Browsing by Author "Farrant, Jill Margaret"

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    Desiccation-driven senescence in the resurrection plant Xerophyta schlechteri (Baker) N.L. Menezes
    (2019) Radermacher, Astrid Lillie; Farrant, Jill Margaret; Mundree, Sagadevan G
    Drought-induced senescence is a degenerative process that involves the degradation of cellular metabolites and photosynthetic pigments and uncontrolled dismantling of cellular membranes and organelles. Angiosperm resurrection plants display vegetative desiccation tolerance and avoid drought-induced senescence in most of their tissues. Developmentally older tissues, however, fail to recover during rehydration and ultimately senesce. Comparison of the desiccation-associated responses of older senescent tissues (ST) with non-senescent tissues (NST) will allow for understanding of mechanisms promoting senescence in the former and prevention of senescence in the latter. In the monocotyledonous resurrection plant Xerophyta schlechteri (Baker) N.L.Menezes, leaf tips senesce following desiccation, whereas the rest of the leaf blade survives. This study characterised structural, metabolic and transcriptional changes in ST and NST at varying water contents during desiccation and rehydration. Light and transmission electron microscopy was used to follow anatomical and subcellular responses, and metabolic differences were studied using gas chromatography-mass spectrometry and colorimetric metabolite assays. These results show that drying below 35% relative water content (0.7 gH2O/g dry mass) in ST resulted in the initiation of age-related senescence hallmarks and that these tissues continue this process after rehydration. Analysis of the transcriptome was done using RNA-Seq, which was subject to differential expression analysis and network analysis to elucidate the potential mechanisms for senescence regulation in this species. Significantly increased transcription of senescence associated genes was observed in the air dry sampling point, indicating that initiation of cellular death occurred below 20% RWC. Network analysis based on Pearson correlation revealed a high degree of clustering of these genes, suggesting co-regulation. The majority of these genes had two enriched motifs in their upstream regions, identified as binding sites for WRKY and other transcription factors. A model integrating these observations is presented, with insights into how senescence is initiated in ST and repressed in NST.
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    Open Access
    Dry and back again: characterization of desiccation-associated differentiation of leaf tissues in Craterostigma pumilum Hochst
    (2023) Du Toit, Stephanus Francois; Farrant, Jill Margaret; Reich, Ziv
    Resurrection plants are a polyphyletic group of angiosperms which display true desiccation tolerance - the ability to survive near complete loss of cellular water for extended periods, while recovering metabolic competence upon watering. This is achieved by employing tailored protection behaviours depending on the relative state of (de)hydration. Recent work has raised interest in desiccation associated changes related to tissue destiny in desiccation tolerant vegetative tissues. In this thesis, physiological and transcriptomic techniques were used to characterize such a phenomenon in the homoiochlorophyllous dicot resurrection plant Craterostigma pumilum. Detailed phenotypic observation and pulse-amplitude-modulation fluorometry were used to identify the critical water contents at which key physiological changes occur in leaves of C. pumilum and how this relates to desiccation-associated differentiation between leaf Tip and Base tissues. This was followed by transcriptomic analyses and comparison between these two tissues, to identify potentially key processes involved in desiccation associated tissue differentiation. All findings were then synthesised with existing information reported on for other resurrection plant species to create a theoretical model of desiccation-associated tissue differentiation. This differentiation phenomenon is shown to be transcriptionally initiated during the desiccation commitment stage of the C. pumilum dehydration cycle but is only realised phenotypically during early rehydration and after initial water movement through the leaf tissues. This work provides strong evidence for the existence of desiccation-associated tissue differentiation in C. pumilum and highlights the potential involvement of the phytohormone auxin in the determination of leaf tissue responses to progressive dehydration and anhydrobiosis in resurrection plants.
