Biology of Plant Hormones
Biology of Plant Hormones HS 702
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HS790A Lecture 9 Brassinosteroid Perception and Signal Transduction Hormone Insensitive Mutants Steve Clouse Department of Horticultural Science North Carolina State University October 15 2008 READING Clouse SD 2008 Brassinosteroid Signaling In L Bogre G Beemster eds Plant Growth Signaling Springer pp 179 197 Go to my website click on Selected Publications click on third reference from the top I Receptors A Receptors are generally proteins or glycoproteins with a speci c three dimensional con guration that recognizes a speci c ligand When ligand is bound a conformational change takes place which leads to activation of a speci c response 6 g phosphorylation in the case of receptor kinases or DNA binding in the case of steroid hormone receptors in animals E Charactensucs of a receptor 7 medrated phenomenon rnclude l Lrgand usually rs ettectrye at very low concentratrons 2 The physrologrcal response can be saturated by rncreasrng hormone concentratrons whrch rndrcates satu u of the receptor wrth lrgand 3 The brndrng of llgnd to receptor rs generally rn the same concentratron range as the physrologrorl sponse measured when exog ous hormone ls applled to the lntact system 4 are smct slructureacuvrty relauonshlps for the hormone l e slrght changes rn structure can cau loss of brologcal acuyrty rmparred brndrng to the receptor c Receptors can be rsolated by several techmques l Erochemrcal a radrolabeled lrgand rs rncubated wrth protern aruacts to determrne lf specl c brndrng rs occumng r ound ys unbound llgnd can be separated by gel lmuon dralysrs absorpuon of free llgnd drrterenual precrprtauon etc rr brndrng rs often analyzed by a Scatchard plot WK 7 EK where E concentratron of bo nd lr and F concentratron of fr e lrgand n total number of brndrng srtes Kd rs the the concentratron of lrgand at whrch halt of the brndrng srtes are occupred equlllbrlum drssocrauon constant b photo af nity labeled ligand for example azido labeled is incubated with protein extract at 180C and irradiated with light of a speci c wavelength Covalent cross linking between ligand and receptor occurs which is then puri ed or analyzed on a gel c the ligand is attached to a matrix such as agarose and used for af nity chromatography to purify the receptor 2 Molecular a a cDNA for a cloned receptor gene from a given species can be used as a probe to screen a library from a different species in order to clone the homologous receptor from that species b the known sequence of a receptor gene from one species can be used to design PCR primers to amplify the homologous receptor from a second species 3 Genetic a A large enough collection of hormone insensitive mutants from a given species will contain individuals with mutations in genes encoding the hormone receptor and other essential components of the signal transduction pathway The mutated gene can be mapped on the chromosome and the corresponding wild type gene can be cloned by several different techniques b Mapped based or positional cloning i The closest molecular markers to the gene are found and used as probes to identify YAC yeast arti cial chromosome clones that contain the gene of interest The DNA is subcloned into plant transformation vectors and introduced into the mutant plant The clone that rescues the phenotype contains the gene of interest which is sequenced The mutant alleles are also sequenced to con rm that they indeed have a mutation c T DNA tagging T DNA mutagenized lines are examined for linkage of T DNA with the phenotype by kanamycin resistance contained on the T DNA and the anking plant DNA is used as a probe to clone the gene of interest d Transposon tagging similar to T DNA tagging except a transposable element is used rather than T DNA BRlnsensitive mutants and BR Signal Transduction 1 Identification of bri1 by root inhibition screen 2 BR1 39 Leucinerich repea receptor kinase 3 Identification and functional analysis of BR1 rylation sites 4 Downstream components of BR signaling 7 Comparison of II Brassinostemid signal twomonthold transduction a bri1 mutant l Aux BR Buffer Buffer BR Aux Root Inhibition Screen for BR Insensitivity Clause et a1 1995 Clause et a1 1996 BRIl TWOmonthOld bril mutant Signal l39vplillv ll39lL ill39 lippmr l nlrrd 39 39sh lnlquot lulvincRii39ll Ripmn tliRRI l RR 2 RR 3 LRR 4 R 5 RR u RR 7 Nmul Unlnnin RR 22 LRR 2 R 24 Hi 25 l nirrd IV lvincx l rlllhlnl39lllhrllllt lhmmln Jquotlunmnhrmw kuuiun Serl hr Kinuw lumuln urlmu I39rrmiunx Clouse et a1 1996 ltgt Multiple leucine rich repeat receptor like kinases LRR RLKs are required for normal Brassinosteroid BR signal transduction BAKlBKKlSERKl Brassinolide BL H0 2x V a l rulinRich Reginn L1 at al 2002 H 12007 2 months 66 a Clouse etal 1996 Imamcum Iwmh an nomewlnm ch eanwbmm l 5522 WT DIM5 urn5 WT brhS lam5 2qusz nthID rmure n A mlInwaxmemmen m an actwatlnn tau lnq 1mm m ant5 Phenntyuean weeknmwantyne eentyuews2 urn5 and nms deI In ulamxarz mwn Scale bar Iemtn I em IS euuwa B baldID partially muurESSESmE Imlnruczncz lEnmh nzmct mum5 nms deIIDulamxhaVE Elnnqatzn pgllnlzx and mene Izathlanzs Mzaxurzmzntxwzre takzn aweek allEr eermmaunn an were avzraqzn 1mm 4 Individual plant main 3 standard emm Stunznt39xt 25 s mmeate thatthz Inllnmxczncz lenth an m ewmm mum5 nanID am unmeanuy nmzrzm39rnmthat mamS wen In nnnnl 2 mum a ma lmzrananh mm Movzrzxurzxxlnn Mam nammnrm n1 gunman K3I7EIn hrl 5hacqununn ruunzn m a nmmantnmatlvz uhennlyue A mumummm YnuuanElhnthhEuhznnl luESnl I v u ranmznlcplantxharhnrlnq e an L em 2002 DIantAntIGFP ya u DzrnxmaXEcnmuqatzn an the mammary antlhnnmx Plant Receptor Kinases ectodumain cymplasmi domain I Dbs ua mEmhmnl mp FGF mm mnasnn mm mm Dunkn39mn ad cl 1er mm Receptor Kinases r LIGAND BINDING WTDPHDSPHDRYLATIDN NUCLEUS PHOSPHORYLAIION GENE XPRESSID TRANSCRIPTION FACTOR Tritiated BR Binds BRI1GFP c Bound 1000 x cpm Fusion Awe m m 0 5a a a dc FA 6 6 on LNE L away ulmg L6 A 1amp0 Ei f wA39OC39 Walg at 3 Nature 2000 EHII gt woman RRZZ um 25 m an and am a 5 fragment D m 21a m b apcs mm a m a 1 dnmams Dun ed fmm 5 call EEHS uDDEr aRn dnmams usem apcs mnan uwer Dane mack mm mdmate Dnsmnns m nundegraded Dmtems Kmusmta 2t 5 Nature 433 15771712uu5 Th2 mjnmy nflml nmmu39nnsclusler in vhzishm l and chase dnxmim 2 rneunchsen n Mquot 2 2 Flam Physlnl 2nnn12312u1256 Hypothetical early events in ER signal transduction and questions to address in wan a a alhe m mam How does an mguhte camplex Xrlnsfer me mammal ol arm slgnai la and am 7 d wnslmnm submrales i iVu mmmum mm w qum Bmssinosteroidr anendent Interaction and Phoshorylation of BRI lBAKl in wm a 31 nmcmn 1635MB mus BRI i but not BAK kinase activity is essential for BRdependent in vivo association of the complex Emma and Emma mhriirFlav aximua arr swap amiss mnaxmrp aL lt 5mm ianmag w GFPWESKEW mbrilrFlag K911E Wang etal ZDEIBDevelnpmen39al Cell mbakerFP K317E u BAKiGFP in vivo phosphorylation is reduced in a bri11 null mutant background and enhanced in a BRI1Flag overexpressing line w I 7 w mm W MW 1 WWW u awerr m i A t J In 7 mam mnmmwm nxiEFP Wang 3131 2mm Developmental Cell nti cation of in m 2 phmphm39ylatiol site Vliednyeold lightgm 39m shaking llquid culture 90 mirrurenr mime treamrenmno nM39 Membrane rracr m maroon x AutlrFia g 11 HRH or An AKU from 39 1P ululs T ed me l llbta e r IMAC quTmpLCvM MS QTUFLCM MS SEQUEbTJMAb T MASch Nhss Spectrometry and Mass Spectrometers ss Spectmmaw s an ana ylma Echmque that 5 used m memory unknuwn Nh umpuunds quamfy Knuwn materwa s and emanate me structura and physma prupemes er mns Mass Spectrometer umzatmnl m I Detectur I Suur I I I z e Ana yzer ActuaHy measure we massrmcharge ram mz nmjustme mass mm mm 15 D M a sum aemms msmwiwm Mass Spectrometry Analysis of Proteins Mass Spectrometry MS analysis of proteins can be used to Accurately determine the molecular mass of a protein eg 2485689 Da Determine amino acid sequence less than 1 sec 100 ms Identify posttranslation modifications Quantify protein abundance and posttranslational modifications Characterize proteinprotein proteinnucleic acid and protein ligand interactions Determine rates of enzymatic reactions and binding affinities 27 Dr MB Goshe BCH 455555 m STATEUNIVERSITV Protein Identification Tandem Mass Spectrometry PrOtei sample Peptides First Stage Mass Spectrum 701 Peptides ProteaseC eluted digestion from LC 300 2200 m elected Precursor Ion Protein Sequence Second Stage fragmentation Mass Spectrum GDVE G lFVQK CAQCHTVEKGGK TGPNLHGLFGR Product ions HKTGPNLHGLFG TGQAPGFTYT Peptide sequence GFGR etc DANKNKGITWKE mapped back to GR FGR ETLMEYLENP Y protein IPGTKMIFAGI R TGPNLHGFGR TEREDLIAYL AT I NE mz 75 2000 28 Dr MB Goshe BCH 455555 uc STA EUNIVEHSITY VvlganHa Eg LCVMSMSidenci cacion H W ofpm 2 b Mb b M2H7 mZ BEIGE W W P Wang el al Plum LeLl June 2005 BRI1 KD Autophosphorylam on n cl 3 a 2 2 33 58 u 39v mm men Oh et 21 2000 Plant thsxology a A quot quot quotquotquotquot39 39quot Acn39van39on loop residues quotquotquot Ame essenu39al for kinase funcn39on Phosphorylau39on of J xcamembmn d Wang et al Plant Cell June 2005 3 1044 and T1049 in the Kinase Domain Activation Loop are essential for BRIl function um m mu am my Wang et 21 Plant Cell June 2005 Identi mtion of in viva and in vitm phosphorylation sites of BAKl by ion tap and QToF LCMSMS analysis pSite m mm m we ERH Tmnsphosphorylauon 5290 13 12 T446 T449 T450 4 7 T455 ns39rsutm Psm m I I a H mm sites in vim BAKl enhances BRIl activity towards a speci c substrate Wang etal 2008 Developmental Cell Functional analysis of BAKl Phosphorylation sites in viva BLedependent pathway 7 briIe5 background Functional analysis of BAKl Phosphorylalion sites in viva BLeindependent pathway 7 bakIbkkl background Wang et al 2008 Developmenml Cell Similar interaction pattern of BRIl and BAKl in vitro and in vivo widenmum a was um um amblll i BRIHIdiIhkkl BAKI IiiI BRII BAKI Enl BRllmhil MlhliklmhiklEIF mlmloBAKl hanlmHh Sequential Lransphosphmylation model ofthe BRIl 39 BAK1 interactlon B Wang etal 2cm Develupmemal Cell HRII he BR R cplm J Rich Repeal Rcccpmr BRASSINOSTEROIDeINSENSITIVE 2 BIN2 KINASE IS A NEGATIVE REGULATOR OF BR SIGNALING Mm amz 1 quot5 w w mm mm u pmum m2 I 4 I Wm H mm Hu mnuSK I I mu amz m 5 7 mm m m humnnESK m m km n humanESK 4 mam 4 JVL nrcskkm Mum w w mum Hp u 2erqu u LLLn r39ns mm v mm 2 Drum r humunZSK 3 quotmy Der B humnnGSK we I w mzns numm x em arm human m BINZ mm WWW m H m J Lietal Science 2951299 1301 2002 Fxgure 2 M271 IS hkely a hypermorphlc mutauon A MW We gene mu dew uonarex hated c o BINZ verexpre glvesnsatoan allehc senesofmep H type s I s wn synth cm m DLhem eplams grown m souun om argome ons E Analysxs ofB N2 ExpressAOn ofthe 1N2 Lransgemc plan RNAs 5 g each Isolated from mid 71 dB N2 nsgemcplantswer 2 b a tad y formaldehydeeoontammg agarose gel bloLEd o nylon membranes and hybnobzeo rst mm a fullelengh BINZ probe and men mm an 183 rDNA probe 42 name 3 A re ucmm m aw gems expressmm pamaHv suppressed aweak um mutatm m7 Seemmg gmwm m vmmetm D7 Mme aw DNAm detectme emingem s aw essages7 Altersmppmg Mamba 22 sa terwa mmsz WM 183 rDNA ombemmmm mum mum 01mm RNAsvmeam Mmemm mager System am murmahzeu sperm218 mm mama s Tm mumber mm s ram betweem a murmahzeu am smax m a gwem mo e am the murmahzeu am mam1 amew mswoe 3 u m 557353 mam m re sa mam E Cymplasm 1 M h Prouasome BESlBZRl39gt 139 39 gt mediaud BINZ BESI BZRI degradation KINASE 11 Nucleus Amman a bmssmnsmmxds respunswe gene expressmn 39 Cell expansm dwlslom and dxffaenuauon Figure I Darn prresses urn thmlvpts am I mmmm mg minmm lm mnr Hrassmaml m m quotwe g r m Ingmaran mman m M mmlum 51mrmnnmrnld plants gmwn m snIl OFWQVI Iarnld dmrqmwnszzdl q mvarM2krnld llangmwn szzqus m M mmquot wnmut MPPQY pans or Mm I w m mum band Dun IND1va Imam af rmrum darqurnwn mman mth absn2 of Drag all M m A Ian In mumth Wm measurer nr anumm 1h amr bars mama nan ard kVImnnJ Y s m2 55 mm cm my mm mm mm am Ammmm he Nudeu m Reivanse m m mum WHd39WpeBES am My pmmW nude u m W um Mamba m mmqu m we nudeusn W mew Newman mu ezamseswdvsmws mzu uMBLmduzeSBESVEFV Mdunzmmuhtmn m an m Amsuzhzzmmuhhan zammue m mm m an an MAst mm am EFVrmdearzzzumuhhan Ymem cm my mm mm 5 Hyman wismmr m m m n m u m m m n Yuma cm my mm mm aim 5 MW Egg 5 Ymem cg m9 mm mm my sumymmmymm unpmspmyymeaxzsy myyya m mwmmymm muhtmnahhstm grmngvmm amywweszsy Emmyng vhmszmtmtmuumbesy m zzzumuhuszth gheyekmvhmm Mezbsenze m A yyegye yummms mom when w yvmeasisy memeth mna umbmm edzzmmuht mvhtedBES n my smmuz unvha mam mm msme e yummymmy unvmsvhmvmedbs mwmm My mm Marmane d0 mummqu mummy ammmyyyma my w my mywymwmmm mvmedxis o smmyzmmmmm Dy unpmspmyvmeaszsy s reduzedm memymmmzkgywm quot nms Negztyyekegummmvsisy bysmz warm essesbmzdwz vhenawvuBath Mamaxvgw awer emznd Metemlvgam My who mm mm msvhv 2 dwz vhermwvumh zamv eu vsuvwessed by new tap mmmaszsy mum vasttwarmbrm my m m WWW expenmemshaws ma mmymbemusmmaszsy my Am mm m aw mmasyrsmmmmn mmmmgasm as u yummy n ygnmamzmwn awis