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. 2006 Jul;98(1):49–55. doi: 10.1093/aob/mcl084

Distribution of G-actin is Related to Root Hair Growth of Wheat

XUE HE 1, YI-MIN LIU 1, WEI WANG 1, YAN LI 1,*
PMCID: PMC2803535  PMID: 16675602

Abstract

• Background and Aims Actin distribution in root hair tips is a controversial topic. Although the relationship between Ca2+ gradient and actin dynamics in plant tip-growth has been a focus of study, there is still little direct evidence on the exact relationship in root hair tip-growth.

• Methods G-actin was labelled by fluorescein isothiocyanate–DNase I. F-actin was labelled by tetramethylrhodamine isothiocyanate–phalloidin. Actin in root hairs of Triticum aestivum (wheat) was investigated using confocal laser-scanning microscopy.

• Key Results Thick F-actin bundles did not extend into a region of approx. 5–10 µm from the tip of the growing root hairs, although they gave off branches of fine actin filaments in the hair tips. A tip-focused G-actin gradient was shown at the extreme apex of growing root hairs. In full-grown wheat root hairs, the tip-focused G-actin gradient disappeared while the thick F-actin bundles extended into the tips. BAPTA-AM, a Ca2+ disruption agent, also caused the tip-focused G-actin gradient to disappear and the diffuse F-actin bundles to appear in the tips of wheat root hairs.

• Conclusions These results suggest that the tip-focused gradient of intracellular G-actin concentration at the extreme apex may be essential for root hair growth, and that preserving the tip-focused gradient needs a high Ca2+ concentration in the root hair tips.

Keywords: G-actin, F-actin, root hairs, plant tip-growth, Ca2+, BAPTA-AM, Triticum aestivum, wheat

INTRODUCTION

Actin plays essential roles in plant tip growth. Root hairs are one type of plant tip-growth cells that have served as a model for plant cytoskeleton studies. F-actin bundles are arranged longitudinally in root hairs, and, together with myosin, are thought to cause cytoplasmic streaming that transports the vesicles to the apex (Mathur and Hülskamp, 2002). However, actin distribution in root hair tips is a controversial topic. Wang et al. (2004) and Ketelaar et al. (2004) found that F-actin distribution was disorganized, with less F-actin in the root hair apex of Arabidopsis thaliana, whereas other reports showed an accumulation or cap of F-actin at the tip of root hairs (Baluška and Volkmann, 2002; Ditengou et al., 2003).

The function of Ca2+ in plant cells is a fascinating research area (Rudd and Franklin-Tong, 2001). An obvious tip-focused gradient of cytosolic Ca2+ was found in root hairs (Mathur and Hülskamp, 2001; Ryan et al., 2001). In vitro, a high concentration of Ca2+ inhibits F-actin polymerization (Pollard and Cooper, 1986), and, in vivo, studies of animal cells have also proved that high concentrations of Ca2+ can lead to F-actin depolymerization while inhibiting its polymerization (Stolz and Bereiter-Hahn, 1988; Constantin et al., 1998). In addition, a proper Ca2+ gradient appears to be essential for root hair tip-growth (Rudd and Franklin-Tong, 2001). Many treatments, including the injection of the Ca2+-specific buffer, dibromo-BAPTA, simultaneously dissipate the tip-focused Ca2+ gradient and inhibit root hair elongation (Carol and Dolan, 2002; Wasteneys and Galway, 2003). Although the relationship between Ca2+ gradient and actin dynamics in plant tip-growth has been a focus of study, there is still little direct evidence on the exact relationship in root hair tip-growth.

In the present study, labelling with fluorescein isothiocyanate (FITC)–DNase I was used to reveal the presence of a tip-focused gradient of intracellular G-actin concentration at the extreme apex of growing wheat root hairs. By contrast, in full-grown wheat root hairs, the tip-focused G-actin gradient disappeared while thick F-actin bundles extended continuously into the tips. As a Ca2+ disruption agent, BAPTA-AM was found to make the tip-focused G-actin gradient to disappear and the F-actin mesh to appear in root hair tips of wheat.

