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DEVELOPMENT AND HORMONE ACTION

The Onset of Gravisensitivity in the Embryonic Root of Flax^1,[OA]

Biology Department, University of Louisiana, Lafayette, Louisiana 70504–2451

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Vertical orientation of emerging roots typically is the first response of plants to gravity. Although root gravitropism has been studied extensively, no conclusive data on the onset of gravisensing exist. We determined the inception of gravisensitivity in flax (Linum usitatissimum) roots by clinorotating germinating seeds after various periods of static orientation (gravistimulation) of imbibed seeds. Gravitropic competency was established about 8 h after imbibition, 11 h prior to germination. The time was determined based on 50% of the newly emerged roots curving in the direction of the gravity vector during static imbibition, despite subsequent clinorotation. The threshold value was affected by the orientation of the seeds. Upward orientation of the micropyle/radicle reduced the number of graviresponding roots to about one-half. Prolonged clinorotation weakened the graviresponse. Gravisensing was accompanied by the development of amyloplasts, but the actin cytoskeleton was not involved because imbibition in Latrunculin B did not affect the onset of gravisensitivity or germination, and the development of F-actin in untreated controls was observed only after the onset of gravisensitivity.

In roots of higher plants, gravity sensing occurs in the central columella cells of the root cap (Juniper et al., 1966; Volkmann and Sievers, 1979; Sack, 1991; Blancaflor et al., 1998). However, there is evidence that gravisensing is not limited to the columella region (Poff and Martin, 1989; Wolverton et al., 2002a, 2002b). Root cap cells display a structural polarity, with the nucleus usually located in the proximal end of the wall and amyloplasts sedimented in response to gravity onto the endoplasmic reticulum (ER) near the distal wall (Sack and Kiss, 1989; Sack, 1991). Such polarity depends on the stage of root cap development and may be disrupted by inversion (Volkmann and Sievers, 1979) and clinorotation (Hensel and Sievers, 1980; Sack, 1991; Smith et al., 1999).

Root gravity sensing and signaling is thought to be regulated by interactions among various cellular structures, such as statoliths, ER, actin cytoskeleton, and vacuoles (Sack, 1997; Zheng and Staehelin, 2001; Blancaflor, 2002; Yano et al., 2003). Models of gravisensing involving a role of actin cytoskeleton suggests that amyloplast sedimentation exerts a pressure on actin filaments connected to the mechanosensitive ion channels in the plasma membrane, leading to channel activation and a subsequent chain of events (Blancaflor, 2002; Perbal and Driss-Ecole, 2003). However, developmentally, it is not known when these structures become functional and what exact roles their interactions play in gravity sensing.

Studies of root gravitropism have been almost exclusively performed on already emerged roots and have shown that gravisensing is correlated with sedimentable, starch-filled amyloplasts in the columella cells of the root cap (Volkmann and Sievers, 1979; Sack, 1991, 1997; Salisbury, 1993; Kiss et al., 1996; Blancaflor et al., 1998). However, gravitropism has also been observed in starchless mutants (Caspar and Pickard, 1989). Although curvature in response to gravity has long been known and the relationship between stimulation and angle of displacement has been described as the sine law (Rawitscher, 1932), the development of gravicompetency has not been investigated. It is likely that such studies will reveal important clues about the development of gravisensitive structures, associated gene activation, development of amyloplasts, and persistence (memory) of the stimulus.

In this study we define the onset of gravisensing as the time needed to induce at least 50% of all the newly emerged roots to grow in the direction of the gravity vector. The time was measured from the onset of imbibition until the beginning of subsequent clinorotation at a rate of approximately one revolution per minute. The threshold time was obtained from a plot of the percentage of positively curving roots against the duration of static stimulus prior to clinorotation. Temporal studies on root cap anatomy and confocal immunofluorescence microscopy of developing actin filaments indicate that gravisensing is accompanied by the development of amyloplasts and that the actin cytoskeleton is not required for gravisensing during seed germination.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Effect of Seed Orientation

When germinating seeds were positioned horizontally with their flat surface either parallel (Fig. 1C ) or perpendicular (Fig. 1D) to the gravity vector, a comparable proportion of the seedling roots curved positively gravitropic (51% and 58%, respectively, after 12-h imbibition; Fig. 2 , P = 0.74). In contrast, seeds with micropyle oriented downward differed significantly from those with the micropyle pointing upward (P < 0.0001). When the micropyle was upwardly oriented, only 25% of the roots curved in a positive direction and 75% exhibited random orientation. In contrast, when the micropyle was pointing downward, 91% of roots grew in the direction of the gravity vector (Fig. 2). This data set indicates that horizontally oriented seeds are best suited to examine gravitropism.

