A comprehensive analysis of carotenoids metabolism in two red-fleshed mutants of Navel and Valencia sweet oranges (Citrus sinensis)

Abstract

Kirkwood Navel and Ruby Valencia are two spontaneous bud mutations of the respective parental lines of sweet orange (Citrus sinensis) Palmer Navel and Olinda Valencia, showing an atypical red pigmentation of the pulp. These red-fleshed varieties are commercially available and highly attractive for consumers but their carotenoid metabolism and the basis of the mutation have not been investigated. The red colour of Kirkwood and Ruby pulp was observed from the very early stages of fruit development until full maturity and associated with an altered carotenoid profiling. The red-fleshed varieties accumulated from 6- up to 1000-times more total carotenoids compared to the standard oranges. Specifically, the pulp of Kirkwood and Ruby accumulated large amounts of phytoene and phytofluene, and moderate contents of lycopene. Moreover, the red-fleshed oranges contained other unusual carotenes as δ-carotene, and lower concentrations of downstream products such as β,β-xanthophylls, abscisic acid (ABA) and ABA-glucosyl ester. This peculiar profile was associated with chromoplasts with lycopene crystalloid structures and round vesicles likely containing colourless carotenes. The flavedo and leaves of Kirkwood and Ruby showed minor changes in carotenoids, mainly limited to higher levels of phytoene. The carotenoid composition in Kirkwood and Ruby fruits was not explained by differences in the transcriptional profile of 26 genes related to carotenoid metabolism, covering the main steps of biosynthesis, catabolism and other processes related to carotenoid accumulation. Moreover, sequence analysis of the lycopene cyclase genes revealed no alterations in those of the red-fleshed oranges compared to the genes of the standard varieties. A striking event observed in Kirkwood and Ruby trees was the reddish coloration of the inner side of the bark tissue, with larger amounts of phytoene, accumulation of lycopene and lower ABA content. These observation lead to the conclusion that the mutation is not only manifested in fruit, affecting other carotenogenic tissues of the mutant plants, but with different consequences in the carotenoid profile. Overall, the carotenoid composition in the red-fleshed mutants suggests a partial blockage of the lycopene β-cyclization in the carotenoid pathway, rendering a high accumulation of carotenes upstream lycopene and a reduced flow to downstream xanthophylls and ABA.

Bioactive Compounds, Nutritional Quality and Antioxidant Capacity of the Red-Fleshed Kirkwood Navel and Ruby Valencia Oranges

“…Thus, the external colorations of the mutant fruits were visually similar to those of the respective ordinary genotypes ( Table 1 and Table 2 ). These results were in good agreement with those of other orange mutants, in which the red-fleshed phenotype is not associated with alterations in the coloration of the peel [ 24 , 25 , 27 , 38 ].…”

“…In the fruits of the N and V, the a/b values were negative and slightly positive, corresponding to green–yellow shades, but in both mutants, the color of pulp showed considerably higher positive values, indicating a red coloration of the pulp. These differences in color parameters were patent from the early stages of development, indicating that the alteration in pulp pigmentation was not linked to the maturation process, as has been previously suggested [ 38 ]. Moreover, in the fruits of both mutants, the a/b values decreased during maturation; this was probably related to dilution caused by the expansion of the pulp ( Table 1 and Table 2 ).…”

“…The second PCA was built with the same variables and levels of carotenoids. The data for total carotenoids for this second PCA analysis were obtained from our previous work [ 38 ], using the same biological samples.…”

“…We recently reported the characterization of carotenoid metabolism during development and maturation in two new red-fleshed orange mutants: Kirkwood Navel (referred in this work as Kirkwood or K) and Ruby Valencia (Ruby or R). These spontaneous bud-mutations are derived from the ordinary Navel and Valencia late oranges, respectively, and are already being commercially propagated in order to check their adaptation to Mediterranean growing and environmental conditions [ 38 ]. The pulps of both mutants contain higher amounts of total carotenoids than the ordinary Navel and Valencia oranges from the early stages of fruit development and, moreover, accumulate large concentrations of phytoene and phytofluene, moderate amounts of lycopene, low levels of β-carotene and minor concentrations of violaxanthin compared to the ordinary varieties [ 38 ].…”

“…These spontaneous bud-mutations are derived from the ordinary Navel and Valencia late oranges, respectively, and are already being commercially propagated in order to check their adaptation to Mediterranean growing and environmental conditions [ 38 ]. The pulps of both mutants contain higher amounts of total carotenoids than the ordinary Navel and Valencia oranges from the early stages of fruit development and, moreover, accumulate large concentrations of phytoene and phytofluene, moderate amounts of lycopene, low levels of β-carotene and minor concentrations of violaxanthin compared to the ordinary varieties [ 38 ]. Hence, the appealing coloration and the high concentration of carotenes make these new orange varieties rich sources of carotenoids for consumption and novel food products with potential benefits for health.…”