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Functional Characterization and Phenotyping of Protoplasts on a Microfluidics-Based Flow Cytometry

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Xingda Dai
Xingda Dai
Xingda Dai
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Zhang Shuaihua at Tianjin University
Zhang Shuaihua
Siyuan Liu at Tianjin University
Siyuan Liu
Hang Qi at Tianjin University
Hang Qi
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Abstract and Figures

A better understanding of the phenotypic heterogeneity of protoplasts requires a comprehensive analysis of the morphological and metabolic characteristics of many individual cells. In this study, we developed a microfluidic flow cytometry with fluorescence sensor for functional characterization and phenotyping of protoplasts to allow an unbiased assessment of the influence of environmental factors at the single cell level. First, based on the measurement of intracellular homeostasis of reactive oxygen species (ROS) with a DCFH-DA dye, the effects of various external stress factors such as H2O2, temperature, ultraviolet (UV) light, and cadmium ions on intracellular ROS accumulation in Arabidopsis mesophyll protoplasts were quantitatively investigated. Second, a faster and stronger oxidative burst was observed in Petunia protoplasts isolated from white petals than in those isolated from purple petals, demonstrating the photoprotective role of anthocyanins. Third, using mutants with different endogenous auxin, we demonstrated the beneficial effect of auxin during the process of primary cell wall regeneration. Moreover, UV-B irradiation has a similar accelerating effect by increasing the intracellular auxin level, as shown by double fluorescence channels. In summary, our work has revealed previously underappreciated phenotypic variability within a protoplast population and demonstrated the advantages of a microfluidic flow cytometry for assessing the in vivo dynamics of plant metabolic and physiological indices at the single-cell level.
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Biosensors2022,12,688.https://doi.org/10.3390/bios12090688www.mdpi.com/journal/biosensors
Article
FunctionalCharacterizationandPhenotypingofProtoplastson
aMicrofluidics‐BasedFlowCytometry
XingdaDai
1,†
,ShuaihuaZhang
2,†
,SiyuanLiu
2
,HangQi
2
,XuexinDuan
2
,ZiyuHan
2,
*andJiehuaWang
1,
*
1
SchoolofEnvironmentalScienceandEngineering,TianjinUniversity,WeijinRd.92,Tianjin300072,China
2
StateKeyLaboratoryofPrecisionMeasuringTechnologyandInstruments,CollegeofPrecisionInstruments
andOptoelectronicsEngineering,TianjinUniversity,WeijinRd.92,Tianjin300072,China
*Correspondence:ziyu_han@tju.edu.cn(Z.H.);jiehuawang@tju.edu.cn(J.W.)
†Theseauthorscontributedequallytothispaper.
Abstract:Abetterunderstandingofthephenotypicheterogeneityofprotoplastsrequiresacom‐
prehensiveanalysisofthemorphologicalandmetaboliccharacteristicsofmanyindividualcells.In
thisstudy,wedevelopedamicrofluidicflowcytometrywithfluorescencesensorforfunctional
characterizationandphenotypingofprotoplaststoallowanunbiasedassessmentoftheinfluence
ofenvironmentalfactorsatthesinglecelllevel.First,basedonthemeasurementofintracellular
homeostasisofreactiveoxygenspecies(ROS)withaDCFH‐DAdye,theeffectsofvariousexternal
stressfactorssuchasH
2
O
2
,temperature,ultraviolet(UV)light,andcadmiumionsonintracellular
ROSaccumulationinArabidopsismesophyllprotoplastswerequantitativelyinvestigated.Second,
afasterandstrongeroxidativeburstwasobservedinPetuniaprotoplastsisolatedfromwhitepet‐
alsthaninthoseisolatedfrompurplepetals,demonstratingthephotoprotectiveroleofanthocya‐
nins.Third,usingmutantswithdifferentendogenousauxin,wedemonstratedthebeneficialeffect
ofauxinduringtheprocessofprimarycellwallregeneration.Moreover,UV‐Birradiationhasa
similaracceleratingeffectbyincreasingtheintracellularauxinlevel,asshownbydoublefluores‐
cencechannels.Insummary,ourworkhasrevealedpreviouslyunderappreciatedphenotypic
variabilitywithinaprotoplastpopulationanddemonstratedtheadvantagesofamicrofluidicflow
cytometryforassessingtheinvivodynamicsofplantmetabolicandphysiologicalindicesatthe
single‐celllevel.
Keywords:microfluidicflowcytometry;protoplasts;fluorescentreporter;UVlights;
anthocyanidin;primarycellwall
1.Introduction
Complexbiologicalprocessesrelyonthedynamicbehaviorofsinglecellsandcell–
cellinteractions.Conventionalbulktissueanalysisobscuresdifferencesincelldiversity
inmostbiological/biomedicalsamples,whereassingle‐cellanalysisallowsaqualitative
and/orquantitativecharacterizationofeachcellinacellpopulation.Itnotonlyreveals
biologicalvariabilitybetweencellpopulations,butalsopavesthewaytonewinsights
intovariouscellularprocessessuchascell‐fatedecisions,physiologicalheterogeneity,or
genotype–phenotypelinkages[1,2].
Withrespecttosingle‐cellphenotyping,flowcytometryhasdemonstrateditsability
forhigh‐throughputquantitativeanalysisandisolationoftargetedbiologicalsamples.
However,conventionalflowcytometryisbulky,complex,andrequireshighlyskilled
personnel.Withthedevelopmentofmicrofluidictechnology,microfluidicflowcytome‐
try(MFCM)hasbeencombinedwithflowcytometrytoachievepowerfulsinglecellfo‐
cusing,detection,andsorting,whichhasbeenproveninvariousbiologicalapplications
[3],includingsingle‐cellRT‐PCR[4],stemcellscreening[5],proteinanalysis[6],etc.
WhileMFCMhasproventobeapowerfultoolforsingle‐cellmanipulationandanalysis
Citation:Dai,X.;Zhang,S.;Liu,S.;
Qi,H.;Duan,X.;Han,Z.;Wang,J.
FunctionalCharacterizationand
PhenotypingofProtoplastsona
Microfluidics‐BasedFlow
Cytometry.Biosensors2022,12,688.
https://doi.org/10.3390/bios12090688
Received:28July2022
Accepted:24August2022
Published:26August2022
Publisher’sNote:MDPIstaysneu‐
tralwithregardtojurisdictional
claimsinpublishedmapsandinsti‐
tutionalaffiliations.
Copyright:©2022bytheauthors.
LicenseeMDPI,Basel,Switzerland.
Thisarticleisanopenaccessarticle
distributedunderthetermsand
conditionsoftheCreativeCommons
Attribution(CCBY)license
(https://creativecommons.org/license
s/by/4.0/).
Biosensors2022,12,6882of14
inmedicaldiagnosticsandanimalcellstudies,similarworkonplant‐cellpropertiesis
stillfarbehind.
Inthisstudy,wedevelopedamicrofluidicflowcytometrythatprovidesasimple,
direct,andcost‐effectivesolutionfortheanalysisofprotoplastsamples.Protoplastsare
plantcellsinwhichthecellwallhasbeenenzymaticallyormechanicallyremoved[7],
andtheyareaveryefficientexperimentalmodelforbiotechnologicalapplicationssuchas
somatichybridizationandgenetictransformation.Protoplastsoffermanycytological
advantagesthatmulticellulartissuesandcellassembliesinsuspensionculturedonot
andarethereforeaninvaluableexperimentalsystemforstudyingcellularprocessessuch
assignaltransduction[8],cellwallregeneration[9],theroleofstressandhormones[10],
andtransientgeneexpression[11].However,aftercellwalldigestion,theresultingpro‐
toplastsareosmosis‐sensitive,fragilestructuresthatrequireextremecaretomaintain
theirintegrity.Inaddition,protoplastsaregenerallylargerindiameterthanmammalian
cellsanddonotadherelikeanimalcells,soanalysisofprotoplastpopulationsusingflow
cytometryrequiressignificantchangesininstrumentconfiguration,andisextremely
difficulttomanagetoachievestableflow[12].
