OeBAS and CYP716C67 catalyze the biosynthesis of health-beneficial triterpenoids in olive (Olea europaea) fruits.

The bioactive properties of olive (Olea europaea) fruits and olive oil are largely attributed to terpenoid compounds, including diverse triterpenoids such as oleanolic, maslinic and ursolic acids, erythrodiol, and uvaol. They have applications in the agri-food, cosmetics, and pharmaceutical industries. Some key steps involved in the biosynthesis of these compounds are still unknown. Genome mining, biochemical analysis, and trait association studies have been used to identify major gene candidates controlling triterpenoid content of olive fruits. Here, we identify and functionally characterize an oxidosqualene cyclase (OeBAS) required for the production of the major triterpene scaffold ß-amyrin, the precursor of erythrodiol, oleanolic and maslinic acids, and a cytochrome P450 (CYP716C67) that mediates 2α oxidation of the oleanane- and ursane-type triterpene scaffolds to produce maslinic and corosolic acids, respectively. To confirm the enzymatic functions of the entire pathway, we have reconstituted the olive biosynthetic pathway for oleanane- and ursane-type triterpenoids in the heterologous host, Nicotiana benthamiana. Finally, we have identified genetic markers associated with oleanolic and maslinic acid fruit content on the chromosomes carrying the OeBAS and CYP716C67 genes. Our results shed light on the biosynthesis of olive triterpenoids and provide new gene targets for germplasm screening and breeding for high triterpenoid content.

Genetics of Plant Metabolism.

This Special Issue is aimed to collect scientific papers that support holistic methodological approaches, both top-down and horizontal, for the correct application of various omics sciences because, when well-integrated, they can contribute to our understanding of the genotypic plasticity of plant species [...].

The eastern part of the Fertile Crescent concealed an unexpected route of olive (Olea europaea L.) differentiation.

Background and Aims: Olive is considered a native plant of the eastern side of the Mediterranean basin, from where it should have spread westward along the Mediterranean shores, while little is known about its diffusion in the eastern direction. Methods: Genetic diversity levels and population genetic structure of a wide set of olive ecotypes and varieties collected from several provinces of Iran, representing a high percentage of the entire olive resources present in the area, was screened with 49 chloroplast and ten nuclear simple sequence repeat markers, and coupled with archaeo-botanical and historical data on Mediterranean olive varieties. Approximate Bayesian Computation was applied to define the demographic history of olives including Iranian germplasm, and species distribution modelling was performed to understand the impact of the Late Quaternary on olive distribution. Key Results: The results of the present study demonstrated that: (1) the climatic conditions of the last glacial maximum had an important role on the actual olive distribution, (2) all Iranian olive samples had the same maternal inheritance as Mediterranean cultivars, and (3) the nuclear gene flow from the Mediterranean basin to the Iranian plateau was almost absent, as well as the contribution of subspecies cuspidata to the diversity of Iranian olives. Conclusions: Based on this evidence, a new scenario for the origin and distribution of this important fruit crop has been traced. The evaluation of olive trees growing in the eastern part of the Levant highlighted a new perspective on the spread and distribution of olive, suggesting two routes of olive differentiation, one westward, spreading along the Mediterranean basin, and another moving towards the east and reaching the Iranian plateau before its domestication.

Overexpression of the olive acyl carrier protein gene (OeACP1) produces alterations in fatty acid composition of tobacco leaves.

Taking into account that fatty acid (FA) biosynthesis plays a crucial role in lipid accumulation in olive (Olea europaea L.) mesocarp, we investigated the effect of olive acyl carrier protein (ACP) on FA composition by overexpressing an olive ACP cDNA in tobacco plants. The OeACP1.1A cDNA was inserted in the nucleus or in the chloroplast DNA of different tobacco plants, resulting in extensive transcription of the transgenes. The transplastomic plants accumulated lower olive ACP levels in comparison to nuclear-transformed plants. Moreover, the phenotype of the former plants was characterized by pale green/white cotyledons with abnormal chloroplasts, delayed germination and reduced growth. We suggest that the transplastomic phenotype was likely caused by inefficient olive ACP mRNA translation in chloroplast stroma. Conversely, total lipids from leaves of nuclear transformants expressing high olive ACP levels showed a significant increase in oleic acid (18:1) and linolenic acid (18:3), and a concomitant significant reduction of hexadecadienoic acid (16:2) and hexadecatrienoic acid (16:3). This implies that in leaves of tobacco transformants, as likely in the mesocarp of olive fruit, olive ACP not only plays a general role in FA synthesis, but seems to be specifically involved in chain length regulation forwarding the elongation to C18 FAs and the subsequent desaturation to 18:1 and 18:3.

