RESUMO
The growth and development of maize (Zea mays L.) largely depends on its nutrient uptake through the root. Hence, studying its growth, response, and associated metabolic reprogramming to stress conditions is becoming an important research direction. A genome-scale metabolic model (GSM) for the maize root was developed to study its metabolic reprogramming under nitrogen stress conditions. The model was reconstructed based on the available information from KEGG, UniProt, and MaizeCyc. Transcriptomics data derived from the roots of hydroponically grown maize plants were used to incorporate regulatory constraints in the model and simulate nitrogen-non-limiting (N+) and nitrogen-deficient (N-) condition. Model-predicted flux-sum variability analysis achieved 70% accuracy compared with the experimental change of metabolite levels. In addition to predicting important metabolic reprogramming in central carbon, fatty acid, amino acid, and other secondary metabolism, maize root GSM predicted several metabolites (l-methionine, l-asparagine, l-lysine, cholesterol, and l-pipecolate) playing a regulatory role in the root biomass growth. Furthermore, this study revealed eight phosphatidylcholine and phosphatidylglycerol metabolites which, even though not coupled with biomass production, played a key role in the increased biomass production under N-deficient conditions. Overall, the omics-integrated GSM provides a promising tool to facilitate stress condition analysis for maize root and engineer better stress-tolerant maize genotypes.
Assuntos
Nitrogênio , Zea mays , Aminoácidos , Biomassa , Raízes de Plantas , Zea mays/genéticaRESUMO
Maize (Zea mays) is an important C4 plant due to its widespread use as a cereal and energy crop. A second-generation genome-scale metabolic model for the maize leaf was created to capture C4 carbon fixation and investigate nitrogen (N) assimilation by modeling the interactions between the bundle sheath and mesophyll cells. The model contains gene-protein-reaction relationships, elemental and charge-balanced reactions, and incorporates experimental evidence pertaining to the biomass composition, compartmentalization, and flux constraints. Condition-specific biomass descriptions were introduced that account for amino acids, fatty acids, soluble sugars, proteins, chlorophyll, lignocellulose, and nucleic acids as experimentally measured biomass constituents. Compartmentalization of the model is based on proteomic/transcriptomic data and literature evidence. With the incorporation of information from the MetaCrop and MaizeCyc databases, this updated model spans 5,824 genes, 8,525 reactions, and 9,153 metabolites, an increase of approximately 4 times the size of the earlier iRS1563 model. Transcriptomic and proteomic data have also been used to introduce regulatory constraints in the model to simulate an N-limited condition and mutants deficient in glutamine synthetase, gln1-3 and gln1-4. Model-predicted results achieved 90% accuracy when comparing the wild type grown under an N-complete condition with the wild type grown under an N-deficient condition.
Assuntos
Modelos Biológicos , Nitrogênio/metabolismo , Folhas de Planta/metabolismo , Zea mays/genética , Zea mays/metabolismo , Disponibilidade Biológica , Biomassa , Perfilação da Expressão Gênica , Genoma de Planta , Metaboloma , Mutação , Nitrogênio/farmacocinética , Proteoma/metabolismoRESUMO
BACKGROUND: To identify the key elements controlling grain production in maize, it is essential to have an integrated view of the responses to alterations in the main steps of nitrogen assimilation by modification of gene expression. Two maize mutant lines (gln1.3 and gln1.4), deficient in two genes encoding cytosolic glutamine synthetase, a key enzyme involved in nitrogen assimilation, were previously characterized by a reduction of kernel size in the gln1.4 mutant and by a reduction of kernel number in the gln1.3 mutant. In this work, the differences in leaf gene transcripts, proteins and metabolite accumulation in gln1.3 and gln1.4 mutants were studied at two key stages of plant development, in order to identify putative candidate genes, proteins and metabolic pathways contributing on one hand to the control of plant development and on the other to grain production. RESULTS: The most interesting finding in this study is that a number of key plant processes were altered in the gln1.3 and gln1.4 mutants, including a number of major biological processes such as carbon metabolism and transport, cell wall metabolism, and several metabolic pathways and stress responsive and regulatory elements. We also found that the two mutants share common or specific characteristics across at least two or even three of the "omics" considered at the vegetative stage of plant development, or during the grain filling period. CONCLUSIONS: This is the first comprehensive molecular and physiological characterization of two cytosolic glutamine synthetase maize mutants using a combined transcriptomic, proteomic and metabolomic approach. We find that the integration of the three "omics" procedures is not straight forward, since developmental and mutant-specific levels of regulation seem to occur from gene expression to metabolite accumulation. However, their potential use is discussed with a view to improving our understanding of nitrogen assimilation and partitioning and its impact on grain production.
