The rice (Oryza sativa) genome harbours three genes encoding CysCysHisCys (CCHC)-type zinc finger-containing glycine-rich RNA-binding proteins, designated OsRZ proteins, but their importance and physiological functions remain largely unknown. Here, the stress-responsive expression patterns of OsRZs were assessed, and the biological and cellular functions of OsRZs were evaluated under low temperature conditions. The expression levels of the three OsRZs were up-regulated by cold stress, whereas drought or high salt stress did not significantly alter its transcript level. OsRZ2 complemented the cold sensitivity of BX04 Escherichia coli cells under low temperatures, and had DNA-melting activity and transcription anti-termination activity, thereby indicating that OsRZ2 possesses an RNA chaperone activity. By contrast, neither OsRZ1 nor OsRZ3 harboured these activities. Ectopic expression of OsRZ2, but not OsRZ3, in cold-sensitive Arabidopsis grp7 knockout plants rescued the grp7 plants from cold and freezing damage, and OsRZ2 complemented the defect in mRNA export from the nucleus to the cytoplasm in grp7 mutant during cold stress. The present findings support the emerging idea that the regulation of mRNA export is one of the adaptive processes in plants under stress conditions, and RNA chaperone functions as a regulator in mRNA export under cold stress conditions.

Among the four cold shock domain proteins (CSDPs) identified in Arabidopsis thaliana, it has recently been shown that CSDP1 harboring seven CCHC-type zinc fingers, but not CSDP2 harboring two CCHC-type zinc fingers, function as a RNA chaperone during cold adaptation. However, the structural features relevant to this differing RNA chaperone activity between CSDP1 and CSDP2 remain largely unknown. To determine which structural features are necessary for the RNA chaperone activity of the CSDPs, the importance of the N-terminal cold shock domain (CSD) and the C-terminal zinc finger glycine-rich domains of CSDP1 and CSDP2 were assessed. The results of sequence motif-swapping and deletion experiments showed that, although the CSD itself harbored RNA chaperone activity, the number and length of the zinc finger glycine-rich domains of CSDPs were crucial to the full activity of the RNA chaperones. The C-terminal domain itself of CSDP1, harboring seven CCHC-type zinc fingers, also has RNA chaperone activity. The RNA chaperone activity and nuclei acid-binding property of the native and chimeric proteins were closely correlated with each other. Collectively, these results indicate that a specific modular arrangement of the CSD and the zinc finger domain determines both the RNA chaperone activity and nucleic acid-binding property of CSDPs; this, in turn, contributes to enhanced cold tolerance in plants as well as in bacteria.

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 longer C-terminal glycine-rich region interspersed with 7 CCHC-type zinc fingers and CSDP2 containing a far shorter glycine-rich region interspersed with 2 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 with a different degree the cold-sensitive glycine-rich RNA-binding protein7 mutant plants from freezing damage, and this rescuing capability was correlated with its complementation ability to 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.

Although high mobility group B (HMGB) proteins have been identified from a variety of plant species, their importance and functional roles in plant responses to changing environmental conditions are largely unknown. Here, we investigated the functional roles of a CsHMGB isolated from cucumber (Cucumis sativus L.) in plant responses to environmental stimuli. Under normal growth conditions or when subjected to cold stress, no differences in plant growth were found between the wild-type and transgenic Arabidopsis thaliana overexpressing CsHMGB. By contrast, the transgenic Arabidopsis plants displayed retarded germination compared with the wild-type plants when grown under high salt or dehydration stress conditions. Germination of the transgenic plants was delayed by the addition of abscisic acid (ABA), implying that CsHMGB affects germination through an ABA-dependent way. The expression of CsHMGB had affected only the germination stage, and CsHMGB did not affect the seedling growth of the transgenic plants under the stress conditions. The transcript levels of several germination-responsive genes were modulated by the expression of CsHMGB in Arabidopsis. Taken together, these results suggest that ectopic expression of a CsHMGB in Arabidopsis modulates the expression of several germination-responsive genes, and thereby affects the germination of Arabidopsis plants under different stress conditions.

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.

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 better understand 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 HMGBs 2, 3 and 4 was up regulated by cold stress, whereas the expression of HMGBs 2 and 3 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 to wild-type plants. Overexpression of HMGB4 had no impact on seed germination and seedling growth of the plants under the stress conditions tested. Contrary to no significant stress-related phenotypes of HMGB5-overexpressing plants, loss-of-function mutants of HMGB5 displayed retarded germination and subsequent growth compared to 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.

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.

As a part of an integrated study of stress-related gene expression, a cDNA clone coding for a protein kinase in the root of Cucumis sativus was isolated and characterized with respect to its sequence and the expression patterns upon various abiotic stress treatments. The predicted polypeptide of 352 amino acid residues contains characteristic features of both the serine/threonine and tyrosine kinase families. In vitro kinase assay confirmed that the isolated protein kinase has autophosphorylation activity. Southern blot analysis showed that the kinase gene is a single-copy gene. Northern blot analysis showed that the kinase gene was more abundantly expressed in the roots and shoots than in the leaves. A quantitative real-time reverse-transcription-polymerase chain reaction analysis revealed that, among the abiotic stresses tested, drought treatment markedly decreased the transcript level of the kinase, whereas the expression of the kinase gene significantly increased by cold treatment. High salinity did not influence its expression. The present report identifies a dual specificity protein kinase in cucumber that shows different responses to abiotic stress treatments.

We demonstrate here by imaging successive surface reactions in self-assembled monolayers (SAMs) on Au(111) at molecular scale with a scanning tunneling microscope (STM):(i) SAM matrices formation with 1-octanethiol on Au(111) in ethanol,(ii) insertion of N-Fmoc-aminooctanethiol into the SAM matrices in ethanol, and (iii) removal of the Fmoc protecting group with tris(2-aminoethyl)amine (TAEA). The total reaction is formation of SAMs containing a small amount of NH2 terminated molecules in the CH3 terminated SAM matrices. After the reaction of the protecting group with TAEA, STM imaging revealed the decrease in heights of the inserted molecules on average. We attributed this observation to removal of the protecting group by taking account of a convolution of electronic and topographic contributions to observed STM heights. Apparent areas of the terminal groups, however, became larger on removal. The increase in the areas was attributed to water adsorption to the NH2 terminal group under air.

Adhesive interaction between a tip and a sample surface was examined on a microscopic scale by pulsed-force-mode atomic force microscopy (PFM-AFM). The signal measured by monitoring pull-off force is influenced by various factors such as topography, elasticity, electrostatic charges, and adsorbed water on surfaces. Here, we focus on the topographic effects on the adhesive interaction. To clarify the topographic influence, the adhesive force measurement of a stretched DNA molecule with a smaller radius of curvature than that of a tip was carried out at low relative humidity (RH) with an alkanethiol-modified tip. The experimental conditions such as low RH and the use of the alkanethiol-modified tip were required to minimise the influence of water capillary force on hydrated DNA strands. The hydrophobic modification of a substrate surface was also important to minimise the adsorbed water effect. The DNA molecules were stretched on the substrate surfaces by an immobilisation process called a dynamic molecular combing method. The two-component vapour-phase surface modification with an alkylsilane mixed with a silane derivative containing an amino end group enhanced the DNA adsorption due to the electrostatic interaction. The experimental results for the topographic effects on the adhesive force mapping were reproducible.