Science, Faculty of
Botany, Department of
DSpace
UBCV
Haslam, Tegan M.
Doctor of Philosophy - PhD
University of British Columbia
Very-long-chain fatty acids (VLCFAs) are essential molecules produced by all plant cells, and are precursors of diverse primary and specialized metabolites. VLCFAs are elongated by a fatty acid elongation (FAE) complex of four core enzymes located on the endoplasmic reticulum, which sequentially adds two carbon units to a growing acyl-CoA chain. Identification and characterization of FAE enzymes in Arabidopsis thaliana has revealed that three of the four enzymes act as generalists, contributing to all metabolic pathways that require VLCFAs. A fourth component, the condensing enzyme, provides substrate specificity and determines the chain length of product synthesized by the entire complex. Chain length is important because it defines what downstream pathway a VLCFA can be used for. In Arabidopsis, characterized condensing enzymes can only elongate VLCFAs up to 28 carbons in length, despite the predominance of 29- and 31-carbon components in plant cuticular wax. This suggests that elongation beyond 28 carbons is unique and requires different protein components. The wax-deficient mutant eceriferum2 (cer2) of Arabidopsis lacks waxes longer than 28 carbons, suggesting that CER2 is specifically required for VLCFA extension past this threshold length. Molecular characterization of the CER2 gene, both in planta and by heterologous expression in Saccharomyces cerevisiae, demonstrates that CER2 is a component of the elongation machinery required to synthesize 30-carbon cuticular wax precursors (Chapter 3). Five homologous CER2-LIKE genes were identified in Arabidopsis; these CER2-LIKEs have similar metabolic functions to CER2, but different expression patterns and substrate specificities (Chapter 4). CER2-LIKEs form a distinct clade of the BAHD acyltransferase superfamily. However, structural predictions and site-directed mutagenesis reveal fundamental differences in the mechanism of activity of CER2-LIKEs relative to characterized BAHDs (Chapter 5). My results suggest that CER2-LIKEs enable condensing enzymes to accept longer VLCFA substrates. I suggest several mechanisms to explain the activity of CER2-LIKEs. This peculiarity of elongation is unique to land plants, and to the production of cuticular wax precursors. Because the acquisition of cuticles as barriers to transpirational water loss was key to plant colonization of land, CER2-LIKE activity is an important specialization of plant lipid metabolism.
!ARABIDOPSIS!ECERIFERUM20LIKE!PROTEINS!ARE!COMPONENTS!OF!THE!FATTY!ACID!ELONGATION!MACHINERY!REQUIRED!FOR!THE!PRODUCTION!OF!CUTICULAR!WAXES!!by!Tegan!M.!Haslam!!B.Sc.,!The!University!of!Calgary,!2009!!A!THESIS!SUBMITTED!IN!PARTIAL!FULFILLMENT!OF!THE!REQUIREMENTS!FOR!THE!DEGREE!OF!!DOCTOR!OF!PHILOSOPHY!in!THE!FACULTY!OF!GRADUATE!AND!POSTDOCTORAL!STUDIES!(BOTANY)!!THE!UNIVERSITY!OF!BRITISH!COLUMBIA!(Vancouver)!!April!2017!!©!Tegan!Haslam,!2017!ii!!Abstract!!VeryTlongTchain!fatty!acids!(VLCFAs)!are!essential!molecules!produced!by!all!plant!cells,!and!are!precursors!of!diverse!primary!and!specialized!metabolites.!VLCFAs!are!elongated!by!a!fatty!acid!elongation!(FAE)!complex!of!four!core!enzymes!located!on!the!endoplasmic!reticulum,!which! sequentially! adds! two! carbon! units! to! a! growing! acylTCoA! chain.! Identification! and!characterization! of! FAE! enzymes! in!Arabidopsis* thaliana* has! revealed! that! three! of! the! four!enzymes! act! as! generalists,! contributing! to! all! metabolic! pathways! that! require! VLCFAs.! A!fourth! component,! the! condensing! enzyme,! provides! substrate! specificity! and! determines! the!chain!length!of!product!synthesized!by!the!entire!complex.!Chain!length!is!important!because!it!defines!what!downstream!pathway!a!VLCFA!can!be!used!for.!In!Arabidopsis,!characterized!condensing!enzymes!can!only!elongate!VLCFAs!up!to!28!carbons!in!length,!despite!the!predominance!of!29T!and!31Tcarbon!components!in!plant!cuticular!wax.!This!suggests!that!elongation!beyond!28!carbons!is!unique!and!requires!different!protein!components.! The!waxTdeficient!mutant!eceriferum2! (cer2)! of! Arabidopsis! lacks! waxes! longer!than!28!carbons,!suggesting! that!CER2! is!specifically! required! for!VLCFA!extension!past! this!threshold! length.! Molecular! characterization! of! the! CER2! gene,! both! in* planta! and! by!heterologous! expression! in! Saccharomyces* cerevisiae,! demonstrates! that! CER2! is! a!component! of! the! elongation! machinery! required! to! synthesize! 30Tcarbon! cuticular! wax!precursors! (Chapter! 3).! Five! homologous! CER2;LIKE* genes! were! identified! in! Arabidopsis]!these!CER2;LIKEs!have!similar!metabolic!functions!to!CER2,!but!different!expression!patterns!and! substrate! specificities! (Chapter! 4).! CER2;LIKEs! form! a! distinct! clade! of! the! BAHD!acyltransferase! superfamily.! However,! structural! predictions! and! siteTdirected! mutagenesis!reveal! fundamental! differences! in! the! mechanism! of! activity! of! CER2;LIKEs! relative! to!characterized! BAHDs! (Chapter! 5).! My! results! suggest! that! CER2TLIKEs! enable! condensing!enzymes! to! accept! longer! VLCFA! substrates.! I! suggest! several! mechanisms! to! explain! the!activity! of! CER2TLIKEs.! This! peculiarity! of! elongation! is! unique! to! land! plants,! and! to! the!production! of! cuticular! wax! precursors.! Because! the! acquisition! of! cuticles! as! barriers! to!transpirational! water! loss! was! key! to! plant! colonization! of! land,! CER2TLIKE! activity! is! an!important!specialization!of!plant!lipid!metabolism.!!iii!!Preface!!A!version!of!Chapter!1!has!been!published!as!a!review!article:!Haslam,!T.M.,!and!Kunst,!L.! (2013)!Extending! the!story!of! veryTlongTchain! fatty!acid!elongation.!Plant*Sci.!210:! 93T107.!Tegan!Haslam!wrote!the!manuscript!with!the!assistance!of!Ljerka!Kunst.!!A! version! of! Chapter! 3! has! been! published! as:! Haslam,! T.M.,! MañasTFernández,! A.,!Zhao,! L.,! and! Kunst,! L.! (2012)! Arabidopsis! ECERIFERUM2! is! a! component! of! the! fatty! acid!elongation!machinery!required!for!fatty!acid!extension!to!exceptional!lengths.!Plant*Physiol.!160:!1164T1174.!Aurora!MañasTFernández,!Ljerka!Kunst,!and!Tegan!Haslam!designed!the!research!experiments,!Tegan!Haslam!performed!all!experiments!except!for!SEM!of!stem!wax,!which!was!carried!out!by!Lifang!Zhao.!Tegan!Haslam!wrote! the!manuscript!with! the!assistance!of!Ljerka!Kunst.!A!portion!of!Chapter!4!has!been!published!as:!Haslam,!T.M.,!Haslam,!R.,!Thoraval,!D.,!Pascal,!S.,!Delude,!C.,!Domergue,!F.,!Mañas!Fernández,!A.,!Beaudoin,!F,!Napier,!J.A.,!Kunst,!L.,! and! Joubès,! J.! (2015)! ECERIFERUM2TLIKE! proteins! have! unique! biochemical! and!physiological! functions! in! veryTlongTchain! fatty! acid! elongation.! Plant* Physiol.! 167:! 682T692.!Ljerka!Kunst,!Aurora!MañasTFernández,!Jérôme!Joubès,!and!Tegan!Haslam!contributed!ideas!for! this!project.!Data! for! the!peerTreviewed!article!were!contributed!by! the!Kunst,!Napier,!and!Joubès! labs,!however! the!data!presented! in! this! thesis!was!produced!by!Tegan!Haslam,!with!the! following!exceptions:!Aurora!MañasTFernández!constructed!plasmids! for! complementation!tests!with!CER2;LIKE1,!and!Jérôme!Joubès’! lab!carried!out! the!expression!profiling!of!CER2;LIKE2! in! flowers,! and! tested! the! activity! of! CER2;LIKEs* with! the! KCSs! specified! in! the!manuscript.! Tegan! Haslam! wrote! the! manuscript! with! the! assistance! of! Ljerka! Kunst! and!Jérôme!Joubès.!A! version! of! Chapter! 5! will! be! submitted! as! a! research! article! for! peerTreview.! The!project!was!conceived!and!interpreted!by!Ljerka!Kunst!and!Tegan!Haslam,!and!Tegan!Haslam!carried!out!the!experiments.!!iv!!Table!of!Contents!!Abstract!........................................................................................................................................!ii!Preface!........................................................................................................................................!iii!Table!of!Contents!.......................................................................................................................!iv!List!of!Tables!............................................................................................................................!viii!List!of!Figures!.............................................................................................................................!ix!List!of!Symbols!and!Abbreviations!..........................................................................................!xi!Acknowledgements!..................................................................................................................!xiv!Dedication!..................................................................................................................................!xv!Chapter!1:!Introduction!...............................................................................................................!1!1.1! VeryTlongTchain!fatty!acids!and!the!plant!cuticle!..........................................................!1!1.2! Cuticular!wax!metabolism!in!Arabidopsis!stems!...........................................................!2!1.3! Discovery!of!plant!elongase!complexes!.......................................................................!6!1.3.1! Condensing!enzymes!................................................................................................!7!1.3.2! βTketoacylTCoA!reductase!.........................................................................................!9!1.3.3! βThydroxyacylTCoA!dehydratase!.............................................................................!10!1.3.4! EnoylTCoA!reductase!...............................................................................................!11!1.4! Plant!condensing!enzymes!.........................................................................................!13!1.4.1! KCS!mechanism!and!catalytic!residues!..................................................................!13!1.4.2! KCS!structure!and!substrate!specificity!...................................................................!16!1.4.3! Biological!functions!of!KCSs!in!Arabidopsis!............................................................!19!1.5! The!eceriferum2!mutant!..............................................................................................!23!1.6! Research!questions,!objectives,!and!significance!of!findings!.....................................!25!Chapter!2:!Materials!and!methods!...........................................................................................!26!2.1! Biological!material!and!growth!conditions!...................................................................!26!2.1.1! Arabidopsis*thaliana!................................................................................................!26!2.1.2! Nicotiana*benthamiana!............................................................................................!27!2.1.3! Saccharomyces*cerevisiae!......................................................................................!27!2.2! Polymerase!chain!reaction!(PCR)!..............................................................................!28!2.2.1! Genotyping!..............................................................................................................!28!2.2.2! RTTPCR!&!qPCR!.....................................................................................................!29!2.3! Constructs,!transformation,!and!selection!..................................................................!30!2.3.1! Stable!expression!in!Arabidopsis!............................................................................!30!v!!2.3.1.1! pGWB4;CER2pro::CER2;GFP*and*pGWB6;35Spro::CER2;GFP!..................!30!2.3.1.2! pGWB4;CER2;LIKE1pro::CER2;LIKE1;GFP* and* pGWB4;CER6pro::CER2;LIKE1;GFP!.....................................................................................................................!30!2.3.2! Constitutive!and!inducible!expression!in!S.*cerevisiae!............................................!32!2.4! SiteTdirected!mutagenesis!..........................................................................................!34!2.5! Sequence!swaps!........................................................................................................!36!2.6! CRISPR/Cas9Tmediated!mutagenesis!.......................................................................!38!2.6.1! Construct!design!and!assembly!..............................................................................!38!2.6.2! Identification!of!CRISPR!mutants!............................................................................!39!2.7! Microscopy!..................................................................................................................!40!2.7.1! Confocal!Imaging!.....................................................................................................!40!2.7.2! SEM!.........................................................................................................................!40!2.7.2.1! Comparison!of!cer2;5!and!wildTtype!stems!.....................................................!40!2.7.2.2! Analysis!of!stems!ectopically!expressing!CER2;LIKE1!...................................!40!2.8! Lipid!analyses!.............................................................................................................!41!2.8.1! GCTFID!analysis!of!wax!load!and!composition!........................................................!41!2.8.2! GCTFID!analysis!of!yeast!FAMEs!............................................................................!41!2.8.3! LC/MS!analysis!of!acylTCoAs!..................................................................................!42!2.9! SplitTluciferase!assay!for!proteinTprotein!interactions!.................................................!43!2.10! CoTimmunoprecipitation!..............................................................................................!44!2.10.1! Microsome!preparation!and!solubilization!...............................................................!44!2.10.1.1! Microsome!preparation!buffer!..........................................................................!45!2.10.2! CoTimmunoprecipitation!...........................................................................................!45!2.10.3! Western!blotting!.......................................................................................................!45!2.10.3.1! Tris!buffer!saline!..............................................................................................!46!2.10.3.2! Bjerrum!SchaferTNielsen!buffer!.......................................................................!46!2.11! HomologyTbased!tertiary!structure!modelling!of!CER2TLIKEs!....................................!46!Chapter! 3:! ECERIFERUM2! is! a! component! of! the! fatty! acid! elongation! machinery!required!for!extension!to!exceptional!lengths!........................................................................!47!3.1! Introduction!.................................................................................................................!47!3.2! Results!........................................................................................................................!49!3.2.1! The!cer2!mutant!is!deficient!in!waxes!longer!than!28!carbons!................................!49!3.2.2! The!CER2!gene!is!expressed!in!epidermal!cells!of!aerial!organs!...........................!51!3.2.3! The!CER2!protein!is!localized!to!the!ER!membrane!...............................................!51!vi!!3.2.4! CER2!modifies!the!output!of!elongation!when!coTexpressed!with!CER6!................!53!3.3! Discussion!..................................................................................................................!55!3.3.1! CER2!localizes!to!the!ER!in!young!epidermal!cells!that!are!active! in!cuticular!wax!biosynthesis!........................................................................................................................!55!3.3.2! CER2!and!CER6!are!necessary!and!sufficient!for!30Tcarbon!VLCFA!synthesis!.....!56!Chapter! 4:! ECERIFERUM20LIKE! proteins! have! unique! biochemical! and! physiological!functions!in!very0long0chain!fatty!acid!elongation!................................................................!57!4.1! Introduction!.................................................................................................................!57!4.2! Results!........................................................................................................................!60!4.2.1! CER2TLIKEs!have!different!expression!patterns!.....................................................!60!4.2.2! Isolation!of!cer2;like!mutants!...................................................................................!65!4.2.3! CER2TLIKE1! has! an! analogous! role! to! CER2! in! the! elongation! of! leaf! wax!precursors!...........................................................................................................................!68!4.2.4! The!acylTCoA!pools!of!cer2;like!mutants!are!unusual!.............................................!69!4.2.5! CER2!and!CER2TLIKE2!contribute!to!male!fertility!.................................................!69!4.2.6! Substitution!of!CER2;LIKEs!is!sufficient!to!modify!the!plant!cuticle!structure!.........!72!4.2.7! CER2TLIKEs! impart! different! substrate! specificities! to! the! same! condensing!!enzyme!...............................................................................................................................!74!4.2.8! CER2TLIKE4!has!a!negative!effect!on!CER6!+!CER2!activity!in!yeast!...................!75!4.2.9! The!cer2;like4*mutant!does!not!have!a!cuticular!wax!phenotype!............................!75!4.3! Discussion!..................................................................................................................!78!4.3.1! CER2TLIKEs!impart!unique!substrate!specificities!to!fatty!acid!elongation!.............!78!4.3.2! Modification!of!KCS!specificity!by!CER2TLIKEs!has!physiological!importance!.......!79!4.3.3! The!function!of!CER2TLIKE4!remains!unknown!......................................................!80!4.3.4! Summary!of!implications!for!models!of!CER2TLIKE!metabolic!function!..................!81!Chapter!5:!Biochemical!characterization!of!ECERIFERUM2!.................................................!82!5.1! Introduction!.................................................................................................................!82!5.2! Results!........................................................................................................................!85!5.2.1! CER2TLIKEs!do!not!require!a!conserved!acyltransferase!motif!for!function!...........!85!5.2.2! CER2!and!CER6!physically!interact!........................................................................!87!5.2.2.1! SplitTluciferase!.................................................................................................!87!5.2.2.2! CoTImmunoprecipitation!...................................................................................!89!5.2.2.3! CER6!is!required!for!correct!localization!of!CER2!...........................................!91!5.2.3! CER2TLIKE!substrate!specificity!is!determined!by!an!NTterminal!sequence!...........!94!vii!!5.2.4! The!NTterminal!sequence!is!not!sufficient!for!CER2TLIKE!activity!...........................!96!5.2.5! Insight!from!CER2TLIKE!structural!models!..............................................................!99!5.2.6! CER2TLIKEs!do!not! require!a!second!histidine! in! the!substrateTbinding!pocket! for!function!.............................................................................................................................!106!5.3! Discussion!................................................................................................................!108!5.3.1! CER2!physically!interacts!with!the!fatty!acid!elongase!complex!...........................!108!5.3.2! CER2TLIKEs!are!not!acyltransferases!...................................................................!109!5.3.3! CER2TLIKE!structures!do!not!suggest!a!catalytic!function!....................................!110!5.3.4! Models!of!paired!CER6TCER2!activity!...................................................................!111!5.3.4.1! Providing!a!physical!extension!of!the!CER6!acylTCoA!binding!pocket!..........!111!5.3.4.2! Manipulation!of!CER6!structure!to!allow!it!to!bind!longerTchain!substrates!...!112!5.3.4.3! Maintaining!a!pool!of!VLCTCoA!substrate!for!CER6!......................................!112!Chapter!6:!Conclusions!..........................................................................................................!115!6.1! Major!findings!of!this!work!........................................................................................!115!6.1.1! Perspective:!why!length!matters!............................................................................!115!6.1.2! Summary:!what!we’ve!learned!about!CER2TLIKEs!...............................................!116!6.2! Future!directions!.......................................................................................................!117!6.2.1! The!origin!of!CER2;LIKE*genes!............................................................................!117!6.2.1.1! BAHD!acyltransferases!..................................................................................!117!6.2.1.2! Metabolic!gene!clusters!.................................................................................!118!6.2.1.3! Acyl!chain!length!and!cuticle!evolution!..........................................................!119!6.2.2! Structure!of!plant!elongase!complexes!.................................................................!120!References!...............................................................................................................................!123!!viii!!List!of!Tables!!Table!1T1:!Summary!of!KCS!genes!of!Arabidopsis*thaliana!......................................................!20!Table!2T1:!Summary!of!mutant!alleles!used!in!this!thesis!..........................................................!26!Table!2T2:!Primers!used!for!genotyping!.....................................................................................!28!Table!2T3:!Primers!used!for!RTTPCR!&!qRTTPCR!.....................................................................!29!Table!2T4:!Constructs!and!primers!used!for!stable!expression!in!Arabidopsis!..........................!31!Table!2T5:!Constructs!and!primers!used!for!constitutive!and!inducible!expression!in!yeast!......!33!Table!2T6:!Constructs!and!primers!used!for!siteTdirected!mutagenesis!.....................................!35!Table!2T7:!Constructs!and!primers!used!for!sequence!swaps!in!yeast!......................................!36!Table!2T8:!Constructs!and!primers!used!for!CRISPR/Cas9!mutagenesis!of!CER2;LIKE4!........!39!Table!2T9:!Constructs!and!primers!for!transient!expression!in!tobacco!.....................................!44!Table!3T1:!Summary!of!characterized!cer2!alleles!of!Arabidopsis!.............................................!48!Table!4T1:!Identities!of!CER2!homologs!from!Arabidopsis!.........................................................!58!Table!5T1:!Percent!identity!matrix!of!CER2TLIKE!proteins!from!Arabidopsis!.............................!94!!ix!!List!of!Figures!!Figure!1T1!Schematic!representation!of!wax!biosynthesis!...........................................................!5!Figure!1T2:!Schematic!representation!of!elongation!.....................................................................!8!Figure!1T3:!Schematic!representation!of!KCS/FAE1Tlike!condensing!enzyme!catalysis!...........!15!Figure!1T4:!Schematic!representation!of!BAHD!acyltransferase!activity!....................................!24!Figure!3T1:!Characterization!of!the!cer2;5!mutant!phenotype!....................................................!50!Figure!3T2:!CER2!expression!determined!by!qPCR!and!CER2pro::CER2TGFP!fluorescence!..!52!Figure!3T3:!CER2!localization!to!the!ER!.....................................................................................!52!Figure!3T4:!CoTexpression!of!CER2!with!plant!condensing!enzymes!in!S.*cerevisiae!...............!54!Figure!4T1:!Phylogeny!of!the!Arabidopsis!BAHD!acyltransferase!family!....................................!59!Figure!4T2:!Expression!pattern!and!localization!of!CER2;LIKE1!................................................!62!Figure!4T3:!Expression!profiling!of!CER2;LIKE2!........................................................................!63!Figure! 4T4:! Schematic! representation! of! TTDNA! insertions! in! cer2;like1,! cer2;like2,! and! cer2;like3!............................................................................................................................................!66!Figure!4T5:!RTTPCR!of!cer2;like1!and!cer2;like2!mutants!.........................................................!66!Figure!4T6:CRISPR/Cas9!target!site!and!mutants!generated!in!CER2;LIKE4!...........................!67!Figure!4T7:!Rosette!leaf!wax!and!acyl!CoAs!of!the!cer2,!cer2;like1,!and!cer2*cer2;like1!mutants....................................................................................................................................................!70!Figure!4T8:!Male!sterility!phenotypes!of!select!cer2;like*mutants!...............................................!71!Figure!4T9:!Ectopic!expression!of!CER2;LIKE1!in!stem!epidermis!............................................!73!Figure!4T10:!FAMEs!profiles!of!yeast!cells!expressing!CER6!and!CER2TLIKE!proteins!...........!76!Figure!4T11:!Phenotypes!of!cer2;like4!mutants!..........................................................................!77!Figure!5T1:!Amino!acid!alignment!of!CER2TLIKE!BAHD!acyltransferases!from!Arabidopsis!.....!84!Figure!5T2:!Phenotypes!of!the!cer2;5!mutant!transformed!with!H166Tmodified!CER2!sequences....................................................................................................................................................!86!Figure!5T3:!Schematic!representation!of!the!split!luciferase!assay!............................................!88!Figure! 5T4:! Split! luciferase! assay! of! CER2! paired! with! core! components! of! the! fatty! acid!elongase!.....................................................................................................................................!88!Figure!5T5:!CoTImmunoprecipitation!of!HA3TCER2!with!MYC3TCER6!........................................!90!Figure!5T6:!Characterization!of!the!ew2!mutant!allele!of!CER6!.................................................!92!Figure!5T7:!Localization!of!CER2TGFP!in!cer2;5!and!cer2;5*ew2!mutant!backgrounds!............!93!Figure!5T8:!CER2TLIKE1!and!CER2TLIKE2!chimeras!used!for!yeast!elongation!assay!with!CER6....................................................................................................................................................!97!x!!Figure!5T9:!Elongation!assay!of!chimeric!CER2TLIKE!proteins!with!CER6!in!S.*cerevisiae!......!98!Figure!5T10:!HomologyTbased!model!of!CER2!tertiary!structure!.............................................!101!Figure!5T11:!HomologyTbased!model!of!CER2TLIKE1!tertiary!structure!..................................!102!Figure!5T12:!HomologyTbased!model!of!CER2TLIKE2!tertiary!structure!..................................!103!Figure!5T13:!HomologyTbased!model!of!CER2TLIKE3!tertiary!structure!..................................!104!Figure!5T14:!HomologyTbased!model!of!CER2TLIKE4!tertiary!structure!..................................!105!Figure!5T15:!Elongation!assay!of!mutagenized!CER2TLIKEs!with!CER6!in!Saccharomyces!..!107!Figure!5T16:!Models!of!paired!CER2TCER6!activity!.................................................................!114!!xi!!List!of!Symbols!and!Abbreviations!!35S! ! ! promoter!from!the!35S!RNA!from!the!cauliflower!mosaic!virus!ACA2! ! ! calcium!ATPase!(Arabidopsis!gene)!ACP! ! ! acyl!carrier!protein!ATP! ! ! adenosine!triphosphate!BAHD! BEAT!(benzylalcohol!OTacetyltransferase)!AHCT!(anthocyanin!OThydroxycinnamoyltransferase)!HCBT!(NThydroxycinnamoyl/benzoyltransferase)!DAT!(deacetylvindoline!4TOTacetyltransferase)!CAPS! ! ! cleaved!amplified!polymorphic!sequences!CAS9! ! ! CRISPRTassociated!protein!9!cDNA! ! ! complementary!DNA!CER! eceriferum!genes!identified!in!Arabidopsis!based!on!glossy,!waxTdeficient!mutant!phenotypes!CoA! ! ! Coenzyme!A!CRISPR! ! clustered!regularlyTinterspaced!short!palindromic!repeats!CSG! ! ! calciumTsensitive!growth!(genes!identified!in!Saccharomyces)! ! !DNA! ! ! deoxyribonucleic!acid!ECR! ! ! enoylTCoA!reductase!ELO! ! ! elongationTdefective!(genes!identified!in!Saccharomyces)!EMS! ! ! ethyl!methanesulfonate!En/Spm! ! enhancer/suppressorTmutator!(transposable!element!system)!ER! ! ! endoplasmic!reticulum!!EW! ! ! epicuticular!wax!(Arabidopsis!genes)!FAE! ! ! fatty!acid!elongase!FAE1! ! ! fatty!acid!elongation!1!(Arabidopsis!gene)!FAME! ! ! fatty!acid!methyl!ester!FAR! ! ! fatty!acyl!reductase!FAS! ! ! fatty!acid!synthase!FID! ! ! flame!ionization!detection!FTA! ! ! flinders!technology!associates!GAL1/GAL10! bidirectional,! galactoseTinducible! promoter! from! Saccharomyces*cerevisiae!xii!!GAPC! glyceraldehydeT3Tphosphate! dehydrogenase! C! subunit! (Arabidopsis!gene)!GC! ! ! gas!chromatography!GFP! ! ! green!fluorescent!protein!GLOSSY! genes!identified!in!maize!based!on!waxTdeficient!mutant!phenotypes!GPD! ! ! glyceraldehydeT3Tphosphate!dehydrogenase!(yeast!gene)!HA! ! ! human!influenza!hemagglutinin!HCD! ! ! 3ThydroxyacylTCoA!dehydratase!HMG1! ! ! 3ThydroxyT3Tmethylglutaryl!CoA!reductase!1!(Arabidopsis!gene)!HCT! ! ! hydroxycinnamoyltransferase!HXXXD! catalytic!motif!of!BAHD!acyltransferases]!histidine,!any!three!amino!acids,!aspartic!acid!KAS! ! ! ketoacylTACP!synthase!KCR! ! ! ketoacylTCoA!reductase!KCS! ! ! ketoacylTCoA!synthase!LC! ! ! liquid!chromatography!LUC! ! ! luciferase!gene!from!firefly!(Photinus*pyralis)!MES! ! ! 2T(NTmorpholino)ethanesulfonic!acid!(buffer)!MS! ! ! mass!spectrometry!MS! ! ! Murashige!and!Skoog!(medium)!MRM! ! ! multiple!reaction!monitoring!MYC/CTMYC! ! avian!myelocytomatosis!viral!oncogene!homolog! ! ! !NADH! ! ! nicotinamide!adenine!dinucleotide!NADPH! ! nicotinamide!adenine!dinucleotide!phosphate!OD! ! ! optical!density!PAM! ! ! protospacer!adjacent!motif!PAS* pasticcino! genes! identified! in! Arabidopsis! in! a! screen! for! hypertrophic!growth!in!response!to!cytokinins!PCR! ! ! polymerase!chain!reaction!PHS1! PTPLA! homolog! involved! in! sphingolipid! biosynthesis! 1! (Arabidopsis!gene)!!PI! ! ! protease!inhibitor!PKS! ! ! polyketide!synthase!PMSF! ! ! phenylmethane!sulfonyl!fluoride!xiii!!PTPLA!! ! protein!tyrosine!phosphataseTlike!!qPCR! ! ! quantitative!polymerase!chain!reaction!QTL! ! ! quantitative!trait!locus!QTRAP! ! quadrupoleTlinear!ion!trap!RNA! ! ! ribonucleic!acid!RT! ! ! room!temperature!RTTPCR! ! reverseTtranscription!polymerase!chain!reaction!SC! ! ! synthetic!complete!SD! ! ! standard!deviation!SEM! ! ! scanning!electron!microscopy!SML! ! ! sucrose!monolaurate!TTDNA!! ! transfer!DNA!Ti! ! ! tumourTinducing!TSC! ! ! temperatureTsensitive!csg2∆!(genes!identified!in!Saccharomyces)!VLCFA! ! veryTlongTchain!fatty!acid!WSD! ! ! bifunctional!wax!ester!synthase/diacylglycerol!acyltransferase!!YPAD! ! ! yeast!extractTpeptoneTadenineTdextrose!medium!!xiv!!Acknowledgements!Many!people!have!contributed! to! this! research!project,! and! I! feel! immensely! fortunate!and!thankful!for!the!scientific!and!personal!support!I!have!received!throughout!my!degree.!Dr.! Ljerka! Kunst,! your! encouragement,! advice,! and! kindness! are! at! the! heart! of! this!thesis.! Thank! you! so!much! for! training!me.! I! cannot! articulate!my! gratitude! for! the! countless!opportunities!you!have!given!me!to!succeed.!!To! my! allTstar! committee,! thank! you! for! making! yourselves! available,! and! for! always!being! helpful! and! constructive.! Dr.! George! Haughn,! thank! you! for! your! logical! insight,! your!criticism,!and!for!teaching!me!how!to!present!my!work.!Thank!you!Dr.!Lacey!Samuels!for!your!enthusiasm! for! this! project,! and! for! the!many! ways! you’ve! supported! and! encouraged!me.! I!would!have!liked!to!thank!Dr.!Carl!Douglas!for!the!patience!and!wisdom!he!shared!with!me!and!with!everyone!in!our!department.!!The!Haughn!and!Kunst! lab!has!been!a!wonderful!place!to!work]!without!exception,! I’m!grateful! for! everyone! I’ve! met! here.! I’d! like! to! give! special! thanks! to! Dr.! Aurora! MañasTFernández! for! her! patient! mentorship,! and! for! teaching! me! to! persevere! and! work! hard!regardless!of!whether!I!feel!that!everything!or!nothing!is!working!at!the!lab!bench.!I’d!also!like!to!thank! Dr.! Gill! Dean! for! technical! advice,! and! for! having! the! absolute! best! attitude! out! of!everyone! I’ve! ever! met.! Thank! you! to! Amy! Harrington,! who! worked! in! our! lab! under! my!supervision! for! one! summer.! Although! Amy’s! work! mapping! the! eceriferum19! mutant! isn’t!included! in! this! thesis,! I’m!grateful! to!have!had!such!a!bright!and! industrious!student! to!work!with.!I! could! not! have! asked! for! a! more! professional! and! knowledgeable! group! of!collaborators! than!my! colleagues! at! Rothamsted.! Thank! you!Dr.! Fred! Beaudoin,! Dr.! Richard!Haslam,! and! Dr.! Johnathan! Napier! for! working! with! me! on! this! project,! and! for! being! such!terrific! hosts.! Thank! you! Dr.! Jérôme! Joubès! for! having! the! resolve! to! turn! our! race! into! a!collaboration]!I’m!pleased!that!we!had!the!opportunity!to!work!together.!Funding! for! this! project! was! provided! in! the! form! of! a!UBC!FourTYear! Fellowship,! an!NSERC!Canada!Graduate!Scholarship,!a!Killam!Doctoral!Scholarship,!and!a!UBC!International!Research!Mobility!Grant.!!Thank! you! to! my! parents,! Dan! and! Susan,! and! my! sister! Bronwyn! for! patience! and!support.!Thank!you!Val,!Jesse,!Kiko,!Susan,!Alex,!Billy,!Chad,!and!Nick!for!friendship,!kindness,!and!for!great!times.!!Thank!you!K.!Adrian!Yip!for!your!love,!companionship,!and!for!having!faith!in!me.!xv!!Dedication!!!!!!!For!my!undergraduate!mentors,!Drs.!Ed!Yeung!and!DaeTKyun!Ro:!Thank!you!for!fostering!my!interest!in!plant!biology,!and!for!giving!me!the!confidence!to!pursue!a!career!in!science.!!1!!Chapter!1:!Introduction!!1.1! Very0long0chain!fatty!acids!and!the!plant!cuticle!Lipid!barriers!are!essential!across!all!domains!of! life.!Glycerolipid!membranes!serve!as!barriers! that! enable! the! organization! and! regulation! of! cellular! processes.! On! a! larger! scale,!lipids!can!form!barriers!that!protect!and!seal!specific!tissues!and!organs.!One!such!barrier!is!the!cuticle,!which!coats!the!primary!aerial!surfaces!of!land!plants.!The!cuticle!consists!of!two!lipidic!components,!cutin!and!cuticular!waxes.!Cutin!is!a!polymer!of!oxidized!fatty!acid!derivatives!and!glycerol,! linked!by!ester!bonds.!Cutin! forms!a!continuous!matrix!across! the!plant!surface! that!provides! resistance! to! pathogens! and! herbivores,! and! prevents! the! fusion! of! epidermal! cells.!Cutin! is! embedded! and! overlaid!with! cuticular!waxes,!which! are! a!mixture! of! veryTlongTchain!fatty!acids!