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. 2022 Oct 8;11(19):2647.
doi: 10.3390/plants11192647.

Tamarix hispida NAC Transcription Factor ThNAC4 Confers Salt and Drought Stress Tolerance to Transgenic Tamarix and Arabidopsis

Meiheriguli Mijiti  1 , Yucheng Wang  2 , Liuqiang Wang  3 , Xugela Habuding  1
Affiliations

Tamarix hispida NAC Transcription Factor ThNAC4 Confers Salt and Drought Stress Tolerance to Transgenic Tamarix and Arabidopsis

Meiheriguli Mijiti et al. Plants (Basel). .

Abstract

Salt and drought are considered two major abiotic stresses that have a significant impact on plants. Plant NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) have been shown to play vital roles in plant development and responses to various abiotic stresses. ThNAC4, a NAC gene from Tamarix hispida involved in salt and osmotic stress tolerance, was identified and characterized in this study. According to a phylogenetic study, ThNAC4 is a member of NAC subfamily II. Subcellular localization analysis showed that ThNAC4 is located in the nucleus, and transcriptional activation experiments demonstrated that ThNAC4 is a transcriptional activator. Transgenic Arabidopsis plants overexpressing ThNAC4 exhibited improved salt and osmotic tolerance, as demonstrated by improved physiological traits. ThNAC4-overexpressing and ThNAC4-silenced T. hispida plants were generated using the transient transformation method and selected for gain- and loss-of-function analysis. The results showed that overexpression of ThNAC4 in transgenic Tamarix and Arabidopsis plants increased the activities of antioxidant enzymes (SOD, POD, and GST) and osmoprotectant (proline and trehalose) contents under stress conditions. These findings suggest that ThNAC4 plays an important physiological role in plant abiotic stress tolerance by increasing ROS scavenging ability and improving osmotic potential.

Keywords: NAC transcription factor; ROS scavenging; Tamarix hispida; abiotic stress.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Phylogenetic analysis of ThNAC4 and NAC proteins from Arabidopsis. The phylogenetic relationship of ThNAC4 and NAC proteins from Arabidopsis. ThNAC4 and the Arabidopsis NACs were aligned; the unrooted NJ tree was constructed using MEGA 7.
Figure 2
Figure 2
Subcellular localization and transcriptional activation of ThNAC4. (A) Subcellular localization analysis of ThNAC4. The 35S-GFP (control) and ThNAC4-GFP fusion proteins were transiently expressed in onion epidermal cells and viewed with a confocal microscope (wavelength 488 nm). (a) 35S-GFP, GFP; (b) 35S-GFP, bright; (c) 35S-GFP, merge; (d) ThNAC4-GFP, GFP; (e) ThNAC4-GFP, bright; (f) ThNAC4-GFP, merge. (B) Transactivation assay of ThNAC4. Full-length or truncated CDSs of ThNAC4 were cloned into the pGBKT7 vector, transformed into Y2HGold cells and grown on SD/-Trp or SD/-Trp/-His/X-α-gal media to assess their transcriptional activation.
Figure 3
Figure 3
Stress tolerance of Arabidopsis overexpressing ThNAC4. (A,B) Seed germination assay of transgenic (OE2, OE8 and OE9) and wild-type (WT) Arabidopsis plants. Seeds were sown on 1/2 MS solid medium containing 150 mM NaCl or 200 mM mannitol and incubated at 22 °C for 7 days. Photographs were taken, and the germination rates were measured. (C–E) Effects of salt and drought stresses on root length and fresh weight. Seven-day-old seedlings were grown on 1/2 MS solid medium containing 150 mM NaCl or 200 mM mannitol for 7 days. Photographs were taken, and the root length and fresh weight were measured. (F) Growth phenotype of transgenic and WT Arabidopsis plants. (G,H) Effects of salt and drought stress treatment on chlorophyll content and water loss. Three-week-old seedlings in soil were treated with 150 mM NaCl or 200 mM mannitol for one week. Photographs were taken, and the chlorophyll content and water loss were measured. The error bars represent the standard deviations of the mean measurements, which were calculated from three independent experiments. * indicates a significant difference (p < 0.05) between transgenic lines and WT plants.
Figure 4
Figure 4
Histochemical staining and physiological analysis of ThNAC4-transformed and WT Arabidopsis plants. (A,B) Detection of ROS levels in ThNAC4-transformed (Lines 2, 8, and 9) and WT plants. Leaves from transgenic lines and WT plants untreated and treated with 150 mM NaCl or 200 mM mannitol for 2 h were used for histochemical staining by NBT and DAB to reveal the accumulation of O2- and H2O2, respectively. (C) Evans blue staining analysis of cell death. Leaves sampled from 4-week-old transgenic and WT seedlings untreated and treated with 150 mM NaCl or 200 mM mannitol for 2 h were used for histochemical staining. (D–I) Physiological analysis of ThNAC4-transformed and WT Arabidopsis plants. Four-week-old seedlings of transgenic lines and WT plants were irrigated with 150 mM NaCl or 200 mM mannitol for 2 days to measure MDA contents (D); SOD (E), POD (F), and GST (G) activities; and proline (H) and trehalose (I) contents. The error bars represent the standard deviations of the mean measurements, which were calculated from three independent experiments. * indicates a significant difference (p < 0.05) between transgenic lines and WT plants.
Figure 5
Figure 5
The expression pattern of the SOD, POD, TPS and TPP genes in transgenic and WT Arabidopsis plants. Four-week-old seedlings of transgenic lines and WT plants were treated with 150 mM NaCl or 200 mM mannitol for 24 h and were harvested for qRT-PCR analysis. The transcription levels of SODs, PODs, TPSs and TPPs in WT plants under the same conditions were set to 1 to calculate their expression in transgenic plants. The error bars were calculated from three replicates.
Figure 6
Figure 6
Histochemical staining and physiological analyses in transgenic T. hispida plants. (A) Detection of ROS levels in transgenic T. hispida plants. Histochemical staining by DAB and NBT revealed the accumulation of H2O2 and O2-, respectively. (B–H) Physiological analysis of transgenic T. hispida plants, including MDA contents (B), chlorophyll contents (C), SOD activity (D), POD activity (E), GST activity (F), proline contents (G) and trehalose contents (H). The plants were grown in 1/2 MS medium or 1/2 MS medium containing 150 mM NaCl or 200 mM mannitol for 24 h and used for analysis. VC: the pROKII vector control transformed T. hispida plants; OE: overexpression of ThNAC4 in T. hispida plants; and IE: ThNAC4 RNAi-silenced T. hispida plants. The error bars represent the standard deviations of the mean measurements, which were calculated from three independent experiments. *, ** indicates a significant difference (* p < 0.05, ** p < 0.01) compared to the control plants.
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