UNIVERSIDAD POLITÉCNICA DE MADRID ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS DEPARTAMENTO DE BIOTECNOLOGÍA MOLECULAR BASIS OF SALT TOLERANCE IN Physcomitrella patens MODEL PLANT: POTASSIUM HOMEOSTASIS AND PHYSIOLOGICAL ROLES OF CHX TRANSPORTERS. Director: Alonso Rodríguez Navarro Profesor Emérito, E.T.S.I. Agrónomos Universidad Politécnica de Madrid TESIS DOCTORAL SHADY ABDEL MOTTALEB MADRID, 2013 MOLECULAR BASIS OF SALT TOLERANCE IN Physcomitrella patens MODEL PLANT: POTASSIUM HOMEOSTASIS AND PHYSIOLOGICAL ROLES OF CHX TRANSPORTERS. Memoria presentada por SHADY ABDEL MOTTALEB para la obtención del grado de Doctor por la Universidad Politécnica de Madrid Fdo. Shady Abdel Mottaleb VºBº Director de Tesis: Fdo. Dr. Alonso Rodríguez-Navarro Profesor emérito Departamento de Biotecnología ETSIA- Universidad Politécnica de Madrid Madrid, Septiembre 2013 To my family i ACKNOWLEDGEMENTS This work would not have been possible without the collaboration and inspiration of many people. First of all, I would like to express my deep appreciation to my supervisor and mentor Alonso Rodríguez Navarro for giving me this once-in-a- lifetime opportunity to both studying what I like most and developing my professional career. For integrating me in his research group, introducing me to novel research topics and for his constant support at both personal and professional level. I owe a debt of gratitude to Rosario Haro for her unconditional help, ongoing support and care during my thesis. For teaching me to pay attention to the details of everything and for her immense efforts in supervising all the experiments of this thesis. I also feel grateful to Begoña Benito for her constant help and encouragement, for her useful advice and for always being available to answer my questions at anytime with enthusiasm and a pleasant smile. I would like to thank a lot Blanca Garciadeblás for her constant encouragement and help in many important experiments of this thesis… and for bearing my constant flow of silly (and not so silly) questions during the past four years! I would also like to thank Mª Antonia Bañuelos for her useful advices and for our interesting conversations. I wish to express my appreciation to my colleagues in the research group. Their help, comments and unique expertise helped me to be accurate, thorough, and balanced throughout the course of this work. Thank you for the great help and for all the good laughs we shared together during the coffee breaks! Ana Claudia Ureta for her help and funny conversations. Marcel Veldhuizen for his help and advice on many useful shortcuts for experimental protocols! Angela Sáez for her support and understanding. Rocío Álvarez for her dedicated help in many experiments, her constant encouragement and for our many funny conversations. A special thanks to my dear friend Ana Fraile for teaching me her experience on the different experimental techniques I used in this work, for her constant help, advices and constructive critics. For being patient with my many questions and for all the after-work activities and trips shared together! Thanks to our neighbor “Rhizobium lab.” members, professors Tomás Argüeso, Pepe Palacios, Juan Imperial, Luis Rey and Belén Brito… and thanks to Bea, Carmen, David, Laura, Marta, Mónica, Rosabel for their collaboration and good times shared during coffee and lunch breaks. A special thanks to my good friend and iii “bus partner” Anabel for all the good times, laughs and conversations we had during our coffee and lunch breaks, in the bus, trips and everywhere! I deeply appreciate the technical help and dedication of Dr. Pablo Melendi in the confocal microscopy experiments. My gratitude is extended to all my friends in the “Plant virus lab.” Antolín, Jean-Michel, Manuel & Manuel, Nils and Pablo, and a special thanks to my “lunch partner” Miguel Ángel for all the good laughs and for teaching me so many aspects of the Spanish culture, typical foods, slang language, sayings and many more! I would also like to thank a lot many people that I was lucky to know in the Centro de Biotecnología y genómica de plantas (CBGP), and had the opportunity to share with them many nice moments: Alessandro, Amir, Angela, Anja, Bahia, Carlos, César, Elena, Eva, Fátima, Jan, Julia, Inés, Irene, Mar, Nancy, Pilar, Ruth, Sara, Simon and Vivi. Many thanks to my CBGP-Basketball team members for all the entertaining matches played under the burning sun, rain or snow! Thanks to Alejandro, Antonio, Chechu, Jorge, Laura, Miguel Ángel, Nacho and Nils. I would also like to thank my housemates and friends in Madrid Alba, Ali, Amr, Basem, Chakib, Gemma, Haytham, Isabel, Jorge, Jose, Juan, Lurdes, Mohamed, Montse, Osama, Vicky and Yasser for keeping my morale high at hard times. It would have been very different without your care and generosity! Finally, I feel extremely lucky to have an exceptional family that trusted me and was so patient and understanding even under all the negative circumstances they have