Physiological responses to Megafol® treatments in tomato plants under drought stress: A phenomic and molecular approach

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ContentslistsavailableatScienceDirect

Scientia

Horticulturae

j o u r n a l ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / s c i h o r t i

Physiological

responses

to

Megafol

®

treatments

in

tomato

plants

under

drought

stress:

A

phenomic

and

molecular

approach

Angelo

Petrozza

a,1

_,

_Antonietta

_Santaniello

b,∗,1

_,

_Stephan

_Summerer

a

_,

Gianluca

Di

Tommaso

c

_,

_Donata

_Di

_Tommaso

c

_,

_Eleonora

_Paparelli

b

_,

_Alberto

_Piaggesi

c

_,

Pierdomenico

Perata

b

_,

_Francesco

_Cellini

a

a_ALSIA_Centro_Ricerche_Metapontum_Agrobios,_s.s._Jonica_106,_km_448,2,_Metaponto,_MT_75010,_Italy

b_PlantLab,_Institute_of_Life_Sciences,_Scuola_Superiore_Sant’Anna,_Piazza_Martiri_della_Libertà_33,_Pisa_56127,_Italy c_Valagro_S.p.A,_via_Cagliari_1,_Atessa,_CH_66041,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received4February2014

Receivedinrevisedform11April2014 Accepted21May2014

Availableonline13June2014 Keywords: Droughtstress Biostimulants Megafol Tomato Scanalyzer3D

a

b

s

t

r

a

c

t

Droughtis oneof themostsigniﬁcant abioticstresses thatlimitsthe growthandproductivityof

cropplants.Weinvestigatedthephysiologicalandmolecularresponsesoftomatoplantstreatedwith

Megafol®_(Valagro_S.p.A),_under_speciﬁc_drought_conditions._The_goal_was_to_evaluate_the_impact_of

Megafol®,abiostimulantcomposedofacomplexofvitamins,aminoacids,proteinsandbetaines,in

atten-uatingthenegativephysiologicalresponsesofdrought.Tomatoplantsweregrowninagreenhouse,and

physiologicalparameterswerecollectedusingScanalyzer3D(LemnaTec,GmbH),aplantphenomics

platform.Usingthistechnologyitispossibletodynamicallystudytheeffectsofbiostimulants,such

asMegafol®,onplantdevelopmentintermsofearlydetectionofphysiologicalplantstressresponses.

Theresultsshowedthatdrought-stressedplantstreatedwithMegafol®_were_healthier_in_terms_of_the

biomassproducedandchlorophyllﬂuorescence,thushighlightingthehighertolerancetostressofthe

treatedplants.TheeffectsofMegafol®werealsostudiedatamolecularlevelbyanalysingtheinduction

ofgenestypicallyinvolvedindroughtstressresponses.OurresultsdemonstratetheefﬁcacyofMegafol®

toreducedrought-stressrelateddamageintomatoplants.

1. Introduction

Multi-spectrumanalysis(infra-redandvisible,ultravioletlight)

ofreﬂected orre-emittedlight fromtheplantcrown, stemand

leavesprovidesinformation onthenutritional,hydrologicaland

physio-pathologicalstateofplants,aswellasontheplant’s

abil-itytointerceptlight. Therelativesimplicityand highefﬁciency

ofsuchimagingtechnologyandthesubsequentdataprocessing

enablesittobesuccessfullyintegratedinexperimentsthatinvolve

manyvariables,ahighnumberofsamplingintervals,and

multi-plecomparisons.Giventhatitcanextractphenotypicdataquickly

and efﬁciently, multi-spectrum analysis has become an

inter-facebetweenmoleculargeneticsandplantphenotyping.Infact,

Abbreviations: ABA,abscissicacid;DHN,dehydrin;HIS,hue,saturationand intensity;LEA,lateembryogenesisabundant;NIR,nearinfra-red;RGB,red,green andblue.

∗ Correspondingauthor.Tel.:+39502216539;fax:+39502216532. E-mailaddress:a.santaniello@sssup.it(A.Santaniello).

1 _Both_the_authors_contributed_equally_to_this_work.

phenotypicanalysisofnewlines hashistoricallybeena

bottle-neckforfundamentalgeneticstudiesinplants(FurbankandTester,

2011).

Withvisiblelightillumination,thereﬂectancespectrumfrom

leavesisparticularlypoor.Infactmostwavelengths(i.e.colours)

are absorbed by the pigmentsof the photosyntheticapparatus

and in particular chlorophyll. Evenwith this reduced emission

spectrum,itisstillpossibletoextractagreatdealofphenotypic

information,suchasthegreenindex,andtheseplantimagesare

valuable for measuring the many morphometric parameters of

plantssuchasheight,width,andbiomass(Rajendranetal.,2009).

