Retrato y Relato

El Caso Alquería fue el nombre con el que se bautizó en 2018 a un supuesto caso de corrupción que sacudió la Diputación valenciana cuando su presidente era Jorge Rodríguez Gramage. Este, que a su vez era también alcalde de Ontinyent, fue acusado de presuntos delitos de prevaricación administrativa, malversación en caudales públicos y falsedad en documento oficial. Todos conocían al político, pero muy pocos a la persona, por eso, este proyecto consiste en descubrir el verdadero retrato que se encuentra detrás de este relato. Es interesante que, aunque Rodríguez dimitió de se puesto como presidente en la Diputación, no lo hizo de la Alcaldía. Sino que, además, revalidó la mayoría absoluta como alcalde tan solo un año después de que estallara este escándalo, en las elecciones de 2019, consiguiendo 17 de los 21 concejales del Ayuntamiento. A día de hoy sigue siendo alcalde, ya que en los comicios de mayo de 2023 consiguió 12 concejalías, volviendo a obtener mayoría absoluta. Sin duda, si algo puede explicar su trayectoria como alcalde, es el apoyo que la gente de su ciudad le ha brindado. Casi todos confiaron en su inocencia y así lo demostraron en las urnas hasta en dos ocasiones, aun sin tener una sentencia firme. Pero no hay que olvidar que, por encima del político, hay una persona que se ha tenido que enfrentar a los peores años de su vida. Incluso perdió a su madre entre toda esta vorágine y siempre ante todos ha mantenido una buena cara. Por eso, a lo largo de este pódcast, se pueden descubrir las inquietudes de su protagonista. Pero sobre todas las cosas, lo que se intenta es comprender lo que esta persona ha tenido que vivir durante los casi cinco años que ha durado la investigación y la resolución del Caso Alquería. Y aunque al final la sentencia ha sido favorable tanto para Rodríguez como para todos los acusados, no se ha cerrado el caso todavía, ya que la Fiscalía, sí decidió recurrir el dictamen del juez. Ahora, no se sabe exactamente cuanto más se extenderá en el tiempo este asunto, pero los investigados ya descansan tranquilos y aliviados, porque todo apunta a que el final de esta pesadilla está muy cerca.The Alquería Case was the name given in 2018 to an alleged corruption case that shook the Valencian Provincial Council when its president was Jorge Rodríguez Gramage. He, who was also the mayor of Ontinyent at the time, was accused of alleged crimes of administrative misconduct, embezzlement of public funds, and forgery of an official document. While many knew the politician, very few knew the person behind the story. This project aims to uncover the true portrait behind this narrative. Interestingly, although Rodríguez resigned from his position as president of the Provincial Council, he did not step down as the Mayor. Furthermore, he managed to secure an absolute majority as mayor just a year after this scandal erupted, in the 2019 elections, winning 17 out of 21 council seats. As of today, he remains the mayor, as he obtained 12 council seats in the May 2023 elections, once again securing an absolute majority. Undoubtedly, what can explain his tenure as mayor is the support that the people of his city have shown him. Almost everyone trusted in his innocence and demonstrated it at the polls on two occasions, even without a firm conviction. However, it's important not to forget that beyond the politician, there is a person who has had to face the worst years of their life. He even lost his mother amidst all this turmoil and always maintained a cheerful demeanor in public. Therefore, throughout this podcast, you can discover the concerns of its protagonist. But above all, the goal is to understand what this person has had to endure during the nearly five years of the Alquería Case investigation and resolution. Although the verdict has been favorable for both Rodríguez and all the accused in the end, the case has not been closed yet, as the Prosecutor's Office decided to appeal the judge's ruling. Now, it is uncertain how much longer this matter will linger, but the investigated parties can now rest assured and relieved, as everything points to the end of this nightmare being very close

Análisis del autocorte ribozimático y la expresión de los retroelementos tipo Penélope en la especie Selaginella moellendorffii

[ES] A principio de los años 80 se descubrieron unas moléculas catalíticas cuyo centro activo estaba compuesto completamente por RNA y que se denominaron RNAs catalíticos o ribozimas. Desde su descubrimiento hasta ahora se han producido muchos avances en este campo que han permitido descubrir diferentes tipos de ribozimas. Entre las ribozimas naturales conocidas, el grupo más numeroso es el de las pequeñas ribozimas de autocorte. Dentro de este grupo, destacan las ribozimas cabeza de martillo o hammerhead (HHR), las primeras en describirse y probablemente las mejor estudiadas. Datos previos a este trabajo indican que las HHR se encuentran distribuidas en multitud de genomas a lo largo de toda la escala biológica. Además, muchos de estos motivos catalíticos se han encontrado con frecuencia en elementos genéticos móviles como son los retrotransposones. En este proyecto se ha trabajado con una primitiva planta vascular, Selaginella moellendorffii, cuyo genoma contiene retrotransposones de la familia de elementos similares a Penélope (PLEs), que se caracterizan por contener en su secuencia ribozimas de cabeza de martillo. Para poder conocer el funcionamiento y las características de los transposones PLE y sus ribozimas en S. moellendorffii, se comprobó inicialmente el poder de autoescisión de dichas ribozimas. Para ello, primero se clonaron en plásmidos varios fragmentos del retrotransposón incluyendo la secuencia de la ribozima HHR. A continuación, se realizaron diversos ensayos de autocorte bajo diferentes condiciones (co-transcripcionales, post-transcripcionales, diversos tiempos de incubación¿) para comprobar la eficiencia catalítica de la ribozima. Los resultados obtenidos in vitro mostraron muy bajas tasas de autocorte de la HHR. Estudios previos apuntaban que las ribozimas de los elementos PLE se disponen como dímeros en tándem. Esta disposición concreta y los resultados obtenidos de baja escisión de manera individual refuerzan la idea de que las ribozimas de los elementos Penélope de S. moellendorffii funcionarían más eficientemente como dímeros. Por tanto, futuros pasos en la investigación de las ribozimas en elementos PLE de S. moellendorffii consistirán en la realización de construcciones diméricas, de las cuales se esperan mayores tasas de escisión. Por otro lado, en este estudio también se comprobó si los elementos Penélope se estaban expresando en planta. Para ello, se extrajo RNA total de plantas de S. moellendorffii y se realizaron ensayos de hibridación Northern usando sondas marcadas con digoxigenina. Mediante estos estudios se comprobó la existencia de una banda de RNA del tamaño esperado para el elemento PLE de S. moellendorffii (~5 kb), lo que sugiere que este elemento transponible realmente se podría estar expresando en la planta. Para completar esta línea de trabajo, será necesario comprobar la amplificación por RT-PCR del RNA detectado por análisis Northern. La elucidación de las tasas de escisión y del funcionamiento de ribozimas que se encuentran en organismos como S. moellendorffii proporciona las bases necesarias para poder comprender el funcionamiento de estos motivos en los genomas de multitud de especies. Con este conocimiento se podrá utilizar con mayor precisión ribozimas artificiales capaces de escindir secuencias específicas. Tanto las ribozimas naturales como las nuevas ribozimas creadas son cada vez más utilizadas en terapia génica para tener un control de la expresión genética.[EN] At the beginning of the 80s, catalytic molecules whose active centre was composed entirely of RNA were discovered and were called catalytic RNAs or ribozymes. Since their discovery until now, many advances have been made in this field that have led to the discovery of different types of ribozymes. Among the known natural ribozymes, the most numerous group is the small self-cleaving ribozymes. Within this group, hammerhead ribozymes (HHR) stand out, the first to be described and probably the best studied. Previous data indicate that hammerhead ribozymes are distributed in multitude of genomes throughout the biological scale. Furthermore, many of these catalytic motifs have been frequently found in mobile genetic elements such as retrotransposons. In this project we have worked with a primitive plant, Selaginella moellendorffii, whose genome contains retrotransposons of the Penelope-like elements family (PLEs), which are characterized by containing hammerhead ribozymes in its sequence. In order to find out how PLE transposon and their ribozymes in S. moellendorffii work and which characteristics they have, the self-cleaving capabilities of these ribozymes were initially tested. For this purpose, several fragments of the retrotransposon including the HHR ribozyme sequence were first cloned into plasmids. Then, several transcription tests were performed under different conditions (co-transcriptionally, post-transcriptionally, different incubation times¿) to check the self-cleaving efficiency of the ribozyme. The results obtained showed very low rates of self-cleaving of this HHR. Previous studies pointed out that ribozymes of PLE elements are arranged as tandem dimers. This particular arrangement and the expected low excision results individually reinforce the idea that ribozymes from S. moellendorffii PLEs would function more efficiently as dimers. Therefore, the next step in the investigation of ribozymes from S. moellendorffii would be to get dimeric constructs, from which higher cleavage rates are expected. On the other hand, in this study we also checked whether the Penelope elements were being expressed in plants. For this purpose, total RNA was extracted from S. moellendorffii plants and Northern hybridization test were performed using probes marked with digoxigenin. These studies proved the existence of an RNA band of the expected size for the PLE of S. moellendorffii (~5kb), suggesting that this transposable element is being expressed in the plant. To complete this work line, it will be necessary to amplify by RT-PCR and to sequence the RNA detected by Northern analysis. The elucidation of the cleavage rates and functioning of ribozymes found in organisms such as Selaginella moellendorffii provides the necessary basis for understanding the functioning of these motifs in the genomes of many other species. With this knowledge, artificial ribozymes capable of cleaving specific sequences could be used more precisely. Both natural and newly created ribozymes are increasingly used in gene therapy to control gene expression.Seguí Pérez, A. (2020). Análisis del autocorte ribozimático y la expresión de los retroelementos tipo Penélope en la especie Selaginella moellendorffii. http://hdl.handle.net/10251/149776TFG

