RESUMO
Aluminum (Al) toxicity and inorganic phosphate (Pi) limitation are widespread chronic abiotic and mutually enhancing stresses that profoundly affect crop yield. Both stresses strongly inhibit root growth, resulting from a progressive exhaustion of the stem cell niche. Here, we report on a casein kinase 2 (CK2) inhibitor identified by its capability to maintain a functional root stem cell niche in Arabidopsis thaliana under Al toxic conditions. CK2 operates through phosphorylation of the cell cycle checkpoint activator SUPPRESSOR OF GAMMA RADIATION1 (SOG1), priming its activity under DNA-damaging conditions. In addition to yielding Al tolerance, CK2 and SOG1 inactivation prevents meristem exhaustion under Pi starvation, revealing the existence of a low Pi-induced cell cycle checkpoint that depends on the DNA damage activator ATAXIA-TELANGIECTASIA MUTATED (ATM). Overall, our data reveal an important physiological role for the plant DNA damage response pathway under agriculturally limiting growth conditions, opening new avenues to cope with Pi limitation.
Assuntos
Alumínio/toxicidade , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Caseína Quinase II/metabolismo , Fosfatos/metabolismo , Alumínio/farmacocinética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Caseína Quinase II/genética , Peptídeos e Proteínas de Sinalização Intercelular , Fosfatos/farmacologia , Fosforilação , Células Vegetais/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Inorganic phosphate (Pi) is the sole source of phosphorus for plants. It is a limiting factor for plant yield in most soils worldwide. Due to economic and environmental constraints, the use of Pi fertilizer is and will be more and more limited. Unfortunately, evaluation of Pi bioavailability or Pi starvation traits remains a tedious task, which often does not inform us about the real Pi plant status. RESULTS: Here, we identified by transcriptomic studies carried out in the plant model Arabidopsis thaliana, early roots- or leaves-conserved molecular markers for Pi starvation, exhibiting fast response to modifications of phosphate nutritional status. We identified their homologues in three crops (wheat, rapeseed, and maize) and demonstrated that they offer a reliable opportunity to monitor the actual plant internal Pi status. They turn out to be very sensitive in the concentration range of 0-50 µM which is the most common case in the vast majority of soils and situations where Pi hardly accumulates in plants. Besides in vitro conditions, they could also be validated for plants growing in the greenhouse or in open field conditions. CONCLUSION: These markers provide valuable physiological tools for plant physiologists and breeders to assess phosphate bio-availability impact on plant growth in their studies. This also offers the opportunity to cope with the rising economical (shortage) and societal problems (pollution) resulting from the management of this critical natural resource.
Assuntos
Arabidopsis , Produtos Agrícolas , Biomarcadores , Produtos Agrícolas/genética , Fenótipo , Arabidopsis/genética , FosfatosRESUMO
STOP1, an Arabidopsis transcription factor favouring root growth tolerance against Al toxicity, acts in the response to iron under low Pi (-Pi). Previous studies have shown that Al and Fe regulate the stability and accumulation of STOP1 in roots, and that the STOP1 protein is sumoylated by an unknown E3 ligase. Here, using a forward genetics suppressor screen, we identified the E3 SUMO (small ubiquitin-like modifier) ligase SIZ1 as a modulator of STOP1 signalling. Mutations in SIZ1 increase the expression of ALMT1 (a direct target of STOP1) and root growth responses to Al and Fe stress in a STOP1-dependent manner. Moreover, loss-of-function mutations in SIZ1 enhance the abundance of STOP1 in the root tip. However, no sumoylated STOP1 protein was detected by Western blot analysis in our sumoylation assay in Escherichia coli, suggesting the presence of a more sophisticated mechanism. We conclude that the sumo ligase SIZ1 negatively regulates STOP1 signalling, at least in part by modulating STOP1 protein in the root tip. Our results will allow a better understanding of this signalling pathway.
