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. 2014 Jan 10;9(1):e84359.
doi: 10.1371/journal.pone.0084359. eCollection 2014.

Novel NAC Transcription Factor TaNAC67 Confers Enhanced Multi-Abiotic Stress Tolerances in Arabidopsis

Free PMC article

Novel NAC Transcription Factor TaNAC67 Confers Enhanced Multi-Abiotic Stress Tolerances in Arabidopsis

Xinguo Mao et al. PLoS One. .
Free PMC article


Abiotic stresses are major environmental factors that affect agricultural productivity worldwide. NAC transcription factors play pivotal roles in abiotic stress signaling in plants. As a staple crop, wheat production is severely constrained by abiotic stresses whereas only a few NAC transcription factors have been characterized functionally. To promote the application of NAC genes in wheat improvement by biotechnology, a novel NAC gene designated TaNAC67 was characterized in common wheat. To determine its role, transgenic Arabidopsis overexpressing TaNAC67-GFP controlled by the CaMV-35S promoter was generated and subjected to various abiotic stresses for morphological and physiological assays. Gene expression showed that TaNAC67 was involved in response to drought, salt, cold and ABA treatments. Localization assays revealed that TaNAC67 localized in the nucleus. Morphological analysis indicated the transgenics had enhanced tolerances to drought, salt and freezing stresses, simultaneously supported by enhanced expression of multiple abiotic stress responsive genes and improved physiological traits, including strengthened cell membrane stability, retention of higher chlorophyll contents and Na(+) efflux rates, improved photosynthetic potential, and enhanced water retention capability. Overexpression of TaNAC67 resulted in pronounced enhanced tolerances to drought, salt and freezing stresses, therefore it has potential for utilization in transgenic breeding to improve abiotic stress tolerance in crops.

Conflict of interest statement

Competing Interests: The authers have declared that no competing interests exist.


