Disclosing the molecular basis of salinity priming in olive trees using proteogenomic model discovery.

Plant responses to salinity are becoming increasingly understood, however, salt priming mechanisms remain unclear, especially in perennial fruit trees. Herein, we showed that low-salt pre-exposure primes olive (Olea europaea) plants against high salinity stress. We then performed a proteogenomic study to characterize priming responses in olive roots and leaves. Integration of transcriptomic and proteomic data along with metabolic data revealed robust salinity changes that exhibit distinct or overlapping patterns in olive tissues, among which we focused on sugar regulation. Using the multi-crossed -omics data set, we showed that major differences between primed and nonprimed tissues are mainly associated with hormone signaling and defense-related interactions. We identified multiple genes and proteins, including known and putative regulators, that reported significant proteomic and transcriptomic changes between primed and nonprimed plants. Evidence also supported the notion that protein post-translational modifications, notably phosphorylations, carbonylations and S-nitrosylations, promote salt priming. The proteome and transcriptome abundance atlas uncovered alterations between mRNA and protein quantities within tissues and salinity conditions. Proteogenomic-driven causal model discovery also unveiled key interaction networks involved in salt priming. Data generated in this study are important resources for understanding salt priming in olive tree and facilitating proteogenomic research in plant physiology.

A Complex Gene Network Mediated by Ethylene Signal Transduction TFs Defines the Flower Induction and Differentiation in Olea europaea L.

The olive tree (Olea europaea L.) is a typical Mediterranean crop, important for olive and oil production. The high tendency to bear fruits in an uneven manner, defined as irregular or alternate bearing, results in a significant economic impact for the high losses in olives and oil production. Buds from heavy loaded ('ON') and unloaded ('OFF') branches of a unique olive tree were collected in July and the next March to compare the transcriptomic profiles and get deep insight into the molecular mechanisms regulating floral induction and differentiation. A wide set of DEGs related to ethylene TFs and to hormonal, sugar, and phenylpropanoid pathways was identified in buds collected from 'OFF' branches. These genes could directly and indirectly modulate different pathways, suggesting their key role during the lateral bud transition to flowering stage. Interestingly, several genes related to the flowering process appeared as over-expressed in buds from March 'OFF' branches and they could address the buds towards flower differentiation. By this approach, interesting candidate genes related to the switch from vegetative to reproductive stages were detected and analyzed. The functional analysis of these genes will provide tools for developing breeding programs to obtain olive trees characterized by more constant productivity over the years.

Changes in volatile organic composition of olive oil extracted from cv. 'Leccino' fruit subjected to ethylene treatments at different ripening stages.

BACKGROUND: Olive, as a non-climacteric fruit, is presumed to be ethylene independent with regard to ripening triggering/coordination. Nevertheless, studies have demonstrated that postharvest ethylene treatments induce changes in composition and properties also of non-climacteric fruits, including aroma profiles, a key quality parameter of extra virgin olive oils. Olive fruit of cv. 'Leccino' harvested at two distinct ripening stages (less advanced ripening, LAR; and more advanced ripening, MAR, with Jaén index of 4.58 and 5.10, respectively) were subjected to ethylene (1000 ppm in air) treatment for 24 h before oil extraction. RESULTS: Based on multivariate analysis of volatile organic compound (VOCs), the effect of ethylene treatment appeared to be more pronounced in MAR samples. However, differences in organoleptic analysis were also detected in ethylene-treated LAR olive oils. Ethylene seems to selectively affect linolenic/linoleic acid metabolism, particularly concerning the C5 pathway, and reduce specific defect-associated compounds. CONCLUSION: Exogenous ethylene applied to cv. 'Leccino' olives before processing was effective in inducing specific changes in the VOC profiles of the resulting oil. The effect was different depending on the ripening stage of the harvested olives. The lipoxygenase pathway (including the production of C5 compounds) and fermentative-related compounds appeared to be affected by the treatment. © 2020 Society of Chemical Industry.

