Browsing by Author "Ingle, Robert"

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    Open Access
    Characterisation of auxin and auxin-related genes in the response of Arabidopsis thaliana to salt stress
    (2019) Cackett, Lee; Donaldson, Lara; Ingle, Robert
    Soil salinization is prominent in agricultural land and has detrimental effects on plant growth and yield. Salt imposes both an osmotic and ionic stress on plants, and the aim of this study was to investigate the molecular basis of the plant response to the ionic component of salinity stress. To do so, transcriptome profiling using microarrays was performed at two developmental stages (two and four weeks post-germination) exposing plants to either NaCl (which imposes an osmotic and ionic stress) or iso-osmolar sorbitol (which imposes osmotic stress only). Clear transcriptomic differences in the plant response to NaCl and sorbitol were observed, allowing the identification of genes that may be involved specifically in the response to the ionic component of salinity stress. Differences in salt-responsive gene expression were also observed between early and later development as well as root and shoot tissues, indicating that there may be both developmental and tissue specific responses to NaCl. ‘Response to auxin’ was a highly enriched gene ontology term associated with genes that are significantly induced specifically in response to ionic stress, suggesting a potential role for this plant growth hormone in ionic stress tolerance. This hypothesis was supported by mass spectrometry analysis which demonstrated that active IAA levels show a significantly greater increase in response to NaCl than to sorbitol, demonstrating an ionic specific auxin response. In planta quantification and spatial distribution of IAA was further analysed in salt stressed plants using two auxin reporter lines, DR5::GUS and DII::VENUS. These analyses showed that IAA levels increased in the shoot and root tip in response to NaCl. Also, local IAA maxima distributed along the primary root of salt stressed seedlings were decreased compared to the untreated control, which may explain the decrease in lateral root number and primary root bending observed in salt stressed plants. There are several different pathways in the tryptophandependent auxin biosynthesis process, each with unique biosynthetic intermediates and ratelimiting enzymes. For this reason, changes in the levels of the different IAA biosynthetic intermediates were analysed in NaCl and sorbitol stressed Arabidopsis to identify a predominant pathway responsible for increasing IAA during ionic stress. The changes in IAN (the IAA biosynthetic intermediate of the IAOx pathway) in response to NaCl and sorbitol mirrored those observed for IAA. Additionally, mRNA levels of Nitrilase 2 (which converts IAN to IAA) were significantly increased in response to NaCl but not sorbitol. Taken together, these results suggest that IAA biosynthesis is increased in response to NaCl via the hydrolysis of IAN to IAA by NIT2. In agreement with this, a Nit2 overexpressing line (35s::Nit2) displayed both improved survival and growth in the presence of NaCl. Furthermore, the Nit2 overexpressor had increased IAA but deceased IAN compared to wild type plants in response to salt stress. Together these results indicate that Nit2 is likely involved in the salt stress response through altering IAA levels in planta. GH3.12 and ILL6, two genes involved in IAA storage and/or degradation via hydrolysis and conjugation to amino acids, were also differentially expressed in response to NaCl at both developmental stages tested. However, mutants for ill6 and gh3.12 were not altered in their salt tolerance, suggesting that IAA storage and/or degradation may not play an important role in modulating active IAA levels in response to salt stress. Finally, preliminary evidence was obtained that the expression of sorghum Nit2 homologs is also increased in response to salt stress, implying that auxin might play a role in salt tolerance across a phylogenetically broad range of plants, making this a potential route to improve salt tolerance in crop plants. Overall, this study provides novel information indicating that IAA levels are altered specifically in response to the ionic component of salinity stress which may contribute to the altered plant growth observed under these conditions, as well as the identification of an auxin-related candidate gene which can improve salt tolerance in plants when overexpressed.
