Electrophoretic Mobility Shift Assay Research Articles

Adenanthin Exhibits Anti-Inflammatory Effects by Covalently Targeting the p65 Subunit in the NF-κB Signaling Pathway

Adenanthin is a structurally unique ent-kaurane diterpenoid isolated from Rabdosia adenantha, a traditional Chinese medicinal plant with potent anti-cancer and anti-inflammatory activities. However, its anti-inflammatory molecular mechanism remains largely elusive to date. Here, we developed an affinity-based label-free protein profiling (ALFPP) to identify potential covalent targets of electrophilic natural products with ketone or aldehyde groups. Using ALFPP, we identified 27 potential covalent targets of adenanthin, among which p65 (RelA) has been associated with its anti-inflammatory activities. Through a series of experiments, including LC-MS/MS, molecular docking, electrophoretic mobility shift assays (EMSA), and genome editing, we demonstrated that adenanthin could covalently modify the Cys38 residue of p65 to affect the binding of DNA to p65, thereby inhibiting the NF-κB signaling pathway. ALFPP will facilitate the target identification of electrophilic carbonylated natural products, especially those containing α, β-unsaturated keto groups. Furthermore, the elucidation of the molecular mechanism of adenanthin will contribute to new drug development of adenanthin to treat inflammations and cancers, enhancing the possibility for its clinical application.

GmSTOP1-3 regulates flavonoid synthesis to reduce ROS accumulation and enhance aluminum tolerance in soybean

Aluminum (Al) toxicity is a significant limiting factor for crop production in acid soils. The functions and regulatory mechanisms of transcription factor STOP1 (Sensitive to Proton Rhizotoxicity 1) family genes in Al-tolerance have been widely studied in many plant species, except for soybean. Here, expression of GmSTOP1-3 was significantly enhanced by Al stress in soybean roots. Overexpression of GmSTOP1-3 resulted in enhanced root elongation and decreased Al content, which was accompanied by increased antioxidant capacity under Al treatment. Furthermore, RNA-seq identified 498 downstream genes of GmSTOP1-3, including genes involved in flavonoid biosynthesis. Among them, the expression of chalcone synthase (GmCHS) and isoflavone synthase (GmIFS) were highly enhanced by GmSTOP1-3 overexpression. Further quantitative flavonoid metabolome analysis showed that overexpression of GmSTOP1-3 significantly increased the content of naringenin chalcone, naringenin, and genistein in soybean roots under Al treatment, which positively correlated with the expression level of the genes relative to flavonoid biosynthesis. Notably, genistein had a significant positive correlation with the expression levels of GmIFS. Combination of Dual Luciferase Complementation (LUC) and Electrophoretic Mobility Shift Assays (EMSA) revealed that GmSTOP1-3 directly bound to the promoters of GmCHS/GmIFS and activated both genes’ transcription. Taken together, these results suggest that GmSTOP1-3 enhances soybean Al tolerance partially through regulating the flavonoid synthesis.

Transcription factor Pofst3 regulates Pleurotus ostreatus development by targeting multiple biological pathways

Pleurotus ostreatus is a popular edible mushroom cultivated worldwide. However, the mechanism of P. ostreatus primordia formation is unclear. Pofst3 is a MHR superfamily transcription factor, which has the function of regulating primordia formation. In this study, the target genes of Pofst3 in P. ostreatus were identified by DAP-Seq approach at the genome level, 1481 peaks were obtained and the Pofst3 binding motif sequence was GARGRVGARGAR. The interaction between transcription factor Pofst3 and this motif was confirmed in vitro and in vivo through electrophoretic mobility shift (EMSA) and yeast one-hybrid screening (Y1H) assays. Among the top 20 GO enrichment results, most were related to transcriptional regulation, and some transcription factor encoding genes, such as HMG-box (gene_5346), MADS-box (gene_86), FOG (gene_6211) and RFX (gene_3496) were obtained. Besides basic metabolism, MAPK signaling pathway, Inositol phosphate metabolism, Glycosylphosphatidylinositol (GPI)-anchor biosynthesis and Pentose phosphate pathway were significantly enriched in the KEGG pathway analysis. The expression levels of randomly selected 11 genes, some transcription factor genes, and genes involved in metabolic pathways in wild and Pofst3 transgenic P. ostreatus strains indicated that target genes likely involved in the development of the P. ostreatus primordia. These results indicated that transcription factor Pofst3 ultimately negatively regulated the development of P. ostreatus primordia very likely through regulating a series of biological pathways.