Dynamics of calcium during in vitro microspore embryogenesis and in vivo microspore development in Brassica napus and Solanum melongena

[EN] Calcium is widely known to have a role as a signaling molecule in many different processes, including stress response and activation of the embryogenic program. However, there are no direct clues about calcium levels during microspore embryogenesis, an experimental process that combines a developmental switch toward embryogenesis and the simultaneous application of different stressing factors. In this work, we used FluoForte, a calcium-specific fluorescent vital dye, to track by confocal microscopy the changes in levels and subcellular distribution of calcium in living rapeseed (B. napus) and eggplant (S. melongena) microspores and pollen grains during in vivo development, as well as during the first stages of in vitro-induced microspore embryogenesis in rapeseed. During in vivo development, a clear peak of cytosolic Ca2+ was observed in rapeseed vacuolate microspores and young pollen grains, the stages more suitable for embryogenesis induction. However, the Ca2+ levels observed in eggplant were dramatically lower than in rapeseed. Just after in vitro induction, Ca2+ levels increased specifically in rapeseed embryogenic microspores at levels dramatically higher than during in vivo development. The increase was observed in the cytosol, but predominantly in vacuoles. Non-embryogenic forms such as callus-like and pollen-like structures presented remarkably different calcium patterns. After the heat shock-based inductive treatment, Ca2+ levels progressively decreased in all cases. Together, our results reveal unique calcium dynamics in in vivo rapeseed microspores, as well as in those reprogrammed to in vitro embryogenesis, establishing a link between changes in Ca2+ level and subcellular distribution, and microspore embryogenesis.This work was supported by grant AGL2014-55177-R to JS from Spanish Ministerio de Economia y Competitividad (MINECO) jointly funded by FEDER. AR was supported by a predoctoral fellowship from the FPI Program of Universitat Politecnica de Valencia.Rivas-Sendra, A.; Calabuig-Serna, A.; Seguí-Simarro, JM. (2017). Dynamics of calcium during in vitro microspore embryogenesis and in vivo microspore development in Brassica napus and Solanum melongena. Frontiers in Plant Science. 8. doi:10.3389/fpls.2017.01177

Development and characterization of an eggplant (Solanum melongena) doubled haploid population and a doubled haploid line with high androgenic response

