Biology subjects

Singh, G.

Publications and source records attributed to Singh, G..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Single-Cell Network Analysis Identifies CLEC4E as a Key Mediator of Proinflammatory mDC Responses in Influenza Infection

The severity of influenza is often driven by an excessive host immune response rather than the virus itself, yet the key molecular drivers within specific immune cells remain poorly understood. While recent single-cell RNA sequencing studies have successfully identified immune populations involved, they have largely not identified the upstream drivers modulating their pro-inflammatory functions. Here we employed an integrated single-cell co-expression network to address this gap. Our analysis identified myeloid dendritic cells (mDCs) as central to pro-inflammatory response during infection. Through a multi-layered key driver analysis, we pinpointed C-type lectin, CLEC4E as a top candidate modulating this pathological inflammatory response. The role of CLEC4E was confirmed in an independent single-cell dataset from influenza-infected patients and further validated in vivo. Pharmacological inhibition of CLEC4E in a murine influenza model significantly reduced disease severity and lower viral titers in the lungs. This study not only clarifies that CLEC4E overexpression in mDCs contributes to pro-inflammatory signaling pathways influencing influenza severity but also shows the power of single-cell network approaches to uncover novel and robust therapeutic targets hidden within complex immune responses.

systems biology↗

bioRxiv2025

Colitis-primed circulating T cells drive small intestinal barrier function through Nod2, microbiota and myosin light chain kinase-dependent mechanisms

Inflammatory bowel diseases (IBD), including Crohns disease (CD) and ulcerative colitis (UC), are characterized by intestinal inflammation and barrier dysfunction. While disruption of the intestinal barrier contributes to the pathogenesis of IBD, yet how colonic inflammation alters small intestinal homeostasis remains poorly defined. Here, we demonstrate that three models of colitis, including 2,4,6-Trinitrobenzenesulfonic acid (TNBS), Dextran Sulfate Sodium (DSS), and oxazolone, induce paracellular barrier dysfunction in the small intestine, with model-specific immune profiles. Th1/Th17-skewed responses (TNBS and DSS) were associated with microbiota-dependent upregulation of myosin light chain kinase (MLCK), ROR{gamma}t CD4 T cell expansion, and elevated expression of Nod2, which was not observed under Th2-dominant (oxazolone) conditions. Inhibition of MLCK restored barrier function and suppressed inflammation in a CD4 T cell-dependent manner. Using Nod2-deficient and 2939insC mutant mice, we showed Nod2 as a critical regulator of small intestinal permeability during colitis. Bone marrow chimeras revealed compartment-specific roles of Nod2, with non-hematopoietic Nod2 sufficient to preserve epithelial integrity, while hematopoietic Nod2 expression is required to limit cytokine-mediated inflammation. Moreover, MLCK inhibition ameliorated intestinal lesions only in mice with Nod2 deficiency in the hematopoietic compartment. Finally, microbiota transfer experiments ruled out a causal role for dysbiosis in driving small intestinal permeability defects in Nod2-deficient mice. These findings uncover a Nod2-MLCK-CD4 T cell axis linking colonic inflammation to small intestinal barrier dysfunction and highlight distinct immune-epithelial-microbial mechanisms shaping intestinal homeostasis during colitis. Graphical AbstractCirculating ROR{gamma}t Foxp3- CD4 T cells from mice with TNBS- or DSS-induced colitis increase small intestinal permeability by promoting cytokine secretion that enhances MLCK activity. This effect is attenuated by Nod2 activation. Specifically, Nod2 expression in hematopoietic cells reduces cytokine secretion and permeability, while activation of Nod2 in non-hematopoietic cells alone is sufficient to prevent increased permeability by directly regulating MLCK activity, independently of cytokine influence. Moreover, the absence of Nod2 is associated with inflammatory lesions in the small intestine of TNBS-treated mice.

immunology↗

bioRxiv2025

Vacuolar protein sorting-associated protein 1 (Vps1) has membrane constricting and severing abilities required for endosomal protein trafficking

