Overexpression of NAC transcription factors from the desert ephemeral plant Eremopyrum triticeum promoted abiotic stress tolerance in Arabidopsis thaliana

doi:10.21203/rs.3.rs-4909198/v1

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Overexpression of NAC transcription factors from the desert ephemeral plant Eremopyrum triticeum promoted abiotic stress tolerance in Arabidopsis thaliana

https://doi.org/10.21203/rs.3.rs-4909198/v1

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Eremopyrum triticeum is a typical spring ephemeral species, which in China mainly distributed in the desert regions of northern Xinjiang, and play an important role in the desert ecosystems. E. triticeum has several adaptive characteristics such as short growth rhythms, high photosynthetic efficiency, high seed production, drought and salt resistance. However, the molecular regulatory mechanism of E. triticeum in responses to abiotic stress resistance is still unknown. In this study, two NAC-like transcription factor-encoding genes, EtNAC1 and EtNAC2, were isolated from E. triticeum. The predicted EtNAC1 and EtNAC2 proteins possess a typical NAC DNA-binding domain at the N-terminal region. The qRT-PCR analysis showed that EtNAC1 and EtNAC2 were highly expressed in mature roots of E. triticeum, and were significantly up-regulated under drought, high salt and abscisic acid (ABA) stresses. Subcellular localization analysis in onion epidermal cells revealed that EtNAC1 and EtNAC2 were located in the nucleus. Expression of EtNAC1 and EtNAC2 in yeast cells improved the survival rate of yeast under low temperature, H₂O₂, high drought and salt stresses. Overexpression of EtNAC1 and EtNAC2 in Arabidopsis thaliana conferred enhanced tolerance to drought and salt stresses, increased ABA sensitivity, and transgenic plants showed higher proline (Pro) content, but lower malondialdehyde (MDA) content, lower chlorophyll leaching, lower water loss rate and stomatal aperture (width/length) than WT plants. In conclusion, EtNAC1 and EtNAC2 play important roles in abiotic stress responses of E. triticeum, which might have significant potential in crop molecular breeding for abiotic stress tolerance.

Eremopyrum triticeum

NAC transcription factor

Multiple stress tolerance

Transgenic Arabidopsis thaliana

Stomatal aperture

Abiotic stresses such as drought, high salinity and extreme temperature significantly affect plant survival and growth, which limit the distribution, yield and quality of crops (Gong et al. 2020). Plants have evolved specific defense mechanisms to deal with various environmental stresses, including morphological, physiological, biochemical, and transcriptional regulating stress-related genes (Xia et al. 2015; Fichman and Mittler, 2021). Several families of transcription factors (TFs) play key roles in regulating stress-related gene expression, such as NAC, WRKY, bHLH, and bZIP (Wang et al. 2020; Li et al. 2020; Jin et al. 2021; Luo et al. 2022). Among these TFs, NAC TFs have been found to play important roles in plant responses to abiotic stress such as drought and salinity (Liu et al. 2011; Zhong et al. 2012; Fang et al. 2015).

NAC proteins, named from these three genes (NAM, ATAF1/2 and CUC2), are one of the largest plant-pacific TF families (Aida et al. 1997). NAC TFs possess a highly conserved N-terminal DNA-binding domain and a diversified C-terminus transcription regulation region (Ernst et al. 2004; Olsen et al. 2005). The DNA binding domain, which is crucial for nuclear localization, possesses around 150 amino acids and divided into five (A-E) subdomains. In contrast, the transcription regulation region at the C-terminal is versatile and can act as a transcriptional activator or repressor (Bian et al. 2020; Olsen et al. 2005).

