Biology subjects

Danek, M.

Publications and source records attributed to Danek, M..

5 recordsLinked to original sources

bioRxiv2026

MILDEW RESISTANCE LOCUS O (MLO) proteins function as trimeric inward calcium channels

Calcium signalling and structural roles are fundamental for plant growth, development, and environmental adaptation. Recent studies have identified MILDEW RESISTANCE LOCUS O (MLO) proteins as novel calcium-permeable channels with roles in root growth, cell wall development, pollen tube growth, and perception. However, the molecular mechanisms underlying MLO function remain unknown. Here, we demonstrate that multimerisation is essential for MLO activity. Chemical crosslinking, split-ubiquitin interaction assays, and single-molecule photobleaching revealed that MLO proteins form stable dimeric and trimeric assemblies at the plasma membrane. Structural modelling uncovered a molecular architecture of the MLO trimer with a central ion-conducting pore, which was further examined by molecular dynamics simulations in a lipid membrane environment. Computational electrophysiology showed preferential inward Ca2+ transport, confirming that MLO proteins function as calcium influx transporters, and identified a conserved set of pore-lining residues that coordinate ion translocation. Functional and structural analyses indicated that the mechanism of calcium permeation is evolutionarily conserved. Our findings provide mechanistic insight into MLO-mediated calcium influx across the plasma membrane and establish multimerisation as a critical determinant of this calcium channels activity.

plant biology↗

bioRxiv2026

The molecular architecture of the Arabidopsis callose synthase complex

ABSTRACTCallose synthase is responsible for the targeted deposition of the {beta}-1,3-glucan polymer, callose which underlines essential plant developmental processes, including cell division, pathogen defense or cell-cell communication. The architecture of the callose synthase complex (CALSC) as well as the molecular mechanisms of callose synthesis remain unknown. Here we report an integrative characterisation of the Arabidopsis CALS complex, with the most enriched subunits, CALS1, CALS2 and CALS3, forming its core. Structurally, CALSC assembles into a trimer, requiring the plant-specific Bag domain to mediate inter-subunit associations. The biological importance of CALSC assembly is highlighted by the simultaneous loss of CALS1 and CALS3, which abolishes plasmodesmal callose deposition and affects symplastic transport. Site-directed mutagenesis and molecular dynamics simulations depict the topology of the CALS1 active site in detail, including the components of the enzymatic reaction. We pinpoint the translocating tunnel through which the nascent glucan is delivered and mechanistically confirm the role of transmembrane helix 8 in regulating glucan export. Our work provides unprecedented insight into the molecular architecture of the CALSC and the distinct changes from maturation to activity at the plasma membrane, while showcasing the mechanisms involved in callose synthesis at the molecular level.

plant biology↗

bioRxiv2025

Mechanisms controlling the plasma membrane targeting and the nanodomain organization of the plant SPFH protein HIR2

HIR2 is a plant-specific protein belonging to the superfamily of SPFH domain-containing proteins that were proposed to play scaffolding functions in membranes. HIR2 organizes in plasma membrane (PM) nanodomains that correspond to nanometric scale structures enriched in specific lipids and proteins acting as signaling/regulation hubs. So far, how PM nanodomains are formed and maintained in plant cells remains largely unknown. Combining state of the art microscopy techniques, we investigated the mechanisms governing the trafficking and the organization into nanodomains of Arabidopsis HIR2 protein. We revealed that the mono S-acylation of HIR2 either on C6 or on C7 was required for HIR2 targeting to the PM of Arabidopsis cells, independently of the conventional secretory pathway. Investigating the mechanisms implicated in the arrangement of HIR2 into nanodomains, we provided evidences that the lipid composition in sterols and very long chain fatty acids of the PM influenced HIR2 organization. HIR2 forms oligomers and we demonstrated here that the C-terminal part of HIR2 is required for self-assembly, similarly to animal SPFH proteins. Interestingly, we highlighted that the oligomerization of HIR2 is essential for its organization in nanodomains and to ensure HIR2 lateral stability in the PM. Overall, we propose that HIR2 nanodomain organization is a complex mechanism relying on different parameters including PM lipid composition and oligomerization. HIR proteins are involved in plant immunity. Here, we revealed that HIR2 nanodomain organization is required to boost the apoplastic ROS burst induced by the bacterial peptide flg22. One sentence summaryS-acylation and oligomerization control the plasma membrane targeting and the organization into nanodomains of the Arabidopsis SPFH-domain containing protein HIR2, respectively.

plant biology↗

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↗

bioRxiv2021

Phospholipase Dα1 acts as a negative regulator of high Mg2+-induced leaf senescence in Arabidopsis

Magnesium is a macronutrient involved in essential cellular processes. Its deficiency or excess is a stress factor for plants, seriously affecting their growth and development and therefore, its accurate regulation is essential. Recently, we discovered that phospholipase D1 (PLD1) activity is vital in the stress response to high-magnesium conditions in Arabidopsis roots. This study shows that PLD1 acts as a negative regulator of high-Mg2+-induced leaf senescence in Arabidopsis. The level of phosphatidic acid produced by PLD1 and the amount of PLD1 in the leaves increase in plants treated with high Mg2+. A knockout mutant of PLD1 (plda1-1), exhibits premature leaf senescence under high-Mg2+ conditions. In pld1-1 plants, higher accumulation of abscisic and jasmonic acid and impaired magnesium, potassium and phosphate homeostasis were observed under high-Mg2+ conditions. High Mg2+ also led to an increase of starch and proline content in Arabidopsis plants. While the starch content was higher in plda1-1 plants, proline content was significantly lower in plda1-1 compared with WT. Our results show that PLD1 is essential for Arabidopsis plants to cope with the pleiotropic effects of high-Mg2+ stress and delay the leaf senescence.

plant biology↗