RESUMO
Homeostasis of the essential micronutrient manganese (Mn) is crucially determined through availability and uptake efficiency in all organisms. Mn deficiency of plants especially occurs in alkaline and calcareous soils, seriously restricting crop yield. However, the mechanisms underlying the sensing and signaling of Mn availability and conferring regulation of Mn uptake await elucidation. Here, we uncover that Mn depletion triggers spatiotemporally defined long-lasting Ca2+ oscillations in Arabidopsis roots. These Ca2+ signals initiate in individual cells, expand, and intensify intercellularly to transform into higher-order multicellular oscillations. Furthermore, through an interaction screen we identified the Ca2+-dependent protein kinases CPK21 and CPK23 as Ca2+ signal-decoding components that bring about translation of these signals into regulation of uptake activity of the high-affinity Mn transporter natural resistance associated macrophage proteins 1 (NRAMP1). Accordingly, a cpk21/23 double mutant displays impaired growth and root development under Mn-limiting conditions, while kinase overexpression confers enhanced tolerance to low Mn supply to plants. In addition, we define Thr498 phosphorylation within NRAMP1 as a pivot mechanistically determining NRAMP1 activity, as revealed by biochemical assays and complementation of yeast Mn uptake and Arabidopsis nramp1 mutants. Collectively, these findings delineate the Ca2+-CPK21/23-NRAMP1 axis as key for mounting plant Mn homeostasis.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Cálcio , Proteínas de Transporte de Cátions , Manganês , Proteínas Quinases , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Homeostase , Manganês/metabolismo , Micronutrientes/metabolismo , Fosforilação , Raízes de Plantas/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/metabolismo , SoloRESUMO
Nitrogen (N) is one of the most essential mineral elements for plants. Brassinosteroids (BRs) play key roles in plant growth and development. Emerging evidence indicates that BRs participate in the responses to nitrate deficiency. However, the precise molecular mechanism underlying the BR signaling pathway in regulating nitrate deficiency remains largely unknown. The transcription factor BES1 regulates the expression of many genes in response to BRs. Root length, nitrate uptake and N concentration of bes1-D mutants were higher than those of wild-type under nitrate deficiency. BES1 levels strongly increased under low nitrate conditions, especially in the non-phosphorylated (active) form. Furthermore, BES1 directly bound to the promoters of NRT2.1 and NRT2.2 to promote their expression under nitrate deficiency. Taken together, BES1 is a key mediator that links BR signaling under nitrate deficiency by modulating high affinity nitrate transporters in plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Ligação a DNA , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Nitratos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Transporte de Ânions/metabolismoRESUMO
Arsenate [As(V)] is a metalloid with heavy metal properties and is widespread in many environments. Dietary intake of food derived from arsenate-contaminated plants constitutes a major fraction of the potentially health-threatening human exposure to arsenic. However, the mechanisms underlying how plants respond to arsenate stress and regulate the function of relevant transporters are poorly understood. Here, we observed that As(V) stress induces a significant Ca2+ signal in Arabidopsis (Arabidopsis thaliana) roots. We then identified a calcium-dependent protein kinase, CALCIUM-DEPENDENT PROTEIN KINASE 23 (CPK23), that interacts with the plasma membrane As(V)/Pi transporter PHOSPHATE TRANSPORTER 1;1 (PHT1;1) in vitro and in vivo. cpk23 mutants displayed a sensitive phenotype under As(V) stress, while transgenic Arabidopsis plants with constitutively active CPK23 showed a tolerant phenotype. Furthermore, CPK23 phosphorylated the C-terminal domain of PHT1;1, primarily at Ser514 and Ser520. Multiple experiments on PHT1;1 variants demonstrated that PHT1;1S514 phosphorylation is essential for PHT1;1 function and localization under As(V) stress. In summary, we revealed that plasma-membrane-associated calcium signaling regulates As(V) tolerance. These results provide insight for crop bioengineering to specifically address arsenate pollution in soils.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Humanos , Arabidopsis/genética , Arabidopsis/metabolismo , Arseniatos/toxicidade , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sinalização do Cálcio , Proteínas de Transporte de Fosfato/metabolismo , Fosfatos/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Membrana Celular/metabolismoRESUMO
