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Plant Physiology and Biochemistry 60 (2012) 88e97

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Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Research article

Loss of membrane fluidity and endocytosis inhibition are involved in rapidaluminum-induced root growth cessation in Arabidopsis thaliana

Jana Krtková a,1, Lenka Havelková a,1, Anna K�repelová b, Radovan Fi�ser c, Stanislav Vosolsob�e a,Zuzana Novotná b, Jan Martinec d, Kate�rina Schwarzerová a,*

aDepartment of Experimental Plant Biology, Faculty of Science, Charles University in Prague, Vini�cná 5, Prague 2, Czech RepublicbDepartment of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, Institute of Chemical Technology Prague, Technická 5, Prague 6, Czech RepubliccDepartment of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Vininá 5, Prague 2, Czech Republicd Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 263, Prague 6, Czech Republic

a r t i c l e i n f o

Article history:Received 26 June 2012Accepted 31 July 2012Available online 9 August 2012

Keywords:Aluminum toxicityArabidopsis thalianaCortical microtubulesEndocytosisPlasma membrane fluidityRoot growth

Abbreviations: Al, aluminum; DMSO, dimethyl sultetraacetic acid; FM4-64, N-(3-triethylammoniumprphenyl)hexatrienyl)pyridinium dibromide; HEPpiperazineethanesulfonic acid; MES, 2-(N-morpholiMurashige and Skoog; MTSB, microtubule-stabilizingered saline.* Corresponding author. Tel.: þ420 221951692; fax

E-mail address: schwarze@natur.cuni.cz (K. Schwa1 These authors contributed equally to this work.

0981-9428/$ e see front matter � 2012 Elsevier Mashttp://dx.doi.org/10.1016/j.plaphy.2012.07.030

a b s t r a c t

Aluminum (Al) toxicity is the main limiting factor in crop production on acid soils. The main symptom ofAl toxicity is a rapid inhibition of root growth, but the mechanism of root growth cessation remainsunclear. Here we examined the earliest changes in the plasma membrane and processes related to themembrane in the Arabidopsis thaliana root tip cells of roots grown in a hydropony. Al suppressed rootgrowth within 2 min, inhibited endocytosis within 10 min of exposure and stabilized cortical microtu-bules within the first 30 min. Spectrofluorometric measurements of the plasma membrane isolated fromArabidopsis plants and labeled with the fluorescent probe laurdan showed that Al induced a reduction inmembrane fluidity. Application of the membrane fluidizer, benzyl alcohol, restored partially membranefluidity and also partially restored root growth during first 30 min of Al treatment. We concluded thatAl-induced loss of membrane fluidity and endocytosis inhibition occurred very early during Al toxicity inplant roots and could be the earliest targets of Al treatment.

� 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

Aluminum (Al) toxicity limits crop production in areas with acidsoils worldwide. Al is bound to soil minerals in an insoluble form,but is released in its toxic form when the pH of the soil solutiondrops. Al is toxic for both plants and animals [1]. The first symptomof Al toxicity in plants has long been known to be a rapid inhibitionof root growth [2e4], with the root tip being themost sensitive part[5]. Long-term exposure to Al results in changes in rootmorphology, such as root thickening, cracking, changes in thecomposition of the cell wall, and eventually death of root cells[6e8].

foxide; EGTA, ethylene glycolopyl)-4-(6-(4-(diethylamino)ES, 4-(2-hydroxyethyl)-1-no)ethanesulfonic acid; MS,buffer; PBS, phosphate-buff-

: þ420 22195 1704.rzerová).

son SAS. All rights reserved.

Although Al toxicity has been recognized as a limiting factorfor plant growth for several decades, the mechanism of the rapidinhibition of root growth remains unclear. Al-induced changes inroots and root cells begin within seconds or minutes of exposure,suggesting that the affected cellular structure is a direct target ofAl. Indeed, Al enters Arabidopsis roots predominantly via meri-stem and distal elongation zone with the highest rate occurringwithin first 30 min of exposure [9]. Recently, the Al3þ transporterwas described in rice [10], which indicates that intracellular Al ispossibly involved in early toxic effects. Changes that becomedetectable after hours or days of exposure, however, need to beinterpreted cautiously. They could be the result of long-termdirect interaction of Al with the given cellular structure, oralternatively a consequence of previous damage of anotherstructure.

Al is known to influence numerous processes and structures inplant cells, raising the possibility that it affects multiple targets.The cell wall, the first barrier for Al entry into plant roots, bindssubstantial amounts of Al, which impairs its plasticity and waterpermeability [11e13]. The rapid response of root growth alsoevokes the involvement of a signaling mechanism. Indeed, Al wasshown to inhibit components of phosphoinositide signalingpathway [14e18] and to disrupt homeostasis of Ca2þ, an

Fig. 1. Root growth cessation by Al. Growth was measured during the first 15 min forroots in standard MS media (pH 5.8) and following perfusion with test media in 1%sucrose. Perfusion with 1% sucrose alone, at either pH 4.0 or pH 5.8, caused rapidincrease in growth speed. Perfusion with 1% sucrose containing 50 mM AlCl3, pH 4.0,substantially reduced the growth speed, whereas perfusion with 1% sucrose containing100 mM AlCl3, pH halted growth almost completely within 2 min of application.

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important second messenger (for review see [19]). The role of thecytoskeleton in Al toxicity has received much attention due toits role in processes of cell elongation and cell shaping. Micro-tubules in root cells were reorganized or disrupted upon Altreatment [20e22], and actin cytoskeleton was influenced as well[23e25].

