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Fluorescent selective probes for reactive oxygen species (ROS) detection in living cells are versatile tools for the documentation of ROS production in plant developmental or stress reactions. We employed high-resolution live-cell imaging and semi-quantitative analysis of Arabidopsis (Arabidopsis thaliana) stained with CM-H2DCFDA, CellROXTM Deep Red and AmplexTM Red for functional characterization of spatiotemporal mode of ROS production, delivery and utilization during root hair formation. Cell viability marker fluorescein diacetate served as a positive control for dye-loading and undisturbed tip growth after staining. Using a colocalization analysis with subcellular molecular markers and two root hair mutants with similar phenotypes of non-elongating root hairs, but with contrasting reasons for this impairment, we found that: i) CM-H2DCFDA is a sensitive probe for ROS generation in the cytoplasm, ii) CellROXTM Deep Red labels ROS in mitochondria, iii) AmplexTM Red labels apoplastic ROS and mitochondria and shows high selectivity to root hairs, iv) the root hair defective 2-1 (rhd2-1) mutant with nonfunctional NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN C/ROOT HAIR-DEFECTIVE 2 (AtRBOHC/RHD2) has a low level of CM-H2DCFDA-reactive ROS in cytoplasm and lacks AmplexTM Red-reactive ROS in apoplasts, v) the ACTIN2-deficient deformed root hairs1-3 (der1-3) mutant is not altered in these aspects. The sensitivity of CellROXTM Deep Red was documented by discrimination between larger ROS-containing mitochondria and small, yet ROS-free pre-mature mitochondria in the growing tip of root hairs. We characterized spatial changes in ROS production and compartmentalization induced by external ROS modulators, ethylene precursor 1-aminocyclopropane-1-carboxylic acid and ionophore valinomycin. This dynamic and high-resolution study of ROS production and utilization opens opportunities for precise speciation of particular ROS involved in root hair formation.
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