158 research outputs found
The role of cytosolic glutamine synthetases in abiotic stress and development in Arabidopsis thaliana
No full textGlutamine (Gln), a major nitrogen source in plants, is considered a central intermediate that coordinates carbon-nitrogen assembly for plant growth and development. To maintain a sufficient Gln supply, plant cells employ glutamine synthetases (GS), including cytosolic GS1 and plastidic GS2 for Gln production. Previous work has shown that the GS1 is responsive to various environmental stresses. This study demonstrated the involvement of GS1s in Gln homeostasis and the role of GS1 in abiotic stress tolerance in Arabidopsis. The GS1 family is comprised of five isoforms in Arabidopsis thaliana. Gene expression profiling showed that GLN1;1, GLN1;3 and GLN1;4 had similar expression patterns and were upregulated by abiotic (salinity and cold) stresses, whereas GLN1;2 exhibited constitutive expression and no GLN1;5 transcript was detected under any of the conditions tested. Null T-DNA insertion mutants for the five GS1 genes were obtained. Only the gln1;1 mutant displayed enhanced sensitivity to a GS inhibitor, phosphinothricin, and to cold and salinity treatments, suggesting a nonredundant role for GLN1;1. Increased stress sensitivity in gln1;1 was associated with accelerated accumulation of reactive oxygen species (ROS), particularly in chloroplasts. To better understand the role of cytosolic GS isoforms, we generated two different triple mutant combinations. Triple mutant gln1;1/gln1;2/gln1;3 showed reduced growth at an early stage. The gln1;1/gln1;3/gln1;4 mutant is pollen lethal, indicating an essential role of Gln in plant gametophyte development. Collectively, our results establish a link between cytosolic Gln production, ROS accumulation, plant stress tolerance and development
Genetic analysis of seed and flower colour in flax (Linum usitatissimum L.) and identification of a candidate gene in the D locus
No full textFlax (Linum usitatissimum L.) is a commercial oilseed crop in Canada. Globally flax is known for industrial oil and fiber. Flaxseed contains Omega 3 fatty acid, lignans like secoisolariciresinol diglucoside (SDG), flavonoids and polysaccharides which offer potential health benefits. Conventional flax cultivars are brown seeded and few mutant lines are yellow seeded. The darkness of seed colour depends on the presence of polymerized proanthocyanidins (PA; condensed tannins) in the seed coat. PAs are the product of the phenylpropanoid pathway. Previous genetic studies by Mittapalli and Rowland (2003) on G1186/94 showed the seed colour trait was governed by the homozygous recessive alleles at D locus and the same locus is closely linked to white or pink flower petals. To start with, single seeds of already developed stable recombinant inbred lines (RILs) (of F8:9 generation) from a cross of yellow seeded European recessive line (G1186/94) and brown seeded CDC Bethune (popular variety) were grown. In this study, seed colour phenotyping was done by measuring seed colour of each RIL in Red-Green-Blue (RGB) values. To understand the genetic basis of flax seed and flower colour, mapping with single sequence repeats (SSRs) and CAPS (Cleaved Amplified Polymorphic Sequences) markers were used. For the first time, a framework genetic linkage map was constructed from populations of CDC Bethune/ G1186/94 containing 19 linkage groups (LGs). LG 1 with four SSR markers was found to be linked with the seed colour locus D. During the fine-mapping, two SSR markers (LuM566 and Lu2351) were found to be linked with the seed colour trait. The D locus has been confined in a 2.8 cM region and the closest marker was LuM566 at a distance of 0.6 cM. This was observed to be a stable locus in two growth trials and in different environments with logarithm of odds (LOD) above 39 and more than 84 % of the trait expressed by the major locus in both trials. As there were no recombinants (off types) for flower colour in F8:9 plants i.e brown-seeded lines produced blue flowers and yellow-seeded lines produced white flowers, the same locus holds well for the flower colour trait. The marker associated with seed and flower colour in G1186/94 (European recessive yellow line) was identified and can be used in flax breeding. Additionally, an interesting putative candidate gene of potential significance was identified through genomics assisted gene search from the flax whole genome sequence database. The gene expression analyses showed lower expression of putative flavonoid 3’ hydroxylase (F3’H) (a gene involved in flavonoid biosynthesis pathway) in both seed coat and flower petal tissues of G1186/94 as compared to CDC Bethune. Therefore, this study represents the first report on genetic mapping based putative candidate gene finding for recessive yellow seed colour mutation in the D locus in flax
Iron and reactive oxygen in wheat-pathogen interactions
