Effects of monosulfuron-ester on metabolic processes of nitrogen-fixing cyanobacteria Anabaena flos-aquae and Anabaena azotica

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

Environmental

Microbiology

Effects

of

monosulfuron-ester

on

metabolic

processes

of

nitrogen-fixing

cyanobacteria

Anabaena

flos-aquae

and

Anabaena

azotica

Jian

Ying

Shen

_,

_Jin

_Zhi

_Liao

_,

_Li

_Li

_Guo

_,

_Rui

_Fang

_Su

a_{ShanghaiJiaotongUniversity,DepartmentofEnvironmentalScienceandResource,Shanghai,China}

b_{FengxianDistrictAgro-TechnologyExtensionCenter,Shanghai,China}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received16June2016 Accepted17October2016 Availableonline11March2017 AssociateEditor:JerriZilli

Keywords: Acetolactatesynthase Aminoacids Cyanobacteria Monosulfuron-ester

a

b

s

t

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a

c

t

Presenceoftherelativelynewsulfonylureaherbicidemonosulfuron-esterat0.03–300nmol/L affectedthegrowthoftwonon-targetnitrogen-fixingcyanobacteria(Anabaenaflos-aquae

andAnabaenaazotica)andsubstantiallyinhibitedinvitroAcetolactatesynthaseactivity,

withIC50of3.3and101.3nmol/LforA.flos-aquaeandA.azotica,respectively.Presenting in30–300nmol/L,itinhibitedproteinsynthesisofthecyanobacteriawithlessaminoacids producedasitsconcentrationincreased.Ourfindingssupporttheviewthat monosulfuron-estertoxicityinbothnitrogen-fixingcyanobacteriaisduetoitsinterferencewithprotein metabolismviainhibitionofbranch-chainaminoacidbiosynthesis,andparticularly Aceto-lactatesynthaseactivity.

©2017PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileirade Microbiologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Monosulfuron-ester is a relatively new sulfonylurea herbi-cidethatwasdevelopedbytheNationalPesticideEngineering Research Center in Tianjin, China.1 _{This herbicide is very}

effective at post-emergence rates of 30–60nmol/L (active ingredients)inawiderangeofcropsincludingcorn(Zeamays L.),wheat(TriticumaestivumL.),rice(OryzasativaL.),andmillet (PanicummiliaceumL.).2_{Themodeofactionofsulfonylureais}

inhibitionofacetolactatesynthase(ALS).3_{TheALSispresent}

inallplantsaswellasinbacteria,fungi,andcyanobacteria,4

butnotpresentinmammals.Becauseoftheirrelativelylow

∗ _{Correspondingauthorat:DepartmentofEnvironmentalScienceandResource,ShanghaiJiaotongUniversity,Shanghai200240,China.}

E-mail:jyshen88@sjtu.edu.cn(J.Y.Shen).

toxicitytomammals,highefficacyandenvironmentalsafety,5

theuseofsulfonylureaherbicideshasincreasedrapidly. How-ever,applicationofselectiveherbicidessuchassulfonylurea incroppingsystemscansubstantiallyreducesoilmicrobial diversityiftheyareoverusedormisused.

Cyanobacteria are one of the largest and most impor-tant groupsofalgaeon earth.Inparticular,nitrogen-fixing cyanobacteriaarevitalphotosyntheticmicroorganismsthat contribute to soil fertility by fixing atmospheric nitrogen, releasing small quantitiesof the major fertilizing product, ammonia, aswell as other small nitrogenouspolypeptides during active growth,6 _{and also because they maintain}

http://dx.doi.org/10.1016/j.bjm.2016.10.028

1517-8382/©2017PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeMicrobiologia.Thisisanopenaccessarticle undertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ecosystemstability.7_{AnabaenaazoticaLeyandAnabaena}

flos-aquae(Lyngb)Brebaretwoofthemostcommonfilamentous

nitrogen-fixing cyanobacteria in China agricultural fields.8

In general, cyanobacteria are very sensitive to herbicides becausetheypossessmanycharacteristicsofhigherplants.9

Previous study showed that cyanobacteria had different degreesofsensitivitytoherbicides.10 _{However,theeffectof}

monosulfuron-ester on nitrogen-fixing cyanobacteria that mayplayaroleinenhancingthefertilityofagriculturalfields hasnotbeenreportedintheliterature.Therefore,the objec-tivesofthisresearchweretodeterminetheeffectsofarange of monosulfuron-ester concentrations on growth, protein content,in vitroand extractableactivityofALS,and amino acidproductionofbothnitrogen-fixingcyanobacteria. Find-ingsfromthisworkshouldprovideabetterunderstandingof thetoxicityanditsmodeofactionofmonosulfuron-esterin thetwoubiquitousnitrogen-fixingcyanobacteria.

