996 resultados para Arabidopsis Proteins


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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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Xanthomonas axonopodis pv. citri, the bacterium responsible for citrus canker, uses effector proteins secreted by a type III protein secretion system to colonize its hosts. Among the putative effector proteins identified for this bacterium, we focused on the analysis of the roles of AvrXacE1, AvrXacE2 and Xac3090 in pathogenicity and their interactions with host plant proteins. Bacterial deletion mutants in avrXacE1, avrXacE2 and xac3090 were constructed and evaluated in pathogenicity assays. The avrXacE1 and avrXacE2 mutants presented lesions with larger necrotic areas relative to the wild-type strain when infiltrated in citrus leaves. Yeast two-hybrid studies were used to identify several plant proteins likely to interact with AvrXacE1, AvrXacE2 and Xac3090. We also assessed the localization of these effector proteins fused to green fluorescent protein in the plant cell, and observed that they co-localized to the subcellular spaces in which the plant proteins with which they interacted were predicted to be confined. Our results suggest that, although AvrXacE1 localizes to the plant cell nucleus, where it interacts with transcription factors and DNA-binding proteins, AvrXacE2 appears to be involved in lesion-stimulating disease 1-mediated cell death, and Xac3090 is directed to the chloroplast where its function remains to be clarified.

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Abstract Background Papaya (Carica papaya L.) is a commercially important crop that produces climacteric fruits with a soft and sweet pulp that contain a wide range of health promoting phytochemicals. Despite its importance, little is known about transcriptional modifications during papaya fruit ripening and their control. In this study we report the analysis of ripe papaya transcriptome by using a cross-species (XSpecies) microarray technique based on the phylogenetic proximity between papaya and Arabidopsis thaliana. Results Papaya transcriptome analyses resulted in the identification of 414 ripening-related genes with some having their expression validated by qPCR. The transcription profile was compared with that from ripening tomato and grape. There were many similarities between papaya and tomato especially with respect to the expression of genes encoding proteins involved in primary metabolism, regulation of transcription, biotic and abiotic stress and cell wall metabolism. XSpecies microarray data indicated that transcription factors (TFs) of the MADS-box, NAC and AP2/ERF gene families were involved in the control of papaya ripening and revealed that cell wall-related gene expression in papaya had similarities to the expression profiles seen in Arabidopsis during hypocotyl development. Conclusion The cross-species array experiment identified a ripening-related set of genes in papaya allowing the comparison of transcription control between papaya and other fruit bearing taxa during the ripening process.

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In Arabidopsis (Arabidopsis thaliana), the blue light photoreceptor phototropins (phot1 and phot2) fine-tune the photosynthetic status of the plant by controlling several important adaptive processes in response to environmental light variations. These processes include stem and petiole phototropism (leaf positioning), leaf flattening, stomatal opening, and chloroplast movements. The PHYTOCHROME KINASE SUBSTRATE (PKS) protein family comprises four members in Arabidopsis (PKS1-PKS4). PKS1 is a novel phot1 signaling element during phototropism, as it interacts with phot1 and the important signaling element NONPHOTOTROPIC HYPOCOTYL3 (NPH3) and is required for normal phot1-mediated phototropism. In this study, we have analyzed more globally the role of three PKS members (PKS1, PKS2, and PKS4). Systematic analysis of mutants reveals that PKS2 (and to a lesser extent PKS1) act in the same subset of phototropin-controlled responses as NPH3, namely leaf flattening and positioning. PKS1, PKS2, and NPH3 coimmunoprecipitate with both phot1-green fluorescent protein and phot2-green fluorescent protein in leaf extracts. Genetic experiments position PKS2 within phot1 and phot2 pathways controlling leaf positioning and leaf flattening, respectively. NPH3 can act in both phot1 and phot2 pathways, and synergistic interactions observed between pks2 and nph3 mutants suggest complementary roles of PKS2 and NPH3 during phototropin signaling. Finally, several observations further suggest that PKS2 may regulate leaf flattening and positioning by controlling auxin homeostasis. Together with previous findings, our results indicate that the PKS proteins represent an important family of phototropin signaling proteins.

