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

Stuart J Conway, Emily Flashman, Daan A Weits, Edward N Smith, Monica Perri, M Shahneawz Khan, Antoine L D Wallabregue, Viktoriia Voloboeva, Amber M Ridgway, Hannah Bolland, Ester M Hammond

Journal: The New phytologist 2025;247(6):2998-3009

PMID: 40344613

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Abstract

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% O) and normoxia (21% O). 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.

Address: Department of Biology, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK.; Department of Biology, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK.; Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford, OX1 3TA, UK.; Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford, OX1 3TA, UK.; Experimental and Computational Plant Development, Institute of Environment Biology, Utrecht University, Padualaan 8, Utrecht, 3584 CH, the Netherlands.; PlantLab, Institute of Plant Sciences, Scuola Superiore Sant'Anna, 56010, Pisa, Italy.; Department of Oncology, University of Oxford, Old Road Campus Research Building, Oxford, OX3 7DQ, UK.; School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, GU2 7AL, UK.; Department of Oncology, University of Oxford, Old Road Campus Research Building, Oxford, OX3 7DQ, UK.; Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford, OX1 3TA, UK.; Department of Chemistry & Biochemistry, University of California Los Angeles, 607 Charles E. Young Drive East, PO Box 951569, Los Angeles, CA, 90095-1569, USA.; Experimental and Computational Plant Development, Institute of Environment Biology, Utrecht University, Padualaan 8, Utrecht, 3584 CH, the Netherlands.

DOI: 10.1111/nph.70202

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MeSH Terms: Fluorescent Dyes, Oxygen, Arabidopsis, Cell Hypoxia, Plant Cells, Seedlings, Plant Roots, Plant Leaves, Biomarkers, Indolequinones, Oxazines, Microscopy, Confocal