Gibberellins (GAs) are phytohormones that regulate many aspects of plant growth and development. The long-term goal of this research is to understand the molecular basis of GA signal transduction in arabidopsis. Loss-of-function spy mutations suppress the phenotypes caused by GA-deficiency; thus, the SPY protein is a suppressor of GA signaling. SPY has extensive sequence similarity to animal O-GlcNAc transferase, which catalyzes the addition of a single O-linked N-acetylglucosamine to specific serine and/or threonine residues of nuclear and cytosolic proteins. O-GlcNAc modification of cytoplasmic and nuclear proteins is about as common as phosphorylation and is believed to be a regulatory modification. The work proposed here will test the model that SPY-catalyzed protein O-GlcNAcylation acts to negatively regulate GA signaling and begin to determine the details of how SPY functions in signaling pathway(s). SPY produced in insect cells is itself O-GlcNAc-modified and has GlcNAc transferase activity toward gp40, a plant nuclear pore protein that is known to undergo terminal O-GlcNAc modification. SPY is a component of a multiprotein complex present in both the nucleus and cytosol of arabidopsis and cauliflower. When GA-deficient plants are treated with GA, a decrease in the GlcNAc modification of several proteins is detectable 1 hr after treatment. spy mutations have similar effects. These data indicate that protein GlcNAcylation plays a role in GA signaling. A number of arabidopsis proteins that putatively interact with SPY's tetratricopeptide repeats in yeast have been identified. One of these proteins is GIGANTEA (GI), a protein involved in a number of processes that are also affected by GA. Consistent with the physical interaction between SPY and GI, with respect to the control of flowering time, spy is weakly epistatic to gi. The goals of this work are to: 1) characterize the protein modification that is produced by SPY and determine the identity of its substrates; 2) determine if the localization or activity of SPY is regulated by determining if changes in GA levels or mutations affecting GA signaling alter the localization, posttranslational modification, protein associations, or activity of SPY; and 3) perform additional tests on the proteins identified in the two-hybrid screens to identify the proteins that physically interact with SPY and then genetically characterize the function of the interacting proteins. As part of the third goal, genetic and physiological studies will be performed to further define the functional interrelationships between SPY and GI.