Caracterização bioquímica e molecular da oxidase alternativa de fruto de graviola (Annona muricata L.) durante o amadurecimento pós-colheita

Abstract

The development and maturation of fruits has received considerable scientific scrutiny of both the uniqueness of such processes to the biology of plants and the importance of fruit as a significant component of the human diet. Molecular and genetic analysis of fruit development, and especially ripening of freshy fruits, has resulted in significant gains in knowledge over recent years finding in areas of fruit primary development regulation and common regulatory mechanisms among climacteric and nonclimacteric ripening in addition to identification of discrete molecular mechanisms specific to unique fruit development traits that differentiate fruiting species. Climacteric fruits are defined by a respiratory burst during the ripening process and this climacteric burst was associated with an enhanced activity of the alternative pathway, that is nonphosphorylating and cyanide-resistant, mediated by alternative oxidase (AOX), which physiological role in plant respiration and metabolism is still largely unknown. In the present work, it has been standardized soursop (Annona muricata L. cv. 'Morada') fruit pulp mitochondria isolation and purification techniques from unripe and ripe fruit. Fruits from commercial orchard in Quixeré, Ceará State/Brazil were divided into six groups of five fruit each and stored at room temperature (24,5 + 0,5°C; 80-85% R.H) for up 8 days. Respiration rate, ethylene production and physical-chemistry and chemical characterization were determined during soursop ripening. Soursop fruit pulp mitochondria were isolated ( purified on Percoll gradient) and characterized concerning biochemistry properties through substrates oxidation, respiratory control and ADP/0 relation. Electron alternative pathway was measured in the presence of cyanide. Oxygen uptake was polarographycally estimated using NADH, succinate and malate/glutamate as a substrates. Molecular and genetic analysis such as genomic DNA extraction, total RNA from unripe and ripe fruit, PCR analysis for amplification of AOX gene using heterologous primers for gene number identification and sequence analysis from cloned PCR product as well as RFLP analysis for investigate the AOX clones polimorphism and analysis of AOX gene expression through immunoblotting using antibodies against AOX Sauromattum guttatum and cDNA obtained through total RNA extraction from unripe and ripe soursop fruit pulp using RT-PCR were also realized. The alternative pathway activity was detected in unripe and ripe soursop fruit mitochondria and its activity was at least 2 times higher in ripe soursop fruit mitochondria than in unripe fruit. The CO2 and ethylene production peak coincided with the climacteric and occurred at the same time as an increase in alternative electron transport. The unripe soursop fruit mitocondria AOX activity was modulated by the pyruvate in the presence of NADH as a substrate. The immunoblotting results showed a presence of only one protein of approximately 34kDa in soursop fruit mitochondria at days 1 to 4 postharvest and from days 5 to 8, three proteins of ca. 32, 34 and 36kDa, were detected. Two bands (32 and 34kDa) were found 5 and 6 days postharvest, and the third band (36kDa) at days 7 and 8 postharvest. The presence of various isoforms detected in the soursop fruit mitochondria isolated during the postharvest ripening suggested possibly the presence of a multigene family for the AOX in soursop fruit which was expressed differentially during development. The increaese of AOX activity comparable to the AOX expression profile and accumulation of new reduced protein bands in the soursop fruit mitochondria isolated during postharvest ripening (1 to 8 days) suggested that there is a AOX modulation associated to the climacteric ripening of this fruit.