RESUMO
Ornithine lipids (OLs) are phosphorus-free lipids found in many bacteria grown under phosphate deprivation, a condition that activates the PhoBR system and leads to phosphate uptake and metabolism. Two OL synthesis pathways have already been described. One depends on OlsB and OlsA acyltransferases to add, respectively, the first and second acyl chains to an ornithine molecule. The other pathway is carried out by OlsF, a bifunctional enzyme responsible for both acylation steps. Although Vibrio cholerae lacks olsBA genes, an olsF homologue (vc0489) was identified in its genome. In this work we demonstrated that V. cholerae produces OLs and expresses vc0489 in response to phosphate depletion, in a PhoBR-dependent manner. In Escherichia coli, under similar condition, vc0489 expression leads to OL accumulation. These results indicate a strong connection between OL synthesis and VC0489 from V. cholerae and, for the first time, a direct regulation of an olsF homologue by the PhoBR system.
Assuntos
Aciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Ornitina/análogos & derivados , Fosfatos/deficiência , Vibrio cholerae/metabolismo , Aciltransferases/genética , Proteínas de Bactérias/genética , Sítios de Ligação , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Lipídeos , Lipídeos de Membrana/química , Mutação , Ornitina/metabolismo , Fosfatos/metabolismo , Vibrio cholerae/genéticaRESUMO
A combined strategy of phosphate (Pi) remobilization from internal and external RNA sources seems to be conserved in plants exposed to Pi starvation. Thus far, the only ribonucleases (RNases) reported to be induced in Nicotiana alata undergoing Pi deprivation are extracellular S-like RNase NE and NnSR1. NnSR1 is a class III non S-RNase of unknown subcellular location. Here, we examine the hypothesis that NnSR1 is an intracellular RNase derived from the self-incompatibility system with specific expression in self-incompatible Nicotiana alata. NnSR1 was not induced in self-compatible Nicotiana species exposed to Pi deprivation. NnSR1 conjugated with a fluorescent protein and transiently expressed in Arabidopsis protoplasts and Nicotiana leaves showed that the fusion protein co-localized with an endoplasmic reticulum (ER) marker. Subcellular fractionation by ultracentrifugation of roots exposed to Pi deprivation revealed that the native NnSR1 migrated in parallel with the BiP protein, a typical ER marker. To our knowledge, NnSR1 is the first class III RNase reported to be localized in ER compartments. The induction of NnSR1 was detected earlier than the extracellular RNase NE, suggesting that intracellular RNA may be the first source of Pi used by the cell under Pi stress.
Assuntos
Regulação da Expressão Gênica de Plantas , Nicotiana/genética , Proteínas de Plantas/genética , Ribonucleases/genética , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Retículo Endoplasmático/metabolismo , Dados de Sequência Molecular , Fosfatos/deficiência , Filogenia , Folhas de Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Protoplastos/metabolismo , Ribonucleases/química , Ribonucleases/metabolismo , Alinhamento de Sequência , Nicotiana/enzimologiaRESUMO
BACKGROUND AND AIMS: Non-S-ribonucleases (non-S-RNases) are class III T2 RNases constitutively expressed in styles of species with S-RNase-based self-incompatibility. So far, no function has been attributed to these RNases. The aim of this work is to examine if NnSR1, a non-S-RNase from Nicotiana alata, is induced under conditions of phosphate (Pi) deprivation. The hypothesis is that under Pi-limited conditions, non-S-RNase functions may resemble the role of S-like RNases. To date, the only RNases reported to be induced by Pi deficiency are class I and class II S-like RNases, which are phylogenetically different from the class III clade of RNases. METHODS: Gene and protein expression of NnSR1 were assayed in plants grown hydroponically with and without Pi, by combining RT-PCR, immunoblot and enzymatic activity approaches. KEY RESULTS: NnSR1 transcripts were detected in roots 7 d after Pi deprivation and remained stable for several days. Transcript expression was correlated based on Pi availability in the culture medium. Antiserum against a peptide based on a hypervariable domain of NnSR1 recognized NnSR1 in roots and stems but not leaves exposed to Pi shortage. NnSR1 was not detected in culture medium and was pelleted with the microsomal fraction, suggesting that it was membrane-associated or included in large compartments. The anti-NnSR1 inhibited selectively the enzymatic activity of a 31-kDa RNase indicating that NnSR1 was induced in an enzymatically active form. CONCLUSIONS: The induction of NnSR1 indicates that there is a general recruitment of all classes of T2 RNases in response to Pi shortage. NnSR1 appears to have regained ancestral functions of class III RNases related to strategies to cope with Pi limitation and also possibly with other environmental challenges. This constitutes the first report for a specific function of class III RNases other than S-RNases.
