Biology subjects

Kacprzyk, J.

Publications and source records attributed to Kacprzyk, J..

7 recordsLinked to original sources

bioRxiv2026

Large-scale forward genetics reveals genetically separable components of plant cell proliferative capacity

Plant cell cultures underpin regeneration, transformation and emerging applications in biotechnology and plant cell agriculture, yet the genetic determinants of proliferative performance remain poorly characterized. Here, we combined large-scale forward genetics with automated image-based phenotyping to quantify 27,648 calli representing 2,880 Arabidopsis thaliana T-DNA insertion lines. Of these, 2,456 lines produced calli smaller than the WT population mean, whereas only 190 produced larger calli, revealing a strongly asymmetric phenotypic distribution. Enhanced callus growth was not consistently associated with increased vegetative growth, indicating that these traits can be genetically uncoupled. Validation of selected high-growth mutants in suspension culture identified lines with enhanced biomass accumulation, while serial propagation revealed independent variation in sustained proliferative capacity. Thus, callus growth, whole-plant growth, short-term suspension biomass accumulation and sustained proliferation represent distinct, not necessarily coupled components of proliferative performance. These findings establish a scalable framework for identifying genetic determinants relevant to the development of improved plant cell production lines.

plant biology↗

bioRxiv2026

The Viability Gambit: An Optimized Sterilisation Protocol for Industrial Hemp ( Cannabis sativa L.) Seeds

Hemp (Cannabis sativa L.) is an increasingly important crop with applications spanning fibre, seed oil and bioactive cannabinoid production, yet the development of reliable tissue culture systems for this species remains a significant challenge. The establishment of axenic seedling cultures is a prerequisite for hypocotyl-based regeneration and future genetic transformation pipelines, but hemp seeds harbour diverse endophytic microbial communities that frequently overwhelm standard surface sterilisation protocols. Here, we present a systematic comparative evaluation of seed sterilisation strategies across seven industrial hemp accessions, examining the effects of sterilisation chemistry, seed provenance and accession identity on both contamination outcome and the subsequent morphogenic competence of hypocotyl explants. Across all treatments and accessions, in-house glasshouse-harvested seeds achieved higher sterility rates than commercially sourced material regardless of protocol applied. This provenance effect, combined with considerable batch-to-batch variation within seed sources, indicates that contamination load is a primary determinant of successful hemp seed sterilisation. Among the sterilisation treatments evaluated, a baseline of 75% ethanol combined with sequential 1% hydrogen peroxide incubation performed consistently well for low-load seed batches, while supplementation with Plant Preservative Mixture (PPMTM) might be necessary to achieve acceptable rates of non-contaminated seedlings from high contamination load batches. Beyond their effect on contamination, sterilisation treatments influenced the morphogenic fate of hypocotyl explants independently of sterility outcomes. Notably, seedling treatment with the Prochloraz-based fungicide Octave promoted shoot and root co-regeneration in the absence of exogenous plant growth regulators in some cases. Hormone-free organogenesis from hypocotyl explants was achievable across multiple hemp accessions, demonstrating that this developmental capacity is broadly distributed within hemp, though its frequency and consistency varied with accession identity and protocol conditions. Together, these findings provide a practical framework for axenic hemp seed culture that can be used as starting point requiring local adaptation based on seed source, batch history and the intended downstream application.

Plant Biology↗

bioRxiv2026

Unveiling Hidden Endophytes by Optimising Identification of Endophytic Bacterial Communities from Wild Grassland Plant Roots

Endophytic bacteria are increasingly recognised for their roles in plant health through symbiosis. However, methodological challenges, such as inconsistent root sterilisation, inefficient microbial DNA extraction, and co-amplification of plant organellar DNA, limit accurate characterisation of these communities, especially in wild grassland plants and non model plant in general. To address this, we developed and tested a streamlined protocol for bacterial endophyte detection from wild grassland plant roots, encompassing surface sterilisation of roots, DNA extraction, clamping of plant internal mitochondrial and chloroplast DNA, and 16S rRNA amplicon sequencing. Our approach minimises plant DNA contamination and yields high-quality microbial profiles. The protocol is adaptable and specific to grassland plant species, offering a standardised foundation for endophyte studies in wild and non-model plants. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=141 HEIGHT=200 SRC="FIGDIR/small/706108v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@157df36org.highwire.dtl.DTLVardef@1ff645aorg.highwire.dtl.DTLVardef@1580ecorg.highwire.dtl.DTLVardef@1c31b89_HPS_FORMAT_FIGEXP M_FIG C_FIG (Haskins and Ajaz, 2026) https://BioRender.com/47gd2xr

plant biology↗

bioRxiv2023

The polyamines spermine and spermidine inhibit or induce programmed cell death in Arabidopsis thaliana in vitro and in vivo in a dose dependent manner.

