Metabolism of guarana (Paullinia cupana Kunth var. sorbilis) plants and fruit production subjected to glyphosate doses.

Guarana (Paullinia cupana Kunth var. sorbilis) is a typically Amazonian plant of high economic value due to the compounds found in its seed. For guarana to reach the maximum productive potential, management practices such as weed control are necessary. The use of herbicides is a viable alternative, however, its drift may lead to adverse effects on the primary and secondary plant metabolisms and cause losses in crop production. This study evaluated the differential drift effects of glyphosate doses on the physiology of guarana plants and the production of compounds of economic interest in their seeds. Glyphosate doses (57.6, 115.2, 230.4, 460.8 g ae ha-1) were applied to adult guarana plants after the flowering period. The photosynthetic functions and metabolism effects were evaluated. Herbicide treatments led to oxidative stress due to increased lipid peroxidation and increased carbohydrate and amino acid in their leaflets. Despite this, glyphosate showed no effect on fruit production or the content of secondary metabolites of commercial interest in seeds.

Fertilization and seasonality influence on the photochemical performance of tree legumes in forest plantation for area recovery in the Amazon.

N-fixing leguminous species can reach atmospheric dinitrogen gas (N2), having an advantage under N-limited degraded environments. These N-fixers are constantly used as facilitative species. Chlorophyll a fluorescence (ChF) acknowledges how different species take up and use light energy during photosynthesis. These techniques assess stress and performance responses to photosynthesis and are used for the selection of species with potential for reforestation. Six Fabaceae species were selected for this study: three nonfixing species (Cenostigma tocantinum, Senna reticulata and Dipteryx odorata) and three N-fixing species (Clitoria fairchildiana, Inga edulis and Acacia spp.). Variations in chlorophyll fluorescence under high vs. low water and nutrient conditions were studied. Multivariate analysis was performed to detect the effects of seasonality and fertilization on dark-adapted ChF two years after the experiment was established. The correlation among ChF variables and growth, photosynthesis and foliar nutrient concentrations was evaluated. Under high water- and nutrient-availability conditions, plants exhibited an enhanced performance index on absorption basis values correlated with electron transport fluxes. Under drought and nutrient-poor conditions, most species exhibit increased energy dissipation as photoprotection. High interspecific variation was found; therefore, species-specific responses should be considered in future ChF studies. Corroborating the ability to colonize high-light environments, N-fixers showed an increased performance index correlated with electron transport and Zn and N foliar concentrations. Negative correlations were found between photosynthesis and trapped fluxes. Diameter growth was positively correlated with electron transport fluxes. Given the different responses among species, ChF is an effective technique to screen for seasonality, fertilization and species effects and should be considered for use during forest restoration. Finally, the addition of fertilization treatments may facilitate tropical forest restoration due to the importance of nutrients in physiological processes. N-fixers showed high photochemical performance and tolerance to abiotic stress in degraded areas and therefore should be included to support ecosystem biomass restoration.

Deposition of lignin in four species of Saccharum.

We used primers designed on conserved gene regions of several species to isolate the most expressed genes of the lignin pathway in four Saccharum species. S. officinarum and S. barberi have more sucrose in the culms than S. spontaneum and S. robustum, but less polysaccharides and lignin in the cell wall. S. spontaneum, and S. robustum had the lowest S/G ratio and a lower rate of saccharification in mature internodes. Surprisingly, except for CAD, 4CL, and CCoAOMT for which we found three, two, and two genes, respectively, only one gene was found for the other enzymes and their sequences were highly similar among the species. S. spontaneum had the highest expression for most genes. CCR and CCoAOMT B presented the highest expression; 4CL and F5H showed increased expression in mature tissues; C3H and CCR had higher expression in S. spontaneum, and one of the CADs isolated (CAD B) had higher expression in S. officinarum. The similarity among the most expressed genes isolated from these species was unexpected and indicated that lignin biosynthesis is conserved in Saccharum including commercial varieties Thus the lignin biosynthesis control in sugarcane may be only fully understood with the knowledge of the promotor region of each gene.

Production of Recombinant Caffeine Synthase from Guarana (Paullinia cupana var. sorbilis) in Escherichia coli.

Caffeine synthase (CS) is a methyltransferase responsible for the last two steps of the caffeine biosynthesis pathway in plants. CS is able to convert 7-methylxanthine to theobromine (3,7-dimethylxanthine) and theobromine to caffeine (1,3,7-trimethylxanthine) using S-adenosyl-L-methionine as the methyl donor in both reactions. The production of a recombinant protein is an important tool for the characterization of enzymes, particularly when the enzyme has affinity for different substrates. Guarana has the highest caffeine content among more than a hundred plant species that contain this alkaloid. Different from other plants, in which CS has a higher affinity for paraxanthine (1,7-dimethylxanthine), caffeine synthase from guarana (PcCS) has a higher affinity for theobromine. Here, we describe a method to produce a recombinant caffeine synthase from guarana in Escherichia coli and its purification by affinity chromatography. The recombinant protein retains activity and can be used in enzymatic assays and other biochemical characterization studies.

Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit.

Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein.

Guarana: revisiting a highly caffeinated plant from the Amazon.

ETHNOPHARMACOLOGICAL RELEVANCE: Guarana (Paullinia cupana Kunth var. sorbilis (Mart.) Ducke) has been traditionally consumed by indigenous communities of the Amazon region. It is valued mainly for its stimulant property because of its high content of caffeine, which can be up to 6% in the seeds. AIM OF THE REVIEW: The purpose of this review is to revisit this typically Brazilian plant, addressing economic considerations, the chemical makeup of the seeds and pharmacological properties so far investigated. RESULTS: Guarana is primarily produced in the Brazilian states of Amazonas and Bahia, and approximately 70% of the production is used by the industry of soft and energy drinks. The other 30% becomes guarana powder for direct consumption in capsules or dilution in water, or it serves as a raw material for the pharmaceutical and cosmetics industries. In addition to its stimulant property, guarana has other therapeutic properties, which have aroused the interest of the scientific community. CONCLUSION: This review shows that other guarana properties may be explored and how scarce are the studies regarding agronomic, plant pathology, physiology and breeding. So far, caffeine has been the main reason to study guarana and still will lead the researches because the demand for this alkaloid by food and pharmaceutical industry, and a strongly growing market related with beauty products. However, guarana has other components and there is great interest in studies designed to elucidate the effects of guarana's bioactive components and their potential pharmacological applications. Significant part of the guarana production in Brazil still comes from Indians tribes in the Amazon State, and any improvement in this plant, in any aspect, may propitiate a positive economic impact in their lives.

Prospeção de inibidores de serinoproteinases em folhas de leguminosas arbóreas da floresta Amazônica / Prospecting serine proteinase inhibitors in leaves from leguminous trees of the Amazon forest

Os inibidores de proteinases são proteínas extensivamente investigadas nos tecidos de estocagem, mas pouco prospectadas em outros tecidos vegetais. O objetivo deste estudo foi detectar a presença de inibidores de serinoproteinases em extratos foliares de quinze espécies de leguminosas arbóreas da Amazônia. As espécies estudadas foram: Caesalpinia echinata, C. ferrea, Cedrelinga cateniformis, Copaifera multijuga, Dinizia excelsa, Enterolobium contortisiliquum, E. maximum, E. schomburgkii, Leucaena leucocephala, Ormosia paraensis, Parkia multijuga, P. pendula, P. platycephala, Swartzia corrugata e S. polyphylla. Folhas foram coletadas, secas a 30ºC durante 48 h, trituradas e submetidas à extração com NaCl (0,15 M, 10 por cento p/v) resultando no extrato total. Ensaios foram executados para determinar a concentração de proteínas e detectar a atividade inibitória contra a tripsina e quimotripsina bovina. Os teores de proteínas bruta e solúvel nos extratos foliares variaram de 7,9 a 31,2 por cento e 1,3 a 14,8 por cento, respectivamente. A atividade inibitória sobre a tripsina e quimotripsina foi observada em todos os extratos foliares. Contudo, nos extratos de E. maximum, L. leucocephala, P. pendula, S. corrugata e S. polyphylla a inibição foi maior sobre a tripsina, enquanto o extrato de P. multijuga foi mais efetivo contra a quimotripsina. Nós concluímos que nos extratos foliares de leguminosas arbóreas têm inibidores de serinoproteinases e exibem potencial aplicações biotecnológicas.

The proteinase inhibitors are proteins extensively investigated in tissue storage, but few prospected in other plant tissues. The aim of this study was to detect the presence of serine proteinase inhibitors in leaf extracts from fifteen species of leguminous trees of the Amazon forest. The species studied were Caesalpinia echinata, C. ferrea, Cedrelinga cateniformis, Copaifera multijuga, Dinizia excelsa, Enterolobium contortisiliquum, E. maximum, E. schomburgkii, Leucaena leucocephala, Ormosia paraensis, Parkia multijuga, P. pendula, P. platycephala, Swartzia corrugata and S. polyphylla. Leaves were collected, dried at 30ºC for 48 h, crushed and subjected to extraction with NaCl (0.15 M, 10 percent w/v), resulting in the total extract. Tests were performed to determine the concentration of proteins and to detect of inhibitory activity against bovine trypsin and chymotrypsin. The content of crude and soluble protein in leaf extracts varied from 7.9 to 31.2 percent and 1.3 to 14.8 percent, respectively. The inhibitory activity on trypsin and chymotrypsin was observed in all leaf extracts. However, in extracts of E. maximum, L. leucocephala, P. pendula, S. corrugata and S. polyphylla, the inhibition was greater on trypsin, while extract of P. multijuga was more effective against chymotrypsin. We conclude that leaf extracts of leguminous trees have serine proteinase inhibitors and show potential biotecnological applications.