JXB Advance Access originally published online on August 1, 2005
Journal of Experimental Botany 2005 56(419):2539-2550; doi:10.1093/jxb/eri248
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
Disruption of the F-actin cytoskeleton limits statolith movement in Arabidopsis hypocotyls
Department of Botany, Miami University, Oxford, OH 45056, USA
* To whom correspondence should be addressed. Fax: +1 513 529 4243. E-mail: kissjz{at}muohio.edu
Received 14 February 2005; Accepted 17 June 2005
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionSummaryReferences |
|---|
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.
Key words: Amyloplast, Arabidopsis, cytoskeleton, F-actin, gravitropism, hypocotyl, latrunculin B, shoot, statolith, stem
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
|---|
Plants sense gravity and maintain the orientation of their organs within the gravitational field through the process of gravitropism. Although gravity acts equally upon all plant cells, gravity sensing occurs in specialized cells known as statocytes, which comprise the columella cells of root tips and the endodermal cells of stems and stem-like organs. These statocytes contain starch-filled amyloplasts, or statoliths, which settle in response to gravity (for review, see Sack, 1991
). The starch–statolith model suggests that gravity is sensed when the statoliths settle within the statocyte in response to changes in the orientation of a plant organ (for review, see Sack, 1997; Kiss, 2000). The mechanical stimulus of intracellular amyloplast redistribution is transduced into a biochemical signal that triggers a differential auxin gradient across the plant organ (Muday and Murphy, 2002; Blancaflor and Masson, 2003; Perbal and Driss-Ecole, 2003). Gravitropism culminates with auxin-mediated curvature of the plant organ toward (roots) or against (stems) the direction of gravitational acceleration.
Exactly how amyloplast sedimentation ultimately elicits differential growth is unknown, but protons seem to mediate the process. Gravicurvature is dependent upon a transient alkalinization of the cytosol and concomitant acidification of the apoplast. The ion flux occurs within seconds after gravistimulation and establishes pH gradients across roots (Scott and Allen, 1999; Fasano et al., 2001) and maize pulvini (Johannes et al., 2001). Inhibition of vacuolar H+-ATPases with bafilomycin A1 enhances root gravicurvature, suggesting a role for the vacuole in this process (Scott and Allen, 1999). Another possible intracellular source for the protons is the endoplasmic reticulum (ER), which might play a significant role in gravitropism of columella cells because the periphery of tobacco columella cells contains a dense ER that may sequester ions and release them in response to amyloplast bombardment or actin-induced tensegrity forces (Yoder et al., 2001; Zheng and Staehelin, 2001). This specialized form of ER has not been observed in stems.
Several studies suggest a more prominent role for the vacuole in the gravitropism of stems compared with roots, because root columella cells have multiple small vacuoles and shoot endodermal cells possess very large central vacuoles that are more likely to restrict the free fall of gravistimulated amyloplasts. In addition, mutants defective in vacuolar formation (Kato et al., 2002a) transport to and from the vacuole (Morita et al., 2002; Yano et al., 2003), and endocytosis (Silady et al., 2004) display impaired amyloplast sedimentation and diminished shoot (but not root) gravitropism. For instance, amyloplasts in inflorescences of agravitropic zig/sgr4 mutants are not enveloped in the tonoplast, are excluded from transvacuolar strands, and do not sediment with gravity. This suggests that intimate amyloplast/tonoplast interactions and/or sedimentation through the central vacuole may be necessary for gravity signal transduction in stems.
The cytoskeleton has also been implicated in the mechanisms of gravitropism. While studies in microgravity indicate that the actomyosin system can reposition statoliths (Hodick et al., 1998; Volkmann et al., 1999; Driss-Ecole et al., 2000; Braun et al., 2002), the exact nature of cytoskeletal involvement in gravity perception remains unknown. The tensegrity model (Ingber, 1993; Ingber et al., 1994; Yoder et al., 2001) envisions a network of actin filaments that forms a mesh through the interior of the statocyte. The amyloplasts presumably percolate through this restraining mesh as they traverse the cell (Yoder et al., 2001), and the gravity signal is transduced when forces of slack and tension in the impacted tensegrity network are related via F-actin to stretch-activated channels that may reside in the plasma membrane (Caspar and Pickard, 1989; Perbal and Driss-Ecole, 2003). The actin tether model (Balu
ka and Hasenstein, 1997) is similar, but suggests that the amyloplasts do not settle passively through the actin network; rather, they are physically tethered to the F-actin.
F-actin disrupting drugs such as cytochalasins and latrunculins have been used to elucidate the role of actin in gravitropism. Gravitropism experiments employing these pharmacological agents often exhibit conflicting results, depending upon the plant species utilized, the organ studied, the drug dosage, and the experimental conditions. Some of these results may reflect subtle differences in the gravitropic mechanisms amongst the various plant organs, as well as variation among species. For instance, while Lat-B inhibits gravicurvature of the snapdragon inflorescence stems (Friedman et al., 2003), it enhances gravicurvature of Arabidopsis inflorescence stems, hypocotyls (Yamamoto and Kiss, 2002), and roots (Hou et al., 2004): however, the concentration of Lat-B sufficient to cause these responses varies by orders of magnitude amongst these organs.
