Milton Massao Shimizu

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.

Decaf and the Steeplechase Towards Decaffito—the Coffee from Caffeine-Free Arabica Plants

Unquestionably, the popularity of the coffee beverage relies on its alerting attribute caffeine. However, susceptibilities to this purine alkaloid, quite frequently associated with health concerns, encouraged a significant market for decaffeinated coffee. The beans of Coffea arabica render the best beverage and a decaffeinated coffee has to preserve the desired organoleptic characteristics of this species. Consequently, besides technical removal of caffeine, the endeavors to attain a decaffeinated Arabica coffee range from traditional studies on genetic variability to advanced techniques to produce genetic modified coffee. The aim of this review is to recover part of this subject focusing mainly on the natural genetic variation for caffeine content in Arabica. We also present historical information about caffeine discovery and briefly discuss molecular approaches to reduce caffeine. We introduce here the term decaffito for coffee derived from Arabica plants with beans naturally low in or almost devoid of caffeine. In the near future, coffee drinkers avoiding caffeine will have the choice between basically three Arabica coffees, namely decaffeinated by (a) selection and breeding, (b) genetic modification and (c) industrial extraction. Although only the last decaf coffee is available for the consumers, we believe that the size of the market of each type will occupy in the future depend on the price and health aspects related to the way the decaffeinated coffee beans are obtained.

Enzyme characterisation, isolation and cDNA cloning of polyphenol oxidase in the hearts of palm of three commercially important species

Heart of palm (palmito) is the edible part of the apical meristem of palms and is considered a gourmet vegetable. Palmitos from the palms Euterpe edulis (Juçara) and Euterpe oleracea (Açaí) oxidise after harvesting, whereas almost no oxidation is observed in palmitos from Bactris gasipaes (Pupunha). Previous investigations showed that oxidation in Juçara and Açaí was mainly attributable to polyphenol oxidase (PPO; EC 1.14.18.1) activity. In this study, we partially purified PPOs from these three palmitos and analysed them for SDS activation, substrate specificity, inhibition by specific inhibitors, thermal stability, optimum pH and temperature conditions, Km and Ki. In addition, the total phenolic content and chlorogenic acid content were determined. Two partial cDNA sequences were isolated and sequenced from Açaí (EoPPO1) and Juçara (EePPO1). Semi-quantitative RT-PCR expression assays showed that Açaí and Juçara PPOs were strongly expressed in palmitos and weakly expressed in leaves. No amplification was observed for Pupunha samples. The lack of oxidation in the palmito Pupunha might be explained by the low PPO expression, low enzyme activity or the phenolic profile, particularly the low content of chlorogenic acid.

Compositional changes of proteins and amino acids in germinating coffee seeds

Endosperm is the main reserve tissue in coffee seeds. Coffee (Coffea arabica L.) seeds were germinated for six weeks and qualitative and quantitative changes in amino acids and proteins were investigated. The total content of free amino acids were reduced during germination, however, protein content remained constant. SDS-PAGE profiles showed that legumin-like proteins became less stained in the last weeks. Asparagine, glutamic acid, aspartic acid, alanine and lysine were the major free amino acids, although serine and glutamine were also significant. Except for tyrosine, which increased with germination, all other amino acids were reduced. Analysis of the amino acid composition of the total soluble protein showed glutamic acid/glutamine and glycine as the main amino acids. However, other amino acids such as leucine, aspartic acid/asparagine, alanine, lysine, serine were also found in reasonable amounts.

Extração e dosagem da atividade da polifenoloxidase do café

A atividade de polifenoloxidase (PFO) tem sido usada como indicadora da qualidade de bebida de cafe. Foram feitas comparacoes entre o metodo tradicionalmente usado para extracao de polifenoloxidase de cafe e outro, onde impediu-se a oxidacao de fenois durante a extracao, com posterior eliminacao por filtracao em coluna de exclusao. Um metodo intermediario entre os dois tambem foi testado. As medicoes de atividade foram feitas por espectrofotometria e por consumo de O2. Os metodos tradicionais de extracao e dosagem da atividade de PFO sofrem forte interferencia de fenois presentes no extrato, nao permitindo reproducao dos dados publicados na literatura. Por espectrofotometria foi possivel diferenciacao apenas entre cafe de bebida Mole de cafes de bebida Dura e Rio, mas nao entre as duas ultimas. Cafe de melhor qualidade, bebida Mole, apresentou maior atividade de PFO. Extracao de PFO na presenca de antioxidantes e complexadores de fenois, seguida pela eliminacao dos mesmos por cromatografia de exclusao e essencial para uma avaliacao correta. Entretanto, usando este procedimento e consumo de O2, a atividade de PFO tambem nao diferenciou os tres cafes testados, exceto o Mole dos dois outros. No lugar de DOPA (3,4 - dihidroxifenilalanina), usado comumente nas dosagens por espectrofotometria, sugere-se o emprego de acido clorogenico (acido 5-cafeoilquinico).

