The Organization of the Actin Cytoskeleton in Vertical And Graviresponding Primary Roots of Maize

Abbreviations: CB, Cytochalasin B; CD, Cytochalasin D; MFs, microfilaments; MTs, microtubules; MBS, m-maleimidobenzoyl N-hydroxysuccinamide ester; NPA, N-1 naphthylphthalamic acid

In order to determine whether actin microfilament (MF) organization is correlated with differential elongation, primary roots of Zea mays, cv. Merit maintained vertically or reoriented horizontally for 15 to 120 min were stained with rhodamine phalloidin and examined with a confocal microscope. Root curvature was measured with a computer controlled video-digitizer. In vertical roots bundles of MFs in the elongation and maturation zone were oriented parallel to the longitudinal axis of cells. MFs in the vascular parenchyma cells were more abundant than in the cortex and epidermis. Epidermal and proendodermal cells in the meristematic region contained transverse cortical MFs. The organization of MFs of graviresponding roots was similar to vertical roots. Application of cytochalasin B (CB) or cytochalasin D; resulted in extensive disruption of MFs in the cortex and epidermis but only partially affected MFs in the stele. Despite the CB-induced depolymerization of MFs, gravicurvature exceeded that of controls. In contrast, the auxin transport inhibitor N-1 naphthylphthalamic acid suppressed root curvature but had no observable effect on the integrity of the MFs. The data indicate that MFs may not be involved in the graviresponse of maize roots.

Differential growth during the root graviresponse is characterized by reduced growth along the lower, concave side and accelerated or sustained growth on the upper, convex side (Ishikawa et al., 1991). The graviresponse is initiated in the zone of post-mitotic, isodiametric growth (PIG, Baluka et al. 1990) or the distal elongation zone (DEZ, Evans and Ishikawa, 1993) and fully expressed in the elongation zone (Nelson and Evans, 1986, Ishikawa et al. 1991). Therefore the response is maximal at a certain distance from the perception site. However, the cellular mechanisms underlying differential elongation during root gravitropism remain to be elucidated. The microtubular component of the cytoskeleton appears to play a role in cell elongation due to their proposed influence on the deposition of cellulose microfibrils (Giddings and Staehelin, 1991). Previous studies with maize roots have shown that cortical microtubules (MTs) are transverse to the direction of cell growth in the elongation zone of vertical roots. Upon gravistimulation MTs become randomly or longitudinally oriented along the slower growing, concave side (Blancaflor and Hasenstein, 1993). This led to the assumption that MT reorientation is involved in cellular growth control during root graviresponse probably by altering the pattern of cellulose microfibril deposition in the slower growing cells located on the lower side of the root. However, the significance of MT reorientation during root graviresponse is limited in view of the observation that MT reorientation occurs only after 45 min of gravistimulation (Blancaflor and Hasenstein, 1995) while the onset of curvature can be detected as early as 20 min after gravistimulation (Ishikawa et al., 1991). Additional evidence for the independence of root gravitropism from the MT cytoskeleton comes from a recent study showing that substances that depolymerize MTs such as colchicine and oryzalin do not inhibit gravicurvature in maize roots (Baluka et al., 1996). Therefore MT reorganization may not be required for root graviresponse but may just be a consequence of reduced or altered growth.

The actin microfilaments (MFs) constitute another component of the plant cytoskeleton (Lloyd, 1989). One function of MFs in the growth control of higher plant cells is illustrated in cells that exhibit tip growth such as pollen tubes (Heslop-Harrison et al., 1988) and root hairs (Lloyd et al., 1987). Tip growth requires the delivery of wall precursor-containing vesicles to the tip-growing zone. These vesicles have been proposed to move along MFs that are aligned parallel to the pollen tube or root hair (Sievers and Schnepf, 1981). In elongating cells of vascular plants, MFs are arranged as thick longitudinal cables connected to a finer array of peripheral MFs which are either oriented longitudinally (Parthasarathy et al., 1985) or transversely (Hasezawa et al., 1989; Traas et al., 1987; Seagull et al., 1987). The actin cytoskeleton may have important implications for cell elongation. For example, Thimann et al. (1992) suggested that the polymerization of actin monomers onto the ends of longitudinally oriented MFs would stretch the polysaccharide matrix of the cell wall and cause the cell to elongate. Since differential elongation is characteristic of graviresponding roots, this system is suitable for testing if MFs are involved in the control of differential cell elongation.

