Genome-Wide Investigation of the NAC Transcription Factor Family in Apocynum venetum Revealed Their Synergistic Roles in Abiotic Stress Response and Trehalose Metabolism

Abstract

NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) are one of the most prominent plant-specific TF families and play essential roles in plant growth, development and adaptation to abiotic stress. Although the NAC gene family has been extensively characterized in many species, systematic analysis is still relatively lacking in Apocynum venetum (A. venetum). In this study, 74 AvNAC proteins were identified from the A. venetum genome and were classified into 16 subgroups. This classification was consistently supported by their gene structures, conserved motifs and subcellular localizations. Nucleotide substitution analysis (Ka/Ks) showed the AvNACs to be under the influence of strong purifying selection, and segmental duplication events were found to play the dominant roles in the AvNAC TF family expansion. Cis-elements analysis demonstrated that the light-, stress-, and phytohormone-responsive elements being dominant in the AvNAC promoters, and potential TFs including Dof, BBR-BPC, ERF and MIKC_MADS were visualized in the TF regulatory network. Among these AvNACs, AvNAC58 and AvNAC69 exhibited significant differential expression in response to drought and salt stresses. The protein interaction prediction further confirmed their potential roles in the trehalose metabolism pathway with respect to drought and salt resistance. This study provides a reference for further understanding the functional characteristics of NAC genes in the stress-response mechanism and development of A. venetum.

Keywords: NAC transcription factor; drought stress; salt stress; trehalose metabolism pathway.

Figure 1

The distribution of AvNACs on chromosomes. Heatmaps on chromosomes represent gene density, from low to high in blue and red, respectively. The gene pairs of tandem duplication are marked with pink connecting lines.

Figure 2

Collinearity analysis of the AvNAC genes. The blue box represents the chromosome, the lines inside the circle indicate collinear blocks within the A. venetum chromosome. The red lines indicate segment duplication events related to AvNAC genes. The blue lines inside the outer blocks indicate gene density.

Figure 3

Synteny analysis of NAC genes between A. venetum and six other plant species (A. thaliana, M. sativa, S. lycopersicum, O. sativa, Z. mays and M. sinensis). The gray lines in the background represent collinear relationships between A. venetum and six other species, while the cyan lines highlight the syntenic NAC gene pairs.

Figure 4

The phylogenetic tree of AvNAC proteins with AtNAC proteins in A. thaliana. The AvNACs are indicated by red color and italic, and the AtNACs are indicated by black color.

Figure 5

Phylogenetic tree, conserved motifs and gene structures of 74 AvNAC TFs. (A) Phylogenetic tree of 74 AvNAC TFs. (B) Conserved motifs in the 74 AvNAC proteins. Different colors represent different motifs. (C) Gene structures of AvNAC genes. Green and yellow boxes indicate the exons and untranslated regions (UTRs), and black lines indicate introns. Pink boxes highlight the NAM domains.

Figure 6

Analysis of cis-elements in AvNAC promoters. Statistics and categories of cis-elements in the promoter regions of AvNAC genes. The numbers of cis-acting element were indicated by numbers and a color gradient.

Figure 7

Expression patterns of AvNAC genes in different tissues (root, stem and leaf). Log₂ (FPKM + 1) values are indicated by the shade of the color, with darker colors representing larger values.

Figure 8

Relative expression of selected AvNAC genes in various tissues of A. venetum under drought stress (0, 5, 10, 20% PEG6000 treatment). (A) Leaf. (B) Stem. (C) Root.

Figure 9

Relative expression of selected AvNAC genes in various tissues of A. venetum under salt stress (0, 50, 100, 200 mM NaCl treatment). (A) Leaf. (B) Stem. (C) Root.

Figure 10

Protein-protein interaction of AvNAC proteins.

Figure 11

The putative transcription factor regulatory network analysis of AvNAC genes. (A) Interaction network of NAC genes and putative transcription factors in A. venetum. The node size represents the number of interactions. (B) The top 10 highly enriched and targeted AvNAC genes.