A fim de eliminar grãos defeituosos do mercado interno, há uma necessidade de propostas alternativas mais atraentes para os produtores de café. O objetivo deste estudo foi avaliar a composição química e bioatividade de grãos oriundos de frutos imaturos (defeito verde) e o efeito dos grãos crus sobre o metabolismo energético de camundongos obesos e eutróficos. Foram utilizados grãos de frutos colhidos no estádio cereja (maduros) e estádio verde (imaturos). Para avaliação da bioatividade do material de alto peso molecular, os grãos foram torrados e foi feito o fracionamento em etanol. Foram avaliados: a composição centesimal, os teores de cafeína, os compostos fenólicos totais, 5-ACQ e a atividade antioxidante (DPPH-IC50). Para o ensaio in vivo, os animais foram divididos em quatro grupos - Eutrófico: alimentados em uma dieta padrão AIN- 93; Eutrófico café: alimentados em uma dieta padrão AIN-93 + 1,0% de defeitos verdes; Obeso: alimentados com uma dieta rica em gordura para induzir obesidade; Obeso café: alimentados em uma dieta rica em gordura para induzir a obesidade + 1,6% de defeitos do café verde. Sangue, tecidos adiposos branco (epidídimo e subcutâneo) e marrom, fígado e músculo gastrocnêmio foram coletados e pesados. Os paramêtros bioquímicos foram dosados no soro e os teores de lipídios totais, triacilglicerol e colesterol total foram dosados também no fígado. Após oito semanas de tratamento, os animais foram submetidos à medição do consumo de oxigênio. Para avaliação da atividade antioxidante in vivo, foram dosados os níveis de hidroperóxidos, MDA, SOD e catalase no tecido adiposo e fígado dos animais. O conteúdo de carboidratos, lipídios e macrominerais não diferiram entre as duas amostras analisadas. Os grãos defeituosos apresentaram quantidades consideráveis de compostos bioativos e atividade antioxidante in vitro. Os grãos defeitos verdes foram capazes de aumentar o gasto energético e reduzir a massa gorda e o aumento de peso ponderal dos animais obesos. A atividade antioxidante in vivo dos grãos foi comprovada pelo aumento da atividade da enzima catalase no tecido adiposo dos animais eutróficos. Para avaliar a bioatividade do material de alto peso molecular (HMWM) dos grãos torrados, foram feitas caracterizações quanto aos teores de: açúcares e ligações glicolíticas, proteínas, melanoidinas, 5-ACQ e cafeína (frações de HMWM) e avaliada a atividade antioxidante (DPPH-IC 50). O material de alto peso molecular (HMWM) do defeito verde possui composição química semelhante ao HMWM do café e expressiva capacidade antioxidante.
Abscisic acid (ABA) is a phytohormone universally conserved in land plants which coordinates several aspects of the plant response to water deficit such as root architecture, seed dormancy and regulation of stomatal closure. A mechanism of ABA signal transduction has been proposed, evolving intracellular ABA receptors (PYR/PYL/RCARs) interacting with PP2Cs phosphatases and SnRK2 protein kinases. The goal of this study was to identify and characterize for the first time the orthologs genes of this tripartite system in Coffea. For this purpose, protein sequences from Arabidopsis, citrus, rice, grape, tomato and potato were chosen as query to search orthologous genes in the Coffee Genome Hub (http://coffee-genome.org/). Differential expression in tissues as leaves, seeds, roots and floral organs was checked through in silico analyses. In vivo gene expression analyses were also performed by RT-qPCR in leaves and roots of drought-tolerant (D T 14, 73 and 120) and drought-susceptible (D S 22) C. canephora Conilon clones submitted (or not) to drought. The expression profiles of the tripartite system CcPYL-PP2C-SnRK2 genes were also analyzed in leaves of C. arabica (Ca) and C. canephora (Cc) plants grown under hydroponic condition and submitted to ABA exogenous treatment (500 μM). This approach allowed the identification and characterization of 24 candidate genes (9 PYL/RCARs, 6 PP2Cs and 9 SnRK2s) in Cc genome. The protein motifs identified in predict coffee sequences enabled characterize these genes as family’s members of PYL/RCARs receptors, PP2Cs phosphatases or SnRK2 kinases of the ABA-dependent response pathway. These families were functionally annotated in the Cc genome. In vivo analyses revealed that eight genes were up-regulated under drought conditions in both leaves and roots tissues. Among them, three genes coding phosphatases were expressed in all (D T and D S ) clones therefore suggesting that they were activated as a general response to cope with drought stress. However, two other phosphatase coding genes were up- regulated only in the D T clones, suggesting that they constitute key-genes for drought tolerance in these clones. The D T clones also showed differential gene expression profiles for five other genes thus reinforcing the idea that multiple biological mechanisms are involved in drought tolerance in Cc. In response to exogenous ABA, 17 genes were expressed in leaves of Cc and Ca plants. Several genes were differentially expressed in the D T clone 14 either in control condition or after 24h with ABA treatment. Under control condition, five genes were higher expressed as in the Cc as in Ca D T plants. The kinase CcSnRK2.6 was highlighted as gene specifically expressed in the Cc plants (D T and D S ) after 72h of ABA treatment. Overall, it was observed that ABA signaling pathway is delayed in the D S C. arabica Rubi. Those molecular evidences corroborated with microscopies analyses which showed that the D T clone 14 was more efficient to control the stomatal closure than other coffee plants in response to ABA treatment. All these evidences will help us to identify the genetic determinism of drought tolerance through ABA pathway essential to obtain molecular markers that could be used in coffee breeding programs.