Polymorphisms in the AOX2 gene are associated with the rooting ability of olive cuttings.

KEY MESSAGE: Different rooting ability candidate genes were tested on an olive cross progeny. Our results demonstrated that only the AOX2 gene was strongly induced. OeAOX2 was fully characterised and correlated to phenotypical traits. The formation of adventitious roots is a key step in the vegetative propagation of trees crop species, and this ability is under strict genetic control. While numerous studies have been carried out to identify genes controlling adventitious root formation, only a few loci have been characterised. In this work, candidate genes that were putatively involved in rooting ability were identified in olive (Olea europaea L.) by similarity with orthologs identified in other plant species. The mRNA levels of these genes were analysed by real-time PCR during root induction in high- (HR) and low-rooting (LR) individuals. Interestingly, alternative oxidase 2 (AOX2), which was previously reported to be a functional marker for rooting in olive cuttings, showed a strong induction in HR individuals. From the OeAOX2 full-length gene, alleles and effective polymorphisms were distinguished and analysed in the cross progeny, which were segregated based on rooting. The results revealed a possible correlation between two single nucleotide polymorphisms of OeAOX2 gene and rooting ability.

Correction: Transcript Analysis and Regulative Events during Flower Development in Olive (Olea europaea L.).

[This corrects the article DOI: 10.1371/journal.pone.0152943.].

Evolution of Extra Virgin Olive Oil Quality under Different Storage Conditions.

The extent and conditions of storage may affect the stability and quality of extra virgin olive oil (EVOO). This study aimed at evaluating the effects of different storage conditions (ambient, 4 °C and -18 °C temperatures, and argon headspace) on three EVOOs (low, medium, and high phenols) over 18 and 36 months, analyzing the main metabolites at six time points. The results showed that low temperatures are able to maintain all three EVOOs within the legal limits established by the current EU regulations for most compounds up to 36 months. Oleocanthal, squalene, and total phenols were affected by storage temperatures more than other compounds and degradation of squalene and α-tocopherol was inhibited only by low temperatures. The best temperature for 3-year conservation was 4 °C, but -18 °C represented the optimum temperature to preserve the organoleptic properties. The present study provided new insights that should guide EVOO manufacturers and traders to apply the most efficient storage methods to maintain the characteristics of the freshly extracted oils for a long conservation time.

Identification of new polymorphic regions and differentiation of cultivated olives (Olea europaea L.) through plastome sequence comparison.

BACKGROUND: The cultivated olive (Olea europaea L.) is the most agriculturally important species of the Oleaceae family. Although many studies have been performed on plastid polymorphisms to evaluate taxonomy, phylogeny and phylogeography of Olea subspecies, only few polymorphic regions discriminating among the agronomically and economically important olive cultivars have been identified. The objective of this study was to sequence the entire plastome of olive and analyze many potential polymorphic regions to develop new inter-cultivar genetic markers. RESULTS: The complete plastid genome of the olive cultivar Frantoio was determined by direct sequence analysis using universal and novel PCR primers designed to amplify all overlapping regions. The chloroplast genome of the olive has an organisation and gene order that is conserved among numerous Angiosperm species and do not contain any of the inversions, gene duplications, insertions, inverted repeat expansions and gene/intron losses that have been found in the chloroplast genomes of the genera Jasminum and Menodora, from the same family as Olea.The annotated sequence was used to evaluate the content of coding genes, the extent, and distribution of repeated and long dispersed sequences and the nucleotide composition pattern. These analyses provided essential information for structural, functional and comparative genomic studies in olive plastids. Furthermore, the alignment of the olive plastome sequence to those of other varieties and species identified 30 new organellar polymorphisms within the cultivated olive. CONCLUSIONS: In addition to identifying mutations that may play a functional role in modifying the metabolism and adaptation of olive cultivars, the new chloroplast markers represent a valuable tool to assess the level of olive intercultivar plastome variation for use in population genetic analysis, phylogenesis, cultivar characterisation and DNA food tracking.

Computational annotation of genes differentially expressed along olive fruit development.

