Acetylation of transcription factor BpTCP20 by acetyltransferase BpPDCE23 modulates salt tolerance in birch.

Teosinte branched 1/Cycloidea/Proliferating cell factor (TCP) transcription factors function in abiotic stress responses. However, how TCPs confer salt tolerance is unclear. Here, we characterized a TCP transcription factor, BpTCP20, that responds to salt stress in birch (Betula platyphylla Suk). Plants overexpressing BpTCP20 displayed increased salt tolerance, and Bptcp20 knockout mutants displayed reduced salt tolerance relative to the wild-type (WT) birch. BpTCP20 conferred salt tolerance by mediating stomatal closure and reducing reactive oxygen species (ROS) accumulation. Chromatin immunoprecipitation sequencing showed that BpTCP20 binds to NeuroD1, T-box, and two unknown elements (termed TBS1 and TBS2) to regulate target genes. In birch, salt stress led to acetylation of BpTCP20 acetylation at lysine 259. A mutated BpTCP20 variant (abolished for acetylation, termed BpTCP20259) was overexpressed in birch, which led to decreased salt tolerance compared with plants overexpressing BpTCP20. However, BpTCP20259-overexpressing plants still displayed increased salt tolerance relative to untransformed WT plants. BpTCP20259 showed reduced binding to the promoters of target genes and decreased target gene activation, leading to decreased salt tolerance. In addition, we identified dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex (BpPDCE23), an acetyltransferase that interacts with and acetylates BpTCP20 to enhance its binding to DNA motifs. Together, these results suggest that BpTCP20 is a transcriptional regulator of salt tolerance, whose activity is modulated by BpPDCE23-mediated acetylation.

BpGRP1 acts downstream of BpmiR396c/BpGRF3 to confer salt tolerance in Betula platyphylla.

Glycine-rich RNA-binding proteins (GRPs) have been implicated in the responses of plants to environmental stresses, but the function of GRP genes involved in salt stress and the underlying mechanism remain unclear. In this study, we identified BpGRP1 (glycine-rich RNA-binding protein), a Betula platyphylla gene that is induced under salt stress. The physiological and molecular responses to salt tolerance were investigated in both BpGRP1-overexpressing and suppressed conditions. BpGRF3 (growth-regulating factor 3) was identified as a regulatory factor upstream of BpGRP1. We demonstrated that overexpression of BpGRF3 significantly increased the salt tolerance of birch, whereas the grf3-1 mutant exhibited the opposite effect. Further analysis revealed that BpGRF3 and its interaction partner, BpSHMT, function upstream of BpGRP1. We demonstrated that BpmiR396c, as an upstream regulator of BpGRF3, could negatively regulate salt tolerance in birch. Furthermore, we uncovered evidence showing that the BpmiR396c/BpGRF3 regulatory module functions in mediating the salt response by regulating the associated physiological pathways. Our results indicate that BpmiR396c regulates the expression of BpGRF3, which plays a role in salt tolerance by targeting BpGRP1.

A reverse chromatin immunoprecipitation technique based on the CRISPR-dCas9 system.

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.

The transcription factor ThDOF8 binds to a novel cis-element and mediates molecular responses to salt stress in Tamarix hispida.

Salt stress is a common abiotic factor that restricts plant growth and development. As a halophyte, Tamarix hispida is a good model plant for exploring salt-tolerance genes and regulatory mechanisms. DNA-binding with one finger (DOF) is an important transcription factor (TF) that influences and controls various signaling substances involved in diverse biological processes related to plant growth and development, but the regulatory mechanisms of DOF TFs in response to salt stress are largely unknown in T. hispida. In the present study, a newly identified Dof gene, ThDOF8, was cloned from T. hispida, and its expression was found to be induced by salt stress. Transient overexpression of ThDOF8 enhanced T. hispida salt tolerance by enhancing proline levels, and increasing the activities of the antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POD). These results were also verified in stably transformed Arabidopsis. Results from TF-centered yeast one-hybrid (Y1H) assays and EMSAs showed that ThDOF8 binds to a newly identified cis-element (TGCG). Expression profiling by gene chip analysis identified four potential direct targets of ThDOF8, namely the cysteine-rich receptor-like kinases genes, CRK10 and CRK26, and two glutamate decarboxylase genes, GAD41, and GAD42, and these were further verified by ChIP-quantitative-PCR, EMSAs, Y1H assays, and ß-glucuronidase enzyme activity assays. ThDOF8 can bind to the TGCG element in the promoter regions of its target genes, and transient overexpression of ThCRK10 also enhanced T. hispida salt tolerance. On the basis of our results, we propose a new regulatory mechanism model, in which ThDOF8 binds to the TGCG cis-element in the promoter of the target gene CRK10 to regulate its expression and improve salt tolerance in T. hispida. This study provides a basis for furthering our understanding the role of DOF TFs and identifying other downstream candidate genes that have the potential for improving plant salt tolerance via molecular breeding.