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    Open Access
    Characterization of the intraspecific variation within the nickel (Ni) hyperaccumulator species Senecio coronatus (Asteraceae): a preliminary analysis of genetic population structure and shoot proteome expression
    (2013) Wolf, Michael; Ingle, Robert; Bishop, Jacqueline; Muasya, Muthama
    Heavy metal (HM) accumulator plants possess the ability to actively hyperaccumulate and detoxify exceptionally high concentrations of metals in their aboveground tissues, without exhibiting any apparent signs of toxicity. Despite nickel (Ni) hyperaccumulator plants representing the largest percentage of known metal accumulator taxa (over 75%), the underlying genetic and molecular basis of Ni accumulation remains unclear. A prominent difficulty in understanding Ni hyperaccumulation has been the severe lack of intraspecific variation in the trait. Hence, the study of a single species exhibiting a significant degree of variation is highly desirable. as it avoids the use of inter-species comparative studies mostly utilized to date. The Ni hyperaccumulator Senecio coronatus (Asteraceae) has been reported to contain a significant degree of phenotypic plasticity with respect to the amount accumulated and subsequent cellular distribution of Ni. This apparent intraspecific variation means that S. coronatus may represent a useful system in which to study Ni hyperaccumulation. No population genetics study has been carried out to date on this species, and the evolutionary relationships between hyper and non- accumulator populations were unknown. Here, results are presented from a genetic analysis of 15 naturally occurring S. coronatus populations. Analysis of molecular variance (AMOVA) and phylogenetic analysis (based on non-coding nuclear and plastid markers) suggest that Ni accumulation may have evolved twice within S. coronatus, as hyperaccumulator plants from site Kaapsehoop, cluster with non-accumulating serpentine populations and demonstrate distinct genetic differentiation from other accumulator populations. Four populations were selected for a preliminary comparative shoot proteome analysis by means of two-dimensional SDS-polyacrylamide gel electrophoresis (2D SDS-PAGE) to identify proteins potentially involved in Ni hyperaccumulation. This analysis identified nine chloroplastic proteins involved in plant energy production and metabolism as overexpressed in hyperaccumulator plants from Agnus Mine and Kaapsehoop, compared to hypertolerant non-accumulator and non-serpentine plants from Galaxy Mine and Pullen Farm, respectively. However, no difference in photosynthetic efficiency, as determined by chlorophyll fluorescence measurements, was detected between these populations.
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    Engineering cyanide-tolerant Arabidopsis thaliana
    (2015) Molojwane, Emang Tsametse Emi; Ingle, Robert
    Cyanide is highly toxic as it inhibits respiration in aerobic organisms by binding to cytochrome c oxidase in the mitochondrial electron transport chain. Plants naturally produce cyanide from the hydrolysis of cyanogenic glycosides and as a by-product of ethylene biosynthesis. β-Cyanoalanine synthase prevents self-poisoning by combining endogenous cyanide with cysteine in the mitochondria to form β-cyanoalanine, which is further hydrolysed to asparagine, or aspartate and ammonia, by plant nitrilase 4 enzymes. β-Cyanoalanine synthase activity enables plants to detoxify limited concentrations of exogenous cyanide. However, phytotoxicity and death occur from exposure to relatively low concentrations of exogenous cyanide. In contrast, some microorganisms have a high capacity for cyanide detoxification due to a number of metabolic pathways including the degradation of cyanide to formate and ammonia; or formamide, by bacterial cyanidase (CynD) and fungal cyanide hydratase (CHT), respectively. Environmental contamination caused by failure to contain cyanide from anthropogenic sources is an important global problem. Hydrometallurgical gold mining utilises cyanide as a lixiviant due to the high affinity of cyanide for gold and the stability of the resulting cyanometallic complexes in aqueous solution, and thus is a significant source of cyanide contamination of soil and water. Biological treatment methods for cyanide, such as phytoremediation, could provide alternatives to the currently used chemical destruction techniques with their associated disadvantages. The use of phytoremediation would require plants to tolerate high concentrations of cyanide in soil. Two attempts have previously been made, with some success, to increase cyanide tolerance in Arabidopsis by genetic engineering: the first, by augmenting the β-cyanoalanine synthase pathway using a microbial nitrilase; and, the second, by introducing a microbial detoxification pathway targeted to the chloroplasts while overexpressing the endogenous enzyme which metabolises the product of the cyanide detoxification reaction. The aim of the current study was to determine whether Arabidopsis thaliana could co-opt the CynD and CHT genes from the cyanide-degrading Bacillus pumilus and Neurospora crassa to detoxify higher levels of cyanide using the encoded enzymes, and whether targeting CynD and CHT to the mitochondria would confer a greater enhancement of cyanide tolerance on plants compared to targeting to the cytoplasm.