mmuumbe vhaspmyvme my Msmsy 2nd May natMBstreyu edbvznmwvakmzse u yymawmwmmyyymmy m 5y MW mameyummyth my D Em mum my Ex mmmb a 3 mm Dswznegzwew mummy Myny Mw d tvvebzzkgmumassszsv 5 my 3 mm pmyvmeaszsuye may bzzkgrmmd mama am am my mm mmmpye a Fxgnle 6 EIMI EIMZ and EIM3 A Invalved m ER szpanxex A Onerweekrald xeedlmgx yawn m xhan day sandman Th2 nnmhen mdma e h avenge hypacatyl mm mm and x andald em wnh a 1m 15 xeedhng manned in each gemtyp Th2 mum we npeamd mm mm bathe afxee and nm ar mum we a tam a mung yaw hxm m lexpa 3 m me an 15m 7 andZEIEI M mm m1th hxmi andEIMOX nxpemvely DE1gh r weekraldplamx afhnLS e andEIMlavuexpnxx nnhnLS hackgummxmoxhms Fumi m mammmmaaxmm mu mmmm mdhmdm mEhnX CWTQ Yin et a Cell 2005 in Flgure 5 EEsl and mm Blnd to and Aeuvate ER Target Gene Promoters In VlvoA j Lmnsgemc plants uslng anurGFP anubodles m M The SAURVACI At4g38850 and SAURVACL m llke Atl g2 promote were detected b 2 rs i 7 PCR ampll eauon and observed by ethldlum ml e stalnln or Sou l n 3 respeeuyely The lower panel lndleates PCR gun o s wth allq ots fszmples b Q m l unopreelpltauon lnpu wrth SAURVACI pnmers E ChIP th 1drtype or BIer in s nEgemC pla u ng anurFLAG 391quot anubodles c Coexpresslon of EESl an un um am a Mr m 1 lldrt a mutant form mm ull Mllmlswnn the of the SAURVACI promoter luclfemse gene D 395 Expenmental Procedures The mutant promoter us an n 5 11 u jltmm Yin et al Cell 2005 l i BRllrsAKl EIN2 asUl RPS l 37m awn Q7 l mm H r EFlrrospons39lvc genes pep pPPw 7 pFPp E boxes mutated D A Nonhem p RNA 10 Mg loaded Fl gure 2 A model for downstream ln e ER srgnal events th sdue on pa way ln s cells the El 2 35m klnase rs Fvveawme V1 PFP Q 4 mmquot u l g ER feedback X m 2R1 regulated tlyrepresse e tr s lpuon msynmellc of ER feedbackrregulzted genes cues su a to adj homeostasls by blndlng to CGTGTCG elements 52 Flgure 3 Models for downELrezm srgnahng Onganl nuclear uans n odel E 2 phosphorylztes EESI d EZRl ln lhe c Up surnulauon promote BRresponses b Nuclear rnodel Em EESL and EZRl are RM nsu y ln the nucl us e acuer ofEESl and EZRl atswml prrrnanly regulaled by Lhelr PW phosphorylauon s L gagsl Mm Altemzuve nucleocyloplzsmlc 55m 1 Hyp osphorylaled EESl a R1 a tandy cycl n WWW b en lhe cylosol and lhe nucleus surnulauon by Enggem conversron of EESl nd EZRl Lo r s l u Lhelr hypophosphorylzted forms Thls rna lead to a greater af nlty to A ndor prevenmne two f uanscnpuon actors from exmng the nucleuE thereby accounung for Lhelr accurnulauon ln the nucleus 53 Exmlwles nmmhldurms k Keg mm Canes elm hum Mlzmznm1m sls HM mumr san n M Wrn runn r rmuwgmluw r are r murmur lan n urnn ss Mm mu z I who 42mlan scamsmmm um r Fum ui 5 uxhldlvSliMll ilcmllmy an m l39mxxllmzvrlrlmvrrguhvu um shunhm w s a hlssgt n as v T EmltsllmmruldmwlamL humus mu rmnl 2 yu nan Mm39lrulm x L 1 n um mslllhnudauzlllleimnsemb Nmmzmmzlcnemlall LCH w x s nu lel um r a seen as gene can run zlm Clouse 2004 Chapter 77 Dames PlanLHormoneE ADDITIONAL READING Clouse SD 2008 Brassinosteroid Signaling In L Bogre G Beemster eds Plant Growth Signaling Springer pp 179197 Wang X Kota U He K Blacbum K Li J Goshe MB Huber SC Clouse SD 2008 Seguential T Imiun of the BRH BAKI Receptor Kinase Complex Impacts Eaer Events in Brassinosteroid Signaling Developmental Cell 15 2207235 Li J 2005 Brassinosteroid signaling from receptor kinases to transcription factors Current Opinion in Plant Biology 8 526531 Y Yin D Vafeados Y Tao S Yoshida T Asami and l Chory 2005 A new class of transcription factors mediates brassinosteroidiregulated gene expression in Arabidopsis Cell 120 2497259 X Wang MB Goshe El Soderblom BS Phinney lA Kuchar J Li T Asami S Yoshida SC Huber and SD Clouse 2005 Identi cation and functional analysis of in vivo phosphorylation sites of the Arabidopsis BRASSINOSTEROIDAINSENSITIVE 1 receptor kinase Plant Cell 17 168i1703 l 39fer L Nemhauser Todd C Mockler and loaIme Chory 2004 Interdependency of Brassinosteroid and Auxin Signaling in Arabidopsis PLoS Biol 9 Clouse SD 2002 Brassinosteroid Signal Transduction Claiifying the Pathway from Ligand Perception to Gene Expression Molecular Cell 10 9737982 Oh MH Ray WK Huber SC Asara JM Gage DA Clouse SD 2000 Recombinant brassinosteroid insensitive 1 receptorilike kinase autophosphorylates on Serine and Threonine residues and phosphorylates a conserved peptide motif in vitro Plant Physiol 124 1715 Clouse SD Langford M and McMorris TC 1996 A brassinosteroidiinsensitive mutant in Arabidopsis thaliana exhibits multiple defects in growth and development Plant Physiol 111 6717678 Clouse SD Hall AF Langford M McMorris TC and Baker ME 1998 Physiological and molecular effects of brassinosteroids on Arabidopsis thaliana J Plant Growth Regul 12 61766 H8702 Lecture 12 Functional Genomics Proteomics and Transgenics in Plant Hormone Research Unixeifsity quotP quotMy 138259 A w Botany 2006 5739 u and BM Simon Preface to special pu39o39ijeomcs gs 9 2 Developrnentg 39n biohggy have when bean Hmi39tsd by the zecha ao cgieg availabie The abn i39igv immense DNA proteins 5 p a y M a major ri e in advancmg our Lia39uderg39iending of u bimcgica q Yl nn39m 1391 x39 v c u v39 uquot GIMME E L Him 391 t H U ILA H W ce uiar 39uunction ET per v Ui ad m lfi d AL Hnr1 4 quot 0 n nf n up 39 u we damageu whim mew A we m A qr L3rmmlcsz pmt m germ n 2y expan er i to Jammie r oymmio jCJI39OtVMS m 09 o and thasueaquot v Systems SA I leelns Elolugy a 1 ca mama Gm SI 4 gum 1 3 Mmmmas wu Wan IECM Proteomics the first decade and beyond 3mm a known mum yr Mhusnkl a39mJnXS ln m Wm pm mm 7 m a Wm Wmng WM WWW mmnmmnmm Wmmaw m m H mm mm mmwm wu umm mmquot hnvx m Wm Mass spectrometrybased proteomics mmmmm mu m mphmmwmmmmwmu atummww vmwmmmm Mn mm uwmmlmmam vmmHM anmsmu39mwul mumw mm quotmm wnm mm mm yMlmmuMwum WWW mfg J quot nil rynlmxixqnm1qy mw L mud m wu mww r quot quot quot Protein Identi cation Mass Spectrometiy m quota A 7 p ximama 39H39H m 3mm rr mme 0mm 1m w Calllsse l 2al puns 2m nmnzsunzsv JMUL it MM may 9 salluuauud 39uao 39llle umzt 39x 39uluzuzo suzsgtz u L a umm ax m u m u H w um ulauml mm mum Jam palqns sanusluuauululanau 1 m angpew u augmom ugede pun mum o pauagqns seams nua mdolaneq Kinetics of synthesis of serine proteases in developing seeds A Fll 4 KIIIElIES n1 Synllless n1 SEIIIIE mlnnunnh MN m an mumsndwelnlunus Is All nm nu n l E t Wm Wm n n callanlnlltl2nn7l MnlCtlPrnlenmlcs 215572179 Global vlew of me compartmentallzatlon of ammo acld blownmetlc pathways between seed tlssues calamn K lzmn Mnl Cell Prnlm 115572179 In Proteomic survey of metal ic pathways in rice Antonius Koller Michael P ashburrr B Markus Lange Nancy L Andon Cosmirr Deciu Paul A Haynes 1 Lara Haysquot David Schieltzquot Ryan Ulaszek Jing 39 Dirk 7 and John R Yates III A systematic proteomic analysis of rice Or za sativa leaf root and seed tissue using two independent teclnrologies twodimensional gel electrophoresis follo ed by tandem mass spectrometr3 and multidimensional protein identi cation technology allowed the detection and identi cation of 2528 unique proteins which represents the most comprehensive proteome exploration to date A comparative display of the expression patterns indicated that enzymes involved in central metabolic pathways are present in all tissues whereas metabolic specialization is re ected in the occurrence of a tissuespeci c enzyme complement For example tissuespeci c and subcellular compar tmentspeci c isoforms of ADP glucose pyrophosphorylase were detected thus providing proteomic con rmation of the presence of distinct regulatory mechanisms involved in the biosynthesis and breakdown of separate starch pools in different tissues In addition several previously characterized allergenic proteins were identi ed in the seed sample indicating the potential of proteomic approaches to survey food samples with regard to the occurrence of allergens Koller Antonius et a1 2002 Proc Natl Acad Sci USA 99 1196911974 39rnhln l Nlllllcl39iclll analysis 01 peptides nnu pmmlm dclcclcd llld lunnrlllcd m the me prolcnllle IDPAGE Mnlll l l39 Combinedi Lan lel Sccd Tlllul Lcul lel Sccd Tulul Lcal39 Rum Seed Tulul Tam pqmd i 1707 700 48 3301 1151 7151 8790 22102 7839 7557 141er7 15423 Unique pcmldcgi KW 415 393 1509 1626 2358 1912 5139 1558 2712 2180 6290 Ll un quotmu h 34X 19 152 55 867 1192 12 1303 1 I22 I 350 2177 1528 L1 V11 ZD PAGE And MudPlT m dcwllbcd 1n MrlmiuA um Millmm The miublc pmlcm cxuucu 1mm CIK391 issuc n1 ncc lcrc lulzlly The sccnnn quotCombinedquot dcmilx he inxcgmlinn of the dillfh CK gcncrilwd 1mm hmh ZDPAGE and MudFlT lnulym at each lune at he ncc pmlcnmc T The null number at SEQL ES39I 2 ml39r and A n rurr rm H 39 r ll ll I l pl pr lv ldcnlllicl ill cucll rlcc limlc 1mm ellcll mClimd 1 Issue la each method lx iliiIZEi In the snhsccnnm Tnml and in the within Combined he 3 pcpudC lcre idcnlmC in multiple l C ulldl llr 1111 buul nk lhod issue 11 mull n39lcllmd h 1iltlCL1 In 11C SlllNCL39UOIh quotTomiquot llnd ln he icCllnn quotCombinedquot he 39 r l r r l r The rnrnl number nfnnlqllc peptide ldcnlriicd 1mm lulu number lll39uniquc peptide I lml uddlullc lk c duak lll t The rmnl number ul unrquc pmlL lm ldcnn ed from r lulul number ul unrquc pl39ulcuh u nur Koller Antonius et a1 2002 Proc Natl Acad Sci USA 99 1196911974 1 1 Fum39 quotmm m n n 31 mm umm 1 11mm 1A1 mpmum mm m 1 1 111 111 11m 311 u 1111 m 11 131 1 111 1139 r y1un vp humvavh mupm x1LF S mm 11 m L quot311 11111113131 11 11 y 113 11111 111 m 1 71113111 113111131 111 lmvdpxmvuu 1111 7 3154 17113911 7 1 111111 1391 1v 1x1 1 15 arr 1 1n111171x1kvn13d u 339v 11 11111111131 39 1 1111131 111111 7 wow EI39 WM WM 11m path 339 1 havvrvdpoman 11 quot11va 1 1121 sallumuud39mg39mw mm M 1n 1 avwn Mdu ow moms Muwv mew pezluqo m ml 0 quotoutrun gum 1 sanuuauud 1m 1m muuzh 39M39fmu mm 1 1 m ummx sax mm HE sasucdsaluvmmg mum njlmmmutmmnnt T W quott W umssamesue mm 19 pmmma r mnmm uunnmm wmw TM t w A Hugh 1 Yznuzad Afamza m m mMa MA da awn Hmunp u kt ssdopmelv u MMMIEII A muzumsuaamuuaumu v 1 M n 121135 pgomsougssmg JO sagluoaloxdoquoqd EZR1 protein is detected by 2D DIGE following iMAc enrichment of phosphoproteins r BZR1IrnIeInIs Hauler by 24 meg imsmmumiei s i i 2 m ii i umuiia NH immiin mum iznmi Mnl cal Prnlmmlcs 7 72mm ERregulated phosphoproteins identi ed in MM fractions by 2D DIGE Swim mm 2mm 2 WW W Dias may M is z w 4 47 m 4 3 quotsky 24 Ding J m m numw iznnm MnlCellPrnIenmIcs E pression of he nbberellin Immivmix Enzyme GA 2 x39idase whh an OmalrSpeci c Prommer Resulu in Sexxnerarf Rice vhhom An39emiL Seed Produmion Wm mm W Mmquot m Mg E pression of he bl whh an erellin Inadi mim Enzyme Gk 2 Iase KEZIIPSPECi C Prommer Resulu in Sexxnerarf Rice mhom An39emim Seed Produmion a WWW r IIJure 4 Phenn39ylm m 01055A20x1lrmsjemc 4 u ncexax mm m Increased 5 berellin bi symhesis in usnsgenle mes pmmmes gmmll b Jmass pr 1 luellon and xylem ber 1e g swellic eugpldm ovel expl essinz an auxin biosynthetic wene lddM try tophan monoxygenase Ilndel the contml of an ovuler speci c pmmoter showed panhenozenic fruit development and produced seedless fruit from emasculated owels Hahn Ell m mm mHunp ammmax mz 39IuIslld mm 3912 a quotA 39D uuuw a m s i a ummasu main Amiseme AL C Symhzse inhibits revexsible ripening in mmam Annual W xlamvmmvmmgq mmmm H87 02 Lectures 7 8 Seed Development Vascular Differentiation Senescence Abscission and Morphogenesis Steve Clouse Department of Horticultural Science North Carolina State University October 7 October 13 Reading Finklestein et al 2008 Molecular Aspects of Seed Dormancy Annual Rev Plant Biol 59 387 415 Fukuda 2004 Signals that control plant vascular cell differentiation Nature Reviews Molecular Cell Biology 5 379 391 1 Seed development is a coordinated set of pathways involving cell division elongation and differentiation and also higher levels of morphogenesis based on cell lineage in the embryo Stages of seed development include a Rapid endosperm and embryo growth after fertilization b Transition from metabolically active to quiescent tissue 0 Dessication and dormancy usually d Imbibition and germination with a return to high metabolic activity Plant hormones are involved in each stage of the process Dormnay induction Dmmmmy maimmuce nounnay m Gummalmn Figure1 Time course of a n 2 om lt g a biosynthetic and catabolic ion depends on locI lnduct combinatio of ABA indep dent maternal an l emb onIc factors and ABA depen e signaling WNW V Release is promoted Maln39ution 5mm Gsmmwm F amiqu i i many environmental factors l l l a 1 him mmlm ML changes in ABAGA WWW mm quot1quot signaling balance eventually WNWH R iquot 7 resulting in wall expansion to Anuu my mm Bml avgxuh permquot radicle emergence P sItive regulationi lndlc ted b and arrows negative regulation by and bars llm39nmnus dun1 wed llm x llllllllt39ll EMEPVDGENYslS i lMlHATu V H 2 MAI UFATlDN Re a jvc Levels ulnr Km l Eaer EMBRYO DEVEOPMquotNT m m mm 21mm mlc rt mm m cmbryu lie 4 r1 ll litlxl cmlllmtl lmmml lt02gtZroo 0 AgtOO UW39U m gttagu u with spa i want mmiaw mm ille tuml m mum n mm m llcmb39yn