The primary sources of nitrogen for plants have been suggested to be nitrate (NO3−) and ammonium (NH4+). However, when both nitrate and ammonium are simultaneously available to plants, it is very difficult to differentially quantify NO3−/NH4+ utilization in culture media or soil. Consequently, the contribution of NO3−/NH4+ to total inorganic nitrogen assimilation cannot be determined. We developed a method called the bidirectional stable nitrogen isotope tracer to differentially quantify the nitrate and ammonium utilization by Orychophragmus violaceus (Ov) and Brassica napus (Bn) plantlets in vitro. The utilization efficiency of nitrate was markedly lower than the utilization efficiency of ammonium for plantlets of both Ov and Bn. In both Ov and Bn, the proportion of NO3−/NH4+ utilization did not show a linear relationship with inorganic nitrogen supply. The Ov plantlets assimilated more nitrate than the Bn plantlets at the lowest inorganic nitrogen concentration. Quantifying the utilization of nitrate and ammonium can reveal the differences in nitrate and ammonium assimilation among plants at different inorganic nitrogen supply levels and provide an alternate way to conveniently optimize the supply of inorganic nitrogen in culture media.

Sulphurous acid derived from sulfur dioxide (SO₂) emission leads to the pollution of irrigation water and the inhibition of plant growth. The safe concentration threshold of NaHSO₃ in plants should be clarified to promote agricultural production. In this study, Orychophragmus violaceus seedlings were used as experimental materials and five NaHSO₃ concentrations (i.e., 0, 1, 2, 5, 10 mmol·L⁻¹) were simultaneously sprayed on the leaf surface of different seedlings separately. Leaf physiology responses under different concentrations were analyzed. The NaHSO₃ did not promote photosynthesis in O. violaceus under the 1 and 2 mmol·L⁻¹ treatments. It was conducive to the net photosynthetic rate (P_N), photorespiration rate (R_p), chlorophyll content, actual photochemical quantum yield (Y_II) and photochemical quenching (qP) under the 5 mmol·L⁻¹ treatment. However, quantum yield of regulated energy dissipation (Y_NPQ) and nonphotochemical quenching (NPQ) were inhibited. Under the 10 mmol·L⁻¹ treatment, P_N, chlorophyll content, Y_II, qP, dark respiration rate (R_d) and electron transport rate (ETR) showed significant decreases, while the photorespiration portion (S_p) significantly increased. Our results demonstrated that NaHSO₃ provided a sulfur source for plant growth and interfered with the redox reaction of the plant itself, and its role as a photorespiratory inhibitor might be masked.

Background: Plantlets grown in vitro with a mixed nitrogen source utilize sucrose and CO₂ as carbon sources for growth. However, it is very difficult to obtain the correct utilization proportions of nitrate, ammonium, sucrose and CO₂ for plantlets. Consequently, the biological effect of ammonium/nitrate utilization, the biological effect of sucrose/CO₂ utilization, and the ammonium/nitrate use efficiency for new C input derived from CO₂ assimilation/sucrose utilization are still unclear for plantlets. Results: The bidirectional stable nitrogen isotope tracer technique quantified the proportions of assimilated nitrate and ammonium in Brassica napus plantlets grown at different ammonium/nitrate ratios. The utilization proportions of sucrose and CO₂ could be quantified by a two end-member isotope mixing model for Bn plantlets grown at different ammonium/nitrate ratios. Under the condition that each treatment contained 20 mM ammonium, the proportion of assimilated nitrate did not show a linear increase with increasing nitrate concentration for Bn plantlets. Moreover, the proportion of assimilated CO₂ did not show a linear relationship with the nitrate concentration for Bn plantlets. Increasing the nitrate concentration contributed to promoting the assimilation of ammonium and markedly enhanced the ammonium utilization coefficient for Bn plantlets. With increasing nitrate concentration, the amount of nitrogen in leaves derived from nitrate assimilation increased gradually, while the nitrate utilization coefficient underwent no distinct change for Bn plantlets. Conclusions: Quantifying the utilization proportions of nitrate and ammonium can reveal the energy efficiency for N assimilation in plantlets grown in mixed N sources. Quantifying the utilization proportion of CO₂ contributes to evaluating the photosynthetic capacity of plantlets grown with variable ammonium/nitrate ratios. Quantifying the utilization proportions of nitrate, ammonium, sucrose and CO₂ can reveal the difference in the ammonium/nitrate use efficiency for new C input derived from CO₂ assimilation/sucrose utilization for plantlets grown at variable ammonium/nitrate ratios.

Poor growth is often observed in artificial young forests due to insufficient inorganic nitrogen in karst soils. However, little is known about the assimilatory demand of the whole plant for nitrate and the partitioning of nitrate assimilation in roots and leaves in woody plants grown in karst habitats. In this study, Broussonetia papyrifera (L.) Vent (B. papyrifera) seedlings were grown under nearly hydroponic conditions. The isotope mass balance approach was employed to quantify the δ¹⁵N values of the N assimilates in plant organs and in whole plants for B. papyrifera seedlings grown at different nitrate concentrations. The δ¹⁵N values of the N assimilates in the whole B. papyrifera seedlings showed a rising trend with increasing nitrate concentration. Increasing the supply of nitrate decreased the leaf–root difference in the δ¹⁵N values of the N assimilates for B. papyrifera seedlings. Quantifying the δ¹⁵N values of N assimilates in the whole B. papyrifera seedlings grown under different nitrate concentrations contributes to estimating the assimilatory demand of the B. papyrifera seedlings for nitrate. The leaf–root difference in the δ¹⁵N values of the N assimilates can be used to estimate the partitioning of nitrate assimilation in the roots and leaves.

Ammonium (NH₄⁺) toxicity impairs plant growth, but nitrate (NO₃⁻) can mitigate this effect through unresolved mechanisms. Using leaf δ¹³C values (photosynthetic capacity) and a bidirectional ¹⁵N tracer (NH₄⁺ assimilation efficiency and source utilization), this study investigated these mechanisms in 35-day-old Orychophragmus violaceus plantlets grown in modified Murashige and Skoog media under varying NH₄⁺:NO₃⁻ ratios. ¹⁵N isotope fractionation during NH₄⁺ (same fixed 20 mM NH₄Cl) assimilation decreased with increasing NO₃⁻ supply (10, 20, and 40 mM NaNO₃). Under 20 mM NH₄⁺ (δ¹⁵N = −2.64‰) at two ¹⁵NO₃⁻-labels (δ¹⁵N-NO₃⁻ = 8.08‰, low ¹⁵N, L) and (δ¹⁵N-NO₃⁻ = 22.67‰, high ¹⁵N, H), increasing NO₃⁻ concentrations enhanced NO₃⁻ assimilation, alleviating acidic stress from NH₄⁺ and improving photosynthesis. Higher NO₃⁻ levels also increased NH₄⁺ utilization efficiency, reducing futile NH₄⁺ cycling and decreasing associated ¹⁵N fractionation during assimilation. Our results demonstrate that NO₃⁻ alleviates NH₄⁺ toxicity primarily by enhancing photosynthetic performance and optimizing NH₄⁺ utilization efficiency.