Enhanced tolerance to ozone and drought stresses in transgenic tobacco overexpressing dehydroascorbate reductase in cytosol

Ascorbate (vitamin C) is a potent antioxidant protecting plants against oxidative damage imposed by environmental stresses such as ozone and drought. Dehydroascorbate reductase (DHAR; EC 1.8.5.1) is one of the two important enzymes functioning in the regeneration of ascorbate (AsA). To examine the protective role of DHAR against oxidative stress, we developed transgenic tobacco plants overexpressing cytosolic DHAR gene from Arabidopsis thaliana . Incorporation of the transgene in the genome of tobacco plants was confirmed by polymerase chain reaction and Southern blot analysis, and its expression was confirmed by Northern and Western blot analyses. These transgenic plants exhibited 2.3–3.1 folds higher DHAR activity and 1.9–2.1 folds higher level of reduced AsA compared with non-transformed control plants. The transgenic plants showed maintained redox status of AsA and exhibited an enhanced tolerance to ozone, drought, salt, and polyethylene glycol stresses in terms of higher net photosynthesis. In this study, we report for the first time that the elevation of AsA level by targeting DHAR overexpression in cytosol properly provides a significantly enhanced oxidative stress tolerance imposed by drought and salt.     

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.     

普通小麦中双脱氢抗坏血酸还原酶(TaDHAR)基因的克隆与生化特性分析

利用同源克隆技术从六倍体普通小麦中获得了两个不同的双脱氢抗坏血酸还原酶(TaDHAR)基因的cDNA克隆。器官表达模式分析表明,这两个TaDHAR基因(暂时命名为TaDHAR1和TaDHAR2)在小麦根、茎、叶、幼穗以及开花后10d、20d和30d的种子中均有表达,为组成型表达基因。原生质体表达实验表明,两个基因的产物均可能定位在细胞质中。在细菌中表达并提纯了两个基因的重组蛋白。体外生化测定表明两个重组蛋白均具有将双脱氢抗坏血酸还原成抗坏血酸的能力,其最适pH为7.5,在37oC时的活性比25oC高,但25oC条件下pH6.0和7.0时,两个DHAR蛋白的活性显著不同。本研究的结果为进一步揭示TaDHAR基因在小麦抗坏血酸代谢中的生理作用奠定了基础。     

Production of dextran in transgenic potato plants

The production of dextran in potato tubers and its effect on starch biosynthesis were investigated. The mature dextransucrase (DsrS) gene from Leuconostoc mesenteroides was fused to the chloroplastic ferredoxin signal peptide (FD) enabling amyloplast entry, which was driven by the highly tuber-expressed patatin promoter. After transformation of two potato genotypes (cv. Kardal and the amylose-free (amf) mutant), dextrans were detected by enzyme-linked immunosorbent assay (ELISA) in tuber juices of Kardal and amf transformants. The dextran concentration appeared two times higher in the Kardal (about 1.7 mg/g FW) than in the amf transformants. No dextran was detected by ELISA inside the starch granule. Interestingly, starch granule morphology was affected, which might be explained by the accumulation of dextran in tuber juices. In spite of that, no significant changes of the physicochemical properties of the starches were detected. Furthermore, we have observed no clear changes in chain length distributions, despite the known high acceptor efficiency of DSRS.     

Overexpression of dehydroascorbate reductase, but not monodehydroascorbate reductase, confers tolerance to aluminum stress in transgenic tobacco

