Stress Response Proteins Research Articles

Deciphering the Proteome and Phosphoproteome of Peanut (Arachis hypogaea L.) Pegs Penetrating into the Soil

Peanut (Arachis hypogaea L.) is one of the most important crops for oil and protein production. The unique characteristic of peanut is geocarpy, which means that it blooms aerially and the peanut gynophores (pegs) penetrate into the soil, driving the fruit underground. In order to fully understand this phenomenon, we investigated the dynamic proteomic and phosphoproteomic profiling of the pegs aerially and underground in this study. A total of 6859 proteins and 4142 unique phosphoproteins with 10,070 phosphosites were identified. The data were validated and quantified using samples randomly selected from arial pegs (APs) and underground pegs (UPs) by parallel reaction monitoring (PRM). Function analyses of differentially abundant proteins (DAPs) and differentially regulated phosphoproteins (DRPPs) exhibited that they were mainly related to stress response, photosynthesis, and substance metabolism. Once the pegs successfully entered the soil, disease-resistant and stress response proteins, such as glutathione S-transferase, peroxidase, and cytochrome P450, significantly increased in the UP samples in order to adapt to the new soil environment. The increased abundance of photosynthesis-associated proteins in the UP samples provided more abundant photosynthetic products, which provided the preparation for subsequent pod development. Phosphoproteomics reveals the regulatory network of the synthesis of nutrients such as starch, protein, and fatty acid (FA). These results provide new insights into the mechanism, indicating that after the pegs are inserted into the soil, phosphorylation is involved in the rapid elongation of the pegs, accompanied by supplying energy for pod development and preparing for the synthesis of metabolites during pod development following mechanical stimulation and darkness.

Efficient degradation of corn straw at low temperature using anovelco-culturedconsortium LHWA.

Straw degradation was slow under low temperature environments. A cold-tolerant consortium LHWA was constructed by Bacillus cereus, Acinetobacter lwoffii, Penicillium griseofulvum, and Talaromyces funiculosus. The consortium and culture conditions were optimized. Under 4°C cultivation, liquid fermentation showed a 55.52% straw weight loss rate after 30 days with inoculum (8.4%, w/v), peptone (0.4%, w/v) and Fe2+ concentration (0.06%, w/v); solid fermentation showed 58.36% straw weight loss rate after 60 days. According to transcriptomic analysis, the mechanism of cold resistance in B.cereus is to improve the fluidity of the cell membrane, including changing the composition of fatty acids, increasing the expression of cold stress response proteins and cold shock proteins. The constructed consortium LHWA significantly improved the straw degradation efficiency under cold environments.

An eCIRP inhibitor attenuates fibrosis and ferroptosis in ischemia and reperfusion induced chronic kidney disease

BackgroundChronic kidney disease (CKD) is a leading cause of death in the United States, and renal fibrosis represents a pathologic hallmark of CKD. Extracellular cold-inducible RNA-binding protein (eCIRP) is a stress response protein involved in acute inflammation, tissue injury and regulated cell death. However, the role of eCIRP in chronic inflammation and tissue injury has not been elucidated. We hypothesize that eCIRP is involved in renal ischemia/reperfusion (RIR)-induced CKD and that C23, an antagonist to eCIRP, is beneficial in attenuating renal fibrosis and ferroptosis in RIR-induced CKD.MethodsC57BL/6 (WT) or CIRP−/− mice underwent renal injury with total blockage of blood perfusion by clamping bilateral renal pedicles for 28 min. In the WT mice at the time of reperfusion, they were treated with C23 (8 mg/kg) or vehicle. Blood and kidneys were harvested for further analysis at 21 days thereafter. In a separate cohort, mice underwent bilateral RIR and treatment with C23 or vehicle and were then subjected to left nephrectomy 72 h thereafter. Mice were then monitored for additional 19 days, and glomerular filtration rate (GFR) was assessed using a noninvasive transcutaneous method.ResultsIn the RIR-induced CKD, CIRP−/− mice showed decreased collagen deposition, fibronectin staining, and renal injury as compared to the WT mice. Administration of C23 ameliorated renal fibrosis by decreasing the expression of active TGF-β1, α-SMA, collagen deposition, fibronectin and macrophage infiltration to the kidneys. Furthermore, intervention with C23 significantly decreased renal ferroptosis by reducing iron accumulation, increasing the expression of glutathione peroxidase 4 (GPX4) and lipid peroxidation in the kidneys of RIR-induced CKD mice. Treatment with C23 also attenuated BUN and creatinine. Finally, GFR was significantly decreased in RIR mice with left nephrectomy and C23 treatment partially prevented their decrease.ConclusionOur data show that eCIRP plays an important role in RIR-induced CKD. Treatment with C23 decreased renal inflammation, alleviated chronic renal injury and fibrosis, and inhibited ferroptosis in the RIR-induced CKD mice.

