Early markers are present in both embryogenesis pathways from microspores and immature zygotic embryos in cork oak, Quercus suber L.

BACKGROUND: In Quercus suber, cork oak, a Mediterranean forest tree of economic and social interest, rapid production of isogenic lines and clonal propagation of elite genotypes have been achieved by developing in vitro embryogenesis from microspores and zygotic embryos respectively. Despite its high potential in tree breeding strategies, due to their recalcitrancy, the efficiency of embryogenesis in vitro systems in many woody species is still very low since factors responsible for embryogenesis initiation and embryo development are still largely unknown. The search for molecular and cellular markers during early stages of in vitro embryogenesis constitutes an important goal to distinguish, after induction, responsive from non-responsive cells, and to elucidate the mechanisms involved in embryogenesis initiation for their efficient manipulation. In this work, we have performed a comparative analysis of two embryogenesis pathways derived from microspores and immature zygotic embryos in cork oak in order to characterize early markers of reprogrammed cells in both pathways. Rearrangements of the cell structural organization, changes in epigenetic marks, cell wall polymers modifications and endogenous auxin changes were analyzed at early embryogenesis stages of the two in vitro systems by a multidisciplinary approach. RESULTS: Results showed that early embryo cells exhibited defined changes of cell components which were similar in both embryogenesis in vitro systems, cellular features that were not found in non-embryogenic cells. DNA methylation level and nuclear pattern, proportion of esterified pectins in cell walls, and endogenous auxin levels were different in embryo cells in comparison with microspores and immature zygotic embryo cells from which embryos originated, constituting early embryogenesis markers. CONCLUSIONS: These findings suggest that DNA hypomethylation, cell wall remodeling by pectin esterification and auxin increase are involved in early in vitro embryogenesis in woody species, providing new evidences of the developmental pattern similarity between both embryogenesis pathways, from microspores and immature zygotic embryos, in woody species.

[Low and non-calorie sweeteners as a tool for reducing the energy density at foodstuffs. An alternative for helping to control and reduce overweight and obesity]. / Los edulcorantes como herramienta de reducción de la densidad energética en los productos alimenticios. Una alternativa para ayudar a poder controlar y reducir el sobrepeso y la obesidad.

Introduction: Introduction: there is more and more scientific data on sweeteners but at the same time there is more dissemination of information and it is sometimes contradictory Methods: observational field study with analysis of data referring to current legislation, approvals by European Union authorities and systematic reviews. Results: the European Union has one of the best systems in the world for the evaluation, approval and authorization of sweeteners and those approved have been immersed since 2010 in a revaluation process, such as that of the other additives. Conclusions: sweeteners are a tool for the reduction and elimination of sugar at foodstuffs. The total diet is the one that must have as a whole a reduction in calories to be effective in the control and reduction of overweight and obesity.

Introducción: Introducción: cada vez existen más datos científicos sobre los edulcorantes, pero a su vez hay más diseminación de información, a veces contradictoria. Material y métodos: estudio de campo observacional con análisis de datos referidos a la legislación vigente, aprobaciones por autoridades de la Unión Europea y revisiones sistemáticas. Resultados: la Unión Europea tiene uno de los mejores sistemas a nivel mundial de evaluación, aprobación y autorización de edulcorantes y los aprobados están inmersos desde el año 2010 en un proceso de reevaluación, como el resto de aditivos. Conclusiones: los edulcorantes son una herramienta para la reducción y eliminación de azúcar en los productos alimenticios. La dieta total es la que debe tener en su conjunto una reducción de calorías para ser efectiva en el control y la reducción del sobrepeso y la obesidad.

Dynamics of Endogenous Auxin and Its Role in Somatic Embryogenesis Induction and Progression in Cork Oak.

