This work was supported by a grant to MEM from the Dept. of Life Sciences and Systems Biology of the University of Turin, Italy. The authors thank G. Gnavi for technical assistance during some experiments. CTR is funded by the Wellcome Trust and the Royal Society (Grant Number 098436/Z/12/Z).

1. Setting of the Triaxial Coils
NOTE: Figure 1 shows the triaxial coils used to reverse the GMF.
<ol>
	<li>Turn on the three-axis magnetometer, whose probe is inserted in the triaxial coils.</li>
	<li>Turn on the computer and launch the magnetometer software that allows data to be collected from three-axis magnetometer.</li>
	<li>Use the component values reported by the magnetometer to calculate the magnitude of the GMF. For instance, with magnetometer values: Bx = 6.39 &#181;T, By = 36.08 &#181;T, Bz = 20.40 &#181;T calculate a field strength of 41.94 &#181;T by using the following equation: B = BGMF + Badditional, where Badditional = (Bx2 + By2 + Bz2)&#189; (i.e., 41.9 &#181;T in the example.)</li>
	<li>Turn on the three DC power supplies (dual range: 0-8V/5A and 0-20V/2.5A, 50W) each one connected to three couples of Helmholtz coils and connected to a computer via a GPIB connection (Figure 1B).</li>
	<li>Set voltages of the power supplies to generate the desired magnetic field with a reversed magnetic field vector. For instance, with BGMF as in step 1.3 and with the coil size of the instrumentation described here, set the voltages to Vx =0.00 V, Vy = 30.52 V, Vz = 0.00 V in order to generate a new resultant B = BGMF + Btriaxial coils = (6.38, -36.08, 20.39) &#181;T. i.e., a new field with the same magnitude as BGMF but pointing to a different direction.</li>
	<li>Verify the new field with the magnetometer software by using the procedure described in 1.3.</li>
	<li>Expose plants to both normal and reversed GMF conditions by using Petri plates as described in section 2.</li>
	<li>Perform sham exposure experiments by keeping the magnitude of the field equal to | BGMF| and keeping the vertical component of the field equal to that of the GMF but altering the direction (i.e., &#8220;North, East or West&#8221;) of the horizontal component of the field with equal currents in the triaxial coils compared to the field reversal condition. Do this by altering the voltage of the coils as described in 1.5. 
	Note: This sham exposure rules out potential subtle heating or vibrational effects from either the coils themselves or from the electronics used to control the coils.</li>
	<li>Run double-blind experiments by applying field conditions blinded from the personnel performing the remainder of the experiments and/or interpreting the data.</li>
</ol>
2. Preparation of Plant Materials and Conditions for Plant Growth
<ol>
	<li>Use seeds of Arabidopsis thaliana, ecotype Columbia 0 (Col 0), place then into a 1.5 ml tube and surface sterilize by treatment with a 5% (w/v) calcium hypochlorite solution and 0.02% (v/v) Triton X-100 in 80% ethanol (EtOH), for 10-12 min at 25-28 &#176;C, with continuous shaking. Then rinse twice with 80% EtOH, wash with 100% EtOH and finally rinse with sterile distilled water.</li>
	<li>Prepare 1 L of Murashige and Skoog 22 (MS) modified medium by adding: 2.297 g of MS (0.5 x MS Basal Salt Mixture), 10 g Sucrose, deionized water up to 1 L, pH 5.8-6.0 adjusted with KOH. Add 16 g of agar and autoclave for 20 min, 120 &#176;C.</li>
	<li>Before solidification, pour 80 ml of medium into each (120 x 120 mm2) square Petri plates. Sow thirty sterile seeds on plate, and then seal plates with a waxy film.</li>
	<li>Vernalize the plates horizontally in darkness at 4 &#176;C for 2 days to potentiate and synchronize germination, and then expose Petri plates to either normal or reversed GMF.</li>
	<li>Expose seeds in a climate controlled environment at 22 &#176;C in a vertical position in parallel experiments both inside the triaxial coils and outside the triaxial coils under a photoperiod schedule of 8 hr darkness and 16 hr light, using Sodium vapor lamps (220 x 10-6 E m-2s-1). Use a blue gelatin film for spotlight to reduce the red component of lamps.</li>
	<li>Expose plants for 10 days before RNA extraction to both normal (control) and reversal (treatment) GMF conditions.</li>
	<li>After exposure, take pictures of Petri dishes.</li>
	<li>Use the ImageJ software to calculate root length and leaf areas.
	<ol>
		<li>Briefly, measure the side of the Petri plate, then open the image of the Petri plate and by using the &#8220;straight&#8221; line option to draw a line that exactly crosses the plate side.</li>
		<li>In the &#8220;analyze&#8221; menu select &#8220;set scale&#8221; and insert the actual distance in the &#8220;known distance&#8221; box (e.g., 120 mm), then insert the unit of length (mm); finally click the &#8220;global&#8221; option to make settings available for all measurements.</li>