Toinvestigatetheperformanceofthedevelopedsystem,wefirstexaminedAra‐
bidopsismesophyllprotoplastsfortheirintracellularaccumulationofreactiveoxygen
species(ROS)inresponsetoavarietyofexternalstimuli.Inplantcells,ROSusuallyrefer
tosuperoxideradicals(O2•−),hydrogenperoxide(H2O2),andhydroxylradicals(•OH),
andasinanimalcells,wheninexcesstheyaregenerallybelievedtohavedeleteriousef‐
fectsoncellmetabolism,leadingtocelldysfunctionanddeath[13,14].Inplants,ROScan
betriggeredbyanumberofabioticandbioticstresses[14,15],suchasbrightlight,cold,
anddesiccation[16–18],andplantshavedevelopedavarietyofantioxidantstrategiesto
scavengeROSbasedonenzymaticreactionsandthedirectradicalscavengingproperty
ofnon‐enzymaticantioxidantssuchasascorbateandglutathione[18,19].Ontheother
hand,ROSalsofunctionasasignalingmoleculemediatingtheinductionand/orresponse
tostress[20–23].Forexample,H2O2regulatesstomatalmovementandplant–pathogen
interactions[14,24],andbothO2andH2O2cantriggerarangeofdefenseresponses
[23,25].Therefore,theeffectsofROSvarywithconcentration,withhighconcentrations
leadingtohypersensitivecelldeath[26],whilelowconcentrationspromotecellcycle
progressionandsecondarycellwalldifferentiation[27].Inthiswork,weuseddichloro‐
dihydrofluoresceindiacetate(DCFH‐DA)fluorescencebiosensorstodeterminethein‐
tracellularROSdynamicsinplantcells.Inadditiontotheresponsesofmesophyllproto‐
plaststovariousenvironmentalstresses,wealsocomparedtheROSresponsesof
non‐photosyntheticprotoplastsisolatedfromPetuniapetaltissuetoultraviolet‐A(UV‐A)
andUV‐Birradiationandvalidatedthelong‐standingquestionofthephotoprotective
effectsofanthocyanins.Asanotherexampleofthefunctionalphenotypingofsingleplant
cells,weexaminedtheregulatoryfunctionofauxin,animportantphytohormone,inthe
processofprimarycellwallregenerationusingwild‐typeandgeneticallydeficientpro‐
toplasts.Wealsodemonstratedthecorrelationbetweenthepromotionofprimarycell
wallregenerationbyUVirradiationandtheregulationofintracellularauxinbydu‐
al‐channelfluorescencedetection.Ourdatapresentedhereareanexampleoftheuseof
microfluidicflowcytometryinsingle‐cellplantresearchandextendourunderstanding
oftwoplant‐specificprocessesatthecellularlevel.Thissystemallowedamoreaccurate
andefficientassessmentofcellstatus,andanunbiasedinterpretationoftheaveragebe‐
haviorofsingleplantcells.Moreimportantly,theresultsobtaineddemonstratethatdue
totheobviousphysiologicalheterogeneitywithinaplantcellpopulation,amanualrep‐
resentativefluorescencephotographofasinglecellcanleadtobiasandanon‐objective
orambiguousinterpretationoftheexperimentalresults.Understandingcellularfunction
inthispost‐genomiceraisagreatopportunityandchallenge.Wehopethatasmicroflu‐
idictechnologiesbecomemoreadvancedandreadilyavailable,newmicrofluidicsystems
willalsoundergosignificantdevelopmentandinnovationinplantcytology.
Biosensors2022,12,6883of14
2.Methods
2.1.DeviceFabricationandOpticalDetectionSetup
Themicrofluidicdevicewasfabricatedinpoly(dimethyl‐siloxane)(PDMS)using
softlithography[28],whichhadachannelheightof60μm,achannelwidthof40μm,
andholesthatwerepunchedforchannelinletsandoutlets.Thefluorescentsignalsof
singleplantcellsweremeasuredusingamicrofluidicflowcytometry.Themeasurement
wassetuponaninvertedmicroscope(IX73,Olympus)using50mW/360nmand50
mW/488nmlasersasexcitationlightsourcescoupledintoabeambyadichroicfilter
(MD416,Thorlabs).Theintensityofthetwolaserswasadjustedusingthevariableme‐
tallicneutraldensityfilters(NDC‐50C‐2M‐A,Thorlabs).Atransmissionlightsource(850
nm)wasmountedontopofthemicrofluidicdeviceforathecamera’sreal‐timeimaging.
Thefluorescenceemissionlightandtransmissionlightweresplitbytwodichroic,
long‐passfilters(DMLP490RandDMLP567R,Thorlabs)andcollectedthroughaside
portofthemicroscopeusingtwoPMTs(H10722‐210,HAMAMATSU)forthesimulta‐
neousacquisitionoftwodifferentcolors,andonehigh‐speedcamera(UX50,Photron).
LabVIEW2016softwareonaPCwithaFPGAdataacquisitioncard(PCIe‐7842R,Na‐
tionalInstruments)wasappliedtorecordthereal‐timeoutputvoltageofthetwoPMTs
whichrevealedthefluorescentintensitiesofsinglecells.Thepeakvalueswerethenex‐
tractedbyaMATLABdataprocessingprogramandnormalizedbysaturationvoltage
(5V)astherelativefluorescenceunits(RFU)fordataanalysis.
2.2.ProtoplastIsolationandDisposal
TheArabidopsisColumbia‐0(Col‐0)ecotype,theauxinexcessmutantsur2,theauxin
deficientmutanttaa1,theDRF‐GFPtransgenicstrainthatrespondstoauxinsignalswith
spontaneousgreenfluorescent,andthewhiteandpurpleflowersofPetuniawereusedin
thisstudy.Theplantsweregrowninagreenhouseat22°Cunderfluorescentwhitelight
witha16‐hlight/8‐hdarkcycle.ThemethodforisolationofArabidopsisleafprotoplasts
wasobtainedwithaslightmodificationofthemethodofYooetal.[11].Inbrief,healthy
andfullyexpandedArabidopsisleaveswerewashed,thelowerepidermiswastornoff
andthentransferredtotheenzymesolution(1.5%cellulaseR10,0.4%macerozymeR10,
0.4Mmannitol,20mMKCl,and10mMMESatpH5.8).Beforeuse,thisenzymesolution
waswarmedat55℃for10min,thencooleddowntoroomtemperatureandcombined
withCaCl2to10mMandBSAto0.1%,andincubatedindarknessat26℃foratleast3h.
Afterenzymaticdigestion,anequalvolumeofW5solution(154mMNaCl,125mM
CaCl2,5mMKCl,and2mMMESatpH5.8)wasusedtostoptheenzymaticdigestion.
Themixturewasthenfilteredthrougha75‐μmnylonmeshintoa50‐mLround‐bottom
tube.Aftercentrifugationat100×gfor6min,theprotoplastswereresuspendedbygentle
swirlinginaculturemedium(0.32%B‐5medium,0.25Mmannitol,and4mMMESatpH
5.8).Afterrepeatedcentrifugationwiththeculturemedium,theprotoplastswereresus‐
pendedintheculturemedium,andtheconcentrationwasdeterminedtobeapproxi‐
mately106cells/mLbyhemocytometer.TheprotoplastextractionfromPetuniawas
modifiedaccordingtothemethodofKangetal.[29].Freshflowersweregentlyrinsed,
cutintothinstripsandtransferredtoanenzymaticdigestionsolution(4.5%cellulaseR10,
1.2%macerozymeR10,0.6Mmannitol,10mMCaCl2,and20mMMESatpH5.8),andthe
processafterenzymaticdigestionwassimilartothatofArabidopsis,exceptthatthe
culturemediumwasreplacedwithabuffer(0.6Mmannitol,10mMCaCl2,and20mM
MESatpH5.8).
