Search Results (254)

Cadmium (Cd) toxicity poses a significant threat to crop production and food safety. Although proline is known to enhance plant tolerance to Cd, the molecular mechanisms regulating Cd detoxification through proline accumulation remain unclear. This study identifies the proline transporter TaPROT2 as a crucial positive regulator of Cd tolerance in wheat. We demonstrate that overexpression of TaPROT2 directly promotes proline accumulation in transgenic wheat while simultaneously activating the antioxidant enzyme system, thereby reducing both Cd accumulation and translocation. Using electrophoretic mobility shift assays (EMSA), yeast one-hybrid (Y1H) assays, and luciferase reporter assays, we confirmed that TaERF3 directly binds to the GCC-box element in the TaPROT2 promoter, thereby activating its transcription. Furthermore, overexpression of TaERF3 enhances the expression of TaPROT2, leading to increased proline accumulation and decreased Cd content. In summary, our study reveals a novel TaERF3-TaPROT2 module that promotes proline accumulation, reduces Cd accumulation, and enhances Cd tolerance, providing a promising target for breeding low-Cd wheat. Full article

Toxigenic Klebsiella oxytoca strains linked to antibiotic-associated hemorrhagic colitis produce the cytotoxins tilimycin and tilivalline, which contribute to intestinal epithelial damage during infection. Tilimycin and tilivalline are synthesized by enzymes encoded within the nonribosomal peptide synthetase (NRPS) operon, yet the regulatory mechanisms controlling operon expression remain poorly understood. SdiA, an orphan LuxR-type quorum-sensing regulator, detects exogenous N-acyl homoserine lactones (AHLs) produced by neighboring bacterial species and modulates gene expression in response to interspecies communication. Although SdiA has been implicated in virulence regulation in several enteric pathogens, its role in K. oxytoca remains unclear. This study demonstrates that SdiA positively regulates npsA, the first gene in the NRPS operon, and that this regulatory effect is enhanced in the presence of exogenous AHL. Electrophoretic mobility shift assays indicate that SdiA directly binds to the upstream regulatory region of npsA, supporting a direct interaction consistent with positive transcriptional regulation. Furthermore, deletion of sdiA significantly reduces cytotoxicity toward HeLa cells under the conditions tested. Collectively, these findings identify SdiA as a quorum-sensing-responsive activator of npsA expression and support its role in modulating cytotoxicity in toxigenic K. oxytoca strains. These results provide new insight into the influence of interspecies quorum-sensing signals on virulence-associated regulatory pathways in K. oxytoca. Full article

N⁶-methyladenosine (m⁶A) is an important epigenetic modification of eukaryotic RNA, playing a significant role in various biological processes. Metasequoia glyptostroboides (M. glyptostroboides) is an ancient tree species in China, with a long history and excellent genetic characteristics. In this study, we identified six MgYTH genes in the genome of M. glyptostroboides, elucidating their phylogenetic relationships, conserved domains, gene structures, conserved motifs, chromosome locations, and prediction of LLPS. The analysis of the cis-regulatory elements in the promoter region suggested that MgYTH genes are associated with drought and the ABA-responsive expression patterns signaling pathway, which was further supported by expression pattern analysis. In addition, to directly evaluate the m⁶A binding ability of MgYTH proteins, we selected MgYTH5 as the representative for homology modeling analysis and electrophoretic mobility shift assay (EMSA). The results demonstrated that MgYTH5 has the ability to bind m⁶A in vitro, thereby providing biochemical evidence that MgYTH5 can bind m⁶A-modified RNA in vitro mRNAs. The subcellular localization results showed that MgYTH5 is located in the cytoplasm. These findings provide new insights into the epigenetic regulation mechanisms in gymnosperms and provide a resource for future functional studies in this species. Full article

