Variations in Fruit Ploidy Level and Cell Size between Small- and Large-Fruited Olive Cultivars during Fruit Ontogeny.

Olive (Olea europaea L.) is one of the major oil fruit tree crops worldwide. However, the mechanisms underlying olive fruit growth remain poorly understood. Here, we examine questions regarding the interaction of endoreduplication, cell division, and cell expansion with olive fruit growth in relation to the final fruit size by measuring fruit diameter, pericarp thickness, cell area, and ploidy level during fruit ontogeny in three olive cultivars with different fruit sizes. The results demonstrate that differences in the fruit size are related to the maximum growth rate between olive cultivars during early fruit growth, about 50 days post-anthesis (DPA). Differences in fruit weight between olive cultivars were found from 35 DPA, while the distinctive fruit shape became detectable from 21 DPA, even though the increase in pericarp thickness became detectable from 7 DPA in the three cultivars. During early fruit growth, intense mitotic activity appeared during the first 21 DPA in the fruit, whereas the highest cell expansion rates occurred from 28 to 42 DPA during this phase, suggesting that olive fruit cell number is determined from 28 DPA in the three cultivars. Moreover, olive fruit of the large-fruited cultivars was enlarged due to relatively higher cell division and expansion rates compared with the small-fruited cultivar. The ploidy level of olive fruit pericarp between early and late growth was different, but similar among olive cultivars, revealing that ploidy levels are not associated with cell size, in terms of different 8C levels during olive fruit growth. In the three olive cultivars, the maximum endoreduplication level (8C) occurred just before strong cell expansion during early fruit growth in fruit pericarp, whereas the cell expansion during late fruit growth occurred without preceding endoreduplication. We conclude that the basis for fruit size differences between olive cultivars is determined mainly by different cell division and expansion rates during the early fruit growth phase. These data provide new findings on the contribution of fruit ploidy and cell size to fruit size in olive and ultimately on the control of olive fruit development.

Hormonal Content and Gene Expression during Olive Fruit Growth and Ripening.

The cultivated olive (Olea europaea L. subsp. europaea var. europaea) is one of the most valuable fruit trees worldwide. However, the hormonal mechanisms underlying the fruit growth and ripening in olives remain largely uncharacterized. In this study, we investigated the physiological and hormonal changes, by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS), as well as the expression patterns of hormone-related genes, using quantitative real-time PCR (qRT-PCR) analysis, during fruit growth and ripening in two olive cultivars, 'Arbequina' and 'Picual', with contrasting fruit size and shape as well as fruit ripening duration. Hormonal profiling revealed that olive fruit growth involves a lowering of auxin (IAA), cytokinin (CKs), and jasmonic acid (JA) levels as well as a rise in salicylic acid (SA) levels from the endocarp lignification to the onset of fruit ripening in both cultivars. During olive fruit ripening, both abscisic acid (ABA) and anthocyanin levels rose, while JA levels fell, and SA levels showed no significant changes in either cultivar. By contrast, differential accumulation patterns of gibberellins (GAs) were found between the two cultivars during olive fruit growth and ripening. GA1 was not detected at either stage of fruit development in 'Arbequina', revealing a specific association between the GA1 and 'Picual', the cultivar with large sized, elongated, and fast-ripening fruit. Moreover, ABA may play a central role in regulating olive fruit ripening through transcriptional regulation of key ABA metabolism genes, whereas the IAA, CK, and GA levels and/or responsiveness differ between olive cultivars during olive fruit ripening. Taken together, the results indicate that the relative absence or presence of endogenous GA1 is associated with differences in fruit morphology and size as well as in the ripening duration in olives. Such detailed knowledge may be of help to design new strategies for effective manipulation of olive fruit size as well as ripening duration.

Characterization of Transcriptome Dynamics during Early Fruit Development in Olive (Olea europaea L.).

