Effects of the myosin ATPase inhibitor 2,3-butanedione monoxime on amyloplast kinetics and gravitropism of Arabidopsis hypocotyls

The actin cytoskeleton is a crucial component in plant gravitropism, and studies confirm that alterations to actin filaments (F-actin) can have dramatic effects on gravitropic curvature in roots and shoots. Many models for gravisensing in higher plants suggest that the key to gravity perception and signal transduction lies in intimate interactions between F-actin and amyloplasts. In this study, we investigated gravitropism in hypocotyls by analyzing the effect of myosin inhibition on gravitropic curvature in order to clarify the role of the actomyosin system in shoot gravitropism. To study amyloplast movement in endodermal cells (i.e., gravity-perceiving statocytes) of living seedlings, we repositioned a confocal laser scanning microscope (CLSM) so that its rotatable stage was oriented vertically. Seedlings containing green fluorescent protein-labeled endodermal amyloplasts were incubated with the ATPase inhibitor 2,3-butanedione monoxime (BDM) and then mounted on the stage so that the hypocotyls were vertical. Using CLSM, we imaged the endodermal amyloplasts, while the hypocotyls were oriented vertically and also after they were reoriented by 90°. Our results show that BDM reduces gravitropic curvature in a concentration-dependent manner. In addition, BDM increases amyloplast movement in hypocotyls of vertical seedlings, but reduces amyloplast movement in hypocotyls of reoriented seedlings, suggesting that myosin may participate in the intracellular transport of amyloplasts in statocytes. These results can be explained in the context of amyloplasts as both noise indicators and gravity susceptors, with BDM producing less coherent amyloplast movement that results in an increased signal-to-noise ratio, which may account for at least part of the observed reduction in gravitopic curvature.     

The role of actin filaments in the gravitropic response of snapdragon flowering shoots

The involvement of the actin and the microtubule cytoskeleton networks in the gravitropic response of snapdragon ( Antirrhinum majus L.) flowering shoots was studied using various specific cytoskeleton modulators. The microtubule-depolymerizing drugs tested had no effect on gravitropic bending. In contrast, the actin-modulating drugs, cytochalasin D (CD), cytochalasin B (CB) and latrunculin B (Lat B) significantly inhibited the gravitropic response. CB completely inhibited shoot bending via inhibiting general growth, whereas CD completely inhibited bending via specific inhibition of the differential flank growth in the shoot bending zone. Surprisingly, Lat B had only a partial inhibitory effect on shoot bending as compared to CD. This probably resulted from the different effects of these two drugs on the actin cytoskeleton, as was seen in cortical cells. CD caused fragmentation of the actin cytoskeleton and delayed amyloplast displacement following gravistimulation. In contrast, Lat B caused a complete depolymerization of the actin filaments in the shoot bending zone, but only slightly reduced the amyloplast sedimentation rate following gravistimulation. Taken together, our results suggest that the actin cytoskeleton is involved in the gravitropic response of snapdragon shoots. The actin cytoskeleton within the shoot cells is necessary for normal amyloplast displacement upon gravistimulation, which leads to the gravitropic bending.     

Microtubule reorientation in shoots precedes bending during the gravitropic response of cut snapdragon spikes

The microtubule reorientation during the gravitropic bending of cut snapdragon (Antirrhinum majus L.) spikes was investigated. Using indirect immunofluorescence methods, we examined changes in microtubule orientation in the cortex, endodermis and pith tissues of the shoot bending zone, in response to gravistimulation. Our results show that dense microtubule arrays were visible throughout the cortical, endodermal and pith shoot tissues, and that the transverse orientation of the microtubules (perpendicular to the growth axis) was specifically associated with the shoot growing bending zone. Microtubules showed gravity-induced kinetics of changes in their orientation, which occurred only in the upper stem flank and preceded shoot bending. While this observation, that the gravity-induced microtubule orientation precedes bending, was previously reported only in special above-ground organs such as coleoptiles and hypocotyls, our present study is the first to show that such patterns of change occur in mature flowering shoots. These changes were exhibited first in the upper flank of the cortex and then in the upper flank of the endodermis. No changes in microtubule orientation were observed in the cortex or endodermis tissues of the lower flanks or in the pith, suggesting that these tissues continue to grow during shoot gravistimulation. Our results imply that microtubules may be involved in growth cessation of the upper shoot flank occurring during the gravitropic bending of snapdragon cut spikes.     