TRIM29 protein partners in cultured immortalized normal basal epithelial cells of the prostate

BACKGROUND: The E3 ubiquitin ligase TRIM29 is involved in basal epithelial development, cellular response to viral infection, and DNA damage. Furthermore, this protein can have both oncogenic and tumor suppressor properties. However, the molecular mechanisms of TRIM29 involvement in such a wide range of biological processes remain unclear. AIM: To identify protein partners of TRIM29 and its truncated forms and to determine the key molecular processes in which it is involved. MATERIALS AND METHODS: Cell cultures of normal prostate basal epithelium with overexpression of a full length TRIM29-FLAG chimeric protein or its truncated forms without the B-box or Coiled-Coil domain were obtained. Subsequently, protein partners of TRIM29 and truncated forms of TRIM29 were identified by protein immunoprecipitation followed by proteomic analysis (high-performance liquid chromatography with tandem mass spectrometry). Results were confirmed by Western blot and immunocytochemistry. RESULTS: TRIM29 binds to 288 proteins in the normal basal epithelium of the prostate. Deletion of the B-box has little effect on TRIM29 protein-protein interactions, whereas deletion of the Coiled-Coil domain deprives TRIM29 of most of its protein partners and impairs its dimerization. TRIM29 was found to localize to both the nucleus and cytoplasm, while deletion of functional domains does not prevent localization to different compartments, but does affect binding to proteins specific to these compartments. TRIM29 binds to cytoskeleton proteins, cellular stress response proteins, and RNA-binding proteins. In addition, TRIM29 is shown to increase cell resistance to genotoxic agents and to affect RNA splicing. CONCLUSION: Proteomic analysis showed that in normal prostate basal epithelium, E3 ubiquitin ligase TRIM29 binds to a relatively large number of proteins that perform different functions in different cell compartments. Our results are consistent with results obtained by other research teams who showed that TRIM29 is actively involved in cytoskeletal remodeling, cellular response to viral infection, and DNA damage. In addition, it was shown for the first time that TRIM29 interacts with stress granule proteins and RNA binding proteins and is able to regulate RNA splicing, and the Coiled-Coil domain of TRIM29 may play a key role in this process.

A microaerobically induced small heat shock protein contributes to Rhizobium leguminosarum/Pisum sativum symbiosis and interacts with a wide range of bacteroid proteins.

During the establishment of the symbiosis with legume plants, rhizobia are exposed to hostile physical and chemical microenvironments to which adaptations are required. Stress response proteins including small heat shock proteins (sHSPs) were previously shown to be differentially regulated in bacteroids induced by Rhizobium leguminosarum bv. viciae UPM791 in different hosts. In this work, we undertook a functional analysis of the host-dependent sHSP RLV_1399. A rlv_1399-deleted mutant strain was impaired in the symbiotic performance with peas but not with lentil plants. Expression of rlv_1399 gene was induced under microaerobic conditions in a FnrN-dependent manner consistent with the presence of an anaerobox in its regulatory region. Overexpression of this sHSP improves the viability of bacterial cultures following exposure to hydrogen peroxide and to cationic nodule-specific cysteine-rich (NCR) antimicrobial peptides. Co-purification experiments have identified proteins related to nitrogenase synthesis, stress response, carbon and nitrogen metabolism, and to other relevant cellular functions as potential substrates for RLV_1399 in pea bacteroids. These results, along with the presence of unusually high number of copies of shsp genes in rhizobial genomes, indicate that sHSPs might play a relevant role in the adaptation of the bacteria against stress conditions inside their host.IMPORTANCEThe identification and analysis of the mechanisms involved in host-dependent bacterial stress response is important to develop optimal Rhizobium/legume combinations to maximize nitrogen fixation for inoculant development and might have also applications to extend nitrogen fixation to other crops. The data presented in this work indicate that sHSP RLV_1399 is part of the bacterial stress response to face specific stress conditions offered by each legume host. The identification of a wide diversity of sHSP potential targets reveals the potential of this protein to protect essential bacteroid functions. The finding that nitrogenase is the most abundant RLV_1399 substrate suggests that this protein is required to obtain an optimal nitrogen-fixing symbiosis.