In the olive (Olea europaea L.), an economically leading oil crop worldwide, fruit size and yield are determined by the early stages of fruit development. However, few detailed analyses of this stage of fruit development are available. This study offers an extensive characterization of the various processes involved in early olive fruit growth (cell division, cell cycle regulation, and cell expansion). For this, cytological, hormonal, and transcriptional changes characterizing the phases of early fruit development were analyzed in olive fruit of the cv. 'Picual'. First, the surface area and mitotic activity (by flow cytometry) of fruit cells were investigated during early olive fruit development, from 0 to 42 days post-anthesis (DPA). The results demonstrate that the cell division phase extends up to 21 DPA, during which the maximal proportion of 4C cells in olive fruits was reached at 14 DPA, indicating that intensive cell division was activated in olive fruits at that time. Subsequently, fruit cell expansion lasted as long as 3 weeks more before endocarp lignification. Finally, the molecular mechanisms controlling the early fruit development were investigated by analyzing the transcriptome of olive flowers at anthesis (fruit set) as well as olive fruits at 14 DPA (cell division phase) and at 28 DPA (cell expansion phase). Sequential induction of the cell cycle regulating genes is associated with the upregulation of genes involved in cell wall remodeling and ion fluxes, and with a shift in plant hormone metabolism and signaling genes during early olive fruit development. This occurs together with transcriptional activity of subtilisin-like protease proteins together with transcription factors potentially involved in early fruit growth signaling. This gene expression profile, together with hormonal regulators, offers new insights for understanding the processes that regulate cell division and expansion, and ultimately fruit yield and olive size.

Transcriptome and Hormone Analyses Revealed Insights into Hormonal and Vesicle Trafficking Regulation among Olea europaea Fruit Tissues in Late Development.

Fruit ripening and abscission are the results of the cell wall modification concerning different components of the signaling network. However, molecular-genetic information on the cross-talk between ripe fruit and their abscission zone (AZ) remains limited. In this study, we investigated transcriptional and hormonal changes in olive (Olea europaea L. cv Picual) pericarp and AZ tissues of fruit at the last stage of ripening, when fruit abscission occurs, to establish distinct tissue-specific expression patterns related to cell-wall modification, plant-hormone, and vesicle trafficking in combination with data on hormonal content. In this case, transcriptome profiling reveals that gene encoding members of the α-galactosidase and ß-hexosaminidase families associated with up-regulation of RabB, RabD, and RabH classes of Rab-GTPases were exclusively transcribed in ripe fruit enriched in ABA, whereas genes of the arabinogalactan protein, laccase, lyase, endo-ß-mannanase, ramnose synthase, and xyloglucan endotransglucosylase/hydrolase families associated with up-regulation of RabC, RabE, and RabG classes of Rab-GTPases were exclusively transcribed in AZ-enriched mainly in JA, which provide the first insights into the functional divergences among these protein families. The enrichment of these protein families in different tissues in combination with data on transcript abundance offer a tenable set of key genes of the regulatory network between olive fruit tissues in late development.

Cell Wall Composition and Ultrastructural Immunolocalization of Pectin and Arabinogalactan Protein during Olea europaea L. Fruit Abscission.

Cell wall modification is integral to many plant developmental processes where cells need to separate, such as abscission. However, changes in cell wall composition during natural fruit abscission are poorly understood. In olive (Olea europaea L.), some cultivars such as 'Picual' undergo massive natural fruit abscission after fruit ripening. This study investigates the differences in cell wall polysaccharide composition and the localization of pectins and arabinogalactan protein (AGP) in the abscission zone (AZ) during cell separation to understand fruit abscission control in 'Picual' olive. To this end, immunogold labeling employing a suite of monoclonal antibodies to cell wall components (JIM13, LM5, LM6, LM19 and LM20) was investigated in olive fruit AZ. Cell wall polysaccharide extraction revealed that the AZ cell separation is related to the de-esterification and degradation of pectic polysaccharides. Moreover, ultrastructural localization showed that both esterified and unesterified homogalacturonans (HGs) localize mainly in the AZ cell walls, including the middle lamella and tricellular junction zones. Our results indicate that unesterified HGs are likely to contribute to cell separation in the olive fruit AZ. Similarly, immunogold labeling demonstrated a decrease in both galactose-rich and arabinose-rich pectins in AZ cell walls during ripe fruit abscission. In addition, AGPs were localized in the cell wall, plasma membrane and cytoplasm of AZ cells with lower levels of AGPs during ripe fruit abscission. This detailed temporal profile of the cell wall polysaccharide composition, and the pectins and AGP immunolocalization in the olive fruit AZ, offers new insights into cell wall remodeling during ripe fruit abscission.

