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.

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.

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.

Poplar PsnICE1 enhances cold tolerance by binding to different cis-acting elements to improve reactive oxygen species-scavenging capability.

Low temperature is a major stress that severely affects plant growth and development. Inducer of CBF expression 1 (ICE1) plays a key role in plant cold tolerance by regulating the expression of cold stress-responsive genes. In the present study, we characterized the function and underlying regulatory mechanism of PsnICE1 from Xiaohei poplar (Populus simonii × Populus nigra). PsnICE1 was significantly induced in response to cold stress in the roots, stems and leaves. PsnICE1 proteins were found to localize to the nucleus and exert transactivation activity via their N-terminal transactivation domain. Compared with non-transgenic poplar, transgenic poplar overexpressing PsnICE1 showed substantially enhanced tolerance to cold stress, with higher survival rates and antioxidant enzyme activity levels and reduced reactive oxygen species (ROS) accumulation. In contrast, plants with RNA inhibition-mediated silencing of PsnICE1 showed the opposite phenotype. PsnICE1 can bind to H-box element and abscisic acid-responsive element (ABRE), and more importantly, it mainly binds to IBS1 (a newly discovered cis-acting element) and E-box elements to regulate stress-related genes involved in ROS scavenging. Overall, these results indicated that PsnICE1 functions as a positive regulator of cold tolerance and serves as a potential candidate gene for plant cold tolerance improvement via molecular breeding.

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.

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.

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.

Phylogenomic synteny network analyses reveal ancestral transpositions of auxin response factor genes in plants.

BACKGROUND: Auxin response factors (ARFs) have long been a research focus and represent a class of key regulators of plant growth and development. Integrated phylogenomic synteny network analyses were able to provide novel insights into the evolution of the ARF gene family. RESULTS: Here, more than 3500 ARFs collected from plant genomes and transcriptomes covering major streptophyte lineages were used to reconstruct the broad-scale family phylogeny, where the early origin and diversification of ARF in charophytes was delineated. Based on the family phylogeny, we proposed a unified six-group classification system for angiosperm ARFs. Phylogenomic synteny network analyses revealed the deeply conserved genomic syntenies within each of the six ARF groups and the interlocking syntenic relationships connecting distinct groups. Recurrent duplication events, such as those that occurred in seed plants, angiosperms, core eudicots and grasses contributed to the expansion of ARF genes which facilitated functional diversification. Ancestral transposition activities in important plant families, including crucifers, legumes and grasses, were unveiled by synteny network analyses. Ancestral gene duplications along with transpositions have profound evolutionary significance which may have accelerated the functional diversification process of paralogues. CONCLUSIONS: The broad-scale family phylogeny in combination with the state-of-art phylogenomic synteny network analyses not only allowed us to infer the evolutionary trajectory of ARF genes across distinct plant lineages, but also facilitated to generate a more robust classification regime for this transcription factor family. Our study provides insights into the evolution of ARFs which will enhance our current understanding of this important transcription factor family.

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.

Transcription Factor-Centered Yeast One-Hybrid Assay.

The interaction between a protein and DNA is involved in almost all cellular functions, and is vitally important in transcriptional regulation. There are two complementary approaches 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 gene-centered system to identify DNA-protein interactions. However, a powerful and simple TF-centered method to study protein-DNA interactions like Y1H is lacking. Here, we provide a TF-centered 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 is generated as the prey DNA sequences to interact with a defined TF as the bait. 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.

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.

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.

An ERF transcription factor from Tamarix hispida, ThCRF1, can adjust osmotic potential and reactive oxygen species scavenging capability to improve salt tolerance.

Ethylene-Responsive Factors (ERFs) are plant-specific transcription factors (TFs) involved in multiple biological processes, especially in abiotic stress tolerance. However, the ERFs from woody halophytes that are involved in salt stress have been little studied. In the present investigation, we characterized a subfamily member of ERF TFs from Tamarix hispida, ThCRF1, which responds to salt stress. ThCRF1 is a nuclear protein that binds to the motifs including TTG, DRE and GCC-box. Transient transformation was performed to generate T. hispida overexpressing ThCRF1 and RNA interference (RNAi)-silenced ThCRF1 to analyze its function using gain- and loss-of-function methods. Overexpression of ThCRF1 in T. hispida significantly improved tolerance to salt-shock-induced stress; by contrast, RNAi-silence of ThCRF1 significantly decreased tolerance to salt-shock-induced stress. Further experiments showed that ThCRF1 induces the expression of genes including those encoding pyrroline-5-carboxylate synthetase (P5CS), trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), superoxide dismutase (SOD) and peroxidase (POD), which lead to enhanced proline and trehalose levels and increased SOD and POD activities. These results were further confirmed by studying transgenic Arabidopsis plants overexpressing ThCRF1. Therefore, the results suggested that ThCRF1 improves tolerance to salt-shock-induced stress by enhancing trehalose and proline biosynthesis to adjust the osmotic potential, and by improving SOD and POD activities to increase reactive oxygen species scavenging capability.

ThNAC13, a NAC Transcription Factor from Tamarix hispida, Confers Salt and Osmotic Stress Tolerance to Transgenic Tamarix and Arabidopsis.

NAC (NAM, ATAF1/2, and CUC2) proteins play critical roles in many plant biological processes and environmental stress. However, NAC proteins from Tamarix hispida have not been functionally characterized. Here, we studied a NAC gene from T. hispida, ThNAC13, in response to salt and osmotic stresses. ThNAC13 is a nuclear protein with a C-terminal transactivation domain. ThNAC13 can bind to NAC recognized sites and calmodulin-binding NAC (CBNAC) binding element. Overexpression of ThNAC13 in Arabidopsis improved seed germination rate and increased root growth and fresh weight gain under salt or osmotic stress. Transgenic T. hispida plants transiently overexpressing ThNAC13 and with RNAi-silenced ThNAC13 were generated for gain- and loss-of-function experiments. Following exposure to salt or osmotic stress, overexpression of ThNAC13 induced superoxide dismutase (SOD) and peroxidase (POD) activities, chlorophyll and proline contents; decreased the reactive oxygen species (ROS) and malondialdehyde levels; and reduced electrolyte leakage rates in both transgenic Tamarix and Arabidopsis plants. In contrast, RNAi-silenced ThNAC13 showed the opposite results in transgenic Tamarix. Furthermore, ThNAC13 induced the expression of SODs and PODs in transgenic Arabidopsis. These results suggest that ThNAC13 improves salt and osmotic tolerance by enhancing the ROS-scavenging capability and adjusting osmotic potential.

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.