RESEARCH ARTICLE

The C2-domain protein QUIRKY and the receptor-like kinaseSTRUBBELIG localize to plasmodesmata and mediate tissuemorphogenesis in Arabidopsis thalianaPrasad Vaddepalli1, Anja Herrmann1,*, Lynette Fulton1, Maxi Oelschner1,‡, Stefan Hillmer2, Thomas F. Stratil3,Astrid Fastner4, Ulrich Z. Hammes4, Thomas Ott3, David G. Robinson2 and Kay Schneitz1,§

ABSTRACTTissuemorphogenesis inplants requirescommunicationbetweencells,a process involving the trafficking ofmolecules through plasmodesmata(PD). PD conductivity is regulated by endogenous and exogenoussignals. However, the underlying signaling mechanisms remainenigmatic. In Arabidopsis, signal transduction mediated by thereceptor-like kinase STRUBBELIG (SUB) contributes to inter-celllayer signaling during tissue morphogenesis. Previous analysis hasrevealed that SUB acts non-cell-autonomously suggesting that SUBcontrols tissue morphogenesis by participating in the formation orpropagation of a downstreammobile signal. A genetic screen identifiedQUIRKY (QKY), encodingapredictedmembrane-anchoredC2-domainprotein, as a component of SUB signaling. Here, we provide furtherinsight into the role ofQKY in this process.We show that likeSUB,QKYexhibits non-cell-autonomywhenexpressed in a tissue-specificmannerand that non-autonomyofQKYextendsacrossseveral cells. Inaddition,we report on localizationstudies indicating thatQKYandSUB localize toPDbut independently of eachother. FRET-FLIM analysis suggests thatSUB and QKY are in close contact at PD in vivo. We propose a modelwhere SUB and QKY interact at PD to promote tissue morphogenesis,thereby linking RLK-dependent signal transduction and intercellularcommunication mediated by PD.

KEY WORDS: Arabidopsis, Plasmodesmata, Signal transduction,Tissue morphogenesis, Receptor-like kinase, STRUBBELIG,QUIRKY

INTRODUCTIONTissue morphogenesis in plants depends on cell-cell communicationas cells and groups of cells divide and expand in a coordinatedfashion. One complication in this process arises from the fact thatplant cells are encased by a semi-rigid cell wall that constitutes abarrier to the communication between plant cells. During evolution,this obstacle has been overcome by at least two types of chemical

signaling mechanisms (Lucas et al., 2009). The first involvesplasmodesmata (PD), plasma membrane-lined channels thattraverse the cell wall interconnecting the cytoplasm of neighboringcells, thereby enabling the intercellular movement of molecules. Thesecond encompasses combinations of small, cell wall-penetratingligands, e.g. peptides or phytohormones, and their receptors.

Inter-cell layer communication during tissue morphogenesis inArabidopsis relies on signal transduction mediated by the atypicalleucine-rich repeat receptor-like kinase (RLK) STRUBBELIG(SUB) (Chevalier et al., 2005; Vaddepalli et al., 2011; Lin et al.,2012). Plants lacking SUB activity exhibit defects in integumentinitiation and outgrowth, altered floral organ shape, stemmorphology and reduced plant height, and asymmetric leaf shape.At the cellular level, SUB participates in division plane control incells of the L2 layer of floral meristems. In addition, SUB, alsoknown as SCRAMBLED (SCM), affects root hair patterning (Kwaket al., 2005). Interestingly, SUB acts in a non-cell-autonomousfashion across several cells in the shoot apex, floral meristem, ovuleand root (Kwak and Schiefelbein, 2008; Yadav et al., 2008). SUB ispresent at the plasma membrane (PM) and does not appear to trafficbetween cells. This indicates that signal perception by SUB resultsin the formation or propagation of a SUB-dependent mobile signal(SMS) that modifies the behavior of cells located several cells away.

Recent efforts have resulted in the identification of additionalgenetic components of SUB signaling, including QUIRKY (QKY)(Fulton et al., 2009; Trehin et al., 2013). Plants lacking SUB or QKYactivities display very similar phenotypes at the gross morphologicaland cellular levels. In addition, flowers of these mutants exhibit asignificant overlap in mis-regulated gene expression. These resultsindicate that QKY represents a central component of SUB signaling.Sequence analysis predicts thatQKY encodes a 121.4 kDa membraneprotein with four C2 domains and a phosphoribosyltransferaseC-terminal domain (PRT_C) containing two putative transmembranedomains (Fulton et al., 2009). As a rule, C2 domains representautonomously folding modules that bind phospholipids in a Ca2+-dependent manner (Rizo and Südhof, 1998). In addition, QKY is thefounding member of a 17-member gene family that includes FT-INTERACTING PROTEIN 1 (FTIP1) a regulator of flowering underlong-day conditions (Liu et al., 2012).

Data presented in this work address the functional connectionbetween QKY and SUB. They reveal that QKY acts in a non-cell-autonomous fashion, similar to SUB. They further show that functionalSUB:EGFP and mCherry:QKY fusion proteins colocalize to PD andthat association with PD occurs independently of each other. Inaddition, the data reveal that PD localization requires the extracellularand transmembrane domains of SUB and the C2C/C2D and thePRT_C domains of QKY, respectively. Finally, the data show thatSUB:EGFP and mCherry:QKY can physically interact in vivo at PD.Received 6 June 2014; Accepted 26 August 2014

1Entwicklungsbiologie der Pflanzen, Wissenschaftszentrum Weihenstephan,Technische Universitat Munchen, Emil-Ramann-Strasse 4, Freising 85354,Germany. 2Plant Cell Biology, Centre for Organismal Studies, University ofHeidelberg, Im Neuenheimer Feld 230, Heidelberg 69120, Germany. 3Institute ofGenetics, Faculty of Biology, Ludwig-Maximilians-University of Munich,Grosshaderner Strasse 2-4, Martinsried 82152, Germany. 4Cell Biology and PlantBiochemistry, Biochemie-Zentrum Regensburg, University of Regensburg,Universitatsstrasse 31, Regensburg 93053, Germany.*Present address: Institute of Plant Biology and Zurich-Basel Plant Science Center,University of Zurich, Zollikerstrasse 107, Zurich CH-8008, Switzerland. ‡Presentaddress: Zentrum fur medizinische Biotechnologie, Universitat Regensburg,Biopark 1, Josef-Engert-Strasse 9, Regensburg 93053, Germany.

§Author for correspondence (kay.schneitz@tum.de)

4139

© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 4139-4148 doi:10.1242/dev.113878

DEVELO

PM

ENT

RESULTSQKY acts in a non-cell-autonomous fashionPrevious genetic data indicated that QKY contributes to SUB-mediated signaling (Fulton et al., 2009; Trehin et al., 2013).Supporting this notion, we found that the expression patterns ofQKY and SUB largely overlap in young flowers and ovules(supplementary material Fig. S1) (Chevalier et al., 2005). Moreover,both genes share similar synergistic interactions with ERECTA inthe regulation of plant height (supplementary material Fig. S2)(Vaddepalli et al., 2011). Non-cell autonomy of SUB (Kwak andSchiefelbein, 2008; Yadav et al., 2008) is another central feature ofthe SUB-dependent signaling pathway. Formally, QKY could eithercontribute to the non-cell autonomy aspect of SUB signaling orparticipate in the perception of the mobile signal. If the formerscenario were true, QKY should also function in a non-cell-autonomous fashion. To address this issue, we generatedqky-8 plants that carried an EGFP:QKY reporter gene under thecontrol of either the WUSCHEL (WUS) or MERISTEM LAYER 1(ML1) promoters, two well-characterized tissue-specific promoters(Sessions et al., 1999; Bäurle and Laux, 2005), and assayed forrescue of the mutant phenotype in aerial tissues. A translationalfusion of EGFP to the N-terminus of QKY results in a functionalEGFP:QKY fusion protein (supplementary material Fig. S3).Seventy-four individual transgenic lines of the genotype pWUS::EGFP:QKY qky-8 were scored. Eighteen exhibited full rescue, 42

showed partial and 14 displayed no phenotypic rescue. With respectto pML1::EGFP:QKY qky-8, we generated 22 independent linesthat all developed as wild-type individuals. Phenotypes werevalidated in the T2 generation.

