Selenium hyperaccumulation offers protection from cell disruptor herbivores

Selenium hyperaccumulation offers protection from cell disruptor herbivores Quinnetal.BMCEcology2010,10:19 Seleniumhyperaccumulationoffersprotection fromcelldisruptorherbivores ColinFQuinn1†,JohnLFreeman1,2,4†,RayJBReynolds1,JenniferJCappa1,SirineCFakra3,MatthewAMarcus3, StormyDLindblom1,ErinKQuinn1,LindsayEBennett1,ElizabethAHPilon-Smits1* Abstract Background:Hyperaccumulation,therarecapacityofcertainplantspeciestoaccumulatetoxictraceelementsto levelsseveralordersofmagnitudehigherthanotherspeciesgrowingonthesamesite,isthoughttobean elementaldefensemechanismagainstherbivoresandpathogens.Previousresearchhasshownthatselenium(Se) hyperaccumulationprotectsplantsfromavarietyofherbivoresandpathogens.Seleniumhyperaccumulatingplants sequesterSeindiscretelocationsintheleafperiphery,makingthempotentiallymoresusceptibletosome herbivorefeedingmodesthanothers.InthisstudyweinvestigatetheprotectivefunctionofSeintheSe hyperaccumulatorsStanleyapinnataandAstragalusbisulcatusagainsttwocelldisruptingherbivores,thewestern flowerthrips(Frankliniellaoccidentalis)andthetwo-spottedspidermite(Tetranychusurticae). Results:AstragalusbisulcatusandS.pinnatawithhighSeconcentrations(greaterthan650mgSekg-1)wereless subjecttothripsherbivorythanplantswithlowSelevels(lessthan150mgSekg-1).Furthermore,inplants containingelevatedSelevels,leaveswithhigherconcentrationsofSesufferedlessherbivorythanleaveswithless Se.Spidermitesalsopreferredtofeedonlow-SeA.bisulcatusandS.pinnataplantsratherthanhigh-Seplants. SpidermitepopulationsonA.bisulcatusdecreasedafterplantsweregivenahigherconcentrationofSe. Interestingly,spidermitescouldcolonizeA.bisulcatusplantscontainingupto200mgSekg-1 dryweight, concentrationswhicharetoxictomanyotherherbivores.Seleniumdistributionandspeciationstudiesusingmicro- focusedX-rayfluorescence(μXRF)mappingandSeK-edgeX-rayabsorptionspectroscopyrevealedthatthespider mitesaccumulatedprimarilymethylselenocysteine,therelativelynon-toxicformofSethatisalsothepredominant formofSeinhyperaccumulators. Conclusions:ThisisthefirstreportedstudyinvestigatingtheprotectiveeffectofhyperaccumulatedSeagainst cell-disruptingherbivores.ThefindingthatSeprotectedthetwohyperaccumulatorspeciesfrombothcell disruptorslendsfurthersupporttotheelementaldefensehypothesisandincreasesthenumberofherbivoresand feedingmodesagainstwhichSehasshownaprotectiveeffect.Becausewesternflowerthripsandtwo-spotted spidermitesarewidespreadandeconomicallyimportantherbivores,theresultsfromthisstudyalsohavepotential applicationsinagricultureorhorticulture,andimplicationsforthemanagementofSe-richcrops. Background showntoreducethechanceofgettingcancer,including Formanyorganisms,includingmammalsandmanyspe- the devastating widespread lung and prostate cancers cies of bacteria and algae, selenium (Se) is an essential [3,4]. In addition, Se plays an essential role in thyroid trace element [1]. These organisms contain selenopro- function [5]. While Se is essential for many organisms, teins, some of which destroy free radicals that damage thereisanarrowmarginbetweendeficiencyandtoxicity DNA [2]. In humans, Se supplementation has been levels.Seleniumtoxicitycanbebothacuteandchronic. Acute Se toxicity leads to “blind staggers” in livestock; the symptoms include staggered walking, impaired *Correspondence:epsmits@lamar.colostate.edu vision, paralysis and sometimes death. Chronic Se poi- †Contributedequally soning leads to hair and nail loss, fatigue, nausea and 1DepartmentofBiology,ColoradoStateUniversity,FortCollins,CO80532, USA eventuallydeath[6]. Fulllistofauthorinformationisavailableattheendofthearticle ?2010Quinnetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited. Page2of11 Quinnetal.BMCEcology2010,10:19 Selenium has noknown essential function for higher to feed on Se hyperaccumulating plants. The effect of plants,andelevatedlevelsofSearetoxictomostplants feeding modeonherbivore susceptibility tohyperaccu- [7].Thistoxicity isduetothechemicalsimilarity ofSe mulated elements is illustrated by the study by Jhee and sulfur (S). Most plants inadvertently assimilate Se et al. [25] who found that the Ni hyperaccumulator intoproteins,leadingtotoxicity[1].Afewplantspecies Streptanthus polygaloides was protected from folivore haveevolvedtoaccumulateunusuallylargeamountsof