Materials

and

methods

Monosulfuron-ester of 99.4% purity was synthesized at the National Pesticide Engineering Research Center in Tianjin, China. Monosulfuron-ester was dissolved in the mixture oforganic solvent dimethylformamide (DMF, N,N-dimethylformamide, Jianshan Chemicals Ltd., China) and surfactant TritonX-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethyleneglycol)(JibishiGeneTechnologyLtd.,China)to formthe herbicideconcentratebelow theconcentration of firsteffect,i.e.,DMF0.5%(v/v)andTritonX-1000.005%(v/v). Thefresh concentratewas filteredand sterilized and then addedtotheculturemediumtopreparethetestsolutionsof thedesiredmonosulfuron-esterdoses.

To measure monosulfuron-ester concentrations in the mediumand in algal cells a HPLCanalysis procedure was developed employing a Agilent 1100 HPLC (Agilent Inc.) equippedwithUV-200detector,ODSDiamonsi(5␮m)column with250×4.6mm.OperatingconditionsfortheHPLC anal-ysiswere:mobilephase-methanol/water(containingH3PO4, pH4.0)(53/47,v/v);mobilephaseflowrate1.0mL/min; sam-pleinjectionvolume10␮L;wavelength236nm;andcolumn temperature 40◦C. Under these conditions the resulting calibrationcurvecorrelatingthemonosulfuron-ester concen-tration tothe apex area was y=91302x+26735 (R2_=0.9953) withanaveragerecoveryof95–103%.

Cultures of A. azotica (FACHB-181) and A. flos-aquae (FACHB-245) two nitrogen-fixing algal species of the Nos-tocaceae,were obtainedfrom theInstitute ofHydrobiology of the Chinese Academy of Sciences in Wuhan, China. Axenic cultures were grown in a sterilized HB-111 liquid medium (0.075g/LK2HPO4, 0.125g/L MgSO4, 0.1g/L CaCO3, 0.005g/L FeC6H5O7, 0.005g/LC6H8O7, 0.0025g/L MoO3·H2O, 0.01g/LNaOH)at30±2◦Cunderconstantfluorescentlightof 36.2␮mol/m2_{/s.Theexperimentalcultureswerefirst grown} in250mLflaskscontaining100mLofthemediumwith0.5–1 millioncellspermillilitersunderthesameconditions.Atthe exponentialgrowthphaseofthealgalcultures,smallaliquots oftheconcentratewereaddedtotheculturemediumflasksto resultin0.003,0.03,0.3,3,30,and300nmol/Lof monosulfuron-esterinthetestsamples.Sterilizedwaterwasaddedinstead

tothesame culturemediatoproducethecontrolsamples. Samples were collected 6d after the herbicide addition to determinetheproteinandaminoacidcontents,andtheALS activity.

Growthratesofcyanobacteriaweremeasuredbyrecording lightabsorbanceofthecultureat448nmusinga spectropho-tometer(ShanghaiBiotechnicLtd.).Standardcurvesrelating Abs448nmwithcellnumberswere developedforcontinuous cultures of A. azotica and A. flos-aquae. During the experi-mentalperiod,sampleswerewithdrawnaftertheherbicide treatmentat1,2,3,4,5,and6daysforgrowthrate measure-ments.Thegrowthrate()ofthecyanobacteriawascalculated usingthefollowingEq.(1).

=lnX1−lnX0 T1−T0

(1)

whereX1andX0areAbs448nmmeasuredattimesT1andT0.To estimatethecell’sdryweight,thecultureswerecentrifuged, and the solid mass waswashed with distilled waterthree times, driedat105◦Cfor8h; thecell’sdry weightwas the averageofthreeweightmeasurements.