In Arabidopsis ovules, the initiating outer integument isseparated from the nucellus by five to seven cells (Schneitzet al., 1995). QKY is expressed throughout young ovules(supplementary material Fig. S1A). WUS expression is restrictedto the nucellus during stages 1 and 2 (stages according to Schneitzet al., 1995) and is no longer detectable around stage 3 (Gross-Hardt et al., 2002). Thus, we tested whether restricting EGFP:QKYexpression to the distal region of the ovule primordium can rescuethe outer integument defects of qky-8. Interestingly, the outerintegument of pWUS::EGFP:QKY qky-8 plants, expressingEGFP:QKY solely in the distal region of the ovule primordium,showed a wild-type appearance (Fig. 1A-D), indicating rescue.This result suggests that QKY can influence the development ofcells, several cells distant from where it is expressed.

QKY is expressed in inflorescence apices and broadly in floralmeristems and flowers (supplementary material Fig. S1). WUSexpression in the inflorescence meristem is limited to a few cellsbeneath the L3 layer (Mayer et al., 1998). In floral meristems,WUSexpression can be monitored up to stage 6 (stages according toSmyth et al., 1990), where its expression is primarily restricted tocells below the L2 layer but is occasionally detected in the L2 layer

Fig. 1. Non-cell autonomy of QKY function. (A,B,E-H,I″-L″)Longitudinal confocal sections. (A,E,G) GFP channel.(B,F,H) Differential interference contrast (DIC) channel overlaidwith GFP channel to outline tissue. (A,B) pWUS::EGFP:QKYreporter in a stage 2-III ovule. Signal is restricted to thenucellus. (B) Arrow indicates early outer integument. Insetshows a scanning electron micrograph of a wild-type ovuleat a similar stage (for details, see Schneitz et al., 1995).(C,D) Scanning electronmicrographs of stage 4 ovules. Inset inC shows awild-type ovule. (D) The qky-8 phenotype is rescuedby pWUS::EGFP:QKY. (E,F) Signal of the pML1::EGFP:QKYreporter in a stage 3 flower. Signal is restricted to the outermostlayer of the tissue. (G,H) Signal of the pWUS::EGFP:QKYreporter in a stage 3 flower. (I-L) Morphology of flowers.(K,L) The qky-8 phenotype is rescued in qky-8 plants carryingthe respective reporters. (L) Partial rescue of floral morphology.(I′-L′) Morphology of siliques (upper panels) and stems (lowerpanels). (I″-L″) Central region of stage 3 floral meristemsstained with pseudo-Schiff propidium iodide (mPS-PI).(I″) Note the regular anticlinal cell divisions in the L1 and L2layers. (J″) Arrows indicate two periclinal cell divisions in the L2layer of a qky-8 mutant. (M,N) Above-ground overallmorphology of qky-8 plants carrying different transgenes. (M)TG: pML1::EGFP:QKY. (N) TG: pWUS::EGFP:QKY.Abbreviations: ii, inner integument; nu, nucellus; oi, outerintegument. Scale bars: 5 μm in A,B; 10 μm in E-H,I″-L″; 50 μmin C,D; 0.5 mm in I-L′.

4140

RESEARCH ARTICLE Development (2014) 141, 4139-4148 doi:10.1242/dev.113878

DEVELO

PM

ENT

itself. Expression of the pWUS::EGFP:QKY reporter can rescuestem and floral development of qky-8 mutants. In lines showingrescue, floral development of pWUS::EGFP:QKY qky-8 plants waslargely indistinguishable from wild type (Fig. 1G,L-L″). In qkymutants, L2 cells of floral meristems divide in an atypical fashion(Fulton et al., 2009) (Fig. 1J″). In pWUS::EGFP:QKY qky-8 plants,the L2 division defects in stage 3 floral meristems were not observed(Fig. 1I″,J″,L″, Table 1). This latter effect, however, may simply bedue to occasional L2 expression of the reporter. Furthermore, stemdevelopment and plant height was rescued in pWUS::EGFP:QKYqky-8 plants, as they appeared wild type in this regard (Fig. 1L′,N).Regular stem and near normal flower development of pWUS::EGFP:QKY qky-8 plants thus provides further evidence thatnon-cell autonomy of QKY extends to several cells.ML1 is expressed in the epidermis of embryos, the L1 of shoot

and floral meristems, and in the epidermis of young floral organs(Lu et al., 1996; Sessions et al., 1999) (Fig. 1E,F). We used thepML1::EGFP:QKY reporter to test whether expression of EGFP:QKY in L1 cells of floral meristems can rescue the L2 divisionplane defects of qky-8 mutants. Indeed, we observed wild-typenumbers of periclinal L2 division planes in pML1::EGFP:QKYqky-8 floral meristems (Fig. 1I″-K″, Table 1), indicating thatEGFP:QKY expression in L1 cells influences the neighboringmutant L2 cells. In addition, pML1::EGFP:QKY qky-8 exhibitednormal floral and stem morphology, and wild-type plant height(Fig. 1K,K′,M).These results suggest that, despite the broad endogenous

expression of QKY within individual tissues, targeted EGFP:QKYexpression in restricted domains, such as the epidermis, is sufficient toconfer regular tissue morphogenesis in qky-8 mutants. In line withprevious SUB:EGFP reporter studies (Yadav et al., 2008), tissuedistribution of EGFP:QKY signal in ovules and floral meristems ofthe tested transgenic lines corresponded to the expression domainsof the WUS and ML1 promoters. Thus, movement of the reporterproteins was undetectable in the assessed tissues. Our results stronglysupport the notion that QKY functions in a non-cell-autonomousfashion and likely influences a downstream mobile signal that canmove across several cells.

EGFP:QKY and SUB:EGFP localize to plasmodesmataTo address the subcellular localization of QKY, we fused EGFP tothe N terminus of QKY under the control of the endogenous QKYpromoter ( pQKY::EGFP:QKY). Thirty out of 33 pQKY::EGFP:QKY qky-8 transgenic lines showed full or partial rescue,indicating the presence of a functional reporter (supplementarymaterial Fig. S3). Despite rescue, the signal could not be detectedin floral organs, indicating its very low expression in those tissues.However, signal could be observed in the root using a confocalmicroscope equipped with high sensitivity detectors (HSD). Wealso selected the UBIQUITIN 10 (UBQ10) promoter to driveexpression of the EGFP:QKY reporter and generated 26 qky-8 and11 Ler lines transgenic for the pUBQ::EGFP:QKY (EGFP:QKY)

reporter. EGFP:QKY qky-8 (24/26) lines showed full rescue of theqky-8 phenotype (supplementary material Fig. S3). In addition,we found no evidence that enhanced expression of EGFP:QKY inqky-8 or Ler backgrounds caused abnormal morphology ordevelopment. Although signal intensity varied the spatialdistribution of the signal did not differ across transgenic lines.Both reporters exhibited identical subcellular signal distributionpatterns.

Using the pQKY::EGFP:QKY reporter, we detected a broad signaldistribution throughout the root (Fig. 2A,B). At the subcellular level, apunctate pattern around the circumference of cells was observedsuggesting labeling of specific sites, such as vesicle docking sites orPD (Fig. 2C,D). ThisEGFP:QKY signal distribution looked similar inall tissues tested (Fig. 2E-G,M-O) (supplementary material Fig. S4).To test whether the punctate EGFP:QKY pattern relates to PD, weinvestigated the root tip and cotyledon epidermis ofEGFP:QKYqky-8seedlings stained with aniline blue (AB), a standard stain for calloseassociated with PD (Bell and Oparka, 2011). As shown in Fig. 2E-H,M-P EGFP:QKY coincides with AB-derived spots, indicating thatEGFP:QKY locates close to or at PD. Using software-based imageanalysis we determined that 89% of EGFP:QKY spots overlappedwith AB punctae in root epidermis cells (Pearson’s correlationcoefficient=0.79).