herbivoresbutnotvascularfeedingherbivores.Inaddi- Se, as much as 1%, or 10,000 mg Se kg-1 dry weight tion,theZnhyperaccumulatorThlaspicaerulescenswas (DW)[8,9]. Theseuniqueplants arecalled Sehyperac- notprotectedfromsnailherbivory[26] cumulatorsandtheyavoidSepoisoningbymethylating This study investigates the protective effect of Se SeCys to form methylselenocysteine (MeSeCys), which hyperaccumulationagainstcelldisruptorherbivorespe- is relatively non-toxic because it does not get incorpo- cies, specifically the two-spotted spider mite (Tetrany- ratedintoproteins[10]. chus urticae) and western flower thrips (Frankliniella Hyperaccumulation is a phenomenon where plants occidentalis).Bothherbivoreshavebeenobservedfeed- accumulate particular elements to levels several orders ing on A. bisulcatus and S. pinnata in the greenhouse of magnitude higher than other plant species growing and both feed by piercing the cell surface with their onthesamesubstrate[11].Someotherelementsbesides mouthpartsandsuckingoutthecellcontents[27].This Sethatcanbehyperaccumulatedbyplantsincludealu- study,thefirsttoexaminecelldisruptorherbivores’sen- minum (Al), arsenic (As), cadmium (Cd), manganese sitivitytoSehyperaccumulation,isecologicallyrelevant (Mn), nickel (Ni) and zinc (Zn) [12]. Feist and Parker becausebothoftheseherbivoressharehabitatswithSe [13] defined Se hyperaccumulation as plants that con- hyperaccumulatingplants[13,28].Interestingly,western tain more than 1,000 mg Se kg-1 DW. Most research flowerthripsandmanySehyperaccumulatingplantspe- investigating the functional significance of hyperaccu- ciesarenativetothewesternUnitedStatesandprotec- mulation has focused on and lent support to the ele- tion against thrips herbivory may have contributed to mentaldefensehypothesis,whichstatesthatplantshave theevolutionofSehyperaccumulation.Bothherbivores evolvedtohyperaccumulatethesevarioustoxicelements arealsoecologicallyimportantpests.Two-spottedspider as protection against herbivore and pathogen attacks mitescanhavedevastatingeffectsoncropyieldsworld- [14]. Hyperaccumulated As,Cd,Ni,ZnandSeallhave wide[29].Outbreaksoftenoccurafterpesticideapplica- been shown to protect plants from herbivores and/or tion inadvertently kills their predators [30]. Western pathogens[15-19]. flower thrips are native to the Western United States, To date, Se hyperaccumulation has been shown to but have been reported on all continents except Asia protect plants fromamammalianherbivore, theblack- and Antarctica [31]. Through a combination of their tailed prairie dog (Cynomys ludovicianus), as well as herbivory andtheirnotoriousability totransfer disease from several arthropod herbivores and two fungal and develop pesticide resistance, western flower thrips pathogens [20-23]. Additionally, Se hyperaccumulating cansignificantlyreducecropyields[32,33].Inthisstudy plantsharboredfewerarthropodsandarthropodspecies we report a significant effect of plant Se accumulation thancomparablenonSehyperaccumulatorsgrowingin ontheresponseoftwoSehyperaccumulators(A.bisul- thesame,seleniferoushabitat[24].Moreover,Sehyper- catus and S. pinnata) to both cell disruptors, the wes- accumulating plants sequester Se in organs andtissues ternflowerthripsandthetwo-spottedspidermite. thataremostsusceptibletoherbivoreattack.Forexam- ple, the Se in hyperaccumulator Astragalus bisulcatus Methods (two-grooved milk vetch) is predominantly present in Plant material the leaf hairs, and Stanleya pinnata (Prince’s plume), SeedsofA.bisulcatuswereobtainedfromplantsgrow- another Se hyperaccumulator, sequesters Se in epider- ing at Pine Ridge Natural Area in Fort Collins, CO, mal cells in the leaf margins [9]. This uneven distribu- USA(40?32.70N,105?07.87W).PineRidgeNaturalArea tion of Se, which leaves some areas of the plant with isaseleniferoushabitat;thepopulationofA.bisulcatus lowerconcentrationsofSethanothers,mayallowsome from which seeds were collected accumulates up to herbivores, depending on their feeding mode, to avoid 10,000 mg Se kg-1 [34]. Seed germination and growth this elemental defense. Indeed, some herbivore species followed an arid western plant growth protocol pre- werefoundlivingonSehyperaccumulatingplantsinthe viously used for Se hyperaccumulating plants and field and apparently were feeding on the Se-rich plant describedbySorsetal.