Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 56
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Comput Struct Biotechnol J ; 19: 5117-5125, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34589187

RESUMO

Co-fractionation mass spectrometry (CF-MS)-based approaches enable cell-wide identification of protein-protein and protein-metabolite complexes present in the cellular lysate. CF-MS combines biochemical separation of molecular complexes with an untargeted mass-spectrometry-based proteomics and/or metabolomics analysis of the obtained fractions, and is used to delineate putative interactors. CF-MS data are a treasure trove for biological discovery. To facilitate analysis and visualization of original or publically available CF-MS datasets, we designed PROMISed, a user-friendly tool available online via https://myshiny.mpimp-golm.mpg.de/PDP1/ or as a repository via https://github.com/DennisSchlossarek/PROMISed. Specifically, starting with raw fractionation profiles, PROMISed (i) contains activities for data pre-processing and normalization, (ii) deconvolutes complex fractionation profiles into single, distinct peaks, (iii) identifies co-eluting protein-protein or protein-metabolite pairs using user-defined correlation methods, and (iv) performs co-fractionation network analysis. Given multiple CF-MS datasets, for instance representing different environmental condition, PROMISed allows to select for proteins and metabolites that differ in their elution profile, which may indicate change in the interaction status. But it also enables the identification of protein-protein and protein-metabolite pairs that co-elute together across multiple datasets. PROMISed enables users to (i) easily adjust parameters at each step of the analysis, (ii) download partial and final results, and (iii) select among different data-visualization options. PROMISed renders CF-MS data accessible to a broad scientific audience, allowing users with no computational or statistical background to look for novel protein-protein and protein-metabolite complexes for further experimental validation.

2.
Sci Rep ; 11(1): 18770, 2021 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-34548590

RESUMO

Mitochondrial malate dehydrogenase (MDH)-citrate synthase (CS) multi-enzyme complex is a part of the Krebs tricarboxylic acid (TCA) cycle 'metabolon' which is enzyme machinery catalyzing sequential reactions without diffusion of reaction intermediates into a bulk matrix. This complex is assumed to be a dynamic structure involved in the regulation of the cycle by enhancing metabolic flux. Microscale Thermophoresis analysis of the porcine heart MDH-CS complex revealed that substrates of the MDH and CS reactions, NAD+ and acetyl-CoA, enhance complex association while products of the reactions, NADH and citrate, weaken the affinity of the complex. Oxaloacetate enhanced the interaction only when it was present together with acetyl-CoA. Structural modeling using published CS structures suggested that the binding of these substrates can stabilize the closed format of CS which favors the MDH-CS association. Two other TCA cycle intermediates, ATP, and low pH also enhanced the association of the complex. These results suggest that dynamic formation of the MDH-CS multi-enzyme complex is modulated by metabolic factors responding to respiratory metabolism, and it may function in the feedback regulation of the cycle and adjacent metabolic pathways.

3.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360938

RESUMO

During seed germination, desiccation tolerance is lost in the radicle with progressing radicle protrusion and seedling establishment. This process is accompanied by comprehensive changes in the metabolome and proteome. Germination of Arabidopsis seeds was investigated over 72 h with special focus on the heat-stable proteome including late embryogenesis abundant (LEA) proteins together with changes in primary metabolites. Six metabolites in dry seeds known to be important for seed longevity decreased during germination and seedling establishment, while all other metabolites increased simultaneously with activation of growth and development. Thermo-stable proteins were associated with a multitude of biological processes. In the heat-stable proteome, a relatively similar proportion of fully ordered and fully intrinsically disordered proteins (IDP) was discovered. Highly disordered proteins were found to be associated with functional categories development, protein, RNA and stress. As expected, the majority of LEA proteins decreased during germination and seedling establishment. However, four germination-specific dehydrins were identified, not present in dry seeds. A network analysis of proteins, metabolites and amino acids generated during the course of germination revealed a highly connected LEA protein network.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Germinação , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Plântula/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Temperatura Alta
4.
Nat Methods ; 18(7): 747-756, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34239102

RESUMO

Mass spectrometry-based metabolomics approaches can enable detection and quantification of many thousands of metabolite features simultaneously. However, compound identification and reliable quantification are greatly complicated owing to the chemical complexity and dynamic range of the metabolome. Simultaneous quantification of many metabolites within complex mixtures can additionally be complicated by ion suppression, fragmentation and the presence of isomers. Here we present guidelines covering sample preparation, replication and randomization, quantification, recovery and recombination, ion suppression and peak misidentification, as a means to enable high-quality reporting of liquid chromatography- and gas chromatography-mass spectrometry-based metabolomics-derived data.


