Effect of different leaf-to-fruit ratios on photosynthesis and fruit growth in olive (Olea europaea L.)

The influence of different leaf-to-fruit (l-t-f) ratios on leaf net photosynthetic rate (P _N) and fruit characteristics in Olea europaea L. cv. Frantoio was evaluated in 2001 and 2002. In both years, at the end of June, at the end of July, and in mid-September (first, second, and third time of treatment, respectively), defoliation or fruit thinning were performed to give l-t-f ratios of 1/1, 3/1, 5/1, and 7/1 (about 5.1, 15.3, 25.6, and 35.8 cm² of leaf area per fruit, respectively) on girdled and ungirdled peripheral shoots. P _N showed substantial seasonal and diurnal variations. In ungirdled shoots, no differences due to the different l-t-f ratios were observed, whereas in girdled shoots P _N tended to be lower in shoots with a high l-t-f ratio. In general, the values of leaf transpiration rate (E), stomatal conductance (g _s), sub-stomatal CO₂ concentration (C _i), and dark respiration rate (R _D) were associated with those of P _N. The starch and reducing sugar contents and area leaf dry mass (ADM) tended to be higher in leaves on girdled shoots with high l-t-f ratio, whereas in ungirdled shoots no differences related to the different l-t-f ratios were observed. The higher saccharide content in the leaves and the lower P _N, in the presence of a high C _i, observed in girdled shoots with a high l-t-f ratio suggests that the depression in P _N in these shoots may be the result of a feedback inhibition of the photosynthetic mechanism that regulates such a process. The l-t-f ratio did not have a substantial effect on fruit drop. In ungirdled shoots, the different l-t-f ratios did not produce significant differences in terms of fruit growth and leaf dry matter and saccharide contents, whereas in girdled shoots fruit growth increased as the l-t-f ratio increased, particularly when treatments were applied at the initial stage of fruit development. The percentage of oil in the pulp, on a dry matter basis, was not substantially influenced by girdling and l-t-f ratio. The abundant availability of assimilates seemed to cause earlier fruit ripening and, at the same time, retard fruit senescence (fruit detachment force). Shoot growth was slightly reduced by girdling. The abundant availability of assimilates, induced by girdling associated with high l-t-f ratio, stimulated flower induction.     

Biochemical characterization of a lipase from olive fruit (Olea europaea L.)

Lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is the first enzyme of the degradation path of stored triacylglycerols (TAGs). In olive fruits, lipase may determine the increase of free fatty acids (FFAs) which level is an important index of virgin olive oil quality. However, despite the importance of virgin olive oil for nutrition and human health, few studies have been realized on lipase activity in Olea europaea fruits. In order to characterize olive lipase, fruits of the cv. Ogliarola, widely diffused in Salento area (Puglia, Italy), were harvested at four stages of ripening according to their skin colour (green, spotted I, spotted II, purple). Lipase activity was detected in the fatty layer obtained after centrifugation of the olive mesocarp homogenate. The enzyme exhibited a maximum activity at pH 5.0. The addition of calcium in the lipase assay medium leads to an increment of activity, whereas in the presence of copper the activity was reduced by 75%. Furthermore, mesocarp lipase activity increases during olive development but declined at maturity (purple stage). The data represent the first contribution to the biochemical characterization of an olive fruit lipase associated to oil bodies.     

Phenological growth stages of olive trees (Olea europaea)

This paper describes the phenological growth stages of olive trees using the BBCH (Biologische Bundesanstalt, Bundessortenamt, Chemische Industrie) scale. Eight principal growth stages for bud, leaf and shoot development, inflorescence emergence, flowering, fruit development, fruit maturity and senescence and 32 secondary growth stages are described. Advantages of the BBCH scale over other methods are discussed.     

Relationship between pollen emission and fruit production in olive (Olea europaea L.)

Aerobiological data of pollen emission concentrations is used to predict fruit production. The principal aim of this work was to study the relationship between pollen emission patterns, emission homogeneity and fruit production in olive (Olea europaea L.). Data of daily pollen concentrations in the atmosphere during the flowering period, collected over a 20‐year period in Perugia (Central Italy), and the corresponding fruit production data were analysed. Correlation and regression analyses on the partial pollen amounts (subdivisions of the whole flowering period), their statistical variability (expressed as coefficients of variation of the daily pollen concentration), and the production values in the different years demonstrate that pollen emission, during the seven to ten day period immediately preceding the maximum pollen emission day, appears to be most closely related to fruit production. Moreover, the pollen emission homogeneity (minimal variability in daily pollen concentrations) during the “critical” flowering period is very important for fertilization.     

