Dual nitrogen species involved in foliar uptake of nitrogen dioxide in Arabidopsis thaliana

ABSTRACT

Foliar uptake of nitrogen dioxide (NO₂) is governed by its reactive absorption mechanism, by which NO₂ 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 NO₂ remains unknown. The involvement of ammonia-inhibitable nitrite transporters in the foliar uptake of NO₂, 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 ¹⁵N-labeled NO₂ for 4 h in light followed by quantification of total nitrogen, reduced nitrogen, and ammonia nitrogen derived from NO₂ using mass spectrometry and capillary electrophoresis. The total nitrogen derived from NO₂ in leaves without MSX treatment was 587.0 nmol NO₂/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 NO₂ by half. Thus, half of the foliar uptake of NO₂ is not attributable to passive diffusion of nitrous acid but to ammonia-inhibitable nitrite transport. Foliar uptake of NO₂ is mediated by a dual mechanism in A. thaliana: nitrous acid-free diffusion and nitrite transporter-mediated transport.

KEYWORDS:

• Arabidopsis thaliana
• methionine sulfoximine (MSX)
• nitrogen dioxide
• ascorbate

Atmospheric nitrogen dioxide (NO₂) can be detrimental or beneficial to plants depending on the concentration and plant species.^1–5 For example, NO₂ at ambient concentrations (10–50 ppb) has been reported to act as a positive growth regulator in plants.^6,7 Thus, the mechanism by which plant leaves uptake NO₂ from ambient air is vital to understanding the physiological relevance of NO₂ in plants.

Despite opposing arguments,⁸ it used to be thought that NO₂ was dissolved in cell wall water of plant cells, where equal amounts of nitrate (NO₃^–) and nitrite (NO₂^–) are formed by disproportionation. In this mechanism, NO₃ ^– and NO₂ ^– were thought to be absorbed into cells through the plasmalemma via respective transporters located on the plasmalemma.⁹ However, the calculated concentrations of NO₃ ^– and NO₂ ^– formed by disproportionation of dissolved NO₂ are seven orders of magnitude lower than the K_m values of high-affinity NO₃^–10 and NO₂ ^– transporters.^11,12 This is largely due to the very low concentrations of NO₂ that dissolve in water (6 × 10^–11 M at 50 ppb NO₂ gas in air) due to its relatively low solubility in water (estimated to be 1.2 × 10^–2 Matm^–1 according to Henry’s law¹³). Therefore, the involvement of NO₃ ^– or NO₂ ^– uptake from disproportionation of dissolved NO₂ in the foliar uptake of NO₂ is unlikely.

In 1993, Ramge et al.⁸ showed that foliar uptake is governed by the reactive absorption mechanism. Based on mathematical models, they showed that foliar uptake of NO₂ via disproportionation of dissolved NO₂ was four orders of magnitude lower than the experimentally determined values of foliar uptake of NO₂ in a variety of plants, but that uptake of NO₂ into mesophyll tissues calculated by a reactive absorption mechanism¹⁴ fit well quantitatively with experimental values.⁸ In this mechanism, following exposure of leaf cells to NO₂, NO₂ molecules diffuse through cell wall layers and simultaneously react with apoplastic ascorbate (Asc–OH) to form dehydroascorbate (Asc–O•) and nitrous acid (HNO₂) (Eq. 1). The product of oxidation, dehydroascorbate, is transported into the cytosol and reduced into ascorbate. Then, the regenerated ascorbate is transferred back into the apoplast.⁸

(1) $\mathrm{A}\mathrm{s}\mathrm{c}-\mathrm{O}\mathrm{H}+\bullet \mathrm{N}{\mathrm{O}}_2\to \mathrm{A}\mathrm{s}\mathrm{c}-\mathrm{O}\bullet +{\mathrm{H}}^{+}\mathrm{N}{{\mathrm{O}}_2}^{-}$(1)

(2) ${\text{HNO}}_2\rightleftarrows {\text{NO}}_2{ }^{-}+{\text{H}}^{+}$(2)

In mesophyll cells of spinach, the permeability coefficients of NO₂ ^– and HNO₂ have been reported to be 5 × 10^–10 and 4 × 10^–5 m/s, respectively,¹⁵ indicating that HNO₂ is freely diffusible, while the spinach plasma membrane is almost impermeable to NO₂^–.¹⁶ This may also be the case for the Arabidopsis thaliana (Arabidopsis) plasma membrane, which may possess permeability values for these compounds similar to those of spinach.

