A Simplified Density Gradient–Free Method for Isolating Functional Symbiosomes From Legume Nodules
一种无需密度梯度的简化方法：从豆科植物根瘤中分离具有功能活性的共生体
(*contributed equally to this work) 发布: 2026年10月05日第16卷第19期 DOI: 10.21769/BioProtoc.5854 浏览次数: 26
评审: Anonymous reviewer(s)
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Abstract
Symbiosomes are plant membrane–bound, organelle-like structures that enclose single or multiple bacteroids (differentiated rhizobia) within legume cells, serving specifically as the site for N2 fixation. Although isolated symbiosomes have been used to study nitrogen fixation, ion transport, and metabolite translocation, conventional density gradient–based isolation methods for their isolation are labor-intensive. Here, we describe a simplified, density gradient–free protocol for rapid enrichment of symbiosome-containing fractions from mature legume nodules using the Medicago truncatula–Sinorhizobium meliloti symbiosis as an example. The method combines razor blade homogenization, filtration through a 40 μm cell strainer, and sequential low-speed centrifugation to remove debris and enrich symbiosome-associated fractions. The resulting material is compatible with downstream mass spectrometry–based proteomics and mutant phenotypic analyses. Compared with conventional approaches, this protocol is rapid, simple, and suitable for higher-throughput biochemical and proteomic studies of symbiosomes.
Key features
• Density gradient–free enrichment of symbiosome-containing fractions from mature legume nodules.
• Rapid workflow using homogenization, filtration, and low-speed centrifugation with minimal specialized equipment.
• Compatible with downstream mass spectrometry–based proteomic analysis of symbiosome-associated proteins.
• Suitable for comparative analysis of wildtype and mutant nodules in symbiotic nitrogen fixation studies.
Keywords: Symbiosome enrichment (共生体富集)
Functional symbiosome (功能性共生体)
Legume nodules (豆科植物根瘤)
Symbiotic nitrogen fixation (共生固氮)
Medicago truncatula (蒺藜苜蓿)
Background
Symbiotic nitrogen fixation in legumes depends on the formation of symbiosomes, specialized membrane-bound compartments that house differentiated rhizobia within infected nodule cells [1,2]. Symbiosomes provide the biochemical environment required for N2 fixation and mediate extensive metabolite and ion exchange between the host plant and bacterial symbiont. Because of their central role in symbiosis, isolated symbiosomes have been used to investigate nitrogen fixation activity, membrane transport, and metabolite exchange [3,4]. Despite the establishment of early symbiosome isolation methods, relatively few studies have subsequently employed isolated symbiosomes for functional analyses, largely due to the fact that conventional density gradient–based procedures are technically demanding, low-throughput, and often require substantial nodule material. Several modified approaches have attempted to simplify the procedure through homogenization and low-speed centrifugation to obtain crude symbiosome or infected-cell fractions [5,6]. Although these methods improve experimental accessibility, the resulting preparations are often heterogeneous and may exhibit reduced structural integrity, and their suitability for downstream physiological analyses has not been extensively evaluated. A recent proteomic study identified 111 symbiosome membrane-associated proteins through purification of intact symbiosomes followed by symbiosome membrane enrichment [7,8]. However, this procedure relied on multiple purification steps and Tris-HCl-based buffers, which may not be optimal for preserving the physiological activity of symbiosomes during downstream in vitro functional assays. Here, we describe a simplified density gradient-free protocol for the enrichment of symbiosome-containing fractions from mature legume nodules using the Medicago truncatula–Sinorhizobium meliloti symbiosis as an example. This protocol was adapted from [3,4] and optimized based on [5]. The method combines mechanical homogenization, filtration, and sequential low-speed centrifugation to reduce cellular debris while enriching symbiosome-associated particles. Although the resulting fractions are less highly purified than those obtained by density gradient centrifugation, they are suitable for downstream mass spectrometry–based proteomics and comparative analyses of wildtype and mutant nodules. In addition, the protocol may facilitate studies of symbiosome-associated protein localization, mutant phenotyping, and regulatory mechanisms involved in symbiotic nitrogen fixation.
