Combined Transcriptomic and Metabolomic Approach Revealed a Relationship between Light Control, Photoprotective Pigments, and Lipid Biosynthesis in Olives.

Olive possesses excellent nutritional and economic values for its main healthy products. Among them, a high content of antioxidant compounds, balanced during the ripening process, are produced under genetic and environmental control, resulting in high variability among cultivars. The genes involved in these complex pathways are mainly known, but despite many studies which indicated the key role of light quality and quantity for the synthesis of many metabolites in plants, limited information on these topics is available in olive. We carried out a targeted gene expression profiling in three olive cultivars, Cellina di Nardò, Ruveia, and Salella, which were selected for their contrasting oleic acid and phenolic content. The -omics combined approach revealed a direct correlation between a higher expression of the main flavonoid genes and the high content of these metabolites in 'Cellina di Nardò'. Furthermore, it confirmed the key role of FAD2-2 in the linoleic acid biosynthesis. More interestingly, in all the comparisons, a co-regulation of genes involved in photoperception and circadian clock machinery suggests a key role of light in orchestrating the regulation of these pathways in olive. Therefore, the identified genes in our analyses might represent a useful tool to support olive breeding, although further investigations are needed.

LPL, FNDC5 and PPARγ gene polymorphisms related to body composition parameters and lipid metabolic profile in adolescents from Southern Italy.

BACKGROUND: Plasma lipid profile and anthropometric variables are known to be under strong genetic control and the identification of genetic variants associated with bioclinical parameters is of considerable public health importance. In this study, a young cohort of healthy individuals was genotyped for genes related to health and pathological conditions, to analyze the association of single nucleotide polymorphisms (SNPs) with different bioclinical parameters, adherence to the Mediterranean Diet (MD) and physical activity, studying the role of lifestyle and body composition parameters on biochemical metabolic profile. METHODS: Association analysis of single variants in the genes of lipoprotein lipase (LPL), fibronectin type III domain containing protein 5 (FNDC5), and peroxisome proliferator-activated receptor-gamma (PPARγ) and haplotype analyses were performed. RESULTS: Multiple (n = 14) common variants in the three genes demonstrated a significant effect on plasma lipoprotein-lipid levels and/or on biochemical parameters in our sample. Specifically, SNPs were related to lipid metabolism (rs3866471, rs4922115, rs11570892, rs248, rs316, rs1059507, rs1801282) or glycemic profile (rs3208305) or anthropometric parameters (rs3480, rs726344, rs1570569) for a total of 26 significant associations (P < 0.01 and/or P < 0.05) and two haplotypes, for the first time, were strongly associated with lipid and body composition parameters. Interestingly, we identified twenty-four new variants not previously described in the literature and a novel significant association between rs80143795 and body composition. CONCLUSIONS: In this study we confirm the association between these SNPs on lipid metabolism and body parameters also in a young cohort, indicating the important role of these genetic factors as determinants of health.

Modeling Phenological Phases across Olive Cultivars in the Mediterranean.

Modeling phenological phases in a Mediterranean environment often implies tangible challenges to reconstructing regional trends over heterogenous areas using limited and scattered observations. The present investigation aimed to project phenological phases (i.e., sprouting, blooming, and pit hardening) for early and mid-late olive cultivars in the Mediterranean, comparing two phenological modeling approaches. Phenoflex is a rather integrated but data-demanding model, while a combined model of chill and anti-chill days and growing degree days (CAC_GDD) offers a more parsimonious and general approach in terms of data requirements for parameterization. We gathered phenological observations from nine experimental sites in Italy and temperature timeseries from the European Centre for Medium-Range Weather Forecasts, Reanalysis v5. The best performances of the CAC_GDD (RMSE: 4 days) and PhenoFlex models (RMSE: 5-9.5 days) were identified for the blooming and sprouting phases of mid-late cultivars, respectively. The CAC_GDD model was better suited to our experimental conditions for projecting pit hardening and blooming dates (correlation: 0.80 and 0.70, normalized RMSE: 0.6 and 0.8, normalized standard deviation: 0.9 and 1.0). The optimization of the principal parameters confirmed that the mid-late cultivars were more adaptable to thermal variability. The spatial distribution illustrated the near synchrony of blooming dates between the early and mid-late cultivars compared to other phases.

