Background

Fluorescent indicators and biosensors have been fundamental in establishing the role of ions and other molecules in the cell physiology of diverse biological systems. The development of a wide range of biosensors for monitoring ions, molecules, and even phytohormones in plants has greatly increased over recent decades (Hilleary et al., 2018; Walia et al., 2018). In particular, the spatiotemporal coordination of ion dynamics is essential for diverse cellular processes and signaling pathways. Genetically encoded probes such as the Ca²⁺ sensor CaMeleon have been extensively used to understand ion dynamics (Miyawaki et al., 1997; Nagai et al., 2004; Monshausen et al., 2008), which became an accessible tool through the generation of stable transgenic lines. The popularization of such approaches has allowed intracellular detection and monitoring, improving our understanding of the extensive role of Ca²⁺, especially in cell signaling, regulation, and communication (Feijó and Wudick, 2018; Walia et al., 2018). The specific expression of biosensors targeted to particular compartments or organelles, together with appropriate live-imaging and genetic tools, has led to the detection and visualization of Ca²⁺ waves in roots (Choi et al., 2014; Evans et al., 2016), propagation of electrical signals in leaves (Nguyen et al., 2018), Ca²⁺-based long-distance signaling (Toyota et al., 2018) and intercellular communication (Ngo et al., 2014). Addressing such phenomena at an organismal level elucidates how these signals are propagated, also showing the physiological integration across tissues and organs.

Although less understood than that of Ca²⁺, the role of pH in intracellular signaling has received increased attention, especially due to the fundamental nature of protons as water constituents and its function as a second messenger, as well as the calcium-proton interplay. Such implication is particularly relevant in pollen tubes, in which the intracellular pH is tightly regulated and the spatial gradient of cytosolic protons plays a major role in apical growth (Certal et al., 2008; Michard et al., 2008; Hoffmann et al., 2020). Substantial progress in the field has been supported by new methodologies enabling live-cell imaging with proper spatiotemporal resolution and timescales, while the quantification of intracellular ion concentration has been made possible by calibrating ratiometric probes with elevated signal-to-noise ratios and a suitable dynamic range of response. Ratiometric probes rely on a single fluorescent protein that emits different wavelengths when bound or unbound to a particular molecule/ion of interest. The ratio analysis is quantitatively reliable, allowing corrections related to image acquisition artifacts and differential expression levels of the probe throughout a cellular compartment. The present protocol describes the intracellular monitoring of protons in pollen tubes of Arabidopsis thaliana and the calibration procedure of the biosensor pHluorin (Miesenbock et al., 1998) after extraction of a cryptic intron and codon usage optimization (Hoffmann et al., 2020). To address the cytosolic proton dynamics in Arabidopsis pollen tubes, a stable transgenic line expressing the pH-sensitive ratiometric pHluorin was generated in wild-type and mutant backgrounds. The mutants investigated in Hoffmann et al. (2020) were of special interest for investigating H⁺ dynamics since they consisted of double- or triple-knockouts of H⁺-ATPases that sustained most of the H⁺ extrusion in the shank region of the pollen tube. The development of minimally invasive protocols enabling live imaging in pollen tubes showed a consistent spatiotemporal gradient, compatible with observations in other species, allowing the identification and characterization of mutant phenotypes (Michard et al., 2008; Certal et al., 2008; Hoffmann et al., 2020). The calibration protocol described herein required quantification of the respective signal coming from both emission channels while varying the cytosolic pH by exposing the biosensor to a diversified pH environment. Principles and experimental details described in the current protocol can be used and adjusted by other researchers to improve image acquisition procedures in diverse systems and calibration of distinct biosensors.