Stress-inducible Peroxidase Research Articles

Transgenic alfalfa plants expressing the sweetpotato Orange gene exhibit enhanced abiotic stress tolerance.

Alfalfa (Medicago sativa L.), a perennial forage crop with high nutritional content, is widely distributed in various environments worldwide. We recently demonstrated that the sweetpotato Orange gene (IbOr) is involved in increasing carotenoid accumulation and enhancing resistance to multiple abiotic stresses. In this study, in an effort to improve the nutritional quality and environmental stress tolerance of alfalfa, we transferred the IbOr gene into alfalfa (cv. Xinjiang Daye) under the control of an oxidative stress-inducible peroxidase (SWPA2) promoter through Agrobacterium tumefaciens-mediated transformation. Among the 11 transgenic alfalfa lines (referred to as SOR plants), three lines (SOR2, SOR3, and SOR8) selected based on their IbOr transcript levels were examined for their tolerance to methyl viologen (MV)-induced oxidative stress in a leaf disc assay. The SOR plants exhibited less damage in response to MV-mediated oxidative stress and salt stress than non-transgenic plants. The SOR plants also exhibited enhanced tolerance to drought stress, along with higher total carotenoid levels. The results suggest that SOR alfalfa plants would be useful as forage crops with improved nutritional value and increased tolerance to multiple abiotic stresses, which would enhance the development of sustainable agriculture on marginal lands.

Overexpression of the IbMYB1 gene in an orange-fleshed sweet potato cultivar produces a dual-pigmented transgenic sweet potato with improved antioxidant activity.

The R2R3-type protein IbMYB1 is a key regulator of anthocyanin biosynthesis in the storage roots of sweet potato [Ipomoea batatas (L.) Lam]. Previously, we demonstrated that IbMYB1 expression stimulated anthocyanin pigmentation in tobacco leaves and Arabidopsis. Here, we generated dual-pigmented transgenic sweet potato plants that accumulated high levels of both anthocyanins and carotenoids in a single sweet potato storage root. An orange-fleshed cultivar with high carotenoid levels was transformed with the IbMYB1 gene under the control of either the storage root-specific sporamin 1 (SPO1) promoter or the oxidative stress-inducible peroxidase anionic 2 (SWPA2) promoter. The SPO1-MYB transgenic lines exhibited higher anthocyanin levels in storage roots than empty vector control (EV) or SWPA2-MYB plants, but carotenoid content was unchanged. SWPA2-MYB transgenic lines exhibited higher levels of both anthocyanin and carotenoids than EV plants. Analysis of hydrolyzed anthocyanin extracts indicated that cyanidin and peonidin predominated in both overexpression lines. Quantitative reverse transcription-polymerase chain reaction analysis demonstrated that IbMYB1 expression in both IbMYB1 transgenic lines strongly induced the upregulation of several genes in the anthocyanin biosynthetic pathway, whereas the expression of carotenoid biosynthetic pathway genes varied between transgenic lines. Increased anthocyanin levels in transgenic plants also promoted the elevation of proanthocyanidin and total phenolic levels in fresh storage roots. Consequently, all IbMYB1 transgenic plants displayed much higher antioxidant activities than EV plants. In field cultivations, storage root yields varied between the transgenic lines. Taken together, our results indicate that overexpression of IbMYB1 is a highly promising strategy for the generation of transgenic plants with enhanced antioxidant capacity.

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.

SCOF-1-expressing transgenic sweetpotato plants show enhanced tolerance to low-temperature stress

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

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.

Expression of Arabidopsis NDPK2 increases antioxidant enzyme activities and enhances tolerance to multiple environmental stresses in transgenic sweetpotato plants

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.

Peptides with dual binding specificity for HLA-A2 and HLA-E are encoded by alternatively spliced isoforms of the antioxidant enzyme peroxiredoxin 5

