PagWOX11/12a positively regulates the PagSAUR36 gene that enhances adventitious root development in poplar.

Adventitious root (AR) development is an extremely complex biological process that is affected by many intrinsic factors and extrinsic stimuli. Some WUSCHEL-related homeobox (WOX) transcription factors have been reported to play important roles in AR development, but their functional relationships with auxin signaling are poorly understood, especially the developmental plasticity of roots in response to adversity stress. Here, we identified that the WOX11/12a-SMALL AUXIN UP RNA36 (SAUR36) module mediates AR development through the auxin pathway in poplar, as well as under salt stress. PagWOX11/12a displayed inducible expression during AR development, and overexpression of PagWOX11/12a significantly promoted AR development and increased salt tolerance in poplar, whereas dominant repression of PagWOX11/12a produced the opposite phenotype. PagWOX11/12a proteins directly bind to the SAUR36 promoter to regulate SAUR36 transcription, and this binding was enhanced during salt stress. Genetic modification of PagWOX11/12a-PagSAUR36 expression revealed that the PagWOX11/12a-PagSAUR36 module is crucial for controlling AR development via the auxin pathway. Overall, our results indicate that a novel WOX11-SAUR-auxin signaling regulatory module is required for AR development in poplar. These findings provide key insights and a better understanding of the involvement of WOX11 in root developmental plasticity in saline environments.

An Efficient Agrobacterium-Mediated Transformation Method for Hybrid Poplar 84K (Populus alba × P. glandulosa) Using Calli as Explants.

A highly efficient Agrobacterium-mediated transformation method is needed for the molecular study of model tree species such as hybrid poplar 84K (Populus alba × P. glandulosa cv. '84K'). In this study, we report a callus-based transformation method that exhibits high efficiency and reproducibility. The optimized callus induction medium (CIM1) induced the development of calli from leaves with high efficiency, and multiple shoots were induced from calli growing on the optimized shoot induction medium (SIM1). Factors affecting the transformation frequency of calli were optimized as follows: Agrobacterium concentration sets at an OD600 of 0.6, Agrobacterium infective suspension with an acetosyringone (AS) concentration of 100 µM, infection time of 15 min, cocultivation duration of 2 days and precultivation duration of 6 days. Using this method, transgenic plants are obtained within approximately 2 months with a transformation frequency greater than 50%. Polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR) and ß-galactosidase (GUS) histochemical staining analyses confirmed the successful generation of stable transformants. Additionally, the calli from leaves were subcultured and used to obtain new explants; the high transformation efficiency was still maintained in subcultured calli after 6 cycles. This method provides a reference for developing effective transformation protocols for other poplar species.

PagWOX11/12a activates PagCYP736A12 gene that facilitates salt tolerance in poplar.

The WUSCHEL-related homeobox (WOX) transcription factors WOX11 and WOX12 regulate adventitious rooting and responses to stress. The underlying physiological and molecular regulatory mechanisms in salt stress tolerance remain largely unexplored. Here, we characterized the roles of PagWOX11/12a from 84K poplar (Populus alba × P. glandulosa) and the underlying regulatory mechanism in salt stress. PagWOX11/12a was strongly induced by salt stress in roots. Overexpression of PagWOX11/12a in poplar enhanced salt tolerance, as evident by the promotion of growth-related biomass. In contrast, salt-treated PagWOX11/12a dominant repression plants displayed reduced biomass growth. Under salt stress conditions, PagWOX11/12a-overexpressed lines showed higher reactive oxygen species (ROS) scavenging capacity and lower accumulation of hydrogen peroxide (H2 O2 ) than non-transgenic 84K plants, whereas the suppressors displayed the opposite phenotype. In addition, PagWOX11/12a directly bound to the promoter region of PagCYP736A12 and regulated PagCYP736A12 expression. The activated PagCYP736A12 could enhance ROS scavenging, thus reducing H2 O2 levels in roots under salt stress in PagWOX11/12a-overexpressed poplars. The collective results support the important role of PagWOX11/12a in salt acclimation of poplar trees, indicating that PagWOX11/12a enhances salt tolerance through modulation of ROS scavenging by directly regulating PagCYP736A12 expression in poplar.

