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Oxidative stress is one of the major causative factors for injury to plants exposed to environmental stresses. Plants have developed diverse defense mechanisms for scavenging oxidative stress-inducing molecules. The antioxidative enzyme 2-cysteine peroxiredoxin (2-Cys Prx) removes peroxides and protects the photosynthetic membrane from oxidative damage. In this study, transgenic potato (Solanum tuberosum L. cv. Atlantic) expressing At2-Cys Prx under control of the oxidative stress-inducible SWPA2 promoter or enhanced CaMV 35S promoter (referred to as SP and EP plants, respectively) was generated using Agrobacterium-mediated transformation. The transgenic plants were tested for tolerance to stress. Following treatment with 3 μM methyl viologen (MV), leaf discs from SP and EP plants showed approximately 33 and 15% less damage than non-transformed (NT) plants. When 300 μM MV was sprayed onto whole plants, the photosynthetic activity of SP plants decreased by 25%, whereas that of NT plants decreased by 60%. In addition, SP plants showed enhanced tolerance to high temperature at 42 °C. After treatment at high temperature, the photosynthetic activity of SP plants decreased by about 7% compared to plants grown at 25 °C, whereas it declined by 31% in NT plants. These results indicate that transgenic potato can efficiently regulate oxidative stress from various environmental stresses via overexpression of At2-Cys Prx under control of the stress-inducible SWPA2 promoter.

Nucleoside diphosphate kinase 2 (NDPK2) is known to regulate the expression of antioxidant genes in plants. Previously, we reported that overexpression of Arabidopsis NDPK2 (AtNDPK2) under the control of an oxidative stress-inducible SWPA2 promoter in transgenic potato and sweetpotato plants enhanced tolerance to various abiotic stresses. In this study, transgenic poplar (Populus alba × Poplus glandulosa) expressing the AtNDPK2 gene under the control of a SWPA2 promoter (referred to as SN) was generated to develop plants with enhanced tolerance to oxidative stress. The level of AtNDPK2 expression and NDPK activity in SN plants following methyl viologen (MV) treatment was positively correlated with the plant's tolerance to MV-mediated oxidative stress. We also observed that antioxidant enzyme activities such as ascorbate peroxidase, catalase and peroxidase were increased in MV-treated leaf discs of SN plants. The growth of SN plants was substantially increased under field conditions including increased branch number and stem diameter. SN plants exhibited higher transcript levels of the auxin-response genes IAA2 and IAA5. These results suggest that enhanced AtNDPK2 expression affects oxidative stress tolerance leading to improved plant growth in transgenic poplar.

Reactive oxygen species (ROS) are produced during seed desiccation, germination, and ageing, leading to cellular damage and seed deterioration and, therefore, decreased seed longevity. The effects of simultaneous over-expression of two antioxidant enzymes on seed longevity and seed germination under stressful conditions were investigated. Transgenic tobacco simultaneously over-expressing the Cu/Zn-superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) genes in plastids showed normal growth and seed development. Furthermore, the transgenic seeds displayed increased CuZnSOD and APX enzymatic activities during seed development and maintained antioxidant enzymatic activity after two years of dried storage at room temperature. The two-year stored non-transgenic seeds (aged NT seeds) had higher levels of ion leakage than the two-year stored transgenic seeds (aged CA seeds), indicating membrane damage caused by ROS was more severe in the aged NT seeds than the aged CA seeds. The aged CA seeds decreased germination rates as compared to newly harvested transgenic and non-transgenic seeds. The aged CA seeds, however, significantly increased germination rates under various abiotic stress conditions as compared to aged NT seeds. These data strongly suggest that simultaneous over-expression of the CuZnSOD and APX genes in plastids improves seed longevity and germination under various environmental stress conditions by attenuating the effects of oxidative stress produced by elongated storage conditions and harsh environmental stresses.

Three sweetpotato cultivars were investigated for their starch content and amylose/amylopectin ratio. Ym starch contains 87.2% amylopectin and 12.8% amylose, when total starch was calculated as 100%. The Zm cultivar contains 33.6% amylopectin and 18.2% amylose, and its total starch was calculated as 51.8% of that of Ym. The Hm cultivar contains 39.1% amylopectin and 30.5% amylose, and its total starch was 69.6%. We analyzed the expression levels of starch and sucrose biosynthesis-related genes including AGPases a, b, and c; sucrose synthases I and II; starch synthase I; GBSS I; and SBEs I and II. All genes tested in this experiment were detected only in Ym, while several genes showed very faint or no expression in Zm and Hm. We also measured tissue-specific expression of these genes in whole plants of Ym. Most of the genes are expressed in the stem and roots of the plants. Expression profiles of starch synthesis-related genes of the sweetpotato leaves were investigated after supplementing the different concentrations of sucrose solution. All genes in Ym were clearly induced by sucrose, but the expression levels of some of these genes did not change in Zm and Hm. The total starch content of Ym, Zm, and Hm gradually increased over time on addition of 3%, 6%, and 9% sucrose concentrations. The greatest accumulation was observed in Ym at 48 h, and it was almost 2.24 times higher than that of the (0%) control, while Zm and Hm showed 1.76 and 1.91 times higher levels of starch, respectively. These results indicate that cooperative expression of all related genes is essential for starch biosynthesis from sucrose. This is the first report on different sucrose contents and the efficiency with which exogenous sucrose switches on gene expression of starch biosynthesis-related genes among cultivars.

Sang-HoonLee *,** ,Ki-WonLee*,** ,KyungHeeKim *,Dae-JinYun *,Sang-SooKwak***andByung-HyunLee *ABSTRACTOxidative stress is the main limiting factor in crop productivity. To solve global environmental problemsusing the plant biotechnology, we have developed on the oxidative stress-tolerant transgenic tall fescueplants via Agrobacterium -mediated genetic transformation method. In order to develop transgenic tall fescue(Festucaarundinacea Schreb.) plants with enhanced tolerance to multiple environmental stresses, nucleotidediphosphate kinase gene under the control of CaMV35S promoter were introduced into genome of tallfescue plants. Proteomic analysis revealed that transgenic tall fescue not only accumulated NDP kinase 2protein in their cells, but also induced several other antioxindative enzyme-related proteins. When leaf discsof transgenic plants were subjected to cold stress, they showed approximately 30% less damage thanwild-type plants. In addition, transgenic tall fescue plants showed normal growth when transgenic plantswere subjected to 4 for 3 days treatments. These results suggest that transgene is important in ROSscavenging by induction of antioxidative proteins, and could improve abiotic stress tolerance in transgenictall fescue plants.(Keywords :Tall fescue, Antioxidant gene, Agrobacterium , Transformation)

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.

