289URBINA ET AL. Biol Res 39, 2006, 289-296Biol Res 39: 289-296, 2006 BRThe Ca2+ pump inhibitor, thapsigargin, inhibits rootgravitropism in Arabidopsis thaliana

DANIELA C URBINA1, 2, HERMAN SILVA1, 2 and LEE A MEISEL1, 2

1 Millennium Nucleus in Plant Cell Biology and Centro de Biotecnología Vegetal, Facultad de Ecología yRecursos Naturales, Universidad Andrés Bello, Santiago, Chile.2 Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.

ABSTRACT

Thapsigargin, a specific inhibitor of most animal intracellular SERCA-type Ca2+ pumps present in thesarcoplasmic/endoplasmic reticulum, was originally isolated from the roots of the Mediterranean plantThapsia gargancia L. Here, we demonstrate that this root-derived compound is capable of altering rootgravitropism in Arabidopsis thaliana. Thapsigargin concentrations as low as 0.1 µM alter root gravitropismwhereas under similar conditions cyclopiazonic acid does not. Furthermore, a fluorescently conjugatedthapsigargin (BODIPY FL thapsigargin) suggests that target sites for thapsigargin are located inintracellular organelles in the root distal elongation zone and the root cap, regions known to regulate rootgravitropism.

Key terms: Arabidopsis thaliana, Ca2+-ATPases, calcium, gravitropism, thapsigargin, root.

Corresponding author: Lee Meisel, Plant Biotechnology Center, Faculty of Ecology and Natural Resources, UniversidadAndrés Bello, Av. República 217, Santiago, Chile, Tel: (56-2) 661-8449, Fax: (56-2) 661-5832, E-mail: lmeisel@unab.cl

Received: October 3, 2005. In revised form: January 2. 2006. Accepted: January 9, 2006.

INTRODUCTION

The ability of a plant to changedevelopmental and metabolic processes inresponse to environmental stimuli isessential for survival. Response togravitational stimuli enables crop plants tosurvive strong weather conditions, such aswind and rainstorms, by directing theirgrowth and development. This responseensures that the root will grow downward inthe soil, thereby optimizing water, mineraland nutrient uptake that are all vital forgrowth and development. Shortly aftergermination, the root senses gravitationalforces and grows towards this gravityvector (gravitropism). Sensing ofgravitational stimuli and subsequentresponse to these stimuli, by alteringgrowth and developmental processes, is achange from a physical force to aphysiological signal, which in turn leads toa physiological response (Blancaflor andMasson, 2003).

It has been demonstrated that denseamyloplasts (statoliths) in specialized rootcolumella (cap) cells (statocytes) sediment inresponse to gravitational forces (physicalforce). This sedimentation of amyloplasts isthen translated into a physiological signal bythe plant, so that the root can grow andelongate in the direction of the newgravitational vector (physiological response)(Moore and Evans, 1986; Sack, 1991; Sack,1997; Chen et al., 1999; Boonsirichai et al.,2002; Morita and Tasaka, 2004). Thetransduction of amyloplasts sedimentation toa physiological signal is poorly understood.It has been proposed that the pulling actiongenerated by amyloplast sedimentation couldstretch specific plasma membranes and/orthe endoplasmic reticulum to activatemechanosensitive channels. The opening ofthese channels leads to a local transientincrease in cytosolic-free Ca2+ levels, whichin turn triggers a signal transduction cascaderesulting in a physiological response(Sievers et al., 1991; Belyavskaya, 1996;

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Chen et al., 1999; Boonsirichai et al., 2002;Kordyum, 2003). Following thephysiological signal (signal transductioncascade), the plant cells must return to abasal level, such that they may respond tofuture stimuli. It has been proposed that Ca2+

pumps (Ca2+-ATPases) participate in thisprocess by returning cytosolic Ca2+ levels tothe basal level (Sze et al., 2000).