Atincreasingthewavelengths,thenearinfra-red(NIR)is

impor-tant in identifyingthe chemical functional groups through the

resonancefrequenciesofbondstretching,contractingand

bend-ing. In detail, wavelength intervals from 1450 to1930nm and

around2500nmhavebeenalreadyusedtostudythedifferences

inthewatercontentlevelsofplants,quantifyingwatermolecules

throughtheOHchemicalbonds(Bergeretal.,2010).Therefore,

thisapproachallowedtomeasuretheinternalwatercontentlevels

withoutdisturbingtheplantrepeatedlyovertime(Petrozzaetal.,

2009).

http://dx.doi.org/10.1016/j.scienta.2014.05.023

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Anotherfundamentalparameterlargelyusedisthechlorophyll

ﬂuorescence,bywhichitispossibletoseesignsofstressinplants

muchearlierthanwithtraditionalphenotypicmethods.Inrecent

years,theanalysisofchlorophyllﬂuorescencehasbecome

ubiqui-tousinplantecophysiologystudies,andtheprincipleunderlying

thistechniqueisrelativelystraightforward.Lightenergyabsorbed

bychlorophyllmoleculescan(1)beusedtodrivephotosynthesis,or

(2)bedissipatedasheator(3)bere-emittedaslight–chlorophyll

ﬂuorescence(Oxborough,2004).Underoptimalgrowthconditions

muchoftheenergyabsorbedfromlightisdirectedinto

photosyn-thesisand plantsemit abasal levelofchlorophyllﬂuorescence.

However, when the photosynthesis apparatus is under abiotic

stress,thebalanceisdisturbedandmoreenergymustbereleased

throughheatandﬂuorescence(Mülleretal.,2001).

Duetotherobustness,relativelylowcost,andreasonable

analy-sistime,theuseofhighthroughputplantimageanalysiscouldbea

veryinterestingtoolforstudyingplants’physiologicalresponsesto

differentenvironmentalconditions,suchasdroughtstress.Water

deﬁciencyis a severe environmentalstress,and hasa negative

impactoncropyieldsworldwide(Boyer,1982;Pennisi,2008).

Con-sideringthatwaterresourcesaredecreasingandclimatechangeis

expectedtoincreasetheamountofdryland,theoutlookfor

agri-cultureisdramatic(BattistiandNaylor,2009).Itispredictedthatin

2050,theglobalpopulationwillincreasefromthecurrent6.7

bil-liontoover9billion,andover3billionwillbethreatenedbywater

shortages.Anincreasingpopulationalsomeansahigherdemand

forfood:maintaininga highyieldin droughtconditions isthus

amajorpriorityasisunderstandingtheresponsesandadaptive

mechanismsofcropstowater deﬁcit.However,the

physiologi-calmechanismsofyieldmaintenance underdroughtconditions

arepoorlyunderstood(TuberosaandSalvi,2006),sincethe

mech-anismsthatplantsusuallyusetomaintaingrowthinlow water

arecomplexandstillnotwellcharacterized.Thestrategiestocope

withwaterscarcitydifferdependingonthespeciesandgenotypes

oftheplants,aswellasonthetypeofdrought.Generally,plants

respondtodrought witha series of physiological mechanisms,

whichincludestomatalclosure,repressionofcellgrowthand

pho-tosynthesis,leadingtoanoverallreductioninplantbiomass.

Furthermore,manychangesatthecellularandmolecularlevel

occur, involvingextensiveincreases in the expressionlevels of

thosegenesthatprotecttheplantfromstressdamage(Shinozaki

andYamaguchi-Shinozaki,2007).Thesegenescanbeclassiﬁedinto

twomajorgroups,accordingtotheirputativefunctions.Theﬁrst

groupincludesearlyresponsetranscriptionalactivators,including

transcriptionfactorsandproteinkinases.Stress-related

transcrip-tionfactorsplayanimportantrole intheregulationofdrought

toleranceandmainlyincludebZIP,WRKY,MYB,andAP2/EREBP

proteins(Abeetal.,1997;FinkelsteinandLynch,2000;Marèetal.,

2004;Songetal.,2005).Thesetranscriptionfactorsrespondto

envi-ronmentalsignals,namelydroughtor highsalinity,alsothough

theinvolvementofabscisicacid.However,itmustbehighlighted

thatbothABA-dependentandindependentpathwaysoperatein

responsetodrought(ShinozakiandYamaguchi-Shinozaki,2007).

Downstreamoftheactivationoftheﬁrstgroupofgenesoperatesa

secondgenegroup,thatincludesgenesthatcodifyfordownstream

effectorsinthestressresponsepathway,includingstructural

pro-teins,osmoregulatoryproteins,antioxidantproteins,aquaporins,

andlateembryogenesisabundant(LEA)proteins(Baillyetal.,2001;