du vlupmtnt n um Seed Ref 9 Chaer G10 P mlle m WNW ll General features of embryogenesls A Femllzahon Pollen enters the embryo through the mlcropyle and releases two sperm nuclel One femllzes the egg to form the dlplold zygote whlch wlll form the embryo w e he other sperm nuclel fuses w two polar nuclel to form the mplold endosperm whlch ls an energy and carbon reserve E Embryogenesls The axrs and polantyof the plant bodyls de ned after the rst lelSlOn when the suspensor basal and embryo ls formed wlth unher dwlslon of the embryo root and shoot aprces and embryomc hssue and organ systems are formed Dunng maturauon storage reserves are accumulated for later germlnatlon The nal stage ls desslcauon and developmental arrest c llormones lAA andc ok mms arelmportantror cell dlylslonln early drops and AEA leyels lncrease dramatlcally The polar transport of lAA m the early embryo ls also essenhal for correct pattem formanon tun ham mung w lr lemm unoTE mm Ra are r mun gt quot3 39ii 7 i f mm 1 1 SeedRB 9 r L t mum cmpmrcm Grunrlurlntt r mulls Wm ll r re r m hn l n hm n wavyq mmu hm m SeedRef M mm v vn m a J va W 5 M L g 1 4 am p m Maygm m SeedRef 1 r f 39 m2 Drnlaymumnf hlpii hm pannrnamHMmh lpwkmdu ng cmbq ngcncm A1 5m an ma mynmmnmlu m gin J mm mm a m a mm mm m cull Tm mm m prmiuccs by damage man m pp J m wum um um chm cm a emmm ulmwx mg mer ohm mpopm or 3 Ac 4 mm um mm a m hvpuphysls n glvc r r milwcn39 rum pmnuvuum 1p 3 m mmrhcrm slzgc m mm Inmardmm Up an vln ijaumxy prmmnh Lle CQJUHngIL m l WW pm de ax m mzoccm um II mm m um m 391 mm M Iwulicx mm uwxmmrmupm mum mm 4 y mm mmquot m W Allhunn glob m ium Wu muss ms w mm mm mm mum pnmouhhm y wnmmhnmj quotm m gmqu me p Nunvbus rcfcr m Magus o cm hvyvgwuxs awarding m Jivgcns m Mm mm Ampm mm ppmqu lmm yevay 2 mm SeedRef 2 71 mm SeedRef 1 Figure 6 Embxyu axis fama mnandanxm nghrmsducnan A Basal damma m manguhpsuge embxyu markzdbybnckzt campuses lzsaf mmw cells m m we embxyumc stelz and a chanctenslwa ydlvlmng lppnnnst suspnsax c2 hypaphyseal39 cell whnse dznwuves farm h QC and gamma mm afthz pnmaryRAM afthz 2min Wemastsuspnsmcellmvldzs ahmnnally DrF Seedhngs 111 am far he presumzd anxm signal mnsductmn gems MONOPTEROS MP AUXIN RESISTANT 6 AXR and EODENLOS EDL are dzfecnve m embxyu axis fama mn muscular duTmmanan Hypucm and mm m msmng m mpandaxr mutants D and E mm vanath xedvced mbd mntnn39s F mm stale missing ndthz afmdlcated gemiyps npmdvced rmm Berlqu n a mum Habbxz a u mum and Hamm n u 1999espeenve1y Seed Ref 3 Polar Auxin Flow in Embryogenesis FIgureZ PW FORMED PW ex ressmn auxmmaxwma and premcted auxm ew uunng stage wumype Embryu Reversms m PW pmanty mamngme Estabhshmem at EU a u subsequenuy auxm aw back mwam me menstem ruuewmg uMEdun mm mm are demarcated wnn aste sks rmure I3Auxntran pnrl and entylennn unsmnn culture nvearly embryn n1 Brdmrdlunredln thE presence Manx n n c Sun Inhlhllnrytnr n 12 4 and 7 nayxrzxuzmvzly mm In et al 1993 SeedRef 8 rmure 15 mm lncallzatlnn and cell unlarny mmunnlncallzatlnn n1 Ixacnrrzlatz I 1 ennmmaten I lncallzatlnn and n1 nverau apicalbasalmll Dnlar y Bar M rrnm stemmann et al 1999 m AEA plays a cntrca1 role m mrd to late embryogenesrs by rnducrng dessrcatron and dormancy whrch rs supported by the followmg a In cultured embryos where AEA levels are low precocrous germrnatron oocurs b Exogenous AEA apphcauon prevents germrnauon orrmmature embryos m vrtro c AEArde cxem mutants have reduced dormancy and seeds germrnate whue sun In the trurt d AEArmsensmve mutants of Arabrdopsrs and marze ambu yryrpary germrnatron wrthout dorman e Apphcauon of undone an AEA brosynthesrsrnhrbrtorresu1tsrn precocrous germrnauon of embryos m mm EA turns on a set of genes LEA rLate Embryogenesrs Abundant that encode smau hydrophrhc proterns that may play a role m membrane protecuon dunng dessrcauon The same genes can be med on m yegetauye ussues such as leaves by dessrcatron or AEA en a w rt ur w M m m 7H u Mrs H m SeedRef 7 u Seed Ref 7 v MM gu V Mk u H mm m per whlmu Mueycgjl39 ulvhevzqn mm Hr mm mu m m h w n r v AEAansmsmve mutants have bem 1denu ed m Ambxdopsxs by the tyof seeds to germmate m the presmce of ABA m3 mutants coaous germmauon des u gene has a and appears 0 moods a tmnscnpuon factor The W1 Vmparous mutant of maxze 1 a homolog of abB and appears to have similar funcums 1m mmpr E Bu 7 A An 9 m2 2 so ma E J A ASH we 5 4 u m m 32 L 2 m 2 19 a 10 100 mm AHA AM SeedRef 9 CmpterlEl v1 Getmtnatton AEA thhtbtts and GA promotes seed getmtnatton The tequttemeht rot GA th gamthatmh ts seen by the fact that GA de cteht mutants mhhot getmthate wtthout exogenous GA treatment The new A EArmSenSlUVe mutant wtu getmthate th 2 GAedehcteht geheuc background Showmg that the tequttemeht for G unhg getmthattort ts to overcome AEA thhtbtttOh Emssmosterolds have also been shown recamy to have a role th getmtnatton eM Steber andPetexMcCam spapxptesems mdznce that dahthtassthastemtdtatt hahhahes dayamte th t t hythe dahthamshes ahsetste aetthaA ahdgthhehethhtGA These Wm hams et antaganlstwd ywnheachmhzx AaAthdtteess ddamaheyth gemthattahsrseeds GAbxeaksse dda ahe ahdpmht te then ehest Ptaht Phystdt Fehhtahyznm Val tzs pp 751769 A Rate taxanssthastemtdsth Gemthandhth Ahahtddpsts Camtn Th y 0 mte th cam m mg eeu odhphehs s have heehhepdnedth t t w that an rescuzs the gemthaodh phehstype afsmx e GA htssyhthene mutahts the pussththtythat an ts heeded fax hanhat gemhattah trmte we vmuld expeet ts deteet gemhattah phehatype th an mutants We ratthd that an mutahts exhthtt a gemthaodh phehstype th the ptesehee afAEA Gemhattahsrhsth the ERbmsymhzoc mn39nmdl rl ahdthe anthsehsmve t th at t ts heeded ts wehemhe thhthmdh argemhattahhyAaA Taheh mgethzx these results pdtht ts a mte fax ERs thsmhtuanhg gemthandh mum Rziuzn39qgrmlnatlnnmGA htnsynthettc mutant h EBR Percent uermmattnn me smherahdMecuurt 2mm Figure 4 Duseresuuuse ulue m atiun in Am umares uerreut uermmauuu u ulw Illtyne hlatk square u det I deter he ur aver and Dr uver aoseeds lur briII From Steber and McCoun 2001 Vll Comrol of cellular drfferermauoh by hormones As arr example we wlll drscuss due role of hormones m vascular dlfferenuzuon here Auxln cylok mln and brasslnosterolds are all requlred for vascular dlfferenuzuon r lurlwl 5 ml om i m r lt u name a Madel Svslenls my sudwvg xueu develapmem a Yvansvevse seursulursusu s stem uem mulled sslue vesm m mesaphvll cell lvansdmevemlallan rue lsvse cell an lue len ls MW mamve and use s dlsllncl scvvr uueresslue sulseem cell sulue Hgmlusl slaned scvv uepssmsu rue lch mmalns same uulsvsplsers and exhlhlls rm Slgns we dl evenllallam quotlav sun vs kuda 2004 Name Rev9W5 Molecua Ce Boogys 3797391 mar cm Fukuda 2004 Name ewews Moecua Ce Boogy 5 3797391 a The vascu ar svstem s composed ot ph oem mammmw embwos In seed mgsl root me stems pro uoe procambwa ceHs to keep the contmmtv of vascu ar bund es ASVW memcaHv transported auxw nref ux camers A stransporte from endomembranes to the p asma brane Th mem 5 process ht be m v the he ura auxm mdo eaceUc acxd 1AA create t dback x w the AAr OW canahzatwon mode 1h procerhbtet ceHS PCS the ooordmated stghehthg by CVtOkH Hn ehd auXH v mduces the expresstoh otgehes that are thvotyed th the am eh cembtet twm s BressthosterotdsBRsththe g e are brosyhthestzed ectwetyth CS ho rete brassmoster dsdo hot s t h tor e amte e s g e e oe o procerhbtet actwmes Instead bressthosterotds th the presehce ot auXH v mtght thtttete drtterehttettoh otprocerhbtet ceHS to precursors of xyterh ceHS pXCs etter reOOgmUOn by e receptor In tsolated mesophyll cells szmmd ezegnnr a undelyused model system for xylem dlffa39enuzuon the process of tmeheary element formauon has been met tnto three stages In Stage L the mesophyll cells dedlfferenuzte after tnduenon by zux m cylokmm and woundtng and s eeme nansenpts are mduoed tneludtng the phenylpropanotd pathmy members pheny1a1amne ammomzalyase and ctnnamate hydroxylase Dunng Stage us phenylpropanotd pathmy gene expresston abates and three dl nsloml networks of aenn maments form In Stage m phenylalamne ammomzalyuse and ctnnamate hydroxylase gene ex esston agatn increase the highlyhgu ed secondary wall is formed and ogammede 1death ensues Current evtdenoe suggests that endogenous 13115 are requtred forenh39ymto Stage m Umconazole an tnhtbttor of both gbbereutn and ER blosynthesxs revented drtterennanon of z ezegnnr mesop yll cells tnto tmeheary elements and Lhs mhbmon ms overcome by ERbut not by gtbbereutn appheatton 7 V W as 2 an 4 Reviews Molecular Cell Bro09y 5 37 973 g 1 u 2 m 72 gm P i A BtuI 3111quot mm m m Rmmmnul V V quotk m mm H Wmmlwm n wllkrl an B zewr 2m zzPllxc p F391 zmvsa Ia wsx um um Po 1mm 2mm 2 15914 cm 2 ZePALlZeFM ZzFALS1DlN ZeFALL my 294142904 vac 15m 1mm 1 mm mm me Fukuda 1997 n WW 39 a n r 1 u w39w grim W CelMaxEvemx measurinan Tmm mmmmmmm Mexaphy Celh m m A 1mm memphy can a Damean c2 c TE pncnlxmcell D Immamn TE 5 a min u u am a 1 Th hammnn hum ag u m ag m appeals m be legula ed by calmm CaCaM cyxmmz HM m f h m um m h m w a q v w u q q wan vc vammle me Fukuda 1997 Evidence that brassinosteroids may be involved in xylem differentiation 39 BR Lausc 39 39 of tracheary elements formed after 24 h of culture in Helianthus tuberosus explants Clouse and Zurek 1991 r 13inifquot lwasaki and Shibaoka 1991 39TCH4 promoterGUS fusion expressed in vascular tissue of roots and shoots Xu et al 1995 39The BRde cient mutant cpd has abnormal xylem development Szekeres et al 1996 39BRs have been isolated from the cambial region of pine Kim et al 1990 BRUl a bmssinostemidrregulated xyloglucan endo ansglycosylase 39 ressed in Vasculartissue in soybean smms m m From Oh etal 1998 Fig 4 In sil hybridization of M1 in elongating soybean epicotyls Lightgrown soybean seedlings were harvested at day 14 and the apical 15 cm portions of the epicotyls were treated with 10 7 M brassinolide for 17 hours to induce elongation One micron cross sections were hybridized with antisense or sense biotinlabeled RNA probes followed by streptavidingold treatment and silver enhancement Sections treated with the antisense probe show labeling throughout the entire section but the strongest reaction appeared in the inner tissues particularly the xylem parenchyma cells and the phloem CO cortex P phloem Pi pith X xylem M1 is expressed at high levels in vascular tissue of elongating soybean epicotyls Panel A shows a one micron cross section hybridized with a uoresceinlabeled M1 antisense probe Strong expression in the phloem P xylem X and starch sheath cells S is observed Panels B antisense probe and C sense probe are higher magni cations Note the strong labeling in the cytoplasm of the parenchyma cells around the vessels arrow heads in panel B From Oh et al 1998 VIII Senescence A Senescence is a form of programmed cell death that results in the salvage of carbon and nitrogen from the senescing tissue prior to abscission In leaves senescence involves breakdown of chloroplasts and chlorophyll which results in yellowing As senescence is initiated there is a reduction in gene expression involved in photosynthesis and an increase in expression of Senescence Associated Genes SAGs These genes are involved in breakdown of proteins nucleic acids and lipids and their transport out of the cell In Arabidopsis SAG12 encodes a protease with homology to proteases involved in programmed cell death in animals and SAG13 encodes a regulatory protein thought to be involved in protease gene expression B Role of hormones in senescence Cytokinins auxins and GAs usually retard senescence while ethylene ABA brassinosteroids and jasmonic acid usually promote senescence The role of cytokinins and ethylene are the most heavily studied mm 9 ma ummn m ugd Prl rgnmum Numb alluvian mum mu unknrm mz yorc r Drunk r r 9 SenesnemeRef M rm LA Schema Hushauun emmymen vecvchng emme an senescence Sameu heve easednumemsNsuchasnmugenave Uanspuned e devempmg seeds andvuung umansanhe shunt apex a Pvugvessmn e1 an senescence m a speees mum senescence Pvuceeds 1mm an mavems mwamme came Nu etha eeus suneuneme the vascmamssuessenesce ve aWEN ale uvammale numemmubmzalmn Yvumamacem seneseme eeus Flam Gan andAmanm 1997 o 9 1mm gm w hm M M Fgun 2 D enmal gen explexxmn dunng 1m xemxceme 37 mxceme dawnrlegulamd gem um wand gem mm m phnmxymlmn xuch 31m chlnmphy hrhmdmg pmm gen 1mm and the Rnhuca xma xnhnnn gem AC gem mm explexxmn npdeguhhd dunng dzvelnpmem hm thu 1m th dmmgxe me Gan andAmanm 1997 A mm Retamauun m an mm W WWW pmuucuuh EIYEWEIKWHS A The s r ecfcsAcz nmmmev 35mm Senescence oymhmms uk 2 mmuucuuh m EWDKVHHS a A Dunlame ms ammegmamw warms sh hm 2 whom m mm W W scunsm 39 n9 oawashhmvpmvmeu by D W 9mm UYAEVDLO The Neme ands me Gan andAmanm 1997 F1gure1 Fluwnhanmdlcaung me senes uf eps mescmceassunaled mhanca39 at beuem shuws an Example fur sneemn Eaehweu cun39ams 711 111 qummhylumbalbfa39yerrgucummde 1m 1mes was EMMAm MW we11 uddrnumb ed ee1umn 1 3 s 7 9 and 11 and 11 a samples are frumthe me pm as areAK new senesan and A4 senesan mp1es 81 Samples mweus Al 112 mmughm 112 are