Functional differentiation of olive PLP_deC genes: insights into metabolite biosynthesis and genetic improvement at the whole-genome level.

KEY MESSAGE: This study identified 16 pyridoxal phosphate-dependent decarboxylases in olive at the whole-genome level, conducted analyses on their physicochemical properties, evolutionary relationships and characterized their activity. Group II pyridoxal phosphate-dependent decarboxylases (PLP_deC II) mediate the biosynthesis of characteristic olive metabolites, such as oleuropein and hydroxytyrosol. However, there have been no report on the functional differentiation of this gene family at the whole-genome level. This study conducted an exploration of the family members of PLP_deC II at the whole-genome level, identified 16 PLP_deC II genes, and analyzed their gene structure, physicochemical properties, cis-acting elements, phylogenetic evolution, and gene expression patterns. Prokaryotic expression and enzyme activity assays revealed that OeAAD2 and OeAAD4 could catalyze the decarboxylation reaction of tyrosine and dopa, resulting in the formation of their respective amine compounds, but it did not catalyze phenylalanine and tryptophan. Which is an important step in the synthetic pathway of hydroxytyrosol and oleuropein. This finding established the foundational data at the molecular level for studying the functional aspects of the olive PLP_deC II gene family and provided essential gene information for genetic improvement of olive.

Autumn Olive (Elaeagnus umbellata Thunb.) Berries Improve Lipid Metabolism and Delay Aging in Middle-Aged Caenorhabditis elegans.

This study evaluated the positive effects of autumn olive berries (AOBs) extract on delaying aging by improving lipid metabolism in middle-aged Caenorhabditis elegans that had become obese due to a high-glucose (GLU) diet. The total phenolic content and DPPH radical scavenging abilities of freeze-dried AOBs (FAOBs) or spray-dried AOBs (SAOBs) were examined, and FAOBs exhibited better antioxidant activity. HPLC analysis confirmed that catechin is the main phenolic compound of AOBs; its content was 5.95 times higher in FAOBs than in SAOBs. Therefore, FAOBs were used in subsequent in vivo experiments. FAOBs inhibited lipid accumulation in both the young adult and middle-aged groups in a concentration-dependent manner under both normal and 2% GLU conditions. Additionally, FAOBs inhibited ROS accumulation in a concentration-dependent manner under normal and 2% GLU conditions in the middle-aged worms. In particular, FAOB also increased body bending and egg production in middle-aged worms. To confirm the intervention of genetic factors related to lipid metabolism from the effects of FAOB, body lipid accumulation was confirmed using worms deficient in the daf-16, atgl-1, aak-1, and akt-1 genes. Regarding the effect of FAOB on reducing lipid accumulation, the impact was nullified in daf-16-deficient worms under the 2% GLU condition, and nullified in both the daf-16- and atgl-1-deficient worms under fasting conditions. In conclusion, FAOB mediated daf-16 and atgl-1 to regulate lipogenesis and lipolysis in middle-aged worms. Our findings suggest that FAOB improves lipid metabolism in metabolically impaired middle-aged worms, contributing to its age-delaying effect.

Leaf Extract from European Olive (Olea europaea L.) Post-Transcriptionally Suppresses the Epithelial-Mesenchymal Transition and Sensitizes Gastric Cancer Cells to Chemotherapy.

