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Foliar uptake of nitrogen dioxide (NO(2)) is governed by its reactive absorption mechanism, by which NO(2) molecules diffuse through cell wall layers and simultaneously react with apoplastic ascorbate to form nitrous acid, which freely diffuses across plasmalemma. However, whether free diffusion of nitrous acid is the sole mechanism of foliar uptake of NO(2) remains unknown. The involvement of ammonia-inhibitable nitrite transporters in the foliar uptake of NO(2), as reported in nitrite transport in Arabidopsis roots, is also unknown. In this study, we treated Arabidopsis thaliana leaves with methionine sulfoximine (MSX) to inhibit incorporation of ammonia into glutamate and exposed them to 4 ppm (15)N-labeled NO(2) for 4 h in light followed by quantification of total nitrogen, reduced nitrogen, and ammonia nitrogen derived from NO(2) using mass spectrometry and capillary electrophoresis. The total nitrogen derived from NO(2) in leaves without MSX treatment was 587.0 nmol NO(2)/g fresh weight, of which more than 65% was recovered as reduced nitrogen. In comparison, MSX treatment decreased the total nitrogen and reduced nitrogen derived from NO(2) by half. Thus, half of the foliar uptake of NO(2) is not attributable to passive diffusion of nitrous acid but to ammonia-inhibitable nitrite transport. Foliar uptake of NO(2) is mediated by a dual mechanism in A. thaliana: nitrous acid-free diffusion and nitrite transporter-mediated transport.

Protein tyrosine nitration is a selective process, as revealed in studies of animals. However, evidence for selective protein nitration in plants is scarce. In this study, Arabidopsis plants were exposed to air with or without nitrogen dioxide at 40 ppm for 8 h in light. Proteins extracted from whole leaves or isolated chloroplasts were subjected to 2D PAGE followed by SYPRO Ruby staining and immunoblotting using an anti-3-nitrotyrosine antibody. We determined the relative intensity of a spot on an immunoblot (designated RISI), and relative intensity of the corresponding spot on SYPRO Ruby gel (designated RISS). Proteins that exhibited a high RISI value and/or a high RISI/RISS ratio were considered selectively nitrated. In whole leaf proteins from exposed plants, all immunopositive spots were identified as PsbO1, PsbO2 or PsbP1 by PMF. Thus, nitration was exclusive to PsbO and PsbP, extrinsic proteins of photosystem II (PSII). Their RISI/RISS ratio was ≤1.5. Non-exposed plants showed very faint nitration. In purified chloroplast proteins, PsbO and PsbP accounted for >80% of the total RISI values, while four non-PSII proteins, including peroxiredoxin II E, exhibited high RISI/RISS ratios (2.5∼6.6). Tyr(9) of PsbO1 was identified as a nitration site. Thus, nitration is selective for two PSII and four non-PSII proteins in Arabidopsis.

Exposure of intact Arabidopsis leaves to 40 ppm nitrogen dioxide (NO2) in light resulted almost exclusively in nitration of PsbO1, PsbO2, and PsbP1 of photosystem II (PSII), with minor nitration of four non-PS II proteins, including peroxiredoxin II E, as reported previously. Our previous findings that light-triggered selective nitration of PsbO1 decreased oxygen evolution and that inhibition of photoelectric electron transport inhibited nitration of PsbO1 implied that the nitratable tyrosine residue of PsbO1 is redox-active. However, whether the nitratable tyrosine residues of PsbO2 and PsbP1 are redox-active is unknown. In this study, we determined the oxygen evolution and maximal photochemical efficiency of PSII in intact Arabidopsis leaves following exposure to 40 ppm NO2 in light and found that these parameters were decreased to 60 and 70% of the non-exposed control, respectively. Because PsbO1, PsbO2, and PsbP1 accounted for > 80% of anti-3-nitrotyrosine antibody signal intensities, observed decreases in the oxygen evolution and maximal photochemical efficiency of PSII were mainly attributable to nitration of the tyrosine residues of these PSII proteins. Thus, it is postulated that nitratable tyrosine residues of PsbO2 and PsbP1 are redox-active, as in the case of PsbO1. A new hypothetical model is proposed.

