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1.
Cell ; 174(2): 448-464.e24, 2018 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-30007417

RESUMO

Land plants evolved from charophytic algae, among which Charophyceae possess the most complex body plans. We present the genome of Chara braunii; comparison of the genome to those of land plants identified evolutionary novelties for plant terrestrialization and land plant heritage genes. C. braunii employs unique xylan synthases for cell wall biosynthesis, a phragmoplast (cell separation) mechanism similar to that of land plants, and many phytohormones. C. braunii plastids are controlled via land-plant-like retrograde signaling, and transcriptional regulation is more elaborate than in other algae. The morphological complexity of this organism may result from expanded gene families, with three cases of particular note: genes effecting tolerance to reactive oxygen species (ROS), LysM receptor-like kinases, and transcription factors (TFs). Transcriptomic analysis of sexual reproductive structures reveals intricate control by TFs, activity of the ROS gene network, and the ancestral use of plant-like storage and stress protection proteins in the zygote.


Assuntos
Chara/genética , Genoma de Planta , Evolução Biológica , Parede Celular/metabolismo , Chara/crescimento & desenvolvimento , Embriófitas/genética , Redes Reguladoras de Genes , Pentosiltransferases/genética , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma
2.
Plant Cell ; 36(5): 1791-1805, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38267818

RESUMO

Polar auxin transport in the Arabidopsis (Arabidopsis thaliana) root tip maintains high auxin levels around the stem cell niche that gradually decrease in dividing cells but increase again once they transition toward differentiation. Protophloem differentiates earlier than other proximal tissues and employs a unique auxin "canalization" machinery that is thought to balance auxin efflux with retention. It consists of a proposed activator of PIN-FORMED (PIN) auxin efflux carriers, the cAMP-, cGMP- and Calcium-dependent (AGC) kinase PROTEIN KINASE ASSOCIATED WITH BRX (PAX); its inhibitor, BREVIS RADIX (BRX); and PHOSPHATIDYLINOSITOL-4-PHOSPHATE-5-KINASE (PIP5K) enzymes, which promote polar PAX and BRX localization. Because of a dynamic PAX-BRX-PIP5K interplay, the net cellular output of this machinery remains unclear. In this study, we deciphered the dosage-sensitive regulatory interactions among PAX, BRX, and PIP5K by their ectopic expression in developing xylem vessels. The data suggest that the dominant collective output of the PAX-BRX-PIP5K module is a localized reduction in PIN abundance. This requires PAX-stimulated clathrin-mediated PIN endocytosis upon site-specific phosphorylation, which distinguishes PAX from other AGC kinases. An ectopic assembly of the PAX-BRX-PIP5K module is sufficient to cause cellular auxin retention and affects root growth vigor by accelerating the trajectory of xylem vessel development. Our data thus provide direct evidence that local manipulation of auxin efflux alters the timing of cellular differentiation in the root.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ácidos Indolacéticos , Proteínas Serina-Treonina Quinases , Ácidos Indolacéticos/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Transporte Biológico , Xilema/metabolismo , Xilema/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética
3.
Plant J ; 115(2): 470-479, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37036146

RESUMO

Chemical inhibitors are often implemented for the functional characterization of genes to overcome the limitations associated with genetic approaches. Although it is well established that the specificity of the compound is key to success of a pharmacological approach, off-target effects are often overlooked or simply neglected in a complex biological setting. Here we illustrate the cause and implications of such secondary effects by focusing on piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H) that is frequently used to investigate the involvement of lignin during plant growth and development. When supplied to plants, we found that PA is recognized as a substrate by GRETCHEN HAGEN 3.6 (GH3.6), an amido synthetase involved in the formation of the indole-3-acetic acid (IAA) conjugate IAA-Asp. By competing for the same enzyme, PA interferes with IAA conjugation, resulting in an increase in IAA concentrations in the plant. In line with the broad substrate specificity of the GH3 family of enzymes, treatment with PA increased not only IAA levels but also those of other GH3-conjugated phytohormones, namely jasmonic acid and salicylic acid. Finally, we found that interference with the endogenous function of GH3s potentially contributes to phenotypes previously observed upon PA treatment. We conclude that deregulation of phytohormone homeostasis by surrogate occupation of the conjugation machinery in the plant is likely a general phenomenon when using chemical inhibitors. Our results hereby provide a novel and important basis for future reference in studies using chemical inhibitors.