Nevertheless, the presence of organ-specific gravitropism mutants as well as the differential Lat-B effects in shoots versus roots (in Arabidopsis with identical experimental conditions; see Yamamoto and Kiss, 2002) suggest that the gravitropic mechanisms exhibit differences between these organs. This study presents novel evidence that there are fundamental differences in the gravity perception mechanisms of hypocotyls as compared to roots of Arabidopsis. The precise kinetics of amyloplast sedimentation were analysed in gravistimulated Arabidopsis hypocotyls preserved by cryofixation, and it is reported here that amyloplasts in endodermal cells of Arabidopsis hypocotyls settle rapidly (within 5 min following reorientation) relative to roots. Furthermore, whereas amyloplasts in gravistimulated Arabidopsis roots settle quicker after cytoskeletal disruption, it was found that amyloplast movement in gravistimulated Arabidopsis hypocotyls is severely limited when the F-actin cytoskeleton is depolymerized with Lat-B. This decreased amyloplast mobility concomitant with cytoskeletal disruption is consistent with an active, actin-mediated mechanism of amyloplast transport within the statocytes of stem-like organs.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
|---|
Plant material and culture conditions
Wild-type (WT) seeds of Arabidopsis thaliana (L.) Heynh. (geographic race Wassilewskija, WS) were surface-sterilized with a 30% (w/w) bleach solution containing 0.1% (v/v) Triton X-100. After 3–5 rinses with distilled water, the seeds were sown onto sterilized nitrocellulose film that was placed on top of nutrient agar [1.2% agar (w/w) at pH 5.5, containing half-strength Murashige and Skoog (MS) solution, 1% (w/v) sucrose] in vertically oriented square Petri dishes. The seedlings were placed under white light (
100 µmol m–2 s–1) for 24 h, and then into darkness for 2.5 d at approximately 22 °C.
Treatment with Lat-B
A 2.0 mM stock solution of latrunculin B (Calbiochem, La Jolla, CA) was prepared in DMSO. Agar containing 2.0 µM Lat-B was prepared immediately prior to use by adding the necessary amount of Lat-B stock solution to sterile, 55 °C liquid nutrient agar [1.2% agar (w/w) at pH 7.3, containing half-strength MS solution and 1% (w/v) sucrose] with continuous stirring. The agar was then poured into square Petri dishes and allowed to solidify. The pH was adjusted to 7.3 in order to maintain the stability of the Lat-B (Yamamoto et al., 2002). For control plates, the pH was also adjusted to 7.3, and an appropriate amount of DMSO (without Lat-B) was added.
When seedlings were 3.5-d-old, they were either transferred to plates containing Lat-B, or to control plates, as described above. This was accomplished by moving the nitrocellulose film containing the seedlings onto the agar surface of the new Petri dish. The seedlings were then inspected to determine if they were vertically oriented and if both the cotyledons and the hypocotyls touched the agar. Blunt tweezers (no. 2A) were used to move all seedlings that lay flat on the agar and were oriented within approximately 20° from the vertical into a directly vertical orientation, and the rest of the seedlings were discarded. After the transfer, the square Petri dishes were sealed with Parafilm®, individually wrapped in aluminium foil, and placed in the dark. Each transfer took approximately 20 s, and each plate was wrapped and placed back in a vertical position within about 1 min. The transfer, screening, and wrapping were all conducted in a dark room under dim green safelights (0.8 µmol m–2 s–1). To allow the seedlings to recover from the transfer, and also to give the Lat-B time to permeate the tissues, the plates were incubated for 2 h in a vertical position in the dark before cryofixation was conducted.
Time-course of gravitropism and growth experiments
Gravitropism experiments were performed as described by Yamamoto and Kiss (2002), except that only controls and 2.0 µM Lat-B concentrations were used. Briefly, when seedlings were 3.5-d-old, they were transferred from the pH 5.5 agar to agar at pH 7.3 containing either 2.0 µM Lat-B or an appropriate amount of DMSO (control). This was accomplished by moving the nitrocellulose film containing the seedlings onto the agar surface of the new Petri dish as described above. The Petri plates were rotated 90°, and photographs were taken under a dim green light at 0, 4, 8, 12, and 24 h using a 35-mm camera equipped with a macro lens and Technical Pan film (ISO 50). Each experiment was performed in triplicate, and values are reported as the mean ±SE. Digital images were captured from the film using Photoshop (version 4.0; Adobe, San Jose, CA), and image analysis was performed using Image Pro Plus (Media Cybernetics, Silver Spring, MD).
Cryofixation experiments
Each Petri dish was reoriented 90° with respect to gravity. Specimens were cryofixed at 0, 0.5, 1, 2.5, 5, and 10 min following reorientation by removing the seedlings from the surface of the agar with no. 2A tweezers and, while maintaining their orientation, plunging them into liquid propane that was cooled by liquid nitrogen (Kiss and Staehelin, 1995). Only the seedlings with cotyledons positioned directly above the hypocotyl after reorientation were chosen for cryofixation. This standardized the specimen rotation and allowed the direction of the new gravity vector to be determined once the specimens were in blocks, since the cotyledons indicate the new ‘up’ direction in all cryofixed specimens. A vertical control was performed as well, and approximately 12 plants were cryofixed for each time point. Three replicates were performed for each of the experimental treatments (control versus Lat-B). The vitrified specimens were placed into cryovials containing a freeze-substitution medium composed of 1% (w/v) osmium tetroxide in 100% glass-distilled acetone. Freeze substitution (Kiss et al., 1990; Kiss and McDonald, 1993) was performed at –78.5 °C by immersing the cryovials into a dry-ice/acetone bath and then storing the bath and vials in a –80 °C freezer for a minimum of 6 d.
