The effect of uni-axial clinostat rotation on germination and root anatomy of Phaseolus vulgaris L.

ABSTRACT

Phaseolus vulgaris L. seed germination and seedling root anatomy were investigated on a slowly rotating clinostat in 1g. Clinostat rotating seeds were oriented as follows: the first group with the longer axis parallel to the rotation pole (horizontal), the other with the longer axis normal to the rotation pole with due attention to the position of the root apex primordium in the dry seeds (vertical). Germination time, percent germination and curvature of developing roots were monitored. Furthermore, the anatomy of the root apex was quantitatively analysed. Seeds placed on the clinostat germinated earlier than controls, and columella cells of roots developed while rotating lost the strict polarity with the nucleus positioned near the proximal periclinal cell wall and amyloplasts sedimented on the distal periclinal wall. Irrespective of seed orientation on the rotation axis, loss of cell polarity occurred as well as a decrease in starch content, modification in cell size, and damage to statocytes whose walls appeared partially digested. Cell size in the elongation zone was also larger in roots rotating on the clinostat than in controls, both in vertically and horizontally placed specimens. Our results demonstrate that prolonged rotation has an effect on the statocyte that continuously perceives gravity from ever-changing directions, although this effect is irrespective of seed position on the rotating axis in P. vulgaris.     

Limitation on the use of the horizontal clinostat as a gravity compensator

If the horizontal clinostat effectively compensates for the influence of the gravity vector on the rotating plant, it should make the plant unresponsive to whatever chronic acceleration may be applied transverse to the axis of clinostat rotation. This was tested by centrifuging plants while they were growing on clinostats. For a number of morphological end-points of development the results depended on the magnitude of the applied g-force. Therefore, gravity compensation by the clinostat was incomplete. This conclusion is in agreement with results of satellite experiments which are reviewed.     

Gravitational compensation and the phototropic response of oat coleoptiles

Avena seedlings were germinated and grown while continuously rotated on the horizontal axis of a clinostat. The coleoptiles of these gravity-compensated plants were phototropically more responsive than those of plants rotated on a vertical axis. When the plants were compensated after unilateral irradiation, phototropic curvature of the shoot progressed for the next 6 hours, with the rate of curving decreasing about 3 hours after irradiation. The decrease in rate was less in the plants gravity-compensated before irradiation than in those vertically rotated. In the period 70 to 76 hours after planting, the growth rate of the compensated coleoptiles was significantly less than that of the vertically rotated seedlings. The greater phototropic curvature, the decreased growth rate, and the slower rate of straightening of the curved, compensated shoot can be correlated with several consequences of compensation: an increase in sensitivity to auxin, a lowering of auxin content in the coleoptile tip, and possibly, from an interaction between compensation and phototropic stimulation, an enhanced difference in auxin transport between the illuminated and shaded halves of the unilaterally irradiated shoot.

The phototropic response of the vertically rotated seedling was significantly different from that of the vertical stationary, indicating the importance of vertically rotated controls in clinostat experiments.

     

