Submitted by Michele Mologni (mologni@unoeste.br) on 2017-08-30T13:15:11Z No. of bitstreams: 1 Fláive Loyze Baldassarini Silva.pdf: 2001999 bytes, checksum: e6f3c3f0169423517f9b8ffac6f5f788 (MD5) / Made available in DSpace on 2017-08-30T13:15:11Z (GMT). No. of bitstreams: 1 Fláive Loyze Baldassarini Silva.pdf: 2001999 bytes, checksum: e6f3c3f0169423517f9b8ffac6f5f788 (MD5) Previous issue date: 2017-06-27 / The association between proline metabolism in the plant and the pentose phosphate pathway has been proposed as a model to stimulate one of the biosynthetic routes of secondary metabolism related to the production of the different phenolic compounds in plants, known as the shikimic acid pathway. In transgenic plants in which overexpression of the P5CS gene Δ1-pyrroline-5-carboxylate synthetase encoding the key enzyme of proline biosynthesis occurs, in addition to increased tolerance to abiotic stresses, there is the possibility of promoting the synthesis of phenolic compounds as a pleiotropic effect. The objective of this work was to evaluate the role of proline "per se" in relation to the production of phenolic compounds in transgenic tobacco plants accumulating this amino acid (Nicotiana tabacum cv. Petit Havana SR1) and to verify if water stress would modify this answer. The experiment was carried out under greenhouse conditions, at Campus II of Universidade do Oeste Paulista, Presidente Prudente - SP. A completely randomized design was used in the factorial scheme 3x2 consisting of 3 genotypes (two transgenic events with constitutive expression 35S::P5CSF129A and untransformed control plants) and two levels of water regime, with daily water replenishment of 100% and 30% of field capacity (water stress). The role of proline "per se" was evaluated by means of biometric growth analyzes (plant height, shoot and root dry mass and leaf number) in addition to proline analyzes, glucose-6-phosphate dehydrogenase activity (G6PDH), phenylalanine ammonia lyase (PAL), total phenolic compounds in leaves and lignin. In a natural response to water stress, a reduction in biometric parameters was observed for all genotypes. The accumulation of proline occurred in a greater proportion in the transgenic plants as expected by the constitutive expression of the P5CS gene, but the activity of the G6PDH enzyme was lower in the transgenic plants. The link between increased proline endogenous content and increased phenol synthesis occurred both under normal hydration conditions (for E2 event) and in the presence of water stress (for both transgenic events), as well as PAL activity. Lignin contents increased in all genotypes in response to stress. Thus, the results of this research lead us to affirm the existence of distinct responses between exogenous application of proline reported in the literature and endogenous cellular metabolism. It was not possible to confirm the hypothesis that the proline metabolism linked to the pentose phosphate pathway induces the synthesis of phenolic compounds, since the activity of the G6PDH enzyme was lower in the transgenic plants in the two water conditions. It is suggested that the precursors to the pathway of phenolic compounds linked to proline may be provided by other metabolic pathways such as glycolysis and the Calvin cycle. Further study will be needed to clarify this issue. / A associação entre o metabolismo de prolina na planta e a via pentose fosfato tem sido proposta como um modelo para se estimular uma das rotas do metabolismo secundário relacionada à produção dos diferentes compostos fenólicos em plantas, denominada via do ácido chiquímico. Em plantas transgênicas nas quais ocorrem a superexpressão do gene P5CS Δ1-pirrolina-5-carboxilato sintetase, que codifica a enzima-chave da biossíntese de prolina, além do aumento de tolerância a estresses abióticos, existe a possibilidade de promoção da síntese de compostos fenólicos como um efeito pleiotrópico. O objetivo deste trabalho foi avaliar o papel da prolina “per se” em relação à produção de compostos fenólicos em plantas transgênicas de tabaco acumuladoras deste aminoácido (Nicotiana tabacum cv. Petit Havana SR1) e verificar se o estresse hídrico modificaria essa resposta. O experimento foi realizado em condições de casa de vegetação, no Campus II da Universidade do Oeste Paulista, Presidente Prudente - SP. Utilizou-se o delineamento experimental em arranjo inteiramente casualizado, no esquema fatorial 3x2, formado por 3 genótipos (dois eventos transgênicos com expressão constitutiva 35S::P5CSF129A e