Overexpression of potato ORANGE (StOR) and StOR mutant in Arabidopsis confers increased carotenoid accumulation and tolerance to abiotic stress.

ORANGE (OR) plays essential roles in regulating carotenoid homeostasis and enhancing the ability of plants to adapt to environmental stress. However, OR proteins have been functionally characterized in only a few plant species, and little is known about the role of potato OR (StOR). In this study, we characterized the StOR gene in potato (Solanum tuberosum L. cv. Atlantic). StOR is predominantly localized to the chloroplast, and its transcripts are tissue-specifically expressed and significantly induced in response to abiotic stress. Compared with wild type, overexpression of StOR increased ß-carotene levels up to 4.8-fold, whereas overexpression of StORHis with a conserved arginine to histidine substitution promoted ß-carotene accumulation up to 17.6-fold in Arabidopsis thaliana calli. Neither StOR nor StORHis overexpression dramatically affected the transcript levels of carotenoid biosynthetic genes. Furthermore, overexpression of either StOR or StORHis increased abiotic stress tolerance in Arabidopsis, which was associated with higher photosynthetic capacity and antioxidative activity. Taken together, these results indicate that StOR could be exploited as a potential new genetic tool for the improvement of crop nutritional quality and environmental stress tolerance.

Anti-obesity activity of anthocyanin and carotenoid extracts from color-fleshed sweet potatoes.

The anti-obesity effects of anthocyanin and carotenoid extracts from color-fleshed potatoes were studied with 3T3-L1 cells in vitro and high-fat diet (HFD)-induced obese mice in vivo. Treatment of 3T3-L1 adipocytes with anthocyanin and carotenoid extracts, respectively, after differentiation induction significantly inhibited fat accumulation by 63.1 and 83.5%. Studies of adipogenesis inhibition showed that the anthocyanin extract acts at intermediate stages, whereas the carotenoid extract influences all the stages. The extracts significantly diminished triglyceride (TG) content and peroxisome proliferator-activated receptor gamma (PPARγ) protein expression during adipogenesis of the intermediate stage. Oral administration of anthocyanin and carotenoid extracts, respectively, to HFD-fed mice significantly reduced weight gain and restored TG levels to normal or lower as compared to the HFD-fed group with improvement of a lipid profile, TG to HDL-C ratio. Histological differences in liver tissues revealed that the extracts protected the liver tissue from adipogenesis by HFD fed. This research presents the first direct demonstration that the two pigment extracts from sweet potato exhibit anti-obesity activities. PRACTICAL APPLICATIONS: Anthocyanins and carotenoids are the main pigments of purple- and orange-fleshed sweet potatoes, respectively, which are highly nutritious foods with antidiabetic and antioxidant properties. Obesity is a rapidly growing health problem that increases major risk factors of several serious diseases including cardiovascular diseases, diabetes, and cancer. The results of this research suggest that anthocyanin and carotenoid-rich extracts from color-fleshed sweet potatoes may be useful as supplementary ingredients for the treatment of obesity and related diseases.

Expressing the sweet potato orange gene in transgenic potato improves drought tolerance and marketable tuber production.

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.

Cloning and characterization of an Orange gene that increases carotenoid accumulation and salt stress tolerance in transgenic sweetpotato cultures.

The Orange (Or) gene is responsible for the accumulation of carotenoids in plants. We isolated the Or gene (IbOr) from storage roots of orange-fleshed sweetpotato (Ipomoea batatas L. Lam. cv. Sinhwangmi), and analyzed its function in transgenic sweetpotato calli. The IbOr gene has an open reading frame in the 942 bp cDNA, which encodes a 313-amino acid protein containing a cysteine-rich zinc finger domain. IbOr was strongly expressed in storage roots of orange-fleshed sweetpotato cultivars; it also was expressed in leaves, stems, and roots of cultivars with alternatively colored storage roots. IbOr transcription increased in response to abiotic stress, with gene expression reaching maximum at 2 h after treatment. Two different overexpression vectors of IbOr (IbOr-Wt and IbOr-Ins, which contained seven extra amino acids) were transformed into calli of white-fleshed sweetpotato [cv. Yulmi (Ym)] using Agrobacterium. The transgenic calli were easily selected because they developed a fine orange color. The expression levels of the IbOr transgene and genes involved in carotenoid biosynthesis in IbOr-Wt and IbOr-Ins transgenic calli were similar, and both transformants displayed higher expression levels than those in Ym calli. The contents of ß-carotene, lutein, and total carotenoids in IbOr-Ins transgenic lines were approximately 10, 6, and 14 times higher than those in Ym calli, respectively. The transgenic IbOr calli exhibited increased antioxidant activity and increased tolerance to salt stress. Our work shows that the IbOr gene may be useful for the biotechnological development of transgenic sweetpotato plants that accumulate increased carotenoid contents on marginal agricultural lands.

Enhanced antioxidant and protective activities on retinal ganglion cells of carotenoids-overexpressing transgenic carrot.