Endosomal protein trafficking pathways are fundamental to maintaining organelle identity and function and are conserved from yeast to humans. The retromer pathway traffics cargo from endosomes to the trans Golgi network and defects in this pathway are linked to a variety of metabolic and neurological disorders. The yeast dynamin Vacuolar protein sorting-associated protein 1 (Vps1) functions in the retromer pathway. Vps1 has been found to associate with tubular endosomes undergoing severing, implying a role in membrane fission. Current biochemical and structural analyses of Vps1 however seem to indicate that it is not sufficient for fission. Here, we analyze Vps1 functions using cell-free reconstitution, live cell imaging and proteomics of isolated yeast vacuoles. We find that the endosomal lipid phosphatidylinositol-3-phosphate (PI(3)P) stabilizes Vps1 to form protein scaffolds around tubular membrane substrates. GTP hydrolysis forces constriction of tubules to a critical [~]7 nm radius, ultimately causing their fission. Furthermore, we discover that the Insert B (InsB) in Vps1 harbor conserved lysine- rich (3K) lipid-binding and phenylalanine-rich (4F) self-assembly motifs. Mutating these motifs renders Vps1 incapable of membrane fission and abrogates its functions in endosomal protein sorting. Quantitative vacuolar proteomic analysis reveals that loss of Vps1 results in the enrichment of a small set of proteins, most of which represent retromer cargo while causing depletion of the bulk of proteins in the vacuole. Vps1 functions in endosomal protein sorting are therefore critical for the global regulation of the vacuole proteome.

biochemistry↗

bioRxiv2025

Transient lung eosinophilia during breakthrough influenza infection in vaccinated mice is associated with protective and balanced Type 1/2 immune responses.

Eosinophils are agile cells that participate in a multitude of homeostatic and inflammatory responses in the lung, ranging from allergic asthma to antiviral defense against respiratory viral infection. In the context of vaccination followed by viral infection, such as breakthrough infection, eosinophils have been linked to aberrant Th2 responses like vaccine-enhanced respiratory disease (VAERD). Here, we demonstrate that the lung immune cell composition, cytokine and chemokine repertoire, histopathological profile, and systemic humoral response of breakthrough influenza infection in mice is distinct from that of primary influenza infection or allergic sensitization, canonical Type 1 and 2 immune responses, respectively. Longitudinal comparison of breakthrough infection with allergic sensitization and primary influenza infection demonstrated major differences in lung immunity between treatment groups in female, BALB/c mice. Breakthrough infection mice exhibit lung eosinophil infiltration that peaks at 7-10 days post-challenge, enriched for the Siglec-Fhi subset, but in the absence of overt pro-inflammatory cytokine/chemokine signals, high viral titers, severe lung lesions, goblet cell hyperplasia, allergic levels of total IgE, or enhanced morbidity. Multiparameter fluorescence imaging corroborated findings from flow cytometry and also unveiled interactions between CD101+Siglec-F+ cells with CD3+ cells in the lung tissue space. Imaging also revealed a marked absence of eosinophil or neutrophil extracellular traps in the lungs of breakthrough infection mice, in contrast with allergic sensitization and primary influenza infection, respectively. Altogether, our findings provide a deeper understanding of the kinetics and cell-cell interplay during non-pathological, balanced Type 1/2 immune responses in vaccinated hosts during breakthrough infection.

immunology↗

bioRxiv2025

p125A (Sec23ip) couples COPII coat assembly with donor-acceptor membrane organization to facilitate tunnel-based traffic.

