Nitrate preference and ammonium sensitivity of Hippuris vulgaris under cold conditions: coordination of nitrogen assimilation, oxidative balance and phytohormone responses.

Low temperature constrains nitrogen (N) acquisition and assimilation in aquatic plants, yet how different inorganic N forms regulate growth and stress physiology under cold conditions remains poorly understood. Here, Hippuris vulgaris was exposed for 40 days to ammonium (NH4+-N) or nitrate (NO3--N) supplied at 0, 2, 5, 10, 20 and 50 mg L-1 N under 2-10°C conditions. A mixed-N experiment with different NH4+-N/NO3--N ratios was further conducted at the same total N concentration to evaluate growth-based N-form preference. Growth traits, tissue N accumulation, N-assimilation enzymes, antioxidant responses, nitric oxide (NO) signal, and phytohormone profiles were examined to identify N-form response. Results showed that H. vulgaris had clear preference for NO3--N over NH4+-N. NH4+-N at ≥10 mg N L-1 significantly inhibited root development and biomass accumulation, whereas NO3--N up to 50 mg N L-1 did not cause comparable growth suppression. After 40 days, weight growth rates under NO3--N treatments were 38.34-141.83% higher than 29.64-63.48% under corresponding NH4+-N treatments, and increasing the NO3--N proportion in mixed-N treatments further promoted fresh weight accumulation. The NO3--N advantage was associated with higher NR, NiR, GS and GOGAT activities and greater tissue N accumulation. Compared with NH4+-N, NO3--N reduced malondialdehyde accumulation, maintained higher antioxidant enzyme activities and enhanced root NO signal. High NH4+-N induced abscisic acid accumulation and was associated with weaker jasmonate-related responses, indicating a shift toward stress-dominated hormonal regulation. The above findings suggest that coordinated N assimilation, oxidative balance and phytohormone reprogramming underlie the contrasting responses of H. vulgaris to NO3--N and NH4+-N, providing physiological evidence for its use in nitrate-dominated cold-season aquatic environments.