(VLCFAs)!and! their!derivatives,!generally!26T34!carbons! in! length.!Cuticular!waxes!restrict! transpiration,! allowing! plants! to! retain! water! and! thrive! in! terrestrial! environments.!Waxes!often!form!unique!structures!and!textures!on!the!surface!of!the!cuticle,!which!can!affect!the! refraction! of! light! on! the! plant! surface,! and! facilitate! or! impede! insect! movement! on! the!plant.!There! are! many! tools! for! studying! cuticular! wax! biosynthesis,! secretion,! and! the!regulation! of! wax! production.! Our! understanding! of! all! of! these! processes! has! been! greatly!improved!by!the!characterization!of!waxTdeficient!“eceriferum”*(“cer”)!mutants!in!the!model!plant!Arabidopsis*thaliana*(Koornneef!et!al.,!1989,!and!Hannoufa!et!al.,!1993),!and!by!reverse!genetic!approaches.!Investigations!of!wax!metabolism!in!Arabidopsis!have!benefitted!not!only!from!the!wealth!of!genetic!tools!available!in!this!model!organism,!but!also!from!the!ease!of!screening!for!waxTdeficient!mutants.!WaxTdeficient!Arabidopsis!stems!are!glossy!and!green!in!comparison!to!the!glaucous!wild!type.!Further,!mutations!affecting!cuticular!wax!metabolism!do!not!affect!plant!viability!or!fitness!unless!they!have!pleiotropic!effects.!The!analysis!of!wax!load!and!composition!is!also!a!relatively!simple!process]!deposited!on!the!outermost!surface!of!the!plant,!waxes!can!be! extracted! by! dipping! the! plant! in! an! organic! solvent.! Finally,! the! study! of! cuticular! wax!biosynthesis!has!benefitted!greatly!from!the!use!of!Saccharomyces*cerevisiae!(S.*cerevisiae,!or!yeast)! as! a! host! for! heterologous! gene! expression! to! test! biochemical! activities.! Given! this!toolkit,!considerable!progress!has!been!made!in!our!field!in!recent!years.!This!progress!has!in!many!instances!laid!the!groundwork!for!further!progress!in!related,!or!broader,!fields!of!biology.!!2!!1.2! Cuticular!wax!metabolism!in!Arabidopsis!stems!Wax!synthesis!begins!in!the!epidermis!with!the!elongation!of!longTchain!fatty!acids!to!veryTlongTchain! fatty! acids! by! a! fatty! acid! elongase! (FAE)! complex.! The! process! of! VLCFA!elongation! is! shared!with! other!metabolic! pathways! in! plants]! VLCFAs! are! also! used! for! the!synthesis! of! sphingolipids,! suberin,! tryphine,! and! in! some! plant! species,! seed! oil.! However,!chain! lengths! in!excess!of!26!carbons!are!used!exclusively! for!cuticular!waxes! (and! tryphine,!which!is!similar!to!wax!in!both!its!composition!and!its!biosynthesis]!discussed!in!Chapter!4).!!As!such,! elongation! is! the! committed! step! in! wax! biosynthesis.! A! small! fraction! of! VLCFAs! are!secreted! to! the! cuticle! directly,! but! these! make! up! only! 1T3%! of! the! total! stem! wax! load! in!Arabidopsis.! Most! fatty! acids! are! processed! by! either! an! acyl! reduction! or! alkaneTforming!pathway!prior!to!secretion!to!the!cuticle.!!The! acyl! reduction! pathway! produces! primary! alcohols! and! wax! esters,! which! together!make!up!10T25%!of!the!total!wax!load!of!Arabidopsis!stems!and!leaves.!VLCFAs!are!reduced!to!primary! alcohols! by! the! reductase! CER4,! which! uses! 24T30Tcarbon! acylTCoAs! as! substrates!(Rowland! et! al.,! 2006).! CER4! is! a! member! of! the! fatty! acyl! reductase! (FAR)! family,! which!catalyze!thioester!cleavage!of!either!acylTACP!or!acylTCoA,!as!well!as!the!subsequent!reduction!of! the! carboxylic! acid! product! to! an! alcohol.! There! are! eight! FARs! in! Arabidopsis,! which!contribute! to! the! synthesis! of! wax,! tryphine,! sporopollenin,! cutin,! suberin,! and! suberinTassociated! waxes! (Rowland! and! Domergue,! 2012).! The! identity! of! CER4/FAR3! as! the!reductase!required!for!the!synthesis!of!cuticular!wax!primary!alcohols!was!determined!based!on!crude!mapping!of!the!glossy!cer4!mutant,!comparison!of!the!rough!chromosomal!location!of!the!cer4! lesion!with!the!established!positions!of! the!FAR!genes,!and!confirmation!of! the!predicted!CER4/FAR3!gene! identity!by!complementation!of! the!cer4*mutant!phenotype! (Rowland!et!al.,!2006).!!While!most!primary!alcohols!are!exported!directly! to! the!cuticle! in!Arabidopsis,! some!are!esterified!to!longTchain!fatty!acids!(LCFAs,!16T18!carbons!in!length)!to!make!wax!esters.!In!wildTtype!Arabidopsis,!wax!esters!42,!44,!and!46!carbons!in!length!are!largely!made!by!esterification!of! a! 16Tcarbon! acyl! group! to! 26T,! 28T,! or! 30Tcarbon! primary! alcohol! (Lai! et! al.,! 2007).! The!Arabidopsis! wax! ester! synthase! was! identified! using! a! reverse! genetic! approach! (Li! et! al.,!2008).!Sequences!of!wax!ester!synthases!(WSs)! from!mammals,! jojoba,!and!bifunctional!wax!ester! synthase/diacylglycerol! acyltransferase! (WS/DGATs)! from! Acinetobacter* calcoaceticus!were!used! to!search! the!Arabidopsis!genome! for!candidate!genes.!One!particular!WS/DGAT,!named! WSD1,! was! selected! for! study! based! on! its! high! expression! in! elongating! stem!epidermal!cells!(Suh!et!al.,!2005).!The!function!of!WSD1!in!the!synthesis!of!cuticular!wax!esters!3!!was!demonstrated!by!mutant! analysis,! and! its! biochemical! function!was! confirmed!by!activity!assays!in!Escherichia*coli*(E.*coli)*and!S.*cerevisiae!(Li!et!al,!2008).!!75T90%! of! stem! and! leaf! waxes! of! Arabidopsis! are! derived! from! the! alkaneTforming!pathway,! which! generates! aldehydes,! alkanes,! secondary! alcohols,! and! ketones.! Early!biochemical!work!by!Cheesbrough!and!Kolattukudy!(1984)!and!Dennis!and!Kolattukudy!(1992)!demonstrated! that! alkanes! are! synthesized! by! reduction! of! acylTCoAs! to! fatty! aldehydes,!followed! by! removal! of! the! carbonyl! group! to! form! alkanes.! Two! coTexpressed!Arabidopsis!genes,!CER1!and!CER3,!were! identified! in! forward!genetic! screens! for!glossy,! cuticular! waxTdeficient!eceriferum!mutants! (Koornneef! et! al,! 1989]! Hannoufa! et! al,!1993).!cer1*and!cer3!mutants!both!have!dramatically! reduced!alkane,!secondary!alcohol,!and!ketone! loads,! providing! evidence! that! these! proteins! are! involved! in! alkane! formation!(Koornneef! et! al,!1989]! Hannoufa! et! al,!1993]! Jenks! et! al,! 1995).! However,! phenotypes! of!the!cer1!and!cer3!mutants! are! not! identical,! as! cer1! accumulates! twice! as! much! 30Tcarbon!aldehyde!as!the!wild!type,!while!cer3!accumulates!only!trace!amounts!of!aldehydes!of!all!chain!lengths.! This! suggests! that! CER3!may! function! as! an! acylTCoA! reductase,! and!CER1! as! an!enzyme!responsible!for!aldehyde!conversion!to!alkanes.!Both!genes!encode!integral!membrane!proteins!of!unknown!function!whose!NTterminal!domains!contain!eight!conserved!histidines!in!a!tripartite!motif! (HX3H,!HX2HH,!HX2HH)! (Aarts!et! al.,! 1995]!Chen!et! al.,! 2003).! In!desaturases!and! related! enzymes,! this! motif! forms! a! diTiron! binding! site! essential! for! redox! catalytic!activity.!Biochemical! confirmation! of! the! roles! of! CER1! and! CER3! in! alkane! formation! was!provided! by! reconstitution! of! the! plant! alkane! biosynthetic! pathway! in! yeast! (Bernard! et! al.,!2012).! Heterologous! expression! of! CER1! and! CER3! in! VLCFATproducing! yeast! cells!demonstrated!that!these!two!genes!are!necessary!and!sufficient!for!alkane!production.!Alkanes!were!not!produced!when!either!gene!was!expressed!alone,!and!aldehydes!were!not!detected!when!either! gene!was!expressed!alone!nor!when!both! genes!were! coTexpressed.!This! study!also! demonstrated! that! the! yield! of! alkanes! produced! by!CER1!and!CER3! in! yeast! could! be!increased! twoTfold!by!coTexpression!of!cytochrome!B5!reductases.!The!authors!proposed! that!CER1,! CER3,! and! cytochrome! B5! reductases! form! a! complex! that! catalyzes! the! twoTstep!conversion!of!acylTCoAs!to!alkanes!in!a!redoxTdependent!manner.!A! portion! of! alkanes! synthesized! by! the! CER3/CER1! complex! is! further! modified! to!secondary! alcohols! and! ketones! by! the! midTchain! alkane! hydroxylase! MAH1,! a! cytochrome!P450!(Greer!et!al.,!2007).!MAH1!was!identified!using!a!reverseTgenetic!approach,!selecting!for!P450s!predicted! to!use! fatty!acidTderived!substrates,!and!with!heightened!expression! in!stem!epidermal!cells.!A!simplified!model!of!wax!biosynthesis!is!presented!in!Figure!1T1.!4!!Though!the!focus!of!this!thesis!will!be!genetic!studies!in!Arabidopsis,!there!has!also!been!rapid!progress! in! the! identification!and!characterization!of!genes! required! for! the!synthesis!of!cuticular!waxes! in!maize,! barley,! rice,! tomato,! and!many!other! plants! in! the! past! decade.!As!cuticular! wax! composition! varies! among! plant! species,! these! studies! have! been! important! in!uncovering!the!breadth!of!metabolic!pathways!that!synthesize!the!acylTlipid!components!of!wax,!as!well! as! triterpenoids!and!phenolics! that!may!also!accumulate! in! the!cuticle.!Such!diversity!underlines!the!fact!that!different!cuticular!waxes!confer!different!properties!to!the!plant!surface.!Further,! that! cuticular! wax! composition! is! consistently! heterogeneous! suggests! that! having! a!diversity!of!wax!components!on!a!given!plant!surface! is!advantageous.!However,!deciphering!what! specific! physical! properties! and! physiological! advantages! different! chain! lengths! and!functional!groups!confer!is!very!much!a!work!in!progress.!It!is!also!of!interest!to!note!that!waxy!surface! layers!are!not!unique! to! the!plant!kingdom.!Animals!also!have!surface! lipids! that!vary!immensely! in! composition,! from! insect! cuticles! that! primarily! consist! of! hydrocarbons!(Golebiowski!et!al.,!2011),!to!lipids!of!human!skin!that!include!triacylglycerols,!wax!esters,!and!squalene!(Pappas,!2014).!Some!prokaryotes!also!secrete!oily!capsules!that!can!be!considered!a! type! of! cuticle.! Among! the! best! studied! is! the! mycolic! acid! capsule! of! the! cell! wall! of!Mycobacterium* tuberculosis,!which!plays!an! important! role! in!pathogenesis! (Asselineau!et!al.,!2002).!One!feature!of!cuticular!wax!composition! that! is!consistent!across! flowering!plant!phyla! is!the!exceptionally!long!chain!length!of!wax!components]!most!waxes!are!derived!from!fatty!acids!28T34! carbons! in! length.! Other! metabolic! pathways! that! incorporate! VLCFAs,! such! as!sphingolipid!and!suberin!synthesis,!use!shorter!acyl!chain!lengths.!The!regulation!of!elongation!is! therefore! of! substantial! developmental! and!physiological! importance.!The!present!work!will!describe!how!extension!of!fatty!acids!from!28!to!34!carbons!is!also!biochemically!distinct!from!elongation! to!shorter! chain! lengths.!Aside! from! the!core!elongation!machinery! involved! in! the!synthesis! of! all! VLCFAs,! an! additional! protein! family,! CER2TLIKEs,! is! uniquely! required! to!generate!VLCFA!precursors!of!cuticular!waxes.!The!following!introduction!will!present!the!core!components! of! the! elongase! with! the! aim! of! setting! a! context! for! the! discovery! and!characterization!of!CER2TLIKEs.!!!!!!5!!!!!!!!!!!!!!!!!!!!!!!!!!Figure!101!Schematic!representation!of!wax!biosynthesis!VLCFAs!are!lengthened!in!twoTcarbon!increments!(black),!and!derivatized!by!one!of!two!pathways.!The!acyl!reduction!pathway!produces!primary!alcohols,!a!portion!of!which!is!esterified!to!fatty!acids!to!make!wax!esters!(blue).!The!alkaneTforming!pathway!produces!aldehyde!intermediates,!from!which!a!carbonyl!group! is! lost! to! form! an! alkane]! alkanes! may! subsequently! undergo! midTchain! oxidation! to! produce!secondary! alcohols! and! ketones! (red).! CER,! ECERIFERUM]! WSD,! bifunctional! wax! ester!synthase/diacylglycerol!acyltransferase]!MAH1,!midTchain!alkane!hydroxylase.!!!!!MAH1CER11° alcohols aldehydesC16C24C28C30C26C32C34wax estersalkanessecondary alcohols, ketonesCER4WSD1CER3elongase6!!1.3! Discovery!of!plant!elongase!complexes!!The!de*novo*synthesis! and!elongation!of! fatty! acids! share!a!basic!mechanism!across!domains!of!life.!This!consists!of!a!sequence!of!four!core!reactions!that!add!two!carbon!units!to!an! acyl! substrate.! First,! either! a! starter!molecule! or! a! growing! acyl! chain! is! condensed! with!malonate,! both! of! which! are! activated! by! conjugation! to! either! acyl! carrier! protein! (ACP)! or!coenzyme! A! (CoA).! This! condensation! reaction! produces! an! activated! βTketoacyl! that! is! two!carbons! longer! than! the! initial! acyl! chain.! Subsequently,! the! βTketo! group! is! reduced! to! an!alcohol,!dehydrated! to!an!enoyl,!and! reduced!again! to! form!an!n+2!acyl!product.!Beyond! this!core!sequence!of!reactions,!there!is!variation!in!the!form!of!substrate!activation,!the!localization!of!biosynthetic!enzymes,!the!mechanisms!determining!substrate!and!product!specificity,!and!in!the!enzymes!involved!in!both!de*novo*synthesis!and!elongation.!!Fatty! acid! synthases! (FAS)! are! enzymes! or! enzyme! complexes! that! catalyze! the! de*novo*synthesis!of!fatty!acids]!they!can!generally!be!grouped!according!to!whether!their!catalytic!activities!reside!on!a!single,!multifunctional!protein,!referred!to!as!a!type!I!fatty!acid!synthase,!or!whether!the!activities!are!associated!with!four!separate,!monofunctional!proteins,!making!up!a!type! II! fatty! acid! synthase.! Plants! have! soluble,! plastidTlocalized! type! II! fatty! acid! synthases,!which!elongate! fatty!acyl!groups!esterified! to!ACP!up! to!18!carbons! in! length.!Plant! fatty!acid!synthases!are!essential!for!the!production!of!membrane!lipids!and!the!longTchain!precursors!of!VLCFAs!(Harwood,!2005).!Early!work!aimed!at! isolating!enzymes!required!for! the!production!of!VLCFAs!in!plants!revealed! that! fatty! acid! elongation! differs!markedly! from! the!de* novo* synthesis! of! fatty! acids!catalyzed!by!the!FAS.!Extensive!biochemical!studies!using!cuticular!waxTproducing!leek!(Allium*porrum)!epidermal!cells!as!a!model!revealed!that!elongation!activity!is!present!predominantly!in!a!microsomal!cell! fraction!(Cassagne!and!Lessire,!1978),!that!the!elongase!uses!malonylTCoA!and!acylTCoA!as! substrates! and!nicotinamide!adenine!dinucleotide!phosphate! (NADPH)!as!a!reductant! (Agrawal!et!al.,!1984),!and! that!elongation!products!are! released! from!the!elongase!as!CoA!thioesters!(Lessire!et!al.,!1985a).!Use!of!chemical!inhibitors!in!this!system!demonstrated!varied!effects!of!chemicals!on!elongase!activity,!suggesting!the!existence!of!multiple!elongases!(Lessire!et!al.,!1985b).!Biochemical!approaches!were!also!crucial!for!our!early!understanding!of!the!nature!of!the!elongase!enzyme(s)]!purification!of!the!leek!elongase!resulted!in!enrichment!of!at!least!three!proteins,!suggesting!that!the!elongase!is!not!a!single!multifunctional!protein!unit,!but!rather!consists!of!multiple!protein!components!(Bessoule!et!al.,!1989).!Additionally,!genetic!analysis!of!cuticular!wax!production!in!barley!demonstrated!that!several!elongase!systems!must!exist! in! a! given! plant,! as! mutagenesis! or! treatment! of! tissues! with! chemical! inhibitors! or!7!!exposure!to!different!environmental!conditions!had!varied!effects!on!the!distribution!of!cuticular!wax! component! chain! lengths! (von! WettsteinTKnowles,! 1982).! Together,! biochemical! and!genetic!evidence!suggested!a!model!where!multiple!heteromeric!fatty!acid!elongase!complexes!carry! out! VLCFA! elongation! in! the! endoplasmic! reticulum,! with! different! elongases! having!substrate!specificity!tailored!to!the!synthesis!of!specific!downstream!products.!!!1.3.1! Condensing!enzymes!The! first! component! of! the! fatty! acid! elongase! to! be! genetically! identified! and!characterized!in!plants!was!FATTY!ACID!ELONGATION!1!(FAE1)!in!a!mutant!screen!for!seed!VLCFA! deficiencies! in!Arabidopsis* thaliana* (James! and!Dooner,! 1990]! Lemieux! et! al.,! 1990]!Kunst!et!al.,!1992).!A!single,!semiTdominant!nuclear!mutation!in!FAE1*was!sufficient!to!block!the!elongation!of! seed!18:1! to!20:1,!20:1! to!22:1,!and!18:0! to!20:0! (Kunst!et!al.,!1992).!Because!three! independent! screens! for! mutants! defective! in! VLCFA! elongation! in! seed! oils! isolated!multiple!alleles!of!only!FAE1,!and!previous!work!by!Bessoule!and!coworkers! (1989)! indicated!that! elongases! consist! of! multiple! protein! components,! a! new! model! consistent! with! both!biochemical! and! genetic! data! designated! FAE1! as! a! single! component! of! a! multimeric!elongase.! In! this! model,! FAE1! would! belong! to! a! family! of! enzymes! with! distinct! substrate!specificities!and!biological!roles,!while!the!enzymes!that!catalyze!the!three!remaining!reactions!required! for! elongation! would! be! generalists.! Knocking! out! any! generalist! component! of! the!elongase! would! compromise! sphingolipid! metabolism]! therefore,! these! mutations! would! be!lethal,!as!suggested!by!their!absence!from!the!mutant!screens.!!Positional! cloning!of! the!FAE1*gene! revealed! that! it! encodes!a!protein!with! sequence!similarity!to!polyketide!synthases!(PKS)!such!as!chalcone!synthase!and!stilbene!synthase,!and!ketoacylTACP! synthase! IIIs! (KASIII)! (James! et! al.,! 1995).! These! enzymes! catalyze! the!condensation!of!malonylTCoA,!or!malonylTACP,!with!a!primer!molecule,!thereby!producing!a!β–ketoacylTCoA/ACP! product.! The! homology! of! FAE1! to! these! monofunctional! condensing!enzymes!provided!strong!support!for!the!hypothesis!that!elongases!are!multimeric!complexes,!and! that! condensing! enzymes! such! as! FAE1! provide! substrate! specificity! as! they! work!alongside! generalist! enzymes! of! the! elongase,! β–ketoacylTCoA! reductase! (KCR),! β–hydroxyacylTCoA!dehydratase!(HCD),!and!enoylTCoA!reductase!(ECR)!(Figure!1T2).!8!!!!Figure!102:!Schematic!representation!of!elongation!MalonylTCoA!(blue)!and!an!acylTCoA!primer!(red)!are!condensed!by!a!ketoacylTCoA!synthase!(KCS)!to!produce!a!βTketoacylTCoA.!The!βTketoacylTCoA!is!reduced!by!a!βTketoacylTCoA!reductase!(KCR)!to!yield!a!βThydroxyacylTCoA,!which!in!turn!is!dehydrated!by!a!βThydroxyacylTCoA!dehydratase!(HCD)!to!produce!an!enoylTCoA.!The!enoylTCoA!is!then!reduced!by!an!enoylTCoA!reductase!(ECR)!to!give!an!acylTCoA!that!is!two!carbons!longer!than!the!initial!acylTCoA!used!as!a!primer.!The!acylTCoA!product!can!then!be!used!as!a!primer! for! the!same!reaction!sequence,!allowing!for!repeated!elongation!of!VLCFAs!in! two!carbon!units.!HOO OS-CoAO OS-CoAOS-CoAECRKCSHCDKCRCycle continuesOS-CoAROH OS-CoAROS-CoARRRCO2 +CoASHNADPH + H+NADP+NADPH + H+NADP+H2O9!!Identification! of! the! FAE1! condensing! enzyme! allowed! for! further! investigation! of! its!activity! and! specificity! in! heterologous! systems.! Millar! and! Kunst! (1997)! demonstrated! that!ectopic!expression!of!FAE1! in!both!Arabidopsis!and! tobacco! (N.* tabacum)! is!sufficient! for! the!synthesis!of!20T!and!22Tcarbon!VLCFAs.!They!also!found!that!FAE1!activity!could!be!observed!when!expressed! in!yeast]! this!was!an! important!discovery!as! it!established!a!simple!and! fast!method! to! determine! KCS! specificity.! Plant! KCSs! are! able! to! function! alongside! generalist!components!of!the!native!yeast!elongase,!and!take!the!place!of!the!yeast’s!native!condensing!enzymes.! It! is! surprising! that! plant! KCSs! function! in! this! system,! as! yeast! ELO! condensing!enzymes! are! not! homologous! to! plant! FAE1Tlike!KCSs.! The! first! component! of! the! elongase!complex! that! was! identified! in! yeast! was! ELONGATION DEFECTIVE 1 (ELO1)! (Toke! and!Martin,!1996),!which!encodes!a!condensing!enzyme!responsible!for!the!extension!of!14Tcarbon!fatty! acids! to! 16! carbons.! Two! homologs,! ELO2 (VBM2/GNS1/FEN1)! and! ELO3 (SRE1/APA1/VBM1/SUR4),!were!subsequently!identified,!with!roles!in!16T!to!24Tcarbon!and!16T!to!26Tcarbon!elongation,! respectively! (Oh!et!al.,!1997).!Yeast!Elo!proteins!are!homologous! to!mammalian!ELOVL!condensing!enzymes.!While!genes!homologous!to!yeast!ELOs!have!been!identified!in!Arabidopsis,!their!biological!functions!remain!largely!unknown.!!While!ELOs!and!KCSs!are!not!homologous,!the!generalist!components!of!the!yeast!and!Arabidopsis! elongases! are.! Elongation! of! VLCFAs! in! plants! shares! many! parallels! with!elongation! in! yeast,! and! it! is! undeniable! that! yeast! research! has! greatly! accelerated! our!progress! in! understanding! fatty! acid! elongation! in! plants.! Because! of! this,! and! because!characterization! of! yeast! elongase! enzymes! has! largely! been! carried! out! using! biochemical!techniques,!while!molecular!genetic!approaches!have!dominated!the!work!done!in!Arabidopsis,!discoveries!of!both!the!yeast!and!Arabidopsis!elongase!systems!will!be!described!here.! !1.3.2! β0ketoacyl0CoA!reductase!The! yeast! βTketoacylTCoA! reductase! gene! YBR159 was! identified! using! the! reverse!genetic! approach! of! searching! the! yeast genome! for! oxidoreductases! of! unknown! function!predicted! to! localize! to! the! ER! (Beaudoin! et! al.,! 2002).! Mutants! of! the! candidate!oxidoreductases! were! then! screened! for! their! inability! to! support! heterologous! activity! of!Caenorhabditis elegans PEA1!(polyunsaturated!fatty!acid!elongating!activity!1)!and!Arabidopsis!FAE1! condensing! enzymes,! which! can! elongate! polyunsaturated! fatty! acids! (PUFAs)! and!monounsaturated! fatty! acids! (MUFAs),! respectively,!when! expressed! in!wildTtype! yeast.!Only!Ybr159p!was!identified!as!essential!for!elongase!activity!using!this!approach,!and!designation!of!Ybr159p! as! a! βTketoacylTCoA! reductase! was! confirmed! by! in vitro assay! (Beaudoin! et! al.,!10!!2002).! Characterization! of! the! ybr159Δ* mutant! revealed! that! cells! have! severely! impaired!growth,! but! are! viable! and! accumulate! residual! amounts! of! VLCFAs! (Beaudoin! et! al.,! 2002).!Ybr159p! is! localized!evenly! throughout! the!endoplasmic! reticulum,!and!coTimmunoprecipitates!with!other!components!of!the!elongase,!Elo3p!and!Tsc13p!(Han!et!al.,!2002).! Beaudoin! et! al.! (2002)! also! identified!a! plant! ortholog!of!YBR159,!β-KETOACYL-COA REDUCTASE 1 (KCR1)!from!Arabidopsis.!The!biochemical!function!of!KCR1 was!confirmed!by!complementation! of! ybr159Δ* mutant! yeast.! KCR1 and! another! putative! βTketoacylTCoA!reductase! in!Arabidopsis,!KCR2,!have!since!been!further!characterized! in planta (Beaudoin!et!al.,! 2009).! Despite! 45%! amino! acid! identity! between! KCR1! and! KCR2,! KCR1! is! the! only! βTketoacylTCoA! reductase! required! for! VLCFA! synthesis! in! Arabidopsis,! as! the! kcr1 mutant! is!lethal,! while! the! kcr2 mutant! has! no! obvious! phenotype.! Ectopic! expression! of!KCR2 cannot!complement!either!the!kcr1 Arabidopsis!mutant!or!the!ybr159Δ*yeast!mutant.!Intriguingly,!both!KCR1!and!KCR2!contain!a!putative!NADH!binding!motif!and!an!essential!SX16YX3K!catalytic!motif,!but!only!KCR1!contains!a!CTterminal!doubleTlysine!endoplasmic!reticulum!retention!motif.!The! double! lysine! motif! is! found! in! several! KCR! orthologs! from! other! plant! species,! and! in!Ybr159p.!!!1.3.3! β0hydroxyacyl0CoA!dehydratase!The! β–hydroxyacylTCoA! dehydratase! Phs1p! (PTPLA! homolog! involved! in! sphingolipid!biosynthesis! 1! [PTPLA:! protein! tyrosine! phosphataseTlike])! was! the! last! component! of! the!elongase! to!be! identified! in!yeast! (Denic!and!Weissman,!2007).!The!phs1Δ*mutation! is! lethal,!indicating!that!it!is!an!essential!component!of!the!yeast!elongase!with!no!redundant!homologs.!Phs1p! interacts! with! other! components! of! the! elongase:! Elops,! Ybr159p,! and! the! enoylTCoA!reductase! Tsc13p! (described! below).! Dehydratase! activity! of! Phs1p! was! demonstrated!genetically!by!analysis!of! fatty!acid!elongation! intermediates! in!microsomes!derived!from!wildTtype! and! PHS1Tdownregulated! cells.! An! in vitro assay! using! purified,! epitopeTtagged! Phs1p!incubated!with!βThydroxyacylTCoA!substrate!resulted!in!the!synthesis!of!the!enoylTCoA!product,!providing!biochemical!evidence!for!Phs1p!function!(Denic!and!Weissman,!2007).! The! initial!characterization!of! the!Arabidopsis!hydroxyacylTCoA!dehydratase!PAS2!was!as!a!proteinTtyrosine!phosphatase!component!of! the!G2/M!cell!cycle!checkpoint! (Bellec!et!al.,!2002]!Da!Costa!et!al.,!2006).!The!PAS2!protein!bears!sequence!homology!to!both!the!yeast!βThydroxyacylTCoA!dehydratase!Phs1p!and!proteinTtyrosine!phosphatases,!and!the!pas2 mutant!has!phenotypes!associated!with!defects!in!both!VLCFA!synthesis!and!cell!cycle!control!(Bach!et!11!!al.,! 2008]! Bellec! et! al.,! 2002).! Further,! PAS2! interacts! with! both! the! enoylTCoA! reductase!CER10! (Bach! et! al.,! 2008)! and! phosphorylated! cyclinTdependent! kinases! (Da! Costa! et! al.,!2006).! How! these! functions! are! connected! remains! very! poorly! understood,! however,! the!separate! roles! of! PAS2! in! regulating! cell! cycle! progression! and! in! elongation! have! been!investigated! in! detail.! Similar! to!kcr1,! lossTofTtranscript! alleles! of!pas2 are! embryo! lethal,! and!knockTdown!mutants!have!reduced!accumulation!of!cuticular!waxes,!seed!oil,!and!sphingolipids!(Bach! et! al.,! 2008).! Ectopic! expression! of! the! yeast! hydroxyacylTCoA! dehydratase! Phs1p! in!Arabidopsis!increased!the!accumulation!of!VLCFAs!relative!to!the!wild!type!(Bach!et!al.,!2008).!The!yeast!dehydratase!was!used!in!this!experiment!to!avoid!any!postTtranslational!mechanisms!of! regulating! dehydratase! activity! in! plants.! This! result! indicates! that! in! addition! to! the!demonstrated! rateTlimiting! role!of!condensing!enzymes! in!elongation! (Millar!and!Kunst,!1997),!dehydratase!activity!can!also!affect!VLCFA!yield.!!Analogous!to!KCR1!and!KCR2,!PAS2!also!has!one!close!homolog,!PTPLA,!which!does!not! have! a! VLCFATdeficient! phenotype,! and! is! unable! to! complement! the! pas2! mutant.!However,! ectopic! PTPLA! expression! in! both! Arabidopsis! and! yeast! increases! VLCFA!accumulation,! and! the! ptpla! mutant! accumulates! hydroxyTacylTCoAs! (Morineau! et! al.,! 2016).!Expression!profiling!of!KCR1,!KCR2,!PAS2,!and!PTPLA!in!roots!revealed!that!KCR1!and!PAS2!are!highly!expressed!in!the!endodermis,!which!may!be!anticipated!as!suberin!is!synthesized!in!this! tissue.! In! contrast,!KCR2! and!PTPLA! expression! is! predominant! in! the! root! vasculature.!The!authors!proposed! that! two! fatty!elongase!complexes!might!exist! in!Arabidopsis,!with!dual!functions!and!expression!profiles.!This!work!underscores!the!fact!that!there!is!much!that!is!not!yet! understood! about! fatty! acid! elongation! in! plants.! The! current! model! of! a! fourTenzyme!complex!is!a!useful!tool,!but!it!is!also!a!simplification!of!the!complexity!that!exists!in!nature.!!1.3.4! Enoyl0CoA!reductase!The! first! identified! generalist! component! of! the! yeast! elongase!was!TEMPERATURE;SENSITIVE* csg2∆* 13! (TSC13 [csg2∆:! calciumTsensitive! growth! 2]),! the! gene! encoding! an!enoylTCoA! reductase,! by! means! of! a! forward! genetic! screen! for! mutants! defective! in!sphingolipid!metabolism! (Kohlwein! et! al.,! 2001]!Dunn! et! al.,! 2000).! Identity! of! Tsc13p! as! the!enoyl!reductase!was!supported!by!four!lines!of!evidence:!(1)!similar!phenotypes!of!the!lossTofTfunction! tsc13-1 mutant! to! the!elo2Δ*and!elo3Δ*single!mutants,! although!null! tsc13Δ*mutants!were! lethal]! (2)! accumulation! of! transT2,3TenoylTCoA! in! the! tsc13-1 mutant]! (3)! homology! of!Tsc13p!with!steroid!5TαTreductases,!which!also!reduce!double!bonds!αTβ! to!a!carbonyl!group]!and!(4)!physical!interaction!of!Tsc13p!with!Elo2p!and!Elo3p.!!12!!Following! the! identification!and!characterization!of!Tsc13p!(Gable!et!al.,!2004)!and!the!identification!of! its!putative!Arabidopsis!ortholog! (Kohlwein!et!al.,!2001),! the!equivalent!enoylTCoA! reductase,! ECERIFERUM10! (CER10),! was! also! the! first! generalist! component! of! the!elongase!to!be!characterized!in!plants!(Zheng!et!al.,!2005).!Surprisingly,!characterization!of!two!mutant!alleles,!cer10T1 and!cer10-2,!revealed!that!the!loss!of!CER10!is!not!lethal!(Zheng!et!al.,!2001)! providing! a! parallel! to! the! nonTlethal! phenotype! of! the! ybr159Δ! yeast!mutants.! In! fact,!both! cer10! mutants! still! accumulate! low! levels! of! VLCFAs! in! seed! oil,! cuticular! waxes,! and!sphingolipids.!Thus,!the!real!puzzle!brought!to!light!by!this!study!is!how!VLCFAs!are!produced!in!the!absence!of!CER10.!As!CER10 has!no!obvious,!closely!related!homologs!in!Arabidopsis,!Zheng!and!coworkers!(2005)!speculated!that!there!could!be!a!functionally!equivalent!enoylTCoA!reductase! existing! in! plants! that! has! no! sequence! homology! to!CER10 or!TSC13.! They! also!suggested!that!alternatively,!another!reductase!with!promiscuous!activity!could!act!on!veryTlongTchain!enoylTCoAs!in!the!absence!of!CER10.!For!example,!steroidT5Treductases!would!be!good!candidates! as! they! also! reduce! double! bonds! αTβ! to! a! carbonyl! group,! and! have! CTterminal!homology!with!Tsc13p!and!CER10.!!13!!1.4! Plant!condensing!enzymes!Condensing!enzymes!are!the!only!core!component!of!the!fatty!acid!elongase!that!differs!markedly!between!yeast!and!plants,!with!yeast!using!Elops,!and!plants!using!FAE1Tlike!KCSs.!However,!genes!with!homology!to!ELOs!have!also!been! identified! in!plant!genomes.!To!date,!the! function! of! these! genes! in! flowering! plants! remains! a! mystery,! however,! some!ELOTlike!genes! have! been! characterized! in! bryophytes. Three! cDNAs! encoding! ELO! homologs! have!been! isolated! from! the! moss! Physcomitrella* patens,! and! mutant! analysis! and! heterologous!expression! in! yeast! have! demonstrated! that! they! elongate! polyunsaturated!VLCFAs! (Zank! et!al.,! 2002]! EiamsaTard! et! al.,! 2013).! These! ELOTlikes! are! thought! to! be! necessary! for! the!synthesis! of! polyunsaturated! VLCFAs! that! are! incorporated! into! diverse! membrane! lipids! in!bryophytes.!Four!ELO!homologs!have!been!identified!in!the!Arabidopsis!genome.!One!homolog!has!been!partially!characterized!(Quist!et!al.,!2009),!however,! the!role!of!plant!ELOs! in!elongation!remains!hotly!contested.!As!the!role!of!KCSs!in!elongation!is!well!established,!and!it!is!clear!that!these! condensing! enzymes! are! the! major! contributors! to! the! elongation! of! cuticular! wax!precursors,!the!remainder!of!this!section!will!focus!on!the!FAE1Tlike!KCS!gene!family!(hereafter!referred!to!as!KCSs).!!1.4.1! KCS!mechanism!and!catalytic!residues!Biochemical!characterization!of!KCSs!has!been!a!challenge!due!to!the!difficulty!inherent!in! studying!membraneTbound! proteins.! However,! homology! of! KCSs! to! soluble! ketoacylTACP!synthase!IIIs!(KASIII)!and!polyketide!synthases!(PKS)!has!been!useful!in!providing!a!precedent!to! guide! structural! and! functional! studies.! KASIIIs,! PKSs,! and! KCSs! have! been! proposed! to!catalyze!similar!threeTstep!reaction!sequences.!First,!an!acyl!group!is!transferred!from!acylTCoA!onto!a!conserved!cysteine!residue!of!the!condensing!enzyme.!Next,!malonylTACP!(KASIIIs)!or!malonylTCoA!(KCSs!and!PKSs)!is!decarboxylated,!producing!an!acetyl!carbanion!intermediate,!which! remains! conjugated! to! its! –CoA! or! –ACP! activating! group.! The! acetyl! carbanion! then!carries!out!a!nucleophilic!attack!on!the!carbonyl!carbon!of!the!acyl!group!esterified!to!cysteine,!producing!an!activated!βTketoTacyl! (Figure!1T3).! It!was!predicted! that!Cys223!of!FAE1/KCS18!binds!the!acyl!substrate!during!fatty!acid!elongation!based!on!the!conservation!of! this!residue!among!condensing!enzymes!(Lassner!et!al.,!1996).!This!prediction!was!later!supported!by!coTcrystallization! of! E.* coli! ketoacylTACP! synthase! III! with! its! acetylTCoA! substrate! (Qiu! et! al.,!1999),!which!demonstrated! that! the!analogous! residue! in! this!enzyme! forms!a! thioester!bond!with! acetate.! Ghanevati! and! Jaworski! (2001)! demonstrated! the! importance! of! Cys223! of!14!!FAE1/KCS18!by!expressing!siteTdirected!mutant!isoforms!of!the!gene!in!yeast,!which!revealed!that!the!C223A!mutation!abolishes!condensation!activity.!However,!evidence!of!direct!binding!of!the!acyl!group!to!Cys223!of!FAE1/KCS18,!and!on!analogous!residues!of!other!KCS!enzymes,!is!lacking.!Ghanevati!and!Jaworski!(2002)!later!developed!a!soluble!in*vitro!assay!system!for!KCSs!that! allowed! them! to! determine! what! specific! catalytic! activities! are! affected! in! siteTdirected!mutants.! In! so! doing,! they! showed! that! the! C223A! mutation! not! only! abolishes! overall!condensation!activity!of!FAE1/KCS18,!but! that! it!also! inhibits!decarboxylation!of!malonylTCoA.!Decarboxylase! activity!was! also! abolished!when! the! assay!was! conducted!with! the!wildTtype!enzyme! in! the! absence! of! acylTCoA! substrate.! These! observations! suggest! a! “PingTPong”!mechanism! for! KCS! enzyme! activity,! wherein! decarboxylation! of! malonylTACP/CoA! is!dependent! on! transfer! of! the! acylTCoA! substrate! to! the! enzyme.! This! mechanism! is! not!conserved! among! all! condensing! enzymes]!Medicago* sativa! chalcone! synthase! (a! PKS)! can!carry! out! decarboxylation! in! the! absence! of! an! acylTCoA! substrate! and! retains! some!decarboxylase! activity! even! when! its! catalytic! cysteine! is! mutated,! although! the! reaction!becomes!much!less!efficient!