Three-year field response of young olive trees (Olea europaea L., cv. Arbequina) to soil salinity: Trunk growth and leaf ion accumulation

Irrigated olive is rapidly increasing in arid and semiarid areas, many of which may be negatively affected by soil salinity. We evaluated changes in trunk growth and leaf Cl^–, Na⁺ and K⁺ concentrations in young Arbequina olives (Olea europaea L.) grown in a saline-sodic field over a three-year period. The trunk diameter was measured at the beginning and the end of the 1999 (70 trees), 2000 (59 trees) and 2001 (42 trees) growing periods. Leaves, sampled in August of each year, were analyzed for Cl^–, Na⁺ and K⁺ concentrations. Soil salinity (apparent electrical conductivity, ECa) of each monitored tree was measured 14 times during the 1999–2001 experimental period with an electromagnetic sensor and converted to root zone electrical conductivity of the soil saturation extract (ECe) based on ECa–ECe calibration curves. Salinity tolerance was determined using the Maas and Hoffman threshold–slope response model. Based on salinity thresholds (ECe_thr), the tolerance of olive in terms of trunk growth was high in 1999 (ECe_thr = 6.7 dS m^–1), but declined with age and time of exposure to salts by 30% in 2000 (ECe_thr = 4.7 dS m^–1) and by 55% in 2001 (ECe_thr = 3.0 dS m^–1). Based on the high absolute slopes obtained in all years (values between 16% and 23% dS^–1 m), olive was classified as very sensitive to ECe values above the threshold. Trunk growth thresholds based on leaf ion concentrations varied, depending on years, between 2.6 and 4.0 mg g^–1 (Cl_thr) and between 1.0 and 1.2 mg g^–1 (Na_thr), indicating that Arbequina olive was less sensitive to leaf Cl^– and much more sensitive to leaf Na⁺ than values reported as toxic in greenhouse studies. Leaf K⁺ slightly decreased with increasing salinity, whereas the K⁺/Na⁺ ratio sharply decreased with increasing salinity. We concluded that the initial salinity tolerance of olive was high, but declined sharply with time of exposure to salts and became quite sensitive due primarily to increasing toxic concentrations of Na⁺ in the leaves.     

Ultrastructural evidence of endoplasmic reticulum changes during the maturation of the olive pollen grain (Olea europaea L., Oleaceae)

Characteristic features of rough endoplasmic reticulum (rER) distribution and proliferation were noted during olive pollen (Olea europaea L.) development, suggesting the physiological significance of this organelle. Initially scarce in the young microspore, ER increases as cytoplasmic vacuoles form. At the vacuolated microspore stage the cytoplasm contains numberous polysomes and elongated rER cisternae arranged preferentially in stacks, with an average intracisternal width of 0.07 µm. Stacks persist in the bicellular pollen grain but consist of fewer, shorter, dilated cisternae (mean intracisternal width 0.1 µm) containing a considerable electron-dense matrix. Cisternae in the mature grain are fragmented, leaving behind an ER of swollen pockets. Pockets of ER containing a material of greater electron density are evenly deposited along the plasmalemma, in close relation with it. A dense material is seen in the tubules of the apertural region, which was lacking in earlier stages. Our results show that ER may be involved in protein transport to the intine.     

In vitro propagation of olive (Olea europaea L.) cv. Koroneiki

Single node explants of 'Koroneiki' olive trees werecultured for one month on a modified Driver-Kuniyuki for Walnut medium, lackinggrowth regulators. The explants were subcultured once a month on a mediumsupplemented with zeatin riboside, 6-(--dimethylallylamino)purine,6-benzyladenine or thidiazuron. Zeatin riboside proved to be superior to othercytokinins in inducing shoot proliferation. The combination of olive knotextract at 25 or 50 mg l^–1 with cytokininssuppressed shoot proliferation. After two months at the proliferation stage,theexplants were cultured for one week in the dark in 1 ml liquidWoody Plant Medium supplemented with IBA, -NAA or IBA+-NAA. Theexplants were then transferred to the same solid medium lacking growthregulators, with a small layer of perlite on the surface. The combination ofthetwo auxins at 1+1 mg l^–1 resulted in almost 76%rooting. The combination of olive knot extract at 50 mgl^–1 with auxins increased the rooting percentage up toalmost 87%. Artificial infection of explants with the bacteriumPseudomonas savastanoi pv. savastanoiinhibited rhizogenesis, even in the presence of auxins. Rooted explants weresuccessfully acclimatised under a mist system, with the survival rate reachingalmost 75%.     

Morphological and cytological development and starch accumulation in hermaphrodite and staminate flowers of olive (Olea europaea L.)

