Abstract
Millets, resilient and nutritionally rich crops, are increasingly recognized for their potential in sustainable agriculture. Ammonium transporter (AMTs) gene family significantly contribute to the absorption and transport of NH4+ form of nitrogen in plants. The information about the structure and function of ammonium transporter genes in millet species is lacking. The millet crops such as pearl millet, proso millet, finger millet, sorghum, foxtail millet and green foxtail millet exhibit genetic variation in AMTs, which can be harnessed to improve NUE. Thus, genomic sequences of the six millet species were used and a total of 53 AMT genes were identified. Further, comprehensive analysis of chromosomal distribution, transmembrane structure prediction, presence of exons and introns, domain and motif organization, phylogeny, and synteny analysis were carried out. The phylogenetic analysis illustrated that millet AMTs belong to two subfamilies AMT1 and AMT2 (AMT2/AMT3/AMT4). Ka/Ks analysis showed that segmental duplications have contributed considerably in the evolution of millet AMTs. Phylogenetic classification of members of Poaceae using the amino acid sequences of AMT1.1 genes confirms the speciation patterns shown by matK gene sequence. Promoter analysis of millet AMTs showed presence of cis-elements related to light response, anaerobic induction, growth hormones, drought stress, biotic stress and several endogenous signals related to plant growth and development. This research provides insights into the structural and functional aspects of ammonium transporter genes in millets, and will serve as a foundation for utilizing AMTs for devising NUE strategies.
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Introduction
Nitrogen (N) is the most commonly required macronutrient for plant growth and development. It is an essential element for the synthesis of biomolecules such as nucleotides, amino acids, proteins, chlorophyll, and several others (Marcos de Leão et al. 2020). Nitrogen in the soil exists in three forms, which include organic nitrogen compounds, ammonium (NH4+), and nitrate (NO3−) ions (Williams and Miller 2001). Plants absorb nitrogen primarily in the ammonium and nitrate forms, and the organic nitrogen compounds need to be converted to these two forms before being taken by the plants. The use of nitrogen by plants involves absorption, assimilation, and remobilization during plant growth and development. In addition to N absorption from the soil, nitrogen use efficiency (NUE) depends on the assimilation of inorganic nitrogen from the soil, and the utilization of nitrogen during the life cycle of a crop plant (Masclaux-Daubresse et al. 2010; Xu et al. 2012). Plants have a preference for ammonium N form over nitrate nitrogen for uptake from the soil due to the direct assimilation of NH4+ into amino acids in plant cells, whereas, NO3− nitrogen has to be reduced to NH4+ before assimilation (Bloom et al. 1992; Jiang et al. 2019; Boschiero et al. 2019). The biological assimilation of nitrogen occurs either through the glutamine synthetase/glutamate synthase pathway (GS/GOGAT) or through glutamate dehydrogenase (GDH), resulting in the synthesis of glutamine which is the substrate for the synthesis of other amino acids via transamination reactions. Ammonium transport is tightly regulated during plant growth and development by the activities of high- and low-affinity ammonium transporters (Loque et al. 2006; Yuan et al. 2007; Kiba and Krapp 2016). Generally, high-affinity ammonium transport is preferred for NH4+ acquisition by plants due to the low ammonium concentration (< 1 mm) in the soil (Hao et al. 2020).
Ammonium transporters (AMTs) involved in the uptake of NH4+ have been identified in varied plant species (Couturier et al. 2007; Yuan et al. 2007; Tang et al. 2020). These AMTs are distributed in the plasma membranes of plant cells and form homo-or heterotrimers complexes for facilitating the passing of NH4+ ions or NH3 through the pore (Shelden et al. 2001; Ludewig et al. 2003). The transport mechanism of plant AMTs could be an NH4+ uniporter, NH4+/H+ symporter, or NH3/H+ co-transporter. Plant AMTs can be divided into the following two subfamilies: the AMT1 subfamily (AMT1 cluster) and the AMT2 subfamily (AMT2/3/4 cluster) (Huang et al. 2022).
The AMT genes were identified both in prokaryotic and eukaryotic organisms (Mcdonald and Ward 2016). The first ammonium transporter genes were identified in Saccharomyces cerevisiae and Arabadopsis thaliana (Marini et al. 1997; Ninnemann et al. 1994). Further, AMT family genes were characterized in several crop species namely; Zea mays (Gu et al. 2013), Glycine max (Kobae et al. 2010), Arabidopsis thaliana (Loqué et al. 2006; Yuan et al. 2007, 2009, 2013; Lanquar et al. 2009; Huang et al. 2015), Lotus japonicas (Guether et al. 2009; Wang et al. 2022), Oryza sativa (Ferreira et al. 2015; Li et al. 2016), Medicago truncatula (Breuillin-Sessoms et al. 2015), Populus trichocarpa (Wu et al. 2015), Triticum aestivum (Duan et al. 2016; Li et al. 2017), Coffea canephora ( Santos et al. 2017), Medicago truncatula (Breuillin-Sessoms et al. 2015), Pinus (Castro-Rodriguez et al. 2016), Solanum lycopersicum (Filiz and Akbudak 2020), and Malus domestica (Huang et al. 2022).
