Journal of Integrative Plant Biology 2010, 52 (11): 952–958

Research Article

Microfilament Dynamics is Required for Root Growthunder Alkaline Stress in ArabidopsisYue Zhou1,2, Zijun Yang3, Guangqin Guo1 and Yan Guo2∗

1Institute of Cell Biology, School of Life Sciences, Lanzhou University, Lanzhou 730000, China2National Institute of Biological Sciences, Beijing, 7 Science Park Road, Zhongguancun Life Science Park, Beijing 102206, China3Agricultural Technology Center, Huadian 132400, China∗Corresponding author

Tel: +86 10 8072 3279; Fax: +86 10 8072 6671; E-mail: guoyan@nibs.ac.cnAvailable online on 1 July 2010 at www.jipb.net and www.wileyonlinelibrary.com/journal/jipbdoi: 10.1111/j.1744-7909.2010.00981.x

Abstract

The microfilament (MF) cytoskeleton has crucial functions in plant development. Recent studies haverevealed the function of MFs in diverse stress response. Alkaline stress is harmful to plant growth;however, it remains unclear whether the MFs play a role in alkaline stress. In the present study, we find thatblocking MF assembly with latrunculin B (Lat B) leads to inhibition of plant root growth, and stabilizationof MFs with phalloidin does not significantly affect plant root growth under normal conditions. In highexternal pH conditions, MF de-polymerization is induced and that associates with the reduction of rootgrowth; phalloidin treatment partially rescues this reduction. Moreover, Lat B treatment further decreasesthe survival rate of seedlings growing in high external pH conditions. However, a high external pH (8.0)does not affect MF stability in vitro. Taken together, our results suggest that alkaline stress may triggera signal that leads the dynamics of MFs and in turn regulates root growth.

Zhou Y, Yang Z, Guo G, Guo Y (2010) Microfilament dynamics is required for root growth under alkaline stress in Arabidopsis. J. Integr. Plant Biol.52(11), 952–958.

Introduction

Intracellular pH plays an important role in root developmentregulation (Evans and Vesper 1980; Mulkey and Evans 1981).A pH change in root cap cells is associated with root gravityperception and appears essential for generating a gravire-sponse signal (Fasano et al. 2001). Root hair development isregulated by apoplastic and cytoplasmic pH gradient (Bibikovaet al. 1998). High external pH is harmful to plant development(Fuglsang et al. 2007), and cell wall relaxing activities arepredicted to decrease at pH ≥ 7.0 (McQueen-Mason et al.1992). Microfilaments (MFs) in pea and rice root depolymerizerapidly at pH 8.0 or above (Andersland and Parthasarathy1993). The cytoskeleton polymerization of animal cells is alsoregulated by changes in pH (Yonezawa et al. 1985; Suprenant1991; Edmonds et al. 1995). It is reported that pH and MFs co-regulate animal cell polarity and growth (Bernstein et al. 2000;Ghosh et al. 2004).

In plants, recent studies have indicated a close relationshipbetween the MFs and stress responses. Short-term and low-concentration salt stress can induce MF assembly; however,long-term and high-concentration salt stress results in MFdisassembly. The survival rate of Arabidopsis seedlings isincreased by phalloidin under salt and osmotic stress, andMF assembly partly rescues the salt phenotype of Arabidopsissalt-sensitive sos2 mutant (Wang et al. 2009). MF disassemblyis also observed in tobacco cells when they are exposed tolow temperature. MFs change from normal organization topartial de-polymerization, form MF rods or dots, and then arecompletely de-polymerized (Pokorna et al. 2004). MF organi-zation is important in low temperature signal transduction; MFstabilizers inhibit the cold acclimatization specific (cas) geneexpression and calcium influx, indicating that MFs play a rolein early low-temperature signal transduction (Orvar et al. 2000).

It is well known that the plasma membrane H+-ATPases (PMH+-ATPases) are important for establishing and maintaining

C© 2010 Institute of Botany, Chinese Academy of Sciences

Microfilament Dynamics is Required for Alkaline Response 953

trans-plasma membrane pH gradient (Fricker et al. 1997;Palmgren 2001). Blue light, fungal elicitors and other factorshave been reported regulating the activity of PM H+-ATPases(Marre 1979; Spalding and Cosgrove 1992; Kinoshita andShimazaki 1999). Moreover, changes of external pH stimulatethe cytosolic Ca2+ current oscillation (Malayev and Nelson1995). Arabidopsis protein kinase PKS5 negatively regulatesthe activity of the PM H+-ATPases and PKS5 is a componentof a Ca2+-dependent signaling pathway elicited by an alkalineenvironment (Fuglsang et al. 2007). Much evidence has shownthat Ca2+ signals and MFs are closely related, and Ca2+

concentration dynamically regulates MF structure in pollentubes (Chen et al. 2003). However, the relationship betweenMFs and alkaline stress remains unclear.

In the present study, we found that MF disassembly withlatrunculin B (Lat B) inhibits root growth. High external pHinduces MF de-polymerizaion, and MF stabilizer (phalloidin)partially rescues the root growth inhibition by high pH possiblythrough declining the MF disassembly rate. These resultssuggest that MF dynamics is important to root growth underalkaline stress.

