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Background: Anthocyanin pigmentation characterizes a number of tissues of Citrus and its relatives. The gain and loss of pigmentation is intriguing and is inherited variously among species. Methods: Citrus germplasm was used to investigate the anthocyanin pigmentation of tissues never before considered, including stamen, style and stigma, and of young leaves, petals, rind and flesh of 28 genotypes belonging to 14 species. Citrus genotypes encompassed citron, lemon, sweet orange, lime, and Citrus relatives included Microcitrus, Murraya, and Severinia. A relative qRT-PCR analysis was carried out on the structural and regulatory genes: phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3&prime, hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), uridine diphosphate glucose flavonoid glucosyl-transferase (UFGT), glutathione S-transferase (GST), Ruby and Noemi. Image analysis and a genomic approach were employed to evaluate how the red pigmentation is inherited among tissues and species. Results: Pigmentation of young leaves and petals is specific to citron and its hybrids. Ruby controls the pigmentation of petals, but not of leaves. The red color of the rind and flesh is a trait that particularly characterizes a diversity of sweet oranges, citron hybrids and Citrus relatives. Color expression depends on external factors and also on developmental stage. The coloration of stamen and style is citron-specific, while a red stigma is exclusive to Moro orange and its hybrids. Conclusion: It is hypothesized that there is a relationship among Citrus species and genes controlling anthocyanin pigmentation.

Sweet oranges are an important source of ascorbic acid (AsA). In this study, the content of AsA in the juice and leaves of four orange clonal selections, different in terms of maturity time and the presence/absence of anthocyanins, was correlated with the transcription levels of the main genes involved in the biosynthesis, recycling, and degradation pathways. Within each variety, differences in the above pathways and the AsA amount were found between the analysed tissues. Variations were also observed at different stages of fruit development and maturation. At the beginning of fruit development, AsA accumulation was attributable to the synergic action of l-galactose and Myo-inositol, while the l-gulose pathway was predominant between the end of fruit development and the beginning of ripening. In leaves, the l-galactose pathway appeared to play a major role in AsA accumulation, even though higher GalUr isoform expression suggests a synergistic contribution of both pathways in this tissue. In juice, the trend of the AsA content may be related to the decrease in the transcription levels of the GME, GDH, MyoOx, and GalUr12 genes. Newhall was the genotype that accumulated the most AsA. The difference between Newhall and the other varieties seems to be attributable to the GLDH, GalUr12, APX2, and DHAR3 genes.

A bstract Four new accessions were obtained by crossing some Sicilian high value cultivars (‘Buscionetto’, ‘Nocellara del Belice’, ‘Nocellara Messinese’ and ‘Tonda Dolce’). The olives were harvested at the green stage and characterized through the evaluation of agronomic and morphological parameters. The DNA was extracted from young leaves of the obtained hybrids and genotyped at 9 nuclear microsatellite loci. The olives were directly brined in order to test the quality of the fermented products. The results of genetic variability clustered all the analyzed samples in two main groups. The carpological parameters of the new hybrids revealed that they are very suitable for table olives. The phenological stages of the new hybrids allow a longer period of commercialization compared to the reference varieties because the ‘C’, ‘E’ and ‘B’ hybrids showed a substantial ripening period advance. The sensory profiles of ‘A’ and ‘C’ hybrids were quite similar, while the other two ones showed different sensory results.

Blood oranges have been cultivated in Italy since the early 19th century. Growers and breeders were able to identify and collect many somatic mutants, which differed in their major pomological traits, such as fruit size and firmness, pulp and peel pigmentation, and ripening period. Recently, in the framework of a genetic resource conservation program, most of the accessions collected at CREA in recent decades were planted in a single block to evaluate their characteristics. The presence of many selections in the same block, of the same age and grafted on the same rootstock is extremely important for a reliable characterization of the pomological characters, particularly pigmentation, which is strongly influenced by both the environment and the rootstock. The collection consists of 88 genotypes, including old lines and nucellar selections. Some commercial Tarocco, Moro and Sanguinello varieties were also included in the collection to facilitate comparison among clones. Moreover, the pomological features of 20 elite Tarocco clones cultivated nearby in the same experimental station were used as a further reference. The pomological characterization, based on 13 traits and performed in two consecutive years, revealed significant variability. Some genotypes showed strong year-to-year variability, mainly in fruit pigmentation, probably due to different climatic conditions during winter. Similarly, other traits such as fruit size, total soluble solids and acidity also varied among and within the varietal groups. These findings have a potential utility in identifying promising clones, which might fulfil the growing market needs such as higher anthocyanin content and longer shelf life.