Ultrastructural aspects of the cytoplasmic origin and accumulation of oil in olive fruit (Olea europaea)

The development of oil bodies and oil droplets in fruits of olive was examined at the ultrastructural level. Both oil bodies that form in young fruits and oil droplets that develop with fruit maturation are cytoplasmic bodies. The formation of the small oil bodies occurs in localized regions of the cytoplasm. These bodies are closely associated and fuse together, forming a small oil droplet that protruded against and indented the vacuolar membrane. As the fruit matures, new oil bodies appear to form and fuse with the oil droplet, resulting in the formation of a single large oil droplet of about 30 μm in diameter in most mature mesocarp cells. The cytoplasmic region where the oil bodies formed had a granulate, ultrastructural appearance, and cytoplasmic components such as membranes and ribosomes were noticeably absent in these regions. The granulate material coated the oil bodies and oil droplets, and appeared as a thin, compressed band between the round inner surface of the droplets and the indented tonoplast. We suggest that this granulate material is involved in the synthesis of the oil and, with enlargement of the oil bodies, this coat becomes thinner in regions where they are closely associated, resulting in zones where confluence of the oil occurs.     

Lipoxygenase pathway in olive callus cultures (Olea europaea)

Stimulation of the lipoxygenase pathway in olive fruit initiates a cascade of reactions that begins with the regio- and stereospecific di-oxygenation of polyunsaturated fatty acids containing a cis, cis-1,4 pentadiene moiety. Later products of the pathway include volatiles that influence the organoleptic properties of harvested olive oil. In this study, we have investigated lipoxygenase activity in olive callus cultures, and found that there is evidence of several isoforms of the enzyme with different pH optima and substrate specificities. Endogenous lipoxygenase activity was detected throughout the growth cycle of olive callus, particularly during the log phase of growth, suggesting that olive lipoxygenases are intimately involved in growth. The most prominent lipoxygenase activity in tissue cultures was found to be soluble but significant activities were detected in the plastid fraction. In addition, hydroperoxide lyase (HPL) activity was measured in the calli; both 13- and 9-HPL activities were found which were particulate.     

Purification, product characterization and kinetic properties of lipoxygenase from olive fruit (Olea europaea L.).

Lipoxygenase from olive fruit was purified to homogeneity for the first time after differential centrifugations and by hydrophobic chromatography. The enzyme had a molecular mass of 98 kDa and exhibited a maximal activity at pH 6. Lipoxygenase had a better affinity for linoleic acid (Km=82.44 microM) than for linolenic acid (Km = 306.26 microM). It is inhibited by linoleate:oxygen oxidoreductase (LOX) inhibitors like nordihydroguaiaretic acid (NDGA) or propyl gallate. The reaction product was 13-hydroperoxy octadecadienoic acid when linoleic acid was used as substrate.     

Effect of polyethylene glycol,urea and sunflower meal on olive (Olea europaea var. europaea) leaf fermentation in continuous fermentors

A continuous culture system, inoculated with rumen liquor from goats or sheep, was used to study fermentation characteristics of olive leaves (OL). The effects of adding polyethylene glycol (PEG 4000 MW; 0, 2 or 20 g/100 g OL) and/or supplementing with urea (U) or sunflower meal (SM) (1.0 g N/100 g OM) were also studied. Olive leaf fermentation promoted low VFA production (35.2 mmol/d), predominantly of acetic acid, and low efficiency of VFA production (4.91 mol/kg digestible carbohydrates, DCHO). Both values increased with N supplementation, but effects of PEG were variable. No differences ascribed to the rumen inoculum origin were observed. The ammonia N concentration was increased only by supplementation with U. Total and amino acid N output was low and increased with N addition, but it was not affected by PEG treatment. No differences ascribed to the inoculum origin were observed concerning microbial N production rate or efficiency (g N/kg DCHO). There was no clear difference between sources of supplementary N regarding bacterial protein synthesis. On the basis of PEG results, the effect of tannins on OL fermentation was not important.     

