Investigation of Ethylene Signal Transduction Mechanisms: Characterizing the Novel Gene AWE1 and Testing Hypothesis of Raf-like CTR1's Function In Vivo

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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.