Differential responses of Arabidopsis thaliana accessions to atmospheric nitrogen dioxide at ambient concentrations

Abstract

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 NO₂ (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.

We reported previously that the exogenous application of nitrogen dioxide (NO₂) influences diverse physiological processes including nutrient uptake, photosynthesis, nitrogen metabolism, cell proliferation, cell enlargement, biomass accumulation, and the acceleration of flowering.^1,2 Therefore, we hypothesized that NO₂ positively regulates plant growth.³ This is based on previous studies using concentrations of 50−200 ppb NO₂, which correspond to those in polluted urban air (OECD 2002).⁴ It is important to understand whether plants respond to ground levels of NO₂ to increase our understanding of the biological and evolutionary relevance of this phenomenon. We reported previously that the Arabidopsis thaliana (Arabidopsis) C24 accession responded positively to treatment with 10 ppb NO_2,¹ which corresponds to the concentrations in rural air. In this study, we confirmed our previous observations using 2 additional Arabidopsis accessions: Columbia (Col-0) and Landsberg erecta (Ler). Importantly, these 3 accessions all possess distinct genetic backgrounds.⁵

Plants were grown and treated with NO₂, and shoot biomass was determined as reported previously.¹ Briefly, Arabidopsis thaliana (L.) Heynh. C24, Col-0, and Ler seeds were surface-sterilized with 1.0% sodium hypochlorite, rinsed in pure water (18.0 MΩ), and sown in a rectangular plastic tray (22 × 5 × 20 cm width, height, and depth, respectively) containing vermiculite and perlite (1:1, v/v), and held in a glass-walled NO₂-exposure chamber (1.3 × 1.0 × 0.65 min width, height, and depth, respectively; NOX-1000-SCII, Nippon Medical and Chemical Instruments Co, Osaka, Japan) in a growth room. The temperature, CO₂ concentration, and relative humidity in the chamber were set at 22 ± 0.1°C, 360 ± 30 ppm, and 70 ± 1.5%, respectively. The air entering the chamber (at 1 L min⁻¹) from outside was stripped of NO, NO₂, and O₃ (to 0 ppb) using activated charcoal and NaMnO₄ (PureliteE30; Nippon Puretec Co, Tokyo, Japan). The tray was placed under fluorescent light (40 µmol photons m² s⁻¹) with a 16:8 h, light:dark cycle, and seeds were allowed to germinate and grow for 1 wk in air lacking NO₂. NO₂ was then added to the air entering the exposure chamber at concentrations of 0, 10 ± 0.2, or 50 ± 0.3 ppb. Seedlings were irrigated twice weekly with half-strength inorganic salts in Murashige and Skoog medium,⁶ and grown for an additional 3−4 wk in the chamber. Plants were then harvested and shoot biomass was calculated. The results are summarized in Table 1.

Table 1. Effects of nitrogen dioxide (NO₂) concentration on shoot biomass yield in Arabidopsis thaliana accessions C24, Columbia (Col-0), and Landsberg erecta (Ler).

NO2 concentration (ppb)	Average dry wt. of shoots (mg per plant) ± SD (n)
	C24	Col-0	Ler
0	25.2 ± 6.8 (4)	14.3 ± 2.5 (5)	11.3 ± 2.6 (5)
10 ± 0.2	80.8 ± 17 (10) ***	20.2 ± 5.5 (15)	26.2 ± 4.0 (15) ***
50 ± 0.3	68.5 ± 9.3 (10) ***	24.2 ± 5.5 (5) *	28.2 ± 2.3 (5) ***

C24, Col-0, and Ler plants were harvested at 5, 4, and 4 wk after sowing, respectively. Statistical significance assessed using the Dunnett t test: *, P < 0.05; ***, P < 0.001. Statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA USA).

All accessions responded positively to treatment with 10 ppb NO₂ with a characteristic response. Shoot biomass in the C24, Col-0, and Ler accessions was increased by 3.2-, 1.4-, and 2.3-fold, respectively, compared with control. This increase was similar to, or even higher than, the increase observed after treatment with 50 ppb: treatment increased shoot biomass by 2.7-, 1.7-, and 2.5-fold in C24, Col-0, and Ler, respectively (Table 1). The increases in shoot biomass, except that in Col, between 0 and 10 ppb were statistically significant (Table 1). Therefore, plants are highly sensitive to NO₂, whereby a concentration as low as 10 ppb significantly stimulated the accumulation of shoot biomass in all 3 Arabidopsis accessions.

The weighted mean NO₂ concentration in cities from over 141 countries is reported to be 50.6 µg/m³ (~27 ppb).⁷ Approximately half of atmospheric NO₂ is derived from human activities such as the combustion of fossil fuels, and the remaining half is derived from non-anthropogenic sources such as soil microbial emissions.⁸ Therefore, it is conceivable that 10 ppb is a reasonable estimate of ground levels of NO₂ derived from natural sources, which is consistent with reported NO₂ values (5−10 ppb) in rural air.^9,10 Our present finding that Arabidopsis plants responded to NO₂ at ambient concentrations suggests that atmospheric NO₂ at the ground level exerts physiological effects on plants by acting as a positive growth signal.

Unlike the current Earth atmosphere, that of primitive Earth is unlikely to have contained nitrogen oxides (nitric oxide [NO] and NO₂), because it contained nitrogen but not free oxygen.^11,12 Geochemical studies indicate that appreciable increases in atmospheric oxygen occurred in the Archean eon ~2.5−3 billion years ago,^11,12,13 and in the Proterozoic eon ~0.75 billion years ago.¹³ The former increase is known as the great oxidation event (GOE), which was triggered by oxygen production by oxygenic photosynthetic cyanobacteria. Furthermore, the ancestors of modern denitrifiers are likely to have arisen before,¹⁴ or at the same time as,¹⁵ the GOE and released NO into the atmosphere. In atmospheric chemistry, the rapid interconversion of NO and NO₂ (the Leighton relationship) involves the oxidation of NO by ozone and the photolysis of NO₂ by sunlight.¹⁶ The ancestors of land plants appeared ~0.4−0.5 billion years ago¹⁷; therefore, atmospheric NO₂ is likely to have influenced the evolution of plants, given that air is important for the coevolution of life and Earth.¹⁸ However, when and how plants evolved traits to use atmospheric NO₂ as a growth signal, and whether the ancestors of terrestrial plants such as green algae¹⁹ possess this trait remains unclear.

The NO₂-induced shoot biomass increase is probably a multigenic trait; therefore, quantitative trait locus (QTL) analysis followed by the identification of the specific genes that underlie QTL is important to understand the molecular mechanisms of this plant behavior. Our present finding of variation in this trait among Arabidopsis accessions might provide an important resource for QTL analysis using recombinant inbred lines¹⁹ to clarify the molecular mechanisms and evolutionary origin of the genes involved in this NO₂-induced plant behavior.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (24580477, 21580403 to MT).