Antidiabetic effect of olive leaf extract on streptozotocin-induced diabetes mellitus in experimental animals / Efecto antidiabético del extracto de hoja de olivo sobre la diabetes mellitus inducida por estreptozotocina en animales de experimentación

INTRODUCTION: recently, a relationship between diabetic complications and oxidative stress has been emphasized. There have been some studies showing the effect of olive leaf on hyperglycemia and diabetic complications due to its antioxidant properties. In many studies the effect of olive leaf on plasma total antioxidant level has been measured by different methods. Our study represents the first time it has been measured by a new method of total thiol disulfide homeostasis. OBJECTIVE: chronic exposure to hyperglycemia and hyperlipidemia contributes to the pathogenesis of diabetic complications through oxidative stress mediators. Thiol is one of the most important antioxidant barriers in humans, and thiol disulfide homeostasis is a new oxidative stress marker. We aimed to investigate the effect of olive leaf extract (OLE) obtained from fresh leaves of Olea europaea, var oleaster on diabetic complications through their hypoglycemic and antioxidant effect in diabetic rats. METHODS: twenty-eight Wistar albino rats aged 12-13 weeks were used in the study. The rats were divided into a control group (C), a diabetic control group (DC), a diabetic group treated with 200 mg/kg OLE (D+200), and a diabetic group treated with 400 mg/kg OLE (D+400), having 7 rats in each group. The treatment groups received OLE by the gavage method for 21 days. At the end of the study, all rats were sacrificed by cervical dislocation. Blood samples collected from the heart were centrifuged and glucose, total cholesterol, triglyceride, urea, uric acid, creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), lipid hydroperoxide (LOOH) level, and thiol-disulfide homeostasis were determined. The hemoglobin A1c (HbA1c) analysis was performed on complete blood. In addition, a tail flick test and hot plate modeling were performed to indicate pain perception loss. RESULTS: it was observed that OLE had no effect on serum glucose and HbA1c levels. On the contrary, OLE reduced the levels of total cholesterol (p < 0.01), urea (p < 0.01) and hot plate latency (p < 0.01) in a significant manner. Also, OLE showed a tendency to reduce LOOH levels and to increase thiol levels in a dose-dependent manner (p > 0.05). CONCLUSION: OLE supplementation for 21 days, at the amounts used, cannot protect against hyperglycemia but may be protective against hypercholesterolemia and tissue damage as caused by diabetes mellitus in rats

