endoplasmic microtubules configure the subapical cytoplasm ... · endoplasmic microtubules...

Endoplasmic Microtubules Configure the SubapicalCytoplasm and Are Required for Fast Growth ofMedicago truncatula Root Hairs1

Bjorn J. Sieberer2, Antonius C.J. Timmers2, Franck G.P. Lhuissier, and Anne Mie C. Emons*

Laboratory of Plant Cell Biology, Wageningen University, Arboretumlaan 4, 6703 BD Wageningen,The Netherlands (B.J.S., F.G.P.L., A.M.C.E.); and Laboratoire de Biologie Moleculaire des RelationsPlantes-Microorganismes, Centre National de la Recherche Scientifique/Institut National de la RechercheAgronomique, BP 27, 31326 Castanet-Tolosan cedex, France (A.C.J.T.)

To investigate the configuration and function of microtubules (MTs) in tip-growing Medicago truncatula root hairs, we usedimmunocytochemistry or in vivo decoration by a GFP linked to a MT-binding domain. The two approaches gave similarresults and allowed the study of MTs during hair development. Cortical MTs (CMTs) are present in all developmental stages.During the transition from bulge to a tip-growing root hair, endoplasmic MTs (EMTs) appear at the tip of the young hairand remain there until growth arrest. EMTs are a specific feature of tip-growing hairs, forming a three-dimensional arraythroughout the subapical cytoplasmic dense region. During growth arrest, EMTs, together with the subapical cytoplasmicdense region, progressively disappear, whereas CMTs extend further toward the tip. In full-grown root hairs, CMTs, theonly remaining population of MTs, converge at the tip and their density decreases over time. Upon treatment of growinghairs with 1 �m oryzalin, EMTs disappear, but CMTs remain present. The subapical cytoplasmic dense region becomes veryshort, the distance nucleus tip increases, growth slows down, and the nucleus still follows the advancing tip, though at amuch larger distance. Taxol has no effect on the cytoarchitecture of growing hairs; the subapical cytoplasmic dense regionremains intact, the nucleus keeps its distance from the tip, but growth rate drops to the same extent as in hairs treated with1 �m oryzalin. The role of EMTs in growing root hairs is discussed.

Root hairs are lateral extensions of epidermal rootcells involved in the uptake of water and nutrients, inanchoring the plant in the soil (Peterson and Far-quhar, 1996; Gilroy and Jones, 2000), and in the in-teraction between nitrogen-fixing rhizobacteria andtheir Fabacean host plants (Mylona et al., 1995; Long,1996). They emerge as bulges from the outer pericli-nal cell wall of epidermal cells and elongate almostperpendicularly to the root axis by tip growth (Medi-cago truncatula: Shaw et al., 2000), which reflects anunderlying polarity of the cytoarchitecture (M. trun-catula: Sieberer and Emons, 2000).

In growing root hairs, cytoplasm, including thenucleus, is concentrated in the subapical region,whereas the basal part is highly vacuolated (vetch[Vicia sativa]: De Ruijter et al., 1998; M. truncatula:Sieberer and Emons, 2000). The subapical cytoplas-mic dense region contains endoplasmic reticulum(ER), mitochondria, plastids, and Golgi bodies

(vetch: Miller et al., 2000). At the base of this subapi-cal cytoplasmic dense region is the nucleus, whichfollows the expanding tip at a certain distance (M.truncatula: Sieberer and Emons, 2000). The extremeapex of the hair looks smooth in the differential inter-ference light microscope, and has been shown withelectron microscopy to be filled with vesicles (freezefixation/freeze substitution: Equisetum hyemale:Emons, 1987; Vicia hirsuta: Ridge, 1988, 1993; Viciavillosa: Sherrier and Van den Bosch, 1994; Arabidopsis:Galway et al., 1997; vetch: Miller et al., 2000).

The subapical cytoplasmic dense region is config-ured by fine bundles of actin filaments, called FB-actin (Miller et al., 1999). It is thought to function inthe transport and/or keeping of Golgi vesicles to thevesicle-rich region in the hair dome. After chemicalfixation and fluorescein-phalloidin staining of grow-ing hairs, this vesicle-rich region at the very tip ap-pears to be devoid of filamentous actin when ob-served with a confocal laser-scanning microscope(CLSM; vetch: Miller et al., 1999). The strongest indi-cation that this typical actin cytoskeleton is involvedin tip growth comes from its reaction to Nod factor,a lipochito-oligosaccharide secreted by rhizobacteria.Nod factor enhances this cytoskeleton configurationin all developmental stages of root hairs (De Ruijteret al., 1999), whereupon hair tips swell and new tipgrowth restarts in those that were arresting growth(De Ruijter et al., 1998). Furthermore, tip growth in

1 This work was supported by the European Community Train-ing and Mobility of Researchers Program (grant no. FMRX CT 980239 to B.J.S. and F.G.P.L.) and by the European Advanced LightMicroscopy Facility of the EMBL (short-term fellowship to A.C.J.T.).

2 These authors contributed equally to the paper.* Corresponding author; e-mail [email protected]; fax

31–317– 485005.Article, publication date, and citation information can be found

at www.plantphysiol.org/cgi/doi/10.1104/pp.004267.

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vetch root hairs is inhibited by cytochalasin D, anactin-depolymerizing drug (Miller et al., 1999).

Most of the earlier work on microtubules (MTs) inroot hairs has dealt with cortical MTs (CMTs) andtheir role in cellulose microfibril orientation (for re-view, see Emons and Mulder, 1998; Ketelaar andEmons, 2000). Endoplasmic MTs (EMTs) have beenobserved only in legume root hairs, and their preciseconfiguration and role remain unclear (Lloyd et al.,1987). Authors have proposed several functions forMTs in root hairs. They may control growth orienta-tion (Arabidopsis: Bibikova et al., 1999), regulate theorganization of actin filaments (Hydrocharis: Tomi-naga et al., 1997), connect the nucleus to the expand-ing tip (V. hirsuta: Lloyd et al., 1987), or determine thewidth of the root hair tube (E. hyemale: Emons et al.,1990). Despite these studies, the functions of MTs intip growth are still less clear than those of actinfilaments. Furthermore, no description exists of MTsduring all stages of root hair development.