Molecular evolution and interaction of ROS with ion transport for plant abiotic stresses

AbstractReactive oxygen species (ROS) serve as crucial signaling molecules in plants, enabling rapid responses to environmental stresses such as abiotic factors. ROS production primarily stems from the activation of enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and peroxidases, as well as disruptions in the respiratory and photosynthetic electron transport chains. This oxidative stress triggers signaling pathways that involve in calcium ion (Ca2+) influx across cell membranes, altering ionic conductance. ROS encompass hydroxyl radicals (OH•) and hydrogen peroxide (H2O2), which activate hyperpolarization‐activated Ca2+ channels and influence ion transport dynamics. Our review focuses on the mechanisms driving ROS generation and ion transport during plant responses to abiotic stress. We explore the regulation, characteristics, and potential structures of ROS‐activated ion channels in plants. Specifically, we examine the molecular responses and evolutionary adaptations of Shaker‐type K+ channels (AKT/KAT/GORK/SKOR) under stress conditions. Comparative genetic analyses highlight the conservation of these channels and other ROS‐regulated proteins (e.g., MDHAR, POX, and RBOH), suggesting their essential roles in plant to adapt to diverse stresses. This study underscores the significance of ROS‐regulated proteins in plant stress responses, advocating for further research to elucidate their fundamental roles.

Whole-Genome Profiling of Endophytic Strain B.L.Ns.14 from Nigella sativa Reveals Potential for Agricultural Bioenhancement.

Endophytic microbes in medicinal plants often possess beneficial traits for plant health. This study focuses on the bacterial endophyte strain B.L.Ns.14, isolated from Nigella sativa leaves, which demonstrated multiple plant growth-promoting properties. In vitro tests showed that B.L.Ns.14 supports plant growth, colonization, and tolerance to abiotic stress. The strain also exhibited antifungal activity against phytopathogens such as Rhizoctonia solani, Colletotrichum acutatum, Verticillium dahliae, and Fusarium oxysporum f. sp. radicis-lycopersici. Whole-genome analysis, supported by ANI and dDDH values, identified B.L.Ns.14 as Bacillus halotolerans. Genome mining revealed 128 active carbohydrate enzymes (Cazymes) related to endophytism and biocontrol functions, along with genes involved in phosphate solubilization, siderophore and IAA production, biofilm formation, and motility. Furthermore, genes for osmolyte metabolism, Na+/H+ antiporters, and stress response proteins were also identified. The genome harbors 12 secondary metabolite biosynthetic gene clusters, including those for surfactin, plipastatin mojavensin, rhizocticin A, and bacilysin, known for their antagonistic effects against fungi. Additionally, B.L.Ns.14 promoted Arabidopsis thaliana growth under both normal and saline conditions, and enhanced Solanum lycopersicum growth via seed biopriming and root irrigation. These findings suggest that Bacillus halotolerans B.L.Ns.14 holds potential as a biocontrol and plant productivity agent, warranting further field testing.

How Small Proteins Adjust the Metabolism of Cyanobacteria Under Stress: The Role of Small Proteins in Cyanobacterial Stress Responses.

Several recently discovered small proteins of less than 100 amino acids control important, but sometimes surprising, steps in the metabolism of cyanobacteria. There is mounting evidence that a large number of small protein genes have also been overlooked in the genome annotation of many other microorganisms. Although too short for enzymatic activity, their functional characterization has frequently revealed the involvement in processes such as signaling and sensing, interspecies communication, stress responses, metabolism, regulation of transcription and translation, and in the formation of multisubunit protein complexes. Cyanobacteria are the only prokaryotes that perform oxygenic photosynthesis. They thrive under a wide variety of conditions as long as there is light and must cope with dynamic changes in the environment. To acclimate to these fluctuations, frequently small regulatory proteins become expressed that target key enzymes and metabolic processes. The consequences of their actions are profound and can even impact the surrounding microbiome. This review highlights the diverse functions of recently discovered small proteins that control cyanobacterial metabolism. It also addresses why many of these proteins have been overlooked so far and explores the potential for implementing metabolic engineering strategies to improve the use of cyanobacteria in biotechnological applications.

