Annals of Botany 100: 791–796, 2007
doi:10.1093/aob/mcm164, available online at www.aob.oxfordjournals.org
Influence of a Weak DC Electric Field on Root Meristem Architecture
WO JC IE C H WAW R E CK I* and BE ATA Z AG ÓR S KA - MA RE K
Institute of Plant Biology, University of Wrocław, Kanonia 6/8, 50-328 Wrocław, Poland
Received: 30 March 2007 Returned for revision: 23 April 2007 Accepted: 12 June 2007 Published electronically: 8 August 2007
† Background and Aims Electric fields are an important environmental factor that can influence the development of
plants organs. Such a field can either inhibit or stimulate root growth, and may also affect the direction of growth.
Many developmental processes directly or indirectly depend upon the activity of the root apical meristem (RAM).
The aim of this work was to examine the effects of a weak electric field on the organization of the RAM.
† Methods Roots of Zea mays seedlings, grown in liquid medium, were exposed to DC electric fields of different
strengths from 0.5 to 1.5 V cm21, with a frequency of 50 Hz, for 3 h. The roots were sampled for anatomical
observation immediately after the treatment, and after 24 and 48 h of further undisturbed growth.
† Key Results DC fields of 1 and 1.5 V cm21 resulted in noticeable changes in the cellular pattern of the RAM. The
electric field activated the quiescent centre (QC): the cells of the QC penetrated the root cap junction, disturbing the
organization of the closed meristem and changing it temporarily into the open type.
† Conclusions Even a weak electric field disturbs the pattern of cell divisions in plant root meristem. This in
turn changes the global organization of the RAM. A field of slightly higher strength also damages root cap initials,
terminating their division.
Key words: Electric field, root apical meristem, quiescent centre, polar auxin transport, Zea mays.
IN TROD UCT IO N
Electromagnetic fields and living organisms
Low frequency electromagnetic fields (EMF) are a ubiquitous factor in the Earth’s environment. They originate
from various sources: geomagnetic fields, electric potential
in the atmosphere and cosmic radiation (Feynman et al.,
2001). All living organisms, including plants, have been
exposed to the Earth’s natural EMF from the beginning
of life. They have adapted to it, over 2 – 3 billion years of
their evolution. Numerous studies show an association
of the internal EMF of plants with many physiological
processes. For example, the electric current is involved in
graviperception (Collings et al., 1992) and root growth
(Souda et al., 1990). Plants use their internal EMF for
determining and controlling their physiological polarity
(Jaffe and Nuccitelli, 1977) and, specifically, for setting
their biological rhythms (Carre, 1996). There are also
many reports describing biological effects of animal and
plant exposure to the external EMF (Blank, 1995;
Rajendra et al., 2004). In plants it usually causes multiple
effects, observed on different levels of plant organization:
changes in growth direction (Wang et al., 1989; Stenz
and Weisenseel 1991; Malhó et al., 1992) and rates of
cell division (Goldsworthy and Rathore, 1985; Rech
et al., 1987), enhancement or inhibition of flowering
(Filek et al., 2003, 2006) and stimulation of embryogenesis
(Dijak et al., 1986).
It was Elfving who, as early as 1882 (cf. Schrank. 1959),
carried out the first experiments with roots growing in an
electric field. He observed that the roots of Lepidium,
Sinapis and Raphanus curved toward the anode, but the
roots of Brassica curved towards the cathode. The electric
* For correspondence. E-mail [email protected]
current used in his experiments was so strong that it
usually caused injury to the roots; thus these reactions
were aberrant rather than typical. More recently it has
again been demonstrated that the roots of Zea mays and
Lepidium sativum respond to an electric field and grow
toward the cathode when the electric current is 1 V cm21
(Stenz and Weisenseel, 1991), and toward the anode
when the field is 8 V cm21 (Stenz and Weisenseel, 1993).
These authors suggested that the latter effect, i.e. growth
toward the anode, might result from damage caused by a
field higher than 1 V cm21. They concluded that the
typical response of uninjured roots was growth toward the
cathode. The more precise observations of root electrotropic
response showed that root curvature occurred in two different regions: in the central elongation zone (CEZ) and in the
distal elongation zone (DEZ) (Wolverton et al., 2000).
Since then, it is known that the electrotropic reaction
occurs in the elongation zone. However, the question
remains of what happens in the most sensitive, meristematic
region of the root, when the field is applied.