COPII-coat proteins play a crucial role in generating small vesicles at endoplasmic reticulum (ER) exit sites (ERES). However, they also assemble at the necks of tunnels and tubules connecting the ER exit site (donor) to the ERGIC/cis-Golgi cisterna (acceptor). How can COPII support these two traffic mechanisms? Through cell-free reconstitutions, we have found that the apposition of donor and acceptor membranes is important for the assembly of the outer layer of the COPII coat (Sec13/31) but had minimal impact on Sar1-induced COPII inner layer (Sec23/24) recruitment. The expression of the adaptor protein p125A, which binds to phosphatidylinositol 4-phosphate (PI4P), Sec31 and Sec23, stabilized the contact between the donor and acceptor membranes and promoted COPII outer layer assembly. A p125A-chimera expressing a Golgi-targeted PI4P-binding domain could also support outer layer assembly. Notably, the C-terminal helical domain of Sec31A, which interacts with p125A, was essential for its assembly at ERES. In cells lacking p125A, the assembly of the COPII outer layer was selectively destabilized. Transcriptome and secretome analyses reveal selective adjustments to extracellular matrix remodeling and collagen secretion, which corresponded with selective inhibition of fibrillar collagen traffic from the ER. Thus, p125A connects the outer COPII layer at ERES with PI4P-rich ERGIC/cis-Golgi membranes, coordinating COPII assembly with tunnel-driven collagen traffic.

cell biology↗

bioRxiv2025

Characterization of the glycoproteins of novel fish influenza B-like viruses

Novel influenza-like virus sequences previously identified in fish and amphibians were found to cluster as a sister clade of influenza B viruses, but have thus far remained uncharacterized. We demonstrate that salamander influenza-like virus (SILV) HA is functionally divergent from influenza B virus HA and does not bind to 2,3- and 2,6-linked sialic acids. However, the HAs of Siamese algae-eater influenza-like virus (SAEILV) and chum salmon influenza-like virus (CSILV) bind to 2,3 linked sialic acid. Furthermore, SAEILV HA binds to sialyated Lewis X, is activated by human airway enzymes and is fusogenic at a wide range of pH conditions. SAEILV NA has a highly conserved active site and a similar structure to other known NAs. We also determined the cryo-electron microscopy structure of the HA of a previously described virus from the same sister clade, the Wuhan spiny eel influenza virus (WSEIV). Importantly, no cross-reactive antibodies against these HAs or NAs were found in the human serum, suggesting that humans are immunologically naive to these viruses. One sentence summaryNovel influenza like-viruses, displayed different target receptor specificity and limited antigenic conservation of the HA and NA relative to influenza B Viruses.

microbiology↗

bioRxiv2025

Modulating populational variance of methyl-guanine methyl transferase expression through miR-181d degradation: a novel mechanism of temozolomide resistance

Intratumoral heterogeneity plays a pivotal role in cancer evolution, providing the substrate for adaptation to selective pressures, including treatment with chemotherapy. Here, we show that micro-RNA regulation of variance in the expression of the DNA repair protein methyl-guanine methyl transferase (MGMT) contributes to this heterogeneity and acquired therapeutic resistance. In cell lines derived from glioblastomas, the most common form of primary brain tumor, treatment with standard-of-care temozolomide chemotherapy triggers a feed-forward loop between polyribonucleotide nucleotidyltransferase 1 (PNPT1) and miR-181d, an MGMT regulating miRNA, expediting miR-181d degradation. This degradation requires the activation of Ataxia Telangiectasia and Rad3-related (ATR) kinase. The degradation of miR-181d in glioblastoma cells increased both the mean and the variance of MGMT expression in the cell population. Subclone reconstituted cell populations with similar populational mean MGMT levels but with differences in the variance of MGMT expression exhibited differential temozolomide sensitivity, with the higher MGMT variance population showing increased resistance. This resistance is suppressed by exogenously transfected miR-181d. These findings suggest a key role for miRNA in regulating intra-tumoral heterogeneity through modulation of key DNA repair enzymes and provide a compelling rationale for miRNA delivery as a platform for glioblastoma therapy. Significance StatementThis study demonstrates a mechanistic link between a feed-forward loop mediating microRNA degradation and cell-to-cell variance in gene expression, and the contribution of this mechanism to intratumoral heterogeneity and therapeutic resistance. We show that when glioblastoma, the most common form of adult primary brain tumor, is treated with standard-of-care chemotherapy, temozolomide, a feed-forward loop between miR-181d and PNPT1 is initiated, causing rapid degradation of miR-181d. This degradation increases the cell-to-cell variability in methyl-guanine methyl transferase (MGMT) expression, expanding intra-tumoral heterogeneity and contributing to acquired temozolomide resistance. This process can be suppressed by therapeutic delivery of microRNA, providing compelling considerations for clinical translation.