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Phyllotaxis, the regular arrangement of leaves and flowers around the stem, is a key feature of plant architecture. Current models propose that the spatiotemporal regulation of organ initiation is controlled by a positive feedback loop between the plant hormone auxin and its efflux carrier PIN-FORMED1 (PIN1). Consequently, pin1 mutants give rise to naked inflorescence stalks with few or no flowers, indicating that PIN1 plays a crucial role in organ initiation. However, pin1 mutants do produce leaves. In order to understand the regulatory mechanisms controlling leaf initiation in Arabidopsis (Arabidopsis thaliana) rosettes, we have characterized the vegetative pin1 phenotype in detail. We show that although the timing of leaf initiation in vegetative pin1 mutants is variable and divergence angles clearly deviate from the canonical 137° value, leaves are not positioned at random during early developmental stages. Our data further indicate that other PIN proteins are unlikely to explain the persistence of leaf initiation and positioning during pin1 vegetative development. Thus, phyllotaxis appears to be more complex than suggested by current mechanistic models.

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Plants have a remarkable potential for sustained (indeterminate) postembryonic growth. Following their specification in the early embryo, tissue-specific precursor cells first establish tissues and later maintain them postembryonically. The mechanisms underlying these processes are largely unknown. Here we define local control of oriented, periclinal cell division as the mechanism underlying both the establishment and maintenance of vascular tissue. We identify an auxin-regulated basic helix-loop-helix (bHLH) transcription factor dimer as a critical regulator of vascular development. Due to a loss of periclinal divisions, vascular tissue gradually disappears in bHLH-deficient mutants; conversely, ectopic expression is sufficient for triggering periclinal divisions. We show that this dimer operates independently of tissue identity but is restricted to a small vascular domain by integrating overlapping transcription patterns of the interacting bHLH proteins. Our work reveals a common mechanism for tissue establishment and indeterminate vascular development and provides a conceptual framework for developmental control of local cell divisions.

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For the efficient translocation of organic nitrogen, small peptides of two to three amino acids are posited as an important alternative to amino acids. A new transporter mediating the uptake of di- and tripeptides was isolated from Arabidopsis thaliana by heterologous complementation of a peptide transport-deficient Saccharomyces cerevisiae mutant. AtPTR1 mediated growth of S. cerevisiae cells on different di- and tripeptides and caused sensitivity to the phytotoxin phaseolotoxin. The spectrum of substrates recognized by AtPTR1 was determined in Xenopus laevis oocytes injected with AtPTR1 cRNA under voltage clamp conditions. AtPTR1 not only recognized a broad spectrum of di- and tripeptides, but also substrates lacking a peptide bond. However, amino acids, omega-amino fatty acids or peptides with more than three amino acid residues did not interact with AtPTR1. At pH 5.5 AtPTR1 had an apparent lower affinity (K-0.5 = 416 mum) for Ala-Asp compared with Ala-Ala (K-0.5 = 54 mum) and Ala-Lys (K-0.5 = 112 mum). Transient expression of AtPTR1/GFP fusion proteins in tobacco protoplasts showed that AtPTR1 is localized at the plasma membrane. In addition, transgenic plants expressing the beta-glucuronidase (uidA) gene under control of the AtPTR1 promoter demonstrated expression in the vascular tissue throughout the plant, indicative of a role in long-distance transport of di- and tripeptides.

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Growth, morphogenesis and function of roots are influenced by the concentration and form of nutrients present in soils, including low molecular mass inorganicN(IN, ammonium, nitrate) and organicN(ON, e. g. amino acids). Proteins, ON of high molecular mass, are prevalent in soils but their possible effects on roots have received little attention. Here, we investigated how externally supplied protein of a size typical of soluble soil proteins influences root development of axenically grown Arabidopsis. Addition of low to intermediate concentrations of protein (bovine serum albumen, BSA) to IN-replete growth medium increased root dry weight, root length and thickness, and root hair length. Supply of higher BSA concentrations inhibited root development. These effects were independent of total N concentrations in the growth medium. The possible involvement of phytohormones was investigated using Arabidopsis with defective auxin (tir1-1 and axr2-1) and ethylene (ein2-1) responses. That no phenotype was observed suggests a signalling pathway is operating independent of auxin and ethylene responses. This study expands the knowledge on N form-explicit responses to demonstrate that ON of high molecular mass elicits specific responses.

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The AXR6 gene is required for auxin signaling in the Arabidopsis embryo and during postembryonic development. One of the effects of auxin is to stimulate degradation of the Aux/IAA auxin response proteins through the action of the ubiquitin protein ligase SCFTIR1. Here we show that AXR6 encodes the SCF subunit CUL1. The axr6 mutations affect the ability of mutant CUL1 to assemble into stable SCF complexes resulting in reduced degradation of the SCFTIR1 substrate AXR2/IAA7. In addition, we show that CUL1 is required for lateral organ initiation in the shoot apical meristem and the inflorescence meristem. These results indicate that the embryonic axr6 phenotype is related to a defect in SCF function and accumulation of Aux/IAA proteins such as BDL/IAA12. In addition, we show that CUL1 has a role in auxin response throughout the life cycle of the plant.