ThHSFA1 Confers Salt Stress Tolerance through Modulation of Reactive Oxygen Species Scavenging by Directly Regulating ThWRKY4.

Heat shock transcription factors (HSFs) play critical roles in several types of environmental stresses. However, the detailed regulatory mechanisms in response to salt stress are still largely unknown. In this study, we examined the salt-induced transcriptional responses of ThHSFA1-ThWRKY4 in Tamarix hispida and their functions and regulatory mechanisms in salt tolerance. ThHSFA1 protein acts as an upstream regulator that can directly activate ThWRKY4 expression by binding to the heat shock element (HSE) of the ThWRKY4 promoter using yeast one-hybrid (Y1H), chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays. ThHSFA1 and ThWRKY4 expression was significantly induced by salt stress and abscisic acid (ABA) treatment in the roots and leaves of T. hispida. ThHSFA1 is a nuclear-localized protein with transactivation activity at the C-terminus. Compared to nontransgenic plants, transgenic plants overexpressing ThHSFA1 displayed enhanced salt tolerance and exhibited reduced reactive oxygen species (ROS) levels and increased antioxidant enzyme activity levels under salt stress. Therefore, we further concluded that ThHSFA1 mediated the regulation of ThWRKY4 in response to salt stress in T. hispida.

WUSCHEL-related homeobox gene PagWOX11/12a responds to drought stress by enhancing root elongation and biomass growth in poplar.

In plants, a large root system improves the uptake of water and nutrients, and is important for responding to drought stress. The poplar WUSCHEL-related homeobox (WOX) transcription factor promotes adventitious rooting, but its regulation of root growth in response to drought stress remains elusive. In this study, we found that PagWOX11/12a from hybrid poplar 84K (Populus alba×Populus glandulosa) is expressed predominantly in the roots and is strongly induced by drought stress. Compared with non-transgenic 84K plants, transgenic poplar plants overexpressing PagWOX11/12a displayed increased root biomass and enhanced drought tolerance, while opposite phenotypes were observed for PagWOX11/12a dominant repression plants. PagWOX11/12a functions as a nuclear transcriptional activator with a transactivation domain at the C-terminus. In addition, PagERF35 was found to specifically bind to a dehydration-responsive element (DRE) within the PagWOX11/12a promoter and activate PagWOX11/12a gene expression. These results indicate that PagERF35 may activate PagWOX11/12a expression in response to drought stress by promoting root elongation and biomass, thereby increasing drought tolerance of poplar.

The auxin receptor TIR1 homolog (PagFBL 1) regulates adventitious rooting through interactions with Aux/IAA28 in Populus.

Adventitious roots occur naturally in many species and can also be induced from explants of some tree species including Populus, providing an important means of clonal propagation. Auxin has been identified as playing a crucial role in adventitious root formation, but the associated molecular regulatory mechanisms need to be elucidated. In this study, we examined the role of PagFBL1, the hybrid poplar (Populus alba × P. glandulosa clone 84K) homolog of Arabidopsis auxin receptor TIR1, in adventitious root formation in poplar. Similar to the distribution pattern of auxin during initiation of adventitious roots, PagFBL1 expression was concentrated in the cambium and secondary phloem in stems during adventitious root induction and initiation phases, but decreased in emerging adventitious root primordia. Overexpressing PagFBL1 stimulated adventitious root formation and increased root biomass, while knock-down of PagFBL1 transcript levels delayed adventitious root formation and decreased root biomass. Transcriptome analyses of PagFBL1 overexpressing lines indicated that an extensive remodelling of gene expression was stimulated by auxin signalling pathway during early adventitious root formation. In addition, PagIAA28 was identified as downstream targets of PagFBL1. We propose that the PagFBL1-PagIAA28 module promotes adventitious rooting and could be targeted to improve Populus propagation by cuttings.

Proteomic Analysis and Identification of Possible Allergenic Proteins in Mature Pollen of Populus tomentosa.