No full textIron is an essential component of various proteins and pigments for both plants and pathogenic fungi. However, redox cycling between the ferric and ferrous forms of iron can also catalyse the production of dangerous free radicals and iron homeostasis is therefore tightly regulated. During pathogen attack, plants quickly produce large amounts of reactive oxygen species at the site of attempted pathogen ingress. This so-called oxidative burst has received considerable attention, but no single enzyme has been shown to account for the phenomenon. Using inductively coupled plasma mass spectrometry and histochemistry, I show that iron is secreted to the apoplast of the diploid wheat Triticum monococcum during attack by the powdery mildew fungus Blumeria graminis f.sp. tritici. This iron accumulates at cell wall appositions synthesised de novo beneath sites of pathogen attack. I further show, using histochemistry and pharmaceutical inhibitors, that this apoplastic iron accumulation is required for production of H2O2 in the oxidative burst. To understand the impact of this massive change in iron homeostasis on gene transcription, I employ a 187 gene targeted macroarray platform and establish that iron overload induces the expression of iron homeostasis-related genes and defence-related genes through iron itself and iron-mediated H2O2 production, respectively. To illustrate how the plant is able to withstand the negative effects of its own oxidative defences, I characterise a novel quinone redox cycle, and show that simultaneous induction of a protective quinone reductase isoform and downregulation of reactive oxygen-producing quinone reductase isoform prevents the spread of reactive oxygen during pathogen attack. Finally, in an effort to understand the impact of iron on fungal pathogenicity, I investigate iron uptake in the head blight pathogen, Fusarium graminearum. Fungi use at least two separate systems to take up iron, one based on enzymatic iron reduction and the other based on the synthesis and secretion of small iron chelators termed siderophores. Using mutants disrupted in either of two modes of iron uptake, I establish that siderophore production is essential for full F. graminearum virulence on wheat. This thesis exposes iron as an important component of both plant defence and fungal virulence
Molecular Cloning and Functional Characterization of Brassica UBC13 Genes
No full textLysine63 (K)-linked polyubiquitination of target proteins is a fundamentally different process from conventional K48-linked polyubiquitination that targets proteins for degradation via the 26S proteosome. Lys63-linked polyubiquitination regulates numerous cellular processes. The unique feature of Ubc13 compared to other ubiquitin-conjugating enzymes (Ubcs) is its ability to form a stable complex with a Ubc-E2 variant (Uev), which promotes the formation of Lys63-linked polyubiquitination. Ubc13 functions in DNA damage tolerance in budding yeast and is involved in several pathways in mammalian cells. Arabidopsis contains two UBC13 genes and four UEV1 genes that are involved in various developmental processes and stress responses including DNA damage response, root development and immunity. Recent studies imply that AtUbc13s contribute to plant susceptibility against soil-borne pathogen such as clubroot, a major disease in Brassica napus. However, there is no published information regarding characterization of B. napus Ubc13s (BnUbc13s). This project aims to understand functions of Ubc13 and Ubc13-Uev1 complexes in canola. As canola is a polyploid and often contains many homologous genes, this study aims to provide guidelines to selectively target a subset of homologous genes by gene editing to protect from clubroot disease. Twelve BnUBC13 genes were identified through genomic data analysis, eight of which encode proteins different from AtUbc13s were cloned and characterized. All eight BnUbc13s were able to physically interact with AtUev1 to form stable complexes. Furthermore, BnUBC13 genes functionally complemented the yeast ubc13 null mutant defects, suggesting that BnUBC13s can replace yeast UBC13 in DNA damage tolerance. Furthermore, a CRIPSR/Cas9 construct was designed to simultaneously target five BnUBC13 genes and was used to transform B. napus cv. Westar (DH12075). Twenty-eight out of thirty regenerated lines were found to contain homozygous or heterozygous mutations in 5 targeted BnUBC13 genes, validating our genomic editing approach in canola. In addition, BnUBC13 transcript levels in resistant and susceptible canola before and after clubroot infection were analyzed based on the in-house RNA-seq data and were found to not fluctuate drastically. This study provides convincing data to support notions that B. napus Ubc13s promotes Ly63-linked polyubiquitination, that BnUbc13s are involved in error-free DNA damage tolerance and that BnUBC13s are housekeeping genes
Suitability of genes in Arabidopsis as above-ground fluorescent reporters to screen for clubroot infection.
