Paclitaxel is an antitumor diterpene from Taxus spp. that binds tubulin, stabilizes microtubules and induces apoptosis in dividing human cells. It was originally isolated from the bark of Taxus brevifolia and approved for clinic uses by the FDA in 1992. Because of its excellent activity in treatment of various cancers, a significant supply shortage has been created by the enormous demand for this natural product. Thus, researchers have been focusing on the development of effective ways to increase the production of paclitaxel and related bioactive molecules. This shortage was initially solved by over-harvesting of T. brevifolia bark; however, it is not an environment-friendly, effective and sustainable way to supply paclitaxel. A semisynthetic route was then developed to convert the more readily available and renewable 10-deacetylbacatin III into paclitaxel. As an alternative, plant cell cultures have been employed to commercially produce paclitaxel and it is a more environment-friendly and sustainable route to end the supply crisis. However, problems associated with plant cell culturing at an industrial scale, such as cell aggregation and variability in yield, significantly affect paclitaxel production. Therefore, a discovery of a better-performing Taxus cell line might be a solution to overcome these culturing-associated problems. A cambial meristematic cell (CMC) line of Taxus cuspidata has been isolated, cultured and demonstrated to be a cost-effective and environmentally friendly platform for the sustainable production of paclitaxel (Lee et al. 2010). Compared to dedifferentiated cell (DDC) lines, CMC lines are undifferentiated cells and proved to have stem cell-like properties. When cultured at an industrial scale, this cell line contains much smaller cell aggregates with many cells appearing as singletons, the biomass of which is still increasing after 22-month culturing, and has much greater paclitaxel production after elicitation (Lee et al. 2010). In my project, we aimed to identify the transcription factors (TFs) that regulate the expression of paclitaxel biosynthesis genes. We performed Illumina Solexa sequencing on cDNA libraries derived from methyl jasmonate (MeJA)-elicitated CMCs to digitally profile gene expression. Analysis of differentially expressed gene (DEG) abundance led to the discovery of 19 putative TFs and bioinformatic analysis further showed that these 19 TFs belong to 5 different TF families. Further, the DNA binding motifs associated with these TFs can be found in the promoters of the two early, taxadiene synthase (TASY) and taxadiene 5α hydroxylase (T5αH), and three late, 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT), phenylpropanoyltransferase (PAM) and 3’-N-debenzoyl-2-deoxytaxol-Nbenzoyltransferase (DBTNBT), paclitaxel biosynthesis pathway genes. Then, yeast one-hybrid analysis, gel shifting assays and plant transient expression assays (TEA) were employed to assay TFs that interact with these promoters. Although Y1H screening did not show any convincing TF-promoter interactions, the attempted plant transient expression assay in the leaves of Nicotiana benthamiana might be a more suitable system to screen the positive regulators. Finally, the elucidation of a TF regulatory network that controls paclitaxel biosynthesis will guide the rational engineering of CMCs to ultimately increase yields of this important pharmaceutical.

Paclitaxel is an anticancer natural product with several biomedical applications produced by Taxus species, with a demand exceeding its supply. We have developed cambial meristematic cells (CMCs) from Taxus cuspidata as high yield source of paclitaxel. The biosynthesis of paclitaxel is predominantly under transcriptional control. Thus, the identification of transcriptional regulators of paclitaxel biosynthesis and their subsequent manipulation may enable further yield enhancement in Taxus CMCs. Previously, Roche 454 sequencing was employed to establish the transcriptome of T. cuspidata CMCs treated with the plant immune activator methyl jasmonate (MeJA). The bioinformatic analysis identified 19 jasmonate related transcription factors (TFs), based on their differential expression. Results of the Arabidopsis thaliana transient assay screen identified two MYB TFs that constitute positive regulators for paclitaxel genes, named MYB3 and MYB4. In this thesis, MYB3 and MYB4 showed in vitro binding to the cis-elements in ten promoters of paclitaxel genes using the electrophoretic mobility shift assay (EMSA). Then, a Taxus CMC protoplasts transient assay demonstrated that the expression of MYB3 and MYB4 trans-activated all tested genes. Further, MYB4 was found to activate the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) gene, key in the mevalonate pathway and precursor of paclitaxel biosynthesis. MYB3 and MYB4 were capable of auto-regulating their own transcription, constituting an important control point for paclitaxel biosynthesis. A possible mechanism for the early activation of MYB3 and MYB4 after MeJA elicitation is proposed. Finally, preliminary results on the expression of MYB3 and MYB4 in unelicited T. baccata CMC protoplasts indicate that their transient expression was sufficient to increase accumulation of paclitaxel and the precursor, 10-deacetyl baccatin III, highlighting their utility for paclitaxel production.