Assuntos
Espectrometria de Massas/métodos , Metabolômica/métodos , Animais , Cromatografia Líquida , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Espectrometria de Massas/normas , Metabolômica/normas , Distribuição Aleatória , Manejo de Espécimes , Fluxo de Trabalho
5.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200446

RESUMO

Ribosome biogenesis is essential for plants to successfully acclimate to low temperature. Without dedicated steps supervising the 60S large subunits (LSUs) maturation in the cytosol, e.g., Rei-like (REIL) factors, plants fail to accumulate dry weight and fail to grow at suboptimal low temperatures. Around REIL, the final 60S cytosolic maturation steps include proofreading and assembly of functional ribosomal centers such as the polypeptide exit tunnel and the P-Stalk, respectively. In consequence, these ribosomal substructures and their assembly, especially during low temperatures, might be changed and provoke the need for dedicated quality controls. To test this, we blocked ribosome maturation during cold acclimation using two independent reil double mutant genotypes and tested changes in their ribosomal proteomes. Additionally, we normalized our mutant datasets using as a blank the cold responsiveness of a wild-type Arabidopsis genotype. This allowed us to neglect any reil-specific effects that may happen due to the presence or absence of the factor during LSU cytosolic maturation, thus allowing us to test for cold-induced changes that happen in the early nucleolar biogenesis. As a result, we report that cold acclimation triggers a reprogramming in the structural ribosomal proteome. The reprogramming alters the abundance of specific RP families and/or paralogs in non-translational LSU and translational polysome fractions, a phenomenon known as substoichiometry. Next, we tested whether the cold-substoichiometry was spatially confined to specific regions of the complex. In terms of RP proteoforms, we report that remodeling of ribosomes after a cold stimulus is significantly constrained to the polypeptide exit tunnel (PET), i.e., REIL factor binding and functional site. In terms of RP transcripts, cold acclimation induces changes in RP families or paralogs that are significantly constrained to the P-Stalk and the ribosomal head. The three modulated substructures represent possible targets of mechanisms that may constrain translation by controlled ribosome heterogeneity. We propose that non-random ribosome heterogeneity controlled by specialized biogenesis mechanisms may contribute to a preferential or ultimately even rigorous selection of transcripts needed for rapid proteome shifts and successful acclimation.


Assuntos
Aclimatação , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Temperatura Baixa , Proteoma/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteoma/análise , Proteínas Ribossômicas/genética , Ribossomos/genética
6.
EMBO J ; 40(15): e106800, 2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34156108

RESUMO

How organisms integrate metabolism with the external environment is a central question in biology. Here, we describe a novel regulatory small molecule, a proteogenic dipeptide Tyr-Asp, which improves plant tolerance to oxidative stress by directly interfering with glucose metabolism. Specifically, Tyr-Asp inhibits the activity of a key glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPC), and redirects glucose toward pentose phosphate pathway (PPP) and NADPH production. In line with the metabolic data, Tyr-Asp supplementation improved the growth performance of both Arabidopsis and tobacco seedlings subjected to oxidative stress conditions. Moreover, inhibition of Arabidopsis phosphoenolpyruvate carboxykinase (PEPCK) activity by a group of branched-chain amino acid-containing dipeptides, but not by Tyr-Asp, points to a multisite regulation of glycolytic/gluconeogenic pathway by dipeptides. In summary, our results open the intriguing possibility that proteogenic dipeptides act as evolutionarily conserved small-molecule regulators at the nexus of stress, protein degradation, and metabolism.

7.
Comput Struct Biotechnol J ; 19: 2170-2178, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34136091

RESUMO

Mining of metabolite-protein interaction networks facilitates the identification of design principles underlying the regulation of different cellular processes. However, identification and characterization of the regulatory role that metabolites play in interactions with proteins on a genome-scale level remains a pressing task. Based on availability of high-quality metabolite-protein interaction networks and genome-scale metabolic networks, here we propose a supervised machine learning approach, called CIRI that determines whether or not a metabolite is involved in a competitive inhibitory regulatory interaction with an enzyme. First, we show that CIRI outperforms the naive approach based on a structural similarity threshold for a putative competitive inhibitor and the substrates of a metabolic reaction. We also validate the performance of CIRI on several unseen data sets and databases of metabolite-protein interactions not used in the training, and demonstrate that the classifier can be effectively used to predict competitive inhibitory interactions. Finally, we show that CIRI can be employed to refine predictions about metabolite-protein interactions from a recently proposed PROMIS approach that employs metabolomics and proteomics profiles from size exclusion chromatography in E. coli to predict metabolite-protein interactions. Altogether, CIRI fills a gap in cataloguing metabolite-protein interactions and can be used in directing future machine learning efforts to categorize the regulatory type of these interactions.

