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1.
PLoS One ; 15(8): e0238319, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866174

RESUMO

Wood is a porous, hygroscopic material with engineering properties that depend significantly on the amount of water (moisture) in the material. Water in wood can be present in both cell walls and the porous void-structure of the material, but it is only water in cell walls that affects the engineering properties. An important characteristic of wood is therefore the capacity for water of its solid cell walls, i.e. the maximum cell wall moisture content. However, this quantity is not straight-forward to determine experimentally, and the measured value may depend on the experimental technique used. In this study, we used a triangulation approach to determine the maximum cell wall moisture content by using three experimental techniques based on different measurement principles: low-field nuclear magnetic resonance (LFNMR) relaxometry, differential scanning calorimetry (DSC), and the solute exclusion technique (SET). The LFNMR data were furthermore analysed by two varieties of exponential decay analysis. These techniques were used to determine the maximum cell wall moisture contents of nine different wood species, covering a wide range of densities. The results from statistical analysis showed that LFNMR yielded lower cell wall moisture contents than DSC and SET, which were fairly similar. Both of the latter methods include factors that could either under-estimate or over-estimate the measured cell wall moisture content. Because of this and the fact that the DSC and SET methods are based on different measurement principles, it is likely that they provide realistic values of the cell wall moisture content in the water-saturated state.


Assuntos
Parede Celular/metabolismo , Água/metabolismo , Madeira/metabolismo , Porosidade
2.
Nat Commun ; 11(1): 4522, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908144

RESUMO

A unique, protective cell envelope contributes to the broad drug resistance of the nosocomial pathogen Acinetobacter baumannii. Here we use transposon insertion sequencing to identify A. baumannii mutants displaying altered susceptibility to a panel of diverse antibiotics. By examining mutants with antibiotic susceptibility profiles that parallel mutations in characterized genes, we infer the function of multiple uncharacterized envelope proteins, some of which have roles in cell division or cell elongation. Remarkably, mutations affecting a predicted cell wall hydrolase lead to alterations in lipooligosaccharide synthesis. In addition, the analysis of altered susceptibility signatures and antibiotic-induced morphology patterns allows us to predict drug synergies; for example, certain beta-lactams appear to work cooperatively due to their preferential targeting of specific cell wall assembly machineries. Our results indicate that the pathogen may be effectively inhibited by the combined targeting of multiple pathways critical for envelope growth.


Assuntos
Infecções por Acinetobacter/tratamento farmacológico , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Infecção Hospitalar/tratamento farmacológico , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Parede Celular/metabolismo , Infecção Hospitalar/microbiologia , Análise Mutacional de DNA , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Testes de Sensibilidade Microbiana , Mutação
3.
Ecotoxicol Environ Saf ; 203: 111007, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32888586

RESUMO

Soil acidification is one of the crucial global environmental problems, affecting sustainable land use, crop yield, and ecosystem stability. Previous research reported the tolerance of crops to acid soil stress. However, the molecular response of woody plant to acid conditions remains largely unclear. Rhododendron L. is a widely distributed woody plant genus and prefers to grow in acidic soils. Herein, weighted gene coexpression network analysis was performed on R. protistum var. giganteum seedlings subjected to five pH treatments (3.5, 4.5, 5.5, 6.0, 7.0), and their ecophysiological characteristics were determined for the identification of their molecular responses to acidic environments. Through pairwise comparison, 855 differentially expressed genes (DEGs) associated with photosynthesis, cell wall, and phenylpropanoid metabolism were identified. Most of the DEGs related to photosynthesis and cell wall were up-regulated after pH 4.5 treatment. Results implied that the species improves its photosynthetic abilities and changes its cell wall characteristics to adapt to acidic conditions. Weighted gene co-expression network analyses showed that most of the hub genes were annotated to the biosynthetic pathways of ribosomal proteins and photosynthesis. Expression pattern analysis showed that genes encoding subunit ribosomal proteins decreased at pH 7.0 treatment, suggesting that pH 7.0 treatment led to cell injury in the seedlings. The species regulates protein synthesis in response to high pH stress (pH 7.0). The present study revealed the molecular response mechanism of woody plant R. protistum var. giganteum to acid environments. These findings can be useful in enriching current knowledge of how woody species adapt to soil acidification under global environmental changes.


