Browsing by Subject "cell biology"

Now showing 1 - 6 of 6
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    Open Access
    Comparative analysis of desiccation responses in three Xerophyta species at two stages of seedling development
    (2025) Ngcala, Mamosa; Illing, Nicola; Ingle Robert; Nikoloski, Zoran; Lyall, Rafe
    Vegetative desiccation tolerance (VDT) is a rare trait among flowering plants, enabling the adult tissues of certain species, known collectively as resurrection plants, to survive near-total water loss and revive upon rehydration. This study used a seedling model to investigate VDT at two developmental stages, pre-leaf and two-leaf stage, across three Xerophyta species: the poikilochlorophyllous Xerophyta schlechteri, and X. humilis, and the homoiochlorophyllous X. elegans. By analyzing changes in cellular ultrastructure, gene expression, and metabolite and lipid concentrations, the aim was to identify core conserved VDT mechanisms in these three Xerophyta species, as well as differences between poikilochlorophyllous and homoiochlorophyllous species in the same genus. Transcriptomic analysis showed that the key seed master maturation transcription factors (TFs) ABI3A and ABI5A, were up-regulated in dry pre-leaf seedlings but no expression was detected in two-leaf seedlings. In contrast, the ABRE binding factor, ABFA, was highly expressed in dry tissues at all developmental stages across all species, suggesting a potential role in VDT. A three-way comparison was used to identify gene orthogroups (OGs) that showed the same response to desiccation across all three Xerophyta species: these 370 up-regulated and 335 down-regulated OGs potentially represented a core set of desiccation-responsive genes in Xerophyta species. Promoter regions of these core OGs were enriched with binding sites for Xerophyta specific expanded TF families, including Heat Shock Factors (HSFs), A-T hook factors (AHLs), and C2H2 Zinc Finger Proteins (ZATs). The gene ontology enrichment showed that the HSF, AHL, and ZAT motif containing OGs that were up-regulated are involved in water deprivation, abscisic acid response, and oxidative stress response processes, whereas the down-regulated OGs are involved in growth and development related processes. Notably, some of the up-regulated HSF motif-containing target OGs included WRKY TFs, stachyose synthase, and solanesyl diphosphate synthase 2, which are involved in stress-related functions enhancing VDT. Metabolite analysis demonstrated that the osmoprotectants proline, sucrose, and trehalose accumulated in dry tissues at both developmental stages of all three Xerophyta species. Moreover, differences in cellular ultrastructure including chloroplast organization were investigated by transmission electron microscopy, comparing a detailed dehydration rehydration time course of poikilochlorophyllous X. schlechteri to homoiochlorophyllous X. elegans. This analysis revealed that desiccation-induced ultrastructural changes, such as vacuolation, chloroplast centralization, and starch degradation, typically observed in adult tissues, were also prevalent in the two-leaf seedlings of X. elegans and X. schlechteri. Notably, complete disassembly of the thylakoid membranes was observed in X. schlechteri but not in X. elegans. These results align with the significant reduction of monogalactosyldiacylglycerol, a major galactolipid involved in chloroplast function. The results of this study highlight differences between the poikilochlorophylly and homoiochlorophylly strategies, as well as conserved molecular responses to desiccation in Xerophyta species, providing insights into the evolution of VDT
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    Open Access
    DDRAD-seq based identification of genetic markers to facilitate the assessment of clonal versus sexual reproduction in dichrostachys cinerea
    (2025) Lumley, Liam Lester; Ingle, Robert; Lenhard, Michael
    Dichrostachys cinerea, commonly referred to as “sicklebush”, is a semideciduous nitrogen-fixing tree species belonging to the Fabaceae family and is native to parts of Africa, India and Northern Australia. In addition to propagation via seed dispersal, the species is capable of asexual reproduction via the process of root suckering. D. cinerea can aggressively colonize grasslands and quickly becomes problematic given its ability to persist through treatments such as fire, chemical treatment and mechanical removal. Due to its shrub-like physiology; propensity for root suckering; and the production of large amounts of seed, removal efforts often fail, especially in areas where D. cinerea has established dense thickets. African savannahs in particular have experienced a rapid rise in woody plant encroachment, with D. cinerea often one of the major species held responsible, and it is a species of concern to local ecologists. While previous studies have investigated the various factors which contribute to woody plant encroachment within Savanna ecosystems, it is still unclear as to what role asexual reproduction plays in this phenomenon and current methods to study this are laborious and time consuming. This study sought to develop a genetic based workflow to allow the detection of clonal individuals in D. cinerea. To facilitate this study, 225 D. cinerea samples were collected across the species range of D. cinerea in South Africa. DNA extracted from these samples allowed for the identification 39 299 SNP markers generated from ddRAD-seq libraries. These SNPs were then used to elucidate the phylogenetic relationships between individuals in order to ascertain whether clonality could be detected within the species, via the use of a genetic-distance-based threshold value which would discriminate between clonal and non-clonal individuals. Findings from this study revealed that across its species range, D. cinerea consisted of a mixed cytotype population, exhibiting a clear phylogenetic divide between diploid and tetraploid individuals. Identity-by-state analysis of the Kruger National Park diploid population confirmed that naturally occurring clonal individuals could be detected via ddRAD-seq generated SNP markers. This workflow will facilitate the study of clonality in D. cinerea in future studies which seek to answer ecologically relevant questions.