Assuntos
Endorribonucleases/metabolismo , Flores/enzimologia , Nicotiana/enzimologia , Fosfatos/deficiência , Proteínas de Plantas/metabolismo , Raízes de Plantas/enzimologia , Caules de Planta/enzimologia , Endorribonucleases/genética , Flores/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Especificidade de Órgãos/efeitos dos fármacos , Fosfatos/farmacologia , Proteínas de Plantas/genética , Raízes de Plantas/efeitos dos fármacos , Caules de Planta/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Nicotiana/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Proper root growth is crucial for anchorage, exploration, and exploitation of the soil substrate. Root growth is highly sensitive to a variety of environmental cues, among them water and nutrient availability have a great impact on root development. Phosphorus (P) availability is one of the most limiting nutrients that affect plant growth and development under natural and agricultural environments. Root growth in the direction of the long axis proceeds from the root tip and requires the coordinated activities of cell proliferation, cell elongation and cell differentiation. Here we report a novel gene, APSR1 (Altered Phosphate Starvation Response1), involved in root meristem maintenance. The loss of function mutant apsr1-1 showed a reduction in primary root length and root apical meristem size, short differentiated epidermal cells and long root hairs. Expression of APSR1 gene decreases in response to phosphate starvation and apsr1-1 did not show the typical progressive decrease of undifferentiated cells at root tip when grown under P limiting conditions. Interestingly, APSR1 expression pattern overlaps with root zones of auxin accumulation. Furthermore, apsr1-1 showed a clear decrease in the level of the auxin transporter PIN7. These data suggest that APSR1 is required for the coordination of cell processes necessary for correct root growth in response to phosphate starvation conceivably by direct or indirect modulation of PIN7. We also propose, based on its nuclear localization and structure, that APSR1 may potentially be a member of a novel group of transcription factors.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Fosfatos/deficiência , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Diferenciação Celular , Ácidos Indolacéticos/metabolismo , Meristema/citologia , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Modelos Moleculares , Mutação , Especificidade de Órgãos , Fenótipo , Filogenia , Epiderme Vegetal/citologia , Epiderme Vegetal/genética , Epiderme Vegetal/crescimento & desenvolvimento , Epiderme Vegetal/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Brotos de Planta/citologia , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Proteínas Recombinantes de Fusão , Plântula/citologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Análise de Sequência de DNA , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Plants have evolved a plethora of responses to cope with phosphate (Pi) deficiency, including the transcriptional activation of a large set of genes. Among Pi-responsive genes, the expression of the Arabidopsis phospholipase DZ2 (PLDZ2) is activated to participate in the degradation of phospholipids in roots in order to release Pi to support other cellular activities. A deletion analysis was performed to identify the regions determining the strength, tissue-specific expression, and Pi responsiveness of this regulatory region. This study also reports the identification and characterization of a transcriptional enhancer element that is present in the PLDZ2 promoter and able to confer Pi responsiveness to a minimal, inactive 35S promoter. This enhancer also shares the cytokinin and sucrose responsive properties observed for the intact PLDZ2 promoter. The EZ2 element contains two P1BS motifs, each of which is the DNA binding site of transcription factor PHR1. Mutation analysis showed that the P1BS motifs present in EZ2 are necessary but not sufficient for the enhancer function, revealing the importance of adjacent sequences. The structural organization of EZ2 is conserved in the orthologous genes of at least eight families of rosids, suggesting that architectural features such as the distance between the two P1BS motifs are also important for the regulatory properties of this enhancer element.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica de Plantas/genética , Fosfatos/deficiência , Fosfolipase D/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis/citologia , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Análise Mutacional de DNA , Dados de Sequência Molecular , Especificidade de Órgãos , Fosfolipase D/metabolismo , Fosfolipídeos/metabolismo , Filogenia , Raízes de Plantas/citologia , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Alinhamento de Sequência , Deleção de SequênciaRESUMO
