Identification and annotation of N. tabacum CKX genes
Aligning the A. thaliana AtCKX sequences with the N. tabacum genome revealed 15 N. tabacum NtCKX genes with similar structural domains. These genes were named NtCKX1–NtCKX15. The basic information of NtCKX gene family is presented in Table 1. The NtCKX gene family has between three–five exons, and the proteins they encode vary between 210–517 amino acids in length and 24,571.53–57,940.98 Da in molecular weight. All the proteins were hydrophilic, and their predicted isoelectric points ranged from 5.58 to 9.21. According to subcellular localization analysis of NtCKX gene expression, NtCKX1 was expressed in the cell membrane; NtCKX4–7, NtCKX10–12, and NtCKX14 were expressed in the extracellular matrix; NtCKX2, NtCKX3, and NtCKX8 were expressed in the vacuoles; NtCKX13 was expressed in the cytoplasm; and NtCKX15 was expressed in the chloroplasts.
Phylogenetic tree, multiple protein sequence alignment, and gene structure analysis
A phylogenetic tree was constructed using MEGA 6.0 for the CKX proteins of N. tabacum, A. thaliana, O. sativa, B. rapa, P. trichocarpa, S. lycopersicum, and P. patens to ascertain the evolutionary relationships of the CKX gene family (Fig. 1A). The phylogenetic tree has a total of eight branches, which are denoted as α, β, γ, δ, ε, ζ, η, and θ. The NtCKX genes are mainly located on the α, β, δ, ζ, and θ branches. The α branch includes NtCKX1–3, NtCKX5–6 and NtCKX8; the β branch includes NtCKX4 and NtCKX7; the δ branch includes NtCKX10–11; the ζ branch includes NtCKX9 and NtCKX12; and the θ branch exclusively consists of the NtCKX13–15 genes. The γ branch consists of eight genes, which are the OsCKX3 and CKX genes of P. patens.
Multiple protein sequence alignment analysis indicated that N. tabacum CKX genes exhibit a high degree of homology and conservativeness with respect to the CKX protein sequences of A. thaliana and S. lycopersicum (Figure S1). We found that genes located on the same branch have similar gene structures (Fig. 1B). For instance, NtCKX3, NtCKX5, NtCKX9 and NtCKX12 have very similar gene structures, and all these genes have five exons that are flanked by four introns. NtCKX10 and NtCKX11 have four exons and three introns, whereas NtCKX6 and NtCKX8 have three exons and two introns. NtCKX14 and NtCKX15 contain four exons and three introns. These NtCKX genes have the highest degree of intra-branch similarity in terms of structure and homology and have the same number of exons and introns. In contrast,NtCKX1, NtCKX2, NtCKX4, NtCKX7, and NtCKX13 genes exhibit intra-branch differences in structure.
Conserved protein motifs of the CKX genes of N. tabacum
The MEME tool analysis of the conserved protein motifs of the CKX genes indicated that NtCKX genes on the same phylogenetic branch have highly similar motifs and that motifs 6, 9, 13, 17, and 20 are present in all the members of the NtCKX gene family (Fig. 2, Table S3). Except for NtCKX15, motifs 2, 4, and 15 are also present in all the members of this family. Motif 14 is present in members of the NtCKX gene family except for NtCKX genes on the α branch. Motifs 10 and 18 are also present in all NtCKX genes except for α-branch NtCKX genes and NtCKX4. Motifs 2 and 16 were annotated as motifs that could be related to the FAD-binding domain and motifs 3, 5, 6, 7, 13, and 17 as possibly related to the cytokinin-binding domain.
Analysis of the protein–protein interaction network of N. tabacum and A. thaliana CKX genes
Figure 3 illustrates the complex network of protein–protein interactions of the N. tabacum and A. thaliana CKX genes predicted using the STRING website. In A. thaliana, AtCKX1 has important functions in the development and morphogenesis of vascular tissues, shoot apical meristems, and axillary meristems; the NtCKX3, NtCKX5, NtCKX6, and NtCKX8 genes that correspond to AtCKX1 could have similar functions. The NtCKX9, NtCKX12, NtCKX13, and NtCKX14 genes that corresponded to AtCKX3 may also autonomously regulate cytokinin levels and meristem cell viability. NtCKX4 and NtCKX7 may function similarly to AtCKX5, which plays a role in regulating stem meristem differentiation and limiting the flow of cytokinins from the stem meristem. NtCKX1 and NtCKX2 could have the same functions as AtCKX6, and play a role in the development of cotyledon, leaf and root vascular systems, and stoma development in N. tabacum.
