SHORT-ROOT Regulates Primary, Lateral, and · SHORT-ROOT Regulates Primary, Lateral, and Adventitious Root Development in Arabidopsis1[C][W][OA] Mikae¨l Lucas, Ranjan Swarup, Ivan

SHORT-ROOT Regulates Primary, Lateral, andAdventitious Root Development in Arabidopsis1[C][W][OA]

Mikael Lucas, Ranjan Swarup, Ivan A. Paponov, Kamal Swarup, Ilda Casimiro, David Lake,Benjamin Peret, Susan Zappala, Stefan Mairhofer, Morag Whitworth, Jiehua Wang, Karin Ljung,Alan Marchant, Goran Sandberg, Michael J. Holdsworth, Klaus Palme, Tony Pridmore,Sacha Mooney, and Malcolm J. Bennett*

Centre for Plant Integrative Biology, University of Nottingham, Nottingham LE12 5RD, United Kingdom(M.L., R.S., K.S., D.L., B.P., S.Z., S. Mairhofer, M.W., A.M., M.J.H., T.P., S. Mooney, M.J.B.); Institute forBiology II, Botany, Center of Biological Signaling Studies, Freiburg Institute of Advanced Studies, Universityof Freiburg, 79104 Freiburg, Germany (I.A.P., K.P.); Universidad de Extremadura, Facultad de Ciencias, 06071Badajoz, Spain (I.C.); and Umea Plant Science Centre, Department of Forest Genetics and Plant Physiology,Sveriges Lantbruksuniversitet, 901 83 Umea, Sweden (J.W., K.L., A.M., G.S.)

SHORT-ROOT (SHR) is a well-characterized regulator of radial patterning and indeterminacy of the Arabidopsis (Arabidopsisthaliana) primary root. However, its role during the elaboration of root system architecture remains unclear. We report that theindeterminate wild-type Arabidopsis root system was transformed into a determinate root system in the shr mutant whengrowing in soil or agar. The root growth behavior of the shr mutant results from its primary root apical meristem failing toinitiate cell division following germination. The inability of shr to reactivate mitotic activity in the root apical meristem isassociated with the progressive reduction in the abundance of auxin efflux carriers, PIN-FORMED1 (PIN1), PIN2, PIN3, PIN4,and PIN7. The loss of primary root growth in shr is compensated by the activation of anchor root primordia, whose tissues areradially patterned like the wild type. However, SHR function is not restricted to the primary root but is also required for theinitiation and patterning of lateral root primordia. In addition, SHR is necessary to maintain the indeterminate growth oflateral and anchor roots. We conclude that SHR regulates a wide array of Arabidopsis root-related developmental processes.

Higher plants exhibit an amazing diversity of rootarchitectures at both the systems and anatomical levels(Waisel et al., 2002). Many dicotyledonous plants like

Arabidopsis (Arabidopsis thaliana) have a primary rootthat repeatedly branches to generate several ordersof lateral roots, whereas the root systems of cerealcrops such as rice (Oryza sativa) and maize (Zea mays)are predominantly composed of adventitious roots(Hochholdinger et al., 2004; Osmont et al., 2007).Despite these anatomical differences, with the excep-tion of the highest orders, roots from all plant speciesexhibit indeterminate growth behavior (i.e. are able tomaintain growth indefinitely; Waisel et al., 2002).

The cellular basis of indeterminate root growth hasbeen best studied in the Arabidopsis primary root. Inthis experimental system, stem cells (also termedinitials) abut specialized organizing cells (often deno-ted as the quiescent center [QC]) within the root apicalmeristem (RAM; Dolan et al., 1993). When initial cellsdivide, they generate two daughter cells; the onedirectly abutting the QC remains a stem cell, whilethe remaining daughter cell (also termed a transitamplifying cell) undergoes a finite number of celldivisions before exiting the meristem and elongating(Scheres, 2007). Mutants that disrupt initial cell/QCfunction often exhibit a determinate root growth phe-notype, resulting in the termination of organ growth(Benfey et al., 1993; Di Laurenzio et al., 1996).

Genetic studies have identified several genes thatregulate indeterminate root growth in Arabidopsis.One of these genes encodes the SHORT-ROOT (SHR)

1 This work was supported by the Biotechnology and BiologicalSciences Research Council and the Engineering Physics ScientificResearch Council Centre for Integrative Systems Biology award tothe Centre for Plant Integrative Biology (to M.L., R.S., M.J.H., T.P., S.Mooney, andM.J.B.); by Marie Curie Fellowship funding (to B.P.); bya 2009 Professorial Research Fellowship award from the Biotechnol-ogy and Biological Sciences Research Council (to M.J.B.); by Uni-versity of Nottingham Interdisciplinary Doctoral Training Centrefunding (to S.Z. and S. Mairhofer); by Collaborative Research Center592; by the Excellence Initiative of the German Federal and StateGovernments (grant no. EXC 294); by Graduiertenkolleg 1305,Bundesministerium fur Forschung und Technik; and by theDeutschesZentrum fur Luft und Raumfahrt and the European Space Agency(to I.P. and K.P.).

* Corresponding author; e-mail [email protected].

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Malcolm J. Bennett ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[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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protein, which has been shown to be essential for RAMfunction (Benfey et al., 1993; Scheres et al., 1995). Inmutant plants lacking SHR, primary roots are dramat-ically shortened (Benfey et al., 1993; Scheres et al.,1995). Another related protein, SCARECROW (SCR),has a similar role alongside SHR (Benfey et al., 1993; DiLaurenzio et al., 1996). SHR and SCR genes encodeclosely related transcription factors belonging tothe GRAS gene family (Di Laurenzio et al., 1996;Helariutta et al., 2000). SHR has been demonstratedto directly regulate the expression of genes includingSCR (Levesque et al., 2006) and a number of cell cyclecomponents including the D-type cyclin, CYCD6;1(Sozzani et al., 2010).The spatiotemporal roles of SHR, SCR, and various

others genes such as PLETHORA1 (PLT1) and PLT2 inestablishing and maintaining the RAM have beenextensively studied. In the model proposed by Blilouet al. (2005), redistribution of polar localized auxinefflux carriers of the PIN-FORMED (PIN) family re-sults in the accumulation of auxin at the prospectivemeristem stem cell niche; this maximum is maintainedthroughout postembryonic elaboration of the root(Sabatini et al., 1999; Aida et al., 2002; Friml et al.,2003, Blilou et al., 2005). The distal auxin maximumthen determines the expression pattern of the tran-scription factors PLT1 and PLT2, which are requiredfor QC identity and stem cell specification from em-bryogenesis onward (Aida et al., 2004). The distalauxin maximum maintained by PIN proteins restrictsPLTexpression to the basal pole of the proembryo; thisfacilitates the activation of the root primordium. Inturn, PLT positively regulates PIN expression (PIN3,PIN4, and PIN7; Blilou et al., 2005), which reinforcesthe flux of auxin into the RAM, thereby maintainingthe postembryonic position of the distal stem cellniche. The radial expression domains of the transcrip-tion factors SHR and SCR participate in the position-ing of the stem cell niche. The SHR gene is transcribedin the stele, but the mobile SHR protein migratesoutward one cell layer to the adjacent QC, cortex-endodermis initial (CEI) cell, and endodermis layer(Helariutta et al., 2000; Nakajima et al., 2001), where itactivates the expression of SCR (Nakajima et al., 2001).The endodermal/QC domain of SHR/SCR proteinexpression overlaps with the distal PLT expressiondomain, with the highest level of both componentsdefining the stem cell niche.Despite of the importance of SHR for regulating