After freeze substitution, the specimens were allowed to warm to room temperature by moving them, in 2 h increments, from the –80 °C freezer to a –20 °C freezer, a 4 °C refrigerator, and finally to room temperature. The acetone was exchanged with 100% ethanol in acetone: ethanol gradients of 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, and 1:4 for 30 min each, followed by two-100% ethanol changes at 1 h each. Specimens were infiltrated with LR White medium grade resin in the following LR White: ethanol increments: 1:3, 1:2, 1:1, 2:1, and 3:1 for 8 h, 16 h, and the rest at 24 h, respectively. Specimens underwent a 100% resin change for 24 h and a second 100% resin change for 2 h before embedding, and were placed on a rotary mixer at 3 rpm during infiltration. Embedding chambers were constructed by attaching gaskets made from 0.015-inch thick polycarbonate film onto glass microscope slides with silicone sealant. The sealant was allowed to cure for 24 h at room temperature before the embedding moulds were used. Specimens were placed in the chamber with fresh LR White resin, and the chambers were covered with 7.8 ml (0.20 mm) Aclar® strips to protect the resin from oxygen. The slides were placed in a 60 °C oven and polymerized for 12–16 h. Specimens were removed from the slides with a razor blade and mounted onto blank resin blocks using epoxy glue. Specimens were cut into 1 mm serial sections using either a glass (6.4 mm thickness) knife or a diamond knife (Histo Jumbo, Diatome). Sections were stained with toluidine blue [0.5% (w/v) in 0.1% (w/v) Na2CO3].
Light microscopy and image analysis
Plastid positions were visualized via brightfield light microscopy on Olympus BH2, Olympus AX70, or Nikon Labophot compound microscopes. Images were captured using either a Kodak DC760 or a Nikon 104 CCD camera, and Image Pro Plus software (Version 5.1; Media Cybernetics, Silver Spring, MD) was used for the analysis. Four endodermal cells were analysed per plant. The endodermal cells were recognized from serial sections based on their relative size and morphology, their proximity to the vascular tissue, and the presence of amyloplasts.
Individual starch grains were used as the base unit of analysis for these experiments (as opposed to analysing the positions of entire amyloplasts). Although amyloplasts can contain many starch grains, a minimum of one starch grain and a maximum of all the starch grains may be visible in any given histological section through an amyloplast. Therefore, it is not possible to examine a histological section and distinguish between one amyloplast containing two visible starch grains, and two closely placed amyloplasts containing one visible starch grain each. It is possible, however, to distinguish starch grains from each other and from other structures that stain with toluidine blue and are visible in the endodermal cell at the light microscopy level (such as the nucleus). Furthermore, because sections were chosen randomly for analysis, there is an equal probability for any number of starch grains to be visible in any given amyloplast. Therefore, the mean position of the starch grains should be an indication of the mean amyloplast position.
Only one photograph from any given endodermal cell was analysed to ensure that no starch grain was counted twice, and only cells directly beneath the hook and across from the cotyledons that also possessed a width-to-length ratio between 1:4 and 1:6 were chosen for analysis. For uniformity, each image was rotated to match the orientation of the plant during fixation—with the long axis of the hypocotyl aligned horizontally and the hook and cotyledons positioned at the upper right of the hypocotyl.
The following information was obtained: cell centroid, starch grain centroid, original gravity vector when the plant was vertically oriented, new gravity vector after reorientation, lower left cell corner, distance from cell centroid to starch grain centroid, distance from cell centroid to cell corner, distance from starch grain centroid to original cell bottom, distance from starch grain centroid to new lower cell wall, average cell length, average cell width, angle that each starch grain made with respect to the gravity vector, angle that each starch grain made with respect to the cell corner, and the fractional cell distance of starch grain from the original cell bottom (vertical specimen) and the new cell bottom (reoriented specimen; Fig. 1).
|
The direction of gravitational acceleration was set at 180° when the plants were vertically oriented (original gravity vector), and at 270° after the specimens were rotated 90°. The line connecting the cell centroid and the lower distal corner of the cell represents the boundary that an amyloplast must pass before it can settle onto the new lower cell wall following 90° reorientation of the seedling. The angle that each plastid made with the cell corner was determined to be negative if the plastid was positioned above the line connecting cell centroid and cell corner, and positive if the plastid resided beneath this line (Fig. 1). Amyloplast distance from the original cell bottom (vertical specimen) and new cell bottom (reoriented specimen) was transformed into a relative (or fractional) cell distance by dividing these distance measurements by the average cell length and width, respectively.
Statistical analyses
The cryofixation experiments were performed in triplicate for each experimental condition (control versus Lat-B). Four plants were analysed from each of the seven time intervals (vertical, 0, 0.5, 1, 2.5, 5, and 10 min following reorientation). Thus, a total of 168 plants were analysed (7 time pointsx4 plants per time pointx2 experimental treatmentsx3 replicates per treatment=168 plants). Four endodermal cells were analysed per plant. One section was analysed per endodermal cell. The endodermal cells contained an average of 2.75 starch grains visible per section. Starch grain positions are reported as the mean ±standard error (SE).
Statistical significance was determined using a mixed model analysis of variance with planned post hoc comparisons. The analysis was performed using a PROC MIXED procedure with SAS for Windows software (version 8.1, SAS Institute, Cary, NC). Post hoc comparisons consisted of determining whether or not a plateau was reached in the data. This was determined by comparing the mean of each successive data point to the collective mean of the remaining data points. When the difference between these two means became not significant (P >0.05), a plateau was reached.
Another test looked at the effect of time on the interaction of the two treatments. That is, if the general behaviour of the curves differed significantly from one time point to the next adjacent time point. This test addressed the behaviour of the amyloplasts, such as the direction of their movement, as opposed to the magnitude of the movement in any direction. Tests of Effects Slices were used to analyse each treatment independently from each other, looking at time as the only factor. This was accomplished by comparing the mean of each time point to the mean of each other time point to determine whether there was a significant time effect for that treatment overall. Lastly, the Least Squares Means Test of Ho: µ=0, versus HA: µ
0 was used to determine if and when the mean amyloplast position became zero. This parameter was used to determine if and when the amyloplasts round the cell corner.