plantas controle não transformadas) e dois níveis de regime hídrico, com reposições diárias de água de 100% e de 30% da capacidade de campo (estresse hídrico). O papel da prolina “per se” foi avaliado por meio de análises biométricas de crescimento (altura de plantas, massas seca de parte aérea e raiz e número de folhas) além das análises de prolina, atividade das enzimas glicose-6-fosfato-desidrogenase (G6PDH), fenilalanina amônia liase (FAL), compostos fenólicos totais nas folhas e lignina. Em resposta natural ao estresse hídrico, foi observado uma redução nos parâmetros biométricos para todos os genótipos. O acúmulo de prolina ocorreu em maior proporção nas plantas transgênicas como esperado pela expressão constitutiva do gene P5CS, porém a atividade da enzima G6PDH foi menor nas plantas transgênicas. A vinculação entre um maior conteúdo endógeno de prolina com a síntese aumentada de fenóis ocorreu tanto em condições normais de hidratação (para o evento E2) como na presença de estresse hídrico (para ambos os eventos transgênicos), assim como a atividade da FAL. Os teores de lignina aumentou em todos os genótipos em resposta ao estresse. Desta forma, os resultados dessa pesquisa levam-nos a afirmar a existência de respostas distintas entre aplicação exógena de prolina relatada na literatura e o metabolismo endógeno celular. Não foi possível confirmar a hipótese de que o metabolismo de prolina vinculado à via pentose fosfato induz a síntese de compostos fenólicos, pois a atividade da enzima G6PDH foi menor nas plantas transgênicas nas duas condições hídricas. Sugere-se que os precursores para a via dos compostos fenólicos ligados a prolina podem ser fornecidos por outras vias metabólicas tais como a glicólise e o ciclo de Calvin. Estudos mais aprofundados serão necessários para esclarecer esta questão.

Le développement et le fonctionnement harmonieux des plantes dépendent de cofacteurs comme le nicotinamide adénine dinucléotide (NAD). Outre ses rôles dans le recyclage redox, le NAD est également impliqué dans les processus de signalisation cellulaire, qui sont des acteurs centraux dans les conditions de stress. Ainsi, le NAD est l'un des principaux déterminants de l'homéostasie de l'énergie des plantes et a un impact important sur la productivité des plantes et la résistance au stress. La L-aspartate oxydase (LASPO) et l'acide nicotinique / nicotinamide mononucléotide adénylyltransférase (N(a)NMNAT) sont deux enzymes importantes dans la biosynthèse du NAD. Par surproduction de ces deux enzymes dans E. coli, nous avons identifié les propriétés biochimiques de ces enzymes. Il ressort que la surexpression de la N(a)MNAT stimule la croissance des plantes en activant les métabolismes photosynthétiques et respiratoires, sans pour autant augmenter les teneurs en NAD. La surproduction de la LASPO augmente la teneur en NAD des plantes, laquelle est corrélée à une augmentation significative de la production de biomasse foliaire et des rendements en graines, et l’inverse est observé lorsque les teneurs en NAD diminuent. La modification des teneurs en NAD affecte les fonctions photosynthétiques et respiratoires, et l’expression de quelques gènes qui pourraient participer au contrôle de la croissance des plantes semble régulée par les histones déacétylases NAD-dépendantes. / The development and smooth functioning of plants depends on co-factors such as nicotinamide adenine dinucleotide (NAD). In addition to its roles in redox recycling, NAD is also involved in cell signaling processes, which are central actors in stress conditions. Thus, NAD is one of the main determinants of plant energy homeostasis and has a significant impact on plant productivity and stress resistance. L-aspartate oxidase (LASPO) and nicotinic acid / nicotinamide mononucleotide adenylyltransferase (N (a) NMNAT) are two important enzymes in the biosynthesis of NAD. By overproducing these enzymes in E. coli, we have identified the biochemical properties of these enzymes. Overexpression of N(a)MNAT stimulates plant growth by activating photosynthetic and respiratory metabolisms, without increasing NAD levels. Overproduction of LASPO enhances NAD content in plants, which is correlated with a significant increase in foliar biomass production and seed yields, and the reverse is observed when NAD levels decrease. Changes in NAD levels affect photosynthetic and respiratory functions, and the expression of a few genes that may be involved in plant growth control appears to be regulated by NAD-dependent histones deacetylases.

