Influence of iron and copper on the activity of laccases in Fusarium oxysporum f. sp. lycopersici

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h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Fungal

and

Bacterial

Physiology

Inﬂuence

of

iron

and

copper

on

the

activity

of

laccases

in

Fusarium

oxysporum

f.

sp.

lycopersici

Wendy

Susana

Hernández-Monjaraz,

César

Caudillo-Pérez,

Pedro

Ulises

Salazar-Sánchez,

Karla

Lizbeth

Macías-Sánchez

∗

InstitutoPolitécnicoNacional,UnidadProfesionalInterdisciplinariadeIngenieríacampusGuanajuato,SilaodelaVictoria,Guanajuato, Mexico

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received28October2017

Accepted22June2018

Availableonline13August2018

AssociateEditor:JerriZilli

Keywords: Chelators Cooper Fusarium Iron Laccases

a

b

s

t

r

a

c

t

Fusariumoxysporumf.sp.lycopersiciisaphytopathogenicfungusthatcausesvascularwilt

intomatoplants.Inthisworkweanalyzetheinﬂuenceofmetalsaltssuchasironand

coppersulphate,aswellasthatofbathophenanthrolinedisulfonicacid(ironchelator)and

bathocuproinedisulfonicacid(copperchelator)ontheactivityoflaccasesintheintra(IF)and

extracellularfractions(EF)ofthewild-typeandthenon-pathogenicmutantstrain(rho1::hyg)

ofF.oxysporum.TheresultsshowthatlaccaseactivityintheIFfractionofthewildand

mutantstrainincreasedwiththeadditionofironchelator(53.4and114.32%;respectively).

Withcopper,itisobservedthatthereisaninhibitionoftheactivitywiththeadditionof

CuSO4fortheEFofthewildandmutantstrain(reductionof82and62.6%;respectively)and

fortheIFofthemutantstrain(54.8%).Withthecopperchelatoralesslaccaseactivityin

theIFofthemutantstrainwasobserved(reductionof53.9%).Theresultsobtainedsuggest

adifferentregulationofintracellularlaccasesinthemutantstraincomparedwiththewild

typeinpresenceofCuSO4andcopperchelatorwhichmaybeduetothemutationintherho

gene.

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Fusariumoxysporumisahighlyrelevantphytopathogenic

fun-gusdue toits economicimportance.15 _The_special_form_of

lycopersiciisthecausalagentofvascularfusariosisdiseasein

tomatoculture.Thisfungusreachesthevascularsystemof

∗ _{Corresponding}_author.

E-mail:kmaciass@ipn.mx(K.L.Macías-Sánchez).

theplantandsubsequentlyachievesthecolonizationofthe

hostplantbyspreadingthroughthexylemvessels.28

Laccases(bencenodiol:oxygenoxidoreductase,EC1.10.3.2)

are a typeof polyphenoloxidase enzymes that are part of

the ligninolytic system and with ascorbate oxidases,

ceru-plasminandotherenzymesbelongamongbluemulticopper

oxidases.23,1 _The_{participation}_of_laccases_is_associated_with

https://doi.org/10.1016/j.bjm.2018.06.002

(2)

many biological processes, such as spore resistance,

pig-mentation, virulence factors,2,37 _{ligniﬁcation} _of _plant _cell

walls,20 _lignin _{biodegradation}5 _and _the _protection _of _the

fungus against phenolic compounds released by plants,

suchasphytoalexins,24 _among_others._It_is_known_that_the

manifestationof laccases in fungiis inﬂuenced byseveral

factors,includingthepresenceofmetals.25_Copper_has_been

demonstrated to regulate the induction transcriptional of

thelaccasesinTrametesversicolorandTrametespubescens,11,14

Pleurotus,32,21_among_others.