Phytochrome-mediated phototropism in maize seedling shoots

Unilateral irradiation with red light (R) or blue light (BL) elicits positive curvature of the mesocotyl of maize (Zea mays L.) seedlings raised under R for 2 d from sowing and kept in the dark for 1 d prior to curvature induction. The fluenceresponse curve for R-induced mesocotyl curvature, obtained by measuring curvature 100 min after phototropic induction, shows peaks in two fluence ranges, designated first positive range (from the threshold to the trough), and second positive range (above the trough). The fluence-response curve for BL is similar to that for R but shifted two orders of magnitude to higher fluences. Blue light elicits the classical first positive curvature of the coleoptile, whereas this response is not found with R. Positive mesocotyl curvature induced by either R or BL is eliminated by R given from above just before the unilateral irradiation, whereas BL-induced coleoptile curvature is not eliminated. The above results collectively offer evidence that phototropic curvature of the mesocotyl is induced by R-sensitive photosystem(s). Mesocotyl curvature in the second positive range is reduced by vertical far-red light (FR) applied after phototropic induction with R, but is not affected by FR applied before R. Unilateral irradiation with FR following vertical irradiation with a high R fluence leads to negative curvature of the mesocotyl. It is concluded that mesocotyl curvature in the second positive range results from a gradient in the amount of the FR-absorbing form of phytochrome (Pfr) established across the plant axis. Mesocotyl curvature in the first positive range is inhibited by vertical FR given either before or after phototropic induction with R. Since the FR used here is likely to produce more Pfr than the very low fluences of R eliciting the mesocotyl curvature in the first positive range, it is assumed that FR reduces the response in this case by adding Pfr at both sides of the plant axis. By rotating seedlings on a clinostat with its axis horizontal, the kinetics of mesocotyl curvature can be studied in the absence of a counteracting gravitropic response. On the clinostat, the R-induced mesocotyl curvature develops after a lag, through two successive phases having different curvature rates, the late phase is slower than the early phase. Negative curvature of the coleoptile can be induced by either R or BL; the BL-induced negative curvature is found at fluences higher than those giving positive curvature. The clinostat experiments show that the negative coleoptile curvature induced by either R or BL is a gravitropic compensation for positive mesocotyl curvature.Abbreviations BL blue light - FR far-red light - Pfr phytochrome in the far-red-absorbing form - Pr phytochrome in the red-absorbing form - R red light C.I.W.-D.P.B. Publication No. 824     

Interpreting Plant Responses to Clinostating: I. MECHANICAL STRESSES AND ETHYLENE

The severe epinasty and other symptoms developed by clinostated leafy plants could be responses to gravity compensation and/or the mechanical stresses of leaf flopping. Epinasty in cocklebur (Xanthium strumarium L.), tomato (Lycopersicon esculentum Mill.), and castor bean (Ricinus communis L.) is delayed by inhibitors of ethylene synthesis and action (aminoethoxyvinylglycine and Ag⁺), confirming the role of ethylene in clinostat epinasty. To test the possibility that clinostat mechanical stresses (leaf flopping) cause ethylene production and, thus, epinasty, vertical plants were stressed with constant, gentle, horizontal, or vertical shaking or with a quick, back-and-forth rotation (twisting). Clinostat leaf flopping was closely approximated but with a minimum of gravity compensation, by turning plants so their stems were horizontal, rotating them quickly about the stem axis, and then returning them to the vertical, repeating the treatment every four minutes (clinostat rotation time). None of these mechanical stresses produced significant epinasties, but vigorous hand-shaking (120 seconds per day) generated minor epinasties, as did Ag⁺ applied daily (concentrations high enough to cause leaf browning). Plants gently inverted every 20 minutes developed epinasty at about the same rate and to about the same extent as clinostated plants, but plants inverted every 20 minutes and immediately returned to the upright position did not become epinastic. It is concluded that clinostat epinasty is probably caused by disturbances in the gravity perception mechanism, rather than by leaf flopping.     

Ethylene as a Cause of Transient Petiole Epinasty in Helianthus annuus during Clinostat Experiments

Petiole curvature and elongation growth in Helianthus annuus L. have been recorded for plants rotating with their stems parallel to the horizontal axis of a clinostat at 8 revolutions per hour over 72 hours. When rotation was continuous, dorso-convex curvature (epinasty) developed in the first 12 hours and was followed by recovery (straightening) in the next 36 hours. Thereafter the petioles remained straight. These changes in shape are due to brief consecutive increases in the elongation growth of the upper and lower halves of the petiole. Plants exposed to 10 μl per liter ethylene after 200 hours on the clinostat, developed similar petiole epinasty, followed by straightening when the exposure to ethylene ceased. Interrupting rotation of the plant for 1 hour in 4, did not change the petiole response, whereas the alternation of 4 hour stationary and rotation periods, delayed the straightening process. The axillary angle between the stem and petiole increased from about 40° to 63° during either continuous rotation or rotation with 1 or 4 hour stationary periods. When detached leaves were inverted, the rate of ethylene release approximately doubled after 4 hours and continued to increase thereafter. The results indicate that the development of transient petiole epinasty on the clinostat, is due to ethylene production caused primarily by the disorientation of the plant, rather than to the rotation process.     

