Phosphorus is an essential macronutrient for plant growth and development, but phosphate resources are limited and rapidly depleting due to massive global agricultural demand. This study identified two genes in the phosphate transporter 2 (PHT2) family of soybean by bioinformatics. The expression patterns of two genes by qRT-PCR at leaves and all were induced by low-phosphate stress. After low-phosphate stress, GmPHT2;2 expression was significantly higher than GmPHT2;1, and the same trend was observed throughout the reproductive period. The result of heterologous expression of GmPHT2 in Arabidopsis knockout mutants of atpht2;1 shows that chloroplasts and whole-plant phosphorus content were significantly higher in plants complementation of GmPHT2;2 than in plants complementation of GmPHT2;1. This suggests that GmPHT2;2 may play a more important role in plant phosphorus metabolic homeostasis during low-phosphate stress than GmPHT2;1. In the yeast backfill assay, both genes were able to backfill the ability of the defective yeast to utilize phosphorus. GmPHT2 expression was up-regulated by a low-temperature treatment at 4 °C, implying that GmPHT2;1 may play a role in soybean response to low-temperature stress, in addition to being involved in phosphorus transport processes. GmPHT2;1 and GmPHT2;2 exhibit a cyclic pattern of circadian variation in response to light, with the same pattern of gene expression changes under red, blue, and white light conditions. GmPHT2 protein was found in the chloroplast, according to subcellular localization analysis. We conclude that GmPHT2 is a typical phosphate transporter gene that can improve plant acquisition efficiency.

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