High level non-carbapenemase carbapenem resistance by overlaying mutations of mexR , oprD, and ftsI in Pseudomonas aeruginosa

ABSTRACT Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a global threat, but the mechanism of non-carbapenemase carbapenem resistance is still unclear. In the current study, we investigated the contributions of point mutations in mexR , oprD , and ftsI to carbapenem resistance in P. aeruginosa during in vivo evolution studies with consecutive clinical isolates. Real-time qPCR and Electrophoretic Mobility Shift Assay demonstrated that MexR (Gln55Pro) mutation increased MexAB efflux pump genes expression by altering MexR’s binding capacity, leading to a four- to eight-fold increase in meropenem MIC in the Pae d1 Green ∆ mexR and PAO1∆ mexR mutants. The OprD (Trp415*) truncation affected porin structure, and the constructed mutant Pae d1 Green oprD Trp415* increased meropenem MIC by 16-fold (from 0.25 to 4 µg/mL). The contribution of ftsI mutation to meropenem resistance was confirmed by clinical linkage analysis and was estimated to cause a two-fold increase in meropenem MIC by comparing the resistant clinical isolate with the Pae d1 Green oprD Trp415*∆ mexR double mutant. The study found that the oprD Trp415* allele alone accounts for the imipenem MIC in clinical isolates, while the ∆ mexR and ftsI Arg504Cys alleles do not contribute to imipenem resistance. In conclusion, we identified and explored the contributions of mexR , oprD, and ftsI mutations to high level non-carbapenemase carbapenem resistance in P. aeruginosa . These findings highlight the interplay of different mutations in causing non-carbapenemase carbapenem-resistance in P. aeruginosa . IMPORTANCE The emergence of carbapenem-resistant Pseudomonas aeruginosa (CRPA) poses a significant global health threat, complicating treatment options for infections caused by this pathogen. Understanding the mechanisms behind non-carbapenemase carbapenem resistance is critical for developing effective therapeutic strategies. This study provides crucial insights into how specific point mutations in key genes- mexR , oprD , and ftsI -contribute to carbapenem resistance, particularly the MexR (Gln55Pro) mutation’s effect on efflux pump expression and the OprD (Trp415*) truncation’s impact on porin structure. The findings elucidate the complex interplay of these mutations, highlighting their roles in conferring high-level resistance, and underscore the imperative for continued research to inform therapeutic strategies against CRPA infections.

