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Oxidative stress derived from excess reactive oxygen species (ROS) is a major damaging factor for plants exposed to environmental stresses. Sweetpotato [ Ipomoea batatas (L.) Lam] has a relatively broad adaptability to harsh environmental conditions compared to other staple crops. In this study, to select stress-tolerant sweetpotato cultivars for sources of molecular breeding on marginal lands, we eval-uated the ion leakage values in 10 different cultivars after treatment of methyl viologen (MV), an ROS-generating non- selective herbicide, to leaf discs. DPPH radical scavenging activity and the contents of total phenolics were also inves-tigated. The ion leakage of each cultivar showed a diverse value, which is well correlated with DPPH radical scavenging activity of each cultivar. DPPH radical scavenging activity also showed a high corelation with the contents of total phenolic contents. Three cultivars of Yanshu 8, Shinhwangmi and Shinzami showed high antioxidant activity. Our results suggest that a simple and efficient DPPH radical scavenging activity would be a suitable method to select potential cul-tivars with enhanced tolerance to multiple environmental stress.

Starch serves not only as an energy source for plants, animals, and humans but also as an environmentally friendly alternative for fossil fuels. Progress in understanding of starch biosynthesis, and the isolation of many genes involved in this process have enabled the genetic modification of crops in a rational manner to produce novel starches with improved functionality. Starch is composed of two glucose polymers, amylose and amylopectin. The amylose and amy- lopectin ratio in starch affects its physical and physic- ochemical properties. Alteration in starch structure can be achieved by modifying genes encoding the enzymes responsible for starch biosynthesis and starch hydrolysis. Here, we describe recent findings concerning the starch modification in sweetpotato. Sweetpotato (Ipomoea batatas (L.) Lam) ranks seventh in annual production among food crops in the world as an important starch source. To develop transgenic sweetpotato plants with modifying starch com- position, we constructed transformation vectors overex- pressing granule bound starch synthase I and inhibiting amylopectin synthesis genes such as starch branching enzyme and isoamylase under the control of 35S promoter, respectively. Transformation of sweetpotato (cv. Yulmi) is

Three peroxidase (POD) cDNAs were isolated from dehydration-treated fibrous roots of sweetpotato (Ipomoea batatas) plant via the screening of a cDNA library, and their expressions were assessed to characterize functions of each POD in relation to environmental stress. Three PODs were divided into two groups, designated the basic PODs (swpb4, swpb5) and the anionic PODs (swpa7), on the basis of the pI values of mature proteins. Fluorescence microscope analysis indicated that three PODs are secreted into the extracellular space. RT-PCR analysis revealed that POD genes have diverse expression patterns in a variety of plant tissues. Swpb4 was abundantly expressed in stem tissues, whereas the expression levels of swpb5 and swpa7 transcripts were high in fibrous and thick pigmented roots. Swpb4 and swpa7 showed abundant expression levels in suspension cultured cells. Three POD genes responded differently in the leaf and fibrous roots in response to a variety of stresses including dehydration, temperature stress, stress-associated chemicals, and pathogenic bacteria.

Porcine epidemic diarrhea virus (PEDV) is an infectious and highly contagious virus of swine. In order to develop the transgenic tobacco culture cells producing PEDV antigen protein, four vectors expressing PEDV spike protein (SP) gene under the control of a CaMV 35S promoter were constructed. Four fragments of the SP region of PEDV, SP1 (444 bp, 1487-1930 bp), SP2 (1.7 kb, 2300-3987 bp), SP3 (1.4 kb, 1559-2950 bp), and SP4 (2.6 kb, 9-2643 bp) were amplified by PCR and then C-MYC tag was fused to the end of each SP gene, respectively. These cassettes are inserted into the pCAMBIA2300 (named as 35S::SP1-M, 35S::SP2-M 35S::SP3-M, and 35S::SP4-M, respectively). Tobacco (cv. BY-2) cultured cells were transformed by co-cultivation with Agrobacterium tumefaciens harboring expression vector. We selected kanamycin-resistant calli and checked for the presence of the introduced SP gene using PCR, resulting 70% of them showed the foreign gene. We selected the lines with high-level expression of PEDV antigen protein based on dot blot analysis. Southern blot analysis confirmed that the PEDV SP gene was integrated into the genome of the tobacco cultured cells. Northern blot analysis showed that the introduced gene was highly expressed in transgenic cultured cells. Transgenic tobacco cultured cells-derived antigen induced immunogenicity in mice as determined by a plaque reduction neutralization assay. These results suggest that the vectors expressing PEDV spike protein gene in this study will be useful for the development of transgenic plants and cultured cells producing PEDV antigene protein.

