Biology subjects

Nowack, M. K.

Publications and source records attributed to Nowack, M. K..

14 recordsLinked to original sources

bioRxiv2025

Plant cells at the organ surface use mechanical cues to activate a specific growth control programme

During morphogenesis of multicellular organs, cells acquire distinct identities that meet specific functional requirements. Epidermal identity is widely considered essential for plant morphogenesis due to the role of the epidermis in both restricting and promoting growth. In the root, epidermal cells are partially covered by a protective root cap, and partially positioned at the organ surface. Here, we propose that epidermal cells at the organ surface have unique requirements for growth control due to high mechanical tension, while covered epidermal cells are mechanically shielded by the root cap. We present in silico and in vivo evidence that plants use surface mechanical cues to activate a cell-type specific growth control programme involving the small GTPase RAB-A5c, allowing them to maintain directional growth at the organ surface. Positional mechanical cues may thus be used to control expression of a sub-population of epidermal genes, linking gene regulation to surface-specific functional requirements.

plant biology↗

bioRxiv2025

Root hair lifespan is antagonistically controlled by autophagy and programmed cell death

Root hairs are tubular tip-growing extensions of root epidermal cells that enhance root surface area for water and nutrient uptake. While mechanisms governing root hair fate, polarity, and tip growth are well understood, the regulation of root hair longevity remains largely unknown. Here, we show that root hair cells employ high levels of autophagy to promote their lifespan. Loss-of-function mutations in the autophagy regulators ATG2, ATG5, or ATG7 induce a premature, cell-autonomous cell death program. This cell death is activated via a gene regulatory network downstream of the NAC transcription factors ANAC046 and ANAC087. Our findings uncover an antagonistic relationship between autophagy and developmentally controlled cell death in root hair lifespan regulation, with potential implications for optimizing plant nutrient and water uptake in crop breeding. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/643910v1_ufig1.gif" ALT="Figure 1"> View larger version (80K): org.highwire.dtl.DTLVardef@1bd570borg.highwire.dtl.DTLVardef@19db5eborg.highwire.dtl.DTLVardef@11279fborg.highwire.dtl.DTLVardef@661c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2024

Programmed cell death and stomatal density regulate anther opening in response to ambient humidity

Anther dehiscence is the process that facilitates pollen release from mature anthers in flowering plants. Despite its crucial importance to reproduction, the underlying molecular regulations and the integration of environmental information remain poorly understood. Using controlled humidity treatments of Arabidopsis thaliana flowers, we show here that high humidity prevents anthers from opening. Lower and higher stomatal densities correlate with slower and faster dehiscence dynamics, respectively, suggesting that controlled transpiration regulates anther opening. Furthermore, analyses of subcellular markers revealed that specific anther tissues undergo a spatially controlled programmed cell death (PCD) process as anthers open. Notably, genetic inhibition of PCD networks delays, whereas precocious PCD induction promotes, anther dehiscence. Our data shed new light on the interplay between ambient humidity, controlled transpiration, and PCD processes in regulating timely pollen release in the model plant Arabidopsis.

plant biology↗

bioRxiv2024

Shedding light on plant proteolysis: genetically encoded fluorescent sensors as tools for profiling protease activities

Proteolysis, a ubiquitous process in living organisms, is driven by proteases that regulate numerous signaling pathways through the hydrolysis of peptide bonds in protein substrates. Understanding the temporal and spatial dynamics of proteolysis and the activation of proteases is crucial for elucidating their roles in biological pathways. Here, we introduce a suite of genetically encoded FRET reporters designed to detect various proteolytic activities in plants. These sensors effectively reported in planta the specific activity of both Tobacco Etch Virus protease and caspase-3. Furthermore, we developed sensors for detecting plant metacaspase activity, validated through both in vitro and in planta experiments. These experiments revealed the spatial dynamics of proteolysis triggered by metacaspase activation following wounding and programmed cell death in roots. The implementation of these tools in plant biology research opens new avenues for investigating proteolytic mechanisms, significantly enhancing the potential for in-depth studies. Our work demonstrates the feasibility of using these sensors to detect diverse protease activities in vivo with high spatiotemporal resolution. These plant proteolytic biosensors hence represent a valuable toolbox for understanding protease functions within their natural context, paving the way for future advancements in plant biology research.

plant biology↗

bioRxiv2023

SMB controls decompartmentalization in Arabidopsis root cap cells to execute programmed cell death