An Insight of Betula platyphylla SWEET Gene Family through Genome-Wide Identification, Expression Profiling and Function Analysis of BpSWEET1c under Cold Stress.

SWEET proteins play important roles in plant growth and development, sugar loading in phloem and resistance to abiotic stress through sugar transport. In this study, 13 BpSWEET genes were identified from birch genome. Collinearity analysis showed that there were one tandem repeating gene pair (BpSWEET1b/BpSWEET1c) and two duplicative gene pairs (BpSWEET17a/BpSWEET17b) in the BpSWEET gene family. The BpSWEET gene promoter regions contained several cis-acting elements related to stress resistance, for example: hormone-responsive and low-temperature-responsive cis-elements. Analysis of transcriptome data showed that BpSWEET genes were highly expressed in several sink organs, and the most BpSWEET genes were rapidly up-regulated under cold stress. BpSWEET1c, which was highly expressed in cold stress, was selected for further analysis. It was found that BpSWEET1c was located on the cell membrane. After 6 h of 4 °C stress, sucrose content in the leaves and roots of transient overexpressed BpSWEET1c was significantly higher than that of the control. MDA content in roots was significantly lower than that of the control. These results indicate that BpSWEET1c may play a positive role in the response to cold stress by promoting the metabolism and transport of sucrose. In conclusion, 13 BpSWEET genes were identified from the whole genome level. Most of the SWEET genes of birch were expressed in the sink organs and could respond to cold stress. Transient overexpression of BpSWEET1c changed the soluble sugar content and improved the cold tolerance of birch.

Construction of two regulatory networks related to salt stress and lignocellulosic synthesis under salt stress based on a Populus davidiana × P. bolleana transcriptome analysis.

KEY MESSAGE: 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.

A 2-Cys peroxiredoxin gene from Tamarix hispida improved salt stress tolerance in plants.

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.

Correction to: A 2-Cys peroxiredoxin gene from Tamarix hispida improved salt stress tolerance in plants.

An amendment to this paper has been published and can be accessed via the original article.

HOS1 regulates Argonaute1 by promoting transcription of the microRNA gene MIR168b in Arabidopsis.

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.

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.

Expression analysis of nine small heat shock protein genes from Tamarix hispida in response to different abiotic stresses and abscisic acid treatment.

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.

Expression profiles of 12 late embryogenesis abundant protein genes from Tamarix hispida in response to abiotic stress.

Twelve embryogenesis abundant protein (LEA) genes (named ThLEA-1 to -12) were cloned from Tamarix hispida. The expression profiles of these genes in response to NaCl, PEG, and abscisic acid (ABA) in roots, stems, and leaves of T. hispida were assessed using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). These ThLEAs all showed tissue-specific expression patterns in roots, stems, and leaves under normal growth conditions. However, they shared a high similar expression patterns in the roots, stems, and leaves when exposed to NaCl and PEG stress. Furthermore, ThLEA-1, -2, -3, -4, and -11 were induced by NaCl and PEG, but ThLEA-5, -6, -8, -10, and -12 were downregulated by salt and drought stresses. Under ABA treatment, some ThLEA genes, such as ThLEA-1, -2, and -3, were only slightly differentially expressed in roots, stems, and leaves, indicating that they may be involved in the ABA-independent signaling pathway. These findings provide a basis for the elucidation of the function of LEA genes in future work.

Phosphorylation of birch BpNAC90 improves the activation of gene expression to confer drought tolerance.

