Cell-to-Cell Transport of Proteins and Fluorescent Tracers via Plasmodesmata during Plant Development Author(s): Patricia Zambryski Reviewed

work(s): Source: The Journal of Cell Biology, Vol. 164, No. 2 (Jan. 19, 2004), pp. 165-168 Published by: The Rockefeller University Press Stable URL: http://www.jstor.org/stable/1621931 . Accessed: 03/04/2012 04:05
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M~iii.eview

Cell-to-cell transport
tracers

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proteins and fluorescent plasmodesmata plant during development

of

Patricia Zambryski
of CA Departmentof Plantand MicrobialBiology,University California, Berkeley, 94720 Berkeley,

Plant cells communicatewith each other via channels called plasmodesmata (PD).PD are not passivechannels, but criticalplayersin gene regulation, controllingintercellulartransport macromolecules of between particular cells duringdevelopment.

Plantcellsareencasedin cellwallsthatformthe plantskeleton, enabling and stabilizingthree-dimensional growth. As plant cells physicallydo not touch, plantshaveevolvedcytoplasmic (PD), spanningcell walls and bridges,called plasmodesmata linking the cytoplasm between adjacent cells. Intercellular communication,as well as temporaland spatialregulationof gene expression,are global mechanismsto control developmental programming multicellular in organisms.Signalscan be transmittedvia receptor-ligandinteractionsin both plant and animalcells. However, PD provideplantswith a unique means of intercellular communication,where each plant cell PD functionduringvegetativedevelopment can form direct conduits to its neighbors,forming domains The vegetative phase is the most prolonged and easiest to of cells sharingcommon components. Historically,PD were access.After seed germination,plants continuouslyproduce seen as facilitating traffic of low molecular weight growth new organs,such as leavesand roots.A largeliterature docuregulators and nutrients, such as the ample products of ments PD function in mature leaf tissues using plant viral photosynthesis.Evidence for macromolecular transportvia and Beachy, 1999). Specificviral moveprobes (Lazarowitz PD was based largelyon piratingof these channelsby plant viruses during infection. Now there is abundant evidence ment proteins enable infectiousspread.As virusesand their proteinswere abovethe (then) predicted 1-kD size exclusion that PD transport endogenousmacromolecules. limit of PD, such studiesrevealedthat PD could function as GenericPD have two majorcomponents, membranesand and 2000; Robertsand Oparka, dilated channels. It was assumed that plant viruses were spaces(Zambryski Crawford, uniquely able to manipulatePD to achieve their ends. The 2003) (Fig. 1). Membranesform the boundariesof the PD advent of improved fluorescent tracers, and noninvasive channel through which transport may occur. The plasma means for their introduction,dramatically shifted this view, membrane (PM) between adjacent cells defines the outer an innate complexityof PD function. limit of PD. The axialcenterof the PD, the desmotubule(D), revealing Studieswith GFP and its fusions revealedthat proteinsof is appressedER. The region between the D and the PM is the cytoplasmicsleeve (CS), the majorconduit for molecular at least 50 kD could trafficthroughcells of the leaf, and the The CS likelycontainscomponents,perhaps anchored extent of such movement was highly dependent on leaf age passage. to the D and PM, that regulateand facilitatePD transport. (Oparkaet al., 1999). Youngerleavesexhibit more extensive cell-to-cell transportthan older leaves. Quantitativestudies Ultrastructural studies of PD revealactin and myosin along demonstrate that the leaf consists of different populations the length and centrinnanofilamentsat the neck region that of cells exhibiting no, low, or high efficiency of GFP Address correspondence to Patricia Zambryski, Department of Plant movement. Thus, there is no single size exclusion limit for and Microbial Biology, 111 Koshland Hall, University of California, leaf PD; insteadthe leaf containsa heterogeneousmixtureof
Berkeley,Berkeley,CA 94720. Tel.: (510) 643-9203. Fax:(510) 642-4995. email: zambrysk@nature.berkeley.edu Key words: transport; cell-to-cell transport; plasmodesmata; plant; development Abbreviationsused in this paper:Apl, Apetalal; CS, cytoplasmic sleeve; D, desmotubule;Lfy, Leafy;PD, plasmodesmata; PM, plasmamembrane; SHR, SHORT-ROOT.