BACKGROUND: Mutations in PIEZO1 (Piezo type mechanosensitive ion channel component 1) cause human lymphatic malformations. We have previously uncovered an ORAI1 (ORAI calcium release-activated calcium modulator 1)-mediated mechanotransduction pathway that triggers lymphatic sprouting through Notch downregulation in response to fluid flow. However, the identity of its upstream mechanosensor remains unknown. This study aimed to identify and characterize the molecular sensor that translates the flow-mediated external signal to the Orai1-regulated lymphatic expansion. METHODS: Various mutant mouse models, cellular, biochemical, and molecular biology tools, and a mouse tail lymphedema model were employed to elucidate the role of Piezo1 in flow-induced lymphatic growth and regeneration. RESULTS: Piezo1 was found to be abundantly expressed in lymphatic endothelial cells. Piezo1 knockdown in cultured lymphatic endothelial cells inhibited the laminar flow-induced calcium influx and abrogated the flow-mediated regulation of the Orai1 downstream genes, such as KLF2 (Krüppel-like factor 2), DTX1 (Deltex E3 ubiquitin ligase 1), DTX3L (Deltex E3 ubiquitin ligase 3L,) and NOTCH1 (Notch receptor 1), which are involved in lymphatic sprouting. Conversely, stimulation of Piezo1 activated the Orai1-regulated mechanotransduction in the absence of fluid flow. Piezo1-mediated mechanotransduction was significantly blocked by Orai1 inhibition, establishing the epistatic relationship between Piezo1 and Orai1. Lymphatic-specific conditional Piezo1 knockout largely phenocopied sprouting defects shown in Orai1- or Klf2- knockout lymphatics during embryo development. Postnatal deletion of Piezo1 induced lymphatic regression in adults. Ectopic Dtx3L expression rescued the lymphatic defects caused by Piezo1 knockout, affirming that the Piezo1 promotes lymphatic sprouting through Notch downregulation. Consistently, transgenic Piezo1 expression or pharmacological Piezo1 activation enhanced lymphatic sprouting. Finally, we assessed a potential therapeutic value of Piezo1 activation in lymphatic regeneration and found that a Piezo1 agonist, Yoda1, effectively suppressed postsurgical lymphedema development. CONCLUSIONS: Piezo1 is an upstream mechanosensor for the lymphatic mechanotransduction pathway and regulates lymphatic growth in response to external physical stimuli. Piezo1 activation presents a novel therapeutic opportunity for preventing postsurgical lymphedema. The Piezo1-regulated lymphangiogenesis mechanism offers a molecular basis for Piezo1-associated lymphatic malformation in humans.
Assuntos
Vasos Linfáticos , Linfedema , Animais , Células Endoteliais/metabolismo , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Vasos Linfáticos/metabolismo , Linfedema/metabolismo , Mecanotransdução Celular/fisiologia , Camundongos , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Azobenzene (Azo) liquid-crystalline polymers are intriguing due to their unique photo-induced isomerization and supramolecular chirality. However, clarification on multicomponent chiral induction towards Azo polymers remains ambiguous and challenging. Herein, chiral solvents and amines were employed to control the chiroptical activity of achiral Azo polymers. Methyl L-/D-lactate was added as the poor solvent and chiral inducer to achieve the first chiral induction in Azo aggregates. Chiral amines were utilized for the second chiral induction based on the acid-base interactions between the carboxyl groups of polymers and amines. The chiral enhancement and inversion of Azo units could be observed through the synergistic or antagonistic effect between solvents and amines. The impacts of solvent, chemical structures, feed ratio, enantiomeric excess, and temperature on supramolecular chirality were systematically studied. Furthermore, this system displayed the chiroptical switching property and chiral recovery under reversible irradiation.
