Sumoylation is a post-translational regulatory process in diverse cellular processes in eukaryotes, involving conjugation/deconjugation of small ubiquitin-like modifier (SUMO) proteins to other proteins thus modifying their function. The PIAS [protein inhibitor of activated signal transducers and activators of transcription (STAT)] and SAP (scaffold attachment factor A/B/acinus/PIAS)/MIZ (SIZ) proteins exhibit SUMO E3-ligase activity that facilitates the conjugation of SUMO proteins to target substrates. Here, we report the isolation and molecular characterization of Oryza sativa SIZ1 (OsSIZ1) and SIZ2 (OsSIZ2), rice homologs of Arabidopsis SIZ1. The rice SIZ proteins are localized to the nucleus and showed sumoylation activities in a tobacco system. Our analysis showed increased amounts of SUMO conjugates associated with environmental stresses such as high and low temperature, NaCl and abscisic acid (ABA) in rice plants. The expression of OsSIZ1 and OsSIZ2 in siz1-2 Arabidopsis plants partially complemented the morphological mutant phenotype and enhanced levels of SUMO conjugates under heat shock conditions. In addition, ABAhypersensitivity of siz1-2 seed germination was partially suppressed by OsSIZ1 and OsSIZ2. The results suggest that rice SIZ1 and SIZ2 are able to functionally complement Arabidopsis SIZ1 in the SUMO conjugation pathway. Their effects on the Arabidopsis mutant suggest a function for these genes related to stress responses and stress adaptation.

Reversible conjugation of the small ubiquitin modifier (SUMO) peptide to proteins (SUMOylation) plays important roles in cellular processes in animals and yeasts. However, little is known about plant SUMO targets. To identify SUMO substrates in Arabidopsis and to probe for biological functions of SUMO proteins, we constructed 6xHis-3xFLAG fused AtSUMO1 (HFAtSUMO1) controlled by the CaMV35S promoter for transformation into Arabidopsis Col-0. After heat treatment, an increased sumoylation pattern was detected in the transgenic plants. SUMO1-modified proteins were selected after two-dimensional gel electrophoresis (2-DE) image analysis and identified using matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We identified 27 proteins involved in a variety of processes such as nucleic acid metabolism, signaling, metabolism, and including proteins of unknown functions. Binding and sumoylation patterns were confirmed independently. Surprisingly, MCM3 (At5G46280), a DNA replication licensing factor, only interacted with and became sumoylated by AtSUMO1, but not by SUMO1ΔGG or AtSUMO3. The results suggest specific interactions between sumoylation targets and particular sumoylation enzymes.