Fluorescence Resonance Energy Transfer-Sensitized Emission of

Fluorescence Resonance Energy Transfer-SensitizedEmission of Yellow Cameleon 3.60 Reveals RootZone-Specific Calcium Signatures in Arabidopsis inResponse to Aluminum and Other Trivalent Cations1[W][OA]

Magaly Rincon-Zachary2*, Neal D. Teaster2, J. Alan Sparks2, Aline H. Valster,Christy M. Motes3, and Elison B. Blancaflor

Department of Biology, College of Science and Mathematics, Midwestern State University, Wichita Falls,Texas 76308 (M.R.-Z.); and Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore,Oklahoma 73401 (N.D.T., J.A.S., A.H.V., C.M.M., E.B.B.)

Fluorescence resonance energy transfer-sensitized emission of the yellow cameleon 3.60 was used to study the dynamics ofcytoplasmic calcium ([Ca2+]cyt) in different zones of living Arabidopsis (Arabidopsis thaliana) roots. Transient elevations of [Ca2+]cytwere observed in response to glutamic acid (Glu), ATP, and aluminum (Al3+). Each chemical induced a [Ca2+]cyt signature thatdiffered among the three treatments in regard to the onset, duration, and shape of the response. Glu and ATP triggeredpatterns of [Ca 2+]cyt increases that were similar among the different root zones, whereas Al3+ evoked [Ca2+]cyt transients thathad monophasic and biphasic shapes, most notably in the root transition zone. The Al3+-induced [Ca2+]cyt increases generallystarted in the maturation zone and propagated toward the cap, while the earliest [Ca2+]cyt response after Glu or ATP treatmentoccurred in an area that encompassed the meristem and elongation zone. The biphasic [Ca2+]cyt signature resulting from Al3+

treatment originated mostly from cortical cells located at 300 to 500 mm from the root tip, which could be triggered in partthrough ligand-gated Glu receptors. Lanthanum and gadolinium, cations commonly used as Ca2+ channel blockers, elicited[Ca2+]cyt responses similar to those induced by Al3+. The trivalent ion-induced [Ca2+]cyt signatures in roots of an Al3+-resistantand an Al3+-sensitive mutant were similar to those of wild-type plants, indicating that the early [Ca2+]cyt changes we report heremay not be tightly linked to Al3+ toxicity but rather to a general response to trivalent cations.

The role of calcium ions (Ca2+) as a ubiquitouscellular messenger in animal and plant cells is wellestablished (Berridge et al., 2000; Sanders et al., 2002;Ng and McAinsh, 2003). Cellular signal transductionpathways are elicited as a result of fluctuations of freeCa2+ in the cytoplasm ([Ca2+]cyt) in response to externaland intracellular signals. These changes in [Ca2+]cytinfluence numerous cellular processes, including ves-icle trafficking, cell metabolism, cell proliferation and

elongation, stomatal opening and closure, seed andpollen grain germination, fertilization, ion tran-sport, and cytoskeletal organization (Hepler, 2005).[Ca2+]cyt fluctuations occur because cells have a Ca2+

signaling “toolkit” (Berridge et al., 2000) composed ofon/off switches and a multitude of Ca2+-binding pro-teins. The on switches depend on membrane-localizedCa2+ channels that control the entry of Ca2+ into thecytosol (Pineros and Tester, 1995, 1997; Thion et al.,1998; Kiegle et al., 2000a; White et al., 2000; Demidchiket al., 2002; Miedema et al., 2008). On the other hand,the off switches consist of a family of Ca2+-ATPasesand Ca2+/H+ exchangers in the plasma membrane orendomembrane that remove Ca2+ from the cytosol,bringing the [Ca2+]cyt down to the initial resting level(Lee et al., 2007; Li et al., 2008).

The numerous cellular processes regulated by Ca2+

have led investigators to ask how specificity in Ca2+

signaling is maintained. It has been proposed thatspecificity in Ca2+ signaling is achieved because aparticular stimulus elicits a distinct Ca2+ signature,which is defined by the timing, magnitude, and fre-quency of [Ca2+]cyt changes. For instance, tip-growingplant cells such as root hairs and pollen tubesexhibit oscillatory elevations in [Ca2+]cyt that partlymirror the oscillatory nature of growth in these celltypes (Cardenas et al., 2008; Monshausen et al., 2008).

1 This work was supported by the Midwestern State University,Faculty Development Research Fund (to M.R.-Z.), The SamuelRoberts Noble Foundation, the National Science Foundation (Re-search Opportunity Award no. DBI–0400580 to E.B.B.), and theDepartment of Energy Biosciences Program (grant no. DE–FG02–05ER15647 to E.B.B.).

2 These authors contributed equally to the article.3 Present address: Forage Improvement Division, The Samuel

Roberts Noble Foundation, Ardmore, OK 73401.* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Elison B. Blancaflor ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

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Another example is nuclear Ca2+ spiking in root hairsof legumes exposed to NOD factors (Oldroyd andDownie, 2006; Peiter et al., 2007). Recently, it wasshown that mechanical forces applied to an Arabidop-sis (Arabidopsis thaliana) root can trigger a stimulus-specific [Ca2+]cyt response (Monshausen et al., 2009).Translating the Ca2+ signature into a defined cellularresponse is governed by a number of Ca2+-bindingproteins such as calreticulin that act as [Ca2+]cyt buffers,which shape both the amplitude and duration of theCa2+ signal or Ca2+ sensors such as calmodulin thatimpact other downstream cellular effectors (Berridgeet al., 2000; White and Broadley, 2003; Hepler, 2005).A deeper understanding of Ca2+ signaling mecha-

nisms in plants has been driven in large part by ourability to monitor dynamic changes in [Ca2+]cyt in thecell. Such measurements have been conducted usingCa2+-sensitive fluorescent indicator dyes (e.g. Indo andFura), the luminescent protein aequorin (Knight et al.,1991, 1996; Legue et al., 1997; Wymer et al., 1997;Cardenas et al., 2008), and more recently the yellowcameleon (YC) Ca2+ sensor, a chimeric protein thatrelies on fluorescence resonance energy transfer(FRET) as an indicator of [Ca2+]cyt changes in the cell(Allen et al., 1999; Miwa et al., 2006; Qi et al., 2006;Tang et al., 2007; Haruta et al., 2008). The YC reporter iscomposed of cyan fluorescent protein (CFP), the Cterminus of calmodulin (CaM), a Gly-Gly linker,the CaM-binding domain of myosin light chain ki-nase (M13), and a yellow fluorescent protein (YFP;Miyawaki et al., 1997, 1999). The increased interactionbetween M13 and CaM upon binding of Ca2+ to CaMtriggers a conformational change in the protein thatbrings the CFP and YFP in close proximity, resulting inenhanced FRET efficiency between the two fluoro-phores (Miyawaki, 2003). Thus, changes in FRET effi-ciency between CFP and YFP in the cameleon reporterare correlated with changes in [Ca2+]cyt.Since it was first introduced, improved versions of

the cameleon reporter have been selected to moreaccurately report [Ca2+]cyt levels in the cell. For in-stance, the YC3.60 version was selected because of itsresistance to cytoplasmic acidification and its higherdynamic range compared with the earlier cameleons.The higher dynamic range of YC3.60 is due to the useof a circularly permutated YFP called Venus (cpVenus)that is capable of absorbing a greater amount of energyfrom CFP (Nagai et al., 2004). Recently, the utility ofYC3.60 for monitoring [Ca2+]cyt was demonstrated inArabidopsis roots and pollen tubes using ratiometricimaging approaches (Monshausen et al., 2007, 2008,2009; Haruta et al., 2008; Iwano et al., 2009). Here, wefurther evaluated YC3.60 as a [Ca2+]cyt sensor in plantsusing confocal microscopy and FRET-sensitized emis-sion imaging. Unlike the direct ratiometric measure-ment of cpVenus and CFP reported in previous studiesusing YC3.60-expressing plants (Monshausen et al.,2008, 2009), the sensitized FRETapproach we describehere involves theuse of donor-only (CFP) and acceptor-only (YFP) controls, allowing us to correct for bleed-

through and background signals from the FRETspecimen (van Rheenen et al., 2004; Feige et al., 2005).

For this study, we focused on monitoring [Ca2+]cytchanges in Arabidopsis seedling roots after alumi-num (Al3+) exposure. Although Ca2+ signaling haslong been implicated in mediating Al3+ responses inplants (Rengel and Zhang, 2003), the [Ca2+]cyt changesevoked by Al3+ reported in the literature havebeen inconsistent, and as such, the significance of these[Ca2+]cyt responses to mechanisms of Al3+ toxicity arenot very clear. For instance, some studies reported thatAl3+ caused a decrease in [Ca2+]cyt in plants (Joneset al., 1998b; Kawano et al., 2004), and others dem-onstrated elevated [Ca2+]cyt upon Al3+ treatment(Nichol and Oliveira, 1995; Lindberg and Strid,1997; Jones et al., 1998a; Zhang and Rengel, 1999;Ma et al., 2002; Bhuja et al., 2004).

