A cana-de-açúcar é uma das principais culturas agrícolas plantadas no Brasil e apresenta significativa importância sócio-econômica e agroindustrial ao país. O cenário mundial encontrase bastante favorável no que concerne à comercialização de seus dois principais produtos derivados, o açúcar e o álcool, impulsionando o desenvolvimento do setor sucroalcooleiro nacional. Neste sentido, o melhoramento genético da cana-de-açúcar aparece como base fundamental para o desenvolvimento de novas variedades para a manutenção e incremento dos agronegócios da agroindústria sucroalcooleira. Técnicas de engenharia genética, como a transformação genética nuclear, estão trazendo excelentes resultados no melhoramento genético da cultura, permitindo diminuir o custo e o tempo de obtenção de novas variedades. Baseando-se na importância em se obter variedades tolerantes a diferentes estresses bióticos e abióticos que induzem perturbações metabólicas e ativam o processo de morte celular programada, o presente trabalho teve por objetivo transformar geneticamente a variedade de cana-de-açúcar RB835089 com o cDNA do gene AtBI-1 isolado de Arabidopsis thaliana, visando suprimir a indução do mecanismo de morte celular sob condição de estresse. Para isto, calos embriogênicos foram utilizados como explante alvo, empregando-se dois métodos de transformação, a cotransformação por biolística, e o mediado por Agrobacterium tumefaciens no qual foram testadas duas técnicas: (a) inoculação direta dos calos em suspensão bacteriana; e (b) agrobiolística que é o bombardeamento dos calos com partículas de tungstênio seguido da inoculação em suspensão bacteriana. A proteína AtBI-1 (Bax inhibitor-1) apresenta homólogos em outros organismos e está localizada na membrana do retículo endoplasmático. Ela apresenta funções citoprotetoras modulando o mecanismo de morte celular programada induzida por estresses bióticos e abióticos. Como resultados deste trabalho, diferentes taxas de eficiência da transformação genética foram obtidas pelo método mediado por A. tumefaciens nas duas técnicas testadas, sendo que suas taxas foram superiores às alcançadas pelo método de co-transformação por biolística. A expressão heteróloga do cDNA do gene AtBI-1 em cana-de-açúcar atenuou a indução das vias de morte celular em presença do antibiótico tunicamicina, indutor do estresse no retículo endoplasmático, sendo comprovado pela maior tolerância ao estresse das plantas transgênicas quando comparadas com as plantas não transformadas que foram afetas no crescimento do sistema radicular, conteúdo de clorofila total, apresentando sintomas típicos de morte celular programada como clorose foliar e morfologia irregular das raízes, com consequente morte do sistema radicular. / Sugarcane is one of the main crops planted in Brazil and presents significant socioeconomic and agribusiness importance to the country. The world scene is quite favorable as regards the marketing of its two main products, sugar and alcohol, driving the development of the national sugar-alcohol sector. Therefore, the sugarcane genetic breeding appears as the fundamental base for developing new varieties for the maintenance and increase of agribusiness in the sugarcane agroindustry. Genetic engineering techniques, such as the nuclear genetic transformation, are providing excellent results in genetic breeding of this crop allowing reducing the cost and time to obtain new varieties. Based on the importance of obtaining varieties tolerant to different biotic and abiotic stresses that induce metabolic disturbances and activate the process of programmed cell death, this work aimed to transform sugarcane variety RB835089 with the cDNA of AtBI-1 gene, isolated from Arabidopsis thaliana, to suppress the induction of the cell death mechanism under stress condition. For this, embryogenic calli were used as target explant, by using two methods of transformation, the cotransformation by biolistic, and mediated by Agrobacterium tumefaciens in which two techniques were tested: (a) direct inoculation of calli in bacterial suspension; (b) agrobiolistic which is the bombardment of calli with tungstein particles followed by inoculation in bacterial suspension. The AtBI-1 protein (Bax inhibitor-1) presents homologs in other organisms and is located in the endoplasmic reticulum membranes. It has cytoprotective functions by modulating the mechanism of programmed cell death induced by biotic and abiotic stresses. As results of this work, different efficiency rates in genetic transformation were obtained in the method mediated by A. tumefaciens in the two techniques tested, and that their rates were higher than those achieved using the cotransformation by biolistic. The heterologous expression of cDNA of AtBI-1 gene in sugarcane attenuated the induction of cell death pathways in the presence of tunicamycin antibiotic, an inducer of stress in the endoplasmic reticulum, being proven by the increased stress tolerance of transgenic plants compared with sugarcane wild type that were affected in the root growth, total chlorophyll content, showing typical symptoms of programmed cell death such as leaf chlorosis and irregular morphology of the roots, with subsequent death of the root system.

