Insight into the bZIP Gene Family in Solanum tuberosum: Genome and Transcriptome Analysis to Understand the Roles of Gene Diversification in Spatiotemporal Gene Expression and Function

Abstract

The basic region-leucine zipper (bZIP) transcription factors (TFs) form homodimers and heterodimers via the coil-coil region. The bZIP dimerization network influences gene expression across plant development and in response to a range of environmental stresses. The recent release of the most comprehensive potato reference genome was used to identify 80 StbZIP genes and to characterize their gene structure, phylogenetic relationships, and gene expression profiles. The StbZIP genes have undergone 22 segmental and one tandem duplication events. Ka/Ks analysis suggested that most duplications experienced purifying selection. Amino acid sequence alignments and phylogenetic comparisons made with the Arabidopsis bZIP family were used to assign the StbZIP genes to functional groups based on the Arabidopsis orthologs. The patterns of introns and exons were conserved within the assigned functional groups which are supportive of the phylogeny and evidence of a common progenitor. Inspection of the leucine repeat heptads within the bZIP domains identified a pattern of attractive pairs favoring homodimerization, and repulsive pairs favoring heterodimerization. These patterns of attractive and repulsive heptads were similar within each functional group for Arabidopsis and S. tuberosum orthologs. High-throughput RNA-seq data indicated the most highly expressed and repressed genes that might play significant roles in tissue growth and development, abiotic stress response, and response to pathogens including Potato virus X. These data provide useful information for further functional analysis of the StbZIP gene family and their potential applications in crop improvement.

Keywords: RNA sequence analysis; Solanum tuberosum; bZIP transcription factor family; drought stress response; gene regulatory networks; genomic survey; heat stress response; potato genome; potato transcriptome; stress physiology and genetics; tuber genome; virus stress response.

Figure 1

Phylogenetic tree constructed using PhyML method (v1.5) and Seaview (v4.7) contains the Arabidopsis and potato basic region-leucine zipper (bZIP) proteins. iTOL (v4) was used to visualize the output, and the diagrams were compiled, labeled, and color-coded using Adobe Photoshop CC (2017). Branch support was assessed with 1000 ultrafast bootstrap approximation and SH-aLRT branch test with 1000 replicates. The legend contains the functional groups assigned according to [8,10]. The bootstrap scores are provided at each node.

Figure 2

Distribution and segmental duplication of StbZIP gene family members in S. tuberosum chromosomes. The location of StbZIP genes on twelve chromosomes. The scale indicates the genome size of S. tuberosum (Mb). Bold lines connect paralogs, and segmental duplication was identified using MCScanX methods. The box identifies two genes representing a tandem duplication event.

Figure 3

Exon/intron organization of StbZIP genes depicted for each group. The legend in Figure 2 explains the color scheme highlighting gene names. Boxes represent exons, and lines represent introns. The numbers “0”, “1”, and “2” identify the splicing phases.

Figure 4

Analysis of amino acid composition for heptads. (A) Depiction of bZIP transcription factor binding DNA and dimeric heptads. (B) Pie chart presenting the frequency (%) of amino acids in the g, a, d, e position of heptads from the 80 StbZIP proteins.

Figure 5

Conserved motifs identified using MEME software suite. All factors, except six, were predicted to have one bZIP domain (p < 0.001). The conserved basic N-X (7)-R/K motif is colored red, and adjoining leucine-rich motifs are shaded blue. Figure S1 provides the details for all motifs in this figure.

Figure 6

Distribution and frequency of cis-regulatory elements (CREs) on StbZIP promoters. Promoter sequences representing 2000 bp from the transcription start site of StbZIP genes were extracted from the DM1-3 516 R44 v 6.1 assembly at Spud DB as described in Materials and Methods Section 3.4. (A) The grid provides the number of sites that contain the CREs involved in the development, hormone signaling, and stress responses. Shades of green, red, and blue point to CREs that are highly represented (dark-colored), moderately represented (medium colored), once or twice represented (light color), or not present (white color). (B) Bar graph demonstrates the distribution frequency of CREs involved in the development, hormone signaling, and stress responses across the 2000 bp sequences analyzed for each StbZIP.

Figure 7

Heat map representation and hierarchical clustering of StbZIP transcript levels across different tissues/organs/developmental stages. The independent gene expression datasets used for this study are provided in Supplementary Table S3. The expression values map to a color gradient from low (blue) to high (red) expression. Values are present using the log2 scale.

Figure 8

Heat map and hierarchical clustering of StbZIP transcript levels in response to the hormone, abiotic stress, and biotic stress. The independent gene expression datasets are detailed in Supplementary Table S3. The relative changes in bZIP gene expression levels were obtained following treatment with: (A) benzyl amino purine (BAP), abscisic acid (ABA), indole-3-acetic acid (IAA), or gibberellic acid (GA3); (B) heat, water stress, salt, mannitol, and wounding; (C) P. infestans, D,L-β-aminobutyric acid (BABA) or Benzo[1–3]thiadiazole-7-carbothionic acid (BTH). The log2 fold change values map to a color gradient from low (blue) to high (red) expression.

Figure 9

Heat map showing StbZIP transcript levels at 2 and 3 dpi with PVX. The transcriptomic analysis is BioPRoject PRJNA679820. The expression values (log2) map to a color gradient from low (blue) to high (red) expression. All values expressed in the heat map were significant (p <0.05).