Disruption of the F-actin cytoskeleton limits statolith movement in Arabidopsis hypocotyls

The F-actin cytoskeleton is hypothesized to play a role in signal transduction mechanisms of gravitropism by interacting with sedimenting amyloplasts as they traverse statocytes of gravistimulated plants. Previous studies have determined that pharmacological disruption of the F-actin cytoskeleton with latrunculin B (Lat-B) causes increased gravitropism in stem-like organs and roots, and results in a more rapid settling of amyloplasts in the columella cells of Arabidopsis roots. These results suggest that the actin cytoskeleton modulates amyloplast movement and also gravitropic signal transduction. To determine the effect of F-actin disruption on amyloplast sedimentation in stem-like organs, Arabidopsis hypocotyls were treated with Lat-B and a detailed analysis of amyloplast sedimentation kinetics was performed by determining amyloplast positions in endodermal cells at various time intervals following reorientation. Confocal microscopy was used to confirm that Lat-B effectively disrupts the actin cytoskeleton in these cells. The results indicate that amyloplasts in hypocotyl endodermal cells settle more quickly compared with amyloplasts in root columella cells. F-actin disruption with Lat-B severely reduces amyloplast mobility within Arabidopsis endodermal statocytes, and these results suggest that amyloplast sedimentation within the hypocotyl endodermal cell is F-actin-dependent. Thus, a model for gravitropism in stem-like organs is proposed in which F-actin modulates the gravity response by actively participating in statolith repositioning within the endodermal statocytes.     

A polarized cell: the root statocyte

In the gravity-perceiving cells (statocytes), located in the centre of the root cap, polarity is expressed in the arrangement of the organelles since, in most genera, the nucleus and the endoplasmic reticulum are maintained at the opposite ends of each cell by actin. Polarity is also evident in the distribution of plasmodesmata, which are more numerous in the transverse walls than in the longitudinal walls. The centre of each statocyte is depleted of microtubules (they are only located at the periphery) but is occupied by numerous amyloplasts (statoliths), denser than the cytoplasm. The amyloplasts do not contribute to the inherent structural polarity since their position is dependent upon the gravity vector. This article focuses on new microscopic analyses and on data obtained from experiments performed in microgravity, which have contributed to our better understanding of the architecture of the actin web implicated in the perception of gravity. Depending upon the plant, the actin network seems to be formed of single filaments arranged in various ways, or, of thin bundles of actin filaments. The amyloplasts are enmeshed in this web of actin and their envelopes are associated with it, but they can have autonomous movement via myosin in the absence of gravity. From calculations of the value of the force necessary to move one amyloplast in the lentil root, and from videomicroscopy performed with living statocytes of maize roots, it is hypothesized that actin microfilaments could be orientated in an overall diagonal direction in the statocyte. These observations could help in understanding how slight amyloplast movements may trigger and transmit the gravitropic signal.     

Inhibition of the gravitropic bending response of flowering shoots by salicylic acid.