Assuntos
Ácidos/farmacologia , Parede Celular/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , Madeira/efeitos dos fármacos , Parede Celular/genética , Parede Celular/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Fotossíntese/genética , Plântula/efeitos dos fármacos , Plântula/metabolismo , Solo/química , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Madeira/genética , Madeira/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(33): 20316-20324, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32737163

RESUMO

Xyloglucan (XyG) is an abundant component of the primary cell walls of most plants. While the structure of XyG has been well studied, much remains to be learned about its biosynthesis. Here we employed reverse genetics to investigate the role of Arabidopsis cellulose synthase like-C (CSLC) proteins in XyG biosynthesis. We found that single mutants containing a T-DNA in each of the five Arabidopsis CSLC genes had normal levels of XyG. However, higher-order cslc mutants had significantly reduced XyG levels, and a mutant with disruptions in all five CSLC genes had no detectable XyG. The higher-order mutants grew with mild tissue-specific phenotypes. Despite the apparent lack of XyG, the cslc quintuple mutant did not display significant alteration of gene expression at the whole-genome level, excluding transcriptional compensation. The quintuple mutant could be complemented by each of the five CSLC genes, supporting the conclusion that each of them encodes a XyG glucan synthase. Phylogenetic analyses indicated that the CSLC genes are widespread in the plant kingdom and evolved from an ancient family. These results establish the role of the CSLC genes in XyG biosynthesis, and the mutants described here provide valuable tools with which to study both the molecular details of XyG biosynthesis and the role of XyG in plant cell wall structure and function.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Parede Celular/metabolismo , Glucanos/biossíntese , Glucosiltransferases/metabolismo , Células Vegetais/metabolismo , Xilanos/biossíntese , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/genética , Mutação , Filogenia
5.
PLoS One ; 15(8): e0237448, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790800

RESUMO

We established that Endosidin2 (ES2) affected the trafficking routes of both newly synthesized and endocytic pools of PIN-FORMED2 (PIN2) in Arabidopsis root epidermal cells. PIN2 populations accumulated in separated patches, which gradually merged into large and compact ES2 aggregates (ES2As). FM4-64 endocytic tracer labeled ES2As as well. Both PIN2 pools also appeared in vacuoles. Accelerated endocytosis of PIN2, its aggregation in the cytoplasm, and redirection of PIN2 flows to vacuoles led to a substantial reduction of the abundance of this protein in the plasma membrane. Whereas PIN-FORMED3 and PIN-FORMED4 also aggregated in the cytoplasm, SYT1 was not sensitive to ES2 treatment and did not appear either in the cytoplasmic aggregates or vacuoles. Ultrastructural analysis revealed that ES2 affects the Golgi apparatus so that stacks acquired cup-shape and even circular shape surrounded by several vesicles. Abnormally shaped Golgi stacks, stack remnants, multi-lamellar structures, separated Golgi cisterna rings, tubular structures, and vesicles formed discrete clusters.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Endocitose/efeitos dos fármacos , Limoninas/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Parede Celular/metabolismo , Citoplasma/metabolismo , Complexo de Golgi/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Transporte Proteico/efeitos dos fármacos , Sinaptotagmina I/metabolismo
6.
Nat Commun ; 11(1): 3703, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32710080

RESUMO

Mycobacterium tuberculosis is a pathogen with a unique cell envelope including very long fatty acids, implicated in bacterial resistance and host immune modulation. FasR is a TetR-like transcriptional activator that plays a central role in sensing mycobacterial long-chain fatty acids and regulating lipid biosynthesis. Here we disclose crystal structures of M. tuberculosis FasR in complex with acyl effector ligands and with DNA, uncovering its molecular sensory and switching mechanisms. A long tunnel traverses the entire effector-binding domain, enabling long fatty acyl effectors to bind. Only when the tunnel is entirely occupied, the protein dimer adopts a rigid configuration with its DNA-binding domains in an open state, leading to DNA dissociation. The protein-folding hydrophobic core connects the two domains, and is completed into a continuous spine when the effector binds. Such a transmission spine is conserved in a large number of TetR-like regulators, offering insight into effector-triggered allosteric functional control.


