Metallomics

Cadmium, being a highly toxic metal, perturbs cellular homeostasis by forming stable complexes with numerous thiol-active proteins, ultimately leading to severe liver and lung damage. Despite its well-documented toxicity, the molecular mechanisms governing cadmium export remain poorly understood. Given the chemical similarity between cadmium and copper, we investigated whether the canonical copper-exporting ATPases, ATP7A and ATP7B participate in cadmium handling. Upon Cd treatment in hepatocytes, ATP7B undergoes trafficking to lysosomes via the retromer complex, as also observed in the case of elevated copper, accompanied by the upregulation of acidic lysosomal populations. In contrast, ATP7A expressed in lung adenocarcinoma cells, though exhibit vesicular redistribution upon Cd exposure, does not mediate lysosomal sequestration, suggesting distinct deployment of late secretory pathways by the two copper ATPases in response to cadmium. We have also observed that ATP7B-/- hepatocytes exhibit increased sensitivity to Cd exposure compared to wild-type cells. Whereas, overexpressing the ATP7B amino-terminal copper-binding domain in bacteria alleviates cadmium-induced stress, indicating its capacity to sequester Cd. Caenorhabditis elegans lacking copper-ATPase cua-1, displayed increased Cd sensitivity, while mutants (glo-1-/-), deficient in lysosome-related organelles (LRO), and (lmp-1-/-), deficient in lysosomal membrane glycoprotein, showed reduced resistance to cadmium toxicity. Treatment of the worm with cadmium increases the abundance of lysosomes marked by elevation in lysosomal biogenesis and functional genes, reinforcing the importance of lysosomal pathways in cadmium detoxification. To summarise, we delineated the non-canonical role of copper ATPases and lysosomes in cadmium-induced cellular toxicity.

Ferredoxins are central to cellular metabolism by mediating electron flow in energy conversion reactions. The focus of this study was to systematically examine twelve ferredoxin and ferredoxin-like proteins from Synechocystis sp. PCC 6803 to identify their properties, activities, and functions in electron transfer. Using electron paramagnetic resonance spectroscopy, we detected cluster types consistent with major ferredoxin families including plant-type [2Fe-2S], adrenodoxin, thioredoxin, and bacterial-type [4Fe- 4S] ferredoxins. In addition, we found that the ssr3184 ferredoxin-like protein exchanged between a [3Fe-4S] or a [4Fe-4S] cluster, pointing to a possible functional change in response to changes in oxygen or cellular redox poise. Electrochemical measurements demonstrated that these ferredoxins constitute a broad potential window, from -243 mV to -520 mV vs SHE. Investigations on their capacity to support electron-transfer focused on reactions with two major redox hubs: Photosystem I and pyruvate:ferredoxin oxidoreductase and included testing of binding interactions with nitrite reductase. Expression profiling under multiple environmental conditions was also used to predict function and revealed distinct regulatory patterns. Collectively, these findings identified a group of core ferredoxins that directly support photosynthetic electron transfer, and more specialized ones that may serve other functions. In summary, Synechocystis utilizes a suite of ferredoxins to maintain cellular redox homeostasis under dynamic environmental conditions.

Zinc is an essential transition metal that participates in many biological processes. In C. elegans, excess zinc is stored in lysosomes in intestinal cells; this process involves increasing the expression of the zinc transporter CDF-2 and remodeling of lysosomes characterized by an increase in the volume of the expansion compartment. To determine if this is a more general property, we investigated other metals. Here we report that lysosomes are remodeled in response to excess copper, manganese, and cadmium, with each metal causing an increase in the volume of the expansion compartment. Mutants with a reduced number of lysosomes were hypersensitive to growth retardation caused by excess copper and manganese, suggesting metal toxicity is prevented by metal sequestration in lysosomes. Using a novel method to analyze isolated lysosomes by X-ray Fluorescence Microscopy we demonstrated that zinc, copper and manganese are detectable in the lumen of lysosomes. To further analyze copper, we examined localization of CUA-1.1, a copper transporter that moves copper into the lumen of lysosomes. Like the zinc transporter CDF-2, CUA-1.1 localizes to both the acidified and expansion compartments in excess copper. These results indicate that the same intestinal lysosomes store zinc, copper and manganese. Lysosome remodeling characterized by an increase in volume of the expansion compartment is not specific to zinc but is a more general phenomenon during metal storage in lysosomes.

