Fusicoccin affects cortical microtubule orientation in the isolated epidermis of sunflower hypocotyls.

Epidermal peels isolated from sunflower hypocotyls provide a convenient model to study the relationship between cortical microtubule orientation and strain rate. Extension of peels can be modulated using chemical treatment and mechanical stress, i.e., by adding a chemical to the incubation medium and applying a load exceeding the yield threshold for irreversible (plastic) strain. In this study, peels were pre-incubated for ca. 12 h (long-term pre-incubation) or for 1 h (short-term pre-incubation). In the long-term pre-incubated peels, fusicoccin applied to the medium neither enhanced the rate of longitudinal plastic strain of loaded peels, nor affected microtubule orientation. However, fusicoccin increased the strain rate of short-term, pre-incubated peels and affected microtubule orientation in both extending (loaded) and non-extending (unloaded) peels. Without fusicoccin, microtubule orientation was generally longitudinal or steep, whereas in fusicoccin-treated unloaded peels it was transverse and oblique microtubules in peel portions corresponding to the apical part of the hypocotyl. Although the frequency of transverse orientation was increased through loading, there was no strong correlation between the rate of fusicoccin-induced strain and microtubule orientation. It is hypothesized that the insensitivity of long-term pre-incubated peels to fusicoccin with respect to strain rate is due to a lack of active plasma membrane H(+) -ATPases. Thus, the sensitivity of short-term, pre-incubated, unloaded (non-extending) peels to fusicoccin, with respect to microtubule orientation, indicates that orientation might be affected by electric currents resulting from fusicoccin stimulation of H(+) -ATPases.

Strain rate does not affect cortical microtubule orientation in the isolated epidermis of sunflower hypocotyls.

A hypothesis exists that external and internal factors affect the orientation of cortical microtubules in as much as these lead to changes in cell elongation rate. Factors that stimulate elongation are proposed to lead to transverse microtubule orientation, whereas factors that inhibit elongation lead to longitudinal orientation. The elongation rate is equal to the rate of longitudinal irreversible strain in cell walls. Incubated epidermis peeled from sunflower hypocotyls does not extend unless it is stretched by loading and the pH of the incubation medium is appropriately low. Thus, peels provide a convenient model to investigate the relationship between longitudinal strain rate and cortical microtubule orientation. In the present study, it was found that peeling affects microtubule orientation. Peels were incubated for several hours in Murashige & Skoog medium (both unbuffered and buffered) to attain a steady state of microtubule orientation before loading. The effects of loading and pH on strain rate and orientation of microtubules under the outer epidermal walls were examined in three portions of peels positioned with respect to the cotyledonary node. Appropriate loading caused longitudinal strain of peels at pH 4.5 but not at pH 6.5. However, no clear effect of strain rate on microtubule orientation in the peels was observed. Independent of applied load and pH of the incubation medium, the microtubule orientation remained unchanged, i.e. orientation was mainly oblique. Our results show that strain rate does not affect cortical microtubule orientation in isolated epidermis of the sunflower hypocotyl model system, although orientation could be changed by white light.

Autonomous changes in the orientation of cortical microtubules underlying the helicoidal cell wall of the sunflower hypocotyl epidermis: spatial variation translated into temporal changes.

Angles (lambda) at which parallel cortical microtubules (cMTs) were oriented with respect to the longitudinal direction were measured in Helianthus annuus hypocotyl epidermal cells. Histograms showing lambda frequencies in cell populations at the instant of epidermis fixation were obtained. Analysis of the histograms indicates that, in a particular position within a cell, the angle lambda changes periodically with time, i.e., there is a cycle of lambda change at that position. This cycle is most likely rotational rather than oscillatory, i.e., the change in lambda has a defined chirality (clockwise or counterclockwise). The full diversity of histograms can be consistently explained by rotational cycles with a variable velocity of lambda change, and with a cMT rebuilding stage taking place at a different phase of the cycle. The rotational cycles also provide the simplest explanation of cMT arrays in which the angle lambda changes along a cell (fixed) and no parallel orientation of cMTs is apparent at a certain position. This explanation assumes a gradient in the phase of the rotational cycle along the cell. The symmetry of the angular characteristics of the rotational cycle, with respect to the morphological directions in cells, leads to the concept that these directions typically represent the principal directions of a certain tensor quantity, which may control the cycling. Possible interactions between the rotational cycle of cMT reorientation and the helicoidal cycle during cell wall formation are discussed.