RAM function and indeterminate primary rootgrowth, no studies have addressed its impact on rootsystem architecture (RSA) to date. The progression of aplant root system from a single meristem-containingprimary root, formed during embryogenesis, to theextensively elaborated RSA of a mature plant involvesnumerous exogenous abiotic and biotic factors plusendogenous genetic factors. One of the most importantfactors determining total RSA is the postembryonicappearance of lateral root primordia (LRP) and ulti-mately lateral roots that branch off from the primary

root (Nibau et al., 2008). Given the fact that lateral rootdevelopment is considered to be a broad recapitula-tion of equivalent processes in the primary root, weinvestigated whether SHR plays a role in LRP devel-opment and, consequently, on global RSA. We reportthat SHR is indeed a key regulator, controlling pri-mary, lateral, and anchor root development at themacroscopic scale and the patterning of lateral (but notanchor) roots at the microscopic scale.

RESULTS

The shr Mutation Causes RSA to Become Determinate

The shr mutants were initially identified based ontheir severely reduced primary root growth and radialpatterning defects (Benfey et al., 1993; Scheres et al.,1995). Nevertheless, shr mutant seedlings are able togrow and complete their life cycle, somehow compen-sating for the dramatic reduction of their primary rootlength.

We initially investigated the morphological effectthe shr mutation had on the development of the rootsystem in soil. X-ray computed tomography (CT) wasemployed to investigate the three-dimensional (3D)root architecture of 3-week-old wild-type and shrseedlings grown in a sandy loam soil (Fig. 1, A–C;Supplemental Movies S1 and S2). This technique relieson discriminating differences in attenuation densitybetween the soil matrix and live roots. Difficulties canarise when discriminating differences in attenuationdensity between a water-filled pore space and liveroots. Nevertheless, the CT scan and subsequent 3Dreconstruction allowed us to retrieve more than 50% ofthe root tissues of the scanned plants. This amountedto a total root volume of 0.3 mm3 for shr and 1 mm3 forthe wild type (root volume was automatically esti-mated by the 3D reconstruction software; for addi-tional details, see “Materials and Methods”). Rootswere relatively straight, with a mean tortuosity of 1.05both in shr and the wild type (tortuosity being the ratioof the root length over the shortest distance betweenthe two extremities of the measured root). However,shr did not exhibit a strong 3D branching phenotypewhen grown in the sandy loam soil compared with thewild type.

To investigate further the capability of shr to developa branched root architecture, we then compared4-week-old compost-grownwild-type and shr seedlings(Fig. 1D). Seedlings grown in compost exhibited amuch bigger root system compared with soil-grownplants. The dimorphism between shr and the wild typewas also amplified compared with soil-grown plants.Whereas wild-type plants had an indeterminate taproot system, the shr mutant exhibited a fibrous deter-minate root system. The shr seedling had numerousshort roots of equivalent lengths, each one thicker thanindividual wild-type roots. Microscopic observation ofthe shr roots revealed a low degree of branching

SHORT-ROOT Is a Key Regulator of Root Development

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toward the apex of roots, with no discernible primor-dia or young laterals along the bottom half of roots(data not shown). Instead, new roots appeared tooriginate and branch near the seedling root-shootjunction. Similar alterations of RSA can be similarlyobserved in agar-grown shr seedlings, which exhibitreduced growth and a characteristic “tripod” archi-tecture after 1 week of growth (Fig. 1E).

The shr Mutant Fails to Reinitiate Cell Division in thePrimary Root following Germination

We next investigated how the shr mutation couldlead to this dramatic change in overall RSA. Tounderstand the basis for the determinacy of the RAMin shr, we studied the mitotic activity of the mutant be-fore and after germination using the cyclinB1:1:GUSmarker (Fig. 2).

As the Arabidopsis embryo develops, almost everycell is continuously dividing, as revealed by the mi-totic marker cyclinB1:1:GUS (Fig. 2A). No major dif-ferences in mitotic marker expression were observedbetween shr and the wild type during embryogenesis.After seedling germination, cell division must bereinitiated in the RAM (Masubelele et al., 2005). SHRand SCR gene products are required to maintain theactivity of this population of root stem cells (Sabatiniet al., 2003). However, we observed that in the shrmutant background, expression of the mitotic markerwas largely absent in cells close to the root apexpostgermination (Fig. 2B). Hence, SHR appears to berequired for the reactivation of the Arabidopsis RAMfollowing germination. Despite this root division de-fect, shr seedlings germinated normally (Supplemental

Fig. S1). However, root length was severely reducedcompared with the wild type or the scr mutant (Sup-plemental Fig. S2).

As a result of the failure to reactivate mitotic activ-ity in the primary root, the RAM became progres-sively smaller (Fig. 2C). This reduction in RAM sizewas monitored using several markers, including theLAX3pro:GUS reporter (Swarup et al., 2008), which isexpressed in stele cells entering the elongation zone.We observed that the LAX3pro:GUS marker movesprogressively closer to the root tip in older shr seed-lings (Fig. 2C). We conclude that as the shr root grows,cells exit the arrested meristem for the elongation zoneuntil the meristem is largely depleted of cells. SHR isrequired to maintain indeterminate growth (and there-fore appears to function as an indeterminacy factor) inthe postembryonic primary root meristem.

The shr Mutant Is Perturbed in Auxin Abundanceand Biosynthesis

The shr mutation disrupts the maintenance of RAMactivity, a process that is closely associated with themitotic signal auxin. To investigate whether there was areduction in the accumulation and/or synthesis of auxinin mutant root tissues, we analyzed indole-3-acetic acid(IAA) concentration and biosynthesis rate in the rootapex of wild-type and shr seedlings during the first 10 dof germination using mass spectrometry (as describedby Edlund et al. [1995] and Ljung et al. [2005]). Surpris-ingly, IAA concentration was significantly elevated inshr root tips compared with the wild type in seedlings at3 and 6 d after germination (DAG; pooled 1-mm roottips; Fig. 3A). However, no significant difference in IAA

Figure 1. RSA becomes determinatein the shr mutant. A and B, 3D x-raymicrotomography analysis of RSA of3-week-old soil-grown shr (A) andwild-type (B) seedlings grown in a sandyloam soil. Bars = 1 mm. C, Quantita-tive description of root architectureextracted from A and B. WT, Wildtype. D, RSA of 4-week-old compost-grown wild-type and shr seedlings.Bar = 1 cm. E, RSA of 12-d-old agar-grown wild-type and shr seedlings. Bar =1 cm.