In this study, we investigate two RpoN homologs in Bradyrhizobium sp. DOA9—chromosomal RpoNc and megaplasmid-borne RpoNp—and their roles in free-living conditions and nitrogen fixation. Phylogenetic analysis showed that RpoNc clusters with RpoN proteins from symbiotic nitrogen-fixing strains, whereas RpoNp forms a distinct clade, consistent with a function in stress responses. RpoNc proved essential for free-living conditions: ΔrpoNc mutants displayed severe growth defects that RpoNp could not compensate for. Transcriptomic comparisons between wild type and mutant RpoN identified 541 differentially expressed genes (DEGs) grouped into three clusters: 100 downregulated, 175 upregulated, and 254 moderately downregulated (with a fold change > 2, and a q-value (FDR, padj) < 0.05). Affected pathways involved nitrogen metabolism, motility, and environmental adaptation. RpoNc controlled major nitrogen fixation genes (nif and fix) along with core growth and stress response functions, while RpoNp mainly influenced stress-adaptation pathways. Genome-wide promoter motif analysis predicted 68 putative RpoNc targets, mainly associated with nitrogen fixation and metabolism, compared with only 22 predicted RpoNp targets, indicating a more restricted regulon. Electrophoretic mobility shift assays (EMSAs) further confirmed that both RpoN proteins directly bind σ⁵⁴-dependent promoters identified from transcriptomic data, supporting their regulatory roles under free-living conditions. Two mutants (ΔrpoNc and ΔrpoNp::ΩrpoNc) showed broad transcriptional disruption across nitrogen fixation, metabolism, and stress responses, underscoring complementary regulation. Overall, RpoNc is the dominant regulator of nitrogen fixation and core metabolism during free-living conditions, whereas RpoNp fine-tunes stress responses, revealing new regulatory insights for DOA9 adaptation. These results clarify how RpoN systems optimize survival across fluctuating conditions. Full article

Streptococcus parauberis is a major pathogen responsible for streptococcosis in both marine and freshwater fish species, causing substantial economic losses in aquaculture. The increasing prevalence of multidrug resistance has highlighted the urgent need for alternative disease control strategies. Interference with bacterial quorum sensing (QS) systems represents a promising approach. This study aimed to identify and biochemically characterize an Rgg-family transcriptional regulator and evaluate its potential as a target for quorum sensing-related regulatory interference in vitro. We hypothesized that this Rgg regulator may function as a quorum sensing-associated transcription factor capable of promoter binding and modulation by small molecules. Bioinformatic analyses were used to identify the rgg gene encoding an Rgg-family transcriptional regulator and predict its structural features. The gene was cloned, heterologously expressed, and purified. Promoter binding activity was examined using electrophoretic mobility shift assay (EMSA), and key amino acid residues were identified through site-directed mutagenesis. The inhibitory effect of the cyclic peptide cyclosporin A (CsA) on Rgg-promoter binding was further assessed. The rgg gene (864 bp) encoding a 287-amino-acid protein (34.1 kDa) was successfully identified and expressed. Purified Rgg specifically bound to its own promoter region in a concentration-dependent manner. Mutations at conserved arginine residues R12 and R15 within the helix-turn-helix DNA-binding domain abolished promoter binding activity. Furthermore, CsA disturbed Rgg-promoter binding in a dose-dependent manner. This study provides the first in vitro characterization of an Rgg-family transcriptional regulator in fish-derived S. parauberis. The findings expand current understanding of Rgg-family regulators potentially associated with quorum sensing in aquatic streptococci and provide a preliminary basis for further investigation of quorum sensing-related regulatory interference strategies for controlling streptococcal diseases in aquaculture. Full article

Acidithiobacillus caldus is perpetually exposed to multiple extreme environmental stresses. CsrA, functioning as a post-transcriptional regulator of physiological metabolism, acts as a differential modulator, facilitating more economical and efficient adaptation to extreme environments. The csrA expression recombinant strain was constructed in A. caldus MTH-04 by conjugative transfer technology pJD215. Physiological characterization revealed enhanced acid tolerance, significantly elongated flagella, elevated extracellular secretion, and altered biofilm composition. Notably, intracellular concentrations of free glutamate and aspartate increased to 24.18 mg/L and 16.07 mg/L, respectively. The secondary structure of CsrA protein was determined in vitro through circular dichroism spectroscopy and size-exclusion chromatography. Electrophoretic Mobility Shift Assay (EMSA) successfully demonstrated in vitro binding activity of CsrA to the rpoS leader mRNA. CsrA suppresses rpoS mRNA translation by competing with ribosomes for binding sites, thereby negatively regulating rpoS expression. Critical binding sites were further validated through site-directed mutagenesis. Through EMSA, RT-qPCR and the translation reporter system, it was also found that CsrA has a dual regulatory function for nearby flagella- and motility-related gene clusters (flgC, 07035, motD, 15040), which also implies the global regulatory role of CsrA. In summary, a potential overall post-transcriptional regulatory mechanism based on CsrA and rpoS by extremophile A. caldus was proposed. Finally, the efficiency of bioleaching application by csrA overexpression strain was improved by 20.81%. Full article