Somatic embryogenesis (SE) is a feasible in vitro regeneration system with biotechnological applications in breeding programs, although, in many forest species, SE is highly inefficient, mainly due to their recalcitrance. On the other hand, SE represents a valuable model system for studies on cell reprogramming, totipotency acquisition, and embryogenic development. The molecular mechanisms that govern the transition of plant somatic cells to embryogenic cells are largely unknown. There is increasing evidence that auxins mediate this transition and play a key role in somatic embryo development, although data on woody species are very limited. In this study, we analyzed the dynamics and possible role of endogenous auxin during SE in cork oak (Quercus suber L.). The auxin content was low in somatic cells before cell reprogramming, while it increased after induction of embryogenesis, as revealed by immunofluorescence assays. Cellular accumulation of endogenous auxin was also detected at the later stages of somatic embryo development. These changes in auxin levels correlated with the expression patterns of the auxin biosynthesis (QsTAR2) and signaling (QsARF5) genes, which were upregulated after SE induction. Treatments with the inhibitor of auxin biosynthesis, kynurenine, reduced the proliferation of proembryogenic masses and impaired further embryo development. QsTAR2 and QsARF5 were downregulated after kynurenine treatment. Our findings indicate a key role of endogenous auxin biosynthesis and signaling in SE induction and multiplication, as well as somatic embryo development of cork oak.

[Bioactive compounds from plants. Development of new or novel food]. / Compuestos bioactivos de origen vegetal: desarrollo de nuevos alimentos.

Introduction: Introduction: substances with physiological effect or bioactive compounds are having a fundamental consideration in the diets of the populations during the last years, especially those that consume high content from plant foods that provide them. Methods: collect the latest scientific data on bioactive compounds from plant origin and develop legislation regulating the nutritional information of foods and beverages, as well as the health claims and reduction of disease risk claims. The approval of novel foods too. Results: there is scientific evidence on the effect of bioactive compounds, and this has meant their incorporation into dietary quality indices, such as the food compass. As well as the approval of 26 health claims and 2 reduction of disease risk claims in the European Union. Several ingredients or foods classified as novel foods have also been approved within European Union food law. Conclusions: bioactive compounds have more and more scientific evidence to be recommended directly or through the foods and beverages that contain them in dietary guidelines and must be considered as other nutrients and incorporated into the nutritional information even if they do not have caloric intake.

Introducción: Introducción: los compuestos bioactivos (sustancias con efecto fisiológico) están teniendo en los últimos años una gran consideración en las dietas de las poblaciones, sobre todo en aquellas que consumen un alto contenido de alimentos vegetales que las aportan. Métodos: recoger los últimos datos científicos sobre compuestos bioactivos de origen vegetal y recopilar la legislación que regula la información nutricional de los alimentos y de las bebidas, así como de las propiedades saludables y de prevención de riesgo de enfermedad y de la aprobación de nuevos alimentos. Resultados: existe evidencia científica sobre el efecto de los compuestos bioactivos, lo que ha supuesto su incorporación en índices de calidad de la dieta (como la brújula alimentaria), así como la aprobación de 26 declaraciones de propiedades saludables y 2 de prevención de riesgo de enfermedad en la Unión Europea. También se han aprobado varios ingredientes o alimentos catalogados como nuevos alimentos dentro de la legislación alimentaria de la Unión Europea. Conclusiones: los compuestos bioactivos tienen cada vez más evidencia científica para estar recomendados, directamente o a través de los alimentos y de las bebidas que los contienen, en las guías alimentarias y deberían considerarse como otros nutrientes e incorporarse a la información nutricional, aunque no tengan aporte calórico.

Production of Doubled Haploid Embryos from Cork Oak Anther Cultures by Antimitotic Agents and Temperature Stress.

Cork oak (Quercus suber L.) is a forest tree species of the family Fagaceae. It is characterized by long life cycles which hamper doubled haploid plant production to obtain homozygotes and pure lines. The time-consuming method of repeated backcrossings by conventional breeding techniques to produce pure lines is impractical in woody species. Nevertheless, biotechnology has offered new tools to make it possible. A doubled haploid plant or embryo is one that is developed by the doubling of a haploid set of chromosomes. A protocol to produce doubled haploids of cork oak has been developed through microspore embryogenesis. By a heat stress treatment, the microspores inside the anther leave the gametophytic pathway and react shifting their development to the sporophytic pathway by means of which haploid embryos are obtained. Chromosome duplication of haploids from cork oak anther cultures occurs either spontaneously or may be induced by the application of antimitotic agents (e.g., colchicine, oryzalin, amiprophos-methyl). Furthermore, a genetic test is designed through microsatellite markers to elucidate whether the diploid embryos obtained are originally haploids which spontaneously duplicated their genome, or alternatively those embryos are generated from the diploid tissue of the anther wall. Here we describe a detailed protocol to produce doubled haploid individuals from cork oak anther cultures by using temperature stress and antimitotic agents.