Cold Stress, Freezing Adaptation, Varietal Susceptibility of Olea europaea L.: A Review.

Olive (Olea europaea L.) is an evergreen xerophytic tree characterizing vegetative landscape and historical-cultural identity of the Mediterranean Basin. More than 2600 cultivars constitute the rich genetic patrimony of the species cultivated in approximately 60 countries. As a subtropical species, the olive tree is quite sensitive to low temperatures, and air temperature is the most critical environmental factor limiting olive tree growth and production. In this present review, we explored the detrimental effects caused of low temperatures on olive cultivars, and analyzed the most frequently experimental procedures used to evaluate cold stress. Then, current findings freezing stress physiology and gene are summarized in olive tree, with an emphasis on adaptive mechanisms for cold tolerance. This review might clear the way for new research on adaptive mechanisms for cold acclimation and for improvement of olive growing management.

Can Ethylene Inhibitors Enhance the Success of Olive Somatic Embryogenesis?

An efficient in vitro morphogenesis, specifically through somatic embryogenesis, is considered to be a crucial step for the application of modern biotechnological tools for genetic improvement in olive (Olea europaea L.). The effects of different ethylene inhibitors, i.e., cobalt chloride (CoCl2), salicylic acid (SA), and silver nitrate (AgNO3), were reported in the cyclic somatic embryogenesis of olive. Embryogenic callus derived from the olive immature zygotic embryos of the cultivar Leccino, was transferred to the expression ECO medium, supplemented with the ethylene inhibitors at 20 and 40 µM concentrations. Among these, the maximum number of somatic embryos (18.6) was obtained in media containing silver nitrate (40 µM), followed by cobalt chloride (12.2 somatic embryos @ 40 µM) and salicylic acid (40 µM), which produced 8.5 somatic embryos. These compounds interfered on callus traits: white friable embryogenic calli were formed in a medium supplemented with 40 µM cobalt chloride and salicylic acid; in addition, a yellow-compact embryogenic callus appeared at 20 µM of all the tested ethylene inhibitors. The resulting stimulatory action of silver nitrate among all the tested ethylene inhibitors on somatic embryogenesis, clearly demonstrates that our approach can efficiently contribute to the improvement of the current SE protocols for olive.

Algerian Olive Germplasm and Its Relationships with the Central-Western Mediterranean Varieties Contributes to Clarify Cultivated Olive Diversification.

Olive tree with its main final product, olive oil, is an important element of Mediterranean history, considered the emblematic fruit of a civilization. Despite its wide diffusion and economic and cultural importance, its evolutionary and phylogenetic history is still difficult to clarify. As part of the Mediterranean basin, Algeria was indicated as a secondary diversification center. However, genetic characterization studies from Maghreb area, are currently underrepresented. In this context, we characterized 119 endemic Algerian accessions by using 12 microsatellite markers with the main goal to evaluate the genetic diversity and population structure. In order to provide new insights about the history of olive diversification events in the Central-Western Mediterranean basin, we included and analyzed a sample of 103 Italian accessions from Sicily and, a set of molecular profiles of cultivars from the Central-Western Mediterranean area. The phylogenetic investigation let us to evaluate genetic relationships among Central-Mediterranean basin olive germplasm, highlight new synonymy cases to support the importance of vegetative propagation in the cultivated olive diffusion and consolidate the hypothesis of more recent admixture events occurrence. This work provided new information about Algerian germplasm biodiversity and contributed to clarify olive diversification process.

Association Study of the 5'UTR Intron of the FAD2-2 Gene With Oleic and Linoleic Acid Content in Olea europaea L.

Cultivated olive (Olea europaea L. subsp. europaea var. europaea) is the most ancient and spread tree crop in the Mediterranean basin. An important quality trait for the extra virgin olive oil is the fatty acid composition. In particular, a high content of oleic acid and low of linoleic, linolenic, and palmitic acid is considered very relevant in the health properties of the olive oil. The oleate desaturase enzyme encoding-gene (FAD2-2) is the main responsible for the linoleic acid content in the olive fruit mesocarp and, therefore, in the olive oil revealing to be the most important candidate gene for the linoleic acid biosynthesis. In this study, an in silico and structural analysis of the 5'UTR intron of the FAD2-2 gene was conducted with the aim to explore the natural sequence variability and its role in the gene expression regulation. In order to identify functional allele variants, the 5'UTR intron was isolated and partially sequenced in 97 olive cultivars. The sequence analysis allowed to find a 117-bp insertion including two long duplications never found before in FAD2-2 genes in olive and the existence of many intron-mediated enhancement (IME) elements. The sequence polymorphism analysis led to detect 39 SNPs. The candidate gene association study conducted for oleic and linoleic acids content revealed seven SNPs and one indel significantly associated able to explain a phenotypic variation ranging from 7% to 16% among the years. Our study highlighted new structural variants within the FAD2-2 gene in olive, putatively involved in the regulation mechanisms of gene expression associated with the variation of the content of oleic and linoleic acid.