Materials and reagents
Biological materials
1. Mature nodules from legume plants (Medicago truncatula nodulated with Sinorhizobium meliloti, harvested 21 days post-inoculation)
Reagents
1. Mannitol (Sigma-Aldrich, CAS: 69-65-8)
2. Ethylene glycol bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma-Aldrich, CAS: 67-42-5)
3. MgSO4·7H2O (Sigma-Aldrich, CAS: 10034-99-8)
4. Dithiothreitol (DTT) (Sigma-Aldrich, CAS: 3483-12-3)
5. Bovine serum albumin (BSA), fatty acid-free (Sigma-Aldrich, CAS: 9048-46-8)
6. Ascorbic acid (Sigma-Aldrich, CAS: 50-81-7)
7. MES (Sigma-Aldrich, CAS: 4432-31-9)
8. KOH (Sigma-Aldrich, CAS: 1310-58-3); 1 M KOH solution was used to adjust MES to pH 7.0
9. KNO3 (Sigma-Aldrich, CAS: 7757-79-1)
10. (Optional) Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 or P9599; recommended for protein/proteomic analyses)
11. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, CAS: 151-21-3); used for protein lysis
12. Ammonium bicarbonate (ABC) (Sigma-Aldrich, CAS: 1066-33-7); 50 mM working solution prepared in water for protein digestion
13. Methanol (Sigma-Aldrich, CAS: 67-56-1); ice-cold, used for chloroform/methanol precipitation
14. Chloroform (Sigma-Aldrich, CAS: 67-66-3); used for protein precipitation
15. Acetone (Sigma-Aldrich, CAS: 67-64-1); 90% (v/v) in water, used for washing protein pellets
16. Urea (Sigma-Aldrich, CAS: 57-13-6); 8 M solution prepared in water for protein solubilization
17. Bicinchoninic acid (BCA) assay kit (Sigma-Aldrich, catalog number: BCA1 or equivalent); used for protein quantification
18. Tris(2-carboxyethyl)phosphine (TCEP) (Sigma-Aldrich, CAS: 51805-45-9); used as reducing agent
19. Chloroacetamide (CAA) (Sigma-Aldrich, CAS: 79-07-2); used for alkylation
20. Trypsin (Sigma-Aldrich, catalog number: T6567 or equivalent); used for protein digestion
21. Trifluoroacetic acid (TFA) (Sigma-Aldrich, CAS: 76-05-1); 20% and 0.1% (v/v) solutions prepared in water for acidification and peptide loading
22. Acetonitrile (ACN), HPLC grade (Sigma-Aldrich, CAS: 75-05-8); used for peptide elution and as mobile phase B for LC-MS/MS
23. Formic acid (FA), HPLC grade (Sigma-Aldrich, CAS: 64-18-6); 0.1% (v/v) solution in water used as mobile phase A and peptide reconstitution buffer
24. Colorimetric peptide assay kit (ThermoFisher Scientific, catalog number: 23275 or equivalent); used for peptide quantification after desalting
Solutions
1. Isolation buffer (see Recipes)
2. Wash buffer (see Recipes)
3. Testing buffer (see Recipes)
Recipes
1. Isolation buffer (100 mL, pH 7.0)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Mannitol | 350 mM | 6.36 g |
| EGTA | 10 mM | 0.38 g |
| MgSO4·7H2O | 10 mM | 0.246 g |
| MES | 25 mM | 0.488 g |
| BSA | 1% | 1 g |
| Ascorbic acid | 20 mM | 0.352 g |
| DTT | 5 mM | 77 mg |
2. Wash buffer (100 mL, pH 7.0)
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Mannitol | 350 mM | 6.36 g |
| MES (pH 7.0) | 25 mM | 0.488 g |
| MgSO4·7H2O | 3 mM | 82 mg |
3. Testing buffer
Prepare by supplementing wash buffer (see Recipe 2) with the substrate of interest, depending on the functional assay to be performed. For example, for nitrate uptake assays, the testing buffer consists of wash buffer containing 2 mM KNO3 as the substrate. The buffer should be prepared fresh on the day of the experiment and kept on ice until use.
Laboratory supplies
1. 1 mL pipette tip (Maisinuo, catalog number: HZX005-1)
2. Double-edged razor blade (Shanghai Feiying Brand, catalog number: FEIYING-DE)
3. 10 × 10 cm Petri dish (LABSTAR, catalog number: BX051)
4. 40 μm cell strainer (WHB, catalog number: WHB-40UM-F)
5. 15 mL sterile centrifuge tube (LABSELECT, catalog number: CT-002-15A)
6. Forceps (Maisinuo, catalog number: HZX186-1)
7. 1.5 mL microcentrifuge tube (Maisinuo, catalog number: HZX018-1)
Equipment
1. Refrigerated centrifuge (Eppendorf, model: 5424 R)
2. Microscope (Olympus, model: BX51)
3. Balance (Mettler Toledo, model: ML203T)
4. 1 mL single-channel pipette (Eppendorf)
Procedure
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文章信息
稿件历史记录
提交日期: Jun 30, 2026
接收日期: Aug 19, 2026
在线发布日期: Sep 30, 2026
出版日期: Oct 5, 2026
版权信息
© 2026 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
如何引用
Li, F., Hou, Y. and Murray, J. D. (2026). A Simplified Density Gradient–Free Method for Isolating Functional Symbiosomes From Legume Nodules. Bio-protocol 16(19): e5854. DOI: 10.21769/BioProtoc.5854.
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