Biology subjects

Geldner, N.

Publications and source records attributed to Geldner, N..

At least 19 recordsLinked to original sources

bioRxiv2026

Genome-wide interrogation of genetic requirements for root colonization by CRISPRi-seq in Bacillus subtilis

Bacillus subtilis is a known plant growth promoting rhizobacterium, yet, the genetic basis of effective root colonization remains understudied. Here, we applied genome-wide CRISPR interference sequencing (CRISPRi-seq) to systematically identify genes required for early Arabidopsis thaliana root colonization by the B. subtilis strain 3610 . Our genome-wide inducible CRISPRi library was used to screen for bacterial fitness during hydroponic root colonization. A complementary RNA-seq in the same conditions further revealed a distinct transcriptional state of root-associated cells. We identified 249 genes impacting root colonization. We reveal a critical role of cell envelope remodeling during early root association, specifically wall teichoic acid synthesis and D-alanylation. Moreover, we observed a metabolic shift toward TCA-cycle-driven aerobic growth with uptake of fructose, active repression of sporulation, competence and prophage programs, as well as induction of secondary metabolite biosynthesis. Our work establishes CRISPRi-seq as a powerful tool for dissecting bacterial-plant interactions relevant to sustainable agriculture.

microbiology↗

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↗

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↗

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↗

bioRxiv2022

A peptide-mediated, multilateral molecular dialogue for the coordination of pollen wall formation

The surface of pollen grains is reinforced by pollen wall components produced non-cell autonomously by tapetum cells that surround developing pollen within the male floral organ, the anther. Here we show that tapetum activity is regulated by the GASSHO (GSO) receptor-like kinase pathway, controlled by two sulfated peptides, CASPARIAN STRIP INTEGRITY FACTOR 3 (CIF3) and CIF4, the precursors of which are expressed in the tapetum itself. Coordination of tapetum activity with pollen grain development depends on the action of subtilases, including AtSBT5.4, which are produced stage-specifically by developing pollen grains. Tapetum-derived CIF precursors are processed by subtilases, triggering GSO-dependent tapetum activation. We show that the GSO receptors act from the middle layer, a tissue surrounding the tapetum and developing pollen. Three concentrically organized cell types therefore cooperate to coordinate pollen wall deposition through a multilateral molecular dialog. Significance StatementPollen viability depends on a tough external barrier called the pollen wall. Pollen wall components are produced by tapetum cells which surround developing pollen grains within the anther. Precise coordination of tapetum activity with pollen grain development is required to ensure effective pollen wall formation. Here we reveal that this is achieved through a multidirectional dialogue involving three distinct cell types. We show that peptide precursors from the tapetum are activated by proteases produced stage-specifically in developing pollen grains. Unexpectedly, we found that activated peptides are perceived not in the tapetum, but in the middle layer, which encloses the developing tapetum and pollen grains, revealing an unsuspected role for this enigmatic cell layer in the control of tapetum development.

plant biology↗

bioRxiv2021

Combined fluorescent seed selection and multiplex CRISPR/Cas9 assembly for fast generation of multiple Arabidopsis mutants

Multiplex CRISPR-Cas9-based genome editing is an efficient method for targeted disruption of gene function in plants. Use of CRISPR-Cas9 has increased rapidly in recent years and is becoming a routine method for generating single and higher order Arabidopsis mutants. To facilitate rapid and efficient use of CRISPR/Cas9 for Arabidopsis research, we developed a CRISPR/Cas9-based toolbox for generating large deletions at multiple genomic loci, using two-color fluorescent seed selection. In our system, up-to eight gRNAs can be routinely introduced into a binary vector carrying either FastRed, FastGreen or FastCyan fluorescent seed selection cassette. Both, FastRed and FastGreen binary vectors, can be co-transformed as a cocktail via floral dip to introduce sixteen gRNAs at the same time. The seeds can be screened either for red or green fluorescence, or for the presence of both colors at the same time. Our approach provides fast and flexible cloning, avoids very big constructs and enables screening different order mutants in the same generation. Importantly, in the second generation after transformation, Cas9 free plants are identified simply by screening the dark, non-fluorescent seeds. Our collection of binary vectors allows to choose between two widely-used promoters to drive Cas enzymes, either the egg cell-specific (pEC1.2) or ubiquitous promoter (PcUBi4-2). Available enzymes are "classical" Cas9, a recently reported, intron-optimized version or Cpf1 (Cas12a). Finally, we have taken care to introduce convenient restriction sites flanking promoter, Cas9 and fluorescent selection cassette in the final T-DNA vectors, thus allowing straightforward swapping of all three elements for further adaptation and improvement of the system.

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

Schengen-pathway controls spatially separated and chemically distinct lignin deposition in the endodermis.

