Abstract
Citrus
is the first tree crop in terms of fruit production. The colour of
Citrus
fruit is one of the main quality attributes, caused by the accumulation of carotenoids and their derivative C
30
apocarotenoids, mainly β-citraurin (3-hydroxy-β-apo-8′-carotenal), which provide an attractive orange-reddish tint to the peel of oranges and mandarins. Though carotenoid biosynthesis and its regulation have been extensively studied in
Citrus
fruits, little is known about the formation of C
30
apocarotenoids. The aim of this study was to the identify carotenoid cleavage enzyme(s) [CCD(s)] involved in the peel-specific C
30
apocarotenoids.
In silico
data mining revealed a new family of five
CCD4-type
genes in
Citrus
. One gene of this family,
CCD4b1
, was expressed in reproductive and vegetative tissues of different
Citrus
species in a pattern correlating with the accumulation of C
30
apocarotenoids. Moreover, developmental processes and treatments which alter
Citrus
fruit peel pigmentation led to changes of β-citraurin content and
CCD4b1
transcript levels. These results point to the involvement of CCD4b1 in β-citraurin formation and indicate that the accumulation of this compound is determined by the availability of the presumed precursors zeaxanthin and β-cryptoxanthin. Functional analysis of CCD4b1 by
in vitro
assays unequivocally demonstrated the asymmetric cleavage activity at the 7′,8′ double bond in zeaxanthin and β-cryptoxanthin, confirming its role in C
30
apocarotenoid biosynthesis. Thus, a novel plant carotenoid cleavage activity targeting the 7′,8′ double bond of cyclic C
40
carotenoids has been identified. These results suggest that the presented enzyme is responsible for the biosynthesis of C
30
apocarotenoids in
Citrus
which are key pigments in fruit coloration.