Assuntos
Ácidos Indolacéticos , Reguladores de Crescimento de Plantas , Ácidos Indolacéticos/farmacologia , Benzoatos , Oxigenases de Função Mista/genética , Cinamatos/farmacologia , Regulação da Expressão Gênica de Plantas
4.
EMBO J ; 39(17): e104238, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32667089

RESUMO

Cell production and differentiation for the acquisition of specific functions are key features of living systems. The dynamic network of cellular microtubules provides the necessary platform to accommodate processes associated with the transition of cells through the individual phases of cytogenesis. Here, we show that the plant hormone cytokinin fine-tunes the activity of the microtubular cytoskeleton during cell differentiation and counteracts microtubular rearrangements driven by the hormone auxin. The endogenous upward gradient of cytokinin activity along the longitudinal growth axis in Arabidopsis thaliana roots correlates with robust rearrangements of the microtubule cytoskeleton in epidermal cells progressing from the proliferative to the differentiation stage. Controlled increases in cytokinin activity result in premature re-organization of the microtubule network from transversal to an oblique disposition in cells prior to their differentiation, whereas attenuated hormone perception delays cytoskeleton conversion into a configuration typical for differentiated cells. Intriguingly, cytokinin can interfere with microtubules also in animal cells, such as leukocytes, suggesting that a cytokinin-sensitive control pathway for the microtubular cytoskeleton may be at least partially conserved between plant and animal cells.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Diferenciação Celular , Proliferação de Células , Citocininas/metabolismo , Microtúbulos/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Animais , Arabidopsis/genética , Citocininas/genética , Microtúbulos/genética , Raízes de Plantas/genética
5.
Plant Cell ; 33(9): 2981-3003, 2021 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-34240197

RESUMO

To overcome nitrogen deficiency, legume roots establish symbiotic interactions with nitrogen-fixing rhizobia that are fostered in specialized organs (nodules). Similar to other organs, nodule formation is determined by a local maximum of the phytohormone auxin at the primordium site. However, how auxin regulates nodule development remains poorly understood. Here, we found that in soybean, (Glycine max), dynamic auxin transport driven by PIN-FORMED (PIN) transporter GmPIN1 is involved in nodule primordium formation. GmPIN1 was specifically expressed in nodule primordium cells and GmPIN1 was polarly localized in these cells. Two nodulation regulators, (iso)flavonoids trigger expanded distribution of GmPIN1b to root cortical cells, and cytokinin rearranges GmPIN1b polarity. Gmpin1abc triple mutants generated with CRISPR-Cas9 showed the impaired establishment of auxin maxima in nodule meristems and aberrant divisions in the nodule primordium cells. Moreover, overexpression of GmPIN1 suppressed nodule primordium initiation. GmPIN9d, an ortholog of Arabidopsis thaliana PIN2, acts together with GmPIN1 later in nodule development to acropetally transport auxin in vascular bundles, fine-tuning the auxin supply for nodule enlargement. Our findings reveal how PIN-dependent auxin transport modulates different aspects of soybean nodule development and suggest that the establishment of auxin gradient is a prerequisite for the proper interaction between legumes and rhizobia.


