>Magister Scientiae - MSc / Nitric oxide (NO) has been shown to orchestrate multiple defense responses to both abiotic and biotic stress. Importantly, elevation of nitric oxide content in plants by using nitric oxide generating compounds has been shown to enhance plant tolerance to abiotic stresses such as salt and drought via up-regulation of genes involved in the regulation of plant responses to abiotic stress. In this study, the effect(s) of nitric oxide (generated from 10 μM of the nitric oxide donor DET/NO) on the expression of a novel soybean cystatin gene (Glyma20g08800), lipid peroxidation, caspase-like activity and cell death in salt (150 mM)-stressed soybean leaves, roots and nodules were investigated. Salt treatment resulted in elevated lipid peroxidation, caspase-like activity and increased cell death in organs studied while the observed detrimental effects of salt stress were reversed by NO treatment. Salt stress suppressed the expression of Glyma20g08800 while the levels of expression of Glyma20g08800 returned towards those of unstressed plants when the salt-stressed plants were supplemented with nitric oxide (DETA/NO). Furthermore, promoter sequences of GmCYS1p626 and three of its homologues (Glyma20g08800, Glyma14g04250 and Glyma18g12240) were analyzed for putative abiotic stress and/NO cisregulatory elements based on co-expression analyses using bioinformatics. Several abiotic stress induced transcription factors (TFs) were identified and were hypothesized to be co-acting either directly or indirectly through additional factors in the regulation of soybean cystatin expression in response to NO and abiotic stress. Taken together, these results highlight the possibility of using NO to drive high levels of expression of cystatins during salt stress and lead to accumulation of the cystatin to levels that are sufficient to inhibit salt stress-induced caspase-like activity, which will inhibit salt stress-induced cell death and thus enhance the tolerance of the plant to salt stress and possibly tolerance to drought stress as well.

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>Magister Scientiae - MSc / In agriculture crop productivity is greatly affected by stresses such as salinity, drought, temperature and honnonal changes of crop plants and responses to these stresses. Studies have shown that a natriuretic peptide based regulatory system responsible for water and ion homeostasis in animals has a hctcrologous equivalent in plants. Plant natriuretic peptide immunoreactants (irPNPs) have been reported to be involved in K+, Na+ and er ions fluxes in plants. Previously, an Arabidopsis thaliana transcript (AtPNP-A) encoding an irPNP (AtPNP-A) has been identified and isolated (Ludidi et al., 2002). The AtPNP-A a novel protein and part of its physiological role is presented here.

Food security is a major concern of the 21st century, given climate change and population growth. In addition, high salt concentrations in soils affect ~20% of irrigated land and cause a substantial reduction in crop yield. Cultivating salt-tolerant crops could enable the use of salt-affected agricultural land, reduce the use of fresh water and alleviate yield losses. Innovative methods need to be developed to study traditional and novel traits that contribute to salinity tolerance and accurately quantify them. These studies would eventually serve for developing new salt tolerant crops, adapted to the harsh arid and semi-arid climate conditions. A study of 200 accessions of the wild tomatoes (Solanum pimpinellifolium) was conducted in field conditions with phenotyping using an unmanned aerial vehicle (UAV)-mounted hyperspectral camera. Six genotypes with different levels of salt tolerance were sampled for leaf pigment analyses, revealing a clear pattern for the high salt tolerant accession M007, where pigment content in the salt-treated plants significantly increased compared to their control counterparts only in harvesting campaigns 3 and 6, each performed two days after the first and second salt stress application events. Moreover, the light harvesting capacity was found to be better maintained under salt stress in the medium (M255) and highly salt tolerant (M007 and M061) accessions. Pigment quantitation data will contribute towards the groundtruthing of hyperspectral imaging for the development of remote sensing-based predictive pigment mapping methods. This work establishes a reliable quantification protocol for correlating pigment content with vegetation indices. Hence, pigment content captured by imaging techniques and validated using biochemical analysis would serve in developing a high-throughput method for pigment quantitation in the field using UAV-based hyperspectral imaging. This would serve as a tool for measuring pigment content in large number of genotypes in the field which would eventually lead to new salt-tolerant genes.