Another main target is the plasmamembrane, which binds largeamounts of Al due to its negative charge [26], resulting in modifi-cations inmembrane properties including disruption of the bindingand transmembrane transport of physiologically important ions(reviewed in [27]). Indeed, disrupted function of ion channels andaberrant ion transport through the membrane have been reportedrepeatedly (for review see [28]). One of the earliest responses to Alis an almost instantaneous depolarization of the plasmamembrane[22,24,29]. Lipid peroxidation has also been described to accom-pany Al toxicity in various plants such as maize and rice [30,31],although this was not regarded as the immediate cause of growthinhibition in pea roots [32]. The central role of plasmamembrane inAl toxicity in biological systems is consistent with the observationof substantial decrease in the fluidity of membranes reconstitutedin vitro from brain cells lipids [33,34]. Importantly, plasmamembrane is a barrier for the transport of Al ions into the sym-plasm. The influx rate of Al ions is rather slow, nevertheless, smallamounts of toxic Al ions are allowed to enter the symplasm withinminutes [27]. Therefore, the understanding Al-induced changes ofthe plasma membrane is essential also for studies of the Al trans-port into the symplasm.

Rapid onset of Al-induced changes in the plasma membranecould initiate some of the symptoms of Al toxicity through themechanism of signaling, which is closely related to plasmamembrane properties. Some components of phosphoinositidesignaling pathways have been shown to be affected by Al-inducedchanges in membrane properties [35,36]. Signaling, in turn,relates to the cytoskeleton. Phospholipase D, for example, isa signaling molecule that is associated with microtubules [37,38].Moreover, cortical microtubules and the actin cytoskeleton areknown to interact functionally and physically with the plasmamembrane [39e41]. It is therefore possible that some changes inthe organization of the cortical cytoskeleton could be the result ofaltered cell walleplasma membraneecytoskeleton interaction[42] rather than direct interaction of Al with cytoskeletalcomponents.

In this study, we focused on early symptoms of Al toxicityrelated to the plasma membrane using hydroponic culture ofArabidopsis thaliana. Using a unique system of root growth in vivoobservation under the microscope, we were able to detect asearly response as the root growth inhibition few minutes after Aladdition to the growth medium. We searched for processes inroot cells that were inhibited or disrupted within the scale ofminutes after Al treatment and we found out that endocytosis inroot cells was inhibited after 10 min and the cortical microtubulecytoskeleton became stabilized against low pH-induced disrup-tion during the first 30 min. Using in vitro root growth observa-tion under the microscope we further showed that drugsdisrupting the vesicle trafficking led to root growth retardation,whereas the drug taxol stabilizing microtubules did not influenceroot growth processes during first 30 min. Further we showedthat Al rapidly reduced the fluidity of isolated plasma membrane.The plasma membrane rigidization could be reversed bymembrane fluidizer, benzyl alcohol, and application of benzylalcohol to Al-treated roots restored partially the root growth.These results are, to our knowledge, first that indicate thatmembrane remodeling and membrane-related processes such asthe vesicle trafficking might be one of the earliest targets of Altoxicity.

2. Results

2.1. Inhibition of root growth occurs within two minutes ofexposure to Al

Arabidopsis seedlings, grown for 3e4 days on standard MSmedia on inclined agar plates, were transferred into miniaturegrowth chambers for hydroponic infiltration with test media andfor microscopic observation. In order to avoid potential interactionsof Al ions with components of MS growth media, we useda minimal medium of 1% sucrose for Al experiments. The pH wasmaintained at 4.0 in all experiments unless stated otherwise.

A typical growth curve for roots of Arabidopsis seedlings grownfor 15 min in standard MS medium (pH 5.8) in the hydroponiccultivation system is shown in Fig. 1. A distinct increase in the rootelongation rate occurred following perfusion with 1% sucrose,which was independent of the pH of medium. Perfusion with 1%sucrose supplemented with aluminum caused dramatic inhibitionof root growth, which became noticeable already within twominutes of application and was sustained over the 30 min of Altreatment. The inhibition caused by 100 mMAlCl3 was stronger thanthe effect of 50 mM AlCl3 that inhibited root growth to a lesserdegree (Fig. 1).

Since Al toxicity is known to be accompanied by depolarizationof the plasma membrane, we applied the voltage-sensitive fluo-rescent probe DiSBAC2(3) [43] to evaluate this effect. Plasmamembrane depolarization occurred almost immediately afterperfusion with 100 mM AlCl3 (see Fig. S1), indicating that Arabi-dopsis roots cultivated in our hydroponic system were suitable forinvestigation of Al toxicity when compared with other methods ofcultivation.

2.2. Al increases plasma membrane rigidity, which contributes tothe inhibition of root growth

Microsomal membranes were prepared from Arabidopsis seed-lings, and the plasma membrane fraction purified by sequentialtwo-phase partitioning according to [44]. Fluidity of the plasmamembrane was evaluated using spectrofluorometry of the

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fluorescence probe laurdan [45]. Generalized polarization spectrashowed that addition of AlCl3, pH 4.0, to the isolated plasmamembranes clearly increased membrane rigidity in a concentra-tion-dependent manner when compared with low pH media only(Fig. 2A). This Al-induced rigidization could be partially reversed byapplying the membrane fluidizer, benzyl alcohol (Fig. 2B).

