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ÐGreen,€KY€421041.€Ìòò3óó€Department€of€Plant€and€Soil€Science,€Molecular€Science€Program,€SouthernÐ À ÐIllinois€University,€Carbondale,€IL€€62901€USA.Ì*€Author€to€whom€correspondence€should€be€addressed€(Fax€618„453„2496;€E„mailÏGA4082@SIU.EDU).ÌÐ  P ÐÔ_Ôò òAbstract€òòó ó€óóÔ€YX: XôYô5ÅÔÔ€ZX: X XYX:ÔThe€abundance€of€the€transcripts€of€certain€plant€genesÐ ° Ðincreases€in€response€to€cytokinin€treatments€in€vitro.€This€studyÏwas€conducted€to€determine€whether€one€such€transcript€€wouldÏrespond€to€cytokinin€treatment€in€planta.€Cytokinin€treatment€ofÏthe€leaves€of€òòPhaseolus€vulgarisóó€L.€plants€grown€in€the€greenhouseÐ  h Ðstimulated€the€accumulation€of€an€mRNA,€designated€L„22,€byÏapproximately€4„fold€within€4€h€in€replicated€experiments.€TheÏextent€of€the€L„22€transcript€accumulation€was€in€logarithmicÏrelation€to€the€dose€of€cytokinin€applied€between€10€ððM€and€1€mM,Ïhowever€the€mRNA€accumulation€was€transient.€Therefore,€transcriptÏabundance€was€regulated€in€response€to€both€the€dose€of€cytokininÏapplied€and€the€event€of€a€change€in€cytokinin€concentration€inÏtreated€leaves.€€The€extent€of€mRNA€accumulation€differed€onlyÏslightly€depending€on€the€structure€of€the€cytokinin€appliedÏindicating€only€small€differences€among€the€activities€ofÏcytokinins€used€in€this€assay.€Among€the€plant€growth€regulatorsÏtested€only€cytokinin€caused€the€mRNA€to€accumulate,€suggesting€theÏresponse€was€cytokinin€specific.€€The€deduced€amino€acid€sequenceÏof€the€L„22€protein€product,€derived€from€the€nucleotide€sequenceÏof€the€L„22€cDNA,€was€up€to€24%€identical€and€49%€similar€to€theÏgamma€subunit€of€mitochondrial€Hòò+óó„transporting€ATP€synthase€(ECÐ h$¸( Ð3.6.1.34)€in€the€conserved,€Ô_Ôcatalytic,€carboxyl„terminal€domain.ÏTherefore,€the€L„22€protein€product€may€have€a€role€in€theÏregulation€of€processes€related€to€plant€respiration€by€Ô_ÔcytokininÔ_Ô.ÌÐ  ¨*ø%0 ÐÔ_Ôò òKey€words€ó ó€ATP€synthase,€chalcone€synthase,€cytokinin,€geneÐ ° Ðexpression,€growth€regulator,€òòPhaseolusóó.Ð à ÐÌò òAbbreviationsó ó€€€ABA,€abcisic€acid;€b,€bases;€bp,€base€pairs;€BA,Ð  P Ðbenzyladenine;€CHS,€chalcone€synthase;€GAòò3óó,€gibberellic€acid;€P,Ð à 0 Ðpichloram;€PGR,€plant€growth€regulator;€T,€thidiazuron;€€Z,€zeatin.Ð  p À  ÐÌÔ€AX3ÝX XZX:Ôò òÌà@996VàIntroductionˆÐ  p ÐÌó óà Ü àThe€physiological€role€of€cytokinins€in€plant€growth€and€development€has€been€extensivelyÐ à 0 Ðreviewed€(Skoog€and€Armstrong,€1970;€Guern€and€Peaud„Lenoel,€1981;€Letham€and€Palni,€1983;ÏMatsubara,€1990),€but€the€molecular€mechanisms€mediating€cytokinin€action€are€less€wellÏcharacterized.€€Several€genes€and€cDNAs€have€been€isolated€that€encode€mRNAs€whose€abundanceÏis€altered€by€cytokinin€treatments;€€these€include,€chlorophyll€òòa/bóó€binding€protein€(Teyssendier€deÐ `° Ðla€Serve€òòet€alóó.,€1985),€ribulose€1,5€bisphosphate€carboxylase€(Flores€and€Tobin,€1986),€the€10€kDaÐ @ ÐPSII€polypeptide€(Watillon€òòet€alóó.,€1991),€phytochrome€(Cotton,€1990),€nitrate€reductase€(Lu€òòet€alóó.,Ð  p Ð1992),€ribosomal€subunits€(Crowell€òòet€alóó.,€1990),€hydroxypyruvate€reductase€(Chen€and€Leisner,Ð P Ð1985),€nodulin€ENOD2€(Dehio€and€de€Bruijn,€1992),€pollen€allergen€homologue€cim€1€(Crowell,Ï1994),€chitinase,€extensin€(Memelink€òòet€alóó.,€1987),€multiple„stimulus€responsive€genes€(Crowell€òòetÐ À Ðalóó.,€1992;€Dominov€òòet€alóó.,€1992)€as€well€as€several€genes€of€unknown€function€(Crowell€òòet€alóó.,Ð  ð Ð1990).€Where€studied€many€of€these€mRNAs€also€accumulate€in€response€to€treatments€with€plantÏgrowth€regulators€other€than€cytokinins€(Shinshi€òòet€alóó.,€1987;€Dominov€òòet€alóó.,€1992;€Lu€òòet€alóó.,Ð `"°  Ð1992),€to€general€stress€(Crowell€òòet€alóó.,€1992)€or€to€adenine€(Dehio€and€de€Bruijn€1992),€thereforeÐ @$" Ðtheir€responses€may€not€be€cytokinin€specific.Ìà Ü à€€Cytokinin€concentration€and€molecular€structure€may€both€vary€in€plant€organs€duringÏdevelopment€(Singh€òòet€alóó.,€1988;€Sossountzov€òòet€alóó.,€1988),€during€daily€fluctuations€in€theÐ à)0%( Ðtranspiration€stream€(Neuman€òòet€alóó.,€1990),€or€during€metabolic€activity€(Nooden€òòet€alóó.,€1993).€Ð À+'* ÐCytokinin€responsive€mRNA€accumulation€has€been€shown€to€be€sensitive€to€differentÏconcentrations€of€cytokinin,€these€dose„responses€include€a€threshold€response€(Dehio€and€DeÏBruijn,€1992;€Crowell,€1994),€an€approximately€linear€response€(Dominov€òòet€alóó.,€1992)€and€anÐ pÀ Ðapproximately€logarithmic€response€(Lu€òòet€alóó.,€1992).€Ð P   Ðà Ü àWe€have€isolated€as€cDNA€an€mRNA€which€accumulated€in€response€to€cytokininÏtreatment€of€bean€cell€cultures€(McDaniel,€1995).€In€this€study€we€characterized€the€transcriptÏaccumulation€in€òòPhaseolus€vulgarisóó€€L.€leaves.€€We€report€the€logarithmic€dose„dependent€natureÐ ð@  Ðof€accumulation€of€the€mRNA,€the€transient€nature€of€mRNA€accumulation€and€the€specificity€ofÏthe€accumulation€of€mRNA€to€cytokinin€treatments.€Furthermore,€the€nucleotide€sequence€of€theÏcDNA€encoded€a€novel€subclass€of€homologue€to€the€gamma€subunit€of€mitochondrial€ATPase.€€€Ð à ÐÐ  pÀ Ðò òÌà@’’9VàRESULTSó óˆÐ à Ðòòò òÌThe€cytokinin€dose€response€of€L„22€mRNA€abundanceóóó ó€Ð P   Ðà Ü àFour€hours€after€cytokinin€treatment€the€abundance€of€L„22€mRNA€in€greenhouse€grown€òòP.Ð 0 € Ðvulgarisóó€leaves€was€altered€maximally€and€in€proportion€to€the€concentration€of€the€cytokininÐ `  Ðapplied€(fig.€1€C).€The€protected€fragment€of€134€b€was€expected€from€the€position€of€the€òòPstóóI€siteÐ ð@  Ðin€the€sequence€of€L„22.€A€protected€fragment€of€114€b€was€also€apparent€and€was€induced€byÏcytokinin€treatment€in€parallel€with€the€expected€fragment€of€L„22.€This€fragment€was€either€aÏdegradation€product€or€editing€intermediate€or€a€homologue€of€L„22€showing€coordinateÏexpression.€If€it€is€a€degradation€product€the€size€of€the€fragment€indicates€the€cleavage€point€wasÏwithin€an€AT€rich€region€of€the€L22€cDNA.Ì€€€€€€The€logarithmic€trend€in€the€dose€response€of€the€L„22€mRNA€4€h€after€zeatin€or€thidiazuronÏtreatment€was€highly€significant€(òòPóó€<€0.002,€òòRóóòò2óó€>€0.73)€(fig.