INTRODUCCIÓN: recientemente se ha resaltado la relación entre las complicaciones diabéticas y el estrés oxidativo. Se han realizado algunos estudios que muestran el efecto de la hoja de olivo sobre la hiperglucemia y las complicaciones diabéticas debido a sus propiedades antioxidantes. En muchos estudios, el efecto de la hoja de olivo sobre el nivel de antioxidantes totales en plasma se ha medido mediante diferentes métodos. En nuestro estudio se ha medido por primera vez mediante un nuevo método de homeostasis total de disulfuro de tiol. OBJETIVO: la exposición crónica a la hiperglucemia y la hiperlipidemia contribuye a la patogénesis de las complicaciones diabéticas a través de mediadores del estrés oxidativo. El tiol es una de las barreras antioxidantes más importantes de los seres humanos y la homeostasis del disulfuro de tiol es un nuevo marcador de estrés oxidativo. El objetivo fue0 investigar el efecto del extracto de hoja de olivo (OLE), obtenido de hojas frescas de Olea europaea var. Oleaster, sobre las complicaciones diabéticas a través del efecto hipoglucémico y antioxidante en ratas diabéticas. MÉTODOS: se utilizaron en el estudio veintiocho ratas albinas Wistar de 12-13 semanas de edad. Las ratas se agruparon en un grupo de control (C), un grupo de control diabético (DC), un grupo diabético tratado con 200 mg/kg de OLE (D+200) y un grupo diabético tratado con 400 mg/kg de OLE (D+400), teniendo 7 ratas en cada grupo. Los grupos de tratamiento recibieron OLE por el método del "gavage" durante 21 días. Al final del estudio, todas las ratas fueron sacrificadas por dislocación cervical. Las muestras de sangre recogidas del corazón se centrifugaron y se determinaron los niveles de glucosa, colesterol total, triglicéridos, urea, ácido úrico, creatinina, alanina-aminotransferasa (ALT), aspartato-aminotransferasa (AST), hidroperóxido de lípidos (LOOH) y homeostasis de tiol disulfuro. El análisis de la hemoglobina A1c (HbA1c) se realizó en sangre entera. Además, se realizaron pruebas de movimiento de la cola y modelado de placa caliente para indicar la pérdida de percepción del dolor. RESULTADOS: se observó que el OLE no tuvo efecto sobre los niveles de glucosa y HbA1c en el suero. Por el contrario, el OLE redujo los niveles de colesterol total (p < 0,01) y urea (p < 0,01), y la latencia de la placa caliente (p < 0,01) de manera significativa. Además, el OLE mostró tendencia a reducir el nivel de LOOH y a aumentar el nivel de tiol de manera dependiente de la dosis (p > 0,05). CONCLUSIÓN: la suplementación con OLE durante 21 días en las cantidades usadas no puede proteger contra la hiperglucemia pero sí puede proteger contra la hipercolesterolemia y el daño tisular causado por la diabetes mellitus en las ratas

Increased firmness and modified cell wall composition by ethylene were reversed by the ethylene inhibitor 1-methylcyclopropene (1-MCP) in the non-climacteric olives harvested at dark green stage - Possible implementation of ethylene for olive quality.

This study aimed to investigate the firmness retention by ethylene treatment in olive fruit, as observed earlier. Ethylene concentrations up to 1000 µL L-1 were applied to dark green 'Konservolia' olives harvested shortly before the green maturation and exposed to 20 °C for up to 9 d. Surprisingly, the results indicated a tendency to fruit firmness increases in concentration-dependent manner in a non-climacteric fruit. The highest concentration increased the firmness within 12 h by approximately 1.35-fold, but transiently for approximately up to 5 d; all ethylene inhibitors tested, either of synthesis (ethoxyvinyl glycine or AVG), or perception (1 -methyl-cyclopropene or 1-MCP, and silver nitrate) prevented the firmness increase. Texture was evaluated by firmness and changes in lignin, cellulose (CL), total pectins (TPC), water soluble pectins (WSP) and total non-cellulosic sugars (total sugars) concentrations, and in pectin esterification degree (DE) in the alcohol insoluble residue (AIR) of 'Konservolia' fruit pericarp during 1.5-d, 5-d and 10-d treatments with 1000 µL L-1 ethylene at 20 °C. Pectins in AIR were also extracted sequentially with cyclohexane-trans-1,2-diaminetetra-acetate (CDTA), Na2CO3, 1 M and 4 M KOH. The results showed that on day 1.5, the increased firmness was consistent with increased CL (crystalline formation, as observed by microscopy), total sugars and DE levels, but reduced WSP, whereas softening reversed the changes and lowered TPC and CDTA-soluble pectins in all fruit on day 10. However, on day 5 ethylene-treated olives exhibited a transitional phase during softening, characterized by retention of high TPC concentration and energy demand, as indicated by elevated respiration rates. The inhibitor 1-MCP, applied before ethylene, did inhibit the responses to ethylene treatment. Ethylene firming effect and the respective cell wall changes in olives are demonstrated for first time. The experiments could be used for research on perception and transcription responses to ethylene in olive, a non-climacteric fruit. In practice, high ethylene concentrations could also be beneficial for firmness increase and/or short storage of dark green olives.

