Genome-Wide Identification and Analysis of the NAC Transcription Factor Gene Family in Garden Asparagus (Asparagus officinalis)

Abstract

As a large plant-specific gene family, the NAC (NAM, ATAF1/2, and CUC2) transcription factor is related to plant growth, development, and response to abiotic stresses. Although the draft genome of garden asparagus (Asparagus officinalis) has been released, the genome-wide investigation of the NAC gene family is still unavailable. In this study, a total of 85 A. officinalis NAC genes were identified, and a comprehensive analysis of the gene family was performed, including physicochemical properties, phylogenetic relationship, chromosome localization, gene structure, conserved motifs, intron/exon, cis-acting elements, gene duplication, syntenic analysis, and differential gene expression analysis. The phylogenetic analysis demonstrated that there were 14 subgroups in both A. officinalis and Arabidopsis thaliana, and the genes with a similar gene structure and motif distribution were clustered in the same group. The cis-acting regulatory analysis of AoNAC genes indicated four types of cis-acting elements were present in the promoter regions, including light-responsive, hormone-responsive, plant-growth-and-development-related, and stress-responsive elements. The chromosomal localization analysis found that 81 NAC genes in A. officinalis were unevenly distributed on nine chromosomes, and the gene duplication analysis showed three pairs of tandem duplicated genes and five pairs of segmental duplications, suggesting that gene duplication is possibly associated with the amplification of the A. officinalis NAC gene family. The differential gene expression analysis revealed one and three AoNAC genes that were upregulated and downregulated under different types of salinity stress, respectively. This study provides insight into the evolution, diversity, and characterization of NAC genes in garden asparagus and will be helpful for future understanding of their biological roles and molecular mechanisms in plants.

Keywords: Asparagus officinalis; NAC transcription factor; gene duplication; gene family; genome-wide analysis; salinity stress; syntenic analysis.

Figure 1

Phylogenetic tree of NAC genes between A. officinalis and A. thaliana. The AoNAC and AtNAC genes are indicated with red and black fonts, respectively. They are divided into 14 subgroups according to the subgroups of Arabidopsis and represented by different colors. The phylogenetic tree was compiled by the Maximum Likelihood (ML) method, with 1000 bootstrap replicates.

Figure 2

Phylogenetic relationship, conserved motifs, and gene structure of AoNAC genes. (A) An unrooted phylogenetic tree was constructed by using the ML method with 1000 bootstrap replicates based on AoNAC protein full-length sequences. (B) The conserved AoNAC protein motifs were predicted by the MEME program. Different colored boxes represent different motifs, and the black lines represent non-conserved sequences. The scale bar is 200 amino acids. (C) The intron/exon structures of AoNAC genes were displayed by using Gene Structure Display Server 2.0 program. The black line represents introns, and the green box represents exons. The intron and exon scale bars are 10 and 1 kb, respectively.

Figure 3

(A) Physical distribution of AoNAC genes on 9 chromosomes (Chr01–Chr09) and an undefined chromosome (Un). Vertical bars represent the chromosome of A. officinalis. The scale is in 15 Mb. (B) Number of AoNAC subgroups on each chromosome.

Figure 4

The cis-acting elements analysis of AoNAC genes. (A) Based on the promoter 2000 bp sequences of 85 AoNAC genes, we analyzed the light-responsive, hormone-responsive, plant-growth-and-development-related, anaerobic inducibility, low-temperature responsive, drought inducibility, defense and stress-responsive, anoxic-specific inducibility, and wound-responsive cis-elements. Different colors represent different cis-acting elements. (B) The number of AoNAC genes in the four types (including six subtypes). (C) The number of the various cis-elements in the stress-responsive element is presented in the bar chart.

Figure 5

Schematic diagram of the duplication patterns of the AoNAC genes. The blue lines indicate segmental duplications of AoNAC gene pairs, and the red lines indicate tandem duplications of AoNAC gene pairs.

Figure 6

Schematic diagram of syntenic analysis. Synteny of the AoNAC genes with the NAC genes of A. thaliana (A), S. indicum (B), and A. comosus (C) was visualized by MCScanX and TBtools software. The gray lines between the chromosomes of the two species indicate all synteny blocks, and the red lines represent the synteny of their NAC genes.

Figure 7

Venn diagram of the identical and different NAC genes among A. officinalis, A. thaliana, S. indicum, and A. comosus.

Figure 8

Differential gene expression of AoNAC genes under different types of salinity stress: (A) non-inoculated A. officinalis plants without salinity stress (NI) and non-inoculated A. officinalis plants subjected to salinity stress (NI + S); (B) inoculated A. officinalis plants without salinity stress (AMF) and inoculated A. officinalis plants subjected to salinity stress (AMF + S). Heatmaps are based on the log10-transformed RPKM values. Red represents a high expression level, and blue represents a low expression level. Volcano plots are based on the significantly adjusted p-value (padj) < 0.05 and an absolute value of log2FC (log of fold change) > 1.