		<li>For root length, follow carefully the shape of the root by using the freehand tool. Measure the length by using the &#8220;measure&#8221; option in the &#8220;analyze&#8221; menu. Continue to measure all roots in the picture and save the file for further statistical analyses.</li>
		<li>For leaf area, from the &#8220;image&#8221; menu use the adjust option and then &#8220;color threshold&#8221;. Select the individual leaf and in the &#8220;analyze&#8221; menu select &#8220;analyze particles&#8221;. Save the individual measurements for statistical analyses.</li>
	</ol>
	</li>
</ol>
3. Arabidopsis Total RNA Extraction, Quantitative Real Time-PCR (qPCR) Reaction Conditions and Primers for Arabidopsis
<ol>
	<li>Collect separately 30 shoots and 30 roots and immediately freeze in liquid nitrogen. Then grind in liquid nitrogen with mortar and pestle.</li>
	<li>Isolate total RNA using a purification kit and RNase-Free DNase treatment kit by using manufacturer&#8217;s instructions.</li>
	<li>Check sample quality and quantity by using an RNA nano kit and capillary gel electrophoresis according to manufacturer&#8217;s instructions. Confirm the quantification of RNA spectrophotometrically.</li>
	<li>Use 2 &#181;g of total RNA and random primers using a cDNA Reverse Transcription Kit to obtain first strand cDNAs according to the manufacturer&#8217;s recommendations.</li>
	<li>Perform all experiments on a Real-Time System using SYBR green I with ROX as an internal loading standard.</li>
	<li>Perform the reaction with 25 &#181;l of mixture consisting of 12.5 &#181;l of 2x SYBR Green qPCR Master Mix, 0.5 &#181;l of cDNA and 100 nM primers. Use the primers listed in Table 1. Include in controls non-RT controls (using total RNA without reverse transcription to monitor for genomic DNA contamination) and non-template controls (water instead of template).</li>
	<li>Calculate primer efficiency for all primers pairs using the standard curve method 23.</li>
	<li>Use the following PCR conditions: CRU3, COTP1, RRTF1. 10 min at 95 &#176;C, 40 cycles of 15 s at 95 &#176;C, 30 sec at 58 &#176;C, and 30 sec at 72 &#176;C; UBP6, eEF1Balpha2, ACT1, GAPC2, CAT3, TAPX, APX1, RbohD, FeSOD1 10 min at 95 &#176;C, 40 cycles of 15 s at 95 &#176;C, 20 sec at 57 &#176;C, and 30 sec at 72 &#176;C.</li>
	<li>Read fluorescence following each annealing and extension phase. For all runs, perform a melting curve analysis from 55 to 95 &#176;C by including the dissociation segment in the thermal profile. Use the dissociation curve screen, accessed through the results tab to view the dissociation profile (plot of the fluorescence as a function of temperature). Ensure that the dataset collected during the dissociation segment of the experiment is selected for analysis using the Analysis/Setup screen.</li>
	<li>Determine the linear range of template concentration to threshold cycle value (Ct value) by performing a tenfold dilution series (1- to 103-fold) using cDNA from three independent RNA extractions analyzed in three technical replicates 24,25.</li>
	<li>Analyze all amplification plots with the Real-Time PCR instrument software to obtain Ct values. Calibrate and normalize relative RNA levels with the level of the best housekeeping genes as follows: 3.11.1) Access the Amplification Plots screen through the Results tab, select the ramp or plateau for which data should be analyzed using the Analysis Selection/Setup screen, and then select the dRn (baseline-corrected normalized fluorescence) from the Fluorescence menu on the command panel. Access the Plate Sample Values screen through the Results tab to display Ct values for the sampled wells.</li>
	<li>Use four different reference genes [e.g., cytoplasmic glyceraldehyde-3-phosphate dehydrogenase, (GAPC2), ubiquitin specific protease 6 (UBP6), Actin1 (ACT1) and the elongation factor 1B alpha-subunit 2 (eEF1Balpha2)] to normalize the results of the real time PCR. Select the top ranked gene using an analysis software 26; and use the most stable gene for normalization.
	<ol>
		<li>Briefly, organize the input data on an Excel sheet with the first column containing the gene names and first row containing the sample names. Then select the analysis software from the menu-bar. Use the dialog box to select the input data.</li>
		<li>Next, check the fields Sample names, gene names and simple output only. Click on Go button to perform the analysis. Select the top ranked gene (which has the smallest stability value) as candidate gene most stably expressed.</li>
	</ol>
	</li>
	<li>Plot data by showing the differential fold change expression in both shoots and roots.</li>
</ol>
4. Statistical Analyses
<ol>
	<li>Express data as mean values &#177; standard error. Compare the control and the treatment groups by performing analysis of variance (ANOVA) and Tukey&#8217;s test with Bonferroni and Dunn-Sidak Adjusted Probability test (0.95% confidence).</li>
</ol>

