Changes in pH are now widely accepted as a signalling mechanism in cells. In plants, proton pumps in the plasma membrane and tonoplast play a key role in regulation of intracellular pH homeostasis and maintenance of transmembrane proton gradients. Proton transport in response to external stimuli can be expected to be finely regulated spatially and temporally. With the ambition to follow such changes live, a new genetically encoded sensor, pHusion, has been developed. pHusion is especially designed for apoplastic pH measurements. It was constitutively expressed in Arabidopsis and targeted for expression in either the cytosol or the apoplast including intracellular compartments. pHusion consists of the tandem concatenation of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP1), and works as a ratiometric pH sensor. Live microscopy at high spatial and temporal resolution is highly dependent on appropriate immobilization of the specimen for microscopy. Medical adhesive often used in such experiments destroys cell viability in roots. Here a novel system for immobilizing Arabidopsis seedling roots for perfusion experiments is presented which does not impair cell viability. With appropriate immobilization, it was possible to follow changes of the apoplastic and cytosolic pH in mesophyll and root tissue. Rapid pH homeostasis upon external pH changes was reflected by negligible cytosolic pH fluctuations, while the apoplastic pH changed drastically. The great potential for analysing pH regulation in a whole-tissue, physiological context is demonstrated by the immediate alkalinization of the subepidermal apoplast upon external indole-3-acetic acid administration. This change is highly significant in the elongation zone compared with the root hair zone and control roots.

Acidification of the apoplastic space facilitates cell wall loosening and is therefore a key step in cell expansion. PSY1 is a growth-promoting secreted tyrosine-sulfated glycopeptide whose receptor directly phosphorylates and activates the plasma membrane H +-ATPase, which results in acidification and initiates cellular expansion. Although the mechanism is not clear, the Rapid Alkalinization Factor (RALF) family of small, secreted peptides inhibits the plasma membrane H +-ATPase, leading to alkalinization of the apoplastic space and reduced growth. Here we show that treating Arabidopsis thaliana roots with PSY1 induced the transcription of genes encoding the RALF peptides RALF33 and RALFL36. A rapid burst of intracellular Ca 2+ preceded apoplastic alkalinization in roots triggered by RALFs, with peptide-specific signatures. Ca 2+ channel blockers abolished RALF-induced alkalinization, indicating that the Ca 2+ signal is an obligatory part of the response and that it precedes alkalinization. As expected, fer mutants deficient in the RALF receptor FERONIA did not respond to RALF33. However, we detected both Ca 2+ and H + signatures in fer mutants upon treatment with RALFL36. Our results suggest that different RALF peptides induce extracellular alkalinization by distinct mechanisms that may involve different receptors.

Genetically encoded fluorescent biosensors have long proven to be excellent tools for quantitative live imaging, but sensor applications in plants have been lacking behind those in mammalian systems with respect to the variety of sensors and tissue types used. How can this be improved, and what can be expected for the use of genetically encoded fluorescent biosensors in plants in the future? In this review, we present a table of successful physiological experiments in plant tissue using fluorescent biosensors, and draw some conclusions about the specific challenges plant cell biologists are faced with and some of the ways they have been overcome so far.

Summary Pathogenic fungi often target the plant plasma membrane (PM) H+‐ATPase during infection. To identify pathogenic compounds targeting plant H+‐ATPases, we screened extracts from 10 Stemphylium species for their effect on H+‐ATPase activity. We identified Stemphylium loti extracts as potential H+‐ATPase inhibitors, and through chemical separation and analysis, tenuazonic acid (TeA) as a potent H+‐ATPase inhibitor. By assaying ATP hydrolysis and H+ pumping, we confirmed TeA as a H+‐ATPase inhibitor both in vitro and in vivo. To visualize in planta inhibition of the H+‐ATPase, we treated pH‐sensing Arabidopsis thaliana seedlings with TeA and quantified apoplastic alkalization. TeA affected both ATPase hydrolysis and H+ pumping, supporting a direct effect on the H+‐ATPase. We demonstrated apoplastic alkalization of A. thaliana seedlings after short‐term TeA treatment, indicating that TeA effectively inhibits plant PM H+‐ATPase in planta. TeA‐induced inhibition was highly dependent on the regulatory C‐terminal domain of the plant H+‐ATPase. Stemphylium loti is a phytopathogenic fungus. Inhibiting the plant PM H+‐ATPase results in membrane potential depolarization and eventually necrosis. The corresponding fungal H+‐ATPase, PMA1, is less affected by TeA when comparing native preparations. Fungi are thus able to target an essential plant enzyme without causing self‐toxicity.