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The major obstacle many promising drugs struggle to overcome is the barrier imposed by the outer cell membrane. In addition to technologies such as liposomes and cell-penetrating peptides, more attention is being given to the class of proteins known as lectins to deliver therapeutic and antigenic proteins to the interiors of cells. Lectins bind to but do not modify sugars, and provide an efficient route to endocytosis. The galactose/N-acetyl-galactosamine specific lectin ricin B-chain (RTB) is especially attractive in possibly fulfilling a carrier role due to its well-characterized endocytotic trafficking and its efficacy over a wide range of cell types. By producing RTB recombinantly in plants it is possible to create a fully active, non-toxic carrier that does not rely on the processing of large amounts of toxic material (e.g. castor bean). Payload molecules such as small molecules and proteins can be attached to RTB via chemical conjugation at primary amine groups, without the loss of lectin or uptake activities. The biotin/streptavidin interaction and direct genetic fusion of polypeptides also provide efficient mechanisms for the attachment of payload proteins to RTB. An immunoglobulin domain-based scaffolding mechanism bridges modified RTB and payload proteins when co-expressed in Agrobacterium-infiltrated plant leaves. Carrier and payload proteins expressed in plants and E. coli, respectively, and purified independently are not able to assemble into an efficient carrier/payload arrangement. These findings show that plant cells are able to correctly produce the two components of the carrier/payload system and assemble them into an efficient and flexible capture and carry technology. / Ph. D.

Las pérdidas causadas por podredumbres durante la post-cosecha de frutos cítricos suelen suponer entre un 5 y un 10 % de la producción, siendo Penicillium digitatum el principal hongo patógeno, responsable de hasta el 80 % de las pérdidas causadas por podredumbres en frutos almacenados a temperatura ambiente. A pesar de la importancia económica de este patógeno nuestro conocimiento sobre los mecanismos de patogenicidad/ virulencia son muy escasos, en contraste con el avance experimentado en los últimos años en el conocimiento de las respuestas de defensa del fruto a la infección por este patógeno. Así, en el grupo de Fisiología y Biotecnología Postcosecha del IATA se está trabajando en la caracterización a nivel bioquímico y molecular de las respuestas de los frutos cítricos frente a la infección por P. digitatum y en el proceso de inducción de resistencia en frutos cítricos frente a la infección. Por este motivo en esta Tesis se han desarrollado un conjunto de herramientas esenciales para poder abordar la caracterización funcional de genes involucrados en virulencia/patogenicidad: transformación de P. digitatum mediada por Agrobacterium tumefaciens, utilización de la proteína verde fluorescente como marcadora, metodología para la obtención de mutantes de deleción de genes específicos, incluyendo mutantes nulos ¿ku80, vectores para silenciamiento génico mediante RNAi y la construcción de una genoteca de DNA genómico de P. digitatum. Se ha secuenciado y analizado el factor de transcripción PacC, que controla la expresión de un grupo de genes regulados por el pH ambiental. En P. digitatum se produce una acidificación del medio para adaptarlo al pH óptimo de su arsenal de enzimas. Se han obtenido mutantes de expresión constitutiva de PacC que presentan una disminución la capacidad infectiva en un 20 %. Mediante el empleo de técnicas de alto rendimiento se ha construido una genoteca substractiva de cDNA para obtener fragmentos de genes de P. digitatum que se inducen durante la infección de frutos de naranja y se ha analizado la expresión génica de los mismos y se ha elaborado una macromatriz conteniendo más de 1330 clones de la genoteca. El grupo de genes con mayor representación en la genoteca y con altos valores de inducción, corresponde a genes que codifican cinco proteasas diferentes. Además, en la genoteca substractiva también hay una alta representación de genes que codifican enzimas de degradación de la pared celular, y otras proteínas implicadas en glucólisis, respuesta a estrés o detoxificación. La ya demostrada importancia de las enzimas de degradación de la pared celular en la virulencia de hongos fitopatógenos, su abundancia y niveles de inducción en la macromatriz nos llevaron a estudiar más en profundidad algunos de estos genes (dos poligalacturonasas y una pectin liasa). Para comprobar su implicación en el proceso de infección se secuenciaron y se obtuvieron mutantes de P. digitatum en los que se eliminó el gen. La disminución de la virulencia de estos mutantes sobre frutos de naranja con respecto a la cepa silvestre fue de aproximadamente un 25 %. Del conjunto de genes relacionados con el metabolismo redox se seleccionó por su patrón de expresión el gen ris1, que codifica una naftaleno dioxigenasa posiblemente implicada en la detoxificación de compuestos aromáticos. A diferencia de los mutantes nulos en los genes de las poligalacturonasas o de la pectin liasa, los mutantes nulos ¿ris1 no presentaron ninguna alteración en su capacidad patogénica. / López Pérez, M. (2013). Estudio de los factores de patogenicidad/virulencia de Penicillium digitatum sobre frutos cítricos [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/34176