The NAC transcription factors (TFs) play important roles in mediating abiotic stress tolerance; however, the mechanism is still not fully known. Here, an NAC gene (BpNAC90) from a gene regulatory network of Betula platyphylla (birch) that responded to drought was characterized. Overexpression and knockout of BpNAC90 displayed increased and reduced drought tolerance, respectively, relative to wild-type (WT) birch. BpNAC90 binds to different DNA motifs to regulate target genes in conferring drought tolerance, such as Eomes2, ABRE and Tgif2. BpNAC90 is phosphorylated by drought stress at Ser 205 by birch SNF1-related protein kinase 2 (BpSRK2A). Mutated BpNAC90 (termed S205A) with abolished phosphorylation, was transformed into birch for overexpression. The transgenic S205A plants displayed significantly reduced drought tolerance compared with plants overexpressing BpNAC90, but still showed increased drought tolerance relative to WT birch. At the same time, S205A showed a decreased capability to bind to motifs and reduced activation of target gene expression, which contributed to the reduced drought tolerance. Additionally, BpSRK2A and BpNAC90 can be induced by drought stress and form a complex to phosphorylate BpNAC90. The results together indicated that phosphorylation of BpNAC90 is necessary in conferring drought tolerance in birch.

An electrophoretic mobility shift assay using the protein isolated from host plants.

BACKGROUND: The electrophoretic mobility shift assay (EMSA) is a common technology to detect DNA-protein interactions. However, in most cases, the protein used in EMSA is obtained via prokaryotic expression, and rarely from plants. At the same time, the proteins expressed from prokaryotic systems usually cannot fold naturally and have no post translationally modification, which may affect the binding of proteins to DNA. RESULTS: Here, we develop a technique to quickly isolate proteins of interest from host plants and then analyze them using fluorescent EMSA. This technology system is called: protein from plants fluorescent EMSA method (PPF-EMSA). In PPF-EMSA, a special transient transformation method is employed to transiently deliver genes into the plant, enabling efficient synthesis the encoded proteins. Then, the target protein is isolated using immunoprecipitation, and the DNA probes were labeled with cyanine 3 (Cy3). Both fluorescent EMSA and super-shift fluorescent EMSA can be performed using the proteins from plants. Three kinds of plants, Betula platyphylla, Populus. davidiana×P. bolleana and Arabidopsis thaliana, are used in this study. The proteins isolated from plants are in a natural state, can fold naturally and are posttranslationally modified, enabling true binding to their cognate DNAs. CONCLUSION: As transient transformation can be performed quickly and not depended on whether stable transformation is available or not, we believe this method will have a wide application, enabling isolation of proteins from host plant conveniently.

Protein profile analysis of tension wood development in response to artificial bending and gravitational stimuli in Betula platyphylla.

Betula platyphylla Suk (birch) is an excellent short-term hardwood species with growth and wood characteristics well suited to wood industries. To investigate the molecular mechanism of wood development in birch, a tension wood (TW) induced system was used to explore the regulatory mechanism at the protein level and identify the key proteins involved in xylem development in birch. The results of dyeing with Safranin O-Fast Green indicated that the cellulose content of TW was significantly higher than that of opposite wood (OW) or normal wood (NW), and the lignin content in TW was significantly lower than that in OW and NW after artificial bending of birch stems. Protein profile analysis of TW, NW and OW by iTRAQ revealed that there were 639 and 460 differentially expressed proteins (DEPs) between TW/OW and TW/NW, respectively. The DEPs were mainly enriched in tyrosine metabolism, glycolysis/gluconeogenesis, phenylalanine and tyrosine metabolism, phenylpropanoid and pyruvate metabolism, the pentose phosphate pathway, the citrate cycle (TCA cycle), fructose and mannose metabolism, carbon fixation in photosynthetic organisms, fatty acid biosynthesis, photosynthesis proteins and other pathways. The proteins in the citrate cycle were upregulated. The expression levels of PGI, PGM and FRK proteins related to cellulose synthesis increased and the expression levels of PAL, 4CL and COMT related to lignin synthesis decreased, leading to an increase in cellulose content and decreased lignin levels in TW. PPI analysis revealed that key DEPs interact with each other, indicating that these proteins form complexes to implement this function, which may provide important insights for wood formation at the molecular level.

The ethylene response factor gene, ThDRE1A, is involved in abscisic acid- and ethylene-mediated cadmium accumulation in Tamarix hispida.

