Verena Ibl

BioStudies – one package for all the data supporting a study

SUMMARY: Positioning of the division plane is a critical step during cellular development. While in animal cells the plane of cell division is defined during metaphase, in plants this decision is made before mitosis and includes the formation of a cytoskeletal structure termed the preprophase band (PPB). Although not essential, the PPB constitutes the earliest mark for the plane of cell division and coincides with the sites where the cell plate fuses with the parental plasma membrane during cytokinesis. Recent studies indicate that endo- and exocytosis are involved in remodelling of the cortical division zone. Here we show that PPBs, phragmoplasts, cell plates and cell walls are misplaced in Arabidopsis mutants of Atvps2.2/hyade (hya) , a plant-specific component of the Endosomal Sorting Complex Required for Transport (ESCRT)-III. We demonstrate that the amino acid substitution Q71P of the hya-3 allele abolishes the homo- and heteromerization with other ESCRT-III components. Functional HYA-GFP fusion proteins are excluded from the canonical location of ESCRT-III complexes at multivesicular bodies (MVBs) and are absent from Brefeldin A (BFA) and Wortmannin (WM) sensitive compartments in Arabidopsis . HYA-GFP neither localize to early nor late endosomes but colocalizes with markers of secretory compartments such as the Qc-SNARE, SYP61 and with trans-Golgi-network (TGN) derived secretory Rab-A3 vesicles. Moreover, HYA-GFP locates to the extracellular space indicating that VPS2.2/HYA is involved in secretion. These results are consistent with a novel, non-canonical function of ESCRT-III in the establishment and maintenance of the cortical division zone via secretion.

Barley lacks wheat’s good gluten-related baking properties, despite containing the wheat gluten homologs D-, B-, C-, and γ-hordeins. It is unknown if hordeins form a disulphide (S-S) bond network like wheat gluten proteins. In this study, gluten networks were isolated from two Hordeum vulgare L. barley varieties, PS3 and Golden Promise, which represent a baking and a brewing variety, respectively. The isolated gluten proteins were processed under non-reducing conditions and measured by liquid chromatography-high-resolution mass spectrometry, resulting in 26 S-S bonded peptides. Based on these, a model of the hordein network was developed, comprising B-, D- and γ-hordeins in an expanded S-S crosslinked network. In the proposed network, B-hordeins functions as elongators, D-hordeins promote network branching, and γ-hordeins act as chain terminators. In this study, no qualitative differences were observed between the S-S network in PS3 and Golden Promise, suggesting other factors differentiating the baking quality.

Salinity, a major abiotic stressor, impacts protein trafficking and alters transport routes. The endosomal sorting complex required for transport (ESCRT)is a key player for membrane protein sorting and is increasingly recognized as pivotal element in abiotic stress responses. However, the functional role of ESCRT proteins in cereal crops under salt stress remains unexplored. Using the as near as possible to nature DRD-BIBLOX (Dark-Root device Brick Black Box), barley ( Hordeum vulgare , L.), although relatively salt-tolerant, exhibits impaired germination, seedling growth, and an altered root system architecture (RSA) under high salinity. Shotgun proteomic analysis revealed spatio-temporal regulation of ESCRT proteins in shoots and roots during seedling development. We identified HvSNF7.1 and HvVPS4 as critical, but differently acting modulators of salt stress responses in barley tissues. In this study we further reveal novel functions of ESCRT-0-like TOM-1-like proteins (TOLs): Previously described as gatekeepers of degradative protein sorting and as regulators of abscisic acid signaling, growth, and heat stress responses in Arabidopsis ( Arabidopsis thaliana ), HvTOLs had not been characterized for their role in salinity adaptation during plant development. Functional analyses demonstrated lethality in Hv toldouble and Hv tolquadruple mutants under standard growth conditions. In Arabidopsis, loss of At TOL3 alone impaired germination and root growth under salt stress, underscoring the essential role of individual TOL proteins in the spatio-temporal regulation of abiotic stress adaptation. These findings highlight ESCRT-mediated trafficking as a key determinant of cereal resilience under saline conditions and provide a molecular framework for improving crop stress tolerance.

SUMMARY: Glycosyl inositol phospho ceramides (GIPC) are the predominant glycosphingolipids in plant membranes, essential for their membrane stability, cell signaling, stress adaptation, and pathogen resistance. However, their complex structures, characterized by a ceramide backbone and a glycan head group, have challenged comprehensive analysis using traditional methods, which often rely on separate glycan or lipid profiling. To overcome these limitations, we developed a glycosphingolipidomics assay using reversed‐phase high‐resolution mass spectrometry including multistage fragmentation (RP‐HRMSⁿ). This method enables direct, detailed structural characterization of GIPC in plants, combining advanced chromatographic separation, multistage fragmentation, and automated annotation using decision rule‐based criteria. Applied to barley grains, the assay identified 102 GIPC species, including A‐, B‐, C‐, and D‐series GIPC, previously unreported glycan branching fragments (421 and 403 m/z), and a huge structural variety in the ceramide moiety. Profiling at different development stages revealed dynamic GIPC regulation during grain development, with an upregulation of B‐ and C‐series towards mature development stages. The application of heat stress induced significant remodeling of GIPC profiles, mainly through upregulation of B‐series species, which emphasizes their roles in maintaining membrane stability and functionality under abiotic stress conditions. The presented glycosphingolipidomics assay enables the first automated analysis of complex GIPC through a decision rule‐based identification approach. By resolving GIPC to the molecular lipid species level, the method provides novel insights into GIPC diversity, homeostasis, and their critical roles in membrane dynamics, stress adaptation, and pathogen resistance, paving the way for advanced research in plant lipidomics and stress biology.

