Perspectives for using genetically encoded fluorescent biosensors in plants

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

Identification of antifungal H⁺-ATPase inhibitors with effect on the plasma membrane potential

ABSTRACT The plasma membrane H + -ATPase (Pma1) is an essential fungal protein and a proposed target for new antifungal medications. The compounds in a small-molecule library containing ∼191,000 commercially available compounds were screened for their ability to inhibit Saccharomyces cerevisiae plasma membranes containing Pma1. The overall hit rate was 0.2%, corresponding to 407 compounds. These hit compounds were further evaluated for ATPase selectivity and broad-spectrum antifungal activity. Following this work, one Pma1 inhibitor series based on compound 14 and analogs was selected for further evaluation. This compound series was able to depolarize the membrane and inhibit extracellular acidification in intact fungal cells concomitantly with a significant increase in intracellular ATP levels. Collectively, we suggest that these effects may be a common feature of Pma1 inhibitors. Additionally, the work uncovered a dual mechanism for the previously identified cationic peptide BM2, revealing fungal membrane disruption, in addition to Pma1 inhibition. The methods presented here provide a solid platform for the evaluation of Pma1-specific inhibitors in a drug development setting. The present inhibitors could serve as a starting point for the development of new antifungal agents with a novel mode of action. </jats:p

Live imaging of intra- and extracellular pH in plants using pHusion, a novel genetically encoded biosensor

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

Assaying the Effect of Peptide Treatment on H⁺-Pumping Activity in Plasma Membranes from Arabidopsis Seedlings

Extracellular acidification or alkalization is a common response to many plant-signaling peptides and microbial elicitors. This may be a result of peptide-mediated regulation of plasma membrane-localized ion transporters, such as the plasma membrane H+-ATPase. Early responses to some signaling peptides can therefore be analyzed by assaying H+-pumping across the plasma membrane. We describe a set-up suited for the purification of plasma membranes by aqueous two-phase partitioning from a small sample of Arabidopsis seedlings. Seedlings are grown in a liquid culture, suited for the analysis of in vivo peptide treatment. Additionally, we describe how to measure the H+-pumping activity of the plasma membrane H+-ATPase using the fluorescent probe ACMA.</p