The Arabidopsis Calcium ATPase,ACA3 (ECA1), has been demonstrated tobe inhibited by cyclopiazonic acid but notby thapsigargin (Liang and Sze, 1998).Cyclopiazonic acid and thapsigargin areboth inhibitors of SERCA Ca2+-ATPases inmammalian and yeast systems (Sorin et al.,1997; Treiman et al. , 1998). Thedifferential sensitivity to cyclopiazonic acidand thapsigargin by plant Ca2+-ATPases hasbeen suggested to evolve becausethapsigargin is a plant-derived compoundand the plant Ca2+-ATPases, therefore, mayhave developed insensitivity to thapsigargin(Liang and Sze, 1998). However, the Ca2+-pump TcSCA from Trypanosoma cruzi alsois sensitive to cyclopiazonic acid andinsensitive to thapsigargin (Furuya et al.,2001), indicating that not only plant-derived Ca2+-ATPases are differentiallysensitive to these inhibitors.

Thapsigargin-sensitive Ca2+-pumpactivity has been identified in pea andcauliflower Golgi-derived vesicles(Ordenes et al. , 2002). Therefore,unidentified thapsigargin-sensitive Ca2+-pumps are present in plants. However, theirphysiological role is poorly understood.

In an effort to determine thephysiological role of thapsigargin-sensitiveCa2+-pumps, we studied the physiologicaleffects that exogenous thapsigargintreatment had on Arabidopsis thaliana rootgrowth and curvature in response togravitropic stimuli. We provide evidencethat thapsigargin inhibits Arabidopsis rootgravitropism, whereas under similarconditions, cyclopiazonic acid does not.Furthermore, our results suggest thatthapsigargin is able to target intracellularorganelles of cells within the distalelongation zone and the root cap, regions ofthe root that play a role in rootgravitropism.

MATERIALS AND METHODS

Plant material and growth conditions

Seven-day-old Arabidopsis thalianaseedlings, ecotype Columbia (Col), weregrown vertically under sterile conditions onplates containing 1/2X MS (pH 5.7), 1%sucrose, 0.9% agar. Plants were grownvertically under 61.6 µmol/m-2s-1 cooledwhite fluorescent light (Philips TLD 30W/33), 16-h light cycle, 25º C, unlessotherwise indicated.

Inhibitor treatment

Thapsigargin (Molecular Probes) andcyclopiazonic acid (Sigma) were dissolvedin dimethyl sulfoxide. Sodium orthovanadate(Sigma) was dissolved in deionized water.The maximum concentration of dimethylsulfoxide in any inhibitor solution used inthese studies did not exceed 0.5% (minimumconcentration in which root growth wasunaffected, data not shown). Control plantsfor thapsigargin and cyclopiazonic acidstudies were treated with DMSO at the sameconcentration used in the highest inhibitorconcentration, respectively. Control plantsfor sodium orthovanadate studies weretreated with H2O.

Seedlings were infiltrated for 15 min inthe dark with Ca2+-ATPase/P-Type ATPaseinhibitors at varying concentrations.Thapsigargin treatments were performed at0, 0.1, 0.5, 1.0, and 5.0 µM. Cyclopiazonicacid treatments were performed at 0, 0.5,1.0, 5.0, and 20 µM. Sodium orthovanadatetreatments were performed at 0, 1.0, 5.0,10, and 50 µM. Post treatment, plants werealigned linearly on 1/2X MS (pH 5.7), 1%sucrose, 0.9% agar containing plates,rotated 90º relative to the gravity vector andplaced in the dark to avoid changes due tophototropism. Six hours post treatment, theangle of root curvature and the length ofroot growth were measured under astereomicroscope. The angle between theplant axis and the tip of the root weremeasured as shown in figure 1D.

Control plants were aligned on the sameplates as the treated plants, thereby servingas controls for solvent treatment as well as

