University of California Riverside

UNIVERSITY OF CALIFORNIA RIVERSIDE Exploring Phosphorylation in the regulation of the WUSCHEL Protein Gradient and Transcription A Thesis submitted in partial satisfaction of the requirements for the degree of Master of Science in Plant Biology by Dariush Ravandi Nejad December 2020 Thesis Committee: Dr. Venugopala Reddy Gonehal, Chairperson Dr. Jaimie Van Norman Dr. Morris Maduro Copyright by Dariush Ravandi Nejad 2020 The Thesis of Dariush Ravandi Nejad is approved: Committee Chairperson University of California, Riverside Acknowledgements I would like to thank Dr. Venu Reddy Gonehal for providing me with the opportunity and his guidance throughout my undergraduate and graduate school career. The projects, training and experiences he has let me been a part of has been invaluable to my development as a scientist and for that I will be forever grateful. I also would like to thank my thesis committee members, Dr. Jaimie Van Norman and Dr. Morris Maduro, who have provided valuable comments and advice throughout my years here. I would like to also thank all my current lab members Dr. Kevin Rodriguez, Alex Plong and Albert Do. Although we were a small group the mentorship, friendship and assistance in all facets throughout graduate school have been immensely helpful and I appreciate all their efforts. Additionally, I want to thank our past postdoctoral researcher, Dr. Andy Snipes, for his amazing work and laying the foundation for my projects. Finally, I would like to thank our exceptional undergraduate student, Hailee Scott, for her incredible work and spearheading many research projects. iv Dedication I feel an overwhelming gratitude for the people in my life who have supported me on this journey. I can’t begin to express how remarkable it feels to be at this point in my career. Although it took an immense amount of hard work, determination and mental fortitude, I know that I could not have done this on my own. I would like to dedicate this thesis to my family for all their love and support. I cannot express how thankful I am for the sacrifices my mother and father made to get me to where I am today but I hope that through my successes I can make that burden they took worth it. I want to thank my brother and sister, I love them more than they know and they inspire me every day to be someone they can look up to. I especially want to thank my love, Jennifer “Jenny” Medina (and her amazing family), for being my partner and greatest friend. Thank you for always cheering me on and picking me up when I’m down. You are my world and I could not imagine making it through all this without you. Finally, I want to thank all the friends I have made during my time at UCR. They were the perfect distraction and have always been there when I needed them. I will always consider “The Boys” my brothers. v Table of Contents Title page………………………………………………………………………………….. Copyright page…………………………………………………………………………… Signature approval page………………………………………………………………… Acknowledgements……………………………………………………………………..iv Dedication………………………………………………………………………………..v Table of Contents……………………………………………………………………….vi List of Figures………………………………………………………………………….viii Chapter 1: Introduction Structural Organization of the SAM…………………………………………...1 Molecular Mechanisms Regulating the SAM…………………………………2 Introduction of WUSCHEL and CLAVATA3 Regulation of the SAM………5 Introduction to Protein Phosphorylation………………………………………9 N-terminal Phosphorylation of the WUSCHEL Protein…………………….11 Introduction to KIN10/KIN11………………………………………………….12 Aims of the Study………………………………………………………………15 vi Chapter 2: Determining the in vivo effects of N-terminus phosphorylation of the WUSCHEL protein Introduction……………………………………………………………………..18 Results………………………………………………………………………….20 Discussion………………………………………………………………………23 Methods…………………………………………………………………………28 Chapter 3: Uncovering the Molecular and Genetic Characterization of SNRK1-WUSCHEL Interaction and its effect on SAM Maintenance Introduction……………………………………………………………………..37 Results………………………………………………………………………….39 Discussion………………………………………………………………………45 Methods…………………………………………………………………………51 References……………………………………………………………………………..61 vii List of Figures Chapter 1: Introduction Fig 1.1 The shoot apical meristem and the signals and process regulating WUSCHEL protein levels……………………………………………………………..16 Fig 1.2 Phosphorylation as a regulatory mechanism for the WUSCHEL protein gradient………………………………………………………………………...17 Chapter 2: Determining the in vivo effects of N-terminus phosphorylation of the WUSCHEL protein Fig 2.1 Mutations of the N-terminal site of phosphorylation on the WUSCHEL protein…………………………………………………………………….32 Fig 2.2 WUS N-terminal phosphorylation decreases the nuclear accumulation of the WUS protein……………………………………………………33 Fig 2.3 Disruption of WUS phosphorylation