Intracellular magnetophoresis of amyloplasts and induction of root curvature

High-gradient magnetic fields (HGMFs) were used to induce intracellular magnetophoresis of amyloplasts. The HGMFs were generated by placing a small ferromagnetic wedge into a uniform magnetic field or at the gap edge between two permanent magnets. In the vicinity of the tip of the wedge the dynamic factor of the magnetic field, (H²/2), was about 10⁹ Oe² · cm^–1, which subjected the amyloplasts to a force comparable to that of gravity. When roots of 2-d-old seedlings of flax (Linum usitatissimum L.) were positioned vertically and exposed to an HGMF, curvature away from the wedge was transient and lasted approximately 1 h. Average curvature obtained after placing magnets, wedge and seedlings on a 1-rpm clinostat for 2 h was 33 ± 5 degrees. Roots of horizontally placed control seedlings without rotation curved about 47 ± 4 degrees. The time course of curvature and changes in growth rate were similar for gravicurvature and for root curvature induced by HGMFs. Microscopy showed displacement of amyloplasts in vitro and in vivo. Studies with Arabidopsis thaliana (L.) Heynh. showed that the wild type responded to HGMFs but the starchless mutant TC7 did not. The data indicate that a magnetic force can be used to study the gravisensing and response system of roots.Abbreviations HGMF high-gradient magnetic field - emu electromagnetic units - Oe Oersted We thank Dr. John Kiss, Miami University, Ohio for providing the Arabidopsis seeds. This work was supported by NASA grant NAGW-3656     

Magnetophoretic induction of curvature in coleoptiles and hypocotyls

Coleoptiles of barley (Hordeum vulgare) were positioned in ahigh gradient magnetic field (HGMF, dynamic factor H²/2 of 10⁹–10¹⁰Oe² cm^–1), generated by a ferromagnetic wedge in a uniformmagnetic field) and rotated on a 1 rpm clinostat. After 4 h90% of coleoptiles had curved toward the HGMF. The cells affectedby HGMF showed clear intracellular displacement of amyloplasts.Coleoptiles in a magnetic field next to a non-ferromagneticwedge showed no preferential curvature. The small size of thearea of non-uniformity of the HGMF allowed mapping of the sensitivityof the coleoptiles by varying the initial position of the wedgerelative to the coleoptile apex. When the ferromagnetic wedgewas placed 1 mm below the coleoptile tip only 58% of the coleoptilescurved toward the wedge indicating that the cells most sensitiveto intracellular displacement of amyloplasts and thus gravitysensing are confined to the top 1 mm portion of barley coleoptiles.Similar experiments with tomato hypocotyls Lycopersicum esculentum)also resulted in curvature toward the HGMF. The data stronglysupport the amyloplast-based gravity-sensing system in higherplants and the usefulness of HGMF to substitute gravity in shoots. Key words: Avena sativa, Hordeum vulgare, Lycopersicon esculentum, curvature, gravitropism, high gradient magnetic field, magnetophoresis     

Lentil root statoliths reach a stable state in microgravity

 The kinetics of the movement of statoliths in gravity-perceiving root cap cells of Lens culinaris L. and the force responsible for it have been analysed under 1 g and under microgravity conditions (S/MM-03 mission of Spacehab 1996). At the beginning of the experiment in space, the amyloplasts were grouped at the distal pole of the statocytes by a root-tip-directed 1-g centrifugal acceleration. The seedlings were then placed in microgravity for increasing periods of time (13, 29, 46 or 122 min) and chemically fixed. During the first 29 min of microgravity there were local displacements (mean velocity: 0.154 μm min⁻¹) of some amyloplasts (first at the front of the group and then at the rear). Nevertheless, the group of amyloplasts tended to reconstitute. After 122 min in microgravity the bulk of amyloplasts had almost reached the proximal pole where further movement was blocked by the nucleus. After a longer period in microgravity (4 h; experiment carried out 1994 during the IML 2 mission) the statoliths reached a stable position due to the fact that they were stopped by the nucleus. The position was similar to that observed in roots grown continuously in microgravity. Treatment with cytochalasin D (CD) did not stop the movement of the amyloplasts but slowed down the velocity of their displacement (0.019 μm min⁻¹). Initial movement patterns were the same as in control roots in water. Comparisons of mean velocities of amyloplast movements in roots in space and in inverted roots on earth showed that the force responsible for the movement in microgravity (F_c) was about 86% less (F_c = 0.016 pN) than the gravity force (F_g = 0.11 pN). Treatment with CD reduced F_c by two-thirds. The apparent viscosity of the statocyte cytoplasm was found to be 1 Pa s or 3.3 Pa s for control roots or CD treated roots, respectively. Brownian motion or elastic forces due to endoplasmic reticulum membranes do not cause the movement of the amyloplasts in microgravity. It is concluded that the force transporting the statoliths is caused by the actomyosin system. Received: 22 March 1999 / Accepted: 18 December 1999     