En este trabajo se ha estudiado la posibilidad de desarrollar un método de regeneración y transformación para producir plantas transgénicas de albaricoquero con cotiledones y secciones de hipocotilo a partir de semillas maduras de las variedades de albaricoquero ‘Canino’, ‘Moniquí’ y ‘Dorada’ y de la subespecie de albaricoquero, Prunus armeniaca L. var. ansu Maxim, cuyas semillas se utilizan para producir patrones paraalbaricoquero y otros frutales en China. Se ha conseguido establecer un protocolo de regeneración con los cotiledones de semillas de ‘Canino’ y del patrón, Prunus armeniaca L. var. ansu Maxim obteniendo porcentajes de regeneración muy elevados (66%) que no se vieron afectados por el genotipo. Sin embargo, la falta de coincidencia de las células que regeneran con las células que son transformadas por Agrobacterium ha impedido la obtención de plantas transformadas hasta el momento. Utilizando los explantos de secciones de hipocotilos de semillas maduras de tres variedades comerciales de albaricoquero, ‘Dorada’, ‘Moniquí’ y ‘Canino’, se ha establecido un protocolo de regeneración eficiente, con un mínimo del 32% de regeneración para las tres variedades estudiadas, que ha permitido abordar experimentos de transformación. Además, se han conseguido plantas transgénicas de las variedades ‘Dorada’ y ‘Moniquí’. El principal interés de la transformación genética de especies frutales reside en la posibilidad de transformar variedades comerciales con interés agronómico y ampliamente aceptadas. Sin embargo, para que la transformación genética se convierta en una herramienta de uso común en los programas de mejora de frutales leñosos se requiere que los procedimientos sean reproducibles, de elevada eficiencia e independientes del genotipo. La utilización de material adulto garantiza la identidad genética del material vegetal y evita los problemas de juvenilidad en aquellas especies en las que estos periodos son muy largos. Con el fin de explorar la posibilidad de desarrollar un protocolo de transformación independiente del genotipo, en esta tesis se ha estudiado el desarrollo de una nueva metodología basada en la transformación de células con capacidad meristemática. Se ha conseguido transformar cuatro variedades de albaricoquero, ‘Helena’, ‘Canino’, ‘Rojo Pasión’ y ‘Lorna’, con eficiencias de transformación que se encuentran entre las más elevadas descritas en especies de Prunus. Las ventajas de esta metodología son la sencillez del protocolo utilizado, su consistencia y rapidez. Sin embargo, hemos detectado que con mucha frecuencia se producen plantas quiméricas al transformar las células meristemáticas. Otro inconveniente en los protocolos de transformación es la utilización de genes marcadores de resistencia a antibióticos o herbicidas. Estos genes producen un rechazo social e incluso existe una directiva europea (2001/18/CE) que regula las plantas transformadas con genes de resistencia a antibióticos. El uso del gen pmi como marcador de selección podría reducir el rechazo de la opinión pública y facilitar los trámites legales para la comercialización de plantas transgénicas, ya que la proteína que codifica este gen no representa un riesgo para la salud humana ni para el medio ambiente. En la presente tesis se ha estudiado el desarrollo de un protocolo de transformación genética de albaricoquero que utiliza el sistema pmi/manosa para seleccionar las plantas transformadas. Con este sistema se ha conseguido producir plantas transgénicas de albaricoquero con una eficiencia similar a la obtenida con genes marcadores de selección que confieren resistencia a antibióticos, demostrando que puede ser un sistema de selección alternativo al uso de antibióticos. / In this Ph.D. thesis a methodology to develop such a procedure has been studied, based in cells with meristematic capability. We have transformed the apricot cultivars ‘Helena’, ‘Canino’, ‘Rojo Pasión’ and ‘Lorna’, with transformation efficiencies, based on PCR analysis, among the highest reported after transformation of other Prunus species. The advantages of this methodology are that is simple, fast and consistent. However, a large frequency of chimerical plants has been detected when transforming meristematic cells. Another problem of most transformation procedures is that they use antibiotic resistance marker genes. These genes produce a social rejection and there is an European law that neither allow deliberate release of plants carrying antibiotic resistance genes, used in veterinary or medicine, after 2004 nor their commercialisation after 2008 (Directive 2001/18/EEC of the European Parliament and the Council of the European Union). Since the PMI protein has revealed no adverse effects on human health and on the environment, the use of the pmi gen as a selectable marker could reduce the social rejection, facilitating legalisation and commercialisation of transgenic plants. In this Ph.D. thesis we have evaluated a transformation procedure using the pmi/mannose system for selecting apricot transgenic plants. Transformation efficiencies were similar to those obtained using antibiotic selection, demonstrating that this system could be an alternative for producing apricot transgenic plants under safe conditions for human health and the environment.