Assuntos
Alumínio/toxicidade , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Ferro/toxicidade , Ligases/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Ligases/genética , Mutação , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Estresse Fisiológico , Sumoilação , Fatores de Transcrição/genéticaRESUMO
Low-phosphate (Pi) conditions are known to repress primary root growth of Arabidopsis at low pH and in an Fe-dependent manner. This growth arrest requires accumulation of the transcription factor STOP1 in the nucleus, where it activates the transcription of the malate transporter gene ALMT1; exuded malate is suspected to interact with extracellular Fe to inhibit root growth. In addition, ALS3 - an ABC-like transporter identified for its role in tolerance to toxic Al - represses nuclear accumulation of STOP1 and the expression of ALMT1. Until now it was unclear whether Pi deficiency itself or Fe activates the accumulation of STOP1 in the nucleus. Here, by using different growth media to dissociate the effects of Fe from Pi deficiency itself, we demonstrate that Fe is sufficient to trigger the accumulation of STOP1 in the nucleus, which, in turn, activates the expression of ALMT1. We also show that a low pH is necessary to stimulate the Fe-dependent accumulation of nuclear STOP1. Furthermore, pharmacological experiments indicate that Fe inhibits proteasomal degradation of STOP1. We also show that Al acts like Fe for nuclear accumulation of STOP1 and ALMT1 expression, and that the overaccumulation of STOP1 in the nucleus of the als3 mutant grown in low-Pi conditions could be abolished by Fe deficiency. Altogether, our results indicate that, under low-Pi conditions, Fe2/3+ and Al3+ act similarly to increase the stability of STOP1 and its accumulation in the nucleus where it activates the expression of ALMT1.
Assuntos
Alumínio/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Ferro/metabolismo , Fosfatos/metabolismo , Fatores de Transcrição/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Regulação da Expressão Gênica de Plantas , Malatos , Transportadores de Ânions Orgânicos/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Fatores de Transcrição/genéticaRESUMO
Phosphate starvation-mediated induction of the HAD-type phosphatases PPsPase1 (AT1G73010) and PECP1 (AT1G17710) has been reported in Arabidopsis (Arabidopsis thaliana). However, little is known about their in vivo function or impact on plant responses to nutrient deficiency. The preferences of PPsPase1 and PECP1 for different substrates have been studied in vitro but require confirmation in planta. Here, we examined the in vivo function of both enzymes using a reverse genetics approach. We demonstrated that PPsPase1 and PECP1 affect plant phosphocholine and phosphoethanolamine content, but not the pyrophosphate-related phenotypes. These observations suggest that the enzymes play a similar role in planta related to the recycling of polar heads from membrane lipids that is triggered during phosphate starvation. Altering the expression of the genes encoding these enzymes had no effect on lipid composition, possibly due to compensation by other lipid recycling pathways triggered during phosphate starvation. Furthermore, our results indicated that PPsPase1 and PECP1 do not influence phosphate homeostasis, since the inactivation of these genes had no effect on phosphate content or on the induction of molecular markers related to phosphate starvation. A combination of transcriptomics and imaging analyses revealed that PPsPase1 and PECP1 display a highly dynamic expression pattern that closely mirrors the phosphate status. This temporal dynamism, combined with the wide range of induction levels, broad expression, and lack of a direct effect on Pi content and regulation, makes PPsPase1 and PECP1 useful molecular markers of the phosphate starvation response.
Assuntos
Proteínas de Arabidopsis/metabolismo , Etanolaminas/metabolismo , Pirofosfatase Inorgânica/metabolismo , Fosfatos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilcolina/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Homeostase , Pirofosfatase Inorgânica/genética , Lipídeos de Membrana/metabolismo , Mutação , Monoéster Fosfórico Hidrolases/genéticaRESUMO
Arabidopsis (Arabidopsis thaliana) absorbs inorganic phosphate (Pi) from the soil through an active transport process mediated by the nine members of the PHOSPHATE TRANSPORTER1 (PHT1) family. These proteins share a high level of similarity (greater than 61%), with overlapping expression patterns. The resulting genetic and functional redundancy prevents the analysis of their specific roles. To overcome this difficulty, our approach combined several mutations with gene silencing to inactivate multiple members of the PHT1 family, including a cluster of genes localized on chromosome 5 (PHT1;1, PHT1;2, and PHT1;3). Physiological analyses of these lines established that these three genes, along with PHT1;4, are the main contributors to Pi uptake. Furthermore, PHT1;1 plays an important role in translocation from roots to leaves in high phosphate conditions. These genetic tools also revealed that some PHT1 transporters likely exhibit a dual affinity for phosphate, suggesting that their activity is posttranslationally controlled. These lines display significant phosphate deficiency-related phenotypes (e.g. biomass and yield) due to a massive (80%-96%) reduction in phosphate uptake activities. These defects limited the amount of internal Pi pool, inducing compensatory mechanisms triggered by the systemic Pi starvation response. Such reactions have been uncoupled from PHT1 activity, suggesting that systemic Pi sensing is most probably acting downstream of PHT1.
