Dynamic changes in proteins during apple (Malus x domestica) fruit ripening and storage

Abstract

A proteomic study, using two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight, was conducted in apple fruit (cv. 'Golden Delicious') starting at 10 days prior to harvest through 50 days in storage. Total protein was extracted using a phenol/sodium dodecyl sulfate protocol. More than 400 protein spots were detected in each gel and 55 differentially expressed proteins (p<0.05) were subjected to matrix-assisted laser desorption/ionization time-of-flight/time-of-flight analysis. Fifty-three of these proteins were finally identified using an apple expressed sequence tag 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%). Proteins involved in several multiple metabolic pathways, including glycolysis, pentose-phosphate pathway, anti-oxidative systems, photosynthesis and cell wall synthesis, were downregulated, 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 upregulated during the ripening process. Some protein spots exhibited a mixed pattern (increasing to maximal abundance followed by a decrease), such as 1-aminocyclopropane-1-carboxylate oxidase, L-ascorbate peroxidase and abscisic acid response proteins. The identification of differentially expressed proteins associated with physiological processes identified in the current study provides a baseline of information for understanding the metabolic processes and regulatory mechanisms that occur in climacteric apple fruit during ripening and senescence.

Figure 1

Identification and differential expression of apple (cv. Golden Delicious) proteins in fruit maintained at 25 °C and sampled during ripening and senescence. 2-DE was performed using 1.8 mg of protein, linear 17 cm IPG strips (pH 4–7) for the first dimension, and 12% SDS–PAGE gels for the second dimension electrophoresis. Gels were stained with colloidal CBB G250. Numbers with arrows indicate the differentially expressed protein spots that were identified in this study. CBB, Coomassie brilliant blue; IEF, isoelectric focusing; IPG, immobilized pH gradient; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

Figure 2

Close-up views of a selected sample of differentially abundant proteins marked in Figure 1. Different ripening stages are displayed above and below the protein images. H-10 is 10 days before the optimal harvest date (H0) and H5–H50 are days of storage at 25 °C after H0. Arrows and numbers indicate the spots with differential protein expression.

Figure 3

Classification of differentially expressed proteins identified in apple fruits at different ripening stages into functional categories. The classification is based on protein descriptions in the GDR, protein annotations in MIPS Functional Catalogue Database and published literature. MIPS, Munich Information Center for Protein Sequences.