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    Identification of the cir1 disease resistance gene in Arabidopsis thaliana
    (2012) Diener, Anastashia; Ingle, Robert
    Plants rely on an elaborate multi-layered defence system to perceive and effectively respond to disease causing pathogens. The defence-related cir1 (constitutively induced resistance 1) mutant was first isolated in an effort to identify components of the Arabidopsis thaliana defence system essential for resistance against pathogens. The cir1 mutant has previously been described as having increased resistance to the virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and oomycete pathogen Hyaloperonospora parasitica Noco2 and was shown to constitutively express salicyclic acid-, jasmonic acid/ethylene- and reactive oxygen intermediate-responsive genes. Genetic analysis and mapping studies of the mutation revealed that it is recessive and may be encoded by one of eight genes located within a 309.10 kb region on the lower arm of chromosome four.
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    Investigating the biological roles of the HSPRO genes in Arabidopsis thaliana
    (2015) Guzha, Delroy Tapiwa; Ingle, Robert
    As a consequence of an immobile lifestyle, plants have had to evolve appropriate perception mechanisms and responses to diverse environmental stresses. Stress can be the result of both biotic and abiotic agents and the ORTHOLOG OF SUGAR BEET HS1 PRO-1 (HSPRO 1) and HSPRO2 genes were previously shown to be induced in response to several stresses including infection with Pseudomonas syringae and drought stress in Arabidopsis thaliana. The aim of this study was to characterise the biological role(s) played by these proteins in Arabidopsis. Several bioinformatics approaches provided evidence that supported function of both genes in response to both biotic and abiotic stresses and identified potential regulatory elements that may drive HSPRO gene expression during stress responses. Accordingly, analysis of null hspro mutants revealed antagonistic functions of the two proteins in PAMP-triggered immunity to P. syringae infections of shoot tissues and osmotic stress tolerance in plant roots. HSPRO proteins have been shown to interact with a central integrator of stress and energy signalling, SUCROSE NON-FERMENTING-1-RELATED KINASE1 (SnRK1) and microarray analysis of the null mutants suggested potential roles in carbohydrate signalling. An array of energy responsive genes including a subset of SnRK1 targets were misregulated in hspro mutants under standard growth conditions supporting involvement of HSPRO in energy signalling. Mutant phenotype and gene expression analysis revealed that HSPRO2 may be of importance in energy perception as hspro2 seeds were hypersensitive to exogenous glucose during germination, and that perception and/or signalling of low energy status may require HSPRO2. Although HSPRO2 expression may be driven via perception of environmental stress cues, promoter-luciferase assays revealed a diurnal expression pattern of the gene that was driven by the circadian clock. However, phenotypic analysis did not reveal a requirement of HSPRO2 for normal clock modulation. Since stress perception typically causes fluctuations in energy levels, it is proposed that HSPRO genes are important for the integration of energy and stress signalling in an effort to maintain a homeostatic balance between coping with environmental stress and normal growth and development.
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    Investigating the variation in susceptibility between Arabidopsis thaliana ecotypes in response to Botrytis cinerea infection
    (2021) Mcalery, John Alexander; Ingle, Robert
    In order to combat the various pathogens that are constantly exposed to, plants have developed a complex multi-layered innate immune system. Arabidopsis thaliana is a widely used model organism for studying the molecular basis of the host defence response to pathogen attack. One reason for this is the phenotypic variation in resistance found across the many Arabidopsis ecotypes, which provides researchers with a rich resource to dissect the molecular basis of resistance (or susceptibility) to a given pathogen. In this study, the resistance of three Arabidopsis ecotypes (Col-0, RLD and No-0) to the necrotrophic fungal pathogen Botrytis cinerea was investigated using detached leaf infection assays, expression profiling of genes known to play a role in defence against B. cinerea, and by generating F1 hybrids from the three ecotypes. The infection assays demonstrated that Col-0 and RLD develop similar sized secondary (spreading) lesions when unable to contain the initial B. cinerea infection, but that RLD was better able to contain the primary lesion, thus preventing a secondary lesion from developing. In contrast the No-0 ecotype was highly susceptible, displaying both larger secondary lesions and no ability to prevent the initial lesion from spreading. These experiments confirmed previously published data on the susceptibility of these three ecotypes. The variation in susceptibility between the three ecotypes is not due to any modulation of circadian clock regulation ofthe host defence response, asincreased resistance to B. cinerea was observed in all three ecotypes when infected at subjective morning versus subjective night. As a first step towards understanding the molecular basis of this phenotypic variation, quantitative real-time PCR analysis of five defence genes was performed on plants infected with B. cinerea. While the expression profile of these five genes wassimilar in Col-0 and RLD, No-0 displayed a markedly different pattern of expression; no induction of ORA59 (a transcription factor which integrates jasmonic acid and ethylene signalling) or PDF1.1 (a defensin with anti-fungal activity) was observed in response to B. cinerea infection in No-0, which correlated with high constitutive expression ofthe JAZ-ZIM repressor JAZ1. Finally, to determine whether the differential susceptibility to B. cinerea exhibited by the three ecotypes falls under the control of a single genetic locus or not, the ecotypes were crossed and infection assays performed on the F1 hybrids. In all three cases the susceptibility displayed by the hybrids was intermediate between that displayed by the parental ecotypes. This pattern is not consistent with control residing in a single locus and so provides further evidence that resistance to B. cinerea is governed by multiple interacting genes in Arabidopsis.