The function of NAC genes responding to abiotic stresses has been extensively investigated in diverse plant species, including Arabidopsis (Ooka et al. 2003), rice (Ooka et al. 2003), soybean (Le et al. 2011), potato (Singh et al. 2013), maize (Shiriga et al. 2014), tartary buckwheat (Liu et al. 2019), and wild emmer wheat (Rui et al. 2023). However, most of the regulatory mechanisms and biological functions of plant TFs still unknown. Studied have shown that overexpressing of three Arabidopsis NAC genes (ANAC072/RD26, ANAC055, and ANAC019) enhanced drought/salt tolerance by stimulating the expression levels of some abiotic stress-regulated genes (Tran et al. 2004). Furthermore, ANAC096 of Arabidopsis could interact with ABF2 and ABF4 to stimulate drought-related genes RD29A transcription, thus involved in regulating dehydration and osmotic stress through an ABA-dependent pathway (Xu et al. 2013). Arabidopsis plants overexpressing ATAF1 exhibit high sensitivity to ABA, high salinity, and oxidative stress (Wu et al. 2009). In rice, overexpression of OsNAC5, OsNAC6, ONAC009, OsNAC10, OsNAC14 and ONAC058 enhances the seedling tolerance to drought, cold and salt stresses, respectively (Jeong et al. 2013; Lee et al. 2017; Nakashima et al. 2007; Hu et al. 2008; Chen et al. 2014; Jeong et al. 2010; Song et al. 2011; Shim et al. 2018). In wheat, overexpression of TaNAC2, TaNAC67 and TaNAC29 in Arabidopsis significantly enhanced tolerance to drought, high salinity and low temperature stress (Mao et al. 2012; Mao et al. 2014; Huang et al. 2015). Overexpression of ONAC022 in rice improved drought and salt stress tolerance by upregulating the expression of ABA-signaling genes (Hong et al. 2016).

Ephemeral plants are a distinctive and crucial part of desert flora which can use snowmelt water and rainwater in early spring to germinate and grow rapidly completing their life cycles in two to three months (Fan et al. 2014; Huang et al. 2016). In China, there are 205 species of ephemeral plants, which mainly distributed in the southern region of the Gurbantunggut Desert, the largest fixed and semi-fixed desert in China (Xiao.et al. 2024). In April, May, and early June, ephemeral plants can cover up to 40% in this region and the ecologists revealed that they play important roles in sand dunes stabilization in spring (Wang et al. 2013). During their life cycle, ephemeral plants are subject to a various of harsh environments, including drought, low temperature and high soil salinity. Therefore, understanding the genetic foundation of the ecological adaptation mechanism in ephemeral plants is an extremely important research area.

Eremopyrum triticeum (Poaceae), a desert ephemeral plant and an important wild relative of wheat (Frederiksen and Bothmer, 1995), is distributed in the desert areas of China, Russia, Iran, Kazakhstan and Kyrgyz. E. triticeum has developed a variety of potential adaptations to cope with harsh environments, including fast growing and reproduction, high photosynthetic capacity, seed yield, low temperature tolerance, drought and salt resistance (Zhang, 2014). Understanding the potential molecular mechanism underling E. triticeum adaptation to extreme environmental conditions is is crucial for using its stress-related genes to enhance crop stress tolerance. In this study, two NAC transcription factor genes, namely EtNAC1 and EtNAC2, were cloned from E. triticeum. Gene expression characteristics demonstrated that EtNAC1/2 responded to drought, high salinity and ABA stresses. Overexpression of EtNAC1/2 in yeast (Saccharomyces cerevisiae) improved tolerance to PEG, salt, cold and H₂O₂ stress. Overexpression of EtNAC1/2 in Arabidopsis improved drought and salt stress tolerance, increased ABA sensitivity, and changed the stomatal aperture under drought conditions. Our data provide new insights into the molecular mechanisms of E. triticeum adapts to abiotic stress in the desert environment and give potential genes for improving crop stress tolerance via genetic engineering.

Plant materials and stress treatments

Seeds of E. triticeum were collected from the desert of the Junggar Basin (Xinjiang, China) in June 2022. Seeds were planted in pots containing nutrient-rich soil, perlite and vermiculite in a ratio of 3:1:1, and seedlings were cultivated in a growth chamber with a 16-h light/8-h dark period at 22°C. Young leaf and root (two-week-old seedlings), and mature root, leaf and stem (four-week-old seedlings) were collected for gene expression analysis. For stress treatments, two-week-old seedlings were subjected to different abiotic stress treatment, namely, drought (15% PEG 6000), salt (200 mM NaCl), and ABA (100 µM), followed by leaf sampling at designated time intervals (1, 3, 6, 12, 24, and 48 hours).