No full textClubroot is a soil-borne disease that affects plants in the Brassicaceae family caused by the obligate parasite Plasmodiophora brassicae. Clubroot infection begins in the roots and leads to gall formation, leading to an overall decrease in plant health. In particular, this can be economically devastating with regards to the Brassica species, which includes major commercial crops such as broccoli, cauliflower, and canola. Currently there are no reliable methods for detecting clubroot infection without digging up the plant to inspect its roots, which is inefficient and impractical when dealing with large numbers of individuals. The objective of this research was to identify potential reporter genes which could be used to screen for early clubroot infection in the shoot on a large-scale basis using a fluorescent non-destructive method. 19 genes in Arabidopsis thaliana, also belonging to the Brassicaceae family, which were either up- or down-regulated during infection according to RNA-Seq data were chosen for testing. A time course consisting of 0, 2, 5, 7, 14, 21, and 28 days post infection was established and gene expression on these days was observed with RT-PCR and RT-qPCR. 8 genes were shown to have coinciding expression trends between the RNA-Seq, RT-PCR, and initial RT-qPCR data, and their promoters were selected to be cloned into reporter vectors. tdTomato and mOrange2 were chosen as fluorescent reporters for their brightness and photostability. A final promoter-reporter construct, GASA6::mOrange2, was transformed into Arabidopsis and T1 seeds generated transgenic lines ginger1 and ginger2. Continued RT-qPCR investigation and cloning were conducted concurrently. Unfortunately, final RT-qPCR data revealed that there was no significant difference in expression between control and infected plants for any of the potential reporter genes. The functions of these genes were discussed to evaluate their connection to clubroot disease, and as possible indicators for other potential infection reporter genes. Though inconclusive, results of this research provide insight into the gene expression dynamics in shoot tissue during clubroot infection
Cell wall appositions associated with glycosylphosphatidylinositol (GPI) modification contributes to penetration resistance in Arabidopsis against fungal pathogen invasion
No full textThe abstract of this item is unavailable due to an embargo
Sclerotinia sclerotiorum necrosis-inducing effectors
No full textStem rot disease in canola caused by Sclerotinia sclerotiorum leads to lodging and severe yield losses in Canada. Sclerotinia sclerotiorum is equipped with small, secreted proteins (effectors) to induce plant cell death to facilitate nutrient uptake. Characterizing cell death/necrosis-inducing effectors might enable devising strategies to identify disease tolerant germplasm that is impervious to select necrosis-inducing effectors. In this study, RNA-Seq analysis was performed with a focus on the events occurring through the early (1 hour) to the middle (48 hours) stages of infection to reveal the gene expression patterns during the course of S. sclerotiorum infection on B. napus. The differentially expressed genes including those encoding hydrolytic enzymes, secreted effectors, enzymes involved in the synthesis of secondary metabolites or their detoxification, signaling, development, as well as oxalic acid and reactive oxygen species production. This investigation provides a broad overview of the sequential expression of virulence/pathogenicity-associated genes during infection of B. napus. To identify candidate necrosis-inducing effectors, the genome of S. sclerotiorum was searched for genes encoding small, secreted, cysteine-rich proteins. These effectors were tested for their ability to induce necrosis in Nicotiana benthamiana via Agrobacterium tumefaciens-mediated transient expression and for their host cellular localization. Six novel necrosis-inducing effectors were discovered, of which all but one required a signal peptide and secretion to the extracellular space for necrotic activity. These five effectors were localized to the endoplasmic reticulum and nucleus, while