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.

A New Perspective for Vineyard Terroir Identity: Looking for Microbial Indicator Species by Long Read Nanopore Sequencing.

Grapevine is one of the most important fruit crops worldwide, being Portugal one of the top wine producers. It is well established that wine sensory characteristics from a particular region are defined by the physiological responses of the grapevine to its environment and thus, the concept of terroir in viticulture was established. Among all the factors that contribute to terroir definition, soil microorganisms play a major role from nutrient recycling to a drastic influence on plant fitness (growth and protection) and of course wine production. Soil microbiome from four different terroirs in Quinta dos Murças vineyard was analysed through long-read Oxford Nanopore sequencing. We have developed an analytical pipeline that allows the identification of function, ecologies, and indicator species based on long read sequencing data. The Douro vineyard was used as a case study, and we were able to establish microbiome signatures of each terroir.

VviAGL11 self-regulates and targets hormone- and secondary metabolism-related genes during seed development.

VviAGL11, the Arabidopsis SEEDSTICK homolog, has been proposed to have a causative role in grapevine stenospermocarpy. An association between a mutation in the coding sequence (CDS) and the seedless phenotype was reported, however, no working mechanisms have been demonstrated yet. We performed a deep investigation of the full VviAGL11 gene sequence in a collection of grapevine varieties belonging to several seedlessness classes that revealed three different promoter-CDS combinations. By investigating the expression of the three VviAGL11 alleles, and by evaluating their ability to activate the promoter region, we observed that VviAGL11 self-activates in a specific promoter-CDS combination manner. Furthermore, by transcriptomic analyses on ovule and developing seeds in seeded and seedless varieties and co-expression approaches, candidate VviAGL11 targets were identified and further validated through luciferase assay and in situ hybridization. We demonstrated that VviAGL11 Wild Type CDS activates Methyl jasmonate esterase and Indole-3-acetate beta-glucosyltransferase, both involved in hormone signaling and Isoflavone reductase, involved in secondary metabolism. The dominant-negative effect of the mutated CDS was also functionally ectopically validated in target induction. VviAGL11 was shown to co-localize with its targets in the outer seed coat integument, supporting its direct involvement in seed development, possibly by orchestrating the crosstalk among MeJA, auxin, and isoflavonoids synthesis. In conclusion, the VviAGL11 expression level depends on the promoter-CDS allelic combination, and this will likely affect its ability to activate important triggers of the seed coat development. The dominant-negative effect of the mutated VviAGL11 CDS on the target genes activation was molecularly validated. A new regulatory mechanism correlating VviAGL11 haplotype assortment and seedlessness class in grapevine is proposed.

Transcriptomic and methylation analysis of susceptible and tolerant grapevine genotypes following Plasmopara viticola infection.

Downy mildew, caused by the biotrophic oomycete Plasmopara viticola, is one of the most economically significant grapevine diseases worldwide. Current strategies to cope with this threat rely on the massive use of chemical compounds during each cultivation season. The economic costs and negative environmental impact associated with these applications increased the urge to search for sustainable strategies of disease control. Improved knowledge of plant mechanisms to counteract pathogen infection may allow the development of alternative strategies for plant protection. Epigenetic regulation, in particular DNA methylation, is emerging as a key factor in the context of plant-pathogen interactions associated with the expression modulation of defence genes. To improve our understanding of the genetic and epigenetic mechanisms underpinning grapevine response to P. viticola, we studied the modulation of both 5-mC methylation and gene expression at 6 and 24 h post-infection (hpi). Leaves of two table grape genotypes (Vitis vinifera), selected by breeding activities for their contrasting level of susceptibility to the pathogen, were analysed. Following pathogen infection, we found variations in the 5-mC methylation level and the gene expression profile. The results indicate a genotype-specific response to pathogen infection. The tolerant genotype (N23/018) at 6 hpi exhibits a lower methylation level compared to the susceptible one (N20/020), and it shows an early modulation (at 6 hpi) of defence and epigenetic-related genes during P. viticola infection. These data suggest that the timing of response is an important mechanism to efficiently counteract the pathogen attack.

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

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

Two bi-functional cytochrome P450 CYP72 enzymes from olive (Olea europaea) catalyze the oxidative C-C bond cleavage in the biosynthesis of secoxy-iridoids - flavor and quality determinants in olive oil.

Olive (Olea europaea) is an important crop in Europe, with high cultural, economic and nutritional significance. Olive oil flavor and quality depend on phenolic secoiridoids, but the biosynthetic pathway of these iridoids remains largely uncharacterized. We discovered two bifunctional cytochrome P450 enzymes, catalyzing the rare oxidative C-C bond cleavage of 7-epi-loganin to produce oleoside methyl ester (OeOMES) and secoxyloganin (OeSXS), both through a ketologanin intermediary. Although these enzymes are homologous to the previously reported Catharanthus roseus secologanin synthase (CrSLS), the substrate and product profiles differ. Biochemical assays provided mechanistic insights into the two-step OeOMES and CrSLS reactions. Model-guided mutations of OeOMES changed the product profile in a predictable manner, revealing insights into the molecular basis for this change in product specificity. Our results suggest that, in contrast to published hypotheses, in planta production of secoxy-iridoids is secologanin-independent. Notably, sequence data of cultivated and wild olives point to a relation between domestication and OeOMES expression. Thus, the discovery of this key biosynthetic gene suggests a link between domestication and secondary metabolism, and could potentially be used as a genetic marker to guide next-generation breeding programs.