The facultative methylotroph model organism Methylorubrum extorquens AM1 is a known lanthanide user, which has shed light on the role of rare-earth metals in biochemistry. The characterization of a methanol dehydrogenase (MDH) protein which requires lanthanides as an enzymatic cofactor outlined the question of how these metals are acquired from the environment. It has been proposed that mesophilic organisms as M. extorquens AM1 can produce siderophore-like molecules, which chelate, transport and traffic rare-earth elements into the microbial cell. Therefore, we performed the bioinformatic and chemical investigation of M. extorquens AM1 by using genome mining, the CAS and arsenazo assay, molecular networking and chemical analytical techniques. Our results showed that indeed Methylorubrum extorquens AM1 harbored a gene cluster to produce metal chelators. The chemical analysis confirmed the production of the known hybrid hydroxamate-citrate siderophores schizokinen A and N-deoxyschizokinen A, which are very likely the side products of the transformation of schizokinen and N-deoxyschizokinen. The determination of the lanthanide chelation activity of the schizokinen siderophores series against three different lanthanides (La, Eu and Lu) showed no coordination activity, thus ruling out the involvement of schizokinen siderophores in rare-earth metal transport.

Pseudomonas aeruginosa relies on the redox cofactor pyrroloquinoline quinone (PQQ) for efficient glucose and ethanol metabolism via periplasmic dehydrogenases (Gcd and ExaA). While PQQ biosynthesis is well-characterized, its uptake mechanisms remain unclear. Here, we identify PA2289 (PqqU), a TonB-dependent transporter, as the primary PQQ importer in P. aeruginosa. Growth assays with PQQ-deficient mutants ({Delta}pqqABCDEH) demonstrated that PqqU is essential for exogenous PQQ uptake, rescuing growth on glucose and ethanol. Genomic analysis across 210 P. aeruginosa and 263 Pseudomonas strains revealed high conservation of PQQ biosynthesis and utilization genes, while PqqU showed lower prevalence (47.7%) in the genus. Transcriptional analyses using fluorescent reporters and qRT-PCR demonstrated that PqqU expression remains unchanged in response to PQQ, varying carbon sources, or iron availability, suggesting constitutive regulation. Comparative proteomics between wild-type and {Delta}pqqABCDEH strains, cultured on glucose or ethanol, uncovered extensive proteomic shifts, underscoring P. aeruginosas metabolic adaptability. Additionally, PQQ-dependent metabolic pathways appear to indirectly influence iron homeostasis, most likely through environmental acidification. Together, these results emphasize the critical role of PqqU in PQQ uptake and its broader significance in shaping the metabolic and environmental versatility of Pseudomonas.

The production of reactive oxygen species (ROS) in response to cadmium (Cd) has been extensively studied, demonstrating that they play a key role in the plants response to this heavy metal. While the role of enzymes like RBOHs has been thoroughly studied, the function of other ROS-producing enzymes, such as peroxisomal glycolate oxidase (GOX), remains largely overlooked. Peroxisomal GOX is a core metabolic enzyme of the photorespiratory pathway occurring in chloroplasts, mitochondria and peroxisomes. Using Arabidopsis (Arabidopsis thaliana) mutants lacking the main peroxisomal GOX genes, GOX1 (gox1-1) and GOX2 (gox2-1) we explored their function in plant response to Cd. Although photosynthetic capacity appears to be affected to the same extent in both mutants under control and Cd stress conditions, GOX2 seems to play a greater role in ROS production in response to the metal. Transcriptomic analyses on WT and gox2-1 pointed to the mitochondrial electron transport chain (mETC) as a target of Cd stress. We further investigated the individual GOX1 and GOX2 functions in mETC regulation and redox state. Although oxidative ratio of mitochondria was higher in both mutants, it was more pronounced in the absence of GOX1. Furthermore, the mETC is affected in both mutants but the regulation of its components differs in each mutant. These results point out the different functions of the two photorespiratory GOX isoforms in Arabidopsis, leading to a better understanding of the photorespiratory pathway.

