A Wheat R2R3-type MYB Transcription Factor TaODORANT1 Positively Regulates Drought and Salt Stress Responses in Transgenic Tobacco Plants

Abstract

MYB transcription factors play important roles in plant responses to biotic and abiotic stress. In this study, TaODORANT1, a R2R3-MYB gene, was cloned from wheat (Triticum aestivum L.). TaODORANT1 was localized in the nucleus and functioned as a transcriptional activator. TaODORANT1 was up-regulated in wheat under PEG6000, NaCl, ABA, and H₂O₂ treatments. TaODORANT1-overexpressing transgenic tobacco plants exhibited higher relative water content and lower water loss rate under drought stress, as well as lower Na⁺ accumulation in leaves under salt stress. The transgenic plants showed higher CAT activity but lower ion leakage, H₂O₂ and malondialdehyde contents under drought and salt stresses. Besides, the transgenic plants also exhibited higher SOD activity under drought stress. Our results also revealed that TaODORANT1 overexpression up-regulated the expression of several ROS- and stress-related genes in response to both drought and salt stresses, thus enhancing transgenic tobacco plants tolerance. Our studies demonstrate that TaODORANT1 positively regulates plant tolerance to drought and salt stresses.

Keywords: MYB; abiotic stress; antioxidation system; stress related genes; wheat.

FIGURE 1

Sequence and phylogenetic tree analysis of TaODORANT1. (A) Phylogenetic relationship of TaODORANT1 with its orthologs from other plant species. (B) Sequence alignment of TaODORANT1 and homologous proteins from other plant species. The black background represents identical amino acid residues in the aligned sequences. Red straight lines indicate the conserved domains of R2R3-MYB.

FIGURE 2

Subcellular localization and transcriptional activity analysis of TaODORANT1. (A) Subcellular localization of TaODORANT1. Recombinant vector ubiqutin::TaODORANT1-GFP and control vector ubiquitin::GFP were transformed into onion epidermal cells and observed with fluorescence microscopy, respectively. (B) Transactivation activity of TaODORANT1 in yeast. Schematic diagrams illustrate the different portions of TaODORANT1 ORF that were introduced into the pGBKT7. Recombined vectors were transformed into yeast strain AH109, and the transformants were screened by SD/-Trp, SD/-Trp/-His+X-α-gal, SD/-Trp/-His/-Ade+X-α-gal media. (C) Binding activity assay of TaODORANT1. Three MYB binding motifs I, II, and IIG were fused with pHIS2 vector, and the TaODORANT1 ORF was fused with pGADT7 vector. Reconstructed pGADT7 and pHIS2 vectors were co-transformed into yeast strain Y187. Transformants were incubated on SD/-Trp/-Leu/-His media with different concentrations of 3-AT. Three independent biological replicates were performed and produced similar results.

FIGURE 3

Expression profiles of TaODORANT1 in wheat. (A) Organ-specific expression assay of TaODORANT1 in wheat (R: seedlings root; S: seedlings stem; L: seedlings leaf; mR: mature root; mS: mature stem; mL: mature leaf; ST: stamen; P: pistil). (B–E) Expression patterns of TaODORANT1 in 14-day-old wheat seedlings after treatment with 20% PEG6000, 200 mM NaCl, 100 μM ABA, and 10 mM H₂O₂, respectively. Three independent biological replicates were performed and produced similar results. Vertical bars refer to ±SE (n = 3).

FIGURE 4

Tolerance analysis of 10-day-old tobacco seedlings. (A–E) Phenotype of seedlings after treatment with mannitol or NaCl for 10 days. (F) Root length statistics of seedlings after treatment with mannitol or NaCl for 10 days. Three independent biological replicates were performed and produced similar results. Vertical bars refer to ±SE (n = 3). Asterisks indicate significant difference between WT and transgenic lines (^∗P < 0.05; ^∗∗P < 0.01).

FIGURE 5

Drought tolerance of TaODORANT1 overexpressed tobacco plants. (A) Phenotype of WT, VC, and OE lines (OE1, OE3, and OE12) after drought treatment. (B) Survival rate statistics after re-watering for 1 week. (C) RWC of leaves after drought stress. (D) Water loss rate of detached leaves. (E) Phenotype of detached leaves after 24 h dehydration. (F) Stomatal aperture after dehydration or ABA treatment. (G) Width/length ratio of stomata. Three independent biological replicates were performed and produced similar results. Vertical bars refer to ±SE (n = 3). Asterisks indicate significant difference between the WT and the transgenic lines (^∗P < 0.05; ^∗∗P < 0.01).

FIGURE 6

Salt tolerance of TaODORANT1 overexpressed tobacco plants. (A) Phenotype of WT, VC and OE lines (OE1, OE3, and OE12) after salt treatment. (B) Survival rate statistical analysis. (C) Chlorophyll content in leaves. (D,E) Na⁺ and K⁺ concentrations in leaves. (F) K⁺/Na⁺ ratio. (G,H) Expression levels of ion transporter genes. Three independent biological replicates were performed and produced similar results. Vertical bars refer to ±SE (n = 3). Asterisks indicate significant difference between the WT and the transgenic lines (^∗P < 0.05; ^∗∗P < 0.01).

FIGURE 7

Oxidative damage of transgenic tobacco plants after drought/salt stress. (A) Histochemical detection of H₂O₂ and O₂^- by DAB staining and NBT staining. (B) MDA content and (C) IL under normal and drought/salt stress. Three independent biological replicates were performed and produced similar results. Vertical bars refer to ±SE (n = 3). Asterisks indicate significant difference between the WT and the transgenic lines (^∗P < 0.05; ^∗∗P < 0.01).

FIGURE 8

Overexpression of TaODORANT1 in tobacco elevated antioxidant enzymes gene expression and activities under drought/salt stress. (A–D) Expression levels of CAT, SOD, POD, and RbohF in tobacco seedlings after 300 mM mannitol treatment for 7 days. (E–H) H₂O₂ content and activities of CAT, SOD, and POD in plant leaves after drought treatment. (I) Expression levels of CAT in tobacco seedlings after 150 mM NaCl treatment for 7 days. (J,K) CAT activity and H₂O₂ content in plant leaves after NaCl treatment. Three independent biological replicates were performed and produced similar results. Vertical bars refer to ±SE (n = 3). Asterisks indicate significant difference between the WT and the transgenic lines (^∗P < 0.05; ^∗∗P < 0.01).

FIGURE 9

Expression analysis of stress-related genes. (A,B) Expression levels of stress-related genes in tobacco seedlings after treatment on 1/2 MS media with 300 mM mannitol or 150 mM NaCl for 1 week. Three independent biological replicates were performed and produced similar results. Vertical bars refer to ±SE (n = 3). Asterisks indicate significant difference between the WT and the transgenic lines (^∗P < 0.05; ^∗∗P < 0.01).