Supplementary Data: The NAC protein from Tamarix hispida, ThNAC7, confers salt and osmotic stress tolerance by increasing reactive oxygen species scavenging capability
Creators: name: Ji, Xiaoyu
Publisher: name: Zenodo
Year: 2019

description: <strong>Figure S1. </strong>Multiple sequence alignment analysis of NAC proteins from 10 other species. ThNAC7: <em>Tamarix hispida </em>(AFN55267.2); PpNAC17: <em>Prunus persica </em>(XP_007201176.1); PaNAC17: <em>Prunus avium </em>(XP_021820479.1); JrNAC17: <em>Juglans regia </em>(XP_018848079.1); MeNAC17: <em>Manihot esculenta </em>(XP_021603416.1); JcNAC17: <em>Jatropha curcas </em>(XP_012073984.1); NaNAC17: <em>Nicotiana attenuate </em>(XP_019238422.1); NtNAC17: <em>Nicotiana tabacum </em>(XP_016438196.1); PtNAC17: <em>Populus trichocarpa </em>(AOF43232.1); NnNAC17: <em>Nelumbo nucifera</em> (XP_010243336.1); and ANAC91: <em>Arabidopsis</em> (AT5G24590.1).

<strong>Figure S2.</strong> Phylogenetic analysis between ThNAC7 and the NAC proteins from <em>T. hispida</em>. The phylogenetic relationship of ThNAC7 and the NAC proteins from the transcriptome of<em> T. hispida</em>. The amino acid sequences of ThNAC7 and 88 <em>T. hispida</em> NAC proteins were aligned; the un-rooted NJ tree was constructed using MEGA 6.06.

<strong>Figure S3.</strong> RT-PCR and qRT-PCR detections of the expression level of exogenous <em>ThNAC7</em> in transgenic <em>Arabidopsis</em> lines.<strong> </strong>Col-0: wild-type <em>Arabidopsis</em> plants; OE1–9: <em>Arabidopsis</em> lines transformed with <em>ThNAC7</em>, M: DL2000 marker.

<strong>Figure S4. </strong>Expression of <em>ThNAC7</em> in OE, IE and control <em>T. hispida</em> plant­s. <em>T. hispida</em> 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 <em>ThNAC7</em>. The expression level of <em>ThNAC7</em> after 48 h of transformation in the control plants was regarded as a calibrator (designated as 1) to standardize the expression of <em>ThNAC7</em> in the other plants. Control: <em>T. hispida</em> plants transformed with empty pROKII; OE: <em>T. hispida</em> plants overexpressing <em>ThNAC7</em>; IE: <em>ThNAC7</em> RNAi-silenced plants. Asterisks indicate * significant (P &lt; 0.05) difference compared with the control plants.

<strong>Figure S5.</strong> Analysis of ROS accumulation and the activities of POD and SOD in OE and Col-0 <em>Arabidopsis </em>plants.<strong> A</strong>,<strong> B</strong>: <em>Arabidopsis</em> plants treated with 150 mM NaCl or 200 mM mannitol, and stained with DAB to visualize H<sub>2</sub>O<sub>2</sub> (<strong>A</strong>), or stained with NBT to visualize O<sub>2</sub><sup>-</sup> (<strong>B</strong>). <strong>C, D</strong>: Measurement of POD and SOD activities. <strong>E</strong>: Measurement of H<sub>2</sub>O<sub>2</sub> levels in <em>Arabidopsis</em> plants. Col-0: wild-type <em>Arabidopsis</em> plants; OE2 and 5: <em>Arabidopsis</em> lines 2 and 5 transformed with <em>ThNAC7</em>.

<strong>Figure S6.</strong> The expression of the <em>PODs</em> and <em>SODs</em> in OE and Col-0 <em>Arabidopsis</em> plants.<strong> </strong>Analysis of <em>POD</em> and <em>SOD</em> gene expression was carried out in <em>Arabidopsis</em> 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 <em>Arabidopsis</em> plants; OE2, 5: the <em>Arabidopsis</em> lines transformed with <em>ThNAC7</em>.

<strong>Figure S7. </strong>Detection of cell death, water loss rates, and MDA content in OE and Col-0 <em>Arabidopsis</em> plants. <strong>A</strong>: Comparison of electrolyte leakage rates. <strong>B</strong>: Water loss rates. <strong>C</strong>: 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 &lt; 0.05) difference compared with Col-0 plants. Col-0: wild-type <em>Arabidopsis</em> plants; OE2 and 5: lines 2 and 5 of <em>Arabidopsis</em> transformed with <em>ThNAC7</em>.

<strong>Figure S8.</strong> Analysis of the biosynthesis of proline in OE and Col-0 <em>Arabidopsis</em> plants. <strong>A</strong>: Analysis of proline levels in OE and Col-0 plants in response to NaCl (150 mM) or Mannitol (200 mM) stress treatments. <strong>B</strong>,<strong> C</strong>: The expression of proline biosynthesis and degradation-related genes. The relative expression levels were log2 transformed. * Significant (P &lt; 0.05) difference compared with Col-0 plants. Col-0: wild-type <em>Arabidopsis</em> plants; OE2, 5: the <em>Arabidopsis</em> lines transformed with <em>ThNAC7</em>.

<strong>Figure S9.</strong> GO classification of the annotated DEGs in salt stress.

<strong>Figure S10.</strong> Comparison of the expression profiles between RNA-seq and qRT-PCR.

<strong>Table S1. </strong>Primer sequences used to construct recombinant plasmids.

<strong>Table S2. </strong>Primer sequences used to construct yeast recombinant plasmids.

<strong>Table S3. </strong>Primer sequences used in qRT-PCR.

<strong>Table S4. </strong>List of differentially expressed genes in the OE5 compared to Col-0 <em>Arabidopsis</em> plants under normal conditions.

<strong>Table S5. </strong>List of differentially expressed genes in the OE5 compared to Col-0 <em>Arabidopsis</em> plants under salt stress conditions.

<strong>Table S6. </strong>The primer sequences of qRT-PCR for validation of the selected DEGs.