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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, ABA-hypersensitivity 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.
To determine whether OsSIZ1 and OsSIZ2 show tissue-specific expression, we analyzed their expression levels in various rice tissues. As shown in Fig. 2, transcripts of OsSIZ1 and OsSIZ2 were detected in all tissues of rice, but to a lesser extent in root. Next, we investigated whether the expression of OsSIZ genes could be induced in response to abiotic, biotic, and hormone stimuli. However, none of these treatments induced transcription of the OsSIZ genes
To identify the subcellular localization of OsSIZ1 and OsSIZ2 in rice, we used GFP and RFP reporter gene fusion constructs in rice protoplasts. The GFP sequence was fused to the C-termini of the OsSIZ1 and OsSIZ2 cDNAs under the control of the CaMV35S promoter (Fig. 3a). Each chimeric OsSIZ1/-2::GFP construct was introduced into rice protoplasts along with an nuclear localization signal (NLS)::RFP construct that represented an authentic nuclear localization marker (Heikal et al. 2000). To further test and monitor potential monocot/dicot differences in localization masked by the heterologous system used, we expressed the GFP fusion constructs in tobacco epidermis cells and in Arabidopsis protoplasts (data not shown). Taken together, the subcellular distributions of each OsSIZ1/-2::GFP signal clearly overlapped with those of the NLS::RFP red fluorescence signals, indicating that OsSIZ1/2 are localized to the nucleus (Fig. 4b).
Similar to most of the characterized and annotated SIZ/PIAS (SAP and MIZ/Protein Inhibitor of Activated STAT) SUMO E3 ligases from all organisms, rice SIZ1 also contains SAP, PINIT, SP-RING, a SUMO binding motif (hhhSXSaaa), a C-terminal nuclear localization signal (NLS) and the plant-specific PHD domain (plant homeodomain with C4HC3-type Zn-finger) (Fig. 3a,b). PINIT (Pro-Ile-Asn-Ile-Thr) and SP-RING, containing a zinc-finger (C2HC3), are essential for SUMO E3 ligase activity, and SAP (scaffold attachment factors SAF-A/B, Acinus, PIAS, a helix–extended loop–helix) forms a helix–extended loop–helix structure probably involved in DNA binding (Aravind & Koonin, 2000). Amino acid comparison revealed that all plant SIZs possess all consensus domains (Fig. 3b). A BlastP search analysis with SIZ1 amino acid sequences used as a query was employed to generate a rooted phylogenetic tree to illustrate the relationship of rice SIZs to their plant orthologs. As shown in Fig. 3c, rice SIZ1 and SIZ2 are positioned in different clades, and AtSIZ1 is closer to rice SIZ1 than to SIZ2. Sequence similarity analysis also confirmed that rice SIZ1 is closely related to AtSIZ1 (Fig. 3d). The amino acid sequences of SIZ1 and SIZ2 showed high similarity to sorghum loci Sb09g002225 and Sb08g000380, with 65.0% and 57.1% identity, respectively (data not shown). This is probably because both rice and sorghum originate from the grass family and have high synteny for sharing similar panicle architecture. You can also add sub-section(s) at will.
Labs working on this gene
1 Division of Applied Life Science (BK21 program), Plant Molecular Biology and Biotechnology Research Center and Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju, 660-701, Korea
2 College of Natural Resources and Life Science, Dong-A University, Busan, 604-714, Korea
3 Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, 47907-2054, USA
4 King Abdullah University of Science and Technology, Thuwal, 23955-6900, Saudi Arabia
5 Departments of Plant Biology and of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
ref 1 PARK, H. C., KIM, H., KOO, S. C., PARK, H. J., CHEONG, M. S., HONG, H., BAEK, D., CHUNG, W. S., KIM, D. H., BRESSAN, R. A., LEE, S. Y., BOHNERT, H. J. and YUN, D.-J. (2010), Functional characterization of the SIZ/PIAS-type SUMO E3 ligases, OsSIZ1 and OsSIZ2 in rice. Plant, Cell & Environment, 33: 1923–1934. doi: 10.1111/j.1365-3040.2010.02195.x