Functional characterization of anion channels of the SLAC/SLAH family in Arabidopsis Thaliana.

! !!!!!!!!!!!!!!!!!!! !!!!!!!!!!!!!!!!!!! ! ! ! ! ! ! ! ! ! Functional characterization of anion channels of the SLAC/SLAH family in Arabidopsis thaliana ! ! ! ! ! ! ! ! ! ! Memoria!que!presenta! la!Licenciada!en!Biología!Paloma&Cubero&Font! para!optar!al!título!de!Doctora!en!Biología! por!la!Universidad!de!Sevilla! ! Sevilla!2017! ! ! ! ! ! ! ! ! ! ! ! El! presente! trabajo! se! ha! realizado! en! el! marco! de! los!proyectos!! AGL2009;08339!y! !AGL2015;71386;R! pertenecientes! al!Plan! Nacional! de! Investigación!y!Desarrollo!(PNIDI!2008;2011),!y!al!Plan!Estatal!de!Investigación! Científica! e! Innovación! (PEICTI! 2013;2016),! respectivamente,! así! como! del! Proyecto! CSIC;2015;40E108! de! la! Agencia! Estatal! CSIC,! con!la! ayuda!de! una! Beca! Predoctoral! JAE;PREDOC!financiada! por!el! Consejo! Superior! de! Investigaciones! Científicas! (CSIC),!así! como! con! una! Ayuda! para! Facilitar! la! Movilidad!de!Investigadores!en!Formación!del!Programa!JAE;PREDOC!del!CSIC,! y!una!Beca!de!Investigación!para!Doctorandos!y!Jóvenes!Científicos!financiada! por!el!Servicio!Alemán!de!Intercambio!Académico!(DAAD).! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! The!results!obtained!during!the!development!of!this!thesis!have!been!presented! in!national!and!international!scientific!congresses!and!have!led!to!a!publication.! ! SCIENTIFIC(PUBLICATIONS( Cubero8Font!P,!Maierhofer!T,!Jaslan!J,!Rosales!MA,!Espartero!J,!Díaz8Rueda!P,!Müller!HM,! Hürter!AL,!Al8Rasheid!KAS,!Marten!I,!Hedrich!R,!Colmenero8Flores!JM,!Geiger!D! (2016)!Silent!S8Type!anion!channel!subunit!SLAH1!gates!SLAH3!open!for!chloride! root8to8shoot!translocation.!Current'Biology!26,!2213–2220.!! ! CONTRIBUTIONS(TO(MEETINGS(AND(SYMPOSIUMS! Franco! JD! Brumós,! Rosales! MA,! Vázquez! A,! Cubero8Font! P,!Rivero! CM,! Talón! M,!! Colmenero8Flores! JM! (2012)(Homeostasis! de! Cl−!y! regulación! del! ! balance! hídrico!en!plantas.!V'Reunión'Nacional'de'la'Red'Española'de'Estrés'Abiótico'de'las' Plantas.(Murcia,!Spain.!Oral!presentation.! Cubero8Font!P,!Rosales!MA,!Díaz!Rueda!P,!Franco8Navarro!JD,!Espartero!J,!Colmenero8 Flores!JM!(2014)!Functional!characterization!of!the!root!anion!channels!SLAH1! and! SLAH4:! involvement! in! Cl−!and! NO3−!nutrition! and! interaction! with!abiotic! stress.!XII' Reunión' de' Biología' Molecular' de' Plantas.(Cartagena,! Murcia,! Spain.! Abstract!book,!234−235.!Poster.! Cubero8Font!P,!Espartero!J,!Maierhofer!T,!Rosales!MA,!Díaz8Rueda!P,!Hedrich!R,!Geiger! D,! Colmenero8Flores! JM! (2016)! Characterization! of!a! root! anion! channel! that! plays! a! pivotal! role! in! plant! chloride! nutrition.!XVI' Simposio' HispanoDLuso' de' Nutrición'Mineral'de'las'Plantas.! San! Pedro! del! Pinatar,! Murcia,! Spain.! Abstract! book,!9−10.!Oral!presentation.! Cubero8Font! P,!Franco8Navarro! JD,!Rosales! MA,! Espartero! J,! Colmenero8Flores! JM! (2016)! Differential! regulation! of! Chloride! vs.! Nitrate! transport! in! plants:! biological! relevance! and! molecular! mechanisms.! RED' DE' EXCELENCIA' 'de' Transportadores'Na'y'K'(KNaTS).!Málaga,!Spain.!Oral!presentation.! Cubero8Font!P,!Barrera!Gavira!JM,(Díaz8Rueda!P,!Colmenero8Flores!JM!(2017)!Canales! aniónicos! SLAH2! y! SLAH4.! RED' DE' EXCELENCIA' 'de' Transportadores' Na' y' K' (KNaTS).!Madrid,!Spain.!Oral!presentation.! Franco8Navarro! JD,! Cubero8Font! P,! Rosales! MA,! Día8Rueda! P,! Espartero! J,! Geiger! D,! Colmenero8Flores!JM!(2017)!Chloride!as!multifunctional!beneficial!ion:!Biological! functions! and! regulation.! XXII' Reunión' de' la' Sociedad' Española' de' Fisiología' Vegetal' (SEFV),' XV' SpanishDPortuguese' Congress' of' Plant' Physyology.! Barcelona! (Spain).!Poster.! ! I! ACKNOWLEDGEMENT ! First&and&foremost,&I&would&like&to&express&my&gratitude&to&my&thesis&director&Dr.&José&Manuel& Colmenero&Flores&(Chema)&for&his&constant&guidance&throughout&these&years&and&for&his&dedication&at& any&time&of&the&day,&at&any&day&of&the&week&all&over&the&year.&& I&truly&appreciate&the&determined&support&of&Prof.&Dr.&Dietmar&Geiger&from&the&Institute&for& Molecular&Plant&Physiology&and&Biophysics&(University&of&Würzburg)&during&my&stay&at&his&Institute.&I& keep& in& mind& the& memory& of& the& first& successful& measurement& of& anion& currents& in& SLAH1PSLAH3& complex.&I&am&really&grateful&for&his&supervision&and&the&opportunity&to&collaborate&in&his&laboratory.&I& would& also& like& to& thank& Dr.& Tobias& Maierhofer& from& the& same& Institute& for& his& training& in& electrophysiology&and&continuous&guidance.& I& would& like& to& express& my& thankfulness& to&Prof.&Dr.& Francisco& Ramos& Morales&from& the& University&of&Sevilla&(US)&for&his&kindness&and&availability&every&time&I&needed&his&help&and&to&Prof.&Dr.& Francisco&Javier&Cejudo&from&the&Institute&of&Plant&Biochemistry&and&Photosynthesis&(IBVF;&CSIC/US)& for&his&support&and&advice.&& Finally,&I&would&like&to&thank&the&Spanish(National(Research(Council&(CSIC),&which&offered&me& the&funds&to&accomplish&my& PhD&studies& at&the&Institute& of&Natural&Resources&and&Agrobiology&of& Seville& (IRNAS,& Seville,& Spain).& I& also& would& like& to& express& my& sincere& gratitude& to& the& German& Academic& Exchange& Service& (DAAD)& for& awarding& me& with& the& Research& Grant& for& Doctoral& Candidates&and&Young&Academics&and&Scientists&at&the&Institute&for&Molecular&Plant&Physiology&and& Biophysics&(University&of&Würzburg,&Würzburg,&Germany).& Gracias& a& mis& compañeros& de& laboratorio,& empezando& por& Sofía& que,& aunque& compartimos& relativamente&poco&tiempo,&lo&disfruté&muchísimo.&A&JuanD&le&agradezco&ser&tan&servicial,&su&alegría&y& todos& los& buenos& ratos& en& el&laboratorio&e&invernadero,& a& Carlos& sus& chistes& malos& con& su& buena& intención&de&hacernos&reír&y&a&Pablo&por&verlo&todo&siempre&tan&sencillo.&Gracias&a&Kino&por&su&buen& humor& y& por& transmitirlo&con& canciones,& pareados,& chistes...& A& Pilar&y& a& Mara& por& amenizar& los& encuentros&fuera&del&laboratorio,&a&Leti&por&su&gran&corazón&y&a&Miriam&por&dedicarme&siempre&un& rato&cuando&nos&encontramos.&Quisiera&agradecerle&a&Alberto&todos&esos&ratos&de&risa&floja&imparable& tan& divertidos;& pero,& sobre& todo,& le& agradezco& enormemente& a& Fran& su& compañerismo,& amistad,& disponibilidad,&ayuda,&atención…&Con&su&llegada&mejoró&el&laboratorio&en&todos&los&aspectos.&& Me&gustaría&expresar&también&mi&agradecimiento&a&mis&amigas&y&compañeras&de&trabajo:&a& Marina&por& todos& los& momentos& que& compartimos,& que& tanto&nos&hacen& disfrutar.& Conocerla&en& Ecuador&fue&lo&mejor&de&aquel& viaje.&Quiero& agradecerle&a&mi&gran&compañera&de&oficina&Elena&el& cambio&que&supuso&su&amistad&en&estos&años&de&tesis.&Desde&que&dejó&su&silla&vacía&la&he&echado& mucho&de&menos&por&su&alegría,&dulzura&y&bondad.&A&Jara&le&agradezco&especialmente&todas&las&risas&y& esos&almuerzos&tan&amenos&al&aire&libre.&Muchas&gracias&a&las&chicas&del&fondo&de&la&segunda&planta:& Marta,&Irene,&Sara,&Ana…&¡Cuánto& se& puede& disfrutar& comiendo& y& limpiándose&los& dientes!& Y& a& mi& querida&Ángela&me&gustaría&agradecerle&su&amabilidad,&sus&detalles&y&todo&el&cariño&que&siempre&me& ha&transmitido,&incluso&desde&la&distancia.& Le& estoy&sinceramente&agradecida&a& Raúl&por& todos& sus& consejos,& que& siempre& me& han& resultado& tan& prácticos.& Espero& seguir& disfrutando& tanto& de& su& sabiduría& como& de& su& sentido& del& ! II! humor.&Asimismo,& quiero&darle&las&gracias&a&Pili&por&animar& el&ambiente&de&trabajo&con&bromas&y& risas,& a& José& María&por& sus&acertijos&y&actividades¶&fomentar&la& atención&y&por&esas&originales& celebraciones&de&cumpleaños&junto&a&Elena,&Marta&y&José&Ramón.&A&la&gente&del&laboratorio&de&la& tercera& planta& del& departamento& de& Biotecnología& Vegetal& (Zaida,& Anna,& Paula,& Belén,& Fran,& Javi),& aunque&ya&no&estén&allí,&les&agradezco&la&ayuda&y&el&compañerismo&dentro&y&fuera&del&laboratorio.&& Muchas&gracias&de&corazón&a&todos&los&que&pertenecen&y&han&pasado&por&el&grupo&de&Riego&y& Ecofisiología&de&Cultivos,&en&el&que&realmente&empecé:&José&Enrique,&Antonio&Díaz,&Torres,&Alfonso,& Celia,& Antonio& Montero,& Vicky,& Carmen,& Virginia,& Rafa,& Adrián,& María& Dolores,& Saray,& Sheren,& Bárbara…& He& disfrutado& mucho&con& ellos,& hasta& quitando& las& malas& hierbas,& y& espero& seguir& haciéndolo&en&cada&uno&de&nuestros&encuentros.&& Gracias& al& pequeño& grupo& fusionado& que& creamos& cuando& empecé& en& el& laboratorio& de& Chema&entre&la&gente&de&éste&(incluyendo& a& Sofy,&Migue,&Pablo&y&Alberto)&y&la&del&laboratorio&de& Heike& (Marta,&María,& Marina,& Lorena& y&Marta& la& cordobesa)& por& los& momentos& tan& necesarios&de& desconexión&laboral.& Me&gustaría&darle&las&gracias&a&todas&esas&personas&del&IRNAS&que&han&hecho&que&con&un& simple& encuentro& por& las& instalaciones& cambie& mi& estado& de& ánimo& favorablemente:& David& y& José& Manuel&de&portería,&Antonio&de&mantenimiento,&Mª&Ángeles&y&Mercedes&del&servicio&de&limpieza,& Luisa,& Mª& Luz& y& Paqui& de& gerencia,& María,& JosAn,& José& María& de& la& Rosa,& Valme,& Isa,& Julia,& Rocío,& Chona,&Jesús&Cambrollé,&Carmen&Trigo,&Celia&Jiménez,&Andrés,&Pepijn,&Esteban,&Maite,&Rosa,&José&Luis,& Marta&Gil,&Pablo,&José&Manuel,&Gabriela,&Manuel…,&y&especialmente&le&agradezco&a&Alicia&el&cariño& que&me&ha&transmitido&siempre&y&su&alegría&permanente.& Quiero&agradecerles&a&algunas&de&las&personas&que&han&pasado&por&el&IRNAS&la&huella&que&han& dejado&en&mí.&A&Silvia&su&cercanía,&amistad&y&todo&lo&que&eso&conlleva,&a&Graciele&su&amabilidad&y& tener&siempre&una&sonrisa&en&la&cara,&a&Rita&por&toda&su&alegría&y&por&el&mejor&cumpleaños&de&mi&vida& y&a&Vincent&por&el&intercambio&de&idiomas&con&el&que&ambos&adquirimos&mayor&fluidez.&& También&me&gustaría&expresar&mi&gratitud&a&la&gente&del&laboratorio&de&Fisiología&Vegetal&del& Departamento&de&Biología&Vegetal&y&Ecología&de&la&Facultad&de&Biología&(US),&donde&me&inicié&en&el& mundo&de&la&investigación,&por&el&buen&trato&que&siempre&me&han&dado.&En&especial,&le&agradezco&a&la& Dr.&Sofía&GarcíaPMauriño&su&firme&apoyo,&y&a&Isa&y&Cire&su&positividad&y&buenas&recomendaciones.& Además,&me&gustaría&darle&las&gracias&a&Irene&del&Departamento&de&Microbiología&de&la&Facultad&de& Biología&(US)&por&su&alegría,&ánimos&y&consejos.& I& big& thank& you&to& everyone& from& Geiger’s& lab:& Sönke,& Jenny,& Julia,& Meli,&Nadin,&Möritz,& Michael…,&especially&to&AnnaPLena&and&Christof&for&their&help,&friendship,&laughs,&talks,&time…&And& thanks&to&the&other&people&working&in&BotanikPI&that&I&met&during&my&stay&in&Germany:&Hanna,&Lena,& Antonella,&Maris,&Katha,&Justyna,&Dawid,&Ayla,&Natalia,&Jens,&Bea,&Alicija,&Martin…&and&particularly&to& Sarah&and&Karimi&for&their&happiness&and&for&being&so&open.& Franca,&herzlichen&Dank&für&alles:&deine&Fröhlichkeit,&deine&Zuneigung,&deine&Hilfe,&das&super& Zusammenleben!&Ich&bedanke&mich&bei&deinen&Eltern&für&die&tolle&Zeit.&Agnes,&Hani&und&Evi:&danke& schön&auch!& Gracias&a&todos&mis&compañeros&y&profesoras&del&Instituto&Cultural&Alemán&(Anna,&Angelika,& Miriam,&Lysann,&Elena,&Mercedes,&Arabia,&Carmen,&Toñi,&Andrea,&Eleanor,&Noelia…)&por&esas&buenas& horas& de& aprendizaje& que& han& hecho& que& tres& horas& de& alemán& un& viernes&sean& más& llevaderas.& Además,& le& agradezco& francamente& a& mi& maestra& de& Taichi& Dª& Mª& Carmen& López& su& vocación,& la& ! III! energía&que&transmite&y&su&sabiduría.&Ha&sido&un&placer&compartir&sus&clases&con&Begoña,&Reyes,&Bea,& Manoli,&Isabel,&Inma,&Carlos,&Rosa,&Miguel&Ángel,&Concha,&Manuela…& Quiero&agradecerles&también&a&mis&amigas&todas&las&experiencias&compartidas&durante&mis& años&de&doctorado.&A&Lucía,&que&fue&mi&sol&en&Alemania&en&los&meses&en&que&éste&no&salió,&por&su& alegría,&simpatía,&cariño&y&tantos&buenos&momentos&que&disfrutamos&y&seguimos&compartiendo.&A&mi& amiga&Isis&por&todas&las&veces&que&ha&venido&a&comer&conmigo&a&Reina&Mercedes¶&compartir&un& ratito& y,& sobre& todo,&por& su& sincera& amistad.& A& mi& amiga&de& la& infancia&Mª& Ángeles&por& tenerme& siempre&presente&y&por&hacer&que,&junto&a&su&estupenda&pareja,&nuestros&encuentros&estén&repletos& de&risas&constantes.&A&Hanah&le&agradezco&su&forma&de&ser,&la&correspondencia&a&lo&largo&de&estos& años&y&que&siempre&que&nos&vemos&reina&el&buen&humor.& A&mi&madre&y&a&mi&padre&les&doy&las&gracias&por&su&confianza,&apoyo&y&amor&inquebrantables&y&a& mi&querido&hermano&Ernesto&le&agradezco,&además,&su&visita&a&Würzburg&en&2014&por&esos&momentos& inolvidables&que&tanto&me&alegra&recordar.&& Quisiera&agradecerles&a&todos&mis&familiares&y&demás&allegados&que&siempre&tengan&tan&buena& disposición¶&reír&y&disfrutar&del&tiempo&que&compartimos.&Eso&siempre&viene&bien,&sobre&todo& para& desconectar& y& disminuir& el& estrés.&A&mi& abuela& Clara& le& quiero& dar& las& gracias& por& su&cálida& sonrisa,&y&a&mi&tíaPabuela&Julia&por&la&energía&y&felicidad&que&siempre&transmite,&que&no&aminora&con& los&años,&y&eso&que&ya&está&rozando&el&siglo.& Finalmente,&y&no&por&ello&menos&importante,&le&agradezco&profundamente&a&mi&queridísimo&Javi,& mi&ideal&compañero&de&vida,&su&alegría&diaria,&sus&bromas,&su&buen&humor,&sus&constantes&detalles&y& su&apoyo&incondicional&desde&que&nos&conocemos,&que&hace&que&siempre&me&sienta&bien&y&tenga&una& sonrisa&en&la&cara.!! ! ! ! ! ! ! ! ! ! ! ! & & & & ! ! IV! & & & & & & & & & & & & & & & & & & & & & & & & & & & & ! XI! LIST OF ABBREVIATIONS ! A!Ampere! ABA!Abscisic!Acid! ABC!ATP0Binding!Cassette! ABI1/2!ABA!Insensitive!1!or!2! ABRE!ABA0Response!Elements! AKT1,Arabidopsis!K+!Transporter!1, Ala!Alanine! ALMT!Aluminum!activated!Malate!Transporter! Arg!Arginine! Asn!Asparagine! Asp!Aspartic!acid! At!Arabidopsis*thaliana! A.!thaliana!!!Arabidopsis*thaliana! AtrbohF!! Respiratory!Burst!Oxidase!Homologue!F! A.!tumefaciens*Agrobacterium*tumefaciens* ! BiFC!Bimolecular!Fluorescence!Complementation! BLAST!Basic!Local!Alignment!Search!Tool! BNS!Basal!Nutrient!Solution!! bp!Base!Pair! BS!Basal!Solution! bZIP!Basic!leucine!Zipper! ! Ca2+!Calcium! CaCl2!Calcium!Chloride! ! Ca(NO3)2!Calcium!Nitrate!! CaSO4!Calcium!Sulfate! CBL!Calcineurin0B0Like!protein!! Cc!Citrus*clementina* CC!Companion!Cell! CCC!Cation!Chloride!Cotransporter! cDNA!Complementary!DNA! CFU!Colony!Forming!Units! CIPK!CBL0Interacting!Protein!Kinase!! Cl–!Chloride! CLC!Chloride!Channel! Col!Columbia! CPK!Calcium0dependent!Protein!Kinase! cRNA!Complementary!RNA! CT!Carboxilo!Terminal! cv! Cultivar! ºC!Degree!Centigrade/Celsius! ! d!Day! das!Days!After!Sowing! DDT!Dithiothreitol! DEPC!Diethyl!pyrocarbonate! DIDS!4,4'0diisothiocyanatostilbene02,2'0disulfonic!acid!! DM!Dry!matter! Dm*Dionaea*muscipula* DNA!Deoxyribonucleic!Acid! dNTP!deoxynucleotide!Triphosphate! dsDNA!Double0Stranded!DNA! DW!Dry!Weight! ! XII! 3D!Three0dimensional! * ΔEn!! Nerst!potential! ECl!Chloride!diffusion!potential! E.!coli!Escherichia*coli* EDTA!Ethylenediaminetetraacetic!acid! e.g.!For!example! eIF4A1!Eukaryotic!translation!Initiation!Factor!4A1! Em!Membrane!potential! etc!et!cetera! * F!! Fardays!constant! F−!Fluoride!! Fig.!! Figure! Figs.!! Figures! FW!Fresh!Weight! ! g!Gramme! ×,g!Units!of!times!gravity! GFP!Green!Fluorescent!Protein! Gly!Glycine! GORK!Guard!cell!Outward!Rectifying!K+!channel! GUS!β0Glucuronidase! ! h!Hour! H+!Protons! HAB1/2!Homology!to!ABI1!or!ABI2!! HCO3−!Carbonate! HI!Harvest!Index! HiTehA!Haemophilus*influenza! HNO3!Nitric!acid! H2O2!! Hydrogen!peroxide! H2SO4!Sulphuric!acid! ! I!Electrical!current!(ampere)! IAA!Indole030acetic!acid! i.e.!That!is! Iinst!Instantaneous!Currents! IJPB!Institut!Jean0Pierre!Bourgin! IM!Infiltration!Medium! IP3!! Inositol030Phosphate! ISS!Steady!State!Currents! IT!! Instantaneous!Currents! Itail!Tail!streams! IVT!In*Vitro!Transcription! IVTVPCR!in*vitro!PCR! ! K!Degree!Kelvin! K+!Potassium! KAT1/2!K+!channel!in!Arabidopsis*thaliana!1/2! Kb!Kilobase! KCl!Potassium!Chloride! K3Fe(CN)6!! Potassium!ferricyanide! K4Fe(CN)6!! Potassium!ferrocyanide! KH2PO4!Potassium!dihydrogen!Phosphate! KNO3!Potassium!Nitrate! KO!Knockout! K2SO4!Potassium!Sulfate! ! ! XIII! l!Litre! LaCl3!Lanthanum!Chloride! LB!Luria0Bertani! Le!Lycopersicon*esculentum! LEA<M!Late0Embryogenesis!Abundant! Leu!Leucine! LiCl!Lithium!Chloride! ! m!Metre! M!Molar! MA2−!Malate! MAE!MOPS0Sodium!Acetate0EDTA! MES!40Morpholineethanesulfonic!acid! mg!Milligramme! MgCl2!Magnesium!Chloride! Mg(NO3)2!Magnesium!Nitrate! MgSO4!Magnesium!Sulfate! min!Minute! ml!Millilitre! mM!Millimolar! MnCl2!Manganese!Chloride! MOPS!40Morpholino!Propanesulfonic!acid! MPa!Megapascal! mRNA!Messenger!RNA! MS!Murashige!and!Skoog!medium! Ms!Millisecond! MscS!Small0conductance!Mechanosensitive!channel! Mt*Medicago*trunculata* mV!Millivolt! ! N!Nitrogen! nA,nanoampere, Na+!Sodium! NaCl!Sodium!Chloride! NADPH!Reduced!Nicotinamide!Adenine!Dinucleotide!Phosphate! NaNO3!Sodium!Nitrate! NaOH!Sodium!Hidroxide! NASC!Nottingham!Arabidopsis!Stock!Centre! NAXT1!Nitrate!Excretion!Transporter!1! NCBI!National!Center!for!Biotechnology!Information! NH3!Ammonia! NH4+!Ammonium! NH4Ac!Ammonium!Acetate! NHX1!Na+/H+!exchanger!1! NKCC!Na+:K+:Cl−!Cotransporter! NO!Nitric!Oxide! NO3–!Nitrate! NPF!Nitrate!Transporter!1/Peptide!Transporter! NRT1/PTR!! Nitrate!Transporter!1/Peptide!Transporter! NRT1V2!Nitrate!Transporter!102! nt!Nucleotide! NT!Amino!Terminal! N.!tabacum!Nicotiana*tabacum! NTP!Nucleotide!Triphosphate! ! OD!Optical!Density! Os!Oryza*sativa* O.!sativa*Oryza*sativa* OST1!Open!Stomata!1,!SnRK2.6! ! XIV! ! P*Permeability! PA!Phosphatidic!Acid! PAR!Photosynthetically0Active!Radiation! ! PBS!Phosphate!buffered!saline! PCR!Polymerase!Chain!Reaction! PEG!Poly!Ethylene!Glycol! Pfu,polymerase!DNA!polymerase!isolated!from!Pyrococcus*furiosus! pH!Potential!of!Hydrogen! Phe!Phenylalanine! PIP3!Phosphatidyl0Inositol030Phosphate! PLC!Phospholipase!C! PLDα1!! Phoshpholipase!Dα1! PM!Plasma!Membrane! PO!Open!Probability! Pp*Physcomitrella*patens* PP2C!Protein!Phosphatases!type!2C! PSII!Photosystem!II! PTR!Peptide!Transporter! Ptt*Populus*tremula*x*P.*tremuloides* PYR!Pyrabactin!Resistance!! PYL!PYR1!Like! PZ!40Phenylazobenzyl0!oxycarbonyl0L0prolyl0L0leucyl0glycyl0L0prolyl0D0arginine! ! qPCR!Quantitative!real!time!PCR! QUAC!Quick!Anion!Channel! * R!Universal!gas!constant! RCAR!Regulatory!Component!of!ABA!Receptor! Rel.,PO!Relative!Open!Probability! RH!Relative!Humidity! RNA!Ribonucleic!Acid! ROS!Reactive!Oxygen!Species! rpm!Revolutions!Per!Minute! RTVPCR!Reverse!Transcriptase!PCR! RVType!Rapidly!activated!anion!channel! ! s!Second! Sb*Solanum*bicolor* SDS!Sodium!Dodecyl!Sulfate! SE!Sieve!Element! Ser!Serine! SKOR!Stelar!K+!Outwardly0Rectifying! SLAC!Slow!Anion!Channel! SLAC1!Slow!Anion!Channel0Associated!1! SLAH1V4!SLAC10Homolog!104! SnRK2!Sucrose!non0fermenting01!Related!protein!Kinase!type02! SO4−!Sulphate! SOB!Super!Optimal!Broth! SOC!Super!Optimal!broth!with!Catabolite!repression!! Spp.,Species! SVType!Slowly!activated!anion!channel! SV!Slow!Vacuolar!channel! * T!! Absolute!temperature! TAE!Tris!Acetate0EDTA!buffer! Taq,polymerase!DNA!polymerase!isolated!from!Thermus*aquaticus! tVDNA!Transfer!DNA!by!A.*tumefaciens*! TVDNA!Transfer!DNA!by!mutagenesis! ! XV! TDT!Tellurite0resistance/C40dicarboxylate! TE!Tris0EDTA! TEVC!Two0Electrode!Voltage!Clamp!! TM!Transmembrane! TPK1,Two0Pore!K+!channel!1! tRNA!Transfer!RNA! ! U!Enzyme!Unit! USER!Uracil0Specific!Excision!Reagent! UTR!Untranslated!Region! ! V!Volt! V½!Half0maximal!activation!potential! Val!Valine! Vc!Voltage!of!command!signal! VDAC!Voltage0Dependent!Anion!Channels! VH!Holding!potential! Vrev!Reversal!Potentials! VK!Vacuolar!K+!channels! Vm!Membrane!potential! vs.!versus! Vv*Vitis*vinifera* ! Ws!Wassilewskija! WT!Wild0type! ! XVIRAC!Xylem!Inward!Rectifying!Anion!Channel! X.!laevis!Xenopus*laevis* XVQUAC!Xylem!Quickly!activating!Anion!Conductance! XVSLAC!Xylem!Slowly!activating!Anion!Conductance! ! YEP!Yeast!Extract!Peptone! YFP!Yellow!Fluorescent!Protein! ! μA!Microampere! μg!Microgramme! μE!Microeinstein! μl!Microlitre! μm!Micrometre! μM!Micromolar! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! XVI! ! ! ! ! ! ! ! ! ! ! ! ! ! ! XVII! LIST OF FIGURES ! Figure'1.1.!Salt!tolerance!mechanisms!in!plants!regulated!by!membrane!transporters!.............!8! Figure' 1.2.!Proposed!model!for!the!symplastic!regulation!of!chloride!(Cl−)!homeostasis!in! the!root!of!a!Cl−!includer!and!a!Cl−!excluder!rootstock!................................................................................!10! Figure'1.3.!Root!pathways!for!nutrients!and!water!uptake!.....................................................................!11! Figure'1.4.!Chloride!(Cl−)!uptake!from!the!soil!and!loading!to!the!shoot!under!nonFnutrient! stressed!conditions!......................................................................................................................................................!13! Figure' 1.5.!Representative! currentFvoltage! curves! for! the! three! plasma! membrane! anion! conductances!from!barley!xylem!parenchyma!cells!.....................................................................................!16! Figure'1.6.!Structure!of!the!sieve!elements!(SEs)!in!Vicia%faba%with!companion!cells!(CCs)!.....!17! Figure'1.7.!Cross!section!of!Mimosa%pudica%leaves!.......................................................................................!17! Figure' 1.8.!Phylogenetic! tree! of! the! CLC! family! from! Arabidopsis%thaliana! (At)! and! Oriza% sativa!(Os)!........................................................................................................................................................................!19! Figure'1.9.!Subcellular!localization!of!CLC!family!members!in!Arabidopsis%thaliana!....................!20! Figure'1.10.!Arabidopsis%thaliana!ALMT!family!phylogenetic!tree!.......................................................!22! Figure'1.11.!Ion!channels!at!intracellular!membranes!in!Arabidopsis%thaliana!..............................!23! Figure' 1.12.!Schematic! representation! of! wholeFcell! activation! and! deactivation! currents! for!(a)!SFtype!currents!and!(b)!RFtype!currents!..............................................................................................!24! Figure' 1.13.!CurrentFvoltage!relationships!of!(a)!SFtype!and!(b)!RFtype!anion!channels!in! Vicia%faba!guard!cell!protoplasts!in!the!absence!and!presence!of!ABA!.................................................!25! Figure'1.14.!SLAC/SLAH!family!phylogenetic!tree!from!different!species!........................................!27! Figure'1.15.!Localization!of!SLAC/SLAH!genes!expression!......................................................................!28! Figure'1.16.!Plasma!membrane!localization!of!SLAC/SLAH!proteins!.................................................!29! Figure'1.17.!SLAC/SLAH!genes!genomic!structure!......................................................................................!29! Figure'1.18.!Homology!model!for!AtSLAC1!channel!...................................................................................!30! Figure'1.19.!Regulation!of!SLAC1!activity!by!different!kinases!..............................................................!31! Figure'1.20.!Regulation!of!SLAH3!activity!by!different!protein!kinases!.............................................!32! Figure'1.21.!Regulation!of!SLAH2!activity!by!different!kinases!.............................................................!33! Figure'1.22.!Guard!cell!signaling!during!stomatal!opening!......................................................................!35! Figure'1.23.!Stomatal!closure!by!ABA!induced!signaling!in!guard!cells!.............................................!37! ' Figure'3.1.!TFDNA!insertion!mutants!in!AtSLAH1!.........................................................................................!55' Figure'3.2.!TFDNA!insertion!mutants!in!AtSLAH3!.........................................................................................!55' Figure'3.3.!TFDNA!insertion!mutant!in!AtSLAH4!...........................................................................................!56' Figure'3.4.!Design!of!USERFcompatible!mutagenesis!primers!................................................................!61' Figure'3.5.!USERTM!cloning!technique!................................................................................................................!70' Figure'3.6.!Gateway®!cloning!technology!.........................................................................................................!72' ! XVIII! Figure'3.7.!Xylem!sap!correlation!with!root!biomass!.................................................................................!80' Figure'3.8.!Diagram!representing!the!principle!of!the!BiFC!assay!........................................................!86! Figure'3.9.!Schematic!representation!of!the!twoFelectrode!voltage!clamp!(TEVC)!technique! on!an!oocyte!....................................................................................................................................................................!87' Figure' 3.10.!Current! traces! of! the! voltage! protocol! used! for! measurement! of! currentF voltage!(IFV)!relations!................................................................................................................................................!88' ' Figure'4.1.!Expression!of!AtSLAH1!and!AtSLAH3!in!the!root!vascular!cylinder!..............................!95' Figure'4.2.!AtSLAH1%and!AtSLAH3%are!coFexpressed!in!the!xylem!pole!pericycle!..........................!96' Figure'4.3.!Identification!and!complementation!of!the!slah1!knockout!line!.....................................!97' Figure' 4.4.!Phenotypic! differences! in! the! root! of! slah1>2% mutant! and! its! wildFtype! (WT)! azygous!line!grown!hydroponically!under!two!different!chloride!treatments!.................................!99' Figure' 4.5.!Vegetative! growth! phenotype! of! the! slah1>2!mutant! and! its! wildFtype! (WT)! azygous!line!grown!hydroponically!under!two!different!chloride!treatments!.................................!100' Figure'4.6.!Differential!shoot!and!inflorescence!stem!growth!of!mutant!(slah1>2)!and!wildF type!(WT)!plants!grown!hydroponically!under!two!different!chloride!feeding!regimens!...........!101' Figure' 4.7.!Differential!rosette!growth!of%slah1>2%KO!line!and!its!wildFtype!(WT)!azygous! line!grown!in!solid!substrate!treated!with!5!mM!chloride!.........................................................................!102' Figure' 4.8.!Differential! main! inflorescence! stem! development! and! siliques! production! of! slah1>2% mutant! and! wildFtype! (WT)! plants! grown! in! solid! substrate! treated! with! 5! mM! chloride!.............................................................................................................................................................................!102' Figure'4.9.!Shoot!chloride!content!was!significantly!lower!in!slah1>2%plants!..................................!103' Figure' 4.10.!Chloride! content! in! shoots! of! slah1>2! TFDNA! knockout! and! wildFtype! (WT)! plants!grown!in!solid!substrate!treated!with!two!different!chloride!feeding!regimens!...............!103! Figure'4.11.!Anion!concentration!in!xylem!sap!of!slah1>2%plants!relative!to!wildFtype!(WT)! plants!at!different!collecting!times!.......................................................................................................................!104' Figure'4.12.!SLAH1!and!SLAH3!are!involved!in!chloride!translocation!into!the!xylem!...............!105' Figure'4.13.!SLAH1!and!SLAH3!regulate!rootFtoFshoot!translocation!of!chloride!.........................!106' Figure'4.14.!SLAH1!is!involved!in!chloride!(Cl−)!transfer!to!the!shoot!...............................................!108' Figure'4.15.!Relative!expression!of!AtSLAH1!and!AtSLAH3!under!abiotic!stress!treatments!...!109' Figure'4.16.!AtSLAH1!gene!expression!is!downFregulated!by!abscisic!acid!......................................!110' Figure' 4.17.!Relative! expression! of! AtSLAH1%and%AtSLAH3%in! response! to! changes! in! the! nutritional!status!of!nitrate!(NO3−)!and!chloride!(Cl−)!.................................................................................!111' Figure' 4.18.!Physical! interaction! of! SLAH1! with! members! of! different! kinase! families! revealed!by!BiFC!experiments!................................................................................................................................!112' Figure'4.19.!SLAH1!remains!electrically!silent!after!coFexpression!with!CPK21!in!chloride! and!nitrate!buffers!.......................................................................................................................................................!113' Figure'4.20.!Sequence!alignment!of!SLAC/SLAH!proteins!from!different!species!.........................!114' Figure'4.21.!SLAH1!F307A!mutant!remains!as!a!silent!channel!............................................................!114' Figure'4.22.!Physical!interaction!between!SFtype!monomers!revealed!by!BiFC!experiments!.!115' Figure'4.23.!Physical!interaction!of!SLAH1!with!SLAC/SLAH!Family!members!.............................!115' ! XIX! Figure'4.24.!Influence!of!SLAH1!on!the!activity!of!SFtype!anion!channels!........................................!116' Figure'4.25.!SLAH1!substitutes!for!kinaseF!and!nitrateF!dependent!activation!of!SLAH3!..........!117' Figure'4.26.!Sequence!alignment!of!SLAC/SLAH!proteins!from!different!species!.........................!118' Figure'4.27.!SLAH1!modifies!the!activation!properties!of!SLAH3!.........................................................!118' Figure' 4.28.!SLAH3! G264D! or! F517L! mutants! did! not! exhibited! a! decrease! in! channel! abundance!.......................................................................................................................................................................!119' Figure'4.29.!Sequence!alignment!of!SLAC/SLAH!proteins!from!different!species!.........................!120' Figure' 4.30.!SLAH1/SLAH3!and!SLAH3!channels!share!the!same!relative!permeability!for! chloride!.............................................................................................................................................................................!120! Figure'4.31.!SLAH1/SLAH3!channels!have!a!higher!chloride!conductance!than!the!kinaseF dependent!SLAH3!channels!.....................................................................................................................................!121' Figure'4.32.!SLAH1!gates!SLAH3!open!without!the!need!of!nitrate!.....................................................!121' Figure'4.33.!AtSLAH1!is!expressed!at!the!flower!..........................................................................................!122' Figure'4.34.!AtSLAH3!expression!within!the!shoot!......................................................................................!123' Figure' 4.35.!Seeds!production!was!significantly!lower!in!slah1>2%line!relative!to!the!wildF type!