The eATP pathway begins a cascade of events which includes the involvement of nitric oxide synthase (NOS) and nitrate reductase (NR) in the production of nitric oxide (NO). Research has shown that SNAP (S-nitroso-N-acetylpenicillamine) and NONOates (diazeniumdiolates) promote the availability of NO and, with the addition of guanylate cyclase, form cyclic guanine monophosphate (cGMP), and root hair growth is promoted. Phosphodiesterases (PDE) break down the cGMP and agents such as IBMX and Viagra inhibit the PDEs thereby inhibiting root hair growth. Several questions remain to be answered. How much cGMP is necessary for the promotion of root hair growth? Is there an optimal concentration of cGMP which stimulates root hair growth, above which is inhibitory, or below which is ineffective? Is there a “non-hydrolyzable analog” of cGMP which is more effective at promoting root hair growth? Is it possible to see inhibition of root hair growth with exposure to a known inhibitor, such as ATPγS, and then reverse the inhibition with a “non-hydrolyzable analog” of cGMP? Answering these vi questions is the substance of this research and the answers will provide direction and understanding to the growth-promoting and regulatory role eATP plays in signal transduction pathways in plants. With the hypothesis asking whether the effects of NO on root hair growth is cGMP-dependent or cGMP-independent we found that there is no consistent concentration of non-hydrolyzable cGMP analog which promotes root hair growth. Additionally we found that the 8-Br-cGMP analog promotes root hair growth more consistently in Arabidopsis thaliana than its counterpart, dibutyryl cGMP. We substantiated previously published results showing an inhibition of root hair growth when root hairs were exposed to high concentrations of ATPγS. Based on these results we believe the promotion of root hair growth in Arabidopsis thaliana to be mediated independently of cGMP. / text

Submitted by Rodolfo Lizcano Toledo (rodolfolizcano@gmail.com) on 2017-04-11T19:04:14Z No. of bitstreams: 1 Dissertação_Rodolfo_Lizcano_Toledo.pdf: 3969406 bytes, checksum: 2ef5dd577215fbca06ae57d5b3bd9983 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-04-11T19:16:06Z (GMT) No. of bitstreams: 1 lizcanotoledo_r_me_jabo.pdf: 3969406 bytes, checksum: 2ef5dd577215fbca06ae57d5b3bd9983 (MD5) / Made available in DSpace on 2017-04-11T19:16:06Z (GMT). No. of bitstreams: 1 lizcanotoledo_r_me_jabo.pdf: 3969406 bytes, checksum: 2ef5dd577215fbca06ae57d5b3bd9983 (MD5) Previous issue date: 2017-03-08 / O experimento foi conduzido em casa de vegetação na FCAV/UNESP. Objetivou-se avaliar a caracterização os sintomas visuais de deficiência de micronutrientes em folhas; e avaliar os efeitos desses micronutrientes no estado nutricional do maracujazeiro amarelo. No experimento foi utilizado solução nutritiva, como substrato vermiculita em extrato úmido. Foram utilizadas mudas de maracujá (Passiflora edulis Sims.) do híbrido comercial BRS Gigante Amarelo do Cerrado. O delineamento experimental foi inteiramente casualizado; com oito tratamentos (solução completa (Controle), omissão de B, omissão de Cu, omissão de Fe, omissão de Mn, omissão de Mo, omissão de Zn, solução completa + Ni); e 5 repetições. Utilizou-se como unidade experimental; vasos com 8 dm-3 de substrato com 1 planta/vaso e 2 vasos por unidade experimental. Após 60 dias do plantio, foi avaliada a matéria seca de cada tratamento, foi feito diagnóstico visual das deficiências nutricionais em folhas. Semanalmente foi avaliado o teor de clorofila para estabelecer regressão linear ou quadrática, entre os teores de clorofila e as deficiências nutricionais de cada tratamento; uma avaliação de variáveis fisiológicas como a transpiração, fotossíntese, condutância estomática e concentração interna e externa de CO2. Avaliaram-se características morfológicas como desenvolvimentos de pêlos radiculares e alterações no mesofilo foliar. A ordem de manifestação das deficiências no experimento foram as seguintes: Fe>Mn>Zn>Cu>Mo>B; a toxicidade de Níquel apareceu aos 40 dias após ao transplante, com sintomas de clorose nas folhas novas, logo um amarelecimento geral e algumas partes brancas. A omissão de B resultou em deformações no limbo foliar e perda da dominância apical, mostrando deformação dos pontos de crescimento; a omissão de Mn ocasionou redução do crescimento e clorose generalizada das folhas jovens com reticulo grosso, a omissão de Fe resultou em uma clorose internerval, com aparência de reticulado fino, sendo Fe e Mn os primeiros nutrientes cuja omissão resulta em sintomas de deficiência nas plantas de maracujá, seguidos pelo zinco e cobre, e