The distribution of MFs in gravistimulated moss protonemata has been recently reported (Walker and Sack, 1995) but there is no corresponding study on the distribution of MFs in higher plant roots during graviresponse. The objective of this study was to determine the organization of MFs in roots and whether MFs are involved in the control of differential elongation and thus necessary for root graviresponse.

Plant Material and Gravistimulation

Caryopses of maize (Zea mays L., cv. Merit, Asgrow Seed Co., Kalamazoo, MI) were soaked in deionized water overnight, planted between wet paper towels, and grown vertically for 3 d between opaque plastic trays in a growth chamber at 24±1^oC. The daily photoperiod consisted of 18 h light (Sylvania cool white 50 W/m² ) and 6 h darkness. Seedlings with straight roots, 2-3 cm in length were selected, mounted in Petri dishes (9 cm diameter, lined with wet filter paper) with caulking compound and maintained in a vertical orientation. After 1 h, seedlings were reoriented by rotating the Petri dishes 90 degrees. Vertical roots and roots reoriented for 15, 30, 45, 60 and 120 min were fixed and processed for microscopy as described below.

Cytochalasin and NPA treatments.

A 50 mM stock solution of cytochalasin B (CB, Aldrich Chemical Co., Milwaukee, WI) or cytochalasin D (CD, Sigma Chemical Co., St. Louis, MO) and 20 mM NPA (Chem Services, Westbury, PA) were prepared in 100% DMSO. Working solutions of 50 µM CB 10 µM CD and 5 µM NPA were made by adding an appropriate volume of the stock solution to 5 mM MES/TRIS buffer, pH 6.5. Seedlings with straight roots were transferred to 1.5 ml microfuge tubes containing working solutions of CB, CD and NPA. The terminal 10 mm of the roots were immersed for 5 h in CB or CD and 3 h in NPA. Roots immersed in buffer with the corresponding concentration of DMSO were used as controls. After treatment, the seedlings were transferred to Petri dishes and placed horizontally. Vertical and horizontally oriented roots treated with CB, CD, and NPA were processed for microscopy.

Rhodamine-Phalloidin Staining of F-actin

After experimentation, the terminal 6 mm of the roots were excised by an oblique cut, the longer side corresponding to the lower side of the root. The excised tissues were fixed for 1 h in a solution of 1.5% formaldehyde in PHEMD buffer (60 mM Pipes; 25 mM Hepes; 10 mM EGTA; 2mM MgCl₂ ; 5% v/v DMSO pH 7.0). After rinsing in PHEMD buffer, roots were mounted on plexiglass blocks with cyanoacrylate (superglue) and sectioned longitudinally on a Vibratome-1000 (Technical Products International, St. Louis) at a thickness of 70 µm. Median sections were selected and transferred to slides coated with albumin adhesive (Fisher Scientific, Orangeburg, NY) and treated with 1% cellulase YC, 0.5% pectolyase Y-23 (Seishin Pharmaceutical Co., Tokyo, Japan) and 0.1% BSA (Sigma Chemical Co., St. Louis, MO) for 5 min. This was followed by incubation in 0.1% Triton X-100 in PHEMD buffer for 30 min. The sections were then incubated for 3 h in rhodamine-phalloidin (Molecular Probes, Eugene, OR). The methanolic stock solution (300 U) was diluted 1:40 in PBS (10 mM sodium phosphate; 150 mM NaCl, pH 7.2). After rinsing in buffer, sections were mounted in 20% Mowiol 4-88 (Calbiochem, La Jolla, CA) in PBS, pH 8.5 containing 0.1% p-phenylenediamine. After 24 h, sections were examined using a confocal microscope (MRC-600; BioRad, Richmond, CA). MFs were observed in sections obtained from at least 5 roots per treatment.

Root Curvature Measurements

Root growth and curvature after gravistimulation was monitored using a computer based video digitizer system (Hasenstein, 1991). The digitizer software was written to monitor the angular position of the root tip against a uniform background. The position of the root tip was continuously determined and recorded in 2 min intervals for a total of 120 min. Data were obtained from at least 10 roots per treatment.

