DNA demethylation by transitory 5-azacytidine treatment improves somatic embryogenesis yield for regeneration and breeding of cork oak Elena Carneros, Esteban M. Díaz‐Luzza, Yolanda Pérez‐Pérez, María Teresa Solís, Pilar S. Testillano Physiologia Plantarum, 2024 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.
Opposite Auxin Dynamics Determine the Gametophytic and Embryogenic Fates of the Microspore Yolanda Pérez-Pérez, María Teresa Solís, Alfonso Albacete, Pilar S. Testillano International Journal of Molecular Sciences, 2023 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 Brassica napus. We analyzed auxin concentration, cellular accumulation, the expression of the TAA1 auxin biosynthesis gene, and the PIN1-like efflux carrier gene, as well as the effects of inhibiting auxin biosynthesis by kynurenine on microspore embryogenesis. During the gametophytic pathway, auxin levels and TAA1 and PIN1-like 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, TAA1 and PIN1-like 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.
Stress-Induced Microspore Embryogenesis Requires Endogenous Auxin Synthesis and Polar Transport in Barley Yolanda Pérez-Pérez, Ahmed-Abdalla El-Tantawy, María Teresa Solís, María C. Risueño, Pilar S. Testillano Frontiers in Plant Science, 2019 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.
Proteases with caspase 3-like activity participate in cell death during stress-induced microspore embryogenesis of Brassica napus Eduardo Berenguer, María-Teresa Solís, Yolanda Pérez-Pérez, Pilar S. Testillano Eurobiotech Journal, 2019 Microspore embryogenesis is a model system of plant cell reprogramming, totipotency acquisition, stress response and embryogenesis initiation. This in vitro system constitutes an important biotechnological tool for haploid and doubled-haploid plant production, very useful for crop breeding. In this process, microspores (cells that produce pollen grains in planta) are reprogrammed toward embryogenesis by specific stress treatment, but many microspores die after the stress. The occurrence of cell death is a serious limiting problem that greatly reduces microspore embryogenesis yield. In animals, increasing evidence has revealed caspase proteolytic activities as essential executioners of programmed cell death (PCD) processes, however, less is known in plants. Although plant genomes do not contain caspase homologues, caspase-like proteolytic activities have been detected in many plant PCD processes. In the present study, we have analysed caspase 3-like activity and its involvement in stress-induced cell death during initial stages of microspore embryogenesis of Brassica napus. After stress treatment to induce embryogenesis, isolated microspore cultures showed high levels of cell death and caspase 3-like proteolytic activity was induced. Treatments with specific inhibitor of caspase 3-like activity reduced cell death and increased embryogenesis induction efficiency. Our findings indicate the involvement of proteases with caspase 3-like activity in the initiation and/or execution of cell death at early microspore embryogenesis in B. napus, giving new insights into the pathways of stress-induced cell death in plants and opening a new way to improve in vitro embryogenesis efficiency by using chemical modulators of cell death proteases.
Pectin de-methylesterification and AGP increase promote cell wall remodeling and are required during somatic embryogenesis of quercus suber Yolanda Pérez-Pérez, Elena Carneros, Eduardo Berenguer, María-Teresa Solís, Ivett Bárány, et al. Frontiers in Plant Science, 2019 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 β-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.
Inhibition of histone H3K9 methylation by BIX-01294 promotes stress-induced microspore totipotency and enhances embryogenesis initiation Eduardo Berenguer, Ivett Bárány, María-Teresa Solís, Yolanda Pérez-Pérez, María C. Risueño, et al. Frontiers in Plant Science, 2017 Microspore embryogenesis is a process of cell reprogramming, totipotency acquisition and embryogenesis initiation, induced in vitro 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 Brassica napus and Hordeum vulgare. 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 BnHKMT SUVR4-like and BnLSD1-like (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.
