Overexpression of sweetpotato swpa4 peroxidase results in increased hydrogen peroxide production and enhances stress tolerance in tobacco

Plant peroxidases (POD) reduce hydrogen peroxide (H₂O₂) in the presence of an electron donor. Extracellular POD can also induce H₂O₂ 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 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 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.     

Changes in reproductive function and white blood cell proliferation induced in mice by injection of a prolactin-expressing plasmid into muscle

Prolactin (PRL) is a pituitary hormone involved in various physiological processes, including lactation, mammary development, and immune function. To further investigate the in vivo and comparative endocrine roles of PRL, mouse PRL cDNA fused to the cytomegalovirus promoter, was introduced into muscle by direct injection. Previously we studied the function of rat PRL using the same protocol. PRL mRNA was detected in the muscle following injection by RT-PCR and subsequent Southern blot analysis. PRL was also detected and Western blot analysis revealed a relatively high level of serum PRL. In the pCMV-mPRL-injected female mice, the estrous cycle was extended, especially in diestrus stage and the uterus thickening that was shown in normal estrous stage was not observed. In the pCMV-mPRL-injected male mice, new blood vessels were first found at 5 weeks of age and fully developed blood vessels were found after 8 weeks in the testis. The number of Leydig cells increased within the testis and the testosterone level in serum was observed high. Finally, the number of white blood cells (WBCs) increased in the pCMV-mPRL-injected mice. The augmentation of WBCs persisted for at least 20 days after injection. When injection was combined with adrenalectomy, there was an even greater increase in number of WBCs, especially lymphocytes. This increase was returned normal by treatment with dexamethansone. Taken together, our data reveal that intramuscularly expressed mouse PRL influences reproductive functions in female, induces formation of new blood vessels in the testis, and augments WBC numbers. Of notice is that the Leydig cell proliferation with increased testosterone was conspicuously observed in the pCMV-mPRL-injected mice. These results also suggest subtle difference in function of PRL between mouse and rat species.     

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 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.     

Enhanced stress-tolerance of transgenic tobacco plants expressing a human dehydroascorbate reductase gene

To analyze the physiological role of dehydroascorbate reductase (DHAR, EC 1.8.5.1) catalyzing the reduction of DHA to ascorbate in environmental stress adaptation, T1 transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants expressing a human DHAR gene in chloroplasts were biochemically characterized and tested for responses to various stresses. Fully expanded leaves of transgenic plants had about 2.29 times higher DHAR activity (units/g fresh wt) than non-transgenic (NT) plants. Interestingly, transgenic plants also showed a 1.43 times higher glutathione reductase activity than NT plants. As a result, the ratio of AsA/DHA was changed from 0.21 to 0.48, even though total ascorbate content was not significantly changed. When tobacco leaf discs were subjected to methyl viologen (MV) at 5 mumol/L and hydrogen peroxide (H2O2) at 200 mmol/L, transgenic plants showed about a 40% and 25% reduction in membrane damage relative to NT plants, respectively. Furthermore, transgenic seedlings showed enhanced tolerance to low temperature (15 degrees C) and NaCl (100 mmol/L) compared to NT plants. These results suggest that a human derived DHAR properly works for the protection against oxidative stress in plants.     

High-level expression in Corynebacterium glutamicum of nitrile hydratase from Rhodococcus rhodochrous for acrylamide production

