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Production of novel mutants with a high ability to mitigate pollutants is important for phytoremediation. We investigated the use of ion beam irradiation to produce mutants of Ficus pumila L. with an improved ability to mitigate atmospheric nitrogen dioxide (NO2). More than 25,000 shoot explants were irradiated with an ion beam (12C5+, 12C6+, or 4He2+), from which 263 independent plant lines were obtained. The plants were analyzed for NO2 uptake by fumigation with 1 ppm 15N-labeled NO2 for 8 h in light, followed by mass spectrometric analysis. The mean NO2 uptake values of each of the 263 lines differed over a 110-fold range. Propagation was attempted using cuttings from 44 lines showing the greatest NO2 uptake; in total, 15 lines were propagated. Two of the 15 lines showed a mean NO2 uptake 1.7- to 1.8-fold greater than that of the wild-type. This increase in NO2 uptake was heritable in both lines; their progenies showed a significantly greater ability to take up and assimilate NO2 than did the wild-type. RAPD analysis demonstrated DNA variation between the progeny plants and the wild type, suggesting that the progeny were true mutants. These mutants of F. pumila may prove useful in mitigating atmospheric NO2. [ABSTRACT FROM AUTHOR]

All plants examined to date possess non-symbiotic hemoglobin whose physiological role remains unclear. The present study explored the catalytic function of three representative classes of the plant hemoglobin from Arabidopsis thaliana: AtGLB1, AtGLB2, and AtGLB3. Purified recombinant proteins of these hemoglobins displayed hydrogen peroxide-dependent oxidation of several peroxidase substrates that was sensitive to cyanide, revealing intrinsic peroxidase-like activity. In the presence of nitrite and hydrogen peroxide, AtGLB1 was the most efficient at mediating tyrosine nitration of its own and other proteins via the formation of reactive nitrogen species as a result of nitrite oxidation. AtGLB1 mRNA significantly accumulated in Arabidopsis seedlings exposed to nitrite, supporting the physiological relevance of its function to nitrite and nitrite-derived reactive nitrogen species. [Copyright &y& Elsevier]

S–Nitrosoglutathione (GSNO), an adduct of nitric oxide (NO) with glutathione, is known as a biological NO reservoir. Heterologous expression in Escherichia coli of a cDNA encoding a glutathione-dependent formaldehyde dehydrogenase of Arabidopsis thaliana showed that the recombinant protein reduces GSNO. The identity of the cDNA was further confirmed by functional complementation of the hypersensitivity to GSNO of a yeast mutant with impaired GSNO metabolism. This is the first demonstration of a plant GSNO reductase, suggesting that plants possess the enzymatic pathway that modulates the bioactivity and toxicity of NO. [Copyright &y& Elsevier]

Particle bombardment has proved to be useful for the transformation of plants. We have previously reported successful transient expression of the β-glucuronidase (GUS) gene in cultured plant cells and tissues and the stable transformation of various plants using a pneumatic particle gun. In this chapter, we describe transient expression of the GUS gene in Arabidopsis thaliana leaves and roots using the pneumatic particle gun. [ABSTRACT FROM AUTHOR]

Summary: The emission of N[sub 2] and N[sub 2]O from intact transgenic tobacco (clone 271) expressing antisense nitrite reductase (NiR) mRNA, and wild-type plants grown aseptically, on NO[sub 3][sup –], NO[sub 2][sup –] or NH[sub 4][sup +] -containing medium was investigated. [sup 15]N contents of gas sampled from gas-sealed pots, in which the plants were grown on [sup 15]N-containing medium, were analyzed by gas chromato- graphy and mass spectrometry (GC–MS). No emission of N[sub 2] was detected in either of the gas samples from plant clone 271 or the wild-type grown on NO[sub 3][sup –]-containing medium. N[sub 2]O emission from clone 271 grown on NO[sub 3][sup –]-containing medium was detected, but not from the wild-type plants. The N[sub 2]O emission rate of clone 271 was 106 ng N[sub 2]O mg[sup –1] incorporated N week[sup –1] and the N[sub 2]O emission was inhibited by tungstate (a nitrate reductase inhibitor). No emission of N[sub 2]O was found from clone 271 or wild-type plants grown on medium containing NH[sub 4][sup +]. Emission of N[sub 2]O also was detected from clone 271 grown on NO[sub 2][sup –]-containing medium and its emission rate increased with increasing NO[sub 2][sup –] levels in plants. We speculate that NO[sub 3][sup –] is reduced to NO[sub 2][sup –] and that a part of NO[sub 2][sup –] is metabolized to N[sub 2]O in clone 271. [ABSTRACT FROM AUTHOR]