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concentration between the wild type and shr was ob-served in root tips from 10-DAG seedlings.In parallel, the IAA synthesis rate in root tips from

wild-type and shr seedlings was also analyzed. Five-day-old excised seedling roots were incubated inliquid medium containing 30% deuterated water. Ex-cised roots were incubated instead of whole seedlingsin order to separate IAA synthesized de novo in theroot system from IAA synthesized in the aerial part ofthe seedling and then transported to the root. After24 h of incubation, 1-mm root tips were pooled andanalyzed (Fig. 3B). We observed a significantly higherIAA synthesis rate in shr root tips compared with thewild type, suggesting that the higher IAA concentra-tion that we observed in the root apex is in part due tohigher root-specific IAA synthesis and is not neces-sarily caused by the accumulation of IAA coming fromthe aerial part of the seedling.We conclude that shr primary root determinancy is

not due to either a reduced level or synthesis of auxin.Instead, mutant RAM tissues exhibit a higher rate of

biosynthesis of auxin and transient accumulation ofthis hormone during the first week after germination.

Root Determinacy Is Associated with a Loss of PINAuxin Carrier Accumulation

In order to understand why auxin accumulates inthe root apex during the first 10 DAG, we monitoredthe expression patterns of AUX1 and PIN classes ofauxin influx and efflux carriers in wild-type versus shrroot apical tissues (Fig. 4).

In wild-type root apical tissues, PIN1 was detectedpredominantly in stele and endodermal cells, and itspattern of expression did not change significantlyduring root development between 2 and 10 DAG(Fig. 4A). In shr root apical tissues, the PIN1 signalwas observed only in stele cells, since this mutant lacksan endodermis (Fig. 4A). Most strikingly, PIN1 abun-dance progressively decreased in shrmutant seedlingsfrom 4 DAG, and the signal had almost completelydisappeared by 10 d (Fig. 4A).

Figure 2. The shr mutation blocks mitotic activity in the RAM following germination. A, CyclinB1:1:GUS expression duringwild-type (WT) and shr embryo development. B, CyclinB1:1:GUS postgermination expression in the RAM of the wild type andshr at different DAG. C, LAX3pro:GUS expression in the RAM of shr at different DAG. The red line indicates the position of the QC,and the black bar indicates the start of the elongation zone coinciding with the LAX3pro:GUS expression.





In the case of PIN2, its signal was significantlyreduced from 4 DAG in shr versus the wild type (Fig.4B). However, the polarity of the PIN2 protein inepidermal cells was the same in shr as in the wild type(i.e. shootward/basipetal [pointing away from the rootapex] in the membrane of epidermal cells and root-ward/acropetal [pointing toward the root apex] in themembrane of cortical cells) until the transition to theelongation zone, where PIN2 switches to a basipetalorientation in the cortex (Supplemental Fig. S3). Nev-ertheless, the switch in PIN2 orientation in the cortexwas shifted toward the RAM in shr (data not shown).This shift is consistent with the reduction of rootmeristem size in the shr mutant (Fig. 2).

In wild-type root apical tissues from 2 to 10 DAG,PIN4 was expressed in the QC, the surrounding ini-tials, and their abutting daughters with a polar local-ization toward the QC (Fig. 4C), consistent with

previous reports (Friml et al., 2002; Blilou et al.,2005). In contrast, PIN4 was absent in the QC of shrseedling roots at 2 DAG but was detected in initialsand their daughter cells plus the stele (Fig. 4C). How-ever, PIN4 was no longer detectable in these shr roottissues from 4 DAG (Fig. 4C). Immunolocalization(Fig. 4D) revealed that PIN7 was one of the first PINproteins to be down-regulated in the shr background,being almost undetectable at 2 DAG. By day 10,neither PIN7 (nor PIN3) proteins were detectable(Fig. 4, D and E).

In addition to PINs, we monitored the expression ofan AUX1pro::AUX1-YFP (for yellow fluorescent pro-tein) translational fusion in the shr background. Con-trary to PINs, AUX1-YFP expression was maintainedin the shr root apex (Fig. 4F). The only significantdifference detected was the absence of AUX1-YFPsignal in the vascular strand and a smaller domain ofexpression in the lateral root cap (reflecting the loss ofprotophloem cells and the smaller size of the meri-stem, respectively).

In summary, shr root apical tissues exhibit a pro-gressive loss of PIN (but not AUX1) auxin transporterexpression, which is likely to negatively impact mer-istem function (Blilou et al., 2005) and may contributeto the loss of primary root indeterminancy.

The scr mutant must clearly reinitiate RAM activity,based on its ability to grow to 50% of the wild-typeroot length (Supplemental Fig. S2). Intriguingly, unlikeshr, the scr mutant does not lose PIN protein expres-sion (Supplemental Fig. S7), consistent with a linkbetween cell proliferation and PIN protein abundance.

SHR Is Not Required for the Initiation and Radial

Patterning of Anchor Root Primordia

We next investigated the impact of the loss of SHRand primary root meristem activity on root architec-ture. The majority of shr seedlings exhibit a distinctivetripod-like root phenotype, with nearly all (89% at15 DAG; n = 394) forming at least one anchor rootoriginating at the root-hypocotyl junction (Fig. 1E). Incontrast, wild-type Arabidopsis seedlings rarely acti-vate anchor root primordia at the root-hypocotyl junc-tion (Fig. 5B). Indeed, only 10% (n = 40) of wild-typeseedlings had formed one or more anchor roots by 8DAG (Fig. 5A). Anchor root activation in the shrseedlings may represent a generic mechanism to com-pensate for the loss of RAM activity. To test this, weexamined the impact of loss of the RAM in wild-typeseedlings by excising the bottom 2 to 3 mm of the wild-type root at 3 DAG, then scoring numbers of anchorroots at 8 DAG (Fig. 5C). The percentage of seedlingsthat formed one or more anchor roots by 8 DAGincreased dramatically from 10% (n = 40) in nonex-cised wild-type seedlings to 97% (n = 39) in RAM-excised wild-type seedlings (Fig. 5C). A representativeimage is shown in Figure 5B. We conclude that thisrepresents a generic mechanism present in wild-typeArabidopsis to compensate for the loss of growth

Figure 3. Auxin abundance and biosynthesis are perturbed in shr rootapical tissues. A, IAA concentration in the root apex of the wild type(WT) and shr at 3, 6, and 10 DAG. B, IAA biosynthesis in the root apexof the wild type and shr at 6 DAG. IAA quantifications and biosynthesismeasurements were performed by gas chromatography-selected reac-tion monitoring-mass spectrometry after 24 h of incubation withdeuterated water and compared using Student’s t test (two-sampleassuming equal variances, two-tailed distribution; ** 0.001, P, 0.01,*** P # 0.001). Values represent means 6 SD.