cancer biology↗

bioRxiv2025

Gliomas organize as liquid crystals: three-dimensional nematic order, disclinations and quasi-long-range order

Active nematic liquid crystals are the main structural phase of gliomas, promoting collective migration and aggression. We establish the existence of nematic order and topological defect lines and loops in 3D in vivo mouse and human glioma brain tumors. As predicted by theory, sections through the disclination lines in 3D appear as {+/-}1/2 topological defects in 2D. In 3D, these defects either persist along disclination lines or twist as they interconvert from -1/2 to +1/2. Cell alignment exhibits quasi-long-range order, spreading throughout the tumor over distances between 300-3000 m. In vitro -1/2 and +1/2 defects display changes in apoptosis levels, suggesting topological defects regulate glioma cell density. The large scale order of gliomas correlates with tumors aggressive behavior. The organization of gliomas as active nematic liquid crystals provides a novel physical foundation of complex solid tumors; their deconstruction signposts potential treatments for deadly cancers.

biophysics↗

bioRxiv2025

Root hair growth phases are coordinated by cytoskeleton, nucleus dynamics and cell mechanics in Arabidopsis

Polar cell growth is a fundamental process across organisms, yet its coordination with nuclear movement and cytoskeleton dynamics remains underexplored. Focusing on Arabidopsis thaliana root hairs, we investigate these processes using high-resolution live imaging within microfluidics-based experiments. By incorporating data on cytoskeletal dynamics, nuclear positioning, and tip growth into a mathematical model, we analyse how their interactions shape the different growth phases that we reveal for the first time in this study. Chemical treatments and mutant analyses further support our model, revealing that timely cytoskeletal changes drive transitions between these growth phases, and correlate with shifts in nuclear movement and morphology. This regulation suggests a microtubule-actin crosstalk in the root hair subapical region. Additionally, we present novel findings on vacuole movement and cell stiffness, further refining our understanding of tip growth dynamics. Collectively, our work provides a comprehensive framework for understanding how transitions between growth phases are orchestrated in plant tip-growing cells.

cell biology↗

bioRxiv2025

PYM1 limits non-canonical Exon Junction Complex occupancy in a gene architecture dependent manner to tune mRNA expression

The Exon Junction Complex (EJC) deposited upstream of exon-exon junctions during pre-mRNA splicing in the nucleus remains stably bound to RNA to modulate mRNA fate at multiple post-transcriptional steps until its disassembly during translation. Here, we investigated two EJC disassembly mechanisms in human embryonic kidney 293 (HEK293) cells, one mediated by PYM1, a factor that can bind both the ribosome and the RBM8A/MAGOH heterodimer of the EJC core, and another by the elongating ribosome itself. We find that EJCs lacking PYM1 interaction show no defect in translation-dependent disassembly but is required for translation-independent EJC destabilization. Surprisingly, PYM1 interaction deficient EJCs are enriched on sites away from the canonical EJC binding position including on transcripts without introns or with fewer and longer exons. Acute reduction of PYM1 levels in HEK293 cells results in a modest inhibition of nonsense-mediated mRNA decay and stabilization of mRNAs that localize to endoplasmic reticulum associated TIS-granules and are characterized by fewer and longer exons. We confirmed the previously reported PYM1-flavivirus capsid protein interaction and found that human cells expressing the capsid protein or infected with flaviviruses show similar changes in gene expression as upon PYM1 depletion. Thus, PYM1 acts as an EJC specificity factor that is hijacked by flaviviruses to alter global EJC occupancy and reshape host cell mRNA regulation.