The proteincontent of the cultureswas determined by usingbovineserumalbumin(BSA)asastandarddescribedby Bradford(1976).11_{One-hundrednanomolesofCoomassie}

Bril-liantBlueG-250wasfirstdissolvedin50mLof95%ethanol andthen100mLof85%(w/v)phosphoricacidwasaddedto thissolution.Theresultingsolutionwasdilutedtoafinal vol-umeof1L.BSAwasdilutedinfiveconcentrations(0.2,0.4,0.6, 0.8,1mg/L)andtheirabsorbanceatthe595nmwavelength weremeasuredafter2minat20◦C.5mLliquidsamplewas takenandcentrifuged(8000rpm)10minutesat4◦Ctocollect algalcells.Addasmallamountofphosphatebuffer(pH7.8), repeated freezingandthawinguntilthealgaefluidbecame blue,thencentrifugedthesampleat8000rmpfor30minat 4◦C and the resulting supernatant was usedto determine proteincontent.0.1mLsupernatantwastakentodetermine theproteincontentaccordingtheregressionequationofthe straightlineofproteinrelatingAbs595nm.

The amino acid components were analyzed using an aminoacidautomaticanalyzer(Hitachi)withanionexchange column afterthe cyanobacterial samplewas hydrolyzedin 6mol/LHClat110±1◦Cfor24h.Theabsorbanceofthe sam-plewasmeasuredat570nmexcept440nmforprolineafter colordevelopmentoftheninhydrinreactionat100◦C.

ALS was extracted from 6d cultivated nitrogen-fixing cyanobacteria of the two test species. The cyanobacteria werecentrifugedat10,000×gfor30minat4◦C(Sorvall Cen-trifuge, DJB Labcare Ltd.) in buffer or medium after cells were sonicated using an ultrasonic cell disruptor (250mv, 3s, 3s)in thedarkat4◦C.Theproteininthe supernatant was precipitatedwith ammonium sulphate,and then cen-trifugedat10,000×gfor30minat4◦Cafter2hincubationat 0◦C.Thepelletwasdissolvedinphosphatebuffer(pH=7.5) containing 200mmol/L sodium pyruvate, 5mmol/L MgCl2, 10mmol/L thiaminepyrophosphate (TPP Sigma Company), 200␮mol/L flavin adenine dinucleotide (FAD Sigma Com-pany), and1mol/Ldl-dithiothreitol (DTTGLBiochem Ltd.). This enzyme solution was kept in ice/water in the dark. Theenzymesolutionwasdissolvedin50mmol/Lphosphate

buffer(pH=7.5)containing20mmol/Lsodiumpyruvateand 0.5mmol/LMgCl2.Avolumeof0.1mLofmonosulfuron-ester wasaddedto0.4mLofenzymeand0.5mLofenzyme reac-tion solution. The mixture was subsequently incubated at 37±1◦C ina water bath for1h in the dark. After adding 0.1mLof3mmol/Lsulfuricacid,themixturewasincubatedat 60±1◦Cfor15mintodecarboxylatetheacetolactateto ace-toin.0.5mLoffreshlyprepared5.0%␣-naphtholin2.5mol/L sodiumhydroxideand0.5mLof0.5%creatinewerethenadded tothemixtureandincubatedat60±1◦Cfor15minforcolor development.Themixturewasthencooledtotheroom tem-perature(25◦C)inawaterbathandabsorbancewasmeasured at525nmusingaspectrophotometer.Thespecificactivityof ALSwasexpressedasnmolofacetoin/nmolofprotein/h.12

To determine the extractable ALS activity, 0.1mL of monosulfuron-esterand0.4mLoftheenzymewere substi-tuted with 0.5mL of the enzyme solution extracted from nitrogen-fixing cyanobacteria that had been cultivated in theculturemediumcontainingmonosulfuron-ester.Six con-centrations of monosulfuron-ester were used as outlined previouslyandthespecificactivityofALSwasdeterminedand expressedasdescribedabove.

