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. 2012 May;63(8):2933-46.
doi: 10.1093/jxb/err462. Epub 2012 Feb 13.

TaNAC2, a NAC-type Wheat Transcription Factor Conferring Enhanced Multiple Abiotic Stress Tolerances in Arabidopsis

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Free PMC article

TaNAC2, a NAC-type Wheat Transcription Factor Conferring Enhanced Multiple Abiotic Stress Tolerances in Arabidopsis

Xinguo Mao et al. J Exp Bot. .
Free PMC article

Abstract

Environmental stresses such as drought, salinity, and cold are major factors that significantly limit agricultural productivity. NAC transcription factors play essential roles in response to various abiotic stresses. However, the paucity of wheat NAC members functionally characterized to date does not match the importance of this plant as a world staple crop. Here, the function of TaNAC2 was characterized in Arabidopsis thaliana. A fragment of TaNAC2 was obtained from suppression subtractive cDNA libraries of wheat treated with polyethylene glycol, and its full-length cDNA was obtained by searching a full-length wheat cDNA library. Gene expression profiles indicated that TaNAC2 was involved in response to drought, salt, cold, and abscisic acid treatment. To test its function, transgenic Arabidopsis lines overexpressing TaNAC2-GFP controlled by the cauliflower mosaic virus 35S promoter were generated. Overexpression of TaNAC2 resulted in enhanced tolerances to drought, salt, and freezing stresses in Arabidopsis, which were simultaneously demonstrated by enhanced expression of abiotic stress-response genes and several physiological indices. Therefore, TaNAC2 has potential for utilization in transgenic breeding to improve abiotic stress tolerances in crops.

Figures

Fig. 1.
Fig. 1.
Sequence alignment of TaNAC2 and NACs in various plant species. (A) Amino acid alignment of TaNAC2 and other NAC family members from selected plant species. Gaps (dashed lines) were introduced for optimal alignment. The numbers on the left indicate amino acid positions. Identical amino acid residues are shaded black. The region underlined indicates the conserved NAC-domain. Alignments were performed using MegAlign of DNAStar. (B) Phylogenetic tree of TaNAC2 and NAC members from other plant species. At, Arabidopsis thaliana; Bn, Brassica napus; Ec, Eleusine coracana; Eg, Elaeis guineensis; Gh, Gossypium hirsutum; Gm, Glycine max; Hv, Hordeum vulgare; Os, Oryza sativa; Ph, Petunia hybrida; Sl, Solanum lycopersicum; Ta, Triticum aestivum; Zm, Zea mays. The tree was constructed with the PHYLIP 3.69 package. Bootstrap values are in percentages.
Fig. 2.
Fig. 2.
Chromosome location of TaNAC2. (A) Genomic origin of TaNAC2 among 16 accessions of wheat and related species. Lanes: 1, 2, Triticum urartu; 3, 4, Triticum monococcum; 5–7, Aegilops speltoides; 8–10, Aegilops tauchii; 11–13, Triticum durum; 14–16, Triticum aestivum; M, 200-bp DNA ladder. (B) Chromosome location of TaNAC2 using 38 nulli-tetrasomic (NT) lines of Chinese Spring. The gene was not amplified by NT5A5B or NT5A5D. M, 200-bp DNA ladder.
Fig. 3.
Fig. 3.
Expression patterns of TaNAC2 in response to stress treatments: abscisic acid (ABA), polyethylene glycol (PEG, –0.5 MPa), salt (NaCl), and low temperature (LT) Two-leaf seedlings of common wheat cv. Hanxuan 10 were exposed to abiotic stresses as described in Materials and methods. The 2−ΔΔCT method was used to measure the relative expression levels of the target gene in stressed and non-stressed leaves. Means were generated from three independent measurements; bars indicate standard errors.
Fig. 4.
Fig. 4.
Subcellular localizations of TaNAC2 in transgenic Arabidopsis root and onion epidermal cells. (A) A construct harbouring 35S::TaNAC2–GFP was introduced into Agrobacterium and transferred into Arabidopsis by floral infiltration. Positive transgenic lines were screened with kanamycin and then examined with a confocal microscope. Images are dark field for green fluorescence (1), bright field (2), and combined (3). (B) Cells were bombarded with constructs carrying GFP or TaNAC2–GFP. GFP and TaNAC2–GFP fusion proteins were transiently expressed under the control of the cauliflower mosaic virus 35S promoter in onion epidermal cells and observed with a laser scanning confocal microscope. Images are dark field (1, 4), bright field (2, 5), and combined (3, 6).
Fig. 5.
Fig. 5.
Comparison of primary root lengths of TaNAC2 plants. Seeds of four TaNAC2 transgenic Arabidopsis lines and wild-type (WT) and green fluorescent protein (GFP) controls were planted on MS agar and cultured under short day conditions. Five seeds of each line were planted in triplicate and root lengths were measured after 8 days. (A) Three typical lines with significantly longer primary root lengths. (B) Comparison of measurements, calculated from three independent assays. *, Significantly different from wild type (P = 0.05).
Fig. 6.
Fig. 6.
TaNAC2 plants have stronger water retention ability. (A) Comparison of water loss rates for detached rosettes between transgenic plants and wild-type (WT) and green fluorescent protein (GFP) controls. (B) Comparison of relative water contents (RWC) of detached rosettes of transformed plants and controls after a 5.5-hour treatment. Ten separate plants were used; values are mean ± SE.
Fig. 7.
Fig. 7.
Comparisons of physiological indices related to abiotic stress response of TaNAC2 transgenic lines under stress conditions. (A) Transgenic TaNAC2 plants had lower osmotic potentials than the wild-type (WT) and green fluorescent protein (GFP) controls. Four TaNAC2 transgenic lines and controls were cultured under well-watered conditions and selected to perform osmotic potential assays. Five plants of each line were pooled as one sample and three samples were measured for each line. *, **, significantly different from wild type: *, P = 0.05; **, P = 0.01. (B) Comparison of photosynthetic potentials of TaNAC2 transgenic lines and controls under high salinity stress. The Fv/Fm ratios of four transgenic lines were significantly higher than the two controls. Twenty plants were measured; values are mean ± SE. **, significantly different from wild type, P = 0.01. (C) Comparison of cell membrane stability (CMS) for TaNAC2 transgenic lines and controls following drought and salt stresses. Fifteen seedlings were pooled as one sample and three samples were measured for each line. *, ** significantly different from wild type: *, P = 0.05; **, 0.01. Values are mean ± SE.
Fig. 8.
Fig. 8.
Transgenic TaNAC2 Arabidopsis lines have enhanced tolerance to drought, salt, and freezing stress. (A) Phenotypes of four TaNAC2 transgenic lines and wild-type (WT) and green fluorescent protein (GFP) controls following drought stress (A), salt stress (B), and freezing stress (C). For drought and salinity stress, ten plants of each line were grown for each treatment. Experiments were performed in triplicate. For freezing stress, pot-cultured transgenic seedlings at 4 weeks were divided into three replicates, and each replicate was stressed at –10 °C for 1.5 h. Twenty plants (five pots) of each line were used for each experiment.
Fig. 9.
Fig. 9.
Comparisons of relative transcript levels of DREBA, RD29A, RD29B, RD22, Rab18, ABI1, ABI2, and ABI5 in control plants and TaNAC2 transgenic lines treated with polyethylene glycol (PEG, –0.5 MPa). Vertical columns indicate relative transcript levels. Seedlings harvested before drought stress were used as controls (CK). Ten seedlings were pooled as a sample and three samples of each line were prepared for quantitative real-time PCR. Values are mean ± SE.

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