A new dehydration responsive element-binding (DREB) protein gene encoding for an AP2/EREBP-type transcription factor was isolated by screening of the cDNA library for dehydration-treated fibrous roots of sweetpotato (Ipomoea batatas). Its cDNA (referred to as swDREB1) fragment of 1206 bp was sequenced from, which a 257 amino acid residue protein was deduced with a predicted molecular weight of 28.17 kDa. A search of the protein BLAST database revealed that this protein can be classified as a typical member of a DREB subfamily. RT-PCR and northern analyses revealed diverse expression patterns of the swDREB1 gene in various tissues of intact sweetpotato plant, and in leaves and fibrous roots exposed to different stresses. The swDREB1 gene was highly expressed in stems and tuberous roots. In fibrous roots, its mRNA accumulation profiles clearly showed strong expression under various abiotic stress conditions such as dehydration, chilling, salt, methyl viologen (MV), and cadmium (Cd) treatment, whereas it did not respond to abscisic acid (ABA) or copper (Cu) treatment. The above results indicate that swDREB1 may be involved in the process of the plant response to diverse abiotic stresses through an ABA-independent pathway.

Human lactoferrin (hLf) is an iron-binding glycoprotein that has been considered to play many biological roles in the human, including the stimulation of the immune system, antimicrobial and anti-inflammatory effects, and regulation of iron absorption. We generated transgenic Siberian ginseng (Acanthopanax senticosus) cell cultures producing a functional hLf protein using the signal peptide sequence from the endoplasmic reticulum and driven by an oxidative stress-inducibleSWPA2 promoter which is highly expressed in plant cell cultures. The production of hLf increased proportionally to cell growth and showed a maximal level (up to 3.6% of total soluble protein) at the stationary phase in suspension cultures. Full-length hLf protein was identified by immunoblot analysis in transgenic cell cultures of Siberian ginseng. Recombinant hLf (rhLf) was purified from suspension cells of Siberian ginseng by ammonium sulfate precipitation, cation-exchange and gel filtration chromatography. N-terminal sequences of rhLf were identical to native hLf (nhLf). The overall monosaccharide composition of rhLf showed the presence of plant specific xylose while sialic acid is absent. Antibacterial activity of purified rhLf was higher than that of nhLf. Taken together, we anticipate that medicinal Siberian ginseng cultured cells, as demonstrated by this study, will be a biotechnologically useful source for commercial production of functional hLf not requiring further purification.

Transformed sweetpotato (Ipomoea batatas (L.) Lam. cv. Yulmi) plants were developed from embryogenic calli following Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105/pCAMBIA2301 harboring genes for intron -glucuronidase (GUS) and kanamycin resistance. Transient expression of GUS gene was found to be higher when embryogenic calli were co-cultivated with Agrobacterium for 2 days. The co-cultured embryogenic calli transferred to selective MS medium containing 1mg/L 2,4-D, 100mg/L kanamycin, and 400mg/L claforan. These embryogenic calli were subcultured to the same selection medium at 4 weeks interval. Kanamycin-resistant calli transferred to hormone-free MS medium with kanamycin gave rise to somatic embryos and then converted into plantlets in the same medium. Southern blot analysis confirmed that the GUS gene was inserted into the genome of the sweetpotato plants. A histochemical assay revealed that the GUS gene was preferentially expressed in the leaf, petiole, and vascular tissue and tip of root.

Low-temperature stress represents one of the principal limitations affecting the distribution and productivity of many plant species, including crops such as sweetpotato. Transgenic sweetpotato (Ipomoea batatas L. cv. Yulmi) plants expressing the soybean cold-inducible zinc finger protein (SCOF-1) under control of an oxidative stress-inducible peroxidase (SWPA2) promoter (referred to as SF plants), were developed and evaluated for enhanced tolerance to low-temperature conditions. Following 4 °C treatment of SF plants, SCOF-1 expression correlated positively with tolerance to low-temperature stress at the leaf disc level. Increased SCOF-1 expression also correlated with enhanced tolerance to different low-temperature treatments at the whole plant level. SF plants treated with low-temperature stress (4 or 10 °C for 30 h) exhibited less of a reduction in photosynthetic activity and lipid peroxidation levels than non-transgenic (NT) plants. Furthermore, the photosynthetic activity and lipid peroxidation levels of SF plants recovered to near pre-stress levels after 12 h of recovery at 25 °C. In contrast, these activities remained at a reduced level in NT plants after the same recovery period. Thus, this study has shown that low-temperature stress in sweetpotato can be efficiently modulated by overexpression of SCOF-1.

