Genetically encoded fluorescent sensors adapted to acidic pH highlight subdomains within the plant cell apoplast.

Monitoring pH is one of the challenges in understanding diverse physiological regulations as well as ionic balance, especially in highly acidic environments such as the apoplast and the vacuole. To circumvent the poor efficiency of pH measurements below pH 5, we designed three genetically encoded sensors composed of two fluorescent proteins in tandem. We selected fluorescent protein pairs of low but sufficiently different pKa so that each protein could differentially sense the imposed pH. The generated tandems, named Acidin2, Acidin3, and Acidin4, were produced in Escherichia coli and extensively characterized. Altogether, these generated tandems cover a pH range of 3-8. The Acidins were targeted either for release in the apoplast (Apo) or for anchoring at the outer face of the plasma membrane (PM-Apo), with the fluorescent part exposed in the apoplast. Apoplastic Acidins in stably transformed Arabidopsis thaliana primary roots responded immediately and reversibly to pH changes, directly reporting physiological conditions related to cell elongation. In addition, membrane-anchored Acidins reveal a gradual acidification from the surface through the anticlinal wall of pavement cells, a process controlled at least partially by H+-ATPase activity.

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

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.