Evolution and functional characterization of Populus salt stress-responsive calcineurin B-like protein-interacting protein kinases.

Identification of salt-responsive calcineurin B-like protein-interacting protein kinases(CIPKs) in Populus. Calcineurin B-like protein-interacting protein kinases (CIPKs) play vital roles in plant growth and abiotic stress responses. Currently, the regulatory mechanisms underlying these processes mediated by CIPK proteins are not completely understood in woody species. This study provided the first systematic analysis of 31 Populus CIPK genes and investigated their evolutionary relationships, gene structures, motif compositions, and salt stress responses. A total of 11 pairs of paralogous PtCIPK genes were identified, of which three pairs may be resulted from whole genome duplication, and two pairs that may be created bytandem duplications. RT-qPCR analysis revealed that 93.5% (29/31) genes showed altered expression levels in roots after salt treatment. Ectopic expression of PdCIPK21 or PdCIPK31 in Arabidopsis resulted in significant increases of seed germination, root elongation and fresh weight under salt stressconditions. Cytological observation revealed that PdCIPK21/31 overexpression lines showed increased number, lumen area and cell wall thickness of xylem vessels, and higher lignin content in stems compared with the wild type, with decreased sensitivity to long-term salt stress treatment. Our results suggest that PdCIPK21/31 serve as candidate genes for improving wood production and enhancing salt tolerance of tree species.

Effects of the Symbiotic Chlorella variabilis on the Host Ciliate Paramecium bursaria Phenotypes.

Paramecium bursaria, a ciliated protist, forms a symbiotic relationship with the green alga Chlorella variabilis. This endosymbiotic association is a model system for studying the establishment of secondary symbiosis and interactions between the symbiont and its host organisms. Symbiotic algae reside in specialized compartments called perialgal vacuoles (PVs) within the host cytoplasm, which protect them from digestion by host lysosomal fusion. The relationship between P. bursaria and symbiotic Chlorella spp. is characterized by mutualism, in which both organisms benefit from this association. Furthermore, symbiotic algae also influence their host phenotypes, and algae-free P. bursaria can be obtained through various methods and reassociated with symbiotic algae, making it a valuable tool for studying secondary endosymbiosis. Recent advancements in genomic and transcriptomic studies on both hosts and symbionts have further enhanced the utility of this model system. This review summarizes the infection process of the symbiotic alga C. variabilis and its effects on the algal infection on number of host trichocysts, mitochondria, cytoplasmic crystals, total protein amount, stress responses, photoaccumulation, and circadian rhythms of the host P. bursaria.

502 The stress-responsive secretory protein adrenomedullin 2 potently induces cell cycle arrest and apoptosis in human hair follicles

502 The stress-responsive secretory protein adrenomedullin 2 potently induces cell cycle arrest and apoptosis in human hair follicles

Molecular Mechanisms of Phosphate Use Efficiency in Arabidopsis via Penicillium olsonii TLL1.

Beneficial fungi are promising tools for enhancing plant growth and crop yield in stressful environments. Penicillium olsonii TLL1 (POT1) was identified as a potential biofertilizer enhancing plant growth and phosphate use efficiency especially under phosphate deficiency stress. Hence, we attempted to explore bioinformatic insights into how POT1 enhances plant growth under phosphate starvation. In our study, wild-type Arabidopsis thaliana Columbia-0 roots and shoots cultivated with POT1 under phosphate-limiting conditions were employed for comparative analyses. By integrating transcriptomic and proteomic data, we identified key molecular pathways regulated by POT1 that influenced phosphate acquisition and plant stress tolerance. Comprehensive RNA-seq analysis revealed significant upregulation of genes involved in phosphate transport, root architecture, and stress-related pathways, while proteome profiling further highlighted proteins associated with lipid remodeling, phosphate metabolism, and phytohormone signaling. Bioinformatic analyses of differentially expressed genes (DEGs) and proteins (DEPs) elucidated the complex regulatory networks at both transcriptional and translational levels, with key contributions from auxin and ethylene signaling. Our study demonstrated that POT1-treated plants exhibited enhanced root development and nutrient uptake under phosphate-deficient conditions, driven by the coordinated regulation of phosphate solubilization genes and stress-responsive proteins. Our findings underscore the potential of multi-omics approaches in unraveling the molecular mechanisms behind plant-microbe interactions, with implications for improving sustainable agricultural practices.

