Recovery of Geotropism after Removal of the Root Cap

Removal of the cap from the primary roots of Zea mays and Triticumaestivum renders the roots unresponsive to gravity. In bothspecies a geotropic response is recovered before a new cap hasstarted to regenerate. Immediately after decapping amyloplastsstart to develop in cells of the root apex and it is proposedthat as the development of amyloplasts continues so they becomefunctional as gravity sensors. It is also suggested that theamyloplasts may be the source of an inhibitor that has beenpostulated to be the intermediary between the perception ofgravity and the geotropic response.     

Amyloplast sedimentation kinetics in gravistimulated maize roots

Amyloplast sedimentation in gravistimulated maize (Zea mays L.) roots was measured using the change in angle from the center of the cell to each amyloplast as an index of sedimentation. Using tissue fixed after gravistimulation, the relationship between mean amyloplast angle and the duration of gravistimulation was found to be linear when plotted on a logarithmic time scale. Extrapolated values for the onset of angular change are 5.9 s after the start of gravistimulation for the entire population of amyloplasts and 11.8 s for lead amyloplasts. By multiplying the instantaneous angular velocity (in radians) by the cell center to amyloplast radius, it is possible to calculate the initial sedimentation velocity to be 19.1 m min^-1 at 5.9 s. During sedimentation, the mean amyloplast angles surpass the calculated cell corner angle of 123° at 2.2 min for all amyloplasts and at 19 s for lead amyloplasts near the new lower wall. Thus, substantial sedimentation occurs within the presentation time, calculated to be 4.1 min. These kinetics are consistent with several hypotheses of graviperception.Symbol t_p presentation time     

Amyloplasts as possible statoliths in gravitropic protonemata of the moss Ceratodon purpureus

The kinetics of gravitropism and of amyloplast sedimentation were studied in dark-grown protonemata of the moss Ceratodon purpureus (Hedw.) Brid. The protonemata grew straight up at a rate of 20–25 m·h^– in nutrient-supplemented agar. After they were oriented to the horizontal, upward curvature was first detected after 1–1.5 h and reached 84° by 24 h. The tip cells exhibited an amyloplast zonation, with a tip cluster of nonsedimenting amyloplasts, an amyloplast-free zone, and a zone with pronounced amyloplast sedimentation. This latter zone appears specialized more for lateral than for axial sedimentation since amyloplasts sediment to the lower wall in horizontal protonemata but do not fall to the basal wall in vertical protonemata. Amyloplast sedimentation started within 15 min of gravistimulation; this is within the 12–17-min presentation time. The data support the hypothesis that some amyloplasts function as statoliths in these cells.This work was supported by the National Aeronautics and Space Administration grant NAGW-780. We thank Professor E. Hartmann and J. Schwuchow for providing Ceratodon cultures, Dr. John Z. Kiss and Jeff Young for valuable discussions, and Professor Rainer Hertel (University of Freiburg, FRG) for bringing this material to our attention.     

Contribution of root cap mucilage and presence of an intact root cap in maize (Zea mays) to the reduction of soil mechanical impedance

BACKGROUND AND AIMS: The impedance to root growth imposed by soil can be decreased by both mucilage secretion and the sloughing of border cells from the root cap. The aim of this study is to quantify the contribution of these two factors for maize root growth in compact soil. METHODS: These effects were evaluated by assessing growth after removing both mucilage (treatment I -- intact) and the root cap (treatment D -- decapped) from the root tip, and then by adding back 2 micro L of mucilage to both intact (treatment IM -- intact plus mucilage) and decapped (treatment DM -- decapped plus mucilage) roots. Roots were grown in either loose (0.9 Mg m(-3)) or compact (1.5 Mg m(-3)) loamy sand soils. Also examined were the effects of decapping on root penetration resistance at three soil bulk densities (1.3, 1.4 and 1.5 Mg m(-3)). KEY RESULTS: In treatment I, mucilage was visible 12 h after transplanting to the compact soil. The decapping and mucilage treatments affected neither the root elongation nor the root widening rates when the plants were grown in loose soil for 12 h. Root growth pressures of seminal axes in D, DM, I and IM treatments were 0.328, 0.288, 0.272 and 0.222 MPa, respectively, when the roots were grown in compact soil (1.5 Mg m(-3) density; 1.59 MPa penetrometer resistance). CONCLUSIONS: The contributions of mucilage and presence of the intact root cap without mucilage to the lubricating effect of root cap (percentage decrease in root penetration resistance caused by decapping) were 43 % and 58 %, respectively. The lubricating effect of the root cap was about 30 % and unaffected by the degree of soil compaction (for penetrometer resistances of 0.52, 1.20 and 1.59 MPa).     

