TsNAC1 Is a Key Transcription Factor in Abiotic Stress Resistance and Growth

Abstract

NAC proteins constitute one of the largest families of plant-specific transcription factors, and a number of these proteins participate in the regulation of plant development and responses to abiotic stress. T. HALOPHILA STRESS RELATED NAC1 (TsNAC1), cloned from the halophyte Thellungiella halophila, is a NAC transcription factor gene, and its overexpression can improve abiotic stress resistance, especially in salt stress tolerance, in both T. halophila and Arabidopsis (Arabidopsis thaliana) and retard the growth of these plants. In this study, the transcriptional activation activity of TsNAC1 and RD26 from Arabidopsis was compared with the target genes' promoter regions of TsNAC1 from T. halophila, and the results showed that the transcriptional activation activity of TsNAC1 was higher in tobacco (Nicotiana tabacum) and yeast. The target sequence of the promoter from the target genes also was identified, and TsNAC1 was shown to target the positive regulators of ion transportation, such as T. HALOPHILA H⁺-PPASE1, and the transcription factors MYB HYPOCOTYL ELONGATION-RELATED and HOMEOBOX12 In addition, TsNAC1 negatively regulates the expansion of cells, inhibits LIGHT-DEPENDENT SHORT HYPOCOTYLS1 and UDP-XYLOSYLTRANSFERASE2, and directly controls the expression of MULTICOPY SUPPRESSOR OF IRA14 Based on these results, we propose that TsNAC1 functions as an important upstream regulator of plant abiotic stress responses and vegetative growth.

Figure 1.

The 130-bp key region of the promoter of TsVP1 is a direct binding target of TsNAC1. A, Multiple amino acid sequence alignment of TsNAC1 and its ortholog in Arabidopsis (RD26). Identical residues are shaded in black, and five subdomains (A–E) are indicated on the sequences. B, Transcriptional activity assay of TsNAC1 in yeast strain YM4271. TsNAC1 was cloned and fused with the GAL4 AD. The promoter of TsVP1 was cloned into pLacZi vector. Streaked transformants are shown on Yeast extract Peptone Dextrose medium with 0.003% Adenine hemisulfate (YPDA) and SD/-uracil medium. The activities of β-galactosidase were examined by X-gal staining. C, Electrophoretic mobility shifts assay (EMSA). Probes, TsNAC1-GST, competitors (130-bp unlabeled fragments), and mutated competitors at 50× and 200× molar excess were present (+) or absent (−) in each reaction. The protein-DNA complexes are marked by black arrowheads.

Figure 2.

TsNAC1 regulated the growth of T. halophila. A, Diagrams of the plant overexpression vector and RNAi vector. B, Phenotypes of TsNAC1 transgenic plants. Shown are seedlings grown on Murashige and Skoog-agar for 4 d after sowing and plants grown in soil for 6 weeks and 14 weeks (after vernalization treatment for 5 weeks). C, Western-blot results of transgenic lines and the wild type (WT). D, Phenotypes of OX, wild-type, and NR T. halophila plants after 48 h of treatment at −4°C with 3,500 lx, after 1 week of drought stress with an 18% PEG6000 solution, after keeping the 600 mm NaCl concentration of culture medium for 2 weeks, and after 36 h of active oxygen stress with 0.1 mL of 3 mm paraquat solution for every pot. E, TsNAC1 expression levels in OX7, OX10, NR10, NR11, and the wild type (the expression was normalized with Actin), and the dry weight of shoots and roots. Bars represent means ± sd, with three biological replicates in the experiment and five plants for each repeat. Significant differences by Student’s t test are indicated with asterisks: **, P < 0.01.

Figure 3.

ChIP-Seq assay of TsNAC1. A, Distribution of TsNAC1 transcription factor-binding sites. B, Identified peak distance from transcription start sites (TSS) for TsNAC1. The peaks were highly enriched from −200 to +100 bp to the transcription start sites. C, ChIP-Seq signals of TP1G13990 (TsVP1) and TP2G25870 (Tub2) on the genome browser. The tag counts were normalized in each bin according to the total number of reads. Short black lines mark regions used for the ChIP-quantitative PCR (qPCR) assay. D, Binding situation of TsNAC1 quantified by the percentage occurrence of the CA(T/A)G motif (x axis) plotted against the log₂ fold enrichment of immunoprecipitation samples compared with the input sample (y axis), with the color of each square mapped to the number of indicated peak motifs. E, Anti-TsNAC1 ChIP-qPCR validation and transcript detection of TsVP1 and TsNAC1. Samples immunized by preimmune serum were used as the negative control in the ChIP-qPCR assay. Bars represent means ± sd, with three biological replicates in the experiment and five plants for each repeat. Significant differences by Student’s t test are indicated with asterisks: **, P < 0.01. F, GO analysis results of the input sample data and data for two immunoprecipitation samples (OX7 and OX10). The dotted rectangles indicate significant and consistent GO enrichment and the hierarchical classification results of biological regulation, developmental processes, and responses to stimuli.

Figure 4.

Validation of candidate downstream genes. A, Downstream target promoter fragments were connected into pLacZi and transformed into the pAD-GAL4-TsNAC1 YM4271 strain. β-Galactosidase (β-GAL) activities were validated by X-gal staining. β-Galactosidase activity was measured with o-nitrophenyl-β-d-galactopyranoside (ONPG) as substrate. B, Binding of TsNAC1 to the promoter of downstream genes as determined by ChIP-PCR. Six-week-old wild-type rosette leaves were used as materials and immunoprecipitated with TsNAC1 antibody and protein A agarose beads. Negative control reactions were performed in parallel using preimmune serum. β-Galactosidase units (Mu) were determined as follows: 1,000 × OD₅₇₄/(t × V × OD₆₀₀), where t is elapsed incubation time (min) and V is volume of culture (mL). C and D, Transient expression of the P35S:TsNAC1 or P35S:RD26 construct with promoter:Luc reporter constructs in T. halophila protoplasts (C) and tobacco leaves (D). Representative bioluminescence images are shown. Bars in B and C represent means ± sd, with three biological replicates in the experiment. Significant differences by Student’s t test are indicated with asterisks: **, P < 0.01.

Figure 5.

Assays of transcription factor activity in the yeast system. A, Binding activity of TsNAC1 and RD26 to the promoter of TsVP1. TsNAC1 and RD26 were cloned and fused with the yeast GAL4 AD. The promoter of TsVP1 was linked into pHis2.1 and pLacZi vectors as reporters. B, Truncation analysis of the transcriptional activation activity of TsNAC1 and RD26. Numbers on the top indicate the positions of amino acids. Subdomains A to E of the NAC DNA-binding domains are colored in black, the ADs are colored in gray, and the mutation of the amino acid is colored in red. The C-terminal AD (164–305 and 181–314), the AD with the repression domain (105–305 and 122–314), and the full-length transcription factor domain (1–305 and 1–314) were cloned and fused with the yeast GAL4 binding domain (BD). Transformants were screened on SD/-Leu or SD/-Trp and SD/-His, and the β-galactosidase (β-GAL) activities were validated by X-gal staining and quantified by ONPG assay. Western blot was executed with the GST antibody. Bars represent means ± sd, with three biological replicates in the experiment. Significant differences by Student’s t test are indicated with asterisks: **, P < 0.01.

Figure 6.

Proposed model for the regulation of plant development and stress responses by TsNAC1. TsNAC1 functions as an upstream regulator of plant osmotic stress (regulates ion transportation), oxidative stress, and cold stress responses and coordinates both vegetative growth (retards cell expansion) and reproductive growth.