Browsing by Author "Mundree, Sagadevan G"
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- ItemOpen AccessCharacterisation of XvPrx2 : a type II peroxiredoxin isolated from the resurrection plant Xerophyta viscosa (Baker)(2006) Govender, Kershini; Mundree, Sagadevan G; Thomson, Jennifer AnnKnowledge of the biochemical and molecular mechanisms by which plants tolerate environmental stresses is necessary for genetic engineering approaches to improve crop performance. A unique feature of resurrection plants, such as Xerophyta viscosa, is their ability to cope with severe water loss of greater than 90%. A full-length cDNA library was synthesised from a cold stressed X viscosa plant. Sequencing and BLAST analysis revealed the identity of sixty genes. A type 2 peroxiredoxin (XvPrx2) was selected for further analyses as it was observed, by northern analyses, to be stress-inducible. The XvPrx2 protein was confirmed to be involved in the stress response by Western analyses. The XvPrx2 gene, which displays highest identity to a rice orthologue, has an open reading frame of 162 amino acids, and codes for a hydrophilic polypeptide of 162 residues with a predicted molecular weight of 17.5 kDa. The XvPrx2 polypeptide displays significant identity with other plant type II Prxs, with an absolutely conserved amino acid sequence proposed to constitute the active site of the enzyme (PGAFTPTCS). The XvPrx2 protein has a single cataly1ic cysteine residue at position 51 similar to Prxs from Oryza sativa and Candida boidinii. A mutated protein (XvV76C) was generated by converting the valine at position 76 to a cysteine resulting in a conformational change as determined by limited proteolysis. An in vitro DNA protection assay showed that, in the presence of either XvPrx2 or XvV76C, DNA protection occurred. In addition, an in vivo assay showed that increased protection was conferred on cell lines over-expressing either XvPrx2 or XvV76C. Several upstream promoter regions were identified for the XvPrx2 gene using the splinkerette method. Southern and two dimensional gel analyses revealed that multiple XvPrx2 homologues exist within the X viscosa genome. These homologues have similar pI values to Arabidopsis orthologues. Immuno-cytochemical data revealed that XvPrx2 is localised to the chloroplast, however, this could be attributed to cross reactivity with a chloroplastic homologue. Using YFP technology, the protein was observed to be expressed in the cytosol, and this location is supported by the absence of an upstream targeting signal in the XvPrx2 sequence. The XvPrx2 activity was maximal with DTT as electron donor and HzOz as substrate with t-BOOH being the next preferred. Using Trx£. coli a 2-15 fold lower enzyme activity was observed. The XvPrx2 activity with GSH was significantly lower and Grx had no measurable effect on this reaction. The XvV76C protein displayed significantly lower activity compared to XvPrx2 for all substrates assessed. Enzymatic kinetic parameter values determined for XvPrx2 using DTT as electron donor and HzOz as substrate were: Km = 45 IlM, V max = 278 Ilmol min-I.mg-I protein, kcat 6.173 x 103 s-1 and kcaJKm = 0.136 X 103 IlM-1.s-l. Based on knowledge-based models of XvPrx2 and XvV76C no structural differences were observed between the two molecules.
- ItemOpen AccessCloning and characterisation of a bZIP transcription factor from a resurrection grass, Eragrostis nindensis(2004) Brocklehurst, David; Farrant, Jill M; Mundree, Sagadevan G; Thomson, Jennifer AnnThe G-box is a plant DNA cis-acting element involved in the regulation of gene expression in response to a range of environmental signals including anaerobiosis, dehydration and light as well as by abscisic acid (ABA). Basic leucine zipper (bZIP) transcription factors have been shown to specifically bind and activate transcription from G-boxes in a dimerized form. A 1.5 kb cDNA for a bZIP class transcription factor, designated EnGBF1, was cloned from a desiccation-tolerant grass, Eragrostis nindensis by degenerate RT-PCR.
- ItemOpen AccessDesiccation-driven senescence in the resurrection plant Xerophyta schlechteri (Baker) N.L. Menezes(2019) Radermacher, Astrid Lillie; Farrant, Jill Margaret; Mundree, Sagadevan GDrought-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.
- ItemOpen AccessEragrostis nindensis: unravelling senescence in an African desiccation tolerant grass(2019) Madden, Christine Frances; Farrant, Jill Margaret; Mundree, Sagadevan GFood 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.