The overall survival of patients with the advanced and recurrent gastric cancer (GC) remains unfavorable. In particular, this is due to cancer spreading and resistance to chemotherapy associated with the epithelial-mesenchymal transition (EMT) of tumor cells. EMT can be identified by the transcriptome profiling of GC for EMT markers. Indeed, analysis of the TCGA and GTEx databases (n = 408) and a cohort of GC patients (n = 43) revealed that expression of the CDH2 gene was significantly decreased in the tumors vs. non-tumor tissues and correlated with the overall survival of GC patients. Expression of the EMT-promoting transcription factors SNAIL and ZEB1 was significantly increased in GC. These data suggest that targeting the EMT might be an attractive therapeutic approach for patients with GC. Previously, we demonstrated a potent anti-cancer activity of the olive leaf extract (OLE). However, its effect on the EMT regulation in GC remained unknown. Here, we showed that OLE efficiently potentiated the inhibitory effect of the chemotherapeutic agents 5-fluorouracil (5-FU) and cisplatin (Cis) on the EMT and their pro-apoptotic activity, as was demonstrated by changes in the expression of the EMT markers (E- and N-cadherins, vimentin, claudin-1) in GC cells treated with the aforementioned chemotherapeutic agents in the presence of OLE. Thus, culturing GC cells with 5-FU + OLE or Cis + OLE attenuated the invasive properties of cancer cells. Importantly, upregulation of expression of the apoptotic markers (PARP cleaved form) and increase in the number of cells undergoing apoptosis (annexin V-positive) were observed for GC cells treated with a combination of OLE and 5-FU or Cis. Collectively, our data illustrate that OLE efficiently interferes with the EMT in GC cells and potentiates the pro-apoptotic activity of certain chemotherapeutic agents used for GC therapy.

The hepatoprotective potentials of Olea europaea L. leaves against carbon tetrachloride-induced hepatic injury in rats.

OBJECTIVE: To examine the therapeutic effects of Olea europaea L. leaves extract on carbon tetrachloride-induced liver injury in rats. Methods: The experimental study was conducted at the Department of Physiology, University of Karachi, Karachi, in July 2021, and comprised Albino Wistar male rats weighing 180-220gm. The animals were divided into control group I, carbon tetrachloride group II, Olea europaea L. + carbon tetrachloride group III and Olea europaea L. group IV. In Vitro model of hepatic toxicity was developed by carbon tetrachloride. A daily dose of 50mg/kg of aqueous extract of olive leaves was administered orally and 0.8ml/kg of carbon tetrachloride was administered twice a week subcutaneously for 28 days. On the 29th day, the animals were sacrificed, and tested for hepatic enzymes, lipid peroxidation markers and histopathology. Data was analysed using SPSS 20. RESULTS: Of the 24 rats, 6(25%) were in each of the 4 groups. Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and total bilirubin levels were significantly reduced (p<0.05) in group II whereas, 4- hydroxynonenal, isoprostane and malondialdehyde levels were significantly increased (p<0.05). However, total antioxidant level increased significantly (p<0.05) in group III compared to group II. Histopathology showed severe liver damage in group II and mild damage in group III. Conclusion: Olea europaea L. leaves extract was found to have profound hepatoprotective effects.

A mathematical model of biofilm growth and spread within plant xylem: Case study of Xylella fastidiosa in olive trees.

Xylem-limited bacterial pathogens cause some of the most destructive plant diseases. Though imposed measures to control these pathogens are generally ineffective, even among susceptible taxa, some hosts can limit bacterial loads and symptom expression. Mechanisms by which this resistance is achieved are poorly understood. In particular, it is still unknown how differences in vascular structure may influence biofilm growth and spread within a host. To address this, we developed a novel theoretical framework to describe biofilm behaviour within xylem vessels, adopting a polymer-based modelling approach. We then parameterised the model to investigate the relevance of xylem vessel diameters on Xylella fastidiosa resistance among olive cultivars. The functionality of all vessels was severely reduced under infection, with hydraulic flow reductions of 2-3 orders of magnitude. However, results suggest wider vessels act as biofilm incubators; allowing biofilms to develop over a long time while still transporting them through the vasculature. By contrast, thinner vessels become blocked much earlier, limiting biofilm spread. Using experimental data on vessel diameter distributions, we were able to determine that a mechanism of resistance in the olive cultivar Leccino is a relatively low abundance of the widest vessels, limiting X. fastidiosa spread.

Highly Efficient Verbascoside Production from Olive (Olea europea L. var. Cellina di Nardò) In Vitro Cell Cultures.

