An improvement of our knowledge on gene composition and expression is essential to investigate the molecular basis of fruit ripening and to define the gene pool involved in lipid and phenol metabolism in an oil crop species as olive, characterized by a peculiar fatty acid and antioxidant composition.
The availability of complete genome sequences and large sets of expressed sequence tags (ESTs) from several plants has recently triggered the development of efficient and informative methods for large-scale and genome-wide analysis of genetic variation and gene expression patterns. The ability to monitor simultaneously the expression of a large set of genes is one of the most important objectives of genome sequencing efforts. In this respect, the 454 pyrosequencing technology [1
] is a rather novel method for high-throughput DNA sequencing, allowing gene discovery and parallel efficient and quantitative analysis of expression patterns in cells, tissues and organs.
In the past few years, several studies based on comparative high throughput sequencing of plant transcriptomes have, indeed, allowed the identification of new gene functions, contaminant sequences from other organisms, alterations of gene expression in response to genotype, tissue or physiological changes, as well as large scale discovery of SNPs (Single Nucleotide Polymorphisms) in a number of model and non model species, such us maize, grapevine and eucalyptus [2
Olive is the sixth most important oil crop in the world, presently spreading from the Mediterranean region of origin to new production areas, due to the beneficial nutritional properties of olive oil and to its high economic value.
It belongs to the family of Oleaceae, order of Lamiales, which includes about 10 families for a total of about 11,000 species. Members of this order are important sources of fragrances, essential oils and phenolics claiming for numerous health benefits, or providing valuable commercial products, such as wood or ornamentals. Information on the genome sequence and transcript profiles of the entire clade are completely lacking.
Olive is a diplod species (2n = 2x = 46), predominantly allogamous, with a genome size of about 1,800 Mb [6
]. In spite of its economical importance and metabolic peculiarities, very few data are available on gene sequences controlling the main metabolic pathways.
Olive accumulates oil mainly in the drupe mesocarp and its content can reach up to 28–30% of total mesocarp fresh weight. Olive oil shows a peculiar acyl composition, particularly enriched in the monounsaturated fatty acid oleate (C18:1), deriving from the desaturation of stearate. Oleate can reach percentages up to 75–80% of total fatty acids, while linoleate (C18:2), palmitate (C16:0), stearate (C18:0) and linolenate (C18:3) represent minor components. The final acyl composition of olive oil varies enormously among varieties. Environmental factors, such as temperature and light during fruit ripening, can deeply influence the balance between saturated and unsaturated fatty acids [8
The chemistry of phenolic oleosides is attracting an increasing interest of pharmacological research and agri-food biotechnology, and the biochemical pathway leading to their biosynthesis and regulation has been recently deeply evaluated [9
], even if the genetic control still remains completely unknown.
Secoiridoids represent the most important class of phenolics and they arise from simple structures, like tyrosol and hydroxytyrosol, to quantitatively more important conjugated forms like oleuropein, demethyloleuropein, 3-4DHPEA-EDA and ligstroside [10
]. Oleuropein is the main secoiridoid, representing up to the 82% of total biophenols, known as the bitter principle of olives and responsible for major effects on human health and for releasing phytoalexins against plant pathogens [10
]. Another secoiridoid with relevant health functions is oleocanthal (deacetoxy ligstroside aglycone) [11
Developing olives contain active chloroplasts capable of photosynthesis, thus representing significant sources of photoassimilates. While chlorophyll is localized mostly in the epicarp, the mesocarp contains significant amounts of other photosynthetic pathway components, such as phosphoenol pyruvate carboxylase [12
Olive fruit development and ripening, takes place in about 4–5 months and includes the following phases: i) fruit set after fertilization, ii) seed development, iii) pit hardening, iv) mesocarp development and v) ripening. During the ripening process, fruit tissues undergo physiological and biochemical changes that include cell division and expansion, oil accumulation, metabolite storage, softening, phenol degradation, colour change (due to anthocyanin accumulation in outer mesocarp cells). Oil synthesis starts after pit hardening, reaching a plateau after 75–90 days, while the phenolic fraction is maximum at fruit set and decreases rapidly along fruit development.
This work is aimed at defining the transcriptome of olive drupes and at identifying ESTs involved in phenolic and lipid metabolism during fruit development. Drupes from two cultivars have been used: a widely cultivated variety characterized by a very high phenolic content, and an oleuropein-lacking natural variant; two developmental stages, at completed fruit set and at mesocarp development, representing diverse sets of expressed genes, were analyzed using 454 pyrosequencing.