(WT)!line!................................................................................................................................................................!124' Figure'4.36.!AtSLAH4%is!expressed!in!root!cortecal!and!epidermal!cells!...........................................!125' Figure'4.37.!AtSLAH4!is!expressed!in!leaves!...................................................................................................!126' Figure'4.38.!Expression!of!AtSLAH4!and!AtSLAH1!in!the!root!during!seedling!development!..!127' Figure'4.39.!slah4>2!line!is!a!knockout!line!.....................................................................................................!128' Figure' 4.40.!Differential! root! growth! of! slah4>2%KO! mutant! and! wildFtype! (ColF0)! plants! grown!hydroponically!under!two!different!chloride!treatments!............................................................!129! Figure'4.41.!Differential!growth!of!slah4>2%KO!mutant!and!wildFtype!(ColF0)!plants!grown! hydroponically!under!two!different!chloride!treatments!..........................................................................!130' Figure'4.42.!Differential!shoot!and!inflorescence!stem!growth!of!mutant!(slah4>2)!and!wildF type!(ColF0)!plants!grown!hydroponically!under!two!different!chloride!treatments!....................!131' Figure' 4.43.!Differential! shoot! and! inflorescence! stem! growth! of! slah4>2%KO! mutant! and! wildFtype!(ColF0)!plants!grown!in!solid!substrate!under!two!different!chloride!treatments!.....!132' Figure'4.44.!Differential!rosette!development!of!slah4>2%KO!mutant!and!ColF0!plants!grown! in!solid!substrate!under!two!different!chloride!treatments!......................................................................!133' Figure' 4.45.!Chloride! content! in! shoots! of! slah4>2! TFDNA! knockout! and! ColF0! wildFtype! plants!growing!with!different!nitrate!(NO3−)!and!chloride!(Cl−)!concentration!treatments!.......!134' Figure'4.46.!Relative!expression!of!AtSLAH4%under!abiotic!stress!treatments!...............................!136' Figure'4.47.!AtSLAH4!expression!is!downFregulated!by!abscisic!acid!(ABA)!...................................!136' Figure'4.48.!Relative!expression!of!AtSLAH4%in!response!to!changes!in!the!nutritional!status! of!nitrate!(NO3−)!and!chloride!(Cl−)!......................................................................................................................!137' Figure' 4.49.!Effect! of! exogenous! auxin! application! on! the! relative! expression! level! of! AtSLAH4%in!roots!and!leaves!of!wildFtype!plants!............................................................................................!138' Figure'4.50.!AtSLAH4!expression!is!downFregulated!by!indoleF3Facetic!acid!(IAA)!in!roots!....!139! ! ! ! XX! Figure'5.1.!AtSLAH1!modulates!AtSLAH3!anion!conductance!................................................................!151' Figure'5.2.!Model!describing!the!hypothetical!function!of!AtSLAH4!in!plant!roots!......................!158' Figure' 5.3.!Model! of! SLAC/SLAH! family! channels! activity! within! the! root! during! early! seedling!development!................................................................................................................................................!159' Figure'5.4.!Model!of!SLAC/SLAH!family!channels!activity!within!the!root!under!favourable! growth!conditions!........................................................................................................................................................!160' Figure' 5.5.!Model! of! SLAC/SLAH! family! channels! activity! within! the! root! under! abiotic! stress!conditions!...........................................................................................................................................................!162! ' ' ' Table&of&contents& & XXVII& 5.2.1.7.!Model&of&SLAH4&biological&function&............................................................................&157& 5.3.&The&integrated&function&of&the&root&SLAH1,&SLAH3&and&SLAH4&channels&in&the& regulation&of&plant&Cl−&homeostasis&......................................................................................................&158& 5.3.1.&Cl−&homeostasis&during&seedling&establishment&.....................................................&159& 5.3.2.&Cl−&homeostasis&in&plants&under&favourable&growth&conditions&......................&160& 5.3.3.!The& homeostatic& model& conforms& to& the& phenotype& of& the& slah13 and& slah43mutant&lines&grown&under&favourable&conditions&.................................................&161& 5.3.4.!Cl−&homeostasis&in&plants&under&abiotic&stress&conditions&.................................&162& 5.4.&The&relevance&of&SLAC/SLAH&anion&channels&vs.3NPF&transporters&..............................&162& 6.&CONCLUSIONS&........................................................................................................................................&165& BIBLIOGRAPHY&...........................................................................................................................................&169& & & ! & INTRODUCTION ! Introduction* * * 3* 1. INTRODUCTION ! 1.1.!CHLORIDE!AS!A!NUTRIENT! Physiological*events*in*plant*metabolism*are*directly*or*somewhat*associated*with*the* mineral*elements.*Therefore*the*attempt*to*understand*the*molecular*mechanisms*behind*their* transport*has*great*importance*in*terms*of*basic*and*applied*plant*sciences.* * 1.1.1.!Chloride!is!an!essential!micronutrient!for!plants! Chloride*(Cl–)*is*an*essential*micronutrient*for*higher*plants*(Broyer*et#al.,*1954;*Johnson* et#al.,* 1957;* Whitehead,* 1985),* involved* in* the* regulation* of*important* cellular* functions* like* enzyme* activities,* membrane* potentials,* pH* gradients* and* electrical* excitability* (Marschner,* 1995;*Teakle*and*Tyerman,*2010;*White*and*Broadley,*2001;*Xu*et#al.,*2000).* As*an* essential*micronutrient,*Cl–*is* involved* in* the* stabilization*of*the* water*splitting* system*at*the*oxidizing*site*of*photosystem*II*(PSII)*in*the*chloroplast*and*in*the*regulation*of* enzyme* activities* such* as* the* asparagine* synthethase,* amylases,* and* the* protonUpumping* ATPase*at*the*tonoplast*(Churchill*and*Sze,*1984;*Kusunoki,*2007).*Important*Cl–*functions*are* also* related* to* the* electrical* charge* balance* of* essential* cations* such* as* potassium* (K+)* and* protons*(H+),*playing*main*roles*in*the*stabilization*of*the*electric*potential*of*cell*membranes* and*the*regulation*of*pH*gradients*(Broadley*et#al.,*2012b;*Hänsch*and*Mendel,*2009;*White*and* Broadley,*2001;*Xu*et#al.,*2000).** In*addition,*Cl–*is*a*major*osmotically*active*solute*in*the*vacuole*involved*in*both*turgor* and*osmoregulation*processes,*such*as*stomatal*movement*(section*1.5.2.2)*and*the*activity*of* motor*cells*controlling*nastic*movements,*like*the*seismonastic*leaf*movement*of*Mimosa#pudica* (section* 1.4.3;* Broadley* et# al.,* 2012b;* Flowers,* 1988;* Hänsch* and* Mendel,* 2009;* Marschner,* 1995;*White*and*Broadley,*2001).* The*tissue*Cl–*concentration*at*which*deficiency*symptoms*are*observed*varies*between* about*0−1*to*5−7*mg*g−1*Dry*Weight*(DW),*around*0.3*mg*g−1*DW*in*most*glycophyte*plants*(Zu* et# al.,* 2000).* Typical* symptoms* of* Cl–*deficiency* include* curling* of* leaflets,* reduction* of* leaf* growth,*bronzing*and*wilting*of*leaves,*as*a*consequence*of*chlorosis*and*necrosis*(Ozanne*et#al.,* 1957;*Smith*et#al.,*1987;*White*and*Broadley,*2001).*Moreover,*roots*become*stunted*and*the* development*of*lateral*roots*is*suppressed*and*fruits*are*decreased*in*number*and*size*(White* and*Broadley,*2001).*Furthermore,*Schwenke*and*colleagues*(2015)*showed*that*Cl–*deficiency* also*delayed*flowering*and*reduced*plant*biomass,*grain*size*and*grain*yield*in*most*durum*and* bread*wheat*varieties*trialled.** Nevertheless,*Cl–*deficiency*rarely*occurs*in*normal*conditions*because*the*biochemical* requirement*of*plants*for*Cl–*is*very*low*and*the*Cl–*concentrations*in*soil*after*rain,*irrigation* and*pollution*are*usually*enough*(Chen*et#al.,*2010;*White*and*Broadley,*2001).*It*becomes*a* limiting*factor*for*plant*growth*in*areas*of*high*precipitation*far*from*the*sea*(Fixen,*1987;*Xu*et# al.,*2000).*Its*value*as*fertiliser*was*recognised*in*the*midU1800s*when*sodium*chloride*(NaCl)* was* applied* to* stiffen* barley* straw* in* England*(Schwenke* et# al.,* 2015).* Researchers* have* reported*inconsistent*yield*responses*to*the*application*of*Cl–*fertiliser*in*winter*cereals*grown* Introduction* * * 4* on* soils* where* tests* indicated* a* likely* response* (DíazUZorita* et# al.,* 2004;* Fixen* et# al.,*1986;* Freeman*et#al.,*2006;*Grant*et#al.,*2001).*The*response*to*Cl–*fertilizers*is*higher*in*plant*species* with*a*high*Cl–*requirement*such*as*kiwifruit*(Buwalda*and*Smith,*1991;*Smith*et#al.,*1987)*and* palm* trees* (Braconnier* and* d’Auzac,* 1990).* In* addition,* Cl–*in* macronutrient* fertilizers* was* found*to*partially*control*a*number*of*diseases*in*different*crop*species*(Fixen,*1993).* ! 1.1.2.!Chloride!as!a!beneficial!macronutrient!! Although* classified* as* a* micronutrient,* our* research* group* has* compiled* abundant* information* indicating* that* Cl–*is* a* multifunctional* beneficial* macronutrient.* Under* moderate* external*Cl–*in*the*soil*medium*(3−5*mM),*plants*accumulate*Cl–*to*levels*of*2−20*mg*g−1*DW*that* are*typical*of*the*content*of*a*macronutrient*(Broadley*et#al.,*2012b;*Brumós*et#al.,*2010;*FrancoU Navarro*et#al.,* 2016a;* Marschner,*1995;*Xu*et#al.,* 2000),* specifically* stimulating* plant* growth* and*leaf*cell*expansion,*improving*tissue*water*balance*and*water*relations*at*the*whole*plant* level* (FrancoUNavarro*et#al.,* 2016a;* Raven,* 2017).* In* addition,* macronutrient* levels* of* Cl–*are* specifically*required*to*allow*optimal*seedling*establishment,*to*enhance*waterUuse*and*nitrateU use*efficiency,*to*increase*drought*tolerance,*and*to*improve*mesophyll*diffusion*conductance*to* CO2*(FrancoUNavarro*et#al.,*2012;!FrancoUNavarro*et#al.,*2016b;*FrancoUNavarro*et#al.,*2016c).* Under*nonUsaline*conditions*Cl–*uptake*via*the*symplastic*pathway*is*an*electrogenic*Cl– /2H+*symport* mechanism,* which* requires* metabolic* energy* (Beilby* and* Walker,* 1981;* Britto* and*Kronzucker,*2006;*Felle,*1994;*Sanders,*1980;!White*and*Broadley,*2001).*Its*uptake*should* implicate* regulatory*mechanisms* since*Cl–*accumulation*to* macronutrient* concentration* requires*a*considerable*use*of*energy*(Brumós*et#al.,*2010).* * 1.2.! CHLORIDE! IN! THE! AGRICULTURE!AND!ITS! INTERACTION! WITH! NITRATE! Chloride*concern*in*the*agriculture*is*related*in*most*of*the*cases*to*its*toxicity*and*also* to*its* antagonism* effect* in*nitrate* (NO3–)* uptake*and* accumulation.* But* Cl–*sensitivity* varies* extensively*within*plant*species*and*the*inhibition*of*NO3–*uptake*by*Cl–*is*determined*also*by* the*plant*species,*as*well*as*the*NO3–*and*Cl–*concentrations*in*the*medium.*However,*according* to*the*results*of*our*research*group,*Cl–*toxicity*thresholds*have*been*underestimated*and*the* antagonistic*effect*of*Cl–*on*the*transport*and*assimilation*of*NO3–*in*higher*plants*has*probably* been*overestimated*(FrancoUNavarro*et#al.,*manuscript*in*preparation).* * 1.2.1.!Chloride!in!the!agriculture! The*negative*effects*of*high*Cl–*concentrations*in*the*soil*and*in*irrigation*water*on*crop* production*are*observed*in*coastal,*arid,*and*semiarid*areas,*where*freshwater*sources*are*often* scarce*and*the*available*groundwater*is*saline*(Xu*et#al.,*2000).** Sensitivity*to*high*Cl–*concentrations*varies*widely*between*plant*species*and*cultivars.* Generally,*most*nonwoody*crops*tolerate*high*levels*of*Cl–,*whereas*many*woody*plant*species* and*beans*are*susceptible*to*Cl–*toxicity*(Maas,*1986).*There*are*agricultural*crops*tolerant*to* high* levels* of* soil* Cl–,* as* corn* (Parker* et# al.,*1985).* Sugar* beet* is* the* crop* with* the* greatest* tolerance*to*Cl–,*that*may*contain*up*to*50.8*mg*g−1*Cl–*in*the*leaves*(Zhou*and*Zhang,*1992).* Introduction* * * 5* Whereas*another*crops*are*sensitive*to*Cl–,*like*the*chinese*cabbage*(Yin*et#al.,*1989)*and*the* soybean*(Parker*et#al.,*1983).*The*least*sensitive*plants*are*large*seeded*beans,*squash,*barley,* maize*and*buckwheat*(White*and*Broadley,*2001).* According* to* the* literature* (reviewed* in* Xu* et# al.,* 2000),* the* critical* toxicity* concentration* is* about* 4–7* and* 15–50* mg* g−1*for* Cl–Usensitive* and* Cl–Utolerant* plant* species,* respectively.** The*data*in*our*laboratory*indicate*that*these*toxicity*thresholds*should*be*modified.*On* the*one*side,*in*the*presence*of*5*mM*Cl–*Nicotiana#tabacum*accumulates*40–50*mg*g−1*Cl−*in*the* leaves*(FrancoUNavarro*et#al.,*2016a).*According*to*Xu*et#al.*(2000),*this*is*the*critical*toxicity* level*of* Cl−Utolerant* species.* The* soUcalled* Cl−Usensitive* Citrus* varieties* Carrizo* citrange* and* rough*lemmon,*treated*for*30*weeks*with*4.5*mM*Cl−,*showed*no*visible*symptoms*of*stress*with* leaf*accumulation*values*of*30–35*mg*g−1*Cl−,*also*typical*of*a*supposed*tolerant*specie*(Walker* et#al.,*1982;*Brumós*et.#al.,*2010).*On*the*other*side,*our*group*has*measured*values*of*5–15*mg* g−1*Cl−*content*in*different*plant*species*growing*with*very*low*amounts*of*Cl−*(75*mM)*during* seedling* establishment* (FrancoUNavarro* et# al.,* manuscript* in* preparation).* According* to* the* aforementioned*ionic*toxicity*thresholds,*these*values*correspond*to*the*critical*toxicity*level*of* Cl−Usensitive*species.*It*seems*therefore*that:*(1)*the*role*of*Cl−*as*a*multifunctional*beneficial* macronutrient* have* yet*to* be* adequately* established;* (2)* the* type* of* treatment* and* the* developmental* state* of* the* plant* are* parameters* that* must* be* taken* into* account* to* define* chloride*toxicity*and*tolerance*thresholds.* * 1.2.2.!Chloride!and!nitrate!interaction! Both*NO3–*and*Cl–*are*essential*for*plant*growth*and*the*most*abundant*anions*in*plants* tissues.*In*addition*to*Cl–,*NO3–*represents*the*major*counterion*for*K+*and*also*plays*an*essential* role* in* plant* cellular* osmoregulation* and* membrane* potential* control* (FrancoUNavarro* et#al.,* 2016a;*Marschner,*1995;*White*and*Broadley,*2001).*These*anions*can*be*transported*through* the*same*or*different*proteins*since*they*are*monovalent*anions*with*a*similar*ionic*radius.* The*macronutrient*NO3–*is*the*major*source*of*nitrogen*(N)*for*protein*and*nucleic*acid* metabolism*and,*after*water,*the*main*limiting*factor*for*growth*of*terrestrial*plants*(Marschner,* 1995).* Uptake* of* sufficient* N* from* the* environment* serves* as* a* key* process* for* plants* to* successfully*grow*and*develop*(Wang*et#al.,*2012).*Nevertheless*the*availability*of*N*is*often*a* limiting* factor* in* plant* growth* and* crop* productivity,* but* the* application* of* N* fertilizers* to* counteract*N*deficiencies*can*have*detrimental*economic*and*environmental*effects.*Plant*roots* can* effectively* absorb* certain* forms* of* N* in* the* soil,* including* organic* (amino* acids)* and* inorganic* molecules,* such* as* NH4+*and* NO3–.* Nitrate* is* the* most* common* form* in* which* N* is* assimilated*by*the*plant,*indicating*that*NO3–*uptake*and*allocation*are*key*factors*in*efficient*N* utilization*(Wang*et#al.,*2012).* As* other* nutrients* like* phosphate* or* ammonium* (NH4+),* NO3–*participates* in* the* regulation*of*lateral*roots*development*and*architecture,*leaf*development,*flowering*induction* and*seed*dormancy*(Alboresi*et#al.,*2005;*Chiu*et#al.,*2004;*Drew*et#al.,*1973;*Drew*and*Saker,* 1975;*Krouk*et#al.,*2010b;*Marin*et#al.,*2011;*Remans*et#al.,*2006;*Ruffel*et#al.,*2011,*2014,*2016;* WalchULiu*et#al.,*2006).** Introduction* * * 6* Besides* its* role* as* a* nutrient,* NO3–*is* also* a*signal* molecule,* which* is* involved* in* the* control*of*many*physiological*processes,*plant*growth*and*crop*yield*(Crawford,*1995;*Krapp*et# al.,*2014;*Vidal*et#al.,*2014).*As*a*signal*molecule,*NO3–*can*induce*the*expression*of*a*number*of* genes*implicated*in*NO3–*transport*and*assimilation*(Bi*et#al.,*2007;*Krouk*et#al.,*2011;*Medici* and*Krouk,*2014;*Wang*et#al.,*2000;*2003,*2004).* The*form*in*which*N*is*taken*up*by*a*plant*(NH4+*or*NO3–)*affects*the*sensitivity*of*the* plant* to* Cl–*salt* stress* (Xu* et# al.,* 2000).* The* antagonism* between* NO3–*and* Cl–*uptake* was* demonstrated* in* many* agronomic* crops,* such* as* avocado* (Bar* et#al.,* 1997;* Wiesman,* 1995),* barley*(Glass*and*Siddiqi,*1985;*Smith,*1973),*broccoli*(Liu*and*Shelp,*1996),*citrus*(Bañuls*et# al.,*1990,*1997;*Bar*et#al.,*1997;*Cerezo*et#al.,*1997;*Chapman*and*Liebig,*1940),*kiwifruit*(Smith* et#al.,*1987),*melon*and*lettuce*(Feigin,*1985;*Wei*et#al.,*1989),*peanut*(Leidi*et#al.,*1992;*Wang* et#al.,*1989),*potato*(James*et#al.,*1970),*strawberry*(Wang*et#al.,*1989),*tobacco*(Fuqua*et#al.,* 1976),*tomato*(Feigin*et#al.,*1987;*Kafkafi*et#al.,*1982;*Zabala,*1984)*and*wheat*(Silberbush*and* Lips,*1991;*Wang*et#al.,*1989).* It* is* well* established* that* the* presence* of* NO3–*in* soils* can* mitigate*the* toxic* effect* of* excessive*Cl–,*apparently*due*to*the*suppressive*effect*that*NO3–*has*on*Cl–*fluxes*into*the*plant,* and* on* Cl–*accumulation* in* plant* tissues* (Adler* and* Wilcox,* 1995;* Bar* et#al.,* 1997;* Glass* and* Siddiqi,*1985;*Kafkafi*et#al.,*1982).*Increasing*concentrations*of*NO3–*decrease*Cl–*concentrations* in*plants*in*a*linear*fashion*(Xu*et#al.,*2000).*The*competition*of*NO3–*versus*Cl–*was*found*to*be* stronger*in*saltUsensitive*plants,*such*as*peanut,*than*in*saltUtolerant*plants,*like*cotton*(Leidi*et# al.,*1992).* Less*clear*is*the*inhibition*of*NO3–*uptake*by*Cl–,*which*has*been*reported*to*depend*on* the*plant*species*and*the*concentrations*of*both*NO3–*and*Cl–*in*the*uptake*medium*(Cerezo*et# al.,* 1997).* Our* research* group* has* shown* that* under* a* moderate* Cl–*treatment* (5mM* Cl–),* resulting* macronutrient* Cl–*accumulation* (40–50* mg* g−1*Cl–)* promote* optimal* NO3–Uuse* efficiency.* Probably,* plants* under* subUoptimal* Cl–*nutrition* require* compartmentalization* of* NO3–*for* maintaining* adequate* osmoregulatory* capacity,* reducing* NO3–*availability* for* N* assimilation*(FrancoUNavarro*et#al.,*2016b).** A* variety* of* NO3–*transporters* have* evolved* in* the* plant* kingdom* with* different* localizations*and*functions:* uptake*of*NO3–*by*roots,*longUdistance*transport*in*the*xylem*and* phloem,* regulation* of* NO3–*content* in* seeds,* and* transport* into* the* vacuole* for* storage* (Dechorgnat*et#al.,*2011;*Forde,*2000;*Hechenberger*et#al.,*1996;*Orsel*et#al.,*2002;*Tsay*et#al.,* 2007;*Wang*et#al.,*2012).*In*Arabidopsis#four*transporter*families*are*involved*in*these*functions:* the*lowUaffinity*nitrate*transporter*1/peptide*transporter*(NRT1/PTR)*family,*recently*named* NPF*(Lerán* et# al.,* 2014),* the* high* affinity* nitrate* transporter* 2* (NRT2)* family*transporters* (Krapp*et#al.,*2014;*Orsel*et#al.,*2002),*the*chloride*channel*(CLC)*family*(BarbierUBrygoo*et#al.,* 2011),*and* the* slow* anion* channelUassociated* homologues* (SLAC/SLAH)* family!(Negi* et# al.,* 2008).*Several*of*these*transporters*also*play*versatile*roles*in*NO3–*sensing,*plant*development,* pathogen* defense,* and* stress* response* (Krapp,* 2015;* Krapp* et#al.,* 2014;* Krouk* et#al.,* 2010a;* Noguero* and* Lacombe,* 2016;* Roeselfma* et# al.,* 2012;* Wang* et# al.,* 2012).*However,* these* transporter*families*are*not*exclusive*for*NO3–*and*in*many*cases*also*transport*Cl–.*Altogether* these*four*families*contain*73*genes,*but*only*35*have*been*characterized*in*detail*and,*among* these,*24*encode*NO3–*transporters*(Krapp*et#al.,*2014).* Introduction* * * 7* On*a*worldwide*basis*Cl–*deficiency*rarely*occurs*in*normal*conditions,*and*the*bigger* concern*about*this*anion*is*due*to*its*toxicity.* * 1.3.!CHLORIDE!TOXICITY!UNDER!SALT!STRESS! The*saline*areas*of*the*world*consist*of*salt*marshes*of*the*temperate*zones,*mangrove* swamps* of* the* subtropics* and* tropics,* and* their* interior* salt* marshes* adjacent* to* salt* lakes.* Saline* soils* are* abundant* in* semiarid* and* arid* regions,* where* the* amount* of* rainfall* is* insufficient* for* substantial* leaching.* In* addition,* salinity* problems* occur* in* nonirrigated* croplands*and*pastures*as*a*result*of*evaporation*and*transpiration*of*saline*underground*water* or*due*to*salt*input*from*rainfall*(Marschner,*1995).* Soil*salinity*is*a*major*abiotic*stress*that*adversely*affects*crop*quality*and*productivity* (Munns,*2002;*Munns*and*Tester,*2008;*Roy*et#al.,*2014).*And*this*problem*is*increasing*due*to* irrigation,*improper*drainage,*seawater*in*coastal*areas,*and*salt*accumulation*in*arid*and*semiU arid*regions*(Rengasamy,*2010).** Saline*soils*are*characterized*by*toxic*levels*of*chlorides*and*sulphates*of*sodium*(Na+).* Salinity* restricts* plant* growth* as* it* causes* nutritional* constraints* by* decreasing* uptake* of* phosphorus,* K+,* NO3−*and* calcium* (Ca2+),* ion* cytotoxicity* and* osmotic* stress,* leading* to* metabolic*imbalances*and*oxidative*stress,*and*thereby*decreasing*the*yield*and*quality*of*crops* (Alscher*et#al.,*1997;*Hirt*and*Shinozaki,*2004;*Munns,*2002;*Munns*and*Tester,*2008;*Munns*et# al.,*2006;*Rengasamy,*2010;*Roy*et#al.,*2014).*! According* to* plant* growth* response* in* saline* soils,* species* can* be* grouped* into* four* categories:*(1)*halophytes*(defined*as*the*native*flora*of*saline*soils),*which*can*be*subdivided* further*into*species*whose*growth*is*stimulated*(e.g.*Sueda#maritima,*Atriplex#numnmlaria)*and* species*whose*growth*is*little*affected*by*200*mM*Cl–*(e.g.*Atriplex#hastata,*Spartina#spp.,*sugar* beet);*(2)*nonUhalophytes*(glycophytes)*whose*growth*is*reduced*substantially*by*100*mM*Cl–,* which*can*be*additionally*subdivided*into*tolerant*(e.g.*Festuca#rubris,*Puccinella#peisonis,*cotton,* barley),*intermediate*(e.g.*tomatoes)*and*sensitive*(e.g.*beans,*soybeans)*species;*and*(3)*very* saltUsensitive* nonUhalophytes* (e.g.* citrus* and* woody* plant* species).* Many* important* cereal,* vegetable*and*fruit*crops*are*susceptible*to*Cl–*toxicity*during*cultivation*(White*and*Broadley,* 2001).* Considering* that*salinity* is* a* common* feature* of* arid* and* semiarid* lands,* plants* have* evolved*mechanisms*to*tolerate*it*through*the*adequate*control*of*several*process*mediated*by* membrane* transporters*(Fig.* 1.1):!(1)*regulation* of* influx* into* cells,* (2)* control* over* long* distance*transport,*and*(3)*compartmentalization*at*both*cellular*and*tissue*levels*(Blumwald,* 2000;*Flowers*and*Colmer,*2008).** Considering*that*NaCl*is*the*most*soluble*and*widespread*salt,*it*is*not*surprising*that*all* plants*have*evolved*mechanisms*to*regulate*its*accumulation*and*to*select*against*it*in*favour*of* other* nutrients* commonly* present* in* low* concentrations,* such* as* K+*and* NO3−*(Munns* and* Tester,*2008).* * Introduction* * * 8* * Figure! 1.1.! Salt! tolerance! mechanisms! in! plants! regulated! by! membrane! transporters.*(1)* Ion* uptake*control*into*cells.* (2)*Ion* movement* control*over*long* distance*transport.* (3)* Regulation*of*the* compartmentalization*at*cellular*level.* * As*mentioned* earlier,* there* are* two* main* components* to* salinity* stress* in* plants:* an* initial*osmotic*stress*in*response*to*the*increase*in*external*osmotic*pressure*and*a*subsequent* accumulation* of* toxic* ions,* which* negatively* affects* cellular* metabolism* (Munns* and* Tester,* 2008;*Munns*et#al.,*2006).*The*osmotic*component*of*salinity*is*caused*by*excess*inorganic*ions,* such*as*Na+*and*Cl−,*in*the*environment*that*decrease*the*osmotic*potential*of*the*soil*solution* and*hence*water*uptake*by*the*root.*Therefore,*uptake*of*abundantly*available*Na+*and*Cl−*offers* a*comparatively*cheap*way*to*lower*the*tissueUosmotic*potential*(Brini*and*Masmoudi,*2012).* To* avoid* the* risk* of* ion* toxicity* associated* with* this* strategy,* Na+*and* Cl−*are* generally* compartmentalized!at*the*cellular*and*intracellular*level,*especially*in*the*vacuole*(section*1.4.4)* and* in* less* sensitive* tissues*to* avoid* toxic* concentrations* within* the* cytoplasm,* especially* in* mesophyll*cells*in*the*leaf*(Munns*and*Tester,*2008;*Roy*et#al.,*2014).*Concurrently,*adjustment* of* the* cytoplasmic* compartment* is* achieved* via* production* of* compatible* osmolytes* such* as* proline,*mannitol,*sorbitol,*and*glycine*betaine*(Brini*and*Masmoudi,*2012).*The*glycine*betaine* additionally*acts*as*antioxidant*and*thus*detoxifies*reactive*oxygen*species*(ROS).*Nevertheless,* when*plants*are*growing*in*high*salt*concentrations,*an*adequate*sequestration*of*ions*in*the* vacuole*can*become*a*limiting*factor,*especially*in*the*case*of*glycophytes.** Under* salinity,* plants* can* accumulate* excessive* amount* of* Na+*in* the* cytosol,* which* negatively*affects*many*aspects*of*cellular*physiology.*The*most*abundant*inorganic*cation* in* the* plant* cytosol* is* K+.* The* physicochemical* similarities* between* Na+*and* K+*lead* to* a* competition* at* transport* and* catalytic* sites* that* usually*bind* the* essential* cation* K+*and* maintaining*a*high*cytosolic*K+/Na+*ratio* is*believed* to* improve*salt*tolerance*(Maathuis*and* Antmann,*1999;*Zhu,*2001).** Similarly*to*the*selectivity*between*Na+*and*K+*to*maintain*favourable*K+/Na+*ratios,*a* parallel*situation*has*been*proposed*for*Cl–*with*respect*to*the*macronutrient*NO3−,*considering* that*Cl−*and* NO3−*compete*for* membrane* transport* mechanisms.* These* anions* can* be* transported* through* the* same* or* different* proteins* since* they* are* monovalent* anions* with* a* similar*ionic*radius*(Li*et#al.,*2016).*However,*contrary*to*K+,*Na+*and*Cl−,*NO3−*is*assimilated*in* plant*anabolic*processes.*In*addition,*plant*NO3−*concentrations*strongly*varies*along*different* developmental*stages,*e.g.*vegetative*versus#reproductive*stages,*indicating*that*NO3−/*Cl−*ratios* might*not*be*an*adequate*predictor*of*Cl−*toxicity*under*salt*stress*conditions*(ColmeneroUFlores* JM,*personal*communication).* Introduction* * * 9* Given*that*Cl−*loading* into*root*xylem*vessels*is*a*key*mechanism*regulating*shoot*Cl−* accumulation*(Brumós*et#al.,*2010;*Gong*et#al.,#2011;*Tregeagle*et#al.,#2010),*xylem*loading*of* NO3−*and*Cl−*has*to*be*tightly*regulated*to*maintain*an*appropriate*balance*between*both*anions.* Taken*into*account*that*the*thermodynamics*of*anion*transport,*release*of*Cl−*and*NO3−*into*the* root*xylem*is*highly*likely*to*be*electrochemically*passive*and*therefore*facilitated* by* plasma* membrane*anion*channels*that*are*not*yet*identified.* Both*Na+*and*Cl−*are*toxic*to*plants*at*high*concentrations,*but*it*has*been*proposed*that* Cl−Usensitive*species*can*control*Na+*transport*better*than*Cl−.*Under*salt*stress*conditions,*Cl−U sensitive,*frequently*woody*perennial*plants*exhibit*toxicity*symptoms*to*Cl−,*rather*than*to*Na+,* accumulation.* This* difference* may* arise* because* Na+*is* accumulated* so* effectively* in* woody* roots*and*stems*that*little*amounts*reach*the*leaves.*Thus*Cl−,*which*continues*passing*to*the* leaves,*becomes*the*more*significant*toxic*component*of*the*saline*solution*(Munns*and*Tester,* 2008;*Teakle*and*Tyerman,*2010;*White*and*Broadley,*2001).*But*this*statement*does*not*imply* that*Cl−*is*more*metabolically*toxic*than*Na+,*rather*that*these*species*are*better*excluding*Na+* from*the*leaf*blades*than*Cl−*(Munns*and*Tester,*2008).*Therefore,*many*economically*valuable* species*in*which*Na+*is*predominantly*retained*in*the*roots*and*stems,*Cl−*is*considered*more* toxic* because*this* ion* is* accumulated* to* high* levels* in* shoot* tissues,* negatively* impacting* on* essential* processes* such* as* photosynthesis* (Brini* and* Masmoudi,* 2012)*and* leading* to* the* disturbance*of*growth*and*productivity*(Geilfus*et#al.,*2015;*Munns,*2001;*Teakle*and*Tyerman,* 2010).* This* phenomenon* has* been* mainly* described* in* fruit* tree* species* that* are* routinely* grown*on*Cl−Uexcluding*rootstocks*like*citrus*(Maas,*1993;*Storey*and*Walker,*1999;*Tadeo*et#al.,* 2008),* stone* fruit* trees* (Bernstein* et#al.,* 1956),* grapevine* (Antcliff* et#al.,* 1983)* and* avocado* (Bar*et#al.,*1997).*But,*Cl−*excess*is*also*harmful*to*a*number*of*herbaceous*species,*including* economically* important* cereals* and* vegetables* as* soybean* (Abel,* 1969),* wheat* (Bernal* et#al.,* 1974)*and*barley*(Greenway*and*Munns,*1980).** In* citrus,* the* evidence* for* the* detrimental* effect* of* Cl−*is* mostly* supported* by* the* association*between*genetic*differences*in*the*rate*of*Cl−*accumulation*in*leaves*and*the*plant* salinity*tolerance*(Cole,*1985;*Moya*et#al.,*2003;*RomeroUAranda*et#al.,*1998;*Storey*and*Walker,* 1999).* According* to* this* information,* Bañuls* and*PrimoUMillo* (1992)* showed* that* NaCl* and* potassium* chloride* (KCl)* stress* had* similar* toxicities,* while!sodium* nitrate* (NaNO3)* resulted* less* harmful.* Higher*correlations* between* shoot* Cl−*accumulation,* than* between* Na+* accumulation,*and*defoliation*were*found*using*different*rootstock*combinations*and*different* chloride*salts*(Bañuls*and*PrimoUMillo,*1995;*Brumós*et#al.,*2009).*Photosynthesis*and*growth* disturbances* were* also*highly* correlated* with* leaf* Cl−*growth*(RomeroUAranda* et# al.,* 1998;* Brumós* et# al.,* 2009).