Theprotoplastsweretransferredtocultureplateswith24wellsandplacedattem‐
peratures(4,20,and37°C),underUVirradiation(UVA,UVB),atdifferentconcentra‐
tionsofH2O2(0,50,100,200,400,600,and800μM),Cd2+(40μM),andIAA(5.7μM)ac‐
cordingtotheexperimentaldesign.
2.3.FluorescenceIntensityObservation
Biosensors2022,12,6884of14
TheROSofprotoplastsweremeasuredwiththefluorescentprobeDCFH‐DA
(S0033M,BeyotimeBiotechnology),andtheprotoplast‐regeneratedprimarycellwalls
werestainedwiththefluorescentbrighteneragentCalcofluorwhite[30].Thedifferent
biologicalresponsesofprotoplastswerequalitativelycharacterizedbyfluorescenceim‐
agestakenwithacamera(DS‐Fi1c,Nikon)mountedonamicroscope(Eclipse50i,Nikon)
influorescencemode(excitation:400nm,emission:450nm;excitation:488nm,and
emission:525nm).
2.4.ROSLocalizationandFlavonoidDetectioninPlantTissues
PlanttissueswerecompletelyimmersedinaDABstainingsolution(1mg/mL,pH
3.8)orNBTstainingsolution(NBTpowderwasdissolvedin50mMsodiumphosphate
solutionandpreparedtoaconcentrationof2mg/mL,pH7.5)andreactedfor12hat
roomtemperatureinthedarktolocateH2O2orO2•−.Theplanttissueswerethenim‐
mersedinanhydrousethanolandheatedinaboiling‐waterbathuntiltheoriginalcolor
wasremovedsothatthestainwasclearlyvisible[31,32].Thedeterminationoftotalfla‐
vonoidsandanthocyaninswasbasedonthemethodofLinetal.andHosuetal.[33,34].
AllunlabeledchemicalswerefromSigma‐Aldrich,withtheexceptionofCellulaseR10
andMacerozymeR10,whicharepurchasedfromYakultPharmaceuticalInd.Co.,Ltd.
(Tokyo,Japan).
3.ResultsandDiscussion
3.1.SystemSetupandMonitoringofIntracellularRedoxStatusofMesophyllProtoplast
Todate,cytosolicROSconcentrationsofprotoplastsmeasuredwithfluorescent
probeshaveoftenbeendeterminedempiricallyonrepresentativecells,whichcarriesthe
riskthattheydonotreflecttheaveragevalue,anddonotallowreliableconclusionstobe
drawn[23,35].Toaddressthisproblemandrevealheterogeneitybetweencellpopula‐
tions,wedevelopedamicrofluidicflowcytometrywithanintegratedfluorescencede‐
tector(Figure1A,B)thatallowscellanalysisatatheoreticalrateof1000cellspersecond.
Inthissystem,theprotoplastsuspensionwasinjectedintothemicrofluidicdeviceata
constantflowrateundercontrolledpressure(FluigentMFCS‐EZ,pressureranges0–1bar
and0–345mbar).Becausesinglecellsmoverapidlythroughamicrofabricatedcon‐
strictionchannelwhosewidthcorrespondstothecellsize(Figure1C),thereal‐time
outputfluorescencesignalsfromthedual‐channelPMTswererecordedsimultaneously
usingaLabVIEWprogramviaadataacquisitioncard(DAQ)(Figure1D).Theacquisition
frequencyoftheDAQwassetat100kHztomaintainsamplingaccuracyandoptimize
storageefficiency.Theprotoplastsuspensionwasrunataflowrateofapproximately1
μL/stomaintainabalancebetweenrelativelyhighflowratesandlowsheath‐fluid
pressure.Toevaluatethefeasibilityofthissystem,wefirsttesteditonisolatedAra‐
bidopsismesophyllprotoplastsandexposedthemtovariousconcentrationsofH2O2for
3,6and9hbeforeadding2,7‐ dichlorodihydrofluoresceindiacetate(DCFH‐DA),a
cell‐permeable,nonfluorescentprobe.Duringthesubsequent5minincubationinthe
dark,thecell‐permeableDCFH‐DAprobepenetratestheprotoplastandpreferentially
distributesinthecytoplasm[23,35],leadingtorapidproductionoftheoxidizedfluores‐
cenceproductsinthepresenceofthecellularROScorrespondingtothesynchronous
peakinthegreenchannel(514.5nm).AsshowninFigure2,underthepresentexperi‐
mentalconditions,exogenouslyappliedH2O2ledtoaprogressiveincreaseinROSac‐
cumulationwithinprotoplastsinatime‐anddose‐dependentmanner.Concentrationsof
lessthan600μMH2O2showedgoodlinearagreementintermsoftheintensityofthein‐
tracellularROSsignal;however,atahigherconcentrationof800μM,therewasnofur‐
therincreaseinintracellularfluorescenceintensity,suggestingthatcellsmayturnon
specificquenchingmechanismstodetoxifyandremovethereactiveintermediates(Fig‐
ure2).Thisresultwasessentiallyinagreementwithapreviousreportthatwhenanon‐
ionsurfacewastreatedwithH2O2,saturationofthefluorescenceresponsewasobserved
Biosensors2022,12,6885of14
at1mM[36].Moreimportantly,ourdatashowthatwiththereliableandrapidacquisi‐
tionofthefluorescenceintensityofeachcellinthepopulation,thecompletedatasetfor
theentirepopulationcancharacterizethepropertiesofthecellpopulationwithhigher
accuracy,andcircumventthepotentialbiasandtimewasteofconventionalmicroscopy
inselectingrepresentativecells.
Figure1.Microfluidicflowcytometry.(A)Schematicdiagramofthedevelopedplatform;(B)
Photographofthedevelopedplatform;(C)Time‐lapseimagesofasingleplantcellpassingthrough
thechannel;(D)Real‐timeresponseofdual‐channelfluorescencedetectionofasingleplantcell.
Biosensors2022,12,6886of14
Figure2.ExogenousH2O2‐mediatedchangesinROScontentofArabidopsisprotoplasts.(A)Fluo‐
rescenceimagesofprotoplastswithascaleof25μm;(B–D)fluorescenceintensitygradientsin‐
ducedbyH2O2concentrationinprotoplastsafter3,6,and9h,respectively;(E)effectofH2O2
treatmenttimeonthefluorescenceintensityofprotoplasts.
3.2.CytosolicRedoxStatusofMesophyllProtoplastuponEnvironmentalStresses
ROSplayanessentialroleinthephysiologicalanddevelopmentalprocessesofplant
growth,aswellasinthedefense‐signalcascadeagainstabioticandbioticstressfactors.
Usingthedevelopedsystem,weinvestigatedtheintracellularROSresponsesofmeso‐
phyllprotoplaststoenvironmentalstressessuchashighandlowtemperatures,UVlight,
andheavy‐metalexposure.First,whenprotoplastsweretreatedatlowtemperature(4
°C)andhightemperature(37°C)for15–150min,theintracellularROSsignalincreasedto
asignificantlyhigherlevel(3.5–5foldsaftera150‐minexposure)thanthatinthecontrol
group(20°C),andtheoxidativepressureinducedbyhightemperaturewassignificantly
higherthanthatinducedbylowtemperature(Figure3A,B).Second,Cdionsatacon‐
centrationof40μMinducedanaccumulationofROSovera9hperiod,withsucha
changeincytosolicredoxstatusoccurringmainlyinthefirst6h(Figure3C,D).Third,
after9hofUV‐AorUV‐Birradiation,theincreaseinROSintwopopulationsofmeso‐
Biosensors2022,12,6887of14
phyllprotoplastsincreasedlinearlywithtimeandshowedalmostparalleltrends,with
theUV‐AresponsebeingstrongerthantheUV‐Bresponse(Figure3E,F).Acomparison
ofthethreetreatmentsshowedthateachstresshasitsownuniqueinductionkineticsin
theaccumulationofreactiveoxygenspeciesinsingleplantcells.Inaddition,treatment
withascorbate(AsA),afree‐radicalscavenger,effectivelysuppressedtheproductionof
ROSinducedbytheabovethreetreatments,suggestingthefluorescencesignaldetected
bythesystemwasindeedfromtheoxidationofprotoplasts(FigureS1,Supplementary
Materials).Insummary,theseresultsconfirmthatoursystemcanbeusedfortherelative
quantificationofbiochemicalandphysiologicalpropertiesinprotoplastsina
high‐throughputmannerandforcomparisonbetweencellpopulationsbyselectingap‐
propriateprobes.