Figure 1
Figure 1. Sequence alignment of TaNAC67 and NACs in various plant species.
A. Amino acid alignment of TaNAC67 and other NAC family members from selected plant species. The numbers on the left indicate amino acid position. Shared amino acid residues are in black background. Gaps, indicated by dashed lines are introduced for optimal alignment. The region underlined indicates the conserved NAC-domain. ▴, conserved amino acid motif (AA sequences in red rectangles). Alignments were performed using the Megalign program of DNAStar. B. Phylogenetic tree of TaNAC67 and NAC members from other plant species. Abbreviations: At, Arabidopsis thaliana; Bd, Brachypodium distachyon; Eg, Elaeis guineensis; Gm, Glycine max; Hv, Hordeum vulgare; Os, Oryza sativa; Sb, Sorghum bicolor; Sl, Solanum lycopersicum; Vv, Vitis vinifera; Zm, Zea mays. The phylogenetic tree was constructed with the PHYLIP 3.69 package, and the bootstrap values are in percent.
Figure 2
Figure 2. Chromosome location of TaNAC67.
A. Genomic origin of TaNAC67 among 20 accessions of wheat and related species. B. Three TaNAC67 genes were identified in the A, B and D genomes of hexaploid wheat. AA, T. urartu; SS, Ae. speltoides; DD, Ae. tauschii; AABB, T. diccocoides; AABBDD, T. aestvium; M, 200 bp DNA ladder. C. Chromosome location of TaNAC67 genes using 41 nulli-tetrasomic (NT) lines of Chinese Spring. Different TaNAC67 genes were missing in each of the NT lines for homoeologous group 6. NT, nulli-tetrasomic line; M, 200 bp DNA ladder. Arrow, pointing at the missing bands in corresponding NT lines.
Figure 3
Figure 3. Expression patterns of TaNAC67 in common wheat.
Expression patterns of TaNAC67 in different tissues at different developmental stages (A) and under stress treatments with ABA, PEG (−0.5 MPa), salt (NaCl) and low temperature (LT) (B). 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 relative expression levels of the target gene in stressed and non-stressed leaves. Three samples were collected for each time point per treatment, and the experiments for each sample were triplicate. Means were generated from three biological replications; bars indicate standard errors. SL, seedling leaves; SR, seedling roots; BS, booting spindles; ES, emerging spikes; PEG, PEG-6000; LT, low temperature.
Figure 4
Figure 4. Subcellular localization of TaNAC67 in onion epidermal cells.
A. The construct harboring 35S::TaNAC67-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), and root outlook (2) and combined in bright field (3). Scale bar  = 300 µm. B. Cells were bombarded with constructs carrying GFP or TaNAC67-GFP as described in Materials and Methods. GFP and TaNAC67-GFP fusion proteins were transiently expressed under control of the CaMV 35S promoter in onion epidermal cells and observed with a laser scanning confocal microscope. Images were taken in dark field for green fluorescence (1, 4), cell outlook (2, 5) and combination (3, 6) in bright field. Scale bar  = 100 µm. At least 30 cells containing each construct were examined.
Figure 5
Figure 5. Comparisons of physiological indices related to abiotic stress response of TaNAC67 transgenics under normal or/and stress conditions.
A. Most TaNAC67 transgenics had higher chlorophyll contents relative to control plants under salt stress. Values are means ± SE (n = 20 plants). *,** significantly different from WT at P = 0.05, 0.01, respectively. B. TaNAC67 transgenics had higher water potentials than the controls. Six TaNAC67 transgenic lines and controls grown in the same containers were subjected to water potential assays performed under well-watered and drought stress conditions. Five plants of each line were collected as one sample, and the experiment consisted of four replications. Values are means ± SE. C. Transgenic TaNAC67 plants had higher osmotic potentials than the controls. Six TaNAC67 transgenic lines, and WT and GFP controls, cultured under well-watered conditions, were subjected to osmotic potential assays. Five plants of each line were pooled as a sample, and the experiment consisted of three replications. Values are means ± SE. D. Comparison of photosynthetic potentials of TaNAC67 transgenics and controls after exposure to high salinity and drought stress. The Fv/Fm ratios of five transgenics were significantly higher than the two controls. The youngest fully expanded leaves were selected to determine chlorophyll fluorescence; three measurements were made for each plant, and 20 plants were used for WT and transgenic lines. Values are means ± SE (n = 20 plants). E. TaNAC67 transgenics have enhanced CMS relative to control plants after exposure to salt stress and water deficit (PEG-6000, −0.5 MPa). Fifteen seedlings were pooled as a sample; three samples were measured for each line. Values are means ± SE.
Figure 6
Figure 6. TaNAC67 transgenics have higher K+ and Na+ ion efflux rates than WT.
A. The transgenics had a higher K+ ion efflux rate than WT plants after a 30 min NaCl shock. Arabidopsis seedlings were pre-incubated in buffer (0.5 mM KCl, 0.1 mM MgCl2, 0.1 mM CaCl2, 0.2 mM Na2SO4, and 0.3 mM MES, pH 6.0) for 30 min and assayed in the same buffer containing 100 mM NaCl at pH 6.0. Five plants were measured for each line. Values are means ± SE. B. TaNAC67 transgenics had higher Na+ ion efflux rates after treatment with 100 mM NaCl. Arabidopsis seedlings were pretreated on MS medium with 100 mM NaCl for 24 h, and then subjected to measurement of Na+ ion flux rates. Five plants were measured for each line, and the values are means ± SE.
Figure 7
Figure 7. Transgenic TaNAC67 Arabidopsis has enhanced tolerance to drought, salt and freezing stresses.
Phenotypes of six TaNAC67 transgenics and WT and GFP controls following drought stress (A), salt stress (B), and freezing stress (C). D, survival rates of transgenic lines under different abiotic stresses. For drought and salinity stress, 11 plants were grown for each line in one treatment, and triplication occurred in three separate plates. For freezing stress, normally pot-cultured transgenic seedlings at 4 weeks were divided into three parts, and each part was stressed at −8±1°C for 1.5 h; 20 plants (5 pots) of each line were used for an experiment. L1-6, six transgenic lines; BF, buffer line. *, ** significantly different at P = 0.05, 0.01, respectively. Values are means ± SE.
Figure 8
Figure 8. Comparisons of relative transcript levels of DREB1A, DERB2A, RD29A, RD29B, Rab18, Cor15, RD22 ABI1, ABI2 and ABI5 in WT and vector control and TaNAC67 overexpressing lines treated for 3 h with PEG-6000 (−0.5 MPa) and assessed by qRT-PCR.
Seedlings harvested before water deficit stress were used as control (CK). Ten seedlings were pooled as a sample, three samples were prepared for qRT-PCR on each line, and the experiments were triplicate. Vertical columns indicate relative transcript levels. Values (and error bars) were calculated using data from three independent assays.

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This study was supported by the National High-tech R&D Project (863 Project, 2012AA10A308), National Basic Research Program of China (973 Program, 2010CB951501) and Beijing Natural Science Foundation (6132030). The funders had no role in study design, data collection and analysis, desision to publish, or prepare of the manuscript.