states alters growth rates and developmental patters…………………………………………………………………34 Fig 2.4 Non-phosphorylatable version of WUS cannot rescue the wus1-1 null mutant line…………………………………………………………………………35 Fig 2.5 Phosphomimic versions of WUS cannot properly accumulate in the clv3-2 null mutant line…………………………………………………………………36 viii Chapter 3: Uncovering the Molecular and Genetic Characterization of SNRK1-WUSCHEL Interaction and its effect on SAM Maintenance Fig 3.1 Mapping the interaction region of WUS and KIN10/KIN11……….54 Fig 3.2 Mapping KIN10/KIN11 interaction to the WUS-Box and EAR-like domains…………………………………………………………………………………55 Fig 3.3 BiFC interaction of WUS-KIN10/KIN11…………………………….56 Fig 3.4 BiFC controls for interaction of WUS-KIN10/KIN11……………….57 Fig 3.5 KIN10OE results in an overproliferation of above ground organs and increased axillary meristem production………………………………………...58 Fig 3.6 KIN10OE results in changes to SAM architecture, pCLV3 and pWUS expression……………………………………………………………………...59 Fig 3.7 KIN10OE decreases pCLV3 expression…………………………...60 ix Introduction: Plant development vastly differs from animal development due to the fact that plants are able to maintain continuous organogenesis throughout their lifespan. This is possible because plants are able to maintain populations of undifferentiated stem cell located at the Shoot Apical Meristem (SAM) and Root Apical Meristem (RAM), which provide a constant supply of cells for tissue and organ development (Barton and Poethig, 1993, Sussex, 1989, Kaufmann et al., 2010). The SAM is responsible for all above ground organ formation producing rosette leaves during the vegetative stage transitioning to producing leaves, flowers and stem during its reproductive phase. On the other hand,the RAM produces all subterranean organs and in sustaining continuous root development and growth. Structural Organization of the SAM The SAM of the model organism Arabidopsis thaliana is a conglomeration of several functional domains organized into three clonally distinct layers of cells. The L1 is the outermost layer that generates the epidermis, the L2 is the subepidermal layer and the L3 is multilayered which is collectively referred to asthe corpus (Sussex, 1989)(Fig. 1.1A). The L1 and L2 layers divide anticlinally, perpendicular to the cell surface, generating clonally distinct monolayers (Fig. 1.1A). The L3 and below differ from the L1 and L2 in that it divides both anticlinally and periclinally, forming its own clonally distinct region (Fig. 1.1A). 1 Simultaneously, there are functional domains of the SAM that arise as cells divide and displace from the center of the L1-L3 outward. At the very center of the L1-L3 near the tip of the SAM there is a population of undifferentiated stem cells that form the Central Zone (CZ) (Reddy, 2008, Steeves and Sussex, 1989) (Fig. 1.1A). Divisions of these cells results in sustained maintenance of the CZ and the lateral displacement of daughter cells into the adjacent Peripheral Zone (PZ) where differentiation begins leading to the development of Organ Primordia (OP) (Fig. 1.1A). Below the CZ is a subset of cells called the Organizing center (OC) that has been shown to provide cues for stem cell specification (Fig. 1.1A). Encompassing the OC and the rest of the L3 and below is the Rib-meristem (RM). Molecular Mechanisms Regulating the SAM Maintenance of SAM organization, gene expression, and transitioning of slow diving stem cells to fast diving differentiating cells requires a wide array of signals and regulatory factors. Specifically, these mechanisms must coordinate to accomplish two basic tasks. First, establish stem cell identity and maintain a core population of stem cells aimed at replenishing themselves to sustain continual growth. Second, allow the timely passage of stem cell daughters into lateral regions and promote differentiation into organs. These two aspects are regulated by using a combination of factors raging from plant hormones to mobile transcription factors. 2 During the embryonic development, the transcription factors WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM) expression is initiated in presumptive SAM cells. WUS can be seen during the 16-cell stage (Mayer et at., 1998). This is followed by the STM expression in presumptive SAM cells and in a small cluster of cells near the boundary of cotyledon primordia during the globular-stage (Long et al., 1996). Both transcription factors work to induce SAM identity and repress genes responsible for differentiation such as AYSMMETRIC LEAVES2, YABBY3, KANADI1 and KANADI2 (Lenhard et al., 2002, Yadav et al., 2013). Mutations in both WUS and STM lead to loss of SAM identity possibly due to a premature differentiation and the SAM fails to grow continuously, further cementing

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