Improve Research Reproducibility A Bio-protocol resource
bpdetail_icon_lock
Peer-reviewed
tooltip

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: 2445

评审: Wenrong HeYao XiaoYe XuAnonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Molecular Plant, featuring study using the protocol.

May 2021

Advertisement

“科研可重复性与创新”系列网络研讨会
Special Issue on Protocols for Organoid Research
Technologies for RNA Detection
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

采用手工匀浆与剪切力匀浆法分离芦荟来源细胞外囊泡的逐步实验方案

采用手工匀浆与剪切力匀浆法分离芦荟来源细胞外囊泡的逐步实验方案

M. Camila Ceballos-Santa [...] Karin Wuertz-Kozak

2026年04月20日 553 阅读

基于质外体液洗脱与尺寸排阻色谱法提取和分离蒌叶来源的细胞外囊泡

基于质外体液洗脱与尺寸排阻色谱法提取和分离蒌叶来源的细胞外囊泡

Izy Sanddy [...] Norhayati Liaqat Ali Khan

2026年05月05日 981 阅读

利用非损伤微测技术（NMT）测定水稻净NH₄⁺流速

利用非损伤微测技术（NMT）测定水稻净NH₄⁺流速

Dong-Wei Di [...] Weiming Shi

2026年08月05日 167 阅读

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.

ris ris Download Citation in RIS Format

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

您对这篇实验方案有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件