Here, we report that Arabidopsis roots expressingthe YC3.60 reporter exhibited transient elevations in[Ca2+]cyt within seconds of Al3+ exposure. The generalpattern of [Ca2+]cyt changes observed after Al3+ treat-ment were distinct from those elicited by ATP or Glu,reinforcing the concept of specificity in [Ca2+]cyt sig-naling. We also observed root zone-dependent varia-tions in the [Ca2+]cyt signatures evoked by Al3+ inregard to the shape, duration, and timing of the [Ca2+]cytresponse. Other trivalent ions such as lanthanum(La3+) and gadolinium (Ga3+), which have been widelyused as Ca2+ channel blockers (Monshausen et al.,2009), also induced a rapid rise in [Ca2+]cyt inroot cells that were similar to those elicited by Al3+.Al3+, La3+, and Gd3+ elicited similar [Ca2+]cyt signaturesin the Al3+-tolerant mutant alr104 (Larsen et al., 1998)and the Al3+-sensitive mutant als3-1 (Larsen et al.,2005), indicating that the early [Ca2+]cyt increases wereport here may not be tightly linked to mechanismsof Al3+ toxicity but rather to a general trivalentcation response. Our study further shows that FRET-sensitized emission imaging of Arabidopsis rootsexpressing YC3.60 provides a robust method fordocumenting [Ca2+]cyt signatures in different root de-velopmental zones that should be useful for futurestudies on Ca2+ signaling mechanisms in plants.

RESULTS

FRET-Sensitized Emission Imaging of YC3.60 Faithfully

Reports [Ca2+]cyt Dynamics in Arabidopsis Root Cells

We generated Arabidopsis plants expressing YC3.60under the control of the cauliflower mosaic virus 35Spromoter (35S::YC3.60) to investigate Ca2+ signalingevents in root cells after Al3+ treatment. All of ourimaging experiments were conducted with one 35S::YC3.60-expressing line that we first established to becapable of displaying Ca2+-dependent fluorescencechanges in response to stimuli that are known toinduce [Ca2+]cyt elevations in plants. The ability of theYC3.60 reporter to monitor changes in [Ca2+]cyt relieson the efficiency of energy transfer between CFP

[Ca2+]cyt Dynamics in Roots Detected by FRET

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and cpVenus upon Ca2+ binding (Nagai et al., 2004).Thus, we utilized a preloaded application wizard fromthe Leica TCS SP2 AOBS confocal microscope tomonitor FRET-sensitized emission in 35S::YC3.60-ex-pressing roots. To efficiently measure FRET-sensitizedemission with the Leica application wizard, weneeded to account for background and bleed-throughartifacts that could compromise accurate FRET effi-ciency readings (van Rheenen et al., 2004; Feige et al.,2005). For these corrections, it was necessary to acquiredonor-only (CFP) and acceptor-only (YFP) fluores-cence. Therefore, we generated Arabidopsis plantsexpressing 35S::CFP or 35S::YFP to serve as referenceplants for the background subtraction and bleed-through correction algorithms needed for measuringFRET-sensitized emission from the 35S::YC3.60-expressing lines (see “Materials and Methods”).

We first confirmed that growing root hairs from ourindependently generated 35S::YC3.60 line displayed thetip-focused [Ca2+]cyt gradients and oscillations that werereported recently using ratiometric cpVenus::CFP imag-ing (Monshausen et al., 2008). When FRET efficiency atthe tip of growing root hairs was plotted over time, weobserved Ca2+-dependent FRETefficiency oscillations atthe root hair tip with a frequency of about two to fourpeaks per min (Supplemental Fig. S1; SupplementalMovie S1). These root hair oscillations were similar tothose reported by Monshausen et al. (2008).

To set the stage for the Al3+ experiments, we nextasked whether we could elicit FRETefficiency changesin YC3.60-expressing roots by applying compoundspreviously shown to induce elevations in [Ca2+]cyt. Wefirst tested whether we could detect global Ca2+-dependent FRET efficiency changes in a region of theroot located 200 to 400 mm from the root cap junction(RCJ; Fig. 1A, white rectangular box). This region ofthe root has been referred to as the distal elongationzone or transition zone and is one of the proposed fourdistinct zones in the Arabidopsis primary root that hascharacteristic cellular activities (Verbelen et al., 2006).We chose this region for our initial Ca2+-dependentFRET efficiency measurements because it has beenshown to be the most responsive to various environ-mental stimuli (Baluska et al., 2001; Verbelen et al.,2006). Glu has been previously shown to causetransient elevations in [Ca2+]cyt in plants (Dennisonand Spalding, 2000; Qi et al., 2006). We thereforeexamined Ca2+-dependent FRET efficiency changes inthe root transition zone of 3- to 4-d-old seedlings inresponse to exogenous Glu. During the conduct of ourexperiments, we found that the consistency of elicitingCa2+-dependent FRET efficiency changes in roots, par-ticularly when it involved exogenous application ofchemicals, was strongly influenced by the manner inwhich seedlings were grown. To efficiently applyexogenous solutions to living Arabidopsis roots, itwas necessary to germinate seeds directly on cover-slips coated with a thin layer of low-melting-pointagarose supplemented with half-strength Murashigeand Skoog (MS) nutrients (see “Materials andMethods”).

This allowed us to securely anchor the root so that it didnot drift away from the microscope field of view uponapplication of the solution. Using this system, weobserved that 1 mM Glu added directly to the rootsurface caused a rise in [Ca2+]cyt in the root transitionzone starting at 19.63 6 1.47 s after application andreached a maximum after 32.18 6 2.80 s (values aremeans 6 SE of 11 roots; Fig. 1, B and C; SupplementalMovie S2). Application of the solvent control solutiondid not induce a rise in [Ca2+]cyt (data not shown).

Since ATP has been shown to induce [Ca2+]cyt in-creases in plants using methods such as aequorin(Demidchik et al., 2003; Jeter et al., 2004), we alsotested the effect of extracellular ATP on [Ca2+]cyt in thetransition zone. When 1 mM ATP was applied toYC3.60-expressing roots, we observed a rapid increasein Ca2+-dependent FRET efficiency in cells of thetransition zone. However, compared with Glu, theonset of the [Ca2+]cyt increase took longer and began at37.5 6 3.87 s after ATP application, with a maximum[Ca2+]cyt response occurring at 63 6 5.84 s (values aremeans 6 SE of 11 roots). Furthermore, unlike the Glu-induced [Ca2+]cyt spike, which declined rapidly afterreaching maximum values (Fig. 1B), the ATP-inducedincrease in [Ca2+]cyt was generally followed by a moregradual decline that spanned a period of approxi-mately 3 to 5 min (Fig. 1D). The shapes of the ATP-induced [Ca2+]cyt signature that we observed weresimilar to those reported in mammalian cells express-ing YC3.60 (Nagai et al., 2004).

Al3+ Induces a Range of [Ca2+]cyt Signatures in theTransition Zone That Are Different from Those Elicitedby Glu and ATP

On the basis of the results described above with Gluand ATP, we were confident that FRET-sensitizedemission measurements with YC3.60-expressingplants presented a simple and highly reproducibleapproach to begin a more detailed evaluation of[Ca2+]cyt signaling in Arabidopsis root cells upon Al3+

treatment. Like Glu and ATP, we first evaluated theeffect of Al3+ on global changes in [Ca2+]cyt in thetransition zone, as this region of the root is a primarytarget of Al3+ (Sivaguru and Horst, 1998). Since thetoxic species of Al3+ forms at low pH (Kinraide andParker, 1987), we prepared a working solution of AlCl3in half-strength MS medium at pH 4.5. Application ofthe low-pH solution alone had no impact on [Ca2+]cyt-dependent FRET efficiency in cells of the transitionzone, but when supplemented with Al3+, we observedtransient [Ca2+]cyt-dependent FRETefficiency increases(Fig. 2). In contrast to the largely uniform shapes of the[Ca2+]cyt signatures induced by Glu or ATP (Fig. 1),the root transition zone showed variations in thepatterns of the [Ca2+]cyt response after Al3+ treatment.We found that 35% of the roots imaged exhibited a[Ca2+]cyt signature that had a single broad peak (Fig.2A), whereas 59% of the roots displayed a [Ca2+]cytsignature that was biphasic (i.e. it had two peaks;

Rincon-Zachary et al.

