Identification and annotations of NAC genes in pearl millet
The HMMER Search with the HMM profile identified 151 NAC genes among 35,757 pearl millet genes. All the identified NAC genes were named by adding the prefix “Pg”, for Pennisetum glaucum, and were numbered according to their chromosomal position, yielding PgNAC001 to PgNAC151. Deduced PgNAC protein sequences exhibited a diverse range of amino acid lengths: the smallest PgNAC was 98 amino acids (PgNAC011) long, whereas the largest was 750 amino acids (PgNAC134) long. The CDD search and the SMART program confirmed the presence of NAC domains in each PgNAC.
Chromosomal distribution and structure of PgNAC proteins
Physical mapping of PgNACs on all 7 pearl millet chromosomes revealed an uneven distribution for the first 134 PgNACs. Among the chromosomes, chromosome 3, which is the largest in size (300.9 Mb), had the largest number of PgNACs (25; 18%). Chromosomes 1 and 6 had the second largest number (15%) of PgNACs. Chromosome 5 had the smallest number (11%) of PgNACs, and its size is 158.7 Mb. PgNACs located on chromosomes 1 and 5 appear to congregate at one end of the arms, while chromosomes 2, 3, and 4 show clusters of PgNACs at both ends. Chromosome 7 has a non-clustered distribution of PgNACs. The remaining PgNACs (PgNAC135 to PgNAC151) mapped to 10 different scaffolds. Among these, scaffold 2474 had the largest number (5) of PgNACs (PgNAC141 to PgNAC145), while scaffolds 1622, 2427, 3470, 3477, 7552 and 8799 each had 1 PgNAC. Twenty-seven (15.56%) PgNACs were found to be tandemly duplicated (Fig. 1 and Additional File 2).
All PgNACs show great variation in size, with the smallest gene being 0.5 kb and the longest being 9 kb. The number of introns in PgNACs ranged from zero to twelve. Thirty-eight PgNACs consist of three exons and two introns. PgNAC101 and PgNAC007 have 13 and 10 exons, respectively. Twenty-nine (40.5%) PgNACs have only one exon (Additional File 3). Three PgNACs (PgNAC073, PgNAC029, and PgNAC151) were predicted to have a single transmembrane helix at the N-terminus (Fig. 2). These three PgNACs are more like membrane-associated NAC proteins from Setaria italica than those from Brachypodium distachyon and Zea mays. Each membrane associated PgNAC was placed in a different NAC protein clade (Fig. 3).
Phylogenetic analysis of PgNACs and conservation of motifs
Comprehensive phylogenetic analyses were performed by aligning the sequences of 145 SiNACs (Setaria italica NAC proteins) and 151 PgNACs. All NACs were grouped into 11 groups (A to K). Group G was the largest group with 32 PgNAC members, while the smallest was group H with 5 members (Fig. 4). Most PgNAC-encoding genes in the same group shared similar exon-intron structures and/or duplication patterns. For instance, all the PgNAC-encoding members in groups D and E have 2 or 3 introns. Group Q has the largest number (12) of duplicated genes.
The MEME suite found 17 motifs in PgNACs. PgNAC N-terminal regions were found to be more conserved than C-terminal regions. Generally, PgNACs in the same groups showed similar motif compositions (Table 1 and Additional File 4), supporting the idea that their functions are also similar.
Table 1. Conserved motifs present in pearl millet PgNACs. Conserved motifs were identified using the online tool MEME with the default parameters.
PgNACs GO Annotation
GO term analysis suggested that the majority of PgNACs are involved in DNA binding (90%). Eight PgNACs (PgNAC009, PgNAC021, PgNAC085, PgNAC125, PgNAC029, PgNAC112, PgNAC118, and PgNAC137) were associated with the GO term “homodimerization”. Approximately 86.25% of PgNACs were associated with the GO term “Nucleus”. Nine PgNACs were associated with the GO term “external and internal stimuli”, and 29 were associated with “response to stress” (Fig. 5).
miRNAs target sites in PgNACs
The results of analyzing miRNAs targeting PgNACs found a total of 88 miRNA target sites in the 59 PgNACs. Among them, PgNAC023 had the most (six) miRNA target sites. PgNAC023 is targeted by miRNA162, miRNA167h, miRNA394a and miRNA394b. While, PgNAC092 had four miRNA target sites, and is targeted by miR165a, miR165b, miR166b and miR166p. In some PgNACs, for example PgNAC117 and PgNAC120 one miRNA target site found. (Additional file: 5). These results suggested that PgNACs function is regulated by multiple miRNAs.
Transcriptomic expression of PgNACs during drought and salinity stress
PgNAC expression was examined using previously published transcriptome data. Seventy-two PgNACs were expressed under these drought-stressed and salinity-stressed conditions. PgNAC108, PgNAC131, PgNAC110, PgNAC146, PgNAC105, PgNAC045, PgNAC113, PgNAC002, PgNAC143, -PgNAC005, -PgNAC125, -PgNAC054, and PgNAC136 were strongly expressed under salinity-stressed conditions, whereas PgNAC137, PgNAC036, PgNAC007, PgNAC020, PgNAC060, PgNAC142, PgNAC074, and PgNAC011 were strongly expressed under drought-stressed conditions. PgNAC093, PgNAC142, PgNAC074, PgNAC020, and PgNAC060 were expressed under both salinity and drought conditions (Fig. 6).
Expression profiling by qRT-PCR
Quantitative RT-PCR was performed to confirm the expression of randomly selected PgNACs under drought and salinity stress. For drought, PgNAC142, PgNAC045, PgNAC105, PgNAC113, PgNAC110, PgNAC072, PgNAC044, PgNAC011, PgNAC022, PgNAC051, PgNAC029, PgNAC094, PgNAC106, PgNAC074, PgNAC033, PgNAC035, and PgNAC081 were studied. The expression of PgNAC081 was 170 times higher in roots under drought-stressed condition than control condition. Under drought-stressed conditions, most PgNACs were more strongly expressed in roots than in leaves (Fig. 7). PgNAC029, PgNAC106, and PgNAC074 were downregulated in both leaf and root tissues. For salinity, PgNAC051, PgNAC005, PgNAC036, PgNAC116, PgNAC146, PgNAC PgNAC131, PgNAC093, PgNAC089, PgNAC050, PgNAC136, PgNAC002, PgNAC113, PgNAC018, PgNAC105, PgNAC045 and PgNAC110 were selected. Most of these PgNACs were upregulated in both root and leaf tissues. PgNAC113 was strongly induced in roots by salinity stress (with 76-fold change) than control condition. (Fig. 8).