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The authors have nothing to disclose.

We recently showed that an amazing correlation exists between GMF reversals and the time when diversion of most of the familial Angiosperm lineages occurred 2. However, despite the stimulating hypotheses and the plethora of studies on the effect of varied GMF intensities, the assumption that GMF reversal may itself induce significant changes in plant gene expression and morphology has never been demonstrated. Here we show for the first time, a method that uses a triaxial octagonal Helmholtz coil to reverse GMF in our laboratory, and that the reversal of the ambient magnetic field can cause phenotypic changes and modulation of gene expression in plants.
In order to obtain GMF reversal (or modification) over a sufficient volume for the plant growth experiments (2 x 2 x 2 m3), we built an octagonal Helmholtz coil system. This system is not commercially available (usually Helmholtz coils are ring-shaped and smaller) and the costs for construction were considerable. Importantly, this system delivers robust field modification, with exceptional time-stability and homogeneity in the modified magnetic fields.
The system is designed and built to reduce the value of the GMF to a thousandth of the normal conditions or to reverse any of the three dimensions of the magnetic field. However, the design of the coils does not allow to generate a high magnetic field strength. Therefore, this instrument in the present form is not suitable for experiments designed to evaluate the effect of high magnetic field strength on plants or other organisms.
In laboratory, alteration of the GMF similar to those described in this method has been obtained by different methods, including shielding by surrounding the experimental zone by ferromagnetic metal plates with high magnetic permeability, which deviate magnetic fields and concentrate them within the metal itself. The advantage of using Helmholtz coils is that the system allows plants to be exposed to more natural conditions (light, air circulation, etc.), thus making it ideal not only for in vitro studies (as with the use of Petri dishes) but also for in vivo plant growth and development experiments. The dimensions of our system create a space that allows a suppression up to &#60;1/1,000th of the natural GMF throughout a 25 x 25 x 25 cm3 spherical volume (see Figure 1A), thus allowing to host several Petri plates or some small pots for plant growth.
The method presented here has been applied to plant biology studies; however, the system allows a wide range of experimentation, including virology and microbiology, as well as studies on nematodes (e.g., Caenorhabditis elegans), arthropods and small animals (including mice and rats). Therefore, tests of the hypothesis that reversal of the GMF is able to induce morphological and transcriptional changes could also be extended to many other living systems, perhaps ultimately even to human cells.
The GMF is constantly changing and fluctuating. Therefore, in our experiments one major challenge is to provide a constant compensation of the GMF in order to obtain the desired new GMF values. This can only be achieved by a continuous control of magnetic fields values through reading of magnetometer values and voltage compensation. Therefore, the system can compensate the slowly varying part of the GMF but it does nothing for higher frequency fluctuations.
In conclusion, the use of triaxial coils to reverse the GMF vector was instrumental to demonstrate that this reversal of the GMF vector is able to induce plant morphological changes and differential gene expression. The results obtained with the presented method provide a compelling evidence in support of the hypothesis that the GMF reversals might have been one of the driving forces for plant evolution over geological timescales 2.