Acompletelyrandomizeddesignwiththreereplications wasusedinallduplicatedexperiments.Analysesofvariance (ANOVA)wereperformedonthenon-transformeddata. Sig-nificantdifferencesweredeterminedusingDuncan’stestat p=0.05levelofsignificance(PROCGLM,SASInstitute,2001). ThevaluesofIC50werecalculatedwiththeprogram Origin-Pro7.5SR1(OriginLabCorporation,USA).

Results

Ingeneral,thegrowthratesofA.flos-aquaeandA.azoticaofthe controldecreasedgraduallyastheculturingtimeincreased; however,thegrowthratesofbothcyanobacteriatreatedwith

500 400 300 200 100 0 0 0.03 0.3 3 30 300

Monosulfuron-ester concentration (nmol/L)

A. azotica A. flos-aquae

Protein content (ug/mg dr

y w

eight)

Fig.2–Effectofmonosulfuron-esterconcentrationon proteincontentoftwocyanobacteria.

monosulfuron-ester(0.03–300nmol/L)exhibitedawave pat-ternofinitialdeclinefollowedbygradualincreases(Fig.1).

The growth rates of A. flos-aquae and A. azotica were reducedby28–97%and41–73%at0.03–300nmol/L,after1day, respectively, relativetothecontrol;themonosulfuron-ester treatment then transientstimulated the growth on second

day.ForA.flos-aquae,thegrowthratesat0.03,0.3,3,30,and

300nmol/Lincreasedby185,184,104,75,38,and9%, respec-tivelyrelativetothecontroltreatmentandthe growthrate increasedeclinedwithhighermonosulfuron-ester concentra-tion; however, forA.azotica, the growth ratesincreasedby 83–90%at3–300nmol/L.Itcanbeseenthatboth cyanobac-teria hadthedifferent responsestomonosulfuron-ester. A.

flos-aquaewasmoresensitivitytothisherbicidethanA.azotica.

Fig. 2 illustrates the effect of monosulfuron-ester con-centration on the protein content in the two cyanobac-teria. Monosulfuron-esterapplied at low concentrationsof 1.8 1.5 1.2 .9 .6 .3 0.0 1 2 3 4 5 1 2 3 Day A. azotica A. flos-aquae Gro wth r ate (/da y x 0.1) Day

– 0.03 nmol/L, – 0.03 nmol/L, – 0.3 nmol/L, – 3 nmol/L, – 30 nmol/L, – 300 nmol/L, – Control. Vertical bar- standard

error of the mean

4 5

180 150 120 A.azotica A.flos-aquae 90 60 30 0 0.003 0.03 0.3 3 30 300 3000

Monosulfuron-ester concentration (nmol/L) The regression lines: y = –5.97×2 – 35.51x + 100.77

(A. flos-aquae, r2=0.98) and y = –1.43×2 – 12.10x + 82.83 (A. azotica, r2

=0.96)

Specific activity (nmol/mg/h)

Fig.3–Effectofmonosulfuron-esterconcentrationon

invitroALSactivity.

0.03–0.3nmol/Lstimulatedtheproductionofproteinsinboth cyanobacteria,butthe reversewasobservedathigher con-centrations (i.e. 30–300nmol/L). The protein content of A.

flos-aquaecellculturestreatedwithmonosulfuron-esterat0.03

and0.3nmol/Lincreased101and89%,respectively relative tothecontrol(p<0.05);itsproteincontentdecreasedsharply as monosulfuron-ester concentration increased, for exam-ple,theproteincontentwas9–16%lowerwith30–300nmol/L monosulfuron-ester.Themonosulfuron-estertreatmentdid notmarkedlyhaveeffectonproteincontentinA.azotica,its proteincontentincreasedbyonly0.8%with monosulfuron-ester0.03nmol/L.ItisthusclearthatA.flos-aquaeexhibited greatersensitivitytomonosulfuron-ester.

TheinvitroALSactivityvariedbetweenthetwo

cyanobacte-rialspeciesfollowingmonosulfuron-esterapplication(Fig.3).

