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Exposing Arabidopsis thaliana (Arabidopsis) seedlings fed with soil nitrogen to 10−50 ppb nitrogen dioxide (NO2) for several weeks stimulated the uptake of major elements, photosynthesis, and cellular metabolisms to more than double the biomass of shoot, total leaf area and contents of N, C P, K, S, Ca and Mg per shoot relative to non-exposed control seedlings. The 15N/14N ratio analysis by mass spectrometry revealed that N derived from NO2 (NO2-N) comprised < 5% of the total plant N, showing that the contribution of NO2-N as N source was minor. Moreover, histological analysis showed that leaf size and biomass were increased upon NO2 treatment, and that these increases were attributable to leaf age-dependent enhancement of cell proliferation and enlargement. Thus, NO2 may act as a plant growth signal rather than an N source. Exposure of Arabidopsis leaves to 40 ppm NO2 induced virtually exclusive nitration of PsbO and PsbP proteins (a high concentration of NO2 was used). The PMF analysis identified the ninth tyrosine residue of PsbO1 (9Tyr) as a nitration site. 9Tyr of PsbO1 was exclusively nitrated after incubation of the thylakoid membranes with a buffer containing NO2 and NO2− or a buffer containing NO2− alone. Nitration was catalyzed by illumination and repressed by photosystem II (PSII) electron transport inhibitors, and decreased oxygen evolution. Thus, protein tyrosine nitration altered (downregulated) the physiological function of cellular proteins of Arabidopsis leaves. This indicates that NO2-induced protein tyrosine nitration may stimulate plant growth. We hypothesized that atmospheric NO2 at ambient concentrations may induce tyrosine nitration of PYR/PYL/RCAR receptors in Arabidopsis leaves, followed by degradation of PYR/PYL/RCAR, upregulation of target of rapamycin (TOR) regulatory complexes, and stimulation of plant growth.

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.

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.

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 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.

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.

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.

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.

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.

• 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.

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.

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.

Assimilatory nitrite reductase (aNiR) reduces nitrite ions (NO(2)(-)) to ammonium ions (NH(4)(+)), whereas assimilatory sulfite reductase reduces sulfite (SO(3)(2-)) to hydrogen sulfide (HS(-)). Although aNiR can also reduce SO(3)(2-), its activity is much lower than when NO(2)(-) is reduced as the substrate. To increase the SO(3)(2-)-reduction activity of aNiR, we performed a N226K mutation of Nii3, a representative aNiR. The resulting Nii3-N226K variant could bind non-native targets, SO(3)(2-), and HCO(3)(-), in addition to its native target, i.e., NO(2)(-). We have determined the high-resolution structure of Nii3-N226K in its apo-state and in complex with SO(3)(2-), NO(2)(-), and HCO(3)(-). This analysis revealed conformational changes of Lys226 and the adjacent Lys224 upon binding of SO(3)(2-), but not NO(2)(-)In contrast, HCO(3)(-) binding induced a conformational change at Arg179. After replacing Asn226 with a positively charged Lys, aNiR showed affinity for several anions. A comparison of all ligand-bound structures for Nii3-N226K revealed that structural changes in the active site depend on the size of the substrate.

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.

Tobacco expresses four isomers of assimilatory nitrite reductase (aNiR), leaf-type (Nii1 and Nii3), and root-type (Nii2 and Nii4). The high-resolution crystal structures of Nii3 and Nii4, determined at 1.25 and 2.3 Å resolutions, respectively, revealed that both proteins had very similar structures. The Nii3 structure provided detailed geometries for the [4Fe-4S] cluster and the siroheme prosthetic groups. We have generated two types of Nii3 variants: one set focuses on residue Met175 (Nii3-M175G, Nii3-M175E, and Nii3-M175K), a residue that is located on the substrate entrance pathway; the second set targets residue Gln448 (Nii3-Q448K), a residue near the prosthetic groups. Comparison of the structures and kinetics of the Nii3 wild-type (Nii3-WT) and the Met175 variants showed that the hydrophobic side-chain of Met175 facilitated enzyme efficiency (k(cat) /K(m) ). The Nii4-WT has Lys449 at the equivalent position of Gln448 in Nii3-WT. The enzyme activity assay revealed that the turnover number (k(cat) ) and Michaelis constant (K(m) ) of Nii4-WT were lower than those of Nii3-WT. However, the k(cat) /K(m) of Nii4-WT was about 1.4 times higher than that of Nii3-WT. A comparison of the kinetics of the Nii3-Q448K and Nii4-K449Q variants revealed that the change in k(cat) /K(m) was brought about by the difference in Residue 448 (defined as Gln448 in Nii3 and Lys449 in Nii4). By combining detailed crystal structures with enzyme kinetics, we have proposed that Nii3 is the low-affinity and Nii4 is the high-affinity aNiR.