Visualization of MFs

Fixation of root tissue in PHEMD buffer containing 1.5% formaldehyde resulted in excellent preservation of MFs (Figs 1-4). Fixation in 2% formaldehyde resulted in poorly preserved MFs in cortical and epidermal cells but was suitable for preservation of Mfs in vascular cells (data not shown). Pretreatment of roots with 100 µM MBS prior to formaldehyde fixation (Sonobe and Shibaoka, 1989) or omitting formaldehyde fixation (Hush and Overall, 1992) also failed to preserve MFs in the cortex and meristematic region (data not shown). Despite successful visualization of MFs with the monoclonal C4 antibody (ICN, Costa Mesa, CA) in other plant cells (McCurdy et al., 1988; Cho and Wick, 1990; Liu and Palevitz, 1992), staining with this antibody yielded poor images of the actin cytoskeleton, particularly in the epidermal and cortical cells (data not shown).

Organization of Microfilaments in Vertical Roots

The organization of MFs in the elongation zone of vertical roots (2 to 4 mm from the root tip) showed thin and thick bundles of MFs parallel to the longitudinal axis of the cell in both the outer (Fig. 1A) and inner cortex (Fig. 1B) often forming straight or curled bundles which typically terminated at the end walls. The maturation zone (6 mm from the root tip) often showed oblique MFs (Fig. 1C). Vascular parenchyma cells contained thick, longitudinal MF bundles that were more abundant than in cortical cells (Fig. 1D). Elongating metaxylem elements between the vascular parenchyma cells were easily discernible because of their larger size and absence of MFs in a large region of the cell with only few discontinuous MF strands (Fig. 1D). Unlike the MFs in the stele or cortex, epidermal cells along the elongation zone were characterized by thin bundles of MFs parallel to the longitudinal axis of the root. Non-preferentially oriented, internal MFs were closely associated with the nucleus (Fig. 1E).

While MFs in cells of the elongation and maturation zone were predominantly longitudinal in orientation, MFs in the meristematic region showed several patterns of organization (Fig. 2). The central and cortical regions of epidermal cells at the root tip exhibited MFs without preferential orientation, but cortical cells in the elongation zone showed longitudinal to non-preferential alinement of cortical MFs and irregularly oriented central MFs. In dividing cells, MFs were detected in the phragmoplast as brightly stained discs (Fig. 2A). Fine arrays of MFs in the cortical cytoplasm of epidermal cells about 0.2 mm from the root tip were aligned perpendicularly to the longitudinal axis of the root (Fig. 2B). Transverse orientation of MFs in the epidermal cells was observed until about 1.5 mm from the root tip. Basal to this region, cortical MFs in the epidermis became irregularly oriented before shifting to a longitudinal orientation at about 2 mm from the root tip (see Fig. 1E). Toward the stele region, MFs in the meristematic zone also exhibited different orientations. The proendodermis and pericycle had irregularly oriented fine internal MFs while vascular parenchyma cells exhibited thick bundles of longitudinal MFs in both the cortical and sub-cortical cytoplasm (Fig. 2C). Highly magnified optical sections of proendodermal cells revealed different patterns of MFs between the periphery and center of these cells. While MFs at the median region (Fig. 2D) showed irregular organization, transverse MFs dominated in the cortical region (Fig. 2E). Transverse cortical MFs in proendodermal cells shifted to disorganized and then to longitudinal with increasing distance from the root tip (data not shown).

The pattern of MFs also appeared to be closely correlated with the differentiation of metaxylem elements in the vascular region of the root. Metaxylem cells can easily be distinguished because they differ in size and shape from other cells in the vascular cylinder. Very close to the root tip, metaxylem cells show the greatest extension perpendicular to the root axis. MFs in these metaxylem cells were sparse with only a ring of highly fluorescent but fragmented MFs around the nucleus (Fig. 2F). With increasing distance from the tip (1 mm), metaxylem cells increased in size and became cuboidal with randomly oriented MFs becoming densely distributed throughout the entire cell (Fig. 2G). At 1.5 mm, the metaxylem elements continued to enlarge but remained square-shaped. However, metaxylem cells at this region began to exhibit fragmented MFs (Fig. 2H). MFs eventually disappeared in all metaxylem cells located greater than 2 mm from the root tip as they began to expand longitudinally. The absence of MFs was characteristic of elongated metaxylem cells (see Fig. 1D).