The upward gravitropic bending of cut snapdragon, lupinus and anemone flowering shoots was inhibited by salicylic acid (SA) applied at 0.5 mM and above. This effect was probably not due to acidification of the cytoplasm, since other weak acids did not inhibit bending of snapdragon shoots. In order to study its mode of inhibitory action, we have examined in cut snapdragon shoots the effect of SA on three processes of the gravity-signaling pathway, including: amyloplast sedimentation, formation of ethylene gradient across the stem, and differential growth response. The results show that 1 mM SA inhibited differential ethylene production rates across the horizontal stem and the gravity-induced growth, without significantly inhibiting vertical growth or amyloplast sedimentation following horizontal placement. However, 5 mM SA inhibited all three gravity-induced processes, as well as the growth of vertical shoots, while increasing flower wilting. It may, therefore, be concluded that SA inhibits bending of various cut flowering shoots in a concentration-dependent manner. Thus, at a low concentration SA exerts its effect in snapdragon shoots by inhibiting processes operating downstream to stimulus sensing exerted by amyloplast sedimentation. At a higher concentration SA inhibits bending probably by exerting general negative effects on various cellular processes.     

An Arabidopsis E3 ligase, SHOOT GRAVITROPISM9, modulates the interaction between statoliths and F-actin in gravity sensing

Higher plants use the sedimentation of amyloplasts in statocytes as statolith to sense the direction of gravity during gravitropism. In Arabidopsis thaliana inflorescence stem statocyte, amyloplasts are in complex movement; some show jumping-like saltatory movement and some tend to sediment toward the gravity direction. Here, we report that a RING-type E3 ligase SHOOT GRAVITROPISM9 (SGR9) localized to amyloplasts modulates amyloplast dynamics. In the sgr9 mutant, which exhibits reduced gravitropism, amyloplasts did not sediment but exhibited increased saltatory movement. Amyloplasts sometimes formed a cluster that is abnormally entangled with actin filaments (AFs) in sgr9. By contrast, in the fiz1 mutant, an ACT8 semidominant mutant that induces fragmentation of AFs, amyloplasts, lost saltatory movement and sedimented with nearly statically. Both treatment with Latrunculin B, an inhibitor of AF polymerization, and the fiz1 mutation rescued the gravitropic defect of sgr9. In addition, fiz1 decreased saltatory movement and induced amyloplast sedimentation even in sgr9. Our results suggest that amyloplasts are in equilibrium between sedimentation and saltatory movement in wild-type endodermal cells. Furthermore, this equilibrium is the result of the interaction between amyloplasts and AFs modulated by the SGR9. SGR9 may promote detachment of amyloplasts from AFs, allowing the amyloplasts to sediment in the AFs-dependent equilibrium of amyloplast dynamics.     

Amyloplasts and vacuolar membrane dynamics in the living graviperceptive cell of the Arabidopsis inflorescence stem

We developed an adequate method for the in vivo analysis of organelle dynamics in the gravity-perceptive cell (endodermis) of the Arabidopsis thaliana inflorescence stem, revealing behavior of amyloplasts and vacuolar membranes in those cells. Amyloplasts in the endodermis showed saltatory movements even before gravistimulation by reorientation, and these movements were confirmed as microfilament dependent. From our quantitative analysis in the wild type, the gravity-oriented movement of amyloplasts mainly occurred during 0 to 3 min after gravistimulation by reorientation, supporting findings from our previous physiological study. Even after microfilament disruption, the gravity-oriented movement of amyloplasts remained. By contrast, in zig/sgr4 mutants, where a SNARE molecule functioning in vacuole biogenesis has been disrupted, the movement of amyloplasts in the endodermis is severely restricted both before and after gravistimulation by reorientation. Here, we describe vacuolar membrane behavior in these cells in the wild-type, actin filament-disrupted, and zig/sgr4 mutants and discuss its putatively important features for the perception of gravity. We also discuss the data on the two kinds of movements of amyloplasts that may play an important role in gravitropism: (1) the leading edge amyloplasts and (2) the en mass movement of amyloplasts.     