suffered during my absence. Their love has been strong, unconditional, and selfless. They have made everything I’ve accomplished possible. I am forever in your debt! iv RESUMEN El suelo salino impone un estrés abiótico importante que causa graves problemas en la agricultura ya que la mayoría de los cultivos se ven afectados por la salinidad debido a efectos osmóticos y tóxicos. Por ello, la contaminación y la escasez de agua dulce, la salinización progresiva de tierras y el aumento exponencial de la población humana representan un grave problema que amenaza la seguridad alimentaria mundial para las generaciones futuras. Por lo tanto, aumentar la tolerancia a la salinidad de los cultivos es un objetivo estratégico e ineludible para garantizar el suministro de alimentos en el futuro. Mantener una óptima homeostasis de K+ en plantas que sufren estrés salino es un objetivo importante en el proceso de obtención de plantas tolerantes a la salinidad. Aunque el modelo de la homeostasis de K+ en las plantas está razonablemente bien descrito en términos de entrada de K+, muy poco se sabe acerca de los genes implicados en la salida de K+ o de su liberación desde la vacuola. En este trabajo se pretende aclarar algunos de los mecanismos implicados en la homeostasis de K+ en plantas. Para ello se eligió la briofita Physcomitrella patens, una planta no vascular de estructura simple y de fase haploide dominante que, entre muchas otras cualidades, hacen que sea un modelo ideal. Lo más importante es que no sólo P. patens es muy tolerante a altas concentraciones de Na+, sino que también su posición filogenética en la evolución de las plantas abre la posibilidad de estudiar los cambios claves que, durante el curso de la evolución, se produjeron en las diversas familias de los transportadores de K+. Se han propuesto varios transportadores de cationes como candidatos que podrían tener un papel en la salida de K+ o su liberación desde la vacuola, especialmente miembros de la familia CPA2 que contienen las familias de transportadores KEA y CHX. En este estudio se intenta aumentar nuestra comprensión de las funciones de los transportadores de CHX en las células de las plantas usando P. patens, como ya se ha dicho. En esta especie, se han identificado cuatro genes CHX, PpCHX1-4. Dos de estos genes, PpCHX1 y PpCHX2, se expresan aproximadamente al mismo nivel que el gen PpACT5, y los otros dos genes muestran una expresión muy baja. La expresión de PpCHX1 y PpCHX2 en mutantes de Escherichia coli defectivos en el transporte de K+ restauraron el crecimiento de esta cepa en medios con bajo contenido de K+, lo que vii sugiere que la entrada de K+ es energizada por un mecanismo de simporte con H+. Por otra parte, estos transportadores suprimieron el defecto asociado a la mutación kha1 en Saccharomyces cerevisiae, lo que sugiere que podrían mediar un antiporte en K+/H+. La proteína PpCHX1-GFP expresada transitoriamente en protoplastos de P. patens co- localizó con un marcador de Golgi. En experimentos similares, la proteína PpCHX2- GFP localizó aparentemente en la membrana plasmática y tonoplasto. Se construyeron las líneas mutantes simples de P. patens ∆Ppchx1 y ∆Ppchx2, y también el mutante doble ∆Ppchx2 ∆Pphak1. Los mutantes simples crecieron normalmente en todas las condiciones ensayadas y mostraron flujos de entrada normales de K+ y Rb+; la mutación ∆Ppchx2 no aumentó el defecto de las plantas ∆Pphak1. En experimentos a largo plazo, las plantas ∆Ppchx2 mostraron una retención de Rb+ ligeramente superior que las plantas silvestres, lo que sugiere que PpCHX2 promueve la transferencia de Rb+ desde la vacuola al citosol o desde el citosol al medio externo, actuando en paralelo con otros transportadores. Sugerimos que transportadores de K+ de varias familias están involucrados en la homeostasis de pH de orgánulos ya sea mediante antiporte K+/H+ o simporte K+-H+. viii ABSTRACT Soil salinity is a major abiotic stress causing serious problems in agriculture as most crops are affected by it. Moreover, the contamination and shortage of freshwater, progressive land salinization and exponential increase of human population aggravates the problem implying that world food security may not be ensured for the next generations. Thus, a strategic and an unavoidable goal would be increasing salinity tolerance of plant crops to secure future food supply. Maintaining an optimum K+ homeostasis in plants under salinity stress is an important trait to pursue in the process of engineering salt tolerant plants. Although the model of K+ homeostasis in plants is reasonably well described in terms of K+ influx, very little is known about the genes implicated in K+ efflux or release from the vacuole. In this work, we aim to clarify some of the mechanisms involved in K+ homeostasis in plants. For this purpose, we chose the bryophyte plant Physcomitrella patens, a nonvascular plant of simple structure and dominant haploid phase that, among many other characteristics, makes it an ideal model.