Biology subjects

Kaufmann, K.

Publications and source records attributed to Kaufmann, K..

At least 19 recordsLinked to original sources

bioRxiv2026

SHANK3-CAMSAP2 interaction links synapses to dendritic microtubule organisation in PV neurons

The microtubule cytoskeleton plays an essential role in establishing and maintaining neuronal polarity. In neurons, microtubules are initially generated at the centrosome which gradually loses this role in development. In mature neurons, microtubules are stabilised by the microtubule minus end-binding protein CAMSAP2. How and where microtubule minus ends are anchored within dendrites of mammalian neurons remains an open question. Here, we show that microtubules are directly attached to the postsynaptic density of excitatory synapses through an interaction of CAMSAP2 with the scaffolding protein SHANK3. This process is particularly relevant in parvalbumin-positive GABAergic neurons, in which excitatory synapses are predominantly located directly on the dendritic shaft in direct proximity to microtubules. Notably, this association is strongly enhanced by the Autism Spectrum Disorder (ASD)-associated SHANK3L68P mutation, leading to an increase of synaptic levels of CAMSAP2. This promotes an increased microtubule association with the synapse and alters microtubule dynamics. Downregulation of CAMSAP2 in SHANK3L68P parvalbumin neurons tips the balance between site-specific microtubule stabilisation and dynamics, reshaping dendritic architecture, connecting SHANK3-CAMSAP2 dependent microtubule regulation to synaptic ASD pathology. TeaserInteraction between synapses and microtubules is enhanced in parvalbumin neurons bearing the ASD-associated SHANK3 L68P mutation.

neuroscience↗

bioRxiv2025

Ca2+ signature-dependent control of auxin sensitivity in Arabidopsis

Plants must continually balance growth with arrest, especially under stress. Auxin signaling acts as a central regulatory hub in this process, yet the mechanisms that dynamically tune early auxin signalling in real time remain unknown. Here, we used the light-gated, Ca2+-permeable Channelrhodopsin 2 variant XXM2.0 to optogenetically impose defined Ca2+ signatures on Arabidopsis root cells. Repetitive light activation triggered cytosolic Ca2+ signals that in turn suppressed auxin-induced membrane depolarization and Ca2+ transients. Moreover, prolonged optogenetic Ca2+ stimulation affects auxin-responsive transcriptional reprogramming. As phenotypic output, reversible inhibition of root growth by suppressing cell division and elongation was observed. We further identify a candidate CaM7-CNGC14 module that likely mediates Ca2+-dependent gating of early auxin signalling. Our study thus introduces a synthetic biology approach to decompose calcium-auxin crosstalk in plant cells, and demonstrates that optogenetically imposed cytosolic Ca2+ signals act as dynamic regulators of auxin susceptibility in roots.

plant biology↗

bioRxiv2025

The role of FRUITFULL controlling cell cycle during early flower development revealed by time series snRNA-seq experiments.

BACKGROUNDStarting from pools of undifferentiated cells, plants generate new organs post-embryonically in response to external and endogenous signals. This requires a dynamic coordination of cell division with cellular growth and differentiation regulatory programs. However, little is known how this coordination is achieved at the molecular level during flower development. RESULTSWe used time-series single-nucleus RNA sequencing (snRNA-seq) experiments of synchronized Arabidopsis thaliana flower developmental stages to characterize the transcriptome dynamics and the connections between cell cycle and developmental regulatory programs during early flower development. The results show a bifurcation between transcriptional trajectories corresponding to cell cycle progression and floral development. We identify the regulation of the cell cycle inhibitor KIP-RELATED PROTEIN 2 (KRP2) by FRUITFULL (FUL) as a key regulatory point on this bifurcation point, and validate the importance of this regulation in vivo. CONCLUSIONSOur work illustrates how time-series snRNA-seq experiments can be used to identify bifurcation points between regulatory programs and to identify candidate regulators on these bifurcations. In particular, we identify the regulation of KRP2 by FUL as an important regulatory point to balance cell division and developmental differentiation in plants.