One answer to this question was given recently in a paper
by Bitonti et al. (2006). They observed a reduction in size
of the quiescent centre (QC) and some disorder in root
cap (RC) cells of the roots of Z. mays exposed to a continuous EMF for 30 h.
Root structure and its physiological basis
One of the most important and dynamic regions of the
plant root is the root apical meristem (RAM), which consists of undifferentiated, rapidly dividing cells. The results
of their activity are the tissues and organs of the postembryonic plant. The meristem also plays a role as the
organizing centre for plant morphogenesis (Kerk and
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792
Wawrecki and Zagórska-Marek — Effects of an Electric Field on Root Architecture
Feldman, 1995). Proximal to the root meristem is the RC,
which protects the underlying RAM when the root traverses
through the soil. In plant species such as maize, a discrete
boundary exists between the RC and the RAM. This boundary is called the root cap junction (RCJ). Roots of this type,
with a so-called closed meristem organization, are contrasted with roots having an open meristem organization,
where there is no sharp boundary between the RC and the
root proper (Clowes, 1981). The type of root architecture
is specific for a given plant, suggesting a decisive role for
the genetic blueprint in setting up processes resulting in
an appropriate RAM cellular phenotype. In the closed
type of RAM, the cells that form the RC and root body
are typically produced by different tiers of cell initials; in
the open type they orginate from a common group of initials. The RCJ arises as the consequence of a high rate of cell
division in the cap initial cells (resulting in a columellar
structure within the cap) and a low rate in the adjacent
tier of cells in the QC. The QC, first discovered by
Clowes (1956) with use of radiolabelled DNA autoradiography, is a distinct central region of the RAM, which consists of slowly dividing cells (Jiang and Feldman, 2005).
On the proximal edge of the QC, a group of mitotically
active cells is located. These are cell initials, which form
the proximal meristem (Clowes, 1956). They give rise to
all cells and tissues of the primary root body (Feldman
and Torrey, 1976). On the distal edge of the QC, the
active cells are root cap initials (RCIs) (Baum and Rost,
1996; Barlow et al., 2001). There are two types of RCI:
cap columella initials, situated at the proximal end of
central cell files forming colummella; and protoderm initials encircling the columella initials (Barlow, 2003).
The status of being an initial is not permanent for a particular cell. This cell’s fate and identity may change over
time, especially when the QC increases or decreases in
size. Thus the cells should be regarded as initials on the
basis of their position relative to the QC (Feldman and
Torrey, 1976).
M AT E R I A L S A N D M E T H O D S
Plant material and medium
All experiments were carried out with 2-d-old seedlings of
Z. mays ‘Limko’. Maize caryopses were soaked in running
tap water for 5 h. Next they were placed in Petri dishes and
stacked between layers of wet paper. The Petri dishes were
placed vertically in a dark chamber at 29 8C and high
humidity. After 48 h, seedlings with straight main roots
about 10– 15 mm long were used for the experiment.
Application of DC electric fields
The seedlings were placed vertically in a plastic chamber
and their roots were submerged in 1 mM MES ( pH 6.0)
(Fig. 1). The seedlings were held in this position attached
to a plastic plate located in the centre of the chamber
(Fig. 1D). The medium was aerated and stirred with an
aquarium pump. Seedlings were allowed to adapt to these
new conditions for at least 1 h before application of the
F I G . 1. Schematic view of DC electric field application to plant roots
growing gravitropically downward in a chamber filled with liquid
medium (grey). A, aerating tube; B, anode; C, cathode; D, plastic plate
with seedlings attached.
electric current. The electric field was generated by two
platinum electrodes placed in the chamber on its opposite
sides (Fig. 1A). The electrodes (Fig. 1B, C) were connected
to a constant current source. The strengths of the field
applied in variants of the experiment were different, but
the frequency of 50 Hz and the timing of root exposure
were always the same. The exposure time (3 h) was
already known from earlier experiments by Stenz and
Weissensel (1993) to be sufficient to cause visible root
bending. The control seedlings were grown in the same conditions as the seedlings exposed to the EMF, but without
voltage. The applied voltage was calculated according
to the equation: J ¼ I/A ¼ sE, where J is the current
density, I is the current, A is the cross-section of the
container, s is the conductivity of the medium and E is
the electric field strength.
