Os10g0445400
OsHCI1 encodes a RING finger E3 ligases, which is specifically induced by heat and cold stress. OsHCI1 can drive nuclear export of multiple protein substrate, and the heterologous overexpression of Arabidopsis can enhance acquired-thermotolerance.
Contents
Annotated Information
Function
OsHCI1 (Oryza sativa Heat and Cold Induced 1) encodes a 246-amino acid protein with a predicted molecular mass of 28.8kDa and harbours a single RING-HC domain in its C-terminal region. It is a rice RING domain E3 ligase, and is highly induced under heat and cold stress conditions, which can lead to adverse outcomes in plant cell functions, including alterations in cellular composition of membrane fluidity and permeability, enzyme activity, metabolism, production of active oxygen species, and gene expression[1][2][3][4][5].
OsHCI1 dynamically moves from the cytoplasm to the nucleus along cytoskeletal tracts under heat shock conditions. OsHCI1 interacts with six substrate proteins and mediates subcellular trafficking of nuclear proteins to the cytoplasm via monoubiquitination.
Arabidopsis overexpressing OsHCI1-EYFP exhibits a heat-tolerant phenotype, suggesting an important role of this protein in the regulation of heat-generated signals in plants.
Expression
The finding that OsHCI1 gene expression patterns are specifically and somewhat rapidly increased by heat and cold stresses but not by salt and drought stresses indicates that the gene is associated closely with thermal stress in rice. And OsHCI1 rapidly responds to hormone treatments,too. (Fig.1)
Fig.1. Expression levels of OsHCl1 in rice plants subjected to four abiotic stresses and four hormonal treatments. Rice seedlings were subjected to abiotic stresses and plant hormone treatments. (A), heat (45 °C), cold (4 °C), NaCl (250mM), and dehydration (dehydration on two pieces of tissues paper); OsHsp90-1, LIP19, OsSalT, and OsbZIP23 were used as reliable stress-inducible genes for each abiotic stress treatment, respectively. (B) 0.1mM abscisic acid (ABA), 0.1mM jasmonic acid (JA), 1mM salicylic acid (SA), 50 μl l–1 ethylene (Ethyl); OsSalT, OsPBZ1, OsPR1b, and OsERF3, were used as reliable genes for each hormonal treatment, respectively. C and T indicate untreated control and stress-treated samples, respectively. The experiments were performed with three biological replicates.
A Y2H screen was performed to identify 6 proteins that interact with OsHCI1. To confirm these positive interactions with OsHCI1, full-length coding sequences of the top six genes, which exhibited strong α-galactosidase activity, were cloned into GAL4 activation domain, respectively. Full-length OsHCI1 and each interacting protein were co-transformed into the Y2H Gold strain and grown on QDO/X/A medium(Fig.2).
Fig2.Identification of OsHCI1 interaction with six proteins. The full-length OsHCI1 was cloned into pGBKT7, and full-length OsPSA7 (Os01g59600, 20S proteasome subunitα7), OsPGLU1 (Os03g53800, periplasmic beta-glucosidase), OsbHLH065, (Os04g41570, basic/Helix-Loop-Helix transcription factor), OsGRP1 (Os05g02770, glycine-rich cell wall structural protein), OsPOX1 (Os07g48020, peroxidase), and Os14-3-3 (Os11g34450, 14-3-3 protein) were cloned into pGADT7. The combination of indicated constructs was co-transformed into the Y2H Gold yeast strain. Yeast cells were dropped onto DDO and QDO/X/A medium, and grown for 5 d separately to test protein-protein interactions. Supplemented BD-murine p53 with AD-SV 40 large T- antigen were used as positive controls (PC). BD-lamin and AD-SV 40 large T-antigen combinations were used as negative controls (NC).
This study also examined the expression patterns of the interacting partner genes with OsHCI1 under two different heat stresses via semi-quantitative RT-PCR with rice seedlings treated by basal or acquired heat shock treatments (Fig.3).These results suggest that heat shock results in high expression of the OsHCI1 transcript or protein, which can affect the transcript levels of its interacting genes.
Fig3. Expression patterns of the response of interacting protein genes with OsHCI1 under heat treatment. Two-week-old rice seedlings were exposed to basal (A) or acquired heat stress (B) and then placed to normal temperature for 2h. Each leaf sample was harvested at different time points.