Cloning and phylogenetic analysis of EtNAC1 and EtNAC2

Total RNA was extracted from samples using RNAprep Pure Plant Kit (TIANGEN, Beijing, China), and first-strand cDNA was synthesized using a SuperScript III first-strand cDNA synthesis kit (Invitrogen). The EtNAC1 and EtNAC2 genes were cloned by PCR using sequence-specific primers (Table S1). The PCR products were purified and linked to pMDTM19-T vector to generate recombinant vector pMD-EtNAC1 and pMD-EtNAC2, and the positive clones were sequenced by Shenggong Biological Engineering Co. (Shanghai, China). The multiple sequence alignment of EtNAC1/2 and 14 NAC proteins from various plant species was carried out using Clustal X version 1.92 and analyzed by ESPript 3 (https://espript.ibcp.fr). The phylogenetic tree of EtNAC1/2 and NAC proteins from Triticum aestivum, Oryza sativa, Arabidopsis and other plant species was constructed using the MEGA 7.0 by the neighbor-joining (NJ) method with a bootstrap value of 1000, and subsequently refined using the iTOL online platform (https://itol.embl.de).

The expression analysis of EtNAC1 and EtNAC2

Quantitative real-time PCR (qRT–PCR) was used to analyze the expression patterns of EtNAC1 and EtNAC2 genes in different tissues of E. triticeum and under different abiotic stress conditions. qRT-PCR was conducted using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) on a 7500 Fast Real-time PCR System (Applied Biosystems, Waltham, USA), and three biological replications were performed. The 18S rRNA gene of E. triticeum (GenBank ID PQ072787) was used as the internal reference. The 2^−∆∆CT method (Livak and Schmittgen 2001) was used for detecting the relative gene expression levels. A list of primers used are given in Table S1.

Subcellular localization of EtNAC1 and EtNAC2

The complete coding regions of EtNAC1 and EtNAC2, excluding the stop codon, were fused with green fluorescent protein (GFP) at the N-terminus in the pBI121 vector to generate the 35S::EtNAC1-GFP and 35S::EtNAC2-GFP constructs, respectively, driven by cauliflower mosaic virus (CaMV) 35S promoter. The recombinant plasmids were then transformed into onion epidermal cells by Agrobacterium-mediated method (Zheng et al. 2012). After 2 days incubation in the dark on MS medium at 28°C, GFP signals were observed using a confocal microscope (Olympus FV1200).

Stress tolerance assays of EtNAC1/2 genes in yeast

The open reading frames (ORFs) of EtNAC1 and EtNAC2 were cloned into the BamH I and Xho I site od yeast expression vector pYES2 (Invitrogen), which is regulated by the GAL1 promoter, to construct the recombinant plasmids pYES2-EtNAC1 and pYES2-EtNAC2. Then, the recombinant vectors and the empty vector (pYES2) were transformed into the yeast strain Saccharomyces cerevisiae (INVSc1) using the lithium acetate method (Kawai et al. 2010), respectively. The transformants were subsequently cultured on uracil-deficient synthetic complete (SCU) media containing 2% (w/v) galactose at 30°C for 36 h. The specific primers with BamH I and Xho I sites are shown in Table S1.

For stress tolerance assays, the yeast strains transformed with pYES2-EtNAC1, pYES2-EtNAC2 or the empty pYES2 vector (control) were induced with 2% galactose and then they were diluted in series: 10⁰, 10^− 1, 10^− 2, 10^− 3, and 10^− 4 times. 2 µL aliquots were spotted onto SCU solid medium with 2% glucose containing or not 30% PEG 6000 (w/v), 2 M NaCl, and 1 M H₂O₂ and incubated at 30°C for 3 days. For cold tolerance testing, the yeast cells were exposed to -20°C for 6 hours in a refrigerator.

Generation of EtNAC1 and EtNAC2 transgenic Arabidopsis

The EtNAC1 and EtNAC2 ORFs were subcloned into the plant expression vector pCAMBIA2300 at the Sma I and Xba I restriction sites, which was driven by the CaMV 35S promoter, to construct the 35S::EtNAC1 and 35S::EtNAC2 vectors, respectively. The recombinant vectors pCAMBIA2300-EtNAC1, pCAMBIA2300-EtNAC2 and pCAMBIA2300 were then introduced into Arabidopsis plants using the floral dipping method (Clough and Bent 1998). Homozygous transgenic Arabidopsis lines were selected on MS medium supplemented with kanamycin (50 mg/L) and verified by genome PCR analysis. The T₃ transgenic overexpressed lines were used for abiotic stress treatment and functional analysis.