Tensile Tissue Stress Affects the Orientation of Cortical Microtubules in the Epidermis of Sunflower Hypocotyl.

In turgid multicellular organs, it is convenient to differentiate between the two kinds of tensile forces acting in cell walls as a result of turgor pressure. The primary forces occur both in situ and in cells isolated from the organ, whereas the secondary forces occur only in situ. The latter are an unavoidable physical consequence of the variation in mechanical parameters of tissues forming layers or strands. The most rigid tissue is under maximal tensile force, whereas the least rigid is under maximal compressive force. These forces cause tissue stresses (that is, certain tissues are under tensile stress, whereas others are under compressive stress in the organ). The primary and secondary forces result in primary and secondary stress in cell walls, respectively. The anisotropy of the primary stress is a function of cell shape. For instance, in cylindric cells the anisotropy expressed as the ratio of longitudinal to transverse stresses is 0.5. The anisotropy of the secondary stress is a function of the compound structure of the organ. For example, in the epidermis of sunflower hypocotyl, the longitudinal secondary stress is much higher than the transverse stress. The primary and secondary stresses are superimposed, and, as a consequence, the stress anisotropy in the outer thick walls of epidermal cells is greater than 1. These outer epidermal walls transmit most of the tissue stress. When the epidermis is peeled but remains turgid, only primary stress remains, but loading of the peel can reestablish the original stress anisotropy. We studied the effect of stress anisotropy changes on the orientation of cortical microtubules (CMTs) in the sunflower hypocotyl epidermis. We showed that changes in stress anisotropy cause the CMT orientation to change in the direction of maximal wall stress. In situ, the relatively high tensile tissue stress in the epidermis causes maximal stress in the longitudinal direction and relatively steep CMT orientation. When the tissue stress is removed from the epidermis by peeling, the CMTs tend to reorient toward the transverse direction, which is the direction of maximal stress in the primary component. On application of external longitudinal stress, to substitute for tissue stress, CMTs tend to reorient in the longitudinal direction. However, a relatively high rate of plastic strain is caused by the stress applied to the peel in an acid medium. This produces a less steep orientation of CMTs. It appears that the change in stress anisotropy orients the CMT in the direction in which the stress is maximal after the change, but there is also some effect of the growth rate on the orientation.

The pattern of acropetal and basipetal cytoplasmic streaming velocities in Chara rhizoids and protonemata, and gravity effect on the pattern as measured by laser-Doppler-velocimetry.

The spatial pattern of acropetal and basipetal cytoplasmic streaming velocities has been studied by laser-Doppler-velocimetry (LDV) in the positively gravitropic (downward growing) rhizoids of Chara globularis Thuill. and for the first time in the negatively gravitropic (upward growing) protonemata. The LDV method proved to be precise and yielded reproducible results even when tiny differences in velocities were measured. In the apical parts of the streaming regions of both cell types, acropetal streaming was faster than basipetal streaming. Starting at the apical reversal point of streaming, the velocity increased basipetally with the distance from that point and became fairly constant close to the basal reversal point; subsequently, the velocity decreased slightly acropetally as the apical reversal point was again approached. There was no change in velocity at the basal reversal point. However, at the apical reversal point there was an abrupt decrease in velocity. The pattern of the ratio of acropetal to basipetal streaming velocity (VR) was a function of the relative distance of the site of measurement from the apical reversal point rather than a function of the absolute distance. Upon inversion of the rhizoids, the VR decreased on average by 3.8% (+/- 0.4%), indicating that the effect of gravity on the streaming velocity was merely physical and without a physiological amplification. Rhizoids that had developed on the slowly rotating horizontal axis of a clinostat, and had never experienced a constant gravity vector, were similar to normally grown rhizoids with respect to VR pattern. In protonemata, the VR pattern was not significantly different from that in rhizoids although the direction of growth was inverse. In rhizoids, oryzalin caused the polar organization of the cell to disappear and nullified the differences in streaming velocities, and cytochalasin D decreased the velocity of basipetal streaming slightly more than that of acropetal streaming. Cyclopiazonic acid, known as an inhibitor of the Ca2+-ATPase of the endoplasmic reticulum, also reduced the streaming velocities in rhizoids, but had slightly more effect on the acropetal stream. It is possible that the endogenous difference in streaming velocities in both rhizoids and protonemata is caused by differences in the cytoskeletal organization of the opposing streams and/or loading of inhibitors (like Ca2+) from the apical/subapical zone into the basipetally streaming endoplasm.