Magnetophoretic induction of curvature in coleoptiles and hypocotyls

Coleoptiles of barley (Hordeum vulgare) were positioned in a high gradient magnetic field (HGMF, dynamic factor gradient of H(2)/2 of 10(9)-10(10) Oe2 cm-1), generated by a ferromagnetic wedge in a uniform magnetic field and rotated on a 1 rpm clinostat. After 4 h 90% of coleoptiles had curved toward the HGMF. The cells affected by HGMF showed clear intracellular displacement of amyloplasts. Coleoptiles in a magnetic field next to a non-ferromagnetic wedge showed no preferential curvature. The small size of the area of nonuniformity of the HGMF allowed mapping of the sensitivity of the coleoptiles by varying the initial position of the wedge relative to the coleoptile apex. When the ferromagnetic wedge was placed 1 mm below the coleoptile tip only 58% of the coleoptiles curved toward the wedge indicating that the cells most sensitive to intracellular displacement of amyloplasts and thus gravity sensing are confined to the top 1 mm portion of barley coleoptiles. Similar experiments with tomato hypocotyls (Lycopersicum esculentum) also resulted in curvature toward the HGMF. The data strongly support the amyloplast-based gravity-sensing system in higher plants and the usefulness of HGMF to substitute gravity in shoots.     

Inhibition of Gravitropism in Etiolated Radish Hypocotyls by Chloramphenicol

The hypocotyls of radish seedlings can grow at an almost normalrate when the roots are immersed in 10^–4 M chloramphenicol(CAP) although there is a reduction in the length of the roots.However, when 4-d-old seedlings are placed horizontally, thehypocotyls of those growing in water bend completely uprightwithin 3 h, whereas those growing in CAP react slowly or notat all. The elongation rate of these gravitropically stimulatedhypocotyls is about 0.87 and 0.72 mm h^–1 in water andin CAP, respectively, and both these values are higher thanthose of unstimulated seedlings, where a rate of 0.29 mm h^–1occurred in both water and CAP treatments. The effect of CAPon gravitropism occurs only when CAP is applied to seedlingsin the first 24 h after sowing. At the cell level the main characteristicof the hypocotyls of CAP-treated seedlings not showing geocurvatureis the absence of starch in the cell layer surrounding the vasculartissues. From a comparison between the presence of starch andgravitropic reaction it is suggested that amyloplasts containedin endodermal cells are the receptors of the gravitational stimulus. Raphanus sativus, radish, gravitropism, amyloplasts, chloramphenicol     

Relocalization of the calcium gradient and a dihydropyridine receptor is involved in upward bending by bulging of Chara protonemata, but not in downward bending by bowing of Chara rhizoids