Assimilatory nitrite reductase (aNiR) reduces nitrite to an ammonium ion and has siroheme and a [Fe(4)S(4)] cluster as prosthetic groups. A reaction mechanism for Nii3, an aNiR from tobacco, is proposed based on high resolution X-ray structures and UV-Vis (ultraviolet-visible) microspectroscopy of Nii3-ligand complexes. Analysis of UV-Vis spectral changes in Nii3 crystals with increasing X-ray exposure showed prosthetic group reductions. In Nii3-NO2(-) structures, X-ray irradiation enhanced the progress of the reduction reaction, and cleavage of the N-O bond was observed when X-ray doses were increased. Crystal structures of Nii3 with other bound ligands, such as Nii3-NO and Nii3-NH(2)OH, were also determined. Further, by combining information from these Nii3 ligand-bound structures, including that of Nii3-NO2(-), with UV-Vis microspectral data obtained using different X-ray doses, a reaction mechanism for aNiR was suggested. Cleavage of the two N-O bonds of nitrite was envisaged as a two-step process: first, the N-O bond close to Lys224 was cleaved, followed by cleavage of the N-O bond close to Arg109. X-ray structures also indicated that aNiR-catalyzed nitrite reduction proceeded without the need for conformation changes in active site residues. Geometrical changes in the ligand molecules and the placement of neighboring water molecules appeared to be important to the stability of the active site residue interactions (Arg109, Arg179, and Lys224) and the ligand molecule. These interactions may contribute to the efficiency of aNiR reduction reactions.

Phylogenetic relationships among eight taxa of seven species of Phaseolus and Vigna (Phaseolus angularis, P. aureus, P. calcaratus, P. coccineus, P. vulgaris, Vigna sesquipedalis and V. sinensis; 2n = 22 each) were studied by the fluorescent chromosome banding technique. Preparations of somatic metaphase chromosomes of each taxon were sequentially stained with Giemsa, GC-specific fluorochrome chromomycin A3 (CMA) and AT-specific fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). On the basis of the fluorescent banding patterns of the 22 chromosomes of each taxon, P. angularis, P. coccineus (from China and Korea) and P. vulgaris were grouped into one group ("Phaseolus group"), P. aureus and two Vigna species were grouped into another ("Vigna group") and P. calcaratus was grouped in an independent group.

Expression of the β-glucuronidase (GUS) gene in shoot primordia of Haplopappus gracilis was obtained by particle bombardment. The efficiency of gene delivery was tested by accelerating the pressure from 115 to 200kg/cm2, the number of bombardments from one to three, the amount of gold particles from 0.1 to 0.4mg per projectile, and the amount of plasmid DNA from 0.2 to 0.8μg/mg gold particles. Double bombardments of 200kg/cm2 with projectiles having 0.2mg of gold particles coated with 8μg DNA/mg of gold gave 884±103 blue spots per petri dish (5.2cm diameter).

