Accumulation of tyrosol glucoside in transgenic potato plants expressing a parsley tyrosine decarboxylase

As part of the response to pathogen infection, potato plants accumulate soluble and cell wall-bound phenolics such as hydroxycinnamic acid tyramine amides. Since incorporation of these compounds into the cell wall leads to a fortified barrier against pathogens, raising the amounts of hydroxycinnamic acid tyramine amides might positively affect the resistance response. To this end, we set out to increase the amount of tyramine, one of the substrates of the hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)-transferase reaction, by placing a cDNA encoding a pathogen-induced tyrosine decarboxylase from parsley under the control of the 35S promoter and introducing the construct into potato plants via Agrobacterium tumefaciens-mediated transformation. While no alterations were observed in the pattern and quantity of cell wall-bound phenolic compounds in transgenic plants, the soluble fraction contained several new compounds. The major one was isolated and identified as tyrosol glucoside by liquid chromatography-electrospray ionization-high resolution mass spectrometry and NMR analyses. Our results indicate that expression of a tyrosine decarboxylase in potato does not channel tyramine into the hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)-transferase reaction but rather unexpectedly, into a different pathway leading to the formation of a potential storage compound.     

Wound-inducible biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine in tryptophan and tyrosine decarboxylase transgenic tobacco lines

The wound-activated biosynthesis of phytoalexin hydroxycinnamic acid amides of tyramine was compared in untransformed and transgenic tobacco (Nicotiana tabacum) lines that express tryptophan decarboxylase (TDC), tyrosine decarboxylase (TYDC), or both activities. Transgenic in vitro-grown tobacco lines expressing TDC activity accumulated high levels of tryptamine but not hydroxycinnamic amides of tryptamine. In contrast, transgenic tobacco lines expressing TYDC accumulated tyramine as well as p-coumaroyltyramine and feruloyltyramine. The MeOH-soluble and cell wall fractions showed higher concentrations of wound-inducible p-coumaroyltyramine and feruloyltyramine, especially at and around wound sites, in TYDC and TDC xTYDC tobacco lines compared to wild-type or TDC lines. All the enzymes involved in the biosynthesis of hydroxycinnamic acid amides of tyramine were found to be similarly wound inducible in all tobacco genotypes investigated. These results provide experimental evidence that, under some circumstances, TYDC activity can exert a rate-limiting control over the carbon flux allocated to the biosynthesis of hydroxycinnamic acid amides of tyramine.     

Enhanced production of reducing sugars from transgenic rice expressing exo-glucanase under the control of a senescence-inducible promoter

We generated transgenic rice plants that express EXG1 exo-glucanase under the control of a senescence-inducible promoter. When a GUS coding sequence was connected to a promoter region of STAY GREEN (SGR) gene of rice and introduced into rice, GUS activity was specifically observed along with senescence. When an EXG1 cDNA was connected to the SGR promoter and introduced into rice, higher cellulase activities were detected after senescence. The EXG1 transgenic plants showed enhanced enzymatic saccharification efficiencies after senescence, but no significant difference of saccharification efficiencies was observed before senescence. The saccharification efficiencies were correlated with the cellulase activities in the transgenic plants. The EXG1 transgenic plants showed neither morphological abnormality nor sterility, both of which were observed when EXG1 was constitutively overexpressed. These results indicate that expression of cell wall degrading enzymes such as cellulase by a senescence-inducible promoter is one of the ways to enhance the saccharification ability of cellulosic biomass without affecting plant growth for efficient production of biofuels.     

High levels of tryptamine accumulation in transgenic tobacco expressing tryptophan decarboxylase

A full-length complementary DNA clone encoding tryptophan decarboxylase (TDC; EC 4.1.1.28) from Catharanthus roseus (De Luca V, Marineau C, Brisson N [1989] Proc Natl Acad Sci USA 86: 2582-2586) driven by the CaMV 35S promoter was introduced into tobacco (Nicotiana tabacum) to direct the synthesis of the protoalkaloid tryptamine from endogenous tryptophan. Young, fully expanded leaves of CaMV 35S-TDC transformed plants had from four to 45 times greater TDC activity than did controls. Tryptamine accumulated in transgenic plants to levels that were directly proportional to their TDC specific activity. Despite their increased tryptamine content, the growth and development of the CaMV 35S-TDC plants appeared normal with no significant differences in indole-3-acetic acid levels between high tryptamine and control plants. Plants with the highest TDC activity contained more than 1 milligram of tryptamine per gram fresh weight, a 260-fold increase over controls.     