DNA methylation dynamics and MET1a-like gene expression changes during stress-induced pollen reprogramming to embryogenesis MT Solis, M Rodriguez-Serrano, M Meijon, MJ Canal, A Cifuentes, ... JOURNAL OF EXPERIMENTAL BOTANY 76 (16), 4729-4729 , 2025 2025
Retraction of: DNA methylation dynamics and MET1a-like gene expression changes during stress-induced pollen reprogramming to embryogenesis MT Solís, M Rodríguez-Serrano, M Meijón, MJ Cañal, A Cifuentes, ... Journal of Experimental Botany 76 (16), 4729 , 2025 2025
Auxin Biosynthesis, Accumulation, Action and Transport are Involved in Stress-Induced Microspore Embryogenesis Initiation and Progression in Brassica Napus (vol 56, pg 1401, 2015) H Rodriguez-Sanz, MT Solis, MF Lopez, A Gomez-Cadenas, MC Risueno, ... PLANT AND CELL PHYSIOLOGY 65 (11), 1900-1900 , 2024 2024
5-azacytidine promotes microspore embryogenesis initiation by decreasing global DNA methylation, but prevents subsequent embryo development in rapeseed and barley (vol 6, 472 … MT Solis, AA El-Tantawy, V Cano, MC Risueno, PS Testillano FRONTIERS IN PLANT SCIENCE 15 , 2024 2024
Corrigendum: 5-azacytidine promotes microspore embryogenesis initiation by decreasing global DNA methylation, but prevents subsequent embryo development in rapeseed and barley MT Solís, AA El-Tantawy, V Cano, MC Risueño, PS Testillano Frontiers in Plant Science 15, 1514893 , 2024 2024
DNA demethylation by transitory 5‐azacytidine treatment improves somatic embryogenesis yield for regeneration and breeding of cork oak E Carneros, EM Díaz‐Luzza, Y Pérez‐Pérez, MT Solís, PS Testillano Physiologia Plantarum 176 (1), e14143 , 2024 2024 Citations: 12
Assembly and phylogeographical analysis of novel Taenia solium mitochondrial genomes suggest stratification within the African-American genotype G Jiménez-Avalos, A Soto-Obando, M Solis, RH Gilman, V Cama, ... Parasites & Vectors 16 (1), 349 , 2023 2023 Citations: 2
Opposite auxin dynamics determine the gametophytic and embryogenic fates of the microspore Y Pérez-Pérez, MT Solís, A Albacete, PS Testillano International Journal of Molecular Sciences 24 (13), 11177 , 2023 2023 Citations: 7
Assembly and phylogeographic analysis of novel Taenia solium mitochondrial genomes reveal further differentiation between and within Asian and African … G Jiménez-Avalos, AS Obando, M Solis, RH Gilman, V Cama, ... bioRxiv, 2022.03. 10.483888 , 2022 2022 Citations: 1
Assessment of dynamics of global DNA methylation during the cryopreservation process of Pinus pinaster embryogenic lines I Mendoza-Poudereux, M Cano, MT Solís, F Esteve-Díaz, PS Testillano, ... bioRxiv, 2022.02. 27.481657 , 2022 2022 Citations: 3
In Situ/Subcellular Localization of Arabinogalactan Protein Expression by Fluorescent In Situ Hybridization (FISH) ML Da Costa, MT Solís, PS Testillano, S Coimbra The Plant Cell Wall: Methods and Protocols, 403-427 , 2020 2020 Citations: 1
Stress-induced microspore embryogenesis requires endogenous auxin synthesis and polar transport in barley Y Pérez-Pérez, AA El-Tantawy, MT Solís, MC Risueño, PS Testillano Frontiers in plant science 10, 1200 , 2019 2019 Citations: 45
Pectin De-methylesterification and AGP Increase Promote Cell Wall Remodeling and Are Required During Somatic Embryogenesis of Quercus suber Y Pérez-Pérez, E Carneros, E Berenguer, MT Solís, I Bárány, B Pintos, ... Frontiers in plant science 9, 1915 , 2019 2019 Citations: 71