plant biology↗

bioRxiv2025

Cellular dynamics and epidermal specialization during Arabidopsis floral development at single-cell resolution

Cellular specialization underlies the functional diversification of floral organs, yet the regulatory networks driving epidermal differentiation remain poorly understood. Here, we constructed a single-nucleus transcriptomic atlas of developing Arabidopsis thaliana flowers by profiling over 70,000 nuclei. For annotation of single-cell identities and differentiation status, we additionally generated tissue- and stage-specific transcriptomic datasets using nine fluorescent marker lines spanning four floral developmental stages. Guided by this integrative approach, we focused specifically on the floral epidermis at single-cell resolution and resolved 22 transcriptionally distinct epidermal cell populations. Gene regulatory network analyses identified key transcription factors essential for epidermal differentiation, highlighting MYB16 as a central regulator within the petal epidermis. Developmental trajectory and chromatin-binding analyses demonstrated that MYB16 dynamically orchestrates gene expression programs involved in epidermal identity establishment, cuticle biosynthesis, and stress resilience. Moreover, we demonstrate MYB16 interacts with DRMY1, a regulator of organ growth robustness. Complementation assays confirmed the non-redundant role of MYB16 in epidermal cell fate specification, distinct from other MYB transcription factors. This study provides a comprehensive cellular and regulatory framework for floral epidermal specialization, advancing our understanding of how temporal transcriptional dynamics integrate with spatial cell fate decisions during flower development.

molecular biology↗

bioRxiv2025

ARABIDOPSIS Bsister and SEEDSTICK MADS-box transcription factors modulate maternal nutrient flow for seed development in Arabidopsis

Successful seed development in angiosperms depends on the coordinated transport and allocation of sugars from maternal tissues to the developing embryo and endosperm. In Arabidopsis thaliana, ovules function as carbohydrate sink organs, accumulating starch in both gametophytic and sporophytic domains prior to fertilization. This stored starch is later mobilized to support early embryogenesis. Despite extensive knowledge of starch metabolism in photosynthetic tissues, the regulatory mechanisms governing sugar transport in reproductive organs remain poorly understood. Recent studies have identified fertilization-dependent changes in nutrient flow, including callose-mediated modulation of symplastic transport at the phloem unloading site. However, the molecular players orchestrating these transitions are largely unknown. Here, we show that the MIKC MADS domain transcription factors ABS/TT16 and STK play critical roles in regulating maternal nutrient flow during ovule maturation and seed development. We dissect their functional redundancy using omics and genetic approaches, underscoring the importance of different ovule tissues in coordinating sugar transport pathways for post-fertilization development. Our findings reveal a previously underappreciated layer of genetic control over nutrient allocation in reproductive tissues and provide new insights into the metabolic reprogramming required for successful seed formation.

plant biology↗

bioRxiv2025

SPOROCYTELESS/NOZZLE acts together with MADS-domain transcription factors to regulate an auxin-dependent network controlling the Megaspore Mother Cell development

The formation of the female gamete is a complex developmental process that begins with the differentiation of the Megaspore Mother Cell (MMC) within the ovule. SPOROCYTELESS/NOZZLE (SPL/NZZ) is the principal regulator of the MMC formation, as mutations in the SPL/NZZ gene lead to the failure of the MMC differentiation. Nonetheless, the SPL/NZZ-dependent regulatory pathway governing the MMC development remains largely unknown. Using a multi-omics approach, we identify direct SPL/NZZ targets and their downstream network. We discovered that SPL/NZZ interacts with ovule-identity MADS-domain transcription factor complexes, to regulate the expression of common target genes. By integrating the omics data with the analysis of either complementation or mutant lines, we describe a comprehensive regulatory mechanism, in which SPL/NZZ determines the differentiation of the MMC by acting on an auxin-dependent downstream network.

plant biology↗

bioRxiv2024

Integration of the DD-genome reshapes gene transcription, chromatin architecture and metabolome of allohexaploid wheat leading to enhanced adaptability

