The influence of root-zone bicarbonate and carbon dioxide enrichment on lettuce, pepper and tomato growth Estibaliz Leibar-Porcel B.Sc Biology, University of Vigo, Spain M.Sc Molecular Biotechnology, University of Barcelona, Spain This thesis is submitted to Lancaster University in partial fulfilment of the requirements for the degree of doctor of philosophy Declaration Except where reference is made to other sources, I declare that the contents in this thesis are my own work and have not been previously submitted, in part or in full, for the award of a higher degree elsewhere. Estibaliz Leibar-Porcel Lancaster University July 2019 i Publications arising from this thesis Leibar-Porcel, E., McAinsh, M.R., Dodd, I.C. Root-zone CO2 enrichment increases biomass accumulation in lettuce and pepper grown hydroponically and aeroponically. Acta Horticulturae. In Press. (Reproduced as Appendix 1) Oral/Poster presentations from this work Conferences: Leibar-Porcel, E. Increasing crop yield and resource efficiency via root-zone CO2 enrichment. The Great British Tomato Conference. Chesford Grange Hotel. 28-29th September 2016. Leibar-Porcel, E. Increasing crop yield and resource efficiency via root-zone CO2 enrichment. Plant & Crop Science Postgraduate Conference, Lancaster University, 4th October 2016. Leibar-Porcel, E. Increasing crop yield and resource efficiency via root-zone CO2 enrichment. AHDB Studentship Conference. Stratford Manor. 6-7th November 2017. Leibar-Porcel, E. Root-zone CO2 enrichment increases biomass accumulation in lettuce and pepper grown hydroponically and aeroponically. Oral presentation at the XXX. International Horticultural Congress. Istanbul, Turkey. 12-16th August 2018. Posters: Leibar-Porcel, E. Increasing crop yield and resource efficiency via root-zone CO2 enrichment. LEC PGR Conference, Lancaster University, 21-22th April 2016. Leibar-Porcel, E. Increasing crop yield and resource efficiency via root-zone CO2 enrichment. 2nd Agriculture and Climate Change Conference, Sitges, 26-28th March 2017 ii Acknowledgments I wholeheartedly thank my supervisor Professor Ian Dodd, for his guidance, patience, motivation, knowledge and endless support through the ups and downs of not just completing my thesis, but also of the journey of life along the way. I could not have had a better supervisor. I am also grateful to my second supervisor Dr. Martin McAinsh for his help, advice and encouragement throughout this journey. A big thanks also to the person who drove me to the starting point of this PhD, Dr.Vincent Vadez, for his advice, knowledge, positivity and for always believing in my research skills. Also, to Dr.Krithika Anbazhagan and Dr.Pushpavalli Raju for their encouragement, advice and friendship regardless the long distance between us! The funding of my project AHDB and Perry Foundation who kindly helped me to finish my thesis are gratefully acknowledged. I would like to thanks all the members of the Plant Physiology group and everyone at Lancaster Environment Center that I have being lucky to meet over the last few years: Shane Rothwell, Dennis Touliatos, Noorliana Mohd Zahn, Bea Burak, Tom Williams, Matias de Felipe, Rachel Baxter, Hend Mandour, Vasilis Giannakopoulos, Joao Pennacchi, Marta Oñate, Ivan Jauregui, Cristina Sale and Thomas Monvain-Monval. Special thanks to Jaime Puertolas, Carlos De Ollas, Sarah Donaldson, Katharina Huntenberg, Rob Kempster, Javi Sierro and Pedro Valdecantos, for your patience, support and for always being there when I needed. I am very lucky to have met precious friends during this period in Lancaster; Luciana and Margherita (Charlie’s Angels),we have passed through many ebbs and flows together. Thank you for being there since day one. Mary and Petros, you are the kindest couple I’ve ever met, many thanks for being there when I needed. Ana, I am very lucky to have met you during this period, thanks for our long chats and for your endless support in every aspect of my life. Pilar, thanks for your support, chats, and mini trips to the beach or the lakes. I will bring you all in my heart wherever I go. A mis amigas galegas, Andrea y Rebeca y a mi mallorquina favorita, Pitu, mil gracias por estar todos estos años a mi lado. Montse, mi primera amistad de los inicios universitarios, gracias por tu ayuda, consejos y amistad. Marta, gracias por todas esas llamadas y apoyo incondicional. Josune, gracias por estar ahí para lo bueno y lo malo durante los últimos 20 años. Muchísimas gracias a Laura, Cris, Maite, Susan, Laura e Isra, Helene…. y a todas las personas que en la lejanía me han apoyado durante los últimos casi cuatro años. Por supuesto, no podría haber llegado hasta aquí sin el apoyo incondicional de mi familia; Ainara, Mikel y Oskar, nadie sabe la suerte que tengo