molecular biology↗

bioRxiv2025

Talk2Biomodels: AI agent-based open-source LLM initiative for kinetic biological models

In this study, we present Talk2Biomodels (T2B), an open-source1, user-friendly, large language model-based agentic AI platform designed to democratize access to computational models of biological interactions and promote the FAIRification (Findability, Accessibility, Interoperability, and Reusability) of these models. T2B enables users to explore and analyse mathematical models of biological systems using natural language. It eschews the traditional graphical user interface (GUI) and minimally adaptable workflow in favour of a modern agentic framework to provide a dynamic and immersive experience to explore mathematical models of biological interactions through conversations in natural language. T2B supports models encoded in the open-source community format Systems Biology Markup Language (SBML) and is integrated with the BioModels database (https://www.ebi.ac.uk/biomodels/), enabling seamless exploration, simulation, and analysis of curated systems biology models. Use cases in precision medicine, epidemiology, and emergent systems properties of biological networks are presented to demonstrate how experts and non-experts in computational biology can benefit from T2B.

systems biology↗

bioRxiv2025

Convergent and clonotype-enriched mutations in the light chain drive affinity maturation of a public antibody

Public antibodies that recognize conserved epitopes are critical for vaccine development, and identifying somatic hypermutations (SHMs) that enhance antigen affinity in these public responses is key to guiding vaccine design for better protection. We propose that affinity-enhancing SHMs are selectively enriched in public antibody clonotypes, surpassing the background frequency seen in antibodies carrying the same V genes, but with different epitope specificities. Employing a human IGHV4-59/IGKV3-20 public antibody as a model, we compare SHM signatures in antibodies also using these V genes, but recognizing other epitopes. Critically, this comparison identified clonotype-enriched mutations in the light chain. Our analyses also show that these SHMs, in combination, enhance binding to a previously uncharacterized viral epitope, with antibody responses to it increasing after multiple vaccinations. Our findings offer a framework for identifying affinity-enhancing SHMs in public antibodies based on convergence and clonotype-enrichment and can help guide vaccine design aimed to elicit public antibodies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/642041v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1c4559eorg.highwire.dtl.DTLVardef@d12202org.highwire.dtl.DTLVardef@fca57org.highwire.dtl.DTLVardef@ad5041_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIClonotype-enriched SHMs are identified in the light chain of a public antibody, M15 C_LIO_LIThese light chain SHMs enhance affinity of M15 C_LIO_LIM15 targets a previously undescribed, conserved viral epitope C_LIO_LISerum antibody levels targeting this epitope increase after repeated vaccinations C_LI

immunology↗

bioRxiv2025

High throughput identification of genetic regulators of microglial inflammatory processes in Alzheimer's disease

Genome-wide association studies (GWAS) have identified over a hundred genetic risk factors for Alzheimers disease (AD), many of which are predominantly expressed in microglia. However, the pathogenic role for most of them remains unclear. To systematically investigate how AD GWAS variants influence human microglial inflammatory responses, we conducted CRISPR inhibition (CRISPRi) screens targeting 119 AD GWAS hits in hiPSC-derived microglia (iMGLs) and used the production of reactive oxygen species (ROS) in response to the viral mimic poly(I:C) as a functional readout. Top hits whose knockdown either increased or decreased ROS levels in response to poly(I:C) were further analyzed using CROP-seq to integrate CRISPRi with single-cell RNA sequencing (scRNA-seq). This analysis identified 9 unique microglial clusters, including a poly(I:C)-driven inflammatory cluster 2. Emerging evidence supports a pathogenic role of viral infections in AD and cross comparison of our scRNA-seq data with iMGLs xenotransplanted into an AD mouse model shows significant overlap between our clusters and AD-relevant microglial clusters. Knockdown of MS4A6A and EED, which resulted in elevated ROS production in the presence of poly(I:C), increased the proportion of cluster 2 cells and induced functionally related changes in gene expression. In addition, KD of MS4A6 led to a reduction in the proportion of iMGLs in the DAM (disease associated microglia) cluster under all conditions, suggesting that this gene may modulate the DAM response. In contrast, KD of INPP5D or RAPEP1 which lead to low levels of ROS in the presence of poly(I:C), did not significantly affect the proportion of cells in cluster 2 but rather shaped the inflammatory response. This included the upregulation of an HLA-associated inflammatory cluster (cluster 6) by INPP5D knockdown under all conditions, independent of poly(I:C) stimulation. Importantly, KD of INPP5D or RAPEP1 had many shared differentially expressed genes (DEGs) under both vehicle and poly(I:C) treated conditions. Overall, our findings demonstrate that despite the diverse biological functions of AD GWAS variants, they converge functionally to regulate human microglial states and shape inflammatory responses relevant to AD pathology.

