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DNA–protein interaction is one of the most crucial interactions in biological processes. However, the technologies available to study DNA–protein interactions are all based on DNA hybridization; however, DNA hybridization is not highly specific and is relatively low in efficiency. RNA-guided DNA recognition is highly specific and efficient. To overcome the limitations of technologies based on DNA hybridization, we built a DNA-binding protein capture technology based on the clustered regularly interspaced palindromic repeats (CRISPR)–dead Cas9 (dCas9) system and transient genetic transformation, termed reverse chromatin immunoprecipitation based on CRISPR–dCas9 system (R-ChIP–dCas9). In this system, dCas9 was fused with Strep-Tag II to form a fusion protein for StrepTactin affinity purification. Transient transformation was performed for the expression of dCas9 and guide RNA (gRNA) to form the dCas9–gRNA complex in birch (Betula platyphylla) plants, which binds to the target genomic DNA region. The dCas9–gRNA–DNA complex was crosslinked, then the chromatin was sonicated into fragments, and purified using StrepTactin beads. The proteins binding to the target genomic DNA region were identified using mass spectrometry. Using this method, we determined the upstream regulators of a NAM, ATAF, and CUC (NAC) transcription factor (TF), BpNAC090, and 32 TFs potentially regulating BpNAC090 were identified. The reliability of R-ChIP–dCas9 was further confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assays, and yeast one-hybrid. This technology can be adapted to various plant species and does not depend on the availability of a stable transformation system; therefore, it has wide application in identifying proteins bound to genomic DNA.

GRAS transcription factors belong to the plant-specific protein family. They are not only involved in plant growth and development but also in plant responses to a variety of abiotic stresses. However, to date, the SCL32(SCARECROW-like 32) gene conferring the desired resistance to salt stresses has not been reported in plants. Here, ThSCL32, a homologous gene of Arabidopsis thaliana AtSCL32, was identified. ThSCL32 was highly induced by salt stress in Tamarix hispida. ThSCL32 overexpression in T. hispida gave rise to improved salt tolerance. ThSCL32-silenced T. hispida plants were more sensitive to salt stress. RNA-seq analysis of transient transgenic T. hispida overexpressing ThSCL32 revealed significantly enhanced ThPHD3 (prolyl-4-hydroxylase domain 3 protein) gene expression. ChIP-PCR further verified that ThSCL32 probably binds to the novel cis-element SBS (ACGTTG) in the promoter of ThPHD3 to activate its expression. In brief, our results suggest that the ThSCL32 transcription factor is involved in salt tolerance in T. hispida by enhancing ThPHD3 expression.

Construction of ML-hGRN for the salt pathway in Populus davidiana × P. bolleana. Construction of ML-hGRN for the lignocellulosic pathway in Populus davidiana × P. bolleana under salt stress. Many woody plants, including Populus davidiana × P. bolleana, have made great contributions to human production and life. High salt is one of the main environmental factors that restricts the growth of poplar. This study found that high salt could induce strong biochemical changes in poplar. To detect the effect of salt treatment on gene expression, 18 libraries were sequenced on the Illumina sequencing platform. The results identified a large number of early differentially expressed genes (DEGs) and a small number of late DEGs, which indicated that most of the salt response genes of poplar were early response genes. In addition, 197 TFs, including NAC, ERF, and other TFs related to salt stress, were differentially expressed during salt treatment, which indicated that these TFs may play an important role in the salt stress response of poplar. Based on the RNA-seq analysis results, multilayered hierarchical gene regulatory networks (ML-hGRNs) of salt stress- and lignocellulosic synthesis-related DEGs were constructed using the GGM algorithm. The lignocellulosic synthesis regulatory network under salt stress revealed that lignocellulosic synthesis might play an important role in the process of salt stress resistance. Furthermore, the NAC family transcription factor PdbNAC83, which was found in the upper layer in both pathways, was selected to verify the accuracy of the ML-hGRNs. DAP-seq showed that the binding site of PdbNAC83 included a "TT(G/A)C(G/T)T" motif, and ChIP-PCR further verified that PdbNAC83 can regulate the promoters of at least six predicted downstream genes (PdbNLP2-2, PdbZFP6, PdbMYB73, PdbC2H2-like, PdbMYB93-1, PdbbHLH094) by binding to the "TT(G/A)C(G/T)T" motif, which indicates that the predicted regulatory network diagram obtained in this study is relatively accurate. In conclusion, a species-specific salt response pathway might exist in poplar, and this finding lays a foundation for further study of the regulatory mechanism of the salt stress response and provides new clues for the use of genetic engineering methods to create high-quality and highly resistant forest germplasms.