The consequences of a non-uniform tension across kinetochores: lessons from segregation of chromosomes in the permanent translocation heterozygote Oenothera.

The alternate (zigzag) configuration of the chromosome ring in oenotheras fulfills the requirement of high tension across kinetochores for stability of the configuration and the progression to anaphase. However, also semialternate configurations (two pairs of adjacent kinetochores interspaced among the zigzag) fulfill the requirement of high tension across kinetochores. If only the magnitude of tensile force acting on a kinetochore pair governs the stability of microtubule attachments, the probability of occurrence of the semialternate configurations would be higher than that of fully alternate configurations. Yet the percentage of irregularity in the zigzag configuration is surprisingly low, which means that the semialternate configurations are corrected. The only difference which distinguishes the fully alternate and the semialternate configurations with respect to the tension across kinetochores is that the tension across a kinetochore alternating with its neighbors is rather uniformly distributed over the kinetochore, while there is a gradient of the tension in the kinetochore having a non-alternating neighbor, with low tension on the side of this neighbor. Apparently, a low tension across a part of a kinetochore brings about correction of its attachment to microtubules. This hypothesis fits with the repeat subunit model of the kinetochore; apparently, each subunit can function autonomously in the tension-governed mechanisms, stabilizing its attachment and controlling the metaphase-to-anaphase transition.

Temporal course of graviperception in intermittently stimulated cress roots.

Gravitropic bending of Lepidium roots caused by intermittent stimulation lasting approximately 1 h was the same for a particular sum of stimulation intervals and was independent of (i) the length of a single stimulation interval (from 1 to 12.2 s) during which the roots were exposed unilaterally and horizontally, and (ii) rest intervals (from 60 to 300 s) during which roots were horizontally rotated at two revolutions per minute on a clinostat. The same effectiveness of equal sums of short stimulations separated by relatively long rest intervals indicates that the signals into which the stimuli are transduced are: (i) additive; (ii) proportional to the duration of a single stimulation; and (iii) stable for at least 5 min. The perception time is shorter than 1 s, the presentation time is approximately 10 s. The effects of intermittent stimulation fit the hypothesis that the gravity-induced movement of statoliths changes asymmetrically the stress in cytoskeletal actin filaments, thereby inducing gravitropic bending.

Graviperception in plants: the role of the cytoskeleton.

NASA: Researchers examine the role of gravity perception in plants. Specific questions are how is the work done by gravity on statoliths transferred to competent cellular structures and what are the structures which transduce the physical stimulus into a biochemical/biophysical signal.^ieng

Graviresponses in herb and trees: a major role for the redistribution of tissue and growth stresses.

Tissue stresses, which occur in turgid herbaceous stems, both elongating and non-elongating. and tree growth stresses (TGSs) which occur in woody stems, are similar in that (i) they form self-equilibrating patterns of stresses (tensile and compressive) in stems, and (ii) the asymmetric, graviresponsive change in the pattern tends to bend the stem. The longitudinal tensile tissue stress (TS) which occurs in the outer layers of turgid stems is a few times higher than the osmotic pressure of cell sap in such a layer. Usually it is considered that TSs originate from the differential growth of tissues in a stem; however, physical analysis of a turgid stem model has shown that TSs are an unavoidable physical consequence of the variation in structural characteristics of cell layers or vascular strands in turgid stems. The model applied to the sunflower hypocotyl gives forces which fit well to those measured. The structural characteristics are sufficient to explain fully the TSs which exist in turgid stems. Differential growth is not necessary in this respect. Examination of the model shows also that the longitudinal elastic strain of all cell walls in a turgid stem is the same at a given stem level regardless of wall thickness, i.e. the structure-based TSs compensate for the variation in turgor-induced wall stress in single cells with variable diameter and wall thickness. The importance of this compensation for the anisotropy of wall stresses is presented. The forces which generate TSs exert bending moments which are high but they sum mutually to zero in a vertical stem. In gravistimulated turgid stems of Reynoutria, the TSs decrease considerably on the lower side while those on the upper side remain unaltered. The consequences of this asymmetric change for gravitropic bending are analysed. Tree growth stresses arise in a process by which new cells added by the cambium to the secondary xylem tend to shrink longitudinally (except compression wood) during maturation of the cell walls. The pattern of TGSs is characterized by tensile or compressive stress in the peripheral or the core wood, respectively. An asymmetrical pattern of TGSs due to asymmetrical deposition of wood results in a bending moment which equilibrates the bending moment caused by the weight of a lateral branch. In response to a gravity-derived stimulus the TGS pattern may be modified by asymmetric formation of reaction wood which differs histologically from normal wood: tension wood in many arborescent angiosperms, and compression wood in conifers. The formation and functioning of the reaction wood is discussed.