€1€D€and€2).€However€at€10€ððM,Ð ` Ðbenzyladenine€treatment€caused€a€reduction€in€L„22€mRNA€abundance€compared€to€the€control€andÏthe€logarithmic€trend€for€benzyladenine€was€not€statistically€significant€(òòPóó€<€0.05),€as€a€result.Ð Ð  Ѐ€€€The€abundance€of€the€CHS„17€mRNA€in€cytokinin„treated€leaves€was€not€significantly€differentÏ(òòPóó€>€0.05)€regardless€of€the€concentration€of€cytokinin€applied€or€the€time€after€application€(fig.€1Ð $à" ÐA).€In€addition€the€mRNA€encoded€by€a€second€anonymous€cDNA€clone€L221€was€significantlyÏreduced€in€abundance€(òòPóó€<€0.01)€by€the€cytokinin€treatments€(Fig.€1B)€(McDaniel,€1996).Ð P( #& ÐÌòòò òThe€effects€of€other€plant€growth€regulators€on€mRNA€abundanceóóÐ ,`'* Ðó óà Ü à€The€abundance€of€the€L„22€mRNA€clearly€increased€in€response€to€most€concentrations€ofÐ ° Ðall€3€cytokinins€tested€(zeatin,€benzyladenine,€and€thidiazuron)€compared€to€controls€(fig.€2).€€€ÏNeither€ABA,€pichloram,€nor€GAòò3óó€treatment€increased€or€decreased€L„22€mRNA€abundance€at€anyÐ pÀ Ðconcentration€or€time€point€tested€(fig.€3).€Co„application€of€cytokinin€with€ABA,€pichloram,€orÏGAòò3óó€did€not€alter€the€cytokinin€response.€Adenine€and€methyl€adenine€treatments€did€not€alter€theÐ 0 € ÐmRNA€abundance€(data€not€shown;€McDaniel,€1995).Ìòòò òÌThe€effects€of€cytokinin€structureó óóóÐ Ð  Ðà Ü àFour€hours€after€cytokinin€treatment€at€one€concentration€(10€ððM),€benzyladenineÏdecreased€the€L„22€mRNA€abundance€2„fold€while€thidiazuron€or€zeatin€treatment€increased€the€L„¼22€mRNA€abundance€2„fold€(fig.€1).€This€phenomenon€was€found€to€be€consistent€in€four€gelsÏfrom€two€replicated€experiments€(fig.€2.).€€At€higher€cytokinin€concentrations€(50€and€100€ððM),Ïthidiazuron€and€zeatin€treatments€increased€the€mRNA€abundance€3„fold€but€benzyladenineÏtreatment€increased€the€mRNA€abundance€only€2€to€2.5„fold.€At€a€concentration€of€1000€ððM,€allÏcytokinins€increased€L„22€mRNA€abundance€4€fold.€If€these€differences€reflected€differences€inÏcytokinin€uptake,€transport€or€stability€rather€than€activity€we€might€expect€mRNA€accumulationÏin€response€to€benzyladenine€to€be€maximal€at€other€time€points.€To€test€this€hypothesis€weÏassayed€mRNA€accumulation€at€2€and€24€h.Ìòòò òÌThe€transient€nature€of€L„22€mRNA€accumulation€in€response€to€cytokininó óóó€à L àTwo€hours€afterÐ P( #& Ðcytokinin€treatment€of€òòP.€vulgarisóó€leaves€the€L„22€mRNA€had€accumulated€up€to€2€fold,€comparedÐ 0*€%( Ðto€4€fold€at€4€h,€but€generally€in€a€concentration„dependent€manner€(fig.€2).€No€evidence€for€theÐ ,`'* Ðreduction€of€mRNA€accumulation€by€10€ððM€benzyladenine€seen€at€4€h€was€apparent€2€h€afterÏtreatment.à 4 àà Œ àÐ à Ðà Ü àTwenty„four€hours€after€most€cytokinin€treatments,€the€abundance€of€L„22€mRNA€returnedÏto€the€level€of€expression€of€the€control€and€no€dose€dependence€was€observed€(fig.€2).€However,Ïcertain€treatments€(zeatin€at€10€ððM,€benzyladenine€at€100€ððM)€caused€slightly€elevated€L„22ÏmRNA€abundance€equivalent€to€the€2€h€treatments.€No€evidence€for€sustained€reduction€of€mRNAÏaccumulation€by€10€ððM€benzyladenine€was€apparent.ÌÌòòò òThe€nucleotide€and€derived€amino€acid€sequence€of€L„22€cDNAó óóó€Ð ° Ðà Ü àThe€nucleotide€sequence€of€the€L„22€cDNA€(fig.€4)€indicated€that€it€contained€the€3'Ïportion€of€the€complete€mRNA.€€A€single€long€open€reading€frame€which€encoded€89€amino€acidsÏwas€detected.€The€3'€untranslated€region€of€L„22€was€short,€at€just€54€b,€and€terminated€in€a€longÏ67€b€poly[A]€tail.€The€transcribed€portion€of€the€3'€untranslated€region€was€extremely€rich€in€AÏand€T€residues€(84%)€and€showed€the€interspersed€blocks€of€poly[T]€and€poly[A]€typical€ofÏextensively€edited€mitochondrial€transcripts€in€kinetoplastid€flagellates€€(Bhat€òòet€alóó.,€1990).Ð ð@ ÐHowever,€it€is€clearly€a€plant€gene€with€one€or€two€homologues€as€judged€by€SouthernÏhybridization€to€total€genomic€DNA€and€replicated€Sòò1óó€nuclease€protection€experiments€with€RNAÐ °"  Ðfrom€tissue€grown€in€axenic€culture€(McDaniel,€1995).Ì€€€€In€the€C„terminal€79€of€the€89€total€residues€the€deduced€amino€acid€sequence€of€L„22€was€24%Ïidentical€and€49%€similar€to€the€gamma€subunit€of€ATP€synthase€(ATPase)€from€mitochondria€ofÏòòTrypanosoma€bruceióó€(Bhat€òòet€alóó.,€1990)€but€only€11%€identical€and€35%€similar€to€the€previouslyÐ 0*€%( Ðidentified€òòVicia€fabaóó€homologue€(fig.€5).€Among€plant€homologues€84€to€92%€similarity€withinÐ ,`'* Ðthis€conserved€region€was€reported€(McFarlane€òòet€alóó.,€1990)€so€that€the€49%€similarity€with€L„22Ð ° Ðis€comparatively€weak.€However,€the€three€proteins€aligned€in€figure€5€were€identical€at€three€ofÏthe€five€residues€conserved€in€all€mitochondrial€ATPase€gamma€subunits€characterized€(Bhat€òòetÐ pÀ Ðalóó.,€1990).€Conservative€changes€were€found€in€the€other€two€positions,€Dòò19óó€instead€of€Eòò19óó€and€Sòò33óóÐ P   Ðin€place€of€Nòò33óó.€Similarity€is€clearly€strongest€between€residues€19€to€43€(fig.€5)€and€this€regionÐ 0 € Ðwas€identified€as€the€strongest€homology€by€the€blast€search€(Altschul€et€al.,€1990).€Independently,Ïthe€same€search€reported€a€second€region€of€homology€between€the€same€proteins€encompassing€L„¼22€residues€56€to€87.€The€two€regions€were€logically€arranged,€correctly€spaced€and€colinear.€TheÏregion€between€L„22€residues€19€to€43€is€postulated€to€be€involved€in€the€critical€subunitÏinteractions€during€catalysis€by€ATPase€(Abrahams€òòet€alóó.,€1994).€Outside€the€carboxyl„terminal€70Ð à Ðresidues€identity€and€similarity€were€much€lower,€as€commonly€observed€with€protein€alignmentsÏof€mitochondrial€ATPase€gamma€subunits€(Schneider€and€Altendorf,€1987).ÌÐ  0€ Ðò òDISCUSSIONó óÐ ° Ѐ€€€The€four€fold€accumulation€of€L„22€mRNA€in€leaves€in€response€to€cytokinin€treatments€isÏrather€modest€compared€to€the€greater€than€10€fold€accumulation€reported€for€other€cytokininÏresponsive€transcripts€in€planta€(Flores€and€Tobin,€1989;€Watillon€òòet€alóó.,€1991;€Dehio€and€DeÐ P   ÐBruijn,€1992;€Lu€òòet€alóó.