Functional and Physiological Characterization of Tyrosine Decarboxylases from Olea europaea L. Involved in the Synthesis of the Main Phenolics in Olive Fruit and Virgin Olive Oil.

The phenolic composition of virgin olive oil (VOO) primarily depends on the phenolic content of the olive fruit. The purpose of this work was to characterize the first metabolic step in the synthesis of tyrosol (Ty) and hydroxytyrosol (HTy), whose derivatives are by far the predominant phenolics in both olive fruit and VOO. To this end, two genes encoding tyrosine/DOPA decarboxylase enzymes, OeTDC1 and OeTDC2, have been identified and functionally and physiologically characterized. Both olive TDC proteins exclusively accept aromatic amino acids with phenolic side chains, such as tyrosine and 3,4-dihydroxyphenylalanine (DOPA), as substrates to produce tyramine and dopamine, respectively. These proteins exhibited a higher affinity for DOPA than for tyrosine, and the catalytic efficiency of both proteins was greater when DOPA was used as a substrate. Both olive TDC genes showed a fairly similar expression profile during olive fruit ontogeny, with OeTDC1 consistently expressed at higher levels than OeTDC2. Expression was particularly intense during the first few weeks after fruit set, coinciding with the active accumulation of Ty and HTy derivatives. The data suggest that both olive TDCs are responsible for the initial step in the synthesis of the most important phenolics, both quantitatively and functionally, in VOO.

Study of the olive ß-glucosidase gene family putatively involved in the synthesis of phenolic compounds of virgin olive oil.

BACKGROUND: Hydrolysis of the fruit phenolic glucosides occurring during the oil extraction process is the main biochemical reaction affecting the biosynthesis and accumulation of secoiridoid compounds in virgin olive oil. An integrated approach at the molecular, biochemical, and metabolic level was used to study the olive ß-glucosidase gene family in seven olive cultivars selected by their different phenolic profiles. RESULTS: Eight ß-glucosidase genes have been identified by in silico analysis of an olive transcriptome. Their expression levels were analyzed by reverse transcription quantitative polymerase chain reaction in olive fruits at different ripening stages: I, green fruits, 16-19 weeks after flowering (WAF); II, yellow-green fruits, 22-25 WAF; III, turning fruits, 28-31 WAF; and IV, fully ripe fruits, 35-40 WAF. Gene expression was compared with the level of ß-glucosidase activity in the fruit and with the phenolic composition of fruits and oils from different olive cultivars. Phylogenetic analysis of the encoded proteins and differences found among the ß-glucosidase genes based on Gene Ontology enrichment analysis data suggests maximum involvement of two genes, OeBGLU1A and OeBGLU1B, in the phenolic composition of virgin olive oil. Positive correlation coefficients were found within each olive cultivar between OeBGLU1A and OeBGLU1B gene expression data and the phenolic content of the oil. CONCLUSION: The results obtained suggest that the expression pattern of specific ß-glucosidase genes may be an accurate predictor for the phenolic content of virgin olive oil that could be used in olive breeding programs. © 2021 Society of Chemical Industry.

Specialized Functions of Olive FAD2 Gene Family Members Related to Fruit Development and the Abiotic Stress Response.

Three different cDNA sequences, designated OepFAD2-3, OepFAD2-4 and OepFAD2-5, encoding three microsomal oleate desaturases (FAD2) have been isolated from olive (Olea europaea cv. Picual). Sequence analysis and functional expression in yeast of the corresponding cDNAs confirm that they encode microsomal oleate desaturases. Gene expression and lipid analysis indicate that these three genes are not involved in the linoleic acid present in seed lipids, while OeFAD2-5, together with OeFAD2-2, contributes mostly to the linoleic acid present in the mesocarp and, therefore, in the olive oil. Our results have also shown that olive FAD2-3, FAD2-4 and FAD2-5 gene expression is not only spatially and temporally regulated in olive fruit, but also is cultivar-dependent, as well as regulated by water regime, temperature, light and wounding. All these data suggest specialized physiological roles for the olive FAD2 gene family members with respect to both aspects of the biosynthesis of the linoleic acid, either present in storage lipids that constitute the olive oil or being part of membrane lipids, which are involved in the response to abiotic stresses, and highlight the differences on FAD2 gene regulation between oilseeds and oil fruits.