Assuntos
Glycine max/crescimento & desenvolvimento , Ácidos Indolacéticos/metabolismo , Proteínas de Plantas/genética , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Transporte Biológico , Proteínas de Plantas/metabolismo , Nódulos Radiculares de Plantas/metabolismo , Glycine max/genética , Glycine max/metabolismo
6.
New Phytol ; 233(1): 329-343, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34637542

RESUMO

Advanced transcriptome sequencing has revealed that the majority of eukaryotic genes undergo alternative splicing (AS). Nonetheless, little effort has been dedicated to investigating the functional relevance of particular splicing events, even those in the key developmental and hormonal regulators. Combining approaches of genetics, biochemistry and advanced confocal microscopy, we describe the impact of alternative splicing on the PIN7 gene in the model plant Arabidopsis thaliana. PIN7 encodes a polarly localized transporter for the phytohormone auxin and produces two evolutionarily conserved transcripts, PIN7a and PIN7b. PIN7a and PIN7b, differing in a four amino acid stretch, exhibit almost identical expression patterns and subcellular localization. We reveal that they are closely associated and mutually influence each other's mobility within the plasma membrane. Phenotypic complementation tests indicate that the functional contribution of PIN7b per se is minor, but it markedly reduces the prominent PIN7a activity, which is required for correct seedling apical hook formation and auxin-mediated tropic responses. Our results establish alternative splicing of the PIN family as a conserved, functionally relevant mechanism, revealing an additional regulatory level of auxin-mediated plant development.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos , Raízes de Plantas/metabolismo , Isoformas de Proteínas/genética
7.
Plant Physiol ; 187(1): 103-115, 2021 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-34618129

RESUMO

Together with auxin transport, auxin metabolism is a key determinant of auxin signaling output by plant cells. Enzymatic machinery involved in auxin metabolism is subject to regulation based on numerous inputs, including the concentration of auxin itself. Therefore, experiments characterizing altered auxin availability and subsequent changes in auxin metabolism could elucidate the function and regulatory role of individual elements in the auxin metabolic machinery. Here, we studied auxin metabolism in auxin-dependent tobacco BY-2 cells. We revealed that the concentration of N-(2-oxindole-3-acetyl)-l-aspartic acid (oxIAA-Asp), the most abundant auxin metabolite produced in the control culture, dramatically decreased in auxin-starved BY-2 cells. Analysis of the transcriptome and proteome in auxin-starved cells uncovered significant downregulation of all tobacco (Nicotiana tabacum) homologs of Arabidopsis (Arabidopsis thaliana) DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1), at both transcript and protein levels. Auxin metabolism profiling in BY-2 mutants carrying either siRNA-silenced or CRISPR-Cas9-mutated NtDAO1, as well as in dao1-1 Arabidopsis plants, showed not only the expected lower levels of oxIAA, but also significantly lower abundance of oxIAA-Asp. Finally, ability of DAO1 to oxidize IAA-Asp was confirmed by an enzyme assay in AtDAO1-producing bacterial culture. Our results thus represent direct evidence of DAO1 activity on IAA amino acid conjugates.


Assuntos
Aminoácidos/metabolismo , Dioxigenases/metabolismo , Nicotiana/enzimologia , Proteínas de Plantas/metabolismo , Oxirredução
8.
Plant J ; 101(3): 619-636, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31610051

RESUMO

Current models of plasma membrane (PM) postulate its organization in various nano- and micro-domains with distinct protein and lipid composition. While metazoan PM nanodomains usually display high lateral mobility, the dynamics of plant nanodomains is often highly spatially restricted. Here we have focused on the determination of the PM distribution in nanodomains for Arabidopsis thaliana flotillin (AtFLOT) and hypersensitive induced reaction proteins (AtHIR), previously shown to be involved in response to extracellular stimuli. Using in vivo laser scanning and spinning disc confocal microscopy in Arabidopsis thaliana we present here their nanodomain localization in various epidermal cell types. Fluorescence recovery after photobleaching (FRAP) and kymographic analysis revealed that PM-associated AtFLOTs contain significantly higher immobile fraction than AtHIRs. In addition, much lower immobile fractions have been found in tonoplast pool of AtHIR3. Although members of both groups of proteins were spatially restricted in their PM distribution by corrals co-aligning with microtubules (MTs), pharmacological treatments showed no or very low role of actin and microtubular cytoskeleton for clustering of AtFLOT and AtHIR into nanodomains. Finally, pharmacological alteration of cell wall (CW) synthesis and structure resulted in changes in lateral mobility of AtFLOT2 and AtHIR1. Accordingly, partial enzymatic CW removal increased the overall dynamics as well as individual nanodomain mobility of these two proteins. Such structural links to CW could play an important role in their correct positioning during PM communication with extracellular environment.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Membrana/metabolismo , Actinas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Parede Celular/metabolismo , Citoesqueleto/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/genética , Microscopia Confocal , Microtúbulos/metabolismo
9.
Plant J ; 100(3): 627-640, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31349380

