Correlation Network Analysis reveals a sequential reorganization of metabolic and transcriptional states during germination and gene-metabolite relationships in developing seedlings of Arabidopsis

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Abstract

Background: Holistic profiling and systems biology studies of nutrient availability are providing more and more insight into the mechanisms by which gene expression responds to diverse nutrients and metabolites. Less is known about the mechanisms by which gene expression is affected by endogenous metabolites, which can change dramatically during development. Multivariate statistics and correlation network analysis approaches were applied to non-targeted profiling data to investigate transcriptional and metabolic states and to identify metabolites potentially influencing gene expression during the heterotrophic to autotrophic transition of seedling establishment.Results: Microarray-based transcript profiles were obtained from extracts of Arabidopsis seeds or seedlings harvested from imbibition to eight days-old. 1H-NMR metabolite profiles were obtained for corresponding samples. Analysis of transcript data revealed high differential gene expression through seedling emergence followed by a period of less change. Differential gene expression increased gradually to day 8, and showed two days, 5 and 7, with a very high proportion of up-regulated genes, including transcription factor/signaling genes. Network cartography using spring embedding revealed two primary clusters of highly correlated metabolites, which appear to reflect temporally distinct metabolic states. Principle Component Analyses of both sets of profiling data produced a chronological spread of time points, which would be expected of a developmental series. The network cartography of the transcript data produced two distinct clusters comprising days 0 to 2 and days 3 to 8, whereas the corresponding analysis of metabolite data revealed a shift of day 2 into the day 3 to 8 group. A metabolite and transcript pair-wise correlation analysis encompassing all time points gave a set of 237 highly significant correlations. Of 129 genes correlated to sucrose, 44 of them were known to be sucrose responsive including a number of transcription factors.Conclusions: Microarray analysis during germination and establishment revealed major transitions in transcriptional activity at time points potentially associated with developmental transitions. Network cartography using spring-embedding indicate that a shift in the state of nutritionally important metabolites precedes a major shift in the transcriptional state going from germination to seedling emergence. Pair-wise linear correlations of transcript and metabolite levels identified many genes known to be influenced by metabolites, and provided other targets to investigate metabolite regulation of gene expression during seedling establishment. © 2010 Allen et al; licensee BioMed Central Ltd.

Figures

  • Figure 1 Trends in differential gene expression. DE genes were determined between each successive day at a threshold cut-off level of 1.5-fold. Each comparative stage, i.e. day, was measured in triplicate and the mean of the hybridization intensities calculated prior to DE analysis. (A) Total number of DE genes and the split between UR and DR genes. (B) The proportion given as percentages of total DE genes comprised by either chloroplast/plastid protein or TF/signaling protein encoding genes as given in the TAIR gene ontology database. Open and closed bars represent UR and DR genes, respectively.
  • Figure 2 Relationships between metabolites. (A) Clusters of metabolites with similar profiles generated by 2D-SOM. Hierarchal and K-means clustering were used to estimate the optimal number of bins for 2D-SOM analysis. Metabolites in cluster 1: sucrose, rhamnose, citrate, alanine, trigonelline, lactate, glucose, threonine, unkS7.37, unkM1.85; Cluster 2: arginine, formate; Cluster 3: fumarate, proline, glutamate, unkD8.0, unkM5.18, unkD3.12; Cluster 4: malate; Cluster 5: valine, isoleucine, leucine, choline, unkD5.69; Cluster 6: fructose, glutamine, unkM7.9. (B) Spring embedding plots showing relationships based on correlations. The plot shows metabolites as nodes and Pearson correlation coefficients over days as connections. The color of the connecting line describes the strength of the correlation between the nodes; a dark red color indicates a strong positive correlation and a dark blue line represents a weaker positive correlation according to the scale of correlation coefficients on the right of the graph. Only correlations above a Bonferroni-adjusted P-value < 0.0001 are shown. (C) Enlargement of the lactate cluster. (D) Enlargement of the valine cluster. Since values start from an initial random configuration, the directions separating cluster in each spring embedding plot are arbitrary, but they provide an indication of distance separating nodes and edges.
  • Figure 3 Day-by-transcript relationships. (A) PCA scores plot of the time points sampled during germination and seedling establishment based on the average transcript levels. Each number 0 to 8 represents one day (24 h) from imbibed seeds (0) to 8 days (8) of age, respectively. (B) Higher order relationships among days based on mean values of transcript levels from the 3 replicates visualized by spring embedding. The plot shows day 0 (d0) to day 8 (d8) as nodes and the relative degree of transcript correlation as edges. Clustering was based on Pearson correlation coefficients at a threshold cut-off of 0.7. The color bar on the right of the figure provides the relative degree of correlation.
  • Figure 4 Day-by-metabolite relationships. (A) PCA scores plot where each number represents one day (24 h) from imbibed seeds (0) to 8 days of age (8). (B) Spring embedding plot where the symbols d0 to d8 correspond to the samples in A. Each point is a node representing the mean value and each line gives the relative degree of correlation. The threshold Pearson correlation coefficient for the spring embedding was 0.7. The color bar on the right of each figure provides the relative degree of coloration. Both types of analysis were based on the mean values (n = 3) of 3 replicates (2 replicates for day 3).
  • Figure 5 A spring embedding model revealing relationships between metabolites and genes from days 0 to 8. Pearson correlation coefficients were determined between every metabolite and gene over the 9 time points. Metabolites are central nodes from which connected genes radiate outwards. The coloured lines represent edges describing the nature of the correlation; a dark red line represents a strong positive correlation whereas a dark blue line represents a strong negative correlation. A total of 237 correlations were identified between 20 metabolites and 209 genes at the threshold cut-off of (p < 0.0001, r > |0.95|). The plots inset show the profiles of the average expression values for the transcription factors IAA14, ARF10 and ABI3 used to calculate correlation coefficients.
  • Table 1: Correlations between metabolites and transcript levels in developing seedlings.
  • Table 2: Identified regulatory genes correlated with metabolites.

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Allen, E., Moing, A., Ebbels, T. M. D., Maucourt, M., Tomos, A. D., Rolin, D., & Hooks, M. A. (2010). Correlation Network Analysis reveals a sequential reorganization of metabolic and transcriptional states during germination and gene-metabolite relationships in developing seedlings of Arabidopsis. BMC Systems Biology, 4. https://doi.org/10.1186/1752-0509-4-62

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