ForA.flos-aquae,theactivityofALSwasreducedintherangeof

0.8–92%astheconcentrationofmonosulfuron-esterincreased from0.003to300nmol/L,andtherewasasignificant differ-enceinactivitybetweeneachconcentration(p<0.05).TheALS activity ofA. azotica atlow monosulfuron-ester concentra-tions(0.003–0.3nmol/L)didnotdiffersignificantlyfromthe sameofthe control;itdeclined notably,by19–75%relative tothe control, asthe concentration ofmonosulfuron-ester increasedfrom3to300nmol/L.CalculatedIC50valuesforA.

flos-aquaeandA.azoticawere3.3and101.3nmol/Lrespectively.

Theseresultsindicatethatmonosulfuron-esterdoesinhibit ALSactivityinthetwonitrogen-fixingcyanobacteria, particu-larlyathigherconcentrations.

Interestingly, the extractable ALS activity in the two cyanobacteriarespondeddifferentlytomonosulfuron-esterin 0.03–300nmol/L(Fig.4);relativetothecontrol,theextractable ALSactivityinA.azoticaincreased28–45%whilethesamein

A.flos-aquaedeclinedby11–33%.Therewasafurtherevidence

thatA.flos-aquaeweremoresensitivetothisherbicidethanA.

azotica.

Table1presentstheeffectofmonosulfuron-ester concen-trationonaminoacids producedin thetwocyanobacteria. AminoacidproductionincellsofA.flos-aquaeandA.azotica

180 150 120 90 60 30 0 0 0.03 0.3

Monosulfuron-ester concentration (nmol/L) The regression lines: y= –0.86×2 – 4.20x + 148.8

(A. flos-aquae, r2_{=0.92) and} y=–1.97×2 +16.87x +94.05 (A. azotica, r2_=0.92)

A.azotica A.flos-aquae

Specific activity (nmol/mg/h)

3 30 300

Fig.4–Effectofmonosulfuron-esterconcentrationon extractableALSactivity.

wasclearlyinhibitedbymonosulfuron-esterinthe character-isticdose-dependentresponse;namely,increasedinhibition ofaminoacidproductionwithincreasingmonosulfuron-ester concentration. The quantity of branch-chain amino acids valine,isoleucine,leucineincellsofA.flos-aquaewasreduced by 56, 67, and 67% at the 0.003nmol/L, respectively rela-tivetothecontrol,and by80,86,and87%atthe30nmol/L (p<0.05). Thevaline,isoleucine,leucinecontent ofA. azot-icacellswasreducedby35,45,and47%atthe0.003nmol/L concentration,andby61,52,and55%atthe30nmol/L con-centration.TheotheraminoacidscontentofA.azoticaandA.

flos-aquae cellssuchasglycine,alanine,phenylalanine,

ser-ine, threonine, methionine,glutarnine, asparticacid,lysine, and argininewere respectively reduced 8–55% and 67–90% at monosulfuron-esterconcentrations 0.003and 30nmol/L, andproline,tyrosine,andhistidinewerebelowdetectionat 30nmol/L.AgainA.flos-aquaewasthemoresensitiveofthe two.

Discussion

The transient stimulative effect of monosulfuron-ester on algal growth on the second day ofthis study issimilar to thefindingsofShenetal.(2005)forbutachlorandacetochlor onseveralAnabaenaspecies.7_{Ourresultthatthe}

correspond-ing growthratesoftheA.flos-aquae andA. azoticaexposed todifferentconcentrationsofmonosulfuron-esterexhibited wavepatternswassimilartothereportofShenetal.(2011).13

Hormesis,thestimulativeeffectofatoxinatsub-toxic con-centrations,followingtheapplicationofotherherbicidesand allelochemicalshasbeendocumented.14

The study has evaluated the effects of herbicides on metabolic pathways of protein synthesis in non-target organisms.15 _{Our findings at low monosulfuron-ester}

con-centrations(0.03–3nmol/L)indicatethattheproteincontent ofcellsfrom thetwocyanobacteriaincreasedsubstantially. Theincreasemayhaveresultedfromstimulationinprotein

Table1–Effectofmonosulfuron-esterconcentrationonaminoacidsproduced.

