Annual Report: CIRAD 2007

Carotenoids are key factors responsible for the organoleptic and nutritional qualities of citrus fruits. They form one of the largest plant pigment classes, and citrus fruits have an especially high and complex carotenoid composition. Over 100 different carotenoid molecules have been isolated in Citrus. CIRAD has carried out a series of studies on the carotenoid composition of different species and its links with the genetic diversity structure within the Citrus genus, the metabolic pathways of these compounds and the environmental conditions.

Substances with a broad range of properties

Carotenoids are responsible for the internal and external yellow, orange or red colour of citrus fruits. For instance, ß-cryptoxanthin gives clementines and mandarin oranges their typical bright orange colour, whereas lycopene gives some pummelo varieties their red colour.The carotenoid composition of citrus fruits is therefore one of the main factors responsible for the organoleptic quality of fresh citrus fruits and juices.

Carotenoids are also micronutrients with many different biological functions. Carotenoids include provitamins A, ie vitamin A precursors that are essential for vision, epithelial tissue preservation, bone growth and reproduction. In developed countries, 25-35% of dietary vitamin A is supplied via carotenoids through fruit and vegetable consumption, while this figure reaches as high as 82% in developing countries. Carotenoids are also antioxidants and thus able to prevent some cancers and cardiovascular diseases. Finally, lutein and zeaxanthin, which are xanthophyllic carotenoids, occur in orange and mandarin pulp and have an important role in vision, especially in the prevention of agerelated macular degeneration.

Diversity associated with the history of citrus crops

CIRAD’s research findings have shown that the carotenoid composition of citrus fruits is structured in three groups: the first includes mandarins and sweet and sour oranges; the second, grapefruits and pummelos; and the third, citrons, lemons and limes. This organization, which is very close to the overall genetic structure of the Citrus genus, suggests that the genetic differentiation that gave rise to the interspecific carotenoid content structure took place before the formation of secondary species—oranges, pummelos, lemons, limes and sour oranges—from three basic taxa of cultivated forms, ie mandarins, pummelos andcitrons.

Carotenoid biosynthesis pathways are well known in plant species. Expressed sequence tags (ESTs) of genes encoding enzymes in this pathway have been isolated and sequenced for different species, including citrus fruits. CIRAD studies have shown that the interspecific carotenoid content diversity structure in citrus fruits is associated with five key steps in this biosynthesis chain. The mandarin and orange group is the only one to explore all the carotenoid biosynthesis pathways with a high level of provitamin A components. Very little carotenoid synthesis takes place in the citron group. Apart from lycopene, they do not synthesize xanthophylls, and stop at ß-cryptoxanthin on the other chain branch. Genotypes of the pummelo group only synthesize carotenes: phytoene, phytofluene, lycopene and ß-carotene, generally in low quantities. These findings suggest that steps that occur in the upper part of the chain are limiting for the grapefruit and citron group. Steps catalysed by lycopene cyclases (LCyb and LcyE) also contribute to this interspecific differentiation, along with ß-carotene hydroxylase (HY-b) and zeaxanthin epoxydase (ZEP), which are involved in the formation of ß-cryptoxanthinand cis-violaxanthin from ß-carotene.

A phylogenetic origin of alleles in cultivated species has been proposed for four out of seven analysed genes. The allelic variability in two genes, ie Lcy-b and Lcy-e, which code for the two lycopene cyclases involved in the formation of a- and ß-carotene from lycopene could be linked to the phenotypic diversity. Variability in the other key steps is likely to be associated with the different expression levels of the corresponding genes. Underexpression of the DXS and PSY genes that encode DOXP synthase and phytoene synthase, which are located in the upper part of the biosynthesis chain, is associated with the overall low carotenoid contents in grapefruit and lemon. Moreover, the absence of ß-cryptoxanthine and corresponding molecules in the pummelo group would be linked withunderexpression of the HY-b gene.

Expression variations according to the environment

Environmental conditions also have a role in determining carotenoid contents. In the mandarin and orange group, Mediterranean cropping conditions boost interspecific differentiation by reinforcing ß-carotene and ß-cryptoxanthine contents in mandarins, as well asluteine, zeaxanthin and violaxanthin contents in sweet oranges. Conversely, lycopene accumulation is greater in Star Ruby pummelos under tropical environmental conditions.

The nutritional quality of citrus fruits and juice will be a major issue for citrus growing in the future, and should thus be taken into account in breeding programmes geared towards developing innovative new varieties. Findings on factors that determine its variability will pave the way for genetic improvements that are targeted to different citrus-growing regions.

Contacts

Patrick Ollitrault, E-mail Genetic Improvement of Vegetatively Propagated Crops (UPR)
Claudie Dhuique-Mayer, Integrated Food Quality System (UMR Qualisud)

Partners

• Institut national de la recherche agronomique (INRA, France)
• Instituto Valenciano de Investigaciones Agrarias (IVIA, Spain)
• University of Corsica
• Corsica Regional Council

For further information

• Dhuique-Mayer C., 2007. Evaluation de la qualité nutritionnelle des jus d’agrumes : estimation in vitro de la biodisponibilité des caroténoïdes. Thèse de doctorat, université Montpellier II, 114 p.
• Dhuique-Mayer C., Borel P., Reboul E., Caporiccio B., Besançon P., Amiot M.J., 2007. Beta-cryptoxanthin from Citrus juices: assessment of bioaccessibility using an in vitro digestion/Caco-2 cell culture model. British Journal of Nutrition, 97 : 883-890.
• Fanciullino A.L., 2007. Etude des déterminants génétiques et moléculaires de la variabilité des compositions en caroténoïdes au sein du genre Citrus . Thèse de doctorat, université de Corse, 188 p.
• Fanciullino A.L., Dhuique-Mayer C., Luro F., Morillon R., Ollitrault P., 2007. Carotenoid biosynthetic pathway in the Citrus genus: number of copies and phylogenetic diversity of seven genes. Journal of Agricultural and Food Chemistry, 55 : 7405-7417.