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potential in the primary root. In the case of shr, theprogressive loss of RAM activity most likely results inthe activation of anchor root primordia. Hence, unlikethe primary RAM, SHR is clearly not required for thepostembryonic activation of anchor root primordia,based on its mutant phenotype (Fig. 1E).We went on to investigate whether SHR was neces-

sary for radial patterning of anchor roots. Confocalimaging of anchor roots in the wild type revealed aradial patterning identical to that observed in primaryand lateral roots (Fig. 5D). Surprisingly, shr anchorroots also contained a wild-type number of layers, asrevealed by the presence of two cell layers between theAUX1-YFP-marked epidermis and pericycle layers inshr AUX1pro::AUX1-YFP seedlings (Fig. 5E). This ob-servation is in contrast to the radial organization ofmutant primary root tissues, where loss of SHR resultsin the loss of a cell layer (Benfey et al., 1993; Schereset al., 1995). Hence, activation and radial patterning of

anchor root primordia appears not to be dependent onSHR function. Nevertheless, shr anchor root growthbehavior is determinate, where the new organ ceasesgrowing at approximately the same length as theprimary root (Fig. 1E). This implies that, as in thecase of primary root meristem, SHR also functions asan indeterminacy factor for anchor root development.

SHR Is Required for Lateral Root Initiation and

Patterning of New Primordia

We next investigated whether SHR plays a roleduring lateral root development. We initially deter-mined whether SHRwas expressed during lateral rootformation employing a GUS reporter regulated by 2.5kb of the native SHR promoter (SHRpro:GUS). SHRpro:GUS was expressed exclusively in the central zone ofthe LRP throughout the initiation and emergenceprocesses (Supplemental Fig. S4, A–E). Upon emer-

Figure 4. The shr mutation causes aprogressive reduction of PIN auxintransporter abundance. A to E, Dy-namic changes of PIN1 (A), PIN2 (B),PIN4 (C), PIN3 (D), and PIN7 (E) ex-pression as shown by immunolocali-zation in the wild type (WT) and shr. F,Expression of AUX1 in the wild typeand shr at 5 DAG. PIN expression in Ato E was assessed by immunolocaliza-tion, while AUX1 expression in F wasinvestigated using the fusion proteinAUX1-YFP.





gence, reporter expression extended into the root cap,in addition to the stele and the base of the lateral root(Supplemental Fig. S4, E–J). SHRpro:GUS expression inthe lateral root then resumed a pattern similar to thatobserved in the primary root (i.e. stele exclusive;Supplemental Fig. S4, K and L). Thus, SHR was ex-pressed during all stages of lateral root develop-ment.

As the SHR protein regulates primary root radialpatterning in a non-cell-autonomous manner by mi-grating from the stele to the QC and future endoder-mis tissues (Nakajima et al., 2001), we examinedwhether SHR also moved to cells outside its expres-sion domain during lateral root development. To dothis, we created a SHR-YFP translational fusion drivenby 2.5 kb of the native SHR promoter (SHRpro::SHR-YFP) for confocal microscopy observation. The SHR-YFP fusion was functional, based on its ability to fullyrescue the shr mutant primary and lateral root pheno-types (Supplemental Fig. S5). The lateral root expres-sion of SHR-YFP in shr SHRpro::SHR-YFP was thenanalyzed by confocal microscopy (Fig. 6). SHR-YFPlocalization was first observed in the lower half ofyoung LRP (Fig. 6, A and B). As primordia developed,the SHR-YFP protein accumulated in the inner celllayers of the primordia, corresponding to the prospec-tive stele and pericycle (Fig. 6, C–E). In addition, theSHR-YFP protein was also targeted to the nuclei andcytoplasm of the flanking cells in the LRP (Fig. 6E,inset). During lateral root emergence, the SHR-YFPprotein was also targeted to the nuclei of the QC andputative endodermis (Fig. 6F, inset). After emergence,the SHR-YFP protein was located in the mature lateralroot as described for the primary root (Nakajima et al.,2001). However, expression was prolonged at the baseof the lateral root following organ outgrowth andelongation (Fig. 6, J and K).

LRP originate from pericycle cells that are consid-ered to represent part of an extended meristem that(unlike other root tissues) retains its mitotic potentialonce exiting the primary root meristem (Peret et al.,2009). Given the impact of the shr mutation on RAMmitotic activity, we investigated whether SHRwas alsorequired for lateral root development. The density oflateral root initiation in wild-type and shr seedlingswas scored from 4 to 14 DAG. Less than 40% (n = 49) of14-d-old mutant seedlings exhibited any lateral rootinitiation events (Fig. 7A). In addition, initiation eventsin shr seedlings that resulted in emerged lateral rootswere greatly reduced compared with the wild type(over a 3-fold reduction; Fig. 7B). Although the densityof emergence events scored after 1 week of growthwasalso reduced in shr, the proportion (density of emer-gence event/density of initiation event) appearedhigher in shr (70%) than in the wild type (40%) after2 weeks of growth (Fig. 7B), consistent with the exis-tence of equilibrium between initiation and emergence(Lucas et al., 2008). However, over 40% (n = 46) ofemerging lateral roots did not develop any furtherfollowing emergence (Fig. 7C).

Figure 5. The shr mutation causes the activation of anchor rootprimordia. A, Percentage of wild-type (WT; n = 40) and shr (n = 394)seedlings having zero (gray bars) or one or more anchor roots (blackbars) at 8 DAG. B, Excision of the RAM of wild-type seedlings.Seedlings were grown vertically, and half the seedlings had 2 to 3mm of the root (from the apex upward) excised at 8 DAG. Represen-tative wild-type (+RAM) and wild-type minus RAM (2RAM) seedlingsare shown at 5 d after excision. Bar = 5 mm. C, Percentage of wild-typecontrol (n = 40) and excised (n = 39) seedlings having zero (gray bars) orone or more anchor roots (black bars) 5 d after excision of the RAM. D,Root structure of wild-type anchor root. Epidermis (ep), cortex (co),endodermis (en), pericycle (pe), and stele (white) tissue structures(inset) are revealed by the LTi6a membrane marker (green) andpropidium iodide (red). Bar = 100 mm. E, Root structure of shr anchorroot. Epidermis, cortex, endodermis, and stele tissue structures arerevealed by propidium iodide (red). Pericycle is revealed by the AUX1-YFP membrane marker. Bar = 100 mm.

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To understand why such a large proportion oflateral roots did not develop in shr, we investigatedlateral root development in the mutant employingscanning electron and confocal microscopy techniques(Fig. 8). Electron microscopic analysis of developinglateral roots revealed that lateral roots emerging fromshr primary roots were approximately double thethickness of wild-type lateral roots (Fig. 8, compareA and B). We employed the AUX1-YFP marker(AUX1pro::AUX1-YFP) in conjunction with confocalmicroscopy to study the cellular organization of LRPin the wild type versus the shrmutant (Fig. 8, C and D).In wild-type plants, primary and lateral roots arepatterned in the following order of cell layers fromthe outside to the inside: epidermis, cortex, endoder-mis, pericycle, stele cylinder. In the shrmutant primaryroot, the cortex/endodermis initials fail to divide,resulting in the cortex and endodermis being replacedby a single ground tissue layer (Scheres et al., 1995).We observed that this is also the case in shr LRP (Fig. 8,

E and F). Confocal imaging revealed that lateral rootcells in the outer tissue layers of the wild type and shrwere of equal dimension (Fig. 8, E and F). However,the increased thickness in developing shr lateral rootappeared to be due to increased radial vascular de-velopment (Fig. 8G). A similar ectopic lignificationphenotype was observed in the shr hypocotyl (Sup-plemental Fig. S6).