Bretonetal.,2003;WangandYe,2006;Shinozakiand Yamaguchi-Shinozaki,2007).Sincetheyincreasemarkedlyinvegetativeorgans

duringwaterdeﬁcits,aprotectiveroleduringwaterlimitationhas

beensuggested(Bies-Etheveetal.,2008;Popelkaetal.,2010).The

DHN(dehydrin)genesalsobelongtotheLEAproteinsfamily,which

areup-regulatedduringdroughtstress(Zhuetal.,2000)and,for

thisreason,havebeenwidelystudiedinhigherplantsinresponse

towater deﬁcits(Ismailetal.,1999;Nylanderetal.,2001).The

mostimportant genomic information concerningtheresponses

ofdifferentplantspecies todroughtstress,collectedsofar,has

beenprovidedbygenome-wideexpressionproﬁlingunderwater

deﬁcits,includingArabidopsis(Sekietal.,2001;Bray,2004;Huang

etal.,2008),rice(Rabbanietal.,2003;Hazenetal.,2005),barley (Talaméetal.,2007;Guoetal.,2009),maize(Hayano-Kanashiro

etal.,2009), sorghum(Prattetal., 2005), andpotato (

Vasquez-Robinetetal.,2008).

Despitetheagronomicandeconomicrelevanceoftomato,this

kindofinformationislacking,thereforethemechanismsthatdrive

responsestowaterdeﬁcitsintomatohavenotbeenwell

character-izedyet,andonlyaverysmallnumberofgenesthatplayarolein

droughttolerancehavebeenidentiﬁed(Atarésetal.,2011;Pineda

etal.,2010).Tomatoisparticularlysensitivetoanumberof

envi-ronmentalstresses,especiallyextremetemperature,droughtand

salinity(Kaloo,1993).

Improvingplantresponsestostressconditionsmightbe

possi-blethroughtreatmentwithspeciﬁcproducts,madewithnatural

productsandknownasbiostimulants.Theapplicationof

biostim-ulantshasapositiveimpactonplantnutritionandplantgrowth,

andalsoprovidesanti-stresseffects(Richardsonetal.,2004).These

important qualities highlight the importance of increasing the

knowledgeregardingtheirphysiologicalfunctions,whichare

cur-rentlyunclear.

Theaimofthisstudywastocharacterizetheresponsetodrought

stressinduced in tomato plants treated withMegafol®, a

bios-timulantprovided by ValagroS.p.A that is knowntopositively

inﬂuencecropyield(Para –dikovi ´cetal.,2011),bycollecting

infor-mationregardingthephysiologicalandmolecularchangesinduced

bythestressandthepotentialpositiveeffects,intermsoftolerance,

duetothetreatment.Thephysiologicalmechanismswere

evalu-atedbyimageanalysis(Scanalyzer3DSystem,LemnaTec),using

visiblelight(RGB),ﬂorescence,andnearinfra-red(NIR)chambers.

2. Materialsandmethods

2.1. Plantmaterialandgrowingconditions

Theexperiments wereperformedin April 2012at thePlant

PhenomicslaboratoryattheALSIAResearchCenterMetapontum

Agrobios,inSouthernItaly.Tomatoplants(cvIkram,SingentaAG)

weregrowninagreenhouseundernaturalambientlight

condi-tions.Fourweeksafterseeding,afterthethirdtrueleafwasfully

expanded,plantsweretransplantedintowhitevases(16cm

diam-eter,20cmheight)fortheactualexperiment.Thevases,volume

max.4L,contained3.5Lofsoilconsistingofa 50:50mixtureof

peatmossandriversand.

Atthemomentoftransplantingintothepots,20unitsof

nitro-gen(N2),40unitsofanhydrousphosphite(P2O5),and20unitsof

potassiumoxide(K2O) wereaddedtothesoilmixture.Drought

stresswasinducedafterthethirdorfourthleaf,non-cotyledon,

hadfullyformedandcontinueduntiltheendoftheexperiment.

Plantsamplesandplantimagesweretakenstartingthreedays

priortothebeginningofthedroughtstress(Day0),between7am

and 9am. Thecontrol plants wereirrigated with 200mLevery

dayboth beforeandduringthedroughtstressexperiment.This

quantityofwater waspreviously determinedtobeoptimalfor

plantgrowthinSouthernItalyinApril,whentheexperimentwas

conducted.Droughtstresswasimposedbywithholdingirrigation.

Forthoseplantssubjectedtodroughtstress,irrigationwas

com-pletelysuspendeduntiltheﬁrstsymptomsofwithering(atday5

ofdroughtstress)when50mLofwaterwasadministered.

Veriﬁ-cationofdroughtstresswasconductedwithaplantleafporometer

(DecagonDevicesSC-1).Conﬁrmationofplantstresswasobtained

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plantsrevealeda5foldlowerlevelofwatertranspirationratewhen

comparedtoregularlyirrigatedplants.

Megafol® treatment was performed on Day 0, by spraying

homogenouslya2mLperlitersolutionovertheleaves.Megafol®

isamixtureofaminoacids(prolineandtryptophan),glycosides,

polysaccharides,organicnitrogenandorganiccarbon(Para –dikovi ´c

etal.,2011).Theapparatususedtospraytheplantswasa6L

agri-culturalpressuresprayer(VOLPI).