291URBINA ET AL. Biol Res 39, 2006, 289-296

Figure 1: Gravitropic root curvature response in the presence of different P-type ATPase inhibitors.A-C, the angle of root curvature in response to gravitropic stimuli was measured 6 h post treatmentwith different concentrations of P-type ATPase inhibitors. A, thapsigargin, a specific inhibitor ofmost animal intracellular SERCA-type Ca2+-pumps but not plant type IIA Ca2+-ATPases (0indicates control plants that have been treated with DMSO at the same concentration used for thethapsigargin-treated plants); B, sodium orthovanadate, a general inhibitor of P-type ATPases, (0indicates control plants that have been treated with water); C, cyclopiazonic acid, a specificinhibitor of animal SERCA type and plant type IIA Ca2+-ATPases, (0 indicates control plants thathave been treated with DMSO at the same concentration used for the cyclopiazonic acid-treatedplants). Bars indicate the mean ± the standard error. Experiments were performed in triplicate withat least ten plants for each experiment. The most representative experiment is shown. D, diagramdemonstrating the angle used to measure root curvature in response to gravitropic stimuli inArabidopsis. Vertically grown Arabidopsis seedlings were treated with different inhibitors,followed by a 90o change in the direction of gravity. Six hours post-treatment, the angle of the rootcurvature, and the length of root growth were measured. α is the angle measured in theseexperiments.

the direction of gravitational forces. Allassays were performed in triplicate, with aminimum of 10 treated and 10 untreatedplants in each assay. All results arepresented as the mean ± standard error.

Statistical significance of the results wasdetermined by performing the Student’s t-test on the mean ± standard error of eachtreatment, with a certainty of 95% anddegree of freedom of n-1.

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Fluorescent analysis

To visualize the cellular and intracellulartargets of thapsigargin, 7-day-old seedlingswere incubated with 1 µM BODIPY FLthapsigargin (Molecular Probes) in 0.1 mMphosphate buffer (pH 7.0) for 2 h in thedark. Samples were subsequently washedthree times with 0.1 mM potassiumphosphate buffer and visualized on anOlympus IX70 epifluorescence microscopewith a Sony DXC-390 3CCD color camera.Differential Interference Contrast imagingwas performed using this equipment and anUPLAN F1 objective. Fluorescenceimaging was performed using a U-N3001FITC filter (excitation D480/30X, dichroic505 DCLP, emission D535/40m). Imageswere processed using Adobe Photoshop.

RESULTS

Calcium ATPase Inhibitors alterArabidopsis root gravitropism

Treatment of Arabidopsis seedlings withlow concentrations of thapsigargin (aspecific inhibitor of most animalintracellular SERCA-type Ca2+ pumps butnot plant type IIA Ca2+-ATPases) showed asignificant inhibition of root curvature inresponse to gravitropic stimuli (Figure 1A).0.1 µM thapsigargin inhibited rootcurvature by 31%. The greatest inhibitionof root curvature was seen with 0.5 µMthapsigargin. This concentration inhibitedroot curvature by 37%. However, higherconcentrations of thapsigargin did notsignificantly inhibit root curvature inresponse to gravitropic stimuli (Figure 1A).To determine if the change in root curvaturepost-thapsigargin treatment was due to achange in the gravitropic response or ageneral inhibition of growth, root growthpost treatment was analyzed (Table 1).Under the conditions utilized in thesestudies, no significant changes wereobserved in the root growth of thapsigargin-treated seedlings.

Arabidopsis seedlings treated with the P-type ATPase inhibitor sodium orthovanadateshowed significant inhibition of root

curvature when 50 µM sodium orthovanadatewas used (Figure 1B). Additionally, rootgrowth increased significantly when seedlingswere treated with this concentration ofsodium orthovanadate (Table 1). Lowerconcentrations of sodium orthovanadate didnot significantly alter root curvature or alterroot growth (Figure 1B, Table 1).

TABLE 1

Effects of Thapsigargin, CyclopiazonicAcid and Sodium Orthovanadate on

Arabidopsis Root Growth

Root Growth (mm)*

Thapsigargin (µM)0 1.8 ± 0.6

0.1 1.8 ± 0.60.5 1.7 ± 0.61 2.4 ± 0.85 2.1 ± 0.5

Sodium Orthovanadate (µM)0 1.8 ± 0.11 1.7 ± 0.15 1.6 ± 0.1

10 1.7 ± 0.250 2.0 ± 0.0

Cyclopiazonic Acid (µM)0 1.6 ± 0.1

0.5 1.4 ± 0.11 1.7 ± 0.25 1.8 ± 0.1

20 1.7 ± 0.1

* Values represent the average root growth ± SE(n=10), 6 hours post treatment. Experiments wereperformed in triplicate. The most representativeexperiment is shown here.