BACKGROUND: Olea europaea L. is a traditional tree crop of the Mediterranean basin with a worldwide economical high impact. Differently from other fruit tree species, little is known about the physiological and molecular basis of the olive fruit development and a few sequences of genes and gene products are available for olive in public databases. This study deals with the identification of large sets of differentially expressed genes in developing olive fruits and the subsequent computational annotation by means of different software. RESULTS: mRNA from fruits of the cv. Leccino sampled at three different stages [i.e., initial fruit set (stage 1), completed pit hardening (stage 2) and veraison (stage 3)] was used for the identification of differentially expressed genes putatively involved in main processes along fruit development. Four subtractive hybridization libraries were constructed: forward and reverse between stage 1 and 2 (libraries A and B), and 2 and 3 (libraries C and D). All sequenced clones (1,132 in total) were analyzed through BlastX against non-redundant NCBI databases and about 60% of them showed similarity to known proteins. A total of 89 out of 642 differentially expressed unique sequences was further investigated by Real-Time PCR, showing a validation of the SSH results as high as 69%. Library-specific cDNA repertories were annotated according to the three main vocabularies of the gene ontology (GO): cellular component, biological process and molecular function. BlastX analysis, GO terms mapping and annotation analysis were performed using the Blast2GO software, a research tool designed with the main purpose of enabling GO based data mining on sequence sets for which no GO annotation is yet available. Bioinformatic analysis pointed out a significantly different distribution of the annotated sequences for each GO category, when comparing the three fruit developmental stages. The olive fruit-specific transcriptome dataset was used to query all known KEGG (Kyoto Encyclopaedia of Genes and Genomes) metabolic pathways for characterizing and positioning retrieved EST records. The integration of the olive sequence datasets within the MapMan platform for microarray analysis allowed the identification of specific biosynthetic pathways useful for the definition of key functional categories in time course analyses for gene groups. CONCLUSION: The bioinformatic annotation of all gene sequences was useful to shed light on metabolic pathways and transcriptional aspects related to carbohydrates, fatty acids, secondary metabolites, transcription factors and hormones as well as response to biotic and abiotic stresses throughout olive drupe development. These results represent a first step toward both functional genomics and systems biology research for understanding the gene functions and regulatory networks in olive fruit growth and ripening.

Physiological, epigenetic and genetic regulation in some olive cultivars under salt stress.

Cultivated olive, a typical fruit crop species of the semi-arid regions, could successfully face the new scenarios driven by the climate change through the selection of tolerant varieties to salt and drought stresses. In the present work, multidisciplinary approaches, including physiological, epigenetic and genetic studies, have been applied to clarify the salt tolerance mechanisms in olive. Four varieties (Koroneiki, Royal de Cazorla, Arbequina and Picual) and a related form (O. europaea subsp. cuspidata) were grown in a hydroponic system under different salt concentrations from zero to 200 mM. In order to verify the plant response under salt stress, photosynthesis, gas exchange and relative water content were measured at different time points, whereas chlorophyll and leaf concentration of Na+, K+ and Ca2+ ions, were quantified at 43 and 60 days after treatment, when stress symptoms became prominent. Methylation sensitive amplification polymorphism (MSAP) technique was used to assess the effects of salt stress on plant DNA methylation. Several fragments resulted differentially methylated among genotypes, treatments and time points. Real time quantitative PCR (RT-qPCR) analysis revealed significant expression changes related to plant response to salinity. Four genes (OePIP1.1, OePetD, OePI4Kg4 and OeXyla) were identified, as well as multiple retrotransposon elements usually targeted by methylation under stress conditions.

Genetic Mapping of the Incompatibility Locus in Olive and Development of a Linked Sequence-Tagged Site Marker.