Inversion of gravitropism by symmetric blue light on the clinostat

Gravitropic stimulation of maize (Zea mays L.) seedlings resulted in a continuous curvature of the coleoptiles in a direction opposing the vector of gravity when the seedlings were rotated on a horizontal clinostat. The orientation of this response, however, was reversed when the gravitropic stimulation was preceeded by symmetric preirradiation with blue light (12.7 mol photons·m^–2). The fluence-response curve of this blue light exhibited a lower threshold at 0.5 mol·m^–2, and could be separated into two parts: fluences exceeding 5 mol·m^–2 reversed the direction of the gravitropic response, whereas for a range between the threshold and 4 mol·m^–2 a split population was obtained. In all cases a very strong curvature resulted either in the direction of gravity or in the opposite orientation. A minor fraction of seedlings, however, curved towards the caryopsis. Furthermore, the capacity of blue light to reverse the direction of the gravitropic response disappeared with the duration of gravitropic stimulation and it depended on the delay time between both stimulations. Thistonic blue-light influence appears to be transient, which is in contrast to the stability observed fortropistic blue-light effects.This work was supported by the Deutsche Forschungsgemeinschaft.     

Cellular basis for the automorphic curvature of rice coleoptiles on a three-dimensional clinostat: possible involvement of reorientation of cortical microtubules

Coleoptiles of rice (Oryza sativa L.) show a spontaneous (automorphic) curvature toward the caryopsis under microgravity conditions. The possible involvement of the reorientation of cortical microtubules in automorphic curvature was studied in rice coleoptiles grown on a three-dimensional clinostat. When rice seedlings that had been grown in the normal gravitational field were transferred to the clinostat in the dark, cortical microtubules of epidermal cells in the dorsal side of the coleoptiles oriented more transversely than the ventral side within 0.5 h. The rotation on the clinostat also increased the cell wall extensibility in the dorsal side and decreased the extensibility in the ventral side, and induced automorphic curvature. The reorientation of cortical microtubules preceded the changes in the cell wall extensibility and the curvature. The irradiation of rice seedlings with white light from above inhibited microtubule reorientation and changes in the cell wall extensibility, as well as curvature of coleoptiles. Also, colchicine, applied to the bending region of coleoptiles, partially inhibited the automorphic curvature. These results suggest that reorientation of cortical microtubules is involved in causing automorphic curvature in rice coleoptiles on the clinostat.     

Autonomic Straightening after Gravitropic Curvature of Cress Roots

Few studies have documented the response of gravitropically curved organs to a withdrawal of a constant gravitational stimulus. The effects of stimulus withdrawal on gravitropic curvature were studied by following individual roots of cress (Lepidium sativum L.) through reorientation and clinostat rotation. Roots turned to the horizontal curved down 62° and 88° after 1 and 5 h, respectively. Subsequent rotation on a clinostat for 6 h resulted in root straightening through a loss of gravitropic curvature in older regions and through new growth becoming aligned closer to the prestimulus vertical. However, these roots did not return completely to the prestimulus vertical, indicating the retention of some gravitropic response. Clinostat rotation shifted the mean root angle −36° closer to the prestimulus vertical, regardless of the duration of prior horizontal stimulation. Control roots (no horizontal stimulation) were slanted at various angles after clinostat rotation. These findings indicate that gravitropic curvature is not necessarily permanent, and that the root retains some commitment to its equilibrium orientation prior to gravitropic stimulation.     

The dynamics of a discrete geotropic sensor subject to rotation-induced gravity compensation

A clinostat achieves gravity compensation by providing circular rotation with uniform speed, about a horizontal axis. The dynamics of an assumed, discrete and free-moving subcellular gravity receptor, subject to clinostat rotation, is analyzed. The results imply that there is an optimum rotation rate; higher speeds result in circular motions with diameters more comparable to thermal noise fluctuations, but with greater linear velocities due to increasing centrifugal forces. An optimizing function is proposed. The nucleolus and mitochondrion is chosen as a gravity receptor for illustrating the use of this theory. The characteristics of their clinostat-induced motions are incorporated with experimental results on Avena plant shoots in an illustrative example.     