Species Concentration(nmol/L) Aminoacid(mg/L)a

VAL ILE LEU LYS THR PHE SER PRO

A.azotica Control 73.8b _{61.7 111.7 62.5 77.3 58 59.4 4.8} 0.003 48.3 33.8 58.8 40.7 51.8 38.7 39.6 0 %c _{34.6 45.2 47.4 34.9 33 33.3 33.3 100} 0.3 49.8 41.2 68.8 44.3 55.3 43.8 42.4 0 % 32.5 33.2 38.4 29.1 28.5 24.5 28.6 100 30 29.1 29.6 50.2 44.3 39.1 31.1 28.3 0 % 60.6 52 55.1 29.1 49.4 46.4 52.4 100 A.flos-aquae Control 250.6 291.2 547.1 298.9 380.6 239.6 271.8 7.8 0.003 110.6 96.5 181.2 95.7 118.5 83.1 91.8 0 % 55.9 66.9 66.9 68 68.9 65.3 66.2 100 0.3 75.2 60 108.2 60.4 75.9 57.7 57 0 % 70 79.4 80.2 79.8 80.1 75.9 79 100 30 48.9 41 72 43.9 54.3 45.6 41.3 0 % 80.5 85.9 86.8 85.3 85.7 81 84.8 100

Species Concentration(nmol/L) Aminoacid(mg/L)a

HIS TYR ARG ALA MET ASP GLU GLY

A.azotica Control 12.7 13.5 81.5 132.4 31.3 138.6 147.4 68.8 0.003 0 7.4 75.3 98.5 24 122.1 110.7 51.9 %c _{100 45.2 7.6 25.6 23.3 11.9 24.9 24.6} 0.3 0 0 70.7 94.7 18.9 120.8 104.5 52.1 % 100 100 13.3 28.5 39.6 12.8 29.1 24.3 30 0 0 36.8 60.9 7 76.2 68.9 37 % 100 100 54.8 54 77.6 45 53.3 46.2 A.flos-aquae Control 87.6 173 492.1 608.3 147.9 676.1 727 329.7 0.003 21.8 46.6 151.1 201.9 47.7 215.6 226.7 107.8 % 75.1 73.1 69.3 66.8 67.7 68.1 68.8 67.3 0.3 11.9 13.8 85 131.6 29.5 139.8 145.9 69.2 % 86.4 92 82.7 78.4 80.1 79.3 79.9 79 30 0 0 58 91 14.9 104.2 106.2 50.7 % 100 100 88.2 85 89.9 84.6 85.4 84.6

a _{Aminoacidisexpressedusingmilligramperliterhydrolyticsolution.} b _{Samplingwascarriedout6dafterherbicidetreatment.}

c _{%ispercentagedecreasecomparedwithcontrol.}

synthesis viaincreased productionofRNA.16 _{Alternatively,}

atlower monosulfuron-esterconcentrations,cyanobacterial cellsmayhaveassimilatedmoreorganiccarbonandnitrogen, which interfered withherbicide uptake,due tothe forma-tionofaninactiveherbicidecomplexwithorganiccarbonand nitrogen,andthusreducedtheherbicide’toxicity;the bacte-riamay alsodegrade the herbicideinto hydrate ofcarbon andaminoacidsmetabolitesresultinginalower toxicity.17

Thelowerproteincontentsofbothcyanobacteriasubjected tomonosulfuron-esterathighconcentrations(30–300nmol/L) mayberelatedtoincreaseofproteaseactivity18_;itmayalso

bethe result ofinhibition ofthe ALSactivity which inter-fered with production of the three branched-chain amino acids(valine,leucine,andisoleucine)essentialprecursorsof proteins.12

The mode of action of the sulfonylurea is inhibition of ALS,which is responsible for catalyzing the biosynthe-sis of the branch-chain amino acids valine, leucine, and isoleucine.3 _{Shen et al. (2009) reported that monosulfuron}

inhibitsvitroALSactivityinthreenitrogen-fixing cyanobac-teriaathigherconcentrations.19 _{Ourstudy issimilartoher}

resultsthatthe ALSactivity wasmorereduced asthe con-centrationmonosulfuron-esterincreased;IC50 valuesof3.3 and101.3nmol/LforA.flos-aquaeandA.azotica,respectively, have provided furtherevidences ofthe negative impactof thisherbicideongrowthofthesecyanobacterialspecies.The strong inhibition of ALS activity found in both species in