The food self-support rate on the basis of cereals in Korea is approximately 27%, which will threaten the national food security. The dramatic increase in population accompanied by rapid industrialization in developing coun-tries has caused imbalances in the supply of food and energy. To cope with these global crises over food and energy supplies as well as environmental problems, it is urgently required to develop new environmentally friendly industrial crop varieties to be grown on marginal lands including desertification areas for sustainable development. Sweet-potato ( Ipomoea batatas (L.) Lam.) ranks seventh in annual production among food crops in the world. Its wide adapt-ability on marginal lands and rich nutritional content provide a high potential for preventing malnutrition and enhancing food security in the developing countries. In addition, sweetpotato can be developed as a bioreactor to produce valuable industrial materials including bio-ethanol, functional feed and antioxidants by molecular breeding. In this respect, we focus on the molecular breeding of sweetpotato with multi-function on marginal lands. The strategies for devel-opment of environmentally friendly industrial sweetpotato will be introduced and discussed.

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

Sweetpotato [ Ipomoea batatas (L.) Lam] leaves are excellent source of low molecular weight antioxidants such as polyphenols, anthocyanins and carotenoids compared to other leafy vegetables. Endogenous antioxidants in sweet-potato help our bodies to prevent ageing, heart diseases and cancer. In this study, to develop the proper cultivars for the functional feed materials, we investigated the contents of anthocyanin, β-carotene, and polyphenols as well as DPPH radical scavenging activity in leaves of 14 different cultivars at the time of the harvest. They showed a diverse antioxidation activity. In DPPH radical scavenging activity, cultivars of Nanjing 9, Yulmi and Shinzami showed higher activity, whereas cv. Huiza 6 showed the lowest. Cultivars of Shinzami and Shinhwangmi had the highest anthocyanin (3.5 mg/g fr wt) and polyphenol (15.8 mg/g fr wt) content, respectively. Interestingly, there was a high correlation between cultivars with colorful pigments in storage roots and antioxidants activity in leaves. These results suggest that sweetpotato leaves with high antioxidant activity at harvest would be suitable for functional feed materials.

In many countries including China and U.S., researchers are developing methods to use sweetpotato as raw material for biofuel. We consider the sweetpotato is not only a source of green fuel, it eventually will provide various material including paper, adhesives, biodegradable plastics, and secondary metabolites. Sweetpotato is one of the high efficiency crop because it yields more calories per unit area than either maize or potato, and it requires the shortest growing cycle of the root crops grown in the tropics. Sweetpotato is the most useful crop for the coming starch- based industry era. Sweetpotato genetic resources are col-lected, characterized, evaluated, and maintained by U.S., China, Japan, and the International Potato Center. New va-rieties of sweetpotato using the proper genetic resources and molecular breeding will be developed to cope with the global food and energy in 21st century. 서 론 고구마의 원산지는 남미의 열대지역으로 알려져 있다. Austin (1988)은 고구마[ Ipomoea batatas (L.) Lam.]의 원산지가 멕시코의 유카탄 반도와 베네수엘라의 오리노코 강 사이의 지역이라고 주장하였고, 그 후 BC 2,500년경까지 카리브 해의 섬들과 남미지역에 전파되면서 품종으로 분화하였다는 가설을 세웠다. 이러한 가설은 최근 Zhang (1998) 등이 AFLP 다형성을 분석한 결과 확인되었다. 우리나라에는 1763년 (영조 39년)에 도입된 것으로 기록되고 있다. 영호(永湖) 조엄(趙儼) 선생이 당시 통신정사(通信正使)로 도일(渡日)하였다가 귀국 길에 대마도(對馬島)에서 고구마를 보고 백성들에게 전래함으로써 구황작물로 활용하였다. 지금도 이러한 그의 업적과 애민정신(愛民精神)을 기리는 행사와 출판물이 끊이지 않고 있다(www.wonju.go.kr, www.heritagebusan.com).우리나라에서 고구마는 식량으로 오랜 기간 이용되어 왔으며, 특히 식량이 부족한 시기나 지역에서 구황작물로 중요한 역할을 하였다. 쌀의 자급 후에는 주정(酒精)의 원료로써 새로운 역할을 하였고, 값싼 수입 타피오카를 주정의 원료로 이용하고부터는 그나마 재배면적과 생산량이 급격히 감소하였다. 그러나 최근 건강식품으로 각광을 받기 시작하면서 소비가 조금씩 늘어나는 추세이다. 우리나라에서 고구마의 용도는 매우 다양하며 외국에서는 보기 힘든 독특한 이용법을 자랑하고 있다. 그 중 대표적인 것은 고구마의 전분을 추출하여 당면을 제조하는 것이다. 또 다른 것은 엽병을 나물로 무쳐 먹는 활용법이다. 대만과 아프리카 일부지역에서 어린잎을 데쳐 먹기도 하지만 엽병을 식용하지는 않는다. 우리 선조들은 고구마를 이용해 다양한 요리법을 구사하였다. 앞으로 고구마는 건강식품으로써의 인기뿐만 아니라 전분의 원료로서 더욱 중요한 자리매김 할 것으로 생각된다. 바이오연료, 분해성 플라스틱, 이차 대사산물 (카로티노이드, 안토시아닌 등)의 생산 및 부산물의 사료 이용 등 다양하고 광범위한 활용 분야가 예상되고 있다. 고구마를 다양한 산업소재로 활용하기 위해서는 목적에 맞는 품종을 육성하여야 할 것이며, 따라서 이미 각국

To understand the adaptability of alfalfa (Medicago sativa L.) to chilling stress, we analyzed the antioxidative mechanism during seed germination. The germination rates of six alfalfa cultivars were studied comparatively at 10°C. Xinmu No. 1 and Northstar were selected as chilling stress-tolerant and stress-sensitive cultivars for further characterization. After chilling treatment, Xinmu No. 1 showed higher seedling growth than Northstar. Xinmu No. 1 exhibited low levels of hydrogen peroxide and lipid peroxidation compared with Northstar. In addition, shoots in Xinmu No. 1 treated with chilling showed higher activities of the superoxide dismutase, ascorbate peroxidase (APX), and catalase than those of Northstar, whereas Xinmu No. 1 showed higher APX activity in roots that Northstar. These results indicated that high antioxidation activity in Xinmu No. 1 under chilling stress is well associated with tolerance to chilling condition during germination.