The integration, through hybridization, of the DD genome into domesticated tetraploid wheat gave rise to allohexaploid wheat, the most cultivated wheat globally growing across diverse environmental conditions. However, the regulatory basis of this integration on increased environmental adaptability in allohexaploid remains largely unexplored. Here, we investigated the change of transcriptome, epigenome as well as the chromatin interactome, and metabolome in three independent polyploidization events representing DD genome integration. Our findings reveal that polyploidization events induce the activation of defense-related genes through comprehensive reorganization of epigenome and chromatin architecture. DD integration not only brings an additional gene copy but also activates the homoeologs existing in the A and B subgenomes through chromatin interactions. Furthermore, secondary metabolites represented by alkaloids and flavonoids that are crucial for environmental adaptation, are significantly enriched following polyploidization. Thus, hexaploid wheat exhibits enhanced tolerance to alkalinity, UV-B light stress and high salt conditions was observed. These results highlight the indispensable role of DD genome integration in the adaptability of allohexaploid wheat during its evolution.

plant biology↗

bioRxiv2024

Cold stress induces a rapid redistribution of the antagonistic marks H3K4me3 and H3K27me3 in Arabidopsis thaliana

When exposed to low temperatures, plants undergo a drastic reprogramming of their transcriptome in order to adapt to their new environmental conditions, which primes them for potential freezing temperatures. While the involvement of transcription factors in this process, termed cold acclimation, has been deeply investigated, the potential contribution of chromatin regulation remains largely unclear. A large proportion of cold-inducible genes carries the repressive mark histone 3 lysine 27 trimethylation (H3K27me3), which has been hypothesized as maintaining them in a silenced state in the absence of stress, but which would need to be removed or counteracted upon stress perception. However, the fate of H3K27me3 during cold exposure has not been studied genome-wide. In this study, we offer an epigenome profiling of H3K27me3 and its antagonistic active mark H3K4me3 during short-term cold exposure. Both chromatin marks undergo rapid redistribution upon cold exposure, however, the gene sets undergoing H3K4me3 or H3K27me3 differential methylation are distinct, refuting the simplistic idea that gene activation relies on a switch from an H3K27me3 repressed chromatin to an active form enriched in H3K4me3. Coupling the ChIP-seq experiments with transcriptome profiling reveals that differential histone methylation correlates with changes in expression. Interestingly, only a subset of cold-regulated genes lose H3K27me3 during their induction, indicating that H3K27me3 is not an obstacle to transcriptional activation. In the H3K27me3 methyltransferase curly leaf (clf) mutant, many cold regulated genes display reduced H3K27me3 levels but their transcriptional activity is not altered prior or during a cold exposure, suggesting that H3K27me3 may serve a more intricate role in the cold response than simply repressing the cold-inducible genes in naive conditions.

plant biology↗

bioRxiv2023

Natural genetic variation in GLK1-mediated photosynthetic acclimation in response to light

GOLDEN-like (GLK) transcription factors are central regulators of chloroplast biogenesis in Arabidopsis and other species. Findings from Arabidopsis show that these factors also contribute to photosynthetic acclimation, e.g. to variation in light intensity, and are controlled by retrograde signals emanating from the chloroplast. However, the natural variation of GLK1-centered gene-regulatory networks is largely unexplored. By evaluating the activities of GLK1 target genes and GLK1 itself in vegetative leaves of natural Arabidopsis accessions grown under standard conditions, we uncovered a remarkable variation in the activity of GLK1 centered regulatory networks. This is linked with ecogeographic origin of the accessions, and can be associated with a complex genetic variation across loci acting in different functional pathways, including photosynthesis, ROS and brassinosteroid pathways. Our results identify candidate upstream regulators that contribute to GLK1 activity in rosette leaves. Indeed, accessions with higher GLK1 activity, arising from habitats with a high monthly variation in solar radiation levels, may show lower levels of photoinhibition at higher light intensities. Our results provide evidence for natural variation in GLK1 regulatory activities in vegetative leaves. This variation is associated with ecogeographic origin and can contribute to acclimation to high light conditions.

