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Gravitational and magnetic field variations synergize to cause subtle variations in the global transcriptional state of Arabidopsis in vitro callus cultures.

Manzano AI, van Loon JJ, Christianen PC, Gonzalez-Rubio JM, Medina FJ, Herranz R - BMC Genomics (2012)

Bottom Line: A high gradient magnetic field can be used to levitate biological material, thereby simulating microgravity and can also create environments with a reduced or an enhanced level of gravity (g), although special attention should be paid to the possible effects of the magnetic field (B) itself.Transcriptomic results confirm that high gradient magnetic fields (i.e. to create μg* and 2 g* conditions) have a significant effect, mainly on structural, abiotic stress genes and secondary metabolism genes, but these subtle gravitational effects are only observable using clustering methodologies.A subtle, but consistent, genome-scale response to hypogravity environments was found, which was opposite to the response in a hypergravity environment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Investigaciones Biológicas (CSIC), C/Ramiro de Maeztu 9, E-28040 Madrid, Spain.

ABSTRACT

Background: Biological systems respond to changes in both the Earth's magnetic and gravitational fields, but as experiments in space are expensive and infrequent, Earth-based simulation techniques are required. A high gradient magnetic field can be used to levitate biological material, thereby simulating microgravity and can also create environments with a reduced or an enhanced level of gravity (g), although special attention should be paid to the possible effects of the magnetic field (B) itself.

Results: Using diamagnetic levitation, we exposed Arabidopsis thaliana in vitro callus cultures to five environments with different levels of effective gravity and magnetic field strengths. The environments included levitation, i.e. simulated μg* (close to 0 g* at B = 10.1 T), intermediate g* (0.1 g* at B = 14.7 T) and enhanced gravity levels (1.9 g* at B = 14.7 T and 2 g* at B = 10.1 T) plus an internal 1 g* control (B = 16.5 T). The asterisk denotes the presence of the background magnetic field, as opposed to the effective gravity environments in the absence of an applied magnetic field, created using a Random Position Machine (simulated μg) and a Large Diameter Centrifuge (2 g).Microarray analysis indicates that changes in the overall gene expression of cultured cells exposed to these unusual environments barely reach significance using an FDR algorithm. However, it was found that gravitational and magnetic fields produce synergistic variations in the steady state of the transcriptional profile of plants. Transcriptomic results confirm that high gradient magnetic fields (i.e. to create μg* and 2 g* conditions) have a significant effect, mainly on structural, abiotic stress genes and secondary metabolism genes, but these subtle gravitational effects are only observable using clustering methodologies.

Conclusions: A detailed microarray dataset analysis, based on clustering of similarly expressed genes (GEDI software), can detect underlying global-scale responses, which cannot be detected by means of individual gene expression techniques using raw or corrected p values (FDR). A subtle, but consistent, genome-scale response to hypogravity environments was found, which was opposite to the response in a hypergravity environment.

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GEDI whole-genome transcriptional status of the samples exposed to different g* and B fields. A 20 × 16 clustering analysis is shown based on the five magnetic experimental conditions (first row panels) and the two mechanical experimental conditions (third row panels) versus the external 1 g control. The panels in the second row have been calculated by extracting the 1 g* panel values (only magnetic effect) from the g* panels immediately above. The vertical colour scale bar indicates the average log2ratio levels of each cluster in the conditions compared to the parallel 1 g control (first and third row) or versus the 1 g* control (second row). The average signal in experimental conditions is slightly higher than the 1 g control (log2ratio equal to 0.03 in the centre of the scale bar) suggesting overall up-regulation. Double up-regulated clusters (with an average log2ratio > 1.03) are saturated to red and those half down-regulated (average log2ratio < -0.97) are saturated to blue. Clusters in between follow a continuous colour scale as indicated. The centre panel indicates the number of probe sets included in each cluster (20 × 16 clusters with an average size of 54 probe sets per pixel) following its own horizontal scale at the bottom. Source GEDI files are available as Additional file 4.
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Figure 4: GEDI whole-genome transcriptional status of the samples exposed to different g* and B fields. A 20 × 16 clustering analysis is shown based on the five magnetic experimental conditions (first row panels) and the two mechanical experimental conditions (third row panels) versus the external 1 g control. The panels in the second row have been calculated by extracting the 1 g* panel values (only magnetic effect) from the g* panels immediately above. The vertical colour scale bar indicates the average log2ratio levels of each cluster in the conditions compared to the parallel 1 g control (first and third row) or versus the 1 g* control (second row). The average signal in experimental conditions is slightly higher than the 1 g control (log2ratio equal to 0.03 in the centre of the scale bar) suggesting overall up-regulation. Double up-regulated clusters (with an average log2ratio > 1.03) are saturated to red and those half down-regulated (average log2ratio < -0.97) are saturated to blue. Clusters in between follow a continuous colour scale as indicated. The centre panel indicates the number of probe sets included in each cluster (20 × 16 clusters with an average size of 54 probe sets per pixel) following its own horizontal scale at the bottom. Source GEDI files are available as Additional file 4.