Carotenoids are considered to act as antioxidants and protect humans from serious disorders such as skin degeneration and ageing, cardiovascular disease, certain types of cancer, and age-related diseases of the eye. In this study, these chemopreventive activities of a carotenoids-overexpressing transgenic carrot were evaluated. The results of DPPH, hydroxyl, and superoxide radical scavenging tests demonstrate that the acetone extract obtained from the taproots of the carrot plants exhibits significant antioxidant activity. A higher activity was detected in the transgenic carrot extract compared with the wild-type extract. A chemopreventive activity test for degenerative diseases of the eye revealed that pretreatment with the carrot extract reduced cell death in a retinal ganglion cell line, RGC-5 cells exposed to 1-buthionine- (R,S)-sulfoximine and L-glutamic acid.

Enhanced salt stress tolerance in transgenic potato plants expressing IbMYB1, a sweet potato transcription factor.

IbMYB1, a transcription factor (TF) for R2R3-type MYB TFs, is a key regulator of anthocyanin biosynthesis during storage of sweet potatoes. Anthocyanins provide important antioxidants of nutritional value to humans, and also protect plants from oxidative stress. This study aimed to increase transgenic potatoes' (Solanum tuberosum cv. LongShu No.3) tolerance to environmental stress and enhance their nutritional value. Transgenic potato plants expressing IbMYB1 genes under the control of an oxidative stress-inducible peroxidase (SWPA2) promoter (referred to as SM plants) were successfully generated through Agrobacterium-mediated transformation. Two representative transgenic SM5 and SM12 lines were evaluated for enhanced tolerance to salinity, UV-B rays, and drought conditions. Following treatment of 100 mM NaCl, seedlings of SM5 and SM12 lines showed less root damage and more shoot growth than control lines expressing only an empty vector. Transgenic potato plants in pots treated with 400 mM NaCl showed high amounts of secondary metabolites, including phenols, anthocyanins, and flavonoids, compared with control plants. After treatment of 400 mM NaCl, transgenic potato plants also showed high DDPH radical scavenging activity and high PS II photochemical efficiency compared with the control line. Furthermore, following treatment of NaCl, UV-B, and drought stress, the expression levels of IbMYB1 and several structural genes in the flavonoid biosynthesis such as CHS, DFR, and ANS in transgenic plants were found to be correlated with plant phenotype. The results suggest that enhanced IbMYB1 expression affects secondary metabolism, which leads to improved tolerance ability in transgenic potatoes.

Down-regulation of ß-carotene hydroxylase increases ß-carotene and total carotenoids enhancing salt stress tolerance in transgenic cultured cells of sweetpotato.

Sweetpotato (Ipomoea batatas Lam.) is an important industrial crop and source of food that contains useful components, including antioxidants such as carotenoids. ß-Carotene hydroxylase (CHY-ß) is a key regulatory enzyme in the beta-beta-branch of carotenoid biosynthesis and it catalyzes hydroxylation into both ß-carotene to ß-cryptoxanthin and ß-cryptoxanthin to zeaxanthin. To increase the ß-carotene content of sweetpotato through the inhibition of further hydroxylation of ß-carotene, the effects of silencing CHY-ß in the carotenoid biosynthetic pathway were evaluated. A partial cDNA encoding CHY-ß was cloned from the storage roots of orange-fleshed sweetpotato (cv. Shinhwangmi) to generate an RNA interference-IbCHY-ß construct. This construct was introduced into cultured cells of white-fleshed sweetpotato (cv. Yulmi). Reverse transcription-polymerase chain reaction analysis confirmed the successful suppression of IbCHY-ß gene expression in transgenic cultured cells. The expression level of phytoene synthase and lycopene ß-cyclase increased, whereas the expression of other genes showed no detectable change. Down-regulation of IbCHY-ß gene expression changed the composition and levels of carotenoids between non-transgenic (NT) and transgenic cells. In transgenic line #7, the total carotenoid content reached a maximum of 117 µg/g dry weight, of which ß-carotene measured 34.43 µg/g dry weight. In addition, IbCHY-ß-silenced calli showed elevated ß-cryptoxanthin and zeaxanthin contents as well as high transcript level P450 gene. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH) in transgenic cells was more than twice that in NT cells. RNA-IbCHY-ß calli increased abscisic acid (ABA) content, which was accompanied by enhanced tolerance to salt stress. In addition, the production of reactive oxygen species measured by 3,3'-diaminobenzidine (DAB) staining was significantly decreased in transgenic cultured cells under salt stress. Taken together, the present results indicate that down-regulation of IbCHY-ß increased ß-carotene contents and total carotenoids in transgenic plant cells and enhanced their antioxidant capacity.

Overexpression of 2-cysteine peroxiredoxin enhances tolerance to methyl viologen-mediated oxidative stress and high temperature in potato plants.

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.

Enhanced tolerance to methyl viologen-induced oxidative stress and high temperature in transgenic potato plants overexpressing the CuZnSOD, APX and NDPK2 genes.

