Comprehensive analysis of NAC transcription factors uncovers their roles during fiber development and stress response in cotton

Abstract

Background: Transcription factors operate as important switches of transcription networks, and NAC (NAM, ATAF, and CUC) transcription factors are a plant-specific family involved in multiple biological processes. However, this gene family has not been systematically characterized in cotton.

Results: Here we identify a large number of genes with conservative NAC domains in four cotton species, with 147 found in Gossypium arboreum, 149 in G. raimondii, 267 in G. barbadense and 283 in G. hirsutum. Predicted membrane-bound NAC genes were also identified. Phylogenetic analysis showed that cotton NAC proteins clustered into seven subfamilies and homologous protein pairs showed similar characteristics. Evolutionary property analysis revealed that purifying selection of NAC genes occurred between diploid and polyploid cotton species, and variation analysis showed GhNAC genes may have been subjected to selection and domestication. NAC proteins showed extensive transactivation and this was dependent on the C-terminus. Some development and stress related cis-elements were enriched in the promoters of GhNAC genes. Comprehensive expression analysis indicated that 38 GhNAC genes were candidates for involvement in fiber development, and 120 in stress responses. Gene co-expression network analysis revealed relationships between fiber-associated NAC genes and secondary cell wall (SCW) biosynthesis genes.

Conclusions: NAC genes were identified in diploid and tetraploid cotton, revealing new insights into their evolution, variation and homology relationships. Transcriptome analysis and co-expression network indicated roles for GhNAC genes in cotton fiber development and stress response, and NAC genes may prove useful in molecular breeding programmes.

Keywords: Cotton; Evolutionary analysis; Fiber development; NAC; Stress response; Transactivation.

Fig. 1

The conserved domain and phylogenetic analysis of NAC proteins in cotton. a Schematic representation of NAC proteins. (i) Typical NAC protein with a highly conserved NAC domain (A-E subdomains) at the N-terminal; (ii) Membrane-bound NAC transcription factors with a transmembrane motif (TM, blue square) at the C-terminal; (iii) Membrane-bound NAC transcription factors with a transmembrane motif (TM, blue square) at the N-terminal. b Phylogenetic tree of NAC proteins in four cotton species. G. hirsutum (red circle), G. barbadense (green square), G. raimondii (blue triangle), G. arboreum (magenta triangle)

Fig. 2

Evolutionary analysis of NAC proteins in cotton. a Statistical analysis of exon numbers of NAC genes in G. hirsutum. b The ratio of non-synonymous to synonymous substitutions (d_N/d_S) of NAC genes in inter-genomic (At Dt) and intra-genomic (A2 At and D5 Dt). c The SNP density of GhNAC genes in Upland cotton populations. The gene body, 2 kb upstream and 2 kb downstream sequences of each GhNAC gene were selected for analysis

Fig. 3

Cis-element analysis of NAC genes in G. hirsutum. Cis-element analysis of GhNAC gene promoter. The 1.5-kb upstream sequence of the start codon of each NAC gene were analyzed by the PLANTCARE database

Fig. 4

Expression analysis of GhNAC genes during fiber development. a The number of highly expressed NAC genes (FPKM ≥20) in different tissues/organs. The expression of NAC family members in different tissues/organs were investigated using transcriptome datasets of G. hirsutum. b Cluster analysis of the GhNAC expression patterns in fiber development. The grey lines indicate the expression levels of genes. c Heat-map of 38 highly and differently expressed NAC genes in fiber development based on transcriptome datasets. The expression values (FPKM) were normalized using Genesis. d The bias expression of fiber related NAC genes in G. hirsutum. e Two gene pairs showed discordant expression changes between G. hirsutum and G. barbadense

Fig. 5

The qRT-PCR and co-expression network analysis of fiber-related GhNAC genes. a Expression analysis of the selected GhNAC genes in fiber development by qRT-PCR. The GhUBQ7 (GenBank accession number: DQ116441) was used as the internal control to calculate and normalize the expression levels. Bars represent means ± standard error (n = 3). The orange lines represent the transcriptome data (FPKM). b The co-expression network with gene expression view of GhNAC genes in fiber development. The gray lines between two nodes indicate co-expression relationships. The purple solid circles represent overlapped genes. c GO enrichment analysis of co-expression genes with GhNAC33, GhNAC125, GhNAC193, GhNAC280

Fig. 6

Expression and co-expression network analysis of GhNAC genes under abiotic stress. a The expression patterns of 120 stress-responsive GhNAC genes under abiotic stress. b Venn diagram showing overlap of different stress-responsive GhNAC genes. c The co-expression network of GhNAC genes with DEGs in the abiotic stress treatment. The gray lines between two nodes indicate co-expression relationship. The purple solid circles represent overlapped genes. d The expression of selected GhNAC genes under salt stress (200 mM NaCl) in leaves by qRT-PCR. e The expression of selected GhNAC genes under osmotic stress (15% PEG) in leaves by qRT-PCR. The GhUBQ7 (GenBank accession number: DQ116441) was used as the internal control. Bars represent means ± standard error (n = 3)

Fig. 7

Transactivation analysis of GhNAC genes. a The transcriptional activity of selected NAC proteins in cotton embryogenic callus protoplasts. Renilla LUC was used as the internal control. Three independent experiments were performed. Asterisks indicate significant differences, ** P ≤ 0.01, student’s t-test. The LUC/Renilla Ratio of each gene compared with control (GAL4DB), respectively. b Transactivation assay of NAC proteins in yeast strain Y2H. The transactivation activities were determined by the growth on SD medium without Tryptophane (Trp) and added X-α-Gal (SD-Trp + X-α-Gal). The photograph was taken after 36 h incubation. Positive control: pGBKT7–53; negative control: pGBKT7-Lam. c Transactivation assay of GhNAC158. Full: the full length of GhNAC158, BD: NAC domain (Binding Domain) of GhNAC158, AD: the C terminal (Activation Domain) of GhNAC158. d Transactivation assay of GhNAC157. △TM: the transmembrane motif (TM) deleted form of GhNAC157

Fig. 8

Model of gene location and functional divergence of fiber development related and stress responsive NAC genes in G. hirsutum. a Gene location of fiber development related and stress responsive NAC genes in G. hirsutum. There are 12 genes (GhNAC9, GhNAC10, GhNAC33, GhNAC34, GhNAC56, GhNAC57, GhNAC59, GhNAC151, GhNAC225, GhNAC226, GhNAC275, GhNAC283) have no distinct chromosome location and could not be accurately mapped. Pink fonts: stress responsive NAC genes; green fonts: fiber development related NAC genes; purple fonts: the common genes involved in these two biological processes. Lines represent homologous genes that are distributed in syntenic blocks. b The NAC genes were analyzed at the transcriptional (tissues/Organs expression and responsiveness to abiotic stress) and protein (transactivation activity) levels. Thirty-eight fiber development related and 120 stress responsive GhNAC genes were identified in this study. There are 27 genes involved in these two biological processes