Looking for the Physiological Role of Anthocyanins in the Leaves of Coffea arabica

The aim of this study was to determine which anthocyanins are related to the purple coloration of young leaves in Coffea arabica var. Purpurascens and assess their impact on photosynthesis as compared to C. arabica var. Catuaí, with green leaves. Two delphinidin glicosides were identified and histological cross‐sections showed they were located throughout the adaxial epidermis in young leaves, disappearing as the leaves mature. Regardless the irradiance level, the photosynthetic performance of Purpurascens leaves did not differ from that observed in leaves of the Catuaí variety, providing no evidence that anthocyanins improve photosynthetic performance in coffee plants. To analyze the photoprotective action of anthocyanins, we evaluated the isomerization process for chlorogenic acids (CGAs) in coffee leaves exposed to UV‐B radiation. No differences were observed in the total concentration of phenolic compounds in either variety before or after the UV treatment; however, we observed less degradation of CGA isomers in the Purpurascens leaves and a relative increase of cis‐5‐caffeoylquinic acid, a positional isomer of one of the most abundant form of CQA in coffee leaves, trans‐5‐caffeoylquinic acid, suggesting a possible protective role for anthocyanins in this purple coffee variety.

Influence of air temperature on proteinase activity and beverage quality in Coffea arabica

Fruits were collected from trees of Coffea arabica cv. Obata grown at Mococa and Adamantina in Sao Paulo State, Brazil, which are regions with marked differences in air temperature that produce coffee with distinct qualities. Mococa is a cooler location that produces high-quality coffee, whereas coffee from Adamantina is of lower quality. The amino acid and protein contents, amino acid profile, and proteinase activity and type in endosperm protein extracts were analysed. Proteinase genes were identified, and their expression was assayed. All results indicate that temperature plays a role in controlling proteinase activity in coffee endosperm. Proteinase activity was higher in the endosperm of immature fruits from Adamantina, which was correlated with higher amino acid content, changes in the amino acid profile, and increased gene expression. Cysteine proteinases were the main class of proteinases in the protein extracts. These data suggest that temperature plays an important role in coffee quality by altering nitrogen compound composition.

Polyphenoloxidase is induced by methyljasmonate and Meloidogyne javanica in soybean roots but is not involved in resistance

The role of the enzyme polyphenoloxidase (PPO) in the response of two soybean varieties, Hartwig (resistant) and Cristalina (susceptible), to Meloidogyne javanica was studied in plants where root systems were exposed to the known PPO inducer, methyljasmonate. Chlorogenic acid was the best substrate for root PPO. Treatment of both varieties with 100 and 400 μM methyljasmonate solutions induced a similar increase in enzyme activity 72 h after treatment. Inoculation of roots with second-stage juveniles (J2) induced PPO increase in cv. Cristalina but not in cv. Hartwig. Moreover, combined treatment of methyljasmonate and J2 inoculation enhanced PPO activity in both varieties. Two PPO cDNAs were isolated from the roots of the resistant variety 48 h after J2 inoculation, and Southern blot experiments in both varieties, using GmPPOJH1 and GmPPOJH2 cDNAs as probes, indicated that PPOs are represented by a multigene family in soybean. RT-PCR assays showed more GmPPOJH1 transcripts in plants treated with methyljasmonate and infected with nematodes, where high PPO activity was also observed. Plants treated with methyljasmonate and infected with J2 showed a marked decrease of nematode population 35 days after inoculation. These findings suggest that methyljasmonate triggers a resistant response in soybean roots to M. javanica but PPO is not involved in the resistance process.