Assuntos
Acil Coenzima A/química , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Mycobacterium tuberculosis/metabolismo , Fatores de Transcrição/química , Acil Coenzima A/metabolismo , Sítio Alostérico , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Cristalografia por Raios X , DNA Bacteriano/química , Proteínas de Ligação a DNA/metabolismo , Ácidos Graxos/metabolismo , Ligantes , Modelos Moleculares , Conformação Proteica , Fatores de Transcrição/metabolismo
7.
Plant Mol Biol ; 104(3): 249-261, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32715397

RESUMO

Secondary cell wall not only provides rigidity and mechanical resistance to plants, but also has a large impact on plant growth and adaptation to environments. Biosynthesis of secondary cell wall is regulated by a complicated signaling transduction network; however, it is still unclear how the transcriptional regulation of secondary cell wall biosynthesis works at the molecular level. Here, we report in rice that OVATE family proteins 6 (OsOFP6) is a positive regulator in modulating expression of the genes related to biosynthesis of the secondary cell wall. Transgenic plants with knock-down of OsOFP6 by RNA interference showed increased leaf angle, which resulted from the thinner secondary cell wall with reduced amounts of cellulose and lignin, whilst overexpression of OsOFP6 in rice led to the thinker secondary cell wall with increased lignin content. Protein-protein interaction analysis revealed that OsOFP6 interacts with Oryza sativa homeobox 15 (OSH15), a class I KNOX protein. The interaction of OsOFP6 and OSH15 enhanced the transcriptional activity of OSH15 which binds to the promoter of OsIRX9 (Oryza sativa IRREGULAR XYLEM 9). Taken together, our study provides insights into the function of OsOFP6 in regulating leaf angle and the control of biosynthesis of secondary cell wall.


Assuntos
Parede Celular/metabolismo , Oryza/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Parede Celular/ultraestrutura , Celulose/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/metabolismo , Lignina/metabolismo , Oryza/genética , Folhas de Planta/anatomia & histologia , Folhas de Planta/citologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Xilema/metabolismo
8.
PLoS One ; 15(7): e0236586, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32726362

RESUMO

Short rotation woody biomass cultivars developed from fast-growing shrub species of willow (Salix spp.) have superior properties as perennial energy crops for the Northeast and Midwest US. However, the insect pest potato leafhopper (PLH) Empoasca fabae (Harris) can cause serious damage and reduce yield of susceptible genotypes. Currently, the willow cultivars in use display varying levels of susceptibility under PLH infestation. However, genes and markers for resistance to PLH are not yet available for marker-assisted selection in breeding. In this study, transcriptome differences between a resistant genotype 94006 (S. purpurea) and a susceptible cultivar 'Jorr' (S. viminalis), and their hybrid progeny were determined. Over 600 million RNA-Seq reads were generated and mapped to the Salix purpurea reference transcriptome. Gene expression analyses revealed the unique defense mechanism in resistant genotype 94006 that involves PLH-induced secondary cell wall modification. In the susceptible genotypes, genes involved in programed cell death were highly expressed, explaining the necrosis symptoms after PLH feeding. Overall, the discovery of resistance genes and defense mechanisms provides new resources for shrub willow breeding and research in the future.