The localization of cytoplasmic free calcium and a dihydropyridine (DHP) receptor, a putative calcium channel, was recorded during the opposite graviresponses of tip-growing Chara rhizoids and Chara protonemata by using the calcium indicator Calcium Crimson and a fluorescently labeled dihydropyridine (FL-DHP). In upward (negatively gravitropically) growing protonemata and downward (positively gravitropically) growing rhizoids, a steep Ca²⁺ gradient and DHP receptors were found to be symmetrically localized in the tip. However, the localization of the Ca²⁺ gradient and DHP receptors differed considerably during the gravitropic responses upon horizontal positioning of the two cell types. During the graviresponse of rhizoids, a continuous bowing downward by differential flank growth, the Ca²⁺ gradient and DHP receptors remained symmetrically localized in the tip at the centre of growth. However, after tilting protonemata into a horizontal position, there was a drastic displacement of the Ca²⁺ gradient and FL-DHP to the upper flank of the apical dome. This displacement occurred after the apical intrusion and sedimentation of the statoliths but clearly before the change in the growth direction became evident. In protonemata, the reorientation of the growth direction started with the appearence of a bulge on that site of the upper flank which was predicted by the asymmetrically displaced Ca²⁺ gradient. With the upward shift of the cell tip, which is suggested to result from a statolith-induced displacement of the growth centre, the Ca²⁺ gradient and DHP receptors became symmetrically relocalized in the apical dome. No major asymmetrical rearrangement was observed during the following phase of gravitropic curvature which is characterized by slower rates of bending. Labeling with FL-DHP was completely inhibited by a non-fluorescently labeled dihydropyridine. From these results it is suggested that FL-DHP labels calcium channels in rhizoids and protonemata. In rhizoids, positive gravitropic curvature is caused by differential growth limited to the opposite subapical flanks of the apical dome, a process which does not involve displacement of the growth centre, the calcium gradient or calcium channels. In protonemata, however, it is proposed that a statolith-induced asymmetrical relocalization of calcium channels and the Ca²⁺ gradient precedes, and might mediate, the rearrangement of the centre of growth, most likely by the displacement of the Spitzenk?rper, to the upper flank, which results in the negative gravitropic reorientation of the growth direction. Received: 13 February 1999 / Accepted: 25 June 1999     

Light Promotion of Hypocotyl Gravitropism of a Starch-Deficient Tobacco Mutant Correlates with Plastid Enlargement and Sedimentation

Dark-grown hypocotyls of a starch-deficient mutant (NS458) of tobacco (Nicotiana sylvestris) lack amyloplasts and plastid sedimentation, and have severely reduced gravitropism. However, gravitropism improved dramatically when NS458 seedlings were grown in the light. To determine the extent of this improvement and whether mutant hypocotyls contain sedimented amyloplasts, gravitropic sensitivity (induction time and intermittent stimulation) and plastid size and position in the endodermis were measured in seedlings grown for 8 d in the light. Light-grown NS458 hypocotyls were gravitropic but were less sensitive than the wild type (WT). Starch occupied 10% of the volume of NS458 plastids grown in both the light and the dark, whereas WT plastids were essentially filled with starch in both treatments. Light increased plastid size twice as much in the mutant as in the WT. Plastids in light-grown NS458 were sedimented, presumably because of their larger size and greater total starch content. The induction by light of plastid sedimentation in NS458 provides new evidence for the role of plastid mass and sedimentation in stem gravitropic sensing. Because the mutant is not as sensitive as the WT, NS458 plastids may not have sufficient mass to provide full gravitropic sensitivity.     

Reduced Gravitropism in Hypocotyls of Starch-Deficient Mutants of Arabidopsis

Gravitropism was examined in dark- and light-grown hypocotylsof wild-type (WT), two reduced starch mutants (ACG 20 and ACG27), and a starchless mutant (ACG 21) of Arabidopsis. In addition,the starch content of these four strains was studied with lightand electron microscopy. Based on time course of curvature andorientation studies, the graviresponse in hypocotyls is proportionalto the amount of starch in a genotype. Furthermore, starch mutationsseem to primarily affect gravitropism rather than differentialgrowth since both phototropic curvature and growth rates amongthe four genotypes are approximately equal. Our results suggestthat gravity perception may require a greater plastid mass inhypocotyls compared to roots. The kinetics of gravitropic curvaturealso was compared following reorientation at 45°, 90°,and 135°. As has been reported for other plant species,the optimal angle of reorientation is 135° for WT Arabidopsisand the two reduced starch mutants, but the magnitude of curvatureof the starchless mutant appears to be independent of the initialangle of displacement. Taken together, the results of the presentstudy and our previous experiments with roots of the same fourgenotypes [Kiss et al. (1996) Physiol. Plant. 97: 237] supporta plastid-based hypothesis for gravity perception in plants. (Received December 16, 1996; Accepted February 7, 1997)     

