An extensive (co-)expression analysis tool for the cytochrome P450 superfamily in Arabidopsis thaliana

Abstract

Background: Sequencing of the first plant genomes has revealed that cytochromes P450 have evolved to become the largest family of enzymes in secondary metabolism. The proportion of P450 enzymes with characterized biochemical function(s) is however very small. If P450 diversification mirrors evolution of chemical diversity, this points to an unexpectedly poor understanding of plant metabolism. We assumed that extensive analysis of gene expression might guide towards the function of P450 enzymes, and highlight overlooked aspects of plant metabolism.

Results: We have created a comprehensive database, 'CYPedia', describing P450 gene expression in four data sets: organs and tissues, stress response, hormone response, and mutants of Arabidopsis thaliana, based on public Affymetrix ATH1 microarray expression data. P450 expression was then combined with the expression of 4,130 re-annotated genes, predicted to act in plant metabolism, for co-expression analyses. Based on the annotation of co-expressed genes from diverse pathway annotation databases, co-expressed pathways were identified. Predictions were validated for most P450s with known functions. As examples, co-expression results for P450s related to plastidial functions/photosynthesis, and to phenylpropanoid, triterpenoid and jasmonate metabolism are highlighted here.

Conclusion: The large scale hypothesis generation tools presented here provide leads to new pathways, unexpected functions, and regulatory networks for many P450s in plant metabolism. These can now be exploited by the community to validate the proposed functions experimentally using reverse genetics, biochemistry, and metabolic profiling.

Figure 1

Expression in the organ and tissue dataset. Microarray data were retrieved from the Genevestigator database. Background was defined for each probe set as the mean intensity of all samples the probe set was called 'absent' (not significantly higher (p < 0.06) than the signal observed with the corresponding mismatch probe set). a) Histogram describing the frequency distribution of P450 genes expressed in the organ and tissue data set. Given in each bin is the number of probe sets representing P450 genes expressed more than twofold above background in 0% to 5%, 5% to 10%, etc., up to 95% to 100% of the 277 organ and tissue hybridization experiments. The number of genes in each bin is given on top of each bin. b) Genes that are expressed in more than 80% of root, whole flower, or leaf samples (>twofold above background), but not in more than 20% of all other samples (from a total of 277 samples) were selected. Shown are expression data of these genes in leaf, root, and flower samples as indicated on top. Expression intensities are compared to background (defined as the mean intensity of all samples called 'absent'

Figure 2

Comparison of expression data between platforms. P450 expression data generated using a spotted microarray covering gene specific PCR products (CYP-array) were retrieved from the 'Functional Genomics of Arabidopsis P450s' web page (Table 1). In this analysis, signal intensities in roots from 1 week old seedlings were generated by comparison to a 'universal RNA' sample [2]. Not detectable intensities were artificially set to a ratio of 0.05 compared to the 'universal control' and after log₂-transformation expression data were mean centered across the experiments. Expression data from published Affymetrix ATH1 array hybridizations were processed as described in Methods. The mean intensities from 17 experiments derived from young roots were selected. To generate a control similar to the 'universal RNA', mean intensities from 69 experiments covering similar samples were calculated and log₂ ratios were generated. Shown is a 2 × 2 plot comparing the mean centered expression ratios [log₂(sample/mean)] from both platforms using data for all P450 genes represented on both array types. Data points following the same trend are shown in black, points which are more than twofold different from the average expression in one platform, but less than twofold different in the other are shown in gray. Red dots indicate genes with opposing expression using the two platforms

Figure 3

Pathogen induced expression of selected P450s. Microarray expression data were retrieved from the 'Genevestigator' database and processed as described in Methods. Selected genes that are up-regulated (>twofold) in more than 30% of at least one treatment group as indicated on top are shown. The complete set of genes fulfilling this criterion is shown in Additional File 2. Background corrected expression intensities were compared to untreated control experiments and log₂-ratios were used for visualization. The resulting heatmap is color coded as indicated. Details on the individual samples can be found in Additional File 2.

Figure 4

Hormone responsive expression. Microarray expression data were retrieved from the 'Genevestigator' database and processed as described in Material and Methods. Background corrected expression intensities were compared to untreated control experiments and log₂-ratios were used. Genes that are up- or down-regulated (>twofold) in more than 30% of each treatment group as indicated were selected. a) Number of P450s which are responsive to each treatment. b) Hierarchical cluster analysis with complete linkage. The resulting heatmap is color coded as indicated.

Figure 5

Expression analysis using CYP98A8 as bait. Data from published Affymetrix microarrays representing 167 organ and tissue samples were retrieved from the Genevestigator database [10]. Background correction and ratio log₂-ratio generation was performed as describe in Methods. The expression vector of CYP98A8 was compared to those of 4,119 genes annotated in diverse databases to be involved in any metabolic pathway using the 'ExpresionAngler' algorithm [9]. Expression profiles of co-expressed genes with a correlation coefficient of more than 0.6 are shown as a heatmap. Groups of samples are indicated on top of the heatmap. Mean-centered signal intensity ratios are color coded as indicated on the bottom of the heatmap. Genes with similarity to enzymes of the phenylpropanoid pathway are highlighted in orange. Genes related to lipid metabolism are highlighted in blue. Detailed information on the co-expressed genes and samples can be found in Additional File 5. To the right a section of the phenylpropanoid pathway is outlined in red and the putative duplicated pathway as hypothesized based on the co-expression analysis of CYP98A8 is outlined in orange.

Figure 6

Co-expression analysis of a P450 associated with plastidial activity: CYP97A3. Microarray expression data were retrieved from the 'Genevestigator' database and processed as described in Methods. The organ expression vector of CYP97A3 was used as bait for co-expression analysis as described in Figure 4. The expression vector of the bait CYP97A3 (first row) is shown across 167 organ and tissue samples. 50 co-expressed genes having a correlation coefficient of r > 0.84 are shown in subsequent rows. The resulting heatmap is color coded as indicated. Highlighted in green are genes from the categories 'plastidial isoprenoids' (BioPath), 'photosystems' (BioPath), 'photosynthesis' (KEGG or FunCat), and 'biogenesis of the chloroplast' (FunCat). Detailed information on the co-expressed genes and samples can be found in Additional File 6. Up to 80 additional P450s in the CYPedia analysis share a similar expression profile and pathway prediction.

Figure 7

Organ expression of co-expressed triterpene synthases (TTPS) and P450s. Microarray expression data were retrieved from the 'Genevestigator' database and processed as described in Methods. Expression vectors from the organ and tissue data sets of five TTPS genes (from A. thalaina was used as a bait for co-expression analysis comparing it's expression with that of all P450 genes. We retained five TTPS genes, which were co-expressed (r > 0.75) with at least one P450 in the organ and tissue expression data set, and the corresponding P450s (Table 2). This set of genes was used visualize expression. TTPS (in bold) and correlated P450 genes with high correlation coefficients are color coded.