Previous work using conventional confocal microscopy revealedthat SUB resides at the plasma membrane (PM) (Kwak andSchiefelbein, 2008; Yadav et al., 2008). In the light of a possiblePD localization of QKY, we re-examined the subcellular signaldistribution of a functional SUB:EGFP reporter driven by theendogenous promoter ( pSUB::SUB:EGFP, gSUB:EGFP), whoseexpression fully overlaps with the reported SUB expression inflowers and roots (Vaddepalli et al., 2011), using a HSD-equippedconfocal microscope. In addition to a visible PM localization, wealso detected punctate signal at the circumference of cells in alltissues tested (Fig. 2I-L,Q-T) (supplementary material Fig. S5).Interestingly, those spots colocalized with signals derived from ABstaining. In root epidermal cells, image analysis revealed that 88percent of the SUB:EGFP spots overlapped with AB punctae(Pearson’s correlation coefficient=0.76). These results indicatethat SUB:EGFP not only localizes to the PM but is also enrichedat PD.

To confirm that EGFP:QKY and SUB:EGFP are positioned atPD, we performed immunogold electron microscopy. Depending onthe section EGFP:QKY-derived signals were seen in either the neckregion or to central domain of a plasmodesma (Fig. 2U,V). Theresults reveal the presence of EGFP:QKY at PD and suggest a broaddistribution in individual PD. Likewise, SUB:EGFP signal was alsoseen in PD (Fig. 2W,X). Thus, EGFP:QKY and SUB:EGFP wereboth detected at PD by immunogold microscopy.

QKY does not affect the subcellular localization ofSUB:EGFPThe data so far presented further strengthen a functional connectionbetween SUB and QKY. Both genes are components of SUBsignaling that contribute to the formation or relay of a non-cell-autonomous mobile signal and the two proteins localize to PD.Next, we investigated at what level SUB and QKY interact. The twogenes do not appear to regulate each other at the transcriptional level(Fulton et al., 2009) (supplementary material Fig. S6). Thus,we tested whether QKY affects the subcellular localization ofSUB:EGFP. We generated 42 gSUB:EGFP qky-8 transgenic linesand showed normal tissue-specific and PM localization of reportersignal in roots and young ovules (Fig. 3A-F). We also examined the

Table 1. Number of periclinal cell divisions in the L2 layer of stage 3floral meristems

Genotype NPCD* Percentage NFM‡

Ler 3 7.3 41qky-8 19 55.9 34ProML1:EGFP:QKY qky-8 3 8.8 34ProWUS:EGFP:QKY qky-8 5 14.7 34

*Number of periclinal cell divisions observed‡Number of floral meristems sampled

4141

RESEARCH ARTICLE Development (2014) 141, 4139-4148 doi:10.1242/dev.113878

DEVELO

PM

ENT

subcellular SUB:EGFP signal in root epidermal cells and confirmedan unaltered punctate pattern (Fig. 3G,H). QKY is therefore notrequired for localizing SUB:EGFP to the PM or PD.

SUB is not an upstream regulator of QKY during SUB-dependent signalingDuring the course of investigating gSUB:EGFP expression in qky-8,we noticed an interesting effect of the gSUB:EGFP transgene on theqky-8 phenotype. Thirty seven out of 42 of the gSUB:EGFP qky-8T1 plants exhibited a mild but reproducible reduction of the qky-8mutant phenotype. Increasing transgene copy number by generatingT2 lines slightly improved this effect, although the qky-8 phenotypewas never fully rescued (Fig. 3I-K). Only 5/42 lines showed norescue. Furthermore, expression of the nonfunctional reportergSUBR599C:EGFP, which carries a mutated SUB kinase domain(Vaddepalli et al., 2011), ablated the observed rescue of the qky-8phenotype (0/18 T1 lines) (Fig. 3L). These genetic results imply thatQKY acts upstream of SUB or that QKY and SUB act in parallel on acommon downstream target (see also below).

SUB does not influence plasmodesmatal localization ofEGFP:QKYOn the basis of the previous findings, one would expect that SUBdoes not affect the subcellular localization of QKY. Indeed, nochanges were detected in the EGFP:QKY signal in roots or carpelwalls of sub-1mutants (Fig. 4A-C) (12 transgenic lines). Moreover,the EGFP:QKY transgene did not rescue the sub-1 phenotype(Fig. 4D-F). These results suggest that the subcellular localization ofEGFP:QKY occurs independently of SUB.

Targeting of SUB:EGFP to PD requires the extracellulardomain in combination with the transmembrane domainA number of functionally characterized RLKs have been reported tobe present at PD, including CRINKLY 4 (CR4) (Tian et al., 2007),ARABIDOPSIS CRINKLY 4 (ACR4) and CLAVATA 1 (CLV1)(Stahl et al., 2013), FLAGELLIN-SENSITIVE 2 (FLS2) (Faulkneret al., 2013) and SUB (this study). In addition, RLKs are frequentlyfound in catalogues of PD proteomes of Arabidopsis and rice(Fernandez-Calvino et al., 2011; Jo et al., 2011). It is unknown,

Fig. 2. Subcellular localization of QKY and SUB. (A-G,I-K,M-O,Q-S) Confocal micrographs of 6-day qky-8 pQKY::EGFP:QKY roots. There is punctate signal distribution along thecircumference of individual cells. (A) Overview of root tip.(B) Overlay with differential interference contrast (DIC)channel. (C,D) Similar to A,B but at higher magnification.(E-G,M-O) Confocal micrographs of qky-8 EGFP:QKY stainedwith Aniline Blue. (I-K,Q-S) Confocal micrographs of sub-1gSUB:EGFP stained with Aniline Blue. (E,I,M,Q) GFP signal.(F,J,N,R) Aniline Blue signal. (G,K,O,S) Overlay of Aniline BlueandGFP channels. (H,L,P,T) Intensity profiles measured alonga line connecting the dots highlighted by the red rectangle inG,K,O,S, respectively. The x-axis marks the distance in μm.The y-axis denotes arbitrary intensity units. (U-X) Immunogoldelectron micrographs. The reporters are indicated.Subepidermal cortical cells in the flank of the root just behindthe meristem of 7-day seedlings are depicted. Arrowheadsindicate signals at plasmodesmata. The asterisk indicates thecell wall. (U) Signal is seen at the neck region of aplasmodesma. (V) Signal is detected in a more central regionof a plasmodesma. (W) Signal can also be seen atmultivesicular bodies (black arrows) and plasma membrane(blue arrow). (X) Signal is detected at plasmodesmata. Scalebars: 5 μm in A-G,I-K,M-O,Q-S; 0.1 μm in U-X.

4142

RESEARCH ARTICLE Development (2014) 141, 4139-4148 doi:10.1242/dev.113878

DEVELO

PM

ENT

however, how RLKs are targeted to PD. To obtain insights into themechanism, we sought to identify domains of SUB that are involvedin this process. We investigated the subcellular localization of a setof mutant cDNA-based SUB:EGFP reporters (cSUBdel:EGFP) instably transformed plants (Fig. 5). All the tested mutations result inthe absence of SUB activity (Vaddepalli et al., 2011). We observednormal signal distribution in lines expressing reporters lackingeither the kinase domain or the entire intracellular domain of SUB(Fig. 5A,B). This result indicates that the intracellular domain isdispensable for PD localization of SUB. In lines carrying a reporter

lacking the extracellular domain (ECD) of SUB, but including thetransmembrane domain (TMD) (cSUBΔECD:EGFP), we observeda stronger signal around the circumference of the cell. However, nopunctate spots were detected. Furthermore, aweak signal distributedin an endoplasmic reticulum (ER)-like pattern was detected(Fig. 5C). The result is compatible with plasma membrane (PM)localization of the reporter and suggests that the ECD is required forlocalization of SUB to PD. It further indicates that absence of theECD renders transport through the secretory pathway inefficient. Totest whether the ECD is sufficient to target SUB to PD we analyzedthe signal distribution of a reporter carrying only the ECD fused toEGFP (cSUBΔTM-Intra:EGFP) (Fig. 5D). We observed aggregatesinside the cell, indicating that the ECD is insufficient for passagethrough the secretory pathway. The targeting of SUB to PD thusrequires the ECD in combination with the TMD.