(Jez!et!al.,!2000).!Witkowski! and! coworkers! (1999)! suggested! that! the! “PingTPong”! dependence! of!decarboxylation!on!acyl!binding!observed! in!some!condensing!enzymes!enforces!a!sequence!on!the!reactions!required!for!condensation.!There!are!several!bacterial!PKSs!that!lack!an!active!site! cysteine,! and! in! its! place!have!a!glutamine! residue! that! structurally!mimics!a!bound!acyl!group]! these! isoforms!very!efficiently!catalyze!malonate!decarboxylation! in! the!absence!of!an!acylTCoA!substrate.!Similarly,!siteTdirected!mutagenesis!of!otherwise!fully!functional!mammalian!fatty! acid! synthase! condensing! enzymes! has! shown! that! a! single! amino! acid! change! from!cysteine! to! glutamine! is! sufficient! to! induce! a! change! from! condensing! activity! to! efficient!decarboxylase!activity!(Witkowski!et!al.,!1999).!While! this!experiment!has!not!been!conducted!with! plant! KCSs,! the! results! of! Ghanevati! and! Jaworski! (2001)! would! suggest! that! a! similar!regulatory! mechanism! could! exist! in! this! enzyme! family.! This! could! serve! to! prevent! the!depletion!of!malonate!in!the!absence!of!an!acyl!substrate.!!15!!!!!!!!!!!!!!!Figure!103:!Schematic!representation!of!KCS/FAE10like!condensing!enzyme!catalysis!(I)!An!acyl!group!is!transferred!from!acylTCoA!(red)!on!to!a!cysteine!residue!of!the!KCS.!(II)!MalonylTCoA!(blue)! is! decarboxylated,! releasing! carbon! dioxide,! and! producing! a! reactive! acetylTCoA! carbanion!intermediate!(which!may!be!stabilized!by!histidine!and!asparagine!residues!of!the!KCS!enzyme).!(III)!The!nucleophilic!carbanion!attacks!the!carbonyl!carbon!of!the!acyl!group!esterified!to!cysteine,!forming!a!new!carbonTcarbon! bond,! releasing! the! acyl! group! from! the! cysteine! of! KCS,! and! regenerating! the! free!enzyme.!!!!!!!!!KCSKCSORHOO OS-CoAOS-CoARO OS-CoARKCSOS-CoA(-)ORCO2IIIIIICoASH16!!Comparison! of! KCSs! to! KASIIIs! and! PKSs! has! also! been! useful! for! predicting! the!mechanism! of! decarboxylation! in! KCSs.! Two! residues,! a! histidine! and! an! asparagine,! are!broadly! conserved! among! these! condensing! enzymes! and! are! thought! to! facilitate! the!decarboxylation!of! the!malonylTACP/CoA!substrate.!Ghanevati!and!Jaworski! (2002)!used!siteTdirected!mutagenesis!to!probe!the!function!of!His391!of!FAE1/KCS18,!predicted!as!a!catalytic!residue!based!on!sequence!alignment!with!KASIIIs!and!PKSs.!Conversion!of!His391!to!several!different! amino! acids! abolished! both! decarboxylation! and! condensation! activities! of! FAE1.! A!basic! residue! in! this! position! has! been! suggested! to! function! in! deprotonating! the! activeTsite!cysteine! to! promote! the! acyl! transfer! reaction! (Abbadi! et! al.,! 2000).! However,! FAE1! His391!substitution! with! a! glutamine! residue,! which! cannot! function! as! a! general! base,! retained!significant! condensation! and! decarboxylation! activities.! A! similar! outcome! was! obtained! with!chalcone! synthase! (Jez! et! al.,! 2000),! where! glutamine! can! also! substitute! for! histidine! and!retain! enzyme! activity.! These! results! suggest! that! the! conserved! histidine! is! not! required! for!proton!abstraction!in!these!enzyme!families.!Jez!and!coworkers!(2000)!suggested,!based!on!the!position!of!the!histidine!in!chalcone!synthase!known!from!its!crystal!structure,!it!could!hydrogen!bond! with! the! carbonyl! group! of! malonylTCoA! to! stabilize! the! reactive! enol! tautomer,! and!thereby!promote!decarboxylation!of!malonylTCoA!to!an!acetyl!anion.!Following!this!reaction,!the!histidine!would!also!be! in!a!good!position!to!stabilize!the!acetyl!anion!product.!The!analogous!histidine!residue!in!plant!KCSs!may!have!a!similar!function.!Work!by!Jez!and!coworkers!(2000)!also!provided!insight!to!the!function!of!an!asparagine!residue! conserved! among! KASIIIs,! PKSs,! and! KCSs]! using! chalcone! synthase! they! showed!that! mutation! of! Asn336! impairs! both! acyl! substrate! transfer! as! well! as! malonylTCoA!decarboxylation.!Ghanevati!and!Jaworski!(2002)!demonstrated!by!siteTdirected!mutagenesis!of!FAE1/KCS18! that! the! equivalent! residue! in! this! protein,! Asn424,! is! required! for! both!condensation! and! decarboxylation,! but! did! not! test! the! effect! of!mutagenizing! asparagine! on!acyl! transfer.! Based! on! the! position! of! Asn336,! Jez! and! coworkers! (2000)! suggested! that! it!could!hydrogen!bond!first!with!the!thioester!carbonyl!of!the!acylTCoA!group,!priming!the!transfer!of! the!acyl!group! from!CoA! to!cysteine.!The!asparagine!could!also!act!similarly! to!histidine! in!hydrogen! bonding!with! the! carbonyl! group! of!malonylTCoA! to! stabilize! the! enol! tautomer! and!thereby!promote!decarboxylation,!and!then!stabilize!the!acetyl!anion!product.!!1.4.2! KCS!structure!and!substrate!specificity!While!there!is!no!crystal!structure!for!any!plant!KCS,!Joubès!and!coworkers!(2008)!used!homology! modelling! to! predict! and! examine! the! tertiary! structure! of! KCS1.! A! sequence!17!!homology!and! threadingTbased!search! for!structural!homologs!with!known!structures! revealed!Medicago* sativa! chalcone! synthase! and! E.* coli! ketoacylTACP! synthase! III! (KASIII/FabH)! as!good!templates!for!determination!of!most!of!the!KCS1!structure.!Homology!modelling!was!then!used!to!extrapolate!the!deduced!structure!of!KCS1!to!the!entire!Arabidopsis!KCS!family.!These!structureTfunction!comparisons!among!KCSs!demonstrated!that!all!members!of!the!family!have!a!similar!structure.!The!NTterminus!of!KCS!proteins!consists!of!either!one!or!two!alpha!helices!that!for!most!members! of! the! family! are! well! positioned! to! serve! as! a! membrane! anchor.! However,! the!position!of! these!helices! in! seven!of! the!21!KCSs! in!Arabidopsis! is! such! that! the!helices!are!poorly! positioned! for! insertion! in! a! membrane! (Joubès! et! al.,! 2008).! In! the! absence! of! a!membrane!anchor,!interactions!between!protein!components!of!the!fatty!acid!elongase!complex!may! be! responsible! for! the! correct! localization! and! orientation! of! KCS! enzymes! in! the!endoplasmic! reticulum.! This! prediction! is! supported! by! (1)! confirmed! endoplasmic! reticulum!localization! of! KCSs! with!membrane! anchors! predicted! to! have! poor! function! (Joubès! et! al.,!2008),! and! (2)! demonstration! that! cleavage! of! the! predicted! transmembrane! domains! from!FAE1/KCS18! is! insufficient! to! solubilize! the! globular! portion! of! the! protein,! and! that! salt!treatment!is!necessary!to!purify!FAE1/KCS18!in!a!soluble!cell!fraction!(Ghanevati!and!Jaworski,!2002).!Domains! involved! in!proteinTprotein! interactions!within! the! fatty!acid!elongase!complex!remain!unknown.!The!remaining,!soluble!core!of!the!KCS!contains!all!of!the!catalytic!residues!(Joubès!et!al.,! 2008).! By! superimposing! the! catalytic! residues! involved! in! both! thioester! bond! formation!with! the!acyl!group! (cysteine)!and!decarboxylation!of!malonylTCoA!(histidine!and!asparagine),!the! authors! were! able! to! evaluate! and! compare! the! sizes! and! shapes! of! substrateTbinding!pockets!of!different!members!of!the!KCS!family.!Though!the!metrics!of!these!comparisons!were!not!made! explicit! in! their! publication,! the! authors! reported! a! general! correlation! between! the!size!of!the!pocket!and!the!length!of!the!acyl!substrate!when!the!latter!was!known!from!previous!biochemical!experiments.!To!identify!additional!features!that!contribute!to!the!substrate!specificity!of!KCSs,!aside!from!the!size!of! the!predicted!substrateTbinding!pocket,!Blacklock!and!Jaworski!(2002)!carried!out!domain!swaps!and!siteTdirected!mutagenesis!using!FAE1/KCS18!from!Arabidopsis!and! its!Brassica* napus* homolog! (BnFAE1).! These! proteins! have! 86%! homology,! and! both! elongate!18:1! to! 20:1! and! 22:1.! However,! the! 22:1/20:1! product! ratio! varies! between! 0.12! for!FAE1/KCS18! and! 0.34! for! BnFAE1! when! the! genes! are! expressed! in! yeast.! The! authors!identified! a! 99Tamino! acid! NTterminal! region! immediately! downstream! of! the! transmembrane!18!!domains!of!FAE1!as!being!sufficient!to!impart!either!Arabidopsis!or!B.*napus!FAE1!specificity.!On!KCS! tertiary! structure!models! (Joubès! et! al.,! 2008)! this! region! lacks! repetitive! secondary!structure,!and!its!sequence!shows!high!variability!within!the!KCS!family!compared!to!the!rest!of!the!protein!sequence.!Another!approach!to!deciphering!structureTfunction!relationships!among!plant!KCSs!was!explored!by!Jasinski!and!coworkers! (2012).!Natural! variation! in! seed!oil! accumulation!among!Arabidopsis!ecotypes!was!used!to!map!quantitative!trait!loci!(QTLs)!that!strongly!impact!VLCFA!production.!A!highTVLCFATproducing!ecotype,!BayT0,!was!crossed!into!a!lowTVLCFATproducing!ecotype,! Shahdara,! and! a! single! genetic! locus! responsible! for! 77%! of! the! observed! genetic!variation! between! the! two! ecotypes! was! identified.! Not! surprisingly,! their! QTL! of! interest!mapped! to! FAE1/KCS18.! This! study!was! not! the! first! of! its! kind]! natural! variation! in! seed! oil!metabolism! among! Arabidopsis! ecotypes! has! been! surveyed,! and! the! genetic! basis! for! this!natural!variation!has!been!investigated!(O’Neill!et!al.,!2003]!Hobbs!et!al.,!2004).!However,! the!work! by! Jasinski! and! coworkers! is! of! particular! interest! because! the! authors! identified! and!discussed! the! specific! amino! acid! substitution! that! strongly! impacts! VLCFA! accumulation! in!Arabidopsis.!The!Shahdara!ecotype!has!a!polymorphism!that!converts!a!wellTconserved!valine!residue! near! the! active! site! to! a! leucine.! The! tertiary! structure! model! (Joubès! et! al.,! 2008)!predicts!that!this!single!amino!acid!substitution!would!induce!conformational!changes!resulting!in!a!25%!reduction!of!the!size!of!the!catalytic!binding!pocket.!Heterologous!expression!of!both!isoforms! in! yeast! confirmed! that! the! valine! to! leucine!polymorphism! in! the!Shahdara!ecotype!reduced! enzyme! activity,! as! yeast! expressing! the! BayT0! isoform! produced! over! 8Tfold! more!VLCFAs! than! yeast! expressing! the! Shahdara! isoform.! A! survey! of! FAE1/KCS18! in! 568!Arabidopsis!ecotypes! revealed! that! this! valine! to! leucine!substitution! is! found! in!35!ecotypes,!and! therefore! accounts! for! substantial! variation! in! VLCFA! production! naturally! observed! in!Arabidopsis.!!There! is! an! additional! feature! common! to! both! KASIIIs! and!PKSs! that! is! of! particular!importance! for! the! discussion! of! KCS! structure]! both! of! these! enzyme! families! are! known! to!function!as!homodimers!on!the!basis!of!crystal!structures!(Qiu!et!al.,!1999]!Qiu!et!al.,!2001]!Jez!et!al.,! 2000)!as!well!as!biochemical!data! (Kreuzaler!et!al.,! 1979).!Considering! the!void! in!our!understanding!of! fatty!acid!elongase!complex!structure!and!stoichiometry,! confirming!whether!this! is!the!case!with!KCSs!would!be!very!interesting.!A!comparison!of!the!crystal!structures!of!free! KASIII! and! ligandTbound! KASIII! revealed! substantial! structural! changes! upon! ligand!binding,!especially!at!the!interface!of!the!two!monomers!(Qiu!et!al.,!2001).!In!the!case!of!both!KASIIIs!and!PKSs,! there! is!a!single!phenylalanine!residue! from!each!monomer! that!protrudes!19!!into! the!active!site!of! the!other,!suggesting! that!dimer! formation!could!play!a! role! in!catalysis.!The!possibility!of!proteinTprotein!interactions!impacting!ligand!binding!is!intriguing,!and!certainly!deserves!further!investigation!in!the!KCS!family.!!1.4.3! Biological!functions!of!KCSs!in!Arabidopsis!There! are! 21! homologous! FAE1Tlike! KCSs! in! the! Arabidopsis! genome.! The!physiological! roles! of! some! of! these! have! been! determined! by! mutant! analysis,! and! the!substrate!specificity!has!been!defined!in!many!using!the!yeast!expression!system.!Our!current!knowledge!of!the!Arabidopsis!KCS!family!based!on!these!studies!is!summarized!in!Table!1T1.!There!is!overlap!in!both!the!expression!patterns!(Joubès!et!al.,!2008]!Suh!et!al.,!2005)!and! substrate! specificities! of!many!Arabidopsis!KCSs! (Paul! et! al.,! 2006]! Tresch! et! al.,! 2012]!Trenkamp!et!al.,!2004]!Blacklock!et!al.,!2006),! suggesting! that! there! is! functional! redundancy!within!the!family.!It!is!particularly!striking!that!the!expression!of!eleven!KCSs!is!upTregulated!in!the!stem!epidermis,!where!cuticular!lipids!are!synthesized!(Suh!et!al.,!2005).!Large!numbers!of!putative!KCS!enzymes!are!also!found!in!barley,!rice,!maize,!and!soybean!(Tresch!et!al.,!2012).!These!broad!observations!suggest!that!there!is!an!advantage!to!having!multiple!specialized,!but!likely!redundant,!KCSs!in!a!genome.!!There!is!inconsistency!in!that!so!many!KCSs!are!expressed!in!stem!epidermal!cells,!but!very! few!KCSs!produce! fatty! acids! longer! than!28! carbons.!Aliphatic!waxes!derived! from!30T!and!32Tcarbon! fatty!acids!account! for! roughly!80%!of! the! total! stem!wax! load! in!Arabidopsis,!and!cuticular!wax!constitutes!roughly!50%!of!the!acylTlipid!output!of!epidermal!cells!(Suh!et!al,!2005).! Therefore,! plants! must! have! a! robust! mechanism! of! synthesizing! the! fatty! acid!precursors!of!these!waxes,!but!this!is!not!evident!from!the!analysis!of!KCS!genes.!20##Table&1(1:&Summary&of&KCS&genes&of&Arabidopsis*thaliana*Gene& Yeast&Expression& Biological&Function& ER&localization&confirmed?&Expression&patterns& Expression&ratio&(e)&Expression&intensity&(e)&At1g01120&(KCS1)&produced#20:0,#20:1,#22:0,#24:0,#26:0#(a)3#substrate#specificity#for#16:0,#16:1,#18:0,#20:13#very#low#activity#with#18:1#(b)3#expressed#in#elo2%elo3#yeast,#but#unable#to#rescue#the#mutant#phenotype#(c)3#produced#20:0,#20:1,#22:0#(d)#required#for#cuticular#wax#accumulation#(Todd#et#al.,#1999)#yes#(Joubès#et#al.,#2008)#expressed#in#all#tissues,#highest#in#siliques,#flowers,#stems#and#cauline#leaves#(Joubès#et#al.,#2008)3#high#expression#in#etiolated#seedlings#(Todd#et#al.,#1999)#4.7# 21,884#At1g04220&(KCS2/DAISY)&produced#22:0,#24:0,#26:0#(a)3#rescued#elo2%elo33#utilized#16:0,#16:1,#18:0,#18:1,#18:2,#20:0,#low#activity#with#22:0#(c)3#produced#20:0,#22:0,#24:0#(d)#required#for#suberin/suberin#associated#wax#biosynthesis#in#roots,#chalaza,#and#in#response#to#environmental#stresses#(Franke#et#al.,#2009)3#has#redundant#roles#in#suberin#and#cuticular#wax#biosynthesis#(Lee#et#al.,#2009)### highest#expression#in#root,#silique,#and#flower#(Lee#et#al.,#20093#Franke#et#al.,#2009)3#enhanced#expression#in#response#to#osmotic#stress#(Lee#et#al.,#2009)3#high#expression#in#stigma,#root#elongation#zone,#epidermis,#chalaza#(Lee#et#al.,#2009)3#expression#detected#in#all#tissues#except#seedling#and#rosette#leaf,#highest#in#silique#(Lee#et#al.,#2009)#8.1# 6,599#At1g07720&(KCS3)&expressed#in#yeast,#but#no#activity#detected#(b,d)#unknown# yes#(Joubès#et#al.,#2008)#expressed#in#all#tissues#except#root,#highest#expression#in#siliques#(Joubès#et#al.,#2008)#9.9# 2,866#At1g19440&(KCS4)&expressed#in#yeast,#but#no#activity#detected#(b,d)3#expressed#in#elo2%elo3#yeast,#but#unable#to#rescue#the#mutant#phenotype#(c),##unknown# ## low#levels#of#expression#in#all#tissues,#highest#in#siliques#(Joubès#et#al.,#2008)#NA# NA#At1g25450&(KCS5/CER60)&produced#20:1,#26:0,#28:0,#30:0#(a)3#expressed#in#elo2%elo3#yeast,#but#unable#to#rescue#the#mutant#phenotype#(c)3#produced#24:0,#26:0,#28:0#(d)#based#on#phylogeny,#heterologous#activity#in#yeast,#and#expression#patterns,#thought#to#be#involved#in#cuticular#lipid#metabolism,#but#not#confirmed#(Fiebig#et#al.,#2000)#yes#(Joubès#et#al.,#2008)#expression#in#all#tissues,#highest#expression#in#siliques#(Joubès#et#al.,#2008)#5.8# 1,527#At1g68530&(KCS6/CER6)&produced#24:0,#26:0,#28:0#(d)# required#for#cuticular#wax#accumulation#throughout#Arabidopsis#shoots#(Millar#et#al.,#19993#Fiebig#et#al.,#2000)#yes#(Joubès#et#al.,#2008)#epidermisTspecific#expression,#enhanced#by#light#and#osmotic#stress#(Hooker#et#al.,#2002)3#expression#in#all#tissues#except#root,#highest#expression#in#siliques#(Hooker#et#al.,#20023#Joubès#et#al.,#2008)#3.1# 19,397#At1g71160&(KCS7)&expressed#in#yeast,#but#no#activity#detected#(b,d)#unknown# ## only#expressed#in#flowers#and#siliques#(Joubès#et#al.,#2008)#NA# NA#At2g15090&(KCS8)&expressed#in#yeast,#but#no#activity#detected#(d)#unknown# yes#(Joubès#et#al.,#2008)#highest#expression#in#cauline#leaves,#some#expression#in#stems,#rosette#leaves,#flowers#and#siliques#(Joubès#et#al.,#2008)##6.6# 296#21##Gene& Yeast&Expression& Biological&Function& ER&localization&confirmed?&Expression&patterns& Expression&ratio&(e)&Expression&intensity&(e)&At2g16280&(KCS9)&rescued#elo2%elo33#elongated#16T22#substrates,#had#lower#activity#for#16:1,#18:1,#and#18:2#substrates3#mostly#produced#24#(c)3#expressed#in#yeast,#but#no#activity#detected#(d)#required#for#suberin,#cuticular#wax,#sphingolipid,#and#phospholipid#precursor#elongation#to#24#(Kim#et#al.,#2013)#yes#(Kim#et#al.,#2013)#expressed#in#all#tissues#tested,#highest#in#stems,#cauline#leaves,#and#siliques#(Joubès#et#al.,#2008)#3.1# 9,337#At2g26250&(KCS10/FDH)&expressed#in#elo2%elo3#yeast,#but#unable#to#rescue#the#mutant#phenotype#(c)3#expressed#in#yeast,#but#no#activity#detected#(d)#required#for#normal#development#of#the#epidermis#(Pruitt#et#al.,#2000)#yes#(Joubès#et#al.,#2008)#expressed#in#all#tissues#except#root,#highest#expression#in#stems#and#siliques#(Joubès#et#al.,#2008)#4# 9,547#At2g26640&(KCS11)&substrate#specificity#for#16:0,#16:1,#18:03#lower#activity#level#with#18:1,#20:0,#20:1#(b)3#expressed#in#elo2%elo3#yeast,#but#unable#to#rescue#the#mutant#phenotype#(c)3#expressed#in#yeast,#but#no#activity#detected#(d)#unknown# ## expressed#at#a#relatively#low,#consistent#levels#in#all#tissues#tested#(Joubès#et#al.,#2008)#NA# NA#At2g28630&(KCS12)&expressed#in#yeast,#but#no#activity#detected#(d)#unknown# yes#(Joubès#et#al.,#2008)#expressed#in#all#tissues#except#root,#highest#expression#in#cauline#leaves#and#siliques#(Joubès#et#al.,#2008)#5.5# 3,857#At2g46720&(KCS13/HIC)&## affects#guard#cell#density#on#leaves3#mutants#show#increased#stomatal#indices#in#high#[CO2],#when#wt#plants#decrease#stomatal#index#in#high#[CO2]#to#enhance#water#use#efficiency#(Gray#et#al.,#2000)### guard#cellTspecific#expression,##low#levels#of#expression#except#in#flowers#and#siliques#(Gray#et#al.,#2000)#NA# NA#At3g10280&(KCS14)&## unknown# ## low#levels#of#expression#except#in#siliques#(Joubès#et#al.,#2008)#NA# NA#At3g52160&(KCS15)&expressed#in#yeast,#but#no#activity#detected#(d)#unknown# ## only#expressed#in#flowers#(Joubès#et#al.,#2008)#NA# NA#At4g34250&(KCS16)&expressed#in#yeast,#but#no#activity#detected#(b)#unknown# ## low#levels#of#expression#except#in#siliques#(Joubès#et#al.,#2008)#NA# NA#At4g34510&(KCS17)&produced#20:0,#20:1,#24:0,#26:0#(a)3#substrate#specificity#for#16:0,#18:0,#20:0,#22:0#(b)3#produced#24:0#(d)####unknown# ## low#levels#of#expression#except#in#flowers#(Joubès#et#al.,#2008)#NA# NA#22##Gene& Yeast&Expression& Biological&Function& ER&localization&confirmed?&Expression&patterns& Expression&ratio&(e)&Expression&intensity&(e)&At4g34520&(KCS18/FAE1)&produced#20:0,#20:1,#22:0,#22:1,#24:0,#24:1,#26:0#(a)3#substrate#specificity#for#16:0,#16:1,#18:0,#18:13#lower#activity#with#20:0,#20:1#(b)3#rescued#elo2#elo33#elongated#16:0,#16:1,#18:0,#18:1,#18:2,#lower#activity#with#20:0#(c)3#produced#20:0,#20:1,#22:0,#22:1#(d)#required#for#20:0,#20:1,#and#22:1#VLCFA#synthesis#for#seed#storage#triacylglycerols#(Kunst#et#al.,#19923#James#et#al.,#1995)### only#expressed#in#seeds#(James#et#al.,#1995)#NA# NA#At5g04530&(KCS19)&expressed#in#yeast,#but#no#activity#detected#(b,d)#unknown# ## low#levels#of#expression#except#in#siliques#(Joubès#et#al.,#2008)#8.7# 254#At5g43760&(KCS20)&produced#22:0,#24:0,#26:0#(a)3#rescued#elo2%elo33#elongated#16:0,#16:1,#18:0,#low#activity#with#18:1,#active#with#20#substrate#(c)3#produced#20:0,#22:0,#24:0#(d)#has#redundant#roles#in#suberin#and#cuticular#wax#biosynthesis#(Lee#et#al.,#2009)### expressed#at#a#relatively#consistent#level#in#all#tissues#tested#except#lower#expression#in#root#(Joubès#et#al.,#2008)#4.8# 9,032#At5g49070&(KCS21)&## unknown# ## only#expressed#in#flowers#(Joubès#et#al.,#2008)#NA# NA###(a)#Trenkamp#et#al.# (2004)#expressed#17#KCS#proteins# in#yeast3#only#6#had#activity# in# this#experiment.#Differentiation#between#plant#and#yeast#condensing#enzyme#products#are#not#clear#as# this#assay#was#carried#out# in#wildTtype#yeast.# (b)#Blacklock#et#al.# (2006)#expressed#selected#HisTtagged#KCS#proteins# in#yeast,#and#assayed# their#activity#by# feeding# the# transgenic#yeast#microsomes# labelled#malonylTCoA#and#assorted#acylTCoAs3#16:0,#16:1,#18:0,#18:1,#20:0,#20:1,#22:0,#22:1.#(c)#Paul#et#al.#(2006)#expressed#select#KCSs#in#elo2%elo3#yeast,#determined#VLCFA#products#in%vivo#by#GC/MS#analysis,#then#carried#out#an#assay#in%vitro#with#substrate#feeding#followed#by#TLC#detection#of#product.#(d)#Tresch#et#al.#(2012)#expressed#17#KCS#proteins#in#InvSc1,#elo2,#and#elo3#yeast,#and#compiled#and#annotated#information#prior#to#reporting#products#of#different#KCSs.#(e)#Suh#et#al.#(2005)#carried#out#microarray#analysis#of#gene#expression#in#stem#epidermis3#the#transcript#ratio#of#top#stem#epidermis/total#stem#top#and#the#mean#signal#intensity#at#the#stem#top#are#presented#here.#23##1.5$ The$eceriferum2$mutant$$In# the#absence#of#a#condensing#enzyme# that#efficiently#elongates# fatty#acids# to# the#chain#lengths#required#to#synthesize#cuticular#waxes,#I#hypothesized#that#there#must#be#other,#as#yet#unknown,# genetic# components# that# assist# in# the#elongation#of# fatty# acids#beyond#28# carbons.#Among# the# waxAdeficient# eceriferum# mutants# described# by# Koornneef# et# al.# (1989),# the#phenotype#of#the#cer2#mutant#strongly#suggests#that#CER2#may#have#a#role#in#the#final#steps#of#elongationK# cer2) has# a# dramatic# reduction# in# all# stem# waxes# longer# than# 28# carbons,# and#increased# accumulation# of# some# wax# components# 28# carbons# and# shorter# (McNevin# et# al.,#1993).#As#cuticular#waxes#derived#from#fatty#acids#30#carbons#and#longer#make#up#over#80%#of#the# total#wax# of#wildAtype#Arabidopsis# stems,# the#cer2)mutant# phenotype# is# obviousK# the#wax#load# of# cer2) is# approximately# one# third# that# of# wild# type,# and# cer2# stems# appear# glossy# and#green#compared#to#the#glaucous#wild#type#(McNevin#et#al.,#1993).##The#cer2#mutation#was#mapped#to#At4g24510#(Negruk#et#al.,#1996K#Xia#et#al.,#1996),#a#gene#homologous#to#plant#BAHD#acyltransferases.#The#BAHD#acyltransferase#family#is#named#for# the# first# four# enzymes# described# in# this# clade:# BEAT,# benzylalcohol#OAacetyltransferaseK#AHCT,# anthocyanin# OAhydroxycinnamoyltransferaseK# HCBT,# NAhydroxycinnamoyl/#benzoyltransferaseK# and#DAT,# deacetylvindoline# 4AOAacetyltransferase.# BAHDs# are# specific# to#plants#and#fungi,#and#have#diverse#roles#in#specialized#metabolic#pathways.#BAHDs#catalyze#the#transfer#of#an#acyl#group#from#a#CoAAthioester#to#an#alcohol#or#amine#acyl#acceptor,#generating#an#ester#or#amide#bond,#respectively#(Figure#1A4).#How#an#acyl#transfer#reaction#could#contribute#to# the#process#of# fatty#acid#elongation#as#we#currently#understand# it# is#not#obvious,#much# less#how#an#acyl#transfer#reaction#could#be#required#for#elongation#exclusively#at#one#particular#chain#length.##24##$$$$$$$$$$$$$$$$$$$$$$$$$$$$$Figure$114:$Schematic$representation$of$BAHD$acyltransferase$activity$$Acyl# transfer# proceeds# via# two# steps.# First,# the# acyl# acceptor# is# deprotonated# by# a# catalytic# histidine#residue#on#the#BAHD.#Second,#the#electronegative#acyl#acceptor#carries#our#a#nucleophilic#attack#on#the#carbonyl#carbon#of#the#acylACoA,#the#acyl#donor.#This#creates#a#new#ester#bond#between#the#acyl#donor#and#acceptor.#+ + : : BAHD BAHD BAHD 25##1.6$ Research$questions,$objectives,$and$significance$of$findings$The# canonical#mechanism# of# elongation# by# FAE# complexes# is# established,# and# the# core#machinery# has# been# thoroughly# studied.# However,# characterized# components# of# the# FAE#complex#are# insufficient# to#produce# fatty#acids# longer# than#28#carbons,#which#are# required# for#wax#synthesis.#The#exceptionally# long#chain# length#of#wax#components# is#one# feature#of#plant#cuticles# that# is# reasonably# consistent# across# flowering# plant# phyla,# with# most# waxes# derived#from# fatty#acids#28A34#carbons# in# length.#Other#metabolic#pathways# that# incorporate#VLCFAs,#such#as#sphingolipid#and#suberin#biosynthesis,#use#chain#lengths#shorter#than#26#carbons.###The#cer2)mutant#is#specifically#deficient#in#waxes#longer#than#28#carbons,#but#little#was#known#about#the#CER2#gene#beyond#this#mutant#phenotype.#Therefore,#my#thesis#research#had#three#objectives:#to#characterize#CER2#(Chapter#3),#to#identify#and#characterize#CER2#homologs#in# Arabidopsis# (Chapter# 4),# and# to# decipher# the# biochemical# activity# of# the# CER2# protein#(Chapter#5).##I# found# that# there# are# five# CER2/LIKE) genes# in# Arabidopsis,# and# that# these# form# a#unique# clade# within# the# BAHD# acyltransferase# family.# CER2ALIKEs# are# necessary# for# the#elongation#of#fatty#acid#precursors#of#cuticular#waxes,#and#establish#the#chainAlength#profiles#of#waxes# on# different# plant# surfaces.# CER2ALIKEs# pair# with# specific# condensing# enzymes# to#elongate# fatty# acids# longer# than# 28# carbons.# Despite# homology# to# BAHD# acyltransferases,#CER2ALIKEs# do# not# appear# to# have# acyl# transfer# function.#While# the# biochemical# function# of#CER2ALIKEs# remains# a# mystery,# this# work# has# uncovered# an# intricacy# in# the# process# of#elongation#that#is#necessary#for#the#synthesis#of#cuticular#waxes.##26##Chapter$2:$Materials$and$methods$$2.1$ Biological$material$and$growth$conditions$#2.1.1$ Arabidopsis0thaliana$Arabidopsis#seed#stocks#were#obtained#from#the#Arabidopsis#Biological#Resource#Center#(Alonso#et#al.,#2003K#http://www.arabidopsis.org/).#Mutants#used#in#this#study#are#summarized#in#Table#2A1.##Table$211:$Summary$of$mutant$alleles$used$in$this$thesis$ATG$ Gene$name$ Line$$ Allele$ Ecotype$ Mutagen$At4g24510# CER2) SALK_084443C# cer2/5) ColA0# TADNA#At4g13840# CER2/LIKE1/CER26) SALK_087857# cer2/like1/1) ColA0# TADNA#At3g23840# CER2/LIKE2/CER26/LIKE) GKA180G04A013602# cer2/like2/1) ColA0# TADNA#At4g29250# CER2/LIKE3) SALK_147254C# cer2/like3/1) ColA0# TADNA#At5g02890# CER2/LIKE4) N/A# cer2/like4/1) ColA0# CRISPR/Cas9#At1g68530# CER6) N/A# ew2) ColA0# En/Spm#transposon##With# the# exception# of#Arabidopsis# plants# grown# for# the# purpose# of# transformation#with#Agrobacterium) tumefaciens,# all# seeds#were# germinated# on#Arabidopsis# (Arabidopsis) thalianaK#AT)# medium# (Somerville# and# Ogren,# 1982)# supplemented# with# 1# %# (w/v)# agar# and,# when#necessary,# with# appropriate# antibiotics# for# transgene# selection.# Seeds# were# stratified# for# 2A4#days#on#AT#plates#stored#in#the#dark#at#4°C,#prior#to#transfer#to#continuous#light#(100#µEmA2sA1#of#photosynthetically# active# radiation)# at# 20°C.# Seven# dayAold# seedlings#were# transferred# to# soil#(Sunshine# Mix# 4# or# 5,# SunGro)# supplemented# with# liquid# AT# medium# under# the# same# light#conditions.# Pots# were# covered# with# cling# wrap# for# 3A4# days# after# transplanting.# Seeds# were#recovered# from# mutants# with# partial# male# sterility# phenotypes# by# either# wrapping# young#inflorescences# in#cling#wrap#for#several#days#or#growing#plants# in#a#chamber#with#high#relative#humidity.#Mutant#lines#were#genotyped#by#PCR#as#described#in#Section#2.2.1.#Insertion#sites#of#TADNAs#and#transposable#elements#were#determined#by#amplifying#the#genetic#sequence#using#one# insertionAspecific# and# one# geneAspecific# primer.# Primers# used# for# this# genotyping# are#specified# in# Table# 2A2.# Plants# were# outAcrossed# manually# to# generate# double,# triple,# and#quadruple#mutants,#and#homozygous#mutants#were# identified# in# the#F2#and#F3#generations#by#PCRAbased#genotyping.#Arabidopsis#seeds#grown#specifically#for#transformation#with#Agrobacterium)tumefaciens#were#suspended#in#a#0.1%(w/v)#agarose#solution,#and#distributed#on#ATAsupplemented#soil#at#a#27##density# of# 100# seeds/6”# pot.# Pots# were# covered# with# cling# wrap# for# 7A10# daysK# plants# were#otherwise#grown#under#the#same#conditions#as#described#above.#$2.1.2$ Nicotiana0benthamiana0Nicotiana) benthamiana# (tobacco)# plants# grown# for# transient# expression# assays# were#sown# directly# on# soil# (Sunshine# Mix# 4# or# 5,# SunGro)# supplemented# with# liquid# AT# medium.#Plants#were#grown#under#16/8#hr#day/night#cycles,#at#100#µEmA2sA1#of#photosynthetically#active#radiation.#Plants#were#transformed#roughly#three#to#four#weeks#after#sowing.##2.1.3$ Saccharomyces0cerevisiae$Saccharomyces) cerevisiae# strain#W3031A# (MATa)ade2/1) his3/11,15) leu2/3,112) trp1/1)ura3/1)can1/100)#were#used#for#all#yeast#expression#experiments.#Cells#were#grown#on#plates#of#yeast#peptone#adenine#dextrose#(YPAD)#or#synthetic#complete#(SC)#dropout#medium#solidified#with# 2%# agar# (Sherman,# 2002).# SC# medium# was# prepared# lacking# nutrients# required# for#selection#of#prototrophic,# transformed#cells.#Cells#were#grown#at#30°C,#and#were#kept#at# room#temperature#for#shortAterm#storage.###28##2.2$ Polymerase$chain$reaction$(PCR)$#2.2.1$ Genotyping$$Plant#DNA#for#genotyping#was#preserved#using#Whatman#FTA#cards.#Insertional#mutants#were#genotyped#using#either#two#geneAspecific#primers#or#one#geneAspecific#and#one#insertionAspecific#primer,#as#listed#in#Table#2A2.##$Table$212:$Primers$used$for$genotyping$Primer$Name$ Sequence$(5'13')$LBb1A3#(SALK#lines)# ATTTTGCCGATTTCGGAAC#LBo8409#(GABIAKAT#lines)# ATATTGACCATCATACTCATTGC#En1#RB# GGGTTTTGGCCGACACTCC#CER2#LP# TGGGAAAAACCAAAACAACAC#CER2#RP# ACGTCGAAGAGTTCACAGTGG#CER2ALIKE1#LP# AGGCCCAACCCTAATCCTCC#CER2ALIKE1#RP# TACTCAGCCGAGACAGCACG#CER2ALIKE2#LP# GACTCATCAAGTTCTGCCCTG#CER2ALIKE2#RP# GATCTACTGCCCCAAAACCTC#CER2ALIKE3#LP# AGGGGTTTTCATGAACCCG#CER2ALIKE3#RP# TTCTTTAACCAGTCTCCCGG#CER2ALIKE4#LP# ACATCTGTATTCGCTGTTGGG#CER2ALIKE4#RP1#(for#CAPS)# AGGCTCTCCCTAAAGATTCC#CER2ALIKE4#RP2#(for#sequencing)# AGAACAATGGAGAGTATTGAGG#CER6#LP# GCCACGCACCATCTACCTCG#CER6#RP# CAACATGGCTCTCTCGTTGCC#29##2.2.2$ RT1PCR$&$qPCR$RNA# was# extracted# from# Arabidopsis# tissues# by# phenol:chloroform:isoamyl# alcohol#separation# and# purified# by# lithium# chloride# and# sodium# acetate# precipitations# (Wilkins# and#Smart,#1995).#Genomic#DNA#was# removed#by# treatment#with#DNase# I# (New#England#Biolabs)#according#to#the#manufacturer’s#protocol.#SingleAstranded#complementary#DNA#was#synthesized#from#equal#amounts#of#RNA#using#Superscript#III#Reverse#Transcriptase#(Invitrogen),#according#to#the#manufacturer’s#protocol.#GAPC1#was#used#as#a#control#for#constitutive#expression.##cDNA# was# quantified# using# iQ# SYBR# green# Supermix# (BioRad),# according# to# the#manufacturer’s#protocol,#in#an#MJ#Mini#Opticon#Personal#Thermocycler#(BioRad).#Four#technical#replicates#were#prepared#for#each#sample.#Expression# levels#were#analyzed#using# the#method#described# by# Pfaffl,# 2001.# Standard# curves# were# used# for# each# qPCR# primer# pair,# and# their#efficiencies#(E#values)#calculated:#CER2,#2.45#(122%)K#CER2/LIKE1,#1.83#(91%)K#CER2/LIKE2,#2.01#(101%)K#GAPC,#2.46#(123%).#Primers#used#for#RTA#and#qPCR#are#listed#in#Table#2A3.###Table$213:$Primers$used$for$RT1PCR$&$qRT1PCR$#Primer$Name$ Sequence$(5'13')$CER2#qPCR#F# GTCTACGATCACGTTCTTGGTCCTG#CER2#qPCR#R## CCCGATACCAGCTGTCCAAGTG#CER2ALIKE1#RT/qAPCR#F# CATCCTATTGGACCGGAATTGACG#CER2ALIKE1#qPCR#R# CTTTTCTCCGGTAATGGCCTTAGC#CER2ALIKE1#RTAPCR#R# CCGACTCCGGCGACTTCCCC#CER2ALIKE2#RT#F# GAGGGAAGCGGTCCTGTTC#CER2ALIKE2#RT#R# CTCCTCCACAACCCCCGG#CER2ALIKE2#qPCR#F# ACCAGTTGGACCAGATTTGGCA##CER2ALIKE2#qPCR#R# CCCACCAGCAAAGGCTTGAGC#GAPC1#RT/qAPCR#F# TCAGACTCGAGAAAGCTGCTA#GAPC1#RT/qAPCR##R# GATCAAGTCGACCACACGG#30##2.3$ Constructs,$transformation,$and$selection$$2.3.1$ Stable$expression$in$Arabidopsis$2.3.1.1$ pGWB4=CER2pro::CER2=GFP0and0pGWB6=35Spro::CER2=GFP0The# genomic# sequence# of# CER2# was# amplified# beginning# from# the# start# codon# and#extending# up# until# but# not# including# the# stop# codon# for# the# pGWB6/35Spro::CER2/GFP#construct,# and# beginning# from# the# end# of# the# 3’# untranslated# region# of# the# gene# directly#upstream# of#CER2# and# extending# up# until# but# not# including# the# stop# codon# of#CER2# for# the#pGWB4/CER2pro::CER2/GFP#construct.#Primers#used#for#both#constructs#are#listed#in#Table#2A4.# Both# sequences# were# amplified# from# ColA0# leaf# genomic# DNA# with# Phusion# polymerase#(NEB),#using#primers#with#Gateway#attB#adaptor#sequences.#The#purified#PCR#products#were#transferred# into# the#Donor# vector#pDONR221#using#BP#Clonase# (Invitrogen),#according# to# the#manufacturer’s# protocol.# Positive# clones# were# identified# by# colony# PCR,# restriction# digestion,#and#sequencing.#The#inserts#were#transferred# into#pGWB4#and#pGWB6#vectors#(T.#Nakagawa#et#al.,#2007),#which#have#CAterminal#GFP#tags,#using#LR#Clonase#(Invitrogen)#according#to#the#manufacturer’s# protocol.# Positive# clones# were# again# identified# by# colony# PCR,# restriction#digestion,# and# sequencing.# Purified# constructs# were# transformed# into# chemically# competent#Agrobacterium)tumefaciens#GV3101#cells#carrying#the#pMP90#Ti#plasmid,#and#verified#by#colony#PCR#and#restriction#digestion.#FiftyAmilliliter#cultures#of# transgenic#A.) tumefaciens# in# log#phase#growth#were#centrifuged,#washed,#and#resuspended#in#5%#sucrose#solution.#FourAweekAold#ColA0# and# cer2/5# plants# were# sprayed# with# the# A.) tumefaciens) suspensions,# and# were# covered#overnight,#while#the#A.)tumefaciens#cells#remained#infectious.#After#plant#senescence,#T1#seeds#were# collected# and# screened# for# hygromycin# resistance# conferred# by# the# TADNA,# and#complementation#of#the#cer2/5#mutant#wax#deficiency.#2.3.1.2$ pGWB4=CER2=LIKE1pro::CER2=LIKE1=GFP0and0 pGWB4=CER6pro::CER2=LIKE1=GFP0The#CER2/LIKE1#coding#region#from#the#start#codon#to#one#codon#directly#upstream#of#the#stop#codon#and# from#2.6#kb#upstream#of# the#CER2/LIKE1#proteinAcoding#sequence# to# the#codon# directly# upstream# of# the#CER2/LIKE1# stop# codon# were# amplified# from# ColA0# genomic#DNA.# The#CER6# promoter,# consisting# of# the# 1.2Akb# fragment# upstream# of# the#CER6# coding#sequence,# was# excised# from# a# pBl101/CER6pro/GUS# construct# (a# kind# gift# from# Dr.