Editorial: Highlights of the 2nd D(dark grown)-root meeting

Roots are the hidden parts of plants, anchoring their above-ground counterparts in the soil. They are responsible for water and nutrient uptake and for interacting with biotic and abiotic factors in the soil. The root system architecture (RSA) and its plasticity are crucial for resource acquisition and consequently correlate with plant performance while being highly dependent on the surrounding environment, such as soil properties and therefore environmental conditions. Thus, especially for crop plants and regarding agricultural challenges, it is essential to perform molecular and phenotypic analyses of the root system under conditions as near as possible to nature (#asnearaspossibletonature). To prevent root illumination during experimental procedures, which would heavily affect root development, Dark-Root (D-Root) devices (DRDs) have been developed. In this article, we describe the construction and different applications of a sustainable, affordable, flexible, and easy to assemble open-hardware bench-top LEGO® DRD, the DRD-BIBLOX (Brick Black Box). The DRD-BIBLOX consists of one or more 3D-printed rhizoboxes, which can be filled with soil while still providing root visibility. The rhizoboxes sit in a scaffold of secondhand LEGO® bricks, which allows root development in the dark and non-invasive root tracking with an infrared (IR) camera and an IR light-emitting diode (LED) cluster. Proteomic analyses confirmed significant effects of root illumination on barley root and shoot proteomes. Additionally, we confirmed the significant effect of root illumination on barley root and shoot phenotypes. Our data therefore reinforces the importance of the application of field conditions in the lab and the value of our novel device, the DRD-BIBLOX. We further provide a DRD-BIBLOX application spectrum, spanning from investigating a variety of plant species and soil conditions and simulating different environmental conditions and stresses, to proteomic and phenotypic analyses, including early root tracking in the dark.

Roots are the hidden parts of plants, anchoring their above ground counterparts in the soil. They are responsible for water and nutrient uptake, as well as for interacting with biotic and abiotic factors in the soil. The root system architecture (RSA) and its plasticity are crucial for resource acquisition and consequently correlate with plant performance, while being highly dependent on the surrounding environment, such as soil properties and therefore environmental conditions. Thus, especially for crop plants and regarding agricultural challenges, it is essential to perform molecular and phenotypic analyses of the root system under conditions as near as possible to nature (#asnearaspossibletonature). To prevent root illumination during experimental procedures, which would heavily affect root development, dark-root (D-Root) devices (DRDs) have been developed. In this article, we describe the construction and different applications of a sustainable, affordable, flexible, and easy to assemble open-hardware bench-top LEGO® DRD, the DRD-BIBLOX (Brick Black Box). The DRD-BIBLOX consists of one or more 3D-printed rhizoboxes which can be filled with soil, while still providing root visibility. The rhizoboxes sit in a scaffold of secondhand LEGO® bricks, which allows root development in the dark as well as non-invasive root-tracking with an infrared (IR) camera and an IR light emitting diode (LED) cluster. Proteomic analyses confirmed significant effects of root illumination on barley root and shoot proteome. Additionally, we confirmed the significant effect of root illumination on barley root and shoot phenotypes. Our data therefore reinforces the importance of the application of field conditions in the lab and the value of our novel device, the DRD-BIBLOX. We further provide a DRD-BIBLOX application spectrum, spanning from investigating a variety of plant species and soil conditions as well as simulating different environmental conditions and stresses, to proteomic and phenotypic analyses, including early root tracking in the dark.

Cereal grain germination provides the basis for crop production and requires a tissue-specific interplay between the embryo and endosperm during heterotrophic germination involving signalling, protein secretion, and nutrient uptake until autotrophic growth is possible. High salt concentrations in soil are one of the most severe constraints limiting the germination of crop plants, affecting the metabolism and redox status within the tissues of germinating seed. However, little is known about the effect of salt on seed storage protein mobilization, the endomembrane system, and protein trafficking within and between these tissues. Here, we used mass spectrometry analyses to investigate the protein dynamics of the embryo and endosperm of barley (Hordeum vulgare, L.) at five different early points during germination (0, 12, 24, 48, and 72 h after imbibition) in germinated grains subjected to salt stress. The expression of proteins in the embryo as well as in the endosperm was temporally regulated. Seed storage proteins (SSPs), peptidases, and starch-digesting enzymes were affected by salt. Additionally, microscopic analyses revealed an altered assembly of actin bundles and morphology of protein storage vacuoles (PSVs) in the aleurone layer. Our results suggest that besides the salt-induced protein expression, intracellular trafficking and actin cytoskeleton assembly are responsible for germination delay under salt stress conditions.