Comparative Analyses of Plant Responses to Drought and Salt Stress in Related Taxa: A Useful Approach to Study Stress Tolerance Mechanisms Mohamad Al Hassan Supervisors: Oscar Vicente Meana Monica Tereza Boscaiu Neagu Valencia, January 2016 Alas, my 3 years journey has come to end, and I find myself in front of the challenging task to summarize my gratitude to those who made this experience end happily. To Pr. Oscar Vicente Meana, a supervisor, a teacher, and forever a good friend. Even before I had the chance to prove myself, you believed in me and gave me a lot of your trust. During these 3 years, I was blessed with your guidance and care that allowed me to attain the aimed title with outmost professionalism, while having the chance to follow my instincts. It is said that “The Mediocre Teacher Tells. The Good Teacher Explains. The Superior Teacher Demonstrates. The Great Teacher Inspires”, and you sir, were and always will be a source of inspiration. I will forever be proud that I was your student. To Dr. Mónica Tereza Boscaiu Neagu, a director, a teacher, and forever a good friend. You were always a great support in every step of this work. Thank you for your trust, guidance and care. I am forever in your debt and will always be proud to have been your student one day. To Dr. Federico Martinelli, a colleague and forever a good friend. Thank you for your time, help and support whenever needed, you are a special person, and I hope that what you and Cristian aspire for, gets fulfilled very soon. To Dr. María Pilar López-Gresa, a colleague and forever a good friend. Thank you for your time, help and support whenever needed. To Dr. Josep V. Llinares. Thank you for your time, help and support whenever needed. To Dr. Mohamad Abbas, a colleague and forever a good friend. Thanks for always being there with me, and for being a brother from another mother, since our first bachelor degree years in Lebanon until the most distant future. I am forever in your debt. To Sorin Traian Schiop, a colleague and forever a good friend. Thank you for being a dear friend whom any person would be blessed to have, on both the personal and academic level. To Robert Paczynski, a colleague and forever a good friend. Thank you for all the great memories, talks and discussions we had, you are a special person, with a unique way of living that makes you such a delightful person to know and to have around. To my colleagues Alexandra, Andrea, Sara, Eduarda, Alejandra, Denise, Raluca, Cristina, Enrico, Orsi, Juliana, Dhikra, Aleksandra, and Sameh. Thank you all, for the good and the bad times. It has been a fun journey and it wouldn’t have reached this outcome if it wasn’t for your contributions, each in their own way. To friends especially Ragab, Mohamad, Abdel Rahman, Victoria, Mercedes, Elena, Bruna, Samantha, Chong Min, Aytana and Moussa. Thank you for being there, and for making this journey full of happy memorable experiences. To my family, Adnan, Iman, Ali, and Mostafa. I had to leave you all in the times of troubles that our country didn’t come out from yet, but still you were with me in every step in the way. I know that I was in your minds and hearts all the time. I never felt alone even though I am more than 1000 miles far, because of your love, trust and confidence. You are my shelter and cradle, and the hard rock that all my dreams could be built upon. Thank you all from the deepest corners of my soul... I love you all. To Mihaela Loredana Morosan, how can I thank you?? I never heard of two lovers thanking each other’s, especially that words would never express what I feel towards you… I will just say that the hope of having you as a part of my future is what keeps a smile on my face every day. Thank you for being the way you are from all of my heart… I love you I would like to thank the evaluators of this work, and the members of the jury tribunal going to assess it as well. It is a privilege to present this work in front of such esteemed scientists. Finally, I would like to present my gratitude to the friendly and lovely people of Valencia, a city that I now consider my second home. Hopefully the coming days will hold more fortune, wealth and the possibility for a brighter future to this lovely city and to the whole of Spain. Abstract Introduction Salinity and drought are the most important environmental stress conditions reducing crop yields worldwide and limiting the distribution of wild plants in nature. Soil salinity, especially secondary salinity caused by anthropogenic practices, such as prolonged irrigation, lead to substantial agricultural yield losses, especially in arid and semiarid regions. Drought, caused by reduced water content in the soil, occurs due to disorders in nature´s water cycle, chiefly when evapotranspiration exceeds precipitation in a certain area, to the point where soil water reserves can no longer support plant growth. Drought and salt stress trigger