Group Title: BioMagnetic Research and Technology 2006, 4:7
Title: High magnetic field induced changes of gene expression in arabidopsis
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Title: High magnetic field induced changes of gene expression in arabidopsis
Series Title: BioMagnetic Research and Technology 2006, 4:7
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Creator: Paul AL
Ferl RJ
Meisel MW
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High magnetic field induced changes of gene expression in
arabidopsis
Anna-Lisa Paul', Robert J Ferl1 and Mark W Meisel*2


Address: 'Department of Horticultural Sciences and The Biotechnology Program, University of Florida, Gainesville, FL 32611-0690, USA and
2Department of Physics and National High Magnetic Field Laboratory, University of Florida, Gainesville, FL 32611-8440, USA
Email: Anna-Lisa Paul alp@ufl.edu; Robert J Ferl robferl@ufl.edu; Mark W Meisel* meisel@phys.ufl.edu
* Corresponding author



Published: 22 December 2006 Received: 04 November 2006
BioMagnetic Research and Technology 2006, 4:7 doi:10.1 186/1477-044X-4-7 Accepted: 22 December 2006
This article is available from: http://www.biomagres.com/content/4/l/7
2006 Paul et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: High magnetic fields are becoming increasingly prevalent components of non-
invasive, biomedical imaging tools (such as MRI), thus, an understanding of the molecular impacts
associated with these field strengths in biological systems is of central importance. The biological
impact of magnetic field strengths up to 30 Tesla were investigated in this study through the use of
transgenic Arabidopsis plants engineered with a stress response gene consisting of the alcohol
dehydrogenase (Adh) gene promoter driving the P-glucuronidase (GUS) gene reporter.
Methods: Magnetic field induced Adh/GUS activity was evaluated with histochemical staining to
assess tissue specific expression and distribution, and with quantitative, spectrofluometric assays to
measure degree of activation. The evaluation of global changes in the Arabidopsis genome in
response to exposure to high magnetic fields was facilitated with Affymetrix Gene Chip
microarrays. Quantitative analyses of gene expression were performed with quantitative real-time
polymerase-chain-reaction (qRT-PCR).
Results: Field strengths in excess of about 15 Tesla induce expression of the Adh/GUS transgene
in the roots and leaves. From the microarray analyses that surveyed 8000 genes, I 14 genes were
differentially expressed to a degree greater than 2.5 fold over the control. These results were
quantitatively corroborated by qRT-PCR examination of 4 of the I 14 genes.
Conclusion: The data suggest that magnetic fields in excess of 15 Tesla have far-reaching effect
on the genome. The wide-spread induction of stress-related genes and transcription factors, and a
depression of genes associated with cell wall metabolism, are prominent examples. The roles of
magnetic field orientation of macromolecules and magnetophoretic effects are discussed as
possible factors that contribute to the mounting of this response.


Background
The possibility that strong, static (non-gradient) magnetic
fields might have an influence on biological processes has
been discussed for many years [1-6], including reports
that implicate high magnetic fields in alterations of the


cleavage plane during cell division [7-9] and other cellular
disorders [10]. Nevertheless, the common viewpoint is
that presently achievable static magnetic fields do not
have a lasting effect on biological systems [3-5]. Indeed,
magnetic resonance imaging (MRI), utilizing static mag-


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netic fields up to 12 Tesla, is a powerful tool for non-inva-
sive in vivo imaging at the molecular level [11,12]. The
demands for more precise in vivo imaging have driven the
field strengths progressively higher, approaching 20 Tesla
[13], yet information regarding the biological impact of
exposing metabolically active cells to fields of this magni-
tude is limited. Herein, we report the effect of high mag-
netic fields on the gene expression profile of the plant
Arabidopsis (Arabidopsis thaliana).

This research was initially driven by an interest in using
magnetic levitation as a ground-based model for the
effects of a reduced gravity environment on plant gene
expression. The utility of using magnetic levitation to
mimic a reduced gravity environment has been explored
in a variety of systems [14-18]. Our research efforts to take
advantage of this venue were initiated with the use of
transgenic Arabidopsis that had been engineered with a
gene reporter shown to be induced by the spaceflight envi-
ronment and named TAGES Transgenic Arabidopsis
Gene Expression System [19]. The TAGES Arabidopsis
plants are engineered with the GUS (P-glucuronidase)
reporter gene driven by the alcohol dehydrogenase (Adh)
gene promoter, which responds to a variety of environ-
mental stresses [20] that initiate transcription of the GUS
reporter gene. The GUS expression can be monitored
qualitatively, by histochemical staining, and quantita-
tively, by biochemical assays of the gene product. The
magnetically levitated plants showed evidence of reporter
gene activation, however, the control plants placed in a
static magnetic field of 19 Tesla showed similar patterns of
transgene expression [21].

