Supplementary Data: The NAC protein from Tamarix hispida, ThNAC7, confers salt and osmotic stress tolerance by increasing reactive oxygen species scavenging capability

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 detections 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 was 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, D: Measurement of POD and SOD activities. E: Measurement of H₂O₂ levels in Arabidopsis plants. 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 and SOD 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.