An Ipomoea batatas Iron-Sulfur Cluster Scaffold Protein Gene,IbNFU1, Is Involved in Salt Tolerance

Iron-sulfur cluster biosynthesis involving the nitrogen fixation (Nif) proteins has been proposed as a general mechanism acting in various organisms. NifU-like protein may play an important role in protecting plants against abiotic and biotic stresses. An iron-sulfur cluster scaffold protein gene, IbNFU1, was isolated from a salt-tolerant sweetpotato (Ipomoea batatas (L.) Lam.) line LM79 in our previous study, but its role in sweetpotato stress tolerance was not investigated. In the present study, the IbNFU1 gene was introduced into a salt-sensitive sweetpotato cv. Lizixiang to characterize its function in salt tolerance. The IbNFU1-overexpressing sweetpotato plants exhibited significantly higher salt tolerance compared with the wild-type. Proline and reduced ascorbate content were significantly increased, whereas malonaldehyde (MDA) content was significantly decreased in the transgenic plants. The activities of superoxide dismutase (SOD) and photosynthesis were significantly enhanced in the transgenic plants. H₂O₂ was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbNFU1 up-regulated pyrroline-5-carboxylate synthase (P5CS) and pyrroline-5-carboxylate reductase (P5CR) genes under salt stress. The systemic up-regulation of reactive oxygen species (ROS) scavenging genes was found in the transgenic plants under salt stress. These findings suggest that IbNFU1gene is involved in sweetpotato salt tolerance and enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and activating ROS scavenging system.     

A sucrose non-fermenting-1-related protein kinase 1 gene from potato, <Emphasis Type="Italic">StSnRK1</Emphasis>, regulates carbohydrate metabolism in transgenic tobacco

Sucrose non-fermenting-1-related protein kinase 1 (SnRK1) has been shown to play an essential role in regulating saccharide metabolism and starch biosynthesis of plant. The regulatory role of StSnRK1 from potato in regulating carbohydrate metabolism and starch accumulation has not been investigated. In this work, a cDNA encoding the SnRK1 protein, named StSnRK1, was isolated from potato. The open reading frame contained 1545 nucleotides encoding 514 amino acids. Subcellular localization analysis in onion epidermal cells indicated that StSnRK1 protein was localized to the nucleus. The coding region of StSnRK1 was cloned into a binary vector under the control of 35S promoter and then transformed into tobacco to obtain transgenic plants. Transgenic tobacco plants expressing StSnRK1 were shown to have a significant increased accumulation of starch content, as well as sucrose, glucose and fructose content. Real-time quantitative PCR analysis indicated that overexpression of StSnRK1 up-regulated the expression of sucrose synthase (NtSUS), ADP-glucose pyrophosphorylase (NtAGPase) and soluble starch synthase (NtSSS III) genes involved in starch biosynthesis in the transgenic plants. In contrast, the expression of sucrose phosphate synthase (NtSPS) gene was decreased in the transgenic plants. Meanwhile, enzymatic analyses indicated that the activities of major enzymes (SUS, AGPase and SSS) involved in the starch biosynthesis were enhanced, whereas SPS activity was decreased in the transgenic plants compared to the wild-type. These results suggest that the manipulation of StSnRK1 expression might be used for improving quality of plants in the future.     

BcPMI2, isolated from non-heading Chinese cabbage encoding phosphomannose isomerase,improves stress tolerance in transgenic tobacco

Phosphomannose isomerase (PMI) is an enzyme that catalyses the first step of the l-galactose pathway for ascorbic acid (AsA) biosynthesis in plants. To clarify the physiological roles of PMI in AsA biosynthesis, the cDNA sequence of PMI was cloned from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) and overexpressed in tobacco transformed with Agrobacterium tumefaciens. The AsA and soluble sugar contents were lower in 35S::BcPMI2 tobacco than in wild-type tobacco. However, the AsA level in BcPMI2-overexpressing plants under stress was significantly increased. The T₁ seed germination rate of transgenic plants was higher than that of wild-type plants under NaCl or H₂O₂ treatment. Meanwhile, transgenic plants showed higher tolerance than wild-type plants. This finding implied that BcPMI2 overexpression improved AsA biosynthetic capability and accumulation, and evidently enhanced tolerance to oxidative and salt stress, although the AsA level was lower in transgenic tobacco than in wild-type tobacco under normal condition.     