These observations lead to the design of additional exper-
iments using transgenic plants as biomonitors of the
effects of high magnetic fields in metabolically active tis-
sues. The evaluation of global changes in the Arabidopsis
genome in response to exposure to high magnetic fields
was facilitated with Affymetrix Gene Chip microarrays.
These arrays allow for the survey of over 8000 genes at a
time and were used for genome-wide characterization of
the effects of exposing Arabidopsis plants to a field of 21
Tesla. The resulting differential patterns of gene expres-
sion from the array data were then used to guide quantita-
tive analyses of gene expression with quantitative real-
time polymerase-chain-reaction (qRT-PCR), which is an
effective means of characterizing an abiotic stress response
[22]. The microarray data indicate that, of the 8000 genes
surveyed, there were 114 genes that were differentially
expressed to a degree greater than 2.5 fold over the con-
trol. These results were quantitatively corroborated by
qRT-PCR examination of 4 of the 114 genes.

The data suggest that magnetic fields in excess of 15 Tesla
have far-reaching effect on the genome. The wide-spread


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induction of stress-related genes and transcription factors,
and a depression of genes associated with cell wall metab-
olism, are prominent examples. The roles of magnetic
field orientation of macromolecules and magneto-
phoretic effects are discussed as possible factors that con-
tribute to the mounting of this response.

Materials and methods
Magnetic fields and local environment control
Four experimental runs were made at the National High
Magnetic Field Laboratory (NHMFL) in Tallahassee using
the magnets housed in Cell 5 (Runs 1, 4 and 5) and in Cell
6 (Run 2). The general physical dimensions of these mag-
nets and typical field profiles are available online [23].
The temperature of incoming/outgoing cooling water for
the magnets was monitored and was regulated to provide
an average temperature of 13 ( 3) C. At a constant mag-
netic field, typically five plants, each individually grown
in plastic tubes, were held in a 50 ml plastic beaker that
was attached to a copper tubing coil assembly. After expo-
sure for either 2.5 or 6.5 hours, two plants were stained
and the other three plants were used for the quantitative,
biochemical assays. Perforations in the bottom of the
beaker allowed for a significant flux of air within the bore
of the magnet at all field strengths. Thermally regulated
water was circulated in the copper tubing, and the temper-
ature near the bottom of the plastic beaker and near the
leaves was monitored with Cu-CuNi thermocouples
whose magneto-response had been calibrated. The ther-
mocouples were monitored every 15 minutes during the
course of a run, and the plants were maintained near 15
( 3)C after cooling from the ambient temperature,
nominally 20 C. The leaves of the plants were spritzed
with water before being inserted into the bore and the
ambient humidity was sufficient to allow water to con-
dense on the copper tubing. The plants were inspected
and photographed after being removed from the magnet,
and in no case was there any evidence of wilting effects
caused by the flux of air through the bore of the magnet.
The plants were inserted so that the center field was at the
shoot/root boundary. Given the finite size of the plants
and their location in the magnet, the largest field gradients
that the plants experienced for the NHMFL experiments
were approximately (0.5 Bo) cm-1, where B0 is the mag-
netic field (in Tesla) at the center of the magnet. The ambi-
ent light down the bore of the magnet was sufficient to
allow the plants to be seen without any difficulty and no
additional lighting sources were used. A similar arrange-
ment was used for an experiment (Run 3) performed with
the superconducting solenoid located at the University of
Florida. However, since the large bore (88 mm diameter)
was accessible at room temperature, the cooling water
assembly was not required, and the specimens always
experienced ambient temperature. For this magnet, the
largest field gradients that the plants experienced were


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approximately (10-4 B0) cm-1. Experimental Runs 1 4
focused on the Adh/GUS studies, and Run 5 was per-
formed to allow the microarray and qRT-PCR analyses.