Microarray data and pathway analyses of peripheral blood mononuclear cells from healthy subjects after a three weeks grape-rich diet.

Using Human Gene Expression Microarrays (Agilent) technologies, we investigated changes of the level of gene expression in peripheral blood mononuclear cells of healthy subjects after 21 days of fresh table grape-rich diet and after an additional 28-day washout. Several hundreds of genes were differentially expressed after grape intake or after washout. The functional analysis of these genes detected significant changes in key processes such as inflammation and immunity, thrombosis, DNA and protein repair, autophagy and mitochondrial biogenesis. Moreover, fresh grape intake was found to influence the expression of many long non-coding RNA genes. The data can be valuable for researchers interested in nutrigenetics and nutrigenomics studies and are related to the research article "Gene expression signature induced by grape intake in healthy subjects reveals wide-spread beneficial effects on PBMCs" [1].

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.

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.

The transcriptional response to the olive fruit fly (Bactrocera oleae) reveals extended differences between tolerant and susceptible olive (Olea europaea L.) varieties.

The olive fruit fly Bactrocera oleae (Diptera: Tephritidae) is the most devastating pest of cultivated olive (Olea europaea L.). Intraspecific variation in plant resistance to B. oleae has been described only at phenotypic level. In this work, we used a transcriptomic approach to study the molecular response to the olive fruit fly in two olive cultivars with contrasting level of susceptibility. Using next-generation pyrosequencing, we first generated a catalogue of more than 80,000 sequences expressed in drupes from approximately 700k reads. The assembled sequences were used to develop a microarray layout with over 60,000 olive-specific probes. The differential gene expression analysis between infested (i.e. with II or III instar larvae) and control drupes indicated a significant intraspecific variation between the more tolerant and susceptible cultivar. Around 2500 genes were differentially regulated in infested drupes of the tolerant variety. The GO annotation of the differentially expressed genes implies that the inducible resistance to the olive fruit fly involves a number of biological functions, cellular processes and metabolic pathways, including those with a known role in defence, oxidative stress responses, cellular structure, hormone signalling, and primary and secondary metabolism. The difference in the induced transcriptional changes between the cultivars suggests a strong genetic role in the olive inducible defence, which can ultimately lead to the discovery of factors associated with a higher level of tolerance to B. oleae.

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.

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.

Olive fruits infested with olive fly larvae respond with an ethylene burst and the emission of specific volatiles.

Olive fly (Bactrocera oleae R.) is the most harmful insect pest of olive (Olea europaea L.) which strongly affects fruits and oil production. Despite the expanding economic importance of olive cultivation, up to now, only limited information on plant responses to B. oleae is available. Here, we demonstrate that olive fruits respond to B. oleae attack by producing changes in an array of different defensive compounds including phytohormones, volatile organic compounds (VOCs), and defense proteins. Bactrocera oleae-infested fruits induced a strong ethylene burst and transcript levels of several putative ethylene-responsive transcription factors became significantly upregulated. Moreover, infested fruits induced significant changes in the levels of 12-oxo-phytodienoic acid and C12 derivatives of the hydroperoxide lyase. The emission of VOCs was also changed quantitatively and qualitatively in insect-damaged fruits, indicating that B. oleae larval feeding can specifically affect the volatile blend of fruits. Finally, we show that larval infestation maintained high levels of trypsin protease inhibitors in ripe fruits, probably by affecting post-transcriptional mechanisms. Our results provide novel and important information to understand the response of the olive fruit to B. oleae attack; information that can shed light onto potential new strategies to combat this pest.

Identification and Characterization of the Iridoid Synthase Involved in Oleuropein Biosynthesis in Olive (Olea europaea) Fruits.

The secoiridoids are the main class of specialized metabolites present in olive (Olea europaea L.) fruit. In particular, the secoiridoid oleuropein strongly influences olive oil quality because of its bitterness, which is a desirable trait. In addition, oleuropein possesses a wide range of pharmacological properties, including antioxidant, anti-inflammatory, and anti-cancer activities. In accordance, obtaining high oleuropein varieties is a main goal of molecular breeding programs. Here we use a transcriptomic approach to identify candidate genes belonging to the secoiridoid pathway in olive. From these candidates, we have functionally characterized the olive homologue of iridoid synthase (OeISY), an unusual terpene cyclase that couples an NAD (P)H-dependent 1,4-reduction step with a subsequent cyclization, and we provide evidence that OeISY likely generates the monoterpene scaffold of oleuropein in olive fruits. OeISY, the first pathway gene characterized for this type of secoiridoid, is a potential target for breeding programs in a high value secoiridoid-accumulating species.

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.