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    Open Access
    Functional divergence of the RNA polymerase II transcription machinery in Plasmodium falciparum
    (2024) Knopp, Jasmin; Oelgeschlger, Thomas
    This thesis describes the functional characterisation of three Plasmodium falciparum general transcription factors - two TBP family proteins designated the TATA-binding protein (TBP) and TBP like protein (TLP), and the P. falciparum orthologue of general transcription factor IIB (TFIIB). Through the biochemical characterisation of protein-DNA interactions, protein-protein interactions and the biomolecular condensation properties of these transcription factors, this study aimed to provide insight into the molecular mechanisms governing promoter recognition and the regulated assembly of the RNA polymerase II transcription initiation complex in P. falciparum, which are hitherto not understood. The work shows that P. falciparum expresses two highly divergent TBP family proteins that do not possess sequence-specific TATA box-binding activity. Both PfTBP and PfTLP bind DNA with a general preference for A/T-rich sequences, mediated through interactions with the DNA minor groove, without detectable preference for specific sequence motifs. PfTBP and PfTLP are thus unlikely to contribute to the recognition of specific promoter regions within the A/T-rich context of the P. falciparum genome. The study further characterises TBP-independent DNA-binding of PfTFIIB, a Plasmodium-specific feature not seen with TFIIB in well-studied eukaryotes. The data presented here show that PfTFIIB binds to DNA in an unspecific manner. Interestingly, PfTFIIB stimulates the DNA binding activity of PfTBP and PfTLP and forms stable PfTBP-PfTFIIB-DNA and PfTLP-PfTFIIB-DNA nucleoprotein complexes. However, these PfTFIIB interactions do not detectably enhance the sequence-selectivity of PfTBP- or PfTLP-DNA interactions. Thus, recognition of genomic regions at which transcription is preferentially initiated must involve additional factors or may depend to a lesser extent on the recognition of specific core promoter elements by the general transcription machinery. Transcription condensates play an important role in eukaryotic transcription regulation. In this study, the potential role of PfTFIIB in the formation and regulation of P. falciparum transcription condensates is investigated by fluorescence microscopy using a panel of fluorescent protein-tagged PfTFIIB fusion proteins. The work demonstrates that PfTFIIB undergoes condensation at nanomolar concentrations, and partitions with PfTBP and PfTLP into mixed phase separated condensates. Furthermore, assembly and properties of PfTFIIB condensates are shown to be strongly influenced by the presence of DNA and RNA. Interestingly, PfTBP and PfTLP are found to localise in discrete foci in cultured P. falciparum blood-stage parasites. Together, these results provide first evidence for the existence of transcription condensates, mediated by transient multivalent interactions between general transcription factors, in the P. falciparum malaria parasite.
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    Open Access
    A Manual of Online Molecular Biology Techniques
    (2014-09-12) Rybicki, Ed
    This resource is a comprehensive manual on practical laboratory and experimental techniques used in molecular and cell biology. This resource is useful for postgraduate students in molecular biology laboratories looking to refine or improve their experimental techniques and the proper use of laboratory equipment.
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    Open Access
    Metabolite profiling of Eragrostis nindensis during desiccation and recovery
    (2024) Baluku, Erikan; Farrant, Jill Margaret; Hilhorst, Henk W M; van der Pas, Llewelyn
    Resurrection plants are a unique group of angiosperms that can withstand cellular water loss of up to 95% and resume full metabolic activity upon rehydration. To withstand extreme water loss, they employ a plethora of molecular, physiological, and biochemical processes including accumulation of metabolites that shield the plant cells from photo-oxidative damage and reactive oxygen species. A global understanding of the whole plant using a multi-omics approach will provide more insights into how different parts of the plant deal with desiccation. This study aimed to identify the different metabolites that are differentially abundant in Eragrostis nindensis at different stages of dehydration and rehydration time points in both desiccation-sensitive senescent (ST) and desiccation-tolerant non-senescent (NST) leaf tissue using gas chromatography-mass spectrometry Furthermore, this study compared the shoot and root systems to unravel similarities and differences at the whole plant level in overcoming desiccation. The metabolomics data from the shoots between NST and ST showed that differentially abundant metabolites in NST act as major drivers for plant desiccation tolerance and also aid the plant post-recovery. The roots accumulated fewer metabolites than the shoots; however, some specific metabolites were shown to accumulate exclusively in the roots. These findings revealed that E. nindensis exhibits a metabolic shift with the abundance of sugars such as raffinose and sucrose, amino acids such as glycine and glutamic acid and organic acids such as alpha-ketoglutaric acid and citric acid during dehydration, resulting in accumulation of desiccation-responsive metabolites predominantly in NST compared to ST. The results demonstrated that the leaves have a different metabolic shift pattern that is more variable, and the roots' metabolome is less affected by desiccation. Post- rehydration, there is an accumulation of amino acids and organic acids to aid in the resumption of metabolism in NST compared to ST and roots. The accumulation of these metabolites may protect E. nindensis from the damage associated with rapid drying, as the accumulation of similar metabolites identified in this study has been reported to function as osmoprotectants, reactive oxygen species quenchers and compatible solutes that replace water during desiccation. The identified metabolites and metabolic process provide a great insight into the goal of improving drought tolerance in orphan and drought-sensitive crops.
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    Open Access
    The regulation of the COL5A1 gene via the 3'-UTR and its impact on Achilles tendinopathy andother exercise-related phenotypes
    (2013) Abrahams, Yoonus; Collins, Malcolm; Prince, Sharon
    The regulation of the COL5A1 gene via the 3'-UTR and its impact on Achilles tendinopathy andother exercise-related phenotypes