membrane
transport
in
plants
Markus
Geisler
Recently,theplanttransportfieldhasshiftedtheirresearch
focustowardamoreintegrativeinvestigationoftransport
networksthoughttoprovidethebasisforlong-rangetransport
routes.Substantialprogresswasprovidedbyofaseriesof
eleganttechniquesthatallowforavisualizationorpredictionof
substratemovementsinplanttissuesincontrasttoestablished
quantitativemethodsofferinglowspatialresolution.These
methodsarecriticallyevaluatedinrespecttotheir
spatio-temporalresolution,invasiveness,dynamicsandoverall
quality.Currentlimitationsoftransportroutepredictions-based
ontransporterlocationsandtransportmodelingare
addressed.Finally,thepotentialofnewtoolsthathavenotyet
beenfullyimplementedintoplantresearchisindicated.
Address
UniversityofFribourg,DepartmentofBiology,CheminduMuse´e10,
CH-1700Fribourg,Switzerland
Correspondingauthor:Geisler,Markus(markus.geisler@unifr.ch)
Introduction
Becauseyouhaveseen me,youhavebelieved;
blessedarethosewhohavenotseenandyethavebelieved.1 John20:29
Thecatalyzedmovementofsubstratesacrossmembranes, commonly referred to as transmembrane transport, has always been a hotspotin plantbiology [1].Throughout
the 80s and90s, the planttransport community concen-trated mainly on biochemical characterization of single transporterisoforms with afocus ona kinetic level (the ‘queen of transport experiments’ [2]). In the last two decades,deepinsightsintotransporterexpression, traffick-ing, regulation and their role in plant physiology were provided.Recently,plantscientistshaveshiftedtheir inter-esttowardamoreintegrativeinvestigationof long-range transportroutesortransportnetworks[3–5]thatisofwater, nutrientsorsomehormones(foranoverview,seeFigure1). Transportwasclassicallyquantifiedonawholeorganor plantlevelusingradiotracers,externalmicroelectrodesor cell-type specific analyses of transport substrates (for reviews on these methods, see Ref. [6]). While these techniquesallowfor ahighleveloftemporalresolution, theyare limited inthe spatialaspect.Therefore,direct andindirecttechniqueshavebeendevelopedinorderto image either local substrate concentrations or intra and intercellulartransportinplanta.Here,wepresentcurrent methods that allow for a quantitative visualization of transmembranetransportandcomparethemwith classi-caltransport measurementsandinsilicotechniques.
Inference
of
transport
routes
by
transporter
imaging
For several substrates, putative transport routes have been deduced from polar transporter localizations [3,5,7–10]. These cell-biological approaches have been partiallybackedupbyanalysesofreporterexpressionand transportmodeling(seebelow).
Thebest-understoodexampleisprobablythepolar trans-port of the auxin, indole-3-acetic acid (IAA), in the Arabidopsis root tip provided by a network of plasma membrane localized im- and exporters of the ABCB, AUX1/LAX and PIN-FORMED (PIN) families [3,11]. Differentdegreesoftissue-specificandpolarlocationsof members of these families have been established by immunolocalizationand fluorescentproteinfusions(see
Figure3)thatareinlinewithcurrentmodelsofareversed fountainauxinfluxpattern(seeFigure1;[8]).Similarly, theboricacidchannel,NIP5;1,and theboronexporter, BOR1, have been localized in a polar fashion in the plasmamembranesfacing towardsoilandstele, respec-tively, and are therefore suspected to function in the uptake and translocationof boron to support growth of various plant species (see Figure 1;[7]). Likewise,the ABCG-typeSLtransporter,PDR1,exhibitsan asymmet-ricallocalizationintheplasmamembraneofpetuniaroot
1Pleasenotethatthisbiblecitationisnotatallmeantasareligious
statement but a stylistic element acknowledging the effort of the
transportfieldtopromotetechniquesallowingforaquantitative
visuali-zationoftransport,whichisthecontentofthisreview.
tips indicatingadirectionalcell-to-celltransportprocess at least in this region of the root (see Figure 1; [5]). However, presenceof atransporteronacellular subdo-maindoes notautomaticallyreflect localactivitylevels. Moreover,thisapproachfailedsofarinthepredictionof transport routes for non-polartransporters known to be involvedin long-distancetransport[12].
Quantification
of
transporter
activity
or
regulation
Abetterproxymightbetherecentlydevelopedtransport activity sensors, such as AmTrac and MepTrac [13,14], NiTrac1 and PepTrac [15]. Although these transport
activitysensorsdonotstrictlymonitortransport,andhave not yet been tested in planta, they will most likely become valuable, especially in the context of analytes for whichnotracersexist.