A Na+ /Ca2+ exchanger of the olive pathogen Pseudomonas savastanoi pv. savastanoi is critical for its virulence.

In a number of compatible plant-bacterium interactions, a rise in apoplastic Ca2+ levels is observed, suggesting that Ca2+ represents an important environmental clue, as reported for bacteria infecting mammalians. We demonstrate that Ca2+ entry in Pseudomonas savastanoi pv. savastanoi (Psav) strain DAPP-PG 722 is mediated by a Na+ /Ca2+ exchanger critical for virulence. Using the fluorescent Ca2+ probe Fura 2-AM, we demonstrate that Ca2+ enters Psav cells foremost when they experience low levels of energy, a situation mimicking the apoplastic fluid. In fact, Ca2+ entry was suppressed in the presence of high concentrations of glucose, fructose, sucrose or adenosine triphosphate (ATP). Since Ca2+ entry was inhibited by nifedipine and LiCl, we conclude that the channel for Ca2+ entry is a Na+ /Ca2+ exchanger. In silico analysis of the Psav DAPP-PG 722 genome revealed the presence of a single gene coding for a Na+ /Ca2+ exchanger (cneA), which is a widely conserved and ancestral gene within the P. syringae complex based on gene phylogeny. Mutation of cneA compromised not only Ca2+ entry, but also compromised the Hypersensitive response (HR) in tobacco leaves and blocked the ability to induce knots in olive stems. The expression of both pathogenicity (hrpL, hrpA and iaaM) and virulence (ptz) genes was reduced in this Psav-cneA mutant. Complementation of the Psav-cneA mutation restored both Ca2+ entry and pathogenicity in olive plants, but failed to restore the HR in tobacco leaves. In conclusion, Ca2+ entry acts as a 'host signal' that allows and promotes Psav pathogenicity on olive plants.

Effects of TNT contaminated soil on vegetation at an explosive range by probing UPLC-qTOF MS profiling method.

Three tree species (Wild olive, Stinkwood and Cape Holy) and a shrub (Dovyalis caffra) were each potted in 20 L pots in order to evaluate the effect of 1,3,5-trinitrotoluene (TNT)-contaminated soil on vegetation. TNT contamination was established by dissolving flake TNT in acetone at 300 and 600 mg per kilogram soil concentrations. One pot for every species was left uncontaminated as control elements. A set of 16 samples, four contaminated, four uncontaminated aerial parts and their corresponding soils, were gathered. These were processed and subjected to a solid phase extraction method to isolate analytes of interest. A laboratory analytical method was applied using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-qTOF MS). For the UPLC-qTOF MS a gradient for the mobile phase was found which allowed the profiling and separation of metabolites in the aerial parts of the vegetation. This method allowed identification and quantification of major changes caused by TNT contaminated soil on vegetation. The Synapt High Definition Mass Spectrometer SYNAPT HDMS G1 was operated using the electrospray ionisation (ESI) technique in both positive and negative mode. A clear comparison of profiles was achieved and this has been demonstrated by the distinct newly-formed metabolites in the TNT contaminated vegetation understudy. The results have also shown that the chlorophyll region in the contaminated profile was also affected by the uptake of TNT degradation products. This has been observed in the contaminated profiles of Wild olive, Stinkwood and Cape Holly extracts indicating enhanced nutrient availability.

Salicylic acid modulates olive tree physiological and growth responses to drought and rewatering events in a dose dependent manner.

