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.     

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.     

Amyloplast Sedimentation in Shoot Statocytes having a Large, Central Vacuole: Further Interpretation from Electron Microscopy

The claim (Lawton, Juniper, and Hawes, 1986) that amyloplastssediment through the central vacuole of geostimulated shootstatocytes has been critically examined. As the result of ourTEM study of Taraxacum statocytes and from theoretical considerationsof amyloplast sedimentation, we conclude that it is possiblefor individual amyloplasts surrounded by a layer of tonoplast-boundedcytoplasm to travel occasionally through the vacuole, but unlikelythat the majority of the amyloplasts in a statocyte sedimentin this manner. We put forward a scheme for amyloplast movementin shoot statocytes which emphasizes the fluidity of the tonoplastmembrane. In this scheme, it is expected that most amyloplastssediment in peripheral cytoplasm down the statocyte cell wall,but amyloplasts may also, as they sediment, create or breaktransvacuolar strands, or move through already existing transvacuolarstrands, or fall through the vacuole while enclosed by somecytoplasm and tonoplast membrane. Finally, it is suggested thatthe tonoplast membrane may have been neglected as a membranesite for detection of the gravity stimulus through interactionwith sedimenting amyloplasts. Key words: Amyloplast sedimentation, statocytes, geotropism, Taraxacum officinale     

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.     

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.     

Amyloplast development in etiolated and ethylene-treated pea epicotyls

The amyloplasts found in the apical hook cells of etiolated pea (Pisum sativum L.) epicotyls were randomly distributed. Sedimentation of endodermal amyloplasts in the direction of gravity became apparent in the transition from the hook to the top of the main axis of the epicotyl. Cortical amyloplasts in this region were not, however, sedimented. These patterns of sedimentation could not be related to changes in amyloplast size, and it is proposed that cytoplasmic properties determine amyloplast behaviour.The differentiation of plastids in the hook differed between the amyloplast-containing endodermal cells and the cortical cells, in which amoeboid plastids predominated over amyloplasts. Amyloplasts disappeared from the cortical cells in the main axis of the epicotyl, but in the endodermal cells sedimented amyloplasts were found throughout the upper epicotyl.Etiolated epicotyls induced to grow horizontally by treatment with ethylene had a normal content of amyloplasts, sedimented in the direction of gravity.     

Statolith Sedimentation Kinetics and Force Transduction to the Cortical Endoplasmic Reticulum in Gravity-Sensing Arabidopsis Columella Cells

The starch statolith hypothesis of gravity sensing in plants postulates that the sedimentation of statoliths in specialized statocytes (columella cells) provides the means for converting the gravitational potential energy into a biochemical signal. We have analyzed the sedimentation kinetics of statoliths in the central S2 columella cells of Arabidopsis thaliana. The statoliths can form compact aggregates with gap sizes between statoliths approaching <30 nm. Significant intra-aggregate sliding motions of individual statoliths suggest a contribution of hydrodynamic forces to the motion of statoliths. The reorientation of the columella cells accelerates the statoliths toward the central cytoplasm within <1 s of reorientation. During the subsequent sedimentation phase, the statoliths tend to move at a distance to the cortical endoplasmic reticulum (ER) boundary and interact only transiently with the ER. Statoliths moved by laser tweezers against the ER boundary experience an elastic lift force upon release from the optical trap. High-resolution electron tomography analysis of statolith-to-ER contact sites indicate that the weight of statoliths is sufficient to locally deform the ER membranes that can potentially activate mechanosensitive ion channels. We suggest that in root columella cells, the transduction of the kinetic energy of sedimenting statoliths into a biochemical signal involves a combination of statolith-driven motion of the cytosol, statolith-induced deformation of the ER membranes, and a rapid release of kinetic energy from the ER during reorientation to activate mechanosensitive sites within the central columella cells.     

A Role for the TOC Complex in Arabidopsis Root Gravitropism

Arabidopsis (Arabidopsis thaliana) roots perceive gravity and reorient their growth accordingly. Starch-dense amyloplasts within the columella cells of the root cap are important for gravitropism, and starchless mutants such as pgm1 display an attenuated response to gravistimulation. The altered response to gravity1 (arg1) mutant is known to be involved with the early phases of gravity signal transduction. arg1 responds slowly to gravistimulation and is in a genetically distinct pathway from pgm1, as pgm1 mutants enhance the gravitropic defect of arg1. arg1 seeds were mutagenized with ethylmethane sulfonate to identify new mutants that enhance the gravitropic defect of arg1. Two modifier of arg1 mutants (mar1 and mar2) grow in random directions only when arg1 is present, do not affect phototropism, and respond like the wild type to application of phytohormones. Both have mutations affecting different components of the Translocon of Outer Membrane of Chloroplasts (TOC) complex. mar1 possesses a mutation in the TOC75-III gene; mar2 possesses a mutation in the TOC132 gene. Overexpression of TOC132 rescues the random growth phenotype of mar2 arg1 roots. Root cap amyloplasts in mar2 arg1 appear ultrastructurally normal. They saltate like the wild type and sediment at wild-type rates upon gravistimulation. These data point to a role for the plastidic TOC complex in gravity signal transduction within the statocytes.     