RESUMO
Benzene exposure is known to cause serious damage to the human hematopoietic system. However, recent studies have found that chronic benzene exposure may also cause neurological damage, but there were few studies in this issue. The aim of this study was to investigate the mechanism of damage to the central nervous system (CNS) by chronic benzene exposure with a multi-omics analysis. We established a chronic benzene exposure model in C57BL/6J mice by gavage of benzene-corn oil suspension, identified the differentially expressed proteins (DEPs) and differentially expressed genes (DEGs) in mice brain using 4D Label-free proteomic and RNA-seq transcriptomic. We observed that the benzene exposure mice had a significant loss of body weight, reduction in complete blood counts, abnormally high MRI signals in brain white matter, as well as extensive brain edema and neural demyelination. 162 DEPs were identified by the proteome, including 98 up-regulated and 64 down-regulated proteins. KEGG pathway analysis of DEPs showed that they were mainly involved in the neuro-related signaling pathways such as metabolic pathways, pathways of neurodegeneration, chemical carcinogenesis, Alzheimer disease, and autophagy. EPHX1, GSTM1, and LIMK1 were identified as important candidate DEGs/DEPs by integrated proteomic and transcriptomic analyses. We further performed multiple validation of the above DEGs/DEPs using fluorescence quantitative PCR (qPCR), parallel reaction monitoring (PRM), immunohistochemistry, and immunoblotting to confirm the reliability of the multi-omics study. The functions of these DEGs/DEPs were further explored and analyzed, providing a theoretical basis for the mechanism of nerve damage caused by benzene exposure.
RESUMO
Cadmium (Cd) is a toxic heavy element for plant growth and development, and plants have evolved many strategies to cope with Cd stress. However, the mechanisms how plants sense Cd stress and regulate the function of transporters remain very rudimentary. Here, we found that Cd stress induces obvious Ca2+ signals in Arabidopsis roots. Furthermore, we identified the calcium-dependent protein kinases CPK21 and CPK23 that interacted with the Cd transporter NRAMP6 through a variety of protein interaction techniques. Then, we confirmed that the cpk21 23 double mutants significantly enhanced the sensitive phenotype of cpk23 single mutant under Cd stress, while the overexpression and continuous activation of CPK21 and CPK23 enhanced plants tolerance to Cd stress. Multiple biochemical and physiological analyses in yeast and plants demonstrated that CPK21/23 phosphorylate NRAMP6 primarily at Ser489 and Thr505 to inhibit the Cd transport activity of NRAMP6, thereby improving the Cd tolerance of plants. Taken together, we found a plasma membrane-associated calcium signaling that modulates Cd tolerance. These results provide new insights into the molecular breeding of crop tolerance to Cd stress.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Cádmio , Cálcio , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cádmio/toxicidade , Cádmio/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismoRESUMO
Manganese (Mn) is an essential micronutrient in plants. However, excessive Mn absorption in acidic soils can cause Mn toxicity, which adversely affects plant growth and crop yields. At present, acidic soils cover c. 30% of the Earth's surface. However, the mechanism underpinning Mn uptake remains largely unknown. We identified cbl1/9 and cipk23 mutants exhibiting high-Mn-sensitive phenotype through the reverse genetics method. Furthermore, we identified the CIPK23 phosphorylated NRAMP1 through a variety of protein interaction techniques and protein kinase assays. Here, we demonstrated that two calcineurin B-like proteins, CBL1/9, and their interacting kinase CIPK23 positively regulated the tolerance of Mn toxicity in Arabidopsis. The cbl1 cbl9 double mutant and cipk23 mutants exhibited high-Mn-sensitive phenotypes, which manifested as decreased primary root length, biomass, and chlorophyll concentration, and higher accumulation of Mn. In addition, CIPK23 interacted with and phosphorylated the Mn transporter NRAMP1 primarily at Ser20/22 in vitro and in vivo, and thereby induced clathrin-mediated endocytosis of NRAMP1 to reduce its distribution on the plasma membrane and enhance plant tolerance to Mn toxicity. In summary, we found that the CBL1/9-CIPK23-NRAMP1 module regulates the tolerance to high-Mn toxicity and provide insight into a mechanism of the tolerance of plants to Mn toxicity.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Manganês , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Manganês/toxicidade , Manganês/metabolismo , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
