(The FASEB Journal. 1999;13:S143-S147.)
© 1999 FASEB
Statoliths motions in gravity-perceiving plant cells: does actomyosin counteract gravity?
DIETER VOLKMANN*1,
FRANTISEK BALUSKA*
,
IRENE LICHTSCHEIDL§,
DOMINIQUE DRISS-ECOLE
and
GÉRALD PERBAL
* Botanisches Institut, Rheinische Friedrich-Wilhelms-Universität Bonn, Germany;
Laboratoire CEMV, Université Pierre et Marie Curie, Paris, France;
Institute of Botany, Slovak Academy of Sciences, Bratislava, Slovakia; and
§ Institut für Pflanzenphysiologie der Universität Wien, Austria
1Correspondence: Botanisches Institut der Universität, Venusbergweg 22, D-53115 Bonn, Germany. E-mail: unb110{at}uni-bonn.de
 |
ABSTRACT
|
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Statocytes from plant root caps are characterized by a polar arrangement
of cell organelles and sedimented statoliths. Cortical microtubules and
actin microfilaments contribute to development and maintenance of this
polarity, whereas the lack of endoplasmic microtubules and prominent
bundles of actin microfilaments probably facilitates sedimentation of
statoliths. High-resolution video microscopy shows permanent motion of
statoliths even when sedimented. After immunofluorescence microscopy
using antibodies against actin and myosin II the most prominent
labeling was observed at and around sedimented statoliths. Experiments
under microgravity have demonstrated that the positioning of statoliths
depends on the external gravitational force and on internal forces,
probably exerted by the actomyosin complex, and that transformation of
the gravistimulus evidently occurs in close vicinity to the statoliths.
These results suggest that graviperception occurs dynamically within
the cytoplasm via small-distance sedimentation rather than statically
at the lowermost site of sedimentation. It is hypothesized that root
cap cells are comparing randomized motions with oriented motions of
statoliths and thereby perceiving gravity.—Volkmann, D.,
Baluska, F., Lichtscheidl, I., Driss-Ecole, D., Perbal, G.
Statoliths motions in gravity-perceiving plant cells: does actomyosin
counteract gravity?
Key Words: statocytes • microgravity
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INTRODUCTION
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GRAVITY-ORIENTED GROWTH of plant organs such as roots
are under the control of at least two signal transduction chains
(1
, 2
). The first of these is located at the
site of stimulus transformation, i.e., in statocytes of the root cap
(3)
; the second is at the site of differential growth
(4-8)
i.e., in cells of the transition zone
(9)
, where a major switch occurs concerning organization
and distribution of cytoskeletal elements (9
,
10
).
In spite of recent controversial discussions of starchless mutants in
relation to the first step of stimulus transformation in root cap
cells, it is mainly accepted that sedimentable statoliths,
starch-filled plastids (amyloplasts), are involved in an intracellular
perception mechanism (11)
. Several sophisticated
experiments on clinostats (12)
and under microgravity
(13)
ruled out endoplasmic reticulum (ER) membranes and
the plasma membrane as cell structures directly involved in the process
of stimulus transformation via membrane contact sites and/or exerted
pressure by statoliths. These results, however, did not exclude these
membranes as mediators of following steps during signal transduction
and transmission. On the other hand, cytoskeletal elements, especially
those composed of the actomyosin complexes, came into debate as the
structures mediating graviperception (14-18)
. One of the
main findings of these investigations was that the position of
statoliths in root cap cells is balanced by two forces, the external
force of gravity and the internal tension force exerted by the network
of cytoskeletal proteins (15
, 16
). As such,
plant statocytes represent a mechanosensing system comparable to the
systems discussed for morphogenetic processes by Ingber's group under
the term tensegrity (19
, 20
).
This review will focus mainly on microgravity experiments (for recent
literature see ref. 21
) supporting the idea that stimulus
transformation occurs via cytoskeletal proteins measuring randomized
versus oriented motions of statoliths.
