The application of proteomics in postharvest refrigerated storage of fruits: a strategy for the identification of protein markers of chilling injury impact and of metabolic pathways affected by low temperature stress

Resumen del trabajo presentado a la 4th International Conference on Analytical proteomics, celebrada en Caparica (Portugal) del 7 al 9 de septiembre de 2015.
[Purpose]: Chilling injury (CI) constitute a set of physiological disorders causing detrimental effects on quality of fruits when stored after harvest at low temperatures to retard postharvest ripening and to extend their shelf-life. We have applied proteomics analysis to identify potential protein markers of CI and cold stress, and to detect biological processes affected by CI or involved in the plant organ response to deal with the cold stress. [Experimental description]: Fruits (bell peppers and tomatoes) were subjected to postharvest storage and posterior reconditioning at room temperature (20ºC). Storage was performed at a low temperature causing CI (1 and 2ºC respectively) for one batch of fruits and at a higher non-chilling temperature (10 and 12ºC respectively) for another batch used as control. Physiological parameters were determined, such as electrolyte leakage, ethylene production by GC-FID, respiration rate by GC-TCD, lipid peroxidation by MDA production, and sugars, organic acids and ascorbic acid by HPLC-RID/UV. Transmission electron microscopy studies were performed in bell pepper fruit sections. After extraction proteins were separated by two-dimensional differential in-gel electrophoresis (2D-DIGE) and proteins spots were identified after trypsin digestion with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The search for peptides was fulfilled in batch mode using GPS Explorer v3.5 software with a licensed version of MASCOT, using viridiplantae and SGN databases. [Results and Conclusions]: A comparative proteomics analysis between fruits stored at chilling and non-chilling temperatures with posterior reconditioning was carried out. In tomato fruits storage at low temperature was ended just before visible CI symptoms appeared, but in bell pepper fruits this storage was extended until development of these symptoms. The proteomic study has allowed for the identification of processes occurring in fruit biology that are critically affected by chilling injury such as the activation of stress defense mechanisms starred by stressresponsive proteins (LEAs, HSPs and PRs), and alterations in carbohydrate/energy metabolism, particularly in enzymes from glycolysis, TCA and Calvin cycle, but also involved in stress signaling such as GAPDH, OGDH, INV and aldose-1-epimerase, this last one a potential new protein marker in cold stress. In tomato a remarkable down-regulation of a chloroplastic ATP synthase occurred, which may lead to depletion of intracellular ATP. In bell pepper fruits where CI progress was more advanced the proteomics analysis revealed alterations in redox homeostasis, protein abundance in the ascorbate-glutathione cycle is altered and CAT down-regulated.
This work was supported by Fundación Séneca (1897/JLI/13).