Tissue expression profile of the NtCKX genes of N. tabacum
To study the functions of the NtCKX genes in further detail, RT-PCR was used to measure the expression of N. tabacum CKX genes in young roots, leaves, and stems; and mature roots, leaves, stems, flowers, and seeds (Fig. 4). NtCKX11 was expressed in all tested tissues with high expression levels in mature roots. NtCKX4 was strongly ex-pressed in young stems, whereas NtCKX6 was expressed abundantly in seeds. NtCKX14 had high expression levels in seeds and mature leaves. NtCKX1 was abundantly expressed in the flowers and mature roots, whereas NtCKX8 and NtCKX13 were strongly expressed in the seeds; these genes were either expressed at low or undetectable levels in all other tissues. NtCXK9 was abundantly expressed in young leaves, and it was also expressed in young roots and stems, mature stems, and seeds. NtCXK5 was only expressed in mature leaves, whereas NtCKX12 was only expressed in the seeds. NtCKX7 was specifically expressed in flowers and mature stems. The expression of NtCKX2, 3, 10 and 15 was very low in all the sampled tissues and organs, so no data on its expression has been detected.
Changes in the expression of N. tabacum NtCKX genes because of ABA- and salt-stress
We also investigated the expression of the NtCKX gene family in plants that were subjected to ABA- and salt-stress (Fig. 5). In the presence of ABA-stress, the expression of NtCKX4 was largely unchanged. The expression of NtCKX5, NtCKX7, NtCKX12, NtCKX13, and NtCKX15 fell to undetectable levels, and the changes in the expression levels of the other NtCKX genes was variable. For instance, the expression levels of NtCKX1, NtCKX2, and NtCKX6 were suppressed at three hour, and then upregulated, with their expression levels peaking at 24 h. The expression of NtCKX3 peaked at 6 h and then fell to undetectable levels. The expression levels of NtCKX8, NtCKX9 and NtCKX14 increased consistently up to 24 h. The expression of NtCKX10 resembled a parabolic curve; it gradually rose from 0 h to 12 h and then gradually decreased. The expression of NtCKX11 was strongly upregulated at 3 h and then decreased gradually, returning to normal levels at 24 h.
In salt-treated plants, NtCKX7, NtCKX12, NtCKX13 and NtCKX15 fell to undetectable levels. The expression of NtCKX4 was continuously down-regulation under salt treatment. The expression of NtCKX8 did not change significantly, whereas NtCKX3 expression was suppressed at 3 h but returned to normal levels shortly after. The expression of NtCKX2 was suppressed starting from 12 h. The expression levels of NtCKX1, NtCKX10, and NtCKX11 were parabola-like, as their expression was upregulated from the point of treatment until 6 h, and then downregulated. The relative transcription levels of NtCKX9 and NtCKX14 gradually increased over time and peaked at 24 hour.
Cis -acting elements in the promoter region of N. tabacum NtCKX genes
The promoter sequences of N. tabacum CKX genes were analyzed to study their response to abiotic stress. The cis-acting elements in the promoter region of each NtCKX gene that were predicted to respond to environmental stress and hormones are shown in Table 2, with the exception of NtCKX14, whose promoter sequences could not be identified. These cis-acting elements include ABA-responsive elements (ABRE), drought-responsive elements (ERE, MBS, and MYB), and salt-responsive elements (DRE). MYC responds to induction by low temperatures, high salinity, and drought. ERE, MBS, and MYB respond to drought conditions and the accumulation of ABA. The promoter regions of NtCKX8 and NtCKX9 contained a large number of MYB elements. ERE was the most abundant element in the promoter region of NtCKX10, which also contained many MYB and MYC elements. The promoter region of NtCKX11 had more MYB elements than any other gene, and it also had a substantial number of ERE, MBS, MYC and DRE elements.