Figure3.RedoxstatusofArabidopsisprotoplastsunderenvironmentalstress.(A)Fluorescence
imagesofprotoplastsatdifferenttemperatureswithascaleof25μm;(B)fluorescenceintensityof
protoplastsatdifferenttemperaturestresses;(C)fluorescenceimagesofprotoplastsunderCd2+
treatmentwithascaleof25μm;(D)fluorescenceintensityofprotoplastsunderCd2+;(E)fluores‐
Biosensors2022,12,6888of14
cenceimagesofprotoplastsunderUVtreatmentwithascaleof25μm;(F)fluorescenceintensityof
protoplastsunderUV.
3.3.ROSAccumulationinPetalCellsIsAssociatedwithAnthocyaninLevelunderUV‐B
Irradiation
UVlightisthoughttocauseoxidativedamageinlivingorganisms[37].Incultured
mammaliancells,UVlighthasbeenshowntostimulateH2O2productionbyphotore‐
ductioninperoxisomesandmitochondria[38].Inplants,strongUVirradiationalsoleads
totheoverproductionofROSinchloroplastsandmitochondria,whichinturnhaspro‐
foundeffectsonotherorganellessuchasperoxisomes,cytosolandvacuoles[39].To
protectthemselvesfromUVstress,plantsbiosynthesizeavarietyofspecializedmetabo‐
litesinspecificintracellularcompartments.However,thefunctionalcharacterizationof
thesenaturalproductsisoftenhamperedbytheirlowabundanceandlimitedavailability
inplanttissues[40],sosingle‐cellanalysisbasedonmicrofluidicscanbeaneffectiveal‐
ternativetool.Inthisstudy,weareinterestedinthepossibleroleofanthocyaninsaspart
ofthecomplexantioxidantdefensesystemthattheplantemploystominimizeoxidative
damagecausedbyUVradiation.Tothisend,wefirstmeasuredthetotalflavonoidand
anthocyanincontentinthewhiteandpurplepetalsofPetunia(Petuniahybrida),respec‐
tively.Thedatashowedthatthepurplepetalshada20‐foldhigherconcentrationofan‐
thocyaninsthanthewhitepetals,butasimilarnumberoftotalflavonoids(Figure4A).
Flavonoidsareclassifiedintochalcones,flavanones,flavonols,flavones,isoflavones,
3‐deoxyflavonoids,and(pro)anthocyanidins[41],andusuallyaccumulateinvacuolesor
thecellwall[42].DuetotheirabsorptionintheUVrange,flavonoidsarethoughttoactas
sunscreens.Inaddition,flavonoidsalsoactasROSscavengersduetothepresenceof
phenolichydroxylgroupsintheirstructure[41].Inthiswork,Petuniaflowersofdifferent
colorswerefirstirradiatedwithUVlightandthenstainedwitheitherdiaminobenzidine
(DAB)forH2O2accumulation[43],ornitrobluetetrazolium(NBT)forsuperoxideradical
(O2•–)accumulation[44].Inbothassays,significantdifferencesinstainingintensitywere
observedbetweenthepurpleandwhiteflowers(Figure4C),suggestingthatthepurple
flowertissueaccumulatesmuchlessROSthantheirwhitecounterparts.Wethenisolated
protoplastsfromthetwotypesofpetalsandagainusedDCFH‐DAdyetomeasuretheir
respectiveintracellularconcentrationsofROSafterUV‐AandUV‐Birradiation(Figure
4D,E).Consistentwiththetissue‐levelresults,boththewhiteandpurpleprotoplasts
showedatime‐dependentincreaseinROScomparedwiththerespectiveuntreatedcon‐
trolcells,butmuchlowerconcentrationsofROSweredetectedinthepurpleprotoplasts
comparedwiththewhiteprotoplastsisolatedfrompetals,consistentwiththephotopro‐
tectiveH2O2scavengingeffectofanthocyanins(Figure4D,E).Thepreincubationofpro‐
toplastswithAsAinthedarkpriortoUVtreatmentdramaticallyreducedtheoxidative
burst,suggestingthatAsAenterscellsandreducesUV‐inducedROSaccumulation
(FigureS1).Thephotoprotectiveeffectofanthocyaninsisthoughttobeduetotheir
UV‐shieldingfunction[42]andtheirabilitytoactaseffectivescavengersofROS[45].
Althoughfurtherstudiesareneededtoelucidatethemechanismofvacuole‐localized
anthocyaninsinscavengingROSinthecytoplasm,ourdataclearlydemonstratethekey
roleofanthocyaninsinthedynamicregulationofintracellularredoxstatusunderUV
lightoverexposureatthecellularlevel.Thisheterogeneityofcellularresponsesalso
providescluesforthereal‐timephenotypicinsituidentificationandclassificationof
subpopulationsofcellsunderspecificstimuli.
Biosensors2022,12,6889of14
Figure4.ResponseofPetuniaprotoplaststoUVirradiation.(A)Totalflavonoidandanthocyanin
contentindifferentcoloredpetals;(B)imagesofpetalsattissueandcelllevels;(C)DABandNBT
Biosensors2022,12,68810of14
stainingofdifferentcoloredpetalsunderUVirradiation;(D)fluorescenceimagesofprotoplasts
ROSwithascaleof25μm;(E)fluorescenceintensityofdifferentcoloredprotoplastsonUV.
3.4.AuxinintheRegulationofPrimaryCellWallRegenerationProcessofProtoplasts
Inhigherplants,theprimarycellwall(PCW)isahighlyorganizedstructurecon‐
sistingofcrystallinecellulosemicrofibrilsembeddedinahydratedmatrixofpectinand
hemicellulose[9].Theprimarycellwallwasstrippedduringprotoplastproductionand
willregenerateoverthenext24to48h[46].Next,weinvestigatedthephysiologicalrole
ofauxinundernormalorUV‐Bconditionsintheprocessofprimarycellwallregenera‐
tion.Auxinisanessentialplanthormonethatplaysacrucialroleinmanyphysiological
anddevelopmentalprocessesatthecellular,tissue,andorganlevels[47–49].Tofurther
investigatetheinfluenceofendogenousauxinonplantcellwallregeneration,wefirst
comparedtheprocessofprimarycellwallregenerationinprotoplastsofsur2andtaa1
mutants,whichhadhigherandlowerauxinlevels[50,51],respectively,withthatof
wild‐typemesophyllcells.Cellwalldigestionandregenerationwereconfirmedby
CalcofluorWhite(CFW)thatspecificallystainscelluloseinthecellwall[30].Asshownin
Figure5,theprocessofcellwallregenerationwasenhancedanddelayed,respectively,in
thesetwomutants(Figure5A,B),suggestingthatauxinisrequiredforPCWregeneration
ofprotoplasts.Asanendogenousauxin,theintracellularconcentrationofindole‐3‐acetic
acid(IAA)dependsonitsinfluxandeffluxmediatedbyseparatemembrane‐transport
processes,andithasbeenreportedthat,undernormalconditions,100pgofIAAispre‐
sentinonemgofArabidopsisrootprotoplastandverylittleIAAescapesfromisolated
protoplasts[52].Tomonitorintracellularauxinlevels,weisolatedmesophyllprotoplasts
fromtheArabidopsisDR5:GFPmarkerline,inwhichtheIAA‐responsivepromoterDR5
drivesfluorescentprotein(GFP)expression,allowingindirectquantificationofIAA
withinthecell[53].AsshowninFigure5C,externallyappliedIAAeffectivelyincreased
bluefluorescenceemittedfromthePCWcomparedwithuntreatedprotoplasts,which
wasaccompaniedbyastrongergreenfluorescencesignalfromtheauxinsignalreporter
DR5,indicatingthatexternalauxinmoleculesenteredthecellthroughtheplasmamem‐
brane(Figure5C).ConsideringthatUVradiationcausesoxidativedamagetoplantpro‐
toplasts,itisreasonabletoassumethatcellsprotectthemselvesagainstthisdamageby
acceleratingtheformationoftheprotectivePCW.Totestthishypothesis,weirradiated
DR5:GFPprotoplastswithUV‐Blightandfoundthatsuchtreatmentindeedaccelerated
theregenerationofPCWformation,whichwasunderlinedbyaconcomitantincreasein
auxinconcentrationwithinthecells(Figure5C),confirmingtheregulatoryinvolvement
ofauxininthisprocess.Therefore,dual‐colorfluorescencecanbeusedtosimultaneously
analyzedifferentphysiologicalprocessesbymonitoringtwosignalsoriginatingfromthe
samecell.AsshowninFigure5D,sixdifferentcellclusterscanbeidentifiedafterthecells
weregroupedbasedontheirdual‐channeldetectionsignals.Amongthem,theaddition
ofexogenousauxinresultedinahigherconcentrationofauxinwithinthecellthanUV‐B
exposure,andthepromotingeffectofUV‐Bonprimarycellwallsynthesisoccurred
mainlyinthefirst12h,andatatimepointof24h,therewaslittledifference,probably
duetothecompletionofthePCWinbothgroups.Basedontheresultsofalargenumber
ofcells,suchasmallanddynamicdifferencewasmorereliableandmeaningfulthan
evaluationbymicroscopicobservation(Figure5D).
