A nitrogen mass balance study in radish (Raphanus sativus L.) grown under nitrate or nitrate and ammonium co-provision

Nitrate and ammonium are the primary nitrogen sources for plants. The process of nitrate reduction, which converts nitrate into ammonium, requires substantial amounts of reducing equivalents. Therefore, ammonium should be considered the preferred nitrogen source due to its lower energy cost. However, ammonium tends to be toxic to plants, especially when supplied as the sole nitrogen source, while the co-provision of nitrate alleviates ammonium toxicity. To better understand the dynamics of nitrate and ammonium absorption and assimilation, we performed a nitrogen mass balance on radish plants grown in hydroponic solutions containing 3.5 mM nitrate as the sole nitrogen source or NH₄⁺/NO₃⁻ combinations (1, 4, 8) with 3.5 mM final nitrogen concentration. All experiments were carried out by enriching the solution with ¹⁵N-NO₃⁻. The results indicated that a small amount of ammonium (NH₄⁺/NO₃⁻ = 1) was beneficial for plant growth, while higher ammonium levels (NH₄⁺/NO₃⁻ = 4 and 8) were detrimental. Similar behaviors were found regarding the accumulation of proteins or products of inorganic nitrogen assimilation within the plant. This suggests that an optimal balance between ammonium and nitrate is crucial for optimizing plant biomass and nitrogen assimilation. Interestingly, under all co-provision conditions, the percentage of the total nitrogen accumulated by the plants derived from nitrate was consistently higher than the percentage of nitrate in the total nitrogen available in the solution. This indicates that plants prioritized nitrate uptake over ammonium uptake. Conversely, under the most balanced ammonium to nitrate ratios (NH₄⁺/NO₃⁻ = 1 and 4), the ¹⁵N/¹⁴N isotope ratio in plant proteins was significantly lower than that measured in total plant nitrogen. This indicates that adequate amounts of nitrate favored the preferential assimilation of the ammonium directly absorbed from the solution. Finally, as the NH₄⁺/NO₃⁻ ratio in the solution increased, there was a noticeable decrease in nitrate accumulation in the plant, accompanied by a dramatic rise in unassimilated ammonium. However, ammonium accumulation at the higher external concentrations was not attributable to its uptake, which was independent of the external concentration. This could be due to its inadequate assimilation, possibly resulting from lower concentrations of organic acids, whose synthesis and accumulation in plant tissues were directly related to nitrate availability.