RESUMO

Auxin concentration gradients are informative for the transduction of many developmental cues, triggering downstream gene expression and other responses. The generation of auxin gradients depends significantly on cell-to-cell auxin transport, which is supported by the activities of auxin efflux and influx carriers. However, at the level of individual plant cell, the co-ordination of auxin efflux and influx largely remains uncharacterized. We addressed this issue by analyzing the contribution of canonical PIN-FORMED (PIN) proteins to the carrier-mediated auxin efflux in Nicotiana tabacum L., cv. Bright Yellow (BY-2) tobacco cells. We show here that a majority of canonical NtPINs are transcribed in cultured cells and in planta. Cloning of NtPIN genes and their inducible overexpression in tobacco cells uncovered high auxin efflux activity of NtPIN11, accompanied by auxin starvation symptoms. Auxin transport parameters after NtPIN11 overexpression were further assessed using radiolabelled auxin accumulation and mathematical modelling. Unexpectedly, these experiments showed notable stimulation of auxin influx, which was accompanied by enhanced transcript levels of genes for a specific auxin influx carrier and by decreased transcript levels of other genes for auxin efflux carriers. A similar transcriptional response was observed upon removal of auxin from the culture medium, which resulted in decreased auxin efflux. Overall, our results revealed an auxin transport-based homeostatic mechanism for the maintenance of endogenous auxin levels. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at http://osf.io/ka97b/.


Assuntos
Ácidos Indolacéticos/metabolismo , Nicotiana/fisiologia , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Linhagem Celular , Homeostase , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Teóricos , Filogenia , Proteínas de Plantas/genética , Nicotiana/genética
10.
J Exp Bot ; 71(11): 3287-3295, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32246155

RESUMO

Auxin, represented by indole-3-acetic acid (IAA), has for a long time been studied mainly with respect to the development of land plants, and recent evidence confirms that canonical nuclear auxin signaling is a land plant apomorphy. Increasing sequential and physiological data show that the presence of auxin transport machinery pre-dates the emergence of canonical signaling. In this review, we summarize the present state of knowledge regarding the origins of auxin transport in the green lineage (Viridiplantae), integrating both data from wet lab experiments and sequence evidence on the presence of PIN-FORMED (PIN), PIN-LIKES (PILS), and AUXIN RESISTANT 1/LIKE-AUX1 (AUX1/LAX) homologs. We discuss a high divergence of auxin carrier homologs among algal lineages and emphasize the urgent need for the establishment of good molecular biology models from within the streptophyte green algae. We further postulate and discuss two hypotheses for the ancestral role of auxin in the green lineage. First, auxin was present as a by-product of cell metabolism and the evolution of its transport was stimulated by the need for IAA sequestration and cell detoxification. Second, auxin was primarily a signaling compound, possibly of bacterial origin, and its activity in the pre-plant green algae was a consequence of long-term co-existence with bacteria in shared ecological consortia.


Assuntos
Clorófitas , Viridiplantae , Transporte Biológico , Clorófitas/genética , Ácidos Indolacéticos , Transdução de Sinais
11.
Int J Mol Sci ; 20(4)2019 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-30791522

RESUMO

The international symposium "Auxins and Cytokinins in Plant Development" (ACPD), which is held every 4⁻5 years in Prague, Czech Republic, is a meeting of scientists interested in the elucidation of the action of two important plant hormones-auxins and cytokinins. It is organized by a group of researchers from the Laboratory of Hormonal Regulations in Plants at the Institute of Experimental Botany, the Czech Academy of Sciences. The symposia already have a long tradition, having started in 1972. Thanks to the central role of auxins and cytokinins in plant development, the ACPD 2018 symposium was again attended by numerous experts who presented their results in the opening, two plenary lectures, and six regular sessions, including two poster sessions. Due to the open character of the research community, which is traditionally very well displayed during the meeting, a lot of unpublished data were presented and discussed. In this report, we summarize the contributions in individual sessions that attracted our attention.


