with TOTAL and PAR samples) would be interesting since it would allow examination of the transcriptional and translational responses from both organisms. The method described here could be helpful for this matter if the extraction of the bacterial RNAs is optimized.
The authors have nothing to disclose.
This research was funded by CSIC I+D 2020 grant No. 282, FVF 2017 grant No. 210, and PEDECIBA (María Martha Sainz).
Plant growth and rhizobia inoculation | |||
Orbital shaker | Daihan Scientific | Model SHO-1D | |
YEM-medium | Amresco | J850 (yeast extract) 0122 (mannitol) | |
Water deficit treatment | |||
KNO3 | Merck | 221295 | |
Porometer | Decagon Device | Model SC-1 | |
Scalpel | |||
Preparation of cytosolic extracts | |||
Brij L23 | Sigma-Aldrich | P1254 | |
Centrifuge | Sigma | Model 2K15 | |
Chloranphenicol | Sigma-Aldrich | C0378 | |
Cycloheximide | Sigma-Aldrich | C7698 | |
DOC | Sigma-Aldrich | 30970 | |
DTT | Sigma-Aldrich | D9779 | |
EGTA | Sigma-Aldrich | E3889 | |
Igepal CA 360 | Sigma-Aldrich | I8896 | |
KCl | Merck | 1.04936 | |
MgCl2 | Sigma-Aldrich | M8266 | |
Plastic tissue grinder | Fisher Scientific | 12649595 | |
PMSF | Sigma-Aldrich | P7626 | |
PTE | Sigma-Aldrich | P2393 | |
Tris | Invitrogen | 15504-020 | |
Triton X-100 | Sigma-Aldrich | T8787 | |
Tween 20 | Sigma-Aldrich | P1379 | |
Weighing dish | Deltalab | 1911103 | |
Preparation of sucrose cushions | |||
Sucrose | Invitrogen | 15503022 | |
SW 40 Ti rotor | Beckman-Coulter | ||
Ultracentrifuge | Beckman-Coulter | Optima L-100K | |
Ultracentrifuge tubes | Beckman-Coulter | 344059 | 13.2 mL tubes |
RNA extraction and quality control | |||
Agarose | Thermo scientific | R0492 | |
Bioanalyzer | Agilent | Model 2100. Eukaryote total RNA nano assay | |
Chloroform | DI | 41191 | |
Ethanol | Dorwil | UN1170 | |
Isopropanol | Mallinckrodt | 3032-06 | |
Glycogen | Sigma | 10814-010 | |
TRIzol LS | Ambion | 102960028 | |
Miscellaneous | |||
Falcon tubes 15 mL | Biologix | 10-0152 | |
Filter tips 10 µL | BioPointe Scientific | 321-4050 | |
Filter tips 1000 µL | BioPointe Scientific | 361-1050 | |
Filter tips 20 µL | BioPointe Scientific | 341-4050 | |
Filter tips 200 µL | Tarsons | 528104 | |
Microcentrifuge tubes 1.5 mL | Tarsons | 500010-N | |
Microcentrifuge tubes 2.0 mL | Tarsons | 500020-N | |
Sequencing company | Macrogen | ||
Sterile 250 mL flask | Marienfeld | 4110207 |