Surprisingly, Arabidopsis seedlingstreated with cyclopiazonic acid (a specificinhibitor of animal SERCA type and planttype IIA Ca2+-ATPases) were notsignificantly altered in their ability to curvein response to gravitropic stimuli (Figure1C). No alterations in root curvature androot growth in response to gravitropicstimuli were detected when plants weretreated with concentrations as high as 20 µMcyclopiazonic acid (Figure 1C, Table 1).

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The cellular and intracellular targets ofthapsigargin

In order to determine whether thapsigargintargets cells in regions known to play a rolein root gravitropism and to determine theintracellular target of thapsigargin,Arabidopsis seedlings were treated with a

Figure 2: Thapsigargin targets regions of the root that are responsible for the root gravitropicresponse. To determine the intracellular target for thapsigargin, 7-day-old Arabidopsis seedlingswere treated with BODIPY FL thapsigargin. Different regions of the root were analyzed forlocalization of the BODIPY FL fluorescence (A and B). Regions of the root analyzed include: A, C,and E, distal elongation zone of the root; B, D, and F, root tip (including root cap, quiescent centerand root meristem); C and D, roots visualized by DIC microscopy; E and F, auto-fluorescence ofuntreated roots. Bar represents 50 µm.

membrane-permeable green fluorescentderivative of thapsigargin, BODIPY FLthapsigargin. This green fluorescentderivative of thapsigargin has been utilizedin various systems to determine the targetof thapsigargin (Csordas and Hajnoczky,2001; Lee and Aarhus, 2000; Abrenica andGilchrist, 2000).

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Fluorescence from the BODIPY FLthapsigargin was detected in the distalelongation zone of the root (Figure 2A) aswell as the root cap (Figure 2B).Interestingly, reduced fluorescence wasdetected in the quiescent center andmeristematic zone of the root (Figure 2B).The BODIPY FL thapsigargin targetedintracellular reticulate structures in thedistal elongation zone of the root (Figure2A) and the root cap (Figure 2B). There isno notable co-localization betweenBODIPY FL thapsigargin and the plasmamembrane in all tissues analyzed. Theintracellular localization of thapsigarginalso has been analyzed in Arabidopsisprotoplasts, onion epidermal cells, andtobacco epidermal cells. All tissues showedsimilar patterns with numerous intracellularorganelles co-localizing with the BODIPYFL thapsigargin (data not shown).

DISCUSSION

In this study, we have demonstrated that theCa2+-ATPase inhibitor, thapsigargin,inhibits root gravitropism in Arabidopsisthaliana. Concentrations of thapsigargin aslow as 0.1 µM, inhibited root gravitropismin Arabidopsis. This concentration is withinthe range utilized to characterizethapsigargin-sensitive Ca2+-ATPaseactivities in several animal studies as wellas in the recently identified Ca2+-ATPaseactivity in Golgi vesicles of etiolated peaseedlings (Simpson and Russell, 1997;Thastrup et al., 1990; Zhong and Inesi,1998; Ordenes et al., 2002). Furthermore,0.1 µM thapsigargin is lower than theconcentration that has been shown to inhibitL-type Ca2+ channels (5 µM) (Nelson et al.,1994; Treiman et al. , 1998). WhenArabidopsis seedlings were infiltrated with5 µM thapsigargin, no significant differencewas detected in the gravitropic responsebetween treated and non-treated plants.This lack of inhibition of root gravitropismwith 5 µM thapsigargin may be due to thesimultaneous inhibition of L-type Ca2+

channels and thapsigargin-sensitive Ca2+-ATPases. This is further evidence that theinhibition of root gravitropism by

thapsigargin at lower concentrations isspecifically due to an inhibition ofthapsigargin-sensitive Ca2+-ATPases andnot due to an inhibition of L-type Ca2+

channels.Thapsigargin is the most widely used

SERCA inhibitor (Treiman et al., 1998). Itis a naturally occurring sesquiterpenelactone, found in several species belongingto the genus Thapsia. Thapsigargin wasisolated originally from the roots of theMediterranean plant Thapsia garganica L.(Linnaeus) (Treiman et al., 1998). Ourfindings suggest that under normalphysiological conditions in planta ,thapsigargin may play a role in signalingand regulating root gravitropism.