Assuntos
Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Família Multigênica , Proteínas de Transporte de Fosfato/metabolismo , Fosfatos/metabolismo , Transdução de Sinais , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Mutação , Proteínas de Transporte de Fosfato/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente ModificadasRESUMO
Compartmentation of the eukaryotic cell requires a complex set of subcellular messages, including multiple retrograde signals from the chloroplast and mitochondria to the nucleus, to regulate gene expression. Here, we propose that one such signal is a phosphonucleotide (3'-phosphoadenosine 5'-phosphate [PAP]), which accumulates in Arabidopsis thaliana in response to drought and high light (HL) stress and that the enzyme SAL1 regulates its levels by dephosphorylating PAP to AMP. SAL1 accumulates in chloroplasts and mitochondria but not in the cytosol. sal1 mutants accumulate 20-fold more PAP without a marked change in inositol phosphate levels, demonstrating that PAP is a primary in vivo substrate. Significantly, transgenic targeting of SAL1 to either the nucleus or chloroplast of sal1 mutants lowers the total PAP levels and expression of the HL-inducible ASCORBATE PEROXIDASE2 gene. This indicates that PAP must be able to move between cellular compartments. The mode of action for PAP could be inhibition of 5' to 3' exoribonucleases (XRNs), as SAL1 and the nuclear XRNs modulate the expression of a similar subset of HL and drought-inducible genes, sal1 mutants accumulate XRN substrates, and PAP can inhibit yeast (Saccharomyces cerevisiae) XRNs. We propose a SAL1-PAP retrograde pathway that can alter nuclear gene expression during HL and drought stress.
Assuntos
Difosfato de Adenosina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Nucleotidases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ascorbato Peroxidases/genética , Ascorbato Peroxidases/metabolismo , Núcleo Celular/genética , Secas , Exorribonucleases/genética , Exorribonucleases/metabolismo , Regulação da Expressão Gênica de Plantas , Luz , Mitocôndrias/metabolismo , Mutação , Nucleotidases/genética , Monoéster Fosfórico Hidrolases , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
Single-molecule fluorescence in situ hybridization (smFISH) is a powerful method for the visualization and quantification of individual RNA molecules within intact cells. With its ability to probe gene expression at the single cell and single-molecule level, the technique offers valuable insights into cellular processes and cell-to-cell heterogeneity. Although widely used in the animal field, its use in plants has been limited. Here, we present an experimental smFISH workflow that allows researchers to overcome hybridization and imaging challenges in plants, including sample preparation, probe hybridization, and signal detection. Overall, this protocol holds great promise for unraveling the intricacies of gene expression regulation and RNA dynamics at the single-molecule level in whole plants.
Assuntos
RNA , Animais , Hibridização in Situ Fluorescente/métodos , RNA/genéticaRESUMO
Intracellular inorganic orthophosphate (Pi) distribution and homeostasis profoundly affect plant growth and development. However, its distribution patterns remain elusive owing to the lack of efficient cellular Pi imaging methods. Here we develop a rapid colorimetric Pi imaging method, inorganic orthophosphate staining assay (IOSA), that can semi-quantitatively image intracellular Pi with high resolution. We used IOSA to reveal the alteration of cellular Pi distribution caused by Pi starvation or mutations that alter Pi homeostasis in two model plants, rice and Arabidopsis, and found that xylem parenchyma cells and basal node sieve tube element cells play a critical role in Pi homeostasis in rice. We also used IOSA to screen for mutants altered in cellular Pi homeostasis. From this, we have identified a novel cellular Pi distribution regulator, HPA1/PHO1;1, specifically expressed in the companion and xylem parenchyma cells regulating phloem Pi translocation from the leaf tip to the leaf base in rice. Taken together, IOSA provides a powerful method for visualizing cellular Pi distribution and facilitates the analysis of Pi signalling and homeostasis from the level of the cell to the whole plant.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Oryza , Fosfatos/metabolismo , Brotos de Planta/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Homeostase/fisiologia , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/metabolismo , Oryza/genética , Oryza/metabolismoRESUMO
