In plants, the generation and supply of methyl units is important in one-carbon (C-1) metabolism, which is essential to all organisms. I have identified a series of cDNA sequences encoding N5, N10-methylenetetrahydrofolate reductase (MTHFR), cobalamin-independent methionine synthase (Met Syn), S-adenosylmethionine synthetase (isoform I, AdoMet Syn2661 and isoform II, AdoMet Syn605), S-adenosylmethionine decarboxylase (SAMDC), serine hydroxymethyltransferase (SHMT) and N5, N10-methenyltetrahydrofolate cyclohydrolase / N5, N10-methylenetetrahydrofolate dehydrogenase (THFC/THFD) in the pathways of generation and supply of methyl units. These are from a cDNA library of mRNA from a susceptible wheat (Triticum monococcum) (Tm) line 441 epidermis, 24 h after inoculation with powdery mildew fungus (Blumeria graminis f. sp. tritici) (Bgt). Phylogenetic tree cluster analysis and subcellular localization prediction by TargetP revealed that MTHFR, Met Syn, AdoMet Syn605, AdoMet Syn2661, SAMDC, and THFC/THFD may be localized in cytosol; SHMT may be localized in mitochondria. Northern blot analysis indicated that expression of MTHFR, Met Syn, AdoMet Syn2661, AdoMet Syn605 and SAMDC genes was up-regulated by powdery mildew infection, abiotic stresses and treatments with stress signal molecules; expression of SHMT and THFC/THFD was either constitutive or down-regulated. These results suggest a close metabolic link between various stresses and the pathways of generation and supply of methyl units in this wheat.

In plants, the generation and supply of methyl units is important in one-carbon (C-1) metabolism, which is essential to all organisms. I have identified a series of cDNA sequences encoding N5, N10-methylenetetrahydrofolate reductase (MTHFR), cobalamin-independent methionine synthase (Met Syn), S-adenosylmethionine synthetase (isoform I, AdoMet Syn2661 and isoform II, AdoMet Syn605), S-adenosylmethionine decarboxylase (SAMDC), serine hydroxymethyltransferase (SHMT) and N5, N10-methenyltetrahydrofolate cyclohydrolase / N5, N10-methylenetetrahydrofolate dehydrogenase (THFC/THFD) in the pathways of generation and supply of methyl units. These are from a cDNA library of mRNA from a susceptible wheat (Triticum monococcum) (Tm) line 441 epidermis, 24 h after inoculation with powdery mildew fungus (Blumeria graminis f. sp. tritici) (Bgt). Phylogenetic tree cluster analysis and subcellular localization prediction by TargetP revealed that MTHFR, Met Syn, AdoMet Syn605, AdoMet Syn2661, SAMDC, and THFC/THFD may be localized in cytosol; SHMT may be localized in mitochondria. Northern blot analysis indicated that expression of MTHFR, Met Syn, AdoMet Syn2661, AdoMet Syn605 and SAMDC genes was up-regulated by powdery mildew infection, abiotic stresses and treatments with stress signal molecules; expression of SHMT and THFC/THFD was either constitutive or down-regulated. These results suggest a close metabolic link between various stresses and the pathways of generation and supply of methyl units in this wheat.

