Effects of anaerobic stress on the proteome of citrus fruit

Exposure of citrus fruit to anaerobic conditions results in induction of anaerobic respiration and accumulation of the off flavor volatiles ethanol and acetaldehyde. In this study, we evaluated the effects of anaerobic stress (exposure to N₂ atmospheres for 24 h) on the proteome of mandarins and grapefruit. With two-dimension polyacrylamide gel electrophoresis (2D-PAGE), we detected more than 400 protein spots in the flavedo tissue and 300 spots in the juice vesicle tissue, that were reproducibly stained in mandarins and grapefruit. Exposure to the anaerobic treatment significantly affected the abundances of 33 different proteins by a factor of at least 1.5. Identification of the citrus anaerobic proteins (ANPs) by mass spectrometry (MS) and annotation according to the Munich Information Center for Protein Sequence (MIPS) revealed tissue- and cultivar-specific differences in the anaerobic response of citrus fruit. In the peel tissue, 64% of the detected ANPs were stress-related proteins involved in cell rescue, defense and virulence and only 6% in energy production, whereas 38% of the ANPs in juice vesicle tissue were involved in energy and 31% in either cell rescue and defense or in cell cycle and protein fate. Furthermore, exposure to N₂ for 24 h had only minor affects on protein abundance in grapefruit juice vesicle tissue (suppression of 5 proteins) but remarkably affected protein accumulation in mandarins, including induction of glycolytic enzymes, a 10-fold increase in the abundance of alcohol dehydrogenase (ADH), and induction of stress proteins, such as heat shock proteins and ascorbate peroxidase. Overall, the present study provides the first 2D-PAGE proteome analysis of fruit tissue responses to anaerobic stress, and the observed data reflect both common as well as citrus-specific anaerobic-response mechanisms.