We made use of green fluorescent protein (GFP)technology to visualize MTs in all developmentalstages of living root hairs of M. truncatula, a legumewell studied for the interaction with rhizobia andmycorrhizal fungi (Cook, 1999). The dynamics of thecytoskeleton in living cells may occlude its clear ob-servation. Compare for instance GFP-talin-labeledactin (Baluska et al., 2000) with fluorescein-phalloidin-stained actin (Miller et al., 1999) in roothairs. Furthermore, we wanted to know whether thesame population of MTs is labeled with the GFP

microtubule-binding domain (MBD) fusion proteinas with immunocytochemistry. The MBD is of animalorigin and thus might not label all MTs in root hairs.Therefore, we compared the results obtained with invivo labeling of MTs by GFP-MBD with results ob-tained with immunocytochemistry after rapid freezefixation/freeze substitution (FF/FS), the most reli-able fixation method (Emons, 1987), also for lightmicroscopy (Baskin et al., 1996; Vos and Hepler,1998). With both methods, we made similar observa-tions and it was possible to monitor the configurationof MTs in all stages of root hair development.

Each stage of root hair development had a specificorganization of MTs. CMTs were present in all stagesof hair development in stage-specific configurations,but EMTs were only present in the subapical regionof vigorously growing root hairs. Studies with theMT-inhibiting drugs oryzalin and taxol gave evi-dence that EMTs are essential in maintaining thespecific cytoarchitecture of growing root hairs, in-cluding a fixed distance between nucleus and hairtip, as well as to keep the growth rate of these hairsat a high level.

RESULTS

We studied the organization of MTs in M. trunca-tula root hairs in all developmental stages usingCLSM. MTs were visualized by immunocytochemis-try after FF/FS, or in vivo by GFP-MBD decoration.With both methods, we find the same configuration

Figure 1. CMTs in trichoblasts before bulge formation (A) and during bulge formation (B–E), visualized with a CLSM inscanning steps of 1 �m. A through C, GFP-MBD; D and E, Immunocytochemistry. A, CMTs are obliquely or longitudinallyoriented to the long axis of the root. Bar � 50 �m. B, Full-stack projection of CMTs. CMTs loop through the tip of the bulgeand are transversely or slightly helically oriented to the long axis of the root in the epidermal part. Bar � 10 �m. C, Projectionof four median sections. There are no detectable EMTs in this developmental stage. D, Full-stack projection of CMTs; forexplanation see B. Bar � 10 �m. E, Note position of the nucleus. For explanation see C.

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of the MT cytoskeleton, but MTs decorated with GFP-MBD appear slightly thicker than MTs labeled withantibodies. Because MTs in living cells are dynamic,they appear thicker in in vivo imaging techniquesthan in fixed specimens. Root hairs of transformedroots developed normally and grew, with similarspeed and pattern, as hairs of nontransformed roots.

Trichoblast (before Bulge Formation)

In M. truncatula, every root epidermal cell has thepotential to form a bulge, and thus, in this species, allepidermal cells are trichoblasts (Sieberer and Emons,2000). This is different from Arabidopsis, for in-stance, where trichoblasts and atrichoblasts are orga-nized in cell files (Dolan et al., 1994). A trichoblast ofM. truncatula has one large main vacuole, which fillsthe cell except for a few cytoplasmic strands travers-ing the vacuole and a thin layer of peripheral cyto-plasm containing all organelles and the nucleus.Within one cell, CMTs are oriented obliquely withdifferent angles and/or transversely to the long axisof the root (Fig. 1A). At the presumptive site of bulgeformation, CMTs are transverse to the root axis andparallel to each other. We have not detected EMTs intransvacuolar cytoplasmic strands of trichoblasts be-fore bulge formation; therefore, in this aspect, theyare the same as other diffuse growing plant cells

(BY-2 tobacco cells: Collings et al., 1998; Granger andCyr, 2000).

Bulge

In M. truncatula, root hair development starts withthe formation of a bulge in the middle of the outerpericlinal wall or slightly toward the root tip. Youngbulges have a triangular shape with the large vacuoleextending into the bulge. The nucleus is located at thesite opposite to the bulge at the inner periclinal wall(Sieberer and Emons, 2000). At the site of the bulge,CMTs are mainly transverse to the long axis of theroot (Fig. 1, B and D). Toward both distal ends of thecell, CMTs may be slightly obliquely oriented at thisstage (Fig. 1, B and D). CMTs in the bulge are con-tinuous with CMTs in the epidermal part of thetrichoblast and pass through the tip of the bulgewhere they loop through (Fig. 1, B and D). At the tipof the bulge, the distance between CMTs increaseswhen the bulge expands. We did not observe anyEMTs between the nucleus and the tip of the bulge(Fig. 1, C and E).

Initiation of Polar Growth

During the transition from bulge to the tip-growingroot hair stage, cytoplasm accumulates at the very tip

Figure 2. MTs during transition from a bulge into a growing root hair, visualized with a CLSM in scanning steps of 1 �m(B–D and F–H). B through D, Immunocytochemistry; F through H, GFP-MBD. In this developmental stage, polar growth isinitiated. A, Corresponding bright-field image to B through D; the bracket indicates a thin layer of cytoplasm at the tip. B,Full-stack projection of MTs. C, Projection of three peripheral sections showing CMTs. A few CMTs still reach the very tipand are net-axially oriented below the tip region. D, Projection of four median sections; EMTs start to appear at this stageof hair development. E, Bright-field image of a hair at a somewhat later stage than shown in A. The cytoplasmic layer in thetip region has increased in length. F, Full-stack projection of MTs. G, Projection of three peripheral sections. Single CMTsstill reach the very tip. H, Projection of four median sections showing EMTs. There is a concomitant increase in the lengthof the subapical cytoplasmic dense region and the density of EMTs. A through D show an earlier stage than E through H.Magnification is the same in all images. Bar � 20 �m. n, Nucleus; v, vacuole; pl, cytoplasmic layer.

