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1.
Methods Mol Biol ; 2554: 21-34, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36178618

RESUMO

Chemical genetics takes advantage of small molecule-protein interactions to explore various biological processes. Although an attractive alternative to classical genetics in plants, the identification of small-molecule targets remains a challenge and limits the broad use of the compounds. The cellular thermal shift assay (CETSA), based on the principle that binding of small molecules could affect the thermal stability of proteins, has been applied for target validation in plant cells. Combined with high-resolution mass spectrometry, CETSA can detect small-molecule targets by monitoring the changes in the protein thermal stability caused by the interactions with small molecules at the proteome level. Here we describe the small-molecule target validation as well as the target identification with mass spectrometry by means of CETSA.


Assuntos
Arabidopsis , Proteoma , Arabidopsis/metabolismo , Espectrometria de Massas , Estabilidade Proteica , Proteoma/metabolismo
2.
J Hazard Mater ; 442: 130092, 2023 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-36303345

RESUMO

In this study, we describe the results obtained in a study of the transgenerational phenotypic effects of chromium (Cr) stress on the model plant species Arabidopsis thaliana. The F1 generation derived from parents grown under chronic and medium chronic stress showed significantly higher levels of the maximal effective concentration (EC50) compared with F1 plants generated from unstressed parents. Moreover, F1 plants from Cr-stressed parents showed a higher germination rate when grown in the presence of Cr. F1 plants derived from parents cultivated under chronic Cr stress displayed reduced hydrogen peroxide levels under Cr stress compared to controls. At lower Cr stress levels, F1 plants were observed to activate promptly more genes involved in Cr stress responses than F0 plants, implying a memory effect linked to transgenerational priming. At higher Cr levels, and at later stages, F1 plants modulated significantly fewer genes than F0 plants, implying a memory effect leading to Cr stress adaptation. Several bHLH transcription factors were induced by Cr stress in F1 but not in F0 plants, including bHLH100, ORG2 and ORG3. F1 plants optimized gene expression towards pathways linked to iron starvation response. A model of the transcriptional regulation of transgenerational memory to Cr stress is presented here, and could be applied for other heavy metal stresses.


Assuntos
Arabidopsis , Metais Pesados , Arabidopsis/metabolismo , Cromo/toxicidade , Cromo/metabolismo , Adaptação Fisiológica , Peróxido de Hidrogênio/metabolismo , Metais Pesados/metabolismo
3.
Semin Cell Dev Biol ; 134: 90-102, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-35317961

RESUMO

Brown algae are a group of multicellular, heterokont algae that have convergently evolved developmental complexity that rivals that of embryophytes, animals or fungi. Early in development, brown algal zygotes establish a basal and an apical pole, which will become respectively the basal system (holdfast) and the apical system (thallus) of the adult alga. Brown algae are interesting models for understanding the establishment of cell polarity in a broad evolutionary context, because they exhibit a large diversity of life cycles, reproductive strategies and, importantly, their zygotes are produced in large quantities free of parental tissue, with symmetry breaking and asymmetric division taking place in a highly synchronous manner. This review describes the current knowledge about the establishment of the apical-basal axis in the model brown seaweeds Ectocarpus, Dictyota, Fucus and Saccharina, highlighting the advantages and specific interests of each system. Ectocarpus is a genetic model system that allows access to the molecular basis of early development and life-cycle control over apical-basal polarity. The oogamous brown alga Fucus, together with emerging comparative models Dictyota and Saccharina, emphasize the diversity of strategies of symmetry breaking in determining a cell polarity vector in brown algae. A comparison with symmetry-breaking mechanisms in land plants, animals and fungi, reveals that the one-step zygote polarisation of Fucus compares well to Saccharomyces budding and Arabidopsis stomata development, while the two-phased symmetry breaking in the Dictyota zygote compares to Schizosaccharomyces fission, the Caenorhabditis anterior-posterior zygote polarisation and Arabidopsis prolate pollen polarisation. The apical-basal patterning in Saccharina zygotes on the other hand, may be seen as analogous to that of land plants. Overall, brown algae have the potential to bring exciting new information on how a single cell gives rise to an entire complex body plan.