An alternative approach might be imaging transporter regulation, such as by protein phosphorylation [16]. AGCkinaseswereshowntophosphorylateandthusalter transport activitiesofauxin transportersof thePINand ABCBfamilies[16–20],buthavenotyetbeenimagedin planta. A future alternative might be offered by novel GFP-based kinase reporters, called SPARK(Separation of Phases-based Activity Reporter of Kinase). SPARK
Figure1
cytokinins strigolactones
abscisic acid auxin boron calcium
stomata
embryo
vasculature
lateral bud
root tip
primary root
Current Opinion in Plant Biology
Illustrationoftrans-membrane,long-rangetransportroutesinplants.
Exemplarylong-rangetransportroutesofindicatedphytohormones,boronandcalciumareindicatedbyarrows.Notethatonlylong-distance
signalingofCa2+whereitactsasasecondmessengerbutnotasanutrientisindicated.Incontrast,boronpathsareindicatingnutritionroutes.
Overallfigureoutlineisbasedon[5];usageofArabidopsismodelwasgrantedbyMaryLouGuerinot.
metabolites) are by nature fluorescent and can be fol-lowed directly, most are not and thus need either be applied as a radiolabeled or fluorescent version (see below)orrequireanindicatingtool[22].Invivo biochem-istry[23]hasprovideduswith aseriesof cell-biological techniques that allow for an imaging of either local substrate concentrations or intraand intercellular trans-portinplanta(seeFigure2).However,itisimportantto keepinmindthatmostofthesetechniquesindicatelocal substrateconcentrationsthatarethenusedasaproxyfor transport.
Chemicalindicators
Chemical indicators are fluorescent molecules that respond to the binding of substrates by changing their fluorescenceproperties([24];seeFigure2).Theyexistfor differentmetalcations(suchaszinc,potassium,sodium, magnesium, calcium), protons and chloride and their loading and membrane targeting has been optimized bygenerating hydrophobicversionsthatare cytoplasmi-callycleavedandactivated.Inplants,themostcommonly used pH-indicator and calcium-indicator are BCECF
indicatorsinplantcellsisthatdyeloadingishinderedby the cell wall or compartmentalization in organelles (mainlyvacuoles)avoidinganequaldistribution[22,26]. Therefore,geneticallyencoded(GE)indicators,reporters andsensors[27–30]havebeendevelopedthatindirectlyor directly indicate local substrate concentrations, respec-tively (Figure 2). They allow for selective targeting to subcellular compartments (by insertion of signal sequences)ortocelltypes(bychoiceofpromoters)[31].
Reporters
Expression-based(orsignaling)reportersconsistof natu-ralorsyntheticpromotersfusedtoareportergene.Many butnotallplant hormones,includingauxin,ABA, cyto-kinin(CK),ethylen,jasmonicacid(JA)andsalicylicacid canbevisualizedin this manner(for reviews,seeRefs. [23,27,30–32]).Sincepublicationoftheprototype artifi-cialauxin-responsiveelement,DR5[33],severalvariants havebeendeveloped (forareview,seeRef.[22]).DR5 activation is slow (ca. 2hour) and reflects an auxin
Figure2
Chemical Indicator
Reporter Sensor Fluorescent substrate Click-chemistry Imaging of radioactivity Principle
Readout type Change of fluorescent properties Promoter activation, reporter degradation FRET, change of fluorescence intensity
Fluorescence Fluorescence (Digital) Radiography, Micro-autoradiography Dynamic readout + +/-1 + + (+) -Subcellular resolution +/- - (+) + + +/-Calibration +/- +/-1 + - - -Noninvasive + (+)2 (+)2 + -
-Current Opinion in Plant Biology
Comparisonofmethodsusedtoderivetransmembrane,long-rangetransportinplants.
Indicatedapproachesareevaluatedwithrespecttotheirreadouttypeanddynamicrange,theirinvasiveness,theirofferedsubcellularresolution
andtheircorrelationbetweentheamountofdetectedtargetmoleculeandreadout(calibration).Targetmoleculesareinblue;fluorescentproteins
aredepictedasroundedrectangles.Notethatprinciplesareillustrativeexamplesandmightbenotcomplete;bracketsindicatelimitationsof
methods.FigureoutlineisinspiredbyRef.[27].1Dynamicrangeandcalibrationvariesbetweenreportertypes;2Reporterandsensorsrequire
geneticaccessandarethusnotsuitablefornon-modelplants(fordetails,seetext).
response maximum,and is thusmainly suitable for the roottip [22,34,35].