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With the aim of analysing the relative importance of sugar supply and nitrogen nutrition for the regulation of sulphate assimilation, the regulation of adenosine 5′‐phosphosulphate reductase (APR), a key enzyme of sulphate reduction in plants, was studied. Glucose feeding experiments with Arabidopsis thaliana cultivated with and without a nitrogen source were performed. After a 38 h dark period, APR mRNA, protein, and enzymatic activity levels decreased dramatically in roots. The addition of 0.5% (w/v) glucose to the culture medium resulted in an increase of APR levels in roots (mRNA, protein and activity), comparable to those of plants kept under normal light conditions. Treatment of roots with D‐sorbitol or D‐mannitol did not increase APR activity, indicating that osmotic stress was not involved in APR regulation. The addition of O‐acetyl‐L‐serine (OAS) also quickly and transiently increased APR levels (mRNA, protein, and activity). Feeding plants with a combination of glucose and OAS resulted in a more than additive induction of APR activity. Contrary to nitrate reductase, APR was also increased by glucose in N‐deficient plants, indicating that this effect was independent of nitrate assimilation. [35S]‐sulphate feeding experiments showed that the addition of glucose to dark‐treated roots resulted in an increased incorporation of [35S] into thiols and proteins, which corresponded to the increased levels of APR activity. Under N‐deficient conditions, glucose also increased thiol labelling, but did not increase the incorporation of label into proteins. These results demonstrate that (i) exogenously supplied glucose can replace the function of photoassimilates in roots; (ii) APR is subject to co‐ordinated metabolic control by carbon metabolism; (iii) positive sugar signalling overrides negative signalling from nitrate assimilation in APR regulation. Furthermore, signals originating from nitrogen and carbon metabolism regulate APR synergistically.

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Essential amino acids cannot be synthesized by humans and animals. They often are limiting in plant-derived foods and determine the nutritional value of a given diet [1]. Seeds and fruits often represent the harvestable portion of plants. In order to improve the amino acid composition of these tissues, it is indispensable to understand how these substrates are transported within the plant. Amino acids result from nitrogen assimilation, which often occurs in leaves, the source tissue. They are transported via the vasculature, the xylem, and the phloem into the seeds, the so-called sink tissue, where they are stored or consumed. In seeds, several tissues are symplasmically isolated [2, 3], i.e., not connected by plasmodesmata, channels in the cell walls that enable a cytoplasmic continuum in plants [4]. Consequently, amino acids must be exported from cells into the apoplast and re-imported many times to support seed development. Several amino acid importers are known, but exporters remained elusive [5, 6]. Here, we characterize four members of the plant-specific UmamiT transporter family from Arabidopsis, related to the amino acid facilitator SIAR1 and the vacuolar auxin transporter WAT1 [7, 8]. We show that the proteins transport amino acids along their (electro)chemical potential across the plasma membrane. In seeds, they are found in tissues from which amino acids are exported. Loss-of-function mutants accumulate high levels of free amino acids in fruits and produce smaller seeds. Our results strongly suggest a crucial role for the UmamiTs in amino acid export and possibly a means to improve yield quality.

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Using Arabidopsis, we analyzed the effect of omission of a nitrogen source and of the addition of different nitrogen-containing compounds on the extractable activity and the enzyme and mRNA accumulation of adenosine 5′-phosphosulfate reductase (APR). During 72 h without a nitrogen source, the APR activity decreased to 70% and 50% of controls in leaves and roots, respectively, while cysteine (Cys) and glutathione contents were not affected. Northern and western analysis revealed that the decrease of APR activity was correlated with decreased mRNA and enzyme levels. The reduced APR activity in roots could be fully restored within 24 h by the addition of 4 mM each of NO3 −, NH4 +, or glutamine (Gln), or 1 mM O-acetylserine (OAS). 35SO4 2− feeding showed that after addition of NH4 +, Gln, or OAS to nitrogen-starved plants, incorporation of 35S into proteins significantly increased in roots; however, glutathione and Cys labeling was higher only with Gln and OAS or with OAS alone, respectively. OAS strongly increased mRNA levels of all three APR isoforms in roots and also those of sulfite reductase, Cys synthase, and serine acetyltransferase. Our data demonstrate that sulfate reduction is regulated by nitrogen nutrition at the transcriptional level and that OAS plays a major role in this regulation.