* A* gradient* of* toxicity* (KCl>NaCl>CaCl2)* that* did* not* correlate* with* the* presence*of*the*toxic*cation*Na+*was*found*when*salinity*damage*was*quantified*in*terms*of*dry* weight*loss*(RomeroUAranda*et#al.,*1998)*or*in*terms*of*global*gene*expression*(Brumós*et#al.,* 2009).* Improving* salt* exclusion* from* the* shoot* is* one* resource*for*enhancing* plant* salinity* tolerance*(Apse*and*Blumwald,*2007;*Guan*et#al.,*2014;*Horie*et#al.,*2009;*Munns*and*Tester,* 2008;* Munns* et# al.,* 2012).*Given* that* Cl−*tolerant* citrus* rootstocks* apparently* possess* more* efficient*root*mechanism*for*Cl−*exclusion*than*sensitive*ones*(FernándezUBallester*et#al.*2003;* Moya*et#al.*2002;*Storey*and*Walker*1999),*Brumós*and*colleagues*(2010)*studied*Cl−*excluder* and* includer* citrus* rootstocks* under* nonUsalinized* conditions* in* order* to* delimit* specific* Cl−* transport*activities*in*the*symplastic*pathway,*Cl−*uptake,*translocation*and*distribution.*They* Introduction* * * 16* * Figure! 1.5.! Representative! currentXvoltage! curves! for! the! three! plasma! membrane! anion! conductances! from! barley! xylem! parenchyma! cells.*XUSLAC,*slowly*activating*anion*conductance;*XU QUAC,* quickly* activating* anion* conductance;* XUIRAC,* inwardly* rectifying* anion* conductance.* EANION,* Equilibrium*potential*for*the*permeant*anion.*Obtained*from*Roberts,*2006.* * Moreover,*Gilliham*and*Tester*(2005)*showed*that*XUIRAC*and*XUQUAC*are*differentially* regulated*by*water*stress*and*Köhler*and*colleagues*(2002)*demonstrated*that*apoplastic*NO3–* modifies* the* voltage* dependent* gating* of* XUQUAC.* The* anion* channel* inhibitor*4,4'U diisothiocyanatostilbeneU2,2'Udisulfonic* acid* (DIDS)* significantly* reduced* xylem* loading* in* barley*seedlings,*indicating*that*anion*channels*contribute*to*the*release*of*anions*into*xylem* vessels*(Köhler*et#al.,*2002;*Schroeder*et#al.,*1993).** To*maintain*an*appropriate*balance*between*the*major*anions*NO3–*and*Cl–*in*the*rootU toUshoot* translocation* process,* xylem* loading* has* to* be* tightly* regulated.* In* response* to* salt* stress,* plants* restrict* the* translocation* of* Cl–*into* the* shoot* by* Cl–*exclusion* from* the* xylem* vessels*in*the*root.*Nevertheless,*the*molecular*nature*of*transporters*loading*the*xylem*with*Cl–* and*the*regulatory*mechanism*leading*to*an*appropriate*balance*between*NO3–*and*Cl–*in*the* xylem*sap*remain*elusive.** * 1.4.3.!Redistribution!via!the!phloem! During*the*growth*of*a*plant,*Cl–*is*translocated*from*the*root*to*the*shoot*via*the*xylem* and* is* redistributed* between* tissues* via* the* phloem.* LongUdistance* transport* in* the* phloem* takes*place*in*living*cells,*the*sieve*tubes*(Fig.*1.6).*The*sieve*element*(SE)/companion*cell*(CC)* complex*is*the*functional*unit*of*sieve*tubes*in*angiosperms.*Whereas*the*SE*is*almost*dead,*the* CC*is*full* of*activity*(Van* Bel,* 2003).* The* highly*specialized*SEs*accommodate*a*mass*flow* of* phloem* sap,* which* is* always* directed* from* sugar* producing* regions* (the* sources)* to* sugar* consuming* regions* (the* sinks).* For* mineral* nutrients,* the* main* sites* for* phloem* loading* are* located*in*the*stem*and*leaves*as*components*of*either*mineral*nutrient*supply*to*growth*sinks* (shoot* apices,* fruits,* roots)* or* of* nutrient* recycling* (Marschner,* 1995).* The* driving* force* for* phloem* transport* of* loading* solutes* at* the* source* and* its* unloading* at* the* sink* is*thought* to* result*in*a*gradient*of*osmotic*potential*along*the*phloem*tubes*(Liesche*and*Schulz,*2013).*The* resulting*gradient*in*turgor*pressure*gives*rise*to*the*movement*of*water*and*solutes*(mass*flow* as*great*as*1*m*h−1)*toward*the*sink*(Oparka*and*Turgeon,*1999;*Patrick,*1997;*Sjölund,*1997).* Introduction* * * 17* * Figure!1.6.!Structure!of!the!sieve!elements!(SEs)!in!Vicia%faba%with!companion!cells!(CCs).*SEs*and* CCs* are* connected* through* numerous* pore/plasmodesma* units* (PPUs).* SieveUelement* plastids* (Pl),* mitochondria*(M),*and*endoplasmic*reticulum*stacks*(ER)*are*partially*positioned*and*evenly*distributed* along*the*SE*plasma*membrane,*whereas*parietal*proteins*(PPs)*are*locally*aggregated.*PPs*and*ER*are* sometimes*located*on*the*sieve*plates*(SPs)*or*at*the*margins*of*the*sieve*pores,*but*do*not*impede*mass* flow.*C,*callose;*CW,*cell*wall;*N,*nucleus;*P,*plastids;*V,*vacuole.#Modified*from*Van*Bel,*2003.* * Phloem*transport*has*a*role*in*cycling*of*mineral*elements.*Potassium*is*the*major*cation* in* the* phloem* and* stimulates* sugar* loading* into* the* phloem* sap* (Giaquinta,* 1980;* Peel* and* Rogers,*1982).*Within*the*phloem,*Cl–*is*relatively*mobile*(Lessani*and*Marschner,*1978)*and*its* recirculation*(defined*as*the*ratio*of*phloem/xylem*nutrient*fluxes)*is*about*20%*in*a*number*of* plants*with*the*phloem*Cl–*concentration*positively*correlating*with*the*Cl–*solution*in* which* plants*are*grown*(White*and*Broadley,*2001).*In*the*phloem*sap,*Cl–*concentration*may*be*in*the* order* of* 120* mM* and* seems* to* play* a* role* in* phloem* loading* and* unloading* of* sugars,* for* example*in*the*osmoregulation*in*the specialized*motor*leaf*organs,*pulvini,*of*Mimosa#pudica# during*the*very*fast*seismonastic*leaf*movement*(Fig.*1.7).*This*movement*is*thought*to*result* from* sudden* Cl–,* K+*and* sugars* unloading* from* the* phloem* into* the*extensor* apoplast.* The* increase* of*osmotic* potential*promotes* water* efflux* from* cells*(Fromm* and* Eschrich,* 1989;! Moran,*2007).*The*osmotic*strength*for*the*leaf*movement*is*powered*by*a*plasma*membrane* H+UATPase,*which*drives*KCl*and*water*fluxes.*The*movement*results*from*different*volume*and* turgor*changes*in*the*two*oppositely*positioned*parts*in*the*pulvinus*(Moran,*2007).* * Figure! 1.7.!Cross!section! of!Mimosa%pudica%leaves.*(a)*Leaf*in*an*open*position.! (b)*Leaf*in*a*folded* state.*Obtained*from*Taiz*and*Zeiger,*2002.*** * Introduction* * * 18* Leaves* are* sink* for* NO3–*during*the* vegetative* growth* stage* meanwhile*assimilated* N* compounds* are* remobilized* for* reuse* in* the* developing* seeds* during* senescence* (Xu* et# al.,* 2012).*Nitrogen* can* be* also* recycled* from* older* (senescent)* leaves* to* sustain* the* growth* of* younger* developing* tissues.* Although* NO3–*is* mostly* transported* by* xylem,* phloem* transport* has*been*proposed*for*local*NO3–*redistribution*in*the*root*(Wang*and*Tsay,*2011)*and*for*NO3–* remobilization* from* older* leaves* to* NUdemanding* tissues,* mostly* under* conditions* of* N* starvation*(Fan*et#al.,*2009;*Kiba*et#al.,*2012).** The*plasma* membrane*low* affinity* NO3–*transporter*NRT1.7*(recently* named* as* AtNPF2.13;*Léran*et#al.,*2014),*which*belongs*to*the*NPF*family,*is*localized*in*the*phloem*SE/CC* complex*and*is*involved*in*NO3–*excess*movement*from*older*leaves*into*developing*tissues*(Fan* et#al.,*2009).*In*addition,*another*NPF*member*called*AtNRT1.9*(recently*named*as*AtNPF2.9;* Léran*et#al.,*2014)*is*expressed*in*phloem*CC*of*roots*and*is*involved*in*local*NO3–*redistribution* in*the*root*(Wang*and*Tsay,*2011).*Furthermore,*the*high*affinity*NRT2.4*transporter*from*the* NRT2*family,*is*expressed*in*phloemUassociated*cells*and*is*involved*in*the*delivery*of*NO3–*into* the*phloem*for*remobilization*under*conditions*of*N*starvation*(Kiba*et#al.,*2012).* As*mentioned*above,*to*mitigate*the*damaging*effects*of*salinity,*the*movement*of*toxic* ions* across* cellular* membranes* is* tightly* regulated.* At* the* wholeUplant* level,* plants* restrict* shoot* ion* accumulation* by* controlling* net* ion* uptake* and* xylem* loading* (Britto* et# al.,* 2004;* Brumós*et#al.,*2010;*Craig*Plett*and*Møller,*2010;*Møller*and*Tester,*2007;*Teakle*and*Tyerman,* 2010).*But*according*to*Davenport*and*colleagues*(2007)*and*Munns*(2002),*recirculation*of* ions*to*the*roots*via*the*phloem*does*not*significantly*contribute*to*the*reduction*of*Na+*and*Cl–* levels*in*leaf*tissue.** Nowadays,*different*NO3–*transporters*involved*in*NO3–*redistribution*within*the*plant* have*been*identified*at*the*phloem*in*Arabidopsis,*but*the*identification*of*membrane*transport* proteins*involved*in*Cl–*allocation*though*the*phloem*remains*unexplored*so*far.* * 1.4.4.! Vacuolar! compartmentalization! and! transport! into! other! organelles! Ion* channels* reside* on* intracellular* membranes* to* regulate* various* organellar* and* cellular*functions*(Stael*et#al.,*2012;*Zampese*and*Pizzo,*2012).*Over*80%*of*transport*processes* occur* inside* the* cells,* but* the* mechanisms* of* ion* flux*across* intracellular* membranes* (the* endoplasmic*reticulum,*Golgi*apparatus,*endosomes,*lysosomes,*mitochondria,*chloroplasts,*and* vacuoles)*are*difficult*to*investigate*and*remain*poorly*understood.** The* large* central* vacuole* serves* as* a* storage* reservoir*that* accumulates* and* releases* ions*as*well*as*metabolites*according*to*demands.*The*physical*and*functional*plasticity*of*the* vacuole* enables* plants* to* efficiently* use* energy* and* nutrients* and* maintain* optimal* physiological* conditions* in* the* cytosol.* Vacuolar* storage* capacity* regulates* intracellular* ion* homeostasis* but* also* influences* wholeUplant* ion* accumulation* and* distribution* (Baetz* et# al.,* 2016).*Plant*vacuoles*store*high*concentrations*of*Ca2+*and*Na+*(Peiter,*2011),*with*Cl–*acting*as* a*major*counteranion*and*osmotically*active*solute*involved*in*both*turgor*and*osmoregulation* processes*(Marschner,*1995).* In*addition,*vacuolar*ion*uptake*is*crucial*to*confer*cellular*tolerance*during*longUterm* salinity*and*also*to* modulate*shoot*ion* accumulation*and*wholeUplant*ion*distribution*during* Introduction* * * 19* early*phases*of*salinity*(Baetz*et#al.,*2016).*During*salinity*the*sequestration*of*Na+*and*Cl–*ions* into*the*vacuole*is*crucial*to*maintain*optimal*metabolic*conditions*in*the*cytosol*and*mitigate* cellular* damage.* Several* transport* proteins* localized* at* the* tonoplast* have* been* suggested* to* facilitate*intracellular*ion*distribution*and*thereby*regulate*cellular*ion*homeostasis*(Martinoia* et# al.,* 2007,* 2012).* However,* only* few* transporters* involved* in* vacuolar* ion* uptake* during* salinity*have*been*studied.* Some*transporter*families*are*related*with*anion*flux*across*intracellular*membranes*in* Arabidopsis,*such*as*the*chloride*channel*(CLC),*the*cationUchlorideUcotransporter*(CCC)*and*the* aluminumUactivated*malate*transporter*(ALMT)*families.* ! 1.4.4.1.!The!CLC!family! Since* the* vacuole* is* moderately* positive* with* reference* to* the* cytoplasm,* part* of* the* vacuolar* Cl–*sequestration* could* proceed* through* ion* channels.* Several* voltageUgated* anion* channels* of*the*chloride* channel*(CLC)* family*(Fig.*1.8)*have*been*located*in* the* tonoplast*of* various*species.*Phylogenetic*and*functional*analyses*have*shown*that*plant*CLC*genes*encode* anion* channels* and* active* (NO3–/Cl–)/H+*antiporters,* localized*in* endosomal* compartments* (Chen,*2005;*Lv*et#al.,*2009;*Zifarelli*and*Pusch,*2010).** * Figure!1.8.!Phylogenetic!tree!of!the!CLC!family!from!Arabidopsis%thaliana!(At)!and!Oriza%sativa!(Os).* Obtained*from*Von*der*Fecht*et#al.,*2010.* * Seven* CLC* members* have* been* identified* in* the* Arabidopsis#genome* (Isayenkov* et#al.,* 2010)*and*all*of*them*are*expressed*in*vascular*tissues*in*both*roots*and*shoots*(De*Angeli*et#al.,* 2007;*Isayenkov*et#al.,*2010;!Lv*et#al.,*2009;*Zifarelli*and*Pusch,*2010).*** The* studies* on* the* subcellular* localizations* of* AtCLCs* have* illuminated* intracellular* anion*transport*mechanisms*in*plant*cells.*In*2006*the*AtCLCa*transporter*was*characterized*as* a*2Anion−/H+*exchanger*that*drives*the*uptake*of*anions*into*vacuoles*of*Arabidopsis*mesophyll* and*guard*cells*(Fig.*1.9;*De*Angeli*et#al.,*2006;*Wege*et#al.,*2016).*AtCLCa*is*permeable*to*Cl−*and* NO3−,* transporting* NO3−*more* selectively* than* Cl−*(De* Angeli* et# al.,* 2006;* Wege* et#al.,* 2010).* Arabidopsis#clca*knockout*mutants*provide*evidence*that*CLCa*functions*in*NO3–*accumulation* into*vacuoles*in*A.#thaliana**(Geelen*et#al.,*2000).*Furthermore,*it*was*shown*that*a*very*steep* Introduction* * * 20* NO3–* gradient* is* maintained*in* plants* accumulating* NO3–* as* N* reserve*(Wege* et# al.,* 2014).* Interestingly,*AtCLCa*plays*a*dual*role*in*guard*cells,*accumulating*anions*in*the*vacuole*during* stomata*opening*in*response*to*light*and*releasing*anions*from*the*vacuole*during*ABAUinduced* stomata*closure.*The*direction*of*NO3–*flux*is*established*by*its*phosphorylation*status*carried* out*by*the*kinase*OST1*(Open*Stomata*1;*also*named*SnRK2.6)*on*its*NUterminal*domain*(Wege* et#al.,*2014).* * Figure! 1.9.! Subcellular! localization! of! CLC! family! members! in!Arabidopsis%thaliana.*AtCLCaUc*and* AtCLCg*are*located*in*the*tonoplast,*AtCLCd*and*AtCLCf*are*present*at*the*plasma*membrane*of*the*Golgi* vesicles,*and*AtCLCe*is*localized*in*the*thylakoid*membranes.*Modified*from*BarbierUBrygoo*et#al.,*2011.* * AtCLCb#is*expressed*in*roots,*shoots*and*in*filaments*and*petals*in*the*flower*(Von*der* FechtUBartenbach*et#al.,*2010).*The*CLCb*transporter*functions*also*as*a*NO3−/H+*antiporter*like* CLCa.* Both* channels* have* a* higher* selectivity* for* NO3−* over* Cl−*and* are* important* for* anion* accumulation* inside* the* vacuole* (Fig.* 1.9;* De* Angeli* et#al.,* 2006;* Geelen* et#al.,* 2000;* Von* der* FechtUBartenbach* et#al.,* 2010).* However,* given* that*no* phenotype* of* CLCb* was* reported,* the* physiological*role*of*CLCb*remains*elusive*(Martinoia*et#al.,*2012).* A*a*single*amino*acid*substitution*can*change*the*selectivity*from*NO3−*to*Cl−*(Bergsdorf* et# al.,* 2009),* raising*the* possibility* that* other* members* of* the* CLC* family* might* actually* be* Cl−/H+*antiporters.*Indeed,*AtCLCc,*which*is*localized*at*the*tonoplast*(Fig.*1.9)*and*expressed*in* guard*cells,*pollen*grains*and*in*the*root*(Jossier*et#al.,*2010),*is*involved*in*the*accumulation*of* Cl−*into*the*vacuole.*TUDNA*insertion*mutants*in*CLCc#presented*lower*Cl−*vacuolar*contents,*and* this*fact*correlated*with*the*impaired*lightUinduced*stomatal*opening*and*ABAUinduced*stomatal* closure*showed*by*these*mutants*(Jossier*et#al.,*2010).*In*addition,*the*clcc*knockout*mutants* were*found*to*be*more*sensitive*to*salt*stress*than*the*wild*type,*and*hence*it*was*suggested*that* CLCc*contributes*to*the*detoxification*of*the*cytosol*by*sequestrating*Cl–*into*the*vacuole*(Jossier* et#al.,*2010).* AtCLCg,*the*closest*homolog*to*AtCLCc,*is*also*expressed*at*the*tonoplast*(Fig.*1.9),*like* CLCa,*CLCb*and*CLCc*(Marmagne*et#al.,*2007;*Nguyen*et#al.,*2016)*and*is*strongly*expressed*in* mesophyll*cells,*but*also*in*epidermal*cells,*hydathodes*and*phloem*within*the*leaf*(Nguyen*et# al.,*2016).*As*CLCc,*CLCg*has*a*physiological*role*in*plants*during*salt*stress*and*both*genes*act* in*the*same*pathway*through*reciprocal*control*of*their*expression*(Nguyen*et#al.,*2016).* Introduction* * * 21* AtCLCd*was*localized*to*the*Golgi*apparatus*(Fig.*1.9)*and*is*involved*in*pH*regulation*in* the*transUGolgi*system*(Von*der!FechtUBartenbach*et#al.,*2007).%AtCLCf*also*reside*in*the*Golgi* membranes,*specifically*al*the*cisUGolgi*(Fig.*1.9;*Marmagne*et#al.,*2007).*And*AtCLCe*is*targeted* to*the*thylakoid*membranes*(Marmagne*et#al.,*2007)*and*to*the*chloroplast*(Fig.*1.9;*Teardo*et* al.,*2005).*The*outer*envelope*membrane*of*the*chloroplast*is*considered*to*be*permeable*to* most* ions* and* metabolites.* In* contrast,* the* inner* envelope* membrane* and* the* thylakoids* harbour*numerous* selective* ion* and* metabolite* transport* pathways,* allowing* regulation* of* optimal*metabolic*activities*and*of*signaling*within*this*organelle*(Eisenhut*et#al.,*2015;*Finazzi* et#al.,*2015;*Weber*and*Linka,*2011).*CLCe*and*CLCf*are*predicted*to*function*as*channels*rather* than* transporters* based* on* their* amino* acid* sequences,* in* particular* by* substitution* of* a* conserved*glutamic*acid,*implicated*in*Cl−/H+*antiporter*activity*(Zifarelli*and*Pusch,*2010).* * 1.4.4.2.!The!CCC!family! AtCCC,*a*member*of*the*cationUchlorideUcotransporter*(CCC)*family* in*Arabidopsis,*is*a* Na+:K+:Cl−* cotransporter* (NKCC)* localized* at* the*plasma* membrane* of* the*transUGolgi* system* involved*in*plant*development*and*ion*homeostasis*(ColmeneroUFlores*et#al.,*2007;*Groen*et#al.,* 2014;*Henderson*et#al.,*2015).*Given*that*AtCCC*affects*rootUtoUshoot*distribution*of*Cl−*and*Na+* in*A.#thaliana*and*that*promoter*GUS*fusions*of*AtCCC*in#planta*displayed*strong*expression*in* the* vasculature* at* the* xylem/symplast* boundary*(ColmeneroUFlores* et# al.,* 2007),* it* was* proposed*to*retrieve*Cl−*from*the*xylem.*But*its*localization*at*the*Golgi/transUGolgi*network*and* its* strong* implication* on* plant* development* suggested*a* function* in* the* Golgi* network!that* impacts*plant*development*and*indirectly*influences*the*ability*of*plants*to*tolerate*high*salinity.* Probably*the*CCC*family*functions*as*NKCC*but*further*studies*to*clarify*its*function*in*plants* should*be*done.* * 1.4.4.3.!The!ALMT!family!! The*ALMT*(aluminumUactivated*malate*transporter)*protein*family*is*specific*to*plants* (Xu*et#al.,*2015)*and*encodes*channels*able*to*mediate*anion*fluxes*across*cellular*membranes* (BarbierUBrygoo*et#al.,*2011).* The*ALMT*protein*family*is*constituted*of*fourteen*members*in* Arabidopsis#(Hoekenga*et#al.,*2006)*and*the*phylogenetic*tree*is*characterized*by*the*presence*of* three*main*branches* (Fig.*1.10;*Kovermann*et#al.,*2007).* Recent*results*show*that*in*clade*II,* ALMT4,*ALMT5,*ALMT6*and*ALMT9*are*localized*at*the*tonoplast,*indicating*that*clade*II*may* consist*of*vacuolar* ALMTs* (Martinoia*et#al.,* 2012).* Furthermore,*ALMT6* and*ALMT9* mediate* anion* fluxes* directed* to* the* vacuolar*lumen,* i.e.,* inwardUrectifying* currents* (De* Angeli* et# al.,* 2013;*Kovermann*et#al.,*2007;*Meyer*et#al.,*2011).** The* MA2−*permeable* channel#AtALMT4* was* recently* known* to* be* targeted* to* the* vacuolar*membrane,*and* the*corresponding*gene*is*highly*expressed*in*leaves*and* roots.*The* atalmt4#knockout*mutants*exhibited*an*impairment*of*drought*tolerance*compared*to*wildUtype* plants*(Zhang,*2013),*but*its*function*has*not*been*precisely*determined*so*far.* * Introduction* * * 22* * Fig!1.10.!Arabidopsis%thaliana!ALMT!family!phylogenetic!tree.*The*AtALMT*dendogram#presents*three* main*groups.*Branch*lengths*are*proportional*to*the*level*of*interfered*evolutionary*change*and*are*given* in* relative* units.* AttDT,* Arabidopsis# thaliana*tonoplast* dicarboxylate* transporter;* TaALMT1,*Triticum# aestivum*aluminumUactivated*malate*transporter*1.*Obtained*from*Kovermann*et#al.,*2007.* * ALMT6*was*found*to*be*expressed*in*Arabidopsis#guard*cells*where*it*was*localized*to* the*tonoplast*(Meyer*et#al.,*2011).*It*is*a*hyperpolarizationUactivated*channel*permeable*to*MA2−* and*fumarate,*which*is*present*in*guard*cells*in*large*quantities.*This*channel*transport*MA2−* into* the* vacuole* in* a* Ca2+U* and* pHUdependent* manner* (Meyer* et# al.,* 2011).* A* detailed* electrophysiological* analysis* revealed* that* vacuolar* pH* and* cytosolic* malate* regulate* the* threshold*of*activation*of*AtALMT6Umediated*currents.*The*interplay*of*these*two*parameters* determines*whether*AtALMT6*functions*as*a*MA2−*influx*or*efflux*channel*(Meyer*et#al.,*2011).** ALMT9* resides* in* the* vacuolar* membrane*of* mesophyll* and* guard* cells*and* it* is* also* localized* in* both* shoots* and* roots.* This* channel*is* permeable* to!MA2−*and* Cl−,* whereby* its* physiological*function*is*linked*to*the*Cl−*conductivity*(De*Angeli*et#al.,*2013;*Kovermann*et#al.,* 2007).*Differently*from*ALMT6,*ALMT9*is*not*regulated*by*cytosolic*Ca2+*(De*Angeli*et#al.,*2013).* ALMT9Umediated*Cl−*currents*across*the*tonoplast*are*activated*by*physiological*concentrations* of* cytosolic* MA2−,* and* mesophyll* vacuoles* of* almt9*knockout*mutants* lacking* the* vacuolar* channel* exhibit* lower* overall* Cl−*currents* (De* Angeli* et#al.,*2013).* Moreover,*ALMT9*channel* mediating* influx* of* Cl−*ions* into* vacuoles* is* essential* for* stomatal*opening,* given* that*almt9* plants*have*reduced*Cl−*uptake*into*vacuoles*of*guard*cells*resulting*in*impaired*lightUdependent* stomatal* opening* and* reduced* wilting* during* drought* stress.* Recently,* Baetz* and* colleagues* (2016)*showed* that* ALMT9* is* highly* expressed* in* the* vasculature* of* shoots* and* roots*and* is* somehow*involved*in*the*regulation*of*longUdistance*transport*of*Cl−*and*Na+.* * Summarizing,* a* plenty* of* metabolite* and* ion* transport* systems* are* found* in* plastids.* Nowadays*the*protein*families*that*are*related*with*Cl−*transport*across*the*plasma*membrane* of*the*vacuole,*Golgi*apparatus*and*chloroplasts*in*Arabidopsis*are*the*chloride*channel*(CLC),* the*cationUchlorideUcotransporter*(CCC)*and*the*aluminumUactivated*malate*transporter*(ALMT)* families*(Fig.* 1.11).* Among* these* families,* the* protein*channels* localized*to* the* tonoplast* (ALMT4,*ALMT6,*ALMT9,*CLCaUc*and*CLCg)*regulate*Cl−*vacuolar*sequestration,*encouraging*Cl−* homeostasis* regulation.*But* further* investigations* to* better* understand* their* regulation* mechanisms*and*to*identify*new*Cl−*transporters*should*be*done.* ! Introduction* * * 23* ! Figure! 1.11.! Ion! channels! at!intracellular! membranes! in! Arabidopsis% thaliana.*CLCaUc,* CLCg* and* ALMT9*are*located*in*the*vacuolar*membrane.*CLCd*and*CCC*are*localized*at*the*transUGolgi*network*and* CLCf*is*also*present*at*the*plasma*membrane*of*the*Golgi,*specifically*al*the*cisUGolgi.*And*CLCe*is*located* in*the*chloroplast.*Modified*from*BarbierUBrygoo*et#al.,*2011.* * 1.5.!PLASMA!MEMBRANE!PROTEINS! Depending* on* the* thermodynamics,* plasma* membrane* proteins* that* mediate* the* transport* of* solutes* across* biological* membranes* can* be* classified* in* active* and* passive* transporters*(Gadsby,*2009).** Active*transporters*catalyse*molecules*translocation*across*cellular*membranes*against* their* electrochemical* gradient,* activity* that* requires* energy* input.* These* transporters* can* be* divided*according*to*their*energy*source*in*primary*and*secondary*active*transporters*(Gadsby,* 2009).*On*the*one*side,*primary*active*transporters*can*be*energized*by*ATP*hydrolysis*(ATPU powered*pumps),*light*and*redox*reactions*that*are*coupled*by*the*protein*to*the*movement*of* molecules* uphill* the* electrochemical* gradient,* like* H+UATPases* (Gadsby,* 2009).* On* the* other* side,* secondary* active* transporters* (carrier* proteins)* use* the* electrochemical* gradient* of* a* chemical* species* as* an* energy* source* and* couple* the* downhill* flux* of* this* species* to* the* movement* of* a* second* species* against* its* electrochemical* gradient.* Thus,* the* activity* of* secondary*active*transporters*is*not*directly*driven*by*ATP*hydrolysis*but*relies*on*the*presence* of* electrochemical* gradients* of* solutes* across* cell* membranes* that* are* generated* and* maintained* by* primary* transporters.*In* higher* plant* cells,* the* energy* exploited* by* secondary* active*transporters*is*generated*by*the*gradient*of*H+*across*cellular*membranes.*Carriers*move* solutes* across* membrane* by* selective* binging* and* significant* conformational* change.* Furthermore,* secondary* active* transporter* can* be* classified* into* symporters* and* antiporters.* While*the*symporters*transport*two*substrates*in*the*same*direction*across*the*membrane,*the* antiporters*transport*the*substrates*in*opposite*directions*(Gadsby,*2009).* Passive* transporters* allow*the* fluxes* of* molecules* across* membranes* along* their* electrochemical* gradient* and* are* dissipative* transport* systems.* Ion* channels* are* a* specific* Introduction* * * 24* category* of* passive* transporters.* They*are* integral* membrane* proteins* that* form* selective* aqueous* pores* through* which* anions* are* rapidly* transported* across* cell* membranes.* Ion* channels* switch* between* open* and* closed* states* according* to* the* factors* that* control* their* gating.*When*an*ion*channel*is*open,*massive*ion*fluxes*occur*according*to*their*electrochemical* gradients.* Ion* movement* across* a* membrane* through* ion* channels* can* be* measured* directly* using*electrophysiological*techniques,*as*twoUelectrode*voltage*clamp*(TEVC).*The*ion*channels* selectivity* depends* on* the* specific* interaction* between* the* permeating* ionic* species* and* the* permeation*pore*of*the*channel*(Dutzler*et#al.,*2002,*2003;*Yamashita*et#al.,*2005;*Zhou*et#al.,* 2001).*Based*on*their*selectivity*characteristics,*ion*channels*are*traditionally*divided*into*anion* and*cation*channels.*In*order*to*fulfil*their*biological*function,*their*activity*is*tightly*regulated* by* different* parameters* such* as* the* membrane* potential,* ligands,* hormones,* light* and* posttranslational*modifications*like*protein*phosphorylation*(Hille,*2001).* Anion* channels*are* present* in* all* plant* membranes,* where* they* are* key* to* a* wide* spectrum* of* physiological* functions* in* plants,* such* as* turgor* regulation,* membrane* voltage* stabilization,* pH*regulation,*signal*propagation,*toxic*metal* tolerance* and* nutrient* acquisition* (BarbierUBrygoo* et# al,* 2011;* Roberts,* 2006;* Roelfsema* et# al,*2012).* Furthermore,* plasma* membrane*channels*are*responsible*for*short*and*long*distance*net*anions*fluxes.* * 1.5.1.!Anion!channels!in!plants! Plant*anion*channels*can*be*broadly*classified*on*the*basis*of*their*voltage*dependence* into*depolarizationU*and*hyperpolarizationUactivated*channels*and*channels*activated*by*light*or* membrane*stretch.** DepolarizationUactivated* anion* channels* can* be* subdivided* further* according* to*their* kinetics* and* gating* properties* into* Rapid* type* (RUtype),* Slow*type* (SUtype),* and* outwardly* rectifying*anion*channels*(Schroeder,*1995;*White*and*Broadly,*2001).** RUtype* and* SUtype* channels* differ* in* the* velocity* of* their* voltageUdependent* activation* and*deactivation*and*both*display*the*lowest*degree*of*activity*at*negative*membrane*potentials* (Vm* <* −150* mV),* but* are*activated* when* the* plasma* membrane* becomes* more* positive* (depolarizes). The*speed*of*RUtype*channel*activation*is*approximately*1000*times*faster*than* activation*of*SUtype*channels*(5–50*ms*for*RUtype*versus*5–50*s*for*SUtype*channels;*Kolb*et#al.,* 1995;*Linder*and*Raschke,*1992).** * Figure!1.12.!Schematic!representation!of!wholeXcell!activation!and!deactivation!currents!for!(a)!SX type! currents! and! (b)! RXtype! currents.*The* dashed* line* represents* zero* current.* EA,* equilibrium* potential*for*the*permeant*anion;*Vm,*membrane*potential.*Obtained*from*Roberts,*2006.** Introduction* * * 25* * RUtype* channels,* which* display* pronounced* voltage* dependence,* are* inactive* at* hyperpolarized*membrane*potentials*but*progressively*activate*upon*depolarization*(Fig.*1.12* b;*Hedrich*and*Marten,*1993;* Kolb* et#al.,* 1995).*In* contrast,!SUtype* channels* are* only* weakly* voltage* dependent* (Fig.* 1.12* a).*The* voltage* dependent* gating* of* both* anion* channel* types* is* strongly* modulated* by* the* external* anion* activity* (Dietrich* and*Hedrich,* 1998;* Geiger*et# al.,* 2009b,*2011;* Hedrich* and*Marten,* 1993;* Hedrich* et# al.,* 1994;* Lohse* and*Hedrich,* 1995;* Maierhofer*et#al.,*2014b).* The* difference* between* the* anion* channel* types* becomes* obvious* when* cells* are* exposed*to*a*gradual*change*in*membrane*potential*(from*−160*mV*to*+80*mV)*within*seconds* (voltage*ramp;*Fig.*1.13).*SUtype*channels*facilitate*inward*(negative)*currents*(anion*efflux)*at* −160* mV* that* decrease* at* more* positive* potentials* and* eventually* the* current* reverses* the* direction*(anion*influx;*Fig.*1.13*a).*The*current–voltage*relation*for*RUtype*channels*is*similar,* except*for*small*currents*at*strongly*negative*potentials*because*of*fast*channel*deactivation*at* these*voltages*(Fig.*1.13*b;*Hedrich*and*Marten,*1993;*Kolb*et#al.,*1995).*The*RUtype*channels* have* a* maximum* activity* at* approximately* −50* mV* and* they* are* rapidly* deactivated* by* hyperpolarization*of*the*plasma*membrane.* * Figure!1.13.!CurrentXvoltage!relationships!of!(a)!SXtype!and!(b)!RXtype!anion!channels!in!Vicia%faba! guard!cell!protoplasts!in!the!absence!and!presence!of!ABA.*The*activities*of*these*two*types*of*anion* channels* are* stimulated* by* the* presence* of* ABA.* The* SUtype* anion* channel* presents* a* nearly* linear* currentUvoltage* relationship,* indicating* weak* voltage* dependence,* whereas* the* RUtype* anion* channel* shows*a*strong*deactivation*at*membrane*potential*more*negative*than*−100*mV.*ABA,*abscisic*acid;*Im,* current;*Vm,*voltage*across*the*membrane.*Obtained*from*Roelfsema*et#al.,*2012* * Being*permeable*to*the*common*anions*MA2–,*NO3–*and*Cl–,*the*RUtype*channel*has*the* peculiarity* to* be* highly* permeable* also* to* sulphate.* However,* SUtype* channels* are*highly* selective* for* NO3−*but* they* are*also* permeable,* in*a* lesser* extent,* to* Cl–*(Geiger* et#al.,* 2009b,* 2011;*Schmidt*and*Schroeder,*1994).** RUtype*and*S* Utype*channels*coexist*in*the*plasma* membrane*of*several*cell* types*and* their* physiological* role* was* elucidated* in* guard* cells* using* electrophysiological* approaches,* where* RU* and* SUtype* channels* were* respectively* named* QUAC* (for* quick* anion* channel)* and* SLAC*(for*slow*anion*channel).*These*channels*are*involved*in*the*early*steps*of*stomatal*closure* in*response*to*the*drought*hormone*abscisic*acid*(ABA;*section* 1.5.2.2;*BarbierUBrygoo*et#al.,* 2011;*Dreyer*et#al.,*2012).** Introduction* * * 32* permeable*to*NO3–*and*Cl–.*In*comparison*to*SLAC1,*SLAH3*exhibits*a*higher*preference*for*NO3–,* being*the*permeability*P(NO3−)/P(Cl−)*ratio*of*10*for*SLAC1*(Geiger*et#al.,*2009b)*and*of*20*for* SLAH3*(Geiger*et#al.,*2011).* * Figure!1.20.!Regulation!of!SLAH3!activity!by!different!protein!kinases.!(a)*BiFCUbased*interaction*of* SLAH3*and*its*regulatory*components*in*Xenopus#oocytes.*Pictures*show*a*quarter*of*an*optical*slice*of*an* oocyte* and* depict* fluorescence* in* the* plasma* membrane.*The* coinjected* plasmids* combinations* are* indicated* in* the* figure.*BiFC* experiments* revealed* that* SLAH3* physically* interacts* with* CPK21* and* CIPK23.