Olive (Olea europea L.) is one of the oldest and most important fruit tree species cultivated in the Mediterranean region. Various plant tissues, drupes, and olive oil contain several phenolics (including verbascoside, although it is present in the plant at a low level) that are well-known for their highly beneficial effects on human health. An in vitro olive cell suspension culture (cultivar Cellina di Nardò, "CdN") was established, characterized for its growth and morphological features. Furthermore, a vital and relatively uniform population of protoplasts was generated from the olive suspension culture to investigate their cellular characteristics during growth. The polyphenolic extract of the in vitro "CdN" olive cells contained almost exclusively verbascoside, as revealed by the UPLC-ESI-MS analysis. The content of verbascoside reached up to 100 mg/g DW, with an average production rate of approximately 50 mg/g DW over one year of culture. This level of production has not been previously reported in a limited number of previous studies. This remarkable production of verbascoside was associated with an exceptionally high antioxidant capacity. The high level of verbascoside production and purity of the extract make this system a promising tool for secondary metabolite production.

Ophthalmic wild olive (ACEBUCHE) oil nanoemulsions exert oculoprotective effects against oxidative stress induced by arterial hypertension.

Oxidative stress plays a key role in several systemic and ocular diseases, including hypertensive eye diseases. In this context, we previously showed that oral administration of wild olive (acebuche, ACE) oil from Olea europaea var. sylvestris can counteract ocular damage secondary to arterial hypertension by modulating excess reactive oxygen species (ROS) produced by the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Therefore, this work describes the development of an ACE oil-based formulation for ocular administration as a local therapy to counteract hypertension-related oxidative damage. Specifically, ACE oil nanoemulsions (NEs) were successfully produced and characterized, exhibiting appropriate features for ophthalmic administration, including a nanometer size (<200 nm), moderate negative ZP, adequate osmolality and pH, and colloidal stability in biorelevant fluids. Likewise, the NEs presented a shear thinning behavior, especially convenient for ocular instillation. In vivo evaluation was performed through either intravitreal injection or topical ophthalmic administration in mice with hypertension induced via administration of Nω-nitro-L-arginine-methyl-ester (L-NAME). Both routes of administration reduced hypertensive morphological alterations and demonstrated a noticeable antioxidant effect thanks to the reduction of the activity/expression of NADPH oxidase in cornea and retina. Thus, an ACE oil ophthalmic formulation represent a promising therapy for ocular pathologies associated with arterial hypertension.

Bacillus G7 improves adaptation to salt stress in Olea europaea L. plantlets, enhancing water use efficiency and preventing oxidative stress.

In addition to genetic adaptative mechanisms, plants retrieve additional help from the surrounding microbiome, especially beneficial bacterial strains (PGPB) that contribute to plant fitness by modulating plant physiology to fine-tune adaptation to environmental changes. The aim of this study was to determine the mechanisms by which the PGPB Bacillus G7 stimulates the adaptive mechanisms of Olea europaea plantlets to high-salinity conditions, exploring changes at the physiological, metabolic and gene expression levels. On the one hand, G7 prevented photosynthetic imbalance under saline stress, increasing the maximum photosynthetic efficiency of photosystem II (Fv/Fm) and energy dissipation (NPQ) and protecting against photooxidative stress. On the other hand, despite the decrease in effective PSII quantum yield (ΦPSII), net carbon fixation was significantly improved, resulting in significant increases in osmolytes and antioxidants, suggesting an improvement in the use of absorbed energy. Water use efficiency (WUE) was significantly improved. Strong genetic reprogramming was evidenced by the transcriptome that revealed involvement of the ABA-mediated pathway based on upregulation of ABA synthesis- and ABA-sensing-related genes together with a strong downregulation of the PLC2 phosphatase family, repressors of ABA-response elements and upregulation of ion homeostasis-related genes. The ion homeostasis response was activated faster in G7-treated plants, as suggested by qPCR data. All these results reveal the multitargeted improvement of plant metabolism under salt stress by Bacillus G7, which allows growth under water limitation conditions, an excellent trait to develop biofertilizers for agriculture under harsh conditions supporting the use of biofertilizers among the new farming practices to meet the increasing demand for food.

