Drought stress improved the capacity of Rhizophagus irregularis for inducing the accumulation of oleuropein and mannitol in olive (Olea europaea) roots.

Olive trees are often subjected to a prolonged dry season with low water availability, which induces oxidative stress. Arbuscular mycorrhizal (AM) symbioses can improve olive plant tolerance to water deficit. This study investigated several aspects related to drought tolerance in AM fungi olive plants. Non-AM and AM plants were grown under well-watered or drought-stressed conditions, and mycorrhizal growth response, neutral lipid fatty acid (NLFA)16:1ω5 and phospholipid fatty acid (PLFA) 16:1ω5 in roots (intraradical mycelium) and in soil (extraradical mycelium), carbohydrates (monosaccharides, disaccharides and polyols) and phenolic compounds (phenolic alcohols, flavonoids, lignans, secoiridoids and hydroxycinnamic acid derivatives) were determined. Results showed that the amounts of PLFA 16:1ω5 and NLFA 16:1ω5 were significantly influenced by drought stress conditions. The NLFA 16:1ω5/PLFA 16:1ω5 ratio showed a dramatic decrease (-62%) with the application of water deficit stress, indicating that AM fungi allocated low carbon to storage structures under stress conditions. Mannitol and verbascoside are the main compounds detected in the roots of well-watered plants, whereas oleuropein and mannitol are the main compounds differentially accumulated in the roots of water-stressed plants. The oleuropein/verbascoside ratio increased in the case of drought-stressed AM plants by 30%, while the mannitol/oleuropein ratio was decreased by 46%, when compared to the non-AM stressed plants. Mycorrhization therefore oriented the flux toward the biosynthetic pathway of oleuropein and the data suggest that sugar and phenolic compound metabolism may have been redirected to the formation of oleuropein in roots of AM stressed plants, that may underlie their enhanced tolerance to drought stress.

Root verbascoside and oleuropein are potential indicators of drought resistance in olive trees (Olea europaea L.).

Polyphenols are constituents of all higher plants. However, their biosynthesis is often induced when plants are exposed to abiotic stresses, such as drought. The aim of the present work was to determine the phenolic status in the roots of olive trees grown under water deficit conditions. The results revealed that roots of water-stressed plants had a higher content of total phenols. The main compound detected in well-watered olive tree roots was verbascoside. Oleuropein was established as the predominant phenolic compound of water-stressed plants. The oleuropein/verbascoside ratio varied between 0.31 and 6.02 in well-watered and water-stressed plants respectively, which could be a useful indicator of drought tolerance in olive trees. Furthermore, this study is the first to provide experimental evidence showing that luteolin-7-rutinoside, luteolin-7-glucoside and apigenin-7-glucoside were the dominant flavonoid glucosides in olive tree roots and showed the most significant variations under water stress.

Long-term field evaluation of the changes in fruit and olive oil chemical compositions after agronomic application of olive mill wastewater with rock phosphate.

The objectives of this study were to determine the long-term effects of agronomic application of olive mill wastewater (OMW) with rock phosphate (RP) in a field of olive trees, on olive fruits and oil quality. The results revealed that olive fruits of OMW-RP amended plants had higher contents of polyphenols and mannitol indicating that agronomic application of OMW with RP generated an oxidative stress. Land spreading of OMW with RP altered the relative proportions of individual sugars in leaves and fruits. Consequently, the oil content decreased significantly, and a marked decrease in the contents of carotenoids and chlorophylls was also observed. Changes also took place in the composition of fatty acids, particularly by the increase of linoleic acid and the decrease of oleic acid. Our results suggested that the use of OMW in combination with RP is expected to have a major negative impact on olive fruit and oil quality.

Modification of pomological characteristics and flavour components of fruits and virgin olive oil following wastewater irrigation and soil tillage.