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    Eragrostis nindensis: unravelling senescence in an African desiccation tolerant grass
    (2019) Madden, Christine Frances; Farrant, Jill Margaret; Mundree, Sagadevan G
    Food security is one of the most important global challenges facing the world today, especially in the context of climate change. Research has been conducted into a unique group of plants, called “resurrection plants”, that can withstand up to 95% tissue water-loss without compromising viability by, inter alia, undergoing extensive metabolic reprogramming and suppressing senescence. In this thesis the African desiccation tolerant grass Eragrostis nindensis (Fical & Hiern) was used as a model plant to identify which biological processes are unique to senescence and critical for desiccation tolerance. When desiccated, the older leaves of E. nindensis senesce, whereas, the younger leaves recover fully upon rehydration, thereby displaying two phenotypes in a single species. Comparing these two tissue types can show how senescence upon abiotic stress is regulated. Differences in transcript abundances between the two tissue types during drying and rehydration was analysed through RNA-seq analysis, coupled with physiological quantitative traits, mass spectrometry analyses and immunoblotting. The transcriptome reflected a transcriptomic reprogramming towards desiccation tolerance by maintaining transcription of genes that control desiccation tolerance traits in both tissue types, however, only the desiccation tolerant (non-senescent) tissue appeared to suppress senescence and maintained translational control. It was hypothesised that the non-senescent tissues regulate and stabilise RNA. The older tissues were unable to suppress senescence, which resulted in cell death. Lipids accumulated in the non-senescent tissue, particularly unsaturated triacylglycerols. It was proposed that lipid droplets that accumulated during drying were stabilised through, in part, the protein expression of oleosin. These lipid droplets appeared to provide a mechanical stabilisation and energy-providing role in the non-senescent tissue. The transcription of genes that control desiccation tolerance traits was generally maintained in both tissue types, however, translation was prevented in the senescent tissue. The non-senescent tissue therefore appeared to engage in a regulation of senescence at the translational level, rather than a fine-tuned transcriptional regulation. The aim of this work was to provide a critical baseline for future studies working on E. nindensis, and desiccation tolerance and senescence in resurrection plants in general. Ultimately, understanding water-deficit stress in the context of senescence can help to improve drought resistance in crops to ensure food security, particularly in Africa.
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    From proteomics to biotechnology. using the resurrection plant eragrostis nindensis to genetically engineer drought tolerant crops
    (2024) Van Der Pas, Llewelyn; Farrant, Jill Margaret; Hitzeroth, Inga; Henk, Hilhorst; Rafudeen, Suhail
    Global climate change is increasingly putting pressure on finding innovative solutions to ensure future food security in particular to developing African nations. Of great relevance are regionally adapted crops, known as orphan crops, which tend to have very little economic value but can provide a source of alternative food security. Vegetative desiccation tolerance is a remarkable feat of selective evolution and is only present in a small number of angiosperms. The ability of some plants, such as Eragrostis nindensis to survive complete cellular water deficit provides an attractive model for discovery-based omics to not only understand the mechanisms involved in driving desiccation tolerance but to explore the feasibility of potential target genes for orphan crop improvement. The work presented herein was aimed at complementing a transcriptomic study using the same leaf tissue from that study to evaluate the changes from RNA to protein and to determine whether there were proteomic signatures that could differentiate the desiccation-tolerant non-senescent (NST) leaves from the desiccation-sensitive senescent (ST) leaves. The data presented here illustrate that several important metabolic pathways are significantly reprogrammed, that only a small subset of proteomic-matching transcripts were translated, and that proteomic differences between the NST and ST were noted despite their being significant similarities between the two in general oxidative and osmotic stress. For instance, the prevention of ferroptosis and accumulation of raffinose synthase and starch synthase in the NST exclusively illustrated that small and subtle increases in protein abundance are likely responsible for enabling resurrection in the NST and not in the ST, which we hypothesise here is likely due to sacrificing of ST upon rehydration as a means to act as a source of nutrition for the NST during resurrection. The study also focussed on functional characterisation of a heat shock 70 protein from E. nindensis as a target for genetic engineering. The selected EnHSP70 was shown to localise to the chloroplast and was able to undergo liquid-liquid phase separation in vitro in a protein concentration and polyethylene glycol dependent manner which could have broad impacts on its role in maintaining proteostasis. In Arabidopsis thaliana, overexpression of EnHSP70 resulted in a stunted germination phenotype whereas expression in BL21 Escherichia coli did not enhance tolerance towards salt or mannitol stress. Furthermore, incubation of EnHSP70 with lactate dehydrogenase did not confer improved thermotolerance. Taken together, the selected HSP70 from E. nindensis did not appear to be involved in stress response and is likely involved with general proteostasis. Lastly, a method for generating embryonic calli from Eragrostis tef is presented with the goal of using this developed protocol for the genetic improvement of the Ethiopian orphan crop.