Simultaneous Overexpression of Citrate Synthase and Phosphoenolpyruvate Carboxylase in Leaves Augments Citrate Exclusion and Al Resistance in Transgenic Tobacco

Phosphoenolpyruvate carboxylase (PEPC) and citrate synthase (CS) are two key enzymes in organic acid synthesis metabolism. In the present study, a cytoplasmic form of CS from tobacco and a mutant (with reduced sensitivity to organic acid inhibition) PEPC from Synechococcus vulcanus were overexpressed simultaneously using a light-inducible promoter in tobacco leaves. The analysis for enzyme activity showed that CS and PEPC enzyme activities were increased by 235% to 257% and 218% to 236% in the selected cs and pepc (double-gene) overexpression lines, respectively, compared with those in the wild-type plants (WT). The measurement for the relative root elongation rate of the tobacco plants exposed to 30???M aluminum (Al) indicated that Al tolerance in the double-gene overexpression lines was stronger than that of the transgenic cs or pepc lines and WT plants. The ¹³C-NMR analysis with NaH¹³CO₃ showed that overexpression of CS and PEPC in the transgenic tobacco successfully constructed a new citrate synthesis pathway. Under the conditions with Al stress, the amount of citrate secreted from the double-transgenic tobacco roots was the largest among the tested plants. When grown on sandy soil supplied with a nutritional solution containing 500???M Al, the growth of the double-transgenic tobacco was better than that of the transgenic cs or pepc tobacco and WT, and their root biomass was the highest among the tested plants. These results demonstrated that construction of a new citrate synthesis pathway by simultaneous overexpression of CS and PEPC in the cytoplasm of transgenic plant leaves could enhance Al resistance in plants.     

Synthesis and accumulation of pea plastocyanin in transgenic tobacco plants

The pea plastocyanin gene in a 3.5 kbp Eco RI fragment of pea nuclear DNA was introduced into tobacco by Agrobacterium-mediated transformation. Regenerated plants contained pea plastocyanin located within the chloroplast thylakoid membrane system. Analysis of seedlings from a self-pollinated transgenic plant containing a single copy of the pea plastocyanin gene indicated that seedlings homozygous for the pea gene contained almost twice as much pea plastocyanin as seedlings hemizygous for the pea gene. Homozygous seedlings contained approximately equal amounts of pea and tobacco plastocyanins. The amount of tobacco plastocyanin in leaves of transgenic plants was unaffected by the expression of the pea plastocyanin gene. The mRNA from the pea gene in tobacco was indistinguishable by northern blotting and S1 nuclease protection from the mRNA found in pea. In both pea and transgenic tobacco, expression of the pea plastocyanin gene was induced by light in leaves but was suppressed in roots. Pea plastocyanin free of contaminating tobacco plastocyanin was purified from transgenic tobacco plants and shown to be indistinguishable from natural pea plastocyanin by N-terminal protein sequencing and ¹H NMR spectroscopy.     

Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation

Pyrroline-5-carboxylate reductase (P5CR) lies at the converging point of the glutamate and ornithine pathways and is the last and critical enzyme in proline biosynthesis. In the present study, a P5CR gene, named IbP5CR, was isolated from salt-tolerant sweetpotato line ND98. Expression of IbP5CR was up-regulated in sweetpotato under salt stress. The IbP5CR-overexpressing sweetpotato (cv. Kokei No. 14) plants exhibited significantly higher salt tolerance compared with the wild-type. Proline content and superoxide dismutase and photosynthetic activities were significantly increased, whereas malonaldehyde content was significantly decreased in the transgenic plants. H₂O₂ was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbP5CR up-regulated pyrroline-5-carboxylate synthase gene and down-regulated proline dehydrogenase and P5C dehydrogenase genes under salt stress. The systemic up-regulation of reactive oxygen species (ROS) scavenging genes was found in the transgenic plants under salt stress. These findings suggest that overexpression of IbP5CR increases proline accumulation, which enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and activating ROS scavenging system. This study indicates that IbP5CR gene has the potential to be used for improving salt tolerance of plants.     

Production of isoflavone genistein in transgenic IFS tobacco roots and its role in stimulating the development of arbuscular mycorrhiza

Flavonoids and isoflavonoids are secondary metabolites in plants. With the goal of obtaining isoflavonoids from a wide range of plants, a few key studies have proven that isoflavonoids can be produced in non-leguminous plants by transgenic engineering. Many earlier studies investigate genistein biosynthesis in leaves and petals of isoflavone synthase (IFS) transgenic tobacco. However, most reports do not attempt to analyze quantification of genistein or do not check the presence of genistein in transgenic plant roots. In addition, little is known about the influence of genistein on arbuscular mycorrhiza (AM). In this paper, we reported that genistein was obtained from transgenic IFS tobacco roots. In addition, we revealed that endogenous genistein and 10???g?g^?1 exogenous genistein enhanced the development of AM symbiosis. We also revealed the relative expression levels of pertinent genes during the development of AM symbiosis. Our results suggest that genistein plays a positive role in the development of AM symbiosis in tobacco roots.     

A novel rice C2H2-type zinc finger protein lacking DLN-box/EAR-motif plays a role in salt tolerance

A cDNA for the gene ZFP182, encoding a C2H2-type zinc finger protein, was cloned from rice by RT-PCR. ZFP182 codes an 18.2 kDa protein with two C2H2-type zinc finger motifs, one nuclear localization signal and one Leu-rich domain. The DLN-box/EAR-motif, which exists in most of plant C2H2-type zinc finger proteins, does not exist in ZFP182. The expression analysis showed that ZFP182 gene was constitutively expressed in leaves, culms, roots and spikes at the adult rice plants, and markedly induced in the seedlings by cold (4 °C), 150 mM NaCl and 0.1 mM ABA treatments. The approximate 1.4 kb promoter region of ZFP182 gene was fused into GUS reporter gene and transformed into tobacco. The histochemical analysis revealed that GUS expression could not be detected in transformed tobacco seedlings under normal conditions, but strongly observed in tobacco leaf discs and the vascular tissue of roots treated with NaCl or KCl. Expression of ZFP182 in transgenic tobacco and overexpression in rice increased plant tolerance to salt stress. These results demonstrated that ZFP182 might be involved in plant responses to salt stress.     

Production and Analysis of Tobacco Transgenic Plants Expressing the Agrobacterial Gene for Tryptophan Monooxygenase

The expression of the agrobacterial iaaM gene for tryptophan monooxygenase, the enzyme catalyzing the first step in the auxin biosynthesis, induced substantial physiological and biochemical changes in transgenic tobacco (Nicotiana tabacum L.) plants. All lines of transgenic plants grown in vitro manifested abnormal phenotypes: enhanced root formation, adventitious roots on stems, and curled leaves. When grown in vivo, plants manifested abnormal, normal, or intermediate phenotype. Under conditions of a greenhouse, the abnormal plants contained the highest amount of auxins in their leaves and manifested an increased number of adventitious roots, poor reproductivity, and the loss in seed germination. Transgenic plants with the normal phenotype did not substantially differ from the wild-type plants in their morphology, and their auxin content was lower than in the abnormal plants. The intermediate-phenotype plants were devoid of some morphological properties characteristic of the abnormal plants. Only the seeds of normal- and intermediate-phenotype transgenic plants germinated at a high rate.