Identification of domains targeting EGFP:QKY to PDThe QKY homolog FTIP1 was reported to reside at PD of phloemcells, where it interacts with the long-day flowering signalFLOWERING LOCUS T (FT) (Liu et al., 2012). Moreover, anadditional five QKY family members have been identified in theArabidopsis PD proteome (Fernandez-Calvino et al., 2011). Thesefindings indicate that the QKY family represents a novel class ofPD-localized proteins. It is unknown how members of this familyget targeted to PD. We investigated whether specific domains ofQKY are required for this process. We generated stable transgenicqky-8 plants carrying a set of reporters encoding progressiveN-terminal deletions of QKY under the control of the endogenousQKY promoter ( pQKY::EGFP:QKYdel). None of the mutantreporters was able to rescue the qky-8 phenotype and the signalwas very weak (not shown). Thus, we used equivalent reportersdriven by the UBQ10 promoter and analyzed the correspondingsubcellular signal patterns in root epidermal cells (Fig. 6).

We observed normal punctate subcellular signal in lines carryingthe EGFP:QKYΔC2A, EGFP:QKYΔC2AL and EGFP:QKYΔA-Breporters (Fig. 6A-C). However, a faint ER-like pattern also became

Fig. 3. Distribution of SUB:EGFP reporter signal in qky-8.(A-H) Longitudinal confocal sections. (A,C,E,F) GFP-basedsignal. (B,D) Overlay with FM4-64-based counterstain.(A-D) Root tips of 6-day seedlings. (E) Stage 2-III ovule.(F) Stage 2-II ovule. The qky-8 and Ler exhibitindistinguishable reporter signals in roots and ovules.(G,H) Root epidermis of a 6-day qky-8 gSUB:EGFP seedling.(G) A punctate pattern is present. (H) Overlay with DICchannel. (I-L) Phenotype of qky-8 plants carrying two copies ofa gSUB:EGFP transgene. Morphology of flowers (upperpanel), stems (central panel) and siliques (bottom panel).(K) Milder phenotype in comparison with J. (L) A qky-8 plantcarrying two copies of amutant gSUBR599C:EGFP transgeneshows no rescue. Scale bars: 10 μm in A-F; 5 μm in G,H;0.5 mm in I-L.

Fig. 4. Localization of EGFP:QKY reporter signal in sub-1. (A-C) Confocalmicrographs revealing EGFP:QKY reporter signal. Signal appears normalacross genotypes and tissues. (A) Root epidermis of a 6-day seedling.(B,C) Carpel wall epidermis of a stage 13 flower. (D-F) Phenotype of sub-1plants carrying EGFP:QKY transgene. Morphology of flowers (upper panel),stems (central panel) and siliques (bottom panel). (F) Stronger sub-1phenotype compared with E. Scale bars: 10 μm in A-C; 0.5 mm in D-F.

4143

RESEARCH ARTICLE Development (2014) 141, 4139-4148 doi:10.1242/dev.113878

DEVELO

PM

ENT

apparent. Those fusion proteins feature progressive deletions of theN-terminal C2 domains (Fig. 6H). Interestingly, in lines carryingthe EGFP:QKYΔC2A-C or EGFP:QKYΔC2A-D fusion proteins,we could still discern a PM-like staining but no punctate pattern(Fig. 6D,E). The ER-like pattern became even more obvious. Theresults indicate that the C2C, and possibly the C2D, domains areimportant for targeting EGFP:QKY to PD. By contrast, theN-terminal region up to and including the C2B domain is notessential for this process. Moreover, the C2 domains appearimportant for efficient transport of QKY to the PM. The resultsfurther suggest that the PRT_C domain is necessary and sufficientfor PM localization. To test this idea, we analyzed lines expressing aEGFP:QKYΔPRT_C reporter that lacks the PRT_C domain. Weobserved both ER-like and cytoplasmic signal distribution, and areduced PM localization (Fig. 6F). This result suggests that thePRT_C domain is necessary for PM localization of EGFP:QKY.The ER-like signal accumulation further indicates that, in theabsence of the PRT_C domain, the remaining fusion protein canassociate with the ER. Finally, we detected a weak and diffusecytoplasmic signal for gEGFP:QKY-L (Fig. 6G). In summary, thecorrect targeting of QKY to PD requires the C2C and/or C2Ddomains in combination with the PRT_C domain.

SUB:EGFP and mCherry:QKY interact physically in plantaSo far the data suggest specific localization of EGFP:QKY to PD,whereas SUB:EGFP is found both at the PM and PD. To testwhether QKY and SUB colocalize to the same PD, we analyzed thesignal distribution in transgenic lines carrying a functional pQKY::mCherry:QKY (mCherry:QKY) reporter (32/35 pQKY::mCherry:QKY qky-8 transgenic lines showed rescue, supplementary materialFig. S3), as well as the gSUB:EGFP construct. We detected signaloverlap in a punctate pattern (Fig. 7A-C), indicating that SUB:EGFP and mCherry:QKY are present at the same PD. This findingraised the possibility that the two proteins are in close contact.To test whether SUB and QKY proteins can interact directly,

we performed a yeast two-hybrid (Y2H) assay using the

C2-domain-containing region of QKY (QKYΔPRT_C, lackingthe PRT_C domain) as bait and either the ECD or the intracellulardomain (ICD) of SUB as prey. On selective medium, yeast growthwas observed when QKYΔPRT_C was combined with the ICD ofSUB (Fig. 7D), demonstrating that QKY can interact with theICD of SUB in this assay, extending previous results (Trehin et al.,2013).

To investigatewhether SUB:EGFPandmCherry:QKYcan interactphysically in planta, we performed Förster Resonance EnergyTransfer – Fluorescence Lifetime Imaging Microscopy (FRET-FLIM) assays using root epidermis cells of 7-day Arabidopsisseedlings stably transformed with the gSUB:EGFP reporter or withgSUB:EGFP and functional pUBQ::mCherry:QKY (mCherry:QKY)reporter constructs (38/40 pUBQ::mCherry:QKY qky-8 transgeniclines showed rescue). Using a pulsed multi-photon laser, FLIMallows determination of the fluorescence lifetime (τ) of the donorfluorophore in a time-resolved manner. In this intensity-independentassay, an interaction between two proteins (distance <10 nm) ischaracterized by a reduction in the lifetime of the donor fluorophore(EGFP) owing to an energy transfer to the acceptor (mCherry). Itshould be noted that the lifetime of the donor greatly depends on thecellular environment and compartment (Nakabayashi et al., 2008; vanManen et al., 2008).

To restrict measurements to plasma membrane-derived fluorescentsignals, we first measured mean fluorescence lifetime values alongthe plasma membranes of multiple root epidermal cells anddetermined a mean τ value of 2263.6±9.1 ps (mean±s.e.m., n=41)in plants that only express SUB:EGFP under control of its nativepromoter. In the presence of mCherry:QKY, an overall τ value of2243.3±6.8 ps (n=42) was found, revealing no significant reductionin fluorescence life time of SUB:EGFP (Student’s t-test, P=0.04;FRET efficiency: 0.9%). However, inspection of the correspondingFLIM CLSM images clearly suggested a non-uniform reduction influorescence lifetime restricted to specific punctae (Fig. 7E). Thesepunctae colocalized with the brighter spots labeled by SUB:EGFPand therefore likely represent PD (Fig. 7F). We re-analyzed the

Fig. 5. Subcellular localization ofmutant cSUB:EGFP reporter signals.(A-E) Confocal micrographs of 6-day sub-1cSUB:EGFPmut roots (left panel) and overlaywith DIC channel (right panel). Mutant reporterconstructs are indicated. Signal is revealed instele cells as this cDNA-based reporter ismoststrongly expressed in this tissue (Vaddepalliet al., 2011). (A,B) There is a punctate patternat the PM. (C) Stronger signal is distributedaround the circumference of cells. Weakersignal is dispersed in an endoplasmicreticulum (ER)-like pattern. (D) Intracellularthread-like signal. (E) Spotty signal pattern incytoplasm. (F) Cartoon depicting the differentdeletions in cSUB:EGFPmut fusion proteins(for details, see Vaddepalli et al., 2011).Abbreviations: CD, catalytic/kinase domain;ECD, extracellular domain; Intra, intracellulardomain; LRR, leucine-rich repeats, PRR,proline-rich region; SP, signal peptide; TMD,transmembrane domain. Scale bars: 5 μm.

