Investigation of two Arabidopsis RING-type E3 ligases, KEG and XBAT32.

Abstract

E3 ubiquitin ligases are a family of proteins that facilitate the covalent attachment of ubiquitin onto target proteins. Protein activity, function, and stability are influenced by ubiquitination and consequently ubiquitination has a major impact on cellular functions and organism physiology. In plant biology, E3 ligases mediate normal growth and development but also facilitate adaptive responses to environmental stress through hormone dependent and independent pathways. Under specific environmental and developmental conditions, E3 ligases mediate the degradation of signalling proteins, transcription factors, biosynthetic enzymes and effector proteins. By using the model plant Arabidopsis thaliana to investigate E3 ligases and their targets, we can better understand how plants control their cellular milieu under normal growth conditions and during stress. Precisely controlled production of the hormone ethylene impacts the growth and development of plants. Knockout lines of the Arabidopsis RING-type E3 ligase, XBAT32, overproduce ethylene which alters lateral root production. In this thesis I present a model whereby XBAT32 regulates ethylene biosynthesis through the ubiquitin-mediated degradation of ethylene biosynthesis proteins. In this work, I show that the stability of ethylene biosynthesis proteins, 1-aminocyclopropane-1-carboxylate synthase (ACS)4 and ACS7, are influenced by XBAT32. I discuss a model wherein XBAT32 ubiquitinates ACS4 and ACS7 to regulate the production of ethylene. Keep on Going (KEG) is a large multidomain E3 ligase that is essential for growth of Arabidopsis seedlings as KEG knockout lines arrest growth after germination. KEG is a regulator of abscisic acid (ABA) signalling and acts by targeting an ABA-responsive transcription factor, ABSCISIC ACID INSENSITIVE 5 (ABI5), for degradation. However, genetic studies suggest that KEG has other ubiquitination targets. In this work, I identified Calcineurin B-like Interacting Protein Kinase (CIPK) 26 as a KEG-interacting protein. Here I provide evidence that CIPK26 is a target of KEG's E3 ligase activity and that CIPK26 acts as part of an ABA signalling cascade. I also provide evidence that the kinase activity of CIPK26 influences its own stability and the stability of KEG. In this thesis, I provide a model wherein KEG and CIPK26 share reciprocal regulation to mediate ABA signalling.