Xanthine dehydrogenase (XDH) is a ubiquitous enzyme involved in purine metabolism which catalyzes the oxidation of hypoxanthine and xanthine to uric acid. Although the essential role of XDH is well documented in the nitrogen-fixing nodules of leguminous plants, the physiological importance of this enzyme remains uncertain in non-leguminous species such as Arabidopsis. To evaluate the impact of an XDH deficiency on whole-plant physiology and development in Arabidopsis, RNA interference (RNAi) was used to generate transgenic lines of this species in which AtXDH1 and AtXDH2, the two paralogous genes for XDH in this plant, were silenced simultaneously. The nearly complete reduction in the total XDH protein levels caused by this gene silencing resulted in the dramatic overaccumulation of xanthine and a retarded growth phenotype in which fruit development and seed fertility were also affected. A less severe silencing of XDH did not cause these growth abnormalities. The impaired growth phenotype was mimicked by treating wild-type plants with the XDH inhibitor allopurinol, and was reversed in the RNAi transgenic lines by exogenous supplementation of uric acid. Inactivation of XDH is also associated with precocious senescence in mature leaves displaying accelerated chlorophyll breakdown and by the early induction of senescence-related genes and enzyme markers. In contrast, the XDH protein levels increase with the aging of the wild-type leaves, supporting the physiological relevance of the function of this enzyme in leaf senescence. Our current results thus indicate that XDH functions in various aspects of plant growth and development.

Our previous study showed that approximately one-third of the nitrogen of 15N-labeled NO2 taken up into plants was converted to a previously unknown organic nitrogen (hereafter designated UN) that was not recoverable by the Kjeldahl method (Morikawa et al., 2004). In this communication, we discuss metabolic and physiological relevance of the UN based on our newest experimental results. All of the 12 plant species were found to form UN derived from NO2 (about 10-30% of the total nitrogen derived from NO2). The UN was formed also from nitrate nitrogen in various plant species. Thus, UN is a common metabolite in plants. The amount of UN derived from NO2 was greatly increased in the transgenic tobacco clone 271 (Vaucheret et al., 1992) where the activity of nitrite reductase is suppressed less than 5% of that of the wild-type plant. On the other hand, the amount of this UN was significantly decreased by the overexpression of S-nitrosoglutathione reductase (GSNOR). These findings strongly suggest that nitrite and other reactive nitrogen species are involved in the formation of the UN, and that the UN-bearing compounds are metabolizable. A metabolic scheme for the formation of UN-bearing compounds was proposed, in which nitric oxide and peroxynitrite derived from NO2 or endogenous nitrogen oxides are involved for nitrosation and/or nitration of organic compounds in the cells to form nitroso and nitro compounds, including N-nitroso and S-nitroso ones. Participation of non-symbiotic haemoglobin bearing peroxidase-like activity (Sakamoto et al., 2004) and GSNOR (Sakamoto et al., 2002) in the metabolism of the UN was discussed. The UN-bearing compounds identified to date in the extracts of the leaves of Arabidopsis thaliana fumigated with NO2 include a ⊿2- 1,2,3-thiadiazoline derivative (Miyawaki et al., 2004) and 4-nitro-β-carotene.

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.

Plants take up inorganic nitrogen and store it unchanged or convert it to organic forms. The nitrogen in such organic compounds is stoichiometrically recoverable by the Kjeldahl method. The sum of inorganic nitrogen and Kjeldahl nitrogen has long been known to equal the total nitrogen in plants. However, in our attempt to study the mechanism of nitrogen dioxide (NO(2)) metabolism, we unexpectedly discovered that about one-third of the total nitrogen derived from (15)N-labeled NO(2) taken up by Arabidopsis thaliana (L.) Heynh. plants was converted to neither inorganic nor Kjeldahl nitrogen, but instead to an as yet unknown nitrogen compound(s). We here refer to this nitrogen as unidentified nitrogen ( UN). The generality of the formation of UN across species, nitrogen sources and cultivation environments for plants has been shown as follows. Firstly, all of the other 11 plant species studied were found to form the UN in response to fumigation with (15)NO(2). Secondly, tobacco ( Nicotiana tabacum L.) plants fed with (15)N-nitrate appeared to form the UN. And lastly, the leaves of naturally fed vegetables, grass and roadside trees were found to possess the UN. In addition, the UN appeared to comprise a substantial proportion of total nitrogen in these plant species. Collectively, all of our present findings imply that there is a novel nitrogen mechanism for the formation of UN in plants. Based on the analyses of the exhaust gas and residue fractions of the Kjeldahl digestion of a plant sample containing the UN, probable candidates for compounds that bear the UN were deduced to be those containing the heat-labile nitrogen-oxygen functions and those recalcitrant to Kjeldahl digestion, including organic nitro and nitroso compounds. We propose UN-bearing compounds may provide a chemical basis for the mechanism of the reactive nitrogen species (RNS), and thus that cross-talk may occur between UN and RNS metabolisms in plants. A mechanism for the formation of UN-bearing compounds, in which RNS are involved as intermediates, is proposed. The important broad impact of this novel nitrogen metabolism, not only on the general physiology of plants, but also on plant substances as human and animal food, and on plants as an integral part of the global environment, is discussed.