Organization of Microfilaments in Graviresponding Roots

MFs in the elongation zone (2-4 mm from the root tip) were examined since differential growth during root gravitropism occurs in this region. In addition, cortical MTs have been shown to reorient in cortical cells on the lower side of the elongation zone after gravistimulation (Blancaflor and Hasenstein, 1993; 1995). The effect of gravistimulation on the organization of MFs of the elongation zone, where curvature takes place, is shown in Fig. 3. Fifteen min after reorientation, the outer cortex on the upper side (Fig. 3A) and lower side (Fig. 3B) contained MFs that were predominantly longitudinal to the cell axis and similar to the organization of MFs in vertical roots. The longitudinal orientation of MFs on the upper (data not shown) and lower side did not change after 30 min (Figure 3C) to 120 min (Fig. 3D) of gravistimulation. MFs in the inner cortex in both the upper and lower side and in the vascular parenchyma cells also retained longitudinally oriented MFs after various times of gravistimulation (15-120 min, data not shown) and were similar in appearance to vertical roots (see Fig. 1).

After 2 h of reorientation, the transition of MFs from transverse to random and eventually to longitudinal in epidermal cells in both the upper and lower side was similar to the sequence in vertical roots. At 1 mm from the root tip, MFs in epidermal cells on the lower side were transverse while cells in the root cortex had thick bundles of cortical longitudinal MFs and finer MFs around the nucleus (Fig. 3E). As in vertical roots, MFs in epidermal cells of graviresponding roots located 2 mm from root tip became oriented parallel to the longitudinal axis of the root and were now co-aligned with the longitudinal MFs in the cortical cells (Fig. 3F).

Curvature and Microfilament Organization of Roots Treated with NPA and Cytochalasin

In order to determine whether the auxin transport inhibitor NPA affects the actin cytoskeleton, roots were pretreated with NPA and the organization of MFs was examined. Pretreatment for 3 h in 5 µM NPA had no effect on MFs in the meristem. Irregularly oriented arrays of cytoplasmic MFs were intact and similar to untreated control roots (Fig. 4A). Similarly, the network of thick MF bundles in the root cortex (Fig. 4B) and vascular parenchyma cells (Fig. 4C) of the elongation zone remained unaffected. MFs of NPA-treated gravistimulated roots remained intact and their organization did not change (data not shown).

To determine whether an intact actin cytoskeleton is necessary for root graviresponse, roots were treated with cytochalsin B or D prior to gravistimulation. Cytochalasins are fungal metabolites that inhibit many actin dependent processes in plants by disrupting the MFs (Hepler and Palevitz, 1974). When maize roots were incubated in 50 µM CB for 5 h, (or 20 µM CD, data not shown) drastic effects on MFs in the epidermis and cortex were observed. The cytoplasm of epidermal and cortical cells in the meristematic region exhibited short rods of MFs and diffuse fluorescence (Fig. 4D). In cortical cells of the elongation zone, the density of MFs was also dramatically reduced with only a few fragmented MF strands remaining (Fig. 4E). In contrast to MFs in epidermal and cortical cells, MFs in the vascular cells appeared more resistant to CB and were only partially affected. In addition to a slight reduction in density, some MF bundles became shorter and appeared less aligned (Fig. 4F).

Even without any obvious effects on the integrity of MFs, pretreatment with NPA strongly inhibited root curvature. For the first 60 min after horizontal reorientation, CB-treated roots curved at a rate similar to controls but the final angle of curvature eventually exceeded that of controls despite the severe disruption of MFs in the root epidermis and cortex (Fig. 5A). CD-treated roots exhibited a ca. 40 min delayed onset of curvature, curved at a slower rate, but completed their graviresponse in about five hours (data not shown). Although NPA and cytochalasins had different effects on the graviresponse of roots both substances caused a similar reduction in root growth rate (Fig. 5B).

