Yeon-Ok Kim

High Cadmium-Binding Ability of a Novel Colocasia esculenta Metallothionein Increases Cadmium Tolerance in Escherichia coli and Tobacco

Experimental evidence in vivo as to the functional roles and binding properties to cadmium (Cd) of type-2 plants metallothionein (MT) has been limited thus far. We investigated the biological role of metallothionein from Colocasia esculenta (CeMT2b) in Escherichia coli and tobacco, and developed a new model for the relationship between Cd tolerance and Cd-binding ability. Heterologous expression of CeMT2b in Escherichia coli greatly enhanced Cd tolerance and accumulated Cd content as compared to control cells. The molecular weight of CeMT2b increased with Cd, and CeMT2b bound up to 5.96±1 molar ratio (Cd/protein). Under Cd stress, transgenic tobacco plants displayed much better seedling growth and high Cd accumulation than the wild type. The presence of an extra CXC motif in CeMT2b contributed to the enhanced Cd-tolerance. The present study provides the first insight into the ability of type-2 plant MT to bind physiological Cd.

Role of pCeMT, a putative metallothionein from Colocasia esculenta, in response to metal stress.

Metallothioneins (MTs) play a major role in metal homeostasis and/or detoxification in plants. In this study, a novel gene, pCeMT, was isolated from Colocasia esculenta and characterized. Our results indicate that Escherichia coli cells expressing pCeMT exhibited enhanced Cd, Cu, and Zn tolerance and accumulation compared with control cells. Furthermore, pCeMT-overexpressing tobacco seedlings displayed better growth under Cd, Cu, and Zn stresses and accumulated more Cd and Zn compared with the wild type. Interestingly, transgenic tobacco displayed markedly decreased hydrogen peroxide (H(2)O(2)) and lipid peroxidation levels under Cd, Cu, and Zn treatments. These results suggest that pCeMT could play an important role in the protection of plant cells from oxidative stress by reactive oxygen species (ROS) scavenging and in the detoxification of free metals by metal binding, leading to improved plant metal tolerance.

The Transgenic Poplar as an Efficient Bioreactor System for the Production of Xylanase

Plants are attractive expression systems for large-scale, low-cost production of high-value proteins. The xylanase 2 gene (Xyn2), encoding an endo-β-1,4-xylanase from Trichoderma reesei, was cloned and expressed in Escherichia coli and the poplar (Populus spp.). The optimal temperature and pH of the recombinant xylanase were 50 °C and 5.0 respectively when expressed in E. coli. The purpose of this study was to produce recombinant xylanase in poplar. The Xyn2 gene was transferred into poplars by Agrobacterium-mediated transformation. The transgenic status and transgene expression of the transformed poplar were confirmed by polymerase chain reaction (PCR) genotyping and reverse transcription (RT)-PCR analysis. The poplar-expressed xylanase was biologically active, with an expression level of up to 14.4% of total leaf soluble protein. In the leaves, the average xylanase content was 1.016 mg per g of leaf fresh weight in the transgenic poplar. We found that the poplar might make possible the large-scale production of commercially important recombinant proteins.

A chloroplast-localized S1 domain-containing protein SRRP1 plays a role in Arabidopsis seedling growth in the presence of ABA.

Although the roles of S1 domain-containing proteins have been characterized in diverse cellular processes in the cytoplasm, the functional roles of a majority of S1 domain-containing proteins targeted to the chloroplast are largely unknown. Here, we characterized the function of a nuclear-encoded chloroplast-targeted protein harboring two S1 domains, designated SRRP1 (for S1 RNA-binding ribosomal protein 1), in Arabidopsis thaliana. Subcellular localization analysis of SRRP1-GFP fusion proteins revealed that SRRP1 is localized to the chloroplast. The T-DNA tagged loss-of-function srrp1 mutants displayed poorer seedling growth and less cotyledon greening than the wild-type plants on MS medium supplemented with abscisic acid (ABA), suggesting that SRRP1 plays a role in seedling growth in the presence of ABA. Splicing of the trnL intron and processing of 5S rRNA in chloroplasts were altered in the mutant plants. Importantly, SRRP1 complemented the growth-defective phenotypes of an RNA chaperone-deficient Escherichia coli mutant at low temperatures and had nucleic acid-melting ability, indicating that SRRP1 possesses RNA chaperone activity. Taken together, these results suggest that SRRP1, the chloroplast-localized S1 domain-containing protein, harboring RNA chaperone activity affects the splicing and processing of chloroplast transcripts and plays a role in Arabidopsis seedling growth in the presence of ABA.