The transfer of Mn-Superoxide Dismutase (SOD2) gene, complex gene (SA) of CuZnSOD and ascorbate peroxidase (APX), and NDP kinase 2 (NDPK2) gene into Korean 4 cultivars (cvs. Millenium White, Glory Blue, Glory Red, and Glory Purple) and 15 purebred lines of petunia was conducted using Agrobaterium-mediated technique. Two (Wongyo A2-16 and A2-36) of 15 purebred lines and one (cv. Glory Red) of 4 cultivars were effective for the transfer of SOD2 gene. The putative transgenic plants survived on the 2nd selection medium were 124. From PCR analysis, 118 (derived from 4 cultivars and 2 purebred lines) of 124 plants were confirmed to contain marker (npt II ) gene, while 58 of 118 plants did not have target genes. There were no plants with both npt II and SA genes. Twenty seven of 28 SOD2 transgenic plants were re-confirmed as transformants by Sothern analysis. SOD2 and NDPK2 genes were expressed in the transgenic petunias as the ratio of 77.8 to 100.0 % and 23.5%, respectively. T1 seeds were obtained from 36 acclimated transgenic plants (SOD2 34 plus NDPK2) in a glasshouse by self-pollination. *Corresponding author Tel 82-31-290-6198 Fax 82-31-290-6219 E-mail: lsy0504@rda.go.kr Introduction Petunia is one of major bedding plants over the world, and it mainly used to be planted on the side of road in company with pansy in Korea from spring to late in summer. A research project for breeding new cultivar of petunia was started in National Horticultural Research Institute (NHRI) (past name of National Institute of Horticultural & Herbal Science, NIHHS), rural development administration (RDA) of Korea, in 1992. After then, 26 cultivars had been developed by conventional breeding program of NHRI until 2002 (National Horticultural Research Institute 2006). One of characteristics, which should be improved in petunia, is resistance to abiotic stresses such as rainfall, humidity, and air pollution. Recently, there have been many reports to develop plants resistant to abiotic stresses through introduction of SOD or APX or NDPK genes (Fang et al. 2002; Haejeong et al. 2003; Tang et al. 2004a; Tang et al. 2004b; Tang et al. 2007; Kim et al. 2005). Successful transformation in most crops relies on genotypes (Shibata 2008), so, it will be necessary to use many genotypes for getting transgenic plant in petunia. By the way, considering patent, if possible, it will be desirable to use Korean domestic purebred lines or cultivars as materials for genetic transformation of petunia. Therefore, this study was executed to obtain transgenic petunia resistant to abiotic stresses through transfer of SOD2 and NDPK2 genes into Korean domestic purebred lines or cultivars of petunia by Agrobacterium-mediated transformation technique. Materials and methods

Sweetpotato (Ipomoea batatas (L). Lam.) is relatively tolerant to unfavorable growth conditions such as drought, yet has not been exploited to provide a better understanding of the molecular basis of drought stress tolerance. We obtained 983 high-quality expressed sequence tags of 100 bp or longer (average length of 700 bp) from cDNA libraries of detached white fibrous root tissues by subjecting them to dehydration for 6 h. The 431 cDNAs were each assigned a function by alignment using the BLASTX algorithm. Among them, three genes associated with various abiotic stresses and nine genes not previously associated with drought stress were selected for expression pattern analysis through detailed reverse transcription-polymerase chain reaction. The direct and indirect relationships of the 12 genes with drought tolerance mechanisms were ascertained at different developmental stages and under various stress conditions.

We have previously demonstrated that overexpression and characterization of Brassica rapa type-l metallothionein gene (BrMT1) in Arabidopsis which showed enhanced resistance to cadmium and ROS. Here, we present the consistent study of our previous report about BrMTs. BrMT3 expressing DTY167 cells showed resistance to Zn and Pb as well as Cd. Thus, we have developed the BrMT3 overexpression Arabidopsis to enhance capacity for metal stresses. Successful expression and localization were achieved using the rubisco transit peptides of RbcS-BrMT3-GFP protein, which was confirmed by western blot analysis with the GFP antibody and green fluorescence signal from the chloroplast. BrMT3 overexpression Arabidopsis plants exhibited a higher resistance to cadmium compared to control plants. This result indicates that BrMT3 would be applicable to the development of plants with enhanced resistance against heavy metal stresses.

To understand the functions of antioxidant enzymes during leaf development in sweetpotato, we investigated the activities of several antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POX), ascorbate peroxidase (APX) and catalase (CAT). Significant increases were observed in the activities of SOD, POX and APX during the late stage of leaf development, whereas CAT activity increased during the early developmental stage. By RT-PCR analysis, various POX and APX genes showed differential expression patterns during leaf development. Four POX genes swpa3, swpa4, swpa6, swpb4 and one APX gene swAPX1 exhibited high levels of gene expression during the senescence stage of leaf development, but two POX genes, swpa1 and swpa7 were preferentially expressed at both the mature green and the late senescence stages of leaf development. These results indicate that hydrogen peroxide (H2O2)-related antioxidant enzymes are differentially regulated in the process of leaf development of sweetpotato.