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.

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.

Citrus tristeza virus (CTV) is the causal agent of various diseases with dramatic effects on citrus crops worldwide. Most Citrus species, grown on their own roots, are symptomless hosts for many CTV isolates. However, depending on different scion-rootstock combination, CTV infection should result in distinct syndromes, being 'tristeza' the more severe one, leading to a complete decline of the susceptible plants in a few weeks. Transcriptomic analyses revealed several genes involved either in defense response, or systemic acquired resistance, as well as transcription factors and components of the phosphorylation cascades, to be differentially regulated during CTV infection in Citrus aurantifolia species. To date little is known about the molecular mechanism of this host-pathogen interaction, and about the rootstock effect on citrus response to CTV infection. In this work, the response to CTV infection has been investigated in tolerant and susceptible scion-rootstock combinations by two-dimensional gel electrophoresis (2DE). A total of 125 protein spots have been found to be differently accumulated and/or phosphorylated between the two rootstock combinations. Downregulation in tolerant plants upon CTV infection was detected for proteins involved in reactive oxygen species (ROS) scavenging and defense response, suggesting a probable acclimation response able to minimize the systemic effects of virus infection. Some of these proteins resulted to be modulated also in absence of virus infection, revealing a rootstock effect on scion proteome modulation. Moreover, the phospho-modulation of proteins involved in ROS scavenging and defense response, further supports their involvement either in scion-rootstock crosstalk or in the establishment of tolerance/susceptibility to CTV infection.

Genes encoding chalcone synthase (CHS), anthocyanidin synthase (ANS), and UDP-glucose-fl avonoid 3-O- glucosyltransferase (UFGT), some of the enzymes of anthocyanin biosynthetic pathway, were assayed in two different experiments using quantitative real-time reverse transcriptase (RT)-PCR, in order to test their transcription levels in the fl esh of blood and common orange (Citrus sinensis (L.) Osbeck) fruit, and to investigate their role in anthocyanin accumulation in the same tissue. The fi rst experiment compared a blood orange and a common orange cultivar during seven different fruit maturation stages. This was followed by the test of 11 different genotypes at the end of the winter season. Data collected from the fi rst experiment, over the blood orange cultivar, were statistically analyzed using the Pearson correlation coeffi cient. Results show that CHS, ANS, and UFGT mRNA transcripts are up- and co-regulated in the blood orange cultivar, whereas they are down-regulated in the common orange cultivar. There is evidence of correspondence between the target genes expression level and the content of the pigment assessed. The second test confi rms this correlation and shows that enzyme synthesis levels and pigment accumulation, in plants grown under the same environmental conditions, are dependent on the differences occurring among the genotypes tested. These results suggest that the absence of pigment in the common orange cultivars may be caused by the lack of induction on the structural genes expression. This is the fi rst report on the characterization of the relationships between biosynthetic genes expression and fruit fl esh anthocyanin content in blood oranges. The blood orange is the typical product of Sicilian citriculture and its red to purple pigmented rind and fl esh makes the fruit more attractive. Red color in blood oranges is due to the pres- ence of anthocyanins, the largest class of pigments among the fl avonoids. They are secondary metabolites, widely spread in higher plants and produced in different organs, in response to a variety of developmental, environmental, and genetic cues (Mol et al., 1996). They serve various reproductive functions (e.g., in fl ower petals to attract pollinators, and in seeds and fruit to aid in seed dispersal (Holton and Cornish, 1995)) and they protect plant tissues against pathogens and damage from environmental conditions (e.g., ultraviolet (UV) irradiation, cold temperature (Dixon and Paiva, 1995) and water stress (Chalker-Scott, 1999)). This class of pigmented molecules also possesses some important therapeutic properties for human health (Saija, 1994), function- ing as antioxidants, anti-infl ammatories, in epithelium repair and protection, and in modulation of capillary permeability. In conferring these properties to blood orange fruit, anthocyanins contribute to their commercial popularity and value, therefore research has been focusing on understanding the biosynthetic pathway for anthocyanins in oranges (Lo Piero et al., 2005

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.