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.     

Actomyosin mediates gravisensing and early transduction events in reoriented cut snapdragon spikes

We investigated the involvement of the actomyosin network in the early events of the gravitropic response of cut snapdragon (Antirrhinum majus L.) spikes. The effects of the actin-modulating drug, cytochalasin D (CD) and/or the myosin inhibitor, 2,3-butanedione-2-monoxime (BDM) on amyloplast displacement, lateral auxin transport and consequently on stem bending were examined. The inhibitory effect on cytoskeleton integrity was studied by using indirect immunofluorescence double-labeling of actin and myosin. Our results demonstrate that no organizational changes in actin filaments occurred in cortical and endodermal cells of the stem bending zone during reorientation. These results suggest that actin depolymerization is not required for amyloplast sedimentation. Unlike the chloroplasts in the cortex, the amyloplasts in the endodermis were surrounded by actin and myosin, indicating that amyloplasts may be attached to the actin filaments via the motor protein, myosin. This suggests the involvement of myosin as part of the actomyosin complex in amyloplast movement in vertical as well as in reoriented stems. This suggestion was supported by the findings showing that: (a) BDM or CD disrupted the normal organization of actin either by altering characteristic distribution patterns of myosin-like protein in the cortex (BDM), or by causing actin fragmentation (CD); (b) both compounds inhibited the gravity-induced amyloplast displacement in the endodermis. Additionally, these compounds also inhibited lateral auxin transport across the stem and stem gravitropic bending. Our study suggests that during stem reorientation amyloplasts possibly remain attached to the actin filaments, using myosin as a motor protein. Thus, gravisensing and early transduction events in the gravitropic response of snapdragon spikes, manifested by amyloplast displacement and lateral auxin transport, are mediated by the actomyosin complex.     

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.     

Joining forces: The interface of gravitropism and plastid protein import

In flowering plants, gravity perception appears to involve the sedimentation of starch-filled plastids, called amyloplasts, within specialized cells (the statocytes) of shoots (endodermal cells) and roots (columella cells). Unfortunately, how the physical information derived from amyloplast sedimentation is converted into a biochemical signal that promotes organ gravitropic curvature remains largely unknown. Recent results suggest an involvement of the Translocon of the Outer Envelope of (Chloro) plastids (TOC) in early phases of gravity signal transduction within the statocytes. This review summarizes our current knowledge of the molecular mechanisms that govern gravity signal transduction in flowering plants and summarizes models that attempt to explain the contribution of TOC proteins in this important behavioral plant growth response to its mechanical environment.Key words: gravitropism, root, amyloplast, TOC complex, TOC132, TOC75     

Disruption of the actin cytoskeleton results in the promotion of gravitropism in inflorescence stems and hypocotyls of Arabidopsis

The actin cytoskeleton is hypothesized to play a major role in gravity perception and transduction mechanisms in roots of plants. To determine whether actin microfilaments (MFs) are involved in these processes in stem-like organs, we studied gravitropism in Arabidopsis inflorescence stems and hypocotyls. Localization studies using Alexa Fluor-phalloidin in conjugation with confocal microscopy demonstrated a longitudinally and transversely oriented actin MF network in endodermal cells of stems and hypocotyls. Latrunculin B (Lat-B) treatment of hypocotyls caused depolymerization of actin MFs in endodermal cells and a significant reduction of hypocotyl growth rates. Actin MFs in Lat-B-treated inflorescence stems also were disrupted, but growth rates were not affected. Despite disruption of the actin cytoskeleton in these two organs, Lat-B-treated stems and hypocotyls exhibited a promotion of gravitropic curvature in response to reorientation. In contrast, Lat-B reduced gravitropic curvature in roots but also reduced the growth rate. Thus, in contrast to prevailing hypotheses, our results suggest that actin MFs are not a necessary component of gravitropism in inflorescence stems and hypocotyls. Furthermore, this is the first study to demonstrate a prominent actin MF network in endodermal cells in the putative gravity-perceiving cells in stems.     

Reduced gravitropism in inflorescence stems and hypocotyls, but not roots, of Arabidopsis mutants with large plastids

The sites of gravity perception are columella cells in roots and endodermal cells in hypocotyls and inflorescence stems. Since plastids are likely to play a role in graviperception, we investigated gravitropism in plastid mutants of Arabidopsis . Previous studies have shown that the arc 6 and arc 12 ( a ccumulation and r eplication of c hloroplasts) mutants have an average of two large plastids per leaf mesophyll cell. In this study, we found that these arc mutants have altered plastid morphology throughout the entire plant body, including the cells involved in gravity perception. There were no major differences in total starch content per cell in endodermal and columella cells of the wild-type (WT) compared to arc 6 and arc 12 as assayed by iodine staining. Thus, the total mass of plastids per cell in arc 6 and arc 12 is similar to their respective WT strains. Results from time course of curvature studies demonstrated that the plastid mutation affected gravitropism only of inflorescence stems and hypocotyls, but not roots. Thus, roots appear to have different mechanisms of gravitropism compared to stems and hypocotyls. Time course of curvature studies with light-grown seedlings were performed in the presence of latrunculin B (Lat-B), an actin-depolymerizing drug. Lat-B promoted gravitropic curvature in hypocotyls of both the WT and arc 6 but had little or no effect on gravitropism in roots of both strains. These results suggest that F-actin is not required for hypocotyl gravitropism.     

