Identification of SiDREBs and phylogenetic analysis
The whole genome of foxtail millet identified 56 DREB family members, which were named SiDREB1-SiDREB56, respectively (Fig. 1; Table S1). To accurately classify the DREB family members in foxtail millet, the protein sequences of 55 DREB family members in Arabidopsis thaliana were aligned with those of 56 DREB family members in foxtail millet and a phylogenetic tree was constructed. The 56 phylogenetic trees of SiDREB genes in foxtail millet could be divided into 6 subfamilies (A1-A6). The SiDREB genes were unevenly distributed among 6 subfamilies. Among them, subfamilies A4 had the most SiDREB genes, subfamilies A1 had only seven SiDREB genes, and subfamilies A3 had no SiDREB genes.
Chromosome localization results showed that 56 SiDREB genes were distributed on 9 chromosomes. By comparison, it was found that the length of amino acids encoded by SiDREBs was 172 aa (SiDREB40) ~ 460 aa (SiDREB03), the molecular weight (MW) was 18,292.24 Da (SiDREB40) ~ 49,022.91 Da (SiDREB03), and the fat coefficient was 48 (SiDREB43) ~ 81.79 (SiDREB49). The value of the theoretical isoelectric point (pI) ranged from 4.26 (SiDREB10) to 11.72 (SiDREB42).
The isoelectric point of 9 proteins was greater than 7, which indicated that only 16% of SiDREB proteins belong to basic proteins. The instability index of proteins changed from 35.31 (SiDREB50) to 86.46 (SiDREB30), and only the SiDREB50 protein has a stability index of less than 40. All proteins were hydrophilic, with the maximum value of SiDREB36 being − 0.104 and the minimum value of SiDREB19 being − 0.738. Detailed data of 56 SiDREB protein sequences were listed (Table S2).
Chromosomal distribution and collinearity analysis of DREB gene families
The results of chromosome distribution indicated that the distribution of SiDREB family gene members across foxtail millet chromosomes shows uneven patterns (Fig. 2; Table S3). Among them, it is most distributed on chromosome III with 12 SiDREB genes. Only 1 SiDREB gene was distributed on chromosome VIII, which was the least distributed chromosome. Given the significant influence of tandem repeats and fragment repeats on the evolution and proliferation of plant gene families[33], then we analyzed the SiDREB gene replication events in the foxtail millet genome. The results showed that 13 gene pairs on 9 chromosomes containing the SiDREB gene were tandem replication types. Specifically, 2 pairs of tandem duplication in the A1 class, 6 pairs in the A2 class, 3 pairs in the A4 class, and 2 pairs in the A5 class. In addition, this study predicted the collateral homology of genes and analyzed the collinearity of the DREB gene in foxtail millet. It was found that the family had obvious duplication, which indicated that it might have doubled during the evolution of foxtail millet (Figure S1). Further analysis of the evolution of the identified SiDREB gene shows that some members of the DREB gene family have collinear relationship: there are 21 collinear gene pairs between 55 Arabidopsis DREB genes and 56 Arabidopsis DREB genes, which are distributed on different chromosomes of Arabidopsis thaliana, indicating that the sources of these 21 pairs of genes are similar and their DREB proteins are closely related (Figure S2).
Gene structure and motif composition of SiDREBs
The conserved sequences of 56 SiDREB genes in foxtail millet were analyzed by MEME online software, and 10 conserved motifs (named motif1-10) were found (Fig. 3A, B). The length of each conserved motif varies from 15 to 50 amino acids (Figure S3). The phylogenetic tree of the protein sequence of SiDREB showed that these proteins could be divided into six categories.
In these six subfamilies, it was found that all the SiDREB genes contained AP2 domains, among which A1 contained seven proteins, and the number of conserved motifs detected was mostly three, only the SiDREB39 protein contained four conserved motifs, mainly motif 1, motif 2 and motif 6 (Fig. 3C). At the same time, it is also found that motif 1, motif 2, and motif 6 always appear adjacent to each other in this class, and motif 6 and motif 2 precede motif 1, and only one Motif 7 is found in this subfamily. The A2 subfamily contains 11 proteins, 9 of which have the same conserved motifs and the most conserved motifs, while only motif 1, motif 2, and motif 6 are found in SiDREB16 and SiDREB37. A3 has no protein; Fourteen proteins in the A4 subfamily all contain motif 1, motif 2, motif 6, and motif 7, and this subfamily contains four motifs on average. The conserved motifs of A5 subfamily members are motif 1, motif 2, and motif 6. The number of A6 subfamily proteins is the same as that of the A2 subfamily, and the number of conserved motifs varies from 1 to 4. Motif 8 is only found in the A6 subfamily. These conserved motifs may be related to the growth of foxtail millet.
Gene structure analysis showed that all SiDREB genes had exon structure, and most of the exon groups in SiDREB gene structure were mainly concentrated in one (Fig. 3D). Only SiDREB04, SiDREB05, and SiDREB06 in the A2 subfamily, SiDREB17 and SiDREB42 in the A4 subfamily, and SiDREB16, SiDREB19, SiDREB23, and SiDREB37 in A6 subfamily contained two or more exons. 50% of the SiDREB genes ( 28 / 56 ) did not contain untranslated regions ( UTR ). SiDREB04 and SiDREB38 only contained upstream regulatory regions. SiDREB01, SiDREB02, SiDREB06, SiDREB08, SiDREB28, SiDREB36, SiDREB37, SiDREB39, SiDREB44 and SiDREB53 contained downstream regulatory regions. The remaining 16 members have neither upstream nor downstream regulatory regions. The protein members of the same family and their coding genes exhibit a high degree of structural and compositional similarity, which lends support to the reliability of phylogenetic development.
