Biology subjects

Ovecka, M.

Publications and source records attributed to Ovecka, M..

12 recordsLinked to original sources

bioRxiv2026

Stomatal movement in Arabidopsis is driven by guard cell-localized and copper-insensitive CSD1 splice variant

Copper (Cu) is an essential micronutrient whose bioavailability is strongly affected by soil physicochemical properties. During evolution, plants have developed mechanisms to flexibly adjust their metabolism to Cu status. Superoxide dismutases (SODs), including Cu/ZnSOD1 (CSD1) and FeSOD1 (FSD1), are key antioxidant enzymes regulated in Cu dependent manner in Arabidopsis thaliana. Examination of CSD1 cellular distribution and activity revealed that CSD1 is a nuclear and cytosolic SOD whose abundance and activity respond to Cu availability inversely to FSD1. Combined microscopic and biochemical analyses of Cu-dependent dynamics revealed that, unlike FSD1, CSD1 localization in guard cells (GCs) remains independent of Cu availability. CSD1 escapes miR398-mediated regulation in GCs through a cell type-specific splice variant (CSD1.2) that carries an altered miR398-binding site. In silico analyses indicate that this mechanism is also present in crop species. Functionally, the csd1 mutant showed reduced sensitivity to abscisic acid (ABA)-induced stomatal closure, a phenotype rescued by reintroducing CSD1. Biochemical and reactive oxygen species (ROS) level analyses indicate that CSD1.2 most likely acts independently of its canonical enzymatic activity in GCs and functions upstream of the ROS burst in the ABA signaling pathway. Together, we present a novel, cell-type-specific mechanism that safeguards ABA-driven stomatal closure under fluctuating Cu supply.

plant biology↗

bioRxiv2025

Protein and genetic interactions between RACK1A and FSD1 modulate plant development and stress granule-dependent response to salt in Arabidopsis.

The generation of reactive oxygen species (ROS) and their regulation by antioxidant enzymes such as IRON SUPEROXIDE DISMUTASE 1 (FSD1) are critical for managing plant responses to salt stress. However, the protein networks modulating ROS levels during salt stress remain incompletely understood. Our co-immunoprecipitation analysis identified the FSD1 as an interaction partner of the scaffolding protein RECEPTOR FOR ACTIVATED C KINASE 1A (RACK1A). Bimolecular fluorescence complementation analyses revealed that RACK1A interacts with FSD1 predominantly in the cytoplasm. Despite elevated FSD1 activity in rack1a mutants, the abundance of FSD1 protein remained unchanged. Computational predictions of interaction interfaces suggested that RACK1A may interfere with the catalytic site of FSD1. Advanced fluorescence microscopy and genetic studies further confirmed localization and relocation patterns of RACK1A and FSD1 during salt stress responses. Additionally, we found that the RACK1A-FSD1 module was involved in root hair tip growth, highlighting the developmental significance of this interaction. While rack1a mutants exhibited salt resilience, fsd1-1 rack1a-1 double mutant displayed reduced salt stress resistance compared to rack1a single mutant, which was substantiated by deregulation of ROS levels. RACK1A and FSD1 accumulated in cycloheximide-sensitive structural condensates in the cytoplasm that colocalized with the stress granule marker protein TSN in roots. However, RACK1A knock-out completely abolished salt-stress-dependent relocation of FSD1 to structural condensates, suggesting that RACK1A recruits FSD1 to these stress granules. Thus, this study uncovers an entirely novel mechanism for the regulation of RACK1/FSD1-dependent antioxidant defense in response to salt stress in Arabidopsis.

plant biology↗

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↗

bioRxiv2022

Genetic manipulation of stress-induced mitogen-activated protein kinase modulates early stages of the nodulation process in Medicago sativa