Treatment of isolated Arabidopsis thaliana thylakoid membranes with nitrogen dioxide (NO2) induces selective nitration of the tyrosine residue at the ninth amino acid (9Tyr) of PsbO1. This selective nitration is triggered by light and is inhibited by photosynthetic electron transport inhibitors. Therefore, we postulated that, similar to 161Tyr of D1 (YZ), 9Tyr of PsbO1 is redox active and is selectively oxidized by photosynthetic electron transport in response to illumination to a tyrosyl radical that is highly susceptible to nitration. This tyrosyl radical may combine rapidly at diffusion-controlled rates with NO2 to form 3-nitrotyrosine. If this postulation is correct, the nitration of 9Tyr of PsbO1 should decrease oxygen evolution activity. We investigated the effects of PsbO1 nitration on oxygen evolution from isolated thylakoid membranes, and found that nitration decreased oxygen evolution to ≥ 0% of the control. Oxygen evolution and nitration were significantly negatively correlated. This finding is consistent with redox active properties of the 9Tyr gene of PsbO1, and suggests that PsbO1 9Tyr acts as an electron relay, such as YZ in the photosystem II oxygenic electron transport chain.

In this communication, we review our work over two decades on air-pollutant-philic plants that can grow with air pollutants as the sole nutrient source. We believe that such plants are instrumental in mitigating air pollution. Our target air pollutant has been atmospheric nitrogen dioxide (NO2), and our work on this subject has consisted of three parts: Variation in plants’ abilities to mitigate air pollutants among naturally occurring plants, genetic improvement of plants’ abilities to mitigate air pollutants, and the plant vitalization effect of NO2. So far, an estimation of the half-life of nitrogen derived from NO2 uptake in plants belonging to the 217 taxa studied to date has shown no plants to be naturally occurring air-pollutant-philic. However, we found that an enormous difference exists in plants’ ability to uptake and assimilate atmospheric NO2. Future studies on the causes of this process may provide an important clue to aid the genetic production of plants that are effectively air-pollutant-philic. Both genetic engineering of the genes involved in the primary nitrate metabolism and genetic modification by ion-beam irradiation failed to make plants air-pollutant-philic, but mutants obtained in these studies will prove useful in revealing those genes critical in doing so. During our study on air-pollutant-philic plants, we unexpectedly discovered that prolonged exposure of plants to a sufficient level of NO2 activates the uptake and metabolism of nutrients that fuel plant growth and development. We named this phenomenon “the plant vitalization effect of NO2” (PVEON). Investigations into the mechanisms and genes involved in PVEON will provide an important clue to making plants air-pollutant-philic in the future.

It is known that when plant leaves are exposed to exogenously applied nitrogen dioxide (NO2), nitrogen derived from NO2 is reduced to amino acid nitrogen. However, whether this is the sole metaboli...

Exposure of Arabidopsis leaves to nitrogen dioxide (NO(2)) results in the selective nitration of specific proteins, such as PsbO1. The 9th tyrosine residue ((9)Tyr) of PsbO1 has been identified as the nitration site. This nitration is triggered by light and inhibited by photosynthetic electron transport inhibitors. During protein nitration, tyrosyl and NO(2) radicals are formed concurrently and combine rapidly to form 3-nitrotyrosine. A selective oxidation mechanism for (9)Tyr of PsbO1 is required. We postulated that, similar to (161)Tyr of D1, (9)Tyr of PsbO1 is selectively photo-oxidized by photosynthetic electron transport in response to illumination to a tyrosyl radical. In corroboration, after reappraising our oxygen evolution analysis, the nitration of PsbO1 proved responsible for decreased oxygen evolution from the thylakoid membranes. NO(2) is reportedly taken into cells as nitrous acid, which dissociates to form NO(2)(−). NO(2)(−) may be oxidized into NO(2) by the oxygen-evolving complex. Light may synchronize this reaction with tyrosyl radical formation. These findings suggest a novel role for PsbO1 in photosynthetic electron transport.