*(b)*WholeUoocyte*current*recordings*in*standard*bath*solution*(3*mM*NO3−,*pH*7.5)*upon*20*s* voltage*pulses*ranging*from*+40*to*−180*mV*in*20*mV*decrements*followed*by*a*3*s*voltage*pulse*to*−120* mV.*The*holding*potential*was*0*mV.*No*current*responses*of*oocytes*expressing*SLAH3*or*CPK21ΔEF* alone* were* recorded.* Coexpression* of* SLAH3* together* with* CPK21ΔEF* resulted* in* macroscopic* anion* currents* slowly* deactivating* at* negative* membrane* potentials*in* the* presence* of* NO3−*only.* (c)* Instantaneous*currents*(IT)*recorded*at*−100*mV*of*oocytes*expressing*SLAH3*in*the*presence*of*OST1,* CPK21ΔEF*and*CPK23ΔEF*in* NO3−Ubased* buffer.*SLAH3*expression* alone* (indicated* as* none)* did* not* result* in* macroscopic* anion* currents.!CPK21ΔEF*and* CPK23ΔEF*are*truncated* CPKs* variant* lacking* a* junction*and*Ca2+Ubinding*domain,*which*renders*these*kinases*Ca2+Uindependent*and*constitutively*active* to*avoid*the*influence*of*different*kinaseUspecific*Ca2+*sensitivities*on*SLAH3*activation.*YFPC,*CUterminal* half*of*the*YFP*fused*to*the*protein;*YFPN,*NUterminal*half*of*the*YFP*fused*to*the*protein.*Modified*from* Geiger*et#al.,*2011*and*Maierhofer*et#al.,*2014a.** * Upon*ABA*activation,*SLAC1*and*SLAH3*release*NO3–*and*Cl–*in*a*concerted*action*in*the* plasma*membrane*of*guard*cells*(section*1.5.2.2.2;*Geiger*et#al.,*2011;*Negi*et#al.,*2008;*Vahisalu* et#al.,*2008).** Besides*its*localization*in*guard*cells,*SLAH3*was*also*shown*to*be*expressed*in*the*root* (Negi*et#al.,* 2008;*Zheng*et#al.,*2015),* where* it* was*recently* shown* that*plays* a* role*in*NO3–U dependent*alleviation*of*NH4+*toxicity*(Zheng*et#al.,*2015).** * Similar*to*SLAH3,*SLAH2*for*becoming*active*requires*phosphorylation*at*its*NUterminal* domain*by*a*Ca2+Udependent*kinase*for*channel*priming*and*the*presence*of*NO3–*in*the*extraU cellular*space*to*gate*open*(Fig.*1.21).*Unlike*SLAC1*and*SLAH3,*which*are*permeable*to*NO3–* and* Cl–,* SLAH2* is* strictly* NO3–*selective,* considering* that* it* has* a* permeability* P(NO3−)/P(Cl−)ratio*of*82.4*(Maierhofer*et#al.,*2014b).** * Introduction* * * 33* * Figure! 1.21.! Regulation! of! SLAH2! activity! by! different! kinases.! (a)*BiFC* experiments* in* Xenopus# oocytes*identified*interacting*proteins*with*SLAH2.*Pictures*show*a*quarter*of*an*optical*slice*of*an*oocyte* and*depict*fluorescence*in*the*plasma*membrane.*The*coinjected*plasmids*combinations*are*indicated*in* the* figure.*BiFC* assays* revealed* that* SLAH2* physically*interacts* with* CPK21* and* CIPK23.* (b)*WholeU oocyte*current*recordings*in*the*presence*or*absence*of*30*mM*NaNO3−*upon*20*s*voltage*pulses*ranging* from* +40* to* −180* mV* in* 20* mV* decrements* followed* by* a* 3* s* voltage* pulse* to* −120* mV.* The* holding* potential* was* 0* mV.* Cooexpression* of* SLAH2* together* with* CIPK23/CBL1* in* oocytes* resulted* in* macroscopic* anion* currents* in* the*presence* of* external* NO3−*only.* The* currents* slowly* deactivated* at* negative*membrane*potentials.!(c)*Instantaneous*currents*(IT)*of*oocytes*in*standard*buffers*containing* different*anions*(gluconate,*chloride*or*nitrate)*plotted*against*the*applied*voltage.*Nitrate*was*required* in*the*extracellular*bath*solution*for*ion*conduction.*CPK21ΔEF,*truncated*CPK*variant*lacking*a*junction* and*Ca2+Ubinding*domain,*which*renders*this*kinase*Ca2+Uindependent*and*constitutively*active*to*avoid* the*influence*of*different*kinaseUspecific*Ca2+*sensitivities*on*SLAH2*activation.*YFPC,*CUterminal*half*of*the* YFP*fused*to*the*protein;*YFPN,*NUterminal*half*of*the*YFP*fused*to*the*protein.*Modified*from*Maierhofer* et#al.,*2014b.** * Interestingly*when*expressed*in*slac1S2*lossUofUfunction*mutant*under*the*control*of*the* SLAC1* promoter,* SLAH1* was* capable* to* rescue* the* SLAC1* stomatal* phenotype* (Negi* et# al.,* 2008),* suggesting* that* this* SUtype* channel* isoform* might* carry* SLAC1Ulike* anion* transport* properties.*However,*the*physiological*role*of*SLAH1*and*SLAH4*in#planta*and*their*functional* characterization*in*heterologous*expression*system*remain*unexplored*so*far.*! * 1.5.2.!Hormonal!regulation!and!signal!transduction!by!ABA! There*are*similarities*between*xylem*loading*and*stomatal*closure,*considering*that*both* are* mechanisms*that* implicate*ion*efflux* and* are* regulated* by* ABA.* However,* whereas* ABA* triggers*SLAC1*and*SLAH3*channel*activation*(opening)*in*guard*cells,*the*stress*phytohormone* negatively* regulates* ion* channel* activities* implicated* in* rootUtoUshoot* translocation* of* Cl–* (Gilliham* and* Tester,* 2005).* This* indicates* that* the* regulation* of* Cl–*ion* efflux* from* cells* surrounding* the* xylem* and* stomatal* guard* cell* is* opposite,* suggesting* the* possibility* that* different*signalling*elements*are*involved*in*both*processes* * 1.5.2.1.!ABA!inhibits!salt!translocation!into!the!xylem! While*little*is*known*about*the*genes*and*proteins*responsible*for*Cl–*loading*into*the* root*xylem*apoplast,*more*is*known*about*the*biological*processes*involved.*ABA*was*shown*to* significantly*inhibit*xylem*loading*of*Cl–,*as*well*as*K+,*while*having*a*limited*effect*on*the*initial* uptake*of*these*ions*from*the*external*medium*(Cram*and*Pitman,*1972;*Pitman,*1977;*Pitman* and*Wellfare,*1978;*Pitman*et#al.,*1974).*Therefore,*abiotic*stress*leads*to*a*reduced*delivery*of* both*Cl–*and*K+*ions*to*the*shoot*and*their*accumulation*in*the*root*(Cram*and*Pitman,*1972;* Pitman,*1977;*Pitman*et#al.,*1974).*The*inhibition*of*ion*transport*following*water*stress*by*ABA* could*facilitate*the*observed*increases*in*the*osmotic*potential,*sustaining*root*growth.** Introduction* * * 34* Two*Arabidopsis*root*K+*channels,*AKT1*and*the*stelar*K+*outwardlyUrectifying*channel* SKOR,*have*been*identified*as*being*involved*in*K+*uptake*from*the*soil*solution*and*K+*release* into*the*xylem*sap,*respectively*(Gaymard*et#al.,*1998;*Hirsch*et#al.,*1998;*Lagarde*et#al.,*1996;* Xu* et# al.,* 2006).*While* SKOR* is* downUregulated* by* ABA* at* both* transcriptional* and* posttranslational*level*(Gaymard*et#al.,*1998;*Roberts,*1998;*Roberts*and*Snowman,*2000),*the* AKT1*expression* in* the* root* cortex* was* unaffected* by*this* hormone* (Roberts* and* Snowman,* 2000).** In* addition,* the* root* stele* NO3–*transporter* AtNRT1.5* (AtNPF7.3)* gene* expression* is* decreased*in*response*to*salt*stress,*decreasing*NO3–*translocation*to*shoot*(Taochy*et#al.,*2015).* The*AtNPF2.4*gene,*which*encodes*for*a*transporter*possibly*involved*in*Cl–*delivery*into* xylem*vessels,*was*also*downUregulated*by*salt*and*ABA*treatment**(Li*et#al.,*2016).* Therefore,* downUregulation* of* genes* expressed* in* vascular* tissues* following* ABA* application,*water*deficit*or*salt*exposure*can*be*used*as*a*framework*to*identify*proteins*that* regulate*Cl–*loading*into*the*root*xylem.* * 1.5.2.2.!ABA!triggers!salt!efflux!from!guard!cells! In* response* to* external* or* internal* stimuli,* including*light,* humidity,* Ozone* or* CO2* concentration,*phytohormones,*drought*and*longUdistance*signals*from*the*root,*turgor*changes* within* guard* cells* promote* stomatal* movement*to* regulate* the* uptake* of* CO2*and* the* transpirational* water* loss* from* leaves* (Assmann* and* Shimazaki,* 1999;* Hetherington* and* Woodward,*2003;*Raschkle,*1987;*Roelfsema*and*Hedrich,*2005;*Schroeder*et#al.,*2001b).** Stomatal*opening*and*closure*are*regulated*through*the*changes*in*guard*cells*turgor,* which*are*the*consequence*of*osmolyte*concentration*changes*within*the*cytosol*(Fischer,*1968;* Pandey*et#al.,*2007).*Solute*influx*and*efflux*take*place*through*plasma*and*vacuolar*membrane* ion* channels* and* transporters,* given* that* there* are* not* plasmodesmata*within* mature* guard* cells* (Kim* et#al.,* 2010;* Pandey* et#al.,* 2007;* Wille* and* Lucas,* 1984).*The* major* active* solutes* involved*in*these*processes*are*K+,*Cl–,*MA2–,* NO3–*and*other*metabolites*like*sugars*(Humble* and* Raschke* 1971;* MacRobbie* and* Lettau* 1980;* Poffenroth* et# al.,*1992;* Talbort* and* Zeiger,* 1998).* Considering* that*osmolytes* and* water* are* accumulated* in* the* vacuole,* all* fluxes* are* coupled*between*the*tonoplast*and*the*plasma*membrane*(Belin*et#al.,*2010;*Ward*et#al.,*1995).** * 1.5.2.2.1.!Stomatal!opening!! Guard* cells* keep* a* cytoplasmic* pH* of* 7.5−7.8,* at* which* the* H+UATPase* presents* a* low* activity.* Stomatal* opening* is* initiated* by* the* activation* of* the* membrane* H+UATPases* by* blue* light,*red*light*or*auxins*(Fig.*1.22;*Assmann*et#al.,*1985;*Schroeder*et#al.,*2001;*Shimazaki*et#al.,* 1986).*The*H+UATPases*cause*a*hyperpolarization*of*the*plasma*membrane*potential*through*the* H+*efflux*from*the* cytoplasm* to* the*apoplast*(Assmann*et#al.,* 1985;* Kinoshita*and*Shimazaki,* 1999;*Shimazaki*et#al.,*1986,*2007).! This*hyperpolarization*activates*inward*rectifying*K+*channels,*such*as*KAT1*(K+*channel* in* Arabidopsis#thaliana* 1)* and* KAT2* (K+*channel* in* Arabidopsis#thaliana* 2),! which*induce*the* passive*influx*of*K+*from*the*apoplast*to*the*cytoplasm*(Fig.*1.22;*Lebaudy*et#al.,*2007;*Pilot*et# al.,*2001;!Wang*and*Wu,*2013).*During*stomatal*opening,*guard*cells*accumulate*Cl–,*NO3–*and* Introduction* * * 35* MA2–*as*counterions*of*K+*(Van*Kirk*and*Raschke,*1978).*Presumably*Cl–*and*NO3–*enter*into*the* cell*by*symport*with*apoplastic*H+,*given*the*outwardUdirected*anion*gradient*and*the*negative* membrane* potential* of* plant* cells* (Dietrich* et# al.,* 2001;* Roelfsema* and* Hedrich,* 2005).*For* example,* the* NO3−/H+*symporter*AtNRT1.1* (also* known* as* CHL1*and* recently* named* as* AtNPF6.3;*Léran*et#al.,*2014)*mediates*NO3−*influx*from*the*apoplast*to*the*cytoplasm*(Fig.*1.22;* Guo*et#al.,*2003;*Tsay*et#al.,*1993).* In*guard*cells,*K+*is*accumulated*in*the*vacuole*by*H+/K+*antiporters,*and*anions*can*be* transported* into* vacuoles* through* lowUaffinity* anion* channels* and* H+/anion* exchange* mechanisms.*The*vacuolar*antiporter*K+/H+*AtNHX1*plays*an*important*role*in*K+*uptake*into* the* vacuole* (Fig.* 1.22;* Barragán* et# al.,* 2012;*Leidi* et# al.,* 2010;* Shi* and* Zhu,* 2002).*The* Cl−* channels*CLCa*and*CLCc*play*a*key*role*in*stomatal*function,*in*the*stomatal*opening*as*well*as* in*the*stomatal*closure.*On*the*one*side,*CLCa*drives*the*uptake*of*Cl−*and*NO3−*into*the*vacuole* during*stomata*opening*(Fig.*1.22;*Wege*et#al.,*2014).*On*the*other*side,*lightUinduced*stomatal* opening* was* impaired* in* AtCLCc* TUDNA* mutants* (Jossier* et# al.,* 2010),* indicating* that* CLCc* participates*in*vacuolar*Cl−*compartmentalization*during*stomatal*opening.* * Figure!1.22.!Guard!cell!signaling!during!stomatal!opening.*Blue*arrows#indicate*activation*or*positive* interactions*between*the*factors.*Grey*arrows#indicate*inhibition.*Dashed*arrows#indicate*ion*movement.* ALMT,* aluminumUactivated* malate* transporter;* CLC,* chloride* channel;* CPK,* Ca2+Udependent* protein* kinase;* KAT1,* K+*channel* in* Arabidopsis# thaliana*1;* NHX1,* Na+/H+*exchanger* 1;* NRT1.1,* nitrate* transporter*1;*PP2C,*protein*phosphatase*2C;*SnRK,*Snf−1*related*protein*kinase.* * Taken*into*account*that*the*RUtype*channel*ALMT6*is*activated*by*hyperpolarization*and* that*vacuolar*pH*and*cytosolic*MA2−*regulate*its*activation*threshold*(Meyer*et#al.,*2011),*ALMT6* will*be*probably*induced,*and*subsequently*MA2−*transport*into*the*vacuole*will*take*place*(Fig.* 1.22).*Similarly,*physiological*concentrations*of*cytosolic*MA2−*activate*ALMT9,*which*is*known* to*mediate*Cl−*currents*across*the*tonoplast*directed*to*the*vacuolar*lumen*(Fig.*1.22;*De*Angeli* et#al.,*2013).* Rise*of*K+,*Cl−*and*NO3−,*together*with*the*production*of*MA2−*from*osmotically*inactive* starch,* increase* the* osmotic* potential* (Fischer,* 1968).* This* process* decreases* the* water* Introduction* * * 36* potential,*driving* water* into* the* cells*that* increase* their*turgor* pressure,* thus* leading* to* the* swelling*of*guard*cells*and*to*the*concomitant*aperture*of*the*stomata.*** During*stomatal*opening*the*typeU2C*protein*phosphatase*(PP2C)*ABI1,*ABI2*and*HAB1* interact*with*different*protein*kinases,*such*as*OST1,*SnRK2.2,*SnRK2.3,*CPK3,*CPK6,*CPK21*and* CPK23,*supressing*their*activity*by*dephosphorylation*(Fig.*1.22;*Fujii*et#al.,*2009;*Imes*et#al.,* 2013;* Scherzer* et# al.,* 2012;* Umezawa* et# al.,* 2009;* Vlad* et# al.,* 2009).*In* addition,* ABI1* also* interacts*and*desphosphorilates*the*NUtermini*of*SLAC1*channel*(Maierhofer*et#al.,*2014a).* As*a*consequence*of*CPK*and*SnRK*dephosphorylation,*a*number*of*downstream*protein* effectors*involved*in*triggering*the*stomatal*closure*remain*deactivated,*like*SUtype,*RUtype*and* CLCa*channels*and*the*NADPH*oxidase*involved*in*ROS*production.* ! 1.5.2.2.2.!Stomatal!closure! Water*deficit,*under*drought*and*high*salinity*conditions,*leads*to*the*synthesis*of*the* plant*stress*hormone*ABA.*This*phytohormone*is*perceived*by*guard*cells,*although*its*origin*is* controversial.* This*hormone*could* travel*from* the* roots* to* the* aerial* parts* through* the* transpiration*stream*via*the*xylem*(Sauter*et#al.,*2001;*Tardieu*and*Davies,*1993;*Wilkinson*and* Davies,*2002;*Wilkinson*et#al.,*2007;*Zhang*et#al.,*1987)*or*ABA*could*be*released*from*internal* stores*(Georgopoulou*and*Milborrow,*2012)*or*newly*synthesized*in*leaves*(Bauerle*et#al.,*2003,* 2004;*Bauer*et#al.,*2013a,*b;*Christmann*et#al.,*2007;*McAdam*and*Brodribb,*2015;*McAdam*et# al.,*2016).* In*guard*cells,*ABA*triggers*a*fast*response*and*a*slow*response.*On*the*one*side,*the*fast* response*lead*to*the*stomatal*closure*within*seconds*or*a*few*minutes*through*the*activation*of* SUtype* and* RUtype*anion* channels* (Roelfsema* et# al.,* 2004)* and* the* repression* of* inwardU rectifying*K+*channels,*such*as*KAT1*and*KAT2.**On*the*other*side,*the*slow*ABA*response*takes* place* within* minutes* to* hours* and* implicates* the* basic* leucine* zipper* (bZIP)* transcription* factors*that*trigger*the*induction*of*genes*involved*in*longUterm*drought*tolerance*(Kline*et#al.,* 2010).** * 1.5.2.2.2.1.!Fast!ABA!signalling!pathway!! The* increased* ABA* level*in* the* leaves* is* perceived* by* members* of* the* ABA* receptor* PYR/PYL/RCAR*family*(Fig.*1.23;*Ma*et#al.,*2009;*Park*et#al.,*2009).*The*interaction*promotes*a* conformational* change* of* the* receptor* that* allows* its* following* interaction* with* a* protein* phosphatase*of*the*PP2C*family*(ABI1,*ABI2,!HAB1;!Miyazono*et#al.,*2009;*Nishimura*et#al.,*2009,* 2010;*Santiago*et#al.,*2009;*Yin*et#al.,*2009),*inhibiting*their*enzymatic*activity*(Ma*et#al.,*2009;* Park* et#al.,* 2009).*Accordingly* to* this* inhibition,* the* PP2C* phosphatases* cannot*suppress* the* SnRK*and*CPK*kinases.** ABA* also* downregulates* the* H+UATPases* located* in* the* plasma* membrane,* inhibiting* stomatal*opening*(Kinoshita*et#al.,*1995;!Schroeder*and*Hagiwara,*1989).* Many*second*messengers*regulate*ABA*signaling*in*guard*cells,*including*reactive*oxygen* species* (ROS),* nitric* oxide* (NO),* phosphatidic* acid* (PA),* phosphatidylUinositolU3Uphosphate* (PIP3)*and*inositolU3Uphosphate*(IP3;*Fan*et#al.,*2004;*Hirayama*and*Shinozaki,*2007;*Israelsson* et#al.,*2006;*Kim*et#al.,*2010;*Schroeder*et#al.,*2001a).* Introduction* * * 37* * Figure!1.23.!Stomatal!closure!by!ABA!induced!signaling!in!guard!cells.*Blue*arrows#indicate*activation* or*positive*interactions*between*the*factors.*Grey*arrows#indicate*inhibition.*Dashed*arrows#indicate*ion* movements.* ABA,* abscisic* acid;* ALMT,* aluminumUactivated* malate* transporter;* CLC,* chloride* channel;* CPK,*calciumUdependent*protein*kinase;*GORK,*guard*cell*outward*rectifying*K+*channel;*IP3,*inositolU3U phosphate;* KAT1,* K+*channel* in* Arabidopsis# thaliana* 1;* PA,* phosphatidic* acid;* PLC,* Phospholipase* C;* PLDα1,*Phoshpholipase* Dα1;*PP2C,*protein*phosphatase*2C;* PYL,*PYR1*like;*PYR,*pyrabactin*resistance;* RCAR,*regulatory*component*of*ABA* receptor;*SLAC1,*slow*anion*channel*associated*1;*SLAH3,*SLAC1U homolog*3;*SnRK,*Snf−1*related*protein*kinase;*TPK1,*twoUpore*K*channel*1.* * ABA* also* induces* an* increase* in* cytosolic* Ca2+,* which* is* also* an* essential* second* messenger*in*plant*signal*transduction*cascades.*Increase*of*cytosolic*Ca2+*is*a*consequence*of* both,*Ca2+*uptake*after*activation*of*plasma*membrane*channels!and*Ca2+*release*from*internal* stores* (Hamilton* et# al.,* 2000;* MacRobbie,* 2000;* Nomura* et# al.,* 2008;* Staxen* et# al.,* 1999).* Following*the*activation*of*the*Phospholipase*C*(PLC)*that*catalyses*inositol*1,4,5Utriphosphate* (IP3),*this*second*messenger*induces*an*increase*in*cytosolic*Ca2+*by*the*activation*of*tonoplast* Ca2+!channels*(Fig.*1.23;*Allen*et#al.,*1995;*Takahashi*et#al.,*2001;*Tang*et#al.,*2007).** Concurrently,*ABA*induces* the*production*of*the*Phospholipase*Dα1*(PLDα1),*which*in* turns*synthetizes*phosphatidic*acid*(PA).*This*PA*binds*to*the*PP2C*ABI1,*inhibiting*its*activity* and*promoting*ABA*signalling*(Fig.*1.23;*Mishra*et#al.,*2006;*Zhang*et#al.,*2004;*Zhao*and*Wang,* 2004).** Cytosolic*Ca2+*rise*initiates*the*processes*required*for*guard*cell*turgor*loss*through*the* modulation*of*ion*channels*and*pumps*in*both*the*plasma*and*vacuolar*membranes*(Fig.*1.23).* Therefore,*inhibition*by*PP2C*phosphatases*(ABI1,*ABI2*and*HAB1),* together*with*the* rise*of*Ca2+,*allow*the*activation*of*distinct*SnRK*and*CPK*protein*kinases.*Among*them,*OST1* together* with*CPK6* and* CPK23* become* active* by* autophosphorylation.*However* CPK3* and* CPK21*additionally*need*Ca2+*to*be*functionally*active.*These*kinases*further*interact*with*the* plasma* membrane* channels* SLAC1* and* SLAH3*(Fig.* 1.23),* phosphorylating*their* cytosolic* NU terminal* moieties* and* likely* these* channels*open*by* a* conformational* change* involving* their* phenylalanine* gate* (section* 1.5.1.2.3;* Geiger* et# al.,*2009b,* 2010,! 2011).* While* SLAC1* can* be* Introduction* * * 38* activated*by*all*of*these*protein*kinases,*SLAH3*is*stimulated* only*by*all*the*CPKs*but*not*by* OST1* (Brandt* et# al.,* 2012;* Geiger* et#al.,*2010,!2011;* Scherzer* et# al.,* 2012).*Therefore* these* channels*are*activated*in*a*Ca2+Udependent*(by*CPK*kinases)*and*Uindependent*(by*OST1*kinase)* manner*in*guard*cells*(Geiger*et#al.,*2010;*Hetherington*and*Brownlee,*2004;*Levchenko*et#al.,* 2005;*Marten*et#al.,*2007;*Mori*et#al.,*2006;*Schroeder*and*Hagiwara,*1989).** OST1* also* activates* the* RUtype*channel*ALMT12*by* phosphorilation,* which* mediates* MA2−*efflux*to*the* apoplast* (Fig.*1.23;*Imes* et#al.,* 2013;* Rasckle* et#al.,* 2003;* Roelfsema* et#al.,* 2004).** In*addition,*OST1*induces*ROS*production*by*activating*NADPH*oxidases*(Fig.*1.23),*such* as*the*plasma*membrane*AtrbohF* (respiratory*burst*oxidase*homologue*F;*Kwak*et#al.,*2003;* Murata*et#al.,*2001;*Sirichandra*et#al.,*2009).*This*ROS*signaling*activates*Ca2+*channels*in*the* plasma*membrane*of*guard*cells,*increasing*intracellular*Ca2+*(Fig.*1.23;*Köhler*et#al.,*2003;*Pei* et#al.,*2000),*and*stimulates*NO*and*PIP3*signaling,*which*in*turn*modulates*the*concentration*of* cytosolic*Ca2+*in*the*cell*(GarciaUMata*and*Lamattina,*2003;*Neill*et#al.,*2003,*2008;*Park*et#al.,* 2000,*2003).*ROS,*such*as*the*hydrogen*peroxide*(H2O2),*reduce*the*activity*of*ABI1*and*ABI2*in# vitro*(Meinhard*and*Grill,*2001;*Meinhard*et#al.,*2002).* Moreover,*OST1*phosphorylates*the*NUterminal*domain*of*AtCLCa,*enhancing*its*activity* in*releasing*Cl−*and*NO3−*from*the*vacuole*(Fig.*1.23;*Wege*et#al.,*2014).*Taken*into*account*that* ABAUinduced* stomatal* closure* of* clcc*knockUout* mutants* was* impaired* (Jossier* et# al.,* 2010),* CLCc*could*also*participate*in*the*stomata*closure*pathway,*transporting*Cl−*out*of*the*vacuole.* As* mentioned* above,* the* hyperpolarizationUactivated* channel* ALMT6* acts* as* a* MA2−* influx*or*efflux*channel,*depending*on*the*vacuolar*pH*and*cytosolic*MA2−*(Meyer*et#al.,*2011).* The*cytosolic*Ca2+*rise*could*activate*this*channel*and*thus*MA2−*would*be*transported*into*the* cytosol*(Fig.*1.23).* The* SUtype* anion* channels* SLAC1* and* SLAH3* inhibit* the* inward* rectifying!K+*channel* KAT1,* which* is* involved* in* stomata* opening.* This* inhibition* takes* place* by*proteinUprotein* interaction,* preventing* stomatal* opening* (Fig.* 1.23;* Wang* et# al.,* 2016).*Additionally,* OST1* inhibits*KAT1*activity*by*phosphorylation*(Sato*et#al.,*2009).*As*a*consequence*the*passive*influx* of*K+*from*the*apoplast*to*the*cytoplasm*is*interrupted.** Following!the*activation*of*anion*channels*(SLAC1,*SLAH3*and*ALMT12)*in*the*plasma* membrane,*Cl–,*NO3–*and*MA2–*are*released*from*guard*cells,*giving*rise*to*depolarization*of*the* plasma* membrane.* This*in* turn* activates* guard* cell* outward* rectifying* K+*channels* (GORK),* leading*to*the*release*of*K+*from*the*cytoplasm*to*the*apoplast*(Fig.*1.23;*Ache*et#al.,*2000;*Hosy* et#al.,*2003;*Kim*et#al.,*2010).** The*cytosolic*Ca2+*activates*the*vacuolar*K+*(VK)*channels*TPK1*(twoUpore*K+*channel*1),* which*drives*the*release*of*K+*from*the*vacuole*into*the*cytoplasm*(Fig.*1.23;*Gobert*et#al.*2007;* Ward*and*Schroeder,*1994).*This*cation*is*subsequently*transported*out*of*the*cell*by*the*GORK* channel.** The* release* of* solutes* from* guard* cells* decreases* the* osmotic* potential,* causing* a* massive* water* movement* out* the* cells.* The* loss* of* guard* cell* turgor* finally* leads* to* stomata* closure,*reducing*transpirational*loss*of*water*from*the*leaf.* * Introduction* * * 39* 1.5.2.2.2.2.*Slow!ABA!signaling!pathway! As* mentioned*above,* the* inhibition* of* the* phosphatase* ABI1* enables* the* activation* of* distinct* SnRK*protein* kinases.* In* Arabidopsis*several* kinases* from* the* SrRK2* subfamily* (SnRK2.2,*SnRK2.3*and*OST1)*phosphorylate*the*bZIP*transcription*factors*of*ABF/AREB*group* in*the*nucleus*(Furihata*et#al.,*2006*Johnson*et#al.,*2002;*LopezUMolina*et#al.,*2001;*Uno*et#al.,* 2000).* The* bZIP* transcription* factors* in* turn* interact* with* the* GUbox* ABAUresponse* elements* (ABRE)*in*the*promoter*of*ABAUresponse*genes,*inducing*their*expression*(Zhu,*2002).** UpUregulated* genes* encode* proteins* involved* in* abiotic* stress* tolerance,* like* transporters,* PYR/PYL/RCAR* receptors,* transcription* factors,* protein* kinases,* protein* phosphatases,* hydrophilins,* molecular* chaperones,* antioxidant* enzymes,* enzymes* of* phospholipid*signaling,*etc.*(Cutler*et#al.,*2010).* * ! ! 40! ! OBJECTIVES Materials)and)Methods) ) ) 48) Table!3.3.!SOB!medium!composition.! Constituent Concentration Tryptone 20+g+l−1+ Yeast+Extract 5+g+l−1+ NaCl 0.58+g+l−1+ KCl 0.18+g+l−1+ MgSO4+2+M ) ) Escherichia!coli!strains)were)grown)at)37ºC.) ) 3.1.1.2.!Agrobacterium)tumefaciens)) GV3101) Agrobacterium!tumefaciens)was) the)only) strain) used) for) obtaining) chemically) competent) cells)(section) 3.5.2.1)) in) order) to) perform) its) transformation) (section) 3.5.2.2)) with) plasmid)DNA.) )The) GV3101) A.! tumefaciens)strain) contains) the) plasmid) pTiC58) with) resistance) to) rifampicin.)Thus,) A.!tumefaciens!was) cultivated) in) LB) medium) (Table) 3.2)) supplemented) with) rifampicin)(50)μg)mlC1).)Solid)LB)medium)was)prepared)as)described)above)(section)3.1.1.1).)) When) the) strain) was) transformed,) the) LB)medium) was) supplemented) with) another) antibiotic)(that)one)to)which)the)plasmid)DNA)provided)resistance).) To) prepare) A.!tumefaciens! chemically) competent) cells) (section) 3.5.2.1)) Yeast) extract) peptone)(YEP))medium)(Table)3.4))was)used)for)growing.) !Table!3.4.!YEP!medium!composition.! Constituent Concentration,(g,l−1 ) Pectone 10 Yeast,extract 10 NaCl 5 ) ) Agrobacterium!tumefaciens!was)grown)at)28ºC.) ) 3.1.2.!Plant!material!and!growth!conditions! 3.1.2.1.!Knockout!(KO)!mutant!lines! Arabidopsis! thaliana)seeds) from) the) slah181)(SALK_039811),) slah381)(GK371G03)) and) slah482) (SALK_091937)) lines) were) obtained) from)the) Nottingham) Arabidopsis) Stock) Centre) (NASC;)Nottingham)University,)UK))and)seeds)from)the)slah182)(INRA_FLAG_329G06))line)from) the) Institut) JeanCPierre) Bourgin) (IJPB;) Versailles,) France).) The) mutant) line) slah384) (SALK_111623))was)provided)by)Dr.)Sheng)Luan)(Zheng)et!al.,)2015).)) The)slah182)line)was)generated)in)the)Wassilewskija)(Ws))background,)whereas)the)other) lines)were)generated)in)the)Columbia)(Col))background.)) ) ) Materials)and)Methods) ) ) 49) 3.1.2.2.!Growth!conditions! )After) 3Cdays)vernalization) at) 4ºC) in) the) dark,) plants) were) grown) according) to) the) experimental)requirements.)Thus)germination)was)synchronized.)) All)irrigation)and)experimental)solutions)were)adjusted)to)pH)5.7.) ) 3.1.2.2.1.!In)vitro!growth!conditions! Seeds)were)surface)sterilized)according)to)the)liquid)sterilization)protocol)from)Clough) and) Bent) (1998)) and) after) vernalization) they) were) placed) on) Petri) dishes) containing)0.5x) Murashige)and)Skoog)(MS))medium)(Murashige)and)Skoog,)1962))supplemented)with)vitamins,) MES)buffer) (Duchefa) Biochemie)) and) 0.5%) sucrose) (Duchefa) Biochemie)) and) solidified) with) 0.8%)plant)agar)(Duchefa)Biochemie).) Plants)were)grown)in)an)in8vitro)growth)chamber)at)24ºC,)with)a)photoperiod)of)16)h) light,)30)μE)m−2)s−1!of)photosyntheticallyCactive)radiation)(PAR))and)without)relative)humidity) (RH))regulation.) For!GUS) (βCGlucuronidase)) and) GFP) (Green) Fluorescent) Protein)) reporter) gene) expression)analyses,)PSLAH1CGFP::GUS,)PSLAH4CGFP::GUS)(section)3.4.2))and)PSLAH3CGUS)(Negi)et!al.,) 2008))transgenic)plants)were)grown)in)Petri)dishes)containing)the)MS)medium)described)above) supplemented) with) 50) µg) mlC1)kanamycin.) Seedlings) were) directly) analysed)after) growing) in) Petri) dishes) or) resistant) seedlings) were) grown) for) longer) time) after) transferring) them) into) hydroponics) medium) (section) 3.1.2.2.3).) In) addition,) resistant) seedlings) were) transplanted) to) soil)to)produce)seeds.!! For) obtaining) independent) slah1/SLAH1)complemented) lines,) seeds) obtained) after) Arabidopsis)transformation)(section)3.6))were)also)sown)on)MS)supplemented)with)20)µg)mlC1) hygromycin.)Resistant)seedlings)were)transplanted)to)soil)to)obtain)seeds.) ) 3.1.2.2.2.!Plant!growth!on!soil! )Peat) was) sieved) and) autoclaved) before) pots) were) filled.) Prior) to) sowing,) peat) was) washed)with)distilled)water)by)irrigation)from)above)to)remove)undesirable)compounds,)and) afterwards)with)the)corresponding)solution.) Plants)were)watered)by)immersion)of)the)base)of)the)pots)every)3)d)approximately)and) were) grown) either) under) long) photoperiod) conditions) in) a) growth) chamber) at) 23ºC/18ºC,) 16h/8h) day/night) photoperiod,) 120) μE) m−2) s−1)of) PAR)and) 60±10%) RH)or) under) short) photoperiod) conditions) in) a) phytotron) chamber) (Ing.) Climas,) Barcelona,) Spain)) at) 23ºC/18ºC,) 8h/16h)day/night)photoperiod,)120)μE)m−2)s−1)of)PAR)and)60±10%)RH.! For)xylem)sap)collection)assays,)peat)was)washed)with)Basal)Solution)(BS),)in)which)Cl−) salts) were) excluded.)BS) was) based) in) 0.25x) Arteca) and) Arteca) (2000)) nutritive) solution,) but) removing)all)Cl−)ions.)Macroelements)composition)of)BS)was)2)mM)KNO3,)0.625)mM)KH2PO4,)0.5) mM)MgSO4)and)0.75)mM)Ca(NO3)2.)Modified)microelements)formulation)was)used,)since)MnSO4) replaced)the)Cl−)salt)in)MnCl2)and)NaCl)was)removed)(Somerville)and)Ogren,)1982).)Therefore,) BS) had) 3.5) mM) NO3−)as) the) sole) nitrogen) source) and) 70) μM) Cl−)(coming) from) contaminant) traces),)enough)to)fulfil)its)requirement)as)a)micronutrient)(FrancoCNavarro)et!al.,)2016a).) Materials)and)Methods) ) ) 50) Potted)plants)of)slah182,)slah381)and)slah384)TCDNA)insertion)mutant)lines)(section)3.7.1),) the) slah18slah3)doubleCKO) mutant) line) (obtained) by) sexual) crosses;) section) 3.7.2),) and) their) corresponding) azygous) segregants) or) wildCtype) (WT)!were) grown) under) long) photoperiod) conditions)and)used)for)xylem)sap)collection)assays.) For!morphological)measurements)assays)(section)3.8.1))to)compare)the)development)of) slah182! and!slah482!with)their) corresponding) azygous) segregating) or) WT) lines,) plants)were) grown)in)pots)under)short)photoperiod)conditions,)but)also)hydroponically)(section)3.1.2.2.3).! Pots)were)watered)by)immersion)of)the)base)of)the)pots)in)a)solution!containing)either)low)Cl−) (approximately) 70) μM) residual) Cl−)) or) 5) mM) Cl−.) Both) solutions) consisted) of) a) modified) BS) medium,)containing)double)of)MgSO4)and)Ca(NO3)2)salts,)supplemented)with)the)following)salt) mixtures)ensuring)the)same)cationic)balance:)2.5)mM)KCl,)0.625)mM)MgCl2,)and)0.625)mM)CaCl2) for)the)5)mM)Cl−)treatment;)and)1.25)mM)KH2PO4,)0.625)mM)K2SO4,)0.625)mM)MgSO4,)and)0.625) mM)CaSO4)for)the)lowCCl−)treatment.)The)microelements)composition)was)the)same)as)the)BS.))) For)slah482!phenotypic)assay)with)different)Cl−)and)NO3−)concentrations,)slah482!and)its) WT)line)(ColC0))plants)were)grown)17)d)with)BS.)By)irrigation)from)above,)peat)was)afterwards) washed)with)the)respective)treatment)solution,)which)consisted)of)a)shared)basal)solution)made) with)0.625)mM)KH2PO4)and)0.5)mM)MgSO4)as)macroelements,)together)with)the)microelements) composition) as) the) BS.)This) solution) was) supplemented) with) different) Cl−)and) NO3−) concentrations)(Table)3.5))to)obtain)5)treatment)solutions)and)another)treatment)solution)also) contained)ammonium)as)N)source.)Nitrate)concentration)was)provided)with)KNO3,)Mg(NO3)2)and) Ca(NO3)2)in) relation) (4:1:1),) and) Cl−)concentration)was) provided) with) the) same) cation) composition,)i.e.)KCl,)MgCl2)and)CaCl2)(4:1:1).)These)plants)were)grown)under)long)photoperiod) condition.) Table!3.5.!Nitrate!(NO3−)!and!chloride!