Sievers and Busch (1992) havedemonstrated that cyclopiazonic acidsensitive Ca2+-ATPases are involved in thetransduction of gravitational stimuli inLepidum sativum L. Cyclopiazonic acid isan indole tetramic acid that has beenreported to be a specific inhibitor of SER-type Ca2+-ATPases (SERCA) (Seidler et al.,1989). The study by Sievers and Busch(1992) demonstrated that Lepidium sativumL. seedlings treated with 20 µMcyclopiazonic acid inhibited root curvaturein response to a change in the gravityvector. However, we have demonstratedthat 20 µM cyclopiazonic acid is unable toinhibit root gravitropism in Arabidopsisafter a 15 min treatment. Sievers and Busch(1992) treated Lepidum sativum L. with 20µM cyclopiazonic acid for 2 h. Longertreatment with cyclopiazonic acid may alterroot gravitropism in Arabidopsis roots.However, such a long treatment also maycause alterations in other physiologicalprocesses that may result in changes in rootcurvature. For example, a change in rootgrowth may give rise to changes in rootcurvature.

To date, a thapsigargin-sensitive Ca2+-ATPase has not been identified inArabidopsis. Rather, two distinct familiesof Ca2+-ATPases have been proposed basedon sequence identities (Geisler et al., 2000).These are type IIA (or ECA for ER- typeCa2+-ATPase) and type IIB (ACA forautoinhibited Ca2+-ATPase). The type IIACa2+-ATPases are insensitive to calmodulin

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and sensitive to cyclopiazonic acid. Whilethe type IIB Ca2+-ATPases are stimulatedby calmodulin. Neither the Ca2+-ATPases,type IIA nor the type IIB have beendemonstrated to be sensitive to thapsigargin(Sze et al., 2000). However, Ordenes et al.(2002) recently have identified athapsigargin-inhibited Ca2+-ATPase activityin Golgi vesicles isolated from peaepicotyls. Similarly, J Watkins, AKCampbell, MR Knight and AJ Trewavasrecently have detected changes inendoplasmic reticulum and nuclear calciumlevels due to thapsigargin treatment(unpublished data). This suggests that athapsigargin sensitive Ca2+-ATPase activityexists in plants. Our results suggest thatthese as yet unidentified thapsigargin-sensitive Ca2+-ATPases are involved inphysiological processes such as rootgravitropism. BODIPY FL thapsigarginlabels numerous cells, including cells in thedistal elongation zone as well as the rootcap. This suggests that these thapsigargin-sensitive Ca2+-ATPases are localized incells that are known to play an importantrole in the root gravitropic response.

We cannot, however, rule out thepossibility that thapsigargin-sensitivetargets, other than Ca2+-ATPases, may existin Arabidopsis and may be playing a role inroot gravitropism. However, our results dodemonstrate that a thapsigargin-sensitivefactor is involved in Arabidopsis rootgravitropism. Furthermore, the target forthapsigargin is localized in reticulateorganelles. This thapsigargin-sensitivefactor may be a Ca2+-ATPase activitylocalized in organelles, such as the Golgiapparatus or the ER, but other moleculartargets for thapsigargin also may exist.Further molecular genetic analyses inArabidopsis to identify the factor(s) that areaffected by thapsigargin, the subcellularlocalization of these factors, and their rolein root gravitropism will help to unveilthese possibilities.

ACKNOWLEDGEMENTS

We would like to thank members of theLaboratory of Plant Molecular Genetics for

their helpful suggestions, Ariel Orellanaand Daniel Wolff for kindly providing uswith sodium orthovanadate andcyclopiazonic acid, Anthony Trewavas andcoworkers for providing us withinformation concerning their unpublishedfindings, Ariel Orellana and Daniel Wolfffor critical reading of this manuscript. Thiswork was supported by research grantsfrom the Fondo Nacional de DesarrolloCientífico y Tecnológico (Fondecyt#1000812), ICM P99-031-F, ICM P02-009-F, and the Departamento de Investigación yDesarrollo de la Universidad de Chile (DID#I010-98/2).

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