Peptides with dual binding specificity for classical HLA class I and non-classical HLA-E molecules have been identified in virus-encoded proteins, but not in cellular proteins from normal or neoplastic cells. Expression screening of a melanoma cDNA library with a CTL clone recognizing an HLA-A2-restricted tumor-specific epitope encoded by mutant peroxiredoxin 5 (Prdx5), a stress-inducible peroxidase, led to the identification of two alternatively spliced isoforms of the same gene. These isoforms, which lack the catalytic cysteine fundamental for enzymatic activity, showed widespread expression in neoplastic and normal tissues but were unstable at the protein level, being detectable, following transient transfection, only after lactacystin treatment to inhibit proteasomal degradation. Isoform-specific sequences which formed, respectively, as result of exon 1 splicing to either exon 3 or 4, encoded two distinct nonapeptides (AMAPIKTHL and AMAPIKVRL, not present in the full-length protein) with anchor residues for HLA-A2 and HLA-E molecules and able to stabilize HLA-A2 and HLA-E cell surface expression. HLA-E+ targets, loaded with these peptides, were not recognized by NK cells expressing CD94/NKG2A inhibitory or CD94/NKG2C activatory receptors. However, both peptides were recognized, although with low avidity, by HLA-E-restricted CD8+ CTL. The nonapeptide AMAPIKVRL was used to elicit HLA-A2-restricted CTL clones that killed peptide-pulsed lymphoblastoid cell lines and melanoma cells expressing the corresponding Prdx5 isoform. Our results suggest that alternatively spliced isoforms of Prdx5, through the generation of HLA-E- and HLA-A2-restricted peptides may be part of immune-mediated stress response contributing to the detection and elimination of damaged normal or neoplastic cells.

엽록체 항산화기구 대사조절에 의한 환경스트레스 내성 식물

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.

담배 배양세포에서 인간 히스톤 단백질 H1.5의 발현

Collagen에 의해 유도되는 류마티스 관절염을 저해하는 효과가 있는 인간 histone 단백질 Hl.5 (hHl.5)를 산화스트레스 유도성 SWPA2 프로모터에 연결하여 형질전환 담배(Nicotiana tabacum L. cv Bright Yellow-2) 배양세포주를 개발하였다. hH1.5 유전자는 Agrobacterium 매개 형질전환 방법으로 담배 BY-2 배양세포에 도입되었다. 형질전환 캘러스는 150mg/L kanamycin과 300mg/L claforan이 포함된 변형된 MS 선발배지에서 선발하여, PCR분석으로 hHl.5 유전자의 도입을 확인하였다. 형질전환 현탁배양세포에서 hH1.5 단백질의 발현은 northern 분석과 Western 분석으로 확인하였는데, 담배배양세포에서 재조합hHl.5 단백질 (42 kDa)은 인간의 것 (32 kDa)과는 다른 크기의 단백질이 확인되었다. 금후 재조합 hH1.5 단백질의 자세한 특성규명이 요구된다. 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.

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.

High expression of a human lactoferrin in transgenic tobacco cell cultures.

Transgenic Nicotiana tabacum cell lines were developed expressing the human lactoferrin gene driven by the oxidative stress-inducible peroxidase (SWPA2) promoter. Western blot analysis showed the accumulation of both the full-length human lactoferrin protein as well as a immuno-reactive truncated fragment. Accumulation of human lactoferrin as monitored by ELISA increased proportionally to cell growth and reached a maximal (up to 4.3% of total soluble proteins) at the stationary phase of growth. Protein extracts from transgenic tobacco cells exhibited antibacterial activity.

A novel oxidative stress-inducible peroxidase promoter from sweetpotato: molecular cloning and characterization in transgenic tobacco plants and cultured cells.

A strong oxidative stress-inducible peroxidase (POD) promoter was cloned from sweetpotato (Ipomoea batatas) and characterized in transgenic tobacco plants and cultured cells in terms of environmental stress. A POD genomic clone (referred to as SWPA2) consisted of 1824 bp of sequence upstream of the translation start site, two introns (743 bp and 97 bp), and a 1073 bp coding region. SWPA2 had previously been found to encode an anionic POD which was highly expressed in response to oxidative stress. The SWPA2 promoter contained several cis-element sequences implicated in oxidative stress such as GCN-4, AP-1, HSTF, SP-1 reported in animal cells and a plant specific G-box. Employing a transient expression assay in tobacco protoplasts, with five different 5'-deletion mutants of the SWPA2 promoter fused to the beta-glucuronidase (GUS) reporter gene, the 1314 bp mutant deletion mutant showed about 30 times higher GUS expression than the CaMV 35S promoter. The expression of GUS activity in transgenic tobacco plants under the control of the -1314 SWPA2 promoter was strongly induced in response to environmental stresses including hydrogen peroxide, wounding and UV treatment. Furthermore, GUS activity in suspension cultures of transgenic cells derived from transgenic tobacco leaves containing the -1314 bp SWPA2 promoter-GUS fusion was strongly expressed after 15 days of subculture compared to other deletion mutants. We anticipate that the -1314 bp SWPA2 promoter will be biotechnologically useful for the development of transgenic plants with enhanced tolerance to environmental stress and particularly transgenic cell lines engineered to produce key pharmaceutical proteins.