Gravitropism in a starchless mutant of Arabidopsis

The starch-statolith theory of gravity reception has been tested with a mutant of Arabidopsis thaliana (L.) Heynh. which, lacking plastid phosphoglucomutase (EC 2.7.5.1) activity, does not synthesize starch. The hypocotyls and seedling roots of the mutant were examined by light and electron microscopy to confirm that they did not contain starch. In upright wild-type (WT) seedlings, starch-filled plastids in the starch sheath of the hypocotyl and in three of the five columellar layers of the root cap were piled on the cell floors, and sedimented to the ceilings when the plants were inverted. However, starchless plastids of the mutant were not significantly sedimented in these cells in either upright or inverted seedlings. Gravitropism of light-grown seedling roots was vigorous: e.g., 10^o curvature developed in mutants rotated on a clinostat following a 5 min induction at 1 · g, compared with 14^o in the WT. Curvatures induced during intervals from 2.5 to 30 min were 70% as great in the mutant as the WT. Thus under these conditions the presence of starch and the sedimentation of plastids are unnecessary for reception of gravity by Arabidopsis roots. Gravitropism by hypocotyls of light-grown seedlings was less vigorous than that by roots, but the mutant hypocotyls exhibited an average of 70–80% as much curvature as the WT. Roots and hypocotyls of etiolated seedlings and flower stalks of mature plants were also gravitropic, although in these cases the mutant was generally less closely comparable to the WT. Thus, starch is also unnecessary for gravity reception in these tissues.Abbreviations PAR photosynthetically active radiation - PAS periodic acid-Schiff's reagent - PGM phosphoglucomutase - WT wild-type     

The MSG1 and AXR1 genes of Arabidopsis are likely to act independently in growth-curvature responses of hypocotyls

Growth-curvature responses of hypocotyls of Arabidopsis thaliana (L.) Heynh. were measured in double mutants between msg1 and axr1, both of which are auxin-resistant and defective in hypocotyl growth curvature induced upon unilateral application of auxin. The msg1 axr1 double mutants showed no auxin-induced growth curvature, that is, they exhibited the msg1 phenotype, though the axr1 defects were partial. Hypocotyls of both the msg1 and axr1 mutants were partially defective in second-positive phototropism, whereas the double mutants lost the response completely. When grown on vertically held agar plates, the axr1 mutant showed normal hypocotyl gravitropism and the mutation did not affect the reduced hypocotyl gravitropism of msg1. Hypocotyls of msg1 and axr1 mutants grew upward like wild-type ones when grown along an agar surface, while they grew more randomly when grown without an agar support, suggesting that axr1 hypocotyls are not completely normal in gravitropism. The extent of defects in growth orientation increased in the order: msg1 axr1 double mutants > msg1 > axr1 > wild type. The hypocotyls of these mutants showed auxin resistance in the order: msg1 axr1 > axr1 > msg1 > wild type. The msg1 mutant had epinastic leaves and axr1 had wrinkled leaves; leaves of the msg1 axr1 double mutants were epinastic and wrinkled. These results suggest that MSG1 and AXR1 act independently in separate pathways of the reactions tested in the present study. In contrast, the phenotype of the msg1 aux1 double mutants shows that AUX1 is not significantly involved in these phenomena. Received: 12 July 1998 / Accepted: 16 August 1998     