Microtubules in Medicago truncatula Root Hairs

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of the bulge and forms a short cytoplasmic denseregion there. The nucleus is still in the epidermal partof the cell (Fig. 2A). CMTs are still oriented trans-versely to the root axis and a few of them still loopthrough the tip of the developing bulge (Fig. 2, B, C,F, and G). It is interesting that within the cytoplasmiclayer at the tip, single EMTs appear (Fig. 2, D and H)and increase in density as the subapical cytoplasmicdense region becomes larger (compare Fig. 2, A withE and D with H). We observed EMTs in living GFP-MBD expressing hairs and in immunolabeled FF/FShairs of this developmental stage.

Growing Root Hairs

Regularly growing root hairs exclusively elongateby tip growth. A growing root hair of M. truncatulahas a characteristic cytoarchitecture (Sieberer andEmons, 2000). It is shown in Figure 3A and it consistsof an apical smooth, vesicle-rich region at the very

tip, which is followed by a subapical cytoplasmicdense region containing the nucleus. The nucleusfollows the expanding tip at a distance of 30 to 40�m, measured from the hair tip to the middle of thenucleus (see below). During this stage of vigorous tipgrowth, the central vacuole never enters the subapi-cal cytoplasmic dense region, although extensions ofthe central vacuole may temporarily penetrate thisregion.

CMTs in the base of a young growing root hair arenet-axially oriented to the root hair axis and arecontinuous with CMTs in the subapex, which havethe same orientation (Fig. 3, B, C, and E). Later, stillduring tip growth, CMTs are longitudinal in thelower part of the hair tube and net-axial in the sub-apex (data not shown). In the CLSM images takenfrom living root hairs and immunolabeled FF/FSsamples, the very tip of a growing root hair is devoidof CMTs (Fig. 3, B, C, and E). In the shank of the root

Figure 3. MTs in growing root hairs visualized with a CLSM in scanning steps of 1 �m (B–F). B through D, Immunocyto-chemistry; E and F, GFP-MBD. A, Bright-field image of a living hair; the small bracket indicates the vesicle-rich region andthe large bracket indicates the subapical cytoplasmic dense region. n, Nucleus; v, central vacuole. Bar � 10 �m. B,Full-stack projection of MTs. C, Projection of three sections of the cell periphery showing net-axially aligned CMTs. CMTsdo not reach the very tip. D, Projection of four median sections showing EMTs. EMTs are abundant close to the nucleus andin the lower part of the subapical cytoplasmic dense region. The density of EMTs in the upper part of the subapicalcytoplasmic dense region is low. Only a few EMTs reach the very tip. E, Projection of three sections of the cell peripheryshowing CMTs. For explanation see C. F, Projection of four median sections; for explanation see D. Magnification in Bthrough F is the same. Bar in B � 10 �m.

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hair, the density of CMTs is higher in growing roothairs than in hairs of other developmental stages.

EMTs are located exclusively in the subapical cy-toplasmic dense region between the basal part of thenucleus and the tip of the hair in a densely structuredthree-dimensional array (Fig. 3, D and F). We ob-served EMTs around and close to the nucleus in ahigh density. To determine whether these EMTs orig-inate from the nuclear envelope, attach to it, or justsurround it was not part of this study and wouldrequire a completely different set of experiments,including, for instance, lifetime imaging during flu-orescence resonance energy transfer. EMTs in thelower part of the subapical cytoplasmic dense regionoccur in a higher density and deviate more from thelong axis of the hair than the EMTs close to the roothair tip. Some EMTs are reaching the very tip (Fig. 3,D and F).

Growth-Arresting Root Hairs

Root hair growth-arrest starts when the main vac-uole has permanently passed the nucleus (Siebererand Emons, 2000). Thin extensions of the vacuole arereaching the very tip; growth rate subsequentlydrops, and eventually cell elongation stops. Duringgrowth arrest, the nucleus does not follow the tip ofthe hair any longer and the cytoplasmic dense regiondecreases progressively in length. While the cyto-plasmic dense region disappears, the central vacuoleexpands toward the tip (Fig. 4A). Growth arrest is agradual process, and root hairs in this developmentalstage are still growing to a certain extent before stop-ping growth (Sieberer and Emons, 2000). In growth-arresting hairs, CMTs (Fig. 4, B, C, and E) are net-axially oriented all along the root hair tube and theyare reaching the very tip. As the subapical cytoplas-mic dense region gets smaller, the area with EMTsgets shorter, but as long as growth continues, EMTsremain present close to the tip (Fig. 4, D and F). Whengrowth stops, all EMTs have disappeared.

Full-Grown Root Hairs

A full-grown hair typically has a thin peripherallayer of cytoplasm around the large central vacuole(Fig. 5A), and the nucleus has a random positionwithin the hair (Sieberer and Emons, 2000). CMTs,the only remaining population of MTs in full-grownroot hairs (Fig. 5, B–F), are net-axial in the hairs thatrecently have stopped growth, and mainly longitu-dinal in the hairs that terminated growth earlier(compare Fig. 5, D with F). CMTs are converging atthe very tip of the hair (Fig. 5, B–E). In full-grownroot hairs, the density of CMTs decreases over time(Fig. 5F), resulting in living hairs with few detectableMTs left. Full-grown root hairs never have EMTs(Fig. 5, C and E).