Analysis of fresh weight, water loss, and chlorophyll leaching rate in Arabidopsis leaves

Arabidopsis seeds from the T₃ generation of wild-type (WT), 35S::EtNAC1 and 35S::EtNAC2 overexpression lines were planted in a soil mix (soil:vermiculite = 3:1). The detached leaves from WT rosettes at 6 weeks of age, EtNAC1 overexpression lines (L2, L3 and L5), and EtNAC2 overexpression lines (L6, L7 and L9) were weighted. For the water loss assay, 6-week-old transgenic Arabidopsis and WT plants were exposed to the dark for 6 h to induce stomatal closure. Then, rosette leaves were detached and weighted every 30 min to evaluate the water loss rate (Patwari et al. 2019). The temperature was 25°C and the humidity was 40%. Three independent biological replicates were performed for each sample. A chlorophyll leaching assay was carried out to assess the epidermal permeability of EtNAC1 and EtNAC2 transgenic Arabidopsis leaves. The rosette leaves of transgenic Arabidopsis and WT (control) from 6-week-old plants were weighed and soaked into 80% ethanol. Chlorophyll leaching at each time point was calculated following the method outlined previously (Lolle et al. 1998).

Drought and salt tolerance assay of EtNAC1/2 transgenic Arabidopsis plants

Homozygous T₃ generation transgenic Arabidopsis with EtNAC1/2 and WT plants were cultivated for 4 weeks under favorable water conditions, and subsequently exposed to drought (withheld watering for 10 days followed by rewatering for 3 days), or salt (irrigated 3 times with 50 mL of 200 mM NaCl at 3-day intervals over a period of 15 days) (Dai et al. 2018). After drought and salt treatments, survival rates, as well as malondialdehyde (MDA) and proline (Pro) content in leaves were assessed using established methods (Wu et al. 2018). Each measurement was performed with three independent biological replicates.

Growth assessments of transgenic Arabidopsis under mannitol, ABA, and salt stress

To evaluate the abiotic stress tolerance of EtNAC1/2 transgenic lines under mannitol, NaCl and ABA stress, Arabidopsis seeds from EtNAC1/2 transgenic lines and WT were planted on solid 1/2 MS medium presence or absence of mannitol (100 mM), ABA (1 µM) and NaCl (75 mM), following protocols outlined by Dixit et al. (2018). The seedlings were cultivated under long-day conditions (16 h light/8 h dark) at 22°C. After 21 days, the length of the primary root was measured, with three independent replicates for each condition.

Stomatal aperture analysis

Stomatal apertures were evaluated according to the method described by Pei et al. (1997). EtNAC1/2 transgenic Arabidopsis and WT plants were exposed to drought for 10 days. Rosette leaves were submerged in solutions containing 10 mM KCl, 50 mM CaCl₂, and 10 mM Mes/Tris (pH 5.6), and subjected to darkness for 2 h followed by 3 h light. Subsequently, the rosette leaves were dried with filter paper and subjected to dehydration for 20 mins. The stomata in epidermal strips from Arabidopsis leaves was observed using a light microscope (Olympus ix71, Tokyo, Japan). The stomatal aperture (width to length ratio) was measured by conducting on 20 randomly selected stomata per sample at a magnification of 400x. The collected data were subsequently processed and analyzed using Image J software (Wang et al. 2019).

Statistical Analysis

All experimental data were derived from a minimum of three independent replicates. Statistical significance was measured using GraphPad Prism 8 (t-test). P < 0.05, 0.01 were considered to be significantly different from the control.

Identification and sequence analysis of the EtNAC1 and EtNAC2

In this study, two NAC genes (EtNAC1 and EtNAC2) were identified from E. triticeum treated with PEG-induced stress. The ORF of EtNAC1 was 927 bp, encoding a protein of 308 amino acids, with a calculated molecular weight of 34.08 kDa. While the ORF of EtNAC2 was 924 bp encoding 307 amino acids with a calculated molecular weight of 34 kDa. The identity of EtNAC1 and EtNAC2 proteins was 93.7%. Then, the sequences of EtNAC1 and EtNAC2 were submitted to the GenBank database with accession numbers PP860406 for EtNAC1 and PP860405 for EtNAC2.