Gravity induced changes in intracellular potentials in statocytes of cress roots.

Two glass microelectrodes were inserted from opposite sides of the root cap into statocytes of Lepidium sativum L. immersed in medium with or without cytochalasin D (CD). Intracellular potentials (Eis) of statocytes were measured with reference to an earthed electrode in the bathing solution. In the absence of CD, Ei values were -160 +/- 2 mV (n = 52) in vertical roots. During the recording of Eis, the roots were tilted from the vertical by 45 degrees so that in a tilted root one electrode was on the upper side and the other on the lower side; after 5 min the roots were returned to the vertical. At approximately 64 s after tilting (lasting 5-15 s) there was a transient lowering of Ei (more negative) by an average of 4.7 mV on both the upper and lower sides (n = 52). In some cases, this decrease in Ei was preceded by a transitory increase. Returning the roots to the vertical resulted in a response similar to that obtained by tilting. In roots treated with CD at a concentration of 3 (microM for 1 h, the initial Ei was -145 +/- 2 mV (n = 43), and the lowering of Ei on position change (tilting or returning) was smaller (2.0 mV) in some statocytes (n = 50) and higher (8.1 mV) in others (n = 14) compared to control roots (without and with DMSO). A higher concentration (10 microM) of CD and longer treatment (2 h) further reduced the decrease in Ei (1.1 mV) on position change (n = 26). The observed effects of CD support the hypothesis that statoliths in statocytes are anchored by actin filaments to the plasma membrane and/or to the cortical endoplasmic reticulum. Movement of statoliths during the first step of graviperception may lead to stress changes in actin filaments, affecting the transmembrane potential and also the Ei.

Variation in velocity of cytoplasmic streaming and gravity effect in characean internodal cells measured by laser-Doppler-velocimetry.

Velocities of cytoplasmic streaming were measured in internodal cells of Nitella flexilis L. and Chara corallina Klein ex Willd. by laser-Doppler-velocimetry to investigate the possibility of non-statolith-based perception of gravity. This was recently proposed, based on a report of gravity-dependent polarity of cytoplasmic streaming. Our measurements revealed large spatial and temporal variation in streaming velocity within a cell, independent of the position of the cell with respect to the direction of gravity. In 58% of the horizontally positioned cells the velocities of acropetal and basipetal streaming, measured at opposite locations in the cell, differed significantly. In 45% of these, basipetal streaming was faster than acropetal streaming. In 60% of the vertically positioned cells however the difference was significant, downward streaming was faster in only 61% of these. When cell positions were changed from vertical to horizontal and vice versa the cells reacted variably. A significant difference between velocities in one direction, before and after the change, was observed in approx. 70% of the measurements, but the velocity was faster in the downward direction, as the second position, in only 70% of the significantly different. The ratio of basipetal to acropetal streaming velocities at opposite locations of a cell was quite variable within groups of cells with a particular orientation (horizontal, normal vertical, inverted vertical). On average, however, the ratio was close to 1.00 in the horizontal position and approx. 1.03 in the normal vertical position (basipetal streaming directed downwards), which indicates a small direct effect of gravity on streaming velocity. Individual cells, however, showed an increased, as well as a decreased, ratio when moved from the horizontal to the vertical position. No discernible effect of media (either Ca(2+)-buffered medium or 1.2% agar in distilled water) on the streaming velocities was observed. The above mentioned phenomenon of graviperception is not supported by our data.

