Beatriz Pintos

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.

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

BackgroundIn 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.ResultsResults 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.ConclusionsThese 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.

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.

Proteomic perspective of Quercus suber somatic embryogenesis.

UNLABELLED 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.

Oak somatic and gametic embryos maturation is affected by charcoal and specific aminoacids mixture

Abstract• Development of both somatic and gametic embryogenesis has many applications in clonal forestry and genetic improvement, for instance as mass-propagation of genetically improved plants and production of pure lines through doubled-haploid plant regeneration from gametic embryos.• The goal of this work was to improve growth, maturation and plantlet regeneration of cork oak (Quercus suber L.) embryos from both somatic and gametic origin.• Activated charcoal promoted a significant increase in growth in terms of relative size and weight of both somatic and doubled-haploid embryos, as well as a more efficient control of secondary somatic embryogenesis during development. A significant interaction was also observed with amino acid nutrition. While some amino acids (i.e., glutamine, arginine or asparagine) did not show significant differences with the controls, a mixture of these three amino acids or gamma amino butyric acid stimulated embryo growth. The highest survival rate during acclimation of plantlets from both somatic and doubled-haploid origin was obtained when the embryos had been previously cultured on basal medium with 3% sucrose and 1% activated charcoal for two months at 4 °C and germinated on medium supplemented with 6-benzylaminopurine and indole-3-butyric acid.• We obtained more than 900 cork oak plantlets acclimated from several embryogenic lines, with a high survival rate, demonstrating that this methodology is applicable for large scale plantlet production. We also report the first regeneration of doubled-haploid plantlets in cork oak.Résumé• Le développement des deux embryogenèses gamétique et somatique a de nombreuses applications dans la foresterie clonale et l’amélioration génétique, par exemple en tant que propagation en masse de plants génétiquement améliorés et la production de lignées pures par régénération de plants haploïdes doublés d’embryons gamétiques.• L’objectif de ce travail était d’améliorer la croissance, la maturation et la régénération des plantules des embryons de chêne-liège (Quercus suber L.) des deux origines gamétique et somatique.• Le charbon actif a favorisé une augmentation significative de la croissance en termes de taille et de poids des embryons haploïdes doublés et somatiques, ainsi qu’un contrôle plus efficace de l’embryogenèse somatique secondaire au cours du développement. Une interaction significative a également été observée avec la nutrition aminoacide. Alors que certains acides aminés (c’est-à-dire, la glutamine, l’arginine ou l’asparagine) ne montrent pas de différences significatives avec les témoins, un mélange de ces trois acides aminés ou l’acide butyrique amino gamma a stimulé la croissance de l’embryon. Le taux de survie le plus élevé au cours de l’acclimatation des plantules d’origine à la fois somatique et haploïde doublé, a été obtenu lorsque les embryons ont été auparavant cultivés sur milieu de base à 3 % de saccharose et de 1 % de charbon actif pendant deux mois à 4 °C et ont germé sur un milieu complété par la 6-benzylaminopurine et l’acide indole-3-butyrique.• Nous avons obtenu plus de 900 plantules de chênes-lièges acclimatés issus de plusieurs lignes embryogéniques, avec un taux de survie élevé, ce qui démontre que cette méthode est applicable pour la production à grande échelle de plantules. Nous annonçons également la première régénération de plantules haploïdes doublés de chêne-liège.

Pro-embryos induction from Olea europaea L. isolated microspore culture

Studies were undertaken with one olive (Olea europaea L.) cultivar to identify buds with microspores competent to embryogenesis in vitro. Isolated microspore cultures were performed for the induction of gametic embryogenesis. Different pollen development stages and stress conditions (heat or cold shock) were evaluated. The correlation of inflorescence, anther morphology and the suitable stage of microspore development were analysed. The morphology of responsive buds was identified which corresponded with microspores from the late uni-nucleate to early bi-nucleate pollen stages. Symmetrical divisions of microspores as well as resulting multinucleate structures and pro-embryos were observed. In this paper, a new method of isolated microspore culture that leads to cell division and pro-embryos in olive, is reported.