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Fig. 2, B and C). Themagnitude of the twopeaks variedfrom root to root such that peakswere either unequal inheight (Fig. 2B), which occurred in most of the rootswith a biphasic response (31%), or roughly similar inheight (28%; Fig. 2C). A representative time-lapsesequence of a heat map of the root transition zoneafter Al3+ application shows the onset of the first andsecond [Ca2+]cyt peaks (Fig. 2E; Supplemental MovieS3). In a few roots (6%), we observed three distinct[Ca2+]cyt peaks during the entire period of imaging (Fig.2D). The onset of the [Ca2+]cyt rise in the transition zonein response to Al3+ generally took longer comparedwith either Glu or ATP. The [Ca2+]cyt increase startedat 66.0 6 6.9 s after Al3+ application, and the firstpeak reached a maximum at 186.5 6 6.9 s (values aremeans 6 SE from 55 roots).

Imaging of FRET-Sensitized Emission Allows

Monitoring of [Ca2+]cyt Changes in Different Root Zones

We extended our sensitized FRETefficiency imagingto themeristem (0–100 mm from the RCJ), root cap, andmaturation zone to determine whether cells in thesedifferent zones also exhibited a [Ca2+]cyt increase in

response to the compounds tested (Fig. 1A; Verbelenet al., 2006). We found that cells in the meristem andcap displayed a Ca2+-dependent increase in FRETefficiency in response to Glu and ATP that had similarshapes as those observed in the transition zone (Fig. 3,A and B). The meristem and root cap exhibited analmost simultaneous elevation in [Ca2+]cyt upon Glutreatment, but the magnitude of FRET efficiency in-creases was lower in the cap (Fig. 3A). The rise in[Ca2+]cyt in the cap started at 16.336 2.29 s, whereas inthe meristem this increase occurred at 15.0 6 1.67 s(values are means 6 SE; n = 10). On the other hand, the[Ca2+]cyt increase in the cap after ATP application wasslightly delayed compared with Glu application, andthe magnitude of the response was also lower com-pared with that of the meristem (Fig. 3B). The rise inthe [Ca2+]cyt in the cap after ATP treatment began at54.0 6 4.29 s, whereas in the meristem the [Ca2+]cytincrease occurred at 40.286 4.48 s (values are means6SE; n = 7). A time-lapse movie sequence of a heat maprepresenting FRET efficiency values shows the almostsimultaneous increase in [Ca2+]cyt in the cap and mer-istem after Glu treatment and a slight delay in theonset of the [Ca2+]cyt rise in the cap compared with the

Figure 1. [Ca2+]cyt-dependent FRET efficiencychanges in Arabidopsis primary roots afterGlu and ATPapplication. A, An imageof a rootfrom a 4-d-old Arabidopsis seedling indicatingdifferent regions of the root. The white rectan-gle marks part of the transition zone of the rootwhere FRETefficiency traceswere obtained. B,Application of 1 mM Glu triggers a rapid in-crease in [Ca2+]cyt-dependent FRET efficiency.C, Pseudocolored time-lapse sequence of cellsin the transition zone after treatment with Glushowing thepeakof the [Ca2+]cyt responseat 30s and its return to basal levels within 60 s.Supplemental Movie S2 shows the completemovie sequence. D, ATP-induced elevation of[Ca2+]cyt-dependent FRET efficiency. Note thebroader shape of the [Ca2+]cyt response of ATP-treated roots comparedwithGlu-treated roots.Arrows in B and D indicate the time ofchemical addition. Bars = 100 mm in A and20 mm in C.





meristem after ATP application. The movies also showthat upon ATP treatment, [Ca2+]cyt remained elevatedfor a longer duration compared with that of Glutreatment (Supplemental Movies S4 and S5).

Unlike in the transition zone, [Ca2+]cyt signatures inthe cap and meristem induced by Al3+ treatment weremore uniform, such that only one [Ca2+]cyt peak wasobserved in all roots imaged (Fig. 3C). Like ATP, theonset of the [Ca2+]cyt increase in the cap in response toAl3+ was delayed compared with that of the meristemand the magnitude of the response was lower. The[Ca2+]cyt increase in the cap after Al3+ treatment startedat 99.4 6 8.5 s, whereas in the meristem this increaseoccurred at 85.26 6.7 s (values are means6 SE; n = 14–15). A time-lapse movie sequence of a heat map forFRET efficiency values shows the [Ca2+]cyt rise in themeristem starting ahead of the [Ca2+]cyt increase in thecap (Supplemental Movie S6).

The maturation zone of the root also displayed a[Ca2+]cyt increase after application of Glu, ATP, or Al3+.The maturation zone of the root can clearly be iden-tified based on the presence of initiating root hairs (Fig.1A). Although all three chemicals triggered a rise in[Ca2+]cyt in the maturation zone, the magnitude of the[Ca2+]cyt increase was noticeably less compared withthat in the meristem and transition zone. The Glu-

induced [Ca2+]cyt spike was similar to that observed inall other regions of the root (Fig. 3D). On the otherhand, with ATP, the majority of the tracings from thematuration zone showed a rise in [Ca2+]cyt followed byoscillations that declined gradually over time (Fig. 3E).Like in the transition zone, Al3+-induced [Ca2+]cytsignatures in the maturation zone were biphasic, butthe [Ca2+]cyt peaks were not as well separated. The first[Ca2+]cyt peak was narrow, followed by a second peakthat was broader compared with the second peakobserved in the transition zone (compare Figs. 3F and2C). The [Ca2+]cyt increase in the maturation zoneevoked by Al3+ was also longer in duration comparedwith that triggered by Glu or ATP (Fig. 3, D–F). Theonset of the [Ca2+]cyt rise in the maturation zoneoccurred at 47.6 6 6.8 s, 26.25 6 2.17 s, and 57.0 64.70 s for Al3+, Glu, and ATP, respectively (values aremeans6 SE; n = 8). Likewise, the duration of the [Ca2+]cytelevation triggered by Al3+ was longer in the matura-tion zone than in the transition zone, meristem, and rootcap. Table I summarizes the timing of the Al3+-triggered[Ca2+]cyt signatures in the different zones of the root.

Based on the onset of [Ca2+]cyt increase in the differ-ent root zones, we were able to infer the likelydirections in which the [Ca2+]cyt wave propagates inresponse to Glu, ATP, and Al3+. In Al3+-treated roots,

Figure 2. [Ca2+]cyt-dependent FRET efficiencychanges in the transition zone of Arabidopsisprimary roots after Al3+ application. A to D,The [Ca2+]cyt signature had one broad peak (A)and two to three peaks (B–D). The dashed linein A is a representative trace after addition ofthe pH-4.5 control solution. B shows tworepresentative tracings with peaks that wereunequal in height, and C shows a tracewith almost two identical peaks. D shows a[Ca2+]cyt signature with three distinct peaks.Numbers in each panel indicate the percent-age of roots imaged that displayed a particular[Ca2+]cyt signature. Arrows indicate the time ofAl3+ application. E, Pseudocolored time-lapsesequence of cells in the transition zone aftertreatment with Al3+. The middle panels showthe timing of two peaks that correspond tomaximum [Ca2+]cyt response. SupplementalMovie S3 shows the complete movie se-quence. Bar = 20 mm.





the fastest [Ca2+]cyt response appeared to occur in thematuration zone, with subsequent [Ca2+]cyt increasespropagating toward the root cap (Table I). Upon Glutreatment, the [Ca2+]cyt increase was fastest in themeristem and root cap and propagated rapidly towardthe transition zone, elongation zone, and maturationzone. Treatment with ATP induced the fastest [Ca2+]cytresponse in the meristem and transition zone, andsubsequent propagation of the [Ca2+]cyt signal appearedto occur toward the root cap and the maturation zone.A summary of the timing and potential directions of[Ca2+]cyt increases in response to Al3+, Glu, and ATPin the primary root is shown in Figure 4.

The Biphasic [Ca2+]cyt Signature Triggered by Al3+ OccursPrimarily in Cells Located at 300 to 500 mm from the RootCap Junction

As shown in Figure 2, the [Ca2+]cyt signatures in theregion designated as the transition zone triggeredby Al3+ were either monophasic or biphasic. Thisvariability in the pattern of [Ca2+]cyt increases promp-ted us to probe further into the reasons behind this

variability. Our initial experiments were con-ducted with a 633 objective with a confocal zoomsetting of 2.0. With such settings, we were only able toacquire data from a 100-mm longitudinal area of theroot. To improve our spatial resolution and askwhether a particular [Ca2+]cyt signature was associatedwith a more defined area of the transition zone, weused a 203 objective with a confocal zoom of 1.0. Atthis magnification, we were able to acquire data thatcovered the apical 500mmof the primary root tip,whichencompassed the cap, meristem, transition zone, and azone before the initiating root hairs, which is likely thezone that Verbelen et al. (2006) refer to as the fastelongation zone. At this low-magnification setting, wefound that the biphasic [Ca2+]cyt rise after Al3+ treatmentwas primarily associatedwith the region of the root thatwas about 300 to 500 mm from the RCJ (Fig. 5A).