Fivedaysafterthecessationofirrigation(indicatedasS1,and

representingthedroughtstress),theplantslostallturgidityandan

emergencyirrigationof50mLwaterperplantwasadministered

(S2).Afurtherﬁvedaysaftertheemergencyirrigation,aperiodof

recoverywasestablishedwithawateringregimeof200mLwater

perplantperday(R).Theexperimentalschememadeuseof13

replicaplantsforeachtestandwascompletelyrandomized.

For the molecular analysis,leaves were harvested and

sub-sequentlytransferredinto liquidnitrogen.Threeleaves(central

position)wereharvestedfromeachofthethreedifferentplants.

Thethreeleaveswerepooledandrepresentasinglesampleoutof

thethreebiologicalreplicates.Plantsusedforsamplingleaveswere

excludedfromsubsequentimagingoradditionalleafsampling.Leaf

samplesweretakenonthesamedaysasimaging.Thepre-drought

sampleswereusedtoestablisha baselineofgeneexpressionof

theplants.Allsamplesweretakenatthesametimeofday(9am),

tominimizeclimaticvariation(i.e.cloudyorsunnydays),sinceat

9amtheimpactofthepresence/absenceofcloudsisminimalfor

thegreenhousemicroenvironment,beingthetimefor

warming-upofthegreenhouseduetohighsunlightlimitedtoafewhours

aftersunset.Inthismannerthehydrationstateoftheplantswas

predominantlyduetotheavailabilityofwaterinthevaseandnot

due topossibletransientincreasesintranspiration atpeak day

temperatures.

2.2. Meteorologicaldataacquisition

Boththeminimum,maximum,andaveragedailytemperatures

andthemaximumdailysolarradiationweremeasured

through-outtheexperimentalperiod.Thesamemeasurementswerealso

collectedduringtheimagingandsamplingprocesses,from7am

to9am.Meteorologicaldatawasmeasuredevery30minwitha

datalogger(WatchdogModel450,SpectrumTechnologies,Inc.)

2.3. Non-destructivemeasurements

Phenotypicmeasurementsoftheplantswerecarriedout

non-destructivelybyplantimaging.Imagesofplantsweretakenover

thecourseoftheexperimentwithaplantimagecapturingsystem

(Scanalyzer3DLemnaTecGmbH).Threeimagingchamberswith

differentlevelsofillumination,nearinfra-red (NIR),whitelight

(RGB),andﬂuorescence(UV),wereused.Foreachchamberthree

imagesweretaken,one fromabove theplantand twolaterally

atanorthogonalangle.TheimagescollectedintheNIRchamber

wereusedtoevaluatetheplantwatercontent.Imagesfromthe

RGBchamberwereusedtoevaluatethestateofhealthoftheplant

viacolourclassiﬁcation(i.e.greenhealthytissue,yellowchlorotic

tissues,and brown necrotic tissue),as wellas for

morphomet-ricmeasurements,suchasdigitalbiovolumeandheight.Finally,

imagesfromtheﬂuorescencechamberwereusedtoevaluatethe

stateofthephotosyntheticapparatus.

2.4. Imageanalysis

Inordertoextractphenotypicmeasurementsfromtheimages,

the planthad to beseparated from the background. This

sep-aration was carried out either by converting the image colour

spacetoHSI(hue,saturationandintensity)orusingtheintensity

channel,iftheimagecontainedasufﬁcientcontrastbetweenthe

plant and the background. Otherwise a mathematical formula,

involvingthethreechannelsoftheRGBcolourspace,wasused

tocalculateanewgrey-scaleimagewithsufﬁcientcontrast.This

grey-scaleimagewasthensubjectedtothresholdingtocreatea

binarymasktoextracttheplantfromtheimage.Theone

excep-tionwasfortheNIRtopviewimages,wherethemaskfromtheRGB

topviewplantimagewasresizedandtransposedtotheNIRimage.

Themeasurementofdigitalbiomass,correlatedwithfreshweight,

wascarriedoutasdescribedbyEberiusandLima-Guerra(2009).

Speciﬁcally,theareasoftheplantinthethreeorthogonalimages

fromtheRGBchamberwereappliedinthefollowingformula:

pixelsideview0◦+

pixelsideview90◦+log

pixeltopview 3

Tomeasureastressindexhistogramofthehuechannel,

pix-els(fromtheHSIcolourspace)fromtheimagesgeneratedinthe

ﬂuorescencechamberwereusedtodeterminethehuevaluewith

thegreatestvaluefornon-stressedandthenfordroughtstressed

plants.Applyingtheformula

Ccontrol−Cstress

Ccontrol+Cstress

anindexvaluefrom−1to+1wascalculated.Thisvalueisindicative

oftheshiftinthemodalvalueofthehistogramoftheﬂuorescence

imagepixels,wherelowervaluesrepresentashifttotherightand

highervaluesashifttotheleft.Thisshiftissigniﬁcantinthatthe

huechannelrepresentsthecolourorwavelengthofthelight,the

scalerunningfrom0to255orredtoviolet.Anindexshifttolower

valuesisrepresentativeofashiftinthehistogramtowardhigher

energyorvioletlightandindicateslowerphotosyntheticefﬁciency,

whichisinterpretedasgreaterstress.Histogramsfromthe

grey-scaleimagesgeneratedintheNIRchamberweredividedinto10

groups,representingadecreaseinwatercontentasthecolourvalue

increasedfrom0to255.Fortheanalysisonlythecategorywiththe

lowestvalue,orthehighestwatercontent,wasconsidered.Inall

cases,comparisonsofthecolourclassdataforthepixelsinRGB,

ﬂuorescence,andNIRweremadewiththeuntreatedcontrol.

