Ammonium (NH₄⁺), as a major inorganic source of nitrogen (N) for tea plant growth, is transported and distributed across membranes by the proteins of ammonium transporters (AMTs). However, the AMT2-type AMTs from tea plants remain poorly understood. In this study, five CsAMT2 subfamily genes were identified in tea plant genomes, and their full-length coding sequences (CDS) were isolated from roots. Then, a NH₄⁺ uptake kinetic comparison of Fudingdabaicha (FD), Huangdan (HD), and Maoxie (MX) showed that FD was a high N efficiency (HNE) cultivar that had a wide range of adaptability to NH₄⁺, HD was a high N efficiency under high N conditions (HNEH) cultivar, in which it was easy to obtain higher yield in a high N environment, and MX was a high N efficiency under low N conditions (HNEL) cultivar, which had a higher affinity for NH₄⁺ than the other two. Tissue-specific expression analysis suggested that CsAMT2.2 and CsAMT2.3 were highly expressed in the roots, indicating that these two members may be unique in the CsAMT2 subfamily. This is further supported by our findings from the temporal expression profiles in the roots among these three different N adaptation cultivars. Expression levels of CsAMT2.2 and CsAMT2.3 in FD and HD were upregulated by a short time (2 h) under high NH₄⁺ treatment, while under low NH₄⁺ treatment, CsAMT2.2 and CsAMT2.3 were highly expressed at 0 h and 2 h in the HNEL-type cultivar-MX. Furthermore, the functional analysis illustrated that CsAMT2.2 and CsAMT2.3 could make a functional complementation of NH₄⁺-defective mutant yeast cells at low NH₄⁺ levels, and the transport efficiency of CsAMT2.3 was higher than that of CsAMT2.2. Thus, we concluded that CsAMT2.2 and CsAMT2.3 might play roles in controlling the NH₄⁺ uptake from the soil to the roots. These results will further the understanding of the NH₄⁺ signal networks of AMT2-type proteins in tea plants.

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