one that did not require signal peptide for necrotizing activity was localized in cytoplasm and nucleus. Virus-induced gene silencing (VIGS) experiments were conducted to reveal the participation of plant receptor-like kinases (RLK) in the induction of cell death. VIGS revealed that these five effectors required the RLKs, BAK1 and SOBIR1, for the induction of necrosis. These results illustrated the importance of necrosis-inducing effectors for S. sclerotiorum virulence and the potential role of host extra-cellular receptor(s) in the perception of S. sclerotiorum effectors. Substitution of cysteine residues with alanine and examination of truncated peptides for one of these effectors suggested that the native protein is necessary for necrotizing activity. These effectors could be applied for effector-assisted breeding of resistance to stem rot disease in canola
Plant growth promotion on and phytoremediation of Athabasca oil sands coarse tailings using the endophytic fungus, Trichoderma harzianum TSTh20-1
No full textThe environmental impact of bitumen mining in the Athabasca region of Canada is of growing concern. Among these concerns is the need and difficulty to remediate and reclaim affected land, including tailing sands (TS), a byproduct of the hot water extraction used to separate bitumen from solid materials. Current reclamation methods consist of multiple steps and take several decades to be effective. The primary reason for the difficulty in reclaiming disturbed land is the harsh environment found within the TS combined with the scale of the problem. TS are extremely nutrient poor, having below-detectable levels of NPK and extremely low C and S. In addition to this TS have pHs outside of environmental normals, and are hydrophobic due to residual hydrocarbons. Previously, an endophytic fungus, Trichoderma harzianum strain TSTh20-1, was isolated from pioneer plants growing naturally on TS sites, and was found to promote plant growth on TS. In my study TSTh20-1 was also found to increase the rate of drought recovery, and to enhance seed germination rates on a variety of soils. Suitable application methods were explored for this endophyte, including seed coatings, granules, as well as direct application to plant/soil. Regardless of method, TSTh20-1 was found to successfully colonize the plants.
Twenty-four species of grasses, forbs, and legumes were tested for their ability to grow on TS. The four most successful species (Trifolium repens, Bouteloua gracilis, Medicago sativa, and Elymus trachycaulus) were put into a seed mixture for use in experiments. In mesocosm-scale experiments, plant health and soil parameters were measured after 2 months of growth. Hydrocarbon analysis of the first mesocosm showed a 2.7-fold increase in total hydrocarbons when TSTh20-1 and plants were present, suggesting degradation of large hydrocarbons beyond the scope of the analysis. A repeat experiment using a different source of tailings did not yield this same result. This is most likely due using a source of tailings that had substantially different chemical characteristics. TSTh20-1 was also analyzed for its ability to produce plant hormones or siderophores, to increase peroxidase enzyme activity, to protect plants from reactive oxygen species, and to solubilize phosphate precipitates from soil. All of these are known mechanisms microbes use to promote plant growth
INVESTIGATING THE ROLES OF PHENYLPROPANOID PATHWAY IN PLANT DEFENSE AGAINST PATHOGEN ATTACK
No full textThe plant phenylpropanoid pathway is initiated from deamination of phenylalanine to form cinnamic acid followed by hydroxylation and methylation of the aromatic ring to generate a variety of phenolic compounds including lignin monomers, flavonoid compounds and sinapate esters. The incorporation of phenylpropanoid metabolism served as a key step in the early land-colonization of plants from aqueous environment since phenolic compounds play important roles in plant development and abiotic/biotic stress responses. Lignin is a heteropolymer of hydroxycinnamyl alcohols that are derived from the major branch of plant phenylpropanoid pathway. The main function of lignin is to enhance