Biology subjects

De Bellis, D.

Publications and source records attributed to De Bellis, D..

13 recordsLinked to original sources

bioRxiv2026

Mechanical cues trigger phellem differentiation during barrier transition

A continuous and uninterrupted barrier tissue is essential for protecting internal tissues from the external environment. In many dicot roots, the endodermis fulfils this role during primary growth; however, the onset of secondary growth in the vascular tissue coincides with the rupture of the overlying endodermis and formation of a new barrier, the periderm, beneath it. Despite the importance of barrier integrity for plant survival, the mechanisms coordinating this barrier transition have remained unexplored. Here, we show that endodermal rupture in Arabidopsis thaliana roots releases mechanical constraints on the underlying properiderm. This release leads to cell expansion and triggers differentiation of the outermost properiderm cells into phellem, a ligno-suberized barrier cell type. Premature relief of mechanical constraints, through hyperosmotic stress or targeted endodermal ablation, is sufficient to trigger phellem differentiation. FERONIA, a cell wall integrity receptor kinase, is required for phellem differentiation in response to mechanical stress. Overall, our findings establish mechanical stress as an instructive cue for phellem differentiation, revealing how plants convert mechanical signals into robust, adaptive protective barriers.

plant biology↗

bioRxiv2025

Imaging of specialized plant cell walls by improved cryo-CLEM and cryo-electron tomography

Cryo-focused ion beam scanning electron microscopy (cryo-FIBSEM) has become essential for preparing electron-transparent lamellae from cryo-plunged and high-pressure frozen specimens. However, targeting specific cellular features within large, complex organs remains challenging. Here we present a series of technical improvements significantly enhancing the efficiency and accessibility of the Serial Lift-Out and SOLIST (Serialized On-grid Lift-In Sectioning for Tomography) procedures that are revolutionizing the field. We were able to extend the cryo-FIBSEM session from 24 hours to 5 days without interruptions. In addition, we describe a modified silver-plated EasyLiftTM needle that eliminates the need of the copper or gold block between the original tungsten needle and the sample. Moreover, we describe a strategy that significantly reduces curtaining effects. Finally, we report a precise routine to target a lamella with a precision of approximately 1 {micro}m in X,Y and Z. Together, these modifications considerably reduce contamination risk and preparation time, making cryo-lift-out techniques more accessible for routine structural biology applications on any type of tissue. Here, we demonstrate the power of our technique by targeting several specific wall structures that are of crucial importance for root function in plants and that were previously inaccessible to cryo-electron tomography (cryo-ET). High-pressure freezing (HPF) of plant tissues presents unique challenges for cryo-electron microscopy sample preparation due to the overall sample size, the individual cells size, their rigid cell wall and finally, their large vacuoles, which contain large amounts of rather diluted water solutions compared to cytosol. The internal root structures targeted are the Casparian strip (CS), suberin lamellae (SL), as well as secondary wall of xylem vessels, requiring reaching a targeting precision of 5 m in a 3 mm long and 80-120 m thick root tip. Our technological improvements for the cryo-correlative light and electron microscopy (cryo-CLEM) workflow enabled successful, targeted cryo-ET in plant roots. We noticed that, despite ice formation in vacuoles and to some degree in the cytosol, the plasma membranes and cell walls are remarkably well preserved, providing stunning insights into the native, hydrated nano-structure of plant cell walls, previously only observable with contrasting agents and in a dehydrated state.

plant biology↗

bioRxiv2025

GPAT2 plays a role in the root cap cuticle formation of Arabidopsis that is not redundant to GPAT4 and GPAT8

During the first days after germination, the root cap protects the seeding with its cuticle, which persists until the embryonic outer root cap cell layer is shed. The polyester constituting the delicate Arabidopsis root cap cuticle of the primary root has an atypical composition, containing unsubstituted very-long-chain fatty acids in addition to the oxygenated fatty acids typically found in cutin. The sn2-glycerol-3-phosphate acyltransferases GPAT4 and GPAT8, which possess an active phosphatase domain, synthesize monoacylglycerols and are crucial for incorporating oxygenated fatty acids into the root cap cutin. Here, we show that the incorporation of very-long-chain fatty acids into cutin requires the glycerol-3-phosphate acyltransferase GPAT2, which lacks an active phosphatase domain and produces lysophosphatidic acids. The GPAT2 knockout alters the properties of the root cap leading to increased mucilage deposition but lower barrier properties. Furthermore, we demonstrate that the long-chain CoA synthetase LACS2 is required solely for the incorporation of oxygenated fatty acids into cutin, similar to GPAT4 and GPAT8. Consequently, two largely non-redundant pathways contribute to root cap cuticle formation in the primary root: both the LACS2/GPAT4/GPAT8- and the GPAT2-dependent pathways contribute non-redundantly to its barrier and surface properties. LACS2 also plays an important role in the formation of the root cap cuticle in emerging lateral roots, whereas GPAT2 has a minor function in this process. These findings underscore the diverse mechanisms by which cuticle synthesis is fine-tuned across plant organs and developmental stages to achieve specialized barrier and surface properties.