MATERIALS AND METHODS

Plant growth

Wheat grains (Triticum aestivum L.) were germinated in a moistened culture dish with filter paper for 2 d in darkness at 26 °C. Seedlings were used after 36 h or 48 h when their roots grew to 0·5–1 cm in length.

BAPTA-AM treatment

BAPTA-AM treatment followed the protocol of Fluo-3/AM treatment reported in Zhang et al. (1998). Briefly, after the wheat grains had germinated, some of them were treated with 65 μm BAPTA-AM (Sigma; A1076, a selective chelator of intracellular Ca2+ stores and membrane-permeable form of BAPTA) in distilled water at 4 °C for 1·5 h. Then, the roots were allowed to recover for 1·5 h at 25 °C. BAPTA-AM was diluted in DMSO as stock solution, and the final concentration of DMSO in treatments did not exceed 0·5 % (v/v).

Fluorescence labelling for G-actin and F-actin

For F-actin labelling, root hairs were fixed as previously described (Li et al., 2001; Wang et al., 2005). To avoid F-actin disruption in root hairs, the whole seedlings including root hairs were fixed directly. Briefly, the seedlings were fixed and vacuum-infiltrated for the first 5 min in a freshly prepared solution of 4 % paraformaldehyde in 50 mm Pipes buffer (pH 6·9), and then kept in the fixation medium for 1 h. Following three washes in 50 mm Pipes buffer, root apices about 5 mm long were cut and incubated in 0·3 μm tetramethylrhodamine isothiocyanate (TRITC)–phalloidin (Sigma), in PBS buffer (0·137 m NaCl, 2·7 mm KCl, 2·7 mm KH2PO4, 8·1 mm Na2HPO4, pH 6·9) containing 1 % DMSO for 1 h. Then the samples were mounted on slides in 50 % glycerol.

For G-actin and F-actin labelling, after fixation and washes, the root tips were incubated in enzyme solution containing 1 % cellulase and 1 % pectinase (50 mM Pipes buffer, pH 6·9) in the dark at 37 °C for 15 min. After three washes in 50 m m Pipes buffer, the samples were incubated in 1 % Triton X-100 (50 mm Pipes buffer, pH 6·9) at room temperature for 1 h. After three washes, the samples were incubated in a mixture of FITC–DNase I (Molecular Probes) and/or TRITC–phalloidin (Sigma; 3 μm FITC–DNase I and/or 0·3 μm TRITC–phalloidin in PBS buffer, containing 1 % DMSO, pH 6·9) for 1 h. After two washes, the samples were mounted on slides in 50 % glycerol. The specificity of FITC–DNase I staining was examined by comparison with controls incubated with 50 µg mL–1 FITC–DNase I previously denatured by boiling for 10 min (Knowles and McCulloch, 1992). In such controls, no specific staining was detected (see Fig. 2G).

For immunofluorescence labelling, after Triton X-100 treatment and three washes, the root tips were incubated with primary antibody for 1 h at 37 °C. The primary antibody was a monoclonal antibody to actin (clone C4 from ICN; diluted 1 : 200 in PBS), which was found to react with actin of root hairs (Braun et al., 1999). Following three washes, the root hairs were incubated in goat anti-mouse FITC-conjugated secondary antibody (Sigma; diluted 1 : 100 in PBS). After three washes, the samples were mounted on slides in 50 % glycerol. In the control the primary antibody was replaced by 3 % BSA, in which case, no staining was detected.

Confocal laser-scanning microscopy and image analysis

Intracellular fluorescence was observed using Zeiss LSM 510 META (Carl Zeiss Far East Co.) or Bio-Rad MRC 1024 (Bio-Rad Laboratories, Inc.) confocal laser-scanning microscopes. All images were projected along the z-axis. Optical sections were obtained at a step of 0·8–1·2 µm. Image analysis was carried out using the standard software supplied with these microscopes.