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STATOCYTE POLARITY AND STATOLITHS BEHAVIOR UNDER 1-gCONDITIONS
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Random motions of amyloplasts versus oriented motions of statoliths
Root caps are characterized by at least three basic cell types
performing different functions: meristematic cells, statocytes that
perceive gravity, and secretory cells that facilitate penetration of
root apices through the soil. Thus, the structure and function of root
cap cells change at least twice, which occurs within a few hours. In
meristematic cells, amyloplasts containing a small amount of starch
mainly surround the nucleus, positioned in the center of the cell. The
main change from the meristematic cell to the statocyte stage is
indicated by cell elongation in the axial direction accompanied by the
development and establishment of a pronounced cell polarity where the
nucleus is always located at the proximal pole. On the other hand, ER
cisternae are mostly arranged at the distal pole, whereas sedimented
amyloplasts (density 
=1.44 g cm-3 versus
=1.03 g cm-3 of the cytoplasm) are located above
the ER and small vacuoles, dictyosomes, and mitochondria are located
mostly between the two poles (for review see ref. 22
). The
development of cell polarity is established by cytoskeletal elements,
especially cortical microtubules and actin microfilaments; this has
been demonstrated directly by microscopic approaches (18
,
23
) and indirectly after inhibitor experiments
(18
, 24
, 25
). Sedimentation of
statoliths is certainly facilitated by the specific cytoskeletal status
of the statocytes, in so far as endoplasmic microtubules and bundles of
actin microfilaments have not been observed in this type of cap cell
(18)
. However, this sedimentation is not just a purely
physical process finalized during cell development; it occurs
continuously, as shown by the fact that it can be observed in root cap
material from different plants through the use of high-resolution video
microscopy. Statoliths are continuously in motion, sometimes even
showing saltatory movements (26)
. This has also been
reported for barium sulfate-filled statoliths (density =4.4 g
cm-3) in the unicellular graviresponding rhizoids of the
green alga Chara (27)
. Due to their small size,
roots from Arabidopsis thaliana are extremely suitable for
observations in the living stage (Fig. 1
) and such observations are also possible with halved root caps
from other Brassicaceae-like Lepidium
sativum or Brassica napus. From those observations it
can be deduced that statolith motions are not randomized, as is typical
with the non-sedimentable amyloplasts surrounding the nucleus, but
occur preferentially correlated to the direction of the gravity vector.
Nevertheless, these tracks may point in any direction, some even
opposite to the gravity vector, comparable to saltatory movements
(26)
. Sometimes even motions of single starch grains are
visible within the statoliths (see ref. 28
). Thus, in
gravity-perceiving cells random motions of amyloplasts are changed into
directed motions that in summary result in their sedimentation. In
other words, oriented gravity-dependent motions overcome the noise of
constitutive random motions.
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Figure 1. Living root cap cells from intact root apices of Arabidopsis
thaliana visualized by high-resolution video microscopy
(micrographs taken from videotapes). Roots have been adapted for 10 min
in a horizontal position under the microscope. The cap cell closest to
the meristem (white star) does not possess starch-filled particles,
whereas the following cell (black star) is characterized by remarkable
large statoliths. Micrographs (A–C) have been selected
every 2 s. Already within 4 s statoliths have moved
remarkably, as indicated by motion tracks (arrows). Bar = 2
µm.
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Cytoskeletal elements as a basis for statoliths motions
Actomyosin-driven transport of cell structures, especially
chloroplasts, is well documented for algae such as Nitella or
Acetabularia (29
, 30
). The latter
species are, due to their large cell size, especially suitable for
injection of marker molecules. With higher plants, visualization of
plant cytoskeletal proteins, especially of actin and its associated
molecules, was hampered by the lack of adequate methods for a long
time. Affinity fluorescence microscopy using rhodamine-phalloidin was
the preferential approach (for recent literature see ref.
31
), although these investigations did not take into
consideration polymerizing effects (32)
of the fungal
toxin on G-actin, and as a consequence the shifting of the ratio
between G- and F-actin toward the filamentous stage. However, specific
antibodies against actin (10
, 33
) and some
associated proteins (34)
are now available and a powerful
immunohistochemical method has been established (10
,
35
) for localization of actin and any other plant protein
(e.g., see ref. 36
). In spite of intensive studies using
different immunocytological techniques, visualization of prominent
actin microfilaments in root statocytes is still lacking (for recent
literature see ref. 17
), whereas other cell types of the
root proper have been shown to be rich in filamentous actin, distinctly
organized in different cell types (10)
. On the other hand,
diffuse actin labeling, especially surrounding the statoliths, suggests
high amounts of short F-actin elements and of G-actin in root
statocytes. Concerning the motor molecule myosin, essential information
is extremely rare for higher plants. Using heterologous antibodies
against myosin II from chicken muscle, labeling is prominent only in
close vicinity to cress root statoliths (37)
. Similar
observations have been reported for statoliths of maize roots
(17)
and Chara rhizoids (38)
.
Thus, in root cap statocytes the actomyosin complex seems to be
restricted to the periphery of statoliths, enabling on the one hand
sedimentation and on the other hand short distance movements.