Assuntos
Citosol/enzimologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Glutamato-Amônia Ligase/genética , Mutação/genética , Zea mays/enzimologia , Zea mays/genética , Regulação Enzimológica da Expressão Gênica , Glutamato-Amônia Ligase/metabolismo , Metabolômica , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Transcriptoma/genética , Zea mays/crescimento & desenvolvimentoRESUMO
Linking plant phenotype to gene and protein expression and also to metabolite synthesis and accumulation is one of the main challenges for improving agricultural production worldwide. Such a challenge is particularly relevant to crop nitrogen use efficiency (NUE). Here, the differences in leaf gene transcript, protein, and metabolite accumulation in maize subjected to long-term nitrogen (N)-deficient growth conditions at two important stages of plant development have been studied. The impact of N deficiency was examined at the transcriptomic, proteomic, and metabolomic levels. It was found that a number of key plant biological functions were either up- or down-regulated when N was limiting, including major alterations to photosynthesis, carbon (C) metabolism, and, to a lesser extent, downstream metabolic pathways. It was also found that the impact of the N deficiency stress resembled the response of plants to a number of other biotic and abiotic stresses, in terms of transcript, protein, and metabolite accumulation. The genetic and metabolic alterations were different during the N assimilation and the grain-filling period, indicating that plant development is an important component for identifying the key elements involved in the control of plant NUE. It was also found that integration of the three 'omics' studies is not straightforward, since different levels of regulation seem to occur in a stepwise manner from gene expression to metabolite accumulation. The potential use of these 'omics' studies is discussed with a view to improve our understanding of whole plant nitrogen economics, which should have applications in breeding and agronomy.
Assuntos
Perfilação da Expressão Gênica , Metaboloma , Nitrogênio/metabolismo , Proteínas de Plantas/genética , Proteoma/genética , Zea mays/genética , Zea mays/metabolismo , Cromatografia Líquida , Análise de Sequência com Séries de Oligonucleotídeos , Espectrometria de Massas em Tandem , Zea mays/crescimento & desenvolvimentoRESUMO
During the grain-filling period of maize, the changes in metabolite content, enzyme activities, and transcript abundance of marker genes of amino acid synthesis and interconversion and carbon metabolism in three lines F2, Io, and B73 have been monitored in the cob and in the kernels. An integrative statistical approach using principal component analysis (PCA) and hierarchical clustering of physiological and transcript abundance data in the three maize lines was performed to determine if it was possible to link the expression of a physiological trait and a molecular biomarker to grain yield and its components. In this study, it was confirmed that, in maize, there was a genetic and organ-specific control of the main steps of nitrogen (N) and carbon metabolism in reproductive sink organs during the grain-filling period. PCA analysis allowed the identification of groups of physiological and molecular markers linked to either a genotype, an organ or to both biological parameters. A hierarchical clustering analysis was then performed to identify correlative relationships existing between these markers and agronomic traits related to yield. Such a clustering approach provided new information on putative marker traits that could be used to improve yield in a given genetic background. This can be achieved using either genetic manipulation or breeding to increase transcript abundance for the genes encoding the enzymes glutamine synthetase (GS), alanine amino transferase (AlaAT), aspartate amino transferase (AspAT), and Δ1-pyrroline-5-carboxylate synthetase (P5CS).
Assuntos
Variação Genética , Nitrogênio/metabolismo , Zea mays/genética , Zea mays/metabolismo , Alanina Transaminase/genética , Alanina Transaminase/metabolismo , Aspartato Aminotransferases/genética , Aspartato Aminotransferases/metabolismo , Análise por Conglomerados , Regulação da Expressão Gênica de Plantas , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Endogamia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Análise de Componente Principal , Locos de Características Quantitativas , Zea mays/química , Zea mays/enzimologiaRESUMO
Cytosolic glutamine synthetase (GS1) is the enzyme mainly responsible of ammonium assimilation and reassimilation in maize leaves. The agronomic potential of GS1 in maize kernel production was investigated by examining the impact of an overexpression of the enzyme in the leaf cells. Transgenic hybrids exhibiting a three-fold increase in leaf GS activity were produced and characterized using plants grown in the field. Several independent hybrids overexpressing Gln1-3, a gene encoding cytosolic (GS1), in the leaf and bundle sheath mesophyll cells were grown over five years in different locations. On average, a 3.8% increase in kernel yield was obtained in the transgenic hybrids compared to controls. However, we observed that such an increase was simultaneously dependent upon both the environmental conditions and the transgenic event for a given field trial. Although variable from one environment to another, significant associations were also found between two GS1 genes (Gln1-3 and Gln1-4) polymorphic regions and kernel yield in different locations. We propose that the GS1 enzyme is a potential lead for producing high yielding maize hybrids using either genetic engineering or marker-assisted selection. However, for these hybrids, yield increases will be largely dependent upon the environmental conditions used to grow the plants.