Mycobacterium tuberculosis Mce3R TetR-like Repressor Forms an Asymmetric Four-Helix Bundle and Binds a Nonpalindrome Sequence†.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a major global health concern. TetR family repressors (TFRs) are important for Mtb's adaptation to the human host environment. Our study focuses on one notable Mtb repressor, Mce3R, composed of an unusual double TFR motif. Mce3R-regulated genes encode enzymes implicated in cholesterol metabolism, resistance against reactive oxygen species, and lipid transport activities important for Mtb survival and persistence in the host and for the cellular activity of a 6-azasteroid derivative. Here, we present the structure of Mce3R bound to its DNA operator, unveiling a unique asymmetric assembly previously unreported. We obtained a candidate DNA-binding motif through MEME motif analysis, comparing intergenic regions of mce3R orthologues and identifying nonpalindromic regions conserved between orthologues. Using an electrophoretic mobility shift assay (EMSA), we confirmed that Mce3R binds to a 123-bp sequence that includes the predicted motif. Using scrambled DNA and DNA oligonucleotides of varying lengths with sequences from the upstream region of the yrbE3A (mce3) operon, we elucidated the operator region to be composed of two Mce3R binding sites, each a 25-bp asymmetric sequence separated by 53 bp. Mce3R binds with a higher affinity to the downstream site with a Kd of 2.4 ± 0.7 nM. The cryo-EM structure of Mce3R bound to the 123-bp sequence was refined to a resolution of 2.51 Å. Each Mce3R monomer comprises 21 α-helices (α1-α21) folded into an asymmetric TFR-like structure with a core asymmetric four-helix bundle. This complex has two nonidentical HTH motifs and a single ligand-binding domain. The two nonidentical HTHs from each TFR bind within the high-affinity, nonpalindromic operator motif, with Arg53 and Lys262 inserted into the major groove. Site-directed mutagenesis of Arg53 to alanine abrogated DNA binding, validating the Mce3R/DNA structure obtained. Among 811,645 particles, 63% were Mce3R homodimer bound to two duplex oligonucleotides. Mce3R homodimerizes primarily through α15, and each monomer binds to an identical site in the DNA duplex oligonucleotide.

OsIAA23 Promotes Heading by Directly Downregulating Ghd7 in rice.

Ghd7 is a central regulator to multiple growth and development processes in rice. While it is not clear how Ghd7 is regulated by upstream factors. To identify its upstream regulator, the truncated Ghd7 promoter fragments were used to screen cis elements binding to rice total nuclear proteins. Electrophoretic mobility shift assays screened one truncated fragment f3 binding to the proteins. Subsequently, the fragment f3 was employed to screen a yeast one-hybrid library, and a transcription factor OsIAA23 was screened as a direct upstream regulator of Ghd7. Dual-luciferase transient assay demonstrated the transcriptional repression effect of OsIAA23 on the activity of Ghd7, and the location of the cis elements binding to OsIAA23 in the region 1264 to 1255bp upstream of ATG. Genetic analysis between the wild type Ghd7-OsIAA23 and single/double mutants further verified that OsIAA23 downregulated Ghd7 expression and led to a delayed heading under long day conditions. Moreover, natural variations in fragment f3 were associated with heading and geographic distribution in rice. This study sheds light on the direct regulatory mechanism of OsIAA23 on Ghd7, which enriches the understanding of the Ghd7 involved flowering regulatory network in rice.

Effects of hap2 deletion on mnp/vp transcription in Pleurotus ostreatus grown on lignocellulosic substrates.

The regulatory mechanisms governing expression of genes encoding lignin-modifying enzymes (LME) in white-rot fungi remain largely unexplored. Although molecular cloning has identified CCAAT-boxes frequently located 5'-upstream of these genes, their role in transcriptional regulation is not well understood. This study examines the function of hap2, a gene encoding a hypothetical protein homologous to a component of the CCAAT-binding Hap complex, in the white-rot fungus Pleurotus ostreatus. Deletion of hap2 resulted in significantly reduced Mn2+-dependent peroxidase activity and lignin-degrading capacity compared to the parental strain 20b grown on beech wood sawdust (BWS) medium. Real-time PCR revealed that vp2 transcript levels were significantly lower in hap2 deletants than in 20b grown when cultured on the three solid media consisting of BWS, holocellulose, or Avicel, but not on yeast-malt-glucose (YMG) agar plates. Additionally, glutathione S-transferase (GST) pulldown and electrophoretic mobility shift assays demonstrated that recombinant P. ostreatus Hap2, Hap3, and Hap5 expressed in Escherichia coli form a complex capable of binding to the CCAAT sequence 5'-upstream of vp2 in vitro. These results suggest that Hap2, as part of the CCAAT-binding complex, is essential for transcriptional upregulation of vp2 in P. ostreatus growing on lignocellulosic substrates. KEY POINTS: • P. ostreatus hap2 deletants were generated. • Lignin-degrading capacity was significantly reduced in the hap2 deletants. • vp2 was significantly downregulated upon hap2 deletion.