Alkaline soil is more challenging factor to grow plants than saline soil based on the size of its affected area. To reveal genetic expression changes by alkaline and saline soil, Leymus chinensis (Trin.) Tzvel. was examined response to Na2CO3 and NaCl stresses by using microarray chips comprising 1642 cDNA clones, previously reported by us. A total of 536 genes were responsive to Na2CO3 and NaCl stresses by up-regulation or down-regulation. We observed transcriptional changes arose more to Na2CO3 stress, or leaves than to NaCl stress or roots, respectively, in L. chinensis genome. Overall, 99 and 59 genes were up-regulated, while 365 and 176 genes were down-regulated in leaves and roots, respectively; demonstrating down-regulation occurs more as the response to the Na2CO3 and NaCl stress. The majority of the down-regulated genes (30.1%) were classified to photosynthesis related proteins while that of the up-regulated genes were categorized into metabolism proteins or noble proteins. Only 37 genes in the stressed leaves and 6 genes in the stressed roots were showed the same expression changing patterns between Na2CO3 and NaCl stresses during three time points of detections. Among those genes, 70% were constantly up-regulated or down-regulated during our detection presenting difference in the gene regulation systems against two difference stress, the saline-alkali stress (Na2CO3) and saline stress (NaCl). In addition, a total of 87 genes detected in this study were characterized to the unclassified proteins, whereas 72 had no similarity to the current GenBank databases which were considered as novel proteins detected in the alkali and saline stressed L. chinensis genome. The four important genes to abiotic stress tolerance, ACC, GDP, hsp70, and elF1 were detected constantly for all of three time points of stressed which are the grand candidates for establishing stress tolerance plants by developing transgenic.

A binary vector devoid of a plant selection-marker gene (designated as pSSA-F) was constructed to overcome bio-safety concerns about genetically modified plants. This vector carried chloroplast-targeted superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes under the control of an oxidative stress-inducible(SWPA2) promoter, and was utilized to transform potato (Solanum tuberosum L.). Integration of these foreign genes into transgenic plants was primarily performed via PCR with genomic DNA. Twelve marker-free transgenic lines were obtained by inoculating stem explants. The maximum transformation efficiency was 6.25% and averaged 2.2%. Successful integration of the SOD and APX genes rendered transgenic plants tolerant to methyl viologen-mediated oxidative stress at the leaf-disc and whole-plant levels. Our findings suggest that this technique for developing selection marker-free transgenic plants is feasible and can be employed with other crop species.

While the phytotoxic responses of arsenic (As) on plants have been studied extensively, based on physiological and biochemical aspects, very little is known about As stress-elicited changes in plants at the proteome level. Hydroponically grown 2-wk-old rice seedlings were exposed to different doses of arsenate, and roots were collected after 4 days of treatment, as well as after a recovery period. To gain a comprehensive understanding of the precise mechanisms underlying As toxicity, metabolism, and the defense reactions in plants, a comparative proteomic analysis of rice roots has been conducted in combination with physiological and biochemical analyses. Arsenic treatment resulted in increases of As accumulation, lipid peroxidation, and in vivo H(2)O(2) contents in roots. A total of 23 As-regulated proteins including predicted and novel ones were identified using 2-DE coupled with MS analyses. The expression levels of S-adenosylmethionine synthetase (SAMS), GSTs, cysteine synthase (CS), GST-tau, and tyrosine-specific protein phosphatase proteins (TSPP) were markedly up-regulated in response to arsenate, whereas treatment by H(2)O(2) also regulated the levels of CS suggesting that its expression was certainly regulated by As or As-induced oxidative stress. In addition, an omega domain containing GST was induced only by arsenate. However, it was not altered by treatment of arsenite, copper, or aluminum, suggesting that it may play a particular role in arsenate stress. Analysis of the total glutathione (GSH) content and enzymatic activity of glutathione reductase (GR) in rice roots during As stress revealed that their activities respond in a dose-dependent manner of As. These results suggest that SAMS, CS, GSTs, and GR presumably work synchronously wherein GSH plays a central role in protecting cells against As stress.

We examined the function of the rice (Oryza sativa L.) antiporter-regulating protein OsARP by overexpressing it in tobacco (Nicotiana tabacum L.). In public databases, this protein was annotated as a putative Os02g0465900 protein of rice. The OsARP gene was introduced into tobacco under the control of the cauliflower mosaic virus 35S promoter. The transformants were selected for their ability to grow on medium containing kanamycin. Incorporation of the transgene in the genome of tobacco was confirmed by PCR, and its expression was confirmed by Western blot analysis. Transgenic plants had better growth and vigor than non-transgenic plants under salt stress in vitro. Overexpression of OsARP in transgenic tobacco plants resulted in salt tolerance, and the plants had a higher rate of photosynthesis and effective PSII photon yield when compared with the wild type. The OsARP protein was localized in the tonoplast of rice plants. Transgenic plants accumulated more Na + in their leaf tissue than did wild-type plants. It is conceivable that the toxic effect of Na + in the cytosol might be reduced by sequestration into vacuoles. The rate of water loss was higher in the wild type than in transgenic plants under salt stress. Increased vacuolar solute accumulation and water retention could confer salt tolerance in transgenic plants. Tonoplast vesicles isolated from OsARP transgenic plants showed Na + /H + exchange rates 3-fold higher than those of wild-type plants. These results suggest that OsARP on the tonoplasts plays an important role in compartmentation of Na + into vacuoles. We suggest that OsARP is a new type of protein participating in Na + uptake in vacuoles.

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.

Potato (Solanum tuberosum L.) is generally considered to be sensitive to drought stress. Even short periods of water shortage can result in reduced tuber production and quality. We previously reported that transgenic potato plants expressing the sweet potato orange gene (IbOr) under the control of the stress-inducible SWPA2 promoter (referred to as SOR plants) showed increased tolerance to methyl viologen-mediated oxidative stress and high salinity, along with increased carotenoid contents. In this study, in an effort to improve the productivity and environmental stress tolerance of potato, we subjected transgenic potato plants expressing IbOr to water-deficient conditions in the greenhouse. The SOR plants exhibited increased tolerance to drought stress under greenhouse conditions. IbOr expression was associated with slightly negative phenotypes, including reduced tuber production. Controlling IbOr expression imparted the same degree of drought tolerance while ameliorating these negative phenotypic effects, leading to levels of tuber production similar to or better than those of wild-type plants under drought stress conditions. In particular, under drought stress, drought tolerance and the production of marketable tubers (over 80g) were improved in transgenic plants compared with non-transgenic plants. These results suggest that expressing the IbOr transgene can lead to significant gains in drought tolerance and tuber production in potato, thereby improving these agronomically important traits.

The complete genome sequence of a South Korean isolate of Rehmannia mosaic virus (ReMV) infecting Rehmannia glutinosa was determined through next-generation sequencing and Sanger sequencing. To our knowledge, this is the first report of a natural infection of R. glutinosa by ReMV in South Korea.