Cadmium (Cd) is a toxic metal that affects the normal growth and development of plants. Roots may directly contact Cd and thus serve as the first barrier in the defense responses of plants. In this study, Tamarix hispida (T. hispida) roots treated with 150 μM CdCl2 were collected for RNA-seq. A total of 2004 differentially expressed genes (DEGs) were identified at different time points. Kyoto Encyclopedia of Genes and Genomes enrichment revealed that the DEGs were significantly enriched in phenylpropanoid biosynthesis, flavonoid biosynthesis and other metabolic pathways. To explore the regulatory role of transcription factors (TFs) involved in the Cd stress response, a multilayer hierarchical gene regulatory network (ML-hGRN) was constructed, including 53 TFs and 54 structural genes in ML-hGRN, with 341 predicted regulatory relationships. Binding of DRE1A, MYC1, FEZ, ERF4 and ERF17 to predicted target genes was detected by ChIP-PCR, and DRE1A, MYC1 and FEZ were transiently overexpressed in T. hispida. The results suggest that these TFs play a key role in the Cd stress response by scavenging reactive oxygen species. In conclusion, this study predicts some Cd-responsive TFs that may have an important function under Cd stress and provides useful information for molecular breeding.

R2R3-MYB transcription factors play an important role in plant development and response to various environmental stresses. In this study, a new R2R3-MYB gene, named ThRAX2, was isolated from T. hispida. ThRAX2 has an open reading frame (ORF) of 1191bp and encodes a protein of 396 amino acids. ThRAX2 was localized in the nucleus. The overexpression of ThRAX2 in Arabidopsis and T. hispida significantly increased Cadmium (Cd) tolerance. Moreover, the accumulation of cadmium in root leaves was significantly reduced. The TF-centred Y1H and Y1H results showed that ThRAX2 was able to specifically bind a new cis-element (MYB-T, CTTCCA). The promoters of some Cd-responsive genes, such as ThSOS1, ThCKX3, ThCAX3A, ThMYB78, ThMIP2, ThTPS4, and ThSOD2, all contained 1-3 MYB-T sequences. Furthermore, chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) and ChIPquantitative (q)PCR showed that the ThRAX2 gene can bind to ThSOS1, ThCKX3, ThCAX3A and ThMYB78 promoter fragments, including the MYB-T motif. Meanwhile, the qRTPCR results also showed that the expression trends of ThSOS1, ThCKX3, ThCAX3A and ThMYB78 were similar to that of ThRAX2. This finding suggests that Cd tolerance of the ThRAX2 gene may regulate the expression of some downstream genes through specific recognition of the MYB-T motif and participate in regulating intracellular ion homeostasis, transport, and protein activity or enhance antioxidant enzyme activity. This study found a novel cis-acting element that binds RAX2 to regulate Cd tolerance, which lays the foundation for the RAX2 regulatory mechanism of Cd stress. This study provides a genetic and theoretical basis for the bioremediation of Cd-contaminated land by cultivating transgenic plants in the future.

The plant vacuolar H+-ATPase (V-ATPase) is a multisubunit complex. In addition to performing basic housekeeping functions, this complex is also involved in abiotic stress resistance in plants. In this study, a V-ATPase c`` subunit gene (ThVHAc``1) from Tamarix hispida Willd was cloned with a 534-bp ORF. Sequence analysis showed that the ThVHAc``1 protein contains four transmembrane helices and lacks a signal peptide. qRT-PCR results showed that ThVHAc``1 was primarily induced by treatments of NaCl, NaHCO3, PEG6000, CdCl2 or ABA in roots, stems and leaves of T. hispida. The expression pattern of ThVHAc``1 was significantly different from that of ThVHAc1 (a V-ATPase c subunit in T. hispida). Furthermore, the cell survival rates and density (OD600) results showed that the transgenic yeast overexpressing ThVHAc``1 exhibited increased tolerance to the above-mentioned abiotic stresses. In addition, the overexpression of ThVHAc``1 confers salt tolerance to transgenic Arabidopsis plants by improving the ROS content and decreasing the accumulation of O2- and H2O2. Similarly, the homologous transformation of the ThVHAc``1 gene into T. hispida also improved salt tolerance. Our results suggest that the ThVHAc``1 gene plays an important role in plant stress tolerance.

Trehalose is a non-reducing disaccharide with high stability and strong water absorption properties that can improve the resistance of organisms to various abiotic stresses. Trehalose-6-phosphate synthase (TPS) plays important roles in trehalose metabolism and signaling. In this study, the full-length cDNA of ThTPS was cloned from Tamarix hispida Willd. A phylogenetic tree including ThTPS and 11 AtTPS genes from Arabidopsis indicated that the ThTPS protein had a close evolutionary relationship with AtTPS7. However, the function of AtTPS7 has not been determined. To analyze the abiotic stress tolerance function of ThTPS, the expression of ThTPS in T. hispida under salt and drought stress and JA, ABA and GA3 hormone stimulation was monitored by qRT-PCR. The results show that ThTPS expression was clearly induced by all five of these treatments at one or more times, and salt stress caused particularly strong induction of ThTPS in the roots of T. hispida. The ThTPS gene was transiently overexpressed in T. hispida. Both physiological indexes and staining results showed that ThTPS gene overexpression increased salt and osmotic stress tolerance in T. hispida. Overall, the ThTPS gene can respond to abiotic stresses such as salt and drought, and its overexpression can significantly improve salt and osmotic tolerance. These findings establish a foundation to better understand the responses of TPS genes to abiotic stress in plants.