Inhibition of the Gravitropic Response of Snapdragon Spikes by the Calcium-Channel Blocker Lanthanum Chloride

The putative Ca²⁺-channel blocker LaCl₃ prevented the gravitropic bending of cut snapdragon (Antirrhinum majus L.) spikes (S. Philosoph-Hadas, S. Meir, I. Rosenberger, A.H. Halevy [1996] Plant Physiol 110: 301–310) and inhibited stem curvature to a greater extent than vertical and horizontal stem elongation at the bending zone. This might indicate that LaCl₃, which modulates cytosolic Ca²⁺, does not influence general stem-growth processes but may specifically affect other gravity-associated processes occurring at the stem-bending zone. Two such specific gravity-dependent events were found to occur in the bending zone of snapdragon spikes: sedimentation of starch-containing chloroplasts at the bottom of stem cortex cells, as seen in cross-sections, and establishment of an ethylene gradient across the stem. Our results show that the lateral sedimentation of chloroplasts associated with gravity sensing was prevented in cross-sections taken from the bending zone of LaCl₃-treated and subsequently gravistimulated spikes and that LaCl₃ completely prevented the gravity-induced, asymmetric ethylene production established across the stem-bending zone. These data indicate that LaCl₃ inhibits stem curvature of snapdragon spikes by preventing several gravity-dependent processes. Therefore, we propose that the gravitropic response of shoots could be mediated through a Ca²⁺-dependent pathway involving modulation of cytosolic Ca²⁺ at various stages.     

Effect of gravity on apical dominance in Pharbitis nil.

When the upper part of main shoot of morning glory (Pharbitis nil) is gently bent down, lateral bud on the bending region is released from apical dominance and starts to elongate. But, clinorotating the bending shoots prevents the release of the lateral bud from apical dominance. These results suggest that gravity affects apical dominance in morning glory. Here we verified the gravity-regulated apical dominance by using a weeping morning glory defective in gravitropic response due to abnormal differentiation of endodermis. That is, bending main shoot of the weeping morning glory hardly caused the lateral bud to elongate. In addition, decapitation of apical bud released the lateral bud from apical dominance, and exogenous auxin applied to the cut surface of the decapitated stem was inhibitory to the outgrowth of the lateral bud in the wild type. However, the effect of auxin was much less in the weeping morning glory. Thus, apical dominance of the weeping morning glory was weaker and less influenced by gravity than that of the wild type, which could occur due to abnormal differentiation of endodermis required for graviperception.     

Regulation of the gravitropic response and ethylene biosynthesis in gravistimulated snapdragon spikes by calcium chelators and ethylene inhibitors

The possible involvement of Ca2+ as a second messenger in snapdragon (Antirrhinum majus L.) shoot gravitropism, as well as the role of ethylene in this bending response, were analyzed in terms of stem curvature and gravity-induced asymmetric ethylene production rates, ethylene-related metabolites, and invertase activity across the stem. Application of Ca2+ chelators (ethylenediaminetetraacetic acid, trans-1,2-cyclohexane dinitro-N,N,N',N'-tetraacetic acid, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid) or a Ca2+ antagonist (LaCl3) to the spikes caused a significant loss of their gravitropic response following horizontal placement. Conversely, the Ca2+ ionophore A23187 or the agonist Bay K-8644 increased gravibending. Longitudinally halved stem sections had significantly higher amounts of ethylene, 1-aminocyclopropane-1-carboxylic acid, and 1-(malonylamino) cyclopropane-1-carboxylic acid compared with vertical controls, with the extra production arising exclusively from the lower half of the stem. trans-1,2-cyclohexane dinitro-N,N,N',N'-tetraacetic acid pretreatment completely abolished the gravity-induced ethylene gradient across the stem, thereby leading to a significant reduction of the curvature. Similarly, reduction of the ethylene produced in the gravistimulated with CoCl2 or inhibition of its action by silver thiosulfate or 2,5-norbornadiene significantly inhibited the subsequent gravibending. Silver thiosulfate and CoCl2 also abolished the gravity-induced gradient of invertase activity across the stem, which is associated with the asymmetric stem elongation. These results suggest that cytosolic Ca2+ may regulate auxin action in snapdragon spikes, manifested as increased ethylene production, which is, in turn, intimately correlated with stem bending. Therefore, both hormones seem to play significant roles in induction and progress of the gravibending of snapdragon spikes.     

