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In this review, we will describe our study on “nitrogen-philic plants”, which can grow with nitrogen dioxide (NO2) as the sole nitrogen source, by screening of naturally occurring plants for the assimilation of nitrogen dioxide and genetic manipulation of plants for those genes involved in the primary nitrate metabolism. Finally, we will briefly describe “Green walls with nitrogen-dioxide-philic plants” for decontamination of pollution in urban areas. [Copyright &y& Elsevier]

In this short review, basic processes for phytoremediation and plant enzymes that are potentially useful in phytoremediation are briefly summarized. The remaining part describes the applications of the basic processes to decontaminate pollutants in the environments that have been done in our laboratory. Our work includes (1) analysis of the capability of various naturally occurring plants to decontaminate atmospheric nitrogen dioxide and selection of nitrogen-dioxide-philic plants that grow with nitrogen dioxide as the sole nitrogen source, (2) production of transgenic plants to improve plants’ capability to decontaminate atmospheric nitrogen dioxide, and (3) analysis of the denitrification process in plants to innovate a gas–gas-converting plants that convert nitrogen dioxide to nitrogen gas. [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]

To gain more insight into the physiological function of nitrogen dioxide ( NO2), we investigated the effects of exogenous NO2 on growth in Arabidopsis thaliana., Plants were grown in air without NO2 for 1 wk after sowing and then grown for 1-4 wk in air with (designated treated plants) or without (control plants) NO2. Plants were irrigated semiweekly with a nutrient solution containing 19.7 mM nitrate and 10.3 mM ammonium., Five-week-old plants treated with 50 ppb NO2 showed a ≤ 2.8-fold increase in biomass relative to controls. Treated plants also showed early flowering. The magnitude of the effects of NO2 on leaf expansion, cell proliferation and enlargement was greater in developing than in maturing leaves. Leaf areas were 1.3-8.4 times larger on treated plants than corresponding leaves on control plants. The NO2-induced increase in leaf size was largely attributable to cell proliferation in developing leaves, but was attributable to both cell proliferation and enlargement in maturing leaves. The expression of different sets of genes for cell proliferation and/or enlargement was induced by NO2, but depended on the leaf developmental stage., Collectively, these results indicated that NO2 regulates organ growth by controlling cell proliferation and enlargement. [ABSTRACT FROM AUTHOR]

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]

Abstract: Atmospheric nitrogen dioxide (NO2) is an environmental oxidant that is removed through direct uptake by foliage, but plant responses to this highly reactive gas are not well understood at the molecular level. From NO2-exposed leaves of a woody azalea (Rhododendron mucronatum), we cloned two cDNAs (RmGLP1 and RmGLP2) for germin-like proteins (GLPs), a group of ubiquitous plant proteins that have been implicated in various plant physiological and developmental processes. Quantitative analysis of mRNA expression, together with immunoblotting data, showed that foliar exposure to NO2 caused a robust induction of these GLP-encoding genes. When produced in tobacco cell culture, recombinant RmGLP2 was secreted into the apoplast, where it exhibited superoxide dismutase activity. RmGLP1 and RmGLP2 represent the first examples of plant genes that are responsive to airborne NO2. These enzymes might have a potential role in extracellular defense mechanisms through attenuation of interactions between reactive nitrogen and oxygen species. [Copyright &y& Elsevier]

We recently discovered that atmospheric nitrogen dioxide (NO2) at ambient levels acts as a signal to Nicotiana plumbaginifolia Viviani, causing these plants to double both their biomass and all of the cell contents. Herein, we addressed whether this effect of NO2 is also observed in various horticultural plants. Lettuce (Lactuca sativa L.), sunflower (Helianthus annuus L.), cucumber (Cucumis sativus L.), and pumpkin (Cucurbita moschata Duch. ex Lam.) were grown with 50, 200, 100, and 200 µL·L–1, respectively, of air supplemented with stable isotope-labelled (15N) NO2, for 5-6 weeks. Control plants were grown without NO2 (<5 µL·L–1). All plants were irrigated with nonlabelled nitrate. The presence of NO2 doubled both the aboveground and belowground biomass in sunflowers compared with their growth in the absence of NO2, whereas lettuce, cucumber, and pumpkin doubled in aboveground biomass only. Contents per shoot of carbon (C), nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg) were almost doubled during the “NO2-enhanced” growth in lettuce, but not in other plants. Mass spectrometry analysis of 15N/14N indicated that only a minor proportion (0.2%–14%) of total plant N was derived from NO2, implying that exogenous NO2 acts as a signal rather than a significant nutrient source in horticultural plants. [ABSTRACT FROM AUTHOR]

