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. 2022 Aug 23;23(17):9522.
doi: 10.3390/ijms23179522.

LjAMT2;2 Promotes Ammonium Nitrogen Transport during Arbuscular Mycorrhizal Fungi Symbiosis in Lotus japonicus

Yanping Wang  1 , Wenqing Zhou  1 , Jiandong Wu  1 , Kailing Xie  1 , Xiaoyu Li  1
Affiliations

LjAMT2;2 Promotes Ammonium Nitrogen Transport during Arbuscular Mycorrhizal Fungi Symbiosis in Lotus japonicus

Yanping Wang et al. Int J Mol Sci. .

Abstract

Arbuscular mycorrhizal fungi (AMF) are important symbiotic microorganisms in soil that engage in symbiotic relationships with legumes, resulting in mycorrhizal symbiosis. Establishment of strong symbiotic relationships between AMF and legumes promotes the absorption of nitrogen by plants. Ammonium nitrogen can be directly utilised by plants following ammonium transport, but there are few reports on ammonium transporters (AMTs) promoting ammonium nitrogen transport during AM symbiosis. Lotus japonicus is a typical legume model plant that hosts AMF. In this study, we analysed the characteristics of the Lotus japonicus ammonium transporter LjAMT2;2, and found that it is a typical ammonium transporter with mycorrhizal-induced and ammonium nitrogen transport-related cis-acting elements in its promoter region. LjAMT2;2 facilitated ammonium transfer in yeast mutant supplement experiments. In the presence of different nitrogen concentrations, the LjAMT2;2 gene was significantly upregulated following inoculation with AMF, and induced by low nitrogen. Overexpression of LjAMT2;2 increased the absorption of ammonium nitrogen, resulting in doubling of nitrogen content in leaves and roots, thus alleviating nitrogen stress and promoting plant growth.

Keywords: AMT; Lotus japonicus; ammonium nitrogen; ammonium transporter; arbuscular mycorrhizal fungi; symbiosis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Bioinformatics analysis of the LjAMT2;2 gene. (A) Evolutionary analysis of the LjAMT family and AMT families in other species. (B) Amino acid sequence alignment of LjAMT2;2 with other reported AMT genes. Red lines indicate conserved ammonium transporter domains, and yellow lines indicate highly conserved domains. (C) Predicted three-dimensional structure of the LjAMT2;2 protein. (D) Distribution of transmembrane domains in LjAMT2;2. Purple represents the inner membrane, red represents the transmembrane, and blue represents the outer membrane.
Figure 2
Figure 2
Subcellular localisation of LjAMT2;2 expression. 35s::GFP is an empty carrier, 35s::LjAMT2;2-GFP is the target gene. Three visual fields were observed: Green channel (GFP), bright channel (bright-field), and merged. Scale bars, 20 μM.
Figure 3
Figure 3
Functional analysis of LjAMT2;2 in ammonium-deficient yeast. (A) Ammonium-deficient yeast supplementation experiment. (B) Yeast growth curves.
Figure 4
Figure 4
Effects of AMF colonisation on the growth of Lotus japonicus under different ammonium concentrations. (A) Trypan blue staining and colonisation rate statistics. H, Hyphae; V, vesicle; A, arbuscule (B) Phenotypes of wild-type plants under different ammonium ion concentrations. (C) Biomasses of aboveground and underground parts of plants under symbiotic and non-symbiotic conditions. Data are means ± standard error (SE; n ≥ 3 biological replicates; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).
Figure 5
Figure 5
Mycorrhizal-inducible expression of the LjAMT2;2 gene. (A) Heatmap of the LjAMT family gene expression induced by AMF. (B) GUS expression induced by AMF in pLjAMT2;2 plants. Arrows indicate Gus expression positions (C) Analysis of cis-acting elements in the LjAMT2;2 gene promoter. (D) Expression of the LjAMT2;2 gene under symbiotic and non-symbiotic conditions. Data are means ± standard error (SE; n ≥ 3 biological replicates; *** p ≤ 0.001).
Figure 6
Figure 6
Comparison of phenotypes between LjAMT2;2-OE and wild-type plants. (A) Growth status of aboveground and underground parts of plants. (B) Biomass statistics and LjAMT2;2 gene expression levels. Data are means ± standard error (SE; n ≥ 3 biological replicates; * p ≤ 0.05, ** p ≤ 0.01, ns: no significant difference).
Figure 7
Figure 7
Comparison of physiological indices between LjAMT2;2-OE and wild-type plants. (A) Total nitrogen content. (B) MDA content. Data are means ± standard error (SE; n ≥ 3 biological replicates; ** p ≤ 0.01, *** p ≤ 0.001).
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Grants and funding

This research was funded by Key research and development Program of Anhui Province (No. 202204c06020021) and National Natural Science Foundation of China (NSFC, No. U21A20235 and No. 31870415).

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