Hepatocellular carcinoma antibodies preferably identify nitro-oxidative-DNA lesions induced by 4-Chloro-orthophenylenediamine and DEANO.

The widespread use of oxidative hair colouring cosmetics threatens public health. Phenylenediamine derivatives serve as the main pigment in permanent hair colours. They interact with biological macromolecules, altering their functional and structural physiology. The study aimed to investigate the effect of a typical synthetic hair dye pigment, 4-Chloro-orthophenylenediamine (4-Cl-OPD), under a nitrating environment of DEANO on the calf thymus DNA molecule. The results showed single-stranded regions, base/sugar-phosphate backbone alterations, molecular changes, and nitro-oxidative lesions. These modifications are referred to as neo-epitopes on the DNA molecule. IgGs from cancer patients with a history of permanent hair dye use were screened for the recognition of neo-epitopes on DNA molecules. Hepatocellular carcinoma IgG showed the highest binding with 56% inhibition in the competition ELISA. The immune complex formation was observed through electrophoretic mobility shift assay. In conclusion, synthetic hair dye users are likely to present with heightened immunological triggers under elevated nitric oxide levels. The study reports chronic hair dye exposure as one of the factors responsible for altering the intricacies of the DNA's microarchitectural structure and inducing neo-epitopes on the molecule. The physiological status of NO may define the susceptibility towards 4-Cl-OPD and humoral response in hair dye users. Persistent nitro-oxidative stress due to 4-Cl-OPD and NO may induce a heightened immune response against neoepitopes in the nitro-oxidatively modified DNA. Therefore, chronic hair dye exposure may be identified as a risk to human health. These findings may contribute to a better understanding and reinforcement of hair dye as one of the modifiable risk factors responsible for the pro-inflammatory carcinogenic environment.

Specific binding between Arabidopsis thaliana phytochrome-interacting factor 3 (AtPIF3) bHLH and G-box originated prior to embryophyte emergence

The basic helix-loop-helix (bHLH) domain via critical amino acid residues on basic region binding to E-box (5′-CANNTG-3′) is known in embryophyte. However, the dictated E-box types selection by bHLH dimers and the significant impact of these critical amino acid residues along embryophyte evolution remain unclear. The Arabidopsis thaliana PIF3-bHLH (AtPIF3-bHLH) recombinant protein and a series of AtPIF3-bHLH mutants were synthesized and analyzed. The reduced DNA binding ability and affinity of AtPIF3-bHLH point-mutation proteins, observed via fluorescence-based electrophoretic mobility shift assay (fEMSA) and isothermal titration calorimetry (ITC), suggest the critical role of these DNA-recognition sites in maintaining the AtPIF3-bHLH–DNA interaction. The purifying selection signals and the DNA-recognition-site conservation at the species level suggest the invariant roles of these sites throughout embryophyte evolution. The G-box outcompeted other types of E-box for binding in our competitive fEMSAs. The dynamic hydrogen bond formed between AtPIF3-bHLH and the G-box core indicates flexible identification of the core region. These features highlight a fast fixation of the bHLH-G-box recognition mechanism through embryophyte evolution and serve as a blueprint for studying DNA recognition determinants of other TF families.

Preparation and characterization of calcium-doped graphene oxide-chitosan Nanocarrier to enhance the gene delivery in MCF-7 cell line

Gene delivery has emerged as a novel and effective method in the treatment of malignancies within medical interventions by applying nanotechnology. Consequently, the development of appropriate nanocarriers is a key focus of this research. Dynamic light scattering (DLS), fourier transform infrared (FT-IR) spectroscopy, x-ray diffraction (XRD), and thermal gravimetric analysis (TGA) were employed for the characterization of the synthesized nanocarrier. Furthermore, to assess the gene transfer capability of the nanocarrier, various techniques such as gel retardation assay, nuclease resistance assay, cytotoxicity assay, flow cytometry, and transfection were employed. The average particle size and zeta potential of the GO-CS@Ca nanocarrier were obtained as 319.8 nm and + 92.8 mv, respectively. In the gel retardation test, it was observed that pDNA was effectively condensed by the GO-CS@Ca nanocarrier. The results of the MTT assay indicated that both GO-CS@Ca nanocarrier and the GO-CS@Ca/pDNA nanoplex with low toxicity. In flow cytometry analysis, it was observed that the complexation of pDNA with the GO-CS@Ca nanocarrier resulted in effective gene delivery to the MCF-7 cell line and consequently increased apoptosis induction.