Oxidative stress is a major damaging factor for plants exposed to environmental stresses. Previously, we have generated transgenic potato (cv. Superior) plants expressing both CuZnSOD and APX genes in chloroplast under the control of an oxidative stress-inducible SWPA2 promoter (referred to as SSA plants) and selected the transgenic potato plant lines with tolerance against methyl viologen (MV)-mediated oxidative stress. When leaf discs of SSA plants were subjected to methyl viologen (MV), they showed approximately 40% less damage than non-transgenic (NT) plants. SSA plantlets were treated with MV stress, SSA plants also exhibited reduced damage in root growth. When 350 MV was sprayed onto the whole plants, SSA plants showed a significant reduction in visible damage, which was approximately 75% less damage than leaves of NT plants. These plants will be used for further analysis of tolerance to environmental stresses, such as high temperature and salt stress. These results suggest that transgenic potato (cv. Superior) plants would be a useful plant crop for commercial cultivation under unfavorable growth conditions.

Transgenic potato plants (Solanum tuberosum L. cv. Superior) with the ability to synthesize glycinebetaine (GB) in chloroplasts (referred to as SC plants) were developed via the introduction of the bacterial choline oxidase (codA) gene under the control of an oxidative stress-inducible SWPA2 promoter. SC1 and SC2 plants were selected via the evaluation of methyl viologen (MV)-mediated oxidative stress tolerance, using leaf discs for further characterization. The GB contents in the leaves of SC1 and SC2 plants following MV treatment were found to be 0.9 and 1.43 micromol/g fresh weight by HPLC analysis, respectively. In addition to reduced membrane damage after oxidative stress, the SC plants evidenced enhanced tolerance to NaCl and drought stress on the whole plant level. When the SC plants were subjected to two weeks of 150 mM NaCl stress, the photosynthetic activity of the SC1 and SC2 plants was attenuated by 38 and 27%, respectively, whereas that of non-transgenic (NT) plants was decreased by 58%. Under drought stress conditions, the SC plants maintained higher water contents and accumulated higher levels of vegetative biomass than was observed in the NT plants. These results indicate that stress-induced GB production in the chloroplasts of GB non-accumulating plants may prove useful in the development of industrial transgenic plants with increased tolerance to a variety of environmental stresses for sustainable agriculture applications.

Plant peroxidases (POD) reduce hydrogen peroxide (H(2)O(2)) in the presence of an electron donor. Extracellular POD can also induce H(2)O(2) production and may perform a significant function in responses to environmental stresses via the regulation of H(2)O(2) in plants. We previously described the isolation of 10 POD cDNA clones from cell cultures of sweetpotato (Ipomoea batatas). Among them, the expression of the swpa4 gene was profoundly induced by a variety of abiotic stresses and pathogenic infections (Park et al. in Mol Gen Genome 269:542-552 2003; Jang et al. in Plant Physiol Biochem 42:451-455 2004). In the present study, transgenic tobacco (Nicotiana tabacum) plants overexpressing the swpa4 gene under the control of the CaMV 35S promoter were generated in order to assess the function of swpa4 in planta. The transgenic plants exhibited an approximately 50-fold higher POD specific activity than was observed in control plants. Both transient expression analysis with the swpa4-GFP fusion protein and POD activity assays in the apoplastic washing fluid revealed that the swpa4 protein is secreted into the apoplastic space. In addition, a significantly enhanced tolerance to a variety of abiotic and biotic stresses occurred in the transgenic plants. These plants harbored increased lignin and phenolic content, and H(2)O(2 )was also generated under normal conditions. Furthermore, they showed an increased expression level of a variety of apoplastic acidic pathogenesis-related (PR) genes following enhanced H(2)O(2) production. These results suggest that the expression of swpa4 in the apoplastic space may function as a positive defense signal in the H(2)O(2)-regulated stress response signaling pathway.

Environmental stress is the major limiting factor in plant productivity. As an effort to solve the global food and environmental problems using the plant biotechnology, we have developed transgenic tall fescue (Festuca arundinacea Schreb.) plants via Agrobacterium-mediated gene transfer method. To develop transgenic tall fescue plants with enhanced tolerance to the environmental stresses, both CuZn superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) genes were incorporated in a pIG121 binary vector and the both of the genes were controlled separately by an oxidative stress-inducible sweet potato peroxidase 2 (SWPA2) premoter expressed in chloroplasts. Leaf discs of transgenic plants showed 10-30% less damage compared to the wild-type when they exposed to a wide range of environmental stresses including methyl viologen (MV), and heavy metals. In addition, when MV was sprayed onto the whole plants, transgenic plants showed a significant reduction of visible damage compared to wild-type plants that were almost damaged. These results suggest that over expression of CuZnSOD and APX genes in transgenic plants might be a useful strategy to protect the crops against a wide range of environmental stresses.

Previous studies have demonstrated the possible role of several aquaporins in seed germination. But systematic investigation of the role of aquaporin family members in this process is lacking. Here, the developmental regulation of plasma membrane intrinsic protein (PIP) expression throughout germination and post-germination processes in rice embryos was analyzed. The expression patterns of the PIPs suggest these aquaporins play different roles in seed germination and seedling growth. Partial silencing of the water channel genes, OsPIP1;1 and OsPIP1;3, reduced seed germination while over-expression of OsPIP1;3 promoted seed germination under water-stress conditions. Moreover, spatial expression analysis indicates that OsPIP1;3 is expressed predominantly in embryo during seed germination. Our data also revealed that the nitric oxide (NO) donors, sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO), promoted seed germination; furthermore, the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, inhibited germination and reduced the stimulative effects of SNP and GSNO on rice germination. Exogenous NO stimulated the transcription of OsPIP1;1, OsPIP1;2, OsPIP1;3 and OsPIP2;8 in germinating seeds. These results suggest that water channels play an important role in seed germination, acting, at least partly, in response to the NO signaling pathway.

