Identification and Functional Divergence Analysis of WOX Gene Family in Paper Mulberry

Abstract

The WOX (WUSCHEL-related homeobox) is a plant-specific transcription factor involved in plant development and stress response. However, few studies have been reported on the WOX gene in woody plants. In this study, 10 BpWOX genes were isolated from paper mulberry by RACE-PCR and categorized into three clades through phylogenetic analysis, ancient, intermediate and WUS clade. Among them, five members had the transcriptional activity detected by yeast one-hybrid and seven were uniquely localized to the nucleus through green fluorescent protein (GFP) observation. The expression patterns of BpWOX genes in different tissues and under diverse treatments were quantified by the qRT-PCR method. Results showed that BpWUS was expressed in the apical bud, stem and root, BpWOX5 and BpWOX7 functioned only in the root tip, and three BpWOXs regulated leaf development redundantly. BpWOX9 and BpWOX10 were induced by indole-3-acetic acid (IAA) or jasmonic acid (JA), while BpWOX2 was repressed by five phytohormones. Interestingly, most BpWOX genes were responsive to the abiotic stress stimuli of drought, salt, cold, and cadmium (CdCl₂). Together, our study revealed that BpWOXs were functionally divergent during paper mulberry development and environmental adaptation, which might be related to their evolutionary relationships. Our work will benefit the systematic understanding of the precise function of WOX in plant development and environmental stress responses.

Keywords: WOX transcription factor; development; paper mulberry; phytohormone; stress response.

Figure 1

The schematic of BpWOX genes structure and homeodomain of BpWOX proteins. (a) The phylogenetic tree of BpWOX genes was reconstructed based on the multiple sequence alignment by using the neighbor-joining method in MEGA 6.0 with 1000 bootstrap replicates; (b) The structures of each BpWOX genes were investigated by the alignment of cDNA sequences and corresponding gDNA sequences. The untranslated regions (UTR) are colored with the gray-box, the exons colored with the green-box, and the black lines represent the introns. Bar = 500 bp; (c) The length of the amino acid of each BpWOX proteins was colored in gray, the homeodomain (red boxes) was across all proteins, the WUS-box (blue boxes) was only presented in the WUS clade, and the ERA domain (green boxes) were found in BpWUS and BpWOX5. Bar = 50 aa.

Figure 2

Phylogenetic analysis of plant WOX family. Evolutionary analysis was conducted in MEGA 6.0, and the bootstrap replicates was 1000. A simplified version of the neighbor joining (NJ) tree was displayed, with 228 sequences of proteins from 19 species from green algae to angiosperms. The tree was divided into three clades; the WUS clade, containing 122 sequences, 16 species (pink); the intermediate clade, containing 59 sequences, 17 species (sky-blue); and the ancient clade, containing 47 sequences, 19 species (navy-blue). Two species, O. lucimarinus (black filled circle) and P. patens (pink filled circle) had members only in the ancient clade. S. moellendorffii (green filled circle) had members in both the ancient clade and intermediate clade. Other species had members in all three clades. The paper mulberry (red filled circle) had two members in the ancient clade and intermediate clade, respectively, and six members in the WUS clade. Mulberry is marked with red blank circle, and the A. thaliana is marked by the black blank box. The information of species used in the phylogenetic tree is shown in Supplementary Table S6.

Figure 3

Phylogenetic tree and conserved motif analysis of WOX family between A. thaliana and paper mulberry. (a) The phylogenetic tree (NJ) was reconstructed by MAGE6 using 24 sequences from A. thaliana and paper mulberry. The WUS clade, intermediate clade, and ancient clade were colored with pink, sky-blue and navy blue, respectively. The BpWOX proteins were represented by black filled boxes, and the AtWOX proteins were represented by black blank boxes; (b) The conserved motifs among the members are highlighted in colored boxes with an arranged number, and the sequences of the motifs are listed in Supplementary Table S7. A total of nine motifs was observed from the motif elicitation tool MEME (Supplementary Figure S12). Motif 1 (red boxes) was homeodomain (HD) and motif 4 (pink boxes) was the WUS-box. Motif 2 (sky-blue boxes) only appeared in the intermediate clade and motif 3 (navy-blue boxes) was unique to the ancient clade. The other motifs are distinguished by colors except for motif 6 (white plaid). Motif 5 is labeled by yellow boxes, motif 7 is labeled by water-red boxes, motif 8 is labeled by white boxes, and motif 9 is labeled by green boxes. Bar = 50 aa.

Figure 4

The transactivation activity of BpWOX family. The full-length ORF of each BpWOX gene was fused with pBridge, and the transformed AH109 yeasts were selected from SD-Trp-His medium containing 0–50 mM 3-aminotriazole (3-AT), which is a competitive inhibitor of HIS3 protein. The empty pBridge (BD) vector was used as a negative control.

Figure 5

Subcellular localization of BpWOX proteins in N. benthamiana. Transient expression of the BpWOXs-GFP fusion protein was performed in tobacco epidermal cells. (a) Subcellular localization of BpWOX1 to BpWOX5; (b) Subcellular localization of BpWOX7 to BpWOX10 and BpWUS; (c) The GFP was used as a positive protein control and was detected in the nucleus and cytoplasm. Green fluorescence was observed using a confocal microscope at 48 h after A. tumefaciens infiltration. From left to right, the images show fluorescent-field illumination, bright-field, and overlay of three illuminations. Bar = 30 µm.

Figure 6

The expression patterns of BpWOX genes in different tissues. The expression level of BpWOX9 (a); BpWOX10 (b); BpWOX7 (c); BpWOX8 (d); BpWUS (e); BpWOX5 (f); BpWOX4 (g); BpWOX1 (h); BpWOX2 (i); and BpWOX3 (j) in the apical bud, leaf, stem, root, and root tip. Transcript levels were determined by qRT-PCR. Expression of BpGAPDH was used as an internal control. Error bars indicate standard deviation of three independent biological replications.

Figure 7

The BpWOX genes respond to the environmental stresses. (a) The expression pattern of BpWOX gene under four environmental conditions. For cold treatment, the seedlings were transferred into a 4 °C growth chamber. For salt and CdCl₂ treatments, the seedlings were washed carefully and transferred into a solution of 200 mM NaCl and 50 µM CdCl₂, respectively. For drought treatment, we used 20% (w/v) PEG solution to simulate drought condition. BpWOX2 was labeled with a red line, BpWOX3 was labeled with a blue line, BpWOX4 was labeled with a yellow line, BpWOX8 was labeled with a green line, BpWOX9 was labeled with a purple line, and BpWOX10 was labeled with a pink line; (b) A heat map of BpWOX genes responding to phytohormones at the transcription level. For phytohormone treatments, 100 µM solution of IAA, GA, SA, Me-JA, ETH, and ABA were sprayed onto the surface of the seedlings, respectively. Transcript levels were determined by qRT-PCR, and the expression of BpGAPDH was used as an internal control.

Figure 8

BpWOX genes might be involved in growth and development in paper mulberry and respond to environmental stresses. (a) BpWOX genes may participate in the tissues development and growth of the paper mulberry. BpWOX genes expressed in a particular tissue were enclosed together with an ellipse or box, BpWOX5 and BpWOX7 (blue text) were highly expressed in the root tip (blue blank box); (b) The network diagram between BpWOX genes and the environmental stresses. The line between the genes and environments demonstrates that the gene responded highly to the environment, genes that showed low response or no response were not displayed in this figure. The red line was up-regulation and green line was down-regulation.