Exogenous Glutathione Enhances Mercury Tolerance by Inhibiting Mercury Entry into Plant Cells

Despite the increasing understanding of the crucial roles of glutathione (GSH) in cellular defense against heavy metal stress as well as oxidative stress, little is known about the functional role of exogenous GSH in mercury (Hg) tolerance in plants. Here, we provide compelling evidence that GSH contributes to Hg tolerance in diverse plants. Exogenous GSH did not mitigate the toxicity of cadmium (Cd), copper (Cu), or zinc (Zn), whereas application of exogenous GSH significantly promoted Hg tolerance during seed germination and seedling growth of Arabidopsis thaliana, tobacco, and pepper. By contrast, addition of buthionine sulfoximine, an inhibitor of GSH biosynthesis, severely retarded seed germination and seedling growth of the plants in the presence of Hg. The effect of exogenous GSH on Hg specific tolerance was also evident in the presence of other heavy metals, such as Cd, Cu, and Zn, together with Hg. GSH treatment significantly decreased H2O2 and O2- levels and lipid peroxidation, but increased chlorophyll content in the presence of Hg. Importantly, GSH treatment resulted in significantly less accumulation of Hg in Arabidopsis plants, and thin layer chromatography and nuclear magnetic resonance analysis revealed that GSH had much stronger binding affinity to Hg than to Cd, Cu, or Zn, suggesting that tight binding of GSH to Hg impedes Hg uptake, leading to low Hg accumulation in plant cells. Collectively, the present findings reveal that GSH is a potent molecule capable of conferring Hg tolerance by inhibiting Hg accumulation in plants.

Comparative expression analysis of genes encoding metallothioneins in response to heavy metals and abiotic stresses in rice (Oryza sativa) and Arabidopsis thaliana

ABSTRACT To get insights into the functions of metallothionein (MT) in plant response to multiple stresses, expressions of 10 rice MT genes (OsMTs) and 7 Arabidopsis MT genes (AtMTs) were comprehensively analyzed under combined heavy metal and salt stress. OsMT1a, OsMT1b, OsMT1c, OsMT1g, and OsMT2a were increased by different heavy metals. Notably, ABA remarkably increased OsMT4 up to 80-fold. Combined salt and heavy metals (Cd, Pb, Cu) synergistically increased OsMT1a, OsMT1c, and OsMT1g, whereas combined salt and H2O2 or ABA synergistically increased OsMT1a and OsMT4. Heavy metals decreased AtMT1c, AtMT2b, and AtMT3 but cold or ABA increased AtMT1a, AtMT1c, and AtMT2a. AtMT4a was markedly increased by salt stress. Combined salt and other stresses (Pb, Cd, H2O2) synergistically increased AtMT4a. Taken together, these findings suggest that MTs in monocot and dicot respond differently to combined stresses, which provides a valuable basis to further determine the roles of MTs in broad stress tolerance. Graphical Abstract MTs in monocot and dicot respond differently to single stress or combined salt and heavy metal, which provides information to generate plants with broad stress tolerance.

An RRM-containing mei2-like MCT1 plays a negative role in the seed germination and seedling growth of Arabidopsis thaliana in the presence of ABA.

