Identification of the NAC Transcription Factor Family during Early Seed Development in Akebia trifoliata (Thunb.) Koidz

Abstract

This study aimed to gain an understanding of the possible function of NACs by examining their physicochemical properties, structure, chromosomal location, and expression. Being a family of plant-specific transcription factors, NAC (petunia no apical meristem and Arabidopsis thaliana ATAF1, ATAF2, and CUC2) is involved in plant growth and development. None of the NAC genes has been reported in Akebia trifoliata (Thunb.) Koidz (A. trifoliata). In this study, we identified 101 NAC proteins (AktNACs) in the A. trifoliata genome by bioinformatic analysis. One hundred one AktNACs were classified into the following twelve categories based on the phylogenetic analysis of NAC protein: NAC-a, NAC-b, NAC-c, NAC-d, NAC-e, NAC-f, NAC-g, NAC-h, NAC-i, NAC-j, NAC-k, and NAC-l. The accuracy of the clustering results was demonstrated based on the gene structure and conserved motif analysis of AktNACs. In addition, we identified 44 pairs of duplication genes, confirming the importance of purifying selection in the evolution of AktNACs. The morphology and microstructure of early A. trifoliata seed development showed that it mainly underwent rapid cell division, seed enlargement, embryo formation and endosperm development. We constructed AktNACs co-expression network and metabolite correlation network based on transcriptomic and metabolomic data of A. trifoliata seeds. The results of the co-expression network showed that 25 AtNAC genes were co-expressed with 233 transcription factors. Metabolite correlation analysis showed that 23 AktNACs were highly correlated with 28 upregulated metabolites. Additionally, 25 AktNACs and 235 transcription factors formed co-expression networks with 141 metabolites, based on correlation analysis involving AktNACs, transcription factors, and metabolites. Notably, AktNAC095 participates in the synthesis of 35 distinct metabolites. Eight of these metabolites, strongly correlated with AktNAC095, were upregulated during early seed development. These studies may provide insight into the evolution, possible function, and expression of AktNACs genes.

Keywords: Akebia trifoliata; NAC family; early seed development; metabolites; transcription factor.

Figure 1

Phylogenetic tree analysis of NAC protein in A. trifoliata (101), grape (71), rice (141), and Arabidopsis thaliana (115). We used the neighbour-joining method to construct the phylogenetic tree, with parameters set to the P-distance, pairwise deletion, and bootstrap value of 1000 replicates, and other parameters by default. The blue, purple, orange, and bottle green represent the NAC proteins of A. trifoliata, Arabidopsis thaliana, grape, and rice respectively.

Figure 2

The conservation of AktNAC amino acid residues was analyzed. The depth of color is used to represent conservation. A darker color indicates a higher conservation of amino acids or nucleotides at that position in the aligned sequences. A, B, C, D, and E are the five subdomains of the NAC domain. The x-axis represents the positions of amino acids, while the y-axis indicates the frequency of each amino acid at a specific position appears in a particular position. Different colors are used to represent different types of amino acids. “N” and “C” typically represent the amino terminus (N-terminus) and carboxyl terminus (C-terminus), respectively.

Figure 3

The chromosome locations of AktNACs genes. The chromosome numbers were indicated at the top of each chromosome. Red indicates tandem-duplicated genes. The location was drafted using Perl 5, version 18, subversion 4 (v5.18.4).

Figure 4

The seed development process of A. trifoliata. (A) Seed microstructure was observed under a laser confocal microscope. Scale bars: 200 µm. SC: Seed coat, EM: Embryo, EN: endosperm. (B) Seed development morphological changes was observed by SMZ800 stereomicroscope. Scale bars: 5 mm. 30DAF, 50DAF, 70DAF, and 150DAF represent 30 days, 50 days, 70 days, and 150 days after flowering, respectively.

Figure 5

The expression levels of AktNACs in early seeds at different developmental stages. Each column in the figure represents a sample and each row represents a gene, normalized by gene expression FPKM values for the rows using the z-score method.

Figure 6

Co-expression network and enrichment analysis of AktNACs during early seed development of A. trifoliata. Co-expression network of AktNAC genes constructed based on transcriptome data during early seed development. Diamonds indicates AktNAC genes, round indicated transcription factors, and color shade indicates the number of associated genes.

Figure 7

Co-expression network analysis of AktNACs and metabolites. Different shapes and colors represent different substances.

Figure 8

Metabolite content in the co-expression network of AktNACs and metabolites. Error bars denote the standard deviation of three replicates (* p < 0.05, ** p < 0.01).

Figure 9

Co-expression of AktNACs, TFs and compounds.

Figure 10

The interaction network of Arabidopsis thaliana NAC homologs proteins. The purple dashed oval indicated the Arabidopsis thaliana homologous genes to the A. trifoliata NAC protein, and the AktNACs were shown in purple below the Arabidopsis thaliana protein.