Plant treatments
A previously developed line of transgenic Arabidopsis
(Arabidopsis thaliana) plants containing the alcohol dehy-
drogenase (Adh) gene promoter linked to the P-glucuoni-
dase (GUS) gene reporter were used throughout. The
histochemical analyses were conducted with 21 day-old
plants grown on the slanted surface of 9 mm diameter
tubes containing a nutrient agar medium. Plants intended
for gene expression analyses were grown vertically on
nutrient agar plates for 9 days before introduction to the
magnetic field [19,22]. After exposure to the magnetic
field, plants to be subjected to subsequent gene expression
or biochemical analyses were harvested to liquid nitrogen,
and plants intended for evaluations of tissue-specific
transgene expression were fixed in histochemical stain.
Control plants were treated similarly; the 21 day-old con-
trol plants for the histochemical and biochemical analyses
were simply maintained on the lab bench for the duration
of the magnetic field exposure. The 9 day-old plate plants
being evaluated for gene expression changes were main-
tained in the bore of the magnet (with zero applied field)
for an equivalent duration of time.

Histochemical staining and biochemical assay ofAdhlGUS
expression
The histochemical stain is composed of the GUS substrate
X-Gluc (2 mM 5-bromo-4-chloro-3-indolylglucuronide),
1% [w/v] dimethylformamide, 0.1 mM K3 [Fe(CNJ)], 0.1
mM K4 [Fe(CN)] 3H20, 1 mM EDTA and 50 mM
NaPO4, pH 7.0. Plants were harvested to stain immedi-
ately following treatment and incubated at room temper-
ature 48 hours. The reaction was stopped and chlorophyll
cleared from plants with several washes of 70% ethanol.
Plants were then photographed to record tissue-specific
deposition of the Adh/GUS transgene product. The sam-
ples frozen for biochemical analysis were homogenized in
extraction buffer (50 mM NaPO4, pH 7.0, 10 mM EDTA,
0.1% sarkosyl, 0.1% [v/v] Triton X-100, and 10 mM P-
mercaptoethanol) and diluted for incubation with a fluor-
ometric substrate. The GUS activity in the lysate was meas-
ured quantitatively on a fluorometer (Shimadzu, Kyoto)
as nanomoles of substrate (4 methylumbelliferyl 3-D-glu-
curonide [4-MUG]) reacted with the GUS enzyme per
microgram total protein per minute [24].

Microarray sample preparation
Five 9 day-old plants (grown as indicated above) were
selected at random from a pool of about 70 plants for
each treatment. The selected plants were placed in the
bore of the magnet, and a field of 21 Tesla was applied for
2.5 hours. The controls were conducted in the bore of the


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magnet in the absence of an applied field. The plants in
the bore were controlled for temperature by circulating
chilled water through copper coils within the plant con-
tainer. The actual temperature was monitored via thermo-
couples and recorded manually (16 18 C). Plants were
harvested to liquid nitrogen immediately upon removal
from the bore and stored at -800C until RNA isolation
procedure. Three of the five plants exposed to 21 Tesla for
6.5 hours (and the comparable control) were combined,
and total RNA was isolated from the pooled samples for
microarray analyses. The RNA was extracted using RNAe-
asyT kits from Qiagen [22]. Purified RNAs were labeled
and prepared for hybridization according to the protocols
outlined in the GeneChip' Expression Analysis Technical
Manual (Revision 1, 2001, Affymetrix, Santa Clara, Ca).

Quantitative qRT-PCR
The two remaining plants from the five exposed to 21
Tesla for 6.5 hours were separated into root and shoot
fractions and the tissue types pooled before RNA extrac-
tion (as previously described). In addition, five plants
exposed to 14 Tesla for 2.5 hours and 21 Tesla for 6.5
hours were similarly treated, as were the remaining con-
trols. Genes to be targeted for quantification were identi-
fied from the microarray results. The quantification was
conducted with Taqman Real Time Reverse Transcriptase
- Polymerase Chain Reaction (qRT-PCR) from Applied
Biosystems (ABI) [25] and evaluated with the ABI Prism'
7700 Sequence Detection Systems. Forward and reverse
primers flanking a ca. 100 bp region of the gene of interest
were obtained from ABI along with the fluorescently
labeled probe sequence that hybridizes between the
primer pairs.