Ontheotherhand,FeSO4ataconcentrationof1mMdid

notinducelaccaseactivitywithrespecttocontrolinPleurotus

pulmonariusat7daysofgrowthbuttherewasaninhibitionof

activityat10daysofgrowth.Asimilarresponsewasobserved

inPleurotusostreatus,sincetherewasaninhibitionof

activ-ityat10 days,butinthiscasethereastimulatoryeffectof

activitywasobservedat7daysofgrowth.33_It_was_obtained

acomplete inhibitionofactivityofapuriﬁedlaccaseofthe

fungusTrematosphaeriamangroveiwithFeSO4at1mM.17

InF.oxysporum f.sp.lycopersicithepresenceofsixgenes

encodinglaccaseproteins(lcc1,lcc2,lcc3,lcc4,lcc5andlcc9)

has been reported. The mutants lcc1, lcc3 and lcc5 were

implemented,inwhichasigniﬁcantdecreaseinthe

extracel-lularactivityoflaccasewasobserved;however,thesemutants

showednolossofpathogenicityontomatoseedlings.8

Toknow theeffect that somemetalsand chelantsmay

haveontheactivityoflaccasesinF.oxysporum,weanalyzed

the effect of FeSO4, the iron chelator

bathophenanthro-linedisulfonic acid (BPDS), CuSO4 and the copper chelator

bathocuproinedisulfonicacid(BCDS)ontheenzymatic

activ-ity of laccase in a wild strain and the mutant rho1::hyg

(non-pathogenic)ofF.oxysporumf.sp.lycopersici.

Materials

and

methods

Isolatedfungiandgrowingconditions

Strain4287(race2;wild-type)andthemutantstrainrho1::hyg

of F. oxysporum f. sp.lycopersici were obtained through Dr.

GonzalezRoncero(UniversityofCordoba,Spain).Themutant

rho1::hyg hasinterruptedrho1 gene(rho1::hyg)and presents

lossofthepathogenicityintomatoseedlings.18

Topreserve thestrains, the wild-type strainwas

inocu-latedinthePDBmediumandthemutantstraininthePDB

mediumwithhygromycin(20␮g/mL;SIGMA,SaintLouis,

Mis-souri,USA)for4–5daysat27◦Cwithshaking.Thegenerated

myceliawere removedthrough ﬁltrationundersterile

con-ditions. Theﬁltrate obtained was centrifuged at 6500g for

10min,andthesedimentcontainingthesporeswaswashed

three times with sterilewater. The strains were stored as

microconidialsuspensionsin30%glycerolat70◦Cuntilits

usage.

For different growth conditions, the germinates were

obtainedfrom thePDB culture,which wasinoculated with

5×106_microconidia_mL−1_for₁₉_h_at₂₇◦_C_under_shaking._The

germsweretransferredtoabasalmedium(BL)(0.4gKH2PO4

L−1,0.2gMgSO4·7H2OL−1,1gNH4NO3L−1,0.01gFeSO4L−1,

0.01gZnSO4L−1 and0.01gMnSO4 L−1, 0.2gKCl L−1,0.25%

sucrose)for6days;BLwithoutcopper(incubationfor3days)

andBLwithoutiron(incubationfor3days)at27◦C,220rpm.

OnthethirddayofgrowthintheBLmediumwithout

cop-per,thecopperchelatorbathocuproinedisulfonicacid(BCDS;

SIGMA, Saint Louis,Missouri, USA)at 1mM and CuSO4 at

25␮Mwasindependentlyaddedinﬂasks.Thesameprocedure

isappliedfortheBLmediumwithoutiron,inwhichtheiron

chelator,bathophenanthrolinedisulfonicacid (BPDS;SIGMA,

SaintLouis,Missouri,USA)wasaddedat1mM;andforthe

duplicateoftheBLmediumwithoutiron,FeSO4wasadded

at25␮M.AftertheadditionofBCDS,CuSO4,BPDSandFeSO4,

theincubationwascontinuedfor3daysat220rpmat27◦C.

Themyceliumandtheextracellularfractionwereobtainedby

centrifugationat6500gfor10minat4◦C.

Obtainmentofthehomogenate

The myceliawere resuspended ina BufferTris–HCl 50mM

solution with protease inhibitors(antipain, leupeptin,

pep-statinandE64at5␮g/mL;SIGMA,SaintLouis,Missouri,USA).

Thecells werefragmented throughsonication for8–10min

for1-minintervals.Subsequentlyaninitialcentrifugationwas

performedat2500×gfor5minatatemperatureof4◦C.The

pelletwasdiscardedandthesupernatanttransferredtoanew

tube,whereasecondcentrifugationwasperformedat6500×g

for45minat4◦C,thepelletdiscardedandthesupernatant

(intracellularfraction)obtained.Theaforementionedprotease

inhibitorswereaddedtotheextracellularfraction.Both

frac-tionswerestoredat−20◦_C_until_its_usage.