Orientador: Douglas Silva Domingues / Resumo: O café é uma das commodities agrícolas tropicais mais comercializadas no mundo. Coffea arabica é a principal espécie utilizada para a produção comercial de café. A espécie é originária da Etiópia. Ela é única espécie alotetraploide do gênero (2n = 4x = 44) e se reproduz predominantemente por autofecundação. As cultivares comerciais de C. arabica possuem baixa diversidade genética, o que indica a necessidade de introgressão de alelos de germoplasma para o melhoramento dessas cultivares. Acessos do centro de origem da espécie possuem maior diversidade que as cultivares comerciais e podem ser utilizados para a identificação de novos alelos. O conteúdo de macronutrientes em grãos do cafeeiro tem impacto direto na qualidade do produto. No entanto, a base molecular da composição mineral de grãos de cafeeiro ainda é pouco conhecida. Com isso, o objetivo desse trabalho foi identificar marcadores SNP possivelmente associados com a composição de macronutrientes em grãos de C. arabica. Para alcance deste objetivo, foram comparados três métodos de imputação de genótipos, bem como foi realizado o mapeamento associativo em estudo de associação genômica ampla (GWAS). Foi utilizado um painel de 110 genótipos de C. arabica, composto por genótipos elite do programa de melhoramento do Instituto Agronômico do Paraná (3), cultivares comerciais (11) e acessos selvagens (96). Foram realizadas análises da composição de cinco macronutrientes (N, P, K, Ca e Mg) em grãos de cafeeiro coletados de 70 e 1... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Coffee is one of the most traded tropical commodities in the world. Coffea arabica is the main species used for commercial production. The species is originally from Ethiopia. In the Coffea genus, C. arabica is the only allotetraploid species (2n = 4x = 44) and it reproduces predominantly by self-fertilization. The commercial cultivars of C. arabica have a narrow genetic base that indicates the need for the introgression of new alleles from germplasm into coffee breeding programs. Wild accessions of C. arabica, from Ethiopia, have higher genetic diversity and can be used to identify new alleles. The macronutrient composition of the coffee grains has a direct impact on grain quality. However, the molecular basis for the mineral composition in coffee grains still poorly understood. Thus, the aim of this work was to perform mapping association analyses using the genome-wide association study (GWAS) technique to identify single nucleotide polymorphisms (SNPs) associated with macronutrient content in coffee grains from C. arabica. We also tested three imputation methods (haplotype missing allele imputation - Beagle, K-nearest neighbors, and Random Forest) in the genotypic data, and mapped it to two C. arabica reference genomes from the cultivar Caturra red and the spontaneous dihaploid Et39. We used a panel of 110 C. arabica genotypes, including elite landraces from the IAPAR coffee breeding program (3), commercial cultivars (11) and wild accessions (96). Analysis of the compositi... (Complete abstract click electronic access below) / Doutor

Abiotic stresses - drought, salinity or extreme temperatures - cause morphological and physiological changes, affect growth, development and plant productivity. In crops, these limitations often reduce yields up to 50%. Plants have developed various mechanisms ensuring some degree of abiotic stress tolerance, which undoubtedly include changes in carbohydrate metabolism. The nature of many defense mechanisms is not yet fully understood, moreover, most findings were obtained by studying model plants and little is known about their validity in crops. This work was therefore focused on woodland strawberry (Fragaria vesca L.) from economically important family Rosaceae. Controlled conditions and targeted exposure to stress factors was obtained through the cultivation in vitro. Strawberry plants and callus cultures were exposed to various stresses and their combinations, better reflecting the natural conditions. At various stages of stress reaction, growth characteristics, changes in the content and the spectrum of soluble carbohydrates and oxidative stress levels were monitored and compared with plants growing under optimal conditions. Abiotic stress responses of strawberry plants were very inhomogeneous, which complicated the obtaining of statistically significant results. The only significant response...