水稻谷蛋白启动子驱动下的ipt基因在转基因烟草中的表达

利用农杆菌系统介导 ,采用叶盘转化法 ,将在水稻谷蛋白启动子驱动下的外源ipt基因导入烟草植株中 ,经过抗生素筛选、PCR与测序分析检测出转基因植株。成熟的转基因烟草种子经过ELISA细胞分裂素试剂盒检测 ,发现iPAs含量为对照的 2 .43倍 ,此外 ,种子的重量也增加了 7.8%。     

Glucose intolerance and reduced islet blood flow in transgenic mice expressing the FRK tyrosine kinase under the control of the rat insulin promoter

The FRK tyrosine kinase has previously been shown to transduce beta-cell cytotoxic signals in response to cytokines and streptozotocin and to promote beta-cell proliferation and an increased beta-cell mass. We therefore aimed to further evaluate the effects of overexpression of FRK tyrosine kinase in beta-cells. A transgenic mouse expressing kinase-active FRK under control of the insulin promoter (RIP-FRK) was studied with regard to islet endocrine function and vascular morphology. Mild glucose intolerance develops in RIP-FRK male mice of at least 4 mo of age. This effect is accompanied by reduced glucose-stimulated insulin secretion in vivo and reduced second-phase insulin secretion in response to glucose and arginine upon pancreas perfusion. Islets isolated from the FRK transgenic mice display a glucose-induced insulin secretory response in vitro similar to that of control islets. However, islet blood flow per islet volume is decreased in the FRK transgenic mice. These mice also exhibit a reduced islet capillary lumen diameter as shown by electron microscopy. Total body weight and pancreas weight are not significantly affected, but the beta-cell mass is increased. The data suggest that long-term expression of active FRK in beta-cells causes an in vivo insulin-secretory defect, which may be the consequence of islet vascular abnormalities that yield a decreased islet blood flow.     

Molecular breeding of transgenic rice plants expressing a bacterial chlorocatechol dioxygenase gene

The cbnA gene encoding the chlorocatechol dioxygenase gene from Ralstonia eutropha NH9 was introduced into rice plants. The cbnA gene was expressed in transgenic rice plants under the control of a modified cauliflower mosaic virus 35S promoter. Western blot analysis using anti-CbnA protein indicated that the cbnA gene was expressed in leaf tissue, roots, culms, and seeds. Transgenic rice calluses expressing the cbnA gene converted 3-chlorocatechol to 2-chloromucote efficiently. Growth and morphology of the transgenic rice plants expressing the cbnA gene were not distinguished from those of control rice plants harboring only a Ti binary vector. It is thus possible to breed transgenic plants that degrade chloroaromatic compounds in soil and surface water.     

Characterization of sunlight-grown transgenic rice plants expressing Arabidopsis phytochrome A

Transgenic rice (Oryza sativa) overexpressing Arabidopsis phytochrome A (phyA) was cultivated up to the T₃ generation in paddy to elucidate the role of phyA in determining the plant architecture and the productivity of sunlight-grown rice plants. PhyA is light-labile and controls plant growth in response to the far-red light-dependent high-irradiance response as well as the very low fluence response. The Arabidopsis phyA gene linked to the rice rbcS promoter was transformed into embryogenic rice calli, and the calli were regenerated to whole plants. Compared to wild-type seedlings, the rbcS::PHYA transgenic seedlings contained more phyA when grown in the dark, and at least 10-fold more phyA when exposed to white light. When grown in paddy, the phyA transgenic plants in general exhibited reduced plant height (dwarfing), larger grain size, higher chlorophyll content, smaller tiller number, and low grain fertility compared to wild-type plants. The heading stage was not significantly changed. However, it is likely that a certain level of phyA is a prerequisite for induction of such changes. It is suggested that phyA overproduction in rice could be a useful tool to improve rice grain productivity by the larger grain size that increases grain yield and the dwarfing that tolerates lodging-associated damage.     