Leguminous plants have established a mutualistic endosymbiotic interaction with nitrogen-fixing rhizobia to secure nitrogen sources in new specialized organs called root nodules. Before nodule formation, the development of early symbiotic structures is essential for rhizobia docking, internalization, targeted delivery, and intracellular accommodation. We have recently reported that overexpression of stress-induced mitogen-activated protein kinase (SIMK) in alfalfa affects root hair, nodule and shoot formation. However, detailed subcellular spatial distribution, activation, and developmental relocation of SIMK during the early stages of alfalfa nodulation remain unclear. Here, we qualitatively and quantitatively characterized SIMK distribution patterns in rhizobium-infected root hairs using live-cell imaging and immunolocalization, employing alfalfa stable transgenic lines with genetically manipulated SIMK abundance and kinase activity. In the SIMKK-RNAi line, showing downregulation of SIMKK and SIMK, we found a considerably decreased accumulation of phosphorylated SIMK around infection pockets and infection threads, which was strongly increased in the GFP-SIMK line, constitutively overexpressing GFP-tagged SIMK. Thus, genetically manipulated SIMK modulates root hair capacity to form infection pockets and infection threads. These results shed new light on SIMK spatio-temporal participation in the early interactions between alfalfa and rhizobia, and its internalization into root hairs, showing that local accumulation of active SIMK indeed modulates nodulation in alfalfa. One sentence summaryGenetic manipulation of SIMK in alfalfa revealed that SIMK modulates root hair capacity to form infection pockets and infection threads during the early interactions between alfalfa and rhizobia.

plant biology↗

bioRxiv2022

Knockout of mitogen-activated protein kinase 3 causes barley root resistance against Fusarium graminearum.

The roles of mitogen-activated protein kinases (MAPKs) in plant-fungal pathogenic interactions are less understood in crops. Here, microscopic, phenotyping, proteomic and biochemical analyses revealed that independent TALEN-based knockout lines of Hordeum vulgare MITOGEN-ACTIVATED PROTEIN KINASE 3 (HvMPK3 KO) were resistant against Fusarium graminearum infection. When co-cultured with roots of the HvMPK3 KO lines, F. graminearum hyphae were excluded to the extracellular space, the growth pattern of hyphae was considerably deregulated, mycelia development was less efficient and number of appressoria and their penetration potential were significantly reduced. Intracellular penetration of hyphae was preceded by the massive production of reactive oxygen species (ROS) in attacked cells of the wild type, but it was mitigated in the HvMPK3 KO lines. Suppression of ROS production in these lines coincided with the elevated abundances of catalase and ascorbate peroxidase. Moreover, differential proteomic analysis revealed downregulation of defense-related proteins in wild type, and the upregulation of peroxidases, lipid transfer proteins, and cysteine proteases in HvMPK3 KO lines after 24h of F. graminearum inoculation. Consistently with proteomic analysis, microscopic observations showed an enhanced suberin accumulation in roots of HvMPK3 KO lines, most likely contributing to the arrested infection by F. graminearum. These results suggest that TALEN-based knockout of HvMPK3 leads to the barley root resistance against Fusarium root rot.

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↗

bioRxiv2020

Cortical microtubule remodelling during strigolactone- and light-mediated growth inhibition of Arabidopsis hypocotyls

Strigolactones are phytohormones involved in shoot branching and hypocotyl elongation. The latter phenomenon was addressed herein by the exogenous application of a synthetic strigolactone GR24 and an inhibitor of strigolactone biosynthesis TIS108 on hypocotyls of wild type Arabidopsis and a strigolactone signalling mutant max2-1 (more axillary growth 2-1). Owing to the interdependence between light and strigolactone signalling, the present work was extended to seedling cultivation under a standard light/dark regime, or under continuous darkness. Given the essential role of the cortical microtubules in cell elongation, their organization and dynamics were characterized under the conditions of altered strigolactone signalling using fluorescence microscopy methods with different spatiotemporal capacities such as confocal laser scanning microscopy and structured illumination microscopy. It was found that the strigolactone-dependent inhibition of hypocotyl elongation correlated with changes in cortical microtubule organization and dynamics, visualized in living wild type and max2-1 seedlings stably expressing genetically-encoded fluorescent molecular markers for microtubules. Quantitative analysis of microscopic datasets revealed that chemical and/or genetic manipulation of strigolactone signalling affected microtubule remodelling, especially under light conditions. The application of GR24 and TIS108 in dark conditions partially alleviated cytoskeletal rearrangement, suggesting a new mechanistic connection between the cytoskeletal behaviour and the light-dependence of strigolactone signalling. HighlightStrigolactones regulate organization and dynamics of cortical microtubules in hypocotyl cells, which contributes to the light-mediated inhibition of hypocotyl growth in Arabidopsis seedlings.

