The covalent attachment of the Small Ubiquitin-like Modifier (SUMO) to other proteins is an essential post-translational modification required in all eukaryotes. This conjugation helps regulate development, cellular homeostasis, and responses to stress through the modification of transcriptional and translation regulators and chromatin modifiers. Particularly influential is the rapid SUMOylation of various nuclear proteins during stress presumably to alter nuclear activities needed for protection. In Arabidopsis thaliana, stress-induced SUMOylation has been connected to hundreds of proteins but the underpinning reasons remain unclear. I investigated three separate aspects of the Arabidopsis SUMO conjugation pathway to further elucidate the roles of SUMOylation in plants. I conducted a phylogenetic study of plant SUMOs and found two types that are universal; a canonical, highly conserved form and a non-canonical form that appears to be lineage specific. These non-canonical SUMO shares little sequence identity among members, implying a role in plant biology that is independent of homology. Plants also have the capacity to assemble concatemers of SUMOs linked internally through lysine-mediated isopeptide linkages. I examined the role(s) of polySUMO chains by studying plants expressing a lysine-null SUMO blocked in chain assembly. Interestingly, the failure to generate SUMO chains has no discernible growth defect under normal and stress conditions, implying that these polymers are not essential. However, the use of lysine-null SUMOs should now facilitate mapping SUMOylation sites by coupling methods to enrich for target peptides bearing a SUMO moiety with identified mass spectrometric (MS) approaches to detect SUMO footprints. Third, I developed a MS method to connect individual SUMO ligases with specific targets, in which the profile of conjugates affinity-purified from wild-type and ligase mutant plants are compared by MS. Using this technique, I identified over a hundred proteins SUMOylated by the ligase SIZ1. These SIZ1-dependent substrates include major transcriptional regulators and chromatin modifiers associated with abiotic and biotic stress, thus connecting the role of SIZ1 in stress protection with a suite of affected processes. In summary, the work completed in this thesis provided insight into specific functions of SUMOylation and methods that will ultimately aid in the study of specific SUMO-dependent functions.