Regulation of auxin transport by aminopeptidases and endogenous flavonoids

 The 1-N-naphthylphthalamic acid (NPA)-binding protein is a putative negative regulator of polar auxin transport that has been shown to block auxin efflux from both whole plant tissues and microsomal membrane vesicles. We previously showed that NPA is hydrolyzed by plasma-membrane amidohydrolases that co-localize with tyrosine, proline, and tryptophan-specific aminopeptidases (APs) in the cotyledonary node, hypocotyl-root transition zone and root distal elongation zone of Arabidopsisthaliana (L.) Heynh. seedlings. Moreover, amino acyl-β-naphthylamide (aa-NA) conjugates resembling NPA in structure have NPA-like inhibitory activity on growth, suggesting a possible role of APs in NPA action. Here we report that the same aa-NA conjugates and the AP inhibitor bestatin also block auxin efflux from seedling tissue. Bestatin and, to a lesser extent, some aa-NA conjugates were more effective inhibitors of low-affinity specific [³H]NPA-binding than were the flavonoids quercetin and kaempferol but had no effect on high-affinity binding. Since the APs are inhibited by flavonoids, we compared the localization of endogenous flavonoids and APs in seedling tissue. A correlation between AP and flavonoid localization was found in 5- to 6-d-old seedlings. Evidence that these flavonoids regulate auxin accumulation in vivo was obtained using the flavonoid-deficient mutant, tt4. In whole-seedling [¹⁴C]indole-3-acetic acid transport studies, the pattern of auxin distribution in the tt4 mutant was shown to be altered. The defect appeared to be in auxin accumulation, as a considerable amount of auxin escaped from the roots. Treatment of the tt4 mutant with the missing intermediate naringenin restored normal auxin distribution and accumulation by the root. These results implicate APs and endogenous flavonoids in the regulation of auxin efflux. Received: 2 December 1999 / Accepted: 16 January 2000     

Gravitropism and starch statoliths in an Arabidopsis mutant

The mutant TC 7 of Arabidopsis thaliana (L.) Heynh. has been reported to be starch-free and still exhibit root gravitropism (T. Caspar and B. G. Pickard 1989, Planta 177, 185–197). This is not consistent with the hypothesis that plastid starch has a statolith function in gravity perception. In the present study, initial light microscopy using the same mutant showed apparently starch-free statocytes. However, ultrastructural examination detected residues of amyloplast starch grains in addition to the starch-depleted amyloplasts. Applying a point-counting morphometric method, the starch grains in the individual amyloplasts in the mutant were generally found to occupy more than 20% and in a few cases up to 60% of the amyloplast area. In the wild type (WT) the starch occupied on average 98 % of the amyloplast area and appeared as densely packed grains. The amyloplasts occupied 13.9% of the area of the statocyte in the mutant and 23.3% of the statocyte area in the WT. Sedimentation of starch-depleted amyloplasts in the mutant was not detected after 40 min of inversion while in the WT the amyloplasts sedimented at a speed of 6 m · h^-1. The gravitropic reactivity and the curvature pattern were also examined in the WT and the mutant. The time-courses of root curvature in the WT and the mutant showed that when cultivated under standard conditions for 60 h in darkness, the curvatures were 83° and 44°, respectively, after 25 h of continuous stimulation in the horizontal position. The WT roots curved significantly more rapidly and with a more normal gravitropic pattern than those of the mutant. These results are discussed in relation to the results previously obtained with the mutant and with respect to the starch-statolith hypothesis.Abbreviation WT wild type This work was supported by grants from Norwegian Research Council for Science and the Humanities (NAVF) which we gratefully acknowledge. We would also like to thank Dr. Timothy Caspar, Michigan State University, East Lansing, USA, for providing us with the seeds of TC 75.     

An alginate-layer technique for culture of Brassica oleracea L. protoplasts

Ten accessions belonging to the Brassica oleracea subspecies alba and rubra, and to B. oleracea var. sabauda were used in this study. Protoplasts were isolated from leaves and hypocotyls of in vitro grown plants. The influence of selected factors on the yield, viability, and mitotic activity of protoplasts immobilized in calcium alginate layers was investigated. The efficiency of protoplast isolation from hypocotyls was lower (0.7 ± 0.1 × 10⁶ ml⁻¹) than for protoplasts isolated from leaf mesophyll tissue (2 ± 0.1 × 10⁶ ml⁻¹). High (70–90%) viabilities of immobilized protoplasts were recorded, independent of the explant sources. The highest proportion of protoplasts undergoing divisions was noted for cv. Reball F1, both from mesophyll (29.8 ± 2.2%) and hypocotyl (17.5 ± 0.3%) tissues. Developed colonies of callus tissue were subjected to regeneration and as a result plants from six accessions were obtained.     