When tomato Craigella is infected with Verticillium dahliae Dvd-E6 (Dvd-E6), a tolerant state is induced with substantial pathogen load, but few symptoms. Unexpectedly, these plants are more robust and taller with Dvd-E6 behaving as an endophyte. Some endophytes can protect plants from virulent pathogens. This research was undertaken to improve understanding of the cellular and molecular nature of Verticillium tolerance in tomato, especially whether infection by Dvd-E6 can protect Craigella from virulent V. dahliae, race 1 (Vd1). To permit mixed infection experiments a restriction fragment length polymorphism (RFLP)-based assay was developed and used for differentiating Dvd-E6 from Vd1, when present in mixed infections. The results suggested that protection involves molecular interplay between Dvd-E6 and Vd1 in susceptible Craigella (CS) tomatoes, resulting in restricted Vd1 colonization. Further studies showed a dramatic reduction of Vd1 spores and mycelia. To examine genetic changes that account for these biological changes, a customized DNA chip (TVR) was used to analyze defense gene mRNA levels. The defense gene response was categorized into four groups. Group 1 was characterized by strong induction of defense genes followed by suppression. However, Vd1-induced gene suppression was blocked by Dvd-E6 in mixed infections. These genes included some transcription factors and PR proteins such as class IV chitinases and beta glucanases which are known to target fungal spores and mycelia. Experiments also were repeated with a Craigella resistant (CR) isoline containing a fully active Ve locus (Ve1+ and Ve2+). The biological results showed that the presence of the Ve1+ allele resulted in restricted Vd1 colonization and, in a mixed infection with Dvd-E6, Vd1 was completely eliminated from the plant stem. Surprisingly, there was no significant increase in defense gene mRNAs. Rather, elevated basal levels of defense gene products appeared sufficient to combat pathogen attack. To investigate functional effects of the genetic changes observed, an inducible RNAi knockdown vector for a defense gene (TUS15G8) with unknown function (pMW4-TUS15G8) as well as the Ve2 resistance gene (pMW-Ve2) was prepared as a initial step for future transformation analyses. Taken together the results reveal intriguing but complex biological and molecular changes in mixed infections, which remain a basis for future experiments and potential agricultural benefits. / Canadian Commonwealth Scholarship and Fellowship Plan

The nuclear pore complex (NPC) is perhaps the largest protein complex in the eukaryotic cell, and controls the movement of molecules across the nuclear envelope. The NPC is composed of up to 30 proteins termed nucleoporins (Nups), each grouped in different sub-complexes. The transmembrane ring sub-complex is composed of Nups responsible for anchoring the NPC to the nuclear envelope. Bioinformatic analysis has traced all major sub-complexes of the NPC back to the last eukaryotic common ancestor, meaning that the nuclear pore structure and function is conserved amongst all eukaryotes. In this study Arabidopsis T-DNA knockout lines for these genes were investigated to characterise gene function. Differences in plant growth and development were observed for the ndc1 knockout line compared to wild-type but gp210 plants showed no phenotypic differences. The double knockout line gp210 ndc1 was generated through crosses to observe plant response to the knockout of two anchoring-Nup genes. No synergistic affect from this double knockout was observed, suggesting that more, as yet unidentified Nups function the transmembrane ring in plants. The sensitivity to nuclear export inhibitor leptomycin B (LMB) was tested also for knockout lines, although growth sensitivity to the drug was not observed. Nucleocytoplasmic transport of knockout lines was measured in cells transformed by particle bombardment. To express fluorescent protein constructs actively transported through the NPC, localisation of protein determined the nucleocytoplasmic transport of the cell. The ndc1single knockout and the double knockout gp210 ndc1 exhibited decreased nuclear export. Further experiments in determining NDC1 localisation and identification of other Nups in the transmembrane ring sub-complex would bring a more comprehensive understanding to the plant NPC.