Gravitropic bending of cress roots without contact between amyloplasts and complexes of endoplasmic reticulum

The polar arrangement of cell organelles in Lepidium root statocytes is persistently converted to a physical stratification during lateral centrifugation (the centrifugal force acts perpendicular to the root long axis) or by apically directed centrifugation combined with cytochalasin-treatment. Lateral centrifugation (10 min, 60 min at 10\g or 50\g) causes displacement of amylplasts to the centrifugal anticlinal cell wall and shifting of the endoplasmic reticulum (ER) complex to the centripetal distal cell edge. After 60 min of lateral centrifugation at 10\g or 50\g all roots show a clear gravitropic curvature. The average angle of curvature is about 40° and corresponds to that of roots stimulated gravitropically in the horizontal position at 1\g in spite of the fact that the gravistimulus is 10-or 50-fold higher. Apically directed centrifugation combined with cytochalasin B (25 g\ml^-1) or cytochalasin D (2.5 g\ml^-1) incubation yields statocytes with the amyloplasts sedimented close to the centrifugal periclinal cell wall and ER cisternae accumulated at the proximal cell pole. Gravitropic stimulation for 30 min in the horizontal position at 1\g and additional 3 h rotation on a clinostat result in gravicurvature of cytochalasin B-treated centrifuged (1 h at 50\g) roots, but because of retarded root growth the angle of curvature is lower than in control roots. Cytochalasin D-treatment during centrifugation (20 min at 50\g) does not affect either root growth or gravicurvature during 3 h horizontal exposure to 1\g relative to untreated roots. As lateral centrifugation enables only short-term contact between the amyloplasts and the distal ER complex at the onset of centrifugation and apically directed centrifugation combined with cytochalasin-treatment even exclude any contact the integrity of the distal cell pole need not necessarily be a prerequisite for graviperception in Lepidium root statocytes.Abbreviations CB cytochalasin B - CD cytochalasin D - ER endoplasmic reticulum - g gravitational acceleration     

Gravity can induce epinastic bending of isolated leaves

Abstract Isolated leaves of Plectranthus fruticosus were grown in cubic plastic cuvettes, and were supplied via their cut petioles with nutrient solution and indole-3-acetic acid (10^?6m ). Holes bored in the cuvette walls allowed the petioles to be oriented at approximately 60°, 90° or 120° to the vertical. Growth of the leaves initially oriented at angles of 60° and 90°, which simulated the situation in the intact plant, did not result in epinastic bending of the petiole. Inversion of the leaves (adaxial surface of the petiole downwards) and orientation of the adaxial/abaxial surfaces of the horizontal petiole parallel to the gravity vector, however, yielded strong epinastic bending of the petioles. In the latter case, this bending was not in the direction of the gravity vector (evidence for point (iii), below). Furthermore, epinastic bending occurred, when the isolated leaves were rotated on a clinostat (petioles parallel to the rotation axis or inclined to the rotation axis at an angle of 30°; 3 r.p.m.). Since a possible influence of the shoot was excluded, it is concluded that (i) perception and response are restricted to the leaf, (ii) gravity alone is sufficient to induce epinasty, (iii) a gravitropic component of the response can probably be excluded. The clinostat induced epinasty may not have been caused by nullifying the effect of gravity but due to continuous gravistimulation of the leaf.     

The Role of Gravity in Apical Dominance : Effects of Clinostating on Shoot Inversion-Induced Ethylene Production, Shoot Elongation, and Lateral Bud Growth

Shoot inversion-induced release of apical dominance in Pharbitis nil is inhibited by rotating the plant at 0.42 revolutions per minute in a vertical plane perpendicular to the axis of rotation of a horizontal clinostat. Clinostating prevented lateral bud outgrowth, apparently by negating the restriction of the shoot elongation via reduction of ethylene production in the inverted shoot. Radial stem expansion was also decreased. Data from experiments with intact tissue and isolated segments indicated that shoot-inversion stimulates ethylene production by increasing the activity of 1-aminocyclopropane-1-carboxylic acid synthase. The results support the hypothesis that shoot inversion-induced release of apical dominance in Pharbitis nil is due to gravity stress and is mediated by ethylene-induced retardation of the elongation of the inverted shoot.     