Role of Synthetic Plant Extracts on the Production of Silver-Derived Nanoparticles.

The main antioxidants present in plant extracts-quercetin, ß-carotene, gallic acid, ascorbic acid, hydroxybenzoic acid, caffeic acid, catechin and scopoletin-are able to synthesize silver nanoparticles when reacting with a Ag NO3 solution. The UV-visible absorption spectrum recorded with most of the antioxidants shows the characteristic surface plasmon resonance band of silver nanoparticles. Nanoparticles synthesised with ascorbic, hydroxybenzoic, caffeic, and gallic acids and scopoletin are spherical. Nanoparticles synthesised with quercetin are grouped together to form micellar structures. Nanoparticles synthesised by ß-carotene, were triangular and polyhedral forms with truncated corners. Pentagonal nanoparticles were synthesized with catechin. We used Fourier-transform infrared spectroscopy to check that the biomolecules coat the synthesised silver nanoparticles. X-ray powder diffractograms showed the presence of silver, AgO, Ag2O, Ag3O4 and Ag2O3. Rod-like structures were obtained with quercetin and gallic acid and cookie-like structures in the nanoparticles obtained with scopoletin, as a consequence of their reactivity with cyanide. This analysis explained the role played by the various agents responsible for the bio-reduction triggered by nanoparticle synthesis in their shape, size and activity. This will facilitate targeted synthesis and the application of biotechnological techniques to optimise the green synthesis of nanoparticles.

Proteomic analysis from haploid and diploid embryos of Quercus suber L. identifies qualitative and quantitative differential expression patterns.

Quercus suber L. is a Mediterranean forest species with ecological, social and economic value. Clonal propagation of Q. suber elite trees has been successfully obtained from in vitro-derived somatic and gametic embryos. These clonal lines play a main role in breeding and genetic studies of Q. suber. To aid in unravelling diverse genetic and biological unknowns, a proteomic approach is proposed. The proteomic analysis of Q. suber somatic and gametic in vitro culture-derived embryos, based on DIGE and MALDI-MS, has produced for the first time proteomic data on this species. Seventeen differentially expressed proteins have been identified which display significantly altered levels between gametic and somatic embryos. These proteins are involved in a variety of cellular processes, most of which had been neither previously associated with embryo development nor identified in the genus Quercus. Some of these proteins are involved in stress and pollen development and others play a role in the metabolism of tannins and phenylpropanoids, which represent two of the major pathways for the synthesis of cork chemical components. Furthermore, the augmented expression levels found for specific proteins are probably related to the homozygous state of a doubled-haploid sample. Proteins involved in synthesis of cork components can be detected at such early stages of development, showing the potential of the method to be useful in searching for biomarkers related to cork quality.

Los edulcorantes como herramienta de reducción de la densidad energética en los productos alimenticios. Una alternativa para ayudar a poder controlar y reducir el sobrepeso y la obesidad / Low and non-calorie sweeteners as a tool for reducing the energy density at foodstuffs. An alternative for helping to control and reduce overweight and obesity

Introducción: cada vez existen más datos científicos sobre los edulcorantes, pero a su vez hay más diseminación de información, a vecescontradictoria.Material y métodos: estudio de campo observacional con análisis de datos referidos a la legislación vigente, aprobaciones por autoridades dela Unión Europea y revisiones sistemáticas.Resultados: la Unión Europea tiene uno de los mejores sistemas a nivel mundial de evaluación, aprobación y autorización de edulcorantes y losaprobados están inmersos desde el año 2010 en un proceso de reevaluación, como el resto de aditivos.Conclusiones: los edulcorantes son una herramienta para la reducción y eliminación de azúcar en los productos alimenticios. La dieta total esla que debe tener en su conjunto una reducción de calorías para ser efectiva en el control y la reducción del sobrepeso y la obesidad. (AU)

Introduction: there is more and more scientific data on sweeteners but at the same time there is more dissemination of information and it issometimes contradictoryMethods: observational field study with analysis of data referring to current legislation, approvals by European Union authorities and systematicreviews.Results: the European Union has one of the best systems in the world for the evaluation, approval and authorization of sweeteners and thoseapproved have been immersed since 2010 in a revaluation process, such as that of the other additives.Conclusions: sweeteners are a tool for the reduction and elimination of sugar at foodstuffs. The total diet is the one that must have as a wholea reduction in calories to be effective in the control and reduction of overweight and obesity. (AU)

Somatic Embryogenesis of Quercus suber L. From Immature Zygotic Embryos.