A precolumn derivatization-HPLC method using 2,4-dinitrophenylhydrazine and 4-nitro-o-phenylenediamine as respective labeling reagents for comprehensive analyses of the reactions catalyzed by acetohydroxyacid synthase (AHAS)/acetolactate synthase (ALS) is developed and evaluated in this research. Comparison with the classic Bauerle' UV assay which can analyze the enzymes only through measurement of acetoin production, the HPLC method shows advantages because it can analyze the enzymes not only via determination of consumption of the substrate pyruvate, but also via measurement of formation of the products including acetoin, 2,3-butanedione, and acetaldehyde in the enzymatic reactions. Thus the results deduced from the HPLC method can reflect the trait of each enzyme in a more precise manner. As far as we know, this is the first time that the reactions mediated by AHAS/ALS using pyruvate as a single substrate are globally analyzed and the features of the enzymes are properly discussed.
Assuntos
Acetolactato Sintase , Acetoína , Cromatografia Líquida de Alta Pressão , Ácido Pirúvico , CatáliseRESUMO
OBJECTIVE: The objective of this study was to compare the clinical efficacy of DRTR (Double Reverse Traction Repositor, DRTR)and traction table in the treatment of femoral shaft fractures with the aid of AN-IMN (Antegrade intramedullary nailing). PATIENTS AND METHODS: In this study, patients with femoral shaft fractures admitted to the Department of Orthopedics at Zhaoqing First People's Hospital from May 2018 to October 2022 were recruited. All patients were treated with anterograde intramedullary nailing, with 23 patients in the DRTR-assisted group and 21 patients in the traction table-assisted group. The demographic characteristics, fracture classification, intraoperative data, postoperative data, and prognostic indicators of the two groups were recorded and analyzed retrospectively. All procedures were performed by the same team of experienced physicians. RESULTS: All the patients in the two groups were followed up for more than 12 months. Both traction methods could provide stable traction for the operator during AN-IMN, and there was no significant difference in demographic characteristics and fracture classification. The intraoperative fluoroscopy times and opening reduction rate of the DRTR group were lower than those of the traction table group (P < 0.05), and the postoperative Harris Hip Score, as well as the Lyshol Lysholm knee function Score of the DRTR group, were significantly higher than the traction table group members (P < 0.05). Postoperative complications such as perineal soft tissue injury and lateral femoral cutaneous nerve injury occurred in the traction table group, but not in the DRTR group. CONCLUSION: DRTR can safely and effectively provide continuous and stable traction in the femoral shaft fractures surgery, and outperforms the traction table in the number of intraoperative fluoroscopy, opening reduction rate, reduction of complications, and postoperative joint function score.
Assuntos
Fraturas do Fêmur , Fixação Intramedular de Fraturas , Humanos , Tração/métodos , Estudos Retrospectivos , Pinos Ortopédicos , Fraturas do Fêmur/diagnóstico por imagem , Fraturas do Fêmur/cirurgia , Fraturas do Fêmur/etiologia , Fixação Intramedular de Fraturas/efeitos adversos , Fixação Intramedular de Fraturas/métodos , Resultado do TratamentoRESUMO
Cells and tissues are exposed to a wide range of mechanical stimuli during development, tissue homeostasis, repair, and regeneration. Over the past few decades, mechanosensitive ion channels (MSCs), as force-sensing integral membrane proteins, have attracted great attention with regard to their structural dynamics and mechanics at the molecular level and functions in various cells. Piezo-type MSC component 1 (Piezo1) is a newly discovered MSC; it is inherently mechanosensitive. However, which type of cells express Piezo1 in vivo remains unclear. To detect and trace Piezo1-expressing cells, we generated and characterized a novel tamoxifen-inducible Cre knock-in mouse line, Piezo1-CreER, which expresses CreER recombinase under the control of the endogenous Piezo1 promoter. Using this genetic tool, we detected the expression of Piezo1 in various cell types at the embryonic, neonatal, and adult stages. Our data showed that Piezo1 was highly expressed in endothelial cells in all the three stages, while the Piezo1 expression in epithelial cells was dynamic during development and growth. In summary, we established a new genetic tool, Piezo1-CreER, to study Piezo1-expressing cells in vivo during development, injury response, and tissue repair and regeneration.