4144

RESEARCH ARTICLE Development (2014) 141, 4139-4148 doi:10.1242/dev.113878

DEVELO

PM

ENT

fluorescence lifetime of SUB:EGFP within these spots. Wedetermined a τ value of 2123.8±6.05 ps (n=205), indicating that thefluorescence lifetime of SUB:EGFP alone in these spots was differentcompared with the overall fluorescence lifetime (P=6.8e−20).Importantly, however, in the presence of mCherry:QKY, wemeasured a τ value of 2075.5±4.98 ps (n=210). Thus, the PD-confined fluorescence lifetime of SUB:EGFP was reduced in astatistically significant manner (P=7.9e−10), revealing a FRETefficiency of 2.3% when compared with 0.9% in the overall plasmamembrane control. Taken together, the data indicate that SUB:EGFPand mCherry:QKY interact in epidermal cells of intact Arabidopsisroots and that the interaction occurs at PD.

DISCUSSIONThe available data support a functional connection betweenSUB and QKY. How do SUB and QKY relate in the context ofSUB signaling? Increasing the copy number of SUB partiallybypasses the requirement for QKY, indicating that QKY actsupstream or in parallel to SUB. The result further suggests thatQKY enables SUB function in a more quantitative rather than astrictly qualitative manner. Transcription of the two genes isregulated independently of each other, as is the subcellularlocalization of SUB and QKY. Our data further indicate that thetwo proteins are in close contact in vivo. Thus, one way toexplain the genetic observation is that a physical interactionbetween QKY and SUB renders SUB more active. In the absenceof QKY function, this condition can be partially circumventedby increasing SUB dose.

Our data suggest that QKY and SUB colocalize to and physicallyinteract at PD. Although the presence of SUB is not constrained toPD, localization of QKY appears to be restricted to PD.With respectto the subcellular localization of QKY, our results differ from arecent study, that reported QKY to be present at the PM (Trehinet al., 2013). Trehin et al., however, used a nonfunctional reportercarrying EGFP at the C terminus of QKY. Here, we fused EGFP tothe N terminus of QKY, which resulted in a functional EGFP:QKYreporter. This discrepancy suggests that a C-terminal fusion ofEGFP to QKY disrupts QKY function and impairs PD localization.It indicates that PD localization of QKY is essential for its function.Our results are also in agreement with findings of a recent proteomicstudy, which identified QKY and SUB as components of PDs(Fernandez-Calvino et al., 2011).

How PD-localized RLKs are targeted to PD is unknown. Ourresults indicate that the ECD, in combination with the TMD, issufficient to target SUB to PD. They differ from findings involvingPLASMODESMATA-LOCATED PROTEIN 1a (PDLP1a) adifferent type I membrane protein that specifically localizes to PD(Thomas et al., 2008). In the case of PDLP1a, the TMD wassufficient to target the protein to PD (Thomas et al., 2008). Theseresults indicate that targeting different types of type I membraneproteins to PD involves different mechanisms. Regarding SUB, ourfindings are in accordance with at least two models. In one scenario,the TMD anchors the SUB RLK to the plasma membrane (PM)while the ECD, through interactions with as yet unknownPD-targeting factors, is instrumental for enriching SUB at PD.Alternatively, PD localization of SUB requires interaction of

Fig. 6. Subcellular localization of mutantEGFP:QKY reporter signal. Confocalmicrographs of qky-8 EGFP:QKYmut roots(left panel) and overlay with DIC channel (rightpanel). Mutant reporter constructs areindicated. (A-C) There is a punctate pattern atthe PM. (D,E) Signal shows a PM-like and anER-like pattern. (F) Signal exhibits an ER-likepattern. PM-like localization is less prominentin comparison with A-E. (G) Signal showsdiffuse cytoplasmic distribution. (H) Cartoondepicting the different deletions in EGFP:QKYmut fusion proteins. Scale bars: 5 μm.

4145

RESEARCH ARTICLE Development (2014) 141, 4139-4148 doi:10.1242/dev.113878

DEVELO

PM

ENT

PD-targeting factors with both the ECD and TMD domains. QKY islikely not among those factors, as our genetic results revealed thatSUB localizes to PD independently of QKY. In future studies, it willbe interesting to identify the PD-targeting factors involved inpositioning SUB at PD.The mechanism that targets QKY family members to PD is also

unknown. With respect to QKY this study could not assign the roleof PD-targeting to any one domain. Rather, like for SUB, a more-complex scenario is implicated. Thus, the PRT_C domain, whichincludes the putative TMDs, may first anchor QKY to the PM, andinteraction of a PD-targeting factor with either C2C alone or acombination of C2C and C2D results in localizing QKY to PD.Alternatively, the hypothetical PD-targeting factor must interactwith both, PRT_C and the C2C/C2D domains. The latter mayexplain the PM localization observed for a nonfunctional reportercarrying a fusion of EGFP to the C terminus of PRT_C(QKY:EGFP) (Trehin et al., 2013).The non-cell autonomy of SUB and QKY indicates that both

genes function in the production and/or propagation of an unknownSUB-dependent mobile signal (SMS). It is curious that the twogenes act non-autonomously, although they are broadly expressed intissues, such as floral meristems or ovule primordia. There isprecedence for this observation, however, because BRI1 and LFY(for example) also function in a non-autonomous fashionnotwithstanding their broad expression (Sessions et al., 2000; Wuet al., 2003; Savaldi-Goldstein et al., 2007). Regarding SUBsignaling, one possible explanation is that SUB andQKYmay exert ahomeostatic function, ensuring distribution throughout the tissue ofimportant factors that potentially occur in very low amounts in a

given cell. This notion may also explain the lack of polarity inSUB/QKY non-cell autonomy.

Several scenarios could principally account for the presence ofSUB and QKY at PD. For example, the specific colocalization ofQKY and SUB at PD is compatible with the notion that SUBsignaling influences trafficking of molecules through PD, as wassuggested for other RLKs (Tian et al., 2007; Stahl et al., 2013).The non-cell autonomy of QKY and SUB indicates that SUBsignaling would support passage of the SMS signal through PD,rather than blocking it. In support of this, there is genetic evidencethat the QKY family member FTIP1 promotes movement of FTthrough PD (Liu et al., 2012). The nature of the proposed SMSsignal is unknown. The transcription factors CAPRICE (CPC) andGLABRA3 (GL3) would be obvious candidates, as the twoproteins are known to travel laterally between root epidermal cellsduring root hair patterning (Wada et al., 2002; Bernhardt et al.,2005; Kurata et al., 2005; Savage et al., 2008). However, SUBdoes not affect the movement and function of CPC or GL3(Kwak and Schiefelbein, 2007). SUB signaling is unlikely torender PD generally more penetrable, as movement of GFP,known to pass through PD by diffusion (Oparka et al., 1999;Crawford and Zambryski, 2000), is not altered in sub or qkymutants (supplementary material Figs S7-S9). Thus, if thishypothesis is valid, SUB signaling must promote selectivepassage of a factor yet to be identified. Alternatively, PD couldsimply provide convenient structural platforms for functionalclustering of SUB and QKY, as the PM within PD exhibits specialcharacteristics, similar to membrane rafts (Maule et al., 2011;Tilsner et al., 2011). In this view, the microenvironment at PDwould foster physical interaction between SUB and QKY,resulting in the subsequent initiation of further downstreamprocesses. One conceivable response could comprise secretion ofthe SMS factor to neighboring cells via vesicular transport andpassage across the cell wall. Another feasible response couldinvolve alteration of cell wall mechanics (Fulton et al., 2009). Itwill thus be interesting to explore the exact mechanism by whichSUB signaling orchestrates tissue morphogenesis in future studies.