>Magister Scientiae - MSc / Agricultural production is one of the most important sectors which provide food for the growing world population which is estimated to reach 9.7 billion by 2050, thus there is a need to produce more food. Climate change, on the other hand, is negatively affecting major global crops such as maize, sorghum, wheat and barley. Environmental factors such as salinity, drought, high temperatures and pathogens affect plant production by oxidatively damaging the physiological processes in plants, leading to plant death. Poor irrigation used to combat drought result in salinasation, which is estimated to affect 50% of arable land by 2050. Plants have developed several mechanisms that protect them against stress and these include overexpression of stress responsive genes and altered signal transduction to change the expression of stress responsive genes, among others. Cyclic 3’5’ guanosine monophosphate (cGMP), a second messenger that is synthesised by guanylyl cyclase (GC), transmit signals to various cellular functions in plants during plant development, growth and response to abiotic and biotic stresses. Arabidopsis thaliana nitric oxide guanylyl cyclase 1 (AtNOGC1) is a guanylyl cyclase which upon activation by nitric oxide (NO) leads to the production of more cGMP. Cyclic GMP further activates protein kinases, ion gated channels and phosphodiesterase which mediate response to various stresses. In this project the role of AtNOGC1 was investigated in response to abiotic and biotic stresses through analysis of its evolutionary relationships, promoter, gene expression and functional analysis via the viability assays in Escherichia coli (E.coli). Phylogenetic tree, exon-intron structure and conserved motifs were analysed using the Molecular Evolutionary Genetics Analysis (MEGA V.7), Gene Structure Display Server 2.0 (GSDS 2.0), and Multiple Expectation Maximisation for Motif Elicitation (MEME) tools respectively. AtNOGC1’s gene expression was analysed by the Real-Time Quantitative Reverse Transcription Polymerase Reaction (qRT-PCR), whereas functional analysis was carried out using the cell viability (liquid and spot) assays to determine its ability to confer stress tolerance to E. coli.

Tall fescue is the most common cool-season grass in the eastern USA, with broad economic and ecological importance to the region. Tall fescue is known to associate with a fungal endophyte, Neotyphodium coenophialum, whose presence can decrease biotic and abiotic stress experienced by the plant. This thesis evaluates the response of tall fescue and the fungal endophyte symbiosis to predicted climate change. I participated in two multi-factor climate change projects where I investigated the response of tall fescue tissue chemistry and growth to various climate change factors. Endophyte-infected (E+) tall fescue had decreased alkaloid production under elevated CO2 but increased alkaloid production under elevated temperatures. Significant differences between E+ and E- (endophyte-free) tall fescue tissue chemistry were also found, suggesting the endophyte interacts with the plant response to abiotic stress. Although several studies have reported benefits of endophyte infection for tall fescue growing under drought stress, my research found no differences between E+ and E- total growth and surprisingly showed increased mortality of E+ individuals under elevated temperature. Taken together, my research indicates that this grass-fungal relationship will respond to climate change, and may produce dramatic and unforeseen results that question the widely believed mutualistic nature of the symbiosis.

Abiotic stresses such as salt stress are the major limiting factors for agricultural productivity, and cause global food insecurity. It is well known that plant associated beneficial microorganisms can stimulate plant growth and enhance resistance to abiotic stresses. In this context, bacterial endophytes are a group of bacteria that colonize the host plant and play a fundamental role in plant growth enhancement under stress condition. Recently, our group reported that the beneficial bacteria Enterobacter sp.SA187 induces plant growth in Arabidopsis under salt stress conditions by manipulation of the plant ethylene signaling pathway. We therefore compared inoculation of plants by SA187 with virulent and non-virulent strains Pst DC3000. Although both strains inhibit plant growth at ambient conditions, Pst DC3000 hrcC-, but not Pst DC3000, induced salt stress tolerance, suggesting that Pst DC3000 hrcC- also contains plant growth promoting activity under stress conditions. Our results indicate that Pst DC3000 hrcC- shares features with beneficial bacteria by inducing salt tolerance through reduction of the shoot and root Na+/K+ ratio. To further elucidate the underlying mechanisms of this interaction with Arabidopsis, RNAseq, hormone and biochemical analyses were performed. Genetic studies also show that Pst DC3000 hrcC- induced salt stress tolerance involving several phytohormone pathways, including auxin, ethylene and salicylic acid. Transcriptome and genetic analyses indicate that glucosinolates play an important role in this beneficial interaction. We found that indolic and alkyl glucosinolates act as negative factors on Pst DC3000 hrcC-, alkyl glucosinolates are positive and indolic glucosinolates negative regulators in SA187 interaction with Arabidopsis. These results reveal that besides a repertoire of effectors, Pst DC3000 hrcC- also produces factors that can be beneficial for plant growth under certain stress conditions, as observed with Enterobacter sp. SA187.

