Hunseung Kang

An Expression Analysis of a Gene Family Encoding Plasma Membrane Aquaporins in Response to Abiotic Stresses in Arabidopsis Thaliana

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 thalianaunder 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 PIPgenes 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;7and the weakly expressed aquaporins such as PIP1;4,PIP2;1,PIP2;4, and PIP2;5were modulated by external stimuli. The analyses of our data revealed that only the PIP2;5was up-regulated by cold treatment, and most of the PIPgenes were down-regulated by cold stress. Marked up- or down-regulation in PIPexpression was observed by drought stress, whereas PIPgenes 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.

Glycine-rich RNA-binding protein7 affects abiotic stress responses by regulating stomata opening and closing in Arabidopsis thaliana

Despite the fact that glycine-rich RNA-binding proteins (GRPs) have been implicated in the responses of plants to environmental stresses, their physiological functions and mechanisms of action in stress responses remain largely unknown. Here, we assessed the functional roles of GRP7, one of the eight GRP family members in Arabidopsis thaliana, on seed germination, seedling growth, and stress tolerance under high salinity, drought, or cold stress conditions. The transgenic Arabidopsis plants overexpressing GRP7 under the control of the cauliflower mosaic virus 35S promoter displayed retarded germination and poorer seedling growth compared with the wild-type plants and T-DNA insertional mutant lines under high salinity or dehydration stress conditions. By contrast, GRP7 overexpression conferred freezing tolerance in Arabidopsis plants. GRP7 is expressed abundantly in the guard cells, and has been shown to influence the opening and closing of the stomata, in accordance with the prevailing stress conditions. GRP7 is localized to both the nucleus and the cytoplasm, and is involved in the export of mRNAs from the nucleus to the cytoplasm under cold stress conditions. Collectively, these results provide compelling evidence that GRP7 affects the growth and stress tolerance of Arabidopsis plants under high salt and dehydration stress conditions, and also confers freezing tolerance, particularly via the regulation of stomatal opening and closing in the guard cells.

Cold shock domain proteins and glycine-rich RNA-binding proteins from Arabidopsis thaliana can promote the cold adaptation process in Escherichia coli

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.

Transgenic Arabidopsis and tobacco plants overexpressing an aquaporin respond differently to various abiotic stresses

Despite the high isoform multiplicity of aquaporins in plants, with 35 homologues including 13 plasma membrane intrinsic proteins (PIPs) in Arabidosis thaliana, the individual and integrated functions of aquaporins under various physiological conditions remain unclear. To better understand aquaporin functions in plants under various stress conditions, we examined transgenic Arabidopsis and tobacco plants that constitutively overexpress Arabidopsis PIP1;4 or PIP2;5 under various abiotic stress conditions. No significant differences in growth rates and water transport were found between the transgenic and wild-type plants when grown under favorable growth conditions. The transgenic plants overexpressing PIP1;4 or PIP2;5 displayed a rapid water loss under dehydration stress, which resulted in retarded germination and seedling growth under drought stress. In contrast, the transgenic plants overexpressing PIP1;4 or PIP2;5 showed enhanced water flow and facilitated germination under cold stress. The expression of several PIPs was noticeably affected by the overexpression of PIP1;4 or PIP2;5 in Arabidopsis under dehydration stress, suggesting that the expression of one aquaporin isoform influences the expression levels of other aquaporins under stress conditions. Taken together, our results demonstrate that overexpression of an aquaporin affects the expression of endogenous aquaporin genes and thereby impacts on seed germination, seedling growth, and stress responses of the plants under various stress conditions.