The genetic control of self-incompatibility (SI) has been recently disclosed in olive. Inter-varietal crossing confirmed the presence of only two incompatibility groups (G1 and G2), suggesting a simple Mendelian inheritance of the trait. A double digest restriction associated DNA (ddRAD) sequencing of a biparental population segregating for incompatibility groups has been performed and high-density linkage maps were constructed in order to map the SI locus and identify gene candidates and linked markers. The progeny consisted of a full-sib family of 229 individuals derived from the cross 'Leccino' (G1) × 'Dolce Agogia' (G2) varieties, segregating 1:1 (G1:G2), in accordance with a diallelic self-incompatibility (DSI) model. A total of 16,743 single nucleotide polymorphisms was identified, 7,006 in the female parent 'Leccino' and 9,737 in the male parent 'Dolce Agogia.' Each parental map consisted of 23 linkage groups and showed an unusual large size (5,680 cM in 'Leccino' and 3,538 cM in 'Dolce Agogia'). Recombination was decreased across all linkage groups in pollen mother cells of 'Dolce Agogia,' the parent with higher heterozygosity, compared to megaspore mother cells of 'Leccino,' in a context of a species that showed exceptionally high recombination rates. A subset of 109 adult plants was assigned to either incompatibility group by a stigma test and the diallelic self-incompatibility (DSI) locus was mapped to an interval of 5.4 cM on linkage group 18. This region spanned a size of approximately 300 Kb in the olive genome assembly. We developed a sequence-tagged site marker in the DSI locus and identified five haplotypes in 57 cultivars with known incompatibility group assignment. A combination of two single-nucleotide polymorphisms (SNPs) was sufficient to predict G1 or G2 phenotypes in olive cultivars, enabling early marker-assisted selection of compatible genotypes and allowing for a rapid screening of inter-compatibility among cultivars in order to guarantee effective fertilization and increase olive production. The construction of high-density linkage maps has led to the development of the first functional marker in olive and provided positional candidate genes in the SI locus.

Usefulness of a New Large Set of High Throughput EST-SNP Markers as a Tool for Olive Germplasm Collection Management.

Germplasm collections are basic tools for conservation, characterization, and efficient use of olive genetic resources. The identification of the olive cultivars maintained in the collections is an important ongoing task which has been performed by both, morphological and molecular markers. In the present study, based on the sequencing results of previous genomic projects, a new set of 1,043 EST-SNP markers has been identified. In order to evaluate its discrimination capacity and utility in diversity studies, this set of markers was used in a representative number of accessions from 20 different olive growing countries and maintained at the World Olive Germplasm Collection of IFAPA Centre 'Alameda del Obispo' (Córdoba, Spain), one of the world's largest olive germplasm bank. Thus, the cultivated material included: cultivars belonging to previously defined core collections by means of SSR markers and agronomical traits, well known homonymy cases, possible redundancies previously identified in the collection, and recently introduced accessions. Marker stability was tested in repeated analyses of a selected number of accessions, as well as in different trees and accessions belonging to the same cultivar. In addition, 15 genotypes from a cross 'Picual' × 'Arbequina' cultivars from the IFAPA olive breeding program and a set of 89 wild genotypes were also included in the study. Our results indicate that, despite their relatively wide variability, the new set of EST-SNPs displayed lower levels of genetic diversity than SSRs in the set of olive core collections tested. However, the EST-SNP markers displayed consistent and reliable results from different plant material sources and plant propagation events. The EST-SNPs revealed a clear cut off between inter- and intra-cultivar variation in olive. Besides, they were able to reliably discriminate among different accessions, to detect possible homonymy cases as well as efficiently ascertain the presence of redundant germplasm in the collection. Additionally, these markers were highly transferable to the wild genotypes. These results, together with the low genotyping error rates and the easy and fully automated procedure used to get the genotyping data, validate the new set of EST-SNPs as possible markers of choice for olive cultivar identification.

High Levels of Variation Within Gene Sequences of Olea europaea L.

Gene sequence variation in cultivated olive (Olea europaea L. subsp. europaea var. europaea), the most important oil tree crop of the Mediterranean basin, has been poorly evaluated up to now. A deep sequence analysis of fragments of four genes, OeACP1, OeACP2, OeLUS and OeSUT1, in 90 cultivars, revealed a wide range of polymorphisms along all recognized allele forms and unexpected allele frequencies and genotype combinations. High linkage values among most polymorphisms were recorded within each gene fragment. The great sequence variability corresponded to a low number of alleles and, surprisingly, to a small fraction of genotype combinations. The distribution, frequency, and combination of the different alleles at each locus is possibly due to natural and human pressures, such as selection, ancestrality, or fitness. Phylogenetic analyses of allele sequences showed distant and complex patterns of relationships among cultivated olives, intermixed with other related forms, highlighting an evolutionary connection between olive cultivars and the O. europaea subspecies cuspidata and cerasiformis. This study demonstrates how a detailed and complete sequence analysis of a few gene portions and a thorough genotyping on a representative set of cultivars can clarify important issues related to sequence polymorphisms, reconstructing the phylogeny of alleles, as well as the genotype combinations. The identification of regions representing blocks of recombination could reveal polymorphisms that represent putatively functional markers. Indeed, specific mutations found on the analyzed OeACP1 and OeACP2 fragments seem to be correlated to the fruit weight.