Immunolocalization of actin in root statocytes of Lens culinaris L

Lentil root statocytes show a strict structural polarity of their organelles with respect to the g vector. These cells are involved in the perception of gravity and are responsible for the orientation of the root. Actin filaments take part in the positioning of their organelles and could also be involved in the transduction of the gravitropic signal. A pre-embedding immunogold silver technique was carried out with a monoclonal antibody in order to study the distribution of actin cytoskeleton in the statocytes at the electron microscopic level. Some areas were never labelled (cell wall, vacuole, nucleoplasm, mitochondria, starch grains of the amyloplasts) or very slightly labelled (stroma of the amyloplasts). The labelling was scattered in the cytoplasm always close to, or on the nuclear and amyloplast envelopes and the tonoplast. Associations of 2 to 6 dots in file were observed, but these short files were not oriented in one preferential direction. They corresponded to a maximum distance of 0.9 micron. This work demonstrated that each statocyte organelle was enmeshed in an actin web of short filaments arranged in different ways. The images obtained by rhodaminephalloidin staining were in accordance with those of immunogold labelling. The diffuse fluorescence of the cytoplasm could be explained by the fact that the meshes of the web should be narrow. The vicinity of actin and of the amyloplasts envelope could account for the movement of these organelles that was observed in spatial microgravity.     

Characterization of the asymmetric growth of gravistimulated snapdragon spikes by stem and cell dimension analyses

Growth patterns of detached spikes of gravistimulated snapdragon (Antirrhinum majus L.) were analyzed in detail. The length increment of 5-mm marked subsections in the upper and lower flanks of the stem-bending zone was measured during gravistimulation using time-lapse photographs. At the onset of bending, a negative relative growth rate of the upper flank was detected, followed by increased relative growth rate in both lower and upper flanks. Consequently, a differential stem growth pattern was obtained during gravistimulation, which was significantly and specifically abolished by calcium antagonists reported previously to inhibit stem curvature of snapdragon. The differential growth patterns resulted from dynamic modifications of the cell dimensions in the epidermal and cortical stem layers. Bending started with both shrinking and widening of the epidermal cells and a parallel decrease in length and height of cortical cells at the upper stem flank. These changes were accompanied with a concomitant increase in length and height of the cortical cells on the lower stem flank, followed by a growth increase of epidermal cells. Our results suggest that both the epidermal and cortical cells play an important role in gravitropic shoot bending of snapdragon.     

Amyloplast displacement is necessary for gravisensing in Arabidopsis shoots as revealed by a centrifuge microscope

The starch‐statolith hypothesis proposes that starch‐filled amyloplasts act as statoliths in plant gravisensing, moving in response to the gravity vector and signaling its direction. However, recent studies suggest that amyloplasts show continuous, complex movements in Arabidopsis shoots, contradicting the idea of a so‐called ‘static’ or ‘settled’ statolith. Here, we show that amyloplast movement underlies shoot gravisensing by using a custom‐designed centrifuge microscope in combination with analysis of gravitropic mutants. The centrifuge microscope revealed that sedimentary movements of amyloplasts under hypergravity conditions are linearly correlated with gravitropic curvature in wild‐type stems. We next analyzed the hypergravity response in the shoot gravitropism 2 (sgr2) mutant, which exhibits neither a shoot gravitropic response nor amyloplast sedimentation at 1  g . sgr2 mutants were able to sense and respond to gravity under 30  g conditions, during which the amyloplasts sedimented. These findings are consistent with amyloplast redistribution resulting from gravity‐driven movements triggering shoot gravisensing. To further support this idea, we examined two additional gravitropic mutants, phosphoglucomutase (pgm) and sgr9, which show abnormal amyloplast distribution and reduced gravitropism at 1  g . We found that the correlation between hypergravity‐induced amyloplast sedimentation and gravitropic curvature of these mutants was identical to that of wild‐type plants. These observations suggest that Arabidopsis shoots have a gravisensing mechanism that linearly converts the number of amyloplasts that settle to the ‘bottom’ of the cell into gravitropic signals. Further, the restoration of the gravitropic response by hypergravity in the gravitropic mutants that we tested indicates that these lines probably have a functional gravisensing mechanism that is not triggered at 1  g .     