Assuntos
Células Endoteliais , Canais Iônicos , Animais , Células Endoteliais/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismo , Camundongos , Camundongos Transgênicos , Transdução de Sinais , Tamoxifeno/farmacologiaRESUMO
Genetic lineage tracing has been widely used for studying in vivo cell fate plasticity during embryogenesis, tissue homeostasis, and disease development. Recent applications with multiple site-specific recombinases have been used in complex and sophisticated genetic fate mapping studies. However, the previous multicolor reporters for dual recombinases had limitations of precise in situ quantification of cell number, which is mainly due to the intermingling of cells in condensed tissues. Here, we generated a dual recombinase-mediated nuclear-localized GFP and tdTomato reporter line, which enables clear, simultaneous quantification of two distinct cell lineages in vivo. Combining this dual genetic reporter with Tbx18-Cre and Cdh5-Dre lines, which genetically trace epicardial and endothelial cells, respectively, we obtained high-resolution images for the anatomic distribution of the descendants of these two distinct cell lineages in the valve mesenchyme during development, remodeling, and maturation stages. This new dual genetic reporter is expected to facilitate fate tracing of two cell lineages and their objective quantification in vivo.
Assuntos
Linhagem da Célula , Núcleo Celular/metabolismo , Genes Reporter , Alelos , Animais , Células Endoteliais/metabolismo , Integrases/metabolismo , Mesoderma/citologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Especificidade de Órgãos , Pericárdio/citologiaRESUMO
BACKGROUND: Strong artificial and natural selection causes the formation of highly conserved haplotypes that harbor agronomically important genes. GWAS combination with haplotype analysis has evolved as an effective method to dissect the genetic architecture of complex traits in crop species. RESULTS: We used the 60 K Brassica Infinium SNP array to perform a genome-wide analysis of haplotype blocks associated with oleic acid (C18:1) in rapeseed. Six haplotype regions were identified as significantly associated with oleic acid (C18:1) that mapped to chromosomes A02, A07, A08, C01, C02, and C03. Additionally, whole-genome sequencing of 50 rapeseed accessions revealed three genes (BnmtACP2-A02, BnABCI13-A02 and BnECI1-A02) in the A02 chromosome haplotype region and two genes (BnFAD8-C02 and BnSDP1-C02) in the C02 chromosome haplotype region that were closely linked to oleic acid content phenotypic variation. Moreover, the co-expression network analysis uncovered candidate genes from these two different haplotype regions with potential regulatory interrelationships with oleic acid content accumulation. CONCLUSIONS: Our results suggest that several candidate genes are closely linked, which provides us with an opportunity to develop functional haplotype markers for the improvement of the oleic acid content in rapeseed.