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    In vitro tissue culture: Towards conservation of threatened desiccation sensitive Encephalartos cycads seeds
    (2019) Malwane, Thembeka Sebenzile Desiree; Farrant, Jill Margaret; Xaba, Phakamani M’Afrika; Donaldson, John
    Approximately 62% of the 355 cycad species in the world are classified as threatened with extinction. The African genus, Encephalartos, has a total of 65 species, approximately 70% of which are threatened. This status emphasizes the need to conserve these species; however, the recalcitrant nature of cycads seeds makes it difficult to conserve using conventional seed banking methods. Recalcitrant seeds have a short lifespan and cannot be dried or stored for prolonged periods as they become non-viable when they lose moisture. While studies on cryopreservation for conserving cycad germplasm and banking these desiccation-sensitive seeds has made some advances, cycad conservation is still limited to ex situ living plant collections. In vitro tissue culture is a promising technique for conserving cycads. While attempts have been made, there have been few reported successes and, to date, there has been no successful regeneration of Encephalartos species. As such, this study was aimed at developing an efficient and successful in vitro regeneration protocol for two Encephalartos species. Embryo regeneration of E. altensteinii and E. manikensis was assessed, testing the effects of plant growth regulators (PGRs) - 0.5 mg/L Kinetin and 0.5 mg/L 6-BAP, alone or in combination, sucrose (0, 15 and 30 g/L) and light. Within six weeks of culture, embryos of both species were able to regenerate, however, each responded differently to the tested variables. While shoot regeneration was evident for both species during this period, this was however not explained by any of the variables assessed in this study. Rooting was highest in the treatments with 0.5 mg/L Kinetin for E. altensteinii, after subculture rooting was favoured by the treatments with 0.5 mg/L Kinetin + 0.5 mg/L 6-BAP. Encephalartos manikensis rooting was significantly higher in PGR-free treatment in the first six weeks of culture. After subculture, rooting was enhanced by the treatment with the highest PGR concentration of 1.0 mg/L Kinetin + 1.0 mg/L 6-BAP. Darkness enhanced rooting while 16h photoperiod enhanced shooting for both species. However, the regeneration of both roots and shoots was more prevalent in the treatments that were initiated in complete darkness as compared to the treatments initiated under 24h of light. Both species responded to sucrose; with increase in sucrose concentration, callus induction increased for E. altensteinii while, necrosis and contamination increased for E. manikensis. In vitro-derived E. altensteinii plantlets acclimatization was unsuccessful and only 3.5% of E. manikensis were successfully acclimatized. This study suggests that although both these species belong to the same genus, in vitro culture protocol should be species specific. The overall regeneration of both species was however low, thus the second study assessed the levels of phytohormones in E. altensteinii seed tissues (embryos and megagametophytes) of the same age as those used in the in vitro regeneration. Phytohormones, as well as multiple phytohormone interactions (i.e. interplay between Abscisic acid (ABA) and Gibberellins (GAs)), play a role in the germination, growth and development of a plant. The high levels of a germination inhibiting ABA compared to the low levels of cytokinins and auxins, as well as the absence of GAs obtained in the assessed seed tissue, suggest that no real germination was taking place. Thus these results suggest that E. altensteinii seeds have a very slow developmental process with the likely chance that at this age they may be immature.