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     

Kinetics of amyloplast sedimentation in gravistimulated maize coleoptiles

Inner mesophyll cells from coleoptiles of Zea mays L. cv. Merit were fixed after varying periods of gravistimulation. A statistically significant amount (17–21%) of amyloplast sedimentation occurred in these cells after 30 s of gravistimulation. The presentation time is approx. 40 s or less. The accumulation of amyloplasts near the new lower wall shows a linear relationship to the logarithm of the gravistimulation time (r=0.92 or higher). The intercept of this line with the baseline value of amyloplasts in vertical coleoptiles indicates that the number of amyloplasts on the new lower wall begins increasing 11–15 s after the onset of gravistimulation. Direct observations of living cells confirm that many amyloplasts sediment within less than 15–30 s. These rapid kinetics are consistent with the classical statolith hypothesis of graviperception involving the sedimentation of amyloplasts to the vicinity of the new lower wall.     

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.     

The Optimum Angle of Geotropic Stimulation and its Relation to the Starch Statolith Hypothesis

Roots which are turned from their normal direction to directions at various angles with the plumb line develop the largest geotropic curvatures during a subsequent klinostat rotation period when the stimulation angle is well above the horizontal. In experiments with roots of Lepidium sativum L., the optimum is located at 120 to 140° when the stimulation time is between 2 and 15 min. If this fact is to be explained by the movements of amyloplasts in the root cap cells, one would expect roots which bad been kept inverted before the stimulation (so that the moveable amyloplasts are accumulated in the opposite end of the cells) to show an optimum angle well below 90°. — Pre-inversion of the roots did suppress the curvatures produced by stimulation at angles larger than 90° when measured after 10 to 30 min of klinostat rotation. This suppression may be taken as a support for the starch statolith hypothesis, since the amyloplasts in pre-inverted roots placed at angles exceeding 90° have a restricted opportunity to slide along the cell walls compared to non-inverted roots placed at the same angles. In pre-inverted roots measured after a period of klinostat rotation, however, no optimum was found at angles below 90°. When the stimulation time was 3.75 min, the response curves were nearly symmetrical about 90°. Stimulation for 15 min, on the other hand, resulted in curvatures which were much larger (although suppressed in comparison with non-inverted roots) when the stimulation angle was 165° than when it was 15°. During the 15 min stimulation period itself, however, pre-inverted roots curved 0.3° when stimulated at 15, but only 3.4° at 165°. This small difference was very highly significant and is in agreement with the starch statolith hypothesis insofar as the amyloplasts in pre-inverted roots placed at 15° have the greatest opportunity to slide along the cell walls. The lack of further development (and the actual decrease) of their curvatures during the subsequent klinostat rotation must then be due to other, depressing, factors, summarily designated as tonic. At angles above 90°, the tonic factors are either absent or even enhancing. Tbe tonic effects cannot be explained by amyloplast movements.     

Movement of Amyloplasts in the Statocytes of Geotropically Stimulated Roots. The Pre-Inversion Effect

Previously inverted Lepidium roots were placed in a horizontal position and the amyloplasts in the statocytes of the root cap allowed to fall through their entire range of movement across the cell. Under these conditions the amyloplasts first follow a mainly downward course for 6 to 8 min at a speed between 0.5 and 0.8 μm per min. For the next 10 min they move slightly more slowly in a direction away from the apical end of the cell, still sinking somewhat, but without reaching the plasmalemma along the lower wall. Previous experiments have shown that conditions assumed to allow the amyloplasts to slide parallel to the longitudinal cell walls are those that give rise to the largest geotropic curvatures. Such conditions are for instance (1) stimulation at 135° (root tips pointing obliquely upward) and (2) inversion of roots for 16 min followed by stimulation at 45°. Treatments assumed not to permit extensive sliding of the amyloplasts produce smaller geotropic curvatures, namely (3) stimulation at 45° without pre-inversion and (4) inversion followed by stimulation at 135°. The location of the amyloplasts after these four kinds of treatment has now been determined on photomicrographs and the assumptions concerning the paths and extent of sliding of the amyloplasts confirmed. Observations on electron micrographs showed that under all conditions the amyloplasts are separated from the plasmalemma by other organelles, such as ER, nucleus or vacuoles. In roots rotated for 15 min parallel to the horizontal axis of the klinostat at 2 rpm, the amyloplasts are not clumped together as densely as in normal, inverted or stimulated roots, but they are not scattered over the entire cell volume. The statolith function of the amyloplasts is discussed in view of these and other observations.     