RESULTS

F-actin in growing root hairs of wheat

TRITC–phalloidin labelling showed that thick F-actin bundles were oriented parallel to the long axis of the growing root hairs (zone I; Heidstra et al., 1994) without extending into a region of approx. 5–10 µm from the tip of the wheat root hairs (Fig. 1A, B). This pattern was similar to the distribution of F-actin bundles in living root hairs of Arabidopsis labelled with GFP-fimbrin 1 (Ketelaar et al., 2004; Wang et al., 2004). The consistency implies that it is likely that the fixative method used here displayed the real distribution of F-actin bundles in root hairs. In addition, when scanned with a stronger laser, a few fine actin filaments branching out of the thick F-actin bundles could also be found in the root hair tip (Fig. 1C, arrow). On the other hand, when the growing root hairs of wheat were stained by immunofluorescence labelling with an actin antibody, apart from the thick F-actin bundles from the sub-apex in the root hairs, a strong fuzzy staining was seen in the hair tips (Fig. 1D). This result was similar to those in previous reports on other plant root hairs labelled by actin antibodies (Braun et al., 1999; Miller et al., 1999; Ketelaar et al., 2002, 2003).

Fig. 1.

Fig. 1.

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Confocal images of F-actin in root hairs of wheat stained by TRITC–phalloidin or actin antibody. (A and B) TRITC–phalloidin labelling for F-actin in short (A) or long root hairs (B) of wheat. These images show that thick F-actin bundles were oriented parallel to the long axis of the root hairs, and disappeared within 5–10 μm from the tip of the root hairs. (C) When scanned with stronger laser power, fine actin filaments branching out of the thick F-actin bundles can also be seen in the root hair tip labelled by TRITC–phalloidin (arrow). (D) Actin stained by immunofluorescence antibody shows, apart from the thick bundles in the sub-apex in the root hair, a strong fuzzy staining in the root hair tip, indicating the presence of more G-actin in this region. Scale bars =10 μm.

G-actin pool in root hairs of wheat

DNase I was found to bind specifically to G-actin in animal cells (Knowles and McCulloch, 1992; Cao et al., 1993; Haugland et al., 1994; Tuvia et al., 1998). Ren et al. (1997) found that plant pollen G-actin could also bind to DNase I and form a high-affinity 1 : 1 complex. Fluorescence-conjugated DNase I was used as a G-actin probe in plant cells and a tip-focused G-actin gradient was shown in pollen tubes of Lilium davidii (Li et al., 2001). Here, using FITC–DNase I labelling coupled with confocal laser-scanning microscopy, a tip-focused gradient of G-actin was also found in the growing root hairs of wheat (Fig. 2A–D). The concentration of G-actin, the highest in the root hair tip, was reduced sharply from the tip to the sub-apex. As measured by confocal laser-scanning microscopy, the relative FITC–DNase I fluorescence intensity, which may represent the relative concentration of G-actin, was approximately twice as high at the tip of the root hairs as in other regions of the hairs (Fig. 2E). Additionally the tip-focused gradient of G-actin disappeared in full-grown root hairs of wheat (Fig. 2F; zone III, Heidstra et al., 1994). In controls, no specific staining was detected (Fig. 2G).

Fig. 2.

Fig. 2.

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Confocal images of G-actin in root hairs of wheat stained by FITC–DNase I. The most intensely fluorescent regions are white, followed by red and yellow, and the least fluorescent regions are blue. (A–D) G-actin forms a tip-focused gradient in the growing root hairs. (E) Comparison of the fluorescence intensity in the regions of the root hairs indicated by the red line, ranging from the tip to other terminals in (D). The relative FITC–DNase I fluorescence intensity, which may represent the relative concentration of G-actin, was approximately twice as high at the tip as in the other regions of the root hairs. (F) The tip-focused G-actin concentration gradient disappeared in a full-grown root hair tip. (G) In control samples incubated with boiling denatured FITC–DNase I, no specific staining was detected. Scale bars = 10 μm.

G-actin related negatively to F-actin in growing root hairs of wheat

Using TRITC–phalloidin and FITC–DNase I double labelling and confocal laser-scanning microscopy, it was found that the distribution of G-actin was related negatively to that of the thick F-actin bundles in growing root hairs of wheat (Fig. 3A and B). In the root hair tips devoid of the thick F-actin bundles (Fig. 3B), the concentration of G-actin was higher (Fig. 3A). These results imply that most F-actin may be depolymerized into G-actin by some factors in the tips of growing wheat root hairs. Conversely, when the tip-focused gradient of G-actin disappeared from the full-grown root hairs of wheat (Fig. 3C), the thick F-actin bundles were found to appear in the tips (Fig. 3D).

