The weightless statoliths in the rhizoids of the green alga Chara move towards the cell base (opposite to the originally acting gravity vector) during TEXUS rocket parabolic flights. After destruction of the cytoskeletal actin filaments by specific drugs, however, the statoliths are not basipetally transported under microgravity. The displacement of statoliths under µg is greater in the axial direction than laterally. Thus it has been proved for the first time that, under 1 g, the statoliths exert tensional forces on actin filaments and that a balance of forces (of gravity and the counteracting force mediated by actin filaments) is responsible for the correct positioning of the statoliths in the rhizoid, guaranteeing the ability to respond to the gravity vector.
The ability of plants to respond to gravity is important for their orientation in space. It influences growth and development: shoots must grow towards the light for photosynthesis and roots must dig into the soil to find water and minerals. Gravity- sensing cells such as the rhizoids of the green alga Chara are highly suitable model systems for the study of graviperception and graviresponse.1, 2
Rhizoids are tubular single cells that attach the alga to the ground. Their polar construction comprises an apex of dense and relatively stationary cytoplasm, including the nucleus, and a base where cytoplasmic streaming occurs. Growing only at the tip, they react gravitropically. When the rhizoids are tilted from the vertical, they change their growth direction according to the gravity vector (Fig. 1a). Under normal vertical orientation, vesicles containing crystals of BaSO4 are localised 10-30 µm above the outermost apical cell wall (Fig. 1b). These vesicles act as statoliths; they sediment on the physically lower cell flank when the rhizoid is tilted from the vertical (Fig. 1c). Bending of the rhizoid is performed by asymmetrical growth of the opposite cell flanks. Without statoliths, fewer statoliths or with statoliths in an unappropriate position, no graviresponse or a reduced response occurs.3, 4, 5
Fig. 1a. Positive gravitropic curvature of Chara rhizoids
tilted several times by 90°. 1b. Rhizoid in normal vertical
orientation with the basal vacuolar part (V) and the apical part
with the nucleus (N) and the statoliths (S). 1c. After tilting
the rhizoid to horizontal position, the statoliths sediment on
the lower cell flank (photographs taken after 3-15 min). The
beginning of curvature can already be seen. The diameter of a
rhizoid is about 25 µm.
A fundamental question in understanding the gravitropic behaviour and the mechanism of graviperception thus concerns the positioning of the statoliths and the celullar structures involved. Experiments with drugs such as cytochalasin6 and the demonstration of actin filaments with rhodamine phalloidin 7 have shown that the statoliths are tethered in a network of actin filaments that prevents them from sedimentation on the apical cell wall in the vertical orientation.3, 8 It has therefore been postulated that a balance of forces is reponsible for the correct positioning of the statoliths within the cell: the gravititational force (Fg) and the counteracting force exerted by actin filaments (Fmf). On Earth, this postulate cannot be proved as the gravitational force always interacts with the endogenous force mediated by actin filaments. Studies under 'reduced' gravity are therefore of primary importance in clarifying these questions and in supporting and proving the postulate. The approximately 6 min of µg provided by the TEXUS flights has for the first time allowed the axial and lateral movements of statoliths in Chara rhizoids to be observed in vivo by videomicroscopy. The position of statoliths at the beginning and end of the µg period has also been determined after the chemical fixation of rhizoid bundles. In addition, the behaviour of statoliths has been analysed after application of cytochalasin D. These experiments were aimed at gaining new insights into the gravi-reaction chain and at verifying and expanding the conclusions drawn from ground experiments. The streaming velocity of the cytoplasm in the rhizoid base towards the tip is greater than towards the base.9 The difference is caused by an endogenous physiological component and a gravitational effect. Discrimination between these two causes was studied under µg conditions for the first time during the TEXUS flights.
