Spatiotemporal distribution of ROS production, delivery and utilization in Arabidopsis root hairs

Abstract

AbstractFluorescent 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 ofArabidopsis thalianastained with CM-H2DCFDA, CellROXTMDeep Red and AmplexTMRed 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. Colocalization analysis with subcellular molecular markers and utilization of two root hair mutants with similar phenotype of non-elongating root hairs, but with contrast reasons for this impairment, we found that: i) CM-H2DCFDA is a sensitive probe for ROS generation in cytoplasm, ii) CellROXTMDeep Red labels ROS in mitochondria, iii) AmplexTMRed labels apoplastic ROS and mitochondria, and shows high selectivity to root hairs, iv)rhd2-1mutant with nonfunctional AtRBOHC/RHD2 has a low level of CM-H2DCFDA-reactive ROS in cytoplasm and lacks AmplexTMRed-reactive ROS in apoplast, v)ACTIN2-deficientder1-3mutant is not altered in these aspects. The sensitivity of CellROXTMDeep 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 new opportunities for precise speciation of particular ROS involved in the root hair formation.One sentence summary:High-resolution live-cell imaging of ROS production and subcellular localization in bulges and growing root hairs of Arabidopsis using CM-H2DCFDA, CellROXTMDeep Red and AmplexTMRed selective probes.