BACKGROUND: The experiment was carried out on olive trees cv. Chemlali, during two successive years (2013/2014). Two irrigation treatments (IT: Trees irrigated with wastewater; TRC: Trees grown under rainfed condition) were combined with two tillage practices (TTS: Trees grown in tilled soil; TNTS: Trees grown in non-tilled soil). RESULTS: The results of the study showed that wastewater irrigation combined with soil tillage improved the pomological characteristics of olive fruits. The tree yield increase was substantial for IT and TTS. However, most of the identified phenolic compounds, especially oleuropein, mainly accumulated in olive fruits of the TC block (TNTS + TRC), suggesting a marked improvement in the nutritional value of these fruits. Moreover, fruits of TNTS had high contents of sugar compounds, required for the synthesis of the fruit storage material. The study also showed that the agronomic practices affected the amounts of some aromatic compounds responsible for the distinctive flavour notes of olive oil. CONCLUSION: Agronomic practices may affect considerably the commercial and nutritional values and the sensorial quality of the commodities. © 2017 Society of Chemical Industry.

Anti-Inflammatory Effect of Dexamethasone Controlled Released From Anterior Suprachoroidal Polyurethane Implants on Endotoxin-Induced Uveitis in Rats.

PURPOSE: Targeted drug delivery to the ocular tissues remains a challenge. Biodegradable intraocular implants allow prolonged controlled release of drugs directly into the eye. In this study, we evaluated an anterior suprachoroidal polyurethane implant containing dexamethasone polyurethane dispersions (DX-PUD) as a drug delivery system in the rat model of endotoxin-induced uveitis (EIU). METHODS: In vitro drug release was studied using PUD implants containing 8%, 20%, and 30% (wt/wt) DX. Cytotoxicity of the degradation products of DX-PUD was assessed on human ARPE-19 cells using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) test. Short-term ocular biocompatibility of suprachoroidal DX-PUD implants was evaluated in normal rat eyes. Endotoxin-induced uveitis was then induced in rat eyes preimplanted with DX-PUD. Clinical examination was performed at 24 hours; eyes were used to assess inflammatory cell infiltration and macrophage/microglial activation. Cytokine and chemokine expression in the iris/ciliary body and in the retina was investigated using quantitative PCR. Feasibility of anterior suprachoroidal PUD implantation was also tested using postmortem human eyes. RESULTS: A burst release was followed by a sustained controlled release of DX from PUD implants. By-products of the DX-PUD were not toxic to human ARPE-19 cells or to rat ocular tissues. Dexamethasone-PUD implants prevented EIU in rat eyes, reducing inflammatory cell infiltration and inhibiting macrophage/microglial activation. Dexamethasone-PUD downregulated proinflammatory cytokines/chemokines (IL-1ß, IL-6, cytokine-induced neutrophil chemoattractant [CINC]) and inducible nitric oxide synthase (iNOS) and upregulated IL-10 anti-inflammatory cytokine. Polyurethane dispersion was successfully implanted into postmortem human eyes. CONCLUSIONS: Dexamethasone-PUD implanted in the anterior suprachoroidal space may be of interest in the treatment of intraocular inflammation.

Characterization of fig achenes' oil of Ficus carica grown in Tunisia.

This work investigated the composition of the oil extract from achenes of "Kholi" variety of Ficus carica, grown in Tunisia. Fatty acid and sterol compositions were analyzed by gas chromatography (GC) coupled to flame ionization detector (FID). Furthermore, the antioxidant capacity in fig achenes' oil was assessed by employing two different in vitro assays such as DPPH, ABTS(+) radical scavenging capacities. Our results indicated that the fig achenes' oil is a rich source of bioactive molecules. The soxhlet n-hexane extraction of these achenes produced a total oil yield of 16.24%. The predominant fatty acid was linolenic acid. Concerning phytosterols, the total amount reached 1061.45 mg/100 g with a predominance of Δ(5,23)-stigmastadienol (73.78%). Regarding antioxidant activities, the half maximal inhibitory concentration (IC50) was 215.86 µg/ml and Trolox equivalent antioxidant capacity (TEAC) was 95.25 mM. These data indicate that fig achenes oil of F. carica could be potentially useful in food and pharmaceutical applications.