Brassinazole-resistant (BZR) transcription factors have important roles in the brassinosteroid (BR) signalling pathway and are widely involved in plant growth and abiotic stress processes. However, there are few studies on the functions and regulatory mechanisms of BZR TFs in birch. In this study, 5 BZR genes were identified from birch. The qRT-PCR results showed that the expression levels of most BpBZRs were significantly downregulated and/or upregulated in at least one organ following NaCl and PEG stress or ABA, GA3 and JA treatments. In particular, BpBZR1 expression was changed in all three organs after exposure to NaCl stress at all time points, indicating that this gene may be involved in salt stress. The BpBZR1 transcription factor was shown to have transcriptional activation activity in a yeast two-hybrid assay. Through a transient transformation system, we found that overexpression of BpBZR1 in birch resulted in lower H2O2 and MDA accumulation, higher SOD and POD activities and maintained a higher photosynthetic intensity and a lower chlorophyll degradation rate than those of the control plants under salt stress. These results preliminarily showed that overexpression of the BpBZR1 gene increased the tolerance of birch to salt stress.

Histone deacetylases (HDACs) play an important regulatory role in plant response to biotic and abiotic stresses. They improve plant stress resistance by increasing the degree of histone acetylation associated with stress-responsive genes. SAP30BP, a human transcriptional regulatory protein, can increase histone deacetylase activity by regulating the deacetylation levels of lysines 9 and 14 in histone H3. In this study, a ThSAP30BP gene was cloned and characterized from Tamarix hispida (a kind of woody halophyte). The expression patterns of ThSAP30BP under different abiotic stresses and hormone treatments were detected by qRT-PCR. The results showed that ThSAP30BP was significantly upregulated at most time points under various stress treatments, suggesting that ThSAP30BP may be related to the abiotic stress response of T. hispida. To further analyze the salt stress resistance function of the ThSAP30BP gene, the plant overexpression vector pROKII-ThSAP30BP was instantaneously constructed and transformed into T. hispida. Meanwhile, the empty vector pROKII was also transformed as a control. The activities of SOD and POD, the contents of H2O2 and MDA, the relative conductance and the staining of NBT, DAB and Evans blue were analyzed and compared under salt stress. The results showed that the overexpression of ThSAP30BP in T. hispida reduced the accumulation of ROS in plants and the cell death rate under salt stress. These results suggested that ThSAP30BP may play an important physiological role in salt tolerance of T. hispida.

Although many genes and biological processes involved in abiotic stress responses have been identified, how they are regulated remains largely unclear. Here, to study the regulatory mechanism of birch (Betula platyphylla) responding to drought induced by polyethylene glycol 6000 (20%, w/v), a partial correlation coefficient-based algorithm for constructing a gene regulatory network (GRN) was proposed, and a three-layer hierarchical GRN was constructed, including 68 transcription factors and 252 structural genes. A total of 1448 predicted regulatory relationships are included, and most of them are novel. The reliability of the GRN was verified by chromatin immunoprecipitation (ChIP)–PCR and qRT–PCR based on transient transformation. About 55% of genes in the bottom layer of the GRN could confer drought tolerance. We selected two TFs, BpMADS11 and BpNAC090, from the top layer and characterized their function in drought tolerance. Overexpression of BpMADS11 and BpNAC090 reduces electrolyte leakage, reactive oxygen species (ROS) and malondialdehyde (MDA) contents, giving greater drought tolerance than wild-type birch. According to this GRN, the important biological processes involved in drought were identified, including ‘signaling hormone pathways’, ‘water transport’, ‘regulation of stomatal movement’, and ‘response to oxidative stress’. This work indicated that BpERF017, BpAGL61, and BpNAC090 are the key upstream regulators of birch drought tolerance. Our data clearly revealed that upstream regulators and transcription factor–DNA interaction regulate different biological processes to adapt to drought stress.

The CONSTANS-LIKE (COL) transcription factor has been reported to play important roles in regulating plant flowering and the response to abiotic stress. To clone and screen COL genes with excellent salt tolerance from the woody halophyte Tamarix hispida, 8 ThCOL genes were identified in this study. The expression patterns of these genes under different abiotic stresses (high salt, osmotic, and heavy metal) and abscisic acid (ABA) treatment were detected using quantitative real-time PCR (qRT-PCR). The expression levels of 8 ThCOL genes changed significantly after exposure to one or more stresses, indicating that these genes were all stress-responsive genes and may be involved in the stress resistance response of T. hispida. In particular, the expression level of ThCOL2 changed significantly at most time points in the roots and leaves of T. hispida under salt stress and after ABA treatments, which may play an important role in the response process of salt stress through a mechanism dependent on the ABA pathway. The recombinant vectors pROKII–ThCOL2 and pFGC5941–ThCOL2 were constructed for the transient transformation of T. hispida, and the transient infection of T. hispida with the pROKII empty vector was used as the control to further verify whether the ThCOL2 gene was involved in the regulation of the salt tolerance response of T. hispida. Overexpression of the ThCOL2 gene in plants under 150 mM NaCl stress increased the ability of transgenic T. hispida cells to remove reactive oxygen species (ROS) by regulating the activity of protective enzymes and promoting a decrease in the accumulation of O2– and H2O2, thereby reducing cell damage or cell death and enhancing salt tolerance. The ThCOL2 gene may be a candidate gene associated with excellent salt tolerance. Furthermore, the expression levels of some genes related to the ABA pathway were analyzed using qRT-PCR. The results showed that the expressions of ThNCED1 and ThNCED4 were significantly higher, and the expressions of ThNCED3, ThZEP, and ThAAO3 were not significantly altered in OE compared with CON under normal conditions. But after 24 h of salt stress, the expressions of all five studied genes all were lower than the normal condition. In the future, the downstream genes directly regulated by the ThCOL2 transcription factor will be searched and identified to analyze the salt tolerance regulatory network of ThCOL2.