Effects of environmentally relevant concentrations of glyphosate and aminomethylphosphonic acid on biotransformation and stress response proteins in the liver of zebrafish (Danio rerio)

Herbicides pose a threat to various non-target organisms, including fish. A widely used herbicide, glyphosate, and its main breakdown product, aminomethylphosphonic acid (AMPA), are quite ubiquitous in freshwater systems. The aim of this work was to analyze changes in the relative abundance of hepatic proteins participating in the biotransformation and response to chemical stress in adult zebrafish Danio rerio exposed to environmentally relevant concentrations of glyphosate (100 μg/L), AMPA (100 μg/L), and their mixture (50 μg/L + 50 μg/L) for two weeks. Proteomic analysis showed that the tested concentrations caused dysregulation of various biotransformation proteins, the most upregulated of which in all treatment groups was the Phase I enzyme cyp27a7. While glyphosate had a more pronounced impact on the biotransformation pathways, AMPA showed stronger interference with redox homeostasis. When acting together, the parent compound and its metabolite were more potent to disturb fish metabolic processes, including nucleotide metabolism and proteasome pathway, and to downregulate proteins known for their roles in protection from oxidative modifications of cellular constituents and disruption of redox signaling.

Carbon nanodots as photosensitizer in photodynamic inactivation of Rickettsia slovaca

Carbon quantum dots (CQDs) are promising therapeutic agent due to their pro-oxidant, antioxidant, antiviral, antibacterial, and anticancer properties when exposed to visible light irradiation. Oxidative stress in bacteria is the main reason for bacteria death after exposure to blue light photoexcited quantum dots. Herein, we present the antibacterial activities of hydrophobic carbon quantum dots/polydimethylsiloxane nanocomposites, hydrophilic citric acid CQDs, and combinations of CQDs with methylene blue. We investigated the antirickettsial effect of hydrophilic and hydrophobic CQDs against Rickettsia slovaca, a tick-borne bacterial pathogen. Photodynamic activity against on rickettsiae reached 99.66% using CQDs with 470 nm blue light irradiation. Combining methylene blue with CQDs further enhanced the effect on rickettsial infection, achieving 99,98% efficacy. The obtained results reveal the in vitro antirickettsial properties of CQDs. Sequencing analysis on the genomic level of control and treated samples showed single nucleotide variants (SNVs). Based on snippy analysis SNVs were assigned to the rRNA genes, 16S rRNA and 30S rRNA genes. By freebayes analysis in treated samples, a stop-lost mutation was detected in pseudogene (RSL_RS06070), while the possible effect on down-stream genes including tsaD, acyl-CoA-desaturase, 30S ribosomal protein S6 and DUF424 family protein. The frameshift mutation was localized within clpB pseudogene belonging to stress-response heat-shock proteins.

Misprocessing of α-Galactosidase A, Endoplasmic Reticulum Stress, and the Unfolded Protein Response.

