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The NAC Protein from Tamarix hispida, ThNAC7, Confers Salt and Osmotic Stress Tolerance by Increasing Reactive Oxygen Species Scavenging Capability

1
State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University), Harbin 150040, China
2
Key Laboratory of Biogeography and Bioresource in Arid Land, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, China
*
Author to whom correspondence should be addressed.
Plants 2019, 8(7), 221; https://doi.org/10.3390/plants8070221
Received: 6 June 2019 / Revised: 5 July 2019 / Accepted: 8 July 2019 / Published: 12 July 2019
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Abstract

Plant specific NAC (NAM, ATAF1/2 and CUC2) transcription factors (TFs) play important roles in response to abiotic stress. In this study, we identified and characterized a NAC protein, ThNAC7, from Tamarix hispida. ThNAC7 is a nuclear localized protein and has transcriptional activation activity. ThNAC7 expression was markedly induced by salt and osmotic stresses. Transiently transformed T. hispida seedlings overexpressing ThNAC7 (OE) or with RNA interference (RNAi) silenced ThNAC7 were generated to investigate abiotic stress tolerance via the gain- and loss- of function. Overexpressing ThNAC7 showed an increased reactive oxygen species (ROS) scavenging capabilities and proline content, which was accomplished by enhancing the activities of superoxide dismutase (SOD) and peroxidase (POD) in transiently transformed T. hispida and stably transformed Arabidopsis plants. Additionally, ThNAC7 activated these physiological changes by regulating the transcription level of P5CS, SOD and POD genes. RNA-sequencing (RNA-seq) comparison between wild-type and ThNAC7-transformed Arabidopsis showed that more than 40 known salt tolerance genes might regulated by ThNAC7, including stress tolerance-related genes and TF genes. The results indicated that ThNAC7 induces the transcription level of genes associated with stress tolerance to enhance salt and osmotic stress tolerance via an increase in osmotic potential and enhanced ROS scavenging. View Full-Text
Keywords: NAC transcription factor; Tamarix hispida; abiotic stress; ROS scavenging; transcription activation; transient expression NAC transcription factor; Tamarix hispida; abiotic stress; ROS scavenging; transcription activation; transient expression
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    Doi: 10.5281/zenodo.3269676
    Link: https://zenodo.org/record/3269676#.XR9UQnbyWZA
    Description: Figure S1. Multiple sequence alignment analysis of NAC proteins from 10 other species. ThNAC7: Tamarix hispida (AFN55267.2); PpNAC17: Prunus persica (XP_007201176.1); PaNAC17: Prunus avium (XP_021820479.1); JrNAC17: Juglans regia (XP_018848079.1); MeNAC17: Manihot esculenta (XP_021603416.1); JcNAC17: Jatropha curcas (XP_012073984.1); NaNAC17: Nicotiana attenuate (XP_019238422.1); NtNAC17: Nicotiana tabacum (XP_016438196.1); PtNAC17: Populus trichocarpa (AOF43232.1); NnNAC17: Nelumbo nucifera (XP_010243336.1); and ANAC91: Arabidopsis (AT5G24590.1). Figure S2. Phylogenetic analysis between ThNAC7 and the NAC proteins from T. hispida. The phylogenetic relationship of ThNAC7 and the NAC proteins from the transcriptome of T. hispida. The amino acid sequences of ThNAC7 and 88 T. hispida NAC proteins were aligned; the un-rooted NJ tree was constructed using MEGA 6.06. Figure S3. RT-PCR and qRT-PCR analysis of the expression level of exogenous ThNAC7 in transgenic Arabidopsis lines. Col-0: wild-type Arabidopsis plants; OE1–9: Arabidopsis lines transformed with ThNAC7, M: DL2000 marker. Figure S4. Expression of ThNAC7 in OE, IE and control T. hispida plants. T. hispida were cultured in 1/2 MS (normal conditions) or 1/2 MS medium containing 150 mM NaCl or 200 mM mannitol for 24 h to determine the expression level of ThNAC7. The expression level of ThNAC7 after 48 h of transformation in the control plants was regarded as a calibrator (designated as 1) to standardize the expression of ThNAC7 in the other plants. Control: T. hispida plants transformed with empty pROKII; OE: T. hispida plants overexpressing ThNAC7; IE: ThNAC7 RNAi-silenced plants. Asterisks indicate * significant (P < 0.05) difference compared with the control plants. Figure S5. Analysis of ROS accumulation and the activities of POD and SOD in OE and Col-0 Arabidopsis plants. A, B: Arabidopsis plants treated with 150 mM NaCl or 200 mM mannitol, and stained with DAB to visualize H2O2 (A), or stained with NBT to visualize O2- (B). C: Measurement of H2O2 levels in Arabidopsis plants. D, E: Measurement of POD (D) and SOD (E) activities. Col-0: wild-type Arabidopsis plants; OE2 and 5: Arabidopsis lines 2 and 5 transformed with ThNAC7. Figure S6. The expression of the PODs and SODs in OE and Col-0 Arabidopsis plants. Analysis of POD (A) and SOD (B) gene expression was carried out in Arabidopsis OE and Col-0 plants in response to NaCl (150 mM) or Mannitol (200 mM) stress treatments. The expression levels of the genes in Col-0 plants grown in the normal condition were regarded as a calibrator (designed as 1) to standardize the relative transcript values. Col-0: wild-type Arabidopsis plants; OE2, 5: the Arabidopsis lines transformed with ThNAC7. Figure S7. Detection of cell death, water loss rates, and MDA content in OE and Col-0 Arabidopsis plants. A: Comparison of electrolyte leakage rates. B: Water loss rates. C: MDA content. The plants were treated with water (control), 150 mM NaCl (NaCl), or 200 mM Mannitol for 24 h for analyses. Data are means ± SD from three independent experiments. * Significant (P < 0.05) difference compared with Col-0 plants. Col-0: wild-type Arabidopsis plants; OE2 and 5: lines 2 and 5 of Arabidopsis transformed with ThNAC7. Figure S8. Analysis of the biosynthesis of proline in OE and Col-0 Arabidopsis plants. A: Analysis of proline levels in OE and Col-0 plants in response to NaCl (150 mM) or Mannitol (200 mM) stress treatments. B, C: The expression of proline biosynthesis and degradation-related genes. The relative expression levels were log2 transformed. * Significant (P < 0.05) difference compared with Col-0 plants. Col-0: wild-type Arabidopsis plants; OE2, 5: the Arabidopsis lines transformed with ThNAC7. Figure S9. GO classification of the annotated DEGs in salt stress. Figure S10. Comparison of the expression profiles between RNA-seq and qRT-PCR. Table S1. Primer sequences used to construct recombinant plasmids. Table S2. Primer sequences used to construct yeast recombinant plasmids. Table S3. Primer sequences used in qRT-PCR. Table S4. List of differentially expressed genes in the OE5 compared to Col-0 Arabidopsis plants under normal conditions. Table S5. List of differentially expressed genes in the OE5 compared to Col-0 Arabidopsis plants under salt stress conditions. Table S6. The primer sequences of qRT-PCR for validation of the selected DEGs.
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He, Z.; Li, Z.; Lu, H.; Huo, L.; Wang, Z.; Wang, Y.; Ji, X. The NAC Protein from Tamarix hispida, ThNAC7, Confers Salt and Osmotic Stress Tolerance by Increasing Reactive Oxygen Species Scavenging Capability. Plants 2019, 8, 221.

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