[EN] We developed an eggplant doubled haploid (DH) population from a commercial hybrid through androgenesis in microspore culture. Morphological variation, reproductive ability and androgenic responsiveness were evaluated. The DH population showed segregation in vegetative traits related to leaf, flower and fruit, and in reproductive traits such as fruit and seed setting or germination rate. The DH population and subsequent generations also presented variation in the androgenic response, with null, low and high response lines. From this population, we were able to identify the first eggplant highly androgenic DH line (DH36), remarkably similar to the donor hybrid in terms of morphology and reproductive ability, but stably producing four times more calli than the hybrid. The segregating DH population is potentially useful for genetic studies and mapping of several traits, whereas the highly androgenic line DH36 may be used as a model line to facilitate the study of eggplant androgenesis and embryogenesis for both basic and applied research.We would like to thank the reviewers of this manuscript for their critical and helpful comments. This work was supported by Grant AGL2014-55177-R to JMSS from Spanish Ministerio de Economia y Competitividad (MINECO) jointly funded by FEDER. ARS is supported by a Predoctoral Fellowship from the FPI Program of Universitat Politecnica de Valencia.Rivas-Sendra, A.; Manuel Campos-Vega; Calabuig-Serna, A.; Seguí-Simarro, JM. (2017). Development and characterization of an eggplant (Solanum melongena) doubled haploid population and a doubled haploid line with high androgenic response. Euphytica. 213(4):1-14. doi:10.1007/s10681-017-1879-3S1142134Alpsoy HC, Seniz V (2007) Researches on the in vitro androgenesis and obtaining haploid plants in some eggplant genotypes. Acta Hortic 729:137–141Başay S, Şeniz V, Ellialtioğlu Ş (2011) Obtaining dihaploid lines by using anther culture in the different eggplant cultivars. J Food Agric Environ 9:188–190Bohanec B (2002) Doubled-haploid onions. In: Rabinowitch HD, Currah L (eds) Allium crop science: recent advances. CABI Publishing, Wallingford, pp 145–157Corral-Martínez P, Seguí-Simarro JM (2012) Efficient production of callus-derived doubled haploids through isolated microspore culture in eggplant (Solanum melongena L.). Euphytica 187:47–61Corral-Martínez P, Seguí-Simarro JM (2014) Refining the method for eggplant microspore culture: effect of abscisic acid, epibrassinolide, polyethylene glycol, naphthaleneacetic acid, 6-benzylaminopurine and arabinogalactan proteins. Euphytica 195:369–382Corral-Martínez P, Parra-Vega V, Seguí-Simarro JM (2013) Novel features of Brassica napus embryogenic microspores revealed by high pressure freezing and freeze substitution: evidence for massive autophagy and excretion-based cytoplasmic cleaning. J Exp Bot 64:3061–3075Daghma DES, Hensel G, Rutten T, Melzer M, Kumlehn J (2014) Cellular dynamics during early barley pollen embryogenesis revealed by time-lapse imaging. Front Plant Sci 5:675Doğramacı-Altuntepe M, Peterson TS, Jauhar PP (2001) Anther culture-derived regenerants of durum wheat and their cytological characterization. J Hered 92:56–64Dumas de Vaulx R, Chambonnet D (1982) Culture in vitro d’anthères d’aubergine (Solanum melongena L.): stimulation de la production de plantes au moyen de traitements à 35°C associés à de faibles teneurs en substances de croissance. Agronomie 2:983–988Dumas de Vaulx R, Chambonnet D, Pochard E (1981) Culture in vitro d’anthères de piment (Capsicum annuum L.): amèlioration des taux d’obtenction de plantes chez différents génotypes par des traitments à +35°C. Agronomie 1:859–864Eudes F, Shim Y-S, Jiang F (2014) Engineering the haploid genome of microspores. Biocatal Agric Biotechnol 3:20–23FAOSTAT (2016). http://faostat.fao.org . Accessed Dec 2016Ferrie A, Caswell K (2011) Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production. Plant Cell Tissue Organ Cult 104:301–309Ferrie AMR, Keller WA (1995) Microspore culture for haploid plant production. In: Gamborg OL, Phillips GC (eds) Plant cell, tissue and organ culture: fundamental methods. Springer, Berlin, pp 155–164Ferrie A, Möllers C (2011) Haploids and doubled haploids in Brassica spp. for genetic and genomic research. Plant Cell Tissue Organ Cult 104:375–386Ferrie AMR, Palmer CE, Keller WA (1995) Haploid embryogenesis. In: Thorpe TA (ed) In vitro embryogenesis in plants. Kluwer Academic Publishers, Dordrecht, pp 309–344Forster BP, Heberle-Bors E, Kasha KJ, Touraev A (2007) The resurgence of haploids in higher plants. Trends Plant Sci 12:368–375Frary A, Doganlar S, Daunay MC (2007) Eggplant. In: Kole C (ed) Vegetables. Springer, Berlin, pp 287–313Germanà MA (2011a) Anther culture for haploid and doubled haploid production. Plant Cell Tissue Organ Cult 104:283–300Germanà MA (2011b) Gametic embryogenesis and haploid technology as valuable support to plant breeding. Plant Cell Rep 30:839–857Hoekstra S, Vanzijderveld MH, Louwerse JD, Heidekamp F, Vandermark F (1992) Anther and microspore culture of Hordeum vulgare L. cv. Igri. Plant Sci 86:89–96Immonen S, Robinson J (2000) Stress treatments and Ficoll for improving green plant regeneration in triticale anther culture. Plant Sci 150:77–84Kaeppler SM, Kaeppler HF, Rhee Y (2000) Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol 43:179–188Kasha KJ, Simion E, Miner M, Letarte J, Hu TC (2003) Haploid wheat isolated microspore culture protocol. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants. Kluwer Academic Publishers, Dordrecht, pp 77–82Li H, Soriano M, Cordewener J, Muiño JM, Riksen T, Fukuoka H, Angenent GC, Boutilier K (2014) The histone deacetylase inhibitor Trichostatin A promotes totipotency in the male gametophyte. Plant Cell 26:195–209Malik MR, Wang F, Dirpaul J, Zhou N, Hammerlindl J, Keller W, Abrams SR, Ferrie AMR, Krochko JE (2008) Isolation of an embryogenic line from non-embryogenic Brassica napus cv. Westar through microspore embryogenesis. J Exp Bot 59:2857–2873Miah MAA, Earle ED, Khush GS (1985) Inheritance of callus formation ability in anther cultures of rice, Oryza sativa L. Theor Appl Genet 70:113–116Miyoshi K (1996) Callus induction and plantlet formation through culture of isolated microspores of eggplant (Solanum melongena L.). Plant Cell Rep 15:391–395Nitsch JP (1972) Haploid plants from pollen. Z Pflanzenzücht 67:3–18Nitsch C, Nitsch JP (1967) Induction of flowering in vitro in stem segments of Plumbago indica L. I. Production of vegetative buds. Planta 72:355Nuez F, Llácer G (2001) La horticultura española. Ediciones de Horticultura, S.L., ReusOleszczuk S, Rabiza-Swider J, Zimny J, Lukaszewski AJ (2011) Aneuploidy among androgenic progeny of hexaploid triticale (XTriticosecale Wittmack). Plant Cell Rep 30:575–586Petolino JF, Jones AM, Thompson SA (1988) Selection for increased anther culture response in maize. Theor Appl Genet 76:157–159Quimio CA, Zapata FJ (1990) Diallel analysis of callus induction and green-plant regeneration in rice anther culture. Crop Sci 30:188–192Rivas-Sendra A, Corral-Martínez P, Camacho-Fernández C, Seguí-Simarro JM (2015) Improved regeneration of eggplant doubled haploids from microspore-derived calli through organogenesis. Plant Cell Tissue Organ Cult 122:759–765Rizza F, Mennella G, Collonnier C, Shiachakr D, Kashyap V, Rajam MV, Prestera M, Rotino GL (2002) Androgenic dihaploids from somatic hybrids between Solanum melongena and S. aethiopicum group Gilo as a source of resistance to Fusarium oxysporum f. sp. melongenae. Plant Cell Rep 20:1022–1032Rotino GL (1996) Haploidy in eggplant. In: Jain SM, Sopory SK, Veilleux RE (eds) In vitro haploid production in higher plants. Kluwer Academic Publishers, Dordrecht, pp 115–141Rotino GL, Restaino F, Gjomarkaj M, Massimo M, Falavigna A, Schiavi M, Vicini E (1991) Evaluation of genetic variability in embryogenetic and androgenetic lines of eggplant. Acta Hortic 300:357–362Rotino GL, Sihachakr D, Rizza F, Vale G, Tacconi MG, Alberti P, Mennella G, Sabatini E, Toppino L, D’Alessandro A, Acciarri N (2005) Current status in production and utilization of dihaploids from somatic hybrids between eggplant (Solanum melongena L.) and its wild relatives. Acta Physiol Plant 27:723–733Salas P, Prohens J, Seguí-Simarro JM (2011) Evaluation of androgenic competence through anther culture in common eggplant and related species. Euphytica 182:261–274Salas P, Rivas-Sendra A, Prohens J, Seguí-Simarro JM (2012) Influence of the stage for anther excision and heterostyly in embryogenesis induction from eggplant anther cultures. Euphytica 184:235–250Sanguineti MC, Tuberosa R, Conti S (1990) Field evaluation of androgenetic lines of eggplant. Acta Hortic 280:177–182Seguí-Simarro JM (2016) Androgenesis in Solanaceae. In: Germanà MA, Lambardi M (eds) In vitro embryogenesis. Springer, New York, pp 209–244Seguí-Simarro JM, Nuez F (2008) Pathways to doubled haploidy: chromosome doubling during androgenesis. Cytogenet Genome Res 120:358–369Seguí-Simarro JM, Corral-Martínez P, Parra-Vega V, González-García B (2011) Androgenesis in recalcitrant solanaceous crops. Plant Cell Rep 30:765–778Snape JW, Sitch LA, Simpson E, Parker BB (1988) Tests for the presence of gametoclonal variation in barley and wheat doubled haploids produced using the miah system. Theor Appl Genet 75:509–513Soriano M, Li H, Jacquard C, Angenent GC, Krochko J, Offringa R, Boutilier K (2014) Plasticity in cell division patterns and auxin transport dependency during in vitro embryogenesis in Brassica napus. Plant Cell 26:2568–2581Toppino L, Mennella G, Rizza F, D’Alessandro A, Sihachakr D, Rotino GL (2008) ISSR and isozyme characterization of androgenetic dihaploids reveals tetrasomic inheritance in tetraploid somatic hybrids between Solanum melongena and Solanum aethiopicum group Gilo. J Hered 99:304–315Uzun S (2007) Effect of light and temperature on the phenology and maturation of the fruit of eggplant (Solanum melongena) grown in greenhouses. N Z J Crop Hortic Sci 35:51–59van der Weerden GM, Barendse GWM (2007) A web-based searchable database developed for the EGGNET project and applied to the Radboud University Solanaceae database. Acta Hortic 745:503–506Veilleux RE (1998) Gametoclonal variation in crop plants. In: Jain SM, Brar DS, Ahloowalia BS (eds) Somaclonal variation and induced mutations in crop improvement. Springer, Dordrecht, pp 123–133Yamagishi M, Yano M, Fukuta Y, Fukui K, Otani M, Shimada T (1996) Distorted segregation of RFLP markers in regenerated plants derived from anther culture of an F1 hybrid of rice. Genes Genet Syst 71:37–41Zhang FL, Takahata Y (2001) Inheritance of microspore embryogenic ability in Brassica crops. Theor Appl Genet 103:254–25

Improved regeneration of eggplant doubled haploids from microspore-derived calli through organogenesis