Pseudomonas aeruginosa is a major causative agent of nosocomial infections worldwide. Carbapenems are the first-line agents for combating severe P. aeruginosa infections. However, the increasing prevalence of carbapenem-resistant P. aeruginosa (CRPA) has developed as a critical threat to global healthcare systems. In this study, we demonstrated that a mutation in the core nitrogen metabolism regulatory gene glnK decreases carbapenem resistance in P. aeruginosa. OprD, the major porin for carbapenem uptake, is upregulated in the glnK mutant, resulting in decreased resistance. We further found that the NtrB/NtrC two-component regulatory system is upregulated in the glnK mutant. An electrophoretic mobility shift assay (EMSA) and genetic studies revealed a direct regulatory role of NtrC on the expression of oprD. Deletion of ntrB, ntrC, or oprD in the glnK mutant restored the bacterial resistance to carbapenems. These results reveal that a GlnK-NtrB/NtrC-OprD regulatory pathway affects carbapenem resistance, shedding light on the regulatory relationship between nitrogen metabolism and carbapenem resistance in P. aeruginosa. Full article

The circadian clock genes in tomato are key regulators of cold stress adaptation. However, the low-temperature regulatory mechanism of the circadian clock gene SlPCL1 remains unclear. In this study, we evaluated the role of SlPCL1 in cold tolerance through low-temperature treatment of transgenic plants. Downstream target genes were identified using RNA-seq, RT-qPCR, yeast-one-hybrid (Y1H), dual-luciferase assays, and electrophoretic mobility shift assay (EMSA), while interacting proteins were characterized using yeast-two-hybrid (Y2H), luciferase complementation imaging (LCI), and pull-down assays, thereby elucidating the molecular mechanism underlying SlPCL1-mediated low-temperature regulation. We identified SlPCL1 as a nuclear-localized circadian clock gene with transcriptional repressor activity. Overexpression of SlPCL1 resulted in a cold-sensitive phenotype, whereas virus-induced gene silencing (VIGS)-mediated silencing of SlPCL1 enhanced cold tolerance. SlNPF4.6 functions as an abscisic acid (ABA) transporter involved in ABA transport. RNA-seq and RT-qPCR identified the ABA transporter SlNPF4.6 as a downstream target. Functional assays confirmed that SlPCL1 binds to the MYB element in the SlNPF4.6 promoter to repress its expression. Meanwhile, VIGS-mediated silencing of SlNPF4.6 decreased cold tolerance. Furthermore, the expression levels of the ABA receptor SlPYLs in the silenced lines were significantly reduced, confirming the decrease in intracellular ABA content. SlSUMO1, a ubiquitin-like protein, can influence gene transcription through noncovalent interactions. In addition, SlSUMO1 was found to interact with the SlPCL1 protein, attenuating SlPCL1 transcriptional repression activity. Together, these findings establish an SlSUMO1-mediated fine control mechanism of the SlPCL1-SlNPF4.6 regulatory module. This integration of circadian clock regulation uncovers new molecular mechanisms of cold tolerance and supports the development of cold-resistant breeding materials. Full article

The tardigrade Ramazzottius varieornatus exhibits extraordinary resilience to extreme environmental stresses, yet the functional diversity of its proteome remains largely unexplored. In this study, the structural and biochemical characterization of RvCA5, an atypical α-carbonic anhydrase (CA) identified in R. varieornatus, is presented. Expression analysis in E. coli revealed the spontaneous formation of a truncated RvCA5 species, which was confirmed to be unrelated to signal peptide cleavage. RvCA5 exhibited distinct structural features, including extended intrinsically disordered regions (IDRs) at both termini. Unlike canonical α-CAs, RvCA5 exhibited negligible CO₂ hydration activity, which was partially enhanced by the removal of the N-terminal IDR, suggesting that this region acts as a dynamic entropic barrier hindering substrate diffusion. RvCA5 possesses multiple surface-exposed reactive cysteine residues, resembling the redox-sensing human CA 3. Notably, consistent with a predicted nuclear localization signal, in silico modeling predicted that RvCA5 can bind DNA via a positively charged patch near the C-terminal IDR. The DNA-binding capability of RvCA5 was experimentally demonstrated by electrophoretic mobility shift assays. Collectively, these findings suggest that RvCA5 potentially functions as a redox-responsive transcriptional regulator. Full article