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    Investigating the physiological and metabolomic effects of Ecklonia maxima-derived biostimulant foliar application in ameliorating the effects of heat shock in tomato plants
    (2025) Dladla, Unathi; Rafudeen, Mohamed Suhail; Farrant, Jill Margaret; Moola, Naadirah
    Tomato (Solanum Lycopersicum) is a globally popular horticultural commodity with great economic importance and is highly susceptible to heat shock and heat stress. Heat shock has evidenced detrimental effects on plant viability and growth, limiting crop productivity and quality. In addition to physical damage, structural damage to plant cell walls and membranes and the overproduction of reactive oxygen species (ROS) also cause metabolic and cellular disturbances. Concurrently, there has been a growing demand for sustainable and affordable agricultural practices using eco-friendly approaches to increase the heat tolerance of crops. Biostimulants are substances or microorganisms that help improve plant growth, yield, nutrient content and quality and can also enhance plant tolerance to different abiotic stresses either as stress priming agents or mitigating the stress directly. In South Africa, commercial biostimulant manufacture is focused on the brown algae (Phaeophyta), Ecklonia maxima (Osbeck) which grows and is harvested along the southern Atlantic coast of Africa. In this study, the aim was to assess and determine whether the prior foliar application of Ecklonia maxima-derived biostimulant on tomato plants could assist in improving the tolerance of tomato plants to subsequent heat shock stress. The focus was on the physiological, biochemical and metabolic responses of tomato plants treated with or without E. maxima-derived biostimulant and subject to heat shock stress. This was achieved through different plant physiological and biochemical approaches that include electrolyte leakage assay, chlorophyll fluorescence and photosynthetic pigment measurements, FRAP (ferric reducing antioxidant power) and DPPH (2,2-diphenyl1-picrylhydrazyl) measurements and lipid peroxidation and proline assays. In addition, changes in the primary metabolites of the treated tomatoes were measured using gas chromatography mass spectrometry (GC-MS) to further elucidate the metabolic pathways involved in the responses to the different treatments. From the findings, it was shown that a prior foliar application of E. maxima-derived biostimulant resulted in better photosynthetic efficiency and a decrease in the amount of electrolyte leakage from plant cells when subsequently exposed to heat shock stress compared to control plants without prior biostimulant application. This indicates improved cell membrane integrity and enhanced thermotolerance of E. maxima treated plants in response to heat shock stress. There was also a reduction in lipid peroxidation and proline content in heat shocked plants treated prior with E. maxima-derived biostimulant, indicating enhanced ROS scavenging and antioxidant systems in these biostimulant treated plants. The metabolic analysis of the shoots from heat shocked plants that were prior treated with E. maxima-derived biostimulant identified key sugars, organic acids and amino acids. These included phenylalanine, valine, proline, threonine, myo-inositol, citric acid, mannitol, and succinic acid. The identified primary metabolites are linked to the promotion of plant growth by increasing chlorophyll content and mitigate stress by assisting in reducing the levels of ROS in plants and improving the antioxidant defence system. This study showed that the E. maxima-derived biostimulant acts as a priming agent to enhance and protect photosynthesis while improving thermotolerance to heat shock stress by directly and/or indirectly enhancing antioxidant capacity in the plants.
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    Investigating the role of phytohormones during desiccation in two evolutionarily distinct resurrection plants
    (2025) Kirchner, Sabine Maye; Farrant, Jill Margaret; Vothknecht, Ute; Van de Pas, Llewelyn
    Most plants encounter challenges brought on by various abiotic stressors which subsequently prompt adaptations to mitigate these challenges. Water deficit brought about by drought conditions is a significant abiotic stressor that impacts crop productivity, ultimately threatening global food security. Significant effort has thus gone into researching the unique adaptations that have allowed a remarkable group of angiosperms termed resurrection plants, to survive extreme water loss. This thesis focused on profiling phytohormone abundances in two evolutionarily distinct resurrection plant species; Craterostigma pumilum and Xerophyta schlechteri, during a dehydration time course. The objective was to elucidate some of the molecular processes that occur in the context of differing desiccation tolerance strategies, namely homoiochlorophylly and poikilochlorophylly. Hormone profiles were then followed up with gene expression analysis of desiccation-responsive genes RD29B and GASA3 in C. pumilum to further explore hormone signalling associated with jasmonates. In order to do this, and because the major findings on the roles of these genes in relation to jasomonate emanate from work on the model plant Arabidopsis thaliana, protocols designed for their analyses in that species were tested against C. pumilum to investigate their effectiveness when implemented in a non-model species. Species-specific hormone profiles were identified not only between the two resurrection plants, but also within their leaf and root tissues. ABA emerged as a central regulator of stress responses, while jasmonic acid (JA) appeared to play more of a supporting role, and the dynamics of OPDA, a precursor of JA, suggested a potential alternative signalling pathway that may occur in resurrection plants during desiccation. Overall, the findings point to species-specific hormone profiles that may be unique to resurrection plants and underscore the complexity of hormonal interactions in plant responses to water deficit stress. Additionally, results highlighted the need for further optimization of laboratory protocols designed with specific species in mind and warn against a potential over-reliance on protocols designed for model species.