the activation of a series of basic stress mechanisms that includes among others, the control of ion transport, exclusion and compartmentalization, as well as the accumulation of compatible solutes (‘osmolytes’), and the activation of antioxidant systems. These mechanisms are conserved in all plants, stress tolerant and sensitive alike, and don’t necessarily confer tolerance. To decipher those mechanisms and have a better understanding on the contribution of different stress responses to the stress tolerance of a given species, we have carried out comparative studies on the responses to drought and salinity in a number of genetically related taxa with different tolerance potentials. Methodology The work concentrated on studying the responses to salt and drought stress in genetically related plants with different tolerance to abiotic stress. The studied taxa included salt-tolerant (halophytes) and salt-sensitive (glycophytes) wild species of two i different genera: Juncus (monocotyledonous) and Plantago (dicotyledonous), as well as plants of crop species: Solanum lycopersicum var. cerasiforme (cherry tomatoes) and different Phaseolus cultivars, one of P. coccineus and three of P. vulgaris. The experimental approach was mostly based on i) establishing the relative tolerance to water and salt stress in the studied species from their distribution in nature (in the case of wild species) and through the relative inhibition of growth in the presence of stress, and ii) correlating changes in the levels of biochemical ‘stress markers’ associated to specific response pathways (ion transport, osmolyte accumulation…) upon stress treatments, with the already established relative tolerance to stress. This strategy proved to be appropriate to distinguish mere general responses to stress from those mechanisms relevant for stress tolerance of the investigated species and cultivars. The work also sheds light on other aspects affected by salt stress, specifically regarding germination and reproductive success or anatomical changes in salt-stressed plants. The expression patterns of the gene NHX1, encoding a vacuolar Na+/H+ antiporter were also studied in the Plantago taxa, as a first step in the full characterisation of this ion transporter, that appears to play an important role in the mechanisms of salt tolerance in this genus. Results and discussion Through the results attained from this work, we have been able to establish which general stress responses are relevant for tolerance in the investigated species or cultivars, and which are not. Thus, we provide clear evidence that, although all plants seem to activate the same mechanisms of defense in response to abiotic stress, their relative contribution to stress tolerance differs widely in different genera and species. ii Moreover, in general, the relative tolerance of the investigated species and cultivars were the same, when referring to salt stress and to water stress, and the same mechanisms – except some related to ion transport and homeostasis – were relevant for tolerance to both stresses. In the studied Phaseolus cultivars, P. vulgaris cv. 'Maxidor' showed the smallest growth inhibition under salt and water stress conditions, and therefore was defined as the most tolerant. 'Maxidor' accumulated lower levels of toxic ions and proline, and recorded higher levels of myo-inositol than the other cultivars. We concluded that blocking ion transport from the roots to the leaves and myo-inositol accumulation, were the mechanisms most relevant for stress tolerance in Phaseolus. Proline is a reliable stress biomarker in this genus, indicating the degree of stress affecting the plants, but is not directly involved in tolerance mechanisms. In the studied Plantago species it was found that the more tolerant taxa transported Na+ and Cl- to the leaves more efficiently than the most sensitive P. major, and tended to accumulate large amounts of proline, albeit only under extreme stress conditions; these responses appear to be the most relevant for tolerance in Plantago. I Toxic ions transported to the leaves are presumably accumulated in vacuoles, which gave incentive to isolating, sequencing and studying the expression of the Na+/H+ vacuolar antiporter gene NHX1 in the studied species. Upon short-term treatments with high NaCl concentrations, the more tolerant species showed higher salt-induced expression of the aforementioned gene, supporting the contribution of the NHX1 antiporter to salt tolerance in Plantago. Meanwhile, the tolerant Juncus species were able to partly inhibit ion transport from the roots to the plants aerial parts and recorded a much larger increment (about 60- fold over the controls) in proline contents, as compared to the stress-sensitive congener. iii Therefore, blocking accumulation of toxic ions and inducing accumulation of proline in the culms appear to be the most important mechanisms of tolerance in Juncus.