Nastic response of maize (Zea mays L.) coleoptiles during clinostat rotation

Rotation of unstimulated maize (Zea mays L.) seedlings on a horizontal clinostat is accompanied by a strong bending response of the coleoptiles towards the caryopsis, yielding curvatures exceding 100°. The corresponding azimuthal distribution shows two peaks, each of which is displayed by 30° from the symmetry axis connecting the shortest coleoptile and caryopsis cross sections. It is argued that this spatial pattern is not the result of two independent bending preferences, but caused by a one-peaked distribution encountering an obstacle in its central part and thus being split into the two subpeaks. The existence of one preferential direction justifies considering this response to be a nastic movement. Its time course consists of an early negative phase (coleoptiles bend away from the caryopsis) followed 2 h later by a longlasting positive bending towards the caryopsis. In light-interaction experiments, fluence-response curves for different angles between blue light and the direction of the nastic response were measured. These experiments indicate that blue light interacts with the nastic response at two levels: (i) phototonic inhibition, and (ii) addition of nastic and phototropic curvatures. It is concluded that phototropic and phototonic transduction bifurcate before the formation of phototropic transverse polarity. The additivity of nastic and phototropic responses was followed at the population level. At the level of the individual seedling, one observes, in the case of phototropic induction opposing nastic movement, three distinct responses: either strong phototropism, or nastic bending, or an avoidance response which involves strong curvature perpendicular to the stimulation plane. With time the nastic bending becomes increasingly stable against opposing phototropic stimulation. This can be seen from a growing proportion of seedlings exhibiting nastic bending when light is applied at variable intervals after the onset of clinostat rotation. At the transition from instability to stability, this type of experiment produces a high percentage of seedlings displaying the avoidance response. However, no cancelling resulting in zero curvature can be observed. It is concluded that the endogenous polarity underlying the nastic response is different in its very nature from the blue-light-elicited stable transverse polarity described earlier (Nick and Schäfer 1988 b).Abbreviation BL blue light (449 nm)     

Effects of submergence on development and gravitropism in the coleoptile of Oryza sativa L.

Caryopses of rice (Oryza sativa L. cv. Sasanishiki) were germinated in air or under water. In submerged seedlings a twofold increase in coleoptile growth rate and an inhibition of root growth was observed. The amount of starch in the amyloplasts of submerged coleoptiles was substantially reduced compared to the air-grown control plants and plastids had a proplastidic character. During the rapid elongation of coleoptiles under water, the osmotic concentration of the press sap remained constant, whereas in air-grown coleoptiles a decrease was measured. Determination of curvature of gravistimulated air-grown and submerged shoots was carried out by placing the coleoptiles horizontally in air of 98% relative humidity. Air-grown coleoptiles reached a vertical orientation within 5 h after onset of gravistimulation. In coleoptiles germinated under water the first signs of consistent negative gravitropic bending occurred after 4–5 h and curvature was complete after 24 h. During the first 5 h of gravistimulation the water-grown coleoptiles grew at an average rate of 0.39 mm·h^–1, whereas in air-grown coleoptiles a rate of 0.27 mm·h^–1 was measured. Concomitant with the delayed onset of gravitropic bending of the water-grown coleoptiles, a change in plastid ultrastructure and an increase in starch content was observed. We conclude that the gravitropic responsiveness of the rice coleoptile depends on the presence of starch-filled amyloplasts.We wish to thank H.-J. Ensikat for technical assistance with the scanning electron microscopy. Supported by the Bundesminister für Forschung und Technologie and the Deutsche Forschungsgemeinschaft.     

Role of Indole-3-acetic Acid in Modification of Geotropic Responses in Clinostat Rotated Avena Seedlings

Oat seedlings were grown in a sand medium on clinostats with horizontal axes of rotation to nullify the directional component of the gravity-force vector. Coleoptile segments from such seedlings showed an enhanced absorption of apically applied exogenous auxin (indole-3-acetic acid), compared to segments from vertically rotated or stationary controls. Absorption of basally applied auxin and auxin transport were unaffected by the gravity treatments. Horizontal rotation did not materially change the amount of auxin produced and transported from excised coleoptile tips: however, plants so rotated showed an enhanced curvature response to unilaterally applied auxin.