Bacterial pathogens must adapt to dynamic host tissue environments to proliferate. Accordingly, elegant regulatory systems evolved to overcome challenges presented by the host and satisfy nutritional requirements. Sulfur is an essential macronutrient and Gram-positive bacteria such as Staphylococcus aureus balance this nutritional requirement by employing the transcriptional repressor, CymR. Previous investigations defined the S. aureus CymR regulon by comparing transcripts generated in a cymR mutant cultured in cystine replete, rich medium to wild type cells. This study defines the S. aureus CymR-dependent and -independent sulfur-starvation response in chemically defined growth conditions. Results demonstrate that the sulfur starvation and sulfur replete CymR regulons exhibit considerable overlap, including previously noted connections between iron acquisition, oxidative stress, and sulfur metabolism. The link between iron acquisition, oxidative stress, and sulfur metabolism is validated further by the finding that sulfur-containing glutathione (GSH) mitigates heme and peroxide toxicity. In addition to GSH, Cys and thiosulfate fulfill the S. aureus sulfur requirement. Transcriptional responses to organic (cysteine, cystine, reduced and oxidized GSH) or inorganic thiosulfate were quantified, revealing sulfur source-specific expression patterns. Thiosulfate induced the largest number of differentially expressed genes. Consequently, the thiosulfate transporter (SAUSA300_RS10985) has been confirmed as essential for S. aureus growth when thiosulfate is the sulfur source. Furthermore, we demonstrate that a hypothetical protein operonic with SAUSA300_RS10985, SAUSA300_RS10980, supports maximal growth on thiosulfate. Collectively, a resourceful transcriptomics framework is provided which underscores the dynamic nature of S. aureus sulfur metabolism.

Glutathione (GSH) is a tripeptide that plays essential roles in redox regulation and stress responses across organisms. In Escherichia coli, the GSH-specific {gamma}-glutamyl cyclotransferase (ChaC) has been characterized biochemically, yet its physiological role remains unclear. Moreover, ChaC has been annotated as a regulator of the Na/H antiporter ChaA based on its genomic association, although experimental evidence supporting this function is limited. In this study, we investigated whether chaC and its co-transcribed gene, chaB, are involved in sodium transport or GSH metabolism. Gene expression analyses revealed that chaA, chaB, and chaC are upregulated under salt stress. Functional analyses using deletion mutants showed that loss of chaA reduced salt tolerance, whereas deletion of chaB enhanced tolerance and decreased intracellular sodium levels. In contrast, deletion of chaC had no significant effect on salt tolerance or sodium accumulation. Overexpression of cha genes further indicated that chaA, but not chaB or chaC, contributed to salt tolerance. Importantly, overexpression of chaC significantly reduced intracellular GSH levels, whereas chaB overexpression had no effect. These results indicate that ChaC primarily functions in GSH degradation rather than in cation transport, and that ChaB does not participate in GSH metabolism. Our findings clarify the distinct physiological roles of ChaC and ChaB and provide new insight into bacterial physiology regarding GSH metabolism and ion transport in E. coli.

