pH biosensing in the plant apoplast-a focus on root cell elongation

Abstract

The pH parameter of soil plays a key role for plant nutrition as it is affecting the availability of minerals and consequently determines plant growth. Although the mechanisms by which root perceive the external pH is still unknown, the impact of external pH on tissue growth has been widely studied especially in hypocotyl and root. Thanks to technological development of cell imaging and fluorescent sensors, we can now monitor pH in real time with at subcellular definition. In this focus, fluorescent dye-based, as well as genetically-encoded pH indicators are discussed especially with respect to their ability to monitor acidic pH in the context of primary root. The notion of apoplastic subdomains is discussed and suggestions are made to develop fluorescent indicators for pH values below 5.0.

Figure 1

Schematic representation of a primary root apex with the different published fluorescent sensors for the apoplast. A, The different zones of the root, from the apex to the maturation zone. B, A detailed transverse section of the maturation zone with the PM-anchored pHluorin, PM-Apo. This sensor is detected down to the stele in living root, and reveals an acidic pH gradient in the apoplast that extends from the surface to the stele. PM-Apo reports pH close to the PM and is insensitive to short-term changes in membrane polarization (Martinière et al., 2018). Epi: epidermis; Cor: cortex; End: endodermis; St: stele. C, A detailed longitudinal section of the epidermis in the transition zone, with pH biosensors in various subdomains of the apoplast, including the “unstirred layer” and the CW. PM-Apo, PM-Apo(bis), and PM-Apo(ter) are PM-anchored sensors; pHluorin is fused to a transmembrane domain and a linker of variable length. SYP122-pHusion is a chimera of the PM SNARE SYP122 and pHusion (Kesten et al., 2019). CBD (Fasano et al., 2001). At5g11420-FP is an uncharacterized CW protein with unknown fine localization (indicated as “?”) that is fused to FPs (Stoddard and Rolland, 2019). Apo-pHluorin is a freely diffusing form of pHluorin.

Figure 2

Schematic excitation spectra associated with the various types of pH biosensors used in plants. Hypothetical spectra are presented to illustrate the effect of pH on the protonated and deprotonated forms of a fluorochrome compared to the spectra at pKa (solid line), based on the assumption that fluorescence intensity increases for pH values above the pKa (dashed line) and decreases for pH lower than the pKa (dotted line). The anionic deprotonated state [R⁻+H⁺] is progressively converted to a protonated state [R−H] as pH decreases. Excitation wavelengths, Ex, Ex1, and Ex2, are indicated for each spectra as ideal wavelengths for either providing pH information (A) or generating a ratiometric pH titration curve (B–D) with Ex2 corresponding to the wavelength with higher pH sensitivity to pH compared to Ex1. Titration curves are obtained by plotting the fluorescence ratio Ex2/Ex1 as a function of applied pH that, in these examples, will increase with pH increase. A, Mono-excitation mono-emission type of spectra as for fluorescein. B and C, Dual-excitation mono-emission spectra as for BCECF and OG, with Ex1 at the isosbestic point (B) or pHluorin, with two peaks with opposite sensitivity to pH (C). D, Mix or fusion of two biosensors with mono-excitation mono-emission spectra, but with high (green) or low (orange) sensitivity to pH, as for pHusion.