Genome-wide characterization and expression profiling of NAC transcription factor genes under abiotic stresses in radish (Raphanus sativus L.)

Abstract

NAC (NAM, no apical meristem; ATAF, Arabidopsis transcription activation factor and CUC, cup-shaped cotyledon) proteins are among the largest transcription factor (TF) families playing fundamental biological processes, including cell expansion and differentiation, and hormone signaling in response to biotic and abiotic stresses. In this study, 172 RsNACs comprising 17 membrane-bound members were identified from the whole radish genome. In total, 98 RsNAC genes were non-uniformly distributed across the nine radish chromosomes. In silico analysis revealed that expression patterns of several NAC genes were tissue-specific such as a preferential expression in roots and leaves. In addition, 21 representative NAC genes were selected to investigate their responses to heavy metals (HMs), salt, heat, drought and abscisic acid (ABA) stresses using real-time polymerase chain reaction (RT-qPCR). As a result, differential expressions among these genes were identified where RsNAC023 and RsNAC080 genes responded positively to all stresses except ABA, while RsNAC145 responded more actively to salt, heat and drought stresses compared with other genes. The results provides more valuable information and robust candidate genes for future functional analysis for improving abiotic stress tolerances in radish.

Keywords: Abiotic stress; Expression profiling; Genome-wide analysis; NAC; Radish; Transcription factor.

Figure 1. Comparative genomic analysis of NAC transcription factors of radish with other species.

There were 17 vascular plants and four lower plants. *Megabases.

Figure 2. Phylogenetic tree among the NAC TFs of radish, Arabidopsis, and rice.

Multiple sequence alignment of full-length NAC proteins was done using ClustalW and the phylogenetic tree was constructed using MEGA6 by the neighbor-joining method with bootstrap replicates, the tree was divided into two major groups (A and B). Group A was further subdivided in into nine phylogenetic subgroups designated A1–A9. Phylogenetic subgroups in-group B were designated as B1 to B10. The different sub-families are marked with different colored backgrounds. The colored shapes next to names represent radish (green circles), Red hexagons (Arabidopsis) and purple hexagons (rice).

Figure 3. Phylogenetic classification and motif analysis of NAC transcription factors of radish.

Multiple sequence alignment of 172 full-length RsNAC proteins was done using ClustalW, and the phylogenetic tree was mapped using MEGA6 by the neighbor-joining method with 1,000 bootstrap replicates. MEME analysis explored conserved motifs of the RsNAC proteins. Gray lines illustrate the non-conserved sequences and each motif is showed by the colored boxes described at the bottom. The different color background indicated different groups. (A and C) multiple alignment, (B and D) conserved motifs.

Figure 4. Phylogenetic tree and Exon/Intron structure of 172 RsNAC genes.

Yellow boxes and black lines represent exons and introns, respectively, while the blue boxes represent the untranslated regions. (A and C) multiple alignment, (B and D) gene structure composition.

Figure 5. Membrane-associated RsNAC subfamily.

Protein structure of membrane-bound NAC TFs poses indicating a conserved NAM domain located at the N-terminal of the proteins, TMs (transmembrane motifs) located at the N-terminal as shown by the blue hexagon.

Figure 6. Chromosomal distribution of RsNAC genes on the genetic map.

(A) A pie chart is indicating the percentage of the anchored and unanchored RsNAC genes and their abundant distribution on each radish chromosome with indication of percentages of RsNACs located on each chromosome. (B) Graphical representation of locations for putative RsNAC genes on each radish chromosome. The vertical red lines on the right of each chromosome indicate tandem duplicated genes. R1–R9 indicates the chromosome numbers. The scale is in megabase.

Figure 7. Expression patterns of RsNAC genes under abiotic stresses treatments.

RT-qPCR was used to analyze the expression profiles of 21 RsNAC genes under cadmium, lead, heat, salt, PEG, and ABA stress. CK is an untreated seedling. The Actin gene was used as an internal control. (A–U) represents expression patterns of the 21 RNAC genes in response to abiotic stresses, respectively. Error bars were obtained from three technical replicates. Asterisks indicate the genes significantly up-regulated or suppressed under abiotic stresses by t-test (* P < 0.05, ** P < 0.01).