Assuntos
Salix/genética , Transcriptoma , Animais , Apoptose/genética , Parede Celular/química , Parede Celular/metabolismo , Produtos Agrícolas , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/genética , Genótipo , Hemípteros/fisiologia , Herbivoria , Interações Hospedeiro-Parasita/genética , Fenótipo , Análise de Componente Principal , RNA de Plantas/química , RNA de Plantas/metabolismo , Salix/parasitologia
9.
Chemosphere ; 260: 127553, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32653748

RESUMO

The impact of ionizing radiation on microorganisms such as microalgae is a topic of increasing importance for understanding the dynamics of aquatic ecosystems in response to environmental radiation, and for the development of efficient approaches for bioremediation of mining and nuclear power plants wastewaters. Currently, nothing is known about the effects of ionizing radiation on the microalgal cell wall, which represents the first line of defence against chemical and physical environmental stresses. Using various microscopy, spectroscopy and biochemical techniques we show that the unicellular alga Chlorella sorokiniana elicits a fast response to ionizing radiation. Within one day after irradiation with doses of 1-5 Gy, the fibrilar layer of the cell wall became thicker, the fraction of uronic acids was higher, and the capacity to remove the main reactive product of water radiolysis increased. In addition, the isolated cell wall fraction showed significant binding capacity for Cu2+, Mn2+, and Cr3+. The irradiation further increased the binding capacity for Cu2+, which appears to be mainly bound to glucosamine moieties within a chitosan-like polymer in the outer rigid layer of the wall. These results imply that the cell wall represents a dynamic structure that is involved in the protective response of microalgae to ionizing radiation. It appears that microalgae may exhibit a significant control of metal mobility in aquatic ecosystems via biosorption by the cell wall matrix.


Assuntos
Chlorella/metabolismo , Metais/metabolismo , Antioxidantes/metabolismo , Biodegradação Ambiental , Biomassa , Parede Celular/metabolismo , Chlorella/efeitos dos fármacos , Ecossistema , Microalgas/metabolismo , Radiação Ionizante , Águas Residuárias
10.
Nat Commun ; 11(1): 2848, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503964

RESUMO

The natural antibiotic teixobactin kills pathogenic bacteria without detectable resistance. The difficult synthesis and unfavourable solubility of teixobactin require modifications, yet insufficient knowledge on its binding mode impedes the hunt for superior analogues. Thus far, teixobactins are assumed to kill bacteria by binding to cognate cell wall precursors (Lipid II and III). Here we present the binding mode of teixobactins in cellular membranes using solid-state NMR, microscopy, and affinity assays. We solve the structure of the complex formed by an improved teixobactin-analogue and Lipid II and reveal how teixobactins recognize a broad spectrum of targets. Unexpectedly, we find that teixobactins only weakly bind to Lipid II in cellular membranes, implying the direct interaction with cell wall precursors is not the sole killing mechanism. Our data suggest an additional mechanism affords the excellent activity of teixobactins, which can block the cell wall biosynthesis by capturing precursors in massive clusters on membranes.


Assuntos
Antibacterianos/farmacologia , Membrana Celular/metabolismo , Depsipeptídeos/farmacologia , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Membrana Celular/ultraestrutura , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Depsipeptídeos/química , Lipossomos/metabolismo , Espectroscopia de Ressonância Magnética , Microscopia de Fluorescência , Estrutura Molecular , Relação Estrutura-Atividade , Uridina Difosfato Ácido N-Acetilmurâmico/química , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismo
11.
Proc Natl Acad Sci U S A ; 117(25): 14444-14452, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513721

RESUMO

Chemical-induced spores of the Gram-negative bacterium Myxococcus xanthus are peptidoglycan (PG)-deficient. It is unclear how these spherical spores germinate into rod-shaped, walled cells without preexisting PG templates. We found that germinating spores first synthesize PG randomly on spherical surfaces. MglB, a GTPase-activating protein, forms a cluster that responds to the status of PG growth and stabilizes at one future cell pole. Following MglB, the Ras family GTPase MglA localizes to the second pole. MglA directs molecular motors to transport the bacterial actin homolog MreB and the Rod PG synthesis complexes away from poles. The Rod system establishes rod shape de novo by elongating PG at nonpolar regions. Thus, similar to eukaryotic cells, the interactions between GTPase, cytoskeletons, and molecular motors initiate spontaneous polarization in bacteria.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Myxococcus xanthus/citologia , Peptidoglicano/metabolismo , Esporos Bacterianos/crescimento & desenvolvimento , Polaridade Celular , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Microscopia Eletrônica , Morfogênese , Myxococcus xanthus/crescimento & desenvolvimento , Myxococcus xanthus/metabolismo , Myxococcus xanthus/ultraestrutura , Peptidoglicano/genética , Esporos Bacterianos/metabolismo , Esporos Bacterianos/ultraestrutura
12.
PLoS Pathog ; 16(6): e1008606, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32569291