# Patricia#Lam,# University# of# British# Columbia).# The# CER2/LIKE1# genomic# sequence# with# the# CER6#promoter#and#the#fullAlength#CER2/LIKE1#genomic#sequence#with#its#native#promoter#were#both#cloned# by# restriction# enzyme# digestion# and# ligation# into# pBluescriptII# SK+# (Stratagene).#31##CER6pro::CER2/LIKE1# and#CER2/LIKE1pro::CER2/LIKE1# were# amplified# from# the# Bluescript#plasmids# using# primers# with# GATEWAY# attB# adaptor# sequences# for# insertion# in# pDONR207,#and#the#constructs#were#confirmed#by#colony#PCR,#restriction#digests,#and#sequencing.#Primer#sequences# are# listed# in# Table# 2A4.# The# CER6pro::CER2/LIKE1# and# CER2/LIKE1pro::CER2/LIKE1# insertions# were# then# transferred# to# the# pGWB4# binary# vector# using# LR# Clonase#(Invitrogen)# with# CAterminal# GFP# tags# (T.# Nakagawa# et# al.,# 2007),# for# stable# expression# in#Arabidopsis.# Purified# constructs# were# transformed# into# chemically# competent# A.) tumefaciens#GV3101# cells# carrying# the# pMP90# Ti# plasmid,# and# verified# by# colony# PCR# and# restriction#digestion.# Arabidopsis# plants# were# transformed# by# the# floral# spray# methodK# the# CER2/LIKE1pro::CER2/LIKE1/GFP# construct# was# transformed# to# wildAtype# ColA0# and# cer2/like1/1#plants,#and# the#CER6pro::CER2/LIKE1/GFP#construct#was# transformed#to#wildAtype#ColA0#and#cer2/5# plants.#Transformed# lines#were#selected#by#growing# the#seedlings#on#hygromycin,#and#genotypes#were#confirmed#by#PCR#in#lines#selected#for#phenotype#analysis.##Table$214:$Constructs$and$primers$used$for$stable$expression$in$Arabidopsis$Construct(s)$ Primer$Name$ Sequence$(5'13')$CER2pro::CER2AGFP#&#35Spro::CER2AGFP#attB1#A#CER2#coding#F# GGGGACAAGTTTGTACAAAAAAGCAGGCTTTATGGAGGGAAGCCCAGTGAC#attB1#A#CER2#promoter#F# GGGGACAAGTTTGTACAAAAAAGCAGGCTTTTCAGGACAACTGGCCTAGTG#attB2#A#CER2#coding,#no#stop#R# GGGGACCACTTTGTACAAGAAAGCTGGGTGTATAATCATATTAGTCACCTCCTCC#CER6p::CER2ALIKE1AGFP#&#CER2ALIKE1p::CER2ALIKE1AGFP#CER6#promoter#F# ??116?36133??361?1?366??6??6CER6#promoter#R# ??111163??36?3661616????11?6CER2ALIKE1#promoter#F# 1111????611??313?16???336?6?311633361?113?11?3?66166CER2ALIKE1#promoter#R# 1111????1163???6636361?3111331113??3??111??3311????133CER2ALIKE1#coding#F# 1111????1163??1?666?361?3?31161131666131666131666?3CER2ALIKE1#coding,#no#stop#R# 1111????3?6316?6636361?3111331113??3??111??3311????133attB1#A#CER6#promoter#F# GGGGACAAGTTTGTACAAAAAAGCAGGCTTTCTTCGATATCGGTTGTTGACG#attB1#A#CER2ALIKE1#promoter#F# GGGGACAAGTTTGTACAAAAAAGCAGGCTTTACTAGTTTCCGTGTCAAGCC#attB2#A#CER2ALIKE1#coding,#no#stop#R# GGGGACCACTTTGTACAAGAAAGCTGGGTGTGGCGCGATCAAACCAAAC#####32##2.3.2$ Constitutive$and$inducible$expression$in$S.0cerevisiae$cDNA#was#prepared#as# described# in#Section# 2.2.2# from#ColA0# stem# tissue.#CER6# and#CER2# coding# DNA# sequences# were# amplified# using# primers# listed# in# Table# 2A5.#CER6# was#cloned# into# the# pESCAURA# yeast# expression# vector# (Agilent)# downstream# of# the# GAL10#inducible#promoter,#and#CER2#was#cloned#into#the#p423#yeast#expression#vector#with#the#HIS3)selection# marker,# downstream# of# the# strong# constitutive# GPD# (glyceraldehydeA3APAdehydrogenase)#promoter#(Mumberg#et#al.,#1995).#Correct#plasmid#construction#was#confirmed#by# colony# PCR,# restriction# digestion,# and# sequencing.# The# yeast# strain# W3031A# was#transformed#using# the#protocol# described#by#Gietz#and#Woods# (2002).#Transgenic# yeast# cells#were#grown#on#synthetic#complete#(SC)#selection#medium#(Sherman,#2002)#lacking#appropriate#amino#acids.#Successful#transformation#was#confirmed#by#colony#PCR.#Three#to#four#individual#cell# lines#were#selected# from#each# transgenic#strain# for# induction#of# transgene#expression#and#lipid#analysis.#Cultures#were#grown#in#5#mL#SC#medium#with#2%#glucose#overnight,#then#plated#on#solid#SC medium#with#2%#galactose#and# incubated#for#2# to#4#d#(strains#harboring#plasmids#grew#slower#than#the#wild#type).#A#similar#method#was#used#for#construction#and#transformation#of#CER2/LIKE#expression#plasmids.#However,#when#none#of#the#transgenes#required#galactose#to# induce#expression,#the#cells#were#streaked#out#and#grown#directly#on#solid#SC#medium#with#2%#glucose#and#the#required#amino#acids.#To# make# CER2# and# CER6# constructs# with# NAterminal# epitope# tags,# the# coding#sequences#of#each#gene#were#amplified#with#an#AvrII#restriction#site#added#3’#to#the#ATG#start#site.#The#coding#sequence#insertions#were#ligated#into#p423#(CER2)#and#p426#(CER6)#vectors.#Triple# hemagglutinin# (HA)# and#MYC# tags#were# excised# from#pRS316AHA3# and#pRS316AMYC3#plasmids#using#SpeI#(a#gift#from#Dr.#Frederic#Beaudoin,#Rothamsted#Research,#UK).#The#p423/AvrII/CER2#and#p426/AvrII/CER6#constructs#were#cut#with#AvrII,#to#allow#for#the#insertion#of#the#SpeIAcut# HA# and# MYC# tags.# However,# a# second# AvrII# site# was# found# in# the# p423# vector#backboneK# this# site# was# not# present# in# the# p423# sequence# downloaded# from# the# ATCC#database,#but#an#AvrII#site# is# found# in# the#HIS3#gene#used#as#a#selection#marker# in# the#p423#vector.# Hence,# the# AvrIIACER2# cassette# was# transferred# into# p426,# cut# with# AvrII,# the# HA3#sequence#was#inserted,#and#then#the#HA3ACER2#sequence#was#cut#and#inserted#back#into#p423.#Correct# plasmid# construction# was# confirmed# by# colony# PCR,# restriction# digestion,# and#sequencing.### Over#the#course#of#my#work#with#yeast,#I#found#that#expression#was#more#consistent#and#fatty#acid#profiles#were#more#reproducible#when#I#expressed#CER6#with#a#constitutive#promoter#such# as#GPD,# instead# of# the# galactoseAinducible# promoter#GAL10.# Therefore,# the# p426AAvrIIA33##CER6#construct#(without#the#MYC3#tag#inserted)#was#used#for#all#yeast#expression#experiments#described# in# Chapters# 4# and# 5.# Further,# the# consistency# provided# by# a# Kozak# consensus#sequence#with#four#adenine#bases#upstream#of#the#ATG#translation#start#site#became#clear#after#some# experience# with# yeast# expression.# Therefore,# the#CER2/LIKE1# and#CER2/LIKE2# yeast#expression#constructs#described# in#Table#2A7#were#used# for# the#yeast#expression#experiments#presented#in#Chapter#4#of#thesis.###Table$215:$Constructs$and$primers$used$for$constitutive$and$inducible$expression$in$yeast$Construct$ Primer$Name$ Sequence$(5'13')$pESCAURAACER6#CER6ANotI#F# AATTGCGGCCGCATGCCTCAGGCACCGATG#CER6ASpeI#R# AATTACTAGTTTAGAGTTTGACAACTTCGGG#p423ACER2#CER2AEcoRI#F# AATTGAATTCATGGAGGGAAGCCCAGTGACC#CER2ASalI#R# AATTGTCGACTTATATAATCATATTAGTCACCTCCTC#p424ACER2ALIKE1#CER2ALIKE1AEcoRI#F# AATTGAATTCATGGGTCGATCTCAAGAACAGGG#CER2ALIKE1ASalI#R# AATTGTCGACTCATGGCGCGATCAAACC#p425ACER2ALIKE2#CER2ALIKE2ASpeI#F# AATTACTAGTATGGGTCTAGTTCAAGAAGAGGG#CER2ALIKE2ASalI#R# AATTGTCGACTTAAACTAACGGCGTGATCAAACC#p423ACER2ALIKE3#CER2ALIKE3ASpeI#F# AATTACTAGTAAAAATGTTTGCTAAAGAGAAGAC#CER2ALIKE3ASalI#R# AATTGTCGACTTATAATTGCTTTGTATCTTCC#p425ACER2ALIKE4#CER2ALIKE4ABamHI#F# AATTGGATCCAAAAATGGAAACCAAAATCCCTAAGTC#CER2ALIKE4ASalI#R# AATTGTCGACTTACTCTTTTGGCAGTGCAAC#p426AAvrIIACER6#CER6ABamHI#F# AATTGGATCCATGCCTAGGCCTCAGGCACCGATGCCAG#CER6AClaI#R# AATTATCGATTTAGAGTTTGACAACTTCGGGAATAAAG#p423AAvrIIACER2#CER2AEcoRI#F# AATTGAATTCATGCCTAGGGAGGGAAGC##CER2ASalI#R# AATTGTCGACTTATATAATCATATTAGTCACCTCCTCC###34##2.4$ Site1directed$mutagenesis$SiteAdirected# mutagenesis# was# carried# out# by# overlap# extension# mutagenesis# as#described#by#Ho#et#al.,#1989.##Different# approaches# were# used# for# the# two# siteAdirected# mutagenesis# experiments#described# in#Chapter#5.#To#mutagenize# the#putative#H166#catalytic# residue#of#CER2# that#was#annotated# based# on# sequence# alignment# with# BAHD# acyltransferases,# the# genomic# CER2#sequence# was# modified,# generating# H166A# and# H166N# artificial# alleles# (Section# 5.2.1).# The#pGWB4/CER2pro::CER2:GFP#construct#was#used#as#a#template.#5’#and#3’#fragments#of#CER2#sequence#were#amplified#using# the#primers#specified# in#Table#2A6,#washed#on#a#DNA#binding#column,#and#then#equal#concentrations#of#each#fragment#were#used#for#overlapAextension#PCR.#5’#and#3’#end#primers#were#added#to#the#overlap#extension#PCR#reaction,#as#this#was#found#to#increase#yield#of#the#final#fullAlength#PCR#product.#The#entire,#unmodified#CER2)sequence#was#also# amplified# from# the# pGWB4/CER2pro::CER2/GFP# construct.# The# constructs# were#assembled# in# the#pGWB4#gateway#vector,# introduced# into#A.) tumefaciens,)and# transformed# to#cer2/5) mutant# Arabidopsis# using# the# method# described# in# Section# 2.3.1.# The# background#genotype#and#the#presence#of#the#transgene#of#all#transformed#plants#selected#for#analysis#were#confirmed#by#PCR#amplification.#The#siteAdirected#mutation#of#the#transgene#was#sequenced#for#one#representative#plant#from#each#transformed#line.#Complementation#of#the#cer2/5)phenotype#was#assessed#by# the#physical# appearance#of# the#plant# stems,# total# stem#wax# load,#and#stem#wax#composition#(section#2.8.1).#To#mutagenize#a#second#putative#catalytic#site#identified#based#on#the#structural#models#of#CER2ALIKE#proteins#(Section#5.2.6),#the#CER2#coding#sequence#was#modified,#generating#an#H38A#artificial#allele.#The#p423/CER2)construct#was#used#as#a#template.#To#mutagenize#CER2/LIKE4# to# mimic# CER2,# the# p425/CER2/LIKE4# construct# was# used# as# a# template,# and# the#GIFRRTV#sequence#of#CER2ALIKE4#was# replaced#with# LHYV# from#CER2.#Overlap#extension#mutagenesis#was#carried#out#as#described#above,#and#detailed#in#Ho#et#al.,#1989.#The#modified#constructs#were#transformed#to#yeast,#with#wildAtype#CER2#and#CER2/LIKE4#cDNA#sequences#as# controls.# Function# of# the# mutagenized# allele# was# assessed# by# FAMEs# analysis# of# the#transgenic#yeast#cell#lines#(section#2.8.2).#35##Table$216:$Constructs$and$primers$used$for$site1directed$mutagenesis$Construct(s)$ Primer$Name$ Sequence$(5'13')$CER2pro::CER2#WT/H166A/H166N#SDM#CER2#genomic#F# GGGGACAAGTTTGTACAAAAAAGCAGGCTTTTCAGGACAACTGGCCTAGTG#CER2#genomic#R# GGGGACCACTTTGTACAAGAAAGCTGGGTGTTATATAATCATATTAGTCACCTCCTCC#CER2#H166A#mutation,#5'#fragment#R# CACGTCTCCAAGAATGGCGGCCCAACTCAA#CER2#H166A#mutation,#3'#fragment#F# TTGAGTTGGGCCGCCATTCTTGGAGACGTG#CER2#H166N#mutation,#5'#fragment#R# TTGAGTTGGGCCAATATTCTTGGAGACGTG#CER2#H166N#mutation,#3'#fragment#F# TTGAGTTGGGCCAATATTCTTGGAGACGTG#p423ACER2#H38A#SDM#CER2#cDNA#F# AATTGAATTCAAAAATGGAGGGAAGCCCAGTGACC#CER2#cDNA#R# AATTGTCGACTTATATAATCATATTAGTCACCTCCTCCTTGAG#CER2#H38A#mutation,#5'#fragment#R# GCTCGGACGTAGGCGAGCTTCATGG#CER2#H38A#mutation,#3'#fragment#F# CCATGAAGCTCGCCTACGTCCGAGC#p425ACER2ALIKE4#SDM#CER2ALIKE4#cDNA#F# AATTGGATCCAAAAATGGAAACCAAAATCCCTAAGTC#CER2ALIKE4#cDNA#R# AATTGTCGACTTACTCTTTTGGCAGTGCAAC#CER2ALIKE4#GIFRRTV>LHYV#mutation,#5'#fragment#R#CGTAACTATGTTGACGTAGTGGAGTACTCCGACGGGATCTAC#CER2ALIKE4#GIFRRTV>LHYV#mutation,#3'#fragment#F#GGAGTACTCCACTACGTCAACATAGTTACGTATTACAAAGAAGC##36##2.5$ Sequence$swaps$CER2/LIKE1# and#CER2/LIKE2# coding# DNA# sequence# fragments# were# amplified# from#p424/p425# yeast# expression# plasmids# described# in# section# 2.3.2,# using# primers# described# in#Table#2.7.#Regions#of#homology#were#selected#as#swap#sites.#5’#and#3’# fragments#amplified# in#an# initial# PCR# reaction# were# combined# in# a# second# PCR# reaction# to# generate# fullAlength,#chimeric# products# by# overlap# extension.# The# chimeras# were# ligated# into# the# p423# vector,#confirmed#and#coAexpressed#in#yeast#with#the#CER6#gene#as#described#in#section#2.3.2.####Table$217:$Constructs$and$primers$used$for$sequence$swaps$in$yeast$Construct$(s)$ Primer$Name$ Sequence$(5'13')$p423ACER2ALIKE1#CER2ALIKE1#SpeI#F# AATTACTAGTAAAAATGGGTCGATCTCAAG#CER2ALIKE1#SalI#R# AATTGTCGACTCATGGCGCGATCAAAC#p423ACER2ALIKE2#CER2ALIKE2#SpeI#F# AATTACTAGTAAAAATGGGTCTAGTTCAAG#CER2ALIKE2#SalI#R# AATTGTCGACTTAAACTAACGGCGTG#p423Achimera#A#CER2ALIKE1#SpeI#F# AATTACTAGTAAAAATGGGTCGATCTCAAG#swap#1#R# GCCCATAAGTTGAAG#swap#1#F# CTTCAACTTATGGGC#CER2ALIKE2#SalI#R# AATTGTCGACTTAAACTAACGGCGTG#p423Achimera#B#CER2ALIKE2#SpeI#F# AATTACTAGTAAAAATGGGTCTAGTTCAAG#swap#1#R# GCCCATAAGTTGAAG#swap#1#F# CTTCAACTTATGGGC#CER2ALIKE1#SalI#R# AATTGTCGACTCATGGCGCGATCAAAC#p423Achimera#C#CER2ALIKE1#SpeI#F# AATTACTAGTAAAAATGGGTCGATCTCAAG#swap#2#R# TAAACCGGGTCATCTGAA#swap#2#F# TTCAGATGACCCGGTTTA#CER2ALIKE2#SalI#R# AATTGTCGACTTAAACTAACGGCGTG#p423Achimera#D#CER2ALIKE2#SpeI#F# AATTACTAGTAAAAATGGGTCTAGTTCAAG#swap#2#R# TAAACCGGGTCATCTGAA#swap#2#F# TTCAGATGACCCGGTTTA#CER2ALIKE1#SalI#R# AATTGTCGACTCATGGCGCGATCAAAC#p423Achimera#E#CER2ALIKE1#SpeI#F# AATTACTAGTAAAAATGGGTCGATCTCAAG#swap#3#R# CATTTCCATATGATTCC#swap#3#F# GGAATCATATGGAAATG#CER2ALIKE2#SalI#R# AATTGTCGACTTAAACTAACGGCGTG#37##Construct$(s)$ Primer$Name$ Sequence$(5'13')$p423Achimera#F#CER2ALIKE2#SpeI#F# AATTACTAGTAAAAATGGGTCTAGTTCAAG#swap#3#R# CATTTCCATATGATTCC#swap#3#F# GGAATCATATGGAAATG#CER2ALIKE1#SalI#R# AATTGTCGACTCATGGCGCGATCAAAC#p423Achimera#G#CER2ALIKE1#SpeI#F# AATTACTAGTAAAAATGGGTCGATCTCAAG#swap#4#R# TCCACGAAACGTGTGCC#swap#4#F# GGCACACGTTTCGTGGA#CER2ALIKE2#SalI#R# AATTGTCGACTTAAACTAACGGCGTG#p423Achimera#H#CER2ALIKE2#SpeI#F# AATTACTAGTAAAAATGGGTCTAGTTCAAG#swap#4#R# TCCACGAAACGTGTGCC#swap#4#F# GGCACACGTTTCGTGGA#CER2ALIKE1#SalI#R# AATTGTCGACTCATGGCGCGATCAAAC#p423Achimera#I#CER2ALIKE1#SpeI#F# AATTACTAGTAAAAATGGGTCGATCTCAAG#swap#5#R# GGTAGTGGAGCTTCATGGC#swap#5#F# GCCATGAAGCTCCACTACC#CER2ALIKE2#SalI#R# AATTGTCGACTTAAACTAACGGCGTG#p423Achimera#J#CER2ALIKE2#SpeI#F# AATTACTAGTAAAAATGGGTCTAGTTCAAG#swap#5#R# GGTAGTGGAGCTTCATGGC#swap#5#F# GCCATGAAGCTCCACTACC#CER2ALIKE1#SalI#R# AATTGTCGACTCATGGCGCGATCAAAC#p423ACER2ALIKE2#truncated#CER2ALIKE2#SpeI#F# AATTACTAGTAAAAATGGGTCTAGTTCAAG#swap#4#R#truncation# AATTGTCGACTCACTCCACGAAACGTGTGCC#p423AASFT#ASFT#SpeI#F# AATTACTAGTAAAAATGGTTGCTGAGAACAATAAAAAC#ASFT#SalI#R# AATTGTCGACTTATATCTGTAAAAACTGTTCTTG#p423Achimera#K#CER2ALIKE2#SpeI#F# AATTACTAGTAAAAATGGGTCTAGTTCAAG#swap#4#R# TCCACGAAACGTGTGCC#swap#4#F#ASFT# GGCACACGTTTCGTGGAAGCAGAAGCAAACTG#ASFT#SalI#R# AATTGTCGACTTATATCTGTAAAAACTGTTCTTG#p423ACER2ALIKE3#CER2ALIKE3#SpeI#F# AATTACTAGTAAAAATGTTTGCTAAAGAGAAGAC#CER2ALIKE3#SalI#R# AATTGTCGACTTATAATTGCTTTGTATCTTCC#p423Achimera#L#CER2ALIKE2#SpeI#F# AATTACTAGTAAAAATGGGTCTAGTTCAAG#swap#4#R# TCCACGAAACGTGTGCC#swap#4#F#CER2ALIKE3# GGCACACGTTTCGTGGAGGCCAGAGCAACC#CER2ALIKE3#SalI#R# AATTGTCGACTTATAATTGCTTTGTATCTTCC#38##2.6$ CRISPR/Cas91mediated$mutagenesis$#2.6.1$ Construct$design$and$assembly$19# and# 20# bp# target# sequences# for# mutagenesis# (protospacers)# were# selected# in# the#CER2/LIKE4#gene#based#on# four#criteria:# (1)# they#are#strictly#unique# to#CER2/LIKE4,# (2)# they#have#an#NGG#protospacer#adjacent#motif#(PAM)#at#the#3’#end#of#the#sequence,#(3)#they#are#in#a#position#likely#to#cause#a#null#mutation,#and#(4)#they#contain#a#restriction#site#within#10#bp#of#the#PAM.##The# target# selected# for# mutagenizing# the# CER2/LIKE4# gene# was#TATCCGTCGTAGATCCCGT(CGG)#which#contains#a#BstYI#digestion#site# (RGATCY)#near# the#PAM.#Target# sites# were# synthesized# as# forward# and# reverse# oligonucleotides# (Table# 2A8)#flanked# by# adaptors# to# allow# for# ligation# into# a# BpiIAdigested# CRISPR# array.# Complementary#oligonucleotides#were#phosphorylated#in#the#following#reaction:#1#µL#oligo#1#(100μM)#1#µL#oligo#2#(100μM)#1#µL#10X#T4#Ligation#Buffer#(NEB)#6.5#µL#ddH2O#0.5#µL#T4#Phosphonucleotide#Kinase#(PNK)#(NEB)#The#mixture#was# incubated#at#37°C#for#30#min,#heated#to#65°C#for#20#min#to# inactivate#the#kinase,#and#then#cooled#at#room#temperature#to#allow#the#complementary#oligonucleotides#to#form#duplexes.##The#pGreenACRISPR#vector#was#digested#with#BpiI#(isoschizomers:#BbsI/BpuAI/BstV2I)#restriction#enzyme# (Thermo),# and# the#digested#vector#was#purified#on#a#DNAAbinding# column,#and#quantitated#on#a#nanodrop#spectrophotometer.#Roughly#50#ng#of# cut#vector#was#used# for#ligation,#with#1#µL#of#the#oligonucleotide#duplex#diluted#1#in#200:#1#µL#(≈50#ng)#digested#pGreenACRISPR#1#µL#1:200#dilution#phosphorylated#primer#dimer#2#µL#10X#T4#ligase#buffer#(NEB)#1#µL#T4#ligase#(NEB)#15#µL#distilled#H2O#The# ligation# was# incubated# at# RT# overnight.# 5A10# µL# of# the# ligation# mixture# was#transformed# to# chemically# competent# DH5!# cells.# Construct# sequences# were# verified,# then#transformed#to#A.)tumefaciens#and# into#Arabidopsis# following#the#protocol#described# in#section#39##2.3.1,# except# that# the# pGreenAbased# plasmid# used# here# was# coAtransformed# with# the# pSoup#helper#plasmid#required#for#replication#of#pGreen.##Table$218:$Constructs$and$primers$used$for$CRISPR/Cas9$mutagenesis$of$CER2=LIKE4$Construct$ Primer$Name$ Sequence$(5'13')#pGreenACRISPRACER2ALIKE4#CRISPRACER2ALIKE4#F# GATTGTATCCGTCGTAGATCCCGT#CRISPRACER2ALIKE4#R# AAACACGGGATCTACGACGGATAC###2.6.2$ Identification$of$CRISPR$mutants$T1,# T2,# and# T3# generations# were# germinated# on# hygromycin# selection# medium# for# the#CRISPR/Cas9#TADNA.#The#BstYI# restriction# site#within# the#CER2/LIKE4# target# sequence#was#used# to# identify# individuals# that# were# mutagenized# by# the# Cas9# nuclease.# DNA# from# all#generations#was# collected# on#Whatman# FTA# cards,# and# a# 408# bp# sequence# surrounding# the#restriction#site#was#amplified#using#primers#listed#in#Table#2.2.#1#µL#of#amplification#product#from#each# reaction# was# digested# in# a# 10# µL# reaction# volume# with# BstYI# for# a# minimum# of# 2# hr.#Product# that# was# not# cleaved# by# BstYI# was# indicative# of# a# putative# cer2/like4# mutant.# The#CER2/LIKE4)gene#was# sequenced# from#candidate#mutants# to# identify# lossAofAfunction# alleles.#Additional# FTA# presses# were# taken# from# each# putative# mutant# for# a# second# CAPS# and#sequencing#screen#to#eliminate#obvious#chimeras.#Homozygous#mutations#were#identified#in#the#T3#and#T4#generations.##40##2.7$ Microscopy$#2.7.1$ Confocal$Imaging$Subcellular# localization# of# GFPAtagged# CER2# and# CER2ALIKE1# was# carried# out# on# a#PerkinAElmer# UltraView# VoX# Spinning# Disk# Confocal# Microscope,# using# a# 633# glycerol#immersion#objective.#Stem#tissue#was#dyed#with#hexyl#rhodamine#B#to#counterstain#the#ER.#GFP#was#excited#with#a#488Anm#laser,#and#hexyl#rhodamine#B#was#excited#with#a#561Anm#laser.#CellAspecific# expression# patterns#were# observed# on# a#Nikon#Eclipse# 80i# Scanning# Laser#Confocal#Microscope# using# a# 203# objective.# GFP# was# excited# with# a# 488Anm# laser,# and# chlorophyll#autofluorescence#was#detected#for#contrast.##2.7.2$ SEM$#2.7.2.1$ Comparison$of$cer2=5$and$wild1type$stems$Segments# from# the#apical#1#cm#of#stem#were#mounted#onto#SEM#stubs,#air#dried,#and#sputter#coated#with#gold#particles#for#10#min#at#40#mA#in#a#SEMPrep2#sputter#coater#(Nanotech).#The# coated# samples# were# viewed# with# a# Hitachi# S4700# field# emission# SEM# using# an#accelerating#voltage#of#5#kV#and#a#working#distance#of#12#mm.##2.7.2.2$ Analysis$of$stems$ectopically$expressing$CER2=LIKE1$One#centimeter#long#stem#segments#approximately#10#cm#from#the#shoot#apex#were#cut,#mounted,#air#dried,#and#sputter#coated#with#gold#particles#to#a#thickness#of#10#nm#using#a#Leica#EM# ACE200# Low# Vacuum# Coater.# Coated# samples# were# viewed# with# a# Hitachi# S4700# field#emission#SEM#using#an#accelerating#voltage#of#5#kV#and#a#working#distance#of#12#mm.#41##2.8$ Lipid$analyses$#2.8.1$ GC1FID$analysis$of$wax$load$and$composition$Wax#analysis#was#carried#out#using#the#top#10#cm#of#3A#to#5AweekAold#primary#stems,#or#3A#to#4AweekAold#entire#rosette#leaves.#Tissues#were#photographed#to#determine#sample#surface#area.#Surface#area#was#established#by#measuring# twoAdimensional#area# in#Adobe#Photoshop,#and# for# stems,# by# multiplying# the# flat# area# by# π.# Tissues# were# submerged# for# 30# s# in#approximately# 8# mL# of# chloroform# containing# 10# µg# tetracosane# as# an# internal# standard.#Chloroform# was# blown# off# under# nitrogen# gas,# and# the# waxes# silylated# by# heating# in# 10# µL#pyridine# and# 10# µL# N,OAbis(trimethylsilyl)trifluoroacetamide# with# 1%# trimethylchlorosilane# at#80°C#for#1#h.#Solvent#was#again#blown#off#under#nitrogen#gas#and#the#waxes#resuspended#in#50#µL# of# chloroform# for# GCAFID# analysis.# Waxes# were# analyzed# on# an# Agilent# 7890A# gas#chromatography#system#with#an#HP1#methyl#siloxane#column,#using# the#method#described#by#Haslam#and#Kunst,#2013.#Leaf#wax#samples#were#analyzed#using#splitless#mode,#and#stem#wax#samples# using# a# 2.7:1# split.# The# program# ran# at# 50°C# for# 2# min,# increased# temperature# by#40°C/min#to#200°C#and#held#for#1#min,#increased#by#3°C/min#to#320°C#and#held#for#15#min.#The#injector#was#set#to#300°C,#and#the#FID#was#set#to#300°C.#H2#was#used#as#a#carrier#gas#with#a#flow# rate# of# 2# mL/min.# Peaks# were# identified# based# on# comparison# of# retention# times# with#standards.#The#analysis#was#repeated#with#four#biological#replicates.##2.8.2$ GC1FID$analysis$of$yeast$FAMEs$Cells#were#scraped#from#plates#and#resuspended#in#2#mL#of#1#N#methanolic#hydrochloric#acid# and# heated# at# 80°C# for# 2# hr# to# transmethylate# fatty# acids.# The# fatty# acid# methyl# esters#(FAMEs)#were#then#extracted#in#2#mL#of#hexane#using#1#mL#of#0.9%#aqueous#sodium#chloride#to# improve# phase# separation.# The# hexane# phase# was# dried# under# a# nitrogen# stream# and#resuspended#in#50#µL#hexane.#FAMEs#were#separated#using#an#Agilent#6890#Plus#System#with#a#DBA23#capillary#column,#using#the#method#described#by#Kunst#et#al.#(1992).#The#injection#was#made#with#a#10:1#split.#The#program#ran#at#180°C#for#1#min,#increased#by#4°C/min#to#240°C#and#held#for#7#min.#The#injector#was#set#to#280°C,#and#the#FID#was#set#to#300°C.#H2#was#used#as#a#carrier# gas# with# a# flow# rate# of# 0.7# mL/min.# Peaks# were# identified# based# on# comparison# of#retention#times#with#standards.#The#analysis#was#repeated#with#four#biological#replicates.#)42##2.8.3$ LC/MS$analysis$of$acyl1CoAs$$AcylACoA# extraction# roughly# followed# the# protocol# described# by# Larson# and# Graham#(2001).#Plant#tissue#or#yeast#cell#pellets#were#ground#in#liquid#nitrogen#and#extraction#buffer#(1#mL#isopropanol#/#1#mL#50#mM#KH2PO4#pH#7.2#/25#µL#glacial#acetic#acid#/#40#µL#50#mg/mL#BSA).#Samples#were#delipidated#by#washing#three#times#with#saturated#petroleum#ether,#and#proteins#were#salted#out#by#addition#of#saturated#(NH4)2SO4.#Samples#were#dried#and#suspended#in#90%#H2O,#10%#acetonitrile,#15#mM#ammonium#hydroxide.#AcylACoAs#were#detected#and#quantitated#using# electrospray# ionizationAtandem#mass# spectrometry# (MS/MS)#MS/MS#+#multiple# reaction#monitoring#(MRM)#or#liquid#chromatography#(LC)AMS/MS#+#MRM#in#positive#ion#mode.#The#LCAMS/MS# +# MRM# analysis# (using# an# ABSciex# 4000# QTRAP)# was# performed# as# described# by#Haynes# et# al.# (2008)# using# an# Agilent# 1200# LC# system# with# a# Gemini# C18# column#(Phenomenex),#2#mm#i.d.,#150#mm#length,#5#mm#particle#size.#For#the#purpose#of#identification#and#calibration,#standard#acylACoA#esters#with#acyl#chain# lengths# from#14# to#20#carbons#were#purchased#from#Sigma#as#free#acids#or#lithium#salts.### $43##2.9$ Split1luciferase$assay$for$protein1protein$interactions$$N.) benthamiana# plants# were# grown# as# described# in# section# 2.1.2# and#A.) tumefaciens#cultures# were# prepared# and# infiltrated# as# described# in# section# 2.3.3.# Genes# of# interest# were#fused# to# firefly# luciferase# gene# fragments# in# the# pCAMBIAAbased# luciferase# vector# system#developed# by# Chen# et# al.# (2008).# The# activity# assay# followed# the# floatedAleaf# luciferase#complementation# imaging# (FLuCI)# protocol# described# by# Gehl# et# al.# (2011).# CER2,) CER6,#KCR1,#PAS2,#CER10,#3/HYDROXY/3/METHYLGLUTARYL)COA)REDUCTASE)1)(HMR1),#and#CALCIUM) ATPASE) 2) (ACA2)# coding# sequences# were# amplified# from# Arabidopsis# cDNA#prepared# as# described# in# section# 2.2.2,# using# primers# described# in# Table# 2A9.# All# genes# of#interest# were# inserted# in# both# the# pCAMBIAANLuc# vector,# with# the# NAterminal# fragment# of#luciferase# tagged# to# the# CAterminus# of# the# gene# of# interest,# and# the# pCAMBIAACLuc# vector,#placing# the# CAterminal# fragment# of# luciferase# at# the# NAterminus# of# the# gene# of# interest.# The#construct# that#produced#the#greatest# luminescence#when#coAexpressed#with#a#CER2#construct#was#interpreted#as#the#best#representation#of#interaction.#Interaction#with#CER6#was#only#tested#with#an#NAterminal#fusion#to#the#CAterminal#half#of#luciferase,#as#previous#work#from#our#lab#has#shown#that#NAterminal#fusions#to#condensing#enzymes#are#more#stable#(Dr.#Hugo#Zheng#and#Dr.#Ljerka# Kunst,# unpublished# data).# Positive# clones# were# identified# by# colony# PCR,# restriction#digestion,#and#sequencing.#Constructs#prepared# for# infiltration#of# tobacco#were# transformed#to#electrocompetent#A.)tumefaciens#by#electroporation.#A.) tumefaciens#cells#were#grown# in#5#mL#cultures#overnight# in#LB#medium#with# antibiotic# selection.#Cultures#were# then#diluted#1/20# in# an# infiltration#medium#containing#10.5#g/L#K2HPO4,#4.5#g/L#KH2PO4,#1.0#g/L#(NH4)2SO4,#0.5#g/L#NaCitrateAH2O,#0.5%#glycerol,#1#mM#MgSO4AH2O,#0.2%#glucose,#50#µM#acetosyringone,#and#10#mM#MES,#and#again#grown#overnight.#Cells#were#then#centrifuged#and#resuspended#in#a#solution#of#4.43#g/L#MS,#10#mM#MES,#and#150#µM#acetosyringone#to#OD600#0.6.#Pairs#of#constructs,#either#CER2ANLuc#or#CLucACER2,# with# every# other# elongase# gene# fused# with# the# opposite# fragment# of# luciferase,#were# mixed# to# make# up# 2# mL# of# culture# and# coAinfiltrated# to# N.) benthamiana) leaves.# The#contours#of#the#infiltrated#tissue#were#marked#with#a#permanent#marker.#After#2A4#days,#infiltrated#tissues#were#excised#using#a#paper#hole#punch.#The#leaf#discs#were#set,#with# the#abaxial#surface# facing#downwards,#on# top#of#50#µL#of# the# following#solution#containing# the#substrate#of# the# firefly# luciferase#enzyme:#1#mM#DAluciferin#dissolved# in#DMSO,#10# mM# MgCl2,# and# 10# mM# MES# pH# 5.6,.# Luminescence# was# detected# on# a# plate# reader.#Luminescence#was#captured#from#each#leaf#disc#for#4#s,#every#4#min,#for#90#min.#The#time#point#with#the#highest#luminescence#reading#from#each#disc#was#used#for#analysis.#44##Table$219:$Constructs$and$primers$for$transient$expression$in$tobacco$Construct$(s)$ Primer$Name$ Sequence$(5'13')$pCAMBIAACER2ANluc# CER2ASacI#F# AATTGAGCTCATGGAGGGAAGCCCAGTG#CER2ASalI#R#ns# AATTGTCGACTATAATCATATTAGTCACCTCCTCC#pCAMBIAAClucACER2# CER2AKpnI#F# AATTGGTACCATGGAGGGAAGCCCAGTG#CER2ASalI#R#s# AATTGTCGACTTATATAATCATATTAGTCACCTCCTCC#pCAMBIAAClucACER6# CER6AKpnI#F# AATTGGTACCATGCCTCAGGCACCGATG#CER6ABamHI#R#s#(ClucA)# AATTGGATCCTTAGAGTTTGACAACTTCGGG#pCAMBIAAKCR1ANluc/#pCAMBIAAClucAKCR1#KCR1AKpnI#F# AATTGGTACCATGGAGATCTGCACTTACTTC#KCR1ASalI#R#ns#(ANluc)# AATTGTCGACTTCTTTCTTCATGGAGTCTTTTTG#KCR1ASalI#R#s#(ClucA)# AATTGTCGACTCATTCTTTCTTCATGGAGTC#pCAMBIAAPAS2ANluc/#pCAMBIAAClucAPAS2#PAS2AKpnI#F## AATTGGTACCATGGCGGGCTTTCTCTCCG#PAS2ASalI#R#ns#(ANluc)# AATTGTCGACTTCCCTCTTGGATTTGGAGAGAGC#PAS2ASalI#R#s#(ClucA)# AATTGTCGACTTATTCCCTCTTGGATTTGGAGAGAGC#pCAMBIAACER10ANluc/#pCAMBIAAClucACER10#CER10AKpnI#F## AATTGGTACCATGAAGGTCACCGTCGTCTCC#CER10ASalI#R#ns#(ANluc)# AATTGTCGACAAGGAATGGAGGAAGTATCACCC#CER10ASalI#R#s#(ClucA)# AATTGTCGACCTAAAGGAATGGAGGAAGTATCACCC#pCAMBIAAHMRANluc/#pCAMBIAAClucAHMR#HMRAKpnI#F# AATTGGTACCATGAAGAAAAAGCAAGCTGGTC#HMRASalI#R#ns#(ANluc)# AATTGTCGACTGTTGTTGTTGTTGTCGTTGTC#HMRASalI#R#s#(ClucA)# AATTGTCGACTCATGTTGTTGTTGTTGTCGTTG#pCAMBIAAACA2ANluc/#pCAMBIAAClucAACA2#ACA2AKpnI#F# AATTGGTACCATGGAGAGTTACCTAAACGAG#ACA2ASalI#R#ns#(ANluc)# AATTGTCGACAACGGGAATCGTCTTCAG#ACA2ASalI#R#s#(ClucA)# AATTGTCGACTCAAACGGGAATCGTCTTC##2.10$ Co1immunoprecipitation$$2.10.1$ Microsome$preparation$and$solubilization$Yeast#cells#carrying#epitopeAtagged#CER6#and/or#CER2#constructs#were#grown#in#5#mL#SD#selection#medium#overnight,#which#was#then#used#to#inoculate#100#mL#cultures#grown#for#a#further#24#hr# to#an#OD600≈1.0.#Cells#were#centrifuged#at#4,500#g#at#RT#for#5#min,#washed#with#water,#and#centrifuged#again.#The#cell#pellets#were#suspended#in#5#mL#microsome#preparation#buffer# (below).#0.5#mm#glass#beads#were#added# to# roughly#1#cm#below#surface#of#buffer,#and#vortexed# for# 1#min,# four# times.# Lysates#were# transferred# to# fresh# tubes,# and# the# glass# beads#were#washed#three#times#with#5#mL#aliquots#of#buffer,#and#the#lysate/washes#for#each#genotype#combined.#The#lysates#were#centrifuged#at#8,000#g#for#10#min#at#4°C#to#remove#cell#wall#debris#and#nuclei.#Supernatants#were#transferred#to#polycarbonate#tubes#and#centrifuged#at#164,000#g#for# 1# hr# at# 4°C# to# pellet# microsomes.# Microsomes# were# resuspended# in# a# mixture# of# 66%#microsome# preparation# buffer# and# 33%# glycerol,# and# the# volume# of# each#microsome# sample#45##was#adjusted#to#obtain#a#final#protein#concentration#of#1.1#mg/mL#(as#determined#by#a#Bradford#assay).#Microsomes#were#flash#frozen#in#liquid#nitrogen#and#stored#at#A80°C.#Microsomes#were#solubilized#by#incubating#at#RT#with#sucrose#monolaurate#(SML)#at#a#final#concentration#of#0.1%#(w/v),#for#15A30#min.#Samples#were#then#centrifuged#at#100,000#g#for#1#hr#at#RT#to#pellet#insoluble#lipids#and#proteins.#The#supernatant#from#this#spin#was#used#for#coAimmunoprecipitations#of# the#now#solubilized,#microsomal#proteins.#The#pellet#and#an#aliquot#of#the#supernatant#were#retained#for#dot#blot#or#western#blot#analysis.#$2.10.1.1$ Microsome$preparation$buffer$50#mM#Tris#pH#7.5#1#mM#EGTA#1#mM#"Amercaptoethanol#1#mM#PMSF#1#tablet#yeast#protease#inhibitor#cocktail##2.10.2$ Co1immunoprecipitation$100#µL#of# solubilized,#microsomal# protein#was#diluted#with#900#µL#of# a#0.1%#SML,#50#mM# Tris# pH# 7.5# solution# (TrisASML).# 2# µL# of# antibody# against# the# epitope# tag# of# one# of# the#proteins#of# interest#(here,#antiAcAMyc,#the#tag#fused#to#CER6)#was#added,#and#the#mixture#was#incubated#at#4°C#on#a#tube#rotator#for#2A4#hr.#20#µL#of#protein#A/GAagarose#beads#(Santa#Cruz)#(4# µL# beads# suspended# in# 16# µL# buffer)# were# added# to# each# immunoprecipitation,# and#incubated#overnight#on#the#tube#rotator#at#4°C.#The#following#day,#beads#were#centrifuged,#and#the#supernatant#kept#for#western#blot#analysis#–#this#was#labelled#as#the#free/unbound#fraction.#The#beads#were#washed#4x#with#TrisASML,#and#retained#as#the#bound#fraction#for#western#blot#analysis.##2.10.3$ Western$blotting$10A20# µL# of# each# protein# fraction# was# loaded# in# 10%# acrylamide# gels# with# 1%# SDS.#Proteins# were# separated# at# a# constant# voltage# of# 200# V# for# 60# min,# and# transferred# to#nitrocellulose# membrane# in# semiAdry# blotting# apparatus# in# Bjerrum# SchaferANielsen# buffer#(recipe#below),#at#a#constant#voltage#of#15#V#for#50#min.#The#membranes#were#blocked#with#5%#skim#milk#powder#in#Tris#buffer#saline#with#0.1%#tween#(TBST)#for#either#30#min#at#RT,#or#at#4°C#overnight.##46##Membranes#were#blotted#with#primary#antibodies#for#1#hr#at#RT.#Both#antiAcAMyc#(mouse)#and#antiAHA#(rat)#antibodies#were#diluted#1:10,000#in#TBST#with#5%#milk.#The#membranes#were#washed#3x#with#TBST,#then#incubated#with#secondary#antibodies#diluted#1:50,000#in#TBST#with#5%#milk#for#1hr#at#RT.#The#blots#were#finally#washed#3x#with#TBST,#and#horseradish#peroxidase#activity#was#detected#with#the#GE#Healthcare#ECL#Prime#western#blotting#detection#kit.##2.10.3.1$ Tris$buffer$saline$50#mM#Tris#150#mM#NaCl#pH#7.6##2.10.3.2$ Bjerrum$Schafer1Nielsen$buffer$48#mM#Tris#39#mM#glycine#20%#methanol#pH#9.2#$2.11$ Homology1based$tertiary$structure$modelling$of$CER21LIKEs$Deduced# amino# acid# sequences# for# the# five# CER2ALIKE# proteins# were# input# into# the#Phyre2#server.#Multiple#structural#homologs#were#found#for#all#of#the#CER2ALIKEs,#however,#the#hydroxycinnamoyltransferase# from# Sorghum) bicolor# (SbHCT)# was# the# best# candidate# for# all#CER2ALIKEs# as# it# had# the# highest# sequence# homology# of# all# proteins# with# experimentally#determined#structures.#Therefore,#models#of#CER2ALIKEs#generated#using#SbHCT#were#used#for#analysis.#Graphics#were#generated,#annotated,#and#analyzed#with#UCFS#Chimera.#Chimera#was# developed# by# the# Resource# for# Biocomputing,# Visualization,# and# Information# at# the#University#of#California,#San#Francisco,#supported#by#NIGMS#P41AGM103311K#Pettersen#et#al.,#2004.##47##Chapter$3:$ECERIFERUM2$is$a$component$of$the$fatty$acid$elongation$machinery$required$for$extension$to$exceptional$lengths$#3.1$ $Introduction$Eceriferum2# was# among# the# most# severe# waxAdeficient# mutants# identified# in# a# visual#screen#for#glossy#stem#wax#phenotypes#in#ethylmethane#sulfonate#(EMS)Ainduced#and#irradiated#mutant# populations# of# Arabidopsis# (Koornneef# et# al.,# 1989).# Several# cer2#mutants# have# been#isolated# in# subsequent# screens# for# wax# deficiencyK# these# are# summarized# in# Table# 3A1.#Characterization# of# the# cer2/4# allele# by#McNevin# et# al.# (1993)# revealed# that# the# cer2# mutant#specifically#lacks#stem#cuticular#waxes#longer#than#28#carbons,#and#accumulates#more#26A#and#28Acarbon#waxes#than#wild#type.#The#authors#suggested#that#the#CER2#gene#could#regulate#the#activity#of#the#elongase#complex,#but#as#the#cer2#mutation#was#mapped#to#a#gene#with#homology#to#BAHD#acyltransferases# (Xia#et#al.,# 1996),# a#mechanism#by#which# this# could#occur#was#not#obvious.#Basic#characterization#of# the#CER2#protein#was#necessary#to#make#predictions#about#its#activity.#Given#that#the#four#characterized#enzymes#of#the#FAE#complex#are#localized#to#the#ER#(Zheng#et#al.,#2005K#Bach#et#al.,#2008K#Joubès#et#al.,#2008K#Beaudoin#et#al.,#2009),# I#predicted#that# the# CER2# gene# product# would# localize# to# the# ER# as# well.# However,# all# characterized#BAHDs#are#soluble,#cytosolic#proteins#(D’Auria,#2006K#Panikashvili#et#al.,#2009K#Rautengarten#et#al.,# 2012),# with# the# exception# of# one# of# three# splice# variants# of# At1g65450,# which# contains# a#nuclear#localization#signal#and#is#localized#to#the#nucleus#(Leshem#et#al.,#2012).#Localization#and#topology#prediction#algorithms#reveal#that#CER2#has#no#transmembrane#domains#and#no#transit#peptide# (AramemnonK#Schwacke#et#al.,#2003).# In# light#of# this# information,#one#might#anticipate#that#CER2#could# localize# to# the#cytosol.# Inconsistent#with#both#of# these#predictions,# the#CER2#protein# was# previously# reported# to# localize# to# the# nucleus,# based# on# cell# fractionation# and#immunoblotting#experiments#(Xia#et#al.,#1997).#Velasco#et#al.