[EN] Doubled haploid (DH) technology allows for the production of pure lines, useful for plant breeding, through a one-generation procedure that reduces considerably the time and resources needed to produce them. Despite the advantages of microspore culture to obtain DHs, this technique is still insufficiently developed in eggplant, where DHs are produced from microsporederived calli through organogenesis. At present, very little is known on the best in vitro conditions to promote this process. This is why in this work we addressed the optimization of the process of regeneration of eggplant DH plants from microspore-derived calli. We evaluated the effect of different media compositions in the induction of organogenesis, in the promotion of shoot growth and elongation, and in root growth. According to our results, we propose the repeated subculture of the calli in MS medium with 0.2 mg/l IAA and 4 mg/l zeatin to produce shoots, and then the repeated subculture of the excised shoots in basal MS medium to promote their conversion into entire plantlets. This procedure yielded 7.6 plants per 100 cultured calli, which represents a *49 increase with respect to previous reports. We also evaluated by flow cytometry and SSR molecular markers the effect of these in vitro culture conditions in the rate of DH plant production, finding that*70 % of the regenerated plants were true DHs. These results substantially improve the efficiencies of DH recovery published to date in eggplant, and may be useful to those working in the field of eggplant doubled haploidy and breeding.We acknowledge Dr. Rosa Peiro for her statistical advice, and the staff of the COMAV greenhouses for their valuable help. This work was supported by the AGL2014-55177-R grant from Spanish MINECO to JMSS.Rivas Sendra, A.; Corral Martínez, P.; Camacho Fernández, C.; Seguí-Simarro, JM. (2015). Improved regeneration of eggplant doubled haploids from microspore-derived calli through organogenesis. Plant Cell, Tissue and Organ Culture. 122(3):759-765. https://doi.org/10.1007/s11240-015-0791-6S7597651223Asif M, Eudes F, Randhawa H, Amundsen E, Spaner D (2014) Phytosulfokine alpha enhances microspore embryogenesis in both triticale and wheat. Plant Cell Tissue Organ Cult 116:125–130Borgato L, Conicella C, Pisani F, Furini A (2007) Production and characterization of arboreous and fertile Solanum melongena plus Solanum marginatum somatic hybrid plants. Planta 226:961–969Castillo AM, Nielsen NH, Jensen A, Vallés MP (2014) Effects of n-butanol on barley microspore embryogenesis. Plant Cell Tissue Organ Cult 117:411–418Corral-Martínez P, Seguí-Simarro JM (2012) Efficient production of callus-derived doubled haploids through isolated microspore culture in eggplant (Solanum melongena L.). Euphytica 187:47–61Corral-Martínez P, Seguí-Simarro JM (2014) Refining the method for eggplant microspore culture: effect of abscisic acid, epibrassinolide, polyethylene glycol, naphthaleneacetic acid, 6-benzylaminopurine and arabinogalactan proteins. Euphytica 195:369–382Dhooghe E, Van Laere K, Eeckhaut T, Leus L, Van Huylenbroeck J (2011) Mitotic chromosome doubling of plant tissues in vitro. Plant Cell Tissue Organ Cult 104:359–373Dumas de Vaulx R, Chambonnet D (1982) Culture in vitro d’anthères d’aubergine (Solanum melongena L.): stimulation de la production de plantes au moyen de traitements à 35°C associés à de faibles teneurs en substances de croissance. Agronomie 2:983–988Dunwell JM (2010) Haploids in flowering plants: origins and exploitation. Plant Biotechnol J 8:377–424Eshaghi ZC, Abdollahi MR, Moosavi SS, Deljou A, Seguí-Simarro JM (2015) Induction of androgenesis and production of haploid embryos in anther cultures of borage (Borago officinalis L.). Plant Cell Tissue Organ Cult 1–9. doi: 10.1007/s11240-015-0768-5Franklin G, Sheeba CJ, Sita GL (2004) Regeneration of eggplant (Solanum melongena L.) from root explants. In Vitro Cell Dev Biol Plant 40:188–191Gisbert C, Prohens J, Nuez F (2006) Efficient regeneration in two potential new crops for subtropical climates, the scarlet (Solanum aethiopicum) and gboma (S. macrocarpon) eggplants. New Zeal J Crop Hort Sci 34:55–62Kaur M, Dhatt AS, Sandhu JS, Gosal SS (2011) In vitro plant regeneration in brinjal from cultured seedling explants. Indian J Hortic 68:61–65Kim M, Park E-J, An D, Lee Y (2013) High-quality embryo production and plant regeneration using a two-step culture system in isolated microspore cultures of hot pepper (Capsicum annuum L.). Plant Cell Tissue Organ Cult 112:191–201Miyoshi K (1996) Callus induction and plantlet formation through culture of isolated microspores of eggplant (Solanum melongena L). Plant Cell Rep 15:391–395Mohinuddin AKM, Chowdhury MKU, Abdullah Zaliha C, Napis S (1997) Influence of silver nitrate (ethylene inhibitor) on cucumber in vitro shoot regeneration. Plant Cell Tissue Organ Cult 51:75–78Moshkov IE, Novikova GV, Hall MA, George EF (2008) Plant growth regulators III: gibberellins, ethylene, abscisic acid, their analogues and inhibitors; miscellaneous compounds. In George EF, Hall MA, De Klerk GJ (eds) Plant propagation by tissue culture, 3 edn, vol 1. Springer, DordrechtParra-Vega V, Renau-Morata B, Sifres A, Seguí-Simarro JM (2013) Stress treatments and in vitro culture conditions influence microspore embryogenesis and growth of callus from anther walls of sweet pepper (Capsicum annuum L.). Plant Cell Tissue Organ Cult 112:353–360Rotino GL (1996) Haploidy in eggplant. In: Jain SM, Sopory SK, Veilleux RE (eds) In vitro haploid production in higher plants, vol 3. Kluwer, Dordrecht, pp 115–141Salas P, Prohens J, Seguí-Simarro JM (2011) Evaluation of androgenic competence through anther culture in common eggplant and related species. Euphytica 182:261–274Seguí-Simarro JM (2015) Androgenesis in solanaceae. In Germanà MA, Lambardi M (eds), In vitro embryogenesis. Springer Science + Business Media, The NetherlandsSeguí-Simarro JM, Nuez F (2006) Androgenesis induction from tomato anther cultures: callus characterization. Acta Hort 725:855–861Seguí-Simarro JM, Corral-Martínez P, Parra-Vega V, González-García B (2011) Androgenesis in recalcitrant solanaceous crops. Plant Cell Rep 30:765–778Sgamma T, Thomas B, Muleo R (2015) Ethylene inhibitor silver nitrate enhances regeneration and genetic transformation of Prunus avium (L.) cv Stella. Plant Cell Tissue Organ Cult 120:79–88Shivaraj G, Rao S (2011) Rapid and efficient plant regeneration of eggplant (Solanum melongena L.) from cotyledonary leaf explants. Indian J Biotechnol 10:125–129Tuberosa R, Sanghineti MC, Conti S (1987) Anther culture of eggplant Solanum melongena L. lines and hybrids. Genética Agrária 41:267–274Veen H, van de Geijn S (1978) Mobility and ionic form of silver as related to longevity of cut carnations. Planta 140:93–96Xing Y, Yu Y, Luo X, Zhang JN, Zhao B, Guo YD (2010) High efficiency organogenesis and analysis of genetic stability of the regenerants in Solanum melongena. Biol Plant 54:231–236Zhang P, Phansiri S, Puonti-Kaerlas J (2001) Improvement of cassava shoot organogenesis by the use of silver nitrate in vitro. Plant Cell Tissue Organ Cult 67:47–5

Effects of growth conditions of donor plants and in vitro culture environment in the viability and the embryogenic response of microspores of different eggplant genotypes

