Functional Characterization of the VvPHT1 Gene and Its Promoter in Vicia villosa

Phosphorus deficiency in the environment induces phosphate (Pi) starvation responses of plants, in which the phosphate transporter is one of the most critical functional genes in this response mechanism. As a prevalent green manure crop in China, Vicia villosa plays a critical role in sustainable agricultural systems, and the expression of its phosphate transporter gene (VvPHT1) is modulated by soil phosphorus availability, highlighting its key adaptive function in nutrient acquisition and utilization under low-Pi conditions. Functional studies of this gene and its promoter contribute to exploring the molecular mechanisms of the tolerance of green manure crops to low phosphorus stress and to improving phosphorus-efficient V. villosa varieties. In this study, analysis of the VvPHT1 promoter sequence revealed a 1524 bp region containing multiple root-specific cis-regulatory elements, including five NODCON2GM, one NODCON1GM, six OSE2ROOTNODULE, one OSE1ROOTNODULE, and fifteen ROOTMOTIFTAPOX1 motifs. Histochemical GUS staining of transgenic Arabidopsis (Arabidopsis thaliana (L.) Heynh.) showed that the VvPHT1 promoter directed root-specific expression of the GUS reporter gene. A fusion expression vector pCAMBIA1300-VvPHT1--GFP was constructed and transformed into tobacco (Nicotiana tabacum L.) cells for subcellular localization analysis, indicating that the protein encoded by VvPHT1 was localized to the plasma membrane. To quantify its expression, VvPHT1 transcript levels in VvPHT1-overexpressing Arabidopsis (OEPHT1) lines were analyzed by quantitative real-time PCR (qRT-PCR) under different phosphorus supply conditions. The results demonstrated that under low-Pi conditions, the expression of VvPHT1 was significantly upregulated in the OEPHT1 lines compared to those of normal-Pi conditions. Furthermore, under low-Pi treatment, the OEPHT1 lines showed significantly increased fresh weight, primary root length, phosphorus content, and chlorophyll content compared to the wild-type Arabidopsis (WT), while no such differences were observed under normal-Pi conditions. In conclusion, the VvPHT1 promoter exhibits root-specific activity, and the VvPHT1 gene encodes a plasma-membrane-localized phosphate transporter that is strongly induced by phosphorus deficiency. Its overexpression enhances phosphorus uptake and plant growth under low-Pi conditions, suggesting that VvPHT1 likely functions as a high-affinity phosphate transporter involved in the adaptation to phosphorus starvation.