We have used two aminoglycosides, G418 and paromomycin, to develop a reliable selection system fornptll transgenic sweet-potato (Ipomoea batatas (L.) Lam.). Embryogenic calli derived from shoot apical meristems were bombarded with gold particles coated with pCAMBIA2301, which contained thenptll andgusA genes. When compared on a kill curve that was based on calli proliferation and cell viability, G413-selection proved to be more efficient and had fewer escapes than kanamycin. These bombarded expiants were then selected on G418-containing media. The total time required from bombardment to plant establishment in soil was seven to nine months. Multiple copies of the transgene were integrated into the sweetpotato genome. Northern analysis confirmed transgene expression in the regenerated plants, and a paromomycin assay demonstrated that thenptll gene was functionally expressed in transformed sweetpotato. These molecular analyses and assays all showed that selection with G418 and paromomycin is reliable. So far, we have produced 69 transgenic events with this system, at a transformation frequency of approx. 1.1%. That efficiency is based on the number of transgenic plants obtained and the amount of calli bombarded. Thus, this selection method that combines G418 with paromomycin is now available for selectingnptll transgenic sweetpotato.

As barley is recalcitrant to transformation with current methods, a new improved system is required to apply genetic transformation in breeding programs. In a previous study, we defined optimal conditions for plant regeneration (PR) using mature embryos. This study was conducted to establish an improved transformation system employing the previously adjusted regeneration conditions. Optimal DNA delivery condition for the embryogenic calli developed from mature embryos was bombardment pressure of 1,100 psi at the target distance of 6 cm. The feasibility of the regeneration and DNA delivery conditions was confirmed by developing transgenic barley plants transformed with the Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) cDNA via particle bombardment of embryogenic calli from mature embryos. Stable integration of AtNDPK2 cDNA into barley genome was confirmed by PCR and Southern blot analysis of AtNDPK2 transgene. Transgenic plants showed about 10% reduction in membrane damage caused by methyl viologen, indicating the expression of AtNDPK2 transgene. The results demonstrated that the transformation system developed in this study employing the PR from mature embryo-derived embryonic callus is applicable in transgenic barley production.

Oxidative stress is one of the major factors causing injury to plants exposed to environmental stress. Transgenic sweetpotato [Ipomoea batatas (L.) Lam. cv. Yulmi] plants with an enhanced tolerance to multiple environmental stresses were developed by expressing the genes of both CuZn superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) under the control of an oxidative stress-inducible SWPA2 promoter in the chloroplasts of sweetpotato plants (referred to as SSA plants). SSA plants were successfully generated by the particle bombardment method and confirmed by PCR analysis. When leaf discs of SSA plants were subjected to 5 μM methyl viologen (MV), they showed approximately 45% less damage than non-transformed (NT) plants. When 200 μM MV was sprayed onto the whole plants, SSA plants showed a significant reduction in visible damage compared to leaves of NT plants, which were almost destroyed. The expression of the introduced CuZnSOD and APX genes in leaves of SSA plants following MV treatment was significantly induced, thereby reflecting increased levels of SOD and APX in the chloroplasts. APX activity in chloroplast fractions isolated from SSA plants was approximately 15-fold higher than that in their counterparts from NT plants. SSA plants treated with a chilling stress consisting of 4°C for 24 h exhibited an attenuated decrease in photosynthetic activity (Fv/Fm) relative to NT plants; furthermore, after 12 h of recovery following chilling, the Fv/Fm of SSA plants almost fully recovered to the initial levels, whereas NT plants remained at a lower level of Fv/Fm activity. These results suggest that SSA plants would be a useful plant crop for commercial cultivation under unfavorable growth conditions. In addition, the manipulation of the antioxidative mechanism in chloroplasts can be applied to the development of various other transgenic crops with an increased tolerance to multiple environmental stresses.

Glycine betaine (GB), a compatible solute, effectively stabilizes the structure and function of macromolecules and enhances abiotic stress tolerance in plants. We generated transgenic poplar plants (Populus alba × Populus glandulosa) expressing a bacterial choline oxidase (codA) gene under the control of the oxidative stress-inducible SWPA2 promoter (referred to as SC plants). Among the 13 SC plants generated, three lines (SC4, SC14 and SC21) were established based on codA transcript levels, tolerance to methyl viologen-mediated oxidative stress and Southern blot analysis. Growth was better in SC plants than in non-transgenic (NT) plants, which was related to elevated transcript levels of auxin-response genes. SC plants accumulated higher levels of GB under oxidative stress compared to the NT plants. In addition, SC plants exhibited increased tolerance to drought and salt stress, which was associated with increased efficiency of photosystem II activity. Finally, SC plants maintained lower levels of ion leakage and reactive oxygen species under cold stress compared to the NT plants. These observations suggest that SC plants might be useful for reforestation on global marginal lands, including desertification and reclaimed areas.

Summary Reversible modifications of target proteins by small ubiquitin-like modifier (SUMO) proteins are involved in many cellular processes in yeast and animals. Yet little is known about the function of sumoylation in plants. Here, we show that the SIZ1 gene, which encodes an Arabidopsis SUMO E3 ligase, regulates innate immunity. Mutant siz1 plants exhibit constitutive systemic-acquired resistance (SAR) characterized by elevated accumulation of salicylic acid (SA), increased expression of pathogenesis-related (PR) genes, and increased resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Transfer of the NahG gene to siz1 plants results in reversal of these phenotypes back to wild-type. Analyses of the double mutants, npr1 siz1, pad4 siz1 and ndr1 siz1 revealed that SIZ1 controls SA signalling. SIZ1 interacts epistatically with PAD4 to regulate PR expression and disease resistance. Consistent with these observations, siz1 plants exhibited enhanced resistance to Pst DC3000 expressing avrRps4, a bacterial avirulence determinant that responds to the EDS1/PAD4-dependent TIR-NBS-type R gene. In contrast, siz1 plants were not resistant to Pst DC3000 expressing avrRpm1, a bacterial avirulence determinant that responds to the NDR1-dependent CCNBS-type R gene. Jasmonic acid (JA)-induced PDF1.2 expression and susceptibility to Botrytis cinerea were unaltered in siz1 plants. Taken together, these results demonstrate that SIZ1 is required for SA and PAD4mediated R gene signalling, which in turn confers innate immunity in Arabidopsis.