€1992).€The€lesser€induction€might€have€been€related€to€endogenousÐ 0 € Ðcytokinin€synthesis€and€transport€in€the€illuminated€whole€plant€leaf€assay€we€used€compared€to€theÏetiolated,€detached€organ€or€cell€culture,€assays.€However,€mRNA€accumulation€was€not€greaterÏthan€4€fold€in€cytokinin„treated€etiolated€cell€cultures€(McDaniel,€1996).€Therefore,€the€L„22ÏmRNA€accumulation€is€not€underestimated€by€the€illuminated€whole€leaf€assay.€However,€in€cellÏculture,€where€endogenous€cytokinin€synthesis€is€lesser,€L„22€mRNA€accumulation€occurred€atÏlower€concentrations€(1€to€10€ððM)€(McDaniel,€1996).Ì€€€€€The€dose€response€of€a€mRNA€to€a€plant€growth€regulator€(PGR)€should€be€a€rapid€and€linearÏfunction€of€the€concentration€applied€if€the€PGR€is€directly€modulating€mRNA€abundance€(KendeÏand€Gardner,€1976).€€Although€the€exogenous€cytokinin€must€pass€through€several€permeabilityÏbarriers€in€leaves,€the€induction€of€L„22€mRNA€abundance€was€rapid,€within€2€to€4€h€afterÏtreatment,€and€linear€when€cytokinin€concentration€was€transformed€by€logòò10óó€(fig.€1).€€Whilst€theÐ Ð  Ðincrease€in€concentration€of€the€cytokinin€in€the€responding€tissue€may€be€much€less€than€theÏconcentration€of€cytokinin€applied,€€the€log„linear€nature€of€the€mRNA€response€indicates€thatÏcytokinin€may€be€directly€modulating€the€L„22€mRNA€abundance.€€A€log„linear€response€toÏcytokinins€was€noted€in€mRNA€accumulation€of€nitrate€reductase€of€barley€(Lu€òòet€alóó.,€1992).€Ð P( #& Ðò ò€€€ó ó€The€response€to€cytokinin€treatments€was€observed€with€the€phenylurea€cytokinin,€thidiazuron,Ð 0*€%( Ðand€with€both€adenine€derived€cytokinins.€Since€all€three€chemically€diverse€cytokinins€wereÐ ,`'* Ðequivalent€in€dose€response€(above€10€ððM)€they€were€equivalent€in€the€sum€of€their€uptake,Ïtransport,€stability€and€their€eventual€recognition€by€the€receptor(s)€that€modulate€L„22€mRNAÏabundance.€The€consistent€reduction€of€L„22€mRNA€abundance€by€10€ððM€BA€but€not€zeatin€orÏthidiazuron€may€indicate€that€the€cytokinins€are€not€equivalent€at€this€concentration.€AdditionalÏexperiments€with€the€cytokinins€in€the€concentration€range€1„100€ððM€will€determine€the€nature€ofÏthe€reduction€of€the€L„22€mRNA€abundance.Ì€€€€€€€L„22€mRNA€abundance€was€not€responsive€to€growth€regulators€from€three€classes,€namelyÏabcisic€acid,€auxin€and€gibberellin.€Therefore,€the€L„22€mRNA€abundance€in€leaves€wasÏspecifically€responding€to€cytokinin,€at€least€among€the€plant€growth€regulators€tested.€Co„¼application€of€cytokinin€and€other€classes€of€plant€growth€regulators€did€not€alter€the€cytokininÏdependent€accumulation€of€mRNA€(data€not€shown).€Therefore,€L„22€mRNA€accumulation€doesÏnot€respond€to€the€ratio€of€two€plant€growth€regulators€unlike€many€cytokinin€responsive€mRNAsÏreported€to€date€(Shinshi€òòet€alóó.,€1987;€Crowell€òòet€alóó.,€1992;€Dominov€òòet€alóó.,€1992;€Lu€òòet€alóó.,Ð 0€ Ð1992).Ìò ò€€€€ó ó€€A€marked€increase€in€mRNA€abundance€was€not€observed€until€4€h€after€cytokinin€application.€Ð ð@ ÐAt€2€h€there€was€lesser€induction€although€dose€dependence€was€evident.€€€€Analysis€of€mRNAÏaccumulation€in€cytokinin„treated€callus€cultures,€where€fewer€permeability€barriers€exist,€was€notÏfaster€but€did€occur€at€lower€concentrations€(1€to€10€ððM)€(McDaniel,€1996).€This€suggests€that€4€hÏwas€required€for€mRNA€accumulation€to€occur.Ì€€€€€€€At€24€h,€the€level€of€expression€of€the€gene€was€returned€to€nearly€the€level€of€non„treatedÏleaves.€€This€decay€in€the€response€may€indicate€that€either€the€plant€was€able€to€metabolize€theÏinflux€of€cytokinin€and€return€cytokinin€concentration€and€gene€expression€to€a€steady€state€or€thatÐ ,`'* Ðthe€responding€cells€were€desensitized€to€cytokinin€concentration€after€the€initial€response€asÏreported€for€other€mRNAs€(Crowell€òòet€alóó.,€1992;€Dominov€òòet€alóó.,€1992).Ð à Ѐ€€€€€The€use€of€the€chalcone€synthase€and€L„221€probes€showed€that€band€intensities€reflected€geneÐ pÀ Ðspecific€mRNA€concentration€and€not€random€variations€in€the€amount€of€RNA€or€proportions€ofÏmRNA€in€total€RNA€in€each€sample.€In€addition€the€treated€plants€were€not€under€cytokinin€stressÏsince€chalcone€synthase€mRNA€abundance€is€induced€by€cytokinin€stress€and€physiologically„¼induced€stresses€(Memelink€òòet€alóó.,€1987;€Ryder€òòet€alóó.,€1987;€€McDaniel,€1996).€Two€cytokinin„Ð ð@  Ðinduced€mRNAs€from€tobacco€and€soybean€€respond€to€both€stress€and€other€plant€growthÏregulators€(Crowell€òòet€alóó.,€1992;€Dominov€òòet€alóó.,€1992).€€L„22€is€clearly€not€a€message€of€thisÐ ° Ðtype.Ì€€€€€€The€L„221€probe€indicated€that€some€gene€specific€reduction€of€mRNA€abundance€occured€atÏ100€and€1000€ððM€cytokinin.€However,€the€pattern€of€reduction€was€not€consistent€within€orÏamong€cytokinin€concentrations.€This€mRNA€appears€to€be€reduced€in€abundance€by€certainÏconcentrations€of€certain€cytokinins€both€in€leaves€(this€work)€and€in€etiolated€cell€cultureÏ(McDaniel,€1996).€Therefore,€L„22€and€L„221€contrast€in€their€regulation€by€cytokinin.Ì€€€€€Polypeptide€sequence€database€searches€suggested€similarity€between€L„22€and€the€gammaÏsubunit€of€mitochondrial€Hòò+óó„transporting€ATPase€was€weak€but€might€be€functionally€significant.Ð °"  ÐAlthough€the€gamma€subunit€polypeptide€sequence€has€been€determined€for€several€plant€speciesÏincluding€òòZea€mays€óó(Dewey€òòet€alóó.,€1985)€and€òòV.€faba€óó(McFarlane€òòet€alóó.,€1990),€the€L„22€sequenceÐ p&À!$ Ðwas€most€similar€to€two€flagellate€kinetoplastid€ATPase€polypeptide€sequences€(Bhat€òòet€alóó.,€1990).€Ð P( #& ÐThe€similarity€with€the€gamma€subunit€was€independently€detected€as€two€separate€regions€thatÏmanual€alignment€showed€were€logically€arranged,€co„linear,€and€from€the€functionally€conservedÐ ,`'* Ðcarboxyl€terminal€70€residues€of€the€ATPase€subunit€(fig.€5;€Schneider€and€Altendorf€1987;ÏAbrahams€òòet€alóó.,€1994).Ð à Ѐ€€€€€Extensive€protein€sequence€divergence€is€common€among€members€of€this€protein€family.