mmmmype Umver ty LSSEIE 1samudel ufFErkmeElma s 1um1neseenee wedmmelers meHe e1a1 2mm nqurez Examl2 m a smzsunq hemvmm a w um i an namequot me re eueeue mare Insm lqmaand vlzn39a en alum menu r an amvllv m asn52msl me enema mmuqh 1 1mmquot 11 zxwuslnn m emz leavzs by Am 01 and snun I emailed wan a nemenemm l23161h2laafshnwn m n s a unnrsmzsdng lean emz trams mtth nMABAfnr h meHe e1a1 2mm nu n whim cm n nf n mmm 397 NM dwrrr quotwnvlnffvn dvyvri WWW WW4 up m Hm Emu1h 125mm Eul mural Shem A A nquot n my m 7 meHeetal zml r mm A A putative Izafsemscence xeguhmxymwmka Arabde 5 GUS expressmnmselsafdu mplzvelcxrc pmmamwg sums G ctnn 1 due m sums sud n maybe regulated hm ax athzx facmxs n 5mm ma um thls us a vexysxmph zd madzl as amused m m m meHe etal 2mm Flgnre 5 RNA gelhlnt E 5 s nmeSV yzllnwmg ax marshy semscmglea slm ma I s pm meHeetal zml meuhh IX Absasnon A Absclsslonls the separahon oflezves ower frults bud stem etc from the parent plant and occurs at a narrowlayer of cells called the abselsslon Zone Ethylene ls thought to promote absclsslon b mducmg genes for oellnlases and peetolyases whlch cleave the mlddle lamella between adjacent layers of cells In the abselsslon zone Un ual ai anslon of the oell layers then creates a mechanlcal force whlch leads to eleamge Auxms retar K1155 nd Jasmonates promote lt Erasslnosterolds have been shown to prevent premature trult drop m grapes and oranges and therefore may be lnvolved m abselsslon as well mu W y Mm ngu y x m Inhinugwn hyn n max Auumu may AbsmssmnRef M AbsmssmnRef 3 AbsmssmnRef 3 mm 2 Pmyzxxwe Change Rammed mm mm Organ Ahxcmmnm Anhxdnpxu A Schzmanc npnxema mnafdzvelnpmentzl pmcexxex mum wuhahxcuxmn Hzavxly xhadzdcuclex npnxem xma dzmelycymplmmc cell apencnclex npnxen arge vammlamd c2 af mam mum hghdyxhadzdavak npnxem expandu gcelh anthz pmmmalface af m mature plan thaunmm uemam mm m pendzrmalxcanuxvz AbsmssmnRef 1 planiphynal law m 5577562 Comparative Study of Ceiluiaaes Assodated with Advenvivious Root Initiation Apical Ends and Leif Flower and Fed Abscision Zones in Soybean Elizabah C Kemmerer and Mark Tudier Cellulase activity was me ured in soybean Glycine max leai abaciaion zones lmer absdssion zones pod abaciaion zones apical buds and adventitious mo ng hypooolyk lnnnimoprecipiiation daia showed that a oell innnimologitally leai ower and pod ahaeiaion zones but 39a not pesent in soybean apical buds or mo ng hypoootyls cDNA and genomic clones or two di erem soybean genes were identified and show Sequence similarity with the bean absdssion cellulase elone pBAcla The emu elone ysacl isolated ran asoybean abae39asion cDNA libiary h 39dizedlolmmcri is in soybean leai ower and pod abae39asion zones Although 39 mo hypooo y uamm39pts were detected in apical bmk nammpts or a second soybean oelldlase gene scz were not detected in any oi the t39asdes surveyed Figure 3 Ndmnern pidts ufpuMA RNA in 5 ug rum pean and suybeantissue extracts Twci identicai pidts were prepared and each was pruped separateNWitnt c n e re A and psAci Bean and szbEari expiarts were treated With 25 uLL etnyiene in airrdr u urAE n Oniyme apscissidn zdne tissue A w cdiiected at u n Frum Aer reated elt iams apscissidn zdnes Al pennies Pi and sterns swere cdiiected Hypridizatidn cdnditidns rdrtne pEAOOrprubEd him were 2 runnarnide 5x SSPE 2x Denharm s suiutiun iau MgmL denatured sairndn spenn DNA at 42 and the rinai wasn was u 2x SSPEi an rnin Hybndizanun cdndindns turtne pSACirpmbEd him were em rdrrnarnide 5x SSPE 2x Denhamt s suiunun iau M mL denatured sairndn spenn DNA at 42 and the rinai wasn was u 2x SSPE u 1 SDS at we term rnin 1713an11me 1995Int55775 2 LAZ FAZ PAZ ARH AB Figures chmpeslle nerlhem mm at pelysemal RNA 5 he 1mm whean lean awer and pen ahselsslen zenes LAZ FAZ and PAL respecnvaly advermtluus munng Wpucutyls ARM and apleal hues AB lms were pmbed wnh eDNA lnsenrmm sAcl LAZ and FAZ RNA arerrem eltplarls vealed wnh amylene far 12 h and RNAfmm PAZ ls rmm explan treated wnh emylenererza h The AB sample ls rmm 117 tn meme plants anelhe ARM sample ls rmm hypeeelyls erexplanls 1mm 1D In meme plants treated wnh 13A 1 hwrerza h quuWEd by WaterfurAE h Hyhrlelzallen eenmllens were em fumamlde 5x SSPE 2x Denharm s sulutlun 15m hgml eenaluree salmen sperm DNA awe and nal wash was 1 2x SSPE u 1 SDS at EI Cfur2EI mln PlamPhyxml 199 m 5577562 The Flam Cell Vel 6 52mm Apnl 1994 The Never Ripe Mutation Blocks Erhylerre Perception in Tomato M B Lanaharh HslaorChlng Yen JJ GIOVBmOm and H J Klee Seedllngs e Inmaiu run npemng mwahe were sereeheel Dr 39helr ablllty ID respo ID 1h 1 he mman39s eseel exeepller DUB Never quotp N7 1 Our resulls malleath Lhatthe lack e M an Vuanm a N fn lexpansmn lnemllzzueh dues heleeeur pedmel abSClSSIDn eeeurs 5 1e 8 days amer h 11 he h 1mm mh A andevelupmem m N plan39s ms helpreeeeeel by pelal seheseehee pe39als andamhers 1 engmeexed lamirvcydopropmel mrboxylam ACC syn39laseruvexexpressmg plan39s lhelealeel that they are phehelyple eppesles uf 1v plan39s Cehsmuuve expmsslun e ACC plahl and rhelueeel peuele eplhasly and premature seheseehee and abscissl n e Powers usually helere anLthls There were he nbvmus effems eh seheseehee m leaves elAcc synthase nverexpressexs suggesung that althnugh ethylene may he Impunam ll ls hm suffment 1e cause Inmaiu leafsenesneme DLherslgnals are elearlymvulveel Ham rna Enaes mE Wene an Absussanand Senesuenue m Yamata Yamatamm nan pawn NrNrenand pawn nrwmgh wsnls s and 2 weeks Yewedwe v anev hveakev saga Yumma mm mm Aw cyae Nywr en Ansa We Nrwmxdd eL and Aw cyae mntnent mantsv vs 3121 bvmkevstage D amatmn saanca E Asdavmdseneum pawn nmna y madame serves22m pawn mmnanaymn a sms devdamng mm TheFlam Ceu Val 6 5217559 Apn11994 G An 1 waeayaem Pearsun NrWr uWEr shuwmg ne stgns etsenescence a se a was re HA137dayrud Pearsun NrWr mEr muvedfurantWm a deve upmgfrmtshmmg hm ted stgns er senescence DA Pearsun NrWr uWErgt15 days cm shuwmg tne type ct pae senescence cnaractensttc er unfemhzed uwers ct tne mutant ThePlantCeu Val 6 5217559 Apnl 1994 x Fhotomolphogenesls A nght ls one of the most lmporlarlt slgnals controlllng development and dlffa39ermauon m plants stem elongauon seed gel mlnauon mmauon owenng leaf development chloro h ll blosynthesls and o expresslon of mar enes and the acuvrt of several enzymes are regulated byllgrt The mteractlorl of llght slgnals and hormones IS a Very acuve area of research E There are at least three photoreoeptors m plants l Elue llght cryptochrome rUVrA absorpuon Involved m phototroplsm and stem elongauon 2 ller 7 lrlvolvedmmdueuon or avorlolds anthocyumns UV protectants ed llght r Phylochrome oorltrols a large number of responses to llgrt lncludlng stem elongauon m am mm quotmm mum mm er mm Nemhauserandchnry zmz Cnlyledon Hypucotyl at nght Skatomovphogenesis Photomorphogenests Mutant analysls has shown that brassrnosterords are actwelyrnyolyed m photomolphogenesls When dlcotyledonous lants such as Araautnunn are uan ey e u chlorophyll AE bmdmg proteln lncrease drarnatlcally To study howllght oontrols developmental pathways a number of Ambldopm rnutants known as m er lolated cop nsutuuve photomotphogenesls and fu fusca have been lderm ed that have charactensucs of llghtrgrown plants even when reduced rnale terulrtyand a rcal domlnance delayed owenng and a delay of leat and chloroplast senescenoe Chery et al 1991 Such a broad spectrum of effects shows that 0512 p llfe le ln s erpanded cotyledons typrcal of a lrgntgrown plant det2 has been shown to be a Eere clent mutant blocked m the step trom carnpesterol to carnpestanol Fgun 1 PhenquES uvoamomwn and Uumremwnduzand Tvp Amme Seedhngs A Savendavrum nammuwn mm m We 02quot and 77 davrum nammuwn M212 mm ENawardequ huhmmwn WHHWPEe and127davrud huhmmwn 4412mm Ea cm me Chrystal 1991 D s a Sralpha steroid reductzse involved in Bmsinoscmid biosymhesis that can be rescued by BR treatment D Light and Hormones overlap in meh effect on many aspects of developmentanda 39 pomntquesd n h nlm o Is whe39herlig tandhormoneactindependendy whedmer me present d2 mutants mus cywkinins lighteregulaued develome y 39mm d or umys of mp mm role in 39 nce ha ownrregulate brassinosberoid biosymhesis genes which would explain meh phehmpy of Jet mumnts Flglll z thmtype afwddetype Anhldnpnx xeedhng am 2 exzweeheryemhmh daran mg emn am mm A Nam ch funnananafa mm wnhxevual have Ear 1 cm me Chmy etal 1994 Nemhauser and Chmy mm X Phototroprsrn A The abrhty of plants to grow toward the hght rs a well known response The Cholodn rWent hypothesrs 1928 states that phototroprsrn results from an asyrnrnemcat drstnbuuon of auxm m response to hght less on hgnted sxde more on shaded srde whrch results m drrrerenuat growth Modern arguernents for and agamst ths hypothesrs are desmbed m your book Some possrb1e rnechanrsrns of asyrnrnetnc drstnbutron of AA 1 Lrght causes net hansport of AA to the shaded srde 2 Lrght causes destrucuon of AA on the hghted srde 3 Less 1AA rs synthesrzed on hgnted stde 4 Lrght decreases rate of AA hansport on hgnted srde hanges m 1AA sensmvxty the rnyotyernent of other hormones and drrect rnhrbmon of cell elongauon by hght are other possrbrhtres The drscoyery of phototroprn I an d u blue hght receptors mth kmase acuvlty suggests that phosphorylauon 15 mvolved m phototroplsm perhaps by a1tenng auxm gradrents nq 2 Prnlzln nructumnnhe Arahl nuxlx hluE um remtars plum and uhntz m and 915 ammn arms rzspzmvzly 1mm nxqun nr vnltaqE lav nnmaln m hnwn m qrzszhz kmaxe nnmalnxywhlch catalyxz the uhnxuhnrylatlnn nvprntem nn szcmc ammn acm mmue thrennme ann smug m thuxcaxe am mnwn nu ma Lxscum znnz Engg and Chum znnz fur all mm mun mypnnss unwanma mg mam mmnsllv mm bluerllq XII Gravitropism A Asymmetric distribution of IAA or changes in IAA sensitivity can also be invoked to explain root and shoot gravitropism Davies has a good section on this XIII Apical dominance Davies describes the roles of auxin cytokinin gibberellins ethylene and abscisic acid in apical dominance Brassinosteroid insensitive or de cient mutants show a decrease in apical dominance suggesting that brassinosteroids are important for apical dominance during normal development The Plant Cell Vol 3 1167 1 175 November 1991 O 1991 American Society of Plant Physiologists An AuxinResponsive Promoter ls Differentially Induced by Auxin Gradients during Tropisms Yi Li Gretchen Hagen and Tom J Guilfoyle Department of Biochemistry 11 7 Schweitzer Hall University of Missouri Columbia Missouri 6521 1 We constructed a chimeric gene consisting of a soybean small auxin up RNA SAUR promoter and leader sequence fused to an Escherichia colt Pglucuronidase GUS open reading frame and a 3 untranslated nopaline synthase sequence from Agrobacteriurn tumefaciens This chimeric gene was used to transform tobacco by Agrobacteriurn mediated transformation In R2 etiolated transgenic tobacco seedlings GUS expression occurred primarily in elongation regions of hypocotyls and roots In green plants GUS was expressed primarily in the epidermis and cortex of stems and petioles as well as in elongation regions of anther laments in developing owers GUS expression was responsive to exogenous auxin in the range of 10398 to 10393 M During gravitropism and phototropismthe GUS activity became greater on the more rapidly elongating side of tobacco stems Auxin transport inhibitors and other manipulations that blocked gravitropism also blocked the asymmetric distribution of GUS activity in gravistimulated stems Light treatment of darkgrown seedlings resulted in a rapid decrease in GUS activity Lightinduced decay in GUS activity was fully reversed by application of auxin Taken together our results add supportfor the formation of an asymmetric distribution of auxin at sites of action during tropism FiguresH1smenem1ea1stammgrureusmme Stem er a Transgem Tubaccu 91am after 4 Hr er Grav1st1mu130un A4eweewu1u mbaccu p1an1was gruwn 1n MQWdarkcymes as descnbed 1n Methuds andmen mated 1n usrmessrur24 m pm 1 cammg uutme grawtrupm expenmem Grawsummatmn was named Dutm me new andme stem was msected and stewed furGUS aemy Mums SeedDeve1epmem 1 Memke DW 1994 Seedaevempmemmmmgpm thalmm InAmmmmm E Meyemwnz c Sememue pp 255295 New ank Ce1e1 Spnng Harbnr Press 115 G 1994 Pammfmmauunmtheembrgm In Ammaopm ee1 E Meyemwnz c Samemlle pp 297512 NewYDrk Ce1e1 SpnngHaxbnr Labnmiurnyess Knumneef M Karssen CM 1994 Seeddnmamyandgemmauun In 5 ms Walbm v Hnlu er N Z1mme an L 1995 L1u CM Xu ZH Chua NH 1995 Auxm pe1amanspems essenual 1390th2 es39abhshment e1 puma symmetry dunng early p1an1 embrgmgenens Flam Ce11 5 621763E 55 1987 Deve1epmema1 Bmlugy Randem Hausa New Ymk Samaucembrgmgenens Flam Ce115 141171425 Dure e1a1 1989 Cummun 21mm and sequenee dnmams mung LheLEA pmueue thgherplan39s PlantMnleculaerlngy 12 475486 T Berleth and s Chat ed 2912 Embxyugemss Panem Fama mn 1mm 5mg ce11 1n The Ammaepss Bunk 1111p Humwasp ngyubhcanunsambmnEBmn 9 Dawes 11 FlantHnmnnes 2nd edmun 1994 Kluwer References Senescence and Abscission 1 Bleecker AB Patterson SA 1997 Last exit senescence Abscission and