[Foxtail millet SiSPX5 negatively regulates the signaling and metabolism of nitrogen and phosphorus].

The synergistic uptake of nitrogen (N) and phosphorus (P) is essential for plant growth, development, and adaptation to environmental stressors. The SPX family proteins are key regulators of the P-starvation response, while their roles in the coordinated regulation of N and P remain unclear. To systematically elucidate the molecular mechanisms by which foxtail millet (Setaria italica) SPX family genes mediate the synergistic uptake of nitrogen and phosphorus, and to characterize their regulatory effects on plant growth and development, we cloned SiSPX5, and systematically characterized its biological function through phylogenetic analysis, tissue-specific expression profiling, subcellular localization, heterologous overexpression in rice, reverse transcription quantitative PCR (RT-qPCR), and protein-protein interaction assays. Our results showed that SiSPX5 was a homolog of OsSPX5.It was predominantly expressed in flag leaves and leaf sheaths, and its encoded protein was localized to both the nucleus and the cytoplasm. Expression of SiSPX5 was induced by low P and high N conditions. Heterologous overexpression of SiSPX5 in rice significantly suppressed the transcript levels of genes involved in N uptake and assimilation (e.g., OsNRT1.2, OsAMT1.1, and OsGS1.1) and low-P response (e.g., OsIPS1, OsPHR2, and OsPHT1.2). Physiological measurements revealed that the transgenic lines exhibited significantly reduced NH4+ and NO3－ influx rates, decreased glutamine synthetase (GS) and nitrate reductase (NR) activities, declined free NH4+ and NO3－concentrations, and reduced total N content. Similarly, both inorganic P and total P content were markedly decreased. Furthermore, SiSPX5 directly interacted with the core P-signal transcription factors SiPHR1 and SiPHR2. Field agronomic trait analysis showed that SiSPX5 overexpression led to significant reductions in plant height, number of tillers, number of grains per panicle, and grain yield of rice, with the yield difference being more pronounced under high N conditions. In conclusion, SiSPX5 simultaneously represses N and P uptake by interacting with SiPHR1/2, thereby inhibiting rice growth and development and reducing the yield. This study defines a negative regulatory role of SiSPX5 in the coordinated control of N and P and suggests that downregulating SiSPX5 expression or abolishing its inhibition on SiPHR1/2 could promote N and P uptake, providing a theoretical basis and valuable gene resources for improving nutrient use efficiency in crops.