Assuntos
Citocininas/metabolismo , Ácidos Indolacéticos/metabolismo , Desenvolvimento Vegetal , Reguladores de Crescimento de Plantas/metabolismo , Transporte Biológico , Meio Ambiente , Redes e Vias Metabólicas , Transdução de Sinais
12.
Ann Bot ; 122(5): 777-789, 2018 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-29293873

RESUMO

Background and Aim: The cytoskeleton plays an important role in the synthesis of plant cell walls. Both microtubules and actin cytoskeleton are known to be involved in the morphogenesis of plant cells through their role in cell wall building. The role of ARP2/3-nucleated actin cytoskeleton in the morphogenesis of cotyledon pavement cells has been described before. Seedlings of Arabidopsis mutants lacking a functional ARP2/3 complex display specific cell wall-associated defects. Methods: In three independent Arabidopsis mutant lines lacking subunits of the ARP2/3 complex, phenotypes associated with the loss of the complex were analysed throughout plant development. Organ size and anatomy, cell wall composition, and auxin distribution were investigated. Key Results: ARP2/3-related phenotype is associated with changes in cell wall composition, and the phenotype is manifested especially in mature tissues. Cell walls of mature plants contain less cellulose and a higher amount of homogalacturonan, and display changes in cell wall lignification. Vascular bundles of mutant inflorescence stems show a changed pattern of AUX1-YFP expression. Plants lacking a functional ARP2/3 complex have decreased basipetal auxin transport. Conclusions: The results suggest that the ARP2/3 complex has a morphogenetic function related to cell wall synthesis and auxin transport.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Parede Celular/metabolismo , Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo
13.
Nature ; 485(7396): 119-22, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22504182

RESUMO

The phytohormone auxin acts as a prominent signal, providing, by its local accumulation or depletion in selected cells, a spatial and temporal reference for changes in the developmental program. The distribution of auxin depends on both auxin metabolism (biosynthesis, conjugation and degradation) and cellular auxin transport. We identified in silico a novel putative auxin transport facilitator family, called PIN-LIKES (PILS). Here we illustrate that PILS proteins are required for auxin-dependent regulation of plant growth by determining the cellular sensitivity to auxin. PILS proteins regulate intracellular auxin accumulation at the endoplasmic reticulum and thus auxin availability for nuclear auxin signalling. PILS activity affects the level of endogenous auxin indole-3-acetic acid (IAA), presumably via intracellular accumulation and metabolism. Our findings reveal that the transport machinery to compartmentalize auxin within the cell is of an unexpected molecular complexity and demonstrate this compartmentalization to be functionally important for a number of developmental processes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Homeostase , Ácidos Indolacéticos/metabolismo , Espaço Intracelular/metabolismo , Família Multigênica , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Retículo Endoplasmático/metabolismo , Genes de Plantas/genética , Germinação , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
14.
Plant Cell Rep ; 37(5): 809-818, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29502206