Multiple sequence alignment indicated that both EtNAC1 and EtNAC2 possessed a highly conserved DNA-binding domain at the N-terminal, including five subdomains (Ⅰ, Ⅱ, Ⅲ, Ⅳ, and Ⅴ), consisting with typical NAC domain characteristics (Fig. 1a). Phylogenetic analysis using NAC genes of 19 plants species showed that EtNAC1 was homologous to AtNAC21 in Aegilops tauschii subsp, and EtNAC2 was homologous to abiotic stress-related TaNAC7 in wheat (Triticum aestivum) (Tang et al. 2012) and BdNAC21 in Brachypodium distachyon (You et al. 2015) (Fig. 1b). Therefore, we hypothesized that EtNAC1 and EtNAC2 have potentially function as stress-responsive genes in E. triticeum.

EtNAC1 and EtNAC2 are induced by ABA, salt and PEG stress

To explore the possible roles of EtNAC1 and EtNAC2 in the abiotic stress response, E. triticeum seedlings (4-week-old) were exposed to PEG 6000 (15%, w/v), NaCl (200 mM), and ABA (100 µM), and their expression patterns were evaluated. qRT-PCR was performed to detect the relative transcript abundance in various tissues, PEG, NaCl and ABA treatment. The results showed that both EtNAC1 and EtNAC2 were mainly expressed in mature root (4-week-old E. triticeum), compared to mature stem, leaf and young (2-week-old E. triticeum) root and leaf (Fig. 2a). EtNAC1 transcription level peaked after 12 h of PEG stress in the leaf, which showed a 45-fold higher expression level compared to the control (Fig. 2b). Under NaCl stress, the expression of EtNAC2 increased gradually, significantly peaking at 1 h, and was 90-fold higher than the control at 3 h (Fig. 2c). In the ABA treatment, both EtNAC1 and EtNAC2 were highly induced at 6 h and 48 h, which showed a 10-fold increase compared to the control (Fig. 2d). These findings indicated that EtNAC1 and EtNAC2 are involved in the response to drought, salt stress, and ABA signaling in plants.

EtNAC1 and EtNAC2 are nuclear proteins

EtNAC1 and EtNAC2 proteins were predicted to be localized in the nucleus using WoLF PSORT (http://wolfpsort.org/). To validate their subcellular localization, the ORFs of EtNAC1 and EtNAC2 were fused to the GFP at the N-terminus in the pBI121 vector to generate the 35S::EtNAC1-GFP and 35S::EtNAC2-GFP constructs driven by the CaMV 35S promoter. Then, the constructs were transiently expressed in onion epidermis cells. As expected, the 35S::EtNAC1-GFP and 35S::EtNAC2-GFP constructs emitted green fluorescence predominantly in nuclei, indicating that the EtNAC1 and EtNAC2 are nuclear proteins (Fig. 3).

Overexpression of EtNAC1 and EtNAC2 enhanced yeast resistance to various abiotic stresses

To elucidate the biological function of EtNAC1 and EtNAC2 in abiotic stress tolerance, the recombinant yeast cells harboring EtNAC1 (pYES2-EtNAC1), EtNAC2 (pYES2-EtNAC2) and the control cells harboring empty pYES2 were treated with PEG 6000 (30%, w/v), NaCl (2 M), H₂O₂ (1 M) or low temperature (-20°C). The findings indicated that the growth rate of yeast cells expressing EtNAC1, EtNAC2 or empty pYES2 had no difference under normal condition (Fig. 4). However, they exhibited noticeable differences after drought, salt, low temperature and oxidative (H₂O₂) treatment. The recombinant yeast cells yeast cells expressing EtNAC1 and EtNAC2 exhibited enhanced tolerance to drought, salinity, low temperature, and oxidative stress compared to the control, suggesting that EtNAC1 and EtNAC2 confers drought, salinity, low temperature and oxidative tolerance to transgenic yeast cells. Hence, these two EtNACs are participated in drought, salt, low temperature and oxidative stress tolerance.