RESUMO

An important effector function of the human complement system is to directly kill Gram-negative bacteria via Membrane Attack Complex (MAC) pores. MAC pores are assembled when surface-bound convertase enzymes convert C5 into C5b, which together with C6, C7, C8 and multiple copies of C9 forms a transmembrane pore that damages the bacterial cell envelope. Recently, we found that bacterial killing by MAC pores requires local conversion of C5 by surface-bound convertases. In this study we aimed to understand why local assembly of MAC pores is essential for bacterial killing. Here, we show that rapid interaction of C7 with C5b6 is required to form bactericidal MAC pores on Escherichia coli. Binding experiments with fluorescently labelled C6 show that C7 prevents release of C5b6 from the bacterial surface. Moreover, trypsin shaving experiments and atomic force microscopy revealed that this rapid interaction between C7 and C5b6 is crucial to efficiently anchor C5b-7 to the bacterial cell envelope and form complete MAC pores. Using complement-resistant clinical E. coli strains, we show that bacterial pathogens can prevent complement-dependent killing by interfering with the anchoring of C5b-7. While C5 convertase assembly was unaffected, these resistant strains blocked efficient anchoring of C5b-7 and thus prevented stable insertion of MAC pores into the bacterial cell envelope. Altogether, these findings provide basic molecular insights into how bactericidal MAC pores are assembled and how bacteria evade MAC-dependent killing.


Assuntos
Atividade Bactericida do Sangue , Membrana Celular/metabolismo , Parede Celular/metabolismo , Complemento C5/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Escherichia coli/metabolismo , Proteínas do Sistema Complemento/metabolismo , Células HEK293 , Humanos
13.
Proc Natl Acad Sci U S A ; 117(27): 15400-15402, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571955

RESUMO

The Casparian strip (CS) is a tight junction-like structure formed by lignin impregnation on the walls of endodermal cells in plant roots. The CS membrane domain (CSDM), demarked by the CASP proteins, is important for orienting lignification enzymes. Here, we report that an endodermis-expressed multicopper oxidase, LACCASE3 (LAC3) in Arabidopsis, locates to the interface between lignin domains and the cell wall during early CS development prior to CASP1 localizing to CSDM and eventually flanks the mature CS. Pharmacological perturbation of LAC3 causes dispersed localization of CASP1 and compensatory ectopic lignification. These results support the existence of a LAC3-based CS wall domain which coordinates with CSDM to provide bidirectional positional information that guides precise CS lignification.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Lacase/metabolismo , Proteínas de Membrana/metabolismo , Raízes de Plantas/metabolismo , Arabidopsis/citologia , Parede Celular/metabolismo , Lacase/genética , Lignina/metabolismo , Proteínas de Membrana/genética , Raízes de Plantas/citologia , Plantas Geneticamente Modificadas , Domínios Proteicos
14.
Proc Natl Acad Sci U S A ; 117(27): 16043-16054, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571919

