Michal Daněk

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 Ca ²⁺ 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 channel’s activity.

: Callose synthase is responsible for the targeted deposition of the β-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.

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 summary S-acylation and oligomerization control the plasma membrane targeting and the organization into nanodomains of the Arabidopsis SPFH-domain containing protein HIR2, respectively.

Macroautophagy is often quantified by live imaging of autophagosomes labeled with fluorescently tagged ATG8 protein (FP-ATG8) in Arabidopsis thaliana. The labeled particles are then counted in single focal planes. This approach may lead to inaccurate results as the actual 3D distribution of autophagosomes is not taken into account and appropriate sampling in the Z-direction is not performed. To overcome this issue, we developed a workflow consisting of immunolabeling of autophagosomes with an anti-ATG8 antibody followed by stereological image analysis using the optical disector and the Cavalieri principle. Our protocol specifically recognized autophagosomes in epidermal cells of Arabidopsis root. Since the anti-ATG8 antibody recognizes multiple AtATG8 isoforms, we were able to detect a higher number of immunolabeled autophagosomes than with the FP-AtATG8e marker, that most likely does not recognize all autophagosomes in a cell. The number of autophagosomes per tissue volume correlated with the intensity of autophagy induction. Compared to the quantification of autophagosomes in maximum intensity projections, stereological methods were able to detect the autophagosomes present in a given volume with higher accuracy. Our novel workflow provides a powerful toolkit for unbiased and reproducible quantification of autophagosomes and offers a convenient alternative to the standard of live imaging with FP-ATG8 markers.

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- At ATG8e marker, suggesting that single At ATG8 isoform markers cannot detect all autophagosomes in a cell. Therefore, immunolabelling provides more precise information as the anti-ATG8 antibody recognizes virtually all At ATG8 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.

Summary: Current models of plasma membrane (PM) postulate its organization in various nano‐ and micro‐domains with distinct protein and lipid composition. While metazoan PM nanodomains usually display high lateral mobility, the dynamics of plant nanodomains is often highly spatially restricted. Here we have focused on the determination of the PM distribution in nanodomains for Arabidopsis thaliana flotillin (AtFLOT) and hypersensitive induced reaction proteins (AtHIR), previously shown to be involved in response to extracellular stimuli. Using in vivo laser scanning and spinning disc confocal microscopy in Arabidopsis thaliana we present here their nanodomain localization in various epidermal cell types. Fluorescence recovery after photobleaching (FRAP) and kymographic analysis revealed that PM‐associated AtFLOTs contain significantly higher immobile fraction than AtHIRs. In addition, much lower immobile fractions have been found in tonoplast pool of AtHIR3. Although members of both groups of proteins were spatially restricted in their PM distribution by corrals co‐aligning with microtubules (MTs), pharmacological treatments showed no or very low role of actin and microtubular cytoskeleton for clustering of AtFLOT and AtHIR into nanodomains. Finally, pharmacological alteration of cell wall (CW) synthesis and structure resulted in changes in lateral mobility of AtFLOT2 and AtHIR1. Accordingly, partial enzymatic CW removal increased the overall dynamics as well as individual nanodomain mobility of these two proteins. Such structural links to CW could play an important role in their correct positioning during PM communication with extracellular environment.

Biologia plantarum, an international journal for experimental botany founded in 1959 by Professor Bohumil N?mec. Covers all branches of experimental botany ranging from molecular biology and biotechnology to whole-plant and stand functioning.

Arabidopsis flotillin 2 (At5g25260) belongs to the group of plant flotillins, which are not well characterized. In contrast, metazoan flotillins are well known as plasma membrane proteins associated with membrane microdomains that act as a signaling hub. The similarity of plant and metazoan flotillins, whose functions most likely consist of affecting other proteins via protein–protein interactions, determines the necessity of detecting their interacting partners in plants. Nevertheless, identifying the proteins that form complexes on the plasma membrane is a challenging task due to their low abundance and hydrophobic character. Here we present an approach for mapping Arabidopsis thaliana flotillin 2 plasma membrane interactors, based on the immunoaffinity purification of crosslinked and enriched plasma membrane proteins with mass spectrometry detection. Using this approach, 61 proteins were enriched in the AtFlot-GFP plasma membrane fraction, and 19 of them were proposed to be flotillin 2 interaction partners. Among our proposed partners of Flot2, proteins playing a role in the plant response to various biotic and abiotic stresses were detected. Additionally, the use of the split-ubiquitin yeast system helped us to confirm that plasma-membrane ATPase 1, early-responsive to dehydration stress protein 4, syntaxin-71, harpin-induced protein-like 3, hypersensitive-induced response protein 2 and two aquaporin isoforms interact with flotillin 2 directly. Based on the results of our study and the reported properties of Flot2 interactors, we propose that Flot2 complexes may be involved in plant–pathogen interactions, water transport and intracellular trafficking.

The non-specific phospholipase C (NPC) is a new member of the plant phospholipase family that reacts to abiotic environmental stresses, such as phosphate deficiency, high salinity, heat and aluminium toxicity, and is involved in root development, silicon distribution and brassinolide signalling. Six NPC genes (NPC1-NPC6) are found in the Arabidopsis genome. The NPC2 isoform has not been experimentally characterized so far. The Arabidopsis NPC2 isoform was cloned and heterologously expressed in Escherichia coli. NPC2 enzyme activity was determined using fluorescent phosphatidylcholine as a substrate. Tissue expression and subcellular localization were analysed using GUS- and GFP-tagged NPC2. The expression patterns of NPC2 were analysed via quantitative real-time PCR. Independent homozygous transgenic plant lines overexpressing NPC2 under the control of a 35S promoter were generated, and reactive oxygen species were measured using a luminol-based assay. The heterologously expressed protein possessed phospholipase C activity, being able to hydrolyse phosphatidylcholine to diacylglycerol. NPC2 tagged with GFP was predominantly localized to the Golgi apparatus in Arabidopsis roots. The level of NPC2 transcript is rapidly altered during plant immune responses and correlates with the activation of multiple layers of the plant defence system. Transcription of NPC2 decreased substantially after plant infiltration with Pseudomonas syringae, flagellin peptide flg22 and salicylic acid treatments and expression of the effector molecule AvrRpm1. The decrease in NPC2 transcript levels correlated with a decrease in NPC2 enzyme activity. NPC2-overexpressing mutants showed higher reactive oxygen species production triggered by flg22. This first experimental characterization of NPC2 provides new insights into the role of the non-specific phospholipase C protein family. The results suggest that NPC2 is involved in the response of Arabidopsis to P. syringae attack.

Plant stomatin/prohibitin/flotillin/HflK/C (SPFH) proteins are represented by prohibitins, flotillins, stomatins, erlins, and hypersensitive induced reaction proteins (HIRs). The purpose of this review is to summarize the current state of knowledge regarding plant flotillins and HIRs and to assign putative functions of plant flotillins and erlins based on the known functions of their mammalian homologs. Similar to human flotillins, plant flotillins are localized in membrane microdomains, and involved in endocytosis, and interact with receptor kinases. HIRs play an important role in plant immunity by promoting the hypersensitive response and binding to leucine-rich repeat proteins. In this way, they participate in resistance to bacterial or fungal pathogens. We further focused on flotillins, HIRs, and erlins in Arabidopsis thaliana and, using public databases, described them in terms of the following: 1) their transcription throughout plant ontogeny and under various environmental conditions; 2) the presence of conserved domains or characteristic motifs in their amino acid sequences; and 3) their potential interactions with other proteins. Based on these data, we hypothesize about their additional functions and properties.