Non-Targeted Spectranomics for the Early Detection of Xylella fastidiosa Infection in Asymptomatic Olive Trees, cv. Cellina di Nardò.

Olive quick decline syndrome (OQDS) is a disease that has been seriously affecting olive trees in southern Italy since around 2009. During the disease, caused by Xylella fastidiosa subsp. pauca sequence type ST53 (Xf), the flow of water and nutrients within the trees is significantly compromised. Initially, infected trees may not show any symptoms, making early detection challenging. In this study, young artificially infected plants of the susceptible cultivar Cellina di Nardò were grown in a controlled environment and co-inoculated with additional xylem-inhabiting fungi. Asymptomatic leaves of olive plants at an early stage of infection were collected and analyzed using nuclear magnetic resonance (NMR), hyperspectral reflectance (HSR), and chemometrics. The application of a spectranomic approach contributed to shedding light on the relationship between the presence of specific hydrosoluble metabolites and the optical properties of both asymptomatic Xf-infected and non-infected olive leaves. Significant correlations between wavebands located in the range of 530-560 nm and 1380-1470 nm, and the following metabolites were found to be indicative of Xf infection: malic acid, fructose, sucrose, oleuropein derivatives, and formic acid. This information is the key to the development of HSR-based sensors capable of early detection of Xf infections in olive trees.

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.

Identification and characterization of long noncoding RNAs in two contrasting olive (Olea europaea L.) genotypes subjected to aluminum toxicity.

Aluminum (Al) toxcity is considered to be the primary factor limiting crop productivity in acidic soil. Many studies indicate that long non-coding RNAs (lncRNAs) fulfil a crucial role in plant growth and responses to different abiotic stress. However, identification and characterization of lncRNAs responsive to Al stress at a genome-wide level in olive tree is still lacking. Here, we performed comparative analysis on lncRNA transcriptome between Zhonglan (an Al-tolerant genotype) and Frantoio selezione (Al-sensitive) responding to Al exposure. A total of 19,498 novel lncRNAs were identified from both genotypes, and 6900 lncRNA-target pairs were identified as cis-acting and 2311 supposed to be trans-acting. Among them, 2076 lncRNAs were appraised as Al tolerance-associated lncRNAs due to their distinctly genotype-specific expression profiles under Al exposure. Target prediction and functional analyses revealed several key lncRNAs are related to genes encoding pectinesterases, xyloglucan endotransglucosylase/hydrolase, WRKY and MYB transcription factors, which mainly participate in the modification of cell wall for Al tolerance. Furthermore, gene co-expression network analysis showed 8 lncRNA-mRNA-miRNA modules participate in transcriptional regulation of downstream Al resistant genes. Our findings increased our understanding about the function of lncRNAs in responding to Al stress in olive and identified potential promising lncRNAs for further investigation.

Biochemical characterization of an alkaline and detergent-stable Lipase from Fusarium annulatum Bugnicourt strain CBS associated with olive tree dieback.

This work describes a novel extracellular lipolytic carboxylester hydrolase named FAL, with lipase and phospholipase A1 (PLA1) activity, from a newly isolated filamentous fungus Ascomycota CBS strain, identified as Fusarium annulatum Bunigcourt. FAL was purified to about 62-fold using ammonium sulphate precipitation, Superdex® 200 Increase gel filtration and Q-Sepharose Fast Flow columns, with a total yield of 21%. The specific activity of FAL was found to be 3500 U/mg at pH 9 and 40°C and 5000 U/mg at pH 11 and 45°C, on emulsions of triocanoin and egg yolk phosphatidylcholine, respectively. SDS-PAGE and zymography analysis estimated the molecular weight of FAL to be 33 kDa. FAL was shown to be a PLA1 with a regioselectivity to the sn-1 position of surface-coated phospholipids esterified with α-eleostearic acid. FAL is a serine enzyme since its activity on triglycerides and phospholipids was completely inhibited by the lipase inhibitor Orlistat (40 µM). Interestingly, compared to Fusarium graminearum lipase (GZEL) and the Thermomyces lanuginosus lipase (Lipolase®), this novel fungal (phospho)lipase showed extreme tolerance to the presence of non-polar organic solvents, non-ionic and anionic surfactants, and oxidants, in addition to significant compatibility and stability with some available laundry detergents. The analysis of washing performance showed that it has the capability to efficiently eliminate oil-stains. Overall, FAL could be an ideal choice for application in detergents.