1.Abiotic stresses like nitrogen deficiency and soil salinity are major factors contributing to low crop yields. The use of selective biofertilizers alleviates both types of stress. In this study, we investigated the biofertilizer activity and plant growth-promoting properties (PGP) of Rhodococcus jialingiae RS1 through cytosolic proteome remodelling. We cultured RS1 under two conditions, i) without and ii) with 6% NaCl, in nitrogen-deficient defined Burks medium. Under dual stress of nitrogen limitation and salt stress, Orbitrap LC-MS/MS proteomics revealed one-quarter of the proteome remodelling, particularly the upregulation of ribosomal synthesis and protein repair systems. As expected, we found high expression of EctC, an ectoine synthase, a key enzyme in osmolyte biosynthesis. Additionally, ribosomal and translational-associated factors, including RpsL, RpsS, RpsT, RpsR1, RplV, RplL, RplA, and elongation factor Tuf, were highly expressed, suggesting enhanced translational fidelity under dual stress. High levels of DNA protection protein, Dps suggest dual stress may lead to DNA damage. Upregulation of chaperones, environmental sensors (KinE), and redox transcriptional factors like WhiB3, Hsp18, AhpC, and MetE suggests protein misfolding and oxidative stress. Metabolic modulations were evident through high expression of IlvA, NAD-dependent glutamate dehydrogenase, lipid/envelope-remodelling enzymes, cutinase/esterases, lipases, endopeptidases like NlpC/P60 and transport systems. In contrast, proteins involved in urease structural components (urea-G), nitrogen regulators and ammonium transporters (GlnK and Amt) were downregulated. Dual stress may lead to an energy crisis, prompting strategic shifts away from high-ATP-dependent ureolytic nitrogen-scavenging pathways towards lower-energy nitrogen-assimilating routes, such as IlvA-mediated deamination and NAD-dependent glutamate dehydrogenation. Genetic manipulations of the above-mentioned genes or their homologues across the genera of microbes, plants, and crops may enhance resilience to abiotic stresses. Our studies reveal stress-responsive genes and biochemical pathways that could be used to improve transgenic efficacy in nitrogen-limited, saline soil and other (a)biotic stresses. Global Proteome Profiling of Rhodococcus jialingiae RS1 to Develop Transgenics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/724437v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1719d80org.highwire.dtl.DTLVardef@1b6b59org.highwire.dtl.DTLVardef@24d367org.highwire.dtl.DTLVardef@1b33224_HPS_FORMAT_FIGEXP M_FIG C_FIG

{gamma}-Glutamyl cyclotransferases (GGCTs) belongs to class of cytosolic enzymes that are responsible for glutathione (GSH) degradation under stress conditions. They regulate GSH homeostasis through the {gamma}-glutamyl cycle which is responsible for maintaining the synthesis of GSH as well as its breakdown, enabling recycling of its constituent amino acids. Although GGCTs have been implicated in enhancing heavy metal (HMs) tolerance in plants, their role in biotic stress remains largely unexplored. Previously, OsGGCT1 was identified as a gene strongly upregulated in Fusarium stress. In this study, the GGCT1 homolog from Oryza sativa japonica was characterized for its role in conferring tolerance to Fusarium oxysporum (F.O.). Similar to abiotic factors, biotic stresses significantly impact crop yield and productivity. The rhizosphere harbors diverse microbial communities, including harmful pathogens such as F. oxysporum. Fusarium causes wilt disease in a variety of plant species, such as: tomato, legumes, rice, and Arabidopsis thaliana. Our results demonstrate that overexpression of OsGGCT1 enhanced tolerance to F. oxysporum in A. thaliana, primarily by reducing fungal spore accumulation. Transgenic plants showed elevated expression of OsGGCT1 along with AtGSH1 and AtGSH2, reduced levels of reactive oxygen species (ROS), improved growth and photosynthetic performance and enhanced activities of the antioxidant enzymes. OsGGCT1 serves as a key component in maintaining GSH homeostasis by supporting glutamate (Glu) regeneration necessary for sustained GSH biosynthesis. Overall, these findings identify OsGGCT1 as an important constituent of the GSH-mediated detoxification pathway against Fusarium oxysporum and provide valuable molecular insights for developing Fusarium-tolerant rice varieties with reduced fungal accumulation.