The Earth&#8217;s magnetic field (or equivalently the geomagnetic field, GMF) is an inescapable environmental factor for all organisms living on the planet, including plants. The GMF has always been a natural feature of the Earth, so during evolution, all living organisms experienced its action. An increasing body of evidence shows that the GMF is able to influence many biological processes 1. The GMF is not uniform and there are significant local differences in its magnitude and direction at the surface of the Earth. The GMF at the Earth&#39;s surface shows a broad range of magnitudes, ranging from less than 30 &#181;T to almost 70 &#181;T. The GMF protects the Earth and its biosphere from the lethal effects of solar wind by deflecting most of its charged particles through the magnetosphere2.
Plants respond to environmental stimuli; and classical responses to abiotic factors such as light and gravity have been thoroughly described by defining the so-called phototropic and gravitropic responses. Very little, or nothing, is known on the mechanisms of perception and responses of plants to magnetic fields, despite the plethora of papers published on this topic and recently reviewed 1. Unlike the gravitational field, the GMF changed consistently during plant evolution thereby representing an important abiotic stress factor that has been recently considered a potential driving force eventually contributing to plant diversification and speciation 2. Geomagnetic reversals (or excursions) are changes in polarity of the GMF. During Earth&#8217;s life-history, GMF reversals occurred several times. These exposed the planet to periods of reduced GMF strength during every polarity transition. Some authors have hypothesized that these transitions periods of low GMF strength might have allowed ionizing radiation from the solar wind to reach the Earth&#8217;s surface, thereby inducing a consistent stress to living organisms, which could have been strong enough to induce gene alterations eventually leading to plant evolution 2.
A detailed analysis of experiments describing the effects of magnetic fields on plants shows a large number of conflicting reports, characterized by a dearth of plausible biophysical interaction mechanisms. Many experiments are simply unrealistic, while others lack a testable hypothesis and, ultimately, are unconvincing 3. Over the past years, the progress and status of research on the effect of magnetic fields on plant has been reviewed 2,4-11. Recently, the effect of both low and high magnetic field has been thoroughly discussed 1, with a particular focus on the involvement of GMF reversal events on plant evolution 2.
The most direct means to substantiate the hypothesis that GMF reversals affect plant evolution is to synthesize a GMF reversal in the laboratory by testing the response of plants to normal and reversed magnetic field conditions. To test the hypothesis, we therefore built a triaxial octagonal Helmholtz coil-pairs magnetic field compensation system (triaxial coils), which is able to accurately reverse the normal GMF conditions.
We used Arabidopsis thaliana as a model plant and we tested the effect of reversed GMF on gene expression of some important genes: CRUCIFERIN 3 (CRU3), that encodes a 12S seed storage protein that is tyrosine-phosphorylated and its phosphorylation state is modulated in response to ABA in Arabidopsis thaliana seeds 12,13; the Copper Transport Protein1 (COTP1), that encodes a heavy metal transport/detoxification superfamily protein with the predominant function in soil Cu acquisition and pollen development 14; and Redox Responsive Transcription Factor1 (RRTF1), that encodes a member of the ERF (ethylene response factor) subfamily B-3 of ERF/AP2 transcription factor family that contains one AP2 domain that facilitate the synergistic co-activation of gene expression pathways and confer cross tolerance to abiotic and biotic stresses 15.
Moreover we also analyzed five genes involved in oxidative stress responses: Fe Superoxide Dismutase1, (FSD1), that encodes a cytoplasmic enzyme that enzymatically and rapidly converts the superoxide anion (O2-) and water (H2O) to hydrogen peroxide (H2O2) and molecular oxygen (O2) 16; Catalase3 (CAT3), that that encodes and enzyme that catalyzes the breakdown of H2O2 into water and oxygen 17,18; Thylakoidal Ascorbate Peroxidase (TAPX), that encodes a chloroplastic thylakoid peroxidase that scavenges H2O2 19;Ascorbate Peroxidase1 (APX1), that encodes a cytosolic peroxidase that scavenges H2O2 and represents one of the potential targets of post-translational modifications mediated by NO-derived molecules 20; and NADPH-Respiratory burst oxidase protein D (RbohD) that encodes an enzyme that generates O2- and plays pivotal roles in regulating growth, development and stress responses in Arabidopsis 21.
Our field-reversal methodology provides the first evidence that GMF reversal can induce a significant change in the morphology and gene expression of A. thaliana roots and shoots. This protocol provides an innovative way to evaluate the effect of GMF reversal on plant morphology and gene expression and can be used to assess the potential effect of GMF reversal on other aspects of plant behavior, and thereby guide discussion of the role of GMF reversal on plant evolution.