Proteases with caspase 3-like activity participate in cell death during stress-induced microspore embryogenesis of Brassica napus. E Berenguer, MT Solís González, Y Pérez-Pérez, P Testillano Sciendo , 2019 2019 Citations: 8
Cell wall remodeling by pectin esterification and AGP expression underlies somatic embryogenesis of cork oak Y Pérez-Pérez, E Carneros, MT Solís, I Bárány, B Pintos, A Gómez-Garay, ... Clonal Trees in the Bioeconomy Age: Opportunities and Challenges , 2018 2018
Autophagy is activated and involved in cell death with participation of cathepsins during stress-induced microspore embryogenesis in barley I Bárány, E Berenguer, MT Solís, Y Pérez-Pérez, ME Santamaría, ... Journal of Experimental Botany 69 (6), 1387-1402 , 2018 2018 Citations: 88
In Vitro evaluation of plant extracts and bio-control agents againstDiplodia seriata SA Calderón, L Martin, MT Solis, BP López, AG Garay International Congress on Grapevine and Wine Sciences: abstracts Book, 93 , 2018 2018
Inhibition of histone H3K9 methylation by BIX-01294 promotes stress-induced microspore totipotency and enhances embryogenesis initiation E Berenguer, I Bárány, MT Solís, Y Pérez-Pérez, MC Risueño, ... Frontiers in plant science 8, 1161 , 2017 2017 Citations: 84
Initiation of leaf somatic embryogenesis involves high pectin esterification, auxin accumulation and DNA demethylation in Quercus alba E Corredoira, V Cano, I Bárány, MT Solís, H Rodríguez, AM Vieitez, ... Journal of plant physiology 213, 42-54 , 2017 2017 Citations: 72
Bn PME is progressively induced after microspore reprogramming to embryogenesis, correlating with pectin de-esterification and cell differentiation in Brassica napus MT Solís, E Berenguer, MC Risueño, PS Testillano BMC plant biology 16 (1), 176 , 2016 2016 Citations: 44
MOST CITED SCHOLAR PUBLICATIONS
A new microspore embryogenesis system under low temperature which mimics zygotic embryogenesis initials, expresses auxin and efficiently regenerates doubled-haploid plants in … D Prem, MT Solís, I Bárány, H Rodríguez-Sanz, MC Risueño, ... BMC Plant Biology 12 (1), 127 , 2012 2012 Citations: 135
5-azacytidine promotes microspore embryogenesis initiation by decreasing global DNA methylation, but prevents subsequent embryo development in rapeseed and barley MT Solís, AA El-Tantawy, V Cano, MC Risueño, PS Testillano Frontiers in plant science 6, 472 , 2015 2015 Citations: 112
DNA methylation dynamics and MET1a-like gene expression changes during stress-induced pollen reprogramming to embryogenesis MT Solís, M Rodríguez-Serrano, M Meijón, MJ Cañal, A Cifuentes, ... Journal of Experimental Botany 63 (18), 6431 , 2012 2012 Citations: 111
Differential expression patterns of arabinogalactan proteins in Arabidopsis thaliana reproductive tissues AM Pereira, S Masiero, MS Nobre, ML Costa, MT Solís, PS Testillano, ... Journal of experimental botany 65 (18), 5459-5471 , 2014 2014 Citations: 98
Autophagy is activated and involved in cell death with participation of cathepsins during stress-induced microspore embryogenesis in barley I Bárány, E Berenguer, MT Solís, Y Pérez-Pérez, ME Santamaría, ... Journal of Experimental Botany 69 (6), 1387-1402 , 2018 2018 Citations: 88
Inhibition of histone H3K9 methylation by BIX-01294 promotes stress-induced microspore totipotency and enhances embryogenesis initiation E Berenguer, I Bárány, MT Solís, Y Pérez-Pérez, MC Risueño, ... Frontiers in plant science 8, 1161 , 2017 2017 Citations: 84
Early markers are present in both embryogenesis pathways from microspores and immature zygotic embryos in cork oak, Quercus suber L H Rodríguez-Sanz, JA Manzanera, MT Solís, A Gómez-Garay, B Pintos, ... BMC plant biology 14 (1), 224 , 2014 2014 Citations: 76