A high affinity transport system (HATS) for nitrate in plants is operated by a two-component NRT2/NAR2 transport system. However, the regulation and localization of NRT2 and NAR2 at protein level are largely unknown and especially so in crop plant species. In this study with barley (Hordeum vulgare), membrane localization, protein expression in the roots, and a direct protein-protein interaction of HvNRT2 and HvNAR2 proteins were investigated. Immunochemical analysis showed that both HvNRT2 and HvNAR2 proteins were co-localized in the plasma membrane of the roots. Expression of HvNRT2 and HvNAR2 proteins was more strongly induced by treatment with higher concentrations of external nitrate, while HATS activity and transcripts for HvNRT2 and HvNAR2 were markedly repressed. An affinity column binding analysis using recombinant proteins suggests that the C-terminus of HvNRT2.1 is possibly involved in its binding to the HvNAR2.3 central region and that the Ser463 present in the HvNRT2.1 C-terminus plays a role in the binding ability.

Atmospheric nitrogen dioxide (NO(2)) 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 NO(2)-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 NO(2) 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 NO(2). These enzymes might have a potential role in extracellular defense mechanisms through attenuation of interactions between reactive nitrogen and oxygen species.

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 microM 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.

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 (–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.

To elucidate the stimulation of leaf growth by atmospheric nitrogen dioxide (NO2), we performed a kinematic analysis of the eighth leaves of Arabidopsis thaliana (accession C24) plants grown for 17–35 days after sowing in the presence or absence of 50 ppb NO2 (designated +NO2 plants and –NO2 plants, respectively). We found that the peak and mean values of the relative rates of leaf expansion, cell division and cell expansion were always greater in +NO2 plants than in –NO2 plants. No evidence for prolonged duration was obtained. Thus, NO2 treatment increased the rates of both cell proliferation and enlargement to increase leaf size. Furthermore, a fold-change analysis showed that cell proliferation and enlargement differentially regulated NO2-induced leaf expansion.

Protein tyrosine nitration is an important post-translational modification. A variety of nitrated proteins are reported in Arabidopsis leaves and seedlings, sunflower hypocotyls, and pea roots. The identities of nitrated proteins are species-/organ-specific, and chloroplast proteins are most nitratable in leaves. However, precise mechanism is unclear. Here, we investigated nitroproteome in tobacco leaves following exposure to nitrogen dioxide. Proteins were extracted, electrophoresed and immunoblotted using an anti-3-nitrotyrosine antibody. Mass spectrometry and FASTA search identified for the first time an exclusive nitration of pathogenesis-related proteins, PR-1, PR-3 and PR-5, which are reportedly located in the apoplast or the vacuole. Furthermore, Tyr36 of thaumatin-like protein E2 was identfied as a nitration site. The underlying mechanism and physiological relevance are discussed.

Exposure of Arabidopsis leaves to nitrogen dioxide (NO2) results in nitration of specific chloroplast proteins. To determine whether NO2 itself and/or nitrite derived from NO2 can nitrate proteins, Arabidopsis thylakoid membranes were isolated and treated with NO2-bubbled or potassium nitrite (KNO2) buffer, followed by protein extraction, electrophoresis, and immunoblotting using an anti-3-nitrotyrosine (NT) antibody. NO2 concentrations in the NO2-bubbled buffer were calculated by numerically solving NO2 dissociation kinetic equations. The two buffers were adjusted to have identical nitrite concentrations. Both treatments yielded an NT-immunopositive band that LC/MS identified as PSBO1. The difference in the band intensity between the two treatments was designated nitration by NO2. Both NO2 and nitrite mediated nitration of proteins, and the nitration ability per unit NO2 concentration was ∼100-fold greater than that of nitrite.

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 0.1 microl l(-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 4 microl l(-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 4 micro l(-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.