In order to test if the increased plasma membrane rigidity couldexplain the inhibition of root growth by Al, we evaluated the effectof adding benzyl alcohol to the Al treatments. Fig. 2C shows thatroots increased their root growth rate to 200% relative to that in theMS medium when grown in 1% sucrose of pH 4.0 or 5.8, indicatingthat the root growth acceleration during first 30 min was not pH-dependent. Addition of benzyl alcohol (10 mM final concentra-tion) to 1% sucrose at pH 4.0 without Al for 30 min did not altersignificantly the growth rate, which reachedw200% relative to thatin the MS medium at pH 5.8. Supplementing the 1% sucrosemedium at pH 4.0 with either 50 mMAlCl3 or 100 mMAlCl3 reducedthe growth rates to w30% or w7%, respectively, relative to those inthe control MS medium at pH 5.8, in agreement with the Al-induced growth inhibition obtained above (Fig. 1). Supplementingthe Al-containing media with benzyl alcohol partly restored the

Fig. 2. Al-induced membrane rigidization is involved in the inhibition of root growth. (A, B)the fluorescent probe laurdan. Membranes were washed in acetate buffer (37 mM sodiumspectrum was recorded (control). Subsequently, AlCl3, benzyl alcohol (BA), or a combinationthe excitation spectra were recorded again. For each sample two excitation spectra were recGeneralized polarization was calculated for each point of the excitation spectrum (see Materfirst 30 min of treatment with 1% sucrose of pH 4.0 and 5.8, and 1% sucrose supplemented wunless stated otherwise. Slopes of growth curves were calculated for each root, with the slopmedium. Error bars ¼ SE, n ¼ 10.

growth rates to w60% and w30% relative to 50 and 100 mM AlCl3treatments, respectively (Fig. 2C).

2.3. Endocytosis in epidemal root cells is inhibited within 10 min ofexposure to Al

Fluorescent probe FM4-64 was used for in vivo observation ofendocytic vesicles formation [46]. In control roots labeled with4 mM FM4-64, the probe incorporated into the plasma membranesof epidermal root cells and gave an intense fluorescence, and theprobe was internalized by means of brightly fluorescing endocy-totic vesicles (Fig. 3A). However, immediately after perfusion witha labeling medium supplemented with 100 mM AlCl3, the fluores-cence in the plasma membranes at the cell periphery becamequenched although fluorescence of endosomes in the cytoplasmappeared to persist (Fig. 3B).

The quenching may be the result of Al-induced changes in theplasma membranes, or alternatively Al may interact directly withthe probe and thereby affect its fluorescence properties. In order toavoid the second possibility, we first pre-treated the roots witha medium containing 100 mM AlCl3. After this, aluminum was

Spectrofluorometric analysis of isolated Arabidopsis thaliana plasma membranes usingacetate, pH 4.5), labeled with laurdan (see Materials and methods) and the excitationof both was added from stock solutions to achieve the desired final concentration, andorded in the range 320e410 nm with different emission wavelengths 430 and 480 nm.ials and methods for details). (C) The rate of root growth of Arabidopsis thaliana duringith AlCl3 and benzyl alcohol at specified concentrations. The pH of the mediumwas 4.0e in the test medium expressed as a percentage of the slope of the same root in the MS

Fig. 3. In vivo observation of fluorescence probe FM4-64 in Arabidopsis root tip cells reveals inhibition of endocytosis within 10 min of exposure to Al. (A, B) FM4-64 quenching inplasma membrane in the presence of Al. (A) A single confocal section through the epidermal cell layer of a root tip treated with 4 mM FM4-64 in 1% sucrose at pH 4.0 for 20 min.Brightly stained plasma membranes and incorporated endosomal vesicles are visible (inset in A). (B) The same root immediately after perfusion with 4 mM FM4-64 in 1% sucrosesupplemented with 100 mM AlCl3, pH 4.0. Al treatment markedly reduced FM4-64 fluorescence in the plasma membranes, although the fluorescence in the endosomes (inset in B)remained unaffected. (CeF) Inhibition of endocytosis by Al. For controls, the roots were pre-incubated with 1% sucrose at pH 4.0 for 30 min, followed by perfusion with 1% sucrosesupplemented with 4 mM FM4-64 (pH 5.8) for 10 min (C). For Al treatments, the roots were pre-incubated with 1% sucrose, pH 4.0, supplemented with 100 mM AlCl3 either for 5 min(D), 10 min (E), or 20 min (F), followed by perfusion with 4 mM FM4-64 in 1% sucrose (pH 5.8) without Al. Images are single confocal sections through the epidermal cell layer of theroot tip. Arrowheads indicate endocytotic vesicles in the cytoplasm. Scale bars ¼ 20 mm.

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washed and the roots were incubated with 4 mM FM4-64 for 10-min and observed under the confocal microscope. A short pre-treatment (5 min) with Al did not inhibit noticeably the internali-zation of FM4-64 into endosomes (Fig. 3D). A 10-min pre-treatmentwith Al, however, partially inhibited the endocytotic processes(Fig. 3E) and 20-min treatment resulted in complete endocytosisinhibition and no endosomes were seen (Fig. 3F). The pre-treatment with medium of low pH of 4.0 did not by itself affectthe endocytotic processes (Fig. 3C). Since the FM4-64 convincinglyincorporated into the plasmamembranes, we concluded that the Alpre-treatment for the minimum of 10 min induced changes in theplasma membrane, which persisted even after the Al removal fromthe medium, and prevented the internalization of the probe byendosomes.

