Properties of nitrogen fertilization are decisive in determining the effects of elevated atmospheric CO₂ on the activity of nitrate reductase in plants

ABSTRACT

The concentration of atmospheric CO₂ is predicted to double by the end of this century. The response of higher plants to an increase in atmospheric CO₂ often includes a change in nitrate reductase (NR) activity. In a recent study, we showed that, under elevated CO₂ levels, NR induction in low-nitrate plants and NR inhibition in high-nitrate plants are regulated by nitric oxide (NO) generated via nitric oxide synthases. This finding provides an explanation for the diverse responses of plants to elevated CO₂ levels, and suggests that the use of nitrogen fertilizers on soil will have a major influence on the nitrogen assimilation capacity of plants in response to CO₂ elevation.

KEYWORDS:

• Elevated CO₂
• NR activity
• nitrate level
• nitrogen form
• NOS

Abbreviations

CO2	=	carbon dioxide
NR	=	nitrate reductase
NO	=	nitric oxide
NOS	=	NO synthase
N	=	nitrogen

Atmospheric CO₂ concentrations are rising at an accelerated rate due to anthropogenic activities; the levels are currently estimated at 400–430 ppm, and are predicted to reach 530–1000 ppm by the end of the 21^st century.¹ CO₂ is an important resource for photosynthesis in plants; thus, CO₂ enrichment has the potential to enhance plant productivity.^2-4 Many studies have focused on the effect of elevated CO₂ on nitrogen assimilation because nitrogen is required by plants in greatest quantity.^5-10 Nitrate reductase (NR), the first enzyme in the nitrate assimilation pathway,¹¹ has proven to be one of the enzymes to undergoes clear changes in response to elevated CO₂.⁶ However, over the past 15 years, many studies have demonstrated that CO₂ enrichment can cause an increase in,^12-16 have no effect on,¹⁷ or even decrease NR activity in plants.^{6,13,16,18-20}

The conflicting results of the effects of elevated CO₂ on NR activity in plants are shown in Table 1. Some studies have shown that elevated CO₂ levels stimulated the activity of NR, e.g., in Arabidopsis,¹³ tobacco,¹² and white pine,²¹ which were treated with 2, 2, and 1.6 mM nitrate, respectively. Conversely, some researchers observed the opposite effect of CO₂ enrichment on NR activity, under high nitrate conditions; Geiger et al.⁶ reported that elevated CO₂ slightly stimulated NR activity in the leaves of tobacco plants fed with 2 mM nitrate, while causing an approximately 30% decrease in leaf NR activity in plants fed with a 6 mM nitrate supply. This result is similar to that of Lekshmy et al.,²² in which CO₂ enrichment increased NR activity (by 11%–24%) in the leaves of wheat seedlings grown in low nitrate conditions (<2 mM), but the opposite occurred when the plants were grown in high nitrate conditions (>5 mM). These findings imply that the effect of elevated CO₂ on NR activity in plants is dependent on the nitrate levels in the growth medium; this was confirmed by our latest report: in low-level nitrate supply conditions, NR activity is stimulated by the basal endogenous nitric oxide (NO) and the NO induced by CO₂ enrichment, via an NOS-dependent pathway.¹⁰ However, in high-level nitrate conditions, NR activity is inhibited by excess NO gathered from high endogenous NO, and NOS-dependent NO generated under elevated CO₂.¹⁰ Therefore, we assumed that an inflection point of nitrate supply may exist for the upregulation or downregulation of NR activity in plants, in relation to elevated CO₂ levels. Such an inflection point might vary with species, physiological state, nutritional availability, and other factors that could affect the basal endogenous NO levels and NOS activity in plants.

Table 1. Studies of NR activity changes under different experimental background in response to elevated CO₂.

