Carbohydrate metabolism during fruit ripening: a functional genomics approach

Abstract

Fruit ripening is a complex phenomena, as a result of the integration and coordination of several different pathways during plant development. The knowledge and understanding of the control points occurring during ripening can provide the scientific basis for the development of new postharvest technologies, for improvement of shelf-life, but also for the enrichment of the nutritional and sensorial attributes of these foods. Changes in carbohydrate metabolism can be pointed out as one of most important metabolic events during ripening, since they account for the typical fruit sweetness and softness. As a consequence, changes in carbohydrate help to make fruit edible but also contribute to mechanical and pathogen spoilage. Previous results obtained in our lab helped to give a better comprehension of the changes in starch composition during ripening of banana, papaya, mango and other non-climacteric fruits. These data were achieved by following the starch degradation process and sucrose synthesis both in intact fruit and hormone infiltrated slices, together with studies involving enzymatic activity and gene expression of some enzymes. As shown by electron microscopy, during banana ripening starch granule degradation proceeds in an axial way, as a result of the concerted action of several enzymes. Alpha-amylase, which can initiate starch degradation, comprises a gene family insensitive to auxins and gibberelins, which is expressed almost at a steady-state level in banana, pulp. However, the level gene expression seems to provide enough enzymatic activity for the initial attack to the granule. On the other side, the B-amylase enzyme, which is present as single-copy gene, is positively stimulated during ripening, although it can be strongly delayed by indol-acetic acid and gibberelin infiltration. Starch phosphorylase is another important enzyme involved in starch degradation, since it can provide substrates for sucrose synthesis in a more favorable way. The results we obtained for phosphorylase activity suggested that metabolic regulation might be more important than gene expression during the climacteric. Because starch is composed by glucose units connected by alfa-1,4 and alfa-1,6 linkage and the amylases provide quite different products, the activity of a-glucosidase and debranching enzymes are essential for the total dismantling of amylose and amylopectin chains. In spite of the concomitant decrease in starch content and the occurrence of the respiration burst during the climacteric, these processes do not seem to so strongly connected. The infiltration of IAA in banana slices delayed the onset of starch degradation and sucrose accumulation, but not the increase in ethylene and CO2 production. The research on fruit brought also some important information regarding the chemical composition, organizational arrangement and dismantling of the cell wall and lamela after harvest. A wide range of chemical, biochemical and molecular biology tools have been employed in our laboratory, the study of some of the main enzymes involved and starch-sucrose and cell wall metabolism as mentioned before. Although these informations contributed to the understanding of the biochemical changes that take place in the amyloplast and cell wall during ripening, new questions arose, mainly because the inherent limitations of this approach. Besides the continuous development of the studies primarily focused on what was perceived as the main enzymes, it is essential the enlargement of the field of analysis seems now to be essential. Because fruit ripening is a complex-phenomena, a wide and integrative approach can give the insight about the main regulatory points of the carbohydrate metabolism. Most of these requisites may be fulfilled by a functional genomics approach, since it considers the analysis at different levels, from transcript to metabolic content, through protein profiling. This integrative approach can reveal genes, proteins or a particular metabolic flow that were hidden in a specific or narrow study. In this way, the main objective of the project is to apply the functional genomic tools to identify some controlling points of the process of fruit ripening, more specifically those related to carbohydrate metabolism. The specific objectives cover studies involving different fruit models and analysis of the fruit composition at different levels: transcriptional, protein, metabolites and also the organizational level arrangement of the amyloplast (in bananas) and cell wall (in papaya). Differential analysis of gene expression during fruit ripening and after treatment with some chemicals, such as methylcyclopropene (MCP). Proteomic analysis for the identification and characterization of genes and proteins differentially expressed during ripening or cell wall development. Heterologous expression of the main enzymes involved in the starch degradation and changes in the cell wall. Analysis of the In situ expression of specific enzymes using antiserum raised against the plant purified enzymes or the recombinant forms, previously obtained. Metabolomic analysis for the differential identification of the main chemical compounds related to the carbohydrate metabolism during ripening. Whole banana and papaya fruits will be used the experimental models for the study of the changes in carbohydrate metabolism during ripening, however, the infiltration of fruit slices will also be employed to study the effects of hormones or labelled compounds. Subcellular fractions, as the amyloplast, cell wall and Golgi apparatus will also be obtained in the specific studies. As a consequence of the large diversity of techniques involved in the project, each member of the project will be responsible for at least, a specific topic of the study. However, since the project is based on an integrative approach, at different levels, as genomic, proteomic and metabolomic, its success will rely on the close collaboration between members and on the cross integration of analysis of the data obtained at the different levels. (AU)