The examined roots showed a complex and extensive network of MFs. Epidermal, cortical and vascular cells in the elongation zone were characterized by thick, predominantly longitudinally oriented MFs. This organization was similar to the organization of MFs in elongating cells of several vascular plants (Parthasarathy et al., 1985) and vascular tissues of gymnosperm (Pesacreta et al., 1982) and maize roots (Vaughan and Vaughn, 1987). Other studies, however, reported the occurrence of fine, more or less parallel cortical MFs in elongating tobacco protoplasts (Hasezawa et al., 1989), carrot (Traas et al., 1987) and alfalfa suspension cells (Seagull et al, 1987), as well as elongating cells of pea roots (Hush and Overall, 1992). In the present study, we demonstrated the occurrence of thin, apparently more fragile, bundles of either longitudinally or irregularly oriented MFs in elongating cells of maize roots in addition to thick bundles. Other protocols for visualization of the actin cytoskeleton used pretreatments with MBS prior to aldehyde fixation (Sonobe and Shibaoka, 1989) or eliminated formaldehyde fixation (Hush and Overall, 1992). We repeated both techniques and neither method revealed transverse cortical MFs in elongating maize cells as has been reported for other systems (Seagull et al., 1987; Hasezawa et al., 1988; Traas et al., 1987), but often resulted in fewer and fragmented MFs in epidermal and cortical cells. Similarly, MBS cross-linking or direct permeabilization with detergents were ineffective in preserving MFs in onion roots (Liu and Palevitz, 1992). Because of the diversity of detected MFs, fixation in 1.5% formaldehyde for 1-2 h appears to be optimal for the preservation of MFs in maize roots.

In the meristematic region of the root, fine cortical MFs were oriented perpendicular to the longitudinal axis of the root but were restricted to epidermal and proendodermal cells. Transverse cortical MFs in cells from the meristematic region of wheat (McCurdy et al., 1988) and onion (Liu and Palevitz, 1992) roots have been documented previously. However, in the above mentioned studies, the cells used for localization of MFs were isolated. Therefore it was not possible to determine the spatial relationship among cells and tissue layers within the root from where the examined cells originated. The similarity of MF organization between epidermis and proendodermis may be related to their common initials (Williams, 1947). In addition, the shift in MF orientation from transverse to longitudinal coinciding with the onset of cell elongation shows that the MF orientation is correlated with the changing shape of the cell. To the best of our knowledge, this is the first report that correlates the localization of transverse cortical MFs with specific tissues and development in the root.

The two central columns of metaxylem elements are a characteristic feature of longitudinal sections of maize primary roots (Luxova, 1981). This study shows for the first time the organization of MFs at different stages of metaxylem development. Similar to cortical cells, the metaxylem cells in maize roots change from an isodiametric to a cylindrical shape. The isodiametric metaxylem cells at the meristem region show abundant arrays of irregularly oriented MFs. Unlike other cells of the root which retain a viable cytoplasm, elongating metaxylem elements lose their cytoplasm at maturity (Lux, 1981). The fragmentation and eventual disappearance of MFs in cylindrical metaxylem vessels are indicative of the initial stages in the breakdown of the cytoplasm (Fig. 1D). In addition, the fine structure of the MFs in these cells emphasizes the high resolution of the MF visualization.

Our data indicate that the graviresponse of maize primary roots is largely independent of the actin cytoskeleton. This conclusion is based on three observations: a) the organization of MFs did not change in cells of the elongation zone during curvature b) CB and CD disrupted MFs without preventing the graviresponse of roots and c) NPA inhibited root graviresponse without changing the organization of MFs.

Recently, Thimann et al. (1992) proposed that the actin cytoskeleton may be involved in plant cell elongation since MFs are disrupted and modified when elongating Avena coleoptile cells are treated with various growth inhibitors. Therefore, it is possible that MFs, like MTs, are reorganized during the root graviresponse due to inhibition of growth on the lower side of the root. The examined time span (15 to 120 min) began prior to the onset of curvature (20 min, see Fig 5a) and continued through the completion of curvature in control- and CB-treated roots and therefore should detect changes correlated with early, continuing and completed curvature. Since the orientation of MFs in both the upper and lower side of graviresponding roots was similar for all tested times and not different from MF pattern in vertically-grown control roots, the organization of MFs appeared unaffected by differential elongation during graviresponse. This result is similar to a recent report showing that upward gravitropic curvature in Ceratodon protonemata did not result in the reorganization of MFs (Walker and Sack, 1995). Furthermore, the determination of a new polarity in wounded pea root cells, which depends on MT reorientation, has been shown not to require MF reorientation (Hush and Overall, 1992).