plant biology↗

bioRxiv2023

MatK impacts Differential Chloroplast Translation of Ribosomal and Photosynthetic genes by limiting spliced tRNA-K(UUU) abundance

The protein levels of chloroplast photosynthetic genes and genes related to the chloroplast genetic apparatus vary to adapt to different conditions. However, the underlying mechanisms governing these variations remain unclear. The chloroplast intron Maturase K is encoded within the trnK intron and has been suggested to be required for splicing several group IIA introns, including the trnK intron. In this study, we employed RNA immunoprecipitation followed by high-throughput sequencing (RIP-Seq) to identify MatKs preference for binding to group IIA intron domains I and VI within target transcripts. Importantly, these domains are crucial for branch point selection, and we discovered alternative branch points in three MatK target introns, the first observed instances of alternative splicing in chloroplasts. The alternative trnK lariat structure showed increased accumulation during heat acclimation. The cognate codon of tRNA-K(UUU) is highly enriched in mRNAs encoding ribosomal proteins and ribosome profiling in a trnK-matK over-expressor exhibited elevated levels of the spliced tRNA-K(UUU). Our analysis revealed a significant up-shift in the translation of ribosomal proteins compared to photosynthetic genes. Our findings suggest the existence of a novel regulatory mechanism linked to the abundance of tRNA-K(UUU), enabling the differential expression of functional chloroplast gene groups.

plant biology↗

bioRxiv2023

SEPALLATA-driven MADS transcription factor tetramerization is required for inner whorl floral organ development

MADS genes encode transcription factors that act as master regulators of plant reproduction and flower development. The SEPALLATA (SEP) subfamily is required for the development of floral organs and plays roles in inflorescence architecture and development of the floral meristem. The SEPALLTAs act as organizers of MADS complexes, forming both heterodimers and heterotetramers in vitro. To date, the MADS complexes characterized in angiosperm floral organ development contain at least one SEPALLATA protein. Whether DNA-binding by SEPALLATA-containing dimeric MADS complexes are sufficient for launching floral organ identity programs, however, is not clear as only defects in floral meristem determinacy were observed in tetramerization impaired SEPALLATA mutants. Here, we used a combination of genome-wide binding studies, high resolution structural studies of the SEP3/AGAMOUS tetramerization domain, structure-based mutagenesis and complementation experiments in sep1 sep2 sep3 and sep1 sep2 sep3 ag-4 plants transformed with versions of SEP3 encoding tetramerization mutants. We demonstrate that while SEP3 heterodimers are able to bind DNA both in vitro and in vivo and recognize the majority of SEP3 wild type binding sites genome-wide, tetramerization is not only required for floral meristem determinacy, but also absolutely required for floral organ identity in the second, third and fourth whorls.

plant biology↗

bioRxiv2023

Strigolactones optimise plant water usage by modulating vessel formation

Wood formation is fundamental for the remarkable growth of plant bodies by continuously providing cells for long-distance transport of water and nutrients1-3. Vessel elements, the water transporting units within woody tissues, are generated from a cylindrical domain of cambium stem cells (CSCs) producing different vascular cell types in a bidirectional manner4-6. However, knowledge on the regulation of CSC activity is limited with unclear cell fate trajectories as the most obscure aspect in this context. Here, via revealing transcriptome signatures of CSCs and their derivatives with single cell resolution in Arabidopsis thaliana, we discover that the strigolactone (SL) signalling pathway modulates cell type composition in vascular tissues and thereby increases drought resistance. In particular, we find that SL signalling negatively regulates vessel element formation and thereby plant water usage. SL signalling is generally associated with differentiating vascular tissues but low in developing vessels and in CSCs implying a local role during fate decisions in CSC-derived cells. Highlighting the importance of vascular tissue composition for the overall plant water balance, alteration of vessel element formation has a direct impact on transpiration rates through leaf stomata. Our results demonstrate the importance of structural alignment of water transporting tissues to unstable water regimes and provide perspectives for a long-term modulation of drought resistance in plants.

