In Vivo Imaging of Ca²⁺, pH, and Reactive Oxygen Species Using Fluorescent Probes in Plants

Abstract

Changes in the levels of Ca(2+), pH, and reactive oxygen species (ROS) are recognized as key cellular regulators involved in diverse physiological and developmental processes in plants. Critical to understanding how they exert such widespread control is an appreciation of their spatial and temporal dynamics at levels from organ to organelle and from seconds to many hours. With appropriate controls, fluorescent sensors can provide a robust approach with which to quantify such changes in Ca(2+), pH, and ROS in real time, in vivo. The fluorescent cellular probes available for visualization split into two broad classes: (a) dyes and (b) an increasingly diverse set of genetically encoded sensors based around green fluorescent proteins (GFPs). The GFP probes in particular can be targeted to well-defined subcellular locales, offering the possibility of high-resolution mapping of these signals within the cell.

Interacting Glutamate Receptor-Like Proteins in Phloem Regulate Lateral Root Initiation in Arabidopsis

“…If GLR-mediated Ca 2+ signals are generated in the phloem as they are in other cell types (Meyerhoff et al, 2005;Qi et al, 2006;Michard et al, 2011;Vincill et al, 2012), which is a potentially testable question given improved fluorescent Ca 2+ reporters (Swanson et al, 2011), and if those Ca 2+ signals affect phloem transport rates by altering sieve plate properties (Knoblauch et al, 2001;Furch et al, 2009), novel mechanisms for appropriately balancing carbon and nitrogen levels at root sinks can be envisioned. Carbohydrate transport rates could be adjusted by GLR3.2/GLR3.4-mediated Ca 2+ signaling according to nitrogen status, which is known to affect lateral root production (Tester, 1990;Forde and Walch-Liu, 2009).…”

A Ratiometric Sensor Using Single Chirality Near‐Infrared Fluorescent Carbon Nanotubes: Application to In Vivo Monitoring

“…[ 24,[37][38][39] Although these fl uorophores enable imaging with cellular resolution, in real time, they present a set of complex problems for in vivo applications. [ 40,41 ] Amplex red is widely used for imaging low concentrations of H 2 O 2 but has rarely been shown to work in living tissues due to its quick photodegradation. [ 42 ] The cell permeable 2′,7′-dichlorodihydrofl uorescein diacetate (H 2 DCF-DA) dye has been demonstrated to detect H 2 O 2 in living organisms despite its relatively nonselectivity to reactive oxygen species (ROS) [ 23 ] and susceptibility to photo-oxidation and photobleaching.…”

“…[ 42 ] The cell permeable 2′,7′-dichlorodihydrofl uorescein diacetate (H 2 DCF-DA) dye has been demonstrated to detect H 2 O 2 in living organisms despite its relatively nonselectivity to reactive oxygen species (ROS) [ 23 ] and susceptibility to photo-oxidation and photobleaching. [ 40 ] Imaging of NO in living systems has been performed with diamionofl uoresceins with the disadvantage that their fl uorescein chromophore is responsive to changes in pH [ 43 ] and reacts with dehydroascorbic and ascorbic acid. [ 41 ] Plants are optically dense living organisms due to thick tissues and photosynthetic pigments, making it diffi cult to detect analytes in vivo.…”

TherolBGene Suppresses Reactive Oxygen Species in Transformed Plant Cells through the Sustained Activation of Antioxidant Defense

“…2,7-Dichlorodihydrofluorescein diacetate (H 2 DCF-DA) is currently the most widely used fluorogenic probe for real-time ROS imaging in plants (Swanson et al, 2011). Subsequent to the cleavage of the diacetate ester by intracellular esterase, this dye reacts with ROS, such as hydrogen peroxide (H 2 O 2 ), peroxyl radicals, and peroxynitrite (Crow, 1997).…”