[35].Plantsweregrownonpre- material, in viewof thefact that they contained higher washed Turface MVP (Profile Products LLC, Buffalo Seconcentrations thanindividuals collected fromnon- Grove,IL)in25cmdiameterpotsingreenhousecondi- hyperaccumulators[24].Thus,itisimportanttoinvesti- tions (24/20?C day/night, 16-h photoperiod, 300 μmol gatetheeffectoffeedingmodeontheherbivores’ability m-2sec-1 photosyntheticphotonflux).Threeweeksafter Page3of11 Quinnetal.BMCEcology2010,10:19 germinationhalfoftheplantsreceivedhigh-Sefertilizer experiments 10 high- or low-Se plants grown as treatments, 1goffertilizer (Miracle-Gro Excel, 15:5:15 described above were placed in 20 L glass tanks that Cal-Mag, The Scotts Co., Marysville, OH) per liter of wereplacedin24/20?Cday/night,16-hphotoperiodand water combined with 20 μM Na2SeO4, while the other 300μmolm-2 sec-1 photosyntheticphotonfluxandcov- halfreceivedlow-Sefertilizertreatments,1goffertilizer ered with 0.2 mm2 mesh. Plants were watered either per liter of water with 2 μM Na2SeO4, three times a with 20 μM Na2SeO4 or without Se 3 times weekly. week. After 20 weeks of growth plants were used for Each plant was inoculated with 100 spider mites that thripsandspidermiteexperimentsasdescribedbelow. were collected from A.bisulcatus plants with high spi- Stanleya pinnata seeds were obtained fromWestern der mite populations. The number of spider mites on NativeSeed(Coaldale,CO,USA)andplantsweregrown eachplantwascountedafter7,14and21daysandthe fromseedinpre-washedTurfaceMVP.Thirty-sixplants percent of the population change was calculated for were grown in a growth room (24?C/20?C, 12 h/12 h each plant. For choice experiments spider mites were light/dark,120μmolm-2s-1photosyntheticphotonflux): given a choice to feed on high- or low-Se plants. One 10 weeks after germination half of the plants were high- and one low-Se A. bisulcatus plant grown as wateredtwiceaweekfor50weekswith1goffertilizer described above was placed in a tank and 100 spider (Miracle-Gro Excel, 15:5:15 Cal-Mag, The Scotts Co., miteswereplacedoneachplant.Thenumberofspider Marysville, OH)perliterofwaterand20μMNa2SeO4, mitesoneachplantwascountedafter7,14and21days theotherhalfwerewateredwith1goffertilizerperliter and percent population change was calculated. This ofwaterasacontrol.Plantswereusedforthripsandspi- experimentwasrepeated7times.LeafSeconcentrations dermiteexperimentsasdescribedbelow. ofyoungestmatureleaveswerecomparedbetweenhigh- andlow-Seplantsasdescribedbelow. Effects ofSeonherbivory ofA.bisulcatus bythrips In addition to choice and non-choice experiments, ToinvestigatethripstoxicitytoSeandtheirpreference low-SeA.bisulcatuspre-infectedwithspidermiteswere tofeedonhigh-orlow-Seplants,bothnon-choiceand provided with Se to determine if adding Se reduces choice experiments were conducted. For non-choice established populationsofspidermites.Attheonsetof experiments high- and low-Se A. bisulcatus were the experiment low-Se A. bisulcatus plants that were infected with western flower thrips by placing three beingtreatedwith2μmSewereinfectedwithlargespi- excised A. bisulcatus leaves previously harboring large dermitepopulations.Forthreeweekseightoftheplants populationsofthripsoneachplant.Thripswereinitially wereprovided with40μmSethree timesaweekwhile acquired from A.bisulcatus plants growing in agreen- eightotherswereprovidedwithwaterasacontrol.The house and suffering thrips infestation. Two high and percent population change in spider mite population low-Se plants were then placed in separate 20 g glass wasrecordedafter7,14and21days.LeafSeconcentra- tanksthatwerekeptin24/20?Cday/night, 16-hphoto- tion of the youngest mature leaves were measured period, 300 μmol m-2 sec-1 photosynthetic photon flux beforeandaftertheexperiment. andwatered3timesaweekeitherwith20μMNa2SeO4 Spidermitesfromhigh-SeA.bisulcatusplantsusedin 2 orwithoutSe.Tankswerecoveredwith0.2mm nylon non-choiceexperimentswerecollectedandanalyzedfor meshtopstopreventthripstransferwhilestillallowing Se speciation. Samples were washed and flash-frozen gas exchange. For choice experiments plants were usingliquidnitrogen.Sampleswerekeptfrozenat-80?C infectedwiththripsasdescribedaboveandahigh-and topreventSemetabolism,andSespeciationwasdeter- alow-Seplantwereplacedinthesameglasstank.After minedusingXANESasdescribedbyMarcusetal.