In addition to the description of differentiation and organization of MFs in roots, the results are also useful to examine the influence of MFs on differential elongation during the graviresponse. According to the Cholodny-Went theory, gravicurvature results from a lateral auxin gradient along the elongation zone of the root. The higher auxin concentration is presumed to cause growth inhibition along the lower flank of the root which leads to downward curvature (Masson, 1995). It is possible that an intact actin cytoskeleton is involved in the root graviresponse, since auxin transport has been linked with the root graviresponse (Hasenstein and Evans, 1988) and the MF disrupter CB (see Fig. 4) moderately inhibits auxin transport (Cande et al., 1973). Maize roots treated with CB showed disrupted MFs similar to the effect reported for maize (Vaughan and Vaughn, 1987), onion (Palevitz, 1988) and pea (Hush and Overall, 1992) root cells. The moderate growth inhibition (fig. 5B) by CB is in line with earlier work (Pope et al., 1979) but these authors used an about 10 fold higher concentration of CB than in the present study. Wendt et al. (1987) observed in cress roots that CD (2.5 µg*ml^-1) neither affected growth nor curvature but CB at 25 µg*ml^-1 reduced curvature, presumably due to growth reduction. Although growth was reduced, curvature of Merit roots was not inhibited with CB but delayed with CD (fig. 5A). Both cytochalasins caused depolymerization of the actin cytoskeleton but CB proved to be a more potent MF disrupter than CD. Since during the entire graviresponse time the MF organization was very similar for the epidermis and outer cortical cells in both vertical roots and the region of maximal curvature (fig. 3) of graviresponding roots, the organization of MFs appears not to be affected by or contribute to differential elongation.

The failure of CB and CD to inhibit root curvature despite their disruptive effect on the MFs argues against the notion that an intact actin cytoskeleton is required for the root graviresponse. However, our data do not rule out a function of the actin cytoskeleton in the process of graviperception. Although staining for filamentous actin of columella cells in maize root caps proved to be unsuccessful (data not shown) the lack of stain does not necessarily rule out that MFs too small to be stained, may function in gravisensing (Baluka and Hasenstein, submitted). Cytochalasins do not fully depolymerize MFs but cause fragmentation (Schliwa 1982, Cooper 1987, fig. 4D, E, F). Therefore cytochalasins may have little effect on gravisensing if relatively small MFs are involved in this process. This assumption is supported by the simultaneous onset and even enhanced curvature in CB treated roots.

The auxin transport inhibitor NPA inhibits asymmetric auxin movement and gravicurvature in maize roots (Lee et al., 1984; Evans et al., 1992, Lomax et al., 1995). A protein with NPA binding activity was assumed to be associated with the cytoskeleton and localized to the peripheral region of the plasma membrane (Cox and Muday, 1994). Since the actin cytoskeleton reorganized in response to signals from outside the cell that impinge on the plasma membrane (Luna and Hitt, 1992), it was hypothesized that one of the effects of NPA leading to the inhibition of root gravicurvature could be the disruption or modification of the organization of MFs. However, since MF-disruption did not inhibit curvature, lateral auxin redistribution appears to be accomplished through a MF-independent pathway. The inhibition of root curvature by NPA despite the absence of any observable effect on MF reorganization disproves this hypothesis and provides evidence for the independence of root graviresponse from the actin cytoskeleton. The isolated nature of the NPA binding site was also demonstrated by Bernasconi et al. (1996) who showed that the NPA binding protein is an integral, and not, as suggested by Cox and Muday (1994) a peripheral membrane protein. Since NPA blocks both basipetal and acropetal auxin transport in maize roots (Hasenstein et al., 1995), the preservation of intact MFs despite NPA treatment also indicates that auxin transport can be blocked without disruption of MFs. However, changes in the actin cytoskeleton other than a reorganization or disruption cannot be ruled out. For example, it was shown that auxin causes a decrease in tension of the actin cytoskeleton in soybean root cells (Grabsky and Schindler, 1996). Nonetheless, a change in tension within the actin network is unlikely to be critical for differential elongation since CB most efficiently depolymerizes MFs in the epidermis and outer cortex where differential tension or compression would have the greatest effect on differential elongation.

In conclusion, there were no dramatic qualitative redistributions in the MFs during graviresponse as visualized by our technique. These results as well as studies on the MT component of the cytoskeleton (Baluka et al., 1996; Blancaflor and Hasenstein, 1995; Hasenstein et al., 1995) show that the plant cytoskeleton does not appear to play a major role in the gravicurvature of maize roots.

