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, 20 (22)

Genome-Wide Characterization, Expression Profile Analysis of WRKY Family Genes in Santalum album and Functional Identification of Their Role in Abiotic Stress

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Genome-Wide Characterization, Expression Profile Analysis of WRKY Family Genes in Santalum album and Functional Identification of Their Role in Abiotic Stress

Haifeng Yan et al. Int J Mol Sci.

Abstract

WRKY proteins are a large superfamily of transcription factors that are involved in diverse biological processes including development, as well as biotic and abiotic stress responses in plants. WRKY family proteins have been extensively characterized and analyzed in many plant species, including Arabidopsis, rice, and poplar. However, knowledge on WRKY transcription factors in Santalum album is scarce. Based on S. album genome and transcriptome data, 64 SaWRKY genes were identified in this study. A phylogenetic analysis based on the structures of WRKY protein sequences divided these genes into three major groups (I, II, III) together with WRKY protein sequences from Arabidopsis. Tissue-specific expression patterns showed that 37 SaWRKY genes were expressed in at least one of five tissues (leaves, roots, heartwood, sapwood, or the transition zone), while the remaining four genes weakly expressed in all of these tissues. Analysis of the expression profiles of the 42 SaWRKY genes after callus was initiated by salicylic acid (SA) and methyl jasmonate (MeJA) revealed that 25 and 24 SaWRKY genes, respectively, were significantly induced. The function of SaWRKY1, which was significantly up-regulated by SA and MeJA, was analyzed. SaWRKY1 was localized in the nucleus and its overexpression improved salt tolerance in transgenic Arabidopsis. Our study provides important information to further identify the functions of SaWRKY genes and to understand the roles of SaWRKY family genes involved in the development and in SA- and MeJA-mediated stress responses.

Keywords: MeJA (methyl jasmonate); SA (salicylic acid); WRKY transcription factor; salt tolerance; sandalwood.

Conflict of interest statement

The authors declare that they have no conflict of interests.

Figures

Figure 1
Figure 1
Phylogenetic tree of WRKY proteins from Santalum album and Arabidopsis thaliana. The 64 SaWRKY proteins and representative Arabidopsis WRKY proteins were aligned by ClustalX 2.0, and the NJ tree was constructed using MEGA6.0 with 1000 bootstrap replicates.
Figure 2
Figure 2
Schematic diagram showing the phylogenetic relationship among conserved motifs of SaWRKY proteins and gene structure of SaWRKY genes. The phylogenetic tree was built on the basis of the full amino acids of 64 SaWRKY proteins whose conserved motifs were identified using MEME.
Figure 3
Figure 3
The 20 top Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathways of potential SaWRKY target genes with at least five W-boxes.
Figure 4
Figure 4
The expression patterns of 41 SaWRKY genes in five tissues of Santalum album. The heat map was generated based on the number of reads per kilobase of exon modelled per million mapped reads (RPKM) of transcriptome data.
Figure 5
Figure 5
Expression profiles of SaWRKY genes following SA and MeJA treatments. The expression of 42 SaWRKY genes was determined under SA (A) and MeJA (B) stimuli at three time points (0, 3, and 6 h) in callus. The heat map was generated based on log2-transformed count value from RT-qPCR data using R language. RT-qPCR data were collected from three biological replicates.
Figure 6
Figure 6
Expression of SaWRKY1 in different tissues of Santalum album. Relative expression levels of SaWRKY1 in four tissues were determined by RT-qPCR with three replicates. Values shown are means ± SE.
Figure 7
Figure 7
Subcellular localization assay of the SaWRKY1 protein.
Figure 8
Figure 8
Phenotypes of two lines of overexpression SaWRKY1 transgenic plants under salt stress. (A) Day 0. (B) Day 3 after irrigation with 300 mM NaCl. (C) Day 5 after second irrigation with 300 mM NaCl.
Figure 9
Figure 9
The chlorophyll content of two lines of overexpression SaWRKY1 transgenic plants under salt stress. All values are means ± SE from at least three independent biological replicates (8 seedlings per replicate). Double asterisk (**) indicates significance at p < 0.01 for each component between wild type and transgenic lines using Duncan’s multiple range test. (A) Day 0. (B) Day 3 after irrigation with 300 mM NaCl. (C) Day 5 after second irrigation with 300 mM NaCl.

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