Accumulation of flavonoids and phenolic compounds in olive tree roots in response to mycorrhizal colonization: A possible mechanism for regulation of defense molecules.

The arbuscular mycorrhizal (AM) fungus promotes plant growth and can alter the production of primary and secondary metabolites. The aim of this work was to determine the influence of AM fungi colonization on the content of phenolic compounds, flavonoids and soluble carbohydrates in olive (Olea europaea L.) tree roots. The results revealed that mycorrhizal plants had a higher content of flavonoids and total phenols. Analysis of sugar contents showed enhanced levels of sucrose and fructose in mycorrhizal roots, while glucose amounts stayed constant. The DPPH radical-scavenging activity of the mycorrhizal root methanolic extracts was higher than that of the non- mycorrhizal root methanolic extracts. These results indicated that olive tree roots contain significant amounts of phenolic compounds, important factors for antioxidant capacity, which can be substantially modified by colonization of olive trees with AM fungi.

Determination of mannitol sorbitol and myo-inositol in olive tree roots and rhizospheric soil by gas chromatography and effect of severe drought conditions on their profiles.

This study reports a method for the analysis of mannitol, sorbitol and myo-inositol in olive tree roots and rhizospheric soil with gas chromatography. The analytical method consists of extraction with a mixture of dichloromethane:methanol (2:1, v/v) for soil samples and a mixture of ethanol:water (80:20) for root samples, silylation using pyridine, hexamethyldisilazane (HMDS) and trimethylchlorosilane (TMCS). The recovery of mannitol sorbitol and myo-inositol (for extraction and analysis in dichloromethane:methanol and ethanol:water) was acceptable and ranged from 100.3 to 114.7%. The time of analysis was <24 min. Among identified polyols extracted from rhizosphere and roots of olive plants, mannitol was the major compound. A marked increase in mannitol content occurred in rhizosphere and roots of water-stressed plants, suggesting a much broader role of mannitol in stress response based on its ability to act as a compatible solute.

Colonization of olive trees (Olea europaea L.) with the arbuscular mycorrhizal fungus Glomus sp. modified the glycolipids biosynthesis and resulted in accumulation of unsaturated fatty acids.

The influence of arbuscular mycorrhizal (AM) fungi colonization on photosynthesis, mineral nutrition, the amount of phospholipids and glycolipids in the leaves of olive (Olea europaea L.) trees was investigated. After six months of growth, the rate of photosynthesis, carboxylation efficiency, transpiration and stomatal conductance in mycorrhizal (M) plants was significantly higher than that of non-mycorrhizal (NM) plants. The inoculation treatment increased the foliar P and Mg but not N. The amount of glycolipids in the leaves of M plants was significantly higher than that of NM plants. However, the amount of phospholipids in the leaves of M plants was not significantly different to that in the leaves of NM plants. Also, we observed a significant increase in the level of α-linolenic acid (C18:3ω3) in glycolipids of M plants. This work supports the view that increased glycolipids level in the leaves of M plants could be involved, at least in part, in the beneficial effects of mycorrhizal colonization on photosynthesis performance of olive trees. To our knowledge, this is the first report on the effect of AM fungi on the amount of glycolipids in the leaves of mycorrhizal plants.

Effect of nutrient-based fertilisers of olive trees on olive oil quality.

