Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Arabidopsis cells cultures undergoing transdifferentiation

ABSTRACT: The formation of vascular tissue occurs when cellulose, hemicellulose, lignin and other wall components are deposited within the primary cell wall. These secondary thickened cells then undergo programmed cell death producing a network of empty cells with which water and ions can be transported throughout the plant. The hormones auxin and cytokinin are the principle signals for vascular tissue initiation. As a consequence cells cultured in-vitro can be converted into vascular tissue with the addition of exogenous auxin and cytokinin. We have created an in-vitro cell system, using callus produced from leaves that can be induced to form vascular tissue. Leaves are callused on induction media for two weeks. The callus is then transferred to liquid media and incubated under optimum conditions resulting in an increase in vascular tissue formation. Approximately 20% of cells will differentiate during the incubation period. The alteration of cytokinin concentration affects the ability of the cultured cells to undergo differentiation. Consequently callus incubated in liquid media, containing lower cytokinin concentrations, will undertake relatively little differentiation. Samples have been isolated from cell cultures at different time points and different hormone concentrations during incubation. Quantitative PCR using the marker AtCesA7, which encodes a cellulose synthase subunit specific to secondary wall deposition, was used as a guide to determine periods of high and low vascular differentiation. This system provides an opportunity to compare gene expression between differentiating and non differentiating cells and allow the identification of genes up regulated during vascular tissue formation. Experiment Overall Design: 6 samples

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Nottingham Arabidopsis Stock Centre (NASC)

PROVIDER: E-GEOD-6148 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The cost of Carica papaya production through seed-based propagation is increased by sex segregation, making in vitro techniques a more appealing option for clonal propagation. Inducing embryogenic callus with 2,4-dichlorophenoxyacetic acid (2,4-D) hold the potential to large-scale cloning, although the molecular mechanisms underlying this process are still not well understood. In this study, we performed a temporal analysis in the proteome of C. papaya callus to identify the key players involved in embryogenic differentiation. Mature zygotic embryos were used as explants and treated with 20 μM 2,4-D to induce embryogenic callus. Total proteins were extracted at 0, 7, 14, and 21 days (T0, T7, T14, and T21), and 1407 proteins were identified using bottom-up proteomic approach. Comparative proteomics revealed 957 differentially accumulated proteins (DAPs) (p<0.05 and log2FC >0.585 or <-0.585) in at least one comparison between the analyzed induction times points. The clustering analysis revealed four clusters with distinct patterns of protein accumulation throughout the embryogenic callus induction treatment. The cluster 1 contains 386 DAPs that accumulated at all analyzed times after treatment with 2,4-D. In contrast, cluster 2 contains 165 DAPs that decrease in abundance during the induction. The cluster 3 contains 251 proteins that are most abundant just after the start of incubation in 2,4-D (T7) and cluster 4 grouped 155 proteins that accumulate after callus formation. Functional analysis revealed that proteins involved with reserve storage and seed maturation were more abundant in the explant at T0 and decreased as callus formation progressed. Biological processes involving carbohydrate and amino acid metabolism, aerobic respiration, and protein catabolic processes were enriched after induction treatment. Regulatory proteins, including histone deacetylase (HDT3) and argonaute 1, were more abundant after the start of induction treatment with 2,4-D, suggesting their role in acquisition of embryogenic competence. Predicted protein-protein networks revealed the regulatory role of proteins 14.3.3 accumulated during callus induction and the association of proteins involved in oxidative phosphorylation, hormone response, and SAM metabolism. Our findings emphasize the modulation of the proteome at different stages during embryogenic callus initiation and identify regulatory proteins that might be involved with the activation of this process.

Project description:The growth of Arabidopsis cell cultures following their sub-culture into fresh media follows standard growth kinetics of a period of exponential increase associated with high rate of cell division, followed by a slowing of the rate of increase as cells approach stationary phase. For the analysis described here, MM2d cells were subcultured into fresh MSS-medium and samples were taken at day 1, day3, day 5 and day7. We have carried out transcriptional profiling analysis with the aim to follow growth stage specific gene expression during unperturbed growth using the near full genome ATH1 arrays (Menges et al., 2003). Journal Absract:; (Plant Molecular Biology: 53, 2003); Plant cell suspension cultures are invaluable models for the study of cellular processes. Here we develop the recently described Arabidopsis suspension culture MM2d as a transcript profiling platform by means of Affymetrix ATH1 microarrays. Analysis of gene expression profiles during normal culture growth, during synchronous cell cycle re-entry and during synchronous cell cycle progression provides a unique integrated view of gene expression responses in a higher-plant system. Particularly striking is that expression of over 14 000 genes belonging to all defined categories can be reliably detected, suggesting that integrated and comparative analysis of data sets derived from transcript profiling of cultures is a powerful approach to identify candidate components involved in a wide range of biological processes. Combinatorial analysis of independent cell cycle synchrony methods allows the identification of genes that are apparently cell-cycle-regulated but are most likely responding to the induction of synchrony. We thus present an integrated genome-wide view of the transcriptional profile of a plant suspension culture and identify a refined set of 1082 cell cycle regulated genes largely independent of synchrony method. Experimenter name = Jim Murray; Experimenter phone = 44 1223 334166; Experimenter fax = 44 1223 334162; Experimenter department = J Murray Laboratory; Experimenter address = University of Cambridge; Experimenter address = Institute of Biotechnology; Experimenter address = Tennis Court Road; Experimenter address = Cambridge; Experimenter zip/postal_code = CB2 1QT; Experimenter country = United Kingdom Experiment Overall Design: 4 samples were used in this experiment

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Transcription profiling of Arabidopsis cells cultures undergoing transdifferentiation

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