Experiments under microgravity have been performed by our groups
showing that the position of statoliths in root cap cells depends on
two forces, the external gravitational force and the internal force
exerted putatively by cytoskeletal proteins, both acting on the
statoliths (16
, 25
). In these experiments,
the possibility of switching from higher g levels to
microgravity was proved to be highly important. The importance of
microgravity as a reference parameter for research in the field of
gravitational biology was clearly demonstrated.
|
STATOCYTE POLARITY AND STATOLITHS BEHAVIOR UNDER MICROGRAVITY
CONDITIONS
|
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Actomyosin counteracts gravity
Experiments investigating cress and lentil roots
(39-42)
demonstrated that in principle the structural
polarity of statocytes is established and maintained under microgravity
conditions, i.e., nucleus and ER membranes are located at the proximal
and distal cell pole of statocytes, respectively. Calculations of the
position of the nucleus showed that its location changed slightly. This
indicates that even the position of the nucleus depends on the
gravitational force (25
, 43
). Astonishingly,
non-sedimented statoliths did not show random distribution, as might be
expected, but showed remarkable shifts in direction of the nucleus,
indicating active motion of the organelles (13
,
40
). In addition, it has been shown that statoliths in
microgravity were grouped near the cell center, whereas they appeared
more dispersed after treatment on the slowly rotating clinostat
(44)
. Direct evidence for actively driven movement of
statoliths has come from experiments on rockets when, after launch
accelerations of some g, gravitational conditions changed
immediately to microgravity. Under these experimental conditions,
statoliths moved in the opposite direction to the originally acting
gravity vector (Fig. 2
) within a few minutes (16)
. These results were also
observed during shuttle experiments (Fig. 3
) (43
, 45
, 46
).
Corresponding behavior of statoliths from the Chara rhizoid
was directly observed in orbit by telecommunication (16)
.
Under cytochalasin D action this motion did not occur and statoliths
remained in their launch position (47
, for review see
48
). Microgravity effects on actin isoforms have been
observed with plant protoplasts. In sounding rocket experiments two of
four actin isoforms nearly disappeared after 6 min of microgravity
(49)
.
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Figure 2. Schematic drawing of the statoliths position (envelope of the
statoliths complex) in root cap statocytes under different experimental
conditions. A) 24 h, 1 g; B)
A + a few seconds 9 g (launch conditions);
C) B + 6 min µg. Adapted from
Volkmann et al. (16)
.
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Figure 3. Schematic drawing of the statoliths position (envelope of the
statoliths complex) in root cap statocytes under different experimental
conditions. A) 28 h, 1 g; B)
28 h CD under 1-g conditions;
C) A + 30 min µg;
D) 28 h µg. Adapted from Driss-Ecole and
Perbal (46).
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Threshold values indicate stimulus transformation in close vicinity
of statoliths
Microgravity experiments offered, for the first time,
possibilities to study important parameters like threshold values under
controlled conditions of sensor physiology. For plant material
cultivated entirely under microgravity conditions, the minimum dose
under continuous stimulation (presentation time
tp; for details see ref. 21
) has
been estimated to be 20–30 g s for cress roots
(50)
, and by extrapolation from microgravity data to be 27
g s for lentil roots (45)
. In contrast, this
threshold value doubled (50–60 g s) when material from the
1-g centrifuge in orbit was used (21
,
50
). Because root elongation is similar both in
microgravity and on the 1-g centrifuge (51)
,
these results indicate that gravisensitivity of roots is larger when
the organs develop in microgravity than when they are growing under
1-g conditions. Correlations of this threshold value with
the position of statoliths show that statoliths moved very slightly in
the direction of the stimulating gravity vector, generally less than 1
µm. Thus one must conclude that stimulus transformation occurs in
close vicinity to the statoliths after short distance movements.
|
CONCLUSIONS
|
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From both ground-based studies and microgravity experiments,
it can be concluded that cytoskeletal proteins, especially actin and
its associated proteins, play an important role in (1)
development and maintenance of structural polarity in root cap
statocytes; (2)
the motion and sedimentation of
statoliths; and probably (3)
the transformation of the
gravity stimulus into the first biochemical signal. So far, prominent
actin microfilament bundles in statocytes have not been demonstrated
but microgravity experiments indicate that actomyosin-driven motion of
statoliths counteracts the sedimentation process caused by the
gravitational force. These results are supported by immunolabeling of
actin and myosin in the close neighborhood of statoliths. On the basis
of this behavior of statoliths, it can be hypothesized that
gravity-perceiving cells are transforming the gravity stimulus by
measuring the position of statoliths via tension within actomyosin
networks. Grouping effects of statoliths as observed under microgravity
might be important in this respect. This hypothesis will be
experimentally tested in the future by investigations on statoliths
motions in living material under microgravity and by parallel
visualization of components of the cytoskeleton.
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ACKNOWLEDGMENTS
|
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D.V. was supported by Deutsches Zentrum für Luft -und
Raumfahrt (Bonn) and Ministerium für Wissenschaft und
Forschung (Düsseldorf), and G. P. by Centre National
d'Études Spatiales (CNES, Paris).
|
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TOP
ABSTRACT
INTRODUCTION