Mosses are small avascular bryophytes with a haploid dominant gametophyte and a diploid sporophyte stage. The gametophyte cells are single layered and lack a protective cuticle, which is the first line of defense in vascular plants. These factors would render them highly susceptible to stress but on the contrary, mosses have flourished on land for the past 450 million years with tolerance to both abiotic and biotic stress. Occurrence of unique lipids in bryophytes was considered as an adaptive means to survive harsh terrestrial condition. A recent study identified a lipid metabolite, anandamide in the Physcomitrella patens. Anandamide (NAE 20:4) belongs to a group of fatty acid ethanolamides or N –acylethanolamines (NAEs). In eukaryotes, NAEs were shown to play an important role in mediating stress responses. In plants, NAE 14:0 has been implicated in biotic stress response; its levels increased up to 50-fold in elicitor-treated tobacco plants, along with induction of defense gene expression and inhibition of alkalization. In animals anandamide acts as an endocannabinoid ligand and mediates several physiological responses including stress. This study aims to use P. patens as the model system because of its available genomic database and prior studies on biotic stress, to examine if NAE 20:4 contributes to their ability to tolerate biotic stress. It is hypothesized that the occurrence of anandamide will play a role in mediating biotic stress tolerance in P. patens. To test this hypothesis, three specific aims are proposed. They are to determine the effect of 1) elicitor-treatment on NAE and fatty acid profile in the moss, 2) anandamide on elicitor-induced morphological and physiological changes in the moss and 3) anandamide on elicitor-induced defense gene expression in moss. Mosses utilize similar defense mechanisms as flowering plants and disease symptoms can easily be studied using microscopy because of their haploid dominant gametophyte stage with monolayer cells. The induction of defense gene expression will be studied by quantitative PCR and changes in lipid profile by selective lipidomics. This study is expected to provide novel insights into the role of anandamide in early land plants, specifically in response to biotic stress.

No description available.

Analysis of SIP68: A UDP Glucosyltransferase for Its Role in Plant Growth and Immunity UDP-glucosyltransferases (GTs) are a group of enzymes that play a crucial role in plant metabolism by transferring glucosyl groups from UDP-glucose to various acceptor molecules. SIP68 is a UDP-glucosyltransferase enzyme that has been identified to interact with SABP2 in a yeast two-hybrid screen. Previous research conducted in our lab has demonstrated that SIP68 is involved in salicylic acid (SA)-mediated defense signaling in tobacco plants. In the current study, we aimed to investigate the potential role of SIP68 in plant development and immune response. Our analysis of SIP68 revealed that this UDP-glucosyltransferase has a gene family, and its gene and protein sequence, molecular attributes, gene structure, and localization in the chromosome, exon-intron distribution, cis-regulatory elements in the promoter region, homology modeling of protein, domain architecture, motif analysis, phylogenetic tree, and protein-protein interaction were analyzed to better understand its potential function in plant metabolism. Our in-silico analysis predicted that SIP68 may play a role in the cytokinin-mediated metabolic pathway, which could affect plant growth and cell proliferation. Specifically, our analysis suggested that SIP68 might transfer glucosyl groups to various acceptor molecules involved in the cytokinin-mediated metabolic pathway. This suggests that SIP68 may play a role in regulating plant growth and development by affecting the cytokinin pathway. To investigate the potential role of SIP68 in plant development, we generated SIP68-deficient transgenic tobacco plants by silencing the SIP68 protein. The observed phenotype of these plants was compared to that of wild-type plants. We found that root, shoot, leaf width, and overall biomass development were all affected in SIP68-deficient plants. This suggests that SIP68 plays a crucial role in regulating various aspects of plant growth and development. This agrees with our previous finding that SIP68 is involved in SA-mediated defense signaling in tobacco plants. Our analysis of protein-protein interactions revealed that SIP68 interacts with various classes of flavanols in-vitro. This interaction provides a starting point for investigating potential targets of SIP68 in tobacco plants. However, the specific in-planta substrate(s) of SIP68 has not yet been identified. Therefore, further investigation is needed to determine the intracellular targets of SIP68 and its specific role in plant metabolism. In conclusion, our study provides insights into the potential role of SIP68 in plant development and immune response. Our findings suggest that SIP68 plays a crucial role in regulating various aspects of plant growth and development. Furthermore, our in-silico analysis predicts that SIP68 may play a role in the cytokinin-mediated metabolic pathway, which could affect plant growth and cell proliferation. Future investigation is needed to determine the intracellular targets of SIP68 and its specific role in plant metabolism. Overall, this study highlights the importance of UDP-glucosyltransferase enzymes (SIP68) in plant development and immune response.