Regulation of sterol content and biosynthetic gene expression during flower opening and early fruit development in olive.

Phytosterols are lipophilic membrane components essential not only for diverse cellular functions but also are biosynthetic precursors of the plant hormone, brassinosteroid (BR). However, the interaction between phytosterol and BR during early fleshy-fruit growth remains largely uncharacterized. In olive, phytosterols are important lipids because they affect oil quality, but phytosterol composition during flowering and early fruit development has not been explored. Here, we first investigated the temporal changes in phytosterol composition, and biosynthetic gene expression that occurred during olive flower opening and early fruit growth. Next, we analyzed the interrelationship between phytosterol and BR, whose levels we manipulated through the application of exogenous BRs (24-epibrassinolide, EBR) or a BR biosynthesis inhibitor (brassinazole, Brz). In this report, the profiling of phytosterol measurement revealed that ß-sitosterol is the most abundant in olive reproductive organs. Our data demonstrate that both OeCYP51 and OeSMT2 genes are upregulated during floral anthesis in good agreement with the rise in cholesterol and ß-sitosterol contents in olive flower. By contrast, the OeCYP51 and OeSMT2 genes displayed different expression patterns during early olive-fruit development. Furthermore, our data show that exogenous EBR enhanced the early olive-fruit growth, as well as the OeSMT2 transcript and ß-sitosterol levels, but decreased the OeCYP51 transcript, squalene, campesterol and cholesterol levels, whereas the Brz treatment exerted the opposite effect. Overall, our findings indicate an up-regulation of ß-sitosterol biosynthesis by BR at the transcriptional level during early olive-fruit growth, providing a valuable tool to unravel the physiological function of SMT2 in future studies.

Modulation of sphingolipid long-chain base composition and gene expression during early olive-fruit development, and putative role of brassinosteroid.

Sphingolipids are abundant membrane components and signalling molecules in various aspects of plant development. However, the role of sphingolipids in early fleshy-fruit growth has rarely been investigated. In this study, we first investigated the temporal changes in sphingolipid long-chain base (LCB) content, composition, and gene expression that occurred during flower opening and early fruit development in olive (Olea europaea L. cv Picual). Moreover, the interaction between sphingolipid and the plant hormone, brassinosteroid (BR), during the early fruit development was also explored. For this, BR levels were manipulated through the application of exogenous BRs (24-epibrassinolide, EBR) or a BR biosynthesis inhibitor (brassinazole, Brz) and their effects on early fruit development, sphingolipid LCB content, and gene expression were examined in olive fruit at 14 days post-anthesis (DPA). We here show that sphingolipid with C-4 hydroxylation and Δ8 desaturation with a preference for (E)-isomer formation are quantitatively the most important sphingolipids in olive reproductive organs. In this work, the total LCB amount significantly decreased at the anthesis stage, but olive sphingosine-1-phosphate lyase (OeSPL) gene was expressed exclusively in flower and upregulated during the anthesis, revealing an association with the d18:1(8E) accumulation. However, the LCB content increased in parallel with the upregulation of the expression of genes for key sphingolipid biosynthetic and LCB modification enzymes during early fruit development in olive. Likewise, we found that EBR exogenously applied to olive trees significantly stimulated the fruit growth rate whereas Brz inhibited fruit growth rate after 7 and 14 days of treatment. In addition, this inhibitory effect could be counteracted by the application of EBR. The promotion of early fruit growth was accompanied by the down-regulation of sphingolipid LCB content and gene expression in olive fruit, whereas Brz application raised levels of sphingolipid LCB content and gene expression in olive fruit after 7 and 14 days of treatment. Thus, our data indicate that endogenous sphingolipid LCB and gene-expression levels are intricately controlled during early fruit development and also suggest a possible link between BR, the sphingolipid content/gene expression, and early fruit development in olive.

Sphingolipid Distribution, Content and Gene Expression during Olive-Fruit Development and Ripening.