In plants, nucleoside diphosphate kinase 2 (NDPK2) is known to regulate the expression of antioxidant genes. In this study, we developed transgenic potato plants (Solanum tuberosum L. cv. Atlantic) expressing Arabidopsis NDPK2 (AtNDPK2) gene in cytosols under the control of an oxidative stress-inducible SWPA2 promoter (referred to as SN plants) or enhanced CaMV 35S promoter (EN plants) and evaluated their tolerance to various environmental stress, including methyl viologen (MV)-mediated oxidative stress, high temperature, and salt stress. When 250 muM MV was sprayed to whole plants, plants expressing NDPK2 showed significantly an enhanced tolerance compared to non-transgenic (NT) plants. SN plants and EN plants showed 51% and 32% less visible damage than NT plants, respectively. Transcript level of AtNDPK2 gene and NDPK2 activity in SN plants following MV treatment well reflected the plant phenotype. Ascorbate peroxidase (APX) activity was also increased in MV-treated SN plants. In addition, SN plants showed enhanced tolerance to high temperature at 42 degrees C. The photosynthetic activity of SN plants after treatment of high temperature was decreased by about 10% compared to the plants grown at 25 degrees C, whereas that of NT plants declined by 30%. When treated with 80 mM NaCl onto the plantlets, both SN plants and EN plants also showed a significant reduced damage in root growth. These results indicate that overexpression of NDPK2 under the stress-inducible SWPA2 promoter might efficiently regulate the oxidative stress derived from various environmental stresses.

BTF3 (betaNAC) was originally isolated as a general transcription factor required for RNA polymerase II-dependent transcription, and later found to be a beta-subunit of nascent-polypeptide-associated complex that has been implicated in regulating protein localization during translation. In this study, virus-induced gene silencing of NbBTF3 encoding a Nicotiana benthamiana homolog of human BTF3 caused leaf yellowing and abnormal leaf morphology without altering the overall growth of the plant. The NbBTF3 gene is constitutively expressed and the NbBTF3-GFP fusion protein is primarily targeted to the nucleus. At the cellular level, downregulation of NbBTF3 expression reduced the chloroplast sizes and chlorophyll contents. The affected cells produced excessive amounts of reactive oxygen species. Furthermore, the transcript level of various plastid- and mitochondria-encoded genes was severely reduced in the NbBTF3-depleted leaf cells. These findings indicate that depletion of NbBTF3 activity preferentially affected development and/or physiology of chloroplasts and mitochondria in plants, possibly by hampering efficient translocation of the nascent organellar proteins into the organelles.

In humans, CHMP1 encodes a protein of dual function that plays a role in both modification of chromatin structure and endosomal vesicle trafficking. Recently, it was found that sal1, a CHMP1 homolog in maize, is important for the development of the aleurone cell layers in maize endosperm. In this study, we investigated the structure and function of a Nicotiana benthamiana CHMP1 homolog designated NbCHMP1. NbCHMP1 encodes a small protein with a bipartite nuclear localization signal at its N-terminus, and good homology with the corresponding genes from diverse plants and animals. NbCHMP1 mRNA was present at comparable levels in stems, roots, flowers, and leaves. A GFP fusion of the full length NbCHMP1 protein was localized to the cytosol in distinct structures, while a GFP fusion of its N-terminal 80 aa was targeted to the nucleus, suggesting dual-targeting of NbCHMP1 in plants. Overexpression of NbCHMP1 in yeast did not affect its growth and the expressed protein was present in the cytosol in particulate form. Virus-induced gene silencing of NbCHMP1 resulted in subtle alterations of leaf morphology and color, without significantly affecting plant viability or development. Thus the CHMP1 homolog apparently does not play an essential role in the development of the vegetative tissues of N. benthamiana, in contrast to the essential role of sail in formation of the aleurone cell layers during maize endosperm development.

The 26S proteasome involved in degradation of proteins covalently modified with polyubiquitin consists of the 20S proteasome and 19S regulatory complex. The NbPAF gene encoding the alpha6 subunit of the 20S proteasome was identified from Nicotiana benthamiana. NbPAF exhibits high sequence homology with the corresponding genes from Arabidopsis, human and yeast. The deduced amino acid sequence of NbPAF reveals that this protein contains the proteasome alpha-type subunits signature and nuclear localization signal at the N-terminus. The genomic Southern blot analysis suggests that the N. benthamiana genome contains one copy of NbPAF. The NbPAF mRNA was detected abundantly in flowers and weakly in roots and stems, but it was almost undetectable in mature leaves. In response to stresses, accumulation of the NbPAF mRNA was stimulated by methyl jasmonate, NaCl and salicylic acid, but not by abscisic acid and cold treatment in leaves. The NbPAF-GFP fusion protein was localized in the cytoplasm and nucleus.