de teneros como hermano/as, gracias por siempre estar ahí. Eva, Maitane e Iñaki, gracias por vuestros consejos y apoyo. Y gracias a todos mis sobris por sacarme siempre una sonrisa. Por último, mi más profundo agradecimiento a mi aita y ama, Eloy y Pilar, por el amor incondicional, por transmitirme tantos y tan buenos valores, por orientarme y apoyarme, y por inculcarme el deseo de superación y lucha. Eskerrik asko. iii Abstract Enhancing atmospheric CO2 levels in commercial glasshouses is a widely used technique to increase productivity, but has high-energy costs and detrimental environmental impacts due to frequent ventilation of the glasshouse (to prevent plant diseases) releasing CO2 into the atmosphere. Previous studies suggest that enrichment of the root zone (RZ) with CO2 (RZ CO2) may be a more economic and sustainable alternative to aerial CO2 enrichment. This thesis aimed to compare the effects of RZ dissolved inorganic carbon (DIC) enrichment by adding either bicarbonate (HCO3-) or gaseous CO2 to hydroponic and aeroponic systems, and to determine the physiological and molecular mechanisms by which plants respond to RZ DIC. Supplying hydroponically grown plants with high bicarbonate concentrations (20 mM) inhibited growth of lettuce, pepper and tomato. However, lower concentrations increased - biomass accumulation in lettuce (10% increase at both 1 mM and 5 mM HCO3 ) and pepper - (10% increase at 1 mM HCO3 ), but had no effect in tomato. Exposing plants to 1 mM 13 - NaH CO3 significantly increased shoot δ13C values over time, therefore confirming the uptake of DIC by the roots. Root δ13C values also significantly increased over time, 13 - however higher values at the beginning of NaH CO3 exposure suggested root-to-shoot transport of DIC. Nutrient solution pH did not affect root carbon uptake, but shoot δ13C values were lower in those plants exposed to lower pH levels (5.8) compared to those exposed to fluctuating pH (between 6.3 and 6.7), suggesting differences in root-to-shoot transport of DIC. Thus, root carbon uptake was independent of the form in which CO2 was - - provided (gaseous CO2 at pH 5.8; HCO3 at higher pHs). Adding 1 mM HCO3 to hydroponically grown plants did not change foliar nutrient content, but K, P, N, Zn, Cu and - Mn concentrations decreased at 20 mM HCO3 , suggesting nutrient deficiencies could limit growth. Applying 2000 ppm RZ CO2 to hydroponically grown lettuce, tomato and pepper did not affect biomass accumulation. Applying 1500 ppm CO2 to the RZ of aeroponically grown lettuce increased shoot biomass between 19-25% (in 4 independent experiments) compared to those grown with 400 ppm RZ CO2. However, leaf gas exchange measurements were inconsistent and therefore increased biomass could not be attributed to higher photosynthetic rates. In another 3 independent experiments, applying 1500 ppm CO2 to the RZ of aeroponically grown lettuce did not stimulate biomass accumulation, iv probably because the plants were exposed to higher night temperatures. Similarly, pepper and tomato did not show any biomass response to elevated RZ CO2, suggesting that the responses to RZ CO2 concentration are environment- and species-dependent. Nutrient analysis indicated that aeration with high RZ CO2 decreased lettuce foliar Mg and S concentrations, whereas root N concentrations were higher than control plants. Multi-hormone analysis of foliar and root tissues revealed that lettuce plants showed few differences in hormone status following RZ CO2 enrichment. High RZ CO2 increased foliar jasmonic acid concentration of lettuce, but the physiological significance of this change is not clear. Pepper plants showed significantly higher foliar 1-aminocyclopropane-1- carboxylic acid and lower trans-zeatin and salicylic acid concentrations, as well as lower root N6-(Δ2-isopentenyl) adenine and higher salicylic acid and gibberellic acid concentrations. These hormonal responses were associated with lower leaf area expansion of pepper plants exposed to elevated RZ CO2. Finally, transcriptome analysis of lettuce plants indicated that fatty acid biosynthesis, amino acid biosynthesis and carbon metabolism appeared to be the major pathways enriched in roots exposed to elevated RZ CO2. In addition, proteins related to the cell walls and membranes seemed enhanced under elevated RZ CO2. Although increased CO2 concentration around the roots caused major transcriptomic restructuring, the aerial parts of the plants showed limited transcriptomic changes. Taken together, this thesis is the first study of the responses of several horticultural species to elevated RZ CO2 within different growing systems in order to decipher the impact that elevated RZ CO2 has on crop productivity. Although bicarbonate enrichment of hydroponics and