The predicted accentuation of drought events highlights the importance of optimize plants capacity to tolerate drought, but also the capacity to recovery from it, especially in species, as olive tree (Olea europaea L.), that grows in particularly susceptible regions. Three different concentrations (10, 100 and 1000 µM) of salicylic acid (SA), a stress signaling phytohormone, was sprayed on 3-year-old potted olive trees subjected to three successive drought and rewatering events. Trees responses to SA application are concentration dependent, being 100 µM the most effective concentration to improve drought tolerance and recovery capacity. During drought events, this effectiveness was achieved by osmolytes accumulation, leaf water status maintenance, reduced photosynthetic systems drought-associated damages, and by optimizing shoot/root ratio. The better plant fitness during drought allowed a fast recovery of the physiological functions upon rewatering and reduced the necessity to invest in extra repair damages, allowing the regrowth. The intense abscisic acid (ABA) signal close to upper epidermis in stressed controls suggests a "memory" of the worst water status displayed by those plants. SA attenuated the limitation of total biomass accumulation imposed by drought, mainly in root system, increased water use efficiency and lead to a higher intense signal of indoleacetic acid (IAA) in leaves during recovery period. In summary, in a suitable concentration, SA demonstrate to be a promising tool to increase drought adaptability of olive trees.

The paradox of oleuropein increase in harvested olives (Olea europea L.).

Olives are non-climacteric fruit. In a previous article, oleuropein (OE) increased substantially in fresh green olives exposed to 20 °C for 7 d, but the increases were lower in preharvest treated fruit with an ethylene synthesis inhibitor. The present aim was to investigate whether phenolic compounds, including OE, were affected by ethylene treatment in green harvested olives. Postharvest treatments with the ethylene perception inhibitor, 1 -methylcyclopropene (1-MCP) at 1.5 µL L-1 for 12 h, and/or ethylene at 1000 µL L-1 at 20 °C for up to 10 d were applied to fruits of 'Konservolia' cultivar. The results showed that ethylene and/or 1-MCP had similar effects on total phenolics (TP), total antioxidant capacity (TAC) and OE and these results are revealed for the first time in olives. Ethylene had no effect on green loss, but 1-MCP prevented it slightly. In all treated fruit, but not in controls, TP and TAC were increased soon after harvest and remained almost stable throughout exposure, whereas OE increased in controls and all treated at later stages (as confirmed by HPLC-DAD-ESI-MS) independently of degreening. The present experiments could be applied to studies of ethylene perception and transcription related responses in these non- climacteric fruit. In practice, harvested olives do not lose their antioxidant capacity, but the OE elevation in short-stored olives at ambient temperature might have an impact on olive products quality.

AOX1-Subfamily Gene Members in Olea europaea cv. "Galega Vulgar"-Gene Characterization and Expression of Transcripts during IBA-Induced in Vitro Adventitious Rooting.

Propagation of some Olea europaea L. cultivars is strongly limited due to recalcitrant behavior in adventitious root formation by semi-hardwood cuttings. One example is the cultivar "Galega vulgar". The formation of adventitious roots is considered a morphological response to stress. Alternative oxidase (AOX) is the terminal oxidase of the alternative pathway of the plant mitochondrial electron transport chain. This enzyme is well known to be induced in response to several biotic and abiotic stress situations. This work aimed to characterize the alternative oxidase 1 (AOX1)-subfamily in olive and to analyze the expression of transcripts during the indole-3-butyric acid (IBA)-induced in vitro adventitious rooting (AR) process. OeAOX1a (acc. no. MF410318) and OeAOX1d (acc. no. MF410319) were identified, as well as different transcript variants for both genes which resulted from alternative polyadenylation events. A correlation between transcript accumulation of both OeAOX1a and OeAOX1d transcripts and the three distinct phases (induction, initiation, and expression) of the AR process in olive was observed. Olive AOX1 genes seem to be associated with the induction and development of adventitious roots in IBA-treated explants. A better understanding of the molecular mechanisms underlying the stimulus needed for the induction of adventitious roots may help to develop more targeted and effective rooting induction protocols in order to improve the rooting ability of difficult-to-root cultivars.

Impact of airborne zinc pollution on the antimicrobial activity of olive oil and the microbial metabolic profiles of Zn-contaminated soils in an Italian olive orchard.