On the other hand, using the short-lived tracer¹³NO^–, Kotur et al.¹¹ reported that NO₂⁻ was transported by a nitrite transporter in Arabidopsis roots. This nitrite transporter is inhibitable by ammonium.¹¹

Given that foliar uptake of NO₂ is governed by the reactive adsorption mechanism, whether foliar uptake of NO₂ is mediated only by free diffusion of HNO₂ remains unknown. Similarly, unknown is the involvement of ammonia-inhibitable nitrite transporters in the foliar uptake of NO₂, as reported in nitrite transport in Arabidopsis roots.¹¹ To clarify these issues, Arabidopsis leaves were first treated (or left untreated as a control) with 1 mM L-methionine sulfoximine (MSX; Sigma), an inhibitor of glutamine synthetase,¹⁷ for 24 h, and then exposed to 4 ppm¹⁵ N-labeled NO₂ for 4 h in the light. The leaves were harvested, rinsed in pure water (18.0 MΩ), lyophilized, ground into a powder, and stored in a desiccator until use.

Approximately 1 mg of powdered leaves were subjected to analysis using an elemental analyzer (EA; EA1108 CHNS/O; Fisons Instruments, Milan, Italy) connected directly to a mass spectrometer (MS; Delta C; Thermo-Finnigan, Bremen, Germany) to determine the total nitrogen content (A in Eq. 3) and the atomic percent of ¹⁵N [¹⁵N/(¹⁵N + ¹⁴N)] (B in Eq. 3) of this fraction.¹⁸

To determine reduced nitrogen, 20 mg of powdered leaves was digested using the Kjeldahl method, and ammonia or Kjeldahl nitrogen (A value) was determined as reported previously.¹⁸ Then, ammonia was concentrated using the Conway diffusion method,¹⁹ and was analyzed using EA-MS to determine the atomic percent of ¹⁵N [¹⁵N/(¹⁵N + ¹⁴N)] (B value) in the fraction.

To determine the ammonium content, powdered leaves (30–50 mg) were homogenized in pure water (18.0 MΩ) using an agate mortar and pestle followed by centrifugation at 18,000 × g for 10 min.²⁰ The supernatant was analyzed for its ammonium content (A value) using capillary electrophoresis as reported previously.²⁰ Next, the ammonia in the supernatant was concentrated with the Conway diffusion method,¹⁹ and the atomic percent of ¹⁵N [¹⁵N/(¹⁵N + ¹⁴N)] (B value) in the fraction was determined with EA-MS.

Using the A and B values for each fraction, the nitrogen derived from NO₂ in each fraction was calculated with the following equation:¹⁸

(3) $\mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{d}\mathrm{f}\mathrm{r}\mathrm{o}\mathrm{m}\mathrm{N}{\mathrm{O}}_2\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{a}\mathrm{c}\mathrm{h}\mathrm{f}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\left(\mathrm{B}-0.3663\right)/100\times \mathrm{A}\times 100/\mathrm{C}$(3)

where C is the atomic percent of¹⁵N in NO₂ gas (51.6 atom%) and the atomic percent of 0.3663 corresponds to the natural abundance of ¹⁵N.²¹

The results are summarized in Table 1. Total nitrogen derived from NO₂ in the leaves without MSX treatment (designated as foliar uptake of NO₂) was 540 μg/g dry weight (dw) [equivalent to 587.0 nmol/g fresh weight (fw)]. MSX treatment decreased both foliar uptake of NO₂ and reduced the nitrogen content in the leaves by about half compared to the untreated control (Table 1). The ammonium nitrogen content in MSX-treated leaves (172.0 ng/g dw) was similar to the reduced nitrogen content (196.0 ng/g dw), suggesting that MSX inhibited ammonia incorporation, and consequently, the free ammonia level was increased following MSX treatment, inhibiting the foliar uptake of NO₂ by half.

Table 1. Total, reduced, and ammonium nitrogen (N) derived from nitrogen dioxide (NO₂) in Arabidopsis leaves treated with methionine sulfoximine (MSX) (or left untreated) and exposed to 4 ppm¹⁵N-labeled NO₂ for 4 h in the light.