por último o boro. O zinco e cobre apresentaram deformação e alongamento das folhas reduzindo consideradamente a altura e desenvolvimento radicular, o molibdênio apresentou uma clorose nas folhas velhas que posteriormente se espalho de forma geral. Os teores dos micros na parte aérea e raiz variaram de acordo ao tratamento, refletindo em problemas morfológicos e a nível ultra-estrutural na célula e fisiológicos, alterando variáveis como a transpiração, fotossíntese, condutância estomática e a relação entre a concentração de CO2 nos espaços intercelulares e a concentração de CO2 no ambiente. A omissão de ferro foi o elemento que mais refletiu problemas pela formação de pêlos radicais, observando-se maior formação destes em condições de deficiência do elemento possivelmente por sua relação com os hormônios etileno e auxinas. Todos os tratamentos apresentaram um desarranjo e desorganização a nível ultra-estrutural, nas organelas como os cloroplastos, parede celular, grânulos de amidos, grânulos de lipídeos, núcleo, afetando o funcionamento da célula e causando problemas fisiológicos que refletem em sintomas visuais e no desenvolvimento do maracujazeiro. / The experiment was conducted in a greenhouse at FCAV / UNESP. The objective was to evaluate the characterization of the visual symptoms of micronutrient deficiency in leaves; And to evaluate the effects of these micronutrients on the nutritional status of yellow passion fruit. In the experiment, a nutrient solution was used, with vermiculite as a substrate in a humid extract. Passion fruit (Passiflora edulis Sims.) Seedlings of commercial hybrid BRS Gigante Amarelo do Cerrado were used. The experimental design was completely randomized; With eight treatments (complete solution (Witness), omission of B, omission of Cu, omission of Fe, omission of Mn, omission of Mo, omission of Zn, complete solution + Ni); And 5 replicates. It was used as experimental unit; Vessels with 8 dm-3 of substrate with 1 plant / pot and 2 pots per experimental unit. After 60 days of planting, the dry matter of each treatment was evaluated, and the nutritional deficiencies were evaluated in leaves. The chlorophyll content was evaluated weekly to establish linear or quadratic regression between chlorophyll levels and nutritional deficiencies of each treatment; besides an evaluation of physiological variables such as transpiration, photosynthesis, stomatal conductance and internal and external CO2 concentration. Morphological characteristics were evaluated by means of optical microscope as were the development of root hair and by means of transmission electron microscopy, changes in leaf mesophyll. The order of manifestation of the deficiencies in the experiment were as follows: Fe> Mn> Zn> Cu> Mo> B; The toxicity of Nickel appeared at 40 days after the experiment was carried out in the vessels with symptoms of chlorosis in the new leaves, thus a general yellowing and some white parts. The omission of B resulted in deformations in the leaf limb and loss of apical dominance, showing deformation of the growth points; The omission of Mn caused reduction of growth and generalized chlorosis of the young leaves with thick reticulum, the omission of Fe resulted in an internerval chlorosis, with the appearance of fine crosslinking, with Fe and Mn being the first nutrients whose omission results in symptoms of plant deficiency Of passion fruit, followed by zinc and copper, and finally boron. The zinc and copper presented deformation and elongation of the leaves, reducing considerably the height and root development, the molybdenum presented a chlorosis in the old leaves that later spread in general form. The contents of the microspheres in the shoot and root varied according to the treatment, reflecting in morphological and ultrastructural problems in the cell and physiological, changing variables such as transpiration, photosynthesis, stomatal conductance and the relation between the concentration of CO2 in the spaces And the concentration of CO2 in the environment. The iron omission was the element that most reflected problems due to the formation of radical hair, being observed a greater formation of iron under conditions of deficiency of the element possibly due to its relation with the hormones ethylene and auxins. The observations in the transmission electron microscope showed in all the treatments a derangement and disorganization at the ultrastructural level, in the organelles as the chloroplasts, cell wall, starch granules, lipid granules, nucleus, affecting the functioning of the cell and causing physiological problems Which reflect on visual symptoms and the development of passion fruit.