Biosensors2022,12,68811of14
Figure5.InvolvementofauxininPCWregenerationprogress.(A)PCWfluorescenceimagesof
protoplastsofdifferentArabidopsisgenotypesatthreetimepointswithascalebarof25μm;(B)
FluorescenceintensityofprotoplastsofdifferentArabidopsisgenotypes;(C)IAAdistributionand
PCWfluorescenceimagesofDRF‐GFPundertheinfluenceofUVBirradiationandexogenousIAA;
(D)Two‐channeldetectionofIAAautofluorescenceandPCWfluorescencefromDRF‐GFPproto‐
plasts.
4.Summary
Inthiswork,wehavedevelopedamicrofluidicmethodfortherapid,efficient,and
directmeasurementoftargetedcellularresponsesinprotoplaststhatprovidesanauto‐
mated,easy‐to‐usetoolforcytochemicalresearchonsingleplantcells.Wevalidatedthe
applicabilityofthismethodforsingle‐cellmeasurementsofROSinthepresenceofava‐
rietyofenvironmentalstimuli,allowingadeeperunderstandingofredoxdynamicsin
vivoduringoxidativestress.Wethenappliedthistooltotheanalysisoftwodifferent
plant‐specificphysiologicalprocesses,andprovidedevidenceforthephotoprotective
roleofanthocyaninlocalizedinthevacuoleandfortheformationoftheprimarycellwall
promotedbyauxinatthecellularlevel.Theplatformwedevelopedissimple,fast,and
hasahigh‐throughputcapacity.Inthefuture,quantitative,ratherthanqualitative,
measurementsofinternalbiophysicalandbiochemicalparametersofplantcellsneedto
befurtherdeveloped.Byintegratingelectrical,optical,andmagneticsensingtechniques
withmicrofluidictechnology,micro‐flowcytometersarecertaintoprovidefurtherin‐
sightintolong‐standingandpressingquestionsinplantscience.
SupplementaryMaterials:Thefollowingsupportinginformationcanbedownloadedat:
https://www.mdpi.com/article/10.3390/bios12090688/s1,FigureS1:FluorescenceimagesofAra‐
bidopsisprotoplastswithandwithoutASAunderdifferentexternalstresstreatments(withascale
barof25um).
Biosensors2022,12,68812of14
AuthorContributions:Conceptualization,J.W.andZ.H.;methodology,X.D.(XingdaDaiand)and
S.Z.;validation,X.D.(XingdaDaiand)andS.Z.;formalanalysis,X.D.(XingdaDaiand)andS.Z.;
software,S.L.;resources,H.Q.;investigation,X.D.(XingdaDaiand)andS.Z.;datacuration,J.W.
andZ.H.;writing–originaldraft,J.W.;writing–reviewandediting,J.W.,Z.H.andX.D.(Xuexin
Duan);visualization,X.D.(XingdaDaiand)andZ.H.;supervision,J.W.andX.D.(XuexinDuan);
projectadministration,Z.H.;fundingacquisition,J.W.andX.D.(XuexinDuan).Allauthorshave
readandagreedtothepublishedversionofthemanuscript.
Funding:ThisworkwassupportedbyNationalNaturalScienceFoundationofChina(32170413).
InstitutionalReviewBoardStatement:Notapplicable.
InformedConsentStatement:Notapplicable.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
References
1. Kallberg,J.;Xiao,W.;VanAssche,D.;Baret,J.‐C.;Taly,V.Frontiersinsinglecellanalysis:Multimodaltechnologiesandtheir
clinicalperspectives.LabChip2022,22,2403–2422.https://doi.org/10.1039/d2lc00220e.
2. Banik,S.;Uchil,A.;Kalsang,T.;Chakrabarty,S.;Ali,M.A.;Srisungsitthisunti,P.;Mahato,K.K.;Surdo,S.;Mazumder,N.The
revolutionofPDMSmicrofluidicsincellularbiology.Crit.Rev.Biotechnol.2022,1–19.
https://doi.org/10.1080/07388551.2022.2034733.
3. Zhang,Y.;Zhao,Y.;Cole,T.;Zheng,J.;Bayin,Q.;Guo,J.;Tang,S.‐Y.Microfluidicflowcytometryforblood‐basedbiomarker
analysis.Analyst2022,147,2895–2917.https://doi.org/10.1039/d2an00283c.
4. Sanchez‐Freire,V.;Ebert,A.D.;Kalisky,T.;Quake,S.R.;Wu,J.C.Microfluidicsingle‐cellreal‐timePCRforcomparativeanal‐
ysisofgeneexpressionpatterns.Nat.Protoc.2012,7,829–838.https://doi.org/10.1038/nprot.2012.021.
5. Liu,L.;Cheung,T.H.;Charville,G.W.;Rando,T.A.Isolationofskeletalmusclestemcellsbyfluorescence‐activatedcellsorting.
Nat.Protoc.2015,10,1612–1624.https://doi.org/10.1038/nprot.2015.110.
6. Liu,L.;Fan,B.;Yang,H.;Chen,D.;Zhang,S.;Wang,J.;Chen,J.Anovelmicrofluidicflow‐cytometryforcountingnumbersof
single‐cellβ‐actins.Nanotechnol.Precis.Eng.2020,3,156–161.https://doi.org/10.1016/j.npe.2020.06.001.
7. Cocking,E.C.Amethodfortheisolationofplantprotoplastsandvacuoles.Nature1960,187,962–963.
8. Sheen,J.SignaltransductioninmaizeandArabidopsismesophyllprotoplasts.PlantPhysiol.2001,127,1466–1475.
9. Leucci,M.R.;DiSansebastiano,G.P.;Gigante,M.;Dalessandro,G.;Piro,G.Secretionmarkerproteinsandcell‐wallpolysac‐
charidesmovethroughdifferentsecretorypathways.Planta2007,225,1001–1017.https://doi.org/10.1007/s00425‐006‐0407‐9.
