Molecular dissection of a rice microtubule-associated RING finger protein and its potential role in salt tolerance in Arabidopsis

Abstract

Although a number of RING E3 ligases in plants have been demonstrated to play key roles in a wide range of abiotic stresses, relatively few studies have detailed how RING E3 ligases exert their cellular actions. We describe Oryza sativa RING finger protein with microtubule-targeting domain 1 (OsRMT1), a functional RING E3 ligase that is likely involved in a salt tolerance mechanism. Functional characterization revealed that OsRMT1 undergoes homodimer formation and subsequently autoubiquitination-mediated protein degradation under normal conditions. By contrast, OsRMT1 is predominantly found in the nucleus and microtubules and its degradation is inhibited under salt stress. Domain dissection of OsRMT1 indicates that the N-terminal domain is required for microtubule targeting. Bimolecular fluorescence complementation analysis and degradation assay revealed that OsRMT1-interacted proteins localized in various organelles were degraded via the ubiquitin (Ub)/26S proteasome-dependent pathway. Interestingly, when OsRMT1 and its target proteins were co-expressed in N. benthamiana leaves, the protein-protein interactions appeared to take place mainly in the microtubules. Overexpression of OsRMT1 in Arabidopsis resulted in increased tolerance to salt stress. Our findings suggest that the abundance of microtubule-associated OsRMT1 is strictly regulated, and OsRMT1 may play a relevant role in salt stress response by modulating levels of its target proteins.

Molecular characterization of rice arsenic‐induced RING finger E3 ligase 2 (OsAIR2) and its heterogeneous overexpression in Arabidopsis thaliana

“…B). Our previous studies revealed that most proteins interacting with the rice RING E3 ligases serve as substrates (Lim et al , Lim et al , Park et al ). Similarly, the present results also show that OsAIR2 ubiquitinates OsKAT1 leading to a reduction in the protein levels of OsKAT1 via 26S proteasome degradation (Fig.…”

“…For example, the O. sativa RING finger protein with microtubule‐targeting domain 1 (OsRMT1) binding to its substrate proteins OsSalT, OsCPA, OsFKBP12, and OsDH1 results in protein degradation via the 26S proteasome system. However, transcript levels of these substrate genes were highly increased in response to salt stress (Lim et al ). Similarly, the partner genes of another gamma ray‐induced E3 ligase OsGIRP1, namely OsrbcL1 and OsrbcS1, were also highly induced when exposed to gamma ray irradiation, followed by the degradation of these substrate proteins by OsGIRP1 via the 26S proteasome pathway (Park et al ).…”

A rice really interesting new gene H2‐type E3 ligase, OsSIRH2‐14, enhances salinity tolerance via ubiquitin/26S proteasome‐mediated degradation of salt‐related proteins

“…RING E3 ligases can interact and regulate multiple substrate proteins in a diverse functional network (Iconomou & Saunders, ; Lim et al, ; Lim et al, ). OsSIRH2‐14 E3 ligase interacts with multiple substrates, including OsSalT, OsPRF2, and OsHKT2;1 (Lim et al, ; Figures and S7).…”

“…These results suggest that the reduced Na + accumulation in OsSIRH2-14-overexpressing plants might lead to decreased oxidative damage and increased capacity for proline and soluble sugars. RING E3 ligases can interact and regulate multiple substrate proteins in a diverse functional network (Iconomou & Saunders, 2016;Lim et al, 2013;Lim et al, 2015). OsSIRH2-14 E3 ligase interacts with multiple substrates, including OsSalT, OsPRF2, and OsHKT2;1 (Lim et al, 2013;Figures 4 and S7).…”

A Vitis vinifera basic helix–loop–helix transcription factor enhances plant cell size, vegetative biomass and reproductive yield

“…Total proteins were extracted from 3-week-old seedlings. Tissue samples (500 mg) were quickly incubated with 1 mL of modified denaturing buffer [50 mM 2-amino-2-(hydroxymethyl)-1,3-propanediol-HCl, pH 7.5, 250 mM NaCl, 0.1% IGEPAL CA-630, 4 M urea, 10 mM NaPO4, pH = 6.0, and 19 protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN)] for 10 min and were centrifuged for 30 min at 16 000 9 g at 4°C for 30 min (Lim et al, 2015). The soluble supernatant fraction was moved to a new microcentrifuge tube, and protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL).…”