Modulation of Anthocyanin Biosynthesis-Related Genes during the Ripening of Olea europaea L. cvs Carolea and Tondina Drupes in Relation to Environmental Factors.

Anthocyanins protect plants against various biotic and abiotic stresses, and anthocyanin-rich foods exert benefits on human health due to their antioxidant activity. Nevertheless, little information is available on the influence of genetic and environmental factors on the anthocyanin content in olive fruits. Based on this consideration, the total anthocyanin content, the genes involved in anthocyanin biosynthesis, and three putative R2R3-MYB transcription factors were evaluated at different ripening stages in the drupes of the Carolea and Tondina cultivars, sampled at different altitudes in the Calabria region, Italy. During drupe ripening, the total anthocyanin content and the transcript levels of analyzed genes gradually increased. In line with the anthocyanin content, a different level of expression of anthocyanin structural genes was observed in 'Carolea' compared to 'Tondina', and in relation to the cultivation area. Furthermore, we identified Oeu050989.1 as a putative R2R3-MYB involved in the regulation of anthocyanin structural genes correlated with the environmental temperature change response. We conclude that anthocyanin accumulation is strongly regulated by development, genotype, and also by environmental factors such as temperature, associated with the altitude gradient. The obtained results contribute to reducing the current information gap regarding the molecular mechanisms on anthocyanin biosynthesis regulation related to the environmental conditions in Olea europaea.

Continuous Production of Dietetic Structured Lipids Using Crude Acidic Olive Pomace Oils.

Crude olive pomace oil (OPO) is a by-product of olive oil extraction. In this study, low-calorie structured triacylglycerols (TAGs) were produced by acidolysis of crude OPO with medium-chain fatty acids (caprylic, C8:0; capric, C10:0) or interesterification with their ethyl ester forms (C8EE, C10EE). These new TAGs present long-chain fatty acids (L) at position sn-2 and medium-chain fatty acids (M) at positions sn-1,3 (MLM). Crude OPO exhibited a high acidity (12.05-28.75% free fatty acids), and high contents of chlorophylls and oxidation products. Reactions were carried out continuously in a packed-bed bioreactor for 70 h, using sn-1,3 regioselective commercial immobilized lipases (Thermomyces lanuginosus lipase, Lipozyme TL IM; and Rhizomucor miehei lipase, Lipozyme RM IM), in solvent-free media at 40 °C. Lipozyme RM IM presented a higher affinity for C10:0 and C10EE. Lipozyme TL IM preferred C10:0 over C8:0 but C8EE over C10EE. Both biocatalysts showed a high activity and operational stability and were not affected by OPO acidity. The New TAG yields ranged 30-60 and the specific productivity ranged 0.96-1.87 g NewTAG/h.g biocatalyst. Lipozyme RM IM cost is more than seven-fold the Lipozyme TL IM cost. Therefore, using Lipozyme TL IM and crude acidic OPO in a continuous bioreactor will contribute to process sustainability for structured lipid production by lowering the cost of the biocatalyst and avoiding oil refining.

Olive pectin-chitosan nanocomplexes for improving stability and bioavailability of blueberry anthocyanins.

