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MYB transcription factors play important roles in transcriptional regulation of many secondary metabolites including anthocyanins. We cloned the R2R3-MYB type IbMYB1 complementary DNAs from the purple-fleshed sweet potato ( Ipomoea batatas L. cv Sinzami) and investigated the expression patterns of IbMYB1 gene with IbMYB1a and IbMYB1b splice variants in leaf and root tissues of various sweet potato cultivars by reverse transcription-polymerase chain reaction. The transcripts of IbMYB1 were predominantly expressed in the purple-fleshed storage roots and they were also detectable in the leaf tissues accumulating anthocyanin pigments. In addition, transcript levels of IbMYB1 gene were up-regulated by treatment with methyl jasmonate or salicylic acid in leaf and root tissues of cv. White Star. To set up the intragenic vector system in sweet potato, we first evaluated the utilization of the IbMYB1 gene as a visible selectable marker. The IbMYB1a was transiently expressed in tobacco leaves under the control of a constitutive cauliflower mosaic virus 35S promoter, a root-specific and sucrose-inducible sporamin promoter, and an oxidative stress-inducible sweet potato anionic peroxidase2 promoter. We also showed that overexpression of IbMYB1a induced massive anthocyanin pigmentation in tobacco leaves and up-regulated the transcript levels of the structural genes in anthocyanin biosynthetic pathway. Furthermore, high-performance liquid chromatography analysis revealed that the expression of IbMYB1a led to production of cyanidin as a major core molecule of anthocyanidins in tobacco leaves. These results suggest that the IbMYB1 gene can be applicable to a visible marker for sweet potato transformation with intragenic vectors, as well as the production of anthocyanin as important nutritive value in other plant species. [ABSTRACT FROM AUTHOR] more...

Plant 2-Cys peroxiredoxins (2-Cys Prx) has both peroxidase and chaperon function. We overexpressed an Arabidopsis 2-Cys Prx in transgenic tall fescue ( Festuca arundinacea) plants to confer tolerance against heat and methyl viologen (MV) stress. Transgenic plants were generated by Agrobacterium-mediated genetic transformation, and integration and expression of the transgene was confirmed by Southern, northern and western blot analyses. Compared to control plants, transgenic plants had significantly less electrolyte leakage and thiobarbituric acid-reactive substances (TBARS) when exposed to heat or MV. Under heat stress (42°C), transgenic plants maintained their chlorophyll fluorescence (Fv /Fm) for 24 h while control plants lost chlorophyll fluorescence very quickly. We conclude that the high levels of 2-Cys Prx proteins in transgenic plants protect leaves from oxidative damage probably due to chaperon activity. [ABSTRACT FROM AUTHOR] more...

Plants synthesize compatible solutes such as glycinebetaine (GB) in response to abiotic stresses. To evaluate the synergistic and protective effect of GB, transgenic potato plants expressing superoxide dismutase (SOD) and ascorbate peroxidase (APX) targeting to chloroplasts (referred to as SSA plants) were retransformed with a bacterial choline oxidase ( codA) gene to synthesize GB in chloroplast in naturally occurring non-accumulator potato plants (including SSA) under the control of the stress-inducible SWPA2 promoter (referred to as SSAC plants). GB accumulation resulted in enhanced protection of these SSAC plants and lower levels of H2O2 compared with SSA and non-transgenic (NT) plants after methyl viologen (MV)-mediated oxidative stress. Additionally, SSAC plants demonstrated synergistically enhanced tolerance to salt and drought stresses at the whole-plant level. GB accumulation in SSAC plants helped to maintain higher activities of SOD, APX and catalase following oxidative, salt and drought stress treatments than is observed in SSA and NT plants. Conclusively, GB accumulation in SSAC plants along with overexpression of antioxidant genes rendered the plants tolerant to multiple environmental stresses in a synergistic fashion. [ABSTRACT FROM AUTHOR] more...

Transgenic sweetpotato ( Ipomoea batatas L. cv. Yulmi) plants expressing the Arabidopsis nucleoside diphosphate kinase 2 ( AtNDPK2) gene under the control of an oxidative stress–inducible peroxidase ( SWPA2) promoter (referred to as SN plants) were developed and evaluated for enhanced tolerance of SN plants under various abiotic stress conditions. 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. Interestingly, we observed that antioxidant enzyme activities such as peroxidase, ascorbate peroxidase, and catalase increased in MV-treated SN plants. In addition, SN plants showed enhanced tolerance to cold, high salinity, and drought stresses by an increase in the activity of H2O2 scavenging enzymes. These results indicate that overexpression of AtNDPK2 in sweetpotato might efficiently modulate oxidative stress from various environmental stresses. [ABSTRACT FROM AUTHOR] more...