Key Points The clinical significance of a number of missense variants of α-galactosidase A is often ambiguous.Defective proteostasis of some missense α-galactosidase A variants induced chronic endoplasmic reticulum stress and the unfolded protein response.Endoplasmic reticulum stress and the unfolded protein response may explain clinical manifestations of non-classic Fabry disease. Background Classic Fabry disease is caused by GLA mutations that result in loss of enzymatic activity of α-galactosidase A, lysosomal storage of globotriaosylceramide, and a resulting multisystemic disease. In non-classic Fabry disease, patients have some preserved α-galactosidase A activity and a milder disease course. Heterozygous female patients may also be affected. While Fabry disease pathogenesis has been mostly attributed to catalytic deficiency of mutated α-galactosidase A, lysosomal storage, and impairment of lysosomal functions, other pathogenic factors may contribute, especially in nonclassic Fabry disease. Methods We characterized the genetic, clinical, biochemical, molecular, cellular, and organ pathology correlates of the p.L394P α-galactosidase A variant that was identified initially in six individuals with kidney failure by the Czech national screening program for Fabry disease and by further screening in an additional 24 family members. Results Clinical findings in affected male patients revealed a milder clinical course, with approximately 15% residual α-galactosidase A activity with normal plasma lyso-globotriaosylceramide levels and abnormally low ratio of these values. None of the four available kidney biopsies showed lysosomal storage. Laboratory investigations documented intracellular retention of mutated α-galactosidase A with resulting endoplasmic reticulum stress and the unfolded protein response, which were alleviated with BRD4780, a small molecule clearing misfolded proteins from the early secretory compartment. We observed similar findings of endoplasmic reticulum stress and unfolded protein response in five kidney biopsies with several other classic and non-classic Fabry disease missense α-galactosidase A variants. Conclusions We identified defective proteostasis of mutated α-galactosidase A resulting in chronic endoplasmic reticulum stress and unfolded protein response of α-galactosidase A expressing cells as a contributor to Fabry disease pathogenesis.

The interplay between LHCSR and PSBS proteins provides photoprotection in Chlamydomonas reinhardtii pgr5 mutant under high light

Cyclic electron transport (CET) is a vital alternative route that protects against photodamage and aids in energy production. This process depends on proton gradient regulation 5 (PGR5) and PGRL1-dependent pathways associated with CET. The exact roles of these proteins in photosystem I photochemistry under prolonged high light conditions are not fully understood. Continuous light adaptation hinges on two critical mechanisms: alterations in the proton motive force (pmf) and adjustments in the ratio of proteins activated by high light that dissipate excess light through non-photochemical quenching (NPQ). To explore this, we studied pgrl1 and pgr5 mutants to gauge their roles in balancing photochemistry and photoacclimation. These mutants showed inhibited growth, reduced photosynthetic efficiency, and a lowered pmf, leading to diminished non-photochemical energy quenching (qE) under high light. Prolonged high light exposure slowed down unregulated energy losses Y(NO), and relaxation helped regulate photosynthetic activity by increasing photoinhibitory quenching (qI), thus preventing further damage to the photosystem. The precise balance between the two pmf components, ΔpH and Δψ, is critical for controlling photochemistry and photoacclimation, yet remains elusive. In pgr5 reduced pmf led to an accumulation of cytochrome b6f under high light, and a decrease in the ΔpH component and increased the Δψ component's role in photosynthetic acclimation. Notably, light-harvesting complex stress response protein 3 (LHCSR3) showed decreased expression in pgrl1, whereas pgr5 exhibited no expression of both LHCSR3 and LHCSR1 under high-light conditions. Moreover, continuous increase in PSBS protein accumulation in pgr5 suggests enhanced photoprotection in the absence of LHCSR3 under high light. The study provides significant insights into how CET regulates photoprotective proteins LHCSR and PSBS, influencing Chlamydomonas' survival strategies.

Stress survival and longevity of Caenorhabditis elegans lacking NCS-1.

Although dysfunctional Ca2+ signaling can trigger biochemical reactions that lead to cell death, the role of calcium-binding proteins (CBPs) in this process is still a topic of debate. Neuronal calcium sensor 1 (NCS-1) is a CBP that is highly conserved and has been shown to increase cell survival against various types of injuries. As such, we hypothesized that NCS-1 could also be a stress-responsive protein with potential effects on survival and longevity. To explore this possibility, we conducted experiments to examine how Caenorhabditis elegans ncs-1 mutant nematodes fared under three different stress conditions: hyperosmotic, thermal, and chemical oxidant challenges. Our results showed that while the lack of NCS-1 had no effect on survival responses to hyperosmotic and thermal stresses, ncs-1 worms demonstrated remarkable resistance to the oxidant paraquat in a dose-dependent manner. Based on these findings, we conclude that C. elegans may employ adaptive mechanisms in the absence of NCS-1 to survive specific oxidative stress stimuli.