BACKGROUND: This work was conducted to determine the effects of two nutrient-based fertilisers on the general physicochemical characteristics (including free fatty acid content, peroxide value and UV spectrophotometric characteristics), fatty acid profile, total phenols, o-diphenols and phytosterol composition of olive oil. Foliar applications were carried out in two successive years and included four treatments: TC (control, without foliar nutrition), T1 (rich in nitrogen, applied at the start of vegetation, 10 days later and 20 days later), T2 (rich in boron, magnesium, sulfur and manganese, applied at the beginning of flowering and 10 days later) and T3 (T1+T2). At the end of the experiment (after 2 years), oils were extracted and analysed. RESULTS: No effect was found on either general physicochemical characteristics or fatty acid composition. Foliar fertilisation caused a significant decrease in both polyphenol and o-diphenol contents. Total sterol content was unaffected by foliar fertilisation. However, the phytosterol composition of the oil, particularly its ß-sitosterol level, was markedly improved after foliar nutrient application. Principal component analysis of the phytosterol composition showed discrimination between the control oil and the oils from T1, T2 and T3 treatments. CONCLUSION: The results of this study extend the current knowledge of such cross-talk between plant nutrition and quality of oil.

Retinal degeneration progression changes lentiviral vector cell targeting in the retina.

In normal mice, the lentiviral vector (LV) is very efficient to target the RPE cells, but transduces retinal neurons well only during development. In the present study, the tropism of LV has been investigated in the degenerating retina of mice, knowing that the retina structure changes during degeneration. We postulated that the viral transduction would be increased by the alteration of the outer limiting membrane (OLM). Two different LV pseudotypes were tested using the VSVG and the Mokola envelopes, as well as two animal models of retinal degeneration: light-damaged Balb-C and Rhodopsin knockout (Rho-/-) mice. After light damage, the OLM is altered and no significant increase of the number of transduced photoreceptors can be obtained with a LV-VSVG-Rhop-GFP vector. In the Rho-/- mice, an alteration of the OLM was also observed, but the possibility of transducing photoreceptors was decreased, probably by ongoing gliosis. The use of a ubiquitous promoter allows better photoreceptor transduction, suggesting that photoreceptor-specific promoter activity changes during late stages of photoreceptor degeneration. However, the number of targeted photoreceptors remains low. In contrast, LV pseudotyped with the Mokola envelope allows a wide dispersion of the vector into the retina (corresponding to the injection bleb) with preferential targeting of Müller cells, a situation which does not occur in the wild-type retina. Mokola-pseudotyped lentiviral vectors may serve to engineer these glial cells to deliver secreted therapeutic factors to a diseased area of the retina.

Retinal stem cells transplanted into models of late stages of retinitis pigmentosa preferentially adopt a glial or a retinal ganglion cell fate.

PURPOSE: To characterize the potential of newborn retinal stem cells (RSCs) isolated from the radial glia population to integrate the retina, this study was conducted to investigate the fate of in vitro expanded RSCs transplanted into retinas devoid of photoreceptors (adult rd1 and old VPP mice and rhodopsin-mutated transgenic mice) or partially degenerated retina (adult VPP mice) retinas. METHODS: Populations of RSCs and progenitor cells were isolated either from DBA2J newborn mice and labeled with the red lipophilic fluorescent dye (PKH26) or from GFP (green fluorescent protein) transgenic mice. After expansion in EGF+FGF2 (epidermal growth factor+fibroblast growth factor), cells were transplanted intravitreally or subretinally into the eyes of adult wild-type, transgenic mice undergoing slow (VPP strain) or rapid (rd1 strain) retinal degeneration. RESULTS: Only limited migration and differentiation of the cells were observed in normal mice injected subretinally or in VPP and rd1 mice injected intravitreally. After subretinal injection in old VPP mice, transplanted cells massively migrated into the ganglion cell layer and, at 1 and 4 weeks after injection, harbored neuronal and glial markers expressed locally, such as beta-tubulin-III, NeuN, Brn3b, or glial fibrillary acidic protein (GFAP), with a marked preference for the glial phenotype. In adult VPP retinas, the grafted cells behaved similarly. Few grafted cells stayed in the degenerating outer nuclear layer (ONL). These cells were, in rare cases, positive for rhodopsin or recoverin, markers specific for photoreceptors and some bipolar cells. CONCLUSIONS: These results show that the grafted cells preferentially integrate into the GCL and IPL and express ganglion cell or glial markers, thus exhibiting migratory and differentiation preferences when injected subretinally. It also appears that the retina, whether partially degenerated or already degenerated, does not provide signals to induce massive differentiation of RSCs into photoreceptors. This observation suggests that a predifferentiation of RSCs into photoreceptors before transplantation may be necessary to obtain graft integration in the ONL.