Abstract  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. [ABSTRACT FROM AUTHOR] more...

Previously, the swpa4 peroxidase gene has been shown to be inducible by a variety of abiotic stresses and pathogenic infections in sweet potato ( Ipomoea batatas). To elucidate its regulatory mechanism at the transcriptional level under various stress conditions, we isolated and characterized the promoter region (2374 bp) of swpa4 (referred to as SWPA4). We performed a transient expression assay in tobacco protoplasts with deletions from the 5′-end of SWPA4 promoter fused to the β-glucuronidase (GUS) reporter gene. The −1408 and −374 bp deletions relative to the transcription start site (+1) showed 8 and 4.5 times higher GUS expression than the cauliflower mosaic virus 35S promoter, respectively. In addition, transgenic tobacco plants expressing GUS under the control of −2374, −1408 or −374 bp region of SWPA4 promoter were generated and studied in various tissues under abiotic stresses and pathogen infection. Gel mobility shift assays revealed that nuclear proteins from sweet potato cultured cells specifically interacted with 60-bp fragment (−178/−118) in −374 bp promoter region. In silico analysis indicated that four kinds of cis-acting regulatory sequences, reactive oxygen species-related element activator protein 1 (AP1), CCAAT/enhancer-binding protein alpha element, ethylene-responsive element (ERE) and heat-shock element, are present in the −60 bp region (−178/−118), suggesting that the −60 bp region might be associated with stress inducibility of the SWPA4 promoter. [ABSTRACT FROM AUTHOR] more...

Abstract  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 μM 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°C. The photosynthetic activity of SN plants after treatment of high temperature was decreased by about 10% compared to the plants grown at 25°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. [ABSTRACT FROM AUTHOR] more...

Abstract  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 μmol/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. [ABSTRACT FROM AUTHOR] more...

Plant peroxidases (POD) reduce hydrogen peroxide (H2O2) in the presence of an electron donor. Extracellular POD can also induce H2>O2 production and may perform a significant function in responses to environmental stresses via the regulation of H2O2 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 ; Jang et al. in Plant Physiol Biochem 42:451–455 ). 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 H2O2 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 H2O2 production. These results suggest that the expression of swpa4 in the apoplastic space may function as a positive defense signal in the H2O2-regulated stress response signaling pathway. [ABSTRACT FROM AUTHOR] more...

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.Cell Research (2007) 17:638–649. doi: 10.1038/cr.2007.34; publication online 24 April 2007 [ABSTRACT FROM AUTHOR] more...

Abstract  The effect of simultaneous expression of genes encoding three antioxidant enzymes, copper zinc superoxide dismutase (CuZnSOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), and dehydroascorbate (DHA) reductase (DHAR, EC 1.8.5.1), in the chloroplasts of tobacco plants was investigated under oxidative stress conditions. In previous studies, transgenic tobacco plants expressing both CuZnSOD and APX in chloroplast (CA plants), or DHAR in chloroplast showed enhanced tolerance to oxidative stresses, such as paraquat and salt. In this study, in order to develop transgenic plants that were more resistant to oxidative stress, we introduced the gene encoding DHAR into CA transgenic plants. Mature leaves of transgenic plants expressing all three antioxidant genes (CAD plants) had approximately 1.6–2.1 times higher DHAR activity, and higher ratios of reduced ascorbate (AsA) to DHA, and oxidized glutathione (GSSG) to reduced glutathione (GSH) compared to CA plants. CAD plants were more resistant to paraquat-induced stress, exhibiting only 18.1% reduction in membrane damage relative to CA plants. In addition, seedlings of CAD plants had enhanced tolerance to NaCI (100 mM) compared to CA plants. These results indicate that the simultaneous expression of multiple antioxidant enzymes, such as CuZnSOD, APX, and DHAR, in chloroplasts is more effective than single or double expression for developing transgenic plants with enhanced tolerance to multiple environmental stresses. [ABSTRACT FROM AUTHOR] more...

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 μ M methyl viologen (MV), they showed approximately 45% less damage than non-transformed (NT) plants. When 200 μ M 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. [ABSTRACT FROM AUTHOR] more...

