| Summary: | <i>Vicia villosa</i>, 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 <i>Vicia villosa</i> varieties, Xu Shao 3 and Soviet <i>Vicia villosa</i>, 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 <i>Vicia villosa</i> 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 <i>SPX</i>, and two <i>PHL</i> genes. In conclusion, Xu Shao 3 exhibited stronger inorganic P accumulation ability and a lesser effect on growth than Soviet <i>Vicia villosa</i> 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 <i>SPX</i> were key DEGs leading to the difference in low P stress tolerance between the two varieties.
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