High yield of cells committed to the photoreceptor fate from expanded mouse retinal stem cells.

The purpose of the present work was to generate, from retinal stem cells (RSCs), a large number of cells committed toward the photoreceptor fate in order to provide an unlimited cell source for neurogenesis and transplantation studies. We expanded RSCs (at least 34 passages) sharing characteristics of radial glial cells and primed the cells in vitro with fibroblast growth factor (FGF)-2 for 5 days, after which cells were treated with the B27 supplement to induce cell differentiation and maturation. Upon differentiation, cells expressed cell type-specific markers corresponding to neurons and glia. We show by immunocytochemistry analysis that a subpopulation of differentiated cells was committed to the photoreceptor lineage given that these cells expressed the photoreceptor proteins recoverin, peripherin, and rhodopsin in a same ratio. Furthermore, cells infected during the differentiation procedure with a lentiviral vector expressing green fluorescent protein (GFP) under the control of either the rhodopsin promoter or the interphotoreceptor retinoid-binding protein (IRBP) promoter, expressed GFP. FGF-2 priming increased neuronal differentiation while decreasing glia generation. Reverse transcription-polymerase chain reaction analyses revealed that the differentiated cells expressed photoreceptor-specific genes such as Crx, rhodopsin, peripherin, IRBP, and phosphodiesterase-alpha. Quantification of the differentiated cells showed a robust differentiation into the photoreceptor lineage: Approximately 25%-35% of the total cells harbored photoreceptor markers. The generation of a significant number of nondifferentiated RSCs as well as differentiated photoreceptors will enable researchers to determine via transplantation studies which cells are the most adequate to integrate a degenerating retina.

BMI1 loss delays photoreceptor degeneration in Rd1 mice. Bmi1 loss and neuroprotection in Rd1 mice.

Retinitis pigmentosa (RP) is a heterogeneous group of genetic disorders leading to blindness, which remain untreatable at present. Rd1 mice represent a recognized model of RP, and so far only GDNF treatment provided a slight delay in the retinal degeneration in these mice. Bmi1, a transcriptional repressor, has recently been shown to be essential for neural stem cell (NSC) renewal in the brain, with an increased appearance of glial cells in vivo in Bmi1 knockout (Bmi1-/-) mice. One of the roles of glial cells is to sustain neuronal function and survival. In the view of a role of the retinal Miller glia as a source of neural protection in the retina, the increased astrocytic population in the Bmi1-/- brain led us to investigate the effect of Bmi1 loss in Rd1 mice. We observed an increase of Müller glial cells in Rd1-Bmi1-/- retinas compared to Rd1. Moreover, Rd1-Bmi1-/- mice showed 7-8 rows of photoreceptors at 30 days of age (P30), while in Rd1 littermates there was a complete disruption of the outer nuclear layer (ONL). Preliminary ERG results showed a responsiveness of Rd1-Bmi1-/- mice in scotopic vision at P35. In conclusion, Bmi1 loss prevented, or rescued, photoreceptors from degeneration to an unanticipated extent in Rd1 mice. In this chapter, we will first provide a brief review of our work on the cortical NSCs and introduce the Bmi1 oncogene, thus offering a rational to our observations on the retina.

Bmi1 loss produces an increase in astroglial cells and a decrease in neural stem cell population and proliferation.

