Ke Wen, Aoqi Peng, Yuanzi Wei, Miao Zhang, Ziyi Wu, Jing Wang, Zhicong You, Weimin Xu, Xiaojun Li, Ying Chen, Yuxuan Ma, Lu Wei, Wenfeng Huang, Baobin Mi, Lingling Xie, Daolong Liao, Wenna Zhang
Nitrogen (N) deficiency is a major constraint on crop productivity, yet the physiological and molecular mechanisms by which rootstocks confer N-use efficiency (NUE) remain unclear. Here, we investigated the responses of wax gourd scions grafted onto pumpkin rootstock (T/H) compared with self-grafted controls (T/T) under low-nitrogen (LN) stress and subsequent recovery. Physiological assays showed that T/H delayed leaf chlorosis, sustained higher chlorophyll content and photosynthetic rates, and promoted shoot nitrogen allocation, thereby maintaining superior NUE relative to T/T. Integrated transcriptomic and metabolomic analyses revealed specific reprogramming of nitrogen assimilation and transport pathways and phenylpropanoid-derived secondary metabolism. T/H seedlings exhibited enhanced expression of nitrate and ammonium transporters (NRT/NPFs, AMTs), glutamate dehydrogenase (GDH), and asparagine synthetase (AS), coupled with increased accumulation of asparagine. In parallel, phenylpropanoid biosynthesis was upregulated, with higher activity of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), and cinnamyl alcohol dehydrogenase (CAD), leading to increased flavonoid and phenolic contents. Functional characterization of heterologous expressed BhiNPF3.1 further demonstrated its plasma membrane localization, nitrogen-responsive expression in roots, and ability to enhance nitrogen accumulation in Arabidopsis, supporting a potential role for rootstock-mediated regulation of N transport. These metabolic and molecular shifts highlight that the pumpkin rootstock appears to sustain carbon-nitrogen balance through improved N partitioning to shoots while modulating phenylpropanoid metabolism in a manner that may contribute to stress adaptation. Together, our results demonstrate that pumpkin rootstock enhances NUE and LN resilience by integrating photosynthetic stability, nitrogen assimilation, transporter activity, and secondary metabolism, providing mechanistic insights into rootstock-mediated NUE.