Perri Monica, Khan M Shahneawz, Wallabregue Antoine L D, Voloboeva Viktoriia, Ridgway Amber M, Smith Edward N, Bolland Hannah, Hammond Ester M, Conway Stuart J, Weits Daan A, Flashman Emily
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.
New Phytol. 2025 Sep;247(6):2998-3009. doi: 10.1111/nph.70202. Epub 2025 May 8.
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.
植物中的低氧信号在发育和应激反应中很重要。由于缺乏可靠检测和量化内源性氧可用性的化学工具，植物细胞和组织中氧水平的测量受到阻碍。我们利用缺氧激活的荧光探针来观察植物细胞和组织中的低氧（缺氧）情况。我们将4-硝基苄基（4NB-）试卤灵和甲基吲哚醌（MeIQ-）试卤灵应用于暴露于缺氧（1% O₂）和常氧（21% O₂）的拟南芥全细胞和幼苗。共聚焦显微镜和荧光强度测量用于观察试卤灵荧光区域。两种探针都能进入拟南芥全细胞，并在缺氧条件下被激活而选择性地发出荧光。同样，与拟南芥幼苗一起孵育导致两种探针都出现缺氧依赖性激活，并在缺氧的根和叶组织中观察到荧光。MeIQ-试卤灵被用于观察常氧条件下拟南芥幼苗侧根原基中的内源性缺氧情况。到目前为止，植物中的氧测量依赖于侵入性探针或基因操作。使用这些化学探针来检测和染色施加的和内源性的缺氧情况，有可能有助于更深入地了解植物细胞和组织中的氧浓度，从而使氧可用性与对缺氧的适应性和发育反应相关联。
