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.

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.

Natural variation of GmFATA1B regulates seed oil content and composition in soybean.

Soybean (Glycine max) produces seeds that are rich in unsaturated fatty acids and is an important oilseed crop worldwide. Seed oil content and composition largely determine the economic value of soybean. Due to natural genetic variation, seed oil content varies substantially across soybean cultivars. Although much progress has been made in elucidating the genetic trajectory underlying fatty acid metabolism and oil biosynthesis in plants, the causal genes for many quantitative trait loci (QTLs) regulating seed oil content in soybean remain to be revealed. In this study, we identified GmFATA1B as the gene underlying a QTL that regulates seed oil content and composition, as well as seed size in soybean. Nine extra amino acids in the conserved region of GmFATA1B impair its function as a fatty acyl-acyl carrier protein thioesterase, thereby affecting seed oil content and composition. Heterogeneously overexpressing the functional GmFATA1B allele in Arabidopsis thaliana increased both the total oil content and the oleic acid and linoleic acid contents of seeds. Our findings uncover a previously unknown locus underlying variation in seed oil content in soybean and lay the foundation for improving seed oil content and composition in soybean.

Polyaniline-modified halloysite nanotubes as high-efficiency adsorbents for removing of naproxen in the presence of different heavy metals.

In this work, novel adsorbent polyaniline-modified halloysite nanotubes (HNT@PA-2) were synthesized successfully by in situ polymerization to increase active adsorption sites. With the increase of the amount of aniline, the adsorption capacity of naproxen becomes higher. The optimal ratio of halloysite nanotubes to aniline was 1 : 2. The effects of adsorption conditions such as pH, mass of HNT@PA-2, time and initial concentration of naproxen were systematically researched. The optimum adsorption for naproxen was pH 9, mass 10 mg and contact time 4 h. The adsorption of naproxen conformed to the pseudo-first-order kinetic model, and the maximum adsorption capacity was 242.58 mg g-1 at 318 K. In addition, the effects of ionic strength and different heavy metals also were studied. Higher ionic strength of the system could influence the adsorption of naproxen. The effects of Al3+, Pb2+, Zn2+ and Co2+ ions on the adsorption of naproxen could be ignored, while Cu2+ and Fe3+ ions inhibited the process. The mechanisms for naproxen adsorbed by the HNT@PA-2 were π-π interaction, hydrogen bonding and hydrophobic reaction. Therefore, the HNT@PA-2 could be used for the treatment of medical wastewater for removing naproxen.

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.

Overexpression of GmWRKY172 enhances cadmium tolerance in plants and reduces cadmium accumulation in soybean seeds.

Introduction: Cadmium (Cd) stress is a significant threat to soybean production, and enhancing Cd tolerance in soybean is the focus of this study. The WRKY transcription factor family is associated with abiotic stress response processes. In this study, we aimed to identify a Cd-responsive WRKY transcription factor GmWRKY172 from soybean and investigate its potential for enhancing Cd tolerance in soybean. Methods: The characterization of GmWRKY172 involved analyzing its expression pattern, subcellular localization, and transcriptional activity. To assess the impact of GmWRKY172, transgenic Arabidopsis and soybean plants were generated and examined for their tolerance to Cd and Cd content in shoots. Additionally, transgenic soybean plants were evaluated for Cd translocation and various physiological stress indicators. RNA sequencing was performed to identify the potential biological pathways regulated by GmWRKY172. Results: GmWRKY172 was significantly upregulated by Cd stress, highly expressed in leaves and flowers, and localized to the nucleus with transcriptional activity. Transgenic plants overexpressing GmWRKY172 showed enhanced Cd tolerance and reduced Cd content in shoots compared to WT. Lower Cd translocation from roots to shoots and seeds was also observed in transgenic soybean. Under Cd stress, transgenic soybean accumulated less malondialdehyde (MDA) and hydrogen peroxide (H2O2) than WT plants, with higher flavonoid and lignin contents, and peroxidase (POD) activity. RNA sequencing analysis revealed that many stress-related pathways were regulated by GmWRKY172 in transgenic soybean, including flavonoid biosynthesis, cell wall synthesis, and peroxidase activity. Discussion: Our findings demonstrated that GmWRKY172 enhances Cd tolerance and reduces seed Cd accumulation in soybean by regulating multiple stress-related pathways, and could be a promising candidate for breeding Cd-tolerant and low Cd soybean varieties.

