! !Angela!Banks!

!Recently,!I!created!a!worksheet!that!tested!the!students’!understanding!of!

regulatory!pathways!that!lead!to!the!closure!of!stomata.!The!stomata!are!small!pores!on!the!underside!of!the!leaf!where!gas!exchange!occurs!into!and!out!of!the!leaf.!This!gas!exchange!is!essential!for!leaves!to!conduct!photosynthesis.!Plants!share!many!structural!similarities!to!the!human!lung.!For!example,!gas!exchange!occurs!internally!in!the!spongy!mesophyll,!which!act!as!the!“lungs”!of!the!plant!and!have!an!increased!surface!area!in!order!to!increase!gas!exchange!(similar!to!the!sacs!in!our!lungs).!The!stomatal!opening!and!closing!is!controlled!by!a!set!of!guard!cells,!which!can!be!compared!to!the!mouth!of!a!human.!These!guard!cells!are!controlled!by!similar!mechanisms!that!occur!in!our!own!body!cells.!Generally!speaking,!a!specific!extracellular!signal!is!received!by!the!cells!receptor,!which!leads!to!a!change!in!membrane!potential!and!a!signal!cascade!that!may!open/close!certain!channels!or!coItransporters,!ultimately!leading!to!a!change!in!cell!activity.!Because!plant!and!animal!cells!work!so!similarly!it!is!important!to!teach!the!mechanisms!that!occur!in!guard!cells!in!order!to!induce!stomatal!opening/closing.!!

In!order!to!study!stomata,!it!is!important!for!students!to!learn!how!to!do!signal!pathway!analysis.!!Many!researchers!perform!experiments!in!order!to!determine!the!pathway!or!the!sequence!of!events!that!lead!to!a!certain!physiological!event!to!occur.!!In!doing!this,!researchers!may!learn!more!about!how!animal!and!plant!cells!work,!which!can!help!lead!to!the!development!of!treatments!for!diseases!in!plants!and!animals.!!Thus,!it!is!a!crucial!skill!for!biology!students!to!be!able!to!look!at!a!set!of!data,!and!determine!the!pathway!or!order!of!events!that!lead!to!a!certain!mechanism.!In!BI!212,!while!teaching!students!information!about!stomata!we!also!make!sure!to!present!them!with!data!from!recent!research!so!they!can!develop!this!skill!of!analyzing!data!in!order!to!determine!pathways.!Specifically!for!stomata,!students!analyze!different!data!in!order!to!determine!what!pathway!must!occur!to!induce!stomatal!opening!or!closing!in!response!to!a!variety!of!environmental!conditions.!

This!worksheet!required!the!students!to!be!able!to!analyze!and!interpret!information!given!by!graphs,!and!practice!their!problem!solving!skills.!We!used!a!recent!paper!(Distefano!et!al.!2012)!as!the!source!of!data!to!use!for!the!worksheet.!In!this!paper!the!scientists!were!studying!the!signaling!events!occurring!downstream!of!ABA,!which!is!known!to!lead!to!stomatal!closure.!Nitric!Oxide!and!the!family!of!phospholipases!(PLDs)!are!both!known!to!act!as!secondary!messengers!in!the!ABAIinduced!stomatal!closure!pathway.!!Previous!studies!have!suggested!that!a!subtype!of!PDA,!PLDIalpha,!occurs!upstream!NO!in!the!ABAIinduced!stomatal!closure!pathway.!!

Previous!studies!have!also!shown!that!NO!leads!to!the!activation!of!PLDs.!The!scientists!who!wrote!this!paper!predicted!that!there!must!be!another!type!of!isoenzyme!downstream!of!NO(other!than!PLDIalpha)!which!acts!in!the!ABAIinduced!pathway.!Another!PLD!subtype,!PLDIgamma,!has!been!shown!to!be!related!to!stomatal!closure!and!dehydration!response,!so!the!scientists!tested!to!see!if!PLDIgamma!lies!downstream!of!NO!in!the!ABAIinduced!stomatal!pathway.!!

