Biology subjects

Beeckman, T.

Publications and source records attributed to Beeckman, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Structure-activity relationship study in soil identifies an effective broad-spectrum inhibitor of archaeal nitrification

Ammonia-oxidizing archaea (AOA) can contribute substantially to nitrification in agricultural soils, yet the portfolio of AOA-targeting nitrification inhibitors (NIs) with demonstrated efficacy under soil conditions remains limited. Moreover, the performance of candidate inhibitors in soil is often difficult to predict from pure-culture assays because their activity is strongly influenced by soil-dependent factors. In this study, we employed a recently developed high-throughput miniaturized soil incubation assay to perform a soil-based structure-activity relationship (SAR) analysis of SIAS (Specific Inhibitor of AOA in Soil), a pyrazolo-pyridine derivative previously identified as an AOA-targeting compound with strong additive nitrification inhibition effects when combined with the AOB-targeting inhibitor DMP (3,4-dimethyl-1H-pyrazole). To identify structural features associated with improved and more robust soil efficacy, a library of 27 SIAS-derived pyrazolo-pyridine analogues was evaluated directly in soil. The SAR analysis showed that inhibitory activity was governed by a specific combination of structural features and led to the discovery of Optimized SIAS (Opt. SIAS), a simplified analogue that consistently outperformed the original lead compound across diverse agricultural soils. Correlation analysis further revealed that soil pH and, particularly, silt content were major determinants of Opt. SIAS efficacy. The strong additive effects obtained through combined application of Opt. SIAS and DMP highlight the contribution of AOA to residual nitrification following AOB inhibition and demonstrate the potential of simultaneously targeting both groups. Together, these results expand the limited portfolio of effective AOA-targeting nitrification inhibitors and provide a foundation for the development of next-generation nitrification inhibition strategies aimed at improving nitrogen-use efficiency and reducing nitrogen losses from agricultural soils.

microbiology↗

bioRxiv2025

Systemic and local regulation of root growth by vascular trehalose 6-phosphate is correlated with re-allocation of primary metabolites between shoots and roots

Trehalose 6-phoshpate (Tre6P) is a key signalling molecule that reflects carbon status and integrates it with developmental decision making. Tre6P has been demonstrated to regulate various developmental processes, including vegetative growth, shoot branching, flowering, and root branching. Here, we investigate how vasculature-derived Tre6P influences root system architecture by expressing heterologous Tre6P synthase (TPS) or Tre6P phosphatase (TPP) specifically in the plant vasculature. Plants with elevated vascular-derived Tre6P levels had smaller root systems, reduced sucrose levels, and lower root metabolite levels, whereas vascular TPP overexpression had the opposite effect. Using reciprocal grafting experiments, we identified shoot-derived vascular Tre6P to be the main driver of these systemic responses. In lines with increased Tre6P in the shoot vasculature, the shoot-to-root metabolite ratios were consistently increased, indicating that Tre6P modulates metabolite allocation and utilization to balance carbon partitioning between shoot and root growth. Our data further suggest that vascular Tre6P contributed to optimizing the carbon-to-nitrogen ratio to support growth and fitness. Besides this systemic function, this study shows that root-derived Tre6P also plays a critical local role in modulating root development. Collectively, our results demonstrate that Tre6P in the vasculature functions both systemically and locally to coordinate metabolic status with root growth and architecture.

plant biology↗

bioRxiv2025

Conserved transcriptional reprogramming in nematode infected root cells

Plant-parasitic nematodes are responsible for important annual losses in crop productivity worldwide1,2. Although the formation of feeding organs within the roots is essential for successful sedentary parasitism3, the molecular mechanisms underlying their development are poorly understood. This is partly because these organs originate from a limited number of root cells4-7, making difficult to capture the transcriptional reprogramming that occurs during the early stages of the infection. Here, we first developed a comparative host-pathogen framework to study the nematode infection process in Arabidopsis and rice. Using a cross-species single-cell transcriptomics approach, we identified a unique molecular signature in infected root cells and show that the cellular reprogramming during these early stages is highly conserved across both host-pathogen interactions. This transcriptional cell reprogramming is associated with stemness acquisition related to de novo organogenesis process. By cell-type specific gene regulatory network analysis, we identified AtATHB2/OsHOX28 as an evolutionary conserved and key regulator of the nematode infection process. Loss-of-function of this regulator in both species results in nematode resistance without affecting root growth. This discovery opens up new avenues for the development of sustainable nematode control strategies that could be translated across crop species.