RESUMO

KEY MESSAGE: Silver ions increase plasma membrane permeability for water and small organic compounds through their stimulatory effect on plasma membrane calcium channels, with subsequent modulation of intracellular calcium levels and ion homeostasis. The action of silver ions at the plant plasma membrane is largely connected with the inhibition of ethylene signalling thanks to the ability of silver ion to replace the copper cofactor in the ethylene receptor. A link coupling the action of silver ions and cellular auxin efflux has been suggested earlier by their possible direct interaction with auxin efflux carriers or by influencing plasma membrane permeability. Using tobacco BY-2 cells, we demonstrate here that besides a dramatic increase of efflux of synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthalene acetic acid (NAA), treatment with AgNO3 resulted in enhanced efflux of the cytokinin trans-zeatin (tZ) as well as the auxin structural analogues tryptophan (Trp) and benzoic acid (BA). The application of AgNO3 was accompanied by gradual water loss and plasmolysis. The observed effects were dependent on the availability of extracellular calcium ions (Ca2+) as shown by comparison of transport assays in Ca2+-rich and Ca2+-free buffers and upon treatment with inhibitors of plasma membrane Ca2+-permeable channels Al3+ and ruthenium red, both abolishing the effect of AgNO3. Confocal microscopy of Ca2+-sensitive fluorescence indicator Fluo-4FF, acetoxymethyl (AM) ester suggested that the extracellular Ca2+ availability is necessary to trigger the response to silver ions and that the intracellular Ca2+ pool alone is not sufficient for this effect. Altogether, our data suggest that in plant cells the effects of silver ions originate from the primal modification of the internal calcium levels, possibly by their interaction with Ca2+-permeable channels at the plasma membrane.


Assuntos
Cálcio/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Espaço Intracelular/metabolismo , Nicotiana/citologia , Nicotiana/metabolismo , Células Vegetais/metabolismo , Prata/farmacologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Ácidos Indolacéticos/metabolismo , Íons , Células Vegetais/efeitos dos fármacos
15.
J Biol Chem ; 291(52): 26794-26805, 2016 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-27810900

RESUMO

Fibrosis, driven by inflammation, marks the transition from benign to progressive stages of chronic liver diseases. Although inflammation promotes fibrogenesis, it is not known whether other events, such as hepatocyte death, are required for the development of fibrosis. Interferon regulatory factor 3 (IRF3) regulates hepatocyte apoptosis and production of type I IFNs. In the liver, IRF3 is activated via Toll-like receptor 4 (TLR4) signaling or the endoplasmic reticulum (ER) adapter, stimulator of interferon genes (STING). We hypothesized that IRF3-mediated hepatocyte death is an independent determinant of chemically induced liver fibrogenesis. To test this, we performed acute or chronic CCl4 administration to WT and IRF3-, Toll/Interleukin-1R (TIR) domain-containing adapter-inducing interferon-ß (TRIF)-, TRIF-related adaptor molecule (TRAM)-, and STING-deficient mice. We report that acute CCl4 administration to WT mice resulted in early ER stress, activation of IRF3, and type I IFNs, followed by hepatocyte apoptosis and liver injury, accompanied by liver fibrosis upon repeated administration of CCl4 Deficiency of IRF3 or STING prevented hepatocyte death and fibrosis both in acute or chronic CCl4 In contrast, mice deficient in type I IFN receptors or in TLR4 signaling adaptors, TRAM or TRIF, upstream of IRF3, were not protected from hepatocyte death and/or fibrosis, suggesting that the pro-apoptotic role of IRF3 is independent of TLR signaling in fibrosis. Hepatocyte death is required for liver fibrosis with causal involvement of STING and IRF3. Thus, our results identify that IRF3, by its association with STING in the presence of ER stress, couples hepatocyte apoptosis with liver fibrosis and indicate that innate immune signaling regulates outcomes of liver fibrosis via modulation of hepatocyte death in the liver.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Estresse do Retículo Endoplasmático , Hepatócitos/patologia , Fator Regulador 3 de Interferon/fisiologia , Cirrose Hepática/etiologia , Proteínas de Membrana/fisiologia , Receptor de Interferon alfa e beta/fisiologia , Animais , Tetracloreto de Carbono/toxicidade , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Feminino , Hepatócitos/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
16.
Biochim Biophys Acta Biomembr ; 1859(5): 686-697, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28108158