Suspension-cultured cells (A-18 line) of the liverwort Marchanta polymorpha were bombarded by a pneumatic particle gun with plasmid pCH harbouring the hygromycin phosphotransferase (HPT) gene ( hpt) under the control of the cauliflower mosaic virus (CaMV) 35 S promoter and the nopaline synthase polyadenylation region. Nine weeks after bombardments, 128 hygromycin-resistant calluses were obtained from an approximate total of 7×10 cells. Ten cell lines chosen randomly were analysed further. Southern blot analysis showed that all of the ten lines contain the hpt gene in the genome, demonstrating that these lines are transformants. An HPT enzyme activity assay confirmed the expression of the gene in all of the transformant lines. [ABSTRACT FROM AUTHOR]

Immature pollen from Nicotiana rustica was bombarded with gold particles coated with plasmid DNA encoding neomycin phosphotransferase II (NPTII) and β-glucuronidase (GUS) genes which, respectively, are under the control of the cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator in the plasmid. Kanamycin-resistant pollen embryoids were selected from the bombarded pollen cells and two independent lines of transgenic plants were regenerated. Enzyme assays showed that one has both NPTII and GUS activities and the other only weak NPTII activity. Southern blot analyses indicated that the former has a DNA fragment corresponding to the intact expression cassettes for both genes in its genome; whereas the latter lacks intact expression cassettes for both genes and has only the intact nptII coding sequence in its genome. The transgenic plants of both lines have 24 chromosomes, confirming haploidy, and they are infertile. These results indicate that transgenic haploid plants can be produced directly by the bombardment-mediated transformation of immature pollen. [ABSTRACT FROM AUTHOR]

Assimilation of nitrogen dioxide in response to fumigation with 15N-labelled nitrogen dioxide was studied in 217 plant taxa. The taxa included 50 wild herbaceous plants collected from roadsides (42 genera, 15 families), 60 cultivated herbaceous plants (55 genera, 30 families) and 107 cultivated woody plants (74 genera, 45 families). Two parameters, the ‘NO2-N content’, or NO2-derived reduced nitrogen content in fumigated plant leaves (mg N g–1 dry weight), and the ‘NO2-utilization index’, or percentage of the NO2-derived reduced nitrogen in the total reduced nitrogen, were determined. The NO2-N content differed 657-fold between the highest (Eucalyptus viminalis; 6·57) and lowest (Tillandsia ionantha and T. caput-medusae; 0·01) values in the 217 taxa; 62-fold in a family (Theaceae) and 26-fold in a species (Solidago altissima). Nine species had NO2-utilization indices greater than 10%, of which Magnolia kobus, Eucalyptus viminalis, Populus nigra, Nicotiana tabacum and Erechtites hieracifolia had NO2-N contents > 4·9. These plants can be considered ‘NO2-philic’ because in them NO2-nitrogen has an important function(s). The Compositae and Myrtaceae had high values for both parameters, whereas the monocots and gymnosperms had low ones. These findings suggest that the metabolic pathway of NO2-nitrogen differs among plant species. The information presented here will be useful for creating a novel vegetation technology to reduce the atmospheric concentration of nitrogen dioxide. [ABSTRACT FROM AUTHOR]

It was found that plasmid DNA (YEp13) can be introduced into intact yeast cells by electric field pulses. The frequency of transformation by this electroinjection method depend upon the initial electric field intensity, the capacitance of the electric discharge capacitor, and the number of pulses applied. A maximum number of transformants (90±20/μgDNA) was obtained by three successive pulses with an initial intensity of 5 KV/cm and with a capacitance of 1 μF. The present electroinjection method is simple, and transformants can be obtained within 2 to 3 days after transformation treatment. [ABSTRACT FROM AUTHOR]

A plasmid pARK 22 harbouring the bar gene encoding phosphinothricin acetyltransferase (PAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator was constructed and introduced into root sections of Arabidopsis thaliana using the pneumatic particle gun. The root sections that had been bombarded with this plasmid gave four to eight times higher yield of drug-resistant calluses than those sections bombarded with pCaMVNEO or pCH, which respectively contain the neomycin phosphotransferase and hygromycin phosphotransferase genes. Among a number of primary transformant (T) plants obtained from independent bialaphos-resistant calluses, three were studied by Southern blot hybridization and PAT enzyme activity analyses, confirming the stable integration of the foreign gene into the Arabidopsis genome and its expression in plants. The progeny analysis showed transmission of the foreign gene and its expression in up to the T generation. Some of the T progeny showed morphological abnormalities. Thus, the bar gene can be used effectively to allow selection of transgenic A. thalianna plants. [ABSTRACT FROM AUTHOR]

Levels of NO-3, NO-2 and NH+4 ions in leaves of six plant species were determined by capillary electrophoresis. Levels of NO-3 ions differed by a factor of more than 350 in the six species. NO-2 and NH+4 ions were detected in all species examined but at lower and more similar respective levels than NO-3 ions. [ABSTRACT FROM AUTHOR]