Growth and phenotype of potato plants expressing an antisense gene of P-protein of glycine decarboxylase under control of a promoter with preference for the mesophyll

A cDNA encoding P‐protein of glycine decarboxylase was expressed in antisense orientation in leaves of potato (Solanum tuberosum cv. Solara) under control of the promoter of a P‐protein gene of glycine decarboxylase from Flaveria pringlei. This promoter targets gene expression preferentially to the leaf mesophyll cells. In two of the transgenic lines, mitochondria oxidise glycine only with extremely low rates. Phenotypically, these transgenic lines were only marginally different from wild type plants under ambient carbon dioxide concentrations and indistinguishable from wild type plants when grown under 800 ppm carbon dioxide. When grown in ambient carbon dioxide, transgenic plants accumulated high amounts of glycine during the light period followed by nearly complete degradation in the following night.     

Fitness of transgenic Anopheles stephensi mosquitoes expressing the SM1 peptide under the control of a vitellogenin promoter

Three transgenic Anopheles stephensi lines were established that strongly inhibit transmission of the mouse malaria parasite Plasmodium berghei. Fitness of the transgenic mosquitoes was assessed based on life table analysis and competition experiments between transgenic and wild-type mosquitoes. Life table analysis indicated low fitness load for the 2 single-insertion transgenic mosquito lines VD35 and VD26 and no load for the double-insertion transgenic mosquito line VD9. However, in cage experiments, where each of the 3 homozygous transgenic mosquitoes was mixed with nontransgenic mosquitoes, transgene frequency of all 3 lines decreased with time. Further experiments suggested that reduction of transgene frequency is a consequence of reduced mating success, reduced reproductive capacity, and/or insertional mutagenesis, rather than expression of the transgene itself. Thus, for transgenic mosquitoes released in the field to be effective in reducing malaria transmission, a driving mechanism will be required.     

Characterization of the root-predominant gene promoter HPX1 in transgenic rice plants

Gene promoter(s) specialized in root tissues is an important component for crop biotechnology. In our current study, we report results of promoter analysis of the HPX1, a gene expressed predominantly in roots. The HPX1 promoter regions were predicted, linked to the gfp reporter gene, and transformed into rice. Promoter activities were analyzed in various organs and tissues of six independent transgenic HPX1:gfp plants using the fluorescent microscopy and q-RT-PCR methods. GFP fluorescence levels were high in root elongation regions but not in root apex and cap of the HPX1:gfp plants. Very low levels of GFP fluorescence were observed in anthers and leaves. Levels of promoter activities were 16- to 190-fold higher in roots than in leaves of the HPX1:gfp plants. The HPX1 promoter directs high levels of gene expression in root tissues producing GFP levels up to 0.39 % of the total soluble protein. Thus, the HPX1 promoter is predominantly active in the root elongation region during the vegetative stage of growth.     

Enhanced drought tolerance of transgenic rice plants expressing a pea manganese superoxide dismutase

We investigated the role that manganese superoxide dismutase (MnSOD), an important antioxidant enzyme, may play in the drought tolerance of rice. MnSOD from pea (Pisum sativum) under the control of an oxidative stress-inducible SWPA2 promoter was introduced into chloroplasts of rice (Oryza sativa) by Agrobacterium-mediated transformation to develop drought-tolerant rice plants. Functional expression of the pea MnSOD in transgenic rice plants (T1) was revealed under drought stress induced by polyethylene glycol (PEG) 6000. After PEG treatment the transgenic leaf slices showed reduced electrolyte leakage compared to wild type (WT) leaf slices, whether they were exposed to methyl viologen (MV) or not, suggesting that transgenic plants were more resistant to MV- or PEG-induced oxidative stress. Transgenic plants also exhibited less injury, measured by net photosynthetic rate, when treated with PEG. Our data suggest that SOD is a critical component of the ROS scavenging system in plant chloroplasts and that the expression of MnSOD can improve drought tolerance in rice.     

Native polyubiquitin promoter of rice provides increased constitutive expression in stable transgenic rice plants

The rice Ubiquitin1 (Ubi1) promoter was tested to evaluate its capacity to express the heterologous gusA gene encoding β-glucuronidase in transgenic rice tissue relative to the commonly used Ubi1 corn promoter and the rice gibberellic acid insensitive (GAI) gene promoter element. Experimental results showed increased expression of gusA gene in rice tissue when driven by the native Ubi1 promoter when compared to the use of corn Ubi1 promoter. Results further indicated that the cis-regulatory elements present in the native promoter element might have been responsible for high expression. However, the gusA gene expression level when driven by the rice GAI promoter was notably lower than both Ubi1 promoters. The present study, thus, for the first time helped to demonstrate that the native Ubi1 promoter is a promising genetic element in transgenic approaches for constitutive expression of any gene in rice tissue.     