[EN] Notwithstanding the importance of eggplant in global horticulture, doubled haploid production in this species is still far from being efficient. Although acknowledged to have a role in the efficiency of androgenesis induction, factors such as the growth conditions of donor plant or the in vitro culture environment have not been deeply explored or not explored at all in eggplant, which leaves room for further improvement. In this work, we investigated the effects of different in vivo and in vitro parameters on the androgenic performance of different eggplant genotypes, including two hybrids and a DH line. The in vivo parameters included the exposure of donor plants to different temperature and light conditions and to increased levels of boron. The in vitro parameters included the use of different concentrations of NLN medium components, sucrose and growth regulators, and the suspension of microspores at different densities. Our results showed that whereas greenhouse temperature variations or boron application did not to have a positive influence, greenhouse lighting influenced their viability, thereby conditioning the embryogenic response. Changes in different sucrose, salts and hormone levels had different effects in the genotypes studied, which correlated with their genetic constitution. Finally, we determined the best microspore density, different from that previously proposed. Our work shed light on the role of different factors involved in eggplant microspore cultures, some of them not yet studied, contributing to make microspore culture a more efficient tool in eggplant breeding.This work was supported by Grant AGL2017-88135-R to JMSS from Spanish MICINN, respectively, jointly funded by FEDER. ARS and CCF were supported by predoctoral fellowships from the FPI Programs of Universitat Politecnica de Valencia and Generalitat Valenciana, respectively.Rivas-Sendra, A.; Corral Martínez, P.; Camacho-Fernández, C.; Porcel, R.; Seguí-Simarro, JM. (2020). Effects of growth conditions of donor plants and in vitro culture environment in the viability and the embryogenic response of microspores of different eggplant genotypes. Euphytica. 216(11):1-15. https://doi.org/10.1007/s10681-020-02709-4S11521611Abdollahi MR, Corral-Martinez P, Mousavi A, Salmanian AH, Moieni A, Seguí-Simarro JM (2009) An efficient method for transformation of pre-androgenic, isolated Brassica napus microspores involving microprojectile bombardment and Agrobacterium-mediated transformation. Acta Physiol Plant 31:1313–1317Aulinger IE (2002) Combination of in vitro androgenesis and biolistic transformation: an approach for breeding transgenic maize (Zea mays L.) lines. Swiss Federal Institute of Technology, Zurich, p 115Borderies G, le Bechec M, Rossignol M, Lafitte C, Le Deunff E, Beckert M, Dumas C, Matthys-Rochon E (2004) Characterization of proteins secreted during maize microspore culture: arabinogalactan proteins (AGPs) stimulate embryo development. Eur J Cell Biol 83:205–212Bueno MA, Gómez A, Sepúlveda F, Seguí-Simarro JM, Testillano PS, Manzanera JA, Risueño MC (2003) Microspore-derived embryos from Quercus suber anthers mimic zygotic embryos and maintain haploidy in long-term anther culture. J Plant Physiol 160:953–960Camacho-Fernández C, Hervás D, Rivas-Sendra A, Marín MP, Seguí-Simarro JM (2018) Comparison of six different methods to calculate cell densities. Plant Methods 14:30Chambonnet D (1988) Production of haploid eggplant plants. Bulletin interne de la Station d’Amélioration des Plantes Maraichères d’Avignon-Montfavet, France, pp 1–10Corral-Martínez P, Seguí-Simarro JM (2012) Efficient production of callus-derived doubled haploids through isolated microspore culture in eggplant (Solanum melongena L.). Euphytica 187:47–61Corral-Martínez P, Seguí-Simarro JM (2014) Refining the method for eggplant microspore culture: effect of abscisic acid, epibrassinolide, polyethylene glycol, naphthaleneacetic acid, 6-benzylaminopurine and arabinogalactan proteins. Euphytica 195:369–382Custers J (2003) Microspore culture in rapeseed (Brassica napus L.). In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants. Kluwer Academic Publishers, Dordrecht, pp 185–193Dunwell JM (1976) A comparative study of environmental and developmental factors which influence embryo induction and growth in cultured anthers of Nicotiana tabacum. Environ Exp Bot 16:109–118Dunwell JM (2010) Haploids in flowering plants: origins and exploitation. Plant Biotechnol J 8:377–424Dutta SS, Pale G, Pattanayak A, Aochen C, Pandey A, Rai M (2017) Effect of low light intensity on key traits and genotypes of hilly rice (Oryza sativa) germplasm. J Exp Biol Agric Sci 5:463–471Esteves P, Clermont I, Marchand S, Belzile F (2014) Improving the efficiency of isolated microspore culture in six-row spring barley: II-exploring novel growth regulators to maximize embryogenesis and reduce albinism. Plant Cell Rep 33:871–879 (in press)Gaillard A, Vergne P, Beckerte M (1991) Optimization of maize microspore isolation and culture conditions for reliable plant regeneration. Plant Cell Rep 10:55–58Höfer M (2004) In vitro androgenesis in apple—improvement of the induction phase. Plant Cell Rep 22:365–370Jouannic S, Champion A, Seguí-Simarro JM, Salimova E, Picaud A, Tregear J, Testillano P, Risueno MC, Simanis V, Kreis M, Henry Y (2001) The protein kinases AtMAP3Kepsilon1 and BnMAP3Kepsilon1 are functional homologues of S. pombe cdc7p and may be involved in cell division. Plant J 26:637–649Kim M, Jang I-C, Kim J-A, Park E-J, Yoon M, Lee Y (2008) Embryogenesis and plant regeneration of hot pepper (Capsicum annuum L.) through isolated microspore culture. Plant Cell Rep 27:425–434Kim M, Park E-J, An D, Lee Y (2013) High-quality embryo production and plant regeneration using a two-step culture system in isolated microspore cultures of hot pepper (Capsicum annuum L.). Plant Cell Tissue Organ Cult 112:191–201Lantos C, Juhasz AG, Vagi P, Mihaly R, Kristof Z, Pauk J (2012) Androgenesis induction in microspore culture of sweet pepper (Capsicum annuum L.). Plant Biotechnol Rep 6:123–132Liu L, Huang L, Li Y (2013) Influence of boric acid and sucrose on the germination and growth of areca pollen. Am J Plant Sci 4:1669–1674Miyoshi K (1996) Callus induction and plantlet formation through culture of isolated microspores of eggplant (Solanum melongena L). Plant Cell Rep 15:391–395Paire A, Devaux P, Lafitte C, Dumas C, Matthys-Rochon E (2003) Proteins produced by barley microspores and their derived androgenic structures promote in vitro zygotic maize embryo formation. Plant Cell Tissue Organ Cult 73:167–176Parra-Vega V, Seguí-Simarro JM (2013) Improvement of an isolated microspore culture protocol for Spanish sweet pepper (Capsicum annuum L.). In: Lanteri S, Rotino GL (eds) Breakthroughs in the genetics and breeding of Capsicum and Eggplant. Universita degli Studi di Torino, Torino, Italy, pp 161–168Peñaloza P, Toloza P (2018) Boron increases pollen quality, pollination, and fertility of different genetic lines of pepper. J Plant Nutr 41:969–979Rivas-Sendra A, Corral-Martínez P, Camacho-Fernández C, Seguí-Simarro JM (2015) Improved regeneration of eggplant doubled haploids from microspore-derived calli through organogenesis. Plant Cell Tissue Organ Cult 122:759–765Rivas-Sendra A, Calabuig-Serna A, Seguí-Simarro JM (2017a) Dynamics of calcium during in vitro microspore embryogenesis and in vivo microspore development in Brassica napus and Solanum melongena. Front Plant Sci 8:1177Rivas-Sendra A, Campos-Vega M, Calabuig-Serna A, Seguí-Simarro JM (2017b) Development and characterization of an eggplant (Solanum melongena) doubled haploid population and a doubled haploid line with high androgenic response. Euphytica 213:89Rivas-Sendra A, Corral-Martínez P, Porcel R, Camacho-Fernández C, Calabuig-Serna A, Seguí-Simarro JM (2019) Embryogenic competence of microspores is associated with their ability to form a callosic, osmoprotective subintinal layer. J Exp Bot 70:1267–1281Robert HS, Grunewald W, Sauer M, Cannoot B, Soriano M, Swarup R, Weijers D, Bennett M, Boutilier K, Friml J (2015) Plant embryogenesis requires AUX/LAX-mediated auxin influx. Development 142:702–711Rotino GL (1996) Haploidy in eggplant. In: Jain SM, Sopory SK, Veilleux RE (eds) In vitro haploid production in higher plants. Kluwer Academic Publishers, Dordrecht, pp 115–141Salas P, Prohens J, Seguí-Simarro JM (2011) Evaluation of androgenic competence through anther culture in common eggplant and related species. Euphytica 182:261–274Salas P, Rivas-Sendra A, Prohens J, Seguí-Simarro JM (2012) Influence of the stage for anther excision and heterostyly in embryogenesis induction from eggplant anther cultures. Euphytica 184:235–250Satpute G, Long H, Seguí-Simarro JM, Risueño MC, Testillano PS (2005) Cell architecture during gametophytic and embryogenic microspore development in Brassica napus. Acta Physiol Plant 27:665–674Saxena N, Johansen C (1987) Adaptation of chickpea and pigeonpea to abiotic stresses. Proceedings of the consultants’ workshop held at ICRISAT Center, India, 19–21 December 1984, ICRISAT, Patancheru, IndiaSeguí-Simarro JM (2010) Androgenesis revisited. Bot Rev 76:377–404Seguí-Simarro JM (2016) Androgenesis in solanaceae. In: Germanà MA, Lambardi M (eds) In vitro embryogenesis. Springer, New York, pp 209–244Seguí-Simarro JM, Nuez F (2008) How microspores transform into haploid embryos: changes associated with embryogenesis induction and microspore-derived embryogenesis. Physiol Plant 134:1–12Seguí-Simarro JM, Corral-Martínez P, Parra-Vega V, González-García B (2011) Androgenesis in recalcitrant solanaceous crops. Plant Cell Rep 30:765–778Sinha R, Eudes F (2015) Dimethyl tyrosine conjugated peptide prevents oxidative damage and death of triticale and wheat microspores. Plant Cell Tissue Organ Cult 122(1):227–237Supena EDJ, Suharsono S, Jacobsen E, Custers JBM (2006) Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper (Capsicum annuum L.). Plant Cell Rep 25:1–10Touraev A, Heberle-Bors E (2003) Anther and microspore culture in tobacco. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants. Kluwer Academic Publishers, Dordrecht, pp 223–228Touraev A, Ilham A, Vicente O, Heberle-Bors E (1996a) Stress-induced microspore embryogenesis in tobacco: an optimized system for molecular studies. Plant Cell Rep 15:561–565Touraev A, Indrianto A, Wratschko I, Vicente O, Heberle-Bors E (1996b) Efficient microspore embryogenesis in wheat (Triticum aestivum L.) induced by starvation at high temperatures. Sex Plant Reprod 9:209–215Tsay H-S (1981) Effects of nitrogen supply to donor plants on pollen embryogenesis in cultured tobacco anthers. J Agric Res China 30:5–13Tsay H-S (1982) Microspore development and haploid embryogenesis of anther culture with five nitrogen doses to the donor tobacco plants. J Agric Res China 31:1–13Tuberosa R, Sanguineti MC, Toni B, Cioni F (1987) Ottenimento di aploidi in melanzana (Solanum melongena L.) mediante coltura di antere. Sementi Elette 3:9–14Żur I, Dubas E, Krzewska M, Janowiak F (2015) Current insights into hormonal regulation of microspore embryogenesis. Front Plant Sci 6:42