#(2002)#used#the#same#technique#to#localize#the#CER2#homolog#of#Zea)mays,#GLOSSY2,#but#found#the#protein#enriched#in#a#cellular#fraction#containing#ER#and#mitochondria,#as#well#as#in#a#soluble,#cytosolic#fraction.#Hence,#there#was#an#obvious#need#to#determine#the#true#localization#of#CER2#in)planta.#Previous#work#has#established#that#FAE#complexes#carry#out#VLCFA#elongation#for#wax#biosynthesis.# Mutant# analysis# in# Arabidopsis# has# demonstrated# that# the# condensing# enzyme#CER6#is#required#to#elongate#VLCFA#precursors#of#cuticular#waxes#greater#than#24#carbons#in#length# (Millar# et# al.,# 1999).# When# expressed# in# yeast,# CER6# elongates# VLCFAs# up# to# 28#48##carbons.# However,# waxes# derived# from# 30A# and# 32Acarbon# VLCFAs# are# by# far# the# most#abundant#constituents#of#Arabidopsis#stem#and#leaf#wax.#No#other#known#condensing#enzymes#from# Arabidopsis# efficiently# elongate# VLCFAs# in# yeast# beyond# 28# carbons.# The# capacity# of#yeast#cells# to#support# the#production#of#VLCFAs# longer# than#28#carbons# is#not# in#question,#as#the# production# of# 30Acarbon# VLCFAs# in# yeast# by# engineered# yeast# Elops# was# previously#reported#(Denic#and#Weissman,#2007).#To# confirm#previous#work#on# the#cer2#mutant# phenotype,# a# new#TADNA#allele,#cer2/5,#was#obtained.#CER2)expression#patterns#were#examined#using#qPCR,#and#a#CER2/GFP#fusion#driven# by# the# native#CER2# promoter.# CER2AGFP# localization#was# determined# using# a# similar#construct# driven# by# the# 35S# promoter.# Finally,# because# yeast# has# proven# to# be# a# powerful#system#for#characterizing#components#of# the#plant# fatty#acid#elongation#machinery,#CER2#was#expressed#in#yeast#cells#to#study#its#function.#$$Table$311:$Summary$of$characterized$cer2$alleles$of$Arabidopsis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esults$#3.2.1$ The$cer2$mutant$is$deficient$in$waxes$longer$than$28$carbons$To#further# investigate#CER2# function,#a#TADNA#insertional#mutant# in#ColA0#ecotype#was#obtained# from# the#Arabidopsis#Biological#Resource#Center# (ABRC),#SALK_084443# (Alonso#et#al.,#2003),#hereafter#referred#to#as#cer2/5.#Homozygous#mutants#were#identified#by#PCRAbased#genotyping,# and# direct# sequencing# of# genomic#DNA# located# a#TADNA# insertion# in# the# second#exon# of# the#CER2# gene# (Figure# 3A1A).#Stems# of#cer2/5# individuals# had# a# glossy# appearance#characteristic# of# eceriferum# mutants# with# decreased# wax# load# (Figure# 3A1B).# This# phenotype#was#further#examined#using#SEMK#stems#of#wildAtype#ColA0#plants#were#densely#coated#with#rodAshaped,#tubular,#and#plateletAshaped#wax#crystals,#whereas#cer2/5#stems#appeared#completely#devoid#of# such#structures# (Figure#3A1C).#Stem#waxes#were#analyzed#by#GCAFID# to#determine#how#wax#load#and#composition#of#the#cer2/5#mutant#compare#with#that#of#the#wild#type#and#other#previously# described# cer2# alleles.# The# total# stem#wax# load# of# cer2/5# was# approximately# 30%#that# of#wild# type# (Figure#3A1D).#All#wax# components# longer# than#28# carbons#were# reduced# to#trace#amounts#in#the#mutant,#and#relative#increases#were#observed#for#several#wax#components#shorter# than#28#carbons#(Figure#3A1E).#These#results#suggest#a#block# in#acyl#chain#elongation#past#28#carbons#in#cer2/5.#The#load#and#composition#of#cer2/5#mutant#rosette#leaf#wax#was#also#examinedK# no# change# compared# with# the# wild# type# was# detected# (data# not# shown).# These#results#agree#with#the#published#data#for#other#cer2#alleles.##Male# sterility# dependent# on# environmental# humidity# is# a# phenotype# associated# with#pollen# coat# deficiencies,# and# has# been# observed# in# several# Arabidopsis# mutants# with#deficiencies#in#cuticular#wax#metabolism#(Koornneef#et#al.,#1989K#Preuss#et#al.,#1993)#including#one#cer2#allele#(Preuss#et#al.,#1993K#Eisner#et#al.,#1998).#The#most#abundant#acyl#lipids#in#pollen#coat#are#29Acarbon#alkane#and#ketone,#and#31Acarbon#alkene#(Jessen#et#al.,#2011),#and#so#it#is#not#surprising#that#the#biosynthesis#of#cuticular#waxes#and#pollen#coat#could#require#the#same#or#similar# enzymes.# No# reduction# in# the# seed# set# of# the# cer2/5# mutant# was# observed.# This#discrepancy# may# be# due# to# subtle# differences# in# growth# conditions,# or# the# different# alleles#selected# for# study.# The# subject# of# VLCFA# elongation# and# pollen# coat# function# is# discussed#further#in#Chapter#4.###50##### ########## # ###Figure$311:$Characterization$of$the$cer2=5$mutant$phenotype$A,#TADNA#insertion#site#in#the#second#exon#of#CER2.#B,#stems#of#wildAtype#ColA0#(left)#and#cer2/5#(right)#plants.#C,#SEM#of#wildAtype#ColA0#(left)#and#cer2/5#(right)#inflorescence#stems.#Scale#bars#=#20#µm.#WildAtype# ColA0# and# cer2/5# total# stem# wax# loads# are# shown# in# D,# and# wax# compositions# in# E.# Both# are#measured#as#the#average#of#four#biological#replicates,#with#error#bars#representing#SD.#Ic,#free#fatty#acidK#ol,# primary# alcoholK# al,# aldehydeK# an,# alkaneK# on,# ketoneK# sol,# secondary# alcohol.# Reproduced# with#permission# from# Haslam# et# al.,# 2012K# Copyright# American# Society# of# Plant# Biologists# ©# 2012#(www.plantphysiology.org)###200bpATG( TAASALK_084443CACATTTTTAATTTTA5’ 3’A051015202530Col)0 cer2)5WaxLoadµg/cm2024681012Col)09WTcer2%5%D E51###3.2.2$ The$CER2$gene$is$expressed$in$epidermal$cells$of$aerial$organs$OrganAspecific# expression# patterns# of# CER2# were# investigated# by# qPCR.# CER2#transcript# was# detected# in# all# tissues# except# roots# (Figure# 3A2A),# suggesting# that# CER2#may#contribute# to#wax#biosynthesis# in#organs#other# than#stem.#To#determine# tissueA#and#cell# typeAspecific#expression#patterns#of#CER2,#GFP#fluorescence#was#examined#in#wildAtype#and#cer2/5#plants# transformed# with# a# CER2/GFP# transgene# driven# by# the# native# CER2# promoter.# This#construct#complemented#the#cer2/5#waxAdeficient#phenotype#(data#not#shown).#In#stems,#siliques,#rosette# leaves,#and#cauline#leaves,#GFP#fluorescence#was#unique#to#epidermal# cells# (Figure# 3A2B,D,E,F).# This# result# supports# the# notion# that# CER2# function# is#specific# to#cuticular# lipid#synthesis,#which#occurs#exclusively# in# the#epidermis,#and# is#similar# to#previous#CER2#expression#patterns#reported#by#Xia#et#al.#(1996,#1997).#GFP#fluorescence#was#very#strong#in#epidermal#cells#near#the#stem#apex#(Figure#3A2B)#and#there#was#a#stark#reduction#in# fluorescence# at# a# distance# of# only# 3# cm# from# the# apex# (Figure# 3A2C).# This# observation#corroborates#previous#findings#that#wax#biosynthesis#occurs#predominantly#at#the#shoot#tip#(Suh#et#al.,#2005).##3.2.3$ The$CER2$protein$is$localized$to$the$ER$membrane$To# determine# the# localization# of# the#CER2# protein,# the# genomic#CER2# sequence#was#fused# to#GFP#and#expressed#downstream#of# the#35S#promoter# in#cer2/5#and#wildAtype#plants.#Multiple# cer2/5# transgenic# individuals# were# recovered# that# displayed# complementation# of# the#cer2/5# waxAdeficient# phenotype# (i.e.# they# had# glaucous# stems,# and# restored# wax# load# and#composition# as# determined# by# GCAFID# analysis).# In# both# genetic# backgrounds,# GFP#fluorescence# strongly# labelled# a# reticulate# structure# in# young,# waxAproducing# stem# epidermal#cells# (Figure# 3A3A).# When# plant# stems# were# stained# with# the# ER# dye# hexyl# rhodamine# B#(Boevink# et# al.,# 1996),# the# stain# and# GFP# fluorescence# showed# clear# coAlocalization# (Figure#3A3B,C).#The#GFP#signal#was#also#observed#in#the#nuclei#of#some#cells,#in#addition#to#the#strong#and#consistent# signal# from# the#ER.#ER# localization#of#CER2#supports#a# role# for#CER2# in#wax#biosynthesis.#52###$Figure$312:$CER2$expression$determined$by$qPCR$and$CER2pro::CER21GFP$fluorescence$A,# qPCR# analysis# of#CER2# expression# patterns# in# the# wild# type# normalized# to#GAPC1,# shown# as# the#average#of# four# technical# replicatesK#error#bars# represent#SD.#CER2pro::CER2/GFP#expression# in#stem#sections#1#cm#from#the#apex#(B),#stem#sections#3#cm#from#the#apex#(C),#developing#siliques#(D),#young#rosette# leaf# (E),# and# young# cauline# leaf# (F).# Scale# bars# =# 50# µm.# Reproduced# with# permission# from#Haslam#et#al.,#2012K#Copyright#American#Society#of#Plant#Biologists#©#2012#(www.plantphysiology.org)##########Figure$313:$CER2$localization$to$the$ER$A,# GFP# fluorescence# in# the# stem# epidermis# of# ColA0# transformed# with# 35S::CER2/GFP.# B,# Hexyl#rhodamine# B# staining.# C,#Merge# of# images# A# and# B,# showing# coAlocalization# of# CER2AGFP# and# hexyl#rhodamine# B.# Scale# bars# =# 10# µm.# Reproduced# with# permission# from# Haslam# et# al.,# 2012K# Copyright#American#Society#of#Plant#Biologists#©#2012#(www.plantphysiology.org)#0100200300400Expression/level/relative/to/rootA53##3.2.4$ CER2$modifies$the$output$of$elongation$when$co1expressed$with$CER6$The#wax#phenotype#of#the#cer2/5#mutant#demonstrates#a#role#for#CER2#in#a#specific#step#of#VLCFA#elongation#required#for#wax#synthesis.#However,#the#biochemical#function#of#CER2#is#unknown.#CER2#could#serve#as#a#condensing#enzyme#in#the#elongation#of#VLCFAs#longer#than#28# carbons,# or# facilitate# acyl# chain# extension# past# 28# carbons# catalyzed# by# a# condensing#enzyme#such#as#CER6.#To#establish#whether#CER2# is#sufficient# for#elongation# to#30#carbons,#CER2#was#expressed#alone#in#yeast,#but#these#cells#showed#no#substantial#change#in#fatty#acid#profile# relative# to# the# wild# type# (Figure# 3A4).# Changes# in# the# yield# of# 26Acarbon# FAME# were#observed# in# some# experimental# replicates.# However,# subtle,# quantitative# changes# in# the# fatty#acid#profile#were#difficult# to# interpret#given#that#the#VLCFA#composition#of#wildAtype#yeast#cells#can#vary#markedly#with#growth#phase,#conditions,#and#growth#medium.##The#phenotype#of# the#cer2/5)mutant#suggests# that#CER2#might# require#28Acarbon#fatty#acid#as#a#substrate# for#elongation,#and#so#CER2#was#expressed# in#yeast#cells# in#combination#with#LfKCS45,#a#condensing#enzyme#from#the#crucifer#Lesquerella)fendleri#(Moon#et#al.,#2004).#LfKCS45# efficiently# elongates# 26Acarbon# fatty# acid# to# 28# carbons,# the# proposed# substrate# for#CER2,#and#also#produces#a#small#amount#of#30Acarbon# fatty#acid.#28Acarbon#FAME#and# trace#amounts# of# 30Acarbon# FAME# were# detected# in# yeast# cells# coAtransformed# with# CER2# and#LfKCS45,#equivalent#to#what#was#produced#when#LfKCS45#was#expressed#alone.#These#results#indicate#that#CER2#is#not#sufficient#for#28Acarbon#VLCFA#elongation.##The# dominant# role# of# the# CER6# condensing# enzyme# in# cuticular# wax# elongation#suggested#that#concerted#activity#of#CER2#and#CER6#might#be#required#for#the#production#of#30Acarbon# VLCFAs.# Therefore,# CER2# and# CER6# were# coAexpressed# in# yeast.# As# previously#reported,# cells# expressing# CER6# alone# accumulated# a# much# greater# amount# of# 28Acarbon#FAME#relative#to#wild#type,#but#no#30Acarbon#FAME.#Remarkably,#in#yeast#cells#expressing#both#CER6#and#CER2,#an#additional#peak#was#detected,#which#was# identified#as#30Acarbon#FAME.#Therefore,# the#combined#activity#of#CER6#and#CER2#is#necessary#and#sufficient# for#acyl#chain#elongation#to#30#carbons.#54###$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$Figure$314:$Co1expression$of$CER2$with$plant$condensing$enzymes$in$S.0cerevisiae$Yeast#total#lipid#profiles#measured#as#FAMEs#extracted#from#W3031A,#W3031A#transformed#with#CER2,#LfKCS45,#LfKCS45+CER2,#CER6,#and#CER6+CER2.#Modified#from#Haslam#et#al.,#2012,#and#reproduced#with# permissionK# Copyright# American# Society# of# Plant# Biologists# ©# 2012# (www.plantphysiology.org).LfKCS45(& CER2LfKCS45CER6CER6(& CER2CER2w3031a55##3.3$ Discussion$#3.3.1$ CER2$localizes$to$the$ER$in$young$epidermal$cells$that$are$active$in$cuticular$wax$biosynthesis$$My# characterization# of# the# cer2/5) mutant# was# consistent# with# previous# reports# from#McNevin# et# al.# (1993)# and# Jenks# et# al.# (1995),# and# suggests# a# function# for# CER2# in# the#elongation#of#cuticular#wax#precursors#from#28A#to#30Acarbons#in#stems.#In#addition#to#the#stem#wax#deficiency#of#cer2) that#was#examined#by#GCAFID,# the#glossy#phenotype#of#cer2#was#also#obvious#on#siliques.#I#analyzed#leaf#wax#of#cer2/5#by#GCAFID#because#wax#deficiency#on#leaves#cannot#be#detected#by#eye,#but#there#was#no#difference#in#composition#relative#to#wild#type,#as#reported#by#Jenks#et#al.#(1995).#qPCR#analysis#of#CER2#expression#revealed#CER2#transcript#in#all#organs#except# root,#and#CER2/GFP#expression#was#observed# in#epidermal#cells#of# stems,#leaves,# and# siliques.# While# the# phenotype# of# cer2# was# only# obvious# on# stems# and# siliques,#CER2)could#also#contribute# to#wax#precursor#elongation# in# leaves#and# in# floral#organs,#but# its#function#may#be#masked#by#genetic#redundancy.#The#enzymes#of#the#FAE#complex#that#generate#VLCFA#wax#precursors,#as#well#as#all#of#the#characterized#wax#biosynthetic#enzymes,#reside#on#ER#membranes#(Samuels#et#al.,#2008).#I#anticipated#that#likewise,#if#CER2#were#involved#in#fatty#acid#elongation,#it#would#localize#to#the#ER.#Confocal#microscopy#performed#on# transgenic# lines#expressing#CER2AGFP#driven#by# the#35S# promoter# confirmed# CER2# localization# to# the# ER# in# epidermal# cells# of# the# inflorescence#stem,#which#actively#synthesize#cuticular# lipids.# In# these#cells,#strong#CER2AGFP#fluorescence#was#clear#throughout#the#ER:#the#reticulate#cortical#ER,#transAvacuolar#strands,#and#the#nuclear#envelope.# Such# localization# is# consistent# with# a# metabolic# function# for# CER2# in# VLCFA#biosynthesis.#Whereas# ER# localization# of# CER2# is# compatible# with# a# biochemical# function# in# wax#synthesis,# it# presents# yet# another# peculiarity# of# CER2# in# the# context# of# homology# to# BAHD#acyltransferases,#as#all#BAHDs#localized#to#date#are#soluble,#cytosolic#enzymes#(D’Auria,#2006K#Panikashvili#et#al.,#2009K#Rautengarten#et#al.,#2012).#Furthermore,#localization#of#maize#GL2#to#both#the#ER#and#the#cytoplasm#suggests#that#CER2#may#reside# in#both#compartments#and#be#both#soluble#and#membrane#bound.#At#present,#it#is#not#clear#how#CER2#associates#with#the#ER.#Further#work#is#needed#to#determine#if#ER#localization#of#CER2#requires#interaction#with#proteins#in#the#FAE#complex#such#as#CER6,#or#other#wax#biosynthetic#enzymes.##56##3.3.2$ CER2$and$CER6$are$necessary$and$sufficient$for$301carbon$VLCFA$synthesis$Plant#KCS#enzymes#expressed# in#yeast#can#work#with#other#components#of# the#native#yeast# FAE# complex# to# synthesize# their# specific# products# (Millar# and#Kunst,# 1997).# Therefore,#yeast#has#been#an# invaluable#system#for# the#functional#characterization#of#KCS#enzymes#from#Arabidopsis.#This#approach#has#revealed#an#inconsistency#in#that#CER6,#the#key#KCS#enzyme#associated#with#VLCFA#elongation# for# cuticular#wax# synthesis,# is# unable# to# elongate#VLCFAs#longer# than#28#carbons.#Arabidopsis#CER60# (Trenkamp#et#al.,#2004)#and#L.) fendleri#LfKCS45#(Moon# et# al.,# 2004)# are# the# only# plant# KCSs# with# demonstrated# 28A# to# 30Acarbon# elongation#activity,#and#both#produce#very#small#amounts#of#30Acarbon#fatty#acid#when#expressed#in#yeast.#When#I#expressed#LfKCS45#alone# in#yeast#cells,#28A#and#a#small#amount#of#30Acarbon#fatty#acid#were#produced,#as#previously#reported#by#Moon#et#al.#(2004).#When#CER2#alone#was#introduced# to# yeast# cells,# no# effect# on# the# fatty# acid# profile#was# obvious# from# the#GC# traces.#However,# analysis# of# the# FAMEs# composition# did# reveal# a# decrease# in# 26Acarbon# FAME# in#CER2/expressing#cells#compared#to#the#wild#type,#in#some#experimental#replicates.#The#reason#for#this#is#unclear,#and#is#particularly#difficult#to#interpret#given#that#the#amount#of#26Acarbon#fatty#acid# accumulating# in#wildAtype# yeast# cells# varies# substantially# between#assays.#The# fatty# acid#profile# of# cells# expressing# CER2# alongside# LfKCS45# was# no# different# from# that# of# cells#expressing#only#LfKCS45,#demonstrating#that#CER2#is#insufficient#for#VLCFA#elongation#past#28#carbons.# Expression# of# CER6# alone# in# yeast# resulted# in# 28Acarbon# FAME# accumulation,# as#expected.#In#contrast,#coAexpression#of#CER2#and#CER6#in#yeast#cells#resulted#in#a#remarkably#high#yield#of#30Acarbon#FAME.#These#findings#demonstrate#that#CER2#and#CER6#are#necessary#and# sufficient# for# 30Acarbon# VLCFA# synthesis.# My# results# also# indicate# that# the# CER6#condensing# enzyme# has# a# property# absent# from# LfKCS45# that# is# necessary# for# CER2Aassociated#elongation.#What#this#property#is#and#how#unique#it#is#to#CER6#remains#unknown.#It#also#remains#to#be#determined#whether#CER2#has#a#distinct#activity#that#contributes#to#VLCFA#elongation,# if# it#plays#a#structural#or#stabilizing#role#necessary#for#the#elongation#of#VLCFAs#by#CER6,#or#has#a#role#in#allosteric#regulation#of#condensing#enzymes.#VLCFAs#longer#than#28#carbons#are#not#common#in#nature,#and#I#am#not#aware#of#any#reports#of#KCSs#that#can#produce#more#than#trace#amounts#of#30Acarbon#fatty#acids#or#longer.#It#would# seem# that# the# canonical# FAE# complexAcatalyzed# mechanism# of# elongation# holds#exceptions#after#28Acarbon#fatty#acid#synthesis.#Previous#phylogenetic#analyses#have#revealed#that#CER2Atype#proteins#are#pervasive#among#flowering#plants#(Yu#et#al.,#2009K#Tuominen#et#al.,#2011).# This# suggests# that# the# CER2# family# has# a# necessary,# conserved# role# in# VLCFA#elongation,#and#that#CER2#activity#is#important#for#cuticular#wax#biosynthesis#in#flowering#plants.#57##Chapter$4:$ECERIFERUM21LIKE$ proteins$ have$ unique$ biochemical$and$physiological$functions$in$very1long1chain$fatty$acid$elongation$$4.1$ Introduction$$In# the#previous#chapter,# the#central# role#of#CER2) in# the#elongation#of# fatty#acids# to#30#carbons# was# demonstrated,# and# the# importance# of# these# VLCFAs# for# the# synthesis# of# stem#cuticular#wax#was#described.#However,#cuticular#waxes#derived#from#fatty#acids#30#carbons#and#longer#coat#all#of#the#primary#aerial#surfaces#of#Arabidopsis.#Similar#lipids#are#also#found#in#the#pollen# coat,# where# they# are# required# for# pollen# hydration# and# germination# on# dry# stigma#(Elleman#et# al.,# 1992K#Preuss#et# al.,# 1993).#Accordingly,# public#microarray#data# (Winter# et# al.,#2007),# qPCR# data,# and# CER2AGFP# expression# patterns# all# indicate# that# the# CER2) gene# is#expressed#in#the#epidermis#throughout#the#shoot.)Yet,#characterization#of#the#cer2/4#mutant#by#Jenks#et#al.#(1995)#showed#that#leaf#waxes#of#cer2/4#do#not#differ#significantly#from#the#wild#type,#and#the#cer2)allele#I#studied#was#fertile.#This#information#suggests#that#other#Arabidopsis#genes#might#contribute#to#the#synthesis#of#VLCFA#precursors#of#leaf#cuticular#wax#and#pollen#coat.#CER2#orthologs#are# found# in#diverse# flowering#plant# lineages,#and#many#species#have#multiple# CER2# paralogs# (Yu# et# al.,# 2009K# Tuominen# et# al.,# 2011).# Previous# analyses# of# the#BAHD#acyltransferase# family#of#Arabidopsis#have# identified#several#genes#with#high#sequence#identity#to#CER2.#Tuominen#and#coworkers#(2011)#reported#that#there#are#two#CER2#homologs#in# Arabidopsis,# At3g23840# and# At4g29250.# Yu# and# coworkers# (2009)# also# reported# the#existence#of#two#CER2#homologs#in#Arabidopsis,#but#identified#At3g23840#and#At4g13840.#The#inconsistency# between# these# publications# was# not# immediately# obvious# to# me,# and# I# began#characterizing#the#“CER2ALIKE”#family#of#Arabidopsis#using#the#gene#identities#specified#by#Yu#et# al.# (2009).# After# noticing# the# discrepancy# between# these# publications,# I# compared# CER2#homologs#that#I#could#find#through#NCBI#and#the#BAHDs#in#the#Yu#et#al.#(2009)#and#Tuominen#et#al.#(2011)#phylogenies,#and#found#that#there#are#in#total#five#CER2ALIKE#genes#in#Arabidopsis.#The# genes# and# their# assigned# names# are# specified# in# Table# 4A1.# A# phylogeny# for# the#Arabidopsis#BAHD#acyltransferase# family# that# includes#all# of# these#proteins#was#published#as#supplementary#material#by#Molina#et#al.,#2009,#and#is#reproduced#in#Figure#4A1.#58###Table$411:$Identities$of$CER2$homologs$from$Arabidopsis$########*HXXXD#is#the#conserved#acyltransferase#catalytic#motif.#H#=#histidine,#D#=#aspartic#acid,#X#=#any#amino#acid.###It# is# not# clear# why# CER2ALIKE3# and# CER2ALIKE4# were# omitted# from# the# phylogeny#published#by#Yu#et#al.#(2009).#CER2ALIKE1#and#CER2ALIKE4#were#excluded#from#the#phylogeny#published# by# Tuominen# et# al.# (2011)# because# both# of# these# proteins# lack# an# HXXXD# motif#characteristic# of# BAHD# acyltransferases.# The# authors# reasoned# that# this# divergent# feature#indicates#that#CER2ALIKE1#and#CER2ALIKE4#cannot#be#true#BAHDs.#My#work#has#since#shown#that# the# CER2ALIKEs# have# biochemical# function# distinct# from# other# characterized# BAHDs#(Chapter#5).#In#light#of#this,#the#absence#of#the#HXXXD#motif#in#two#CER2ALIKEs#reflects#novelty#in# the# CER2ALIKE# clade,# and# does# not# preclude# CER2ALIKE1# and# CER2ALIKE4# from# having#common#function#with#CER2,#CER2ALIKE2,#and#CER2ALIKE3.##It# is#necessary# to#characterize# the#metabolic#and#physiological# functions#of#CER2ALIKE#proteins.#Beyond#the#value#of#knowing#the#specific#roles#of#each#homolog,#such#an#investigation#has#the#potential#to#elucidate#the#biochemical#nature#of#CER2ALIKE#activity.#The#present#chapter#will# address# the# following# questions:# (1)# Can# CER2ALIKE# proteins# function# with# condensing#enzymes#other#than#CER6,#and#if#so,#is#the#effect#of#the#CER2ALIKE#constant?#(2)#Do#different#CER2ALIKE#proteins#have#different#effects#on#the#substrate#specificity#of# the#same#condensing#enzyme?#(3)#What#is#the#physiological#relevance#of#the#subtle#changes#in#acyl#lipid#chain#length#that#CER2ALIKE#proteins#induce?#$ $ Identified$by$ Gene$locus$ Name$ Yu$et$al.$ Tuominen$et$al.$ HXXXD*$At4g24510# CER2) Y# Y# HILDG At4g13840# CER2/LIKE1) Y# N# NIIDG At3g23840# CER2/LIKE2) Y# Y# HIMGD At4g29250# CER2/LIKE3) N# Y# HLLAD At5g02890# CER2/LIKE4) N# N# ILIAD 59##Figure$411:$Phylogeny$of$the$Arabidopsis$BAHD$acyltransferase$family$Numbers#on#branches#represent#bootstrap#values#as#percentages.#Roman#numerals#correspond#to#BAHD#clades# designated# by# D’Auria,# 2006.# Several# BAHDs# are# labelled:# AACT1:# Anthocyanin# 5Aaromatic#acyltransferase1K#AT5MAT:#OAmalonyltransferaseK#CHAT:#acetylACoA:(Z)A3AhexenA1Aol#acetyltransferaseK#HCT:# hydroxycinnamoylACoA:shikimate/quinate# hydroxycinnamoyltransferase.# Reproduced# with#permission# from# Molina# et# al.,# 2009K# Copyright# American# Society# of# Plant# Biologists# ©# 2009#(www.plantphysiology.org)$60##4.2$ Results$#4.2.1$ CER21LIKEs$have$different$expression$patterns$$Based#on#the#phylogeny#of#Yu#et#al.#(2009),#my#work#initially#focused#on#only#three#of#the#five# CER2/LIKEs:# CER2,# CER2/LIKE1,# and# CER2/LIKE2.# Characterization# of# CER2) was#presented# in# the# previous# chapter.# To# guide# phenotypic# analyses# of#cer2/like1)and#cer2/like2)mutants,#gene#expression#patterns#were#examined.#OrganAspecific#expression#patterns#of#both#genes#were# investigated#by#qPCR,#as#described# in#Section#2.2.2.#CER2/LIKE1# transcript#was#detected# in# seedlings,# rosette# and# cauline# leaves,# and# siliques# (Figure# 4A2A).# CER2/LIKE2#transcript# was# detected# in# seedlings,# rosette# and# cauline# leaves,# and# flowers# (Figure# 4A3A).#Expression# of# both# genes# in# leaves# is# consistent# with# the# notion# that# they#may# contribute# to#cuticular# wax# biosynthesis.# High# expression# levels# of#CER2/LIKE2# in# flowers# suggest# that# it#could#be#required#for#the#production#of#the#unique#cuticular#lipids#on#floral#organs,#the#synthesis#of#lipidic#components#of#the#pollen#coat,#or#sporopollenin.#A#CER2/LIKE1/GFP#fusion#driven#by#the#native#CER2/LIKE1#promoter#was#constructed#to#examine#cellAtype#specific#expression#patterns.#This#construct#complemented# the#cer2/like1#leaf# wax# phenotype# (complementation# not# shown).# Unfortunately,# GFP# fluorescence# was# too#weak# to# discern# cellAtype# specific# fluorescence#patterns# in# handAsectioned#material.#However,#this# construct# was# useful# as# localization# of#CER2/LIKE1/GFP# to# the# ER# in# intact# rosette# leaf#pavement#cells#was#easily#observed#(Figure#4A2B).##Characterization#of#CER2/LIKE1)and#CER2/LIKE2)was#carried#out# in#collaboration#with#Dr.# Jérôme#Joubès’# lab#at# the#University#of#Bordeaux,#France.#Dr.# Joubès’# lab#used#qPCR# to#examine#CER2/LIKE2#expression#throughout#floral#development#following#the#staging#described#by#Smyth#et#al.#(1990).#Temporal#patterns#of#CER2/LIKE2#expression#were#compared#to#those#of#FATTY)ACYL)REDUCTASE2#(FAR2),#CER1,#and#CER2#(Figure#4A3B).#FAR2#contributes#to#the#synthesis#of# fatty#alcohols# in#sporopollenin# (Aarts#et#al.,#1997).#CER1#encodes#an#alkaneAforming#enzyme#required#for#cuticular#wax#biosynthesis#(Bernard#et#al.,#2012).#CER2#and#CER1#are#both#expressed# in#different# floral#organs# throughout#development#and#contribute# to#cuticle#formation#(Bourdenx#et#al.,#2011K#Pascal#et#al.,#2013).#Further,#specific#mutant#alleles#of#both#of#these# CER# genes# cause# conditional# male# sterility# at# low# humidity# (Koornneef# et# al.,# 1989K#Preuss#et#al.,#1993),#suggesting#that#they#contribute#to#the#synthesis#of#pollen#coat#lipids.#FAR2#transcript# could# be# detected# only# until# stage# eight,# which# fits# with# a# role# for# FAR2# in#sporopollenin#synthesis.#CER1#and#CER2#were#expressed#throughout#all#of#the#developmental#stages#profiled,#consistent#with# functions#of#both#genes# in#both# floral# cuticular#wax#and#pollen#61##coat# lipid#biosynthesis.#CER2ALIKE2#expression#was#highest# in# flower#buds#collected#at#stage#eight# or# younger,# decreased# to# approximately# 25%# of# that# level# at# stage# nine,# and# was# not#detected#later#in#development.##Dr.# Joubès’# lab# further# examined# patterns# of#CER2/LIKE2# expression# using# a#CER2/LIKE2pro::GUS#construct#stably#expressed#in#wildAtype#ColA0#plants#(Figure#4A3C).#When#whole#inflorescences#were#stained,#GUS#activity# could#be#detected#only# in# young#anthers# (Figure#4A3C[a]).#Flowers#were# fixed,#embedded,#and#cut# in#semiAthin#sections#at#developmental#stages#eight,#nine,#and#eleven#to#observe#cell#typeAspecific#expression#patterns#(Figure#4A3C,#[b–d]).#In#stage# eight# flowers,# GUS# activity# was# present# in# the# tapetum# and# on#microspores# within# the#locule.#I#was#skeptical#of#microspore#staining,#as#several#promoters#that#drive#gene#expression#uniquely# in# the# tapetum# have# resulted# in# blue#microspores#when# fused# to#GUS# (Yokoi# et# al.,#1997K# Kapoor# et# al.,# 2002K# Choi# et# al.,# 2014).# It# is# possible# that# secretion# of# either# the#GUS#protein# or# its#ClBrAindigo# product,# or# the# release# of# either# upon# degeneration# of# tapetal# cells,#may# have# caused# the# observed# staining# of#microspores.# In# stage# nine# flowers,#GUS# staining#was#exclusive#to#tapetal#cells,#and#no#staining#was#observed#in#stage#eleven#flowers.#Together,#the# spatial# and# temporal# expression#patterns#of#CER2/LIKE2# suggest# that# its#gene#product# is#required#for#pollen#coat#or#sporopollenin#biosynthesis.#While#CER2/LIKE3#and#CER2/LIKE4#were#not#extensively#characterized#in#the#present#work,# their# expression# profiles# can# be# generally# described# based# on#microarray# data# publicly#available#in#the#Arabidopsis#eFP#browser#(Winter#et#al.,#2007).#CER2/LIKE3#expression#is#only#detected#in#young#flower#buds,#between#developmental#stages#9A11.#CER2/LIKE4#expression#is#broader,# with# transcript# detected# in# leaves,# stems,# floral# organs,# roots,# and# siliques.# Notably,#expression# is#upAregulated# in#young#stem#epidermal#cells,# in# the#developing#seed#coat,#and# in#the#L1#layer#of#the#shoot#meristem.###62##$###Figure$412:$Expression$pattern$and$localization$of$CER2=LIKE10(A)#qPCR#analysis#of#CER2/LIKE1#expression#patterns#in#the#wild#type#normalized#to#GAPC1,#shown#as#the# average# of# four# technical# replicatesK# error# bars# represent# SD.# Reproduced# with# permission# from#Haslam#et#al.,# 2012K#Copyright#American#Society#of#Plant#Biologists#©#2012# (www.plantphysiology.org)#(B)# SubAcellular# localization# of# CER2ALIKE1# in# rosette# leaf# pavement# cells# of# the# cer2/like1) mutant,#complemented#with#CER2/LIKE1pro::CER2/LIKE1/GFP.#Scale#bar#=#18#µm.#0200400600800Expression/Level/Relative/to/Root CER2%LIKE1A63###Figure$413:$Expression$profiling$of$CER2=LIKE2##(cont’d#on#following#page)#0300600900Expression.Level.Relative.to.Root CER2%LIKE2A0.00.51.01.52.02.5<9 9;10 11 12 13 14 15 16Relative.Transcript.AbundanceStage.of.flower.developmentCER2CER2%LIKE2CER1FAR2(a) (b)(c)(d)BC64##(A)#qPCR#analysis#of#CER2/LIKE2#expression#patterns#in#the#wild#type#normalized#to#GAPC1,#shown#as#the# average# of# four# technical# replicatesK# error# bars# represent# SD.# Reproduced# with# permission# from#Haslam#et#al.,# 2012K#Copyright#American#Society#of#Plant#Biologists#©#2012# (www.plantphysiology.org)#(B)# Differential# expression# analysis# of# CER2,) CER2/LIKE2,) CER1) and) FAR2# genes# in# Arabidopsis#flowers.# Gene# expression# level# was# determined# by# realAtime# PCR# analysis.# The# relative# transcript#abundance#of#ACT2#and#eIF/4A/1)in#each#sample#was#determined#and#used#to#normalize#for#differences#of# total# RNA# amount.# Results# are# presented# as# transcript# abundance# for# each# gene# compared# to#expression#levels#at#stage#<9.#The#data#represent#the#means#±SD#of#three#replicates.#(C)$AntherAspecific#staining#of#GUS#in#flowers#in#transgenic#Arabidopsis#plants#harbouring#the#CER2/LIKE2)promoter#fused#to#the#GUS#reporter#gene.#Promoter#activity#was#visualized#through#histochemical#GUS#staining#of#a#flower#bud#(a)#and#on#semiAthin#sections#of#anthers#at#stage#8#(a),#9#(b)#and#11#(c)#of#flower#development.#Scale#bar#=#0.5#mm.#B#and#C#reproduced#with#permission#from#Haslam#et#al.,#2015K#Copyright#American#Society#of#Plant#Biologists#©#2015#(www.plantphysiology.org)#####65##4.2.2$ Isolation$of$cer2=like$mutants$TADNA#mutant#alleles#of#CER2ALIKE1,#CER2ALIKE2,#and#CER2ALIKE3#were#obtained#to#investigate# the#biological# functions#of#CER2/LIKE#genes# in#Arabidopsis.#The#cer2/like1#mutant#was#ordered#directly#from#ABRC,#and#the#cer2/like2)and#cer2/like3)mutants#were#kind#gifts#from#Dr.# Carl# Douglas# and# Lexcy# Li.# Individual# plants# were# genotyped# to# identify# homozygous#mutants,#and#the#precise#sites#of#TADNA#insertions#were#determined#by#PCR#and#sequencing,#as#described# in#Section#2.2# (Figure#4A4).#cer2)cer2/like1,#cer2)cer2/like2,#and#cer2/like1)cer2/like2# double# mutants# and# a# cer2) cer2/like1) cer2/like2) triple# mutant# were# generated# to#investigate#some#of#the#possible#overlapping#biological#roles#within#this#gene#family.#Because#the#TADNA#insertion#in#the#cer2/like1)mutant#is#situated#in#an#intron,#and#the#TADNA# insertion# in# cer2/like2# is# situated# near# the# 3’# end# of# the# gene,# RTAPCR# was# used# to#determine#whether#fullAlength#transcript#was#produced#in#either#mutant.#No#fullAlength#transcript#was#detected#in#either#line#(Figure#4A5).#No#TADNA#lines#were#available#with# insertions# in# the#coding#sequence#of#CER2/LIKE4,#so#the#CRISPR/Cas9#system#was#used#to#generate#lossAofAfunction#mutants.#A#19#bp#sequence#located#100#bp# from#the#start#of# the#coding#region#was#selected#as#a# target.#Two#alleles#were#chosen#for#analysis,#which#were#named#cer2/like4/1#and#cer2/like4/2.#Both#had#small#deletions#that#caused#frameAshift#mutations.#The#mutations#in#these#genes#are#shown#in#Figure#4A6.##66###Figure$414:$Schematic$representation$of$T1DNA$insertions$in$cer2=like1,$cer2=like2,$and$cer2=like30Coding#sequences#are#represented#as#black#boxes,#UTRs#as#grey#boxes,#and#introns#and#unAtranscribed#flanking#genomic#DNA#as#a#straight#black#line.#The#TADNA#insertion#sites#were#determined#by#PCR#and#sequencing.# Modified# and# reproduced# with# permission# from# Haslam# et# al.,# 2012K# Copyright# American#Society#of#Plant#Biologists#©#2012#(www.plantphysiology.org)#$$$$$$$$$Figure$415:$RT1PCR$of$cer2=like1$and$cer2=like2$mutants$#(A)#RTAPCR#of#CER2/LIKE1#expression# in#rosette# leaves#of# the#wild# type#and# in# the#cer2/like1)mutant.#CER2/LIKE1Aspecific#primers#annealed#to#the#first#and#second#exons,#flanking#the#intron#that#contains#the#TADNA.#30#amplification#cycles#were#used# for#GAPC,#32# for#CER2/LIKE1.#Reproduced#with#permission#from# Haslam# et# al.,# 2012K# Copyright# American# Society# of# Plant# Biologists# ©# 2012#(www.plantphysiology.org)#)(B)#RTAPCR#of#CER2/LIKE2#expression#in#cauline#leaves#of#the#wild#type#and#in# the# cer2/like2)mutant.#CER2/LIKE2Aspecific# primers# annealed# near# the# start# and# stop# of# the# open#reading# frame.# 28# amplification# cycles# were# used# for# GAPC,# and# 30# for#CER2/LIKE2.)All# primers# are#specified#in#Table#2A3.#Reproduced#with#permission#from#Haslam#et#al.,#2015K#Copyright#American#Society#of#Plant#Biologists#©#2015#(www.plantphysiology.org)#   -   2 201    2 22 2   SALK_147254C,cer2%like1cer2%like2cer2%like367############$Figure$416:CRISPR/Cas9$target$site$and$mutants$generated$in$CER2=LIKE4$(A)# Schematic# representation# of# CER2/LIKE4# and# the# target# sequence# selected# for# CRISPR/Cas9Amediated#mutagenesis.#The#protospacer#adjacent#motif#(PAM)#is#in#bold#type.#(B)#Mutations#in#cer2/like4/1#and#cer2/like4/2.#Eight#base#pairs#are#deleted#in#cer2/like4/1,#and#five#are#deleted#in#cer2/like4/2,#both#causing#frame#shift#mutations.#Cas9%target:%TATCCGTCGTAGATCCCGTCGG wild2type%(Col20): TATCCGTCGTAGATCCCGTCGGcer2%like4%1: TATCCGTC--------CGTCGGcer2%like4%2: TATCCGTCGTAG-----GTCGGAB68##4.2.3$ CER21LIKE1$ has$ an$ analogous$ role$ to$ CER2$ in$ the$ elongation$ of$ leaf$ wax$precursors$Because#CER2# is# expressed# in# leaves,# but#cer2)has#no# leaf#wax#phenotype,# possible#roles#of#CER2/LIKEs#in#rosette#leaf#cuticular#wax#metabolism#were#examined.#Wax#composition#of# cer2/like1# rosette# leaves# was# analyzed# first.# Strikingly,# cer2/like1# leaves# accumulated#approximately#60%#less#31Acarbon#alkane#and#four#times#more#29Acarbon#alkane#than#the#wild#type# (Figure# 4A7A).# Increased# accumulation# of# 30Acarbon# primary# alcohol# and# aldehyde# was#also# observed.# The# shift# in# wax# component# chain# length# distribution# suggests# a# function# for#CER2ALIKE1#in#the#elongation#of#fatty#acids#from#30#to#32#carbons#in#leaf,#parallel#to#the#role#of#CER2#in#the#elongation#of#fatty#acids#from#28#to#30#carbons#in#stems.