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Figure 1. Organization of microfilaments in the elongation and maturation zone of vertically-grown maize roots. The root tip is toward the bottom of the page for all images. Cells in the outer (A) and inner (B) cortex (3-4 mm from the root tip) were characterized by thick cables and thin arrays of longitudinally oriented MFs that formed straight or curled bundles. (C) Longitudinal to helical arrays of MF bundles were observed in the maturation zone (6 mm from the tip). (D) Parenchyma cells in the vascular region of the root had a more extensive array of longitudinally oriented MF bundles. Metaxylem elements (x) which lose their cytoplasm at maturity show disintegrating MFs (arrows). (E) The cortical cytoplasm of epidermal cells throughout the elongation zone had fine arrays of longitudinally oriented MFs while sub-cortical MFs were randomly oriented and appeared to encircle the nucleus (n) Bar in D: 25 µm, for A-D; Bar in E: 10 µm.

Figure 2. Organization of MFs in the meristematic region of vertically-grown maize roots. The root tip is toward the bottom of the page for all images. (A) Epidermal cells (e) at the root tip region showed irregular arrays of internal (arrowhead) and cortical MFs (arrow). Cells in the root cortex had random cytoplasmic MFs (*) and longitudinal cortical MFs (double arrows). Actin organization in the phragmoplast appeared as a double disc (double arrowheads). (B) At 0.25 mm from the root tip, cortical MFs in the epidermal (e) cells became highly aligned and were perpendicular to the longitudinal axis of the root. Internal MFs in the root cortex (c) showed no preferential orientation. (C) Random arrays of internal MFs were observed in the proendodermis (en) and pericycle (p), while thick longitudinal MFs were characteristic of all parenchyma cells in the vascular region (v). (D) MFs in endodermal cells at the plane of the nucleus (n) were random in orientation while (E) peripheral MFs were transverse to the longitudinal axis of the root. (F) Metaxylem elements (x) very close to the root tip only had a ring of short MFs surrounding the nucleus (arrows). (G) At 1 mm from the root tip MFs in metaxylem cells were randomly oriented and increased in density. (H) At 1.5 mm from the root tip, enlarging metaxylem cells showed fragmentation of MFs. Bar in D & E: 10 µm, all other bars: 25 µm.

Figure 3. Organization of MFs along the elongation zone of maize roots 15, 30 and 60 min after reorientation. The root tip is to the left and the gravity vector is toward the bottom of the page. 15 min after gravistimulation, MFs in the outer cortex on the upper (A) and lower (B) side were parallel to the longitudinal axis of the cells. The orientation of MFs in the outer cortex of the lower side did not change even after 30 min (C) and 1 h (D) of gravistimulation. After 120 min of reorientation (E, F)., the cells 1 mm from the root tip cortex contain thick bundles of longitudinally oriented peripheral MFs and irregular and finer internal MFs (arrow, E). MFs in the epidermis (e) are preferentially transverse in orientation. 2 mm from the root tip, longitudinally oriented peripheral MFs in the root cortex become more abundant while the thinner arrays of MFs in the epidermis (e) shift to a longitudinal orientation (F). Bar: 25 µm

Figure 4. MFs in maize roots treated for 3 h with 5 µM NPA (A-C) and 5 h with 50 µM cytochalasin B (D-F). The appearance of MFs in NPA treated roots in the meristematic region (A), cortex (B) and stele vascular parenchyma (C) at 3 mm from the root tip were similar to untreated control roots. CB treatment caused a disruption of MFs in the meristem (D), cortex (E) while causing only a partial depolymerization of MF bundles in the vascular parenchyma (F). Bars: 25 µm.

Figure 5. (A) Comparison of gravicurvature in control roots () with roots pretreated for 3 h with 5 µM NPA (), 5 h with 50 µM CB () or 5 h with 10 µM CD (). Roots treated with CB curved at a similar rate to controls but the final angle of curvature eventually exceeded that of controls. CD treatment caused a delay in the onset of curvature but depolymerized actin less than CB while NPA blocked root curvature without any observable effect on MFs. (B) Elongation rate of vertically growing control roots () and roots treated with 5 µM NPA () or 50 µM CB () or 10 µM CD (). Both CB and NPA showed a similar reduction of elongation growth compared to controls but only NPA blocked the graviresponse. CD () caused stronger reduction in growth despite being less effective than CB in depolymerizing MFs. Means ± SE, n=10.

This work was supported by NASA grant NAGW-3565 and a Sigma Xi Grant in Aid of Research to EBB.