Programmed cell death (PCD) is a fundamental cellular process crucial to development, homeostasis, and immunity in multicellular eukaryotes. In contrast to our knowledge on the regulation of diverse animal cell death subroutines, information on execution of PCD in plants remains fragmentary. Here we make use of the accessibility of the Arabidopsis thaliana root cap to visualize the execution process of developmentally controlled PCD. We identify a succession of selective decompartmentalization events and ion fluxes that are controlled by a gene regulatory network downstream of the NAC transcription factor SOMBRERO (SMB). Surprisingly, breakdown of the large central vacuole is a relatively late and variable event, preceded by an increase of intracellular calcium levels and acidification, release of mitochondrial matrix proteins, leakage of nuclear and endoplasmic reticulum lumina, and release of fluorescent membrane reporters into the cytosol. Elevated intracellular calcium levels and acidification are sufficient to trigger cell death execution specifically in cells that are rendered competent to undergo PCD by SMB activity, suggesting that these ion fluxes act as PCD-triggering signals.

plant biology↗

bioRxiv2023

Seed Longevity is Controlled by Metacaspases

To survive extreme desiccation, seeds enter dormancy that can last millennia. This dormancy involves the accumulation of protective but structurally disordered storage proteins through unknown adjustments of proteolytic surveillance mechanisms. Mutation of all six types II metacaspases (MCAs)-II in the model plant Arabidopsis revealed their essential role in modulating these proteolytic mechanisms. MCA-II mutant seeds fail to properly target at the endoplasmic reticulum (ER) the AAA ATPase Cell Division Cycle 48 (CDC48) to dispose of misfolded proteins. MCA-IIs cleave a CDC48 adaptor, the ubiquitination regulatory X domain-containing (PUX) responsible for localizing CDC48 to the lipid droplets. When cleaved, CDC48-PUX is inactivated and allows a lipid droplet-to-ER shuttling of CDC48, an important step in the regulation of seeds lifespan. In sum, we uncover antagonism between proteolytic pathways bestowing longevity. One-Sentence SummaryMetacaspase proteases confer seed longevity by antagonizing CDC48 activity.

plant biology↗

bioRxiv2023

Root cap cell corpse clearance limits microbial colonization

Programmed cell death occurring during plant development (dPCD) is a fundamental process integral for plant growth and reproduction. Here, we investigate the connection between developmentally controlled PCD and fungal accommodation in Arabidopsis thaliana roots, focusing on the root cap-specific transcription factor ANAC033/SOMBRERO (SMB) and the senescence-associated nuclease BFN1. Mutations of both dPCD regulators increase colonization by the beneficial fungus Serendipita indica, primarily in the differentiation zone. smb-3 mutants additionally exhibit hypercolonization around the meristematic zone and a delay of S. indica-induced root-growth promotion. This demonstrates that root cap dPCD and rapid post-mortem clearance of cellular corpses represent a physical defense mechanism restricting microbial invasion of the root. Additionally, reporter lines and transcriptional analysis revealed that BFN1 expression is downregulated during S. indica colonization in mature root epidermal cells, suggesting a transcriptional control mechanism that facilitates the accommodation of beneficial microbes in the roots. Key findings in bullet pointsO_LIThe process of programmed cell death in root development (dPCD) influences the extent and outcomes of fungal symbiosis C_LIO_LIFungal colonization of the root tip and differentiation zone is restricted by SMB-mediated clearance of dead cells, which preserves the meristem and regulates symbiosis C_LIO_LIExpression of plant nuclease BFN1, which is associated with senescence, is modulated to facilitate root accommodation of beneficial microbes C_LI

plant biology↗

bioRxiv2022

Autophagy promotes programmed cell death and corpse clearance in specific cell types of the Arabidopsis root cap

Autophagy is a conserved quality control pathway that mediates the degradation of unnecessary or dysfunctional cellular components by targeting them to the lysosomes or central vacuoles. Autophagy has been implicated in the regulation or execution of regulated cell death processes in a wide range of eukaryotes. However, its function in developmentally controlled programmed cell death (dPCD) in plants remains little studied and controversial. Here, we investigated the role of autophagy in dPCD using the Arabidopsis root cap as an accessible and genetically tractable model system. We show that autophagic flux is induced prior to dPCD execution in both root cap tissues, the columella and the lateral root cap (LRC), and impaired in autophagy-deficient mutants. These mutants show a strongly delayed cell death and an absence of corpse clearance in the columella during and after their shedding into the rhizosphere. However, autophagy deficiency does not affect dPCD execution or corpse clearance in LRC cells at the distal end of the root cap. Our results demonstrate that autophagy promotes dPCD in a highly cell-type specific manner, and present the root cap as a powerful model system to study organ-specific autophagy in vivo.

plant biology↗

bioRxiv2022

Spatial and Temporal Regulation of Parent-of-Origin Allelic Expression in the Endosperm

