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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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Analysis of enriched GO groups in genes showing significant altered expression in the different experimental conditions by GO domains. A) GO Cellular component, B) GO Biological process &C) GO Molecular function. We have use the symbols +/- when the list of genes used as input for the BINGO GO tool were filtered using limma p value and ↑/↓ when the lists were filtered using the FDR-rankprod p value, analyzing up- (+/↑) and down-regulated (-/↓) gene lists separately. The following GO groups are significantly overrepresented in each sample as determined using BINGO 2.3 with default settings (one symbol indicate a p < 0.001 and two symbols a p < 0.0005 using a hypergeometric test with BH-FDR algorithm). We use shading to indicate if this GO term is overrepresented using limma p value input list (soft shaded) only, both lists (dark shaded) or FDR rankprod p value list only (black shaded, white letters).
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Figure 3: Analysis of enriched GO groups in genes showing significant altered expression in the different experimental conditions by GO domains. A) GO Cellular component, B) GO Biological process &C) GO Molecular function. We have use the symbols +/- when the list of genes used as input for the BINGO GO tool were filtered using limma p value and ↑/↓ when the lists were filtered using the FDR-rankprod p value, analyzing up- (+/↑) and down-regulated (-/↓) gene lists separately. The following GO groups are significantly overrepresented in each sample as determined using BINGO 2.3 with default settings (one symbol indicate a p < 0.001 and two symbols a p < 0.0005 using a hypergeometric test with BH-FDR algorithm). We use shading to indicate if this GO term is overrepresented using limma p value input list (soft shaded) only, both lists (dark shaded) or FDR rankprod p value list only (black shaded, white letters).

Mentions: Figure 2 reflects the number of genes whose signal level changes in the different altered gravity/magnetic field environments, compared with the 1 g controls outside the simulators with a raw limma p value < 0.01 (above the diagonal) and a FDR corrected RankProd p value < 0.05 (below the diagonal). To determine the effects of the magnetic field alone we needed to pay attention to the effects on the internal 1 g* control and common genes in other positions. Using a raw limma p value, after 200 min in the magnet 96 genes showed significant alterations in the 1 g* position (in which B = 16.5 T without changing the effective g force. See Additional file 2: Table S1 for quantitative gene expression data) equally distributed between up- and down-regulated genes. On studying the gene ontologies (GOs) affected in this group of genes (Figure 3) we found significant enrichment in some biosynthetic and metabolic processes including thylakoid-related genes, all of them over-expressed. Comparison between different positions in the magnet (values out of the main diagonal in Figure 2) offers a low number of common genes (below 5%) except for the μg* vs 0.1 g* and the μg* vs 2 g* positions. Importantly, all miss-regulated genes in the magnet samples behaved similarly under both conditions (second and third values between brackets are 0) suggesting that these gene expression variations are related more with the high magnetic field (> 10 T in any position) than with the differential net force between them. Analysis of these common genes suggests a general stress response involving multiple enzyme activities that can be related to the presence of the high magnetic field (tranferases and peroxidases, see Additional file 3: Table S2). Using stringent statistical tests (RankProd FDR p value < 0.05) we obtained a lower number of affected genes (14), but equally distributed between up- and down-regulated genes, and enriched GO groups (Figure 3).


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)

Analysis of enriched GO groups in genes showing significant altered expression in the different experimental conditions by GO domains. A) GO Cellular component, B) GO Biological process &C) GO Molecular function. We have use the symbols +/- when the list of genes used as input for the BINGO GO tool were filtered using limma p value and ↑/↓ when the lists were filtered using the FDR-rankprod p value, analyzing up- (+/↑) and down-regulated (-/↓) gene lists separately. The following GO groups are significantly overrepresented in each sample as determined using BINGO 2.3 with default settings (one symbol indicate a p < 0.001 and two symbols a p < 0.0005 using a hypergeometric test with BH-FDR algorithm). We use shading to indicate if this GO term is overrepresented using limma p value input list (soft shaded) only, both lists (dark shaded) or FDR rankprod p value list only (black shaded, white letters).
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Figure 3: Analysis of enriched GO groups in genes showing significant altered expression in the different experimental conditions by GO domains. A) GO Cellular component, B) GO Biological process &C) GO Molecular function. We have use the symbols +/- when the list of genes used as input for the BINGO GO tool were filtered using limma p value and ↑/↓ when the lists were filtered using the FDR-rankprod p value, analyzing up- (+/↑) and down-regulated (-/↓) gene lists separately. The following GO groups are significantly overrepresented in each sample as determined using BINGO 2.3 with default settings (one symbol indicate a p < 0.001 and two symbols a p < 0.0005 using a hypergeometric test with BH-FDR algorithm). We use shading to indicate if this GO term is overrepresented using limma p value input list (soft shaded) only, both lists (dark shaded) or FDR rankprod p value list only (black shaded, white letters).
Mentions: Figure 2 reflects the number of genes whose signal level changes in the different altered gravity/magnetic field environments, compared with the 1 g controls outside the simulators with a raw limma p value < 0.01 (above the diagonal) and a FDR corrected RankProd p value < 0.05 (below the diagonal). To determine the effects of the magnetic field alone we needed to pay attention to the effects on the internal 1 g* control and common genes in other positions. Using a raw limma p value, after 200 min in the magnet 96 genes showed significant alterations in the 1 g* position (in which B = 16.5 T without changing the effective g force. See Additional file 2: Table S1 for quantitative gene expression data) equally distributed between up- and down-regulated genes. On studying the gene ontologies (GOs) affected in this group of genes (Figure 3) we found significant enrichment in some biosynthetic and metabolic processes including thylakoid-related genes, all of them over-expressed. Comparison between different positions in the magnet (values out of the main diagonal in Figure 2) offers a low number of common genes (below 5%) except for the μg* vs 0.1 g* and the μg* vs 2 g* positions. Importantly, all miss-regulated genes in the magnet samples behaved similarly under both conditions (second and third values between brackets are 0) suggesting that these gene expression variations are related more with the high magnetic field (> 10 T in any position) than with the differential net force between them. Analysis of these common genes suggests a general stress response involving multiple enzyme activities that can be related to the presence of the high magnetic field (tranferases and peroxidases, see Additional file 3: Table S2). Using stringent statistical tests (RankProd FDR p value < 0.05) we obtained a lower number of affected genes (14), but equally distributed between up- and down-regulated genes, and enriched GO groups (Figure 3).

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