1O_LIZinc (Zn) deficiency limits rice productivity and poses a risk to human health, particularly in populations reliant on rice-based diets. Although rice germplasm exhibits wide variation in Zn-deficiency tolerance, the underlying physiological mechanisms remain poorly resolved. Evidence across the literature for Zn-deficiency-induced secretion of 2'-deoxymugineic acid (DMA) is inconsistent. This study clarifies the role of DMA secretion as a Zn-deficiency stress response. C_LIO_LIWe developed and validated a sensitive LC-ESI-Q-TOF-MS method for selective detection of DMA in rice root exudates. Five rice genotypes with contrasting Zn-deficiency tolerance were grown hydroponically and DMA secretion measured. C_LIO_LIZn-deficiency increased DMA exudation across all genotypes, with sensitive genotypes also showing higher secretion compared with control, supporting DMAs role as a general response to Zn stress rather than being restricted to efficient genotypes. C_LIO_LIFold-change responses exceeded previous studies, likely due to more severe stress exposure. Our results confirm that DMA secretion is induced under Zn-deficiency in rice as part of the micronutrient stress response. However, the lack of increased Zn uptake indicates that additional tolerance mechanisms are involved. These findings reconcile inconsistencies in the literature and position DMA secretion as an important, but not exclusive, component of Zn-deficiency adaptation in rice. C_LI

Organic germanium, particularly carboxyethyl germanium sesquioxide (Ge-132), has been investigated for decades in relation to diverse biological effects, with a strong emphasis on its antioxidant properties. However, the available literature remains dispersed, encompassing heterogeneous experimental models and endpoints that limit mechanistic interpretation. While antiglycative activity has been described at the biochemical level, its downstream gene regulatory consequences under glycative stress remain inconsistently characterized. Here, we combined systematic review of the literature of experimental studies with targeted molecular analysis in a standardized cellular model. The literature mapping was used to guide pathway selection rather than to establish quantitative associations. Based on patterns emerging from literature, we focused on pathways associated with glycative stress responses, including carbonyl stress, inflammatory signaling, and autophagy regulation. Gene expression analysis revealed a limited and selective modulation of regulatory pathways under glycative stress conditions, consistent with a context-dependent effect rather than broad transcriptional reprogramming. In parallel, protein analysis showed reduced intracellular accumulation of advanced glycation end products (AGEs) in Ge-132-treated cells under glycative stress conditions. Importantly, these findings support a dissociation between glycative damage reduction and cellular stress-response pathways. This combined approach helps interpretation of previously fragmented observations across the literature and highlights gene regulation under glycative stress as a relevant but still unresolved aspect of organogermanium biology.

The invasive fungal pathogen Pseudogymnoascus destructans is responsible for the collapse of several North American bat species through an infectious fungal skin disease known as White-Nose Syndrome (WNS). Recent transcriptomic studies have suggested that trace copper ion acquisition is essential for P. destructans propagation on its animal hosts. However, little is known about the mechanistic details of P. destructans adaptation occurring at the protein level. In this study, we report the global proteomic adaptation of P. destructans under chronic Cu-stress growth conditions employing chemically defined media. We identify 4340 P. destructans proteins, or approximately 47.8% of the predicted proteome, spanning a dynamic intensity range of six orders of magnitude. Chronic Cu-withholding stress leads to substantial alterations in the proteome, with 1398 differentially abundant proteins (DAPs) exhibiting statistically significant (p < 0.05) changes in protein levels compared to control growth conditions. We find that Cu-withholding stress induces increased levels of proteins associated with high-affinity Cu-acquisition, changes in intracellular superoxide dismutase (SOD) levels, and alterations in mitochondrial proteins related to aerobic respiration. In contrast, chronic Cu-overload stress leads to 390 DAPs (p < 0.05), which are more widely distributed across the proteome, with several DAPs associated with genomic stability and basic metabolism. Additionally, in this report, we present assessment of antisera products against intracellular and cell-surface protein targets of P. destructans that are effective for indicating Cu-withholding stress by western blotting.