[36], three weeks of herbivory the percentage of young usingknownselenocompoundsasstandards. (matureleavesfromthetopfivenodes),medium(leaves frommiddle nodes) andold (leaves fromthebottom 3 Sespeciation, X-ray microprobe measurements nodes)leavesandthepercentageofleafletsperleafwith Spider mites from high-Se A.bisulcatus plants used in visualsignsofthripsherbivorywerecalculatedoneach non-choiceexperimentswerecollectedandanalyzedfor plant. Non-choice experiments were repeated 6 times Se speciation. Samples were washed and flash-frozen andchoiceexperimentswererepeated4timesforhigh- usingliquidnitrogen.Sampleswerekeptfrozentopre- Seand4timesforlow-Setreatments.Seleniumconcen- vent Se metabolism, andSespeciation was determined trations for young, medium and old leaves were using XANES as described earlier [21,36], using well measuredasdescribedbelow. characterizedselenocompoundsasstandards. Effects ofSeonherbivory ofA.bisulcatus byspider mites Effects ofSeonherbivory ofS.pinnata bythrips Spider mite non-choice and choice experiments were Stanleyapinnata,anotherSehyperaccumulatingspecies, also conducted using A. bisulcatus . For non-choice was also used to determine if Se protects against cell Page4of11 Quinnetal.BMCEcology2010,10:19 disrupting herbivores. Thrips were given a choice to Results feed on either high- or low-Se S. pinnata. Eighteen Effects ofSeonherbivory ofA.bisulcatus bythrips high-Seand18low-Seplantsgrownasdescribedabove Toinvestigate iftheSehyperaccumulator A.bisulcatus wereintermixedandplacedinagrowingroom(12h/12 wasprotected from western flower thrips, both choice h light/dark, 120 μmol m-2 s-1 photosynthetic photon andnon-choicestudieswereconductedwithplantscon- flux) heavily infested with thrips. Plants were watered taininghighandlowconcentrationsofSe(thethripsare either with 20 μM Na2SeO4 or without Se 3 times displayed in Figure 1A, B; high- and low-Se leaflets weekly.After4weeksofbeingexposedtothripsherbiv- exposedtothripsherbivoryareshowninFigure1C,D). ory the percentage of leaves with visual signs of thrips Thenon-choiceexperimentsrevealedthatthefractionof herbivorywascomparedbetweenplantswithandwith- leaves with herbivory wassignificantly less forhigh-Se outSe.Inaddition,sincewithinoneS.pinnataplantSe plantsthanlow-Seplants(p=0.018,t=-2.832,n=6for isunevenlydistributedwedeterminedSeconcentration both high- andlow-Se experiments), andthat younger ofleaveswiththripsherbivoryandleaveswithoutthrips leavessufferedlessherbivorythanolderleavesfromboth herbivoryfromthesamehigh-Seplant.ForsixoftheS. high-andlow-Se plants (Figure 2A).Inaddition, fewer pinnata plants treated with Se two similar-aged leaves leafletsperleafsufferedthripsherbivoryonhigh-Sethan per plant, one with herbivory and one without herbiv- ory,werecollected andanalyzed forelemental concen- trations using ICP-AES, as described below. To determine the variation in Se concentration of leaf age inplantsnotsufferingherbivory,threeleavesfromcon- secutive nodes on the same high-Se S. pinnata plants weretestedforSeconcentration.Thiswasrepeatedfor 6plants. Effects ofSeonherbivory ofS.pinnata byspider mites Ten high-Se and nine low-Se S. pinnata grown as described above were interspersed in a50 cm× 50 cm area ona greenhouse bench. Eachplant was watered 3 timesweeklyeitherwith20μMNa2SeO4orwithoutSe and was infected with spider mites by placing three leaves fromotherplants thatharboredhighconcentra- tionsofspidermitesonthecenterofeachplant.Spider miteswereallowedtoforagefortwoweeks,andherbiv- ory was then scored by counting the number of leaves on each plant with and without visual signs of spider mite herbivory. The youngest mature leaves were col- lected from each plant and analyzed for Se concentration. Elemental analysis Elemental concentrations inleaves weredetermined by digestingapproximately100mgDWofleafmaterialin 1 ml of nitric acid as described by Zarcinas et al. [37]. Usingdistilledwaterthesamplesweredilutedto10ml and elemental concentrations were determined using Inductively Coupled Plasma Atomic Emission Spectro- metry(ICP-AES)asdescribedbyFassel[38]. Data analysis The software package JMP-IN (3.2.6, SAS Institute, Figure 1 Western flower thrips feeding on A. bisulcatus. A, B: Cary,NC)wasusedforalldataanalysis.Student’ st-test ThripspiercingA.bisulcatusleaflets,asviewedfromthetop(A)and