Genome-wide identification and comprehensive analysis of the NAC transcription factor family in Sesamum indicum

Abstract

The NAM, ATAF1/2, and CUC2 (NAC) family constitutes a large family of plant-specific transcription factors, involved in many aspects of physiological processes and a variety of abiotic stresses. There is little information concerning the NAC family in Sesamum indicum. In this study, 87 sesame NAC genes were identified and phylogenetically clustered into 12 groups with Arabidopsis NAC genes. A total of 83 SiNAC genes were distributed non-randomly on the 16 linkage groups in sesame. Four and 49 SiNACs were found to be tandemly and segmentally duplicated, respectively. Expression profiles of SiNAC genes in different tissues (root, stem, leaf, flower, seed, and capsule) and in response to drought and waterlogging stresses by using RNA-seq data demonstrated that 23 genes were highly expressed in all tissues, 18 and 31 SiNACs respond strongly to drought and waterlogging stresses, respectively. In addition, the expression of 30 SiNAC genes distributed in different subgroups was analyzed with quantitative real-time RT-PCR under cold, osmotic, and salt stresses, revealed that their expression patterns vary in response to abiotic stresses. SiNAC genes displayed diverse expression patterns among the different tissues and stress treatments, suggested that their contribution to plant growth and development in sesame and multiple stress resistance in sesame. In this study, NAC transcription factors were analyzed in sesame and some specific candidate SiNAC genes in response to abiotic stress for functional study were identified. This study provides valuable information to deepen our understanding of the abiotic stress responses by NAC transcription factors in sesame.

Fig 1. Distribution of SiNAC genes on 16 linkage groups (LGs).

Vertical bars represent the LGs of sesame. The LG number is on the top of each LG. The scale is in 2 Mb.

Fig 2. Phylogenetic relationships, motif compositions, and gene structure of SiNAC TFs.

(A) The phylogenetic tree. The amino acid sequences of the SiNAC were aligned using ClustalX 2.1, and the phylogenetic tree was generated using MEGA 5.0 by the neighbor-joining method with 1000 bootstrap replicates. (B) Schematic representation of the conserved NAC proteins motifs from sesame elucidated by MEME. The colored boxes indicate the motifs. The black lines indicate the non-conserved sequences. The scale bar represents 100 aa. (C) Intron/exon structures of SiNAC genes. The black lines and green boxes indicate introns and exons, respectively. The scale bar represents 1.0 kb.

Fig 3. Phylogenetic tree of NAC proteins from sesame and Arabidopsis.

The amino acid sequences of the NAC proteins were aligned using ClustalX 2.1, and the phylogenetic tree was generated using the neighbor-joining method with 1000 bootstrap replicates in the MEGA 5.0. The red dots and green triangles represent sesame and reported Arabidopsis NACs, respectively.

Fig 4. Phylogenetic relationships of membrane-bound NACs of sesame, Arabidopsis, and O. sativa.

The full-length amino acid sequences of MTFs were aligned by Clustal X 2.1, and the phylogenetic tree was constructed using MEGA 5.0 by the neighbor-joining method with 1000 bootstrap replicates.

Fig 5. Expression patterns of SiNAC genes in different tissues.

(A) Hierarchical clustering of expression profile of SiNAC genes in different tissues. Log₁₀-based RPKM values were used to create the heat map with clustering. The relative signal intensity of RPKM values was represented The scale. (B) Number of genes specifically in each tissue. (C) An overview of SiNAC gene numbers in six tissues. (D) Number of genes: specific (1) or shared by 2, 3… tissues.

Fig 6. Expression profiles of SiNAC genes in roots of drought-tolerant (DT) and drought-sensitive (DS) cultivars after drought treatment.

d0 (control), d1, d2, and d3 indicated the samples harvested with the soil water content at 35%, 15%, 9%, and 6%, respectively. Log₂-transformed values of the relative expression levels of the SiNAC genes under drought stress were used to create the heat map. Changes in gene expression are shown in color as the scale.

Fig 7. Expression profiles of SiNAC genes in roots of waterlogging-tolerant (WT) and waterlogging-sensitive (WS) cultivars after waterlogging treatment.

Log₂-transformed values of the relative expression levels of the SiNAC genes under waterlogging stress were used to create the heat map. Changes in gene expression are shown in color as the scale.

Fig 8. Expression profiles of SiNAC genes in leaves upon exposure to various abiotic stresses.

14-day-old seedlings were treated to osmotic (15% PEG 6000), salt (150 mM NaCl), and cold (4°C) stresses. Relative expression levels of the SiNACs were analyzed by qRT-PCR, and the heat maps were created by log2-based fold-change values (mean of three biological replicates) (original data were shown in S5 Fig).