10. Pasternak,T.P.;Prinsen,E.;Ayaydin,F.;Miskolczi,P.;Potters,G.;Asard,H.;VanOnckelen,H.A.;Dudits,D.;Feher,A.The
Roleofauxin,pH,andstressintheactivationofembryogeniccelldivisioninleafprotoplast‐derivedcellsofalfalfa.Plant
Physiol.2002,129,1807–1819.https://doi.org/10.1104/pp.000810.
11. Yoo,S.D.;Cho,Y.H.;Sheen,J.Arabidopsismesophyllprotoplasts:Aversatilecellsystemfortransientgeneexpressionanaly‐
sis.Nat.Protoc.2007,2,1565–1572.https://doi.org/10.1038/nprot.2007.199.
12. Antoniadi,I.;Skalicky,V.;Sun,G.;Ma,W.;Galbraith,D.W.;Novak,O.;Ljung,K.Fluorescenceactivatedcellsorting‐Aselec‐
tivetoolforplantcellisolationandanalysis.Cytom.PartA2021.https://doi.org/10.1002/cyto.a.24461.
13. Noctor,G.;Reichheld,J.P.;Foyer,C.H.ROS‐relatedredoxregulationandsignalinginplants.Semin.CellDev.Biol.2018,80,3–
12.https://doi.org/10.1016/j.semcdb.2017.07.013.
14. Apel,K.;Hirt,H.Reactiveoxygenspecies:Metabolism,oxidativestress,andsignaltransduction.Annu.Rev.PlantBiol.2004,55,
373–399.https://doi.org/10.1146/annurev.arplant.55.031903.141701.
15. Mittler,R.;Vanderauwera,S.;Gollery,M.;VanBreusegem,F.Reactiveoxygengenenetworkofplants.TrendsPlantSci.2004,9,
490–498.https://doi.org/10.1016/j.tplants.2004.08.009.
16. Yang,S.;Yu,Q.;Zhang,Y.;Jia,Y.;Wan,S.;Kong,X.;Ding,Z.ROS:TheFine‐TunerofPlantStemCellFate.TrendsPlantSci.
2018,23,850–853.https://doi.org/10.1016/j.tplants.2018.07.010.
17. delRio,L.A.;Pastori,G.M.;Palma,J.M.;Sandalio,L.M.;Sevilla,F.;Corpas,F.J.;Jimenez,A.;Lopez‐Huertas,E.;Hernandez,J.A.
Theactivatedoxygenroleofperoxisomesinsenescence.PlantPhysiol.1998,116,1195–1200.
https://doi.org/10.1104/pp.116.4.1195.
18. Niyogi,K.K.PHOTOPROTECTIONREVISITED:GeneticandMolecularApproaches.Annu.Rev.PlantPhysiol.PlantMol.Biol.
1999,50,333–359.https://doi.org/10.1146/annurev.arplant.50.1.333.
19. Soares,C.;Carvalho,M.E.A.;Azevedo,R.A.;Fidalgo,F.Plantsfacingoxidativechallenges—Alittlehelpfromtheantioxidant
networks.Environ.Exp.Bot.2019,161,4–25.
20. Allan,A.C.;Fluhr,R.TwoDistinctSourcesofElicitedReactiveOxygenSpeciesinTobaccoEpidermalCells.PlantCell1997,9,
1559–1572.https://doi.org/10.1105/tpc.9.9.1559.
21. Karpinski,S.;Reynolds,H.;Karpinska,B.;Wingsle,G.;Creissen,G.;Mullineaux,P.Systemicsignalingandacclimationin
responsetoexcessexcitationenergyinArabidopsis.Science1999,284,654–657.https://doi.org/10.1126/science.284.5414.654.
22. Mittler,R.ROSAreGood.TrendsPlantSci.2017,22,11–19.https://doi.org/10.1016/j.tplants.2016.08.002.
Biosensors2022,12,68813of14
23. Smirnoff,N.;Arnaud,D.Hydrogenperoxidemetabolismandfunctionsinplants.NewPhytol.2019,221,1197–1214.
https://doi.org/10.1111/nph.15488.
24. Rhee,S.G.Cellsignaling.H2O2,anecessaryevilforcellsignaling.Science2006,312,1882–1883.
https://doi.org/10.1126/science.1130481.
25. Cerny,M.;Habanova,H.;Berka,M.;Luklova,M.;Brzobohaty,B.HydrogenPeroxide:ItsRoleinPlantBiologyandCrosstalk
withSignallingNetworks.Int.J.Mol.Sci.2018,19,2812.https://doi.org/10.3390/ijms19092812.
26. Jabs,T.;Dietrich,R.A.;Dangl,J.L.InitiationofrunawaycelldeathinanArabidopsismutantbyextracellularsuperoxide.Sci‐
ence1996,273,1853–1856.https://doi.org/10.1126/science.273.5283.1853.
27. Potikha,T.S.;Collins,C.C.;Johnson,D.I.;Delmer,D.P.;Levine,A.Theinvolvementofhydrogenperoxideinthedifferentiation
ofsecondarywallsincottonfibers.PlantPhysiol.1999,119,849–858.https://doi.org/10.1104/pp.119.3.849.
28. Xia,Y.;Whitesides,G.M.SoftLithography.Angew.Chem.Int.Ed.Engl.1998,37,550–575.
https://doi.org/10.1002/(SICI)1521‐3773(19980316)37:5<550::AID‐ANIE550>3.0.CO;2‐G.
29. Kang,H.;Naing,A.H.;Park,S.K.;Chung,M.Y.;Kim,C.K.Protoplastisolationandtransientgeneexpressionindifferentpe‐
tuniacultivars.Protoplasma2022,1–10.https://doi.org/10.1007/s00709‐022‐01776‐9.
30. Nagata,T.;Takebe,I.Cellwallregenerationandcelldivisioninisolatedtobaccomesophyllprotoplasts.Planta1970,92,301–
308.
31. Wu,T.‐M.;Huang,J.‐Z.;Oung,H.‐M.;Hsu,Y.‐T.;Tsai,Y.‐C.;Hong,C.‐Y.H2O2‐basedmethodforrapiddetectionof
transgene‐freericeplantsfromsegregatingCRISPR/Cas9genome‐editedprogenies.Int.J.Mol.Sci.2019,20,3885.
32. GrelletBournonville,C.F.;Díaz‐Ricci,J.C.QuantitativedeterminationofsuperoxideinplantleavesusingamodifiedNBT
stainingmethod.Phytochem.Anal.2011,22,268–271.
33. Lin,J.‐Y.;Tang,C.‐Y.Determinationoftotalphenolicandflavonoidcontentsinselectedfruitsandvegetables,aswellastheir
stimulatoryeffectsonmousesplenocyteproliferation.FoodChem.2007,101,140–147.
34. Hosu,A.;Cristea,V.‐M.;Cimpoiu,C.Analysisoftotalphenolic,flavonoids,anthocyaninsandtanninscontentinRomanian
redwines:Predictionofantioxidantactivitiesandclassificationofwinesusingartificialneuralnetworks.FoodChem.2014,150,
113–118.
35. Exposito‐Rodriguez,M.;Laissue,P.P.;Yvon‐Durocher,G.;Smirnoff,N.;Mullineaux,P.M.Photosynthesis‐dependentH2O2
transferfromchloroplaststonucleiprovidesahigh‐lightsignallingmechanism.Nat.Commun.2017,8,49.
https://doi.org/10.1038/s41467‐017‐00074‐w.
36. Kristiansen,K.A.;Jensen,P.E.;Møller,I.M.;Schulz,M.Monitoringreactiveoxygenspeciesformationandlocalisationinliving
cellsbyuseofthefluorescentprobeCM‐H(2)DCFDAandconfocallasermicroscopy.Physiol.Plant2009,136,369–383.
37. Babu,T.S.;Akhtar,T.A.;Lampi,M.A.;Tripuranthakam,S.;Dixon,D.G.;Greenberg,B.M.Similarstressresponsesareelicited
bycopperandultravioletradiationintheaquaticplantLemnagibba:Implicationofreactiveoxygenspeciesascommonsig‐
nals.PlantCellPhysiol.2003,44,1320–1329.https://doi.org/10.1093/pcp/pcg160.
