Spain has been a fruitful locus for the European imagination for centuries, and it has been most often perceived in black-and-white oppositions -- either as a tyrannical and fanatical force in the ...early modern period or as an imaginary geography of a ‘Romantic’ Spain in later centuries. However, the image of Spain, its culture and its inhabitants did not evolve inexorably from negative to positive. From the early modern period onwards, it responded to an ambiguous matrix of conflicting Hispanophobic and Hispanophilic representations. Just as in the nineteenth century latent negative stereotypes continued to resurface, even in the Romantic heyday, in the early modern period appreciation for Spain was equally undeniable. When Spain was a political and military superpower, it also enjoyed cultural hegemony with a literary Golden Age producing internationally hailed masterpieces. Literary Hispanophobia and Hispanophilia in Britain and the Low Countries (1550-1850) explores the protracted interest in Spain and its culture, and it exposes the co-existent ambiguity between scorn and fascination that characterizes Western historical perceptions, in particular in Britain and the Low Countries, two geographical spaces with a shared sense of historical connectedness and an overlapping, sometimes complicated, history with Spain.
Somatic embryogenesis (SE) is a crucial biotechnological tool for large‐scale mass propagation of selected material, genetic transformation and breeding, with many advantages for forest tree ...improvement. However, the application of SE in forest species is limited because of their recalcitrance. SE is also a valuable system for studying cell reprogramming, acquisition of totipotency and embryo development. Increasing evidence reveals that epigenetic reprogramming takes place during SE through DNA methylation, although there is scarce information on forest species. In this work, we have evaluated DNA methylation dynamics during SE and the effects of the DNA demethylating agent 5‐azacytidine (AzaC) in cork oak. After induction and early stages of SE, a reduced DNA methylation level is observed, followed by an increase of methylation during embryo differentiation. These changes in DNA methylation during SE progression were associated with expression profiles of DNA methyltransferase genes QsMET1, QsDRM2 and QsCMT3, and DNA demethylase QsDME‐like. Treatment with AzaC reduced DNA methylation, promoted SE induction rate and proembryogenic masses proliferation, and induced the expression of the SE marker gene QsSERK1‐like. However, continuous AzaC treatment hindered embryo differentiation, suggesting that DNA methylation is needed for further embryo development. Interestingly, AzaC removal from the culture medium of embryogenic masses restored embryo development and led to a significant increase in somatic embryo production compared with untreated cultures. These findings open new possibilities using transitory treatments with small molecule epigenetic modulators, as AzaC, to enhance SE yields for forestry breeding programs.
•Heterogeneous organization was found, particularly in embryogenic callus.•Calli with different competencies showed similar global H3K9 methylation levels.•H3K9me2 signal was lower in cells showing ...embryogenic-like features.•Increasing H3K9 methylation and H3K9me2 signal were observed in developing embryos.•H3K9 methylation was found involved in tamarillo somatic embryo development.
The capacity to regenerate is intrinsic to plants and is the basis of natural asexual propagation and artificial cloning. Despite there are different ways of plant regeneration, they all require a change in cell fate and pluripotency reacquisition, in particular somatic embryogenesis. The mechanisms underlying somatic cell reprogramming for embryogenic competence acquisition, expression and maintenance remain not fully understood. These complex processes have been often associated with epigenetic markers, mainly DNA methylation, while little is known about the possible role of histone modifications. In the present study, the dynamics of global levels and distribution patterns of histone H3 methylation at lysine 9 (H3K9), a major repressive histone modification, were analyzed in somatic embryogenesis-induced cell lines with different embryogenic capacities and during somatic embryo initiation, in the woody species Solanum betaceum. Quantification of global H3K9 methylation showed similar levels in the three types of proliferating calli (embryogenic, long-term and non-embryogenic), kept in high sucrose and auxin-containing medium. Microscopic analyzes revealed heterogeneous cell organization and different cell types, particularly evident in embryogenic callus. The H3K9 dimethylation (H3K9me2) immunofluorescence signal was lower in nuclei of cells showing embryogenic-like and proliferating features, while labeling was higher in vacuolated, non-embryogenic cells with higher proliferation rates. By auxin removal, somatic embryo development was promoted in the embryogenic cell line. During the initiation of this process, increasing levels of global H3K9 methylation were found, together with increasing H3K9me2 immunofluorescence signals, especially in cells of the developing embryo. These results suggest that H3K9 methylation is involved in somatic embryo development, a developmental pathway in which this epigenetic mark could play a role in the gene transcription variation that is associated with embryogenic competence expression in S. betaceum. Altogether, these data provide new insights into the role of this epigenetic mark in somatic embryogenesis in trees, where scarce information is available.