Quercus suber L., cork oak, is a forest tree of high social and economic value. The cork is traditionally used in the wine industry to produce bottle stoppers, but it is also a very good material for both thermal and acoustic insulation in construction. Since its harvest does not harm the tree, the use of cork in the industry has a positive impact on the environment.Somatic embryogenesis is considered a feasible system for in vitro regeneration procedures, with many advantages in woody species. Classical genetic breeding programs have important limitations in forest trees, like cork oak, due to their long life span and difficulties of seed conservation and vegetative reproduction. Therefore, somatic embryogenesis has a great potential for large-scale propagation and cryopreservation of elite genotypes, as well as for transformation strategies. In the case of Q. suber, several in vitro propagation systems through somatic embryogenesis have been reported, with different efficiency rates.In the present chapter, updated information is reported about an efficient protocol for induction of somatic embryogenesis of Q. suber from immature zygotic embryos, as well as methods for proliferation and maturation of somatic embryos, germination, plantlet regeneration, and acclimatization.

Pectin De-methylesterification and AGP Increase Promote Cell Wall Remodeling and Are Required During Somatic Embryogenesis of Quercus suber.

Somatic embryogenesis is a reliable system for in vitro plant regeneration, with biotechnological applications in trees, but the regulating mechanisms are largely unknown. Changes in cell wall mechanics controlled by methylesterification of pectins, mediated by pectin methylesterases (PMEs) and pectin methyl esterase inhibitors (PMEIs) underlie many developmental processes. Arabinogalactan proteins (AGPs) are highly glycosylated proteins located at the surface of plasma membranes, in cell walls, and in extracellular secretions, with key roles in a range of different processes. In this study, we have investigated changes in two cell wall components, pectins and AGPs, during somatic embryogenesis in Quercus suber, a forest tree of high economic and ecologic value. At early embryogenesis stages, cells of proembryogenic masses showed high levels of esterified pectins and expression of QsPME and QsPMEI genes encoding a PME and a putative PMEI, respectively. At advanced stages, differentiating cells of heart, torpedo and cotyledonary embryos exhibited walls rich in de-esterified pectins, while QsPME gene expression and PME activity progressively increased. AGPs were detected in cell walls of proembryogenic masses and somatic embryos. QsLys-rich-AGP18, QsLys-rich-AGP17, and QsAGP16L1 gene expression increased with embryogenesis progression, as did the level of total AGPs, detected by dot blot with ß-glucosyl Yariv reagent. Immuno dot blot, immunofluorescence assays and confocal analysis using monoclonal antibodies to high- (JIM7, LM20) and low- (JIM5, LM19) methylesterified pectins, and to certain AGP epitopes (LM6, LM2) showed changes in the amount and distribution pattern of esterified/de-esterified pectins and AGP epitopes, that were associated with proliferation and differentiation and correlated with expression of the PME and AGP genes analyzed. Pharmacological treatments with catechin, an inhibitor of PME activity, and Yariv reagent, which blocks AGPs, impaired the progression of embryogenesis, with pectin de-esterification and an increase in AGP levels being necessary for embryo development. Findings indicate a role for pectins and AGPs during somatic embryogenesis of cork oak, promoting the cell wall remodeling during the process. They also provide new insights into the regulating mechanisms of somatic embryogenesis in woody species, for which information is still scarce, opening up new possibilities to improve in vitro embryo production in tree breeding.

Antimitotic agents increase the production of doubled-haploid embryos from cork oak anther culture.