Quantiﬁcationofenzymeactivity

Thelaccaseactivity was determined using2,2

-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) [ABTS; SIGMA, Saint

Louis, Missouri, USA] as a substrate.7 _The _laccase _activity

wasquantiﬁedthroughtheoxidationoftheABTS substrate

(1mM)in20mMsodiumacetatepH3.5buffer,byincreasing

theabsorbanceat436nm.14_Final_reaction_mixture_contained

100␮Lofthesampleand2mLoftheABTSsubstrate,16

mea-suringthereactionat0and10min.Aunitoflaccaseactivityis

deﬁnedastheamountofenzymeneededtooxidizea␮molof

ABTSmin−1at25◦C.14_All_assays_were_performed_in_triplicate

andthreeindependentbatcheswereperformed.

Statisticanalysis

Thedataonenzymeactivitywerecomparedbetweenthetwo

strainsusingANOVAandTukey’stests,withMinitabver.15

software(MinitabInc.,StateCollege,PA,USA),consideringa

levelofsigniﬁcanceat0.05.

Results

Inﬂuenceofironontheenzymaticactivityoflaccases

From three independentbatches,the enzymatic activityof

laccases wasmeasured inthe intracellular and

extracellu-larfractionofthewildandmutantstrainF.oxysporumf.sp.

lycopersici, inthepresenceofironandthe BPDSiron

(3)

Table1–Relationbetweentheactivityinthemediumof

therho1::hygmutantandinthatofthewild-typestrain

inthecontrolandinthepresenceofBPDSandFeSO4.

Fraction Condition rhol::hygactivity/wild-type

activity Intracellular Control 2.035218 BPDS 2.813707 FeSO4 3.288776 Extracellular Control 2.246674 BPDS 2.162287 FeSO4 2.022699

demonstratedhigherextracellularandintracellularenzyme activity(inthecontrolcondition,inthepresenceofBPDSand FeSO4)comparedtothewild-typestrain.Withanormal

distri-butionofthedata(p-value>0.1)itisconsideredthatthe activ-itybetweenbothstrainswillalwaysbegreaterforthemutant strainunderthesameconditionsoftheexperiment(Table1).

According to the statistical analysis through the Tukey

method,whichallowsustoanalyzethedifferencein

enzy-maticactivitybetweentheconditionsstudiedforeachstrain

(controlgroup,inthepresenceofBPDSchelatorandinthe

presenceofFeSO4),it was observedthat inthe

extracellu-larfraction ofthe wild strainand themutant strain there

existsnodifferencebetweentheenzymaticactivityofthe

con-trolgroupwithrespecttothegroupinthepresenceofBPDS

orthe groupinthepresenceofFeSO4.Withrespecttothe

enzymaticactivityoftheintracellularfraction,inbothstrains

(wildandmutant)abiggeractivityinthegroupwiththeBPDS

chelatorwasobservedwithrespecttocontrolgroup(53.4and

114.32%,respectively),whereasnodifferenceofintracellular

enzymaticactivitywasobservedinbothstrainsbetweenthe

controlgroupandthegroupwithFeSO4(Fig.1).

Inﬂuenceofcopperontheenzymaticactivityoflaccase

From threeindependentbatches,the enzymatic activity of

laccaseswasmeasuredintheintracellularandextracellular

6000 5000 4000 3000 2000 1000

BPDSControlFeSO4 BPDSControlFeSO4 BPDSControlFeSO4 BPDS Control FeSO4 rho1::hy g Wild-ty pe rho1::hy g Wild-ty pe

Extracellular Intracellular

Enzymatic activity (U/mg)

Fig.1–Enzymaticactivityintheextracellularand intracellularfractionsofwild-typeandrho1::hygofF. oxysporumf.sp.lycopersicistrainswithorwithoutFeSO4 andwithBPDS.Thedataweregroupedbytheresultsof Tukey’stestusingaconﬁdencelevelof95%.

Table2–Relationbetweentheactivityinthemediumof

therho1::hygmutantandinthatofthewild-typestrain

inthecontrolandinthepresenceofBCDSandCuSO4.

Fraction Condition rhol::hygactivity/wild-type

activity Intracellular Control 5.144615 BCDS 2.424381 CuSO4 1.850917 Extracellular Control 6.184008 BCDS 6.805616 CuSO4 12.58875

fractionofthewild andmutantstrainofF.oxysporumf.sp.

lycopersici, in the presenceofcopper and the BCDS copper chelator.Inbothfractions,theANOVAtestperformedonall thelotsallowedustoknowthatthemutantstrainrho1::hyg

had a higher enzymatic activity for the different growth conditionscomparedtothewildstrain.Thedatamaintained anormaldistribution(p-value>0.1)(Table2).