- ItemOpen AccessGene expression associated with drought tolerance in Xerophyta viscosa Baker(2000) Ndima, Tozama Beauty; Mundree, Sagadevan G; Farrant, Jill M; Thomson, Jennifer AnnHerophyta viscosa (Baker) is a monocytyledonous resurrection plant that can tolerate extremes of dessication. Upon rewatering, it rehydrates completely and assumes its full physiological activities. Studies on changes in gene expression associated with dehydration stress tolerance were conducted. A cDNA library constructed from m RNA isolated from dehydrated (85%, 37% and 5% relative water content) X. viscosa leaves, was differently screened. Of the 192 randomly selected cDNAs screened, 30 showed higher expression levels when X. viscosa was dehydrated while 20 showed lower expession. XVLEA, XVDH and XVLEC represent three cDNAs that were upregulated during dehydration stress. XVLEA showed the highest identity at the amino acid level with a late embryogenesis abundant protein, LEA29G, from Gossipium hirsutum (30%) and LEA D-29 from cotton (50%). XVDH exhibited significant identity to dehydrin proteins from Arabidopsis thaliana (45%) and Pisum sativum (43%) at the amino acid level. It encodes a glycine-rich protein (27kDa) which is largely hydrophilic and contains a hydrophobic segment at the C-terminus. XVLEC showed 28% identity and 50% similarity to a lectin-like protein from Arabidopsis thaliana. Southern blot analysis confirmed the presence of the three cDNAs in the X.viscosa genome. Both XVLEA and XVDH transcripts were highly expressed during dehydration- (37% RWC) and rehydration (4%, 32%, 72% RWC) treatment of the plant ͌ 1.0kb was observed. However, with XVDH a transcript of ͌ 1.0 kb and 1.09 kb were observed. XVDH transcripts accumulated in X. viscosa plants in response to low temperature, heat and dehydration stresses, as well as to exogenous supply of abscisic acid, ethylene and methyl jasmonate. Localization studies of the XVDH encoded protein showed that XVDH is located in the plasma membrane-cell wall region.
- ItemOpen AccessGenetic effects of prolonged UV-B exposure in a Namaqualand daisy - Dimorphotheca sinuata(2001) Mpoloka, Sununguko Wata; Thomson, Jennifer Ann; Abratt, Valerie Rose; Mundree, Sagadevan G; Riddoch, BruceThis thesis describes investigations into the genetic effects of long term UV-B exposure in Namaqualand daisies (Dimorphotheea sinuata) grown for several generations under ambient and enhanced UV-B levels. Enhanced UV-B radiation was found to have a major effect on the biochemical composition of the chloroplast accompanied by impairment of photosynthetic function, involving a down-regulation of photosynthetic genes and an up-regulation of flavonoid biosynthesis.
- ItemOpen AccessIsolation and characterisation of an Hsp90 homologue from the resurrection plant Xerophyta viscosa(2002) Walford, Sally-Ann; Mundree, Sagadevan G; Thomson, Jennifer Ann; Farrant, Jill MPrior to this study, a eDNA library of dehydrated Xerophyta viscosa was differentially screened and several genes were found to be upregulated during dehydration. One of these cDNAs was found to share a high degree of sequence identity with the ER-Iocated Hsp90 or Grp94 family of proteins (hereafter referred to as XVGrp94) and forms the basis of this work. The XVGrp94 eDNA was found to be truncated at the 5· terminus and a full length eDNA was isolated using SMART-RACE™ (§witching Mechanism gt 5' end of RNA Iranscript- Random ~mplification of Complementary .!;rids). This eDNA was sequenced and appeared to be a representative of the Hsp90 family of genes. The putative gene contained an ORF (Open Reading frame) potentially coding for an 812 amino acid protein with a calculated size of 92.83 kDa. It shares 85% homology with other Hsp90s from plants and it contains several characteristic features of these proteins. Additionally, it contains the ER (endoplasmic reticulum) targeting and retention signals. Southern blot analysis confirmed the presence of the gene in the X. viscosa genome possibly as a member of a family of closely related genes. Northern blot analysis revealed a transcript size of 2.8 kb, however, expression patterns of the transcript could not be established. Western blot analysis showed that the XVGrp94 concentration increased significantly in response to heat and dehydration, and a slight increase was observed in response to conditions of high salt, but no response was seen in response to high light, cold or exogenous ABA (abscisic acid) application. The XVGrp94 open reading frame was cloned into the pProEX HTa expression vector and expressed in E. coli, but purification of the recombinant protein was not successful.