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In many organisms, including plants, nucleic acid bases and derivatives such as caffeine are transported across the plasma membrane. Cytokinins, important hormones structurally related to adenine, are produced mainly in root apices, from where they are translocated to shoots to control a multitude of physiological processes. Complementation of a yeast mutant deficient in adenine uptake (fcy2) with an Arabidopsis cDNA expression library enabled the identification of a gene, AtPUP1 (for Arabidopsis thaliana purine permease1), belonging to a large gene family (AtPUP1 to AtPUP15) encoding a new class of small, integral membrane proteins. AtPUP1 transports adenine and cytosine with high affinity. Uptake is energy dependent, occurs against a concentration gradient, and is sensitive to protonophores, potentially indicating secondary active transport. Competition studies show that purine derivatives (e.g., hypoxanthine), phytohormones (e.g., zeatin and kinetin), and alkaloids (e.g., caffeine) are potent inhibitors of adenine and cytosine uptake. Inhibition by cytokinins is competitive (competitive inhibition constant Ki = 20 to 35 μM), indicating that cytokinins are transported by this system. AtPUP1 is expressed in all organs except roots, indicating that the gene encodes an uptake system for root-derived nucleic acid base derivatives in shoots or that it exports nucleic acid base analogs from shoots by way of the phloem. The other family members may have different affinities for nucleic acid bases, perhaps functioning as transporters for nucleosides, nucleotides, and their derivatives.

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Singlet oxygen is a prominent form of reactive oxygen species in higher plants. It is easily formed from molecular oxygen by triplet–triplet interchange with excited porphyrin species. Evidence has been obtained from studies on the flu mutant of Arabidopsis thaliana of a genetically determined cell death pathway that involves differential changes at the transcriptome level. Here we report on a different cell death pathway that can be deduced from the analysis of oep16 mutants of A. thaliana. Pure lines of four independent OEP16-deficient mutants with different cell death properties were isolated. Two of the mutants overproduced free protochlorophyllide (Pchlide) in the dark because of defects in import of NADPH:Pchlide oxidoreductase A (pPORA) and died after illumination. The other two mutants avoided excess Pchlide accumulation. Using pulse labeling and polysome profiling studies we show that translation is a major site of cell death regulation in flu and oep16 plants. flu plants respond to photooxidative stress triggered by singlet oxygen by reprogramming their translation toward synthesis of key enzymes involved in jasmonic acid synthesis and stress proteins. In contrast, those oep16 mutants that were prone to photooxidative damage were unable to respond in this way. Together, our results show that translation is differentially affected in the flu and oep16 mutants in response to singlet oxygen.