Enzyme and Biological Activities of the Water Extracts from the Plants Aesculus hippocastanum, Olea europaea and Hypericum perforatum That Are Used as Folk Remedies in Turkey.

Phenolic compounds are secondary metabolites that are found ubiquitously in plants, fruits, and vegetables. Many studies have shown that regular consumption of these compounds could have a positive effect on our health. The aim of this study was to compare the phytochemical contents of the water extracts from three different plants used as folk remedies in Turkey: Aesculus hippocastanum, Olea europaea, and Hypericum perforatum. A liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) analysis was performed to explore the phenolic profiles. The biological activities of these extracts were also evaluated in terms of their antioxidant activities (2,2-diphenyl-1-picrylhydrazyl DPPH, 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid ABTS, Ferric Reducing Antioxidant Power Assay FRAP, cupric ion reducing antioxidant capacity CUPRAC, ß-carotene, phosphomolybdenum, and metal chelating) and enzyme inhibitory properties (against acetylcholinesterase, butyrylcholinesterase, and tyrosinase). The aqueous extract of H. perforatum showed the highest levels of total phenolic, flavonoid, and saponin contents. Protocatechuic acid, vanillic acid, verbascoside, hesperidin, hyperoside, apigenin 7-hexosides, and quercetin were the most common compounds found in this species. The results confirm that A. hippocastanum, O. europaea, and H. perforatum represent a potential source of natural-derived molecules with positive properties that could be used as valid starting point for new food supplements, and drugs in the pharmaceutical, cosmetic, and food industries.

Two novel methylesterases from Olea europaea contribute to the catabolism of oleoside-type secoiridoid esters.

MAIN CONCLUSION: Two newly identified phytohormone cleaving esterases from Olea europaea are responsible for the glucosidase-initiated activation of the specialized metabolites ligstroside and oleuropein. Biosynthetic routes leading to the formation of plant natural products are tightly orchestrated enzymatic sequences usually involving numerous specialized catalysts. After their accumulation in plant cells and tissues, otherwise non-reactive compounds can be enzymatically activated, e.g., in response to environmental threats, like pathogen attack. In olive (Olea europaea), secoiridoid-derived phenolics, such as oleuropein or ligstroside, can be converted by glucosidases and as yet unidentified esterases to oleoside aldehydes. These are not only involved in pathogen defense, but also bear considerable promise as pharmaceuticals or neutraceuticals. Making use of the available olive genomic data, we have identified four novel methylesterases that showed significant homology to the polyneuridine aldehyde esterase (PNAE) from Rauvolfia serpentina, an enzyme acting on a distantly related metabolite group (monoterpenoid indole alkaloids, MIAs) also featuring a secoiridoid structural component. The four olive enzymes belong to the α/ß-hydrolase fold family and showed variable in vitro activity against methyl esters of selected plant hormones, namely jasmonic acid (MeJA), indole acetic acid (MeIAA), as well as salicylic acid (MeSA). None of the identified catalysts were directly active against the olive metabolites oleuropein, ligstroside, or oleoside 11-methyl ester. When employed in a sequential reaction with an appropriate glucosidase, however, two were capable of hydrolyzing these specialized compounds yielding reactive dialdehydes. This suggests that the esterases play a pivotal role in the activation of the olive secoiridoid polyphenols. Finally, we show that several of the investigated methylesterases exhibit a concomitant in vitro transesterification capacity-a novel feature, yielding ethyl esters of jasmonic acid (JA) or indole-3-acetic acid (IAA).