<table>
	<tbody>
		<tr>
			<td>three-axis magnetometer</td>
			<td>Bartington&#160;</td>
			<td>Mag-03MC</td>
			<td>triaxial fluxgate magnetometer</td>
		</tr>
		<tr>
			<td>Magnetometer power supply</td>
			<td>Bartington&#160;</td>
			<td>Mag-03PSU</td>
			<td>triaxial fluxgate magnetometer</td>
		</tr>
		<tr>
			<td>Magnetometer software</td>
			<td>Bartington&#160;</td>
			<td>Mag03DAM</td>
			<td>triaxial fluxgate magnetometer</td>
		</tr>
		<tr>
			<td>DC power supply&#160;</td>
			<td>Agilent Technologies</td>
			<td>E3642A</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium hypochlorite&#160;</td>
			<td>Sigma</td>
			<td>211389</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Sigma</td>
			<td>X100&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol&#160;</td>
			<td>Sigma</td>
			<td>2860</td>
			<td></td>
		</tr>
		<tr>
			<td>GroLux Sodium vapor lamps&#160;</td>
			<td>OSRAM Sylvania</td>
			<td>600W</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Plant RNA kit&#160;</td>
			<td>Qiagen</td>
			<td>74903</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-Free DNase&#160;</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer</td>
			<td>Agilent Technologies</td>
			<td>G2938B</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop ND-1000&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>not available</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit&#160;</td>
			<td>Applied Biosystems</td>
			<td>4368813</td>
			<td></td>
		</tr>
		<tr>
			<td>Mx3000P</td>
			<td>Agilent Technologies</td>
			<td>401512</td>
			<td></td>
		</tr>
		<tr>
			<td>2X MaximaTM SYBR Green qPCR Master Mix&#160;</td>
			<td>Fermentas International, Inc</td>
			<td>K0221</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma</td>
			<td>P7793-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige and Skoog Basal Medium</td>
			<td>Sigma</td>
			<td>M5519&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish square (120x120)</td>
			<td>Sigma</td>
			<td>Z692344&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

geomagnetic field (gmf) and plant evolution: investigating the effects of gmf reversal on arabidopsis thaliana development and gene expression

One of the most stimulating observations in plant evolution is a correlation between the occurrence of geomagnetic field (GMF) reversals (or excursions) and the moment of the radiation of Angiosperms. This led to the hypothesis that alterations in GMF polarity may play a role in plant evolution. Here, we describe a method to test this hypothesis by exposing Arabidopsis thaliana to artificially reversed GMF conditions. We used a three-axis magnetometer and the collected data were used to calculate the magnitude of the GMF. Three DC power supplies were connected to three Helmholtz coil pairs and were controlled by a computer to alter the GMF conditions. Plants grown in Petri plates were exposed to both normal and reversed GMF conditions. Sham exposure experiments were also performed. Exposed plants were photographed during the experiment and images were analyzed to calculate root length and leaf areas. Arabidopsis total RNA was extracted and Quantitative Real Time-PCR (qPCR) analyses were performed on gene expression of CRUCIFERIN 3 (CRU3), copper transport protein1 (COTP1), Redox Responsive Transcription Factor1 (RRTF1), Fe Superoxide Dismutase 1, (FSD1), Catalase3 (CAT3), Thylakoidal Ascorbate Peroxidase (TAPX), a cytosolic Ascorbate Peroxidase1 (APX1), and NADPH/respiratory burst oxidase protein D (RbohD). Four different reference genes were analysed to normalize the results of the qPCR. The best of the four genes was selected and the most stable gene for normalization was used. Our data show for the first time that reversing the GMF polarity using triaxial coils has significant effects on plant growth and gene expression. This supports the hypothesis that GMF reversal contributes to inducing changes in plant development that might justify a higher selective pressure, eventually leading to plant evolution.