Millets, a group of small-seeded grains, have gained recognition as a pivotal component in achieving global food security and contribute to agricultural sustainability. Millet species include pearl millet (Pennisetum glaucum), finger millet (Eleusine coracana), green foxtail millet (Setaria viridis), foxtail millet (Setaria italica), great millet (Sorghum bicolor), proso millet (Panicum miliaceum), kodo millet (Paspalum scrobiculatum), Japanese barnyard millet (Echinocloa esculenta), Indian barnyard millet (Echinocloa frumentacea), and little millet (Panicum sumatrense), among others (Goron and Raizada 2015). Millets are hardy, resilient crops that thrive in diverse agro-climatic conditions, making them an essential resource for enhancing agricultural sustainability. Their exceptional nutritional profile, including high levels of protein, fiber, and essential micronutrients, placed millets as key contributors to improving food security, especially in regions grappling with malnutrition and food scarcity (Ceasar 2023). Improving the NUE of the cereals is essential to enhance yields under low-nutrient soils and conserve the fertility of the soils (Baligar et al. 2001; Bariya and Ahish 2014; Naeem et al. 2017; Nieves-Cordones et al. 2020). Several investigations have been conducted during the last decade by the wider scientific community, employing various molecular genetic tools to study and improve the NUE of crop plants. These include the utilization of genome-wide association study (GWAS) approach, molecular marker-assisted breeding (MAB), nutrient transporter characterization and functional genomics approaches. These investigations have been reported for model crop plants to improve NUE (Hawkesford 2012; Avin-Wittenberg et al. 2018). However, genome-based and forward genetic research may not be easy for millets with limited genomic resources.
Complete annotated genome sequence information is lacking for many millet species, which limits the understanding of gene sequences involved in determining the NUE traits in these crops. Nevertheless, the genome sequences of six millets, namely; Setaria viridis, Setaria italica, Eleusine coracana, Sorghum bicolor, Pennisetum glaucum and Panicum miliaceum are available for sequence analyses. These genome sequences provide a valuable resource to understand the structural and functional domains of genes coding for plant productivity, consequently enabling us to identify the AMT genes in the millet genome. Scanty reports for the AMT gene family characterization in millets are available in the literature, with limited coverage of genome sequence databases. Inadequate analysis of two AMTs in S. bicolor (Koegel et al. 2013), followed by an EcAMT1 study with other nutrient transporters (Maharajan et al. 2022), and a brief report about S. italica AMTs (SiAMT1.1 and SiAMT1.3) phylogeny and level of expression (Ahmad et al. 2018; Ceasar et al. 2023). Whereas, S. viridis, P. glaucum and P. miliaceum AMTs have not been taken into account for defining the structural and functional domains of this gene family.
After discussing all the above, in this study, six millet species are taken into consideration. We performed detailed analyses of the sequence characteristics, gene structures, chromosome distribution, motif compositions, and evolutionary relationships of millet AMT genes. In this context, the characterization of AMT genes from these six species and their comparative analysis to study and improve the NUE in millets may be helpful for further research.
Material and methods
Identification of AMT genes in different species
The genomic sequences, protein sequences, coding sequences (CDS) and genomic feature files (GFF) of six millet crops (S. viridis, S. italica, E. corocana, S. bicolor, P. miliaceum, and P. glaucum) obtained from Phytozome database (https://phytozome-next.jgi.doe.gov/ accessed on June1, 2023), National Genomics Data Center (NGDC) (https://ngdc.cncb.ac.cn/gwh/ accessed on June 1 2023) and the International Pearl Millet Genome Sequencing Consortium (IPMGSC) (https://cegsb.icrisat.org/ipmgsc/index.html accessed on June 1 2023). The Hidden Markov model (HMM) of all the conserved protein domain file Pfam-A.hmm was downloaded from InterPro (https://www.ebi.ac.uk/interpro/download/pfam/ accessed on 5 June, 2023). A simple HMM search of the TBtools software (Chen et al. 2020) was used to obtain ammonium transporters in different species. Pfam Id of ammonium transporter (Ammonium_transp—PF00909) was used for this study. Proteins with e-values of less than 5E-40 were included in further analyses. Different splicing variants of one gene and the incomplete genes were discarded. We searched for the ammonium-domain in all of the collected proteins using Interproscan (http://www.ebi.ac.uk/ Tools/pfa/iprscan/ accessed on June 6, 2023) and SMART software (Letunic et al. 2012).
Physicochemical properties and transmembrane structure analysis of AMT proteins
The theoretical molecular weight (kDa) and isoelectric point (pI) of millet AMTs were calculated using the ExPASy server (http://web.expasy.org/compute_pi/ accessed on June 6, 2023) (Gasteiger et al. 2003). The evaluation of the grand average of hydropathicity (GRAVY) of all identified proteins was measured through the GRAVY calculator (https://www.gravy-calculator.de/ accessed on June 6, 2023) (Gasteiger 2003). Predictions of subcellular localization of the concerned proteins were verified with the help of Plant-mPloc tool (http://www.csbio.sjtu.edu.cn/cgi-bin/PlantmPLoc.cgi/ accessed on June 6, 2023) (Chou and Shen 2010). The TMHMM server v. 2.0 (https://services.healthtech.dtu.dk/services/TMHMM-2.0/ accessed on June 6, 2023) (Krogh et al. 2001) was used for the prediction of transmembrane helices in AMT proteins. Individually, the physical locations of millet AMTs genes were obtained from the millet database (S. viridis, S. italica, E. coracana, S. bicolor, P. miliaceum, and P. glaucum) genome, and the map to locate genes on chromosomes of all six millet species was constructed through the PhenoGram (http://visualization.ritchielab.org/phenograms/plot accessed on June 10, 2023 (Wolfe et al. 2013).