Histological preparation
The roots were sampled for anatomical investigation
immediately after the treatment with the electric field and
after 24 or 48 h of subsequent growth. In one experiment,
the roots before sampling were allowed to grow for
5 d. Roots were cut at 3– 5 mm above the tip and fixed in
FAA (formalin – alcohol – acetic acid) or 4 % paraformaldehyde (PFA) at room temperature for 24 h. They were
then dehydrated in a graded series of ethanol, and
embedded in paraplast or paraffin. Root tips were sectioned
at 4 and 7 mm with a Leica RM 2135 rotary microtome. The
sections were stained with fuchsin [in a periodic acid –
Schiff (PAS) reaction], as in Figs 3A, B and 4D, or with
Safranin/Fast green (Figs 3 C, D and 4A – C) and calcofluor
(Fig. 3E). Images of the sections were taken and archived
with a light microscope (Olympus SZX9) and a fluorescent
microscope (Olympus BX50) connected to a Silicon
Graphics INDY Workstation.
R E S U LT S
In all experiments where the plant roots were exposed to the
EMFs, the protocol of Stenz and Weissensel (1993) was
Wawrecki and Zagórska-Marek — Effects of an Electric Field on Root Architecture
followed, with some minor modifications. MES-buffered
medium, without salt, was used. It was aerated and stirred
by air from the aquarium pump. Only a weak electric
field was used, which did not cause injury to the roots.
Only those roots which either did not show any sign of
damage (as a change in colour of the root tip) or were
able to grow continuously after disconnection of the field
were analysed.
Exposure of the root to a DC electric field of 0.5 V cm21
In the first series of experiments the roots exposed to the
electric field were growing, gravitropically downward.
When the field was disconnected, the roots were allowed
to grow further and they continued evidently undisturbed
growth downward. Anatomical observations of the roots
sampled immediately after the electric field had been shut
off and after 24 h from that moment showed that there
was no variation in the RAM cellular structure in comparison with the control (Fig. 3A, B).
Exposure of the root to a DC electric field of 1 V cm21
These experiments showed that almost all of the roots
bent toward the cathode, clearly demonstrating an electrotropic reaction (Fig. 2A). At the end of the action of the
EMF, the average curvature of the root tips was approximately 308. After disconnecting the field, the roots continued their growth in the direction attained for 24 h. Then
they started growing gravitropically, i.e. downward
(Fig. 2B). The architecture of the RAM showed some
changes 24 h after disconnecting the field. This resulted
from periclinal divisions of the cells forming the first tier
of the root body. A new layer of cells appeared between
the tip of the procambial cylinder and the RCJ. At 48 h
after disconnecting the field, the expansion of the root
proper cells began toward the RC area (Fig. 3E). During
that process, the boundary between these two regions maintained its integrity in .90 % of the roots. It bent, however,
resulting in formation of a very characteristic mammillary
apex (Fig. 3C, D). In the remaining 10 % of the roots, the
integrity of the boundary was already destroyed, by a few
cells of the root proper protruding actively and deeply
into the RC. Cells of the cortex – epidermis complex,
793
formerly belonging to the root proper, slowly converted
into the RCIs and a new epidermis became constituted laterally from the outer layer of the former cortex. This process
started slowly defining a new RCJ boundary, now located
deeper within the former root body. By 48 h, the previous
RCJ was faint or almost completely disappeared as a
result of periclinal divisions of epidermal derivatives. The
root meristem organization became at this stage clearly
open (Fig. 4A). In order to see the long-term effects of
RAM reconstruction, some roots were allowed to grow for
5 d after the treatment. It was observed that in these roots
a new cap had been produced and the cells of the old disintegrating cap were already dead and highly compressed
(Fig. 4B, D). Between the RC and root proper a new RCJ
was arising, with the meristem becoming closed again.
Neither of the above effects was observed in control roots.
Exposure of the root to a DC electric field of 1.5 V cm21
When this field was applied to the roots, they also
reoriented their growth toward the cathode; however,
some of them stopped growing after the treatment.
Changes in RAM architecture were similar to those in the
former experiment. The cells of the root body invaded the
RC territory. However, a new anomaly was noted in a
region of the RC: some columellar initials failed to divide
(Fig. 4C). The starch granules appeared in their cytoplasm,
which is normally observed only in the derivatives of the
initials. Again, all these changes were absent in roots
from a control experiment.
DISCUSSION
In the normal gravitropic reaction, a plant root typically
changes the direction of its growth but the cellular pattern
of the RAM remains undisturbed. When the roots of
maize in the present experiments had been stimulated by
an EMF of even low strength, but sufficient to induce
bending, the treatment profoundly affected the pattern of
cell division, changing the global organization of the RAM.