Reporters-basedontheprinciple of SCF-mediated pro-teasomaldegradationof transcriptionalrepressorsinthe signaling pathway of several hormones weredeveloped increasing the temporal (but also spatial) resolution of expression-based reporters. The blueprint consisted of theDIIdegronsequenceofAux/IAArepressorproteinsof auxin signaling, which was fused to a nuclear targeted VENUS [36].This moduleprovidesa decreaseof DII-VENUS reporterfluorescence in tens of minutes upon auxin perception and is therefore suitable to image unequal auxindistribution in bending roots [36]. Com-biningthedegronmotivefusionwithanauxin-insensitive reportergeneresultedinratiometricreporters.For exam-ple,R2D2,nowofferssemi-quantitativeauxinimagingat very reasonable spatio-temporalresolution[37,38,39]. Degradation-based reporters have also been developed forGA(GFP-RGA;[40]),JA(Jas9-VENUS;[41])andSL (StrigoQuant; [42]).
Despite tremendous improvements in expression and degradation-based reporters with respect to speed and sensitivity,theirindirectmodeofactionallowsonlyfor— at best — semi-quantitative in vivo imaging of local substrate concentrations.Further,reportersdisplayfinal outputs that integrate over transport, biosynthesis and
metabolism. An evenmore limiting aspectis that their sensitivity isprimarilydeterminedbythe expressionof the employed signaling machinery and thus cell and tissue-specific[34].
Sensors
Aseriesofgeneticallyencodedactivitysensorsthatareable to reportdirectly andwithinseconds changesin calcium, proton, energy metabolite, heavy metal or macronutrient concentrations have been established [27,31]. Like reporters, sensorareusedtoassessresidentsubstrateconcentrations thatarethenoftenequalizedastransportactivities[27,43]. The photoprotein aequorin, from jellyfish has been widelyusedinplantsformorethantwodecadesinorder tostudycalciumdynamicsbymeansofemitted biolumi-nescence[44].However,duetoitslowcellularsensitivity, aequorin was slowly replaced by FRET-based sensors (Figure 2; [45]), such as Cameleon [24]. Versions of Cameleon with improved calcium binding affinity and dynamic range [23,27,28] (YC3-60, [46] and YC-nano), wererecentlyusedfordemonstratinglong-rangecalcium signalingintherootandbetweenshootandroot, respec-tively [47–49]. The intensiometric calcium sensor, R-GECO1, exhibits anincreased (>10)sensitivity com-pared with YC3.6 in response to elicitors of the innate immune response to pathogens, flagelin22 and chitin [50],andR-wasusedtoimageauxin-inducedcalcium
Figure3 measured DII predicted DII measured IAA IAA influx WT root geometry WT root geometry transporter location
lrc
ci
5 0 190 0tip
Current Opinion in Plant Biology
148
0 0.0
14.2
Comparisonofimaged,modeledandmeasuredauxintransportintheroottip.
MeasuredDII-VENUSsignalsandtransporterlocations(here:PINs)wereextractedfromconfocalimagesandarecomparedwithmodeled
DII-VENUSdistributionandpredictedauxinfluxes(alltakenfromRef.[89]).Asreference,imagedauxin(transporter)distributionsarecomparedwith
heatmappresentationofIAAinfluxprofilesmeasuredusinganIAA-specificmicroelectrode[97]orofhigh-resolutionIAAcontentsmeasuredby
MSaftercell-typesorting[97,98].Notethatthelattertwoquantitativetransporttechniquesarenotdescribedinthisreview.Celltypeslabeledon
theschematicsofwild-typerootgeometry(leftandright)aregiven;foridentityrefertoRefs.[89]and[97].Notethatthealignmentofschematics
isbasedonpositionofroottips,columellainitials(ci)andendoflateralrootcaps(lrc).
plasmic and apoplastic pH [28]. Recently, a pHusion variant allowed measuring pH in and outside of the TGN/early endosome network demonstrated the role oftheV-ATPaseforproteinsecretion[54].
The Frommer group was driving forward the develop-ment of FRET sensors for of a wide range of sugars [23,27,31]. Beside a quantification of intracellular con-centrations, these sensors also permitted the kinetic characterization of individual sugar transporters [1] and theidentification of novel transporters[55].Sensors for transportofATP;[56,57],ROS,heavymetals(eCALWY) (for references, see Refs. [27,31]) and different macro-nutrients have been developed (for a review, see Ref. [58]). Very recently, ATP sensing in living plant cells usingtheFRET-basedsensor, ATeam1.03-nD/nA[56], revealed concentration gradients in plant tissues and stress-relateddynamicsofenergyphysiology[57]. Hormoneimagingbysensorshasbeenachievedfirstfor ABA: two independent groups have developed sensors consisting of a FRET pair that is linked by an ABA-responsive sensory module, called ABACUS [59] and ABAleon[60],respectively.Bothsensorshaveprovento beusefulindemonstratingkineticsofABAtransportand dynamicsupon applicationof externalABA,[30,32,59]. ThenuclearlocalizedGAsensor,GIBBERELLIN PER-CEPTIONSENSOR1(GPS1),allowedsensingof nano-molarconcentrationsinArabidopsis[61].