the strength of plant cell wall and waterproof the vascular system for long-distance transportation of water and solutes. In addition, lignin is also involved in protecting plants against pathogen attack. My Ph.D. research is to investigate how lignin biosynthesis contributes to plant immunity. The results showed that the expression of major lignin biosynthetic genes was induced upon host fungal pathogen infection. Moreover, a mutant disrupted in the lignin gene F5H1 showed enhanced susceptibility when challenged with several fungal pathogens. F5H1 encodes a ferulic acid 5-hydroxylase that is uniquely present in angiosperm plants, leading to the biosynthesis of syringyl lignin monomer, which is not present in gymnosperm plants. Subsequent research demonstrated that f5h1 mutation impaired the penetration (pre-invasion) resistance but did not impact post-invasion resistance. Furthermore, the pathogen-induced expression of lignin genes was independent of well-characterized defensive signaling pathways, and regulated by a novel regulating mechanism. F5H1 contributes to pmr2-mediated resistance but acts independently of other molecular components of penetration resistance including PEN1, PEN2, and PEN3. In contrast to f5h1, a knockout mutant of flavonoid pathway gene chalcone isomerase (CHI/TT5) showed enhanced resistance to host anthracnose pathogen Colletotrichum higginsianum in a salicylic acid (SA)-dependent manner. Taken together, our results for the first time provide genetic evidence demonstrating that lignin biosynthetic gene F5H1 plays critical roles in plant penetration resistance and that an uncharted pathway in flavonoid metabolism confers an SA-dependent resistance pathway in Arabidopsis
Locust Flight Muscle Activity and Body Orientation in Response to Objects Moving within Different Backgrounds
No full textLocusts are ideal model systems to study complex behaviours, such as flight responses to objects approaching on a collision course. Previous studies have described the flight muscle activity, wing kinematics and aerodynamic forces during collision avoidance behaviour of locusts flying in the wind tunnel. Neural recordings have revealed the influence of backgrounds on the responses of the descending contralateral movement detector (DCMD). Flow field backgrounds delay DCMD responses to looming stimuli that are known to evoke collision avoidance and muscle asynchrony during flight. Therefore, I hypothesize that the flow field background will delay behavioural responses and affect the timing of flight muscle activity. To test for this hypothesis, I placed a loosely-tethered flying locust inside a flight simulator, and presented visual stimuli composed of disks looming within different backgrounds. Concurrent electromyogram (EMG) and videos were recorded before and during the approach. My results show that against both simple and flow field backgrounds, the locust performed collision avoidance behaviours, by exiting the area defining the pre-response epoch, in most of the trials. The time of behaviour (TOB) varied among trials, and neither depressor asymmetry (DA) nor LM97 firing rate changed at TOB. The linear correlation between rotational degrees of freedom [RDOF, including roll (η), pitch(χ), and yaw(ψ)], or the changes in RDOF from the previous frame (ΔRDOF), and DA was calculated to investigate the relationship between depressor asymmetry and behaviours. In the presence of a simple background, more trials showed a significant correlation between RDOF and DA, compared to a flow field background. In the simple background, the time when the correlation between RDOF/ΔRDOF (η, Δη, Δχ, and Δψ) and DA became significant occurred during the interquartile range of TOB. In the flow field background, the correlation between certain RDOF/ΔRDOF (η, ψ, Δη) and DA became significant, while the correlation between DA and χ became insignificant, during the interquartile range of TOB. These results suggest that the background types affected the correlation between RDOF (or ΔRDOF) and DA, and the time when the significance of the correlation changes could be related to TOB
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