Ilka Nacif Abreu

Brazilian coffee genome project: an EST-based genomic resource

Coffee is one of the most valuable agricultural commodities and ranks second on international trade exchanges. The genus Coffea belongs to the Rubiaceae family which includes other important plants. The genus contains about 100 species but commercial production is based only on two species, Coffea arabica and Coffea canephora that represent about 70 % and 30 % of the total coffee market, respectively. The Brazilian Coffee Genome Project was designed with the objective of making modern genomics resources available to the coffee scientific community, working on different aspects of the coffee production chain. We have single-pass sequenced a total of 214,964 randomly picked clones from 37 cDNA libraries of C. arabica, C. canephora and C. racemosa, representing specific stages of cells and plant development that after trimming resulted in 130,792, 12,381 and 10,566 sequences for each species, respectively. The ESTs clustered into 17,982 clusters and 32,155 singletons. Blast analysis of these sequences revealed that 22 % had no significant matches to sequences in the National Center for Biotechnology Information database (of known or unknown function). The generated coffee EST database resulted in the identification of close to 33,000 different unigenes. Annotated sequencing results have been stored in an online database at http://www.lge.ibi.unicamp.br/cafe. Resources developed in this project provide genetic and genomic tools that may hold the key to the sustainability, competitiveness and future viability of the coffee industry in local and international markets.

Induction of pilocarpine formation in jaborandi leaves by salicylic acid and methyljasmonate

Jaborandi seedlings were subjected to different treatments in order to study the induction of pilocarpine in the leaves. In addition four extraction methods were assessed to extract the alkaloid from dried leaves. The highest yielding extraction and recovery was observed when dried leaves were first treated with base and then extracted with chloroform. Salt stress (NaCl), wounding, hypoxia, and N and K omission of the nutrient soln caused reductions in pilocarpine contents. Whereas complete nutrient soln and P omission maintained normal levels of the alkaloid. Salicylic acid and methyljasmonate induced a 4-fold increase of pilocarpine, but this increase was dependent on the concentration and time after exposure.

Production of pilocarpine in callus of jaborandi (pilocarpus microphyllus stapf)

SummaryJaborandi (Pilocarpus microphyllus) is the only known source of pilocarpine, and although this alkaloid is the only natural compoud used to treat glaucoma, very little is known about its metabolism. Calluses obtained from petioles of P. microphyllus leaves were partially immersed in MS (Murashige and Skoog) liquid medium containing different pH levels (4.8, 5.8, and 6.8), nutrient concentration (MS normal basal medium concentration, absence of N, P, and K and three times normal concentrations), histidine and threonine (0.05, 0.15 and 0.75 mM) NaCl (25 and 75 mM) and polyethylene glycol (5 and 15%). Exposure to methyljasmonic acid (MJ) vapor was also investigated. The calluses were subjected to these conditions for 4 and 8d under gentle agitation in the dark. Some calluses were also kept under continuous light. Pilocarpine was identified in the liquid medium by liquid chromatography-mass spectrometry/mass spectrometry. The alkaloid quantifications in the media and cells were carried out by high performance liquid chromatography (HPLC). The calluses maintained in the dark released the greatest quantities of pilocarpine into the medium. Methyljasmonate inhibited the release of pilocarpine in the medium. High pH (6.8), absence and excess of N, excess of P, and 0.75 mM of histidine and threonine induced the highest production of the alkaloid.

Propagacao in vivo e in vitro de Cissus sicyoides, uma planta medicinal

O estudo da propagacao de especies utilizadas na medicina popular tem sido intensificado nos ultimos anos devido ao crescente investimento em pesquisas para a descoberta de novos farmacos e da utilizacao da fitoterapia como um meio alternativo. O objetivo do trabalho foi a propagacao in vivo e in vitro (estabelecimento e multiplicacao) de Cissus sicyoides. Plantas mantidas em casa de vegetacao forneceram estacas com 10 e 20 cm de comprimento, as quais foram tratadas com 0, 80 ou 160 mg/l de AIB, com ou sem sacarose + acido borico, por duas horas. Para o estabelecimento in vitro, apos desinfestacao, segmentos nodais com 10 mm de comprimento foram inoculados em meio de cultura solido (MS), com diferentes concentracoes de cinetina, BAP e ANA. Para a multiplicacao in vitro, segmentos nodais com 10 mm foram inoculados em meio MS, suplementado com diferentes concentracoes de BAP e ANA, e ANA e cinetina. Na propagacao in vivo as estacas com 10 cm de comprimento apresentaram maior eficiencia no enraizamento quando tratadas com 160 mg/l de AIB. In vitro os explantes foram melhor estabelecidos e multiplicados em meio de cultura suplementado com cinetina e ANA, que proporcionaram maior inducao de gemas, crescimento em altura e ausencia de calos na base das plântulas.

HPLC-ESI-MS/MS of Imidazole Alkaloids in Pilocarpus microphyllus

Pilocarpine, an important imidazole alkaloid, is extracted from the leaves of Pilocarpus microphyllus (Rutaceae), known in Brazil as jaborandi and used mainly for the treatment of glaucoma. Jaborandi leaves also contain other imidazole alkaloids, whose pharmacological and physiological properties are unknown, and whose biosynthetic pathways are under investigation. In the present study, a HPLC method coupled with ESI-MSn was developed for their qualitative and quantitative analysis. This method permits the chromatographic separation of the imidazole alkaloids found in extracts of jaborandi, as well as the MS/MS analysis of the individual compounds. Thus two samples: leaves of P. microphyllus and a paste that is left over after the industrial extraction of pilocarpine; were compared. The paste was found to contain significant amounts of pilocarpine and other imidazole alkaloids, but had a slightly different alkaloid profile than the leaf extract. The method is suitable for the routine analysis of samples containing these alkaloids, as well as for the separation and identification of known and novel alkaloids from this family, and may be applied to further studies of the biosynthetic pathway of pilocarpine in P. microphyllus.