8.
Nat Commun ; 12(1): 3426, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103516

RESUMO

Adaptive plasticity in stress responses is a key element of plant survival strategies. For instance, moderate heat stress (HS) primes a plant to acquire thermotolerance, which allows subsequent survival of more severe HS conditions. Acquired thermotolerance is actively maintained over several days (HS memory) and involves the sustained induction of memory-related genes. Here we show that FORGETTER3/ HEAT SHOCK TRANSCRIPTION FACTOR A3 (FGT3/HSFA3) is specifically required for physiological HS memory and maintaining high memory-gene expression during the days following a HS exposure. HSFA3 mediates HS memory by direct transcriptional activation of memory-related genes after return to normal growth temperatures. HSFA3 binds HSFA2, and in vivo both proteins form heteromeric complexes with additional HSFs. Our results indicate that only complexes containing both HSFA2 and HSFA3 efficiently promote transcriptional memory by positively influencing histone H3 lysine 4 (H3K4) hyper-methylation. In summary, our work defines the major HSF complex controlling transcriptional memory and elucidates the in vivo dynamics of HSF complexes during somatic stress memory.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição de Choque Térmico/metabolismo , Resposta ao Choque Térmico/genética , Complexos Multiproteicos/metabolismo , Transcrição Genética , Proteínas de Arabidopsis/genética , Epistasia Genética , Genes de Plantas , Loci Gênicos , Fatores de Transcrição de Choque Térmico/genética , Histonas/metabolismo , Cinética , Lisina/metabolismo , Metilação , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
9.
Patterns (N Y) ; 2(4): 100235, 2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33982025

RESUMO

The growth of plant organs is driven by cell division and subsequent cell expansion. The transition from proliferation to expansion is critical for the final organ size and plant yield. Exit from proliferation and onset of expansion is accompanied by major metabolic reprogramming, and in leaves with the establishment of photosynthesis. To learn more about the molecular mechanisms underlying the developmental and metabolic transitions important for plant growth, we used untargeted proteomics and metabolomics analyses to profile young leaves of a model plant Arabidopsis thaliana representing proliferation, transition, and expansion stages. The dataset presented represents a unique resource comprising approximately 4,000 proteins and 300 annotated small-molecular compounds measured across 6 consecutive days of leaf growth. These can now be mined for novel developmental and metabolic regulators of plant growth and can act as a blueprint for studies aimed at better defining the interface of development and metabolism in other species.

10.
Plant Physiol ; 2021 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-33742682

RESUMO

VC generated transgenic plants. XC, ET, JR, SM and MU performed western blot analyses. UA and SM performed co-immunoprecipitations. MU generated cTP-YFP fusions and localized fusions using confocal microscopy. SM performed BN-PAGE analysis. JE and IF performed MS analysis on BN slices. MU produced recombinant protein in E. coli and performed size exclusion chromatography together with ML and AS. ES and AS performed MS analysis of recombinant protein. SM, MU and ET performed Chl a fluorescence measurements during light fluctuations. MAS measured 77K Chl a fluorescence emission spectra, ECS kinetics and Cytf redox state. MAS and UA performed Chl a fluorescence and P700 light response curves and steady state measurements, respectively. TvB carried out simultaneous CO2 assimilation and Chl a fluorescence measurements. UA wrote manuscript with help from all authors.