Arabidopsis thaliana was transformed with a gene encoding a nitroreductase (NTR, E.C. 1.6.99.7) with activity against a wide range of nitroaromatic compounds. The gene was transferred from Escherichia coli by an Agrobacterium-mediated in planta method. The ob­tained seeds were sowed to produce T1 plants, and they were assayed for the integration of the transgene in the plant genome. Transgenic plants that were positive with the PCR analysis were self-pollinated to produce T2 generation plants. Seven lines obtained were assayed for the NTR activity. While the noil-transformed wild-type plants showed no detectable NTR activity, the enzyme activity of the transgenic plant lines was approx. 20 times higher. Using the line with the highest NTR activity, the phytoremediation characteristics of plants against 2,4,6-trinitrotoluene (TNT) was investigated. While the wild-type plants did not grow in the presence of 0.1 mᴍ TNT, the transgenic plants grew almost normally in this condition. The uptake of TNT by seedlings of transgenic plants increased by 7 to 8 times when they were floated on TNT solution. HPLC analysis showed that the peak due to TNT taken up into plant body was much smaller in the transgenic plants as compared with that of the wild type, and that a number of peaks attributable to the degradation products of TNT, including 4-amino-2,6-dinitrotoluene, were detected in the extract from the transgenic plants. This indi­cates that the expression of bacterial NTR improved the capability of plants to degrade TNT.

Shoot explants cut from aseptically grown Ficus thunbergii plants were irradiated with 12 C 5� , 12 C 6� and 4 He 2� ion beams of 10 to 200 Gray (Gy). After irradiation, the explants were allowed to regenerate. The regeneration frequency (RF), or percent explants bearing regenerated shoots to the total number of explants irradiated, decreased in a dose- dependent manner. Morphological changes in leaves and stems were observed in regenerants. The morphological change frequency (MF), or percent regenerants bearing morphologically changed shoots to the total number of regenerants peaked at 15 to 25% at the dose of each ion beam that gave an RF of about 80% (except for 4 He 2� ). A negative correlation was observed between the linear energy transfer (LET) and ion beam dose both in RF and MF. The present results strongly suggest that ion beam irradiation can induce mutations in the genome of F. thu nbergii.

A 507-bp nuclear matrix attachment region (MAR) sequence (designated TJ1) was flanked at both at 5� and 3� ends of the expression cassette for a modified green fluorescent protein (GFP) gene of the 35 W-sGFP(S65T) in four different orientations. A plasmid p507TH bearing a 507-bp non-MAR sequence from yeast at both ends of the cassette also was constructed. These five plasmids and 35 W-sGFP(S65T) were separately delivered into cultured tobacco BY-2 cells by particle bombardment. The yield of stable transformants that showed a uniform green fluorescence was determined. Southern blot analysis confirmed the integration of the GFP gene in tobacco genome. Transformant yields of the two TJ1- lacking plasmids were not significant. Transformant yields appeared to be increased by more than 2 times by the presence of TJ1-MAR sequences. The orientation of TJ1-MAR sequences did not influence the transformant yield.

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.

The production of a novel "nitrogen-dioxide-philic plant" that can grow with NO 2 as the sole nitrogen source was investigated as a possible solution to pollution. 217 plant taxa were screened for the assimilation of NO 2 , among them 50 wild herbaceous plants collected from roadsides, 60 cultivated herbaceous plants and 107 cultivated woody plants. It was discovered that the NO 2 -derived reduced nitrogen [μg N/mg dry weight] differed by a factor of 657 between the highest (Eucalyptus viminalis, 6.57) and lowest (Tillandsia ionantha and T. caputmedusae, 0.01) level in the 217 taxa. The capability of plants to assimilate NO 2 was also found to change in a family-dependent manner. Compositae, Myrtaceae, Salicaceae and Solanaceae had taxa with high NO 2 assimilation capability, while none of the Gramineae, Theaceae and Rosaceae plants examined showed high NO 2 assimilation capability. The 217 taxa included two species of the Solanaceae family (tobacco and petunia), both of which showed a high NO 2 assimilation and their reduced nitrogen derived from NO 2 was the third and twenty-sixth highest. This suggested that the Solanaceae family includes plants of high NO 2 assimilation. Therefore, in this study eight taxa of the Solanaceae family and four taxa of other families were investigated for NO 2 assimilation in the same manner as reported previously by the fumigation of plants with 1 5 N-labelled NO 2 followed by the mass spectroscopic analysis based upon the reduced nitrogen derived from NO 2 . The eight taxa Solanaceae included tobacco (Nicotiana tabacum) and petunia (Petunia X hybrida), ground cherry (Physalis alkekengi var. franchetii), tomato (Lycopersicon esculentum), datura (Datura innoxia), potato vine (Solanum jasminoides), red pepper (Capsicum annuum) and green pepper (Capsicum annuum var. grossum SENDTN.). The four taxa included two ivy cultivars (Hedera helix "Pittsburgh" and Hedera helix "Wonder"), an evergreen creeper (Ficus thunbergii) and kenaf (Hibiscus cannabinus). Among the twelve taxa, kenaf, tobacco and ground cherry showed the highest, second and third highest values, respectively, of reduced nitrogen derived from NO 2 in plants, which reflects the capability to assimilate NO 2 -nitrogen. The quantitative significance of these findings is discussed in relation to the reduction of nitrogenous pollution by the atmospheric NO 2 pollution.