Polyamines are ubiquitous biomolecules with a number of established functions in eukaryotic cells. In plant cells, polyamines have previously been linked to abiotic and biotic stress tolerance, as well as to the modulation of programmed cell death (PCD), with contrasting reports on their pro-PCD and pro-survival effects. Here, we used two well established platforms for the study of plant PCD; Arabidopsis thaliana suspension cultures cells and the root hair assay, to examine the roles of the polyamines spermine and spermidine in the regulation of PCD. We demonstrate that both polyamines can trigger PCD when applied exogenously at higher doses, whereas at lower concentrations they inhibit PCD induced by both biotic and abiotic stimuli. Furthermore, we show that concentrations of polyamines resulting in inhibition of PCD generated a transient ROS burst in our experimental system, and activated the expression of oxidative stress- and pathogen response-associated genes. Finally, we examined PCD responses in existing Arabidopsis polyamine synthesis mutants, and identified a subtle PCD phenotype in Arabidopsis seedlings deficient in thermo-spermine. The presented data show that polyamines can have a role in PCD regulation, however that role is dose-dependent and consequently they may act as either inhibitors, or inducers, of PCD in Arabidopsis.

plant biology↗

bioRxiv2023

Transcriptional signatures associated with waterlogging stress responses and aerenchyma formation in barley root tissue

The negative impact of soil waterlogging on crop production is expected to increase due higher frequencies of extreme rainfall events arising from climate change. Consequently, understanding the molecular mechanisms that enable plants to mitigate waterlogging stress is critical for breeding programmes, particularly in the case of waterlogging-susceptible crop species, such as barley (Hordeum vulgare). Aerenchyma formation is a key morphological adaptation allowing plants to cope with waterlogging stress and hypoxic conditions, however, the genetic regulation of its development in barley remains largely unresolved. In this study, two barley cultivars with contrasting waterlogging tolerance (Franklin and Yerong) were subjected to waterlogging stress, followed by analysis of phenotypic traits including root aerenchyma formation, and transcriptomic profiling of root tissue samples. Differential expression analyses identified genes transcriptionally responsive to 24 and 72 h of waterlogging in both cultivars, and highlighted metabolic adaptations, regulation of ROS signalling and management of stress responses as key elements of the waterlogging response. The results revealed large intra-individual variations in root aerenchyma formation, and these variations were exploited to isolate 81 candidate aerenchyma-associated genes from the generated RNA-seq datasets. Network analyses suggest the involvement of the DNA damage response gene, DRT100 and cell wall modifying genes, XHT16 and XHT15 as regulatory hub genes in aerenchyma formation. Collectively, the generated data provide insights into transcriptional signatures associated with the barley root responses to waterlogging and aerenchyma formation, informing our understanding of strategies that plants employ to cope with the negative impacts of heavy rainfall.

plant biology↗

bioRxiv2023

Duplications of human longevity-associated genes across placental mammals

Natural selection has shaped a wide range of lifespans across mammals, with a few long-lived species showing negligible signs of ageing. Approaches used to elucidate the genetic mechanisms underlying mammalian longevity usually involve phylogenetic selection tests on candidate genes, analyses of differential gene expression between age cohorts or species, and measuring age-related epigenetic changes. However, the link between gene duplication and evolution of mammalian longevity has not been widely investigated. Here, we explored the association between gene duplication and mammalian lifespan by analysing 287 human longevity-associated genes across 37 placental mammals. We estimated that the expansion rate of these genes is eight times higher than their contraction rate across these 37 species. Using phylogenetic approaches, we identified 43 genes whose duplication levels are significantly correlated with longevity quotients (FDR < 0.05). In particular, strong correlation observed for four genes (CREBBP, PIK3R1, HELLS, FOXM1) appears to be driven mainly by their high duplication levels in two ageing extremists, the naked mole rat (Heterocephalus glaber) and the greater mouse-eared bat (Myotis myotis). Further sequence and expression analyses suggest that the gene PIK3R1 may have undergone a convergent duplication event, whereby the similar region of its coding sequence was independently duplicated multiple times in both of these long-lived species. Collectively, this study identified several candidate genes whose duplications may underlie the extreme longevity in mammals, and highlighted the potential role of gene duplication in the evolution of mammalian long lifespans.

evolutionary biology↗

bioRxiv2023

Arabidopsis cell suspension culture and RNA sequencing reveal regulatory networks underlying plant programmed cell death.

Programmed cell death (PCD) facilitates targeted elimination of redundant, damaged, or infected cells via genetically controlled pathways. In plants, PCD is often an essential component of normal development and can also mediate responses to abiotic and biotic stress stimuli. However, studying the transcriptional regulation of this fundamental process is hindered by difficulties in sampling small groups of cells undergoing PCD that are often buried within the bulk of living plant tissue. We addressed this challenge by using RNA sequencing (RNA-Seq) of Arabidopsis thaliana suspension cells, a system that allows precise monitoring of PCD activation and progression. The use of three PCD-inducing treatments (salicylic acid, heat and critical dilution), in combination with three cell death modulators (3- methyladenine, lanthanum chloride and conditioned medium), allowed isolation of candidate core and stimuli-specific PCD genes, inference of underlying gene regulatory networks and identification of putative transcriptional regulators. This analysis underscored cell cycle disturbance and the repression of both pro-survival stress responses and mitochondrial retrograde signalling as key elements of the PCD-associated transcriptional signature in plants. Further, phenotyping of twenty Arabidopsis T-DNA insertion mutants in selected candidate genes confirmed a role for several in PCD and stress tolerance regulation, and validated the potential of these generated resources to identify novel genes involved in plant PCD pathways and/or stress tolerance in plants.

plant biology↗