F-actin labelling and confocal microscopy
Fluorescence staining and visualization of F-actin by confocal microscopy were performed as described by Yamamoto and Kiss (2002). Briefly, 3.5-d-old seedlings were dissected longitudinally using a scalpel, immersed in PME buffer (50 mm 1,4-piperazinediethanesulphonic acid [PIPES], 4 mm MgSO4, 10 mm EGTA) buffer (pH 6.9) containing 300 µM MBS (3-maleimidobenzoyl-N-hydroxy-succinimide ester) for 30 min, then incubated for 15–20 min in PME buffer containing 0.1 µM Alexa Fluor 488 phalloidin (Molecular Probes, Eugene, OR), 0.3 M mannitol, and 2% (v/v) glycerol. The samples were washed with PME buffer and mounted on slides directly prior to confocal microscopy analysis with a Nikon PCM-2000 confocal laser scanning microscope. Images were captured using a x40 objective (numerical aperture=0.75), and a 50 µm pinhole. Between 5 and 10 scans were performed and then averaged for each image. The Alexa Fluor 488 was excited with an argon laser at 488 nm, and emission from 500–530 nm was collected. Images were processed using Corel Photo Paint (version 8; Corel Corporation, Ottawa, Ontario, Canada).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
|---|
Latrunculin B enhances gravitropic curvature in etiolated Arabidopsis hypocotyls
Time-course of curvature experiments demonstrate that Lat-B enhances gravicurvature of etiolated Arabidopsis hypocotyls (Fig. 2). Growth rate for control specimens was 0.090±0.008 mm h–1 (n=108), whereas the growth rate for specimens exposed to 2 µM Lat-B was reduced to 0.019±0.002 mm h–1 (n=108). In spite of this growth reduction, curvature at 24 h after reorientation increased from 44.2±4.8° (mean ±SE) in controls to 92.9±5.7° (mean ±SE) in Lat-B-treated specimens (Fig. 2). Thus, although exposure to 2.0 µM Lat-B greatly reduced growth, a 2-fold increase in curvature was observed, compared with controls. These results confirm the more detailed studies of Lat-B effects on gravitropism, including dose–response experiments, reported in Yamamoto and Kiss (2002)
.
|
Cryofixation is necessary to effectively preserve plastid position in graviperceptive endodermal cells
In this paper, cryofixation was used to fix amyloplast positions in etiolated Arabidopsis hypocotyls. In previous studies, chemical fixation has been used to immobilize plastids in gravistimulated root tissues (Sack et al., 1985
; MacCleery and Kiss, 1999). In contrast to plastids in columella cells of Arabidopsis roots, amyloplasts in the endodermal cells of Arabidopsis hypocotyls settle too rapidly for conventional chemical fixation to preserve their positions adequately (Fig. 3). Amyloplasts were settled at the bottom of endodermal cells when seedlings were conventionally fixed in a vertical orientation (Fig. 3A). However, immediately following reorientation, the plastids were no longer in the distal portion of the endodermal cell when specimens were immersed in chemical fixatives immediately following reorientation (Fig. 3B), suggesting that chemical fixatives do not penetrate the tissues of stem-like organs quickly enough to capture the early stages of amyloplast sedimentation in endodermal cells.
|
Because of these observations, cryofixation was employed to preserve plastid position in endodermal cells since this method has the potential to arrest plastid movement on the order of milliseconds (Kiss and Staehelin, 1995
). Amyloplasts in the hypocotyls of vertical seedlings were settled at the cell bottom when these specimens were prepared by cryofixation (Fig. 3C). In contrast to plastids in chemically fixed specimens, the plastids in cryofixed plants appeared to remain static when specimens were cryofixed directly following reorientation (Fig. 3D), and the mean plastid positions in these specimens did not differ significantly from vertical controls (P <0.05). These results underscore the utility of cryofixation as an adequate method for instantaneously capturing amyloplast positions in endodermal cells of gravistimulated Arabidopsis hypocotyls.
Utilizing cryofixation, it was determined that amyloplasts in gravistimulated Arabidopsis hypocotyls begin settling toward the new cell bottom within 30 s, round the endodermal cell corner within 1 min, and are effectively settled into the lower half of the endodermal cell by 5 min following reorientation of the seedling. On the other hand, amyloplasts in Arabidopsis roots settle more slowly, arriving at the columella cell corner at 5 min following reorientation (MacCleery and Kiss, 1999). This increased rate of settling in shoots compared with roots has been reported previously for maize (Sack et al., 1986). Indeed, these findings add to the mounting evidence suggesting that the cellular mechanisms of gravitropism differ between roots and shoots.
Plastids in endodermal cells do not exhibit significant movement following Lat-B treatment
In order to get a more global perspective on plastid positions following seedling reorientation, qualitative observations of endodermal cells were made in whole mounts of Arabidopsis seedlings at various intervals following reorientation. Immediately after reorientation of the seedlings, amyloplasts present in the endodermal cells of the hypocotyls remained in their original positions near the distal cell wall (0 min; Fig. 4A). At 1 min following reorientation, amyloplast positions had changed within the endodermal cells (Fig. 4B), and by 5 min after reorientation, the plastids were settled to the new cell bottom (Fig. 4C). By contrast, after depolymerization of the F-actin cytoskeleton with Lat-B, amyloplast sedimentation kinetics were dramatically altered compared with control specimens. Whereas the plastids were at their original position near the distal cell wall immediately after reorientation (0 min; Fig. 4D), they did not appear to move from their original positions after 1 min (Fig. 4E), 5 min (Fig. 4F), or 10 min (data not shown) following reorientation.