GRAS (GAI, RGA and SCR) domain proteins are plant-specific transcriptional regulators that play roles in developmental processes and stress responses. However, the function and molecular regulatory networks of woody plant GRAS under various physiological conditions are still unclear. As a step toward understanding the GRAS function in plants under diverse environmental stimuli, a total of 12 GRAS genes from Tamarix hispida were identified and characterized. Sequence analysis showed that most of these ThGRASs contained a complete GRAS DNA-binding domain and a variable N-terminal region. Phylogenetic analysis revealed that these GRAS members were divided into PAT1, SHR, LISCL, DLT and DELLA groups. The expressions of each individual GRAS under various abiotic stress conditions or hormone treatments were investigated by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR). qRT-PCR results showed that these ThGRASs were all differentially expressed in the roots or leaves of T. hispida under abiotic stress and hormone treatment, especially ThGRAS3, ThGRAS5, ThGRAS6, ThGRAS7 and ThGRAS10 genes, indicating that these genes were involved in abiotic stress responses and signaling pathways. This study provides a basis for the elucidation of the function of GRAS genes for future works.

The MYB (v-myb avian myeloblastosis viral oncogene homolog) family is one of the largest transcription factor families in plants, and is widely involved in the regulation of plant metabolism. In this study, we show that a MYB4 transcription factor, BpMYB4, identified from birch (Betula platyphyllaSuk.) and homologous to EgMYB1 fromEucalyptus robustaSmith and ZmMYB31 fromZea maysL. is involved in secondary cell wall synthesis. The expression level ofBpMYB4was higher in flowers relative to other tissues, and was induced by artificial bending and gravitational stimuli in developing xylem tissues. The expression of this gene was not enriched in the developing xylem during the active season, and showed higher transcript levels in xylem tissues around sprouting and near the dormant period.BpMYB4also was induced express by abiotic stress. Functional analysis indicated that expression ofBpMYB4in transgenic Arabidopsis (Arabidopsis thaliana) plants could promote the growth of stems, and result in increased number of inflorescence stems and shoots. Anatomical observation of stem sections showed lower lignin deposition, and a chemical contents test also demonstrated increased cellulose and decreased lignin content in the transgenic plants. In addition, treatment with 100 mM NaCl and 200 mM mannitol resulted in the germination rate of the over-expressed lines being higher than that of the wild-type seeds. The proline content in transgenic plants was higher than that in WT, but MDA content was lower than that in WT. Further investigation in birch using transient transformation techniques indicated that overexpression ofBpMYB4could scavenge hydrogen peroxide and O2.–and reduce cell damage, compared with the wild-type plants. Therefore, we believe that BpMYB4 promotes stem development and cellulose biosynthesis as an inhibitor of lignin biosynthesis, and has a function in abiotic stress resistance.

The salt overly sensitive (SOS) signal transduction pathway is one of the most highly studied salt tolerance pathways in plants. However, the molecular mechanism of the salt stress response in Tamarix hispida has remained largely unclear. In this study, five SOS genes (ThSOS1–ThSOS5) from T. hispida were cloned and characterized. The expression levels of most ThSOS genes significantly changed after NaCl, PEG6000, and abscisic acid (ABA) treatment in at least one organ. Notably, the expression of ThSOS3 was significantly downregulated after 6 h under salt stress. To further analyze ThSOS3 function, ThSOS3 overexpression and RNAi-mediated silencing were performed using a transient transformation system. Compared with controls, ThSOS3-overexpressing transgenic T. hispida plants exhibited greater reactive oxygen species (ROS)-scavenging capability and antioxidant enzyme activity, lower malondialdehyde (MDA) and H2O2 levels, and lower electrolyte leakage rates under salt stress. Similar results were obtained for physiological parameters in transgenic Arabidopsis, including H2O2 and MDA accumulation, superoxide dismutase (SOD) and peroxidase (POD) activity, and electrolyte leakage. In addition, transgenic Arabidopsis plants overexpressing ThSOS3 displayed increased root growth and fresh weight gain under salt stress. Together, these data suggest that overexpression of ThSOS3 confers salt stress tolerance on plants by enhancing antioxidant enzyme activity, improving ROS-scavenging capability, and decreasing the MDA content and lipid peroxidation of cell membranes. These results suggest that ThSOS3 might play an important physiological role in salt tolerance in transgenic T. hispida plants. This study provides a foundation for further elucidation of salt tolerance mechanisms involving ThSOSs in T. hispida.