RESUMO

The plant-specific DREPP protein family comprises proteins that were shown to regulate the actin and microtubular cytoskeleton in a calcium-dependent manner. Our phylogenetic analysis showed that DREPPs first appeared in ferns and that DREPPs have a rapid and plastic evolutionary history in plants. Arabidopsis DREPP paralogues called AtMDP25/PCaP1 and AtMAP18/PCaP2 are N-myristoylated, which has been reported as a key factor in plasma membrane localization. Here we show that N-myristoylation is neither conserved nor ancestral for the DREPP family. Instead, by using confocal microscopy and a new method for quantitative evaluation of protein membrane localization, we show that DREPPs rely on two mechanisms ensuring their plasma membrane localization. These include N-myristoylation and electrostatic interaction of a polybasic amino acid cluster. We propose that various plasma membrane association mechanisms resulting from the evolutionary plasticity of DREPPs are important for refining plasma membrane interaction of these signalling proteins under various conditions and in various cells.


Assuntos
Membrana Celular/química , Proteínas de Membrana/química , Proteínas de Plantas/química , Evolução Biológica , Ácido Mirístico/metabolismo , Filogenia , Eletricidade Estática
17.
J Exp Bot ; 68(15): 4185-4203, 2017 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-28922768

RESUMO

The volatile two-carbon hormone ethylene acts in concert with an array of signals to affect etiolated seedling development. From a chemical screen, we isolated a quinoline carboxamide designated ACCERBATIN (AEX) that exacerbates the 1-aminocyclopropane-1-carboxylic acid-induced triple response, typical for ethylene-treated seedlings in darkness. Phenotypic analyses revealed distinct AEX effects including inhibition of root hair development and shortening of the root meristem. Mutant analysis and reporter studies further suggested that AEX most probably acts in parallel to ethylene signaling. We demonstrated that AEX functions at the intersection of auxin metabolism and reactive oxygen species (ROS) homeostasis. AEX inhibited auxin efflux in BY-2 cells and promoted indole-3-acetic acid (IAA) oxidation in the shoot apical meristem and cotyledons of etiolated seedlings. Gene expression studies and superoxide/hydrogen peroxide staining further revealed that the disrupted auxin homeostasis was accompanied by oxidative stress. Interestingly, in light conditions, AEX exhibited properties reminiscent of the quinoline carboxylate-type auxin-like herbicides. We propose that AEX interferes with auxin transport from its major biosynthesis sites, either as a direct consequence of poor basipetal transport from the shoot meristematic region, or indirectly, through excessive IAA oxidation and ROS accumulation. Further investigation of AEX can provide new insights into the mechanisms connecting auxin and ROS homeostasis in plant development and provide useful tools to study auxin-type herbicides.


Assuntos
Aminoácidos Cíclicos/metabolismo , Arabidopsis/metabolismo , Herbicidas/química , Ácidos Indolacéticos/metabolismo , Quinolonas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Etilenos/metabolismo , Expressão Gênica , Homeostase , Plântula/metabolismo
18.
Liver Int ; 37(7): 968-973, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28345165

RESUMO

BACKGROUND & AIMS: Inflammation and impaired hepatocyte regeneration contribute to liver failure in alcoholic hepatitis (AH). Interleukin (IL)-1 is a key inflammatory cytokine in the pathobiology of AH. The role of IL-1 in liver regeneration in the recovery phase of alcohol-induced liver injury is unknown. METHODS: In this study, we tested IL-1 receptor antagonist to block IL-1 signalling in a mouse model of acute-on-chronic liver injury on liver inflammation and hepatocyte regeneration in AH. RESULTS: We observed that inhibition of IL-1 signalling decreased liver inflammation and neutrophil infiltration, and resulted in enhanced regeneration of hepatocytes and increased rate of recovery from liver injury in AH. CONCLUSION: Our novel findings suggest that IL-1 drives sustained liver inflammation and impaired hepatocyte regeneration even after cessation of ethanol exposure.


