Zachary L. Nimchuk, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Plant cell proliferation is restricted to specialized regions called meristems. Stem cell populations within meristems give rise to all tissues of the adult plant. Cells generated in the shoot meristems are eventually incorporated into above ground lateral organs. In order to prevent depletion of shoot meristems, the loss of cells to lateral organs is balanced with the rate of stem cell proliferation. Mutations in the CLAVATA loci disrupt this balance and lead to a hyper-accumulation of stem cells, extra organ production and a reduction in fertility and plant growth. CLAVATA1 (CLV1) is a conserved receptor-like kinase (RLK) defined first in Arabidopsis and proposed to act as a receptor for the secreted peptide CLV3. After 20 years of genetic analysis we still do not know how CLV1 functions at the cellular level. Genetic evidence suggests that CLV1 signaling is regulated by CLV3-mediated internalization (LME). LME is an important mechanism for attenuating RLK signaling in plants and animals. Understanding CLV1 LME at the molecular level would allow us to address where in the meristem CLV1 is active, how CLV1 activation is regulated by other potential signaling partners and how CLV1 might activate downstream pathways that regulate stem cell production. Recent advances in live imaging in the Meyerowitz lab now allow this to be dissected. This proposal aims to understand CLV1 LME at the cellular level in Arabidopsis. In specific, an active CLV1 YFP fusion protein will be used to address where CLV1 is localized in the cell and how localization is regulated to CLV3 perception. This will be examined by using clv3 mutants, exogenous CLV3 peptides and known inhibitors of LME. The requirement for different domains of CLV1 in LME will be addressed using mutant versions of CLV1 -YFP . In addition, a system for creating conditionally inhibitable kinase versions of CLV1 will be detailed which will allow us to test if CLV3 activates CLV1 directly and will provide a tool to explore the effects of CLV1 inhibition in real time in live tissue. Lastly, I will detail a genetic strategy to identify mutants in key regulators of CLV1 LME which have so far proved elusive. This research will greatly expand our knowledge of RLK activation in plants and will serve as a model for studying signaling by other CLV1 or CLV3 family members. The work will provide our first knowledge of how CLV1 functions at the cellular level, a key roadblock for understanding how stem cell pathways are regulated in plants. This work will enlighten our knowledge of how this kingdom differs in stem cell biology from humans and other animals. This knowledge may lead to novel approaches for studying stem cell biology in general and may lead to novel approaches to treating disease in humans. [unreadable] [unreadable] [unreadable]

All plant biomass on the planet is derived ultimately from the production of stem cells, populations of self-renewing cells that drive plant development in every tissue. The maintenance of plant stem cell populations depends on communication pathways between the stem cells and neighboring accessory cells. Determining how these communication pathways function is critical to understanding how plant stem cell function has been altered during domestication and evolution, and how it might be harnessed to drive further crop improvement. This project aims to understand the signaling pathways that control communication between stem cells and neighboring cells. It uses novel genetic systems and biochemical approaches to look at key regulators of receptors that control stem cell production in order to elucidate how they control receptor signaling. This project is also identifying critical signals that are involved after receptors in stem cell control.

Plant domestication and evolution are associated with changes in stem cell proliferation. Despite the importance of plant stem cells, there is little understanding of the signaling pathways that control plant stem cell proliferation. This research is helping fill that gap by identifying signaling components and target genes that regulate plant stem cell production in the model plant Arabidopsis thaliana. It is creating genome-wide datasets and identifying new loci that will help dissect how stem cell pathways have shaped plant form and function. Altering plant development, through the manipulation of stem cell activity, has the promise to boost crop and biofuel production. This research focuses directly on this target, by identifying key regulators of plant stem cell production that can ultimately be deployed to enhance crop growth and yield. In addition, the project is advancing scientific outreach by creating a high school learning program to educate students on the coming global food crisis, and about how scientists are using biotechnology and plant development to create solutions to this challenge. This project will also train a graduate student and multiple undergraduate students.

Project Summary The long term goal of the proposed research is to decipher how cell to cell signaling networks control stem cell maintenance. Because stem cell activity is critical to human development, and a current goal of the field is to harness the properties of stem cells for disease treatment, an understanding of the molecular networks controlling stem cell function is critical for diverse public health challenges. We have chosen a highly tractable model plant system, Arabidopsis thaliana, to take a multi-level approach to dissect the receptor kinase pathways that control shoot stem cell proliferation. Arabidopsis is easy to transform, mutagenize, and there is a wealth of mutants and transgenic lines that affect stem cell function, all acquired during decades of study. In addition, the stem cell niche in the shoot is easily visualized, allowing living stem cells to be imaged at cellular and sub-cellular levels. The proposal will support a series of projects that collectively aim to comprehensively understand the function of receptor signaling in stem cell regulation. The projects will identify the transcriptional regulators that act downstream of receptor activation, tie their function to signaling cascades, and identify the suite of transcriptional outputs they regulate. The proposal will also define the mechanism of receptor activation and complex formation at the plasma membrane and elucidate the signaling components that act as intermediates through a combination of novel genetic and biochemistry approaches. The project will assess how stem cell network components have diversified and been co-opted into new developmental modules outside of the shoot stem cell niche. Lastly, the project will take a broad evolutionary approach to assess how stem cell networks have been altered by evolution and domestication to shape form and function. The project will last five years, but will impact the direction of the lab beyond the scope of the proposed research. The projects will train scientists at the post-doctoral, graduate and undergraduate levels. This work will benefit from collaboration with several expert groups using different model systems with the aim of amplifying and diversifying data sets, and exchanging skill sets. Ultimately, the projects aim to define network architecture and specificity at a level that allows the creation of synthetic pathways with the potential to alter plant growth and be deployed in heterologous systems, potentially including therapeutic applications.