PlZAT10 binds to the ABA catabolism gene PlCYP707A2 promoter to mediate seed dormancy release in Paeonia lactiflora.

PlZAT10-PlCYP707A2 module promotes Paeonia lactiflora seeds germination. The herbaceous peony (Paeonia lactiflora) seeds exhibit double dormancy in the epicotyl and hypocotyl, which significantly inhibits the process of cultivation and breeding of new varieties. Nevertheless, the molecular mechanism underlying seed dormancy release in P. lactiflora remains to be fully identified.In this current study, we analyzed differentially expressed genes based on transcriptome data and selected the abscisic acid catabolic gene PlCYP707A2 for further investigation. The conserved domain of the protein indicated that PlCYP707A2 possessed a cytochrome P450 monooxygenase domain. Subcellular localization indicated that PlCYP707A2 was localized on the cytoplasm and cell membrane. Overexpression of PlCYP707A2 in P. lactiflora seeds decreased ABA contents and promoted seeds germination. The silencing of PlCYP707A2 resulted in seed dormancy and an alteration in the content of ABA. Moreover, yeast one-hybrid, electrophoretic mobility shift and dual-luciferase reporter assay revealed that PlZAT10 bound to the promoter of PlCYP707A2. In conclusion, the results demonstrated the mechanism of the PlZAT10-PlCYP707A2 module in regulating the dormancy release of P. lactiflora seeds, enriching relevant theories on seed dormancy and having significant implications for the herbaceous peony industry developing.

Regulatory Effect of Transcriptional Regulator TetR on the Synthesis of Conjugated Linoleic Acid in Lactiplantibacillus plantarum AR195.

Conjugated linoleic acid (CLA) possesses anticancer, anti-inflammatory, and antiobesity properties, making it a significant research focus. In this study, we identified TetR, a TetR/AcrR family transcriptional regulator encoded by AR1031, as the transcriptional regulator of CLA synthesis in Lactiplantibacillus plantarum AR195. TetR binds to the promoter regions of the CLA synthesis genes, including the cla operon and cla-hy, thereby enhancing CLA biosynthesis. Knockout of tetR led to the downregulation of cla-hy by 68%, cla-dh by 86%, and cla-dc by 33%, resulting in reduced CLA yield. Further bioinformatic analysis and segmented electrophoretic mobility shift assay (EMSA) experiments revealed that TetR recognizes a conserved sequence (5'-TGTAGATTG-n4-CTTCA-3') and binds to multiple sites within the regulatory region of the cla operon and cla-hy, thus promoting CLA biosynthesis. Overexpression of tetR increased the CLA yield by 7%. These findings provide precise insights into the regulation of CLA biosynthesis and suggest strategies for enhancing CLA production.

Transcriptomics analysis reveals the regulatory role of PsERF3 in anthocyanin biosynthesis of ‘Taoxingli’ plum fruit in response to MeSA treatment

Mature plum fruit (Prunus salicina cv. Taoxingli) exhibits an attractive red appearance and significant nutritional value due to anthocyanin accumulation. Some ‘Taoxingli’ plums are harvested before reaching full ripeness to prevent fruit cracking caused by hot and rainy weather. At this stage, plum fruit has not reached its optimal quality. In this study, methyl salicylate (MeSA) treatment promoted anthocyanin accumulation in earlier-harvested ‘Taoxingli’ plum fruit during postharvest. Transcriptomic techniques were employed to unravel the regulatory mechanisms. A weighted gene co-expression network analysis (WGCNA) was constructed based on RNA-seq data, and 22 co-expression modules were constructed. In the specific module, seven synthetic genes were identified. Thus, a transcription factor (TF)-target gene regulatory network related to anthocyanin biosynthesis was built. Yeast one-hybrid assays and electrophoretic mobility shift assay (EMSA) indicated that PsERF3 could directly bind to the promoter of PsANS via RAA motif. Transient over-expression of PsERF3 accelerated anthocyanin biosynthesis in apple through inducing structural genes expression. This work offers fresh perspectives on the regulatory mechanisms of anthocyanin accumulation in ‘Taoxingli’ plum fruit.