Embryogenic competence of microspores is associated to their ability to form a callosic, osmoprotective subintinal layer

[EN] Microspore embryogenesis is an experimental morphogenic pathway with important applications in basic research and applied plant breeding, but its genetic, cellular, and molecular bases are poorly understood. We applied a multi-disciplinary approach using confocal and electron microscopy, detection of Ca2+, callose, and cellulose, treatments with caffeine, digitonin, and endosidin7, morphometry, qPCR, osmometry, and viability assays in order to study the dynamics of cell wall formation during embryogenesis induction in a high-response rapeseed (Brassica napus) line and two recalcitrant rapeseed and eggplant (Solanum melongena) lines. Formation of a callose-rich subintinal layer (SL) was common to microspore embryogenesis in the different genotypes. However, this process was directly related to embryogenic response, being greater in high-response genotypes. A link could be established between Ca2+ influx, abnormal callose/cellulose deposition, and the genotype-specific embryogenic competence. Callose deposition in inner walls and SLs are independent processes, regulated by different callose synthases. Viability and control of internal osmolality are also related to SL formation. In summary, we identified one of the causes of recalcitrance to embryogenesis induction: a reduced or absent protective SL. In responding genotypes, SLs are markers for changes in cell fate and serve as osmoprotective barriers to increase viability in imbalanced in vitro environments. Genotype-specific differences relate to different responses against abiotic (heat/osmotic) stresses.Thanks are due to the Electron Microscopy Service of Universitat Politecnica de Valencia, Marisol Gascon (IBMCP Microscopy Service), Dr Kim Boutilier (WUR, Wageningen) for hosting ARS at her lab, and Dr Samantha Vernhettes (INRA Versailles) for kindly providing us with S4B. This work supported by grants AGL2014-55177-R and AGL2017-88135-R to JMSS from MINECO jointly funded by FEDER.Rivas-Sendra, A.; Corral Martínez, P.; Porcel, R.; Camacho-Fernández, C.; Calabuig-Serna, A.; Seguí-Simarro, JM. (2019). Embryogenic competence of microspores is associated to their ability to form a callosic, osmoprotective subintinal layer. Journal of Experimental Botany. 70(4):1267-1281. https://doi.org/10.1093/jxb/ery458S12671281704Abramova, L. I. (2003). Russian Journal of Plant Physiology, 50(3), 324-329. doi:10.1023/a:1023866019102Adkar-Purushothama, C. R., Brosseau, C., Giguère, T., Sano, T., Moffett, P., & Perreault, J.-P. (2015). Small RNA Derived from the Virulence Modulating Region of the Potato spindle tuber viroid Silences callose synthase Genes of Tomato Plants. The Plant Cell, 27(8), 2178-2194. doi:10.1105/tpc.15.00523Cordewener, J., Bergervoet, J., & Liu, C.-M. (2000). Changes in Protein Synthesis and Phosphorylation during Microspore Embryogenesis in Brassica napus. Journal of Plant Physiology, 156(2), 156-163. doi:10.1016/s0176-1617(00)80300-4Corral-Martínez, P., García-Fortea, E., Bernard, S., Driouich, A., & Seguí-Simarro, J. M. (2016). Ultrastructural Immunolocalization of Arabinogalactan Protein, Pectin and Hemicellulose Epitopes Through Anther Development inBrassica napus. Plant and Cell Physiology, 57(10), 2161-2174. doi:10.1093/pcp/pcw133Fortes, A. M., Testillano, P. S., Del Carmen Risueño, M., & Pais, M. S. (2002). Studies on callose and cutin during the expression of competence and determination for organogenic nodule formation from internodes of Humulus lupulus var. Nugget. Physiologia Plantarum, 116(1), 113-120. doi:10.1034/j.1399-3054.2002.1160114.xFurch, A. C. U., Hafke, J. B., Schulz, A., & van Bel, A. J. E. (2007). Ca2+-mediated remote control of reversible sieve tube occlusion in Vicia faba. Journal of Experimental Botany, 58(11), 2827-2838. doi:10.1093/jxb/erm143Grewal, R. K., Lulsdorf, M., Croser, J., Ochatt, S., Vandenberg, A., & Warkentin, T. D. (2009). Doubled-haploid production in chickpea (Cicer arietinum L.): role of stress treatments. Plant Cell Reports, 28(8), 1289-1299. doi:10.1007/s00299-009-0731-1Hoekstra, S., van Bergen, S., van Brouwershaven, I. ., Schilperoort, R. ., & Wang, M. (1997). Androgenesis in Hordeum vulgare L.: Effects of mannitol, calcium and abscisic acid on anther pretreatment. Plant Science, 126(2), 211-218. doi:10.1016/s0168-9452(97)00096-4Hong, Z., Delauney, A. J., & Verma, D. P. S. (2001). A Cell Plate–Specific Callose Synthase and Its Interaction with Phragmoplastin. The Plant Cell, 13(4), 755-768. doi:10.1105/tpc.13.4.755Jacobs, A. K., Lipka, V., Burton, R. A., Panstruga, R., Strizhov, N., Schulze-Lefert, P., & Fincher, G. B. (2003). An Arabidopsis Callose Synthase, GSL5, Is Required for Wound and Papillary Callose Formation. The Plant Cell, 15(11), 2503-2513. doi:10.1105/tpc.016097Jacquard, C., Mazeyrat-Gourbeyre, F., Devaux, P., Boutilier, K., Baillieul, F., & Clément, C. (2008). Microspore embryogenesis in barley: anther pre-treatment stimulates plant defence gene expression. Planta, 229(2), 393-402. doi:10.1007/s00425-008-0838-6Jensen, W. A. (1968). Cotton embryogenesis: The zygote. Planta, 79(4), 346-366. doi:10.1007/bf00386917Joosen, R., Cordewener, J., Supena, E. D. J., Vorst, O., Lammers, M., Maliepaard, C., … Boutilier, K. (2007). Combined Transcriptome and Proteome Analysis Identifies Pathways and Markers Associated with the Establishment of Rapeseed Microspore-Derived Embryo Development. Plant Physiology, 144(1), 155-172. doi:10.1104/pp.107.098723KAY, R., CHAN, A., DALY, M., & MCPHERSON, J. (1987). Duplication of CaMV 35S Promoter Sequences Creates a Strong Enhancer for Plant Genes. Science, 236(4806), 1299-1302. doi:10.1126/science.236.4806.1299Ochatt, S., Pech, C., Grewal, R., Conreux, C., Lulsdorf, M., & Jacas, L. (2009). Abiotic stress enhances androgenesis from isolated microspores of some legume species (Fabaceae). Journal of Plant Physiology, 166(12), 1314-1328. doi:10.1016/j.jplph.2009.01.011Park, E., Díaz-Moreno, S. M., Davis, D. J., Wilkop, T. E., Bulone, V., & Drakakaki, G. (2014). Endosidin 7 Specifically