Assuntos
Brassica napus/genética , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Genes de Plantas/genética , Estudo de Associação Genômica Ampla/métodos , Ácido Oleico/metabolismo , Brassica napus/classificação , Brassica napus/metabolismo , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Ligação Genética , Haplótipos , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do Genoma/métodosRESUMO
The plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) play a vital role in the response to drought stress. Here, we report a lipid-anchored NACsa TF in Medicago falcata MfNACsa is an essential regulator of plant tolerance to drought stress, resulting in the differential expression of genes involved in oxidation reduction and lipid transport and localization. MfNACsa is associated with membranes under unstressed conditions and, more specifically, is targeted to the plasma membrane through S-palmitoylation. However, a Cys26-to-Ser mutation or inhibition of S-palmitoylation results in MfNACsa retention in the endoplasmic reticulum/Golgi. Under drought stress, MfNACsa translocates to the nucleus through de-S-palmitoylation mediated by the thioesterase MtAPT1, as coexpression of APT1 results in the nuclear translocation of MfNACsa, whereas mutation of the catalytic site of APT1 results in colocalization with MfNACsa and membrane retention of MfNACsa. Specifically, the nuclear MfNACsa binds the glyoxalase I (MtGlyl) promoter under drought stress, resulting in drought tolerance by maintaining the glutathione pool in a reduced state, and the process is dependent on the APT1-NACsa regulatory module. Our findings reveal a novel mechanism for the nuclear translocation of an S-palmitoylated NAC in response to stress.
Assuntos
Núcleo Celular/metabolismo , Lactoilglutationa Liase/metabolismo , Medicago/fisiologia , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Membrana Celular/metabolismo , Cisteína/metabolismo , Desidratação , Secas , Regulação da Expressão Gênica de Plantas , Glutationa/metabolismo , Metabolismo dos Lipídeos , Lipídeos/química , Lipoilação , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Transporte Proteico , Fatores de Transcrição/genéticaRESUMO
Cold acclimation is an important process by which plants respond to low temperature and enhance their winter hardiness. C-REPEAT BINDING FACTOR1 (CBF1), CBF2, and CBF3 genes were shown previously to participate in cold acclimation in Medicago truncatula In addition, MtCBF4 is transcriptionally induced by salt, drought, and cold stresses. We show here that MtCBF4, shown previously to enhance drought and salt tolerance, also positively regulates cold acclimation and freezing tolerance. To identify molecular factors acting upstream and downstream of the MtCBF4 transcription factor (TF) in cold responses, we first identified genes that are differentially regulated upon MtCBF4 overexpression using RNAseq Digital Gene Expression Profiling. Among these, we showed that MtCBF4 directly activates the transcription of the COLD ACCLIMATION SPECIFIC15 (MtCAS15) gene. To gain insights into how MtCBF4 is transcriptionally regulated in response to cold, an R2R3-MYB TF, MtMYB3, was identified based on a yeast one-hybrid screen as binding directly to MYB cis-elements in the MtCBF4 promoter, leading to the inhibition of MtCBF4 expression. In addition, another MYB TF, MtMYB61, identified as an interactor of MtMYB3, can relieve the inhibitory effect of MtMYB3 on MtCBF4 transcription. This study, therefore, supports a model describing how MtCBF4 is regulated by antagonistic MtMYB3/MtMYB61 TFs, leading to the up-regulation of downstream targets such as MtCAS15 acting in cold acclimation in M. truncatula.
Assuntos
Aclimatação/genética , Congelamento , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Sequência de Bases , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Medicago truncatula/genética , Medicago truncatula/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/classificação , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Benzene is known to be a common toxic industrial chemical, and prolonged benzene exposure may cause nervous system damage. At present, there were few studies on benzene-induced neurological damage. This research aimed to identify the protein biomarkers to explore the mechanism of nervous system damage caused by benzene. We established a benzene poisoning model of C57 mice by gavage of benzene-peanut oil suspension and identified differentially expressed proteins (DEPs) in brain tissue using tandem mass tag (TMT) proteomics. The results showed a significant weight loss and decrease in leukocyte and neutrophil counts in benzene poisoning mice compared to the control group. We also observed local cerebral oedema and small vessel occlusion in the cerebral white matter of benzene poisoning mice. TMT proteomic results showed that a total 6,985 proteins were quantified, with a fold change (FC) > 1.2 (or < 1/1.2) and P value <0.05 were considered as DEPs. Compared with the control group, we identified 43 DEPs, comprising 14 upregulated and 29 downregulated proteins. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis results showed that the candidate proteins were mainly involved in cholesterol metabolism, complement and coagulation cascades, african trypanosomiasis, PPAR signaling pathway, and vitamin digestion and absorption. Three proteins, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (UGT8), Apolipoprotein A-I (APOA1) and Complement C3 (C3) were validated using immunoblotting and immunohistochemical. In conclusion, our study preliminarily investigated the mechanism of benzene toxicity to the nervous system by analyzing DEPs changes in the brain.