MATERIALS AND METHODSPlant workArabidopsis thaliana (L.) Heynh. var. Columbia (Col-0) and var. Landsberg(erecta mutant) (Ler) were used as wild-type strains. Plants were grownessentially as described previously (Fulton et al., 2009). The EMS-inducedsub-1 and qky-8 mutations (Ler) and the T-DNA induced qky-11(SALK_043901, Col) mutations have been described previously(Chevalier et al., 2005; Fulton et al., 2009). Wild-type, sub-1 and qky-8plants were transformed with different constructs using Agrobacteriumstrain GV3101/pMP90 (Koncz and Schell, 1986) and the floral dip method(Clough and Bent, 1998). Transgenic T1 plants were selected on eitherkanamycin (50 μg/ml) or hygromycin (20 μg/ml) plates and transferred tosoil for further inspection.

Recombinant DNA technologyFor DNA and RNA work, standard molecular biology techniques wereused. PCR fragments used for cloning were obtained using Phusion high-fidelity DNA polymerase (New England Biolabs, Frankfurt, Germany) orTaKaRa PrimeSTAR HS DNA polymerase (Lonza, Basel, Switzerland).PCR fragments were subcloned into pJET1.2 using the CloneJET PCRcloning kit (Fermentas) or into pCRII-TOPO (Invitrogen). All PCR-basedconstructs were sequenced. The plasmid pCAMBIA2300 (www.cambia.org) and the Gateway-based (Invitrogen) destination vector pMDC32(Curtis and Grossniklaus, 2003) were used as binary vectors. Primersequences used in this work are listed in supporting supplementarymaterial Table S1.

Fig. 7. Interaction between SUB and QKY. (A-C) Confocal micrographs of6-day root epidermis cells of plants transgenic for both gSUB:EGFP andpQKY::mCherry:QKY reporters. (A) gSUB:EGFP signal. (B) pQKY::mCherry:QKY signal. (C) Overlay of A and B. Punctate signals overlap. (D) Yeast two-hybrid assay involving a QKY variant, including all four C2 domains but lackingthe PRT_C domain (QKYΔPRT_C) fused to the GAL4 activating domain(GAD) and the extracellular domain (ECD) or intracellular domain (ICD) of SUBfused to the GAL4 DNA-binding domain (GBD), respectively. Growth on –LWpanel indicates successful transformation of both plasmids and on –LWHpanel indicates presence or absence of interaction. (E,F) FRET-FLIM analysisin root epidermal cells of Arabidopsis plants stably transformed with SUB:EGFPandmCherry:QKY reporters. (E) Fluorescence lifetime image. Color bardenotes the false color code for SUB:EGFP fluorescence lifetimes (τ).(F) Confocal micrograph depicting SUB:EGFP signal. Scale bars: 5 μm.

4146

RESEARCH ARTICLE Development (2014) 141, 4139-4148 doi:10.1242/dev.113878

DEVELO

PM

ENT

Reporter constructsFor plasmid pQKY::GUS 1.2 kb of promoter sequence spanning genomicDNA up to the 3′ end of the next gene was amplified from Ler genomicDNA using primers Kpn1_Qky::_F/Qky::_Asc1_R and cloned intopCAMBIA2300. The GUS-coding sequence was amplified frompCAMBIA 2301 using primers Asc1_Gus-plus_F/Gus-plus_spe1_R andcloned behind the QKY promoter sequence. All SUB:EGFP reporters havebeen described previously (Vaddepalli et al., 2011). To obtain the EGFP:QKY reporter (pUBQ10::EGFP:QKY), a 2 kb promoter fragment ofUBQ10 (At4g05320) (up to the 3′ end of the next gene) and the QKY(At1g74720) coding sequence were amplified from Ler genomic DNAusing primers P-UBQ (Kpn1)_For/P-UBQ (Asc1)_rev and Ala_Qky_F/Qky_Xba1_R, respectively. The EGFP-coding sequence was amplifiedfrom plasmid gSUB:EGFP. EGFP and QKY sequences were fusedby overlap PCR and cloned into pCAMBIA 2300 followed by inclusionof the UBQ10 promoter fragment. The pWUS::EGFP:QKY andpML1::EGFP:QKY reporters were obtained via Gateway cloning(Invitrogen). The EGFP:QKY fragment was amplified from pUBQ10::EGFP:QKY and cloned into pDONR207. Subsequently, the EGFP:QKYfragment was subcloned into destination vector pMDC32 containingthe pWUS [5.6 kb, obtained by AscI/KpnI digestion from plasmidpWUS::SUB:3xmyc (Yadav et al., 2008)] and pML1 [3.4 kb, amplifiedfrom plasmid pML1::SUB:EGFP (Yadav et al., 2008) using primersAtML1proHindIIIfor/AtML1proHindIIIrev] promoter sequences. Togenerate deletion constructs of QKY, different fragments of QKY wereamplified with the following primers: C2A/Del_F, C2AB/Del_F, C2B/ala/spe1_F, C2ABC/Del_F, C2ABCD/Del_F and qky/Pst1/R. Amplified PCRproducts were digested with SpeI/PstI and subcloned into ProUBQ10:EGFP:QKY. For EGFP:QKY-L, the linker fragment was amplified withQKY linker/Spe1_F, QKY linker_R and the 3′UTR was amplified withQKY 3′UTR_F and QKY/Pst1/R. Fragments were fused by overlappingPCR using primers QKY linker/Spe1_F and QKY/Pst1/R and cloned intoProUBQ10:EGFP:QKY. All clones were verified by sequencing.

Yeast two-hybrid assaypGBKT7 plasmids (Matchmaker, Clontech) containing either the SUBextracellular (ECD) or intracellular (ICD) domain (Bai et al., 2013) wereco-transformed with pGADT7-QKYΔPRT_C plasmid [obtained by PCR-based cloning using primers Nde1/QKY(-TM)_F and QKY(-TM)Xma1_R]into the yeast strain AH109. After 3 days, transformants were selected onsynthetic complete (SC) medium lacking leucine and tryptophan (-LW) at30°C. To examine Y2H interactions, the transformants were grown on solidSCmedium lacking leucine and tryptophan (SC-LW) or leucine, tryptophanand histidine (-LWH), and supplemented by 2 mM 3-amino-1,2,4-triazole(3-AT) for 2 days at 30°C.

Microscopy, in situ hybridization and art workConfocal laser scanning microscopy using staining with Aniline Blue orwith pseudo-Schiff propidium iodide (mPS-PI), or detection of EGFP andFM4-64 was performed primarily as described (Schneitz et al., 1997; Sagiet al., 2005; Truernit et al., 2008; Yadav et al., 2008; Fulton et al., 2009). Insome instances, confocal high sensitivity detection (HSD) was employedinvolving two gallium arsenide phosphide photomultipliers (GaAsP PMTs)mounted equidistantly to the probe. Colocalization studies were carried outusing the respective module of the confocal microscope software (Olympus,Fluoview 4.1a). Approximately 14 ROIs (rectangle selection tool, length of5-8 μm) were randomly placed across different image areas depictingplasma membrane regions. Histochemical localization of β-glucuronidase(GUS) activity in whole-mount tissue and scanning electron microscopywere performed as reported previously (Jefferson et al., 1986; Schneitzet al., 1997; Sieburth and Meyerowitz, 1997). High-pressure freezing andimmunogold electron microscopy have also been previously described(Scheuring et al., 2011; Hillmer et al., 2012). Immunogold labeling wasperformed as described previously (Scheuring et al., 2011) using a GFPantiserum at a dilution of 1:600 or 1:1000 in PBS. Sections were observed ina JEOL JEM1400 transmission electron microscope operating at 80 kV andimages were taken with a Fastscan-F214 CCD camera (TVIPS, Gauting,Germany). Contrast and brightness were adjusted in 16-bit images using the