Actomyosin promotes cell plate alignment and late lateral expansion in <Emphasis Type="Italic">Tradescantia</Emphasis> stamen hair cells

Cytokinesis in higher-plant cells involves the formation of a cell plate in the interzone between the separating chromatids. The process is directed by the phragmoplast, an array of microtubules, actin filaments, and membranous elements. To determine if the role of actin in cytokinesis is dependent on myosin, we treated stamen hair cells of Tradescantia virginiana L. with 2,3-butanedione monoxime (BDM), an inhibitor of myosin ATPase and ML-7, a specific inhibitor of myosin light-chain kinase. Treatment with BDM resulted in a tilted cytokinetic apparatus during early initiation and a wavy cell plate with curved phragmoplasts during late lateral expansion. Treatment with ML-7 also resulted in inefficient late lateral expansion of the cell plate, with effects ranging from slower expansion to complete inhibition. Taken together, these results implicate myosin in the control of cell plate expansion and alignment.     

The Lazy Mutation in Rice Affects a Step between Statoliths and Gravity-Induced Lateral Auxin Transport

Abstract: Compared to wild type, the lazy mutant in Oryza sati-va L. shows a reduced gravitropic response. In order to locate the lesion in the stimulus-response chain, coleoptile segments of lazy rice were investigated with respect to auxin transport. Gravity-induced lateral movement of radiolabelled indoleacetic acid (IAA) was strongly inhibited by the lazy mutation compared to wild type while uptake and longitudinal transport of IAA, as well as amyloplast sedimentation, were not significantly affected. These findings suggest that LAZY controls a step in the signalling chain between statoliths and auxin secretion.     

Actin-facilitated assembly of smooth muscle myosin induces formation of actomyosin fibrils

To identify regulatory mechanisms potentially involved in formation of actomyosin structures in smooth muscle cells, the influence of F-actin on smooth muscle myosin assembly was examined. In physiologically relevant buffers, AMPPNP binding to myosin caused transition to the soluble 10S myosin conformation due to trapping of nucleotide at the active sites. The resulting 10S myosin-AMPPNP complex was highly stable and thick filament assembly was suppressed. However, upon addition to F-actin, myosin readily assembled to form thick filaments. Furthermore, myosin assembly caused rearrangement of actin filament networks into actomyosin fibers composed of coaligned F-actin and myosin thick filaments. Severin-induced fragmentation of actin in actomyosin fibers resulted in immediate disassembly of myosin thick filaments, demonstrating that actin filaments were indispensable for mediating myosin assembly in the presence of AMPPNP. Actomyosin fibers also formed after addition of F-actin to nonphosphorylated 10S myosin monomers containing the products of ATP hydrolysis trapped at the active site. The resulting fibers were rapidly disassembled after addition of millimolar MgATP and consequent transition of myosin to the soluble 10S state. However, reassembly of myosin filaments in the presence of MgATP and F-actin could be induced by phosphorylation of myosin P-light chains, causing regeneration of actomyosin fiber bundles. The results indicate that actomyosin fibers can be spontaneously formed by F-actin-mediated assembly of smooth muscle myosin. Moreover, induction of actomyosin fibers by myosin light chain phosphorylation in the presence of actin filament networks provides a plausible hypothesis for contractile fiber assembly in situ.     