2.5. Statisticalanalysisofdata

Phenotypingresultswereanalyzedusingone-wayanalysisof

variance(ANOVA),andthemeanswerecomparedusingthe

Dun-can’sNewMultipleRangeTest(MRT;p<0.05).Datawerereported

inTable1.

2.6. Molecularanalysis

Total RNA was extracted from each sample using the

SpectrumTM _Plant _Total _RNA _Kit _(Sigma _Aldrich,_St _Louis, _MO,

USA),accordingtothemanufacturer’sinstructions.

Electrophore-sis using a 1% agarose gel was performed for all the RNA

Table1

Phenotypingresultsoftheexperimentallytreatedplantsforthedigitalbiomassand highwatercontentindexattheendoftheexperiment,andforthestressindextwo daysaftertreatment.

Tesis Digital biomass Highwater contentindex Stress index Untreatedcontrol 383617a 0.292a 0.541a

Megafolcontrol 400213a 0.292a 0.495a

Untreateddroughtstress 279845c 0.258c 0.128c Megafoldroughtstress 309353b 0.278b 0.314b Foreachcolumnmedianvalueswithsigniﬁcantdifferences,ascalculatedbythe Duncan’sNewMultipleRangeTestwithp<0.05,indicatedbydifferentletter group-ing.

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samplesto check for RNA integrity, followed by spectrophoto-metricquantiﬁcation.ContaminatingDNAwasremovedusinga TURBODNA-freekit(Ambion,www.ambion.com).RNAwasthen

reverse-transcribed usingan iScriptTM _cDNA _synthesis _kit

(Bio-Rad Laboratories, Hercules, CA; www.bio-rad.com). Expression

analysisoftomatodroughtstressmarkergenes(Solyc02g084840

andSolyc03g025810) wasperformed byreal-time PCRusing an

ABIPrism7000sequencedetectionsystem(AppliedBiosystems).

Thesegeneswereselectedbecausetheyareorthologousof

Ara-bidopsisRAB18and RD29Bgenes, respectively (Lang and Palva,

1992; Cutler et al., 2010).Both RAB18 and RD29B are strongly

upregulatedbydrought(Dingetal.,2011).QuantitativePCRwas

performedusing40ngcDNAandiQTM_SYBR®_Green_Supermix

(Bio-radLaboratories,Hercules,CA; www.bio-rad.com), accordingto

themanufacturer’sinstructions.SlEF1A(S.lycopersicumelongation

factor1-alpha)andSlUBQ(S.lycopersicumubiquitin)wereusedas

referencegenes.Therelativequantizationofeachindividualgene

expressionwasperformedusingthegeometricaveragingmethod

(geNorm;http://medgen.ugent.be/∼jvdesomp/genorm/).

Thefollowingspeciﬁcprimerswereused:Solyc02g084840,5

-ATGGAACTCAGGCTGGACAC-3 (forward), 5

-TCTTCCTTCTACCGC-CATGT-3 (reverse); Solyc03g025810, 5

-ACACGAGCAGCATTCAC-AAG-3 (forward), 5-GCCCATTGACACAACTTCCT-3 (reverse);

SlEF1a, 5-GCTGCTGTAACAAGATGGATGC-3 (forward), 5

-GG-GGATTTTGTCAGGGTTGTAA-3 (reverse); SlUBQ, 5

-CACCAAG-CCAAAGAAGATCA-3 (forward), 5-TCAGCATTAGGGCACTCCTT-3

(reverse).

3. Results

3.1. Environmentalconditionsduringtreatments

Theminimum,maximum,andaveragedailytemperatures

mea-sured throughout the experiment are shown in Fig. 1A, while

Fig.1Breportsthemaximumdailysolarradiation.Twodaysafter

Megafol®treatment(Day2,S1),theﬁrstfulldayofdroughtstress,

theclimaticconditionswereparticularlywarm,accompaniedby

highsolar radiation(500Wm−2), creating a particularlystrong

combinationofheatanddroughtstress.Otherhightemperature

daysandsolarradiationoccurredduringtherecoveryphase(R),so

thesymptomsofstresswerelesspronounced.Inaddition,the

sec-onddayaftertreatmentalsoexperiencedthegreatestdailychange

intemperature.ThesixthdayaftertheMegafol® treatmentwas

particularlycloudyand humid,thechangein temperature

dur-ingthedaywasinsigniﬁcantandthedailysolarradiationwaslow

(100Wm−2).Therelativemeteorologicaldataduringimagingand

samplingoftheplantsarereportedinFig.2.

