Vicia villosa, a high-quality green manure crop, helps to increase the content of soil phosphorus (P) by returning to the field. Soil P deprivation is a severe constraint on crop productivity that triggers the low P stress response in plants, which is controlled by various transcriptional regulatory network pathways. Identifying key genes from these transcriptional regulatory networks can help in developing low P-tolerant crops. In this study, we performed physiological observations and transcriptome sequencing of seedlings from the two Vicia villosa varieties, Xu Shao 3 and Soviet Vicia villosa, under P starvation conditions. The results showed that the main root length, plant height, shoot dry weight, root acid phosphatase activity, and inorganic P content of Xu Shao 3 were significantly higher than those of Soviet Vicia villosa under low P conditions. Based on transcriptome data analysis, 183 (shoot) + 144 (root) differential genes (DEGs) between the two varieties were identified; 144 (shoot) + 79 (root) were upregulated, and 69 (shoot) + 65 (root) were downregulated. KEGG analysis found that DEGs in shoots were significantly enriched in photosynthesis pathways, such as vitamin B6 and riboflavin metabolism. Meanwhile, DEGs in roots were enriched in plant signal transduction, fatty acid degradation, citric acid cycle, pentose, glucuronic acid conversion, etc. GO enrichment analysis suggested that DEGs in shoots were significantly enriched in biological processes, including cell response to P stress, intracellular ion homeostasis, etc., and molecular functions, including phosphate ester hydrolase, phosphatase, acid phosphatase activity, etc. Furthermore, DEGs associated with low P tolerance included three acid phosphatases, a phosphoesterase, a sulfoquinovosyl diacylglycerol synthase, a phosphoenolpyruvate carboxylase, six phosphate transporters and glycerol-3-phosphate transporters, eight SPX, and two PHL genes. In conclusion, Xu Shao 3 exhibited stronger inorganic P accumulation ability and a lesser effect on growth than Soviet Vicia villosa under low P conditions, which might result from photosynthesis, sugar, and P metabolism differences between the two varieties. Acid phosphatase, phosphoesterase, phosphoenolpyruvate carboxylase, sulfoquinovosyl diacylglycerol synthase, phosphate transporter, glycerol-3-phosphate transporter, and SPX were key DEGs leading to the difference in low P stress tolerance between the two varieties.

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.