To characterize the biochemical differences in paraquat-resistant and -susceptible biotypes of Erigeron canadensis L. collected from Korea, we investigated the constitutive levels of various antioxidants such as antioxidant enzymes and low molecular weight antioxidants in leaves, as well as after paraquat treatment. The activities of superoxide dismutase, peroxidase, ascorbate peroxidase, and catalase were higher in the paraquat-resistant biotype than in the paraquat-susceptible biotype. Reduced ascorbic acid content was higher in the resistant biotype, but the content of reduced glutathione was higher in the susceptible biotype. These results indicate that one of the paraquat-resistant mechanisms in E. canadensis in the present study might be related to protecting the activities of antioxidant enzymes, such as superoxide dismutase, peroxidase ascorbate peroxidase, and catalase, as well as the contents of low molecular weight antioxidants such as ascorbate and glutathione. [ABSTRACT FROM AUTHOR] more...

A vector system has been developed to express isoenzyme A1 of sweet potato peroxidase (POD) and was introduced into Saccharomyces cerevisiae. The system contains the signal sequence of Aspergillus oryzae α-amylase to facilitate the extracellular secretion of peroxidase under the control of constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter. In a batch culture using YNBDCA medium (yeast nitrogen base without amino acids 6.7 g l-1, Casamino acids 5 g l-1 and glucose 20 g l-1), the recombinant strain expressed the swpa1 gene giving a secretion yield of POD activity of ca. 90% of total expressed peroxidase. Supplementation with PMSF (0.05 mM) and Casamino acids (5 g/50 ml) increased extracellular POD activity to nearly 10 kU ml-1, equivalent to 1.5 kU g-1 cell dry wt. This is 9 fold higher than that obtained in medium without PMSF. From SDS-PAGE and native-PAGE analyses POD has an Mr of 53 kDa. [ABSTRACT FROM AUTHOR] more...

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. [ABSTRACT FROM AUTHOR] more...

Plants have diversified their leaf morphologies to adapt to diverse ecological niches. The molecular components responsible for regulating leaf morphology, however, have not been fully elucidated. By screening Arabidopsis activation-tagging lines, we identified a dominant mutant, which we designated longifolia1-1D (lng1-1D). lng1-1D plants were characterized by long petioles, narrow but extremely long leaf blades with serrated margins, elongated floral organs, and elongated siliques. The elongated leaves of the mutant were due to increased polar cell elongation rather than increased cell proliferation. Molecular characterization revealed that this phenotype was caused by overexpression of the novel gene LNG1, which was found to have a homolog, LNG2,in Arabidopsis. To further examine the role of the LNG genes, we characterized lng1 and lng2 loss-of-function mutant lines. In contrast to the elongated leaves of lng1-1D plants, the lng1 and lng2 mutants showed slightly decreased leaf length. Furthermore, the lng1-3 lng2-1 double mutant showed further decreased leaf length associated with less longitudinal polar cell elongation. The leaf widths in lng1-3 lng2-1 mutant plants were similar to those in wild type, implying that the role of LNG1 and LNG2 on polar cell elongation is similar to that of ROTUNDIFOLIA3 (ROT3). However, analysis of a lng1-3 lng2-1 rot3-1 triple mutant and of a lng1-1D rot3-1 double mutant indicated that LNG1 and LNG2 promote longitudinal cell elongation independently of ROT3. Taken together, these findings indicate that LNG1 and LNG2 are new components that regulate leaf morphology by positively promoting longitudinal polar cell elongation independently of ROT3 in Arabidopsis. [ABSTRACT FROM AUTHOR] more...

Sweetpotato [Ipomoea batatas (L.) Lam], which contains high levels of antioxidants such as ascorbate and carotenoids in its storage root, is one of the healthiest foods, as well as one of the best starch crops for growth on marginal lands. In plants, carotenoid pigments are involved in light harvesting for photosynthesis and are also essential for photo-protection against excess light. As dietary antioxidants in humans, these compounds benefit health by alleviating aging-related diseases. The storage root of sweetpotato is a good source of both carotenoids and carbohydrates for human consumption. Therefore, metabolic engineering of sweetpotato to increase the content of useful carotenoids represents an important agricultural goal. This effort has been facilitated by cloning of most of the carotenoid biosynthetic genes, as well as the Orange gene involved in carotenoid accumulation. In this review, we describe our current understanding of the regulation of biosynthesis, accumulation and catabolism of carotenoids in sweetpotato. A deeper understanding of these topics should contribute to development of new sweetpotato cultivars with higher levels of nutritional carotenoids and abiotic stress tolerance. [ABSTRACT FROM AUTHOR] more...