Genomic imprinting promotes differential expression of parental alleles in the endosperm of flowering plants, and is regulated by epigenetic modification such as DNA methylation and histone tail modifications in chromatin. After fertilization, the endosperm develops through a syncytial stage before it cellularizes and becomes a nutrient source for the growing embryo. Both in early and late endosperm development regional compartmentalization has been shown, and different transcriptional domains suggest divergent spatial and temporal regional functions. The analysis of the role of parent-of-origin allelic expression in the endosperm as a whole and also investigation of domain specific functions has been hampered by the availability of the tissue for high-throughput transcriptome analyses and contamination from surrounding tissue. Here we have used Fluorescence-Activated Nuclear Sorting (FANS) of nuclear targeted eGFP fluorescent genetic markers to capture parental specific allelic expression from different developmental stages and specific endosperm domains. This RNASeq approach allows us to successfully identify differential genomic imprinting with temporal and spatial resolution. In a systematic approach we report temporal regulation of imprinted genes in the endosperm as well as region specific imprinting in endosperm domains. Our data identifies loci that are spatially differentially imprinted in one domain of the endosperm while biparentally expressed in other domains. This suggests that regulation of genomic imprinting is dynamic and challenges the canonical mechanisms for genomic imprinting.

genetics↗

bioRxiv2022

Evolutionary PTEN Gene Divergence Underpins the Remodeling of Plant Vacuolar Compartments

Membrane fusion and fission are fundamental processes in sustaining cellular compartmentalization. Fission of a lipid bilayer requires a furrow formation that brings membranes in close proximity prior to a contiguous membrane cleavage. Although plant ancestors abandoned cleavage furrow-mediated cytokinesis more than 500 million years ago, here we show that plants still employ this mechanical principle to divide embryonic vacuoles. The evolutionary divergence in PHOSPHATASE AND TENSIN HOMOLOG DELETED ON CHROMOSOME TEN (PTEN) enzymes was required to coordinate this process, as Arabidopsis loss-of-function pten2a pten2b mutants contain hyper compartmentalized embryonic vacuoles. In contrast, PTEN2 overexpression hinders lytic and secretion cellular pathways downstream of TGN in xylem cells. These processes are critical for the formation of secondary cell walls in xylem cells and depend on a poorly characterized and evolutionarily novel N-terminal domain in PTEN2s. The PTEN2 subfamily appeared with the emergence of the Phragmoplastophyta clade, when vacuolar compartments enlarged and cleavage furrow-mediated cytokinesis became extinct. Together, our work suggests that the evolutionary innovation of the PTEN family is conserved across terrestrial plants and central to vacuolar remodelling.

plant biology↗

bioRxiv2021

GPI anchor remodeling by the plant PGAP1 ortholog HLD1 is essential for Papaver self-incompatibility

In eukaryotes, glycosylphosphatidylinositol anchored proteins (GPI-APs) are tethered to the outer leaflet of the plasma membrane where they function as key regulators of a plethora of biological processes. Self-incompatibility (SI) plays a pivotal role regulating fertilization in higher plants through recognition and rejection of self pollen. Here we used Arabidopsis thaliana lines engineered to be self-incompatible by expression of Papaver SI determinants for an SI suppressor screen. We identify HLD1, an ortholog of human GPI-inositol deacylase PGAP1, whose mutation completely abolishes the SI response. We show that HLD1 functions as a GPI-inositol deacylase and that this GPI-remodeling activity is essential for SI. Using GFP-SKU5 as a representative GPI-AP, we show that HLD1 mutation does not affect GPI-AP production and targeting, but alters the configuration of mature GPI-APs. This prevents GPI-AP release from the plasma membrane, suggesting that this process plays a critical role in the regulation of SI. Our data not only identify GPI anchoring as a new pathway of SI providing new directions to investigate SI mechanisms, but identifies for the first time a function for GPI-AP remodeling by inositol deacylation in plants. One sentence summaryThe Papaver self-incompatibility response requires GPI-anchor modification by HLD1, an ortholog of the mammalian PGAP1.

plant biology↗

bioRxiv2020

Efficient simultaneous mutagenesis of multiple genes in specific plant tissues by multiplex CRISPR

Multiplex CRISPR approaches enable mutating multiple genes in plants, however it is unclear how feasible this is in tissue-specific mutagenesis. Here we simultaneously mutated six genes either ubiquitously or exclusively in the root cap of Arabidopsis. The mutation frequencies for all target genes were positively correlated and unaffected by the order of gRNAs in the vector, indicating that efficient higher-order mutagenesis in specific plant tissues can be readily achieved.

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↗

bioRxiv2018

CRISPR-TSKO facilitates efficient cell type-, tissue-, or organ-specific mutagenesis in Arabidopsis

Detailed functional analyses of many fundamentally-important plant genes via conventional loss-of-function approaches are impeded by severe pleiotropic phenotypes. In particular, mutations in genes that are required for basic cellular functions and/or reproduction often interfere with the generation of homozygous mutant plants, precluding further functional studies. To overcome this limitation, we devised a CRISPR-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis, CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The underlying modular cloning system allows for efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens new avenues to discover and analyze gene functions in spatial and temporal contexts of plant life while avoiding pleiotropic effects of system-wide loss of gene function.

plant biology↗