plant biology↗

bioRxiv2020

The interplay of HSP90s with YDA regulates main body axis formation during early embryogenesis in Arabidopsis

The YODA kinase (YDA) pathway is intimately associated with the control of Arabidopsis thaliana embryo development but little is known regarding its regulators. Using genetic analysis, HEAT SHOCK PROTEINS 90 (HSP90s) emerge as potent regulators of YDA in the process of embryo development and patterning. This study is focused on the characterization and quantification of early embryonal traits of single and double hsp90 and yda mutants. The mutant analysis was supported by expression analyses of cell-specific WUSCHEL-RELATED HOMEOBOX 2 (WOX2) and WOX8 genes during early embryonic development. Chromatin immunoprecipitation assays corroborated the involvement of YDA and HSP90s in the epigenetic control of chromatin remodeling during early embryogenesis. Genetic interactions among HSP90s and members of the YDA signaling pathway affected the development of both embryo proper and suspensor. Impaired function of HSP90s or YDA had an impact on the spatiotemporal expression of WOX8 and WOX2 suggesting their essential role in cell fate determination and interference with auxin distribution. Hence, the interplay between HSP90s and YDA signaling cascade mediates the epigenetic control regulating the transcriptional networks shaping early embryo development.

plant biology↗

bioRxiv2020

FSD1: a plastidial, nuclear and cytoplasmic enzyme relocalizing to the plasma membrane under salinity

Here, we aimed to resolve the developmental expression and subcellular localization of Arabidopsis iron superoxide dismutase FSD1, which belongs to the family of superoxide dismutases (SODs), prominent enzymes decomposing superoxide anion and determining abiotic stress tolerance. We found that fsd1 knockout mutants exhibit reduced lateral root number and that this phenotype was complemented by proFSD1::GFP:FSD1 and proFSD1::FSD1:GFP constructs. Light sheet fluorescence microscopy revealed a temporary accumulation of FSD1-GFP at the site of endosperm rupture during seed germination. In emerged roots, FSD1-GFP showed the highest abundance in cells of the lateral root cap, columella, and endodermis/cortex initials. The largest subcellular pool of FSD1-GFP was localized in the plastid stroma, while it was also located in the nuclei and cytoplasm. FSD1 is crucial for seed germination and salt stress tolerance, which is tightly coupled with FSD1-GFP subcellular relocation to the plasma membrane. FSD1 is most likely involved in superoxide decomposition in the periplasm. This study suggests a new osmoprotective function of SODs in plants.

plant biology↗

bioRxiv2018

Environment and HSP90 modulate MAPK stomatal developmental pathway

Stomatal ontogenesis is a key element of plant adaptation aiming to control photosynthetic efficiency and water management under fluctuating environments 1,2,3. Development of stomata is guided by endogenous and environmental cues and is tightly coupled to overall plant growth 1,2,3. YODA signaling pathway is essential to stomatal lineage specification4,5,6 since it regulates the activities of transcription factors such as SPEECHLESS (SPCH)7,8,9,10. Heat-shock proteins 90 (HSP90s) are evolutionarily conserved molecular chaperones implicated in a broad range of signalling pathways being integrated in interaction networks with client proteins11,12,13,14. Herein, based on genetic, molecular, biochemical, and cell biological evidence we report that heat-stress conditions affect phosphorylation and deactivation of SPCH and modulate stomatal density. We show that genetic and physical interactions between HSP90s and YODA control stomatal patterning, distribution and morphology. We provide solid evidence that HSP90s play a major role in transducing the heat-stress response since they act upstream and downstream of YODA signalling, regulate the activity and nucleocytoplasmic distribution of MAPKs, and the activation of SPCH. Thus, HSPs control the stomatal development both under normal temperature and acute heat-stress conditions. Our results demonstrate that HSP90s couple stomatal formation and patterning to environmental cues providing an adaptive mechanism of heat-stress tolerance response and stomatal formation in Arabidopsis.

plant biology↗