Nitrogen (N) fertilization is necessary for obtaining high rice yield. But excessive N fertilizer reduces rice plant N efficiency and causes negative effects such as environmental pollution. In this study, we assembled key genes involved in different nodes of N pathways to boost nitrate and ammonium uptake and assimilation, and to strengthen amino acid utilization to increase grain yield and nitrogen use efficiency (NUE) in rice. The combinations OsNPF8.9a × OsNR2, OsAMT1;2 × OsGS1;2 × OsAS1, and OsGS2 × OsAS2 × OsANT3 optimized nitrate assimilation, ammonium conversion, and N reutilization, respectively. In co-overexpressing rice lines obtained by co-transformation, the tiller number, biomass, and grain yield per plant of the OsAMT1;2 × OsGS1;2 × OsAS1-overexpressing line exceeded those of wild-type ZH11, the OsNPF8.9a × OsNR2 × OsGS1;2 × OsAS1-overexpressing line, and the OsGS2 × OsAS2 × OsANT3-overexpressing line. The glutamine synthase activity, free amino acids, and nitrogen utilization efficiency (NUtE) of the OsAMT1;2 × OsGS1;2 × OsAS1-overexpressing line exceeded those of ZH11 and other lines that combined key genes. N influx efficiency was increased in the OsAMT1;2 × OsGS1;2 × OsAS1-overexpressing line and OsNPF8.9a × OsNR2 × OsGS1;2 × OsAS1-overexpressing line under a low ammonium and a low nitrate treatment, respectively. We propose that combining overexpression of OsAMT1;2, OsGS1;2, and OsAS1 is a promising breeding strategy for systematically increasing rice grain yield and NUE by focusing on key nodes in the N pathway.

Rice has a large number of nitrate or peptide transporter family (NPF) genes, but the effects of most members on rice growth and development are unknown. We report that OsNPF5.16, a nitrate transporter gene with natural variation in its promoter sequence, is essential for rice growth and yield. The promoter sequence showed various differences between indica and japonica cultivars, and higher expression of OsNPF5.16 was found in indica cultivars with higher plant weight and more tillers than japonica cultivars. OsNPF5.16 was highly expressed in roots, tiller basal parts, and leaf sheaths, and its protein was localized on the plasma membrane. In cRNA-injected Xenopus laevis oocytes, OsNPF5.16 transport of nitrate at high nitrate concentration depended on pH. Overexpression of OsNPF5.16 increased nitrate content and total nitrogen content in leaf sheath as well as biomass and tiller bud length in rice. Elevated expression of OsNPF5.16 increased rice tiller number and grain yield by regulating cytokinin levels. Inhibition of OsNPF5.16 expression showed the opposite effects. Regulating OsNPF5.16 expression has potential for improving rice grain yield.