Aquaporin belongs to a highly conserved group of membrane proteins called major intrinsic proteins that facilitate water transport across biological membranes. The genome of Arabidopsis encodes 35 aquaporin genes with 13 homologs in the plasma membrane intrinsic protein (PIP) subgroup. However, the function of each individual aquaporin isoform and the integrated function of plant aquaporins under various physiological conditions remain unclear. As a step toward understanding the aquaporin function in plants under various environmental stimuli, the expressions of a gene family encoding 13 PIPs in Arabidopsis thaliana under various abiotic stress conditions including drought, cold, and high salinity, or abscisic acid (ABA) treatment were investigated by a quantitative real-time reverse transcription-PCR analysis. Several PIP genes were predominantly expressed either in the roots or in the flowers. The expressions of both the highly expressed aquaporins including PIP1;1, PIP1;2, and PIP2;7 and the weakly expressed aquaporins such as PIP1;4, PIP2;1, PIP2;4, and PIP2;5 were modulated by external stimuli. The analyses of our data revealed that only the PIP2;5 was up-regulated by cold treatment, and most of the PIP genes were down-regulated by cold stress. Marked up- or down-regulation in PIP expression was observed by drought stress, whereas PIP genes were less-severely modulated by high salinity. The responsiveness of each aquaporin to ABA were different, implying that the regulation of aquaporin expression involves both ABA-dependent and ABA-independent signaling pathways. Together, our comprehensive expression profile of the 13 members of the PIP gene family provides novel basis to allocate the stress-related biological function to each PIP gene.

Despite the fact that cold shock domain proteins (CSDPs) and glycine-rich RNA-binding proteins (GRPs) have been implicated to play a role during the cold adaptation process, their importance and function in eukaryotes, including plants, are largely unknown. To understand the functional role of plant CSDPs and GRPs in the cold response, two CSDPs (CSDP1 and CSDP2) and three GRPs (GRP2, GRP4 and GRP7) from Arabidopsis thaliana were investigated. Heterologous expression of CSDP1 or GRP7 complemented the cold sensitivity of BX04 mutant Escherichia coli that lack four cold shock proteins (CSPs) and is highly sensitive to cold stress, and resulted in better survival rate than control cells during incubation at low temperature. In contrast, CSDP2 and GRP4 had very little ability. Selective evolution of ligand by exponential enrichment (SELEX) revealed that GRP7 does not recognize specific RNAs but binds preferentially to G-rich RNA sequences. CSDP1 and GRP7 had DNA melting activity, and enhanced RNase activity. In contrast, CSDP2 and GRP4 had no DNA melting activity and did not enhance RNAase activity. Together, these results indicate that CSDPs and GRPs help E.coli grow and survive better during cold shock, and strongly imply that CSDP1 and GRP7 exhibit RNA chaperone activity during the cold adaptation process.

A glycine-rich RNA-binding protein4 (GR-RBP4), one of the eight GR-RBP family members in Arabidopsis thaliana, was investigated for its stress-related expression, nucleic acid-binding property, and functional roles in plants subjected to various stresses including cold, high salinity, and dehydration. Real-time RT-PCR and GUS expression analyses showed that GR-RBP4 was abundantly expressed in young plants, root tips, and flowers, but weakly in mature leaves and stems, implying that GR-RBP4 is highly expressed in actively proliferating organs. The transcript level of GR-RBP4 increased markedly with cold stress, decreased significantly with salt stress, and decreased slightly with dehydration stress. In vitro nucleic acid-binding assays revealed that GR-RBP4 protein binds sequence non-specifically to RNAs and DNAs. Characterization of the transgenic Arabidopsis plants overexpressing GR-RBP4 under the control of the 35S promoter revealed that 35S::GR-RBP4 lines displayed retarded germination compared with the wild type under salt or dehydration stress. Despite the marked up-regulation of GR-RBP4 expression by cold stress, the 35S::GR-RBP4 lines did not show any noticeable changes in cold or freezing tolerance compared with wild-type plants. These results indicate that GR-RBP4 contributes differently to altered germination and seedling growth of Arabidopsis plants under various stress conditions.

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.

High mobility group B (HMGB) proteins found in the nuclei of higher eukaryotes play roles in various cellular processes such as replication, transcription and nucleosome assembly. The Arabidopsis thaliana genome contains eight genes encoding HMGB proteins, the functions of which remain largely unknown in the transcriptional regulation of plant stress responses. To understand better the functions of HMGB proteins in the responses of plants to environmental stimuli, we examined the effect of various abiotic stresses on germination and growth of transgenic Arabidopsis plants that overexpress a single isoform of HMGB. The expression of HMGB2, HMGB3 and HMGB4 was up-regulated by cold stress, whereas the expression of HMGB2 and HMGB3 was markedly down-regulated by drought or salt stress. Under salt or drought stress, the transgenic Arabidopsis plants that overexpress HMGB2 displayed retarded germination and subsequent growth compared with wild-type plants. Overexpression of HMGB4 had no impact on seed germination and seedling growth of the plants under the stress conditions tested. In contrast to no significant stress-related phenotypes of HMGB5-overexpressing plants, loss-of-function mutants of HMGB5 displayed retarded germination and subsequent growth compared with wild-type plants under stress conditions. Although transcript levels of various stress-responsive genes were not modulated by the expression of HMGB2, expression of several germination-responsive genes was modulated by HMGB2 under salt stress. Taken together, these results provide a novel basis for understanding the biological functions of HMGB protein family members that differently affect germination and seedling growth of Arabidopsis plants under various stress conditions.