Effect of Low Concentrations of MT Inhibitors onGrowing Root Hairs

To study the function of EMTs, they were depoly-merized with oryzalin and stabilized with taxol.Oryzalin is a dinitroaniline herbicide that binds rap-idly and reversibly to plant tubulin with high affinity(Hugdahl and Morejohn, 1993) and depolymerizesplant MTs (Morejohn et al., 1987). It binds to tubulin

Figure 4. MTs in growth-arresting hairs visualized with a CLSM inscanning steps of 1 �m (B–F). B through D, GFP-MBD; E and F,immunocytochemistry. At this developmental stage, the subapicalcytoplasmic dense region has almost disappeared and the distancetip-nucleus has increased. A, Bright-field image of a living hair; thebracket indicates the remaining subapical cytoplasmic dense regionand the smooth (vesicle rich) region at the very tip. Bar � 10 �m(A–D). B, Full-stack projection of MTs. C, Projection of three periph-eral sections showing CMTs. CMTs are net-axially oriented. D, Pro-jection of four median sections. Region of EMTs has decreased inlength (bracket). E, Projection of three peripheral sections showingCMTs. For explanation see C. Bar � 10 �m. F, Projection of fourmedian sections; for explanation see D. n, Nucleus; v, vacuole.





heterodimers in the cytoplasm, and thereby preventsfurther growth of MTs, leading to depolymerizationof MTs, beginning with the more dynamic ones (forreview, see Anthony et al., 1999). Thus, the use of lowconcentrations of oryzalin and the observation of itseffect in time may enable us to discriminate betweenmore and less stable MTs. We applied oryzalin inconcentrations of 0.25, 0.5, and 1 �m to growing roothairs that had a length of 100 to 150 �m by the timeof drug application. None of these concentrationsaffected the viability of the root hairs or the cytoplas-mic streaming. In all these concentrations, oryzalindid not inhibit bulge formation (data not shown).Furthermore, with all these concentrations, EMTsdisappeared within 5 to 10 min, whereas CMTs re-mained present. Figure 6 shows this for 1 �m oryza-lin in a GFP-MBD-expressing root hair and an immu-nolabeled sample. EMTs, which normally areabundant in the subapical cytoplasmic dense regionof growing hairs, were completely absent in oryzalin-treated growing root hairs, whereas CMTs in thesame hairs were not obviously affected.

Taxol, on the other hand, is a drug that binds toMTs and causes free tubulin in the cytoplasm toassemble into MTs, thus stabilizing MTs (Morejohn,1991). We found that the concentration of a rangetested (0.25, 0.5, and 1 �m) that had a clear effect ongrowth rate but did not affect cell viability was 1 �m.We used taxol in a concentration that had a similareffect on root hair growth rate as 1 �m oryzalin,which appeared to be 1 �m. However, at 1 �m, taxol

root hairs completely recovered their growth ratewithin 2 to 3 h. Therefore, we refreshed the taxolevery 120 min.

Oryzalin, at a concentration of 1 �m, had strikingeffects on the cytoarchitecture of growing root hairs(Fig. 7, A–C). Within minutes after application oforyzalin, growing root hairs lost their typical cytoar-chitecture. The subapical cytoplasmic dense regiondisappeared gradually within 5 to 10 min, and thevacuole passed the nucleus and expanded toward thetip of the hair. An oryzalin-treated hair finally had nosubapical cytoplasmic dense region, the tip-nucleusdistance had increased (see below), and the smoothregion containing the Golgi vesicles was still presentat the very tip and became even slightly longer overtime. The smooth region in an oryzalin-treated haircould even reach a length of 15 to 20 �m instead ofthe normal length of approximately 3 �m (Siebererand Emons, 2000). As long as oryzalin was notwashed out of the growth medium, the growing roothairs did not reestablish their typical cytoarchitec-ture. Hairs were observed up to 7 h after drugapplication.

The nucleus of an oryzalin-treated growing roothair lost its typical position of 30 to 40 �m from thetip and moved slowly backward in the root hair tubewithin the first 90 to 120 min after treatment (Fig.8A). After that time, the nucleus was again followingthe expanding root hair tip at a significantly largerdistance (Fig. 8A). A nuclear movement at the pacewith cell elongation was observed in all hairs, but the

Figure 5. MTs in full-grown root hairs visualized with a CLSM in scanning steps of 1 �m. B and C, Immunocytochemistry;D through F, GFP-MBD. A, Bright-field image of a living hair. Note position of the nucleus (n). Bar � 10 �m. B, Full-stackprojection of CMTs. CMTs, the only remaining population of MTs in this developmental stage, are longitudinally oriented;they converge at the very tip. Bar � 10 �m (B–F). C, Projection of two median sections. Full-grown hairs have no EMTs. D,Full-stack projection of CMTs. The hair has stopped growth recently and the density of CMTs, which are net-axially oriented,is similar to previous developmental stages. E, Projection of two median sections; for explanation see C. F, Full-stackprojection of CMTs in a hair of a later stage than shown in D. The density of CMTs is lower than in hairs that have justterminated growth.

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distance between the nucleus and the tip was not thesame in all hairs. In approximately 70% of the hairs,this distance was 140 � 30 �m over a period of 4 to5 h. The other 30%, not represented in Figure 8A, hada smaller (minimally 80 �m) or larger (maximally 250�m) nucleus-tip distance, but also in these hairs, thenucleus followed the tip. The exact distance is notrelevant here, but the fact that the nucleus keepsfollowing the growing root hair tip is.

Taxol at a concentration of 1 �m had no effect onthe cytoarchitecture (Fig. 7, D–F) or the position ofthe nucleus (Fig. 8A). The distance nucleus-tip of 30to 40 �m remained constant. Observed with bright-field microscopy over 7 h, the subapical cytoplasmicdense region did not show any obvious changes in

cytoarchitecture and length compared with controls.Immunocytochemistry showed that 1 �m taxol didnot change the configuration of EMTs (data notshown).