In addition to an extravascular development pat-terning defect, we observed that around half of devel-oping shr LRP exhibited severe patterning defectscompared with the wild type (Fig. 8, H and I). Priorto emergence, such LRP lacked the cellular organiza-tion of wild-type LRP (Fig. 8I) and exhibited disorga-nized expression of the AUX1-YFP marker (Fig. 8H).Postemergence, we distinguished two types of abortedlateral roots. In the first category, termed “globularLRP” due to their symmetrical shape, the AUX1-YFPmarker was not significantly expressed and propidiumiodide rapidly stained the nuclei of lateral root cells

Figure 6. SHR is expressed throughout lateralroot development. Confocal images of LRP andlateral roots of 8-d-old shr SHRpro::SHR-YFP Arab-idopsis are shown. The SHR-YFP fusion protein isfunctional and rescues the shr mutant when ex-pressed under the control of the SHR promoter(Supplemental Fig. S5). A and B, The SHR protein(green) is expressed in the lower half of theprimordium during initial developmental stages.C to E, As the primordium acquires its domeshape, the SHR protein accumulates in the futurestele of the primordium and nuclei of the flankingcells of the primordia (insets). F and G, Uponemergence, the SHR protein is targeted to thenuclei and cytoplasm of the QC and groundtissues (inset). H and I, Once the lateral root isfully emerged, the SHR protein is targeted to anannulus of cells around the base of the newvascular bundle neighboring the flanking cells ofthe original primordium (insets). J and K, The SHRprotein is located in the mature lateral root as it islocated in the primary root. Localization in theflanking cells of the young primordium is main-tained along the ground tissue following lateralroot outgrowth. Root tissue structure is revealedby propidium iodide counterstaining (red). Bars =50 mm.





(Fig. 9, A–D), indicating that cells were no longerviable. In the second category, AUX1-YFP expressionwas maintained and revealed that despite their alteredouter structure (Fig. 9, E and H), these aborted organsretained some internal organization (Fig. 9, F and I).

In contrast to globular LR, the second class of lateralroot formed clusters of fused LRP (Fig. 9, F and I).These “cluster LRP” expressed the AUX1-YFP markerin a pattern similar to normal LRP (Fig. 9, G and J).

We conclude that SHR is required for the initiation,radial patterning, and organization of new LRP. In theabsence of SHR, lateral roots exhibit radial and vas-cular patterning defects. This may reflect, as observedin the primary root, that shr mutant cells differentiateinto a state where they are no longer competent to fullyform lateral roots.

DISCUSSION

The shrmutant was originally identified based on itsradial patterning and determinate RAM phenotype(Benfey et al., 1993). However, the impact of the shrmutation on other root developmental programs isunclear. In this paper, we describe the important rolesplayed by SHR during primary, lateral, and adventi-tious root development.

SHR Is Essential for Mitotic Reactivation of the RAMafter Germination

SHR has been described to be essential for main-taining RAM activity (Benfey et al., 1993; Di Laurenzioet al., 1996; Fig. 1). In mutant plants lacking SHR,primary root growth is severely reduced (Benfey et al.,1993; Scheres et al., 1995; Fig. 1; Supplemental Fig. S2).Mutant roots exhibit QC defects and loss of activity ofsurrounding stem (initial) cells, resulting in the dis-ruption of formative cell division events (Sozzani et al.,2010) and the depletion of proliferating cells in theRAM (Sabatini et al., 2003).

SHR is also required for reinitiating mitotic activityin the RAM following germination. We report that inthe absence of SHR, mutant RAM cells fail to expressthe mitotic marker, cyclinB1:1:GUS (Fig. 2B; Ferreiraet al., 1994). Consistent with a role as a key root mitoticregulator, Sozzani et al. (2010) recently reported thatSHR controls the expression of a number of genesencoding components of the cell cycle machinery.Direct targets of SHR include the D-type cyclin geneCYCD6;1 and the cyclin-dependent kinase genesCDKB2;1 and CDK2;2 (Sozzani et al., 2010). Whileectopic expression of two of these genes in shrwas ableto partially restore formative divisions, it was not ableto rescue mutant root growth (Sozzani et al., 2010).Hence, further genes are likely to be required. Indeed,266 genes were identified by Sozzani et al. (2010) to bedirect targets of SHR, of which 65 were differentiallyexpressed in a SHR-inducible system. In total, theexpression of approximately 2,500 genes was observedto change over a 12-h time course following theinduction of SHR expression in a shr background.Hence, SHR appears to control the expression of a verylarge number of genes, consistent with its role as a key

Figure 7. The shr mutation disrupts lateral root initiation, patterning,and emergence. A, Time course of the percentage of wild-type (WT)and shr seedlings having initiated at least one lateral root (n = 49). B,Time course of densities of lateral root initiation and lateral rootemergence events in wild-type and shr seedlings with at least oneinitiation event (in events per mm; n = 20). Values represent means 6SD. C, Percentage of abnormal lateral roots after emergence in shrSHRpro::SHR-YFP and shr seedlings (n = 46). [See online article for colorversion of this figure.]

Lucas et al.




regulator linking patterning and growth (Sozzaniet al., 2010).We report that the shrmutant exhibits a progressive

reduction and eventual loss of detectable PIN auxinefflux carrier protein following germination (Fig. 4).PIN protein localization was performed up to 10DAG on seedling roots from multiple independentshr null alleles isolated from different accessions (fordetails, see “Materials and Methods”). Every nullallele tested was observed to exhibit a progressiveloss of PIN1, PIN2, PIN3, PIN4, and PIN7 proteins(Fig. 4). Hence, PIN protein abundance appeared tobe dependent on SHR activity. None of these PIN

genes were identified among the direct targets of SHRregulation (Levesque et al., 2006; Sozzani et al., 2010);hence, protein abundance was likely to be regulatedindirectly by SHR. Surprisingly, quantitative reversetranscription (qRT)-PCR profiling revealed that SHRdoes not regulate PIN abundance at the transcrip-tional level, since their mRNAs are still detectable(Supplemental Fig. S8). Our qRT-PCR results areconsistent with recently published array data for shrand scr root apical tissues, which detected PIN ex-pression in both mutant backgrounds (Sozzani et al.,2010). Hence, SHR must regulate PIN abundance atthe posttranscriptional level. As in the wild type, PIN