The intensiometric calcium sensor, R-GECO1, was shown to exhibit an increased (>10) sensitivity com-paredwithclassicalYC3.6inresponsetoelicitorsof the innate immune response to pathogens, flagelin22 and chitin[50].R-GECO1wasusedtoimageauxin-induced calciumgradientsinroot-hairtipsbut alsolong-distance calciumwavesbetweenrootcells[51].
Fluorescentlylabelledsubstrates
Fluorescentlylabeledsubstrateshaveonlyrecentlybeen used in plants (Figure 2). The fluorescent BR analog, Alexa Fluor 647-castasterone (AFCS) [62], GA-fluores-cein [63] and different fluorescent auxins [34,35,64] have been developed. Moreover, a quantum dot-based fluorescent probe, CdTe-JA, was employed to labelJA bindingsitesintissuesectionsofmungbeanand Arabi-dopsisseedlings[65].Recently,differentfluorescentSL analogsweresuccessfully designed:while EGO10A-BP and CISA-1 [66] are constitutively fluorescent variant making it first choice for transport studies,
designedfluorescent auxinanalogsto be actively trans-portedbuttobeinactiveforauxinsignaling[34],while veryrecentlynewfluorescentlylabeledauxinsexhibitan anti-auxinactivity[64].Theformerisimportantbecause auxinsignaling is knownto influenceauxin transporter expressionand location[34].NBD-IAA (7-nitro-2,1,3-benzoxadiazole-IAA)revealedasimilarauxindistribution as found with DII-based reporters [34] but lacked signals in the quiescent center known to be mainly produced by auxin biosynthesis and not transport. A limitationof NBD-auxins is thatthey are for unknown reasons only working in root and not in shoots [34]. Recently,NBD-NAA,whosenuclearimportisrestricted, wasusedasatooltodissectcytoplasmicandERdelivery of nuclear auxin concentrations [39]. This indicates theirusefulnessinanalyzingintracellulartransport,which isusuallyhardtouncoverusingclassicaltools.
Click-chemistryoftransportsubstrates
Inanimalmodels, theproblemofbulkyfluorescenttags interferingwithbindingtotransporter(orreceptor) pro-teinshasbeen partiallysolvedby‘clickchemistry’[68]. This techniquetriggers theselective, covalent connec-tionbetweenamoleculeofinterestandadetectiontagin situ(Figure2).Avarietyoflabelingtagscanbeusedthen formicroscopyoranalytics.
Toourknowledgeonlyonestudyreportedonthe distri-bution of a mobile substrate using this technology in plants: the azido derivative of indole-3-propionic acid (IPA, an active auxin), IPA-N3, was used as an auxin
tracer and provided some evidence of the presence of auxin binding sites in the apoplast of elongating cells [69]. IPA-N3 was detected mainly in the outer cell
layersoftheroot tipand doesthus notfullyreflectthe auxinaccumulationsites.Thisseemsto belessa pene-tration but a fixation problem [69]. However, these technical issues will be overcome and as such ‘click chemistry’ mightsoonbe themethodofchoice despite thefactthatitrequiresafixationsteppreventinginvivo imaging.
Imagingofradiolabeledsubstrates
Radio-autographicanalysisofthedistributionoftransport hasalongtradition inplants (e.g.[70]).Recently,polar auxintransportofArabidopsisstemsegmentswas visual-ized by detecting 14C-IAA with a double-sided silicon strip detector [71].In recent years, the micro-autoradi-ographymethod(MAR)hasbeenestablishedtovisualize thedistributionof109Cdand 33Pwithinsectionsofrice
tissues [72].Currently, onlyfixed materialcanbeused, preventinganydynamicinformationaboutsubstrate dis-tributions.As14C(and3H)emitbetaparticlesatverylow energy,mosttransportsubstratescouldbelabelledwith suitable isotopes for autoradiography, which would expand the application properties of MAR (Figure 2; [72]).
