Enrichment analysis of DEGs induced by salt treatment
To further understand the biological function of DEGs, gene ontology (GO) enrichment analysis was performed and 30 significant enrichment GO terms are presented. As shown in Fig. 2, the significantly enriched GO terms of Biological Process (BP) included the “stress responses” and “response to chemical”. The most enriched Cellular Component (CC) was “extracellular region” revealed the plant needed to modulate metabolism to cope with external salt stress. In addition, the significantly enriched GO terms of molecular function (MF) were “DNA-binding transcription factor activity” and “transcription regulator activity” suggested that transcriptional regulation were mainly activated by salt stimulus.
Next, we performed enrichment analyses of DEGs based on Kyoto Encyclopedia of Genes and Genomes (KEGG) which revealed that the DEGs were mainly enriched in 96 pathways (Supplemental Table 3). Among them, DEGs were most significantly enriched in the MAPK signaling pathway (Fig. 3).
Genome-wide characterization of the Mitogen-activated protein kinase kinases in Seteria italica (SiMKKs)
The transcriptome analysis indicated that MAPK signaling pathway was essential for the responses to salt stress in foxtail millet. However, few investigations were focus on the MAPK signaling of foxtail millet and the components of MAPK cascades had not yet been identified. Therefore, we performed a comprehensive identification to confirm Mitogen-activated protein kinase kinases in Seteria italica (SiMKKs). In this assay, the amino acid sequences of ten Arabidopsis MKKs (TAIR: https://www.arabidopsis.org/) were used as queries for BLAST searches of the foxtail millet genome (https://phytozome-next.jgi.doe.gov/). As results, a total number of fourteen SiMKKs genes were identified and the physicochemical characteristics of the corresponding proteins were predicted (Table 1). SiMKKs were distributed on seven chromosomes: three each on chromosome 1, chromosome 5 and chromosome 9; two on chromosome 4; and one each on chromosome 2, chromosome 3 and chromosome 6 (Supplemental Fig. 1). MAPK cascades are conserved in plants and MKK genes have been identified in other species. To study the phylogenetic relationship between foxtail millet and other species, we constructed a phylogenetic tree of MKK proteins from Seteria italica (Si), Arabidopsis thaliana (At), Oryza sativa (Os). As shown in Supplemental Fig. 2, the phylogenetic analysis suggested that MKKs from different species all could be divided into four groups (A, B, C and D). MKKs in foxtail millet (SiMKKs) were named based on the result of phylogenetic analysis.
Gene structural and conserved domain analysis
To further clarify the evolutionary relationships and explore the structural diversity of SiMKKs, the distribution of exon-intron structure was analyzed and displayed. As shown in Fig. 4A, SiMKK1, SiMKK3-1 and SiMKK3-2 contained eight introns; SiMKK3-3 contained nine introns; both SiMKK6-1 and SiMKK6-2 had seven introns; SiMKK10-4 had one intron and the other SiMKKs didn’t contain any introns. It was showed that members in same group had similar exon-intron structures. On the other hand, we performed promoter analysis of SiMKKs which predicted the cis-elements in the 2.0 kb promoter region upstream from the start codon (Fig. 4B). All SiMKKs contains more than one MYB-binding sites and MYC-binding sites. The numbers of CGTCA-motif, TGACG-motif and G-box were significantly higher than other motifs. For phytohormone response element, the results showed there were more ABA response elements (ABRE) than auxin response elements (ARE) in the promoters of SiMKKs, which indicated SiMKKs might play critical roles in stress responses.
Next, we predicted the conserved domains of SiMKKs with an online tool (https://prosite.expasy.org/prosite.html). As the result shown in Fig. 4C, all SiMKKs contained protein kinase domain, ATP binding region and kinase activate sites. Meanwhile, members of group B (SiMKK3-1, SiMKK3-2 and SiMKK3-3) contained nuclear transport factor 2 domain at the C-terminal of these proteins.
SiMKK genes expression patterns in response to salt stress
To explore the response of SiMKK genes to salt stress, we treated seedlings of YG1 with 200 mM NaCl and detected the expression patterns of 14 SiMKKs genes within 12 hours after salt treatment. Except for SiMKK4-2, SiMKK10-2 and SiMKK10-5, the expression level of the other 11 SiMKKs up-regulated within 12 h of salt treatment. Among genes with up-regulation expression, the expression levels SiMKK3-1, SiMKK10-1 and of all three genes in group A increased at least fivefold. In addition, the expression level of SiMKK6-2 increased by more than 20 times 3 hours after salt treatment which was the highest in SiMKK gene family.
Root development of heterologous overexpression lines of SiMKK1, SiMKK6-1 and SiMKK6-2 in Arabidopsis
As the expression levels of the three genes in subgroup A were all significantly increased under salt treatment, and 6 − 2 was the largest increase among all family members. Next, we took a closer look at these three genes. Through the existing transcriptome data, we analyzed the expression patterns of 14 MKK genes in different tissues and different growth periods, and the data showed that the expression levels of the three genes in group A were high in multiple tissues such as root and leaf. In addition, we also predicted the interacting proteins of the three proteins, and the results showed that the predicted interacting proteins of 6 − 1 and 6 − 2 were the same. SiMKK1 shares many common interacting proteins with 6 − 1 and 6 − 2, with 8 of the 10 proteins with the highest likelihood of interacting being the same. Therefore, we hypothesized that the three proteins might have very similar functions.
We cloned CDS of these three genes, constructed 35S promoter-driven expression vectors, and transformed them into Arabidopsis thaliana to obtain heterologous overexpression lines. Through phenotypic observation, we found that all overexpressed transgenic strains could have more developed roots under normal growth conditions, and the number of lateral roots was significantly higher than that of wild Arabidopsis. We planted the plants on a medium containing 100mM NaCl, and the results showed that under salt stress, the number of lateral roots of the transgenic plants was also significantly higher than that of the wild type. And two separate lines of the same gene showed similar phenotypes.
In addition, we also tested whether these three genes were involved in the regulation of taproot length. We grew wild-type plants and overexpressed strains on MS medium and medium containing 100mM NaCl respectively, and then measured the root length. The results showed that taproot length had no significant difference when there was no salt stress. The root length of transgenic plants was significantly longer than that of the wild type. Through calculation of relative root length (the ratio of average root length under salt stress to average root length under normal growth), we found that overexpression of these three millet MKK genes could significantly reduce the shortening of taproots under salt stress.