Arrests Late Cytokinesis and Inhibits Callose Biosynthesis, Revealing Distinct Trafficking Events during Cell Plate Maturation. Plant Physiology, 165(3), 1019-1034. doi:10.1104/pp.114.241497Parra-Vega, V., Corral-Martínez, P., Rivas-Sendra, A., & Seguí-Simarro, J. M. (2015). Induction of Embryogenesis in Brassica Napus Microspores Produces a Callosic Subintinal Layer and Abnormal Cell Walls with Altered Levels of Callose and Cellulose. Frontiers in Plant Science, 6. doi:10.3389/fpls.2015.01018Paul, D. C., & Goff, C. W. (1973). Comparative effects of caffeine, its analogues and calcium deficiency on cytokinesis. Experimental Cell Research, 78(2), 399-413. doi:10.1016/0014-4827(73)90085-2Pauls, K. P., Chan, J., Woronuk, G., Schulze, D., & Brazolot, J. (2006). When microspores decide to become embryos — cellular and molecular changesThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology. Canadian Journal of Botany, 84(4), 668-678. doi:10.1139/b06-064Reynolds, T. L. (1990). Interactions between calcium and auxin during pollen androgenesis in anther cultures of Solanum carolinense L. Plant Science, 72(1), 109-114. doi:10.1016/0168-9452(90)90192-qReynolds, T. L. (2000). Effects of calcium on embryogenic induction and the accumulation of abscisic acid, and an early cysteine-labeled metallothionein gene in androgenic microspores of Triticum aestivum. Plant Science, 150(2), 201-207. doi:10.1016/s0168-9452(99)00187-9Rivas-Sendra, A., Calabuig-Serna, A., & Seguí-Simarro, J. M. (2017). Dynamics of Calcium during In vitro Microspore Embryogenesis and In vivo Microspore Development in Brassica napus and Solanum melongena. Frontiers in Plant Science, 8. doi:10.3389/fpls.2017.01177Rivas-Sendra, A., Campos-Vega, M., Calabuig-Serna, A., & Seguí-Simarro, J. M. (2017). Development and characterization of an eggplant (Solanum melongena) doubled haploid population and a doubled haploid line with high androgenic response. Euphytica, 213(4). doi:10.1007/s10681-017-1879-3Rivas-Sendra, A., Corral-Martínez, P., Camacho-Fernández, C., & Seguí-Simarro, J. M. (2015). Improved regeneration of eggplant doubled haploids from microspore-derived calli through organogenesis. Plant Cell, Tissue and Organ Culture (PCTOC), 122(3), 759-765. doi:10.1007/s11240-015-0791-6Saidi, Y., Finka, A., Muriset, M., Bromberg, Z., Weiss, Y. G., Maathuis, F. J. M., & Goloubinoff, P. (2009). The Heat Shock Response in Moss Plants Is Regulated by Specific Calcium-Permeable Channels in the Plasma Membrane. The Plant Cell, 21(9), 2829-2843. doi:10.1105/tpc.108.065318Samuels, A. L., & Staehelin, L. A. (1996). Caffeine inhibits cell plate formation by disrupting membrane reorganization just after the vesicle fusion step. Protoplasma, 195(1-4), 144-155. doi:10.1007/bf01279193Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., … Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods, 9(7), 676-682. doi:10.1038/nmeth.2019Schl�pmann, H., Bacic, A., & Read, S. (1993). A novel callose synthase from pollen tubes of Nicotiana. Planta, 191(4). doi:10.1007/bf00195748Shi, X., Sun, X., Zhang, Z., Feng, D., Zhang, Q., Han, L., … Lu, T. (2014). GLUCAN SYNTHASE-LIKE 5 (GSL5) Plays an Essential Role in Male Fertility by Regulating Callose Metabolism During Microsporogenesis in Rice. Plant and Cell Physiology, 56(3), 497-509. doi:10.1093/pcp/pcu193Slewinski, T. L., Baker, R. F., Stubert, A., & Braun, D. M. (2012). Tie-dyed2 Encodes a Callose Synthase That Functions in Vein Development and Affects Symplastic Trafficking within the Phloem of Maize Leaves. Plant Physiology, 160(3), 1540-1550. doi:10.1104/pp.112.202473Sun, F., Fan, G., Hu, Q., Zhou, Y., Guan, M., Tong, C., … Wang, H. (2017). The high-quality genome ofBrassica napuscultivar ‘ZS11’ reveals the introgression history in semi-winter morphotype. The Plant Journal, 92(3), 452-468. doi:10.1111/tpj.13669Tan, H., Yang, X., Zhang, F., Zheng, X., Qu, C., Mu, J., … Zuo, J. (2011). Enhanced Seed Oil Production in Canola by Conditional Expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in Developing Seeds. Plant Physiology, 156(3), 1577-1588. doi:10.1104/pp.111.175000Töller, A., Brownfield, L., Neu, C., Twell, D., & Schulze-Lefert, P. (2008). Dual function of Arabidopsis glucan synthase-like genes GSL8 and GSL10 in male gametophyte development and plant growth. The Plant Journal, 54(5), 911-923. doi:10.1111/j.1365-313x.2008.03462.xVerma, D. P. S. (2001). CYTOKINESIS ANDBUILDING OF THECELLPLATE INPLANTS. Annual Review of Plant Physiology and Plant Molecular Biology, 52(1), 751-784. doi:10.1146/annurev.arplant.52.1.751Verma, D. P. S., & Hong, Z. (2001). Plant Molecular Biology, 47(6), 693-701. doi:10.1023/a:1013679111111Vithanage, H. I. M. V., Gleeson, P. A., & Clarke, A. E. (1980). The nature of callose produced during self-pollination inSecale cereale. Planta, 148(5), 498-509. doi:10.1007/bf00552666Waldmann, T., Jeblick, W., & Kauss, H. (1988). Induced net Ca2+ uptake and callose biosynthesis in suspension-cultured plant cells. Planta, 173(1), 88-95. doi:10.1007/bf00394492WHITE, P. J. (2003). Calcium in Plants. Annals of Botany, 92(4), 487-511. doi:10.1093/aob/mcg164Xie, B., Deng, Y., Kanaoka, M. M., Okada, K., & Hong, Z. (2012). Expression of Arabidopsis callose synthase 5 results in callose accumulation and cell wall permeability alteration. Plant Science, 183, 1-8. doi:10.1016/j.plantsci.2011.10.015Ling You, X., Seon Yi, J., & Eui Choi, Y. (2006). Cellular change and callose accumulation in zygotic embryos of Eleutherococcus senticosus caused by plasmolyzing pretreatment result in high frequency of single-cell-derived somatic embryogenesis. Protoplasma, 227(2-4), 105-112. doi:10.1007/s00709-006-0149-3Yu, Y., Jiao, L., Fu, S., Yin, L., Zhang, Y., & Lu, J. (2016). Callose Synthase Family Genes Involved in the Grapevine Defense Response to Downy Mildew Disease. Phytopathology®, 106(1), 56-64. doi:10.1094/phyto-07-15-0166-rZhang, C., Guinel, F. C., & Moffatt, B. A. (2002). A comparative ultrastructural study of pollen development in Arabidopsis thaliana ecotype Columbia and male-sterile mutant apt1-3. Protoplasma, 219(1-2), 59-71. doi:10.1007/s00709020000