plant biology↗

bioRxiv2023

Comparisons of two receptor pathways in a single cell-type reveal features of signalling specificity.

To respond appropriately to diverse stimuli, cells harbour numerous receptor pathways yet share downstream signalling components. Mitogen-Activated Protein Kinase (MPK) cascades, present in all eukaryotes, act as central hubs shared amongst diverse signalling pathways. How signalling specificity is maintained within a single plant cell-type is not well understood. We engineered a genetic background for direct comparisons of a developmental and an immunity pathway in the Arabidopsis root endodermis. We show the two pathways maintain distinct outputs despite similar MPK phosphorylation patterns. Systematic activation of all individual MPK Kinases (MKKs) in the endodermis revealed that different MKK groups can drive distinct outputs. We propose that specificity is achieved through a balance of combinatorial activation and cross-pathway inhibition within the MPK cascade. Our findings demonstrate the ability of MPK cascades to generate diverse outputs in one plant cell-type, providing new insights into the mechanisms contributing to signalling specificity.

plant biology↗

bioRxiv2023

Air channels create a directional light signal to regulate hypocotyl phototropism

In light-limiting conditions, aerial organs of most plants reorient their growth towards the light to improve photosynthesis, through a process known as phototropism1-3. The blue light receptors phototropin control phototropic responses through light-induced protein kinase activity4. Current models posit that asymmetric activation of these sensory receptors across a unilaterally illuminated organ leads to asymmetric distribution of the growth hormone auxin ultimately leading to growth re-orientation4,5. However, the tissue properties required to generate a light gradient across the stem triggering phototropism remain unclear1. Here we show that inter-cellular air channels6,7 are required for an efficient phototropic response. These channels enhance light scattering (refraction and reflection) in Arabidopsis hypocotyls thereby enhancing the light gradient across the photo-stimulated organ. We identify an embryonically expressed ABC transporter that is required to keep air in inter-cellular spaces in seedlings and for efficient phototropism. Our work suggests that this transporter shapes cell wall properties to maintain air between cells. Moreover, we establish the functional importance of inter-cellular air channels in the hypocotyl for phototropism.

plant biology↗

bioRxiv2022

Regulation and Function of a Polarly Localized Lignin Barrier in the Exodermis

Multicellular organisms control interactions with their environment through the development of specialized barriers in specific cell types. A conserved barrier in plant roots is the endodermal Casparian strip (CS). The CS is made of polymerized lignin and forms a ring-like structure that seals the apoplastic space between the endodermal cells. Most angiosperms also have another root cell type, the exodermis, that is reported to form a barrier. Our understanding of exodermal developmental and molecular regulation, as well as function, is limited as this cell type is absent from the model species Arabidopsis thaliana. Using tomato (Solanum lycopersicum) as a model system we demonstrate that in this species, the exodermis does not form a CS. Instead, it forms a polar lignin cap with an equivalent barrier function to the endodermal CS. We demonstrate that although endodermal regulators are conserved between Arabidopsis and tomato, exodermal differentiation occurs by a distinct regulatory pathway involving the SlSCZ and SlEXO1 transcription factors. Although the exodermis and endodermis both produce barriers that restrict mineral ion uptake, they have unique and overlapping roles in their selectivity. Whether conservation and similarities between the endodermis and exodermis exist in other species remains to be determined. Nonetheless, in tomato, these distinct lignin structures have a convergent function with different genetic regulations.