Synthesis of Nano-Paramagnetic Oleuropein to Induce KRAS Over-Expression: A New Mechanism to Inhibit AGS Cancer Cells.

Background and objectives: Human gastric adenocarcinoma (AGS) is one of the most common malignant cancers worldwide. The present study aimed to transfer oleuropein into cancer cells using synthetic paramagnetic nanoparticles and study their effect on the AGS (ATCC® CRL1739™) cell line. Materials and Methods: Paramagnetic nano-oleuropein was synthesized using four-stage co-precipitation by developing NH-connected bridges and was evaluated by EDS, SEM and FTIR methods. Different concentrations of magnetic oleuropein (0, 0.15, 0.45, 1.37, 4.12, 12.35, 37.04, 111.11, 333.33, 1000 µg/mL) were used to treat the AGS cell line in a completely randomized design using a statistical framework with three replicates. The relative expression rate of miR-200 and KRAS oncogenes was evaluated using real-time PCR. The inhibition rate of the AGS cells was assessed using the MTT test at 24, 48 and 72 h intervals. Results: The results showed that there was a significant difference between the inhibition rates of magnetic nano-oleuropein at IC50-24h (23.6 µg/mL), IC50-48h (15.2 µg/mL) and IC50-72h (9.2 µg/mL). Real-time PCR indicated that the relative expression of KRAS and miR-200 genes was highest at IC50 at these intervals. Conclusions: Magnetic nano-oleuropein can be subjected to objective testing and clinical evaluations as a natural antioxidant to prevent and treat gastric adenocarcinoma.

Identification of novel superoxide dismutase isoenzymes in the olive (Olea europaea L.) pollen.

BACKGROUND: Among antioxidant enzymes, the superoxide dismutase (SOD) family is a major actor in catalysing the disproportionation of superoxide. Apart from its role as antioxidant, these enzymes have a role in cell signalling, and Cu,Zn-SOD proteins are also major pollen allergens. In order to deepen our understanding of the SOD isoenzymes present in olive pollen and to analyse the molecular variability of the pollen Cu,Zn-SOD family, we carried out biochemical, transcriptomic and localization studies of pollen grains from different olive cultivars and other allergenic species. RESULTS: Olive pollen showed a high rate of total SOD activity in all cultivars assayed, which did not correlate with pollen viability. Mass spectrometry analysis together with activity assays and Western blotting experiments enabled us to identify new forms of Cu,Zn-SOD enzyme (including chloroplastidic and peroxisomal forms) as well as differentially expressed Mn-, Fe- and Cu,Zn-SOD isoenzymes among the pollen of different olive cultivars and allergenic species. Ultrastructural localization of Cu,Zn-SOD revealed its plastidial localization in the pollen grain. We also identified the occurrence of a shorter form of one of the cytosolic Cu,Zn-SOD enzymes, likely as the result of alternative splicing. This shorter enzyme showed lower SOD activity as compared to the full length form. CONCLUSIONS: The presence of multiple SOD isoenzymes in the olive pollen could be related to the need of finely tuning the ROS metabolism during the transition from its quiescent condition at maturity to a highly metabolically active state at germination.

The C-Domain of Oleuropein ß-Glucosidase Assists in Protein Folding and Sequesters the Enzyme in Nucleus.

Oleuropein, a terpene-derived glycosylated secoiridoid biosynthesized exclusively by members of the Oleaceae family, is involved in a two-component defense system comprising a ß-glucosidase that activates oleuropein into a toxic glutaraldehyde-like structure. Oleuropein and its deglycosylated derivatives have high pharmaceutical interest. In this study we determined that the in planta heterologous expressed OeGLU, an oleuropein-specific ß-glucosidase from olive (Olea europaea), had enzymatic kinetics similar to the olive native enzyme. The C terminus encompassing the nuclear localization signal sequesters the enzyme in the nucleus, and predetermines the protein-protein recognition and homodimerization. Biochemical analysis revealed that OeGLU is a homomultimer with high Mr In silico prediction modeling of the complex structure and bimolecular fluorescence complementation analyses revealed that the C terminus of OeGLU is essential for the proper assembly of an octameric form, a key conformational feature that determines the activity of the enzyme. Our results demonstrate that intrinsic characteristics of the OeGLU ensure separation from oleuropein and keep the dual-partner defensive system conditionally inactive. Upon cell destruction, the dual-partner defense system is activated and olive massively releases the arsenal of defense.

