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Isolation of Intact Vacuoles from Arabidopsis Root Protoplasts and Elemental Analysis

拟南芥根原生质体完整液泡的分离及元素分析

CJ Chuanfeng Ju
DF Dali Fu
Cun Wang Cun Wang
ZZ Zhenqian Zhang

发布: 2023年03月05日第13卷第5期 DOI: 10.21769/BioProtoc.4625 浏览次数: 1014

评审: Wenrong HeYao XiaoYe XuAnonymous reviewer(s)

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Abstract

The vacuole is one of the most conspicuous organelles in plant cells, participating in a series of physiological processes, such as storage of ions and compartmentalization of heavy metals. Isolation of intact vacuoles and elemental analysis provides a powerful method to investigate the functions and regulatory mechanisms of tonoplast transporters. Here, we present a protocol to isolate intact vacuoles from Arabidopsis root protoplasts and analyze their elemental content by inductively coupled plasma mass spectrometry (ICP-MS). In this protocol, we summarize how to prepare the protoplast, extract the vacuole, and analyze element concentration. This protocol has been applied to explore the function and regulatory mechanisms of tonoplast manganese (Mn) transporter MTP8, which is antagonistically regulated by CPK4/5/6/11 and CBL2/3-CIPK3/9/26. This protocol is not only suitable for exploring the functions and regulatory mechanisms of tonoplast transporters, but also for researching other tonoplast proteins.


Graphical abstract


Keywords: Isolation of intact vacuoles (完整液泡的分离) search Arabidopsis protoplasts (拟南芥原生质体) search Elemental analysis (元素分析) search Mn transporter (锰转运体) search MTP8 (MTP8) search

Background

The central vacuole is the largest compartment of a mature plant cell and participates in plenty of physiological processes. The determination of its elemental concentrations is essential for researching the function and regulation of plant tonoplast transporters. The transport rates of different anions and the factors affecting the uptake of chloride ions across the tonoplast were detected in isolated barley vacuoles (Martinoia et al., 1986). Transport of phosphate across the tonoplast was also detected in intact vacuoles, which were isolated from suspension-cultured cells of Catharanthus roseus (L.) G. Don (Massonneau et al., 2000). Shimaoka et al. (2004) modified the method for extracting intact vacuoles, which is summarized in this article, and detected the tonoplast proteins combined with proteomic analysis. The function and regulatory mechanisms of the tonoplast manganese (Mn) transporter MTP8 were resolved via this method (Eroglu et al., 2016; Zhang et al., 2021; Ju et al., 2022). In conclusion, this protocol is feasible and also applied for the research to investigate tonoplast transporters in the future.

Materials and Reagents

  1. 40 μm cell strainer (Corning, catalog number: 431750)

  2. 50 mL tubes (Sangon Biotech, catalog number: F602788)

  3. Cellulase R10 (Yakult, catalog number: L0012)

  4. Macerozyme R10 (Yakult, catalog number: L0021)

  5. D-mannitol (Sigma-Aldrich, catalog number: M9647)

  6. MES hydrate (Sigma-Aldrich, catalog number: M2933)

  7. Tris-base (Thermo Fisher Scientific, catalog number: BP152-5)

  8. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670)

  9. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266)

  10. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333)

  11. Percoll (GE Healthcare, catalog number: 17-0891-09)

  12. HEPES (Sigma-Aldrich, catalog number: H3375)

  13. EGTA (Sigma-Aldrich, catalog number: H3889)

  14. Sucrose (Sigma-VETEC, catalog number: V900116)

  15. K-gluconate (Sigma-Aldrich, catalog number: G4500)

  16. D-sorbitol (Sigma-Aldrich, catalog number: S1876)

  17. MnSO4 (Sigma-Aldrich, catalog number: M7634)

  18. HNO3 (GHTECH, catalog number: 1.14003.018)

  19. H2O2 (Sinopharm Chemical Reagent, catalog number: 10011208)

  20. Murashige and Skoog (MS) base salts with vitamins (Phytotech Labs, catalog number: M519)

  21. Agar (Sigma-Aldrich, catalog number: A1296)

  22. KNO3 (Sinopharm Chemical Reagent, catalog number: 10017218)

  23. Ca(NO3)2·4H2O (Sigma-Aldrich, catalog number: SC278601)

  24. NH4H2PO4 (Sinopharm Chemical Reagent, catalog number: 10002808)

  25. MgSO4·7H2O (Sinopharm Chemical Reagent, catalog number: 10013018)

  26. H3BO3 (Sinopharm Chemical Reagent, catalog number: 10004808)

  27. MnCl2·4H2O (Sigma-Aldrich, catalog number: SM500501)

  28. (NH4)6Mo7O24·4H2O (Sinopharm Chemical Reagent, catalog number: 10002318)

  29. ZnSO4·7H2O (Sinopharm Chemical Reagent, catalog number: 10024018)

  30. CuSO4·5H2O (Sinopharm Chemical Reagent, catalog number: 10008218)

  31. EDTA-Fe(III)Na (Biotopped, catalog number: Q0028-100g)

  32. BSA (Sigma-Aldrich, catalog number: V900933)

  33. β-mercaptoethanol (14.3 M) (Millipore, catalog number: 444203)

  34. 1/2 MS medium (see Recipes)

  35. 1/5 Hoagland solution (see Recipes)

  36. Protoplasting solution (see Recipes)

  37. Medium B (see Recipes)

  38. Medium I (see Recipes)

  39. Medium II (see Recipes)

  40. Medium III (see Recipes)

  41. Medium IV (see Recipes)

  42. Medium V (see Recipes)

  43. Medium VI (see Recipes)

  44. Medium VII (see Recipes)

  45. Medium VIII (see Recipes)

Equipment

  1. Artificial illumination incubator (PERCIVAL, model: LT-36VL)

  2. Horizontal centrifuge (Eppendorf, model: Centrifuge 5810R)

  3. Dissolver (LabTech, model: DigiBlock ED54)

  4. ICP-MS (Thermo Fisher Scientific, model: ICAP Qc)

  5. Perfluoroalkoxy alkane (PFA) vessels (LabTech, model: GC-36-L)

  6. Fuchs-Rosenthal hemacytometer (Marienfeld, model: Dark-Line 0650010)

Procedure

English
中文翻译

文章信息

版权信息

© 2023 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

Ju, C., Fu, D., Wang, C. and Zhang, Z. (2023). Isolation of Intact Vacuoles from Arabidopsis Root Protoplasts and Elemental Analysis. Bio-protocol 13(5): e4625. DOI: 10.21769/BioProtoc.4625.

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分类

植物科学 > 植物细胞生物学 > 细胞器分离

植物科学 > 植物生物化学 > 其它化合物

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