Assuntos
Insuficiência Hepática Crônica Agudizada/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Hepatite Alcoólica/tratamento farmacológico , Hepatócitos/efeitos dos fármacos , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Interleucina-1/antagonistas & inibidores , Regeneração Hepática/efeitos dos fármacos , Fígado/efeitos dos fármacos , Insuficiência Hepática Crônica Agudizada/metabolismo , Insuficiência Hepática Crônica Agudizada/patologia , Insuficiência Hepática Crônica Agudizada/fisiopatologia , Animais , Modelos Animais de Doenças , Feminino , Hepatite Alcoólica/metabolismo , Hepatite Alcoólica/patologia , Hepatite Alcoólica/fisiopatologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Interleucina-1/metabolismo , Fígado/metabolismo , Fígado/patologia , Fígado/fisiopatologia , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos/efeitos dos fármacos , Recuperação de Função Fisiológica , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo
19.
Alcohol Alcohol ; 52(4): 414-424, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28482064

RESUMO

BACKGROUND: Innate immunity plays a critical role in the development of alcohol-induced liver inflammation. Understanding the inter-relationship of signals from within and outside of the liver that trigger liver inflammation is pivotal for development of novel therapeutic targets of alcoholic liver disease (ALD). AIM: The aim of this paper is to review recent advances in the field of alcohol-induced liver inflammation. METHODS: A detailed literature review was performed using the PubMed database published between January 1980 and December 2016. RESULTS: We provide an update on the role of intestinal microbiome, metabolome and the gut-liver axis in ALD, discuss the growing body of evidence on the diversity of liver macrophages and their differential contribution to alcohol-induced liver inflammation, and highlight the crucial role of inflammasomes in integration of inflammatory signals in ALD. Studies to date have identified a multitude of new therapeutic targets, some of which are currently being tested in patients with severe alcoholic hepatitis. These treatments aim to strengthen the intestinal barrier, ameliorate liver inflammation and augment hepatocyte regeneration. CONCLUSION: Given the complexity of inflammation in ALD, multiple pathobiological mechanisms may need to be targeted at the same time as it seems unlikely that there is a single dominant pathogenic pathway in ALD that would be easily targeted using a single target drug approach. SHORT SUMMARY: Here, we focus on recent advances in immunopathogenesis of alcoholic liver disease (ALD), including gut-liver axis, hepatic macrophage activation, sterile inflammation and synergy between bacterial and sterile signals. We propose a multiple parallel hit model of inflammation in ALD and discuss its implications for clinical trials in alcoholic hepatitis.


Assuntos
Microbioma Gastrointestinal , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/microbiologia , Metaboloma , Humanos , Inflamassomos/metabolismo , Inflamação/metabolismo , Inflamação/microbiologia , Mediadores da Inflamação/metabolismo , Ativação de Macrófagos
20.
Int J Mol Sci ; 18(11)2017 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-29109378

RESUMO

Coordination of plant development requires modulation of growth responses that are under control of the phytohormone auxin. PIN-FORMED plasma membrane proteins, involved in intercellular transport of the growth regulator, are key to the transmission of such auxin signals and subject to multilevel surveillance mechanisms, including reversible post-translational modifications. Apart from well-studied PIN protein modifications, namely phosphorylation and ubiquitylation, no further post-translational modifications have been described so far. Here, we focused on root-specific Arabidopsis PIN2 and explored functional implications of two evolutionary conserved cysteines, by a combination of in silico and molecular approaches. PIN2 sequence alignments and modeling predictions indicated that both cysteines are facing the cytoplasm and therefore would be accessible to redox status-controlled modifications. Notably, mutant pin2C-A alleles retained functionality, demonstrated by their ability to almost completely rescue defects of a pin2 null allele, whereas high resolution analysis of pin2C-A localization revealed increased intracellular accumulation, and altered protein distribution within plasma membrane micro-domains. The observed effects of cysteine replacements on root growth and PIN2 localization are consistent with a model in which redox status-dependent cysteine modifications participate in the regulation of PIN2 mobility, thereby fine-tuning polar auxin transport.


Assuntos
Proteínas de Arabidopsis/metabolismo , Sequência Conservada , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Cisteína/genética , Ácidos Indolacéticos/metabolismo , Microdomínios da Membrana/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Transporte Proteico
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