Fig. 3.

Fig. 3.

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Confocal images of G-actin and F-actin in growing or full-grown root hairs of wheat. G-actin was stained by FITC–DNase I, and F-actin by TRITC–phalloidin. (A) Tip-focused gradient of G-actin in growing root hairs. (B) The image shows the disappearance of the thick F-actin bundles within approx. 10 μm from the hair tip, indicating a negative correlation between the distribution of the thick F-actin bundles and that of G-actin in the root hairs. (C) G-actin concentration gradient disappeared in a full-grown root hair tip. (D) F-actin bundles extended into the tip of a full-grown root hair. The most intensely fluorescent regions are red or yellow and the least fluorescent regions are blue in (A) and (C). Scale bars = 10 μm.

BAPTA-AM disrupts the G-actin pool in root hairs of wheat

BAPTA has been proven to disrupt Ca2+ distribution in root hairs by causing a rapid dissipation of the tip-focused Ca2+ gradient and thereby inhibits root hair growth (Herrmann and Felle, 1995; Felle and Hepler, 1997). Here it was found that the tip-focused G-actin gradient also disappeared when the growing root hairs of wheat were treated with 65 μm BAPTA-AM (Fig. 4A). At the same time, a dense F-actin mesh was found in the root hair tip (Fig. 4B). We also found that the BAPTA-AM treatment could disrupt obviously the tip-focused Ca2+ gradient in growing root hairs of wheat (see supplementary material). These results indicate that a high Ca2+ concentration is essential for maintaining the G-actin pool in growing root hair tips of wheat.

Fig. 4.

Fig. 4.

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Confocal images of G-actin and F-actin in BAPTA-AM-treated root hairs of wheat (zone I), showing simultaneously (A) the disappearance of G-actin tip-focused gradient and (B) the appearance of a dense F-actin mesh in the hair tip, both in the same cell. The most intensely fluorescent regions are red or yellow and the least fluorescent regions are blue. Scale bar = 10 μm.

DISCUSSION

In the study reported here, TRITC–phalloidin labelling of the root hairs of wheat showed that thick F-actin bundles run parallel to the long axis of the hairs, without extending into the region approx. 5–10 µm from the tip. In addition, a few fine actin filaments branching out of the thick F-actin bundles could also be found in the growing root hair tip. These results support the view that large organelles move on the thick F-actin bundles in the root hair while Golgi-derived vesicles move on the fine actin filaments in the root hair tip (Ketelaar and Emons, 2001; Mathur and Hülskamp, 2002). Additionally, actin antibody labelling showed a strong fuzzy staining in the growing root hair tips of wheat. The present results in root hairs of wheat are consistent with previous reports on other plant root hairs (Braun et al., 1999; Miller et al., 1999; Ketelaar et al., 2002, 2003, 2004). Since phalloidin labels only F-actin and actin antibody labels both F-actin and G-actin in cells, it is speculated that there might be more G-actin in the growing root hair tips of wheat.

Analysis of the G-actin pool helps us understand the actin dynamics in cells. Cramer et al. (2002) found that when animal cells were fixed in formaldehyde, fluorescently-labelled DNase I specifically stained G-actin. Moreover, a tip-focused G-actin gradient was found with fluorescent–DNase I labelling in pollen tubes (Li et al., 2001), indicating that fluorescence–DNase I could be used as a probe for G-actin labelling in plant cells. Here using FITC–DNase I labelling, a tip-focused G-actin gradient was also found in the growing tips of root hairs. Using phalloidin and DNase I double labelling, we found that in the root hair tips devoid of the thick F-actin bundles, the concentration of G-actin was higher. These results imply that most F-actin may be depolymerized into G-actin by some factors in the root hairs, supporting the view that actin is very dynamic in the tips of plant tip-growth cells (Fu et al., 2001; Wasteneys and Galway, 2003).