The microspore can follow two different developmental pathways. In vivo microspores follow the gametophytic program to produce pollen grains. In vitro, isolated microspores can be reprogrammed by ...stress treatments and follow the embryogenic program, producing doubled-haploid embryos. In the present study, we analyzed the dynamics and role of endogenous auxin in microspore development during these two different scenarios, in
We analyzed auxin concentration, cellular accumulation, the expression of the
auxin biosynthesis gene, and the
efflux carrier gene, as well as the effects of inhibiting auxin biosynthesis by kynurenine on microspore embryogenesis. During the gametophytic pathway, auxin levels and
and
expression were high at early stages, in tetrads and tapetum, while they progressively decreased during gametogenesis in both pollen and tapetum cells. In contrast, in microspore embryogenesis,
and
genes were upregulated, and auxin concentration increased from the first embryogenic divisions. Kynurenine treatment decreased both embryogenesis induction and embryo production, indicating that auxin biosynthesis is required for microspore embryogenesis initiation and progression. The findings indicate that auxin exhibits two opposite profiles during these two microspore developmental pathways, which determine the different cell fates of the microspore.
Inhibition of GSK-3 activity by novel small molecules enhances somatic embryogenesis efficiency by activating the brassinosteroid pathway, an innovation with high potential to improve in vitro plant ...regeneration for crop and forest breeding techniques.
Abstract
Plant in vitro regeneration systems, such as somatic embryogenesis, are essential in breeding; they permit propagation of elite genotypes, production of doubled-haploids, and regeneration of whole plants from gene editing or transformation events. However, in many crop and forest species, somatic embryogenesis is highly inefficient. We report a new strategy to improve in vitro embryogenesis using synthetic small molecule inhibitors of mammalian glycogen synthase kinase 3β (GSK-3β), never used in plants. These inhibitors increased in vitro embryo production in three different systems and species, microspore embryogenesis of Brassica napus and Hordeum vulgare, and somatic embryogenesis of Quercus suber. TDZD-8, a representative compound of the molecules tested, inhibited GSK-3 activity in microspore cultures, and increased expression of embryogenesis genes FUS3, LEC2, and AGL15. Plant GSK-3 kinase BIN2 is a master regulator of brassinosteroid (BR) signalling. During microspore embryogenesis, BR biosynthesis and signalling genes CPD, GSK-3-BIN2, BES1, and BZR1 were up-regulated and the BAS1 catabolic gene was repressed, indicating activation of the BR pathway. TDZD-8 increased expression of BR signalling elements, mimicking BR effects. The findings support that the small molecule inhibitors promoted somatic embryogenesis by activating the BR pathway, opening up the way for new strategies using GSK-3β inhibitors that could be extended to other species.
