Uncovering pH at both sides of the root plasma membrane interface using noninvasive imaging

Abstract

Building a proton gradient across a biological membrane and between different tissues is a matter of great importance for plant development and nutrition. To gain a better understanding of proton distribution in the plant root apoplast as well as across the plasma membrane, we generated plants expressing stable membrane-anchored ratiometric fluorescent sensors based on pHluorin. These sensors enabled noninvasive pH-specific measurements in mature root cells from the medium-epidermis interface up to the inner cell layers that lie beyond the Casparian strip. The membrane-associated apoplastic pH was much more alkaline than the overall apoplastic space pH. Proton concentration associated with the plasma membrane was very stable, even when the growth medium pH was altered. This is in apparent contradiction with the direct connection between root intercellular space and the external medium. The plasma membrane-associated pH in the stele was the most preserved and displayed the lowest apoplastic pH (6.0 to 6.1) and the highest transmembrane delta pH (1.5 to 2.2). Both pH values also correlated well with optimal activities of channels and transporters involved in ion uptake and redistribution from the root to the aerial part. In growth medium where ionic content is minimized, the root plasma membrane-associated pH was more affected by environmental proton changes, especially for the most external cell layers. Calcium concentration appears to play a major role in apoplastic pH under these restrictive conditions, supporting a role for the cell wall in pH homeostasis of the unstirredsurface layer of plasma membrane in mature roots.

Integrating pathway elucidation with yeast engineering to produce polpunonic acid the precursor of the anti-obesity agent celastrol

“…Yeast is generally regarded as a suitable host for enzymes of plant origin [24]. However, several factors, including differences in intracellular pH [59,60], membrane lipid composition of the ER [30,31] (relevant for ER-membrane-anchored OSCs and P450s), and availability of dedicated co-factors, such as, for example, specialized cytochrome b5 enzymes [32], are generally regarded to cause a plant enzyme not to function properly in a yeast cell. To minimize the risk of failing to identify enzymes in the celastrol pathway because of suboptimal conditions, we performed functional screening of enzymes by transient co-expression in tobacco.…”

Pathogen-induced pH changes regulate the growth-defense balance of plants

“…Our work adds a detailed layer of spatial resolution to the analysis of subcellular pH and demonstrates the high level of cell compartmentalization, as ΔpH changes were restricted to the PM interface and did not affect the pH cyto , probably due to a buffering effect of the tonoplast (Pittman, 2012) . The pH apo and pH cortical sensors are novel molecular tools, which allow for reliable measurement of apoplastic and PM-cortical pH with little intracellular background (Gjetting et al , 2012;Martinière et al , 2018) . They enabled us to perform real time measurements of fast, fungal-induced acidification of the root cell apoplast and ultimately cortex, which derived from the upregulation of AHAs (Fig 2).…”

“…To test the above hypothesis, we sought to measure the pH of plant roots at different developmental zones, from meristem to mature regions, and various cell layers in response to alive Fo5176 hyphae and elicitors. As previously reported, the apoplastic pH sensor apo-pHusion suffers from high background signal in the endoplasmic reticulum (ER), which compromises precise pH measurements in the apoplast (Gjetting et al , 2012;Martinière et al , 2018) . Therefore, we generated a pH apo probe with as low as possible intracellular background signal by fusing the ratiometric probe pHusion (Gjetting et al , 2012) to the C-terminus of the PM-localized SNARE (soluble N-ethyl-maleimide sensitive factor attachment protein receptor) protein Syntaxin of Plants 122 (SYP122; (Assaad et al , 2004;Uemura et al , 2004) ).…”

Biochemical pH clamp: the forgotten resource in membrane bioenergetics

“…() on transgenic Arabidopsis expressing pHluorin, a GFP derivative directed to the apoplast. When pHluorin was directly anchored to the external face of the plasma membrane, pH o in the root cortex was in the same range ( c. pH 6.4; Martinière et al ., ). The bulk measurements indicate a ΔpH of 1.5 to 2 units across the plasma membrane; the method of Martinière et al .…”

“…The bulk measurements indicate a ΔpH of 1.5 to 2 units across the plasma membrane; the method of Martinière et al . () which allows a more precise measurement of the actual ΔpH across the membrane, renders values between 1.0 and 1.7, increasing with radial distance from the root surface.…”