side(B).C,D:Representativeleafletsfromhigh-Se(C)andlow-Se was used to compare differences in herbivory between (D)A.bisulcatusplantsafterexposuretothripsherbivory.Herbivory high-Se and low-Se plants and to compare elemental damageisapparentaswhitepatcheswithblackspots. concentrationsofleafsamples. Page5of11 Quinnetal.BMCEcology2010,10:19 Figure2SeleniumreducesthripsherbivorytoA.bisulcatusinbothchoiceandnon-choiceexperiments.A-C:Thripsnon-choicefeeding experimentwherethripswereofferedonlyhigh-Seorlow-Seplants.HerbivorywasquantifiedasthepercentageofentireA.bisulcatusyoung, mediumandoldleavesthatshowedherbivory(A)andasthepercentageofleafletsperleafthatshowedherbivory(B).TheleafSe concentrationofthehigh-Seandlow-Seplantsusedinthenon-choicestudiesisshowninpanelC.D-F:Thripschoicefeedingexperiments wherethripswereprovidedwithachoicebetweenhigh-Seandlow-Seplants.HerbivorywasquantifiedasthepercentageofA.bisulcatus young,mediumandoldleavesthatshowedherbivory(D)andasthepercentageofleafletsperleaf(E)thatsufferedherbivory.TheleafSe concentrationoftheplantsusedinthechoicestudyisshowninpanelF.Valuesaremeans+/-SE.Anasteriskaboveapairofbarsrepresentsa significantdifferencebetweenthehigh-Seandlow-Setreatments(t-tests,a=0.05,n=6forbothhigh-Seandlow-Senon-choiceexperiments, n=4forchoiceexperiments). Page6of11 Quinnetal.BMCEcology2010,10:19 low-Seplants(Figure2B;p=0.011,t=-3.095,n=6for plants nottreated withSeincreased by50%during the bothhigh-andlow-Seexperiments).Youngleavesfrom sametimeperiod(Figure4A,p=0.004,t=3.416,n=8 high-Se plants contained roughly 1.5-fold and 5-fold high- and8 low-Se plants). Three weeks after the start higher Se concentrations than medium-aged and old of theSe treatment thespider mite populations on the leaves (respectively) of the same plants, ranging from high-Se plants had decreased by almost 80% while the 3,945mgSekg-1foryoungleavesand812mgSekg-1for populations of spider mites on low-Se plants still oldleaves,whileleavesfromlow-Seplantsdidnotreach showed an increase of 50% (Figure 4A, p < 0.001, t = above11mgSekg-1(Figure2C). 12.807, n= 8high- and8low-Se plants). Prior tocon- When thrips were given a choice tofeed on high- or ducting the experiment, all A. bisulcatus plants con- low-Se plants they showed a significant preference to tained between 100 - 200 mg Se kg-1 DW. After the colonizelow-Seplants.Inthesechoiceexperimentslow- three-week experiment the high-Se plants contained Seleavesandleafletssufferedmoreherbivorythanhigh- almost800mgSekg-1 DWandthelow-Seplants con- Se leaves and leaflets (Figure 2D p = 0.001, t = -5.926; tained 100mgSe kg-1 (Figure 4B,p= 0.010, t= 2.870, Figure2E,p<0.001,t=-6.443n=4pairsofhigh-and n=8high-and8low-Seplants). low-Seplants).Onhigh-Seplantsyoungleavessuffered Since the spider mites appeared to tolerate plant Se lessherbivorythanoldleaves(Figure2D,p=0.001,t= concentrationsupto150mgSekg-1 wecollectedspider 5.913,n=4pairsofhigh-andlow-Seplants).Similarto mitesoffSe-treatedplantstoinvestigatethemechanism whatwasfoundforplantsusedinthenon-choicethrips of their relatively high Se tolerance at the biochemical experiments, young leaves of the high-Se plants con- level. Selenium speciation studies using Se K-edge (X- tained moreSethan oldleaves, 3,000 mgSekg-1 com- ray absorption near-edge structure (XANES)) spectro- pared to 1,350 mg Se kg-1, respectively (Figure 2F). scopyandleast squarelinearcombinationfitting (LCF) Whileinthechoicestudyhigh-Seplantshadmanyfold of the XANES spectra using standard compounds higherSeconcentrationsthanlow-Seplants,leavesfrom revealed that spider mites store Se primarily as an low-Se plants also contained around 100 mg Se kg-1 organic C-Se-C form similar to methylselenocysteine DW in young leaves and approximately 50 mg Se kg-1 (MeSeCys)(Figure5A-C). DWinmedium-agedandoldleaves(Figure2F). Effects ofSeonherbivory ofS.pinnata bythrips Tofurtherinvestigate ifSehyperaccumulators arepro- Effects ofSeonherbivory ofA.bisulcatus byspider mites Non-choiceandchoice experimentswereconductedto tected fromcell disrupting herbivores weused another determine ifSeeffectively protected A.bisulcatus from Se hyperaccumulating plants species, S. pinnata , and anothercelldisruptorherbivore,thetwo-spottedspider againusedthripsinachoiceherbivoryexperiment.The mite. During the non-choice study spider mite