In olive (Olea europaea L.), the formation of functionally staminate flowers rather than fully functional hermaphrodites is one of the major factors limiting fruit set, as flowers with aborted pistils are incapable of producing fruit. Studies conducted on various angiosperm species have shown a correlation between flower abortion and starch content. Thus, it is important to know if starch content plays a role in regulating pistil development in olive and if so, what mechanism regulates starch distribution. Cyto-histological observations of staminate and hermaphrodite olive flowers show that pistil development in staminate flowers is interrupted after the differentiation of the megaspore mother cell. At that stage, starch grains were only detected in the ovary, style and stigma of the hermaphrodite flowers. No starch was observed in the pistils of the staminate flowers. This finding suggests a tight correlation between starch content and pistil development. The secondary origin of starch within the flower is indicated by low chlorophyll content in the gynoecium, undetectable Rubisco activity in the pistils of these two kinds of flowers and by the ultrastructure of the plastids observed by transmission electron microscope analysis. The plastids have few thylakoid membranes and grana and in the staminate flowers appeared very similar to proplastids. Considering differences in starch content between staminate and hermaphrodite flowers and the secondary origin of the starch, differences in pistil development in the staminate and hermaphrodite flowers could be related to differences in the sink strength of these two types of flowers.     

Aerobiological and meteorological factors’ influence on olive (Olea europaea L.) crop yield in Castilla-La Mancha (Central Spain)

Knowledge of the main biological and climate factors influencing final harvest is becoming increasingly necessary in order to obtain reliable crop estimates and, thus, ensure optimised, effective private crop management. This knowledge is also of great value to public agricultural institutions for the planning of government subsidies. Castilla-La Mancha (Central Spain) is the second largest olive-oil-producing region in Spain, the highest olive-oil-producing country in the world. This study sought to identify the main factors influencing olive fruit production in this region, including atmospheric pollen as an index of flowering intensity, and meteorological data over the flowering and fruiting seasons in two main olive-producing provinces of the region: Ciudad Real and Toledo. Statistical analysis indicated that the annual pollen index (PI) was the variable influencing most the final olive crop in both provinces. The maximum temperature in March was the meteorological variable affecting most the annual olive crop. Also, the rainfall registered in October influences the final fruit production. The integration of aerobiological and meteorological data represents an important step forward in the development of future crop forecasting models in the region of Castilla-La Mancha.     

Vegetative growth retardation,improved rooting and viability of olive cuttings in response to application of growth retardants

Spray and soil treatments of paclobutrazol and uniconazole were applied to young and mature olive plants and olive cuttings. Two clear phases, were found in the growth response of olive shoots to growth retardants: an early phase, which retards and even inhibits growth considerably; and a later phase, during which the shoots are released from the retardation and start to elongate rapidly. A somewhat slower response of the plants to soil application than to spray application of growth retardants was noticed. Paclobutrazol enhanced the rooting of cv. Manzanillo cuttings, whether applied to the mother plants or to the cuttings themselves. Indole-3-butyric acid (IBA) was needed in both cases. Sprouting was shown to reduce rooting. Paclobutrazol significantly inhibited sprouting and increased the meristematic activity in the base of the cuttings. Rooting of the hard-to-root cv. Kalamata was not enhanced by the treatments although callus formation was induced and viability was prolonged.     

Ultrastructural and cytochemical examination of the nuclear crystalloid inclusions of Olea europaea L. mesophyll cells

Summary The nuclei of mesophyll cells of olive trees contain numerous sizeable crystalloid inclusions. Cytochemical examination using epoxy resin-embedded, semithin-sectioned tissue indicated the presence of proteins and oligoor polysaccharides in these inclusions. Their electron microscopical analysis revealed a crystalline substructure consisting of intersected subunits of high order. The spacing of the lattice fibrils and the angles of intersection were determined and used to establish a model of the unit cell of crystallization. It is suggested that the nuclear crystalloids of olive trees consist of glycoprotein molecules. They differ from the intranuclear crystalloids observed in other species predominantly in the high density of their subunit arrangement.     

Identification of ancient Olea europaea L. and Cornus mas L. seeds by DNA barcoding

The analysis of ancient DNA (aDNA) provides archaeologists and anthropologists with innovative, scientific and accurate data to study and understand the past. In this work, ancient seeds, found in the "Mora Cavorso" archaeological site (Latium, Central Italy), were analyzed to increase information about Italian Neolithic populations (plant use, agriculture, diet, trades, customs and ecology). We performed morphological and genetic techniques to identify fossil botanical species. In particular, this study also suggests and emphasizes the use of DNA barcode method for ancient plant sample analysis. Scanning electron microscope (SEM) observations showed seed compact structure and irregular surface but they did not permit a precise nor empirical classification: so, a molecular approach was necessary. DNA was extracted from ancient seeds and then it was used, as template, for PCR amplifications of standardized barcode genes. Although aDNA could be highly degraded by the time, successful PCR products were obtained, sequenced and compared to nucleotide sequence databases. Positive outcomes (supported by morphological comparison with modern seeds, geographical distribution and historical data) indicated that seeds could be identified as belonging to two plant species: Olea europaea L. and Cornus mas L.     

Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination

Background and Aims

A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized.

Methods

The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods.

Key Results

Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles.

Conclusions

In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma.     

De Novo Assembly and Functional Annotation of the Olive (Olea europaea) Transcriptome

Olive breeding programmes are focused on selecting for traits as short juvenile period, plant architecture suited for mechanical harvest, or oil characteristics, including fatty acid composition, phenolic, and volatile compounds to suit new markets. Understanding the molecular basis of these characteristics and improving the efficiency of such breeding programmes require the development of genomic information and tools. However, despite its economic relevance, genomic information on olive or closely related species is still scarce. We have applied Sanger and 454 pyrosequencing technologies to generate close to 2 million reads from 12 cDNA libraries obtained from the Picual, Arbequina, and Lechin de Sevilla cultivars and seedlings from a segregating progeny of a Picual × Arbequina cross. The libraries include fruit mesocarp and seeds at three relevant developmental stages, young stems and leaves, active juvenile and adult buds as well as dormant buds, and juvenile and adult roots. The reads were assembled by library or tissue and then assembled together into 81 020 unigenes with an average size of 496 bases. Here, we report their assembly and their functional annotation.     

Heat requirement for the onset of the Olea europaea L. pollen season in several sites in Andalusia and the effect of the expected future climate change

Olives are one of the largest crops in the Mediterranean region, especially in Andalusia, in southern Spain. A thermal model has been developed for forecasting the start of the olive tree pollen season at five localities in Andalusia: Cordoba, Priego, Jaen, Granada and Malaga using airborne pollen and meteorological data from 1982 to 2001. Threshold temperatures varied between 5°C and 12.5°C depending on bio-geographical characteristics. The external validity of the results was tested using the data for the year 2002 as an independent variable and it confirmed the models accuracy with only a few days difference from predicted values. All the localities had increasingly earlier start dates during the study period. This could confirm that olive flower phenology can be considered as a sensitive indicator of the effects of climate fluctuations in the Mediterranean area. The theoretical impact of the predicted climatic warming on the olives flowering phenology at the end of the century is also proposed by applying Regional Climate Model data. A general advance, from 1 to 3 weeks could be expected, although this advance will be more pronounced in mid-altitude inland areas.     

Stomatal and photosynthetic responses of olive (Olea europaea L.) leaves to water deficits

The leaf gas exchange of mature olive trees (Olea europaea L.) was characterized over a wide range of water deficits in the field during 1998, in Cordoba, Spain. Leaf photosynthesis (A) and stomatal conductance (g_l) responded diurnally and seasonally to variations in tree water status and evaporative demand. In the absence of water stress, A and g_l were generally high during autumn and low in days of high vapour pressure deficits (VPD). Leaf A varied between 0 and 2 µmol m^?2 s^?1 under severe water deficits that lowered the stem water potential (Ψ_x) to ?8·0 MPa, but recovered rapidly following rehydration. Transpiration efficiency (TE) was curvilinearly related to VPD and not influenced by water deficits except in cases of severe water stress, where low TE values were observed at Ψ_x below ?4 MPa. Three models of leaf conductance were calibrated and validated with the experimental data; two were based on the model proposed by Leuning (L) and the other was derived from the widely used Jarvis (J) model. The L models performed better than the J model in two validation tests. The scatter of the predictions and the limited accuracy of all three models suggest that, in addition to the physiological and environmental variables considered, there are additional endogenous factors influencing the g_l of olive leaves.     

Kinetin regulation of growth and secondary metabolism in waterlogging and salinity treated Vigna sinensis and Zea mays

Waterlogging mostly increased fresh weight and water content in shoots and roots of Vigna sinensis and Zea mays while salinity seemed to have a decreasing effect. There was a marked induction of proline in shoots and roots of both plants by salinity with lower values in logged plants. In addition, anthocyanin content was increased in Vigna sinensis by both treatments and in Zea mays only by salinity. Meanwhile the treatments significantly accumulated phenolic compounds in plant shoots. Also there were increased activities of phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) in shoots and roots of both plants. Foliar application of kinetin equilibrated, if any, the effects of both treatments on contents of proline, anthocyanin and phenolic compounds as well as activities of PAL and TAL in shoots and roots of treated plants. These findings reveal that kinetin alleviates the stress symptoms and regulates the changes in phenolic metabolism of waterlogged or salinity treated Vigna sinensis and Zea mays.     