Pollen grains from Populus tomentosa, a widely cultivated tree in northern area of China, are considered to be an important aeroallergen causing severe allergic diseases. To gain insight into their allergenic components, mature Populus tomentosa pollen proteins were analyzed by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF MS). A total of 412 spots from mature pollen were resolved on pH 4-7 immobilized pH gradient (IPG) strips and 159 distinct proteins were identified from 242 spots analyzed. The identified proteins were categorized based on their functional role in the pollen, which included proteins involved in energy regulation, protein fate, protein synthesis and processing, metabolism, defense/stress responses, development and other functional categories. Moreover, among the identified proteins, 27 proteins were identified as putative allergens using the Structural Database of Allergenic Proteins (SDAP) tool and Allergen Online. The expression patterns of these putative allergen genes indicate that several of these genes are highly expressed in pollen. The identified putative allergens have the potential to improve specific diagnosis and can be used to develop vaccines for immunotherapy against poplar pollen allergy.

Identification of novel cis-elements bound by BplMYB46 involved in abiotic stress responses and secondary wall deposition.

Transcription factors (TFs) play vital roles in various biological processes by binding to cis-acting elements to control expressions of their target genes. The MYB TF BplMYB46, from Betula platyphylla, is involved in abiotic stress responses and secondary wall deposition. In the present study, we used a TF-centered yeast one-hybrid technology (TF-centered Y1H) to identify the cis-acting elements bound by BplMYB46. We screened a short-insert random library and identified three cis-elements bound by BplMYB46: an E-box (CA(A/T/C)(A/G/C)TG) and two novel motifs, a TC-box (T(G/A)TCG(C/G)) and a GT-box (A(G/T)T(A/C)GT(T/G)C). Chromatin immunoprecipitation (ChIP) and effector-reporter coexpression assays in Nicotiana tabacum confirmed binding of BplMYB46 to the TC-box, GT-box, and E-box motifs in the promoters of the phenylalanine ammonia lyase (PAL), peroxidase (POD), and superoxide dismutase (SOD) genes, which function in abiotic stress tolerance and secondary wall biosynthesis. This finding improves our understanding of potential regulatory mechanisms in the response to abiotic stress and secondary wall deposition of BplMYB46 in B. platyphylla.

Expression of the MYB transcription factor gene BplMYB46 affects abiotic stress tolerance and secondary cell wall deposition in Betula platyphylla.

Plant MYB transcription factors control diverse biological processes, such as differentiation, development and abiotic stress responses. In this study, we characterized BplMYB46, an MYB gene from Betula platyphylla (birch) that is involved in both abiotic stress tolerance and secondary wall biosynthesis. BplMYB46 can act as a transcriptional activator in yeast and tobacco. We generated transgenic birch plants with overexpressing or silencing of BplMYB46 and subjected them to gain- or loss-of-function analysis. The results suggest that BplMYB46 improves salt and osmotic tolerance by affecting the expression of genes including SOD, POD and P5CS to increase both reactive oxygen species scavenging and proline levels. In addition, BplMYB46 appears to be involved in controlling stomatal aperture to reduce water loss. Overexpression of BplMYB46 increases lignin deposition, secondary cell wall thickness and the expression of genes in secondary cell wall formation. Further analysis indicated that BplMYB46 binds to MYBCORE and AC-box motifs and may directly activate the expression of genes involved in abiotic stress responses and secondary cell wall biosynthesis whose promoters contain these motifs. The transgenic BplMYB46-overexpressing birch plants, which have improved salt and osmotic stress tolerance, higher lignin and cellulose content and lower hemicellulose content than the control, have potential applications in the forestry industry.

ThWRKY4 from Tamarix hispida Can Form Homodimers and Heterodimers and Is Involved in Abiotic Stress Responses.