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    Label-free proteomic analysis of Xerophyta schlechteri leaf tissue under dehydration stress
    (2021) Gabier, Hawwa; Rafudeen, Mohamed; Farrant, Jill Margaret
    Most higher plants cannot withstand severe water loss, except for a small group of angiosperms called resurrection plants. They can survive severe water loss without the loss of viability by employing mechanisms that aid them in desiccation tolerance. Desiccation tolerance in resurrection plants is a complex and multifaceted phenomenon and allows the plant to implement various strategies for survival. The focus of this study was a label-free proteomic analysis of Xerophyta schlechteri, a monocotyledonous and poikilochlorophyllous resurrection plant, in response to desiccation. The study investigated some of the physiological, morphological and biochemical changes of X. schlechteri leaf tissue in response to dehydration followed by proteomic analyses using a spectral counting approach. The differentially expressed proteins were identified and quantified and then subjected to gene ontological analyses to identify relevant biological processes involved in desiccation tolerance. The proteomic data was finally correlated to and validated using metabolomic analyses. X. schlechteri was subjected to a controlled dehydration stress treatment, in which changes in the relative water content (RWC) of leaf tissues, the associated changes in processes outlined above and further expanded on below, were determined. Three physiological stages were tentatively identified, namely, the early response to drying (ERD) which represents ~ 80 - 70% RWC (1.61 gH2O g ̄ˡ dwt -1.5 gH2O g ̄ˡ dwt), a mid-response to drying (MRD) represented by ~ 60 - 40% RWC (1.5 gH2O g ̄ˡ dwt -1.0 gH2O g ̄ˡ dwt) and a late response to drying (LRD), represented by ~ 40 - 10% RWC (1.0 gH2O g ̄ˡ dwt - 0.5 gH2O g ̄ˡ dwt). Morphological changes in the late stages of drying were marked by loss of green chlorophyll, increased purple anthocyanin production and leaf folding along the midrib with the abaxial surface exposed to light. Chlorophyll content analyses showed a significant decrease in chlorophyll content in the dehydrated leaf tissue as compared to the fully hydrated state. Biochemical assays to measure the activity of enzymatic antioxidants, namely, ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR) and superoxide dismutase (SOD) were done at selected RWC points. There was a significant increase in antioxidant enzyme activity for APX, CAT, GR and SOD in the dehydrated plant tissue. The label-free proteomics approach utilized, identified a total of 3125 unique proteins in the X. schlechteri leaf tissue across the dehydration treatment of which a combined 517 proteins were significantly differentially expressed in response to drying. Amongst the differentially expressed proteins, 253 proteins were upregulated, and 264 proteins were downregulated. This was followed by functional analyses and classification of gene ontologies using bioinformatics tools such as Blast2GO, MapMan and KEGG. This allowed the identification of certain biological processes and pathways involved in the X. schlechteri desiccation response. Key biological processes and molecular processes were differentially expressed across the drying stages, these included photosynthesis, cellular respiration and antioxidant activity, respectively. The proteomic analysis was complemented and validated using metabolomics approaches based on GC MS/MS and LC/MS. The abundance of specific sugars, sugar alcohols, fatty acids, organic acids, phytohormones and amino acids of X. schlechteri during desiccation were investigated. Sugars such as raffinose and sucrose are known to play a protective role in desiccation and were found to be abundant in MRD and LRD leaf tissue while, L-histidine, an amino acid which plays a critical role in plant growth, was found to be more abundant in LRD tissue as compared to MRD. The phytohormone abscisic acid, invoked in desiccation tolerance was found to be abundant at LRD and less abundant at ERD. The metabolomic data suggested that the regulation of metabolites was towards reducing possible toxic metabolites while increasing the expression of metabolites that help and protect plant cell integrity from the negative effects of desiccation. The use of a label-free proteomics approach complemented with metabolomics allowed the identification and validation of biological processes and pathways potentially involved in establishing desiccation tolerance in X. schlechteri. As far as we are aware, this is the first label-free proteomic analysis of X. schlechteri in response to dehydration.