Proteome regulation during Olea europaea fruit development.

BACKGROUND: Widespread in the Mediterranean basin, Olea europaea trees are gaining worldwide popularity for the nutritional and cancer-protective properties of the oil, mechanically extracted from ripe fruits. Fruit development is a physiological process with remarkable impact on the modulation of the biosynthesis of compounds affecting the quality of the drupes as well as the final composition of the olive oil. Proteomics offers the possibility to dig deeper into the major changes during fruit development, including the important phase of ripening, and to classify temporal patterns of protein accumulation occurring during these complex physiological processes. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we started monitoring the proteome variations associated with olive fruit development by using comparative proteomics coupled to mass spectrometry. Proteins extracted from drupes at three different developmental stages were separated on 2-DE and subjected to image analysis. 247 protein spots were revealed as differentially accumulated. Proteins were identified from a total of 121 spots and discussed in relation to olive drupe metabolic changes occurring during fruit development. In order to evaluate if changes observed at the protein level were consistent with changes of mRNAs, proteomic data produced in the present work were compared with transcriptomic data elaborated during previous studies. CONCLUSIONS/SIGNIFICANCE: This study identifies a number of proteins responsible for quality traits of cv. Coratina, with particular regard to proteins associated to the metabolism of fatty acids, phenolic and aroma compounds. Proteins involved in fruit photosynthesis have been also identified and their pivotal contribution in oleogenesis has been discussed. To date, this study represents the first characterization of the olive fruit proteome during development, providing new insights into fruit metabolism and oil accumulation process.

Olive phenolic compounds: metabolic and transcriptional profiling during fruit development.

BACKGROUND: Olive (Olea europaea L.) fruits contain numerous secondary metabolites, primarily phenolics, terpenes and sterols, some of which are particularly interesting for their nutraceutical properties. This study will attempt to provide further insight into the profile of olive phenolic compounds during fruit development and to identify the major genetic determinants of phenolic metabolism. RESULTS: The concentration of the major phenolic compounds, such as oleuropein, demethyloleuropein, 3-4 DHPEA-EDA, ligstroside, tyrosol, hydroxytyrosol, verbascoside and lignans, were measured in the developing fruits of 12 olive cultivars. The content of these compounds varied significantly among the cultivars and decreased during fruit development and maturation, with some compounds showing specificity for certain cultivars. Thirty-five olive transcripts homologous to genes involved in the pathways of the main secondary metabolites were identified from the massive sequencing data of the olive fruit transcriptome or from cDNA-AFLP analysis. Their mRNA levels were determined using RT-qPCR analysis on fruits of high- and low-phenolic varieties (Coratina and Dolce d'Andria, respectively) during three different fruit developmental stages. A strong correlation was observed between phenolic compound concentrations and transcripts putatively involved in their biosynthesis, suggesting a transcriptional regulation of the corresponding pathways. OeDXS, OeGES, OeGE10H and OeADH, encoding putative 1-deoxy-D-xylulose-5-P synthase, geraniol synthase, geraniol 10-hydroxylase and arogenate dehydrogenase, respectively, were almost exclusively present at 45 days after flowering (DAF), suggesting that these compounds might play a key role in regulating secoiridoid accumulation during fruit development. CONCLUSIONS: Metabolic and transcriptional profiling led to the identification of some major players putatively involved in biosynthesis of secondary compounds in the olive tree. Our data represent the first step towards the functional characterisation of important genes for the determination of olive fruit quality.

Molecular interactions between the olive and the fruit fly Bactrocera oleae.

BACKGROUND: The fruit fly Bactrocera oleae is the primary biotic stressor of cultivated olives, causing direct and indirect damages that significantly reduce both the yield and the quality of olive oil. To study the olive-B. oleae interaction, we conducted transcriptomic and proteomic investigations of the molecular response of the drupe. The identifications of genes and proteins involved in the fruit response were performed using a Suppression Subtractive Hybridisation technique and a combined bi-dimensional electrophoresis/nanoLC-ESI-LIT-MS/MS approach, respectively. RESULTS: We identified 196 ESTs and 26 protein spots as differentially expressed in olives with larval feeding tunnels. A bioinformatic analysis of the identified non-redundant EST and protein collection indicated that different molecular processes were affected, such as stress response, phytohormone signalling, transcriptional control and primary metabolism, and that a considerable proportion of the ESTs could not be classified. The altered expression of 20 transcripts was also analysed by real-time PCR, and the most striking differences were further confirmed in the fruit of a different olive variety. We also cloned the full-length coding sequences of two genes, Oe-chitinase I and Oe-PR27, and showed that these are wound-inducible genes and activated by B. oleae punctures. CONCLUSIONS: This study represents the first report that reveals the molecular players and signalling pathways involved in the interaction between the olive fruit and its most damaging biotic stressor. Drupe response is complex, involving genes and proteins involved in photosynthesis as well as in the production of ROS, the activation of different stress response pathways and the production of compounds involved in direct defence against phytophagous larvae. Among the latter, trypsin inhibitors should play a major role in drupe resistance reaction.