meristem arrest in Arabidopsis Plant Cell 9 1 1691179 2 Gan S Amasino RM 1997 Making sense of senescence Plant Physiol 113313 319 3 Brown KM 1997 Ethylene and abscission Physiologia Plantarum 100567576 4 Arteca RN 1996 Plant growth substances ChapmanHall New York 5 Yuehui He Weining Tang Johnnie D Swain Anthony L Green Thomas P Jack and Susheng Gan 2001 Networking SenescenceRegulating Pathways by Using Arabidopsis Enhancer Trap Lines Plant Physiol 126 707716 References Mothogenesis 1 Chory J Nagpal P Peto CA 1991 Phenotypic and genetic analysis of det2 a new mutantthat affects lightr m at d eedh na in A quotd r 39 Plant Cell 3445 459 Chory J Susek RE 1994 Light signal transduction and the control of seedling development In Ambidopsis Meyerowitz E and Sommerville C eds New York Cold Spring Harbor Laboratory Press pp 579614 Chory J Reinecke D Sim S Washbum T Brenner M 1994 A role for cytokinins in deietiolation in Ambmopsis Plant Physiol 1043397347 Li J Nagpal P Vitart V McMorris TC Chory J 1996 A role for brassinosteroids in light dependent development of Ambmopsis Science 272 3987401 Cotton et al 1990 Downregulation of phytochrome mRNA abundance by red light and benzyladenine in etiolated cucmber cotyledons Plant Mol Biol 147077714 Iino M 1995 Gravitropism and Phototropism of maize coleoptiles Plant Cell Physiol 36 3617367 Li Y Hagen G Guilfoyle TJ 1991 An auxiniresponsive promoter is differentially induced by auxin gradients during tropisms Plant Cell 3 116771175 Emmanuel Liscum 2002 Phototropism Mechanisms and Outcomes In The Arabidopsis Book httpwww a nh quot 39 39 39 Nemhauser and Chory 2002 Photomorphogenesis In The Arabidopsis Book httDINWWW a nh quot 39 39 39 rfm WR Briggs and JM Christie 2002 Phototropins l and 2 versatile plant bluelight receptors Trends in Plant Science 7204210 rfm H87 02 Lectures 1314 Practical Applications of Plant Hormones and their Derivatives Tissue Culture be dew 7 g 7 JAMM A ll Some of characteristics 039 culture are I a occurs on micro scale l3 performed under optimized physical and nutritional conditions c microorganisms and pests are excluded 1 01 normal part quot s of development are often disrupted eg the f a 397 a 39tu reutiated or cle cli erentiatecl callus cultures on agar medium e it gives the a ility to grow arid manipulateindividual plant cells f many plants liare the capacity for regeneration of a mature plants from a tissue or single cell toupotency Tissue Culture 111 T pes of plant cell and ti 1e culture 39 Callus culture Single cell suspension cell culture Organ culture 7 l meristem 7 2 shoottip 7 3 root 7 4 anther e Embryo f Intact plan s I m mm mar whim um 0mm wt Jmlr w m m Drczdm wk mum lmu or nul mm mm ol m Mrmlcm mm m Ia Smut Im a mum us twin mum Herrrum mm ulmvr m 10 n CnHuL2mpmucn m H mm x2 x 7 7 1 Jun 1 ml Emmy of main and mm mm m 21 M munenmmnmhh mu pn mm ubxmlnn a1 Klaus m 1 mum Immune on I mmplnu cullwv m l m mumquot ulnmm mum V Chronology of important events in the development of plant cell and tissue culture techniques l902 Haberlanc lt originates the concept of cell culture and was the rst to attempt cultivation o isolated plant cells i Vitro failed 1927 in Vitro germination of orchid seeds l922 Robbins develops rootutip culture and maintained maize roots for 20 weeks by subculture l934 Successr al culture of tomato roots White 1935 Discovery of the plant hormone auxin Went Thimarm Snow l93 6 Ember culture of gyinnospcrrns l939 First continuously growing callus culture Gautheret Nobecourt White from cambial explants of carrot required lnclole Acetic Acirl 1AA an auxin for growth 19M linvitro culture crown gall tissue Braun First tobacco culture Skoog ee dalilia nroduced by istem culture l953 Haplorcl callus from pollen produced 1955 Discovery of kinetin a cytokinin by Miller 1957 Regulation of rootshoot formation by changing cytokininauxin ratios Slmog amp lWller Low auxf 391 V Chronology of important events in the Cl lopment of plant cell 39 e t eelmiques 1960 Cocking uses enzymes to obt large 7 bers of protoplasts l960 Veget ive propagation of orclnrls by ineristem culture 1964 First haploid plants produced from pollen grains 1969 lsolation of protoplasts from suspension cell culture l970 Selection of biochemical mutants in Vitro l970 Protoplast fusion l97l rirst Plants regener l97l lnterspeci c protop ast i lsion 1973 to l998 Plant cell and tissue culture becomes indispenwble for plant molecular biology and genetic engineering a The media used for plant tissue culture and the plants themselves are ideal substrates for the growth of bacteria fungi and Viruses These must be eliminated for successful culture of plant cells and tissues Most experiments are performed in a laminar flow hood which lters microbes out of the room air The surface of the lam39nar f 39 ow hood is erilizecl with alcohol andor li ht Plant parts seeds stems explants etc are sterilzed with ethanol or dilute bleach sodium liypochloride and rinsed with sterile water zation teclmiques used in plant cell and tissue a b Types of sterilization procedures 7 1 physical destruction gtgt i Dry hot air metal insturments and soil can be sterilized in an oven ii Steam the autoclave is essential for sterilizing liquid components as well as instruments glassware some plastics and soil mixes The advartcges of autoclaving are speed implicity effectiveness and destruction of viruses The quot at e the ti componens suc as zeatin gibberellins Vitamins B1 B1 panotlienic acid and C otics and enzymes lt can also cause sucrose to car i which toxic to plantcells salts can precinitate and volatile substances are lost a r 0v 3 Jl luL gtgt iii irradiatiori plasticwareand surfaces can be sterilized by If V or gamma irradiation V Sterilization techniques used in pl i culture 7 2 Chemical destruction gtgt i ethylene oxide 7 gas used to steri e glass and plastic gtgt ii alcohol 7 ethanol used to Wipe counter tops and steiilize seeds and plant parts iii hypoclilorite diluted commercial bleach is used to sterilize pla A ssue and seeds iv antibiotics kanamycin nampicin arnpicilliri chloramphenicol genie n streptomycin etc are ccasionally added to med a to prevent bacterial gro Nth 7 3 Fi ation nylon or nitrocellulose lters with 02 or 04 micron pores are used to remove bacteria ard ungal spores from media that cannot be autoclaved Does not quotnove most viruses J 19 VI Nutritional requiremen of an la 11 ti e growing in culture are otten ditTe ent than a whole plant growing in oil The component of 39 ue culture medium can he cla 39 iet gani ugar amin 1 aci vitamin and growth regulatorsl inorganic macronutnen s P K Ca Mg SL inorganic micronutrients e 1 n Ni B Al Mn Mo Cu 1 unde ned ated plant 1 or 390 a e ac p extract c ein ljtl ot39 r 1 el The are a number ofwellkno 39 ndard culture met ia 5 me of which are lited helow Otten th need to be modi ed depending on the plant eci e ant 39 l e type lmwT mmvmwmw m39 39 quotL quotm n u Mama u mm a p i m 39III Optimizing media component a 39 r mammal in in 1 mm axiEWplam IX A callus may be defined as an amorphous mass of loosely arranged thinwalled parenchyma cells arising from the proliferating cells of the parent tissue Callus cultures are grown on solid agar medium and consist of a mass of undifferentiated cells a Source Material Theoretically all multicellular plant material should form callus In p1 ctice immature dicot tissue is much better than mature woody ti ue or monocots Especially good are s ybean cotyledons bean hypocon potato tubers carrot cambium 7 tobacco pith 7 sweet potato storage root 7 radish hypocotyl b Callus cultures must be transferred at regular intervals to fresh medium for continued grov h Th subculturing I Grmull rusponst m 1 l alum Lulruzc 39Hus wilth Fig 3 r callus should l s xubculmrcd a ly at rhu umu unlitch lxy I l 000 900 BOO 39 ngm m gurus lmql J 2 16 20 Days a cumin Tnssue culture is used 0 propogme disease free honiculmral crops 4 m Cm umm m Effems ofauxin and cymkinin on propagmion of roses in vino Sammie emb ogenesis IS anmher imponam is us WWW 9 pmwuw lw ill 1 a quotmy 7 mmnmmmw rquot Pullman a and Pam 1 x w u amabxln u may 31 E 11 somum nmnmgmm m Inmum pine 1 9111mm 31 E b ur xnmananxnwmtex and me namceu Mm warm r Megagameluphyk EunyExmmm Lme Exhusxnn human Wm I mm The use of auxin to enhance routing in V gemive propngmion WWW Q The use of auxin to enhanur naming in Vegetative propagmion The use of auxin 0 enhance mo in vegemuve propaga on msmuuun mum rm 5 liLI iD S of bmssinostemlds enhance V 39e l m mu rmquotmmmommmu I Dmnm n quotmum Emu um 17 M m NW km mm mm mm mo m m m 7w an A hid y w m m v 1 mam nqvmu m mmquot quot7 m mmth H mm canons of bmssinostemids enhanur yield H87 02 Lectures 1314 Practical Applications of Plant Hormones and their Derivatives x 1quot45 1 A s Ay4ul A A r a lm fi39 UJJUSL 44 499 at 9 m 5 i1 1 Fppigeapp Mumwmm I X r epiquam re M04313 90 CD9 11 nunc1 mm s antk aurene Clmam I u V nclnhulmzci entrKau renolc Aold niconaznlerP lnaben de GA Zfldehyde n exadioneca Tnnexapaeethv Danunazide A Onium compounds Inhibit the cyclization of geranylgeranyl pyrophosphate to copallyl pyrophosphate eventing the biosynthesis of gibberellins Treated plants have shorter internodes and thicker greener leaves 0 Used as anti lodging agents in cereal production and for the reduction of excess vegetative growth in cotton mm 2 pm gm mam x tummy nmtmmnmchlcmle lhlmozlmlinen cc 4 Memqum hlan e Dianelhylylper mulm chloridleFC am 1 n V p u ma lrcnlunxllnll39lnxL Mandi e I dol and Fluqnimidol which are thought to inhiblting the cytochrome P7450 w lie 1 controls ox dation of kaurene to kaurenoic acid during GA biosynthesi Simr cyt hmme 1145th are also involved in brassinostemid biosynthesls these compounds are li e 39 to in 1i i bios vnthesis as well Us l on omamentals and turf gras B Pyrimidines 7 The two most often used at nc Triazoles 7 the most common are Paclobutrazol and Uniconazole which are cytochmne P7450 39nhibitms which lead to blockage of GA BR and steml biosynthesis Brussim mums fruit mes nd ornamentals are comnnn uses of es t tnazol aquot A lt I A H OICOQHZNOS QIDVWQS quot 1 x 1m 412mg D Cyclohexanetriones cimectacarb and prohexadione calcium inhibit later stages of GA biosynthesis ie hyd Kylation at th and 3f Figure 4 Structural mimics of 2 oxoglutaric acid I Prohexadione calcium ll Trinexapacethyl lll Daminozide IV 2Oxoglutaric acid Rademacher Like Cutless Primo MAXX 1L trinexapac ethyl is a Type II PGR that inhibits GA biosynthesis However Primo interrupts biosynthesis late in the pathway via the 3Bhydroxylase enzyme Blocking this enzyme inhibits formation of active GAs Unlike Cutless Primo is foliar absorbed thus irrigation should be delayed for at least one hour following application This should be done to ensure adequate foliar penetration The rate of Primo typically used on Tifway bermudagrass maintained at fairway height ranges from 6 oz to 12 oz of product per acre 042 kg to 084 kg product per ha Periods of rapid growth andor excessive rainfall could require use of higher rates or shorter application intervals The cost of Primo at 128 oz of product per acre and based on seven applications per season is approximately 245 per acre annually SePRO 2004 Bene ts of Primo use include increases in turf color promotes lateral stem and root mass development and reduces vertical growth SePRO 2005quot Table 1 Types of plant growth regulators and rate used by Noth Carolina bedding plant growers Crap Chemical Type Agernmm Geranium Cyrocelquot Seed F Spray Rates number of times applied 5000 ppm 1500 ppm 10 ppm 20 ppm weekly 30 ppm as needed 30 ppm applied once 3 m 3 ppm 3C00 ppm once once 3020 ppm 30 ppm applied once DI me if needed l 0 ppm apphed every 10 days if needed Stage of Development Chemical Applied Used as needed to one and harden Apij 39 3 Weeks a er transplant Applied Men plants are 354m ushed size to hold 2 l0 3 weeks Applied 3 weeks after uansplammg 28 plugs Apply um weeks after uansplamng 384 plugs Applied as needed depending on plan growth and weather days me Hansplanu39ng 381 plugs L39sed as needed to tone and harden Bonn apphed 5 l0 6 weeks lam Sm applxcacimls 2 weeks after unnsplam Cycotel 1500 ppm applied once Cytocel 1000 ppm or Applied after mplanr 3mm 3 ppm or 39 alter transplant Floxel 350 ppm Applied a ex transplant F1151 BSine Followed by Eonzi 000 ppm applied mice 5 ppm Applied 2 and 4 weeks after mspm when plans are Sums nished ste 0 hold 2 w 3 weeks uzmnn Illuh rule 1 39r m39cl 14300 mun um tam 1 uu39m quott m mm Wm H m A http u u vawces110su eduu depts11011quot o cultureu Plorexu BP EZOPGR 120Rates202 pdf Paclos Bonzi and Piccolo Courtesy of Brian Geranium Paolo Sprays ppm Piccolo Pansy Paolo Sprays ppm PotSunflower Paolo Drenches mg ai 7 j 3936 Top or Pimp imjdo Courtesy of Brian Whipker or Pre plant Soak Dr nchsss Courtesy of Brian Whipker Caladium Sumagic Drench I Caladium Piccolo Drench May require 15 mg drench for control Caladium Top or Top or Drenche s Dahlia Dren of 1 to 2 mg