Caracterização das reservas de carbono e metabolismo de lipídeos em Pisolithus

• Autor(a) principal: Campos, André Narvaes da Rocha
• Data de Publicação: 2008
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/1569
• topic: Microorganismo do solo; Pisolithus; Glicogênio; Ácidos graxos; Glioxilatos; Lipídeos - Oxidação; Soil Microorganism; Pisolithus; Glycogen; Fatty acids; Glyoxylates; Lipids - Rust; CNPQ::CIENCIAS AGRARIAS::AGRONOMIA::CIENCIA DO SOLO::MICROBIOLOGIA E BIOQUIMICA DO SOLO

The mobilization of carbon storage compounds is an important metabolic step in processes such as basidiosporogenesis, spore germination, and establishment of the ectomycorrhizal association in Pisolithus. The objective of this work was to study the composition of the carbon reserve compounds during the development of Pisolithus basidiocarp as well as to investigate the mobilization pathways of these compounds in the mycelium, mycorrhizas, and fruit bodies. Histochemical analysis of the basidiocarps showed the presence of glycogen inside basidia and during spore development. Total RNA and proteins were detected in young basidia, although only RNA was found in the spores. The presence and relevance of different biomolecules in Pisolithus basidiocarp was demonstrated, confirming that lipids are the most important storage compound in this strutucture. Fatty acid analyses during different developmental stages of the basidiocarps showed that lipid composition is not influenced by morphology or geographic origin of the basidiocarp. The fatty acid 18:2w6,9 is the most abundant in the beginning of development, while 18:1w9 is the most important in matures spores. Analysis of EST databases from funcional ectomycorrhizas of Pisolithus led to the identification of genes coding for lipid metabolism enzymes as well as glycolysis and glyconeogenesis. The genes coding the glyoxylate cycle enzymes, isocitrato lyase and malate synthase, were identified using degenerated primers and the full length sequences were obtained by Genome Walking. Genes coding both isocitrate lyase and malate synthase, are interrupted by 5 introns, in ORFs presenting 1897 e 1880 bp respectively, and proteins of 540 aa. Also, the localization of isocitrato lyase is peroxissomal. Malate synthase localization cannot be predicted based on the amino acid sequence obtained. The expression analysis of lipid metabolism, glyoxylate cycle, and gluconeogenesis genes was done by Real Time RT-PCR. The patterns of gene expression in functional mycorrhizas of Pisolithus with Eucalyptus globulus or Populus trichocarpa were similar. The exception was the up regulation of the genes coding enzymes with a role in the synthesis and modification of fatty acids in P. microcarpus-E. globulus ectomycorrhiza. In Pisolithus basidiocarp gene expression patterns were similar in undifferentiated and young peridioles, and the most important changes were observed in mature peridioles and enclosed spores. The up-regulation of β-oxidation and glyoxylate cycle genes in mature peridioles indicates that lipids are used in the synthesis of precursor compounds during spore formation. Also, the up-regulation of the genes coding enzymes participating in the synthesis and modification of fatty acids in enclosed spores indicates that synthesis of storage lipids takes places inside these propagules. The expression of genes coding for glyoxylate cycle and gluconeogenesis in the vegetative mycelium were up-regulated in the presence of acetate and repressed by glucose. However, mycelium was not capable of using [2-13C] sodium acetate in its metabolism, accumulating this compound in the hyphae. After 144 hours of glucose starvation, the vegetative mycelium induced genes coding for β-oxidation, glyoxylate cycle, and gluconeogenesis enzymes. Under these conditions, the consumption of lipid reserves was observed in the hyphae stained with red Nile. The importance of transcriptional regulation of fatty acids metabolism is demonstrated by the analysis of gene expression, microscopy, and gas-chromatography. The results obtained in this work represent an important advance in the comprehension of lipid metabolism in the ectomycorrhizal fungus Pisolithus sp.