#To# explore# the# possibility# of# functional# redundancy# between#CER2# and#CER2/LIKE1,#rosette#leaf)wax#composition#of#a#cer2)cer2/like1)double#mutant#was#examined.#cer2)cer2/like1#rosette#leaf#wax)accumulated#roughly#triple#the#wildAtype#load#of#28Acarbon#primary#alcohol)and#VLCFA,#and#roughly#85%#less#31Acarbon#alkane.)Unlike#the#cer2/like1)single#mutant,#the#double#mutant# did# not) accumulate# excess# 29Acarbon# alkane# (Figure# 4A7A).# Overall,# this# result# was#interpreted#as#an#accumulation#of#leaf#cuticular)waxes#28Acarbons#and#shorter,#and#a#reduction#in# waxes) longer# than# 28# carbons# in# cer2) cer2/like1.# That# the# block# in# leaf# wax) elongation#occurred#at#28#carbons#in#the#double#mutant)suggests#that#CER2/LIKE1,#like#CER2,#contributes#to#the#elongation#of)28Acarbon#fatty#acids#to#30.##The# fact# that# the# cer2/like1# mutant# does# accumulate# some# 31# and# 33# carbon# alkane#implies# that# there#are#more#genes# that# contribute# to# the#elongation#of#VLCFAs# from#30# to#34#carbons.#Because#CER2/LIKE2#is#also#expressed#in#rosette#leaves#(Figure#4A3A,#Section#4.2.1),#CER2/LIKE2#or)one#of#the#many#condensing#enzymes#expressed#in#leaves#could#contribute#to#the#elongation#of# these#VLCFAs.#Analysis#of# the#rosette# leaf#cuticular#wax#of#a#cer2)cer2/like1)cer2/like2) triple#mutant#revealed#that#while#the#triple#accumulated#slightly# less#30#to#34#carbon#waxes# than# the# cer2) cer2/like1# double#mutant,# these#waxes# could# still# be# detected# (data# not#shown).#This#might#be#in#part#due#to#presence#of#truncated,#but#functional,#CER2ALIKE2#protein,#as# the#TADNA#insertion#site# in# the#GK180AG04#allele#was# located#near# the#3’#end#of# the#gene.#Alternatively,#other#condensing#enzymes#or#CER2ALIKEs#could#contribute# to#elongation# in# leaf#epidermal#cells.#Surprisingly,# the# total# leaf# wax# load# of# the# cer2/like1# single# mutant# was# nearly# 50%#greater# than# that# of# the#wild# type.# Although# there# is# considerable# variability# in# total# wax# load#measurements#between# individual#plants,# this#difference#was#statistically# significant# (Figure#4A7A).# The# reason# for# increased# wax# load# in# the# mutant# is# unclear.# Perhaps# it# reflects# a#69##compensatory#effort#by# the#plant# to#build#an#effective#cuticular#barrier#by#producing#more#wax#when# the# cuticle# structure# is# compromised# by# altered# composition.# Alternatively,# the# alkane#synthetic# pathway# that# produces# most# of# the# rosette# leaf# wax# of# Arabidopsis# may# have#preference# for# the# 30Acarbon# acylACoA# substrate# that#might# accumulate#when#CER2,# but# not#CER2ALIKE1,#is#present.##4.2.4$ The$acyl1CoA$pools$of$cer2=like$mutants$are$unusual$$The# cuticular# wax# phenotypes# of# cer2/like# mutants# and# the# activity# of# CER2# in# yeast#suggest# that# CER2ALIKE# proteins# modify# the# substrate# specificity# of# fatty# acid# elongation.#Therefore,# characterization# of# cer2/like# mutants# based# on# their# wax# composition,# though#convenient,#is#indirect.#Subsequent#modification#of#VLCAacylACoAs#to#different#wax#components,#and#secretion#of#the#waxes#to#the#cuticle#might#obscure#the#precise#effect#of#cer2/like#mutations#on# elongation.# Hence,# the# phenotypes# of# cer2,# cer2/like1,# and# cer2) cer2/like1# mutants# were#further#investigated#by#profiling#the#acylACoA#pools#of#rosette#leaves#by#LCAMS#(Figure#4A7B).##The# leaf# acylACoA# profile# of# cer2# was# no# different# from# the# wild# type.# The# cer2/like1#mutant# had# a# 3Afold# increase# in# 30Acarbon# acylACoA,# a# 50%# reduction# in# the# amount# of# 32Acarbon#acylACoA,#and#only# trace#amounts#of#34Acarbon#acylACoA.#cer2)cer2/like1#had#a#2Afold#increase# in# the# amount# of# 28Acarbon# acylACoA,# no# difference# compared# to# wild# type# in# the#amount# of# 30Acarbon# acylACoA,# and# the# same# reduction# in# 32A# and# 34Acarbon# acylACoAs# as#observed# in# the#cer2/like1#single#mutant.#These# results#are#consistent#with#conclusions#drawn#based#on#cuticular#wax#phenotypes:#CER2#and#CER2/LIKE1#both#contribute#to#the#elongation#of#acylACoAs# from#28# to#30#carbons# in# leaf,# and#CER2/LIKE1# is# involved# in#elongation#up# to#34#carbons.#Accumulation#of#28Acarbon#acylACoAs# is#much#more#dramatic# than# the#cuticular#wax#phenotype#for#the#double#mutant.##4.2.5$ CER2$and$CER21LIKE2$contribute$to$male$fertility$The#cer2)cer2/like2#double#mutant#and# the#cer2)cer2/like1)cer2/like2# triple#mutant#had#stunted# siliques,# which# were# largely# devoid# of# viable# seeds# at# maturity# (Figure# 4A8A).# This#phenotype#was#rescued#by#growing#the#mutants#in#high#humidity,#suggesting#that#they#may#be#male#sterile#due#to#a#pollen#coat#defect.#To#investigate#this#possibility,#reciprocal#crosses#were#made# between# the# triple# mutant# line# and# the# wild# type.# Normal# seed# set# and# siliques# were#produced#when#wildAtype#pollen#was#applied#to#triple#mutant#pistils#(Figure#4A8B),#but#few#or#no#seeds#could#be# recovered#with# the# reciprocal# cross# (Figure#4A8C).#These# results# indicate# that#CER2#and#CER2/LIKE2#have#redundant#functions#required#for#male#fertility.#70##Figure$417:$Rosette$leaf$wax$and$acyl$CoAs$of$the$cer2,$cer2=like1,$and$cer20cer2=like1$mutants$(A)# Load# and# composition# determined# by# GCAFID.# Values# are# averages# of# at# least# four# biological#replicates,# error# bars# are# SD.# Asterisk# denotes# significant# difference# between# cer2/like1# and# wild# type#determined# by# a#Student’s# TAtest,# ***p<0.001.# (B)#AcylACoA#profiles# determined# by# LCAMS.#Values# are#averages# of# at# least# four# biological# replicates,# error# bars# are# SD.# Reproduced# with# permission# from#Haslam#et#al.,#2015K#Copyright#American#Society#of#Plant#Biologists#©#2015#(www.plantphysiology.org)$((((    - 824 24 867424 24 8674)/ 901  13!529 totalalkanes ketone) )    )   )  01  13!529 VLCFAs aldehydesalcohols0%10%20%30%20:0 22:0 24:0 24:1 26:0 28:0 30:0 32:0 34:0%;of;total;acyl=CoA;poolAcyl=CoA;chain;lengthAB***71#########################Figure$418:$Male$sterility$phenotypes$of$select$cer2=like0mutants$(A)#Mature#siliques#of#single,#double,#and#triple#mutants.#Siliques#of#cer2/5)cer2/like2/1#and#cer2/5)cer2/like1/1)cer2/like2/1#are#stunted#and#contain#few#or#no#seeds#at#maturity.#(B)$Siliques#of#cer2/5)cer2/like1/1) cer2/like2/1# triple# mutant# manually# pollinated# with# wildAtype# pollen.# (C)# Siliques# of# wildAtype# plants#manually#pollinated#with#cer2/5)cer2/like1/1)cer2/like2/1#triple#mutant#pollen.#Reproduced#with#permission#from# Haslam# et# al.,# 2015K# Copyright# American# Society# of# Plant# Biologists# ©# 2015#(www.plantphysiology.org)##Col$0cer2%like1cer2%like2cer2cer2%like1*cer2%like2cer2*cer2%like1cer2*cer2%like2cer2*cer2%like1*cer2%like2BAC72###4.2.6$ Substitution$of$CER2=LIKEs$is$sufficient$to$modify$the$plant$cuticle$structure$The#phenotypes#of# the#cer2/like#mutants#do#not# reveal#whether# the#activities#of#CER2ALIKE#proteins#are#competitive,#nor#whether#they#can#substitute#for#the#functions#of#one#another#in) planta.# To# address# these# unknowns,# a# CER2/LIKE1/GFP# transgene# was# ectopically#expressed# in# the# stem# epidermis# of# wild# type# and# cer2/5#mutants,# using# the# promoter# of# the#condensing#enzyme#gene#CER6# (Hooker#et#al.,#2002).#GCAFID#analysis#of#stem#wax#revealed#that#there#was#no#significant#difference#in#the#total#wax#load#when#CER2/LIKE1#was#expressed#in# wildAtype# plants# (Figure# 4A9A).# CER2/LIKE1# expression# in# the# cer2/5# mutant# background#recovered# more# than# oneAhalf# the# wildAtype# wax# load# (Figure# 4A9B).# The# wax# chain# length#profiles#of#the#transgenics#indicate#that#CER2ALIKE#activity#is#sufficient#to#modify#the#specificity#of# elongation# in) plantaK# when# CER2/LIKE1# was# expressed# in# both# wildAtype# and# cer2/5#backgrounds,# the# ratio# of# 31:29Acarbon#wax# components#was# greater# than# 1,#whereas# it#was#0.03#in#ColA0#wild#type.#The#increased#accumulation#of#31A#and#33Acarbon#waxes#at#the#expense#of# 29Acarbon#waxes# in#wildAtype#background# suggests# that# the# activities# of#CER2#and#CER2ALIKE1#are#competitive.##Plants#carrying#the#CER2/LIKE1/GFP#transgene#in#either#wildAtype#or#cer2/5#background#appeared# glossy,# despite# the# fact# that# they# had# completely# or# partially# recovered# stem# wax#loads,# respectively.#A#similar#observation#has#been#made#when#cer2/like1#was#complemented#with#a#35Spro::CER2/LIKE1#construct#(Pascal#et#al.,#2013).#Examination#of#the#progeny#of#three#independent# transgenic# lines# in# each# background# by# scanning# electron# microscopy# (SEM)#revealed#that#their#stems#had#reduced#wax#crystal#density#relative#to#wild#type.#Crystals#that#did#form#on#the#stems#of#transgenic#plants#consisted#of#irregularly#shaped,#thin#flakes#(Fig.#4A9C).##73###Figure$419:$Ectopic$expression$of$CER2=LIKE1$in$stem$epidermis$(A)$Expression#of#CER2/LIKE1# in#wildAtype#stem.# (B)#Expression#of#CER2/LIKE1# in#cer2/5# stem.#Total#wax# load# includes# all# aliphatic# wax# components,# and# the# alkane# composition# shows# the# chain# length#profile.#Values#represent#averages#of#3#(A)#and#4#(B)#independent#T1#lines.#Error#bars#represent#standard#deviation.#(C)#SEM#images#of#stems#from#transgenics#in#wildAtype#(top)#and#cer2/5)(bottom)#background.#Each#image#was#taken#of#a#different#T1#line.#Modified#and#reproduced#with#permission#from#Haslam#et#al.,#2015K#Copyright#American#Society#of#Plant#Biologists#©#2015#(www.plantphysiology.org)##50μmCol(0cer2%5background independent6CER6pro::CER2%LIKE1%GFP5linesC024681005101520Wax6Load6(µg/cm2 )A 56  - 1 2 2 024681025 27 29 31 3305101520TotalWax6Load6(µg/cm2 )Balkane6chain6length 2 56  - 1060674###4.2.7$ CER21LIKEs$ impart$ different$ substrate$ specificities$ to$ the$ same$ condensing$enzyme$$$The# cer2/like# phenotypes# indicate# that# different# CER2ALIKEs# have# distinct# roles# in#VLCFA# elongation.# These# differences# could# be# due# to# either# (1)# each# CER2ALIKE# protein#functioning#with#a#different#condensing#enzyme,#or#(2)#CER2ALIKEs#imparting#their#own#unique#substrate# specificities# to# the# same# condensing# enzyme.# To# differentiate# between# these# two#possibilities,#each#of# the# five#Arabidopsis#CER2/LIKEs#was#coAexpressed#with#CER6# in#yeast,#and# yeast# FAMEs# were# analyzed# by# GCAFID.# As# reported,# CER2# and# CER6# coAexpression#produced# 30Acarbon# fatty# acid# (Figure# 4A10A).# Yeast# cells# coAexpressing# CER2/LIKE1# with#CER6#accumulated#30A,#32A,#and#34Acarbon#fatty#acids.#The#specificity#of#CER2/LIKE1#observed#here# is# in# agreement# with# the# fact# that# the# cer2/like1# mutant# rosette# leaf# wax# has# reduced#amounts#of#31A,#32A,#and#33Acarbon#waxes,#which#are#abundant#in#wildAtype#leaf#cuticular#wax.#Yeast# cells# coAexpressing#CER6# and#CER2/LIKE2# synthesized# 30A,# and# traces# of# 32Acarbon#fatty#acids.#CER6#with#CER2/LIKE3#had#a#similar#FAMEs#profile#to#CER6)with#CER2,#producing#30Acarbon# fatty#acids.#Finally,#CER2/LIKE4#was#unique#among# the#homologs# in# that# it#did#not#extend# the# substrate# specificity# of# CER6,# and# only# reduced# the# accumulation# of# 26Acarbon#FAMEs# detected# when#CER6# was# expressed# alone.# CER2,# CER2ALIKE1,# CER2ALIKE2,# and#CER2ALIKE3#have#a#similar#negative#effect#on# the#accumulation#of#26Acarbon# fatty#acid#when#coAexpressed#with#CER6,#however,#this#may#simply#be#due#to#26Acarbon#fatty#acid#being#used#as# substrate# for# elongation# to# longer# chain# lengths.#Why# less# 26Acarbon#VLCFA#accumulates#when#CER2ALIKE4#is#coAexpressed#with#CER6#is#less#clear.#Curiously,#coAexpression#of#each#of#the#CER2ALIKEs#except#CER2ALIKE4#with#CER6#reduced# the#accumulation#of#28Acarbon# fatty#acid#in#comparison#to#CER6#alone.#Together,# these# results# demonstrate# that# CER2/LIKE# proteins# have# unique# substrate#specificities# in#the#presence#of# the#same#condensing#enzyme.#CoAexpression#of#CER2,#CER2/LIKE1,#and#CER6#produced#VLCFA#profiles#similar# to#CER2/LIKE1#and#CER6# (Figure#4A10B),#indicating#that#there#is#no#synergistic#effect#of#having#multiple#CER2ALIKE#proteins.##CER2# can# alter# chain# length# specificity# of# the# elongation# complex# when# it# is# coAexpressed#with#CER6,#but#not#when# it# is#coAexpressed#with# the#LfKCS45#condensing#enzyme#from# the# crucifer# L.) fendleri# (Chapter# 3).# This# implies# that# a# particular# feature# of# CER6# is#required#for#CER2#activity,#but#how#unique#this#feature#is#to#CER6#is#unknown.#To#determine#if#other# KCS# enzymes# can# pair# with# CER2ALIKEs# to# produce# VLCFAs# 30# carbons# and# longer,#another# suite# of# yeast# expression# experiments# was# devised.# This# work# was# done# in#75##collaboration# with# Dr.# Jérôme# Joubès# at# the# University# of# Bordeaux.# Dr.# Joubès’# lab# coAexpressed#CER2,)CER2/LIKE1,#and#CER2/LIKE2#with#the#following#condensing#enzyme#genes#from#Arabidopsis:#ECERIFERUM60#(CER60/KCS5K#Fiebig#et#al.,#2000),#FIDDLEHEAD#(KCS10K#Pruitt#et#al.,#2000),#KCS1# (Todd#et#al.,#1999),#KCS9# (Lü#et#al.,#2012),#and#KCS20# (Lee#et#al.,#2009).# These# condensing# enzymes# were# selected# because# their# null# mutants# have# cuticle#defects,#they#belong#to#different#subclasses#of#the#FAE1Alike#KCS#family#(Joubès#et#al.,#2008),#and#because#they#have#demonstrated#activity#in#yeast#(Tresch#et#al.,#2012).#CER2ALIKE#activity#was# not# tested# with# ELOALIKE# proteins.# All# three# CER2ALIKEs# tested# had# an# effect# on# the#specificity#of#CER60,#which#is#88#%#identical#to#CER6,#and#had#the#same#substrate#specificity#as#CER6# in# this# assay.# The# effects# of# CER2ALIKEs# on#CER60#were# identical# to# their# effects# on#CER6.#None#of#the#other#KCSs#tested#had#different#activity#when#paired#with#CER2ALIKEs#(data#not#shownK#Haslam#et#al.,#2015).###4.2.8$ CER21LIKE4$has$a$negative$effect$on$CER6$+$CER2$activity$in$yeast$The# effect# of# CER2ALIKE4# on#CER6# activity# in# yeast# was# unique# in# that#CER2/LIKE4#expression# reduced# the# accumulation# of# 26Acarbon# fatty# acids,# but# was# not# correlated# with#increased#accumulation#of#any#longer#fatty#acids.#It# is#unknown#whether#and#how#CER2ALIKE4#might# contribute# to# wax# metabolism.# The# Arabidopsis# eFP# browser# (Winter# et# al.,# 2007)#indicates# that#CER2/LIKE4# is#expressed# in#both#stem#and# leaf#epidermal#cells.#As#only#CER2#among#the#remaining#CER2/LIKEs#is#expressed#in#stem,#CER6,#CER2,#and#CER2/LIKE4#were#coAexpressed# in# yeast# to# mimic# the# Arabidopsis# stem# epidermal# elongase# (Figure# 4A10C).#Accumulation#of# 26Acarbon# fatty# acid#was#not# substantially# different# in# cells# expressing#CER6)and#CER2# compared# to# cells# expressing#CER6,#CER2,# and#CER2/LIKE4.# However,#CER2/LIKE4# did# have# a# negative# effect# on# elongation# in# that# 50%# less# 30Acarbon# fatty# acid#accumulated#in#CER6,#CER2,#and#CER2/LIKE4Aexpressing#cells)than#in#yeast#cells#expressing#only#CER6#and#CER2.#Accumulation#of#28Acarbon#fatty#acid#was#maintained#at#similar#levels#as#in#cells#transformed#with#either#CER6#or#CER6#and#CER2/LIKE4.###4.2.9$ The$cer2=like40mutant$does$not$have$a$cuticular$wax$phenotype$To#investigate#the#in)planta#function#of#CER2ALIKE4,#cer2/like4#mutants#were#generated#using# the#CRISPR/Cas9#system.#Preliminary#analyses#of# two)cer2/like4#mutant#alleles#did#not#reveal#any#obvious#phenotype.#The#stems#of# the#mutants#appeared#glaucous,#and#analysis#by#GC/FID#did#not#uncover#any#significant#changes#in#the#total#wax#load#or#chain#length#distribution#of#wax#components#(Figure#4A11).##76##Figure$4110:$FAMEs$profiles$of$yeast$cells$expressing$CER6$and$CER21LIKE$proteins$Values#represent#averages#of#four#biological#replicatesK#error#bars#represent#standard#deviation.$#0123426:0 28:0 30:0 32:0 34:0Percent/total/FAMEsw3031aCER6CER6%+%CER2CER6%+%CER2(LIKE1CER6%+%CER2(LIKE2CER6%+%CER2(LIKE3CER6%+%CER2(LIKE40123426:0 28:0 30:0 32:0 34:0Percent/total/FAMEsw3031aCER6CER6%+%CER2CER6%+%CER2(LIKE1CER6%+%CER2%+%CER2(LIKE1AB0123426:0 28:0 30:0 32:0 34:0Percent/total/FAMEsFAME/lengthw3031aCER6CER6%+%CER2CER6%+%CER2(LIKE4CER6%+%CER2%+%CER2(LIKE4C77########Figure$4111:$Phenotypes$of$cer2=like4$mutants$(A)#Stems#of#wildAtype#and#cer2/like4#mutant#plants.# (B)#Total# load#and#(C)#alkane#composition#of#stem#cuticular# wax# for# wild# type# and# cer2/like4# mutants.# Values# are# averages# of# at# least# four# biological#replicatesK#error#bars#represent#standard#deviation.### #Col$0cer2%like4%1cer2%like4%20510152025Wax,Load,µg/cm2Axis,TitleTotal024681027an 29an 31an 33an27, 29 31 33Col$0cer2%like4%1cer2%like4%2Alkane,chain,lengthACB78##4.3$ Discussion$#4.3.1$ CER21LIKEs$impart$unique$substrate$specificities$to$fatty$acid$elongation$Cuticular# wax# and# acylACoA# profiles# of# cer2,# cer2/like1,# and# cer2) cer2/like1# mutants#demonstrate#that#CER2#and#CER2ALIKE1#contribute#to#the#elongation#of#fatty#acids#to#different#chain# lengths.# Building# off# the# conclusions# of# Chapter# 3,# this# observation# suggests# two#hypotheses:# (1)# different# CER2ALIKE# proteins# could# associate# with# and# thereby# alter# the#specificity#of#different#condensing#enzymesK#and/or#(2)#different#CER2ALIKE#proteins#could#have#unique# effects# on# the# specificity# of# the# same# condensing# enzyme.# These# possibilities# are# not#mutually#exclusive,#but#at#least#one#of#them#must#be#true#to#account#for#the#different#phenotypes#observed.##CoAexpression#of#each#of#the#CER2/LIKE)genes#with#CER6# in#yeast#demonstrated#that#all# of# the#CER2ALIKEs# have# unique# product# specificity.# CER2ALIKE1# and#CER2ALIKE2# amino#acid#sequences#are#66%#identical,#and#are#the#most#similar#among#the#CER2ALIKE#proteins.#As#such,#I#was#surprised#to#find#that#CER2ALIKE1#uniquely#supports#elongation#up#to#34#carbons,#while# CER2ALIKE2# is# similar# to# CER2# and# CER2ALIKE3# in# primarily# contributing# to# the#accumulation# of# 30Acarbon# fatty# acids.# This# observation# will# be# valuable# in# designing#experiments#to#dissect#the#basis#of#substrate#specificity#of#CER2ALIKE#proteins.#When# CER6,# CER2,# and# CER2ALIKE1# are# coAexpressed,# the# yeast# FAMEs# profile# is#similar# to# that#of#only#CER2ALIKE1#and#CER6# together# in# that# fatty#acids#up# to#34#carbons# in#length#are#produced.#CER6#+#CER2#+#CER2ALIKE1#cells#accumulate#less#30Acarbon#fatty#acid#than# only# CER6# +# CER2,# but# substantially# more# than# CER6# +# CER2ALIKE1.# This# result#suggests# that# there# could# be# competition# among# CER2ALIKEs# for# activity# alongside# the# core#elongation#machinery.#This#idea#is#further#supported#by#the#dramatic#accumulation#of#30#carbon#acylACoAs#and#29Acarbon#waxes#in#the#cer2/like1#single#mutant,#which#greatly#exceeds#the#loss#of#32Acarbon#acylACoAs#and#31Acarbon#waxes#observed#in#this#mutant.#When# CER2,# CER2ALIKE1,# and# CER2ALIKE2# were# coAexpressed# with# several#condensing# enzymes# from# Arabidopsis# known# to# contribute# to# the# synthesis# of# cuticular# lipid#precursors,#they#only#had#activity#with#CER6#and#CER60/KCS5.#The#specificity#of#CER2ALIKEs#was#not#changed#by# the# identity#of# the#condensing#enzyme.#That# the#CER2ALIKEs# functioned#with# CER6# and# CER60# was# not# surprising,# as# the# amino# acid# sequences# of# these# two#condensing#enzymes#are#88.6%#identical#and#they#belong#to#the#same#clade#within#the#FAE1Alike#KCS#family#(Joubès#et#al.,#2008).#I#conclude#that#CER2ALIKE#proteins#are#active#only#when#they#are#coAexpressed#with#an#appropriate#condensing#enzyme#partner.#Although# the# identical#79##effects#of#CER2ALIKEs#on#CER6#and#CER60#suggest# that# the#specificity#of#CER2ALIKEs#does#not#vary#with#different#condensing#enzymes,#it#remains#a#possibility#that#CER2ALIKE#activity#may#be#different#with#other#condensing#enzymes# that#were#not# investigated#here.#Without#knowing#what#specific#property#of#CER6#and#CER60#enables#them#to#pair#with#CER2ALIKEs,#it#is#difficult#to#make#logical#predictions#and#design#experiments#to#further#explore#this#question.##4.3.2$ Modification$of$KCS$specificity$by$CER21LIKEs$has$physiological$importance$To# better# understand# the# physiological# importance# of# CER2ALIKEs,# the# impact# of# a#modified#acylAchain# length#profile#on# the#downstream#synthesis#of# cuticular#waxes#and#on# the#assembly#of#a#cuticular#barrier#was#investigated.#The#strong,#epidermisAspecific#CER6#promoter#was#used#to#drive#expression#of#a#CER2/LIKE1/GFP# transgene#in#stems#of#both#wildAtype#and#cer2#plants.#This#resulted#in#a#substantial#increase#in#31A#and#33Acarbon#waxes,#and#a#decrease#in#29Acarbon#waxes#in#the#transgenic#plants#compared#to#both#genetic#backgrounds.#The#CER2/LIKE1/GFP#construct#was#able#to#partially#complement#the#total#wax#load#deficiency#of#the#cer2/5#mutant,#demonstrating# that#CER2ALIKE#proteins#can#substitute# for#one#another’s# function# in)vivo.# The# total# wax# load# of# the# transgenics# in# the# wildAtype# background# did# not# differ#substantially# from# the# nonAtransformed# controls.# This# was# somewhat# counterintuitive,# as# the#stems# of# the# transgenics# of# both# genetic# backgrounds# were# glossy,# suggesting# a# wax# load#deficiency.# As# the# changes# in# relative# amounts# of# different# types# of# acyl# lipids# (e.g.# ketones,#alkanes,#secondary#alcohols,#etc.)#were#minor#compared#to#the#relative#changes#in#amounts#of#acyl#chain#lengths,#this#result#suggests#that#wax#component#chain#length#affects#the#formation#of#wax#crystals.#This#is#in#agreement#with#the#fact#that#Arabidopsis#leaf#cuticular#waxes,#which#are#predominantly# composed# of# 31Acarbon# alkanes,# do# not# appear# white# or# waxy,# while# stem#cuticular#wax#that#is#largely#composed#of#29Acarbon#alkanes,#secondary#alcohols,#and#ketones,#imparts#a#glaucous#appearance#to# the#stem.#Hence,# the#effects#of#CER2ALIKE#proteins#on#the#final#steps#of#VLCFA#elongation#affect#the#assembly#and#physical#properties#of#the#cuticle.##Male#sterility#dependent#on# low#environmental#humidity# is#a#phenotype#associated#with#pollen#coat#deficiencies,#and#has#been#observed# in#several#Arabidopsis#waxAdeficient#mutants#(Koornneef# et# al.,# 1989K# Preuss# et# al.,# 1993)# including# one# cer2# allele# (Preuss# et# al.,# 1993K#Eisner#et#al.,#1998).#The#most#abundant#acyl#lipids#in#the#pollen#coat#are#29Acarbon#alkane,#29Acarbon# ketone,# and# 31Acarbon# alkene# (Jessen# et# al.,# 2011),# so# it# is# not# surprising# that# the#biosynthesis# of# cuticular# waxes# and# pollen# coat# could# require# the# same# or# related# gene#products.# I#did#not#detect#any# reduction# in#seed#set# in# the#single#cer2/5#mutant# relative# to# the#wild# type.# This# discrepancy# with# previous# work# could# be# due# to# subtle# differences# in# growth#80##conditions,#or#the#different#alleles#selected#for#study.#However,#I#did#observe#that#inactivation#of#both#CER2#and#CER2/LIKE2#caused#male#sterility#in#lowAhumidity#environments.)This#suggests#that# the# chain# length# of# acyl/lipid# pollen# coat# components# is# relevant# to# function,# or) that# the#chain#length#of#VLCFAs#is#important#for)their#conversion#to#alkanes#and#other#aliphatics#found#in)the#pollen#coat.#The#precise#role#of#VLCAacyl# lipids# in#the)pollen#coat#remains#unknown.#It#has#been#suggested)that#they#could#be#required#to#create#physical#contact#between)the#pollen#grain#and#stigmatic#papillae#to#enable#the)diffusion#of#signalling#molecules,#that#they#could#provide#a)matrix# that# could# stabilize# other# pollen# coat# components,) or# that# VLCAacyl# lipids# could#themselves#function#as#signalling#molecules#(Preuss#et#al.,#1993).#Regardless#of#which#of#these#functions#VLCAacyl# lipids#fulfill# in# the#pollen#coat,#my#data#show#that#CER2ALIKEs#are#required#for#fertilization.##4.3.3$ The$function$of$CER21LIKE4$remains$unknown$The# role# of# CER2ALIKE4# in# VLCFA# metabolism# remains# unknown.# CoAexpression# of#CER6)with#CER2/LIKE4#in#yeast#resulted#in#reduced#26Acarbon#fatty#acid#accumulation,#and#coAexpression#of#CER6,#CER2,#and#CER2/LIKE4#resulted#in#lower#accumulation#of#30Acarbon#fatty#acid.#This#suggests#that#CER2/LIKE4#could#function#as#a#negative#regulator#of#wax#biosynthesis#by# restricting# the# production# of# VLCFA# precursors# of# cuticular# wax.# However,# as# previously#discussed,#quantitative#changes# in# fatty#acid#production# in# the#heterologous#yeast# system#are#difficult# to# interpret,# and# are# arguably# meaningless# in# the# absence# of# a# cer2/like4# mutant#phenotype.##Unfortunately,# no# change# in# stem#cuticular#wax# load#and# composition#was#detected# in#cer2/like4#mutant#plants.#It#remains#a#possibility#that#CER2ALIKE4#could#affect#wax#biosynthesis#under# stress# conditions,# or# that# CER2ALIKE4# could# contribute# to# or# regulate# VLCFA# flux# into#other# lipid# biosynthetic# pathways.# Cursory# evaluation# of# CER2/LIKE4# expression# patterns#reported#on#the#Arabidopsis#eFP#browser#(Winter#et#al.,#2007)#reveals#that#its#expression#is#upAregulated# in# the# L1# layer# of# the# shoot# apical# meristem.# In# these# rapidly# dividing,# polarized#epithelial# cells,# I#expect# that#both#wax#and#sphingolipid#biosynthesis#are#highly#active.#Recent#work#has#demonstrated#that#hydroxylated#24A#and#26Aacyl#chains#incorporated#in#ceramides#are#required# to# support# polarized# secretion# in# young# root# epithelial# cells# (WatteletABoyer# et# al.,#2016).# I# anticipate# that# similar# sphingolipids# could# be# required# in# the# L1# of# the# shoot# apical#meristem# to# support# polar# secretion# of# cuticular# lipids# and# cell# wall# carbohydrates.# Because#ceramides# incorporate#shorter#VLCFAs# than#are#used# for#wax#metabolism,#one#hypothesis# for#the# function#of#CER2ALIKE4# is# that# it#could#serve#as#a# ‘brake’# that# limits#substrate#channelling#81##into#wax#biosynthesis#by#reducing#the#production#of#VLCFAs#26#carbons#and#longer.#This#would#ensure# that# sufficient#VLCFAs#are#available# for#ceramide#synthesis,#and#would#be#particularly#important#when#wax#metabolism# is# upAregulated# under# stress# conditions.# Therefore,# it# will# be#important# to# examine# the# cer2/like4# wax# and# sphingolipid# phenotypes# of# plants# subjected# to#conditions#known#to#upAregulate#wax#production,#including#drought,#cold,#and#high#light#intensity.#An#alternative#hypothesis#is#that#CER2ALIKE4#could#support#the#production#of#28Acarbon#fatty#acids.#This#is#suggested#by#the#fact#that#yeast#cells#expressing#CER6,#CER2,#and#CER2ALIKE4# accumulated# more# 28Acarbon# FAME# than# cells# coAexpressing# CER6# and# CER2.# 28Acarbon# acylACoAs# are# preferentially# used# by# the# fatty# acyl# reductase,# CER4/FAR3,# which#generates# cuticular# fatty# alcohols.#While# this# hypothesis# is# not# supported# by# wax# analysis# of#cer2/like4# mutants,# it# is# possible# that# a# reduction# in# primary# alcohol# load#might# be# observed#when#the#cer2/like4#mutation#is#introduced#to#a#genotype#and#cell#type#that#accumulates#more#of#these# particular# waxes,# such# as# stems# of# the# cer2# single# mutant.# Analysis# of# the# cer2/like4#phenotype#in#stems#and#leaves#of#cer2)cer2/like1)cer2/like2#mutants#is#currently#underway.##4.3.4$ Summary$of$implications$for$models$of$CER21LIKE$metabolic$function$While#the#focus#of#this#chapter#has#been#a#descriptive#characterization#of#the#breadth#of#biological#functions#of#CER2ALIKEs,#I#can#also#make#inferences#from#these#results#that#pertain#to# the# biochemical,# mechanistic# contribution# of# these# proteins# to# elongation.# CER2ALIKEs#contribute#to#the#chainAlength#specificity#of#elongation,#and#functionally#partner#only#with#specific#condensing#enzymes.#Further,# the# functions#of#CER2ALIKEs#are# unique#and# competitive,#with#individual# proteins# having# the# capacity# to# positively# or# negatively# affect# elongation.# These#conclusions#will# be# incorporated# in#models#of#activity#of#CER2ALIKEs#developed#at# the#end#of#Chapter#5.#82##Chapter$5:$Biochemical$characterization$of$ECERIFERUM2$$#5.1$ Introduction$Based# on# sequence# homology,# CER2# was# annotated# as# a# BAHD# acyltransferase.#BAHDs#transfer#an#acyl#group#from#a#CoAAthioester#to#either#an#alcohol#or#amine#acyl#acceptor,#generating#an#ester#or#amide#bond,#respectively#(Figure#1A4).#BAHD#acyltransferases#are#plantA#and# fungiAspecific# enzymes# that# use# diverse# substrates# in# a# wide# variety# of# specialized#metabolic# pathways,# are# soluble,#monomeric,# and# are# localized# to# the# cytosol.# Their# catalytic#mechanism# has# been# characterized# extensively# using# protein# modelling# and# siteAdirected#mutagenesis#(reviewed#in#D’Auria,#2006).#While#CER2#is#homologous#to#this#family#of#enzymes,#and#was,#in#fact,#one#of#the#first#members#of#the#family#to#be#identified,#CER2#differs#markedly#from#other#characterized#BAHDs#in#two#respects.##First,# CER2# with# CAterminally# fused# green# fluorescent# protein# (GFP)# localizes#predominantly#to#the#ER#(Section#3.2.3).#This#makes#biological#sense#in#that#the#ER#is#the#site#of#VLCFA#elongation.#However,#it#is#unusual#for#a#BAHD#acyltransferase,#particularly#since#CER2#does#not#have#any#predicted#transmembrane#domains#or#an#ER#targeting#signal#(AramemnonK#Schwacke#et#al.,#2003).#Therefore,#either#postAtranslational#modifications#of#the#CER2#protein#or#interactions# between# CER2# and# ER# membraneAlocalized# proteins# may# be# involved# in# CER2#localization.# It# is# tempting#to#speculate#that#CER2#interaction#with#other#protein#components#of#the#fatty#acid#elongase#complex#may#account#for#CER2#association#with#the#ER.##Second,# two# conserved# motifs# have# been# described$ for# BAHDs:# carboxyAterminal#DFGWG,#which#is#predicted#to#have#a#function#in#retaining#structural#stability#of#the#enzyme#but#is#not#present# in#all#BAHDs,#and#HXXXD,#which#catalyzes# the#acyl# transfer# reaction# (D’Auria,#2006).# All# of# the# CER2ALIKEs# that# I# identified# lack# the# putatively# stabilizing# DFGWG# motif.#However,# this#may# not# be# surprising# given# that# BAHDs# are# soluble# enzymes# (D’Auria,# 2006K#Panikashvili#et#al.,#2009K#Rautengarten#et#al.,#2012),#while#CER2#and#CER2ALIKE1# localize# to#the# ER# membrane.# Perhaps# the# DFGWG# motif# is# not# required# when# these# proteins# are# in#association#with#the#ER.#83##SiteAdirected# mutagenesis# experiments# on# anthocyanin# malonyltransferase# of# Salvia)splendens#(Suzuki#et#al.,#2003),#vinorine#synthase#of#Rauvolfia)serpentina#(Bayer#et#al.,#2004),#hydroxycinnamoyltransferases# of# Coffea) canefora# (Lallemande# et# al.,# 2012),# and# Sorghum)bicolor# (Walker# et# al.,# 2013)# have# demonstrated# that# the# histidine# residue# within# the# HXXXD#motif# is# essential# for# enzyme# activity.# The# histidine# residue# is# thought# to# be# responsible# for#deprotonating# the# alcohol# or# amine# acyl# acceptor,# creating# a# nucleophile# that# attacks# the#carbonyl#carbon#of#the#acylACoA,#and#resulting#in#the#release#of#CoASH#and#formation#of#a#new#ester# or# amide# bond# (Figure# 1A4).# The# importance# of# the# conserved# histidine# residue# is#supported# by# the# crystal# structures# of# vinorine# synthase# from# Rauvolfia# and# anthocyanin#malonyltransferase#from#Chrysanthemum,# in#which#the#histidine#residue#is#ideally#positioned#at#the#junction#of#the#acyl#donor#and#acyl#acceptors#within#the#active#site#(Ma#et#al,#2005K#Unno#et#al,#2007).#$Both#CER2/LIKE1) and#CER2/LIKE4) lack# a# histidine# in# their# predicted# HXXXD# motifs#(Figure#5A1).#Genetic#analysis#of#a#null#cer2/like1)mutant#(Chapter#4)#revealed#that#despite#this#abnormality,#CER2ALIKE1)has#an#analogous#role#to#CER2#in#VLCFA#elongation.#This,#and#the#absence# of# an# obvious# purpose# of# an# acyl# transfer# reaction# in# the# elongation# of# VLCFAs# to#specific#chain#lengths,#led#me#to#question#whether#CER2ALIKEs#have#acyl#transfer#activity.##Beyond#the#transferase#domain#and#its#catalytic#motif,#there#are#no#recognizable#features#in#CER2ALIKE#proteins#that#suggest#a#biochemical#function.#I#took#several#approaches#to#search#for#clues#as#to#how#CER2#might#participate#in#elongation.#First,#I#used#siteAdirected#mutagenesis#to#determine#whether#CER2#requires# its#HXXXD#motif# for# function.#I#characterized# interactions#between#CER2#and#components#of#the#core#elongase#complex#using#the#splitAluciferase#system,#and# used# coAimmunoprecipitation# to# confirm# CER2ACER6# interaction.# I# also# tested# whether#interaction#with#CER6#was#required#for#CER2#targeting#to#the#ER#by#comparing#the#localization#of#CER2AGFP# in#cer2#and#cer2)cer6#genetic#backgrounds.# I#generated#sequence#chimeras#of#CER2ALIKE1# and# CER2ALIKE2,# the# two# CER2ALIKEs# with# the# greatest# sequence# identity# in#Arabidopsis,#to#investigate#what#features#determine#their#distinct#substrateAspecificities.#Finally,#I#used# homologyAbased# tertiary# structure# models# to# compare# CER2ALIKE# structures# to# each#other,#and#to#BAHDs#with#confirmed#acyl#transfer#function.##84##Figure$511:$Amino$acid$alignment$of$CER21LIKE$BAHD$acyltransferases$from$Arabidopsis$The#HXXXD#motif#of#CER2#is#marked#with#a#black#box.#The#predicted#catalytic#histidine#residues#of#all#five#homologs# are# shaded# red.# The# sequence# swap# sites# discussed# in# Section# 5.2.3# are#marked# S#.# The#histidine#residues#discussed#in#Section#5.2.4#are#highlighted#in#blue.##S5 S4 S4 S3 S2 S1 85##5.2$ Results$#5.2.1$ CER21LIKEs$do$not$require$a$conserved$acyltransferase$motif$for$function$SiteAdirected#mutagenesis# was# used# to#modify# the# putatively# catalytic# histidine# 166# of#CER2# to# determine# if# it# is# required# for# CER2# function.# Histidine# was# replaced# with# alanine#(H166A),# to# knock# out# any# catalytic# activity# at# this# site,# and# asparagine# (H166N),# to# mimic#CER2ALIKE1.#Constructs#with#these#mutant#alleles,#as#well#as#a#wildAtype#control#(H166H),#were#transformed# into#cer2)mutant# plants.# In#each# case,# insertion#of# the# transgene#and# the#mutant#background# were# confirmed# by# PCRAbased# genotyping,# and# Sanger# sequencing# of# the#transgene# verified# the#mutations.# Plants#were# visually# examined# for# glossiness/glaucousness,#and# wax# load# and# composition# were# measured# by# GCAFID.# Both# mutant# alleles# fully#complemented# the# background# mutant# phenotype,# indicating# that# CER2# does# not# have# the#described# catalytic#mechanism#of# characterized#members#of# the#BAHD#acyltransferase# family#(Figure#5A2).