Assuntos
Arabidopsis , Feófitas , Animais , Zigoto , Feófitas/genética , Feófitas/metabolismo , Polaridade Celular , Divisão Celular , Plantas
4.
Chemosphere ; 310: 136881, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36257391

RESUMO

Pollution of the environment by heavy metals (HMs) has recently become a global issue, affecting the health of all living organisms. Continuous human activities (industrialization and urbanization) are the major causes of HM release into the environment. Over the years, two methods (physical and chemical) have been widely used to reduce HMs in polluted environment. However, these two methods are inefficient and very expensive to reduce the HMs released into the atmosphere. Alternatively, researchers are trying to remove the HMs by employing hyper-accumulator plants. This method, referred to phytoremediation, is highly efficient, cost-effective, and eco-friendly. Phytoremediation can be divided into five types: phytostabilization, phytodegradation, rhizofiltration, phytoextraction, and phytovolatilization, all of which contribute to HMs removal from the polluted environment. Brassicaceae family members (particularly Arabidopsis thaliana) can accumulate more HMs from the contaminated environment than those of other plants. This comprehensive review focuses on how HMs pollute the environment and discusses the phytoremediation measures required to reduce the impact of HMs on the environment. We discuss the role of metal transporters in phytoremediation with a focus on Arabidopsis. Then draw insights into the role of genome editing tools in enhancing phytoremediation efficiency. This review is expected to initiate further research to improve phytoremediation by biotechnological approaches to conserve the environment from pollution.


Assuntos
Arabidopsis , Metais Pesados , Poluentes do Solo , Arabidopsis/metabolismo , Biodegradação Ambiental , Proteínas de Membrana Transportadoras/metabolismo , Metais Pesados/análise , Plantas/metabolismo , Solo/química , Poluentes do Solo/análise
5.
J Hazard Mater ; 443(Pt A): 130141, 2023 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-36241498

RESUMO

Phosphine (PH3) is an ideal fumigant alternative on methyl bromide (MB) as MB has been classified as an ozone-depleting substance. However, several challenges limit its efficient use in crop production, including the emergence of PH3-resistant insect pests and the incidence of phytotoxic effects on nursery plants. Therefore, this study aims to elucidate the mechanism underlying PH3 phytotoxicity in plants using transcriptomic techniques. Fumigation with 2 g/m3 PH3 induced phytotoxic effects in A. thaliana, as evidenced by a decrease in growth and vegetation indices compared to the control group. Transcriptomic analysis revealed that PH3 fumigation phytotoxicity responses in A. thaliana involve genes related to hypoxia stress and energy metabolism. Additionally, pretreatment with ethylene induced pre-adaptation to hypoxia under light conditions during fumigation effectively suppressed the phytotoxic effects of PH3 in A. thaliana by increasing the expression of hypoxia-adaptive genes. Moreover, the phytotoxicity of PH3 was also confirmed in pumpkin (Cucurbita moschata Duch.), and was dependent on light. Overall, our findings showed that fumigation under light conditions and ethylene pretreatment could be used to minimize PH3-induced phytotoxic effects in plants.


Assuntos
Arabidopsis , Cucurbita , Fosfinas , Arabidopsis/genética , Transcriptoma , Fosfinas/toxicidade , Hipóxia , Metabolismo Energético , Etilenos
6.
Methods Mol Biol ; 2594: 1-12, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36264484

RESUMO

The TARGET system allows for the rapid identification of direct regulated gene targets of transcription factors (TFs). It employs the transient transformation of plant protoplasts with inducible nuclear entry of the TF and subsequent transcriptomic and/or ChIP-seq analysis. The ability to separate direct TF-target gene regulatory interactions from indirect downstream responses and the significantly shorter amount of time required to perform the assay, compared to the generation of transgenics, make this plant cell-based approach a valuable tool for a higher throughput approach to identify the genome-wide targets of multiple TFs, to build validated transcriptional networks in plants. Here, we describe the use of the TARGET system in Arabidopsis seedling root protoplasts to map the gene regulatory network downstream of transcription factors-of-interest.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fatores de Transcrição/genética , Células Vegetais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Redes Reguladoras de Genes
7.
Methods Mol Biol ; 2594: 29-43, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36264486