Plant sphingolipids are involved in the building of the matrix of cell membranes and in signaling pathways of physiological processes and environmental responses. However, information regarding their role in fruit development and ripening, a plant-specific process, is unknown. The present study seeks to determine whether and, if so, how sphingolipids are involved in fleshy-fruit development and ripening in an oil-crop species such as olive (Olea europaea L. cv. Picual). Here, in the plasma-membranes of live protoplasts, we used fluorescence to examine various specific lipophilic stains in sphingolipid-enriched regions and investigated the composition of the sphingolipid long-chain bases (LCBs) as well as the expression patterns of sphingolipid-related genes, OeSPT, OeSPHK, OeACER, and OeGlcCerase, during olive-fruit development and ripening. The results demonstrate increased sphingolipid content and vesicle trafficking in olive-fruit protoplasts at the onset of ripening. Moreover, the concentration of LCB [t18:1(8Z), t18:1 (8E), t18:0, d18:2 (4E/8Z), d18:2 (4E/8E), d18:1(4E), and 1,4-anhydro-t18:1(8E)] increases during fruit development to reach a maximum at the onset of ripening, although these molecular species decreased during fruit ripening. On the other hand, OeSPT, OeSPHK, and OeGlcCerase were expressed differentially during fruit development and ripening, whereas OeACER gene expression was detected only at the fully ripe stage. The results provide novel data about sphingolipid distribution, content, and biosynthesis/turnover gene transcripts during fleshy-fruit ripening, indicating that all are highly regulated in a developmental manner.

Localization of Sphingolipid Enriched Plasma Membrane Regions and Long-Chain Base Composition during Mature-Fruit Abscission in Olive.

Sphingolipids, found in membranes of eukaryotic cells, have been demonstrated to carry out functions in various processes in plant cells. However, the roles of these lipids in fruit abscission remain to be determined in plants. Biochemical and fluorescence microscopy imaging approach has been adopted to investigate the accumulation and distribution of sphingolipids during mature-fruit abscission in olive (Olea europaea L. cv. Picual). Here, a lipid-content analysis in live protoplasts of the olive abscission zone (AZ) was made with fluorescent dyes and lipid analogs, particularly plasma membrane sphingolipid-enriched domains, and their dynamics were investigated in relation to the timing of mature-fruit abscission. In olive AZ cells, the measured proportion of both polar lipids and sphingolipids increased as well as endocytosis was stimulated during mature-fruit abscission. Likewise, mature-fruit abscission resulted in quantitative and qualitative changes in sphingolipid long-chain bases (LCBs) in the olive AZ. The total LCB increase was due essentially to the increase of t18:1(8E) LCBs, suggesting that C-4 hydroxylation and Δ8 desaturation with a preference for (E)-isomer formation were quantitatively the most important sphingolipids in olive AZ during abscission. However, our results also showed a specific association between the dihydroxylated LCB sphinganine (d18:0) and the mature-fruit abscission. These results indicate a clear correlation between the sphingolipid composition and mature-fruit abscission. Moreover, measurements of endogenous sterol levels in the olive AZ revealed that it accumulated sitosterol and campesterol with a concomitant decrease in cycloartenol during abscission. In addition, underlying the distinct sterol composition of AZ during abscission, genes for key biosynthetic enzymes for sterol synthesis, for obtusifoliol 14α-demethylase (CYP51) and C-24 sterol methyltransferase2 (SMT2), were up-regulated during mature-fruit abscission, in parallel to the increase in sitosterol content. The differences found in AZ lipid content and the relationships established between LCB and sterol composition, offer new insights about sphingolipids and sterols in abscission.

Effects of several applications of digested sewage sludge on soil and plants.

Sewage sludge production has significantly increased during the last years in European Union (EU) countries, being primarily used for agricultural purposes. In this study, digested sewage sludge was applied to greenhouse soil over a three-year period (2001--2003), with three sludge treatments in the first two years (2, 4, and 6 kg m(-2)) and three more applications using a greater quantity in the last year (6, 8, and 10 kg m(-2)). The effects of sewage sludge application on soil and on a leafy crop (Lactuca sativa L.) were studied. Mineral elements, organic matter, pH, and heavy metals were measured in soil and plant tissues. Pathogen and indicator microorganism dynamics in soil were also determined after each sludge application. Results showed that sewage sludge applications increased organic matter, P, and N Ca content in soil. Furthermore, Zn, Pb, Ni, and Cu content increased in soils, primarily after high doses of sludge. The highest yield value was obtained in the second-year harvest, since the last sludge application did not increase yield. Fecal coliform numbers decreased significantly one month after sludge application. However, total coliforms, Clostridium sulphite-reducers and Salmonella, were present in soils three months after sludge application.

