Investigating Water Responsive Actuation Using the Resurrection Plant Selaginella Lepidophylla As a Model System

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Investigating Water Responsive Actuation Using the Resurrection Plant Selaginella Lepidophylla As a Model System Investigating Water Responsive Actuation using the Resurrection Plant Selaginella lepidophylla as a Model System Véronique Brulé Department of Biology McGill University, Montréal Summer 2018 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy © Véronique Brulé 1 ! ABSTRACT Nature is a wealth of inspiration for biomimetic and actuating devices. These devices are, and have been, useful for advancing society, such as giving humans the capability of flight, or providing household products such as velcro. Among the many biomimetic models studied, plants are interesting because of the scope of functions and structures produced from combinations of the same basic cell wall building blocks. Hierarchical investigation of structure and composition at various length-scales has revealed unique micro and nano-scale properties leading to complex functions in plants. A better understanding of such micro and nano-scale properties will lead to the design of more complex actuating devices, including those capable of multiple functions, or those with improved functional lifespan. In this thesis, the resurrection plant Selaginella lepidophylla is explored as a new model for studying actuation. S. lepidophylla reversibly deforms at the organ, tissue, and cell wall level as a physiological response to water loss or gain, and can repeatedly deform over multiple cycles of wetting and drying. Thus, it is an excellent model for studying properties leading to reversible, hierarchical (i.e., multi length-scale) actuation. S. lepidophylla has two stem types, inner (developing) and outer (mature), that display different modes of deformation; inner stems curl into a spiral shape while outer stems curl into an arc shape. In depth investigation of S. lepidophylla revealed that morphological and compositional gradients result in hierarchical stiffness gradients leading to differential tissue swelling/shrinking and, ultimately, directional stem (un)curling. Morphological 2 ! gradients are observed at the tissue level between adaxial and abaxial stem sides, and include changes in tissue density, cell shape, size, and cell angle relative to the stem axis. Compositional gradients are observed along the length of the stem from tip to base at both tissue and cell wall levels and include changes in lignification and xylan distribution. Comparison between inner and outer S. lepidophylla stem types showed that morphological gradients are most likely involved in directional stem bending while compositional gradients appear to contribute to the degree of stem curling. 3 ! RÉSUMÉ La biologie est source d’inspiration pour la production d’appareils biomimétiques et actionnement qui font avancer la technologie. Parmi les modèles biologiques étudiés, les plantes sont des sujets intéressants parce qu’elles présentent plusieurs types de fonctions et structures dérivées des mêmes matériaux de base (i.e., les polymères des parois cellulaires). Des enquêtes hiérarchiques ont démontré que des traits de morphologie et composition au niveau micro et nano sont responsables de fonctions complexes dans les plantes. En étudiant des modèles hiérarchiques pour mieux comprendre les traits qui provoquent le mouvement, on serait en mesure de les reproduire dans des matériaux synthétiques avec des fonctions plus complexes, ou avec une durée de fonction prolongée. Cette thèse explore les espèces Selaginella, une ‘plante de la résurrection’, comme nouveau modèle d’actionnement. Les plantes de la résurrection changent de forme réversiblement à plusieurs échelles de longueur (organe, cellule, paroi cellulaire) en réponse aux fluctuations de contenu en eau. Le changement de forme se répète sur plusieurs cycles de déshydratation et réhydratation. Donc, ces espèces sont des modèles idéaux pour étudier les traits qui provoquent l’actionnement hiérarchique et répétable. De plus, les tiges de S. lepidophylla démontrent des modalités de mouvement différentes. En déshydratant, les tiges intérieures se recourbent sur elles-mêmes en spirale, et les tiges extérieures se courbe en forme d’arc. Une enquête compréhensive de S. lepidophylla a démontré que des changements gradés en morphologie et composition résultent en des changements gradés et 4 ! hiérarchiques de rigidité responsables de l’engorgement et l’assèchement différentiels des tissus, ce qui explique le mouvement directionnel des tiges. Les changements gradés en morphologie s’observent au niveau du tissu entre les régions adaxiale et abaxiale de la tige incluant des changements dans la densité du tissu ainsi que l’angle cellulaire relatif à l’axe de la tige. Les changements gradés en composition sont observables au long de la tige, au niveau du tissu et la paroi cellulaire, et incluent des changements en lignification et en distribution de xylane. En comparant des tiges vivantes et mortes de S. lepidophylla, il a été démontré que les changements en morphologie sont responsables pour du mouvement directionnel de la tige, et que les changements en composition déterminent le point où la tige peut courber. 