#86##########Figure$512:$Phenotypes$of$the$cer2=5$mutant$transformed$with$H1661modified$CER2$sequences$(A)# Inflorescence# stems# of# (left# to# right)# ColA0# wild# type,# cer2/5,# cer2/5# complemented# with# wildAtype,#H166A,# and# H166N# CER2# sequences.# (B)# Total# wax# load# of# inflorescence# stems# from# the# same#genotypes.#Values# are# the# average# of# four# biological# replicates.#Error# bars# represent#SD.# The# asterisk#denotes#significant#difference#between#the#total#wax#loads#as#determined#by#a#Student’s#TAtest#(p<0.05).#Reproduced#with#permission#from#Haslam#et#al.,#2012K#Copyright#American#Society#of#Plant#Biologists#©#2012#(www.plantphysiology.org).##87##5.2.2$ CER2$and$CER6$physically$interact$#5.2.2.1$ Split1luciferase$Interactions#between#CER2#and#the#core#elongase#complex#were#investigated#using#the#splitAluciferase#system#in#Nicotiana)benthamiana)(tobacco)#leaf.#All#of#the#elongase#components#were#cloned#to#produce#either#NA#or#CAterminal#fusions#to#either#the#CAterminal#or#NAterminal#half#of# the# firefly# luciferase# enzyme,# respectively,# and# coAexpressed# with# CER2# fused# with# the#opposite#luciferase#half.#Luminescence#is#produced#when#the#two#coAexpressed#fusion#proteins#interact#or# come# in# such#close#physical# proximity#as# to#allow# the# luciferase#enzyme#halves# to#reconstitute#the#entire,#lightAproducing#enzyme.#Luminescent#signal#was#anticipated#even#when#observing#negative#controls,#as#all#of#the#proteins# were# overAexpressed,# and# localize# to# the# ER.# Another# complication# of# working# in#tobacco#leaf#is#that#the#capacity#for#infiltration,#and#competency#for#transformation,#varies#greatly#among# plants,# and# between# different# stages# of# leaf# development.# Hence,# each# technical#replicate#for#each#putative#interaction#pair#was#normalized#against#a#negative#control#interaction#pair# infiltrated# symmetrically# to# the# same# tobacco# leaf.# HydroxymethylglutarylACoA# reductase#(HMG1)#was#used#as#a#negative#control#as#it#is#not#expected#to#interact#with#CER2,#but#is#similar#to#elongase#enzymes#in#that# it# is#an#ERAlocalized#transmembrane#protein#(Leivar#et#al.,#2005).#Protein#pairs#that#truly#interact#were#expected#to#produce#more#luminescence#than#their#coupled#negative#control#pair.#The#validity#of#the#HMG1#+#CER2#negative#control#was#confirmed#using#a#second# negative# control,# ACA2,# to# ensure# that# both# HMG1# and# ACA2# produce# equal#luminescence# levels#when#coAexpressed#with#CER2.#ACA2# is#CALCIUM)ATPASE2,#a#calcium#transporter# localized# to# the# ER# (Hong# et# al.,# 1999).# A# sample,# hypothetical# experiment# is#presented#in#Figure#5A3#to#illustrate#how#data#was#collected.#Sources#of#error#that#produce#falsely#negative#results,#such#as#protein#degradation,#misAfolding,# or# misAtargeting# were# anticipated,# but# these# were# expected# to# vary# among# different#proteins,#and#different#protein#fusions.#Therefore,#while#there#are#two#construct#pairs#possible#for#each# interaction# tested# (e.g.# CLucACER2# +# CER6ANLuc# vs.# CER2ANLuc# +# CLucACER6),#whichever#pair#produced# the#greatest# luminescence#was#considered# to#be# the#most#accurate,#and#is#presented#in#Figure#5A4.#Interaction#was#detected#between#CER2#and#all#components#of#the#core#elongase#complex#(Figure#5A4).#CER2#interactions#with#CER6,#PAS2,#and#CER10#were#all#7A#to#9Afold#greater#than#the#negative#control,#and#CER2AKCR1#interaction#was#nearly#5Afold#greater# than# the# negative# control.# CER2# coAexpressed# with# itself,# in# the# opposite# luciferase#fusion,#also#interacted,#producing#roughly#7Afold#more#luminescence#than#the#negative#control.##88###Figure$513:$Schematic$representation$of$the$split$luciferase$assay$Each#technical#replicate#was#normalized#using#the#CER2AHMG1#interaction#pair# infiltrated#symmetrically#to#the#same#N.)benthamiana# leaf.#While#luminescence#of#the#CER2ACER6#pairs#is#different#between#the#two#technical#“replicates”#shown#here#at#the#left#and#centre,#the#ratio#between#light#emitted#by#the#test#and#control# pairs# is# the# same.# On# the# right,# the# second# negative# control# used,# ACA2,# produces# the# same#luminescence#paired#with#CER2#as#does#HMG1.####Figure$514:$Split$luciferase$assay$of$CER2$paired$with$core$components$of$the$fatty$acid$elongase$$NL=#NAterminal#fragment#of# luciferase,#CL=#CAterminal#fragment#of# luciferase.#All#proteins#were#fused#to#both# NA# and# CAterminal# fragments# of# luciferaseK# the# pair# that# showed# the# greatest# luminescence# is#presented.#ACA2#=#calcium#ATPase,#an#ERAlocalized#protein#used#as#a#negative#control.#Bars#are#mean#+/A#SD,#n=3,#asterisks#denote# significance# in#a#Student’s#TAtest# compared# to# corresponding#assay#with#CER2#paired#with#ACA2,#p<0.05.#CER2%NLuc*+*Cluc%CER6CER2%NLuc*+*Cluc%HMG1CER2%NLuc*+*Cluc%CER6CER2%NLuc*+*Cluc%HMG1CER2%NLuc*+*Cluc%ACA2CER2%NLuc*+*Cluc%HMG1051015CER2(NL+CL(CER2CL(CER2+CER2(NLCER2(NL+CL(CER6CL(CER2+KCR1(NLCL(CER2+PAS2(NLCER2(NL+CL(CER10CER2(NL+CL(ACA2CL(CER2+ACA2(NLLuminescence+relative+to+N/C(CER2+++N/C(HMG(CoA+reductase * ** ***89##5.2.2.2$ Co1Immunoprecipitation$To#confirm#that#CER2#and#CER6#physically#interact,#I#attempted#to#coAimmunoprecipitate#the#two#proteins#from#solubilized#microsomes#of#yeast#cells#coAexpressing#epitopeAtagged#CER2#and#CER6.#A#triple#hemagglutinin#tag#was#fused#to#the#NAterminus#of#CER2,#and#a#triple#cAMYC#tag# was# fused# to# the# NAterminus# of# CER6.# Both# were# constitutively# expressed# in# yeast# cells#using#the#GPD#promoter.#Microsomes#were#prepared#and#solubilized#from#cells#coAtransformed#with#HA3ACER2#and#MYC3ACER6#constructs.##Solubilization# of# the# microsomes# was# inconsistent.# Frequently,# more# protein# was#detected# in# the# insoluble# fraction# compared# to# the# soluble# fraction# used# for# the#immunoprecipitation,#and#the#efficiency#of#CER2#and#CER6#solubilization#did#not#always#match.#Therefore,#the#efficiency#of#each#solubilization#was#checked#by#dot#blot#before#proceeding#with#immunoprecipitation#and#western#blot.##antiAcAMYC# antibody# coupled# with# proteinAA/G# beads# were# used# to# immunoprecipitate#MYC3ACER6.#MYC3ACER6#was#not# consistently#pulled#downK# some#MYC3ACER6#could#almost#always# be# detected# in# the# free,# unbound# fraction.# However,# when# MYC3ACER6# was#immunoprecipitated,#so#was#HA3ACER2#(Figure#5A5).#This#result#confirms#that#CER2#and#CER6#interact.# Further,# given# that# HA3ACER2# and#MYC3ACER6# were# heterologously# expressed# and#isolated# from# yeast# cells,# coAimmunoprecipitation# of# CER2# indicates# that# the# two# proteins#interact#directly.####90#########Figure$515:$Co1Immunoprecipitation$of$HA31CER2$with$MYC31CER6$Insoluble,#soluble,# free#and#bound# fractions#of#yeast#microsomes#separated#by#SDSAPAGE#and#probed#with# either# antiAMYC# (top)# or# antiAHA# (bottom).# The# soluble# fraction# was# the# input# for# coAimmunoprecipitation.#The#band#visible#below#CER6# in# the#bound#product# lane# is# the#heavy#chain#of# the#immunoglobulin#used#for#the#immunoprecipitation.####HA3$CER2MYC3$CER691###5.2.2.3$ CER6$is$required$for$correct$localization$of$CER2$Localization#of#CER2#to#the#ER#membrane#is#curious#in#that#CER2#does#not#have#an#ERAtargeting# signal,# nor# any# transmembrane# domains.# Additionally,# other#members# of# the# BAHD#acytransferase# family# have# all# been# found# to# be# soluble,# cytosolic# proteins.# I# suggested# in#Chapter#3#that#CER2#interactions#with#CER6#might#be#required#for#correct#CER2#localization#to#the#ER.#To# test# this# hypothesis,# localization#of# the#CER2AGFP#protein# (construct# described# in#section# 2.3.1,# initial# experiment# in# section# 3.2.2)# was# examined# in# a# null# cer6# mutant#background,#ew2.#The#ew2)mutant#was#a#gift#from#Dr.#Ellen#WismanK#the#mutant#was#generated#in# a# screen# for# phenylpropanoidAdeficient#mutants# using# the#En/1# transposable# element# as# a#mutagen# (Wisman#et#al.,# 1998).# In#ew2,# the# transposable#element# is# located#782#bp# from# the#start# codon#of#CER6# (Figure#5A6A),# and#no# fullAlength#CER6# transcript# is#produced# (Figure#5A6B).# The# En/1) transposable# element# is# mobile# in# Arabidopsis,# but# no# reverting# or# chimeric#progeny#were#observed.#Analysis#of#the#stem#cuticular#wax#of#ew2#revealed#a#97%#reduction#in#the#stem#cuticular#wax#load#(Figure#5A6C).#The# ew2# mutant# was# crossed# with# cer2# mutants# carrying# the# 35Spro::CER2/GFP#construct.# F2# progeny# carrying# the# transgene#were# selected# by# sowing# seeds# on# hygromycin,#and# genotypes# of# the# ew2) and# cer2/5# mutants# were# determined# by# PCRAbased# genotyping.#Three# double# mutant# lines# derived# from# independent# 35Spro::CER2/GFP# transgenic# plants#were# selected# for# analysis,# as# well# as# three# cer2/5# single# mutants# (also# independent# lines#carrying#the#35Spro::CER2/GFP)transgene)#for#comparison.##As# described# in# section# 3.2.3,# GFP# fluorescence# in# the# cer2/5# single# mutant# was#consistently# observed# in# a# fine,# bright,# reticulate# pattern.# In# the# cer2/5) ew2# double# mutant#background,# GFP# fluorescence# was# diffuse,# taking# shape# in# the# contours# of# organelles.# This#pattern#of# fluorescence#matched#that#of# free,#cytosolic#GFP.#This#result# indicates#that#CER6#is#needed#for#localization#of#CER2#to#the#ER.#92######Figure$516:$Characterization$of$the$ew2$mutant$allele$of$CER6$A,#En/1# insertion#site# in# the# first#exon#of#CER6.#The#En/1# transposable#element# is# represented#with#an#open#white#box.#The#dark#grey#box#marked#with#an#asterix#adjacent#to#En/1#represents#additional,#chimeric#sequence#that#was# found# inserted#between#En/1#and#the#CER6#gene.#A#part#of# this# fragment# is# identical# to#nonAcoding#sequence# downstream# of# the# gene# At1g68490,# and# a# second# part# is# identical# to# nonAcoding# sequence#downstream#of#At1g68875.#Coding#sequence#of#CER6# is#marked#with#a#solid#black#box,#and#the#untranslated#regions# (UTRs)# are# marked# in# pale# grey# boxes.# Introns# and# unAtranscribed# flanking# genomic# DNA# is#presented#as#a#straight#black# line.#B,#RTAPCR#of# fullAlength#CER6# transcript# in#wildAtype#ColA0#(left)#and#ew2)(right).#No#fullAlength#transcript#was#detected#in#the#ew2#mutant.#C,#WildAtype#and#ew2#total#stem#wax#loads#and,#D,# stem#wax# compositions.# Load#and# composition# are# both#measured#as# the# average#of# at#least#four#biological#replicates,#with#error#bars#representing#SD.#200bp5’ 3’ATG+ TAAEn#1ACGCCTAATTACTATC *A0246810121416Col*0ew20510152025303540Col*0 ew2/Wax/Load/µg/cm2e 2DC93######Figure$517:$Localization$of$CER21GFP$in$cer2=5$and$cer2=50ew2$mutant$backgrounds$cer2/5) and# cer2/5) ew2# individuals# derived# from# the# same# lines# carrying# the# CER2/GFP) transgene.#Images#were#taken#10#cm#from#the#stem#apex,#where#fine,#reticulate#ER#can#be#easily#and#consistently#observed.#Scale#bar#=#10#µm.#94##5.2.3$ CER21LIKE$substrate$specificity$is$determined$by$an$N1terminal$sequence$Conserved#domains#and#motifs#often#help#predict#protein#function.#The#only#domain#annotated# for# the#CER2ALIKE#proteins# is#a# transferaseK# this#designation# is#questionable# in#light#of#the#fact#that#the#HXXXD#catalytic#motif#of#this#domain#is#not#required#for#CER2ALIKE#function.# To# break# down# the# amino# acid# sequences# of# CER2ALIKEs# into# functional# units,#sequence# swaps# were# carried# out# between# CER2/LIKE1# and# CER2/LIKE2# coding#sequences.# The# chimeric# genes# were# then# expressed# in# a# yeast# elongation# assay# to#determine#what#sequences#affect#CER6/CER2ALIKE#product#specificity.##CER2ALIKE1#and#CER2ALIKE2#were#selected#for#this#experiment#because#they#have#the# greatest# sequence# identity# among# the#CER2ALIKE#proteins# of#Arabidopsis# (Table# 5A1)#but# they# have# distinct# substrate# specificities.# Percent# identity#was# determined#with#Clustal#2.1.##Table$511:$Percent$identity$matrix$of$CER21LIKE$proteins$from$Arabidopsis$######### Primers#used#for#domain#swaps#are#listed#in#Table#2A7.#All#CER2ALIKE#chimera#and#control# constructs#were#cloned# in# the#p423#vector#with#histidine#selection,#using# the#same#restriction#sites#and#with#identical#AAAA#Kozak#consensus#sequences#inserted#upstream#of#the# ATG# start# codon# (S.# Nakagawa# et# al.,# 2007).# All# of# the# chimeras# generated# for# this#experiment# are# illustrated# in# Figure# 5A8,# and# the# swap# sites# are#marked# in# the# sequence#alignment# presented# at# the# beginning# of# this# chapter# (Figure# 5A1).# The# swap# sites# were#selected# in# regions# of# identical# sequence# for# CER2ALIKE1# and# CER2ALIKE2,# so# that#chimeras# could# be#generated#by#overlap#extension#PCR.#All# chimeras#were# coAexpressed#with# the#CER6#condensing#enzyme.#Unmodified#CER2ALIKE1#and#CER2ALIKE2#constructs#were#used#as#controls.#The# first#pair#of#chimeras,#A#and#B,#have#sequence#swap#sites# just#downstream#of#the# predicted# HXXXD# motif.# Chimera# A# has# the# NAterminus# of# CER2ALIKE1# and# the# CAterminus# of# CER2ALIKE2K# Chimera# B# has# the# NAterminus# of# CER2ALIKE2,# and# the# C#$$ CER2$ CER21LIKE1$ CER21LIKE2$ CER21LIKE3$ CER21LIKE4$CER2$ 100.00# 36.23# 37.37# 21.90# 19.82#CER21LIKE1$ 36.23# 100.00# 65.55# 21.46# 21.43#CER21LIKE2$ 37.37# 65.55# 100.00# 22.36# 20.61#CER21LIKE3$ 21.90# 21.46# 22.36# 100.00# 25.57#CER21LIKE4$ 19.82# 21.43# 20.61# 25.57# 100.00#95##terminus# of#CER2ALIKE1.#Chimera#A# had# no# activityK# the# yeast# cells# coAexpressing#CER6#and#chimera#A#had#the#same#FAMEs#profile#as#cells#transformed#with#only#CER6.#Chimera#B#had# the#same#substrate#specificity#as#CER2ALIKE2,#producing#30Acarbon#VLCFA,#and#a#lesser# amount# of# 32Acarbon#VLCFA# (Figure# 5A9).# The# position# of# the# sequence# swap# site#might# have# produced# an# unstable# chimera# A,# so# a# second# set# of# chimeras# was# created,#chimeras#C/D/E/F,# with# the# swap# site#moved# both# upA# and# downAstream# of# the# swap# site#selected#for#chimeras#A#and#B.#Chimeras#C# and#D# have# a# swap# site# positioned# one# third# of# the# coding# sequence#downstream#from#the#ATG,#and#chimeras#E#and#F#have#a#swap#site#positioned#two#thirds#of#the# coding# sequence#downstream# from# the#ATG.#C#and#E#have# the#NAterminus#of#CER2ALIKE1#and# the#CAterminus#of#CER2ALIKE2.#Both#had#activity# similar# to#CER2ALIKE1#when#coAexpressed#with#CER6#in#yeastK#that#is,#they#produced#small#amounts#of#both#32A#and#34Acarbon#VLCFAs.#D#and#F,#on#the#other#hand,#have#the#NAterminus#of#CER2ALIKE2#and#the#CAterminus#of#CER2ALIKE1,#and#both#had#similar#activity#to#CER2ALIKE2.#The#major#product#of#elongation#with#these#chimeras#was#30Acarbon#fatty#acid,#with#some#32Acarbon#fatty#acid#accumulating# as# well# (Figure# 5A9).# These# results# indicate# that# product# specificity# is#determined#by#the#NAterminal#third#of#the#CER2ALIKE1#and#CER2ALIKE2#protein#sequences.#Another# set# of# chimeras# was# generated# to# narrow# down# the# NAterminal# sequence#required#for#product#specificity.#Chimeras#G#and#H#have#a#swap#site#positioned#roughly#100#amino# acids# from# the# start# codon.# Chimera# G# has# the# 100# NAterminal# amino# acids# from#CER2ALIKE1,# and# displayed# activity# similar# to# CER2ALIKE1.# Chimera# H,# which# has# the#opposite# composition,# had# activity# similar# to# CER2ALIKE2# (Figure# 5A9).# The# last# set# of#chimeras,#I#and#J,#finally#broke#this#trend,#as#neither#had#activity#that#could#be#described#as#similar#to#either#CER2ALIKE1#or#CER2ALIKE2.#Chimera#I,#which#has#only#the#NAterminal#50#amino#acids#of#CER2ALIKE1#and#the#remaining#sequence#from#CER2ALIKE2,#was#similar#to#CER2ALIKE2# in# that# its#major# product#was# 30Acarbon#VLCFA#with# a# lesser# amount# of# 32Acarbon# VLCFA.# However,# the# yield# of# both# of# these# products# with# chimera# I# was# roughly#50%#that#of#CER2ALIKE2.#Chimera#J,#which#has#the#NAterminal#50#amino#acids#from#CER2ALIKE2#and# the# remaining#sequence# from#CER2ALIKE1,#produced#30Acarbon#VLCFA#and#a#large# amount# of# 32Acarbon# VLCFA,# which# is# not# a#major# product# for# any# of# the# wildAtype#Arabidopsis# CER2ALIKE# proteins# (Figure# 5A9).# These# results# indicate# that# NAterminal# 100#amino# acid# fragments# of# CER2ALIKE1# and# CER2ALIKE2# are# necessary# to# confer# product#specificity,#and#that#further#divisions#within#these#proteins#disrupt#specificity.###96##5.2.4$ The$N1terminal$sequence$is$not$sufficient$for$CER21LIKE$activity$The#100#NAterminal#amino#acids#of#CER2ALIKE1#and#CER2ALIKE2#are#necessary#to#confer# their# product# specificity#when#paired#with# the#CER6#condensing#enzyme.#However,#whether#this#sequence#is#sufficient#for#CER2ALIKE#activity#is#unclear.#To#test#this,#the#CER2ALIKE2# protein# was# truncated# after# only# the# first# 100# amino# acids,# and# coAexpressed# with#CER6#in#yeast.#Not#surprisingly,#the#truncated#CER2ALIKE2#was#not#functional#(Figure#5A9).##Cleaving# the#CAterminal# fragment# from#CER2ALIKE2,#which#accounts# for#more# than#75%#of# the#protein,# is#a#very#drastic#modification.#The# tertiary#structure#of# the#NAterminus,#and#by#extension#any#putative#function#of# the#NAterminus,#could#easily#be#compromised#by#this#truncation.#Therefore,#the#NAterminal#fragment#of#CER2ALIKE2#was#tagged#onto#CER2ALIKE3# and# aliphatic# suberin# feruloylAtransferase# (ASFT)# proteins# with# their# NAterminal#fragments#removed.#This#was#done#in#an#attempt#to#provide#the#NAterminal#peptide#fragment#of# CER2ALIKE2# some# structural# support.# CER2ALIKE3# and# ASFT# were# selected# as# they#have#high#and# low# levels#of# sequence# identity#with#CER2ALIKE2,# respectively.#Unmodified#ASFT# and#CER2ALIKE3#were# also# coAexpressed#with#CER6# as# controls# in# the# elongation#assay.#At#the#time#that#this#experiment#was#designed,#it#was#not#known#that#CER2ALIKE3#is#a# CER2ALIKE.# It# was# only# clear# that# the# gene# encoding# CER2ALIKE3,# At4g29250,# was#homologous#to#BAHDs,#was#not#characterized,#and#had#relatively#high#sequence#identity#to#CER2ALIKE2.# Because# the# phylogenetic# tree# used# as# reference# (Yu# et# al.,# 2009)# did# not#cluster#At4g29250#with#the#other#CER2ALIKEs,#it#was#not#expected#to#have#similar#activity.#)The# entire# ASFT# protein# did# not# function# with# CER6,# and# neither# did# chimera# K,#which# has# the# NAterminal# sequence# of# CER2ALIKE2# tagged# to# the# CAterminus# of# ASFT.#Unmodified#At4g29250#elongated#VLCFAs# to# 30Acarbons.#This# experiment# serendipitously#revealed# that# there# was# something# important# missing# from# the# phylogeny# I# used# as#reference.# Chimera# L,# which# has# the# NAterminus# of# CER2ALIKE2# tagged# on# to# the# CAterminus#of#At4g29250,#was#nonAfunctional#(Figure#5A9).#This#demonstrates#that#the#role#of#the# 100#NAterminal# amino# acids# in# product# specificity# is# a# character# unique# to# the#CER2ALIKE1#A#CER2ALIKE2#pair.##97### #Figure$518:$CER21LIKE1$and$CER21LIKE2$chimeras$used$for$yeast$elongation$assay$with$CER6$Chimeras#were#generated#by#overlap#extension#PCR.#CER2ALIKE1#sequence#is#represented#in#blue,#CER2ALIKE2#sequence#in#red,#ASFT#in#gold,#and#CER2ALIKE3#in#green.#CER2-LIKE1 CER2-LIKE2 A B C D G H I J S5 S4 S2 S1 47 aa 106 aa 151 aa 180 aa E F 270 aa S3 LKAt4g29250 ASFT 98###Figure$519:$Elongation$assay$of$chimeric$CER21LIKE$proteins$with$CER6$in$S.0cerevisiae$Values#are#averages#of#at#least#four#biological#replicates,#and#error#bars#represent#standard#deviation.99##5.2.5$ Insight$from$CER21LIKE$structural$models$$Crystal# structures#have#been# solved# for# several# plant#BAHD#acyltransferasesK# vinorine#synthase# from# Rauvolfia) (Ma# et# al.,# 2005),# anthocyanin# malonyltransferase# from#Chrysanthemum# (Unno# et# al.,# 2007),# hydroxycinnamoyl# transferases# from# Sorghum) bicolor#(Walker# et# al.,# 2013)# and# Coffea) canefora# (Lallemand# et# al.,# 2012),# and# a# promiscuous#malonyltransferase# from# tobacco# (Manjasetty#et#al.,# 2012)# (Protein#Databank).#There#are#also#crystal# structures# available# for# several# unrelated# acyltransferases# that# have# similar# catalytic#functions# to# BAHDs.# While# there# are# no# structures# available# for# CER2ALIKEs,# homology#modelling# based# on# known# BAHD# structures# has# the# potential# to# reveal# similarities# and#differences# both# among# CER2ALIKEs# and# between# CER2ALIKEs# and# BAHDs# that# might# cast#light#on#CER2ALIKE#metabolic#function.##Phyre2#(protein#homology/analogy#recognition#engine)#was#used#to#model#all#five#CER2ALIKEs#from#Arabidopsis#(Kelley#and#Sternberg,#2009).#Phyre2#compares#a#query#sequence#with#sequences# of# proteins# with# known# crystal# structures.# A# protein# that# will# serve# as# the# best#structural#template#for#the#query#is#selected#based#on#sequence#homology,#secondary#structure#homology,# and# the# presence# of# insertions# and# deletions.# An# alignment# between# the# template#and# the# query# is# then# used# to# generate# a# tertiary# structure# prediction# for# the# query.# Phyre2#identified#hydroxycinnamoylACoA#shikimate#hydroxycinnamoyl#transferase#from#Sorghum)bicolor#(SbHCT)#as#the#best#structural#template#for#CER2,#and#generated#a#model#for#CER2#based#on#SbHCT#that#covers#96%#of#the#CER2#sequence#(Figure#5A10).##The#position#of#the#NAterminal#sequence#that#affects#substrate#specificity#(Section#5.2.3)#was# examined# on# the# CER2# model.# Largely,# this# peptide# sequence# was# positioned# on# the#surface#of#the#protein,#removed#from#a#channel#running#through#its#core.#That#a#surface#feature#of#CER2# could# determine# its# product# specificity# supports# the# notion# that# the# activity# of# CER2#could# be#mediated# by# physical# interaction# with# other# proteins,# namely# the# CER6# condensing#enzyme.#Structural# models# for# all# of# the# remaining# CER2ALIKE# proteins# were# also# generated#based#on#SbHCT#(Figures#5A11,#5A12,#5A13,#5A14).#With#the#exception#of#CER2ALIKE4,#all#of#the#CER2ALIKE#models# had# comparable# shapes,# with# channels# that# passed# through# the# core# of#each#protein.#The#NAterminal#portions#of#the#peptides#were#largely#positioned#on#the#surface#of#each#protein.#A# channel# running# through# the# core# the# protein# was# evident# in# CER2# model.#Interestingly,#the#predicted#catalytic#histidine#of#the#HXXXD#motif#(H166)#was#positioned#with#its#imidazole# side# chain# pointing# inwards# near# the# center# of# the# channel,# similar# to# true# BAHD#100##acyltransferases.# Examination# of# other# residues# lining# the# channel# revealed# the# presence# of#another#histidine,#H38,#positioned# in#very#close#proximity#and#with#similar#orientation# to#H166,#despite# being# distant# from# the# catalytic# motif# in# the# amino# acid# sequence.# Notably,# H38# lies#within#the#NAterminal#sequence#that#affects#product#specificity.#It# is# clear# from# the# CER2ALIKE# sequence# alignment# (Figure# 5A1)# that# CER2ALIKE1,#CER2ALIKE2,# and# CER2ALIKE3# all# have# NAterminal# histidine# residues# that# align# with# H38# of#CER2K# the# only# CER2ALIKE# that# lacks# this# NAterminal# histidine# residue# is# CER2ALIKE4.#Importantly,#the#positions#of#the#NAterminal#histidines#in#each#CER2ALIKE#structural#model#were#also#consistently# in#close#proximity# to# the#HXXXD#histidine# (Figures#5A10,#5A11,#5A12,#5A13,#5A14).# It#seems#possible# that# these#NAterminal#histidines#could# take# the# role#ascribed# to#HXXXD#histidine#residues#based#on#sequence#homology,#and#act#as#base#catalysts#for#an#acyl#transfer#reaction.# This# would# explain# why# the# HXXXD# histidine# is# absent# from# some# CER2ALIKEs#(CER2ALIKE1)# and# is# simply# not# required# for# function# in# others# (CER2).# However,# it# remains#completely#obscure#how#an#acyl# transfer# could#be#useful# in# the#process#of#elongation,# should#CER2ALIKEs#have#such#activity.#To#determine#if#this#secondAsite#NAterminal#histidine#is#required#for#CER2# function# in#VLCFA#elongation,# and#by#extension,#whether#CER2ALIKEs#are# likely# to#contribute# to# elongation# via# acyl# transfer# function,# siteAdirected#mutagenesis# on#H38#of#CER2#was#performed#(Section#5.2.6).#101###Figure$5110:$Homology1based$model$of$CER2$tertiary$structure$The#NAterminal# 100#amino#acids# are# colored# blue,# the# histidine# of# the# predicted#HXXXD#catalytic#motif#(H166)# is# colored# orange,# and# a# second,# NAterminal# histidine# residue# (H38)# is# colored# magenta.# Side#chains#of#both#labelled#histidines#point#in#towards#a#predicted#channel.#102###Figure$5111:$Homology1based$model$of$CER21LIKE1$tertiary$structure$The#NAterminal# 100#amino#acids#are# colored#blue,# the#asparagine#which# substitutes# for# histidine# in# the#predicted#HXXXD#catalytic#motif#is#colored#orange,#and#a#second,#NAterminal#histidine#residue#is#colored#magenta.#Side#chains#of#both#labelled#residues#point#in#towards#a#predicted#channel.##103###Figure$5112:$Homology1based$model$of$CER21LIKE2$tertiary$structure$The#NAterminal#100#amino#acids#are#colored#blue,#the#histidine#of#the#predicted#HXXXD#catalytic#motif#is#colored# orange,# and# a# second,# NAterminal# histidine# residue# is# colored# magenta.# Side# chains# of# both#labelled#histidines#point#in#towards#a#predicted#channel.##104###Figure$5113:$Homology1based$model$of$CER21LIKE3$tertiary$structure$The#NAterminal#100#amino#acids#are#colored#blue,#the#histidine#of#the#predicted#HXXXD#catalytic#motif#is#colored# orange,# and# a# second,# NAterminal# histidine# residue# is# colored# magenta.# Side# chains# of# both#labelled#histidines#point#in#towards#a#predicted#channel.##105###Figure$5114:$Homology1based$model$of$CER21LIKE4$tertiary$structure$The#NAterminal# 100#amino#acids#are# colored#blue,# the#asparagine#which# substitutes# for# histidine# in# the#predicted#HXXXD#catalytic#motif#is#colored#orange.####106##5.2.6$ CER21LIKEs$do$not$require$a$second$histidine$in$the$substrate1binding$pocket$for$function$SiteAdirected#mutagenesis#was#used#to#establish#whether#the#NAterminal#H38#of#CER2#is#required#for#its#function#in#VLCFA#elongation.#H38#was#replaced#in#the#CER2#coding#sequence#with#an#alanine#(H38A)#to#abolish#any#catalytic#activity.# I#also#attempted#to#generate#a#gainAofAfunction#mutant# allele# of#CER2ALIKE4# by# inserting# a# short# peptide# sequence# from#CER2# that#includes#H38,#and#the#surrounding#fragment#of#the#NAterminal#sequence#that#protrudes#into#the#channel#of#CER2.#The#sequence#GIFRRTV#of#CER2ALIKE4#was#replaced#by#LHYV#from#CER2#by#overlap#extension#mutagenesis.#The#gainAofAfunction#experiment#was#undertaken#with#some#skepticism,# as# the# predicted# structure# of# CER2ALIKE4# is# very# obviously# unlike# that# of# other#CER2ALIKEs.# There# are#many# possible# reasons# beyond# the# presence# or# absence# of# putative#catalytic#residues#why#CER2ALIKE4#might#not#contribute#to#elongation.#The#mutant#alleles#were#cloned# into#p42X#yeast#expression#vectors.#The#mutant#alleles,#and#unmodified#controls,#were#then#coAtransformed#with#a#CER6#expression#construct#into#yeast.#The# H38A# allele# of# CER2# retained# its# activity# in# a# yeast# fatty# acid# elongation# assay.#Further,#there#was#no#gainAofAfunction#for#the#mutagenized#CER2ALIKE4#protein#(Figure#5A15).#I#conclude#that,#like#the#HXXXD#histidine,#the#NAterminal#histidine#of#CER2#is#not#required#for#its#activity# in# VLCFA# elongation.# This# experiment# strongly# suggests# that# the# role# of# CER2,# and#CER2ALIKEs,#does#not#involve#acyl#transfer#activity.###107###############Figure$5115:$Elongation$assay$of$mutagenized$CER21LIKEs$with$CER6$in$Saccharomyces$The#H38#residue#does#not#affect#CER2#activity#in#VLCFA#elongation.#####00.511.522.533.526:0 28:0 30:0Percent0total0FAMEs w3031aCER6CER60+0CER20WTCER60+0CER20SDMCER60+0CER2ALIKE40WTCER60+0CER2ALIKE40SDM108###5.3$ Discussion$#5.3.1$ CER2$physically$interacts$with$the$fatty$acid$elongase$complex$The# phenotypes# of# Arabidopsis# cer2/like# mutants# indicate# that# this# gene# family# is#necessary# for# the# elongation# of#VLCFA#precursors# of# cuticular#waxes.# That#CER2ALIKEs# and#CER6#function#together#in#yeast#to#produce#30Acarbon#VLCFAs#(and#longer)#demonstrates#that#their#combined#activity#is#sufficient#for#VLCFA#elongation.#How#CER2ALIKEs#affect#elongation#is#not#clear,#but#given#that#condensing#enzymes#determine#the#substrate#specificity#of#the#fatty#acid#elongase,#and# that#CER2#requires#CER6#for#activity# in#yeast,#physical# interaction#between#the#two#proteins#is#likely.#Interaction#would#be#necessary#if#CER2ALIKEs#were#to#somehow#alter#the#substrate# binding# capacity# of#CER6,# or# to# feed# substrate# to#CER6.#Here,# physical# interaction#between#CER6#and#CER2#was#demonstrated#using#three#experimental#approaches.#A# split# luciferase# assay# was# used# to# test# CER2# interactions# with# all# four# core#components# of# the# fatty# acid# elongase.# I# paired# CER2# with# CER6,# and# the# three# generalist#components#of#the#elongase:#ketoacylACoA#reductase#1#(KCR1),#hydroxyacylACoA#dehydratase#(PAS2),# and# enoylACoA# reductase# (CER10).# CER2# interacted# with# all# of# these# proteins,#suggesting#that#CER2#is#in#close#physical#proximity#to#the#entire#elongase#complex.#Notably,#a#positive#result#in#the#splitAluciferase#assay#does#not#indicate#a#direct#proteinAprotein#interaction,#as#the#two#halves#of#luciferase#could#reconstitute#the#functional#enzyme#if#the#proteins#to#which#they#are#tagged#are#simply#in#close#proximity.#CER2ACER6# interactions# were# next# examined# by# coAimmunoprecipitation# of# the#ectopicallyAexpressed# and# epitopeAtagged# proteins# from# yeast.# The# epitopeAtagged# proteins#were# functional# in# an# elongation# assay.#While# solubilization# and# precipitation# of# CER6#was# a#constant#challenge,#CER2#did#consistently#coAprecipitate#with#CER6.# It# is#possible# that#CER6ACER2#interaction#is#mediated#by#a#third#interacting#partner.#However,#in#that#case,#highly#similar#orthologs#of#such#a#protein#would#need#to#be#present# in#Arabidopsis#and#in#yeast.#This#seems#improbable# given# that# CER2ALIKEs# are# not# found# in# yeast.# Thus,# CER2ACER6# interaction# is#likely#direct.#Finally,#to#determine#whether#physical#interaction#between#CER2#and#CER6#is#the#basis#for# CER2# localization# to# the# ER,# localization# patterns# of# CER2AGFP# in# the# presence# and#absence#of#CER6#protein#were#compared.# Introduction#of#CER2AGFP#into#cer2#complemented#the#mutant# phenotype,# and# resulted# in# a# distinct,# tight,# webAlike# pattern# of# GFP# fluorescence#characteristic# of# ER# localization.# In# contrast,# CER2AGFP# in# the# cer2) ew2# double# mutant#109##background# had# a# more# diffuse# pattern# of# fluorescence# that# strongly# resembled# free# GFP#fluorescence# patterns# in# the# cytosol# of# stem# epidermal# cells.# This# result# demonstrates# that#CER6#is#required#for#CER2#association#with#the#ER.###5.3.2$ CER21LIKEs$are$not$acyltransferases$The# presence# of# a# conserved# HXXXD# motif# is# nearly# ubiquitous# among# BAHD#acyltransferases,#and#there#is#robust#evidence#that#the#histidine#residue#in#this#motif#is#essential#for#acyl#transfer#activity.#SiteAdirected#mutagenesis#of#the#predicted#catalytic#histidine#residues#of#anthocyanin#malonyltransferase#of#Salvia)splendens#(Suzuki#et#al.,#2003),#vinorine#synthase#of#Rauvolfia) serpentina# (Bayer# et# al.,# 2004),# hydroxycinnamoyltransferase# of# Sorghum) bicolor#(Walker# et# al.# 2013),# and# hydroxycinnamoyltransferase# of#Coffea) canefora# (Lallemand# et# al.,#2012)# have# all# demonstrated# that# this# residue# is# essential# for# metabolic# function.# The# only#exception# to# this# rule# that# I# am# aware# of# is# the# baccatin# III:3AaminoA3Aphenylpropanoyltransferase#(BAPT)#enzyme#from#Taxus,#which#is#required#for#the#synthesis#of#taxol.# In# this#protein,# the#catalytic#histidine# is#substituted#by#a#glycine.#An#amine#group#beta# to#the# CoA# of# the# acyl# donor# has# been# suggested# to# functionally# replace# the# histidine# and#deprotonate# the# hydroxyl# group# of# the# acyl# acceptor# to# create# a# nucleophile# (Walker# et# al.,#2002).## Histidine#166#of#the#CER2#amino#acid#sequence#was#predicted#to#be#a#catalytic#residue#based# on# alignment# to# BAHD# acyltransferase# protein# sequences.# Structural#models# of# CER2#also#placed#H166#in#a#similar#position#to#that#described#for#characterized#BAHDs.#SiteAdirected#mutagenesis#converting# the#H166#of#CER2# to#an#alanine#or#an#asparagine# revealed# that,# just#like#CER2ALIKE1,#CER2#does#not# require# the#HXXXD#sequence# for#activity.#Therefore,#CER2#and# the# CER2ALIKEs# represent# a# clade# with# biochemical# functions# distinct# from# those# of#characterized#BAHD#family#members.#Arguably,#they#may#not#be#acyltransferases#at#all.#There#are#no#histidine#residues#neighboring#H166#in#the#CER2#amino#acid#sequence#–#the#closest#histidine#upstream#of#H166# is#32#amino#acids#away,#and# the#nearest#downstream#histidine# is# 22# amino# acids# away.#However,# tertiary# structure#models# of# CER2,# CER2ALIKE1,#CER2ALIKE2,# and#CER2ALIKE3# all# suggested# that# a# histidine# residue# from# the# NAterminus# of#each# sequence# is# positioned# in# very# close# physical# proximity# to# the# HXXXD# histidine,# within#channels#running#through#the#core#of#each#protein.#The#only#CER2ALIKE#that#lacks#this#residue#is#CER2ALIKE4,#which#does#not#appear#to#enhance#elongation.#This#second,#NAterminal#histidine#presented# a# pressing# reason# to# doubt#my# previous# conclusion# that# CER2ALIKEs# entirely# lack#acyl#transfer#activity.##110##I#mutagenized# the#NAterminal#histidine#of#CER2#cDNA,#H38,#and#substituted# it#with#an#alanine.# I#also#swapped# the#short#amino#acid#sequence# from#CER2# that#contains#H38,#LHYV,#into#CER2ALIKE4,#removing#the#sequence#GIFRRTV.#The#exact#regions#used#for#this#sequence#swap#were#selected#based#on#superposition#of#the#structural#models#of#CER2#and#CER2ALIKE4.#Both#mutagenized# CER2# (H38A)# and# CER2ALIKE4# (GIFRRTV>LHYV)# retained# the# activity# of#their# wildAtype# sequences# when# coAexpressed# in# yeast# with# the# CER6# condensing# enzyme.#Therefore,#neither#H166#nor#H38#are#required#for#CER2#function#in#elongation.##Remarkably,# there# is# a# precedent# for# an#NAterminal# histidine# residue# (H35)# situated# in#close# physical# proximity# to# a# true,# midAchain# catalytic# histidine# (H153),# in# the# BAHD#hydroxycinnamoyltransferase#from#Coffea)canefora)(robusta#coffee)#(CcHCT)#(Lallemand#et#al.,#2012).