Post-transcriptional regulation of gene expression is influenced by RNA-binding proteins (RBPs) and small non-coding RNAs that bind to conserved mRNA sequences to modulate mRNA processing. These regulatory molecules affect the structural conformation of mRNAs, creating formations like G-quadruplexes (G4s), which alter translation initiation and regulatory-factor site accessibility. Recent studies have highlighted Nuclear factor erythroid 2–related factor 2 (NRF2) as a key regulator of cellular redox homeostasis and cellular response to oxidative stress. An intriguing feature of NRF2 is the structural formation of its 5′ untranslated region (UTR), which may promote or inhibit translation initiation depending on the cellular context. In this study with minigenes, we provide in vitro evidence of RNA G4s in the NRF2 mRNA’s 5′ UTR under basal (no stress) conditions. Achieved via electrophoretic mobility shift assay and fluorescence spectra in the presence of Pyridostatin. Understanding how structural motifs within NRF2 5′ UTRs influence mRNA function provides insights into a common molecular mechanism underlying diseases where NRF2 is dysregulated, like cancers, cardiovascular disease, and neurodegeneration, and highlights potential therapeutic avenues through regulation of NRF2. Full article

Lead (Pb) severely impairs plant growth, yet the role of WRKY transcription factors in Pb tolerance in maize remains largely unknown. Here, we identified a Pb-responsive WRKY transcription factor, ZmWRKY4, whose transcript levels were rapidly and strongly induced in maize leaves following Pb exposure. Physiological and biochemical analyses showed that overexpression of ZmWRKY4 substantially enhanced Pb tolerance in maize. Transgenic lines exhibited significantly lower malondialdehyde (MDA) levels and reduced electrolyte leakage than wild-type plants. In addition, ZmWRKY4 overexpression increased catalase (CAT) activity and effectively limited H₂O₂ accumulation. Further analyses revealed that ZmWRKY4 positively regulates ZmCAT1, a key antioxidant gene involved in H₂O₂ scavenging, under Pb stress. Electrophoretic mobility shift assays and ChIP-qPCR collectively confirmed that ZmWRKY4 directly binds to W-box elements within the ZmCAT1 promoter in vivo and in vitro, thereby activating its transcription. Together, these findings define a previously uncharacterized ZmWRKY4-ZmCAT1 regulatory module that enhances antioxidant capacity and mitigates oxidative damage during Pb stress. This work provides new insights into the molecular mechanisms underlying heavy metal tolerance in maize and identifies a promising genetic target for developing Pb-resilient crop varieties. Full article

As cold-sensitive fruits, loquats easily develop chilling injury (CI) during cold storage, which leads to quality deterioration and economic losses. Our prior research indicated that exogenous melatonin (MT) treatment can mitigate CI in postharvest loquats by regulating reactive oxygen species (ROS) metabolism, but the underlying molecular mechanism remains unclear. The primary objective of this study is to decipher the molecular regulatory pathway by which MT alleviates CI in postharvest loquats, focusing on the role of MYB transcription factors (TFs) in modulating antioxidant enzyme genes. Here, MT treatment remarkably reduced CI severity in loquat fruits, as reflected by lower CI index, reduced cell membrane permeability, decreased firmness, lower a* and b* values, and higher L* value, compared with the control group. Moreover, a cold-induced MYB TF, designated EjMYB15, was identified. Compared to non-treated fruits, the expression level of EjMYB15 was maintained at higher levels in MT-treated loquats. Subcellular localization and transactivation assays demonstrated that EjMYB15 is a nuclear-localized transcriptional activator. Electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter (DLR) assays showed that EjMYB15 binds the MYB-binding sites (MBS) in the promoters of four antioxidant enzyme genes (EjCAT1, EjCAT2, EjGST1, and EjGST2), thereby activating their transcription. Taken together, these findings indicate that EjMYB15 positively regulates cold tolerance of loquat fruits by improving ROS scavenging capacity. These results elucidate the regulatory pathway by which MYB TFs mitigate CI and provide new theoretical support for the application of MT in alleviating CI in postharvest fruits. Full article