Forecasting olive (Olea europaea) crop production by monitoring airborne pollen

Forecasting harvests of olives destined for the production of olive oil can be based on counts of airborne olive pollen, and meteorological and agronomic observations. This study was carried out during six consecutive years (1990–1995) in the Campiña Alta (an olive-producing region in the province of Córdoba, south-west Spain). Olive pollen totals are the annual sum of the concentrations recorded for the periods that the filters of a Cour trap were exposed. The meteorological data are the values of accumulated rainfall between 1 September and the following 15 April (a date prior to the beginning of olive flowering). The agronomic data are the forecast and actual productions for the province of Córdoba, supplied by the Board of Agriculture of the Andalusian government, and the actual production of the Campiña Alta, supplied at the end of harvest by private olive-growing co-operatives. The data were combined, and four mathematical equations were obtained to forecast the crop 6 months in advance, with varying degrees of reliability. The reliability was very high for an appropriate agricultural area. The most accurate equation isY=?1.90×10⁴+2.35X+53.94 (which forecasts the production of the Campiña Alta), whereY is the olive production (MT),X the olive pollen count,Z the rainfall prior to flowering, anda, b andc are constants. The least accurate equation is that relating olive pollen concentrations with olive production in the province of Córdoba.     

Isolation,culture and division of olive (Olea europaea L.) protoplasts

Protoplasts from Olea europaea L. have been compared in terms of their yield, viability, cell division and callus differentiation. Viable protoplasts were isolated from in vitro cultured leaves and cotyledons by an overnight incubation in an enzyme solution containing 1–1.5% driselase and 0.5M sucrose. This method allowed high yield of purified protoplasts, which floated and formed a dark green band at the meniscus, after centrifugation. Purified protoplasts were diluted to 3×10⁴ protoplasts·ml^–1 in culture medium. After cell wall regeneration, protoplasts gradually increased their volumes under appropriate conditions. The first divisions occurred during the second week in culture. Division efficiency ranged from 5.2 to 9.8% after 20 days in culture. Two weeks later visible microcolonies developed only from cotyledon protoplasts. After 6 weeks in culture, the microcalli were transferred to a solidified culture medium with 0.6% agarose, which induced active callus growth.Abbreviations OM olive proliferation medium, Rugini 1984 - Omg OM for the germination of olive embryos - OMr=OM for root induction - OMp=OM for protoplasts - OMc=OM for callus - BN Bourgin and Nitsch medium 1967 - IBA indol-3-butyric acid - NAA naphthalene acetic acid - 2,4-D dichlorophenoxyacetic acid.     

Molecular identification of Greek olive (Olea europaea) cultivars based on microsatellite loci

Olea europaea is one of the oldest species of domesticated trees. We used microsatellite markers for fingerprinting and for evaluation of genetic similarity and structure of 26 Greek olive cultivars, which cover most of the olive cultivation regions of Greece, including previously undescribed denominations from northern Greece. Eighty-one alleles were revealed with six SSR loci that were selected as most informative of 10 SSR primers that were initially investigated. The number of alleles per locus varied from 7 to 20 (mean, 13.5). Heterozygosity ranged from 0.240 at locus DCA-3 to 0.826 at locus UDO99-9, with a mean value of 0.600. Analysis of 104 trees representing 26 denominations (four trees per denomination) revealed 26 distinct SSR profiles, indicating 26 olive cultivars; no intracultivar variability was observed. Genetic and geographic distances were not significantly correlated, based on the Mantel test. These SSR loci allowed unequivocal identification of all the cultivars and will be useful for future breeding and olive germplasm management efforts.     

Glycosyl-linkage composition of tomato fruit cell wall hemicellulosic fractions during ripening

Hemicelluloses were extracted from isolated tomato ( Lycopersicon esculentum Mill. cv. Rutgers) pericarp cell wall material at 3 different stages of ripeness with 4 M and 8 M KOH. Little change in molecular weight or composition of 4 M KOH-extracted material was observed during ripening. However, the composition of 8 M KOH-extracted material changed, and a relative increase in polymers of < 40 kDa was observed during ripening. Changes in glycosyl linkage composition of the 8 M KOH hemicellulosic material were detected, including increases in 4-linked mannosyl, 4,6-linked mannosyl, and 4-linked glucosyl, and decreases in 5-linked arabinosyl residues in polymers of < 40 kDa, and decreases in terminal glocosyl residues in polymers of > 40 kDa. These data may indicate that de novo hemicellulose synthesis occurs throughout tomato fruit ripening, even at the red ripe stage.     