The growing of microbial resistance leads to a great interest about some natural alternatives to synthetic compounds. This study was carried out in two olive orchards (Olea europaea L., cv. Coratina) South Italy (Basilicata region), one located in a polluted area near a fertilizers factory releasing Zn and the other in a control unpolluted site, both managed with similar cultivation techniques. Olive oil samples were studied from both areas during 2014 and 2015. The soil microbiological status of the polluted and unpolluted orchards has been characterized and the antimicrobial effects of olive oils extracted from polluted plants (PP) and control plants (CP) against some phytopathogens have been explored. Results showed that the antibacterial activity of PP oil was significantly higher than CP and this could be due to the high content of some phenolic compounds elicited by air and soil Zn pollution (especially in the layer 0-20 cm). There is no detectable antifungal activity of the studied oils. The metabolic activity (both total and for each carbon substrate group), diversity and evenness of PP soil bacterial communities were significantly different from CP soil, while the effects of soil depth was negligible. The same parameters measured on soil fungal communities are lower in PP soil at 0-20 cm soil depth. The current research clarified the impact of atmospheric Zn pollution on the antimicrobial activity of olive oil and the soil microbial metabolic profiles. The bioactive substances extracted from olive oils growing in Zn-polluted area might be used as antibiotics.

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.

Effect of osmotic dehydration of olives as pre-fermentation treatment and partial substitution of sodium chloride by monosodium glutamate in the fermentation profile of Kalamata natural black olives.

This study examined the effect of osmotic dehydration of Kalamata natural black olives as pre-fermentation treatment in combination with partial substitution of NaCl by monosodium glutamate (MSG) on the fermentation profile of olives. Osmotic dehydration was undertaken by immersing the olives in 70% (w/w) glucose syrup overnight at room temperature. Further on, three different mixtures of NaCl and MSG with/without prior osmotic dehydration of olives were investigated, namely (i) 6.65% NaCl - 0.35% MSG (5% substitution), (ii) 6.30% NaCl - 0.70% MSG (10% substitution), (iii) 5.95% NaCl - 1.05% MSG (15% substitution), and (iv) 7% NaCl without osmotic dehydration (control treatment). Changes in the microbial association (lactic acid bacteria [LAB], yeasts, Enterobacteriaceae), pH, titratable acidity, organic acids, sugars, and volatile compounds in the brine were analyzed for a period of 4 months. The final product was subjected to sensory analysis and the content of MSG in olives was determined. Results demonstrated that osmotic dehydration of olives prior to brining led to vigorous lactic acid processes as indicated by the obtained values of pH (3.7-4.1) and acidity (0.7-0.8%) regardless of the amount of MSG used. However, in non-osmotically dehydrated olives, the highest substitution level of MSG resulted in a final pH (4.5) that was beyond specification for this type of olives. MSG was degraded in the brines being almost completely converted to γ-aminobutyric acid (GABA) at the end of fermentation. Finally, the sensory assessment of fermented olives with/without osmotic dehydration and at all levels of MSG did not show any deviation compared to the control treatment.

Leaf biochemical responses and fruit oil quality parameters in olive plants subjected to airborne metal pollution.

This study was carried out in two olive orchards (Olea europaea L., cv. Chemlali) located in a polluted area near a fertilizers factory and in a control unpolluted site, managed with similar cultivation techniques. The aim was to investigate the physiological and biochemical responses of polluted plants (PP), exposed to atmospheric metal contamination (Cd, Cu, Fe, Mn, Ni and Pb) as compared to control plants (CP). Leaves, roots and fruits of PP showed a depression of their non-enzymatic and enzymatic antioxidant defences and a disruption of their hormonal homeostasis. The anomalous physiological status of PP was also demonstrated by the lower values of pigments in leaves and fruits, as compared to CP. Atmospheric metals negatively affected olive oil chemical and sensory quality. However, despite metal deposition on fruit surfaces, the accumulation of potentially toxic metals in olive oil was negligible. Considering that olive oil is an important food product worldwide and that many productive olive orchards are exposed to several sources of pollution, this work could contribute to clarify the effects of atmospheric metal pollution on olive oil quality and its potential toxicity for humans.