38. Hockberger,P.E.;Skimina,T.A.;Centonze,V.E.;Lavin,C.;Chu,S.;Dadras,S.;Reddy,J.K.;White,J.G.Activationoffla‐
vin‐containingoxidasesunderlieslight‐inducedproductionofH2O2inmammaliancells.Proc.Natl.Acad.Sci.USA1999,96,
6255–6260.https://doi.org/10.1073/pnas.96.11.6255.
39. Noctor,G.;Foyer,C.H.IntracellularRedoxCompartmentationandROS‐RelatedCommunicationinRegulationandSignaling.
PlantPhysiol.2016,171,1581–1592.https://doi.org/10.1104/pp.16.00346.
40. Carqueijeiro,I.;Guimaraes,A.L.;Bettencourt,S.;Martinez‐Cortes,T.;Guedes,J.G.;Gardner,R.;Lopes,T.;Andrade,C.;Bispo,
C.;Martins,N.P.;etal.IsolationofCellsSpecializedinAnticancerAlkaloidMetabolismbyFluorescence‐ActivatedCellSort‐
ing.PlantPhysiol.2016,171,2371–2378.https://doi.org/10.1104/pp.16.01028.
41. FalconeFerreyra,M.L.;Serra,P.;Casati,P.RecentadvancesontherolesofflavonoidsasplantprotectivemoleculesafterUV
andhighlightexposure.Physiol.Plant2021,173,736–749.
42. Winkel‐Shirley,B.Flavonoidbiosynthesis.Acolorfulmodelforgenetics,biochemistry,cellbiology,andbiotechnology.Plant
Physiol.2001,126,485–493.https://doi.org/10.1104/pp.126.2.485.
43. Myouga,F.;Hosoda,C.;Umezawa,T.;Iizumi,H.;Kuromori,T.;Motohashi,R.;Shono,Y.;Nagata,N.;Ikeuchi,M.;Shinozaki,
K.Aheterocomplexofironsuperoxidedismutasesdefendschloroplastnucleoidsagainstoxidativestressandisessentialfor
chloroplastdevelopmentinArabidopsis.PlantCell2008,20,3148–3162.https://doi.org/10.1105/tpc.108.061341.
44. Kawai‐Yamada,M.;Ohori,Y.;Uchimiya,H.DissectionofArabidopsisBaxinhibitor‐1suppressingBax‐,hydrogenperoxide‐,
andsalicylicacid‐inducedcelldeath.PlantCell2004,16,21–32.https://doi.org/10.1105/tpc.014613.
45. Isah,T.Stressanddefenseresponsesinplantsecondarymetabolitesproduction.Biol.Res.2019,52,39.
https://doi.org/10.1186/s40659‐019‐0246‐3.
46. Chen,L.C.;Han,Z.Y.;Fan,X.T.;Zhang,S.H.;Wang,J.H.;Duan,X.X.Animpedance‐coupledmicrofluidicdeviceforsingle‐cell
analysisofprimarycellwallregeneration.Biosens.Bioelectron.2020,165,112374.https://doi.org/10.1016/j.bios.2020.112374.
47. Hagen,G.Auxinsignaltransduction.EssaysBiochem.2015,58,1–12.https://doi.org/10.1042/bse0580001.
48. Millner,P.A.Theauxinsignal.Curr.Opin.CellBiol.1995,7,224–231.https://doi.org/10.1016/0955‐0674(95)80032‐8.
49. Weijers,D.;Jurgens,G.Funnelingauxinaction:Specificityinsignaltransduction.Curr.Opin.PlantBiol.2004,7,687–693.
https://doi.org/10.1016/j.pbi.2004.09.006.
50. Delarue,M.;Prinsen,E.;VanOnckelen,H.;Caboche,M.;Bellini,C.Sur2mutationsofArabidopsisthalianadefineanewlocus
involvedinthecontrolofauxinhomeostasis.PlantJ.1998,14,603–611.https://doi.org/10.1046/j.1365‐313X.1998.00163.x.
Biosensors2022,12,68814of14
51. Stepanova,A.N.;Robertson‐Hoyt,J.;Yun,J.;Benavente,L.M.;Xie,D.‐Y.;Dolezal,K.;Schlereth,A.;Juergens,G.;Alonso,J.M.
TAA1‐mediatedauxinbiosynthesisisessentialforhormonecrosstalkandplantdevelopment.Cell2008,133,177–191.
https://doi.org/10.1016/j.cell.2008.01.047.
52. Petersson,S.V.;Johansson,A.I.;Kowalczyk,M.;Makoveychuk,A.;Wang,J.Y.;Moritz,T.;Grebe,M.;Benfey,P.N.;Sandberg,
G.;Ljung,K.AnauxingradientandmaximumintheArabidopsisrootapexshownbyhigh‐resolutioncell‐specificanalysisof
IAAdistributionandsynthesis.PlantCell2009,21,1659–1668.https://doi.org/10.1105/tpc.109.066480.
53. Ulmasov,T.;Murfett,J.;Hagen,G.;Guilfoyle,T.J.Aux/IAAproteinsrepressexpressionofreportergenescontainingnatural
andhighlyactivesyntheticauxinresponseelements.PlantCell1997,9,1963–1971.https://doi.org/10.1105/tpc.9.11.1963.
... Seedlings from different treatment groups were immersed in a pH 6.5 Tris-KCl buffer (10 mM Tris, 50 mM KCl) supplemented with 20 μM DCFH-DA for 48 h under salt stress conditions (Medeiros et al. 2017). Subsequent to that, vacuum permeation was conducted at room temperature for 30 min, followed by multiple washes with distilled water to remove any excessive solution (Dai et al. 2022). Fluorescence accumulation in the leaves was visualized through laser confocal scanning microscopy while employing ImageJ software to calculate the average optical density of guard cells. ...
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Flow cytometry has proven its capability for rapid and quantitative analysis of individual cells and the separation of targeted biological samples from others. The emerging microfluidics technology makes it possible to develop portable microfluidic diagnostic devices for point-of-care testing (POCT) applications. Microfluidic flow cytometry (MFCM), where flow cytometry and microfluidics are combined to achieve similar or even superior functionalities on microfluidic chips, provides a powerful single-cell characterisation and sorting tool for various biological samples. In recent years, researchers have made great progress in the development of the MFCM including focusing, detecting, and sorting subsystems, and its unique capabilities have been demonstrated in various biological applications. Moreover, liquid biopsy using blood can provide various physiological and pathological information. Thus, biomarkers from blood are regarded as meaningful circulating transporters of signal molecules or particles and have great potential to be used as non (or minimally)-invasive diagnostic tools. In this review, we summarise the recent progress of the key subsystems for MFCM and its achievements in blood-based biomarker analysis. Finally, foresight is offered to highlight the research challenges faced by MFCM in expanding into blood-based POCT applications, potentially yielding commercialisation opportunities.
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Fluorescence activated cell sorting—A selective tool for plant cell isolation and analysis
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Full-text available
  • May 2021
Instrumentation for flow cytometry and sorting is designed around the assumption that samples are single‐cell suspensions. However, with few exceptions, higher plants comprise complex multicellular tissues and organs, in which the individual cells are held together by shared cell walls. Single‐cell suspensions can be obtained through digestion of the cells walls and release of the so‐called protoplasts (plants without their cell wall). Here we describe best practices for protoplast preparation, and for analysis through flow cytometry and cell sorting. Finally, the numerous downstream applications involving sorted protoplasts are discussed.