! In!order!to!study!pldIgammas!role!in!the!abaIinduced!stomatal!closure!pathway!the!scientists!ran!first!measured!pldIgamma!expression!in!WT!Arabidopsis*epidermal!peels!and!leaves.!They!found!that!PLDIgamma!is!expressed!in!leaves!and!guard!cells!and!is!induced!by!ABA.!Following!the!initial!experiment,!the!scientists!created!and!isolated!several!Arabidopsis*knockIout!mutants(pldIalphaI,!pldIgammaI,!and!pldIalphaI/pldIgammaI)!and!!then!exposed!them!to!different!conditions.!They!carried!out!experiments!to!see!if!the!different!mutants!closed!stomata!after!NO!treatment.!The!results!showed!that!PLDIalphaI!mutants!were!able!to!close!stomata!after!treatment,!PLDIgamma!were!not!able!to!close!stomata,!and!the!double!mutant!of!PLDIalpha,!and!PLDIgamma!were!not!able!to!close!stomata.!Previous!studies!have!already!determined!PLDIalpha!to!work!upstream!from!NO,!but!this!experiment!suggests!that!PLDIgamma!works!downstream!NO,!since!NO!treatment!could!not!regain!WT!phenotype!in!the!PLDIgamma!mutant!plants.!!! ABA!has!been!shown!to!lead!to!NO!synthesis,!so!the!scientists!also!ran!experiments!to!see!if!the!mutants!could!synthesize!NO!after!being!treated!with!ABA.!The!scientists!used!fluorescent!analysis!in!order!to!detect!the!presence!of!NO!in!each!mutant.!As!expected,!the!PLDIgamma!loss!of!function!plants!were!able!to!synthesize!the!same!amount!of!NO!as!WT!plants,!further!supporting!the!conclusion!that!PLDIgamma!works!downstream!from!NO.!! This!study!provides!a!clear!order!of!events!in!ABAIinduced!stomatal!closure.!ABA!leads!to!NOIinduced!stomatal!closure,!so!NO!must!work!downstream!from!the!pathway.!The!results!of!this!experiment!as!well!as!previous!experiments!have!concluded!that!PLDIalpha!works!upstream!from!NO!and!PLDIgamma!works!downstream!from!NO.!Although!the!study!provided!useful!information!that!the!students!could!learn,!it!would!not!be!practical!or!a!good!use!of!class!time!to!make!the!students!read!through!the!article!and!analyze!each!experiment!in!order!to!determine!the!ABA/NO!pathway!depicted.!Instead,!the!graphs!and!data!provided!by!the!study!were!utilized!in!order!to!create!a!simplified!worksheet.!The!worksheet!still!accurately!described!the!results!shown!in!the!paper,!however!it!was!presented!in!a!way!that!students!could!analyze!the!information!quickly!in!order!to!determine!the!pathway!for!themselves.!Some!of!the!graphs!from!the!original!paper!used!to!create!the!worksheet!are!shown!below:!!

!Figure!2!shows!that!the!NO!donor!GSNO!induces!stomatal!closure!in!WT!plants,!but!fails!to!have!the!same!effect!on!pldIgamma!loss!of!function!mutants.!This!indicates!NO!is!upstream!from!pldIgamma!in!the!ABA/NO!stomatal!closure!pathway.!=!