(Cl−)!concentrations!of!the! treatment! solutions! for!the! slah472) phenotypic! assay.! A) treatment)also)contained)ammonium)(NH4+)!! Treatment(solution NO3 1((mM) Cl1((mM) NH4 +((mM) 15.00 1%*1% 22.00 3.00 % 30.25 4.75 % 45.00 5.00 % 50.75 5.00 % 60.25 4.75 1.75 ) )*)70)μM)Cl−)coming)from)contaminant)traces.! ) ) For)Arabidopsis)transformation)(section)3.6))A.!thaliana)ColC0)plants)were)grown)under) short)day)conditions)with)an)optimal)solution.) ) 3.1.2.2.3.!Plant!growth!in!hydroponic!culture! For) adequate) normalization) of) ion) translocation) rates) in) xylem) sap) collection) assays,) Arabidopsis)ColC0) plants) were) grown) hydroponically) in) the) BS) described) above) under) long) photoperiod) conditions.) Plants) were) germinated) in) 1.5) ml) bottomless) Eppendorf) tubes) filled) with)rockwool)previously)imbibed)in)distilled)water.) Materials)and)Methods) ) ) 51) ) For!morphological)measurements)assays)(section)3.8.1))to)compare)the)development)of) slah182!and!slah482)with)their)corresponding)WT)lines,)plants)were)also)grown)in)hydroponics) conditions)under) short) and) long) photoperiod) conditions,) respectively.) The) solution) described) above!containing)either)low)Cl−)(70)μM)residual)Cl−))or)5)mM)Cl−)(section)3.1.2.2.2))was)replaced) every)3)d.) For) reverse) transcriptase) PCR) (RTCPCR))studies) (section) 3.2.4.2),) slah181,) slah182)and) slah482) TCDNA) insertion) mutant) lines) (section) 3.7.1),) the) slah1/SLAH1! complemented) lines) (section)3.4.2))and)their)corresponding)azygous)segregating)or)WT)lines)were)grown)in)liquid) medium) containing) 0.25x) MS)solution) supplemented) with) vitamins) and) MES) buffer) (Duchefa) Biochemie))under)short)photoperiod)conditions.)) For!GUS) and) GFP) reporter) gene) expression) analyses,) PSLAH1CGFP::GUS,) PSLAH4CGFP::GUS) (section) 3.4.2)) and) PSLAH3CGUS) (Negi) et! al.,) 2008)) transgenic) seedlings) were) transferring) into) hydroponics) medium) containing) 0.25x) MS) supplemented) with) vitamins) and) MES) buffer) after) growing)in)Petri)dishes)to)obtain)older)plants)for)further)assays.) For!quantitative) real) time) PCR)(qPCR)) analyses) (section) 3.2.4.3),) Arabidopsis!thaliana) ColC0) plants) were) grown) under) short) photoperiod) conditions) in) hydroponic) medium) with) different)solutions:) 1.)Plants)grown)for)6)weeks)in)the)0.25x)MS)solution)described)before)were)used)for)the) salt) treatment,) the) water) deficit) treatment) and) for) the) application) of) ABA.) For) the) saltCstress) treatment,) plants) were) transferred) to) hydroponics) jars) containing) 150) mM) NaCl) in) 0.25x) MS,) meanwhile)for)the)waterCdeficit)treatment,)plants)were)transferred)to)0.25x)MS)with)287)g)l−1) PEGC8000)(equivalent)to)−1)MPa)of)osmotic)potential).)And)plants)were)transferred)to)0.25x)MS) supplemented)with)100)μM)ABA)for)the)exogenous)ABA)application.)Plants)used)as)control)were) transferred)to)0.25x)MS)medium.) 2.)Plants)grown)for)2)weeks)in)0.25x)MS)and)6)weeks)more)in)BS)solution)supplemented) with)either)5)mM)Cl−!or)70)µM)Cl−!(using)KCl:MgCl2:CaCl2)in)a)ratio)2:1:1))were)subjected)either) to) Cl−)supply) and) starvation) assays) or)to)indoleC3Cacetic) acid) (IAA)) treatment.) For) chloride) supply)treatment,)plants)grown)in)optimal)solution)with)70)µM)Cl−)were)transferred)to)5)mM)Cl−) solution,) while) for) chloride) starvation) treatment,) plants) grown) in) the) solution) with) 5) mM) Cl−) were)transferred)to)the)70)µM)Cl−)solution.)And)for)the)exogenous)auxin)application,)plants)were) transferred) to) hydroponics) jars) containing) 0.25x) MS,) with) either) 5) mM) Cl−!or) 70) µM) Cl−,) supplemented)with)100)μM)IAA.)Plants)used)as)control)were)transferred)to)the)same)nutrient) solution)in)which)they)were)growing.) 3.)Plants)grown)for)5)weeks)in)0.25x)MS)and)3)weeks)more)in)a)solution)based)in)the)BS,) sharing) the) microelements) composition) with) the) BS) but) removing) all) NO3−)ions) from) the) macroelements.) Thus) the) macroelements) composition) was) 0.625) mM) KH2PO4,) 0.5) mM) MgSO4) and) 0.5) mM) CaSO4,) and) was) supplemented) with) either) 5) mM) NO3−) ions) (provided) with) KNO3:Mg(NO3)2:Ca(NO3)2)in)a)ratio)4:1:1))or)2.5)mM)ammonium)succinate.)Plants)grown)in)these) two)solutions)with)different)nitrogen)source)were)subjected)to)either)NO3−)supply)or)starvation) assays.) For) nitrate) starvation) treatment,) plants)grown) in) the) solution) containing) NO3−)as) N) source) were) transferred) to) the) nitrateCfree) solution,) and) for) nitrate) supply) treatment) plants) grown) in) nitrateCfree) solution) were) transferred) to) the) solution) containing) NO3−)as) N) source.) Materials)and)Methods) ) ) 52) Plants) used) as) control) were) transferred) to) the) same) nutrient) solution) in) which) they) were) growing) Three)and) 24)h) after)treatments)application,) roots) and) rosette) leaves) were)harvested,) quickly)washed)with)distilled)water)and)immediately)frozen)with)liquid)nitrogen)and)stored)at) −80°C.)) ) 3.2.!DNA!ANALYSES! 3.2.1.!DNA!extraction!!!! )In)order)to)genotype)Arabidopsis)plants!(section)3.7.1),)DNA)extraction)from)leaves)was) performed)with)the)REDExtractCNCAmpTM)Plant)PCR)kit)(SigmaCAldrich)Co.,)MO,)USA).)For)each) plant,)a)disk)of)leaf)tissue)of)around)3)mm)was)incubated)in)10)µl)of)the)extraction)solution)at) 95°C)for)10)min.)To)neutralize)inhibitory)substances,)an)equal)volume)of)dilution)solution)was) added.)For)genotyping)by)polymerase)chain)reaction)(PCR;)section)3.7.1),)1)µl)of)the)extracts)was) used.) In) addition,) to) obtain) template) DNA) for) performing) PCR) (section) 3.2.4.1),)DNA) was) extracted)from)Arabidopsis)fresh)or)frozen)leaves)through)the)iCgenomic)Plant)DNA)Extraction) Mini) Kit)(Intron)Biotechnology) Inc.,) KyeonggiCdo,) Korea)) following) the) manufacturer’s) instructions.)) Through)the)miniCpreparations)protocols,)the)plasmid)DNA)was)isolated)from)bacteria.)) The)miniprep)was)performed)the)following)day)of)growing)single)colonies)in)2−3)ml)of) LB) liquid) medium) (section) 3.1.1)) containing) the) appropriate) antibiotic.) The)saturated) culture) was)centrifuged)at)12,100)×)g)for)2)min.)The)supernatant)was)discarded)and)the)bacteria)were) resuspended) with) 125)μl)of) cold) Solution) I)(Table)3.6).) The)samples) were) shaked)with) a) Bio) Vortex)V1)(Biosan,)Riga,)Latvia).)Subsequently)225)μl)of)freshly)prepared)Solution)II)(Table)3.6)) was)added)and)the)samples)were)gently)mixed)and)placed)on)ice)for)3)min.)Afterwards)118)μl)of) cold)Solution)III)(Table)3.6))was)added.)) Table!3.6.!Composition!of!solutions!I,!II!and!III.! !!!!!!!!!!!!!!!!!!!!!!!!!!!Solution!I !!!!!!!!!!!!!!!!!!!!!!!!!!!Solution!II !!!!!!!!!!!!!!!!!!!!!!!!!Solution!III Constituent Concentration!(M) Constituent Concentration Constituent Concentration!(M) Glucose 0.050 Sodium/hidroxide 0.2/M Potassium/acetate 3 Tris:Cl/1M/(pH/8.0) 0.025 SDS 1:%Glacial/acetic/acid 5 EDTA/0.5/M/(pH/8.0) 0.010 ) ) The) samples) were) gently) mixed) by) inversion) and) placed) on) ice) for) at) least) 10)min)to) allow) most) of) the) protein,) high) molecular) weight) RNA) and) chromosomal) DNA) to) precipitate.) Then)20)μl)of)chloroformCisoamyl)alcohol) (24:1))was)added)to)the)samples)and)after)a)gently) shaked,) they) were) centrifuged) 5) min) at) 12,100)×)g.) The) supernatant) was) transferred) into) a) second)microcentrifuge) tube) and) 560)μl)of) phenolCchloroformCisoamyl) alcohol)(24:24:1)) was) added.) The) samples) were) shaked) and) centrifuged) 5) min)at) 12,100)×)g.) The) supernatant) was) transferred)into)a)new)microcentrifuge)tube)and)after)adding)500)μl)of)cold)chloroformCisoamyl) alcohol)the)samples)were)shaked)and)centrifuged)3)min)at)12,100)×)g.)The)clear)supernatant)was) transferred)into)a)new)microcentrifuge)tube)and)two)volume)of)iceCcold)100%)ethanol)was)used) to) precipitate) the) plasmid) DNA.) The) samples) were) maintained) at) least) 10) min) at) −20ºC.) Materials)and)Methods) ) ) 53) Afterwards)samples)were)centrifuged)15)min,)the)supernatant)was)discarded)and)1)ml)of)cold) 70%) ethanol) was) used) for) washing) them.) After) a) centrifugation) of) 5) min)at) 12,100) ×) g,) the) ethanol) was) discarded) and) pellets)were) dried) at) 40ºC.)Finally,) pellets) were) resuspended) in) 20−30) μl)water) containing) RNase) A) (20) μg) ml−1).) An) aliquot) of) the) plasmid) DNA) was) subsequently) digested) with) restriction) enzymes) (section) 3.2.7)) and) analysed) in) agarose) gels) (section)3.2.5).) The)Qiaprep) spin) Miniprep) kit) (Qiagen,) Germany))and) the) Plasmid) PLUS) DNA) Purification)Mini)Spin)Column)kit)(Genaxxon)Bioscience,)Ulm,)Germany))were)used)for)plasmid) DNA)extraction)from)E.!coli)previously)transformed)(section!3.5.1.2))with)USER)cloning)(section) 3.4.1).) The)day) before)single) bacterial) colonies) were) picked) into) a) vial) of) 5) ml)of)LB)medium) (section)3.1.1.1))supplemented)with)ampicillin)(50)μg)mlC1))and)shaked)overnight)at)37ºC.)After) centrifuging)at) 6,800) ×) g)for) 1−3) min)at) room) temperature,) the) obtained) pellet) was) used) according)to)the)manufacturer’s)protocols.)Finally)the)concentration)of)the)50)μl)extracted)DNA) samples)was)quantified)(section)3.2.3))and)its)size)was)verified)by)electrophoresis)in)agarose)gel) (section)3.2.5).)After)obtaining)the)right)sequence)(section)3.2.8),)the)DNA)samples)were)used) for)the)IVTCPCR)(section)3.2.4.5)) Together)with)miniCpreparations,)maxiCpreparations)are)a)way)for)longCterm)storage)of) plasmid)vectors)and)the)constructs)contained)therein)at)−20ºC.) For) maxiCpreparations,) a)colony) of) E.! coli)containing) the) plasmid) of) interest,) was) inoculated)in)100)ml)of)LB)medium)(section!3.1.1.1))and)grown)overnight)at)37°C)with)shaking.) Culture) was) centrifuged) 5) min)at) 4,300) ×) g.) Supernatant) was) discarded) and) cell) pellet) was) washed)with)20)ml)distilled)milliCQ)water,) gently) shaked) and)centrifuged)3) min)at)4,300) ×) g.) Water)was)discarded)and)the)pellet)was)well)resuspended)with)5)ml)of)Solution)I)(Table)3.6))by) shaking)in)the)Bio)Vortex)V1)(Biosan).)The)sample)was)subsequently)maintained)10)min)on)ice.) Afterwards,)10) ml) of)freshly)prepared)Solution)II)(Table)3.6))was)added)and)mixed)by)gentle) inversion)and)then)maintained)10)min)on)ice.)Following)the)addition)of)7.5)ml)of)cold)Solution)III) (Table)3.6),)the)sample)was)mixed)by)gentle)inversion)and)incubated)30−60)min)on)ice)to)allow) the)precipitation)of)proteins,)high)molecular)weight)RNA)and)chromosomal)DNA.)Then)800)μl)of) chloroformCisoamyl)alcohol)(24:1))was)added)to)the)sample)and)after)a)quickly)shaked,)it)was) centrifuged)15)min)at)14,600)×)g)for)at)4°C.)The)supernatant)was)transferred)into)a)new)tube) and)one) volume) of) isopropanol) was) added.) The) sample) was)incubated) 10) min) at) −20ºC) to) precipitate) the)nucleic) acids.) A) centrifugation) of) 10) min)at) 6,700)×) g)was) performed) and) afterwards)the)supernatant)was)discarded.)The)pellet)was)resuspended)with)2.5)ml)TrisCEDTA) (TE;!Table)3.7))buffer)by)shaking)in)the)vortex,)followed)by)the)addition)of)5)ml)of)4)M)LiCl.)) !Table!3.7.!TE!buffer!composition.!! Constituent Concentration,(M) Tris%Cl(1M((pH(8.0) 0.010 EDTA(0.5(M((pH(8.0) 0.001 ) ) The)sample)was)incubated)15)min)on)ice.)The)RNA)was)precipitated)by)centrifuging)10) min)at)6,700)×)g.)The)supernatant)was)transferred) into) a)new)microcentrifuge)tube) and)after) adding)1ml)isopropanol)and)centrifuging)10)min)at)6,700)×)g)the)DNA)was)precipitated.)The)DNA) was)resuspended)with)1.6)ml)TE)buffer,)containing)RNase)A)(20)μg)ml−1),)by)shaking)in)a)vortex) and) the) sample) was) incubated) 15) min)at) 37ºC.)Then) 500) μl) of) phenolCchloroformCisoamyl) Materials)and)Methods) ) ) 54) alcohol)(24:24:1))was)added.)The)sample)was)mixed)and)centrifuged)5)min)at)6,700)×) g.) The) supernatant) was) mixed) with) 500) μl)and) centrifuged) 3) min.) Afterwards,) the) DNA) of) the) supernatant)was)precipitated)with)0.1)volume)of)3)M)Sodium)acetate)together)with)a)volume)of) isopropanol.)The)sample)was)gently)mixed)and)following)the)incubation)for)20)min)on)ice,)it)was) centrifuged)10)min)at)6,700)×)g.)The)pellet)was)washed)with)cold)70%)ethanol,)which)was)then) discarded) and) the) pellet)was) dried) at) 42ºC.) Finally) the)pellet,) containing) the) DNA,) was) resuspended)with)15)μl)milliCQ)water.)) A)microliter)was)used)for)quantifying)DNA)concentration)(section)3.2.3)) ! 3.2.2.!Complementary!DNA!(cDNA)!synthesis!from!RNA!) One) microgram) of) total) RNA) obtained) from) plant) tissues) (section) 3.3.1)) was) used) as) template) for) the) first) strand)cDNA) synthesis) in) a) 10Cμl) reaction) using) the) iScriptTM)cDNA) synthesis)kit)(BioCRad)Laboratories,)Hercules,)CA,)USA).)) Each)reaction)mix)(Table)3.8))had)a)final)volume)of)10)μl)and)was)incubated)5)min)at) 25ºC)followed)by)30)min)at)42ºC)and)5)min)at)85ºC.) !Table!3.8.!cDNA!synthesis!reaction.!! Components Volume,(μl) 5x#iScript#reaction#mix 2 iScript#reverse#transcriptase 0.5 RNA#template 1 Nuclease:free#water 6.5 ) ) cDNA)samples)were)used)either)for)determining)the)integrity)of)the)AtSLAH)mRNA)in)the) TCDNA)insertion)mutant)lines)through)RTCPCR)(section)3.2.4.2))and)to)perform)qPCR)analyses) (section)3.2.4.3))with)the)samples)obtained)from)Arabidopsis)ColC0)(WT))line.) ) 3.2.3.!DNA!quantification!) The) determination) of) the) DNA) concentration) of) the) samples) was) performed) with) 1) μl) using) either) the) NanoDrop®)NDC1000) or) the) NanoDrop®)2000c) spectrophotometer)(Thermo) Fisher)Scientific).) ! 3.2.4.!DNA!Amplification! 3.2.4.1.!Conventional!and!high!fidelity!Taq!polymerase!reactions! For)genotyping)by)PCR,)1)µl)of)DNA)extracted)from)leaves)(section)3.2.1))of)Arabidopsis! TCDNA)insertion)mutant)lines)(section)3.1.2.1))and)double)mutants)(section)3.7.2))was)used.)) Materials)and)Methods) ) ) 55) ) Figure!3.1.!TYDNA!insertion!mutants!in!AtSLAH1.! ! Homozygous) and) azygous) segregating) Arabidopsis! plants) were) screened) by) using) primers)suitable)for)the)left)border)of)the)TCDNA)for)the)corresponding)gene)and)geneCspecific) primers) flanking) the) position) of) the) TCDNA) inserts) (Fig.)3.1,) 3.2) and) 3.3).!The) design) of) the) primers)was)conducted)using)Primer3)(Rozen)and)Skaletsky,)2000))and)OligoCalc)software.) ) ) Figure!3.2.!TYDNA!insertion!mutants!in!AtSLAH3.! ! The)PCRs)(Table)3.9))were)performed)with)the)MaximeTM!Taq)DNA)polymerase)(Intron) Biotechnology)Inc.))in)a)BIOER)XP)Cycler)(Bioer)Technology)Co.,)Hangzhou,)China).) ) Materials)and)Methods) ) ) 56) ) Figure!3.3.!TYDNA!insertion!mutant!in!AtSLAH4.! ! For)genotyping)the)TCDNA)insertion)mutants)in)SLAH1)and)SLAH4)genes,)58ºC)and)20)s) in) the) annealing) step) were) used,) meanwhile) 60ºC) and) 25) s) were) used) for) TCDNA) insertion) mutants)in)SLAH3.) !Table!3.9.!Cycling!parameters!for!genotyping.!! PCR$cycle Temperature$(ºC) Time$(s) Initial'denaturation''''''''' 94 120 Denaturation 94 20 35'cycles Annealing 58/62 20/25 Extension 72 60 Final'extension''''''''''''''' 72 300 ) ) Each)reaction)mix)contained)1)μl)of)DNA,)0.5)μM)of)both)forward)and)reverse)primers) (Table)3.10),)0.2)mM)dNTPs,)2)μl)of)10x)PCR)buffer)and)0.4)μl)of)MaximeTM)Taq)DNA)polymerase) (5)U)μl−1))in)a)volume)of)20)μl.) !Table!3.10.!Primers!used!for!genotyping.!! Name Sequence*(5'*!*3') S1#UTR#5' ACCATAGAGCCAAACAAGCCAAT S1#E1#3' CACGAGGTTTGCGATTACCGA S1P#F0 CACCGGGCAAAAGGTTTCAATGG S1P#R0 GGTTTATCGATGAAAAAGAGGTATTG Slah30NT ACCCCATTTCCACCTTCGGTATG Slah30CT GGATAATGGTGGTCACGAGCAG S4#UTR#5' ATTGAGAAGGGCTCATGGTCAAC S4#E2#3' TCCCGCATGGAGACTACTCAAAA LBb1 GCGTGGACCGCTTGCTGCAACT LB4#2 CCGTCGGCTATTGGTAATAGGAC LB#GK2 CCCATTTGGACGTGAATGTAGACAC ) ) PCR) products) were) run) in) 1%) agarose) gel) by) electrophoresis) (section)3.2.5)) to) verify) DNA)fragment)sizes.) ) Materials)and)Methods) ) ) 57) High) fidelity) Taq) polymerases) were) used) for) genes) amplification) for) the) purpose) of) Gateway®)technology)cloning)in)the)BIOER)XP)Cycler)(Bioer)Technology)Co.).)Genomic)DNA)from) Arabidopsis)ecotype)Columbia)was)used)as)template)for)amplifications)(section)3.2.1).) The) primer) design) (Table)3.11)) was)performed)according)to)the)selected) entry) vector) and)the)manufacturer’s)instructions.)The)pENTR/DCTOPO®)vector)(Invitrogen),)which)contains) the) attL) sites,) was) used) as) entry) vector) for) SLAH1!promoter) cloning.) Hence)four) bases) were) added) to) the) forward) primer) sequence) designed) for)generating)a) 3’) bluntCend) DNA) complementary) to) a) region) in) the) vector) generated) by) restriction) enzymes,) achieving) directionally) cloning.)Nevertheless,) pDONRTM201) was) the) entry) vector) chosen) for) SLAH4! promoter) and) AtSLAH1)gene) cloning.) Therefore)25) bp)attB) sites) were) incorporated) in) both) primers.!! !Table!3.11.!Primers!used!in!Gateway®!cloning.!! Name Sequence*(5'*!*3') S1P$F& CACCGGGCAAAAGGTTTCAATGG S1P$R& GGTTTATCGATGAAAAAGAGGTATTG S4PattB1$F GGGGACAAGTTTGTACAAAAAAGCAGGCTCTTGTTTTATCACCTTTTCCTACGTG S4PattB2$R GGGGACCACTTTGTACAAGAAAGCTGGGTGCTTATTTTGCTCTATGTTGTGCT S1gen$F GGGGACAAGTTTGTACAAAAAAGCAGGCTGGGCAAAAGGTTTCAATGGTTAAG S1gen$R GGGGACCACTTTGTACAAGAAAGCTGGGTCTAGTTTTGGTTAGTCGCATTGAG ) )) For) constructing) the) SLAH1! promoterCGFG::GUS) reporter) fusion,) a) genomic) fragment) including)1.25)kb)upstream)of)the)AtSLAH1)gene)start)codon,)containing)the)promoter)and)the)5’C UTR)region)of)the)AtSLAH1)gene)was)amplified)by)PCR)(Table)3.12))using)the)suitable)primers) (S1PCF)and)S1PCR;)Table)3.11))and)the)iCPfu)DNA)polymerase)(Intron)Biotechnology)Inc.).)) Each)reaction)mix)contained)1)μl)of)template)DNA,)0.5)μM)of)both)forward)and)reverse) primers,)0.2)mM)dNTPs,)3)μl)of)10x)DNA)polymerase)buffer)and)0.5)μl)of)iCPfu)DNA)polymerase) (2.5)U)μl−1))in)a)volume)of)30)μl.) Table! 3.12.! Cycling! conditions! for! SLAH1) promoter! amplification!by!iYPfu!DNA!polymerase.!! PCR$cycle Temperature$(ºC) Time$(s) Initial'denaturation''''''''' 94 120 Denaturation 94 20 35'cycles Annealing 58 15 Extension 70 180 Final'extension''''''''''''''' 72 300 ) ) For)constructing)the)SLAH4!promoterCGFG::GUS)reporter)fusion,)a)2.91)kb)DNA)fragment) (1.313) kb) longer) than) the) construction) used) by) Zheng) and) colleagues) [2015]),) containing) the) promoter)and)the)untranslated)5CUTR')region)of)the)AtSLAH4)gene,)was)amplified)by)PCR)(Table! 3.13))using)the)suitable)primers)(S4PattB1CF)and)S4PattB2CR;)Table)3.11)))and!the)Velocity)DNA) polymerase)(Bioline,)London,)UK).)) Each)reaction)mix)contained)1)μl)of)DNA,)0.5)μM)of)both)forward)and)reverse)primers,) 0.2)mM)dNTPs,)6)μl)of)5x)DNA)polymerase)buffer)and)0.6)μl)of)Velocity)polymerase)(2)U)μl−1))in)a) volume)of)30)μl.) Materials)and)Methods) ) ) 64) from)Xenopus.)From)the)pNBlu)vectors)numbers)18−21)(Table)3.29;)section)3.4.1),)the)sequence) of)the)reporter)gene)YFP)(necessary)for)BiFC;)section)3.11))was)also)amplified.) !Table!3.22.!IVT!primers!sequences.!! Name Sequence*(5'*!*3') IVT$fwd CAA$GGC$GAT$TAA$GTT$GGG$TAA$C IVT$rev GAT$TAC$GCC$AAG$CTA$TTT$AGG$TG ) ) The) DNA) amplification) was) performed) with) the) proofCreading) Phusion) Cx)polymerase) (Thermo) Fisher) Scientific),) as) in) the) Mutagenesis) PCR.) The) reaction) mix) composition) and) the) cycling)parameters)were)as)described)for)the)Mutagenesis)PCR)(section)3.2.4.4).) Afterwards,)the)linearized)DNA)samples)were)checked)in)an)electrophoresis)1%)agarose) gel)(section)3.2.5))and)subsequently)purified)(section)3.2.6))for)being)used)as)template)in)the)in! vitro!transcription)(IVT;)section)3.3.3.).) ) 3.2.5.!DNA!electrophoresis!! For)DNA)electrophoresis,)agarose)gel)contained)0.7−2%)agarose)melted)in)1x)TAE)buffer) (Table)3.23))was)used)for)the)separation)of)differently)sized)nucleic)acid)fragments.)In)order)to) visualize)the)fragments)within)the)gel,)either)Red)Safe)(50)μl)l)−1;)Intron)Biotechnology)Inc.))or) GelGreenTM)(0.05)μl)l)−1;)Biotium,)Hayward,)CA,)USA))was)added.) Different) agarose) concentrations) were) used) for) different) fragment) sizes.) For) DNA) fragments)larger)than)700)bp,)1%)agarose)gel)was)used,)meanwhile)1.5−2%)agarose)was)utilised) for)DNA)strands)with)smaller)length.) The)samples)were)mixed)with)6x)DNA)buffer)(Table)3.23))and)loaded)into)the)pockets)of) the) agarose) gel.) The) 6x) DNA) loading) buffer) contained) glycerol) to) give) the) samples) a) higher) density)compared)to)the)TAE)run)buffer,)leaving)them)in)the)gel)packs)and)not)diffusing)into)the) buffer.) !Table!3.23.!TAE!buffer!and!DNA!loading!buffer!composition.!)) !!!!!!!!!!!!!!!!!!!!!!!!!1x!TAE!Buffer !!!!!!!!!!!!!!!!!!!!!!!!!!6x!Loading!Buffer Constituent Amount Constituent Concentration!(%) Tris%base 0.04%M Bromophenol%blue 0.25 Glacial%acetic%acid 1.14%ml Glycerol 30.0 EDTA%(pH%8.0) 0.01%M ) ) As) a) reference) for) the) length) of) the) samples,) either) a) DNA) Ladder) from) different) companies)(Intron)biotechnology)Inc.;)Favorgen)Biotech)Corp.,)Kaohsiung,)Taiwan))or)a)λCPstI) marker)obtained)by)the)digestion)of)the)DNA)of)the)λ)phage)with)the)restriction)enzyme)PstI)was) used.) The) agarose) gel) was) run)in) a) gel) electrophoresis) chamber) with) 1x) TAE) buffer) at) 70−120V.)) Materials)and)Methods) ) ) 65) The)samples)migrate)through)the)gel)along)the)applied)electric)field.)As)DNA)molecules) are)negatively)charged)due)to)the)phosphate)backbone)of)the)strands,)they)migrate)in)an)electric) field)to)the)positive)pole)(anode))in)the)electrophoresis)chamber.) After) electrophoresis) the) gel) was) photographed) with) either) the)ChemiDocTM)XRS) Gel) documentation) system) (BioCRad)Laboratories)) or) the)transiluminator) Intas) UV) Imager) (Intas,) Göttingen,)Germany).) ) 3.2.6.!DNA!purification) DNA)purification)from)either)agarose)gel)or)PCR)products)was)performed)with)either)the) FavorPrepTM)gel/PCR)purification)kit)(Favorgen)Biotech) corp.))or)the)MEGAquickCspinTM)Total) Fragment) DNA) Purification) Kit) (Intron) Biotechnology) Inc.),) both) according) to) manufacturers’) protocols.)The)DNA)was)finally)eluted)with)40−50)μl)of)the)corresponding)elution)buffer.) ) After)the)IVTCPCR)(section)3.2.4.5),)DNA)products)were)purified)with)the)QIAquick)PCR) purification)kit)(Qiagen))following)the)manufacturer’s)protocol.)The)DNA)was)eluted)with)35)μl) of)elution)buffer.)The)purified)DNA)samples)were)further)sequenced)(section)3.2.8))and)used)for) cloning)(section)3.4.1))or)cRNA)synthesis)by)the)in!vitro!transcription)(IVT;)section)3.3.3).) ! 3.2.7.!DNA!digestion!with!restriction!enzymes!! In) order) to) verify) the) presence) and) the) appropriate) size) of) the) insert) in) the) bacterial) transformed) cells) (sections) 3.51.2) and) 3.5.2.2)) restriction) enzyme)analyses)with) the) suitable) enzymes)(Thermo)Fisher)Scientific))were)done)after)plasmid)DNA)extraction)(section)3.2.1).) Each)reaction)mix)contained)3)μl)of)plasmid)DNA,)1.5)μl)of) the)appropriate)10x)buffer)) (Table)3.24))and)0.4−0.5)μl)of)restriction)enzyme)(10)U)μl−1))in)a)volume)of)15)μl.)The)enzymatic) reaction)including)the)NotI)enzyme)also)contained)1.5)μl)of)10x)BSA)and)1.5)μl)of)10x)Triton.) Restriction)reactions)with)two)enzymes)were)performed)with)a)proper)buffer)for)both)of)them.) Reactions) were) incubated) overnight) at) the) optimal) temperature)for) the) activity) of) the) corresponding) enzyme.)Apart) from) SmaI,) whose) optimal) activity) temperature) is) 30ºC,) all) the) enzymes)were)incubated)at)37ºC.) Table!3.24.!Restriction!enzymes!and!their!suitable!buffer.! Restriction*enzyme Buffer EcoRI EcoRI HindIII R"(red) MluI R"(red) PstI O"(orange) SmaI TangoTM XhoI R"(red) ) ) Following) the) DNA) digestion) with) restriction) enzymes,) DNA) digestion) products) were) separated)by)electrophoresis)in)agarose)gels)(section)3.2.5).) Materials)and)Methods) ) ) 66) In) the) Gateway) cloning)(section) 3.4.2),) DNA) digestion) with) restriction) enzymes) was) performed)after)bacteria)transformation)(sections)3.51.2)and)3.5.2.2))to)verify)the)presence)of) the) insert.) HindIII)and) MluI) were) the) enzymes) used) for) SLAH1! promoterCpENTR/DCTOPO®) restriction)analyses,)EcoRI)and)PstI)for)SLAH4!promoterCpDONRTM201)and)PstI,)together)with)a) double)reaction)of)XhoI)and)PstI,)were)used)for)AtSLAH1)geneCpDONRTM201)restriction)analyses.) In)addition,)SmaI)and)HindIII)were)used)for)SLAH!promoterCpKGWFS7)restriction)analyses)and) HindIII,) PstI,) and) a) double) reacton) PstI/XhoI)were) used) for) AtSLAH1)geneCpK7WG)restriction) analyses.) DpnI)restriction)enzyme)(Thermo)Fisher)Scientific))was)used)after)the)MutagenesisCPCR) (section)3.2.4.4))and)before)the)USER)reaction)(section)3.4.1).)DpnI)eliminates)the)plasmid)DNA) used)in)the)MutagenesisCPCR)as)template)because)it)digests)methylated)DNA.)One)microliter)of) this) enzyme) was) added) directly) in) the) PCR) products,) without) buffer.) After) incubating) the) reactions)1)h)at)37ºC,)the)DpnI)enzyme)was)inactivated)by)maintaining)the)sample)20)min)at) 80ºC.) ! 3.2.8.!DNA!sequencing! )Purified) DNA) fragments)(section) 3.2.6)) that) were) produced)by) high) fidelity) Taq) polymerase)amplification)(section)3.2.4.1))and)the)isolated)plasmid)DNA)(section)3.2.1))obtained) after) transformation) during)the) Gateway®)cloning) method)were) sequenced) by) Secugen) S.L.) (Madrid,)Spain))and)Stab)Vida)(Caparica,)Portugal).) To)verify)the)amplified)and)cloned)genes)obtained)through)the)USERTM)cloning,)isolated) plasmid)DNA)(section)3.2.1))and)purified)DNA)samples)(section)3.2.6))were)sequenced)by)GATCC Biotech)(Germany).)) All)DNA)samples)were)diluted)according)to)the)company)protocol.)Only)after)a)positive) complete)sequencing,)DNA)samples)were)used)for)further)work)steps.) ) 3.2.9.!Amino!acid!sequences!alignments! )Database) searches) looking) for) sequence) similarities) were) performed) using) the) Basic) Local)Alignment)Search)Tool)(BLAST))program)(Altschul)et!al.,)1997))from)the)National)Center) for) Biotechnology) Information) (NCBI;) https://blast.ncbi.nlm.nih.gov/Blast.cgi).) Multiple) sequence)alignments)were)performed)using)the)ClustalW2)program)(Larkin)et!al.,)2007).)) ) 3.3.!RNA!ANALYSES!! 3.3.1.!RNA!extraction!! RNA)was)extracted)from)Arabidopsis)ColC0)WT)plants)in)order)to)perform)qPCR)analyses) (section) 3.2.4.3)) and) also) from)the) TCDNA) insertion)mutant) lines)and) the) two) independent) slah1/SLAH1)complemented)lines)(P1C12)and)P1C31))to)determine)the)integrity)of)the)AtSLAH) mRNA)through)RTCPCR)(section)3.2.4.2).) Plant) RNA) extraction) was) performed) either) by) the) Isolate) II) RNA) Plant) kit) (Bioline),) following)the)manufacturers’)protocol,)or)by)TRIsureTM)reagent)procedure)(Bioline).) Materials)and)Methods) ) ) 67) Through) TRIsureTM)reagent) procedure) total) RNA) was) extracted) from) frozen) tissues.) Following)the)homogenization)of)frozen)samples)with)1)ml)TRIsureTM)per)50−100)mg)of)tissue) by) shaking) in) a)Bio) Vortex) V1) (Biosan),) insoluble) material) was) removed) by) centrifugation) at) 12,000)×)g!for)10)min)at)4ºC)and)the)clear)supernatant)was)transferred)to)a)new)microcentrifuge) tube.)Afterwards,)200)μl)of)chloroform)per)millilitre)of)used)TRIsureTM)was)added)to)the)samples) and)they)were)shaked)vigorously)by)hand)for)15)s.)After)incubating)the)samples)3)min)at)room) temperature,)they)were)centrifuged)at)12,000)×)g!for)15)min)at)4ºC.)The)aqueous)phase,)which) contained)the)RNA,)was)transferred)into)a)new)microcentrifuge)tube.)The)RNA)was)subsequently) precipitated)by)mixing)the)samples)with)600)μl)of)cold)isopropyl)alcohol)per)millilitre)of)used) TRIsureTM.)After) 10)min)of)incubation)at)room)temperature,)the) samples)were) centrifuged)10) min) at) 12,000) ×) g! at) 4ºC)and) the) supernatant) was) discarded) and) the) pellet) was) dried.) Afterwards,) the) pellet) was) washed) with) 1ml) of) 70%) coldCice) ethanol) per) millilitre) of) used) TRIsureTM.)Following)the)shaked)of)the)samples)with)the)Vortex,)they)were)centrifuged)5)min)at) 7,500) ×) g!at) 4ºC.)The) ethanol) was) then) discarded) and) the) pellet) died.) Finally,) the) pellet) was) resuspended)in)20−30)µl)milliCQ)water)treated)with)0.1%)DEPC.) The)RNA)concentration)of)the)samples)was)quantified)(section)3.3.4))and)the)quality)of) them)was)verified)by)electrophoresis)in)1%)agarose)gel)(section)3.3.2).) Previously)to)perform)the)RNA) retrotranscription)(section) 3.2.4.2),) contaminated)DNA) was)eliminated)by)RNAseCfree)DNase)I)(Thermo)Fisher)Scientific))according)to)manufacturers’) protocol)and) RNases) were) also) inhibited) using) RiboLock) RNase) Inhibitor) (Thermo) Fisher) Scientific).)Each)reaction)mix)(Table)3.25))had)a)final)volume)of)10)μl)and)was)incubated)30)min) at)37ºC)followed)by)10)min)at)65ºC.)) !Table!3.25.!DNase!reaction!mix.!! Components Volume,(μl) 10x$Reaction$buffer$with$MgCl21 DEPC;treated$water 6.75 RNA 1 Dnase$I,$Rnase;free$(1U$μl−1)1 Ribolock$Rnase$Inhibitor$(40Uμl−1)0.25 EDTA$0.5$M$(pH$8.0) 1 ) ) 3.3.2.!RNA!electrophoresis!! )Agarose)gel)was)also)used)for)RNA)separation)by)electrophoresis)to)verify)the)purity)and) degree)of)denaturation)of)the)RNA.) For) RNA) electrophoresis,) agarose) gel) contained) 1%) agarose) melted) in) 1x) MAE) buffer) (Table)3.26))and)3.7%)formaldehyde.) !Table!3.26.!1x!MAE!buffer.! Constituent Concentration,(mM) MOPS 20 Sodium,acetate,(pH,7.0) 5 EDTA,(pH,8.0) 1 ) ) Materials)and)Methods) ) ) 68) In)order)to)visualize)the)fragments)within)the)gel,)ethidium)bromide)was)added)to)the) RNA) loading) buffer) (Table) 3.27).) RNA) samples) were)mixed) with) 1.25x) RNA) loading)buffer,) heated)at)56ºC)for)15)min)to)open)its)secondary)structures)and)immediately)placed)on)ice.)) Table!3.27.!1.25x!RNA!loading!buffer!composition.! Constituent Concentration Formamide 55.0,% Formaldehyde 7.37,% 10x,MAE 1.25,% Bromophenol,blue 0.5,g,l−1, Glycerol 8.0,% Ethidium,bromide 40,g,l−1, ) ) The) samples) were)charged) into) the)gel) and) the) electrophoresis) was)carried) out) at) a) voltage)of)70−80)V.)RNA)molecules)are)negatively)charged,)like)DNA)molecules,)and)thus)migrate) to)the)anode)in)the)electrophoresis)chamber.) Finally) the) gel) was) photographed) with) either) the)ChemiDocTM)XRS) Gel) documentation) system)(BioCRad)Laboratories))or)the)transiluminator)Intas)UV)Imager)(Intas).) ) 3.3.3.!Complementary!RNA!