Fig 1.
Phylogenetic tree of ZF-HDs predicted in wheat and those previously identified in maize, rice, and Arabidopsis.
All sequences were aligned using ClustalW2, and the phylogenetic tree was constructed using the Neighbor-joining (NJ) method with 1000 bootstrap replications. Different group was distinguished by different color. ZF-HDs from different species are distinguished with different color dots.
Table 1.
Characterization of ZF-HD proteins in wheat.
Fig 2.
Phylogenetic tree, gene structure and motif analysis of wheat ZF-HD family.
(A) The phylogenetic tree of TaZF-HDs. The tree was created using the Neighbor-joining (NJ) method. (B) The exon-intron structure of the TaZF-HDs. Exon-intron structure analyses were conducted using the GSDS server. The untranslated regions (UTRs) are indicated by blue boxes, the exons are indicated by yellow boxes and the introns are indicated by black lines. (C) The motifs of TaZF-HDs were identified by MEME tool. Each motif is indicated with a specific color. (D) Tertiary structure prediction of TaZF-HD proteins.
Fig 3.
Analysis of cis-acting elements in the promoter sequences of TaZF-HD genes.
(A) Phylogenetic analysis of wheat ZF-HD family; (B) Cis-acting elements in the promoters of TaZF-HD genes. The different colors and numbers of grids indicate the numbers of different promoter elements. (C) The histograms of different colors represent the sum of the cis-acting elements in each category.
Fig 4.
Chromosomal locations, Ka/Ks analysis of TaZF-HDs and orthologous analysis between wheat and other species.
(A) Chromosomal locations of TaZF-HDs. The scale on the left indicates the length of the wheat chromosome, different colors of the genes represent different subfamily, red letters represent the genes of the ZHD subfamily, and the blue letters represent the genes of the MIF subfamily; Chr represents Chromosome, the number on the left indicates the length of the chromosome in megabase (Mb). (B) Ka/Ks values for duplicated TaZF-HD gene pairs. (C) Phylogeny of wheat and its genome ancestors. The phylogenetic tree was constructed using a Neighbor-joining (NJ) method. ZF-HDs from different species were distinguished with different color dots. (D) Homologous relationships between wheat and its three genome ancestors. The chromosomes of different species were shown in different color boxes. The numbers along each chromosome box indicate sequence lengths in Mb. Lines between two chromosomes represent the homologous relationships. (E) Synteny analysis of ZF-HD genes between wheat and four representative plant species. Including two dicotyledonous plants (Arabidopsis and tomato), and two monocotyledonous plants (rice and maize). Gray lines in the background indicate the collinear blocks within wheat and other plant genomes, while the red lines highlight the syntenic ZF-HD gene pairs.
Fig 5.
Protein interaction and miRNA targeting analysis.
(A) The picture of the regulatory network relationships between the putative miRNAs and their targeted wheat ZF-HD genes. Different colored circles represent different types, blue represents miRNA, purple indicates predicted miRNA target genes. (B) Interaction networks of the wheat ZF-HD proteins.
Fig 6.
Multi-conditional transcriptome analysis of TaZF-HDs in different tissues and different biotic/abiotic stress environments.
The R package ‘pheatmap’ was used to generate the heat maps. (A) Transcript level of TaZF-HDs responding to abiotic stresses; (B) Transcript level of TaZF-HDs responding to biotic stresses. (C) Transcript level of TaZF-HDs in wheat different tissues and development stages.
Fig 7.
qRT-PCR analysis of TaZF-HD genes under biotic/abiotic stresses.
The data were analyzed by three independent repeats, and standard deviations were shown with error bars. The expression level of TaZF-HDs genes was drawn using Origin software. * indicates significant differences that compared with control at p < 0.05.
Fig 8.
Subcellular localization and verification of transcriptional autoactivation activity of TaMIF4-5D and TaZHD6-3B.
(A) Subcellular localization of TaMIF4-5D and TaZHD6-3B. The figure shows the fluorescence signal of GFP, the fluorescence signal after DAPI staining, the bright field and the fusion image, respectively. The fluorescent signal of GFP in the figure is used to mark the expression position of the fusion proteins. The fluorescent signal after DAPI staining is used to show the position of the cell nucleus. Scale bar = 50 μm. (B) Verification of TaMIF4-5D and TaZHD6-3B transcriptional autoactivation activity. The concentrations (cells/ml) of yeast transformants were adjusted to 106, 105,104,103, respectively.