View larger version (88K): [in this window] [in a new window]   Figure 1. Experimental setup. Seed cassettes with seeds aligned on paper were positioned in one of four orientations. 1, Seed cassettes with the edge (*) positioned downward. This position contained seeds in the top row in upright (A) and seeds in the bottom row in downward orientation (B). 2, Seed cassettes positioned on the short side (** downward) resulted in the horizontal edge position of seeds (C, looking at micropyle). 3, Seed cassettes in a horizontal position kept the seeds in horizontal position (D, viewing the micropyle). The seeds were kept in their respective position during various time periods of imbibition before clinorotation. The design of the cassettes enabled root emergence without touching any surface. The diagrams indicate the scoring of the experiments: Root tips located in the shaded volume were marked as positive, tips in r were scored as random, and tips in n were designated negative.

View larger version (13K): [in this window] [in a new window]   Figure 2. Positional effects of gravistimulation. Imbibed seeds were gravistimulated for 12 h in horizontal (flat or on edge) or vertical (micropyle up or downward oriented; see Fig. 1), and clinorotated for the remainder of 36 h. Thereafter, the percentage of roots that grew in the direction of gravity during imbibition was determined. Statistically significant differences are indicated by different letters. Each data point is based on 150 to 192 germinated seeds from three replicates, ±SE.

The onset of gravisensing was determined as the time of static imbibition after which 50% of the roots grew in the direction of the gravity vector during imbibition. This time point was obtained from a plot of the percentage of positively curving roots from horizontally oriented seeds (as in Fig. 1D) against the duration of initial, static stimulus prior to clinorotation (Fig. 3 ). The distribution of curvature by classification (positive, negative, or random) was similar to an assessment of curvature of clinostated roots (data not shown) but eliminated negative values and difficulties in assessing contorted or damaged roots, which were common after extended clinorotation (Fig. 4C ).

View larger version (21K): [in this window] [in a new window]   Figure 3. Determination of the onset of gravisensitivity in germinating flax seeds. Seeds were imbibed in either water (solid lines) or 1 µM Lat B (dashed lines) for various periods before being clinorotated for the remainder of a 24-h (squares) or 36-h (triangles) period. The percentage of roots that curved in the direction of the gravity vector during imbibition at termination of clinorotation was evaluated. The 50% value (horizontal line) was set as threshold for the onset of gravisensitivity. The intercept with the regression lines (stippled) of the data points between 6 and 12 h determined the threshold value of 8 h for 24-h clinorotated roots and 12 h for 36-h clinorotation (vertical arrows). Compared to controls, Lat B-imbibed seeds showed the same slope but a reduced threshold (10.9 h [Lat B, dashed vertical arrow] versus 12 h [controls]) for the 36-h experiment. n = 150 to 192 seeds, three replicates, ±SE. The inset illustrates the length of controls (solid line) and Lat B-treated flax roots (dashed line) over time. Based on regression lines, the growth rate differed but the time of root emergence was identical (19.2 and 19 h for controls and Lat B-treated roots, respectively). Each data point is based on 40 to 44 germinated seeds from three replicates, ±SE.

View larger version (185K): [in this window] [in a new window]   Figure 4. The development of the columella during germination of flax roots. Root cap cells contained no starch-filled amyloplasts after 3 h of imbibition (A). Amyloplast development and sedimentation in roots after 10 h of imbibition represents the developmental state of amyloplasts when 75% of the roots exhibited gravicompetency (B). Clinorotation of seeds from the beginning of imbibition had no effect on amyloplast formation or integrity of the root cap during the first 12 h, but caused disintegration of amyloplasts and loss of an entire story of columella cells by 24 h (C, seen in all seven roots examined). Amyloplasts and cap remained intact in roots that were clinorotated after 12 h of static imbibition for 12 h (D) or 24 h (E). Roots at 24 h of imbibition showed large amyloplasts most prominent in the two upper cell layers (F). The arrow indicates the direction of the gravity vector in A, B, and F. Root caps depicted in C, D, and E were clinorotated. Bars = 15 µm.