Amyloplasts are necessary for full gravitropic sensitivity in roots of Arabidopsis thaliana

The observation that a starchless mutant (TC7) of Arabidopsis thaliana (L.) Heynh. is gravitropic (T. Caspar and B.G. Pickard, 1989, Planta 177, 185–197) raises questions about the hypothesis that starch and amyloplasts play a role in gravity perception. We compared the kinetics of gravitropism in this starchless mutant and the wild-type (WT). Wild-type roots are more responsive to gravity than TC7 roots as judged by several parameters: (1) Vertically grown TC7 roots were not as oriented with respect to the gravity vector as WT roots. (2) In the time course of curvature after gravistimulation, curvature in TC7 roots was delayed and reduced compared to WT roots. (3) TC7 roots curved less than WT roots following a single, short (induction) period of gravistimulation, and WT, but not TC7, roots curved in response to a 1-min period of horizontal exposure. (4) Wild-type roots curved much more than TC7 roots in response to intermittent stimulation (repeated short periods of horizontal exposure); WT roots curved in response to 10 s of stimulation or less, but TC7 roots required 2 min of stimulation to produce a curvature. The growth rates were equal for both genotypes. We conclude that WT roots are more sensitive to gravity than TC7 roots. Starch is not required for gravity perception in TC7 roots, but is necessary for full sensitivity; thus it is likely that amyloplasts function as statoliths in WT Arabidopsis roots. Furthermore, since centrifugation studies using low gravitational forces indicated that starchless plastids are relatively dense and are the most movable component in TC7 columella cells, the starchless plastids may also function as statoliths.Abbreviations S2 story two - S3 story three - WT wild-type     

Curvature Induced by Amyloplast Magnetophoresis in Protonemata of the Moss Ceratodon purpureus

After gravistimulation of Ceratodon purpureus (Hedw.) Brid. protonemata in the dark, amyloplast sedimentation was followed by upward curvature in the wild-type (WT) and downward curvature in the wwr mutant (wrong way response). We used ponderomotive forces induced by high-gradient magnetic fields (HGMF) to simulate the effect of gravity and displace the presumptive statoliths. The field was applied by placing protonemata either between two permanent magnets at the edge of the gap, close to the edge of a magnetized ferromagnetic wedge, or close to a small (<1 mm) permanent magnet. Continuous application of an HGMF in all three configurations resulted in plastid displacement and induced curvature in tip cells of WT and wwr protonemata. WT cells curved toward the HGMF, and wwr cells curved away from the HGMF, comparable to gravitropism. Plastids isolated from protonemal cultures had densities ranging from 1.24 to 1.38 g cm⁻³. Plastid density was similar for both genotypes, but the mutant contained larger plastids than the WT. The size difference might explain the stronger response of the wwr protonemata to the HGMF. Our data support the plastid-based theory of gravitropic sensing and suggest that HGMF-induced ponderomotive forces can substitute for gravity.The force exerted by gravity is proportional to an object''s volume and density. Therefore, objects denser than the surrounding medium fall or sediment. Much evidence suggests that gravity sensing in higher plants depends on the sedimentation of dense, starch-filled amyloplasts inside specialized cells, so-called statocytes (Sack, 1991, 1997; Kuznetsov and Hasenstein, 1996, 1997b; Balu$\breve{s}$ka and Hasenstein, 1997).Dark-grown protonemata of the moss Ceratodon purpureus are tip-growing cells that are negatively gravitropic, i.e. they grow upward (Fig. ​(Fig.1).1). The wwr mutant (wrong way response) of C. purpureus is positively gravitropic, with reaction kinetics similar to the WT (Wagner et al., 1997). In horizontal WT (Fig. ​(Fig.1)1) and wwr protonemata, amyloplasts sediment in a specific zone located behind the apical dome. Plastid sedimentation is probably responsible for gravity sensing in both genotypes because it precedes curvature and because the recovery of gravitropism after basipetal centrifugation correlates with the return and sedimentation of amyloplasts (Walker and Sack, 1990, 1991; Wagner et al., 1997; Sack et al., 1998). Figure 1Gravitropic curvature and amyloplast sedimentation (arrowheads) in WT protonemata of C. purpureus that were rotated from the vertical to the horizontal 4 to 5 h before fixation.To study further the possible role of amyloplast sedimentation in gravity sensing, it is helpful to displace amyloplasts without reorienting the cell in the gravitational field. This can be achieved by exposing cells to an HGMF, thereby inducing the intracellular magnetophoretic displacement of starch-containing plastids (Kuznetsov and Hasenstein, 1996, 1997a, 1997b).Dense plastids such as amyloplasts and the cytoplasm differ in their chemical composition and physical properties, including their magnetic characteristics. When subjected to a nonuniform magnetic field, magnetically heterogeneous systems experience ponderomotive forces that depend on their relative magnetic susceptibilities (Kuznetsov and Hasenstein, 1996). Therefore, a magnetic field of sufficient intensity and gradient should be able to displace plastids inside the cell and provide an excellent test for plastid-based gravity sensing.If gravity sensing is plastid dependent, negatively gravitropic WT protonemata should curve toward stronger field intensities. In contrast, wwr cells should curve toward lower field intensities or in a positive gravitropic sense, similar to previous experiments with positively gravitropic roots (Audus, 1960; Kuznetsov and Kuznetsov, 1989; Kuznetsov and Hasenstein, 1996) and negatively gravitropic shoots (Schwarzacher and Audus, 1973; Kuznetsov and Hasenstein, 1997b). These experiments suggest that intracellular magnetophoresis is equivalent to plastid-based gravity sensing. However, these experiments were performed with higher plant organs, where the sites for perception and response are different, rather than with single cells that are capable of both sensing and responding to gravity. Moreover, the small size of moss protonemata and the availability of genotypes with opposite gravitropic responses warrants the use of HGMFs to study the possible involvement of plastid-based sensing in C. purpureus. If gravitropic sensing depends on the amyloplast sedimentation, then exposure to a magnetic field should induce both amyloplast displacement and the curvature of the tip cells in directions that are genotype dependent.This hypothesis was tested using several configurations to produce magnetic fields of different intensities and geometries. We report here that exposure to HGMF caused magnetophoretic displacement of amyloplasts and induced curvature in both WT and wwr protonemata in the predicted directions.     