2.4. Al stabilizes cortical microtubules against low pH-induceddisorganization

Immunofluorescence visualization of the microtubule cyto-skeleton using anti-a-tubulin antibody and confocal microscopyshowed that microtubules in lateral root-cap cells of the root tip inthe control MS medium at pH 5.8 formed typical, transverselyoriented parallel arrays (Fig. 4A). Perfusion with MS medium at pH4.0 for 30 min resulted in substantial randomization and depoly-merization of microtubules (Fig. 4B) whereas perfusion with 1%sucrose at pH 5.8 had no effect (Fig. 4C), suggesting low pH ratherthan medium composition is responsible for disruption of themicrotubule array. Indeed, perfusion with 1% sucrose at pH 4.0resulted in substantial disorganization of the array within 30 min(Fig. 4D) followed by almost complete disorganization or loss ofmicrotubules within 1 h (Fig. 4E). In contrast, most of the micro-tubules remained in ordered transverse orientation in the presenceof 100 mM AlCl3 at pH 4.0 for 30 min (Fig. 4F). Transverse order wasmaintained beyond 30 min, although some diffuse fluorescence,

indicative of microtubule depolymerization, began to appearthroughout the cytoplasm after 1 h incubation and fewer micro-tubules in thinner bundles remained (Fig. 4G). Almost completemicrotubule depolymerization together with diffuse cytoplasmicfluorescence were seen after 6 h incubation (Fig. 4H). Clearly, Alstabilizes cortical microtubules in the lateral root-cap cells againstlow pH-induced randomization during the first 30 min of exposure,although gradual disintegration of microtubules occurs subse-quently during prolonged exposure. This effect was observed alsoin epidermal cells of the root tip (data not shown).

2.5. Inhibition of microtubule dynamics does not influence the rootgrowth of A. thaliana within 30 min of treatment

Al induced root growth inhibition within 2 min in our experi-ments, which suggested that a fast process or processes needed forroot growth were immediately affected by Al. We further observedthat Al clearly stabilized microtubules and inhibited endocytosiswithin first 30 min of treatment of Arabidopsis roots. Could changesin the microtubule network and the inhibition of endocytosis beinvolved in such a rapid root growth inhibition? To answer thisquestion, we next determined the impact of microtubules stabili-zation and the vesicle trafficking inhibition on the root growthwithin first 30 min. An inhibitor of microtubule dynamics (taxol)and an inhibitor of vesicle trafficking (brefeldin A) were used. Ourresults showed that the stabilization of microtubules with 40 mMtaxol had no effect on root growth rate during first 30 min (Fig. 5A).Roots increased their growth rate tow200%when perfusedwith 1%sucrose, pH¼ 5.8 with the addition of taxol, which was comparableto respective controls (see Fig. 2C for comparison). Consistentlywith this, the root growth rate in the medium supplemented withtaxol always showed control-like near-linear course (Fig. 5B).Treatment with 50 mM brefeldin A, however, led to the root growthretardation when compared to control (Fig. 5A; compare to 1%

Fig. 4. Al-treated root cells display transverse microtubules during first 30 min. Indirect immunofluorescence visualization of microtubules lateral root cap cells of Arabidopsisthaliana seedlings using anti-a-tubulin antibody and confocal microscopy. Images are optical sections through the cortical region of the cells. Roots treated with MS medium, pH 5.8(A), and pH 4.0 (B), 1% sucrose, pH 5.8 (C), 1% sucrose, pH 4.0 (D) for 30 min, 1% sucrose, pH 4.0 for 1 h (E), 1% sucrose supplemented with 100 mM AlCl3, pH 4.0 for 30 min (F), 1 h (G),and 6 h (H). Scale bars ¼ 20 mm.

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sucrose, pH ¼ 5.8 in Fig. 2C). Each root grown in the test mediumwith brefeldin A displayed a clear tendency of gradual growthretardation with heavily retarded growth at the end of the studiedperiod of 30 min (Fig. 5C).

Fig. 5. Inhibition of vesicle trafficking, but not microtubule stabilization, results in root growfirst 30 min of treatment with 1% sucrose supplemented with 40 mM taxol or 50 mM brefeldinin the test medium expressed as a percentage of the slope of the same root in the MS mediumbrefeldin A suppressed the root growth during first 30 min of the treatment. Error bars ¼ S50 mM brefeldin A (C). Growth was measured during the first 15 min for roots in standard Mroots showed near linear course of the growth rate, (B), whereas brefeldin A-treated roots

Since vesicle trafficking inhibition by brefeldin A resulted in rootgrowth retardation within 30 min, we concluded that processes ofvesicle transport, which include endocytosis as well, are necessaryfor rapid root growth in 1% sucrose during first 30 min. On the

th retardation within 30 min. (A) The rate of root growth of Arabidopsis thaliana duringA (BFA), pH ¼ 5.8. Slopes of growth curves were calculated for each root, with the slope. Whereas taxol treatment did not affect root growth that was comparable to controls,

E, n ¼ 6. (B, C). Growth curves of representative roots treated with 40 mM taxol (B) orS media (pH 5.8) and following perfusion with test media in 1% sucrose. Taxol-treateddisplayed gradual growth retardation during the test period of 30 min (C).

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contrary, our experiments showed that the inhibition of microtu-bule dynamics does not influence the root growth rate during first30 min of the treatment.