The regulation of intracellular pyrophosphate (PPi) level is crucial for proper morphogenesis across all taxonomic kingdoms. PPi is released as a byproduct from ~200 metabolic reactions, then hydrolyzed by either membrane-bound (H+-PPase) or soluble pyrophosphatases (PPases). In Arabidopsis, the loss of the vacuolar H+-PPase/FUGU5, a key enzyme in PPi homeostasis, results in delayed growth and a number of developmental defects, pointing to the importance of PPi homeostasis in plant morphogenesis. The Arabidopsis genome encodes several PPases in addition to FUGU5, such as PPsPase1/PECP2, VHP2;1 and VHP2;2, although their significance regarding PPi homeostasis remains elusive. Here, to assess their contribution, phenotypic analyses of cotyledon aspect ratio, palisade tissue cellular phenotypes, adaxial side pavement cell complexity, stomatal distribution, and etiolated seedling length were performed, provided that they were altered due to excess PPi in a fugu5 mutant background. Overall, our analyses revealed that the above five traits were unaffected in ppspase1/pecp2, vhp2;1 and vhp2;2 loss-of-function mutants, as well as in fugu5 mutant lines constitutively overexpressing PPsPase1/PECP2. Furthermore, metabolomics revealed that ppspase1/pecp2, vhp2;1 and vhp2;2 etiolated seedlings exhibited metabolic profiles comparable to the wild type. Together, these results indicate that the contribution of PPsPase1/PECP2, VHP2;1 and VHP2;2 to PPi levels is negligible in comparison to FUGU5 in the early stages of seedling development.
RESUMO
New regulatory functions in plant development and environmental stress responses have recently emerged for a number of apocarotenoids produced by enzymatic or nonenzymatic oxidation of carotenoids. ß-Cyclocitric acid (ß-CCA) is one such compound derived from ß-carotene, which triggers defense mechanisms leading to a marked enhancement of plant tolerance to drought stress. We show here that this response is associated with an inhibition of root growth affecting both root cell elongation and division. Remarkably, ß-CCA selectively induced cell cycle inhibitors of the SIAMESE-RELATED (SMR) family, especially SMR5, in root tip cells. Overexpression of the SMR5 gene in Arabidopsis induced molecular and physiological changes that mimicked in large part the effects of ß-CCA. In particular, the SMR5 overexpressors exhibited an inhibition of root development and a marked increase in drought tolerance which is not related to stomatal closure. SMR5 up-regulation induced changes in gene expression that strongly overlapped with the ß-CCA-induced transcriptomic changes. Both ß-CCA and SMR5 led to a down-regulation of many cell cycle activators (cyclins, cyclin-dependent kinases) and a concomitant up-regulation of genes related to water deprivation, cellular detoxification, and biosynthesis of lipid biopolymers such as suberin and lignin. This was correlated with an accumulation of suberin lipid polyesters in the roots and a decrease in nonstomatal leaf transpiration. Taken together, our results identify the ß-CCA-inducible and drought-inducible SMR5 gene as a key component of a stress-signaling pathway that reorients root metabolism from growth to multiple defense mechanisms leading to drought tolerance.
RESUMO
Aluminum (Al) is a major limiting factor for crop production on acidic soils, inhibiting root growth and plant development. At acidic pH (pH < 5.5), Al3+ ions are the main form of Al present in the media. Al3+ ions have an increased solubility at pH < 5.5 and result in plant toxicity. At higher pH, the free Al3+ fraction decreases in the media, but whether plants can detect Al at these pHs remain unknown. To cope with Al stress, the SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) transcription factor induces AL-ACTIVATED MALATE TRANSPORTER1 (ALMT1), a malate-exuding transporter as a strategy to chelate the toxic ions in the rhizosphere. Here, we uncoupled the Al signalling pathway that controls STOP1 from Al toxicity using wild type (WT) and two stop1 mutants carrying the pALMT1:GUS construct with an agar powder naturally containing low amounts of phosphate, iron (Fe), and Al. We combined gene expression [real-time PCR (RT-PCR) and the pALMT1:GUS reporter], confocal microscopy (pSTOP1:GFP-STOP1 reporter), and root growth measurement to assess the effects of Al and Fe on the STOP1-ALMT1 pathway in roots. Our results show that Al triggers STOP1 signaling at a concentration as little as 2 µM and can be detected at a pH above 6.0. We observed that at pH 5.7, 20 µM AlCl3 induces ALMT1 in WT but does not inhibit root growth in stop1 Al-hypersensitive mutants. Increasing AlCl3 concentration (>50 µM) at pH 5.7 results in the inhibition of the stop1 mutants primary root. Using the green fluorescent protein (GFP)-STOP1 and ALMT1 reporters, we show that the Al signal pathway can be uncoupled from the Al toxicity on the root. Furthermore, we observe that Al strengthens the Fe-mediated inhibition of primary root growth in WT, suggesting an interaction between Fe and Al on the STOP1-ALMT1 pathway.