WRKY proteins are a large family of transcription factors that are involved in diverse developmental processes and abiotic stress responses in plants. However, our knowledge of the regulatory mechanisms of WRKYs participation in protein-protein interactions is still fragmentary, and such protein-protein interactions are fundamental in understanding biological networks and the functions of proteins. In this study, we report that a WRKY protein from Tamarix hispida, ThWRKY4, can form both homodimers and heterodimers with ThWRKY2 and ThWRKY3. In addition, ThWRKY2 and ThWRKY3 can both bind to W-box motif with binding affinities similar to that of ThWRKY4. Further, the expression patterns of ThWRKY2 and ThWRKY3 are similar to that of ThWRKY4 when plants are exposed to abscisic acid (ABA). Subcellular localization shows that these three ThWRKY proteins are nuclear proteins. Taken together, these results demonstrate that ThWRKY4 is a dimeric protein that can form functional homodimers or heterodimers that are involved in abiotic stress responses.

ThERF1 regulates its target genes via binding to a novel cis-acting element in response to salt stress.

Ethylene responsive factors (ERFs) are plant-specific transcription factors that are involved in a variety of biological processes. We previously demonstrated that an ERF gene from Tamarix hispida, ThERF1, encodes a protein binding to GCC-box and DRE motifs and negatively modulates abiotic stress tolerance. In the present study, microarray analysis was performed to study the genes regulated by ThERF1 on a genomic scale. There were 154 and 307 genes (respectively representing 134 and 260 unique genes) significantly up- and downregulated by ThERF1 under salt stress conditions, respectively. A novel motif, named TTG, was identified to be recognized by ThERF1, which commonly presents in the promoters of ThERF1-targeted genes. The TTG motif is also bound by other ERFs of a different subfamily from T. hispida and Arabidopsis, indicating that it is commonly recognized by ERF proteins. The binding affinities of ERFs to the TTG motif are significantly induced by salt stress. The TTG motif is more enriched than the GCC-box and DRE motifs in the promoters of ThERF1-targeted genes. Taken together, these studies suggested that the TTG motif plays an important role in the gene expression regulated by ERFs in response to salt stress.

A novel method to identify the DNA motifs recognized by a defined transcription factor.

The interaction between a protein and DNA is involved in almost all cellular functions, and is vitally important in cellular processes. Two complementary approaches are used to detect the interactions between a transcription factor (TF) and DNA, i.e. the TF-centered or protein-DNA approach, and the gene-centered or DNA-protein approach. The yeast one-hybrid (Y1H) is a powerful and widely used system to identify DNA-protein interactions. However, a powerful method to study protein-DNA interactions like Y1H is lacking. Here, we developed a protein-DNA method based on the Y1H system to identify the motifs recognized by a defined TF, termed TF-centered Y1H. In this system, a random short DNA sequence insertion library was generated as the prey DNA sequences to interact with a defined TF as the bait. Using this system, novel interactions were detected between DNA motifs and the AtbZIP53 protein from Arabidopsis. We identified six motifs that were specifically bound by AtbZIP53, including five known motifs (DOF, G-box, I-box, BS1 and MY3) and a novel motif BRS1 [basic leucine zipper (bZIP) Recognized Site 1]. The different subfamily bZIP members also recognize these six motifs, further confirming the reliability of the TF-centered Y1H results. Taken together, these results demonstrated that TF-centered Y1H could identify quickly the motifs bound by a defined TF, representing a reliable and efficient approach with the advantages of Y1H. Therefore, this TF-centered Y1H may have a wide application in protein-DNA interaction studies.

A novel ethylene-responsive factor from Tamarix hispida, ThERF1, is a GCC-box- and DRE-motif binding protein that negatively modulates abiotic stress tolerance in Arabidopsis.

Ethylene-responsive factor (ERF) family is one of the largest families of plant-specific transcription factor that can positively or negatively regulate abiotic stress tolerance. However, their functions in regulating abiotic stress tolerance are still not fully understood. In this study, we characterized the functions of an ERF gene from Tamarix hispida, ThERF1, which can negatively regulate abiotic stress tolerance. The expression of ThERF1 was induced by salinity, PEG-simulated drought and abscisic acid (ABA) treatments. ThERF1 can specifically bind to GCC-box and DRE motifs. Overexpression of ThERF1 in transgenic Arabidopsis plants showed inhibited seed germination, and decreased fresh weight gain and root growth compared with wild-type (WT) plants. In addition, the transcript levels of several superoxide dismutase (SOD) and peroxidase (POD) genes in transgenic plants were significantly inhibited compared with in WT plants, resulting in decreased SOD and POD activities in transgenic plants under salt and drought stress conditions. Furthermore, the reactive oxygen species (ROS) levels, malondialdehyde (MDA) contents and cell membrane damage in ThERF1-transformed plants were all highly increased relative to WT plants. Our results suggest that ThERF1 negatively regulates abiotic stress tolerance by strongly inhibiting the expression of SOD and POD genes, leading to decreased ROS-scavenging ability.

