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UC Riverside UC Riverside Electronic Theses and Dissertations Title The Effects of Fruit Load, Cytokinin, WUSCHEL and CLAVATA3 on Meristem Maintenance Permalink https://escholarship.org/uc/item/3c13699m Author DeVries, Aaron Edward Publication Date 2015 Supplemental Material https://escholarship.org/uc/item/3c13699m#supplemental Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA RIVERSIDE The Genetics of Plant Meristem Development in Response to Alternate Bearing, WUSCHEL, and Cytokinin A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Plant Biology by Aaron Edward DeVries December 2015 Dissertation Committee: Dr. G. Venugopala Reddy, Chairperson Dr. Patricia S. Springer Dr. Morris M. Maduro Copyright by Aaron Edward DeVries 2015 The Dissertation of Aaron Edward DeVries is approved: Committee Chairperson University of California, Riverside Acknowledgments The work presented in this document would not have been possible without the contributions and support from many others. First is for Dr. Harley M.S. Smith who readily accepted me into his lab and introduced me to the interesting world of field research. Much of the work in his lab also would not have been possible without the support of the Hofshi foundation, which generously donated trees for our experiments, and Mary Lou Arpaia, whose vast technical expertise helped precipitate many the ideas discussed in chapter 2. I am further indebted to Dr. G. Venu Reddy, who was kind enough to accept me into his lab when I had nowhere else to go, and who taught me the art of scientific pointillism. Special thanks go to Dr. Sean Cutler, who adjusted his schedule upon short notice to participate in my oral exam committee, along with Dr. Linda Walling, Dr. Morris Maduro, and Dr. Julia Bailey-Serres. I must also thank the National Science Foundation for providing my IGERT funding, which considerably eased my financial burden for several years. I also owe my experiences in the Chemical Genomics program to Dr. Bailey-Serres (again), and thank Matthew Collins and Ronly Schlenk for their support while completing my IGERT requirements. Within the department of Botany, I’d like to thank Diedra Kornfeld for helping me through registration issues in my first year, in addition to Dr. Edith Allen and the Department chair Mikeal Roose for arranging supplemental funding for my final year. Finally, there is Dr. Norman Ellstrand, who very patiently helped guide me through my first teaching experience. Among my lab mates, I owe thanks to Dr. Kaori Miyawaki for introducing me to my future work with cytokinin signaling, and whom also provided the ahk2/3/4 triple mutant data in shown Figure 22. Likewise Mariano Perales, Steven Snipes, Kevin Rodriguez, and Ricardo Muir, who generously donated their seeds and provided many stimulating conversations. I am also indebted to Shruti Lal for providing her microarray data, which made the completion of chapter 1 possible. Though iv I have not had the pleasure of meeting them personally, I must also thank Dr. Bruno Mueller in the Harvard Medical School for providing the pTCSn1:mGFP5-ER seed line. Finally, I am also indebted to Dr. Patricia Springer and Dr. Morris Maduro for serving on my dissertation committee. v ABSTRACT OF THE DISSERTATION The Genetics of Plant Meristem Development in Response to Alternate Bearing, WUSCHEL, and Cytokinin by Aaron Edward DeVries Doctor of Philosophy, Graduate Program in Plant Biology University of California, Riverside, December 2015 Dr. Gonehal Venugopala Reddy, Chairperson The ability of plants to produce new leaves and flowers depends largely on a small population of stem cells found at the apex of the stem, which is known as the Shoot Apical Meristem (SAM). The activity of the SAM can be influenced by the development of immature fruit, often causing a biennial pattern in yield called Alternate Bearing (AB). In order to understand how the SAM responds to the fruit at a genetic level, the expression profile of inflorescence meristems from Arabidopsis thaliana were studied under high fruit load conditions. The pattern of responses was found to resemble carbohydrate starvation, supporting the competition hypothesis. Several additional discussions are provided to revise old concepts, offer suggestions for improvement, and describe how genetic tools could be best applied to alternate bearing research. On a more basic level, the SAM is maintained by the WUSCHEL-CLAVATA feedback loop, where WUS is thought to directly activate CLV3 expression. The nature of the CLV3 regulome was studied by combining phylogenetic footprinting with large promoter deletions. This found that the 5’ CLV3 promoter is less than 70bp long, and is likely to be regulated in part by an auxin vi response element. In addition, three large cis-regulatory modules were found in the 3’ enhancer, two of which were found in a naturally occurring transposon. The role of transposition in the evolution of the WUS-CLV3 feedback loop is discussed. Finally, the role of cytokinin hormones on WUS regulation was investigated by adjusting native cytokinin levels with receptor mutants and genetic constructs, in order to observe changes in WUS, CLV3 and cytokinin fluorescent reporters. The results found that cytokinin had little or no effect on the transcription, nuclear transport, or protein degradation of either gene, while auxin responses rapidly reduced WUS protein levels. Surprisingly, the complete absence of cytokinin responses in the central zone was found to be critical for meristem maintenance, and the response-free zone existed independently of WUS-CLV3 feedback loop. vii Table of contents: Preface. .xiv Section 1 Introduction to Alternate bearing What is alternate bearing? . 1 The basic anatomical model. 4 Related phenomena: Fruit drop. .8 The competition hypothesis. 10 The inhibitor hypothesis. .11 The fruit dominance hypothesis. 14 Chapter 1 High fruit load and the response of the inflorescence meristem Introduction. 16 Microarray results. 17 Characterization of meristem activities. .25 Discussion. 31 Materials and Methods. .39 Works Cited . .45 Section 2 Introduction to the shoot apical meristem (for chapters 2 and 3) SAM anatomy. .63 Hormone regulation . .67 The WUS-CLV3 feedback loop. .68 viii Chapter 2 CLV3 promoter characterization Introduction. 71 Results: . 72 Predicted cis-motifs. .72 Phylogenetic footprinting. .76 CLV3 promoter deletions . .85 Discussion. 87 Methods . .97 Chapter 3 Hormones and their influence on WUS distribution patterns Introduction. 109 Results: . 111 Cytokinin-response-free zone. 111 WUS transcription is independent of Cytokinin. 119 Cytokinin affect WUS protein distribution . .106 Cytokinin do not affect WUS nuclear localization. 121 Cytokinin and WUS protein stability. .124 Auxin and WUS degradation . 125 Discussion. 127 Materials and methods. 136 Section 2 Works cited. .140 ix Figures Section 1 overview Figure 1.0 Alternate bearing branch structure . .9 Chapter 1 Figure 1.1 Rate of Immature flower buds. 26 Figure 1.2 Meristem activity, in buds/day. 27 Figure 1.3 Rosette leaf initiation rate. 27 Figure 1.4 Senescent SAM phenotype. .28 Figure 1.5 Leaf tip removal and IM growth rates. 29 Figure 1.6 De-fruited plants and IM growth rates. 30 Figure 1.7 Avocado gene expression in response to fruit load . .31 Section 2 overview Figure 2.0 SAM structure. .61 Chapter 2 Figure 2.1 CLV3 expression pattern . .73 Figure 2.2 MEME identified motifs. .75 Figure 2.3 CLV3 protein alignment. .77 Figure 2.4 Phylogenetic footprinting. .80 Figure 2.5 AtCLV3 consensus of conserved areas, with cis-motifs. 86 Figure 2.6 Deletion analysis of the AtCLV3 regulatory regions. .88 Figure 2.7 Map of pCLV3m:H2B-YFP used for deletions. 108 Chapter 3 Figure 3.1 Location of cytokinin responses in WUS/CLV3 mutants. 113 Figure 3.2 Time course of ectopic cytokinin induction. ..