3.2. StressIndex

Thecalculatedstressindexforalltheexperimentalplantgroups

showedstatisticallyinsigniﬁcantdifferencesuptothedayof

treat-mentanddroughtstress(Day0,Fig.3).Plantsthatweretreated

withMegafol®didnotshowadrought-dependentdecreaseinthe

stressindexwhencomparedtothecontrolsonedayafter

irriga-tionwasstopped.Ontheseconddayofdrought,thestressindex

ofMegafol® treatedplants waslower thanthatofcontrols, but

higherthantheonerecordedforuntreated,droughtstressedplants.

TheMegafol®treatedplantskeptahigherstressindexalongthe

durationof theS1phase (Fig.3)until Day4 of droughtstress,

whenthestressindexofMegafol® treatedplantswasthesame

astheoneofthe“untreateddrought”plants(Fig.3).Atthispointit

wasdecidedtoadminister50mLwatertotheseverelydehydrated

plants(S2).WiththisemergencyirrigationtheMegafol® treated

plantsrespondedbetterthantheuntreatedonesforthefollowing

Fig.1.Meteorologicaldataduringtheexperiment.Minimum,maximum,and aver-agedaily temperatures(A), andthemaximum daily solarradiation(B) were measuredthroughouttheexperimentaltimeframe.

twodays,afterwhichdifferencesbecamestatisticallyinsigniﬁcant.

Thenormallyirrigatedplants(R),whetherMegafol®treatedornot,

didnotshowstatisticaldifferencesintheirstressindex.Daily

vari-ationsinthestressindexwereduetovariationsinthedailysolar

radiation.Thisistobeexpectedasthecalculatedstressindexwas

obtainedfromchlorophyllﬂuorescence,andvariationsinthe

avail-ablelightwouldnaturallyaffectthephotosyntheticapparatusof

theplants.

3.3. BiomasschangesinresponsetodroughtstressandMegafol®

treatment

ThecalculateddigitalbiomassoftheplantsisshowninFig.4A.

Clearly, the plants treated with Megafol® showed a delayed

responsetothedrought stress(Day2after treatment)in

com-parisonwiththeuntreatedplants.Thisresponsemanifesteditself

asareductioninthecalculateddigitalbiomass.Notonlywasthe

responsedelayed,buttheMegafol® treatedplantsmaintaineda

greaterdigitalbiomassthroughouttherestoftheexperiment.This

indicatesthattheMegafol®treatedplantsweremoreresistantto

droughtstress.NodifferencesbetweentheMegafol®andcontrol

plantswerenotedfortheunstressedplants.Thedigitalbiomass

canbecorrelatedwiththeweightoftheplants,but isstrongly

inﬂuencedbytheprojectedareainthetwodimensionalimages.

Thesetwodimensionalimageswereusedtocalculateavolume:

whenplantsaresubjectedtodroughtstressandlooseturgor

pres-sure,areduced digital biomassresults. Thissensitivitytoplant

volumecanbeseen whenthefreshweightresults(Fig.4B)are

comparedtothecalculateddigitalbiomass(Fig.4A).Plantsthat

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Fig.2.Meteorologicaldatameasuredduringtheperiodofimagingandsampling, from7amto9am.Minimum,maximum,andaveragedailytemperatures(A),and theaveragesolarradiation(B).

intheirdigitalbiomass(whetherornottreatedwithMegafol®).

Thedrought-stressedplantsshowed,instead,amuchlower

digi-talbiomass(Fig.4A)withconcomitantfreshweightlosses,when

comparedtotheircontrols.Forthedroughtstressedplants,the

higherdigitalbiomassdetectedintheMegafol®treatedplantswas

maintainedthroughouttheexperiment,evenafterirrigationofthe

plantswasreinstatedforarecoveryphase.

3.4. Megafol®inﬂuencesthehighwatercontentclassindex

Thefractionofplantareawiththehighestwatercontentclass

isreportedinFig.5.Asexpected,theirrigatedplantsshowedmuch

Fig.3. Stressindexduringtheexperiment.Thestressindex(SI)isameasureofthe stateofhealthofthephotosyntheticapparatusoftheplantsandiscalculatedwith theformula(Ccontrol−Cstress)/(Ccontrol+Cstress)whichresultsinavaluefrom−1to+1.