The nhhBAG gene of Rhodococcus rhodochrous M33 that encodes nitrile hydratase (NHase), converting acrylonitrile into acrylamide, was cloned and expressed in Corynebacterium glutamicum under the control of an ilvC promoter. The specific enzyme activity in recombinant C. glutamicum cells was about 13.6 μmol/min/mg dry cell weight (DCW). To overexpress the NHase, five types of plasmid variants were constructed by introducing mutations into 80 nucleotides near the translational initiation region (TIR) of nhhB. Of them, pNBM4 with seven mutations showed the highest NHase activity, exhibiting higher expression levels of NhhB and NhhA than wild-type pNBW33, mainly owing to decreased secondary-structure stability and an introduction of a conserved Shine-Dalgarno sequence in the translational initiation region. In a fed-batch culture of recombinant Corynebacterium cells harboring pNBM4, the cell density reached 53.4 g DCW/L within 18 h, and the specific and total enzyme activities were estimated to be 37.3 μmol/min/mg DCW and 1,992 μmol/min/mL, respectively. The use of recombinant Corynebacterium cells for the production of acrylamide from acrylonitrile resulted in a conversion yield of 93 % and a final acrylamide concentration of 42.5 % within 6 h when the total amount of fed acrylonitrile was 456 g.     

The crucial role and regulations of miRNAs in zebrafish development

To comprehend the events during developmental biology, fundamental knowledge about the basic machinery of regulation is a prerequisite. MicroRNA (miRNAs) act as regulators in most of the biological processes and recently, it has been concluded that miRNAs can act as modulatory factors even during developmental process from lower to higher animal. Zebrafish, because of its favorable attributes like tiny size, transparent embryo, and rapid external embryonic development, has gained a preferable status among all other available experimental animal models. Currently, zebrafish is being utilized for experimental studies related to stem cells, regenerative molecular medicine as well drug discovery. Therefore, it is important to understand precisely about the various miRNAs that controls developmental biology of this vertebrate model. In here, we have discussed about the miRNA-controlled zebrafish developmental stages with a special emphasis on different miRNA families such as miR-430, miR-200, and miR-133. Moreover, we have also reviewed the role of various miRNAs during embryonic and vascular development stages of zebrafish. In addition, efforts have been made to summarize the involvement of miRNAs in the development of different body parts such as the brain, eye, heart, muscle, and fin, etc. In each section, we have tried to fulfill the gaps of zebrafish developmental biology with the help of available knowledge of miRNA research. We hope that precise knowledge about the miRNA-regulated developmental stages of zebrafish may further help the researchers to efficiently utilize this vertebrate model for experimental purpose.     

Extraction of indole alkaloids fromCatharanthus roseus by using supercritical carbon dioxide

Summary Indole alkaloids, particularly vindoline and catharanthine, were extracted from the leaves ofCatharanthus roseus by supercritical extraction with CO₂. The contents of vindoline and catharanthine in the extracts were determined by HPLC and identified by LC/MS. About 52 %(w/w) of the initial vindoline content, 1.5 mg vindoline/g dry wt leaves, was recovered after extracting this material for 10 h with the CO₂ flow rate of 400 ml/min at 40°C and 150 bar. Vindoline concentration in the extract was 67 %(w/w).     

Transgenic cucumber fruits that produce elevated level of an anti-aging superoxide dismutase

Superoxide dismutase (SOD) plays an important role in cellular defense against oxidative stress in aerobic organisms. To generate cucumber (Cucumis sativus L.) fruits producing high yields of SOD for an anti-aging cosmetic material as a plant bioreactor, the CuZnSOD cDNA (mSOD1) from cassava was introduced into cucumber fruits by Agrobacterium-mediated transformation using the ascorbate oxidase promoter with high expression in fruits. The bialaphos-resistant shoots were selected on medium containing MS basal salts, 2 mg l^–1 BA, 0.1 mg l^–1 IAA, 300 mg l^–1 claforan, and 2 mg l^–1 bialaphos. After 6 weeks of culture on the selection medium, the shoots were transferred to MS medium containing 1 mg l^–1 IAA, 300 mg l^–1 claforan, 2 mg l^–1 bialaphos to induce roots. Southern blot analysis confirmed that the mSOD1 gene was properly integrated into the nuclear genomes of three cucumber plants tested. The mSOD1 gene was highly expressed in the transgenic cucumber fruits, whereas it was expressed at a low level in the transgenic leaves. The SOD specific activity (units/mg protein) in transgenic fruits was approximately 3 times higher than in those of non-transgenic plants.