Oryzalin, at the above-mentioned concentration of1 �m, had a significant effect on the growth rate ofgrowing root hairs, but did not inhibit tip growth perse (Fig. 8B). Treated hairs did grow, but with a lowergrowth rate than untreated growing root hairs. Wefollowed growing root hairs treated with oryzalinover a time span of 7 h. During this period, we didnot observe any changes in shape and width of theroot hair tube. Furthermore, these hairs always main-tained one point of growth (i.e. the expanding tip).Shortly after application of 1 �m oryzalin, most roothairs exhibited a single deviation from their normalgrowth axis, which is perpendicular to the root. Insome root hairs, several deviations from the hairs’growth axis occurred irregularly over time in thepresence of oryzalin. However, we never observed awavy growth pattern of root hairs after MT inhibitortreatment, as described for Arabidopsis (Bibikova etal., 1999). An important difference with that work isthat these authors depolymerized all MTs, whereasin our approach, only EMTs were depolymerized andnot the CMTs. It is not known whether Arabidopsishas EMTs; they have not been reported.

When oryzalin was washed out after 7 h by replac-ing the medium with fresh plant growth medium(PGM), 35% to 40% of the growing root hairs fullyrecovered their cytoarchitecture, but their growthrate reached a maximum 70% of the initial growthrate (data not shown). This recovery took place overa period of 4 to 6 h.

Taxol, at a concentration of 1 �m, had a similareffect on the growth rate of growing root hairs asoryzalin (Fig. 8B). Although the growth rate in taxol-treated growing root hairs dropped to approximately40% of the initial growth rate, the distance nucleus-tip of 30 to 40 �m remained constant. In 1 �m taxol,root hairs maintained one single point of growth,always kept the same directionality of growth, andalways had the same shape as control root hairs.

Oryzalin in concentrations of 10 and 30 �m had thesame effect on the cytoarchitecture of growing M.truncatula root hairs as a concentration of 1 �m had(data not shown). Growing root hairs that had alength of 100 to 150 �m by the time of drug applica-tion were observed for at least 5 h. Within minutes oftreatment, the distance between nucleus and hair tipincreased, whereas the subapical cytoplasmic denseregion disappeared. The apical vesicle-rich regionremained unchanged and tip growth itself pro-ceeded, but growth rate dropped in a similar way asin hairs treated with 1 �m oryzalin. Using immuno-cytochemistry after a 30-min treatment with 30 �moryzalin, we could not detect EMTs or intact CMTs inthese hairs, whereas they kept on growing for hours.

Figure 6. MTs in growing root hairs treated with 1 �M oryzalin. Aand B, GFP-MBD; C and D, immunocytochemistry. A, Hair beforetreatment; a full-stack projection shows abundant MTs in the subapi-cal region (bracket). B, Ten minutes after treatment. The subapicalregion contains less MTs and the MTs (cortical) in the shank of thehair are unchanged. C, Hair 15 min after treatment; a full-stackprojection shows CMTs. D, A projection of four median sections ofthe hair in C shows that EMTs and the subapical cytoplasmic denseregion have completely disappeared, but the vesicle-rich region stillis present at the very tip (see also Fig. 7, B and C). Bar in A � 10 �m;bar in C � 10 �m.





DISCUSSION

In this paper, we report changes in the spatialconfiguration of the MT cytoskeleton during devel-opment of M. truncatula root hairs and the effect ofMT inhibitors on the MT configuration, the cytoar-chitecture, the nuclear position, and the growth rateof growing hairs. The results obtained with the twoapproaches used, cells expressing a GFP-MBD fusionprotein (Marc et al., 1998) and immunolabeled fixedwhole-mount samples, were similar. Thus, we con-clude that the GFP-MBD construct decorates thesame set of MTs as does the anti-�-tubulin mousemonoclonal antibody clone DM 1�. We identifiedtwo different populations of MTs in root hairs:CMTs, present in all developmental stages, and moredynamic and labile EMTs, unique for growing roothairs. Furthermore, we did not observe any measur-able effect on root hair development, cytoarchitec-ture, growth pattern, and growth rate of GFP-MBD-expressing root hairs.

CMTs during Root Hair Development

All diffuse growing interphase plant cells haveCMTs, which are transverse to the direction of elon-gation of the cell. They become less frequent andobliquely aligned when cell elongation decelerates(for review, see Traas et al., 1985; Cyr, 1994;Wasteneys, 2000). These MTs are involved in cellelongation and appear often, but not always, in thesame orientation as nascent cellulose microfibrils (forreview, see Sugimoto et al., 2000; Wasteneys, 2000).As expected, the diffuse growing trichoblasts have

CMTs transverse to the root axis. The time of bulgeformation seems to be the time these cells begin tostop growing. At their upper and lower edges, MTsare becoming helical when bulges first appear. How-ever, at the site of bulge formation, the CMTs aretransverse, and after bulge appearance, they are stillin this orientation running over the tip of the bulge.During bulge formation, the distance between CMTsincreases; they seem to passively part at the bulge tip.In the presence of oryzalin, bulges are being formednormally. In addition, from studies on the cytoskel-eton (actin: Miller et al., 1999; actin and MTs: Baluskaet al., 2000), ultrastructure (vetch: Miller et al., 2000),and cytoplasmic [Ca2�] gradients in wild-type Ara-bidopsis and the rhd-2 mutant (Wymer et al., 1997), itwas concluded that bulge formation is a distinctlydifferent process than tip growth of the root hairproper.

CMTs have been found in root hairs of all speciesexamined in helical or mostly net-axial orientations (forreview, see Ketelaar and Emons, 2000, 2001). The func-tion of CMTs in root hairs is still not clear. Arabidopsisroot hairs with depolymerized MTs (Bibikova et al.,1999) or a mutant with disorganized CMTs (Whitting-ton et al., 2001) continue to elongate by tip growth,but in a slightly wavy pattern. Thus, CMTs do not seemto be required for growth per se, but appear tobe involved in determining direction of elongation(Bibikova et al., 1999). Because CMTs are often found tobe aligned with microfibrils, it is often thought thatCMTs are responsible for directing nascent cellulosemicrofibrils. However, contradictory examples havebeen reported, and other mechanisms have been pro-

Figure 7. The effect of treatment with oryzalin or taxol on the cytoarchitecture of growing root hairs. A, Before treatment.B, Thirty minutes after 1 �M oryzalin. C, Sixty minutes after 1 �M oryzalin. D, Before treatment. E, Thirty minutes after 1 �M

taxol. F, Sixty minutes after 1 �M taxol. Bright-field images from living root hairs. Large bracket indicates the subapicalcytoplasmic dense region, and the small bracket indicates the smooth (vesicle-rich) region. n, Nucleus. Magnification is thesame in all images. Bar � 20 �m.