Gd3+ and La3+ Trigger Increases in [Ca2+]cyt That AreSimilar to Those Induced by Al3+

To evaluate whether the increases in [Ca2+]cytwere specific to Al3+ and not due to a general effect

Figure 3. [Ca2+]cyt-dependent FRETefficiency changes in the cap, mer-istematic zone, and maturationzone of Arabidopsis primary rootsafter Glu, ATP, and Al3+ applica-tion. A to C, The shape of the[Ca2+]cyt response after Glu (A),ATP (B), and Al3+ (C) applicationwas roughly similar between thecap and the meristem, but themagnitude of the response wasgenerally lower in the cap. Arrowsindicate the time of treatmentapplication. Insets show pseudo-colored images before and afterchemical application. Supplemen-tal Movies S4 to S6 show the com-plete movie sequences for theinsets. Yellow rectangles in A indi-cate areas corresponding to the capand meristem where [Ca2+]cyttraces were obtained. D, Glu ap-plication triggered a [Ca2+]cyt in-crease in the maturation zone thathad similar shapes as those elicitedin the cap and meristem. E, ATPapplication induced a [Ca2+]cyt sig-nature in the maturation zone thatoscillated slightly while graduallydeclining to basal levels. F, In thematuration zone, Al3+ treatmentinduced a [Ca2+]cyt response witha sharp peak and a second broaderpeak. Bars in insets = 20 mm.





of trivalent ions, we analyzed the effect of Gd3+ andLa3+ on Ca2+-dependent FRET efficiency changes inthe root transition zone. Both Gd3+ and La3+ inducedCa2+-dependent FRETefficiency transients in root cellsof the transition zone that were similar to those trig-gered by Al3+. Like Al3+, La3+ and Gd3+ treatmentsinduced both biphasic and monophasic [Ca2+]cytsignatures (Supplemental Fig. S2). Because the var-iability of La3+- and Gd3+-induced [Ca2+]cyt signatureswas reminiscent of those induced by Al3+, we resortedto low-magnification imaging to determine whether aparticular [Ca2+]cyt signature was associated with aspecific part of the transition zone. Like Al3+ treatment,the originof thebiphasic [Ca2+]cyt responseafterLa

3+andGd3+ application occurred primarily in cells located at300 to 500 mm from the root tip (Fig. 5, B and C).

The Biphasic [Ca2+]cyt Signature Induced by TrivalentCations Originated from Epidermal and Cortical Cells

In order to further resolve the origin of the biphasic[Ca2+]cyt response in the transition zone triggered byAl3+, Gd3+, and La3+, we acquired FRET efficiency datain a region of the root that was approximately 400 to500 mm from the tip, where we consistently observedthe biphasic [Ca2+]cyt signature (Fig. 5). For theseexperiments, we used a 633 objective and a confocalzoom setting of 2.0 to obtain better cellular resolution.We imaged the root at a depth that allowed us toclearly distinguish epidermal cells from the underly-ing cortical cells. This was only possible if the root wasgrowing flat across the surface of the coverslip. Thisensured that the outlying epidermal cells, which weremostly vacuolated, were in the same focal plane as thecytoplasm-rich cortical cells. We collected FRET effi-ciency traces from a region of the root that corre-sponded to epidermal or cortical cells (Fig. 6A, whiterectangles). In a majority of the roots imaged (61%), thebiphasic [Ca2+]cyt response after Al3+ treatment wasassociated with cortical cells, whereas the [Ca2+]cytsignature from the epidermal cells had only onepeak (Fig. 6A). We found in 39% of the roots thatboth epidermal and cortical cells exhibited the bi-

phasic [Ca2+]cyt signature (Fig. 6B). Similar observa-tions were made when the roots were treated withGd3+ (Fig. 6C) or La3+ (data not shown).

EGTA, BAPTA, Verapamil, La3+, and Gd3+ PretreatmentDisrupt the Shape, Amplitude, and Timing of theTrivalent Al3+-Triggered Biphasic [Ca2+]cyt Signature

To further characterize the nature of the biphasic[Ca2+]cyt response to Al3+, we pretreated the roots withreagents that block Ca2+ channels or chelate Ca2+ fromthe external medium. We first utilized verapamil,which is known to block plasma membrane voltage-dependent Ca2+ channels (Demidchik et al., 2002).Roots pretreated with the solvent control solution for20 min and then with Al3+ displayed the typical Al3+-induced biphasic [Ca2+]cyt response (Fig. 7A). In rootspretreated with 1 mM verapamil, Al3+ evoked only asingle [Ca2+]cyt peak (Fig. 7B). In roots pretreated withcompounds that chelate free Ca2+ ions, such as EGTAor 1,2-bis(2-aminophenoxy)ethane-N,N,N#,N#-tetraaceticacid (BAPTA), the Al3+-induced biphasic [Ca2+]cyt wasgenerally dampened (Fig. 7, C and D). Similar obser-

Table I. Time analysis of Al3+-induced [Ca2+]cyt elevations in cells from different developmental regions of Arabidopsis roots expressing YC3.60

Values are means 6 SE from at least three independent experiments. Number of replicates is in parentheses. N/A, Not applicable.

Parameter Root Cap Meristem Transition Zone Maturation Zone

Response onset 99.4 6 8.5 (n = 14) 85.2 6 6.7 (n = 15) 66.0 6 6.9 (n = 55) 47.6 6 6.8 (n = 8)Time to reach maximum [Ca2+]cyt

elevation (first peak) 211.8 6 10.4 (n = 14) 197.0 6 5.3 (n = 15) 186.5 6 6.9 (n = 55) 115.9 6 8.7 (n = 8)

Time to reach maximum [Ca2+]cytelevation from onset (first peak) 67.4 6 12.0 (n = 14) 66.8 6 5.3 (n = 15) 66.4 6 5.2 (n = 55) 23.3 6 2.6 (n = 8)

Time to reach maximum [Ca2+]cytelevation (second peakwhen observed)

N/A N/A 260.5 6 12.5 (n = 33) 11.0 6 37.2 (n = 6)

Time to reach maximum [Ca2+]cytelevation from onset ofsecond peak

N/A N/A 31.5 6 2.9 (n = 33) 28.5 6 4.9 (n = 6)

Total duration of [Ca2+]cyt elevation 282.6 6 31.3 (n = 14) 197.7 6 23.6 (n = 15) 280.8 6 15.7 (n = 55) 416.6 6 41.4 (n = 8)

Figure 4. Summary of the onset of the [Ca2+]cyt increase in the root afterGlu, ATP, and Al3+ application. Numbers indicate the average time forthe onset of the [Ca2+]cyt rise 6 SE. Arrows indicate the proposeddirection of the [Ca2+]cyt wave based on the timing of the [Ca2+]cyt risefor each root developmental zone. Bar = 100 mm.





vations were made on Gd3+- and La3+-treated rootsafter pretreatment with verapamil, EGTA, or BAPTA(data not shown).Since La3+ and Gd3+ are commonly used as Ca2+

channel blockers (Sivaguru et al., 2003; Monshausenet al., 2009), we investigated whether pretreatment ofroots with these two trivalent cations had any impacton the Al3+-induced [Ca2+]cyt transients. We found thatpretreatment with La3+ or Gd3+ for 20 min pro-duced obvious effects on the pattern of the Al3+-elici-ted [Ca2+]cyt signatures in the root transition zone, butthese effects were quite variable (Supplemental Fig.S3). In some experiments, pretreatment with eithercation appeared to completely inhibit any [Ca2+]cytincrease after Al3+ application (Supplemental Fig.S3B), whereas in other experiments, either the first orsecond [Ca2+]cyt peak was dampened or inhibited(Supplemental Fig. S3, B and C). In the majority ofthe tracings, pretreatment with La3+ or Gd3+ followedby Al3+ application triggered a [Ca2+]cyt rise with abroad peak (Supplemental Fig. S3B). An Al3+-inducedbiphasic signature was rarely observed when the rootwas pretreated with La3+ or Gd3+. In one case (6% oftracings), we observed a biphasic [Ca2+]cyt signature inGd3+-pretreated roots, but the peaks were widelyseparated and smaller in area (Supplemental Fig.S3C). The basis for this variability is unclear, but it isevident that these two cations altered the Al3+-induced[Ca2+]cyt signatures. Further experiments are needed touncover the reasons for these observations.In another set of experiments, roots were treated

with elevated external Ca2+ by applying 10 mM CaCl2and [Ca2+]cyt-dependent FRETchanges weremonitoredshortly thereafter. Application of 10 mM CaCl2 alonedid not trigger a rise in [Ca2+]cyt in the root transitionzone (Supplemental Fig. S4A). When Al3+ was added

to CaCl2-pretreated roots, we observed a rapid in-crease in [Ca2+]cyt that remained elevated throughoutthe 10-min sampling period (Supplemental Fig. S4B).These results indicate that roots have a mechanism toprevent Ca2+ influx into the cytosol despite high Ca2+ inthe externalmedium,which is altered byAl3+ application.