RESUMO

Functional cis-regulatory elements (CREs) act as precise transcriptional switches for fine-tuning gene transcription. Identification of CREs is critical for understanding regulatory mechanisms of gene expression associated with various biological processes in eukaryotes. It is well known that CREs reside in open chromatin that exhibits hypersensitivity to enzyme cleavage and physical shearing. Currently, high-throughput methodologies, such as DNase-seq, ATAC-seq, and FAIRE-seq, have been widely applied in mapping open chromatin in various eukaryotic genomes. More recently, differential MNase (micrococcal nuclease) treatment has been successfully employed to map open chromatin in addition to profiling nucleosome landscape in both mammalian and plant species. We have developed a MNase hypersensitivity sequencing (MH-seq) technique in plants. The MH-seq procedure includes plant nuclei fixation and purification, differential treatments of purified nuclei with MNase, specific recovery of MNase-trimmed small DNA fragments within 20~100 bp in length, and MH-seq library construction followed by Illumina sequencing and data analysis. MH-seq has been successfully applied for global identification of open chromatin in both Arabidopsis thaliana and maize. It has been proven to be an attractive alternative for profiling open chromatin. Thus, MH-seq is expected to be valuable in probing chromatin accessibility on a genome-wide scale for other plants with sequenced genomes. Moreover, MHS data allow to implement footprinting assays to unveil binding sites of transcription factors.


Assuntos
Arabidopsis , Cromatina , Animais , Cromatina/genética , Nucleossomos , Nuclease do Micrococo/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , DNA/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Plantas/genética , Fatores de Transcrição/metabolismo , Mamíferos/genética
8.
Food Chem ; 404(Pt B): 134657, 2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-36327512

RESUMO

Both cytokinin and NAC transcription factors were reported to involve in leaf senescence. However, the mechanism of NAC transcription factors how to regulate cytokinin-delayed leaf senescence is still unknown. In this study, application of N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU), a cytokinin analogue, significantly delayed leaf senescence and maintained cytokinin content of Chinese flowering cabbage during storage. Meanwhile, the expression of an NAC transcriptional activator (BrNAC029) was increased but suppressed by CPPU treatment. Furthermore, BrNAC029 activated the expressions of chlorophyll catabolic genes BrPAO and BrSGR2, cytokinin oxidase gene BrCKX1 and senescence maker gene BrSAG113 by binding to their promoters. Additionally, overexpressions of BrNAC029 in tobacco and Arabidopsis accelerated leaf senescence and up-expressed the related genes. Taken together, it was suggested that BrNAC029 may serve as a transcriptional activator to activate the transcriptions of these related genes to eventually accelerate leaf senescence of Chinese flowering cabbage by promoting chlorophyll degradation and reducing endogenous cytokinin level.


Assuntos
Arabidopsis , Brassica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica de Plantas , Citocininas , Senescência Vegetal , Folhas de Planta/metabolismo , Brassica/genética , Brassica/metabolismo , Clorofila/metabolismo , Arabidopsis/metabolismo , China , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
9.
Gene ; 851: 146931, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36244548

RESUMO

APETALA1 (AP1), CAULIFLOWER (CAL) and FRUITFULL (FUL) were homologous genes with redundant functions in the process of flower transformation and floral development in Arabidopsis. Two CALs genes, MiCAL1 and MiCAL2, were cloned from mango (Mangifera indica L.). Their full-length sequences contained 717 bp and 714 bp, encoding 239 and 238 amino acids, respectively. Both the MiCAL1 and MiCAL2 proteins contained typical MADS-box and K-box domains and therefore belonged to the CAL-like protein family. MiCAL1 and MiCAL2 were expressed in all tissues at the inflorescence elongation stage and flowering stage, with the highest expression in the leaves at the flowering stage. They had similar expression patterns during flower development, with the highest expression levels in leaves during flower differentiation and the lowest expression levels during fruit development. Overexpression of MiCAL1 and MiCAL2 resulted in significantly earlier flowering in Arabidopsis. Overexpression of MiCAL1 resulted in terminal flowers with normal flower organs, while overexpression of MiCAL2 induced partially variation in floral organs but had no effect on inflorescences. Yeast two-hybrid (Y2H) experiments showed that MiCAL1 and MiCAL2 can interact with several flower-related proteins as well as stress response proteins, such as SEP1, SVP1, SVP2, SOC1G and Di19-4. These results suggest that these two MiCALs genes may have an important influence on mango flowering.