Accordingtothestatisticalanalysisperformedthroughthe

Tukeymethod,itwasobservedthatintheextracellular

frac-tion, the enzymatic activity inthe wild and mutant strain

presented less activity inthe group with CuSO4 compared

totheirrespectivecontrolgroups(areductionoftheactivity

of82and62.6%withrespectthecontrolgroup,respectively).

Therewasnoeffectbetweenthecontrolgroupandthegroup

withadditionoftheBCDSchelatoroftheextracellular

frac-tion(inbothstrains).Ontheotherhand,intheintracellular

fraction ofthe wild-type strain there wasno difference in

laccaseactivity between the controlgroup, the groupwith

CuSO4 and the group with addition of the BCDS chelator;

however,inthemutantstraintheCuSO4groupdemonstrated

less activity withrespect controlgroup(a reductionofthe

activity of 54.8%). Also in the same fraction, the mutant

strain showedless activity oflaccaseinthe BCDSchelator

group compared with controlgroup (a reductionof 53.9%)

(Fig.2). 6000 7000 8000 9000 5000 4000 3000 2000 1000 0

BCDS Control CuSO4 BCDS Control CuSO4 BCDS Control CuSO4 BCDS Control CuSO4 rho1::hyg Wild-ty pe rho1::hyg Wild-ty pe

Extracellular Intracellular

Enzymatic activity (U/mg)

Fig.2–Enzymaticactivityintheextracellularand intracellularfractionsofwild-typeandrho1::hygofF. oxysporumf.sp.lycopersicistrainswithorwithoutCuSO4 andwithBCDS.Thedataweregroupedbytheresultsof Tukey’stestusingaconﬁdencelevelof95%.

(4)

Fig.3–OxidationofABTSbylaccase.ABTScationradical (a)andABTSdication(b).22

Discussion

Laccases are a typeofphenoloxidase enzymes widely

dis-tributedinnature,astheyhavebeenfoundinplants,bacteria

andfungi.19_Among_the_functions_attributed_to_laccases_are

ligniﬁcation,20_the_protection_of_the_fungus_against_phenolic

compounds,24_among_others._ABTS_is_an_organic_redox

medi-atorusedinthelaccasereaction.ABTSundergoestwostep

oxidationreactionduringtheenzymaticreactionbylaccase

(ﬁrstfortheobtentionofABTS cationradicaland thenthe

ABTSdication)22_(Fig._3).

InstudiesbyCa ˜neroandRonceroin20088_demonstrated

theexpressionofvariouslaccasegenesduringtheinfection

processofthetomatoplantwithF.oxysporum f.sp.

lycoper-sici (lcc1, lcc3 and lcc9). Inaddition, the location ofeach of

the laccaseproteinsthrough theaminoacidsequence was

identiﬁed, demonstratingthatLcc1andLcc2havean

intra-cellularlocalization,Lcc3istransmembraneand Lcc4,Lcc5

and Lcc9have anextracellular localization.An analysisof

thepromoterregionsofthelcc1,lcc2,lcc3,lcc4,lcc5andlcc9

genesdemonstratemetal-responsiveelements(MRE),

stress-responsiveelements(STRE),xenobiotic-responsiveelements

(XRE),anACE1copper-responsivetranscriptionfactoranda

PacCambientpHresponsefactor.Laccaseactivityhasbeen

reportedtoincreasewithlowconcentrationsofsucrose,

glyc-erolandpolygalacturonicacid.8_Therefore,_in_this_work_a_basal

medium with a low concentration of sucroseas a carbon

sourcewasused.