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    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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    The functional role of root-associated microbiome and metabolome of myrothamnus flabellifolia
    (2025) Tebele, Shandry Mmasetshaba; Farrant, Jill Margaret
    Global climate change is predicted to increase the occurrence and severity of drought, particularly in Africa, which will negatively impact crops and food production. Drought is the leading factor that adversely affects agricultural productivity and yield. Over the last four decades, extensive research on resurrection plants has yielded valuable insights into the mechanisms these plants employ to adapt during desiccation. Despite this, the role of the microbiome in desiccation tolerance, particularly in resurrection plants, remains a relatively unexplored area. Myrothamnus flabellifolia, a resurrection plant, stands out for its remarkable ability to endure severe desiccation, making it an ideal model for investigating the contributions of the plant microbiome to desiccation tolerance. Recognising the significance of root-associated microbes in stress tolerance opens up promising opportunities for enhancing drought resilience in crucial crops. However, the intricate dynamics of these interactions under severe water limitations have not been comprehensively investigated. Consequently, a primary objective of this study was to unravel the beneficial root-associated microbiome of M. flabellifolia and delineate their functions in the context of water deficit conditions. The intricate tripartite interplay involving plant roots, soil, and microorganisms remains enigmatic and demands further exploration. This study delved into the microbiome of belowground zones—bulk soil, rhizosphere soil, and endosphere of M. flabellifolia. Metagenomic analysis unveiled prevalent bacterial phyla (Acidobacteriota, Actinobacteriota, Chloroflexota, Planctomycetota, and Pseudomonadota) and dominant fungal phyla (Ascomycota and Basidiomycota) across all zones. While the bulk soil hosted numerous beneficial root-associated microbes, it exhibited lower functional diversity than the rhizosphere, which showcased the highest diversity of bacteria and fungi. Conversely, the endosphere exhibited lower microbial abundance and diversity. These findings suggest that M. flabellifolia may recruits soil microbes from bulk soil to rhizosphere and subsequently to the endosphere. Metatranscriptomic analysis has revealed crucial insights into the dynamics of plant-microbe interactions and the adaptive mechanisms employed by root-associated bacteria during desiccation in M. flabellifolia. The transcriptional activity of bacteria involved both monoderm and diderm lineages, consistent with the bacterial phyla identified in metagenomic analysis. However, the dominance of the Pseudomonadota phyla at the transcriptional activity was observed. Root-associated bacteria showed distinct transcriptional responses during dehydration and rehydration, suggesting dynamic shifts in microbial activity under fluctuating water availability. The expression of differentially expressed genes (DEGs) under dehydration conditions showcased the activation of proteins associated with antioxidant enzymes, molecular chaperones, protein kinases, and biosynthesis of sugars and amino acids. This implies a coordinated response to counteract damage and enhance survival. Intriguingly, the upregulation of genes encoding protein kinases, antioxidant enzymes, and trehalose synthase in root-associated bacteria reflects a common strategy for surviving desiccation stress. This suggests a potential case of convergent evolution in desiccation tolerance within microbiomes. The observed upregulation of genes related to plant growth and enhanced plant-microbe interaction under rehydration conditions suggests a resumption of microbial activity. Exploring the rhizosphere soil metabolome provided insights into the metabolic changes during drought stress in M. flabellifolia. Dehydrated rhizosphere soil exhibited increased levels of sugars (e.g., trehalose), organic acids (malic acid), and phytohormones (indole-3-acetic). Conversely, rehydrated rhizosphere samples showed significantly higher amino acid levels compared to desiccated samples, indicating a shift in biochemical processes in both the plant roots and rhizosphere microbiome. While rhizosphere metabolites are typically attributed to root exudates and microbial activity, this study revealed that many were possibly produced by rhizospheric bacteria. The upregulation of bacterial genes associated with metabolite biosynthesis under dehydration conditions, such as trehalose, further substantiated the the notion that drought serves as a selective pressure driving convergent evolution in species with desiccation tolerance. These findings indicates that the microbiome's adaptability under harsh environmental stress. Furthermore, inoculating maize plants with rhizospheric bacteria from M. flabellifolia's rhizosphere significantly improved drought tolerance, physiological, and morphological traits. The study concludes that root-associated microbiomes play a crucial role in M. flabellifolia's desiccation tolerance and plant growth-promoting microbes have a potential to be used as a biostimulant. This innovative research has implications for enhancing food security, developing resilient agricultural systems, and promoting sustainability.