Elevated tyrosine decarboxylase and tyramine hydroxycinnamoyltransferase levels increase wound-induced tyramine-derived hydroxycinnamic acid amide accumulation in transgenic tobacco leaves

Feruloyltyramine (FT) and 4-coumaroyltyramine (4CT) participate in the defense of plants against pathogens through their extracellular peroxidative polymerization, which is thought to reduce cell wall digestibility. Hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110) and tyrosine decarboxylase (TYDC; EC 4.1.1.25) are purported to play key roles in the stress-induced regulation of tyramine-derived hydroxycinnamic acid amide (HCAAT) metabolism. Transgenic tobacco (Nicotiana tabacum cv. Xanthi) was engineered to constitutively express tobacco THT. A T₁ plant over-expressing THT was crossbred with T₁ tobacco expressing opium poppy TYDC2, to produce a T₂ line with elevated THT and TYDC activities compared with wild type plants. The effects of an independent increase in TYDC or THT activity, or a dual increase in both TYDC and THT on the cellular pools of HCAAT pathway intermediates and the accumulation of soluble and cell wall-bound FT and 4CT were examined. Increased TYDC activity resulted in a larger cellular pool of tyramine and lower levels of L-phenylalanine in transgenic leaves. In contrast, elevated THT activity reduced tyramine levels. HCAAT levels were low in healthy leaves, but were induced in response to wounding and accumulated around wound sites. Similarly, endogenous THT and TYDC activities were wound-induced. The rate of wound-induced HCAAT accumulation was highest in transgenic plants with elevated THT and TYDC activities showing that both enzymes exert control over the flux of intermediates involved in HCAAT biosynthesis under some conditions.     

Aberrant chloroplasts in transgenic rice plants expressing a high level of maize NADP-dependent malic enzyme

 NADP-dependent malic enzyme (NADP-ME) is a major decarboxylating enzyme in NADP-ME-type C₄ species such as maize and Flaveria. In this study, chloroplastic NADP-ME was transferred to rice (Oryza sativa L.) using a chimeric gene composed of maize NADP-ME cDNA under the control of rice light-harvesting chlorophyll-a/b-binding protein (Cab) promoter. There was a 20- to 70-fold increase in the NADP-ME activity in leaves of transgenic rice compared to that in wild-type rice plants. Immunocytochemical studies by electron microscopy showed that maize NADP-ME was mostly localized in chloroplasts in transgenic rice plants, and that the chloroplasts were agranal without thylakoid stacking. Chlorophyll content and photosystem II activity were inversely correlated with the level of NADP-ME activity. These results suggest that aberrant chloroplasts in transgenic plants may be caused by excessive NADP-ME activity. Based on these results and the known fact that only bundle sheath cells of NADP-ME species, among all three C₄ subgroups, have agranal chloroplasts, we postulate that a high level of chloroplastic NADP-ME activity could strongly affect the development of chloroplasts. Received: 27 January 1999 / Accepted: 20 January 2000     

Anthranilate synthase and chorismate mutase activities in transgenic tobacco plants overexpressing tryptophan decarboxylase fromCatharanthus roseus

TransgenicNicotiana tabacum L. Petit Havana SR1 F₁-plants expressing tryptophan decarboxylase cDNA (tdc) fromCatharanthus roseus (L.) G. Don under the control of the CaMV 35S promoter and terminator exhibited tryptophan decarboxylase (TDC) enzyme activity and accumulated tryptamine. The plants with the highest TDC activity contained 19 pkat per mg of protein. The influence of transgenic expression oftdc on the activities of anthranilate synthase (AS) and chorismate mutase (CM) were examined in 10 transgenic tobacco plants. The specific activities of these two chorismate-utilizing enzymes were not significantly affected by expression oftdc, despite their important functions as branch point enzymes in the shikimate pathway. The results indicate that the normal route of tryptophan biosynthesis in plants is sufficient to supply a considerable amount of this essential amino acid for the biosynthesis of secondary metabolites. Despite their increased tryptamine content, the growth and development of the transgenic tobacco plants expressingtdc appeared normal.