Cytological analysis of early microspore divisions leading to gametic embryo formation in Quercus suber L. anther cultures

The correlation between the phenologic stage of the inflorescence and the microspore development stage was studied. Cytological examinations of the development of microspores during in vitro anther culture of cork oak (Quercus suber L.), were carried out during the first four weeks of culture. To observe the division occurring in the microspores, anthers were taken randomly from the cultures after heat shock treatment and were stained with DAPI. Most of the anthers responding to a heat stress treatment contained 91 % vacuolated microspores, indicating that this developmental stage is responsive to embryogenesis induction in cork-oak microspores. After the heat shock treatment some cork-oak microspores were induced and initiated the embryogenic pathway with the occurrence of numerous symmetric mitosis, producing structures with two to ten or more nuclei. These lead to the formation of high numbers of multicellular cork-oak microspores (pro-embryos). Twenty-forty days after induction, small white globular and cotyledonal embryos were observed, which further developed root and shoot, regenerating plantlets.

Embryogenic Response in Different Medicago arborea L. Explants Depending on Cytokinin/Auxin Balances

Summary This paper analyses the effect of various combinations of 2,4-dichlorophenoxyacetic acid and a number of cytokinins on the induction of somatic embryogenesis in hypocotyl, cotyledon, petiole and leaf explants of Medicago arborea. 2,4-dichlorophenoxyacetic acid was used at concentrations of 1 and 2 mg L −1 . The cytokinins tested in combination with 2,4-dichlorophenoxyacetic acid were kinetin (0.25, 0.5 and 2 mg L −1 ), 6-benzylaminopurine (2 mg L −1 ) or thidiazuron (2 mg L −1 ). Significant differences have been found in embryogenic response, both among different media and among different explants. The medium with the highest concentration of 2,4-dichlorophenoxyacetic acid (2 mg L −1 ) and kinetin (2 mg L −1 ) was optimal for embryogenic calli induction, whereas in the media containing kinetin or 2,4-dichlorophenoxyacetic acid alone, the mentioned response was absent. Among the explants analysed, hypocotyl and petiole show a significantly greater capacity to evoke an embryogenic response. In general, an increase in the kinetinl2,4-dichlorophenoxyacetic acid balance has a positive effect on the embryogenic response in all of the different explants analysed. The results obtained seem to suggest the possible existence of an endogenous hormonal gradient from the first trifoliate leaf to the hypocotyl, which can affect embryogenic capacity.

Induction of Quercus ilex L. haploid and doubled-haploid embryos from anther cultures by temperature-stress

Abstract This paper describes a method to obtain haploid and doubled-haploid (DH) embryos using anther cultures of holm oak (Quercus ilex L.). The production of haploids and DH through gametic embryogenesis provides an attractive biotechnological tool for developing homozygous lines from heterozygous parents, which is important in breeding programs, as well as in genetic studies. As a consequence, protocols to produce homozygous plants have a significant impact on forest tree improvement. Anthers were subjected to different temperature treatments for embryo induction: a cold pre-treatment (4°C) from 3 to 7 days was carried out at the beginning, followed by a heat shock (33°C) from 2 to 5 days. Most anthers responding to these stress treatments contained vacuolated microspores, indicating that this developmental stage is responsive to embryogenesis induction in holm-oak microspores. In all cases, embryos grew from the interior of the anthers, breaking through the degenerating anther walls. Under these conditions, embryo formation occurred in 31 anthers between 46 and 95 days after culture initiation. Embryo analysis performed with flow-cytometry and DNA-microsatellite markers showed haploid profiles and/or spontaneous doubling of the chromosomes during early regeneration stages. This is, to our knowledge, the first published report on gametic embryogenesis in holm oak.

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.