Biology subjects

Krupar, P.

Publications and source records attributed to Krupar, P..

3 recordsLinked to original sources

bioRxiv2023

A novel workflow for unbiased quantification of autophagosomes in 3D in Arabidopsis thaliana roots

Macroautophagy is frequently quantified by live imaging of autophagosomes decorated with a marker of fluorescently tagged ATG8 protein (FT-ATG8) in Arabidopsis thaliana. This requires generation of suitable plant material by time-consuming crossing or transformation with FT-ATG8 marker. Autophagosome quantification by image analysis often relies on their counting in individual focal planes. This approach is prone to deliver biased results due to inappropriate sampling of the regions of interest in the Z-direction, as the actual 3D distribution of autophagosomes is usually not taken into account. To overcome such drawbacks, we have developed and tested a workflow consisting of immunofluorescence microscopy of autophagosomes labelled with anti-ATG8 antibody followed by stereological image analysis employing the optical disector and the Cavalieri principle. Our immunolabelling protocol specifically recognized autophagosomes in epidermal cells of A. thaliana root. Higher numbers of immunolabelled autophagosomes were observed when compared with those recognized with FT-AtATG8e marker, suggesting that single AtATG8 isoform markers cannot detect all autophagosomes in a cell. Therefore, immunolabelling provides more precise information as the anti-ATG8 antibody recognizes virtually all AtATG8 isoforms. The number of autophagosomes per tissue volume determined by stereological methods correlated with the intensity of autophagy induction treatment. Compared to autophagosome quantifications in maximum intensity projections, stereological methods detected autophagosomes present in a given volume with higher accuracy. Our novel application of immunolabelling combined with stereological methods constitutes a powerful toolbox for unbiased and reproducible quantification of autophagosomes and offers a convenient alternative to the standard of live imaging using FP-ATG8 marker.

plant biology↗

bioRxiv2023

Chitosan stimulates root hair callose deposition and inhibits root hair growth

Although angiosperm plants have a general capacity to react after the immunity elicitor chitin or chitosan treatment by the cell wall callose deposition, this response in particular cell types and its evolutionary conservation is not understood. Here we show that also the growing root hairs (RHs) of Arabidopsis can respond to a mild (0.001%) chitosan treatment by the callose deposition and by a deceleration of the RH growth. We demonstrate that the glucan synthase-like 5 (GSL5)/PMR4 is vital for chitosan-induced callose deposition but not for RH growth inhibition. Upon the higher chitosan concentration (0.01%) treatment, RHs do not deposit callose, while growth inhibition is prominent. To understand the specificities of the low and high concentration chitosan treatments, we analysed the corresponding PTI signalling components, gene expression, and RH cellular endomembrane and cytoskeleton modifications. Importantly, chitosan-induced callose deposition is also present in the functionally analogous and evolutionarily only distantly related RH-like structures rhizophores (lycophytes) and rhizoids (bryophytes). Our results point to the RH callose deposition as a conserved strategy of soil-anchoring plant cells (rhizoids/rhizophores/RHs) to deal with mild biotic stress. At the same time, high chitosan concentration prominently disturbs intracellular dynamics, tip-localised endomembrane compartments and RH growth, precluding callose deposition.

plant biology↗

bioRxiv2022

An ultra-fast, proteome-wide response to the plant hormone auxin

The plant signaling molecule auxin controls growth and development through a simple nuclear pathway that regulates gene expresion. There are however several cellular and physiological responses to auxin that occur within seconds, far too rapid to be mediated by transcriptional changes, for which no molecular mechanism has yet been identified. Using a phosphoproteomic strategy in Arabidopsis thaliana roots, we identify an ultra-rapid auxin response system that targets over 1700 proteins, many within 30 seconds. Auxin response is chemically specific, requires known auxin-binding proteins, and targets various pathways. Through exploring its temporal dynamics, we infer auxin-triggered kinase-substrate networks and identify apoplastic pH changes as a target of signaling and as part of a relay mechanism. By generating a variety of phosphoproteomic datasets, integrated with structural information in a web-app, and by demonstrating analysis and inference strategies, we offer a resource to explore rapid and dynamic signaling in plants.

plant biology↗