plant biology↗

bioRxiv2023

Arabidopsis thaliana BBX14 is a target of GLK1 and involved in high-light acclimation, photomorphogenesis and GUN-type retrograde signaling

Development of photosynthetically competent seedlings requires both light and retrograde biogenic signaling pathways. The transcription factor GLK1 functions at the interface between these pathways, and receives input from the biogenic-signaling integrator GUN1. BBX14 was previously identified, together with GLK1, in a core module that mediates the response to high light levels and biogenic signaling. To gain insight into the function of BBX14, we generated BBX14 overexpressors and CRISPR/Cas-mediated bbx14 mutant plants, conducted high-light, RT-qPCR and ChIP-Seq experiments, measured photosynthetic parameters, chlorophyll contents and growth rates, and analyzed alterations in transcriptomics. We found that, although overexpression of BBX14 is deleterious under normal growth conditions, BBX14 is needed to acclimate plants to high light stress. BBX14 is a direct target of GLK1, and RNA-Seq analysis suggests that BBX14 is involved in the circadian clock. Knockout of BBX14 results in a long-hypocotyl phenotype that depends on a retrograde signal, and BBX14 expression during biogenic signaling requires GUN1. Finally, we clarify the role of BBX14 in GUN-type biogenic signaling. We conclude that BBX14 is an integrator of photomorphogenetic and biogenic signals, and suggest that BBX14 is a nuclear target of retrograde signals downstream of the GUN1/GLK1 module.

plant biology↗

bioRxiv2022

A primary patient-derived model for investigating functional heterogeneity within the human Leukemic Stem Cell Compartment

The ability of leukemic stem cells (LSC) to evade therapy and fuel leukemic progression causing relapse impedes therapeutic success in acute myeloid leukemia (AML). The LSC pool within a patient sample is not homogenous but comprises distinct LSC subsets that vary in self-renewal and propagation properties. The stemness programs that underlie LSC types are poorly understood since human LSC studies require primary patient samples where LSC numbers are low and isolation methods impure. To overcome these challenges, we developed a patient-derived AML model system (OCI-AML22) displaying a functionally, transcriptionally and epigenetically defined cellular hierarchy driven by functional LSCs that can be immunophenotypically identified and isolated. Through single cell and functional approaches, the OCI-AML22 LSC fraction was found to contain distinct LSCs that vary in proliferative and differentiation properties. OCI-AML22 represents a valuable resource to decipher mechanisms driving stemness and the multiple layers of heterogeneity within LSCs.

cancer biology↗

bioRxiv2021

A 3D gene expression atlas of the floral meristem based on spatial reconstruction of single nucleus RNA sequencing data

Identity and functions of plant cells are influenced by their precise cellular location within the plant body. Cellular heterogeneity in growth and differentiation trajectories results in organ patterning. Therefore, assessing this heterogeneity at molecular scale is a major question in developmental biology. Single-cell transcriptomics (scRNA-seq) allows to characterize and quantify gene expression heterogeneity in developing organs at unprecedented resolution. However, the original physical location of the cell is lost during the scRNA-seq procedure. To recover the original location of cells is essential to link gene activity with cellular function and morphology. Here, we reconstruct genome-wide gene expression patterns of individual cells in a floral meristem by combining single-nuclei RNA-seq with 3D spatial reconstruction. By this, gene expression differences among meristematic domains giving rise to different tissue and organ types can be determined. As a proof of principle, the data are used to trace the initiation of vascular identity within the floral meristem. Our work demonstrates the power of spatially reconstructed single cell transcriptome atlases to understand plant morphogenesis. The floral meristem 3D gene expression atlas can be accessed at http://threed-flower-meristem.herokuapp.com

plant biology↗

bioRxiv2021

The Intervening Domain Is Required For DNA-binding and Functional Identity of Plant MADS Transcription Factors

