AP Biology: Unit 4: Energy Pod 2: Energy Exchange

Adapted from D. Knuffke, L. Ferguson, & B Williamson

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ShoeBoxLab:Photosynthesis EssentialQuestion: How do you designanexperimenttoquantifytheeffectofanenvironmentalvariable(ex.pH,temperature,lightintensity,ageofleaves,etc.)ontherateofphotosynthesis.Background:Photosynthesisisananabolicprocessusedbyallphotoautotrophstocapturelightenergyandconvertittothechemicalenergyincarbohydrates.Itcanbemeasuredinavarietyofways.Althoughnumerousintermediaryreactionsareinvolved,theoverallphotosyntheticreactionissimple:carbondioxidecombineswiththehydrogenfromwaterproducingacarbohydrate–thesix-carbonsugarglucose–andoxygengas.

6CO2+6H2O+sunlight→C6H12O6+6O2Thephotosyntheticproductionofoxygenandknowledgeofleafanatomywillallowfortheconstructionofasimplesystemthatcanbeusedtoexperimentallyinvestigatemanyofthephotosyntheticvariables.Manyextracellularspacesexistwithinplantleavesthatarenormallyfilledwithairforpurposesofgasexchange.Thisiswhyleaveswillfloatonthesurfaceofbodiesofwater.Butwouldyouhappenifalltheairisforcedoutoftheairspacesintheleaf?Whatwilltheleafdothen?Ifbasicrequirementsforphotosynthesisaresupplied,theoxygentheleafproduceswillformgasbubblesandtheleafwouldre-float.Inessence,thisistheexperimentalmethod,however,smalldiskscutfromleaveswillbeusedinsteadofwholeleavestoperformthefloatingleafdiskassay(FLDA).Thisassayofphotosynthesismaybeusedtoanswermanyquestions,including:Howdochangesinlightintensity,wavelength,orCO2concentrationaffecttherateofphotosynthesis?Oneprobleminmeasuringarateofphotosynthesisisthatthereisacompetingprocessoccurringatthesametime,cellularrespiration,aprocessthatusesoxygen.TheFLDAactuallymeasurestherateofphotosyntheticoxygenproductionminustherateofrespiratoryoxygenuseduringthesameperiod.TheFLDAmeasuresthenetrateofphotosynthesis,theenergetic“profit”madebytheplant.Actualphotosyntheticactivityisgreaterthanthisandiscalledthegrossrateofphotosynthesis.Ifcellularrespirationcanbemeasuredseparately,asimplecalculationcandeterminegrossphotosynthesis.Materials:

• Sodiumbicarbonate(Bakingsoda)• LiquidSoap• Plasticsyringe(10ccorlarger)—removeanyneedle!• Leafmaterial• Holepunch

• Plasticcups• Timer• Groupspecificmaterialsasneeded• Lightsource

SafetyandHousekeeping:TheprimarysafetyissuesinthislabhavetodowithsolutionsNEARelectriclights.TakeCAUTIONtoobservepropercarewithsolutionsnearlights.Ifyouwillbeworkingincloseproximitytoexposedlightbulbs,makesuretowearEYEPROTECTIONintheformofsafetyglasses.Moreover,somehigh-intensitylightsourcesgetextremelyHOT.Ifyouareusingthese,beADVISEnottodripwateronthem(shatterhazard)ortoleanagainstalight(burnhazard).

AP Biology: Unit 4: Energy Pod 2: Energy Exchange

Adapted from D. Knuffke, L. Ferguson, & B Williamson

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TheFloatingLeafDiskAssayforPhotosyntheticActivity:Forthisexperiment,youwillusethe“FloatingLeafDiskAssay”forphotosyntheticactivity.Adetailedprotocolforthisprocedureisattached.Essentially,theassayfollowsthefollowingsteps:

1. Smalldisksectionsofleaftissuearepunchedfromaleaf.2. Thedisksareinfiltratedwithabicarbonatesolution.Theinfiltrationservestwomajorpurposes:

a. Itincreasesthedensityoftheleafdiskssothattheysinkb. Itsuppliesthediskswithacarbonsource(thebicarbonateion)forthepurposeofphotosynthesis.

3. Afterinfiltration,thedisksareplacedatthebottomofacontainer(wewillusepetridishes)ofthebicarbonatesolution.

4. Whenexposedtolight,thediskswillproduceOxygengas,decreasingtheirdensitytothepointthattheywillfloattothetopofthecontainer.

5. Thetimeittakesfordiskstofloatisdirectlyrelatedtotherateofphotosyntheticactivitytakingplaceintheleafdisks.

ProceduralConstraints&Suggestions:TheNeedforAControl:

• Youwouldn’twanttorunanexperimentwithout(atleastonecontrol).DeterminationoftherateofPhotosynthesisANDtheRateofRespiration:

• TherateofrespirationneedstobedeterminedinordertocalculatetherateofgrossphotosynthesisStepstocompletebeforebeginningyourexperiment:

• Developadetailedprotocol&clearexperimentalplanforyourexperiment.• Reviewthesafetyandhousekeepingitemsfortheexperiment.• Developadatatableforyourexperiment&Determineanycalculationsthatwillbenecessaryforyourdata.• Thinkabouthowyouwillgraphyourresults(atleastonegraphisrequiredforthislab).