Statoliths pull on microfilaments: experiments under microgravity.

Previous videomicroscopy of Chara rhizoids during parabolic flights of rockets showed that the weightless statoliths moved basipetally. A hypothesis was offered that the removal of gravity force disturbed the initial balance between this force and the basipetally acting forces generated in a dynamic interaction of statoliths with microfilaments (MFs). The prediction of this hypothesis that the statoliths would not be displaced basipetally during the microgravity phase (MG-phase) after disorganizing the MFs was tested by videomicroscopy of a rhizoid treated with cytochalasin D (CD) immediately before the flight. The prediction was fully supported by the flight experiment. Additionally, by chemical fixation of many rhizoids at the end of the MG-phase it was shown that all rhizoids treated with CD before the flight had statoliths at the same location. i.e., sedimented an the apical cell wall, while all untreated rhizoids had statoliths considerably displaced basipetally from their normal position. Thus, a dynamical interaction involving shearing forces between MFs and statoliths appears highly probable.

How well does the clinostat mimic the effect of microgravity on plant cells and organs?

The effect of clinostatting and microgravity on plant cells and organs is considered on the basis of distinguishing two types of gravistimulation: static and dynamic. The former is switched off both by clinostatting and microgravity, the latter is switched off by microgravity but occurs inevitably during clinostatting and may be perceived by cells if the rotation is not fast enough. Effects of clinostatting and microgravity on different examples of static gravistimulation (tonic effects, formation of compression wood, growth of "grass nodes," compensation of epinasty, stabilization of cellular polarity) are considered. The mechanism of the dynamic stimulation is presented; it is related to the displacement of the gravity sensing masses in the cell containing them, and involves disturbance of cytoskeletal tension. The low threshold for gravity perception and short minimal time of dynamic stimulation are emphasized. Only a relatively fast rotating clinostat, on which the radial distance of the cells from the rotational axis is small enough to keep the centrifugal force low, can effectively compensate gravity. However, one must take into account the extreme sensitivity of plants to mechanical stresses that may appear during clinostatting at different levels of plant organization.

Oriented movement of statoliths studied in a reduced gravitational field during parabolic flights of rockets.

During five rocket flights (TEXUS 18, 19, 21, 23 and 25), experiments were performed to investigate the behaviour of statoliths in rhizoids of the green alga Charo globularia Thuill. and in statocytes of cress (Lepidium sativum L.) roots, when the gravitational field changed to approx. l0(-4) g (i.e. microgravity) during the parabolic flight (lasting for 301-390 s) of the rockets. The position of statoliths was only slightly influenced by the conditions during launch, e.g. vibration, acceleration and rotation of the rocket. Within approx. 6 min of microgravity conditions the shape of the statolith complex in the rhizoids changed from a transversely oriented lens into a longitudinally oriented spindle. The center of the statolith complex moved approx. 14 micrometers and 3.6 micrometers in rhizoids and root statocytes, respectively, in the opposite direction to the originally acting gravity vector. The kinetics of statolith displacement in rhizoids demonstrate that the velocity was nearly constant under microgravity whereas it decreased remarkably after inversion of rhizoids on Earth. It can be concluded that on Earth the position of statoliths in both rhizoids and root statocytes depends on the balance of two forces, i.e. the gravitational force and the counteracting force mediated by microfilaments.

Cytoplasmic streaming in Chara rhizoids: studies in a reduced gravitational field during parabolic flights of rockets.

In-vivo videomicroscopy of Chara rhizoids under 10(-4)g demonstrated that gravity affected the velocities of cytoplasmic streaming. Both, the acropetal and basipetal streaming velocities increased on the change to microgravity. The endogenous difference in the velocities of the oppositely directed cytoplasmic streams was maintained under microgravity, yet the difference was diminished as the basipetal streaming velocity increased more than the acropetal streaming velocity. Direction and structure of microfilaments labeled by rhodamine-phalloidin had not changed after 6 min of microgravity.

Oriented movement of statoliths studied in a reduced gravitational field during parabolic flights of rockets.