Assuntos
Clima , Regulação da Expressão Gênica de Plantas , Glutamato-Amônia Ligase/genética , Proteínas de Plantas/genética , Sementes/crescimento & desenvolvimento , Tempo (Meteorologia) , Zea mays/fisiologia , Alelos , Citosol , Glutamato-Amônia Ligase/metabolismo , Hibridização Genética , Melhoramento Vegetal , Proteínas de Plantas/metabolismo , Sementes/genética , Estados Unidos , Zea mays/enzimologia , Zea mays/genéticaRESUMO
Modification of the Medicago truncatula root proteome during the early stage of arbuscular mycorrhizal symbiosis was investigated by comparing, using two-dimensional electrophoresis, the protein patterns obtained from non-inoculated roots and roots synchronized for Glomus intraradices appressorium formation. This approach was conducted in wild-type (J5), mycorrhiza-defective (TRV25, dmi3), and autoregulation-defective (TR122, sunn) M. truncatula genotypes. The groups of proteins that responded to appressorium formation were further compared between wild-type and mutant genotypes; few overlaps and major differences were recorded, demonstrating that mutations in DMI3 and SUNN modified the appressorium-responsive root proteome. Except for a chalcone reductase, none of the differentially displayed proteins that could be identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry previously was known as appressorium responsive. A DMI3-dependent increased accumulation of signal transduction-related proteins (dehydroascorbate reductase, cyclophilin, and actin depolymerization factor) was found to precede mycorrhiza establishment. Differences in the accumulation of proteins related to plant defense reactions, cytoskeleton rearrangements, and auxin signaling upon symbiont contact were recorded between wild-type and hypermycorrhizal genotypes, pointing to some putative pathways by which SUNN may regulate very early arbuscule formation.
Assuntos
Genes de Plantas/genética , Mutação/genética , Micorrizas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Proteoma/análise , Eletroforese em Gel Bidimensional/métodos , Espectrometria de Massas/métodos , Medicago truncatula/genética , Medicago truncatula/metabolismo , Medicago truncatula/microbiologia , Proteínas de Plantas/análise , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , Proteômica/métodos , Simbiose/fisiologia , Fatores de TempoRESUMO
Crenate broomrape (Orobanche crenata) is a parasitic plant that threatens legume production in Mediterranean areas. Pea (Pisum sativum) is severely affected, and only moderate levels of genetic resistance have so far been identified. In the present work we selected the most resistant accession available (Ps 624) and compared it with a susceptible (Messire) cultivar. Experiments were performed by using pot and Petri dish bioassays, showing little differences in the percentage of broomrape seed germination induced by both genotypes, but a significant hamper in the number of successfully installed tubercles and their developmental stage in the Ps 624 compared to Messire. The protein profile of healthy and infected P. sativum root tissue were analysed by two-dimensional electrophoresis. Approximately 500 individual protein spots could be detected on silver stained gels. At least 22 different protein spots differentiated control, non-infected, Messire and Ps 624 accessions. Some of them were identified by MALDI-TOF mass spectrometry and database searching as cysteine proteinase, beta-1,3-glucanase, endochitinase, profucosidase, and ABA-responsive protein. Both qualitative and quantitative differences have been found among infected and non-infected root extracts. Thus, in the infected susceptible Messire genotype 34 spots were decreased, one increased and three newly detected, while in Ps 624, 15 spots were increased, three decreased and one newly detected. In response to the inoculation, proteins that correspond to enzymes of the carbohydrate metabolism (fructokinase, fructose-bisphosphate aldolase), nitrogen metabolism (ferredoxin-NADP reductase) and mitochondrial electronic chain transport (alternative oxidase 2) decreased in the susceptible check, while proteins that correspond to enzymes of the nitrogen assimilation pathway (glutamine synthetase) or typical pathogen defence, PR proteins, including beta-1,3-glucanase and peroxidases, increased in Ps 624. Results are discussed in terms of changes in the carbohydrate and nitrogen metabolism an induction of defence proteins in response to broomrape parasitism.