Ventilación mecánica en el estado de mal asmático. Experiencia de 5 años en la unidad de cuidados intensivos / Mechanical ventilation in status asthmaticus. A 5-year experience. Intensive Care Units

Se hace un estudio retrospectivo de los enfermos ingresados por status asmaticus en la Unidad de Cuidados Intensivos del Hospital Provincial Docente Clinicoquirúrgico de Pinar del Río, en un período de 5 años y que fueron sometidos a ventilación mecánica, los cuales representaron el 32,65 por ciento del total de ingresos por esa entidad. El 57 por ciento fueron mujeres con una media de edad de 48 años. La media de tiempo de intubación fue de 60 horas. Se hizo un estudio de su situación clínica al ingreso, las causas de intubación y los parámetros ventilatorios que se vienen utilizando hasta la actualidad y se analizaron las complicaciones y la mortalidad que ascendió a 9,38 por ciento. Todos los resultados fueron comparados con los de otros autores y la literatura nacional e internacional

Ventilacion mecanica en el estado de mal asmatico. Experiencia de 5 anos en la unidad de cuidados intensivos / Mechanical ventilation in status asthmaticus. A 5-year experience. Intensive Care Units

Se hace un estudio retrospectivo de los enfermos ingresados por status asmaticus en la Unidad de Cuidados Intensivos del Hospital Provincial Docente Clinicoquirurgico de Pinar del Rio, en un periodo de 5 anos y que fueron sometidos a ventilacion mecanica, los cuales representaron el 32,65 por ciento del total de ingresos por esa entidad. El 57 por ciento fueron mujeres con una media de edad de 48 anos. La media de tiempo de intubacion fue de 60 horas. Se hizo un estudio de su situacion clinica al ingreso, las causas de intubacion y los parametros ventilatorios que se vienen utilizando hasta la actualidad y se analizaron las complicaciones y la mortalidad que ascendio a 9,38 por ciento. Todos los resultados fueron comparados con los de otros autores y la literatura nacional e internacional

Morbilidad por parotiditis y rubeola / Mups and rubella morbidity

Se estudió la totalidad de los casos afectados por parotiditis en los meses de enero a septiembre de 1986 (1 767 casos) y 1987 (124 casos), así como los enfermos de rubeola notificados en el mismo período de 1986 (8584 casos) y 1987 (5 casos) en la provincia de inar del Río. Se destacó el alza en la tasa de incidencia con base anual, por 100 000 habitantes que han tenido ambas entidades durante 1986 en los diferentes municipios. Se comenta la tendencia francamente descendente que han tenido las 2 enfermedades en 1987 después de la vacunación triple viral (93

) para la parotiditis y el 99,15

para la rubeola. Se comprobó que las entidades en estudio afectaron más a los niños en la edad escolar (5-14 años). Se demostró que ambas enfermedades no son frecuentes por debajo de un año de edad (76,4 y 120,1 casos por 100 000 habitantes respectivamente). Se comprobó el tanto por ciento de efectividad de la vacuna, el 97,4

para la parotiditis y eliento por ciento para la rubeola (AU)

Morbilidad por parotiditis y rubeola / Mumps and rubella morbidity

Se estudió la totalidad de los casos afectados por parotiditis en los meses de enero a septiembre de 1986 (1 767 casos) y 1987 (124 casos), así como los enfermos de rubeola notificados en el mismo período de 1986 (8584 casos) y 1987 (5 casos) en la provincia de inar del Río. Se destacó el alza en la tasa de incidencia con base anual, por 100 000 habitantes que han tenido ambas entidades durante 1986 en los diferentes municipios. Se comenta la tendencia francamente descendente que han tenido las 2 enfermedades en 1987 después de la vacunación triple viral (93%) para la parotiditis y el 99,15% para la rubeola. Se comprobó que las entidades en estudio afectaron más a los niños en la edad escolar (5-14 años). Se demostró que ambas enfermedades no son frecuentes por debajo de un año de edad (76,4 y 120,1 casos por 100 000 habitantes respectivamente). Se comprobó el tanto por ciento de efectividad de la vacuna, el 97,4% para la parotiditis y eliento por ciento para la rubeola