Uncovering Olive Biodiversity through Analysis of Floral and Fruiting Biology and Assessment of Genetic Diversity of 120 Italian Cultivars with Minor or Marginal Diffusion.

The primary impetus behind this research was to provide a boost to the characterization of the Italian olive biodiversity by acquiring reliable and homogeneous data over the course of an eight-year trial on the floral and fruiting biology of 120 molecularly analyzed cultivars, most of which have either low or very low diffusion. The obtained data highlighted a considerable variability to almost all of the analyzed parameters, which given the uniformity of environment and crop management was indicative of a large genetic variability in the accessions under observation, as confirmed through the molecular analysis. Several cases of synonymy were reported for the first time, even among plants cultivated in different regions, whilst all of the varieties examined, with only one exception, showed very low percentages of self-fruit-set, indicating a need for the employment of suitable pollinator plants. Eventually, a fitted model allowed us to evaluate the clear effects of the thermal values on blossoming, particularly in the months of March and April, whereas the climatic conditions during the flowering time had only a modest effect on its duration.

Traceability of "Tuscan PGI" Extra Virgin Olive Oils by ¹H NMR Metabolic Profiles Collection and Analysis.

According to Coldiretti, Italy still continues to hold the European Quality record in extra virgin olive oils with origin designation and protected geographical indication (PDO and PGI). To date, 46 Italian brands are recognized by the European Union: 42 PDO and 4 PGI (Tuscan PGI, Calabria PGI; Tuscia PGI and PGI Sicily). Specific regulations, introduced for these quality marks, include the designation of both the geographical areas and the plant varieties contributing to the composition of the olive oil. However, the PDO and PGI assessment procedures are currently based essentially on farmer declarations. Tuscan PGI extra virgin olive oil is one of the best known Italian trademarks around the world. Tuscan PGI varietal platform is rather wide including 31 specific olive cultivars which should account for at least 95% of the product. On the other hand, while the characteristics of other popular Italian extra virgin olive oils (EVOOs) cultivars from specific geographical areas have been extensively studied (such as those of Coratina based blends from Apulia), little is still known about Tuscan PGI EVOO constituents. In this work, we performed, for the first time, a large-scale analysis of Tuscan PGI monocultivar olive oils by ¹H NMR spectroscopy and multivariate statistical analyses (MVA). After genetic characterization of 217 leaf samples from 24 selected geographical areas, distributed all over the Tuscany, a number of 202 micro-milled oil samples including 10 PGI cultivars, was studied. The results of the present work confirmed the need of monocultivar genetically certified EVOO samples for the construction of ¹H-NMR-metabolic profiles databases suitable for cultivar and/or geographical origin assessment. Such specific PGI EVOOs databases could be profitably used to justify the high added value of the product and the sustainability of the related supply chain.

GBS-derived SNP catalogue unveiled wide genetic variability and geographical relationships of Italian olive cultivars.

Information on the distribution of genetic variation is essential to preserve olive germplasm from erosion and to recover alleles lost through selective breeding. In addition, knowledge on population structure and genotype-phenotype associations is crucial to support modern olive breeding programs that must respond to new environmental conditions imposed by climate change and novel biotic/abiotic stressors. To further our understanding of genetic variation in the olive, we performed genotype-by-sequencing on a panel of 94 Italian olive cultivars. A reference-based and a reference-independent SNP calling pipeline generated 22,088 and 8,088 high-quality SNPs, respectively. Both datasets were used to model population structure via parametric and non parametric clustering. Although the two pipelines yielded a 3-fold difference in the number of SNPs, both described wide genetic variability among our study panel and allowed individuals to be grouped based on fruit weight and the geographical area of cultivation. Multidimensional scaling analysis on identity-by-state allele-sharing values as well as inference of population mixtures from genome-wide allele frequency data corroborated the clustering pattern we observed. These findings allowed us to formulate hypotheses about geographical relationships of Italian olive cultivars and to confirm known and uncover novel cases of synonymy.