Ethephon induced oxidative stress in the olive leaf abscission zone enables development of a selective abscission compound.

BACKGROUND: Table olives (Olea europaea L.), despite their widespread production, are still harvested manually. The low efficiency of manual harvesting and the rising costs of labor have reduced the profitability of this crop. A selective abscission treatment, inducing abscission of fruits but not leaves, is crucial for the adoption of mechanical harvesting of table olives. In the present work we studied the anatomical and molecular differences between the three abscission zones (AZs) of olive fruits and leaves. RESULTS: The fruit abscission zone 3 (FAZ3), located between the fruit and the pedicel, was found to be the active AZ in mature fruits and is sensitive to ethephon, whereas FAZ2, between the pedicel and the rachis, is the flower active AZ as well as functioning as the most ethephon induced fruit AZ. We found anatomical differences between the leaf AZ (LAZ) and the two FAZs. Unlike the FAZs, the LAZ is characterized by small cells with less pectin compared to neighboring cells. In an attempt to differentiate between the fruit and leaf AZs, we examined the effect of treating olive-bearing trees with ethephon, an ethylene-releasing compound, with or without antioxidants, on the detachment force (DF) of fruits and leaves 5 days after the treatment. Ethephon treatment enhanced pectinase activity and reduced DF in all the three olive AZs. A transcriptomic analysis of the three olive AZs after ethephon treatment revealed induction of several genes encoding for hormones (ethylene, auxin and ABA), as well as for several cell wall degrading enzymes. However, up-regulation of cellulase genes was found only in the LAZ. Many genes involved in oxidative stress were induced by the ethephon treatment in the LAZ alone. In addition, we found that reactive oxygen species (ROS) mediated abscission in response to ethephon only in leaves. Thus, adding antioxidants such as ascorbic acid or butyric acid to the ethephon inhibited leaf abscission but enhanced fruit abscission. CONCLUSION: Our findings suggest that treating olive-bearing trees with a combination of ethephon and antioxidants reduces the detachment force (DF) of fruit without weakening that of the leaves. Hence, this selective abscission treatment may be used in turn to promote mechanized harvest of olives.

Differential Contribution of Endoplasmic Reticulum and Chloroplast ω-3 Fatty Acid Desaturase Genes to the Linolenic Acid Content of Olive (Olea europaea) Fruit.

Linolenic acid is a polyunsaturated fatty acid present in plant lipids, which plays key roles in plant metabolism as a structural component of storage and membrane lipids, and as a precursor of signaling molecules. The synthesis of linolenic acid is catalyzed by two different ω-3 fatty acid desaturases, which correspond to microsomal- (FAD3) and chloroplast- (FAD7 and FAD8) localized enzymes. We have investigated the specific contribution of each enzyme to the linolenic acid content in olive fruit. With that aim, we isolated two different cDNA clones encoding two ω-3 fatty acid desaturases from olive (Olea europaea cv. Picual). Sequence analysis indicates that they code for microsomal (OepFAD3B) and chloroplast (OepFAD7-2) ω-3 fatty acid desaturase enzymes, different from the previously characterized OekFAD3A and OekFAD7-1 genes. Functional expression in yeast of the corresponding OepFAD3A and OepFAD3B cDNAs confirmed that they encode microsomal ω-3 fatty acid desaturases. The linolenic acid content and transcript levels of olive FAD3 and FAD7 genes were measured in different tissues of Picual and Arbequina cultivars, including mesocarp and seed during development and ripening of olive fruit. Gene expression and lipid analysis indicate that FAD3A is the gene mainly responsible for the linolenic acid present in the seed, while FAD7-1 and FAD7-2 contribute mostly to the linolenic acid present in the mesocarp and, therefore, in the olive oil. These results also indicate the relevance of lipid trafficking between the endoplasmic reticulum and chloroplast in determining the linolenic acid content of membrane and storage lipids in oil-accumulating photosynthetic tissues.