The survival of plants, as sessile organisms, depends on a series of postembryonic developmental events that determine the final architecture of plants and allow them to contend with a continuously changing environment. Modulation of cell differentiation and organ formation by environmental signals has not been studied in detail. Here, we report that alterations in the pattern of lateral root (LR) formation and emergence in response to phosphate (Pi) availability is mediated by changes in auxin sensitivity in Arabidopsis thaliana roots. These changes alter the expression of auxin-responsive genes and stimulate pericycle cells to proliferate. Modulation of auxin sensitivity by Pi was found to depend on the auxin receptor TRANSPORT INHIBITOR RESPONSE1 (TIR1) and the transcription factor AUXIN RESPONSE FACTOR19 (ARF19). We determined that Pi deprivation increases the expression of TIR1 in Arabidopsis seedlings and causes AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) auxin response repressors to be degraded. Based on our results, we propose a model in which auxin sensitivity is enhanced in Pi-deprived plants by an increased expression of TIR1, which accelerates the degradation of AUX/IAA proteins, thereby unshackling ARF transcription factors that activate/repress genes involved in LR formation and emergence.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácidos Indolacéticos/farmacologia , Fosfatos/deficiência , Fosfatos/fisiologia , Raízes de Plantas/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The preg gene encodes a cyclin-like protein that is implicated in the derepression of nucleases and phosphatases that scavenge phosphate from the environment. To better understand the regulatory role of the preg gene product, the differential display reverse transcriptase - polymerase chain reaction was used to isolate transcripts differentially expressed in the pregc mutant strain of the mold Neurospora crassa grown under phosphate starvation, at pH 7.8. Two transcripts, whose differential expressions were confirmed by Northern blotting, were downregulated in a strain of N. crassa carrying a loss-of-function mutation in the preg gene (preg(c) allele). These transcripts revealed genes coding for enzymes involved in the thymidine salvage pathway (iso-orotate decarboxylase) and in the biosynthesis of coenzyme Q (ubiquinone C-methyltransferase), which may be relevant to a further understanding of the molecular events involved in the phosphorus sensing in N. crassa.
Assuntos
Carboxiliases/genética , Proteínas Fúngicas/genética , Mutação , Neurospora crassa/genética , Fosfatos/deficiência , Timidina/biossíntese , Northern Blotting , Carboxiliases/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/metabolismo , Fosfatos/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
Proteoid roots play a major role in enabling white lupin (Lupinus albus L.) to adapt to phosphate (Pi) deficiency. Such roots release citrate from proteoid rootlets, which allows this species to mobilize Pi from sparingly soluble Pi sources. Release of citrate is preceded by a significant accumulation of organic acids, in which a Pi deficiency-inducible phosphoenolpyruvate carboxylase (PEPC) activity has been involved. To gain an insight into this adaptive mechanism, the expression of three different transcripts coding for PEPC was examined in proteoid rootlets of Pi-starved and Pi-starved-and-rescued white lupin. Semi-quantitative reverse transcriptase (RT)-PCR experiments performed with gene-specific primers targeted to the 3'-end region of the corresponding cDNAs revealed that the transcripts for these three PEPCs differentially accumulate in both Pi-starved and Pi-starved-and-rescued proteoid rootlets. Semi-quantitative RT-PCR analysis in Pi-starved proteoid rootlets sampled at different times after being rescued from Pi deficiency showed that Pi levels differentially down-regulated the three PEPC transcripts. RT-PCR experiments were further extended to Pi-starved and Pi-fed whole roots, cotyledons, and leaves on which a tissue-specific, Pi-dependent PEPC expression was observed. These results indicate that there exists at least three different transcripts coding for PEPC in proteoid root clusters of white lupin, whose expression are differentially regulated by Pi.