popula- thripspreferredtofeedonS.pinnataplantswithoutSe tionsonlygraduallyincreasedinsizeonhigh-Seplants, when given a choice between high- and low-Se plants whereasplantspre-treated withalowSeconcentration (Figure6A,p<0.001,t=-10.333,n=18high-and18 showed an 800% spider mite population growth rate low-Seplants).WithintheSe-treatedplants,leaveswith over three weeks (Figure 3A; p < 0.001, t = 5.306, n = elevated Se suffered less herbivory than similar-aged 10high-and10low-Seplants).Whenspidermiteswere leaves on the same plants with lower Se levels (Figure given a choice to feed on high- or low-Se plants they 6B, p = 0.012, t = -3.056, n = 6 high- and 6 low-Se preferredlow-Seplants.TheprotectiveeffectofSewas plants).Theleavesthatwerecomparedhadsimilarcon- alreadydetectableafteroneweek,aspopulationsofspi- centrationsofotherelementsbesideSe(Figure6C).To dermitesonhigh-Seplantsdecreasedinsizeovertime determineifthedifferenceinSeconcentrationfoundin while populations on low-Se plants increased by over eachpairofleaveswasaresponsetoherbivoryorrather 200%afterthreeweeks(Figure3B;p<0.001,t=6.004, a leaf age-related difference in Se concentration to n = 7 high- and 7 low-Se plants). High-Se plants con- which the herbivore responded, Se concentration as a tained over 2,200 mg Se kg-1 DW and low-Se plants function of leaf age was investigated in more detail in contained110mgSekg-1 DW(p=0.009,t=-4.792,n plantswithoutherbivory.Threeleavesfromconsecutive =3high-and3low-Seplants). nodes on each of six high-Se plants were analyzed for Anotherexperimentwasconductedtoinvestigatethe Se.Theyoungestofthethreeleavescontainedahigher effectofaddingSetoA.bisulcatuspre-infestedwithspi- Seconcentrationthantheoldest-agedleaf(Figure7). der mites. Half ofthe plants infested with spider mites was treated with Seandtheother half wasgiven water Effects ofSeonherbivory ofS.pinnata byspider mites as a control. After seven days the Se treatment had Spidermitesweregivenachoicetofeedoneitherhigh- resulted ina50%reduction inthepopulation ofspider Seorlow-SeS.pinnatatodetermineifelevatedSecon- mites; in contrast, the population of spider mites on centrations protected S. pinnata from spider mite Page7of11 Quinnetal.BMCEcology2010,10:19 Figure 4 Selenium added to spider mite-populated A. Figure 3 Growth of spider mite populations feeding on high- bisulcatusplantsreducedspidermitepopulationgrowth.A: Seorlow-SeA.bisulcatusoverthecourseofanon-choice Percentpopulationchangeofestablishedspidermitepopulations feedingstudy(A)andachoicefeedingstudy(B).Valuesare onA.bisulcatusoverthecourseofa3-weekhigh-Seorlow-Se means+/-SE.Anasteriskbetweendatapointsinthenon-choiceor treatment.B:Seleniumconcentrationofplantsatthebeginningand choicefeedingexperimentsrepresentsasignificantdifference endoftheexperiment.Valuesshowmeans+/-SE.Anasterisk betweenhigh-Seandlow-Seplants(t-tests,a=0.05). betweendatapoints(A)orbars(B)representsasignificant differencebetweenthehigh-andlow-Setreatments(t-test,a= 0.05,n=10fornon-choiceexperiments,n=7forchoice herbivory.OnplantswithelevatedSeonly35%ofleaves experiments). suffered spidermiteherbivorywhileover75%ofleaves from low-Se plants suffered spider mite herbivory (p = 0.002, t = 3.617, n = 10 high and 9 low-Se plants). species, S. pinnata and A. bisulcatus , are protected High-Se plants contained 420 mg Se kg-1 compared to against two ecologically relevant and economically lowSeplants,whichonlyhad50mgSekg-1 (p=0.007, importantcelldisruptorherbivores,thetwo-spottedspi- dermiteandthewesternflowerthrips,onlywhencon- t=-3.078,n=10highand9low-Seplants). taining elevated Se concentrations. The non-choice studies showed that high-Se plants suffered less spider Discussion These results expand on previous studies investigating mite and thrips herbivory. The choice studies demon- the functional significance of Se hyperaccumulation. stratedthatspidermitesandthripspreferredlow-SeA. TheearlierstudieshaveshownthatelevatedSecanpro- bisulcatus and S. pinnata plants over high-Se plants. tect plants from arthropod folivore herbivores (grass- Furthermore, within a single plant, low-Se leaves suf- hoppers, caterpillars), grazing mammalian herbivores feredmorethripsherbivorythanhigh-Seleaves.Studies (prairie dogs), phloem-feeding arthropods (aphids) and usingA.bisulcatusshowedthatthripspreferredtofeed