Assuntos
Orobanche/fisiologia , Pisum sativum/genética , Proteínas de Plantas/análise , Raízes de Plantas/metabolismo , Proteômica , Bases de Dados de Proteínas , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Genótipo , Pisum sativum/microbiologia , Mapeamento de Peptídeos , Coloração pela Prata , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Plant cells have specific microtubule structures involved in cell division and elongation. The tonneau1 (ton1) mutant of Arabidopsis thaliana displays drastic defects in morphogenesis, positioning of division planes, and cellular organization. These are primarily caused by dysfunction of the cortical cytoskeleton and absence of the preprophase band of microtubules. Characterization of the ton1 insertional mutant reveals complex chromosomal rearrangements leading to simultaneous disruption of two highly similar genes in tandem, TON1a and TON1b. TON1 proteins are conserved in land plants and share sequence motifs with human centrosomal proteins. The TON1 protein associates with soluble and microsomal fractions of Arabidopsis cells, and a green fluorescent protein-TON1 fusion labels cortical cytoskeletal structures, including the preprophase band and the interphase cortical array. A yeast two-hybrid screen identified Arabidopsis centrin as a potential TON1 partner. This interaction was confirmed both in vitro and in plant cells. The similarity of TON1 with centrosomal proteins and its interaction with centrin, another key component of microtubule organizing centers, suggests that functions involved in the organization of microtubule arrays by the centrosome were conserved across the evolutionary divergence between plants and animals.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Sequência de Aminoácidos , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/genética , Centrossomo/metabolismo , Citoesqueleto/metabolismo , Imunofluorescência , Regulação da Expressão Gênica de Plantas , Proteínas Associadas aos Microtúbulos/classificação , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Dados de Sequência Molecular , Filogenia , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-HíbridoRESUMO
Wheat proteins and specially gluten proteins have been well studied and are closely associated with baking products. Amphiphilic proteins (proteins that are soluble using nonionic detergent Triton X-114 ) also play an important role in wheat quality. Some of them, like puroindolines, are lipid binding proteins, and are strongly linked to dough foaming properties and to fine crumb texture. However many amphiphilic proteins are still unknown and both their physiological and technological functions remain to be analysed. In order to explore these proteins, proteomic analysis was carried out using 81 F9 lines, progeny obtained from an interspecific cross "W7984"x"Opata", and already used to built a map of more than 2000 molecular markers (International Triticeae Mapping Initiative, ITMImap). Two-dimensional electrophoresis (immobilized pH gradient (pH 6-11)x sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed on amphiphilic proteins with three to five replicates for each line. Silver stained gels were analysed using Melanie 3 software. Genetic determinism was carried out on 170 spots segregating between the two parental hexaploïd wheats. Many of these spots were mapped on different chromosomes of the ITMImap. Spots of interest were identified using matrix-assisted laser desorption/ionization-time of flight and some of them were partly sequenced using electrospray ionization-tandem mass spectrometry. This proteomic approach provided some very useful information about some proteic components linked to bread wheat quality and particularly to kernel hardness.
Assuntos
Proteínas/química , Proteínas/genética , Proteoma/química , Triticum/genética , Triticum/metabolismo , Mapeamento Cromossômico , Cruzamentos Genéticos , Detergentes/farmacologia , Eletroforese em Gel Bidimensional , Genes de Plantas , Espectrometria de Massas , Modelos Genéticos , Octoxinol , Polietilenoglicóis/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Puroindolines are endosperm lipid binding proteins, which are separated by reversed phase-high-performance liquid chromatography or cation exchange chromatography into two isoforms, puroindoline-a (PIN-a) and puroindoline-b (PIN-b). Being very basic and close in molecular weight, PIN-a and PIN-b have never been separated using conventional isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A two-dimensional electrophoresis method, linear immobiline pH gradient (IPGxSDS-PAGE), was developed, using 6-11 linear immobiline Dry Strips in the first dimension, which allowed the puroindolines to be focused between isoelectric point 10.5 and 11. Immunoblotting revealed that both PIN-a and PIN-b were each composed of several spots. Two-dimensional patterns from unrelated wheat varieties revealed that several spots can be highlighted among varieties. Matrix-assisted laser desorption/ionization-time of flight spectrometry allowed the majority of the spots revealed in the puroindoline zone to be identified. The two-dimensional IPGxSDS-PAGE of these very basic wheat endosperm proteins, puroindolines and related grain softness proteins should facilitate the identification of the proteins associated with wheat endosperm texture that have a strong effect on milling, dough properties and end-uses of wheats.
Assuntos
Eletroforese em Gel Bidimensional/métodos , Proteínas de Plantas/química , Western Blotting , Cátions , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Immunoblotting , Espectrometria de Massas , Polimorfismo Genético , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Triticum/metabolismoRESUMO
We have developed a protocol in which proteins and mRNA can be analyzed from single root samples. This experimental design was validated in arbuscular mycorrhiza by comparing the proteins profiles obtained with those from a classical protein extraction process. It is a step forward to make simultaneous proteome and transcriptiome profiling possible.