Biology subjects

Kubenova, L.

Publications and source records attributed to Kubenova, L..

4 recordsLinked to original sources

bioRxiv2023

Remodelling of cytoskeleton and plasma membrane proteins contributes to drought sensitivity of Arabidopsis rhd2 mutant

NADPH oxidases are enzymes localised in the plasma membrane and emitting superoxide to the extracellular space. By production of superoxide as one type of reactive oxygen species (ROS), they exert pleiotropic functions in plant development and various stress responses. Arabidopsis thaliana RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN C/ROOT HAIR DEFECTIVE 2 (AtRBOHC/RHD2) is an NADPH oxidase with preferential gene expression in roots. Polar localisation and ROS production by this enzyme are essential for root hair elongation. However, the proteome-wide and physiological consequences of RBOHC/RHD2 mutations are unknown. To find out potential new functions of AtRBOHC/RHD2, we employed a differential proteomic analysis of Arabidopsis rhd2-1 mutant, carrying a loss-of-function mutation in RBOHC/RHD2. Proteomic analyses that were validated with independent biochemical, phenotypical and advanced microscopy methods, showed quantitative deregulation of proteins involved in abiotic and biotic stress response, metabolism, vesicular transport and cell wall modification. Considerable differences in the differential proteomes between roots and above-ground parts were found in the mutant. The altered abundance of aquaporins and homeostasis of transmembrane pumps and transporters most likely determine the higher sensitivity of Arabidopsis rhd2-1 mutant to drought. HighlightProteomics and advanced microscopy reveal that the drought sensitivity of Arabidopsis mutant in ROOT HAIR DEFECTIVE 2 is linked to altered homeostasis of plasma membrane proteins and cytoskeleton remodelling.

plant biology↗

bioRxiv2023

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

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 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. 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) CellROXTM Deep Red labels ROS in mitochondria, iii) AmplexTM Red labels apoplastic ROS and mitochondria, and shows high selectivity to root hairs, iv) rhd2-1 mutant with nonfunctional AtRBOHC/RHD2 has a low level of CM-H2DCFDA-reactive ROS in cytoplasm and lacks AmplexTM Red-reactive ROS in apoplast, v) ACTIN2-deficient 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 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, CellROXTM Deep Red and AmplexTM Red selective probes.

plant biology↗

bioRxiv2021

AtRBOHC/RHD2 vesicular delivery to the apical plasma membrane domain during root hair development

Arabidopsis root hairs develop as long tubular extensions from the rootward pole of trichoblasts and exert polarized tip growth. The establishment and maintenance of root hair polarity is a complex process involving the local apical production of reactive oxygen species (ROS) generated by NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN C/ROOT HAIR DEFECTIVE 2 (AtRBOHC/RHD2). It has been shown that loss-of-function rhd2 mutants have short root hairs that are unable to elongate by tip growth, and this phenotype was fully complemented by GFP-RHD2 expressed under the RHD2 promoter. However, the spatiotemporal mechanism of AtRBOHC/RHD2 subcellular redistribution and delivery to the plasma membrane (PM) during root hair initiation and tip growth are still unclear. Here, we used advanced microscopy for detailed qualitative and quantitative analysis of vesicular compartments containing GFP-RHD2 and characterization of their movements in developing bulges and growing root hairs. These compartments, identified by an independent marker such as the trans-Golgi network (TGN), deliver GFP-RHD2 to the apical PM domain, the extent of which correlates with the stage of root hair formation. Movements of TGN/early endosomes, but not late endosomes, were affected in the bulging domains of the rhd2-1 mutant. Finally, we reveal that accumulation in the growing tip, docking, and incorporation of TGN compartments containing GFP-RHD2 to the apical PM of root hairs requires structural sterols. These results help clarify the mechanism of polarized AtRBOHC/RHD2 targeting, maintenance, and recycling at the apical PM domain, coordinated with different developmental stages of root hair initiation and growth. One-sentence summaryAdvanced microscopy and quantitative analysis of vesicular TGN compartments revealed that delivering GFP-RHD2 to the apical plasma membrane domains of developing bulges and growing root hairs requires structural sterols.

plant biology↗

bioRxiv2020

Single amino acid exchange in ACTIN2 confers increased tolerance to oxidative stress in Arabidopsis der1-3 mutant

Single-point mutation in the ACTIN2 gene of der1-3 mutant revealed that ACTIN2 is an essential actin isovariant required for root hair tip growth, and leads to shorter, thinner and more randomly oriented actin filaments in comparison to wild-type C24 genotype. Actin cytoskeleton has been linked to plant defence against oxidative stress, but it is not clear how altered structural organization and dynamics of actin filaments may help plants to cope with oxidative stress. In this study, we characterized seed germination, root growth, plant biomass, actin organization and antioxidant activity of der1-3 mutant under oxidative stress induced by paraquat and H2O2. Under these conditions, plant growth was better in der1-3 mutant, while actin cytoskeleton in der1-3 carrying pro35S::GFP:FABD2 construct showed lower bundling rate and higher dynamicity. Biochemical analyses documented lower degree of lipid peroxidation, elevated capacity to decompose superoxide and hydrogen peroxide. These results support the view that der1-3 mutant is more resistant to oxidative stress. Single amino acid exchange in mutated ACTIN2 protein (Cys to Arg at the position 97) is topologically exposed to the protein surface and we propose that this might alter protein post-translational modifications and/or protein-protein interactions, leading to enhanced tolerance of der1-3 mutant against oxidative stress. HighlightTopological position of one amino acid exchanged in the ACTIN2 protein structure in der1-3 mutant enhanced tolerance to oxidative stress through increased capacity to decompose reactive oxygen species, lower bundling and enhanced dynamicity of the actin cytoskeleton.

plant biology↗