ÏHowever,€only€partial€conservation€of€the€five€residues€indicated€in€figure€2€is€very€unusualÏ(Steinemann€and€Lill,€1994)€as€they€form€part€of€a€critical€site€of€subunit€interaction€(Abrahams€etÏal.,€1994).€Other€explanations€for€the€highly€divergent€amino€acid€sequence€may€be€that€thisÏcDNA€is€encoded€by€an€ancient€òòatp6óó€pseudogene€that€has€transferred€to€the€nuclear€genome€orÐ ð@  Ðrepresents€convergent€evolution€in€an€unrelated€gene.€Future€experiments€will€concentrate€onÏidentification€and€localization€of€the€full€length€cDNA,€gene€and€protein€product.€Ì€€€€€€Cytokinin„modulation€of€a€subunit€of€an€ATPase,€or€analagous€enzyme,€could€partly€explainÏhow€cytokinin€treatments€can€control€plant€growth€and€development€(for€a€review€see€Musgrave,Ï1994).€Several€studies€have€linked€cytokinins€with€mitochondrial€function,€either€as€inhibitors€ofÏcyanide„resistant€electron€transport€in€the€inner€mitochondrial€membrane€(Miller,€1982;Ð 0€ ÐDizengremel€òòet€alóó.,€1982)€or€in€stimulating€mitochondrial€development€(Miller,€1982;€Morohashi,Ð ` Ð1984).€If€the€L„22€gene€product€can€be€proven€to€be€a€mitochondrial€protein€in€the€future,€the€L„22ÏcDNA€may€provide€a€novel€analytical€tool€for€these€phenomena.€Ð Ð  ÐÐ  °"  Ðà@[[9Vàò òMETHODSˆÐ ° ÐòòPlant€material€and€growth€conditions.ó óóó€òòPhaseolus€vulgarisóó€L.€cv.€'Great€Northern'€plants€wereÐ à Ðgrown€from€seed,€sown€2€to€a€3€gallon€nursery€pot€in€Promix€òòTMóó€in€a€greenhouse.€€Plants€wereÐ pÀ Ðwatered€daily€and€fertilized€weekly€with€Peter'sòòTMóó€plant€food€consisting€of€20%€nitrogen,€10%Ð P   Ðphosphorus,€and€20%€potassium.€€Temperatures€in€the€greenhouse€ranged€from€20òòoóóC€(night)€toÐ 0 € Ð30òòoóóC€(day).€€Leaf€treatments€were€performed€on€8€week„old€plants€with€6€vegetative€nodes.Ð `  Ðòòò òÌLeaf€treatmentsó óóó.€The€abaxial€surfaces€of€randomly€chosen€leaflets€of€separate,€greenhouse„grownÐ Ð  ÐòòP.€vulgarisóó€seedlings€were€painted€with€0,€10,€50,€100€or€1000€ððM€zeatin,€benzyladenine,Ð ° Ðthidiazuron,€adenine€or€methyladenine€which€were€dissolved€in€100€ððl€of€1€N€KOH,€diluted€in€20Ïml€of€water€and€adjusted€to€pH€7.8.€€As€a€surfactant,€Tween€20€was€added€to€1%€(v/v).€€€LeafletsÏon€separate€plants€were€treated€with€0,€10,€50,€or€1000€ððM€ABA,€auxin€(pichloram€as€auxin),€orÏGAòò3óó€dissolved€in€100€ððl€of€ethanol,€diluted€in€20€ml€of€water€and€adjusted€to€pH€7.8.€Tween€20Ð 0€ Ðwas€added€as€a€surfactant€to€1%€(v/v).€€Leaflets€were€harvested€at€0,€2,€4€or€24€h€after€treatment.Ìòòò òÌRNA€isolationsó óóó.€Leaf€RNA€was€extracted€in€buffer€(4.0€M€guanidinium€thiocyanate,€0.1€MÐ Ð  ÐTris.HCl,€1%€(v/v)€ „mercaptoethanol)€and€centrifuged€(30€min,€20,000xg€at€10òòoóóC).€€The€aqueousÐ °"  Ðlayer€was€removed,€mixed€with€10€ml€phenol/chloroform€(1/1,€v/v),€and€centrifuged€(15€min,Ï15,000xg€at€10òòoóóC).€€The€aqueous€layer€was€removed€and€loaded€onto€a€5.7€M€cesium€chlorideÐ p&À!$ Ðcushion€and€centrifuged€(35,000xg,€12€h€at€15òòoóóC).€Ð P( #& ÐÌòòò òIsolation€of€cDNA€clonesó óóó.€The€€cDNA€clones,€pL„22€and€L221€were€isolated€from€a€libraryÐ ,`'* Ðsynthesized€from€mRNA€from€the€leaves€of€non„treated€21€d€old€òòP.€vulgarisóó€seedlings€as€describedÐ ° Ðby€Lightfoot€òòet€alóó.,€(1988)€except€that€double„stranded€€cDNA€was€inserted€into€the€òòPstóó€I€site€ofÐ à ÐpUC9€vector€by€homopolymer€tailing€(Maniatis€et€al.,€1992).€€Annealants€were€transformed€intoÏòòEscherichia€colióó€JM83€and€single€colonies€picked€into€microtiter€plates.€€DNA€was€prepared€fromÐ P   Ð1/2€each€culture€plate€as€a€mixed€DNA€pool.€€Plasmids€were€then€used€to€transform€òòE.colióó€strainÐ 0 € ÐXL1€blue.€Plasmid€DNA€from€43€single€colonies€and€10€known€gene€probes€was€extracted€andÏused€to€synthesize€probes€which€were€used€for€slot€blot€hybridization€to€RNA€from€cytokinin„¼treated€cell€cultures€(Crowell€òòet€alóó.,€1990).€Probes€which€appeared€to€detect€mRNAs€thatÐ Ð  Ðaccumulated€in€response€to€cytokinin€treatment€were€selected€for€gene€specific€analysis€by€S1Ïnuclease€protection.Ð à ÐÌòòò òSòò1óó€nuclease€protection€assays.ó óóó€The€cDNA€inserts€were€€labeled€using€the€probe€synthesis€reactionÐ P  Ðof€T4€DNA€polymerase€(Maniatis€òòet€alóó.,€1992).€For€L„22€specific€probes,€the€172€bp€òòEcoóóRI/òòPstóóIÐ 0€ Ðfragment€containing€134€bp€of€L„22€cDNA€coding€region,€14€bp€of€Poly[C]€and€24€bp€of€theÏpUC9€multi„cloning€site€(Fig.€1)€was€used.€For€L„221€specific€probes,€the€283€bp€òòHindóóIII/òòHaeóóIIIÐ ð@ Ðfragment€containing€253€bp€of€L„221€cDNA€coding€region,€4€bp€of€Poly[C]€and€26€bp€of€theÏpUC9€multi„cloning€site€was€used.€For€chalcone€synthase€specific€probes,€the€372€bp€€òòPvuóóIIÐ °"  Ðfragment€from€coding€region€of€the€€CHS„17€cDNA€€was€used€(Ryder€òòet€alóó.,€1987).€ExonucleaseÐ $à" Ðdigestion€of€1€ððg€of€each€restriction€fragment€by€1€unit€of€T4€DNA€polymerase€occurred€at€27€òòoóóCÐ p&À!$ Ðfor€5€min.€Probe€synthesis€was€initiated€by€the€addition€of€dATP,€dTTP€and€dGTP€to€0.25€mM,Ï1MBq€of€[òò32óóP]„dCTP€(1€Tbq€mMòò„1óó)€and€proceeded€at€37òòoóóC€for€1€h.€To€ensure€completeÐ 0*€%( Ðpolymerization€additional€dATP,€dCTP,€dGTP€and€dTTP€were€added€to€0.5€mM€and€the€reactionÐ ,`'* Ðincubated€for€another€15€min.€€The€reaction€was€stopped€by€the€addition€of€50€mM€EDTA€and€theÏlabelled€DNA€was€purified€by€ethanol€precipitation.Ìà Ü à€Aliquots€of€10€ððg€total€RNA€were€dried€with€20,000€cpm€of€each€labeled€probe,Ïresuspended€and€hybridized€overnight€in€10€ððl€of€50%€formamide,€0.4€M€NaCl,€2€mM€EDTA,€20ÏmM€€3„(N„morpholino)propanesulfonic€acid€at€pH€7.2.€€The€single€stranded€nucleic€acids€wereÏdigested€by€1000€units€of€Sòò1óó€nuclease€for€90€min€at€20òòoóóC€in€0.3€M€NaCl,€30€mM€sodium€acetateÐ `  Ð(pH€4.6),€1€mM€ZnSOòò4óó,€20€ððg€mlòò„1óó€calf€thymus€DNA.€€The€protected€fragments€were€purified€byÐ ð@  Ðethanol€precipitation€and€separated€by€electrophoresis€on€a€6%€polyacrylamide,€7€M€urea,Ïsequencing€gel€for€4€h€at€1800€V.€Gels€were€fixed,€dried€and€used€to€expose€presensitized€film€at€„¼70òò€oóóC€for€1€h€to€3€d.€€Scanning€laser€densitometry€was€used€to€compare€intensities€ofÐ à Ðautoradiograph€bands.€€The€peak€areas€were€calculated€in€arbitrary€units.