Localization
BiFC technology was employed to visualize the interactions between OsHCI1 and each of the interaction partners in living cells [6]. Full-length coding sequences of OsHCI1 and each of the six interacting protein genes were cloned into the 35S-HA-SPYCE(M) and 35S-c-myc-SPYNE(R)173 vectors, respectively. All of the YFP signals except that of OsPSA7 appeared to associate with the cytoplasm and nucleus; however, the OsPGLU1-, OsbHLH065-, and OsGRP1-DsRed2 alone protein signals were detected only in the nucleus (Fig. 4B–D). In contrast, the OsHCI1 BiFC complex with OsPSA7 was localized to the cytoplasm with a punctuate complex (Fig. 5).
Fig. 4. Subcellular localization of six interacting proteins. The full-length OsPSA7 (A), OsPGLU1 (B), OsbHLH065 (C), OsGRP1 (D), OsPOX1 (E), Os14-3-3 (F), and empty EYFP (G) were tagged with DsRed2 and transiently expressed with p19 in Nicotiana leaves. Images were captured and merged by z-series optical sections after 5 days of agro-infiltration.
Fig.5. BiFC assay for six substrate proteins confirms the interaction with OsHCI1 in living cells. Full-length OsPSA7 (A), OsPGLU1 (B), OsbHLH065 (C), OsGRP1 (D), OsPOX1 (E), and Os14-3-3 (F) were cloned into pSPYNE(R) and OsHCI1 was cloned into pSPYCE(M). Combinations of each construct and SPYNE(R):empty (G, negative control) with OsHCI1:SPYCE(M) were transiently expressed with p19 in Nicotiana leaves. Images were captured and merged by z-series optical sections after 5 days of agro-infiltration.
Wild type vs. Mutant
Several independent Arabidopsis transgenic lines (T3) were developed with strong OsHCI1 gene expression and compared to plants without the gene (35S:EYFP), which served as controls. The OsHCI1-overexpressing lines showed strikingly high survival rates; however, most control plants did not recover (Fig. 6).
Fig. 6. Thermotolerance phenotype of 35S:OsHCI1-EYFP. Seven-day-old seedlings of 35S:EYFP (empty vector, EV) and 35S:OsHCI1-EYFP T3 transgenic plants (three independent lines) were grown on agar in the light for 7d and heated to 38 °C for 90min, cooled 24 °C for 2h, then heated to 45 °C for 3h (acquired thermotolerance) or heated to 45 °C for 60min (basal thermotolerance). A, RT-PCR analysis of seven independent Col-0/35S:OsHCI1 T3 transgenic plants, control wild type (WT), and EV. (B) The phenotypes of EV and three independent OsHCI1-overexpressed plants were treated at various high temperatures. Images were captured 5 days after heat shock treatment. (C) Percentage of surviving plants relative to the control (EV) on the same plate was determined 5 days after heat shock. Bars indicate standard deviation from the mean over all experiments (n = 30). The experiments were performed with four biological replicates.
Labs working on this gene
1.Department of Applied Plant Sciences, Kangwon National University, Chuncheon 200-713, Korea.
References
- ↑ Kampinga HH, Brunsting JF, Stege GJ, Burgman PW, Konings AW. 1995. Thermal protein denaturation and protein aggregation in cells made thermotolerant by various chemicals: role of heat shock proteins. Experimental Cell Research 219, 536–546.
- ↑ Alfonso M, Yruela I, Almarcegui S, Torrado E, Perez MA, Picorel R. 2001. Unusual tolerance to high temperatures in a new herbicide-resistant D1 mutant from Glycine max (L.) Merr. cell cultures deficient in fatty acid desaturation. Planta 212, 573–582.
- ↑ Larkindale J, Knight MR. 2002. Protection against heat stress-induced oxidative damage in Arabidopsis involves calcium, abscisic acid, ethylene, and salicylic acid. Plant Physiology 128, 682–695.
- ↑ Larkindale J, Huang B. 2004. Thermotolerance and antioxidant systems in Agrostis stolonifera: involvement of salicylic acid, abscisic acid, calcium, hydrogen peroxide, and ethylene. Journal of Plant Physiology 161, 405–413.
- ↑ Larkindale J, Hall JD, Knight MR, Vierling E. 2005. Heat stress phenotypes of Arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerance. Plant Physiology 138, 882–897.
- ↑ Waadt R, Kudla J . 2008. In planta visualization of protein interactions using bimolecular fluorescence complementation (BiFC). CSH Protocols 2008, pdb prot4995.