RESUMO
Canola is the largest self-produced vegetable oil source in China, although excessive levels of cadmium, lead, and arsenic seriously affect its yield. Therefore, developing methods to identify canola materials with good heavy metal tolerance is a hot topic for canola breeding. In this study, canola near-isogenic lines with different oil contents (F338 (40.62%) and F335 (46.68%) as the control) and heavy metal tolerances were used as raw materials. In an experiment with 100 times the safe standard values, the superoxide dismutase (SOD) and peroxidase (POD) activities of F335 were 32.02 mmol/mg and 71.84 mmol/mg, while the activities of F338 were 24.85 mmol/mg and 63.86 mmol/mg, exhibiting significant differences. The DEGs and DAPs in the MAPK signaling pathway of the plant hormone signal transduction pathway and other related pathways were analyzed and verified using RT-qPCR. SAUR36 and SAUR32 were identified as the key differential genes. The expression of the SAUR36 gene in canola materials planted in the experimental field was significantly higher than in the control, and FY958 exhibited the largest difference (27.82 times). In this study, SOD and SAUR36 were found to be closely related to heavy metal stress tolerance. Therefore, they may be used to screen for new canola materials with good heavy metal stress tolerance for canola breeding.
RESUMO
In animal cells, the Golgi apparatus serves as the central hub of the endomembrane secretory pathway. It is responsible for the processing, modification, and sorting of proteins and lipids. The unique stacking and ribbon-like architecture of the Golgi apparatus forms the foundation for its precise functionality. Under cellular stress or pathological conditions, the structure of the Golgi and its important glycosylation modification function may change. It is crucial to employ suitable methodologies to study the structure and function of the Golgi apparatus, particularly when assessing the involvement of a target protein in Golgi regulation. This article provides a comprehensive overview of the diverse microscopy techniques used to determine the specific location of the target protein within the Golgi apparatus. Additionally, it outlines methods for assessing changes in the Golgi structure and its glycosylation modification function following the knockout of the target gene.
RESUMO
As the central hub of the secretory pathway, the Golgi apparatus plays a crucial role in maintaining cellular homeostasis in response to stresses. Recent studies have revealed the involvement of the Golgi tether, GORASP2, in facilitating autophagosome-lysosome fusion by connecting LC3-II and LAMP2 during nutrient starvation. However, the precise mechanism remains elusive. In this study, utilizing super-resolution microscopy, we observed GORASP2 localization on the surface of autophagosomes during glucose starvation. Depletion of GORASP2 hindered phagophore closure by regulating the association between VPS4A and the ESCRT-III component, CHMP2A. Furthermore, we found that GORASP2 controls RAB7A activity by modulating its GEF complex, MON1A-CCZ1, thereby impacting RAB7A's interaction with the HOPS complex. The assembly of both STX17-SNAP29-VAMP8 and YKT6-SNAP29-STX7 SNARE complexes was also attenuated without GORASP2. These findings suggest that GORASP2 helps seal autophagosomes and activate the RAB7A-HOPS-SNAREs membrane fusion machinery for autophagosome maturation, highlighting its membrane tethering function in response to stresses.Abbreviations: BafA1: bafilomycin A1; ESCRT: endosomal sorting complex required for transport; FPP: fluorescence protease protection; GEF: guanine nucleotide exchange factor; GFP: green fluorescent protein; GORASP2: golgi reassembly stacking protein 2; GSB: glucose starvation along with bafilomycin A1; HOPS: homotypic fusion and protein sorting; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; PBS: phosphate-buffered saline; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; PK: proteinase K; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SIM: structured illumination microscopy; UVRAG: UV radiation resistance associated.