(cRNA)!synthesis!by!in)vitro!transcription) (IVT))! Since) the) optimal) templates) for) in! vitro! transcription) are) linear) doubleCstranded) DNA) (dsDNA)) molecules)with) 5’Cprotuding) ends,) the) linearized) IVTCPCR) products)(section)3.2.4.5),) which)were)purified)(section)3.2.6),)were)used)as)templates)for)the)complementary)RNA)(cRNA)) synthesis.) Taking) into) account) that)all) the) vectors) contained) the) T7) promoter) of) the) T7) RNA) polymerase)from)the)T7)bacteriophage,)T7)RNA)polymerase)was)used)to)synthesise)cRNAs)from) the) different) purified) constructs) using) the) AmpliCapCMax™) T7) High) Yield) Message) Maker) Kit) (Cellscript,)Madison,)US).) Each)IVT)reaction)mix)(Table)3.28))had)a)final)volume)of)10)μl)and)was)incubated)90)min) at)37ºC.)) !Table!3.28.!IVT!reaction!mix.! Component Volume+(μl) 10x$AmpliCap,Max$T7$Transcription$Buffer 1 100$mM$Dithiothreitol$(DTT) 1 AmpliCap,Max$Cap/NTP$PreMix 4 ScriptGuard$Rnase$Inhibitor 0.25 AmpliCap,Max$T7$Enzyme$Solution 1 Linearized$template$DNA$with$T7$RNAP$promoter 2.75 ) ) The) transcription) products) were) purified) using) ammonium) acetate) (NH4Ac)) precipitation) that) selectively) precipitates) RNA,) while) leaving) most) of) the) protein,) DNA) and) unincorporated)NTPs)in)the)supernatant.)A)volume)of)60)μl)of)5)M!NH4Ac)was)added)to)each) Materials)and)Methods) ) ) 69) reaction) and) they) were) stored) at) −20ºC) for) at) least) 1) h.) The) cRNAs) were) precipitated) by) centrifugation)at) 20,000)×)g)for)1)h)at)4ºC.)The) supernatant)was)removed)with)a)pipette)and) gently)washed)with)75%)iceCcold)ethanol.)The)cRNAs)were)dried)and)subsequently)resuspended) in)16)μl)RNaseCfree)water.) The)concentration)of)the)samples)was)quantified)(section)3.3.4))and)the)quality)of)them) was)verified)by)electrophoresis)in)1%)agarose)gel)(section)3.3.2).) ) 3.3.4.!RNA!quantification!) The)determination)of)the)concentration)of)the)cRNA)samples)obtained)from)the)in!vitro! transcription) (IVT;) section) 3.3.3)) was) performed) with) 1) μl) in) a) NanoDrop®)2000c) spectrophotometer)(Thermo)Fisher)Scientific).)The)concentration)of)the)samples)was)generally) adjusted)to)500)ng)μl−1)in)order)to)electrophysiologically)examine)the)clones)generated)by)the) twoCelectrode)voltage)clamp)(TEVC))technique)(section)3.12),)) The) concentration) of) RNA) samples) obtained) from) plant) tissues) (section) 3.3.1)) was) measured) using) the) spectrophotometer) NanoDrop®)NDC1000) (Thermo) Fisher) Scientific).) The) samples) whose) RNA) concentration) was) higher) than) 1.5) μg) μl−1,) were) adjusted) to) a) final) concentration)of)1.5)μg)μl−1)with)milliCQ)water)treated)with)0.1%)DEPC.) ) 3.4.!CLONING!METHODS!AND!PLASMID!CONSTRUCTION! )Through) cloning,) a) DNA) fragment) is) integrated) into) a) vector.) This) construct) can) be) transformed) into) different) hosts,) like) Escherichia! coli.)Plasmid) vectors) are) DNA) molecules) replicated)and)amplified)in)bacterial)cells.)) In) conventional) cloning) techniques,) restriction) endonucleases) are) used) to) cut) plasmid) vectors)at)the)cloning)site)so)that)singleCstranded)overhangs)are)formed)at)the)interfaces.)Thus,) DNA)fragments)with)complementary)overhangs)can)be)subsequently)inserted)at)these)sites.)The) disadvantage)of)this)cloning)technique)is)that)two)different)restriction)sites)are)required)in)both) the)DNA)of)interest)and)the)vector)for)directional)cloning.)And)through)conventional)restriction) enzymes) only) short) overhangs) of) 2−4) bases) are) formed,) which) require) an) additional) ligation) step)during)cloning.) ) 3.4.1.!USERTM!cloning!! The)USERTM)(uracilCspecific)excision)reagent))cloning)technique)(NourCEldin)et!al.,)2006)) allows) fast) and) very)efficient) cloning) of) a) PCR) fragment) without) a) ligation) step.) The) cloning) method) is) based) on) the) fact) that) 8) nucleotides!(nt)) long) complementary) overhangs) are) generated)at)the)3’)ends)of)a)PCR)amplified)DNA)fragment)(insert)with)the)point)mutation))and)a) linearized)destination)vector)to)make)a)stable)hybridization)product)without)ligation)(Fig.)3.5).) On) the) one) side,) a) 38) bpClong) PacICUSER) cassette) makes) vectors) compatible) with) the) USER) process) (NourCEldin) et!al.,) 2006).) Treatment) with) the) restriction) enzyme) PacI) (Fig.) 3.5;) New)England)Biolabs)Inc.,)UK))cut)the)vectors)and)together)with)the)enzyme)Nt.BbvCI)(Fig.)3.5;) New)England)Biolabs)Inc.),)which)only)cuts)one)strand)of)the)doubleCstranded)DNA,)3’)overhangs) Materials)and)Methods) ) ) 70) of)8)bp)are)produced,)which)are)not)complementary)between)each)others.)This)fact)prevents)a) rehybridization)of)the)vector)and)also)ensures)a)directed)insertion.) On) the) other) side,) PCR) products) must) have) 5’) overhangs) complementary) to) the) 3’) overhangs)of)the)vectors)(Fig.)3.5).)These)overhangs)are)generated)on)PCR)fragments)by)adding) to)the)geneCspecific)sequence)8)bp)sequences)to)both)overhangs,)whose)eighth)nucleotide)is)a) uracil) residue) (U).) For) this) purpose,) a) specific) sequence) is) contained) in) the) forward) (5’C GGCTTAAUC3’))and) the)reverse)(5’CGGTTTAAUC3’)) primer) at) the) 5’) region.) Following) the) DNA) amplification)by)a)DNA)polymerase)that)does)not)regard)uracil)as)a)defect)in)a)DNA)molecule) (section)3.2.4.4),)the)resulting)PCR)product)is)treated)with)an)USER)enzyme)mix)(New)England) Biolabs)Inc.))consisting)of)a)uracil)DNA)glycosylase)and)a)DNA)glycosylaseClyase)Endo)VIII)(New) England)Biolabs)Inc.).)These)enzymes)remove)the)uracil)residues)and)enable)the)dissociation)of) the) singleCstranded) fragments) lying) upstream) from) the) cleavage) sites) (Fig.) 3.5).) Thus) vectorC compatible)singleCstranded)overhangs)are)formed.) ) Figure!3.5.! USERTM!cloning! technique.) A) 38) bpClong) PacI) cassette) (upper) left) corner)) contained) in) a) USER)vector)is)digested)with)the)restriction)enzymes)PacI)and)Nt.BbvCI)to)produce)8)nt)singleCstranded)3’) overhangs.) An) amplified) PCR) fragment) containing)uracil) (pink)) is) added) to) the) linearized) vector) and) through) the)USER)enzyme)Mix,)the) uracils)are) removed) and)subsequently) the)vector) and)the) fragment) hybridize.)Yellow)and)green)mark)single)base)differences)between)the)generated)3’)overhangs,)which)are) responsible)for)the)directional)insertion)of)the)PCR)fragment.)Obtained)from)NourCEldin)et!al.,)2006.) ) The)vectors)(Table)3.29))used)in)this)study)for)expression)in)the)heterologous)expression) system) Xenopus!laevis)oocytes) were) provided) by) Prof.) Dr.) Dietmar) Geiger) (JuliusCvonCSachs) Institute,)Würzburg)University,)Germany).)All)the)vectors)contained)the)T7)promoter)of)the)T7) RNA)polymerase)together)with)the)Xenopus)βCglobin)UTRs)and)the)ampicillin)resistance)gene)in) the) USER) cassette) (NourCEldin) et! al.,)2006).) The) vector) pNBlu) number) 16) was) used) for) constitutive)gen)expression)in)Xenopus)oocytes)and)TEVC)recordings)(section!3.12))and)the)rest) Materials)and)Methods) ) ) 71) of)the)vectors)were)also)used)for)performing)the)protein)interaction)assays)with)the)bimolecular) fluorescence)complementation)(BiFC))analyses)(section!3.11).)) Table!3.29.! USERTM!vectors! used! for! expression! in! Xenopus)laevis)oocytes.!Nomenclature) and) description) of) the) oocytes) expression) vectors.) ) For) the) BiFC) experiments,) the) Yellow) Fluorescent)Protein)(YFP))was)cleaved)into)the)NCterminal)(amino)acids)1C155))and)CCterminal) (amino)acids)156C239))halves.) Vector' number Name Description 16 pNBlu Xenpus(oocytes(expression(vector( made(USER(compatible 17 pNBlu(N=term(YC C=terminal(half(of(YFP(placed( upstream(of(the(USER(cassette 18 pNBlu(C=term(YC C=terminal(half(of(YFP(placed( downstream(of(the(USER(cassette 19 pNBlu(N=term(YN N=terminal(half(of(YFP(placed( upstream(of(the(USER(cassette 20 pNBlu(C=term(YN N=terminal(half(of(YFP(placed( downstream(of(the(USER(cassette 21 pNBlu(N=term(YFP YFP(placed(upstream(of(the(USER( casette ) ) Therefore,)to)perform) the)insertion)of)the)gene)of)interest)into)the)desired)vector,)the) DNA)sequence)of)this)gene)previously)obtained)(section)3.2.4.4))was)cloned)in)the)pNBlu)vector) through)the)USER)enzyme)mix)described)above.)The)USER)reaction)(Table)3.30))was)incubated) at)37ºC)for)25)min)followed)by)25)min)at)25ºC)in)a)Mastercycler)Personal)(Eppendorf).) Table!3.30.!Components!of!the!USER!cloning!reaction.! Component Volume+(μl) Vector 1.5 PCR-product 4 TE-Buffer 3.5 USER9Enzyme-Mix-(1-U-μl−1) 1 ) ) As)explained)before,)siteCdirected)mutations)(section)3.2.4.4))could)be)introduced)in)the) gene)of)interest)by)a)modified)USER)fusion)method)(DadaczCNarloch)et!al.,)2011).))In)this)case,) the)USER)reaction)contained)(Table)3.31))the)two)PCR)products)that)constitute)the)two)halves)of) a) pNBlu) vector) with) the) gene) of) interest.) The) samples) were)incubated) at) 37ºC) for) 25) min) followed)by)25)min)at)25ºC.) Table!3.31.!Components!of!the!modified!USER!cloning!reaction.! Component Volume+(μl) PCR$product$1 2 PCR$product$2 2 TE$Buffer 5 USER6Enzyme$Mix$(1$U$μl−1) 1 ) ) The) whole)vector) produced) in) the) USER) reaction) was) immediately) transformed) into) chemically)competent)E.!coli)cells)(section)3.5.1.2).)After)bacteria)selection)in)LB)supplemented) Materials)and)Methods) ) ) 72) with) ampicillin)(section) 3.11),) single) colonies) were) picked) to) perform) the) DNA) plasmid) extraction)(section)3.2.1).)After)obtaining)the)right)sequence,)the)DNA)samples)were)used)for)the) IVTCPCR)(section)3.2.4.5))by)which)the)resulting)pNBlu)plasmid)with)the)insert)of)interest)was) linearized.) The) product) reaction) was) purified) (section) 3.2.6)) and) the) DNA) concentration) was) determined) (section) 3.2.3).) Afterwards,) the) dsDNA) was) in! vitro)transcribed) (section) 3.3.3)) followed)by)a)verification)of)the)RNA)purity)(section)3.3.2).) ) 3.4.2.!Gateway®!technology!! The)Gateway®)cloning)method)(Invitrogen))is)based)on)the)siteCspecific)recombination) system)of)bacteriophage)lambda)(λ))(Landy,)1989),)which)facilitates)the)integration)of)lambda) into)the)E.!coli)chromosome,)and)provides)a)quick)and)efficient)way)to)transfer)DNA)sequences) between)different)vectors)for)different)objectives,)like)functional)analysis)or)protein)expression) (Hartley)et!al.,)2000).)) The)recombination)occurs)between)specific)DNA)recombination)sequences)(att!sites))by) a)mixture)of)lambda)and)E.!coliCencoded)recombination)proteins)that)catalyse)the)recombination) reaction.)The)DNA)segments)flanking)the)recombination)sites)are)switched,)so)the)att!sites)are) hybrid)sequences)comprised)of)sequences)donated)by)each)parental)vector)after)recombination.) The) crossover) occurs) between) homologous) 15) bp) core) regions) on) the) two) sites,) but) the) surrounding)sequences)are)essential)since)they)contain)the)binding)sites)for)the)recombination) enzymes)(Landy,)1989).) ) Figure!3.6.!Gateway®!cloning! technology.)The)BP)reaction)generates)the)entry)clone)(with)attL)sites)) after)the)recombination)between)the)entry)vector)(containing!attP)sites))and)the)DNA)of)interest)flanked) by)attB)sites)or)an)expression)clone.)In)the)LR)reaction)takes)places)the)recombination)of)the)entry)clone) with)a)destination)vector)(with)the)attR)sites),)leading)to)an)expression)clone)containing!attB)sites.)Genes) can) be) transferred) between) different) vectors) once) it) is) cloned) in) a) Gateway) vector.) The) byCproduct) molecules)containing)the)ccdB)gene)do)not)generate)negative)clones.) ) Two) recombination) reactions) constitute) the) basis) of) this) cloning) method.) In) the) BP) reaction)takes)places)the)recombination)of)a)PCR)product)flanked)by)attB)sites)(or)a)linearized) attB)expression)clone))with)an)entry)vector!containing!attP)sites,)leading)to)an)attLCcontaining) entry)clone)(Fig.)3.6).)And)the)LR)reaction)leads)to)an)attBCcontaining)expression)clone)after)the) Materials)and)Methods) ) ) 73) recombination)of)an)attLCcontaining)entry)clone)with)a)destination)vector)containing)the)attR) sites)(Fig.)3.6).)In)the)design)of)the)primers,)the)attB)sites)have)to)be)incorporated)to)generate)a) suitable)PCR)product.) The)att!sites)flank) a) cassette) that) contains) the) chloramphenicol) resistance) gene) (CmR)) and)also)the)ccdB)gene,)which)allow)negative)selection)in)E.!coli!since)the)CcdB)protein)interferes) with) the!DNA) gyrase,) supressing) its) growth) when) the) recombination) does) not) take) place) (Bernard) and)Couturier,)1992).)After)each)recombination)reaction)this)cassette)is)replaced)by) the)gene)of)interest.) ) 3.4.2.1.!Producing!entry!clones! The) AtSLAH1)promoter) (section) 3.2.4.1)) was) cloned) into) the) entry) vector) pENTR/DC TOPO®)using)the)pENTRTM/DCTOPO®)Cloning)kit)(Invitrogen).)The)cloning)reaction)(Table)3.32)) was)performed)following)the)manufacturer’s)instructions.)) Table!3.32.!TOPO®!cloning!reaction!composition.! Components Volume,(μl) PCR$Product 4 pENTR/D2TOPO$vector 1 Salt$Solution 1 ! ) The)AtSLAH4)promoter)(section)3.2.4.1))for)constructing)the)SLAH4!promoterCGFG::GUS) reporter)fusion)and!the)AtSLAH1)gene)(section)3.2.4.1))for!complementation)of)the)slah182)TC DNA) insertion) mutant) were) cloned) into) the) pDONRTM201) entry) vector)using) the) Gateway) BP) ClonaseTM)II)Enzyme)Mix)kit)(Table)3.33);)Invitrogen).)) !Table!3.33.!BP!recombination!reaction.!! Components Volume,(μl) PCR$Product 5 pDONR201$vector 1.5 TE$Buffer,$pH$8.0 1.5 BP$Clonase$II$enzyme$mix 2 Proteinase$K$solution 1 ) ) The) reaction) products) from) SLAH1! promoterCpENTR/DCTOPO®,!SLAH4! promoterC pDONRTM201) and) AtSLAH1CpDONRTM201)were) used) for) transforming) TOP10)or) Mach1TMCT1) chemically) competent) E.!coli)cells) (section) 3.5.1.2).) Following) the) bacteria) selection) in) LB) (section)3.1.1))supplemented)with)50)µg)mlC1)kanamycin,)single)colonies)were)picked)to)perform) the) DNA) plasmid) extraction) (section) 3.2.1).)In) order) to) verify) the) presence) of) the) insert) enzymatic) restriction) analyses) were) performed) (section) 3.2.7).)The) DNA) plasmid) was) then) sequenced)(section)3.2.8).)After)obtaining)the)right)sequences,)the)LR)reaction)(section)3.4.3.2)) was)performed)with)these)DNA)plasmids.)) ) ) ) Materials)and)Methods) ) ) 80) mutant)lines.)In)addition,)the)slah18slah3)doubleCKO)mutant,)obtained)by)sexual)crosses!(section) 3.7.2),)and)its)azygous)segregating)line)(ColC0)x)WS))were)also)used.) When)the)first)bolt)appeared,)it)was)removed)to)promote)further)bolting.)Plants)were) used) with) secondary) bolts) 5−15) cm) tall.)For) collection) of) xylem) sap) all) rosette) leaves) were) removed)with)blunted)scissors)to)prevent)loss)of)xylem)solution)from)the)cut)area.)In)addition,) floral)stems)were)cut)with)a)scalpel,)allowing)secretion)of)xylem)sap)droplets)from)the)base)of) the)stem.)The)container)was)sealed)to)maintain)saturating)humidity.)Drops)of)xylem)sap)at)the) cutting)surface)of)the)stem)were)then)collected)using)a)micropipette)(Gaymard)et!al.,)1998;)Shi)et! al.,)2002;)Sunarpi)et!al.,)2005).)Xylem)sap)was)collected)at)different)times)(1)h,)2)h,)3)h)and)5)h) after)starting)the)assay))and)Cl−)and)NO3−)concentrations)were)quantified)(section)3.8.4.2).)) We)observed)that)xylem)sap)was)more)efficiently)and)abundantly)extracted)from)potted) plants) than) from) hydroponically) grown) plants.) Ion) translocation) rate) values) are) usually) normalized)with)root)biomass)but)it)was)difficult)to)obtain)intact)roots)from) plants)grown)in) solid) substrate.) Under) the) premise) that) the) volume) of) exuded) xylem) sap) maintains) a) linear) relationship) with) the) root) biomass,) we) used) plants) grown) in) hydroponics) medium) (section) 3.1.2.2.3)) to) test) this) hypothesis.) The) linear) correlation) between) the) volume) of) xylem) sap) secreted) per) plant) and) the) root) biomass) of) hydroponically) grown) plants) was) confirmed) (Fig.) 3.7).)Therefore)we)decided)to)normalize)ion)translocation)rate)with)the)total)volume)of)xylem) sap) secreted) per) potted) plant.)For) further) xylem) sap) ion) content) quantifications,) only) results) from)assays)where)the)sap)volume)of)mutant)and)WT)plants)were)comparable)were)used.))) ) Figure!3.7.!Xylem!sap!correlation!with!root!biomass.!Correlation)of)sap)flux)with)root)biomass.)WildC type)(WT))plants)were)grown)hydroponically)with)Basal)Solution)(BS).)Xylem)sap)was)collected)one)hour) after)shoot)trimming.))Sap)secretion)volume)was)linearly)correlated)with)root)biomass.)! ! To)quantify)the)Cl−)translocation)into)the)xylem,)sixCweekCold)plants)grown)in)pots)with) BS) solution) (containing) 70) μM) Cl−;) section) 3.1.2.2.2)) were) transferred) to) a) container) and) submerged)in)the)same)solution)supplemented)with)5)mM)Cl−)almost)up)to)substrate)level.) Moreover,)to)perform)different)Cl−)salt)stress)treatments)with)slah182)plants,)pots)were) transferred)to)BS)solution)supplemented)with)different)Cl−)salt)concentrations)(15,)30,)60)and) 120)mM)Cl−).)To)avoid)interference)caused)by)sodium)toxicity,)the)following)combination)of)Cl−) salts)was)used)KCl:MgCl2:CaCl2)in)a)ratio)2:1:1.)Xylem)sap)from)these)plants)was)collected)3)h) after) starting) the) salt) exposure)and) its) Cl−)concentration)was) afterwards) quantified) (section) 3.8.4.2).) Materials)and)Methods) ) ) 81) 3.8.3.!Growth!at!different!Cl−!and!NO3−!concentrations! For) slah482! phenotypic) assay) with) solutions) containing) different) Cl−)and) NO3−) concentrations,) slah482! and) its) WT) line) (ColC0)) plants) were) first) grown) with) BS) (section) 3.1.2.2.2))to)allow)their)proper)beginning)development)and)17)days)after)sowing!(das))Cl−)and) NO3−!treatments)started)(section)3.1.2.2.2;)Table)3.5).) slah482! and) WT)plants) were) harvested) according) to) their) developmental) stage) at) different)times:)) T1:)Vegetative)phase:)plants)with)growing)rosette)without)inflorescence.)) T2:)Reproductive)onset:)beginning)of)bolting)stem)elongation.) T3:)Flowering)stage:)plants)with)abundant)flowers)and)few)siliques.) T4:)Fruit)maturation)stage:)plants)with)abundant)siliques)and)few)flowers.) )Plants)were)dried)in)an)oven)Selecta)(JP)Selecta)SA,)Barcelona,)Spain))at)70ºC)until)the) determination)of)Cl−)content)(section)3.8.4.1).) ) 3.8.4.!Chloride!and!nitrate!contents!quantification! 3.8.4.1.!Anion!content!in!plants! slah182)KO)mutants)and)WT)plants)harvested)at)the)end)of)the)phenotypic)assay)where) different)morphological)measurements)were)took)(section)3.8.1),!were)dried)in)an)oven)Selecta) (JP)Selecta)SA))to!determine)its)Cl−!content.)Dried)root)and)shoots!were)ground)to)a)fine)powder) with) mortar) and) pestle.) The) powder) was) incubated) in) an) acidic) solution) (0.1) M) HNO3,) 10%) glacial)acetic)acid))with)gentle)shaking)overnight)at)28ºC)in)the)dark.)Afterwards,)samples)were) centrifuged)5)min)at)1,500)rpm)in)the)centrifuge)5403)(Eppendorf))and)100)μl)of)the)samples) were)used)to)determine)the)Cl−)concentration)by)silver)ionCtitration)according)to)Gilliam)(1971),) using)a)chloride)meter)Corning)926)(Sherwood)Scientific)Ltd.,)Cambridge,)UK).) Dried!slah482)KO)mutant)and)WT)(ColC0))plants)from)the)phenotypic)assay)with)different) Cl−)and)NO3−)concentrations)(section)3.8.3))were)used)for)Cl−!content) determination.)Samples! were) ground) to) a) fine) powder) with) mortar) and) pestle) and) the) dry) powder) was) incubated) in) milliCQ) water)with) gentle) shaking.) Samples) were) centrifuged) 5) min) at) 1,500) rpm) in) the) centrifuge)5403)(Eppendorf))and)the)content)of)Cl−!was)determined)by)colorimetric)assays,)as)it) is)described)for)xylem)sap)samples)in)the)following)section)(section)3.8.4.2).) ) 3.8.4.2.!Anion!content!in!xylem!sap!samples!! Nitrate) and) Cl−)content) in) xylem) sap) samples) (section) 3.8.2)) was) determined) by) colorimetric) assays) with) the)spectrophotometer!SPECTROstar®)Omega,) (BMG) Labtech) GmbH,) Ortenberg,)Germany).)Nitrate)concentration)was)determined)according)to)Cataldo)et!al.)(1975)) and)Cl−)concentration)as)described)in)Frankenberger)et!al.)(1996).)) For)NO3−)content)determination,)5)μl)of)the)sample)was)mixed)with)20)μl)of)10%)sodium) salicylate)(dissolved)in)sulphuric)acid),)forming)a)complex)of)NO3−)with)salicylic)acid)under)this) highly)acidic)condition.)Afterwards,)200)μl)of)4.75)M)NaOH)were)added)and)the)samples)were) incubated)at)room)temperature)for)1)h)in)the)dark.)Nitrate)content)was)obtained)measuring)the) Materials)and)Methods) ) ) 82) samples)at)410)nm)by)the)spectrophotometer.)To)transform)the)absorbance)measurements)into) NO3−)content,) 13Cfold) dilution) series) of) 50) mM) KNO3)dilutions)were)used)as) standard) curves.) Absorbance)of)the)chromophore)is)directly)proportional)to)the)amount)of)NO3−)present)in)the) sample.) For)determination)of)Cl−)content,)each)reaction)mix)contained)40)μl)of)the)sample,)20)μl) of)0.4)%)mercury)thiocyanate)(dissolved)in)methanol),)20)μl)of)20.2%)ferric)nitrate)and)170)μl)of) milliCQ)water,)in)a)volume)of)250)μl.)Based)on)known)concentrations)of)mercury)thiocyanate)and) ferric) nitrate,) 10Cfold) dilution) series) were) prepared) and) used) as) standard) curves.) This) colorimetric) method) is)based) on) the) displacement) of) the) thiocynate) (from) the) mercury) thiocyanate))by)Cl−)ions,)leading)to)mercury)chloride)and)ferric)thiocyanate)(colored)product).) The) absorbance) of) the) resulting) colored) compound) is) measured) at) 480) nm)by) the) spectrophotometer.) The) anion) content) in) the) xylem) sap) was) used) afterwards) to) obtain) the) translocation) efficiency)for)Cl−)and)NO3−)and)the) NO3−/Cl−)selectivity)in) the)different)ecotypes)assayed)(WS,) ColC0,)and)the)WS)x)ColC0)hybrid).)The)translocation)efficiency)for)Cl−)and)NO3−)was)calculated)as) the) ratio) between) the) anion) content) in) the) xylem) sap) and!the) anion) content) in) the) nutrient) solution.)And)the)NO3−/Cl−)selectivity)was)obtained)by)dividing)the)translocation)efficiency)for) NO3−)by)that)for)Cl−)in)each)ecotypes)assayed.) ) 3.9.!HISTOLOGICAL!TECHNIQUES! 3.9.1.!Histochemical!analysis!of!βYGlucuronidase!(GUS)!activity!! Independent)transgenic)lines)of)PSLAH1CGFP::GUS)(five)lines),)PSLAH3CGUS)(two)lines;)Negi) et! al.,) 2008)) and) PSLAH4CGFP::GUS) (eight)lines)) were) tested) for) GUS) histochemical) activity) (Jefferson,)1987))to)localise)the)promoter)expression.) Samples) at) various) developmental) stages) were) incubated) 20) min) in) 90%) acetone,) vacuumCinfiltrated) for) 5) min) in) the) βCGlucuronidase) (GUS)) staining) solution) (Table)3.37)) and) incubated) at) 37) °C) in) the) dark) for)either)3) h) (PSLAH1CGFP::GUS) and) PSLAH3CGUS)) or) 5) h) (PSLAH4C GFP::GUS).) !Table!3.37.!GUS!reaction!composition.! Components Concentration X"Gluc'(5"bromo"4"chloro"3"indolyl'β"D"glucuronide) 1'mM K4Fe(CN)62'mM K3Fe(CN)62'mM Triton'X"10 2"% NaPO4'(pH'7.2) 50'mM ) ) During)the)activity)of)the)βCGlucuronidase,)an) uncoloured)product)is)produced,)but)its) oxidative)dimerization)leads)to)an)insoluble)blue)product.)) To) stop) the) staining) and) remove) chlorophyll,) samples) were) cleared) with) a) series) of) ethanol)washings,)ending)with)70%)ethanol.)) Materials)and)Methods) ) ) 83) Samples) were) analysed) under) the) stereomicroscope) Olympus)SZCCTV) (Olympus)) and) micrographs)of)GUSCstained)tissues)were)obtained)with)a)Zeiss)Axioskop)microscope)(Carl)Zeiss,) Jena)Gmbh,)Jena)Germany))and)the)AxioVision)Release)4.8.2.)Zeiss)software)(Carl)Zeiss).) Independent)transgenic)lines)of)SLAH)promoterCGUS)showing)consistent)staining)pattern) were)selected)for)further)analyses.)) ) ) 3.9.1.1.!GUS!after!hormonal!assays! To)elucidate)the)possible)regulation)of)abscisic)acid)(ABA))and)indoleC3Cacetic)acid)(IAA)) in)SLAH)gene)expression,)Arabidopsis!plants)expressing)the)GUS)reporter)gene)under)the)control) of)SLAH1,)SLAH3)or)SLAH4)promoter)were)used.)) ThirteenCdayCold) seedlings) of) PSLAH1CGFP::GUS,) PSLAH3CGUS) and) PSLAH4CGFP::GUS) lines) grown)in)Petri)dishes)(section)3.1.2.2.1))were)sprayed)with)either)100)μM)ABA)or)the)control) (water))solution)using)an)airbrush)spray)device)(Pintuc)Screw)Compressors,)Barcelona,)Spain).) Additionally,) given) that) SLAH4! gene) was) expressed) in) organs) associated) with) high) primary)auxin)production,)PSLAH4CGFP::GUS)line)seedlings)were)also)subjected)to)100)μM)IAA)to) further)explore)its)promoter)expression)regulation.) After)15)h)incubation,)plants)were)subjected)to)GUS)staining)as)described)previously.)) ) 3.9.2.!Cryostat!sectioning!of!plants!expressing!GUS!! Following) GUS) staining,)cross) sections) of) roots) using) a) cryostat) were) performed.)For) sectioning,)samples)were)rehydrated)and)then)fixed)with)formaldehyde)(Table)3.38))for)4)h)at) 4ºC.)To)stop)the)fixation,)samples)were)washed)with)sodium)phosphate)buffer)pH)7.2.) !Table!3.38.!Fixing!solution.! Components Concentration Formaldehyde 4,% NaPO41(pH17.2) 1001mM ) ) Samples) were) embedded) in) tissue) freezing) medium) (Leica) Biosystems,) Nussloch,) Germany)) and) frozen) at) −20ºC.) Sections) (15) μm)) were) prepared) using) a) cryomicrotome) (Cryostat) Leica) CM1950,) Leica) Biosystems).) They) were) afterwards) transferred) to) microscope) slides)and)covered)with)coverCslip)once)embedded)in)glycerol:PBS)(80:20).) Samples)were)analysed)with)a)Zeiss)Axioskop)microscope)(Carl)Zeiss))and)the)AxioVision) Release)4.8.2.)Zeiss)software.) ) 3.10.!Xenopus)laevis!AS!HETEROLOGOUS!EXPRESSION!SYSTEM! The)ease)of)obtaining)large)number)of)Xenopus!laevis)oocytes)together)with)their)size)and) robust)nature)have)made)them)a)suitable)system)to)investigate)transport)processes)on)a)cellular) level.)The)oocytes)of)the)South)Africa)clawed)toad)Xenopus!laevis)have)been)extensively)used)in) biological)and)pharmacological)research)(Weber,)1999).))The)mature)oocytes)have)a)diameter)of) Materials)and)Methods) ) ) 84) 1−1.3)mm)(which)facilitates)its)handling))and)pigmentation)with)marked)polarization)into)the) dark)animal)hemisphere)and)the)beige)vegetative)hemisphere.) During) oogenesis,) oocytes) accumulate) large) quantities) of) proteins) and) organelles) that) form)a)reserve)for)early)embryonic)development.)Among)them)the)ribosomes,)tRNAs)and)other) components) important) for) translation) may) be) used) to) translate) exogenous) RNA) (Theodoulou) and)Miller,)1995).)They)also)carry)out)most)postCtranslational)modifications)to)foreign)proteins) as)well)as)their)correct)incorporation)into)the)cell)membrane.) The)heterologous)expression)system)of)the)Xenopus)oocytes)allows)the)characterization) of) proteins) isolated) from) their) natural) environment.) This) expression) system) has) been) widely) employed)to)study)the)structure)and)function)of)ion)channels)and)receptors)(Guan)et!al.,)2013).) The) oocyte) system) allows) performing) a) whole) plethora) of) different) techniques) for) the) investigation) of) ion) channels)and) transporters) (Weber,) 1999))like) the) twoCelectrode) voltage) clamp)(TEVC))technique)and)the)bimolecular)fluorescence)complementation)(BiFC))analysis.) Although) the) X.! laevis! oocyte) is) endogenously) equipped) with) a) host) of) ion) channels,) transport) systems) and) receptors,) heterologously) expressed) ion) channels) can) be) clearly) distinguished) from) the) endogenous) ones.) Most) of) the) time,) currents) produced) by) the) endogenous) ion) channels) are) small) compared) with) the) currents) produced) by) the) expressed) channels)(Sigel,)1990;)Weber,)1999)) Today) this) expression) system) is) one) of) the) recognized) standard) methods) for) the) translation)and)expression)of)animal)and)plant)ion)channels,)transporters)and)receptors)and)for) their)electrophysiological)investigation.) ) 3.10.1.!Oocytes!preparation))) Xenopus!laevis)female)frogs)were)kept)in)aquaria)at)a)water)temperature)of)20−22ºC.)A) cover)was)used)to)prevent)escape.)The)frogs)were)fed)twice)weekly)and)the)tanks)were)cleaned) every)week.)The)oocytes)were)obtained)from)adult)frogs.) The) frog) was) hypodermically) anesthetized) with) tricaine!(0.75) g) l−1)) and) cooled) down) with)ice.)The)abdominal)skin)and)the)underlying)muscle)tissue)were)surgically)opened)and)the) ovary)lobes)were)removed)with)tweezers)and)collected)in)the)ND96)solution)(Table)3.39).)After) removing)the)oocytes,)the)abdomen)of)the)frog)was)sewn.)) !Table!3.39.!ND96!solution!composition.! Constituent Concentration,(mM) NaCl 96 KCl 2 MgCl2+1 HEPES 10 pH+was+adjusted+to+7.4+with+NaOH Osmolarity+was+adjusted+to+220+mosmol+kg−1+with+sorbitol ) ) The) oocytes) were) subsequently) divided) into) smaller) portions) as) carefully) as) possible) under) the) stereomicroscope) Olympus) SZC40) (Olympus)) with) fine) watchmaker) tweezers) to) improve)the)accessibility)of)the)oocytes)to)the)collagenase)in)the)following)step.)After)several) Materials)and)Methods) ) ) 85) washes)of)the)oocytes)in)Ca2+Cfree)ND96)solution,)they)were)subjected)to)a)collagenase)digestion) (0.25−0.27)PZ)U)mg−1,)12)mg)ml−1))to)remove)the)remaining)connective)tissues,)blood)capillaries,) and)the)follicular)layer,)thus)isolating)the)oocytes)(Guan)et!al.,)2013).)The)digestion)in)Ca2+Cfree) solution)was)necessary)because)the)collagenase)shows)an)uncontrollable)activity)in)the)presence) of)Ca2+,)which)would)prevent)uniform)oocyte)digestion.)The)oocytes)were)incubated)for)90)min) at)room)temperature)with)gentle)shaking.)Afterwards,)the)oocytes)were)washed)several)times) with)the)ND96)solution)in)order)to)remove)the)collagenase)and)the)residues)of)the)digestion.)) Finally,)the)individual)oocytes)were)placed)in)ND96)solution)supplemented)with)1)mM) Ca2+)(1mM)CaCl2))and)gentamycin)(50)µg)ml−1))and)were)stored)at)16ºC.))) Since)not)all)oocytes)were)suitable)for)further)use)due)to)different)size)or)injuries,)they) were)sorted)according)to)optical)characteristics)and)only)the) undisturbed)oocytes)were) used.) The)oocytes)were)screened)daily)to)remove)either)nonChealthy)looking)or)damaged)oocytes)and) to)change)the)culture)medium)when)it)was)necessary.) ) 3.10.2.!cRNA!injection!in!oocytes) After)obtaining)glass)capillary)by)the)Narishige)PCC10)micropipette)puller)(Narishige)Co.,) Tokio,) Japan)) with) single) stage) pulling,) their) tip) was) obliquely) cut) off) with) the) aid) of) a) Hund) Wetzlar) microscope) (Helmut) Hund) GmbH,) Wetzlar,) Germany)) in) order) to) not)damage) the) membrane)of)the)oocyte.)The)diameter)of)the)truncated)tip)of)the)microinjection)needles)was) 10−20)μm.) The) microinjection) needle) was) completely) filled) with) mineral) oil) containing) a) small) amount)of)Sudan)(enough)to)impart)a)distinct)red)colour).)The)oilCsoluble)dye)was)incorporated) to)facilitate)the)visualization)of)the)interface)between)the)RNA)and)the)mineral)oil)used)in)the) needle)(Smart)and)Krishek,)1995).)It)is)essential)to)avoid)the)introduction)of)air)bubbles)in)the) microinjection)needle.) The)cRNA)obtained)from)the)in!vitro)transcription)(IVT;)section)3.3.3))was)injected)into) the) oocytes) with) the) NanoliterC2000) Injector) (World) Precision) Instruments,) Berlin,) Germany)) mounted) on) a) micromanipulator) (Drummond) Scientific) Co.,) Broomall,) PA,) US)) under) a) stereomicroscope)Olympus)SZC40)(Olympus).)To)draw)cRNA)into)the)glass)needle,)a)drop)of)oil) was) previously) forced) out) of) the) needle.) The) oocytes) were) located) in) a) specially) prepared) chamber) in) individual) wells) to) stabilize) them.) The) chamber) was) filled) with) ND96) solution) supplemented)with)1)mM)Ca2+)and)gentamycin.)For)injection,)the)needle)was)inserted)into)the) oocytes)by)means)of)the)micromanipulator,)and)each)oocyte)was)injected)with)50)nl)of)cRNA.) Water)was)injected)as)a)negative)control.) For) oocyte) BiFC) and) electrophysiological)experiments,) 10) ng) each) of) SLAC1,) SLAH1,) SLAH2,)SLAH3,)SLAH4,)SLAH)mutants,)CPKs,)SnRKs)and)CBLs)cRNA)and)5)ng)of)CIPKs)cRNA)was) injected.)Electrical)recordings)were)done)with)native)proteins)(without)any)fusions).)To)avoid) differences) in) the) kinase) activity) due) to) different) Ca2+Cdependencies) and) calcium) levels) in) Xenopus)oocyte)batches,)truncated)kinases)were)used,)i.e.)Ca2+)independent)mutants)of)CPK3)and) 21) (CPK3ΔEF,) CPK21ΔEF,) [Geiger) et!al.,) 2010,) 2011;) Maierhofer) et!al.,) 2014a;) Scherzer) et!al.,) 2012]).)) Materials)and)Methods) ) ) 86) The) injected) oocytes) were) subsequently) stored)in) Petri) dishes)containing) the) same) solution)at)16ºC)and)after)2−3)d)they)were)used)either)in)the)TEVC)technique)(section)3.12))or)in) the)BiFC)analyses)(section)3.11).) ) 3.11.!PROTEIN!INTERACTION!STUDIES!THROUGH!THE!BIMOLECULAR! FLUORESCENCE!COMPLEMENTATION!