|
Angular measurements confirm the limited movement of plastids following Lat-B treatment
In addition to the qualitative observations described above (Fig. 4), quantitative measurements of plastid positions in endodermal cells were made according to several parameters. The first parameter, the radial movement of the plastids around the endodermal cells, was quantified using angular measurements according to MacCleery and Kiss (1999)
, because angular data more accurately represent plastid movement in two dimensions, compared with linear sedimentation data. For control and Lat-B-treated specimens, the amyloplasts were positioned initially, as expected, along the original cell bottom (see Fig. 1 and Materials and methods) at mean angles of approximately 180° (Fig. 5). In the control specimens, the angle reached 247.1±7.5° (mean ±SE) at 10 min after reorientation. By contrast, amyloplasts in endodermal cells treated with Lat-B ended at a mean angle of 180.2±0.52°, which is not significantly different (P >0.05) from their initial angle prior to reorientation. Thus, the radial movement of amyloplasts around the cell was significantly inhibited in seedlings with disrupted F-actin.
|
Because amyloplasts with diminished radial mobility may still settle to the new cell bottom, the angle was also measured that the amyloplasts made with respect to the one cell corner, which they must pass if they reach the new cell bottom following reorientation of the seedling (see Fig. 1 and ‘Materials and Methods’). Amyloplasts in endodermal cells of both control and Lat-B-treated specimens were initially positioned above the corner (Fig. 6). For control specimens, the mean angle became positive between 0.5 min and 1 min after reorientation, and continued to increase until it reached 59.2±6.9° after 10 min. After cytoskeletal disruption, the mean plastid angle with respect to the cell corner did not change significantly (P >0.05) within 10 min following reorientation (Fig. 6). Thus, depolymerization of the F-actin cytoskeleton with Lat-B prevents amyloplasts from settling past the endodermal cell corner and onto the new cell bottom of reoriented Arabidopsis hypocotyls.
|
Linear distance that plastids move following reorientation is also limited following disruption of F-actin cytoskeleton with Lat-B
In addition to determining the rate of amyloplast settling (angular measurements), it is also important to determine the extent to which the amyloplasts settle because previous studies have shown that increased amyloplast sedimentation is correlated with increased gravity response (reviewed in Kiss, 2000
). To this end, linear measurements were obtained because angular measurements do not present an accurate gauge of the distance that plastids cover as they traverse the cell. Relative amyloplast distance from the original cell bottom was measured in order to determine how far the plastids move from their original positions along the distal end of the cell (see Fig. 1 and Materials and methods). The plastids began at a relative cell distance of 0.124±0.009 (mean ±SE) in the control specimens and at 0.063±0.004 in the Lat-B-treated specimens (Fig. 7). However, the relative distance from the original cell bottom increased to 0.435±0.026 for control specimens, yet did not change significantly (0.057±0.003, P >0.05) for the Lat-B-treated ones (Fig. 7). These measurements indicate that amyloplasts were settled near the original cell bottom (in vertically oriented specimens), and, after reorientation, amyloplasts in control specimens moved away from the original cell bottom. However, amyloplasts did not move significantly in reoriented specimens after the F-actin cytoskeleton was depolymerized with Lat-B.
|
It was not only important to establish that the amyloplasts in gravistimulated cells moved away from the original cell bottom, but also that they moved toward the new cell bottom after reorientation. Therefore, the amyloplast distance from the new cell bottom was also measured (see Fig. 1 and ‘Materials and Methods’). The plastids began at a relative cell distance of 0.473±0.021 (mean ±SE) in the control specimens and at 0.450±0.020 in the Lat-B-treated specimens. However, relative plastid distance from the new cell bottom decreased to 0.266±0.019 for control specimens, but did not change significantly (0.465±0.020, P >0.05) for the Lat-B-treated ones (Fig. 8). These measurements indicate that gravistimulated amyloplasts move from the original cell bottom toward the new cell bottom in control specimens, but fail to do so in specimens with disrupted F-actin.
|
Latrunculin B disrupts the F-actin cytoskeleton in endodermal cells of Arabidopsis hypocotyls
Alexa Fluor-phalloidin staining was employed to confirm that the drug dosage and experimental conditions employed effectively disrupt F-actin in the endodermal cells of Arabidopsis hypocotyls. The endodermis of control specimens stained with Alexa Fluor-phalloidin displayed thick, longitudinally oriented actin cables and thin, transverse actin filaments (Fig. 9A). Treatment with 2 µM Lat-B disrupted the F-actin network, and only short actin fragments and punctate fluorescence were visible in the endodermis of Lat-B-treated specimens (Fig. 9B). It was therefore concluded that 2.0 µM Lat-B, when applied as described in our methods, effectively fragments F-actin (Fig. 9B) and causes the enhanced gravitropic curvature observed in etiolated Arabidopsis seedlings (Fig. 2). These results confirm the more detailed studies of Lat-B effects on the F-actin cytoskeleton and tropistic responses from our laboratory (Yamamoto and Kiss, 2002
; Yamamoto et al., 2002).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
|---|
Amyloplast sedimentation kinetics in hypocotyls compared with roots
One of the earliest events of gravitropism is the movement of amyloplasts, which function as statoliths in specialized cells of roots and stems. Most studies of statolith function have focused on roots, and there have been relatively few reports regarding stems and stem-like organs. Since this is the first detailed study of amyloplast movement in hypocotyls of Arabidopsis following gravistimulation, it is important to evaluate amyloplast sedimentation in accordance with established criteria. In this paper, the methods and standards of MacCleery and Kiss (1999)
were used, which consist of both angular and linear measurements, because this work was performed using the same organism grown under similar experimental conditions.
A comparison of results from the present study to that of the previous paper (MacCleery and Kiss, 1999) indicates that amyloplasts settle more quickly in Arabidopsis hypocotyls compared with roots. Data from other Arabidopsis root studies also support this conclusion. For instance, Blancaflor et al. (1998) measured amyloplast velocities in Arabidopsis roots over a 5 min period following 135° rotation with respect to gravity, and the amyloplast velocities ranged from 0.64–1.20 µm min–1 in the most graviperceptive columella cells possessing the fastest sedimentation rates. By contrast, in this study amyloplast velocity was found to range from 1.88 to 3.24 µm min–1 in Arabidopsis hypocotyls during the 5 min period following 90° rotation with respect to gravity.