Antioxidative response of olive to air emissions from tire burning under various zinc nutritional treatments.

Uniform 2-year old seedlings of a commercial olive cultivar (Olea europaea L., cv. Mahzam) were exposed or unexposed to the air pollution from the controlled burning of waste tires. The plants were supplied with zinc sulfate (ZnSO4) or synthesized Zn(Glycine)2 (Zn-Gly) or unsupplied with Zn. Exposure to air pollution resulted in oxidative damage to the olive, as indicated by the higher production of malondialdehyde (MDA). Supplement with Zn partly alleviated oxidative damage induced by the air emissions on the olive. Leaf concentration of MDA was higher at the active period of tire burning than that of the inactive one. Exposure to the emissions from tire burning significantly increased leaf ascorbate peroxidase (APX) activity. Supplement with Zn increased APX activity in plants exposed to the air pollution. According to the results, Zn nutrition was effective in alleviating oxidative stress induced by air pollution on the olive. APX seemed to play a significant role in alleviating oxidative damages induced by air emissions from tire burning on the olive; however, the role of other antioxidant enzymes should be addressed in future studies.

Salt stress induces differential regulation of the phenylpropanoid pathway in Olea europaea cultivars Frantoio (salt-tolerant) and Leccino (salt-sensitive).

Olive tree (Olea europaea L.) is an important crop in the Mediterranean Basin where drought and salinity are two of the main factors affecting plant productivity. Despite several studies have reported different responses of various olive tree cultivars to salt stress, the mechanisms that convey tolerance and sensitivity remain largely unknown. To investigate this issue, potted olive plants of Leccino (salt-sensitive) and Frantoio (salt-tolerant) cultivars were grown in a phytotron chamber and treated with 0, 60 and 120mM NaCl. After forty days of treatment, growth analysis was performed and the concentration of sodium in root, stem and leaves was measured by atomic absorption spectroscopy. Phenolic compounds were extracted using methanol, hydrolyzed with butanol-HCl, and quercetin and kaempferol quantified via high performance liquid-chromatography-electrospray-mass spectrometry (HPLC-ESI-MS) and HPLC-q-Time of Flight-MS analyses. In addition, the transcripts levels of five key genes of the phenylpropanoid pathway were measured by quantitative Real-Time PCR. The results of this study corroborate the previous observations, which showed that Frantoio and Leccino differ in allocating sodium in root and leaves. This study also revealed that phenolic compounds remain stable or are strongly depleted under long-time treatment with sodium in Leccino, despite a strong up-regulation of key genes of the phenylpropanoid pathway was observed. Frantoio instead, showed a less intense up-regulation of the phenylpropanoid genes but overall higher content of phenolic compounds. These data suggest that Frantoio copes with the toxicity imposed by elevated sodium not only with mechanisms of Na+ exclusion, but also promptly allocating effective and adequate antioxidant compounds to more sensitive organs.

Assessing ambient ozone injury in olive (Olea europaea L.) plants by using the antioxidant ethylenediurea (EDU) in Saudi Arabia.