Segments of Chara globularis Thuill. were cultivated for invivo videomicroscopy in flat cuvettes in 1.2% agar in distilled water at room temperature and under incandescent light for 4-6 days.10, 11 The cuvettes (constructed by the mechanical workshop of the Botanisches Institut, Universität Bonn, and MBB/ERNO, Bremen) consisted of a frame of V2A steel covered by a Plexiglas slab on either side and sealed against vacuum. Aluminium sheets with observation windows protected the cuvette against pressure disturbances. The rhizoids were observed on the stage of a horizontal microscope in the flight module (MBB/ERNO, module TEM 06-16) and video-recorded with a CCD camera. In order to observe the lateral movement of statoliths, rhizoids growing perpendicularly to the cuvette's long axis under 1 µg were gravistimulated 45 min before launch by tilting to the horizontal position. The statoliths sedimented on the lower cell flank, and under µg their lateral displacement could be studied. In order to test whether forces are indeed exerted on the statoliths by interaction with actin filaments, the actin-specific drug cytochalasin D (CD, 5.5 µg/ml) was applied 30 min before launch and the statoliths' behaviour observed under µg.12 For statistical analyses, control rhizoids as well as rhizoids treated with CD were fixated with glutaraldehyde and studied after recovery of the flight module.12 Streaming velocities were measured by streak-photography using video-records.9, 1
Photographs of the rhizoid's apex recorded on the ground and during the µg period in the vertical orientation are presented in Fig. 2. The typical polar organisation of the cell has been maintained under the short µg period.11 During µg, the statolith complex moved basipetally with an average spped of 2.4 µm/min and became spindle-like. The distance of the centre of the statolith complex from the apical cell wall almost doubled after 6 min of µg. The nucleus, however, remained in its positon. These phenomena were statistically confirmed by different TEXUS flights.
Fig. 2. Axial (basipetal) displacement of statoliths in a
Chara rhizoid during 6 min of µg. As control, the position
of the statolith complex (S) under 1 g (-187 s before the µg
phase) is shown; pictures taken after 122 s in µg and at the
end of the µg phase (after 394 s) demonstrate the basipetal
displacement of statoliths.
After treatment with CD, the statoliths sedimented under 1 g and they did not move basipetally under µg.12 This result was found to be significant, as shown in bundles of rhizoids fixated with glutaraldehyde. Partial inactivation of the function of the actin filaments by CD prevents basipetal transport of the statoliths under µg. It can therefore be concluded that, on the ground, the actin filaments in cooperation with myosin 13 indeed pull on statoliths and vice versa, and counteract the gravity force.
The lateral displacement of statoliths under µg is not as strong as the axial displacement (Fig. 3). The statolith complex moved laterally only for some µm during the µg period, but simultaneously further in the basipetal direction. Thus it follows that the organisation and function of the actin filaments are different as regards their efficiencies in the axial and lateral directions. For the first time, such a specification of cytoskeletal elements has been demonstrated in the polarly structured rhizoid. Experiments using optical tweezers to dislocate the statoliths in Chara rhizoids have recently confirmed that forces of different strength are necessary to move the statoliths axially and laterally.5
Fig. 3. Lateral and simultaneously basipetal displacement of
statoliths in a Chara rhizoid under µg. The statolith
complex has sedimented on the lower apical cell flank under 1 g;
after 19 s, 129 s and 369 s in µg (pictures from top to
bottom), the lateral displacement of statoliths is small compared
to the basipetal displacement.
Based on the short µg observations, a model has been developed incorporating the forces acting on statoliths under 1 g and µg.14, 11 The results from the TEXUS experiments have provided the basic and fundamental data for this model on the cellular mechanisms involved in graviperception and graviresponse in Chara rhizoids.
The individual data from ground and µg experiments verify the model's validity. The statoliths' movement and the kinetic of this process indicate that shearing forces exist between statoliths and actin filaments counteracting the gravity force.11 Moreover, these findings also shed light on the mechanism of the first steps of graviperception in higher plants.15, 14, 16, 11
Simultaneous records of both directions of cytoplasmic streaming during the µg period have been used to measure the streaming velocities. Under 1 g, the difference between the up and down velocities was about 10%. Under µg, however, this difference diminished to 5.3%. The basipetal streaming velocity increased compared to the acropetal streaming velocity. Thus, it has been demonstrated under µg that gravity affects the streaming velocity of the cytoplasm in rhizoids. The ground-based experiments have been verified; this is a clear case of a gravity effect in cells without the use of specialised gravity-sensors such as statoliths.10
Financial support by the AGRAVIS project (DARA, Bonn, and Ministerium für Wissenschaft und Forschung, Düsseldorf) is greatly acknowledged. MBB/ERNO, Bremen, and the teams at the authors' institute deserve special thanks for their skillful technical support and excellent cooperation, as do the staff at ESRANGE, Sweden.
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