Lignin is a complex polymer precisely deposited in the cell wall of specialised plant cells, where it provides essential cellular functions. Plants coordinate timing, location, abundance and composition of lignin deposition in response to endogenous and exogenous cues. In roots, a fine band of lignin, the Casparian strip encircles endodermal cells. This forms an extracellular barrier to solutes and water and plays a critical role in maintaining nutrient homeostasis. A signalling pathway senses the integrity of this diffusion barrier and can induce over-lignification to compensate for barrier defects. Here, we report that activation of this endodermal sensing mechanism triggers a transcriptional reprogramming strongly inducing the phenylpropanoid pathway and immune signaling. This leads to deposition of compensatory lignin that is chemically distinct from Casparian strip lignin. We also report that a complete loss of endodermal lignification drastically impacts mineral nutrients homeostasis and plant growth.

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

Spatially Restricted Immune Responses Allow for Root Meristematic Activity During Bacterial Colonisation

Plants circumscribe microbe-associated molecular pattern (MAMP)-triggered immune responses to weak points of the roots. This spatially restricted immunity was suggested to avoid constitutive responses to rhizosphere microbiota. To demonstrate its relevance, we combined cell-type specific expression of the plant flagellin receptor (FLS2) with fluorescent defence markers and mapped immune competency at cellular resolution. Our analysis distinguishes cell-autonomous and non-cell autonomous responses and reveals lignification to be tissue-independent, contrasting cell-type specific suberisation. Importantly, our analysis divides the non-responsive meristem into a central zone refractory to FLS2 expression, and a cortex that becomes highly sensitised by FLS2 expression, causing meristem collapse upon MAMP exposure. Meristematic epidermal expression generates super-competent lines that detect native bacterial flagellin and bypass the absence of response to commensals, providing a powerful tool for studying root immunity. Our precise manipulations and read-outs demonstrate incompatibility of meristematic activity and defence and the importance of cell-resolved studies of plant immunity.

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

Tissue-autonomous phenylpropanoid production is essential for establishment of root barriers

Plants deposit polymeric barriers in their root cell walls to protect against external stress and facilitate selective nutrient uptake. The compounds that make up these barriers originate from the fatty acid- and phenylpropanoid biosynthetic pathways. Although the machinery responsible for production of the barrier constituents is well-char-acterized, our pathway models lack spatiotemporal resolution - especially in roots - and the source tissue is often not clear due to the apoplastic nature of barriers. Insights into how the individual root tissues or cells contribute to forming apoplastic barriers is important for elucidation of their ultrastructure, function and development. Manipulation of the associated biosynthesis is delicate, as mutants often display pleiotropic phenotypes due to the broad role of the underlying metabolites. Here, we address these issues by creating a genetic tool that allows in vivo repression of the phenylpropanoid pathway with both spatial and temporal control. We provide strong evidence that tissue-auton-omous production of phenylpropanoids is essential for establishment of the endodermal Casparian strip. Moreover, we find that in order to maintain deposition and attachment of a coherent suberin matrix to the cell wall, cells require continuous production of aromatic constituents. This process is especially crucial in the suberized endodermis where we find that repression of phenylpropanoid production leads to active removal of suberin.

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↗

bioRxiv2019

ROS production by localized SCHENGEN receptor module drives lignification at subcellular precision

Production of reactive-oxygen species (ROS) by NADPH oxidases (NOXs) impacts many processes in animals and plants and many plant receptor pathways involve rapid, NOX-dependent increases of ROS. Yet, their general reactivity has made it challenging to pinpoint the precise role and direct cellular targets of ROS. A well-understood ROS target in plants are lignin peroxidases in the cell wall. Lignin can be deposited with exquisite spatial control, but the underlying mechanisms have remained elusive. Here we establish a full kinase signaling relay that exerts direct, spatial control over ROS production and lignification within the cell wall. We show that polar localization of a single kinase component is crucial for pathway function. Our data indicates that an intersection of more broadly localized components allows for micrometer-scale precision of lignification and that this system is triggered through initiation of ROS production as a critical peroxidase co-substrate.

plant biology↗

bioRxiv2019

A two-way molecular dialogue between embryo and endosperm required for seed development

The plant embryonic cuticle is a hydrophobic barrier deposited de novo by the embryo during seed development. At germination it protects the seedling from water loss and is thus critical for survival. Embryonic cuticle formation is controlled by a signaling pathway involving the protease ALE1 and the receptor-like kinases GSO1 and GSO2. We show that a sulfated peptide, TWISTED SEED1 (TWS1) acts as a GSO1/GSO2 ligand. Cuticle surveillance depends on the action of ALE1 which, unlike TWS1 and GSO1/2, is not produced in the embryo but in the neighboring endosperm. Cleavage of an embryo-derived TWS1 precursor by ALE1 releases the active peptide, triggering GSO1/2-dependent cuticle reinforcement in the embryo. A bidirectional molecular dialogue between embryo and endosperm thus safeguards cuticle integrity prior to germination.\n\nOne Sentence SummarySubtilase-mediated activation of the TWISTED SEED1 peptide provides spatial cues during embryo cuticle integrity monitoring.

plant biology↗