The MYB (v-myb avian myeloblastosis viral oncogene homolog) family is one of the largest transcription factor families in plants, and is widely involved in the regulation of plant metabolism. In this study, we show that a MYB4 transcription factor, BpMYB4, identified from birch (Betula platyphylla Suk.) and homologous to EgMYB1 from Eucalyptus robusta Smith and ZmMYB31 from Zea mays L. is involved in secondary cell wall synthesis. The expression level of BpMYB4 was higher in flowers relative to other tissues, and was induced by artificial bending and gravitational stimuli in developing xylem tissues. The expression of this gene was not enriched in the developing xylem during the active season, and showed higher transcript levels in xylem tissues around sprouting and near the dormant period. BpMYB4 also was induced express by abiotic stress. Functional analysis indicated that expression of BpMYB4 in transgenic Arabidopsis (Arabidopsis thaliana) plants could promote the growth of stems, and result in increased number of inflorescence stems and shoots. Anatomical observation of stem sections showed lower lignin deposition, and a chemical contents test also demonstrated increased cellulose and decreased lignin content in the transgenic plants. In addition, treatment with 100 mM NaCl and 200 mM mannitol resulted in the germination rate of the over-expressed lines being higher than that of the wild-type seeds. The proline content in transgenic plants was higher than that in WT, but MDA content was lower than that in WT. Further investigation in birch using transient transformation techniques indicated that overexpression of BpMYB4 could scavenge hydrogen peroxide and O2.– and reduce cell damage, compared with the wild-type plants. Therefore, we believe that BpMYB4 promotes stem development and cellulose biosynthesis as an inhibitor of lignin biosynthesis, and has a function in abiotic stress resistance.

The ETHYLENE INSENSITIVE 3-LIKE (EIL) transcription factor (TF) family plays a key role in the ethylene signal transduction pathway. However, it is still unknown how EIL proteins mediate salt tolerance in Tamarix hispida. Here, we cloned and functionally characterized the TF ThEIL1 from T. hispida. ThEIL1 is a nuclear protein possessing transcriptional activation activity. In addition to binding to the PERE motif (GGATTCAA), a novel EIL protein-DNA interaction was identified, in which ThEIL1 binds to GCC-box (AGCCGCC). Gain- and loss-of-function analysis in T. hispida showed that ThEIL1 regulates the expression of trehalose-6-phosphate synthase (TPS), pyrroline-5-carboxylate synthetase (P5CS), and trehalose-6-phosphate phosphatase (TPP) to mediate the biosynthesis of proline and trehalose, respectively, thereby elevating osmotic potential. Additionally, ThEIL1 controls the activities of peroxidase (POD) and superoxide dismutase (SOD) by regulating the expression of genes encoding SODs and PODs, leading to increased reactive oxygen species scavenging capability. Moreover, overexpression of ThEIL1 in Arabidopsis significantly increased root length and fresh weight under salt treatment conditions. Furthermore, the physiological traits of T. hispida were consistent with those of Arabidopsis stably overexpressing ThEIL1. Thus, ThEIL1 serves as a TF that mediates salt tolerance by elevating osmotic potential and improving ROS scavenging capability.

Background Peroxiredoxins (Prxs) are a large family of antioxidant enzymes that respond to biotic and abiotic stress by decomposing reactive oxygen species (ROS). In this study, the stress tolerance function of the Th2CysPrx gene was further analysed. It lays a foundation for further studies on the salt tolerance molecular mechanism of T. hispida and improved salt tolerance via transgenic plants. Results In this study, the stress tolerance function of the Th2CysPrx gene was further analysed. The results of transgenic tobacco showed higher seed germination rates, root lengths, and fresh weight under salt stress than wild-type tobacco. Simultaneously, physiological indicators of transgenic tobacco and T. hispida showed that Th2CysPrx improved the activities of antioxidant enzymes and enhanced ROS removal ability to decrease cellular damage under salt stress. Moreover, Th2CysPrx improved the expression levels of four antioxidant genes (ThGSTZ1, ThGPX, ThSOD and ThPOD). Conclusions Overall, these results suggested that Th2CysPrx enhanced the salt tolerance of the transgenic plants. These findings lay a foundation for further studies on the salt tolerance molecular mechanism of T. hispida and improved salt tolerance via transgenic plants.

The plant vacuolar H

Background Peroxiredoxins (Prxs) are a large family of antioxidant enzymes that respond to biotic and abiotic stress by decomposing reactive oxygen species (ROS). In this study, the stress tolerance function of the Th2CysPrx gene was further analysed. It lays a foundation for further studies on the salt tolerance molecular mechanism of T. hispida and improved salt tolerance via transgenic plants. Results In this study, the stress tolerance function of the Th2CysPrx gene was further analysed. The results of transgenic tobacco showed higher seed germination rates, root lengths, and fresh weight under salt stress than wild-type tobacco. Simultaneously, physiological indicators of transgenic tobacco and T. hispida showed that Th2CysPrx improved the activities of antioxidant enzymes and enhanced ROS removal ability to decrease cellular damage under salt stress. Moreover, Th2CysPrx improved the expression levels of four antioxidant genes (ThGSTZ1, ThGPX, ThSOD and ThPOD). Conclusions Overall, these results suggested that Th2CysPrx enhanced the salt tolerance of the transgenic plants. These findings lay a foundation for further studies on the salt tolerance molecular mechanism of T. hispida and improved salt tolerance via transgenic plants.