€mRNA€abundance€wasÏdetermined€from€the€mean€values€from€2„4€experimental€replications€and€2„3€gel€replications€perÏexperiment.€Data€was€expressed€in€arbitrary€units€of€optical€density€relative€to€control€samples.Ì€€€€€€The€quantitative€nature€of€the€Sòò1óó€assays€were€ensured€with€several€safeguards.€FirstlyÐ ` Ðsaturation€experiments,€where€increasing€concentrations€of€L„22,€L221€and€CHS„17€were€added€toÏknown€concentrations€of€RNA€and/or€unlabelled€probe€DNA,€were€conducted€for€each€cDNA€toÏensure€that€probe€DNA€was€in€€greater€than€10„fold€molar€excess€to€target€mRNA€and€toÏapproximately€quantify€the€band€intensities€observed.€Secondly,€three€concentrations€of€the€sameÏRNA€sample€were€hybridized€with€a€probe€and€the€band€intensities€were€shown€to€be€linear€withÏincreasing€concentrations€of€RNA€used.€Thirdly,€samples€were€loaded€in€a€randomized€array€toÏexclude€gel€effects€on€band€intensities.Ìòòò òÐ ,`'* ÐStatistical€analysisóó.€ó óStatistical€significance€was€calculated€using€one„way€analysis€of€variance€orÐ ° Ðlinear€regression€performed€with€SAS€(SAS€Institute€Inc.,€Cary,€NC).€€The€results€wereÏconsidered€significantly€different€from€the€control€when€òòPóó€had€a€value€less€than€0.01.Ð pÀ ÐÌòòò òDNA€sequence€analysis.óóó ó€Sequence€determinations€were€performed€by€Oregon€State€UniversityÐ 0 € ÐCenter€for€Gene€Research€and€Biotechnology€(Corvallis,€OR)€by€the€dideoxy€chain„terminationÏmethod.€€Homology€searches€were€carried€out€using€NCBI€blast€e„mail€server€(Altschul€òòet€alóó.,Ð ð@  Ð1990)€to€search€both€non„redundant€nucleotide€and€peptide€sequence€databases.€ConservativeÏchanges€were€determined€using€the€PAM€120€matrix€(Altschul,€1993)€for€pairwise€amino€acidÏcomparisons€scoring€neutral€or€positive€bits€as€similarity.ÌÔ€ZX: XXAX3ÝÔÐ  pÀ ÐÔ€Zô5Åô XZX:ÔÔ€Yô5ÅôôZô5ÅÔà@bb7VàREFERENCESˆÐ ° ÐÓ€Óò òAbrahams€J.P.,€Leslie€A.G.W.,€Lutter€R.€and€Walker€J.E.,€1994ó ó.€Structure€at€2.8€AÐ @ Ðresolution€of€Fòò1óó„ATPase€from€bovine€heart€mitochondria.€òòNatureóó,€ò ò370ó ó,621„628.Ð l¼ ÐÌò òAltschul€S.F.,€Gish€W.,€Miller€W.,€Myers€E.W.€and€LipmanD.J.,€1990.ó ó€Basic€localÐ Ä  Ðalignment€search€tool.€€òòJ.€Mol.€Biol.óó,€215,403„410.Ð ð @  ÐÌò òAltschul€S.F.,€1993.ó ó€A€protein€alignment€tool€sensitive€at€all€evolutionaryÐ H ˜ Ðdistances.€òòJ.€Mol.€Evol.óó,€ò ò36ó ó,290„300.Ð tÄ  ÐÌò òBhat€G.J.,€Kostowsky€D.J.,€Feagin€J.E.,€Smiley€B.L.€and€Stuart€K.,€1990.ó ó€AnÐ Ì  Ðextensively€edited€mitochondrial€transcript€in€kinetoplastids€encodes€a€proteinÏhomologous€to€ATPase€subunit€6.€€òòCell,óó€ò ò61ó ó,885„894.Ð $t ÐÌò òChen€C.€and€Leisner€S.,€1985.ó ó€€Cytokinin„modulated€gene€expression€in€excisedÐ |Ì Ðpumpkin€cotyledons.€€òòPlant€Physiol.,óó€€ò ò77ó ó,99„103.€Ð ¨ø ÐÌò òCotton€J.,€Ross€C.,€Byrne€D.€and€Colbert€J.,€1990.ó ó€€Down„regulation€ofÐ P" Ðphytochrome€mRNA€abundance€by€red€light€and€benzyladenine€in€etiolated€cucumberÏcotyledons.€€òòPlant€Mol.€Biol.,óó€€ò ò14ó ó,707„714.€Ð X¨& ÐÌò òCrowell€D.N.,€1994.ó ó€Cytokinin€regulation€of€a€soybean€pollen€allergen€gene.€Ð °* ÐòòPlant€Mol.€Biol.óó,€ò ò25ó ó,829„835.Ð Ü,, ÐÌò òCrowell€€D.N.,€Kadlecek€A.T.,€Manorama€C.J.€and€Amasino€R.,€1990.ó ó€€Cytokinin„Ð 4!„0 Ðinduced€mRNAs€in€cultured€soybean€cells.€€òòProc.€Natl.€Acad.€Sci.€USAóó,€ò ò87ó ó,8815„Ð `"°2 Ð8819.€ÌÌò òCrowell€D.N.,€Maliyakal€J.E.,€Russell€D.€and€Amasino€R.M.,€1992.ó ó€CharacterizationÐ ä%4!8 Ðof€a€stress„induced,€developmentally€regulated€gene€family€from€soybean.€€òòPlantÐ '`": ÐMol.€Biol.,óó€€ò ò18ó ó,459„466.€Ð <(Œ#< ÐÌò òDehio€C.€and€de€Bruijn€F.,€1992ó ó.€€The€early€nodulin€gene€òòSrEnod2óó€from€òòSesbaniaÐ ”*ä%@ Ðrostrataóó€is€inducible€by€cytokinin.€òò€Plant€J.óó,€€ò ò2ó ó,117„128.Ð À+'B Їò òDewey€R.E.,€Levings€III€C.S.€and€Timothy€D.H.,€1985.ó ó€Nucleotide€sequence€ofÐ ° ÐATPase€subunit€6€gene€of€maize€mitochondria.€ò òPlant€Physiol.,ó ó€79,914„919.Ð Ü, ÐÌò òDizengremel€P.,€Chauveau€M.€and€Roussaux,€J.,€1982.ó ó€Inhibition€by€adenineÐ 4„ Ðderivatives€of€the€cyanide„insensitive€electron€transport€pathway€of€plantÏmitochondria.€òòPlant€Physiol.,óó€ò ò70ó ó,585„593.Ð Œ Ü  ÐÌò òDominov€J.,€Stenzler€L.,€Lee€S.,€Schwarz€J.,€Leisner€S.€and€Howell€S.,€1992ó ó.€Ð ä 4 ÐCytokinins€and€auxins€control€the€expression€of€a€gene€in€òòNicotianaÐ `  Ðplumbaginifoliaóó€cells€by€feedback€regulation.€€òòPlant€Cellóó,€ò ò4ó ó,451„461.€Ð <Œ  ÐÌò òFeierabend€J.,€1980.ó ó€Influence€of€cytokinin€on€plastid€biogenesis€in€rye€leaves.Ð ”ä  ÐIn€òòMetabolism€and€molecular€activities€of€cytokininsóó,€Guern€J.€and€Peaud„LenoelÐ À ÐC.,€ed.,€Springer„Verlag,€Berlin,€252„60.€ÌÌò òFlores€S.€and€Tobin€E.M.,€1989.€ó ó€Benzyladenine€modulation€of€the€expression€ofÐ D” Ðtwo€genes€for€nuclear„encoded€chloroplast€proteins€in€òòLemna€gibbaóó:€€ApparentÐ pÀ  Ðpost„transcriptional€regulation.€€òòPlantaóó,€ò ò168ó ó,340„349.€Ð œì" ÐÌò òGuern€J.€and€Peaud„Lenoel€C.,€1981.ó ó€Metabolism€and€molecular€activities€ofÐ ôD& Ðcytokinins,€Spring„Verlag,€New€York,€USA.€ÌÌò òKende€H.€and€Gardner€G.,€1976.€ó ó€Hormone€binding€in€plants.€€òòAnnu.€Rev.€PlantÐ xÈ, ÐPhysiol.,óó€€ò ò27ó ó,267„290.€Ð ¤ô. ÐÌò òLetham€D.S.€and€Palni€L.M.S.,€1983ó ó.€The€biosynthesis€and€metabolism€ofÐ ü!L2 Ðcytokinins.€€òòAnnu.€Rev.€Plant€Physiol.óó,€€ò ò34ó ó,163„197.€ò òÐ (#x4 ÐLightfoot€D.A.,€Green€N.K.€and€Cullimore€J.V.,€1988.ó ó€€The€chloroplast€locatedÐ T$¤6 Ðglutamine€synthetase€of€òòPhaseolusóó€òòvulgarisóó€L.:€€nucleotide€sequence,€expressionÐ €%Ð 8 Ðin€different€organs€and€uptake€into€isolated€chloroplasts.€òòPlant€Molec.€Biol.óóÐ ¬&ü!: Ðò ò11ó ó,191„202.Ð Ø'(#< Ðò òÌLu€J.,€Ertl€J.€and€Chen€C.,€1992.ó ó€Transcriptional€regulation€of€nitrate€reductaseÐ 0*€%@ ÐmRNA€levels€by€cytokinin„abscisic€acid€interactions€in€etiolated€barley€leaves.€Ð \+¬&B ÐòòPlant€Physiol.,óó€ò ò98ó ó,1255„1260€Ð ° ÐÌò òManiatis€T.,€Sambrook€J.,€and€Fritsch€E.F.€1992ó ó.€Molecular€cloning:€€A€laboratoryÐ X Ðmanual.€€Cold€Springs€Harbor€Laboratory,€Cold€Springs€Harbor,€N.Y.,€USA,€117„121.ÌÌò òMatsubara€S.,€1990.