Overexpressing EtNAC1 and EtNAC2 in Arabidopsis improved rosette growth, decreased water loss and cuticular permeability

To study the functions of EtNAC1 and EtNAC2 in vivo, transgenic Arabidopsis plants overexpressing these two genes were constructed, and three independent lines of 35S::EtNAC1 (L2, L3, and L5) and 35S::EtNAC2 (L6, L7 and L9) were used for further analysis. The growth of the transgenic Arabidopsis lines was analyzed under normal condition. After 6 weeks of growth in pots, we observed the phenotype of transgenic Arabidopsis and observed that the rosette leaf size was greater in transgenic lines compared to WT plants (Fig. 5a). Next, we quantified the fresh weight of rosette leaves from WT plants and EtNAC1 and EtNAC2 overexpressing lines, and found that the overexpression lines showed increased fresh weight compared to WT (Fig. 5b). The water retention capacity was also evaluated by determining the water loss rate from detached rosette leaves. As shown in Fig. 5c, d, the water loss rates in the EtNAC1/2 overexpression lines were notably lower compared to WT across all time points, suggesting that transpiration occurred more slowly from the EtNAC1/2 overexpression lines compared to WT. Furthermore, we investigated the cuticular permeability and water loss rate in transgenic lines, and found that the chlorophyll leaching rate from EtNAC1 and EtNAC2 overexpressing lines was significantly reduced compared to WT leaves (Fig. 5e, f), suggesting decreased cuticular permeability in the overexpressing Arabidopsis plants.

Overexpression of EtNAC1/2 in Arabidopsis increased osmotic stress tolerance and enhanced ABA sensitivity in MS media

To detect the osmotic stress tolerance of EtNAC1/2 transgenic lines, 75 mM NaCl, 100 µM mannitol and 1 µM ABA were supplemented on 1/2 MS medium. After three weeks, the root length of Arabidopsis was measured. The results indicated that the transgenic lines and WT plants showed no clear differences in root length without stress, while the root length of EtNAC1 and EtNAC2 transgenic lines was significantly longer than that of WT under mannitol and NaCl treatments (Fig. 6a, b, c and d). The results indicated that overexpressing EtNAC1 and EtNAC2 improved drought and salt tolerance in transgenic Arabidopsis plants. However, under ABA treatment, the root length of EtNAC1 and EtNAC2 transgenic lines was severely inhibited compared to WT plants, suggesting that overexpression of EtNAC1 and EtNAC2 in Arabidopsis increased ABA sensitivity (Fig. 6a, b, c and d). These results suggested that EtNAC1 and EtNAC2 may play a role in salt and drought stress response by ABA-dependent pathway.

EtNAC1 and EtNAC2 improved tolerance to drought and salt stress in transgenic Arabidopsis

To investigate the function of EtNAC1 and EtNAC2 in abiotic stress tolerance, WT plants and transgenic lines overexpressing EtNAC1 and EtNAC2 were exposed to drought stress (withholding water for 10 days followed by rewatering for 3 days), or salt stress (irrigated with 200 mM NaCl at 3-day intervals for 15 days). Then, the water loss rate, MDA content and Pro content were evaluated. After 10 days of drought conditions and rewatered for 3 days, the leaves of WT plants exhibited more wilting symptoms compared to those of the EtNAC1 overexpression lines. (L2, L3, and L5) and EtNAC2 overexpression lines (L6, L7, and L9) (Fig. 7a). Following 200 mM NaCl treatment, both WT and transgenic lines experienced leaf yellowing and wilting; however, WT plants incurred more severe damage (Fig. 7b). After drought and salt stress, the transgenic lines exhibited significantly increased Pro levels and decreased MDA levels compared to WT plants (Fig. 7c, d, e, f). Furthermore, after drought and salt stress, higher survival rates were found in EtNAC1 and EtNAC2 overexpression lines compared to WT plants (Fig. 7g, h). The above results suggest that EtNAC1 and EtNAC2 involved in plant drought and salt tolerance.