Gene structure, conserved motif and conserved protein domain analyses of AMTs
The gene structures (CDS/exon/intron) of all the AMT genes were determined using the Gene Structure Display Server (CSDS) (http://gsds.gao-lab. org/ accessed on June 8, 2023) (Hu et al. 2015). For these analyses, the predicted coding sequence (CDS) of AMT genes and their corresponding genomic DNA sequences were used. The MEME (Multiple Em for Motif Elicitation) online tool (Bailey and Elkan 1994; Bailey et al. 2009) was used to identify the conserved motifs in the promoter regions of AMT genes and AMT amino acid sequences (https://meme-suite.org/meme/tools/meme accessed on June 8, 2023). The conserved domains of AMT proteins were analysed by the NCBI-CD (National Center for Biotechnology Information- Conserved Domain) search tool (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi accessed on June 8, 2023) (Marchler-Bauer and Bryant 2004). The TBtools software was used to integrate the phylogenetic tree, conserved motifs of and domains of millet AMT proteins (Chen et al. 2020).
Phylogenetic tree analysis of AMTs
The full-length amino acid sequences of AMTs from Arabidopsis thaliana, Triticum aestivum, Oryza sativa, Zea mays, Brachypodium distachyon, and Hordium vulgare were downloaded from the Phytozome database (Goodstein et al. 2012). AMT amino acid sequences of two bacteria, viz. Escherichia coli and Nitrosomonas europaea are also downloaded from NCBI (National Center for Biotechnology Information) (https://blast.ncbi.nlm.nih.gov/ accessed on June 6, 2023). To gain a deeper understanding of the taxonomical classification of the poaceae family, the chloroplast maturase K (matK) amino acid sequences were also downloaded. The amino acid sequences of AMTs as well as matK were aligned by MEGA-XI software (Koichiro et al. 2021), and a total of four phylogenetic trees were constructed by the maximum-likelihood method (ML). Bootstrap analysis was calculated for 1000 replicates. The evolutionary tree was visualized on the web-based tool Interactive Tree of Life (iTOL, https://itol.embl.de/ accessed on June 15, 2023) (Letunic and Bork 2021).
Ka/Ks analyses
The synonymous (Ks) and non-synonymous (Ka) substitution rates of the paralogs genes were further investigated by using the Ka_Ks calculator 2.0 (Zhang et al. 2006). A circular ideogram was made by Circos (Krzywinski et al. 2009) using TBTool software (Chen et al. 2020) to facilitate the display of relationships between paralogous pairs by the use of coloured lines. These encode the position, size, and orientation of related genomic elements in the Circos plots.
Synteny analysis of AMT genes
For visualization of protein sequence similarity between these six millet AMT genes, an online visualization tool named Circoletto (http://tools.bat.infspire.org/circoletto/ accessed on June 16, 2023) (Darzentas 2010) was used, which provides fast and informative overview of sequence similarity of search results. These results provide an essential first glimpse of the relationship between protein sequences.
Cis-element analysis of millet AMT promoter regions
The 2 kb upstream genomic DNA sequences of all six millet AMT genes were used for promoter analysis, and the cis-regulatory elements were predicted using the PlantCARE online website (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ accessed on 18 June 2023) (Lescot et al. 2002). The data were visualised by TBtools (Chen et al. 2020).
Results
Identification of AMT genes in different species
After validation of AMTs by HMM search, a total of 53 AMT protein sequences (S. viridis-7; S. italica-9; E. corocana -12; S. bicolor-5; P. glaucum -8 and P. miliaceum -12) were identified from six millet genomes. The AMTs of these species were termed as per existing rules of nomenclature for gene symbols, such as SvAMTs (derived from S. viridis), SiAMTs (derived from S. italica), EcAMTs (derived from E. corocana), SbAMTs (derived from S. bicolor), PgAMTs (derived from P. glaucum) and PmAMTs (derived from P. glaucum) genes throughout the study.
Physicochemical properties and transmembrane structure analysis of AMT proteins
The physiochemical properties of AMT proteins have been established using parameters such as chromosome location, strand, protein length, molecular weight (MW), isoelectric point (pI), prediction of the hydrophobicity (GRAVY), subcellular location and their family. The lengths of the millet AMT proteins ranged from 304 (SbAMT 2.2b; Sobic.003G344700) to 632 (EcAMT 2.1; ELECO.r07.5BG0417460) amino acids, with molecular weight ranging from 32.07 kD (PmAMT4.1b; GWHPAAEZ055444) to 67.61 kD (EcAMT 2.1; ELECO.r07.5BG0417460) and theoretical pI values ranging from 5.37 (SbAMT 3.3; Sobic.004G173200) to 8.84 (PmAMT1.3b; GWHPAAEZ069937). Subcellular localization prediction showed that all millet AMTs were localized to the cell membrane with few exceptions such as, SbAMT 3.3 (Sobic.004G173200), PmAMT1.3a (GWHPAAEZ021947), PmAMT3.2 (GWHPAAEZ054634), and PmAMT3.3 (GWHPAAEZ070534) are located both in cell membrane as well as vacuole. And the PgAMT1.1 (Pgl_GLEAN_10009225) is located in both cell membrane and mitochondrion whereas PmAMT3.2 (GWHPAAEZ054634) found in cell membrane, vacuole and mitochondrion (Table 1). The grand average of hydropathy (GRAVY) value was calculated for all the millet AMT proteins.