This was a somewhat unexpected result as the roots at first
sight did not differ from those which ahd gravitropically
stimulated and, most importantly, were still capable of
growth. Reconstruction of the cellular pattern of the RAM
F I G . 2. Response of different maize roots to application of a DC electric field of 1 V cm21 over 3 h. The root on the left was photographed 3 h after
beginning the field application (A) and that on the right 24 h after disconnecting the field (B). The root in B has just started growing downward
again. The extent of root growth deviation from the vertical course, caused by the field, is indicated by the angle drawn on the photograph on the left.
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Wawrecki and Zagórska-Marek — Effects of an Electric Field on Root Architecture
F I G . 3. The response of maize root apices subjected to various treatments, as seen on longitudinal central sections. The control root, growing without
application of the electric field (A), shows a closed meristem organization, with a clearly demarcated root cap junction separating the root cap and root
body. A similar pattern is present in the root exposed to an electric field of 0.5 V cm21 (B). Mammillary root apex developing 24 h after disconnecting the
field of 1 V cm21 (C and D). Formation of a new cap in the root treated with the same field strength as in C, but sampled 48 h after disconnecting the field
(E). Scale bar ¼ 100 mm (A–D), 200 mm (E).
most probably was a result of a reaction or even damage in
the most sensitive RCIs, as indicated by the lower rate or
the entire cessation of their division. When the field action
is limited in time, the changes are reversible – the RC
cells became replaced by others, produced by numerous
cell divisions in the activated QC. The new distinct regions
of root cellular pattern, such as a new RC and new tiers of
initials, emerged from deeper, more proximally located
strata of cells. The sequence of changes observed in these
experiments resembled reconstruction of the root cellular
pattern after surgical removal of the RC (Barlow, 1974).
It is well known that RCIs are extremely sensitive to
environmental factors. Increasing sensitivity of the cells,
the derivatives of which are constantly discarded, is sensible from the point of view of adaptive strategies. On the
other hand, the agents usually affecting rapidly dividing
cells, e.g. X-irradiation, triiodobenzoic acid (TIBA) and
low temperature, accelerate cell divisions in the QC, resulting in perturbation of the organization of the RAM
(Clowes, 1963; Clowes and Stewart, 1967; Kerk and
Feldman, 1994).
The sequence of these events and their possible causal
relationship with auxin transport is not entirely clear. Kerk
and Feldman (1994) suggested that all structural changes
result from damage of cap initials, which secondarily disrupts
the polar auxin transport. Such damage in the present experiments is suggested by the fact that the initials are pushed out
and replaced by new cells adopting their function, located
deeper in the region of the root proper.
Morris (1980) hypothesized, in turn, that a small DC
electric current might directly inhibit polar auxin transport.
When the current is switched off, freshly supplied indole
acetic acid (IAA) should be again transported normally.
This means that functions of auxin polar transport are not
permanently inhibited. According to this view, perturbation
of auxin movement alters positional signalling, i.e. changes
not only the condition of the QC but also the signal, which
flows from the QC and controls the activity of RCIs. This
Wawrecki and Zagórska-Marek — Effects of an Electric Field on Root Architecture
795
F I G . 4. Longitudinal sections of the root apices exposed to a DC electric field of 1 V cm21 (A, B, D) and 1.5 V cm21 (C). At 48 h after disconnecting the
field, the new root cap develops, which is manifested by clear bending of the RCJ clonal border (arrows) (A). Five days after disconnecting the field,
the complex of two root caps is still present. The new root cap formation is well advanced and the old cap is pushed aside (B). At 24 h after disconnecting
the field, the cells stop dividing in the collumellar region and become axially elongated. The displacement of the amyloplast toward the anode is clearly
visible in these cells. (C). On the edge of the newly formed root cap, the cells of the old root cap are crushed and become compressed (D). Scale bar ¼
40 mm (A, D), 100 mm (C) and 200 mm (D).
causes aberration in root meristem organization (van der
Berg et al., 1997). This hypothesis explains the cessation
of cell division and, especially, accumulation of starch
granules in the RCIs observed in the present experiments.
These cells would not be expected to differentiate properly,
as they do develop the clear phenotype of a statocyte, if
lethally damaged by the field.
Thus, despite a growing body of experimental data, the
first target of the action of an EMF in the RAM still
remains unknown and the question of whether the
changes in cell divisions are due to the damage to sensitive
RCIs or to the polar auxin transport being altered by the
field still awaits an answer.
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