Many plants are highly sensitive to salt in the soil, and their growth and yield can be greatly hindered by as little as less than 1% salt concentration in the soil. Additionally, soil salinity is a growing issue globally and affects significant areas in Utah. Halophytes are plants that are adapted to grow in saline soils and have been widely studied for their physiological and molecular characteristics, but little is known about their associated microbiomes. Bacteria were isolated from the rhizosphere and as root endophytes of Salicornia rubra, Sarcocornia utahensis, and Allenrolfea occidentalis, three native Utah halophytes. Several strains of halophilic bacteria have been isolated and screened for the ability to stimulate plant growth in saline conditions despite the high salt concentrations. Halomonas, Bacillus, and Kushneria species were consistently isolated both from the soil and as endophytes from roots of all three plant species at all collection times. Of the isolates tested for the ability to stimulate growth of alfalfa under saline conditions, Halomonas and Kushneria strains stimulated plant growth in the presence of 1% NaCl. The same bacteria used in the inoculation were recovered from surface sterilized alfalfa roots, indicating the ability of the inoculum to become established as an endophyte. This raises the question of whether these plant associated halophilic isolates contain genes that aid in plant growth promotion. We are interested in genomic sequencing of our Halomonas and Kushneria strains and performing genomic analysis to determine if there is a difference in genes between plant associated and non-plant associated halophilic isolates. We explored the hypothesis that certain bacterial properties have been selected for to aid plant growth. This was accomplished by performing whole genome sequencing of 26 Kushneria and Halomonas strains, both plant and non-plant associated. These strains came from freezer stocks of previously collected isolates as well as field trips to collect more samples. Halophilic bacteria were isolated from bulk soil, rhizosphere, and halophyte tissues (root and shoot tissues). The non-plant associated (bulk soil) halophilic Kushneria and Halomonas strains aided in determining if there are specific bacterial genes that are expressed in plant associated strains. Whole genome sequencing of the isolates was performed on the Oxford Nanopore platform. The sequence data was then assembled and annotated. The genomes were then included in a genome wide association study was performed. The results from the GWAS show that there is not a significant difference between plant and non-plant associated isolates, disproving our hypothesis. The results also show that few known genes for phytohormone synthesis were present in the pangenome, highlighting the need for further research to determine how these halophilic isolates aid in plant growth promotion in saline soils.

In many arid and semiarid regions worldwide, high levels of soil salinity is a key driver of land degradation, as well as a key impediment to re-establishing plant cover. Combating land degradation and erosion associated with soil salinity requires experimental determination of plant species that can grow in soils with high levels of salinity and can be used to re-establish plant cover. Herein, we evaluated the responses of untested candidate cultivars of two halophytic grass species to high soil salinity: alkali sacaton (Sporobolus airoides Torr.) and seashore paspalum (Paspalum vaginatum Swartz). We evaluated the growth responses of both species in a greenhouse under control (no-salt) and various levels of NaCl salinity (EC 8, 16, 24, 32, 40, and 48dSm(-1)) using Hoagland solution in a hydroponics system in a randomized complete block design trial. At all salinity levels, sacaton grass had a greater shoot height, shorter root length, lower shoot fresh and dry weights, and poorer color and general quality compared to seashore paspalum. The shoot fresh and dry weights of both grasses were greatest at the low to medium levels of salinity, with the greatest response observed at EC 16dSm(-1). At the highest level, salinity significantly reduced shoot fresh and dry weights of both grasses. Because growth of both halophytic species exhibited high tolerance to salinity stress and were stimulated under low to medium levels of salinity, both species could be considered suitable candidates for re-establishing plant cover in drylands to combat desertification and land degradation associated with high levels of soil salinity.