Figure 8. The shr mutant exhibits radial and vascular patterning defects. A and B, Electron microscopy images of Col-0 (A) andshr (B) emerging lateral roots. Emerging lateral roots in shr are approximately two times wider than wild-type lateral roots. C andD, Confocal images of Col-0 AUX1pro::AUX1-YFP (C) and shr AUX1pro::AUX1-YFP (D). E, Root structure of lateral root in shr.Lateral roots in shr exhibit the same radial patterning defect as the primary root, with a single ground tissue layer (gt) replacingcortex and endodermis between pericycle and epidermis. Stele tissues are marked in white. Root structure is revealed byconfocal imaging with propidium iodide staining (red). F, Root structure of lateral root in Col-0. From outer to inner layer:epidermis (ep), cortex (co), endodermis (en), pericycle (pe), stele tissues (white). Root structure is revealed by confocal imagingwith propidium iodide staining (red). G, Confocal images of shr AUX1pro::AUX1-YFP primary (bottom left) and lateral (top right)roots. The thickness gain in the developing lateral root of shr is linked with excessive radial development of vascular tissues (bluebar), with no visible changes in the thickness of outer tissue layers (green and yellow bars). H and I, Confocal images of AUX1pro::AUX1-YFP in shr and the wild type. Propidium iodide (red) stains cells in the outer tissue layers of young LRP, while the AUX1-YFP marker (green) is expressed in lateral root stele cells. Overlaying the two markers reveals that outer cells in shr LRP arenonviable (H, insets) compared with the wild type (I, inset). Bars = 100 mm.





abundance is maintained in the scr mutant (Supple-mental Fig. S7), suggesting that the abundance ofthese proteins is regulated via a gene product(s)controlled by SHR (but not SCR). It is intriguingthat PIN abundance in shr root apical cells decreases(Fig. 4), despite elevated auxin abundance and bio-synthesis in the mutant (Fig. 3) and contrary to theobserved auxin inducibility of PIN expression (Vietenet al., 2005). One simple explanation is that the loss ofPIN abundance in shr (but not scr) may represent anindirect consequence of mutant RAM cells differen-tiating as a result of their failure to reinitiate celldivision following germination.

SHR Is Required for Lateral Root Formation in a Manner

Distinct from the Primary Root

LRP in Arabidopsis originate from the pericycle(Malamy and Benfey, 1997). LRP initiation involves theasymmetric division of xylem pole pericycle foundercells (Dubrovsky et al., 2001). Based on data showingthat the pericycle remains in G1 phase of the cell cycle(Beeckman et al., 2001) and that the xylem polepericycle continues to cycle after leaving the RAM(Dubrovsky et al., 2000), it has been postulated that thepericycle acts as an extended monolayered meristem(Casimiro et al., 2003). While pericycle division is firstvisible in the differentiation zone well above thedividing RAM, xylem pole pericycle founder cells firstneed to be primed in the basal meristem (close to theelongation zone) in order to trigger LRP initiation (De

Smet et al., 2007). Despite the loss of mitotic activityin the shr RAM, pericycle cells retain their ability toinitiate new LRP (Figs. 1 and 7). Nevertheless, thenumber of newly initiated LRP in shrwas significantlyreduced, suggesting that SHR facilitates (rather than isessential for) lateral root initiation.

LRP development and patterning are generally con-sidered to be a postembryonic recapitulations of theequivalent processes in the primary root (Malamy andBenfey, 1997; Scheres et al., 2002). We investigatedthe expression of SHR employing transcriptional andtranslational reporter fusions during LRP develop-ment. We first observed that SHR-YFP moved toadjacent QC, CEI, and endodermal cell layers whenthe dome-like structure of the LRP is formed (stagesIV/V onward; Fig. 6). This expression event coincideswith the restricted expression domain of SCR inemerging primordia and prior to LRP tissues adoptinga radial organization equivalent to the primary root(Malamy and Benfey, 1997). After LRP emergence, theexpression domain of the SHR protein appears iden-tical to that described for SHR in the RAM (Fig. 6;Nakajima et al., 2001).

Our study also revealed that, in addition to its radialpatterning function, SHR is required to coordinatethe overall morphology of new LRP. In the absence ofSHR, over half of the initiated LRP exhibited severepatterning defects (Fig. 9). These LRP were eitherclassified as cluster LRP or globular LRP. Cluster LRPwere composed of multiple fused LRP, while globularLRP failed to develop any properly organized inner

Figure 9. SHR is required for lateral root patterning and viability. Upon emergence, approximately 50% of shr lateral roots fail toelongate. Two types of patterning defects can be distinguished within these abnormal lateral roots. A to D, Globular lateral rootslack inner structure (B and D, schematic representations of the inner organization of the lateral root), and their cells appear to benonviable based on their permeability to propidium iodide (red, strong nuclei staining). Images of shr AUX1pro::AUX1-YFP(green) were taken by confocal microscopy with propidium iodide counterstaining. E to J, Fused lateral roots appeared outwardlyamorphous (E and H) yet contained internal structures (F and I). Optical sectioning by confocal microscopy of shr AUX1pro::AUX1-YFP with propidium iodide counterstaining reveals multiple fused LRP (G and J, schematic representation of the fusedLRP). The LRP comprising the fused lateral roots exhibit standard patterns of AUX1pro::AUX1-YFP expression (epidermis, lateralroot cap, columella, and stele tissues). Bars = 100 mm.

Lucas et al.




architecture or boundaries. Consistent with such func-tions, in addition to being expressed in inner LRPtissues, the SHR-YFP marker was also detected in thenuclei and cytoplasm of the outermost cells flankingthe LRP as early as stage III/IV (Fig. 6). This noveldomain of SHR expression in cells flanking the LRPcould play a role in the specification of primordiaboundaries, akin to its regulation of radial patterningin QC, CEI, and endodermal cell layers. Hence, SHRappears to regulate lateral root patterning in an over-lapping yet distinct manner to the primary root.

SHR Is Not Required for Correct Patterning of Anchor

Root Tissues

SHR is best known as a regulator of radial pattern-ing in Arabidopsis primary root apical tissues (Benfeyet al., 1993). The SHR gene is first transcribed in thestele, and then the SHR protein moves outward one

cell layer to adjacent QC, CEI, and endodermal cells(Nakajima et al., 2001). SHR triggers SCR expression,causing CEI cells to divide longitudinally and generateendodermal and cortical layers. In the absence of SHR,mutant roots form a single layer of cells with a corticalidentity (Benfey et al., 1993; Scheres et al., 1995).Hence, SHR is necessary for radial patterning of roottissues plus specifying endodermal cell fate. In con-trast to defective radial patterning in the shr RAM,mutant anchor roots retain a wild-type-like tissueorganization (Fig. 5). Nevertheless, SHR is requiredfor radial patterning of LRP tissues (Fig. 8). Thesedifferences are unlikely to reflect that SHR is essentialfor radial patterning only in root-derived organs, sincethis gene product is also required for the formation ofthe starch-sheath layer in shoot tissues (Fukaki et al.,1998). Instead, it is most likely that another GRASfamily member (Pysh et al., 1999) performs a SHR-likepatterning function in this tissue.

Figure 10. Comparative summary ofroot system development of the wildtype and shr. The inset lists the mainevents of root system development im-pacted by the shr mutation.