[ES] El calcio (Ca2+) es un catión esencial que juega un papel fundamental en todos los organismos vivos. Desde el punto de vista funcional, el Ca2+ actúa como un segundo mensajero que regula distintos procesos celulares. Trabajos anteriores indican que la señalización mediante Ca2+ podría estar implicada en las primeras etapas de la inducción de la embriogénesis in vitro de las plantas, pero el verdadero papel del Ca2+ en este proceso es aún desconocido. Por eso, el principal objetivo de la presente Tesis es el estudio del papel del Ca2+ en la embriogénesis in vitro mediante dos sistemas in vitro: la embriogénesis somática y la embriogénesis de microsporas. Para determinar la importancia de la homeostasis del Ca2+ en la inducción de la embriogénesis y las dinámicas de los niveles de Ca2+ durante la inducción y el establecimiento de embriones somáticos y derivados de microsporas, se utilizaron tratamientos químicos y se detectaron los niveles de Ca2+ mediante sondas fluorescentes y sensores cameleon codificados genéticamente, visualizados con microscopía fluorescente y confocal. Observamos que el aumento de Ca2+ es un marcador temprano en la inducción de la embriogénesis in vitro y que los niveles de Ca2+ durante la embriogénesis in vitro son dinámicos en todos los sistemas estudiados. Además, las oscilaciones en los niveles de Ca2+ podrían estar relacionadas con los procesos de diferenciación que ocurren en las células inducidas una vez une el Ca2+ a la calmodulina. Mostramos que un aumento de Ca2+ dentro de un rango definido de concentración tiene un efecto positivo, dependiendo del sistema, en la producción de embriones, siendo más sensibles aquellos sistemas basados en suspensiones de células aisladas que aquellos que usan tejidos como explantes. Finalmente, estudiamos el papel de la calosa durante la embriogénesis somática, observando que la inhibición de la deposición de calosa impide el desarrollo embrionario, lo que sugiere una relación entre la formación de una barrera de calosa y el establecimiento de la identidad embrionaria en las células somáticas. / [CAT] El calci (Ca2+) és un catió essencial que juga un paper fonamental en tots els organismes vius. Des del punt de vista funcional, el Ca2+ actua com a un segon missatger que regula diferents processos cel·lulars. Treballs anteriors indiquen que la senyalització mitjançant el Ca2+ podria estar implicada en les primeres etapes de la inducció de l'embriogènesi in vitro de les plantes, però el paper real del Ca2+ en aquest procés encara és desconegut. Per això, el principal objectiu de la present Tesi és l'estudi del paper del Ca2+ en l'embriogènesi in vitro mitjançant dos sistemes in vitro: l'embriogènesi somàtica i l'embriogènesi de micròspores. Per tal de determinar la importància de l'homeòstasi del Ca2+ en la inducció de l'embriogènesi i les dinàmiques dels nivells de Ca2+ durant la inducció i l'establiment d'embrions somàtics i derivats de micròspores, es van utilitzar tractaments químics i es van detectar els nivells de Ca2+ mitjançant sondes fluorescents i sensors de cameleon codificats genèticament, visualitzats amb microscòpia fluorescent i confocal. Vam observar que l'augment de Ca2+ és un marcador primerenc en la inducció de l'embriogènesi in vitro i que els nivells de Ca2+ durant l'embriogènesi in vitro són dinàmics en tots els sistemes estudiats. A més, les oscil·lacions en els nivells de Ca2+ podrien estar relacionades amb els processos de diferenciació que tenen lloc en les cèl·lules induïdes una vegada uneix el Ca2+ a la calmodulina. Vam mostrar que un augment de Ca2+ dins d'un rang definit de concentració té un efecte positiu, depenent del sistema, en la producció d'embrions, essent més sensibles aquells sistemes basats en suspensions de cèl·lules aïllades que aquells que usen teixits com a explants. Finalment, vam estudiar el paper de la cal·losa durant l'embriogènesi somàtica, i vam observar que la inhibició de la deposició de cal·losa impedeix el desenvolupament embrionari, la qual cosa suggereix una relació entre la formació d'una barrera de cal·losa i l'establiment de la identitat embrionària en les cèl·lules somàtiques. / [EN] Calcium (Ca2+) is an essential cation that plays fundamental roles in all living organisms. From a functional point of view, Ca2+ acts as a second messenger that regulates different cellular processes. Previous works point to the fact that Ca2+ signaling may be involved in the early stages of induction of in vitro plant embryogenesis, but the actual role of Ca2+ in this process remained unveiled. Thus, the main goal of the present Thesis is to study the role of Ca2+ in in vitro embryogenesis using two in vitro systems: somatic embryogenesis and microspore embryogenesis. Chemical treatments and detection of Ca2+ with fluorescent probes and genetically-encoded cameleon sensors imaged by fluorescence and confocal microscopy were performed to determine the importance of Ca2+ homeostasis for induction of embryogenesis and the dynamics of Ca2+ levels during the induction and establishment of somatic and microspore-derived embryos. We observed that Ca2+ increase is an early marker of induction of in vitro embryogenesis and Ca2+ levels during in vitro embryogenesis are dynamic in all the systems we studied. Moreover, Ca2+ oscillations might be related to the differentiation processes that take place in the induced cells upon binding to calmodulin. We showed that Ca2+ increase within a defined range has system-specific positive effects in embryo yield, being more sensitive those systems using isolated cell suspensions rather than those using tissues as explants. Finally, we studied the role of callose during somatic embryogenesis, and we observed that inhibiting callose deposition prevents embryo development, which suggests a relationship between the formation of a callose barrier and the establishment of embryo identity in somatic cells. / Calabuig Serna, A. (2023). Multidisciplinary study of the role of calcium in plant in vitro embryogenesis [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/196022