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

Abstract

O_LILow 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. C_LIO_LIWe 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. C_LIO_LIBoth 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. C_LIO_LIOxygen measurement in plants until now has relied on invasive probes or genetic manipulation. Use of these chemical probes to detect applied and endogenous hypoxia has the potential to facilitate a greater understanding of oxygen dynamics in plant cells and tissues, allowing correlation of oxygen concentrations with adaptive and developmental responses to hypoxia. C_LI

Code and data availability

The supplied blocks describe probe synthesis, plant growth, hypoxia treatments, confocal imaging, and ImageJ/Prism/R analyses, but contain no public phenotype dataset, image deposit, author analysis code, or data availability statement with a URL. No paper-specific public asset is identified.

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