The antiozonant chemical, ethylenediurea (N-[2-(2-oxo-1-imidazolidinyl)ethyl]-N'-phenylurea, abbreviated as EDU), was applied as stem injections or soil drenches to 5-year-old containerized plants of olive (Olea europaea L. cultivar Kalamata) in growth chambers in order to assess its ameliorative effects against realistic ozone (O3) stress. Visible injury symptoms were reduced greatly in individuals treated with EDU, with injection applications having greater protection than soil drenches. EDU application caused increases in the measured ecophysiological parameters compared to untreated individuals. In particular, the stem injection protected plants against photosynthetic impairment (unchanged net photosynthetic rates and intercellular CO2 concentration, in comparison to plants grown in filtered air). EDU application increased the protection of PSII from ambient O3 oxidative stress, although it did not retain the proportion of redox state of QA, pigment composition of photosynthetic apparatus and size of light-harvesting complex of PSII. However, the stem injection of plants with EDU induced lower non-photochemical quenching (NPQ) values in comparison to ambient air (-2 %), indicating a better photoprotection of PSII in comparison to soil drench application. EDU application caused increases in the morphological and biometric parameters compared to individuals exposed to ambient air. To the best of our knowledge, this is the first study highlighting the protection of Kalamata olive trees due to EDU in terms of growth, yield, visible injury, and photosynthetic performance. Furthermore, this study proved that EDU could be a low-cost and a low-technology efficient tool for assessing O3 effects on plant performances in the field in Saudi Arabia.

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.

454 Pyrosequencing of Olive (Olea europaea L.) Transcriptome in Response to Salinity.

Olive (Olea europaea L.) is one of the most important crops in the Mediterranean region. The expansion of cultivation in areas irrigated with low quality and saline water has negative effects on growth and productivity however the investigation of the molecular basis of salt tolerance in olive trees has been only recently initiated. To this end, we investigated the molecular response of cultivar Kalamon to salinity stress using next-generation sequencing technology to explore the transcriptome profile of olive leaves and roots and identify differentially expressed genes that are related to salt tolerance response. Out of 291,958 obtained trimmed reads, 28,270 unique transcripts were identified of which 35% are annotated, a percentage that is comparable to similar reports on non-model plants. Among the 1,624 clusters in roots that comprise more than one read, 24 were differentially expressed comprising 9 down- and 15 up-regulated genes. Respectively, inleaves, among the 2,642 clusters, 70 were identified as differentially expressed, with 14 down- and 56 up-regulated genes. Using next-generation sequencing technology we were able to identify salt-response-related transcripts. Furthermore we provide an annotated transcriptome of olive as well as expression data, which are both significant tools for further molecular studies in olive.

Isolation, molecular characterization and functional analysis of OeMT2, an olive metallothionein with a bioremediation potential.

Metallothioneins are essential in plants for metal detoxification in addition to their other roles in plant life cycle. This study reports the characterization of an olive (Olea europaea L. cv. Ayvalik) metallothionein with respect to molecular and functional properties. A cDNA encoding a type 2 metallothionein from olive was isolated from a leaf cDNA library, characterized and named OeMT2 after its molecular and functional properties. OeMT2 was expressed in Escherichia coli, and a single protein band was confirmed by protein gel blot analysis. Metal tolerance ability of bacterial cells expressing OeMT2 was determined against 0.2 mM CdCl2, 0.4 mM CdCl2 and 1 mM CuSO4 in the growth medium. Metal ion contents of bacterial cells expressing OeMT2 were measured by ICP. Metal tolerance assays and ICP measurements suggested that OeMT2 effectively binds Cu and Cd. Molecular analysis of OeMT2 revealed two introns, three exons, a short 3' UTR and a long 5' UTR. Comparing the genomic sequences from 14 olive cultivars revealed OeMT2 had both intron and exon polymorphisms dividing the cultivars into three groups. Real-time PCR analysis demonstrated that OeMT2 expresses more or less the same amounts in all tissues of the olive tree examined. The genomic copy number of OeMT2 was also determined employing real-time PCR which suggested a single copy gene in the olive genome while three other MT2 members were determined from the draft olive genome sequences of Ayvalik cultivar and that of wild olive. This is the first report on molecular and functional characterization of an olive metallothionein and shows that OeMT2 expressed in E. coli has the capability of effectively binding toxic heavy metals. This may suggest that OeMT2 plays an important role in metal homeostasis in addition to a good potential for environmental and industrial usage.