In this study, the predictions of the hydrophobicity of the deduced amino acid sequences indicated that the GRAVY of all millet AMT proteins were above zero, this led to conclusion that these amino acids are polar in nature (Table 1). Further, distribution of all six millet AMTs on chromosomes was analysed. It was observed that AMTs of S. viridis were located on five chromosomes viz. Chr1, Chr3, Chr5, Chr7 and Chr9 (Online Resource: S1), AMTs of S. italica located on five scaffolds with number 1, 3, 5, 7, 9 (Online Resource: S2), AMTs of E. corocana placed on Chr 1A, Chr 1B, Chr 2A, Chr 3A, Chr 3B, Chr 4B and Chr 5B. (Online Resource: S3), AMTs of S. bicolor located on three chromosomes viz, Chr1, Chr3 and Chr4 (Online Resource: S4). PgAMTs were present on three chromosomes (Chr 1, Chr 3 and Chr 6) and on the scaffold 2474 (Fig. 1). Whereas, PmAMTs were located on six chromosomes viz. Chr 1, Chr 3, Chr 4, Chr 5, Chr 6 and Chr 12 (Online Resource: S5). Transmembrane domain analysis of all millet AMTs showed occurrence of conserved transmembrane domains (Fig. 2 and Online Resource: S6-S10). These transmembrane domains regulate membrane localization and transport activity of a protein. Generally, millet AMTs have 11–12 transmembrane domains, whereas, these PgAMT1.1, PmAMT4.1b, SbAMT 2.2, SbAMT 3.3, PmAMT3.2, PgAMT1.2b, PmAMT2.2, PgAMT2.2, PmAMT1.3b and PgAMT1.2a have transmembrane domains varying from 6 to 10) which may be due to the small size of their protein sequences (Table 1).
Representative figure chromosomes of Pennisetum glaucum showing distribution of PgAMT genes. The chromosome number is listed below each chromosome while the numbers on the left represent location of the PgAMT genes. (AMTs chromosomal locations for other five millet species are given in Online Resource: S1-S5)
Representative figure of transmembrane structure prediction AMT proteins of Pennisetum glaucum. *Orange line represents outside, purple line indicates on transmembrane and blue line represents inside transmembrane position. (Transmembrane structure prediction of AMTs of other five millet species are given in Online Resource: S6-S10)
Gene structure, conserved motif and conserved protein domain analyses of AMTs
Structural analysis of the AMT proteins of six millet species were carried out expending the conserved domains and motifs based on the evolutionary relationships (Fig. 3). Gene structures of millet AMT proteins were predicted by using their CDS and genomic sequences. The graphical representation derived using GSDS showed that the AMT1 superfamily has less number of introns whereas in AMT2 the presence of introns is very common (Fig. 3). The domain analysis prediction showed that, the ammonium transporter (Ammonium_transp; PFam ID: PF00909, InterPro ID: IPR001905) structural domains are present in all the query proteins. This Ammonium_transp domains have found to relate the cl03012 protein superfamily and are mainly associated with transporting NH4 + across the membrane. In MEME server, the number of motif finder parameter was set to 20, so that upto 20 putative conserved motifs were found from each of the query protein sequences. Throughout the motif analysis it was found that, the subfamilies AMT1 and AMT2 had variable motif compositions. Also, proteins in the same subgroup showed identical motif components. Some of the motifs usually range from 1 to 5 were found present in AMTs of all the species this indicated that there are characteristic motifs of ammonium transporters.
Representation of the millet AMT genes structure showing exons, upstream/downstream regions of the gene and introns with the pink box, blue and black lines, respectively. Scale bar indicates number of nucleic acids (bp)
Noticeably, presence of a small motif can cause differences in subgroups and which may give an idea about the evolution of AMTs. In S. viridis AMT proteins, four motifs (motif-3, 6, 7 and 9) were found commonly present in all the AMT proteins of this species (Online Resource: S11). In S. italica AMT proteins, the five motifs (motif-5, 6, 8, 9 and 11) were found common in all AMTs (Online Resource: S12). Whereas in E. corocana, only single motif (motif-1) was found common in both the AMT1 and AMT2 families (Online Resource: S13). Similarly, in P. miliaceum, a single motif (motif-4) was observed common in all the AMTs (Online Resource: S15). Furthermore, in S. bicolor three motifs (motif-1, 2 and 19) are common in all the AMT proteins, (Online Resource: S14). Motif analysis of P. glaucum AMTs showed four common motifs in both the protein subfamily (AMT 1 and AMT 2) (Fig. 4).