The Analysis of cis-acting elements of SiDREB promoter
To further investigate the regulatory mechanism of SiDREBs in abiotic stress, the promoter region (2 000 bp) of the SiDREB genes were analyzed for cis-acting elements (Fig. 4; Table S4). The results demonstrated that the promoter region of 56 foxtail millet SiDREB genes exhibited the presence of 20 distinct cis-acting elements, including low temperature, anaerobic induction, defense and stress responsiveness elements, light responsiveness elements, hormone response elements related to IAA, ABA, MeJA, GA and SA. As well as elements related to growth regulation and circadian rhythms, including cell cycle regulatory elements, seed-specific regulatory elements, and endosperm expression elements, and it also included a few wound-responsive elements (Fig. 4; Table S4). Among them, all SiDREB gene promoters contain defense and stress responsive elements, and hormone responsive elements were widely found in SiDREB family members. Furthermore, except SiDREB20, SiDREB21, SiDREB22, SiDREB24, SiDREB25, SiDREB26, SiDREB39, and SiDREB54, all the other DREB family members of foxtail millet have been observed to contain ABRE elements within their promoter regions. It is hypothesized that members of the DREB family may contribute to the stress response of foxtail millet via the ABA-dependent pathway.
Analysis of tissue expression pattern of DREB gene in foxtail millet
Different genes play different functions in different tissues of plants. By detecting the expression of 26 SiDREB genes in 21 tissues, the function of SiDREB genes was further studied (Fig. 5; Table S5). The results showed that there were some differences in the expression of SiDREB gene in the same kind of different organs. In addition, many SiDREB genes were highly expressed in neck-panicle-internodes and stem-top-second. Some SiDREB genes had a high abundance of transcripts in foxtail millet. SiDREB09 and SiDREB12 were strongly expressed in various parts. However, the transcription abundance of SiDREB19 gene was very low. In addition, the expression of the DREBs gene was different in different tissues. The expression of some SiDREB genes varied with different genes and tissues, showing tissue specificity. Such as SiDREB07, SiDREB08, SiDREB47, and SiDREB48, which were highly expressed in neck-panicle-internodes and stem-top-second of foxtail millet during the grain filling stage. The expression of SiDREB52 was relatively high in developing spikelets and seeds, especially in spikelets. The expression of SiDREB54 in 21 tissues was slightly or even not expressed. Additionally, there were significant differences in the expression of the same gene in different tissues and organs at the same growth stage. SiDREB09 was highly expressed in leaves and roots and almost not expressed in immature seed S5, and SiDREB12 expression was high in leaves and stems. During the grain filling stage, SiDREB09 demonstrated notable expression in leaves and roots, whereas SiDREB12 displayed particularly high expression in flag leaves. The results of this study elucidated the intricate role of SiDREB genes in the growth and development of foxtail millet, highlighting their tissue-specific expression patterns.
Expression analysis of DREB gene family in foxtail millet under ABA and abiotic stress
To detect the response pattern of the SiDREB gene family to abiotic stress, the relative expression of this family gene in foxtail millet leaves under low temperature (4°C), 100 µM ABA, and osmotic stress (20% PEG 6000) was analyzed by qRT-PCR technology. Subsequently, we constructed a gene expression heat map (Fig. 6). Generally, under hormone treatment and stress, the expression of SiDREB genes did not show consistent characteristics. Under low temperature stress, SiDREB48 and SiDREB53 gene expression decreased obviously, and the expression level of SiDREB08, SiDREB09, SiDREB11, SiDREB13, SiDREB14, SiDREB15, SiDREB28, SiDREB33, SiDREB35, SiDREB39, SiDREB41, SiDREB45, SiDREB47, and SiDREB52 were remarkably increased. The expression levels of SiDREB11 and SiDREB47 increased first and then decreased. The SiDREB08, SiDREB09, SiDREB11, SiDREB14, SiDREB15, SiDREB41, and SiDREB47 genes reached a high level at 3 h. The expression of SiDREB39 was the highest at 72 h, which was about 25.5 fold that at 0 h. The expression peak of SiDREB13, SiDREB33, SiDREB45, and SiDREB52 appeared at 96 h (Fig. 6A).
After ABA treatment, the expression of SiDREB09, SiDREB33, and SiDREB41 were decreased. The expression of genes SiDREB07, SiDREB08, SiDREB09, SiDREB15, SiDREB28, SiDREB35, and SiDREB39 exhibited notable enhancement, and the expression was exceeded 0 h. At 6 hours, the expression peaks of SiDREB07 and SiDREB39 appeared. The expression levels of SiDREB13, SiDREB14, SiDREB18, and SiDREB45 genes were the highest at 48 h, and their expression levels were 20.0 times, 15.0 times, 2.7 times, and 4.9 times higher than those of 0 h, respectively. The expression peak of SiDREB53 appeared at 96 h (Fig. 6B). Furthermore, they could respond to osmotic stress. For example, the expression of SiDREB08, SiDREB09, SiDREB13, SiDREB15, SiDREB35, and SiDREB52 could be up-regulated by osmotic stress. The expression of SiDREB09, SiDREB15, SiDREB28, and SiDREB47 were highest at 3 h. The expression peaks for SiDREB08, SiDREB39, SiDREB41, and SiDREB45 appeared at 12 h. The expression levels of SiDREB07, SiDREB14, SiDREB18, and SiDREB19 increased and then decreased. The expression of SiDREB11, SiDREB13, and SiDREB52 changed significantly at 96 hours (Fig. 6C). Some SiDREBs genes showed down-regulated expression patterns under stress, and the expression patterns of the SiDREB genes differ in response to various forms of stress, likely due to their involvement in disparate defence mechanisms against these stressors.