3. Discussion

3.1. Al rapidly inhibits Arabidopsis root growth and inducesdepolarization in root tip cells

Although Al toxicity has been studied for many years, theimmediate cause of Al-induced root growth inhibition is notknown. Using Arabidopsis seedlings and a miniature hydroponiccultivation system, we examined the earliest changes in plasmamembrane fluidity, endocytosis, and organization of the corticalmicrotubule cytoskeleton in the root tip. The hydroponic cultiva-tion enabled us to change quickly the growth medium withoutinducing mechanical stress in the roots, it ensured rapid anduniform access of test solutions to the roots, and it facilitatedimmediate observation of the roots under a microscope. For short-term treatments (minutes), the solution of 1% sucrose was used inorder to avoid possible interactions of Al ions with components ofthe growth media. Under the test conditions, the concentration ofpositively charged Al forms in the medium was >80 mM and>40 mM for 100 mM and 50 mM AlCl3, respectively.

Inhibition of root growth and depolarization of the plasmamembrane represent two of the earliest phenomena of Al toxicitydescribed in roots of various plant species [34,47]. In our hydro-ponic cultivation system, a dramatic inhibition of root growthbecame apparent during the first two minutes of exposure to100 mM AlCl3, which is, to our knowledge, the fastest growthresponse to Al reported so far. Membrane depolarization, detectedby increased fluorescence of the transmembrane potential fluo-rescence probe DiSBAC2(3), was likewise detectable during the firstfew minutes of treatment (see Fig. S1). The fluorescence probe forAl-induced membrane depolarization was first used by Sivaguruet al. [48] who detected extensive depolarization of plasmamembrane after 60min of Al treatment in suspension tobacco cells.Our measurements with DiSBAC2(3) showed almost immediatedepolarization of plasmamembrane in root tip epidermal cells of A.thaliana, which corresponds to fast changes of plasma membraneobserved in root cells in response to Al [22,24]. Therefore, theeffects of Al observed in our experimental model are in goodaccordance with the effects described in other plant species andexperimental conditions.

The dramatic inhibition of root growth by Al was in sharpcontrast with the acceleration of growth in the minimal medium of1% sucrose without Al, which produced almost 200% increase ingrowth rate relative to that in MS medium. The acceleration ofgrowth could be the result of the sudden change of replacing theMSmediumwith the osmotically less active minimal medium of 1%sucrose, leading to turgor pressure-driven acceleration in cellexpansion. After 24 h of cultivation, plants cultivated in 1% sucroseshowed reduced root length relative to that in MS medium (seeFig. S2), consistent with the observation that the acceleration ofgrowth was transient. Nevertheless, considering the initial accel-eration of growth, the Al-induced inhibition was remarkablyeffective.

3.2. Al-induced membrane rigidization contributes to the inhibitionof root growth

Al was reported to increase membrane rigidity of membranesreconstituted in vitro from synthetic lipids or lipids isolated frombrain tissues [33]. Similarly, our spectrofluorometric measurementsshowed that Al increased the rigidity of A. thaliana plasma

membranes in vitro, which is consistent with the above report.During in vitro spectrofluorometric measurements, the membranefluidity decrease occurred immediately after Al addition. The effectof Al on membrane fluidity is immediate also in vivo, because theaddition of benzyl alcohol, which alleviates the rigidization effect ofAl, resulted in immediate root growth reconstitution (Fig. 2C).These results demonstrated that Al-inducedmembrane rigidizationcontributes substantially to the rapid cessation of root growthduring Al stress. The ameliorating effect of benzyl alcohol occurringin roots treated with all concentrations of Al used in our studyclearly indicates that Al concentrations used were physiologicallyacceptable and pertinent for this type of study.

Since the application of benzyl alcohol only partially restoredroot growth, membrane rigidization was probably not the onlycause of root growth inhibition. Indeed, the ameliorating effect ofbenzyl alcohol was absent during prolonged cultivation of roots inAl-containing medium (see Fig. S2). This can be explained by thefact that the effect of Al on plasma membrane fluidity could triggersome other effects during early phases of Al stress. For instance,oxidative stress was reported to occur early during Al exposure inroot cells. As demonstrated by Kaneko et al. [49] and Verstraetenand Oteiza [34], Al-induced packing of lipids in the membranes isa contributing factor in the enhancement and propagation of lipidperoxidation. Oxidative stress response observed in plant cells thusmay be the result of increased membrane rigidity induced by Al.

Reduced membrane fluidity may also directly influence phos-phoinositide metabolism in the plasma membrane. For example,PIP2 hydrolysis and PI-PLC activity in in vitro reconstituted lipo-somes were inhibited due to the Al-induced loss of membranefluidity [36]. Therefore, we suggest that the plasma membranerigidization probably lies at the beginning of the cascade of eventsthat occur during Al stress in root cells. Considering the biologicalimportance of the plasma membrane, we hypothesize that theeffect of Al on its properties could underlie Al toxicity in eukaryotesin general.

3.3. Endocytosis is inhibited within 10 min of Al stress

In order to search for another early effect of Al-treatment thatcould be responsible, together with the loss of membrane fluidity,for rapid root growth inhibition, we studied the process of endo-cytosis in epidermal cells of the root tip using the fluorescentmarker FM4-64 [50]. Pre-treatment with 100 mM Al for 10 min wassufficient to induce changes at the plasma membrane that resultedin the inhibition of endosomes formation even after the Al washout.Al-induced inhibition of FM4-64 internalization by endosomes inArabidopsis root cells was previously reported by Illes et al. [51] asan absence of brefeldin A-induced, fused endosomal compartmentsfollowing a 90-min treatment with Al. In root meristematic cells ofAl-sensitive variety of maize, however, the formation of brefeldin Acompartments was blocked within 30 min of Al treatment [25].Together with our experiments, the results suggest that Al-inducedblockade of endocytosis is a fast process depending on plantmaterial sensitivity and experimental conditions of Al toxicity.