#Analysis#of#the#crystal#structure#of#CcHCT#revealed#piAstacking#interactions#between#the#imidazole# rings# of# the# two# histidine# residues,# which# prompted# the# authors# of# this# study# to#suggest# that# the#NAterminal# histidine# served# to# help# orient# the# catalytic# histidine# for# catalysis.#This# hypothesis# is# supported# by# siteAdirected# mutagenesis:# mutagenesis# of# H153# abolished#CcHCT# activity,# and# mutagenesis# of# H35# reduced# activity.# In# light# of# this# I# infer# that# the# NAterminal#H38#of#CER2#does#not#substitute#for#the#function#formerly#ascribed#to#H166,#but#that#it#is#another#remnant#of#ancestral#BAHD#function.##The# only# evidence# remaining# that# CER2ALIKEs# are# BAHD# acyltransferases# is# their#homology#to#this#protein#family.#However,#examination#of#the#most#conserved#features#of#BAHD#sequences# puts# this# classification# in# question.# CER2ALIKEs# entirely# lack# the# DFGWG# motif#present#at# the#CAterminus#of#most#BAHDs.#The#midApeptide#HXXXD#motif# is#present# in#some,#but#not#all,#CER2ALIKEs,#but#I#have#shown#with#siteAdirected#mutagenesis#that#this#motif# is#not#required#for#CER2ALIKE#function.#I#examined#models#of#CER2ALIKE#structure#for#other#histidine#residues#that#might#function#as#base#catalysts#for#an#acyl#transfer#reaction,#and#identified#H38.#Because# siteAdirected# mutagenesis# demonstrated# that# this# residue# is# also# not# required# for#function,# I# am# left# without# any# evidence# in# support# of# the# notion# that# CER2ALIKEs# are#acyltransferases.# I# conclude# that# while# CER2ALIKEs# are# clearly# related# to# the# BAHD#acyltransferase# family,# the# CER2ALIKE# clade# has# evolved# a# new# function# distinct# from#characterized#members#of#this#diverse#protein#family.###5.3.3$ CER21LIKE$structures$do$not$suggest$a$catalytic$function$The# CER2ALIKE# sequence# chimeras# generated# using# CER2ALIKE1# and# CER2ALIKE2#revealed#that#the#product#specificity#of#elongation#is#determined#by#the#NAterminus#of#these#two#CER2ALIKE#proteins.#This#fragment#could#be#reduced#to#100#amino#acids#without#substantially#111##altering#the#specificity#or#efficacy#of#CER2ALIKE1/CER2ALIKE2#function.#However,#the#truncated#NAterminal#fragment#of#CER2ALIKE2#was#not#sufficient#to#maintain#function,#nor#was#it#functional#when# swapped# onto# two# other# proteins,# the# BAHD# ASFT# or# CER2ALIKE3,# which# both# have#lower# homology# to# CER2ALIKE2# than# does# CER2ALIKE1.# Perhaps# the# NAterminal# peptide#fragment#requires#the#structure#of#the#remainder#of#the#protein#to#support#its#function.#Structural#models#of#all#five#CER2ALIKE#proteins#except#CER2ALIKE4#were#very#similar.#Most#of#the#NAterminal#peptide#was#positioned#on#the#exterior#surfaces#of#the#protein,#but#a#loop#of# each# NAterminal# fragment# extended# into# the# channel.# These# models# cannot# provide#conclusive# information#about# the#biochemical# function#of#CER2ALIKEs,#but# rather#suggest# that#either#an#activity#involving#binding#of#a#ligand,#or#activity#mediated#by#the#surface#of#the#protein,#could#be#the#basis#of#CER2ALIKE#function#in#veryAlongAchain#fatty#acid#elongation.###5.3.4$ Models$of$paired$CER61CER2$activity$At#present,#how#CER2ALIKEs#contribute#to#elongation#is#unclear.#Several#models#can#be#proposed,# however,# based# on# the# results# of# this# chapter# and# prior# characterization# of# CER2ALIKEs# (Figure# 5A16).# In# addition# to# suggesting# biochemical# functions# for# CER2ALIKEs,# I# have#tried#to#account#for#two#related,#outstanding#questions#regarding#the#metabolism#of#cuticular#wax#precursors.#First,#why#do#plant#KCSs#not#produce#acylACoAs#longer#than#28#carbons,#and#why#are#differences#in#KCS#expression#and#activity#alone#insufficient#to#determine#the#upper#limit#of#elongation?#Second,#how#does#an#epidermal#cell#accommodate#the#massive#quantities#of#VLCAacylACoAs#required#to#support#cuticular#wax#biosynthesis?##5.3.4.1$ Providing$a$physical$extension$of$the$CER6$acyl1CoA$binding$pocket$AcylACoA# substrate# specificity# of# plant# KCS# enzymes# has# primarily# been# investigated#using# molecular# genetic# approaches.# Domains# affecting# specificity# have# been# identified# in#domain#swaps#between#orthologous#genes# (Blacklock#and#Jaworski,#2002),#and# residues# that#affect#substrate#specificity#have#been#found#by#QTL#mapping#in#wild#accessions#(Jasinski#et#al.,#2014).# The# structure# of# acylACoA# binding# pockets# of# KCSs# have# only# been# described# using#homologyAbased#models#(Joubès#et#al.,#2008).#Therefore,#while#relative#size#predictions#can#be#compared#between#KCSs,#the#absolute#depths#of#substrate#binding#pockets#are#unknown.#It# is#not#clear#whether#the#substrateAbinding#pocket#of#CER6#can#support#binding#of#26A,#28A,#30A,#or#32A#carbon#acylACoAs.#In#this#first#model#of#CER2ALIKE#activity,#I#propose#that#CER6#alone#can#only#accept#acylACoA#substrates#up#to#26#carbons#in#length,#based#on#the#fact#that#CER6#only#generates#28Acarbon#product#when#expressed#alone#in#yeast#cells.#112##Structural#models#predict# channels# in#all#CER2ALIKEs#except#CER2ALIKE4.#This# is#not#surprising#given# that# the#models#were#generated#based#on#crystal#structures#of#BAHDs.#Since#CER2ALIKEs# are# derived# from# BAHD# acyltransferases# that# bind# acylACoAs,# it# does# seem#reasonable# that# these# channels# could# exist# within# CER2ALIKEs,# and# that# CER2ALIKEs# could#bind#acylACoA#substrates.#Assuming#that#CER2ALIKEs#have#indeed#retained#the#capacity#to#bind#acylACoA#groups,#it#becomes#a#possibility#that#CER2ALIKEs#could#serve#as#a#physical#extension#of# the#substrateAbinding#pocket#of#KCSs#such#as#CER6.#CER2ALIKEs#could#thereby#determine#the#upper#limit#of#the#length#of#acylACoA#that#CER6#accepts#for#condensation#with#malonylACoA#(Figure# 5A16A).# This#model# depends# heavily# on# direct#CER2ALIKEACER6# interaction,#which# is#supported#by#CoIP,#splitAluciferase,#and#CER2AGFP#localization#data.#Structural#determination#of#CER2,#CER6,#and#mapping#of# the# interacting#domains#of#both#proteins#will#be#required# to# test#this#hypothesis.###5.3.4.2$ Manipulation$of$CER6$structure$to$allow$it$to$bind$longer1chain$substrates$Again,# assuming# that# the# size# of# the# CER6# substrateAbinding# pocket# limits# the# chain#length#of#VLCAacylACoA# it# can#accept# for#condensation,#CER2ALIKEs#could#serve#as#allosteric#modifiers#of#CER6#specificity.#CER2ALIKE#binding#could#alter# the# tertiary#structure#of#CER6# in#such#a#way#as#to#reshape#or#expand#the#size#of#the#substrateAbinding#pocket.#Different#CER2ALIKEs# would# interact# in# subtly# different# ways# with# CER6# to# account# for# the# unique# product#specificities#of#CER2ALIKEs#(Figure#5A16B).#This#model#does#not#require#that#CER2ALIKEs#bind#acylACoAs.# However,# it# remains# a# possibility# that# VLCAacylACoAs# or# malonylACoA# could# be#bound#by#CER2ALIKEs#and#affect#protein#structure#or#activity.#This#model#requires#support#in#the#form#of#structural#determination#of#CER6#alone#and#in#the#presence#of#CER2.##5.3.4.3$ Maintaining$a$pool$of$VLC1CoA$substrate$for$CER6$AcylACoAs# are# amphipathic#molecules# that# partition# into# lipid#membranes# (Jolly# et# al.,#1997K# Gossett# et# al.,# 1996),# and# the# affinity# of# acylACoAs# for# membranes# increases# with#increasing#chain#length#(Boylan#and#Hamilton,#1992).#Given#that#greater#than#95%#of#longAchain#acylACoAs#partition#into#microsomes#(Jolly#et#al.,#1997),#I#anticipate#that#28A,#30A,#and#32Acarbon#veryAlongAchain# acylACoA# precursors# of# cuticular# wax# biosynthesis# would# partition# almost#entirely#into#lipid#membranes.##The#structure#of# the# fatty#acid#elongase# is#obscureK#crystal#structures#are#not#available#for#any#of#the#core#elongase#components,#and#the#quaternary#structure#and#stoichiometry#of#the#complex# is# a# complete# mystery.# It# is# not# known# how# substrate# is# received# and# transferred#113##between# elongase# components# within# and# between# elongation# cycles.# However,# it# seems#unlikely# that#VLCAacylACoA#substrate# is#provided# to#plant#KCSs#directly# from#cell#membranes.#Cuticular#lipids#constitute#roughly#50%#of#the#acylAlipid#output#of#stem#epidermal#cells#(Suh#et#al.,#2005),# and,# as# shown# in# Figure# 4A7,# the# 28A,# 30A# and# 32A# carbon# acylACoA# precursors# of#cuticular#waxes#make#up#nearly#20%#of#the#acylACoA#pool#of#whole#rosette#leaves.#If#all#of#the#saturated,# veryAlongAchain# acylACoA# precursors# of# cuticular# wax# were# integrated# in# ER#membranes,# concentrated# around# the# elongase,# I# would# anticipate# that# the# cohesion# and#stability#of#the#membrane#would#be#compromised.#AcylACoA#binding#proteins# (ACBPs)#play#a# role# in# lipid#metabolism#by#solubilizing#acylACoAs,# thereby# making# them# available# for# subAcellular# transport# and# lipid# synthesis,# and#preventing# hydrolysis# of# the# CoA# group.# In# Arabidopsis,# there# are# six# characterized# ACBPs,#which#have#different#physiological# roles#and#binding#affinities# (Xiao#and#Chye,#2011).#Notably,#ACBP1#has#been#found#to#bind#VLCAacylACoAs#up#to#26#carbons#in#length,#but#binds#these#with#lower#affinity#than#longAchain#acylACoAs#(Xue#and#Xia#et#al.,#2014).#ACBP1#has#not#been#tested#with# substrates# longer# than#26# carbons.#This#unknown# is#not# for# lack#of# interestK# longer#VLCAacylACoA#substrates#are#effectively# insoluble#in#aqueous#media,#and#are#therefore#very#difficult#to# work# with# in) vitro.# Nevertheless,# the# low# affinity# of# ACBPs# for# VLCAacylACoAs# in# general#suggests#that#other#proteins#might#be#required#to#make#these#substrates#soluble#in#the#cell.#If#CER2ALIKE#proteins#do#bind#acylACoAs#as#suggested,#they#might#serve#as#specialized#VLCAacylACoA#binding#proteins# that#solubilize#substrate# for#CER6#in# the#cytosol.#Based#on#the#fact#that#CER2#interacts#with#CER6,#VLCAacylACoA#binding#by#CER2ALIKEs#could#also#serve#to#concentrate# substrate# around# the# fatty# acid# elongase# complex.# In# this# model,# specificity# of#CER2ALIKEs# for# chain# lengths# of# acylACoAs#would# determine#what# chain# lengths# of# substrate#would#be#available#for#use#by#CER6#(Figure#5A16C).#Unlike#the#previous#two#models#presented,#this#one#implies#that#CER6#constitutively#accepts#chainAlengths#of#acylACoAs#up#to#32#carbons,#but#is#limited#by#the#availability#of#these#VLCAacylACoAs#in#the#cytosol.#Investigation#of#the#effect#of#CER2/LIKEs# on# the# acylACoA# pool,# the# capacity# of# CER2ALIKEs# and# CER6# to# bind# acylACoAs,#and#distribution#of#acylACoAs#in#different#subAcellular#fractions,#will#be#essential#to#further#explore#this#hypothesis.####114#############################Figure$5116:$Models$of$paired$CER21CER6$activity$#(A)# Active# site# extension.# A# hypothetical# acylACoA# binding# site# in# CER2# could# physically# extend# the#substrateAbinding# pocket# of#CER6.# (B)#Allosteric#modulation.# ProteinAprotein# interaction# between#CER2#and# CER6# could# modify# the# structure# of# the# substrateAbinding# pocket# of# CER6,# enabling# it# to# accept#longer# acyl# chains.# (C)# Substrate# solubilization# and# concentration.# CER2# could# bind,# solubilize,# and#concentrate#acylACoAs#around#the#fatty#acid#elongase.##CER6CER2KCR1PAS2CER10 CER2CER2ER lumen cytosolKCR1PAS2CER10CER6ER cytosolKCR1PAS2CER10CER6ER cytosolCER2KCR1PAS2CER10CER6ER cytosolCER2KCR1PAS2CER10CER6ER cytosol28 carbon acyl-CoA26 carbon acyl-CoAABC115##Chapter$6:$Conclusions$$6.1$ Major$findings$of$this$work$#6.1.1$ Perspective:$why$length$matters$Fatty#acid#elongation# is#a#cornerstone# in#our#model#of#cuticular#wax#metabolism.#While#wax# composition# varies#immensely# among#individual# plant# species,# the# exceptional# length# of#cuticular#waxes#is#a#common#feature#of#these#lipids#in#many#flowering#plants.#Elongation#of#fatty#acids#past#26# carbons# is# the# committed# step# in# cuticular# wax# synthesis# in# Arabidopsis,# with#shorter# chain# lengths# of# VLCFAs# shared# among# wax,# suberin,# seed# oil,# and# sphingolipid#synthetic#pathways.#UpAregulation#of#elongation#beyond#26#carbons# in#young#shoot#epidermal#cells#is#necessary#to#support#the#assembly#of#an#effective#cuticular#barrier,#and#to#fuel#increased#wax#production#in#response#to#environmental#stresses#such#as#cold,#drought,#and#high#light.#At#the#same#time,#elongation#up#to#26#carbons#must#be#precisely#coordinated#to#ensure#sufficient#VLCFA#substrate#is#retained#for#sphingolipid#metabolism,#without#which#the#plant#will#die.###Within#the#range#of#VLCFA#chain#lengths#used#to#make#cuticular#waxes,#some#are#more#readily#taken#up#by#either#the#acyl#reduction#or#alkaneAsynthetic#pathway.#Current#work#on#FARs#(summarized# in# Rowland# and#Domergue,# 2012)#and# CER1# homologs#(unpublished# results#reported# at# the# 2016# International# Symposium#on#Plant# Lipids# and# the# 2015#Plant#Apoplastic#Diffusion# Barriers# Meeting# by# Dr.#Jérôme#Joubès)# indicate# that#enzymes# from# each# of# these#families# have# their# own# substrate# chainAlength# specificities.#Consequently,# the# chain# length#profile#of# the#pool#of#VLCFA#wax#precursors#will# affect# substrate#partitioning#between# the# two#pathways.#It#remains#unclear#what#specific#properties#of#the#cuticle#may#be#attributed#to#different#wax# functional# groups.# It# is# also# unknown# how# specific# chain# lengths# of# waxes# contribute# to#cuticle# structure# and# function,# independent# of# their# downstream# modification.# However,# the#phenotype#of#the#CER6pro::CER2/LIKE1)overAexpression#line#is#evidence#that#changes#in#chain#length#profile#alone#are#sufficient#to#alter#cuticle#structure.#Considering#how#profoundly#chain#length#affects#substrate#channelling#into#cuticular#wax#biosynthesis,#partitioning# of# substrate# between# wax# modification# pathways,# and# the# physical#properties# of# the# plant# cuticle,# understanding# how# the# plant# cell# produces# and# regulates# the#metabolism# of# these# exceptionally# long# acylACoAs# is# key# to# understanding# cuticular# wax#biosynthesis.# Despite# extensive# work# dissecting# the# elongation# of# wax# precursors# in#Arabidopsis,# glaring# deficiencies# in# our# understanding# of# this# process# remain.# For# example,#the#biochemical# functions# of# CER2ALIKEs# remain# unknown.# It# is# also# unclear# why# the#116##elongation#of#VLCFAs#by#KCSs#cannot#exceed#28#carbons.#Further,#it’s#not#apparent#how#plant#cells#can#accumulate#the#large#quantities#of#VLCAacylACoAs#required#to#produce#waxes.#In#this#thesis,# I# have#characterized# the# CER2ALIKE# proteins# from# Arabidopsis# in# an# attempt#to#determine# their# biochemical# function# and# address# some# of# these# other# pressing# questions#concerning#fatty#acid#elongation.###6.1.2$ Summary:$what$we’ve$learned$about$CER21LIKEs$Previous#characterization#of#several#cer2)mutant#alleles# revealed# that#CER2)is# required#for# the# production# of# cuticular# waxes# longer# than# 28# carbons# (McNevin#et# al.,# 1993).#The#CER2)gene#was#cloned#by#position#and#found#to#encode#a#protein#with#homology#to#BAHD#acyltransferases#(Xia#et#al.,#1996).###Similar# to# condensing# enzymes,# CER2# activity# can# be# assayed# in# yeast# (Chapter# 3).#When#coAexpressed#with#the#CER6#condensing#enzyme#that#elongates#VLCFAs#to#28#carbons,#CER2# increases# the#chainAlength#specificity#of#elongation# to#30#carbons.# It# is#clear# that#CER2#cannot# function# independently# to#elongate# fatty#acids# from#28#to#30#carbons,#as#CER2#has#no#activity#when#coAexpressed#with#another#condensing#enzyme#that#produces#28Acarbon#VLCFAs,#LfKCS45.# This# simple# experiment# demonstrates# that# CER2# and# CER6# are# necessary# and#sufficient# to# produce# 30Acarbon# VLCFAs,# which# are# essential# to# cuticular# wax# biosynthesis.#Further,# it# reveals# that#CER6#has#a#property# lacking# from#LfKCS45# that#enables# it# to# function#with#CER2.###There# are# five# CER2/LIKE# genes# in# Arabidopsis,# and# all# of# them# affect# elongation#(Chapter# 4).# In# yeast,# each# CER2ALIKE# has# a# unique# effect# on# the# product# specificity# of#elongation,# and# affects# elongation# only# when# paired# with# the# same# specific# condensing#enzymes.# Analysis# of# cer2/like# mutant# phenotypes# show# that# in# the# plant,# CER2ALIKEs# are#required# to# produce# the# bulk# of# VLCFA# precursors# of# cuticular# lipids.# Further,# the# unique#substrate# specificities# of# different# CER2ALIKEs# fineAtune# elongation# to# determine# the# precise#chainAlength# profile# of# cuticular# waxes# and# pollen# coat# lipids.# Condensing# enzymes# are#absolutely#required#to#produce#VLCFAs#and#until#now#their#activity#and#expression#were#thought#to# control# the#substrate#specificity#of#elongation.#The#contribution#of#CER2ALIKEs# to#substrate#specificity# demonstrated# here# does# not# contradict# this#model,# but# introduces# another# layer# of#complexity#to#our#understanding#of#elongation#and#its#regulation.##Though#biochemical#activities#of#CER2ALIKEs# remain#unknown,#experiments#described#in#this#thesis#have#provided#clues#about#their#function#(Chapter#5).#First,#while#CER2ALIKEs#are#homologous# to# BAHD# acyltransferases,# siteAdirected# mutagenesis# shows# that# CER2# cannot#117##possibly#use#the#same#mechanism#described#for#other#characterized#BAHDs.#CER2/LIKEs#form#a#unique#clade#within#the#BAHD#family#that#modify#the#substrate#specificity#of#elongation#by#an#unknown# mechanism,# which# is# unlikely# to# involve# acyl# transfer.#Second,# the# splitAluciferase#assay,#coAimmunoprecipitation,#and#localization#patterns#of#CER2AGFP#demonstrate#that#CER2#and#CER6#physically#interact.#This#proteinAprotein#interaction#in#and#of#itself#neither#proves#nor#disproves# anything# about# CER2# function.# However,# interaction#between# these# two# proteins#is#requisite#support#for#all#three#of#the#CER2#activity#models#proposed#(Figure#5A16).###6.2$ Future$directions$I# investigated# the# roles# of# CER2ALIKEs# in# an# attempt# to#better# understand# how# plants#synthesize#and#regulate#the#production#of#the#VLCAacylACoA#precursors#of#waxes,#but#my#efforts#fell#short#of#elucidating# their#biochemical# function.#Models# for#paired#CER2ACER6#activity#were#suggested#in#Chapter#5,#along#with#experimental#approaches#that#could#be#taken#to#test#them.#Additionally,# in# Chapter# 4,# next# steps# to# explore# the# physiological# function# of# the# anomalous#CER2ALIKE4#were#proposed.#As#these#immediate#steps#were#discussed#in#previous#chapters,#in#the# final# section# of# this# thesis,# I# will# focus# on# related# questions# that# could# be# pursued# in#future#research. #6.2.1$ The$origin$of$CER2=LIKE0genes$#6.2.1.1$ BAHD$acyltransferases$The#BAHD#acyltransferase# family# is# diverseK# BAHDs# have# a# broad# range# of# substrate#specificities,#and# there# is# very# low# common# sequence# identity# among# these# proteins.# This#breadth# of# form# reflects# the# diversity# of# specialized# metabolic# pathways# that# BAHDs# are#associated#with.#Genes# involved# in#specialized#metabolism#more#readily#adapt#under#selective#pressure#than#genes#associated#with#primary#metabolic#pathways,#as#they#can#neofunctionalize#with# or#without# prior# gene# duplication# (Pichersky# and# Lewinsohn,# 2011K# Pichersky# and#Gang,#2000).#Perhaps# it# is#not#entirely#surprising# then# that#CER2ALIKEs#have#diverged#so#profoundly#from# their# BAHD# ancestors# as# to# lose# acyl# transfer# activity,# and# to# acquire# an# entirely# new#function.# In# fact,# another# example# of# BAHDs# losing# their# catalytic# function# was# recently#published.##Two# hydroxycinnamoyltransferases# (HCTs)# from# maize# have# been# reported# to# have#functions# in# plant# defence,# independent# of# acyl# transfer# activity# (Wang# et# al.,# 2015).# Both#proteins,# HCT1806# and# HCT4918,# interact# with# and# downAregulate# the# activity# of# a#118##constitutivelyAactive# positive# regulator# of# the# hypersensitive# response,# Rp1AD21.# SiteAdirected#mutagenesis# of# both# HCTs# revealed# that# their# predicted,# catalytic# histidine# residues# are# not#required# for# their#negative# regulation#of# the#hypersensitive# response# (Wang#et#al.,# 2015).#The#authors#of#this#study#suggested#that,#as#phenylpropanoid#metabolism#is#sometimes#upAregulated#in#response#to#pathogen#attack,#some#HCT#proteins#may#be#targets#of#pathogen#effectors.#In#a#healthy# plant,# HCT1806# and# HCT4918# would# interact# with# and# disable# Rp1AD21# activity# by#defaultK# upon# infection,# HCT1806# and# HCT4918# would# serve# as# decoys,# in# that# their#modification#by#pathogen#effectors#would#cause#them#to#dissociate#from#Rp1AD21,#and#thereby#enable#the#hypersensitive#response#(Wang#et#al.,#2015K#Wang#et#al.,#2016).##While# this#model# of# HCT# function# is# distinct# from#my#models# for# CER2ALIKE# function,#both#are#examples#of#loss#of#acyl#transfer#function#within#the#BAHD#family.#There#may#be#other#instances#of#BAHDs#losing#their#acyl#transfer#function#that#have#yet#to#be#characterized.#It#will#be#interesting# to# investigate# how# these# divergent# clades# of# BAHDs# arose# from# true# BAHD#acyltransferases,#and#what#their#ancestral#functions#were.###6.2.1.2$ Metabolic$gene$clusters$The#activity#of#CER2ALIKEs#and#other#genes#involved#in#cuticular#wax#metabolism#exist#in# a# grey# area# between# primary# and# specialized# metabolic# processes.# Specialized# metabolic#pathways#have#historically#been# investigated# in#nonAmodel#plants#with#no#sequenced#genome#available.#Hence,# the# identification#and#characterization#of# genes# involved# in# these#processes#has#largely#relied#on#transcriptomic#data.#As#genomic#data#becomes#available#for#an#increasing#number#of#species,#it#is#becoming#apparent#that#genes#required#for#the#synthesis#of#specialized#metabolites#are#often#arranged#in#clusters#reminiscent#of#prokaryotic#operons#–#more#frequently#than# they# are# found# randomly# dispersed# throughout# the# genome# (Osbourn# and# Field,# 2009K#Nützmann#and#Osbourn,#2014).#This#emerging#aspect#of#metabolic#evolution#will#be#particularly#exciting# to# explore# in# the# context# of# cuticular# wax#metabolism.#While# there# is# no# evidence# of#clustering# among# genes# known# to# contribute# to# wax# biosynthesis# in# Arabidopsis,# two# recent#studies# identified# metabolic# gene# clusters# required# for# the# synthesis# of# diketone# waxes# in#Hordeum#vulgare# (barley)#and#Triticum#aestivum# (wheat)# (Schneider#et#al.,#2016K#HenAAvivi#et#al.,#2016).#It#will#be#interesting#to#examine#whether#gene#clusters#are#involved#in#the#synthesis#of#unique# waxes# in# other# organisms,# and# whether# there# are# mechanisms# that# regulate# the#expression#of#entire#clusters.##BAHD#acyltransferases#have#been#found#in#several#metabolic#gene#clusters#(Medeema#and# Osbourn,# 2016).# If# divergent# BAHDs# such# as# CER2ALIKEs# have# maintained# linkage# to#119##genes# from# an# ancestral# cluster,# this#may# shed# light# on# their# evolutionary# history.#CER2# and#CER2ALIKE4#are#both#positioned#adjacent#to#CYP450#genes#with#unknown#functions.#CYP450s#are#ubiquitous#in#plant#metabolism,#and#nearly#all#plant#metabolic#gene#clusters#identified#to#date#include#one#or#more#CYP450.#Examining#the#phylogeny#of#the#CYP450s#adjacent#to#CER2#and#CER2/LIKE4#may#help#trace#the#evolution#of#the#CER2/LIKE#clade.##6.2.1.3$ Acyl$chain$length$and$cuticle$evolution$To# date,# molecular# genetic# studies# of# cuticular# wax# metabolism# have# primarily# been#conducted# with# model# flowering# plants# such# as# Arabidopsis,# maize,# and# barley.# However,#chemical# analysis# of# cuticular# wax# composition# has# been# carried# out# in# diverse# plant# phyla.#Chemical# descriptions# of# waxes# on# bryophytes,# the# extant# relatives# of# early# land# plants,# are#cause# for# further# studies#of# these#organisms.#While# the#bulk#of#wax#present# in# the# cuticles#of#angiosperms#is#composed#of#29A31#carbon#components,#predominant#waxes#in#bryophytes#vary#substantially#from#species#to#species.#In#the#model#moss#Physcomitrella)patens#(P.)patens),#the#most#abundant#waxes#are#primary#alcohols#and#VLCFAs#24,#26,#and#28#carbons#in#length,#and#wax#esters#42A50#carbons#in#length#(dimers#from#a#VLCFA#and#an#alcohol)#(Buda#et#al.,#2013).#Similar# patterns# were# found# across# twelve# species# of# the# peat# bog# moss# genus,#Sphagnum#(Baas#et#al.,#2000).#A#study#that#examined#cuticular#waxes#of#both#endohydric#(Pogonatum)#and#ectohydric# (Andreaea)# mosses# had# similar# results,# with# 22A28# carbon# waxes# predominating#(Haas,# 1982).# While# wax# chain# lengths# up# to# 32# carbons# could# be# detected# in# all# of# these#mosses,#shorter#chain#lengths#were#consistently#more#abundant.#It#is#possible#that#shorter#chain#lengths#of#waxes#offer#a#selective#advantage#to#mosses,#such#as#allowing#water#to#be#absorbed#through#shoot#tissues,#but#because#the#roles#of#different#wax#chain#lengths#are#not#known,#such#an#advantage#is#difficult#to#infer.#Another#explanation#could#be#that#mosses,#and#perhaps#other#bryophytes,# lack# the#biosynthetic#capacity# to#efficiently#produce#waxes#of# longer#chain# lengths#that#predominate#in#the#cuticles#of#flowering#plants.##Molecular#genetic# investigation#of#cuticle#biosynthetic#machinery# in#bryophytes#was#not#attempted#until#quite#recently.#Completion#of#genome#sequencing#for#P.)patens#(Rensing#et#al.,#2008)# has# allowed# the# identification# and# characterization# of# an# ABC# transporter# required# for#cuticular#wax#deposition#in#P.)patens#(Buda#et#al.,#2013),#and#will#surely#accelerate#future#work#on#wax#biosynthesis#in#this#species.#It#will#be#fascinating#to#investigate#wax#metabolism#in#these#extant#relatives#of#the#earliest#land#plants,#and#to#compare#the#functionality#of#bryophyte#cuticles#to# the# cuticles# of# vascular# plants.# The# phylogeny# of# both#KCSs# and#CER2/LIKEs# is# of# clear#interest.#In#Arabidopsis,#the#elongation#machinery#required#to#make#waxes#up#to#28#carbons#in#120##length#does#not# include#CER2ALIKEs,#suggesting#that#mosses#might#have#shorter#chain# length#profiles#because#they#lack#a#CER2#homolog.##6.2.2$ Structure$of$plant$elongase$complexes$# This# thesis# has# highlighted# several# unknowns# regarding# the# elongase# structure# that# are#relevant#to#its#function.#One#glaring#issue#is#that#the#stoichiometry#of#the#elongase#components#remains#unknown.#The#simplest#model# is#a#heterotetramer,#with#one#condensing#enzyme#and#one#of#each#of#the#generalist#enzymes#in#a#single#complex.#Different#elongase#heterotetramers#could# have# different# condensing# enzymes,#with# substrate# passed# from# one# entire# complex# to#another# to# generate# longer# and# longer# acyl# chain# lengths.# Alternatively,# multiple# condensing#enzymes#could#function#with#the#same#suite#of#generalist#components,#allowing#for#elongation#of#a#given#fatty#acid#from#start#to#finish.#A#sequence#of#condensing#enzymes#could#be#swapped#in#and#out#of#the#complex#as#longer#and#longer#fatty#acids#are#generated,#or#multiple#condensing#enzymes# could# simultaneously# associate# with# the# same# complex.# Finally,# none# of# these#streamlined# models# is# necessarily# trueK# elongases# could# exist# as# large# aggregations# with#multiple#copies#of#any#or#all#of#their#protein#components,#including#condensing#enzymes#and#the#generalist#enzymes.#Determining#whether#multiple#KCS#enzymes#simultaneously#associate#with#the#same#elongation#complex#could#be#easily#studied#using#molecular#genetic#assays#such#as#splitAluciferase,# or# coAimmunoprecipitations# on# cells# coAexpressing# multiple,# tagged# KCSs.#Resolving# the# stoichiometry# of# the# entire# fatty# acid# elongation# complex# could# be# achieved# by#twoAdimensional#blue#native#PAGE#followed#by#SDSAPAGE#(Swamy#et#al.,#2006).### Regardless#of#the#quaternary#structure,#there#must#be#a#mechanism#for#transferring#acylACoAs# between# components# within# a# given# elongase# complex,# and# perhaps# among# different#elongase# complexes.# As# previously# discussed,# acylACoA# binding# proteins# could# facilitate#transfer# of# acylACoA# substrates# and# intermediates.# These# proteins# could# either# be# archetypal#ACBPs,# or# nonAhomologous# proteins# with# similar# function# to# ACBPs.# Analysis# of# acylACoA#profiles#of#soluble#and#microsomal#cell#fractions#might#help#resolve#the#localization#of#elongation#substrates,# products,# and# intermediates.# Additionally,# as# described# in# Section# 5.3.4.3,# further#investigation# of# the# roles# of# ACBPs# and# CER2ALIKEs# in# elongation# may# provide# a# clear#explanation#of#how#metabolites#are#provided#to#the#elongase#complex.### Several#genes#have#been# identified# in#Arabidopsis# that# resemble#elongase#components,#but#which#have#nebulous# functions# in#VLCFA#biosynthesis.#These# include#KCR2# and#PTPLA,#ELOAlike# KCSs,# and# PAS1.# The# similarities# and# differences# between# the# ketoacylACoA#reductases# KCR2# and# KCR1,# and# hydroxyacylACoA# dehydratases# PTPLA# and# PAS2,# were#121##summarized#briefly#in#the#introduction#to#this#thesis,#and#compelling#evidence#was#presented#by#Morineau#et#al.#(2016)#for#the#existence#of#dual#elongase#complexes.#While#it#would#appear#that#the# different# ketoacylACoA# reductases# and# hydroxyacylACoA# dehydratases# make# different#contributions# to# VLCFA# elongation# in# different# tissues,# what# function# this# serves# in# unclear,#given#that#KCSs#alone#typically#determine#substrate#specificity.### Similarly,# the# fact# that# there# are# two# families# of# ketoacylACoA# synthases# in#Arabidopsis,#and# presumably# in# other# plants,# is# puzzling.# The# FAE1Alike# family# that# CER6# belongs# to#predominates#in#plants,#and#so#throughout#this#thesis#I’ve#referred#to#this#family#simply#as#KCSs.#The#FAE1Alike# family# is# homologous# to# a# condensing# enzyme# discovered# in# association# with#seed#oil#biosynthesis#(Kunst#et#al.,#1992K#James#et#al.,#1995K#Lassner#et#al.,#1996).#The#ELOAlike#family# is#homologous# to# the#yeast#condensing#enzymes# responsible# for#sphingolipid#synthesis#(Dunn# et# al.,# 2004),# also# characteristic# of# animal# metabolism.# While# many# of# the# FAE1Alike#KCSs# from# plants# have# been# characterized,# the# functions# of# ELOAlike# KCSs# are# almost#completely# unknown.# Obvious# approaches# to# characterizing# these# genes# include# analysis# of#Arabidopsis#mutants,#and#heterologous#expression#of#Arabidopsis#ELOs#in#yeast#cells.#The#fact#that#some#ELOs#have#been#characterized# from#a#bryophyte# (Zank#et#al.,#2002K#EiamsaAard#et#al.,# 2013)# suggests# that# phylogenetic# analysis# of# ELO# gene# distribution# and# analysis# of# the#contributions#of#ELOs#to#metabolism#in#different#plant#lineages#could#be#insightful.### Finally,# PAS1# is# homologous# to# immunophilins# identified# in# animal# systems,# has# been#shown#to#physically#interact#with#the#elongase#in#the#ER,#and#is#required#for#normal#production#of# VLCFAs# and# sphingolipids# (Roudier# et# al.,# 2010).# How# this# gene# contributes# to# elongase#function#is#unknown,#but#Roudier#and#coworkers#(2010)#suggested#it#might#serve#as#a#molecular#scaffold#for#the#protein#complex.### The#fact#that#so#many#genes#have#been#discovered#that#affect#VLCFA#production,#but#do#not# fit# into# the# current#model# of# elongation,# suggests# that# our#model# needs# improvement# and#that# there# are# very# likely# other# components# of# the# elongation#machinery# that# we# are# not# yet#aware#of.#Many#of# the#genes# involved# in#elongation#have#been# identified#based#on# sequence#homology,# such# as# the#KCRs,# which# were# identified# based# on# homology# to# the# yeast# gene,#YBR159.# However,# elongation# in# plants# is# clearly# unique,# and# homology# searches# based# on#other#organisms#are#blind# to# the# intricacies#of#plant#metabolism.#Diverse#mutant#screens#have#yielded#insight#into#the#process#of#elongation.#The#screen#for#pasticcino#mutants#targeted#genes#involved# in# cytokinin# responses,# but# yielded# the# PAS1# immunophilin,# the# hydroxyacylACoA#dehydratase#PAS2,#and#the#pas3#mutant#allele#of#ACETYL/COA)CARBOXYLASE#(Faure#et#al.,#1998).#Screens# for#waxAdeficient#mutants#have#also# led# to# the# identification#of# components#of#122##the# elongase# such# as# CER2# and# CER10,# the# enoylACoA# reductase# of# the# complex.# That#elongation# defects# have# been# investigated# through# such# different# screens# underscores# the#broad#influence#of#VLCFAs#on#plant#health.#As#the#core#components#of#the#elongase#have#now#been# identified,# further# suppressor# and# enhancer# mutant# screens# are# possible,# again# using#phenotypes# such# as# cuticular# wax# load# or# growth# as# tools.# HighAthroughput# screens# for#interacting#proteins#are#also#possible#using#proteomics,#or#yeastAtwo#hybrid#systems.## The#core#components#of# the#plant# fatty#acid#elongation#machinery#have#been# identified,#characterized,# and# used# to# build# a#model# that# has# served# our# research# community#well.# This#model# presents# the# elongase# as# a# complex# of# four# enzymes#with# distinct# activities.#However,#there# have# been# many# clues# that# suggest# that# this# model# is# an# oversimplification.# The#requirement#of#CER2ALIKE#proteins#for#the#synthesis#of#cuticular#wax#precursors#is#one#example#of# an# intricacy# that# refines# the# activity# of# the# elongase,# and# strongly# influences# downstream#metabolic# pathways.# There# is# no# doubt# that# tracing# the# evolution# of# fatty# acid# elongation# in#plants,# examining# the# structure# of# the# fatty# acid# elongase,# and# screening# for# genes# with#peripheral#functions#in#elongation#will#reveal#a#much#more#elaborate#process.####123##References$#Aarts,$MG,$Keijzer,$CJ,$Stiekema,$WJ,$and$Pereira,$A$(1995).#Molecular#characterization#of#the#CER1#gene#of#Arabidopsis#involved#in#epicuticular#wax#biosynthesis#and#pollen#fertility.#Plant#Cell$7:#2115–2127.#Aarts,$MG,$Hodge,$R,$Kalantidis,$K,$Florack,$D,$Wilson,$ZA,$Mulligan,$BJ,$Stiekema,$WJ,$Scott,$R,$Pereira,$ A$ (1997)# The# Arabidopsis# MALE$ STERILITY# 2# protein# shares# similarity# with# reductases# 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Thesis/Dissertation
eng
Botany
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Arabidopsis ECERIFERUM2-LIKE proteins are components of the fatty acid elongation machinery required for the production of cuticular waxes
Text