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.

When hypocotyl sections of Raphiolepis umbellata L. were cultured in a woody plant medium containing 8.9μM benzylaminopurine and 1.1μM α-naphthaleneacetic, adventitous shoots were formed. Fully developed plants were obtained in 3 months of the culture. A chimeric plasmid pENiR harboring cDNA of Arabidopsis thaliana nitrite reductase gene (nii) under the control of an enhanced promoter consisting of sequences from both cauliflower mosaic virus 35S promoter and tobacco mosaic virus omega regions and a nopaline synthase terminator was constructed and introduced to the hypocotyl sections by particle bombardment. A plasmid pCH bearing hygromycin phosphotransferase gene (hph) was cointroduced. Transformed shoots were selected for hygromycin resistance. Polymerase chain reaction analysis showed the presence of hph gene in 16 out of 2572 bombarded hypocotyl sections, and two of which were found to bear Arabidopsis nii gene. Translation of this gene in transgenic plants was confirmed by Western blot analysis.

Protein tyrosine nitration is a selective process, as revealed in studies of animals. However, evidence for selective protein nitration in plants is scarce. In this study, Arabidopsis plants were exposed to air with or without nitrogen dioxide at 40 ppm for 8 h in light. Proteins extracted from whole leaves or isolated chloroplasts were subjected to 2D PAGE followed by SYPRO Ruby staining and immunoblotting using an anti-3-nitrotyrosine antibody. We determined the relative intensity of a spot on an immunoblot (designated RISI), and relative intensity of the corresponding spot on SYPRO Ruby gel (designated RISS). Proteins that exhibited a high RISI value and/or a high RISI/RISS ratio were considered selectively nitrated. In whole leaf proteins from exposed plants, all immunopositive spots were identified as PsbO1, PsbO2 or PsbP1 by PMF. Thus, nitration was exclusive to PsbO and PsbP, extrinsic proteins of photosystem II (PSII). Their RISI/RISS ratio was ≤1.5. Non-exposed plants showed very faint nitration. In purified chloroplast proteins, PsbO and PsbP accounted for80% of the total RISI values, while four non-PSII proteins, including peroxiredoxin II E, exhibited high RISI/RISS ratios (2.5∼6.6). Tyr(9) of PsbO1 was identified as a nitration site. Thus, nitration is selective for two PSII and four non-PSII proteins in Arabidopsis.

In order to select individuals of Rhododendron mucronatum plants that have a high capacity to decontaminate nitrogen dioxide (NO 2 ), which is a major air pollutant, a number of lines prepared by cutting propagation were analyzed for their uptake of NO 2 . In 1996, 1452 different lines (referred to as the 96-plants) were fumigated with 4 μl/l 15 NO 2 for 8 hours, and the total nitrogen content of the leaves, derived from NO 2 , was determined and reflects the plant's NO 2 uptake capacity. A 56-fold difference was observed between the individuals with the highest and lowest capacities. About 100 lines each from the high-, middle- and low-capacity groups were propagated and assayed for NO 2 uptake in the following year (the 97-plants). The maximum variation in NO 2 uptake was 5.6 times. In 1998, 227 lines propagated from the 96-plants and 95 lines from the 97-plants were assayed for NO 2 uptake (the 98-plants). The variations in NO 2 uptake in 97- and 98-plants were a maximum of 4.9 and 5.2 times, respectively. The reason(s) for the disappearance of the initial large variation in NO 2 uptake is (are) not yet known. Those lines showing high and low NO 2 uptake capacities will be useful when studying the genes involved in this important roadside tree.