Impact of proline application on cadmium accumulation, mineral nutrition and enzymatic antioxidant defense system of Olea europaea L. cv Chemlali exposed to cadmium stress.

Proline plays an important role in plant response to various environmental stresses. However, its involvement in mitigation of heavy metal stress in plants remains elusive. In this study, we examined the effectiveness of exogenous proline (10 and 20 mM) in alleviating cadmium induced inhibitory effects in young olive plants (Olea europaea L. cv. Chemlali) exposed to two Cd levels (10 and 30 mg CdCl2 kg(-1) soil). The Cd treatment induced substantial accumulation of Cd in both root and leaf tissues and a decrease in gas exchange, photosynthetic pigments contents, uptake of essential elements (Ca, Mg and K) and plant biomass. Furthermore, an elevation of antioxidant enzymes activities (superoxide dismutase, catalase, glutathione peroxydase) and proline content in association with relatively high amounts of hydrogen peroxide, thiobarbituric acid reactive substances and electrolyte leakage were observed. Interestingly, the application of exogenous proline alleviated the oxidative damage induced by Cd accumulation. In fact, Cd-stressed olive plants treated with proline showed an increase of antioxidant enzymes activities, photosynthetic activity, nutritional status, plant growth and oil content of olive fruit. Generally, it seems that proline supplementation alleviated the deleterious effects of young olive plants exposed to Cd stress.

Evaluation of Olive Fruit Lipoxygenase Extraction Protocols on 9- and 13-Z,E-HPODE Formation.

In plant tissues, enzymes implicated in the lipoxygenase (LOX) pathway are responsible for the hydroperoxydation of polyunsaturated fatty acids, ultimately leading to the production of small chemical species involved in several physiological processes. During industrial olive oil production, these enzymes are activated upon crushing and grinding of olive fruit tissue, subsequently leading to the synthesis of volatile compounds responsible for the positive aroma and flavor of the oil. An investigation of LOX activity during olive fruit ripening and malaxation could assist in the production of oils with favorable aroma and taste. Therefore, a reliable method for olive LOX purification is crucial. Here we report a critical review of six LOX extraction protocols, two of which have shown minimum enzyme activity, possibly leading to misconceptions in the interpretation of experimental data. Future research concerning olive LOX should employ extraction methods that preserve enzyme activity.

Comparison Between Different Flavored Olive Oil Production Techniques: Healthy Value and Process Efficiency.

Three different flavoring methods of olive oil were tested employing two different herbs, thyme and oregano. The traditional method consist in the infusion of herbs into the oil. A second scarcely diffused method is based on the addition of herbs to the crushed olives before the malaxation step during the extraction process. The third innovative method is the implementation of the ultrasound before the olive paste malaxation. The objective of the study is to verify the effect of the treatments on the quality of the product, assessed by means of the chemical characteristics, the phenol composition and the radical scavenging activity of the resulting oils. The less favorable method was the addition of herbs directly to the oil. A positive effect was achieved by the addition of herbs to the olive paste and other advantages were attained by the employment of ultrasound. These last two methods allow to produce oils "ready to sell", instead the infused oils need to be filtered. Moreover, the flavoring methods applied during the extraction process determine a significant increment of phenolic content and radical scavenging activity of olive oils. The increments were higher when oregano is used instead of thyme. Ultrasound inhibited the olive polyphenoloxidase, the endogenous enzyme responsible for olive oil phenol oxidation. This treatment of olive paste mixed with herbs before malaxation was revealed as the most favorable method due to the best efficiency, reduced time consumption and minor labor, enhancing the product quality of flavored olive oil.

