RESUMO
A quantitative and robust bioassay to assess plant defense response is important for studies of disease resistance and also for the early identification of disease during pre- or non-symptomatic phases. An increase in extracellular pH is known to be an early defense response in plants. In this study, we demonstrate extracellular alkalinization as a defense response in potatoes. Using potato suspension cell cultures, we observed an alkalinization response against various pathogen- and plant-derived elicitors in a dose- and time-dependent manner. We also assessed the defense response against a variety of potato pathogens, such as protists (Phytophthora infestans and Spongospora subterranea) and fungi (Verticillium dahliae and Colletotrichum coccodes). Our results show that extracellular pH increases within 30 min in proportion to the number of pathogen spores added. Consistently with the alkalinization effect, the higher transcription level of several defense-related genes and production of reactive oxygen species was observed. Our results demonstrate that the alkalinization response is an effective marker to study early stages of defense response in potatoes.
RESUMO
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.
RESUMO
Cancer stem cells reside in a distinct region within the tumor microenvironment that it is believed to play a fundamental role in regulating stemness, proliferation, survival, and metabolism of cancer cells. This study aimed to analyze the effect of extracellular alkalinization on metabolism and survival of human CD24-/CD44+ breast cancer stem cells (BCSCs). BCSCs were cultured in alkalinized DMEM-F12 and incubated at 37°C, 5% CO2, and 20% O2 for 30 min, 6, 24, and 48 h. After each incubation period, we analyzed the modulation of various mRNA expressions related to pH and cellular metabolic regulation using the qRT-PCR. Metabolic state was measured using colorimetric and fluorometric assays. To examine cell proliferation and apoptosis, we used trypan blue and annexin V/propidium iodide assay, respectively. This study demonstrated that alkalinization could stimulate extracellular carbonic anhydrase (CAe) activity, as well as CA9 and HIF1α expression. Under alkaline pH and HIF1α regulation, glucose consumption, extracellular lactate production, and LDH activity of BCSCs were upregulated while O2 consumption was downregulated. These metabolic shifts seemed to promote apoptosis and suppress the proliferation of BCSCs. To conclude, modulation of the extracellular environment through alkalinization could change the metabolic states of BCSCs, which in turn affect the cell survival.