Angular and linear sedimentation data were also gathered in order to characterize amyloplast movement in the endodermis of gravistimulated Arabidopsis hypocotyls. The angular sedimentation data indicate that amyloplasts in vertically oriented plants are situated at the distal end (= original bottom) of the endodermal cells. In response to seedling reorientation, amyloplasts traverse the cell in a radial fashion, reaching a plateau along the new cell bottom after 2.5 min. Linear measurements were used to determine the extent of amyloplast movement away from the original cell bottom, and also their movement toward the new cell bottom. As with the radial data, the linear data indicate that amyloplasts move away from the original cell wall, reaching a plateau by 2.5 min following reorientation. However, amyloplast proximity to the new cell bottom tended to oscillate, which can be explained by the characteristic saltatory movements of amyloplasts that has been reported previously (Sack et al., 1986). These results provide baseline data on amyloplast dynamics in Arabidopsis hypocotyls, suggesting that, under normal circumstances, amyloplasts reorient with respect to the gravity vector and undergo dynamic movement within the cell.
Disruption of the actin cytoskeleton reduces amyloplast mobility in hypocotyls
The actin cytoskeleton has been implicated in signal transduction mechanisms of gravitropism for both roots (Sack et al., 1984; Kiss, 2000) and stem-like organs (Yamamoto and Kiss, 2002). F-actin disrupting drugs such as cytochalasins and latrunculins have been used in attempts to elucidate the role of actin in gravitropism. Depolymerization of the F-actin cytoskeleton using Lat-B causes enhanced gravitropic curvature in Arabidopsis roots (Hou et al., 2004) and shoots (Yamamoto and Kiss, 2002; Yamamoto et al., 2002). It was confirmed that Lat-B enhances hypocotyl gravicurvature in etiolated Arabidopsis seedlings (Fig. 1), despite causing a significant reduction in growth.
In order to determine why F-actin disruption causes increased gravicurvature in shoots, amyloplast sedimentation was analysed in gravistimulated hypocotyls with and without an intact cytoskeleton. It was found that the bulk movement of amyloplasts in hypocotyls is arrested by F-actin disruption with 2 µM Lat-B, suggesting an active mechanism of amyloplast movement in the cell. An occasional ‘rogue’ amyloplast was also observed located away from the cluster of amyloplasts. Saito et al. (2005) observed a similar phenomenon in Arabidopsis inflorescence stems treated with 0.2 µM Lat-B. However, Friedman et al. (2003) observed only a partial inhibition of amyloplast motility in snapdragon inflorescence stems treated with 12 µM Lat-B, and nanomolar concentrations of Lat-B caused an increased plastid sedimentation rate in Arabidopsis roots (Hou et al., 2004). As mentioned previously, the differing impact of cytoskeletal disruption in these studies may be due to differences in plant species, plant organs, or experimental conditions. Nevertheless, differential Lat-B effects have been observed in shoots, stems, and roots of Arabidopsis under identical experimental conditions (Yamamoto and Kiss, 2002), suggesting that the gravitropic mechanisms vary amongst these organs, and that amyloplast sedimentation may not be necessary for gravity perception in hypocotyls or stems.
In addition to amyloplast sedimentary movements, the characteristic saltatory movements typically exhibited by amyloplasts (Sack et al., 1986) are also affected by Lat-B. These dynamic movements cease upon F-actin disruption, as is indicated by the comparatively small standard error in the Lat-B-treated specimens compared with the controls (Figs 5–8
). A similar loss of saltatory movement has been observed in root statocytes (Hou et al., 2004) and inflorescence stems (Saito et al., 2005) after treatment with Lat-B. What role (if any) these saltations play in gravitropism is unknown, but the movements are clearly F-actin-dependent, suggesting that the amyloplasts are actively propelled inside the statocyte.
An active mechanism of gravity perception in stem-like organs
The loss of amyloplast mobility observed after F-actin disruption suggests an active, actomyosin-mediated mechanism of amyloplast transport within the hypocotyl endodermal cell. A potential caveat to this conclusion is the possibility that high concentrations of F-actin fragments increase cytosolic viscosity, so much so that the amyloplasts fail to sediment or undergo saltations. This scenario is unlikely for several reasons. First, Saito et al. (2005) observed that a few amyloplasts still sedimented in endodermal cells of gravistimulated inflorescence stems following F-actin disruption, and, as mentioned previously, similar ‘rogue’ amyloplasts were observed in this study. An occasional amyloplast retaining the ability to sediment in Lat-B-treated cells suggests that F-actin disruption does not affect cytosolic viscosity enough to alter amyloplast movement, and the phenomenon is better explained by the fact that F-actin was disrupted, but not entirely depleted, in each of these studies. Furthermore, other reports suggest that Lat-B may actually decrease cytosolic viscosity in Chara rhizoids (Kuznetsov and Hasenstein, 2001). Lastly, a dominant actin mutation reportedly produces similar effects on gravitropism to that produced by Lat-B (Morita and Tasaka, 2004).
Regardless of whether Lat-B affects cytosolic viscosity, the robust gravicurvature observed in Lat-B-treated specimens suggests that amyloplast sedimentation and an intact actin cytoskeleton might not be necessary for gravity perception to occur in endodermal cells of etiolated Arabidopsis seedlings. This raises the following questions: (i) if amyloplast sedimentation is not required to initiate gravicurvature, what role do these organelles play in shoot gravitropism; and (ii) if aerial plant organs still exhibit gravitropic curvature without an intact cytoskeleton, what is the function of F-actin in shoot gravitropism? Based on these results, it seems that amyloplast sedimentation and an intact F-actin cytoskeleton may be necessary for modulating the rate of organ curvature and for determining when to cease organ curvature.