A defence-related Olea europaea ß-glucosidase hydrolyses and activates oleuropein into a potent protein cross-linking agent.

Oleuropein, the major secoiridoid compound in olive, is involved in a sophisticated two-component defence system comprising a ß-glucosidase enzyme that activates oleuropein into a toxic glutaraldehyde-like structure. Although oleuropein deglycosylation studies have been monitored extensively, an oleuropein ß-glucosidase gene has not been characterized as yet. Here, we report the isolation of OeGLU cDNA from olive encoding a ß-glucosidase belonging to the defence-related group of terpenoid-specific glucosidases. In planta recombinant protein expression assays showed that OeGLU deglycosylated and activated oleuropein into a strong protein cross-linker. Homology and docking modelling predicted that OeGLU has a characteristic (ß/α)8 TIM barrel conformation and a typical construction of a pocket-shaped substrate recognition domain composed of conserved amino acids supporting the ß-glucosidase activity and non-conserved residues associated with aglycon specificity. Transcriptional analysis in various olive organs revealed that the gene was developmentally regulated, with its transcript levels coinciding well with the spatiotemporal patterns of oleuropein degradation and aglycon accumulation in drupes. OeGLU upregulation in young organs reflects its prominent role in oleuropein-mediated defence system. High gene expression during drupe maturation implies an additional role in olive secondary metabolism, through the degradation of oleuropein and reutilization of hydrolysis products.

The C-terminal domains of two homologous Oleaceae ß-1,3-glucanases recognise carbohydrates differently: Laminarin binding by NMR.

Ole e 9 and Fra e 9 are two allergenic ß-1,3-glucanases from olive and ash tree pollens, respectively. Both proteins present a modular structure with a catalytic N-terminal domain and a carbohydrate-binding module (CBM) at the C-terminus. Despite their significant sequence resemblance, they differ in some functional properties, such as their catalytic activity and the carbohydrate-binding ability. Here, we have studied the different capability of the recombinant C-terminal domain of both allergens to bind laminarin by NMR titrations, binding assays and ultracentrifugation. We show that rCtD-Ole e 9 has a higher affinity for laminarin than rCtD-Fra e 9. The complexes have different exchange regimes on the NMR time scale in agreement with the different affinity for laminarin observed in the biochemical experiments. Utilising NMR chemical shift perturbation data, we show that only one side of the protein surface is affected by the interaction and that the binding site is located in the inter-helical region between α1 and α2, which is buttressed by aromatic side chains. The binding surface is larger in rCtD-Ole e 9 which may account for its higher affinity for laminarin relative to rCtD-Fra e 9.

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.

Olive seed protein bodies store degrading enzymes involved in mobilization of oil bodies.

The major seed storage reserves in oilseeds are accumulated in protein bodies and oil bodies, and serve as an energy, carbon, and nitrogen source during germination. Here, the spatio-temporal relationships between protein bodies and several key enzymes (phospholipase A, lipase, and lipoxygenase) involved in storage lipid mobilization in cotyledon cells was analysed during in vitro seed germination. Enzyme activities were assayed in-gel and their cellular localization were determined using microscopy techniques. At seed maturity, phospholipase A and triacylglycerol lipase activities were found exclusively in protein bodies. However, after seed imbibition, these activities were shifted to the cytoplasm and the surface of the oil bodies. The activity of neutral lipases was detected by using α-naphthyl palmitate and it was associated mainly with protein bodies during the whole course of germination. This pattern of distribution was highly similar to the localization of neutral lipids, which progressively appeared in protein bodies. Lipoxygenase activity was found in both the protein bodies and on the surface of the oil bodies during the initial phase of seed germination. The association of lipoxygenase with oil bodies was temporally correlated with the appearance of phospholipase A and lipase activities on the surface of oil bodies. It is concluded that protein bodies not only serve as simple storage structures, but are also dynamic and multifunctional organelles directly involved in storage lipid mobilization during olive seed germination.