software of the camera. Procedures for FRET-FLIM analysis andmeasurements were adapted from (Toth et al., 2012). FLIM wasperformed using a Leica SP5 confocal microscope with an integratedpulsed (80 MHz) Ti:Sapphire laser (Mai Tai) for multi-photon excitationand a fast FLIM photomultiplier (Becker & Hickl, Berlin, Germany). Two-photon excitation of the EGFP donor fluorophore was achieved at 900 nmand fluorescence was detected at 500-580 nm. Time correlated spectralcounting was performed for a maximum of 5 min and 64 scanning cycleswith a spatial resolution of 256×256 pixel. Fluorophore lifetimes at theplasma membranes were determined using SPCImage software (Becker &Hickl) and image-adapted settings. A total of 40 images from a minimum of12 independent roots were acquired and evaluated. For PD-confined lifetimeanalysis, five individual spots were analyzed per image, resulting in over200 measurements per genotype. Data were statistically analyzed usingStudent’s t-test. In situ hybridization with digoxigenin-labeled probes havebeen described previously (Sieber et al., 2004). To generate the QKY probe,a 2.1 kb fragment, spanning the 5′ part of the coding sequence, wasamplified from genomic Ler DNA using primers Pst1_Qky_F/Qky-TM_Kpn1_R and cloned into pBluescript SK (Agilent) and pRSET-C(Invitrogen) to yield pSK:QKY (antisense probe) and pRSET-C:QKY(sense control), respectively. Slides were viewed with an Olympus BX61upright microscope using DIC optics. Images were adjusted for color andcontrast using Adobe Photoshop CS5 (Adobe) software.

AcknowledgementsWe thank Keiko Torii for the pKUT196 construct, Xuelin Wu andDetlef Weigel for thepML1::2xGFP and pML1::NLS:GFP plasmids, Christine Faulkner and AndrewMaule for the pPDLP3::PDLP3:GFP construct, and Ruth Stadler for the pSUC2::GFP and pSUC2::tmGFP9 plasmids. We further acknowledge Ruth Stadler forhelpful comments on themanuscript andmembers of the Schneitz lab for stimulatingdiscussions.

Competing interestsThe authors declare no competing financial interests.

Author contributionsP.V., U.Z.H., T.O., D.G.R. and K.S. designed the research. P.V., A.H., L.F., M.O.,T.F.S., A.F. and S.H. performed research. P.V., A.H., S.H., U.Z.H., T.O, D.G.R. andK.S. analyzed the data. K.S. wrote the paper.

FundingThis work was funded the German Research Council (DFG) [SCHN 723/6-1 andSFB924 (TPA2) to K.S., SFB924 (TPB4) to T.O. and SFB924 (TPA8) to U.Z.H.] andby the Free State of Bavaria.

Supplementary materialSupplementary material available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.113878/-/DC1

ReferencesBai, Y., Vaddepalli, P., Fulton, L., Bhasin, H., Hulskamp, M. and Schneitz, K.

(2013). ANGUSTIFOLIA is a central component of tissue morphogenesismediated by the atypical receptor-like kinase STRUBBELIG. BMC Plant Biol.13, 16.

Baurle, I. and Laux, T. (2005). Regulation of WUSCHEL transcription in the stemcell niche of the Arabidopsis shoot meristem. Plant Cell 17, 2271-2280.

Bell, K. and Oparka, K. (2011). Imaging plasmodesmata. Protoplasma 248, 9-25.Bernhardt, C., Zhao, M., Gonzalez, A., Lloyd, A. and Schiefelbein, J. (2005). The

bHLH genes GL3 and EGL3 participate in an intercellular regulatory circuit thatcontrols cell patterning in the Arabidopsis root epidermis. Development 132,291-298.

Chevalier, D., Batoux, M., Fulton, L., Pfister, K., Yadav, R. K., Schellenberg, M.and Schneitz, K. (2005). STRUBBELIG defines a receptor kinase-mediatedsignaling pathway regulating organ development in Arabidopsis. Proc. Natl. Acad.Sci. USA 102, 9074-9079.

Clough, S. J. and Bent, A. F. (1998). Floral dip: a simplified method forAgrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16,735-743.

Crawford, K. M. and Zambryski, P. C. (2000). Subcellular localization determinesthe availability of non-targeted proteins to plasmodesmatal transport. Curr. Biol.10, 1032-1040.

Curtis, M. D. and Grossniklaus, U. (2003). A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol. 133, 462-469.

4147

RESEARCH ARTICLE Development (2014) 141, 4139-4148 doi:10.1242/dev.113878

DEVELO

PM

ENT

Faulkner, C., Petutschnig, E., Benitez-Alfonso, Y., Beck, M., Robatzek, S.,Lipka, V. and Maule, A. J. (2013). LYM2-dependent chitin perception limitsmolecular flux via plasmodesmata. Proc. Natl. Acad. Sci. USA 110, 9166-9170.

Fernandez-Calvino, L., Faulkner, C., Walshaw, J., Saalbach, G., Bayer, E.,Benitez-Alfonso, Y. and Maule, A. (2011). Arabidopsis plasmodesmalproteome. PLoS ONE 6, e18880.

Fulton, L., Batoux, M., Vaddepalli, P., Yadav, R. K., Busch, W., Andersen, S. U.,Jeong, S., Lohmann, J. U. and Schneitz, K. (2009). DETORQUEO, QUIRKY,and ZERZAUST represent novel components involved in organ developmentmediated by the receptor-like kinase STRUBBELIG in Arabidopsis thaliana.PLoSGenet. 5, e1000355.

Gross-Hardt, R., Lenhard, M. and Laux, T. (2002).WUSCHEL signaling functionsin interregional communication during Arabidopsis ovule development. GenesDev. 16, 1129-1138.

Hillmer, S., Viotti, C. and Robinson, D. G. (2012). An improved procedure for low-temperature embedding of high-pressure frozen and freeze-substituted planttissues resulting in excellent structural preservation and contrast. J. Microsc. 247,43-47.

Jefferson, R. A., Burgess, S. M. and Hirsh, D. (1986). beta-Glucuronidase fromEscherichia coli as a gene-fusion marker. Proc. Natl. Acad. Sci. USA 83,8447-8451.

Jo, Y., Cho, W. K., Rim, Y., Moon, J., Chen, X.-Y., Chu, H., Kim, C. Y., Park, Z.-Y.,Lucas, W. J. and Kim, J.-Y. (2011). Plasmodesmal receptor-like kinasesidentified through analysis of rice cell wall extracted proteins. Protoplasma 248,191-203.

Koncz, C. and Schell, J. (1986). The promoter of TL-DNA gene 5 controls thetissue-specific expression of chimaeric genes carried by a novel type ofAgrobacterium binary vector. Mol. Gen. Genet. 204, 383-396.

Kurata, T., Ishida, T., Kawabata-Awai, C., Noguchi, M., Hattori, S., Sano, R.,Nagasaka, R., Tominaga, R., Koshino-Kimura, Y., Kato, T. et al. (2005). Cell-to-cell movement of the CAPRICE protein in Arabidopsis root epidermal celldifferentiation. Development 132, 5387-5398.

Kwak, S.-H. and Schiefelbein, J. (2007). The role of the SCRAMBLED receptor-like kinase in patterning the Arabidopsis root epidermis. Dev. Biol. 302, 118-131.

Kwak, S.-H. and Schiefelbein, J. (2008). A feedback mechanism controllingSCRAMBLED receptor accumulation and cell-type pattern in Arabidopsis. Curr.Biol. 18, 1949-1954.

Kwak, S.-H., Shen, R. and Schiefelbein, J. (2005). Positional signaling mediatedby a receptor-like kinase in Arabidopsis. Science 307, 1111-1113.

Lin, L., Zhong, S.-H., Cui, X.-F., Li, J. and He, Z.-H. (2012). Characterization oftemperature-sensitive mutants reveals a role for receptor-like kinaseSCRAMBLED/STRUBBELIG in coordinating cell proliferation and differentiationduring Arabidopsis leaf development. Plant J. 72, 707-720.

Liu, L., Liu, C., Hou, X., Xi, W., Shen, L., Tao, Z., Wang, Y. and Yu, H. (2012).FTIP1 is an essential regulator required for florigen transport. PLoS Biol. 10,e1001313.