During five rocket flights (TEXUS 18, 19, 21, 23 and 25), experiments were performed to investigate the behaviour of statoliths in rhizoids of the green alga Chara globularia Thuill. and in statocytes of cress (Lepidium sativum L.) roots, when the gravitational field changed to approx. 10(-4) · g (i.e. microgravity) during the parabolic flight (lasting for 301-390 s) of the rockets. The position of statoliths was only slightly influenced by the conditions during launch, e.g. vibration, acceleration and rotation of the rocket. Within approx. 6 min of microgravity conditions the shape of the statolith complex in the rhizoids changed from a transversely oriented lens into a longitudinally oriented spindle. The center of the statolith complex moved approx. 14 µm and 3.6 µm in rhizoids and root statocytes, respectively, in the opposite direction to the originally acting gravity vector. The kinetics of statolith displacement in rhizoids demonstrate that the velocity was nearly constant under microgravity whereas it decreased remarkably after inversion of rhizoids on Earth. It can be concluded that on Earth the position of statoliths in both rhizoids and root statocytes depends on the balance of two forces, i.e. the gravitational force and the counteracting force mediated by microfilaments.

Propagated fluctuations of the electric potential in the apoplasm of Lepidium sativum L. roots.

The electric potential on the surface of the Lepidium sativum L. root apex was recorded by means of six non-polarizable electrodes. Nonevoked fluctuations of the potential with amplitudes below 0.1 mV were observed. The fluctuations could be reversibly inhibited either by ether vapor or by anoxia caused by N2. They did not occur in killed roots. Cross-correlation analysis of the fluctuations from six electrodes located one above another along the 3-mm apical region showed a pattern of time delay which indicates that the fluctuations may be the consequence of signals propagated in the root with a velocity of 3-9 mm · s(-1) in a basipetal direction from the root cap. We hypothesize that the fluctuations are due to signals of an unknown nature propagated along an intrasymplasmic continuous system, the "symreticulum", composed of the cortical ER of individual cells and desmotubules passing through the plasmodesmata.

Differential growth resulting in the specification of different types of cellular architecture in root meristems.

Symplastic growth of plant organs may be described by a continuous growth tensor field. In tensorial analysis of meristems, the trajectories of periclinal and anticlinal cell walls represent trajectories of the principal directions of growth (PDGs); this follows from the maintenance of mutual orthogonality between periclinal and anticlinal wall trajectories during growth. Periclinal and anticlinal cell divisions are also oriented in the principal planes of growth. The growth tensor for the root apex is specified in such a way that the principal directions of the tensor fit the pattern of periclinal and anticlinal walls in the apex, and that the grid formed by material particles aligned along PDG trajectories preserve this alignment during growth. Two growth tensors are formulated--one giving a maximum and the other giving a minimum of the volumetric relative elemental growth rate at the region of the initial cell(s). Temporal sequences of deformation of a grid formed by lines coinciding with the principal directions of growth are shown. The formation of cellular patterns in root apices is simulated. Two types of patterns are obtained: one with an apical cell and merophytes, and another with files of cells converging towards a quiescent centre.

Induced fluctuations of electric potentials in the apoplast of leaves.

Extracellular recordings of electrical potential in leaves of different species by means of band-pass amplifiers showed the occurrence of fast, small changes (spikes) with an amplitude below 1 mV. Local illumination of leaves induced temporal patterns of spikes outside the illuminated region. The light-induced patterns recorded by a given electrode in a particular experimental setup were similar for successive illuminations. The patterns recorded at different sites on the same leaf were different. Locally repetitive patterns of spikes at the electrode outside the illuminated region indicate the occurrence of some signals transmitted from this region to the cells in the neighborhood of the electrode.

Regulation of the position of statoliths in Chara rhizoids.

The behavior of statoliths in rhizoids differently oriented with respect to the gravity vector indicates that there are cytoskeleton elements which exert forces on the statoliths, mostly in the longitudinal directions. Compared to the sum of the forces acting on a statolith, the gravitational force is a relatively small component, i.e., less than 1/5 of the cytoskeleton force. The balance is disturbed by displacing the rhizoid from the normal vertical orientation. It is also reversibly disturbed by cytochalasin B such that some statoliths move against the gravity force. Phalloidin stabilizes the position of the statoliths against cytochalasin B. We infer that microfilaments are involved in controlling the position of statoliths, and that there is a considerable tension on these microfilaments. The vibration frequency of the microfilaments corresponding to this tension is in the ultrasonic range.