- ItemOpen AccessIsolation of the aldose reductase gene (XvAld1) from the resurrection plant Xerophyta viscosa, and characterisation of the gene product and transgenic plants expressing the gene(2007) Maredza, Alice T; Thomson, Jennifer Ann; Farrant, Jill M; Mundree, Sagadevan GThe Xerophyta viscosa aldose reductase cDNA (XvAld1) was isolated from a dehydration library. Gene transcripts that are upregulated during stress are normally involved in protection and/ or adaptation, leading to stress tolerance. The genomic organisation of XvAld1 was characterised using Southern blot analysis and DNA sequencing. The results revealed more than one copy of the gene with a complex banding pattern that was partially resolved by sequencing. The sequencing of PCRamplified genomic clones showed that the gene is organised into nine exons and eight introns spanning ~2.9 kb. The observed nucleotide differences between the sequenced clones could reflect polymorphisms between different copies of the gene. An 870-bp clone of the 5′ untranslated region, matching the 5′ leader sequence on the XvAld1 cDNA was analysed for cis-acting response elements. Many of the sequence motifs matched those for hormonal regulation, organ specific expression, dehydration, high or low temperature responses, light and phytochrome responsiveness, wounding, as well as G-box, CAAT and TATA-boxes.
- ItemOpen AccessMolecular characterization of ALDRXv4, an aldose reductase orthologue isolated from xerophyta viscosa, in response to abiotic stress(2005) Davis, Emily; Thomson, Jennifer Ann; Farrant, Jill M; Mundree, Sagadevan GIncludes bibliographical references (leaves 61-70).
- ItemOpen AccessMolecular characterization of XvlNO1, a myo-inositol 1-phosphate synthase gene from Xerophyta viscosa(2005) Chopera, Denis Rutendo; Mundree, Sagadevan G; Farrant, Jill M; Thomson, Jennifer AnnMyo-inositol I-phosphate synthase (INO 1) catalyses the conversion of glucose-6-phosphate to myo-inositol I-phosphate, which is subsequently dephosphorylated to myo-inositol. Myo-inositol is a precursor for a number of important metabolites that include membrane components, storage molecules, phytohormones and a variety of osmoprotectants. Xerophyta viscosa Baker (Family Velloziaceae) is a monocotyledonous angiosperm which has the ability to resume full physiological function after desiccation. The full-length cDNA for INO1 from X viscosa was isolated using the RACE technique.
- ItemOpen AccessMolecular characterization of XvPer1, a novel antioxidant enzyme from the resurrection plant Xerophyta viscosa, and AC3, a LEA-like protein from Arabidopsis thaliana(2005) Mowla, Shaheen B; Mundree, Sagadevan G; Thomson, Jennifer Ann; Farrant, Jill M; Foyer, C H; Theodoulou, FBy differential screening of a cDNA library of the resurrection plant Xerophyta viscosa, a cDNA termed XvPer I was isolated for its over-expression under dehydration stress. Analysis of the cDNA sequence indicated a hydrophilic protein of a predicted Mw of 24.2 kDa with high identity to plant l-Cys peroxiredoxins, a novel antioxidant enzyme. Southern blot analysis revealed that XvPer I was most probably a single copy gene. The polypeptide sequence had significant identities (-70%) with other recently identified plant I-Cys peroxiredoxins with an absolutely conserved active site (PVCTTE). The protein sequence also had a putative bipartite nuclear localization signal.