DNA sequences of transgenic loci in the transgenes of tobacco and Arabidopsis were analyzed. Almost all transgenic loci studied contained cis elements characteristic of nuclear matrix attachment regions (MARs). A MAR sequence isolated from one such locus of tobacco cells was cloned into the original transformation vector, and introduced into tobacco cells. The presence of the MAR sequence resulted in a five- to tenfold increase in the transformant yields, and it appeared to stimulate both the expression of the transgene and its integration into the host genome. A search using the BLAST program revealed that the transgenic loci contained junction region sequences that may have originated from different chromosomes of the nuclear genome and from the chloroplast genome.

Prolonged exposure of plants to a sufficient level of atmospheric nitrogen dioxide (NO2) activates the uptake and metabolism of nutrients that fuel plant growth and development, a phenomenon termed the plant vitalization effect of NO2. This study examined the effect of NO2 on the fruit yield of tomato (Solanum lycopersicum L. cv. Micro-Tom). Double exposure chambers, designated ±NO2 chambers, were placed in a confined greenhouse. The air entering the −NO2 chamber was scrubbed of nitrogen oxides including NO2, and the air entering the +NO2 chamber was supplemented with NO2 (50 ± 10 ppb). Two-week-old tomato seedlings that had been grown in the −NO2 chamber after sowing were then grown in a +NO2 or −NO2 chamber for the remainder of the growth period until 96 days after sowing. Growth in the +NO2 chamber led to a significant increase in fruit yield of approximately 40% as compared to growth in the −NO2 chamber. This increase in fruit yield was accompanied by acceleration of flowering time by 3.2 days and an increase in flower number per plant of up to 60%. These results show that exposure to NO2 increases fruit yield via stimulation of flowering in tomato.

Transgenic plants of Arabidopsis bearing the spinach (Spinacia oleracea) nitrite reductase (NiR, EC 1.7.7.1) gene that catalyzes the six-electron reduction of nitrite to ammonium in the second step of the nitrate assimilation pathway were produced by use of the cauliflower mosaic virus 35S promoter and nopaline synthase terminator. Integration of the gene was confirmed by a genomic polymerase chain reaction (PCR) and Southern-blot analysis; its expression by a reverse transcriptase-PCR and two-dimensional polyacrylamide gel electrophoresis western-blot analysis; total (spinach + Arabidopsis) NiR mRNA content by a competitive reverse transcriptase-PCR; localization of NiR activity (NiRA) in the chloroplast by fractionation analysis; and NO2 assimilation by analysis of the reduced nitrogen derived from NO2(NO2-RN). Twelve independent transgenic plant lines were characterized in depth. Three positive correlations were found for NiR gene expression; between the total NiR mRNA and total NiR protein contents (r = 0.74), between the total NiR protein and NiRA (r = 0.71), and between NiRA and NO2-RN (r = 0.65). Of these twelve lines, four had significantly higher NiRA than the wild-type control (P < 0.01), and three had significantly higher NO2-RN (P < 0.01). Each of the latter three had one to two copies of spinach NiR cDNA per haploid genome. The NiR flux control coefficient for NO2 assimilation was estimated to be about 0.4. A similar value was obtained for an NiR antisense tobacco (Nicotiana tabacum cv Xanthi XHFD8). The flux control coefficients of nitrate reductase and glutamine synthetase were much smaller than this value. Together, these findings indicate that NiR is a controlling enzyme in NO2assimilation by plants.