Project Summary The long term goal of the proposed research is to decipher how cell to cell signaling networks control stem cell maintenance. Because stem cell activity is critical to human development, and a current goal of the field is to harness the properties of stem cells for disease treatment, an understanding of the molecular networks controlling stem cell function is critical for diverse public health challenges. We have chosen a highly tractable model plant system, Arabidopsis thaliana, to take a multi-level approach to dissect the receptor kinase pathways that control shoot stem cell proliferation. Arabidopsis is easy to transform, mutagenize, and there is a wealth of mutants and transgenic lines that affect stem cell function, all acquired during decades of study. In addition, the stem cell niche in the shoot is easily visualized, allowing living stem cells to be imaged at cellular and sub-cellular levels. The proposal will support a series of projects that collectively aim to comprehensively understand the function of receptor signaling in stem cell regulation. The projects will identify the transcriptional regulators that act downstream of receptor activation, tie their function to signaling cascades, and identify the suite of transcriptional outputs they regulate. The proposal will also define the mechanism of receptor activation and complex formation at the plasma membrane and elucidate the signaling components that act as intermediates through a combination of novel genetic and biochemistry approaches. The project will assess how stem cell network components have diversified and been co-opted into new developmental modules outside of the shoot stem cell niche. Lastly, the project will take a broad evolutionary approach to assess how stem cell networks have been altered by evolution and domestication to shape form and function. The project will last five years, but will impact the direction of the lab beyond the scope of the proposed research. The projects will train scientists at the post-doctoral, graduate and undergraduate levels. This work will benefit from collaboration with several expert groups using different model systems with the aim of amplifying and diversifying data sets, and exchanging skill sets. Ultimately, the projects aim to define network architecture and specificity at a level that allows the creation of synthetic pathways with the potential to alter plant growth and be deployed in heterologous systems, potentially including therapeutic applications.

Plant development is driven in part by chemical communication among cells that signal and co-ordinate growth. Some of these signals are small molecules, called peptides, that are perceived by other "receptor" molecules in the plant cell. A cascade of developmental information is transmitted when peptides find their receptors, similar to the opening of a door by a key in a lock. Surprisingly, scientists discovered that receptors are also used by plants to detect signals from invading pathogens. Upon detection, plants can then ward off further pathogen attack by activating an arsenal of molecular defenses. How these related and similar receptors function in normal plant development and also plant defense is puzzling and remains an important unanswered question in plant biology. This research tackles the problem by identifying the molecular basis of peptide recognition and by deciphering how receptors control both development and defense. Specifically, the research addresses the question of how the same receptor family can distinguish between very different peptide signals to activate different cell signaling pathways. The research brings together a diverse and international group of scientists with complementary technical skills to address these questions. Challenges to plant health from pathogens remain a pressing problem facing national food security, as diseases spread widely across the globe. The outcomes of this research provide foundational information that can be applied to improving disease resistance in plants. In the process, students and post-doctoral researchers are trained in cutting edge interdisciplinary science in the area of basic plant development and practical plant health.

Peptide perception in plant pathogen interactions and development are mediated primary receptors belonging to the leucine-rich repeat transmembrane kinase superfamily. Individual primary receptors mediate specific peptide binding, but it is becoming clear that diverse receptors can use specific and often shared co-receptors for peptide recognition and downstream signaling. The molecular basis of specificity in both recognition and downstream signaling is unclear. This research studies the structural basis of how related receptors use co-receptors to recognize specific peptide ligands. In addition, the project aims to use phosphoproteomics and quantitative biochemistry to determine how specific ligand perception is transduced into specific downstream signaling outputs. The project will also decipher the function of a clade of receptor interactors in the control of both development and defense receptor signaling using a combination of biochemical and genetic methods. It is envisioned that this work will provide a molecular framework for understanding how specificity is encoded at the molecular level in receptor kinase signaling, setting the stage for engineering these pathways in crops in the future as one of the major broader impacts of the reseach. The program also ensures a productive future by training the next generation of scientists in inter-disciplinary and international research.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.