leafandstem/rootfungalpathogens[18,20,21,23]. This onolderleaves,whichcontainedlessSe.StudieswithS. isthefirststudytoshowthatSeprotectshyperaccumu- pinnatashowedthatleaveswithhighconcentrationsof latingplantsfromcelldisruptingherbivores.Thisstudy Se suffered less thrips and spider mite herbivory than provides evidence that two Se hyperaccumulating low-Se leaves andthat younger leaves, even when only Page8of11 Quinnetal.BMCEcology2010,10:19 A B 96% MeSeCys 0.5 mm 4% SeO3 C MeSeCys Figure 6 Selenium prevents thrips herbivory to S. pinnata in bothchoiceandnon-choicestudies.A:Choicefeeding experimentquantifyingthripsherbivoryofS.pinnataplantstreated withorwithoutSe,quantifiedaspercentageofleavesperplant Spider mite showingherbivory.B:Seleniumconcentrationinplantstreatedwith Se,comparingleavesthatexperiencedthripsherbivorywithleaves showingnoherbivory.C:ElementalconcentrationofFe,Mg,Mn, Mo,SandZninplantstreatedwithSe,comparingleavesthat experiencedthripsherbivorywithleavesshowingnoherbivory. Valuesaremeans+/-SE.Anasteriskbetweenapairofbars representsasignificantdifferencebetweenthetwotreatments(t- tests,a=0.05,n=18). support to the hypothesis that Se hyperaccumulation serves as protection against herbivore attacks, and 12.64 12.68 12.72 12.76 12.80 expands the list of herbivores against which Se is Energy (keV) effective. Figure 5 Selenium speciation in spider mites collected from Herbivorefeedingmodecanbeanimportantfactorin Se-richA.bisulcatusplants.X-rayanalysisofnear-edgespectra plant-herbivoreinteractions.Itislikelythatsomeherbi- (XANES)revealedthattwo-spottedspidermitescollectedfromSe- vores can circumvent plant defenses, including richA.bisulcatusplants(showninpanelA)containedprimarily methylselenocysteine(B,C).TheXANESSespectraofthespider mitesandofthemethylselenocysteinestandardcompoundare showninpanelC. onenodeapart,hadhigherSeconcentrationsthanolder leaves.Thoseresultssuggestthattheseplantspreferen- tiallysequesterSeintheiryoungerleaves,whichmaybe more valuable than older leaves because of higher photosynthesisrates[39],andindoingsoaresuccessful in protecting whatmaybeconsidered their morevalu- ablepartsagainsttheseherbivores.Interestingly,innon- choicestudieswedidnotfindsignificant differences in thrips herbivory damage on young leaves of high- and low-Se plants but did find more thrips herbivory on low-Semediumagedandoldleaves comparedtohigh- Semediumagedandoldleaves(Figure2A,B).Itispos- sibly that these young leaves contain more nutrients Figure 7 S. pinnata plants show an age-related difference in than older leaves, which is true in other plant species leafSeconcentration.ShownisSeconcentrationinthreeyoung leavesfromconsecutivenodes.Valuesaremeans+/-SE.(Tukey- [40],andthereforeareamoreattractivefoodsourcefor Kramertest,a=0.05,n=6). herbivores. The results of this study lend further Page9of11 Quinnetal.BMCEcology2010,10:19 elemental defense, as a result of feeding modes [41,42] Conclusions and that different hyperaccumulating plants are pro- Herbivores with different feeding modes may respond tectedfromdifferentgroupsofherbivores.Forexample, differently tohyperaccumulation in plants, as was sug- Nihyperaccumulationdoesnotappeartoprotectplants gestedbyJheeetal.[25].BecauseSehyperaccumulating from xylem and phloem feeding herbivores [25], while plantspreferentiallyallocateSetospecificlocationsthey elevated Se, evenatconcentrations aslowas 10mgSe may leave other locations vulnerable to herbivore kg-1 DW, can protect plants from the phloem-feeding attacks. This study shows that Se hyperaccumulating greenpeachaphid[18].StudiesinvestigatingSedistribu- plants are protected from two economically important tion in Se hyperaccumulating plants suggests that they cell disrupting herbivores. Thewestern flower thrips is are better protected from some feeding modes than consideredamajorpestbecauseitisknowntofeedon others.LeavesofS.pinnatasequesterSeintheperiph- plantsinover62differentfamiliesincludingmanycrop ery of the leaves, in the epidermal cell layer, which is species[44],theyeffectivelytransfervirusestocropspe- expected to be particularly effective against many foli- cies [45] and they rapidly develop pesticide resistance vores, like grasshoppers andcaterpillars [9]. Astragalus [46,47].Two-spottedspidermitesarealsoknowntotar- bisulcatus leaves sequester Seintrichomes, whichmay