La mise en place des interactions symbiotiques, existant entre les Légumineuses et des microorganismes, sont des processus finement régulés, tant sur le plan moléculaire que cellulaire. Dans le cas de la symbiose avec des bactéries fixatrices d'azote, ou rhizobia, ces modifications se déroulent dans les poils racinaires. Plusieurs arguments tendent à montrer que la perception de contraintes mécaniques pourrait avoir un rôle dans la régulation de ces étapes. Par conséquent, nous nous sommes intéressés à une famille de protéines impliquées dans la mécanotransduction: les MSL (MscS-Like). Il s'agit de canaux capables de s'ouvrir en réponse à une tension mécanique appliquée sur la membrane plasmique. Nous avons exploré leurs fonctions dans les poils racinaires, lors de la mise en place de la symbiose entre la légumineuse modèle Medicago truncatula et des rhizobia. Des études in silico du génome de M. truncatula nous ont permis de définir la famille des MtMSL. Les analyses de l'expression de ces candidats ont montré que seuls deux d'entre eux sont transcrits dans les poils racinaires: MtMSL2.1 et MtMSL2.4, ce dernier étant majoritaire. Néanmoins, leur expression ne semble pas modifiée par un traitement aux facteurs Nod, molécules bactérienne induisant les premières étapes de la signalisation symbiotique. Parallèlement, des analyses en microscopie confocale, indiquent la présence de MtMSL2.4 à la membrane plasmique et dans des endomembranes. De plus, des mesures électrophysiologiques ont confirmé leur nature de canal à forte conductance activé par la tension de membrane. Enfin, plusieurs analyses phénotypiques ont été menées sur des mutants Mtmsl2.4, tant sur différents aspects de l'interaction avec les rhizobia, que sur la croissance racinaire ou celle des poils racinaires, pour laquelle une méthode de mesure semi-automatique a été développée. Cependant aucune différence avec les contrôles n'a pu être observée. Ces résultats laissent penser que le canal mécanosensible MtMSL2.4 aurait un rôle lors d'autres phénomènes qu'il serait intéressant de découvrir. / The first steps of symbiosis occurring between Legumes and microorganisms are highly regulated processes, both at the molecular level and at the cellular level. During symbiosis with nitrogen fixing bacteria, called rhizobia, these modifications occur in root hairs. Several arguments have shown that mechanical constraints may regulate these steps. Hence, the involvement of physical sensors during these early events is worth considering. We focused here on one of these sensor families, the MSL (MscS-Like). These proteins are channels able to open upon mechanical stretching. We look for their role during first steps of symbiosis between the model Legumes Medicago truncatula and rhizobia. In silico studies of M. truncatula's genome allowed us to define a MtMSL family. Transcript analyses showed that only two of them were expressed in root hairs: MtMSL2.1 and MtMSL2.4, the latter being the most expressed. However, this expression is not modified by Nod factor treatments; molecules produced by rhizobia that induces the first symbiotic signalization steps. In parallel, confocal microscopy analyses show plasma membrane and endomembrane localization of MtMSL2.4. Moreover, electrophysiological measurements confirmed that this candidate is a high conductance channel mechanically activated. Finally, we performed several phenotypical studies with Mtmsl2.4 mutants in different conditions. No differences were observed between the mutants and WT during rhizobia symbiotic interaction and root growth. There were also no differences observed in root hair development, for which a partly automatic measurement system was set up. These results suggest MtMSL2.4 may have a role in other phenomena, which could be interesting to understand further.