Immunolocalizationoftransportsubstrates
ABA[73,74],IAA[22]andCK[75]havebeenvisualized in differentplantspeciesbywhole-mount immunoloca-lization using polyclonal and monoclonal antisera ( Fig-ure2;[76]).Despiterequiringoffixation,someofthese antisera have become successful tools for the in situ localization of local maxima[75,77] or thetranslocation ofhormonesinplants[78–82].Anti-IAAantibodysignals werereportedtorecapitulatesignalsfoundwiththeDR5 reporterinthematureandlateralroot[77,79].However,a partofthesepublicationshavebeenheavilydebatedwith respecttotheirinterpretationandtechnicalquality[83].
Modeling
of
transmembrane
transport
over
tissues
Early auxin transportmodels, called flux-basedmodels, wereinspiredbyTsviSachsinthelate1960sand hypoth-esized that the flux of auxin through cell membranes reinforces itself [84,85]. The integration of experimen-tallydeterminedpatternsoftransporterlocalizationinto simulations confirmed this in Arabidopsis [86–88,89]. Gradient-based models were introduced [90,91], pro-vided support for the assumed roles of auxin maxima in shootdevelopment(foran excellentreview,seeRef. [92]).
The primarymotivationof mostofthesestudieswasto predict the movement of a transported substrate (here: auxin;Figure3)withinadevelopingtissue.Inthisregard, thedevelopmentoftheOnGuardplatformformodeling transport processesin stomata has provensuccessful in uncoveringpreviouslyunexpectedfeaturesofguardcell physiology [93].However, today’s interest lies more in testing if a hypothesized role for a substrate can be supportedbyinsilicodata[94].Arecentstudyemploying data-driven modeling of intracellular auxin fluxes between theER or cytoplasm and thenucleus, respec-tively, indicated that the ER has a dominant role in controlling nuclearauxin uptake[39]. This and other approachesarguethatcomputermodelscanprovide(or even go beyond) a ‘proof of principle’. However, any conclusions-basedonhuman-made,and as such subjec-tive, algorithms will always require an experimental validation.
Conclusions
and
perspectives
Recently, the plant transport field has made a major conceptual shift away from a single transporter analysis toward a more integrative transporterapproach, aimed at
exploring transporter networks in order to understand theirinterplayandtheirincorporationintoplant physiol-ogy.Thisprogresshasbeenmadepossiblebythe devel-opmentofaseriesoftechniquesthatallowvisualization of local concentrations and movements of substrates in plant tissues. While early quantitative methods (like radiolabeled substrates, micro-electrodes and cell-type specificanalysesoftransportsubstrates)providetemporal resolution,visualizationoftransportbymeansofchemical indicators, fluorescent analogs and especially reporters and sensorsprovides goodspatial information with rea-sonable resolution for many substrates.In that respect, doubting Thomas was right: seeing is better than believing.
Mosttools havetheirindividualadvantagesand limita-tions(seeFigure2).Someallowonlyfor (semi)-quanti-tative analyses withhigh spatial butonlylowtemporal resolution (such as reporters and sensors), while some indicators, sensorsorfluorescent analogswerefoundto interferewithplantphysiologybyhighsubstratebinding affinities or by competing with endogenous signaling [24,32,57].However, thesekinds ofproblemsmaytoa certaindegreebeovercomebyusingnon-plantsubstrate binding domains[95].
As usual, newtechnology and insightprovidesus with new challenges. For example, the transfer of sensors from singlecellapproaches totheplantlevel hasbeen difficult due tothewideaffinity/sensitivityrange (nM-mM) that is required for some substrates. A solution might be the co-expression of spectrally non-overlap-ping, intensiometric sensors with different affinity ranges. Further, in contrast to intercellular transport, transmembrane transportbetweensubcellular compart-ments hasproven difficult tomeasure directly. Inthat respect, a directed targeting of sensors to individual subdomains of interest as shown recently [54] and design of selective permeable fluorescent dyes [39] willbehelpful.
Another issueis theinadequate separationof transport from signaling/biosynthesisbysomeimagingtools,like reporters orsensors. In that respect it appearsas if we have not yet fully used the potential of fluorescent transport substrates or click chemistry. In the future, structure-activity design will allow us topredict fluor-ophore positions and linker length [63,64] in order to direct uptake, stability and distribution of fluorescent substrates [96].
Acknowledgements
TheauthorwouldliketothanktoMartindiDonatoandAure´lienBaillyfor
discussionandcomments.Theconstructiveinputofreviewersandeditorsis
highlyappreciated.WorkinthegeislerLabiscurrentlyfundedbytheSwiss
NationalFunds(project31003A_165877/1)andtheEuropeanSpace
Association(CORA-GBFprojectLIRAT).
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