Method for obtaining high-resolution proteomic analysis from pericarps of guarana.

Guarana has great agricultural potential and is largely used therapeutically and in the production of non-alcoholic energy drinks. Genomic and proteomic studies are crucial to identify proteins that play central roles in the maintenance and viability of fruits, as well as to identify proteins related to the main metabolic pathways. However, the success of any protein analysis starts with the protein extract preparation, which needs to offer an extract that is free of contaminants. This study aimed to evaluate different extraction methods to obtain high-quantity and high-quality extracts that are compatible with analysis by 2-dimensional electrophoresis and tandem mass spectrometry protein identification. Three different methods were tested: trichloroacetic acid (TCA)/acetone, sodium dodecyl sulfate (SDS)/phenol, and polyvinylpolypyrrolidone (PVPP)/SDS/phenol. The extract obtained from the TCA/acetone precipitation presented low solubility and contamination with lipids and carbohydrates. On the other hand, the quality of the extract gradually improved after using phenol and PVPP/phenol, enabling a yield up to 2 mg/g macerated tissues and the detection of 457 spots by 2-dimensional electrophoresis. The effectiveness of the procedure used was validated by identification of 10 randomly selected proteins by mass spectrometry. The procedure described here can be a starting point for applications using tissues of other organs of guarana or tissues of species that are similar to guarana.

Electrophoresis and spectrometric analyses of adaptation-related proteins in thermally stressed Chromobacterium violaceum.

Chromobacterium violaceum is a Gram-negative proteobacteria found in water and soil; it is widely distributed in tropical and subtropical regions, such as the Amazon rainforest. We examined protein expression changes that occur in C. violaceum at different growth temperatures using electrophoresis and mass spectrometry. The total number of spots detected was 1985; the number ranged from 99 to 380 in each assay. The proteins that were identified spectrometrically were categorized as chaperones, proteins expressed exclusively under heat stress, enzymes involved in the respiratory and fermentation cycles, ribosomal proteins, and proteins related to transport and secretion. Controlling inverted repeat of chaperone expression and inverted repeat DNA binding sequences, as well as regions recognized by sigma factor 32, elements involved in the genetic regulation of the bacterial stress response, were identified in the promoter regions of several of the genes coding proteins, involved in the C. violaceum stress response. We found that 30 °C is the optimal growth temperature for C. violaceum, whereas 25, 35, and 40 °C are stressful temperatures that trigger the expression of chaperones, superoxide dismutase, a probable small heat shock protein, a probable phasing, ferrichrome-iron receptor protein, elongation factor P, and an ornithine carbamoyltransferase catabolite. This information improves our comprehension of the mechanisms involved in stress adaptation by C. violaceum.

Seasonal shifts in isoprenoid emission composition from three hyperdominant tree species in central Amazonia.

Volatile isoprenoids regulate plant performance and atmospheric processes, and Amazon forests comprise the dominant source to the global atmosphere. Still, there is a poor understanding of how isoprenoid emission capacities vary in response to ecophysiological and environmental controls in Amazonian ecosystems. We measured isoprenoid emission capacities of three Amazonian hyperdominant tree species - Protium hebetatum, Eschweilera grandiflora, Eschweilera coriacea - across seasons and along a topographic and edaphic environmental gradient in the central Amazon. From wet to dry season, both photosynthesis and isoprene emission capacities strongly declined, while emissions increased among the heavier isoprenoids: monoterpenes and sesquiterpenes. Plasticity across habitats was most evident in P. hebetatum, which emitted sesquiterpenes only in the dry season, at rates that significantly increased along the hydro-topographic gradient from white sands (shallow root water access) to uplands (deep water table). We suggest that emission composition shifts are part of a plastic response to increasing abiotic stress (e.g. heat and drought) and reduced photosynthetic supply of substrates for isoprenoid synthesis. Our comprehensive measurements suggest that more emphasis should be placed on other isoprenoids, besides isoprene, in the context of abiotic stress responses. Shifting emission compositions have implications for atmospheric responses because of the strong variation in reactivity among isoprenoid compounds.