ó ó€€Structure„activity€relationships€of€cytokinins.€€òòCrit.€Rev.Ð Œ Ü  ÐPlant€Sci.óó,€ò ò9ó ó,17.€Ð ¸   ÐÌò òMcDaniel€K.L.,€1996.ó ó€Cytokinin€modulation€of€gene€specific€mRNA€abundance:€TheÐ `  Ðeffects€of€different€cytokinins€in€òòPhaseolusóó€òòvulgarisóó€òòL.óó€òòPh.D.€Thesisóó,€SouthernÐ <Œ  ÐIllinois€University€at€Carbondale,€USA.ÌÌò òMcFarlane€J.L.,€Wahleithner€J.A.€and€Wolstenholme€D.R.,€1990ó ó.€A€broad€beanÐ À Ðmitochondrial€òòatp6óó€gene€with€an€unusually€simple,€non„conserved€5'€region.€òòCurr.Ð ì< ÐGenet.,óó€ò ò18ó ó,87„91.Ð h ÐÌò òMemelink€J.,€Hoge€J.H.C.€and€Schilperoort€R.A.,€1987.ó ó€Cytokinin€stress€changesÐ pÀ  Ðthe€developmental€regulation€of€several€defence„related€genes€in€tobacco.€€òòEMBOÐ œì" ÐJ.,óó€€ò ò12ó ó,3579„3583.€Ð È$ ÐÌò òMiller€C.O.,€1982.ó ó€Cytokinin€modification€of€mitochondrial€function.€òòPlantÐ  p( ÐPhysiol.,óó€ò ò69ó ó,1274„1277.€€Ð Lœ* ÐÌò òMorohashi€Y.,€1984.ó ó€Effect€of€benzyladenine€on€the€development€of€mitochondrialÐ ¤ô. Ðactivities€in€imbibed€black€gram€cotyledons.€òòJ.€Plant€Physiol.óó,€ò ò116ó ó,235„240.Ð Ð 0 ÐÌò òMusgrave€M.E.,€1994ó ó.€Cytokinins€and€oxidative€processes.€In€òòCytokinins:Ð (#x4 ÐChemistry,€action€and€function,óó€Mok€D.W.S.€and€Mok€M.,€CRC€Press,€Boca€Raton,Ð T$¤6 Ð167„178.ÌÌò òNeuman€D.S.,€Rood€S.B.€and€Smit€B.A.,€1990.ó ó€Does€cytokinin€transport€from€root„Ð Ø'(#< Ðto„shoot€in€the€xylem€sap€regulate€leaf€responses€to€root€hypoxia?€òò€J.€Exp.€Bot.,óóÐ )T$> Ðò ò41ó ó,1325„1333.€Ð 0*€%@ ÐÐ \+¬&B Ðò òNooden€L.D.€and€Letham€D.S.,€1993.ó ó€€Cytokinin€metabolism€and€signalling€in€theÐ ° Ðsoybean€plant.€òòAust.€J.€Plant€Physiol.,óó€ò ò20ó ó,639„653.Ð Ü, ÐÌò òParthier€B.,€Lerbs€S.€and€Klyachko€N.L.,€1980.ó ó€Plastogenesis€and€cytokininÐ 4„ Ðaction.€€Cytokinin€and€light€interactions€in€plastid€enzyme€formation€of€detachedÏòòCucurbitaóó€cotyledons.€€In€€€òòMetabolism€and€Molecular€Activities€of€Cytokininsóó,Ð Œ Ü  ÐGuern€J.€and€Peaud„Lenoel€C.,€ed.,€Springer„Verlag,€€New€York,€274„286.€€ÌÌò òRyder€J.B.,€Hendrick€S.A.,€Bell€J.N.,€Liang€X.,€Clouse€D.€and€Lamb€C.J.,€€1987.ó óÐ `  ÐOrganization€and€differential€activation€of€a€gene€family€encoding€plant€defenseÏenzyme€chalcone€synthase€in€òòPhaseolus€vulgarisóó.€€òòMol.€Gen.€Genet.,óó€€ò ò210ó ó,219„233.€Ð h¸  ÐÌò òSchneider€E.€and€Altendorf€K.,€1987ó ó.€Bacterial€adenosine€5'„triphosphate€synthaseÐ À Ð(Fòò1óóFòò0óó):€Purification€and€reconstitution€of€Fòò0óó€complexes€and€biochemical€andÐ ì< Ðfunctional€characterization€of€their€subunits.€òòMicrobiol.€Rev.,óó€ò ò51ó ó,477„497.Ð h ÐÌò òShinshi€H.,€Mohnen€D.€and€Meins€F.,€1987.ó ó€€Regulation€of€a€plant€pathogenesis„Ð pÀ  Ðrelated€enzyme:€€Inhibition€of€chitinase€and€chitinase€mRNA€accumulation€inÏcultured€tobacco€tissues€by€auxin€and€cytokinin.€€òòProc.€Natl.€Acad.€Sci.€€USA,óóÐ È$ Ðò ò84ó ó,89„93.€Ð ôD& ÐÌò òSingh€S.,€Letham€D.S.,€Jameson€P.E.,€Zhang€R.,€Parker€C.W.,€Bandencoch„Jones€J.Ð Lœ* Ðand€Nooden€L.D.,€1988.ó ó€Cytokinin€biochemistry€in€relation€to€leaf€senescence.Ð xÈ, ÐòòPlant€Physiol.,óó€ò ò88ó ó,788„794.€Ð ¤ô. ÐÌò òSkoog€F.€and€Armstrong€D.,€1970.ó ó€Cytokinins.€€òòAnnu.€Rev.€Plant€Physiol.,óó€€ò ò21ó ó,359„Ð ü!L2 Ð384.€ÌÌò òSossountzov€L.,€MaldineyR.,€Sotta€B.,€Sabbagh€I.,€Habricot€Y.,€Bonnet€M.€andÐ €%Ð 8 ÐMiginiac€E.,€1988ó ó.€Immunocytochemical€localization€of€cytokinins€in€òòCraigellaóóÐ ¬&ü!: Ðtomato€and€a€sideshootless€mutant.€€òòPlantaóó,€ò ò175ó ó,291„304.€Ð Ø'(#< ÐÌò òSteinemann€D.€and€Lill€M.,€1994.ó ó€€The€gamma€subunit€of€ATPase€of€òòSpirulinaÐ 0*€%@ Ðplatensisóó:€€A€new€principle€of€thiol€modulation€of€Fòò0óóFòò1óó€ATP€synthase?€òòGenbank,Ð \+¬&B ÐAccessionóó,€ò òZ46799ó ó,€Locus,€SPATSGSU.Ð ° ÐÌò òTeyssendier€de€la€Serve€B.,€Axelos€M.,€and€Peaud„Lenoel€C.,€1985.ó ó€€CytokininÐ X Ðmodulates€the€expression€of€genes€encoding€the€protein€of€light„harvestingÏchlorophyll€òòa/bóó€complex.€òòPlant€Mol.€Biol.,óó€€ò ò5ó ó,155„163.€Ð ` ° ÐÌò òWattillon€B.,€Kettman€R.,€Boxus€P.€and€Burny€A.,€1991.ó ó€Characterization€of€twoÐ ¸   Ðgene€transcripts€modulated€by€cytokinins€in€micropropagated€apple€(òòMalusÐ ä 4 Ðdomesticaóó€[L.]€Borkh)€plantlets.€òòPlant€Physiol.óó,€ò ò96ó ó,479„485Ð `  ÐÓ€ÓÔ€YX: XôYô5ÅÔÔ€ZX: X XYX:ÔÐ  <Œ  Ðò òFigure€1.ó ó€€L„22€mRNA€accumulation€at€various€doses€ofÏcytokinin€in€òòòòóóP.€vulgarisóó€leaves€4€h€after€treatment.€AÏquantitative€Sòò1óó€nuclease€protection€assay€of€CHS„17Ï(A),€L„221€(B)€and€L„22€(C)€mRNA€abundance.€UndigestedÏprobe€was€372€bp€(A),€283€bp€(B)€and€172€bp€(C).€TheÏprotected€fragment€was€372€b€(A),€253€b€(B)€and€134€bÏ(C).€The€114€b€fragment€(C)€may€be€an€exonucleolyticÏproduct,€processing€intermediate€or€L„22€homologue.€L„¼22€mRNA€abundance€was€estimated€by€densitometry€fromÏthe€autoradiogram,€compared€to€the€cytokinin€doseÏplotted€as€a€logòò10óó„linear€scale€(D).€€€ÌÇò òFigure€2.€ó ó€L„22€mRNA€accumulation€at€various€doses€andÏtimes€after€treatment€with€cytokinins.€A,ÏBenzyladenine;€B,€Thidiazuron;€C,€Zeatin.€DensitometricÏquantification€from€Sòò1óó€nuclease€protection€assays€of€L„¼22€mRNA€abundance€in€leaves€treated€with€differentÏconcentrations€of€cytokinins€at€2,€4€and€24€h€afterÏtreatment.€Mean€data€from€4€replicated€experiments€areÏshown,€T€bars€indicate€the€variation€observed,€*Ïindicates€a€treatment€was€significantly€different€fromÏthe€control€(òòPóó<0.01).ÌÇò òFigure€3.ó ó€€L„22€mRNA€accumulation€at€various€doses€andÏtimes€after€treatment€with€an€auxin,€gibberellin€andÏabcisic€acid.€Densitometric€quantification€from€Sòò1óóÏNuclease€protection€assays€of€L„22€mRNA€abundance€inÏleaves€treated€with€an€auxin€(pichloram),€abcisic€acidÏ(ABA),€and€gibberellin€(GAòò3óó).€(A),€4€h€and€(B),€24€hÏafter€PGR€treatment.ò òó óò òó óò òó óò òó ó€Mean€data€from€4€replicatedÏexperiments€are€shown.€T€=€+/„€1€unit€indicates€thatÏthe€T€bars€represent€the€variation€observed.€No€*Ïindicates€that€no€treatment€was€significantly€differentÏfrom€the€control€(òòPóó<0.01).Çò òFigure€4.ó ó€Nucleotide€and€deduced€amino„acid€sequencesÏof€the€L„22€cDNA.€The€òòPstóóI€site€used€in€probe€synthesisÏis€underlined.