Aluminum (Al) inhibits plant growth partly by causing oxidative damage that is promoted by reactive oxygen species and can be prevented by improving antioxidant capacity. Ascorbic acid (AsA), the most abundant antioxidant in plants, is regenerated by the action of monodehydroascorbate reductase (MDAR) and dehydroascorbate reductase (DHAR). We investigated the role of MDAR and DHAR in AsA regeneration during Al stress using transgenic tobacco (Nicotiana tabacum) plants overexpressing Arabidopsis cytosolic MDAR (MDAR-OX) or DHAR (DHAR-OX). DHAR-OX plants showed better root growth than wild-type (SR-1) plants after exposure to Al for 2 weeks, but MDAR-OX plants did not. There was no difference in Al distribution and accumulation in the root tips among SR-1, DHAR-OX, and MDAR-OX plants after Al treatment for 24 h. However, DHAR-OX plants showed lower hydrogen peroxide content, less lipid peroxidation and lower level of oxidative DNA damage than SR-1 plants, whereas MDAR-OX plants showed the same extent of damage as SR-1 plants. Compared with SR-1 plants, DHAR-OX plants consistently maintained a higher AsA level both with and without Al exposure, while MDAR-OX plants maintained a higher AsA level only without Al exposure. Also, DHAR-OX plants maintained higher APX activity under Al stress. The higher AsA level and APX activity in DHAR-OX plants contributed to their higher antioxidant capacity and higher tolerance to Al stress. These findings show that the overexpression of DHAR, but not of MDAR, confers Al tolerance, and that maintenance of a high AsA level is important to Al tolerance.     

Photoinhibition of photosystem II in tobacco plants overexpressing glutathione reductase and poplars overexpressing superoxide dismutase

We studied photoinhibition in two cultivars of tobacco ( Nicotiana tabacum L.) expressing the bacterial gor gene in the cytosol and in four lines of poplar ( Populus tremula × P. alba ) expressing the FeSOD gene of Arabidopsis thaliana in the chloroplast. The respective total activities of glutathione reductase (EC 1.6.4.2) in leaves of gor tobaccos and superoxide dismutase (EC 1.15.1.1) in the FeSOD poplars were 5–8 times higher than in the respective untransformed control plants. Leaves of control and transformed plants were subjected to high-light stress at 20°C, and photoinhibition of photosystem II (PSII) was measured by oxygen evolution and chlorophyll fluorescence. The leaves were illuminated both in the presence and absence of lincomycin, which inhibits chloroplast protein synthesis. In both cases, the time course of loss of PSII activity was identical in plants overproducing superoxide dismutase (SOD) and in the untransformed controls, suggesting that the ability to convert superoxide to hydrogen peroxide is not a limiting factor in protection against photoinhibition, or in the repair of photoinhibitory damage or that the site of O₂⁻ production is not accessible to the transgene product. The rate constant of photoinhibition, measured in lincomycin-treated leaves, was smaller in glutathione reductase (GR) overproducing tobacco cv. Samsun than in the respective wild-type, but this difference was not seen in cv. Bel W3. The steady-state level of PSII activity measured when the PSII repair cycle was allowed to equilibrate with photoinhibitory damage under high light was not higher in the GR overproducing cv. Samsun, suggesting that the repair of photoinhibitory damage was not enhanced in plants overproducing GR in the cytosol.     

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.     

Cytokinins and cytokinin-binding sites in transgenic potato plants

The introduction of the gene for cytokinin biosynthesis into the potato genome led to a manifold increase in the level of cytokinins (zeatin and zeatin riboside) in transgenic plants grown in vitro. The high amount of endogenous cytokinins in 20-day-old plants of clone 1339-3A correlated with high cytokinin-binding capacity of ribosomes that is presumably attribute to a cytokinin receptor.     

Enhancing the scopolamine production in transgenic plants of Atropa belladonna by overexpressing pmt and h6h genes

Atropa belladonna is officially deemed as the commercial plant to produce scopolamine in China. In this study we report the simultaneous overexpression of two functional genes involved in biosynthesis of scopolamine, which encode the upstream key enzyme putrescine N-methyltransferase (PMT) and the downstream key enzyme hyoscyamine 6β-hydroxylase (H6H), respectively, in transgenic herbal plants Atropa belladonna. Analysis of gene expression profile indicated that both pmt and h6h were expressed at a higher level in transgenic lines, which would be favorable for biosynthesis of scopolamine. High-performance liquid chromatography result suggested that transgenic lines could produce higher accumulation of scopolamine at different levels compared with wild-type lines. Scopolamine content increased to 7.3-fold in transgenic line D9 compared with control lines. This study not only confirms that co-overexpression of pmt and h6h is an ideal method to improve the biosynthetic capacity of scopolamine but also successfully cultivates the transgenic line D9, which significantly enhanced the scopolamine accumulation. Our research can serve as an alternative choice to provide scopolamine resources for relative industry, which is more competitive than conventional market.     