may provide contractile elements to regulate PD aperture. To date, no unique PD componentshave been identified. In the last few years, there have been two major breakFirst,native (nonvirallyperturbed) throughsin PD research. PD are no longer consideredstatic but instead fluctuate in aperturein different cell types during development and in responseto the environment.Second, evidencethat PD can increaseddramatically. transport(nonviral)macromolecules Numerous reports demonstrate intercellularmovement of both exogenous proteins (such as GFP) and endogenous transcription factors (cited below) and RNAs, such as mRNA (Ruiz-Medranoet al., 1999; Kim et al., 2001), and gene silencing signals (Baulcombe, 1999; Voinnet, 2001). Recent studies on protein and fluorescenttracermovement during the three major stages of plant development are summarizedbelow.

@ The Rockefeller Press,0021-9525/2004/01/165/4 $8.00 University The Journalof Cell Biology,Volume 162, Number2, January 2004 165-168 19, http://www.jcb.org/cgi/doi/10.1083/jcb.200310048

165

166 The Journal of Cell Biology I Volume 162, Number 2, 2004

Figure 1. Diagram of longitudinal section of PD spanning the cell wall between adjacentcells. CR,centralrod; CW,cell wall;SP,spoke-likeconnections between the D and PMthat may control aperture.Blue and orange circles represent hypothetical PD-specific proteins. and Oparka,2003. Figurefrom Roberts
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cells where PD aperturesfluctuate according to changes in the environment(Crawfordand Zambryski,2001). There are two general types of protein movement, nontargeted and targeted (Fig. 2) (Crawfordand Zambryski, 2000). Nontargeted movement is exemplifiedby the diffusive patternof GFP movement following its expressionin a single cell; GFP is detected as a gradient from high in the initial cell to low in surroundingcells to which it has moved. GFP is not targetedto particular cellularsites, but ratherdisuniform fluorescencethroughoutthe cell. To date, tarplays geted movement is best illustratedby fusing GFP to proteins known to interactspecificallywith PD, such as viral movement proteins; such GFP fusions localize to punctate foci aroundcell perimetersthat correspondto PD. Obviouslyprotein cargosize and the apertureof PD phystraffic.Given these two criteically govern macromolecular ria are met, can all macromoleculesmove cell to cell like GFP? If such rampantexchange occurs, how are cells protected againstloss of cell-specificcomponents?While GFP is a soluble cytoplasmicprotein that can move freelyvia PD, GFP fusions targeted to the ER or anchored to actin filaments do not move cell to cell (Crawfordand Zambryski, 2000) (Fig. 3). Further,as the nucleus is a soluble compartment, GFP carryingan NLS localizes to the nucleus while retaining the ability to move between cells. Double-sized GFP carryingan NLS did not move cell to cell, presumably as nuclear-cytoplasmicshuttling is limited to proteins <40 kD (transcriptionfactors likely are retainedin the nucleus by binding to specific chromatin sites; Fig. 4). While nonbetween cells may be targetedmovement of macromolecules the default state, cells likely protect against loss by specifically anchoringor sequesteringproteinswithin the cell. The vascularsystem, immediatelyconnected to surrounding tissues by PD, provides an exceptionallyvaluable conduit for macromolecular dispersal.Elegantstudies to induce GFP expressionspecificallyin cells immediatelyabuttingthe vascularsystem revealthat GFP generallymoves toward regions of new growth, such as new leavesand newly emerging floral organs (Imlau et al., 1999). Such movement implies that endogenous macromolecular signalsmay trafficthe vascularhighwayto facilitatenew development. PD transportin other vegetativetissueshighlightsamazing The maizeKZNOTTED1 precisionto intercellular trafficking.