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posed (for review, see Emons and Mulder, 2000). Theconfiguration of CMTs we now find in growing roothairs is net-axial in the upper part of growing root hairsand helical in the basal part. A cell wall study has yet tobe performed for M. truncatula.

In M. truncatula root hairs, CMTs are absent fromthe extreme tip in the GFP-MBD plant and afterFF/FS immunolabeling. In electron microscopy im-ages of FF/FS root hairs of other species, CMTs werevery close to the tip. They have been observed in thearea where the Golgi vesicles are located (E. hyemale:Emons, 1989; Arabidopsis: Galway et al., 1997). Theabsence of CMTs from the very tip could be ex-plained by insufficient labeling of MTs at this place.The specific binding sites of CMTs close to the tip ofa growing root hair may not be fully accessible forGFP-MBD or antibodies because CMTs at this site arehighly decorated with MT-binding proteins. There-fore, the absence of CMTs in M. truncatula should beconfirmed with electron microscopy.

EMTs during Root Hair Development

EMTs are unique for the subapical region in tip-growing root hairs of M. truncatula. From our exper-iments with low concentrations of oryzalin, it is clearthat EMTs are far more sensitive to depolymerizationthan CMTs and thus are more dynamic than CMTs(Anthony and Hussey, 1999).

Until now, EMTs have only been reported for le-gume root hairs, and not for any other species. Lloydet al. (1987) found EMTs in growing root hairs of V.hirsuta that were hypothesized to connect the migrat-ing nucleus to the root hair tip, where they fountainout upon the cortex. We have not seen similar struc-

tures in M. truncatula. Lloyd et al. (1987) used chem-ical fixation and immunolocalization to visualizeMTs and, due to the limited resolving power of thefluorescence microscope as compared with a CLSM,the precise configuration of EMTs is still a matter ofspeculation. In our hands, EMTs appear at the roothair tip when tip growth starts from bulges, theyremain present in the tip region during cell elongation,and they disappear upon growth arrest. In growingroot hairs, EMTs form a densely structured three-dimensional array close to and in front of the nucleus.They extend throughout the subapical cytoplasmicdense region toward the tip region, appearing lessdense there. A few EMTs reach the very tip. What isthe function of these EMTs? Are they involved in cellelongation or the determination of growth direction,nuclear positioning, regulation of tube width, or actinfilament configuration? We discuss the function ofEMTs in cell elongation and in configuring cell archi-tecture, including nuclear position.

Function of EMTs in Configuring the SubapicalCytoplasm, Including the Localization of theNucleus at a Certain Distance from the Hair Tip

The MT-depolymerizing drug, oryzalin, at a con-centration of 1 �m, caused a dramatic change in thecytoarchitecture of the subapex of growing M. trun-catula root hairs, whereas taxol, an MT-stabilizingdrug, left the cytoarchitecture intact. EMTs, but notCMTs, were depolymerized by 1 �m oryzalin. Themost straightforward conclusion from these experi-ments is that EMTs must contribute directly or indi-rectly to the cytoarchitecture of growing root hairs.Therefore, we conclude that EMTs are involved in

Figure 8. Nuclear position (A) and root hair growth rate (B) in growing M. truncatula root hairs after control treatment andtreatment with 1 �M oryzalin or 1 �M taxol. In controls and taxol-treated hairs, the nucleus kept a distance of 30 to 40 �mto the tip over time, but it was significantly increased in oryzalin (A). Growth rate in the hairs remained high in controls, butdropped by approximately 60% in oryzalin and taxol (B). Results for each treatment are presented as the means of 10 hairswith their SD. Results are representative for three independent replicates for each treatment.





supporting the organization and maintenance of thesubapical cytoplasmic dense region. The questionthen arises: What is the relationship with the actincytoskeleton? The actin cytoskeleton forms a specificconfiguration of FB-actin in the subapical cytoplas-mic dense region (Miller et al., 1999). An interestingoption for the function of EMTs is the one suggestedby Tominaga et al. (1997) for Hydocharis morsus-ranaeroot hairs. From their experiments in which theycombined actin and MT drugs, these authors suggestthat MTs regulate the organization of actin filamentsin the cortex of root hairs. In diverse cell types,functional interactions may exist between the actinand MT cytoskeleton (for review, see Goode et al.,2000) and have been suggested for tip-growing plantcells (for review, see Kropf et al., 1998).

Their role in the positioning of the nucleus at adistance of 30 to 40 �m to the growing root hair tip isrelated to the role of EMTs in the formation of thesubapical cytoarchitecture. In an oryzalin-treatedhair, the nucleus first loses its position, but after aperiod of backward movement, it is again activelyfollowing the (now more slowly) growing tip at alarger but again fixed distance. For Arabidopsis roothairs, it has been reported that MT inhibitors such asoryzalin and taxol had no effect on nuclear move-ment (Chytilova et al., 2000). The question, not onlyfor drug-treated cells, but also for control cells is:What keeps the nucleus following the root hair tip?Our results show that EMTs are necessary for keep-ing the nucleus at a certain position to the root hairtip, but are not sufficient for nuclear movement.