Glycine-rich RNA-binding proteins are functionally conserved in Arabidopsis thaliana and Oryza sativa during cold adaptation process

Contrary to the increasing amount of knowledge regarding the functional roles of glycine-rich RNA-binding proteins (GRPs) in Arabidopsis thaliana in stress responses, the physiological functions of GRPs in rice (Oryza sativa) currently remain largely unknown. In this study, the functional roles of six OsGRPs from rice on the growth of E. coli and plants under cold or freezing stress conditions have been evaluated. Among the six OsGRPs investigated, OsGRP1, OsGRP4, and OsGRP6 were shown to have the ability to complement cold-sensitive BX04 E. coli mutant cells under low temperature conditions, and this complementation ability was correlated closely with their DNA- and RNA-melting abilities. Moreover, OsGRP1 and OsGRP4 rescued the growth-defect of a cold-sensitive Arabidopsis grp7 mutant plant under cold and freezing stress, and OsGRP6 conferred freezing tolerance in the grp7 mutant plant, in which the expression of AtGRP7 was suppressed and is sensitive to cold and freezing stresses. OsGRP4 and OsGRP6 complemented the defect in mRNA export from the nucleus to the cytoplasm in grp7 mutants during cold stress. Considering that AtGRP7 confers freezing tolerance in plants and harbours RNA chaperone activity during the cold adaptation process, the results of the present study provide evidence that GRPs in rice and Arabidopsis are functionally conserved, and also suggest that GRPs perform a function as RNA chaperones during the cold adaptation process in monocotyledonous plants, as well as in dicotyledonous plants.

Hydrogen peroxide permeability of plasma membrane aquaporins of Arabidopsis thaliana

Although aquaporins have been known to transport hydrogen peroxide (H2O2) across cell membranes, the H2O2-regulated expression patterns and the permeability of every family member of the plasma membrane intrinsic protein (PIP) toward H2O2 have not been determined. This study investigates the H2O2-regulated expression levels of all plasma membrane aquaporins of Arabidopsis thaliana (AtPIPs), and determines the permeability of every AtPIP for H2O2 in yeast. Hydrogen peroxide treatment of Arabidopsis down-regulated the expression of AtPIP2 subfamily in roots but not in leaves, whereas the expression of AtPIP1 subfamily was not affected by H2O2 treatment. The growth and survival of yeast cells that expressed AtPIP2;2, AtPIP2;4, AtPIP2;5, or AtPIP2;7 was reduced in the presence of H2O2, while the growth of yeast cells expressing any other AtPIP family member was not affected by H2O2. These results show that only certain isoforms of AtPIPs whose expression is regulated by H2O2 treatment are permeable for H2O2 in yeast cells, and suggest that the integrated regulation of aquaporin expression by H2O2 and the capacity of individual aquaporin to transport H2O2 are important for plant response to H2O2.

MicroRNA402 Affects Seed Germination of Arabidopsis thaliana under Stress Conditions via Targeting DEMETER-LIKE protein3 mRNA

The functional roles of miR402 in Arabidopsis thaliana were investigated under abiotic stress conditions. Overexpression of miR402 accelerated the seed germination and seedling growth of Arabidopsis under salt stress conditions, while its overexpression promoted only seed germination but not seedling growth of Arabidopsis under dehydration or cold stress conditions. The expression of DEMETER-LIKE protein3 mRNA was down-regulated in miR402-overexpressing transgenic plants. These results imply that miR402 plays a role as a positive regulator of seed germination and seedling growth of Arabidopsis under stress conditions, and that microRNA-guided regulation of DNA demethylation is an adaptive process of plants to stress conditions.

Cold shock domain proteins affect seed germination and growth of Arabidopsis thaliana under abiotic 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.

Functional Characterization of DEAD-Box RNA Helicases in Arabidopsis thaliana under Abiotic Stress Conditions

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.

Plant RNA chaperones in stress response

Post-transcriptional regulation of RNA metabolism is a key regulatory process in diverse cellular processes, including the stress response of plants, during which a variety of RNA-binding proteins (RBPs) function as central regulators in cells. RNA chaperones are RBPs found in all living organisms and function by providing assistance to the correct folding of RNA molecules during RNA metabolism. Although our understanding of the role of RNA chaperones in plants is far less advanced than in bacteria, viruses, and animals, recent progress in functional characterization and determination of RNA chaperone activity of several RBPs has shed new light on the emerging roles of RNA chaperones during the stress response of plants.