In order to answer a question if endocytosis could be respon-sible for quick root growth inhibition within minutes, we used thedrug brefeldin A that interferes with the vesicle trafficking to studyits early effect on root growth. Brefeldin A inhibits anterogradetransport from endoplasmic reticulum to Golgi apparatus andinduces internalization of rapidly recycling plasma membraneproteins and their accumulation in brefeldin A-induced compart-ments, thus inhibiting also endocytic recycling of plasmamembrane proteins [52,53]. Our experiments using 50 mM bre-feldin A showed that the inhibition of vesicle transport in root cellsresulted in root growth retardation during first 30 min of the

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treatment. Root growth cessation was gradual with distinct retar-dation at the end of 30 min (a typical growth rate curve is shown inFig. 5C). This suggests that active processes of vesicle traffickingwere necessary for root growth. It is important to mention thatbrefeldin A is a general inhibitor of vesicle trafficking, whereas ourexperiments showed that endocytotic pathway is affected by Al andother pathways were not investigated. Nevertheless, our resultsclearly indicated that vesicle trafficking might be one of theprimary targets of Al stress that could contribute to Al-induced rootgrowth inhibition during first 30 min.

3.4. Al stabilizes microtubules against low pH-induced disruption

Cortical microtubules are cytoskeletal structures found underthe plasma membrane. The tight interaction of cortical microtu-bules with the plasma membrane is necessary for the control of thecell wall remodeling in growing cells [54]. As dynamic structures,microtubules can quickly response to extracellular factors and thusmediate a fast growth response. Several studies have reportedreorganization, disorganization, or stabilization of microtubulesduring Al treatment in various plant cells and tissues[7,20,22,24,55]. This variation can be attributed to several factorsincluding tissue-specificity, species-specificity, physiologicalmaturity of the tissue, and the severity and duration of the Altreatment. For example, Sivaguru et al. [24] reported that 90 mM Altreatment disrupted microtubules in cells of the distal part of thetransition zone in maize roots, while no changes in microtubularorganization were observed in cells of elongating zone after 1 h. InArabidopsis roots treated with 100 mM Al, Shen et al. [56] did notobserve substantial changes in cortical microtubules after 2 h ofincubation. In our experiments, the treatment with 100 mM Alclearly stabilized cortical microtubules in lateral root cap cells orepidermal cells in the root tip during the first 30 min of exposureagainst low pH-induced randomization. The randomization ordepolymerization of microtubules in response to low pH wasdescribed in Nitella internodes [57] and lettuce roots [58]. In thelatter study, low pH-induced randomization of microtubulesinduced the formation of root hairs [58], presumably because themicrotubule reorganization is needed for root hair initiation [59].The reorganization of microtubules induced by low pH can betherefore considered as a physiological reaction, whichwas blockedby Al in our experiments. The Al-induced initial stabilization ofmicrotubules in our experiments was followed by gradual disor-ganization and depolymerization over the period of 6 h, after whichonly few microtubules remained.

A question arose if fast changes in cortical microtubule arraysinduced by Al can be associated with the fast root growth inhibitionby Al. Our further experiments suggested that it was not the case.First, the randomization and depolymerization of microtubulesoccurred at low pH irrespective of the type of the medium, whereasacceleration of root growth occurred only in the minimal mediumof 1% sucrose in our experiments. Therefore, the cell expansionwasuncoupled from the organization of the cortical microtubule arrayin this case and transversally oriented cortical microtubules werenot required for rapid root growth. Similar phenomenon was sug-gested also by Sainsbury et al. [41] and Sugimoto et al. [60]. Second,in order to study a possible involvement of microtubule stabiliza-tion in the root growth inhibition, we exposed roots to 40 mM taxol.We observed that during first 30 min of the treatment, no retar-dation of the growth occurred and the root growth rate increased tonearly 200%, suggesting that root growth processes were uncou-pled from the organization of microtubule arrays also in this case.

Al-induced destabilization of microtubules later during Al stresscould play an important role during prolonged Al stress (hours todays of exposure). Similarly to oryzalin-induced destabilization of

microtubules that resulted in changes of root morphology [61], theloss of microtubules during prolonged exposure to Al could berelated to changes in root morphology observed in long-termexperiments with Al toxicity. However, our experiments sug-gested that neither the loss of microtubules observed in rootsgrown in the medium with low pH, nor microtubule stabilizationwith 40 mM taxol at pH 5.8 had an immediate effect on the rootgrowth rate during first 30 min of exposure. Under both conditions,roots exhibited increased root growth comparable to controlmedium of 1% sucrose, pH 5.8. Therefore we concluded that Alinduced early changes in cortical microtubules in Arabidopsis rootcells, but that this effect was probably not responsible for rootgrowth inhibition during first 30 min of Al treatment. The potentialsignificance of microtubule polymer status in relation to phos-pholipase D signaling and its role in vesicular transport [61]remains to be established.

3.5. Multiple target sites of Al in plant cells

Using themethod of precise timing of the root tip treatment andthe root growth rate measurements we showed that the loss ofplasma membrane fluidity and endocytosis inhibition wereinvolved in quick root growth inhibition under Al stress. The rela-tionship between Al-induced membrane fluidity changes andendocytosis remains to be tested, however, our results support thehypothesis that Al may interfere with target multiple processes andsites in plant cells that may be responsible for quick inhibition ofthe root growth. For example, it is clear that Al targets apoplasticstructures as well (for review see [27]), where pectins were shownto bind substantial portion of Al [62] and their Al-induced redis-tribution coincided with root growth inhibition in maize [63]. Moreresearch is needed to identify all factors contributing to root growthinhibition under Al stress.