Ecophysiological parameters of non-transgenic sweetpotato (NT) and transgenic sweetpotato (SSA) plants were compared to evaluate their resistance to multiple environmental stresses. Stomatal conductance and transpiration rate in NT plants decreased markedly from Day 6 after water was withheld, whereas those values in SSA plants showed relatively higher level during this period. Osmotic potential in SSA plants was reduced more negatively as leaf water potential decreased from Day 8 after dehydration treatment, while such reduction was not shown in NT plants under water stressed condition. SSA plants showed less membrane damage than in NT plants. As water stress and high light stress, were synchronously applied to NT and SSA plants maximal photochemical efficiency of PS II () in NT plants markedly decreased, while that in SSA plants was maintained relatively higher level. This trend of changes in between SSA plants and NT plants was more conspicuous as simultaneously treated with water stress, high light and high temperature stress. These results indicate that SSA plants are more resistive than NT plants to multiple environmental stresses and the enhanced resistive characteristics in SSA plants are based on osmotic adjustment under water stress condition and tolerance of membrane.

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.

Oxidative stress is a major damaging factor for plants exposed to environmental stresses. In order to develop transgenic potato plants with enhanced tolerance to environmental stress, the genes of both Cu/Zn superoxide dismutase and ascorbate peroxidase were expressed in chloroplasts under the control of an oxidative stress-inducible SWPA2 promoter (referred to as SSA plants). SSA plants showed enhanced tolerance to 250 microM methyl viologen, and visible damage in SSA plants was one-fourth that of non-transgenic (NT) plants that were almost destroyed. In addition, when SSA plants were treated with a high temperature of 42 degrees C for 20 h, the photosynthetic activity of SSA plants decreased by only 6%, whereas that of NT plants decreased by 29%. These results suggest that the manipulation of the antioxidative mechanism of the chloroplasts may be applied in the development of industrial transgenic crop plants with increased tolerance to multiple environmental stresses.

The calmodulin as a Ca-binding protein mediates cellular Ca signals in response to a wide array of stimuli in higher eukaryotes. Plants produce numerous calmodulin isoforms that exhibit differential gene expression patterns and sense different Ca signals. SCaM-5 is a soybean calmodulin that is involved in plant defense signaling. Here, we constructed a SCaM-5 CDNA under control of CaMV 35S promoter and transformed it into tomato (Lycopersicon esculentum). The constitutive over-expression of SCaM-5 in tomato plants exhibited a high levels of pathogenesis-related (PR) gene expression, and conferred an enhanced resistance to two fungal pathogen (Phytophthora capsici, Fusarium oxysporum), and a bacterial pathogen, Pseudomonas syringae pv. tomato DC3000. Thus, this results collectively suggest that SCaM-5 plays an important role in plant defense of tomato.

Leymus chinensis (Trin.) Tzvel., a xerophilous grass, is adapted to high pH (8.5–11.5) sodic soils. Consequently, the species must have developed molecular and physiological mechanisms to adapt itself under such stress conditions during the course of evolution. In order to identify and characterize the complexity of this adaptation, it is important to identify genes by large-scale sequencing of cDNA clones or expressed sequence tags (ESTs). cDNA libraries were constructed from leaf tissue collected from the plants growing in the natural habitat, and from root by subjecting the seedlings to Na 2 CO 3 stress. Of the 998 cDNA clones from each leaf and root tissues, 869 and 773, respectively, showed high quality sequences. In the leaf cDNA library, 574 clones were non-redundant, of which 295 had homology to the known transcripts, and 183 to unclassified proteins. The remaining 96 ESTs had no homology in the databases searched. Similar analysis of root cDNA library revealed that, of the 654 non-redundant clones, 269 had homology to the known transcripts, whereas 148 were novel. Sequence comparison of these clones using BLASTX led to the identification of 39 and 31 putative abiotic stress related genes from leaf and root cDNA library, respectively. The putative ESTs will serve as useful resource to understand the molecular basis of high pH sodic soil tolerance in plants.

Salt stress is one of major environmental factors influencing plant growth and development. To identify salt tolerance determinants in higher plants, a large-scale screen was conducted with a bialaphos marker-based T-DNA insertional collection of Arabidopsis ecotype C24 mutants. One line for salt stress-sensitive mutant (referred to as ssm1) exhibited increased sensitivity to both ionic (NaCl) and nonionic (mannitol) osmotic stress in a root growth assay. This result suggests that ssm1 mutant is involved in ion homeostasis and osmotic compensation in plant. Molecular cloning of the genomic DNA flanking T-DNA insert of ssm1 mutant was achieved by mutant genomic DNA library screening. T-DNA insertion appeared in the first exon of an open reading frame on F3M18.7, which is the same as AtSYP61. SSM1 is SYP61/OSM1 that is a member of the SNARE superfamily of proteins required for vesicular/target membrane fusions and factor related to abiotic stress.

Injury caused by reactive oxygen species (ROS), known as oxidative stress, is one of the major damaging factors in plants exposed to environmental stress. Chloroplasts are specially sensitive to damage by ROS because electrons that escape from the photosynthetic electron transfer system are able to react with relatively high concentration of in chloroplasts. To cope with oxidative stress, plants have evolved an efficient ROS-scavenging enzymes such as superoxide dismutase (SOD) and ascorbate peroxidase (APX), and low molecular weight antioxidants including ascorbate, glutathione and phenolic compounds. To maintain the productivity of plants under the stress condition, it is possible to fortify the antioxidative mechanisms in the chloroplasts by manipulating the antioxidation genes. A powerful gene expression system with an appropriate promoter is key requisite for excellent stress-tolerant plants. We developed a strong oxidative stress-inducible peroxidase (SWPA2) promoter from cultured cells of sweetpotato (Ipomoea batatas) as an industrial platform technology to develop transgenic plants with enhanced tolerance to environmental stress. Recently, in order to develop transgenic sweetpotato (tv. Yulmi) and potato (Solanum tuberosum L. cv. Atlantic and Superior) plants with enhanced tolerance to multiple stress, the genes of both CuZnSOD and APX were expressed in chloroplasts under the control of an SWPA2 promoter (referred to SSA plants). As expected, SSA sweetpotato and potato plants showed enhanced tolerance to methyl viologen-mediated oxidative stress. In addition, SSA plants showed enhanced tolerance to multiple stresses such as temperature stress, drought and sulphur dioxide. Our results strongly suggested that the rational manipulation of antioxidative mechanism in chloroplasts will be applicable to the development of all plant species with enhanced tolerance to multiple environmental stresses to contribute in solving the global food and environmental problems in the 21st century.

