Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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Item Investigation of Ethylene Signal Transduction Mechanisms: Characterizing the Novel Gene AWE1 and Testing Hypothesis of Raf-like CTR1's Function In Vivo(2009) Kendrick, Mandy Danielle; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ethylene is a gaseous plant hormone affecting multiple plant processes. Sixteen years ago the first components of the ethylene signaling pathway, the receptor ETR1 and Raf-like kinase CTR1, were identified. Since then many additional components of the pathway have been elucidated through genetic screens. Recent discoveries suggest ethylene signaling, once thought to be a linear pathway from ethylene perception at the endoplasmic reticulum to transcriptional activation at the nucleus, is more complex with multiple auto-feedback loops and potential parallel kinase cascades downstream of the receptors. Although the genetic backbone of the pathway is well established, the signaling mechanisms of the components remain unclear. ETR1 displays histidine kinase activity in vitro and physically interacts with the next-known downstream component of the pathway, CTR1. However the histidine kinase activity of ETR1 is mostly dispensable for signaling to CTR1. How then is CTR1 activated? I proposed that additional proteins, like AWE1, play a role in ETR1 to CTR1 signaling, and that the non-catalytic, amino-terminal region of CTR1 is required both for activation through direct interaction with the ETR1 receptor complex and for auto-inhibition of CTR1 kinase activity. ASSOCIATES-WITH-ETR1 (AWE1) was isolated in a yeast-two-hybrid screen for ETR1-interacting proteins and was of specific interest because the AWE1 clone also interacted with a portion of CTR1. Protein-protein interaction studies and genetic analysis of an awe1 mutant support a role of AWE1 in repressing ethylene responses. However double mutant analysis, over-expression analysis, and protein sub-cellular localization studies suggest that AWE1's function in hypocotyl elongation and cell expansion is more general. AWE1's function may require ETR1 for proper regulation but is likely to lie outside of the direct step from ETR1 to CTR1. To investigate a role of the CTR1 amino-terminal region in CTR1 regulation, I constructed transgenes consisting of truncated ETR1 receptors fused to truncated or full length CTR1 and examined how those transgenes carrying the truncated CTR1 (kinase domain only) affected Arabidopsis seedling growth compared to those transgenes expressing full length CTR1. I concluded that the CTR1 amino-terminal region may have a role in autoregulation, but additional components are required for regulation of CTR1 signaling.Item REVERSION-TO-ETHYLENE-SENSITIVITY1: A Novel Regulator of Ethylene Receptor Function in Arabidopsis thaliana(2006-11-25) Resnick, Josephine; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ethylene is a plant hormone that has profound effects on plant growth and development. Genetic analysis has been central in the elucidation of the ethylene-signaling pathway, made possible through the isolation of ethylene-response mutants in Arabidopsis. This thesis focuses on elucidating the function of the Arabidopsis REVERSION-TO-ETHYLENE-SENSITIVITY1 (RTE1) locus, which was identified in a genetic screen for suppressors of the ethylene-insensitive receptor mutant etr1-2. The RTE1 gene was cloned by positional cloning and found to encode a novel integral membrane protein with homologs in plants and animals, but with no known molecular function. Our studies show that RTE1 is a negative regulator of the ethylene-response pathway, specifically acting as a positive regulator of the ETR1 ethylene receptor. Loss-of-function mutations in the RTE1 gene suppress the etr1-2 ethylene-insensitive phenotype, and genetic analysis suggests that loss of RTE1 results in a largely non-functional ETR1-2 mutant receptor. Similarly, wild-type ETR1 function appears to be greatly reduced in the absence of RTE1. Overexpression of the RTE1 gene confers weak ethylene insensitivity that is largely dependent on ETR1. rte1 mutations do not appear to affect the other four ethylene receptors of Arabidopsis, indicating that RTE1 specifically regulates ETR1. Sequence analysis revealed regions of conserved cysteine and histidine residues, and one rte1 loss-of-function mutant contains a point mutation at Cys161. Since such residues are common in metal binding proteins, we explored the possibility that RTE1 may be involved in facilitating the binding of an essential copper cofactor to the ETR1 receptor. However, experimental evidence suggests that this is not the likely role of RTE1. Interestingly, rte1 was unable to suppress the ethylene insensitive mutant etr1-1, indicating that the differences between etr1-2 and etr1-1 may hold a clue as to how RTE1 regulates ETR1. A suppression analysis of eleven additional etr1 insensitive mutants suggests that RTE1 plays a role in regulating signaling by the transmitter domain of ETR1. A possible role for RTE homologs in non-plant systems is also discussed, although more work is required to elucidate a detailed biochemical model for RTE1 action.