Collectively, these experiments indicate that enhanced plant responses to horizontal clinostat rotation, where rates of rotation are sufficient to nullify the directional component of the gravity-force vector, are caused primarily by increases in metabolism and not by a modification of auxin availability. These data do not support recently advanced hypotheses that the polarity of auxin transport is based on gravitational sedimentation of cell inclusions.

     

Initial phases of gravity-induced lateral auxin transport and geotropic curvature in corn coleoptiles

Summary Wild-type corn coleoptiles showed an initial downward bending upon transfer from the vertical to the horizontal position. Strong upward curvature started only 15–30 min after the begin of horizontal exposure.Little, if any at all, initial downward geotropic bending was found with amylomaize coleoptiles at 1 X g. With stronger stimuli (10 or 20 X g) the amylomaize mutant reacted initially strongly in the wrong direction, i.e. opposite to the later response.When wild-type coleoptiles had been symmetrically prestimulated for 60 min with alternating 2-min horizontal exposures from opposite sides, no initial downward bending occurred if the plane of horizontal exposure was maintained from pretreatment to the continuous horizontal stimulation of the test. If, however, the coleoptiles were rotated 90° around their long axis between pretreatment and test, the initial downward bending reaction developed as in the non-prestimulated controls. Thus changes in reactivity remained localized to the site of stimulation.Following the same pretreatments used for the curvature measurements, lateral ³H-IAA transport was measured in coleoptile segments for 10 or 12.5 min. The auxin distribution found was strikingly parallel to the bending for all pretreatments.The dependence of reaction pattern on the duration of prestimulation in the same plane was tested. The function indicates a half life of 10–20 min for the change in sensitivity. The findings are discussed in view of a model of overstimulation and adaptation.     

Circumnutation of rice coleoptiles: its relationships with gravitropism and absence in lazy mutants

Although circumnutation occurs widely in higher plants, its mechanism is little understood. The idea that circumnutation is based on gravitropism has long been investigated, but the reported results have been controversial. We used dark-grown coleoptiles of rice (Oryza sativa L.) to re-investigate this issue. The following results supported the existence of a close relationship between gravitropism and circumnutation: (1) circumnutation disappears on a horizontal clinostat; (2) circumnutation is interrupted by a gravitropic response and re-initiated at a definable phase after gravitropic curvature; (3) circumnutation can be re-established by submergence and a brief gravitropic stimulation in the coleoptiles that have stopped nutating in response to red light; and (4) lazy mutants show no circumnutation. In spite of these results, however, there were cases in which gravitropism and circumnutation could be separated. Firstly, the non-circumnutating lazy coleoptile showed nearly a wild-type level of gravitropic responsiveness in its upper half, although this part was an active site of both gravitropism and circumnutation in wild-type coleoptiles. Secondly, coleoptiles could nutate without overshooting the vertical when developing phototropic curvature. It is concluded that gravitropism influences, but it is not directly involved in the process of circumnutation. It is further suggested that a gravity signal, shared with gravitropism, contributes to the maintenance of circumnutation.     

The phototropic response of Triticum aestivum coleoptiles under conditions of low gravity

Wheat (Triticum aestivum L. cv Broom) coleoptiles were stimulated by unilateral blue light pulses, the duration of which varied between 3 s and 30 min, under microgravity conditions on the IML-1 Spacelab mission and in ground controls. The stimuli covered first positive, indifferent and second positive response regions. When phototropic responses were observed under low-gravity conditions, slightly fewer seedlings responded compared with 1 g conditions. The latent period was similar in flight and 1g treatments (10–20 min), except for the response to 300,μmol m^?2 in the indifferent response region, where a positive response in flight plants followed a 130 min latent period, while no response was observed in 1 g plants. First positive responses at Og were slightly enhanced, both in magnitude and in duration, compared to the 1 g controls, but not to the extent predicted by clinostat studies. The response kinetics in the second positive region at 1g showed a plateau at 120 min, in contrast to the single maximum at Og. The fluence-response relationship was similar in both flight and ground controls. Only the responses to 4 and 6μmolm-2 showed significantly greater curvatures at 9g. This contrasts with previous clinostat studies, which reported substantial response enhancement at all fluence levels.