The aim of this protocol is to provide a method to assess whether reversal of the geomagnetic field (GMF) may affect plant development and gene expression of Arabidopsis thaliana ecotype Col 0. Triaxial coils as shown in Figure 1A are used to reverse the GMF when set with the appropriate drive voltages (Figure 1B), obtained as described in step 1.5 in the protocol. The dimensions of the triaxial coils are ~2 x 2 x 2 m3, which allowed sufficient space with reversed GMF conditions to host several Petri plates. Controls were grown in the same environmental conditions and at normal GMF values. After 10 days of exposure to normal and reversed GMF conditions, the phenotype of plants showed evident morphological alterations. As shown in Figure 2, control plants (i.e., grown in normal GMF conditions) showed root lengths with significantly (Dunn-Sidak and Bonferroni Adjusted Prob &#60;0.001; Student&#8217;s t = 10.68, df = 31) higher values (29.41 mm; SEM = 1.04; N = 32) with respect to plants exposed to reversed GMF (17.53 mm; SEM = 0.58; N = 36). In GMF-reversed plants, the morphology of shoots was also altered by showing a reduced development of leaflet expansion. Plants exposed to normal conditions showed an average leaf area of 4.95 mm2 (SEM 0.025, N = 54), whereas plants exposed to reversed GMF conditions showed significantly (Dunn-Sidak and Bonferroni Adjusted Prob = &#60;0.001; student&#8217;s t = 31.32, df = 53) lower leaf area values (3.71 mm2; SEM = 0.032; N = 54). Therefore, exposure of Arabidopsis to reversed GMF conditions induced a reduction in both root length and leaf area.
Leaf expansion and root growth are dependent on both the division and the elongation of cells 27. Therefore, plant development, productivity and overall fitness are dependent on an optimal shoot- and root-system architecture 28. The reduced root length and leaf size of plants exposed to reversed GMF conditions indicate the presence of a sensing system able to not only perceive variations in magnetic field intensity, but also to respond to changes in the magnetic field &#8220;direction&#8221; compared to gravity. The hypothesis that GMF reversal may affect plant growth finds compelling evidence in our experiments, which demonstrate that GMF reversal conditions can significantly affect plant development.
The morphological changes were also accompanied by changes in gene expression. Among housekeeping genes, the most stable gene was the elongationfactor1B alpha-subunit 2. The first group of genes (CRU3, COTP1, RRTF1) showed a dramatic alteration in the gene expression (Figure 3). Shoot expression of all three genes was significantly increased (P &#60;0.05) by about 2.5-fold in plants exposed to reserved GMF conditions. Root expression of CRU3 was upregulated in the roots in plants exposed to normal GMF conditions, but was significantly (P &#60;0.05) downregulated in reversed GMF conditions. The opposite was found for COTP1 and RRTF1, which were downregulated in normal conditions and upregulated in the presence of GMF reversal (Figure 3).
Cruciferin (a 12 S globulin) is the most abundant storage protein in the seeds of A. thaliana and other crucifers and is synthesized as a precursor in the rough endoplasmic reticulum. It is then transported to the protein storage vacuoles 13. Seedling germination requires the breakdown of cruciferin, which is used as an initial source of nitrogen. Down-regulation of cruciferin degradation reduces embryos development by impairing cell structures or cell components development 29,30. Our results show that upregulation of CRU3 correlates with a lower leaf expansion and a reduced root length, thus indicating that this gene in sensitive to GMF reversal and that its overexpression may contribute to the reduction of plant development. Moreover, GMF reversal induces a significant downregulation of CRU3 in roots, which correlates with a reduced root length. Copper is an essential cofactor for key processes in plants, but it exerts harmful effects when in excess; thus, overexpressing copper transport compromises plant growth. The effect of GMF reversal was a significant overexpression of COTP1 in both shoots and roots, thus explaining the reduced plant growth. Ion stress impairs chloroplast metabolism, which is tightly linked to the redox state of the cell. In Arabidopsis the transcription factor RRTF1 is important for the expression of genes associated to the ability to adjust to redox changes 31. Therefore, when plants are exposed to external stimuli able to alter their physiological and developmental programs an overexpression of this important transcription factor is expected. Reversal of the GMF induced a significant overexpression of RRTF1 in both shoots and roots, thus indicating higher oxidative stress responses of plants to reversed GMF conditions.