Les systèmes de sécrétion de NO3– de la membrane plasmique des cellules végétales jouent un rôle important dans l'activité stomatique et la réponse des plantes à des stress biotiques et abiotiques. Malgré quelques avancées récentes, ces systèmes restent mal connus sur le plan moléculaire. Mon travail de thèse a consisté à caractériser le rôle physiologique de NAXT2, un membre du sous-groupe NAXT (NitrAte eXcretion Transporter) de la famille des transporteurs NRT1/PTR chez Arabidopsis thaliana. Mes résultats montrent que NAXT2 est un transporteur de NO3– et qu'il est principalement exprimé dans les cellules du péricycle de la racine, au voisinage des vaisseaux xylémiens. Sous contrainte saline et comparativement aux plantes sauvages (WT), un mutant knock-out pour NAXT2 (naxt2-1) présente un défaut de distribution du NO3– vers les feuilles et de sécrétion du NO3– dans la sève xylémienne, qui se traduisent par une diminution des teneurs en NO3– foliaires. NAXT2 est donc impliqué dans la charge du xylème en NO3– sous contrainte saline. Aucune des différences phénotypiques mises en évidence entre le mutant et WT sous contrainte saline n'a été observée sous stress osmotique ou en condition standard, suggérant que NAXT2 est essentiellement impliqué dans la réponse à la composante ionique du stress salin. Enfin, après un traitement salin prolongé, la biomasse foliaire de naxt2-1 est inférieure à celle de WT, ce qui indique que NAXT2 joue un rôle important dans l'adaptation des plantes aux contraintes salines modérées. Dans l'ensemble, ce travail suggère que NAXT2 est impliqué dans une fonction physiologique majeure, la translocation du NO3–, point de contrôle de la distribution du nitrate, et dans l'adaptation de la plante aux contraintes salines. / NO3– secretion systems at the plasma membrane of plant cells play an important role in stomata activity and plant response to biotic and abiotic stresses. Despite of few recent advances, these systems are still poorly known at the molecular level. During my thesis, I worked on the characterization of the physiological role of NAXT2, a member of the NAXT (NitrAte eXcretion Transporter) sub-group from the large NRT1/PTR transporters family in Arabidopsis thaliana. The results presented here show that NAXT2 is a NO3– transporter and that it is mainly expressed in root pericycle cells, close to the xylem vessels. Under salinity constraint and relatively to wild type plants (WT), a NAXT2 knock-out mutant (naxt2-1) displayed a defect in NO3– distribution towards the shoots and in NO3– secretion into the xylem sap, which lead to a decrease in shoot NO3– content. Thus, NAXT2 is involved in NO3– xylem loading under salinity constraint. None of the phenotypic differences described in this work between WT and mutant was observed under osmotic stress or standard culture conditions, suggesting that NAXT2 is specifically involved in response to the ionic component of salt stress. Finally, after a prolonged salt treatment, naxt2-1 shoot biomass was lower than that of WT, indicating that NAXT2 plays an important role in plant adaptation to mild salinity constraint. Altogether, this work suggests that NAXT2 is involved in a major physiological function, the NO3– translocation, control point of nitrate distribution and in plant adaptation to salinity constraint.

The exposure of plants to high salt concentrations causes accumulation of sodium ions. This leads to the inability of the plants to uptake water, a disturbance of ion homeostasis, a decrease in photosynthesis and oxidative stress. As a result of the salt stress, the availability of NADPH decreases. The adaptation to the concentrations of salt depends on plant's ability to compensate for the decreased availability of NADPH, which can be further used in antioxidative cycles and the synthesis of antioxidative compounds and osmoprotectants. In this work, the reduction of relative water content, a decrease in the Rubisco enzyme activity, an increase of Hsp70 in the leaves and an increase in the accumulation of sodium ions was shown in cucumber plants (Cucumis sativa L. convar. Jogger F1) exposed to salt stress (100 mM NaCl). As a consequence of salt stress, an increase in the activity of NADPH providing enzymes was found. Particularly on the second and third day of salt stress, an increase in the activity (up to 270 %) of: NADP-isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, NADP-malic enzyme, non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase in leaves was detected. The activity of less abundant NADP-dehydrogenases (glucose 1-dehydrogenase, gluconate 2-dehydrogenase, galactose...

