Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Arabidopsis ecotypes Landsberg erecta (Ler) and Landsberg to assess the effect of ERECTA on gene expression

ABSTRACT: To assess the effect of ERECTA on gene expression in Arabidopsis, genome wide transcript abundance was measured in Landsberg erecta (Ler) compared to Landsberg. For the differentially expressed genes, genetic variation in gene expression in the Ler/Cvi recombinant inbred line (RIL) population, resulted in the construction of a putative genetic regulatory network downstream of ERECTA.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Inez Terpstra

PROVIDER: E-TABM-627 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Plant cells are characterized by a high degree of compartmentalization and a diverse proteome and metabolome. Only a very limited number of studies has addressed combined subcellular proteomics and metabolomics which strongly limits biochemical and physiological interpretation of large-scale omics data. Our study presents a methodological combination of non-aqueous fractionation, shotgun proteomics, enzyme kinetics and metabolomics to reveal subcellular diurnal dynamics of plant metabolism. Subcellular marker protein sets were identified and enzymatically validated to resolve metabolism in a 4-compartment model comprising chloroplasts, cytosol, vacuole and mitochondria. These marker sets are now available for future studies which aim to monitor subcellular metabolome and proteome dynamics. Comparing subcellular dynamics in wild type plants and (hexokinase) HXK1-deficient gin2-1 mutants revealed a strong impact of HXK1 activity on metabolome dynamics in multiple compartments. Glucose accumulation in the cytosol of gin2-1 was accompanied by diminished vacuolar glucose levels. Subcellular dynamics of pyruvate, succinate and fumarate concentrations were significantly affected in gin2-1 which coincided with differential mitochondrial proteome dynamics. Lowered mitochondrial glycine and serine concentrations in gin2-1 together with reduced abundance of photorespiratory proteins indicated an effect of the gin2-1 mutation on photorespiratory capacity. Our findings highlight the necessity to resolve plant metabolism to a subcellular level in order to provide a causal relationship between metabolites, proteins and metabolic pathway regulation. --- In an attempt to draw a full picture of abiotic stress responses a complete set of time resolved, compartmentalized metabolomics and proteomics data under different stress conditions was generated. To investigate interactions of stress responses with primary metabolism, the gin2-1 mutant, was compared to its wildtype Landsberg erecta (Ler) under high irradiance and at low temperature. Chlorophyll fluorescence parameters proved the well-known sensitivity of the gin2-1 mutant to high irradiance. Dynamics of subcellular metabolite redistribution upon stress were delayed in gin2-1, and this correlated with massive reduction in proteins belonging to the ATP producing electron transport chain. Evidence for compounds specifically protecting compartments, e.g. maltose in plastids and myo-inositol in mitochondria, was obtained in Ler, but gin2-1 failed to respond or responded only slowly to high irradiance, while no delay was obtained in the cold. Whole-cell concentrations of the amino acids glycine, and serine, provided strong evidence for an important role of the photorespiratory pathway during stress exposure and may contribute to the slow growth of the gin2-1 mutant under high irradiance.

Project description:We carried out a five-generation selection experiment in which two Arabidopsis accessions (Ler and Cvi) and 17 Recombinant Inbred Lines (RILs) derived from them were subjected in 24 replicated populations to four levels of micro-landscape fragmentation and two levels of disturbance, factorially crossed with each other to yield 8 experimental selection regimes. We used 19 lines (including the two parental Ler and Cvi) in generation 1 of our experiment, nine of which contained the erecta mutation and ten of which did not. Lines expressing the erecta mutation are short and compact and are likely to disperse seeds over shorter distances. Both the non-erecta and the erecta lines spanned the full available range of seed sizes from the original 162 RILs in the collection. We set up 24 experimental landscapes consisting of 2, 4, 8, or 16 patches where the total area was constrained to be equal. Plants that germinated outside the designated suitable patches were removed. In half of the landscapes which we termed static the top 2-3 mm of seeds and soil were removed from the surface of the suitable patches after 8 weeks of growth and reproduction. In the other half of the landscapes which we termed dynamic a number of Petri dishes, equivalent in size and number to the existing suitable patches, were distributed around the landscape at random, to catch dispersing seeds. The seeds collected by these two methods were cold-treated for one week while all patches were replenished with fresh substrate. Seeds were then returned to the surface of the new patches at the beginning of the next generation. Thus, in dynamic landscapes, seeds must disperse to have any chance of entering the next generation, while in static landscapes, only seeds that do not disperse have any chance of further propagation. After five generations of such selection, a single pod from approximately 70 individuals from each landscape was removed. Single individuals derived from such pods were reared under standardized conditions to measure several phenotypic characters, such as seed mass, height, presence of the erecta mutation, flowering time etc. Clear phenotypic differences are apparent between the different landscapes. However, we have no information as to the underlying genes, which might have been selected or the epigenetic modifications that might have occurred. Material for DNA extraction from each population (? 50 plants/population) was collected for subsequent genetic analyses. The allele frequencies of Ler and Cvi alleles was determined at the genome-wide level using ATH1 Affymetrix GeneChips® (each in quadruplicate = 8 parental microarrays). 26638 single feature polymorphisms (SFPs) between these two accessions could be detected. The relative contribution of Ler and Cvi alleles in a population at each locus, for which a single feature polymorphism can be detected, was assessed. We then analyzed the DNA isolated from the experimental populations (24 landscapes of generation 5 = 24 microarrays & original populations in triplicate = 3 microarrays) and determine the relative contributions of Ler and Cvi alleles for the 26638 identified SFPs (i.e. allele frequency shifts).

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Transcription profiling of Arabidopsis ecotypes Landsberg erecta (Ler) and Landsberg to assess the effect of ERECTA on gene expression

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