EtNAC1 and EtNAC2 participated in drought stress-induced stomatal closure

Stomatal closure is a crucial adaptive response of plants to drought stress, effectively reducing water loss through transpiration pathways. Previous research has highlighted the role of NAC TF genes, such as VvNAC17 and ZmNAC49, in regulating stomatal density and enhancing drought tolerance in grapevine and maize (Ju et al. 2020; Xiang et al. 2021). To explore whether EtNAC1 and EtNAC2 influence water loss through stomatal modulation, we observed the stomatal movement in transgenic lines under drought stress conditions. Under normal conditions, no significant differences of stomatal aperture were found between EtNAC1/2 overexpression lines and WT plants. However, after 10 days of drought stress, the stomatal aperture (width/length ratio) in EtNAC1/2 overexpression lines was significantly reduced (Fig. 8a, b). These results suggest that EtNAC1 and EtNAC2 play important roles in promoting stomatal closure and reducing transpiration to prevent water loss under drought stress.

Several NAC TFs are known to participate in various physiological processes in plants, including growth and development, senescence, as well as responses to biotic and abiotic stresses (Iuchi et al. 2001; Fang et al. 2015). E. triticeum, a representative desert ephemeral species, plays a crucial role in maintaining ecosystem stability and environmental conservation in the Gurbantunggut Desert. It can generate substantial forage biomass in harsh environments, and could serve as an excellent resource for studying stress-responsive genes like NAC to promote growth and maintaining yield under stress conditions, which may be be absent in numerous cultivated crops. In this study, we isolated two new NAC TFs, EtNAC1 and EtNAC2, from E. triticeum. EtNAC1 and EtNAC2 was expressed in response to drought, high salinity, and exogenous ABA treatments. Furthermore, EtNAC1 and EtNAC2 contain a conserved NAC domain at the N-terminus (Fig. 1) and are localized in the nucleus (Fig. 3). Phylogenetic analysis indicated that EtNAC1 and EtNAC2 were closely related to T. aestivum TaNAC7 and B. distachyon BdNAC21, both of which have been reported to participate in the response to abiotic stress (Tang et al. 2012; You et al. 2015). Therefore, the results above imply that EtNAC1 and EtNAC2 are multifunctional NAC TFs, involved in responses to abiotic stress and influencing plant growth and development.

Numerous NAC transcription factors have been documented to participate in plant responses to various abiotic stresses such as salt, drought, heat, and cold treatments (Hu et al. 2008; Fang et al. 2015; Wang et al. 2021). Expression analysis indicated that both EtNAC1 and EtNAC2 exhibited high expression levels in mature roots of E. triticeum (Fig. 2a), indicating their potential significant roles in root development. Furthermore, the expression of EtNAC1 and EtNAC2 were significantly upregulated in response to PEG, NaCl, and exogenous ABA treatment (Fig. 2b, c, d), indicating that they may involve in salt and drought signal transduction pathways. Heterologous expression of EtNAC1 and EtNAC2 in yeast improved tolerance to H₂O₂, drought, low temperature, and salinity (Fig. 4). To gain deeper insights into the function of EtNAC1/2, we ectopically expressed these two genes individually in A. thaliana. We observed that the overexpression of EtNAC1/2 in A. thaliana improved tolerance to mannitol, salt, and exogenous ABA (Fig. 6, Fig. 7). Transgenic lines showed higher survival rate (Fig. 7g, h) and longer root length (Fig. 6) than control plants under salt and mannitol stress. A robust root system enables plants to absorb greater amounts of water from the soil, thereby minimizing damage from water loss and enhancing plant osmotic tolerance (Guo et al. 2015; Wei et al. 2018). Under stress conditions, the transgenic plants displayed elongated roots, indicating that EtNAC1/2 may confer enhanced resistance by modulating the root system.

Drought stress impacts both plant growth and development, whereas drought-resistant plants possess intricate mechanisms for osmotic regulation to survive under drought conditions (Ozturk et al. 2021). Water scarcity can alter chlorophyll levels in plants, and severe water stress can damage chloroplast lamellar structures, reducing chlorophyll content, diminishing photosystem II activity, impairing the Hill reaction, and curtailing electron transport and photosynthetic phosphorylation, ultimately decreasing photosynthesis (Manivannan et al. 2007). Therefore, variation in chlorophyll content under drought conditions can partially reflect the ability for stress tolerance in plants, and increases in chlorophyll content indicate enhanced stimulation of cortical photosynthesis in response to drought (Rustioni et al. 2021). In this study, we analyzed chlorophyll leaching and leaf dehydration rates in WT and EtNAC1/2 overexpression lines during drought stress, demonstrating that both parameters were lower in the EtNAC1/2 overexpression lines compared to the WT (Fig. 5c, d, e, f). Hence, we hypothesize that the decrease in chlorophyll leakage and water loss might due to the reduced tissue permeability in transgenic lines. These findings imply that EtNAC1/2 have a close relationship with cell permeability, chlorophyll leakage, and water loss in plants, might have crucial roles in drought tolerance.

