Hypoxia-activated fluorescent probes as markers of oxygen levels in plant cells and tissues.

Low oxygen signalling in plants is important in development and stress responses. Measurement of oxygen levels in plant cells and tissues is hampered by a lack of chemical tools with which to reliably detect and quantify endogenous oxygen availability. We have exploited hypoxia-activated fluorescent probes to visualise low oxygen (hypoxia) in plant cells and tissues. We applied 4-nitrobenzyl (4NB-) resorufin and methyl-indolequinone (MeIQ-) resorufin to Arabidopsis thaliana whole cells and seedlings exposed to hypoxia (1% O2) and normoxia (21% O2). Confocal microscopy and fluorescence intensity measurements were used to visualise regions of resorufin fluorescence. Both probes enter A. thaliana whole cells and are activated to fluoresce selectively in hypoxic conditions. Similarly, incubation with A. thaliana seedlings resulted in hypoxia-dependent activation of both probes and observation of fluorescence in hypoxic roots and leaf tissue. MeIQ-Resorufin was used to visualise endogenous hypoxia in lateral root primordia of normoxic A. thaliana seedlings. Oxygen measurement in plants until now has relied on invasive probes or genetic manipulation. The use of these chemical probes to detect and stain applied and endogenous hypoxia has the potential to facilitate a greater understanding of oxygen concentrations in plant cells and tissues, allowing the correlation of oxygen availability with acclimative and developmental responses to hypoxia.