Concetta Licciardello

Proteome analysis of Citrus sinensis L. (Osbeck) flesh at ripening time

A combination of 2-DE and LC-MSMS approaches was used to identify the differentially expressed proteome of a pigmented sweet orange (Citrus sinensis, cv. Moro) in comparison with a common cultivar (Cadenera) at ripening time. The comparison of the protein patterns of Moro and Cadenera showed 64 differential expressed protein spots. Fifty-five differentially expressed proteins were identified. Proteins were classified according to their putative function and known biosynthetic pathways. Most of the proteins related to sugar metabolism were overexpressed in Moro, while those related to stress responses were overexpressed in Cadenera. The abundance of proteins belonging to Unknown/Unnamed and Hypothetical classes could be associated to the incomplete data available on the Citrus genome. The relative abundance of Secondary metabolism and Oxidative process proteins substantiated the key role of the anthocyanin pathway in Moro, which is characterized by a strong pigmentation at ripening time. The potential role of protein differential expression between Moro and Cadenera fruits was discussed, and proteomic results were compared with the known variations of transcripts of the same fruits. The latter analyses highlighted many discrepancies, confirming the necessity to associate both proteomic and transcriptomic approaches in order to achieve a more complete characterization of the biological system.

Identification of differentially expressed genes in the flesh of blood and common oranges

The objective of this research was the identification of genes differentially expressed in blood oranges compared to common oranges and the identification of anthocyanin pathway genes that are up-regulated in flesh of blood oranges. A subtracted complementary DNA library of 1,248 clones was constructed using RNA from the flesh of a nucellar line (58-8D-1) of Moro (a blood orange) as tester and from Cadenera (a common orange) as driver. After screening by reverse Northern, a total of 230 clones were found to be up-regulated in blood orange, while 30 were up-regulated in the common blond one. Sequence analysis identified genes involved in the anthocyanin pathway including genes encoding biosynthetic enzymes like phenylalanine ammonialyase, chalcone synthase, dihydroflavonol-4-reductase, anthocyanidin synthase, UDP:glucose flavonoid 3-O-glucosyltransferase, glutathione S-transferase, and a regulatory gene encoding a basic Helix-Loop-Helix protein, while others were related to primary metabolism, flavor biosynthesis, signal transduction mechanisms, and defense. Some sequences were classified as unknown and unnamed and some others were unclassified. Semiquantitative reverse transcription-polymerase chain reaction (PCR) and quantitative real-time PCR were used to confirm the differential expression patterns of selected candidate genes of different functional classes. Correlations between the expression of some genes and the processes involved in the ripening of blood oranges were identified.

Use of a custom array to study differentially expressed genes during blood orange (Citrus sinensis L. Osbeck) ripening

A flesh-specific oligonucleotide custom array was designed to study gene expression during blood orange ripening. The array included 301 probes derived from a subtracted SSH library, a cDNA-AFLP collection, and a set of regulatory genes from the Harvest citrus database. The custom array was hybridized using samples of Moro, a pigmented cultivar, and Cadenera, a common cultivar, at three different ripening stages: the immature phase, the halfway point of maturation (corresponding to the start of Moro pigmentation) and the full ripening. Of the 301 probes, 27 in total, corresponding to 20 different transcripts, indicated differential expression in stage-to-stage and/or cultivar-to-cultivar comparisons. Transcripts encoding for anthocyanin biosynthesis represented most of the total over-expressed probes. The remaining differentially expressed transcripts were functionally associated with primary metabolism as flavor biosynthesis, defense and signal transduction. The expressed products associated with probes indicating differential expression were confirmed by qRT-PCR. The microarray was designed considering a small collection of sequences useful for monitoring specific pathways and regulatory genes related to fruit ripening and anthocyanin pigmentation. The main novelty of this customization is the use of expressed sequences specifically derived from blood orange flesh to study different cultivars and ripening stages, and the provision of further information about processes related to anthocyanin pigmentation in citrus fruit flesh.