Chronic hepatitis B virus (HBV) infection remains a global public health challenge, and the currently approved medications can not achieve a cure. Synthetic triterpenoids have shown promising therapeutic potential for liver pathologies. In our search for novel antiviral agents against HBV, we found that two triterpenoids, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and CDDO-ethyl amide (CDDO-EA), significantly inhibited HBV DNA replication. Further mechanistic investigation indicated that these two compounds did not significantly alter the levels of total HBV pgRNA, but dramatically reduced extracellular pgRNA and intracellular encapsidated pgRNA in a dose-dependent manner. Western blot analysis indicated minimal effects on core protein expression. Interestingly, using a particle gel assay, we observed that CDDO and CDDO-EA promoted the formation of empty capsids with no alteration in electrophoretic mobility. Moreover, we demonstrated that both compounds modulated the phosphorylation status of the core protein. Further cellular thermal shift assay (CETSA), surface plasmon resonance (SPR) assay, and molecular docking analyses collectively suggested that CDDO and CDDO-EA could bind directly to the dimer–dimer interfaces of HBV core protein. Finally, a synergistic effect was observed between CDDO-EA and lamivudine in reducing intracellular and extracellular HBV DNA levels. Our findings indicate that triterpenoids CDDO and CDDO-EA are new mechanistically type of HBV capsid assembly modulators and warranted for further development as lead compounds against HBV. Full article

Cell membrane glycosylations play a critical role in regulating cell signaling, adhesion, and immune recognition. Abnormal glycosylation is a hallmark of cancer and promotes tumor progression. Sulfatide (SM4), a glycosphingolipid synthesized by galactosylceramide sulfotransferase (CST), is frequently altered in cancers, including breast cancer. Our previous studies identified SM4 as an adhesive molecule that enhances metastasis through interactions with platelets and endothelium; however, its elevated levels increase apoptotic sensitivity and reduce tumorigenicity. Here, we elucidate the molecular mechanisms linking sulfatide metabolism to apoptosis and gene regulation. Using MDA-MB-231 and MDA-MB-468 breast cancer cells with altered CST/SM4 levels, RNA sequencing and functional analyses revealed that overproduction of the CST/SM4 significantly downregulated BOLA2, a gene in the CIAPIN1 pathway involved in apoptosis. RT-qPCR and Western blot confirmed an inverse relationship between CST/SM4 and BOLA2. Overexpressing BOLA2 provided resistance to doxorubicin-induced apoptosis, suggesting that SM4-mediated repression of BOLA2 increases apoptotic sensitivity. Luciferase assays showed reduced BOLA2 promoter activity in SM4-enriched cells. Transcription factor profiling and Electrophoretic Mobility Shift Assay demonstrated that SM4 suppresses STAT5 activation, which directly binds and regulates the BOLA2 promoter. SM4 also altered integrin profiles by upregulating β4/β5 and downregulating β1 subunits. Reintroducing β1 integrin restored STAT5 activation and BOLA2 expression, positioning β1 integrin upstream of STAT5. Collectively, these findings identify a novel sulfatide-dependent β1 integrin–STAT5–BOLA2 pathway controlling apoptosis in breast cancer cells. SM4 suppresses β1 integrin and STAT5-mediated BOLA2 transcription, promoting apoptosis, while β4/β5 upregulation may facilitate invasion. This pathway represents a potential therapeutic target in breast cancer. Full article

As reported in earlier work, when a pea apyrase, psNTP9 (PS), and a modified version of it, psNTP9-DM (DM), are expressed in Saccharomyces cerevisiae, they localize to nuclei, binding to largely non-overlapping promoter regions of chromatin. PS- and DM-expressing yeast also exhibit different expression profiles for potentially regulated target genes, consistent with observed phenotypes. In the present study, we use ChIP-seq assays to show that PS and DM also associate with largely different promoter regions of Arabidopsis genes, with similar non-overlapping expression profiles for potential target genes. Functional studies, using electrophoretic mobility shift assays (EMSA), verified PS-specific binding to yeast or plant promoter binding sites. DM binding to both heterologous dsDNA and to PS-specific binding site sequences was minimal. AlphaFold3 modeling of PS protein binding to a yeast PHM6 promoter sequence identified potential DNA-binding residues and a potential binding site motif (5′-(G/T)GG(G/T)A-3′) that is also present in two Arabidopsis promoter binding sites. These novel findings extend the previously known functions of PS and other plant apyrases in the Golgi or extracellular matrix, and support their potential function as DNA-binding proteins that can regulate gene expression in both yeast and Arabidopsis. Full article