Rice U-box E3 Ub ligases (OsPUBs) are implicated in biotic stress responses. However, their cellular roles in response to abiotic stress are poorly understood. In this study, we performed functional analyses of two homologous OsPUB2 and OsPUB3 in response to cold stress (4°C). OsPUB2 was up-regulated by high salinity, drought, and cold, whereas OsPUB3 was constitutively expressed. A subcellular localization assay revealed that OsPUB2 and OsPUB3 were localized to the exocyst positive organelle (EXPO)-like punctate structures. OsPUB2 was also localized to the nuclei. OsPUB2 and OsPUB3 formed a hetero-dimeric complex as well as homo-dimers in yeast cells and in vitro. OsPUB2/OsPUB3 exhibited self-ubiquitination activities in vitro and were rapidly degraded in the cell-free extracts with apparent half-lives of 150–160 min. This rapid degradation of OsPUB2/OsPUB3 was delayed in the presence of the crude extracts of cold-treated seedlings (apparent half-lives of 200–280 min). Moreover, a hetero-dimeric form of OsPUB2/OsPUB3 was more stable than the homo-dimers. These results suggested that OsPUB2 and OsPUB3 function coordinately in response to cold stress. OsPUB2- and OsPUB3-overexpressing transgenic rice plants showed markedly better tolerance to cold stress than did the wild-type plants in terms of survival rates, chlorophyll content, ion leakage, and expression levels of cold stress-inducible marker genes. Taken together, these results suggested that the two homologous rice U-box E3 Ub ligases OsPUB2 and OsPUB3 are positive regulators of the response to cold stress.

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Few plant species can survive in Antarctica, the harshest environment for living organisms. Deschampsia antarctica is the only natural grass species to have adapted to and colonized the maritime Antarctic. To investigate the molecular mechanism of the Antarctic adaptation of this plant, we identified and characterized D. antarctica C-repeat binding factor 4 (DaCBF4), which belongs to monocot CBF group IV. The transcript level of DaCBF4 in D. antarctica was markedly increased by cold and dehydration stress. To assess the roles of DaCBF4 in plants, we generated a DaCBF4-overexpressing transgenic rice plant (Ubi:DaCBF4) and analyzed its abiotic stress response phenotype. Ubi:DaCBF4 displayed enhanced tolerance to cold stress without growth retardation under any condition compared to wild-type plants. Because the cold-specific phenotype of Ubi:DaCBF4 was similar to that of Ubi:DaCBF7 (Byun et al., 2015), we screened for the genes responsible for the improved cold tolerance in rice by selecting differentially regulated genes in both transgenic rice lines. By comparative transcriptome analysis using RNA-seq, we identified 9 and 15 genes under normal and cold-stress conditions, respectively, as putative downstream targets of the two D. antarctica CBFs. Overall, our results suggest that Antarctic hairgrass DaCBF4 mediates the cold-stress response of transgenic rice plants by adjusting the expression levels of a set of stress-responsive genes in transgenic rice plants. Moreover, selected downstream target genes will be useful for genetic engineering to enhance the cold tolerance of cereal plants, including rice.

The Antarctic flowering plant Deschampsia antarctica is highly sensitive to climate change and has shown rapid population increases during regional warming of the Antarctic Peninsula. Several studies have examined the physiological and biochemical changes related to environmental stress tolerance that allow D. antarctica to colonize harsh Antarctic environments; however, the molecular mechanisms of its responses to environmental changes remain poorly understood. To elucidate the survival strategies of D. antarctica in Antarctic environments, we investigated the functions of actin depolymerizing factor (ADF) in this species. We identified eight ADF genes in the transcriptome that were clustered into five subgroups by phylogenetic analysis. DaADF3, which belongs to a monocot-specific clade together with cold-responsive ADF in wheat, showed significant transcriptional induction in response to dehydration and cold, as well as under Antarctic field conditions. Multiple drought and low-temperature responsive elements were identified as possible binding sites of C-repeat-binding factors in the promoter region of DaADF3, indicating a close relationship between DaADF3 transcription control and abiotic stress responses. To investigate the functions of DaADF3 related to abiotic stresses in vivo, we generated transgenic rice plants overexpressing DaADF3. These transgenic plants showed greater tolerance to low-temperature stress than the wild-type in terms of survival rate, leaf chlorophyll content, and electrolyte leakage, accompanied by changes in actin filament organization in the root tips. Together, our results imply that DaADF3 played an important role in the enhancement of cold tolerance in transgenic rice plants and in the adaptation of D. antarctica to its extreme environment.