PagWOX11/12a from hybrid poplar enhances drought tolerance by modulating reactive oxygen species.

WOX11/12 is a homeobox gene of WOX11 and WOX12 in Arabidopsis that plays important roles in crown root development and growth. It has been reported that WOX11/12 participates in adventitious root (AR) formation and different abiotic stress responses, but the downstream regulatory network of WOX11/12 in poplar remains to be further investigated. In this study, we found that PagWOX11/12a is strongly induced by PEG-simulated drought stress. PagWOX11/12a-overexpressing poplar plantlets showed lower oxidative damage levels, greater antioxidant enzyme activities and reactive oxygen species (ROS) scavenging capacity than non-transgenic poplar plants, whereas PagWOX11/12a dominant repression weakened root biomass accumulation and drought tolerance in poplar. RNA-seq analysis revealed that several differentially expressed genes (DEGs) regulated by PagWOX11/12a are involved in redox metabolism and drought stress response. We used RT-qPCR and yeast one-hybrid (Y1H) assays to validate the downstream target genes of PagWOX11/12a. These results provide new insights into the biological function and molecular regulatory mechanism of WOX11/12 in the abiotic resistance processes of poplar.

Comprehensive transcriptional profiling of NaHCO3-stressed Tamarix hispida roots reveals networks of responsive genes.

Root tissue is the primary site of perception for stress from soil, and is the main tissue involved in stress response. Tamarix hispida is a woody halophyte that is highly tolerant to salt and drought stress, but little information available about gene expression in roots in response to abiotic stress. In this study, eight transcriptomes from roots of T. hispida treated with NaHCO3 for 0, 12, 24 and 48 h (two biological replicates were set at each time point) were built. In total, 47,324 unigenes were generated, and 6,267 differentially expressed genes (DEGs) were identified. There were 2,510, 3,690, and 2,636 genes significantly differentially expressed after stress for 12, 24 and 48 h, respectively. Co-expressed DEGs were clustered into ten classes (P < 0.001). Gene ontology enrichment analysis showed that 13 pathways were highly enriched, such as signal transduction, cell wall, phosphatase activity, and lipid kinase activity, suggesting that these pathways play important roles in the saline-alkaline response. Furthermore, the genes involved in lignin metabolic processes and biosynthesis of proline and trehalose are found closely involved in NaHCO3 stress response. This systematic analysis may provide an in-depth view of stress tolerance mechanisms in T. hispida.

Characterization of a eukaryotic translation initiation factor 5A homolog from Tamarix androssowii involved in plant abiotic stress tolerance.

BACKGROUND: The eukaryotic translation initiation factor 5A (eIF5A) promotes formation of the first peptide bond at the onset of protein synthesis. However, the function of eIF5A in plants is not well understood. RESULTS: In this study, we characterized the function of eIF5A (TaeIF5A1) from Tamarix androssowii. The promoter of TaeIF5A1 with 1,486 bp in length was isolated, and the cis-elements in the promoter were identified. A WRKY (TaWRKY) and RAV (TaRAV) protein can specifically bind to a W-box motif in the promoter of TaeIF5A1 and activate the expression of TaeIF5A1. Furthermore, TaeIF5A1, TaWRKY and TaRAV share very similar expression pattern and are all stress-responsive gene that functions in the abscisic acid (ABA) signaling pathway, indicating that they are components of a single regulatory pathway. Transgenic yeast and poplar expressing TaeIF5A1 showed elevated protein levels combined with improved abiotic stresses tolerance. Furthermore, TaeIF5A1-transformed plants exhibited enhanced superoxide dismutase (SOD) and peroxidase (POD) activities, lower electrolyte leakage and higher chlorophyll content under salt stress. CONCLUSIONS: These results suggested that TaeIF5A1 is involved in abiotic stress tolerance, and is likely regulated by transcription factors TaWRKY and TaRAV both of which can bind to the W-box motif. In addition, TaeIF5A1 may mediate stress tolerance by increasing protein synthesis, enhancing ROS scavenging by improving SOD and POD activities, and preventing chlorophyll loss and membrane damage. Therefore, eIF5A may play an important role in plant adaptation to changing environmental conditions.