Le remodelage de la chromatine et la modification des histones jouent des rôles très importants dans l’établissement et la reprogrammation de l’état de l’expression génique. Il reste largement inconnu concernant les mécanismes de la régulation de ces processus chromatiniens dans le contrôle de l’expression génique impliquée dans le développement de la plante et son adaptation à l’environnement. Mon sujet de thèse se focalise sur l’analyse fonctionnelle d’un facteur de remodelage de la chromatine de type Chromodomain/Hélicase/DNA-binding 1 (CHD1) d’Arabidopsis, appelé CHR5 et une histone démethylase qui est spécifiquement impliquée dans la démethylation de l’histone H3 lysine 4 (H3K4), appelée JMJ15. Dans la première partie de cette étude, nous avons montré que le gène CHR5 est activé au cours de l’embryogénèse et que son expression se maintient élevé dans les tissues/organes en développement. L’analyse de mutants révèle que la perte de fonction de ce gène fait réprimer l’expression de gènes régulateurs de la maturation de l’embryon tels que LEC1, ABI3 et FUS3 pendant le développement des graines, et fait baisser l’accumulation des protéines de réserve. L’analyse de double mutants a permis de démontrer une fonction antagoniste entre CHR5 et PKL, une protéine du groupe « CHD3 », dans l’activité du promoteur de gènes régulateurs du développement de l’embryon et l’accumulation de réserve de graine. Nous avons montré que la protéine CHR5 s’associe directement avec les promoteurs d’ABI3 et FUS3 et que la mutation du gène CHR5 conduit à l’augmentation de présence de nucléosome dans la région du départ de transcription. Ces résultats suggèrent que CHR5 est impliquée dans le positionnement de nucléosome pour stimuler l’expression de gènes de la maturation de l’embryon, ce qui est contrebalancé par l’action de PKL au cours du développement de l’embryon. La deuxième partie de cette étude a permis de montrer que l’expression du gène de l’histone démethylase JMJ15 manifeste une forte spécificité tissulaire. L’analyse de mutants du gène a permis de l’identification de 2 allèles de gain de fonction (avec surexpression du gène), et un allèle de perte de fonction. La surexpression du gène réduit la croissance d’hypocotyle et de tige de la plante avec accumulation de lignine dans la tige, mais le perte de fonction du gène ne produise pas de phénotype apparent. Par ailleurs, la surexpression du gène renforce la tolérance de la plante au stress salin, alors la perte de fonction du gène rend la plante plus sensible. L’analyse du transcriptome a révélé beaucoup plus de gènes réprimés qu’activés par la surexpression du gène JMJ15. Ces gènes réprimés sont préférentiellement marqué la H3K4me2 ou H3K4me3, parmi lesquels beaucoup codent de facteurs de transcription. Ces données suggèrent que l’induction de JMJ15 pourrait réguler le programme de l’expression génique qui coordonne la restriction de la croissance de la plante et la tolérance au stress. Ces travaux de thèse a permis ‘identifier quelques nouveaux éléments dans la compréhension de la fonction de régulateurs chromatiniens dans l’expression génique de la plante. / Chromatin remodeling and histone modification play important roles in the establishment and dynamic regulation of gene expression states. However, little is known regarding to the regulatory mechanism of chromatin modification and remodeling that control gene expression involved in plant development and responses to environmental cues. My thesis work concerns functional analysis of an Arabidopsis Chromodomain/Helicase/DNA-binding 1 (CHD1) type chromatin remodeling gene known as CHR5 and a histone demethylase gene that specifically removes methyl groups from methylated histone H3 lysine 4 (H3K4me), called JMJ15 in regulating chromatin structure or in resetting chromatin modifications that control the expression of plant developmental and stress responsive genes. In the first part of the study we found that CHR5 expression is activated during embryogenesis and remained to be expressed in developing organs/tissues. Analysis of mutants revealed that loss-of-function of the genes led to decreased expression of key embryo maturation genes LEC1, ABI3 and FUS3 in developing seeds and reduced seed storage protein accumulation. Analysis of double mutants revealed an antagonistic function between CHR5 and PKL, a CHD3 gene, in embryo gene promoter activity and seed storage protein accumulation. CHR5 was directly associated with the promoters of ABI3 and FUS3 and chr5 mutations led to increased nucleosome occupancy near the transcriptional start site. The results suggest that CHR5 is involved in nucleosome occupancy to regulate embryo identity genes expression, which is counterbalanced by PKL during embryo development. The second part of this study showed that expression of JMJ15 was restricted to a few tissues during vegetative growth. The jmj15 gain-of-function mutations reduced the length of seedling hypocotyls and inflorescence stems with higher accumulation of lignin in the stem, while the loss-of-function mutants did not show any visible phenotype. The gain-of-function mutants enhanced salt tolerance, whereas the loss-of-function mutants were more sensitive to salt. Transcriptomic analysis revealed a much higher number of genes down-regulated in JMJ15 over-expression plants, which are highly enriched for H3K4me3 and H3K4me2. Among the down-regulated genes, many encode transcription regulators of stress responsive genes. The data suggest that increased JMJ15 levels may regulate the gene expression program that may coordinate plant growth restrains and enhances stress tolerance. Taken together, my thesis work brought a few new elements to the current understanding of chromatin regulators function in plant gene expression.