It was reported earlier that there was a tip-focused G-actin gradient in the pollen tube tips (Li et al., 2001). Here a tip-focused G-actin gradient was also found in the growing root hair tips. In addition, the tip-focused G-actin gradient disappeared in full-grown root hairs, as well as when the growth of the root hairs was inhibited by BAPTA-AM. On the other hand, caffeine treatments that inhibited pollen tube tip-growth also led to the disappearance of the G-actin pool from the pollen tube tip (Li et al., 2001). These results suggest that the tip-focused G-actin gradient was related to plant tip-growth. Previous reports also indicated that actin dynamics were essential for plant tip-growth (Jiang et al., 1997; Fu et al., 2001). Based on these results, it is suggested that the tip-focused G-actin pool could provide enough monomer actin for its dynamics to support the tip-growth in both root hairs and pollen tubes.

A high apical Ca2+ gradient was visualized only in growing but not in full-grown root hairs (de Ruijter et al., 1998). Similarly, in the present study, the tip-focused G-actin gradient was found only in growing root hairs of wheat. Thus, the distribution of G-actin is consistent with that of Ca2+ in root hairs. On the other hand, BAPTA is a specific chelating agent for Ca2+. It was found that BAPTA treatments could cause the tip-focused Ca2+ gradient to disappear while inhibiting the tip-growth of root hairs (Herrmann and Felle, 1995; Felle and Hepler, 1997). Here it was found that the BAPTA-AM treatment could also cause the tip-focused Ca2+ gradient to disappear in growing root hairs of wheat (see supplementary material). Additionally, the study reported here shows that BAPTA-AM could also cause the tip-focused G-actin gradient to disappear in root hairs of wheat. Taken together, these results suggest that high Ca2+ concentration may be a key factor for maintaining a tip-focused G-actin gradient by controlling the actin dynamics of polymerization and/or depolymerization in root hair tips.

An interesting question is how Ca2+ maintains a tip-focused G-actin pool in plant tip-growth cells. Since a high concentration of Ca2+ inhibits the F-actin polymerization both in vitro and in vivo (Pollard and Cooper, 1986; Stolz and Bereiter-Hahn, 1988; Constantin et al., 1998), it is possible that in plant tip-growth cells a high concentration of Ca2+ directly causes F-actin depolymerization and thereby maintains the G-actin pool. On the other hand, although Ca2+ was proven to affect F-actin polymerization directly, it could also exert its effect indirectly by controlling actin-binding protein (ABP) activity in vivo (Pollard and Cooper, 1986; Pollard et al., 1994). A few ABPs have indeed been found in plant cells (McCurdy et al., 2001). In addition, profilin and actin-depolymerizing factor (ADF) were also found in the root hair tip (Jiang et al., 1997; Braun et al., 1999; Baluška et al., 2000). Hussey et al. (2002) suggested that ADF could sever F-actin and lead to its depolymerization into G-actin in root hair tips. Kovar et al. (2000) showed that the actin-sequestering activity of plant profilins depended on the concentration of free calcium. They suggested that profilin altered cellular concentrations of actin polymers in response to fluctuations in cytosolic calcium concentration, which could account for the decreased amount of F-actin at the extreme apex of actively growing pollen tubes. Some authors also suggested that ADF and villin might regulate F-actin dynamics in both root hairs and pollen tubes (Tominaga et al., 2000; Smertenko et al., 2001; Allwood et al., 2002; Chen et al., 2002, 2003). It was also found that the activity of villin and ADF was controlled by Ca2+ concentration in plant cells (Gibbon, 2001; McCurdy et al., 2001). Based on these results, it is suggested that a high Ca2+ concentration may cause F-actin depolymerization by controlling ABPs (e.g. profilin, ADF or villin) activity while maintaining a tip-focused G-actin gradient in tips of root hairs and pollen tubes.

SUPPLEMENTARY MATERIAL

Four confocal images of Ca2+ in growing root hairs of wheat are available online at http://aob.oxfordjournals.org, showing Ca2+ forming a tip-focused gradient, and the disappearance of that gradient after treatment with BAPTA-AM.

Acknowledgments

We thank Ms Shi-Wen Li and Mr ChengYuan for their kind help with the confocal microscope observations. This study was supported by the National Natural Science Foundation of China (30421002 and 30470109).

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