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La resistencia de las plantas a los hongos necrótrofos como Plectosphaerella cucumerina es genéticamente compleja y depende de la activación coordinada de distintas rutas de señalización (Llorente et al, 2005; Sanchez-Vallet et al, 2010). Entre éstas se encuentran las mediadas por la proteína G heterotrimérica, un complejo formado por tres subunidades (Gα, Gβ y Gγ) que regula tanto la respuesta de inmunidad a diferentes patógenos como distintos procesos de desarrollo (Temple and Jones, 2007). En esta Tesis hemos demostrado que, en Arabidopsis, el monómero funcional formado por las subunidades Gβ y Gγ1/Gγ2 es el responsable de la regulación de la respuesta de defensa, ya que mutantes nulos en estas subunidades (agb1 y agg1 agg2) presentan una alta susceptibilidad al hongo P. cucumerina. Además, hemos identificado varios aminoácidos (Q102, T188 y R235) de la proteína AGB1 esenciales en la interacción con los efectores correspondientes para la regulación de la respuesta inmune (Jiang et al, enviado). Para determinar las bases moleculares de la resistencia mediada por la proteína G heterotrimérica, llevamos a cabo un análisis transcriptómico comparativo entre los genotipos agb1 y Col-0, el cual reveló que la resistencia mediada por AGB1 no depende de rutas defensivas implicadas en la resistencia a hongos necrotrofos, como las mediadas por el ácido salicílico (SA), etileno (ET), jasmónico (JA) o ácido abscísico (ABA), o la ruta de biosíntesis de metabolitos derivados del triptófano. Este estudio mostró que un número significativo de los genes desregulados en respuesta a P. cucumerina en el genotipo agb1 respecto a las plantas silvestres codificaban proteínas con funciones relacionadas con la pared celular. La evaluación de la composición y estructura de la pared de los mutantes de las subunidades de la proteína G heterotrimérica reveló que los genotipos agb1 y agg1 agg2 presentaban alteraciones similares diferentes de las observadas en plantas silvestres Col-0, como una reducción significativa en el contenido de xilosa en la pared. Estos datos sugieren que la proteína G heterotrimérica puede modular la composición/estructura de la pared celular y contribuir, de esta manera, en la regulación de la respuesta inmune (Delgado- Cerezo et al, 2011). La caracterización del interactoma de la proteína G heterotrimérica corroboró la relevancia funcional que presenta en la regulación de la pared celular, ya que un número significativo de las interacciones identificadas estaban comprendidas por proteínas relacionadas directa o indirectamente con la biogénesis y remodelación de la pared celular (Klopffleisch et al, 2011). El papel en inmunidad de algunos de estos potenciales efectores ha sido validado mediante el análisis de la resistencia a P. cucumerina de los mutantes de pérdida de función correspondientes. Con el objetivo de caracterizar las rutas de señalización mediadas por AGB1 e identificar efectores implicados en esta señalización, llevamos a cabo una búsqueda de mutantes supresores de la susceptibilidad de agb1 a P. cucumerina, identificándose varios mutantes sgb (supressor of Gbeta). En esta Tesis hemos caracterizado en detalle el mutante sgb10, que presenta una activación constitutiva de las rutas de señalización mediadas por SA y JA+ET y suprime el fenotipo de susceptibilidad de agb1. SGB10 y AGB1 forman parte de rutas independientes en la regulación de la respuesta inmune, mientras que interaccionan de forma compleja en el control de determinados procesos de desarrollo. La mutación sgb10 ha sido cartografiada entre los genes At3g55010 y At3g56408, que incluye una región con 160 genes. ABSTRACT Plant resistance to necrotrophic fungi Plectosphaerella cucumerina is genetically complex and depends on the interplay of different signalling pathways (Llorente et al, 2005; Sanchez-Vallet et al, 2010). Among others, the heterotrimeric G protein complex has a relevant role. The G protein that is formed by three subunits (Gα, Gβ and Gγ) is a pleiotropic regulator of immune responses to different types of pathogens and developmental issues (Temple and Jones, 2007). Throughout the Thesis, we have demonstrated that Arabidopsis’ functional monomer formed by the Gβ and Gγ1/Gγ2 subunits is a key regulator of defense response, as null mutants (agb1 and agg1 agg2) are equally hypersusceptible to P. cucumerina infection. In addition we have identified several AGB1 aminoacids (Q102, T188 y R235) essentials to interact with specific effectors during the regulation of immune response (Jiang et al, sent).To determine the molecular basis of heterotrimeric G protein mediated resistance we have performed a microarray analysis with agb1-1 and wild type Col-0 plants before and after P. cucumerina challenge. A deep and exhaustive comparative transcriptomical analysis of these plants revealed that AGB1 mediated resistance does not rely on salicilic acid (SA), ethylene (ET), jasmonates (JA), abscisic acid (ABA) or triptophan derived metabolites biosynthesis. However the analysis revealed that a significant number of cell wall related genes are misregulated in the agb1 mutant after pathogen challenge when compared to wild-type plants. The analysis of cell wall composition and structure showed similar cell wall alterations between agb1 and agg1 agg2 mutants that are different from those of wild-type plants, so far the mutants present a significant reduction in xylose levels. All these results suggest that heterotrimeric G protein may regulate immune response through modifications in the cell wall composition/structure (Delgado-Cerezo et al, 2011). The characterization of Heterotrimeric G protein interactome revealed highly connected interactions between the G-protein core and proteins involved in cell wall composition or structure (Klopffleisch et al, 2011). To test the role in immunity of several effectors identified above, we have performed resistance analysis of corresponding null mutants against P. cucumerina. In order to characterize AGB1 mediated signalling pathway and identify additional effectors involved in AGB1-mediated immune response against P. cucumerina, we have performed a screening to isolate mutants with suppression of agb1 phenotype. One of the mutants, named sgb10, has been characterized during the Thesis. The mutant shows constitutive expression of SA, JA+ET-mediated defense signaling pathways to suppres agb1 hypersusceptibility. SGB10 and AGB1 proteins seem to be part of independent pathways in immunity, however its function during development remains unclear. At present, we have mapped the sgb10 mutation between At3g55010 and At3g56408 genes. This region contains 160 genes.