Induction of gravity-dependent plasmatic responses in root statocytes by short time contact between amyloplasts and the distal endoplasmic reticulum complex

Statocytes from roots of Lepidium sativum L., which developed after a 2-min soaking on a horizontal clinostat (2 rotations per min) for 44 h, exhibit the same polarity as in vertically grown roots, as indicated by a complex of endoplasmic reticulum (ER) cisternae at the distal cell pole. Amyloplasts are distributed randomly. The kinetics of graviresponse (=curvature) of such roots are identical to those of normally grown roots. Ten-minute exposure of the root, after 24 h development on the clinostat with gravity acting towards the root's basis (inversion), induces no changes in statocyte ultrastructure. However, corresponding exposure in normal orientation leads to subsequent disintegration of the distal ER complex, loss of amyloplast starch, confluence of lipid droplets, and an increase of the lytic compartment. These ultrastructural events thus appear to be induced by a physical contact — however short — between amyloplasts and the distal ER complex.Abbreviation ER endoplasmic reticulum Dedicated to Professor Dr. W. Haupt on the occasion of his 60th birthday     

Some aspects of geotropism in coleoptiles

Summary Auxin transport was studied in coleoptile sections that were stimulated geotropically. The early time course of auxin-transport asymmetry was measured. An initial phase in which more IAA was delivered into the receptor for the upper half was found after 5 min of horizontal exposure. After about 15 min this was followed by the expected known asymmetry in which more auxin flows in the lower side of the coleoptile. Upon return of the coleoptile to a vertical position, this asymmetry disappeared within 30 min.Earlier correlations of geosensitivity of the auxin transport system with sedimentation of amyloplasts in comparisons of wild type corn and an amylomaize mutant were confirmed and extended. It was also shown that, in contrast to the geotropic effect, phototropically induced lateral auxin asymmetry was not significantly different in wild type and amylomaize. Eleven other single-gene endosperm starch mutants of corn were compared to their corresponding normals. In all pairs, if a difference in geosensitivity of lateral auxin transport was present, it was correlated with a parallel difference in amyloplast sedimentation: e.g., sugary 1 (67) had an amyloplast asymmetry index of 0.32 and a 13% gravity effect on auxin transport; the paired wild-type had both a greater amyloplast asymmetry (0.61) and a greater gravity effect on transport (23%).Correlations between gravity effects on auxin transport and amyloplasts were also shown in comparisons of apical and basal sections of corn, oat and Sorghum coleoptiles.Further results, confirming the increased effect of centrifugal acceleration greater than 1xg on lateral auxin transport and on curvature, are in agreement with the hypothesis that the pressure exerted by amyloplasts, acting as statoliths, locally stimulates the auxin transport system in the individual cells.with participation by Charles steele and Vicky fan     

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 .     

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.     

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.     

Geoperception in the lentil root cap

Previous analysis showed that, in its initial phase, the geotropic response of Lens culinaris L. roots cannot be explained by a simple action by sliding, pressure or contact of amyloplasts on a sensitive surface located along the longitudinal wall. In this study another mode of action is tested by considering the following parameters as functions of the roots inclination: (1) the distance (d) which the amyloplasts move; (2) their number of contacts (mean c) with parietal cytoplasm; (3) the variable (sin alpha) of the transversal component of the statolith weight (mean M x g sin alpha). It is shown that the initial rate of curvature (mean V), at the various angles, is related to the sedimentation of the amyloplasts by the equation mean V = a log b mean d mean c sin alpha (where a and b are constants). The results obtained prove that the geotropic stimulation is dependent upon the sine of the angle (alpha) of the root inclination and explain the sine rule deviation. The role of statoliths is discussed in the light of recent literature on growth inhibitors which are involved in the geotropic reaction.     

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     

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.     

Abscisic acid and the response of the roots of Zea mays L. seedlings to gravity

Summary Exogeneous application of abscisic acid (ABA) to intact roots of LG 11 maize seedlings inhibits root elongation and induces bending of the root in response to gravity in darkness, even though the roots of these seedlings are not normally positively geotropic in the dark. ABA cannot, however, induce geotropic curvature in dark-exposed decapped roots, thus confirming that the root cap is the site of graviperception in the intact root.Abbreviation ABA abscissic acid     

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.     

Salt-induced Changes in the Distribution of Amyloplasts in the Root Cap of Excised Pea Roots in Culture

The effects of 120 mM NaCl on the anatomy and ultrastructureof the root tip of cultured excised pea roots was investigatedafter 24 h exposure to salinity. In the meristematic cells mitochondrialdamage was apparent and these cells showed increased vacuolation.The root cap was already severely affected after 24 h exposureto salinity and clumping of the cap amyloplasts around the cellnuclei was apparent. The possibility that salinity may affectroot gravitropic responses is discussed. Pisum sativum L. cv. Alaska, salinity, roots, root culture, amyloplasts, ultrastructure