RESUMO
Soil availability of inorganic ortho-phosphate (PO43-, Pi) is a key determinant of plant growth and fitness.1 Plants regulate the capacity of their roots to take up inorganic phosphate by adapting the abundance of H+-coupled phosphate transporters of the PHOSPHATE TRANSPORTER 1 (PHT1) family2 at the plasma membrane (PM) through transcriptional and post-translational changes driven by the genetic network of the phosphate starvation response (PSR).3-8 Increasing evidence also shows that plants integrate immune responses to alleviate phosphate starvation stress through the association with beneficial microbes.9-11 Whether and how such phosphate transport is regulated upon activation of immune responses is yet uncharacterized. To address this question, we first developed quantitative assays based on changes in the electrical PM potential to measure active Pi transport in roots in real time. By inserting micro-electrodes into bulging root hairs, we were able to determine key characteristics of phosphate transport in intact Arabidopsis thaliana (hereafter Arabidopsis) seedlings. The fast Pi-induced depolarization observed was dependent on the activity of the major phosphate transporter PHT1;4. Notably, we observed that this PHT1;4-mediated phosphate uptake is repressed upon activation of pattern-triggered immunity. This inhibition depended on the receptor-like cytoplasmic kinases BOTRYTIS-INDUCED KINASE 1 (BIK1) and PBS1-LIKE KINASE 1 (PBL1), which both phosphorylated PHT1;4. As a corollary to this negative regulation of phosphate transport by immune signaling, we found that PHT1;4-mediated phosphate uptake normally negatively regulates anti-bacterial immunity in roots. Collectively, our results reveal a mechanism linking plant immunity and phosphate homeostasis, with BIK1/PBL1 providing a molecular integration point between these two important pathways.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismo , Fosfatos/metabolismo , Raízes de Plantas/metabolismoRESUMO
Plants are constantly adapting to ambient fluctuations through spatial and temporal transcriptional responses. Here, we implemented the latest-generation RNA imaging system and combined it with microfluidics to visualize transcriptional regulation in living Arabidopsis plants. This enabled quantitative measurements of the transcriptional activity of single loci in single cells, in real time and under changing environmental conditions. Using phosphate-responsive genes as a model, we found that active genes displayed high transcription initiation rates (one initiation event every ~3 s) and frequently clustered together in endoreplicated cells. We observed gene bursting and large allelic differences in single cells, revealing that at steady state, intrinsic noise dominated extrinsic variations. Moreover, we established that transcriptional repression triggered in roots by phosphate, a crucial macronutrient limiting plant development, occurred with unexpectedly fast kinetics (on the order of minutes) and striking heterogeneity between neighbouring cells. Access to single-cell RNA polymerase II dynamics in live plants will benefit future studies of signalling processes.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Fosfatos/metabolismo , Células Vegetais/metabolismo , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia , Transcrição Gênica , Regulação da Expressão Gênica de Plantas , Cinética , RNA Polimerase II/genéticaRESUMO
Environmental cues profoundly modulate cell proliferation and cell elongation to inform and direct plant growth and development. External phosphate (Pi) limitation inhibits primary root growth in many plant species. However, the underlying Pi sensory mechanisms are unknown. Here we genetically uncouple two Pi sensing pathways in the root apex of Arabidopsis thaliana. First, the rapid inhibition of cell elongation in the transition zone is controlled by transcription factor STOP1, by its direct target, ALMT1, encoding a malate channel, and by ferroxidase LPR1, which together mediate Fe and peroxidase-dependent cell wall stiffening. Second, during the subsequent slow inhibition of cell proliferation in the apical meristem, which is mediated by LPR1-dependent, but largely STOP1-ALMT1-independent, Fe and callose accumulate in the stem cell niche, leading to meristem reduction. Our work uncovers STOP1 and ALMT1 as a signalling pathway of low Pi availability and exuded malate as an unexpected apoplastic inhibitor of root cell wall expansion.