Gravity sensing in tip-growing cells

In addition to the statocytes of roots and shoots, a number of tip-growing cells also sense gravity, which influences the cells' growth and development. Since these tip-growing cells are highly suitable for observations in vivo, the movement and sedimentation of their statoliths can be studied in detail. Experimental manipulation by centrifugation, drug application, optical tweezers or microgravity can be monitored by light microscopy. The statoliths are localized in distinct cytoplasmic areas by interactions with actin filaments or microtubules, and their sedimentation seems to be narrowly confined. Since gravisensing and the graviresponse take place within the same cell, the gravitropic signal transduction chain is not complicated by signal transmission between sensing and responding cells. Studies on tip-growing cells have now enabled the formulation of models explaining positive and negative gravitropism.     

Mechanotransduction molecules in the plant gravisensory response: Amyloplast/statolith membranes contain a β1 integrin-like protein

Summary It has been hypothesized that the sedimentation of amyloplasts within root cap cells is the primary event in the plant gravisensory-signal transduction cascade. Statolith sedimentation, with its ability to generate weighty mechanical signals, is a legitimate means for organisms to discriminate the direction of the gravity vector. However, it has been demonstrated that starchless mutants with reduced statolith densities maintain some ability to sense gravity, calling into question the statolith sedimentation hypothesis. Here we report on the presence of a ₁ integrin-like protein localized inside amyloplasts of tobacco NT-1 suspension culture, callus cells, and whole-root caps. Two different antibodies to the ₁ integrin, one to the cytoplasmic domain and one to the extracellular domain, localize in the vicinity of the starch grains within amyloplasts of NT-1. Biochemical data reveals a 110-kDa protein immunoprecipitated from membrane fractions of NT-1 suspension culture indicating size homology to known ₁ integrin in animals. This study provides the first direct evidence for the possibility of integrin-mediated signal transduction in the perception of gravity by higher plants. An integrin-mediated pathway, initiated by starch grain sedimentation within the amyloplast, may provide the signal amplification necessary to explain the gravitropic response in starch-depleted cultivars.Abbreviations BA 6-benzylaminopurine - ETOH ethyl alcohol - LP liquid propane - LR London Resin - PBST phosphate-buffered saline with Tween - TEM transmission electron microscopy - OSM optical-sectioning microscopy     

Amyloplasts that sediment in protonemata of the moss Ceratodon purpureus are nonrandomly distributed in microgravity

Little is known about whether or how plant cells regulate the position of heavy organelles that sediment toward gravity. Dark-grown protonemata of the moss Ceratodon purpureus displays a complex plastid zonation in that only some amyloplasts sediment along the length of the tip cell. If gravity is the major force determining the position of amyloplasts that sediment, then these plastids should be randomly distributed in space. Instead, amyloplasts were clustered in the subapical region in microgravity. Cells rotated on a clinostat on earth had a roughly similar non-random plastid distribution. Subapical clusters were also found in ground controls that were inverted and kept stationary, but the distribution profile differed considerably due to amyloplast sedimentation. These findings indicate the existence of as yet unknown endogenous forces and mechanisms that influence amyloplast position and that are normally masked in stationary cells grown on earth. It is hypothesized that a microtubule-based mechanism normally compensates for g-induced drag while still allowing for regulated amyloplast sedimentation.     

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.     

Cytoplasmic streaming affects gravity-induced amyloplast sedimentation in maize coleoptiles

Living maize (Zea mays L.) coleoptile cells were observed using a horizontal microscope to determine the interaction between cytoplasmic streaming and gravity-induced amyloplast sedimentation. Sedimentation is heavily influenced by streaming which may (1) hasten or slow the velocity of amyloplast movement and (2) displace the plastid laterally or even upwards before or after sedimentation. Amyloplasts may move through transvacuolar strands or through the peripheral cytoplasm which may be divided into fine cytoplasmic strands of much smaller diameter than the plastids. The results indicate that streaming may contribute to the dynamics of graviperception by influencing amyloplast movement.     