plant biology↗

bioRxiv2025

Unified nonparametric analysis of single-molecule spatial omics data using probabilistic indices

Spatial omics technologies localize individual molecules at subcellular resolution, shedding light on the spatial micro-organisation of living organisms. Yet the development of analysis methods struggles to keep pace with growing numbers of molecules, features and replicates being measured, and with new scientific questions arising on single molecules localization patterns. To meet this need, we present smoppix, a nonparametric analysis method based on the probabilistic index, which unifies tests for several univariate and bivariate localization patterns, such as aggregation of transcripts or colocalization of transcript pairs, in a single framework. These tests can be performed across tissues as well as within cells, while accounting for nested design structures. The high-dimensionality of the data is exploited for variance weighting and for providing a meaningful background null distribution, unique for every individual molecule. smoppix sidesteps segmentation, warping, edge correction and density estimation, and scales to high numbers of molecules and replicates thanks to an exact permutation null distribution. We demonstrate its power by unearthing spatial patterns in four published datasets from different kingdoms, and validate some findings experimentally on Selaginella moellendorfii roots. Our method is available from Bioconductor as the R-package smoppix.

bioinformatics↗

bioRxiv2024

Cell wall-mediated maternal control of apical-basal patterning of the kelp Undaria pinnatifida

The role of maternal tissue in the control of embryogenesis remains enigmatic in many complex organisms. Here, we investigate the contribution of maternal tissue to apical-basal patterning in the kelp embryo. Using a modified kelp fertilisation protocol which yields synchronously developing kelp embryos, we show that detachment from maternal tissue leads to compromised robustness of apical-basal patterning. Detached embryos are rounder and often show aberrant morphologies. Furthermore, absence of contact with maternal tissue increases parthenogenesis, highlighting the critical role of maternal signals in the initial stages of kelp development. When zygotes are detached from the female gametophyte while part of the oogonial cell wall still remains attached to the egg, the proper apical-basal patterning is rescued showing a key role for the connection to the maternal cell wall in developmental patterning in kelps. This observation is reminiscent of another brown alga, Fucus, where the cell wall has been shown to play a key role in cell fate determination. In the case of kelps, the maternal oogonium mediates basal cell fate determination by providing an extrinsic patterning cue in its extracellular matrix to the future embryo. Our findings suggest a conserved mechanism across phylogenetically distant oogamous brown algal lineages, where localised secretion of sulphated F2 fucans mediate establishment of the apical-basal polarity.

developmental biology↗

bioRxiv2024

New high-throughput screening assays for nitrification inhibitors identify an oxazolidine-thione as biological nitrification inhibitor

Nitrification is a microbial two-step process whereby ammonia (NH3) is first oxidized into nitrite (NO2-), by ammonia-oxidizing microorganisms, and then into nitrate (NO3-) by nitrite-oxidizing bacteria (NOB) in a mutualistic symbiosis. Due to high nitrogen (N) input in agriculture, this process needs to be slowed down as NO2- and NO3- easily leach, becoming unavailable for plants and leading to eutrophication. Moreover, they may be further processed into the very strong greenhouse gas nitrous oxide (N2O). Nitrification thus results in inefficient use of fertilizer and leads to detrimental environmental impact. Inhibitors can be used to mitigate these negative effects and are shown to positively affect plant growth and crop yield and strongly reduce nitrogen (N) pollution. However, currently, only a limited portfolio of nitrification inhibitors is commonly available. These show a variable efficiency and come with logistic complexities. As such, there is a high demand for a broader portfolio of novel molecules. To contribute to the discovery phase of new inhibitors and enable the evaluation of a large number of molecules, we developed miniaturized high-throughput screening assays on the soil-borne ammonia-oxidizing bacteria (AOB) Nitrosomonas europaea and Nitrosospira multiformis. We give a detailed overview of the procedure and illustrate its use by testing structural variants of oxazolidine-thiones that were previously discovered as nitrification inhibitors. This led to the discovery of the natural compound goitrin as a new biological nitrification inhibitor (BNI). Overall, the presented assays are promising discovery tools, as demonstrated by the newly discovered BNI, and may contribute to the advancement of a more sustainable agriculture.