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.

One (T.Y.) of the authors studied NO2 uptake and metabolism in plants from 1976 to 1979 in the National Institute of Environmental Studies, Tsukuba, and most of the results were published by 1980. After about 17 years, we have been asked to write a review on NO2 metabolism in plants. The task has become considerably easier with the joining of the coauthors (H.M., H.S.S.), who have been actively engaged in the study of plant responses to NO2 during the past. It has been interesting to discuss what was known up to 1980 and what has become known since then about NO2 metabolism and detoxification in plants. In addition, another nitrogen-containing but reduced form of gas, NH3, is also included in this chapter in the hope that a comparison of the behavior and fate of oxidized and reduced gases in plants may provide further insight into their metabolic and regulatory pathways.

A 1980 estimate puts the total natural and anthropogenic emissions of nitrogen oxides, which include nitric oxide (NO) and nitrogen dioxide (NO2) as the major components, at 150 million tons per year. Road transport, the major anthropogenic source of nitrogen oxides in many developed countries, accounted for up to 75% of the nitrogen oxides in some metropolitan cities in 1984. This value is still rising because of the increase in volume of road traffic. In many developing countries, petrofueled motor vehicles are also reported to be the principal source of nitrogen oxides (Yunus et al. 1996).

A negative correlation has consistently been reported between the change in flowering time and the change in leaf number at flowering in response to environmental stimuli, such as the application of exogenous compounds, cold temperature, day length and light quality treatments in Arabidopsis thaliana (Arabidopsis). However, we show here that the application of exogenous nitrogen dioxide (NO2) did not change the number of rosette leaves at flowering, but actually accelerated flowering in Arabidopsis. Furthermore, NO2 treatment was found to increase the rate of leaf appearance. Based on these results, reaching the maximum rosette leaf number earlier in response to NO2 treatment resulted in earlier flowering relative to controls.

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.

Nitrite reductase (NiR) is the second enzyme in the primary assimilation pathway of nitrate to catalyze six-electron reduction of nitrite to ammonia, and thus this enzyme is essential in assimilatory nitrate reduction in plants. We have been studying the assimilation of nitrate and nitrogen dioxide in transgenic Arabidopsis thaliana plants that overexpress NiR gene. Interestingly, our data implied that plants of zero NiR activity could reduce nitrate and nitrogen dioxide. Therefore, we have studied nitrogen assimilation in transgenic plants that express antisense NiR RNA and hence lack or have much reduced NiR activity.

Nitrogen dioxide (NO2) is a major atmospheric pollutant, and plants act as a sink for it (1, 2, 3, 4, 5, 6, 7). NO2 molecules taken up into plant leaves through stomata are, at least in part, converted to organic nitrogens such as amino acids or proteins through the nitrate assimilation pathway. Formation of 15N-labeled nitrate, nitrite, and amino acids in plants fumigated with 15N-labeled NO2 has been reported by the previous authors, and since then it has been assumed that NO2 nitrogen is metabolized similarly to the nitrate in plant cells. Whether a stoichiometric relationship exists between the total nitrogen (N) derived from NO2 and the sum of reduced and nitrate/nitrite N has not, however, been studied. We therefore used 15N-labeled NO2 (15NO2) and potassium nitrate (K15NO3) to clarify whether the total 15N taken up in the leaves of Arabidopsis thaliana plants is equal to the amount of reduced 15N plus nitrate/nitrite 15N.

Atmospheric nitric oxide (NO) and nitrogen dioxide (NO2) have long been recognized as either detrimental or beneficial for plant development. Recent research has established that NO is a phytohormone. Our present knowledge of the physiological role of NO2 is incomplete. We do know, however, that exogenous NO2 positively regulates the vegetative and reproductive growth of plants. We may therefore postulate that NO2 is a positive growth regulator for plants. We are now in a position to coherently summarize what is known of NO2 physiology; collated information on the topic is presented here.