The global industrialization and rapid urbanization elevated the risk of toxic pollutant exposure, which affects human health specially during pregnancy. Pregnant mothers are daily exposed to bisphenol-A (BPA), which is a common plastic leachate and a prominent toxic pollutant present in our environment. BPA act as an endocrine disrupting chemical (EDCs) by altering feto-placental homeostasis. This persistent and potent exposure of BPA during gestation can trigger placental damage affecting trophoblast cell function and survival. BPA even disrupts specific signalling cascades by altering post translational protein phosphorylation. However, this BPA mediated dysregulation of signalling nodes in early trimester placenta is still unexplored. Therefore, this study investigates the global proteome changes in post-BPA exposed extravillous trophoblast (EVTs) cells, which revealed a BPA mediated dynamic regulation of phosphoproteome-signatures and their associated kinases. Further inspection showed that the altered phosphorylation of c-JUN (S63) and GSK3 (Y279) is associated with BPA toxicity in EVTs and placenta. This altered phosphorylation affects the cellular signalling downstream, imparting damage upon the growing feto-placental unit. This highlights an altered phosphorylation mediated mechanism of BPA toxicity in placenta which can cause an onset of adverse pregnancy outcome. Data are available via ProteomeXchange with the identifiers PXD074780.

Staphylococcus aureus (S. aureus) is an opportunistic pathogen that is a global health concern for its ability to cause a wide spectrum of clinical infections. Due to the emergence of resistance to commonly used antibiotics, there has been interest in exploring the use of antimicrobial peptides to treat S. aureus infections. However, changes in the lipid composition of the lipid bilayer membrane can alter the activity of peptides, and S. aureus is able to induce variations in lipid composition in response to environmental stress. Here, we explore how the main lipid components in S. aureus are altered when exposed to LL-37, a human cathelicidin involved in primary immune response, and ATRA-1, a short antimicrobial peptide derived from the snake Naja atra venom. A lipidomic study is conducted through HPLC-MS-MS (LC-ESI-MS/MS) to quantify phosphatidylglycerol, cardiolipin, lysyl-phosphatidylglycerol, monogalacto- and digalacto-diacylglycerol, and carotenoids. In addition, menaquinones, responsible for electron transport during oxidative phosphorylation, were also quantified. Biophysical properties such as membrane electric surface potential and lipid packing were assessed. We find that lipid adaptation is specific to the type of antimicrobial peptide, where ATRA-1 mainly induces changes in the electric surface potential through variations in Lysyl-PG, while exposure to LL-37 changes carotenoid levels, inducing an increase in membrane rigidity as measured by FTIR. In addition, both peptides induce a reduction in menaquinone and DGDG levels. These findings highlight the role of membrane lipid remodeling as a peptide-specific response mechanism in S. aureus, with implications for the development of AMP-based therapies. HighlightsO_LIStaphylococcus aureus responds through shifts in lipid composition and membrane biophysical properties to exposure to the antimicrobial peptides LL-37 and ATRA-1. C_LIO_LIBoth LL-37 and ATRA-1 lead to shifts in the glycolipids MGDG and DGDG; two lipids involved in regulating negative membrane curvature stress and responsible for shifting resistance to antimicrobial peptide activity in Staphylococcus aureus. C_LIO_LILL-37 treatment leads to an overall reduction in carotenoid content in Staphylococcus aureus, including the carotenoid end-product staphyloxanthin and the precursor 4,4-diaponeurosporenoic acid. Both lipids regulate membrane biophysical properties and protect Staphylococcus aureus from oxidative stress. C_LIO_LIBoth LL-37 and ATRA-1 lead to a reduction in menaquinone levels, which are involved in the electron transport chain during oxidative phosphorylation. Reduction in these menaquinones have been associated to the formation of small colony variants that are often observed in chronic Staphylococcus aureus infections. C_LI

