A Systems Biology Approach to Browning in Apple: Proteomics

During long term storage of apple (Malus Domestica), many things can go wrong with their quality. Several internal defects are known, which are characterized by flesh browning. The degree of browning is cultivar, batch and season dependent and is caused by a combination of preharvest growing and postharvest storage factors resulting in losses up to 40% of the fruit. The cultivars Braeburn and Kanzi are commercial cultivars in Belgium that are prone to this browning disorder. The aim of this project is to generate insight in the pre and postharvest factors influencing browning through a systems biology approach. This contribution presents our findings on mapping the proteome differences between healthy, sound and brown apple tissue. Gas exchange plays a fundamental role in the storage of pomme fruit, covering both respiratory activity and transport of gasses from the storage atmosphere into the fruit and visaversa. Diffusion limitations of the respiratory gasses O2 and CO2 is key to the development of browning disorders. Also, the balance between oxidative and reductive processes in the cells, is important. Under normal conditions harmful reactive oxygen species (ROS) are efficiently removed by the antioxidant system. However, under stress conditions, ROS production increases, causing abnormalities in the cellular metabolism resulting in, for example, loss of membrane integrity. Due to this membrane damage, polyphenoloxidase (PPO) starts oxidating phenolic compounds, forming o-quinones, which form brown coloured polymers. Apples (Malus Domestica, cv. Kanzi) were picked in the orchard of pcfruit in Sint-Truiden (Belgium) and pre cooled during 14 days, after which they were stored under controlled atmosphere (CA) conditions of 2°C, 2.5% O2 and 0.7% CO2. After 5 months storage, tissue samples were taken from brown, sound and healthy tissue. To extract the proteins, a phenol extraction/methanol-ammonium acetate precipitation protocol was used, while the Bradford method was applied to determine protein quantification. Because of the complexity of the samples, proteins were separated using SDS-PAGE before trypsin digestion took place. Masses of the peptides were measured with a Q-TOF LC-MS, and statistically analyzed. The peptides characteristic for the different stages of browning were identified with LC-MS/MS.status: publishe