通过含有pH敏感指示剂溴甲酚紫的凝胶评估根部pH值的变化

Aparecida L. Silva; Keini  Dressano; Paulo H. O. Ceciliato; Juan Carlos  Guerrero-Abad; Daniel S. Moura

doi:10.21769/BioProtoc.2796

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Peer-reviewed

Evaluation of Root pH Change Through Gel Containing pH-sensitive Indicator Bromocresol Purple

通过含有pH敏感指示剂溴甲酚紫的凝胶评估根部pH值的变化

Aparecida L. Silva

KD Keini Dressano

Paulo H. O. Ceciliato

Juan Carlos Guerrero-Abad

Daniel S. Moura email

发布: 2018年04月05日第8卷第7期 DOI: 10.21769/BioProtoc.2796 浏览次数: 8953

评审: Arsalan DaudiLaia ArmengotVinay Panwar

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Abstract

The Rapid Alkalinization Factor (RALF) is a plant hormone peptide that inhibits proton transport causing alkalinization of the extracellular media. To detect the alkalinization response elicited by RALF peptides in root cells, Arabidopsis seedlings are carefully transferred to a gel containing the pH-sensitive indicator bromocresol purple, treated with the peptide and photographed after 30 min. Herein the protocol is optimized for evaluation of exogenous treatment, described in detail and expected results are presented.

Keywords: pH indicator (pH指示剂)

Alkalinization (碱化)

RALF (RALF)

Bromocresol purple (溴甲酚紫)

Background

Proton transport is induced by a myriad of signals and is used by plants to coordinate growth, defense and development. Some plant hormone peptides can affect proton transport, causing a strong alkalinization of the extracellular medium (Felix and Boller, 1995; Pearce et al., 2001a). The 5 kDa peptide hormone Rapid Alkalinization Factor (RALF), after being secreted, binds to its receptor FERONIA, and causes the phosphorylation of the plasma membrane H⁺-Adenosine triphosphatase 2, inhibiting proton transport and alkalinizing the extracellular media (Pearce et al., 2001b; Haruta et al., 2014).

Growth media containing the pH indicator bromocresol purple is an effective method to visualize alkalinization or acidification in the media around the roots. This method has been used previously to show that NaRALF is required for regulating root hair extracellular pH (Wu et al., 2007), and that roots of plants overexpressing AtRALF23 have reduced capacity to acidify the rhizosphere (Srivastava et al., 2009). Growth medium with the pH indicator bromocresol purple was also used by Masachis et al. (2016) to demonstrate that RALF homologs produced by fungal pathogens induced alkalinization of media around the roots of tomato plants.

We have optimized the pH indicator bromocresol purple protocol and using the improved protocol we were able to visualize the alkalinization effect around Arabidopsis roots after AtRALF1 treatment in wild type and mutant seedlings (Dressano et al., 2017). Here we demonstrate how this assay can provide qualitative information on the extracellular pH that surrounds Arabidopsis roots.

Materials and Reagents

Biological material
1. Arabidopsis thaliana seeds and seedlings (Ecotype Columbia, Col-0)
2. In-house produced 6xHis AtRALF1 recombinant peptide
Chemicals and materials for seed sterilization and growth
1. Square Petri dish with Grid 100 mm W x 15 mm H, sterile (Electron Microscopy Sciences, catalog number: 70691 )
2. Clear plastic wrap
3. Graduated cylinder 1,000, 100 and 10 ml (Uniglas)
4. 1,000 µl filter tips (NEST Scientific, catalog number: NPT1000-B-B )
5. 200 µl filter tips (NEST Scientific, catalog number: NPT0200-B-Y )
6. 10 µl filter tips (NEST Scientific, catalog number: 301001 )
7. Gellan Gum Powder (Culture Gel TM Type I- BioTech Grade) (PhytoTechnology Laboratories, catalog number: G434 )
8. Murashige & Skoog Basal Salt Mixture (PhytoTechnology Laboratories, catalog number: M524 )
9. Sterile distilled water
10. Sodium hypochlorite solution
11. Sodium hypochlorite (NaClO) solution 50% (v/v) (see Recipes)
Chemicals and materials for gel containing the pH-sensitive indicator bromocresol purple
1. Graduated cylinder 100 ml (Uniglas)
2. Petri dish 150 x 20 mm sterile
3. Bromocresol purple free acid reagent Grade (C₂H₁₆Br₂O₅S, AMRESCO, catalog number: 0531-25G )
4. Calcium sulfate dihydrate (CaSO₄·2H₂O, Merck, catalog number: 102161 )
5. Potassium hydroxide (KOH)
6. Hydrochloric acid (HCl)
7. Agarose RA (Biotechnology Grade, AMRESCO, catalog number: N605-500G )
8. Sterile distilled water

Equipment

Weighing balance (RADWAG Wagi Elektroniczne, model: AS 220/C/2 )
Magnetic stirrer (Fisher Scientific)
pH meter (Thermo Orion, PerpHecT LongR meter, model: 350 )
Pipettes (Nichipet EX, P1000, P200, P10)
Beaker 1,000 ml (PHOX, Boro 3.3)
Beaker 250 ml (APRX, Boro 3.3)
Autoclave (Sercon, model: HS 1-0101 )
Laminar flow hood (Pachane, model: PA 440 )
Growth chamber/Controlled environment room
Erlenmeyer (PIREX)
Microwave (BRASTEMP, catalog number: BMJ38ARANA )
Tweezers
Digital camera (Cyber-shot, Sony, model: DSC-H300 )

Procedure

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如何引用

Silva, A. L., Dressano, K., Ceciliato, P. H. O., Guerrero-Abad, J. C. and Moura, D. S. (2018). Evaluation of Root pH Change Through Gel Containing pH-sensitive Indicator Bromocresol Purple. Bio-protocol 8(7): e2796. DOI: 10.21769/BioProtoc.2796.