Genome-Wide Analysis of HECT E3 Ligases Members in Phyllostachys edulis Provides Insights into the Role of PeHECT1 in Plant Abiotic Stress Response

Homology to E6-AP Carboxy Terminus (HECT) E3 ubiquitin ligases play pivotal roles in plant growth, development, and responses to abiotic stresses. However, the function of HECT genes in Phyllostachys edulis (P. edulis) remains largely uninvestigated. In this study, a comprehensive genome-wide analysis of the HECT E3 ubiquitin ligases gene family in P. edulis was conducted, aiming to elucidate its evolutionary relationships and gene expansion. Analysis of gene structure, conserved motifs and domains, and synteny genome regions were performed. Furthermore, cis-elements in HECT gene promoters that respond to plant hormones and environmental stresses were identified and corroborated by expression data from diverse abiotic stress conditions and hormone treatments. Based on the co-expression network of PeHECTs under cold and dehydration stresses, PeHECT1 was identified as a key candidate gene associated with abiotic stress tolerance. Overexpression of PeHECT1 in tobacco leaves significantly upregulated genes related to reactive oxygen species (ROS) detoxification and polyamine biosynthesis. Yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), and dual-luciferase (dual-LUC) assays suggested that the transcription factor ETHYLENE RESPONSE FACTOR 3 (PeERF3) bound to the dehydration-responsive element (DRE) of the promoter of PeHECT1 and activated its transcription activity. Phylogenetic analysis indicated that PeHECT1 in P. edulis exhibited a close association with the diploid herbaceous bamboo Olyra latifolia, followed by the divergence of rice and bamboo. In summary, this study enhances our comprehensive understanding of the HECT E3 ubiquitin ligases gene family in P. edulis and highlights the potential role of PeHECT1 in plant abiotic stress response.

Unexpected proteins involved in the regulation of secondary metabolism in Myxococcus xanthus DK1622.

Regulating secondary metabolite (SM) in Myxococcus xanthus bears the potential to influence the formation of important natural products with various biological activities. The authors of this study have previously found that the detectable levels of two proteins (4-hydroxyphenylpyruvate dioxygenase [HppD] and a Hsp90-like protein [HtpG]) are affected by ROK inactivation. As evidence, the current study was designed to elucidate the possible role of these two proteins in regulating the SMs' biosynthesis in this bacterium. To begin with, inactivation of the corresponding genes was carried out, and two mutant strains (M. xanthus hppD- and htpG-) were constructed. Subsequently, high-performance liquid chromatography coupled with mass spectrometry analysis for the metabolic extracts of the mutants revealed a significant reduction in the production of several SMs, like DKxanthene, myxalamide A, and myxochromide A, in comparison to the wild type. Furthermore, electrophoretic mobility shift assays using purified ROK protein suggested a direct binding on the genes' promoter region encoding the two proteins under study. It is therefore possible to conclude that hppD and htpG genes are implicated in the bacterium SMs' biosynthetic regulatory cascade, which seems to be directly regulated by the ROK protein. The present study provides additional evidence to a previous investigation showing the pleiotropic regulatory role of ROK on the production of SMs in M. xanthus.

Wheat TaPYL9-involved signalling pathway impacts plant drought response through regulating distinct osmotic stress-associated physiological indices.