Summary The emission of N2 and N2O from intact transgenic tobacco (clone 271) expressing antisense nitrite reductase (NiR) mRNA, and wild-type plants grown aseptically, on NO3–, NO2– or NH4+ -containing medium was investigated. 15N contents of gas sampled from gas-sealed pots, in which the plants were grown on 15N-containing medium, were analyzed by gas chromato- graphy and mass spectrometry (GC–MS). No emission of N2 was detected in either of the gas samples from plant clone 271 or the wild-type grown on NO3–-containing medium. N2O emission from clone 271 grown on NO3–-containing medium was detected, but not from the wild-type plants. The N2O emission rate of clone 271 was 106 ng N2O mg–1 incorporated N week–1 and the N2O emission was inhibited by tungstate (a nitrate reductase inhibitor). No emission of N2O was found from clone 271 or wild-type plants grown on medium containing NH4+. Emission of N2O also was detected from clone 271 grown on NO2–-containing medium and its emission rate increased with increasing NO2– levels in plants. We speculate that NO3– is reduced to NO2– and that a part of NO2– is metabolized to N2O in clone 271.

Particle bombardment has proved to be useful for the transformation of plants. We have previously reported successful transient expression of the beta-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.

Assimilation of nitrogen dioxide in response to fumigation with 15 N-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 'NO 2 -N content', or NO 2 -derived reduced nitrogen content in fumigated plant leaves (mg N g -1 dry weight), and the 'NO 2 -utilization index', or percentage of the NO 2 -derived reduced nitrogen in the total reduced nitrogen, were determined. The NO 2 -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 NO 2 -utilization indices greater than 10%, of which Magnolia kobus, Eucalyptus viminalis, Populus nigra, Nicotiana tabacum and Erechtites hieracifolia had NO 2 -N contents > 4.9. These plants can be considered 'NO 2 -philic' because in them NO 2 -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 NO 2 -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.

• 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.

Suspension-cultured cells (A-18 line) of the liverwortMarchanta 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 7×106 cells. Ten cell lines chosen randomly were analysed further. Southern blot analysis showed that all of the ten lines contain thehpt 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.

Root explants ofArabidopsis thaliana ecotype C24 were bombarded with the plasmid pCH harboring the hygromycin phosphotransferase gene (hpt). A selection condition with post-bombardment culture of 3 days followed by culture with 20 mgl−1 hygromycin gave the highest yield of transformants. More than 44% of explant clumps formed transformant shoots.

Transgenic phalaenopsis plants have been obtained by particle bombardment with a pneumatic particle gun device. All promoters of cauliflower mosaic virus (CaMV) 35S, maize ubiquitin and rice actin genes could be expressed in phalaenopsis using transient assay of β-glucuronidase (gus) gene as a reporter. Bialaphos resistance gene (bar) which confers tolerance to herbicide bialaphos, was used as a selectable marker. Protocormlike bodies (PLBs) of phalaenopsis derived from the leaf segment culture were bombarded by gold particles coated with pMSP38 and pWI-GUS DNA containing the bar gene and the gus gene respectively driven by 35S promoter. Newly-formed PLBs were selected on the medium including bialaphos. Finally seven transgenic plants resistant to bialaphos were obtained, and one of them contained and expressed gus gene. PCR analysis confirmed their transgenic nature and western analysis and histochemical GUS assay showed the expression of bar and gus gene respectively.

Root explants of Eustoma grandiflorum were bombarded with plasmid pBI221 harboring the uidA gene encoding β-glucuronidase (GUS) driven by a cauliflower mosaic virus 35S promoter, and the effects of pre- and post-bombardment culture in various starvation media on the number of blue spots of GUS-expressing cells were studied. It was found that pre- and post-bombardment culture in nitrogen-depleted media increased the expression frequency of the transgene, by 4- to 8-times that of the control, and that 3-h pre-culture in the nitrogen-depleted medium gave the highest expression frequency.

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 x 10(5) and about 3 x 10(6) copies microg(-1) of total RNA before and after nitrate treatment, respectively) and in roots (3-4 x 10(4) and 3-6 x 10(5) copies microg(-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 x 10(5) and 8 x 10(6) copies microg(-1) of total RNA before and after nitrate treatment, respectively), and in leaves (5-9 x 10(4) and 4 x 10(5) copies microg(-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.