Background: Trehalose is a nonreducing disaccharide with high stability and strong water absorption properties that can improve the resistance of organisms to various abiotic stresses. Trehalose-6-phosphate synthase (TPS) plays important roles in trehalose metabolism and signaling. Results: A full-length cDNA of ThTPS was cloned from Tamarix hispida. The phylogenetic tree among ThTPS and 11 AtTPS in Arabidopsis indicates that the ThTPS protein had a close evolutionary relationship with AtTPS7. However, the function of AtTPS7 has not been determined. To analyze the abiotic stress tolerance function of ThTPS, the expression patterns of ThTPS were monitored under salt and drought stress and JA, ABA and GA3 hormone stimulation in T. hispida by qRT-PCR. The results showed that ThTPS expression was clearly induced by these 5 kinds of treatments at at least one studied point. Particularly under salt stress, ThTPS was highly induced in the roots of T. hispda. Furthermore, the ThTPS gene was transiently overexpressed in T. hispida. The results of physiological indexes and staining showed that overexpression of the ThTPS gene increased T. hispida salt and drought stress tolerance. Conclusion: The ThTPS gene can respond to abiotic stress such as salt and drought, and overexpression of ThTPS gene can significantly improve salt and drought tolerance. These findings establish a foundation to better understand the response of TPS genes to abiotic stress in plants.

HSPs are a large family of proteins, including HSP100, HSP90, HSP70, DnaJ and various sHSPs, which are important for plants to resist abiotic stress. However, the precise functions of HSPs involved in heat stress have not been thoroughly elucidated in birch (Betula platyphylla). In this study, 21 HSP genes in birch (named BpHSP1-BpHSP21) were cloned and characterized. The expression patterns of BpHSPs in response to high temperature stress were monitored by quantitative real time-polymerase chain reaction (qRT-PCR). The results showed that most of the BpHSPs were significantly upregulated or downregulated in response to high temperature, indicating that these BpHSPs may be involved in birch heat stress. Among the BpHSPs, the transcription of BpHSP9 was upregulated in stems and leaves under heat stress throughout all study periods, suggesting that this protein may play an important role under heat stress. Furthermore, BpHSP9-overexpressing, RNAi-silencing and empty pROKII vector (as the control) birch plants were generated by transient transformation. Under heat stress, overexpression BpHSP9 transgentic birch plants increased reactive oxygen species (ROS) scavenging, the activities of some antioxidant enzymes and non-enzymatic antioxidants. These effects led to diminished O2− and H2O2 content and reduced electrolyte leakage rates. However, the results were reversed when BpHSP9 was transiently silenced by RNAi. Taken together, these results strongly suggest that BpHSP9 may plays an important physiological role in the heat stress tolerance of birch plants.

Highlights ● The functional characterization of ThSOS genes were investigated by bioinformatics analysis and molecular characterization. ● ThSOS genes can respond to abiotic stresses (salt and drought) and hormone treatment (ABA). ● ThSOS3 gene overexpression increased ROS-scavenging capability and decreasing lipid peroxidation in cell membrane. ● ThSOS3 could effectively enhance the tolerance of transgenic T. hispida and Arabidopsis to salt stress.

The MYB transcription factors (TFs) is a plant TF families, which involves in hormone signal transduction, and abiotic stress tolerance, etc. However, there are few studies on the MYB TFs family and its regulatory mechanism in Tamarix hispida. In this study, 14 MYB genes (named ThMYB1 - ThMYB14) were cloned and characterized from T. hispida. The transcription profiles of ThMYBs in T. hispida under different abiotic stress conditions were monitored using qRT-PCR. Most of studied ThMYBs were significantly downregulated and/or upregulated by salt and osmotic stress, ABA, GA3 and JA treatments in at least one organ. Especially, ThMYB13 was induced in the leaves and roots of T. hispida when exposed to NaCl treatment at all study periods, indicating that it may involve in salt stress. To further study ThMYB13 function, ThMYB13 overexpression and knock-down plants and control plants transformed with an empty pROKII were obtained using a transient transformation system. Overexpression of ThMYB13 in T. hispida displayed the lowest O2-, H2O2 and MDA accumulation, minimal cell death, the most stable K+/Na+ ratio and the lowest electrolyte leakage rate among the three kinds of transient expression in T. hispida. Conversely, the RNAi-silencing, transiently transformed plants displayed the opposite physiological changes. Therefore, ThMYB13 might play a role in salt stress tolerance in transgenic T. hispida plants.

High mobility group (HMG) proteins play an important role in plant responses to abiotic stresses. However, to date, few studies have focused on the functional characterization of HMG genes in woody plants. In this study, 11 BpHMG genes named BpHMG1-BpHMG11 were cloned and characterized from Betula platyphylla. The expression patterns of BpHMG genes under two types of abiotic stress (osmotic and salt stress) and ABA (abscisic acid) treatments were detected by qRT-PCR. The results showed that the expression of 11 BpHMG genes in birch roots, stems and leaves changed in at least one stress treatment time point under all three treatments, suggesting that 11 BpHMG genes could respond to salt, osmotic stress and ABA treatment. In particular, the expression of BpHMG6 in birch stems was significantly upregulated at all studied time points under NaCl stress. The highest level was increased up to 118-fold compared with the control, indicating that this gene may be involved in salt stress responses. To further study the salt tolerance function of BpHMG6, the overexpression vector pROKII-BpHMG6 and silencing expression vector pFGC-BpHMG6 were constructed and transiently transformed into birch. The empty vector pROKII was also transformed as a control. POD activity, H2O2 and MDA levels, relative electrolyte and NBT, DAB and Evans blue staining were analysed and compared between transgenic and control plants under salt stress. The results indicated that the overexpression of BpHMG6 in birch not only increased the activity of protective enzymes under salt stress but also enhanced the ROS scavenging capacity to reduce cell damage and death caused by salt stress. Conversely, the silenced transiently transformed plants displayed the opposite physiological changes. Therefore, BpHMG6 potentially plays a role in salt stress tolerance in transgenic birch plants.