Ionic signaling in plant responses to gravity and touch

Touch and gravity are two of the many stimuli that plants must integrate to generate an appropriate growth response. Due to the mechanical nature of both of these signals, shared signal transduction elements could well form the basis of the cross-talk between these two sensory systems. However, touch stimulation must elicit signaling events across the plasma membrane whereas gravity sensing is thought to represent transformation of an internal force, amyloplast sedimentation, to signal transduction events. In addition, factors such as turgor pressure and presence of the cell wall may also place unique constraints on these plant mechanosensory systems. Even so, the candidate signal transduction elements in both plant touch and gravity sensing, changes in Ca2+, pH and membrane potential, do mirror the known ionic basis of signaling in animal mechanosensory cells. Distinct spatial and temporal signatures of Ca2+ ions may encode information about the different mechanosignaling stimuli. Signals such as Ca2+ waves or action potentials may also rapidly transfer information perceived in one cell throughout a tissue or organ leading to the systemic reactions characteristic of plant touch and gravity responses. Longer-term growth responses are likely sustained via changes in gene expression and asymmetries in compounds such as inositol-1,4,5-triphosphate (IP3) and calmodulin. Thus, it seems likely that plant mechanoperception involves both spatial and temporal encoding of information at all levels, from the cell to the whole plant. Defining this patterning will be a critical step towards understanding how plants integrate information from multiple mechanical stimuli to an appropriate growth response.     

Statoliths and microfilaments in plant cells

Microfilaments have been demonstrated in rhizoids of Chara fragilis Desvaux by labelling of actin with rhodamine-conjugated phalloidin. Each rhizoid contains thick microfilament-bundles arranged longitudinally in the basal region. In the subapical and apical regions, much thinner bundles exist which contact the statoliths and encircle them in the form of a dense envelope. In root statocytes from Lepidium sativum L. the presence of an actin network is indicated by the fact that application of cytochalasin B (25 g·ml^-1 for 4 h) results in an approximately threefold increase in the rate of statolith (amyloplast) sedimentation relative to controls. It is concluded that in gravity-perceiving plant cells statoliths may trigger the transduction mechanism via actin filaments.Abbreviation CB cytochalasin B - ER endoplasmic reticulum - MF microfilament     

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.     

Effects of prolonged omnilateral gravistimulation on the ultrastructure of statocytes and on the graviresponse of roots

Statocytes of vertically growing roots of Lepidium sativum L. exhibit a strict polarity: The nucleus is positioned near the proximal periclinal cell wall, amyloplasts are sedimented on a complex of rough endoplasmic reticulum (ER) consisting of parallel cisternae near the distal periclinal cell wall.When 24 h old, vertically grown roots are rotated for an additional 20 h on a horizontal clinostat, this polarity is destroyed. Furthermore, the prolonged omnilateral stimulation leads to a damage of the statocytes, which in some cases ends in the self-destruction of the sensitive cells. The different components of the ultrastructural respones of the statocytes are: Displacement of the nucleus; changes in amount and distribution of the ER; loss of amyloplast starch; confluence of lipid droplets to large aggregates: a considerable increase of the lytic compartment. In addition, even anticlinal cell walls may be lysed up to small stumps. As all these effects are clearly restricted to the statocytes, only these cells are able to respond to the continuously changing direction of the gravity vector, thus perceiving gravity as such.After being exposed horizontally, the graviresponse of rotated roots is delayed as compared to the controls. About 20% of the rotated roots do not respond (curve) at all, but grow perpendicular in relation to the gravity vector. Perception of gravity is inevitably correlated with the polarity and the integrity of the statocytes.Abbreviation ER endoplasmic reticulum A preliminary report was presented at the Fall Meeting of the German Society for Cell Biology in Salzburg, Austria, September 1979 (Hensel and Sievers 1979)This paper represents part of a dissertation (D 5) of W. H.     

Root gravitropism

When a plant root is reoriented within the gravity field, it responds by initiating a curvature which eventually results in vertical growth. Gravity sensing occurs primarily in the root tip. It may involve amyloplast sedimentation in the columella cells of the root cap, or the detection of forces exerted by the mass of the protoplast on opposite sides of its cell wall. Gravisensing activates a signal transduction cascade which results in the asymmetric redistribution of auxin and apoplastic Ca²⁺ across the root tip, with accumulation at the bottom side. The resulting lateral asymmetry in Ca²⁺ and auxin concentration is probably transmitted to the elongation zone where differential cellular elongation occurs until the tip resumes vertical growth. The Cholodny-Went theory proposes that gravity-induced auxin redistribution across a gravistimulated plant organ is responsible for the gravitropic response. However, recent data indicate that the gravity-induced reorientation is more complex, involving both auxin gradient-dependent and auxin gradient-independent events.     

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.