The Al3+-Induced Biphasic [Ca2+]cyt Signature Is Modifiedby an Antagonist of Neuronal Glu Receptors and anAnion Channel Blocker

It was previously reported that the impact of Al3+ onArabidopsis root growth and the cytoskeleton couldbe mediated in part by interactions with Glu signalingpathways (Sivaguru et al., 2003). We asked wheth-er imaging of [Ca2+]cyt using sensitized FRETof YC3.60could reveal additional insights into the nature of thisinteraction. We first asked how Glu pretreatment af-fected the Al3+-induced biphasic [Ca2+]cyt signature inthe root transition zone. The transition zone of controlroots (i.e. roots pretreated with pH-4.5 solvent controlsolution) displayed the characteristic biphasic [Ca2+]cytsignature after Al3+ application (Fig. 8A). When rootswerepretreatedwithGlufor20minprior toapplicationofAl3+, only a single [Ca2+]cyt peak was observed thatroughly coincided with the second peak of control roots(Fig. 8B). When Al3+ and Glu were applied simulta-neously (Fig. 8C), roots exhibited [Ca2+]cyt signatures thatmirrored those of roots treated with Glu alone (Fig. 1B).

We next asked whether the Al3+-induced [Ca2+]cyttransients could be facilitated through ionotropic Glureceptors, which are known to conduct cations acrossthe plasma membrane upon binding to Glu (Mayer,2005). We pretreated roots with 2-amino-5-phospho-nopentanoate (AP-5), an antagonist of neuronal Glureceptors (Davies et al., 1981), which was shown by

Figure 5. [Ca2+]cyt-dependent FRETefficiency changes in the apical 500 mm of Arabidopsis primary roots after Al3+ (A), La3+ (B),and Gd3+ (C) application. FRET efficiency imaging and calibration were conducted using a 203 objective. Representative FRETefficiency tracings were acquired from the regions indicated by the black rectangles. Cations were applied 45 s after the start ofimaging. A clear biphasic [Ca2+]cyt signature was obtained primarily from the region 300 to 500 mm from the root cap junctionafter exposure to all three trivalent cations. FRET efficiency measurements are representative of at least eight independentexperiments. Bar = 100 mm.





Sivaguru et al. (2003) to block Al3+-induced microtu-bule disruption and membrane depolarization. Inter-estingly, 1 mM AP-5 on its own was able to inducerapid [Ca2+]cyt transients resembling that of rootstreated with Glu alone (Fig. 8D). When Al3+ wasapplied to roots pretreated with AP-5, only a singlebroad [Ca2+]cyt peak was observed in the transitionzone, which was comparable to the Al3+-induced[Ca2+]cyt peak of Glu-pretreated roots (Fig. 8, compareE and B). Consistent with its function as an anta-gonist of ionotropic Glu receptors, AP-5 pretreatmentcompletely blocked the Glu-induced [Ca2+]cyt increasesin the root transition zone (Fig. 8F).

We also tested whether the Al3+-induced biphasic[Ca2+]cyt signature in the root transition zone wasaffected by the anion channel blocker 5-nitro-2-(3#-phenylpropyl-amino) benzoate (NPPB), since thiscompound was also shown to prevent Al3+-triggeredcellular changes in Arabidopsis roots (Sivaguru et al.,2003). NPPB alone did not evoke any [Ca2+]cyt changesin the root transition zone (Fig. 8G), but pretreatmentwith this compound altered the shape and amplitudeof the Al3+-induced [Ca2+]cyt signature. Treatment withthis anion channel inhibitor either abolished the firstAl3+-induced [Ca2+]cyt peak while dampening the sec-ond peak or caused a late rise in [Ca2+]cyt that wassustained throughout the entire 10-min sampling pe-riod (Fig. 8H). Like AP-5, NPPB pretreatment inhib-ited the Glu-induced [Ca2+]cyt spike (Fig. 8I).

Trivalent Cations Elicit Similar Early [Ca2+]cyt Increases inan Al3+-Resistant and an Al3+-Sensitive Mutant

The observation that La3+ and Gd3+ elicited [Ca2+]cytsignatures similar to those evoked by Al3+ indicatesthat these early [Ca2+]cyt changes represent a generalresponse to trivalent cations rather than to mecha-nisms that are specific to Al3+ toxicity or signaling. Toaddress this issue further, we transformed one Al3+-resistant mutant (alr104; Larsen et al., 1998)and one Al3+-sensitive mutant (als3-1; Larsen et al.,2005) with the YC3.60 reporter and evaluated the[Ca2+]cyt response after treatment with Al3+, La3+, orGd3+. We again focused our imaging on the region ofthe root that exhibited the biphasic [Ca2+]cyt response,since this pattern of [Ca2+]cyt change appears to bethe most distinguishing and common feature of thetrivalent cation response. We observed similar triva-lent-induced biphasic [Ca2+]cyt signatures in the roottransition zone in the wild type and the two Al3+

response mutants (Fig. 9, A–F). Like wild-type roots,roots of alr104 and als3-1 pretreated with 1 mM verap-amil for 20 min, followed by Al3+ addition, displayedonly a single [Ca2+]cyt peak (Fig. 9, G and H).

Growth Analysis of Arabidopsis RootsExpressing 35S::YC3.60

Since alr104 and als3-1 mutants expressing theYC3.60 reporter did not show any obvious differences

Figure 6. Epidermal and cortical cell measurements of [Ca2+]cyt-dependent FRET efficiency changes in the root transition zone. Mea-surements were obtained from the areas marked by the white rectanglesto delineate epidermal and cortical cells. Measurements were obtainedat 400 to 500 mm from the tip, where the biphasic [Ca2+]cyt signature isconsistently observed. Cations were applied 45 s after the start ofimaging. In traces with a biphasic [Ca2+]cyt response, two generalpatterns were observed. A, Cortical cells exhibited biphasic [Ca2+]cytsignatures, whereas epidermal cells displayed a monophasic [Ca2+]cytsignature. B, Both epidermal and cortical cells displayed biphasic[Ca2+]cyt responses. Numbers in A and B indicate the total number ofroots displaying a particular cell type response over the total number ofroots imaged. C, Representative [Ca2+]cyt signature traces after Gd3+

treatment showing biphasic and monophasic signatures in the corticaland epidermal cells, respectively. Bars = 20 mm.





in their [Ca2+]cyt response as compared with YC3.60-expressing wild-type roots after exposure to Al3+, weconducted short- and long-term growth assays on allthree genotypes to determine whether YC3.60 trans-formation altered the tolerance and sensitivity of theselines to Al3+. We followed the growth kinetics of wild-type Arabidopsis primary roots expressing the 35S::YC3.60 reporter maintained under the identical con-ditions used for [Ca2+]cyt measurements (i.e. the same1 mM Al3+ concentration and volume used to elicit the[Ca2+]cyt changes reported here). Images of growingwild-type roots were obtained at 1-min intervals for 60min. Arabidopsis primary roots growing unperturbedon the coverslip system for 10 min elongated at a rateof 2.50 6 0.17 mm min21 (n = 11). When treated with1 mM Al3+, the growth rate dropped to an average of1.46 6 0.20 mm min21 (n = 11) for the next 50 min ofimaging (Supplemental Fig. S5A). We also monitoredthe growth kinetics of alr104 and als3-1 lines express-ing YC3.60. The growth rates of untreated alr104 andals3-1 were 3.08 6 0.40 (n = 6) and 2.30 6 0.37 mmmin21 (n = 6), respectively. Upon application of 1 mM

Al3+, the growth rates of alr104 and als3-1 dropped toan average of 1.62 6 0.1 (n = 6) and 1.09 6 0.07 mmmin21 (n = 6), respectively, during the next 50 min ofimaging (Supplemental Fig. S5A). Based on ANOVA,these growth rates were not statistically differentamong the three genotypes. However, two-sample ttests show that there were statistical differences be-tween the growth rates before adding Al3+ (0–10 min)and after (10–60 min) in all three genotypes.For long-term Al3+ exposure growth assays, we

followed the protocol of Sivaguru et al. (2003), as thisproved to be the most effective method for long-termapplication of Al3+ on Arabidopsis seedlings growing

on agar plates. Six-day-old seedlings expressingYC3.60 of the wild type, alr104, and als3-1 were trans-planted onto agar plates perfused with a range of Al3+

concentrations. The position of the root tip wasmarked on the back of the petri dish at 0 and 24 hafter transplanting, and the increment between markswas measured (Sivaguru et al., 2003). The data wereanalyzed using ANOVA and Duncan’s multiple com-parison test. In the absence of Al3+, there was nostatistical difference in root growth among the threegenotypes. As expected, als3-1 was more sensitive tothe growth inhibitory effects of Al3+ compared withthe wild type at 40 and 80 mM Al3+, whereas alr104wasmore resistant at 40 to 160 mM Al3+ (Supplemental Fig.S5B). The differences in growth were statistically sig-nificant at a = 0.05 as determined by Duncan’s mul-tiple comparison test. Thus, these data indicate thatdifferences in growth among the three genotypes aremost strongly manifested after long-term exposure toAl3+ and that expressing the YC3.60 in the mutantbackgrounds did not alter the tolerance or sensitivenature of these plants to Al3+.