Function of EMTs in Cell Elongation

Oryzalin, at a concentration at which only EMTsare depolymerized, caused a completely different cy-toarchitecture, but had the same effect on growth rateas taxol at a concentration that kept the cells viable.Oryzalin caused the disappearance of the subapicalcytoplasmic dense region, but in taxol-treated hairs,the cytoarchitecture of apex and subapex remainedunaltered. After treatment with oryzalin, the smoothvesicle-rich region at the very tip remained presentand even increased in length. What is the same inboth treatments is that the vesicle-rich region at theroot hair tip remains present. This is the one andmost important prerequisite for tip growth. The fu-sion of the vesicles with the plasma membrane is theactual cell elongation process, which of course cannottake place if the vesicles are not there. We concludethat exocytotic vesicles are still being produced anddelivered to the vesicle-rich region, and they fusewith the plasma membrane in growing root hairstreated with oryzalin or taxol. Tip growth proceeds,though at a low rate. From electron microscopy ex-periments, we know that the subapical region oflegume root hairs contains longitudinal cisternae ofthe ER, mitochondria, and Golgi bodies (vetch: Miller

et al., 2000). Actin labeling has shown the occurrenceof subapical net-axial FB-actin (vetch: Miller et al.,1999). Because hairs in which the subapical EMTs arenot present anymore (oryzalin) or stabilized (taxol)do still grow, EMTs, possibly together with or inrelation to FB-actin, configure the subapical cyto-plasm, but the vesicle transport system appears to beactin based. The combined EMTs, FB-actin, and ERcell structure may form a subapical buffer reservoirfor exocytotic vesicles in transit to the apical vesicle-rich region.

In the presence of oryzalin and taxol, the growthrate of root hairs drops by 60%. The simplest expla-nation for this in the case of oryzalin is that thedelivery of exocytotic vesicles to the vesicle-rich re-gion is inefficient because there is no buffer of vesi-cles. Along the same line, this delivery may be inef-ficient in the presence of taxol because the EMTs arenot functioning properly. In addition, there are roothairs of Limnobium stoloniferum (A.M.C. Emons, un-published data) or H. morsus-ranae (see figures inTominaga et al., 1997), for instance, that do not havea subapical cytoplasmic dense region or that have anextremely short one. Of course, they do possess avesicle-rich region. Growth rates of root hairs of spe-cies with and without a subapical cytoplasmic denseregion have not been compared.

One should have an open mind for other possibleexplanations. One of the important observations isthat although the subapical cytoplasmic dense regiondisappears in oryzalin-treated hairs, the vesicle-richregion remains present and even enlarges. The factthat upon oryzalin treatment, the vesicle-rich regionenlarges, whereas growth rate is tempered, suggeststhat the drug may interfere with the growth process,exocytosis, itself. In that case, taxol should interferewith exocytosis in a similar fashion.

Relevance of Endoplasmic MTs for Legumes

It is striking that EMTs have only been reported forlegume root hairs, although MTs in root hairs ofseveral species have been studied (for review, seeKetelaar and Emons, 2000). Several of these studieswere designed to investigate the relationship ofCMTs with cellulose microfibrils. Therefore, only thecell cortex was investigated, and EMTs may havebeen missed.

However, we should consider that legume roothairs might be special. Legumes have developedsymbiosis with rhizobia. During the root hair curlingaround rhizobia, one of the first steps during theinfection process, the cytoplasm (cytoskeleton, ER,Golgi bodies, etc.) mediates the curling and the for-mation of an infection thread through which thebacteria traverse the hair toward the root cortex. Onecan imagine that for curling and infection threadformation, EMTs are important.

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MATERIAL AND METHODS

Plant Culture

Seeds of Medicago truncatula cv Jemalong (Fabaceae) were scarified with97% (w/v) sulfuric acid for 10 min and were surface sterilized with amixture of 96% (v/v) ethanol and 30% (v/v) hydrogen peroxide (ethanol:hydrogen peroxide [1:1, v/v]) for 3 min. After rinsing with sterile water,seeds were imbibed for 4 h at 30°C in sterile water and then placed on 1%(w/v) agarose plates. Plates were sealed and stored upside down in the darkfor 2 to 4 d at 4°C to synchronize germination. During germination for 6 to8 h at 24°C, the seeds were protected from light. Seedlings were transferredonto wet filter paper and were grown in vertical orientation for 24 to 48 hunder sterile conditions at 24°C with a 16-h day length. The PGM, whichwas used to humidify the filter paper, contained 1.36 mm CaCl2, 0.97 mmMgSO4, 1.12 mm Na2PO4, 1.36 mm KH2PO4, and 20 �m Fe-citrate, pH 6.5(Miller et al., 1999).

In a different setup, seedlings were grown at an angle of approximately25o for 24 to 30 h in microchambers in between a coverslip and a glass slide(Fåhraeus slides: Fåhraeus, 1957; Heidstra et al., 1994) in sterile conditions at24°C with a 16-h day length. Each Fåhraeus slide contained two seedlingsand 3 mL of PGM.

Whole-Mount Immunocytochemistry of MTs

For rapid FF, seedlings were plunged into liquid propane cooled to�180°C with liquid nitrogen and were kept there for at least 20 s. Roots wereexcised and transferred into cryogenic vials, freeze substituted in water-freemethanol containing 0.05% (v/v) glutaraldehyde for 48 h at �90°C, andallowed to warm to room temperature over a 24-h period. Samples wererehydrated in a graded series of methanol in phosphate-buffered saline(PBS; 137 mm NaCl, 2.7 KCl, 1.5 mm KH2PO4, and 8.1 mm Na2HPO4, pH 7.4)containing fixative (0.1% [v/v] glutaraldehyde and 4% [w/v] paraformal-dehyde). After rehydration, a partial cell wall digestion was carried out ina saturated suspension of driselase (Fluka, Buchs, Switzerland) and mac-erocyme R10 (Serva, Heidelberg) in 100 mm MES for 40 min at 35°C and pH6.15. The specimens were then washed two times for 5 min in PBS and, toblock unspecific sites, were incubated for 5 min in PBS containing 0.1%(w/v) acetylated bovine serum albumin (BSAac; Aurion, Wageningen, Neth-erlands) and 0.05% (v/v) Triton X-100 (BDH Laboratory Supplies, Poole,UK). The samples were incubated for 12 h at 4°C in the monoclonal primaryantibody anti-�-tubulin mouse, clone DM 1� (Sigma, St. Louis). The primaryantibody was diluted 1:300 (v/v) in PBS containing 0.1% (w/v) BSAac and0.05% (v/v) Triton X-100. After washing three times for 5 min in PBS, thesamples were incubated for 12 h at 4°C in the secondary antibody goatagainst mouse/IgG/Alexa 488 (Molecular Probes, Eugene, OR). The sec-ondary antibody was diluted 1:300 (v/v) in PBS containing 0.1% (w/v)BSAac and 0.05% (v/v) Triton X-100. Specimens were washed twice for 5min with PBS and for 5 min with CITIFLUOR PBS solution (Citifluor,London), and were mounted in an antifading medium (PROLONG ANTI-FADE; Molecular Probes).