PILOCARPINE CONTENT AND MOLECULAR DIVERSITY IN JABORANDI

Pilocarpine is an imidazol alkaloid exclusively found in Pilocarpus genus and P. microphyllus accumulates its highest content in the leaves. There is no report in the literature on the variability of the pilocarpine content in this genus. A population of 20 genotypes of P. microphyllus from the state of Maranhao, Brazil, was analyzed for Random Amplification of Polymorphic DNA (RAPD) markers and pilocarpine content. Although it was not possible to establish any correlation between these features, the absence or presence of some markers could indicate in some genotypes a possible association with the content of the alkaloid.

Indução de metabólitos secundários em plântulas de Hypericum brasiliense Choisy crescendo in vitro

The production of rutin, quercetin, 1,5-dihydroxyxanthone and betulinic acid was investigated in plantlets of H. brasieliense in vitro, and exposed to salycilic acid, poliethylene glycol, NaCl, 24-epibrassinolide, benzothiadiazole (BION), methyljasmonate and increased concentrations of boron and nitrogen in the liquid culture medium. Evaluations of the contents were carried out after 5 and 10 days of treatments. The highest increase was observed in quercetin in the salycilic acid and B treatments after 5 days of exposure, and in 24-epibrassinolide and BION after 10 days.

Metabolic alterations in different developmental stages of Pilocarpus microphyllus.

Pilocarpine is an imidazole alkaloid that has been used for more than a century in glaucoma treatment. It is present in several species of the Pilocarpus genus (jaborandi), with its highest concentrations in P. microphyllus. In addition to pilocarpine, pilosine--an imidazole alkaloid without pharmacological use--is produced in high concentrations in mature plants. A metabolomic study was carried out on juvenile and mature plants to obtain information about pilocarpine metabolism at different developmental stages. Methanol-water and alkaloid extracts were analyzed by ¹H NMR and ESI-MS. Metabolic profiles from both techniques showed clear differences between various developmental stages. Intense signals in the aromatic region of the ¹H NMR spectrum and ions from pilosine and related alkaloids by ESI/MS were found only in extracts from mature plant. Two new imidazole alkaloids were identified by MS(n). Our results suggest that pilosine is produced exclusively in mature developmental stage, and juvenile plant material seems to be appropriate for further studies on pilocarpine biosynthesis.

Nitrogênio e fósforo na produção vegetal e na indução de mucilagem em plantas de insulina

The influence of nitrogen and phosphorus fertilization on production of biomass and mucilage in Cissus sicyoides plants was evaluated. 45-day old plants obtained from cuttings were planted in pots with capacity for 5 Kg of soil, having as substrate allic cambisol soil, medium texture from Nazareno (Brazil). Basic fertilization with macro and micronutrients was done. Nitrogen doses (0; 40; 80; 160 and 200 mg of N/Kg of soil) and phosphorus doses (50 and 150 mg of P/Kg of soil) were evaluated. The experiment was laid out in a complete randomised factorial scheme of 5 x 2, with four replications. The research was carried out in a greenhouse during 90 days, being evaluated the dry matter of leaves, stalks and roots; chemicals characteristics and mucilage were determined. Between nitrogen and phosphorus doses and the production of dry matter of leaves was observed interaction. Higher biomass gain occurred with higher nitrogen dose of 150 mg P/Kg of soil. The mucilage production increased with increasing doses of nitrogen and phosphorus, reaching a higher content at the same conditions that gave a higher leaves biomass gain and total aerial part.

Characterisation of the membrane transport of pilocarpine in cell suspension cultures of Pilocarpus microphyllus

Pilocarpine is an alkaloid obtained from the leaves of Pilocarpus genus, with important pharmaceutical applications. Previous reports have investigated the production of pilocarpine by Pilocarpus microphyllus cell cultures and tried to establish the alkaloid biosynthetic route. However, the site of pilocarpine accumulation inside of the cell and its exchange to the medium culture is still unknown. Therefore, the aim of this study was to determine the intracellular accumulation of pilocarpine and characterise its transport across membranes in cell suspension cultures of P. microphyllus. Histochemical analysis and toxicity assays indicated that pilocarpine is most likely stored in the vacuoles probably to avoid cell toxicity. Assays with exogenous pilocarpine supplementation to the culture medium showed that the alkaloid is promptly uptaken but it is rapidly metabolised. Treatment with specific ABC protein transporter inhibitors and substances that disturb the activity of secondary active transporters suppressed pilocarpine uptake and release suggesting that both proteins may participate in the traffic of pilocarpine to inside and outside of the cells. As bafilomicin A1, a specific V-type ATPase inhibitor, had little effect and NH4Cl (induces membrane proton gradient dissipation) had moderate effect, while cyclosporin A and nifedipine (ABC proteins inhibitors) strongly inhibited the transport of pilocarpine, it is believed that ABC proteins play a major role in the alkaloid transport across membranes but it is not the exclusive one. Kinetic studies supported these results.