O aumento contínuo das emissões de gases causadores do efeito estufa resulta em níveis elevados de aquecimento do planeta. Estes efeitos, relacionados à mudança do clima, representam impactos na saúde humana, na produção de alimentos, nos ecossistemas e no abastecimento hídrico. Com o abastecimento hídrico afetado, as águas de menor qualidade para fins de irrigação, tornam-se importantes fontes hídricas para produção de alimentos. Dessa forma, objetivou-se estudar a tolerância da mini melancia à salinidade, bem como a resposta da cultura ao incremento de CO2 atmosférico, sob irrigação salina e não salina. O primeiro experimento foi realizado na Escola Superior de Agricultura Luiz de Queiroz (ESALQ-Piracicaba), em casa de vegetação. Cultivaram-se plantas de mini melancia, cv. Smile, irrigadas com água de diferentes condutividades elétricas- CEa (1, 2, 3, 4 e 5 dSm-1) com o objetivo de estudar a tolerância das plantas, em função do estresse salino. O segundo experimento foi realizado no Centro Nacional de Pesquisa de Informática Agropecuária (CNPTIA-Campinas) em duas câmaras de crescimento. Com objetivo de estudar como o incremento do CO2 atmosférico afeta a tolerância à salinidade da mini melancia cv. Smile. Na primeira câmara de crescimento (C1), cultivaram-se as plantas irrigadas com águas de diferentes condutividades elétricas- CEa (1 e 5 dSm-1), com aumento da concentração atmosférica de CO2 para 800 ppm. Na segunda câmara de crescimento (C2), cultivaram-se as plantas irrigadas com as mesmas condutividades elétricas da C1, entretanto com a concentração de CO2 atmosférico de 400 ppm. A salinidade afetou negativamente e o aumento da concentração de CO2 afetou positivamente a massa e o tamanho dos frutos da mini melancia. Assim, conclui-se que a mini melancia cv. Smile é moderadamente sensível à salinidade e que o aumento da concentração de CO2 atmosférico favorece o desenvolvimento dos frutos irrigados com água salina e não salina. / The continued increase in emissions of greenhouse gas effect inducing gases results in the warming of the planet. These climate change-related effects impact human health, food production, ecosystems and water supply. With the water supply affected, lower quality water becomes a possible water source for food production. Thus, the purpose of this analysis was to study the tolerance of mini watermelon to salinity and the crop response to increasing atmospheric CO2 in saline and non-saline irrigation. The first experiment was conducted at the School of Agriculture \"Luiz de Queiroz\" (ESALQ-Piracicaba), under greenhouse conditions. Mini watermelon plants were cultivated and irrigated with water of different electrical conductivities-ECw (1, 2, 3, 4 and 5 dSm-1) with the purpose of observing the behavior of plants affected by salt stress. The second experiment was carred out at the National Center of Agricultural Informatics Research (CNPTIA-Campinas) in two different growth chambers. In the first growth chamber (C1), the plants were cultivated and irrigated with different electrical conductivities-ECw (1 and 5 dSm-1), with an increase of atmospheric CO2 concentration to 800 ppm. In the second growth chamber (C2), plants were grown and irrigated with the same electrical conductivities as in C1, though the atmospheric concentration was kept at CO2 400 ppm. The salinity negatively affected the mass and the size of the fruits of mini watermelon while the increased CO2 concentration had a positive effect. Thus, it is possible to conclude that the cv. Smile mini watermelon is moderately sensitive to salinity whereas the increasing atmospheric CO2 concentration favors the development of irrigated fruit with both saline and non-saline water.