MYB transcription factors are important in abiotic stress responses; however, the detailed mechanisms are unclear. Tamarix hispida contains multiple MYB genes. The present study characterized T. hispida MYB8 (ThMYB8) during salt stress using transgenic T. hispida and Arabidopsis assays. ThMYB8 overexpression and ThMYB8 RNAi analysis demonstrated that ThMYB8 enhanced the salt stress tolerance. Transgenic Arabidopsis ectopic expression of ThMYB8 significantly increased root growth, fresh weight, and seed germination rate compared with that of the wild-type under salt stress. Physiological parameters analysis in T. hispida and Arabidopsis showed that ThMYB8 overexpressing plants had the lowest levels of O2, H2O2, cell death, malondialdehyde, and electrolyte leakage. Overexpression of ThMYB8 regulated Na+ and K+ concentrations in plant tissues while maintaining K+/Na+ homeostasis. Analysis using qRT-PCR and ChIP-PCR identified possible downstream ThMYB8-regulated genes. ThMYB8 regulated the expression of ThCYP450-2 (cytochrome p450-2), Thltk (leucine-rich repeat transmembrane protein kinase), and ThTIP (aquaporin TIP) by binding to the MBSI motif ('CAACTG') in their promoters. The results indicated that ThMYB8 enhanced salt stress tolerance in T. hispida by regulating gene expression related to the activation of stress-associated physiological changes, such as enhanced reactive oxygen species scavenging capability, maintaining K+/Na+ homeostasis, and decreasing the malondialdehyde content and lipid peroxidation cell membranes.

Molecular analysis of a zeta subfamily GST gene from T. hispida involved in ABA and methyl viologen tolerance in transgenic Arabidopsis and Tamarix. Glutathione S-transferase (GST) genes are important for the improvement of plant abiotic stress tolerance, and our previous study demonstrated that the ThGSTZ1 gene from Tamarix hispida improves plant salt and drought tolerance. To further understand the role of ThGSTZ1 in the response of plants to abscisic acid (ABA) and oxidative stress, three ThGSTZ1-overexpressing transgenic Arabidopsis thaliana lines were analyzed in the current study. The results showed that the transgenic lines exhibited higher biomass accumulation, higher activities of GST and other protective enzymes, and less reactive oxygen species (ROS) and cell damage than wild-type (WT) plants under ABA and methyl viologen (MV) stress. In addition, the analysis of a transgenic T. hispida line transiently expressing ThGSTZ1 confirmed these results. The activities of GST, glutathione peroxidase, and superoxide dismutase were markedly higher in the ThGSTZ1-overexpressing lines compared with the control lines under both ABA and MV treatments, and the transgenic lines also exhibited a lower degree of electrolyte leakage (EL) and a decreased H2O2 content. All these results suggested that ThGSTZ1 can also improve plant ABA and oxidation tolerance by regulating ROS metabolism and that ThGSTZ1 represents an excellent candidate gene for molecular breeding to increase plant stress tolerance.

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.

Proper accumulation and function of miRNAs is essential for plant growth and development. While core components of the miRNA biogenesis pathway and miRNA-induced silencing complex have been well characterized, cellular regulators of miRNAs remain to be fully explored. Here we report that High Expression Of Osmotically Responsive Genes1 (HOS1) is a regulator of an important miRNA, mi168a/b, that targets the Argonaute1 (AGO1) gene in Arabidopsis. HOS1 functions as an ubiquitin E3 ligase to regulate plant cold-stress responses, associates with the nuclear pores to regulate mRNA export, and regulates the circadian clock and flowering time by binding to chromatin of the flowering regulator gene Flowering Locus C (FLC). In a genetic screen for enhancers of sic-1, we isolated a loss-of-function Arabidopsis mutant of HOS1 that is defective in miRNA biogenesis. Like other hos1 mutant alleles, the hos1-7 mutant flowered early and was smaller in stature than the wild-type. Dysfunction in HOS1 reduced the abundance of miR168a/b but not of other miRNAs. In hos1 mutants, pri-MIR168b and pre-MIR168b levels were decreased, and RNA polymerase II occupancy was reduced at the promoter of MIR168b but not that of MIR168a. Chromatin immunoprecipitation assays revealed that HOS1 protein is enriched at the chromatin of the MIR168b promoter. The reduced miR168a/b level in hos1 mutants results in an increase in the mRNA and protein levels of its target gene, AGO1. Our results reveal that HOS1 regulates miR168a/b and AGO1 levels in Arabidopsis by maintaining proper transcription of MIR168b.