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.     

Localization of cells containing sedimented amyloplasts in the shoots of normal and lazy rice seedlings.

We have examined the localization of the cells containing sedimented amyloplasts (putative statocytes) and its relation to the graviresponding sites in the shoots of normal and lazy rice seedlings. All graviresponsive organs of the shoots of normal rice seedlings, the mesocotyl, the coleoptile and the leaf-sheath base, were found to possess the statocytes. This is the first indication that mesocotyl senses gravity by its own cells in inducing gravitropic bending in rice seedlings. In lazy-Kamenoo, although the shoots lost their gravitropic response with the advance of age, sedimentation of amyloplasts itself might not be attributable to the agravitropic growth of the shoots, because, including those of the leaf-sheath bases that had lost their response to gravity, sedimented amyloplasts appeared to be identical to those of normal Kamenoo and of younger seedlings of lazy-Kamenoo whose gravitropism is still apparent.     

Gravitropism in the starch excess mutant of Arabidopsis thaliana

Amyloplasts are hypothesized to play a key role in the cellular mechanisms of gravity perception in plants. While previous studies have examined the effects of starch deficiency on gravitropic sensitivity, in this paper, we report on gravitropism in plants with a greater amount of starch relative to the normal wild type. Thus, we have studied the sex1 (starch excess) mutant of Arabidopsis thaliana, which accumulates extra starch because it is defective in a protein involved in the regulation of starch mobilization. Compared to the wild type (WT), sex1 seedlings contained excess starch in cotyledons, hypocotyls, the root-hypocotyl transition zone, the body of the root, root hairs, and in peripheral rootcap cells. Sedimented amyloplasts were found in both the WT and in sex1 in the rootcap columella and in the endodermis of stems, hypocotyls, and petioles. In roots, the starch content and amyloplast sedimentation in central columella cells and the gravitropic sensitivity were comparable in sex1 and the WT. However, in hypocotyls, the sex1 mutant was much more sensitive to gravity during light-grown conditions compared to the WT. This difference was correlated to a major difference in size of plastids in gravity-perceiving endodermal cells between the two genotypes (i.e., sex1 amyloplasts were twice as big). These results are consistent with the hypothesis that only very large changes in starch content relative to the WT affect gravitropic sensitivity, thus indicating that wild-type sensing is not saturated.     

Role of endodermal cell vacuoles in shoot gravitropism

In higher plants, shoots and roots show negative and positive gravitropism, respectively. Data from surgical ablation experiments and analysis of starch deficient mutants have led to the suggestion that columella cells in the root cap function as gravity perception cells. On the other hand, endodermal cells are believed to be the statocytes (that is, gravity perceiving cells) of shoots. Statocytes in shoots and roots commonly contain amyloplasts which sediment under gravity. Through genetic research with Arabidopsis shoot gravitropism mutants, sgr1/scr and sgr7/shr, it was determined that endodermal cells are essential for shoot gravitropism. Moreover, some starch biosynthesis genes and EAL1 are important for the formation and maturation of amyloplasts in shoot endodermis. Thus, amyloplasts in the shoot endodermis would function as statoliths, just as in roots. The study of the sgr2 and zig/sgr4 mutants provides new insights into the early steps of shoot gravitropism, which still remains unclear. SGR2 and ZIG/SGR4 genes encode a phospholipase-like and a v-SNARE protein, respectively. Moreover, these genes are involved in vacuolar formation or function. Thus, the vacuole must play an important role in amyloplast sedimentation because the sgr2 and zig/sgr4 mutants display abnormal amyloplast sedimentation.     

Plastid movement in statocytes of the arg1 (altered response to gravity) mutant

The ability of a plant to respond to gravity is crucial for growth and development throughout the life cycle. A key player in the cellular mechanisms of gravitropism is ARG1 (altered response to gravity), a DnaJ-like protein that associates with components of the vesicular trafficking pathway and carries a C-terminal domain with similarities to cytoskeleton-associated proteins. The arg1-2 mutant of Arabidopsis thaliana has reduced and delayed gravitropism in roots, shoots, and inflorescence stems when grown in the light or dark. We performed light microscopic studies of plastid movement in the gravity-perceiving statocytes (endodermal cells) of hypocotyls of arg1-2 and WT light-grown seedlings following reorientation to better characterize the role of ARG1 in gravitropism. Cryofixation/freeze substitution procedures were used because they provide a reliable indication of rapid cellular events within the statocytes. Our results suggest that ARG1 affects gravitropism by reducing plastid movement/sedimentation, a process known to be essential for early phases of signaling cascades in the statocytes.     

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 .     

