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, Alkalinization, RALF, Bromocresol purple
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
Equipment
Procedure
Data analysis
Notes
Recipes
Acknowledgments
This research was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). DSM is a research fellow of CNPq. This protocol was adapted from Wu et al. (2007). The authors declare that they do not have any conflicts of interest or competing interests.
References
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Warner I do not put direct bromocresol blue in MS medium because of the sensitivity of the medium with blue bromocresol reagent in pH changes, also because to visualize the extracellular medium alkalinization caused by the RALF1 peptide we need to concentrate the roots on a hard surface, to facilitate visualization of alkalinization.Sincerely