DISCUSSION

In this investigation, we employed a preloadedapplication wizard from the Leica TCS SP2 AOBSconfocal microscope to monitor Ca2+-dependent FRETefficiency in Arabidopsis primary roots expressing theYC3.60 sensor. The Leica application wizard takes intoaccount spectral bleed-through (i.e. the amount oflight detected in the FRET channel that is not due toenergy transfer) and normalizes for differences indonor and acceptor levels (van Rheenen et al., 2004;

Figure 7. [Ca2+]cyt-dependent FRET efficiencychanges in the transition zone of Arabidopsisprimary roots pretreated with verapamil,EGTA, or BAPTA for 20 min prior to Al3+

application. Imaging was conducted at 400to 500 mm from the root cap junction, whichconsistently shows a biphasic [Ca2+]cyt signa-ture in response to Al3+. A, Pretreatment withdeionized water (solvent control) does notaffect the Al3+-induced biphasic [Ca2+]cyt sig-nature. B, Only a monophasic [Ca2+]cyt signa-ture is observed in verapamil-pretreated rootsafter Al3+ application. Note that the peak ofthe [Ca2+]cyt signature in verapamil-pretreatedroots coincides with the second peak of sol-vent control-pretreated roots. EGTA (C) andBAPTA (D) dampened the amplitude of theAl3+-elicited [Ca2+]cyt signature. Arrows indi-cate the time of treatment application. FRETefficiency measurements are representative ofat least eight independent experiments.





Feige et al., 2005) for collecting FRET-sensitized emis-sion images. Using this approach, we were able todemonstrate [Ca2+]cyt-dependent FRET efficiency os-cillations in growing root hair tips (Supplemental Fig.S1; Supplemental Movie S1). We were also able to elicit[Ca2+]cyt-dependent FRET efficiency changes in rootcells after treatment with Glu and ATP, two com-pounds that are known to cause rapid spikes in [Ca2+]cytin plant cells (Fig. 1). In contrast to previousstudies with Glu and ATP, which relied mostlyon luminescence-based [Ca2+]cyt measurements withaequorin-expressing plant cells (Dennison and Spald-ing, 2000; Demidchik et al., 2003; Jeter et al., 2004; Qiet al., 2006), the improved spatial resolution of FRET-based confocal imaging of YC3.60 allowed us to defineregions of the root from which a particular [Ca2+]cytresponse originated. In doing so, we showed that alldevelopmental regions of the root exhibited a [Ca2+]cyt

response to Glu and ATP. Furthermore, based on thetiming of the [Ca2+]cyt rise, we were able to infer thedirection in which the [Ca2+]cyt signal propagatedin the root (Figs. 1, 3, and 4; Supplemental Movies S4and S5).

Having established that imaging of FRET efficiencycan be used to monitor [Ca2+]cyt transients in rootsexpressing YC3.60, we asked whether this [Ca2+]cytdetection method would allow us to gain additionalinsight into the early responses of plants to Al3+ andthe underlying mechanisms behind the Al3+-induced[Ca2+]cyt changes that are prevalent in the literature.Increased [Ca2+]cyt in response toAl3+ has been reportedin intact rye (Secale cereale; Ma et al., 2002), barley(Hordeum vulgare; Nichol and Oliveira, 1995), andwheat (Triticum aestivum; Zhang and Rengel, 1999;Bhuja et al., 2004) roots. In these studies, the Al3+-induced [Ca2+]cyt increase was observed 10 to 20 min

Figure 8. [Ca2+]cyt-dependent FRET efficiency changes in the transition zone of Arabidopsis primary roots pretreated with Glu,AP-5, and NPPB for 20 min prior to Al3+ application. Imaging was conducted at 400 to 500 mm from the root cap junction, whichconsistently shows a biphasic [Ca2+]cyt signature in response to Al3+. A, Pretreatment with the solvent control solution does notaffect the Al3+-induced biphasic [Ca2+]cyt signature. B, Only a monophasic [Ca2+]cyt signature is observed in Glu-pretreatedroots after Al3+ application. C, Simultaneous application of Glu and Al3+ triggers a [Ca2+]cyt response that resembles that inducedby Glu alone. D, AP-5 treatment evokes a [Ca2+]cyt transient similar to that induced by Glu alone. E and F, Preatreatment withAP-5 appears to block or dampen the first Al3+-induced [Ca2+]cyt peak (E) while inhibiting the Glu-induced [Ca2+]cyt increase (F).G, The anion channel inhibitor NPPB does not cause a [Ca2+]cyt response when applied on its own. H and I, NPPB pretreatmentmodifies both the Al3+-induced (H) and Glu-induced (I) [Ca2+]cyt response. Arrows indicate the time of treatment application.FRET efficiency measurements are representative of at least eight independent experiments.





(Zhang and Rengel, 1999; Ma et al., 2002) or hours(Nichol and Oliveira, 1995; Bhuja et al., 2004)after Al3+ treatment. Here, we show an Al3+-induced[Ca2+]cyt elevation after 48 to 99 s of application (Fig.2; Table I). Lindberg and Strid (1997) observed in-creases in [Ca2+]cyt within approximately the sametime frame as those reported here using wheat rootprotoplasts loaded with the Ca2+-sensitive dyeFura 2. However, with constitutive expression ofthe YC3.60 reporter, we were able to expand on theseearlier observations by demonstrating that differentregions of an intact root have distinct responses toAl3+ application with regard to the onset, shape, andduration of the [Ca2+]cyt response (Figs. 2–5; Table I).Furthermore, we showed that the [Ca2+]cyt rise afterAl3+ treatment generally begins in the maturationzone and propagates toward the root cap (Fig. 4).The manner of propagation of the [Ca2+]cyt response

is distinct from that elicited by ATP or Glu (Fig. 4).The reason for differences in [Ca2+]cyt responsesamong the different root developmental zones uponAl3+ application remains unclear. However, one pos-sibility is that cells from each root developmentalregion differ in their efficiency in removing Ca2+

because of differences in the expression of Ca2+-ATPases in the plasma membrane and/or endoplas-mic reticulum and Ca2+/H+ exchangers in theplasma membrane and tonoplast (Sze et al., 2000;Sanders et al., 2002). Another possibility is that cellsin the different root regions express certain receptorproteins that perceive the applied chemical signal,which in turn activate a specific type of Ca2+ channel(Kiegle et al., 2000a). Nonetheless, our ability todistinguish unique patterns of [Ca2+]cyt increases indifferent root developmental zones in response tovarious chemical treatments paves the way for un-

Figure 9. [Ca2+]cyt-dependent FRET efficiency mea-surements in alr104 and als3-1 mutants after treat-ment with 1 mM Al3+, La3+, and Gd3+. Imaging wasconducted at 400 to 500 mm from the root cap junc-tion, which consistently shows a biphasic [Ca2+]cytsignature in response to the trivalent cations. Bothalr104 (A–C) and als3-1 (D–F) show similar [Ca2+]cytsignatures as the wild type. Like wild-type roots, only amonophasic [Ca2+]cyt signature is observed in verapamil-pretreated roots of alr104 (G) and als3-1 (H) after Al3+

application. Cations were applied 45 s after the startof imaging. FRET efficiency measurements are rep-resentative of at least five independent experimentsfor each genotype.





raveling spatial [Ca2+]cyt signaling mechanisms withina growing, intact root.

The majority of our measurements of Al3+-induced[Ca2+]cyt elevations in the transition zone of the rootwere biphasic (Fig. 2). The spatial resolution providedby FRET efficiency imaging allowed us to narrowdown the region of the root from where the biphasic[Ca2+]cyt signature originated to cells located approx-imately 300 to 500 mm from the RCJ (Fig. 5). However,the significance of the Al3+-induced biphasic [Ca2+]cytresponse is unclear. From our results, it seems likelythat the first Al3+-induced [Ca2+]cyt peak is linkedto Ca2+ influx across the plasma membrane throughCa2+-permeable channels (Demidchik et al., 2002;Miedema et al., 2008) and that the second peakis triggered by the release of Ca2+ from internal stores(e.g. vacuole, endoplasmic reticulum, mitochondria,or plastids; Trewavas and Malho, 1998; Sanders et al.,2002; Nomura et al., 2008). In this context, the initialrise in [Ca2+]cyt may activate Ca2+ channels located inthe tonoplast, endoplasmic reticulum, and other inter-nal Ca2+ stores, resulting in the appearance ofthe second peak. Indeed, such a phenomenon of Ca2+-induced Ca2+ release from internal stores has beendescribed in plant cells (Ng and McAinsh, 2003). It isalso possible that the observed biphasic elevation in[Ca2+]cyt represents [Ca

2+]cyt transients originating fromtwo cell types, which have [Ca2+]cyt signatures thatdiffer in their amplitude and timing. To address thishypothesis, we collected [Ca2+]cyt-dependent FRET ef-ficiency data from epidermal and cortical cells in theregionof the root that consistently exhibited the biphasic[Ca2+]cyt response upon Al3+ treatment. Our mea-surements of FRET efficiency showed that the biphasic[Ca2+]cyt signature could be traced to either epidermal orcortical cells, with the majority of the biphasic [Ca2+]cytelevations originating from the cortex (Fig. 6). However,we cannot rule out the possibility that other cell types,such as vascular, endodermal, or pericycle cells, con-tribute to the biphasic [Ca2+]cyt signature. Expressing theYC3.60 reporter in various root cell types (Kiegle et al.,2000b) combinedwith imaging of FRETefficiency in theintact root should help uncover the precise nature of theAl3+-evoked biphasic [Ca2+]cyt signature.