ai Topflor pflor Top or J Courtesy of Brian Whipker Geranium 6inch Samba Pot Mum Duluth Control T25 1 x T25 2x ppm Bonzi or 5 ppm Sumagic T25 T5 B30 85 Asa i T and Ynsllidz a 1 Wmummmw v Siw wv amt a r mum pm am39nshidas mm mm x J 1 alum mm 1 1 T Uh hgln nix 1m dad HS702 Lecture 11 Cytokinin and Ethylene Signal Transduction Regulation of Gene Expression by Plant Hormones Steve Clouse Department of Horticultural Science North Carolina State University October 20 2008 READING To and Kieber 2008 Cytokinin signaling two components and more Trends in Plant Science 13 85 92 Etheridge et a1 Progress report ethylene signaling and responses Planta 2006 223 387 391 TWO component phosphorelay systems are involved in cytokinin and ethylene signaling A A classical TCS E coli osmotic regulation Signal Requ EnvZ P ompR E 9 Output Sensor Hiskinm HK Rsponse Regulators RR B A mullisup phosphorelay via HPI E cab39 anaerobic regulation quotPl Rm P Ach p p e A A G3 gt L gt0uipul Hybrid Sens Hisldnm HK Spam Rgulalors RR C A TCS in eukaryotes S pambe oxidative stress response Signal Receiver v P PhklZf 0 Spyl Mcs4 P qp gt p gt or Hybrid Sensnr Hiskinnsa HK HPl R45an Rgulnlms RR Mizuno 2005 Biosci Bioteclmol Biochem 69 226372276 t M 1m 4 mm am my u m p humpunner m w 7 mm M K my mmmm am n w mm mquot WWW mum m er mm mm w my nmmnmmw mm M 0mm yam mm memm man 12 t wasan M xx Mn W vmn Mum mm cympmm mummyquot ucleus A l C D Mam Inactive lurm mam mllvz Venn H AW 2 a Targu gems mm mm m we CRE MHKA may quot mum if gk 0 1 2 pm 1mm B USIIIVC re mm regulator in L tokinin signaling Induction of callus fornmion and shoot initiation on hypocolyl segments from Col arr1 arr10l12 and arr1l10l12 on different concentrations oftranszmtin ZAD stmw VIAMin 19M lt Fl the anlllEllIZ mmant A Eighteenrdayrold plants ofCol an arrlU arr im an aan am12 arrlEl arrlZ arriEllZ an arrlEl arrlZ mum12 audath 31116 er am5m mutants Theywere grown onMS agar plates under long day nonmuons 16 h light8 hdark Wentyeightrdayrold lam ofCol anlllEl were e smcesswe 51hqu and rst Overexpression of the typeA response regulator R6 abolished shoot regeneration i 39 suggesting that OsRR6 acts as a negative regulator of cytokinin signaling OsRR remesses shnnl muenemllnn m nce call Morphologles of transgenic plants overexpre rpl xm pl39 a mu um daulcal quot151211 522 quotIn wm n an r A Sncla m 1 r Advancement n15u2ncePhnln by K quot1 smu nu erhman sgam uvzrx yJ mfm 1m 3 m w my gm 539 quotw W mm mm nmw n m LDquun o 1 mt um mm mm m o m r u rdnimm Wm W mm mm m m HHJHV Mn r m m n o a mum ma 3 asmunmamnmmagnummumyumvammr am hi mr to am an 39uresslnn Izrdnre t rrnws and mm Im mm quotmm 21mm um mama murmur amnn nu ma mummy am mmamm wmrzas mum I hm nn 1 r 1mmquot shmvntn 0urthmllglmlren m g 1 mm 1v 1 cnnmr aalJmamznnssanMs w pm vm m Saluh mmmmm n m mm m a E F mm mm m m xhlr mm quot1 mm crp HAHF and Am mm mam r elal Ann amznns ugnL 5 Repress thylcnt res m1er CHEN vrr a al Ann am 2nn5 959n17915 p Jun mm mm u m WNW m pm 11 Amy Wm Muw m m Hhxdynwhuuiul nvxnudw mlmvhmdn Manama 1 W vmm lwydwlm 1 w TF I n quotmuquot m u j u x 1 Amman quota u F mum uvv1xmvuw 139 TH y hm mm pr xn u nm In quot 139 mm M min1m wm lw w my mm m lmmx may quot TF I mm mm 9 m r n a 1 may W 0 mm H up app p ada m mm n 31 3mm H mm mam MAPKKK J quot2323222 HHIHHW H m an summ awm qmnscnpnm am 5 Tramswp ons cascade wequot u smmg me x gmmmnbvwzdwlaw 1w eumene B Regulation of genes by ethylene during senescence of carnation flower petals l The best known gene in this system is the pSRS gene which encodes glntathione Stransferase A A M r i i v p c 4 A 140 op sequence from the prommer 1s necessary ano sufncrent lor ethylene regulation in transient assays in carnation petals 3 A nuclear protein from petals was found to interact with this region C Regulation of genes by ethylene luring tomato fruit ripening l Ethylene induces a set of genes during ripening in tomato fruit E4 which has homology to methionine sulfoxide reductase which appears to be a dioxygenase a F enees tor ethylene regulatiOn and regulation t ripe clear protein that binds to both the l l and E8 promoter has been identi ed and the corresponding gene has been cloned 6 L XUI ESSL Cl lnun m math Wu rum mmg 1 m am mm mm mm n w 1 v m r4523 w 1 v77a mm V Luxx xm u mun w wn39 mm mm h 1 mi N mg a um ch H dn39mwxvaj 31 w 1 qm mum u x mum maximu M a 1 h v m axujHFTpmvvuu m gnu Bade a d have we r a mu ppuuhe meehamsms uf physm ugma prpee sea as a resu he he h the g uba appruach arm the uup ed thh a u dempher p ex re y have the resuurces m El stare dat re atmnsmps whewuax mves ugaturs ra purchase the Eqmpment reqmred furtms type uf ana ys s a dt us became prumment craarray u iStTL39C DH clst or 0335 CONE 0L TISSUE RENEW PCR Ampli cation Isolaw mRN A w J Txeaued glass slide wiLh 1 Upw 10020 5 pl spots mm be of DNA 973 00 HYBREMZE LASB SCAN K r39rm1 ltcn HivIn lmm39rvui Ala rmnw IA 13mm 35 J Prepare cDNA Probes Uniniec ed Infected N xxx W Reverse Transcription Label with Fluorescent Dyes G Fl omblne Equal Amounts Hybridize Probes lo Micmarmy I Prepare Microarray G D Gene Transcription in Sporulating Yeast C1111 et 211 L 23 OCTOBER 7998 VOL 282 SINCE Gene Transcription in Sporulating Yeast AHm I 5 01122 5 1 911 Iagmrupruum Iala 1 c z a 390 1131an use 3 o Genes medic cream mags lenpuml pm lea HEWquot Eulzl arm II C1111 et a1 vizNH mum Mlddh GRII AFLH m quot1115 23 OCTOBER 998 VOL 282 711an Newquot wow MW m39yum Humansqu m 53H1Vuu ew 1mm HM mm my m mm 44 m I Plant2ll m I5 ISnlrlnnuulv um i mi mm a 39 W Lquot quotEmu m mm wa Table 2 GA nesuunsm Gen n es lmulltated m ell Elunuatlun my Mnumn m urwm nun n caringllama gum and Metalmlm mum x mm 2 mm m m an pallzl h graphs at m h yam mgr 12 n 39 T 5 ga n 1941 We ailev rammmlm quotm 3 mm mm p nlmmsmn a m 24 2 4a a me any mtlbmth nu Plant2ll nl I5I5 llrlnll41uv 2m m 1o 2 quota 5 museums rm antsr wnmumulv In l2135 12136 12 351215 mm on comm 5AA Tum zmr Ireaxmanl h Twme allevuealmenl h 3 GR hIEBYlIUIE germs MGAZIJDXZ R595I6II3 and itGAZIJuxGquot M59072 entude GAZIII ux e C EUIYIEIIE himynthesis and resuunse germs 4639 9mm udes am as lulluw MZgIE Y ERSI MZE I M V and HUI Rt 1375 The Plant 2mm I5 15911534 JulyZl D Auxln Tranpang m cm AUX emu 7 quotmason 7 wm 5 1a 2 32 a Txmc mm mummn 74 can rm 5A4 me any Ilealmavv n m cm Imulmatzd auxm transport AGI at mks are asvnuunw AUXI Augamm PlNzEl l A g57m x 3 pm Augzanan m gumssaom1uwznn3 E Transcription Factor za 513 Daznzszlmndmg putatwotranirlutlml rtqulatmsvmnse Exurzssmlwas requlatul lavaswan a fordar y g 55ml um lzsarz splam 5293mm gunman AGI qtquot ndzsarz asvnuunw AI Ilmuzndnmamlzu up pram 4 mm AlMVB MAYKI mm in nonzaquumm ngzasm 5u mum xn Auqsmzm mumwan 1Ath I475 pm Augum n z fulqerl Augznan 3 2 nger Atzgzrmn mm m gumssaom1uwznn3 ArM YBSB WE F PIF4 A Zinc nger 1 fr Zinc nger 2 om r 39 quot V 39 0 8 18 24 32 40 48 Time after imbibition h The Plant Cell Vol 15 15911604 July 2003 Comprehensive Comparis 11 0f Auxin Regulated and Brassinosteroid Regulated Genes in Arabidopsis H Goda S Sawa T Asami S Fujioka Y Shimada and S Yoshida Plant Physiol 2004 1341555 73 Although numerous physiological studies have addressed the interactions between brassinosteroids and auxins little is known about the underlying molecu ar mechanisms Using an Affymetrix GeneChip representing approximately 8300 Arabidopsis genes we studied comprehensive transcript profiles over 24 h in response to indole 3 acetic acid IAA and brassinolide BL We identified 409 genes as BL inducible 276 genes as IAA inducible and 637 genes in total These two hormones regulated only 48 genes in common suggesting that most ofthe actions ofeach hormone are mediated by gene expression that is unique to each IAA up regulated genes were enriched in genes regulated in common They were induced quickly by IAA and more slowly by BL suggesting divergent physiological roles Expraaakm Laval Signal Lug nan 39bLzail Figure l Gene expression patterns in response to BL ang lAAtreatment sevengayolg m 39 eretreateg with IAA or getz seeglings weretr39 ateg wi h BL T hen transcript apungance 39 39 gapouta rapigopsisgenesAlhe e genes listeg inlaples llll Colors reg to plu gefl to the left orthe column representthe ma nituge oringuction 39n san lues relativeto mocktreateg amples Genes were clustereg h39 rarchically using Gene 39 ott ort 39 column the trees atthe top in blue s 39onal categories ingicateg in D an estaplisheg orputative runctionslhe genes inguceg more than 2fold within 3 h orhormone treatment are genneg as early inguciple genes angthose inguceg petween l2 h ang 24 h are genneg as late inguciple geneslhe numpers orlAA B or BL regulateg genes 0 areshown the frequencies orgenes regulateg py IRA or BL D or both E areshown 55 Au w c Number 039 IM regulated genes m J H mmqu W m M39 D39BL39WQV ME 99quot Genes r mam ham MA and E c m a a u 9 5 3 m a 5 n E z nypocolyl length mm 1 m a m or a 39ucca 39 yucca b v bri WT yucca ygf a bm F39 nre 2 Enlnancerl Hypocotyl Elongation of yucca Mutants Requires Functional BRIl r stage hypoco 1 lengths of 3elleokl plants Error Im39s represent standard error 1 hTeuedzlyeold Vuccayucubri1e116andbriellliseadli r Nemhausel and Chol auxin UP B R down 39 slgnal lransouclion I unknown l cell wallmembrane metahullsm l humlane regulnlion Nemhausel and Chol v D WTYB WTVB BAY EIA A I Nemhauser and Chery 2004 lmcck mm Bn mum and ER Ll Au GarW70 mgmm Mglm39n m mHNAuhlqu n Nemhauser and Chery 2004 Figure Genomic Eff ec 1 ap n shared genes reveal a potential gr0 th hich show elevatedlevel e shown in orange those with dec ea d levels ar in blue and those tran ri s whose lev a 39 e not changed are shown in yellow C Relative ratios 39ere deri ed from the follo ng comparisons from left to right BR r1quot plants B auxin v mock treatment Vl plants A us V1quot Y The three columns to the left are BRiupregulated genes md the three columns to ie right are Br do nregulated genes Among the Br 117 egulated genes there are a large number that are al 0 inducede auxin treatment or 1 a yu background Few Ber el 39 L 11 61 H i nc no change D Effect 39 a hackgrounc Relative r treatment in Vl plants 39 1cca mutant BRiregulated genes 39ere not Quantitative PCR sho that shar 39lt ith both auxin and BRs Ath6lquot Q g E C Nemhauser and Chmy 2004 Plos Biol 2258 Biosynthesis Transport and Metabolism of Plant Hormones AuXins and Cytokinins HS 702 Lecture 2 Steve Clouse Department of Horticultural Science North Carolina State University September 26 2008 RT RECENT EWC Zhao Y 2008 The role of local biosynthesis of auxin and cytokinin in plant development Current Opinion in Plant Biology 1 1 16 22 There are a number of ways to dynamically regulate hormone levels in a given plant tissue including de novo synthesis catabolism conjugation and deconjugation and transport into and out of the tissue In the next three lectures we will examine these processes for each hormone Tip dependent Trpmdepmdent 6 y 6 I Perception 1AA n wRReSPm e 0 1k Storage Protection aminn adds peptidES Transport m sugars mymnosltol Fig 1 Factors affecting 1AA metabolism in plants Arrows 39 cat targets for developmental and tissuespeci c regulnliun quot1 39 r39AI 1 39 muamnma c an by the dashed arrowsV may be limited alrid tissue speci c From Nonnanly 1997 II Auxins Milestones in early auxin research 1 1880 Darwin proposed that a chemical signal in the tip of coleoptiles of canary grass was transported basipetally and mediated bending of the coleoptile towards the light 1885 Salkowski discovered 1AA from non plant fermentation media 1926 Vent describes isolation of an active principle by placing Avena coleoptile tips 011 agar blocks 1928 1935 Thimann isolates 1AA from cultures of Rhizopus mums ent develops Avena coleoptile bioassay 19 16 Haagen Smit et al isolate pure 1AA from endosperm of immature corn seeds Avena coleoptile bioassay Went 1928 Based on the ability of auxin to promote curvature when applied in an agar disk 11 r I I I Id I m gt Mo lipid4N wllmulm humnu Maximaw 4me Wanml yummdm uwi lwkpnll 7 l Ann um m gt WWW Warm ou1Ds sziosi999 3391214199199951 Plili219591995999 Yf 1 92339 As summarized in chapter Bl auxin is synthesized in leaf primordia young leaves and developing seeds Basipetal auxin transport occurs cell to cell in a polar manner and also can occur via the ph oem Auxin promotes cell elongation and division vascular i 39 M J quot root initiation bending in response to light and gravity apical dominance and g 7th of ower rat s Auxin delays leaf senescence and may ei ner promote or inhibit fruit abscission lntlole 3 ace tic acid 1AA is the primary ac ive auxin in plants although indole 3 butyric acid lBA an acetic acid 4 Cl lAA are also found it 39 lAA and lBA can he conjugated via amide linkage to amino acids such as aspartate and by ester linkages to glucose and myo inositol Conjugated forms of lAA may be present at much gher levelsthan free MA and are consr ed to be inactive storage andor transport forms of the hormone Their enzymati release to free lA is an important