A LEA gene regulates Cadmium tolerance by mediating physiological responses.

In this study, the function of a LEA gene (TaLEA1) from Tamrix androssowii in response to heavy metal stress was characterized. Time-course expression analyses showed that NaCl, ZnCl(2), CuSO(4), and CdCl(2) considerably increased the expression levels of the TaLEA1 gene, thereby suggesting that this gene plays a role in the responses to these test stressors. To analyze the heavy metal stress-tolerance mechanism regulated by TaLEA1, TaLEA1-overexpressing transgenic poplar plants (Populus davidiana Dode × P. bollena Lauche) were generated. Significant differences were not observed between the proline content of the transgenic and wild-type (WT) plants before and after CdCl(2) stress. However, in comparison with the WT plants, the TaLEA1-transformed poplar plants had significantly higher superoxide dismutase (SOD) and peroxidase (POD) activities, and lower malondialdehyde (MDA) levels under CdCl(2) stress. Further, the transgenic plants showed better growth than the WT plants did, indicating that TaLEA1 provides tolerance to cadmium stress. These results suggest that TaLEA1 confers tolerance to cadmium stress by enhancing reactive oxygen species (ROS)-scavenging ability and decreasing lipid peroxidation. Subcellular-localization analysis showed that the TaLEA1 protein was distributed in the cytoplasm and nucleus.

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

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.

WUSCHEL-related homeobox gene PagWOX11/12a is involved in drought tolerance through modulating reactive oxygen species scavenging in poplar.

WUSCHEL-related homeobox (WOX) transcription factors play essential roles in key developmental processes and in response to different abiotic stresses. In a recent study, we have refined a molecular regulation mechanism that drought-induced PagERF35 directly activates the expression of PagWOX11/12a thus to promote root elongation and biomass, especially under drought conditions, and resulting in enhanced drought tolerance in poplar. In this study, we further found that PagWOX11/12a overexpression significantly enhanced drought tolerance and improved survival rate. Interestingly, transgenic poplars overexpressing PagWOX11/12a exhibited higher ability in scavenging reactive oxygen species (ROS) under drought stress. Combined with these and previous findings, we proposed the mechanism that PagWOX11/12a could not only promote root elongation and biomass growth to increase drought tolerance but also improve plant drought tolerance by regulating ROS level through possibly modulating the expression of ROS scavenging related genes.

ThNAC12 from Tamarix hispida directly regulates ThPIP2;5 to enhance salt tolerance by modulating reactive oxygen species.

NAC (NAM, ATAF1/2 and CUC2) transcription factors play critical roles in plant development and abiotic stress responses, and aquaporins have diverse functions in environmental stress responses. In this study, we described the salt-induced transcriptional responses of ThNAC12 and ThPIP2;5 in Tamarix hispida, and their regulatory mechanisms in response to salt stress. Using yeast one-hybrid (Y1H), chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays, we identified that ThNAC12 directly binds to the NAC recognition sequence (NACRS) of the ThPIP2;5 promoter and then activates the ThPIP2;5 expression. Subcellular localization and transcriptional activation assays demonstrated that ThNAC12 was a nuclear protein with a C-terminal transactivation domain. Compared with the corresponding control plants, transgenic plants overexpressing ThNAC12 exhibited enhanced salt tolerance and displayed increased reactive oxygen species (ROS) scavenging capability and antioxidant enzyme activity levels under salt stress. All results suggested that overexpression of ThNAC12 in plants enhanced salt tolerance through modulation of ROS scavenging via direct regulation of ThPIP2;5 expression in T. hispida.