Arabinogalactan protein profiles and distribution patterns during microspore embryogenesis and pollen development in Brassica napus AA El-Tantawy, MT Solís, ML Da Costa, S Coimbra, MC Risueño, ... Plant Reproduction 26 (3), 231-243 , 2013 2013 Citations: 75
Initiation of leaf somatic embryogenesis involves high pectin esterification, auxin accumulation and DNA demethylation in Quercus alba E Corredoira, V Cano, I Bárány, MT Solís, H Rodríguez, AM Vieitez, ... Journal of plant physiology 213, 42-54 , 2017 2017 Citations: 72
Pectin De-methylesterification and AGP Increase Promote Cell Wall Remodeling and Are Required During Somatic Embryogenesis of Quercus suber Y Pérez-Pérez, E Carneros, E Berenguer, MT Solís, I Bárány, B Pintos, ... Frontiers in plant science 9, 1915 , 2019 2019 Citations: 71
Changes in histone methylation and acetylation during microspore reprogramming to embryogenesis occur concomitantly with BnHKMT and BnHAT expression and are associated with … H Rodríguez-Sanz, J Moreno-Romero, MT Solís, C Köhler, MC Risueño, ... Cytogenetic and Genome Research 143 (1-3), 209-218 , 2014 2014 Citations: 68
Early markers of in vitro microspore reprogramming to embryogenesis in olive (Olea europaea L.) MT Solís, B Pintos, MJ Prado, MÁ Bueno, I Raska, MC Risueño, ... Plant Science 174 (6), 597-605 , 2008 2008 Citations: 66
Changes in DNA methylation levels and nuclear distribution patterns after microspore reprogramming to embryogenesis in barley AA El-Tantawy, MT Solís, MC Risueño, PS Testillano Cytogenetic and genome research 143 (1-3), 200-208 , 2014 2014 Citations: 65
Auxin Biosynthesis, Accumulation, Action and Transport are Involved in Stress-Induced Microspore Embryogenesis Initiation and Progression in Brassica napus H Rodríguez-Sanz, MT Solís, MF López, A Gómez-Cadenas, MC Risueño, ... Plant and Cell Physiology 56 (7), 1401-1417 , 2015 2015 Citations: 62
Epigenetic changes accompany developmental programmed cell death in tapetum cells MT Solís, N Chakrabarti, E Corredor, J Cortés-Eslava, ... Plant and Cell Physiology 55 (1), 16-29 , 2014 2014 Citations: 58
Stress-induced microspore embryogenesis requires endogenous auxin synthesis and polar transport in barley Y Pérez-Pérez, AA El-Tantawy, MT Solís, MC Risueño, PS Testillano Frontiers in plant science 10, 1200 , 2019 2019 Citations: 45
Bn PME is progressively induced after microspore reprogramming to embryogenesis, correlating with pectin de-esterification and cell differentiation in Brassica napus MT Solís, E Berenguer, MC Risueño, PS Testillano BMC plant biology 16 (1), 176 , 2016 2016 Citations: 44
The 5‐methyl‐deoxy‐cytidine (5mdC) localization to reveal in situ the dynamics of DNA methylation chromatin pattern in a variety of plant organ and tissue cells during development PS Testillano, MT Solís, MC Risueño Physiologia Plantarum 149 (1), 104-113 , 2013 2013 Citations: 41
DNA demethylation by transitory 5‐azacytidine treatment improves somatic embryogenesis yield for regeneration and breeding of cork oak E Carneros, EM Díaz‐Luzza, Y Pérez‐Pérez, MT Solís, PS Testillano Physiologia Plantarum 176 (1), e14143 , 2024 2024 Citations: 12
Proteases with caspase 3-like activity participate in cell death during stress-induced microspore embryogenesis of Brassica napus. E Berenguer, MT Solís González, Y Pérez-Pérez, P Testillano Sciendo , 2019 2019 Citations: 8