Plants are exposed to many adverse factors during their life cycles. Abiotic stresses are significantly limiting plant growth and development. Abiotic stress response mechanisms involve compatible solute synthesis (e.g. sugars, sugar alcohols and amino acids). The aim of this study was to characterise the responses of plants from Plantago genus to different abiotic stresses (drought, salinity, cold and stress combination). The complete plants grew under in vitro conditions. I compared morphological parameters, selected metabolic parameters (carbohydrates balance and proline accumulation) under optimal conditions and stress exposure. This study was focused on plants from genus Plantago, because they differ from each other in their tolerance to the salinity. Both, the glycophyte and the halophyte species are described within this genus. Plantains produce besides widespread soluble carbohydrates (sucrose, glucose, fructose) also sugar alcohol sorbitol, which has been reported as a significant component of the stress response. In addition, the reaction of plants to different carbon and energy sources was tested. More severe growth inhibition of the glycophyte Plantago lanceolata compared to the halophyte P. maritima was observed under salt treatment. Significant accumulation of sorbitol was observed...

Plants are exposed to the stress of the environment throughout their entire live. They therefore had to develop sophisticated mechanisms to avoid or tolerate stress. In some plants, repeated exposure to stress increases tolerance, which is known as stress memory. The aim of this study is to assess stress memory in terms of oxidative stress level. Content of malondialdehyde (MDA), reactive oxygen species (ROS) and the electrolyte leakage were chosen as markers of oxidative stress. Fresh weight, plant height and water content were also evaluated. The level of oxidative stress was assessed in several genotypes of Solanum tuberosum subspecies, Andigena and Tuberosum. In vitro culture did not work well for the evaluation of stress memory, ex vitro partially did. The content of MDA in 66B was significantly lower than in cv. Lada (Tuberosum) in root (in vitro) and shoot (ex vitro) in control groups as well as during water shortage. Treatment of acclimation-inducing cold before the stress-inducing cold had a variable effect on growth and electrolyte leakage according to the culture conditions. In vitro, the effect on electrolyte leakage in roots of cv. Lada was positive, in leaves of 66B (Andigena) it was negative. In ex vitro, a positive effect was found on electrolyte leakage in 66B leaves, but at the...