microbiology↗

bioRxiv2023

The selective estrogen receptor modulator clomiphene inhibits sterol biosynthesis in Arabidopsis thaliana

Sterols are produced via complex, multistep biosynthetic pathways involving similar enzymatic conversions in plants, animals and fungi, yielding a variety of sterol metabolites with slightly different chemical properties to exert diverse and specific functions. The role of plant sterols has been studied in the context of cell biological processes, signaling and overall plant development, mainly based on mutants. Due to their essential nature, genetic interference with their function causes pleiotropic developmental defects. An important alternative is to use a pharmacological approach. However, the current toolset for manipulating sterol biosynthesis in plants remains limited. Here, we probed a collection of inhibitors of mammalian cholesterol biosynthesis to identify new inhibitors of plant sterol biosynthesis. We provide evidence that imidazole-type fungicides, bifonazole, clotrimazole and econazole inhibit the obtusifoliol 14-demethylase CYP51, that is highly conserved among eukaryotes. Surprisingly, we found that the selective estrogen receptor modulator, clomiphene, inhibits sterol biosynthesis, in part by inhibiting the plant-specific cyclopropyl-cycloisomerase CPI1. These results demonstrate that rescreening of the animal sterol biosynthesis pharmacology is an easy approach for identifying novel inhibitors of plant sterol biosynthesis. Such molecules can be used as entry points for the development of plant-specific inhibitors of sterol biosynthesis that can be used in agriculture.

plant biology↗

bioRxiv2023

Structure-function analysis of a calcium-independent metacaspase reveals a novel proteolytic pathway for lateral root emergence

Metacaspases are part of an evolutionarily broad family of multifunctional cysteine proteases, involved in disease and normal development. Despite the extensive study of metacaspases in the two decades since their discovery, the structure-function relationship of metacaspases remains poorly understood. Furthermore, previous studies on their function have been thwarted by the redundancy in gene copy number and potential phenotypic suppression of genetic mutations, especially in plants. Here, we have solved the X-ray crystal structure of an Arabidopsis thaliana type II metacaspase (AtMCA-IIf) that belongs to a particular sub-group that does not require calcium ions for activation. Compared to crystal structures of other metacaspases and caspases, the AtMCA-IIf active site is structurally similar and poses a conundrum for the catalytic mechanism of the cysteine-histidine dyad. To study metacaspase activity in plants, we developed an in vitro chemical screen to identify small molecule metacaspase inhibitors. Several hits with a minimal thioxodihydropyrimidine-dione (TDP) structure were identified, some being specific inhibitors of AtMCA-IIf. We provide a mechanistic basis for inhibition by the TDP-containing compounds through molecular docking onto the AtMCA-IIf crystal structure. Finally, a TDP-containing compound (TDP6) was effective at inhibiting lateral root emergence in vivo, likely through the inhibition of metacaspases that are specifically expressed in the endodermal cells overlaying developing lateral root primordia. In the future, the small compound inhibitors and crystal structure of AtMCA-IIf can be used to study metacaspases in various other species, such as important human pathogens including those causing neglected diseases.

plant biology↗

bioRxiv2022

GOLVEN peptides regulate lateral root spacing as part of a negative feedback loop on the establishment of auxin maxima

The initiation of lateral roots in Arabidopsis requires the accumulation of auxin in lateral root founder cells, yielding a local auxin maximum. The positioning of these auxin maxima along the primary root determines the density and spacing of lateral roots. The GOLVEN6 (GLV6) and GLV10 signaling peptides and their receptors have been established as regulators of lateral root spacing via an inhibitory effect on lateral root initiation. However, it remained unclear how these GLV peptides interfere with auxin signaling or homeostasis. Here we show that GLV6/10 signaling regulates the expression of a subset of auxin response genes during lateral root initiation, downstream of the canonical auxin signaling pathway, while simultaneously inhibiting the establishment of auxin maxima in xylem-pole pericycle cells. We present genetic evidence that this inhibitory effect relies on the activity of the PIN3 and PIN7 auxin export proteins. Furthermore, GLV6/10 peptide signaling was found to enhance PIN7 abundance in the plasma membrane of xylem-pole pericycle cells, which likely stimulates auxin efflux from these cells. Based on these findings, we propose a model in which the GLV6/10 signaling pathway serves as a negative feedback loop that contributes to the robust patterning of auxin maxima along the primary root.

