Unlike the well-known functions of cold shock proteins in prokaryotes during cold adaptation, the biological functions of cold shock domain proteins (CSDPs) in plants remain largely unknown. Here, we examined the functional roles of two structurally different CSDPs, CSDP1 harboring a long C-terminal glycine-rich region interspersed with seven CCHC-type zinc fingers and CSDP2 containing a far shorter glycine-rich region interspersed with two CCHC-type zinc fingers, in Arabidopsis thaliana under stress conditions. CSDP1 overexpression delayed the seed germination of Arabidopsis under dehydration or salt stress conditions, whereas CSDP2 overexpression accelerated the seed germination of Arabidopsis under salt stress conditions. CSDP1 and CSDP2 rescued the cold-sensitive glycine-rich RNA-binding protein 7 mutant plants from freezing damage to a different degree, and this rescuing capability was correlated with their ability to complement the cold-sensitive Escherichia coli BX04 mutant at low temperatures. The nucleic acid-binding properties of CSDPs varied depending on the N-terminal cold shock domain and the C-terminal glycine-rich zinc finger region. Collectively, these results showed that CSDP1 and CSDP2 perform different functions in seed germination and growth of Arabidopsis under stress conditions, and that the glycine-rich region interspersed with CCHC-type zinc fingers is particularly important for its nucleic acid-binding activities and function.

DEAD-box RNA helicases have been implicated to have a function during stress adaptation processes, but their functional roles in plant stress responses remain to be clearly elucidated. Here, we assessed the expression patterns and functional roles of two RNA helicases, AtRH9 and AtRH25, in Arabidopsis thaliana under abiotic stress conditions. The transcript levels of AtRH9 and AtRH25 were up-regulated markedly in response to cold stress, whereas their transcript levels were down-regulated by salt or drought stress. Phenotypic analysis of the transgenic plants and T-DNA-tagged mutants showed that the constitutive overexpression of AtRH9 or AtRH25 resulted in the retarded seed germination of Arabidopsis plants under salt stress conditions. AtRH25, but not AtRH9, enhanced freezing tolerance in Arabidopsis plants. Both AtRH9 and AtRH25 complemented the cold-sensitive phenotype of BX04 Escherichia coli mutant cells, but AtRH25 had much more prominent complementation ability than AtRH9. An in vitro nucleic acid binding assay showed that AtRH9 binds equally to all homoribopolymers, whereas AtRH25 binds preferentially to poly(G). Taken together, these results demonstrate that AtRH9 and AtRH25 impact on the seed germination of Arabidopsis plants under salt stress conditions, and suggest that the difference in cold tolerance capability between AtRH9 and AtRH25 arises from their different nucleic acid-binding properties.

AtAIRP2 is a cytosolic RING-type E3 ubiquitin ligase that positively regulates an abscisic acid (ABA) response in Arabidopsis (Arabidopsis thaliana). Yeast two-hybrid screening using AtAIRP2 as bait identified ATP1 (AtAIRP2 Target Protein1) as a substrate of AtAIRP2. ATP1 was found to be identical to SDIRIP1, which was reported recently to be a negative factor in ABA signaling and a target protein of the RING E3 ligase SDIR1. Accordingly, ATP1 was renamed ATP1/SDIRIP1. A specific interaction between AtAIRP2 and ATP1/SDIRIP1 and ubiquitination of ATP1/SDIRIP1 by AtAIRP2 were demonstrated in vitro and in planta. The turnover of ATP1/SDIRIP1 was regulated by AtAIRP2 in cell-free degradation and protoplast cotransfection assays. The ABA-mediated germination assay of 35S:ATP1/SDIRIP1-RNAi/atairp2 double mutant progeny revealed that ATP1/ SDIRIP1 acts downstream of AtAIRP2. AtAIRP2 and SDIR1 reciprocally complemented the ABA-and salt-insensitive germination phenotypes of sdir1 and atairp2 mutants, respectively, indicating their combinatory roles in seed germination. Subcellular localization and bimolecular fluorescence complementation experiments in the presence of MG132, a 26S proteasome inhibitor, showed that AtAIRP2 and ATP1/SDIRIP1 were colocalized to the cytosolic spherical body, which lies in close proximity to the nucleus, in tobacco (Nicotiana benthamiana) leaf cells. The 26S proteasome subunits RPN12a and RPT1 and the molecular chaperones HSP70 and HSP101 were colocalized to these discrete punctae-like structures. These results raised the possibility that AtAIRP2 and ATP1/SDIRIP1 interact in the cytosolic spherical compartment. Collectively, our data suggest that the down-regulation of ATP1/SDIRIP1 by AtAIRP2 and SDIR1 RING E3 ubiquitin ligases is critical for ABA and highsalinity responses during germination in Arabidopsis.

SignificanceThe essential roles of cytoplasmic E3 ligases in the protein quality control (PQC) pathways have been increasingly highlighted in yeast and animal studies. However, in plants, only CHIP E3 ligase has been characterized, while the knowledge of cytoplasmic PQC E3 ligases remains rudimentary. Misfolded Protein Sensing RING E3 ligase 1 (MPSR1), a self-regulatory sensor system that functions only in the occurrence of misfolded proteins, is an identified cytoplasmic PQC E3 ligase in plants that directly recognizes emergent misfolded proteins independently of chaperones. In addition, MPSR1 sustains the integrity and activity of the 26S proteasome under proteotoxic stress. Given that MPSR1 RING E3 ligase is well conserved in eukaryotes, this study sheds light on a PQC pathway that is present particularly in plants and beyond.

Plants are constantly exposed to a variety of abiotic stresses, such as drought, heat, cold, flood, and salinity. To survive under such unfavorable conditions, plants have evolutionarily developed their own resistant-mechanisms. For several decades, many studies have clarified specific stress response pathways of plants through various molecular and genetic studies. In particular, it was recently discovered that ubiquitin proteasome system (UPS), a regulatory mechanism for protein turn over, is greatly involved in the stress responsive pathways. In the UPS, many E3 ligases play key roles in recognizing and tethering poly-ubiquitins on target proteins for subsequent degradation by the 26S proteasome. Here we discuss the roles of RING ligases that have been defined in related to abiotic stress responses in plants.