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    Transcriptional regulation of seasonal desiccation tolerance in the fronds and rhizome of the fern Anemia caffrorum
    (2023) Khan, Farrah; Farrant, Jill Margaret; Rafudeen Mohamed
    Agriculture in drought-prone regions of the world is at increasing risk as climate change causes more intense and extended droughts. Desiccation tolerant plants are capable of surviving long periods of severe water loss (> 95% of cellular water) and are therefore prominent models for the production of climate smart crops. The fern Anemia caffrorum is a particularly interesting model as it is capable of seasonal desiccation tolerance. In winter, when water availability is high, desiccation sensitive (DS) fronds are produced by the rhizome. In summer, when water availability is low, desiccation tolerant (DT) fronds are produced. Seasonal desiccation tolerance conferred to crops would allow for the maximization of food yields when water is available and prioritization towards survival when water is scarce. In this study transcriptional regulation of A. caffrorum desiccation tolerance in the fronds and rhizomes was explored. In particular, rhizomes were of interest as they have been postulated as the regulator of frond phenotype (DS/DT). Fronds and rhizomes were subjected to desiccation and tissues of different water contents were collected. Tissue from the fronds of both phenotypes (DS/DT) at full turgor (approximately 100% RWC), 55% RWC, 30% RWC, 10% RWC and 24-hours post rehydration were chosen. Tissue from the rhizomes producing DS fronds (winter rhizomes) and those producing DT fronds (summer rhizomes) was collected at 100% RWC and 30% RWC. Methods for isolating total RNA from all tissue types, of sufficient purity and yield, for next generation sequencing (NGS) experimentation were established. These samples were used to conduct short- and long-read sequencing for transcriptome assembly and gene expression studies. Transcriptome assembly using long read data outperformed de novo short read assembly as it resulted in greater rates of annotation and yielded a less fragmented assembly. The transcriptome was determined to be of a high-quality and was used to perform differential expression (DE) studies. DE studies showed that DS fronds had initiated senescent processes by 55% RWC which was only realised after rehydration. DT fronds were able to survive a desiccation event through several constitutive and inducible effects that were more reminiscent of desiccation tolerance in angiosperms than that of bryophytes. However, a relatively low transcriptional response compared to angiosperm desiccation tolerant plants, was a feature of bryophytic-type desiccation tolerance. Therefore A. caffrorum, like other ferns, demonstrated a mixed model of desiccation tolerance. DE studies of the rhizome provided several insights into canonical mechanisms of desiccation tolerance of the underground organ, alternate seasonal strategies, the role of the organ in frond phenotype regulation and cross-organ dynamics. Firstly, at 30% RWC rhizomes were postulated to be fully prepared for desiccation 3 and achieved this status through many common features observed in angiosperm resurrection plants. The strong presence of chloroplastic protective proteins in this response highlighted the likely transport of such transcripts to newly emerging fronds. In the reverse direction, rhizomes appeared to acquire sucrose and transcripts required for the recruitment of endophytic fungi from the fronds. Finally, severely dehydrated rhizomes showed prominent upregulation of genes that are responsible for leaf senescence, leaf development and initiation of spore production which was highly correlated with ABA-regulation. Also, under the control of ABA, was the production of transcripts that ultimately initiate the production of orange, trichomelike scales which are a key feature of the DT phenotype. Not under the control of ABA regulation, but relevant to the DT phenotype, were genes that promote dwarfism and reduced photosynthetic potential in fronds. Altogether, the study has provided possible candidate genes that may govern the DT phenotype and has underscored the significant interplay between these organs in achieving seasonal tolerance.