Fig.4.Digital(A)andfreshbiomass(B)throughouttheexperimentaltimeframe. Circles representtheuntreated (ﬁlled)anddrought stressed(empty) samples, whereastrianglesrepresenttheMegafol®controls(ﬁlled)andstress-treatedplants (empty).

higherlevelsduringthedroughtstressperiodwhichwasalready

evidentonDay1inS1.Thedroughtstressedplantsshowedsmall

butsigniﬁcantdifferenceswithorwithouttheMegafol®treatment,

althoughwiththeapplicationof50mLwater(emergency

irriga-tion,S2) a much greaterdifferencein highwater contentclass

levels wasobserved. Interestingly, the Megafol® treated plants

Fig.5.Highwatercontentindexintomatoplantsthroughouttheexperimentaltime frame.Thewatercontentisreportedasahighwatercontentindex.Thepixelsfrom thegrey-scaleimagesgeneratedintheNIRchamberweredividedintoequidistant classesaccordingtotheirintensity.Thegroupslevelsindicatetheabsorbanceof NIRlight,whichisdirectlyrelatedtothequantityofwaterintheplanttissue.For thisexperimentonlyvaluesfrom105to255wereconsideredanddividedinto10 classes.Thehighwatercontentindexwascalculatedbyaddingtherelativearea fortheﬁrstthreeclasses,thoseofthehighestwatercontent,andisreportedasa fractionofthetotalarea.

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Fig.6.PatternofexpressionofSolyc02084840(A)andSolyc03g025810(B)intomatoplantsunderdroughtstress.Ontheleftforeachgenethecomparisonbetweenstressed (emptycircles)andnon-stressedplants(ﬁlledcircles).OntherightthecomparisonbetweensamplestreatedwithMegafol®_(time₀_time_of_Megafol®_treatment)_before_the

waterstressinduction(emptytriangles)andstressedwithoutbiostimulanttreatment(ﬁlledtriangles).Thepatternisanalysedduringatimecourse,splittingthetotaltime oftreatmentsintothreephases:S1–totalstopofwatering,S2–partialreplacementofwatering(50mLday−1₎_and_R_–_total_watering_recovery₍₂₀₀_mL_day−1_)._The_stressed

tomatoplantspre-treatedwithMegafol®showedalowergeneinduction,suggestingahardeningeffectinducedbythebiostimulant.

weremuchbetteratincreasingandmaintainingtheirleafwater

content.Thiswasprobablyduetoanincreasedmetaboliccapacity

oftheMegafol®treatment,resultinginagreaterleafarea(digital

biomassinFig.4A)andagreaterwaterdistributionintheleaves

(Fig.5).Finally,duringtherecoveryatnormalirrigation(R),the

droughtstressedplantsreachedalmostthesamefractionofhigh

watercontentclassastheunstressedplants.Attheendofthe

recov-eryphase,thewatercontentofthestressedplantsnottreatedwith

Megafol®wasslightlybutstillsigniﬁcantlylowerthantheother

experimentalconditions.

3.5. Geneexpressionanalysis

Plantsrespondactivelytostressconditionsthroughthe

per-ception of stress signals, resulting in the enhanced expression

of related genes leading to the activation cascade of

stress-responsivegenes(ShinozakiandYamaguchi-Shinozaki,2007).The

study of the behaviour of these genes might help in

under-standingthemolecularmechanismsgoverningstressresponses

andtolerancein tomato plantsafter treatmentwitha

biostim-ulantproduct, such as Megafol®. In order to study therole of

Megafol®,weanalysedintomatoplantstreatedwiththe

biostimu-lantandthensubjectedtowaterstressthetranscriptlevelsoftwo

droughtstressmarkergenes:(1)thedehydration-induciblegene

Solyc02g084840,encodingforalow-temperature-inducedprotein,

and(2)Solyc03g025810,adehydrin.Thesegenesareorthologousof

ArabidopsisRAB18andRD29Bgenes,respectively(LangandPalva,

1992;Cutleretal.,2010).Wethencomparedtheseresultswith

thoseobtainedforstressedplantswithoutpre-treatingthemwith

thebiostimulant.Intriguingly,theexpressionofbothgenes

dra-maticallyincreased(Fig.6AandB)onthethirddayofwaterstress

(S1),when the maximumstress perceptionis achieved.

Subse-quently,relativetranscriptlevelsprogressivelydecreasedduring

emergencywatering(S2),untiltheydisappearedintherecovery

phase(R).Theexpressionpatternofthetwogeneswasverysimilar

underthecontrolconditions.Overall,thelowerexpressionlevels

ofbothgenesintheMegafol®-treatedplants(Fig.6)suggestthat

theseplantswereexperiencingamuchlowerdroughtstress.On

thewhole,theseresultsconcerningtheMegafol®treatmentarein

fullagreementwiththephenomicdatadescribedabove.

4. Discussion

Biostimulantsrepresentaclassofproductsusedinagriculture

thatisattractingtheinterestofthemarketandtheresearch

com-munity(Santanielloetal.,2013).AsdeﬁnedbyDuJardin(2012),

“plantbiostimulantsaresubstancesandmaterials,withthe

excep-tionofnutrientsandpesticide,whichwhenappliedtoplants,seeds

orgrowingsubstrateinspeciﬁcformulations,havethecapacity

tomodifyphysiologicalprocessesofplantsinawaythatprovides

potentialbeneﬁtstogrowth,developmentand/orstressresponse”.