Results

MF disassembly results in inhibition of Arabidopsisroot growth

In order to test the MF organization change under high pHconditions, we used a transgenic Arabidopsis plant expressing

Figure 1. Microfilaments (MFs) are important to root growth.

(A) Four-d-old wild type (WT) seedlings grown for 5 d on pH 5.8 MS media containing 1 µM Lat B (left panel), no drug (middle panel), or

1.5 µM phalloidin (right panel).

(B) Relative primary root length of seedlings on pH 5.8 MS media with different MF-targeting drug or no treatment.

Bar indicates 1 cm (A); values are means ± SE (B).

a marker that green fluorescent protein (GFP) was fused toboth the C- and N- termini of ABD2 (Actin-Binding Domain2) of Arabidopsis fimbrin 1 and controlled by a 35S promoter(Wang et al. 2008) as the wild-type (WT) plants in this study.

To confirm that the MF dynamics plays a role in root growth,we treated the seedlings with MF-targeting drugs (disruptionof MF with Lat B and stabilization of MF with phalloidin).Consistent with previous findings, the root growth was remark-ably reduced by Lat B, and it was little affected by phalloidintreatment (Figure 1A).

When the seedlings were treated with 1.5 µM phalloidin, theMF organization had no significant change compared with thatof wild-type plants (Figure 2C). This result is consistent with theobservation that primary root growth is similar with or withoutphalloidin treatment (Figure 1B). The MF was de-polymeriziedby 1 µM Lat B, the disassembly rate at 6 h was 27.7%, andincreasing to 94.4% after 12 h treatment (Figure 2B, C). Theseresults suggest that inhibition of root growth by Lat B is due tothe MF disassembly.

Alkaline stress induces MF cytoskeletonde-polymerizaion in vivo

Actin filaments polymerize in a pH 8.0 buffer, suggestingthat high external pH did not affect MF stability in vitro(Figure 3A); however, high external pH (such as pH 8.0)reduced root growth (Fuglsang et al. 2007). To determine ifthe inhibition is associated with the changes of MF organi-zation under alkaline stress response, 4-d-old seedlings were

954 Journal of Integrative Plant Biology Vol. 52 No. 11 2010

Figure 2. Microfilament (MF) organization is affected by MF-targeting drugs.

(A) MF organization of 4-d-old seedlings in a root cell.

(B) Disassembly rate of MF treated by 1.0 µM Lat B at 3, 6, 12 and 18 h.

(C) MF organization in root cells was observed at 12 h after transferred to pH 5.8 MS media in the presence of 1.0 µM Lat B (left panel),

1.5 µM phalloidin (right panel), or no treatment (middle panel).

Bars indicate 10 µm (A and C); values are means ± SE (B).

transferred to pH 8.0 MS media at different time points and MForganization was observed accordingly. The MF organizationin root mature and elongation zone was observed as a finecytoplasmic network in normal conditions (Figure 3B, C). After6 h incubation on MS medium with pH 8.0, 23.3% of MFswere de-polymerized in root mature and elongation zone cells,and this number reached to 92.2% after 18 h incubation(Figure 3D–F). ACTIN transcription level is very stable and it iswidely used as the internal control to monitor changes of othergene expression. Taken together, our results indicate that highexternal pH induced MF de-polymerization.

Alkaline tolerance requires MF assembly

To further confirm that the inhibition of root growth by alkalinestress is associated with the change of MF organization, LatB and phalloidin were used in the assay. Lat B acceleratedthe MF de-polymerization under high pH conditions (Figure

3D), and this further decreased the survival rate comparedwith that under either high external pH (Figure 4A). However,phalloidin treatment not only significantly reduced the MF de-polymerization by alkaline stress treatment (Figure 3D), but

also reduced the inhibition of root growth (Figure 4C). Ourresults indicate that MF organization plays a vital role in plantresponse to alkaline stress. High external pH did not affectMF stability in vitro but induced MF de-polymerization in vivo,suggesting that alkaline stress may trigger a signal that led toMF reorganization.

Discussion

Microfilaments play important roles in many cellular processes,such as nuclear division and cytokinesis (Staehelin and Hepler1996), polarized growth (Baskin and Bivens 1995), root hairgrowth (Schiefelbein and Somerville 1990) and pollen tubegrowth (Bedinger 1992), and are essential for plant develop-ment (Kost et al. 1999; Volkmann and Baluska 1999; Staiger2000; Staiger and Blanchoin 2006). In the present study, wefound that MFs play an important role in root growth underalkaline stress response.

Alkaline stress leads to plant growth inhibition (Fuglsanget al. 2007). Plant regulates the potential gradients across theplasma membrane mainly by pH PM H+-ATPases (Palmgren2001). Through studying the pks5 mutant, researchers found

Microfilament Dynamics is Required for Alkaline Response 955

Figure 3. High external pH treatment depolymerizes actin filaments in Arabidopsis but does not affect microfilament (MF)

organization in vitro.

(A) F-actin polymerize in pH 8.0 buffer in vitro.