As reported previously, atmospheric nitrogen dioxide (NO2) at an ambient level increased plant size and the contents of cell constituents. We investigated this effect of atmospheric NO2 on decontamination of cadmium (Cd) by kenaf (Hibiscus cannabinus). Seventeen-day-old seedlings of kenaf were grown in air either with NO2 or without NO2. (Plants were exposed to 100 ± 50 ppb NO2 for 10 d under irrigation of 0.1% Hyponex supplemented with 20 μM CdCl2.) Plants were then harvested and the biomass of stems, leaves, and roots, as well as the content of Cd in the organs, was determined. The stem and root biomass per plant were 1.25-1.27-fold greater in +NO2 plants than in -NO2 plants. The Cd content per stem was more than 30% greater in +NO2 plants than in -NO2 plants. [ABSTRACT FROM AUTHOR]

Abstract: In order to screen for the best species for mitigating nitrogen dioxide (NO2) by plants at urban levels, we investigated assimilation of nitrogen dioxide by 70 taxa of woody plants that are mostly utilized as roadside trees. They were fumigated with 15N-labeled NO2 at 0.1μll−1 for 8h, and the amount of reduced nitrogen derived from NO2 (in mg Ng−1 dry weight) in the leaves (designated NO2 assimilation capability hereafter) were determined. Data were analyzed in the comparison with the previously reported ones obtained at 4μll−1 NO2. Among the 70 taxa, the value of NO2 assimilation capability differed by a factor of 122 between the highest (Prunus yedoensis; 0.061) and the lowest (Cryptomeria japonica; 0.0005). Based on the analysis of NO2 assimilation capability values at 0.1 and 4μl−1 NO2, the 70 taxa of woody plants appeared to be classified into four types; those of high NO2 assimilation and high NO2 resistance, those of high NO2 assimilation but low NO2 resistance, those of low NO2 assimilation and low NO2 resistance, and those of low NO2 assimilation but high NO2 resistance. The first, second, third and fourth types include 13, 11, 35 and 11 taxa, respectively. The broad-leaf deciduous trees may have advantages of high biomass and fast growth as compared with woody plants of other habits. Thus, four broad-leaf deciduous species, Robinia pseudo-acacia, Sophora japonica, Populus nigra and Prunus lannesiana, were concluded here to be the best phytoremediators for the urban air. [Copyright &y& Elsevier]

• We report the unexpected novel finding that exogenously supplied atmospheric NO2 at an ambient concentration is a plant vitalization signal to double shoot size and the contents of cell constituents. • When seedlings of Nicotiana plumbaginifolia were grown for 10 wk under natural light and irrigation with 10 m m KNO3 in air containing (+NO2 plants) or not containing (–NO2 plants) 15NO2 (150 ± 50 ppb), shoot biomass, total leaf area, and contents per shoot of carbon (C), nitrogen (N), sulphur (S), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), free amino acids and crude proteins were all approximately 2 times greater in +NO2 plants than in –NO2 plants. • In mass spectrometric analysis of the 15N/14N ratio, it was found that NO2-derived N (NO2-N) comprised < 3% of total plant N, indicating that the contribution of NO2-N to total N was very minor. • It thus seems very likely that the primary role of NO2 is as a multifunctional signal to stimulate plant growth, nutrient uptake and metabolism. [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]