Drought and salt stress induce oxidative stress in plants, leading to lipid peroxidation and membrane degradation, resulting in an increase in MDA content, which is a widely used indicator to assess the severity of membrane lipid damage (Hu et al. 2012; Moore and Roberts,1998). Our results demonstrated that under drought and salt stress conditions, the EtNAC1/2 overexpression lines exhibited significantly reduced MDA levels compared to the control group. (Fig. 6a, b, c and d), suggesting that the EtNAC1/2 overexpression plants may undergo reduced lipid peroxidation and membrane damage. Proline is also closely associated with osmotic adjustment in plants, serving as a signaling molecule in response to abiotic stress and participating in the expression regulation of stress-responsive genes (Ghosh et al. 2022). The A. thaliana plants overexpressing EtNAC1/2 accumulated significantly higher level of proline under drought and salt stress conditions compared to the WT plants (Fig. 7e, f). These findings demonstrated that EtNAC1/2 may play important roles in enhancing the resistance ability of plants to drought and salt stress.

Several previous studies have documented that plants can minimize water loss through the regulation of stomatal closure, a process that is influenced by ABA (Wei et al. 2018; Butt et al. 2017; Hura et al. 2022). As the gateway for water and gas exchange between plant leaves and the surrounding environment, stomata play an important role in sensing environmental changes. The status of opening and closing of the stomata is crucial for plants to adapt to the external environment (Hetherington et al. 2003). To verify whether the improved drought tolerance of the transgenic plants was linked to the regulation of stomatal movement, we detected the stomatal behavior in the leaves of EtNAC1/2 overexpressing lines and WT plants after exposure to drought stress. The results showed that the leaves of EtNAC1/2 overexpression lines exhibited reduced stomatal aperture after the drought stress (Fig. 8), suggesting that EtNAC1 and EtNAC2 help minimize water loss through transpiration via a stomatal closure pathway. Further investigations are required to elucidate the molecular mechanisms of EtNAC1/2 in regulating the stomatal movement.

In this study, two typical NAC TF genes, EtNAC1 and EtNAC2, were cloned from E. triticeum and functionally verified in overexpressing yeast and A. thaliana. Overexpressing EtNAC1 and EtNAC2 in A. thaliana enhanced drought and salt tolerance by preventing the water loss and chlorophyll loss of leaves, reducing cellular membrane damage, and stimulating root elongation. EtNAC1 and EtNAC2 contribute to enhancing drought resistance in transgenic plants through the regulation of stomatal closure. This study indicated that EtNAC1 and EtNAC2 act as positive regulators of drought and salt stress, suggesting their potential for enhancing stress tolerance in crops through transgenic breeding.

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical statement

No Ethical statement was reported.

Author Contribution

L and X conceived and designed the experiments. Material preparation, data collection and analysis were performed by X, J, M and X. All authors read and approved the final manuscript.

Acknowledgements

This work was supported by the Third Xinjiang Scientific Expedition Program (grant no. 2022xjkk1503).

Data Availability

All the data in this study are included in this manuscript.

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Overexpression of NAC transcription factors from the desert ephemeral plant Eremopyrum triticeum promoted abiotic stress tolerance in Arabidopsis thaliana

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Abstract

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Introduction

Materials and Methods

Plant materials and stress treatments

Subcellular localization of EtNAC1 and EtNAC2

Analysis of fresh weight, water loss, and chlorophyll leaching rate in Arabidopsis leaves

Growth assessments of transgenic Arabidopsis under mannitol, ABA, and salt stress

Stomatal aperture analysis

Statistical Analysis

Result

EtNAC1 and EtNAC2 are nuclear proteins

Discussion

Conclusions

Declarations

Ethical statement

Author Contribution

Acknowledgements

Data Availability

References

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Supplementary Files

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Version 1