Assuntos
Arabidopsis , Brassica , Mangifera , Arabidopsis/metabolismo , Mangifera/genética , Mangifera/metabolismo , Regulação da Expressão Gênica de Plantas , Expressão Ectópica do Gene , Brassica/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Flores , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas de Domínio MADS/genética
10.
Life Sci Alliance ; 6(1)2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36265897

RESUMO

The coordination of cell division with stress response is essential for maintaining genome stability in plant meristems. Proteins involved in pre-mRNA splicing are important for these processes in animal and human cells. Based on its homology to the splicing factor SART1, which is implicated in the control of cell division and genome stability in human cells, we analyzed if MDF has similar functions in plants. We found that MDF associates with U4/U6.U5 tri-snRNP proteins and is essential for correct splicing of 2,037 transcripts. Loss of MDF function leads to cell division defects and cell death in meristems and was associated with up-regulation of stress-induced genes and down-regulation of mitotic regulators. In addition, the mdf-1 mutant is hypersensitive to DNA damage treatment supporting its role in coordinating stress response with cell division. Our analysis of a dephosphomutant of MDF suggested how its protein activity might be controlled. Our work uncovers the conserved function of a plant splicing factor and provides novel insight into the interplay of pre-mRNA processing and genome stability in plants.


Assuntos
Arabidopsis , Ribonucleoproteína Nuclear Pequena U5 , Humanos , Animais , Fatores de Processamento de RNA/genética , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Divisão Celular/genética , Instabilidade Genômica
11.
Methods Mol Biol ; 2581: 83-92, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36413312

RESUMO

SUMO conjugation is a conserved process of eukaryotes, and essential in metazoa. Similar to ubiquitylation, a SUMO-activating enzyme links to the SUMO carboxyl-terminal Gly in a thioester bond, and a SUMO-conjugating enzyme accepts activated SUMO and can transfer it to substrates. Unlike ubiquitylation, this transfer can also occur, in an unspecified number of cases, in the absence of ligase-like enzymes. Different isoforms of SUMO are present in eukaryotic genomes. Saccharomyces cerevisiae has only one SUMO protein, humans have four, and Arabidopsis thaliana has eight, the main isoforms being SUMO1 and SUMO2 with about 95% identity. Functionally similar to human SUMO2 and SUMO3, Arabidopsis SUMO1 and 2 can be transferred to substrates as single moieties, but can also form SUMO chains, a process enhanced by chain-forming ligases. By combined action with SUMO chain recognizing ubiquitin ligases, chains can channel substrates into the ubiquitin-dependent degradation pathway.A method is described to sumoylate substrates and to generate SUMO chains, using plant enzymes produced in E. coli. In vitro SUMO chain formation may serve for further analysis of SUMO chain functions. It can also provide an easy-to-synthesize substrate for SUMO-specific proteases.


Assuntos
Arabidopsis , Sumoilação , Humanos , Escherichia coli/metabolismo , Arabidopsis/metabolismo , Ligases/metabolismo , Isoformas de Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismo
12.
Methods Mol Biol ; 2581: 93-108, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36413313

RESUMO

Plant SUMO conjugation is an essential posttranslational modification involved in plant development and responses to environmental stress. Most likely, this biological diversification is supported by a functional specialization of the different isoforms of the SUMO conjugation machinery. For instance, the two essential Arabidopsis SUMO isoforms, SUMO1/2, display higher conjugation rate than SUMO3 and 5, which are not essential, linking their specific biochemical properties to their biological role. To study the biochemical properties of plant SUMO conjugation systems, quantitative biochemical assays must be performed. We will present a detailed protocol for reconstituting an in vitro SUMO conjugation assay covering all steps from protein preparation to assay development.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Sumoilação , Cinética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Isoformas de Proteínas/metabolismo
13.
Methods Mol Biol ; 2581: 69-79, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36413311