3.6. Conclusion

In conclusion, we have shown that Al induced loss of fluidity ofthe plasma membrane, the inhibition of endocytosis in theepidermal cells, and induced changes in microtubular organizationin the lateral root cap cells and epidermal cells of the Arabidopsisroot tip within the scale of minutes. We further showed that theloss of plasma membrane fluidity was involved in quick rootgrowth inhibition within 30 min and we propose that the plasmamembrane and membrane e related processes such as vesicletrafficking are early targets of Al toxicity in root cells.

4. Materials and methods

4.1. Hydroponic cultivation of Arabidopsis seedlings

A. thaliana Col-0 seeds were surface-sterilized, stratified insterile distilled water at 4 �C for 3 days, sown on an inclined agargrowth medium of half MS (2.2 g l�1 of MurashigeeSkoog [MS]salts, 5 mg/l of thiamine, 0.5 mg l�1 of nicotinic acid, 50 mg l�1 ofinositol, 0.5 g l�1 of pyridoxine, 500 mg l�1 of MES, 10 g l�1 ofsucrose and 16 g l�1 of agar, pH¼ 5.8), and cultivated at 25 �C under16-h day length for 3e4 days. For microscopic observations, seed-lings were transferred into miniature growth chambers (‘mini-chambers’) assembled under sterile conditions using a microscopeslide and a coverslip separated by two strips of folded parafilm,making sufficient space for root growth [64]. The mini-chamberswith seedlings were placed in a vertical position into a glasscontainer with liquid growth medium as above but without theaddition of agar, and the seedlings maintained for one day under

J. Krtková et al. / Plant Physiology and Biochemistry 60 (2012) 88e97 95

the same conditions as above to allow resumption of normal rootgrowth.

4.2. Treatment with Al

For Al treatments, a stock solution of 1 mM AlCl3 in distilled H2Owas used to prepare the final concentrations and pH was adjustedto 4.0e4.5. To avoid possible interactions of Al ions withcomponents of the growth media, we routinely employed a testmedium 1% sucrose with pH adjusted to 4.0e4.5. Under theseconditions almost all Al in the solution is expected to be present inthe monomeric form [65]. Concentrations of positively chargedforms of Al in the test mediumwere measured using the separationon the catex as described by Navrátil [66]. According to repeatedmeasurements the concentration of positively charged Al formswas >80 mM for solutions prepared as 100 mM and >40 mM forsolutions prepared as 50 mM.

4.3. Root growth measurement

Mini-chambers with Arabidopsis seedlings were placed inOlympus Provis AX 70 microscope (Olympus Optical Co., Ltd.,Japan) and perfused with fresh growth medium. The growing roottip was then scanned for 15 min at 1-min intervals using the imageacquisition analysis software NIS Elements (Laboratory ImagingPrague, Czech Republic). Seedlings were then perfusedwith the testsolution and the growth of root tips recorded again at 1-minintervals for a total of 30 min. Test solutions containing inhibitorswere prepared from stock solutions of 10 mM taxol (Paclitaxel; MPBiomedicals, Irvine, California, USA) in DMSO, or 20 mM BFA(Sigma) in ethanol by adding appropriate volumes directly to testmedia to obtain the required final concentrations.

The data for increase in root length obtained by the NISElements were plotted in a graph (Fig. 1), and the slopes of growthcurves under control (MS) medium and test (Al) conditions wereseparately calculated. For each root, the slope obtained with testmedium was expressed as a percentage of the slope in the controlmedium, taking the control slope as 100% (Fig. 3C).

4.4. Monitoring of endocytosis

Endocytosis in root cells was monitored using the fluorescenceprobe FM4-64 (N-(3-triethylammoniumpropyl)-4-(6-(4-(dieth-ylamino)phenyl)hexatrienyl)pyridinium dibromide; Invitrogen,Life Technologies). A stock solution of 10 mM FM4-64 in DMSOwasdiluted in the test medium to 4 mM final concentration. Roots ofseedlings in themini-chamber were perfused with the test solutionand incubated for specified times. Subsequently, the test solutionwas replaced by perfusion with 1% sucrose (pH 5.8) supplementedwith 4 mM FM4-64, incubated for 10 min, and root tip epidermalcells were observed with a laser scanning microscope (Leica TCSSP2) at 514 nm excitation, 650e750 nm emission, and a 63x Pla-nApo NA 1.2 water immersion objective lens.

4.5. Immunostaining of microtubules

Arabidopsis seedlings grown in a modified sterile hydroponiccultivation system were used. A large coverslip was laid on amicroscopic slide and secured together with two threads. 2% (w/v)agar solution in distilled water was pipetted along the longer edgeof the coverslip and surface-sterilized WT seeds were sown ontothe agar edge. Microscopic slides were placed vertically into a glasscultivation box, liquid MS medium was poured into the cultivationbox up to the level of the agar edge with seeds, and cultivated for4e5 days under the same conditions as described above. For Al

exposure, microscopic slides with seedlings were placed in a newcultivation box so that the roots were immersed in the liquid testmedium, and incubated for specified times.