5 ! ACKNOWLEDGMENTS I would like to begin by thanking Dr. Tamara Western for her all her guidance and support during my research project. Tamara, I feel privileged to have learned so much from you. Not only did you show me how to perform research, but you fostered my love for teaching and helped me to grow as an instructor. Thanks for all our conversations, from the serious ones right down to cat stories and chats about writing. Thank you to my collaborators in engineering: Dr. Damiano Pasini, Dr. Ahmad Rafsanjani, and Dr. Meisam Asgari. Mechanics was a steep learning curve, but you showed me how interesting a field it is and how to navigate it best from a biologist’s point of view. I appreciate all your insight and guidance, and am grateful to have worked alongside you. I would also like to thank my committee members, Dr. Tom Bureau and Dr. Alejandro Rey, for your excellent feedback on my project. Thanks to my awesome labmates: Mike Ogden, Jonathan Palozzi, Lydiane Gaborieau, and Bronwen Froward. Coming to the lab was guaranteed to be fun with all of you around! And to Joe, Frank Anne-Marie, and Kathy – thanks for all the great conversations and laughs! You made Stewart Bio a memorable place. MB, this journey would have been a lot more difficult without your daily smiles, words of encouragement, and all your homemade bread! Manimal and Taya, you are THE best furry thesis co-writers. Your love and snuggles kept me sane. Mom B and Pop B, I am forever grateful for all your love, support and your belief in me. You gave me the strength and confidence to see this through. Finally, thank you to everyone else whose feedback or kind words helped me on this PhD journey! ! 6 ! AUTHOR CONTRIBUTION AND CHAPTER OVERVIEW This thesis contains four chapters, three of which are original research. Chapter 1 is a literature review that provides an overview of the various topics explored in this research project. Chapters 2-4 contain original contribution to our understanding of the hierarchical compositional and morphological features, as well as the mechanical forces arising from these features, that drive stem deformation in the resurrection plant Selaginella lepidophylla. Appendix 1 includes a meta-analysis of stiffness in Arabidopsis thaliana as it relates to plant function, and also highlights other plant models used to study stiffness in relation to plant deformation and actuation. Appendices 2-3 include small experiments that complement the work presented in Chapters 2-4, but that will not be included in any manuscripts. Finally, Appendix 4 preliminarily explores the relationship between water movement and corresponding vegetative tissue deformation in both Selaginella lepidophylla and Myrothamnus flabellifolius. Chapter 2: This chapter is published. I performed the microscopy, timelapse video capture, and weight loss quantification, while Dr. Ahmad Rafsanjani performed the computational modelling. We both performed tensile testing experiments. The manuscript was co-written by Dr. Rafsanjani and I, and Drs. Tamara Western and Damiano Pasini reviewed and provided feedback on the manuscript. Chapter 3: This chapter is in preparation for publication and is expected to be submitted within the next month. I carried out all the presented work, with the following exceptions: Dr. Rafsanjani performed the micro-computed x-ray tomography experiments described 7 ! in the text and presented in Figure 3.2. Dr. Meisam Asgari performed the atomic force microscopy imaging and indentation presented in Figure 3.3. Again, Drs. Tamara Western and Damiano Pasini reviewed and provided feedback on the manuscript. Chapter 4: This chapter is in preparation for publication for NanoLetters (which combines results and discussion into a single section), and is expected to be submitted within the next month. Dr. Asgari performed the atomic force microscopy experiments while I carried out the other microscopy (brightfield, immunofluorescence, and scanning electron microscopy) presented in the chapter. Drs. Tamara Western and Damiano Pasini reviewed and provided feedback on the manuscript. Appendix 1: This work is published. Dr. Tamara Western and I wrote the bulk of the manuscript, with contributions from Drs. Damiano Pasini and Ahmad Rafsanjani. Appendices 2-3: I performed all these experiments myself. Appendix 4: The data presented here will be prepared for publication, along with x-ray tomography data collected from
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