Regulation of photosynthesis and stomatal and mesophyll conductance under water stress and recovery in olive trees: correlation with gene expression of carbonic anhydrase and aquaporins.

The hypothesis that aquaporins and carbonic anhydrase (CA) are involved in the regulation of stomatal (g s) and mesophyll (g m) conductance to CO2 was tested in a short-term water-stress and recovery experiment in 5-year-old olive plants (Olea europaea) growing outdoors. The evolution of leaf gas exchange, chlorophyll fluorescence, and plant water status, and a quantitative analysis of photosynthesis limitations, were followed during water stress and recovery. These variables were correlated with gene expression of the aquaporins OePIP1.1 and OePIP2.1, and stromal CA. At mild stress and at the beginning of the recovery period, stomatal limitations prevailed, while the decline in g m accounted for up to 60% of photosynthesis limitations under severe water stress. However, g m was restored to control values shortly after rewatering, facilitating the recovery of the photosynthetic rate. CA was downregulated during water stress and upregulated after recovery. The use of structural equation modelling allowed us to conclude that both OePIP1.1 and OePIP2.1 expression could explain most of the variations observed for g s and g m. CA expression also had a small but significant effect on g m in olive under water-stress conditions.

Cold perception and gene expression differ in Olea europaea seed coat and embryo during drupe cold acclimation.

FAD2 and FAD7 desaturases are involved in cold acclimation of olive (Olea europaea) mesocarp. There is no research information available on cold acclimation of seeds during mesocarp cold acclimation or on differences in the cold response of the seed coat and embryo. How FAD2 and FAD7 affect seed coat and embryo cold responses is unknown. Osmotin positively affects cold acclimation in olive tree vegetative organs, but its role in the seeds requires investigation. OeFAD2.1, OeFAD2.2, OeFAD7 and Oeosmotin were investigated before and after mesocarp acclimation by transcriptomic, lipidomic and immunolabelling analyses, and cytosolic calcium concentration ([Ca(2+)](cyt)) signalling, F-actin changes and seed development were investigated by epifluorescence/histological analyses. Transient [Ca(2+)](cyt) rises and F-actin disassembly were found in cold-shocked protoplasts from the seed coat, but not from the embryo. The thickness of the outer endosperm cuticle increased during drupe exposure to lowering of temperature, whereas the embryo protoderm always lacked cuticle. OeFAD2 transcription increased in both the embryo and seed coat in the cold-acclimated drupe, but linoleic acid (i.e. the product of FAD2 activity) increased solely in the seed coat. Osmotin was immunodetected in the seed coat and endosperm of the cold-acclimated drupe, and not in the embryo. The results show cold responsiveness in the seed coat and cold tolerance in the embryo. We propose a role for the seed coat in maintaining embryo cold tolerance by increasing endosperm cutinization through FAD2 and osmotin activities.

New insights into the early steps of oil body mobilization during pollen germination.

In some plants, pollen grains accumulate storage lipids that serve as energy supply during germination. Here, three enzymes involved in early steps of oil body mobilization in the male gametophyte were functionally characterized for the first time. The effect of extracellular sugars on pollen performance and oil body dynamics was also analysed. Olive pollen oil bodies showed phospholipase A, lipase, and lipoxygenase activities on their surface. Enzyme activity levels increased during germination with a maximum after 3h. Removal of extracellular sugars from the germination medium did not affect pollen performance but increased enzyme activity rates and sped up oil body mobilization. Inhibitors seriously hampered pollen germination and pollen tube growth, leading to a characteristic accumulation of oil bodies in the germinative aperture. It can be concluded that storage lipids are sufficient for proper olive pollen germination. A lipase and a lipoxygenase are likely involved in oil body mobilization. Extracellular sugars may modulate their function, while a phospholipase A may promote their access to the storage lipids.