Comparative Proteomics of a Lycopene-accumulating Mutant Reveals the Important Role of Oxidative Stress on Carotenogenesis in Sweet Orange (Citrus Sinensis [L.] Osbeck)

A spontaneous sweet orange (Citrus sinenesis [L.] Osbeck) mutant 'Hong Anliu' is of high value due to lycopene accumulation in the pulp. In this study, we analyzed the proteomic alterations in the pulp of 'Hong Anliu' versus its wild type (WT) at four maturing stages by using 2-DE combined with MALDI-TOF-TOF MS. Among the 74 differentially expressed proteins identified, the majority are predicted to be involved in stress response, carbohydrate/energy metabolism and regulation, or protein fate, modification and degradation. Particularly, expression levels of six anti-oxidative enzymes were altered by the mutation; and assays of their respective enzymatic activities indicated an enhanced level of oxidative stress in 'Hong Anliu', implying a regulatory role of oxidative stress on carotenogenesis. This conclusion was further confirmed by our observation that treatment of fruit pulps with tert-butylhydroperoxide (a ROS progenitor) induced lycopene accumulation in 'Hong Anliu' only. Gene expression showed that genes predicted to function upstream of lycopene biosynthesis were generally upregulated in juice sacs, but downregulated in segment membranes in both 'Hong Anliu' and its WT. The result suggests an important role of post-transcriptional regulation on carotenogenesis since lycopene was induced in 'Hong Anliu' but not WT. The result also implies that carotenogenesis in juice sacs and segment membranes of citrus fruits may be regulated by different mechanisms.