<p>Nitrogen and phosphorus are the two macronutrients which play important roles in the plant, both structurally and functionally, e.g., starting from being constituents of cellular integrity to being signal molecules in signal transduction. Since they are required by plants in higher concentrations, it becomes indispensable to replenish their pools in soils by the application of chemical fertilizers. However, this practice is not only costly, the sources of Phosphorus and Nitrogen are not renewable and the excessive application in the form of fertilizers is not environmentally sustainable. Therefore, it warrants a better understanding of the plant responses during the nutrient deficiency because such knowledge will help implement strategies for breeding crops with more efficient use of minerals.</p><p>Most prior efforts in studying the molecular and physiological responses to low minerals were focused on roots. However, recently it has been found that shoot-to-root long distance signaling plays an important role in the adaptation of roots to low nitrogen or phosphorus. Here, we measured different physiological and morphological parameters and used RNA-Seq to elucidate the physiological and molecular responses in the vascular tissues of <i>Plantago major</i>, a new model species established in our laboratory, to low nitrogen, low phosphate or combined nitrogen and phosphate starvation<i>. </i>In this study, <i>P major </i>showed reduced photosynthesis and Fv/Fm, increased catalase and ascorbate peroxidase activity, reduced phosphate and nitrate contents in respective treatments. In addition, assessment of root morphological parameters revealed that nutrient deficiencies could lead to higher root densities and increased root to shoot ratios.</p><p>For molecular analysis of transcriptome changes, 24 hours of nutrient starvation exhibited an alteration of 33, 221, and 329 genes for the deficiencies of phosphorus, nitrogen and combined nitrogen and phosphorus, respectively. Our study helped to dissect several novel pathways associated with the vascular system in response to the deficiencies of major macronutrients. </p>

Rice (Oryza sativa) is the staple food for more than 3.5 billion people worldwide. As the population continues to grow, rice yield will need to increase by 1% every year for the next 30 years to keep up with the growth. In the US, Arkansas accounts for more than 50% of rice production. Over the last 68 years, rice production has continued to grow in Mississippi, placing it in fourth place after Arkansas, Louisiana, and California. Due to increasing rice acreage, regionally and worldwide, the need to develop abiotic stress-tolerant rice has increased. Unfortunately, current rice breeding programs lack genetic diversity, and many traits have been lost through the domestication of cultivated rice. Currently, stressors stemming from the continued effects of climate change continue to impact rice. To counteract the impacts of climate change, research has shifted to evaluating wild and weedy relatives of rice to improve breeding techniques. Weedy rice (Oryza sativa ssp.) is a genetically similar, noxious weed in rice with increased competitive ability. Studies have demonstrated that weedy rice has increased genetic variability and inherent tolerance to abiotic stressors. The aims of this study were to 1) screen a weedy rice mini-germplasm for tolerance to cold, heat, and complete submergence-stress, 2) utilize simple sequence repeat (SSR) markers and single nucleotide polymorphisms to evaluate the genetic diversity of the weedy rice population, and 3) use genome-wide association (GWAS) to identify SNPs associated with candidate genes within the population.

Hevea brasiliensis est cultivé pour le caoutchouc naturel contenu dans le latex. Une exploitation intensive combinée aux stress environnementaux affectent la production de latex. L'encoche sèche ou Tapping Panel Dryness (TPD) est déclenché par un désordre physiologique complexe au sein des laticifères. Il est responsable d'une perte annuelle de production de 10 à 40% en fonction de l'âge de la plantation et des clones d'hévéa utilisés Le stress oxydatif, point de départ de la maladie, affecte l'écoulement, par la coagulation in situ des particules du caoutchouc. Chez les plantes, la réponse adaptative aux stress abiotiques dépend de la finesse de la régulation de l'expression des gènes. Ce contrôle se fait au niveau transcriptionnel mais également au niveau post-transcriptionnel. Les micro-ARNs jouent un rôle crucial en menant les ARN messagers cibles à la dégradation ou au blocage de leur traduction L'objectif de cette thèse est de comprendre et d'identifier la régulation du stress oxydatif en étudiant l'implication des micro-ARNs en réponse aux stress abiotiques et suite à l'apparition du TPD chez l'hévéa. L'isolement et le séquençage à haut débit de petits-ARNs ont permis l'identification de micro-ARNs d'hévéa. Dans un premier temps, une banque de petits ARNs a été effectuée à partir de vitroplants soumis ou non à des stress abiotiques, à laquelle s'est ajoutée dans