Blueberry anthocyanins (ANCs) are natural dietary bioactive colorants, but are unstable and easily degraded. To improve their stability, we constructed the nanocarriers for ANCs through an electrostatic self-assembly method, using chitosan (CS) and olive pectin (PC). Results showed that the CS-ANCs-PC nanocomplexes had nanoscale particle size (81.22 ± 0.44 nm), and an encapsulation efficiency of 91.97 ± 0.33% at pH 3.0, 1:1:5 ratio (m/v) of CS: ANCs: PC. Fourier transform infrared and UV-visible spectra demonstrated that ANCs can be embedded into the CS-PC carrier through electrostatic interaction. CS-ANCs-PC with stacked spherical particle structure had good thermal stability by scanning electron microscope and thermogravimetric analysis. Compared with free anthocyanins, CS-ANCs-PC possessed better DPPH· and ·OH scavenging activities, stronger environmental stability, and better targeted release in vitro digestion. This study may provide an important fundamental basis for improving the stability of anthocyanins in the blueberry industry.

Effect of micronization on olive pomace biotransformation in the static model of colonic fermentation.

The effect of granulometric fractionation and micronization of olive pomace (OP) on the biotransformation of phenolic compounds by intestinal microbiota was investigated in vitro. Three types of powdered OP samples were incubated with human feces to simulate colonic fermentation, after a sequential static digestion: non-fractionated OP (NF), granulometrically fractionated OP (GF) and granulometrically fractionated and micronized OP (GFM). GF and GFM favored the release of hydroxytyrosol, oleuropein aglycone, apigenin and phenolic acid metabolites in the first hours of colonic fermentation compared to NF (up to 41-fold higher). GFM caused higher release of hydroxytyrosol than GF. GFM was the only sample to release tyrosol and sustained tyrosol levels up to 24 h of fermentation. Micronization associated with granulometric fractionation was more efficient than granulometric fractionation alone to increase the release of phenolic compounds from the OP matrix during simulated colonic fermentation and can be further studied for nutraceutical purposes.

The impact of biosynthesized ZnO nanoparticles from Olea europaea (Common Olive) on Pseudomonas aeruginosa growth and biofilm formation.

There is a limitation in the range of effectual antibiotics due to the Pseudomonas aeruginosa (PA) infection due to its innate antimicrobial resistance. Researchers have therefore been concentrating their efforts to discover advanced and cost effective antibacterial agents among the ever-increasing PA bacterial resistance strains. It has been discovered that various nanoparticles can be employed as antimicrobial agents. Here, we evaluated the antibacterial properties of the Zinc Oxide nanoparticles (ZnO NPs), which was biosynthesized, being examined on six hospital strains of PA alongside a reference strain (ATCC 27853). A chemical approach was applied to biosynthesize the ZnO NPs from Olea europaea was performed, and confirmed by using X-ray diffraction and Scanning Electron Microscopes. The nanoparticles then applied their antibacterial properties to examine them against six clinically isolated PA strains alongside the reference strain. This process tested for the results of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). The Growth, biofilm formation and eradication were analyzed. The influence of the differentiating degrees ZnO NPs in regard to Quorom sensing gene expression were further examined. The ZnO NPs exhibited a crystalline size and diameter (Dc) of 40-60 nm and both the MIC and MBC tests revealed positive outcomes of concentrations of 3 and 6 mg/ml for each PA strain, respectively. At sub inhibitory concentration, The ZnO NPs were found to significantly inhibit the growth and biofilm formation of all PA strains and decreases in the biomass and metabolic behavior of PA established biofilms; these decreases varied depending on the dosage. At ZnO NPs concentrations of 900 µg/ml, the expression of majority of quorum sensing genes of all strains were significantly reduced, at ZnO NPs concentrations of 300 µg/ml, few genes were significantly impacted. In conclusion, the treatment of PA and could be other antibiotic resistant bacteria can therefore be approached by using ZnO NPs as it has been uncovered that they withhold advanced antibacterial properties.

Analysis of Antioxidant and Antiviral Effects of Olive (Olea europaea L.) Leaf Extracts and Pure Compound Using Cancer Cell Model.