The polycomb transcriptional repressor Bmi1 promotes cell cycle progression, controls cell senescence, and is implicated in brain development. Loss of Bmi1 leads to a decreased brain size and causes progressive ataxia and epilepsy. Recently, Bmi1 was shown to control neural stem cell (NSC) renewal. However, the effect of Bmi1 loss on neural cell fate in vivo and the question whether the action of Bmi1 was intrinsic to the NSCs remained to be investigated. Here, we show that Bmi1 is expressed in the germinal zone in vivo and in NSCs as well as in progenitors proliferating in vitro, but not in differentiated cells. Loss of Bmi1 led to a decrease in proliferation in zones known to contain progenitors: the newborn cortex and the newborn and adult subventricular zone. This decrease was accentuated in vitro, where we observed a drastic reduction in NSC proliferation and renewal because of NSC-intrinsic effects of Bmi1 as shown by the means of RNA interference. Bmi1(-/-) mice also presented more astrocytes at birth, and a generalized gliosis at postnatal day 30. At both stages, colocalization of bromodeoxyuridine and GFAP demonstrated that Bmi1 loss did not prevent astrocyte precursor proliferation. Supporting these observations, Bmi1(-/-) neurospheres generate preferentially astrocytes probably attributable to a different responsiveness to environmental factors. Bmi1 is therefore necessary for NSC renewal in a cell-intrinsic mode, whereas the altered cell pattern of the Bmi1(-/-) brain shows that in vivo astrocyte precursors can proliferate in the absence of Bmi1.

NB-3/Notch1 pathway via Deltex1 promotes neural progenitor cell differentiation into oligodendrocytes.

Neurons and glia in the vertebrate central nervous system arise in temporally distinct, albeit overlapping, phases. Neurons are generated first followed by astrocytes and oligodendrocytes from common progenitor cells. Increasing evidence indicates that axon-derived signals spatiotemporally modulate oligodendrocyte maturation and myelin formation. Our previous observations demonstrate that F3/contactin is a functional ligand of Notch during oligodendrocyte maturation, revealing the existence of another group of Notch ligands. Here, we establish that NB-3, a member of the F3/contactin family, acts as a novel Notch ligand to participate in oligodendrocyte generation. NB-3 triggers nuclear translocation of the Notch intracellular domain and promotes oligodendrogliogenesis from progenitor cells and differentiation of oligodendrocyte precursor cells via Deltex1. In primary oligodendrocytes, NB-3 increases myelin-associated glycoprotein transcripts. Thus, the NB-3/Notch signaling pathway may prove to be a molecular handle to treat demyelinating diseases.

Non-neural regions of the adult human eye: a potential source of neurons?

PURPOSE: Because it is known that both melanocytes and neurons are generated from neural crest stem cells and their derived precursors, the current study was undertaken to evaluate whether adult human ocular tissues, containing melanocytes, have the capacity to generate neuronlike cells in vitro. METHODS: Choroid and Sclera cells from adult human eyes were separately dissociated and cultivated in the presence of epidermal growth factor and 10% fetal bovine serum. No retinal pigmented epithelial cells were detected. After cell growth, cells were transferred under conditions known to induce neuronal differentiation. Cells were plated on laminin in the presence of fibroblast growth factor-2 or brain-derived neurotrophic factor. RESULTS: Cells derived from the sclera and the choroid of 15 donors proliferated to attain a 10(8)-fold increase in the number of cells within 4 months. At each passage, groups of cells differentiated into cells with neuronal morphology, expressing neuronal markers confirmed by immunocytochemistry and RT-PCR analyses, such as beta-tubulin-III, neurofilament, and tau. Parallel to neuronlike formation, glialike cells, revealed by expression of vimentin and P0, were generated in large amounts. Although, absent from choroid and sclera tissues, nondifferentiated cells appeared in cultures. CONCLUSIONS: The adult human eye conserves cells able to recapitulate certain neural developmental features. This observation opens new perspectives to study human neurogenesis and to provide an important source of neurons for transplantation studies in the retina and other regions of the central nervous system.