Mentions: Although the changes mentioned above affected a relatively low number of genes, we wanted to evaluate the overall outcome of the transcriptional profile of Arabidopsis callus exposed to these anomalous environments. We analysed the microarray data with the "Gene Expression Dynamics Inspector" (GEDI) program [42]. GEDI is a "Self Organizing Map" based software that allows the visualization of whole genome expression patterns in mosaics of n × m tiles. Each tile corresponds to a cluster of genes that share a similar gene expression pattern across conditions (centroid). Different colours reflect the expression intensity of a centroid in each condition (in our case the average ratio of intensities compared to 1 g controls). Additionally, GEDI places similar centroids close to each other in the mosaic, creating an image of the transcriptome and allowing its analysis as an entity by simple visualization and through different conditions. For this analysis we avoided filtering the data with any p value that could hide information, which meant normalizing the expression data and removing probe sets without at least a 0.5 fold change in any condition. Accordingly, 17419 of 44562 probe-sets were finally used for the GEDI analysis. They were placed in 20 × 16 mosaics with an average of 54 genes per centroid. Figure 4, shows examples of similar analyses [29,43]. When comparing the transcriptional status panels with the magnetic simulator conditions (versus parallel external 1 g control) we observed similar but not identical patterns related to the high magnetic fields (10.1 to 16.5 Tesla, Figure 4 first row). In order to minimize the magnetic field effects on the altered gravity panels we subtracted the 1 g* panel signal ratios from the altered gravity panels (xg*-1 g* → xg, Figure 4 second row). The panels obtained after this simple operation corroborated two ideas. First, the effect of the magnetic field is greater than the effect of altered gravity in this context. Second, the μg/0.1 g and 1.9 g/2 g panels are very similar to each other, but partially opposite to the hypogravity and hypergravity panels (blue repressed areas are substituted by yellow/red areas).


Gravitational and magnetic field variations synergize to cause subtle variations in the global transcriptional state of Arabidopsis in vitro callus cultures.

Manzano AI, van Loon JJ, Christianen PC, Gonzalez-Rubio JM, Medina FJ, Herranz R - BMC Genomics (2012)

GEDI whole-genome transcriptional status of the samples exposed to different g* and B fields. A 20 × 16 clustering analysis is shown based on the five magnetic experimental conditions (first row panels) and the two mechanical experimental conditions (third row panels) versus the external 1 g control. The panels in the second row have been calculated by extracting the 1 g* panel values (only magnetic effect) from the g* panels immediately above. The vertical colour scale bar indicates the average log2ratio levels of each cluster in the conditions compared to the parallel 1 g control (first and third row) or versus the 1 g* control (second row). The average signal in experimental conditions is slightly higher than the 1 g control (log2ratio equal to 0.03 in the centre of the scale bar) suggesting overall up-regulation. Double up-regulated clusters (with an average log2ratio > 1.03) are saturated to red and those half down-regulated (average log2ratio < -0.97) are saturated to blue. Clusters in between follow a continuous colour scale as indicated. The centre panel indicates the number of probe sets included in each cluster (20 × 16 clusters with an average size of 54 probe sets per pixel) following its own horizontal scale at the bottom. Source GEDI files are available as Additional file 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 4: GEDI whole-genome transcriptional status of the samples exposed to different g* and B fields. A 20 × 16 clustering analysis is shown based on the five magnetic experimental conditions (first row panels) and the two mechanical experimental conditions (third row panels) versus the external 1 g control. The panels in the second row have been calculated by extracting the 1 g* panel values (only magnetic effect) from the g* panels immediately above. The vertical colour scale bar indicates the average log2ratio levels of each cluster in the conditions compared to the parallel 1 g control (first and third row) or versus the 1 g* control (second row). The average signal in experimental conditions is slightly higher than the 1 g control (log2ratio equal to 0.03 in the centre of the scale bar) suggesting overall up-regulation. Double up-regulated clusters (with an average log2ratio > 1.03) are saturated to red and those half down-regulated (average log2ratio < -0.97) are saturated to blue. Clusters in between follow a continuous colour scale as indicated. The centre panel indicates the number of probe sets included in each cluster (20 × 16 clusters with an average size of 54 probe sets per pixel) following its own horizontal scale at the bottom. Source GEDI files are available as Additional file 4.
Mentions: Although the changes mentioned above affected a relatively low number of genes, we wanted to evaluate the overall outcome of the transcriptional profile of Arabidopsis callus exposed to these anomalous environments. We analysed the microarray data with the "Gene Expression Dynamics Inspector" (GEDI) program [42]. GEDI is a "Self Organizing Map" based software that allows the visualization of whole genome expression patterns in mosaics of n × m tiles. Each tile corresponds to a cluster of genes that share a similar gene expression pattern across conditions (centroid). Different colours reflect the expression intensity of a centroid in each condition (in our case the average ratio of intensities compared to 1 g controls). Additionally, GEDI places similar centroids close to each other in the mosaic, creating an image of the transcriptome and allowing its analysis as an entity by simple visualization and through different conditions. For this analysis we avoided filtering the data with any p value that could hide information, which meant normalizing the expression data and removing probe sets without at least a 0.5 fold change in any condition. Accordingly, 17419 of 44562 probe-sets were finally used for the GEDI analysis. They were placed in 20 × 16 mosaics with an average of 54 genes per centroid. Figure 4, shows examples of similar analyses [29,43]. When comparing the transcriptional status panels with the magnetic simulator conditions (versus parallel external 1 g control) we observed similar but not identical patterns related to the high magnetic fields (10.1 to 16.5 Tesla, Figure 4 first row). In order to minimize the magnetic field effects on the altered gravity panels we subtracted the 1 g* panel signal ratios from the altered gravity panels (xg*-1 g* → xg, Figure 4 second row). The panels obtained after this simple operation corroborated two ideas. First, the effect of the magnetic field is greater than the effect of altered gravity in this context. Second, the μg/0.1 g and 1.9 g/2 g panels are very similar to each other, but partially opposite to the hypogravity and hypergravity panels (blue repressed areas are substituted by yellow/red areas).