RESUMO

In the indeterminate nodules of a model legume Medicago truncatula, ∼700 nodule-specific cysteine-rich (NCR) peptides with conserved cysteine signature are expressed. NCR peptides are highly diverse in sequence, and some of these cationic peptides exhibit antimicrobial activity in vitro and in vivo. However, there is a lack of knowledge regarding their structural architecture, antifungal activity, and modes of action against plant fungal pathogens. Here, the three-dimensional NMR structure of the 36-amino acid NCR044 peptide was solved. This unique structure was largely disordered and highly dynamic with one four-residue α-helix and one three-residue antiparallel ß-sheet stabilized by two disulfide bonds. NCR044 peptide also exhibited potent fungicidal activity against multiple plant fungal pathogens, including Botrytis cinerea and three Fusarium spp. It inhibited germination in quiescent spores of B. cinerea In germlings, it breached the fungal plasma membrane and induced reactive oxygen species. It bound to multiple bioactive phosphoinositides in vitro. Time-lapse confocal and superresolution microscopy revealed strong fungal cell wall binding, penetration of the cell membrane at discrete foci, followed by gradual loss of turgor, subsequent accumulation in the cytoplasm, and elevated levels in nucleoli of germlings. Spray-applied NCR044 significantly reduced gray mold disease symptoms caused by the fungal pathogen B. cinerea in tomato and tobacco plants, and postharvest products. Our work illustrates the antifungal activity of a structurally unique NCR peptide against plant fungal pathogens and paves the way for future development of this class of peptides as a spray-on fungistat/fungicide.


Assuntos
Antifúngicos/farmacologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacologia , Simbiose , Sequência de Aminoácidos , Botrytis/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Cisteína/química , Fusarium/metabolismo , Lycopersicon esculentum/metabolismo , Lycopersicon esculentum/microbiologia , Espectroscopia de Ressonância Magnética , Medicago truncatula/microbiologia , Pichia/metabolismo , Doenças das Plantas/microbiologia , Tabaco/metabolismo , Tabaco/microbiologia
15.
Food Chem ; 330: 127252, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32585587

RESUMO

Mycoprotein is a food ingredient from filamentous fungi rich in protein and fibre. This study investigated the protein bioaccessibility from the fungal cells by colourimetric assays in different mycoprotein formulations, following extraction methods and in vitro gastrointestinal digestion. The methods effects were further analysed by static laser light scattering, SDS-PAGE and optical-fluorescence microscopy. The extraction methods released a comparable proportion of protein (30 wt%) independent of sample concentration (10 wt% and 25 wt%), whereas the simulated digestions endpoints released a higher proportion of protein from the less concentrated (46 wt%). Furthermore, mechanical/physical processing had only a minor impact. Intestinal proteases promoted the most efficient protein release but without causing any apparent damage to the cell walls when viewed by microscopy. This suggested that the enzymes can diffuse through the cell walls, due to its porosity/permeability, and are the main factors responsible for the hydrolysis and bioaccessibility of protein from mycoprotein.


Assuntos
Proteínas Fúngicas/metabolismo , Animais , Disponibilidade Biológica , Parede Celular/química , Parede Celular/metabolismo , Fibras na Dieta/metabolismo , Digestão , Proteínas Fúngicas/química , Fungos/química , Fungos/metabolismo
16.
PLoS Genet ; 16(6): e1008847, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32559234

RESUMO

Plant cell growth requires the coordinated expansion of the protoplast and the cell wall, which is controlled by an elaborate system of cell wall integrity (CWI) sensors linking the different cellular compartments. LRR-eXtensins (LRXs) are cell wall-attached extracellular regulators of cell wall formation and high-affinity binding sites for RALF (Rapid ALkalinization Factor) peptide hormones that trigger diverse physiological processes related to cell growth. LRXs function in CWI sensing and in the case of LRX4 of Arabidopsis thaliana, this activity was shown to involve interaction with the transmembrane Catharanthus roseus Receptor-Like Kinase1-Like (CrRLK1L) protein FERONIA (FER). Here, we demonstrate that binding of RALF1 and FER is common to most tested LRXs of vegetative tissue, including LRX1, the main LRX protein of root hairs. Consequently, an lrx1-lrx5 quintuple mutant line develops shoot and root phenotypes reminiscent of the fer-4 knock-out mutant. The previously observed membrane-association of LRXs, however, is FER-independent, suggesting that LRXs bind not only FER but also other membrane-localized proteins to establish a physical link between intra- and extracellular compartments. Despite evolutionary diversification of various LRX proteins, overexpression of several chimeric LRX constructs causes cross-complementation of lrx mutants, indicative of comparable functions among members of this protein family. Suppressors of the pollen-growth defects induced by mutations in the CrRLK1Ls ANXUR1/2 also alleviate lrx1 lrx2-induced mutant root hair phenotypes. This suggests functional similarity of LRX-CrRLK1L signaling processes in very different cell types and indicates that LRX proteins are components of conserved processes regulating cell growth.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Parede Celular/metabolismo , Hormônios Peptídicos/metabolismo , Fosfotransferases/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Genes de Plantas , Mutação , Fosfotransferases/genética , Raízes de Plantas/citologia , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Pólen/citologia , Pólen/crescimento & desenvolvimento , Domínios Proteicos/genética , Mapas de Interação de Proteínas , Plântula/citologia , Plântula/crescimento & desenvolvimento , Transdução de Sinais/genética
17.
J Vis Exp ; (159)2020 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-32510485