- ItemOpen AccessA molecular physiological review of vegetative desiccation tolerance in the resurrection plant Xerophyta viscosa (Baker)(Springer, 2015-08) Farrant, Jill M; Cooper, Keren; Hilgart, Amelia; Abdalla, Kamal O; Bentley, Joanne; Thomson, Jennifer Ann; Dace, Halford; Mundree, Sagadevan G; Rafudeen, Mohamed SXerophyta viscosa (Baker) is a monocotyledonous resurrection plant from the family Vellociacea that occurs in summer-rainfall areas of South Africa, Lesotho and Swaziland. It inhabits rocky terrain in exposed grasslands and frequently experiences periods of water deficit. Being a resurrection plant it tolerates the loss of 95 % of total cellular water, regaining full metabolic competency within 3 days of rehydration. In this paper, we review some of the molecular and physiological adaptations that occur during various stages of dehydration of X. viscosa, these being functionally grouped into early and late responses, which might be relevant to the attainment of desiccation tolerance. During early drying (to 55 % RWC) photosynthesis is shut down, there is increased presence and activity of housekeeping antioxidants and a redirection of metabolism to the increased formation of sucrose and raffinose family oligosaccharides. Other metabolic shifts suggest water replacement in vacuoles proposed to facilitate mechanical stabilization. Some regulatory processes observed include increased presence of a linker histone H1 variant, a Type 2C protein phosphatase, a calmodulin- and an ERD15-like protein. During the late stages of drying (to 10 % RWC) there was increased expression of several proteins involved in signal transduction, and retroelements speculated to be instrumental in gene silencing. There was induction of antioxidants not typically found in desiccation-sensitive systems, classical stress-associated proteins (HSP and LEAs), proteins involved in structural stabilization and those associated with changes in various metabolite pools during drying. Metabolites accumulated in this stage are proposed, inter alia, to facilitate subcellular stabilization by vitrification process which can include glass- and ionic liquid formation.
- ItemRestrictedA novel stress-inducible antioxidant enzyme identified from the resurrection plant Xerophyta viscosa Baker.(Springer, 2002) Mowla, Shaheen B; Thomson, Jennifer A; Farrant, Jill; Mundree, Sagadevan GA cDNA corresponding to 1-Cys peroxiredoxin, an evolutionarily conserved thiol-specific antioxidant enzyme, was isolated from Xerophyta viscosa Baker, a resurrection plant indigenous to Southern Africa and belonging to the family Velloziaceae. The cDNA, designated XvPer1, contains an open reading frame that encodes a polypeptide of 219 residues with a predicted molecular weight of 24.2 kDa. The XvPer1 polypeptide shows significant sequence identity (approx. 70%) to other recently identified plant 1-Cys peroxiredoxins and relatively high levels of sequence similarity (approx. 40%) to non-plant 1-Cys peroxiredoxins. The XvPer1 cDNA contains a putative polyadenylation site. As for all 1-Cys peroxiredoxins identified to date, the amino acid sequence proposed to constitute the active site of the enzyme, PVCTTE, is highly conserved in XvPer1. It also contains a putative bipartite nuclear localization signal. Southern blot analysis revealed that there is a single copy of XvPer1 in the X. viscosa genome. All angiosperm 1-Cys peroxiredoxins described to date are seed-specific and absent in vegetative tissues even under stress conditions; therefore, XvPer1 is unique in that it is expressed in the vegetative tissues of X. viscosa. The XvPer1 transcript was absent in fully hydrated X. viscosa tissue but levels increased in tissues subjected to abiotic stresses such as dehydration, heat (42 °C), high light intensity (1,500 µmol photons m–2 s–1) and when treated with abscisic acid (100 µM ABA) and sodium chloride (100 mM NaCl). Western blot analyses correlated with the patterns of expression of XvPer1 transcripts under different stress conditions. Immunofluorescence analyses revealed that XvPer1 is localized in the nucleus of dehydrated X. viscosa leaf cells. These results suggest that XvPer1 is a stress-inducible gene, which may function to protect nucleic acids within the nucleus against oxidative injury.
- ItemOpen AccessXvERD15, an early-responsive gene to stress from Xerophyte viscosa(2005) Lee, Ming-Yi; Mundree, Sagadevan G; Thomson, Jennifer Ann; Farrant, Jill MGenes that are upregulated in the early response to stress are not well understood. ERD15 (early-responsive to dehydration) in Arabidopsis and its homologues in various other plants have been shown to be upregulated within I hr post-exposure to dehydration and high salinity stress treatments. There is however limited literature on the functionality of ERD15. A cDNA showing homology to ERD15 was isolated from a library generated by low temperature stress treatment of Xerophyta viscosa and was subsequently named XvERD15.
- ItemOpen AccessXvVHA-c``1- a novel stress responsive V-ATPase subunit c`` homologue isolated from the resurrection plant Xerophyta viscosa Baker(2004) Marais, Saberi; Mundree, Sagadevan G; Thomson, Jennifer Ann; Farrant, Jill MIncludes bibliographical references.