In this study poplar trees have been examined under different stress conditions. Apart from the detailed descriptions above two main conclusions might be drawn: i) A small plant like Arabidopsis thaliana is highly susceptible to stress situations that might become life-threatening compared to a tree that has extremely more biomass at its disposal. Such an organism might be able to compensate severe stress much longer than a smaller one. It seems therefore reasonable that a crop like Arabidopsis reacts earlier and faster to a massive threat. ii) In poplar both tested stress responses seemed to be regulated by hormones. The reactions to abiotic salt stress are mainly controlled by ABA, which also has a strong impact upon cold and drought stress situations. The term commonly used for ABA is “stress hormone” and is at least applicable to all abiotic stresses. In case of herbivory (biotic stress), jasmonic acid appears to be the key-player that coordinates the defence mechanism underlying extrafloral nectary and nectar production. Thus the presented work has gained a few more insights into the complex network of general stress induced processes of poplar trees. Future studies will help to understand the particular role of the intriguing indirect defence system of the extrafloral nectaries in more detail. / In dieser Arbeit wurden Pappelbäume unter verschiedenen Stressbedingungen untersucht. Zusammenfassend und zusätzlich zu den obigen Beschreibungen lassen sich zwei Schlussfolgerungen ziehen: i.) Eine kleine Pflanze wie Arabidopsis ist viel empfindlicher für Stresssituationen, die möglicherweise lebensbedrohlich werden könnten, im Gegensatz zu einem Baum mit wesentlich grösserer Biomasse . Solch ein Organismus kann schwerwiegendem Stress viel länger kompensieren als ein kleinerer Organismus. Es erscheint daher sinnvoll, dass eine Pflanze wie Arabidopsis viel früher und schneller auf eine massive Bedrohung reagiert. ii.) In Pappeln scheinen beide untersuchten Arten von Stressreaktion durch Hormone reguliert zu werden. Die Reaktionen auf abiotischen Salzstress werden hauptsächlich durch ABA kontrolliert, welches auch einen starken Einfluss auf Kälte- und Trockenstressszenarien hat. Üblicherweise wird für ABA der Ausdruck "Stress-Hormon" verwendet, was zumindest für abiotischen Stress zutreffend ist. Im Fall von Herbivorie (biotischer Stress) scheint Jasmonsäure die Schlüsselrolle zu spielen, die die Abwehrmechanismen koordiniert, die den extrafloralen Nektarien und der Nektarproduktion zu Grunde liegt. Demzufolge hat die vorliegende Arbeit ein paar neue Einsichten in das komplexe Netzwerk der Stress-induzierten Prozesse der Pappel ermöglicht. Zukünftige Studien werden dazu beitragen die besondere Rolle des faszinierendem indirektem Abwehrmechanismus der extrafloralen Nektarien en detail zu verstehen.