neuroscience↗

bioRxiv2025

Pathogenicity and transmissibility of bovine-derived HPAI H5N1 B3.13 virus in pigs

Since the first emergence of highly pathogenic avian influenza (HPAI) H5N1 viruses in dairy cattle, the virus has continued to spread, reaching at least 17 states and at least 970 dairy herds in the United States. Subsequently, spillovers of the virus from dairy cattle to humans have been reported. Pigs are an important reservoir in influenza ecology because they serve as a mixing vessel in which novel reassortant viruses with pandemic potential can be generated. Here, we show that oro-respiratory infection of pigs resulted in productive replication of a bovine-derived HPAI H5N1 B3.13 virus. Infectious virus was mainly identified in the lower respiratory tract of principal infected pigs, and sero-conversion was observed in most of the principal pigs at later time points. In one animal, we detected the emergence of a mutation in hemagglutinin (HA) previously associated with increased affinity for "mammalian-type" 2,6-linked sialic acid receptors, but this mutation did not reach consensus levels. Sentinel contact pigs remained sero-negative throughout the study, indicating lack of transmission. The results support that pigs are susceptible to a bovine-derived HPAI H5N1 B3.13 virus, but this virus did not replicate as robustly in pigs as mink-derived HPAI H5N1 and swine-adapted influenza viruses.

microbiology↗

bioRxiv2025

Advancing Cardiac Graft Assessment Methods During Normothermic Machine Perfusion to Achieve Improved Transplantation Outcomes

Heart failure, a leading global health challenge, affects over 23 million people worldwide, with heart transplantation being the gold standard for end-stage disease. However, the scarcity of viable donor hearts presents a significant barrier, with only one-third of available grafts used due to stringent selection criteria. Machine perfusion technologies, particularly normothermic machine perfusion (NMP), offer promise in improving graft preservation and assessment, yet their full potential for predicting transplantability remains underexplored. This study investigates three assessment methods to enhance human heart evaluation during NMP, focusing on mitochondrial function, left ventricular (LV) performance, and inflammatory markers. First, resonance Raman spectroscopy (RRS) is employed to assess mitochondrial redox state as a proxy for metabolic competency, offering a non-invasive and dynamic evaluation of mitochondrial function during ex vivo preservation. Second, LV function is quantified using intraventricular balloons, providing critical insights into graft viability and performance. Third, inflammatory markers and endothelial activation are assessed from perfusate to predict post-transplant outcomes. These methods were tested on human donor hearts declined for transplantation, preserved via static cold storage (SCS) and subsequently assessed with NMP in Langendorff mode. The results demonstrate that these parameters can be easily integrated into existing clinical perfusion workflows and hold potential for improving heart transplantation outcomes by enhancing graft selection and optimizing donor heart use. Future studies will further validate these biomarkers across different preservation techniques and evaluate their clinical applicability.

bioengineering↗

bioRxiv2025

Monoclonal antibody to Filarial serpin Wb123 impedes the urokinase plasminogen activator receptor-mediated Alternative activation of macrophages