We succeeded expressing the streptomyces cholesterol oxidase gene (choA), a potent insecticidal protein active against boll weevil larvae, in tobacco cells. A plasmid, pBC4, containing the choA gene under the control of the 35S promoter of cauliflower mosaic virus, was introduced into tobacco cells (BY-2) using a pneumatic particle gun. Stable integration of the choA gene in the genome of the transformed callus line was confirmed by Southern hybridization analysis. Northern blot analysis revealed material corresponding in size to the choA transcript in the transformed cells. Cholesterol oxidase activity in transformed callus was about eightfold higher than that in control cell line.

PsbO1 is exclusively nitrated when isolated thylakoid membranes are incubated in a buffer bubbled with nitrogen dioxide (NO2) containing NO2 and nitrite. NO2 is the primary intermediate for this selective nitration. Isolated thylakoid membranes were incubated in NO2-bubbled buffer at 25°C in the light or dark. Protein analysis confirmed the selective nitration of PsbO1. Illumination was found to be essential in PsbO1 nitration. A nitration mechanism whereby nitratable tyrosine residues of PsbO1 are, prior to nitration, selectively photo-oxidized by photosynthetic electron transport to tyrosyl radicals to combine with NO2 to form 3-nitrotyrosine was hypothesized. We tested the electron transport inhibitors 3-(3,4-dichlorophenyl)-1,1- dimethylurea, sodium azide, and 1,5-diphenylcarbazide and found distinct inhibition of nitration of PsbO1. We also propose a possible nitration mechanism.

Immature pollen fromNicotiana 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 intactnptII 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.

A highly repeated DNA sequence with a repeating unit of approximately 380bp was found in EcoRV digests of the total genomic DNA of Allium fistulosum. Three independent clones containing this unit were isolated, and their repeating units sequenced. These units showed more than 94% sequence homology, and the copy number was estimated to be about 2.8×10(6) per haploid genome. In situ hybridization, with the repeating unit as a probe, and C-banding analyses indicated that the repeated DNA sequence of A. fistulosum is closely associated with the major C-heterochromatin in the terminal regions of all 16 chromosomes at mitotic metaphase. The characters of the repeating unit are similar to those of the A. cepa unit, which is taxonomically closely related to A. fistulosum.

Phylogenetic relationships between four Phaseolus (P. angularis, P. calcaratus, P. coccineus and P. vulgaris (including six cultivars)) and two Vigna (V. sesquipedalis and V. sinensis) species were studied by restriction fragment length polymorphism (RFLP) analysis using 15 restriction enzymes and two ribosomal DNA fragments from rice and mung bean as probes. Based on the RFLP data, phylogenetic trees were constructed by the distance matrix and maximum parsimony methods. The two trees constructed by the distance matrix method and one tree constructed by the maximum parsimony method had congruent topology and recognized three clusters: one containing V. sesquipedalis and V. sinensis; a second containing P. angularis; and a third containing P. calcaratus, P. coccineus and six cultivars of P. vulgaris. Grouping of the six species based on the molecular taxonomic data obtained is compared with grouping based on previously reported cytological data.

Various plasmid constructs containing bacterial genes of arylsulfatase (ats) and β-glucuronidase (gus) were introduced into tobacco cells (BY-2) using a pneumatic particle gun device. A 3.6-kb atsB-atsA operon or a 1.8-kb atsA gene was inserted between the cauliflower mosaic virus (CaMV) 35S promoter and the gus gene in the pBI121 vectors. The frequency of transient GUS expression of the vector containing the atsA-gus genes was comparable to that of pBI121 without the atsA gene. The gus gene was also expressed in BY-2 cells with the vector containing the atsB-atsA operon and gus gene, although the degree of transient GUS expression was less than that of the vector with atsA. However, no Ats activity was observed in these transformants. Stable integration of the gus and atsA-gus genes in transformed callus lines was confirmed by Southern hybridization analysis. Northern blot analysis showed 1.9-kb and 3.7-kb transcripts that were identical in size to the gus and atsA-gus genes, respectively. These results suggest that the atsA-gus genes are transcribed polycistronically under the control of the CaMV 35S promoter. Protein gel assay showed two bands with GUS activity in extracts from transformed cells with the vector containing atsA-gus. These results and deletion analysis suggest that the polycistronic transcripts are translated from at least two initiation codons upstream of the gus gene.