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.

Genome-wide identification, expression and salt stress tolerance analysis of the GRAS transcription factor family in Betula platyphylla.

The GRAS gene family is a plant-specific family of transcription factors and play a vital role in many plant growth processes and abiotic stress responses. Nevertheless, the functions of the GRAS gene family in woody plants, especially in Betula platyphylla (birch), are hardly known. In this study, we performed a genome-wide analysis of 40 BpGRAS genes (BpGRASs) and identified typical GRAS domains of most BpGRASs. The BpGRASs were unevenly distributed on 14 chromosomes of birch and the phylogenetic analysis of six species facilitated the clustering of 265 GRAS proteins into 17 subfamilies. We observed that closely related GRAS homologs had similar conserved motifs according to motif analysis. Besides, an analysis of the expression patterns of 26 BpGRASs showed that most BpGRASs were highly expressed in the leaves and responded to salt stress. Six BpGRASs were selected for cis-acting element analysis because of their significant upregulation under salt treatment, indicating that many elements were involved in the response to abiotic stress. This result further confirmed that these BpGRASs might participate in response to abiotic stress. Transiently transfected birch plants with transiently overexpressed 6 BpGRASs and RNAi-silenced 6 BpGRASs were generated for gain- and loss-of-function analysis, respectively. In addition, overexpression of BpGRAS34 showed phenotype resistant to salt stress, decreased the cell death and enhanced the reactive oxygen species (ROS) scavenging capabilities and proline content under salt treatment, consistent with the results in transiently transformed birch plants. This study is a systematic analysis of the GRAS gene family in birch plants, and the results provide insight into the molecular mechanism of the GRAS gene family responding to abiotic stress in birch plants.

UNFERTILIZED EMBRYO SAC 12 phosphorylation plays a crucial role in conferring salt tolerance.

Arabidopsis (Arabidopsis thaliana) UNFERTILIZED EMBRYO SAC 12 (AtUNE12) belongs to the basic helix-loop-helix DNA-binding superfamily of proteins. However, its function is not well known. Here, we found that AtUNE12 plays an important role in mediating salt tolerance. AtUNE12 is a transcriptional activator located in the nucleus whose expression is induced by NaCl, mannitol, and abscisic acid. In addition to binding to the G-box "CACGTG", AtUNE12 also binds to the low temperature responsive element 15 (LTRE15) "CCGAC". Furthermore, the serine residue at position 108 of AtUNE12 is phosphorylated during the salt stress response, enabling AtUNE12 to trigger gene expression by binding to G-box and/or LTRE15 motifs. Phosphorylated AtUNE12 regulates the expression of the genes involved in ion transport leading to reduced Na+ accumulation and K+ loss. At the same time, phosphorylation of AtUNE12 also induces the expression of AtMYB61 to decrease stomatal aperture, leading to a reduced transpiration rate. Overall, AtUNE12 serves as a transcriptional activator that is induced and phosphorylated upon salt stress, and the induction and phosphorylation of AtUNE12 in turn activate the salt-overly-sensitive pathway and decrease the stomatal aperture, enabling improved salt tolerance.

Revealing the salt tolerance mechanism of Tamarix hispida by large-scale identification of genes conferring salt tolerance.

The identification of genes conferring salt tolerance is important to reveal plant salt tolerance mechanisms. Here, we employed yeast expression system combined with high-throughput sequencing to identify genes conferring salt tolerance from Tamarix hispida Willd. A total of 1224 potential genes conferring salt tolerance were identified. Twenty-one genes were randomly selected for functional characterization using transient transformation in T. hispida and stable transformation in Arabidopsis thaliana (L.) Heynh. More than 90% of studied genes are found to confer tolerance to salt stress, indicating that the identified genes are reliable. More than 75% of the identified genes were highly expressed in roots rather than in leaves, suggesting roots play an important role in salt tolerance. The genes belonging to 'response to stimulus' were highly accumulated , and these accounted for 32% of the total identified genes. In addition, the processes of 'protein translation', 'osmotic adjustment', 'scavenging of free radicals', 'photosynthesis, detoxification of cells', 'protection of cellular macromolecules' and 'maintenance of cellular pH' play important roles in salt tolerance. This study provides useful information on the salt tolerance mechanism of T. hispida and offers a valuable resource for exploring genes used in salt tolerance breeding.