is in transcript expressed the innercell layersof the shoot meristem, but not the outermostcell layer.However,immunolocalizationstudies revealKN1 protein in outermostcells (Lucas et al., 1995). Recentstudieswith GFP-KN1 fusionsreveal directional traffic of KN1 in different tissues (Kim et al., 2003). SHORT-ROOT(SHR) mRNA and protein are expressedin the stele cells of the centralregion of the root tip. the Remarkably, Shr proteinmoves to a single file of adjacent cells (Nakajimaet al., 2001) (Fig. 4). While Shr has a diffuse cytoplasmiclocalizationin stele cells, it nuclearlocalizesafter to transport adjacentcells. This latterlocalizationthen allows Shr to induce expressionof a relatedgene (SCARECROW) that inducesdifferentiation two cell types. of

PD functionduringreproductivedevelopment
One of the major differencesbetween plants and animals is their reproductivestrategy.In animals, reproductiveorgans are set aside early,during embryogenesis. contrast,plants In matureto adultsbeforeproducingreproductive organs.Durroots and leaves are produced ing vegetative development, continuouslyfrom meristematic regions.The shoot meristem, at the plant apex, contains a dome of undifferentiated cells that give rise to new leaf primordiaoff its flanks.Late in life, in responseto developmentaland environmental signals,the adult plant reprograms this apical meristemto stop making leaves and instead make floral organs. This developmental switch resultsin a dramaticreorganization the meristem; of
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2. Figure Nontargetedand targetedcell-to-cellproteinmovement. Panel a exemplifies nontargetedmovement of GFP,and panel b shows targetedmovement,GFPfusedto a plantviralprotein.Figure fromZambryski Crawford, and 2000.

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Figure3. Subcellular localization determines the availability of proteins for PD transport. GFPfused to an ERretention signal (a), the actin filament binding domain of talin (b), or an NLS(c). (d) Double-sized GFPfused to an NLS.Figurefrom Crawfordand 2000. Zambryski,

4. Shrproteinlocalizationin Arabidopsis roots.Transgenic Figure plants expressingShrfused to GFPreveal Shr in the inner cells of the stele (Ste)as well as adjacentendodermal(End) cells, while SHR (GFPunderthe controlof the SHRpromoterin inset)are transcripts only presentin stele cells. Cor, cortex; Epi,epidermis;Cei, cortex/ endodermisinitial; 50 QC, quiescentcenter.Bars: and 25 pm(inset). FigurefromNakajimaet al., 2001.

instead of giving rise to single leaf primordia,the meristem which in turn producemultinow producesfloralmeristems, to ple primordia corresponding the four floralorgantypes. The importantresource developmentally of competentcells in the meristemis expectedto be highly sensitiveto signals that trafficcell to cell. Indeed,temporaland spatialchangesin cell-to-celltransportoccur at the shoot apex as a function of developmentalage (Gisel et al., 2002). Apices of vegetative 1,3,6 trisulfonicacid plants reveal tracer (8-hydroxypyrene [HPTS]; 520 D) in the outer cell layersof the meristemand in very young primordia.Surprisingly, fluorescence signal in the entire shoot apical meristemregion decreasesbefore the onset of flowering.After floral morphogenesisis underway, floralapicesagaintraffictracer.Such studiessuggestthat cellvia of to-cell transport PD is an additionalparameter the multifactorialcontrol of the transitionto reproductive developof ment, and supporta model where reductionof transport a floral repressor(s) contributesto the induction of flowering. As the apicalmeristemundergoesprofoundchangesin archiit tectureand concomitantsignalingand expression pathways, into from intercellular may be advantageous be sequestered of put duringestablishment the reproductive program. studies on the initiation and control of floral Independent developmentpredominantlyaim to define which genes control this pathway.Many floral regulatory genes encode tranfactors that affect floral patterningdepending on scription their specific temporal and spatial patterns of expression. However,when and where their specificRNAs are expressed is not the end point of their regulation.It is now clear that factorstransitPD. Such trafficis criticalfloraltranscriptional as some, but not all, meristem-expressed highly regulated factors move cell to cell (Sessions et al., 2000; Kim et al., factors, 2003; Wu et al., 2003). Two essentialtranscription

Apetalal (Apl) and Leafy (Lfy),were each expressedfrom a promoter driving outer cell layer-specificexpression.While Lfy could move down into internalcell layers,Ap l remained in the outer cell layer.As suggestedabove,nontargeted movement (Lfy) may be the default state for macromolecules, unless they are specificallyretained (Apl). Perhapsnontargeted proteinmovementprovidescoordinationbetweenlarge groups of cells, whereas proteins with more limited movement programmore specific pathways.Such studies impliof cate PD not only as regulators overalltrafficpotential,but also as regulators the specificityof "cargo" of macromolecules that interactwith and transitthese channels.

