Genome-wide organization and expression profiling of the NAC transcription factor family in potato (Solanum tuberosum L.)

Abstract

NAC [no apical meristem (NAM), Arabidopsis thaliana transcription activation factor [ATAF1/2] and cup-shaped cotyledon (CUC2)] proteins belong to one of the largest plant-specific transcription factor (TF) families and play important roles in plant development processes, response to biotic and abiotic cues and hormone signalling. Our genome-wide analysis identified 110 StNAC genes in potato encoding for 136 proteins, including 14 membrane-bound TFs. The physical map positions of StNAC genes on 12 potato chromosomes were non-random, and 40 genes were found to be distributed in 16 clusters. The StNAC proteins were phylogenetically clustered into 12 subgroups. Phylogenetic analysis of StNACs along with their Arabidopsis and rice counterparts divided these proteins into 18 subgroups. Our comparative analysis has also identified 36 putative TNAC proteins, which appear to be restricted to Solanaceae family. In silico expression analysis, using Illumina RNA-seq transcriptome data, revealed tissue-specific, biotic, abiotic stress and hormone-responsive expression profile of StNAC genes. Several StNAC genes, including StNAC072 and StNAC101that are orthologs of known stress-responsive Arabidopsis RESPONSIVE TO DEHYDRATION 26 (RD26) were identified as highly abiotic stress responsive. Quantitative real-time polymerase chain reaction analysis largely corroborated the expression profile of StNAC genes as revealed by the RNA-seq data. Taken together, this analysis indicates towards putative functions of several StNAC TFs, which will provide blue-print for their functional characterization and utilization in potato improvement.

Keywords: Illumina RNA-seq; NAC transcription factor; abiotic stress; genome-wide analysis; potato.

Figure 1.

Chromosomal distribution of 105 potato NAC genes identified in this study. The chromosome number is indicated on the top of each chromosome. Values in parenthesis following each gene represent its position on the chromosome. Arrows pointing downward and upward represents forward and reverse orientation of the respective gene, respectively, on the chromosome. Sixteen clusters of StNAC genes are indicated in boxes. Grey bars on chromosome 1, 2, 5 and 12 represent known gaps in the chromosome assembly.

Figure 2.

Depiction of segmentally duplicated StNAC genes on 12 potato chromosomes. Grey lines indicate collinear blocks in whole potato genome, and black lines indicate duplicated StNAC gene pairs.

Figure 3.

Phylogenetic relationship and conserved motif compositions of StNAC proteins. (A) Multiple sequence alignment of 136 full-length StNAC proteins was done using ClustalW2, and the phylogenetic tree was constructed using MEGA5.05 by the Neighbor-joining method with 1000 bootstrap replicates. The tree was divided into 12 phylogenetic subgroups designated as I to XII marked with different colour backgrounds. (B) Schematic representation of the conserved motifs in the StNAC proteins as revealed by MEME analysis. Grey lines represent the non-conserved sequences, and each motif is represented by a box numbered at the bottom. The length of protein can be estimated using the scale at the bottom.

Figure 4.

Phylogenetic tree of NAC proteins of potato, Arabidopsis and rice. Multiple sequence alignment of full-length NAC proteins was done using ClustalW2, and the phylogenetic tree was constructed using MEGA5.05 by the Neighbor-joining method with 1000 bootstrap replicates. The tree was divided into 18 phylogenetic subgroups, designated as NAC-a to NAC-r. Members of potato, Arabidopsis and rice were denoted by triangle, circle and diamond respectively. Subgroup NAC-q represents the TNAC subgroup, which seems restricted to Solanaceae.

Figure 5.

Membrane-bound potato NAC proteins. (A) Protein structure of membrane-bound NAC TFs. The highly conserved NAM domain is shown at the N-terminal of the proteins. α-helical TMs located at the C-terminal are shown as open box. The number of total amino acid residues in each protein is shown at the right side of each protein structure. (B) Phylogenetic relationship of membrane-bound NAC proteins of potato with that of Arabidopsis and rice. Multiple sequence alignment of full-length NAC MTF proteins was done using ClustalW2, and the phylogenetic tree was constructed using MEGA5.05 by the Neighbor-joining method with 1000 bootstrap replicates. The tree was divided into five phylogenetic subgroups designated as I to V. The scale at the bottom represents relative divergence of the sequences examined, and bootstrap values are displayed next to the branch.

Figure 6.

Heat map representation and hierarchical clustering of StNAC genes across different tissues and developmental stages. The Illumina RNA-seq data were reanalyzed, and the FPKM values were log₂ transformed and heat map generated using TIGR MeV v4.1.1 software. Bar at the bottom represents log2 transformed values, thereby values 0, 1.5 and 4.0 represent low, intermediate and high expression, respectively.

Figure 7.

Heat map representation and hierarchical clustering of StNAC genes during (A) abiotic stress, (B) biotic stress and (C) hormone treatments. The Illumina RNA-seq data were reanalyzed, and the relative expression was calculated with respect to respective control (untreated) samples. Heat maps were generated using the TIGR MeV v4.1.1 software. Bar at the bottom of each heat map represents relative expression values, thereby values 0, 1.0 and 2.0 represent downregulated, unaltered and upregulated expression, respectively.

Figure 8.

The relative expression ratio of 24 representative StNAC genes in young leaf (YL), old leaf (OL), stem and root tissues of potato determined using qRT-PCR. Relative expression ratios in different tissue samples have been calculated with reference to tissue sample in which the respective transcript exhibited the lowest expression. The relative expression values were log₁₀ transformed. qRT-PCR data were normalized using potato elongation factor 1-α gene. The name of the gene is written on the top of each bar diagram. (Error bars indicate standard deviation.)

Figure 9.

The relative expression ratio of 16 representative StNAC genes analysed by qRT-PCR under stress treatments for 4 h (grey bars) and 24 h (black bars). The relative expression ratio of each gene was calculated relative to its expression in control sample. qRT-PCR data were normalized using potato elongation factor 1-α gene. The name of the gene is written on the top of each bar diagram. (Error bars indicate standard deviation.)