Results
Histochemical and biochemical indications
In a series of experiments, 21 day-old plants were exposed
for durations of 2.5 hours per run in the 50 mm diameter
bore resistive magnets at the National High Magnetic
Field Laboratory (NHMFL) in Tallahassee, and in the 88
mm diameter bore superconducting solenoid located at
the University of Florida. As described in the Materials
and Methods section, the local environmental variables of
temperature, light intensity, atmosphere, and humidity
were controlled, and did not impact transgene expression.
Figure 1 provides qualitative examples of the stress
response mounted by the plants exposed to high magnetic
fields. The histochemical staining shown in Figure 1 illus-
trates that GUS is being expressed in both leaf and root tis-
sues in these plants. The baseline corrected, quantitative
results are provided in Figure 2. Each data point in Figure
2 represents the average of the results from three plants,
and the standard deviation is given by the uncertainty lim-
its. Nonparametric statistical methods have been used to
test the hypothesis of an association between the GUS


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activity and the magnetic field. For the leaves (Figure 2a),
the Spearman correlation coefficient of 0.58 (P = 0.001)
indicates that there exists a significant association
between these two variables [26,27]. For the roots (Figure
2b), the Spearman correlation coefficient of 0.40 (P
0.033) suggests a somewhat weaker association [26,27].

In a second step, a statistical model was formulated,
hypothesizing a log-normal relationship (y = A exp [-
ln2(x/xj)/2w21) for the effect of the magnetic field (x) on
the level of GUS activity (y). A log-normal functional form
might be anticipated as the presence of the magnetic field,
above a threshold value, initiates a stress response, while
subsequently stronger magnetic fields further perturb all
gene activation, causing the GUS activity to decline. The
data for the leaves (Figure 2a) were analyzed using nonlin-
ear weighted least squares [28,29], with the weights com-
puted as the reciprocals of the variance estimates, yielding
parameter estimates ( standard errors) ofA = 11 2, xc =
19.9 0.4, and w = 0.12 0.02. This result is plotted as the
black line in Figure 2a, with the 95% confidence limits
represented by the grey lines. Standard techniques (i.e.
residual analysis, evaluation of correlation matrix, R2
measure) were used for checking the goodness of fit of the
model. Finally, it is important to note that neither simple-
linear nor quadratic dependence are represented in the
data. These two functional forms might be anticipated if
the stress response was linked to some other magnetic
field factor, e.g. a small amount of sinusoidal field ripple,
rather than just the strength of the static field.


E ,fA






b13


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Gene expression analyses
Three week-old plants were exposed to field strengths of
14 Tesla for 2.5 hours and of 21 Tesla for durations of 2.5
and 6.5 hours. The control experiments were conducted in
the bore of the magnet in the absence of an applied field.
Figure 3 shows scatterplots comparing the gene expression
patterns of plants exposed to 21 Tesla for 6.5 hours and
control plants maintained in ambient field strength
within the bore of the magnet for the equivalent amount
of time. The levels of differential expression for the 8000
genes represented on the microarray are indicated by dots
in Figure 3a, and regions of high density become saturated
with data points. Figure 3b, a topographical (or "relief")
version of Figure 3a, provides a sense of the density of the
data points [30] and indicates that most of the 8000 rep-
resentative genes show less than a 2-fold difference in
expression between treatment and control. The yellow
spots in Figure 3a represent genes that are typically unaf-
fected by abiotic stress (i.e. "housekeeping" genes), and as
expected, these housekeeping genes show little evidence
of differential expression. There are, however, 114 genes
that were differentially expressed to a degree greater than
2.5 fold over the control. In this group, many genes asso-
ciated with a variety of stress responses were induced
(heat, cold, drought, touch), as were genes encoding pro-
teins that are involved with ion transport functions (chlo-
ride, sulfate, ammonium). The down regulated set
included a number of genes involved in cell wall biosyn-
thesis (e.g. Xtr7). A final large category is populated by
genes that encode transcription factors (e.g. Athbl2). A


Figure I
Qualitative examples of GUS expression. Histochemical staining of the controls indicates that Adh/GUS was not expressed in
these plants (a b). An increase in magnetic field strength induces expression of the Adh/GUS transgene (e.g. 20 Tesla for 2.5
hours, c d). The increased magnification of the plants shown in b and d (second row) provide closer inspection of GUS local-
ization in the roots and leaves of these samples. The middle panel (e) provides a top-view of the five 21 day-old plants just
prior to insertion into the bore of the magnet. The right hand panel (f) shows the plants from the side.