PD function duringembryonicdevelopment
The correspondencebetween levels of cell-to-cell transport and differentiationnoted above predictsthat other essential programs,such as embryogenesis, .also alter their intercellular transport capacity to induce or regulate development. While small fluorescent tracer (HPTS) traffics uniformly during all stages of embryogenesis in Arabidopsis,larger tracer(10-kD fluorescentdextran)trafficsonly during early embryogenesis.F-dextranceasestrafficduring mid-embryogenesiswhen primitiveroot and leavesbegin to form. Thus, there is a developmentaldown-regulationof PD apertureat this stage (Kim et al., 2002). Embryogenesis providesa simple accessiblesystem to dissect PD function.

of Identification PD components
PD researchis at a juncture.The dynamic and criticalroles of PD are established.It is now essentialto uncover the intricacies of the mechanisms involved in PD structureand function during development. To understandPD requires the identificationof specific structuralor regulatory components that control PD function. Several recent approaches are highlighted.

168 The Journalof Cell Biology I Volume 162, Number2, 2004

A biochemical approach used proteins known to traffic via PD as bait for the affinity purification of interaction partners contained within PD-enriched cell wall extracts (Lee et al., 2003). One protein, NCAPP1, is a highly basic domain that localizes protein with an ER trans-membrane to the cell periphery. The generationof plant cDNA-GFP fusions and the subof patternsof GFP in vivo sequentobservation the expression of revealscellularaddresses the proteinfusions (Cutleret al., 2000). A high throughputplant viral expressionsystem allowed the screeningof infection foci expressionof >20,000 independentGFP fusions. 12 fusion proteinsspecificallylocalized to PD (Escobaret al., 2003). Localizationwas observed as puncta (as in Fig. 2 b), strongly supportingthat or candidatesare bone fide PD structural regulatory components. Half of the encoded proteinsexhibit no similarityto novel PD factors. known proteinsand may represent a classic method to identify PD-specific compoFinally, nents is genetics.A majorobstacleto this approachis that alterations of PD function likely will have severe defects in growth and not produceviable plants. However, PD defects areexpectedto manifestfirstas defectsduringembryodevellines can be propagatedas heterozyopment. Embryo-lethal that segregate phenotypes wild-type and embryo-lethal gotes in their seedpods. Determination of developmentaltransitions in PD function during embryogenesisprovides entry points to identify mutantswith alteredcell-to-celltransport. Indeed, screening embryo-defectivelines individually by fluorescencemicroscopyidentifiedlines alteredin their abil(Kim et al., ity to traffic tracersduring mid-embryogenesis of the affected genes and their 2002). Map-based cloning should provide insights into intercellular characterization communicationin plants.