Phototropism of rice (Oryza sativa L.) coleoptiles: fluence-response relationships, kinetics and photogravitropic equilibrium

Phototropism of rice (Oryza sativa L.) coleoptiles induced by unilateral blue light was characterized using red-light-grown seedlings. Phototropic fluence-response relationships, investigated mainly with submerged coleoptiles, revealed three response types previously identified in oat and maize coleoptiles: two pulse-induced positive phototropisms and a phototropism that depended on stimulation time. The effective ranges of fluences and fluence rates were comparable to those reported for maize. Compared with oats and maize, however, curvature responses in rice were much smaller and coleoptiles straightened faster after establishing the maximal curvature. When stimulated continuously, submerged coleoptiles developed curvature slowly over a period of 6 h, whereas air-grown coleoptiles, which showed smaller phototropic responsiveness, established a photogravitropic equilibrium from about 4 h of stimulation. The plot of the equilibrium angle against log fluence rates yielded a bell-shaped optimum curve that spanned over a relatively wide fluence-rate range; a maximal curvature of 25° occurred at a fluence rate of 1 μmol · m⁻² · s⁻¹. This optimum curve apparently reflects the light sensitivity of the steady-state phototropic response. Received: 28 June 1996 / Accepted: 30 July 1996     

A microtechnique for the analysis of free and conjugated indole-3-acetic acid in milligram amounts of plant tissue using a benchtop gas chromatograph-mass spectrometer

A microtechnique was developed for the quantification of indole-3-acetic acid (IAA) in plant samples of one milligram fresh weight or less. The method permitted quantification of both free and conjugated IAA using a benchtop gas chromatograph-mass spectrometer. New methods for sample purification with high recovery at microscale levels, together with simple changes that result in enhanced sensitivity of the instrumentation, allowed for a significant reduction in the amount of plant material required for analysis. Single oat (Avena sativa L.) coleoptile tips could be studied with this method and were found to contain free and total IAA levels of 137 and 399 pg · mg⁻¹ fresh weight, respectively. A single 5-d-old Arabidopsis thaliana (L.) Heynh. seedling was shown to contain 61 pg · mg⁻¹ fresh weight free IAA and 7850 pg · mg⁻¹ fresh weight of total IAA following basic hydrolysis. This microtechnique provides a way to accurately measure IAA levels in very small structures and individual seedlings, thus making it a valuable research tool for elucidating the role and distribution of auxin in relation to growth and development. Received: 1 May 1994 / Accepted: 25 June 1997     