Microscopic Observation

Anti-�-tubulin-labeled MTs were visualized with a CLSM (MCR 600;Bio-Rad, Hertfordshire, UK) with an argon-krypton ion laser attached to aninverted microscope (DIAPHOT300; Nikon Europe B.V., Badhoevedorp,The Netherlands) equipped with a 60� FL 1.4 n.a. oil immersion objective(Nikon). Samples were scanned in subsequent steps of 1 �m. Images wereacquired and projected with Confocal Assistant, version 4.02 (Bio-Rad,written by Todd Clark Brelje) and were processed with Scion Image Beta4.0.2. (Scion Corporation, Frederick, MD) and Adobe Photoshop 5.5 (AdobeSystems, Mountain View, CA).

GFP-MBD Decoration of MTs/TransgenicM. truncatula Plants

The GFP-MBD fusion gene with expression under the control of the 35Spromotor was provided in a pUC18 vector by Richard Cyr (Marc et al.,1998). The complete insert from this plasmid was transferred to pCam-bia1390 (CAMBIA, Canberra, Australian Capitol Territory, Australia) by

using the HindIII-EcoRI sites, and was kindly provided by W.J. TheodorusGadella, Jr. (Wageningen University, Wageningen, The Netherlands).

Plant Transformation and Culture

Transformed roots of M. truncatula cv Jemalong were obtained by usingAgrobacterium rhizogenes according to the protocol described by Boisson-Dernier et al. (2001). About 3 to 4 weeks later, plants with transformed rootswere put into square 12-cm plastic dishes (Greiner Labortechnik, Krems-munster, Austria) with one of the four sides containing a round perforationin the middle of about 5 mm in diameter. Each individual plant was put inthe perforation in such a way that the root was inside on Fåhraeus mediumcontaining 0.8% (w/v) agar and the stem part was outside the plate. Plateswere put vertically in a culture room at 25°C and an 18-h day length.

Microscopic Observations

For observation, the roots growing on agar were submerged in sterilewater and were covered with a gas-permeable plastic foil (bioFOLIE 25;Sartorius AG, Vivascience Support Center, Gottingen, Germany) to preventthem from drying. The opened dish was put on the microscope stage of aZEISS LSM510 (Carl Zeiss SA, Le Pecq, France), and observations werecarried out with a 40�/0.8 WPH2 Achroplan or a 63�/0.9 WPH3 Achroplanobjective. In general, optical sections were made of whole root hairs with aseparating distance of 1 �m between subsequent sections. Image projectionswere made with ZEISS LSM Image Examiner (Zeiss), and images wereprocessed with Image-Pro plus (Media Cybernetics, Silver Spring, MD).

Light Microscopy

Root hairs were observed with a 20� 0.4 n.a. or a 40� 0.55 n.a. objective(Nikon) on an inverted microscope with Hoffman modulation contrastsystem (DIAPHOT200; Nikon) equipped with a CCD camera (DXC-95OP;Sony, Tokyo). Image recording and processing and quantitative live mea-surements were done with a real-time digital contrast and low-light en-hancement image processor (ARGUS-20; Hamamatsu Photonics,Hamamatsu City, Japan). To prevent light-induced stress, low light andgreen filters were used during quantitative live measurements and relatedimage recording.

Drug Studies

Oryzalin (Greyhound Chromatography, Birkenhead, UK) was dissolvedin dimethyl sulfoxide (DMSO; Merck, Darmstadt, Germany) as a 10 mmstock solution and was used at 0.25, 0.5, 1, 10, and 30 �m in PGM. Taxol(paclitaxel; Sigma) was dissolved in DMSO as a 10 mm stock solution andwas used at 0.25, 0.5, and 1 �m in PGM.

In Fåhraeus slides, drug solutions were applied on the microscope stagewith a constant flow of 1 mL min�1, gradually replacing the PGM in theslides. The final volume in each slide was 3 mL. Although oryzalin wasapplied only once, taxol was refreshed every 120 min in the same way as itwas applied the first time (see above). To prevent evaporation of waterduring observation, the slides were covered in the microscope stage with alarge plastic petri dish. Measurements on growth rate and nuclear positionwere done on growing root hairs of nontransformed M. truncatula cv Jema-long roots. Growing root hairs had a length of 100 to 150 �m whenmeasurements were started. Control roots were treated with PGM onlycontaining the same amount of DMSO as the drug solutions.

Distribution of Materials

Upon request, all novel materials described in this publication will bemade available in a timely manner for noncommercial research purposes,subject to the requisite permission from any third-party owner of all parts ofthe material. Obtaining any permission will be the responsibility of therequestor.





ACKNOWLEDGMENTS

A.C.J.T. would like to thank Rainer Pepperkok, Jens Rietdorf, TimoZimmermann, and Andreas Girod of the Advanced Light Microscopy Fa-cility for their hospitality and help during his stay at the EMBL. B.J.S. wouldlike to thank Jan Vos for stimulating discussion and helpful comments onthe manuscript.

Received February 15, 2002; returned for revision April 9, 2002; acceptedJune 24, 2002.

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