un second temps deux banques fabriquées à partir de latex d'arbres en exploitation atteints ou non par le TPD. L'analyse de la population de petits ARNs montre une diminution de la taille des séquences en réponse à la maladie, la majorité des séquences de petits ARNs de latex étant de 21 nucléotides chez les arbres malades et 24 nucléotides chez les arbres sains. En combinant le pipeline LeARN et les données transcriptomiques, soixante huit familles de micro-ARNs conservés entre les espèces et quinze nouvelles familles de micro-ARNs ont été identifiées chez l'hévéa. Les gènes codant pour la voie de biogenèse des micro-ARNs sont présents dans le latex, suggérant leur production dans ce compartiment cellulaire particulier. L'identification des séquences de trente précurseurs de micro-ARNs ont permis d'étudier l'expression des gènes MIR en réponse aux stress abiotiques et en réponse au TPD. Les gènes MIR étudiés sont différentiellement exprimés chez des hévéas immatures en réponse au stress abiotiques et aux traitements par l'éthylène et le méthyl-jasmonate. L'abondance relative de transcrits des gènes MIR est fortement réduite par le TPD dès 5% de longueur d'encoche sèche à l'exception d'un gène.Les cibles potentielles des 83 familles de micro-ARNs ont été prédites. Ces micro-ARNs sont impliqués dans les voies de détoxication des espèces activées de l'oxygène, dans les voies de biosynthèse du caoutchouc naturel, dans les voies de biosynthèse et de signalisation de l'éthylène et du jasmonate. Trois cibles ont été validées expérimentalement dont la CuZnSOD chloroplastique, enzyme importante du système antioxydant. / Hevea brasiliensis is cultivated for natural rubber produced in latex cells. Intensive harvesting systems combined with environmental cues affect latex production. The Tapping Panel Dryness (TPD), a complex physiological disorder, causes a loss of production of 10-40%. Oxidative stress, starting point of the disease, affect latex flow because of in situ coagulation of rubber particles. In plants, the adaptation to abiotic stress relies on the fine tuning of the gene expression at the transcriptional and post-transcriptional levels. MicroRNAs play a crucial roles leading to mRNAs degradation or repression of their translation.The aim of this thesis is to understand and identify the regulation of the oxidative stress by studying the involvement of microRNAs in the regulation of abiotic stress and TPD occurrence in Hevea. Isolation and high-throughput sequencing of small RNAs allowed identifying Hevea microRNAs. Firstly, a small RNA library was constructed from in vitro plantlets subjected or not to abiotic stress, and secondly, two others small RNA libraries were constructed with latex from healthy and TPD-affected trees. Analyses of the small RNA population showed a decrease in the size of the reads in response to TPD, the majority of the small RNAs from latex being 21 nucleotides in TPD-affected trees and 24 nucleotides in healthy trees. Combining the LeARN pipeline and transcriptomic data, sixty eight microRNAs families conserved between plant species and fifteen new families were identified in Hevea. Genes involved in microRNA biogenesis are present in latex suggesting their production in this particular cellular compartment. Identification of thirty precursors of microRNAs allowed the expression analyses of the corresponding MIR genes in response to abiotic stress and upon TPD occurrence. MIR genes are differentially expressed in young plants in response to abiotic stress and in response to ethylene and methyl jasmonate treatments. Moreover, relative transcript abundance of MIR genes is strongly repressed upon TPD occurrence a soon as 5% of dry cut length except for one MIR gene.Putative targets were predicted for the 83 families. MicroRNAs are involved in ROS detoxification, natural rubber biosynthesis, ethylene and jasmonate biosynthesis and signalling pathways. Three targets were experimentally validated including the chloroplastic isoform of CuZnSOD, which is an important enzyme of the ROS-scavenging system.

Plants respond to environmental stress by altering their gene expression. Under stress conditions some genes are activated and some genes are repressed. Even though a lot of work has been done to understand mechanisms of gene activation under abiotic stress very little information is available on how stress responsive genes are kept repressed under normal growth conditions. Recent work has revealed that plants use transcriptional repression as common mechanism of gene repression. Transcriptional repression is achieved by recruitment co-repressor complexes to the target genes. Recent studies have revealed that the co-repressor LUH complexes with SLK1 and SLK2 to silence Arabidopsis thaliana stress responsive genes. However, the transcription factors involved in the recruitment of this complex to its target genes are not known. In this study, we identified SLK2INT1, as a novel transcription factor that is involved in silencing of select Arabidopsis thaliana stress responsive genes by recruiting the LUH-SLK2 complex.