Illustration of conserved protein motifs and conserved domain of AMTs in Pennisetum glaucum. An unrooted phylogenetic tree represents AMT1 and AMT2 subfamilies with their respective motifs are represented using different colours and conserved domains are shown by yellow boxes. (Conserved protein motifs and conserved domain analysis of AMTs of other five millet species are given in Online Resource: S11-S15)
Phylogenetic tree analysis of AMTs
A total of 53 millet AMT protein sequences have been identified using sequence search and alignment and used to understand the evolutionary relationships among AMT genes (Fig. 5). The common feature among AMTs of all the millet crops showed that they belong to two subfamilies i.e. AMT1 and AMT2 (AMT2/AMT3/AMT4). The major evolutionary conservation among the transporters exhibited that all millet AMTs stemmed from two major AMT transporter groups necessitates confirmation and further exploration of the evolutionary relationships with the AMTs from other species. The AMT protein sequences of twelve plant species (Setaria viridis, Setaria italica, Eleusine corocana, Sorghum bicolor, Pennisetum glaucum, Panicum miliaceum, Arabidopsis thaliana, Oriza sativa, Zea mays, Hordium vulgare, Triticum aestivum and Brachypodium distachyon) and two bacteria namely, Escherichia coli and Nitrosomonas europaea (ammonia oxidizing bacterium) were used to construct phylogenetic tree (Fig. 6). The phylogenetic tree structure clearly demonstrated the association of AMTs of Poaceae family which include all millets and other cereal species. The AMTs of Poaceae family have some degree of similarity with AMT genes from Arabadopsis thaliana, however, formed a different clade and clearly indicated divergence of the monocot transporters from the dicot ammonium transporters. The exclusion of bacterial group AMTs (E. coli and N. europaea) as an outgroup further, confirmed the AMT relationships and divergences in the phylogenetic grouping.
Phylogenetic tree analysis of AMTs from six millet species Setaria viridis (Sv), Setaria italica (Si), Eleusine coracana (Ec), and Sorghum bicolor (Sb), Pennisetum glaucum (Pg) and Panicum miliacium (Pm). *Different colours of circles represent different clusters
Phylogenetic tree analysis of AMTs of Setaria viridis (Sv), Setaria italica (Si), Eleusine corocana (Ec), Sorghum bicolour (Sb), Arabidopsis thaliana (At), Oriza sativa (Os), Zea mays (Zm), Hordium vulgare (Hv), Triticum aestivum (Ta) and Brachypodium distachyon (Bd). Two bacterial AMT namely, Escherichia coli (Eco) and Nitrosomona seuropaea (Ne) showing out grouping. *Different colors of circles represent different clusters
Ka/Ks analyses
Nucleotide substitutions in the coding regions may or may not result into amino acid change in the protein. One of the parameters i.e. Ka/Ks ratio which is the measure of the number of nonsynonymous substitutions per nonsynonymous site (Ka) and the number of synonymous substitutions per synonymous site (Ks). This Ka/Ks ration is the measure of selection pressure a gene has experienced during evolution. In the millet AMT family, analysis of selection types of duplicate gene pairs in the SvAMTs, SiAMTs, EcAMTs, SbAMTs, PgAMTs and PmAMTs genes were carried out using the Ka/Ks ratio. Majority of duplicated gene pairs has Ka/Ks ration less than one hence, less nonsynonymous substitutions taken place and the most of millet AMT genes have undergone negative selection. Majority of AMT genes are the resultant products of purifying selection during evolution (Table 2). However, the gene pair SbAMT1.2 and SbAMT4.1 showed that it had undergone neutral selection (Ka/Ks = 1). Schematic representations of the chromosomal distribution and inter chromosomal relationships was studied for all the six millet species by making circos plot of each species separately. These graphical representations showed gene duplication events in circular format. Among the 7 SvAMTs genes, three segmental duplication pairs (SvAMT1.2/SvAMT1.1, SvAMT2.2/SvAMT2.1 and SvAMT3.1/SvAMT3.2 were identified (Online Resource: S16). In S. italica, out of 9 SiAMTs genes only one tandem repeat pair (SiAMT2.3/SiAMT2.2) was found and other 3 pairs (SiAMT4.1/SiAMT2.1, SiAMT3.2/SiAMT3.1 and SiAMT1.1/SiAMT1.2) showed segment duplication (Online Resource: S17). Further, the 12 EcAMTs, 5 segment duplication pairs (EcAMT3.1b/EcAMT3.1a, EcAMT2.2c/EcAMT2.2b, EcAMT2.2d /EcAMT2.2a, EcAMT1.1a/EcAMT1.2a and EcAMT4.2/EcAMT4.1) were identified (Online Resource: S18). In S. bicolor, two segmental duplication pairs were observed out of 5 SbAMTs genes (Online Resource: S19). In P. glaucum, out of 8 genes, one PgAMT pair (PgAMT1.2b/PgAMT1.2a) found as tandem while other two pairs (PgAMT3.2/PgAMT3.3 and PgAMT2.2/PgAMT2.1) showed segmental duplications (Fig. 7). Whereas, in case of P. miliaceum, all the five pairs (PmAMT4.1b/PmAMT4.1a, PmAMT2.1/PmAMT2.3, PmAMT1.3a/PmAMT1.3b, PmAMT1.2b/PmAMT1.2a and PmAMT3.3/PmAMT3.1) among twelve genes showed segmental duplications (Online Resource: S20). The frequency of occurrence of segmental duplication events in the AMTs of these millet species suggest that these duplications plays bigger role in evolution of these genomes. Several research reports mentioned the significant role of tandem and segmental duplication events in gene family expansion and evolution of their genomes (Canon et al. 2004; Panchy et al. 2016; Kuo et al. 2019).