(BiFC)!TECHNIQUE) This)method)is)based)on)two)nonCfluorescent)fragments)of)the)yellow)fluorescent)protein) (YFP))that)can)reconstitute)the)fluorophore)when)fused)to)proteins)that)interact)with)each)other) (Hu) et!al.,) 2002;) Kerppola,) 2006;) 2008).) The) association) of) the) two) halves) of) the) YFP) (the) CC terminal)and)the)NCterminal)half),)because)of)the)nearness)of)the)examined)proteins,)cause)the) formation) of) a) bimolecular) fluorescent) complex) (Fig.)3.8).) BiFC) analysis) allows) direct) visualization)of)protein)interactions)in)living)cells)environment.) ) Figure!3.8.!Diagram!representing!the!principle!of!the!BiFC!assay.!Two)fragments,)the)NCterminal)(NT)) and) the) CCterminal) (CT)) halves,)of) the) yellow) fluorescent) protein) (YFP)) are) fused) to) two) putative) interaction)partners.)In)the)absence)of)an)interaction)between)the)proteins,)the)fluorophore)halves)remain) nonCfunctional.)But)the)interaction)between)the)proteins)facilitates)the)association)between)the)two)YPF) fragments)and)a)functional)fluorophore)is)constituted,)exhibiting)emission)of)fluorescence)upon)excitation) with)an)appropriate)wavelength.)Modified)from)Hu)et!al.,)2002.! ! The)BiFC)method)has)the)limitation)that)fluorescent)protein)fragments)can)associate)with) each)other)when)they)are)nearby,)independently)of)an)interaction)between)the)proteins)fused)to) the) fragments,) obtaining) false) positives) (Kerppola,) 2006).) Generally,) this) disadvantage) can) be) alleviated)by)expressing)the)fusion)proteins)at)concentrations)approximating)their)endogenous) counterparts)or)testing)alternative)fusions.) The)expression)of)the)examined)protein)in)the)pNBlu)vector)numbers)17)and)18)(Table) 3.29;)section)3.4.1)!enabled)the)fusion)of)the)protein)to)the)CCterminal)end)of)the)YFP,)as)well)as) the) expression) in) the) vector) numbers) 19) and)20) enabled) the) fusion) of) the) protein) to) the) NC terminal)end)of)the)YFP.) The)DNA)of)the)examined)proteins)(SLAH/SLAC)channels)and)kinases))was)previously) cloned) into) a) Xenopus!oocyte) expression) vector) by) the) USERTM)cloning) (section) 3.4.1).) FortyC eight)hours)after)cRNA)injection,) oocytes) images) were) taken.) The)oocytes)expressing)the)YFP) where)focused)on)the)equatorial)plane)and)pictures)were)taken)with)a)Leica)SP5)confocal)laser) scanning) microscope) (Leica) Microsystems) CMS) GmbH,) Mannheim,) Germany)) equipped) with) a) Leica)HCX)IRAPO)L25x/0.95W)objective.) ) Materials)and)Methods) ) ) 87) 3.12.!TWOYELECTRODE!VOLTAGE!CLAMP!(TEVC)!RECORDINGS) To) investigate) the) properties) of) ion) channels) in) Xenopus! laevis)oocytes,) the) electrophysiological)technique)of)twoCelectrode)voltage)clamp)(TEVC))was)used.)) In)this)technique)two)microelectrodes)are)inserted)into)the)oocytes)to)set)the)membrane) potential)at)desired)values)and)record)the)membrane)current)to)analyse)ion)channel)activities) (Fig.)3.9;)Guan)et!al.,)2013).)) The) difference) in) electric) potential) between) the) cytosolic) side) of) the) oocyte) and) the) surrounding) liquid) medium)is)the) membrane) potential) (Vm).)The) voltage) electrode) senses)the) membrane) potential) in) relation) to) the) potential) of) the) reference) electrode) in) the) bath) (conventionally)set)to)zero))and)the)current)electrode)injects)the)current)to)set)the)membrane) potential)at)the)desired)value.) ) Figure!3.9.!Schematic!representation!of!the!twoYelectrode!voltage!clamp!(TEVC)!technique!on!an! oocyte.)The)membrane)potential)(Vm))is)monitored)by)connecting)the)voltage)electrode)to)the)input)of)a) voltage) follower.) The) clamping) amplifier) compares) the) Vm)with) the) voltage) command) signal) (Vc).) The) resulting)current)flows)through)the)current)electrode)into)the)oocyte.) ) The) clamping) amplifier) (Fig.)3.9)) compares) the) membrane) potential) (Vm)) with) the) voltage)of)command)signal)(Vc))applied)to)the)input)terminal)and)the)difference)between)them) results) in) a) output) current) which) is) injected) in) the) cell) by) the) current) electrode) (Guan) et!al.,) 2013).)The)applied)current)for)maintaining)the)membrane)potential)in)the)oocyte)is)therefore) directly) proportional) to) the) current) flowing) through) a) transporter) protein) at) the) plasma) membrane)of)the)oocyte.) In) order) to) characterize) a) channel,) rapid) voltage) changes) are) applied) to) the) oocyte) membrane) and) the) resulting) currents) are) recorded.) These) currents) are) composed) of) the) instantaneous)capacitive)current)and)the)current)flowing)through)the)transport)proteins.)) The)capacitive)current)results)from)very)fast)charge)transitions)in)the)membrane)and)can) only)be)recorded)in)a)short)period)of)time)(1−2)ms))after)the)voltage)change.)By)means)of)an) offset) reading) of) the) measured) voltages,) these) rearrangement) effects) can) be) avoided) and) therefore)does)not)interfere)with)the)characterization)of)the)channel.) ) ) Materials)and)Methods) ) ) 88) 3.12.1.!Measurement!setup! Both) the) voltage) and) current) microelectrodes) were) placed) on) micromanipulators) (Marzhauser) MM33,) Drummond) Scientific) Co.)) located) on) opposite) sides) of) the) oocyte.) The) reference)electrode)was)connected)to)the)ground)of)the)preamplifier)of)the)voltage)electrode)and) placed) in) the) measuring) chamber) in) the) close) to) the) oocyte.) The) measuring) computer) was) connected)to)the)clamping)amplifier)(Turbo)TEC)10CD,)NPI)Electronics)GmbH,)Tamm,)Germany).) Different) voltage) pulse) protocols) and) data) acquisition) were) established) with) the) PatchMaster) software)(HEKA)Elektronik,)Lambrecht,)Germany).)The)recorded)data)were)evaluated)with)both) the)software)Excel)(Microsoft))and)IGOR)(WaveMetrics).) The) oocyte) was) registered) in) a) measuring) chamber) that) was) uniformly) perfused) with) different)bath)solutions)(section)3.12.3).)) Borosilicate)glass)capillaries)(KwikCFillTM,)World)Precision)Instruments))were)obtained) by)the)Narishige)PCC10)micropipette)puller)(Narishige)Co.))with)two)stage)pulling)and)they)were) filled)with)3)M)KCl)solution.)The)reference)electrode)was)placed)in)a)polyvinyl)tube,)which)also) contained)3)M)KCl)solution.)An)agar)plug)in)the)tube)opening,)consisting)of)3)M)KCl)and)2%)agar,) prevented) the) highly) concentrated) electrolyte) solution) from) flowing) into) the) lowCmolar) bath) solution)without)showing)too)much)electrical)resistance.)) ) 3.12.2.!Voltage!protocols,!data!collection!and!data!analysis! Before) starting) the) measurement,) the) electrodes) were) immersed) in) the) bath) solution.) Since) the) measuring) solutions) always) had) a) lower) chloride) concentration) than) the) electrode) solution,)a)potential)gradient)occurred,)which)was)adjusted)to)zero.)) The)impalement)of)the)electrodes)into)the)oocyte)was)controlled)both)microscopically) and)by)an)acoustic)signal.)) In)the) wholeCcell)configuration,)the)membrane)potential)of)the)oocytes) was)measured.) Following)a) holding)potential)(VH))of)0)mV,)the)instantaneous)currents)(Iinst))at)single)voltage) pulses)of)50Cms)in)10)mV)decrements)from)+70)to)−150)mV)were)extracted.) ) Figure!3.10.)Current!traces!of!the!voltage!protocol!used!for!measurement!of!currentYvoltage!(IYV)! relations.)At)the)beginning)the)cell)is)clamped)to)a)holding)potential)of)0)mV.)Afterwards,)single)voltage) pulses)of)20)s)were)applied)in)20)mV)decrements)from)+60)to)−200)mV.)The)equilibrium)currents)(ISS))are) obtained)after)19)s.)In)a)subsequent)pulse)at)−120)mV,)the)tail)streams)(Itail))were)obtained.) ) For) recording) representative) current) traces,) steady) state) currents) (ISS)) and) for) calculating)the)voltage)dependent)relative)open)probability)(rel.)PO),)standard)voltage)protocol) Materials)and)Methods) ) ) 89) (Fig.)3.10))was)as)follows:)Starting)from)a)VH)of)0)mV,)single)voltage)pulses)were)applied)in)20) mV)decrements)from)+60)to)−200)mV.)The)steady)state)currents)(ISS)!were)extracted)at)the)end) of)this) test) pulses)lasting)20)s.)In) a)subsequent)pulse)at)−120)mV,)the)tail)streams)(Itail))were) obtained,)which)was)used)for)determining)the)relative)open)probability)(rel.)PO).) For) an) ion)channel) in) which) only) one) state) is) conductive,) the) whole) cell) current) amplitude) (I)) measured) at) a) membrane) voltage) (V)) across) the) oocyte) membrane) can) be) expressed)by)the)following)equation:)) I)(V))=)PO)(V))·)N)·)i)(V))) where,)N)is)the)total)number)of)activated)channels,)i)is)the)single)channel)current))(i.e.,)amplitude)of)the) current)flowing)through)the)single)channel),)and)PO)is)the)open)probability)of)the)channel.) ) Since) both) the) number) of) channels) (N)) and) the) singleCchannel) current) (i)) remain) unchanged)during)the)constant)voltage)at)the)representative)current)traces)recordings)(Fig.)3.9),) the)open)probability)(PO))can)be)directly)attributed)from)the)tail)streams)(Itail))obtained)at)−120) mV:) I)(V))=)PO)(V)) )The)experimental)data)points)were)fitted)with)a)single)Boltzmann)equation)(Hoth)et!al.,) 1997).) The) currents) were) normalized) to) the) saturation) value) of) the) calculated) Boltzmann) distribution.)The)halfCmaximal)activation)potential)(V½))was)obtained)from)the)curve)obtained) after)applying)the)Boltzmann)equation.) The)reversal)potentials)(Vrev))used)for)the)calculation)of)the)relative)permeability)were) recorded)in)the)currentCclamp)mode,!in)which)the)current)across)the)membrane)is)clamped)to) zero,!to)avoid)loading)of)oocytes)with)NO3−)and)to)keep)the)cytosolic)anion)composition)constant) for)correct)reversal)potential)determination)in)response)to)either)nitrate)concentration)changes) in)the)external)medium)or)during)anion)selectivity)measurements.)For)determination)of)Vrev)for) the)respective)anion,)oocytes)were)preincubated)in)50)mM)NO3−)to)gain)full)activity)of)SLAH3.) The)permeability)relative)to)nitrate)for)gluconate)and)chloride)of)the)expressed)channels)were) based)on)measurements)of)current)reversal)potential)in)the)presence)of)different)solutions,)each) containing) only) one) anion) species.) The) GoldmanCHodgkinCKatz!equation) (Goldman,) 1943;) Hodgkin) and) Katz) 1949)) describes) the) dependence) of) the) membrane) potential) on) all) the) different)permeable)ions:! ) where)Vm!is)the)membrane)potential,)P!!is)the)permeability)coefficient)of)the)individual)ion,)[x+],[y−])and) [z−]!are) the) concentrations) of) the) individual) ions) outside) (o)) or) inside) (i)) the) cell,) F! is) the) Faraday’s) constant,)R!is)the)gas)constant,)and)T!is)the)absolute)temperature.)) ) Considering)that)channels)were)examined)in)the)presence)of)a)single)permeating)anion) so) the) oocyte) membrane) was)permeable) only) for) this)species,) the) equation) above) can) be) simplified)with)the)Nerst)equation)for)monovalent)anions)as:) ) Results' ' ' 96' AtSLAH3'(dIf)'native'promoter'regions.'(a)'AtSLAH1'is'expressed'in'the'root,'but'no'expression'could'be' detected'at'the'apical'meristems.'(b)'The'expression'of'AtSLAH1'within'the'root'is'located'at'the'vascular' cylinder.'(c)'AtSLAH1'is'not'expressed'in'emerging'secondary'roots.''(d)'Similarly'to'AtSLAH1,'AtSLAH3(is' expressed' in' the' root,' but' not' in' the' apical' meristem.' (e)'AtSLAH3'is' also' expressed' in' the' root' vasculature.'(f)'AtSLAH3'is'not'expressed'in'emerging'secondary'roots.'Scale'bars,'200'μm'(a,'d),'50'μm'(b,' c,'e,'f).' ' For'localization'of'AtSLAH3'expression,'two'independent'transgenic'lines'of'A.(thaliana( expressing'the'GUS'gene'under'the'regulation'of'the'AtSLAH3(promoter'were'obtained'from'Dr.' Negi’s'laboratory.'Contrary'to'the'previously'published'results,'AtSLAH3'expression'was'located' in' the' root' vasculature'(Fig.' 4.1' dIf),' exhibiting' an' expression' pattern' very' similar' to' that' of' AtSLAH1'(Fig.'4.1'aIc).'Gene'expression'of'both'AtSLAH1(and'AtSLAH3(genes'was'absent'from' apical'meristems'(Fig.'4.1'a,'d)'and'emerging'lateral'root'primordia'(Fig.'4.1'c,'f).' Confocal'images'of'GFP'fluorescence'showed'that'AtSLAH1(expression'was'associated'to' cells'adjacent'to'xylem'vessels'(Fig.'4.2'aIc),'an'expression'pattern'similar'to'that'exhibited'by' the'AtSLAH3'gene'according'to'histochemical'GUS'activity'obtained'in'longitudinal'root'sections' (Fig.' 4.2' e).'A' closer' inspection' using' cross' sections' of' GUSIexpressing' roots'(section' 3.9.2)' identified'the'xylemIpole'pericycle'as'the'specific'cell'type'where'AtSLAH1'and'AtSLAH3'were' coIexpressed'in'the'root'(Fig.'4.2'd,'f).' ' Figure!4.2.!AtSLAH1(and!AtSLAH3(are!co>expressed!in!the!xylem!pole!pericycle.!(a>c)!Localization'in' xylemIassociated'cells'of'green'fluorescent'protein'(GFP)'activity'in'roots'of'transgenic'lines'harbouring' the' construction' PSLAH1IGFP::GUS.' (d)'Localization' in' xylemIpole' pericycle' cells' of' GUS' activity' in' cross' sections'of'the'root'of'transgenic'lines'harbouring'the'construction'of'PSLAH1IGFP::GUS.'(e)'Localization'in' xylemIassociated'cells'of'GUS'activity'in'longitudinal'sections'of'the'root'of'transgenic'lines'harbouring' the'construction'PSLAH3IGUS.'(f)'Localization'in'xylemIpole'pericycle'cells'of'GUS'activity'in'cross'sections' of'the'root'of'transgenic'lines'harbouring'the'construction'PSLAH3IGUS.'E:'epidermis;'C:'cortex;'asterisk:' endodermis;'red'spot:'protoxylem;'blue'spot:'metaxylem.'Scale'bars,'100'μm'(a),'50'μm'(b),'20'μm(c,'e),' 10'μm'(d,'f).' Results' ' ' 97' ' CoIexpression'of'AtSLAH1(and(AtSLAH3(genes'in'xylemIpole'pericycle'cells'suggests'that' both'SItype'anion'channels'might'be'involved'in'the'regulation'of'NO3−'and/or'Cl−'secretion'into' root'xylem'vessels.' ' 4.1.1.2.! Identification! of! AtSLAH1! T>DNA! knockout! and! slah1> complemented!lines! Arabidopsis( thaliana'seeds' from' the' slah171'(SALK_039811)' and' slah172' (INRA_FLAG_329G06)'lines'were'obtained'from'the'Nottingham'Arabidopsis'Stock'Centre'and' the' Institut' JeanIPierre'Bourgin,'respectively'(section'3.1.2.1).'The'slah171'and' slah172(TIDNA' insertions' were' generated' in' the' Columbia' (Col)' and' the' Wassilewskija' (Ws)' backgrounds,' respectively.'Following'the'extraction'of'DNA'from'leaves'of'segregating'plants'(section'3.2.1),' Arabidopsis( lines,' homozygous' and' azygous' for' the' TIDNA' insertions,' were' screened' by' PCR' (section' 3.2.4.1)' using' primers' suitable' for' the' left' border' of' the' TIDNA' and' SLAH1Ispecific' primers'flanking'the'position'of'the'TIDNA'inserts'(Fig.'4.3).' ' Figure! 4.3.! Identification! and! complementation! of! the! slah1!knockout! line.! (a)'Gene'structure'of' AtSLAH1'and' localization' of' the' TIDNA' insertions' in' the' slah171( (SALK_039811)' and' slah172' (INRA_FLAG_329G06)'transgenic'lines.'Closed'boxes'represent'exons'(E)'and'solid'lines'represent'introns' (I).'PCR'amplification'primers'used'for'genotyping'and'retroItranscription'of'total'RNA'are'indicated'with' arrows.'(b)'AtSLAH1'transcript'integrity'determined'by'RTIPCR'analysis'of'total'RNA'isolated'from'roots' of:' wildItype' plants' (WS,' ColI0),' homozygous' KO' mutant' lines' (slah171'and' slah172),' an' azygous' line' obtained'from'segregation'of'a' plant'heterozygous'for'the'TIDNA'insertion'of'the'slah172(line'(SLAH1I 2.72),' and' slah172(complemented' plants' (P1I12,' P1I31).' The' housekeeping' translation' initiation' factor' eIF4A1'expression'level'was'used'as'RNAI'or'cDNAI'loading'control.'' ' Results' ' ' 98' To'complement'the'slah172(KO'mutant'line,'plants'homozygous'for'the'TIDNA'insertion' were'transformed'(section'3.6)'with'a'construct'containing'the'whole'AtSLAH1'gene,'including' its'own'promoter'region.'The'segregating'progeny'was'screened'by'PCR'as'explained'above.' The' mRNA' integrity' of' genotyped' plant' lines,' homozygous' for' the' TIDNA' insertion,' slah171( (SALK_039811),' slah172( (INRA_FLAG_329G06),'together' with' two' independent' slah1/SLAH1'complemented' lines,' was' determined' through' its' retrotranscription' to' cDNA' (section' 3.2.2)' and' amplification'by' Reverse' Transcriptase' PCR' (RTIPCR)'(section' 3.2.4.2).' In' addition'to'the'homozygous'plants,'an'azygous'line'(SLAH1I2.72)'and'wildItype'lines'(WS,'ColI 0)'were'also'screened.' The'TIDNA'insertion'in'the'slah171(line'mapped'into'the'promoter'region'of'the'AtSLAH1( gene'(Fig.'4.3'a),'meanwhile'in'the'slah172'line'the'TIDNA'insertion'mapped'into'the'ExonI1'(E1;' Fig.' 4.3'a).' RTIPCR' analysis' of' total' RNA' isolated' from' roots' showed' that' the' slah171( line' maintained' the' ability' to' synthesize' the'native'AtSLAH1'transcript'(Fig.' 4.3'b),' whereas'the( AtSLAH1(transcript'was'disrupted'in'the'slah172'line'(Fig.'4.3'b).'' Since' no' additional' knockout' lines' were' obtained,' the' slah172'line' was'complemented' with' the' AtSLAH1( gene' as' previously' explained' (section' 3.4.2).' RTIPCR' analysis' of' he' slah1/SLAH1'complemented' lines' P1I12' and' P1I31' revealed' that' both' complemented' lines' synthesized'the'AtSLAH1'transcript'(Fig.'4.3'b).' For'further'studies'of'the'slah172(mutant'lines'phenotype,'the'segregating'azygous'line' SLAH1I2.72'lacking'the'TIDNA'insertion,'thereafter'named'WT,'was'used'as'a'control.' ' 4.1.1.3.!Loss!of(AtSLAH1(function!impairs!shoot!growth!! Given'the'localization'of'AtSLAH1(gene'expression'in'cells'facing'xylem'vessels,'one'could' predict'that'the'SIType'anion'channel'feeds'the'xylem'sap'ascending' from' root'to'shoot'with' NO3−'and/or'Cl−'anions.'To'test'this'assumption,'the'AtSLAH1'gene'KO'mutant'line,'slah172,'was' inspected'under'two'chloride'feeding'regimes'(70'µM'and'5'mM'Cl−)'in'plants'growing'under' hydroponic'conditions'(section'3.1.2.2.3)'and'in'pots'(section'3.1.2.2.2)'under'short'photoperiod' conditions.' The' development' of' the' slah172'KO' line' was' compared' with' its' WT' line' by' taking' morphological'measurements'every'3'd'approximately'(section'3.8.1).' No' differences' in' the' root' length' of' slah172' mutant' and' WT' lines' grown'in'the' low' chloride'nutrient'solution'(70'µM'Cl−)'were'observed'(Fig.'4.4'a).'However,'in'the'high'chloride' solution'(5'mM'Cl−)'the'root'of'slah172(KO'plants'was'significantly'shorter'than'the'root'of'WT' plants'since' 44' days' after' sowing' (Fig.' 4.4'b).' When' the' root' length' of' both' genotypes' was' represented'according'to'the'Cl−'nutrition'regime,'no'differences'were'observed'between'low' chloride'(70'µM'Cl−)'and'high'chloride'(5'mM'Cl−)'treatments'in'the'WT'line'(Fig.'4.4'd),'and'only' small'differences'could'be'appreciated'in'the'slah172(mutant'line'(Fig.'4.4'c).'( At' the' end' of' the' phenotypic' characterization,' plant' organs' were' harvested.' No' differences' between' slah172'and' WT' lines' were' observed' in' either' root' biomass' (data' not' shown)'or'root'Cl−'content'(Fig.'4.4'e,'f).' Results' ' ' 99' ' Figure!4.4.! Phenotypic! differences! in! the! root! of! slah1-2(mutant!and! its! wild>type! (WT)! azygous! line! grown! hydroponically! under! two! different! chloride! treatments.' Plants' were' grown' hydroponically'in'nutrient'solutions'containing'either'low'(70'μM)'or'high'(5'mM)'chloride'(Cl−).'In'the' low' chloride'solution,'Cl−'salts'were'replaced'with'sulphate'and' phosphate'salts,'maintaining'the'same' balance' of' cations' in' comparison' to' the' 5' mM' chloride' solution.' (a)'Root'length' of' slah172( versus'WT' plants'grown'in'a'nutrient'solution'containing'low'chloride'(70'μM'Cl−).'(b)!Root'length'of'slah172(vs.'WT' plants'grown'in'a'nutrient'solution'containing'high'chloride'(5'mM'Cl−).'(c)'Root'length'of'slah172(plants' alternatively' grown' under' low' (70' μM' Cl−)' or' high' (5' mM' Cl−)' chloride.' (d)'Root'length' of' WT(plants' alternatively'grown'under'low'(70'μM)'or'high'(5'mM)'chloride.'(e)'Root'Cl−'content'in'slah172(and'WT' plants'grown'in'a'nutrient'solution'containing'low'chloride'(70'μM'Cl−).'(f)'Root'Cl−'content'in'slah172(and' WT' plants' grown' in' a' nutrient' solution' with' containing' high' chloride' (5' mM' Cl−).' Asterisks' indicate' statistically' significant' differences' (oneIway' ANOVA,' *P'<' 0.05,' n≥' 5).' Different' letters' indicate' a' statistically'significant'difference'in'slah172'and'WT'plants'development'over'time'(linear'mixed'models,'P' <'0.05).' ' No' significant' differences' were' observed' in' the' rosette' diameter' of' slah172( and' WT' plants'grown'hydroponically'in'either'low'chloride'(70'µM'Cl−;'Fig.'4.5'a)'or'high'chloride'(5'mM' Cl−;'Fig.'4.5'b)'nutrient'solutions.'However,'the'growth'of'the'rosettes'in' both'genotypes'was' higher'with'high'chloride'(5'mM'Cl−;'Fig.'4.5'c)'compared'to'the'low'chloride'treatment'(70'µM' Cl−;'Fig.'4.5'd),'indicating'that'macronutrient'content'of'Cl−'stimulates'leaf'growth.' The'slah172'mutant'exhibited'significantly'fewer'number'of'rosette'leaves'than'the'WT' under'the'low'Cl−'treatment'(Fig.'4.5'e).'However'this'difference'was'abolished'when'the'mutant' line'was'grown'in'5'mM'Cl−'(Fig.'4.5'f),'indicating'that'the'slah172'mutant'line'was'specifically' more'sensitive' to' low' Cl−'nutrition'than' the'WT'line.'This'finding'was'more'clearly'observed' when' the' number' of' leaves' in' highI' and' lowICl−'solutions' was'compared' separately' in' each' genotype.'Whereas'the'number'of'leaves'is'distinctly'reduced'by'the'low'chloride'treatment'(70' µM' Cl−)' in' the' slah172( mutant' line' (Fig.' 4.5' g),' no' differences' between' the' two' different' chloridefeeding'regimes'in'the'WT'line'were'observed'(Fig.'4.5'h).'' Similarly,' significant' differences' in' rosette' biomass' linked' to' Cl−'nutrition' were' specifically'observed'in'the'slah172(mutant'line'(Fig.'4.5'k),'but'not'observed'in'the'WT'line'(Fig.' 4.5'l),'suggesting'again'higher'sensitivity'to'Cl−'nutrition'in'the'slah172(mutant'line,'possibly'as'a' consequence' of'the'reduced'Cl−'content'close' to'the'nutrient' deficiency'threshold'(0.2'mg'g−1' DW;'Xu'et(al.,'2000).'! Results' ' ' 100' ' Figure!4.5.(Vegetative!growth!phenotype!of!the!slah1-2!mutant!and!its!wild>type!(WT)!azygous!line! grown! hydroponically! under! two! different! chloride! treatments.' Plants' grown' hydroponically' in' a' solution' containing' either' 70' μM' or' 5' mM' chloride' (Cl−).' In' the' low' chloride' solution,' Cl−'salts' were' replaced'with'sulphate'and'phosphate'salts,'maintaining'the'same'balance'of'cations'in'comparison'to'the' 5'mM'chloride'solution.'(a)'Rosette'diameter'of'slah172(versus'WT'plants'watered'with'a'nutrient'solution' containing'low'chloride'(70'μM'Cl−).'(b)!Rosette'diameter'of'slah172(vs.'WT'plants'watered'with'a'nutrient' solution'with'high'chloride'(5'mM'Cl−).'(c)'Rosette'diameter'of'slah172(plants'alternatively'watered'with' low'(70'μM'Cl−)'or'high'(5'mM'Cl−)'chloride.'(d)'Rosette'diameter'of'WT(plants'alternatively'watered'with' low'(70'μM)'or'high'(5'mM)'chloride.'(e)'Number'of'rosette'leaves'of'slah172(versus'WT'plants'watered' with'a'nutrient'solution'containing'low'chloride'(70'μM'Cl−).'(f)'Number'of'rosette'leaves'of'slah172(vs.'WT' plants'watered'with'a'nutrient'solution'containing'high'chloride'(5'mM'Cl−).'(g)!Number'of'rosette'leaves' of'slah172'plants'alternatively'watered'with'low'(70'μM'Cl−)'or'high'(5'mM'Cl−)'chloride.'(h)'Number'of' rosette'leaves'of'WT'plants'alternatively'watered'with'low'(70'μM)'or'high'(5'mM)'chloride.'(i)'Rosette' fresh'weight'(FW)'of'slah172(versus'WT'plants'grown'in'a'nutrient'solution'containing'low'chloride'(70'μM' Cl−).'(j)'Rosette'fresh'weight'of'slah172(vs.'WT'plants'grown'in'a'nutrient'solution'containing'high'chloride' (5'mM'Cl−).'(k)!Rosette'fresh'weight'of'slah172'plants'alternatively'grown'under'low'(70'μM'Cl−)'or'high' (5'mM'Cl−)'chloride.'(l)'Rosette'fresh'weight'of'WT'plants'alternatively'grown'under'low'(70'μM)'or'high' (5'mM)'chloride.'Asterisks'indicate'statistically'significant'differences'(oneIway'ANOVA,'*P'<'0.05,'n≥'5).' Different'letters'indicate'a'statistically'significant'difference'in'slah172'and'WT'plants'development'over' time'(linear'mixed'models,'P'<'0.05).' ' No' differences' in' the' length' of' the' inflorescence' were' observed' when' slah172'mutant' plants'were'compared'with'WT'plants'grown'hydroponically'in'either'low'(70'µM'Cl−;'Fig.'4.6'a)' or'high'(5'mM'Cl−;'Fig.'4.6'b)'chloride'treatments.'In'addition,'the'concentration'of'chloride'in' the'nutrient'solution'did'not'affect'the'length'of'the'inflorescence'in'either'slah172'(Fig.'4.6'c)'or' WT'(Fig.'4.6'd)'lines.'However,'the'number'of'siliques'was'stimulated'by'high'Cl−'addition'in'the' WT'line,'but'not'in'the'slah172'mutant'line''(Fig.'4.6'eIf).'This'finding'was'clearly'visible'when' the'number'of'siliques'in'highI'and'lowICl−'solutions'is'compared'separately'in'each'genotype' (Fig.' 4.6' gIh),' suggesting' that' SLAH1' could' be' involved' in' flower' fertilization' of' seed' developmental' processes.' Moreover,' the' significantly' higher' biomass' of' inflorescences' from' plants' treated' with' high' chloride' (5' mM' Cl−)' compared' to' low' chloride' (70' µM' Cl−)' in' both' genotypes'(Fig.'4.6'kIl)'point'to'a'role'of'Cl−'nutrition'in'plant'reproductive'development'and' fertilization'(as'it'is'proposed'in'section'4.1.2.1).' Results' ' ' 101' ' ' Figure!4.6.! Differential! shoot! and! inflorescence! stem! growth! of! mutant! (slah1-2)! and! wild>type! (WT)! plants! grown! hydroponically! under! two! different! chloride!feeding! regimens.!Plants' were' grown'hydroponically'with'nutrient'solutions'containing'70'µM'or'5'mM'chloride'(Cl−).'In'the'70'µM'Cl−' solution,' Cl−'salts' were' replaced' with' sulphate' and' phosphate' salts' maintaining' the' same' balance' of' cations'of'the'5'mM'Cl−'solution.'(a)'Main'inflorescence'stem'length'of'slah172(versus'WT'plants'grown'in'a' nutrient'solution'containing'low'chloride'(70'μM'Cl−).'(b)!Main'inflorescence'stem'length'of'slah172(vs.'WT' plants' grown' in' a' nutrient' solution' containing' high' chloride' (5' mM' Cl−).' (c)'Main' inflorescence'stem' length'of'slah172(plants'alternatively'grown'under'low'(70'μM'Cl−)'or'high'(5'mM'Cl−)'chloride.'(d)'Main' inflorescence'stem'length'of'WT(plants'alternatively'grown'under'low'(70'μM)'or'high'(5'mM)'chloride.' (e)'Number'of'siliques'produced'by'slah172(versus'WT'plants'grown'in'a'nutrient'solution'containing'low' chloride' (70' μM' Cl−).' (f)'Number' of' siliques' produced' by(slah172( vs.'WT' plants' grown' in' a' nutrient' solution' containing' high' chloride' (5' mM' Cl−).' (g)'Number' of' siliques' produced' by(slah172( plants' alternatively'grown'under'low'(70'μM'Cl−)'or'high'(5'mM'Cl−)'chloride.'(h)'Number'of'siliques'produced' by'WT(plants'alternatively'grown'under'low'(70'μM)'or'high'(5'mM)'chloride.'(i)'Inflorescence'stems'dry' weight'(DW)'of'slah172(versus'WT'plants'grown'in'a'nutrient'solution'containing'low'chloride'(70'μM'Cl−).' (j)'Inflorescence'stems'dry'weight'of(slah172(vs.'WT'plants'grown'in'a'nutrient'solution'containing'high' chloride'(5'mM'Cl−).'(k)'Inflorescence'stems'dry'weight'of(slah172(plants'alternatively'grown'under'low' (70' μM' Cl−)' or' high' (5' mM' Cl−)' chloride.' (l)'Inflorescence' stems' dry' weight' of(WT(plants'alternatively' grown'under'low'(70'μM)'or'high'(5'mM)'chloride.'Asterisks'indicate'statistically'significant'differences' (oneIway'ANOVA,'*P'<'0.05,'n≥'5).'Different'letters'indicate'a'statistically'significant'difference'in'slah172' and'WT'plants'development'over'time'(linear'mixed'models,'P'<'0.05).' ' Altogether' the' results' show' a' clear' interaction' between' SLAH1' function' and' chloride' nutrition' that' impacts' the' development' of' shoot' organs,' indicating' that' this' anion' channel' is' involved'in'chloride'nutrition'in'plants.' When' monitoring' the' phenotype' of' the'Arabidopsis'mutant' and' the' WT' lines' growing' with'5'mM'Cl−'in'pots,'the'slah172'mutant'exhibited'significantly'smaller'rosettes'(Fig.'4.7'a)'and' significantly'lower'number'of'leaves'than'the'WT!(Fig.'4.7'b).'However,'the'rosette'development' of'mutant'plants'reached'the'WT'rosette'development'(Fig.'4.7'a),'indicating'that'the'mutant'line' phenotype' consisted' on' a' delayed' rather' than' a' reduced' growth' compared' to' the' WT,' which' began'the'senescence'stage'earlier.'Thus'it'is'not'surprising'that'slah172'KO'mutant'plants'had' Results' ' ' 102' significantly'higher'rosette'fresh'and'dry'weight'(Fig.'4.7'cId)'relative'to'WT'plants'when'plants' were'harvested'at'the'end'of'the'development'cycle.' ' Figure!4.7.'Differential!rosette!growth!of(slah1-2(KO!line!and!its!wild>type!(WT)!azygous!line!grown! in!solid! substrate!treated!with!5!mM!chloride.!(a)!Rosette'diameter'of'slah172(versus'WT'plants.'(b)! Number'of'rosette'leaves'of'slah172(vs.'WT'plants.!(c)! Rosette'fresh'weight'(FW)'of'slah172(versus'WT' plants.!(d)!Rosette'dry'weight'(DW)'of'slah172(versus'WT'plants.'Asterisks'indicate'statistically'significant' differences'(oneIway'ANOVA,'*P'<'0.05,'n≥'10).' ' Inflorescence'development'of'the'slah172'mutant'line'grown'in'pots'with'5'mM'Cl−'was' lower'at'the'beginning'of'the'reproductive'phase'in'relation'with'the'development'of'WT'plants' (Fig.'4.8'a),'although'it'reached'the'WT'stem'development'(75'days'after'sowing)'and'became' even'significantly'higher,'similarly'to'the'rosette'development'observed'(Fig.'4.7'a).'However,'no' differences'in'the'production'of'siliques'between'both'lines'were'observed'(Fig.'4.8'b).' ' Figure!4.8.'Differential!main!inflorescence!stem!development!and! siliques!production!of! slah1-2( mutant!and!wild>type!