ARRA: Molecular Genetic Analysis of a Novel Feedback Inhibition Mechanism in the Cytokinin Response Pathway

Cytokinins (CKs) are plant hormones that regulate cell division, elongation and differentiation, and are therefore essential for every aspect of plant growth and development (Mok, 1994; Shani et aL, 2006; Kyozuka, 2007; Delio loio et aL, 2008). For example, CKs control the development of meristems and vasculature, and play an important role in senescence and nutrient allocation (Mok, 1994; Gan and Amasino, 1995; Matsumoto-Kitano et aL, 2008; Nieminen et aL, 2008). Due to the importance of CKs for the plant life cycle, the CK signal transduction cascade has been a focus of intense studies (Kakimoto, 2003; MOiler and Sheen, 2007; To and Kieber, 2008). These studies have addressed two critical questions: (1) what are the essential components of the signaling pathway and (2) what are the control mechanisms that allow the CK signal to initiate a robust and tightly regulated switch in gene expression. Similar to other signal transduction cascades, CK signaling includes negative feedback control mechanisms that reduce or suppress the effects of the input signal thus limiting the intensity and duration of the response. Currently described negative feedback controls depend on the CK-induced accumulation of response inhibitors (Hwang and Sheen, 2001; To et aI., 2007). However, one of the most effective ways to attenuate a response is to degrade the response activators (Behar et aL, 2007). Our preliminary data show that a positive regulator of CK responses, the type-B response regulator ARR1, is a target for CK-induced and ubiquitin/proteasome system (UPS)-dependent proteolysis. This suggests that plant cells employ a proteolysis-based desensitization mechanism to ensure fast adaptability to changes in CK signal intensity. The long-term goals of our research are to understand this negative feedback mechanism at the molecular level, and to determine if it also controls the activities of other type-B ARR family members. To achieve these goals, we propose the following: Aim 1 - Define the ARR1 destabilization domain: The region of ARR1 that is required for CK-induced degradation will be determined by testing the degradation rates of a series of C- and N-terminally truncated ARR1 proteins. Aim 2 - Identify and characterize the Ub ligase(s) that target ARR1 for degradation: UPS substrates need to be polyubiquitinated before they can be degraded by the 26S proteasome. The main specificity determinants of the polyubiquitination reaction are Ub ligases or E3s. To identify the E3(s) that target ARR1, we propose (1) to study E3 enzymes encoded by CK-inducible genes and (2) to perform a screen for ARR1-binding proteins. Aim 3 - Test if other type-B ARR family members are CK-dependent UPS targets: ARR1 belongs to a family of response activators whose members have similar - if not identical - functions. We will test if the protein stability of other type-B ARR family members is regulated in a CK- and UPS-dependent manner.