Schematic representations of the chromosomal distribution and inter-chromosomal relationships among AMT genes of Pennisetum glaucum. *Duplication events occurred in AMT gene family of P. glaucum are represented by blue, green, and yellow lines. *Chromosomes are represented in sky blue colors with the chromosomal number indicated inside each chromosome. (Visualization of chromosomal distribution and inter chromosomal relationships of other five millet species are given in the Online Resource: S16-S20)
Synteny analysis of AMT genes
In this study, synteny analysis between six millet AMT proteins (S. viridis, S. italica, E. corocana, S. bicolor, P. glaucum and P. miliaceum) with other species of poaceae family (O. sativa, Z. mays, H. vulgare, T. aestivum and B. distachyon) was performed by circoletto tool to understand the evolutionary history of genomes. In this analysis, amino acid sequences of AMTs of one species were used as query and while the rest of all the AMT sequences used as comparative files. ‘E-value’ and ‘score/max’ ratio parameter was considered to produce the colour bands and the colour of the bands indicate the sequence similarities (blue ≤ 0.25, green ≤ 0.50, orange ≤ 0.75, and red > 0.75). Based on the synteny analyses, maximum high synteny blocks (maximum red coloured bands > 0.75) were identified between AMTs of millets and other poaceae members/species. Considering these maximum red coloured synteny blocks, it can be concluded that, the AMT genes are more conserved in terms of evolutionary and genomic architecture in poaceae family.
Based on best score match parameter in circoletto tool, the best matched AMT sequences of different species showed synteny blocks. The AMTs of the species S. viridis and S. italica showed best synteny association (Online Resource: S21, S22). In case of E. corocana AMTs, 12 best score synteny blocks were identified between E. corocana AMTs and S. viridis, S. italica and S. bicolor AMTs (Online Resource: S23), while in case of S. bicolor 5 synteny blocks with best score was found between S. bicolor and Zea mays, S. viridis, S. italica (Online Resource: S24). In P. glaucum, eight best score synteny blocks were found in between P. glaucum and S. viridis, S. italica, S. bicolor AMTs (Fig. 8). Twelve best score synteny blocks were found to be associated with P. miliaceum AMTs and P. glaucum and S. viridis, S. italica (Online Resource: S25). Genomic dynamicity and evolutionary improvement along mobile elements in the genome of these six studied millet species were determined in these syntenic circles.
Visualization of the sequence similarity of AMT genes between Pennisetum glaucum with other millet AMTs as well as AMTs of different species of poaceae family (Oryza sativa, Zea mays, Hordeum vulgare, Triticum aestivum and B. distachyon). a Representation of synteny of PgAMTs and other AMTs. b Synteny blocks in ‘best score’ matching parameter of circoletto showing best matches between AMTs of P. glaucum and AMTs belongs to same tribe (S. italica, S. viridis and P. miliaceum). (Visualization of synteny analysis of other five millets are given in Online Resource: S21-S25)
Cis-element analysis of millet AMT promoter regions
To study the expression characteristics and potential functions of millet AMT genes, 2000 bp upstream sequences of start codons of the AMT genes of the six studied species were obtained as promoter sequences and used to analyse their cis-acting elements. The comprehensive results showed that millet AMT promoters have numerous cis-elements that respond to endogenous signals related to plant growth and development (viz. zein metabolism, circadian control, endosperm and meristem expression, root-seed-palisade mesophyll cells regulations), growth hormones (mainly auxin, gibberellin, abscisic acid, salicylic acid and methyl jasmonate), and environmental stresses (e.g. light response elements, low temperature stress-related elements, defense and stress, wound, anaerobic induction, anoxic specific induction and drought stress) (Fig. 9, Online Resource: S26-S30, Table. S1). All the millet AMT promoters have cis-elements responsive to light, suggesting an essential role of these AMT genes in plant growth and metabolism. From the data, it was evident that each gene promoter contains response element (s) to different phytohormone (s) with varied numbers ranging from 1 to 20, indicating that these AMT genes are under the regulation of hormone (s) and are involved in the hormone-mediated biological processes. Cis-elements involved in regulation of anaerobic induction are also common in all the millet AMT promoters, suggesting their possible role in plant growth and metabolism in anaerobic conditions. Almost all the promoter sequences have binding site for MYB-transcription factors related to many biological processes, such as plant growth and development, primary and secondary metabolic reactions, different physiological activity and responses to environmental stresses. Cis-elements related to drought- inducibility are also present adequate amount in almost all the promoters.