Treated seedlings were processed for immunofluorescencelocalization of microtubules using a modified method described in[60]. The seedlings were fixed with 4% paraformaldehyde in MTSBbuffer (50 mM PIPES, 5 mM EGTA, 5 mMMgSO4$2H2O; 0.1% Triton-X100, pH 7.0) under low pressure (400e500 kPa) for 2.5 h. Seed-lings were washed with MTSB (3� 10 min) and digested for 20 minwith 0.05% pectolyase Y-23 (Kyowa Chemical Products Co., Ltd,Osaka, Japan) in MTSB containing 0.4 M mannitol and proteaseinhibitors (0.3 mM PMSF, 10 mM Leupeptin, and 1.25 mM Pepstatin).After washing with MTSB (3 � 10 min), the seedlings were incu-bated in methanol at �20 �C for 15 min and rehydrated for 10 minwith PBS (NaCl 8 g l�1; KCl 0.2 g l�1; KH2PO4 0.158 g l�1; NaH-PO4$12H2O 2.31 g l�1). After pre-incubation with 2% bovine serumalbumin in PBS for 30 min, the preparations were incubated over-night at 4 �C with monoclonal mouse anti-a-tubulin DM1a(SigmaeAldrich, St Louis, Mo, USA) diluted 1:1000 in PBS.Following further washing with PBS (3 � 5 minutes), the prepara-tions were incubated with TRITC-conjugated anti-mouse antibodydiluted 1:300 in PBS for 3 h at 37 �C. Finally, the seedlings werewashed with PBS (5 � 5 minutes) and embedded in 50% glycerolin PBS.

4.6. Purification of plasma membranes

Microsomal fractions were isolated from 14-day-old A. thalianaseedlings by two-phase partitioning using an aqueousdextranepolyethylene glycol system [44]. The plant material wassuspended at the ratio of 1:3 (w/v) in homogenization buffer(50 mM HEPESeNaOH, pH 7.5, 0.4 M sucrose, 0.1 M KCl., 0.1 MMgCl2) supplemented with protease inhibitors (0.23 mM PMSF,0.83 mM benzamidine, 0.7 mM pepstatin, 1.1 mM leupeptin, and77 nM aprotinin), and homogenized by ultrasonication (4 �C,6 � 40 s). The homogenate was pre-centrifuged at 10 000 g for10min, themicrosomalmembranes then pelleted by centrifugationat 100 000 g for 1 h, and the pellets re-suspended in 5 mMpotassium phosphate buffer, pH 7.8. About 13 ml of microsomalfraction were obtained from 100 g of fresh tissue, and furtherprocessed for plasma membrane purification.

Aqueous two-phase system was formed using Dextran (Fluka)and polyethylene glycol 3350, both at a final concentration of 6.1%(w/w) in 0.43 mM phosphate buffer (pH 7.8) supplemented with3 mM KCl and 0.22 M sucrose. 2 g of the microsomal fraction wereadded to 14 g of the system in a cuvette, the content slowly mixed40 times, allowed to stabilize for 1e2 h at 4 �C, and then centrifugedat 1500 g for 5 min. The plasma membrane fraction (upper phase)was further purified by sequential partitioning against freshlyprepared lower-phase. Finally the upper phase was diluted 5 timeswith HEPESeTris buffer (5 mM HEPESeTris, pH 7.8), centrifuged at100 000 g for 1 h, and the purified plasma membrane pellet re-suspended in fresh HEPESeTris buffer.

4.7. Membrane fluidity measurements

Isolated membranes (proteins concentration 50 mg ml�1) werecentrifuged, washed with acetate buffer (37 mM sodium acetate,pH 4.5), and incubated with 5 mM laurdan (Invitrogen) from a 1mMstock solution in DMSO for 90 min at 25 �C in dark; the finalconcentration of DMSO in the samples did not exceed 0.5%. Themembranes were then washed again with acetate buffer to removeunbound probe, and 50 ml of this sample were placed in a 3� 3mmquartz fluorometric cuvette (Helma GmbH & Co, Müllheim,Germany). After recording of excitation spectra (see below), AlCl3

J. Krtková et al. / Plant Physiology and Biochemistry 60 (2012) 88e9796

was added from a stock solution of either 5mM or 1mM (pH 4.5) toachieve the specified final AlCl3 concentration, and the excitationspectra were recorded again. For membrane fluidization, 12 ml ofa 50 mM stock solution of benzyl alcohol in acetate buffer (pH 4.5)were added to achieve 10 mM final concentration. Subsequentlythe fluorometric measurement was repeated.

Fluorescence measurements were performed at 25 �C usingFluoroMax-3 fluorometer (Horiba Jobin Yvon). For each sample,two excitation spectra were recorded in the range 320e410 nm(bandwidths 1.5 nm) with emission wavelengths at 430 or480 nm. Each point of the generalized polarization (GP) excitationspectrum was calculated as GP ¼ (Ig � Il)/(Ig þ Il), where Ig and Ilare the fluorescence intensities at the maximum emission wave-lengths in the gel (430 nm) and in the liquid-crystalline phase(480 nm) of the membrane, respectively. GP spectra were correctedfor background intensities of unlabeled membranes.

Acknowledgments

The authors wish to thank Dr. Jan Marc (University of Sydney)for helpful discussion and for critical reading of the manuscript andto Dr. Tomá�s Navrátil (Institute of Geology, Academy of Science ofthe Czech Republic) for measurements of Al concentration in testmedia. Financial support: Czech Science Foundation 522/05/0340and P207/12/P890, Ministry of Education, Youth, and Sports of theCzech Republic MSM 0021620858, and Charles University in PragueSVV 265203/2012.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.plaphy.2012.07.030.

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