The MADS transcription factors (TF) are an ancient protein family with a high degree of sequence identity that bind almost identical DNA sequences across all eukaryotic kingdoms of life, yet fulfill dramatically different physiological roles. In plants, the family is divided into two main lineages, type I and II, based on sequence conservation of the DNA-binding MADS-box domain (M domain) with yeast and animal M domains. Here, we demonstrate that DNA binding in both lineages absolutely requires a short amino acid sequence C-terminal to the M domain called the Intervening domain (I domain) in type II MADS. Structural elucidation of the MI domains from the floral regulator, SEPALLATA3 (SEP3), shows a highly conserved MADS-box fold with the I domain forming an alpha helix and acting to stabilize the M domain. Based on secondary structure prediction, sequences fulfilling the same function as the SEP3 I domain can be found in both lineages of plant MADS TFs, suggesting the I domain is a conserved and required part of the DNA-binding domain. Using the floral organ identity MADS TFs, SEP3, APETALA1 (AP1) and AGAMOUS (AG), domain swapping demonstrate that the I domain alters DNA-binding specificity based on seq-DAP-seq experiments. Yeast 2-hybrid experiments further revealed the role of the I domain in dimerization specificity. Surprisingly, introducing AG carrying the I domain of AP1 in the Arabidopsis ap1 mutant, resulted in a high degree of complementation and restoration of first and second whorl organs. Taken together, these data demonstrate that the I domain acts both as an integral part of the DNA-binding domain and strongly contributes to the functional identity of the MADS TF.

plant biology↗

bioRxiv2020

Single-nuclei RNA-sequencing of plants

Single-cell genomics provides unprecedented potential for research on plant development and environmental responses. Here, we introduce a generic procedure for plant nuclei isolation combined with nanowell-based library preparation. Our method enables the transcriptome analysis of thousands of individual plant nuclei. It serves as alternative to the use of protoplast isolation, which is currently a standard methodology for plant single-cell genomics, although it can be challenging for some plant tissues. We show the applicability of our nuclei isolation method by using different plant materials from several species. The potential of our snRNA-seq method is shown through the characterization of transcriptomes of seedlings and developing flowers from Arabidopsis thaliana. We evaluated the transcriptome dynamics during the early stages of anther development, identify stage-specific transcription factors regulating this process and the prediction of their target genes. Our nuclei isolation procedure can be applied in different plant species and tissues, thus expanding the toolkit for plant single-cell genomics experiments. SIGNIFICANCE STATEMENTWe introduce an optimized plant nuclei isolation procedure followed by single nuclei RNA-seq that can be applied to different plant tissues without the need for protoplast isolation.

genomics↗

bioRxiv2020

A functional screen for optimization of short hairpin RNA biogenesis and RISC loading

Gene silencing via short hairpin mediated RNAi (shRNA) is a valuable experimental tool and has promise as a therapeutic strategy. Several shRNA platforms make use of the loop and flanking sequences from the endogenous microRNA (miRNAs) miR-30a or other miRNAs to provide an RNA structure for efficient and accurate biogenesis of the RNA trigger. However, the stem regions of these shRNAs are typically designed as perfect duplex structures which is an uncommon feature for endogenous miRNA precursors. A limitation of these designs is that shRNAs with perfect duplex stems undergo extensive stem cleavage analogous to the Dicer independent miRNA miR-451, destroying the shRNA trigger sequence that is present in the 3P arm. We employed an unbiased screen of > 9000 shRNA structures to identify features that prevent stem cleavage and promote canonical biogenesis and loading into the effector complex RISC. We find that a central stem bulge or kink reduces central stem cleavage and improves accuracy of Dicer processing. Furthermore, 9 - 10 GC nucleotides in the guide strand improves shRNA efficiency. These design rules enable more effective shRNA tools and are compatible with existing sets of optimized guide/target sequences.

molecular biology↗