Despite the difference in the direction of propaga-tion of the [Ca2+]cyt wave between Al3+- and Glu-treated roots (Fig. 4), there is reason to believe thatsome aspects of the [Ca2+]cyt response evoked by eachchemical occurs through a common signaling path-way. Sivaguru et al. (2003) found that Al3+-inducedmicrotubule reorganization and membrane depolari-zation could involve Glu receptors, since both cellularresponses were blocked by pretreatment with AP-5,a specific neuronal Glu receptor antagonist. Further-more, they showed that Al3+ and Glu applied simulta-neously did not have an additive effect on microtubulereorganization and membrane depolarization. Inagreement with this study, we found that Glu andAl3+ applied together evoked [Ca2+]cyt transients thatwere similar to those induced by Glu alone, whereas

Glu and AP-5 pretreatment inhibited the onset ofthe first Al3+-induced [Ca2+]cyt peak (Fig. 8). These re-sults reinforce the notion that an ionotropic Glu re-ceptor that conducts cation transport across themembrane partly contributes to the Al3+-induced Ca2+

influx in the root transition zone and broadly supportthe findings of Sivaguru et al. (2003).

The inhibitor studies depicted here reveal additionalinsights into the nature of the Al3+-evoked bi-phasic [Ca2+]cyt signature in the root transition zone,since pretreatment with either AP-5 or Glu inhibitedonly the first Al3+-induced [Ca2+]cyt peak (Fig. 8). Thissuggests that in addition to the yet to be identifiedplant Glu receptor(s), the biphasic Al3+-evoked [Ca2+]cytsignature in the transition zone could be a reflectionof Al3+ acting on a cellular target that triggersthe release of Ca2+ from internal stores rather thanCa2+-induced Ca2+ release. This notion is supported bythe fact that most of the inhibitors we used in thisstudy, including the plasma membrane Ca2+ channelblocker verapamil and the Ca2+ chelators EGTA andBAPTA, dampened or abolished the first Al3+-induced[Ca2+]cyt peak, with minimal or no effect on the secondpeak. Although our experiments with AP-5, verapa-mil, and Ca2+ chelators support the idea that the Al3+-induced [Ca2+]cyt elevations are linked to Ca2+ entryacross the plasma membrane, it is possible that otherchannels could also contribute to Ca2+ influx, includ-ing hyperpolarization-activated Ca2+ channels anddepolarization-activated Ca2+ channels (Kiegle et al.,2000a; Miedema et al., 2008). Al3+ has been shown tohyperpolarize (Kinraide, 1993, and refs. therein)or depolarize the plasma membrane of root cells(Sivaguru et al., 2003; Illes et al., 2006). This couldtrigger both hyperpolarization-activated Ca2+ chan-nels and depolarization-activated Ca2+ channels toopen, resulting in Ca2+ influx into the cytoplasm andcontributing to the [Ca2+]cyt transients in the root.Pretreatment with NPPB, an anion channel inhibitor,also prevented the initial Al3+-induced [Ca2+]cyt peak inthe transition zone, similar to its effect on mem-brane depolarization and microtubule organization(Sivaguru et al., 2003). Since Glu-induced membranedepolarization was not blocked by NPPB, Sivaguruet al. (2003) hypothesized that Al3+ might facilitate theopening of a channel through which Glu permeates,triggering a [Ca2+]cyt rise upon binding to its receptor.Since we show that NPPB also prevented the Glu-induced [Ca2+]cyt increase (Fig. 8), a more likely scenariowould be Al3+ acting on a plasma membrane receptor,which then triggers a Ca2+ influx. Electrophysiologicalstudies in conjunction with [Ca2+]cyt imaging and phar-macological approaches would be a fruitful area ofresearch to better understand the precise nature of theearly Al3+-induced [Ca2+]cyt increases in the root.

Gd3+ and La3+ also triggered increases in [Ca2+]cytthat were similar to those induced by Al3+, particularlywith regard to the biphasic [Ca2+]cyt response in theroot transition zone (Fig. 5). These trivalent cationshave been shown to inhibit specific types of Ca2+





currents in plants (Kiegle et al., 2000a; Demidchiket al., 2002; Qu et al., 2007) and as such have beencommonly used as Ca2+ channel blockers. Thus, it wassurprising to observe elevations in [Ca2+]cyt in responseto all three trivalent cations. This indicates that, at leastin Arabidopsis, the biphasic [Ca2+]cyt changes we ob-served are part of a more general signal transductionpathway in response to trivalent cations and are notdirectly linked to mechanisms of Al3+ toxicity. Despitethe ability of La3+ and Gd3+ to trigger early [Ca2+]cytincreases in the root, we found that a 20-min pretreat-ment with these trivalent cations had obviousbut inconsistent effects on the Al3+-triggered biphasic[Ca2+]cyt signature (Supplemental Fig. S3). Althoughsuch observations somewhat validate the use ofLa3+ and Gd3+ as Ca2+ signaling antagonists, additionalstudies are needed to clarify the mode of action ofthese cations on [Ca2+]cyt responses. Pretreatment withLa3+ and Gd3+, particularly at the concentrations usedhere, could on their own have a negative impact on thegrowth of the root. Thus, any subsequent chemicalapplication on pretreated roots will display an altered[Ca2+]cyt signature that could be more reflective of thegrowth status of the root. For example, the broad Al3+-induced [Ca2+]cyt shapes that we occasionally observedin the transition zone of La3+- or Gd3+-pretreated roots(Supplemental Fig. S3B) might be indicative of re-sponses of nongrowing cells like those observed in themature zone or differential effects on specific cell types(Figs. 3 and 6).An additional observation indicating that the early

[Ca2+]cyt signatures shown here are not specificallyrelated to Al3+ toxicity is the fact that [Ca2+]cyt changeswere first initiated in the maturation and elongationzones, which are less sensitive to Al3+ compared withthe transition zone (Sivaguru and Horst, 1998; Illeset al., 2006). This notion is further supported by thefact that alr104 and als3-1 mutants, which were previ-ously shown to be resistant and sensitive to Al3+,respectively (Larsen et al., 1998, 2005), displayed thesame [Ca2+]cyt responses to all three trivalent cations aswild-type seedlings (Fig. 9). The als3-1 mutant, whichis disrupted in a gene that encodes a phloem-localizedATP-binding cassette-like transporter, was hypersen-sitive to Al3+ but not to La3+ (Larsen et al., 1998),indicating that the trivalent ion-induced [Ca2+]cyt risein Arabidopsis root cells is unrelated to the Al3+

response mechanism involving the ALS3 gene. To thebest of our knowledge, the ALR104 gene has not yetbeen cloned, but given the similar nature of the Al3+-induced [Ca2+]cyt response between the wild typeand alr104, these early [Ca2+]cyt responses might beindependent of ALR104. However, since significantdifferences in sensitivity to Al3+ among all three geno-types arose hours after Al3+ application (SupplementalFig. S5B), we cannot rule out the possibility thatdifferences in [Ca2+]cyt responses will be more stronglymanifested at later time points and other root zones.Careful documentation of [Ca2+]cyt responses or oscil-lations in all root developmental regions during long

periods ofAl3+ exposurewill allowus tomorepreciselydescribe the relationship between the Al3+-sensitiveand -resistant natures of the mutants to Ca2+ signaling.

In conclusion, we demonstrate an alternativemethod for imaging dynamic changes in [Ca2+]cyt inliving roots of Arabidopsis seedlings expressing theYC3.60 reporter. This method is based on a standard-ized FRET-sensitized emission approach involvingthe use of donor-only and acceptor-only control linesto correct for optical cross talk and spectral bleed-through. Using this approach, we were able to monitorelevations in [Ca2+]cyt in different growth zones of intactArabidopsis roots treated with Al3+. The results wereport here provide evidence that the rapid [Ca2+]cyttransients induced by Al3+ are not tightly linked tomechanisms of Al3+ toxicity but rather are a feature ofa common signaling pathway in response to trivalentcations, part of which involves modulation by a Glureceptor.