plant biology↗

bioRxiv2022

A suberized exodermis is required for tomato drought tolerance

Plant roots integrate environmental signals and developmental programs using exquisite spatiotemporal control. This is apparent in the deposition of suberin, an apoplastic diffusion barrier, which regulates the entry and exit of water, solutes and gases, and is environmentally plastic. Suberin is considered a hallmark of endodermal differentiation, but we find that it is absent in the tomato endodermis during normal development. Instead, suberin is present in the exodermis, a cell type that is absent in the model organism Arabidopsis thaliana. Here, we uncover genes driving exodermal suberization and describe its effects on drought responses in tomato, unravelling the similarities and differences with the paradigmatic Arabidopsis endodermis. Cellular resolution imaging, gene expression, and mutant analyses reveal loss of this program from the endodermis, and its co-option in the exodermis. Functional genetic analyses of the tomato MYB92 transcription factor and ASFT enzyme demonstrate the importance of exodermal suberin for a plant water-deficit response. Controlling the degree of exodermal suberization could be a new strategy for breeding climate-resilient plants.

plant biology↗

bioRxiv2022

Directed growth and fusion of membrane-wall microdomains requires CASP-mediated inhibition and displacement of secretory foci

Casparian strips (CS), the main extracellular diffusion barrier in plant roots, are precisely localized cell wall lignin-impregnations, contrasting animal tight-junctions. The CS membrane domain (CSD) proteins 1-5 (CASP1-5) define and accumulate at the CS associated membrane domains displaying matrix adhesion and protein exclusion. A full CASP knock-out (caspQ) now reveals that CASPs are not needed for localization of lignification or lignin-polymerizing enzymes, since correctly aligned spots still form in the mutant. Ultra-structurally, however, these spots appear as highly disorganized secretory foci, with neither exclusion zone nor membrane attachment and excessive cell wall growth. Biotin proximity labelling identifies RabA-GTPases as potential CASP-interactors. We confirm their localisation and function at the CSD, similar to exocyst subunits, known Rab effectors. Our work reveals that CASPs enforce displacement of initial secretory foci through exclusion of vesicle tethering factors, thereby ensuring rapid fusion of microdomains and effective sealing of the cell wall space.

plant biology↗

bioRxiv2021

Extracellular membrane tubules involved in suberin deposition in plant cell walls

Suberin is a fundamental plant biopolymer, found in protective tissues, such as seed coats, exodermis and endodermis of roots, the outer layers of stems and roots with secondary growth, as well as in wound-induced tissues. Its presence allows organs to resist various environmental stresses, such as pathogen attack, drought or excessive salt concentrations. Suberin is a mostly aliphatic polyester of long-chain fatty acids and alcohols, often co-occurring with lignin-like polymers in the same cells. Most suberizing cells appear to deposit suberin in the form of lamellae just outside of the plasma membrane, below the primary cell wall. The monomeric precursors of suberin are thought to be glycerated fatty acids, synthesized at the endoplasmic reticulum. However, it has remained obscure how these monomers are transported outside of the cell, where they will be polymerized to form suberin lamellae. Here, we demonstrate that extracellular vesicular-tubular structures accumulate specifically in suberizing cells. By employing various, independent mutational and hormonal challenges, known to affect suberization in distinct ways, we demonstrate that their presence correlates perfectly with root suberization. Surprisingly, no endosomal compartment marker showed any conspicuous changes upon induction of suberization, suggesting that this compartment might not derive from endosomal multi-vesicular bodies, but possibly form directly from endoplasmic reticulum subdomains. Consistent with this, we could block formation of both, suberin deposition and vesicle accumulation by a pharmacogenetic manipulation affecting early steps in the secretory pathway. Whereas many previous reports have described extracellular vesicle occurrence in the context of biotic interactions, our results suggest a developmental role for extracellular vesicles in suberin formation. One Sentence SummarySuberin lamellae formation is associated with extracellular membrane tubules.

plant biology↗

bioRxiv2020

CASP microdomain formation requires cross cell wall stabilization of domains between neighbors and non-cell autonomous action of LOTR1 cell wall protein

Efficient uptake of nutrients in both animal and plant cells requires tissue-spanning diffusion barriers separating inner tissues from the outer lumen/soil. However, we poorly understand how such contiguous three-dimensional superstructures are formed in plants. Here, we show that correct establishment of the plant Casparian Strip (CS) network relies on local neighbor communication. We show that positioning of Casparian Strip membrane domains (CSDs) is tightly coordinated between neighbors in wild-type and that restriction of domain formation involves the putative extracellular protease LOTR1. Impaired domain restriction in lotr1 leads to fully functional CSDs at ectopic positions, forming "half strips" LOTR1 action in the endodermis requires its expression in the stele. LOTR1 endodermal expression cannot complement, while cortex expression causes a dominant-negative phenotype. Our findings establish LOTR1 as a crucial player in CSD positioning acting in a directional, non-cell-autonomous manner to restrict and coordinate CS positioning.