Sometraceelementsareessentialforfungalmetabolism;

however,athighconcentrationstheycouldbetoxic.6,4_Some

traceelementssuchasFe,Znandothersactthrough

metal-responsive promoter interaction, for post-transcriptional

regulation.34,13,10_In_fungi,_it_is_known_that_regulation_in_the

manifestationoflaccasescanbemediatedbythepresenceof

variousmetals.25

Inthe resultsobtainedinthiswork,ahigherenzymatic

activityoflaccasewasobservedintherho1::hygmutantstrain

comparedtothewild-typestrainunderalltestedconditions

(controlgroup,presenceofiron,copper,andBPDSandBCDS

chelators).Theseresultscorrespondwithtothereportedby

Reyes-MedinaandMacías-Sánchez,26_since_under_the

condi-tionsutilizedinthatworktheyalsodetectedgreateractivity

inthemutantstrainwithrespecttothewild-type,

suggest-ing that this effect could be inﬂuenced by the absence of

Rho protein. The results obtained atthe extracellular and

intracellular levels suggest that the presenceof FeSO4 has

no apparent effect on the enzymatic activity oflaccasein

the wild strain ofF. oxysporum f.sp. lycopersiciin

compari-sontoitsbasalcondition,whichcouldbeduetoFeSO4not

actingasaninductoroftheextracellularlaccase(Lcc4,Lcc5

andLcc9)orintracellularlaccases(Lcc1andLcc2),orthatthe

concentrationused(25␮M)isnottheconcentrationnecessary

togeneratethe inductionoflaccases.Asimilarbehavioris

observedforthemutantstrain,wheretheadditionofFeSO4

doesnotappeartoaffectlaccaseactivity.TheeffectofFeon

laccaseactivitydiffersdependingonthemicroorganismand

theconcentrationused;forexample,theeffectofthismetal

ataconcentrationof2mMontheactivityofthepuriﬁed

lac-caseonAspergillusnidulansdemonstratedarelativeactivityof

139%withrespecttothatobtainedinthecontrolcondition.36

However,inStreptomycescyaneusareductionofthe activity

oflaccasepuriﬁedunderthe presenceofFeSO4

(concentra-tions since0–50mM) was observed.3 _On _the _other _hand,38

observed inTrametesvelutinaan increaseinthe

extracellu-larenzymaticactivitywhenaddingironfromaconcentration

of0.04mM.27_tested_the_effect_of_FeSO

4ataconcentrationof

5mMon laccaseactivity inChalara(syn. Thielaviopsis)

para-doxa,observinganinhibitionofthe activitywithrespectto

thecontrol.

InP.ostreatusanincreaseinlaccaseactivitywasobserved

in the presence of FeSO4 at 7 days of incubation;

how-ever, at10 days ofincubation there was less activity with

respect to its control. On the other hand, the activity of

laccase in Pleurotus pulmonarius was similar in presence

(5)

and there was a decrease of the activity in the

pres-ence of Fe compared to the control group at 10 days of

incubation.33

TheBPDSisknownasapotentironchelatingagent.31_In

thepresentstudy,nodifferenceswereobservedbetweenthe

controlgroupandthegroupwithadditionofBPDSinthe

extra-cellularfractionofthewild andmutantstrain. Incontrast,

intheintracellularfraction,therewasobservedthatinboth

strainstheBPDSgrouppresentedgreateractivitycomparedto

thecontrolgroup,whichwouldindicatethataloweriron

con-centrationisrequiredthantheusedinthecontrolcondition

toinducetheactivityofthelaccases.

Copperplaysanimportantroleinthe regulationof

lac-casessinceitisknownthattheadditionoflowconcentrations

of copper to the culture medium can stimulate laccase

production21_;_in_addition,_laccases_have_four_copper_atoms_in

theircatalyticcenter,whichhavedifferentproperties.23_With

thedataobtainedinthiswork,inthegroupwiththe

addi-tionofCuSO4,indicate adecrease intheactivity oflaccase

intheextracellular fractionofthewild and mutantstrain,

aswellasintheintracellularfractionofthemutantstrain,

indicating that CuSO4 at aconcentration of25␮Minhibits

the activity of laccases in F. oxysporum. f. sp. lycopersici.11

reported that an increase in the concentration of copper

causesanincrementinthelaccases activityinT.versicolor.