Representation of promoter cis-element analysis of AMT genes in P. glaucum. a Promoter position information. The different colored markers indicate different predicted cis-acting elements. b Promoter number analysis. The color scale to the right of the heat map represents the number of promoters. (Promoter cis-element analysis of AMT genes of other five millets are given in Online Resource: S26-S30)
Discussion
In plants, the Arabidopsis thaliana AtAMT gene was first recognized as an ammonium transporter (Ninnemann et al. 1994; Sohlenkampet al. 2000). Further analysis in Arabidopsis thaliana, proved that these AMTs also act as ammonium sensors that can sense the signal for cell–cell communication during plant growth and promote root to shoot ammonium translocation (Giehl et al. 2017). Genetic and molecular analysis in rice AMTs also proved that it acts in cell–cell communication and enhance the crown root formation in plants (Luo et al. 2022). In poaceae, several AMT homologues have been reported to play important roles in ammonium transport, such as Triticum aestivum (Li et al. 2017; Jiang et al. 2019), Oriza sativa (Li et al. 2009; Su-mei et al. 2012), Zea mays (Gu et al. 2013; Dechorgnat et al. 2019), Hordium vugare (Han et al. 2016) and Saccharum spontaneum (Wu et al 2021). In some millets, these AMT genes were also identified and are predicted to be associated with plant growth and development via ammonium transport (Maharajan et al. 2022; Ceasar et al. 2023). In Sorghum bicolor, induction of AMTs by arbuscular mycorrhizal fungi was studied which enhances the ammonium transport in plant parts (Koegel et al. 2013). The results suggested that, this AMT gene family has been involved in many biological processes in poaceae family. Millets are highly nutritious cereal crops and realizing their potential as nutraceutical food, much emphasis is given to improvement of these crops. Understanding the genomic loci involved in response, uptake and utilization of the nitrogen, a major nutrient in millet growth and production has utmost significance. There are two transporters involved in nitrogen uptake, the NRTs and AMTs in crop plants. Extensive research on in-silico analysis of NRTs has been carried out in millets. However, information about AMTs in millet crops is scanty. Hence, we performed an in-silico characterization of millet AMT genes that belong to two subfamilies viz. AMT1 and AMT2 (AMT2/AMT3/AMT4). Generally, the approximate length of members AMT gene family are between 400–450 amino acids and the structure can range from 45 to 50 kDa (Ninnemann et al. 1994; Blakey et al. 2002). The present study involved AMTs of six millet species and the length of the amino acids ranged from 304 to 632, and molecular weights ranging from 32.07 to 67.61 kDa are in consensus with earlier research.
Structural analysis AMT genes of millet revealed that the two subfamilies AMT1 and AMT2 exhibit divergent exon–intron patterns (Fig. 3). The structure of AMT genes of millet are highly conserved, among all the studied millets. AMT1 of P. glaucum (PgAMT1.2b, PgAMT1.2a, PgAMT1.1) and P. miliaceum (PmAMT1.2b, PmAMT1.3a) have introns in it, others are intronless. Similar research in MdAMT1 of Apple and GmAMT1 of Soybean reported absence of introns in AMT1 sub family (Huang et al. 2022; Yang et al. 2023). In Populus, Lotus japonicus, chilli pepper, most AMT1 genes have no introns in it, with the exception of LjAMT1.1, PtAMT1.7 and CaAMT1.1 that have one intron (Wu et al. 2015; Wang et al. 2022; Fang et al 2023). Millet AMT2 genes contain introns (ranges from 1 to 3), exons, and UTRs. The lengths of the UTRs, exons and introns vary among these AMT2 genes. Introns are usually involved in the regulation of gene expression and/or RNA stability (Shaul 2017). Mutations in critical regions in gene structure, including upstream region and coding sequence site may alter the expression patterns of members of gene family under evolution events (Heidari et al. 2022; Yaghobi and Heidari 2023). The lack of introns in the AMT1 subfamily genes suggests that the expression of these genes is essentially regulated at the transcriptional level. Large variations in the length and number of introns in different AMT2 subfamily genes indicate that these genes may undergo more complicated regulation, such as mRNA transport, alternative splicing, or chromatin assembly, which have been reported previously (Zhao et al. 2014; Jo and Choi 2015).
The phylogenetic analysis for ammonium transporters genes of six millets (Fig. 5) revealed that S. viridis, S. italica, P. glaucum and P. miliaceum shares a close relationship after alignment of retrieved proteins sequences of all the AMTs. This could be due to taxonomic commonality for instance, these four species (S. viridis, S. italica, P. glaucum and P. miliaceum) belong to the same tribe Paniceae (Li and Bruntnell 2011). Further, combined phylogenetic analysis using AMT proteins of all cereals (millets, rice, wheat, maize, barley and brachypodium), arabidopsis and bacterial AMTs (E. coli and N. europaea) evidently identified close association among six millets species for two ammonium transporter subfamilies. The maize transporter (ZmAMTs) were found closely related with millet AMTs as Zea mays (maize) is a member of Andropogoneae, which is a sister tribe to millet family, the Paniceae (Li and Brutnell 2011). The phylogenetic analysis of all millet AMTs and the combined phylogenetics involving AMTs of rice, wheat, maize, arabadopsis AMTs and bacterial AMTs (E. coli and N. europaea) clustered into conspicuous two subgroups of AMTs and similar findings of AMTs grouping has been reported in several investigations carried on other crops such as, soybean (Yang et al. 2023), poplus (Wu et al. 2015) and cassava (Xia et al. 2023).
