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It is known that when plant leaves are exposed to exogenously applied nitrogen dioxide (NO 2 ), nitrogen derived from NO 2 is reduced to amino acid nitrogen. However, whether this is the sole metabolic fate of exogenously applied NO 2 is unclear. In this study, Arabidopsis leaves were exposed to 4 ppm 15 N-labeled NO 2 for 4 h in light, followed by capillary ion analysis and elemental analysis-mass spectrometry with an elemental analyzer connected directly to a mass spectrometer. We found that leaf cells exposed to 15 N-labeled NO 2 accumulated a large amount of 15 N-labeled nitrate. Neither 15 N-labeled nitrite nor endogenous nitrite was present in exposed leaves. It is likely that exogenously applied NO 2 is first converted to nitrite, and that nitrite is oxidized to nitrate in Arabidopsis leaf cells. The complete disappearance of nitrite derived from exogenously applied NO 2 and endogenous nitrite supports this mechanism.

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.

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.

Exposure of intact Arabidopsis leaves to 40 ppm nitrogen dioxide (NO 2 ) 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 NO 2 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 (NO 2 ) induces selective nitration of the tyrosine residue at the ninth amino acid ( 9 Tyr) of PsbO1. This selective nitration is triggered by light and is inhibited by photosynthetic electron transport inhibitors. Therefore, we postulated that, similar to 161 Tyr of D1 (Y Z ), 9 Tyr 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 NO 2 to form 3-nitrotyrosine. If this postulation is correct, the nitration of 9 Tyr 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 9 Tyr gene of PsbO1, and suggests that PsbO1 9 Tyr acts as an electron relay, such as Y Z in the photosystem II oxygenic electron transport chain.

PsbO1 is exclusively nitrated when isolated thylakoid membranes are incubated in a buffer bubbled with nitrogen dioxide (NO 2 ) containing NO 2 and nitrite. NO 2 is the primary intermediate for this selective nitration. Isolated thylakoid membranes were incubated in NO 2 -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 NO 2 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.

Exposure of Arabidopsis leaves to nitrogen dioxide (NO 2 ) results in nitration of specific chloroplast proteins. To determine whether NO 2 itself and/or nitrite derived from NO 2 can nitrate proteins, Arabidopsis thylakoid membranes were isolated and treated with NO 2 -bubbled or potassium nitrite (KNO 2 ) buffer, followed by protein extraction, electrophoresis, and immunoblotting using an anti-3-nitrotyrosine (NT) antibody. NO 2 concentrations in the NO 2 -bubbled buffer were calculated by numerically solving NO 2 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 2 treatments was designated nitration by NO 2 . Both NO 2 and nitrite mediated nitration of proteins, and the nitration ability per unit NO 2 concentration was ∼100-fold greater than that of nitrite.

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, Tyr(36) of thaumatin-like protein E2 was identfied as a nitration site. The underlying mechanism and physiological relevance are discussed.

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.

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.

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.

To better understand the response of plants to atmospheric nitrogen dioxide (NO2), we investigated biomass accumulation in 3 accessions of Arabidopsis thaliana: C24, Columbia (Col-0), and Landsberg erecta (Ler). Plants were grown in NO2-free air for 1 week after sowing, followed by 3 (Col-0 and Ler) to 4 (C24) weeks in air with or without NO 2 (10 or 50 ppb). NO2 treatment increased the biomass of all 3 accessions to varying extents. Treatment with 10 ppb NO2 increased shoot biomass in C24, Col-0, and Ler by 3.2-, 1.4-, and 2.3-fold, respectively, compared with control. Treatment with 50 ppb gave similar increases, except in C24 (2.7-fold). The physiological, evolutionary, and genetic significance of these results are discussed below.