11.
Front Plant Sci ; 12: 624365, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33613605

RESUMO

Recently, we published a set of tobacco lines expressing the Daucus carota (carrot) DcLCYB1 gene with accelerated development, increased carotenoid content, photosynthetic efficiency, and yield. Because of this development, DcLCYB1 expression might be of general interest in crop species as a strategy to accelerate development and increase biomass production under field conditions. However, to follow this path, a better understanding of the molecular basis of this phenotype is essential. Here, we combine OMICs (RNAseq, proteomics, and metabolomics) approaches to advance our understanding of the broader effect of LCYB expression on the tobacco transcriptome and metabolism. Upon DcLCYB1 expression, the tobacco transcriptome (~2,000 genes), proteome (~700 proteins), and metabolome (26 metabolites) showed a high number of changes in the genes involved in metabolic processes related to cell wall, lipids, glycolysis, and secondary metabolism. Gene and protein networks revealed clusters of interacting genes and proteins mainly involved in ribosome and RNA metabolism and translation. In addition, abiotic stress-related genes and proteins were mainly upregulated in the transgenic lines. This was well in line with an enhanced stress (high light, salt, and H2O2) tolerance response in all the transgenic lines compared with the wild type. Altogether, our results show an extended and coordinated response beyond the chloroplast (nucleus and cytosol) at the transcriptome, proteome, and metabolome levels, supporting enhanced plant growth under normal and stress conditions. This final evidence completes the set of benefits conferred by the expression of the DcLCYB1 gene, making it a very promising bioengineering tool to generate super crops.

12.
Commun Biol ; 4(1): 181, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568709

RESUMO

Protein-metabolite interactions are of crucial importance for all cellular processes but remain understudied. Here, we applied a biochemical approach named PROMIS, to address the complexity of the protein-small molecule interactome in the model yeast Saccharomyces cerevisiae. By doing so, we provide a unique dataset, which can be queried for interactions between 74 small molecules and 3982 proteins using a user-friendly interface available at https://promis.mpimp-golm.mpg.de/yeastpmi/ . By interpolating PROMIS with the list of predicted protein-metabolite interactions, we provided experimental validation for 225 binding events. Remarkably, of the 74 small molecules co-eluting with proteins, 36 were proteogenic dipeptides. Targeted analysis of a representative dipeptide, Ser-Leu, revealed numerous protein interactors comprising chaperones, proteasomal subunits, and metabolic enzymes. We could further demonstrate that Ser-Leu binding increases activity of a glycolytic enzyme phosphoglycerate kinase (Pgk1). Consistent with the binding analysis, Ser-Leu supplementation leads to the acute metabolic changes and delays timing of a diauxic shift. Supported by the dipeptide accumulation analysis our work attests to the role of Ser-Leu as a metabolic regulator at the interface of protein degradation and central metabolism.


Assuntos
Metabolismo Energético , Fosfoglicerato Quinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Glicólise , Metaboloma , Metabolômica , Fosfoglicerato Quinase/genética , Mapas de Interação de Proteínas , Proteólise , Proteoma , Proteômica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
13.
Sci Rep ; 11(1): 2410, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33510206

RESUMO

Arabidopsis REIL proteins are cytosolic ribosomal 60S-biogenesis factors. After shift to 10 °C, reil mutants deplete and slowly replenish non-translating eukaryotic ribosome complexes of root tissue, while controlling the balance of non-translating 40S- and 60S-subunits. Reil mutations respond by hyper-accumulation of non-translating subunits at steady-state temperature; after cold-shift, a KCl-sensitive 80S sub-fraction remains depleted. We infer that Arabidopsis may buffer fluctuating translation by pre-existing non-translating ribosomes before de novo synthesis meets temperature-induced demands. Reil1 reil2 double mutants accumulate 43S-preinitiation and pre-60S-maturation complexes and alter paralog composition of ribosomal proteins in non-translating complexes. With few exceptions, e.g. RPL3B and RPL24C, these changes are not under transcriptional control. Our study suggests requirement of de novo synthesis of eukaryotic ribosomes for long-term cold acclimation, feedback control of NUC2 and eIF3C2 transcription and links new proteins, AT1G03250, AT5G60530, to plant ribosome biogenesis. We propose that Arabidopsis requires biosynthesis of specialized ribosomes for cold acclimation.


Assuntos
Aclimatação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Temperatura Baixa , Regulação da Expressão Gênica de Plantas , Ribossomos/metabolismo , Citosol/metabolismo , Mutação , Especificidade de Órgãos , Desenvolvimento Vegetal/genética , Biossíntese de Proteínas , Proteômica/métodos , Subunidades Ribossômicas/metabolismo , Transcrição Genética
14.
FEBS J ; 288(1): 281-292, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32301545