Glucocorticoid receptor intestinal epithelial knockout mice show attenuated colonic inflammatory response but unaffected permeability in early experimental sepsis

Introduction: Sepsis is defined as an organic dysfunction that threatens the life of patients due to an abnormally regulated response to infection [1]. The initial phase of sepsis is dominated by an increased production of proinflammatory cytokines, which leads to augmented capillary permeability, extravasation, hypercoagulability and myelopoiesis. One of the main sources of infection in sepsis is believed to be the intestinal microbiota via traslocation through the mucosa to the bloodstream. Systemic inflammation weakens intestinal barrier function (IBF) in animal models, resulting in increased bacterial traslocation [2]. Even if the management of sepsis has advanced in the last decades, mortality is still high and there are blanks in terms of pathological systems and long-term consequences. Thus, the search for effective treatments is clearly justified. Glucocorticoids (GC) are part of the drugs used in sepsis, but they have only shown a moderate therapeutic effect. This fact may be caused by harmful effects of GCs on IBF, whose compromise may limit GC clinical benefit by facilitating luminal translocation of microorganisms. Besides, GC treatment impairs epithelial healing in experimental colitis in mice [3]. Previous results of our research group have shown that mice with induced deletion of the GC receptor (GR) in intestinal epithelial cells (i.e. NR3C1ΔIEC mice) are protected against dextran sulphate sodium (DSS)-induced colitis [4]. In turn, gene deletion results in a short lived inflammatory response in the colon [5]. Objective: Understanding the role of the intestinal epithelial GR and its involvement in IBF regulation in experimental sepsis, with the ultimate goal of improving the management of sepsis with GCs. Matherial and methods: The cecal ligation and puncture (CLP) model of sepsis was applied to WT C57BL/6J and NR3C1ΔIEC mice. Ceacum-exposed mice were used as control (Sham). Mice were sacrificed 24 hours after surgery. Four hours before sacrifice, mice were administered 4 kD FITC-dextran, a fluorescent marker of permeability. Colon, jejunum, adrenes, kidney and liver RT-qPCRs were performed as well as determination of plasma FITC-dextran and corticosterone plasma levels. Results: After 24 h, CLP mice exhibited elevated corticosterone plasma levels with hypoglycemia and splenomegaly. Intestinal barrier function was weakened, as indicated by increased FITC-dextran plasma levels. A modest increase in inflammatory markers (S100a8, Cxcl1) was noted in the colon and jejunum. The expression of Tjp1, involved in barrier function, was downregulated in CLP mice. Similarly, the colonic expression of Cyp11a1 and Lrh1, involved in local steroidogenesis, was lower in CLP mice, regardless of genotype. Markers of inflammation were also augmented in the lung and kidney. CLP mice exhibited hypercorticosteronemia, which was associated to increased Cyp11a1 in the adrenes. Of note, both parameters were less pronounced in KO mice. The latter also exhibited dampened inflammatory response in the colon but not the jejunum. FITC-dextran plasma levels were similarly increased in WT and KO mice. Conclusions: In the early stages of the CLP model of sepsis the colon and jejunum are inflamed, and epithelial deletion of the glucocorticoid receptor appears to modulate inflammation in the former, with no change in barrier function. Further studies will characterize the microbiota composition and phenotype in later stages and in the response to glucocorticoid treatment

Induction of embryogenesis in Brassica napus microspores produces a callosic subintinal layer and abnormal cell walls with altered levels of callose and cellulose

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2015.01018The induction of microspore embryogenesis produces dramatic changes in different aspects of the cell physiology and structure. Changes at the cell wall level are among the most intriguing and poorly understood. In this work, we used high pressure freezing and freeze substitution, immunolocalization, confocal and electron microscopy to analyze the structure and composition of the first cell walls formed during conventional Brassica napus microspore embryogenesis, and in cultures treated to alter the intracellular Ca2+ levels. Our results revealed that one of the first signs of embryogenic commitment is the formation of a callose-rich, cellulose-deficient layer beneath the intine (the subintinal layer), and of irregular, incomplete cell walls. In these events, Ca2+ may have a role. We propose that abnormal cell walls are due to a massive callose synthesis and deposition of excreted cytoplasmic material, and the parallel inhibition of cellulose synthesis. These features were absent in pollen-like structures and in microspore-derived embryos, few days after the end of the heat shock, where abnormal cell walls were no longer produced. Together, our results provide an explanation to a series of relevant aspects of microspore embryogenesis including the role of Ca2+ and the occurrence of abnormal cell walls. In addition, our discovery may be the explanation to why nuclear fusions take place during microspore embryogenesis.We want to express our thanks to the staff of the Electron Microscopy Service of Universitat Politecnica de Valencia. Thanks are also due to Dr. Kim Boutilier (PRI Wageningen, The Netherlands) for her help during the stays of VPV and ARS at her lab, to Dr. Samantha Vernhettes (INRA Versailles, France) for her kind gift of S4B staining, and especially to Prof. L. A. Staehelin for his help and friendship during the stay of JMSS at UC Boulder. This work was supported by grants BEST/20081154 from Generalitat Valenciana and AGL2014-55177-R from Spanish MINECO to JMSS.Parra Vega, V.; Corral Martínez, P.; Rivas-Sendra, A.; Seguí-Simarro, JM. (2015). Induction of embryogenesis in Brassica napus microspores produces a callosic subintinal layer and abnormal cell walls with altered levels of callose and cellulose. Frontiers in Plant Science. 6(1018):1-17. https://doi.org/10.3389/fpls.2015.01018S1176101

Papel de la enzima fosfatasa alcalina no específica de tejido (TNAP) en el epitelio intestinal en la inflamación

Introducción: la fosfatasa alcalina (AP) es una familia de enzimas que ha sido relacionada con la protección frente a inflamación intestinal. Se ha descrito que una de sus isoformas, la fosfatasa alcalina intestinal (IAP), es capaz de desfosforilar diferentes antígenos bacterianos, de tal forma que la enzima regula el crecimiento de la microbiota e impide el paso de antígenos activos. En cuanto a la isoforma TNAP, se ha observado que su expresión se encuentra incrementada en la colitis experimental, no solo debido a la infiltración de células del sistema inmunológico, sino también por el incremento de expresión de esta enzima en las células del epitelio intestinal. Objetivo: conocer el papel de la TNAP en la inflamación intestinal. Métodos y resultados: se ha generado un modelo de ratón con deleción condicional inducible del gen que codifica TNAP (Alpl) en el epitelio intestinal (ratones AlplIEC-/-). El silenciamiento específico de TNAP en IECs en inflamación por DSS (7 días) supuso una pérdida mayor de peso en los ratones, sin observarse diferencias en el índice de actividad de la enfermedad (DAI). A nivel histológico se observó un mayor nivel de infiltración en la submucosa en el colon de los ratones sin TNAP. Los ratones AlplIEC-/- presentaron una expresión reducida de marcadores inflamatorios en el colon, como S100a8, Il6 y Tnf. Por el contrario, la deficiencia en TNAP en el epitelio intestinal supuso un aumento en la expresión de la fosfatasa alcalina intestinal global (Akp6) en el colon, sugiriendo que podría existir algún mecanismo de compensación. Además, la ausencia de TNAP en el epitelio intestinal provocó un aumento de expresión de genes relacionados en el mantenimiento de la función de barreara, como Muc4, Tjp1 y Tff3. Conclusión: los ratones AlplIEC-/- presentan un fenotipo mixto, con mayor infiltración y daño histológico pero menor expresión de marcadores inflamatorios en la colitis por DSS. Perspectivas futuras: se realizarán estudios de transcriptómica para conocer el efecto de la TNAP presente en el intestino sobre la función de barrera intestinal y sobre la microbiota