Oxidative stress is a major threat for plants exposed to various environmental stresses. Previous studies found that transgenic potato plants expressing both copper zinc superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) (referred to as SSA plants), or nucleoside diphosphate kinase 2 (NDPK2) (SN plants), showed enhanced tolerance to methyl viologen (MV)-induced oxidative stress and high temperature. This study aimed to develop transgenic plants that were more tolerant of oxidative stress by introducing the NDPK2 gene into SSA potato plants under the control of an oxidative stress-inducible peroxidase (SWPA2) promoter to create SSAN plants. SSAN leaf discs and whole plants showed enhanced tolerance to MV, as compared to SSA, SN or non-transgenic (NT) plants. SSAN plants sprayed with 400 µM MV exhibited about 53 and 83% less visible damage than did SSA and SN plants, respectively. The expression levels of the CuZnSOD, APX and NDPK2 genes in SSAN plants following MV treatment correlated well with MV tolerance. SOD, APX, NDPK and catalase antioxidant enzyme activities were also increased in MV-treated SSAN plants. In addition, SSAN plants were more tolerant to high temperature stress at 42°C, exhibiting a 6.2% reduction in photosynthetic activity as compared to plants grown at 25°C. In contrast, the photosynthetic activities of SN and SSA plants decreased by 50 and 18%, respectively. These results indicate that the simultaneous overexpression of CuZnSOD, APX and NDPK2 is more effective than single or double transgene expression for developing plants with enhanced tolerance to various environmental stresses.

Simultaneous expression of choline oxidase, superoxide dismutase and ascorbate peroxidase in potato plant chloroplasts provides synergistically enhanced protection against various abiotic stresses.

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 H(2)O(2) 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.

Stress-induced expression of choline oxidase in potato plant chloroplasts confers enhanced tolerance to oxidative, salt, and drought stresses.

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.

Enhanced tolerance of transgenic potato plants overexpressing nucleoside diphosphate kinase 2 against multiple environmental stresses.

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.

Molecular characterization of a cDNA encoding DRE-binding transcription factor from dehydration-treated fibrous roots of sweetpotato.

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 1206bp was sequenced from, which a 257 amino acid residue protein was deduced with a predicted molecular weight of 28.17kDa. 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.

Enhanced tolerance of transgenic potato plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against oxidative stress and high temperature.

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.

Molecular characterization of a cDNA encoding copper/zinc superoxide dismutase from cultured cells of Manihot esculenta.

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.

Bax-induced cell death of Arabidopsis is meditated through reactive oxygen-dependent and -independent processes.

An Arabidopsis protoplast system was developed for dissecting plant cell death in individual cells. Bax, a mammalian pro-apoptotic member of the Bcl-2 family, induces apoptotic-like cell death in Arabidopsis. Bax accumulation in Arabidopsis mesophyll protoplasts expressing murine Bax cDNA from a glucocorticoid-inducible promoter results in cytological characteristics of apoptosis, namely DNA fragmentation, increased vacuolation, and loss of plasma membrane integrity. In vivo targeting analysis monitored using jellyfish green fluorescent protein (GFP) reporter indicated full-length Bax was localized to the mitochondria, as it does in animal cells. Deletion of the carboxyl-terminal transmembrane domain of Bax completely abolished targeting to mitochondria. Bax expression was followed by reactive oxygen species (ROS) accumulation. Treatment of protoplasts with the antioxidant N -acetyl- -cysteine (NAC) during induction of Bax expression strongly suppressed Bax-mediated ROS production and the cell death phenotype. However, some population of the ROS depleted cells still induced cell death, indicating that there is a process that Bax-mediated plant cell death is independent of ROS accumulation. Accordingly, suppression of Bax-mediated plant cell death also takes place in two different processes. Over-expression of a key redox-regulator, Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) down-regulated ROS accumulation and suppressed Bax-mediated cell death and transient expression of Arabidopsis Bax inhibitor-1 (AtBI-1) substantially suppressed Bax-induced cell death without altering cellular ROS level. Taken together, our results collectively suggest that the Bax-mediated cell death and its suppression in plants is mediated by ROS-dependent and -independent processes.

Transgenic ginseng cell lines that produce high levels of a human lactoferrin.

In order to produce a human lactoferrin (hLf) protein in cultured plant cells, we developed Korean ginseng (Panax ginseng) cell line using an oxidative stress-inducible peroxidase (SWPA2) promoter and characterized the production of human lactoferrin in cultured cells. A construct containing a targeting signal peptide from tobacco endoplasmic reticulum fused to human lactoferrin cDNA under the control of SWPA2 promoter was engineered. Transgenic Korean ginseng cell lines that produced a recombinant hLf protein were successfully generated and confirmed by PCR and Southern blot analyses. Western blot and ELISA analyses showed that hLf protein was synthesized in the transgenic cells. The production of hLf showed a maximal level (up to 3.0% of total soluble protein) in the stationary phase of callus cultures. These results suggest that the transgenic cell lines in this study will be biotechnologically useful for the commercial production of hLf protein in cell cultures, with no need for purification.