!Figure!3!shows!the!results!of!a!double!mutant!experiment!in!which!the!scientists!treated!Aribidopsis*plants!with!NO!treatment.!The!results!show!that!pldIalpha!mutants!close!stomata!in!response!to!NO!treatment!(indicating!pldIalpha!is!

upstream!from!NO!in!the!pathway),!however!the!pldIgamma/pldIalpha!double!mutant!plants!fail!to!close!stomata.!This!indicates!pldIgamma!is!downstream!from!pldIalpha!in!the!ABA/NO!stomatal!closure!pathway.!Figure!3!shows!the!results!of!a!double!mutant!experiment!in!which!the!scientists!treated!Aribidopsis*plants!with!NO!treatment.!The!results!show!that!pldIalpha!mutants!close!stomata!in!response!to!NO!treatment,!however!the!pldIgamma/pldIalpha!double!mutant!plants!fail!to!close!stomata.!This!indicates!pldIgamma!is!downstream!from!pldIalpha!in!the!ABA/NO!stomatal!closure!pathway.!!The!results!shown!by!these!figures!as!well!as!others!in!the!paper!were!used!to!create!the!following!worksheet:!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

!!BI!212!Worksheet!3! ! ! ! ! Lab!Section:___________________!

!!Name:__________________!

!!

!Experiments!were!conducted!measuring!the!stomatal!pore!size!of!wild!type!(WT)!and!two!loss!of!function!mutant!plants!PldaI!and!plddI.!These!experiments!were!carried!out!with!and!without!ABA!(abscisic!acid)!and!NO(nitric!oxide)!treatments.!Another!set!of!experiments!was!carried!out!to!measure!the!amount!of!NO!generated!by!wild!type!and!mutant!plants,!with!and!without!ABA!treatment.!The!data!presented!in!the!graphs!above!allowed!the!researchers!to!propose!a!regulatory!pathway!model!that!includes!the!roles!of!NO,!PLDa!and!PLDd!in!stomatal!closure.!!!

1) How!does!Nitric!Oxide!affect!stomatal!aperture?!What!information!helped!you!make!that!conclusion?!!!!!

!2) What!is!required!for!NO!synthesis?!What!does!this!tell!you!about!its!position!in!the!pathway?!

!!

!3) Indicate!the!order!of!activity!of!the!components!of!ABA!and!NO!regulation!of!

stomatal!closure!by!filling!in!the!blanks!and!turning!the!dotted!lines!into!arrows!indicating!activation!or!inhibition.!!

ABA→ !pldα!!IIII!!NO!IIII!pldδ!IIII!Closure !!

!!!

!

KEY:%%BI!212!Worksheet!3! ! ! ! ! Lab!Section:___________________!

!!Name:___________________!!

!!

!Experiments!were!conducted!measuring!the!stomatal!pore!size!of!wild!type!(WT)!and!two!loss!of!function!mutant!plants!PldαI!and!pldδI.!These!experiments!were!carried!out!with!and!without!ABA!(abscisic!acid)!and!NO(nitric!oxide)!treatments.!Another!set!of!experiments!was!carried!out!to!measure!the!amount!of!NO!generated!by!wild!type!and!mutant!plants,!with!and!without!ABA!treatment.!The!data!presented!in!the!graphs!above!allowed!the!researchers!to!propose!a!regulatory!pathway!model!that!includes!the!roles!of!NO,!PLDα!and!PLDδ!in!stomatal!closure.!!!

1) How!does!Nitric!Oxide!affect!stomatal!aperture?!What!information!helped!you!make!that!conclusion?!!A:!Nitric!oxide!treatment!leads!to!stomatal!closure.!You!can!find!this!information!by!comparing!WT!bar!in!the!top!left!graph!(no!treatment)!to!the!bottom!left!graph(with!nitric!oxide!treatment).!When!treated!with!nitric!oxide!the!WT!stomatal!opening!is!reduced.!

2) What!is!required!for!NO!synthesis?!What!does!this!tell!you!about!its!position!in!the!pathway?!A:!This!information!can!be!derived!from!the!top!right!graph.!By!comparing!the!WT!to!the!WT+ABA!bar!graph!you!can!infer!that!ABA!must!be!added!in!order!to!have!NO!synthesis.!Taking!a!look!at!the!plbαI!+ABA!bar!on!the!graph!you!see!that!when!pldα!is!not!working!then!NO!can!no!longer!be!synthesized!even!though!ABA!is!added,!thus!pldα!is!required!for!NO!synthesis.!In!contrast,!you!can!see!in!the!pldδI+ABA!bar!when!pldδ!is!not!working!nitric!oxide!synthesis!can!still!occur,!so!it!is!not!required!for!NO!synthesis.!Since!plbα!is!required!for!NO!synthesis!it!must!come!BEFORE!NO!in!the!pathway,!and!since!pldδ!is!not!required!for!NO!synthesis!is!must!be!further!downstream!in!the!pathway!than!NO.!!!