The abscisic acid (ABA) signalling pathway plays a crucial role in plants' response to drought stress. In this study, we aimed to characterize the impact of an ABA signalling module, which consisted of TaPYL9 and its downstream partners in Triticum aestivum, on plant drought adaptation. Our results showed that TaPYL9 protein contains conserved motifs and targets plasma membrane and nucleus after being sorted by the endoplasmic reticulum. In addition, TaPYL9 transcripts in both roots and leaves were significantly upregulated in response to drought stress. We conducted glucuronidase (GUS) histochemical staining analysis for transgenic plants carrying a truncated TaPYL9 promoter, which suggested that cis-elements associate with ABA and drought response, such as ABRE, DRE and recognition sites MYB and MYC, regulating the gene transcription under drought conditions. Using protein interaction assays (i.e., yeast two-hybrid, bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP) and in vitro pull-down), we demonstrated interactions between the intermediate segment of TaPYL9, the intermediate segment of TaPP2C6, the N-terminus of TaSnRK2.8 and the C-terminus of the transcription factor TabZIP1 in wheat, indicating the involvement of TaPYL9 in the constitution of an ABA signalling module, namely TaPYL9/TaPP2C6/TaSnRK2.8/TabZIP1. Transgene analysis revealed that TaPYL9, TaSnRK2.8 and TabZIP1 positively regulated drought response, while TaPP2C6 negatively regulated it, and that these genes were closely associated with the regulation of stomata movement, osmolyte accumulation and ROS homeostasis. Electrophoretic mobility shift (EMSA) and transcriptioal activation assays indicated that TabZIP1 interacted promoters of TaP5CS2, TaSLAC1-1 and TaCAT2 and activated transcription of these genes, which regulated proline biosynthesis, stomata movement and ROS scavenging upon drought signalling, respectively. Furthermore, we found that the transcripts of TaPYL9 and stress-responsive genes were positively correlated with yields in wheat cultivars under field drought conditions. Altogether, our findings suggest that the TaPYL9-involved signalling pathway significantly regulates drought response by modulating osmotic stress-associated physiological processes in T. aestivum.

Genome-wide associated study identifies FtPMEI13 gene conferring drought resistance in Tartary buckwheat.

Tartary buckwheat is known for its ability to adapt to intricate growth conditions and to possess robust stress-resistant properties. Nevertheless, it remains vulnerable to drought stress, which can lead to reduced crop yield. To identify potential genes involved in drought resistance, a genome-wide association study on drought tolerance in Tartary buckwheat germplasm was conducted. A gene encoding pectin methylesterase inhibitors protein (FtPMEI13) was identified, which is not only associated with drought tolerance but also showed induction during drought stress and abscisic acid (ABA) treatment. Further analysis revealed that overexpression of FtPMEI13 leads to improved drought tolerance by altering the activities of antioxidant enzymes and the levels of osmotically active metabolites. Additionally, FtPMEI13 interacts with pectin methylesterase (PME) and inhibits PME activity in response to drought stress. Our results suggest that FtPMEI13 may inhibit the activity of FtPME44/FtPME61, thereby affecting pectin methylesterification in the cell wall and modulating stomatal closure in response to drought stress. Yeast one-hybrid, dual-luciferase assays, and electrophoretic mobility shift assays demonstrated that an ABA-responsive transcription factor FtbZIP46, could bind to the FtPMEI13 promoter, enhancing FtPMEI13 expression. Further analysis indicated that Tartary buckwheat accessions with the genotype resulting in higher FtPMEI13 and FtbZIP46 expression exhibited higher drought tolerance compared to the others. This suggests that this genotype has potential for application in Tartary buckwheat breeding. Furthermore, the natural variation of FtPMEI13 was responsible for decreased drought tolerance during Tartary buckwheat domestication. Taken together, these results provide basic support for Tartary buckwheat breeding for drought tolerance.