vitro confirms that the toxic effects ofmonosulfuron-ester

is indeed of inhibition to ALS as is the case with other sulfonylurea herbicides.20 _{Shen et al. (2009) also pointed}

outthatmonosulfuronstimulatedtheextractableALS

activ-ity in A. azotica, but the specific activity in A. flos-aquae

decreased athigh concentrationofmonosulfuron.19 _Inour

study, withmonosulfuron-esterpresent at0.03–300nmol/L, theextractableALSactivityinA.azoticaincreasedby28–45%, whilethespecificactivityofA.flos-aquaedecreasedby3–11%. It is possible that differences in specific activity between

in vitro and in vivo responses of the two cyanobacteria

were due to the presences of different levels of herbicide detoxifyingenzymes(e.g.glutathioneS-transferase(GST)and mixed-functionoxidase(MFO)).21_{Otherpossiblereasonsfor}

speciesonthe extractableALSspecificactivityatthesame monosulfuron-esterconcentrationincludeabsorptionofthe herbicideandthedifferentdegreesofALSinhibitionbetween thespecies.

That A. flos-aquae exhibited greater sensitivity to

monosulfuron-ester than A. azotica may be related to its greater capacityfortakingup theherbicide.Suchdifferent responses may be due to different biological properties of the cyanobacteria22 _{and may be exploited to select for}

cyanobacteria that are more resistant to monosulfuron-esterinthecroppingsystems.Forinstance,theculturingof beneficial nitrogen-fixing cyanobacteria in paddy fields as ‘bio-fertilizers’isahighlydesirableoptionforincreasingrice yieldand preventing any negativeeffects ofthis cropping systemontheenvironment.10

Conclusion

Application of monosulfuron-ester at 0.03–300nmol/L affected the growth rates of A. flos-aquae and A. azotica inthe wavepatterns, andhad an inhibitoryeffecton pro-tein synthesis at higher concentrations (30–300nmol/L). Theproduction ofamino acids was reduced with increas-ing monosulfuron-ester concentration. Monosulfuron-ester substantially inhibited in vitro Acetolactate synthase (ALS) activityasevidencedbytheIC50valuesof3.3and101.3nmol/L

forA.flos-aquaeandA.azotica,respectively.Theresultsshow

that the normal agricultural use of monosulfuron-ester at post-emergenceratesof30–60nmol/Linricefieldswilllikely betoxictothebothubiquitousnitrogen-fixingcyanobacteria; themonosulfuron-estertoxicitywasduetoits interference withproteinmetabolismviainhibitionofbranch-chainamino acidbiosynthesis,especiallytheALSactivity.Wealsosuggest that inrice cropping systems wherethe new sulfonylurea herbicide monosulfuron-ester is frequently applied, the beneficial nitrogen-fixing cyanobacteria, A. azotica, may be employedas“biofertilizers”sinceitismoreresistanttothe herbicidethanA.flos-aquae.

Conflicts

of

interest

Theundersignedauthor(s)state(s)thateveryoneinvolvedin the publication process (authors, reviewers, editorial board members,andeditorialstaff)mustbefreefrom conflictsof interestthatcouldadverselyinfluencetheirjudgment, objec-tivityorloyaltytothearticleandassignments.

Acknowledgment

FundingforthisworkwasprovidedbytheNationalNatural ScienceFoundationofChina(21377086).

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e

s

1.LiZM,JiaGF,WangLX.Sulfonylureacompoundsandits herbicideusage.ChinesepatentCNI:ZL94118793.1994.

2.WangW.Applicationofnewsulfonylureaherbicidesinrice directseedlingfields.ShanghaiAgricSci.2006:12–16. 3.RiarDS,NorsworthyJK,SrivastavaV,etal.Physiologicaland

molecularbasisofacetolactatesynthase-inhibiting

herbicideresistanceinbarnyardgrass(Echinochloacrus-galli).J AgricFoodChem.2012;61(2):278–289.

4.YuQ,NelsonJK,ZhengMQ,etal.Molecularcharacterisation ofresistancetoALS-inhibitingherbicidesinHordeum leporinum,biotypes.PestManageSci.2007;63(9): 918–927.