In synteny analysis, the high score synteny blocks (red > 0.75) reinforce the idea that, AMT genes of poaceae are conserved in this family (Fig. 8, Online Resource: S21-S25). Five studied millets (S. viridis, S. italica, P. glaucum, P. miliaceum and S. bicolor) belongs to the subfamily panicoideae showed the best score synteny blocks frequently, imparting knowledge about the conservation of AMT genes in this subfamily. Again, AMTs of S. viridis, S. italica, P. glaucum and P. miliaceum exhibit maximum best scores synteny blocks as they belong to the same tribe paniceae. Furthermore, best score synteny blocks were found between S. bicolor and Z. mays, which again supporting the concept that AMTs are also conserved in tribes, as those two belongs to the identical tribe andropogoneae. In best match synteny analysis of E. corocana synteny blocks were also appeared between E. corocana and other millet AMTs, but the frequency is low. E. corocana belongs to chloridoideae subfamily, which is a close relative of subfamily panicoideae, and this may suggest that, there are resemblance of AMT genes between two closely related sister subfamilies. No best score synteny blocks were found between millet AMTs and other members of poaceae viz. O. sativa (subfamily: oryzoideae), B. distachyon, T. aestivum and H. vulgare (subfamily: pooideae) considered for this study, as they shared distant relationship from panicoideae subfamily. The phylogenetic tree generated by chloroplast matK genes of all the species of poaceae family in this study gives a depiction of taxonomic classification of poaceae family (Fig. 10), (Sorenget al. 2015, 2017, 2022). Interestingly, the phylogenetic tree constructed using AMT1.1 gene of all the previously studied Poaceae family crops reflected precisely the same pattern as proposed in their taxonomic classification (Fig. 10). This suggests that, in the course of evolution, AMT genes were also evolved by means of gene flow, natural selection, mutation or genetic drift.
Phylogenetic classification of Poaceae family by using matK (A) and AMT1.1 (B) gene sequences
A promoter is a region of DNA upstream of a gene where relevant proteins viz. RNA polymerase and transcription factors have to bind and initiate transcription of that gene (Hernandez-Garcia and Finer 2014). The level of transcriptional activation in eukaryotes is coordinated by upstream cis-acting elements in the regulation of gene expression, which are key links in plant environmental responses. Plant gene promoters contain a variety of important cis-acting elements that are involved in regulating the expression of corresponding downstream genes at the transcriptional level, thereby enabling plants to resist environmental stresses (Li et al. 2020). Cis-acting regulatory element analysis of millet AMTs promoter regions revealed a great abundance of light responsive elements, which implies that AMT gene expression is closely associated with photosynthesis and might be diurnally regulated. In research with Arabidopsis AMTs, AtAMT1.3 exhibited a typical diurnal pattern of change in expression; absorption of ammonium increased significantly towards the end of the day's light, and decreased as light intensity decreased (Gazzarrini et al. 1999). Additionally, two tomato AMTs (LeAMT1.2 and LeAMT1.3) also demonstrated rhythmic regulation (Von Wirén et al. 2000). Further, all the AMT genes share cis-elements responsive to anaerobic conditions. This has functional application in rice where AMTs has been widely studied for their role to uptake and utilize ammonium form of nitrogen in anaerobic conditions (Konishi and Feng. 2021). Cis-element analysis of AMT genes in majority of millet species showed involvement of at least one cis element in host defense response to the various biotic stresses in this study. It has been revealed in wheat and rice that ammonium transporters 1.1, 1.3, and 2.3 are associated with defense response to pathogens (Wu et al. 2022; Li et al. 2017; Jiang et al. 2019). Similarly evidences in support of role of AMTs in plant–microbe symbiosis e.g. LjAMT2.1 and LjAMT2.2 of Lotus japonicus and MtAMT2.3 of Medicago truncatula could be involved in ammonium transport from the host plants to nitrogen-fixing rhizobia and arbuscular mycorrhizae (Simon-Rosin et al. 2003; Guether et al. 2009; Breuillin-Sessoms et al. 2015). In addition, these AMT genes are under the control of different phytohormone (s) during the development and their response varies under diverse environmental conditions, thereby co-ordinately regulating ammonium uptake and metabolism.
Conclusion
The ammonium transporter gene (AMT) family plays a key role in the acquisition and transport of NH4 + forms of nitrogen in plants. This study identified a total of 53 AMT genes in the genomic sequences of the six millet species and classified them into two subfamilies, AMT1 and AMT2 (AMT2/AMT3/AMT4), based on phylogenetic analysis. The expansion of millet AMTs is the outcome of segmental and tandem duplication events in evolution. Syntenic conservation was observed in the structure and function of ammonium transporters in members of Poaceae. Promoter analysis of millet AMTs showed the presence of cis-elements regulating light response, anaerobic induction, growth hormones, drought stress, biotic stress, and several endogenous signals related to plant growth and development. This study provides in-depth information about the ammonium transporter gene family in millets, which would assist in improving nitrogen use efficiency through genomic manipulation of the expression patterns of these transporters.
Data availability
All data generated or analysed during this study are available from the corresponding author upon request.
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Sarkar, T., Bakshi, S. Ammonium transporter genes in millets: insights into structure, function, evolutionary conservation, divergence, and phylogenetic analysis. Genet Resour Crop Evol (2024). https://doi.org/10.1007/s10722-024-02092-2
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DOI: https://doi.org/10.1007/s10722-024-02092-2