The objective of this study is to induce the nuclear DNA duplication of anther-derived embryos of cork oak (Quercus suber L.) to obtain doubled-haploid plants. Anther culture of this species produces a low percentage (7.78%) of spontaneous diploids, as assessed by flow cytometry. Therefore, three antimitotic agents, colchicine, oryzalin and amiprophos-methyl (APM), were applied in vitro to anther-derived cork oak haploid embryos from six genotypes at different concentrations and for different treatment durations. Antimitotic toxicity was determined by embryo survival. Efficiency in inducing chromosome doubling of haploid embryos was evaluated by flow cytometry measurements and differences were observed between treatments. Nuclear DNA duplication and embryo survival of cork oak haploid embryos was most efficiently induced with oryzalin 0.01 mM for 48 h. Around 50% diploid embryos were obtained. The rate of chromosome duplication induced by APM 0.01 mM was also acceptable but lower than that induced by oryzalin, regardless of the duration of the treatment. Colchicine 1.3 or 8.8 mM was the least efficient, with the induction of necrosis and only a small rate of nuclear DNA duplication.

Compuestos bioactivos de origen vegetal: desarrollo de nuevos alimentos / Bioactive compounds from plants. Development of new or novel food

Introducción: los compuestos bioactivos (sustancias con efecto fisiológico) están teniendo en los últimos años una gran consideración en las dietas de las poblaciones, sobre todo en aquellas que consumen un alto contenido de alimentos vegetales que las aportan. Métodos: recoger los últimos datos científicos sobre compuestos bioactivos de origen vegetal y recopilar la legislación que regula la información nutricional de los alimentos y de las bebidas, así como de las propiedades saludables y de prevención de riesgo de enfermedad y de la aprobación de nuevos alimentos. Resultados: existe evidencia científica sobre el efecto de los compuestos bioactivos, lo que ha supuesto su incorporación en índices de calidad de la dieta (como la brújula alimentaria), así como la aprobación de 26 declaraciones de propiedades saludables y 2 de prevención de riesgo de enfermedad en la Unión Europea. También se han aprobado varios ingredientes o alimentos catalogados como nuevos alimentos dentro de la legislación alimentaria de la Unión Europea. Conclusiones: los compuestos bioactivos tienen cada vez más evidencia científica para estar recomendados, directamente o a través de los alimentos y de las bebidas que los contienen, en las guías alimentarias y deberían considerarse como otros nutrientes e incorporarse a la información nutricional, aunque no tengan aporte calórico. (AU)

Introduction: substances with physiological effect or bioactive compounds are having a fundamental consideration in the diets of the populations during the last years, especially those that consume high content from plant foods that provide them. Methods: collect the latest scientific data on bioactive compounds from plant origin and develop legislation regulating the nutritional information of foods and beverages, as well as the health claims and reduction of disease risk claims. The approval of novel foods too. Results: there is scientific evidence on the effect of bioactive compounds, and this has meant their incorporation into dietary quality indices, such as the food compass. As well as the approval of 26 health claims and 2 reduction of disease risk claims in the European Union. Several ingredients or foods classified as novel foods have also been approved within European Union food law. Conclusions: bioactive compounds have more and more scientific evidence to be recommended directly or through the foods and beverages that contain them in dietary guidelines and must be considered as other nutrients and incorporated into the nutritional information even if they do not have caloric intake. (AU)

Changes in pectins and MAPKs related to cell development during early microspore embryogenesis in Quercus suber L.

The occurrence and significance of changes in cell wall components and signalling molecules has been investigated during early microspore embryogenesis in cork oak (Quercus suber L.) in relation to cell proliferation and cell differentiation. Microspore embryogenesis has been induced in in vitro anther cultures of Q. suber by the application of a stress treatment of 33 degrees C. After the treatment, microspores at the responsive developmental stage of vacuolate microspore switched towards proliferation and the embryogenesis pathway to further produce haploid plantlets. Ultrastructural and immunocytochemical analysis revealed changes in cell organisation after induction at different developmental stages, the cellular features displayed being in relation to the activation of proliferative activity and the beginning of differentiation in young and late proembryos. Immunogold labelling with JIM5 and JIM7 antibodies showed a different presence of pectin and level of its esterification in cell walls at different developmental stages. Non-esterified pectins were found in higher proportions in cells of late proembryos, suggesting that pectin de-esterification could be related to the beginning of differentiation. The presence and subcellular distribution of Erk 1/2 MAPK homologues have been investigated by immunoblotting, immunofluorescence and immunogold labelling. The results showed an increase in the expression of these proteins with a high presence in the nucleus, during early microspore proembryos development. The reported changes during early microspore embryogenesis are modulated in relation to proliferation and differentiation events. These findings provided new evidences for a role of MAPK signalling pathways in early microspore embryogenesis, specifically in proliferation, and would confer information for the cell fate and the direction of the cell development.