The Effect of Latrunculin B

Latrunculin (Lat) B treatment neither expedited nor delayed the onset of gravisensitivity (P = 0.0928). When seeds were imbibed and germinated in 1 µM Lat B, gravisensitivity developed at the same time as in controls (Fig. 3). However, under conditions of prolonged clinorotation (36 h), Lat B-treated seeds exhibited a greater percentage of gravitropism in response to gravistimulus received during the first 12 h of static imbibition than controls (Fig. 3, P = 0.0028) and, thus, an earlier onset of gravisensitivity, even though gravitropic response started to level off after 12 h of gravistimulation. The 28% reduction in root growth between 24 and 36 h (Fig. 3, inset) indicates that Lat B was taken up by the seeds. The constant slope (Fig. 3, inset) indicates that Lat B remained effective for the duration of the experiment.

Effect of Clinorotation

Possible effects of clinorotation were examined by comparing the responses of incremental gravistimulation after 24-h clinorotation with data from seedlings that were clinorotated for an additional 12 h. Longer clinorotation (i.e. until 36 h after imbibition) significantly delayed the 50% threshold by about 4.5 h (Fig. 3, P < 0.0001), but the interpolated onset of gravisensing still preceded root emergence.

Root Cap Anatomy

The examination of the root cap structure of embryonic roots at times before and after the onset of gravisensitivity, as well as of emerged roots, indicated that the appearance of the amyloplasts in the central columella cells was a gradual process that was linked to the initiation of gravisensitivity (Fig. 4). During the first 3 h of imbibition, amyloplasts were not detectable (Fig. 4A). Amyloplasts began to appear 10 to 12 h after imbibition, when the majority of the primary roots were found to be graviperceptive (Fig. 4B).

On average, the diameter of amyloplasts in the central columella cells was 0.86 ± 0.03 µm at 10 to 12 h after imbibition, and reached 1.52 ± 0.07 µm in emerged roots at 24 h (Fig. 4F). Although the amyloplasts in embryonic roots were 44% smaller than those found in mature columella cells, their uneven distribution (Fig. 4B) indicated that they were able to respond to the gravistimulus. Despite their small size, the amyloplasts accumulated near the lower cell wall after reorientation.

Clinorotation of germinating seeds from the onset of imbibition caused disintegration of amyloplasts and damaged the root cap. We commonly (in all seven examined roots) observed a disruption between the fourth and fifth layer of columella cells from the tip (Fig. 4C). In contrast, when germinating seeds were kept static for 12 h prior to subsequent clinorotation for 12 h (Fig. 4D) or 24 h (Fig. 4E), the root cap remained intact and the amyloplasts were of similar size as in nonclinostated roots. However, clinorotation led to a dispersal of amyloplasts within the columella cells (Fig. 4, D and E). Emerged roots contained the largest amyloplasts in the upper two stories of the columella (Fig. 4F).

Development of the Actin Cytoskeleton

The actin cytoskeleton did not fully develop in columella cells within the first 48 h after imbibition (Fig. 5 ). After 12 h of imbibition, the external layer of the root cap began to exhibit fine actin filaments. Approximately 28 h after germination (48 h after imbibition), actin filaments became more readily observable in the peripheral root cap cells (Fig. 5A). The gradual appearance of stainable actin filaments also pertained to the root cortex (Fig. 5B). Lat B-treated roots showed only diffuse labeling, and no individual actin filaments could be detected (data not shown).

View larger version (86K): [in this window] [in a new window]   Figure 5. The actin cytoskeleton of flax roots 48 h after imbibition shows a gradual development. Older cells in the root cap (A) contain stainable, filamentous actin. Cortical cells (B, 50–250 µm from quiescent center) show a gradient of F-actin formation (arrows) toward the elongation zone. Bars = 20 µm.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

The examination of the root cap structure of embryonic roots before and after the onset of gravisensitivity, as well as of emerged roots, indicated that the appearance of the amyloplasts in the central columella cells was a gradual process (Fig. 4, A, B, and F). Amyloplasts were not detectable during the first 3 h of imbibition but appeared 10 to 12 h after imbibition, when the majority of the primary roots were found to be graviresponsive. The delay between the development of gravicompetence and the subsequent response, i.e. curvature after germination, indicates that the two processes are independent. The development of the amyloplasts in the columella became first noticeable in the central layers where the highest gravisensitivity was reported (Blancaflor et al., 1998). As the columella cells matured, amyloplasts enlarged and became more numerous. Smaller and thus less massive amyloplasts are likely to generate weaker signals. Therefore, stimulation must be effective for a longer time or it may not reach the critical threshold until sufficiently massive amyloplasts have developed (Kiss et al., 1989; Sack, 1994). The development of amyloplasts has also been reported as prerequisite for gravisensitivity in lateral roots (Kiss et al., 2002).