Irrigation effects on daily and seasonal variations of trunk sap flow and leaf water relations in olive trees

Irrigation effects on whole-plant sap flow and leaf-level water relations were characterised throughout a growing season in an experimental olive (Olea europaea L.) orchard. Atmospheric evaporative demand and soil moisture conditions for irrigated and non-irrigated olive trees were also monitored. Whole-plant water use in field-grown irrigated and rain fed olive trees was determined using a xylem sap flow method (compensation heat-pulse velocity). Foliage gas exchange and water potentials were determined throughout the experimental period. Physiological parameters responded diurnally and seasonally to variations in tree water status, soil moisture conditions and atmospheric evaporative demand. There was a considerable degree of agreement between daily transpiration deduced from heat-pulse velocity and that determined by calibration using the Penman–Monteith equation in the field. Summer drought caused decreasing leaf gas exchange and water potentials, and a progressive increase in hydraulic conductance (stronger in non-irrigated than irrigated trees), probably attributable to modifications in hydraulic properties at the soil-root interface. Negligible hysteresis, attributable to low plant capacitance, was observed in the relationship between leaf water potential and sap flow. A proportional decrease in maximum daily leaf conductance with increasing vapour pressure deficit was observed, while mean daytime canopy stomatal conductance decreased with the season. As a result, plant water use was limited and excessive drought stress prevented. Non-irrigated olive trees recovered after the summer drought, showing a physiological behaviour similar to that of irrigated trees. In addition to physiological and environmental factors, there are endogenous keys (chemical signals) influencing leaf level parameters. Olive trees are confirmed to be economical and sparing users of soil water, with an efficient xylem sap transport, maintenance of significant gas exchange and transpiration, even during drought stress.     

Clinical significance of allergic sensitization toOlea europaea L. pollen in Naples area,Italy

Summary The aim of this study is the evaluation of clinical significance of the allergic sensitization toOlea europaea L. pollen allergens in the Naples area, because in the last 10 years an increasing frequency of IgE mediated sensitization to these allergens has been observed. 4124 patients of both sexes (2989 adults and 1153 children) consecutively examined in our Allergological Center from January 1, 1988 to December 30, 1989 have been considered in this study. The patients were selected on the basis of a history of rhinitis (R) and/or asthma (A) and were evaluated by clinical examination, SPT, RAST and specific or aspecific BPT. Among 2267 adults and 879 children sensitized to almost one aeroallergen we found respectively 306 (13.49%) and 75 (8.53%) subjects with an allergic sensitization toOlea europaea L. pollen. The frequency of clinical manifestation was respectively R+A (58.16%), R (39.54%), A (2.28%) in adults; R+A (61.33%), R (26.66%), A (12%) in children. A monosensitization toOlea europaea L. pollen was found in 1.33% of children and in 2.28% of adults examined. The association with other allergens especially with mites and/or other pollen was very common.     

Electrical resistance changes during exposure to low temperature measure chilling and freezing tolerance in olive tree (Olea europaea L.) plants

Electrical resistance changes in different organs of four olive tree (Olea europaea L.) varieties, characterized by different tolerance to chilling and freezing, were examined, during exposure to low temperature. Apparent critical temperatures (CT) and freezing temperatures (T_fr) were identified on the basis of the electrical resistance changes. Both temperatures were lower for the more chilling‐tolerant genotypes. From the apparent critical temperatures, the absolute critical temperature (CT_abs) and the time delay of the chilling signal transduction process were calculated. In shoots, CT_abs varied from 8·8 °C for Ascolana (chilling‐tolerant variety) to 13·6 °C for Coratina (chilling‐sensitive variety). The magnitude of the transduction time was very similar (about 2 min) for the three genotypes that are more sensitive to chilling, whereas it was significantly higher (about 3 min) for the most tolerant genotype. Different freezing temperatures were observed for different organs. It would appear from this experiment that the order of sensitivity is roots > leaves > shoots > vegetative buds. Accord was found between the absolute critical temperature of electrical resistance and the critical temperature of membrane potential. The occurrence of electrical resistance changes in the tissues of the olive trees exposed to low temperature suggests the use of this experimental procedure as a quick, easy and non‐destructive tool to screen plant tissues for chilling tolerance. The strong dependence of the electrical resistance on low temperature, and the critical temperature of around 10 °C, can yield interesting information about the lowest thermal limits for the continuation of normal physiological processes and therefore about the adaptability of plants to particular environments.