Loss of SHR Activity Impacts RSA

We report that the loss of SHR activity leads to majorchanges in RSA (summarized in Fig. 10). Micro-CTimaging of RSA in soil revealed that the wild-type taproot architecture is converted to a dwarfed fibrous-likeroot system in shrmutants (Fig. 1). This dramatic changein shr RSA is caused by changes in the relative contribu-tion of RAM, lateral root, and anchor roots (Fig. 10). Wehave reported that the shr RAM fails to reinitiate mitoticactivity postembryonically (Fig. 2). Consequently, themutant primary root elongates until the pool of cells atits RAM is depleted and no longer sufficient to maintaingrowth. Loss of RAM activity in shr promotes the initi-ation of anchor root primordia (Fig. 7). We propose thatthis activation represents a mechanism to compensate forRAM death, as it could be phenocopied in the wild typefollowing RAM excision. The underlying factors respon-sible for anchor root activation upon loss of the RAMremain unclear. It is possible that an as yet unidentifiedinhibitory signal(s) is produced in the RAM that nor-mally acts at the root-hypocotyl junction to preventanchor root formation in the presence of an activeRAM. Alternatively, anchor root activation may simplybe a response to the supply of nutrients and/or prolif-erative signals from the aerial portions of the plant thatare in excess of that able to be consumed by the deter-minate mutant primary root. The activation of determi-nate anchor roots leads to repeated ramification near thebase of the root-hypocotyl junction, as observed in soil-grown shr (Fig. 1). Moreover, shr exhibits lower density oflateral root initiation, with a significant fraction of initi-ated (globular and fused) lateral roots aborting postemer-gence. The activation of anchor roots and the lower levelof lateral root initiation and development give rise to thefibrous architecture of the shr mature root system, withhigh branching density near the base of the root systemand low branching density along the roots (Fig. 10).

In summary, our results reveal that SHR function isnot restricted to the primary root. Instead, SHR isrequired for the initiation and patterning of LRP andfor maintaining the indeterminate growth of primary,lateral, and adventitious roots.

MATERIALS AND METHODS

Plant Lines and Growth Conditions

Arabidopsis (Arabidopsis thaliana) seeds were surface sterilized for 8 min in

50% (v/v) bleach and then washed twice in 0.1% (v/v) Triton X-100. The seeds

were then plated on square plates containing 0.53 Murashige and Skoog salt

mixture in 1% (w/v) bacto agar. The plates were cold treated for 2 d at 4�C in

the dark to synchronize germination. Plates were then incubated in a nearly

vertical position at 23�C in 150 mmol m22 s21 constant light with a cycle of 18 h

of light/6 h of dark. For the analysis of the loss of the RAM on ecotype

Columbia (Col-0) seedlings, 2 to 3 mm of the root tip was excised with a sterile

razor blade at 3 DAG, and the seedlings were returned to growth conditions.

To generate the SHRp::SHR-YFP construct, 2-kb upstream sequence of the

SHR gene (AT4G37650) was PCR amplified and cloned into BamHI and XhoI

sites of pBluescript SK1 (Stratagene) to create pBS-SHRpro. Subsequently,

full-length SHR gene (including the 3# untranslated region) was PCR ampli-

fied with an in-frame XhoI restriction enzyme site at the N terminus and

cloned between XhoI and KpnI sites of pBS-SHRpro to create pBS-SHR-X. Full-

length EYFP sequence (excluding the stop codon) was then PCR amplified

using primers with in-frame XhoI restriction enzyme sites at the ends and

subsequently cloned in frame at the engineered XhoI site of the SHR gene to

create pBS-SHR-YFP. The SHR-YFP sequence was then cloned into a binary

vector (pMOG; Mogen International) between BamHI and KpnI to create

pMOG-SHR-YFP. Transformation of Agrobacterium tumefaciens (C58) and

Arabidopsis was done as described by Swarup et al. (2005).

X-Ray CT Scanning

Plants (shr and wild-type Col-0) were grown in a sandy loam soil

(Dunnington Heath series; Stagno-gleyic Luvisol) from the University of

Nottingham experimental farm at Sutton Bonington (52.5�N 1.3�W), sieved to

2 mm, and packed loosely (bulk density approximately 1.1 g cm23) into 30-

mm-diameter, 55-mm-high columns. The base of the column was covered

with “micropore” tape and placed in a tray to allow watering from below to

prevent gas entrapment. Seeds were sown directly on the soil surface after

surface watering, and columns were subsequently watered from below. The

plants were provided with supplemental nutrients by adding 1 mL of

commercial Miracle-Gro (diluted as per instructions) to the top of the columns

on two occasions, 11 and 25 d after sowing. Plants were grown in a controlled-

environment room with 16-h day/8-h night and temperature of 23�C day and

18�C night. The columns with live plants were scanned in a Nanotom X-Ray

Micro-Computed Tomography scanner (Phoenix X-Ray). Samples were scan-

ned at 80 mV and 220 mA, with 1,440 images collected over a 50-min period.

The spatial resolution was set at 18 mm pixel21. All samples were scanned at

an approximated field capacity soil moisture status. Image slices were

reconstructed into 3D volumes using DatosX Rec version 1.5 (Phoenix

X-Ray) with beam-hardening and ring artifact reduction algorithms applied

and then manipulated and analyzed in VGStudioMax 2.0 (Volume Graphics).

The 3D volume was median filtered, and the roots were manually segmented

through a region growing tool and visual identification. Tortuosity is defined

here as the ratio of the root length over the shortest distance between the two

extremities of the measured root.

Phenotypic Analysis

Primary root length was determined from digital images of the plates by

measuring from root tip to hypocotyl base using ImageJ 1.40 software (http://

rsb.info.nih.gov/ij/). Emerged lateral and anchor roots were counted using a

binocular. Two-sample unpaired t test was used for comparison of primary

root length and lateral root density, using a confidence interval of 95%.

GUS Assay and Microscopy

GUS activity of LAX3pro:GUS Col-0 and shr seedlings was assayed by

incubating whole seedlings in a staining solution comprising 1 mM 5-bromo-4-

chloro-3-indolyl b-D-glucuronide in 0.5% (v/v) dimethylformamide, 0.5% (v/v)

Triton X-100, 1 mM EDTA (pH 8), and 500 mM phosphate buffer (Na2PO4, pH 7)

for 2 to 3 h at 37�C. To limit the diffusion of the blue stain and therefore maintain

high staining specificity, 0.5 mM K3Fe(CN)6 and K4Fe(CN)6 were added. Both

stained and unstained roots were cleared by immersion in 20% (v/v) methanol/

4% (v/v) hydrochloric acid at 57�C for 20 min, followed by immersion in 7%

(w/v) NaOH/60% (v/v) ethanol at room temperature for 15 min. Roots were

then rehydrated for 5 min each in 40%, 20%, and 10% (v/v) ethanol and in-

filtrated in 5% (v/v) ethanol/25% (v/v) glycerol for 15 min. Roots were

mounted in 50% (v/v) glycerol on glass microscope slides and were imaged

using a Nikon differential interference contrast optics microscope.

For analysis of AUX1pro::AUX1-YFP and SHRpro::SHR-YFP seedlings, whole

mounts were prepared on microscope slides in water, and the roots were imaged

either on a Leica SP2 confocal microscope using the 514-nm line of the argon laser

or a Nikon eC1/TE2000U inverted confocal microscope using the 488-nm line of

the argon laser. For general morphological analysis, seedlings were stained by

immersion in 5 mL mL21 propidium iodide for 5 min, whole mounted in water,

and imaged using a Nikon eC1/TE2000U inverted confocal microscope.