Genome-wide analyses of Ariadne family genes reveal their involvement in abiotic stress responses in apple

E3 ligases are essential for ubiquitination and play a role in regulating various aspects of eukaryotic life. Ariadne (ARI) proteins, a subfamily of RBR (RING-between-RING) proteins, have been recognized as a new class of RING-finger E3 ligases. Recent research has shed light on their potential involvement in plants’ responses to abiotic stress. However, comprehensive studies on ARI genes in apple (Malus domestica) are still lacking. This study identified ten MdARI genes in the apple genome, and examined intraspecific and interspecific collinearity to explore the evolutionary relationships of ARI family members. Phylogenetic analyses classified MdARIs into two subfamilies (A and B), and by integrating gene structure, conserved motifs, and sequence comparison results, subfamily B was further divided into two subgroups (I and II). Tissue expression analyses revealed varied expression patterns of MdARI genes in different tissues, and subcellular localization showed that MdARI1-1, MdARI1-2, and MdARI9-1 were located in the nucleus, while the other seven MdARIs were distributed throughout the cell. Analyses of promoter cis-elements and expression patterns under cold, salt, and drought treatments indicated the involvement of MdARIs in abiotic stress responses. Several proteins crucial to the plant stress response were predicted to be potential MdARIs-interacting proteins based on the protein interaction network. Additionally, the interaction between UBC11 (E2) and MdARI7-2 was confirmed by a yeast two-hybrid (Y2H) experiment, suggesting that MdARI7-2 may function as an E3. These findings will greatly benefit future research on the role and mechanisms of ARI proteins in apple stress response.

Characterization of the Ubiquitin-Modified Proteome of Recombinant Chinese Hamster Ovary Cells in Response to Endoplasmic Reticulum Stress.

Chinese hamster ovary (CHO) cells remain the most widely used host cell line for biotherapeutics production. Despite their widespread use, understanding endoplasmic reticulum (ER) stress conditions in recombinant protein production remains limited, often creating bottlenecks preventing improved production titers and product quality. Ubiquitination not only targets substrates (e.g., misfolded proteins) for proteasome degradation but also has important regulatory control functions including cell cycle regulation, translation, apoptosis, autophagy, etc. and hence is likely to be central to understanding and controlling the productivity of recombinant biotherapeutics. This study aimed to uncover differentially expressed ubiquitinated proteins following artificial induction of ER-stress in recombinant CHO cells. CHO cells were treated with the stress inducer tunicamycin and the proteasome inhibitor MG132, followed by LC-MS/MS proteomic analysis. We identified >4000 ubiquitinated peptides from CHO-DP12 cells under ER stress conditions and proteasome inhibition. Moreover, data analysis showed altered abundance levels of >900 ubiquitinated proteins under the combination of ER stress and proteasome inhibition compared to untreated controls. Gene Ontology (GO) analysis of these ubiquitinated proteins resulted in a significant enrichment of key pathways involving the proteasome, protein processing in the ER, N-glycan biosynthesis, and ubiquitin-mediated proteolysis. ER stress response proteins such as GRP78, HSP90B1, ATF6, HERPUD1, and PDIA4 were found to be highly ubiquitinated and exhibited a significant increase in abundance following induction of ER-stress conditions. This study broadens our comprehension of the roles played by protein ubiquitination in CHO cell stress responses, potentially revealing targets for tailored cell line engineering aimed at enhancing stress tolerance and production efficiency.

Unravelling the Significance of Seed Proteomics: Insights into Seed Development, Function, and Agricultural Applications.

Seeds are essential for plant reproduction, ensuring species survival and dispersal while adapting to diverse environments throughout a plant's life. Proteomics has emerged as a powerful tool for deciphering the complexities of seed growth, germination, and stress responses. Advanced proteomic technologies enable the analysis of protein changes during germination, dormancy, and ageing, enhancing our understanding of seed lifespan and vitality. Recent studies have revealed detailed insights into metabolic processes and storage protein profiles across various plant species. This knowledge is crucial for improving seed storage, conserving quality, and maintaining viability. Additionally, it contributes to sustainable agriculture by identifying stress-responsive proteins and signalling pathways that can mitigate stress and enhance farming practices. This review highlights significant advancements in seed proteomics over the past decade, discussing critical discoveries related to storage proteins, protein interactions, and proteome modifications due to stress. It illustrates how these insights transform seed biology, boosting productivity, food security, and environmentally friendly practices. The review also identifies existing knowledge gaps and provides direction for future research, underscoring the need for continued interdisciplinary collaboration in this dynamic field.