Cultivar classification of Apulian olive oils: Use of artificial neural networks for comparing NMR, NIR and merceological data.

The development of an efficient and accurate method for extra-virgin olive oils cultivar and origin authentication is complicated by the broad range of variables (e.g., multiplicity of varieties, pedo-climatic aspects, production and storage conditions) influencing their properties. In this study, artificial neural networks (ANNs) were applied on several analytical datasets, namely standard merceological parameters, near-infra red data and 1H nuclear magnetic resonance (NMR) fingerprints, obtained on mono-cultivar olive oils of four representative Apulian varieties (Coratina, Ogliarola, Cima di Mola, Peranzana). We analyzed 888 samples produced at a laboratory-scale during two crop years from 444 plants, whose variety was genetically ascertained, and on 17 industrially produced samples. ANN models based on NMR data showed the highest capability to classify cultivars (in some cases, accuracy>99%), independently on the olive oil production process and year; hence, the NMR data resulted to be the most informative variables about the cultivars.

New Technologies for Insect-Resistant and Herbicide-Tolerant Plants.

The advent of modern molecular biology and recombinant DNA technology has resulted in a dramatic increase in the number of insect-resistant (IR) and herbicide-tolerant (HT) plant varieties, with great economic benefits for farmers. Nevertheless, the high selection pressure generated by control strategies for weed and insect populations has led to the evolution of herbicide and pesticide resistance. In the short term, the development of new techniques or the improvement of existing ones will provide further instruments to counter the appearance of resistant weeds and insects and to reduce the use of agrochemicals. In this review, we examine some of the most promising new technologies for developing IR and HT plants, such as genome editing and antisense technologies.

Fad7 gene identification and fatty acids phenotypic variation in an olive collection by EcoTILLING and sequencing approaches.

The ω-3 fatty acid desaturases (FADs) are enzymes responsible for catalyzing the conversion of linoleic acid to α-linolenic acid localized in the plastid or in the endoplasmic reticulum. In this research we report the genotypic and phenotypic variation of Italian Olea europaea L. germoplasm for the fatty acid composition. The phenotypic oil characterization was followed by the molecular analysis of the plastidial-type ω-3 FAD gene (fad7) (EC 1.14.19), whose full-length sequence has been here identified in cultivar Leccino. The gene consisted of 2635 bp with 8 exons and 5'- and 3'-UTRs of 336 and 282 bp respectively, and showed a high level of heterozygousity (1/110 bp). The natural allelic variation was investigated both by a LiCOR EcoTILLING assay and the PCR product direct sequencing. Only three haplotypes were identified among the 96 analysed cultivars, highlighting the strong degree of conservation of this gene.

Recovery of useful traits from isolates inhabiting an agricultural soil cultivated with herbicide-resistant poplars.

The aim of this study was to investigate the culturable bacteria living in soil cultivated with Basta-tolerant transgenic white poplars (Populus alba L. 'Villafranca'). Plate Count Agar medium containing phosphinothricin, the active component of Basta, was used to isolate the herbicide-resistant bacteria (HRB). No significant changes in the size of the soil microbial flora following herbicide treatment were observed. The characterization of HRB isolates by 16S rDNA-based taxonomy revealed a predominance of Pseudomonas and Bacillus species. The screening carried out on soil samples allowed for the recovery of isolates with useful properties for biotechnological and agronomical purposes, particularly in relation to root development. Among the tested isolates, only HRB-1b, HRB-1c, and HRB-7 showed remarkable swarming ability, a valuable trait supporting the beneficial plant-microbe interactions. HRB-1c was also characterized by consistent production of indoleacetic acid (17.8 +/- 0.09 microg x mL-1 x (OD600 unit)-1), and it was able to stimulate the in vitro growth of Villafranca explants. Since novel tools are constantly required to enhance productivity of perennial species and to expand their use for practical purposes, the availability of bacteria that support tree growth, such as the HRB-1c isolate, represents a significant advantage.