Measurement of H+ Flux in Rice by Non-invasive Micro-test Technology
无创显微技术测定水稻中的H+流
发布: 2015年12月05日第5卷第23期 DOI: 10.21769/BioProtoc.1649 浏览次数: 9526
评审: Wenrong HeCindy AstZhaohui Liu
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The authors used this protocol in:
Apr 2015
Abstract
Rice plants release proton (H+) from root cells into rhizosphere area leading to the acidification of the rhizosphere and increased solubility of ferric iron complexes on the cell membrane, which is important for iron uptakes. Here, we present a detailed protocol to measure H+ flux in root hairs of transgenic rice seedlings and transgenic rice protoplasts by the Non-invasive Micro-test Technique (NMT). The NMT system is based on a non-invasive microelectrode technology that is automatically controlled by a computer, to achieve a three-dimensional, real-time, dynamic characterization of the concentration, velocity, and direction of a variety of molecules or ions. Because there is no need to directly contact the measured cells that could cause cell damage, we are able to obtain accurate and real time information on ion concentration. This is the first protocol that describes the non-invasive micro measurement technique of both root hairs and protoplasts in rice. In NMT, voltage differences are measured at two excursion points that are manipulated using a computer. Voltage differences can be converted into H+ fluxes using the ASET 2.0 (The imFlux® software) and JCal v3.2.1 Software. Analysis of the H+ fluxes provides a simultaneous measure of the crossing of a localized region of the root surface in response to stress, which provides real-time in-situ detection of net ion transport across membranes. This method will promote use of NMT in plant biology.
Keywords: Proton flux (质子通量)
Non-invasive Micro-test Technique (NMT) (非损伤微测技术（NMT）)
Rice (水稻)
Microelectrode (微电极)
Materials and Reagents
- Cell strainer (400 mesh)
- Centrifuge tube (1.5 ml and 10 ml) (Corning Inc., catalog number: MC-150-C )
- Round Petri dish (35 mm in diameter) (Corning Inc.)
- Square Petri dish (100 mm, pym-100-10) (Xinshengke)
- Plastic strips
- Constructed vector (Li et al., 2015)
- Transgenic Secretory 24 (OsSEC24) (Li et al., 2015) rice seedlings and plasma membrane (PM) ATPase 2 (OsPMA2)-mCherry (Li et al., 2015) plus OsSEC24-GFP double-transgenic rice protoplasts
- Cellulase (Onozuka, catalog number: 130419-01 )
- Macerozyme (Japan) (catalog number: 121207-01)
- M519 (catalog number: 14C0519105B)
- Mannitol
- MES (pH 5.7)
- KCl
- NaCl
- CaCl2
- MgCl2
- Sucrose
- 1%-1.5% agar
- Calibration medium buffer (pH 7.0) for NMT (see Recipes)
- Calibration medium buffer (pH 5.0) for NMT (see Recipes)
- Incubation solution (see Recipes)
- Mmg solution (see Recipes)
- MS medium (see Recipes)
- NT medium (see Recipes)
- Protoplasting enzyme solution (see Recipes)
- Polyethylene glycol (PEG)-calcium solution (see Recipes)
- Standard medium buffer (pH 6.0) for NMT (see Recipes)
- Washing buffer (see Recipes)
Equipment
- Centrifuge (SIGMA Laborzentrifugen GmbH, model: 3K15 )
- Glass beaker (100 ml) (Bomex)
- Growth chamber
- Liquid ion exchanger (LIX) Holder (YoungerChina)
- Microelectrodes (1-2 μm, 4-5 μm) (YoungerUSA)
- Micropipettor (100-1,000 μl and 10-100 μl) (Eppendorf)
- Non-invasive Micro-test System (YoungerUSA, model: NMT100 series )
- Shaker (Focus Technology Co., Shanghai Meditry Instrument, model: THZ-C-1 )
Software
- NIS-Elements AR software (available at http://www.youngerusa.com)
- ASET 2.0 software (The imFlux® software) (available at http://www.youngerusa.com)
- JCal v3.2.1 (available at http://www.youngerusa.com)
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Zhang, X., Zhang, X. and Yin, L. (2015). Measurement of H+ Flux in Rice by Non-invasive Micro-test Technology. Bio-protocol 5(23): e1649. DOI: 10.21769/BioProtoc.1649.
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分类
植物科学 > 植物生理学 > 离子分析
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