Assuntos
Regulação da Expressão Gênica de Plantas/fisiologia , Lupinus/enzimologia , Fosfatos/deficiência , Fosfoenolpiruvato Carboxilase/biossíntese , Raízes de Plantas/enzimologia , Sequência de Bases , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Isoenzimas/biossíntese , Lupinus/genética , Dados de Sequência Molecular , Fosfoenolpiruvato Carboxilase/genética , RNA Mensageiro/metabolismo , Alinhamento de SequênciaRESUMO
We retrieved a series of measurements made 35 years ago of the concentration of inorganic phosphate (P) in the serum from 56 cases of severe protein-energy malnutrition at the Tropical Metabolism Research Unit, Jamaica. There is no record of whether or not the cases were randomly selected. The samples were obtained within 4 days of admission and except in 3 cases there was no follow-up. The average age was 12 months. The children have been classified retrospectively from the notes as marasmus (11 cases), kwashiorkor (22 cases) and marasmic kwashiokor (23 cases). In all 11 children died (fatality rate 20 percent), eight of them from the group with marasmic kwashiorkor. Weight-for-age, length-for-age and weight-for-length have been calculated as Z-scores. Nearly all serum phosphate concentrations were low (mean 1.41 mmol.1-1, SD 0.444, range 0.50-2.45) compared with the normal value at this age of about 2 mmol.1-1. The serum P was significantly less depressed in the marasmic children (P=0.042), but there was no relation between serum P and any of the anthropometric measurements, nor with outcome (death or survival). There was, however, a significant relationship with the degree of oedema. Death was related to age - the children who died were younger (mean difference 3.8 months; P=0.01; 95 percent confidence interval 0.23-6.43). It took about 3 weeks of feeding a milk-based diet for serum phosphate to reach normal levels. There have been few previous measurements of serum P in malnutrition. We agree with previous authors that the low serum values are evidence of phosphate depletion and suggest that phosphate might be added to the electrolyte solutions used in the early stages of recovery. However, reports of adverse effects indicate that this should be done with great care (AU)
Assuntos
Lactente , Humanos , Transtornos da Nutrição Infantil/sangue , Fosfatos/sangue , Desnutrição Proteico-Calórica/sangue , Estatura , Peso Corporal , Transtornos da Nutrição Infantil/classificação , Transtornos da Nutrição Infantil/dietoterapia , Transtornos da Nutrição Infantil/mortalidade , Intervalos de Confiança , Seguimentos , Fosfatos/deficiência , Desnutrição Proteico-Calórica/classificação , Desnutrição Proteico-Calórica/dietoterapia , Desnutrição Proteico-Calórica/mortalidade , Valores de Referência , Estudos Retrospectivos , Índice de Gravidade de Doença , Taxa de Sobrevida , Fatores EtáriosRESUMO
We treated a 10 6/12 year old prepubertal male with hypophosphatemic rickets, who was growing poorly despite appropriate treatment with calcitriol and phosphate, with exogenous growth hormone (for an initial trial period of 4 months, followed by 14 months of continuous treatment at a dose of 4 IU three times weekly) even though his growth hormone testing proved to be normal. His growth rate increased significantly during treatment with synthetic growth hormone (from a basal rate of 3.9 cm/yr to 9 cm/yr during the first 4 months of therapy and from 2.7 cm/yr to 6.0 cm/yr during next 14 months of treatment) and his predicted adult height increased as well. Slight metabolic changes were detected in this patient during treatment, with an increase in serum phosphorus and a decrease in twenty-four hour urine calcium concentrations. It would seem reasonable to evaluate the growth hormone status of children with hypophosphatemic rickets who are growing poorly despite appropriate therapy with calcitriol and phosphate and to consider a trial period of therapy with growth hormone in some of them.