ÌThe€nucleotide€and€amino€acid€sequences€were€submittedÏto€GenBank€and€assigned€accession€number€U28921.ÌÇò òFigure€5.ó ó€Comparison€of€the€amino€acid€sequenceÏpredicted€for€the€L„22€cDNA€with€the€C„Terminal€aminoÏacid€sequence€of€the€gamma€subunit€of€ATPase€fromÏmitochondria€of€òòVicia€fabaóó€(Vif)€and€òòTrypanosoma€bruceióóÏ(Tyb).€Amino€acids€which€are€identical€(*)€or€conservedÏ(+)€in€the€PAM€120€matrix€(Altschul,€1990,€1993)€areÏindicated.€Amino€acids€conserved€across€all€reportedÏsequences€(ðð)€are€indicated.€Gaps€introduced€toÏmaximize€the€alignment€with€the€consensus€plant€proteinÏsequence€(„)€are€indicated€(positioned€after€Bhat€òòetÏalóó.,€1990).ÌÇÓÓÌ1€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€Ï€€60ÌAAGAGTTATAATATTTTGCAAAAATACAGGGAATGCTGTAGATTATACAGCTCAA¼GATATÌ€€E€€V€€I€€I€€F€€C€€K€€N€€T€€G€€N€€A€€V€€D€€Y€€T€€A€€Q€ÏD€€I€€Ì€€€€Ì61€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€Ï€€120ÌTCAAAGTTTCATCCCTCCACATACATCATATGTGAGGTTGTTTTGTTCTTTAAGA¼GTTTCÌ€€Q€€S€€F€€I€€P€€P€€H€€T€€S€€Y€€V€€R€€L€€F€€C€€S€€L€€R€ÏV€€S€€€€ÌÌ121€€€€€€€_òòPstóóI_€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€Ï€€180ÌCCATTTCAGTCTGCAGATGACAGTACACTCATTAATGCCATTTTGCACTTTATTG¼TGCCGÌ€€H€€F€€S€€L€€Q€€M€€T€€V€€H€€S€€L€€M€€P€€F€€C€€T€€L€€L€ÏC€€RÌÌ181€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€Ï€€240ÌAATTATATTTGAATCAGGAATTTTTCTTGTTCATCTCTGTGTAATGTTTTTAATT¼AACATÌ€€I€€I€€F€€E€€S€€G€€I€€F€€L€€V€€H€€L€€C€€V€€M€€F€€L€€I€ÏN€€IÌÌ241€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€Ï€€300ÌTATGATCAAATATAGCTACTCTAAACAGTAATATTAAACTTTTTATTTGACCAAA¼AAAAAÌ€€M€€I€€K€€Y€€S€€Y€€S€€K€€Q€stopÌÌ301€€€€€€€€€€€€€€€€€€€€389€€€€€€€€€€€€€€€€€€€€€ÌAAAAATAAAATATTATGGAGTG[A]òò67óóÌÌÇÑ  ÑÑ  ÑÑ\RØ'3\ÑÌVif€€(173)€FLLPAGVPLPLAPFLVLLELISYCFRALSLGIRLFANMMAGHSLVKILSGFÌ€€€€€€€€€€€€++€€€€€€€€*€€€+€€++€*€€+€+€+€€+***€++€++*€€+++€€ÌL„22€(001)€EVIIFCKNTGNAVDYTAQDIQS„FIPPHTSYVRLFCSLRVSHFSLQMTVHSÌ€€€€€€€€€€€€€€€*€€€€€€€€€€€€€+€€*€+€€€€++++****+*€€***€*€*€€€€ÌTyb€€(114)€VGDSFMDVFFIRFLLCFLECFSLLCRCLSTFLRLFCNLLSSHFLLLMFFDFÌ€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ðð€€ðð€€€€€€€€€€ðððð€€ððÌ€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€ÌÌVif€€(225)€AWTMLCMNDLLYFIGDLGPLFIVLALTGPELGVAISQAHVSTISICIYLNDATNLHQTCLLFIYNÌ€€€€€€€€€€€€€€€€€€€€€€€+€€€+€€€€*€*+€€€€€+€++*€€€€€*€€€€€€€€€€€ÌL„22€(51)€€LMPFC„„„„„TLLCRIIFESGIFLVHLCVMFLINIM„IKYSYSKQÌ€€€€€€€€€€€€€€+€€€€€€+++€€€€*€€€*+€++€**+**€€*+€€€+€+€€€€€€€€ÌTyb€€(166)€FYFIF„„„„„VFFFLWCFLLLIYFIYFCVLFLFIIL„CVFIFVGFICRHITVIYFLÌÇÑ \ ÑÑ , ÑÑ \ ÑÑ , ÑÑ  ÑÑ  ÑÑ\R3Ø'\ÑÓÓÑ#€ZZXd#ÑAcknowledgements.€We€thank€Dr.€N.€Bassil,€Dr.€M.C.€MokÏDr.€D.W.S.€Mok€and€Dr.€J.€Preece€for€helpfulÏdiscussions€during€the€initial€stages€of€this€project.ÏFor€financial€support€we€thank€the€Department€of€PlantÏBiology€for€the€T.A.€to€KMcD;€the€ISPOB€for€a€researchÏenhancement€award;€and€the€IBHE€for€MBI€hatch€funds.ÌÌÌÇÌÌÌÌÌภ..¸ àà..ààh..hààÀ..Àà€€€à..à€€€òòA:€4€hóóÌÌÌÌÌÌÌÌÌÌÌÌÌÌÌÌÌÌÌÌÌÌÌÌÌ̇ÌÑ€[Ñภ..¸ àà..ààh..hààÀ..Àà€€à..à€€€€òòB:€24€hóóÇÌÌÌÌÌภ..¸ àà..ààh..hààÀ..Àà€€òòA:€Benzyladenine.óóÌÌÌÌÌÌÌÌÌÌÌÌÌÌÌÌÌÌÌÑ€\Ñภ..¸ àà..ààh..hààÀ..Àà€€€òòB:€Thidiazuron.óóÌÌÌÌÌÌ̇ÌÌÌÌÌÌÌÌÌÌÌÑ€]Ñภ..¸ àà..ààh..hààÀ..Àà€€à..àòòC:€Zeatin.óóÇÔ€¼¼¼ÔÔU€A*Ͼ¦ `(CG TimesScalableUÔÌÌÌÌÌÌÌÌÌDecember€27,€1994ÌÌJan€A.D.€Zaevert,€Editor,ÌPlant€Physiology,ÌMSU„DOE€Plant€Research€Laboratory,ÌMichigan€Stat€University,ÌEast€Lansing,ÌMI€48824„1312.ÌÌÌDear€Dr.€Zaevert,ÌÌplease€find€enclosed€a€revised€version€of€a€paper€submitted€to€youÏin€April,€1994.€We€feel€we€have€made€major€improvements€asÏsuggested€by€the€reviewers€and€feel€it€is€now€a€solid€contribution.€Would€you€be€so€kind€as€to€review€it€before€sending€it€out€forÏreview€so€that€we€might€not€delay€further.€I€enclose€the€reviewersÏcomments€for€your€information.Ì€€€In€addition€I€enclose€a€second€manuscript€for€the€short€reportÏsection.€Co„publication€of€the€two€manuscripts€would€be€useful€toÏyour€readers.€Many€Thanks.ÌÌSincerely€yours,ÌÌÌDavid€A.€LightfootÌAssistant€Professor€ÌòòòòóóóóÇÌÌÌÌÌÌÌMarch€30,€1995ÌÌÌPlant€Physiology,ÌDeborah€Weiner,€Managing€Editor,€€ÌAmerican€Society€of€Plant€Physiologists,Ì15501€Monona€Drive,ÌRockville,€€€€ÌMD€20855„2768ÌÌDear€Dr.€Weiner,ÌÌplease€find€enclosed€a€revised€version€of€a€paper€submitted€to€youÏin€April,€1994.€We€feel€we€have€made€major€improvements€asÏsuggested€by€the€reviewers€and€feel€it€is€now€a€solid€contribution.ÌÌSincerely€yours,̇ÌDavid€A.€LightfootÌAssistant€Professor€ÌòòòòóóóóÇÌÌÌÌÌÌÌMay€30,€1995ÌÌÌPlanta,ÌProfessor€R.€L.€Jones,€€€€€€€€€€€€ÌUniversity€of€California,€€€ÌDepartment€of€Plant€Biology,ÌBerkeley,€€€€€€ÌCA€94720€USA.ÌÌDear€Prof.€Jones,ÌÌplease€find€enclosed€a€paper€for€submission€to€the€journal€Planta.ÏFrom€the€list€of€editors€E.€Tobin€or€P.B.€Green€might€be€familiarÏwith€the€research€field€covered€by€this€manuscript.ÌÌSincerely€yours,̇ÌDavid€A.€LightfootÌAssistant€Professor€ÌòòòòóóóóÇÌÌÌÌÌÌÌOctober€20,€1995ÌÌÌPlant€Physiology€and€Biochemistry,ÌProfessor€Mark€Jacobs,€€€€€€€€€€€€ÌDepartment€of€Biology,ÌSwarthmore€College,€€€€€€ÌSwarthmore,ÌPennsylvania€19081,ÌUSA.ÌÌDear€Prof.€Jacobs,ÌÌplease€find€enclosed€a€paper€for€submission€to€the€journal€PlantÏPhysiology€and€Biochemistry.€It€represents€the€first€paper€from€theÏPh€D.€thesis€of€K.€Mc€Daniel.€I€received€your€instructions€forÏauthors€whilst€attending€the€International€Nitrogen€Assimilation€Symposium€in€Darmstadt€and€have€become€an€avid€reader€of€yourÏJournal.ÌÌSincerely€yours,ÌÌÌDavid€A.€LightfootÌAssistant€Professor€ÌòòòòóóóóÇÌÌÌÌÌÌÌMay€20,€1996ÌÌÌPlant€Physiology€and€Biochemistry,ÌProfessor€Mark€Jacobs,€€€€€€€€€€€€ÌDepartment€of€Biology,ÌSwarthmore€College,€€€€€€ÌSwarthmore,ÌPennsylvania€19081,ÌUSA.ÌÌDear€Prof.€Jacobs,ÌÌplease€find€enclosed€a€paper€for€publication€by€the€journal€PlantÏPhysiology€and€Biochemistry.€It€represents€the€first€paper€from€theÏPh€D.€thesis€of€K.€Mc€Daniel.€I€include€below€a€point€by€pointÏresponse€to€the€reviewers€comments.ÌAlso€a€copyright€release€form€for€figures€1„3€for€the€book€chapterÏto€be€published€in€1997.