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A novel microfluidic flow-cytometry for counting numbers of single-cell β-actins
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Full-text available
  • Jul 2020
As a house keeping protein with stable expressions, β-actin is used as a loading control in normalization of western blotting. However, the actual numbers of β-actins at the single-cell level remain elusive. Based on a home-developed flow cytometry, single-cell numbers of β-actin from 8 cell types (subtypes) and 2 tumour patient samples were quantified as 9.62 ± 4.29 × 10⁵ (A549, Ncell = 14,242), 6.46 ± 3.34 × 10⁵ (Hep G2, Ncell = 35,932), 1.58 ± 0.90 × 10⁶ (MCF 10A, Ncell = 16,650), 1.08 ± 0.48 × 10⁶ (HeLa, Ncell = 26,151), 7.60 ± 4.34 × 10⁵ (PC3, Ncell = 11,922), 1.10 ± 0.72 × 10⁶ (SACC-83, Ncell = 13,616), 8.58 ± 4.54 × 10⁵ (CAL 27, Ncell = 7271), 9.00 ± 4.69 × 10⁵ (CAL 27-LN2, Ncell = 6222), 8.26 ± 4.48 × 10⁵ (Oral Tumour Patient I, Ncell = 359), and 8.19 ± 5.12 × 10⁵ (Oral Tumour Patient II, Ncell = 175), and were analyzed by statistical approaches including one-way analysis of variance, neural network based pattern recognition and Bayesian estimation, with varied expressions of β-actins among different cell types located. The dataset reported in this study may serve as a reference in future studies of quantitative protein analysis.
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H2O2-Based Method for Rapid Detection of Transgene-Free Rice Plants from Segregating CRISPR/Cas9 Genome-Edited Progenies
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Full-text available
Genome-editing techniques such as CRISPR/Cas9 have been widely used in crop functional genomics and improvement. To efficiently deliver the guide RNA and Cas9, most studies still rely on Agrobacterium-mediated transformation, which involves a selection marker gene. However, several limiting factors may impede the efficiency of screening transgene-free genome-edited plants, including the time needed to produce each life cycle, the response to selection reagents, and the labor costs of PCR-based genotyping. To overcome these disadvantages, we developed a simple and high-throughput method based on visual detection of antibiotics-derived H2O2 to verify transgene-free genome-edited plants. In transgenic rice containing hygromycin phosphotransferase (HPT), H2O2 content did not change in the presence of hygromycin B (HyB). In contrast, in transgenic-free rice plants with 10-h HyB treatment, levels of H2O2 and malondialdehyde, indicators of oxidative stress, were elevated. Detection of H2O2 by 3,3′-diaminobenzidine (DAB) staining suggested that H2O2 could be a marker to efficiently distinguish transgenic and non-transgenic plants. Analysis of 24 segregating progenies of an HPT-containing rice plant by RT-PCR and DAB staining verified that DAB staining is a feasible method for detecting transformants and non-transformants. Transgene-free genome-edited plants were faithfully validated by both PCR and the H2O2-based method. Moreover, HyB induced overproduction of H2O2 in leaves of Arabidopsis, maize, tobacco, and tomato, which suggests the potential application of the DAB method for detecting transgenic events containing HPT in a wide range of plant species. Thus, visual detection of DAB provides a simple, cheap, and reliable way to efficiently identify transgene-free genome-edited and HPT-containing transgenic rice.
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Stress and defense responses in plant secondary metabolites production
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Full-text available
  • Dec 2019
Abstract In the growth condition(s) of plants, numerous secondary metabolites (SMs) are produced by them to serve variety of cellular functions essential for physiological processes, and recent increasing evidences have implicated stress and defense response signaling in their production. The type and concentration(s) of secondary molecule(s) produced by a plant are determined by the species, genotype, physiology, developmental stage and environmental factors during growth. This suggests the physiological adaptive responses employed by various plant taxonomic groups in coping with the stress and defensive stimuli. The past recent decades had witnessed renewed interest to study abiotic factors that influence secondary metabolism during in vitro and in vivo growth of plants. Application of molecular biology tools and techniques are facilitating understanding the signaling processes and pathways involved in the SMs production at subcellular, cellular, organ and whole plant systems during in vivo and in vitro growth, with application in metabolic engineering of biosynthetic pathways intermediates.
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The revolution of PDMS microfluidics in cellular biology
Article
  • Apr 2022
Microfluidics is revolutionizing the way research on cellular biology has been traditionally conducted. The ability to control the cell physicochemical environment by adjusting flow conditions, while performing cellular analysis at single-cell resolution and high-throughput, has made microfluidics the ideal choice to replace traditional in vitro models. However, such a revolution only truly started with the advent of polydimethylsiloxane (PDMS) as a microfluidic structural material and soft-lithography as a rapid manufacturing technology. Indeed, before the "PDMS age," microfluidic technologies were: costly, time-consuming and, more importantly, accessible only to specialized laboratories and users. The simplicity of molding PDMS in various shapes along with its inherent properties (transparency, biocompatibility, and gas permeability) has spread the applications of innovative microfluidic devices to diverse and important biological fields and clinical studies. This review highlights how PDMS-based microfluidic systems are innovating pre-clinical biological research on cells and organs. These devices were able to cultivate different cell lines, enhance the sensitivity and diagnostic effectiveness of numerous cell-based assays by maintaining consistent chemical gradients, utilizing and detecting the smallest number of analytes while being high-throughput. This review will also assist in identifying the pitfalls in current PDMS-based microfluidic systems to facilitate breakthroughs and advancements in healthcare research.
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Recent advances on the roles of flavonoids as plant protective molecules after UV and high light exposure
Article
  • Aug 2021
Flavonoids are plant specialized metabolites that consist of one oxygenated and two aromatic rings. Different flavonoids are grouped according to the oxidation degree of the carbon rings; they can later be modified by glycosylations, hydroxylations, acylations, methylations or prenylations. These modifications generate a wide collection of different molecules which have various functions in plants. All flavonoids absorb in the UV wavelengths, they mostly accumulate in the epidermis of plant cells and their biosynthesis is generally activated after UV exposure. Therefore, they have been assumed to protect plants against exposure to radiation in this range. Some flavonoids also absorb in other wavelengths, for example anthocyanins, which absorb light in the visible part of the solar spectrum. Besides, some flavonoids show antioxidant properties, i.e., they act as scavengers of reactive oxygen species that could be produced after high fluence UV exposure. However, to date most reports were based on in vitro studies, and there is very little in vivo evidence of how their roles are carried out. In this review we first summarize the biosynthetic pathway of flavonoids and their characteristics, and we describe recent advances on the investigation of the role of three of the most abundant flavonoids: flavonols, flavones and anthocyanins, protecting plants against UV exposure and high light exposure. We also present examples of how using UV-B supplementation to increase flavonoid content, it is possible to improve plant nutritional and pharmaceutical values. This article is protected by copyright. All rights reserved.
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An impedance-coupled microfluidic device for single-cell analysis of primary cell wall regeneration
Article
  • Jun 2020
  • BIOSENS BIOELECTRON
Primary cell wall (PCW) is a rigid yet flexible cell wall surrounding plant cells and it plays key roles in plant growth, cell differentiation, intercellular communication, water movement and defence. As a technique widely used to study the characteristics of mammalian cells, electrical impedance spectroscopy (EIS) is rarely used in plant science. In this work, we designed and fabricated an EIS based biosensor coupled with microfluidic platform to investigate the formation process of primary cell wall (PCW) at the single-cell level. Arabidopsis mesophyll cells with completely regenerated PCW showed significantly higher impedance values compared to the nascent protoplasts without PCW, demonstrating that PCW formation caused a dramatic change in cell electrical properties. The device could also discriminate plant mutant cells with modified PCW compositions, thus provided a novel tool for physical phenotyping of plant cells. The dose-dependent effects of exogenously applied auxin on PCW regeneration were corroborated on this platform which revealed its potential to sensitively detect the influences of in vitro stimuli. This work not only provided one novel application of impedance-based biosensor to characterize a plant-specific developmental event, but also revealed the promises of EIS integrated microfluidic system as a sensitive, time-effective and low-cost platform to characterize single plant cells and make new scientific discoveries in plant science.
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