!!

3) Indicate!the!order!of!activity!of!the!components!of!ABA!and!NO!regulation!of!stomatal!closure!by!filling!in!the!blanks!and!turning!the!dotted!lines!into!arrows!indicating!activation!or!inhibition.!!

ABA→pldα!!!!!NO!!!pldδ!!!!Closure !!

!We!have!already!determined!from!question!2!that!pldα!is!upstream!from!NO!in!the!pathway!and!pldδ!is!downstream.!You!can!doubleIcheck!this!conclusion!by!taking!a!look!at!the!bottom!left!graph.!When!there!is!a!loss!of!function!mutation!in!pldα,!but!it!is!treated!with!NO!the!phenotype!still!matches!the!WT!phenotype.!So!pldα!must!be!further!upstream!in!the!pathway,!because!even!when!it!is!broken!if!NO!is!added!then!stomatal!closure!still!occurs.!Using!the!same!logic!you!can!conclude!that!pldδ!is!further!down!the!pathway!since!stomatal!closure!cannot!occur!if!pldδ!is!broken!even!when!NO!is!added.!!!As!far!as!filling!in!the!arrows!of!activation!or!inhibition:!Looking!at!the!bottom!left!graph!we!know!pldδ!activates!stomatal!closure,!because!a!loss!of!function!mutation!leads!to!open!stomata!even!when!NO!is!present.!We!concluded!that!NO!leads!to!stomatal!closure!in!question!1,!so!NO!must!activate!pldδ.!We!saw!in!question!2!that!pldα!is!required!for!NO!synthesis,!or!in!other!words!it!activates!NO.!!!!!!!!!!!!!!!!!!!

As!you!can!see!the!graphs!in!this!worksheet!are!simplified!from!the!graphs!provided!in!the!experiment.!This!allows!the!students!to!compare!the!data!easily!between!each!graph.!In!addition,!the!bars!on!each!graph!had!two!options,!they!were!either!at!their!maximum!height!(indicating!stomatal!opening)!or!a!shortened!height(indicating!stomatal!closure).!Thus,!the!students!would!only!have!to!determine!if!each!mutant!led!to!stomatal!opening!or!closing!in!each!treatment,!further!simplifying!the!data.!!The!nitric!oxide!treatment!graph!was!simplified!in!a!similar!way.!

The!students!were!required!to!predict!the!chemical!pathway!of!ABA/NOIinduced!stomatal!closure!using!the!simplified!graphs!given!to!them.!In!order!to!help!them!solve!this!I!broke!up!the!problem!into!several!questions!to!guide!them!along.!First!they!were!required!to!figure!out!how!nitric!oxide!affects!stomatal!aperture!(they!would!need!to!be!able!to!compare!the!no!treatment!and!NO!treatment!graph).!This!question!would!give!them!the!information!that!both!ABA!and!NO!lead!to!stomatal!closure,!as!stated!in!Distelfano’s!article.!!