get many crops, such as fruit trees and vegetables, and actasaninitial defense mechanismagainst avariety of can also develop resistance to pesticides [48]. The feedingtypes[9]. results of this study provide support for the elemental Interestingly, itappearsthatspidermitescantolerate defense hypothesis and have implications for manage- plant Se concentrations in hyperaccumulators up to mentofseleniferous habitats andSephytoremediation. ~150 mgSekg-1 DW,concentrations that aretoxic to SeleniummayactasanaturalpesticideinSe-richcrops manyotherherbivores (Figure4A,B)[18,22].Selenium and plants used for Se phytoremediation in areas such speciationstudiesrevealedthatthespidermitesaccumu- asthewesternUnitedStates,wheretwo-spottedspider- lated an organic form of Se indistinguishable from mites, western flower thrips andSehyperaccumulators MeSeCys (Figure 5B, C). This form of Se is less toxic all occur and where Se-rich agriculture is present. The thanmanyotherformsofSebecause itisnotincorpo- observedavoidanceofSe-richplantsbyherbivoresmay ratedintoproteins[10].ThesameformofSewasfound alsoreducetheprobabilityofSemovementandbiocon- inSehyperaccumulator plants aswellasinSe-tolerant centrationinthefoodchain.However,theabilityofspi- herbivores foundfeedingonhyperaccumulators [21].If dermitestotolerate150mgSekg-1 mayallowtransfer thespider mitesaccumulate MeSeCysaswell,thismay of Se into higher trophic levels, which is an area that contributetotheirtoleranceofrelativelyhighconcentra- needstobefurtherinvestigated. tions of Se. It should benoted, however, that XANES doesnoteffectively distinguish betweenvariousC-Se-C Acknowledgements compounds,includingMeSeCys,selenomethionine,and This research was supported by grants #IOB-0444471 Se-cystathionine [9]andthereforeitispossiblethatthe and #IOS-0817748 from the National Science Founda- mitesaccumulatedamoretoxicformofSe,oramixture tion to EAHPS. We thank David Steingraeber, Arathi oftheseorganic selenocompounds. Thiswouldexplain Seshadri and Mark Paschke for helpful comments on why,athigherSelevels(around420mgSekg-1DWfor the manuscript. We would also like to thank 2 anon- S.pinnataand800mgSekg-1DWforA.bisulcatus)Se ymous reviewers for helpful comments that improved effectivelyprotectedtheplants,evenagainstspidermites. the manuscript. The Advanced Light Source is sup- These results have important implications for mana- ported bytheOffice of Science, Basic Energy Sciences, gingSe-richagriculturalornaturalareasandSephytor- DivisionofMaterialsScienceoftheU.S.Departmentof emediation or biofortification crops. Crops in Energy(DE-AC02-05CH11231). seleniferoushabitatsandplantsusedforSephytoreme- diation often do not accumulate more than 150 mg Se Authordetails kg-1 [43]. Whilethese plantsmaybeprotected bytheir 1DepartmentofBiology,ColoradoStateUniversity,FortCollins,CO80532, lowSelevelsfromfolivorearthropods,theymaystillbe USA. 2AgriculturalResearchService,U.S.DepartmentofAgriculture,Parlier, CA93648,USA. 3AdvancedLightSource,LawrenceBerkeleyNational susceptibletospidermiteherbivory.Ontheotherhand, Laboratory,Berkeley,CA94720,USA. 4CaliforniaStateUniversityFresno, Se hyperaccumulating plants, which typically contain CenterforIrrigationTechnology,FresnoCA93740,USA. more than 1,000 mg Se kg-1 DW [24], likely are pro- Authors’contributions tected against both folivores and spider mites. This CFQ,JLF,RJBRandEAHPSconceivedandcoordinatedtheexperimentsin combined protective effect of Se accumulation against thisstudy.SCFperformedμXRFandμXASdataanalyses.Allauthorsassisted such a wide variety of herbivores may have been an withtheexperiments.CFQ,JLFandEAHPSdraftedthemanuscript.All authorsreadandapprovedthefinalmanuscript. important driving force for the evolution of Se hyperaccumulation. Page10of11 Quinnetal.BMCEcology2010,10:19 Received:28May2010 Accepted:27August2010 22. QuinnCF,FreemanJL,GaleasML,KlamperEM,Pilon-SmitsEAH:Theroleof Published:27August2010 seleniuminprotectingplantsagainstprairiedogherbivory:implications fortheevolutionofseleniumhyperaccumulation.Oecologia2008, 155:267-275. References 23. FreemanJL,QuinnCF,LindblomSD,KlamperEM,Pilon-SmitsEAH: 1. StadtmanTC:Seleniumbiochemistry.AnnuRevBiochem1990,59:111-127. SeleniumprotectsthehyperaccumulatorStanleyapinnataagainstblack- 2. 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