Treatment of dark-grown Arabidopsis thaliana with a brassinosteroid-biosynthesis inhibitor, brassinazole, induces some characteristics of light-grown plants

When a brassinosteroid biosynthesis inhibitor, brassinazole (Brz), was applied at concentrations ranging from 0.1 to 2 μM, Arabidopsis thaliana (L.) Heynh seedlings grown in the dark exhibited morphological features of light-grown plants, i.e. short hypocotyls, expanded cotyledons, and true leaves, in a dose-dependent manner. Control (non Brz-treated) seedlings grown in the dark for 40 d did not develop leaf primordia. However, treatment with the lowest concentration of Brz induced the development of leaf buds, although it hardly induced any short hypocotyls, and treatment with the highest concentration of Brz induced both short hypocotyls and leaves. Labeling experiments with the thymidine analogue 5-bromo-2′-deoxyuridine revealed that amplification of cell nuclei and organellar nucleoids is activated in the shoot apical meristems of dark-grown Brz-treated seedlings. These results suggest that Brz-treatment induces development of true leaves. Furthermore, condensation and scattering of plastid nucleoids, which is known to occur during the differentiation of etioplasts into chloroplasts, was observed in the plastids of dark-grown Brz-treated cotyledons. In addition, high levels of ribulose-1,5-bisphosphate carboxylase-oxygenase proteins accumulated in the plastids of the cotyledons. Electron microscopy showed that the plastids were etioplasts with a prolamellar body and few thylakoid membranes. These results suggest that Brz treatment in the dark induces the initial steps of plastid differentiation, which occur prior to the development of thylakoid membranes. This is a novel presumed function of brassinosteroids. These cytological changes seen in Brz-treated Arabidopsis were exactly the same as those seen in a brassinosteroid-biosynthesis-deficient mutant, det2, supporting the hypothesis that Brz has no side-effects except inhibiting brassinosteroid biosynthesis, and should prove a useful tool in clarifying the role of brassinosteroids. Received: 10 February 2000 / Accepted: 11 April 2000     

Light intensity, gibberellin content and the resolution of shoot growth in Brassica

The role of gibberellins (GAs) in the regulation of shoot elongation is well established but the phytohormonal control of dry-matter production is poorly understood. In the present study, shoot elongation and dry-matter production were resolved by growing Brassica napus L. seedlings under five light intensities (photon flux densities) ranging from 25 to 500 μmol m⁻² s⁻¹. Under low light, plants were tall but produced little dry weight; as light intensity was increased, plants were progressively shorter but had increasing dry weights. Endogenous GAs in stems of 16- and 17-d-old plants were analyzed by gas chromatography-selected ion monitoring with [²H₂] internal standards. The contents of GAs increased dramatically with decreasing light intensity: GA₁, GA₃, GA₈ and GA₂₀ were 62, 15, 16 and 32 times higher, respectively, under the lowest versus highest light intensities. Gibberellin A₁₉ was not measured at 25 μmol m⁻² s⁻¹ but was 9␣times greater in the 75 compared to 500 μmol m⁻² s⁻¹ treatment. Shoot and hypocotyl lengths were closely positively correlated with (log) GA concentration (for example: r ² = 0.93 for GA₁ and hypocotyl length) but shoot dry matter was negatively correlated with GA concentration. The application of gibberellic acid (GA₃) produced elongation of plants grown under high light, indication that their low level of endogenous GA was limiting shoot elongation. Although endogenous GA₂₀ showed the greatest influence of light treatment, metabolism of [³H]GA₂₀ and of [³H]GA₁ was only slightly influenced by light intensity, suggesting that neither 2β- nor 3β-hydroxylation were points of metabolic regulation. The results of this study indicate that GAs control shoot elongation but are not directly involved in the regulation of shoot dry weight in Brassica. The study also suggests a role of GAs in photomorphogenesis, serving as an intermediate between light condition and shoot elongation response. Received: 18 June 1998 / Accepted: 29 July 1998