5.ReddyKN,WhitingK.Weedcontrolandeconomic

comparisonsofglyphosate-resistant,sulfonylurea-tolerant, andconventionalsoybean(Glycinemax)systems.Weed Technol.2014;14(1):204–211.

6.GreweCB,PulzO.TheBiotechnologyofCyanobacteria.Ecologyof CyanobacteriaII;2012:707–739.

7.ShenJY,LuYT.Effectsofherbicidesonbiodiversityofrice fieldsinChina.In:ImpactAssessmentofFarmChemicalsRun OfffromPaddyConference.Japan:NationalInstitutefor Agro-EnvironmentalSciences,Japan(NIAES)/National InstituteofAgriculturalScienceandTechnology,Korea (NIAST);2005:56–67.

8.VijaiKA.Anabaenaflos-aquae.CritRevEnvironSciTechnol. 2014;44(18):1995–2037.

9.DaiG,DebloisCP,LiuS,etal.Differentialsensitivityoffive cyanobacterialstrainstoammoniumtoxicityandits inhibitorymechanismonthephotosynthesisofrice-field cyanobacteriumGe–Xian–Mi(Nostoc).AquatToxicol. 2008;89(2):113–121.

10.ShenJY,LuYT,ChengG.Effectsofchemicalherbicideson toxicityofnon-targetnitrogen-fixingCyanobacteriainpaddy fields.In:20thAsian-PacificWeedSci.Conf.Vietnam:WeedSci. Soc.Asian-Pacific.2005:665–670.

11.BradfordMM.Arapidandsensitivemethodforthe

quantitationofmicrogramquantitiesofproteinutilizingthe principleofprotein-dyebinding.AnalBiochem.

1976;72:248–254.

12.FanZJ,AiYW,QianCF,LiZM.Herbicideactivityof Monosulfuron-esteranditsmodeofaction.JEnvironSci (China).2005;17:399–403.

13.ShenJY,LuoW.Effectsofmonosulfuronongrowth: photosynthesis,andnitrogenaseactivityofthree nitrogen-fixingcyanobacteria.ArchEnvironContamToxicol. 2011;60(1):34–43.

14.CalabreseEJ.Paradigmlost,paradigmfound:the

re-emergenceofhormesisasafundamentaldoseresponse modelinthetoxicologicalsciences.EnvironPollut.

2005;138:379–412.

15.OkmenG,TurkcanO,ErdalP.Effectofherbicideson chlorophyll-a,␤-caroten,phycocyaninandallophycocyanin contentofAnabaenasp.JApplBiolSci.2012;7:

20–27.

16.GruenhagenRD,MorelandDE.EffectsofherbicidesonATO levelsinexcisedsoybeanhypocotyls.WeedSci.

1971;19:319–323.

17.NagaiT,IshiharaS,YokoyamaA,etal.Effectsoffourrice paddyherbicidesonalgalcellviabilityandtherelationship withpopulationrecovery.EnvironToxicolChem.

2011;30(8):1898–1905.

18.BhuniaAK,BasuNK,RoyD,ChakrabartiA,BanerjeeSK. Growth,chlorophyllacontent,nitrogen-fixingability,and certainmetabolicactivitiesofNostocmuscorum:effectof methylparathionandbenthiocarb.BullEnvironContam Toxicol.1991;471:43–50.

19.ShenJY,AntonioDT,ShenM,etal.Molecularbasisfor differentialmetabolicresponsestomonosulfuroninthree nitrogen-fixingcyanobacteria.WeedSci.2009;57(2): 133–141.

20.ChaleffRS,MauvaisCJ.Acetolactatesynthaseisthesite actionoftwosulfonylureaherbicidesinhigherplants. Science.1984;224:1443–2144.

21.ChenJ,ZhengHW,ShongTY,XiGZ,TongLF.Thegenetic diversityofAnabaenaazollaebasedonRAPDanalysis.Acta HydrobiolSin.2001;25:531–534.

22.CumminsI,WortleyDJ,SabbadinF,etal.Keyrolefora glutathionetransferaseinmultiple-herbicideresistancein grassweeds.ProcNatlAcadSciUSA.2013;110(15): 5812–5817.