Eukaryotic translation initiation factor 1A (eIF1A) functions as an mRNA scanner and AUG initiation codon locator. However, few studies have clarified the role of eIF1A in abiotic stress. In this study, we cloned eIF1A (TheIF1A) from Tamarix hispida and found its expression to be induced by NaCl and polyethylene glycol (PEG) in roots, stems and leaves. Compared to a control, TheIF1A root expression was increased 187.63-fold when exposed to NaCl for 6 h, suggesting a potential abiotic stress response for this gene. Furthermore, transgenic tobacco plants overexpressing TheIF1A exhibited enhanced seed germination and a higher total chlorophyll content (tcc) under salt and mannitol stresses. Increased superoxide dismutase (SOD), peroxidase (POD), glutathione transferase (GST) and glutathione peroxidase (GPX) activities, as well as decreased electrolyte leakage (EL) rates and malondialdehyde (MDA) contents, were observed in TheIF1A-transgenic tobacco and T. hispida seedlings under salt and mannitol stresses. Histochemical staining suggested that TheIF1A improves reactive oxygen species (ROS) scavenging in plants. Moreover, TheIF1A may regulate expression of stress-related genes, including TOBLTP, GST, MnSOD, NtMPK9, poxN1 and CDPK15. Moreover, a 1352-bp promoter fragment of TheIF1A was isolated, and cis-elements were identified. Yeast one-hybrid assays showed that ThDof can specifically bind to the Dof motif present in the promoter. In addition, ThDof showed expression patterns similar to those of TheIF1A under NaCl and PEG stresses. These findings suggest the potential mechanism and physiological roles of TheIF1A. ThDof may be an upstream regulator of TheIF1A, and TheIF1A may function as a stress response regulator to improve plant salt and osmotic stress tolerance via regulation of associated enzymes and ROS scavenging, thereby reducing cell damage under stress conditions.

WRKY transcription factors play important roles in many biological processes, and mainly bind to the W-box element to regulate gene expression. Previously, we characterized a WRKY gene from Tamarix hispida, ThWRKY4, in response to abiotic stress, and showed that it bound to the W-box motif. However, whether ThWRKY4 could bind to other motifs remains unknown. In this study, we employed a Transcription Factor-Centered Yeast one Hybrid (TF-Centered Y1H) screen to study the motifs recognized by ThWRKY4. In addition to the W-box core cis-element (termed W-box), we identified that ThWRKY4 could bind to two other motifs: the RAV1A element (CAACA) and a novel motif with sequence of GTCTA (W-box like sequence, WLS). The distributions of these motifs were screened in the promoter regions of genes regulated by some WRKYs. The results showed that the W-box, RAV1A, and WLS motifs were all present in high numbers, suggesting that they play key roles in gene expression mediated by WRKYs. Furthermore, five WRKY proteins from different WRKY subfamilies in Arabidopsis thaliana were selected and confirmed to bind to the RAV1A and WLS motifs, indicating that they are recognized commonly by WRKYs. These findings will help to further reveal the functions of WRKY proteins.

Although plant glutathione transferase (GST) genes are reported to be involved in responses to abiotic stress, few GST genes have been functionally characterized in woody halophytes. In the present study, a GST gene from Tamarix hispida, designated ThGSTZ1, was cloned and functionally characterized. Expression of ThGSTZ1 was downregulated by drought and salinity stress, and abscisic acid. Transgenic Arabidopsis thaliana plants with constitutive expression of ThGSTZ1 showed increased survival rates under drought and salinity stress. These transgenic Arabidopsis plants exhibited increased levels of GST, glutathione peroxidase, superoxide dismutase and peroxidase activity, along with decreased malondialdehyde content, electrolyte leakage rates and reactive oxygen species (ROS) levels under salt and drought stress conditions. Transgenic T. hispida that transiently overexpressed ThGSTZ1 showed increased GST and GPX activities under NaCl and mannitol treatments, as well as improved ROS scavenging ability. These results suggest that ThGSTZ1 can improve drought and salinity tolerance in plants by enhancing their ROS scavenging ability. Therefore, ThGSTZ1 represents a candidate gene with potential applications for molecular breeding to increase stress tolerance in plants.

Heat shock proteins (HSPs) play important roles in protecting plants against environmental stresses. Furthermore, small heat shock proteins (sHSPs) are the most ubiquitous HSP subgroup with molecular weights ranging from 15 to 42 kDa. In this study, nine sHSP genes (designated as ThsHSP1-9) were cloned from Tamarix hispida. Their expression patterns in response to cold, heat shock, NaCl, PEG and abscisic acid (ABA) treatments were investigated in the roots and leaves of T. hispida by real-time RT-PCR analysis. The results showed that most of the nine ThsHSP genes were expressed at higher levels in roots than in leaves under normal growth condition. All of ThsHSP genes were highly induced under conditions of cold (4 °C) and different heat shocks (36, 40, 44, 48 and 52 °C). Under NaCl stress, all nine ThsHSPs genes were up-regulated at least one stress time-point in both roots and leaves. Under PEG and ABA treatments, the nine ThsHSPs showed various expression patterns, indicating a complex regulation pathway among these genes. This study represents an important basis for the elucidation of ThsHSP gene function and provides essential information that can be used for stress tolerance genetic engineering in future studies.