Next!the!students!were!required!to!figure!out!what!was!needed!for!NO!synthesis;!this!information!can!be!obtained!by!looking!at!the!NO!synthesis!graph.!By!comparing!each!of!the!different!bars!the!students!could!infer!that!pldIalpha!activates!NO!synthesis!(since!NO!isn’t!synthesized!in!the!pldIalpha!mutant)!and!pldIgamma!is!not!needed!for!NO!synthesis!(since!NO!is!synthesized!even!in!the!pldIgamma!mutant).!Using!the!stepwise!and!logical!analysis!the!student!should!be!able!to!conclude!pldIalpha!is!upstream!from!NO!and!pldIgamma!is!downstream!from!NO!in!the!pathway.!Finally,!as!long!as!the!students!completed!questions!1!and!2!correctly,!they!had!all!the!information!they!needed!to!complete!the!final!question!of!the!worksheet,!which!was!to!add!positive!or!negative!arrows!to!indicate!the!ABAIinduced!stomatal!closure!pathway.!!

Although!the!worksheet!was!broken!up!in!steps!in!order!to!guide!students!to!determine!the!correct!pathway,!it!still!proved!to!be!a!difficult!task!for!most!students!when!we!implemented!it.!We!had!hoped!that!this!worksheet!could!be!an!exercise!in!active!learning,!because!if!any!students!had!difficulty!solving!any!of!the!problems!they!could!work!together!to!help!each!other.!However,!in!reality!we!found!that!there!was!a!general!separation!of!students!in!the!classroom:!the!students!(primarily!at!the!front)!who!were!able!to!solve!the!assignment!very!quickly,!and!many!students!at!the!middle!and!nearly!all!at!the!back!of!the!classroom!who!needed!most!of!class!to!solve!the!assignment,!if!they!could!solve!it!at!all.!Because!these!two!groups!of!students!were!mostly!separated!in!their!position!in!the!classroom,!the!students!were!not!able!to!help!each!other,!and!it!was!up!to!the!TA’s!to!help!groups!of!students!who!were!struggling.!In!consequence,!the!assignment!took!much!longer!than!expected,!and!there!were!a!number!of!students!who!didn’t!accept/receive!any!help!and!it!turned!out!they!could!not!complete!the!assignment!correctly.!

In!retrospect!there!may!have!been!a!few!things!we!could!have!done!differently!in!our!implementation.!We!could!have!compiled!a!set!of!slides!that!provided!information!to!get!the!struggling!students!started!on!each!question.!For!example,!after!a!certain!amount!of!time!we!could!have!posted!up!a!slide!that!pointed!out!which!graph!the!students!had!to!take!a!look!at,!and!maybe!even!which!bars!to!compare!in!each!graph.!If!the!students!were!still!struggling!with!the!question!we!

could!have!added!one!additional!piece!of!information!that!would!help!them!further!along.!This!process!could!have!been!repeated!with!each!question!in!order!to!help!the!students!get!through!the!assignment!quicker.!The!problem!with!this!approach!is!that!the!reasoning!is!then!done!for!the!student.!But!if!the!first!“how!to”!example!is!given,!maybe!the!next!would!occur!to!more!students.!

For!future!inIclass!assignments,!in!order!to!promote!active!learning!we!could!have!recruited!the!students!who!seem!to!solve!the!assignment!with!ease.!For!instance,!if!we!were!to!have!used!this!method!during!this!inIclass!assignment!the!students!who!finished!the!assignment!early!could!have!been!asked!to!walk!around!the!classroom!to!help!those!who!were!struggling.!We!could!also!establish!groups!before!the!students!started!the!assignment,!in!order!to!make!sure!there!was!a!mixture!of!students!who!knew!the!material!well,!and!those!who!didn’t.!By!using!this!approach!we!can!minimize!the!problems!we!had!with!TA’s!being!unable!to!help!struggling!students.!Students!who!excel!at!the!next!worksheet!can!help!their!fellow!peers,!which!will!help!more!students!to!progress!through!the!worksheet!while!also!promoting!active!learning!in!the!class!room.!!!!!!!!!!!!!!!!!!!!!!!!!Cited!Sources!!Distefano!A.,!Scruffi!D.,!GarciaIMata!C.,!Lamattina!L.,!Laxalt!A.!(2012)!Phospholipase!DIgamma!is!involved!in!nitric!oxidIinduced!stomatal!closure.!Planta!236:!1899I1907.!