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1) a Leaves (2.5 hr exposure)
20 Run 1 NHMFLCell 5
.C Run 2- NHMFL Cell 6
E A Run 3 UF Supercon.
U Run 4- NHMFL Cell 5
C
a 10 Log-normal Function
-- 95% limits
O
I



(D


Sb Roots (2.5 hr exposure)
-5 40 v- Run1-NHMFLCell5
E Run 2 NHMFLCell 6
C A Run 3 UF Supercon.
U Run 4- NHMFL Cell 5
.-20 -

0 -



4D _:: ..1I ..I :.I_


0 5 10 15 20 25

Magnetic Field (Tesla)

Figure 2
Quantitative GUS activity as a function of applied magnetic field. The amount of GUS activity, as measured by biochemical/
fluorometric procedures, is plotted as a function of applied magnetic field. Each data point represents the average of three
plants and the uncertainty limits are given by the standard deviation. Three data sets were collected at the NHMFL and one
was obtained in a UF superconducting solenoid capable of reaching 9 Tesla. Nonparametric statistical methods have been used
to test the hypothesis about an association between the GUS activity and the magnetic field. For the leaves (a), the Spearman
correlation coefficient of 0.58 (P = 0.001) indicates that there exists a significant association between these two variables. For
the roots (b), the Spearman correlation coefficient of 0.40 (P = 0.033) suggests a somewhat weaker association. A statistical
model was formulated, hypothesizing a log-normal relationship for the effect of the magnetic field on the level of GUS activity
(see text). The data for the leaves (a) were analyzed using nonlinear weighted least squares, and the result is plotted as the
black line, with the 95% confidence limits given by the grey lines.



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listing of all 114 genes is given in the supplementary
material [See Additional file 1].

Quantitative qRT-PCR was used to corroborate the expres-
sion patterns of a select number of genes indicated by the
arrays, and these genes are designated by the boxes in the
scatterplot of Figure 3. Figure 4 shows the results the
results of utilizing quantitative RT-PCR (ABI) to deter-
mine the differential expression levels of expression for
four genes: Athb 12 (AF001949, a homeobox transcription
factor), Xero2 (U19536, a dehydrin), Xtr7 (U43489,
xyloglucan endotransglycoslase) and Cor78 (L22567,
cold regulated 78). Values within each treatment are
depicted as fold induction or repression relative to the
control. The controls represent plants held within the
bore of the magnet without the application of an exoge-
nous magnetic field. All conditions for the controls and
the subsequent runs were identical, with the exception of
field strength, and the exposure time was 2.5 hours. The
genes Athb 12, Xero2 and Cor78 display an increase in the
level of expression when in a strong magnetic field,
whereas expression of Xtr7 decreases with increasing field.
The patterns of gene expression determined with quanti-
tative qRT-PCR, Figure 4, reflect the trends in expression
indicated by the array data, Figure 3.

Discussion
The results indicate that high magnetic fields have far-
reaching effects on the genome. The biological impact of
high magnetic fields on Arabidopsis, as quantified by
microarray and qRT-PCR analyses, is stronger than was
reported for microarry data obtained for TCA cycle-related
genes of budding yeast (Saccharomyces cerevisiae) exposed
to fields up to 14 Tesla [31]. Although a detailed under-
standing of the results will require additional studies, per-
haps involving isolated in vitro processes [32], hints of the
underlying mechanism generating the effects may be
gleaned from the strength of the response as a function of
applied magnetic field. For example, the data in Figures 2
and 4 suggest that while a minimum threshold field can
initiate a stress response that is manifested as either an
induction or a repression of select genes, higher fields may
compromise some aspects of the transcriptional machin-
ery, and effectively arrest the process. This field depend-
ence may suggest that magnetic orientation or
magnetophoresis plays a role in the seemingly dual nature
of the response.