RESUMO

Proteogenic dipeptides are intermediates of proteolysis as well as an emerging class of small-molecule regulators with diverse and often dipeptide-specific functions. Herein, prompted by differential accumulation of dipeptides in a high-density Arabidopsis thaliana time-course stress experiment, we decided to pursue an identity of the proteolytic pathway responsible for the buildup of dipeptides under heat conditions. By querying dipeptide accumulation versus available transcript data, autophagy emerged as a top hit. To examine whether autophagy indeed contributes to the accumulation of dipeptides measured in response to heat stress, we characterized the loss-of-function mutants of crucial autophagy proteins to test whether interfering with autophagy would affect dipeptide accumulation in response to the heat treatment. This was indeed the case. This work implicates the involvement of autophagy in the accumulation of proteogenic dipeptides in response to heat stress in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Relacionadas à Autofagia/genética , Dipeptídeos/genética , Regulação da Expressão Gênica de Plantas , Resposta ao Choque Térmico/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/biossíntese , Autofagia , Proteínas Relacionadas à Autofagia/biossíntese , Dipeptídeos/biossíntese , Luz , Mutação , Proteólise , Espécies Reativas de Oxigênio/metabolismo , Transcriptoma , Triglicerídeos/metabolismo
15.
Front Plant Sci ; 11: 595792, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33224174

RESUMO

Plants exhibit different physiological and molecular responses to adverse changes in their environment. One such molecular response is the sequestration of proteins, RNAs, and metabolites into cytoplasmic bodies called stress granules (cSGs). Here we report that, in addition to cSGs, heat stress also induces the formation of SG-like foci (cGs) in the chloroplasts of the model plant Arabidopsis thaliana. Similarly to the cSGs, (i) cpSG assemble rapidly in response to stress and disappear when the stress ceases, (ii) cpSG formation is inhibited by treatment with a translation inhibitor (lincomycin), and (iii) cpSG are composed of a stable core and a fluid outer shell. A previously published protocol for cSG extraction was successfully adapted to isolate cpSG, followed by protein, metabolite, and RNA analysis. Analogously to the cSGs, cpSG sequester proteins essential for SG formation, dynamics, and function, also including RNA-binding proteins with prion-like domain, ATPases and chaperones, and the amino acids proline and glutamic acid. However, the most intriguing observation relates to the cpSG localization of proteins, such as a complete magnesium chelatase complex, which is involved in photosynthetic acclimation to stress. These data suggest that cpSG have a role in plant stress tolerance.

16.
Autophagy ; : 1-16, 2020 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-32967551

RESUMO

In nature, plants are constantly exposed to many transient, but recurring, stresses. Thus, to complete their life cycles, plants require a dynamic balance between capacities to recover following cessation of stress and maintenance of stress memory. Recently, we uncovered a new functional role for macroautophagy/autophagy in regulating recovery from heat stress (HS) and resetting cellular memory of HS in Arabidopsis thaliana. Here, we demonstrated that NBR1 (next to BRCA1 gene 1) plays a crucial role as a receptor for selective autophagy during recovery from HS. Immunoblot analysis and confocal microscopy revealed that levels of the NBR1 protein, NBR1-labeled puncta, and NBR1 activity are all higher during the HS recovery phase than before. Co-immunoprecipitation analysis of proteins interacting with NBR1 and comparative proteomic analysis of an nbr1-null mutant and wild-type plants identified 58 proteins as potential novel targets of NBR1. Cellular, biochemical and functional genetic studies confirmed that NBR1 interacts with HSP90.1 (heat shock protein 90.1) and ROF1 (rotamase FKBP 1), a member of the FKBP family, and mediates their degradation by autophagy, which represses the response to HS by attenuating the expression of HSP genes regulated by the HSFA2 transcription factor. Accordingly, loss-of-function mutation of NBR1 resulted in a stronger HS memory phenotype. Together, our results provide new insights into the mechanistic principles by which autophagy regulates plant response to recurrent HS.Abbreviations: AIM: Atg8-interacting motif; ATG: autophagy-related; BiFC: bimolecular fluorescence complementation; ConA: concanamycinA; CoIP: co-immunoprecipitation; DMSO: dimethyl sulfoxide; FKBP: FK506-binding protein; FBPASE: fructose 1,6-bisphosphatase; GFP: green fluorescent protein; HS: heat stress; HSF: heat shock factor; HSFA2: heat shock factor A2; HSP: heat shock protein; HSP90: heat shock protein 90; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; 3-MA: 3-methyladenine; NBR1: next-to-BRCA1; PQC: protein quality control; RFP: red fluorescent protein; ROF1: rotamase FKBP1; TF: transcription factor; TUB: tubulin; UBA: ubiquitin-associated; YFP: yellow fluorescent protein.