Although the amyloplasts in embryonic roots were much smaller than those in mature columella cells, their localization at the lower cell wall suggests that they sedimented and were suitable gravisensors. About 4 h past root emergence, the top two layers of columella cells contained the largest amyloplasts (Fig. 4F). Although the size of the amyloplasts was comparable between clinorotated and static roots, clinorotation dispersed the amyloplasts within the columella cells. The lack of constant gravistimulation or the continuously changing gravity vector could induce secondary responses that lead to the hydrolysis of organelle, cell wall, and tissue. The loss of an entire story of the columella after excessive clinorotation (Fig. 4C) adds to earlier reports on the impact of clinorotation on roots. Wall deterioration and reduction in amyloplasts was observed in Trifolium (Smith et al., 1999), and in Lepidium the root cap disintegrated, and statocytes lost polarity and exhibited occasional self-destruction (Hensel and Sievers, 1980). These observations suggest that the destroyed layer of the columella contains the most gravi- and mechanosensitive cells (Blancaflor et al., 1998) and that overstimulation of young cells leads to apoptosis.

For the development of gravisensitivity, several factors may be important. In addition to the sufficient accumulation of starch in amyloplasts, the columella cells may have to reach a sufficient size and maturity. Maturity may include suitable cytoplasmic viscosity, differentiation of special ER membrane domains, and possibly the development of a functional cytoskeleton (Ingber, 1993; Zheng and Staehelin, 2001). The latter point is especially important in light of the postulated function of the actomyosin system (Volkmann et al., 1993; Baluka and Hasenstein, 1997). As has been demonstrated earlier, the disruption of actin did not reduce gravisensing (Blancaflor and Hasenstein, 1997; Yamamoto and Kiss, 2002) but interfered with the termination of the graviresponse (Hou et al., 2004) and inhibited amyloplasts sedimentation (Palmieri and Kiss, 2005). Similar to these reports, our data indicate that actin is not required for the establishment of gravisensing because germination in the presence of a high concentration of Lat B did not delay the onset of gravisensing (Fig. 3), and the actin cytoskeleton was not detectable in primordial columella cells but developed in cortical and distal cap cells (Fig. 5) long after gravicompetency was established. Although staining of F-actin in columella cells is difficult, the data support the Lat B results that gravisensing occurs in the absence of F-actin (Blancaflor and Hasenstein, 1997; Palmieri and Kiss, 2005). Because of the seed orientation along the edge of the germination paper (see "Materials and Methods"), the emerging root did not contact the substrate and therefore cannot take up Lat B. Thus, we conclude that the Lat B-induced growth inhibition, despite identical root emergence (Fig. 3, inset), originated from Lat B that was taken up during imbibition.

Extended clinorotation decreased the number of graviresponding roots probably because of autostraightening, the tendency of expanding tissue to grow in a straight trajectory (Chapman et al., 1994; Stankovic et al., 1998, 2001). However, when seeds were imbibed in Lat B and exposed to prolonged clinorotation, the percentage of roots exhibiting positive gravitropism increased compared to roots that were not exposed to Lat B (Fig. 3). The observation that Lat B weakens the effect of extended clinorotation suggests either that the formation of the actin cytoskeleton inhibits gravisensing and/or that Lat B promoted the retention (memory) of the gravitropic set point (Digby and Firn, 1995). The reduced Lat B effect in 12- to 18-h imbibed seeds may be related to the Lat B-induced growth inhibition (Fig. 3, inset). However, despite the inhibitory effect of extended clinorotation, the onset of gravisensing still preceded root emergence. Although reduced, the shorter clinorotation is likely to have had similar effects on graviresponse as extended clinorotation. Therefore, the value of 8 h represents a conservative estimate of the onset of gravisensitivity. The same data set suggests that the gravity-induced signal persists for a finite time. The memory of the original signal is likely to be overpowered by endogenous events that favor straight-growing expansion. This point is supported by the reduction of roots that curve in the correct direction from approximately 25% after 24-h clinorotation to close to 0% after 36-h clinorotation. Assuming a random distribution of curving roots, about one-quarter (25%) should grow in the positive (originally downward) direction, as shown in the shorter clinorotation times (Fig. 3). The reduction to 0% indicates that roots grew straight and that the additional 12 h of clinorotation overcame the randomness of curvature for below-threshold stimulations.