Colletotrichum sublineola (Cs), the hemibiotrophic fungus that causes sorghum anthracnose, impacts sorghum grain and biomass crop production worldwide. Although nutrient availability is known to influence development in filamentous fungi, including Colletotrichum species, how in vitro nutrient limitation reprograms the Cs cellular state remains unclear. We cultured Cs on full-strength, half-strength, and one-tenth-strength potato dextrose agar (PDA) to define responses across a nutrient gradient. Nutrient limitation induced a pronounced high-sporulation phenotype, with one-tenth-strength PDA producing the strongest conidiation response, followed by half-strength PDA. To study the underlying molecular programs in each condition, we employed a multiplexed metabolite, protein, and lipid extraction (MPLEx) protocol for global proteomics and metabolomics. Global proteomics resulted in 4,590 protein identifications, including 204 unique to one-tenth-strength PDA. Among them are proteins linked to sporulation, vesicular transport, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, and common in fungal extracellular membrane (CFEM)-domain proteins. Differential abundance and pathway analyses revealed a broad reduction of central carbon and energy metabolism, including glycolysis/gluconeogenesis, pentose phosphate, pyruvate metabolism, and glyoxylate pathways, together with increased ribosome-related processes, cAMP signaling, and cell-surface remodeling in one-tenth-strength PDA conditions. In addition, correlative metabolomics supported selective metabolic depletion and resource reallocation toward stress adaptation, membrane remodeling, and conidiation, supporting proteomics findings. Together, these data support a starvation-adapted Cs developmental state associated with enhanced sporulation, cellular pathway reprogramming, and potential virulence linked preparedness under nutrient-limited growth conditions in vitro. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/724728v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@f6ceb2org.highwire.dtl.DTLVardef@17c4836org.highwire.dtl.DTLVardef@68e995org.highwire.dtl.DTLVardef@1bf3983_HPS_FORMAT_FIGEXP M_FIG C_FIG

Abscisic acid (ABA) is involved in Cd tolerance in Arabidopsis, but the underlying mechanisms are unclear. In this study, we revealed that the ABI5-WRKY45-LSU1 axis confers the tolerance of Arabidopsis to Cd stress. Under Cd stress, the biosynthesis of ABA is increased, and the expression of transcription factor ABI5 is upregulated. Accordingly, the abi5-8 mutants show increased Cd sensitivity. ABI5 directly binds the ABRE element in the WRKY45 promoter to activate its transcription. Overexpression of WRKY45 rescues the Cd-hypersensitive phenotype of the abi5-8 mutant, placing WRKY45 downstream of ABI5. Transcriptome analyses identified LSU1 as a potential WRKY45 target. qRT-PCR, DUAL-LUC and EMSA experiments verified that WRKY45 binds the W-box cis-element in the LSU1 promoter to activate its expression. Overexpression of LSU1 enhances Cd tolerance by promoting the biosynthesis of non-protein thiols (NPT), glutathione (GSH), and phytochelatins (PC). Moreover, overexpression of LSU1 suppresses Cd sensitivity in the wrky45 mutant, confirming LSU1 acts downstream of WRKY45. On the other hand, we found that ATP sulfurylase 1 (APS1) interacts with LSU1 based in vitro and in vivo evidences. LSU1 stabilizes APS1, slows its degradation, and enhances APS1 activity, thus leading to increased NPT, GSH, and PC accumulation and improved Cd detoxification. Notably, overexpressing LSU1 did not rescue the Cd sensitivity of the aps1-1 mutant, indicating that LSU1 acts upstream of and depends on APS1. In short, we demonstrated a novel ABI5-WRKY45-LSU1 axis that regulates Cd tolerance through sulfur assimilation and phytochelatin synthesis. HighlightsO_LICadmium stress triggers ABA biosynthesis and ABI5 expression; ABI5 directly binds to ABRE motifs in the WRKY45 promoter and activates its transcription. C_LIO_LIWRKY45 transcriptionally activates LSU1, and LSU1 interacts with APS1 to stabilize it and elevate ATP sulfurylase activity, acting in an APS1-dependent manner. C_LIO_LIThe ABI5-WRKY45-LSU1 module enhances Arabidopsis Cd tolerance by boosting sulfur assimilation and GSH/PC-mediated Cd detoxification, rather than reducing Cd uptake. C_LI