RESUMO

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in molecular and imaging technologies allowed us to gain insights into how biochemical signals influence morphogenetic events. However, it is increasingly evident that apart from biochemical signals, mechanical cues also impact several aspects of cell and tissue growth. The Arabidopsis shoot apical meristem (SAM) is a dome-shaped structure responsible for the generation of all aboveground organs. The organization of the cortical microtubule cytoskeleton that mediates apoplastic cellulose deposition in plant cells is spatially distinct. Visualization and quantitative assessment of patterns of cortical microtubules are necessary for understanding the biophysical nature of cells at the SAM, as cellulose is the stiffest component of the plant cell wall. The stereotypical form of cortical microtubule organization is also a consequence of tissue-wide physical forces existing at the SAM. Perturbation of these physical forces and subsequent monitoring of cortical microtubule organization allows for the identification of candidate proteins involved in mediating mechano-perception and transduction. Here we describe a protocol that helps investigate such processes.


Assuntos
Arabidopsis/citologia , Citoesqueleto/metabolismo , Fenômenos Mecânicos , Microtúbulos/metabolismo , Imagem Molecular , Arabidopsis/crescimento & desenvolvimento , Fenômenos Biomecânicos , Sobrevivência Celular , Parede Celular/metabolismo , Celulose/metabolismo , Meristema/citologia , Meristema/crescimento & desenvolvimento , Morfogênese
18.
PLoS One ; 15(5): e0227591, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32433654

RESUMO

Plants emit high rates of methanol (meOH), generally assumed to derive from pectin demethylation, and this increases during abiotic stress. In contrast, less is known about the emission and source of acetic acid (AA). In this study, Populus trichocarpa (California poplar) leaves in different developmental stages were desiccated and quantified for total meOH and AA emissions together with bulk cell wall acetylation and methylation content. While young leaves showed high emissions of meOH (140 µmol m-2) and AA (42 µmol m-2), emissions were reduced in mature (meOH: 69%, AA: 60%) and old (meOH: 83%, AA: 76%) leaves. In contrast, the ratio of AA/meOH emissions increased with leaf development (young: 35%, mature: 43%, old: 82%), mimicking the pattern of O-acetyl/methyl ester ratios of leaf bulk cell walls (young: 35%, mature: 38%, old: 51%), which is driven by an increase in O-acetyl and decrease in methyl ester content with age. The results are consistent with meOH and AA emission sources from cell wall de-esterification, with young expanding tissues producing highly methylated pectin that is progressively demethyl-esterified. We highlight the quantification of AA/meOH emission ratios as a potential tool for rapid phenotype screening of structural carbohydrate esterification patterns.