The orientation of the seeds during germination also affected the results. The response was most stable and strongest in vertical, downward-oriented roots, intermediate in horizontally orientated, and weakest in inverted seeds. The discrepancy between up- and downward-oriented seeds suggests that the statocyte endomembranes have different sensitivities, develop differentially, or exhibit different signal transduction capabilities (Volkmann and Sievers, 1975). Because the development of amyloplasts was not affected, it is unlikely that the development of columella cells is orientation specific. Although the actin cytoskeleton is not involved in the initial development of gravity sensing before seed germination, it is likely to play a regulatory role in gravitropism after root emergence (Sievers et al., 1996; Baluka and Hasenstein, 1997; Nick et al., 1997; Hou et al., 2004) because of its mechanical properties (Ingber, 1993). If the sensing mass (amyloplasts) is actively moved or lifted by the cytoskeleton, the system would benefit from amplification of weak signals by noise or stochastic resonance (Douglass et al., 1993; Kondrachuk, 2001). Actin-dependent cellular motion (noise) may explain why starchless mutants are capable of proper orientation in response to gravity even though they do not have the benefit of massive, sedimenting amyloplasts (Caspar and Pickard, 1989). This concept of dynamic sensing is supported by saltatory movements of amyloplasts (Sack et al., 1984; Saito et al., 2005) and assumes that the cellular activity regularly probes the cells' orientation and contributes to the static signal that is likely to originate from sedimented statoliths.

Because our data imply that actin is not involved in the gravisensing step, it is important to reconcile this statement with the widely held view that the actomyosin system serves as signal transduction system for gravitropism. Actomyosin-independent gravisensing mechanisms in young roots (this article) may rely on the sedimentation of amyloplasts on the ER membrane system (Volkmann 1974; Sievers and Heydercaspers, 1983) without participation of the actomyosin system. However, roots with a functional actomyosin system could use continuous (dynamic) actin-mediated lifting and subsequent sedimentation or impinging of amyloplasts to sense gravity. The redundancy of this system assures robust gravity sensing.

The temporal analysis of the onset of gravisensing as illustrated in this report will permit a better understanding of the contribution of the cytoskeleton, amyloplasts, and membranes to gravisensing. The time dependency of gravitropic sensitivity will further help identify so-far elusive genes that are necessary for gravisensing. Our data suggest that plant graviresponse is not the consequence of a single mechanism or event, but the result of developmental processes that integrate environmental stimuli.

Because of the effects of chronic clinorotation on the polarity and integrity of columella and statocytes (Hensel and Sievers, 1980; Smith et al., 1999), the usefulness of clinostats to mimic microgravity or study graviperception has been questioned (Brown et al., 1996; Hoson et al., 1997; Smith et al., 1999). However, clinorotation does not hinder the formative stages of the gravisensory apparatus, nor is the detrimental effect of clinorotation universal (Kraft et al., 2000). Our investigations show that clinostats, in addition to their classical role of averaging gravity effects, can provide mechanostimulation based on rate and duration of rotation and thus may be useful for studies of cellular apoptosis and tissue repair processes.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Material

Flax (Linum usitatissimum) seeds were soaked in distilled water for 10 min. Eight seeds were aligned to the long edges of germination paper (40 x 16 mm), with the micropyle perpendicular to and pointing away from the paper. The seeds adhere to the paper because of the mucilage produced during soaking. The paper strips were allowed to dry completely and then placed in cassettes and secured with an overlay of a mesh (polypropylene, 109-µm pore size; Spectramesh) and covered with removable lids (Fig. 1). These cassettes were chosen to allow roots to grow in any direction without contacting a surface, which could induce thigmotropic reactions.

Imbibition, Gravistimulation, and Clinorotation

The cassettes were inserted into slots of a light-protected chamber and the dispensation of 0.35 mL of deionized water started the experiment. The chambers were positioned in one of three ways such that the seeds were oriented horizontal flat, horizontal on edge, or vertical (micropyle down or up; Fig. 1). A second volume of 0.15 mL was applied 2 h later. After 3 to 18 h, the chamber was clinorotated at 1 rpm for the remainder of either 24 or 36 h. All experiments were performed at room temperature (23°C–24°C).