Calmodulin (CaM) influences many cellular processes by interacting with various proteins. Here, we isolated AtBAG6, an Arabidopsis CaM-binding protein that contains a central BCL-2-associated athanogene (BAG) domain. In yeast and plants, overexpression of AtBAG6 induced cell death phenotypes consistent with programmed cell death (PCD). Recombinant AtBAG6 had higher affinity for CaM in the absence of free Ca2 + than in its presence. An IQ motif (IQXXXRGXXXR, where X denotes any amino-acid) was required for Ca2 +-independent CaM complex formation and single amino-acid changes within this motif abrogated both AtBAG6-activated CaM-binding and cell death in yeast and plants. A 134-amino-acid stretch, encompassing both the IQ motif and BAG domain, was sufficient to induce cell death. Agents generating oxygen radicals, which are known to be involved in plant PCD, specifically induced the AtBAG6 transcript. Collectively, these results suggest that AtBAG6 is a stress-upregulated CaM-binding protein involved in plant PCD.

Superoxide dismutase (SOD) cDNA, mSOD2, encoding cytosolic copper/zinc SOD (CuZnSOD) cDNA was isolated from suspension-cultured cells of cassava (Manihot esculenta Crantz) by cDNA library screening, and its expression was investigated in relation to environmental stress. mSOD2 is 774 bp in length with an open reading frame (ORF) of 152 amino acids, corresponding to a protein of predicted molecular mass 15 kDa and a pI of 5.22. One copy of the mSOD2 gene was found to be present in the cassava genome by Southern analysis using an mSOD2 cDNA-specific probe. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed diverse expression patterns for the mSOD2 gene in various tissues of intact cassava plants, at various stages of the growth in suspension cultures, and in the leaf tissues exposed to different stresses. The mSOD2 gene was highly expressed in suspension-cultured cells and in the stems of intact plants. However, it was expressed at low levels in leaves and roots. During suspension cell growth, the mSOD2 transcript progressively increased during culture. Moreover, the mSOD2 gene in excised cassava leaves responded to various stresses in different ways. In particular, it was highly induced in leaf tissue by several abiotic stresses, including high temperature (37 degrees C), chilling (4 degrees C), methyl viologen (MV) exposure, and wounding treatment. These results indicate that the mSOD2 gene is involved in the antioxidative process triggered by oxidative stress induced by environmental change.

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.

Cultured plant cells are a good system for the study of antioxidant mechanisms and for the mass production of antioxidants, because they can be grown under conditions of high oxidative stress. Alterations in the intracellular and extracellular activities of three antioxidant enzymes, superoxide dismutase (SOD), guaiacol-type peroxidase (POD), and glutathione peroxidase (GPX), were investigated in suspension cultures of sweetpotato (Ipomoea batatas) during cell growth. Intracellular SOD activities (units/mg protein) at 15 days after subculture (DAS) and 30 DAS were 10 and 20 times higher, respectively, compared with the SOD activity at 1 DAS, whereas intracellular specific POD and GPX activities did not significantly increase until after 15 DAS, when they rapidly increased. The extracellular activities of the three enzymes in culture medium were much higher than were the intracellular activities. The change in extracellular SOD activity was similar to that of extracellular GPX during cell growth. Those activities showed high levels until 5 DAS and then significantly decreased. Extracellular POD activity had an almost constant level regardless of the cell growth stage. In addition, intracellular SOD and POD isozymes were quite different from those isozymes in the culture medium. The changes in SOD and POD isozymes observed here suggest that different isozymes might modulate the levels of reactive oxygen intermediates during cell growth. Characterization of extracellular antioxidant enzymes discovered here would provide a new understanding for defense mechanism in plants.

To establish high frequency shoot formation from two cultivars (Cheongyangjaerae and Myungan) of boxthorn (Lycium chinense Mill.), hypocotyl segments with cotyledons from seedlings were used as explants. High frequency adventitious shoot formation (more than 80%) were obtained from hypocotyl segments with cotyledon on MS medium supplemented with 1.0 mg/L zeatin, when precultured for 3 weeks under dark conditions followed by transfer to light conditions. But there was no shoot induction in the explants cultured without preculture under dark conditions. Roots were induced from the shoots when transferred to rooting medium supplemented with 1.0 mg/L IAA for 4 weeks. Regenerated plantlets were grown to normal mature plants in soil.

Arabidopsis NDPK2 (AtNDPK2) is a key singaling component that regulate cellular redox state and known to enhance multiple stress tolerance when over-expressed in Arabidopsis plant (Moon et al. 2003). In order to develop transgenic potato plants with enhanced tolerance to multiple stresses, we placed an AtNDPK2 cDNA under the control of a stress-inducible SWPA2 promoter or enhanced CaMV 35S promoter. Transgenic potato plants (cv. Superior and Atlantic) were generated using an Agrobacterium-mediated transformation system and selected on MS medium containing 100 mg/L kanamycin. Genomic Southern blot analysis confirmed the incorporation of AtNDPK2 cDNA into the potato genome. When potato leaf discs were treated with methyl viologen (MV) at 10 M, transgenic plants showed higher tolerance to MV than non-transgenic or vector-transformed plants. The NDPK2 transgenic potato plants will be further used for analysis of stress-tolerance to multiple environmental stresses.

Transgenic tobacco (Nicotiana tabacum L. cv Bright Yellow-2) cell lines expressing a human histone H1.5 (referred to as hH1.5), which suppress collagen-induced rheumatoid arthritis, were developed under the oxidative stress-inducible peroxidase (SWPA2) promoter. Tobacco BY-2 cells were transformed by Agrobacterium-mediated method. The kanamycin-resistant calli were selected on the modified MS medium containing 150mg/L kanamycin and 300mg/L claforan. Transgenic cell lines were confirmed by PCR and northern blot analysis. Recombinant hH1.5 (rhH1.5) protein (42 kDa) was also detected by Western blot analysis, showing a different molecular weight of human hH1.5 (32 kDa). These results suggested that a hH1.5 gene was properly introduced in tobacco cultured cells under the control of SWPA2 promoter. The further characterization of rhH1.5 protein remains to be studied.