The age of monumental olive trees (Olea europaea) in northeastern Spain

Trees can reach ages that in some cases amount to thousands of years. In the Mediterranean region, olive trees (Olea europaea) have traditionally been considered a particularly long-lived species. The main objective of this study was to assess the age of large olive trees considered to be millenarian and classified as monumental trees in northeastern Spain. We extracted cores of 14 individuals and obtained 8 sections of trees which had already been cut in the area where the largest olive trees in the northeastern Iberian Peninsula are found. The age of the sampled olive trees was assessed by counting the number of annual growth rings. Tree rings did not cross-date well, neither within nor between individuals, but boundaries between likely annual rings were clearly distinct. We found a linear relationship between DBH and tree age (in years) (Age = 2.11 × diameter(cm) + 88.93, R² = 0.80), which was used to estimate the age of unsampled olive trees. The maximum estimated age (627 ± 110 years) is among the greatest ages reported for olive trees around the world (700 years) and among the oldest trees in Mediterranean ecosystems.     

Stem and whole-plant hydraulics in olive (Olea europaea) and kiwifruit (Actinidia deliciosa)

A field study and an experiment under controlled conditions using pressure-flux relationships were conducted to compare the stem and whole-plant conductance in olive (Olea europaea) and kiwifruit (Actinidia deliciosa) species. Anatomical observations were also made on one-year-old stem to determine the conductive area of vessels (A _ves) and the total xylem area (A _xyl). Results show that A _ves of kiwifruit twigs was ~2.5-fold of that in olive twigs, and the hydraulically weighted mean diameter was up to threefold that of the olive ones. One-year-old olive twigs had lower hydraulic conductivity (k) than the kiwifruit, while values of leaf-specific conductivity (i.e. k normalised per unit leaf area) were higher than the kiwifruit (i.e. ~49 and 29 × 10^?6 kg m^?1 s^?1 MPa^?1, respectively). In the field experiment, the flux of sap (heat balance method) and differences in water potential through the soil–plant system (ΔP) were used for both species to calculate the whole-plant conductance that was normalised per unit leaf area (leaf-specific whole-plant conductance, K _plant,LA). Values of K _plant,LA are attributable to the combined effect of the ΔP and anatomical features of conduits. Olive species showed a larger ΔP (2.4 MPa at midday) than the kiwifruit (0.5 MPa) which contributed to lower K _plant,LA in Olea than the Actinidia plants. This information, combined with vessel density data, contributes to explain differences amidst olive and kiwifruit species, in terms of susceptibility to some drought-related hydraulic impairments induced by the Mediterranean environment.     

Low soil temperatures induce water deficits in olive (Olea europaea) trees

Olive trees are often subjected to low temperatures during winter. To quantify the effects of low temperatures on the water relations of olive trees, we studied the responses to low soil temperatures on winter days of variable evaporative demand (ET₀) in 1-year-old potted olive (Oleo europaea L. cv. Picual) trees in 1996 and 1997. Low night (2.5 and 5.2°C) but ambient day soil temperatures (above 10°C) did not affect stomatal conductance (g_s), leaf (Ψ_leaf) and stem (Ψ_stem) water potentials. Soil temperature levels inducing water stress in olive trees were determined for winter days with ET₀ typical for southern Spain (ET₀= 1.5 ± 0.3 mm day^?1). Leaf and stem water potential decreased and root hydraulic resistance (r_root) increased when trees were exposed to night and day soil temperatures below 10°C. Stomatal conductance was not affected at soil temperatures between 6.4 and 10°C, but decreased at temperatures below 6.4°C. The soil temperature levels affecting the water uptake of olive trees remained relatively constant over the range of ET₀ of 1-2 mm day^?1 during winter and early spring months. However, the soil temperature influencing gs appeared to be more variable and was affected by ET₀. Olive tree recovery from low soil temperature stress depended on stress duration and severity and interacted with ET₀. Recovery of ψ started already during the stress period, probably induced by stomatal closure and high r_root, thus allowing tree rehydration overnight. Root hydraulic resistance contributed the major part of whole-tree hydraulic resistance in response to cold stress, accounting for 76 and 89% at 6.4 and 4.6°C, respectively; which indicates that r_root is the primary control of the water status in olive trees under low temperatures.     