Microspore embryogenesis is a process of cell reprogramming, totipotency acquisition and embryogenesis initiation, induced
by stress treatments and widely used in plant breeding for rapid production ...of doubled-haploids, but its regulating mechanisms are still largely unknown. Increasing evidence has revealed epigenetic reprogramming during microspore embryogenesis, through DNA methylation, but less is known about the involvement of histone modifications. In this study, we have analyzed the dynamics and possible role of histone H3K9 methylation, a major repressive modification, as well as the effects on microspore embryogenesis initiation of BIX-01294, an inhibitor of histone methylation, tested for the first time in plants, in
and
. Results revealed that microspore reprogramming and initiation of embryogenesis involved a low level of H3K9 methylation. With the progression of embryogenesis, methylation of H3K9 increased, correlating with gene expression profiles of
and
(writer and eraser enzymes of H3K9me2). At early stages, BIX-01294 promoted cell reprogramming, totipotency and embryogenesis induction, while diminishing bulk H3K9 methylation. DNA methylation was also reduced by short-term BIX-01294 treatment. By contrast, long BIX-01294 treatments hindered embryogenesis progression, indicating that H3K9 methylation is required for embryo differentiation. These findings open up new possibilities to enhance microspore embryogenesis efficiency in recalcitrant species through pharmacological modulation of histone methylation by using BIX-01294.
Stress-induced microspore embryogenesis is a model in vitro system of cell reprogramming, totipotency acquisition, and embryo development. After induction, responsive microspores abandon their ...developmental program to follow an embryogenic pathway, leading to in vitro embryo formation. This process is widely used to produce doubled-haploid lines, essential players to create new materials in modern breeding programs, particularly in cereals, although its efficiency is still low in many crop species, because the regulating mechanisms are still elusive. Stress signaling and endogenous hormones, mainly auxin, have been proposed as determinant factors of microspore embryogenesis induction in some eudicot species; however, much less information is available in monocot plants. In this study, we have analyzed the dynamics and possible role of endogenous auxin during stress-induced microspore embryogenesis in the monocot Hordeum vulgare, barley. The results showed auxin accumulation in early proembryo cells, from embryogenesis initiation and a further increase with embryo development and differentiation, correlating with the induction and expression pattern of the auxin biosynthesis gene HvTAR2-like. Pharmacological treatments with kynurenine, inhibitor of auxin biosynthesis, and α-(p-chlorophenoxy)-isobutyric acid (PCIB), auxin antagonist, impaired embryogenesis initiation and development, indicating that de novo auxin synthesis and its activity were required for the process. Efflux carrier gene HvPIN1-like was also induced with embryogenesis initiation and progression; auxin transport inhibition by N-1-naphthylphthalamic acid significantly reduced embryo development at early and advanced stages. The results indicate activation of auxin biosynthesis with microspore embryogenesis initiation and progression, in parallel with the activation of polar auxin transport, and reveal a central role of auxin in the process in a monocot species. The findings give new insights into the complex regulation of stress-induced microspore embryogenesis, particularly in monocot plants for which information is still scarce, and suggest that manipulation of endogenous auxin content could be a target to improve in vitro embryo production.Stress-induced microspore embryogenesis is a model in vitro system of cell reprogramming, totipotency acquisition, and embryo development. After induction, responsive microspores abandon their developmental program to follow an embryogenic pathway, leading to in vitro embryo formation. This process is widely used to produce doubled-haploid lines, essential players to create new materials in modern breeding programs, particularly in cereals, although its efficiency is still low in many crop species, because the regulating mechanisms are still elusive. Stress signaling and endogenous hormones, mainly auxin, have been proposed as determinant factors of microspore embryogenesis induction in some eudicot species; however, much less information is available in monocot plants. In this study, we have analyzed the dynamics and possible role of endogenous auxin during stress-induced microspore embryogenesis in the monocot Hordeum vulgare, barley. The results showed auxin accumulation in early proembryo cells, from embryogenesis initiation and a further increase with embryo development and differentiation, correlating with the induction and expression pattern of the auxin biosynthesis gene HvTAR2-like. Pharmacological treatments with kynurenine, inhibitor of auxin biosynthesis, and α-(p-chlorophenoxy)-isobutyric acid (PCIB), auxin antagonist, impaired embryogenesis initiation and development, indicating that de novo auxin synthesis and its activity were required for the process. Efflux carrier gene HvPIN1-like was also induced with embryogenesis initiation and progression; auxin transport inhibition by N-1-naphthylphthalamic acid significantly reduced embryo development at early and advanced stages. The results indicate activation of auxin biosynthesis with microspore embryogenesis initiation and progression, in parallel with the activation of polar auxin transport, and reveal a central role of auxin in the process in a monocot species. The findings give new insights into the complex regulation of stress-induced microspore embryogenesis, particularly in monocot plants for which information is still scarce, and suggest that manipulation of endogenous auxin content could be a target to improve in vitro embryo production.
