Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-04-26T14:05:04Z No. of bitstreams: 1 2012 - Patricia Gon?alves Galv?o.pdf: 5289496 bytes, checksum: 8dcb41bc971793cc8b4cdf383401ade4 (MD5) / Made available in DSpace on 2017-04-26T14:05:05Z (GMT). No. of bitstreams: 1 2012 - Patricia Gon?alves Galv?o.pdf: 5289496 bytes, checksum: 8dcb41bc971793cc8b4cdf383401ade4 (MD5) Previous issue date: 2012-03-01 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The objective of this study was to evaluate the protein profile expression of G. diazotrophicus PAL5 and its defective mutant in the indole compound production (lao-) grown in the presence or absence of tryptophan through 2DE-PAGE technique. The spectrometric analysis allowed the identification of 24 differentially expressed proteins. The majority of the proteins down regulated in the wild type PAL5 cultivated with tryptophan as compared to the cultivation without the amino acid belonged to the category of transductional modification, protein turnover and chaperones. For the mutant lao- grown in the same conditions, the majority of the proteins that presented differential expression belonged to the category of production and conversion of energy. In addition, the majority of the protein differentially expressed in the mutant lao- as compared to the wild-type PAL5 strains belonged to carbohydrates metabolism and transport. On the other hand, no proteins related to the tryptophan biosynthesis were detected in any condition, possibly due to the low yield of the proteins during the spectrometric analysis. Furthermore, mutants lao- and nif- of G. diazotrophicus were used for inoculation of micropropagated sugarcane plants in order to determine the influence of auxins produced by the bacteria in the plant growth promotion in comparison with PAL5. The first experiment, carried out in hydroponic conditions for 10 days showed a significant inoculation effect of the wild type on plant shoot. The other experiment was conducted in a period of 120 days in pots containing sand:vermiculite substrate fertilized with 30 and 60 ppm with ammonium sulphate enriched with 15N. The plants were inoculated in vitro with the wild type and mutants lao- and nif-, and the results showed a visual difference in the roots inoculated with PAL5 that showed higher volume suggesting a higher number of secondary roots and root hairs. On the other hand, the plants inoculated with the lao- mutant were ticker and showed lower number of secondary roots and root hairs. The shoot biomass of plants inoculated with PAL5 was higher than those inoculated with the mutant strains for both N dose, however the difference was not significant. Plants grown with 60 kg N dose and inoculated with the mutants showed lower accumulation of dry shoot mass than plants inoculated with the wild type strain. In conclusion, the present study showed the occurrence of several differentially expressed proteins either in the wild type strain or in the mutant lao- grown in LGI-P with and without tryptophan. The role played by these proteins in the metabolism of the bacteria requires additional studies, including different growth conditions. In addition, the inoculation of micropropagated sugarcane plants suggested a hormonal effect of the bacteria mainly on the root development e consequently in the N use efficiency. However, the size of the pots may have limited the plant development, suggesting that new experiments should be carried out in more appropriated conditions to confirm the influence of the indol production and the BNF during the association of the G. diazotrophicus and sugarcane plants / Este estudo teve por objetivo avaliar o perfil de express?o de prote?nas de G. diazotrophicus PAL5 e seu mutante defectivo na produ??o de compostos ind?licos (lao-) cultivados na presen?a e aus?ncia de triptofano atrav?s da t?cnica de 2DE-PAGE. A an?lise por espectrometria de massa permitiu a identifica??o de 24 prote?nas diferencialmente expressas. A maioria das prote?nas com a express?o diminu?da em PAL5 cultivada em meio com triptofano em rela??o ao meio de cultivo sem esse amino?cido pertenceu ? categoria modifica??o p?s-traducional, turnover de prote?nas e chaperonas. No mutante lao- cultivado nas mesmas condi??es, a maioria das prote?nas que apresentaram express?o diferencial pertencia ? categoria produ??o e convers?o de energia. Em adi??o, a maioria das prote?nas que foram diferencialmente expressas no mutante lao- em compara??o com a estirpe selvagem PAL5 pertencia ? categoria metabolismo e transporte de carboidratos. Por outro lado, n?o foram observadas prote?nas relacionadas ? bioss?ntese de triptofano em nenhuma condi??o analisada possivelmente devido ao baixo rendimento das identifica??es por espectrometria. Al?m das an?lises dos perfis de prote?nas, os mutantes lao- e nif- de G. diazotrophicus foram inoculados em plantas de cana-de-a??car micropropagadas com o objetivo de determinar a influ?ncia das auxinas na promo??o do crescimento dessa cultura em compara??o com a estirpe selvagem PAL5. O primeiro experimento, conduzido em condi??es de hidroponia pelo per?odo de 10 dias, mostrou efeito significativo da inocula??o da estirpe selvagem na promo??o de crescimento da parte ?rea das plantas, enquanto que o mutante lao-, n?o diferiu estatisticamente do controle n?o inoculado. O outro experimento, foi conduzido por 120 dias em vasos com substrato areia:vermiculita contendo 30 ou 60 ppm de sulfato de am?nio enriquecido com 15N e as pl?ntulas foram inoculadas in vitro. Os resultados mostraram uma diferen?a visual nas ra?zes das plantas inoculadas com PAL5, que se mostraram mais volumosas, aparentando um n?mero mais elevado de ra?zes secund?rias e p?los radiculares. J? as plantas inoculadas com lao- apresentaram ra?zes mais grossas, com um n?mero muito reduzido de ramifica??es ou p?los radiculares. A biomassa seca da parte a?rea das plantas inoculadas com PAL5 foi superior ?quelas inoculadas com as estirpes mutantes para as duas doses de nitrog?nio, por?m essa diferen?a n?o foi significativa. N?o foram observadas evid?ncias de contribui??o da FBN, por?m as plantas inoculadas com PAL5 foram menos eficientes na recupera??o do N fertilizante. Em conclus?o, o presente estudo mostra a ocorr?ncia de diversas prote?nas diferencialmente expressas tanto na estirpe selvagem como em lao- quando crescidas na presen?a e aus?ncia do amino?cido triptofano. A defini??o do papel dessas prote?nas no metabolismo da bact?ria requer estudos adicionais, inclusive em diferentes condi??es de cultivo. Em adi??o, a inocula??o dessas bact?rias em plantas de cana-de-a??car mostrou o efeito hormonal da bact?ria no desenvolvimento das ra?zes e, por conseguinte na maior efici?ncia de uso do N aplicado. Entretanto, dado a limita??o de espa?o f?sico dos vasos para o desenvolvimento das plantas, sugere-se a realiza??o de novos experimentos, em condi??es mais apropriadas, para confirmar a influ?ncia da produ??o de ?ndoles e da FBN durante a associa??o da bact?ria com as plantas de cana-de-a??car.