RESUMO

Deubiquitylating enzymes, or DUBs, are important regulators of ubiquitin homeostasis and substrate stability, though the molecular mechanisms of most of the DUBs in plants are not yet understood. As different ubiquitin chain types are implicated in different biological pathways, it is important to analyze the enzyme characteristic for studying a DUB. Quantitative analysis of DUB activity is also important to determine enzyme kinetics and the influence of DUB binding proteins on the enzyme activity. Here we show methods to analyze DUB activity using immunodetection, Coomassie brilliant blue staining, and fluorescence measurement that can be useful for understanding the basic characteristic of DUBs.


Assuntos
Arabidopsis , Enzimas Desubiquitinantes , Enzimas Desubiquitinantes/metabolismo , Arabidopsis/metabolismo , Ubiquitina/metabolismo
14.
Methods Mol Biol ; 2581: 179-199, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36413318

RESUMO

Protein stability influences many aspects of biology, and measuring their stability in vivo can provide important insights into biological systems.This chapter describes in detail two methods to assess the stability of a specific protein based on its transient expression in Arabidopsis protoplasts. First, a pulse-chase assay based on radioactive metabolic labeling of cellular proteins, followed by immunoprecipitation of the protein of interest. The decrease in radioactive signal is monitored over time and can be used to determine the protein's half-life.Alternatively, we also present a nonradioactive assay based on the use of reporter proteins, whose ratio can be quantified. This assay can be used to determine the relative stability of a protein of interest under specific conditions.


Assuntos
Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Protoplastos/metabolismo , Estabilidade Proteica , Imunoprecipitação , Proteínas/metabolismo
15.
Methods Mol Biol ; 2581: 255-265, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36413323

RESUMO

Protein phosphorylation is one of the most widely studied posttranslational modifications, and its role in signal transduction has gained particular attention. The relatively low abundance of the phosphorylated form of proteins makes identification by mass spectrometry challenging in the absence of selective enrichment. Titanium oxide-based enrichment of phosphopeptides in the presence of acidic modifiers is highly selective and makes it technically possible to detect thousands of phosphopeptides in a single sample by liquid chromatography-mass spectrometry. In this chapter, we describe a detailed protocol for the selective enrichment of microsomal and cytosolic phosphopeptides from Arabidopsis seedlings. The resulting phosphopeptide fractions enable routine identification of several thousands of phosphopeptide spectra per sample by mass spectrometry.


Assuntos
Arabidopsis , Plântula , Fosfopeptídeos , Cromatografia de Afinidade
16.
Methods Mol Biol ; 2581: 13-29, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36413307

RESUMO

The posttranslational attachment of the small protein modifier ubiquitin (Ub) is best known for its function in targeting proteins for degradation by the proteasome. However, ubiquitination also serves as a signal determining protein localization, activity, and interaction. Ubiquitination requires the sequential activity of E1 ubiquitin-activating enzyme (UBA), E2 ubiquitin-conjugating enzyme (UBC), and E3 ubiquitin ligase. Recognition of a target protein by an Ub-E2-E3 complex can result in its mono-ubiquitination (attachment of a single Ub moiety) or poly-ubiquitination, i.e., attachment of Ub chains. While the E3 ligase is important for the reaction specificity, the E2s catalyze the attachment of Ub to the target and to Ub itself to generate chains. In Arabidopsis thaliana, there are two E1s, 37 UBCs (and two ubiquitin-like conjugating enzymes) and more than 1400 E3 ligases, working in a combinatorial way. Therefore, in order to understand E3 ligase function, it is important to frame it within its possible E2s interactors. In this chapter, we propose a two-step identification and characterization of physiological E2-E3 pairs. In a first step, in vivo interacting E2s are identified through bimolecular fluorescence complementation (BiFC) using transient expression in Arabidopsis protoplast. In the second step, the activity of E2-E3 pairs is analyzed by a synthetic biology approach in which autoubiquitination is reconstituted in bacteria.