Submitted by Francine Silva (francine.silva@unipampa.edu.br) on 2016-09-28T20:54:58Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Análise Transcricional de Physcomitrium Acutifolium Broth. Por meio da Técnica de RNA-SEG um enfoque sobre o estresse por frio em plantas.pdf: 2456403 bytes, checksum: f125855c91a08aa6e84cb53239871aa3 (MD5) / Made available in DSpace on 2016-09-28T20:54:58Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Análise Transcricional de Physcomitrium Acutifolium Broth. Por meio da Técnica de RNA-SEG um enfoque sobre o estresse por frio em plantas.pdf: 2456403 bytes, checksum: f125855c91a08aa6e84cb53239871aa3 (MD5) Previous issue date: 2015-05-29 / Os estresses abióticos são responsáveis pela indução de adaptações em plantas. Estas quando submetidas ao estresse, respondem através de mecanismos de sinalização nas rotas fisiológicas, desencadeando um processo de aclimatação. Entretanto, nem sempre este potencial de adaptação é expresso, mas se persistir ao longo do desenvolvimento da planta, torna-se uma adaptação oriunda de mudança genética. Consequentemente, essas alterações incipientes possam ser identificadas em nível transcricional, os precursores de algumas alterações genéticas importantes tais como splicing alternativo. Em ambientes polares a expressão de genes permitiu a adaptação das plantas a temperaturas de congelamento. Entre estas plantas estão os musgos, presentes nos ambientes de climas contrastantes, isto sugere que estes organismos tenham plasticidade fenotípica e genotípica. Ainda que haja poucos estudos destes organismos em relação a diferentes agentes estressores, é amplamente difundido que estes possuem potenciais de resistência aos estresses ambientais. Para descobrir estes potenciais de resistências é necessário estudar os genes relacionados especificamente com o fator estressor em questão, neste caso o estresse ao frio. Para tanto é necessário um processo de sequenciamento dos genes expressos quando a planta é submetida ao estresse. Neste estudo foram realizados testes com explantes cultivados in vitro do musgo Physcomitrium acutifolium Broth. em diferentes temperaturas, com 6 tratamentos variando de 0 a 25 ºC, seguidos das análises fenotípicas e posteriormente genômicas, incluindo processo de sequenciamento e identificação de genes expressos. Os resultados sugerem relação do estresse por baixas temperaturas e o potencial de expressão de genes relacionados ao estresse por frio neste musgo, principalmente por uma identificação de maiores ocorrências de splicing alternativo nas plantas cultivadas a temperatura mais baixa testada. Assim, o uso potencial de espécies de musgo em estudos relacionados à resistência ao congelamento em plantas, torna-se como uma alternativa interessante em processos de biotecnologia vegetal. / The abiotic stresses are responsible for inducing adaptations in plants. When subjected to stress, plants respond by signaling mechanisms in physiological pathways, starting a process of acclimatization. However, not every adaptation potential is identified in phenotypic level, but if the selection pressure led by the stress persist over plant development, there may be a change in genotypic level. Consequently, these incipient changes can be identified in transcriptional level, the precursors of some important genetic changes such as alternative splicing. In polar environments are noted that the plants were adapted to survive at low temperatures, this adjustment is related to the expression of genes, which confer them resistance to freezing. Among these plants are mosses, present in the environments of contrasting climates, this suggests that these organisms have phenotypic and genotypic plasticity. Even if there are few studies of these organisms in relation to different stressors, we know that these have the potential for resistance to environmental stresses. To discover these potential resistance is necessary to study the genes specifically related to the stressor in question, this study is the cold stress. Then you need a sequencing process is necessary genes expressed when the plant is subjected to stress. In this study were performed tests with cultured explants in vitro moss Physcomitrium acutifolium Broth. at different temperatures, with 6 treatments ranged from 0°C to 25°C, followed by phenotypic analysis and subsequently transcriptome analysis based in RNA sequencing process aiming to identify a differential gene expression. The results suggest stress relationship for low temperatures and the potential for expression of genes related to cold stress in this moss, mainly by an identification of a higher occurrences of alternative splicing in plants growing at lowest temperature tested. Thus, the potential use of moss species in studies related to plant resistance to freeze temperatures becomes as an interesting alternative in plant biotechnology processes.