Role of vacuoles in gravity perception mechanism
It has been proposed that the central vacuole may play a role in the gravity signal transduction pathway in shoot endodermal cells (Clifford et al., 1989; Morita et al., 2002; Haswell, 2003). An ultrastructural comparison of columella and endodermal cells reveals that the central columella cells in roots typically have several relatively small vacuoles, whereas shoot endodermal cells contain large, centrally located vacuoles that occupy a majority (>90%) of the total volume (reviewed in Kiss, 2000). Thus, while the settling of plastids is relatively unimpeded in columella cells, the substantially-sized vacuole in endodermal cells presents a significant obstacle to amyloplast sedimentation in the shoot endodermis (Volkmann et al., 1993).
In addition, amyloplasts in endodermal cells interact intimately with the tonoplast and the vacuole. The tonoplast has been shown to undulate when amyloplasts impact it, possibly ejecting ions in the process because the vacuole sequesters and extrudes ions (Volkmann et al., 1993; Morita et al., 2002). However, if amyloplast bombardment causes the vacuole to extrude ions that are necessary for gravity signal transduction, it is difficult to explain how the immobile amyloplasts in Lat-B-treated endodermal cells evoke an enhanced gravicurvature response. It is possible that proteins in the amyloplast membrane interact with proteins or other molecules at the tonoplast in order to elicit a gravity response. If this is the case, then arrested amyloplast movement may lead to the enhanced gravity response due to extended contact at the tonoplast interface, even if these amyloplasts do not impart a directional force to the vacuole.
Settling endodermal amyloplasts not only impact the tonoplast, but also they pass through the vacuole in thin strands of cytoplasm that traverse the vacuole (Clifford et al., 1989; Morita et al., 2002), and mutations affecting vacuolar formation and/or tonoplast function that result in amyloplast exclusion from the vacuole also inhibit shoot gravitropism. Examples include a variety of shoot gravitropism mutants such as sgr2, sgr3, and sgr4 (sgr4 was renamed zig because of the zig-zag phenotype of the stems). These strains have mutations in genes whose expression products function in vacuolar formation (Morita et al., 2002) and in the vacuolar transport pathway from the Golgi apparatus to the vacuole (Zheng et al., 1999; Kato et al., 2002a; Yano et al., 2003). They contain aberrant vacuole-like structures (Kato et al., 2002b) and exhibit abnormal amyloplast sedimentation (Fukaki et al., 1996; Zheng et al., 1999).
The SGR4/ZIG gene encodes a donor vesicle SNARE (v-SNARE), AtVTI11p, which is involved with vesicle transport processes – including transport to the vacuole. The SGR3 gene encodes a target membrane SNARE (t-SNARE), AtVAM3p, which localizes to the prevacuolar compartment and the vacuole, and it has also been shown to associate with AtVI11 in endodermal cells (Yano et al., 2003). The SGR2 protein is a PA-phospholipase A1 enzyme (Kato et al., 2002a) that localizes to the tonoplast and may play a role in gravitropic signal transduction, either by affecting amyloplast distribution through the vacuolar membrane, or by producing fatty acids and/or lysophospholipids to act as signalling molecules.
The altered response to gravity (ARG) protein affects both root and hypocotyl gravitropism (Fukaki et al., 1997; Sedbrook et al., 1999). It possesses a DNAJ domain that may interact with the cytoskeleton, and alterations to ARG affect the pH gradient that occurs in roots in response to gravistimulation (Boonsirichai et al., 2003). This pH transient has been linked to the transport of auxin carriers, the redistribution of which results in an asymmetric auxin gradient and subsequent bending of the plant organ in response to gravity (for review, see Moore, 2002). Evidence suggests that cytoskeletal disruption attenuates the dissipation of this lateral proton gradient in roots (Hou et al., 2004). Whether this pH transient is common to stem-like structures in plants is unknown, and the effect of F-actin disruption on this phenomenon has not been studied for shoots. Future studies should focus on determining if and how latrunculin B affects this pH transient in shoots.
Lastly, if the gravity signal is transduced in part by interactions between amyloplast membrane proteins and proteins in the tonoplast, then it is possible that amyloplast saltatory movements prevent these discrete connections from forming. If so, then it seems plausible that the abolition of these saltations causes extended interactions between the proteins, which might result in an amplified gravity signal. Considering this scenario, it is difficult to understand how directionality resulting in gravicurvature is imparted to the gravity signal. However, it has been established that a few amyloplasts sediment even after F-actin has been disrupted, and it may be that these amyloplasts establish the directional component as they pass through the vacuole or sediment onto the new cell bottom. What makes these ‘rogue’ amyloplasts capable of sedimentation when the rest do not is unknown. However, since F-actin is merely disrupted and not completely depolymerized in these experiments, an occasional filament might reform from the fragments and allow actomyosin transport of an amyloplast to occur.
|
Summary |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
|---|
In these studies, cryofixation was used to analyse amyloplast sedimentation over time in endodermal cells of etiolated Arabidopsis seedlings before and after disruption of the F-actin cytoskeleton with Lat-B. It was determined that the amyloplasts in the untreated controls settle to the new endodermal cell bottom within 5 min following reorientation. Furthermore, an intact actin cytoskeleton is essential for amyloplast mobility within the endodermal statocyte because amyloplast positions are not significantly altered in endodermal cells of gravistimulated seedlings once the F-actin cytoskeleton is disrupted with Lat-B. Since depolymerization of F-actin results in increased gravitropic curvature of Arabidopsis hypocotyls (Yamamoto and Kiss, 2002
; Yamamoto et al., 2002), these results indicate that amyloplast sedimentation is not a requirement for graviperception in endodermal cells in Arabidopsis seedlings. However, an intact cytoskeleton is required for a normal gravitropic response, and it is suggested that F-actin plays an important role in gravity signal transduction, possibly by modulating the gravity response by actively participating in statolith repositioning within the endodermal statocytes. |
Acknowledgements |
|---|
This work was supported by NASA grants NCC2-1200 and NGT5-50480. The authors also wish to thank Kaz Yamamoto for his helpful contributions regarding the kinetics of gravitropism and confocal microscopy experiments.