plant biology↗

bioRxiv2022

Phase separation-based visualization of protein-protein interactions and kinase activities in plants

Protein activities depend heavily on protein complex formation and dynamic post-translational modifications, such as phosphorylation. Their dynamic nature is notoriously difficult to monitor in planta at cellular resolution, often requiring extensive optimization and high-end microscopy. Here, we generated and exploited the SYnthetic Multivalency in PLants (SYMPL)-vector set to study protein-protein interactions (PPIs) and kinase activities in planta based on phase separation. This technology enabled easy detection of inducible, binary and ternary protein-protein interactions among cytoplasmic, nuclear and plasma membrane proteins in plant cells via a robust image-based readout. Moreover, we applied the SYMPL toolbox to develop an in vivo reporter for SnRK1 kinase activity, allowing us to visualize tissue-specific, dynamic SnRK1 activation upon energy deprivation in stable transgenic Arabidopsis plants. The applications of the SYMPL cloning toolbox lay the foundation for the exploration of PPIs, phosphorylation and other post-translational modifications with unprecedented ease and sensitivity.

plant biology↗

bioRxiv2022

Cellular and gene expression patterns associated with root bifurcation in Selaginella

The roots of lycophytes branch through dichotomy or bifurcation, which means that the root apex splits into two daughter roots. This is morphologically distinct from lateral root (LR) branching in the extant euphyllophytes, where LRs develop along the root axis at different distances from the apex. The process of root bifurcation is poorly understood, while such knowledge can be important, as it may represent an evolutionarily ancient strategy that roots recruited to form new stem cells or meristems. In this study, we examined root bifurcation in the lycophyte Selaginella moellendorffii. We characterized an in vitro developmental time-frame based on repetitive apex bifurcations, allowing us to sample different stages of dichotomous root branching and analyze the root meristem and root branching in S. moellendorffii at the microscopical and transcriptional level. Our results show that, in contrast to previous assumptions, initial cells in the root meristem are mostly not tetrahedral but rather show an irregular shape. Tracking down the early stages during root branching argues for the occurrence of a symmetric division of the single initial cell resulting in two apical stem cells allowing for root meristem bifurcation. Moreover, we generated a S. moellendorffii root branching transcriptome, which resulted in the delineation of a subset of core meristem genes. The occurrence of multiple meristem-related orthologues in this dataset, including inversely correlated expression profiles of a SCARECROW (SCR) versus a RETINOBLASTOMA-RELATED1 (RBR1) homologue suggests the presence of conserved pathways in the control of meristem and root stem cell establishment or maintenance. One-sentence summaryThe root of the spike moss Selaginella moellendorffii bifurcates following a symmetric cell division of the single stem cell and involves conserved genetic modules known from angiosperm roots.

plant biology↗

bioRxiv2021

ABA represses TOR and root meristem activity through nuclear exit of the SnRK1 kinase

The phytohormone abscisic acid (ABA) promotes plant tolerance to major stresses like drought, partly by modulating plant growth. However, the underlying mechanisms are poorly understood. Here, we show that cell proliferation in the Arabidopsis thaliana root meristem is controlled by the interplay between three kinases, SNF1-RELATED KINASE 2 (SnRK2), the main driver of ABA signaling, the SnRK1 energy sensor, and the growth-promoting TARGET OF RAPAMYCIN (TOR) kinase. Under favorable conditions, the SnRK11 catalytic subunit is enriched in the nuclei of root cells and this is accompanied by normal cell proliferation and meristem size. Depletion of SnRK2s in a snrk2.2 snrk2.3 double mutant causes constitutive cytoplasmic localization of SnRK11 and a reduction in meristem size, suggesting that, under non-stress conditions, SnRK2s enable growth by retaining SnRK11 in the nucleus. In response to elevated ABA levels, SnRK11 translocates to the cytoplasm and this is accompanied by inhibition of TOR, decreased cell proliferation and meristem size. Blocking nuclear export with leptomycin B abrogates ABA-driven SnRK11 relocalization to the cytoplasm and the inhibition of TOR. Fusion of SnRK11 to an SV40 nuclear localization signal leads to defective TOR repression in response to ABA, demonstrating that SnRK11 nuclear exit is a premise for this repression. Altogether, we demonstrate that SnRK2-dependent changes in SnRK11 subcellular localization are crucial for the regulation of TOR activity and root growth in response to ABA. Such swift relocalization of key regulators may represent a more general strategy of sessile organisms like plants to rapidly respond to environmental changes.

