Depletion of the heaviest stable N isotope is associated with NH4+/NH3 toxicity in NH4+-fed plants

Background: In plants, nitrate (NO3-) nutrition gives rise to a natural N isotopic signature (delta N-15), which correlates with the delta N-15 of the N source. However, little is known about the relationship between the delta N-15 of the N source and the N-14/N-15 fractionation in plants under ammonium (NH4+) nutrition. When NH4+ is the major N source, the two forms, NH4+ and NH3, are present in the nutrient solution. There is a 1.025 thermodynamic isotope effect between NH3 (g) and NH4+(aq) which drives to a different delta N-15. Nine plant species with different NH4+-sensitivities were cultured hydroponically with NO3- or NH4+ as the sole N sources, and plant growth and delta N-15 were determined. Short-term NH4+/NH3 uptake experiments at pH 6.0 and 9.0 (which favours NH3 form) were carried out in order to support and substantiate our hypothesis. N source fractionation throughout the whole plant was interpreted on the basis of the relative transport of NH4+ and NH3. Results: Several NO3--fed plants were consistently enriched in N-15, whereas plants under NH4+ nutrition were depleted of N-15. It was shown that more sensitive plants to NH4+ toxicity were the most depleted in N-15. In parallel, N-deficient pea and spinach plants fed with (NH4+)-N-15 showed an increased level of NH3 uptake at alkaline pH that was related to the N-15 depletion of the plant. Tolerant to NH4+ pea plants or sensitive spinach plants showed similar trend on N-15 depletion while slight differences in the time kinetics were observed during the initial stages. The use of RbNO3 as control discarded that the differences observed arise from pH detrimental effects. Conclusions: This article proposes that the negative values of delta N-15 in NH4+-fed plants are originated from NH3 uptake by plants. Moreover, this depletion of the heavier N isotope is proportional to the NH4+/NH3 toxicity in plants species. Therefore, we hypothesise that the low affinity transport system for NH4+ may have two components: one that transports N in the molecular form and is associated with fractionation and another that transports N in the ionic form and is not associated with fractionation.