항산화효소 유전자를 이용한 산업용 형질전환식물체 개발

각종 환경스트레스에 의해 생체 내에서 과량으로 생성되는 독성의 활성산소종 (ROS)은 산화스트레스를 유발시켜 식물의 질병, 노화 및 세포사멸을 촉진시킨다. 연구팀은 21세기 당면한 지구규모의 환경, 식량 및 보건문제 해결에 기여할 수 있는 기반기술 (plateform technology)를 개발하기 위하여 식물세포의 항산화기구 규명. 산화스트레스 유도성 항산화효소 유전자 개발. 스트레스 내성식물 개발에 관한 연구를 수행하고 있다. 여기에서는 항산화효소 유전자를 이용한 산업용식물체 개발에 관한 연구팀의 최근 연구결과를 중심으로 소개하였다. SOD와 APX 유전자를 엽록체에 동시에 발현시킨 담배 식물체는 MV, 건조 등 여러 스트레스에 대한 내성을 나타내어, 복합 스트레스내성 농작물개발에 활용이 기대된다 인체 DHAR 유전자를 엽록체에 도입시킨 담배식물체는 정상적으로 DHAR 유전자를 발현시켰으며, MV 등 여러 스트레스에 대한 내성을 나타내었다. 피부 노화방지 등에 관여하여 ROS를 제거하는 SOD를 과실에 과발현시킨 형질전환오이를 성공적으로 개발하여. SOD 오이는 기능성화장품의 용도로 제품개발이 기대된다. 또한 고구마에서 산화스트레스에 특이적으로 발현하는 POD (SWPA2.) promoter를 개발하였다. SWPA2 promoter는 스트레스내성 및 의료용 단백질 등 고부가가치 생리활성물질을 생산할 수 있는 산업용 형질전환 식물체 및 배양세포주 개발에 이용될 수 있을 것으로 기대된다. Oxidative stress derived from reactive oxygen species (ROS) is one of the major damaging factors in plants exposed to environmental stress. In order to develop the platform technology to solve the global food and environmental problems in the 21st century, we focus on the understanding of the antioxidative mechanism in plant cells, the development of oxidative stress-inducible antioxidant genes, and the development of transgenic plants with enhanced tolerance to stress. In this report, we describe our recent results on industrial transgenic plants by the gene manipulation of antioxidant enzymes. Transgenic tobacco plants expressing both superoxide dismutase (SOD) and ascorbate peroxidase (APX) in chloroplasts were developed and were evaluated their protection effects against stresses, suggesting that simultaneous overexpression of both SOD and APX in chloroplasts has synergistic effects to overcome the oxidative stress under unfavorable environments. Transgenic tobacco plants expressing a human dehydroascorbate reductase gene in chloroplasts were showed the protection against the oxidative stress in plants. Transgenic cucumber plants expressing high level of SOD in fruits were successfully generated to use the functional cosmetic purpose as a plant bioreactor. In addition, we developed a strong oxidative stress-inducible peroxidase promoter, SWPA2 from sweetpotato (lpomoea batatas). We anticipate that SWPA2 promoter will be biotechnologically useful for the development of transgenic plants with enhanced tolerance to environmental stress and particularly transgenic cell lines engineered to produce key pharmaceutical proteins.

Identification of the Heat Shock Protein of Neurospora crassa Corresponding to the Stress-Inducible Peroxidase

Heat shock and other stress treatments, resulting in thermotolerance in Neurospora crossa cells, stimulate the induction of a peroxidase at a high level. The putative gene encoding this heat shock-induced peroxidase (hspp) has been cloned, using a cDNA clone of the manganese peroxidase of Phanerochaete chrysosporium, as a probe. Northern blot analysis of total RNA from heat-shocked cell showed the stress-dependent accumulation of a ∼10 kb transcript. The identity of the HSP, corresponding to the heat-induced peroxidase, was established using hybrid-arrested in vitro translation.