Extracellular Alkalinization Assay for the Detection of Early Defense Response

Abstract

Plant recognition of invading organisms occurs through identification of foreign molecules associated with attackers and of self‐derived, damage‐associated molecules. Perception of these molecules activates signaling processes including dynamic changes in ion balance, production of second messengers such as reactive oxygen species and nitric oxide, increased levels of plant hormones, and map kinase cascade activation. Together these signaling events stimulate transcriptional changes to initiate plant defense responses. Among the earliest detectable signaling events is a rapid increase in apoplastic pH, i.e., extracellular alkalinization. Here, an assay for quantification of this alkalinization response using suspension‐cultured cell lines for Arabidopsis, potato, and maize is described. This assay is an inexpensive, fast, simple, and reproducible method to quantify defense signaling output, providing a powerful tool for evaluating early plant responses to elicitors and pathogens. Results from the alkalinization assay are comparable to other more costly and time‐consuming methods for assessing defense signaling, such as measurement of the oxidative burst, calcium influx, and marker gene expression. This bioassay is a quantitative and robust method for evaluation of plant defense output. © 2017 by John Wiley & Sons, Inc.

Bioassays for Identifying and Characterizing Plant Regulatory Peptides

“…Suspensioncultured potato cells are highly sensitive to molecular stress factors and alkalinize the culture medium in response to their action, similar to that observed in tobacco cultivated cells [94]. The alkalinization bioassay based on plant suspension cell cultures is proposed as a marker system to study the early stages of plant defense reactions [95]. This highly sensitive cellular approach can be used to screen newly identified and putative signaling peptides for their signaling potential during stress and defense reactions.…”

Mass spectrometry in the discovery of peptides involved in intercellular communication: From targeted to untargeted peptidomics approaches

“…The tobacco hydroxyproline‐rich systemin (HypSys) I and II eliciting wounding responses and Arabidopsis thaliana plant peptide elicitor 1 (AtPep1) triggering systemic antipathogen responses were isolated by screening peptides for inducing extracellular alkalinization and characterized by Edman sequencing and MALDI‐MS (Huffaker et al, 2006; Pearce et al, 2001). Extracellular alkalinization is one of the major plant defense responses triggered by different types of stresses, including wounding, pathogens, and herbivore pests (Felix et al, 1999; Moroz et al, 2017). To assay the separated peptide for inducing extracellular alkalinization, the medium pH of plant cell culture was measured after the peptide treatment.…”

Toward the Discovery of Host-Defense Peptides in Plants

“…root growth inhibition are hallmarks of-and are commonly used as a readout for-the activation of plant immunity (30,31). This suggests that these three peptides can directly control immunity, potentially working as a ligand to an immune receptor, as hypothesized for RISP (32).…”