Dynamic Changes In Proteins During Apple (Malus X Domestica) Fruit Ripening And Storage

A proteomic study, using 2-DE and MALDI-TOF-TOF, was conducted in apple fruit (cv.’Golden Delicious’) during maturation and different stages of ripening. The main interestes cocused were the identification, functional and bioinformatics analysis of differently expressed proteins in order to further explain the regulatory mechanisms of apple fruit during maturation, ripening and senescence. The results were as following:1. An effective two-dimensional gel electrophoresis (2-DE) system was developed for total protein from apple fruits, by comparing different extracted methods in terms of total protein, different pH ranges strips and different sample volume. The high quality 2-DE protein map with over 400 protein spots was achieved by optimized conditions of modified phenol extraction,17 cm IPG strip with pH 4～7,12% SDS-PAGE and detecting proteins with modified colloidal coomassie brilliant blue G-250 staining. Most of protein spots were found between pH 5-7 and most of relative molecular weight of protein spots were between 15to95kDa.2. The results showed that there were differentially expressed proteins during maturation and different stages of ripening.55 differentially expressed proteins were revealed by comparing the 2-DE maps between the optimal date of harvest and different ripening stages. After using MALDI-TOF/TOF analysis,53 protein spots of these proteins were finally identified according to an apple EST database downloaded from Genome Database for Rosaceae and placed into six categories. The categories, and the percentage of proteins placed in each category were stress response and defense (49.0%), energy and metabolism (34.0%), fruit ripening and senescence (5.6%), signal transduction (3.8%), cell structure (3.8%) and protein synthesis (3.8%).53 spots are identified by their subcellular localization analysis, there are 27 in the cytoplasm, eight located in the chloroplast, four located in the mitochondria,6 in the nucleus. Proteins involved in glycolysis, the pentose-phosphate pathway (PPP), antioxidative systems, photosynthesis, and cell wall synthesis, were down-regulated, especially during the climacteric burst in respiration and during the senescent stages of fruit development. Proteins classified as allergens or involved in cell wall degradation were up-regulated during the ripening process. Some protein spots exhibited a mixed pattern similar to respiratory climacteric, such as 1-aminocyclopropane-l-carboxylate oxidase (ACO), L-ascorbate peroxidase (APX), and abscisic acid response proteins.