Responses of MxPPO overexpressing transgenic tall fescue plants to two diphenyl-ether herbicides, oxyfluorfen and acifluorfen

We generated transgenic tall fescue (Festuca arundinacea Schreb. cv. Kentucky-31) plants harboring a synthetic Myxococcus xanthus protoporphyrinogen oxidase (MxPPO) gene through Agrobacterium-mediated gene transfer. Successful integration of the transgene into the genome of transgenic plants confirmed by polymerase chain reaction (PCR) and Southern blot analysis, and the functional expression of the MxPPO gene at the mRNA level in transgenic lines was validated by Northern blot analysis. Responses of transgenic and non-transgenic tall fescue plants to diphenyl-ether herbicides such as oxyfluorfen and acifluorfen have been evaluated in respect of various physiological and biochemical parameters. Differential responses were observed in chlorophyll content, in vivo H₂O₂ deposition and lipid peroxidation in both transgenic and non-transgenic plants exposed to oxyfluorfen or acifluorfen. Isozyme profiles of four antioxidant-enzymes, including peroxidase (POD), catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX), were also investigated in transgenic and non-transgenic plants using native PAGE analysis. Compared to the transgenic lines, higher staining activities of the examined antioxidant-enzymes observed in non-transgenic plants subjected to 100 μM of oxyfluorfen or acifluorfen suggests that non-transgenic plants are unable to prevent the photodynamic induced oxidative stress caused by herbicides. In addition, both transgenic and non-transgenic plants exposed to oxyfluorfen exhibited proportionally increased band-staining patterns in contrast to acifluorfen, which suggests that oxyfluorfen has relatively greater or more rapid effects on leaves than acifluorfen. Both Ki-Won Lee and Nagib Ahsan have contributed equally to this work.     

Polyamine homeostasis in transgenic plants overexpressing ornithine decarboxylase includes ornithine limitation

It was reported recently that overexpression of human ornithine decarboxylase (ODC) cDNA in transgenic rice plants resulted in increased steady-state concentration of polyamines, i.e., enough biosynthetic control is invested at this step to enable adjustment of polyamine levels. To investigate critically whether constitutive overexpression of ODC is sufficient to control steady-state polyamine levels, we expressed an ODC cDNA from Datura stramonium in transgenic tobacco plants. Transgenic progeny of self-fertilised primary transformants exhibited increases in ODC activity of 25-fold in leaves and 5-fold in flower buds. However, the increase in putrescine levels was only 1.5- to 2.1-fold in leaves and 1.1- to 1.3-fold in flower buds. Emphatically, no changes to spermidine or spermine steady-state levels or to soluble or insoluble hydroxycinnamic acid-conjugated polyamines were observed. Ornithine feeding to cell suspension cultures derived from the transgenic plants indicated that putrescine accumulation was limited in part by ornithine availability. These results demonstrate that a large increase in the capacity of the tobacco plants to decarboxylate ornithine does not result in a comparable increase in the level of free or conjugated polyamines. Plant polyamine homeostatic mechanisms efficiently accommodate increased ODC activity, suggesting that polyamine biosynthetic control is invested at multiple interdependent steps.     

Differential regulation of two ABA-inducible genes from Craterostigma plantagineum in transgenic Arabidopsis plants

In Craterostigma plantagineum the CDeT-6-19 and CDeT-27-45 genes are expressed following desiccation and/or ABA treatment. Their promoters were fused to the -glucuronidase reporter gene (GUS) and tested in transgenic Arabidopsis. GUS activity was measured in mature Arabidopsis seeds, and the responsiveness to ABA in vegetative tissue was found to be limited to the early developmental stages. When transgenic plants were crossed with plants over-expressing the ABI3 gene, it was observed that ABI3 is not required for ABA induction of the CDeT-6-19 promoter, whereas it is crucial for expression of the CDeT-27-45 promoter.     