Macrophages play a multifaceted role in mounting an immune response against pathogens through phagocytosis, antigen presentation and cytokine production. Activated macrophages are critical for the initiation, maintenance, and resolution of inflammation. They can either be classically activated (Th1 response), which promotes inflammation or alternatively activated (Th2 response), which resolves inflammation. The alternate activation of macrophages, along with diminished T cell response, is thought to be an important immune evasion strategy employed by the filarial parasites. However, the mechanism behind filaria-induced alternate activation remains unclear. In this study, by using in-silico approach we identified filarial serpins which are highly expressed in the infective L3 larval stages. We characterized the filarial serine protease inhibitor, Wb123 and established its role in alternate activation. We observed that Wb123 impairs nitric oxide (NO) and reactive oxygen species (ROS) expression, which plays a crucial role in mounting a strong immune response. Surprisingly, we also observed elevated IL6 expression and phosphorylation of STAT3, indicating IL-6-dependent alternate activation. Our findings indicate that the urokinase plasminogen activator receptor (uPAR) is essential for Wb123-induced alternate activation. Furthermore, we demonstrated that the monoclonal antibody MAbG8 impedes the Wb123-induced alternate activation, as shown by reduced CD163 expression along with increased ROS and uPA expression in response to lipopolysaccharide (LPS) and interferon-gamma (IFN-{gamma}). Overall, our study identifies the filarial serpin Wb123 and highlights its potent role in alternative activation. This interaction reveals, for the first time a mechanism exploited by filarial parasites to evade a strong pro-inflammatory immune response.

cell biology↗

bioRxiv2025

Integrative spatial transcriptomic analysis pinpoints the role of TaMCO3 encoding ferroxidase in wheat root tip iron mobilization

Roots play an critical role in the sensing and absorption of essential minerals from the rhizosphere. Iron (Fe) deficiency, for example, triggers a well-known series of physiological and molecular responses within roots that facilitate uptake, which differs between monocots and dicots. In monocots, little is known about molecular responses that occur within specific root development zones in response to iron deprivation, and how these differences results in overall nutrient uptake. Here, we conducted a transcriptome analysis of wheat root tips under Fe deficiency (-Fe) and performed a comparative transcriptome analysis with the previous datasets generated from the whole root. Gene ontology analysis of differentially expressed genes highlighted the significance of oxidoreductase activity and metal/ion transport in the root tip, which are critical for Fe mobilisation. Interestingly, wheat, an allohexaploid species consisting of three different genomes (A, B, and D) displayed varying gene expression levels arising from the three genomes that contributed to similar molecular functions. Detailed analysis of oxidoreductase function at the root tip revealed multiple multi-copper oxidase (MCO) proteins, such as Fe-responsive TaMCO3, that likely contribute to the overall ferroxidase activity. Detailed characterisation of TaMCO3 shows that it complements the yeast FET3 mutant and rescues the -Fe sensitivity phenotype of Arabidopsis atmco3 mutants by enhancing vascular Fe loading. Transgenic wheat lines overexpressing TaMCO3 exhibited increased root Fe accumulation and improved tolerance to -Fe by augmenting the expression of Fe-mobilizing genes. Our findings highlight the role of spatially resolved gene expression in -Fe responses, suggesting strategies to reprogram cells for improved nutrient stress tolerance.

plant biology↗

bioRxiv2025

Machine learning segmentation tool trained on synthetic data for tracking cytoskeleton polymerisation and depolymerisation

The cytoskeleton is important in controlling the growth and morphology of plant cells, so tracking its morphological changes is essential. Here, we develop a new machine learning based segmentation tool for microtubules (MTs), which can distinguish between polymerised and depolymerised fibres. To circumvent the low abundance of data, we trained on synthetic images of microtubules from a computational micro-tubule model, pre-processed to reproduce microscope effects and partial depolymerisation. We used this tool to investigate how the MT network in an Arabidopsis thaliana root hair cell repolymerises after depolymerisation under Oryzalin (OZ) drug treatments. Specifically, we show the network initially repolymerises from the shank region. This work demonstrates the viability of using synthetic data to train machine learning systems handling cytoskeletal image data.

plant biology↗