Uptake of CeO2 nanoparticles and its effect on growth of Medicago arborea In vitro plantlets.

The present study analyzes some effects of nano-CeO2 particles on the growth of in vitro plantlets of Medicago arborea when the nanoceria was added to the culture medium. Various concentrations of nano-CeO2 and bulk ceric oxide particles in suspension form were introduced to the agar culture medium to compare the effects of nanoceria versus ceric oxide bulk material. Germination rate and shoot dry weight were not affected by the addition of ceric oxide to the culture media. Furthermore, no effects were observed on chlorophyll content (single-photon avalanche diode (SPAD) measurements) due to the presence of either nano- or micro-CeO2 in the culture medium. When low concentrations of nanoceria were added to the medium, the number of trifoliate leaves and the root length increased but the root dry weight decreased. Also the values of maximum photochemical efficiency of PSII (F(v)/F m) showed a significant decrease. Dark-adapted minimum fluorescence (F 0) significantly increased in the presence of 200 mg L(-1) nanoceria and 400 mg L(-1) bulk material. Root tissues were more sensitive to nanoceria than were the shoots at lower concentrations of nanoceria. A stress effect was observed on M. arborea plantlets due to cerium uptake.

Identification of DNA-microsatellite markers for the characterization of somatic embryos in Quercus suber.

Nuclear DNA-microsatellite markers led the possibility to characterize individually both Quercus suber trees and somatic embryos. The genotype inferred by SSR markers opens the possibility to obtain a fingerprint for clonal lines identification. Furthermore, allow to infer the origin of somatic embryos from haploid cells (microspores) or from diploid tissues. Using few SSR markers from other Quercus species and an automatic system based in fluorescence, it is possible to obtain a high discrimination power between genotypes. This method is sufficient to assign tissues to an individual tree with high statistical certainty. Nevertheless, it is necessary to take care to select the adequate DNA extraction method to avoid PCR inhibitors present in diverse Q. suber tissues.

Proteomic perspective of Quercus suber somatic embryogenesis.

Quercus suber L. is a forest tree with remarkable ecological, social and economic value in the southern Europe ecosystems. To circumvent the difficulties of breeding such long-lived species like Q. suber in a conventional fashion, clonal propagation of Q. suber elite trees can be carried out, although this process is sometimes unsuccessful. To help decipher the complex program underlying the development of Q. suber somatic embryos from the first early stage until maturity, a proteomic approach based on DIGE and MALDI-MS has been envisaged. Results highlighted several key processes involved in the three developmental stages (proliferative, cotyledonary and mature) of Q. suber somatic embryogenesis studied. Results show that the proliferation stage is characterized by fermentation as an alternative energy source at the first steps of somatic embryo development, as well as by up-regulation of proteins involved in cell division. In this stage reactive oxygen species play a role in proliferation, while other proteins like CAD and PR5 seem to be implied in embryonic competence. In the transition to the cotyledonary stage diverse ROS detoxification enzymes are activated and reserve products (mainly carbohydrates and proteins) are accumulated, whereas energy production is increased probably to participate in the synthesis of primary metabolites such as amino acids and fatty acids. Finally, in the mature stage ethylene accumulation regulates embryo development. BIOLOGICAL SIGNIFICANCE: Quercus suber L. is a forest tree with remarkable ecological, social and economic value in the southern Europe ecosystems. To circumvent the difficulties of breeding such long-lived species like Q. suber in a conventional fashion, clonal propagation of Q. suber elite trees can be carried out, although this process is sometimes unsuccessful. To help decipher the complex program underlying the development of Q. suber somatic embryos from the first early stage until maturity, in deep studies become necessary. This article is part of a Special Issue entitled: Translational Plant Proteomics.