Hydrogenation of phenol to cyclohexanone in aqueous phase, under mild conditions, has been carried out using palladium on different supports such as hydroxyapatite (HA), carbon (C), alumina (γ
-Al
2O
...3) and Al
2O
3
-CWE synthesized with high surface area. High activity (100%) and selectivity (98%) to cyclohexanone was obtained using Pd/ Al
2O
3
-CWE catalyst in 50
min under mild reaction conditions.
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► Liquid phase hydrogenation of phenol. ► High selectivity to cyclohexanone. ► Metal nanoparticles on high surface area aluminas.
Autophagy is activated and plays a role in stress-induced cell death during microspore embryogenesis, along with cathepsin-like C1A protease activities. Autophagy inhibition reduces cell death, ...improving embryogenesis induction efficiency.
abstract
Microspores are reprogrammed towards embryogenesis by stress. Many microspores die after this stress, limiting the efficiency of microspore embryogenesis. Autophagy is a degradation pathway that plays critical roles in stress response and cell death. In animals, cathepsins have an integral role in autophagy by degrading autophagic material; less is known in plants. Plant cathepsins are papain-like C1A cysteine proteases involved in many physiological processes, including programmed cell death. We have analysed the involvement of autophagy in cell death, in relation to cathepsin activation, during stress-induced microspore embryogenesis in Hordeum vulgare. After stress, reactive oxygen species (ROS) and cell death increased and autophagy was activated, including HvATG5 and HvATG6 up-regulation and increase of ATG5, ATG8, and autophagosomes. Concomitantly, cathepsin L/F-, B-, and H-like activities were induced, cathepsin-like genes HvPap-1 and HvPap-6 were up-regulated, and HvPap-1, HvPap-6, and HvPap-19 proteins increased and localized in the cytoplasm, resembling autophagy structures. Inhibitors of autophagy and cysteine proteases reduced cell death and promoted embryogenesis. The findings reveal a role for autophagy in stress-induced cell death during microspore embryogenesis, and the participation of cathepsins. Similar patterns of activation, expression, and localization suggest a possible connection between cathepsins and autophagy. The results open up new possibilities to enhance microspore embryogenesis efficiency with autophagy and/or cysteine protease modulators.
We investigate whether key audit matter (KAM) paragraphs disclosed in extended audit reports—paragraphs in which the auditor highlights significant risks and critical judgments of the ...company—contribute to assess corporate credit ratings. This assessment is a complicated and expensive process to grade the reliability of a company, and it is relevant for many stakeholders, such as issuers, investors, and creditors. Although credit rating evaluations have attracted the interest of many researchers, previous studies have mainly focused only on financial ratios. We are the first to use KAMs for credit rating modelling purposes. Applying four machine learning techniques to answer this real-world problem—C4.5 decision tree, two different rule induction classifiers (PART algorithm and Rough Set) and the logistic regression methodology—, our evidence suggests that by simply identifying the KAM topics disclosed in the report, any decision-maker can assess credit scores with 74% accuracy using the rules provided by the PART algorithm. These rules specifically indicate that KAMs on both external (such as going concern) and internal (such as company debt) aspects may contribute to explaining a company’s credit rating. The rule induction classifiers have similar predictive power. Interestingly, if we combine audit data with accounting ratios, the predictive power of our model increases to 84%, outperforming the accuracy in the existing literature.
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