OsATL38 mediates mono-ubiquitination of the 14-3-3 protein OsGF14d and negatively regulates the cold stress response in rice.

One of the major regulatory pathways that permits plants to convert an external stimulus into an internal cellular response within a short period of time is the ubiquitination pathway. In this study, OsATL38 was identified as a low temperature-induced gene that encodes a rice homolog of Arabidopsis Tóxicos en Levadura RING-type E3 ubiquitin (Ub) ligase, which was predominantly localized to the plasma membrane. OsATL38-overexpressing transgenic rice plants exhibited decreased tolerance to cold stress as compared with wild-type rice plants. In contrast, RNAi-mediated OsATL38 knockdown transgenic progeny exhibited markedly increased tolerance to cold stress relative to that of wild-type plants, which indicated a negative role of OsATL38 in response to cold stress. Yeast two-hybrid, in vitro pull-down, and co-immunoprecipitation assays revealed that OsATL38 physically interacted with OsGF14d, a rice 14-3-3 protein. An in vivo target ubiquitination assay indicated that OsGF14d was mono-ubiquitinated by OsATL38. osgf14d knockout mutant plants were more sensitive to cold stress than wild-type rice plants, indicating that OsGF14d is a positive factor in the response to cold stress. These results provide evidence that the RING E3 Ub ligase OsATL38 negatively regulates the cold stress response in rice via mono-ubiquitination of OsGF14d 14-3-3 protein.

OsPUB41, a U-box E3 ubiquitin ligase, acts as a negative regulator of drought stress response in rice (Oryza Sativa L.).

KEY MESSAGE: OsPUB41 plays a negative role in drought stress response through the mediation of OsUBC25 and interacts with OsCLC6, suggesting a putative substrate. The notable expansion of Plant U-Box E3 ligases (PUB), compared with those in mammals, implies that PUB proteins have evolved to perform plant-specific functions. OsPUB41, a potential ortholog of CMPG1, was recently reported to regulate the cell wall degrading enzyme (CWDE)-induced innate immune response in rice. Here, we characterized the OsPUB41 gene, which encodes a dual-localized cytosolic and nuclear U-box E3 ligase in rice. OsPUB41 expression was specifically induced by dehydration among various abiotic stresses and abscisic acid (ABA) treatments. Furthermore, we revealed that the core U-box motif of OsPUB41 possesses the E3 ligase activity that can be activated by OsUBC25 in rice. The Ubi:RNAi-OsPUB41 knock-down and ospub41 suppression mutant plants exhibited enhanced tolerance to drought stress compared with the wild-type rice plants in terms of transpirational water loss, long-term dehydration response, and chlorophyll content. Moreover, the knock-down or suppression of the OsPUB41 gene did not cause adverse effect on rice yield-related traits. Yeast two-hybrid and an in vitro pull-down analyses revealed that OsCLC6, a chloride channel, is a putative substrate of OsPUB41. Overall, these results suggest that OsPUB41 acts as a negative regulator of dehydration conditions and interacts with OsCLC6, implying that it is a substrate of OsPUB41.

OsBZR1 turnover mediated by OsSK22-regulated U-box E3 ligase OsPUB24 in rice BR response.

Oryza sativa BRASSINAZOLE RESISTANT 1 (OsBZR1) is the closest rice homolog of the Arabidopsis BZR1 and bri1-EMS-SUPPRESSOR 1 (BES1)/BZR2 transcription factors. OsBZR1 plays a central role in the rice brassinosteroid signaling pathway. Despite its functional importance, the control mechanism by which the cellular stability of OsBZR1 is regulated has not yet been fully elucidated. Here, we report that a rice U-box E3 ubiquitin (Ub) ligase OsPUB24 acts as a negative regulator in the BR signaling pathway via the 26S proteasome-dependent degradation of OsBZR1. The ospub24 T-DNA knock-out mutant and Ubi:RNAi-OsPUB24 knock-down rice plants displayed enhanced seedling growth, increased lamina joint bending, and hypersensitivity to brassinolide (BL). The expressions of the BR biosynthetic genes suppressed by BR in a negative feedback loop were lower in the mutant progeny than in the wild-type rice plants, which indicated increased BR responses in the mutant line. OsPUB24 ubiquitinated OsBZR1, resulting in the proteasomal degradation of OsBZR1. In addition, the stability of OsPUB24 was downregulated by BL and bikinin, an inhibitor of Oryza sativa Shaggy/GSK3-like kinase 22 (OsSK22). OsSK22, the homolog of Arabidopsis BRASSINOSTEROID INSENSITIVE 2 (BIN2) protein kinase, phosphorylated OsPUB24 and elevated the cellular stability of OsPUB24. Our findings suggest that OsPUB24 participates in OsBZR1 turnover, and that the regulatory networks of OsPUB24, OsSK22 and OsBZR1 are crucial for fine-tuning the BR response in rice.