Selection of appropriate reference genes for quantitative real-time reverse transcription PCR in Betula platyphylla under salt and osmotic stress conditions.

Selecting appropriate reference genes is vital to normalize gene expression analysis in birch (Betula platyphylla) under different abiotic stress conditions using quantitative real-time reverse transcription PCR (qRT-PCR). In this study, 11 candidate birch reference genes (ACT, TUA, TUB, TEF, 18S rRNA, EF1α, GAPDH, UBC, YLS8, SAND, and CDPK) were selected to evaluate the stability of their expression in different tissues and under different abiotic stress conditions. Three statistical algorithms (GeNorm, NormFinder, and BestKeeper) were used to analyze the stability of the 11 candidate reference genes to identify the most appropriate one. The results indicated that EF-1α was the most stable reference gene in different birch tissues, ACT was the most stable reference gene for normal conditions, ACT and TEF were the most stable reference genes for salt stress treatment, TUB was the most stable reference gene for osmotic stress treatment, and ACT was the most appropriate choice in all samples of birch. In conclusion, the most appropriate reference genes varied among different experimental conditions. However, in this study, ACT was the optimum reference gene in all experimental groups, except in the different tissues group. GAPDH was the least stable candidate reference gene in all experimental conditions. In addition, three stress-induced genes (BpGRAS1, BpGRAS16, and BpGRAS19) were chosen to verify the stability of the selected reference genes in different tissues and under salt stress. This study laid the foundation for the selection of appropriate reference gene(s) for future gene expression pattern studies in birch.

The NAC Protein from Tamarix hispida, ThNAC7, Confers Salt and Osmotic Stress Tolerance by Increasing Reactive Oxygen Species Scavenging Capability.

Plant specific NAC (NAM, ATAF1/2 and CUC2) transcription factors (TFs) play important roles in response to abiotic stress. In this study, we identified and characterized a NAC protein, ThNAC7, from Tamarix hispida. ThNAC7 is a nuclear localized protein and has transcriptional activation activity. ThNAC7 expression was markedly induced by salt and osmotic stresses. Transiently transformed T. hispida seedlings overexpressing ThNAC7 (OE) or with RNA interference (RNAi) silenced ThNAC7 were generated to investigate abiotic stress tolerance via the gain- and loss- of function. Overexpressing ThNAC7 showed an increased reactive oxygen species (ROS) scavenging capabilities and proline content, which was accomplished by enhancing the activities of superoxide dismutase (SOD) and peroxidase (POD) in transiently transformed T. hispida and stably transformed Arabidopsis plants. Additionally, ThNAC7 activated these physiological changes by regulating the transcription level of P5CS, SOD and POD genes. RNA-sequencing (RNA-seq) comparison between wild-type and ThNAC7-transformed Arabidopsis showed that more than 40 known salt tolerance genes might regulated by ThNAC7, including stress tolerance-related genes and TF genes. The results indicated that ThNAC7 induces the transcription level of genes associated with stress tolerance to enhance salt and osmotic stress tolerance via an increase in osmotic potential and enhanced ROS scavenging.

Identifying modulation formats through 2D Stokes planes with deep neural networks.

A lightweight convolutional (deep) neural networks (CNNs) based modulation format identification (MFI) scheme in 2D Stokes planes for polarization domain multiplexing (PDM) fiber communication system is proposed and demonstrated. Influences of the learning rate of CNN is discussed. Experimental verifications are performed for the PDM system at a symbol rate of 28GBaud. Six modulation formats are identified with a trained CNN from images of received signals. They are PDM-BPSK, PDM-QPSK, PDM-8PSK, PDM-16QAM, PDM-32QAM, and PDM-64QAM. By taking advantage of computer vision, the results show that the proposed scheme can significantly improve the identification performance over the existing techniques.

SMAC: Simultaneous Mapping and Clustering Using Spectral Decompositions.

We introduce a principled approach for simultaneous mapping and clustering (SMAC) for establishing consistent maps across heterogeneous object collections (e.g., 2D images or 3D shapes). Our approach takes as input a heterogeneous object collection and a set of maps computed between some pairs of objects, and outputs a homogeneous object clustering together with a new set of maps possessing optimal intra- and inter-cluster consistency. Our approach is based on the spectral decomposition of a data matrix storing all pairwise maps in its blocks. We additionally provide tight theoretical guarantees for the accuracy of SMAC under established noise models. We also demonstrate the usefulness of our approach on synthetic and real datasets.