Assuntos
Proteínas de Arabidopsis/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Fosfatos/metabolismo , Raízes de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Crescimento Celular , Parede Celular/genética , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Ferro/metabolismo , Malatos/metabolismo , Meristema/citologia , Meristema/genética , Meristema/metabolismo , Transportadores de Ânions Orgânicos/genética , Oxirredutases/genética , Oxirredutases/metabolismo , Peroxidase/genética , Peroxidase/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Transdução de Sinais/genética , Fatores de Transcrição/genéticaRESUMO
AtHMA4 is an Arabidopsis thaliana P1B-ATPase which transports Zn and Cd. Here, we demonstrate that AtHMA4 is localized at the plasma membrane and expressed in tissues surrounding the root vascular vessels. The ectopic overexpression of AtHMA4 improved the root growth in the presence of toxic concentrations of Zn, Cd and Co. A null mutant exhibited a lower translocation of Zn and Cd from the roots to shoot. In contrast, the AtHMA4 overexpressing lines displayed an increase in the zinc and cadmium shoot content. Altogether, these results strongly indicate that AtHMA4 plays a role in metal loading in the xylem.
Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Cobre/metabolismo , Zinco/metabolismo , Arabidopsis/genética , Sequência de Bases , Transporte Biológico , Clonagem Molecular , Primers do DNA , Tolerância a Medicamentos , Éxons/genética , Perfilação da Expressão Gênica , Íntrons/genéticaRESUMO
The objective of the present study was to compare two configurations of the hepatocyte model namely suspensions (SH) and conventional primary cultures (CPC) for their ability to predict the hepatic clearance in vivo in the rat and, to investigate the impact of serum on the prediction accuracy. The metabolic competences of several cytochrome P450 isoenzymes were investigated both in CPC and SH in the presence or absence of serum. Under the same conditions, the in vitro intrinsic clearance of six test compounds metabolised by a variety of phase I and phase II enzymes (antipyrine, RO-X, mibefradil, midazolam, naloxone and oxazepam) were derived from Vmax/Km scaled up to the corresponding in vivo hepatic metabolic clearance. CYP activities were shown to be stable in both CPC and SH for up to 6 h of incubation, except for the CYP 3A1 activity that decreased in CPC even in the presence of serum. Moreover, the clearances predicted from SH in the presence of serum were closer to the in vivo values than those obtained from CPC. SH represent a convenient model to assess the hepatic metabolism of xenobiotics, the presence of serum in the incubation medium significantly improved in several instances the quality of the predictions.
Assuntos
Meios de Cultura/química , Hepatócitos/metabolismo , Preparações Farmacêuticas/metabolismo , Farmacocinética , Animais , Células Cultivadas , Cromatografia Líquida , Sistema Enzimático do Citocromo P-450/metabolismo , Hepatócitos/enzimologia , Técnicas In Vitro , Fígado/enzimologia , Masculino , Espectrometria de Massas , Microssomos Hepáticos/enzimologia , Valor Preditivo dos Testes , Ligação Proteica , Ratos , Ratos WistarRESUMO
Repeated administration of thioacetamide (TA), either intraperitoneally or in drinking water, produced liver cirrhosis in normal Sprague-Dawley rats (SDR) with significant histological alterations similar to those observed in human cirrhosis. In the present study, we evaluated the ability of TA to induce liver cirrhosis in mutant Nagase analbuminaemic SDR. Thioacetamide was administered either intraperitoneally up to 4 months or in drinking water up to 6 months to normal and to Nagase analbuminaemic SDR. Nagase analbuminaemic rats (NAR) were also administered TA in drinking water up to 10 months. Liver cirrhosis development was determined by macroscopic and microscopic analysis. In contrast to normal SDR, no histological characteristics of cirrhosis could be observed in NAR submitted to a 4 or 6 months treatment with TA. Such failure to induce cirrhosis in Nagase rats was confirmed even after prolonged TA administration in drinking water for up to 10 months. In contrast, fibrosis and cholangiolar proliferation occurred in the 10-month TA-treated analbuminaemic rats, suggesting that the mechanisms involved in cirrhosis induction are different from those involved in fibrosis development and carcinogenesis. It is unlikely that the protective effect against TA-induced cirrhosis observed in analbuminaemic rats is related to the absence of albumin in this rat strain, since a co-administration of TA with albumin in analbuminaemic rats did not restore the potential for TA to induce cirrhosis in this rat strain. In conclusion, the fact that induction of cirrhosis by TA is prevented in the inherently hyperlipidaemic and hypercholesterolaemic analbuminaemic rats could be considered for potential application in the treatment of clinical cirrhosis.
