Biology subjects

Miras, M.

Publications and source records attributed to Miras, M..

4 recordsLinked to original sources

bioRxiv2025

Making plant tissue accessible for cryo-electron tomography

Cryo-Electron Tomography (cryo-ET) allows to visualize the molecular architecture of pristinely preserved cells and tissues. The workflow of sample preparation for cryo-ET is rather complex; it involves vitrification by rapid freezing followed by cryo-Focused Ion Beam (FIB) milling rendering the volumes of interest thin enough for cryo-ET data acquisition. The established protocols for single cells grown on or deposited on EM-grids are not suitable for multicellular plant tissues. Plunge-freezing does not yield vitrified samples in most cases and must be replaced by high-pressure freezing. This, in turn, necessitates extensive modifications of the subsequent FIB milling procedures. In this communication we describe procedures for sample screening, targeted FIB milling guided by cryo-fluorescence microscopy and a novel lamella trimming step that allows to obtain homogenously thin lamellae suitable for cryo-ET. We have tested all the steps along the workflow with a variety of plant tissues including the moss Physcomitrium patens and tissues of Arabidopsis thaliana and Limonium bicolor. We could demonstrate that the workflow optimized for plant tissues allows to attain subnanometer resolution in cases where subtomogram averaging is applicable.

plant biology↗

bioRxiv2024

Identification of nuclear pore proteins at plasmodesmata

Plasmodesmata (PD) mediate intercellular exchange of small molecules, RNAs and proteins between plant cells with an apparent exclusion limit for passive non-specific transport, and transport of specific cargo mediated by mediators. PD and nuclear pore complexes (NPC) are nanometer sized micropores with strikingly similar properties. Cargo translocation through NPC is mediated by phase separating FG-nucleoporins (FG-NUP). Here, bioinformatics, proteomics and fluorescence imaging identified FG-NUPs at PD. Transient expression of GFP fusions at low and intermediate expression levels supported dual localization of 12 NUPs to NPC and PD. Structured illumination microscopy detected the transmembrane anchor NUP CPR5 close to orifices of PD. cpr5 mutants showed reduced intercellular short-root (SHR) transport. However, transport defects cannot be excluded due to indirect effects in the mutants. Identification of FG-NUPs at PD is consistent with the recruitment of NUPs to form a PD pore gating complex consistent with phase separation domains as diffusion barriers at PD. Further analyses will be required to determine whether NUPs are bona fide PD components, or accumulate at PD in certain conditions, or may serve intermediate NPC storage.

cell biology↗

bioRxiv2023

Rice Yellow Mottle Virus resistance by genome editing of the Oryza sativa L. ssp. japonica nucleoporin gene OsCPR5.1 but not OsCPR5.2

Rice yellow mottle virus (RYMV) causes one of the most devastating rice diseases in Africa. Management of RYMV is challenging. Genetic resistance provides the most effective and environment-friendly control. The recessive resistance locus rymv2 (OsCPR5.1) had been identified in African rice (O. glaberrima), however, introgression into O. sativa ssp. japonica and indica remains challenging due to crossing barriers. Here, we evaluated whether CRISPR/Cas9 genome editing of the two rice nucleoporin paralogs OsCPR5.1 (RYMV2) and OsCPR5.2 can be used to introduce RYMV resistance into the japonica variety Kitaake. Both paralogs had been shown to complement the defects of the Arabidopsis atcpr5 mutant, indicating partial redundancy. Despite striking sequence and structural similarities between the two paralogs, only oscpr5.1 loss-of-function mutants were fully resistant, while loss-of-function oscpr5.2 mutants remained susceptible, intimating that OsCPR5.1 plays a specific role in RYMV susceptibility. Notably, edited lines with short in-frame deletions or replacements in the N-terminal domain (predicted to be unstructured) of OsCPR5.1 were hypersusceptible to RYMV. In contrast to mutations in the single Arabidopsis AtCPR5 gene, which caused severely dwarfed plants, oscpr5.1 and oscpr5.2 single knockout mutants show neither substantial growth defects nor symptoms indicative of programmed cell death, possibly reflecting functional redundancy of the isoforms regarding other important functions. The specific editing of OsCPR5.1, while maintaining OsCPR5.2 activity, provides a promising strategy for generating RYMV-resistance in elite Oryza sativa lines as well as for effective stacking with other RYMV resistance genes or other traits.

plant biology↗

bioRxiv2020

Unique and distinct identities and functions of leaf phloem cells revealed by single cell transcriptomics

AO_SCPLOWBSTRACTC_SCPLOWThe leaf vasculature plays a key role in solute translocation. Veins consist of at least seven distinct cell types, with specific roles in transport, metabolism, and signaling. Little is known about the vascular cells in leaves, in particular the phloem parenchyma (PP). PP effluxes sucrose into the apoplasm as a basis for phloem loading; yet PP has only been characterized microscopically. Here, we enriched vascular cells from Arabidopsis leaves to generate a single-cell transcriptome atlas of leaf vasculature. We identified [≥]19 cell clusters, encompassing epidermis, guard cells, hydathodes, mesophyll, and all vascular cell types, and used metabolic pathway analysis to define their roles. Clusters comprising PP cells were enriched for transporters, including SWEET11 and SWEET12 sucrose and UmamiT amino acid efflux carriers. PP development occurs independently from APL, a transcription factor required for phloem differentiation. PP cells have a unique pattern of amino acid metabolism activity distinct from companion cells (CC), explaining differential distribution/metabolism of amino acids in veins. The kinship relation of the vascular clusters is strikingly similar to the vein morphology, except for a clear separation of CC from the other vascular cells including PP. In summary, our scRNA-seq analysis provides a wide range of information into the leaf vasculature and the role and relationship of the leaf cell types.

plant biology↗