plant biology↗

bioRxiv2021

The pH-dependent processivity of Arabidopsis AtPME2 can control cell wall mechanical properties

Pectin methylesterases (PMEs) modify homogalacturonans (HG) chemistry and play a key role in regulating primary cell wall mechanical properties. How PME activity can fine-tune pectin structure in the growing plant has remained elusive. Here we report on the Arabidopsis AtPME2, which we found to be highly expressed during lateral root emergence and dark-grown hypocotyl elongation. We produced the mature active enzyme using heterologous expression in Pichia pastoris and characterized it through the use of a generic plant PME antiserum suitable for detecting recombinant and native enzyme independent of species source. At neutral pH AtPME2 is preferentially active on pectins with a degree of 55-70% methylesterification and can be inhibited by PME inhibitor protein (PMEI). We show that the mode of action for AtPME2 can switch from full processivity (at pH 8), creating large blocks of unmethylated galacturonic acid, to low processivity (at pH 5) and relate these observations to the differences in electrostatic potential of the protein at acidic and alkaline pH. To assess the role of AtPME2 in development, we characterized two knock-out lines. We show that in the context of acidified apoplast, low-processive demethylesterification by AtPME2 can loosen the cell wall, with consequent increase in cell elongation and etiolated hypocotyl length. Our study brings insights into how the pH-dependent regulation by PME activity could affect pectin structure and associated cell wall mechanical properties in expansion. One sentence summaryThe processivity of AtPME2, a pectin methylesterase that fine-tunes cell wall pectins is modulated by pH in vitro and impacts the mechanical properties of the wall, affecting development in planta.

plant biology↗

bioRxiv2021

Auxin-triggered changes in the Arabidopsis root tip (phospho)proteome reveal novel root growth regulators

Auxin plays a dual role in growth regulation and, depending on the tissue and concentration of the hormone, it can either promote or inhibit division and expansion processes in plants. Recent studies revealed that, beyond transcriptional reprogramming, alternative auxin-controlled mechanisms regulate root growth. Here, we explored the impact of different auxin concentrations on the root tip proteome and phosphoproteome, generating a unique resource. From the phosphoproteome data we pinpointed (novel) growth regulators, such as the RALF34-THE1 module. Our results together with previously published studies suggest that auxin, H+-ATPases, cell wall modifications and cell wall sensing receptor-like kinases are tightly embedded in a pathway regulating cell elongation. Furthermore, our study assigned a novel role to MKK2 as a regulator of primary root growth and a (potential) regulator of auxin biosynthesis and signalling, and suggests the importance of the MKK2 Thr31 phosphorylation site for growth regulation in the Arabidopsis root tip. ONE SENTENCE SUMMARYAn auxin-triggered Arabidopsis root tip (phospho)proteome reveals novel root growth regulators

plant biology↗

bioRxiv2021

Phellem translational landscape throughout secondary development in Arabidopsis roots