However, the use of different concentrations of the metal

on the induction of laccases in other microorganisms has

demonstratedthatatacertainconcentrationamaximumis

reachedintheactivityandafterthistheactivityconsiderably

decreasesandinsomecasesitiscanceledcompletely.Inthe

Grammothelesubargenteafungus,itwasobservedthatinthe

concentrationrangeof0.6–1.2mMofCuSO4majorenzymatic

activity of laccase occurred, whereas at higher

concentra-tionsofthemetal(1.5and1.8mM),theactivitydecreases30_;

asimilarsituationisobservedinT.versicolor,wherethe

lac-caseactivityisinducedinthepresenceofCuSO4upto17%;

however, itdecreases signiﬁcantly withhigh levelsofCu2+

(80mM).16 _A _particular _case _is _that _of _Trametes _hirsuta, _in

which there was observed the irregular behavior of

enzy-maticactivity underinductionconditionswith CuSO4 with

respectto the culture time and the used concentration of

themetal;inthat experimentbeingpossibletodistinguish

severalpeaksofactivity,followedbyperiodsofinhibition.29

In this work, there is no difference in the activity of the

laccaseofthe controlgroupand the group withcopper in

theintracellular fractionofthe wild strain, wherepossibly

ahigher concentrationofcopperis requiredtoobserve an

effect on the enzymatic activity of the laccases. This also

indicatesthatthelaccasespresent intheintracellular

frac-tionofthewildstraindonotpresentasmuchsensitivityto

copper,unlikethelaccases presentintheintracellular

frac-tion ofthe mutantstrain, and inthe extracellular fraction

(wildandmutantstrain).

BCDSisachelatingagentforcopperions.35_Endo_et_al.12

used a chelator concentration similar to this work. They

observedareductionoftheenzymaticactivity betweenthe

conditionand its control. Withthe resultsobtainedinour

work,inthepresenceoftheBCDSchelatorinthe

extracel-lularfraction,nodifferenceswere observedwithrespectto

the control for the two strains utilized, as well as in the

intracellularfractionofthe wildstrain,notbeingthesame

caseforthe mutantstrain, wherethereisadiminishingin

the activity in the group withthe addition ofBCDS, being

the onlycasewherethe inhibitionofthe enzymatic

activ-ityisobservedwiththepresenceofthischelator.9_analyzed

theinﬂuenceofcopperintheexpressionoflaccasegenesin

F. oxysporumutilizing CuSO4 andBCDS;inbothcasesthere

wereseennodifferencesintheexpressionofthelcc1,lcc2,

lcc3,lcc4,lcc5andlcc9genes;however,iftherewasahigher

extracellular enzymatic activity in the wild-type strain in

the presence of CuSO4 compared to the medium without

CuSO4 (growthfor5daysinaminimalmediumcontaining

sucrose with and without 250␮M of CuSO4), these results

differfrom those obtainedinthiswork,sinceweobserved

lessactivityinthepresenceofcopper,eventhoughasmaller

amount ofCuSO4 (25␮M) was utilized; however, the

incu-bation wasperformedforashorterperiodoftime(3days),

where it is possible to observe a different behavior over

time, which may be dueto regulation mechanisms of the

metal.

In this work, it was of interest to learn about the

invitrobehaviorofthelaccaseenzymes’activityintheintra

and extracellular fraction of the wild strain of F.

oxyspo-rum f. sp. lycopersici (pathogen strain), and of the mutant

strain rho1::hyg (non-pathogen strain), and its response

to inductors like Fe and Cu, and with the iron chelator

(bathophenanthrolinedisulfonic acid), and copper chelator

(bathocuproinedisulfonic acid). Nevertheless, these do not

constitute a ﬁeld treatment for the control of fusariosis.

However, knowledge of the behavior of laccaseactivity in

the wild and mutant strains under these conditions may

be used as a guide in the analysis and modulation of

these metallic ions in the soil, thus creating soil

condi-tions that canbeless appropriatefortheestablishmentof

fusariosis.

Withtheresultsobtainedinthisworkitcanbesuggested

thattheintracellularlaccasesofthewildandmutantstrain

demonstrateadifferentialresponsetothepresenceofcopper

ions.Thedifferenceobservedcouldberegulatedthroughof

rho1gene.Becausetheroleofcopperinthefunctionoflaccase

proteins,thisresponsecouldbeattributedtoitsinsertionin

theinactiveapoprotein.Asperspectivesofthiswork,the

con-ditionsthatpresentedabiggereffectonthelaccaseactivity

canbeusedtoobservetheeffectthatpresentinthe

sporu-lation,pigmentation,andexpressionoftranscriptionfactors,

amongotherprocesses.

Funding

ThisstudywasfundedbytheInstitutoPolitécnicoNacional,

SecretaríadeInvestigaciónyPosgrado(20131056).

Conﬂicts

of

interest

(6)

Acknowledgements

ThisresearchwassupportedbytheSecretaríadeInvestigación

dePosgradooftheInstitutoPolitécnicoNacional(20131056).

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