Bottom Line: A high gradient magnetic field can be used to levitate biological material, thereby simulating microgravity and can also create environments with a reduced or an enhanced level of gravity (g), although special attention should be paid to the possible effects of the magnetic field (B) itself.Transcriptomic results confirm that high gradient magnetic fields (i.e. to create μg* and 2 g* conditions) have a significant effect, mainly on structural, abiotic stress genes and secondary metabolism genes, but these subtle gravitational effects are only observable using clustering methodologies.A subtle, but consistent, genome-scale response to hypogravity environments was found, which was opposite to the response in a hypergravity environment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Investigaciones Biológicas (CSIC), C/Ramiro de Maeztu 9, E-28040 Madrid, Spain.

ABSTRACT

Background: Biological systems respond to changes in both the Earth's magnetic and gravitational fields, but as experiments in space are expensive and infrequent, Earth-based simulation techniques are required. A high gradient magnetic field can be used to levitate biological material, thereby simulating microgravity and can also create environments with a reduced or an enhanced level of gravity (g), although special attention should be paid to the possible effects of the magnetic field (B) itself.

Results: Using diamagnetic levitation, we exposed Arabidopsis thaliana in vitro callus cultures to five environments with different levels of effective gravity and magnetic field strengths. The environments included levitation, i.e. simulated μg* (close to 0 g* at B = 10.1 T), intermediate g* (0.1 g* at B = 14.7 T) and enhanced gravity levels (1.9 g* at B = 14.7 T and 2 g* at B = 10.1 T) plus an internal 1 g* control (B = 16.5 T). The asterisk denotes the presence of the background magnetic field, as opposed to the effective gravity environments in the absence of an applied magnetic field, created using a Random Position Machine (simulated μg) and a Large Diameter Centrifuge (2 g).Microarray analysis indicates that changes in the overall gene expression of cultured cells exposed to these unusual environments barely reach significance using an FDR algorithm. However, it was found that gravitational and magnetic fields produce synergistic variations in the steady state of the transcriptional profile of plants. Transcriptomic results confirm that high gradient magnetic fields (i.e. to create μg* and 2 g* conditions) have a significant effect, mainly on structural, abiotic stress genes and secondary metabolism genes, but these subtle gravitational effects are only observable using clustering methodologies.

Conclusions: A detailed microarray dataset analysis, based on clustering of similarly expressed genes (GEDI software), can detect underlying global-scale responses, which cannot be detected by means of individual gene expression techniques using raw or corrected p values (FDR). A subtle, but consistent, genome-scale response to hypogravity environments was found, which was opposite to the response in a hypergravity environment.

Show MeSH
Related in: MedlinePlus