Ammonium transporters (AMTs) are responsible for ammonium absorption and utilization in plants. As a high-nitrogen-demand crop and a legume, soybean can also obtain ammonium from symbiotic root nodules in which nitrogen-fixing rhizobia convert atmospheric nitrogen (N₂) into ammonium. Although increasing evidence implicates vital roles of ammonium transport in soybean, no systematic analyses of AMTs in soybean (named GmAMTs) or functional analyses of GmAMTs are available. In this study, we aimed to identify all GmAMT family genes and gain a better understanding of the characteristics of GmAMT genes in soybean. Here, due to the improved genome assembly and annotation of soybean, we tried to generate a phylogenetic tree of 16 GmAMTs based on new information. Consistent with reported data, GmAMT family members can be divided into two subfamilies of GmAMT1 (6 genes) and GmAMT2 (10 genes). Interestingly, unlike Arabidopsis, which has only one AMT2, soybean has substantially increased the number of GmAMT2s, suggesting enhanced demand for ammonium transport. These genes were distributed on nine chromosomes, of which GmAMT1.3, GmAMT1.4, and GmAMT1.5 were three tandem repeat genes. The gene structures and conserved protein motifs of the GmAMT1 and GmAMT2 subfamilies were different. All the GmAMTs were membrane proteins with varying numbers of transmembrane domains ranging from 4 to 11. Promoter analysis found that these GmAMT genes have phytohormone-, circadian control-, and organ expression-related cis-elements in their promoters, and notably, there were nodulation-specific and nitrogen-responsive elements in the promoters of the GmAMT1 and GmAMT2 genes. Further expression data showed that these GmAMT family genes exhibited different spatiotemporal expression patterns across tissues and organs. In addition, GmAMT1.1, GmAMT1.2, GmAMT2.2, and GmAMT2.3 were responsive to nitrogen treatment, while GmAMT1.2, GmAMT1.3, GmAMT1.4, GmAMT1.5, GmAMT1.6, GmAMT2.1, GmAMT2.2, GmAMT2.3, GmAMT3.1, and GmAMT4.6 showed circadian rhythms in transcription. RT-qPCR validated the expression patterns of GmAMTs in response to different forms of nitrogen and exogenous ABA treatments. Gene expression analysis also confirmed that GmAMTs are regulated by key nodulation gene GmNINa, indicating a role of GmAMTs in symbiosis. Together, these data indicate that GmAMTs may differentially and/or redundantly regulate ammonium transport during plant development and in response to environmental factors. These findings provide a basis for future research on the functions of GmAMTs and the mechanisms through which GmAMTs regulate ammonium metabolism and nodulation in soybean.

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