MATERIALS AND METHODS

Preparation of Plant Material

Surface sterilization and planting of transgenic Arabidopsis (Arabidopsis

thaliana) seeds (Columbia ecotype) were conducted as described by Wang

et al. (2004). The seeds were planted on sterile 48-3 64-mm coverslips layered

with 2 mL of 0.5% NuSieve agarose (FMC BioProducts) containing half-

strength MS salts (Caisson Laboratories) and vitamins. The coverslips were

placed inside square petri dishes and placed in a growth chamber at 24�C and

40% humidity with a 16-h-light (124 mE m22 s21)/8-h-dark cycle for 3 to 4 d.

The petri dishes were vertically positioned to promote root growth along and

against the coverslip (Wang et al., 2004).

Subcloning of the YC3.60 andArabidopsis Transformation

The plasmid pcDNA3 containing the gene YC3.60 (GenBank accession no.

AB178712) was kindly provided by Dr. Atsushi Miyawaki (RIKEN Brain

Science Institute). The pcDNA3 plasmid was cut with EcoRI (New England

Biolabs), and the recessed 3# ends were filled in using the large Klenow

fragment (Promega) of the DNA PolI. The linearized plasmid was purified

using a PCR purification kit (Qiagen) and then digested with NcoI (New

England Biolabs). The products were separated by 1% agarose gel electro-

phoresis, and the YC3.60 construct was recovered from the gel and purified

using a Qiagen gel purification kit. The YC3.60 construct was subcloned into

the NcoI/PmlI cloning sites of the plant binary vector 35S/pCAMBIA1390

using standard protocols. 35S::YC3.60/pCAMBIA1390 plasmid was isolated

from overnight cell cultures and introduced into competent Agrobacterium

tumefaciens cells (C58C1 strain). Flowering Arabidopsis plants were trans-

formed by dipping them in medium containing A. tumefaciens cells harboring

35S::YC3.60/pCAMBIA1390 as described by Clough and Bent (1998). Simi-

larly, A. tumefaciens harboring 35S::eCFP/pCAMBIA1390 or 35S::eYFP/

pCAMBIA1390 was used to obtain transgenic Arabidopsis expressing eCFP

or eYFP only. Plants were then transferred to a growth chamber and grown at

24�C/22�C day/night temperature, 50% relative humidity, under a cycle of

16 h of light (87 mE m22 s21) and 8 h of dark.

Confocal Laser Microscopy and Measurement ofFRET-Sensitized Emission

Imaging was done with an inverted Leica TCS SP2 AOBS confocal laser

scanning microscope (Leica Microsystems) with an argon ion laser using a

203 dry lens (numerical aperture 70) or a 633 water-immersion lens (nu-

merical aperture 1.2). Excitation wavelengths were 458 and 514 nm for CFP

and YFP, respectively. Images were taken every 3 s for 10 min without any line





averaging. Images were captured at a scanning speed of 200 MHz and a pixel

resolution of 256 3 256. Single optical sections were taken at a depth of about

20 mm from the root epidermal cell layer. At this focal plane, both epidermal

and cortical cells in longitudinal view were visible. Exogenous application of

chemicals did not affect the focus of the roots during imaging, since roots were

securely anchored on the agarose that coated the coverslips. In cases where

the root drifted slightly, focus was restored by quickly rotating the fine-

adjustment knob of the microscope. Roots that drifted significantly during

imaging were not included in the analysis.

Because the spectra of CFP and YFP overlap, the sensitized emission must

be corrected for bleed-through of the emission of the donor (CFP) into the

acceptor (YFP or cpVenus) channel and for direct excitation of YFP during the

excitation of CFP (van Rheenen et al., 2004; Feige et al., 2005). Although bleed-

through ratios are theoretically constant, they can vary with fluorophore

intensities. Thus, corrections to account for bleed-through and for the differ-

ences in intensity between the donor and the acceptor must be performed.

The Leica application wizard for FRET-sensitized emission imaging was

used to make the necessary corrections. For this, Arabidopsis roots expressing

YC3.60 and either CFP or YFP alone were used each time to obtain calibration

images. To maintain consistency in imaging, calibration was done for each

objective used (203 versus 633) and root developmental region. Also, new

calibrations were always performed on the day of a new experiment (i.e. old

calibration images from the previous day were not used for an experiment

conducted on another day). In this procedure, an image of the root zone of

interest expressing YC3.60 was acquired first. The image was taken after

optimizing settings (pinhole, laser intensity, and photomultiplier gain) for the

CFP, FRET, and YFP channels. Then, images of reference roots (CFP and YFP

alone) were obtained using the same settings and used to generate calibration

coefficients. FRET efficiency was calculated by the Leica software using the

formula described by van Rheenen et al. (2004):

EA ¼ B2A3b2C3ðc2 a3bÞC

where EA is the apparent FRETefficiency; A, B, and C are the intensities of the

three channels (CFP, FRET, and YFP); and a, b, and c are the calibration

coefficients. The CFP-only reference generates the correction factor b, which is

CFP cross talk in the FRET image, b = B/A. The YFP-only reference generates

correction factors a and c. The ratio A/C is equal to a, which corrects for YFP

cross-excitation in the CFP image; c = B/C and corrects for YFP cross-excitation

in the FRET channel.

Chemical Treatments

Three- to 4-d-old wild-type and mutant seedlings expressing YC3.60 were

kept in the agarose growth medium on coverslips and carefully placed on the

stage of the confocal microscope. Stock solutions of 1 M AlCl3, LaCl3, GdCl3,

and Glu were made in deionized water, and a 10 mM stock solution was made

for ATP. Half-strength MS medium (pH 5.7 or 4.5 adjusted with 1.5 M Tris)

without vitamins was used to prepare all treatment solutions. A 1mMworking

solution was used for all treatments. Using a Hamilton syringe, 25 to 50 mL of

treatment solution was delivered gently on top of the agarose layer where the

root was embedded. All experiments were replicated and repeated at least five

times.

Stock solutions of 50 mM verapamil (Sigma-Aldrich), 10 mM EGTA, 10 mM

BAPTA, 10 mM AP-5, and 10 mM NPPB were prepared in deionized water and

further diluted in half-strengthMSmedium (pH 4.5) to a final concentration of

1 mM. NPPB was used at a final concentration of 100 mM. Pretreatment with

half-strength MS medium (pH 4.5) was used as a control for the inhibitor

work.

Root Growth Assessment and Statistical Analysis

For short-term growth measurements, 4-d-old seedlings of the wild type,

als3-1, and alr104 expressing YC3.60 were grown on agarose-coated coverslips

as described above. The time course of root growth was monitored using a

Nikon Eclipse TE300 microscope equipped with a Hamamatsu image proces-

sor CCD camera (Hamamatsu Photonics). Images were taken every 60 s for 60

min. Twenty-five microliters of 1 mM Al3+ solution was applied at 10 min after

starting the time course. Change in root elongation was measured from

individual frames using Metamorph 6.3r5 software (Molecular Devices). For

long-term growth analysis, 6-d-old seedlings of the wild type, als3-1, and

alr104 expressing YC3.60 were transplanted onto agar plates with or without

Al3+ according to the methods of Sivaguru et al. (2003). ANOVA, Duncan’s

multiple comparison tests, and two-sample t tests were performed using the

software package Number Cruncher Statistical System 97 (JL Hintze).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Time course of the Ca2+-dependent FRET effi-

ciency oscillations at the tip of a growing Arabidopsis root hair from a

root expressing YC3.60.

Supplemental Figure S2. Ca2+-dependent FRET efficiency changes in

Arabidopsis primary roots after La3+ and Gd3+ application.

Supplemental Figure S3. Ca2+-dependent FRET efficiency changes in the

root transition zone induced by Al3+ after La3+ or Gd3+ pretreatment.

Supplemental Figure S4. Al3+-induced Ca2+-dependent FRET efficiency

changes in the root transition zone pretreated with 10 mM CaCl2.

Supplemental Figure S5. Short- and long-term growth responses of wild-

type, als3-1, and alr104 roots to Al3+.

Supplemental Movie S1. Ca2+-dependent FRET efficiency oscillations at

the tip of a growing root hair.

Supplemental Movie S2. Ca2+-dependent FRET efficiency increase in the

root transition zone induced by Glu.


root transition zone induced by Al3+.


root cap and meristematic zone induced by Glu.


root cap and meristematic zone induced by ATP.


root cap and meristematic zone induced by Al3+.

ACKNOWLEDGMENTS

We are grateful to Dr. Atsushi Miyawaki (Laboratory for Cell Function

and Dynamics, Brain Science Institute, RIKEN) for providing the YC3.60

construct and to the Arabidopsis Biological Resource Center for seed of the

alr104 and als3-1 mutants. We also thank Dr. Maria Monteros (The Samuel

Roberts Noble Foundation) for her critical review of the manuscript.

Received September 11, 2009; accepted January 3, 2010; published January 6,

2010.

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