plant biology↗

bioRxiv2020

GDSL-domain containing proteins mediate suberin biosynthesis and degradation, enabling developmental plasticity of the endodermis during lateral root emergence

ABSTRACTRoots anchor plants and deliver water and nutrients from the soil. The root endodermis provides the crucial extracellular diffusion barrier by setting up a supracellular network of lignified cell walls, called Casparian strips, supported by a subsequent formation of suberin lamellae. Whereas lignification is thought to be irreversible, formation of suberin lamellae was demonstrated to be dynamic, facilitating adaptation to different soil conditions. Plants shape their root system through the regulated formation of lateral roots emerging from within the endodermis, requiring local breaking and re-sealing of the endodermal diffusion barriers. Here, we show that differentiated endodermal cells have a distinct auxin-mediated transcriptional response that regulates cell wall remodelling. Based on this data set we identify a set of GDSL-lipases that are essential for suberin formation. Moreover, we find that another set of GDSL-lipases mediates suberin degradation, which enables the developmental plasticity of the endodermis required for normal lateral root emergence.Competing Interest StatementThe authors have declared no competing interest.View Full Text

plant biology↗

bioRxiv2020

High-order mutants reveal an essential requirement for peroxidases but not laccases in Casparian strip lignification

The invention of lignin has been at the heart of plants capacity to colonize land, allowing them to grow tall, transport water within their bodies and protect themselves against various stresses. Consequently, this polyphenolic polymer, that impregnates the cellulosic plant cell walls, now represents the second most abundant polymer on Earth, after cellulose itself. Yet, despite its great physiological, ecological and economical importance, our knowledge of lignin biosynthesis in vivo, especially the crucial last steps of polymerization within the cell wall, remains vague. Specifically, the respective roles and importance of the two main polymerizing enzymes classes, laccases and peroxidases have remained obscure. One reason for this lies in the very high numbers of laccases and peroxidases encoded by 17 and 73 homologous genes, respectively, in the Arabidopsis genome. Here, we have focused on a specific lignin structure, the ring-like Casparian strips (CS) within the endodermis of Arabidopsis roots. By reducing the number of possible candidate genes using cellular resolution expression and localization data and by boosting the levels of mutants that can be stacked using CRISPR/Cas9, we were able to knock-out more than half of all laccases in the Arabidopsis genome in a nonuple mutant - abolishing the vast majority of laccases with detectable endodermal-expression. Yet, we were unable to detect even slight defects in CS formation. By contrast, we were able to induce a complete absence of CS formation in a quintuple peroxidase mutant. Our findings are in stark contrast to the strong requirement of xylem vessels for laccase action and indicate that lignin in different cell types can be polymerized in very distinct ways. We speculate that cells lignify differently depending on whether they deposit lignin in a localized or ubiquitous fashion, whether they stay alive during and after lignification as well as the composition of the cell wall.

plant biology↗

bioRxiv2020

Phosphorylation by Aurora B kinase regulates caspase-2 activity and function

Mitotic catastrophe (MC) is an important oncosuppressive mechanism that serves to eliminate cells that become polyploid or aneuploidy due to aberrant mitosis. Previous studies have demonstrated that the activation and catalytic function of caspase-2 are key steps in MC to trigger apoptosis and/or cell cycle arrest of mitotically defective cells. However, the molecular mechanisms that regulate caspase-2 activation and its function are unclear. Here we identify six new phosphorylation sites in caspase-2 and show that a key mitotic kinase, Aurora B kinase (AURKB), phosphorylates caspase-2 at the highly conserved residue S384. We demonstrate that phosphorylation at S384 blocks caspase-2 catalytic activity and apoptosis function in response to mitotic insults, without affecting caspase-2 dimerisation. Moreover, molecular modelling suggests that phosphorylation at S384 may affect substrate binding by caspase-2. We propose that caspase-2 S384 phosphorylation by AURKB is a key mechanism that controls caspase-2 activation during mitosis.

cell biology↗