1. ! !
Angela!Banks!
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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!PLDI
gamma!lies!downstream!of!NO!in!the!ABAIinduced!stomatal!pathway.!!

2. ! 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!PLDI
gamma!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:!
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3. !
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.!
=!
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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!

4. 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/pldI
alpha!double!mutant!plants!fail!to!close!stomata.!This!indicates!pldIgamma!is!
downstream!from!pldIalpha!in!the!ABA/NO!stomatal!closure!pathway.!
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The!results!shown!by!these!figures!as!well!as!others!in!the!paper!were!used!to!
create!the!following!worksheet:!
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5. !
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BI!212!Worksheet!3! ! ! ! ! Lab!Section:___________________!
!!Name:__________________!
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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.!!
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1) How!does!Nitric!Oxide!affect!stomatal!aperture?!What!information!helped!you!make!that!
conclusion?!!
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2) What!is!required!for!NO!synthesis?!What!does!this!tell!you!about!its!position!in!the!pathway?!
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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.!
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ABA→ !pldα!!IIII!!NO!IIII!pldδ!IIII!Closure !
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6. KEY:%
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BI!212!Worksheet!3! ! ! ! ! Lab!Section:___________________!
!!Name:___________________!
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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.!!
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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.!
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7. !
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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.!
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ABA→ pldα!!!!!NO!!!pldδ!!!!Closure !
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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.!!
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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.!!!
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8. 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/NOI
induced!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!pldI
gamma!is!not!needed!for!NO!synthesis!(since!NO!is!synthesized!even!in!the!pldI
gamma!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!ABAI
induced!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!

9. 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.!
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Cited!Sources!
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Distefano!A.,!Scruffi!D.,!GarciaIMata!C.,!Lamattina!L.,!Laxalt!A.!(2012)!Phospholipase!
DIgamma!is!involved!in!nitric!oxidIinduced!stomatal!closure.!Planta!236:!1899I1907.!