(B) MF organization of 4-d-old seedlings in root mature zone.

(C) MF organization of 4-d-old seedlings in root elongation zone.

(D) Disassembly rate of MF on pH 8.0 MS media with different MF-targeting drugs or no drugs at 3, 6, 12 and 18 h.

(E) MF organization in root mature zone cells were observed at 6 and 18 h on pH 8.0 MS media with MF-targeting drug or no drug treatments.

(F) MF organization in root elongation zone cells were observed at 6 and 18 h on pH 8.0 MS media with MF-targeting drug or no drug

treatments.

Bars indicate 10 µm (A) and 50 µm (B, C, E and F) and; values are means ± SE (D).

956 Journal of Integrative Plant Biology Vol. 52 No. 11 2010

Figure 4. Microfilament (MF) dynamics is important in response to alkaline stress.

(A) The survival rate of 4-d-old seedlings after transferred to pH 8.0 MS media containing 1.0 µM Lat B or not for 5 d.

(B) Relative root growth after transferred to pH 5.8 MS media containing 1.5 µM phalloidin or not for 24 h.

(C) Relative root growth after transferred to pH 8.0 MS media containing 1.5 µM phalloidin or not for 24 h. Significance is determined by

T-test (∗P < 0.01). Values are means ± SE (A, B and C).

that PKS5 phosphorylates the PM H+-ATPase AHA2 in theC-terminal regulatory domain to inhibit AHA2 activity. Highexternal pH triggers an increased concentration of cytoplasmicfree Ca2+ · PKS5 interacts with the SOS3-like Ca2+ bindingprotein SCaBP1 and they perhaps form a part of a calcium-signaling pathway under alkaline stress (Fuglsang et al. 2007).We found that MF stability is not affected in high pH bufferin vitro; however, MF de-polymerization was observed whenseedlings were grown on high pH MS media, which triggersan increase in cytoplasmic pH (Fuglsang et al. 2007). Theseresults suggested that MF dynamics is the downstream eventof alkaline stress response, and that in turn regulates to theroot growth under this stress.

Stabilization of MF organization increases plantalkaline stress tolerance

Recent studies have shown that MF dynamics is associatedwith plant stress responses. A plasma membrane ion ex-changer, NHE1, associates with actin binding proteins andregulates MF organization (Denker et al. 2000). TaADF, ho-mologous to the members of the actin-depolymerizing factor(ADF)/cofilin family, is expressed only in cold-acclimating con-ditions and its protein activity is modulated by low temperature,suggesting that MF rearrangement and cold stress share thesame regulatory protein (Ouellet et al. 2001). Our studiesdemonstrate that MF organization indeed plays a vital rolein alkaline stress tolerance. Survival rate is decreased onpH 8.0 MS media with Lat B treatment. However, the rootgrowth is better on pH 8.0 MS media in the presence ofphalloidin than the absence of this drug, indicating that MFstabilization is important to plant alkaline stress tolerance. It isconsistent with that MF disassembly rate in root that declinedon pH 8.0 media by adding phalloidin.

Materials and Methods

Plant materials

The 35S::GFP-ABD2-GFP line (Wang et al. 2008) was used inall experiments. Seedlings were germinated and grown on MSmedia (pH 5.8) at 23 ◦C under continual illumination. Seedlings(4-d-old) were transferred to different pH MS media with or with-out MF-targeting drugs. All of the seedlings were per-incubatedin actin buffer (100 mM 1,4-Piperazinediethanesulfonic acid(PIPES) , 10 mM ethylene glycol tetraacetic acid (EGTA), 5 mMMgSO4 and 0.3 M mannitol, pH 6.9), containing 1% (w/v)glycerol before being transferred (Olyslaegers and Verbelen1998). The root growth length was measured at 24 h aftertransfer; the survival seedlings were recorded at 5 d aftertransfer.

Confocal laser scanning microscopy

Immunofluorescence images of the MF organization in rootmature and elongation zones were observed at gradient timesand captured with a Zeiss LSM 5 Pascal Confocal. The sampleswere excited at 488 nm using a krypton-argon laser line anddetected using a 505–530 nm bandpass filter. Thirty root cellsof each seedling were observed, and the cells with fine MForganization were recorded. Then the disassembly rate of MF incells was calculated. Statistics for each assay, at least 100 cellsof three seedlings were observed.

F-actin polymerize in vitro

Actin was purified in G buffer (5 mM Tris-HCl, pH 8.0,0.2 mM ATP, 0.1 mM CaCl2, 0.5 mM dithiothreitol (DTT), and0.01% NaN3) using rabbit skeletal muscle acetone powder as

Microfilament Dynamics is Required for Alkaline Response 957

described (Pardee and Spudich 1982). 4 µM F-actin were poly-merized in the presence of equal molar rhoddamine-phalloidinin G buffer. Then the F-actin was diluted and observed.

Acknowledgements

This work was supported by the State Key Basic Researchand Development Program of China (2006CB100100) and theNational High Technology Research and Development Programof China (863) (2003AA210100) to Y.G.

Received 8 Apr. 2010 Accepted 13 Jun. 2010

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