Assuntos
Arabidopsis , Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo
17.
Methods Mol Biol ; 2581: 267-284, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36413324

RESUMO

Due to their endosymbiotic origin, chloroplasts harbor several subcompartments and membrane systems. Each of these has a different protein and lipid composition that dynamically changes either naturally during plant development or induced by environmental stimuli. Here, we describe a protocol for chloroplast envelope membrane subfractionation via discontinuous sucrose gradients, which offers the possibility to separate the different plastid subcompartments for several downstream applications. It is a strong tool for protein sublocalization studies as well as for tracking dynamic movement patterns. Furthermore, it can be combined with in vitro import studies of radioactively labeled proteins, which allows sublocalization of putative envelope proteins independent of the availability of specific antisera.


Assuntos
Arabidopsis , Ervilhas , Cloroplastos , Membranas , Plastídeos
18.
Methods Mol Biol ; 2581: 31-42, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36413308

RESUMO

CRL (Cullin-Ring ubiquitin ligases) are the major class of plant E3 ubiquitin ligases. Immunoprecipitation-based methods are useful techniques for revealing interactions among Cullin-Ring Ligase (CRL) subunits or between CRLs and other proteins, as well as for detecting poly-ubiquitin modifications of the CRLs themselves. Here, we describe two immunoprecipitation (IP) procedures suitable for CRLs in Arabidopsis: (1) a procedure for IP analysis of CRL subunits and their interactors and a second procedure for in vivo ubiquitination analysis of the CRLs. Both protocols can be divided into two major steps: (1) preparation of cell extracts without disruption of protein interactions and (2) affinity purification of the protein complexes and subsequent detection. We provide a thorough description of all the steps, as well as advice on how to choose proper buffers for these analyses. We also suggest a series of negative controls that can be used to verify the specificity of the procedure.


Assuntos
Arabidopsis , Arabidopsis/metabolismo , Plântula/metabolismo , Proteínas Culina/metabolismo , Ubiquitina/metabolismo , Imunoprecipitação
19.
Methods Mol Biol ; 2581: 323-335, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36413328

RESUMO

Many peptide hormones and growth factors in plants, particularly the small posttranslationally modified signaling peptides, are synthesized as larger precursor proteins. Proteolytic processing is thus required for peptide maturation, and additional posttranslational modifications may contribute to bioactivity. To what extent these posttranslational modifications impact on processing is largely unknown. Likewise, it is poorly understood how the cleavage sites within peptide precursors are selected by specific processing proteases, and whether or not posttranslational modifications contribute to cleavage site recognition. Here, we describe a mass spectrometry-based approach to address these questions. We developed a method using heavy isotope labeling to directly compare cleavage efficiency of different precursor-derived synthetic peptides by mass spectrometry. Thereby, we can analyze the effect of posttranslational modifications on processing and the specific sequence requirements of the processing proteases. As an example, we describe how this method has been used to assess the relevance of tyrosine sulfation for the processing of the Arabidopsis CIF4 precursor by the subtilase SBT5.4.


Assuntos
Arabidopsis , Hormônios Peptídicos , Hormônios Peptídicos/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Arabidopsis/metabolismo , Isótopos/metabolismo , Peptídeo Hidrolases/metabolismo
20.
Methods Mol Biol ; 2581: 367-383, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36413331

RESUMO

ABSTACT: Plant sumoylation research has seen significant advances in recent years, particularly since high-throughput proteomic strategies have enabled the discovery of more than one thousand SUMO targets. In the present chapter, we update the previously reported SUMO (small ubiquitin-related modifier) gene network (SGN) to its v4 iteration. SGN is a curated assembly of Arabidopsis thaliana genes that have been functionally associated with sumoylation, from SUMO pathway components to targets and interactors. The enclosed tutorial helps interpret and manage these datasets and details bioinformatic tools that can be used for in silico-based hypothesis generation. The latter include tools for sumoylation site prediction, comparative genomics, and gene network analysis.


Assuntos
Arabidopsis , Redes Reguladoras de Genes , Biologia Computacional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteômica , Arabidopsis/genética , Arabidopsis/metabolismo
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