An order of magnitude comparison between the strength
of magnetic orientation and magnetophoresis can be
made for the experimental conditions of our experiments.
It is important to stress that although these two effects are
present in our experiments, the current results do not
identify them as the source of the effects on gene expres-
sion. However, these types of effects have been detected in


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experimental configurations evaluating the magnetic ori-
entation or magnetophoresis on molecules, so they are
relevant for the present discussion. Specifically, the
biomacromolecules involved in signal transduction and
gene regulation may experience forces and/or torques that
are induced by the presence of the magnetic field. For
example, one such torque arises from the anisotropy of
the diamagnetic susceptibility [33,34] of the molecule
and attempts to magnetically orient the macromolecule.
This effect has been known for some time, but it has only
recently been exploited in NMR structural determinations
of large molecules as the measurements evolved from 14
Tesla to 17.5 Tesla [35,36]. In addition, the macromole-
cules may experience magnetophoresis due to forces gen-
erated by inhomogeneities in the applied magnetic field.


More specifically, in a magnetic field B (f ), where f is
the vector identifying the spatial coordinates, the mag-
netic energy of an object possessing a magnetic suscepti-
bility tensor may be written as [36,37]


E= B(f) -B(f), (1)

where [t is the permittivity of the material and a reasona-
ble approximation is that [t = .0, the permittivity of free
space. Variations of this energy arise from anisotropies of
the susceptibility and of the magnetic field, such that the
dominant effects are given by


6E = (B(F) 6 B(f)+ 2B(T) AZ 6B(f)}.
2o0


The first term on the right hand side of Equation 2 is the
energy due to anisotropy of the magnetic susceptibility
and causes macromolecules to orient to minimize this
energy. The second term on the right hand side of Equa-
tion 2 is the energy due to inhomogeneities in the mag-
netic field and differences of the susceptibilities of the
molecules and their surroundings. In order to provide an
order of magnitude comparison of the sizes of the two
effects, the ratio, R, of these two terms may be written as


R 2B A- 6B 2AX6B
-B 5xB (3)
The inhomogenetiy of the magnetic field is largest at the
top of the leaves and the bottom of the roots where (SB/
B) = 5 x 10-3. On the other hand, a reasonable bound for
the anisotropy of the susceptibility for biomacromole-
cules is 10-1< (S6/AX) < 1 [1,15,36-38]. Consequently, 10-
2 < R < 10-1, and the effect of magnetic orientation domi-
nates the magnetophoretic effects in our experiments.
Although the sum of the variations of the magnetic energy


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10 100


1000 10000


Control


Control


Figure 3
Scatterplots of the Affymetrix ATH I Arabidopsis Array for magnetic field data. The 9 day-old plants were exposed to a field of
21 Tesla for 6.5 hours and compared to control plants that had been maintained in the bore of the magnet for the same
amount of time without an applied field. In (a), each data point represents the level of differential expression for each of the
8000 genes represented on the microarray, and the axes are logarithmic. Genes of interest that were chosen for further quan-
tification are indicated with boxes, see text. The large yellow spots represent "housekeeping" genes that are typically unaf-
fected by abiotic stress. The two parallel lines represent the 2-fold increase or decrease limits. The data in (a) replotted in (b)
using a topographical routine [30], where the color gradient designates the number of genes appearing in a given localized
region of the graph.


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10000



1000


100


10


1


-14

-12

-10

-8


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BioMagnetic Research and Technology 2006, 4:7


T


0
C()
(C)
a)

X
LU
a)
a)

-0
a)
N


L0
O
Z


Controls


14 Tesla


21 Tesla


-20 I I
Figure 4
Quantitative qRT-PCR analyses of selected genes and treatments. Four genes were chosen for additional quantification:
Athb 12, Xero2, Xtr7, and Cor78. The normalized gene expression levels represent fold-induction or fold-repression relative
to the controls. The exposure to the magnetic fields was for 2.5 hrs.


is less than 100 ppm of the ambient thermal energy (=
kBT, where kB is the Boltzmann constant and T is the tem-
perature = 290 K), the magnetic orientation is readily
observable [1,36-38], and magnetophoretic effects, aris-
ing from BSB ranges similar to the ones present in our
work, have been reported [39].