Assuntos
Cirrose Hepática/induzido quimicamente , Albumina Sérica/deficiência , Tioacetamida/toxicidade , Administração Oral , Animais , Ductos Biliares Intra-Hepáticos/efeitos dos fármacos , Ductos Biliares Intra-Hepáticos/patologia , Divisão Celular/efeitos dos fármacos , Colangite/induzido quimicamente , Colangite/patologia , Modelos Animais de Doenças , Hipercolesterolemia/genética , Injeções Intraperitoneais , Cirrose Hepática/genética , Cirrose Hepática/patologia , Cirrose Hepática/prevenção & controle , Ratos , Ratos Mutantes , Ratos Sprague-Dawley , Albumina Sérica/genética , Tioacetamida/administração & dosagem , Abastecimento de ÁguaRESUMO
BACKGROUND: Mutations in the FRY1/SAL1 Arabidopsis locus are highly pleiotropic, affecting drought tolerance, leaf shape and root growth. FRY1 encodes a nucleotide phosphatase that in vitro has inositol polyphosphate 1-phosphatase and 3',(2'),5'-bisphosphate nucleotide phosphatase activities. It is not clear which activity mediates each of the diverse biological functions of FRY1 in planta. PRINCIPAL FINDINGS: A fry1 mutant was identified in a genetic screen for Arabidopsis mutants deregulated in the expression of Pi High affinity Transporter 1;4 (PHT1;4). Histological analysis revealed that, in roots, FRY1 expression was restricted to the stele and meristems. The fry1 mutant displayed an altered root architecture phenotype and an increased drought tolerance. All of the phenotypes analyzed were complemented with the AHL gene encoding a protein that converts 3'-polyadenosine 5'-phosphate (PAP) into AMP and Pi. PAP is known to inhibit exoribonucleases (XRN) in vitro. Accordingly, an xrn triple mutant with mutations in all three XRNs shared the fry1 drought tolerance and root architecture phenotypes. Interestingly these two traits were also complemented by grafting, revealing that drought tolerance was primarily conferred by the rosette and that the root architecture can be complemented by long-distance regulation derived from leaves. By contrast, PHT1 expression was not altered in xrn mutants or in grafting experiments. Thus, PHT1 up-regulation probably resulted from a local depletion of Pi in the fry1 stele. This hypothesis is supported by the identification of other genes modulated by Pi deficiency in the stele, which are found induced in a fry1 background. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the 3',(2'),5'-bisphosphate nucleotide phosphatase activity of FRY1 is involved in long-distance as well as local regulatory activities in roots. The local up-regulation of PHT1 genes transcription in roots likely results from local depletion of Pi and is independent of the XRNs.
Assuntos
Arabidopsis , Exorribonucleases/metabolismo , Mutação , Monoéster Fosfórico Hidrolases/genética , Raízes de Plantas/enzimologia , Alelos , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Exorribonucleases/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Genes de Plantas/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Mutação/fisiologia , Fenótipo , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Inanição/genética , Inanição/patologiaRESUMO
RNA transcript levels of Arabidopsis plants, infected by the rhizobacterium Pseudomonas thivervalensis (strain MLG45), and axenic control plants were compared using cDNA microarrays representing approximately 14 300 genes. The analysis revealed an increase of defence-related transcripts in the shoots of bacterized plants relative to control (axenic) plants. These modifications of transcript levels were confirmed by physiological experiments. Plants infected with P. thivervalensis were more resistant to subsequent infections by the virulent pathogen P. syringae pv. tomato (strain DC3000) than control plants. In addition, photosynthesis rates were repressed consistently with the reduced growth of plants colonized by P. thivervalensis. These results highlight the value of molecular phenotyping to predict physiological changes.