Elucidation of the genetic architecture of self-incompatibility in olive: Evolutionary consequences and perspectives for orchard management.

The olive (Olea europaea L.) is a typical important perennial crop species for which the genetic determination and even functionality of self-incompatibility (SI) are still largely unresolved. It is still not known whether SI is under gametophytic or sporophytic genetic control, yet fruit production in orchards depends critically on successful ovule fertilization. We studied the genetic determination of SI in olive in light of recent discoveries in other genera of the Oleaceae family. Using intra- and interspecific stigma tests on 89 genotypes representative of species-wide olive diversity and the compatibility/incompatibility reactions of progeny plants from controlled crosses, we confirmed that O. europaea shares the same homomorphic diallelic self-incompatibility (DSI) system as the one recently identified in Phillyrea angustifolia and Fraxinus ornus. SI is sporophytic in olive. The incompatibility response differs between the two SI groups in terms of how far pollen tubes grow before growth is arrested within stigma tissues. As a consequence of this DSI system, the chance of cross-incompatibility between pairs of varieties in an orchard is high (50%) and fruit production may be limited by the availability of compatible pollen. The discovery of the DSI system in O. europaea will undoubtedly offer opportunities to optimize fruit production.

Transcript Analysis and Regulative Events during Flower Development in Olive (Olea europaea L.).

The identification and characterization of transcripts involved in flower organ development, plant reproduction and metabolism represent key steps in plant phenotypic and physiological pathways, and may generate high-quality transcript variants useful for the development of functional markers. This study was aimed at obtaining an extensive characterization of the olive flower transcripts, by providing sound information on the candidate MADS-box genes related to the ABC model of flower development and on the putative genetic and molecular determinants of ovary abortion and pollen-pistil interaction. The overall sequence data, obtained by pyrosequencing of four cDNA libraries from flowers at different developmental stages of three olive varieties with distinct reproductive features (Leccino, Frantoio and Dolce Agogia), included approximately 465,000 ESTs, which gave rise to more than 14,600 contigs and approximately 92,000 singletons. As many as 56,700 unigenes were successfully annotated and provided gene ontology insights into the structural organization and putative molecular function of sequenced transcripts and deduced proteins in the context of their corresponding biological processes. Differentially expressed genes with potential regulatory roles in biosynthetic pathways and metabolic networks during flower development were identified. The gene expression studies allowed us to select the candidate genes that play well-known molecular functions in a number of biosynthetic pathways and specific biological processes that affect olive reproduction. A sound understanding of gene functions and regulatory networks that characterize the olive flower is provided.

High genetic diversity detected in olives beyond the boundaries of the Mediterranean Sea.

BACKGROUND: Olive trees (Olea europaea subsp. europaea var. europaea) naturally grow in areas spanning the Mediterranean basin and towards the East, including the Middle East. In the Iranian plateau, the presence of olives has been documented since very ancient times, though the early history of the crop in this area is shrouded in uncertainty. METHODS: The varieties presently cultivated in Iran and trees of an unknown cultivation status, surviving under extreme climate and soil conditions, were sampled from different provinces and compared with a set of Mediterranean cultivars. All samples were analyzed using SSR and chloroplast markers to establish the relationships between Iranian olives and Mediterranean varieties, to shed light on the origins of Iranian olives and to verify their contribution to the development of the current global olive variation. RESULTS: Iranian cultivars and ecotypes, when analyzed using SSR markers, clustered separately from Mediterranean cultivars and showed a high number of private alleles, on the contrary, they shared the same single chlorotype with the most widespread varieties cultivated in the Mediterranean. CONCLUSION: We hypothesized that Iranian and Mediterranean olive trees may have had a common origin from a unique center in the Near East region, possibly including the western Iranian area. The present pattern of variation may have derived from different environmental conditions, distinct levels and selection criteria, and divergent breeding opportunities found by Mediterranean and Iranian olives.These unexpected findings emphasize the importance of studying the Iranian olive germplasm as a promising but endangered source of variation.