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    Keeping time on the plant-pathogen arms race : a role for the plant circadian clock in immune response
    (2011) Bhardwaj, Vaibhav; Roden, Laura; Ingle, Robert
    In this study, Arabidopsis thaliana (Arabidopsis) in the Columbia-0 (Col-0) background showed time-of-day variation in susceptibility to the plant-pathogen Pseudomonas syringae DC3000 pathovar tomato (P. syringae DC3000) when infected under constant light and temperature conditions. Wild type plants showed least susceptibility at circadian time (CT) 26 and 50, which correspond to "subjective" morning. Plants were most susceptible when infected at CT42 and CT66, "subjective" night.
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    Molecular biological studies on neuropeptides of the adipokinetic hormone/red pigment-concentrating hormone family and the neuroparsin family in the arthropod sister groups of insects and crustaceans
    (2012) Anders, Lance; G?de, Gerd; Marco, Heather G; Ingle, Robert
    This study describes the identification of three novel precursor transcripts which includes the adipokinetic hormone (AKH) / red pigment-concentrating hormone (RPCH) and the neuroparsin (NP) from the South African spiny lobster, Jasus lalandii, the RPCH and the RPCH receptor (RPCHR) from the water flea D. pulex and the NP from the southern green stinkbug, Nezara viridula. The study also investigates the localisation and expression profiles of the AKH/RPCH and NP transcripts within crustaceans and insects.
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    Non-coding RNA networks regulating leaf vegetative desiccation tolerance in the resurrection plant Xerophyta humilis.
    (2018) Milborrow, Evan; Illing, Nicola; Ingle, Robert
    Common to orthodox seeds, desiccation tolerance (DT) is exceedingly rare in the vegetative tissues of modern angiosperms, being limited to a small number of "resurrection plants". While the molecular mechanisms of DT, as well as the transcription factors regulating the seed and vegetative DT programmes, have been identified, very little is known with regards to the role of regulatory noncoding RNAs (ncRNAs). To investigate the presence and roles of possible ncRNA players, RNA-Seq was performed on desiccating Xerophyta humilis leaves and a bioinformatic pipeline assembled to identify the potential decoy lncRNAs and miRNAs present. Interaction mapping was performed, identifying a number of small regulatory networks each regulating a small subset of the desiccation transcriptome. Predicted networks were screened for function related to DT and expression consistent with functional regulatory interactions. Of the predicted networks, two appear highly promising as potential regulators of key DT response genes. The results indicate that differentially expressed (DE) desiccation response ncRNAs are present in the vegetative tissues of X. humilis and likely play a key role in the regulation of DT. This suggests that ncRNAs appear to play a more important role in DT than previously thought, and may have facilitated the evolution of vegetative DT through reprogramming of seed DT programs in vegetative tissues.