Summary A 12.5-kb DNA fragment with junction regions between the transgene and genomic DNA was cloned from a transgenic tobacco cell line obtained by microprojectile bombardment of plasmid pCaMVNEO. Nucleotide sequence analysis of the fragment (DDBJ accession no. D84238) showed that it carried a 7.7-kb core sequence (concatemer of a complete pCaMVNEO and a partial pCaMVNEO) and two identical 1.3-kb junction sequences that flanked both the 5′ and 3′ ends of the core sequence and had inverted orientations. These sequences had topoisomerase II (Topo II) cleavage sites and adenine and thimine-rich sequences known to be specific to nuclear scaffold-attachment regions (SARs). An in vitro binding assay showed that a 507-bp fragment (designated TJ1) from the 1.3-kb sequence had the ability to bind to nuclear scaffold preparations of cultured tobacco cells, confirmation that the 1.3-kb sequence is an SAR. Insertion of TJ1at the 5′ and 3′ sides of the expression cassette for the npt II gene increased transformant yields 5- to 10-fold and the NPT II enzyme activity per copy of the gene 5-fold. TJ1enhances the integration or expression of the transgene, or both. Clearly, TJ1is very useful for producing transgenic plants. This is the first report on an SAR-like sequence that is located in the transgene locus and enhances transformation efficiency in eukaryotic cells. The possible role of TJ1-SAR in the molecular evolution of plant genome is discussed. [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]

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]

Tobacco (Nicotiana tabacum L. cv. Xanthi XHFD8) possesses four nitrite reductase (NiR) genes: nii1, nii2, nii3, and nii4. Their differential expression in leaves and roots was investigated by quantitative competitive RT-PCR using gene-specific primer pairs. These results appear to contradict existing views on the expression of these NiR genes: (i) the mRNA of each of the four NiR genes was distinguishable both in leaves and roots; (ii) nitrate treatment increased nii1 and nii3 mRNA in leaves and roots by at least 4-fold (at least 5-fold in nii2 and nii4 mRNA); and (iii) the steady-state levels of nii1 and nii3 mRNA were almost the same in leaves (6–7×105 and about 3×106 copies μg−1 of total RNA before and after nitrate treatment, respectively) and in roots (3–4×104 and 3–6×105 copies μg−1 of total RNA before and after nitrate treatment, respectively). Very similar relationships were obtained for the steady-state levels of nii2 and nii4 mRNA in roots (2–4×105 and 8×106 copies μg−1 of total RNA before and after nitrate treatment, respectively), and in leaves (5–9×104 and 4×105 copies μg−1 of total RNA before and after nitrate treatment, respectively). These results demonstrate that nii1 and nii3 transcripts are a dominating, but not exclusive, NiR mRNA in leaves, and the same is true for nii2 and nii4 transcripts in roots. [ABSTRACT FROM PUBLISHER]

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]

A correction to the article "Atmospheric nitrogen dioxide at ambient levels stimulates growth and development of horticultural plants," that was published on the National Research Council (NRC) Research Press Web site on March 20, 2008 is presented.

Summary The importance of nitric oxide (NO) as a signaling molecule to various plant physiological and pathophysiological processes is becoming increasingly evident. However, little is known about how plants protect themselves from nitrosative and oxidative damage mediated by NO and NO-derived reactive nitrogen species (RNS). Peroxynitrite, the product of the reaction between NO and superoxide anion, is considered to play a central role in RNS-induced cytotoxicity, as a result of its potent ability to oxidize diverse biomolecules. Employing heterologous expression in bacteria and yeast, we investigated peroxynitrite-scavenging activity in plants of 2-Cys peroxiredoxin (2CPRX), originally identified as a hydroperoxide-reducing peroxidase that is ubiquitously distributed among organisms. The putative mature form of a chloroplast-localized 2CPRX from Arabidopsis thaliana was overproduced in Escherichia coli as an amino-terminally hexahistidine-tagged fusion protein. The purified recombinant 2CPRX, which was catalytically active as peroxidase, efficiently prevented the peroxynitrite-induced oxidation of a sensitive compound. We also examined in vivo the ability of the Arabidopsis 2CPRX to complement the 2CPRX deficiency of a Saccharomyces cerevisiae mutant. Functional expression in the mutant strain of the Arabidopsis 2CPRX not only increased cellular tolerance to hydrogen peroxide, but also complemented the hypersensitive growth defect induced by nitrite-mediated cytotoxicity. The complemented cells significantly enhanced the capacity to reduce RNS-mediated oxidative damages. The results presented here demonstrate a new role of plant 2CPRX as a critical determinant of the resistance to RNS, and support the existence of a plant enzymatic basis for RNS metabolism. [ABSTRACT FROM AUTHOR]

Indicates that all correspondence concerning 'The Plant Journal' paper titled 'Emission of nitrous oxide (N[sub 2]O) from transgenic tobacco expressing antisense NiR mRNA' should be addressed to Hiromichi Morikawa, Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.