MSX	Total N derived from NO2 (ng/g dry weight)	Reduced N derived from NO2 (ng/g dry weight)	Ammonium N derived from NO2 (ng/g dry weight)
–	540.0	357.6	31.7
+	286.0	196.0	172.0

As passive permeation of HNO₂ is not inhibited by ammonia, around half of the foliar uptake of NO₂ is attributable not to the free diffusion of nitrous acid, but to the ammonia-inhibitable transport system. The NO₂ ^– influx transporter located on the plasma membrane in Arabidopsis roots is reportedly specifically inhibited by up to 21.8% by ammonium compared to the control.¹¹ NO₂ ^– transport in barley root has been reported to be inhibited by ammonium.²² Therefore, half of the foliar uptake of NO₂ in Arabidopsis leaves can be attributed to the ammonia-inhibitable NO₂ ^– transporter. This finding is congruent with the report by Kotur et al.¹¹ that nitrite transport in Arabidopsis roots is mediated by ammonia-inhibitable nitrite transporters.

Nitrous acid (HNO₂; pKa = 3.4)²³ reversibly dissociates into NO₂ ^– and a hydrogen ion (Eq. 2). The small fraction of undissociated HNO₂ (pKa = 3.4) in the cell wall (pH 5.0) and cytoplasm (pH 7.4), equal to about 2.5% and 10^–4%, respectively, can be compensated by its significantly higher membrane permeability compared with NO₂^–.²⁴ Thus, a uniform concentration of HNO₂ in the cell wall and cytoplasm is rapidly established.

The rate of foliar NO₂ uptake was calculated as 587.0 nmol/g fw divided by 4 h (= 40.8 pmol/g fw/s). This was comparable to that of spinach leaves (6 pmol/g fw/s at 8 ppm NO₂).²⁵ Assuming that NO₂ ^– concentrations in both the apoplast and cell are the same in A. thaliana and that 1,000 g fw corresponds to 1,000 cm³, an exposure time of 1.3 h (186/0.0408 s ≈ 1.3 h) to 4 ppm NO₂ was determined to elevate the apoplastic NO₂ ^– concentration to the K_m value (186 μM) of the NO₂ ^– transporter¹⁰ located on the plasma membrane of A. thaliana leaves.

Cytoplasmic NO₂ ^– is transported into the chloroplast stroma via the NO₂ ^– transporter CsNitr1-L⁵. Chloroplastic NO₂ ^– is metabolized into ammonia by NO₂ ^– reductase, and ammonia is assimilated into glutamic acid to form glutamine via glutamine synthetase. The rate of uptake of the nitrite transporter CsNitr1-L located on the inner chloroplast envelope membrane has been estimated to be about 1 nmol/g fresh cells,¹² which is 25 times greater than the rate of NO₂ ^– uptake by foliar cells. The reaction rate of NO₂ ^– reductase (41.6–83.2 nmol NO₂^–/g fw/s)²⁶ was three orders of magnitude faster than the NO₂ ^– uptake rate by foliar cells. Meanwhile, the reaction rate of glutamine synthetase (~150 μmol glutamine/g fw)²⁶ was three orders of magnitude higher than that of NO₂ ^– reductase.

In summary, during the first 1.3 h after starting exposure to NO₂, NO₂ is mainly taken up into foliar cells by free diffusion of HNO₂ via the reactive absorption mechanism. As free diffusion of HNO₂ proceeds, the apoplastic NO₂ ^– concentration is gradually elevated, reaching the K_m value (186 μM) of the NO₂ ^– transporter after about 1.3 h of exposure. Thereafter, the foliar uptake of NO₂ in Arabidopsis leaves is dually mediated by both the passive diffusion of HNO₂ and active transport of NO₂ ^– by NO₂ ^– transporters.^10,11

Notably, the uptake of NO₂ in human respiratory tract cells is governed not by the direct uptake of disproportionation-formed NO₃ ^– or NO₂^–, but by a reactive absorption mechanism, as is the case in plants.¹⁷ Once inhaled into the respiratory tract, NO₂ first encounters the aqueous phase of the epithelial lining fluid (ELF) that covers the epithelial surface of the respiratory tract,²⁷ into which NO₂ molecules dissolve and diffuse. They simultaneously react with substrates such as ascorbate and glutathione in the ELF to form HNO₂, which freely enters respiratory tract cells.²⁷ However, it is unknown whether ammonia-inhibitable transport like that in Arabidopsis is involved in the uptake of NO₂ in human respiratory tract cells.²⁷

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Professor Masaaki Takahashi, Osaka Prefecture University for his interest in this work, invaluable discussions, and critical reading of the manuscript.

Additional information

Funding

This work was supported by a grant from the Nippon Life Insurance Foundation; Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science [15710149]; Nissan Science Foundation; Grant-in-Aid for Creative Scientific Research from the Japan Society for the Promotion of Science [13GS0023].