Conclusion
To summarize, although each of these magnetic-field-
induced effects is quite small compared to the randomiz-
ing ambient thermal energy of the plant, we conjecture
that they are sufficient to perturb the delicate conforma-
tional dynamics involved in aspects of gene regulation,


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60


Athbl2
Xero2
Xtr7
Cor78


50


40


30


20


10


0


-10


I I


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BioMagnetic Research and Technology 2006, 4:7



thereby resulting in the differential expression of a variety
of genes in the plant. Compared to each other, the mag-
netic orientation effects are estimated to be 10 to 100
times larger than the magnetophoretic forces for our
experiments, thus it is likely that magnetophoresis plays a
minor role in the induction or repression of gene expres-
sion. Our results indicate that nominally 15 Tesla is the
threshold of field strength required to initiate the stress
response. It is interesting to note that reports of magnetic
orientation effects during NMR studies of macromole-
cules intensified when the fields were increased from 14
Tesla to 17.5 Tesla [35,36]. These observations are conso-
nant with the idea that macromolecular orientation plays
a role in stress-gene activation in field strengths greater
than 15 Tesla. On a macroscopic scale, static magnetic
fields of 10 Tesla are sufficient to align the cleavage planes
in developing frog eggs [ 7-9 ]. However, on the basis of our
results, we cannot determine if the observed effects are
related to assemblies of molecules or simple conforma-
tional flexing/bending of single molecules. There are
other processes (e.g. chemical reactions [40]) that might
be perturbed by the presence of the magnetic field, and
these perturbations may impact local chemistry of any of
several signaling pathways that are connected to the dif-
ferential expression of any number of genes.

The induction of the Adh/GUS transgene was the first indi-
cation that gene expression processes may be sensitive to
high magnetic fields. The GUS gene induction is a highly
sensitive histochemical reaction that can indicate even
subtle tissue-specific changes in gene expression. Subse-
quent experiments surveyed genome-wide changes in
gene expression in response to exposing Arabidopsis
plants to 21 Tesla. Of the 8000 genes surveyed, there were
114 genes that were differentially expressed to a degree
greater than 2.5 fold over the control.

In conclusion, exposure to magnetic fields above nomi-
nally 15 Tesla is accompanied by differential gene expres-
sion responses in Arabidopsis plants. These data provide
evidence for the perturbation of metabolic processes in
the presence of strong magnetic fields and may be useful
for guiding future research designed to calibrate safe expo-
sure standards for living organisms [5].

Abbreviations
Adh: alcohol dehydrogenase

Athb12: AF001949, a homeobox transcription factor

Cor78: L22567, cold regulated 78

EDTA: ethylenediaminetetraacetic acid

GUS: P-glucuronidase


http://www.biomagres.com/content/4/1/7


NHMFL: National High Magnetic Field Laboratory

NMR: nuclear magnetic resonance

MRI: magnetic resonance imaging.

TAGES: Transgenic Arabidopsis Gene Expression System

TCA: tricarboxylic acid

qRT-PCR: quantitative real-time polymerase-chain-reac-
tion

Xero2: U19536, a dehydrin

Xtr7: U43489, xyloglucan endotransglycoslase

Competing interests
The authors) declare that they have no competing inter-
ests.

Authors' contributions
ALP and RJF designed and executed the work directly
related to the plants and the associated analyses. MWM
designed the magnetic studies and generated the analysis
related to the comparison of the competing magnetic
interactions. ALP and MWM drafted the manuscript, and
all authors read and approved the final version.

Additional material


Additional file 1
Supplementary Material. Excel Worksheet, and two plots clustered by
function, of the 114 genes that were Im . i ,, expressed, between
treatment in 21 Tesla and zero-field bore controls, to a degree greater than
2.5 fold. Plants were held at 21 Tesla or at zero-field, in the bore of the
magnet, for 6.5 hours. (Paul-Ferl-Meisel-Supplementary-Material.xls)
Click here for file
[http://www.biomedcentral.com/content/supplementary/1477-
044X-4-7-S1.xls]



Acknowledgements
This work was supported, in part, by the National Science Foundation and
the State of Florida through support and operation of the National High
Magnetic Field Laboratory (NHMFL) and by the NHMFL In-House
Research Program. We gratefully acknowledge the early contributions
made byJ. S. Brooks, B. Klingenberg, A. N. Morgan, and J. Yowtak. We
thank J. Ch. Davis for his assistance in preparing Figure 3. We have bene-
fited from conversations with S. J. Hagen, M. Iwasaka and T. H. Mareci.

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