Plants	Position	N source	Concentration	Cultivation methods	Seedling age	Time of CO2 treatment	Changes in NR level	References
Tobacco (Nicotian atabacum L)	Leaves	NO3^−	2 mM	Vermiculite	45 d	45 d	↑	6
			6 mM				↓
White pine (Pinus strobus)	Leaves	NO3^−	1.6 mM	Nutrition solution	105 d	105 d	↑	21
Tobacco nd (Nicotian atabacum L)	Leaves	NO3^−	2 mM	Nutrition solution	39 d	25 d	↑	12
Arabidopsis (Arabidopsis thaliana)	Shoots	NO3^−	2 mM	Nutrition solution	35 d	7 d	↑	13
Wheat (Triticum aestivum L)	Shoots	NO3^−	0.02–2 mM	Nutrition solution	20 d	20 d	↑	22
			4–10 mM				↓
Arabidopsis (Arabidopsis thaliana)	Leaves	NO3^−	<3 mM	Nutrition solution	35 d	4 h	↑	10
			>4 mM				↓
Tobacco (Nicotian atabacum L)	Leaves	NO3^−	Fertilized twice daily with a nutrient solution containing 12 mM nitrate.	Quartz crystal sand	35 d	11 d	↑	23
Cucumber (Cucumissativus L)	Leaves	NO3^−	Irrigated daily with a nutrient solution containing 10 mM nitrate	Perlite vermiculite	21 d	2 h	↑	24
Barley (Hordeum vulgare L)	Leaves	NO3^−	Watered twice a week with a nutrient solutioncontaining 20 mM nitrate	Perlite and vermiculite	16 d	16 d	↑	25
Tobacco (Nicotian atabacum L)	Leaves	NH4NO3	1 mM	Vermiculite	45 d	45 d	↓	6
			3 mM				↓
			10 mM				↓
Tobacco (Nicotian atabacum L)	Leaves	NH4NO3	1 mM	Nutrition solution	39 d	25 d	↓	12
Loblolly pine (Pinus taeda)	Leaves	NH4NO3	5.6 g N m^−2 year^−1	Acidic soils	22 years	7 years	↓	19
Arabidopsis (Arabidopsis thaliana)	Shoots	NH4^+	2 mM	Nutrition solution	35 d	7 d	−(no change)	13
Barley (Hordeum vulgare L)	Shoots	NH4 NO3	1 mM	Nutrition solution	27 d	15 d	↓	20

However, some of the data in Table 1 ^23–25 is inconsistent with this suggested mechanism, e.g., NR activity under enhanced CO₂ conditions were increased in the leaves of tobacco, cucumber, and barley plants growing on high nitrate at 12,²³ 10,²⁴ and 20 mM,²⁵ respectively. These contrasting results may be attributable to differences in plant culturing; the plants in these studies were grown in quartz crystal sand or perlite and vermiculite, and were watered to field capacity with a complete nutrient solution containing nitrate, either once²⁴ or twice each day²³ or weekly.²⁵ In other words, although the level of nitrate used was high, the supply regime was intermittent, rather than sustained; therefore, the plants could be considered to have grown in an overall low nitrate supply. In this context, these studies were also consistent with our hypothesis. However, we cannot completely exclude the possibility that elevated CO₂ levels increase NR activity in the case of high nitrate (> 10 mM), since the nitrate levels in our study only ranged from 0.2–10 mM.¹⁰

Furthermore, under high nitrate and ammonium conditions, elevated CO₂ decreased NR activity regardless of the nitrogen supply levels. For example, elevated CO₂ led to a significant (30%–50%) inhibition of NR activities in plants grown at 1, 3, and 10 mM NH₄NO₃ conditions.⁶ These results are similar to the findings of Matt et al.¹² and Wang et al.,²⁰ in which elevated CO₂ decreased NR activity in tobacco and barley growing at 1 mM NH₄NO₃ conditions. These results may be associated with the fact that CO₂ enrichment preferentially stimulates ammonium uptake and assimilation, leading to an accumulation of reduced nitrogen and repression of NR.⁶

All of these findings indicated that the nitrogen source—concentration, availability, and form—might be a decisive factor directly affecting the rate of nitrate assimilation for plants during an increase in atmospheric CO₂. This may have profound implications for research on plant responses to elevated CO₂. Although elevated CO₂ increased the yield of plants grown on high nitrate conditions,⁶ excessive use of nitrate fertilizer may result in a low efficiency of nitrogen use. Additionally, in the presence of ammonium, nitrate will also be unutilized. Therefore, a relatively low concentration of nitrate fertilizers may improve NR activity, and consequently promote nitrogen assimilation, alleviating nitrogen pollution and economic losses for farmers.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was financially supported by the Zhejiang Province Natural Science Foundation (No. LY14C130001), the Natural Science foundation of China (Grant Nos 30900170).