Eun-Woon Noh

Transgenic poplar trees expressing yeast cadmium factor 1 exhibit the characteristics necessary for the phytoremediation of mine tailing soil.

Genetic engineering of plants for phytoremediation is thought to be possible based on results using model plants expressing genes involved in heavy metal resistance, which improve the plants tolerance of heavy metals and accumulation capacity. The next step of progress in this technology requires the genetic engineering of plants that produce large amounts of biomass and the testing of these transgenic plants in contaminated soils. Thus, we transformed a sterile line of poplar Populus alba X P. tremula var. glandulosa with a heavy metal resistance gene, ScYCF1 (yeast cadmium factor 1), which encodes a transporter that sequesters toxic metal(loid)s into the vacuoles of budding yeast, and tested these transgenic plants in soil taken from a closed mine site contaminated with multiple toxic metal(loid)s under greenhouse and field conditions. The YCF1-expressing transgenic poplar plants exhibited enhanced growth, reduced toxicity symptoms, and increased Cd content in the aerial tissue compared to the non-transgenic plants. Furthermore, the plants accumulated increased amounts of Cd, Zn, and Pb in the root, because they could establish an extensive root system in mine tailing soil. These results suggest that the generation of YCF1-expressing transgenic poplar represents the first step towards producing plants for phytoremediation. The YCF1-expressing poplar may be useful for phytostabilization and phytoattenuation, especially in highly contaminated regions, where wild-type plants cannot survive.

Identification of genes upregulated by pinewood nematode inoculation in Japanese red pine

Pine wilt disease caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, has destroyed huge areas of pine forest in East Asia, including Japan, China and Korea. No protection against PWN has been developed, and the responses of pine trees at the molecular level are unrecorded. We isolated and analyzed upregulated or newly induced genes from PWN-inoculated Japanese red pine (Pinus densiflora Sieb. et Zucc.) by using an annealing control primer system and suppression subtractive hybridization. Significant changes occurred in the transcript abundance of genes with functions related to defense, secondary metabolism and transcription, as the disease progressed. Other gene transcripts encoding pathogenesis-related proteins, pinosylvin synthases and metallothioneins were also more abundant in PWN-inoculated trees than in non-inoculated trees. Our report provides fundamental information on the molecular mechanisms controlling the biochemical and physiological responses of Japanese red pine trees to PWN invasion.

Drought, salt and wounding stress induce the expression of the plasma membrane intrinsic protein 1 gene in poplar (Populus alba × P. tremula var. glandulosa)

Water uptake across cell membranes is a principal requirement for plant growth at both the cellular and whole-plant levels; water movement through plant membranes is regulated by aquaporins (AQPs) or major intrinsic proteins (MIPs). We examined the expression characteristics of the poplar plasma membrane intrinsic protein 1 gene (PatPIP1), a type of MIP, which was isolated from a suspension cell cDNA library of Populus alba×P. tremula var. glandulosa. Examination of protoplasts expressing the p35S-PatPIP1::sGFP fusion protein revealed that the protein was localized in the plasma membrane. Northern blot analysis revealed that the gene was strongly expressed in poplar roots and leaves. Gene expression was inducible by abiotic factors including drought, salinity, cold temperatures and wounding, and also by plant hormones including gibberellic acid, jasmonic acid and salicylic acid. Since we found that the PatPIP1 gene was strongly expressed in response to mannitol, NaCl, jasmonic acid and wounding, we propose that PatPIP1 plays an essential role in the defense of plants against water stress.

Isolation and characterization of osmotic stress-induced genes in poplar cells by suppression subtractive hybridization and cDNA microarray analysis

Osmotic stress induces changes in the expression of various genes including those associated with drought tolerance, cell wall metabolism and defense. We isolated 852 cDNA clones, the expression of which is induced by osmotic stress, from cells of a hybrid poplar (Populus alba x Populus tremula var. glandulosa) by suppression subtractive hybridization after mannitol treatment. We examined how stress affected their expression using cDNA microarray analysis, which identified 104 genes significantly up-regulated by osmotic stress. These include genes with functions related to transcription, signal transduction, cell wall metabolism and defense. Other gene transcripts encoding cysteine protease and aquaporin are also up-regulated during osmotic stress. The function of about one-third of the genes in poplar cells that were significantly up-regulated by stress is not known, suggesting that the cell suspension may offer an opportunity of finding novel genes otherwise never expressed and that we still need more information at the molecular level.

