Objectives Phosphate starvation response (PHR) plays an important role in plant root development and phosphorus (P) nutrition. This study was conducted to clarify the biological functions of the PHR1 gene in Vicia villosa and provide a theoretical basis for cultivating high-phosphorus efficiency green manure crops.

Methods We obtained the VvPHR1 gene sequence by RNA-seq technique and verified the transcriptional activation of the gene by using a yeast single hybridization method. The overexpression vector of the VvPHR1 gene was constructed and genetically transformed into the wild-type and mutant (Atphr1) Arabidopsis thaliana by the pollen-tube pathway method. Expressions of the VvPHR1 gene and downstream phosphate transporter in wild-type and transgenic Arabidopsis thaliana were grown for 30 days in normal (1 mmol/L Pi) and low Pi (1 μmol/L Pi) media and analyzed by real-time fluorescent quantitative PCR. Phenotype and physiological indices of transgenic Arabidopsis thaliana, such as main root length, fresh weight, total P and Pi contents were analyzed.

Results  There were 13 PHR genes in Vicia villosa transcriptome, and transcripts 129590, 96227, and 120424 had the highest similarity with the PHR1 gene of Arabidopsis thaliana. The expression level of transcript 120424 was the highest under low Pi stress; thus, it was named the VvPHR1 gene. The full length of VvPHR1 cDNA was 1008 bp, encoding 335 amino acid proteins with a putative molecular weight of 36.5 KD and an isoelectric point of 6.04. The phylogenetic tree results showed that this gene was closely clustered with the PHR1 gene from Medicago truncatula and Glycine max. Subcellular localization analysis showed that this gene was localized in the cell nucleus. Yeast single hybridization experiments indicated that the VvPHR1 gene had transcriptional auto-activation and could activate downstream reporter gene expression. The results of real-time PCR showed that the expression of VvPHR1 in transgenic Arabidopsis thaliana was (P<0.05) enhanced under low Pi treatment, and the expression of downstream phosphate transporters in transgenic Arabidopsis thaliana was (P<0.05) upregulated compared with the wild-type Arabidopsis thaliana. Further phenotypic and physiological analysis of transgenic Arabidopsis thaliana revealed no significant differences (P>0.05) in plant growth, main root length, total P and Pi contents between wild-type and overexpressed VvPHR1 Arabidopsis thaliana, as well as between mutant and mutant back-complemented Arabidopsis thaliana in normal Pi treatments. In low Pi treatments, main root length, total P, and Pi of overexpressed VvPHR1 and mutant back-complemented Arabidopsis thaliana (P<0.05) increased compared with the wild-type and mutant Arabidopsis thaliana.

Conclusions The VvPHR1 gene is localized in the cell nucleus and has a transcriptional auto-activation function, a specific biological characteristic of transcription factors. Expression of the VvPHR1 gene is upregulated in transgenic Arabidopsis thaliana under low Pi conditions. The VvPHR1 gene has a similar function to the AtPHR1 gene and could promote root elongation and increase Pi uptake in plants under low Pi treatment.