The plastid gene rpoB encodes a plastid-specific, DNA-dependent RNA polymerase. A study of a barley mutant, albostrians, indicated that rpoB itself is transcribed by a nuclear-encoded RNA polymerase. However, the molecular nature of the nuclear-encoded RNA polymerase and mechanisms of the transcriptional regulation of rpoB expression have not been elucidated. Using an in vivo transient assay system for gene expression in plastids, the 5′-flanking region of rpoB from Arabidopsis thaliana in plastids of cultured BY-2 tobacco cells was analyzed. A series of deletion analyses of the 5′-flanking region indicated that there were two positive and one negative regulatory regions for rpoB expression. Comparison of the 5′-flanking sequences of rpoB among several plant species revealed the existence of three conserved motifs in these regions.

A number of A/T-rich sequences and a CATGCAT/A sequence are contained in the 5'-upstream regions of the genes encoding concanavalin A (Con A) and canavalin, two major seed storage proteins of Canavalia gladiata D.C. To study the role of these sequences in the seed-specific gene expression, we constructed 5'-deletion mutants and examined the transient expression of beta-glucuronidase reporter gene by particle bombardment and the stable expression by Agrobacterium-mediated transformation of tobacco plants. Positive regulatory elements were located in the -894/-602 and -602/-74 regions of the Con A gene, and in the -428/-376, -281/-155 and -155/-50 regions of the canavalin gene. In addition, the results suggested that the A/T-rich sequences in the 5'-upstream region of the Con A gene play a role in transcriptional activation, but that those of the canavalin gene have little effect on the gene expression. The CATGCAT/A sequence was not sufficient by itself for high levels of expression of both the Con A and canavalin genes. The canavalin polypeptide amounted to about 1% of the total extractable protein in the transgenic tobacco seeds, but the Con A polypeptide was not detected in the extractable protein.

Stable transformation of suspension-cultured cells of tobacco with plasmid DNA (pCaMV-NEO) harbouring the neomycin phosphotransferase II (npt-II) gene was achieved using a previously described gas-pressure-driven particle acceleration device. The cells were bombarded with DNA-coated gold particles accelerated by the device, and callus containing the introduced gene was selected in the presence of geneticin disulphate. The geneticin-resistant callus exhibited npt-II enzyme activity, and Southern analysis confirmed the stable integration of the foreign gene into the tobacco genome. Transformants were obtained at a rate of more than 1.9 × 10−4.

When stirred with powdered samarium and iodine in methanol at ice-cold temperature, ethyl 1,2,3-thiadiazole-4-carboxylate 5 underwent unusual reduction involving the dimeric ring enlargement with a sulfur addition, giving dimethyl 1,2,5-trithiepan-4,6-dicarboxylates 7a,b as a cis/trans-isomeric mixture in acceptable yield. The 1,2,3-thiadiazole ring of 5 proved to resist reduction by ordinary reducing agents, where the only choice in most cases was either recovery of the unchanged substrate or decomposition to an intractable mixture of unidentified products and tar.

Exploded wood lignin (EXWL) was extracted from moist steam hydrolysed aspen (Populustremuloides) wood using methanol. The lignin was examined by elemental analysis, gel permeation chromatography, infrared spectroscopy, proton and 13C nmr. The observed nmr spectra were compared with those of aspen milled wood lignin (MWL). Typical composition of the material is C9H9.2O2.7(OCH3)1.1 Assignment of all the signals in the nmr spectrum led to the conclusion that the explosion process causes cleavage of the β-aryl-ether bond. The weight average molecular weight was found to be 1700–1900 and the polydispersity 2.6. The finely divided exploded wood was soluble in 90% methanol/water mixtures to well beyond 20% by weight at room temperature.

Experimental evidence has been presented that the non-ionic, acid-labile and base-stable bonds involving pectic carboxylate groups, both oriented and not-oriented, play an important role in holding pectic substances in the cell wall of Nitella. Participation of hydroxyl groups in the bonds has been confirmed by the vapor phase deuteration method. Irreversible acid-degradation of the cell wall ultrastructure associated with the carboxylate groups has also been demonstrated by acid-base titrimetry.