Enhanced drought and salt tolerance by expression of AtGSK1 gene in poplar

A GSK3/shaggy-like kinase (AtGSK1) has been implicated in the regulation of drought and salt tolerance. We transferred AtGSK1 from Arabidopsis thaliana to a hybrid poplar (Populus albaxa0×xa0P. tremula var. grandulosa) to determine the effect of the transgene expression in the transgenic trees. The results from northern blot and RT-PCR analyses showed that the expression level varied among the transgenic lines. During their culture on tissue culture media, the transgenic poplars formed vigorous growing roots even in the presence of 125xa0mM NaCl and callus in the presence of 150xa0mM NaCl. When the transgenic poplars were growing in pots and provided with NaCl solution, they stayed much healthier than did nontransgenic poplars, showing higher rates of photosynthetic rates, stomatal conductance, and evaporation rates under the stress. Whereas the total level of leaf Na+ level increased dramatically in transgenic poplars under severe saline conditions (150xa0mM NaCl), that of leaf K+ decreased in the same plants under the same conditions. Total root Na+ level increased in nontransgenic poplars under severe saline conditions. In contrast, total root K+ level decreased in the same plants under the same conditions. The chloride content and relative electrical conductivity of the transgenic poplars after salt stress treatment were lower than those of nontransgenic poplars. The transgenic poplars were also tolerant to up to 20xa0% PEG remaining significantly healthy when compared with nontransgenic poplars with necrosis and chlorosis symptoms. Another dramatic feature of the transgenic poplars was wilting tolerance for prolonged drought treatment up to 2xa0weeks. The results provide evidence that the expression of AtGSK1 gene conferred drought and salt tolerance in the transgenic poplars.

Microarray and suppression subtractive hybridization analyses of gene expression in hybrid poplar (Populus alba × Populus tremula var. glandulosa) cell suspension cultures after exposure to NaCl.

The gene expression profiles of hybrid poplar (Populus albaxa0×xa0Populus tremula var. glandulosa) cells in suspension culture after exposure to salinity (NaCl) induced stress were examined by constructing two suppression subtractive hybridization (SSH) libraries. cDNA from non-treated cells was used as a driver and cDNA samples from cell suspension cultures exposed to 150xa0mM NaCl for 2 or 10xa0h were used as testers. Randomly selected clones from each SSH library were sequenced and 727 high-quality expressed sequence tags (ESTs) were obtained and analyzed. Four novel ESTs were identified. Between the two libraries, 542 unique SSH clones were selected for placement on a cDNA microarray. In total, 18 differentially expressed genes were identified with 4 and 12 genes being significantly differentially expressed 2 and 10xa0h after the treatment, respectively. Genes related to metabolism and protein synthesis and several genes whose protein products are implicated in salt or other abiotic stress-related responses were expressed in the salt-stressed cells.

A Deletion in Fungal Ras Promoter in Two Korean Strains of Oak Mushroom (Lentinula edodes)

This study unexpectedly detected a deletion in the promoter region of ras gene in two Korean strains of oak mushrooms, Lentinula edodes (Berk.). Sequencing of the promoter regions revealed that one type consisting of two strains had a 113 bp deletion in the region. The pas promoter region of Korean strains differed by 16 bases from that of the Japanese strains. Between the two types of Korean strains, except for the deleted portion, only a single site appeared to be different.

Transgenic poplar expressing AtNDPK2 exhibits enhanced biomass in the LMO field

Abstract Nucleoside diphosphate kinase 2 (NDPK2) is known to regulate the expression of antioxidant genes and auxin-responsive genes in plants. Previously, it was noted that the overexpression of Arabidopsis NDPK2 (AtNDPK2) under the control of an oxidative stress-inducible SWPA2 promoter in transgenic poplar (Populus alba × P. tremular var. glandulosa) plants (referred to as SN plants) enhanced tolerance to oxidative stress and improved growth (Plant Biotechnol J 9: 34-347, 2011). In this study, growth of transgenic poplar was assessed under living modified organism (LMO) field conditions in terms of biomass in the next year. The growth of transgenic poplar plants increased in comparison with non-transgenic plants. The SN3 and SN4 transgenic lines had 1.6 and 1.2 times higher dry weight in stems than non-transgenic plants at 6 months after planting, respectively. Transgenic poplar also exhibited increased transcript levels of auxin-response genes such as IAA1, IAA2, IAA5 and IAA6. These results suggest that enhanced AtNDPK2 expression increases plant biomass in transgenic poplar through the regulation of auxin-response genes.Keywords Transgenic poplar, Nucleoside diphosphate kinase, Inducible promoter, Auxin-response genes, Biomass, Field conditions

Selection of transgenic Solanum nigrum L. used environmental remediation expressing organomercurial lyase

Methylmercury, an organic derivative, is the principal form of mercury that biomagnifies and causes neurodegenerative symptoms in animals. In recent years, living modified organism (LMO) resulting from biotechnology has played a highly visible and controversial role. Despite the potential benefits of this technology, public concerns have been raised about the environmental risk of LMO. The concern on the risk from LMO release has urged efforts to evaluate and manage the risks of the LMO. To build up the capacity building of risk assessment method for LMO used environmental remediation, we engineered Solanum nigrum L, expressing the modified bacterial gene, merB, encoding organomercurial lyase. Two independently isolated transgenic lines produced merB RNA. Transgenic Solanum nigrum leaf discs expressing merB gene showed organic mercury resistance, forming shoots well on growth medium containing methylmercury (II) chloride and phenylmercuric acetate while control plants breached. Transgenic merB seeds germinated and grew on growth medium containing methylmercury (II) chloride and phenylmercuric acetate. The merB transgenic plants will be used for risk assessment of natural environment.