Potato and Bambara groundnut ammonium transporter (AMT1) structure and variation in expression level in potato leaf tissue in response to nitrogen form and availability

Abstract

Plants require nitrogen (N) to support desired production levels. Nitrogen fertilization strategy is a major consideration in field management with regard to achieving both economic and environmental objectives. For instance, in potato, insufficient N supply reduces tuber size and overall yield while excessive N supply can reduce tuber quality and increase environmental risk through nitrate (NO3-) leaching and nitrous oxide emission. Selection of an adequate N fertilizer application rate for crops is difficult, due to marked variations in soil N supply and crop N demand in both the field and over time. This research was conducted to characterise the ammonium transporter gene (AMT1) of Bambara groundnut and potato using molecular biology and bioinformatics methods. Nucleotide database sequences were used to design AMT1-specific primers which were used to amplify and sequence the core-region of the gene from Bambara groundnut and potato. Bioinformatics techniques were used to predict the structure and infer properties of the proteins. Nucleotide sequence alignment and phylogenetic analysis indicate that BgAMT1 and PoAMT1 are indeed from the AMT1 family, due to the clade and high similarity they respectively shared with other plant AMT1 genes. Amino acid sequence alignment showed that BgAMT1 is 92%, 89% and 87% similar to PvAMT1.1, GlycineAMT1 and LjAMT1.1 respectively, while PoAMT1 is 92%, 83% and 76% similar to LeAMT1.1, LjAMT1.1 and LeAMT1.2 respectively. BgAMT1 and PoAMT1 fragments were shown to correspond to the 5th - 10th transmembrane spanning-domains. Mutation of Bg W1A-L and S28A (for BgAMT1) and Po S70A (for PoAMT1) is predicted to enhance ammonium (NH4+) transport activity. Residues Bg D23 (for BgAMT1) and Po D16 (for PoAMT1) must be preserved otherwise NH4+ transport activity is inhibited. In all, BgAMT1 and PoAMT1 play a role in N uptake from the root while BgAMT1 may contribute more in different steps of rhizobia interaction. In an investigation of the correlation between AMT1 gene expression levels and leaf chlorophyll content index (CCI) with plant N status, potato plants were grown in a hydroponic greenhouse with 0.75 or 7.5 mM NO3- and 0.75 or 7.5 mM NH4+ as forms of N supply in a completely randomized design. Leaf CCI as measured by chlorophyll content meter, showed that an increase in N supply results in increased leaf CCI in response to both forms of N. Total RNA was isolated from leaf sampled at 28 days after treatment and expression level of the AMT1 gene was determined by reverse transcription-qPCR using a second set of primers designed for qPCR. The results showed that expression levels of AMT1 increased from 8.731 ± 2.606 when NO3- supply was high to 24.655 ± 2.93 when NO3- supply was low. However, there was no significant response in AMT1 expression levels to changes in NH4+. This result suggested that AMT1 transports NO3- less efficiently than NH4+, and thus more transport channels are required in the cell membrane when NO3- levels are low. Such variation in AMT1 expression levels are not necessary for NH4+ transport since the transport mechanism for NH4+ is efficient even at low NH4+ levels.