Mechanism of inhibition of cytoplasmic streaming by a myosin inhibitor, 2,3-butanedione monoxime

Summary On the basis of the inhibition of myosin by 2,3-butanedione monoxime (BDM), the protein's involvement in various cell activities is discussed. However, it has not been established whether BDM inhibits plant myosin. In the present study, the effect of BDM on isolated plant myosin was analyzed in vitro. The sliding between myosin from lily (Lilium longiflorum) pollen tubes and actin filaments from skeletal muscle was inhibited to 25% at a concentration of 60 mM, indicating that BDM can be used as a myosin inhibitor for plant materials. Cytoplasmic streaming was completely inhibited by BDM at 30 mM in lily pollen tubes and at 70 mM in short root hair cells, and at 100 mM in long root hair cells ofHydrocharis dubia. However, BDM at high concentrations induced the disorganization of actin filament bundles in lily pollen tubes and short root hair cells. In addition, cortical microtubules were also fragmented in short root hair cells treated with BDM, suggesting a possible side effect of BDM.Abbreviations AF actin filament - BDM 2,3-butanedione monoxime - MT microtubule     

Organelle Sedimentation in Gravitropic Roots of Limnobium is Restricted to the Elongation Zone

Roots of the aquatic angiosperm Limnobium spongia (Bosc) Steud.were evaluated by light and electron microscopy to determinethe distribution of organelle sedimentation towards gravity.Roots of Limnobium are strongly gravitropic. The rootcap consistsof only two layers of cells. Although small amyloplasts arepresent in the central cap cells, no sedimentation of any organelle,including amyloplasts, was found. In contrast, both amyloplastsand nuclei sediment consistently and completely in cells ofthe elongation zone. Sedimentation occurs in one cell layerof the cortex just outside the endodermis. Sedimentation ofboth amyloplasts and nuclei begins in cells that are in theirinitial stages of elongation and persists at least to the levelof the root where root hairs emerge. This is the first modernreport of the presence of sedimentation away from, but not in,the rootcap. It shows that sedimentation in the rootcap is notnecessary for gravitropic sensing in at least one angiosperm.If amyloplast sedimentation is responsible for gravitropic sensing,then the site of sensing in Limnobium roots is the elongationzone and not the rootcap. These data do not necessarily conflictwith the hypothesis that sensing occurs in the cap in otherroots, since Limnobium roots are exceptional in rootcap originand structure, as well as in the distribution of organelle sedimentation.Similarly, if nuclear sedimentation is involved in gravitropicsensing, then nuclear mass would function in addition to, notinstead of, that of amyloplasts.Copyright 1994, 1999 AcademicPress Limnobium spongia, gravitropism, roots, sedimentation, cortex     

Effects of myosin ATPase inhibitor 2,3-butanedione 2-monoxime on distributions of myosins, F-actin, microtubules, and cortical endoplasmic reticulum in maize root apices

2,3-Butanedione 2-monoxime (BDM) is a general inhibitor of myosin ATPases of eukaryotic cells, and its effects on animal and yeast cells are well described. Using immunofluorescence and electron microscopy, we have analyzed the impacts of BDM on distributions of plant myosins, actin filaments (AFs), microtubules (MTs), and cortical endoplasmic reticulum (ER) elements in various cell types of maize root apices. Treatment of growing maize roots with BDM altered the typical distribution patterns of unconventional plant myosin VIII and of putative maize homologue(s) of myosin II. This pharmacological agent also induced a broad range of impacts on AFs and on cortical ER elements associated with plasmodesmata and pit fields. BDM-mediated effects on the actomyosin cytoskeleton were especially pronounced in cells of the root transition zone. Additionally, BDM elicited distinct reactions in the MT cytoskeleton; endoplasmic MTs vanished in all cells of the transition zone and cortical MTs assembled in increased amounts preferentially at plasmodesmata and pit-fields. Our data indicate that AFs and MTs interact together via BDM-sensitive plant myosins, which can be considered as putative integrators of the plant cytoskeleton. Morphometric analysis revealed that cell growth was prominently inhibited in the transition zone and the apical part, but not the central part, of the elongation region. Obviously, myosin-based contractility of the actin cytoskeleton is essential for the developmental progression of root cells through the transition zone.