Effects of drought stress on phenolic accumulation in greenhouse-grown olive trees (Olea europaea)

The present study evaluates the effects of severe drought stress on the content of phenolic compounds in olive leaves, namely hydroxytyrosol, tyrosol, p-hydroxybenzoic acid, catechin, luteolin 7-O-rutinoside, luteolin 7-O-glucoside, apigenin 7-O-glucoside, quercetin, apigenin, pinoresinol, oleuropein and verbascoside in greenhouse-grown plantlets. The results showed that oleuropein, verbascoside, luteolin 7-O-glucoside and apigenin 7-O-glucoside were the most important phenolic compound of stressed olive plants and can represent up to 84% of the total amount of the identified phenolic compounds. Application of drought stress caused a significant increase in the level of oleuropein (87%), verbascoside (78%), luteolin 7-O-glucoside (72%) and apigenin 7-O-glucoside (85%), when compared to the control. The elevated values of these phenolic compounds can help controlling the water status of olive plants and avoiding serious oxidative damage induced by water deficit stress. To our knowledge, this is the first report to show the boost in the concentrations of verbascoside, luteolin 7-O-glucoside and apigenin 7-O-glucoside in the leaves of olive trees after water deficit stress.     

Identification of simple sequence repeats (SSRs) in olive ( Olea europaea L.)

A small insert genomic library of Olea europaea L., highly enriched in (GA/CT)n repeats, was obtained using the procedure of Kandpal et al. (1994). The sequencing of 103 clones randomly extracted from this library allowed the identification of 56 unique genomic inserts containing simple sequence repeat regions made by at least three single repeats. A sample of 20 primer pairs out of the 42 available were tested for functionality using the six olive varieties whose DNA served for library construction. All primer pairs succeeded in amplifying at least one product from the six DNA samples, and ten pairs detecting more than one allele were used for the genetic characterisation of a panel of 20 olive accessions belonging to 16 distinct varieties. A total of 57 alleles were detected among the 20 genotypes at the ten polymorphic SSR loci. The remaining primer pair allowed the amplification of a single SSR allele for all accessions plus a longer fragment for some genotypes. Considering the simple sequence repeat polymorphism, 5.7 alleles were scored on average for each of the ten SSR loci. A genetic dissimilarity matrix, based on the proportion of shared alleles among all the pair-wise combinations of genotypes, was constructed and used to disentangle the genetic relationships among varieties by means of the UPGMA clustering algorithm. Graphical representation of the results showed the presence of two distinct clusters of varieties. The first cluster grouped the varieties cultivated on the Ionian Sea coasts. The second cluster showed two subdivisions: the first sub-cluster agglomerated the varieties from some inland areas of Calabria; the second grouped the remaining varieties from Basilicata and Apulia cultivated in nearby areas. Results of cluster analysis showed a significant relationship between the multilocus genetic similarities and the geographic origin of the cultivars. Received: 2 February 2001 / Accepted: 1 June 2001     

LTR retrotransposon dynamics in the evolution of the olive (Olea europaea) genome

Improved knowledge of genome composition, especially of its repetitive component, generates important information for both theoretical and applied research. The olive repetitive component is made up of two main classes of sequences: tandem repeats and retrotransposons (REs). In this study, we provide characterization of a sample of 254 unique full-length long terminal repeat (LTR) REs. In the sample, Ty1-Copia elements were more numerous than Ty3-Gypsy elements. Mapping a large set of Illumina whole-genome shotgun reads onto the identified retroelement set revealed that Gypsy elements are more redundant than Copia elements. The insertion time of intact retroelements was estimated based on sister LTR’s divergence. Although some elements inserted relatively recently, the mean insertion age of the isolated retroelements is around 18 million yrs. Gypsy and Copia retroelements showed different waves of transposition, with Gypsy elements especially active between 10 and 25 million yrs ago and nearly inactive in the last 7 million yrs. The occurrence of numerous solo-LTRs related to isolated full-length retroelements was ascertained for two Gypsy elements and one Copia element. Overall, the results reported in this study show that RE activity (both retrotransposition and DNA loss) has impacted the olive genome structure in more ancient times than in other angiosperms.     

Ethylene evolution from various developing organs of olive (Olea europaea) after excision

Ethylene evolution from leaves, stems, inflorescences and fruits of the olive plant ( Olea europaea L.) cv. Manzanillo was studied at various stages of their development. Mature non-growing organs, particularly leaves, have a constant, low, and uniform rate of ethylene evolution. Ethylene evolution from detached mature olive leaves was constant during the first 12 h after excision. Leaves on shoots maintained in vitro kept a constant rate of ethylene evolution for at least the first 5–6 days. Leaf injury significantly increased ethylene evolution. Ethylene evolution from injured and non-injured control leaves could be markedly inhibited aminoethoxyvinylglycine (AVG) applied to the leaves or fed to the shoot. The use of excised olive shoots and leaves as an in vitro model system for studies of induced metabolic processes such as abscission and developing water stress was suggested.