An agravitropic mutant of Arabidopsis, endodermal-amyloplast less 1, that lacks amyloplasts in hypocotyl endodermal cell layer

We have isolated a new recessive mutant of Arabidopsis thaliana for gravitropism, endodermal-amyloplast less 1 (eal1). eal1 shows reduced gravitropism in hypocotyl, and completely lacks gravitropism in inflorescence stems; root gravitropism is not affected. Starch staining with I-KI solution reveals almost no amyloplasts in eal1 hypocotyls when grown on a sucrose-free medium, though the root columella cells contain as many amyloplasts as wild type. On a medium containing 1% sucrose, eal1 hypocotyls contain as many starch granules as those of wild type, suggesting that starch synthesis is not affected in eal1. The endodermal cell layer which is thought to function as statocytes in hypocotyls is present in eal1. These results suggest that differentiation or development of gravity-responsive amyloplasts are affected in eal1 hypocotyls.     

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.     

Time-lapse analysis of gravitropism in Ceratodon protonemata

The tip cell of the protonema of the moss Ceratodon purpureus (Hedw.) Brid. is negatively gravitropic when grown in the dark on supplemented agar. Gravitropism, plastid distribution, and plastid movement were studied in living cells using time-lapse video microscopy and infrared light. A wrong-way (downward) curvature preceded upward curvature and was detected as early as 2 minutes after reorientation. Upward curvature began 30-45 minutes after reorientation to the horizontal. Cell division temporarily reversed upward curvature, but did not inhibit wrong-way curvature. Since significant amyloplast sedimentation always occurred before the start of upward curvature, it is possible that these amyloplasts function as statoliths for upward curvature. However, no significant amyloplast sedimentation occurred before wrong-way curvature. Thus, this early phase of gravitropism cannot require plastid sedimentation for gravity sensing. Most plastids moved within and between zones, and plastid zonation was highly dynamic. Plastids moved toward the apex and toward the base of the cell at rates much slower than cytoplasmic streaming. Despite the dynamic nature of plastid movement and zonation, during upward curvature the distance between sedimented plastids and the apex stayed constant. Time-lapse analysis has revealed intriguing events not readily seen previously using destructive sampling.     

Tropic responses of hypocotyls from normal tomato plants and the gravitropic mutant Lazy-1

Abstract Etiolated hypocotyls from normal tomato plants show a negative gravitropic response within 20 min of stimulation. In contrast, etiolated hypocotyls from the gravitropic mutant Lazy-l do not reorientate after gravistimulation. Etiolated hypocotyls from both types of plant are positively phototropic, however, Lazy-l seedlings achieve a greater final angle of bending following phototropic stimulation compared to normal plants. Anatomical studies reveal that etiolated hypocotyls from normal plants contain sedimenting amyloplasts located within the endodermal cells. Such sedimenting amyloplasts are absent in Lazy-l tissue. It is hypothesized that the hypocotyl of Lazy-l is agravitropic since it is unable to perceive a gravistimulus.     

Characterization and genetic analysis of a lettuce (Lactuca sativa L.) mutant,weary, that exhibits reduced gravitropic response in hypocotyls and inflorescence stems

A lettuce (Lactuca sativa L.) mutant that exhibits a procumbent growth habit was identified and characterized. In two wild type (WT) genetic backgrounds, segregation patterns revealed that the mutant phenotype was controlled by a recessive allele at a single locus, which was designated weary. Hypocotyls and inflorescence stems of plants homozygous for the weary allele exhibited reduced gravitropic responses compared with WT plants, but roots exhibited normal gravitropism. Microscopic analysis revealed differences in the radial distribution of amyloplasts in hypocotyl and inflorescence stem cells of weary and WT plants. Amyloplasts occurred in a single layer of endodermal cells in WT hypocotyls and inflorescence stems. By contrast, amyloplasts were observed in several layers of cortical cells in weary hypocotyls, and weary inflorescence stem cells lacked amyloplasts entirely. These results are consistent with the proposed role of sedimenting amyloplasts in shoot gravitropism of higher plants. The phenotype associated with the weary mutant is similar to that described for the Arabidopsis mutant sgr1/scr, which is defective in radial patterning and gravitropism.     

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.     

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.     

Actin microfilaments in presumptive statocytes of root caps and coleoptiles

Rhodamine-phalloidin was used to determine the distribution of actin microfilament bundles (mfb) in cells thought to be the site of gravity perception (statocytes) in coleoptiles and root caps of Zea mays and Hordeum vulgare. In coleoptile cells, amyloplasts were usually observed in close proximity to thick mfb, which often appeared to divide into finer mfb adjacent to individual amyloplasts. The nucleus in these cells was surrounded by an extensive network of mfb, which were connected to thicker transvacuolar mfb. Columella cells of the root cap contained an extensive reticulum of fine mfb throughout the protoplast, but lacked the much thicker mfb seen in coleoptile cells. The distribution and extent of mfb observed in fixed cells correlates with patterns of streaming and amyloplast movement seen in living cells. A possible role for actin mfb in the perception of gravity is discussed.