O_LIThe phellem is a specialized boundary tissue providing the first line of defense against abiotic and biotic stresses in organs undergoing secondary growth. Phellem cells undergo several differentiation steps, which include cell wall suberization, cell expansion and programmed cell death. Yet, the molecular players acting particularly in phellem cell differentiation remain poorly described, particularly in the widely used model plant Arabidopsis thaliana. C_LIO_LIUsing specific marker lines we followed the onset and progression of phellem differentiation in A. thaliana roots, and further targeted the translatome of new developed phellem cells using Translating Ribosome Affinity Purification followed by mRNA sequencing (TRAP-SEQ). C_LIO_LIWe showed that phellem suberization is initiated early after phellogen (cork cambium) division. The specific translational landscape was organized in three main domains related to energy production, synthesis and transport of cell wall components, and response to stimulus. Novel players in phellem differentiation, related to suberin monomer transport and assembly, as well as novel transcription regulators were identified. C_LIO_LIThis strategy provided an unprecedented resolution of the transcriptome of developing phellem cells, giving a detailed and specific view on the molecular mechanisms controlling cell differentiation in periderm tissues of the model plant Arabidopsis. C_LI Significance statementTo improve the understanding of phellem differentiation into a suberized protective layer, we followed the establishment of periderm in Arabidopsis roots and sequenced the phellem-specific translatome. We found that phellem suberization occurs shortly after pericycle cell divisions with the induction of pivotal suberin biosynthesis genes. In parallel, we detected the activation of three central genetic modules acting throughout the phellem differentiation. This study provides a unique and targeted genetic resource for further functional studies of phellem tissues.

plant biology↗

bioRxiv2020

Pharmacological and genetic manipulations of Ca2+ signaling have contrasting effects on auxin-regulated trafficking

A large part of a plants developmental plasticity relies on the activities of the phytohormone auxin and the regulation of its own distribution. This process involves a cohort of transcriptional and non-transcriptional effects of auxin on polar auxin transport, regulating the abundancy, biochemical activity and polar localization of the molecular components, predominantly PIN auxin exporters. While the transcriptional auxin signaling cascade has been well characterized, the mechanism and role of non-transcriptional auxin signaling remains largely elusive. Here, we addressed the potential involvement of auxin-induced Ca2+ signaling in auxins inhibitory effect on PIN endocytic trafficking. On the one hand, exogenous manipulations of Ca2+ availability and signaling effectively antagonized auxin effects suggesting that auxin-induced Ca2+ signaling is required for inhibition of internalization. On the other hand, we addressed the auxin-mediated inhibition of PIN internalization in the auxin signaling (tir1afb2,3) or Ca2+ channel (cngc14) mutants. These mutants were strongly defective in auxin-triggered Ca2+ signaling, but not in auxin-inhibited internalization. These data imply that, while Ca2+ signaling may be required for normal PIN trafficking, auxin-mediated increase in Ca2+ signaling is not a direct part of a downstream mechanism that mediates auxin effects on Brefeldin A-visualized PIN intercellular aggregation. These contrasting results obtained by comparing the mutant analysis versus the exogenous manipulations of Ca2+ availability and signaling illustrate the critical importance of genetics to unravel the role of Ca2+ in a process of interest.

cell biology↗

bioRxiv2020

ABCB-mediated auxin transport in outer root tissues regulates lateral root spacing in Arabidopsis

Root branching is an important strategy to explore efficiently large volumes of soil. To economize this process, lateral roots (LR) are formed along the growing root at discrete positions that are instructed by oscillating auxin signals derived from the lateral root cap (LRC). This assumes that auxin moves from the LRC across multiple layers to accumulate in the pericycle. Here, we identified, using gene silencing and CRISPR based approaches, a group of five genetically linked, closely related ABCBs that control LR spacing by modulating the amplitude of the auxin oscillation. The transporters localize to the plasma membrane and reveal significant auxin export activity. These ABCBs are mainly expressed in the LRC and epidermis where they contribute to auxin transport towards the root oscillation zone. Our findings highlight the importance of auxin transport in the outer tissues of the root meristem to regulate LR spacing.

plant biology↗

bioRxiv2019

The use of mutants and inhibitors to study sterol biosynthesis in plants

Sterols are very well known for their important roles in membranes and signaling in eukaryotes. Plants stand out among eukaryotes by the large variety of sterols that they can produce, and employing them across a wide spectrum of physiological processes. Therefore, it is critical to understand the wiring of the biosynthetic pathways by which plants generate these distinct sterols, to allow manipulating them and dissect their precise physiological roles. Many enzymatic steps show a deep evolutionary conservation, while others are executed by completely different enzymes. Here, we review the complexity and variation of the biosynthetic routes of the most abundant phytosterols in the green lineage and how different enzymes in these pathways are conserved and diverged from humans,yeast and even bacteria. Based on their evolutionary conservation we discuss the use of human and yeast sterol biosynthesis inhibitors in plants, as an argument for the development of plant-tailored inhibitors of sterol biosynthesis.

plant biology↗