Despite an increasing understanding of the essential role of the Mei2 gene encoding an RNA-binding protein (RBP) in premeiotic DNA synthesis and meiosis in yeasts and animals, the functional roles of the mei2-like genes in plant growth and development are largely unknown. Contrary to other mei2-like RBPs that contain three RNA-recognition motifs (RRMs), the mei2 C-terminal RRM only (MCT) is unique in that it harbors only the last C-terminal RRM. Although MCTs have been implicated to play important roles in plants, their functional roles in stress responses as well as plant growth and development are still unknown. Here, we investigated the expression and functional role of MCT1 (At1g37140) in plant response to abscisic acid (ABA). Confocal analysis of MCT1-GFP-expressing plants revealed that MCT1 is localized to the nucleus. The transcript level of MCT1 was markedly increased upon ABA treatment. Analysis of MCT1-overexpressing transgenic Arabidopsis plants and artificial miRNA-mediated mct1 knockdown mutants demonstrated that MCT1 inhibited seed germination and cotyledon greening of Arabidopsis plants under ABA. The transcript levels of ABA signaling-related genes, such as ABI3, ABI4, and ABI5, were markedly increased in the MCT1-overexpressing transgenic plant. Collectively, these results suggest that ABA-upregulated MCT1 plays a negative role in Arabidopsis seed germination and seedling growth under ABA by modulating the expression of ABA signaling-related genes.

A chloroplast-targeted cabbage DEAD-box RNA helicase BrRH22 confers abiotic stress tolerance to transgenic Arabidopsis plants by affecting translation of chloroplast transcripts

Although the roles of many DEAD-box RNA helicases (RHs) have been determined in the nucleus as well as in cytoplasm during stress responses, the importance of chloroplast-targeted DEAD-box RHs in stress response remains largely unknown. In this study, we determined the function of BrRH22, a chloroplast-targeted DEAD-box RH in cabbage (Brassica rapa), in abiotic stress responses. The expression of BrRH22 was markedly increased by drought, heat, salt, or cold stress and by ABA treatment, but was largely decreased by UV stress. Expression of BrRH22 in Arabidopsis enhanced germination and plantlet growth under high salinity or drought stress. BrRH22-expressing plants displayed a higher cotyledon greening and better plantlet growth upon ABA treatment due to decreases in the levels of ABI3, ABI4, and ABI5. Further, BrRH22 affected translation of several chloroplast transcripts under stress. Notably, BrRH22 had RNA chaperone function. These results altogether suggest that chloroplast-transported BrRH22 contributes positively to the response of transgenic Arabidopsis to abiotic stress by affecting translation of chloroplast genes via its RNA chaperone activity.

Rice DEAD-box RNA helicase OsRH53 has negative impact on Arabidopsis response to abiotic stresses

DEAD-box RNA helicases (RHs) play key roles in the regulation of RNA metabolism at the posttranscriptional level. In this study, the expression patterns under abiotic stresses and the functions of a rice (Oryza sativa) RH, OsRH53, in stress response were determined using transgenic Arabidopsis plants. The level of OsRH53 decreased upon abiotic stress treatment, including, drought, salt, cold, and UV stress, and by abscisic acid (ABA). Although OsRH53 contains a putative chloroplast transit peptide at the N-terminal end, confocal analysis of OsRH53–GFP fusion proteins transiently expressed in tobacco leaves revealed that OsRH53 is localized to the nucleus. OsRH53-expressing transgenic Arabidopsis displayed retarded germination and reduced growth under high salinity or dehydration stress but not under cold stress. OsRH53 negatively affected the growth and cotyledon greening of seedlings upon ABA application by activating the genes related to ABA signaling such as ABI3 and ABI4. The ability of OsRH53 to recover growth-defect phenotype of Escherichia coli mutant, and both in vitro and in vivo base pair-breaking ability confirmed that OsRH53 harbors RNA chaperone activity. Collectively, these results suggest that OsRH53 negatively affects plant abiotic stress responses via modulating RNA metabolism through its RNA chaperone activity.