Kyung Jin Kwak

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.

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.

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.

Zinc finger-containing glycine-rich RNA-binding protein in Oryza sativa has an RNA chaperone activity under cold stress conditions

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.

Structural determinants crucial to the RNA chaperone activity of glycine-rich RNA-binding proteins 4 and 7 in Arabidopsis thaliana during the cold adaptation process

Although glycine-rich RNA-binding proteins (GRPs) have been determined to function as RNA chaperones during the cold adaptation process, the structural features relevant to this RNA chaperone activity remain largely unknown. To uncover which structural determinants are necessary for RNA chaperone activity of GRPs, the importance of the N-terminal RNA recognition motif (RRM) and the C-terminal glycine-rich domains of two Arabidopsis thaliana GRPs (AtGRP4 harbouring no RNA chaperone activity and AtGRP7 harbouring RNA chaperone activity) was assessed via domain swapping and mutation analyses. The results of domain swapping and deletion experiments showed that the domain sequences encompassing the N-terminal RRM of GRPs were found to be crucial to the ability to complement cold-sensitive Escherichia coli mutant cells under cold stress, RNA melting ability, and freezing tolerance ability in the grp7 loss-of-function Arabidopsis mutant. In particular, the N-terminal 24 amino acid extension of AtGRP4 impedes the RNA chaperone activity. Collectively, these results reveal that domain sequences and overall folding of GRPs governed by a specific modular arrangement of RRM and glycine-rich sequences are critical to the RNA chaperone activity of GRPs during the cold adaptation process in cells.

The Arabidopsis U12-Type Spliceosomal Protein U11/U12-31K Is Involved in U12 Intron Splicing via RNA Chaperone Activity and Affects Plant Development

Correct splicing of U12 introns is essential for constitutive and regulated gene expression in eukaryotes. This study provides evidence that U11/U12-31K, a U12-type spliceosomal protein in Arabidopsis thaliana, is an RNA chapereone that is indispensible for proper U12 intron splicing and for normal growth and development of plants. U12 introns are removed from precursor-mRNA by a U12 intron-specific spliceosome that contains U11 and U12 small nuclear ribonucleoproteins. Although several proteins unique to the U12-type spliceosome have been identified, the manner by which they affect U12-dependent intron splicing as well as plant growth and development remain largely unknown. Here, we assessed the role of U11/U12-31K, a U12-type spliceosomal protein in Arabidopsis thaliana. T-DNA–tagged homozygote lines for U11/U12-31K could not be obtained, and heterozygote mutants were defective for seed maturation, indicating that U11/U12-31K is essential for the normal development of Arabidopsis. Knockdown of U11/U12-31K by artificial microRNA caused a defect in proper U12 intron splicing, resulting in abnormal stem growth and development of Arabidopsis. This defect in proper splicing was not restricted to specific U12-type introns, but most U12 intron splicing was influenced by U11/U12-31K. The stunted inflorescence stem growth was recovered by exogenously applied gibberellic acid (GA), but not by cytokinin, auxin, or brassinosteroid. GA metabolism-related genes were highly downregulated in U11/U12-31K knockdown plants. Importantly, U11/U12-31K was determined to harbor RNA chaperone activity. We propose that U11/U12-31K is an RNA chapereone that is indispensible for proper U12 intron splicing and for normal growth and development of plants.

Expression of Arabidopsis glycine-rich RNA-binding protein AtGRP2 or AtGRP7 improves grain yield of rice (Oryza sativa) under drought stress conditions.

Although posttranscriptional regulation of RNA metabolism is increasingly recognized as a key regulatory process in plant response to environmental stresses, reports demonstrating the importance of RNA metabolism control in crop improvement under adverse environmental stresses are severely limited. To investigate the potential use of RNA-binding proteins (RBPs) in developing stress-tolerant transgenic crops, we generated transgenic rice plants (Oryza sativa) that express Arabidopsis thaliana glycine-rich RBP (AtGRP) 2 or 7, which have been determined to harbor RNA chaperone activity and confer stress tolerance in Arabidopsis, and analyzed the response of the transgenic rice plants to abiotic stresses. AtGRP2- or AtGRP7-expressing transgenic rice plants displayed similar phenotypes comparable with the wild-type plants under high salt or cold stress conditions. By contrast, AtGRP2- or AtGRP7-expressing transgenic rice plants showed much higher recovery rates and grain yields compared with the wild-type plants under drought stress conditions. The higher grain yield of the transgenic rice plants was due to the increases in filled grain numbers per panicle. Collectively, the present results show the importance of posttranscriptional regulation of RNA metabolism in plant response to environmental stress and suggest that GRPs can be utilized to improve the yield potential of crops under stress conditions.