Determination of Root Orientation following Clinorotation

After removal of the seed cassettes, the paper strips with attached seedlings were immediately photographed from two different angles to record the orientation of the roots. Based on their orientation prior to clinorotation, roots were classified as positive, random, or negative (see Fig. 1). The number of positively curving roots or roots growing in the direction of the g-vector was then reported as percentage of all germinated seeds. The onset of gravisensitivity was determined from the curvature of roots from seeds that were imbibed in a horizontal, flat orientation. The data set determined when 50% of the roots curved after clinorotation in the direction of the gravity vector during imbibition.

Effect of Lat B on the Onset of Gravisensitivity and Root Growth

In separate experiments, seeds were imbibed in 1 µM Lat B, a potent actin-depolymerizing agent (Geldner et al., 2001; Hou et al., 2004), gravistimulated with their flat surface horizontal (Fig. 1D), and clinorotated until either 24 or 36 h after imbibition. The onset of gravisensitivity in the presence of Lat B was determined as described above. In addition, growth rates of Lat B-treated and nontreated roots were digitally recorded with time-lapse imaging, and root length was measured with the public domain NIH Image J program (version 1.32j, developed at the United States National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image).

Microscopy

Seeds were imbibed with their flat surface horizontal, vertically, or clinorotated after 3, 10, 12, or 24 h and then fixed in 4% (v/v) formaldehyde, 60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2, and 5% (v/v) dimethyl sulfoxide, pH 7. Samples were dehydrated in a graded ethanol series and 100% acetone, and embedded in Spurr's resin. Longitudinal median sections (0.5 µm thick) were cut on an ultra-microtome (Sorvall MT2-B) and stained with toluidine blue (0.1% [w/v] in 0.1% [w/v] boric acid). The serial sections were photographed with a digital camera (Sony DKC-ST5).

Detection of Actin Filaments and Confocal Microscopy

Seeds imbibed for 6, 12, or 48 h were fixed for 2 h in 3.7% (v/v) formaldehyde in phosphate-buffered saline (PBS; 135 mM NaCl, 24 mM KCl, 11 mM Na₂HPO₄, pH 7.2). Roots (embryonic or emerged) were then washed in PBS buffer and sectioned on a vibratome (OTS-3000; EMS) at a thickness of 60 µm. Median longitudinal sections were selected under a dissecting microscope, transferred to slides coated with Mayer's albumin adhesive, then covered by a thin agarose-gelatin film (Brown and Lemmon, 1995). Sections were treated with a solution containing 0.2% (v/v) Triton X-100, 0.5% (w/v) pectolyase, 1% (w/v) cellulase, 1% (w/v) bovine serum albumin, and 10% (v/v) glycerol for 30 min, and incubated overnight in monoclonal anti-pea actin antibody (gift of Dr. Richard Cyr; Andersland et al., 1994). Sections were washed with PBS buffer three times, followed by incubation with secondary antibody (goat anti-mouse IgG; Accurate Chemical & Scientific) conjugated to fluorescein isothiocyanate for 2 h. After three washes in PBS buffer, slides were mounted with ProLong Anti-fade kit (Molecular Probes) and observed using a confocal laser scanning electron microscope (MRC-1024 ES; Bio-Rad), with excitation at 488 nm and emission at 520 nm. Images were processed with Adobe Photoshop 7.0.

Statistics

All data were examined by ANOVA and regression analyses using the General Linear model (PROC GLM, SAS, version 9.1; SAS Institute, Cary, NC) with arc sin square root transformed percentage values. Comparisons were based on Tukey's adjusted multiple comparison tests. Significant differences are indicated by letters in figures and probabilities in the text.

	ACKNOWLEDGMENTS

 
We thank Dr. T.C. Pesacreta, Dr. R.C. Brown, and Q. Jin for assistance with the microscopy; and Dr. S. Mopper for help with the statistical analysis.

Received October 24, 2005; returned for revision November 15, 2005; accepted November 15, 2005.

	FOOTNOTES

 
¹ This work was supported by the National Aeronautics and Space Administration (grant nos. NAG10–190 and NNA04CK48G).

² Present address: Division of Science, Truman State University, Kirksville, MO 63501.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Karl H. Hasenstein (hasenstein{at}louisiana.edu).

^[OA] Open Access articles can be viewed online without a subscription.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.073296.

^* Corresponding author; e-mail hasenstein{at}louisiana.edu; fax 337–482–5834.

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