Lu, P., Porat, R., Nadeau, J. A. and O’Neill, S. D. (1996). Identification of ameristem L1 layer-specific gene in Arabidopsis that is expressed duringembryonic pattern formation and defines a new class of homeobox genes.Plant Cell 8, 2155-2168.

Lucas, W. J., Ham, B.-K. and Kim, J.-Y. (2009). Plasmodesmata - bridging the gapbetween neighboring plant cells. Trends Cell Biol. 19, 495-503.

Maule, A. J., Benitez-Alfonso, Y. and Faulkner, C. (2011). Plasmodesmata -membrane tunnels with attitude. Curr. Opin. Plant Biol. 14, 683-690.

Mayer, K. F. X., Schoof, H., Haecker, A., Lenhard, M., Jurgens, G. and Laux, T.(1998). Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shootmeristem. Cell 95, 805-815.

Nakabayashi, T., Wang, H.-P., Kinjo, M. and Ohta, N. (2008). Application offluorescence lifetime imaging of enhanced green fluorescent protein tointracellular pH measurements. Photochem. Photobiol. Sci. 7, 668-670.

Oparka, K. J., Roberts, A. G., Boevink, P., Santa Cruz, S., Roberts, I., Pradel,K. S., Imlau, A., Kotlizky, G., Sauer, N. and Epel, B. (1999). Simple, but notbranched, plasmodesmata allow the nonspecific trafficking of proteins indeveloping tobacco leaves. Cell 97, 743-754.

Rizo, J. and Sudhof, T. C. (1998). C2-domains, structure and function of a universalCa2+-binding domain. J. Biol. Chem. 273, 15879-15882.

Sagi, G., Katz, A., Guenoune-Gelbart, D. and Epel, B. L. (2005). Class 1 reversiblyglycosylated polypeptides are plasmodesmal-associated proteins delivered toplasmodesmata via the Golgi apparatus. Plant Cell 17, 1788-1800.

Savage, N. S., Walker, T., Wieckowski, Y., Schiefelbein, J., Dolan, L. and Monk,N. A. M. (2008). A mutual support mechanism through intercellular movement of

CAPRICE and GLABRA3 can pattern the Arabidopsis root epidermis. PLoS Biol.6, e235.

Savaldi-Goldstein, S., Peto, C. and Chory, J. (2007). The epidermis both drivesand restricts plant shoot growth. Nature 446, 199-202.

Scheuring, D., Viotti, C., Kruger, F., Kunzl, F., Sturm, S., Bubeck, J., Hillmer, S.,Frigerio, L., Robinson, D. G., Pimpl, P. et al. (2011). Multivesicular bodiesmature from the trans-Golgi network/early endosome in Arabidopsis. Plant Cell23, 3463-3481.

Schneitz, K., Hulskamp, M. and Pruitt, R. E. (1995). Wild-type ovule developmentin Arabidopsis thaliana: a light microscope study of cleared whole-mount tissue.Plant J. 7, 731-749.

Schneitz, K., Hulskamp, M., Kopczak, S. D. and Pruitt, R. E. (1997). Dissection ofsexual organ ontogenesis: a genetic analysis of ovule development inArabidopsisthaliana. Development 124, 1367-1376.

Sessions, A., Weigel, D. and Yanofsky, M. F. (1999). The Arabidopsis thalianaMERISTEM LAYER 1 promoter specifies epidermal expression in meristems andyoung primordia. Plant J. 20, 259-263.

Sessions, A., Yanofsky, M. F. and Weigel, D. (2000). Cell-cell signaling andmovement by the floral transcription factors LEAFY and APETALA1. Science 289,779-781.

Sieber, P., Gheyeselinck, J., Gross-Hardt, R., Laux, T., Grossniklaus, U. andSchneitz, K. (2004). Pattern formation during early ovule development inArabidopsis thaliana. Dev. Biol. 273, 321-334.

Sieburth, L. E. and Meyerowitz, E. M. (1997). Molecular dissection of theAGAMOUS control region shows that cis elements for spatial regulation arelocated intragenically. Plant Cell 9, 355-365.

Smyth, D. R., Bowman, J. L. and Meyerowitz, E. M. (1990). Early flowerdevelopment in Arabidopsis. Plant Cell 2, 755-767.

Stahl, Y., Grabowski, S., Bleckmann, A., Kuhnemuth, R.,Weidtkamp-Peters, S.,Pinto, K. G., Kirschner, G. K., Schmid, J. B., Wink, R. H., Hulsewede, A. et al.(2013). Moderation of Arabidopsis root stemness by CLAVATA1 andARABIDOPSIS CRINKLY4 receptor kinase complexes. Curr. Biol. 23, 362-371.

Thomas, C. L., Bayer, E. M., Ritzenthaler, C., Fernandez-Calvino, L. and Maule,A. J. (2008). Specific targeting of a plasmodesmal protein affecting cell-to-cellcommunication. PLoS Biol. 6, e7.

Tian, Q., Olsen, L., Sun, B., Lid, S. E., Brown, R. C., Lemmon, B. E., Fosnes, K.,Gruis, D., Opsahl-Sorteberg, H.-G., Otegui, M. S. et al. (2007). Subcellularlocalization and functional domain studies of DEFECTIVE KERNEL1 in maizeand Arabidopsis suggest a model for aleurone cell fate specification involvingCRINKLY4 and SUPERNUMERARY ALEURONE LAYER1. Plant Cell 19,3127-3145.

Tilsner, J., Amari, K. and Torrance, L. (2011). Plasmodesmata viewed asspecialised membrane adhesion sites. Protoplasma 248, 39-60.

Toth, K., Stratil, T. F., Madsen, E. B., Ye, J., Popp, C., Antolin-Llovera, M.,Grossmann, C., Jensen, O. N., Schussler, A., Parniske, M. et al. (2012).Functional domain analysis of the Remorin protein LjSYMREM1 in Lotusjaponicus. PLoS ONE 7, e30817.

Trehin, C., Schrempp, S., Chauvet, A., Berne-Dedieu, A., Thierry, A.-M., Faure,J.-E., Negrutiu, I. and Morel, P. (2013). QUIRKY interacts with STRUBBELIGand PAL OF QUIRKY to regulate cell growth anisotropy during Arabidopsisgynoecium development. Development 140, 4807-4817.

Truernit, E., Bauby, H., Dubreucq, B., Grandjean, O., Runions, J., Barthelemy,J. and Palauqui, J.-C. (2008). High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of phloemdevelopment and structure in Arabidopsis. Plant Cell 20, 1494-1503.

Vaddepalli, P., Fulton, L., Batoux, M., Yadav, R. K. and Schneitz, K. (2011).Structure-function analysis of STRUBBELIG, an Arabidopsis atypical receptor-like kinase involved in tissue morphogenesis. PLoS ONE 6, e19730.

van Manen, H.-J., Verkuijlen, P., Wittendorp, P., Subramaniam, V., van denBerg, T. K., Roos, D. and Otto, C. (2008). Refractive index sensing of greenfluorescent proteins in living cells using fluorescence lifetime imaging microscopy.Biophys. J. 94, L67-L69.

Wada, T., Kurata, T., Tominaga, R., Koshino-Kimura, Y., Tachibana, T., Goto, K.,Marks, M. D., Shimura, Y. and Okada, K. (2002). Role of a positive regulator ofroot hair development, CAPRICE, in Arabidopsis root epidermal celldifferentiation. Development 129, 5409-5419.

Wu, X., Dinneny, J. R., Crawford, K. M., Rhee, Y., Citovsky, V., Zambryski, P. C.andWeigel, D. (2003). Modes of intercellular transcription factor movement in theArabidopsis apex. Development 130, 3735-3745.

Yadav, R. K., Fulton, L., Batoux, M. and Schneitz, K. (2008). The Arabidopsisreceptor-like kinase STRUBBELIGmediates inter-cell-layer signaling during floraldevelopment. Dev. Biol. 323, 261-270.

4148

RESEARCH ARTICLE Development (2014) 141, 4139-4148 doi:10.1242/dev.113878

DEVELO

PM

ENT