References
Baulcombe, D.C. 1999. Gene silencing: RNA makes RNA makes no protein. Curr.Biol. 9:R599-R601. Crawford, K.M., and P.C. Zambryski. 2000. Subcellular localization determines the availabilityof non-targeted proteins to plasmodesmataltransport. Curr. Biol. 10:1032-1040. Crawford, K.M., and P.C. Zambryski. 2001. Non-targeted and targeted protein movement through plasmodesmatain leaves in different developmental and physiological states. Plant Physiol. 125:1802-1812. Cutler, S.R., D.W. Ehrhardt,J.S. Griffitts, and C.R. Somerville. 2000. Random GFP::cDNA fusions enable visualization of subcellularstructuresin cells of at Arabidopsis a high frequency.Proc. Natl. Acad Sci. USA. 97:3718-3723. Escobar, N.M., S. Haupt, G. Thow, P. Boevink, S. Chapman, and K. Oparka. 2003. High throughput viral expression of cDNA-green fluorescent protein fusions revealsnovel subcellularaddressesand identifies unique proteins that interact with plasmodesmata.Plant Cell. 15:1507-1523. Gisel, A., F.D. Hempel, S. Barella, and P. Zambryski. 2002. Leaf-to-shoot apex movement of symplastictraceris restrictedcoincident with flowering in Arabidopsis.Proc. Natl. Acad. Sci. USA. 99:1713-1717. Imlau, A., E. Truernit, and N. Sauer. 1999. Cell-to-cell and long-distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues. Plant Cell 11:309-322. Kim, I., F.D. Hempel, K. Sha, J. Pfluger, and P.C. Zambryski. 2002. Identification of a developmental transition in plasmodesmatal function during emthaliana. Development.129:1261-1272. bryogenesisin Arabidopsis Kim, J.-Y., Z. Yuan, and D. Jackson. 2003. Developmental regulation and significance of KNOX protein trafficking in Arabidopsis. Development. 130: 4351-4362. Kim, M., W. Canio, S. Kessler,and N. Sinha. 2001. Developmental changes due to long-distance movement of a homeobox fusion transcriptin tomato. Science.293:287-289. Lazarowitz,S.G., and R.N. Beachy. 1999. Viral movement proteins as probes for intracellularand intercellulartraffickingin plants. Plant Cell. 11:535-548. Lee, J.Y., B.C. Yoo, M.R. Rojas, N. Gomez-Ospina, L.A. Staehelin, and W.J. Lucas. 2003. Selective traffickingof non-cell-autonomous proteins mediated by NtNCAPP1. Science.299:392-396. Lucas, W.J., S. Bouche-Pillon, D.P. Jackson, L. Nguyen, L. Baker, B. Ding, and S. Hake. 1995. Selective trafficking of KNOTTED1 homeodomain protein and its mRNA through plasmodesmata.Science.270:1980-1983. Nakajima, K., G. Sena, T. Nawy, and P.N. Benfey. 2001. Intercellularmovement of the putative transcription factor SHR in root patterning. Nature. 413: 307-311. Oparka, K.J., A.G. Roberts, P. Boevink, S. Santa Cruz, I. Roberts, K.S. Pradel,A. Imlau, G. Kotlizky, N. Sauer, and B. Epel. 1999. Simple, but not branched, plasmodesmata allow the nonspecific trafficking of proteins in developing tobacco leaves. Cell. 97:743-754. Roberts, A.G., and K.J. Oparka. 2003. Plasmodesmataand the control of symplastic transport.Plant Cell Environ.26:103-124. Ruiz-Medrano, R., B. Xoconostle-Cazares, and W.J. Lucas. 1999. Phloem longdistance transportof CmNACP mRNA: implications for supracellularregulation in plants. Development.126:4405-4419. Sessions, A., M.F. Yanofsky, and D. Weigel. 2000. Cell-cell signaling and movement by the floral transcription factors LEAFY and Science. APETA1LA. 289:779-782. Voinnet, O. 2001. RNA silencing as a plant immune system againstviruses. Trends Genet. 17:449-459. Wu, X., J.R. Dinnemy, K.M. Crawford, Y. Rhee, V. Citovsky, P.C. Zambryski, and D. Weigel. 2003. Modes of intercellulartranscriptionfactor movement apex. Development.130:3735-3745. in the Arabidopsis Zambryski, P., and K. Crawford.2000. Plasmodesmata:gatekeepersfor cell-to-cell transport of developmental signals in plants. Annu. Rev. Cell Dev. Biol. 16: 393-421.

Perspectives
PD are essentialgatekeepersfor plant cell-to-cell transport. As PD have the innate ability to transportmacromolecules, developmental transitions in PD function and aperture of likely play criticalroles in the transmission macromolecular signals to coordinate differentiationpathways.Obvious moleculessignal questionsfor the futureare:what regulatory PD to open or close; what structuralelements of PD respond to such regulatorymolecules;do PD in differentcell types have different specific components; what changes in occur as a cell matures;how is PD components/architecture the number of PD per specific cell type determined;and with cell division? how is PD formationsynchronized
P. Zambryskithanks colleagues and the National Institutes of Health (GM45244)for support. 9 Submitted: October2003 2003 24 Accepted: November