17.
Curr Protoc Plant Biol ; 5(3): e20118, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32946676

RESUMO

Stress granules (SGs) are ubiquitous nonmembrane-bound assemblies of protein and mRNA formed under stress conditions associated with stalled translation. SGs are evolutionarily conserved across eukaryotes. The canonical function of SGs is to selectively protect mRNAs and proteins from unfolding and prevent degradation induced by diverse environmental stresses. Moreover, sequestration into SGs provides an elegant way to regulate protein activities. Disassembly of SGs upon stress recovery is accompanied by the reactivation of protein translation and protein activities. The regulatory importance of SGs has been corroborated by recent studies describing the multiomics analysis of the composition of SGs from yeast, animal, and plant cells. Herein, we describe an isolation protocol of SGs that allows for the identification of proteins, mRNA, and metabolites sequestered into SG cores. Furthermore, the described protocols can be used to isolate other SG-like foci. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of SG-enriched fraction from plant material Basic Protocol 2: Affinity purification to isolate SGs Basic Protocol 3: Simultaneous extraction of proteins and metabolites from affinity-purified beads Basic Protocol 4: Protein digestion on affinity-purified beads Basic Protocol 5: Data analysis.


Assuntos
Grânulos Citoplasmáticos , Estresse Fisiológico , Animais , RNA Mensageiro , Saccharomyces cerevisiae
18.
Int J Mol Sci ; 21(16)2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32781659

RESUMO

Lolium multiflorum/Festuca arundinacea introgression forms have been proved several times to be good models to identify key components of grass metabolism involved in the mechanisms of tolerance to water deficit. Here, for the first time, a relationship between photosynthetic and antioxidant capacities with respect to drought tolerance of these forms was analyzed in detail. Two closely related L. multiflorum/F. arundinacea introgression forms distinct in their ability to re-grow after cessation of prolonged water deficit in the field were selected and subjected to short-term drought in pots to dissect precisely mechanisms of drought tolerance in this group of plants. The studies revealed that the form with higher drought tolerance was characterized by earlier and higher accumulation of abscisic acid, more stable cellular membranes, and more balanced reactive oxygen species metabolism associated with a higher capacity of the antioxidant system under drought conditions. On the other hand, both introgression forms revealed the same levels of stomatal conductance, CO2 assimilation, and consequently, intrinsic water use efficiency under drought and recovery conditions. However, simultaneous higher adjustment of the Calvin cycle to water deficit and reduced CO2 availability, with respect to the accumulation and activity of plastid fructose-1,6-bisphosphate aldolase, were clearly visible in the form with higher drought tolerance.


Assuntos
Adaptação Fisiológica , Antioxidantes/metabolismo , Secas , Festuca/fisiologia , Lolium/fisiologia , Fotossíntese , Água , Festuca/genética , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Peroxidação de Lipídeos , Lolium/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estômatos de Plantas/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Solo/química , Superóxidos/metabolismo
19.
Plants (Basel) ; 9(7)2020 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-32674508

RESUMO

Conventional preparation methods of plant ribosomes fail to resolve non-translating chloroplast or cytoplasmic ribosome subunits from translating fractions. We established preparation of these ribosome complexes from Arabidopsis thaliana leaf, root, and seed tissues by optimized sucrose density gradient centrifugation of protease protected plant extracts. The method co-purified non-translating 30S and 40S ribosome subunits separated non-translating 50S from 60S subunits, and resolved assembled monosomes from low oligomeric polysomes. Combining ribosome fractionation with microfluidic rRNA analysis and proteomics, we characterized the rRNA and ribosomal protein (RP) composition. The identity of cytoplasmic and chloroplast ribosome complexes and the presence of ribosome biogenesis factors in the 60S-80S sedimentation interval were verified. In vivo cross-linking of leaf tissue stabilized ribosome biogenesis complexes, but induced polysome run-off. Omitting cross-linking, the established paired fractionation and proteome analysis monitored relative abundances of plant chloroplast and cytoplasmic ribosome fractions and enabled analysis of RP composition and ribosome associated proteins including transiently associated biogenesis factors.

20.
Trends Plant Sci ; 25(7): 627-630, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32349926

RESUMO

Unlike the situation for humans and microbes, the active multiprotein assemblies of plants have not been systematically defined. A recent report by McWhite et al. remedies this by analyzing the protein complexes of 13 plant species, thereby defining core assemblies and providing an essential resource for interpreting the genotype-phenotype space of plants.


Assuntos
Complexos Multiproteicos , Proteínas de Plantas , Humanos , Fenômenos Fisiológicos Vegetais , Proteínas de Plantas/genética , Plantas/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...