Resveratrol Inhibits Nucleosome Binding and Catalytic Activity of PARP1

The natural polyphenol resveratrol is a biologically active compound that interacts with DNA and affects the activity of some nuclear enzymes. Its effect on the interaction between nucleosomes and poly(ADP-ribose) polymerase-1 (PARP1) and on the catalytic activity of PARP1 was studied using Western blotting, spectrophotometry, electrophoretic mobility shift assay, and single particle Förster resonance energy transfer microscopy. Resveratrol inhibited PARP1 activity at micro- and sub-micromolar concentrations, but the inhibitory effect decreased at higher concentrations due to the aggregation of the polyphenol. The inhibition of PARP1 by resveratrol was accompanied by its binding to the enzyme catalytic center and a subsequent decrease in PARP1 affinity to nucleosomal DNA. Concurrent binding of talazoparib to the substrate binding pocket of PARP1, which occurs in the presence of resveratrol, restores the interaction of PARP1 with nucleosomes, suggesting that the binding sites of resveratrol and talazoparib overlap. The data suggest that resveratrol can be classified as a natural inhibitor of PARP1.

Eggplant NAC domain transcription factor SmNST1 as an activator promotes secondary cell wall thickening.

NAC-domain transcription factors (TFs) are plant-specific transcriptional regulators playing crucial roles in plant secondary cell wall (SCW) biosynthesis. SCW is important for plant growth and development, maintaining plant morphology, providing rigid support, ensuring material transportation and participating in plant stress responses as a protective barrier. However, the molecular mechanisms underlying SCW in eggplant have not been thoroughly explored. In this study, the NAC domain TFs SmNST1 and SmNST2 were cloned from the eggplant line 'Sanyue qie'. SmNST1 and SmNST2 expression levels were the highest in the roots and stems. Subcellular localization analysis showed that they were localized in the cell membrane and nucleus. Their overexpression in transgenic tobacco showed that SmNST1 promotes SCW thickening. The expression of a set of SCW biosynthetic genes for cellulose, xylan and lignin, which regulate SCW formation, was increased in transgenic tobacco. Bimolecular fluorescence and luciferase complementation assays showed that SmNST1 interacted with SmNST2 in vivo. Yeast one-hybrid, electrophoretic mobility shift assay (EMSA) and Dual-luciferase reporter assays showed that SmMYB26 directly bound to the SmNST1 promoter and acted as an activator. SmNST1 and SmNST2 interact with the SmMYB108 promoter and repress SmMYB108 expression. Altogether, we showed that SmNST1 positively regulates SCW formation, improving our understanding of SCW biosynthesis transcriptional regulation.

The PHD transcription factor ThPHD5 regulates antioxidant enzyme activity to increase salt tolerance in Tamarix hispida.

PHD proteins are an important class of transcription factors (TFs) that are widely distributed in eukaryotes and play crucial roles in many aspects of plant growth, development and response to stress. We identified a transcription factor, ThPHD5, from the PHD family in Tamarix hispida based on its potential involvement in abiotic stress response processes. In this study, the salt tolerance function of ThPHD5 from T. hispida was further characterized. The qRTPCR results showed ThPHD5 was expressed in response to NaCl, PEG and ABA treatments. Transient transformation analysis revealed that ThPHD5 improved salt tolerance in T. hispida by increasing POD and SOD activity and decreasing the MDA, total ROS content and electrolyte leakage. To explore the salt tolerance mechanism of the ThPHD5 TF, its binding DNA motifs and potential downstream regulatory genes were analyzed. The results showed that ThPHD5 affect the expression of 7 antioxidant enzyme-related genes. The Yeast one-hybrid (Y1H) and Electrophoretic Mobility Shift Assay (EMSA) results indicated ThPHD5 could bind to ABRE, MYB and Dof cis-acting elements. ChIP-PCR further confirmed ThPHD5 exercise its regulatory function by directly binding motifs on the ThPOD16, ThSOD and ThSOD1 promoters. Taken together, these findings indicate the ThPHD5 TF improves salt tolerance in T. hispida by regulating the expression of antioxidant enzyme-related genes to increase antioxidant enzyme activity, enhance the ROS scavenge ability, reduce ROS accumulation and cellular damage.