Proper stem cell maintenance and organ formation in the Arabidopsis shoot meristem requires the activity of the CLAVATA loci (CLV1, CLV2, CLV3). Plants homozygous for mutations in any of the CLV loci accumulate massive numbers of stem cells as an apparent consequence of defects in organ formation. The CLV loci encode signal transduction components - CLV1 encodes a receptor kinase, CLV2 a receptor-like protein, and CLV3 a ligand. Our previous work has characterized inactive and active CLV1 protein complexes in vivo, and many of the components of these complexes, including a protein phosphatase and a Rho-related protein. We seek to determine the mechanisms of receptor activation, how signal is relayed across the plasma membrane, and how the cytoplasmic components interact with each other to produce a signal that modifies gene expression and cell fate. This understanding will not only be central to understanding meristem development, stem cell maintenance, and organ formation, but may also provide one paradigm for receptor-mediated signaling in plants. In addition, components and features of signaling common between plants and animals will also be elucidated, perhaps illuminating features of animal signaling as yet unidentified. This research proposal is designed to get us closer to these goals through genetic and biochemical studies of key aspects and components of CLV1 signaling. Phosphorylation sties in CLV1 will reveal how the kinase domain becomes activated, where effectors bind, and what role each effector plays in vivo. Inducible activation of CLV1 signaling may allow us to dissect the early activation events, as well as monitor the activation of downstream kinases as well as regulation of WUS. POL will provide us with a critical downstream position from which to identify the cytoplasmic and nuclear portion of the signal transduction cascade, with preliminary indications of a significant role for MAPKs. Finally, determining the role and interactions of the redundant receptor-kinases BAM1 and BAM2 may be central to getting at the mechanism of CLV1 activation.
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