ÌÌSincerely€yours,ÌÌÌDavid€A.€LightfootÌAssistant€Professor€ÌòòòòóóóóÌÌReviewer€1.ÌÌ1.€Page€10,€Line€9.€A€line€was€added€to€make€clear€that€L„22Ïinduction€was€not€greater€in€etiolated,€detached€organ€or€cellÏculture€assays€than€in€the€illuminated,€whole€plant,€leaf€assay€weÏreported€here.ÌÌ2.€Page€15€Line€20.€I€added€enough€description€of€the€cDNAÏsynthesis€that€using€the€reference€material€the€clone€isolation€wouldÏbe€reproducible.ÌÌ3.€Page€9€lines€8„10.€Page€12€Line€20.€Added€descriptions€of€theÏidentification€of€the€homologous€region€as€two€separate€regions€byÏBLAST€and€the€discovery€the€were€linearly€and€logically€arranged.Ì€€€Page€13€line€2.€We€describe€why€a€longer€cDNA€will€not€beÏinformative€on€the€identity€of€the€protein€product,€at€least€as€far€asÏhomology€to€gamma„ATPase.ÌÌÌReviewer€2.ÌÌ1.€Figure€1A.€I€would€prefer€to€keep€panel€A€since€that€mRNAÏwas€not€repressed€by€cytokinin€as€was€the€mRNA€in€panel€B€atÏcertain€concentrations.€In€addition€we€did€several€mRNA€minusÏcontrols€with€this€probe€to€be€sure€that€the€band€was€protectedÏmRNA€and€not€just€reannealed€probe.ÌÌ2.€€Page€9.€Modified€to€moderate€claims€of€positive€identification.ÌÌ3.€Change€in€cytokinin€implied€the€plant€cell€sensed€the€alterationÏin€cytokinin€separately€from€the€concentration,€hence€the€responseÏwas€transient.ÌÌ4.€Deleted€the€word€an.ÌÌÌEditors€Comments.ÌÌ1.€Discussion.€Rewritten€to€be€more€modest€(shorter€too).ÌÌ2.€Fig€1A.€I€strongly€feel€this€should€be€retained€see€point€1Ïreviewer€2.€In€our€paper€describing€the€responses€to€cytokinin€inÏetiolated€cell€culture€that's€in€preparation€we€show€L221€isÏrepressed€by€cytokinins.€Hence€it€isn't€a€very€perfect€control€(noteÏsome€"repression"€in€panel€B).ÌÌ3.€Same€as€1.€Being€clear€and€explicit€is€always€harder€than€itÏsounds.ÌÇò òó óภ..¸ àSòò1óó€nuclease€protection€assays€were€used€to€quantify€L„22€andÏchalcone€synthase„17€(CHS)€mRNA€abundance.€€Since€CHS€wasÏnot€cytokinin„responsive€and€is€a€stress€response€gene,€it€was€usedÏto€confirm€that€observed€differences€in€mRNA€abundance€were€notÏdue€to€cytokinin€stress.€€Both€cDNAs€were€used€in€labelingÏdifferent€labeling€reactions€(endlabeling€by€Klenow,€probe€synthesisÏof€T4€DNA€Polymerase,€and€exchange€reaction€of€T4€DNAÏPolymerase)€to€determine€which€labeling€method€best€facilitated€theÏincorporation€of€the€radiolabel.€€€ÌÌòòóóÌòòóóÌTherefore€developing€organs€or€photosynthetic€organs€might€bothÏexpress€genes€that€normally€respond€to€fluctuations€in€cytokininÏconcentration€and/or€structure.€€Such€genes€might€be€involved€inÏplant€development€or€diurnal€rhythmicity.ÌÌMethods€RemovedÌ€€€The€cDNA€insert,€L„22òòóó,€was€end„labeled€with€the€KlenowÏfragment€of€òòE.€colióó€DNA€polymerase€Iòòóó€(Maniatis€òòet€alóó.,€1992).€ÏThe€172€bp€òòEcoóóRI/òòPstóóI€fragment€òòóóòòóócontaining€134€bp€of€L„22€cDNAÏcoding€region,€14€bp€of€Poly[C]€and€24€bp€of€the€pUC9Ïmulticloning€site€(Fig.€1)€was€used.€The€labeling€reaction€òòóócontainedÏof€0.5€mM€dATP,€0.5€mM€dGTP,€0.5€mM€dTTP,€30€ððCi€òò32óóP„ºdCTP€(3000€Ci€mmolòò„1óó),€2€units€of€Klenow€fragment,€1€ððg€of€DNAÏand€was€incubated€at€37€òòoóóC€for€1€h.€€The€DNA€was€purified€fromÏunincorporated€label€by€ethanol€precipitation.Ìภ..¸ àThe€372€bp€€òòPvuóóII€fragment€from€coding€region€of€theÏchalcone€synthase,€CHS„17òòóó,€cDNA€(Ryder€òòet€alóó.,€1987)€wasÏlabeled€using€the€probe€synthesis€reaction€of€T4€DNA€PolymeraseÏ(Kornberg,€1980;€Maniatis€òòet€alóó.,€1992).€With€1€ððg€of€DNA€and€1Ïunit€T4€DNA€polymerase€exonuclease€digestion€occurred€at€27€òòoóóCÏfor€5€min.,€after€which€dATP,€dTTPòòóó€and€dGTP€were€added€to€0.25ÏmM€òòóówith€30€ððCi€of€òò32óóP„dCTP€(3000€mCi€mMòò„1óóòòóó).€€The€reaction€wasÏincubated€for€1€h€at€37€òòoóóC€then€dATP,€dCTP,€dGTP€and€dTTPÏwere€added€to€0.5€mM€and€the€reaction€incubated€for€another€15Ïmin.€€The€reaction€was€stopped€by€the€addition€of€50€mM€EDTAÏand€the€labelled€DNA€was€purified€by€ethanol€precipitation.ÌÌòòSlot€Blot€HybridizationóóÌò òó óภ..¸ àTotal€RNA€(1„3€ððg)€was€denatured€with€50%€formamide€andÏthen€transferred€to€a€nylon€Hybond€membrane€using€vacuumÏsuction€(White€and€Bancroft,€1982).€€The€membrane€was€air€driedÏand€heated€to€80òòoóóC€for€2€h€under€vacuum€in€a€gel€drier.€Ìภ..¸ àThe€entire€cDNA€inserts€were€isolated€and€radio„labeled€byÏmultiprime€synthesis€(Feinberg€and€Vogelstein,€€1984).€€TemplateÏDNA€(25ng)€was€dissolved€in€10€ððL€of€water€and€boiled€for€10Ïminutes€with€5ððL€of€primer.€€Labeling€buffer€(5ððL)€containing€0.5€mM€unlabelled€dATP,€dGTP,€dTTP,€òò32óóPòò„óódCTP€(3ððL),€and€2€unitsÏof€Klenow€fragment€(2ððL)€were€added€to€the€DNA,€mixed€andÏincubated€at€37òòoóóC€for€1h.€€The€labelled€DNA€was€purified€byÏethanol€precipitation.ÌÌòòDetection€of€Cytokinin€Responsive€mRNAsÌóóò òó óภ..¸ àNorthern€hybridization€of€51€single€gene€probes€to€separateÏfilters€of€total€mRNA€from€cytokinin€treated€cell€cultures€€resultedÏin€the€identification€of€2€cytokinin„responsive€cDNA€clones.€€OneÏcDNA€encoded€an€mRNA€that€was€induced€to€accumulate€byÏcytokinin€treatment€and€was€found€to€be€constitutive€in€the€roots,Ïleaves€and€stems€of€òòPóó.€òòvulgarisóó€plants.€€The€cDNA,€L„22,€was€452Ïbp€with€an€internal€òòPstóó€1€site€(Fig.€1).€€Data€bank€inquiries€into€theÏidentity€of€the€sequence€revealed€similarity€with€the€Ô€XXXÔÔU€A*Ͼ¦ `(CG TimesScalableUÔgÔ€XXXÔÔU€A*Ͼ¦ `(CG TimesScalableUÔ€subunit€of€aÏHòò+óó„transporting€ATPase€(Fig.€2).€€Among€the€53€clones€tested€those€thatÏhybridized€to€mRNAs€that€do€not€show€a€cytokinin€response€in€cell€cultureÏwere€the€small€subunit€of€ribulose€1,5€bisphosphate€carboxylase,€òòóóòòòòóóòòóóòòóóòòóóóóchlorophyllÏa/b€binding€protein,€glutamine€synthetase,€ferredoxin,€ferredoxin€NADP„Ïreductase,€chalcone€synthase,€phenylalanine€ammonia€lyase,€leghemoglobin,Ïphytohemaglutenin,€and€actin€sequences€(Data€not€shown).òòóóÌòòóóòòóóÌò òMusgrave€M.E.€and€Siedow€J.N.,€1985.ó ó€A€relationship€between€cyanideÏresistant€respiration€and€plant€responses€to€cytokinins.€òòPlant€Physiol.óó,€ò ò64ó ó,161„¼169.Ì