Assuntos
Ácido Acético/metabolismo , Parede Celular/metabolismo , Metanol/metabolismo , Folhas de Planta/metabolismo , Acetilação , Atmosfera , Hidrolases de Éster Carboxílico/metabolismo , Esterificação , Metilação , Pectinas/metabolismo , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Populus/efeitos dos fármacos , Populus/crescimento & desenvolvimento , Populus/metabolismo , Estresse Fisiológico/genética
19.
PLoS One ; 15(5): e0231881, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32357186

RESUMO

The sequencing and bioinformatics analysis of bacteriophages infecting mycobacteria has yielded a large amount of information on their evolution, including that on their environmental propagation on other genera such as Gordonia, closely related to Mycobacterium. However, little is known on mycobacteriophages cell biology such as the nature of their receptor(s) or their replication cycle. As part of our on-going screening for novel mycobacteriophages, we herein report the isolation and genome bioinformatics analysis of Weirdo19ES, a singleton Siphoviridae temperate mycobacteriophage with a 70.19% GC content. Nucleotide and protein sequence comparison to actinobacteriophage databases revealed that Weirdo19ES shows low homology to Gordonia phage Ruthy and mycobacteriophages falling in clusters Q and G and to singleton DS6A.Weirdo19ES also displays uncommon features such as a very short Lysin A gene (with only one enzymatic domain) and two putative HNH endonucleases. Mycobacterium smegmatis mutants resistant to Weirdo19ES are cross- resistant to I3. In agreement with that phenotype, analysis of cell envelope of those mutants showed that Weirdo19ES shares receptors with the transducing mycobacteriophage I3.This singleton mycobacteriophage adds up to the uncommonness of local mycobacteriophages previously isolated by our group and helps understanding the nature of mycobacteriophage receptors.


Assuntos
Genoma Viral , Glicolipídeos/genética , Micobacteriófagos/genética , Mycobacterium smegmatis/virologia , Composição de Bases , Parede Celular/metabolismo , Análise por Conglomerados , Uso do Códon , Hibridização Genômica Comparativa , Glicolipídeos/deficiência , Micobacteriófagos/classificação , Micobacteriófagos/isolamento & purificação , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/isolamento & purificação , Fenótipo , Filogenia
20.
PLoS One ; 15(5): e0229700, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379829

RESUMO

One of the most important and exclusive characteristics of mycobacteria is their cell wall. Amongst its constituent components are two related families of glycosylated lipids, diphthioceranates and phthiocerol dimycocerosate (PDIM) and its variant phenolic glycolipids (PGL). PGL have been associated with cell wall impermeability, phagocytosis, defence against nitrosative and oxidative stress and, intriguingly, biofilm formation. In bacteria from the Mycobacterium tuberculosis complex (MTBC), the biosynthetic pathway of the phenolphthiocerol moiety of PGL depends upon the expression of several genes encoding type I polyketide synthases (PKS), namely ppsA-E and pks15/1 which constitute the PDIM + PGL locus, and that are highly conserved in PDIM/PGL-producing strains. Consensus has not been achieved regarding the genetic organization of pks15/1 locus and knowledge is lacking on its transcriptional signature. Here we explore publicly available datasets of transcriptome data (RNA-seq) from more than 100 MTBC experiments in 40 growth conditions to outline the transcriptional structure and signature of pks15/1, using a differential expression approach to infer the regulatory patterns involving these and related genes. We show that pks1 expression is highly correlated with fadD22, Rv2949c, lppX, fadD29 and, also, pks6 and pks12, with the first three putatively integrating into a polycistronic structure. We evidence dynamic transcriptional heterogeneity within the genes involved in phenolphtiocerol and phenolic glycolipid production, most exhibiting up-regulation upon acidic pH and antibiotic exposure and down-regulation under hypoxia, dormancy, and low/high iron concentration. We finally propose a model based on transcriptome data in which σD positively regulates pks1, pks15 and fadD22, while σB and σE factors exert negative regulation at an upper level.


Assuntos
Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Glicolipídeos/biossíntese , Glicolipídeos/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Policetídeo Sintases/genética , Transcriptoma , Parede Celular/metabolismo , Simulação por Computador , Redes Reguladoras de Genes , Loci Gênicos , Genoma Bacteriano/genética , Ligases/genética , RNA-Seq , Virulência/genética
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