Interesting results are obtained by analyzing the five genes involved in oxidative stress. In general, all genes extracted and analyzed in shoots did not show significant differences (P &#62;0.05) when plants were grown in normal or reversed GMF conditions (Figure 4 and Figure 5). However, a significant down-regulation was always observed in roots of plants exposed to reversed GMF conditions. In particular, CAT3 showed the highest downregulation (Figure 5), followed in order of downregulation by APX1, FSD1, RBOHD and TAPX (Figure 4).
Cross tolerance to abiotic and biotic stress is provided by the activation of different genes involved in several biochemical pathways. RRTF1 transcription factor facilitates the synergistic co-activation of gene expression of these pathways 15,31, and can be potentially involved in oxidative stress 32. Therefore, upregulation of RRTF1 is expected when oxygen scavenging is reduced. Downregulation of root scavenging enzymes correlates with the upregulation of RRTF1, which acts in response to increased oxidative stress. The dramatic root downregulation of CAT3, APX1 and TAPX indicates the reduced ability of root cells to scavenge H2O2, which is accompanied by the reduced ability to dismutate the superoxide anion by downregulation of FSD1. The oxidative stress responses is higher in roots, which appear to be the main site of reversed GMF perception.
<img alt="Figure 1" src="/files/ftp_upload/53286/53286fig1.jpg" /> 
Figure 1.&#160;Geomagnetic field Compensation system. (A) triaxial coils (comprising a pair of octagonal coils for each of three perpendicular axes) used to reverse the geomagnetic field vector. (B) A computer-controlled power supply is connected to each pair of Helmholtz coils. (Voltages in these figures are arbitrary) <a href="https://www.jove.com/files/ftp_upload/53286/53286fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53286/53286fig2.jpg" /> 
Figure 2.&#160;Effects of geomagnetic field reversal on Arabidopsis morphology. After ten days of exposure, control plants (i.e., those exposed to normal GMF conditions) show a significantly greater root length and more expanded leaflets compared to plants that were exposed to reversed GMF conditions. Metric bar = 18 mm. <a href="https://www.jove.com/files/ftp_upload/53286/53286fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53286/53286fig3.jpg" /> 
Figure 3.&#160;Effects of geomagnetic field reversal on Arabidopsis gene expression. After ten days of exposure, total RNA of control and treated plants was extracted and analysed by Real-Time PCR for expression analysis. The effect of reversal of the GMF was to induce a drastic change in the gene expression of all genes that were tested. CRU3, Cruciferin 3; COTP1, Copper Transport Protein1; RRTF1, Redox Responsive Transcription Factor1. Bars indicate standard error; asterisks indicates significant (P &#60;0.05) differences between plants exposed to reversed and normal GMF conditions. <a href="https://www.jove.com/files/ftp_upload/53286/53286fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53286/53286fig4.jpg" /> 
Figure 4.&#160;Effects of geomagnetic field reversal on Arabidopsis antioxidant-related gene expression. After ten days of exposure, total RNA of control and treated plants is isolated and employed for gene expression analysis using Real-Time PCR. The effect of reversal of the GMF was to induce no significant changes in shoot gene expression; however, a drastic downregulation was observed in root gene expression of plants grown under reversed GMF conditions. TAPX, Thylakoidal Ascorbate Peroxidase; APX1, Ascorbate Peroxidase1; FSD1, Fe Superoxide Dismutase1; RbohD, NADPH/Respiratory burst oxidase protein D. Bars indicate standard error; asterisks indicates significant (P &#60;0.05) differences between plants exposed to reversed and normal GMF conditions. <a href="https://www.jove.com/files/ftp_upload/53286/53286fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53286/53286fig5.jpg" /> 
Figure 5. Effects of the geomagnetic field reversal on Arabidopsis Catalase 3 (CAT3) gene expression. After ten days of exposure, total RNA of control and treated plants is isolated and employed for gene expression analysis using Real-Time PCR. The effect of reversal of the GMF was to induce no significant changes in shoot gene expression; however, a drastic downregulation was observed in root gene expression of plants grown under reversed GMF conditions. Bars indicate standard error; asterisks indicates significant (P &#60;0.05) differences between plants exposed to reversed and normal GMF conditions. <a href="https://www.jove.com/files/ftp_upload/53286/53286fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Geomagnetic Field Gmf and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression at JoVE.com