IAA Quantification and Biosynthesis Measurements

Seedlings were grown on vertical agar plates under long-day conditions as

described by Ljung et al. (2005). IAA quantification was performed on shr and

Lucas et al.




wild-type seedlings at 3, 6, or 10 DAG. For IAA synthesis measurements,

excised roots (cut at the hypocotyl-root junction) coming from 5-d-old shr and

wild-type seedlings were transferred to liquid medium containing 30%

deuterated water and incubated in long days for 24 h. The most apical

1 mm of wild-type and shr seedling roots was pooled for IAA quantification

(50 sections per sample, five to eight replicates) and biosynthesis measure-

ments (100 sections per sample, four replicates). IAA quantifications and

biosynthesis measurements were done by gas chromatography-selected reac-

tion monitoring-mass spectrometry (Edlund et al., 1995; Ljung et al., 2005) and

compared using Student’s t test (two-sample assuming equal variances, two-

tailed distribution; ** 0.001 , P , 0.01, *** P # 0.001).

PIN Localization

Seedlings of the wild type, scr, and shr were sampled at different stages of

development (see “Results”). PIN localization was investigated in three

different alleles of shr mutants (shr1 [ecotype Wassilewskija], shr3, and tpd

[Col-0]). All alleles demonstrated similar dynamics of changes in PIN local-

ization. Immunolocalization in roots was performed as described (Friml et al.,

2002). Rabbit anti-PIN1 (Galweiler et al., 1998), anti-PIN2 (Muller et al., 1998),

affinity-purified anti-PIN3 (Friml et al., 2002), anti-PIN4 (Friml et al., 2002),

and mouse anti-PIN7 (Paponov et al., 2005) antibodies were diluted 1:500,

1:400, 1:100, 1:400, and 1:50, respectively. The secondary antibody, Alexa 488-

conjugated anti-rabbit or anti-mouse (for PIN7) antibody, was diluted 1:400.

Solutions during the immunolocalization procedures were changed using a

pipetting robot (Insitu Pro; Intavis).

Quantitative PCR

Plants were grown on 0.53 Murashige and Skoog and 1% bacto agar at

23�C and 150 mmol m22 s21 in long days (16 h of light). Total RNA was

extracted from 6-DAG roots using the Qiagen RNeasy Plant Mini Kit with on-

columnDNase treatment (RNase free DNase set; Qiagen). Poly(dT) cDNAwas

prepared from 2 mg of total RNA using the Transcriptor first-strand cDNA

synthesis kit (Roche). Quantitative PCR was performed using SYBR Green

Sensimix (Quantace) on a Stratagene Mx3005P apparatus. PCR was carried

out on 96-well optical reaction plates heated for 5 min to 95�C, followed by

40 cycles of denaturation for 10 s at 95�C and annealing-extension for 30 s at

60�C. Target quantifications were performed with the following specific

primer pairs: for TIR1 (AT3G62980), TIR1forward (5#-CCTAAACTGCAGCGCC-TCT-3#) and TIR1reverse (5#-GGTTGAAGCAAGCACCTCA-3#); for AFB1

(AT4G03190), AFB1forward, (5#-ACTGATGGTATCGCTGCTATTG-3#) and

AFB1reverse (5#-AGTTGAACTCTCTGGAAAATAGCTAAG-3#); for AFB2

(AT3G26810), AFB2forward (5#-CGTGCCTCGAAGGAGAAAC-3#) and AF-

B2reverse (5#-TTTGGAGACCTAGCAACAAGC-3#); for AFB3 (AT1G12820),

AFB3forward (5#-TGATAAACTTTACCTCTACCGAACAG-3#) and AFB3re-

verse (5#-CCTAACATATGGTGGTGCATCTT-3#); for PIN3 (AT1G70940),

PIN3forward (5#-CCCAGATCAATCTCACAACG-3#) and PIN3reverse

(5#-CCGGCGAAACTAAATTGTTG-3#); for PIN7 (AT1G23080), PIN7forward

(5#-TGGGCTCTTGTTGCTTTCA-3#) and PIN7reverse (5#-TCACCCAAACT-

GAACATTGC-3#); for PIN1 (AT1G73590), PIN1forward (5#-CCTCAGGG-

GAATAGTAACGACA-3#) and PIN1reverse (5#-TCATCGTCTTTGTTACC-

GAAACT-3#); for PIN2 (AT5G57090), PIN2forward (5#-GGCGAAGAAAG-

CAGGAAGA-3#) and PIN2reverse (5#-GGTGGGTACGACGGAACA-3#); forPIN4 (AT2G01420), PIN4forward (5#-TTGTCTCTGATCAACCTCGAAA-3#)and PIN4reverse (5#-ATCAAGACCGCCGATATCAT-3#); and for IAA2

(AT3G23030), IAA2forward (5#-GAAGAATCTACACCTCCTACCAAAA-3#)and IAA2reverse (5#-CACGTAGCTCACACTGTTGTTG-3#).

Expression levels were normalized to UBA (AT1G04850) using the fol-

lowing primers: UBAforward (5#-AGTGGAGAGGCTGCAGAAGA-3#) and

UBAreverse (5#-CTCGGGTAGCACGAGCTTTA-3#). All qRT-PCR experiments

were performed in triplicate, and the values presented represent means 6 SD.

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession numbers At4g37650 (SHR), At3g62980 (TIR1),

At4g03190 (AFB1), At3g26810 (AFB2), At1g12820 (AFB3), At1g70940 (PIN3),

At1g23080 (PIN7), At1g73590 (PIN1), At5g57090 (PIN2), At2g01420 (PIN4),

At3g23030 (IAA2), and At1g04850 (UBA).

Supplemental Data

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

Supplemental Figure S1. Germination of the shr mutant is comparable

with the wild type.

Supplemental Figure S2. Root length in shr is severely reduced compared

with the wild type and scr.

Supplemental Figure S3. Inversion of polarity of PIN2 in the cortical layer.

Supplemental Figure S4. SHRpro:GUS expression pattern during lateral

root development.

Supplemental Figure S5. The SHRpro::SHR-YFP transgene rescues the shr

mutation.

Supplemental Figure S6. The shr mutation causes ectopic lignification in

the hypocotyl.

Supplemental Figure S7. The loss of auxin carrier expression in shr is

independent of SCR.

Supplemental Figure S8. Expression of auxin-related genes in wild-type,

shr, and scr 6-d-old seedlings.

Supplemental Movie S1. 3D movie of a 3-week-old shr seedling grown in

sandy loam.

Supplemental Movie S2. 3D movie of a 3-week-old wild-type seedling

grown in sandy loam.

ACKNOWLEDGMENTS

We thank Philip Benfey (Duke University) for providing seeds for shr1

(Wassilewskija) and shr3 alleles and Laurent Laplaze (IRD Montpellier) and

members of our team for their helpful comments on the manuscript.

Received September 1, 2010; accepted October 26, 2010; published October 27,

2010.

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SHORT-ROOT Regulates Primary, Lateral, and · SHORT-ROOT Regulates Primary, Lateral, and Adventitious Root Development in Arabidopsis1[C][W][OA] Mikae¨l Lucas, Ranjan Swarup, Ivan