Tamarix hispida is highly tolerant to salt, drought and heavy metal stress and is a potential material for the remediation of cadmium (Cd)-contaminated soil under harsh conditions. In this study, T. hispida growth and chlorophyll content decreased, whereas flavonoid and carotenoid contents increased under long-term Cd stress (25 d). The aboveground components of T. hispida were collected for RNA-seq to investigate the mechanism of Cd accumulation. GO and KEGG enrichment analyses revealed that the differentially expressed genes (DEGs) were significantly enriched in plant hormone-related pathways. Exogenous hormone treatment and determination of Cd2+ levels showed that ethylene (ETH) and abscisic acid (ABA) antagonists regulate Cd accumulation in T. hispida. Twenty-five transcription factors were identified as upstream regulators of hormone-related pathways. ThDRE1A, which was previously identified as an important regulatory factor, was selected for further analysis. The results indicated that ThABAH2.5 and ThACCO3.1 were direct target genes of ThDRE1A. The determination of Cd2+, ABA, and ETH levels indicated that ThDRE1A plays an important role in Cd accumulation through the antagonistic regulation of ABA and ETH. In conclusion, these results reveal the molecular mechanism underlying Cd accumulation in plants and identify candidate genes for further research.

Bp-miR408a participates in osmotic and salt stress responses by regulating BpBCP1 in Betula platyphylla.

The microRNAs, which are small RNAs of 18-25 nt in length, act as key regulatory factors in posttranscriptional gene expression during plant growth and development. However, little is known about their regulatory roles in response to stressful environments in birch (Betula platyphylla). Here, we characterized and further explored miRNAs from osmotic- and salt-stressed birch. Our analysis revealed a total of 190 microRNA (miRNA) sequences, which were classified into 180 conserved miRNAs and 10 predicted novel miRNAs based on sequence homology. Furthermore, we identified Bp-miR408a under osmotic and salt stress and elucidated its role in osmotic and salt stress responses in birch. Notably, under osmotic and salt stress, Bp-miR408a contributed to osmotic and salt tolerance sensitivity by mediating various physiological changes, such as increases in reactive oxygen species accumulation, osmoregulatory substance contents and Na+ accumulation. Additionally, molecular analysis provided evidence of the in vivo targeting of BpBCP1 (blue copper protein) transcripts by Bp-miR408a. The overexpression of BpBCP1 in birch enhanced osmotic and salt tolerance by increasing the antioxidant enzyme activity, maintaining cellular ion homeostasis and decreasing lipid peroxidation and cell death. Thus, we reveal a Bp-miR408a-BpBCP1 regulatory module that mediates osmotic and salt stress responses in birch.

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.

A DNA-binding protein capture technology that purifies proteins by directly isolating the target DNA.

DNA-protein interactions are critical to almost all cellular functions, and identification of the proteins that bind to an DNA site of interest (gene-centered approach) is an important investigation area. However, gene-centered methods are mainly based on DNA hybridization to isolate target proteins, which is complex and inefficient. Here, we built a gene-centered approach involving direct isolation of target DNA, termed protein capture based on biolistic transformation (PCaB). The target DNA was labeled with biotin and cyanine 3 (Cy3) at its 5' and 3' DNA ends, respectively, and introduced into the host plants through biolistic transformation. The DNA and its binding proteins were crosslinked using formaldehyde. The labeled DNAs were obtained using gel excision and biotin-Streptavidin affinity according to the indication of Cy3 fluorescence, which make harvest of target DNA with a low background. The DNA-binding proteins were identified using mass spectrometry analysis. The PCaB method allowed us to identify and confirm 16 putative upstream regulators of the BpERF3 gene from Betula platyphylla. Theoretically, PCaB could be adapted to all plant species that can be transformed using biolistic bombardment, and captures DNA-binding proteins quickly with a low background. Therefore, PCaB will provide a powerful tool to discover DNA-protein interactions.

The R2R3-MYB transcription factor ThRAX2 recognized a new element MYB-T (CTTCCA) to enhance cadmium tolerance in Tamarix hispida.

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 1191 bp 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 roots and 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 ThRAX2 to regulate Cd tolerance, which lays the foundation for the ThRAX2 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.