OsDIRP1, a Putative RING E3 Ligase, Plays an Opposite Role in Drought and Cold Stress Responses as a Negative and Positive Factor, Respectively, in Rice (Oryza sativa L.).

As higher plants are sessile organisms, they are unable to move to more favorable places; thus, they have developed the ability to survive under potentially detrimental conditions. Ubiquitination is a crucial post-translational protein modification and participates in abiotic stress responses in higher plants. In this study, we identified and characterized OsDIRP1 (Oryza sativa Drought-Induced RING Protein 1), a nuclear-localized putative RING E3 ubiquitin (Ub) ligase in rice (Oryza sativa L.). OsDIRP1 expression was induced by drought, high salinity, and abscisic acid (ABA) treatment, but not by low temperature (4°C) stress, suggesting that OsDIRP1 is differentially regulated by different abiotic stresses. To investigate its possible role in abiotic stress responses, OsDIRP1-overexpressing transgenic rice plants (Ubi:OsDIRP1-sGFP) were generated, and their phenotypes were analyzed. The T4 Ubi:OsDIRP1-sGFP lines showed decreased tolerance to drought and salt stress as compared to wild-type rice plants. Moreover, Ubi:OsDIRP1-sGFP progeny were less sensitive to ABA than the wild-type during both germination and post-germination growth. In contrast, Ubi:OsDIRP1-sGFP plants exhibited markedly higher tolerance to prolonged cold (4°C) treatment. These results suggest that OsDIRP1 acts as a negative regulator during drought and salt stress, whereas it functions as a positive factor during the cold stress response in rice.

CaPUB1, a Hot Pepper U-box E3 Ubiquitin Ligase, Confers Enhanced Cold Stress Tolerance and Decreased Drought Stress Tolerance in Transgenic Rice (Oryza sativa L.).

Abiotic stresses such as drought and low temperature critically restrict plant growth, reproduction, and productivity. Higher plants have developed various defense strategies against these unfavorable conditions. CaPUB1 (Capsicum annuum Putative U-box protein 1) is a hot pepper U-box E3 Ub ligase. Transgenic Arabidopsis plants that constitutively expressed CaPUB1 exhibited drought-sensitive phenotypes, suggesting that it functions as a negative regulator of the drought stress response. In this study, CaPUB1 was over-expressed in rice (Oryza sativa L.), and the phenotypic properties of transgenic rice plants were examined in terms of their drought and cold stress tolerance. Ubi:CaPUB1 T3 transgenic rice plants displayed phenotypes hypersensitive to dehydration, suggesting that its role in the negative regulation of drought stress response is conserved in dicot Arabidopsis and monocot rice plants. In contrast, Ubi:CaPUB1 progeny exhibited phenotypes markedly tolerant to prolonged low temperature (4°C) treatment, compared to those of wild-type plants, as determined by survival rates, electrolyte leakage, and total chlorophyll content. Cold stress-induced marker genes, including DREB1A, DREB1B, DREB1C, and Cytochrome P450, were more up-regulated by cold treatment in Ubi:CaPUB1 plants than in wild-type plants. These results suggest that CaPUB1 serves as both a negative regulator of the drought stress response and a positive regulator of the cold stress response in transgenic rice plants. This raises the possibility that CaPUB1 participates in the cross-talk between drought and low-temperature signaling pathways.