Althoughthescientiﬁcliteraturereportsevidencefortheefﬁcacyof

biostimulants(Para –dikovi ´cetal.,2011),littleisknownabouttheir

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the effects of biostimulants at the molecularlevel (Santaniello

etal.,2013),highlightingtheabilityofrawmaterialsthatareused

toformulatebiostimulantstoinducetheexpressionofvarioussets

ofgenes.Remarkably,amongthegenefamiliesthatwereshownto

beup-regulated,severalbelongedtofunctionalcategoriesrelated

toabioticstresstoleranceandresponsestoABA(Santanielloetal.,

2013).These evidences suggest that biostimulants may act by

priming the treated crops against abiotic stresses. To test this

hypothesis we performed the integrated molecular/phenotypic

analysis we described here.To our knowledge this is the ﬁrst

experimentalevidence for themechanism behindthe function

ofbiostimulantssuchas Megafol®.Analysisoftheresultsfrom

theNIR,ﬂuorescence,andwhitelightimaging,demonstratedthat

theMegafol® pre-treatment of theplants confersresistanceto

theeffectsofdroughtstress,inagreementwithaprimingeffect

of this treatment.In addition to drought stress resistance, the

Megafol®-treatedplantswereabletorecovermorequicklywhen

theyhadaccesstowater.Thebiostimulantappearstodirectlyand

positivelyinﬂuenceplantresponsetodrought,aneffectthat

per-sistsuntiltheendofthedroughtstress.Photosyntheticefﬁciency

anda greaterlevelofplantwatercontentunderdroughtstress

conditionsindicateanimprovedmetabolicactivityofplantsunder

difﬁcultconditions.Thisresultsinplantswithagreaterbiomass.

Bydelayingthenegativeeffectsofdroughtstress,theMegafol®

-treatedplantsareabletousethescarcewaterresourceavailable

more efﬁciently. Under normal irrigation conditions, Megafol®

treatment appears to have no inﬂuence on the plant growth,

photosyntheticefﬁciency(stressindex),orplantwatercontent.

ThepositiveeffectsoftheMegafol®treatmentsonthestressed

plants wereconﬁrmedat themolecularlevel. Byanalysingthe

expressionlevelsofdroughtstressmarkergenesintomatoplants,

we observed that plants pre-treated with Megafol® showed a

lower expressionof drought-related genes even when strongly

water-stressed. Considering the functional role of the marker

genesanalysed,whichtypicallyaccumulateandup-regulate

dur-ingdroughtstress(Ismailetal.,1999),theassociationbetweenthe

accumulationofmembersoftheLEAproteinfamilyandtoleranceto

stressisreasonableandhasalreadybeenshowninseveralspecies

(Cellieretal.,1998;Lopezetal.,2003).

ThelowerexpressionofthesegenesintheMegafol®-treated

tomatoplantshighlightsthefactthattheseplantsareindeed

expe-riencinga lower level ofwater stress, asa consequence ofthe

Megafol®treatmentitself.Thisresultwasconﬁrmedinouranalysis

ofthestress-relatedparameters.Megafol®containsbetaineswhich

couldexplaintheresultsdescribedinthissetofexperiments.The

roleofthesemoleculesinreducingdroughtstresscellulardamage

inplantsiswelldocumented(AshrafandFoolad,2007;Hoqueetal.,

2007;Parketal.,2006;ChenandMurata,2008).Ofthevarious

pro-tectivemechanismsagainstcellulardamageinducedbydrought

stress,theaccumulationofsmallorganicmetabolitesisimportant.

Thisdiffersamongspecies,butismostlyrepresentedbypolyols,

sugars,aminoacids,betainesandrelatedcompounds.Betainesare

synthesizedinabioticstressresponsesandtheirleveliscorrelated

withtheincreasedtolerancebyplants(RhodesandHanson,1993).

Tomatoplantsdonotnaturallyaccumulatebetaines(WynJones

andStorey,1981),butthereismuchevidencethatexogenous

appli-cationsimprovethegrowthandsurvivalofnumerousspeciesunder

stress(Zhaoetal.,1992;Aliaetal.,1998;Allardetal.,1998;Mäkelä

etal.,1998;Jokinenetal.,1999;Chenetal.,2000)andintomato,

whenappliedtotheleaves,theyaretakenupreadily(Parketal.,

2006).ThebetainecomponentsofMegafol®probablyreducethe

sensitivityoftheplantstowaterdeﬁciency,resultinginalower

perceptionofthestresssignalandinweakenednegative

physio-logicalconsequences.Althoughadditionalresearchisnecessaryto

furtherclarifythemolecularandphysiologicalmechanismswhich

exploitbiostimulants,suchasMegafol®,toovercomehighlevels

ofabioticstress,thepresentresearchdemonstratedthat

biostimu-lantsexertaclear,measurablealleviationofabioticstressinjuries.

Acknowledgement

WewouldliketothankProf.AlbertoPardossi(UniversityofPisa,

Italy)forhisinvaluablediscussion.

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