of lAA metabolis n C lAA biosynthesis 1AA is fanned via the shikirnic acid pathway in which anthtam39lic acid is synthesized from sugar precursors This is the pathway of aromatic amino acid hiosynthesis tryptophan phenylalanine tyrosine lAA can be synthesized directly from tryptophan or by a tryptophan independent pathway Potential pathways of IAA biosy hesis in arabidopsis 14mm h yhmnoirlrdeaxy buloieSrphosphmc quot rip r J s cm r 2 quot7 m 2 i Trpdbcarbmvlnsc e0 v w a iTMi HL i i lt1 m i induibrfrwcmmoxme mm v 1 my HAN 3 r r WOODWARD A W et al Ann Bot 2005 95707735 doi101093aobmci083 Proposed pathways for IAA bio rnthesis Figure 1 Proposed Trp dependent pathways are shown and key intermediates and enzymes discussed in WWWM the text are in bold Trp VV We independent IAA biosynthesis 35333 x is indicated by the dashed arrow and Agrobacterium quotw W A pathway for IAA biosynthesis is indicated by the dotted mm M Pquot arrow IAA indole3acetic NHquot r UN T IAOX i CVPBSE acid IAAId indole3 acetaldehyde IAM indole3 acetamide IAN indole3 I M5 Md I acetonitrile IAOx indole3 acetaldehyde G indole quggx glucosinolate IGP indole3 glycerol phosphate IPA indole3 pyruvic acid NHT Nhydroxyl tryptamine NIE 1acinitro2indonIethane TAM tryptamine Trp tryptophan i a Cold Spmu Harbor Laboramry Press INSERTIONAL MUTAGENESIS Pi I M gem X quotUOMie 1 anquot M A a A w oss 0L 3 memmv39mquot L L n 00 redumu QM ACTIVATION TAGGING T DNA ENHAI JCER yucca hypocotyls are longer than Wildtype yucca has increased lateral root formation compared to Wildtype 0 MJ keg L akb39A39 f M39 Testing auxin levels in the mutant gt free IAA pgmg tissue 0 4 wild type yucca Science l2 Examining the biochemical properties of YUCCA A Fig 3 YUCCA is a ofthe FMohke family A Amino acid onUCCA The putative FAD and NADPH binding motifs near the 09 NH2 terminus and in the middle of the protein VV E quotAD43615 respectively are indicated in bold B Phylogenetic tree of YUC CA and related Arabia opsis genes along with human FMOs Hs we FMO 124 GenBank accession numbers are HEW shown Proteins analyzed here are indicated in SW 5 bold C Tryptamine is a substrate for YUCCA in yitro Reaction mixture with tryptamine as the V isq 130NquotZ DH substrate was separated on a TLC plateThe arrow H points to the enzymatic product 1 is the control and 2 is the reaction The product was characterized by mass spectrometry Y Zhao eta Science 291306 309 2001 13 Published by AAAS 5 5 5 1 AC 022522 AA612577 Intensity cps Ctochrome P450s are revalent in hormone biosnthesis Often catalyze hydroxlation Via molecular oxygen CYP RH NADPH 02 H ROH NADP H20 Often membrane bound endoplasmic reticulum membrane An iron atom Fe3 is part of the active site Occur as very large multi gene families in plants Phenotype of C P79B2 overexpression Figure 2 CYP7932 overexpressors display phenotypes consistent with increased IAA synthesis A CYP79320X line 1D3 shows elongated hypocotyls and epinastic cotyledons compared with Col 0 and appears more similar to known IAA overproducers rooty and yucca B CYP79520x lines have longer hypocotyls than Col 0 From Zhao et 31 gt 8 Overexpression of CYP 79B2 leads to increased endogenous IAA levels on am From 21130 et 2 Free IAA ngg issue Figure 4 Quanti cation oflAA and IAN in CYP79320X lines Free IAA A and IAN B were measured for CYP79320X lines 1D1 1D3 113 and Col 0 grown in low light 18 30 E m 2sec for 6 d after germination at 21 C Values are given in nanograms per gram fresh weight oftissue presented as the mean SEM from three orfour independent samples 16 Hypocolyl Is m m 00 a I OOH 01477932 cyp79l32 43137983 00 0 cyp7953 ggggg Figure 6 Growth phenotypes orthe cyp7952 cyp7953 double mutant A cyp7952 cyp7953 and wild type plants from the Col 0 and ws accession were rown at 21 c under continuous light 2535 m m 2sec and were pho ra hed at 25 d alter germination 50 The cyp7952 cyp7953 double mutant has decreased hypocotyl length compared with wild type ol o po a er germination and were grown at 2 c D s gro cyp7953 douhle mutant plants d ype nt Plantw re grown at 193 under con ous light 2535 m m 2sec and were photographed at the days alter germination indicated have smaller rosette leaves From Zhao et a Metabolism of IAA mph 3 Unvulmxac lump inn Coxlllmscquot r um cm i bomtlalwn A M A m s WDDDWARD A W et al Ann Bot 2005 95707735 doi101093laoblmciw3 m mmp A mm Ar mvm m a m m m a Mum n mm WMMMW x1 mum au m and up w Vrmpmmplnhahnu39 m A mmma inhxvxma r a r uh rm 2 Manel nnlle urntem that men W MW u ex Much enmam an Integral r IAuxlntrallsunrlIxunlarmArahmnuxlx ln DIE llyunenty lullnrexeenee aml ntller 12m ISSlIES auxm quotWIVES m a mule nlauextnwamxthz wmw rants mavemem mm mm m mighlmnmnnln t lrnmtlle uaxe malmal ungtlle rnuetal mm W lncallzallnn nnlle ar er x I emu tnwar enmulex II 1 a IrEct n rnllmlczllxn DIE emex almnr DIE Eumzrmlx Halequot l l quot2 ll Il ludav and l Fig5Mnd2l1nrlh2 elven ntt llncllalasm n all unlar auxm rallsunrl ln eel r dzlery Mung hzlwzznthE ulasma ue u I I msueemeu l en cellular enmuamuem ln cytnellalasm n a Entxarz lraamemeu and unlar auxm lrallSunrl lSrElIl ee ln lamquot Slructura mmm ralmnmlze DIE lncallzallnn nnlle elllux camer enmulex by dlxruutlnu cn meal amquot almnr urevemmg amnmematea veaele Cycling he MPINI a plasma rane protein vssn memhrane segments five and six uure llhe mutation olthe Arabidopsis HldI dnd AIPIHI gen hangesthe phenotypes usis plan dal Y renily laheled ies 39 Sign lluores antibad d gt rha rEr in these B mutants eler v Cytokinirls A Milestones in early cytokinin research 1 1941 Van Overbeek et al showed hat a natura39ly occurring suhsanoe in coconut milk promoted cell prolifera ion in carrot embryos 2 1955 Miller et al isolated 6rfurfurylaminopurine from herring sperm DNA which they named kinetin became of its ability to promote cell division cytokinesis in tobacco pith tissue 1961 Mille reported the identi cation of a kinezirlrlike mpbund in maize zeatin 4 1963Lethamshdwed tha zeatin from rnaize induced cell division aid determined its struaure Naturally occurring cytokinins are derivatives of adenine and are synthesized in root tips and developing seeds Transport is via the xylem from roots to shoots Cytokinins promote cell division and vascular differentiation growth of lateral buds leaf expansion and chloroplast division cytoklnins delay leaf senescence Biosynthesis The basics of thokinin biosynthesis are known but a complete description of tho min biosynthesis has not been obtained in any plant system Cytoki 1in biosynthesis in 39es transfer of isopentenyl pyrophosphate to adenine which is either free or within a hydrolysis and reduction Glucose hose and amino acid conjugates can be formed and cleaved palticularly glucosides which may be storage forms of cytokinins and dihydrozeatin the reduced form of zeatin is commonly found in plant tissue Oxidation The side chain can be cleaved via oxidation to yield free adenine an13 1Ie 2 butenal 35 Structure of Various Cytokinins lsoprenoid CKs V AAOH HN HN N N N N Q0 I gt u kw H N Visopentenylkdenine lranszeatin UP 1 MC HN K N N H ciszeatin OZ r a y manlemnvm and m Jam H m allv2nrll39 atzd V s ummmmumrza annd nnnummjhm am vq ml Hm n m m a m m m w n m n left or m ug mlr p92 Jamnnzuo uaaq mou sun 5 91 qu uyumouio 11 511915 111129 lump m saunizug 1123 1 11ng napI Bymuad n angaaul op ng The LOG gene of rice is e 39 in shoot apical meristems F39gure 3 aquot b39 LOG expresslon In the SAM of wildtype meristems P1 h c e f and m a 50 Kurakawa et al 2007 Nature445652655 200W 39 lug mutants are defective in the maintenance of shoot meiistems leading to a severe reduction of the particle size and abnormal branching patterns the number of oral organs is decreased in L mutants and o ers often contain only one stamen but no pistil thus 39L77lt Kurakawa at al 2007 Nature445652655 LOG directly converts inactive cytokinin nucleotides to the freebase ronns w ich are biologica ly act39 y its cytokininspeci c phosphoribohydrolase actiinty tn inn on i i v a l mu To mw law 2 my nm 3 l V l Altering cytokinin metabolism can affect grain yield in rice Most agriculturally important traits are regulated by genes known as quantitative trait loci QTLS derived from namral allelic variations We here show that a that increases grain productivity in rice Cu or is 39 OsClQQ an enzyme that degrades the plrytohormone cytokinin of OSCKX 2 causes in inflorescencemeristerns and increases the number of 4 77 repro suctrve organs resulting in References Woodward AW and Bartel B 2005 AuXin regulation action and interaction Ann Bot Lend 95 707735 Benjamins R Mat er ca N and Lusclinig C 2005 Regulating the regulator the control of auxin transport Bioessays 27 12461255 Blakeslee J 1 Peer WA and Murphy AS 2005 Auxin transport Curr Opin Plant Biol 8 494500 Leyser O 2005 AuXin distribution and plant pattern formation how many angels can dance on the point of PEN Cell lZl 8l9822 Cohen JD Slovin IP and Hendrickson AM 2003 Two genetically discrete pathways convert tryiitophan to auxin more redundancy in auxin biosynthesis Trends Plant Sci 8 l97l99 Ljung et a 2002 Biosynthesis conjugation catabolism a d homeostasis of indoleBacetic acid in Arabidopsis thaliana Plant Mol Biol 50 309 332 Zhao Y etal 2002 Trydependent aum39n biosyntlresis in Arabidopsisinvolvementof cytochrome P450s CYP79B2 and CYP79B3 GenesDevJa 3100 3112 Muday GK andDeLong A 200i Polar uxin transport Controlling where and how much Trend39s Plant Sci 6 535 Mud39ty39a G K and Murphy AS 2002 An Emerging lVlodel of Auxin Transport Regi atio Plant Cell 1442935299 Normanly J and Bartel B 1999 Redundancy as a way of life 1AA metabolism Cun Opin Plant Biol 2 207 2 13 Normanly J 1997 Auxin metabolism Physiol Plant 100 431 142 Palme K and Galweiler L 1999 PINpointing the molecular basis of auxin transport Current Opinion in Plant Biology 2 375381 C tokii 1s Sakakibara H 2006 Cytokinins activity biosynthesis and translocation Annu Rev Plant Biol 57 431449 Zubko E Adams CJ Machaekova I Malbeck J Scollan C andMeyer P 2002 Activation tagging identi es a gene from Petunia In brida responsible for the production of active cytokinins in plants The Plant Journal 29 7 7 808 Mok DVV Mok MC 2001 Cytokinin metabolism and action Annu Rev Plant Phys1ol Plant Mol Biol 52 89 1 18 Taliei K Saliakibara H Sugiyama T 2001 Identi cation of genes encoding adenylate isopentenyltransferase a cytokinin biosynthesis enzyme in Arabidopsis IIIaIz39ana J Biol Chem 276 26405 26410 Haberer G Kieber JJ 2002 Cytokinins New insights into a classic phytohomlone Plant Physiol 128 354362 Kakimoto T 2001 Identi cation of plant cytokinin biosynthetic enzymes as dimethylally diphosphate ATPu39ADP isopentenyltransferases Plant Cell Physiol 42 677685 HS702 Lecture 6 Cell Expansion and Division Steve Clouse Department of Horticultural Science North Carolina State University October 6 2008 Reading Davies Plant Hormones 2004 Chapter C1 del Pozo et al 2005 Hormonal control of the plant cell cycle PHYSIOLOGIA PLANTARUM 123 173 183 BR IAA or GA application at nM to MM levels causes pronounced elongation of hypocotyls epicotyls and peduncles of dicots as well as r and of Young vegetative tissue is particularly responsive to hormones While both BR and IAA promote elongation their kinetics are quite different Auxin generally shows a very short lag time of 10 to 15 minutes between application and the onset of elongation with maximum rates of elongation reached within 30 to 45 minutes In contrast BR has lag times of at least 45 minutes with elongation rates continuing to increase for several hours Other differences in the effect of auxin and BR on elongation have been observed in physiological and molecular studies Synergisms between BR and auxins occur in many systems Additive effects on elongation are often seen with gibberellins Inhibitory effects of cytokinins abscisic acid and ethylene on BR induced elongation have been described for stem tissue BRade cient dwf4 has shorter cells than wild type 92 7 WV 15 Stem from a 57 weewuxg WHdrtype mam Average 2 Engm s 79 2 a m 10 Hypumerum a 77 dayrmd m4 mam AVEng 2 Engm s 32 2 WW 1m Stem mm a Sweewum m4 mam Average 2 Engm s 15 8 WW A and 1c are atme same he same magnmeanun Ears m A anth1EIEI WW 3 a m g 7k W a 0 5 E s a E a g a Figure 3 BRdoserresponse curve The standard soybean epxcotyl assay was performed on 20 rephcate 15rcm secuons for the mdmared umes atvanous BRconcenLrauons Ermrbars 35 From Clouse eta 1992 mm W me ln Figure l Cunnnunus reccrdrng DfBernduced gruunhrnscybean eprcctyl secncns Gmunh ms lmually recorded rnmsc butler Amer l h BR ms addedtn the rncubaucnmedrum nal cuncen Lm lnM and a gradual accelerancn cl gmunh began after appmmmately 5 mm Flve or mare hnurs were requrrecltn acheve mammum smadyrs39ats Bernduced growth me Zureketal 1994 ll The plant cell wall forms a hrghly crossrllnked rrgd mamxthat opposes cell expanslon and dlfferentlauon ln order forelongtlon and other morphogeneth processes to oocun the cell wall must be modr lied r e