|
Footnotes |
|---|
Abbreviations: ARG and ARG1, altered response to gravity; AtVAM3p, Vacuolar Morphogenesis t-SNARE 3 (encoded by SGR3); AtVTI11p, Vesicle Transport v-SNARE 11 (encoded by SGR4/ZIG); CCD, charge coupled device; DMSO, dimethyl sulphoxide; EGTA, ethylenebis(oxyethylenenitrilo)tetra-acetic acid; F-actin, filamentous actin; ISO, International Standards Organization; Lat-B, latrunculin B; MBS, 3-maleimidobenzoyl-N-hydroxy-succinimide ester; MS, Murashige and Skoog; PA phospholipase A1, phosphatidic acid preferring phospholipase A1; PIPES, 1,4-piperazinediethanesulphonic acid; PME, 1,4-piperazinediethanesulphonic acid (PIPES)/MgSO4/EGTA; PROC MIXED, Mixed Procedure; SAS, Statistical Analysis Software; sgr2, sgr3, and sgr4 (sgr4 is zig, zig-zag), shoot gravitropism mutants; SNARE, Soluble N-ethyl-maleimide-sensitive-factor Attachment-factor Receptor; t–SNARE, target membrane–SNARE; v–SNARE, donor vesicle–SNARE; zig, zig-zag, see sgr4.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryReferences |
|---|
Balu
ka F, Hasenstein KH. 1997. Root cytoskeleton: its role in perception of and response to gravity. Planta 203, S69–S78.[CrossRef][ISI][Medline]
Blancaflor EB, Fasano JM, Gilroy S. 1998. Mapping the functional roles of cap cells in the response of Arabidopsis primary roots to gravity. Plant Physiology 116, 213–222.
Blancaflor EB, Masson PH. 2003. Plant gravitropism. Unraveling the ups and downs of a complex process. Plant Physiology 133, 1677–1690.
Boonsirichai K, Sedbrook JC, Chen RJ, Gilroy S, Masson PH. 2003. ALTERED RESPONSE TO GRAVITY is a peripheral membrane protein that modulates gravity-induced cytoplasmic alkalinization and lateral auxin transport in plant statocytes. The Plant Cell 15, 2612–2625.
Braun M, Buchen B, Sievers A. 2002. Actomyosin-mediated statolith positioning in gravisensing plant cells studied in microgravity. Journal of Plant Growth Regulation 21, 137–145.[Medline]
Caspar T, Pickard BG. 1989. Gravitropism in a starchless mutant of Arabidopsis: implications for the starch-statolith theory of gravity sensing. Planta 177, 185–197.[CrossRef][ISI][Medline]
Clifford PE, Douglas S, McCartney GW. 1989. Amyloplast sedimentation in shoot statocytes having a large, central vacuole: further interpretation from electron-microscopy. Journal of Experimental Botany 40, 1341–1346.
Driss-Ecole D, Jeune B, Prouteau M, Julianus P, Perbal G. 2000. Lentil root statoliths reach a stable state in microgravity. Planta 211, 396–405.[CrossRef][ISI][Medline]
Fasano JM, Swanson SJ, Blancaflor EB, Dowd PE, Kao TH, Gilroy S. 2001. Changes in root cap pH are required for the gravity response of the Arabidopsis root. The Plant Cell 13, 907–921.
Friedman H, Vos JW, Hepler PK, Meir S, Halevy AH, Philosoph-Hadas S. 2003. The role of actin filaments in the gravitropic response of snapdragon flowering shoots. Planta 216, 1034–1042.[ISI][Medline]
Fukaki H, Fujisawa H, Tasaka M. 1996. SGR1, SGR2, and SGR3: Novel genetic loci involved in shoot gravitropism in Arabidopsis thaliana. Plant Physiology 110, 945–955.[Abstract]
Fukaki H, Fujisawa H, Tasaka M. 1997. The RHG gene is involved in root and hypocotyl gravitropism in Arabidopsis thaliana. Plant and Cell Physiology 38, 804–810.
Haswell ES. 2003. Gravity perception: how plants stand up for themselves. Current Biology 13, R761–R763.[CrossRef][ISI][Medline]
Hodick D, Buchen B, Sievers A. 1998. Statolith positioning by microfilaments in Chara rhizoids and protonemata. Advances in Space Research 21, 1183–1189.[CrossRef][ISI][Medline]
Hou GC, Kramer VL, Wang YS, Chen RJ, Perbal G, Gilroy S, Blancaflor EB. 2004. The promotion of gravitropism in Arabidopsis roots upon actin disruption is coupled with the extended alkalinization of the columella cytoplasm and a persistent lateral auxin gradient. The Plant Journal 39, 113–125.[CrossRef][ISI][Medline]
Ingber DE. 1993. Cellular tensegrity: defining new rules of biological design that govern the cytoskeleton. Journal of Cell Science 104, 613–627.[ISI][Medline]
Ingber DE, Dike L, Hansen L, et al. 1994. Cellular tensegrity: exploring how mechanical changes in the cytoskeleton regulate cell-growth, migration, and tissue pattern during morphogenesis. International Review of Cytology 150, 173–224.[ISI][Medline]
Johannes E, Collings DA, Rink JC, Allen NS. 2001. Cytoplasmic pH dynamics in maize pulvinal cells induced by gravity vector changes. Plant Physiology 127, 119–130.
Kato T, Morita MT, Fukaki H, Yamauchi Y, Uehara M, Niihama M, Tas