Gama-aminobutyric acid (GABA) plays an important role in regulating plant growth and response to various stresses. Here, we studied the natural variation of GABA transporter gene OsGAT3 in rice germplasm and found four primary promoter haplotypes. OsGAT3 was highly expressed in roots, culms, and leaf blades and was strongly induced in roots by GABA, salt, drought, and low temperature. OsGAT3 expression level was positively correlated with tiller number and nitrogen utilization efficiency (NUtE). OsGAT3 was localized in the plasma membrane. Overexpression of OsGAT3 significantly promoted GABA accumulation in basal parts suggesting it boosted axillary bud formation, tillering, and grain yield. OsGAT3 overexpression also enhanced tolerance to drought and salt stress. Transcriptome analysis revealed enriched phenylpropanoid and pentose phosphate pathways, and MAPK signaling underlining tolerance to salt stress, whereas drought tolerance was attributed to enhanced photosynthesis, glyoxylate and dicarboxylate metabolism, and carotenoid biosynthesis. Taken together, our data support the potential application of the gene for increasing yield under abiotic tolerance.

Amino acids are the primary form of nitrogen utilization in higher plants, mainly transported by amino acid transporters. In this study, we analyzed the natural variation of amino acid transporter-like 4 (OsATL4) in rice germplasm resources, identified its spatiotemporal expression characteristics, determined its substrate transport, and validated its function using transgenic plants. We found that the promoter sequence of OsATL4 varied across 498 rice varieties. The expression level of OsATL4 was higher in japonica rice, which was negatively correlated with tiller number and grain yield. OsATL4 was highly expressed in the basal part, leaf sheath, stem, and young panicle, with its two splicing variants localized to the cell membrane. OsATL4a (the long splicing variant) had a high affinity for transporting Ser, Leu, Phe, and Thr, while OsATL4b (the short splicing variant) had a high affinity for transporting Ser, Leu, and Phe. Blocking OsATL4 promoted axillary bud outgrowth, rice tillering, and grain yield, whereas overexpression lines exhibited the opposite phenotype. Exogenous application of low concentrations of Ser promoted axillary bud outgrowth in overexpression lines, while high concentrations of Ser inhibited it. Conversely, the mutant lines showed the opposite response. Altered expression of OsATL4 might affect the expression of genes in nitrogen, auxin, and cytokinin pathways. We propose that two splicing variants of OsATL4 negatively regulate rice tillering and yield by mediating the transport of amino acids, making it a significant target for high-yield rice breeding.

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.