Ascorbic acid in plants: biosynthesis and function

Ascorbic acid (vitamin C) is an abundant component of plants. It reaches a concentration of over 20 mM in chloroplasts and occurs in all cell compartments, including the cell wall. It has proposed functions in photosynthesis as an enzyme cofactor (including synthesis of ethylene, gibberellins and anthocyanins) and in control of cell growth. A biosynthetic pathway via GDP-mannose, GDP-L-galactose, L-galactose, and L-galactono-1,4-lactone has been proposed only recently and is supported by molecular genetic evidence from the ascorbate-deficient vtc 1 mutant of Arabidopsis thaliana. Other pathways via uronic acids could provide minor sources of ascorbate. Ascorbate, at least in some species, is a precursor of tartrate and oxalate. It has a major role in photosynthesis, acting in the Mehler peroxidase reaction with ascorbate peroxidase to regulate the redox state of photosynthetic electron carriers and as a cofactor for violaxanthin de-epoxidase, an enzyme involved in xanthophyll cycle-mediated photoprotection. The hypersensitivity of some of the vtc mutants to ozone and UV-B radiation, the rapid response of ascorbate peroxidase expression to (photo)-oxidative stress, and the properties of transgenic plants with altered ascorbate peroxidase activity all support an important antioxidative role for ascorbate. In relation to cell growth, ascorbate is a cofactor for prolyl hydroxylase that posttranslationally hydroxylates proline residues in cell wall hydroxyproline-rich glycoproteins required for cell division and expansion. Additionally, high ascorbate oxidase activity in the cell wall is correlated with areas of rapid cell expansion. It remains to be determined if this is a causal relationship and, if so, what is the mechanism. Identification of the biosynthetic pathway now opens the way to manipulating ascorbate biosynthesis in plants, and, along with the vtc mutants, this should contribute to a deeper understanding of the proposed functions of this multifaceted molecule.     

Stacking of antimicrobial genes in potato transgenic plants confers increased resistance to bacterial and fungal pathogens

Solanum tuberosum plants were transformed with three genetic constructions expressing the Nicotiana tabacum AP24 osmotine, Phyllomedusa sauvagii dermaseptin and Gallus gallus lysozyme, and with a double-transgene construction expressing the AP24 and lysozyme sequences. Re-transformation of dermaseptin-transformed plants with the AP24/lysozyme construction allowed selection of plants simultaneously expressing the three transgenes. Potato lines expressing individual transgenes or double- and triple-transgene combinations were assayed for resistance to Erwinia carotovora using whole-plant and tuber infection assays. Resistance levels for both infection tests compared consistently for most potato lines and allowed selection of highly resistant phenotypes. Higher resistance levels were found in lines carrying the dermaseptin and lysozyme sequences, indicating that theses proteins are the major contributors to antibacterial activity. Similar results were obtained in tuber infection tests conducted with Streptomyces scabies. Plant lines showing the higher resistance to bacterial infections were challenged with Phytophthora infestans, Rhizoctonia solani and Fusarium solani. Considerable levels of resistance to each of these pathogens were evidenced employing semi-quantitative tests based in detached-leaf inoculation, fungal growth inhibition and in vitro plant inoculation. On the basis of these results, we propose that stacking of these transgenes is a promising approach to achieve resistance to both bacterial and fungal pathogens.     

转基因植物中标记基因的消除

随着转基因植物的商业化,植物遗传转化技术将为农业生产带来一场新的革命,新的基因转化程序要求转基因为单拷贝,不带有标记基因,并在不同的转化体中表达一致,稳定遗传,本文讨论了转基因植物中有关标记基因及其安全性和标记基因消除的方法等问题。