Stepstocompleteduringyourexperiment:

• Recordthetimeittakesfortheleafdiskstofloat.• Takeapictureofyourexperiment.Makesuretocaptureoneofyouandyourpartner!• Recordanythingthattakesplacethatcapturesyourattentionduringtheexperiment

Stepstocompleteafteryourexperiment:

• Usinggoogleslides,produceawriteupthatincludesthefollowing:o ExperimentalDesignandProtocol.Makesuretoincludepicturesofyouconductingtheexpiremento Claim,Evidence(DataTable,Graph&Calculations),Reasoning(OneSlideEach)o Analysisquestions:

§ Whywasthesoapneededintheprotocol?§ ExplainthespecificpurposeoftheBicarbonateIonsintheleafdiskphotosynthesis(inother

words,whatmoleculeisBicarbonatestandinginfor).§ Whatisthesourceofelectronsfortheleafdiskphotosynthesis?§ Proposetwootherwaysofmeasuringtherateofphotosynthesisinaphotoautotroph(any

organismyouwant).§ Whyisitimportanttousetheaveragerateofphotosynthesis(orrespiration)?§ Whatistheimportanceofestablishingacontrolforthisexperiment?§ Whymustweconsiderrespirationwhenperformingthisactivity?§ Whyisitimportanttostudyphotosyntheticrateofplants?

• SubmittheGoogleSlidesviaGoogleClassroomandprintacopyforyourBILL

AP Biology: Unit 4: Energy Pod 2: Energy Exchange

Adapted from D. Knuffke, L. Ferguson, & B Williamson

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TheFloatingLeafDiskAssayforInvestigatingPhotosynthesis

Introduction:Tryingtofindagood,quantitativeprocedurethatstudentscanuseforexploringphotosynthesisisachallenge.Thestandardproceduressuchascountingoxygenbubblesgeneratedbyanelodeastemtendtonotbe“student”prooforreliable.Thisisaparticularproblemifyourlaboratoryinstructionemphasizesstudent-generatedquestions.Overtheyears,Ihavefoundthefloatingleafdiskassaytechniquetobereliableandunderstandabletostudents.Oncethestudentsarefamiliarwiththetechniquetheycanreadilydesignexperimentstoanswertheirownquestionsaboutphotosynthesis. Thebiologybehindtheprocedure:Leafdisksfloat,normally.Whentheairspacesareinfiltratedwithsolutiontheoveralldensityoftheleafdiskincreasesandthedisksinks.TheinfiltrationsolutionincludesasmallamountofSodiumbicarbonate.Bicarbonateionservesasthecarbonsourceforphotosynthesis.Asphotosynthesisproceedsoxygenisreleasedintotheinterioroftheleafwhichchangesthebuoyancy--causingthediskstorise.Sincecellularrespirationistakingplaceatthesametime,consumingoxygen,theratethatthedisksriseisanindirectmeasurementofthenetrateofphotosynthesis.

Procedure:

1. Thebicarbonateservesasanalternatedissolvedsourceofcarbondioxideforphotosynthesis.Preparea0.2%solution.(Thisisnotverymuchitisonlyabout1/8ofateaspoonofbakingsodain300mlofwater.)

2. Add1dropofdiluteliquidsoaptothissolution.The

soapwetsthehydrophobicsurfaceoftheleafallowingthesolutiontobedrawnintotheleaf.It’sdifficulttoquantifythissinceliquidsoapsvaryinconcentration.Avoidsuds.Ifyoursolutiongeneratessudsthendiluteitwithmorebicarbonatesolution.

AP Biology: Unit 4: Energy Pod 2: Energy Exchange

Adapted from D. Knuffke, L. Ferguson, & B Williamson

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3. Cut10ormoreuniformleafdisksforeachtrial.Singleholepunchesworkwellforthisbutstoutplasticstrawswillworkaswell.

4. Choiceoftheleafmaterialisperhapsthemostcriticalaspectof

thisprocedure.Theleafsurfaceshouldbesmoothandnottoothick.Avoidplantswithhairyleaves.Ivy,freshspinach,WisconsinFastPlantcotyledons--allworkwell.Ivyseemstoprovideveryconsistentresults.Manydifferentplantleavesworkforthislab.Myclasseshavefoundthatinthespring,Pokeweedmaybethebestchoice.Avoidmajorveins.

5. Infiltratetheleafdiskswithsodiumbicarbonatesolution.a. Removethepistonorplungerandplacetheleafdisks

intothesyringebarrel.b. Replacetheplungerbeingcarefulnottocrushtheleaf

disks.Pushontheplungeruntilonlyasmallvolumeofairandleafdiskremaininthebarrel(<10%).

c. Pullasmallvolumeofsodiumbicarbonatesolutionintothesyringe.Tapthesyringetosuspendtheleafdisksinthesolution

e. Holdingafingeroverthesyringe-opening,drawbackontheplungertocreateavacuum.Holdthisvacuumforabout10seconds.Whileholdingthevacuum,swirltheleafdiskstosuspendtheminthesolution.

f. Letoffthevacuum.Thebicarbonatesolutionwillinfiltratetheairspacesintheleafcausingthediskstosink.

g. Youwillprobablyhavetorepeatthisprocedure2-3timesinordertogetthediskstosink.Ifyouhavedifficultygettingyourdiskstosinkafterabout3evacuations,itisusuallybecausethereisnotenoughsoapinthesolution.Addafewmoredropsofsoap.

7. Pourthedisksandsolutionintoaclearplasticcup.Addbicarbonatesolutiontoadepthofabout3centimeters.Usethesamedepthforeachtrial.Shallowerdepthsworkjustaswell.

8. Foracontrolinfiltrateleafdiskswithasolutionofonlywaterwithadropofsoap--nobicarbonate.

9. Placeunderthelightsourceandstartthetimer.Attheendof

eachminute,recordthenumberoffloatingdisks.Thenswirlthediskstodislodgeanythatarestuckagainstthesidesofthecups.Continueuntilallofthedisksarefloating.