by will relaxatron or loosemng and by mcorporauon of new polymers mto the enmdlng wall to mar wal mte Xyloglucans are major constrtuents of the hemlcelluloslc component of the pnmary cell wall m drcotyledonous plants and are thought to be the tethers that attach adjacent cellulose mrcro bnls page or breakage of these tethers would be cnuml to allow cell expansron 7 Thls alterauon of the Xyloglucans must ether be reverslble andor acoompamed by 42 mo synthesrs and moorporatron of new Xyloglucans m order to marntarn wall mtegnty dunng growth Several protelns wth posslble roles 111 cell wall rnods hcauon processes have been ldenu ed lncludlng e gucanases e xyloglucan andotransglyoosylases XETs NOTE xETs have been renamed recentlyas xylugluczn endutxansglucusylasehydmlases XTHs Expanslns Whlle the complauty of the wall has made a de nluve molecular model of wall extenslon eluslve a plauslble scenano has been presented by Cosgrove m whlch e arpanslns are pnrnanlyresponslble for wall relaxauon e gucanases and xETs affect the extent of expansln acuvrty by altenng the Vlsooslty of the hemlrcellulose rnamx e XETs mayfuncuon to lncorporate new xylogucan lnto the growlng wall 7 oellulose blosynthesls would also be expected to occur lt ls known that IAA GAs and ERs alter the blophyslcal propemes of plant oell walls and also lncrease the abundance of mRNA hansmpts for walla rnodltylng protelns such as Km and expansms 7 From Carplta and Glbeaut 1995 a b C d EA UiL r ALL 06 f 1 TP MPa 0 05 03 05 Davies Chapter C1 Figure 5 4i 24z771 lt a 9mcruvm Lazaruer zpblk m 42 me Fassmuxa andey 1992 me Cnsgmve 1997 an al pmcesses are mdmatgdwlth bmken lmes Aquot the mt rpumnual lilffereme dnvmg cellular mm uptake umsllulm r Al len ls a slmpmed mm m m2 mwmg wall lepesema l uysla me mlumltllls m Dellumse ma Wlm nmuelll a Wl 5i eg h 2 F01 new mlya l 2 WWW hmlcalulnses ave Shawn T 252 3 pulymevv gl Flgmz Wmv m l ml m um Wm M Msmvwls m r r m tune me Cusgmve l997 ma 2 up apgwmalaly nan m m2 nmshuwn In Acld GroWLh Theory hr the early mos rt was proposed that protohs may be lnvolved lh Auxmrlnduoed gromh Evldmce for thls theory lhcluded A auxlnrtreated stem segments secrete protons whlch lower the pll of the apoplast cell wall space E treatment of these ussue wth acrdlc buffer pH 5 0 can muse auxm c nemml buffers can lhhlblt auxlnrmduced groth D the fungi toxm fuslcoccln acrdlhes the cell wall and also leads to rapld cell elohgtloh lt was proposed that the lowered pll acuyated wallrloosemng enzymes The propos d mechahlsm of acrd gromh wast lhcreased ATPase lev membrane 6 hat auxin els or actmty by phosphorylauon etc m the whlch resulted lh lhcreased protoh pumplhg to the apoplast ERs may also be lnvolved lh add groth slhce Eernduced expahsloh ls accompahled by proton emusloh and hyperpolahzauoh of cell membranes x r y mum Davies 2004 Chapter C1 Figure 6 mm mil 4 n39bg s 3 f f39ex 51 W a W xw Ms aL El me Raer andCleland 1992 IV Hormones affect mechanical m erties of cell walls Beginning in the 196039s plant physiologists began apply techniques orrowe rom metal engineering LNSTRON stressrstrain analysis in Whlch be swe w a device that created a own ountof e s s then rele dw ea n I lame extensibility of the walls Hormones generally increased plastic extensibility which maimed they were causing wallrloosening ie disruption of bonds in the wall matrix Figure 35mm ufBRun p1asue m11egtaens1b11ny A Snybean cm ep1ee1y1seeuens 15 were 1 mu 139 Lhmkness see quotMatenals and Medunds D213 pmms represent 3 vexege e1 1n repheaue the a segmeme SE 13 Suybean ep1ee1y1seeuens 15 em were Lreamd nth the 1 med cnmentmunns e1 013924 h e r and p1asue exembuny ms me Zurek e1a1 1994 mu Namnmnswhuem pmcwmiunwswnwlHuhm1hx 91w We 1111mm mum m1 mkr r u e w m JHI wmw 1mmw1 21 1111111mm1 rum mMmum imam arm m1 511171 rm mum 14 p 1mm 1mm me Cleland 1986 V Xyloglucan endotransglycosylases XETs now called xyloglucan endotransglucosylasehydrolase XTH The discovery of XET an enzyme that cleaves xyloglucan speci cally and H attaches the cut ends to new xyloglucan acceptors provided a candidate for a reversible wall loosening enzyme involved in expansion growth While no direct biophysical or biochemical evidence is available proving the role of XETs in wall loosening circumstantial evidence for the involvement of xyloglucans and XETs in cell expansion can be presented Both auxins and Ht which induce growth also result in a decrease in mean xyloglucan molecular weight 2 Lectins or antibodies speci c to xyloglucans inhibit elongation 3 XET activity and mRNA levels are often but not always associated with areas of enhanced cell elongation and are often stimulated by growth promoting hormones However an attempt to show a direct effect of XETs on wall loosening in isolated cell wall preparations was unsuccessful while the activity of expansins another class of wall affecting proteins had a direct effect Therefore the role of XETs as wall loosening enzymes remain uncertain but the widespread distribution of XETs and their presence as differentially regulated multi gene families indicates that these enzymes are likely to play an important role in some critical process involving cell walls 19 While much of the accumulated evidence shows a positive correlation between growth and XET transcript and enzyme levels there are numerous exceptions It is often the case that XET levels remain high in tissues when elongation has ceased It is possible that changes in wall structure such as peroxidase mediated phenolic cross linking may prevent the action of XET even though it continues to be regulated by BR GA3 or auxin in non elongating tissue However it is also possible that XET is not involved in wall loosening during elongation but in strengthening of the expanded wall by incorporation of new xyloglucans It has also been proposed that XETs and endoglucanases modify the walls to allow access by expansins Possible roles of XET in Growth 1Waquot 39 sequotquot V V V two 00 cellulose microfibril 390 3 0Aquot A 0A 2 Incorporation of new xyloglucan into growing wall 3 Wall modification during fruit ripening and abscission 4 Formation of perforation plates and sieve plates in developing xylem and phoem 5 Creating access for expansins to reach the cellulose xyloglucan interface 21 In elongating soybean epicotyls BR application resulted in increased plastic extensibility of the walls within two hours with a concomitant increase in the mRNA level of a gene named BRU 1 which encodes an XET Moreover increasing concentrations of applied BR during early stages of elongation lead to a linear increase in extractable XET activity in the epicotyls The BR U1 gene was regulated speci cally by BRs during early stages of elongation and increased expression was not simply the consequence of enhanced elongation Therefore BRUl is likely to play an important role in BR promoted epicotyl elongation in soybean The role of expansins in this system has yet to be examined 5xeu c me Zurek andClnuse 1994 seams me Zurek et a1 1 E Figure 7 umreated RNA was 6 Cumpansnn e the effect m p perfumed m dupueaue densnumetry resul39s were avenged mu Expressmn m seyuearr p ants Tuta rsenatee rre varmus szbEan ergarrs and Rnase pmtecnun as rgure e Seedhngs were frum 2 enmated spruuts stem apmes and rent Ups were rrem we prams ana yzed by desmbed W Va n e s were we apma nuns Epmu were we apma sectmns EpmuMs H were Me n basa sectmns EpmuMs m were 2w apma seeuerrs seeds seed pens and uwers were rrem 757 u p ants The RNasE prutectmn ex enmem was penermee m mphcate and me eerrsrternetry resuus were averaged mam menus rim comm Fig 2 Effect uf bmslncshamld an xyluglucan endmnsglymsylase a W m abuse Wm 1quot epmmyls Lightrgmwn snybean edl re can an wmtwmaan ini me Oh etai 1998 measuremenis r BrassinosteroidR egulated Xyloglucan Endotransglycosylases Gene Specles Methods Reference ERU1 soybean Nortnems RMase Protection zurek and clouse1 in situ hybridization nuclear zurek et al1554 runon on etaI155e 1cm Arabldopsls Nomerns promoterGUS xu et a15551556 LeXet Tomato Nomerns in situ nyb cataia et a1 1557 LeER1 Tomato Nortnems Koka et al 2000 554 TCH4 ENCODES A BRREGULATED XET IN ARABIDOPSIS 39KMENRQEEQWUKE XVAQRLUQWWTWVRQEW 39 WWW 51 RR MB T 39w Hugh MW Q WHORE 39PAW J 399 FM 2 31 th 955 Wlamimhwmmww am MUM xu 2 zl1995PLAMI CELL 7155171567 1 Kzusmmznn 2 2 we Flam 4 97n1r713 VI Expansms Exparsms are pmuams that respo m and pH by affecung 39he mechanmal pmpemes a cell walls and are Lhnught m be a crucial numpunsnt a the cell mu elungauun machmery 1 Assa nfamddndmed thawed and abraded In tam heave eeu mus I e eel dead bu the m1 ah the lsare ve a g tnetueeel extensten rst anuw hut thts may nestuned hy the semen Fm 0 ng 1998 expansmsennndanuw n nt MA at mnnarmnnnrtnerqunervmr heme s et en m mgetxivtymuqozms m t39tt m39m atutmn 39 0151 new Ust mt w e t t an WWW WM mmmtm Wenm ntmttmm mtwtmmm hum Met whslve etennents nspcnvely These hennaneexesphnswe etennents an rmwwwwm penntheses tnateete the dmmce the From Lee et 31 2001 tnmntmnstte quot5 2 w 211 am my 2339 a F me Darleyetal 2mm mama af cm exhypa atny A mm hm Frgwl Th2 an h c Film 5 maimed cucum claw E man 17 mRNA abundance m mp Nanhzm him pmhed mm 14mm demmn manly am acmylxecnam by wand pnclpnaunn dzmmnng gel 11 mzmhnnz by capillary mama ham24 l A maul 13m lama ntvr r lnmrm wauupnmmbvaaw mg was gtltmrltrmrlt zwamlmam mm mamv quotEma pn puma V Microtubule and cellulose micro bril orientation In order for elongation to occur cellulose micro brils must be transversely oriented not longitudinally Cell expansion also depends on an adequate supply of wall components and recent work con rmed the inhibition of BR induced elongation in stem tissue by inhibitors of cellulose biosynthesis or microtubule orientation Brassinolide alone or in combination with auxin enhanced the percentage of transversely oriented cortical microtubules The orientation of cortical microtubules which generally correlates with the orientation of micro brils follows a cyclic pattern and phosphorylation of proteins possibly those linking the microtubules to the plasmalemma is an essential component in the maintenance of transversely oriented microtubules BR induced elongation also depends on such phosphorylation VI Control of cell division in eukaryotes A Cell division in eukaryotes consists of I The S phase in which DNA replication takes place I The G2 phase in which the cell prepares for mitosis I The M phase in which mitosis chromosome condensation nuclear envelope breakdown assembly of mitotic apparatus sister chromatid segregation cell plate formation takes place I And the G1 phase which is a resting state between M and S B The cycling between these phases is controlled by cyclin dependent protein kinases CDK and protein regulatory subunits called cyclins Neither one is active by itself The CDKcyclin complex is controlled by transcription phosphorylation state and cellular localization which are in turn controlled by signal transduction pathways for growth factors and hormones C Auxin and cytokinins and sometimes gibberellins and brassinosteroids are known to control cell division in plants Cell cycle modes during development n m uferauan Aai eremiar n cs Dime m an 39 endaredupucaum mm 1 cm aye made man dzvelnpmzm Dnrmg the yuw hafa muhcelhdar arganum mm c2 aye pmyamx ale mum Typmuy mwnngand 12x 03 mm c2 c mad accnlx hywhlch Mann md apmhiuauve ya mancelkale g e Mm Mum nuarceupmmmmn mmmdwu 1y gunman c vex hefme camphm wnhdlawa m ce c 1 and Eye Haw hum y slang wnh m enn a an many p an mm can with mm 3 mm c2 aye m an endandnphca an cycle In which m mlclzar DNA became quotpuma mum xuhxequzm nuclear and um dwumn leadmg m palyplmd cell as Vemest 2 2 Flam Physlnl 2nn5s 1391nw711n6 quot pg 1 pathway mmmumg 5 39M m tthmcexxexleqmledfm WW my W mmmmmsr lt a 1mm 2 J azahxcmcacdeXEMand cm m V Mama n ce cyclzngula n W cm amman Wm quotBquot m mzc l awn mwxpmmmpamyum m m ZS havemwuheen f L Mammy m zruum m a 13mm qwnan CDKracnvanng lame JAKE f WWW cD39n Cd m mgquot me Guuemezetal mm 35 0 3 From DeWi e Murra CYCD3 T CDKBT EZFc p 7 n I In A39 39A ll cells in G1 H associzu39on EZFrrgulztedgenes including CD06 CYCD31 Ech and CD are thereby aclivzte e conuol incorporates posiu39ve and negzu39ve feedback loops because CYCD31 an 12212 target as a the Ech which lacks 37 may m m m Leann m x 4w W mmn 93 3 upsm m or m a K m aw hum WW mm mmmnlm 38 WISH CchZ1 Cch3l CyCDS m Luading comm mum 3 Grthuhamnwznnzm unnenua ry ErsuhculturE r39 Name that 0103392 IS appmxumately m In 2nvnm mm abundantthan the nthEr twn LYclInx me Smell etal 1999 m u quotmy quot wwwwmw n D EmisswnoA39CYCD mm dunrg mm 3911 Ce Eye m mm an m me Smell etal 1999 w An md psls Nanhem rm H mm 21 9 adznmz 1mm 91am m E a mama byakadmc a punnz s hmpemenyhdzmm z wenmc n ma unmawa cell suspnsmn animus 14 mm 22 Wu pmbed wnhCchZ Early smmmxyce s a9 7 m a absence a m y a c 2mm 15 dOA F Addmnnafl by um um stanms mumcs mm 25mm ulnanxsmeAmS D blac bath me RmuKhamlmh etal 1999 me RmuKhamlmh etal 1999