Geomagnetic Field Gmf and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

This protocol describes a method to test the hypothesis that the reversal of the effective geomagnetic field (GMF) induces differential gene expression and alters the morphology of Arabidopsis thaliana. A triaxial octagonal system of Helmholtz coil-pairs was used to artificially generate reversed GMF conditions in the plant growth chamber.

Geomagnetic Field (Gmf) and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

One of the most stimulating observations in plant evolution is a correlation between the occurrence of geomagnetic field (GMF) reversals (or excursions) ...

geomagnetic-field-gmf-plant-evolution-investigating-effects-gmf

Research

JoVE Journal

Developmental Biology

13.1K Views.  University of Turin. This protocol describes a method to test the hypothesis that the reversal of the effective geomagnetic field (GMF) induces differential gene expression and alters the morphology of Arabidopsis thaliana. A triaxial octagonal system of Helmholtz coil-pairs was used to artificially generate reversed GMF conditions in the plant growth chamber.

Video: Geomagnetic Field Gmf and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

We present transient transgenesis and gene knockdown in Ciona intestinalis, a chordate sister group to vertebrates, using microinjection and electroporation techniques. Such methods facilitate functional genomics in this simple invertebrate that features rudimentary characteristics of vertebrates, including notochord and head sensory epithelia, and many orthologs of human disease associated genes.

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and subcellular processes, high resolution time-lapse microscopy strategies that preserve physiological conditions are required. Plant tissues must have adequate nutrient and water supply with sustained gaseous exchange but, when submerged and immobilized under a coverslip, they are particularly susceptible to anoxia. One strategy that has been successfully employed is the use of a perfusion system to maintain a constant supply of oxygen and nutrients. However, such arrangements can be complicated, cumbersome, and require specialized equipment. Presented here is an alternative strategy for a simple imaging system using perfluorodecalin as an immersion medium. This system is easy to set up, requires minimal equipment, and is easily mounted on a microscope stage, allowing several imaging chambers to be set up and imaged in parallel. In this system, lateral root growth rates are indistinguishable from growth rates under standard conditions on agar plates for the first two days, and lateral root growth continues at reduced rates for at least another day. Plant tissues are supplied with nutrients via an agar slab that can be used also to administer a range of pharmacological compounds. The system was established to monitor lateral root development but is readily adaptable to image other plant organs such as hypocotyls and primary roots.

Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and ...

A Method for Characterizing Embryogenesis in Arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, early stage plant embryos are small and contain few cells, making them difficult to observe and analyze. A method is described here for characterizing pattern formation in plant embryos under a microscope using the model organism Arabidopsis. Following the clearance of fresh ovules using Hoyer's solution, the cell number in and morphology of embryos could be observed, and their developmental stage could be determined by differential interference contrast microscopy using a 100X oil immersion lens. In addition, the expression of specific marker proteins tagged with Green Fluorescent Protein (GFP) was monitored to annotate cell identity specification during embryo patterning by confocal laser scanning microscopy. Thus, this method can be used to observe pattern formation in wild-type plant embryos at the cellular and molecular levels, and to characterize the role of specific genes in embryo patterning by comparing pattern formation in embryos from wild-type plants and embryo-lethal mutants. Therefore, the method can be used to characterize embryogenesis in Arabidopsis.

A Method for Characterizing Embryogenesis in Arabidopsis

This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday products. They are classified as endocrine disrupting compounds (EDC) with estradiol-like properties. Long-term exposure to EDCs, even at low doses, has been linked with various health defects including cancer, behavioral disorders, and infertility, with greater vulnerability during early developmental periods. To study the effects of BPA on the development of neuronal function, we used an in vitro neuronal network derived from the early chick embryonic brain as a model. We found that exposure to BPA affected the development of network activity, specifically spiking activity and synchronization. A change in network activity is the crucial link between the molecular target of a drug or compound and its effect on behavioral outcome. Multi-electrode arrays are increasingly becoming useful tools to study the effects of drugs on network activity in vitro. There are several systems available in the market and, although there are variations in the number of electrodes, the type and quality of the electrode array and the analysis software, the basic underlying principles, and the data obtained is the same across the different systems. Although currently limited to analysis of two-dimensional in vitro cultures, these MEA systems are being improved to enable in vivo network activity in brain slices. Here, we provide a detailed protocol for embryonic exposure and recording neuronal network activity and synchrony, along with representative results.

Exposure to environmental toxins can acutely impact developing embryos. Endocrine disrupting chemicals such as bisphenols are known to adversely affect the nervous system. Here we describe a protocol using an in vitro vertebrate (chick embryo) neuronal network model to study the functional impact of toxin exposure on early embryos.

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...