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Comparative Study
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Comparative Genomic Analysis of the WRKY III Gene Family in Populus, Grape, Arabidopsis and Rice

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Comparative Study

Comparative Genomic Analysis of the WRKY III Gene Family in Populus, Grape, Arabidopsis and Rice

Yiyi Wang et al. Biol Direct.

Abstract

Background: WRKY III genes have significant functions in regulating plant development and resistance. In plant, WRKY gene family has been studied in many species, however, there still lack a comprehensive analysis of WRKY III genes in the woody plant species poplar, three representative lineages of flowering plant species are incorporated in most analyses: Arabidopsis (a model plant for annual herbaceous dicots), grape (one model plant for perennial dicots) and Oryza sativa (a model plant for monocots).

Results: In this study, we identified 10, 6, 13 and 28 WRKY III genes in the genomes of Populus trichocarpa, grape (Vitis vinifera), Arabidopsis thaliana and rice (Oryza sativa), respectively. Phylogenetic analysis revealed that the WRKY III proteins could be divided into four clades. By microsynteny analysis, we found that the duplicated regions were more conserved between poplar and grape than Arabidopsis or rice. We dated their duplications by Ks analysis of Populus WRKY III genes and demonstrated that all the blocks were formed after the divergence of monocots and dicots. Strong purifying selection has played a key role in the maintenance of WRKY III genes in Populus. Tissue expression analysis of the WRKY III genes in Populus revealed that five were most highly expressed in the xylem. We also performed quantitative real-time reverse transcription PCR analysis of WRKY III genes in Populus treated with salicylic acid, abscisic acid and polyethylene glycol to explore their stress-related expression patterns.

Conclusions: This study highlighted the duplication and diversification of the WRKY III gene family in Populus and provided a comprehensive analysis of this gene family in the Populus genome. Our results indicated that the majority of WRKY III genes of Populus was expanded by large-scale gene duplication. The expression pattern of PtrWRKYIII gene identified that these genes play important roles in the xylem during poplar growth and development, and may play crucial role in defense to drought stress. Our results presented here may aid in the selection of appropriate candidate genes for further characterization of their biological functions in poplar.

Figures

Fig. 1
Fig. 1
Chromosomal location of WRKY III genes. The distribution of WRKY III genes among the chromosomes in each species is diverse. The chromosome number is indicated at the top of each chromosome
Fig. 2
Fig. 2
Phylogenetic tree of full-length WRKY III proteins from Populus, grape, Arabidopsis and rice. The tree was constructed using the neighbor-joining (NJ) method with MEGA 6.0. Dicotyledonous (Populus, grape and Arabidopsis) and monocotyledonous (rice) WRKY III proteins are marked with colored dots. The tree was also divided into four shared clades (clades 1–4) according to the bootstrap support and evolutionary distances. The gene names are listed in Additional file 1: Table S1
Fig. 3
Fig. 3
Phylogenetic relationship of WRKY III proteins and the exon-intron structure of WRKY III genes From Populus, grape, Arabidopsis and rice. Left panel: an unrooted phylogenetic tree constructed using MEGA 6.0 by the N-J method. Clades of WRKY III genes (1–4) are highlighted with different colored backgrounds. Right panel: exon-intron structure. The exons and introns are indicated by green rectangles and thin lines, respectively. The untranslated regions (UTRs) are indicated by thick blue lines
Fig. 4
Fig. 4
Distribution of conserved motifs in the WRKY III family members. All motifs were identified by MEME using the complete amino acid sequences of 57 Populus, grape, Arabidopsis and rice WRKY III proteins documented in Fig. 4. Names of all members among the defined gene clusters and combined P-values are shown on the left side of the figure, and motif sizes are indicated at the bottom of the figure. The positions of zn-finger domains predicted by the SMART tool. Database are indicated by vertical tick marks below each protein model. The different-colored boxes represent different motifs and their position in each WRKY III sequence. The length of protein can be estimated using the scale at the bottom. For details of the motifs see Additional file 1: Table S1
Fig. 5
Fig. 5
Extensive microsynteny of WRKY III regions across Populus, Grape, Arabidopsis and Rice. Populus chromosomes labeled Ptr, are indicated by rose red boxes. The Grape, Arabidopsis and Rice chromosomes, shown in different colors, are labeled Vv, At and Os, respectively. Numbers along each chromosome box indicate sequence lengths in megabases. The whole chromosomes of these four species, harboring WRKY regions, are shown in a circle. Black lines represent the syntenic relationships between WRKY regions
Fig. 6
Fig. 6
Microsynteny related to WRKY III families in (a) Populus; (b) grape; (c) Arabidopsis; (d) rice. a, b, c, d: The genomic fragments are represented by a series of triangles that represent a gene in a family and its flanking genes. The genes in the same fragment show the same color, except the gene in a family that is shaded by a black triangle. The triangle also indicates the gene’s orientation. A gray line connects the homologous genes on two fragments
Fig. 7
Fig. 7
Microsynteny related to WRKY III families in (a) clade 1; (b) clade 2; (c) clade 3; (d) clade 4. a, b, c, d: The genomic fragments are represented by a series of triangles that represent a gene in a family and its flanking genes. The genes in the same fragment show the same color, except the gene in a family that is shaded by a black triangle. The triangle indicates the gene’s orientations. A gray line connects the homologous genes on two fragments
Fig. 8
Fig. 8
Scatter plots of the Ka/Ks ratios of duplicated WRKY III genes in Populus. The Y- and X-axes denote the Ka/Ks ratio and synonymous distance for each pair, respectively
Fig. 9
Fig. 9
Sliding window plots of representative duplicated WRKY III genes in Populus. As shown in the key, the gray blocks indicate the positions of the WRKY domain. The window size was 150 bp, and the step size was 9 bp
Fig. 10
Fig. 10
qRT-PCR expression levels of selected PtrWRKY genes following SA (100uM), and different tissues. The Y-axis indicates the relative expression levels; 0, 1, 3, 6, 9, 12, and 24 (X-axis) indicate hours of treatment. Mean values and standard deviations (SDs) were obtained from three biological and three technical replicates. a Expression patterns of WRKY III genes from Populus in various tissues. R, roots; YL, young leaves; ML, mature leaves; ST, stems; X, xylem; Phl, phloem. b Expression levels of selected PtrWRKY genes under SA treatment. Horizontal discontinuous lines marks the 1.0 value
Fig. 11
Fig. 11
qRT-PCR expression levels of selected PtrWRKY genes following PEG-6000 (25 %) treatment, and ABA (100uM) treatments. The Y-axis indicates the relative expression levels; 0, 1, 3, 6, 9, 12, and 24 (X-axis) indicate hours of treatment. Mean values and standard deviations (SDs) were obtained from three biological and three technical replicates. a Expression levels of selected PtrWRKY genes under PEG treatment. b Expression levels of selected PtrWRKY genes under ABA treatment. Horizontal discontinuous lines marks the 1.0 value

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