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    The genetic basis of plumage polymorphism in the black sparrowhawk
    (2022) Rodseth, Edmund; Ingle, Robert; Amar, Arjun
    This study aimed to examine the molecular basis of several aspects of plumage colouration in the black sparrowhawk (Accipiter melanoleucus), an African diurnal bird of prey. The adults of this species occur in two discrete morphs, light and dark. Light morph individuals were found to differ from dark morph individuals in the concentration of eumelanin in contour feathers from the breast feather tract, with light morph breast feathers containing no detectable eumelanin, while dark morph breast feathers contained similar amounts of eumelanin to the uniformly dark-coloured back feathers. No polymorphisms associated with morph were found in the coding regions of the melanogenesis genes melanocortin 1 receptor (MC1R), agouti signalling protein (ASIP), or proopiomelanocortin (POMC). However, the expression levels of several genes involved in melanogenesis differed between the two morphs, with light morph developing breast feathers showing significantly higher levels of expression of ASIP and lower expression of downstream melanogenesis genes tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and microphthalmia-associated transcription factor (MITF) than the dark morph. The black sparrowhawk also shows distinct rufous juvenile plumage, which is a particularly common trait in raptors. The genetic basis of juvenile plumage was investigated, and eumelanin and pheomelanin levels were found to be tightly correlated in the breast and back contour feathers of juvenile black sparrowhawks. The amount of both eumelanin and pheomelanin produced was negatively correlated with expression of ASIP, as was expression of downstream melanogenesis genes TYR, MITF, and TYRP1, the same genes that appear to determine adult plumage morph. Pheomelanin levels were found to correlate with fault bar formation, and thus may reflect acute or chronic stress experienced during feather development, but not with condition or parental plumage morph. Finally, possible pleiotropic effects of morph were investigated. Dark morphs were found to have a lower haemoparasite infection intensity than light morphs, confirming previously published results, but differences in telomere length were not associated with parasite infection or morph in this species. However, sex was found to be significantly associated with telomere length, with males having longer telomeres than females.
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    The MYC transcription factors are involved in regulating the time-of-day variations in susceptibility to Botrytis cinerea in Arabidopsis thaliana
    (2021) Joseph, Rageema; Ingle, Robert; Roden, Laura
    Plants are exposed to pathogens at specific, yet predictable times of the day-night cycle. The circadian clock, an endogenous timing mechanism, allows plants to anticipate recurring changes in pathogen abundance. A recent study showed that in Arabidopsis thaliana (hereafter: Arabidopsis), the circadian clock influences temporal differences in susceptibility to the necrotrophic pathogen, B. cinerea. Plants were less susceptible following inoculation at subjective dawn compared to subjective midnight. Based on the time required for B. cinerea spores to germinate and hyphae to penetrate host tissue and infect plants, infection takes place approximately 12 hours after inoculation. The jasmonic acid (JA) pathway regulates immune responses against B. cinerea. The paralogous basic helix-loop-helix transcription factors, MYC2, MYC3 and MYC4 are primary regulators of the JA pathway and have been implicated in immune responses against B. cinerea but it is not known whether any or all of these transcription factors are involved in regulating time-of-day variations in susceptibility. This study aimed to investigate whether MYC2, MYC3, and MYC4 are involved in the timeof-day differences in susceptibility to B. cinerea, and if they serve as molecular links to regulate interactions between the JA pathway and the circadian clock in Arabidopsis. In this work, the wild-type temporal variation in susceptibility was abolished in the myc234 triple mutant coupled with increased susceptibility following inoculation at both times of the day. The presence of MYC2, MYC3 or MYC4 alone was sufficient to maintain the wild-type time-of-day differences in susceptibility. Constitutive expression of MYC2, MYC3 or MYC4 abolished time-of-day differences in susceptibility but had different effects on overall susceptibility; constitutive expression of MYC2 and MYC4 decreased susceptibility while constitutive expression of MYC3 increased susceptibility to B. cinerea. Gene expression analyses performed on leaves revealed that the transcripts of MYC2 and MYC3 but not MYC4 are expressed rhythmically with peak transcript abundance timed to discrete times of the day under both light-dark and constant light conditions. Constitutive expression of circadian clock genes CCA1 and TOC1 abolished rhythmic MYC expression. The circadian clock modulates rhythmic leaf movement and rhythmic leaf movement was abolished in plants with constitutive MYC2 and MYC3 expression but not in plants with constitutive MYC4 expression and/or in the myc234 triple mutant. Altogether, the data suggest that the MYCs are involved in circadian-driven defence responses against B. cinerea. More specifically, MYC2, MYC3 and MYC4 function redundantly in regulating time-of-day differences in defence responses. In addition, the MYCs function as a point of convergence between the JA pathway and the circadian clock. MYC2 and MYC3 (but not MYC4) may be involved in reciprocal interactions between the JA pathway and the circadian clock.