(WT)!plants!grown!in!solid!substrate!treated!with!5!mM!chloride.!(a)!Main' inflorescence'stem'length'of'slah172(versus'WT'plants.'(b)'Number'of'siliques'produced'by(slah172(vs.'WT' plants.'Asterisks'indicate'statistically'significant'differences'(oneIway'ANOVA,'*P'<'0.05,'n≥'10).''' ' Results' ' ' 103' After' plant' harvesting''(93'days' after' sowing),' chloride' content' in' the' shoot' was' determined' (section' 3.8.4.1).' The' chloride' contents' of'slah172'plants' were' significantly' lower' than'that'of'WT'plants'grown'in'pots'treated'with'5'mM'Cl−'(Fig.'4.9).'' ' Figure!4.9.!Shoot!chloride!content!was!significantly!lower!in!slah1-2(plants.!Plants'grown'in'pots'and' watered'with'a'nutrient'solution'containing'5'mM'chloride'(Cl−)'were'harvested'at'lateIsenescence'stage.! slah172'KO'plants'had'significantly'lower'chloride'content'in'shoots!than'WT'plants.' Asterisks'indicate' statistically'significant'differences'(oneIway'ANOVA,'*P'<'0.05,'n≥'10).! ! To' find' out' whether' and' how' the' loss' of'function' of' the'AtSLAH1'gene' affects' the' Cl−' content'of'Arabidopsis'plants,'slah172'mutant'and'WT'lines'were'grown'in'pots'under'the'two' feeding'regimes,'70'µM'and'5'mM'Cl−.'' Plants' were' harvested' at' lateIsenescence' stage' (130'days' after' sowing)' and' shoot' chloride'content'was'determined'(section'3.8.4.1).'It'could'be'observed'that'slah172'KO'mutant' plants'accumulated'significantly'less'Cl−'in'the'shoot'than'the'WT'plants,'independent'from'the' chloride'treatment'(Fig.'4.10'aIb).' ' Figure!4.10.'Chloride! content! in! shoots! of! slah1-2! T>DNA! knockout! and! wild>type! (WT)! plants' grown!in!solid!substrate!treated!with!two!different!chloride!feeding!regimens.!Plants'were'watered' with'nutrient'solutions'containing'70'µM'or'5'mM'chloride'(Cl−).'In'the'70'µM'Cl−'solution,'Cl−'salts'were' replaced' with' sulphate' and' phosphate' salts' maintaining' the' same' balance' of' cations' of' the' 5' mM' Cl−' solution.!(a)!Chloride'content'of'slah172(and'WT'plants'watered'with'a'nutrient'solution'containing'70'µM' Cl−.'(b)'Chloride'content'of'slah172(and'WT'plants'watered'with'a'nutrient'solution'containing'5'mM'Cl−.' Asterisks'indicate'statistically'significant'differences'(oneIway'ANOVA,'*P'<'0.05,'**P'<'0.01,'n≥'6).! ! Altogether'the'results'indicate'that'AtSLAH1'regulates'Cl−'homeostasis'at'the'wholeIplant' level,'probably'through'the'regulation'of'rootItoIshoot'Cl−'transport.' ' ' Results' ' ' 104' 4.1.1.4.! SLAH1,! together! with! SLAH3,! contributes! to! chloride! feeding!of!root!xylem!! The' lower' shoot' Cl−'content' of' the' AtSLAH17mutant' line' suggested' that' SLAH1' was' engaged'in'Cl−'homeostasis.'To'reach'the'shoot,'nutrients'that'enter'the'root'have'to'be'loaded' into' the' root' xylem' vessels.' Since'AtSLAH1'and' AtSLAH3'expression' was' localized' in' the' pericycle'cells'facing'the'xylem'vessels'in'the'root'(Fig.'4.2),'the'pericycle'cells'should'contribute' to'the'anion'composition'of'the'ascending'xylem'sap.' To' inspect' whether' AtSLAH1'and' its' coIexpressed' homolog' AtSLAH3'are' involved'in' anion' transport' from' xylem' parenchyma' cells' into' xylem' vessels,' genotyped' KO' plant' lines' (section' 3.2.4.1),'homozygous'for'the'TIDNA'insertion,' together'with'the'respective' wildItype' lines'were'grown'in'pots'under'long'photoperiod'conditions'and'watered'with'Basal'Solution' (BS;'section'3.1.2.2.2)'and'used'for'xylem'sap'collection'assays'(section'3.8.2).'Afterwards,'Cl−' and'NO3−'contents'in'the'xylem'sap'were'colorimetrically'quantified'(section'3.8.4.2).' For'quantification'of'rootItoIshoot'anion'translocation'rates,'Cl−'and'NO3−'concentration' was'measured'in'the'root'sap'exudates'of'WT'and'slah172'mutant'line'(Fig.'4.11).'To'collect'sap' ascending' from' the' root' xylem,' the' shoot' had' to' be' removed.' After' trimming' the' shoot,' sap' droplets'were'collected'at'different'times'(1'h,'2'h,'3'h'and'5'h'after'starting'the'assay).'The'Cl−' and'NO3−'concentration'obtained'from'this'samples'was'plotted'at'each'collection'point.'It'could' be' observed' that' Cl−'concentration' in' the' xylem' sap' of' both'lines'decreased' gradually' after' trimming'the' shoot,'meanwhile'NO3−'concentration'increased'within'the'first'2−3'h,'and'then' decreased' (Fig.' 4.11).' The' gradually' drop' of' Cl−'concentration' in' the' xylem' sap' observed' in' slah172( plants' ' (Fig.'4.11)' could' be' due' to' the' negative' regulation' of' the' AtSLAH1( gene' expression'by'stress.'It'could'be'also'observed'that'xylem'sap'Cl−'concentration'of'slah172(plants' was'significantly'lower'that'that'of'WT'plants'(Fig.'4.11).' ' Figure!4.11.!Anion!concentration!in!xylem!sap!of!slah1-2(plants!relative!to!wild>type!(WT)!plants!at! different! collecting! times.! Plants' were' grown' hydroponically' with' Basal' Solution' (BS).' Evolution' of' chloride'(Cl−)'and'nitrate'(NO3−)'content'in'xylem'sap'after'shoot'trimming'represented'as'percentage'of' the'WT'in'the'first'collection'point.'Asterisks'indicate'statistically'significant'differences'(Student’s't'test,' *P'<'0.05,'n'='12'experiments;'mean'±'SE).''! ! Therefore,'for'minimizing'artifactual'responses'inherent'to'the'experimental'technique' (e.g.'the'response'to'mechanical'damage'caused'by'cutting'the'rosette),'only'the'xylem'sap'anion' content'of'the'first'collecting'time'was'represented'in'subsequent'assays.' Results' ' ' 105' For'quantification'of'rootItoIshoot'anion'translocation'rates,'Cl−'and'NO3−'concentration' was'measured'in'the'root'sap'exudates'of'slah172'and'slah371'mutant'lines'and'their'respective' WT'lines'(section'3.8.4.2).'Only'assays'where'the'sap'exudation'rate'of'mutant'and'WT'plants' were'comparable,'indicating'similar'root'biomass,'were'used'(Fig.'4.12'a,'f).' Xylem'sap'Cl−'concentration'was'significantly'lower'in'both'slah172(and'slah371(mutant' lines'in'relation'to'their'respective'WT'lines'(Fig.'4.12'b,'g),'meanwhile'NO3−'concentration'of'the' mutant'lines'remained'similar'to'WT'values'(Fig.'4.12'd,'i).'' RootItoIshoot' translocation' rates' were' obtained'through' multiplying' their' concentrations'in'the'exudates'(Fig.'4.12'b,'d,'g,'i)'by'the'sap'exudation'rates'of'each'plant'(Fig.' 4.12'a,'f)' and' then' compared' with' their' respective' WT' lines.' Reduction' of' Cl−'content' in' the' xylem'sap'of'plants'lacking'functional'copies'of'AtSLAH1(or'AtSLAH3'genes'was'a'consequence'of' a'lower'rootItoIshoot'translocation'rate'of'Cl−'(Fig.'4.12'c,'h),'meanwhile'the'NO3−'translocation' rate'did'not'significantly'change'(Fig.'4.12'e,'j).'' ' Figure!4.12.!SLAH1!and!SLAH3!are!involved!in!chloride!translocation!into!the!xylem.'(a)'Exudation' rate'of'wildItype'(WT)'and'slah172(plants'used'to'quantify'anion'translocation'into'the'xylem.'(b)'Chloride' (Cl−)'concentration'in'the'xylem'sap'of'WT'and'slah172(plants.'(c)'RootItoIshoot'translocation'rate'of'Cl−'in' WT'and'slah172(plants.'(d)'Xylem' sap' nitrate'(NO3−)' concentration' of' WT' and' slah172'plants.'(e)'NO3−' rootItoIshoot' translocation' rate' of' WT' and' slah172'plants.'(f)'Exudation'rate'of' WT'and'slah371(plants' used'for'Cl−'and'NO3−'quantification'in'the'xylem'sap.'(g)'Xylem'sap'Cl−'concentration'of'WT'and'slah371( plants.' (h)'Cl−'rootItoIshoot' translocation' rate' of' WT' and' slah371'plants.' (i)'NO3−'concentration' in' the' xylem'sap'of'WT'and'slah371(plants.'(j)'RootItoIshoot'translocation'rate'of'NO3−'in'WT'and'slah371(plants.! The' translocation' rates' were' obtained' by' multiplying' their' concentrations' in' the' exudates' by' the' sap' exudation'rates.!Asterisks'indicate'significant'differences'between'WT'and'either'the'slah172'mutant'line' (Student’s't'test,'*P'<'0.05,'n≥'12,'n'='12'experiments;'mean'±'SE)'or'the'slah371'mutant'line'(oneIway' ANOVA,'*P'<'0.05,'**P'<'0.01,'n≥'6,'n'='2'experiments;'mean'±'SE).! ! Given'the'absence'of'a'second'independent'mutant'line,'to'confirm'that'the'reduction'of' Cl−'content'in'the'xylem'sap'of'slah172'plants'was'specifically'due'to'the'lack'of'AtSLAH1(gene' function,' xylem' sap' of'the' complemented' lines,' P1I12' and' P1I31,' expressing' the' wildItype' AtSLAH1(gene'under'the'control'of'its'native'promoter'(section'3.4.2),'was'inspected.'With'the' same'purpose,'another'independent'AtSLAH3Imutant'line,'slah374,'was'used'to'confirm'that'the' reduction' of' Cl−'content' in' the' xylem' sap' was' specifically' due' to' the' lack' of' AtSLAH3( gene' function'in'the'SLAH37mutant'background.' Results' ' ' 112' ' 4.1.1.6.!SLAH1!is!a!silent!anion!channel! Kinetic'and'regulatory'properties'of'the'SLAH1'and'SLAH3'anion'channels'were'studied' through' functional' expression' of' the' corresponding' genes' in' the' heterologous' expression' system(of'Xenopus'oocytes'(section'3.10)'in'combination'with'the'twoIelectrode'voltage'clamp' (TEVC)'technique'(section'3.12).' In'previous'studies'the'SItype'anion'channel'function'of'SLAC1,'SLAH2,'and'SLAH3'was' studied'in'Xenopus'oocytes'after'expressing'them'alone'or'with'activating'kinases'from'different' (Ca2+Idependent'and'independent)'families'(Brandt'et(al.,'2012;'Geiger'et(al.,'2009b,'2010;'Lee' et(al.,'2009;'Maierhofer'et(al.,'2014a,b;'Scherzer'et(al.,'2012).'' To' find' interacting' kinases' of' SLAH1' and' to' study' its' regulatory' properties,' the' anion' channel'was'fused'to'the'CIterminal'half'of'the'yellow'fluorescent'protein'(YFP)'and'candidate' kinases'to'the'NITerminal'half'of'YFP.'Besides'the'kinases'that'are'known'to'activate'channels'of' the' SLAC/SLAH' family' (CPK3,' CPK6,' CPK21,' CPK23,' CIPK23,' SnRK2.6' (OST1);' Brandt' et( al.,' 2012;' Geiger' et( al.,' 2009b,' 2010;' Lee' et( al.,' 2009;' Maierhofer' et( al.,' 2014a,b;' Scherzer' et( al.,' 2012)'or'that'interact'with'them'(SnRK2.3,'SnRK2.8;'Geiger'et(al.,'2009),'several'members'of'the' same' kinase' families,' whose' expression' is' predicted' in' the' root' pericycle' (www.genevestigator.org),' were' tested.' Following' the' expression' in' Xenopus'oocytes,' bimolecular'fluorescence'complementation'(BiFC;'section'3.11)'analyses'revealed'pronounced' YFP' signals!with' the' Ca2+Idependent' kinases!CPK6' and' CPK21'(Fig.'4.18),' but' only' weak' fluorescence'with'CPK23'and'CIPK23/CBL1,'as'well'as'with'the'Ca2+Iindependent'SnRK2'kinases' SnRK2.2,'SnRK2.3,'SnRK2.6'(OST1),'SnRK2.7'and'SnRK2.8'(Fig.'4.18).'In'addition,'no'YFP'signal' was'obtained'with'CPK3'(Fig.'4.18).'The'fluorescence'obtained'suggested'a'physical'interaction' between'SLAH1'and'different'potential'regulatory'kinases.'' ' Figure!4.18.!Physical!interaction!of!SLAH1!with!members!of!different!kinase!families!revealed!by! BiFC!experiments.!The'SLAH1'anion'channel'was'fused'with'the'CIterminal'half'of'the'yellow'fluorescent' protein'(YFPCT)'and'candidate'kinases'to'the'NITerminal'half'of'YFP'(YFPNT)'previously'to'coIexpression' in'Xenopus'oocytes.'Representative'images'of'oneIquarter'of'Xenopus(oocytes'are'shown.'When'the'coI expressed'components'interact'between'them,'the'YFP'can'be'reconstituted,'emitting'fluorescence'given' its' excitation' with' an' appropriate' wavelength.' Ca2+Iindependent' truncated' kinases' of' CPK3' (CPK3ΔEF)' and'CPK21'(CPK21ΔEF)'were'used.' ! To' test' whether' these' kinases,' that' activate' other'SLAC/SLAH' SItype' anion' channels,' could'also' activate' SLAH1'in' Xenopus( oocytes,' the' channel' was' coIexpressed' with' SnRK2.2,' Results' ' ' 113' SnRK2.3,'SnRK2.6,'SnRK2.7,'SnRK2.8,'CIPK23/CBL1'or'with'one'of'the'Ca2+Idependent'protein' kinases'CPK3,'CPK6,'CPK21'or'CPK23.'To'avoid'differences'in'the'kinase'activity'due'to'different' Ca2+Idependencies' and' calcium' levels' in' Xenopus'oocyte' batches,' truncated' Ca2+Iindependent' mutants' of' CPK3' and' CPK21' were' used' (CPK3ΔEF,' CPK21ΔEF,' Geiger' et( al.,' 2010,' 2011;' Maierhofer'et(al.,'2014a,b;'Scherzer'et(al.,'2012).'However,'neither'SLAH1'expression'alone'or'in' combination' with'the' kinases' that' were' effective' with' SLAC1,' SLAH2' or' SLAH3' resulted' in' macroscopic'anion'currents'in'Xenopus'oocytes'(data'not'shown).'' SLAH3,' which' coIexpresses' with' SLAH1' in' root' pericycle'cells'facing' xylem' vessels,' requires'a'Ca2+Idependent'kinases'such'as'CPK21'to'conduct'NO3−'and'Cl−'anions,'but'only'when' gated' open' by' NO3−'at' the' extracellular' entrance' of' the' channel' pore' (Geiger' et( al.,' 2011;' Maierhofer'et(al.,'2014b).'Nevertheless,'oocytes'expressing'SLAH1'in'the'absence'or'presence'of' CPK21ΔEF'did'not'respond'to'extracellular'Cl−'nor'NO3−'(Fig.'4.19'aIb),'meanwhile'it'could'be' observed'that'the'kinase'activation'and' the'NO3−'in' the' external' medium' were' enough' to' get' SLAH3'channel'open'(Fig.'4.19'cId).' ' Figure!4.19.! SLAH1! remains! electrically! silent! after! co>expression! with! CPK21! in! chloride! and! nitrate!buffers.!(a)!WholeIoocyte'currents'of'Xenopus(oocytes'expressing'SLAH1'alone'or'together'with' CPK21ΔEF'in'the'presence'of'30'mM'chloride'(Cl−)'or'nitrate'(NO3−)'(representative'cells'are'shown).!(b)' Instantaneous'currents'(Iinst)'of'oocytes'coIexpressing'SLAH1'in'the'absence'or'presence'of'CPK21ΔEF'in' standard'buffers'containing'either'100'mM'Cl−'or'NO3−.'(c)!WholeIoocyte'currents'of'oocytes'expressing' SLAH3'alone'or'with'CPK21ΔEF'in'the'presence'of'30'mM'Cl−'or'NO3−'(representative'cells'are'shown).!(d)' Iinst'of'Xenopus(oocytes'coIexpressing'SLAH3'in'the'absence'or'presence'of'CPK21ΔEF'in'standard'buffers' containing'either'100'mM'Cl−'or'NO3−.'Ca2+Iindependent'truncated'kinase'of'CPK21'(CPK21ΔEF)'was'used' (n'≥'3;'mean'±'SE).!!' ' Chen' and' colleagues' (2010)' crystallized' the' bacterial' homologue' of' SLAC1' from' Haemophilus( influenza'(HiTehA)' and' found' a' central' conserved' phenylalanine' (Phe)' residue' (F450)'in'the'pore'region'of'SLAC1'linked'to'the'anion'gate.'Replacement'of'this'pore'residue'by' alanine'(Ala)'turned'AtSLAC1'into'a'constitutively'open'anion'channel,'even'in'the'absence'of'an' activating'kinase.'This'residue'is'conserved'in'the'entire'SLAC/SLAH'protein'family'(Chen'et(al.,' 2010;'Dreyer'et(al.,'2012)'and'the'mutation'of'this'Phe'in'the'Arabidopsis(SLAC1Ihomolog'SLAH2' by'replacing'this'residue'by'alanine'(F402A)'also'led'to'an'anion'conducting'state'(Maierhofer'et( al.,'2014b).'Furthermore'it'has'been'recently'shown'that'this'openIgate'mutation'shifts'kinaseI insensitive'Arabidopsis'SLAC1Ihomologues'from'alga'and'liverwort'(Lind'et(al.,'2015)'into'active' channels.' Results' ' ' 114' Based' on' multiple' alignments' of' amino' acid' sequences' (section' 3.2.9)' and' homology' models' of' AtSLAH1' and' AtSLAH3' to' the' crystal' structure' of' HiTehA' (Chen' et( al.,' 2010),' the' corresponding'Phe'residues'were'identified'at'the'transmembrane'(TM)'domain!9'(Fig.'4.20).' ' Figure!4.20.! Sequence! alignment! of! SLAC/SLAH! proteins! from! different! species.' Shading' levels' indicate' degree' of' conservation' (black,' denotes' very' high' conservation;' dark' grey,' mediumIhigh' conservation,' light' grey,'moderate' conservation;' and' white,' no' conservation).' Tm9,'Transmembrane' domain' 9.'Phe,' Phenyalanine.'At,' Arabidopsis( thaliana;' Cc,' Citrus( clementina;' Os,' Oryza( sativa;' Pp,' Physcomitrella(patens;'Sb,'Solanum(bicolor;'Vv:'Vitis(vinivera.' ' The'Phe'residues'were'mutated'to'Ala'in'SLAH1'(F307A)'and'SLAH3'(F517A)'to'obtain' deregulated' open' channels.'After' their' expression' in' Xenopus'oocytes' it' could' be' observed' through'TEVC'recordings'that'while'SLAH3'F517A'mutant'led'to'an'open'conducting'state,'the' SLAH1'F307A'anion'channel'remained'electrically'silent'(Fig.'4.21).'' ! Figure!4.21.!SLAH1!F307A!mutant!remains!as!a!silent!channel.!Instantaneous'currents'(Iinst)'recorded' at' −100' mV' of' Xenopus( oocytes' expressing' the' “gating' mutants”' SLAH1' F307A' or' SLAH3' F517A' in' comparison'to'WT'SLAH1'and'WT'SLAH3'±'CPK21ΔEF'in'100'mM'nitrate'buffer'(n'≥'4'experiments;'mean' ±'SE).'' Results' ' ' 115' 4.1.1.7.!SLAH1!and!SLAH3!form!heteromeric!anion!channels! Given'that'the'threeIdimensional' (3D)' homology' model' of' HiTehA' revealed' a' trimeric' structure' with' three' subunits,' each' forming' an' individual' pore' (section'1.5.1.2.3;' Chen' et( al.,' 2010),' the' physical' interaction' within' SLAH1' homomeric' channel' complexes,' as' well' as' in' SLAH3,'was'verified'through'BiFC'technique'after'coIexpressing'the'anion'channel'subunit'fused' to'the'CIterminal'half'of'the' YFP'(YFPCT)'or'to'the'NITerminal'half'of'YFP'(YFPNT)'in'Xenopus' oocytes.'A'strong'interaction'between'individual'SLAH1'subunits,'as'well'as'between'individual' SLAH3'subunits,'was'observed'(Fig.'4.22).'' ! Figure!4.22.! Physical! interaction! between! S>type! monomers! revealed! by! BiFC! experiments.! SLAC/SLAH' subunits' are' able' to' assemble,' leading' to' homomeric' channel' complexes.' The' CIterminal' (YFPCT)'or'NIterminal'(YFPNT)'half'of'YFP'was'fused'to'the'NIterminus'of'SLAH1'or'SLAH3.'Representative' images'of'oneIquarter'of'Xenopus(oocytes'are'shown.' ' Similar'to'the'situation'with'the'Shaker'K+'channel'subunits'AKT1'and'ATKC1,'which'are' expressed'in'the'same'root'cells'in'A.(thaliana(and'form'heteromeric'complexes'(Geiger'et(al.,' 2009a;'Jeanguenin'et(al.,'2011),'the'SIType'anion'channel'subunits'SLAH3'and'SLAH1'coIexpress' in'the'same'cell'type.'To'test'whether'SLAC/SLAH'subunits'also'assemble'to'heteromeric'anion' channels'and'whether'SLAH1'might'function'as'a'channel'modulator'rather'than'an'autonomous' SItype' anion' channel,' SLAH1' was' coIexpressed' together' with' its' Arabidopsis'homologues' in' Xenopus'oocytes.'' Using'BiFC'constructs'with'one' half' of' YFP'fused' to' the' NIterminus' of' SLAH1'and'the' other' half' fused' to' the' NIterminus' to' each' of' the' other' SLAC/SLAH' family' members,' YFP' fluorescence'signals'pointed'to'an'interaction'between'all'tested'SItype'anion'channel'subunits' (Fig.'4.23),' indicating' that' SLAC/SLAH' subunits' are' able' to' assemble' heteromeric' channel' complexes.' ' Figure!4.23.! Physical! interaction! of! SLAH1! with! SLAC/SLAH! Family! members.! BiFC' experiments' revealed'that'SLAC/SLAH'subunits'are'able'to'assemble,'leading'to'heteromeric'channel'complexes.'The'CI terminal'(YFPCT)'or'NIterminal'(YFPNT)'half'of'YFP'was'fused'to'the'NIterminus'of'SLAC1,'SLAH1,'SLAH2,' SLAH3'or'SLAH4.'Representative'images'of'oneIquarter'of'Xenopus(oocytes'are'shown.' ' ' ' Results' ' ' 116' 4.1.1.7.1.!SLAH1!substitutes!for!kinase>!and!nitrate>!dependent!activation!of! SLAH3!' Given'that'BIFC'images'showed'that'SLAC/SLAH'subunits'could'constitute'heteromeric' channels'with'SLAH1,'each'member'of'the'Arabidopsis'SLAC/SLAH'anion'channel'family'was'coI expressed' with' SLAH1' in' Xenopus( oocytes' to' further' explore' their' putative' functional' interaction.' TEVC' recordings' revealed' that' only' the' interaction' between' SLAH1' and' SLAH3' dramatically' changed' the' electrical' current' characteristics'(Fig.' 4.24' a),' since' macroscopic' currents'could'be'measured'in'the'absence'of'an'activating'kinase'with'similar'amplitudes'as' those'recorded'with'kinaseIactivated'SLAH3'(e.g.'with'CPK21ΔEF,'Fig.'4.24'b)'and'without'the' need'of'NO3−'in'the'bath'solution'(Fig.'4.24'bIc).'In'contrast,'coIexpression'of'SLAH1'with'SLAC1,' SLAH2'or'SLAH4'did'not'result'in'macroscopic'anion'currents'(Fig.'4.24'a)'compared'with'the' activation'of'the'channels'SLAC1,'and'SLAH2,'by'CPK21ΔEF'(Fig.'4.24'b).' ! Figure!4.24.!Influence!of!SLAH1!on!the!activity!of!S>type!anion!channels.!(a)!Instantaneous'currents' (Iinst)'of'Xenopus(oocytes'coIexpressing'SLAH1'with'other'SLAC/SLAH'channels'recorded'at'−100'mV'in' the'presence'of'either'100'mM'chloride'or'100'mM'nitrate'(n'≥'3;'mean'±'SE).'(b)!Instantaneous'currents' (Iinst)'of'oocytes'expressing'SLAC1'or'SLAH3'in'the'presence'of'SLAH1'or'CPK21ΔEF'in'standard'buffers' containing' 100' mM' chloride' (Cl−)' or' 100' mM' nitrate' (NO3−).' Currents' are' plotted' against' the' applied' voltage'(n'≥'3;'mean'±'SE).'(c)!WholeIoocyte'currents'of'Xenopus(oocytes'expressing'SLAH1'with'SLAH3' in'the'presence'of'30'mM'chloride'or'nitrate'(representative'cells'are'shown).' ' Results' ' ' 117' Oocytes' expressing' SLAH1/SLAH3' with' different' Ca2+Iindependent' SnRK2' kinases' (SnRK2.2'and'SnRK2.3)'were'recorded'by'TEVC'technique'but,'in'the'same'way'as'NO3−'did'not' change'the'current'amplitude'associated'with'constitutively'active'SLAH1/SLAH3'heteromeric' complexes' (Fig.' 4.24' bIc),' coIexpression' of' SLAH1/SLAH3' with' these' kinases' did' not' further' increase'the'amplitude'(Fig.'4.25),'suggesting'that'the'channels'complex'was'already'fully'active.' ' Figure!4.25.! SLAH1!substitutes! for! kinase>! and! nitrate>! dependent! activation! of! SLAH3.! Instantaneous' currents' (Iinst)' of' Xenopus( oocytes' expressing' SLAH1/SLAH3' with' SnRK2.2' and' Snrk2.3' kinases' recorded' at' −100' mV' in' the' presence' of' either' 100' mM' chloride' or' 100' mM' nitrate' show' SLAH1/SLAH3'heteromer'is'kinaseIindependent'(n'≥'3;'mean'±'SE).! ! The'fact'that'the'coIexpression'of'SLAH1'with'SLAC1,'SLAH2'or'SLAH4'did'not'result'in' macroscopic'anion'currents'in'the'absence'of'an'activating'kinase'indicates'that'SLAH1ISLAH3' interaction'and'activation'is'SItype'channel'subunit'specific.'' ' 4.1.1.7.2.!SLAH1!is!a!modulatory!subunit!' The' observed' kinaseIindependent' activity' of'the' heteromeric' channel' complex' SLAH1/SLAH3,' as' well' as' the' NO3−Iindependent' activation,' could' be' the' consequence' of' the' addition'of'a'SLAH1IintrinsicIchloride'conductance'superimposing'on'the'NO3−/Cl−'conductance' of'SLAH3'or'alternatively'SLAH1'might'represent'a'silent'subunit'that'modulates'the'properties' of'SLAH3.''To'test'the'contribution'of'each'interaction'partner'to'the'observed'anion'currents,' inactive'channel'mutants'of'SLAH3'and'SLAH1'were'originated.' The' 3D' homology' model' of' SLAC1' predicted' that' the' substitution' of' the' central' conserved'Phe'residue'(F450)'in'the'pore'region'by'leucine'(F450L),'as'well'as'the'exchange'of' the' glycine' (Gly)' residue' G194'present' in' the' transmembrane'domain' 1' (Fig.' 4.26)' to' a' negatively'charged'aspartate'(Asp),'would'block'the'pore.'Indeed,'it'has'been'shown'in'AtSLAC1' that'the'point'mutations'F450L'and'G194D'lead'to'a'nonIfunctional'channel'(Chen'et(al.,'2010;' Negi'et(al.'2008).' Based'on'multiple'alignments'of'amino'acid'sequences'(section'3.2.9)'and'on'homology' models'of'SLAH1'and'SLAH3,'the'respective'mutants'in(AtSLAH1'(F307L'and'R47D;'Fig.'4.26)' and'AtSLAH3'(F517L'and'G264D;'Fig.'4.26)'were'generated.' Results' ' ' 118' ' Figure!4.26.! Sequence! alignment! of! SLAC/SLAH! proteins! from! different! species.' Shading' levels' indicate' degree' of' conservation' (black,' denotes' very' high' conservation;' dark' grey,' mediumIhigh' conservation,' light' grey,' moderate' conservation;' and' white,' no' conservation).' Tm1,' Transmembrane' domain'1.'Arg,'Arginine;'Gly,'Glycine.'At,'Arabidopsis(thaliana;'Cc,'Citrus(clementina;'Os,'Oryza(sativa;'Pp,' Physcomitrella(patens;'Sb,'Solanum(bicolor;'Vv:'Vitis(vinivera.' ' Using' the' poreIblocking' mutants,'TEVC' recordings' were' performed' after' their' expression' in' Xenopus'oocytes.' Neither' the' coIexpression' with' CPK21ΔEF' nor' coIexpression' with' SLAH1' could' elicit' macroscopic' currents' in' SLAH3' F517L'and' G264D' (Fig.' 4.27).' In' contrast,'the'mutations'at'the'equivalent'positions'in'SLAH1'(SLAH1'F307L'and'R47D)'did'no' significantly' affect' the' anion' currents' mediated' by' the' SLAH1/SLAH3' channel' complex' when' they'were'coIexpressed'with'SLAH3'(Fig.'4.27).'' The' background' currents' of' the' SLAH3' F517L' mutant,' when' coIexpressed' with' CPK21ΔEF,' SLAH1' WT,' or' SLAH1' mutants' (Fig.' 4.27),' result' from' the' residual' activity' of' the' SLAH3'mutant'rather'than'from'a'conductivity'of'SLAH1.' ' Results' ' ' 119' Figure!4.27.! SLAH1! modifies! the! activation! properties! of! SLAH3.! Instantaneous' currents' (Iinst)' recorded'at'−100'mV'in'Xenopus(oocytes'expressing'different'combinations'of'SLAH1'WT'or'mutants'and' SLAH3'WT'or'mutants'as'indicated'in'the'figure'in'100'mM'nitrate'buffer'solution'(n'≥'3;'mean'±'SE).' ' To'exclude'the'possibility'that'the'loss'in'anion'conductance'with'the'SLAH3'G264D'or' F517L' mutants' was' due' to' a' reduction' in' the' channel' abundance,' YFP' was' fused' to' the' NI terminus' of' either' SLAH3' WT' or' mutants' and' they' were' expressed' in' oocytes.' BiFC' analysis' revealed'that'the'fluorescence'signals' associated'with'the'Xenopus'oocyte'expression'of'these' constructs'had'similar'intensity'and'plasma'membrane'localization'(Fig.'4.28).' ' Figure!4.28.!SLAH3!G264D!or!F517L!mutants!did!not!exhibited!a!decrease!in!channel!abundance.! BiFC'experiments'showed'that'the'fluorescence'signal'associated'with(oocytes'expressing'SLAH3'G264D' or'F517L'mutants'was'similar'to'that'associated'to'SLAH3'WT.! ! The' finding' that' nonIfunctional' channel' mutants' of' SLAH3' did' not' elicit' macroscopic' currents'when'coIexpressed'with'either'CPK21ΔEF'or'SLAH1,'while'the'equivalent'mutants'in' SLAH1'did'no'significantly'modify'the'anion'currents'mediated'by'the'SLAH1/SLAH3'channel' complex,'in'combination'with'the'fact'that'the'replacement'of'the'conserved'Phe'residue'by'Ala' in' SLAH1' (F307A)' to' obtain' a' constitutively' open' anion' channel' did' not' elicited' macroscopic' currents,'indicate'that'rather'than'adding'a'separate'anion'conductance'to'the'conductance'of' SLAH3,'SLAH1'modifies'the'electrical'properties'of'SLAH3,'making'it'independent'of'kinase'and' NO3−'activation.' ' 4.1.1.7.3.! The! SLAH1/SLAH3! heteromeric! complex! exhibits! higher! Cl−! conductance!than!the!SLAH3!homomer' Since' the' chloride' currents' of' the' SLAH1/SLAH3' heteromeric'complex'were'markedly' increased' compared' to' the' currents' of' SLAH3' activated' by' CPK21' (Fig.' 4.24' b),' the' relative' permeability'to'nitrate'(Pj/PNO3−)'of'this'complex'was!calculated'for'Cl−'after'the'coIexpression'of' both'channels'in'oocytes.'Nevertheless,'it'could'be'observed'that'the'permeability'for'Cl−'relative' to'NO3−'(PCl−/PNO3−)'of'the'SLAH1/SLAH3'heteromer'was'unaltered'with'respect'to'the'SLAH3' homomer'(Fig.'4.29).'' Based' on' the' homology' model' of' SLAH2' and' SLAC1,' the' exchange' of' the' polar' serine' (Ser)' at' position' 228' in' SLAH2' to' a' nonpolar' valine' (Val),' the' amino' acid' in' SLAC1' at'the' respective'position,'turned'the'nitrateIselective'SLAH2'into'a'chloride/nitrateIpermeable'anion' channel' (Maierhofer' et( al.,' 2014b).' Taken' this' finding' into' account' to' further' explore' the' selectivity' of' the' SLAH1/SLAH3' heteromer,' the' corresponding'Ser' residue' in' SLAH1' was' substituted' to' the' nonpolar' Val' (S130V)'according' to'multiple' alignments' of' amino' acid' sequences'(Fig.'4.29;'section'3.2.9)'and'on'homology'models.' Results' ' ' 120' ' Figure!4.29.! Sequence! alignment! of! SLAC/SLAH! proteins! from! different! species.' Shading'levels' indicate' degree' of' conservation' (black,' denotes' very' high' conservation;' dark' grey,' mediumIhigh' conservation,' light' grey,' moderate' conservation;' and' white,' no' conservation).' Tm3,'Transmembrane' domain' 3.' Ser,' Serine;' Val,' Valine.' At,' Arabidopsis( thaliana;' Cc,' Citrus( clementina;' Os,' Oryza( sativa;' Pp,' Physcomitrella(patens;'Sb,'Solanum(bicolor;'Vv:'Vitis(vinivera.' ' The' relative' permeability' of' SLAH3' coIexpressed' with' this' SLAH1' mutant' was' then' checked'in'oocytes.'However'the'substitution'of'the'Ser'130'residue'in'SLAH1'to'Val'(S130V)'did' not'modify'the'anion'selectivity'of'the'SLAH1/SLAH3'channel'complex'(Fig.'4.30).' ' Figure!4.30.!SLAH1/SLAH3!and!SLAH3!channels!share!the!same!relative!permeability!for!chloride.' Relative'permeability'of'SLAH3'coIexpressed'with'SLAH1,'CPK21ΔEF'or'SLAH1'S130V'mutant'in'Xenopus( oocytes'(permeability'for'nitrate'was'set'to'1).'Standard'bath'solution'contained'50'mM'of'the'respective' anion'(n'≥'4;'mean'±'SE).! ! The'finding'that'the'relative'permeability'for'Cl−'obtained'in'SLAH3'(coIexpressed'with' CPK21)' remained' unchanged' compared' with' its' coIexpression' with' SLAH1' indicates' that' the' elevated'Cl−'currents'mediated'by'the'SLAH1/SLAH3'heteromeric'complex'result'from'the'NO3−I independent'activation'of'SLAH3'rather'than'from'an'increased'Cl−'permeability.'To'elucidate' Results' ' ' 121' this' assumption' the' chord' conductance' for' Cl−'in' SLAH3' homomer' and' SLAH1/SLAH3' heteromer'was'obtained.'It'could'be'observed'that'while'the'NO3−'conductance'did'not'change' between'oocytes'expressing'SLAH1/SLAH3'and'SLAH3'with'CPK21,'the'conductance'for'Cl−'was' seven' times' higher' in' oocytes' expressing' SLAH1/SLAH3' compared' with' the' kinaseIactivated' SLAH3'channels'(Fig.'4.31).' ' Figure!4.31.! SLAH1/SLAH3! channels! have! a! higher! chloride! conductance! than! the! kinase> dependent!SLAH3!channels.'Chord'conductance'of'Xenopus'oocytes'coIexpressing'SLAH3'together'with' either'CPK21ΔEF' or' SLAH1.' Standard' bath' solution' contained' 100' mM' chloride' or' nitrate' (n' ≥' 4' experiments;'mean'±'SE).'! ! To'investigate'the'molecular'mechanism'underlying'the'enhanced'Cl−'current'amplitudes' of'the'SLAH1/SLAH3'heteromeric'complex,'the'NO3−Idependent'gating'properties!of'the'kinaseI activated'SLAH3'homomeric'channel'compared'to'the'SLAH1/SLAH3'heteromer'were'analysed.' On' the' one' side,' the' relative' open' probability' (rel.' PO)' of' oocytes' coIexpressing' SLAH3' and' CPK21ΔEF'exposed'to'varying'Cl−'concentrations'shifted'to'more'negative'membrane'potentials' with' increasing' Cl−'concentrations.' However,' even' with' 30' mM' Cl−'in' the' external' buffer,' the' halfImaximal'(V½)'open'probability'did'not'reach'the'physiological'membrane'potential'range' (Fig.'4.32;'between'–80'and'–200'mV'[Fromm'and'Lautner,'2007]).'In'contrast,'with'30'mM'of' external'NO3−'the'rel.'PO'of'SLAH3'markedly'shifted'to'hyperpolarized'(physiological)'membrane' potentials' (Fig.' 4.32).' On' the' other' side,' the' rel.' Po' of' SLAH1/SLAH3' channel' complexes' appeared' independent' from' the' external' anion' composition' (Fig.' 4.32),' indicating' that' interaction'of'SLAH1'with'SLAH3'already'gated'SLAH3'open'without'the'need'of'NO3−.'' ! Figure!4.32.!SLAH1!gates!SLAH3!open!without!the!need!of!nitrate.'Relative'voltageIdependent'open' probabilities'(rel.'Po)'derived'from'oocytes'coIexpressing'SLAH3'with'either'CPK21ΔEF'or'SLAH1'in'the'