Copper is widely used as a fast-acting antimicrobial, yet the strategies that allow bacteria to survive copper stress remain incompletely understood. Here, we characterize the transcriptional responses of Escherichia coli MG1655 to excess ionic copper using RNA sequencing and a genome-wide GFP-based promoter library. We applied 2 mM copper, which slows growth, and 8 mM copper, a near-lethal concentration. RNA-seq revealed extensive transcriptome remodeling, with 487 genes upregulated at 2 mM and 364 at 8 mM. Both concentrations strongly induced canonical copper-responsive systems, oxidative stress defenses, histidine biosynthesis, and multiple iron acquisition pathways - including enterobactin biosynthesis and transport - despite external iron failing to reduce copper toxicity. At 2 mM copper, additional pathways were activated, including heat-shock and protein-folding functions as well as lipid A, methionine and arginine biosynthesis. Copper exposure also repressed large gene sets: 486 genes at 2 mM, enriched for biofilm formation and pH elevation, and 217 genes at 8 mM, enriched for anaerobic metabolism. In contrast to the robust RNA seq results, we investigated the Horizon Discovery E. coli genome-wide GFP based promoter library as an alternative screening tool. However, in our experiments it showed low signal to noise ratios, limiting its suitability for large scale gene expression screening.

Bisphenol A (BPA) and Bisphenol S (BPS) exposure disrupt ovarian function and granulosa cell (GC) steroidogenesis. Extracellular vesicles (EVs) and their miRNA cargo, as mediators of cellular response to environmental stimuli, might be involved in fertility and folliculogenesis. This study explored modulation of microRNA expression after 48h BPA or BPS exposure (10 {micro}M) in ovine primary GC and EVs from corresponding conditioned medium (CM EVs). Small RNA sequencing of control (0h) and 48h treated GC, CM EVs as well as follicular fluid EVs allowed identification of 533 ovine miRNAs, including 129 new sequences. BPA did not alter miRNA expression in GC, while BPS decreased cellular oar-24b miR. In contrast, BPA modified expression of 4 miRNAs in CM-EVs, including 3 new sequences, and two miRNAs were modified by BPS. Both compounds reduced expression of sequence homologous to miR-1306. Further studies are required to decipher their roles in bisphenol toxicity in GC.

Mitochondrial morphology and function are critical determinants of neuronal function and survival, with disruptions in mitochondrial dynamics often preceding the overt neuronal dysfunction seen in neurodegenerative diseases such as Alzheimers disease, Huntingtons disease and Parkinsons disease. The kynurenine pathway accounts for 95% of dietary tryptophan catabolism and many of the metabolites are neuroactive, including redox-active 3-hydroxykynurenine (3-HK). 3-HK is present under normal physiological conditions in the central nervous system (CNS) and is elevated during inflammation. While supraphysiological levels of 3-HK have been associated with neurotoxicity, the effects of physiological concentrations on neuronal cells, and specifically their mitochondria, remain poorly understood. Here we assessed viability, ATP levels and redox status to determine cellular health and function in neuronal cells exposed to physiological levels of 3-HK, alongside confocal imaging and transcriptomic profiling, finding significant alterations in mitochondrial function and morphology. Interestingly, a biphasic influence of 3-HK on mitochondrial morphology was observed, with an elongated network as well as decreased surface area and volume being observed only at the lowest concentration of 3-HK, reflecting normal physiological levels. At the highest 3-HK concentration tested, reflecting an inflammatory situation, an increased number of mitochondria were present, accompanied by increased activation of caspase-3/7 and enhanced production of mitochondrial superoxide. These results highlight a previously unknown role for 3-HK in regulating mitochondrial function and structure, possibly through altered fission and fusion events, suggesting that subtle changes in kynurenine pathway metabolism may contribute to early mitochondrial dysfunction in neurological disease.