The present study aims to assess the antioxidant and antiviral effectiveness of leaf extracts obtained from Olea europaea L. var. sativa and Olea europaea L. var. sylvestris. The total antioxidant activity was determined via both an ammonium phosphomolybdate assay and a nitric oxide radical inhibition assay. Both extracts showed reducing abilities in an in vitro system and in human HeLa cells. Indeed, after oxidative stress induction, we found that exposition to olive leaf extracts protects human HeLa cells from lipid peroxidation and increases the concentration of enzyme antioxidants such as catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase. Additionally, OESA treatment affects viral DNA accumulation more than OESY, probably due to the exclusive oleuropein content. In fact, subtoxic concentrations of oleuropein inhibit HSV-1 replication, stimulating the phosphorylation of PKR, c-FOS, and c-JUN proteins. These results provide new knowledge about the potential health benefits and mechanisms of action of oleuropein and oleuropein-rich extracts.

Characterization of Transcriptome Dynamics during Early Fruit Development in Olive (Olea europaea L.).

In the olive (Olea europaea L.), an economically leading oil crop worldwide, fruit size and yield are determined by the early stages of fruit development. However, few detailed analyses of this stage of fruit development are available. This study offers an extensive characterization of the various processes involved in early olive fruit growth (cell division, cell cycle regulation, and cell expansion). For this, cytological, hormonal, and transcriptional changes characterizing the phases of early fruit development were analyzed in olive fruit of the cv. 'Picual'. First, the surface area and mitotic activity (by flow cytometry) of fruit cells were investigated during early olive fruit development, from 0 to 42 days post-anthesis (DPA). The results demonstrate that the cell division phase extends up to 21 DPA, during which the maximal proportion of 4C cells in olive fruits was reached at 14 DPA, indicating that intensive cell division was activated in olive fruits at that time. Subsequently, fruit cell expansion lasted as long as 3 weeks more before endocarp lignification. Finally, the molecular mechanisms controlling the early fruit development were investigated by analyzing the transcriptome of olive flowers at anthesis (fruit set) as well as olive fruits at 14 DPA (cell division phase) and at 28 DPA (cell expansion phase). Sequential induction of the cell cycle regulating genes is associated with the upregulation of genes involved in cell wall remodeling and ion fluxes, and with a shift in plant hormone metabolism and signaling genes during early olive fruit development. This occurs together with transcriptional activity of subtilisin-like protease proteins together with transcription factors potentially involved in early fruit growth signaling. This gene expression profile, together with hormonal regulators, offers new insights for understanding the processes that regulate cell division and expansion, and ultimately fruit yield and olive size.

RNA Polymerases IV and V Are Involved in Olive Fruit Development.

Transcription is carried out in most eukaryotes by three multimeric complexes (RNA polymerases I, II and III). However, plants contain two additional RNA polymerases (IV and V), which have evolved from RNA polymerase II. RNA polymerases II, IV and V contain both common and specific subunits that may specialise some of their functions. In this study, we conducted a search for the genes that putatively code for the specific subunits of RNA polymerases IV and V, as well as those corresponding to RNA polymerase II in olive trees. Based on the homology with the genes of Arabidopsis thaliana, we identified 13 genes that putatively code for the specific subunits of polymerases IV and V, and 16 genes that code for the corresponding specific subunits of polymerase II in olives. The transcriptomic analysis by RNA-Seq revealed that the expression of the RNA polymerases IV and V genes was induced during the initial stages of fruit development. Given that RNA polymerases IV and V are involved in the transcription of long non-coding RNAs, we investigated their expression and observed relevant changes in the expression of this type of RNAs. Particularly, the expression of the intergenic and intronic long non-coding RNAs tended to increase in the early steps of fruit development, suggesting their potential role in this process. The positive correlation between the expression of RNA polymerases IV and V subunits and the expression of non-coding RNAs supports the hypothesis that RNA polymerases IV and V may play a role in fruit development through the synthesis of this type of RNAs.