An?lise prote?mica comparativa em Gluconacetobacter diazotrophicus estirpe PAL5 em resposta a presen?a de per?xido de hidrog?nio e efeitos do estresse na coloniza??o de plantas de arroz

• Autor(a) principal: Ferreira, J?ssica de Paula
• Data de Publicação: 2016
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da UFRRJ
• Texto Completo: https://tede.ufrrj.br/jspui/handle/jspui/3257
• topic: Prote?mica diferencial; Estresse oxidativo; Inocula??o; Proteomics differential; Oxidative stress; Inoculation; Fisiologia

Nitrogen is the nutrient uptake in larger quantities by the plants and, despite being abundant in the atmosphere, it is found a non-assimilable form. Among the alternatives of input of N to ecosystems, nitrogen fixation (BNF) is considered as the most viable process, since it reduces the costs and damage to the environment, reduces the dependence of nitrogen fertilizers and contributes to soil fertility. This process is performed by nitrogenase, which requires large amounts of ATP provided by aerobic respiration. During this process, reactive oxygen species (ROS) may be generated, damaging the cells, inhibiting the activity of nitrogenase and affecting the processes of photosynthesis and colonization. To accomplish FBN and colonize plants, endophytic bacteria must resist efficiently or repair the damage caused by reactive oxygen species. Gluconacetobacter diazotrophicus is an endophytic nitrogen fixing bacteria which, when subjected to oxidative stress, express proteins that directly eliminate the oxidant. This study analyzed the effect of oxidative stress induced by H2O2 in protein expression profile and the colonization of G. diazotrophicus strain PAL5 in rice plants. To this end, the wild type G. diazotrophicus was grown in biological triplicate in modified LGI-P, followed by exposure of cells to treatment with the presence and the absence of H2O2 (control). Cells were collected at different time intervals after induction with H2O2 or not, sampling was performed 15, 30 and 60 minutes. From these cultures were obtained total protein from each treatment which were used in liquid chromatography analysis coupled to mass spectrometry (ESI-LC-MS/MS). Analysis of mass spectrometry for protein identifications in various cellular processes such as replication, transcription, cell division, proteolysis, biosynthetic processes, transport, among others. As proteins involved in cell and oxidative control were identified after 30 minutes of exposure to H2O2, with increased abundance of Peroxidase and Superoxide Dismutase. In addition proteins, several other proteins related to the oxidation-reduction processes were also observed with greater differential abundance. In addition, a study was conducted to verify whether the oxidative stress induced by H2O2 affects the colonization of PAL5 strain when inoculated into rice plants. Rice plant variety IAC4440 was inoculated or not with mcherry PAL5 strain containing the reporter gene inserted in plasmid pMP7604. The roots were collected and examined in confocal microscope and the bacteria associated with roots were counted on plates medium modified LGI-P. The results showed that oxidative stress induced by H2O2 did not affect the colonization of the bacteria in rice plants since there was no difference in the number of CFU among treatments and the microscopic analysis showed that endophytic colonization of the roots by the strain PAL5 it was not altered by oxidative stress. The results of proteomics associated with colonization studies suggest that PAL5 bacteria triggers protective mechanisms to oxidative stress to rapidly decrease the levels of reactive oxygen species so that they do not affect their development and endophytic colonization.