Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Oral Communication Abstract - S1e

Cloning, sequencing and expression levels of several genes involved in the anthocyanins biosynthesis pathway in Citrus genus

Cotroneo p. s. *, Lo piero a. r.**, ciuni m. *, consoli a. **, puglisi i. **, morana l. **, russo m. p. *, reforgiato recupero g. *, petrone g. **

*) Istituto Sperimentale per l’Agrumicoltura, Acireale (CT)

**) DACPA, Facoltà di Agraria, Università di Catania

Anthocyanins, cDNA cloning, orange flesh, Real Time RT-PCR, Citrus spp. L.

Among citrus fruits anthocyanins are expressed exclusively in blood oranges and their hybrids. Blood oranges represent the typical sicilian citriculture production and their healthy value contributes to raise the commercial popularity of these selections. Anthocyanin greatly varying content is strictly related to genotype and environmental conditions, moreover expression levels vary considerably among flesh and rind, these facts causing trouble to marketing of the product. Even if the biosynthetic pathway to anthocianins has been deeply investigated in some crops (maize, Anthirrinum, Perrilla, Malus etc.), concerning Citrus studies have just started. This paper is to refer on preliminary results from analysis of some genes related to anthocyanins biosynthesis.

Two of the enzymes of the biosynthetic pathway to anthocyanins in Citrus: chalcone syntase (CHS), assumed to catalyze the reaction from 4-cumaroyl-CoA to chalcone, in the early steps of the pathway, and anthocyanidine synthase (ANS), postulated to catalyse the reaction from colorless leucoanthocyanidine to the colored anthocyanidine, at the end of the pathway, have been submitted to molecular analysis. In order to achieve some information about the expression levels of genes involved in the anthocyanins biosynthesis pathway, partial clones coding for CHS and ANS were isolated from a blood orange flesh cDNA library. Assigment of function to the isolated cDNA clones was confirmed by comparing the nucleotide sequence with the corresponding structural genes from other species. The obtained sequences have been employed to evaluate expression levels of the enzymes during blood and blond fruits maturation by Real Time RT-PCR. RNA from seven different maturation stages for each cultivar were tested, using as internal reference the elongation factor (EF), present in Citrus at constant level. Preliminary results show that CHS and ANS are present, at low concentration, in both blood and blond oranges, in early stages of maturation, their quantity increasing in pigmented ones in the first four stages and maintaining the same level in the last three ones. In blond oranges CHS showed always the same concentration in all stages with slight decreases in the 4^th and 5^th, while ANS showed continuous decreasing. The two enzymes concentrations are related to each other in every maturation stage of the blood cultivars and from the first to the fifth sampling of the blond ones, while in the last two stages of the latter, CHS shows slight increasing never followed by ANS which goes on decreasing.